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Clinical pharmacology of ertapenem in the treatment of multidrug-resistant tuberculosisvan Rijn, Sander Pascal
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Clinicalpharmacologyof
ertapeneminthetreatmentof
Multidrug-resistantTuberculosis
SanderPascalvanRijn
VanRijnS.P.ClinicalpharmacologyofertapeneminthetreatmentofMultidrug-resistantTuberculosis
Thesis,UniversityofGroningen,TheNetherlandsPublicationof this thesiswas financially supportedbyUniversity ofGroningen,UniversityMedical Center Groningen, Graduate School of Medical Sciences, KNCV tuberculosisFoundation, Royal Dutch Pharmacists Association (KNMP), Stichting Beatrixoord Noord-Nederland,PharmIntelandTerWelle&Associés(TW&A).
Cover MaartenKarremans–MardoniCreativeProductionLay-out SandervanRijnPrintedby GVOdrukkers&vormgeversB.V.ISBN 978-94-6332-473-1©Copyright2018.S.P.vanRijn,Groningen,TheNetherlandsAllrightsreserved.Nopartsofthisthesismaybereproduced,storedinaretrievalsystem,ortransmitted in any form or by any means, electronical, mechanical, by photocopying,recordingorotherwise,withoutthepriorwrittenpermissionoftheauthor.
Clinical pharmacology of ertapenem in the treatment of
Multidrug-resistant Tuberculosis
Proefschrift
ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen
op gezag van de rector magnificus prof. dr. E. Sterken
en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op
woensdag 10 april 2019 om 12.45 uur
door
Sander Pascal van Rijn
geboren op 12 februari 1988 te Bochum (BRD)
VanRijnS.P.ClinicalpharmacologyofertapeneminthetreatmentofMultidrug-resistantTuberculosis
Thesis,UniversityofGroningen,TheNetherlandsPublicationof this thesiswas financially supportedbyUniversity ofGroningen,UniversityMedical Center Groningen, Graduate School of Medical Sciences, KNCV tuberculosisFoundation, Royal Dutch Pharmacists Association (KNMP), Stichting Beatrixoord Noord-Nederland,PharmIntelandTerWelle&Associés(TW&A).
PharmIntel Cover MaartenKarremans–MardoniCreativeProductionLay-out SandervanRijnPrintedby GVOdrukkers&vormgeversB.V.ISBN 978-94-6332-473-1©Copyright2018.S.P.vanRijn,Groningen,TheNetherlandsAllrightsreserved.Nopartsofthisthesismaybereproduced,storedinaretrievalsystem,ortransmitted in any form or by any means, electronical, mechanical, by photocopying,recordingorotherwise,withoutthepriorwrittenpermissionoftheauthor.
Clinical pharmacology of ertapenem in the treatment of
Multidrug-resistant Tuberculosis
Proefschrift
ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen
op gezag van de rector magnificus prof. dr. E. Sterken
en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op
woensdag 10 april 2019 om 12.45 uur
door
Sander Pascal van Rijn
geboren op 12 februari 1988 te Bochum (BRD)
Promotores
Prof.dr.J.W.C.Alffenaar
Prof.dr.T.S.vanderWerf
Prof.dr.J.G.W.Kosterink
Beoordelingscommissie
Prof.dr.B.Wilffert
Prof.dr.R.vanCrevel
Prof.dr.J.W.A.Rossen
Tableofcontents
Chapter1 GeneralIntroduction
Chapter2 Evaluation of Carbapenems for Treatment of Multi- and Extensively Drug-
ResistantMycobacteriumTuberculosis
Chapter3 QuantificationandValidationofErtapenemUsingaLiquidChromatography-
TandemMassSpectrometryMethod
Chapter4 Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant
Tuberculosis
Chapter5 Susceptibility Testing of Antibiotics That Degrade Faster than the Doubling
Time of Slow-Growing Mycobacteria: Ertapenem Sterilizing Effect Versus
MycobacteriumTuberculosis
Chapter6 Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of
TuberculosisandImplicationsonClinicalDosing
Chapter7 PharmacokineticModellingandLimitedSamplingStrategiesBasedonHealthy
VolunteersforMonitoringofErtapeneminPatientswithMultidrug-resistant
Tuberculosis
Chapter8 GeneralDiscussionandFuturePerspectives
Chapter9 Summary
Chapter10 SamenvattingDankwoordAbouttheAuthorPublicationList
Promotores
Prof.dr.J.W.C.Alffenaar
Prof.dr.T.S.vanderWerf
Prof.dr.J.G.W.Kosterink
Beoordelingscommissie
Prof.dr.B.Wilffert
Prof.dr.R.vanCrevel
Prof.dr.J.W.A.Rossen
Tableofcontents
Chapter1 GeneralIntroduction
Chapter2 Evaluation of Carbapenems for Treatment of Multi- and Extensively Drug-
ResistantMycobacteriumTuberculosis
Chapter3 QuantificationandValidationofErtapenemUsingaLiquidChromatography-
TandemMassSpectrometryMethod
Chapter4 Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant
Tuberculosis
Chapter5 Susceptibility Testing of Antibiotics That Degrade Faster than the Doubling
Time of Slow-Growing Mycobacteria: Ertapenem Sterilizing Effect Versus
MycobacteriumTuberculosis
Chapter6 Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of
TuberculosisandImplicationsonClinicalDosing
Chapter7 PharmacokineticModellingandLimitedSamplingStrategiesBasedonHealthy
VolunteersforMonitoringofErtapeneminPatientswithMultidrug-resistant
Tuberculosis
Chapter8 GeneralDiscussionandFuturePerspectives
Chapter9 Summary
Chapter10 SamenvattingDankwoordAbouttheAuthorPublicationList
CHAPTER 1
General Introduction
CHAPTER 1
General Introduction
8
Chapter 1
GeneralIntroduction
Tuberculosis (TB) is caused byMycobacterium tuberculosis. TB is the deadliest infectious
diseaseworldwide. Itmostlyaffects the lungs,butcanalsoattackotherorgans; together,
theseformsofTBarereferredtoasextrapulmonary.Lymphnodes,thepleuralandperitoneal
space;theaxialskeleton;thegut;theurogenitalsystem;andthemostlethalform:thecentral
nervous system can all be affected. In 2017, 10 million new cases were reported, and
approximately1.6millionpeoplediedofTB[1].Anestimated1millionchildrenfellillwith
TB,aquarterofwhomdied.TBhasaglobalimpact,howeverover95%ofTBdeathsoccurin
low-andmiddle-incomecountries,with61%ofallnewcasesreportedinAsiaand26%ofnew
casesinAfrica,withsixcountriesaccountingfor60%ofthistotal.Anincreaseinrefugeeflow,
increasedtravellingandglobalization,socialinequalityandpoverty,lackofsafewater,poor
sanitationandpoorhygieneservicesareimportantriskfactorsforTB.Asimmigrationand
internationaltraveliscommoninaffluentregions,TBoughttobeaconcernforhighincome
countries[1-3].
Tuberculosisistransmittedviarespiratorydroplets,microbescarriedindropletsoraerosols
loadedwithM.tuberculosis froman infectedpersonviaclosepersonalcontact,coughing,
sneezingandlaughing.Onlyasmallminority–anestimated5-10%ofallinfectedindividuals
-willeverdevelopactiveTBwithsymptomssuchasproductivecoughwithpurulentsputum
thatmaycontainblood,weightloss,fatigueandnightsweats;besides,dependingonthesite
of disease manifestation, people may experience chest pain, back pain, abdominal pain,
headache,andseizures.TheimmuneresponseofmostpeoplesuccessfullyfightsoffTBbacilli
thatmaybekilledbyactivatedmacrophageimmunecells,withTBbacilliinthephagosome-
lysosome.TBbacillimayhoweveralsosurvivewithinthephagosomeofthesemacrophages,
andinthelattercase,thesepeoplearesaidtobelatentlyinfected.Theirbacillisurviveina
hibernatingmode,controlledbyageneticsystemreferredtoasdosRregulon[4].Thisisa
geneticprogramcontrolledbyasetof48genes thatallows the tuberclebacilli tosurvive
understressconditions;duringactiveimmunesuppression,hibernatingorganismsarecalled
‘latent’whileduringdrugtreatment,theseorganismsarecalled‘persistent’.Latentlyinfected
individualstypicallyhaveonlylimitednumbersof livingbacterialcellsthatslowlyreplicate
and are hardly metabolically active. People with latent TB infection feel well, have no
symptomsandarenotcontagious[5].
M. tuberculosis belongs to a small group of highly pathogenic bacteria (M. tuberculosis
complex)intheverylargefamilyofmycobacteria,characterizedbyathickcellwall,consisting
ofseveraldifferentspecificlipidmoleculesincludinglipoarabinomannanandmycolicacids.
Themajorityofcrosslinksinthepeptidoglycanlayerareformeddifferentlycomparedtogram
positivebacteria,makingmycobacteriamoreresistanttochemicaldamageandhydrophilic
antibiotics. As the replication rate ofM. tuberculosis – even if actively replicating and
metabolicallyactive-isveryslow(≈20h)comparedtootherbacteria(≈20min),TBrequires
morespecificantibioticsandprolongedtreatment,asmostantibioticsonlyworkonactively
replicatingbacteria[5].Typically,rapidlydividingmetabolicallyactivebacillicanbereduced
rapidlywithinweeks; severalhighlyactiveagentshavebactericidalproperties.Dueto the
slowreplicationrateofM.tuberculosis,especiallyofdifficulttoeradicatepersisterphenotype
bacteria. TB treatment needs to last long to obtain a sterilising effect. TheWorld Health
Organisation (WHO) therefore advises to threat TB with a standard first-line treatment
consistingofisoniazid(H),Rifampicin(R),pyrazinamide(Z)andethambutol(E)–HRZE–And
therebyintensivelydecreasethebacterialload(intensivephase).Followedbytheintensive
phase, a four-month continuationwith isoniazid and rifampicin is needed to provide the
opportunity to eliminate the last TB bacteria which are in a persistent state of being
capsulatedbymacrophages.
MDRTB-AntimicrobialresistanceofTB
Unfortunately, our world is facing a public health crisis and security threat due to the
treatmentofTBbecomingincreasinglychallengingwiththeemergenceofresistancetofirst-
linedrugs.Multidrug resistant (MDR)-TB is definedas an infectiousdisease causedbyM.
tuberculosisthatisresistanttoatleastisoniazidandrifampicin,whicharethecornerstone
drugs of drug-susceptible TB treatment. Extensively drug resistant (XDR)-TB is defined as
MDR-TBwithadditionalresistancetoatleastoneofthefluoroquinolonesandtoatleastone
oftheinjectablesecondlinedrugs[6].WHOestimatesthattherewere558.000newcases
withresistancetorifampicin,themosteffectivefirstlinedrug[2].
Development of new drugs is slow and expensive due to the obligatory market access
regulationssuchasrandomizedclinicaltrials.BedaquilineandDelamanid,bothwithanovel
mechanismofaction,were included intheWHOguidelinesonMDR-TB,afterapprovalby
9
1
General Introduction
GeneralIntroduction
Tuberculosis (TB) is caused byMycobacterium tuberculosis. TB is the deadliest infectious
diseaseworldwide. Itmostlyaffects the lungs,butcanalsoattackotherorgans; together,
theseformsofTBarereferredtoasextrapulmonary.Lymphnodes,thepleuralandperitoneal
space;theaxialskeleton;thegut;theurogenitalsystem;andthemostlethalform:thecentral
nervous system can all be affected. In 2017, 10 million new cases were reported, and
approximately1.6millionpeoplediedofTB[1].Anestimated1millionchildrenfellillwith
TB,aquarterofwhomdied.TBhasaglobalimpact,howeverover95%ofTBdeathsoccurin
low-andmiddle-incomecountries,with61%ofallnewcasesreportedinAsiaand26%ofnew
casesinAfrica,withsixcountriesaccountingfor60%ofthistotal.Anincreaseinrefugeeflow,
increasedtravellingandglobalization,socialinequalityandpoverty,lackofsafewater,poor
sanitationandpoorhygieneservicesareimportantriskfactorsforTB.Asimmigrationand
internationaltraveliscommoninaffluentregions,TBoughttobeaconcernforhighincome
countries[1-3].
Tuberculosisistransmittedviarespiratorydroplets,microbescarriedindropletsoraerosols
loadedwithM.tuberculosis froman infectedpersonviaclosepersonalcontact,coughing,
sneezingandlaughing.Onlyasmallminority–anestimated5-10%ofallinfectedindividuals
-willeverdevelopactiveTBwithsymptomssuchasproductivecoughwithpurulentsputum
thatmaycontainblood,weightloss,fatigueandnightsweats;besides,dependingonthesite
of disease manifestation, people may experience chest pain, back pain, abdominal pain,
headache,andseizures.TheimmuneresponseofmostpeoplesuccessfullyfightsoffTBbacilli
thatmaybekilledbyactivatedmacrophageimmunecells,withTBbacilliinthephagosome-
lysosome.TBbacillimayhoweveralsosurvivewithinthephagosomeofthesemacrophages,
andinthelattercase,thesepeoplearesaidtobelatentlyinfected.Theirbacillisurviveina
hibernatingmode,controlledbyageneticsystemreferredtoasdosRregulon[4].Thisisa
geneticprogramcontrolledbyasetof48genes thatallows the tuberclebacilli tosurvive
understressconditions;duringactiveimmunesuppression,hibernatingorganismsarecalled
‘latent’whileduringdrugtreatment,theseorganismsarecalled‘persistent’.Latentlyinfected
individualstypicallyhaveonlylimitednumbersof livingbacterialcellsthatslowlyreplicate
and are hardly metabolically active. People with latent TB infection feel well, have no
symptomsandarenotcontagious[5].
M. tuberculosis belongs to a small group of highly pathogenic bacteria (M. tuberculosis
complex)intheverylargefamilyofmycobacteria,characterizedbyathickcellwall,consisting
ofseveraldifferentspecificlipidmoleculesincludinglipoarabinomannanandmycolicacids.
Themajorityofcrosslinksinthepeptidoglycanlayerareformeddifferentlycomparedtogram
positivebacteria,makingmycobacteriamoreresistanttochemicaldamageandhydrophilic
antibiotics. As the replication rate ofM. tuberculosis – even if actively replicating and
metabolicallyactive-isveryslow(≈20h)comparedtootherbacteria(≈20min),TBrequires
morespecificantibioticsandprolongedtreatment,asmostantibioticsonlyworkonactively
replicatingbacteria[5].Typically,rapidlydividingmetabolicallyactivebacillicanbereduced
rapidlywithinweeks; severalhighlyactiveagentshavebactericidalproperties.Dueto the
slowreplicationrateofM.tuberculosis,especiallyofdifficulttoeradicatepersisterphenotype
bacteria. TB treatment needs to last long to obtain a sterilising effect. TheWorld Health
Organisation (WHO) therefore advises to threat TB with a standard first-line treatment
consistingofisoniazid(H),Rifampicin(R),pyrazinamide(Z)andethambutol(E)–HRZE–And
therebyintensivelydecreasethebacterialload(intensivephase).Followedbytheintensive
phase, a four-month continuationwith isoniazid and rifampicin is needed to provide the
opportunity to eliminate the last TB bacteria which are in a persistent state of being
capsulatedbymacrophages.
MDRTB-AntimicrobialresistanceofTB
Unfortunately, our world is facing a public health crisis and security threat due to the
treatmentofTBbecomingincreasinglychallengingwiththeemergenceofresistancetofirst-
linedrugs.Multidrug resistant (MDR)-TB is definedas an infectiousdisease causedbyM.
tuberculosisthatisresistanttoatleastisoniazidandrifampicin,whicharethecornerstone
drugs of drug-susceptible TB treatment. Extensively drug resistant (XDR)-TB is defined as
MDR-TBwithadditionalresistancetoatleastoneofthefluoroquinolonesandtoatleastone
oftheinjectablesecondlinedrugs[6].WHOestimatesthattherewere558.000newcases
withresistancetorifampicin,themosteffectivefirstlinedrug[2].
Development of new drugs is slow and expensive due to the obligatory market access
regulationssuchasrandomizedclinicaltrials.BedaquilineandDelamanid,bothwithanovel
mechanismofaction,were included intheWHOguidelinesonMDR-TB,afterapprovalby
10
Chapter 1
FederalDrugAdministration(FDA)andEuropeanMedicinesAgency(EMA)almostfiveyears
ago.Anindividualpatientdatameta-analysisrevealedthatofalldrugsusedtotreatMDR-TB,
theaddedvalueoftheinjectableagents’kanamycinandcapreomycinwasactuallyassociated
with poor outcome. Bedaquiline; the fluoroquinolones levofloxacin, gatifloxacin and
moxifloxacin;andlinezolidwereassociatedwithbeneficialoutcome[7].Basedonthismeta-
analysis,WHOissuedarapidcommunicationupdatingtheprovisionalguidelinesforMDR-TB
treatment [8]. Bedaquiline has now obtained a position in Group A; together with
FluoroquinolonesandLinezolid,thisdrugisnowconsideredamongthemostpowerfulagents
tofightMDR-TB.Unfortunately,resistancetothesenovelagentshasalreadybeendetected
[9].Obviously,thecostsofthesehighlyeffectivenovelagentsareaconstraintforuseinlow-
resourcedsettings.
The challenges to eradicate TB by 2030 are vast;many of the second line drugs are also
associatedwith toxicityandadverseeffects;and there is thereforeadesire foradditional
drugs with low inherent toxicity. Apart from developing additional new drugs, the
repurposingofdrugsthatarealreadyavailable forother indicationswouldbeanasset to
improveandextendcurrenttreatmentoptions,bydevelopingmoreactive–sterilizing-anti
TBdrugs[10-11].Multiplepartnershipshavebeeninitiatedwiththejointgoaloferadicating
resistancebydevelopingandproducingnewdrugsandrediscoveringolddrugs.
Rediscoveryofolddrugs
Oneparticularlyeffectivestrategy isrediscoveryofolddrugsasnewagentsfortreatment
against multidrug resistant tuberculosis. Linezolid and moxifloxacin already have been
exploredasnewagentsagainstMDR-TB[12-15].Benefitsofrepurposingoldantibioticsisthat
thesedrugsare commonly cheapandclinicalexperience is substantial resulting inawell-
establisheddrugsafetyprofile.TounlocktheirpotentialasnewTBagentsandobtainmarket
approval, efficacy, safety and toxicity profile needs to be established. Therefore, a
pharmacokinetic andpharmacodynamicsprofileneeds tobeestablishedanddose-finding
studiesareneededtoestablishrequireddoseinTBpatients.
Nowadays, beta-lactam antimicrobial drugs arewidely used drugs for the treatment of a
rangeofinfections[16].BeginningwiththediscoveryofpenicillinbyAlexanderFleminginthe
late1920s,antibioticschangedtreatmentofbacterialinfections,savingmillionsoflives.By
mid-1940 itbecameclearthatpenicillinwasnoteffectiveatkillingM.tuberculosis. Inthe
1960s it became clear that M. tuberculosis produced an enzyme, called beta-lactamase
(BLaC),whichrapidlyhydrolysis’sthebeta-lactamring.Carbapenemactivityhavetherefore
longbeenconsideredtobeoflimiteduse.However,morethanadecadeago,researchers
showedthatclavulanateirreversiblyblockedbeta-lactamaseenzymeofM.tuberculosis[17].
Recent studies suggest that beta-lactams, using clavulanate/clavulanic acid, show more
activityagainstM.tuberculosisandcouldbebeneficialinthetreatmentofTB[18-22].
CarbapenemsareearmarkedaspotentiallyactivedrugsforthetreatmentofM.tuberculosis.
Imipenem-cilastatinandmeropenemhavebeenlistedasadd-ondrugsintheupdatedWHO
treatment guidelines. Ertapenem, approved in 2001 by the FDA, an old drugwidely used
againstgrampositiveandnegativebacteriahasshowntobeactive inMDR-TB [23-25]. In
general,ertapenemappearstobefavourableandahighlypromisingdrugforthetreatment
ofMDR-TBthatwarrantsfurtherinvestigation.
Aimofthethesis
TobetterunderstandthepotentialroleofertapenemforthetreatmentofM/XDR-TB,the
aimofthisthesiswastoevaluatecurrentliterature,invitroactivity,andpharmacokinetics
andsafetyinTBpatients.
11
1
General Introduction
FederalDrugAdministration(FDA)andEuropeanMedicinesAgency(EMA)almostfiveyears
ago.Anindividualpatientdatameta-analysisrevealedthatofalldrugsusedtotreatMDR-TB,
theaddedvalueoftheinjectableagents’kanamycinandcapreomycinwasactuallyassociated
with poor outcome. Bedaquiline; the fluoroquinolones levofloxacin, gatifloxacin and
moxifloxacin;andlinezolidwereassociatedwithbeneficialoutcome[7].Basedonthismeta-
analysis,WHOissuedarapidcommunicationupdatingtheprovisionalguidelinesforMDR-TB
treatment [8]. Bedaquiline has now obtained a position in Group A; together with
FluoroquinolonesandLinezolid,thisdrugisnowconsideredamongthemostpowerfulagents
tofightMDR-TB.Unfortunately,resistancetothesenovelagentshasalreadybeendetected
[9].Obviously,thecostsofthesehighlyeffectivenovelagentsareaconstraintforuseinlow-
resourcedsettings.
The challenges to eradicate TB by 2030 are vast;many of the second line drugs are also
associatedwith toxicityandadverseeffects;and there is thereforeadesire foradditional
drugs with low inherent toxicity. Apart from developing additional new drugs, the
repurposingofdrugsthatarealreadyavailable forother indicationswouldbeanasset to
improveandextendcurrenttreatmentoptions,bydevelopingmoreactive–sterilizing-anti
TBdrugs[10-11].Multiplepartnershipshavebeeninitiatedwiththejointgoaloferadicating
resistancebydevelopingandproducingnewdrugsandrediscoveringolddrugs.
Rediscoveryofolddrugs
Oneparticularlyeffectivestrategy isrediscoveryofolddrugsasnewagentsfortreatment
against multidrug resistant tuberculosis. Linezolid and moxifloxacin already have been
exploredasnewagentsagainstMDR-TB[12-15].Benefitsofrepurposingoldantibioticsisthat
thesedrugsare commonly cheapandclinicalexperience is substantial resulting inawell-
establisheddrugsafetyprofile.TounlocktheirpotentialasnewTBagentsandobtainmarket
approval, efficacy, safety and toxicity profile needs to be established. Therefore, a
pharmacokinetic andpharmacodynamicsprofileneeds tobeestablishedanddose-finding
studiesareneededtoestablishrequireddoseinTBpatients.
Nowadays, beta-lactam antimicrobial drugs arewidely used drugs for the treatment of a
rangeofinfections[16].BeginningwiththediscoveryofpenicillinbyAlexanderFleminginthe
late1920s,antibioticschangedtreatmentofbacterialinfections,savingmillionsoflives.By
mid-1940 itbecameclearthatpenicillinwasnoteffectiveatkillingM.tuberculosis. Inthe
1960s it became clear that M. tuberculosis produced an enzyme, called beta-lactamase
(BLaC),whichrapidlyhydrolysis’sthebeta-lactamring.Carbapenemactivityhavetherefore
longbeenconsideredtobeoflimiteduse.However,morethanadecadeago,researchers
showedthatclavulanateirreversiblyblockedbeta-lactamaseenzymeofM.tuberculosis[17].
Recent studies suggest that beta-lactams, using clavulanate/clavulanic acid, show more
activityagainstM.tuberculosisandcouldbebeneficialinthetreatmentofTB[18-22].
CarbapenemsareearmarkedaspotentiallyactivedrugsforthetreatmentofM.tuberculosis.
Imipenem-cilastatinandmeropenemhavebeenlistedasadd-ondrugsintheupdatedWHO
treatment guidelines. Ertapenem, approved in 2001 by the FDA, an old drugwidely used
againstgrampositiveandnegativebacteriahasshowntobeactive inMDR-TB [23-25]. In
general,ertapenemappearstobefavourableandahighlypromisingdrugforthetreatment
ofMDR-TBthatwarrantsfurtherinvestigation.
Aimofthethesis
TobetterunderstandthepotentialroleofertapenemforthetreatmentofM/XDR-TB,the
aimofthisthesiswastoevaluatecurrentliterature,invitroactivity,andpharmacokinetics
andsafetyinTBpatients.
12
Chapter 1
OUTLINEOFTHETHESIS
In this thesis, we plan to evaluate the pharmacology of ertapenem in the treatment of
multidrugresistanttuberculosis.
inchapter2,Weplantostudyliteraturetoevaluatecurrentknowledgeoninvitro,invivo
andhumanactivityofcarbapenems
In chapter 3 we aim to develop a simple validated LC-MS/MS for the validation and
quantificationofertapenemrequiredforfuturepharmacokineticstudies.
Inchapter4weplantoevaluatepharmacokineticsandsafetyofertapenemusedtocomplete
atreatmentregimenforMDRTBpatients
Inchapter5weaimtodevelopasuitableexperiment toevaluate thesusceptibilityofM.
tuberculosisforertapenemasthecurrentlyusedassaysarenotsuitablebecauseertapenem
degradesfastunderstandardconditions(37C)
Inchapter6,weintendtostudythesterilizingeffectofertapenem-clavulanateinahollow
fibermodeloftuberculosistoselectadoseforfutureclinicalstudies
Inchapter7,weproposetodevelopapharmacokineticmodelandalimitedsamplingstrategy
whichcouldbeusedforafuturephaseIIstudy
References
1. Tuberculosisfactsheet.2018;Availableat:http://www.who.int/en/news-room/fact-
sheets/detail/tuberculosis
2. WHOreport:GlobalTuberculosisreport2017.2017.http://www.who.int/tb/en/
3. National Institute for Public Health and the Environment (RIVM). 2017. State of
infectiousdiseasesinTheNetherlands,2016.NationalInstituteforPublicHealthand
theEnvironment,Bilthoven,TheNetherlands.
4. VanAltena,DuggiralaS,GröschelMI,vanderWerf.2011.Immunologyintuberculosis:
challengesinmonitoringofdiseaseactivityandidentifyingcorrelatesofprotection.
CurrPharmDes.2011;17(27):2853-62.
5. WorldHealthOrganization.GuidelinesfortreatmentofTuberculosis.Fourthedition
ed.Geneva,Switserland:WorldHealthOrganization;2010.
6. FalzonD, SchünemannHJ, Harausz E, González-Angulo L, Lienhardt C, Jaramillo E,
Weyer K. 2017World Health Organization treatment guidelines for drug-resistant
tuberculosis,2016update.EurRespirJ.Mar22;49(3).
7. Collaborative Group for the Meta-Analysis of Individual Patient Data in MDR-TB
treatment–2017, Ahmad N, Ahuja SD, Akkerman OW, Alffenaar JC, Anderson LF,
BaghaeiP,BangD,BarryPM,BastosML,BeheraD,BenedettiA,BissonGP,Boeree
MJ,BonnetM,BrodeSK,BrustJCM,CaiY,CaumesE,CegielskiJP,CentisR,ChanPC,
ChanED,ChangKC,CharlesM,CiruleA,DalcolmoMP,D'AmbrosioL,deVriesG,Dheda
K,EsmailA,FloodJ,FoxGJ,Fréchet-JachymM,FregonaG,GayosoR,GegiaM,Gler
MT,Gu S,Guglielmetti L,Holtz TH,Hughes J, Isaakidis P, Jarlsberg L, KempkerRR,
KeshavjeeS,KhanFA,KipianiM,KoenigSP,KohWJ,KritskiA,KuksaL,KvasnovskyCL,
KwakN,LanZ,LangeC,Laniado-LaborínR,LeeM,LeimaneV,LeungCC,LeungEC,Li
PZ,LowenthalP,MacielEL,MarksSM,MaseS,MbuagbawL,MiglioriGB,MilanovV,
MillerAC,MitnickCD,ModongoC,MohrE,MonederoI,NahidP,NdjekaN,O'Donnell
MR,PadayatchiN,PalmeroD,PapeJW,PodewilsLJ,ReynoldsI,RiekstinaV,RobertJ,
RodriguezM,SeaworthB,SeungKJ,SchnippelK,ShimTS,SinglaR,SmithSE,SotgiuG,
SukhbaatarG,TabarsiP,TiberiS,TrajmanA,TrieuL,UdwadiaZF,vanderWerfTS,
VezirisN,ViikleppP,VilbrunSC,WalshK,WestenhouseJ,YewWW,YimJJ,ZetolaNM,
Zignol M, Menzies D. Treatment correlates of successful outcomes in pulmonary
13
1
General Introduction
OUTLINEOFTHETHESIS
In this thesis, we plan to evaluate the pharmacology of ertapenem in the treatment of
multidrugresistanttuberculosis.
inchapter2,Weplantostudyliteraturetoevaluatecurrentknowledgeoninvitro,invivo
andhumanactivityofcarbapenems
In chapter 3 we aim to develop a simple validated LC-MS/MS for the validation and
quantificationofertapenemrequiredforfuturepharmacokineticstudies.
Inchapter4weplantoevaluatepharmacokineticsandsafetyofertapenemusedtocomplete
atreatmentregimenforMDRTBpatients
Inchapter5weaimtodevelopasuitableexperiment toevaluate thesusceptibilityofM.
tuberculosisforertapenemasthecurrentlyusedassaysarenotsuitablebecauseertapenem
degradesfastunderstandardconditions(37C)
Inchapter6,weintendtostudythesterilizingeffectofertapenem-clavulanateinahollow
fibermodeloftuberculosistoselectadoseforfutureclinicalstudies
Inchapter7,weproposetodevelopapharmacokineticmodelandalimitedsamplingstrategy
whichcouldbeusedforafuturephaseIIstudy
References
1. Tuberculosisfactsheet.2018;Availableat:http://www.who.int/en/news-room/fact-
sheets/detail/tuberculosis
2. WHOreport:GlobalTuberculosisreport2017.2017.http://www.who.int/tb/en/
3. National Institute for Public Health and the Environment (RIVM). 2017. State of
infectiousdiseasesinTheNetherlands,2016.NationalInstituteforPublicHealthand
theEnvironment,Bilthoven,TheNetherlands.
4. VanAltena,DuggiralaS,GröschelMI,vanderWerf.2011.Immunologyintuberculosis:
challengesinmonitoringofdiseaseactivityandidentifyingcorrelatesofprotection.
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Weyer K. 2017World Health Organization treatment guidelines for drug-resistant
tuberculosis,2016update.EurRespirJ.Mar22;49(3).
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treatment–2017, Ahmad N, Ahuja SD, Akkerman OW, Alffenaar JC, Anderson LF,
BaghaeiP,BangD,BarryPM,BastosML,BeheraD,BenedettiA,BissonGP,Boeree
MJ,BonnetM,BrodeSK,BrustJCM,CaiY,CaumesE,CegielskiJP,CentisR,ChanPC,
ChanED,ChangKC,CharlesM,CiruleA,DalcolmoMP,D'AmbrosioL,deVriesG,Dheda
K,EsmailA,FloodJ,FoxGJ,Fréchet-JachymM,FregonaG,GayosoR,GegiaM,Gler
MT,Gu S,Guglielmetti L,Holtz TH,Hughes J, Isaakidis P, Jarlsberg L, KempkerRR,
KeshavjeeS,KhanFA,KipianiM,KoenigSP,KohWJ,KritskiA,KuksaL,KvasnovskyCL,
KwakN,LanZ,LangeC,Laniado-LaborínR,LeeM,LeimaneV,LeungCC,LeungEC,Li
PZ,LowenthalP,MacielEL,MarksSM,MaseS,MbuagbawL,MiglioriGB,MilanovV,
MillerAC,MitnickCD,ModongoC,MohrE,MonederoI,NahidP,NdjekaN,O'Donnell
MR,PadayatchiN,PalmeroD,PapeJW,PodewilsLJ,ReynoldsI,RiekstinaV,RobertJ,
RodriguezM,SeaworthB,SeungKJ,SchnippelK,ShimTS,SinglaR,SmithSE,SotgiuG,
SukhbaatarG,TabarsiP,TiberiS,TrajmanA,TrieuL,UdwadiaZF,vanderWerfTS,
VezirisN,ViikleppP,VilbrunSC,WalshK,WestenhouseJ,YewWW,YimJJ,ZetolaNM,
Zignol M, Menzies D. Treatment correlates of successful outcomes in pulmonary
14
Chapter 1
multidrug-resistant tuberculosis: an individual patient data meta-analysis. Lancet.
2018Sep8;392(10150):821-834.doi:10.1016/S0140-6736(18)31644-1.Review.
8. WHO report: Rapid Communication: Key changes to treatment of multidrug- and
rifampicin-resistant tuberculosis (MDR/RR-TB) 2018. Available at:
http://www.who.int/tb/publications/2018/rapid_communications_MDR/en/
9. NguyenTVA,AnthonyRM,BañulsAL,VuDH,AlffenaarJC.Bedaquilineresistance:Its
emergence, mechanism and prevention. Clin Infect Dis. 2017 Nov 8. doi:
10.1093/cid/cix992.
10. WorldHealthOrganization(WHO(ed.).2014.TheendTBstrategy:Globalstrategyand
targets for tuberculosis prevention, care and control after 2015. World Health
OrganizationGeneva,Switzerland.
11. United nations development Programme: Goal 3; Good-health and well-being:
http://www.undp.org/content/undp/en/home/sustainable-development-goals/goal-
3-good-health-and-well-being.html
12. MillardJ,PertinezH,BonnettL,HodelEM,DartoisV,JohnsonJL,CawsM,TiberiS,
Alffenaar JC, Davies G, Sloan DJ. 2018. Linezolid pharmacokinetics in MDR-TB: a
systematic review, meta-analysis and Monte Carlo simulation. J Antimicrob.
Chemother.
13. SotgiuG,CentisR,D’AmbrosioL,SpanevelloA,MiglioriGB;InternationalGroupfor
the study of Linezolid. 2013. Linezolid to treat extensively drug-resistant TB:
retrospective data are confirmed by experimental evidence. Eur Respir J.
Jul;42(1):288-90.
14. PrangerAD,AlffenaarJW,AarnoutseRE.2011.Fluoroquinolones,thecornerstoneof
treatmentofdrug-resistant tuberculosis:apharmacokineticandpharmacodynamic
approach.CurrPharmDes.2011;17027):2900-30
15. PrangerAD,vanAltenaR,AarnoutseRE,vanSoolingenD,UgesDR,KosterinkJG,van
der Werf TS, Alffenaar JW. Evaluation of moxifloxacin for the treatment of
tuberculosis:3yearsofexperience.EurRespirJ.201138(4):888-894
16. YatesTA,KhanPY,KnightGM,etal.ThetransmissionofMycobacteriumtuberculosis
inhighburdensettings.LancetInfectDis2016;16(2):227-38.
17. Hugonnet JE, Blanchard JS. 2007. Irreversible inhibition of the Mycobacterium
tuberculosis beta-lactamase by clavulanate. Biochemistry. 46:11998-12004. doi:
10.1021/bi701506h.
18. HugonnetJE,TremblayLW,BoshoffHI,Barry3rdCE,BlanchardJS.2009.Meropenem-
clavulanate is effective against extensively drug-resistant Mycobacterium
tuberculosis. Science. 323:1215-1218. doi: 10.1126/science.1167498;
10.1126/science.1167498.
19. DeshpandeD,SrivastavaS,ChapagainM,MagombedzeG,MartinKR,CirrincioneKN,
Lee PS, Koeuth T, Dheda K, Gumbo T. 2017. Ceftazidime-avibactam has potent
sterilizing activity against highly drug-resistant tuberculosis. Sci Adv. Aug
30;3(8):e1701102
20. DeshpandeD,SrivastavaS,BendetP,MartinKR,CirrincioneKN,LeePS,Pasipanodya
JG,DhedaK,GumboT.2018.AntibacterialandSterilizingEffectofBenzylpenicillinin
Tuberculosis.AntimicrobAgentsChemother.Jan25;62(2).
21. Cynamon,M.H.,andG.S.Palmer.1983.Invitroactivityofamoxicillinincombination
with clavulanic acid against Mycobacterium tuberculosis. Antimicrob Agents
Chemother.24:429-431.
22. DeshpandeD,SrivastavaS,BendetP,MartinKR,CirrincioneKN,LeePS,Pasipanodya
JG,DhedaK,GumboT.2018.AntibacterialandSterilizingEffectofBenzylpenicillinin
Tuberculosis.AntimicrobAgentsChemother.Jan25;62(2).
23. KaushikA,MakkerN,PandeyP,ParrishN,SinghU,LamichaneG.2015.Carbapenems
and Rifampicin exhibit synergy against Mycobacterium tuberculosis and
Mycobacterium abscessus. Antimicrob Agents Chemother 59:6561-6567.
Doi:10.1128/AAC.01158-15
24. Veziris, N., C. Truffot, J. L.Mainardi, and V. Jarlier. 2011. Activity of carbapenems
combined with clavulanate against murine tuberculosis. Antimicrob. Agents
Chemother.55:2597-2600.doi:10.1128/AAC.01824-10;10.1128/AAC.01824-10.
25. TiberiS,D’AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,Alffenaar JWC,
MiglioriGB.2015. Ertapenem in the treatmentofmultidrug-resistant tuberculosis:
firstclinicalexperience.EurRespirJ47:333-336.Doi:10.1183/13993003.01278-2015
15
1
General Introduction
multidrug-resistant tuberculosis: an individual patient data meta-analysis. Lancet.
2018Sep8;392(10150):821-834.doi:10.1016/S0140-6736(18)31644-1.Review.
8. WHO report: Rapid Communication: Key changes to treatment of multidrug- and
rifampicin-resistant tuberculosis (MDR/RR-TB) 2018. Available at:
http://www.who.int/tb/publications/2018/rapid_communications_MDR/en/
9. NguyenTVA,AnthonyRM,BañulsAL,VuDH,AlffenaarJC.Bedaquilineresistance:Its
emergence, mechanism and prevention. Clin Infect Dis. 2017 Nov 8. doi:
10.1093/cid/cix992.
10. WorldHealthOrganization(WHO(ed.).2014.TheendTBstrategy:Globalstrategyand
targets for tuberculosis prevention, care and control after 2015. World Health
OrganizationGeneva,Switzerland.
11. United nations development Programme: Goal 3; Good-health and well-being:
http://www.undp.org/content/undp/en/home/sustainable-development-goals/goal-
3-good-health-and-well-being.html
12. MillardJ,PertinezH,BonnettL,HodelEM,DartoisV,JohnsonJL,CawsM,TiberiS,
Alffenaar JC, Davies G, Sloan DJ. 2018. Linezolid pharmacokinetics in MDR-TB: a
systematic review, meta-analysis and Monte Carlo simulation. J Antimicrob.
Chemother.
13. SotgiuG,CentisR,D’AmbrosioL,SpanevelloA,MiglioriGB;InternationalGroupfor
the study of Linezolid. 2013. Linezolid to treat extensively drug-resistant TB:
retrospective data are confirmed by experimental evidence. Eur Respir J.
Jul;42(1):288-90.
14. PrangerAD,AlffenaarJW,AarnoutseRE.2011.Fluoroquinolones,thecornerstoneof
treatmentofdrug-resistant tuberculosis:apharmacokineticandpharmacodynamic
approach.CurrPharmDes.2011;17027):2900-30
15. PrangerAD,vanAltenaR,AarnoutseRE,vanSoolingenD,UgesDR,KosterinkJG,van
der Werf TS, Alffenaar JW. Evaluation of moxifloxacin for the treatment of
tuberculosis:3yearsofexperience.EurRespirJ.201138(4):888-894
16. YatesTA,KhanPY,KnightGM,etal.ThetransmissionofMycobacteriumtuberculosis
inhighburdensettings.LancetInfectDis2016;16(2):227-38.
17. Hugonnet JE, Blanchard JS. 2007. Irreversible inhibition of the Mycobacterium
tuberculosis beta-lactamase by clavulanate. Biochemistry. 46:11998-12004. doi:
10.1021/bi701506h.
18. HugonnetJE,TremblayLW,BoshoffHI,Barry3rdCE,BlanchardJS.2009.Meropenem-
clavulanate is effective against extensively drug-resistant Mycobacterium
tuberculosis. Science. 323:1215-1218. doi: 10.1126/science.1167498;
10.1126/science.1167498.
19. DeshpandeD,SrivastavaS,ChapagainM,MagombedzeG,MartinKR,CirrincioneKN,
Lee PS, Koeuth T, Dheda K, Gumbo T. 2017. Ceftazidime-avibactam has potent
sterilizing activity against highly drug-resistant tuberculosis. Sci Adv. Aug
30;3(8):e1701102
20. DeshpandeD,SrivastavaS,BendetP,MartinKR,CirrincioneKN,LeePS,Pasipanodya
JG,DhedaK,GumboT.2018.AntibacterialandSterilizingEffectofBenzylpenicillinin
Tuberculosis.AntimicrobAgentsChemother.Jan25;62(2).
21. Cynamon,M.H.,andG.S.Palmer.1983.Invitroactivityofamoxicillinincombination
with clavulanic acid against Mycobacterium tuberculosis. Antimicrob Agents
Chemother.24:429-431.
22. DeshpandeD,SrivastavaS,BendetP,MartinKR,CirrincioneKN,LeePS,Pasipanodya
JG,DhedaK,GumboT.2018.AntibacterialandSterilizingEffectofBenzylpenicillinin
Tuberculosis.AntimicrobAgentsChemother.Jan25;62(2).
23. KaushikA,MakkerN,PandeyP,ParrishN,SinghU,LamichaneG.2015.Carbapenems
and Rifampicin exhibit synergy against Mycobacterium tuberculosis and
Mycobacterium abscessus. Antimicrob Agents Chemother 59:6561-6567.
Doi:10.1128/AAC.01158-15
24. Veziris, N., C. Truffot, J. L.Mainardi, and V. Jarlier. 2011. Activity of carbapenems
combined with clavulanate against murine tuberculosis. Antimicrob. Agents
Chemother.55:2597-2600.doi:10.1128/AAC.01824-10;10.1128/AAC.01824-10.
25. TiberiS,D’AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,Alffenaar JWC,
MiglioriGB.2015. Ertapenem in the treatmentofmultidrug-resistant tuberculosis:
firstclinicalexperience.EurRespirJ47:333-336.Doi:10.1183/13993003.01278-2015
CHAPTER 2
Antimicrob. Agents Chemother. 2019 Jan 29; 63(2).PMID: 30455232.
*Both authors contributed equally
S.P. van Rijn*M.A. Zuur*R. AnthonyB. Wilffert
R. van AltenaO.W. Akkerman
W.C.M. de LangeT.S. van der WerfJ.G.W. KosterinkJ.W.C. Alffenaar
Evaluation of Carbapenems for Treatment of Multi- and Extensively
Drug-Resistant Mycobacterium Tuberculosis
CHAPTER 2
Antimicrob. Agents Chemother. 2019 Jan 29; 63(2).PMID: 30455232.
*Both authors contributed equally
S.P. van Rijn*M.A. Zuur*R. AnthonyB. Wilffert
R. van AltenaO.W. Akkerman
W.C.M. de LangeT.S. van der WerfJ.G.W. KosterinkJ.W.C. Alffenaar
Evaluation of Carbapenems for Treatment of Multi- and Extensively
Drug-Resistant Mycobacterium Tuberculosis
18
Chapter 2
Abstract
M/XDR-TB has become an increasing threat in high burden countries but also in affluent
regionsduetoincreasedinternationaltravelandglobalization.Carbapenemsareearmarked
aspotentiallyactivedrugs for the treatmentofM.tuberculosis.Tobetterunderstand the
potential of carbapenems for the treatment ofM/XDR-TB, the aim of this reviewwas to
evaluatetheliteratureoncurrentlyavailableinvitro,invivoandclinicaldataoncarbapenems
inthetreatmentofM.tuberculosisanddetectionofknowledgegaps,inordertotargetfuture
research.InFebruary2018,asystematicliteraturesearchofPubMedandWebofSciencewas
performed.Overalltheresultsofthestudiesidentifiedinthisreview,whichusedavarietyof
carbapenemsusceptibilitytestsonclinicalandlabstrainsofM.tuberculosis,areconsistent.
In vitro the activity of carbapenems againstM. tuberculosis is increased when used in
combinationwith clavulanate, a BLaC inhibitor.However, clavulanate is not commercially
available alone, and therefore is it practically impossible to prescribe carbapenems in
combinationwith clavulanateat this time. Few in vivo studieshavebeenperformed,one
prospective, two observational and seven retrospective clinical studies to assess
effectiveness,safetyandtolerabilityofthreedifferentcarbapenems(imipenem,meropenem
andertapenem).Presentlywefoundnoclearevidencetoselectoneparticularcarbapenem
among the different candidate compounds, to design an effective M/XDR-TB regimen.
Therefore, more clinical evidence and dose optimization substantiated by hollow fiber
infection studies are needed to support repurposing carbapenems for the treatment of
M/XDR-TB.
Introduction
Treatmentoftuberculosis(TB),adiseasecausedbyMycobacteriumtuberculosis,hasbecome
morechallengingwiththeemergenceofmultidrugresistant(MDR)-TBandextensivelydrug
resistant(XDR)-TBamongpreviouslyandnewlydetectedcases(1).M/XDR-TBhasbecomean
increasing threat in high burden countries but also in affluent regions due to increased
internationaltravelandglobalization.
MDR-TBisdefinedasaninfectiousdiseasecausedbyM.tuberculosisthatisresistanttoat
leastisoniazidandrifampicin.XDR-TBisdefinedasMDR-TBwithadditionalresistancetoat
least one of the fluoroquinolones and to at least one of the injectable second line drugs
(amikacin, capreomycinor kanamycin).NewTBdrugs,with anovelmechanismof action,
includebedaquilineanddelamanid thathave recentlybeenapprovedand included in the
World Health Organization guidelines on MDR-TB as add-on agents (2). Unfortunately,
resistancetotheseagentshasalreadybeendetected(3).Explorationofcurrentlyavailable
drugs for their potential effect against TB, may be an additional source for potential
candidatestobeusedincaseofextensiveresistancetotrytocomposeatreatmentregimen
(4-5).
Beta-lactam antimicrobial drugs are widely used drugs for the treatment of a range of
infections.Also,imipenem-cilastatinandmeropenemhavebeenlistedasadd-ondrugsinthe
updatedWHOtreatmentguidelines(6).Carbapenemactivityhaslongbeenconsideredtobe
of limited use, due to rapid hydrolysis of the beta -lactam ring by broad-spectrum
mycobacterialclassAbeta-lactamases(BLaC).TheadditionoftheBLaCinhibitorclavulanate
suggests that beta-lactams combined with BLaC inhibitors could be beneficial in the
treatmentofTB(7).Recentstudiessuggestthatbeta-lactams,usingclavulanate/clavulanic
acid,showmoreactivityagainstM.tuberculosis(7-14).
Thebacterialactivityofbeta-lactamsisduetotheinactivationofbacterialtranspeptidases,
commonlyknownaspenicillinbindingproteins(PBP),whichinhibitthebiosynthesisofthe
peptidoglycanlayerofthecellwallofbacteria(8,15).Polymerizationsofthepeptidoglycan
layer in most bacteria are predominantly cross-linked by D,D-transpeptidases (DDT), the
19
2
Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis
Abstract
M/XDR-TB has become an increasing threat in high burden countries but also in affluent
regionsduetoincreasedinternationaltravelandglobalization.Carbapenemsareearmarked
aspotentiallyactivedrugs for the treatmentofM.tuberculosis.Tobetterunderstand the
potential of carbapenems for the treatment ofM/XDR-TB, the aim of this reviewwas to
evaluatetheliteratureoncurrentlyavailableinvitro,invivoandclinicaldataoncarbapenems
inthetreatmentofM.tuberculosisanddetectionofknowledgegaps,inordertotargetfuture
research.InFebruary2018,asystematicliteraturesearchofPubMedandWebofSciencewas
performed.Overalltheresultsofthestudiesidentifiedinthisreview,whichusedavarietyof
carbapenemsusceptibilitytestsonclinicalandlabstrainsofM.tuberculosis,areconsistent.
In vitro the activity of carbapenems againstM. tuberculosis is increased when used in
combinationwith clavulanate, a BLaC inhibitor.However, clavulanate is not commercially
available alone, and therefore is it practically impossible to prescribe carbapenems in
combinationwith clavulanateat this time. Few in vivo studieshavebeenperformed,one
prospective, two observational and seven retrospective clinical studies to assess
effectiveness,safetyandtolerabilityofthreedifferentcarbapenems(imipenem,meropenem
andertapenem).Presentlywefoundnoclearevidencetoselectoneparticularcarbapenem
among the different candidate compounds, to design an effective M/XDR-TB regimen.
Therefore, more clinical evidence and dose optimization substantiated by hollow fiber
infection studies are needed to support repurposing carbapenems for the treatment of
M/XDR-TB.
Introduction
Treatmentoftuberculosis(TB),adiseasecausedbyMycobacteriumtuberculosis,hasbecome
morechallengingwiththeemergenceofmultidrugresistant(MDR)-TBandextensivelydrug
resistant(XDR)-TBamongpreviouslyandnewlydetectedcases(1).M/XDR-TBhasbecomean
increasing threat in high burden countries but also in affluent regions due to increased
internationaltravelandglobalization.
MDR-TBisdefinedasaninfectiousdiseasecausedbyM.tuberculosisthatisresistanttoat
leastisoniazidandrifampicin.XDR-TBisdefinedasMDR-TBwithadditionalresistancetoat
least one of the fluoroquinolones and to at least one of the injectable second line drugs
(amikacin, capreomycinor kanamycin).NewTBdrugs,with anovelmechanismof action,
includebedaquilineanddelamanid thathave recentlybeenapprovedand included in the
World Health Organization guidelines on MDR-TB as add-on agents (2). Unfortunately,
resistancetotheseagentshasalreadybeendetected(3).Explorationofcurrentlyavailable
drugs for their potential effect against TB, may be an additional source for potential
candidatestobeusedincaseofextensiveresistancetotrytocomposeatreatmentregimen
(4-5).
Beta-lactam antimicrobial drugs are widely used drugs for the treatment of a range of
infections.Also,imipenem-cilastatinandmeropenemhavebeenlistedasadd-ondrugsinthe
updatedWHOtreatmentguidelines(6).Carbapenemactivityhaslongbeenconsideredtobe
of limited use, due to rapid hydrolysis of the beta -lactam ring by broad-spectrum
mycobacterialclassAbeta-lactamases(BLaC).TheadditionoftheBLaCinhibitorclavulanate
suggests that beta-lactams combined with BLaC inhibitors could be beneficial in the
treatmentofTB(7).Recentstudiessuggestthatbeta-lactams,usingclavulanate/clavulanic
acid,showmoreactivityagainstM.tuberculosis(7-14).
Thebacterialactivityofbeta-lactamsisduetotheinactivationofbacterialtranspeptidases,
commonlyknownaspenicillinbindingproteins(PBP),whichinhibitthebiosynthesisofthe
peptidoglycanlayerofthecellwallofbacteria(8,15).Polymerizationsofthepeptidoglycan
layer in most bacteria are predominantly cross-linked by D,D-transpeptidases (DDT), the
20
Chapter 2
enzymesinhibitedbybeta-lactams(8,16).Themajorityofcrosslinksinpeptidoglycanappear
tobeformedbythenon-classicalL,D-transpeptidases(LDT)inM.tuberculosis(17-23).Several
studiesrevealedthestructuralbasisandtheinactivationmechanismofLDTandtheactive
roleofcarbapenems,providingabasisforthepotentialuseofcarbapenemsininhibitingM.
tuberculosis(24-28).
Beta-lactams show time-dependent activity, carbapenems have been shown to have
bactericidalactivitywhenthefreedrugplasmaconcentrationexceedstheMICforatleast40
%ofthetimeinnon-TBbacterialspecies(29-30).
CarbapenemsareearmarkedaspotentiallyactivedrugsforthetreatmentofM.tuberculosis.
TobetterunderstandthepotentialofcarbapenemsforthetreatmentofM/XDR-TB,theaim
ofthisreviewwastoevaluatetheliteratureoncurrentlyavailableinvitro,invivoandclinical
dataoncarbapenemsinthetreatmentofM.tuberculosisanddetectionofknowledgegaps,
inordertotargetfutureresearch.
Methods
Prisma
ThissystematicreviewwasconductedinaccordancewiththePreferredReportingItemsfor
SystematicReviewsandMeta-Analyses(PRISMA)statement.
Search
In February2018, a systematic literature searchof PubMedandWebof Science,without
restrictions with respect to publication date was employed using the key words
(´Carbapenem’ OR ‘Carbapenems’ OR ‘Imipenem’ OR ‘Meropenem’ OR ‘Ertapenem’ OR
‘Doripenem’ OR ‘Faropenem’ OR ‘Biapenem’ OR ‘Panipenem’ OR ‘Tebipenem’) AND
(‘Tuberculosis’ORTBORMycobacteriumtuberculosis)asMeShTerms.Retrievedstudiesand
abstractsfrombothPubMedandWebofSciencewerepooledandduplicateswereremoved.
Titlesandabstractsofretrievedarticleswerescreened.Reviews,case-reportsorstudieson
otherspeciesthanTBorstudiesonotherdrugsthancarbapenemswereexcluded.Studies
werescreenedforeligibility.Ifeligible,thefull-textwasreadbyaresearcher(SvR).Asecond
researcher(MZ)independentlyrepeatedthearticlesearchandselection.Discrepancieswere
resolved by discussion, or a third researcher was consulted (JWA). Full text papers were
subdividedintothreesections;invitro,invivoandclinicaldata.Fulltextpapersforinvitro
data were eligible for inclusion if an M. tuberculosis strain was studied and minimum
inhibitoryconcentrationswere reported.Full textpapers for invivodatawereeligible for
inclusioniftreatmentofM.tuberculosisinfectionswithcarbapenemswerestudiedinanimal
models,andifcolonyformingunitsand/orsurvivaldatawerereported.Fulltextpapersfor
clinical data were eligible for inclusion if pharmacokinetics of carbapenems or safety or
responsetotreatmentmeasuredassurrogateendpoints(sputumconversion)orclinicalend
pointswerestudiedandreported.Referencesofallincludedarticleswerescreenedbyhand.
Thesamesystematic searchwasperformedusingclinicaltrials.gov to findongoing studies
investigatingcarbapenemsinTBpatients(Feb2018).
Dataextraction
Aresearcher(SvR)performeddataextractionfirstbyusingastructureddatacollectionform.
Asecondresearcher(MZ)verifiedthedataextractionindependently.Dataweresubdivided
intothreesections;invitro,invivoandclinicaldata.Variablesinthesection‘invitro’included;
M. tuberculosis strain, experimental methods, drug of interest. Minimal inhibitory
concentration,minimal inhibitory concentrationwith clavulanic acid,minimal bactericidal
concentrationandcolonyformingunits(CFU)wereextractedfromtheincludedarticles.For
thesection‘invivo’thefollowingdatawereincluded;M.tuberculosisstrain,mice,routeof
infection, drug of interest with or without clavulanic acid, dose, and treatment, colony
formingunits and survival rate,were retrieved from the includedarticles. For the clinical
section,weextracteddatafromtheincludedarticlesontypeofstudypopulation,numberof
subjects, study design, drug of interest, and dosage. Sputum smear, sputum culture,
treatment success, adverseeventsand interruptiondue toadverseeventswerenotedas
outcomes.AUC,Peakdrugconcentration(Cmax),half-life(t1/2),Distributionvolume(Vd),and
clearancewereextracted.Possibilityofpoolingdatafromincludeddatawasassessedondata
presentation.
21
2
Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis
enzymesinhibitedbybeta-lactams(8,16).Themajorityofcrosslinksinpeptidoglycanappear
tobeformedbythenon-classicalL,D-transpeptidases(LDT)inM.tuberculosis(17-23).Several
studiesrevealedthestructuralbasisandtheinactivationmechanismofLDTandtheactive
roleofcarbapenems,providingabasisforthepotentialuseofcarbapenemsininhibitingM.
tuberculosis(24-28).
Beta-lactams show time-dependent activity, carbapenems have been shown to have
bactericidalactivitywhenthefreedrugplasmaconcentrationexceedstheMICforatleast40
%ofthetimeinnon-TBbacterialspecies(29-30).
CarbapenemsareearmarkedaspotentiallyactivedrugsforthetreatmentofM.tuberculosis.
TobetterunderstandthepotentialofcarbapenemsforthetreatmentofM/XDR-TB,theaim
ofthisreviewwastoevaluatetheliteratureoncurrentlyavailableinvitro,invivoandclinical
dataoncarbapenemsinthetreatmentofM.tuberculosisanddetectionofknowledgegaps,
inordertotargetfutureresearch.
Methods
Prisma
ThissystematicreviewwasconductedinaccordancewiththePreferredReportingItemsfor
SystematicReviewsandMeta-Analyses(PRISMA)statement.
Search
In February2018, a systematic literature searchof PubMedandWebof Science,without
restrictions with respect to publication date was employed using the key words
(´Carbapenem’ OR ‘Carbapenems’ OR ‘Imipenem’ OR ‘Meropenem’ OR ‘Ertapenem’ OR
‘Doripenem’ OR ‘Faropenem’ OR ‘Biapenem’ OR ‘Panipenem’ OR ‘Tebipenem’) AND
(‘Tuberculosis’ORTBORMycobacteriumtuberculosis)asMeShTerms.Retrievedstudiesand
abstractsfrombothPubMedandWebofSciencewerepooledandduplicateswereremoved.
Titlesandabstractsofretrievedarticleswerescreened.Reviews,case-reportsorstudieson
otherspeciesthanTBorstudiesonotherdrugsthancarbapenemswereexcluded.Studies
werescreenedforeligibility.Ifeligible,thefull-textwasreadbyaresearcher(SvR).Asecond
researcher(MZ)independentlyrepeatedthearticlesearchandselection.Discrepancieswere
resolved by discussion, or a third researcher was consulted (JWA). Full text papers were
subdividedintothreesections;invitro,invivoandclinicaldata.Fulltextpapersforinvitro
data were eligible for inclusion if an M. tuberculosis strain was studied and minimum
inhibitoryconcentrationswere reported.Full textpapers for invivodatawereeligible for
inclusioniftreatmentofM.tuberculosisinfectionswithcarbapenemswerestudiedinanimal
models,andifcolonyformingunitsand/orsurvivaldatawerereported.Fulltextpapersfor
clinical data were eligible for inclusion if pharmacokinetics of carbapenems or safety or
responsetotreatmentmeasuredassurrogateendpoints(sputumconversion)orclinicalend
pointswerestudiedandreported.Referencesofallincludedarticleswerescreenedbyhand.
Thesamesystematic searchwasperformedusingclinicaltrials.gov to findongoing studies
investigatingcarbapenemsinTBpatients(Feb2018).
Dataextraction
Aresearcher(SvR)performeddataextractionfirstbyusingastructureddatacollectionform.
Asecondresearcher(MZ)verifiedthedataextractionindependently.Dataweresubdivided
intothreesections;invitro,invivoandclinicaldata.Variablesinthesection‘invitro’included;
M. tuberculosis strain, experimental methods, drug of interest. Minimal inhibitory
concentration,minimal inhibitory concentrationwith clavulanic acid,minimal bactericidal
concentrationandcolonyformingunits(CFU)wereextractedfromtheincludedarticles.For
thesection‘invivo’thefollowingdatawereincluded;M.tuberculosisstrain,mice,routeof
infection, drug of interest with or without clavulanic acid, dose, and treatment, colony
formingunits and survival rate,were retrieved from the includedarticles. For the clinical
section,weextracteddatafromtheincludedarticlesontypeofstudypopulation,numberof
subjects, study design, drug of interest, and dosage. Sputum smear, sputum culture,
treatment success, adverseeventsand interruptiondue toadverseeventswerenotedas
outcomes.AUC,Peakdrugconcentration(Cmax),half-life(t1/2),Distributionvolume(Vd),and
clearancewereextracted.Possibilityofpoolingdatafromincludeddatawasassessedondata
presentation.
22
Chapter 2
Dataquality
No validated tool for risk of bias assessment for in vitro studies, in vivo studies and
pharmacokinetic studieswasavailable.Tobeable toassess thequalityofeachstudy,we
verifiedifeachstudyreportedonkey-elementsrequiredforadequatedatainterpretation.If
studiesreportedadequatelyonthekey-elements,riskofbiaswasconsideredtobelow.If
studieshadmissingdataorifprocedureswerenotclearornotmentioned,riskofbiaswas
considered to be high. The following key-elements were identified for in vitro studies;
descriptionof laborclinical strains,minimalsamplesizeof>10strains,>3concentrations
testedperdrug,MIC/CFUdeterminedusingtheproportionmethod,evaluationendpointof
minimal inhibitory concentration (MIC 50 orMIC 90), evaluation of endpoint of minimal
bactericidal concentration (MBC99) and CFU reduction, for in vivo studies; description of
laboratory or clinical strains, type ofmice, route of administration of the drug, dose and
treatment duration, MIC/CFU determined using the proportion method, evaluation of
endpointofCFUandsurvivalrateandforclinicalstudies;forhumanstudies;studydesign,
patientpopulation(TB/MDR-TB;HIVco-infection),numberofstudyparticipants,endpoints
tested,definedassputumsmearconversion,sputumcultureconversion,treatmentsuccess,
adverse events. The following components were checked for pharmacokinetic studies:
samplesize,typeofpatients,typeofassay,numberofplasmasamplesdrawnperpatient,
samplehandling,useofvalidatedanalyticalmethodsandmethodofAUCcalculation.
Results
Basedon theselectioncriteria,250articleswereretrieved inPubMedand260 inWebof
Science.Afterremovalof146duplicates,364articlesremainedforscreening.Afterscreening
ofthetitleandabstract,46articlesremainedforfulltextevaluation.Reasonsforexclusion
included;notavailable(n=6),otherdrugs(n=2),noMIC(n=1),case-report(n=1),other(n=1).
Afterthisprocess,35relevantarticleswereincludedinthisstudy(Flowchart;Fig1).Dueto
lownumberandhighdiversityofstrains,analyticalmethodsandstudydesigns,presenceof
biochemicalinstabilityofthedrugsofinterest,theshorthalf-lifeofdrugsofinterestinmice
andthediversity inMICdetermination,wedidnothaveenoughdata toperformameta-
analysis.RiskofbiasoftheincludedstudiesisshownintableS1.Studiesonclinicaltrials.gov
areshowninS2.
Figure1.Flowchart
Invitro
Resultsoftheinvitrostudiesreportingoncarbapenemsarepresentedintable1.
Imipenem
Susceptibility testing of imipenem, using various analyticalmethods against strainH37Rv,
H37Ra,ErdmanandclinicalisolatesofM.tuberculosisshowedarangeofMIC’sbetween2–
32mg/LwithoutclavulanicacidandarangeofMIC’sbetween0.16–32withclavulanicacid.
(8,32-37).WhenImipenemwascombinedwithclavulanateitshoweda4-16-foldlowerMIC
againsttheM.tuberculosisH37Rvreferencestrain(8,33-35).
23
2
Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis
Dataquality
No validated tool for risk of bias assessment for in vitro studies, in vivo studies and
pharmacokinetic studieswasavailable.Tobeable toassess thequalityofeachstudy,we
verifiedifeachstudyreportedonkey-elementsrequiredforadequatedatainterpretation.If
studiesreportedadequatelyonthekey-elements,riskofbiaswasconsideredtobelow.If
studieshadmissingdataorifprocedureswerenotclearornotmentioned,riskofbiaswas
considered to be high. The following key-elements were identified for in vitro studies;
descriptionof laborclinical strains,minimalsamplesizeof>10strains,>3concentrations
testedperdrug,MIC/CFUdeterminedusingtheproportionmethod,evaluationendpointof
minimal inhibitory concentration (MIC 50 orMIC 90), evaluation of endpoint of minimal
bactericidal concentration (MBC99) and CFU reduction, for in vivo studies; description of
laboratory or clinical strains, type ofmice, route of administration of the drug, dose and
treatment duration, MIC/CFU determined using the proportion method, evaluation of
endpointofCFUandsurvivalrateandforclinicalstudies;forhumanstudies;studydesign,
patientpopulation(TB/MDR-TB;HIVco-infection),numberofstudyparticipants,endpoints
tested,definedassputumsmearconversion,sputumcultureconversion,treatmentsuccess,
adverse events. The following components were checked for pharmacokinetic studies:
samplesize,typeofpatients,typeofassay,numberofplasmasamplesdrawnperpatient,
samplehandling,useofvalidatedanalyticalmethodsandmethodofAUCcalculation.
Results
Basedon theselectioncriteria,250articleswereretrieved inPubMedand260 inWebof
Science.Afterremovalof146duplicates,364articlesremainedforscreening.Afterscreening
ofthetitleandabstract,46articlesremainedforfulltextevaluation.Reasonsforexclusion
included;notavailable(n=6),otherdrugs(n=2),noMIC(n=1),case-report(n=1),other(n=1).
Afterthisprocess,35relevantarticleswereincludedinthisstudy(Flowchart;Fig1).Dueto
lownumberandhighdiversityofstrains,analyticalmethodsandstudydesigns,presenceof
biochemicalinstabilityofthedrugsofinterest,theshorthalf-lifeofdrugsofinterestinmice
andthediversity inMICdetermination,wedidnothaveenoughdata toperformameta-
analysis.RiskofbiasoftheincludedstudiesisshownintableS1.Studiesonclinicaltrials.gov
areshowninS2.
Figure1.Flowchart
Invitro
Resultsoftheinvitrostudiesreportingoncarbapenemsarepresentedintable1.
Imipenem
Susceptibility testing of imipenem, using various analyticalmethods against strainH37Rv,
H37Ra,ErdmanandclinicalisolatesofM.tuberculosisshowedarangeofMIC’sbetween2–
32mg/LwithoutclavulanicacidandarangeofMIC’sbetween0.16–32withclavulanicacid.
(8,32-37).WhenImipenemwascombinedwithclavulanateitshoweda4-16-foldlowerMIC
againsttheM.tuberculosisH37Rvreferencestrain(8,33-35).
24
Chapter 2
Tabl
e1.
Resultsoftheinvitrostudiesreportingoncarbapenems.
Firs
taut
hor
(ref
).St
rain
N
Met
hod
Carb
apen
em(s
)β-
Lact
amas
ein
hibi
tor(s)
Valu
e(s)
[mg/
liter
;med
ian
(ran
ge)]fo
r:
Carbapenem
CarbapenemwithCLV(2.5mg/L)
MIC
MIC
50
MIC
90
MIC
MIC
50
MIC
90
MBC
99
ΔlogCFU
reduction
Cham
bers
et
al(3
2)
H37Ra,
H37Rv,
clinical
isolates
7
BactecTB
system
Imipenem
None
(2–4)
Cohe
net
al
(38)
H37Rv,
Clinical
isolates
91
Microplate
alamarBlue
assay
Meropenem
Clavulanate
22
(2–
32)
5.4(0.5–32)
Cava
naug
het
al
(39)
Clinical
isolates
15
3
Resazurin
microdilution
assay
Meropenem
Clavulanate
(<0.12–>16)
1
8
Des
hpan
de
eta
l(47
) H37Ra,THP1
monocytes
1
Resazurin
microdilution
assay,CFU
counts
Faropenem
None
1
2.71log
Dha
reta
l(4
9)
H37Rv,
Erdman
2
96W
ellflat-
bottom
polystyrene
microtiter
plate
Faropenem,
meropenem,
imipenem
Clavulanate
1.3;
2.5;
2.5
1.3;
0.3;
0.5
Engl
and
eta
l(4
0)
H37Rv,
macrophages
1
CFUcounts
Meropenem
Clavulanate
2log
Fors
man
eta
l(4
1)
H37Rv,
Clinical
isolates
69
Broth
microdilution
Meropenem
Clavulanate
(0.125–32)
1
2log
Gon
zalo
eta
l(4
2)
H37Rv,
Clinical
isolates
28
960M
GIT
system
Meropenem
None
Resistan
t at
5
mg/L
(1.28–2.56)
Gur
umur
thy
eta
l(48
) H37Rv
1
96W
ellsplate
Faropenem
Clavulanate,
avibactama
(5–10)
20
0log
Hor
itae
tal
(43)
H37Rv,
Clinical
isolates
42
Broth
microdilution
Meropenem
Biapenem
Tebipenem
Clavulanate
(1–32),
(1–32),
(0.25–8)
16,
16,
4
32,
32,
8
(0.063–8),
(0.25–8),
(0.063–8)
2,
2,
1
4,
4,
1
Hug
onne
tet
al(8
)Erdman,
H37Rv,
Clinical
isolates
15
Broth
microdilution
Imipenem
Meropenem
Clavulanate
0.16,
(0.23–1.25)
Kaus
hik
eta
l(3
3)
H37Rv,
Clinical
isolates
1
Broth
microdilution
Imipenem,
meropenem,
ertapenem,
doripenem,
biapenem,
faropenem,
tebipenem,
panipenem
None
(40–80),
(5–10),
(10–20),
(2.5–5),
(2.5–5),
(2.5–5),
(1.25–2.5)
>80
(20–40),
(2.5–5),
(5–10),
(1.25–2.5),
(0.6–1.2),
(2.5–5),
(0.31–0.62),
ND
ND
80
ND
20
20
20
10
ND
Kaus
hik
eta
l(5
1)
H37Rv,strain
115R,
strain
124R
3
Broth
microdilution
Biapenem
Clavulanate
(2–16)
Sala
eta
l(44
)18bcells
1
Serial
dilutions,CFU
counts
Meropenem
Clavulanate
Sola
pure
et
al(3
4)
H37Rv,
18b
cells
1
Resazurin
microdilution
assay,CFU
counts
Imipenem,
meropenem,
faropenem
Clavulanate
4,8,4
0.5,1,2
4
2
4
2log
Sriv
asta
vae
tal
(45)
H37Ra
1
Resazurin
microdilution
assay
Ertapenem
Clavulanate
0.6
Veziris
eta
l(3
5)
H37Rv
1
Broth
microdilution
Imipenem,
meropenem,
ertapenem
16,8,
16
1,1,
4
2.38log10
a.MICvaluesforcarbapenemswithavibactamarenotshowninthistable.
N:numberofstrains,MIC:Minimalinhibitoryconcentration(mg/L),M
IC50:Minimalinhibitoryconcentrationrequiredtoinhibitgrowthof50%
oftheorganisms,MIC90:Minimalinhibitoryconcentrationrequiredtoinhibitgrowthof90%oftheorganisms,CLV:clavulanate(mg/L),M
BC99:
minimalbactericidalconcentrationthatkills99%ofreplicationculture(mg/L),CFU:colonyform
ingunits(Log/(CFU/ml))
25
2
Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis
Tabl
e1.
Resultsoftheinvitrostudiesreportingoncarbapenems.
Firs
taut
hor
(ref
).St
rain
N
Met
hod
Carb
apen
em(s
)β-
Lact
amas
ein
hibi
tor(s)
Valu
e(s)
[mg/
liter
;med
ian
(ran
ge)]fo
r:
Carbapenem
CarbapenemwithCLV(2.5mg/L)
MIC
MIC
50
MIC
90
MIC
MIC
50
MIC
90
MBC
99
ΔlogCFU
reduction
Cham
bers
et
al(3
2)
H37Ra,
H37Rv,
clinical
isolates
7
BactecTB
system
Imipenem
None
(2–4)
Cohe
net
al
(38)
H37Rv,
Clinical
isolates
91
Microplate
alamarBlue
assay
Meropenem
Clavulanate
22
(2–
32)
5.4(0.5–32)
Cava
naug
het
al
(39)
Clinical
isolates
15
3
Resazurin
microdilution
assay
Meropenem
Clavulanate
(<0.12–>16)
1
8
Des
hpan
de
eta
l(47
)H37Ra,THP1
monocytes
1
Resazurin
microdilution
assay,CFU
counts
Faropenem
None
1
2.71log
Dha
reta
l(4
9)
H37Rv,
Erdman
2
96W
ellflat-
bottom
polystyrene
microtiter
plate
Faropenem,
meropenem,
imipenem
Clavulanate
1.3;
2.5;
2.5
1.3;
0.3;
0.5
Engl
and
eta
l(4
0)
H37Rv,
macrophages
1
CFUcounts
Meropenem
Clavulanate
2log
Fors
man
eta
l(4
1)
H37Rv,
Clinical
isolates
69
Broth
microdilution
Meropenem
Clavulanate
(0.125–32)
1
2log
Gon
zalo
eta
l(4
2)
H37Rv,
Clinical
isolates
28
960M
GIT
system
Meropenem
None
Resistan
t at
5
mg/L
(1.28–2.56)
Gur
umur
thy
eta
l(48
)H37Rv
1
96W
ellsplate
Faropenem
Clavulanate,
avibactama
(5–10)
20
0log
Hor
itae
tal
(43)
H37Rv,
Clinical
isolates
42
Broth
microdilution
Meropenem
Biapenem
Tebipenem
Clavulanate
(1–32),
(1–32),
(0.25–8)
16,
16,
4
32,
32,
8
(0.063–8),
(0.25–8),
(0.063–8)
2,
2,
1
4,
4,
1
Hug
onne
tet
al(8
) Erdman,
H37Rv,
Clinical
isolates
15
Broth
microdilution
Imipenem
Meropenem
Clavulanate
0.16,
(0.23–1.25)
Kaus
hik
eta
l(3
3)
H37Rv,
Clinical
isolates
1
Broth
microdilution
Imipenem,
meropenem,
ertapenem,
doripenem,
biapenem,
faropenem,
tebipenem,
panipenem
None
(40–80),
(5–10),
(10–20),
(2.5–5),
(2.5–5),
(2.5–5),
(1.25–2.5)
>80
(20–40),
(2.5–5),
(5–10),
(1.25–2.5),
(0.6–1.2),
(2.5–5),
(0.31–0.62),
ND
ND
80
ND
20
20
20
10
ND
Kaus
hik
eta
l(5
1)
H37Rv,strain
115R,
strain
124R
3
Broth
microdilution
Biapenem
Clavulanate
(2–16)
Sala
eta
l(44
)18bcells
1
Serial
dilutions,CFU
counts
Meropenem
Clavulanate
Sola
pure
et
al(3
4)
H37Rv,
18b
cells
1
Resazurin
microdilution
assay,CFU
counts
Imipenem,
meropenem,
faropenem
Clavulanate
4,8,4
0.5,1,2
4
2
4
2log
Sriv
asta
vae
tal
(45)
H37Ra
1
Resazurin
microdilution
assay
Ertapenem
Clavulanate
0.6
Veziris
eta
l(3
5)
H37Rv
1
Broth
microdilution
Imipenem,
meropenem,
ertapenem
16,8,
16
1,1,
4
2.38log10
a.MICvaluesforcarbapenemswithavibactamarenotshowninthistable.
N:numberofstrains,MIC:Minimalinhibitoryconcentration(mg/L),M
IC50:Minimalinhibitoryconcentrationrequiredtoinhibitgrowthof50%
oftheorganisms,MIC90:Minimalinhibitoryconcentrationrequiredtoinhibitgrowthof90%oftheorganisms,CLV:clavulanate(mg/L),M
BC99:
minimalbactericidalconcentrationthatkills99%ofreplicationculture(mg/L),CFU:colonyform
ingunits(Log/(CFU/ml))
26
Chapter 2
Meropenem
Multiplestudiesreportedthatmeropeneminpresenceofclavulanateisactiveinvitroagainst
clinicalandlabstrains,H37RvandH37Ra,ofM.tuberculosis,showingMIC’s≤1mg/L.Invitro
studiesreportingsusceptibilityofmeropenemofM.tuberculosisreferencestrainandclinical
isolatesshowedMICvaluesbetween1-32mg/L(8,33-44).Meropenemincombinationwith
clavulanicacidwasshowntohaveaMICbetween0.063–32mg/L(33-35,38,43)Meropenem
in combinationwith clavulanate killed the non-replicating ss18b strain ofM. tuberculosis
moderatelyandwasshowntohaveaMICof0.125–2.56mg/LagainstM.tuberculosisH37Rv
strains(8,34-35,40).Adecreaseof2log10CFUsoversixdayswasreportedinM.tuberculosis-
infectedmurinemacrophages(40).
Ertapenem
InclinicalstrainsofM.tuberculosistheMICofertapenem,assingleagent,was16mg/Land
when combinedwith clavulanate 4mg/L (33,35). Another study showed ertapenemwas
unstabledegradingfasterthanthedoublingtimeofM.tuberculosisinthegrowthmediaused,
suggestingpreviouspublishedMICsofertapenemarelikelytobefalselyhigh(45).Inahollow
fibermodelwith supplementationofertapenem inabrothmicrodilution test,ertapenem
showedaMICof0.6ml/L(46).A28-dayexposure-responsehollowfibermodelofTBstudy
tested8differentdosesofertapenem in combinationwith clavulanateand identified the
ertapenemexposureassociatedwithoptimalsterilizingeffectforclinicaluse.MonteCarlo
simulationwith10,000MDR-TBpatientsidentifiedasusceptibilitybreakpointMICof2mg/L
foranintravenousdoseof2gonceadaythatachievedorexceeded40%T>MIC(46)
Faropenem
Faropenemshoweda4-foldreductionwhencombinedwithclavulanicacid(33,34),resulting
inaMICrangebetween1-5mg/L(33-34,47-49)Inahollowfibermodel,theoptimaltarget
exposurewasidentifiedtobeassociatedwithoptimalefficacyinchildrenwithdisseminated
TB using Monte Carlo simulations; the predicted optimal oral dose was 30 mg/kg of
faropenem-medoxomil3-4timesdaily.Theexposuretargetforfaropenem-medoxomilwas
60%Tfree>MIC(50).
Othercarbapenems
Othercarbapenems,suchasdoripenem,biapenemandtebipenemshowedatleasta2-fold
reductioninMICwhencombinedwithclavulanicacid(33,37,43,51).
Invivo
Resultsoftheinvivostudiesreportingoncarbapenemsarepresentedintable2.
Imipenem
Thebacterialburdeninimipenem-treatedCD-1femalemice(twicedaily(BID)100mg/kg),
infectedwithM.tuberculosisstrainH37Rv,wasreducedby1.8log10insplenictissueand1.2
log10inlungtissueafter28days,showingananti-mycobacterialeffectaswellasimproved
survivalinthismousemodel(52).InanotherstudySwissmice,infectedwithM.tuberculosis
strainH37Rv,weretreatedwithasubcutaneousadministrationof100-mg/kgimipenemin
combinationwithclavulanateonceadaytosimulateahumanequivalentdose.TheCFUcount
after28daysoftreatmentincreasedcomparedtotheCFUcountatstartoftreatment.There
onlywasasignificantdifferenceintheimipenem-clavulanatetreatedmice(35).
Meropenem
It has been reported that 300mg/kgBIDmeropenemalone, and in combinationwith 50
mg/kgclavulanatebothresultedinasignificant,thoughmodestreduction, inCFUsin lung
andspleentissuesinC57BL/6mice(40).Vezirisetal.reportedaCFUincreasecomparedto
startofthetreatmentofmeropenemwhengivenasmono-therapyorincombinationwith
clavulanateinadoseof100mg/kg,onCFUs,spleenweights,orlunglesionsinSwissmice
(35).Meropeneminadoseof300mg/kgincombinationwithclavulanate,75mg/kgthrice-
dailygiventoBALB/cmiceshowedmarginalreductioninCFUcountsintheacutemodeland
noreductioninthechronicmodel(34).Meropenem,givensubcutaneously300mg/kgthree
timesaday,showedaCFUcountreductionof1.7loginthelungsofTF3157DHP-1deficient
mice(53).
27
2
Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis
Meropenem
Multiplestudiesreportedthatmeropeneminpresenceofclavulanateisactiveinvitroagainst
clinicalandlabstrains,H37RvandH37Ra,ofM.tuberculosis,showingMIC’s≤1mg/L.Invitro
studiesreportingsusceptibilityofmeropenemofM.tuberculosisreferencestrainandclinical
isolatesshowedMICvaluesbetween1-32mg/L(8,33-44).Meropenemincombinationwith
clavulanicacidwasshowntohaveaMICbetween0.063–32mg/L(33-35,38,43)Meropenem
in combinationwith clavulanate killed the non-replicating ss18b strain ofM. tuberculosis
moderatelyandwasshowntohaveaMICof0.125–2.56mg/LagainstM.tuberculosisH37Rv
strains(8,34-35,40).Adecreaseof2log10CFUsoversixdayswasreportedinM.tuberculosis-
infectedmurinemacrophages(40).
Ertapenem
InclinicalstrainsofM.tuberculosistheMICofertapenem,assingleagent,was16mg/Land
when combinedwith clavulanate 4mg/L (33,35). Another study showed ertapenemwas
unstabledegradingfasterthanthedoublingtimeofM.tuberculosisinthegrowthmediaused,
suggestingpreviouspublishedMICsofertapenemarelikelytobefalselyhigh(45).Inahollow
fibermodelwith supplementationofertapenem inabrothmicrodilution test,ertapenem
showedaMICof0.6ml/L(46).A28-dayexposure-responsehollowfibermodelofTBstudy
tested8differentdosesofertapenem in combinationwith clavulanateand identified the
ertapenemexposureassociatedwithoptimalsterilizingeffectforclinicaluse.MonteCarlo
simulationwith10,000MDR-TBpatientsidentifiedasusceptibilitybreakpointMICof2mg/L
foranintravenousdoseof2gonceadaythatachievedorexceeded40%T>MIC(46)
Faropenem
Faropenemshoweda4-foldreductionwhencombinedwithclavulanicacid(33,34),resulting
inaMICrangebetween1-5mg/L(33-34,47-49)Inahollowfibermodel,theoptimaltarget
exposurewasidentifiedtobeassociatedwithoptimalefficacyinchildrenwithdisseminated
TB using Monte Carlo simulations; the predicted optimal oral dose was 30 mg/kg of
faropenem-medoxomil3-4timesdaily.Theexposuretargetforfaropenem-medoxomilwas
60%Tfree>MIC(50).
Othercarbapenems
Othercarbapenems,suchasdoripenem,biapenemandtebipenemshowedatleasta2-fold
reductioninMICwhencombinedwithclavulanicacid(33,37,43,51).
Invivo
Resultsoftheinvivostudiesreportingoncarbapenemsarepresentedintable2.
Imipenem
Thebacterialburdeninimipenem-treatedCD-1femalemice(twicedaily(BID)100mg/kg),
infectedwithM.tuberculosisstrainH37Rv,wasreducedby1.8log10insplenictissueand1.2
log10inlungtissueafter28days,showingananti-mycobacterialeffectaswellasimproved
survivalinthismousemodel(52).InanotherstudySwissmice,infectedwithM.tuberculosis
strainH37Rv,weretreatedwithasubcutaneousadministrationof100-mg/kgimipenemin
combinationwithclavulanateonceadaytosimulateahumanequivalentdose.TheCFUcount
after28daysoftreatmentincreasedcomparedtotheCFUcountatstartoftreatment.There
onlywasasignificantdifferenceintheimipenem-clavulanatetreatedmice(35).
Meropenem
It has been reported that 300mg/kgBIDmeropenemalone, and in combinationwith 50
mg/kgclavulanatebothresultedinasignificant,thoughmodestreduction, inCFUsin lung
andspleentissuesinC57BL/6mice(40).Vezirisetal.reportedaCFUincreasecomparedto
startofthetreatmentofmeropenemwhengivenasmono-therapyorincombinationwith
clavulanateinadoseof100mg/kg,onCFUs,spleenweights,orlunglesionsinSwissmice
(35).Meropeneminadoseof300mg/kgincombinationwithclavulanate,75mg/kgthrice-
dailygiventoBALB/cmiceshowedmarginalreductioninCFUcountsintheacutemodeland
noreductioninthechronicmodel(34).Meropenem,givensubcutaneously300mg/kgthree
timesaday,showedaCFUcountreductionof1.7loginthelungsofTF3157DHP-1deficient
mice(53).
28
Chapter 2
Table2.Resultsofthe
invivostud
iesr
eportin
gon
carbap
enem
s.
Firsta
utho
r(ref).
Stra
in
Miceof
miceof
ot
hera
nimal
mod
el
Infection
Drug
Do
se
Infection
mod
el
Trea
tmen
tEn
d-po
int
Organ
sCF
Ure
duction
Surviva
lrate
CFU/
clv
Cham
bers
etal(52
) H3
7Rv
CD-1Fem
ale
mice
su
bcutan
eous
ly
Imipen
em
Bid10
0mg/kg
ND
28
day
sCF
Uco
unt,
Survivalra
te
Spleen
,lung
s 1.8log
65%
ND
Dhareta
l(49)
H37R
vad
ultC
57BL
/6J
mice
intratrach
eal
Farope
nem
500mg/kg
ac
uteTB
8da
ys
CFUco
unt
Lung
sredu
ctionof
CFU:
10^
5-
10^6
ND
ND
Englan
det
al(4
0)
H37R
vC5
7BL/6Mice
su
bcutan
eous
ly
Merop
enem
bid30
0mg/kg
Ch
ronic
stag
e 2wee
ks
CFUco
unt
Spleen
,lung
s 1log
ND
1log
NewZea
land
whitera
bbits
intrav
enou
sbo
lus
Merop
enem
75
mg/kg
12
5mg/kg
ND
ND
PK
data
ND
ND
ND
ND
Kaus
hiket
al(5
1)
H37R
vBA
LB/cm
ice
Aeroso
lBiap
enem
20
0mg/kg
BID
300mg/kg
BID
Latepha
se
acuteTB
rifam
picin
resis
tantTB
8wee
ks
4wee
ks
CFUco
unt
Lung
s1log
ND
ND
ND
Rulla
seta
l(53)
H37R
vTF
3157
DHP
-IKO
su
bcutan
eous
ly
Merop
enem
Fa
rope
nem
TID30
0mg/kg
TID50
0mg/kg
AcuteTB
mod
el
21day
sCF
Uco
unt
Lung
s1.7log
2log
ND
ND
ND
ND
Solapu
reet
al(3
4)
H37R
vBA
LB/cm
ice
Aeroso
lMerop
enem
TID30
0mg/kg
Ac
utean
dch
ronic
mod
el
4wee
ks
CFUco
unt
lung
sno
redu
ction
ND
no
redu
ction
Vezir
isetal
(35)
H37R
vFe
maleSw
iss
mice
Intrav
enou
sly
Imipen
em
Merop
enem
Ertape
nem
100mg/kg
10
0mg/kg
10
0mg/kg
prev
entiv
emod
el
28day
sCF
Uco
unt,
Survivalra
te
Spleen
,lung
s >1
.2lo
g*
>1.8lo
g*
>1.7lo
g*
1de
ad
3de
ad
3de
ad
>0.9lo
g*
>1.4lo
g*
>1.6lo
g*
*Th
erewasaCFU
increa
sein
thegrou
psw
ithand
with
outc
lavu
lana
teco
mpa
redtoth
estarto
fthe
trea
tmen
t.
MIC:M
inim
alin
hibitorycon
centratio
n(m
g/L),C
LV:c
lavu
lana
te(m
g/L),MBC
:minim
albacteric
idalcon
centratio
n(m
g/L),C
FU:c
olon
yform
ing
units
,od:onc
eada
y,bid:twice
aday
,tid:three
timesaday
,qid:fou
rtim
esaday
,ND:notdescribed
.
Ertapenem
InamurineTBmodel infectedwithH37Rv,adoseof100mg/kgoncedailyertapenemas
monotherapyorincombinationwithclavulanatehadneitherbactericidalnorbacteriostatic
activityinlungsandspleensofTB-infectedmice.Spleenweightandlunglesionsremained
similarcomparedtotheuntreatedgroupofmice.TherewasanincreaseinCFUscompared
totheCFUsatthestartofthetreatment(35).
OtherCarbapenems
Anoraldoseof500mg/kgfaropenem-medoxomil,giventhreetimesdaily,gaveareduction
of2logCFUcountinthelungsofTF3157DHP-1deficientmice(53).Neitherinvivonorclinical
studiesforothercarbapenemsaspartofamulti-drugregimenagainstTBwereretrieved.
Clinicalstudies
Resultsoftheclinicalstudiesreportingoncarbapenemsarepresentedintable3.
Imipenem
Ten patients were treated with imipenem in combination with two or more other
antimicrobialagents.Itwasreportedthatitwaslikelythat1gofimipenem(BID)contributed
to sputum culture conversion in these patients (52). A prospective study evaluated 1000
mg/day imipenem/clavulanateatadoseofoncedaily in12patients,11of thesepatients
receivedlinezolid-containingregimens.Allpatientsshowedsputumandcultureconversion
within180days.Noadverseeventswerereportedforimipenem/clavulanate(54).Inalarge
observational study, the clinical outcomes of 84 patients, treated with 500 mg
imipenem/clavulanatefourtimesaday,werecomparedwithresultsfrom168controls.The
studyshowedthatimipenem-containingregimensachievedcomparableresultscomparedto
theimipenemsparingregimens,whilesuccessratesweresimilartomajorinternationalMDR-
TBcohorts(55).
Meropenem
A regimen includingmeropenem-clavulanate given to 18 patientswith severe pulmonary
XDR-TB led to sputum culture conversion in 15 patients, of which 10 has successfully
completedandfivepatientswereconsideredcuredaccordingtoWHOguidelines.Long-
29
2
Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis
Table2.Resultsofthe
invivostud
iesr
eportin
gon
carbap
enem
s.
Firsta
utho
r(ref).
Stra
in
Miceof
miceof
ot
hera
nimal
mod
el
Infection
Drug
Do
se
Infection
mod
el
Trea
tmen
tEn
d-po
int
Organ
sCF
Ure
duction
Surviva
lrate
CFU/
clv
Cham
bers
etal(52
)H3
7Rv
CD-1Fem
ale
mice
su
bcutan
eous
ly
Imipen
em
Bid10
0mg/kg
ND
28
day
sCF
Uco
unt,
Survivalra
te
Spleen
,lung
s1.8log
65%
ND
Dhareta
l(49)
H37R
vad
ultC
57BL
/6J
mice
intratrach
eal
Farope
nem
500mg/kg
ac
uteTB
8da
ys
CFUco
unt
Lung
sredu
ctionof
CFU:
10^
5-
10^6
ND
ND
Englan
det
al(4
0)
H37R
vC5
7BL/6Mice
su
bcutan
eous
ly
Merop
enem
bid30
0mg/kg
Ch
ronic
stag
e2wee
ks
CFUco
unt
Spleen
,lung
s1log
ND
1log
NewZea
land
whitera
bbits
intrav
enou
sbo
lus
Merop
enem
75
mg/kg
12
5mg/kg
ND
ND
PK
data
ND
ND
ND
ND
Kaus
hiket
al(5
1)
H37R
vBA
LB/cm
ice
Aeroso
lBiap
enem
20
0mg/kg
BID
300mg/kg
BID
Latepha
se
acuteTB
rifam
picin
resis
tantTB
8wee
ks
4wee
ks
CFUco
unt
Lung
s1log
ND
ND
ND
Rulla
seta
l(53)
H37R
vTF
3157
DHP
-IKO
su
bcutan
eous
ly
Merop
enem
Fa
rope
nem
TID30
0mg/kg
TID50
0mg/kg
AcuteTB
mod
el
21day
sCF
Uco
unt
Lung
s1.7log
2log
ND
ND
ND
ND
Solapu
reet
al(3
4)
H37R
vBA
LB/cm
ice
Aeroso
lMerop
enem
TID30
0mg/kg
Ac
utean
dch
ronic
mod
el
4wee
ks
CFUco
unt
lung
sno
redu
ction
ND
no
redu
ction
Vezir
isetal
(35)
H37R
vFe
maleSw
iss
mice
Intrav
enou
sly
Imipen
em
Merop
enem
Ertape
nem
100mg/kg
10
0mg/kg
10
0mg/kg
prev
entiv
emod
el
28day
sCF
Uco
unt,
Survivalra
te
Spleen
,lung
s>1
.2lo
g*
>1.8lo
g*
>1.7lo
g*
1de
ad
3de
ad
3de
ad
>0.9lo
g*
>1.4lo
g*
>1.6lo
g*
*Th
erewasaCFU
increa
sein
thegrou
psw
ithand
with
outc
lavu
lana
teco
mpa
redtoth
estarto
fthe
trea
tmen
t.
MIC:M
inim
alin
hibitorycon
centratio
n(m
g/L),C
LV:c
lavu
lana
te(m
g/L),MBC
:minim
albacteric
idalcon
centratio
n(m
g/L),C
FU:c
olon
yform
ing
units
,od:onc
eada
y,bid:twice
aday
,tid:three
timesaday
,qid:fou
rtim
esaday
,ND:notdescribed
.
Ertapenem
InamurineTBmodel infectedwithH37Rv,adoseof100mg/kgoncedailyertapenemas
monotherapyorincombinationwithclavulanatehadneitherbactericidalnorbacteriostatic
activityinlungsandspleensofTB-infectedmice.Spleenweightandlunglesionsremained
similarcomparedtotheuntreatedgroupofmice.TherewasanincreaseinCFUscompared
totheCFUsatthestartofthetreatment(35).
OtherCarbapenems
Anoraldoseof500mg/kgfaropenem-medoxomil,giventhreetimesdaily,gaveareduction
of2logCFUcountinthelungsofTF3157DHP-1deficientmice(53).Neitherinvivonorclinical
studiesforothercarbapenemsaspartofamulti-drugregimenagainstTBwereretrieved.
Clinicalstudies
Resultsoftheclinicalstudiesreportingoncarbapenemsarepresentedintable3.
Imipenem
Ten patients were treated with imipenem in combination with two or more other
antimicrobialagents.Itwasreportedthatitwaslikelythat1gofimipenem(BID)contributed
to sputum culture conversion in these patients (52). A prospective study evaluated 1000
mg/day imipenem/clavulanateatadoseofoncedaily in12patients,11of thesepatients
receivedlinezolid-containingregimens.Allpatientsshowedsputumandcultureconversion
within180days.Noadverseeventswerereportedforimipenem/clavulanate(54).Inalarge
observational study, the clinical outcomes of 84 patients, treated with 500 mg
imipenem/clavulanatefourtimesaday,werecomparedwithresultsfrom168controls.The
studyshowedthatimipenem-containingregimensachievedcomparableresultscomparedto
theimipenemsparingregimens,whilesuccessratesweresimilartomajorinternationalMDR-
TBcohorts(55).
Meropenem
A regimen includingmeropenem-clavulanate given to 18 patientswith severe pulmonary
XDR-TB led to sputum culture conversion in 15 patients, of which 10 has successfully
completedandfivepatientswereconsideredcuredaccordingtoWHOguidelines.Long-
30
Chapter 2
Tabl
e3.
Resultsoftheinhum
anstudiesreportin
gon
carbapenems.
Firs
taut
hor(
ref)
Year
of
publ
icat
ion
Coun
try
Stud
ypo
pula
tion
Stud
yde
sign
Drug
Do
sage
Pa
tient
sPa
edi
atric
Sp
utum
Sm
ear
Sput
um
cultu
re
Trea
tmen
tsuc
ces
Adve
rse
even
ts
inte
rrup
tion
due
AE
Arbexetal(54)
2016
Brazil
2013-2
015
Observatio
nal,
retrospective
Imipenem
1goc
12
No
12/12
12/12
7/12
0/12
0/12
Cham
bersetal
(52)
2005
USA
ND
Prospective
Imipenem
1gbid
10
No
ND
8/10
7/10
ND
ND
DeLorenzoetal
(58)
2014
Italy,
The
Netherla
nds
2001-2012
Observatio
nal
case-con
trol
Merop
ene
m
1gtid
37
No
28/32
31/37
ND
5/37
2/5
Payenetal(57)
2018
Belgium
2009-2016
Retrospective
casese
ries
Merop
ene
m
2g
tid
(thenbid)
18
No
16/18
16/18
15/18
0/18
0/18
Palmero
etal
(59)
2015
Argentina
2012-2013
Retrospective
Merop
ene
m
2g
tid
(then
1g
tid)
10
No
ND
8/10
3/6
0/10
ND
Van
Rijnetal
(10)
2016
The
Netherla
nds
2010-2013
Retrospective
Ertapenem
1goc
18
yes
ND
15/18
15/18
2/18
3/18
Tiberietal(61)
2016
Italy,
2008-2015
Retrospective,
coho
rt
Ertapenem
1goc
5No
3/5
3/5
4/5
0/5
0/5
Tiberietal(60)
2016
Multicentred
in3cou
ntrie
s 2003-2015
Observatio
nal,
retrospective,
coho
rt
Merop
ene
m
1gtid
(2g
tid)
96
No
55/58
55/58
55/96
6/93
8/94
Tiberi
etal(11,
55)
2016
Multicentred
in8cou
ntrie
s 2003-2015
Observatio
nal
retrospective
case-con
trol
Imipenem
500mgqid
84
No
51/64
51/64
34/57
3/56
4/55
od:onceaday,bid:twiceaday,tid:th
reetim
esadat,qid:fou
rtimesaday.PK:pharm
acokinetic,N
D:notdescribed
termsafetywasnotaprobleminthisstudyasnoadverseeventswerereported(56-57).The
first study, that evaluated efficacy, safety and tolerability,was a case-control study in 37
patients, who received meropenem/clavulanate as part of a linezolid based multi-drug
regimen.Thisisthefirststudythatshowedanaddedvalueofmeropenem/clavulanateina
multi-drug regimen. The meropenem/clavulanate containing regimen showed a sputum
microscopy conversion of 87.5 % and a sputum culture conversion of 83.8%, while the
meropenem/clavulanatesparingregimenshowedasputummicroscopyconversionof56.3%
andasputumcultureconversionof62.5%after90daysoftreatment(58).Inanotherstudy,
10XDRandpre-XDRfemalepatientswere treatedwithmulti-drugregimensandreceived
meropenem/clavulanatefor6monthsormore.Eightpatientsachievedsputumconversion
after 6 months, while two patients died. (59). Pharmacokinetic parameters of 1 g
meropenem/clavulanategiven intravenouslyover5minutes showedaserumpeakof112
mg/mlandaconcentrationof28.6mg*h/L (39). Inanobservational retrospectivecohort-
study,efficacyandsafetywereevaluatedin96patientstreatedwithmeropenem/clavulanate
containingregimensandcomparedwith168controls.Sputumsmearandcultureconversion
rates were found to be similar (60) In an observational study comparing therapeutic
contribution, such as sputum smear and culture conversion rates and success rates, of
imipenem/clavulanate andmeropenem/ clavulanate in a background regimen, suggested
thatmeropenem/clavulanatecancontributetotheefficacyofaregimenintreatingM/XDR-
TBpatients(11).
Ertapenem
Thefirstreportonclinicalexperiencewithertapenempresenteddatafromfivepatientswho
were treated with an intravenous injection of 1 g ertapenem once daily in a multi-drug
regimen.Threeofthesepatientsshowedsputumsmearandcultureconversion;fouroffive
patientshadasuccessfultreatmentoutcome.Twopatientsinterruptedtreatmentduetoan
adverseevent.Theseadverseeventswereconsideredunrelatedtothestudydrug(61).Inan
observationalstudy18patientsweretreatedwith1gertapenemoncedaily;fifteenofthese
patientshadasuccessfultreatmentoutcomewerecured.Threepatientswere lostdueto
follow-up.Threepatientsstoppedertapenemtreatmentduetoertapenemunrelatedadverse
events.Pharmacokineticparameterswereevaluated in12patients, showingameanpeak
plasmaof127.5(range73.9-277.9)mg/LandanAUCof544.9(range390-1130)mg*h/L.
31
2
Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis
Tabl
e3.
Resultsoftheinhum
anstudiesreportin
gon
carbapenems.
Firs
taut
hor(
ref)
Year
of
publ
icat
ion
Coun
try
Stud
ypo
pula
tion
Stud
yde
sign
Drug
Do
sage
Pa
tient
sPa
edi
atric
Sp
utum
Sm
ear
Sput
um
cultu
re
Trea
tmen
tsuc
ces
Adve
rse
even
ts
inte
rrup
tion
due
AE
Arbexetal(54)
2016
Brazil
2013-2
015
Observatio
nal,
retrospective
Imipenem
1goc
12
No
12/12
12/12
7/12
0/12
0/12
Cham
bersetal
(52)
2005
USA
ND
Prospective
Imipenem
1gbid
10
No
ND
8/10
7/10
ND
ND
DeLorenzoetal
(58)
2014
Italy,
The
Netherla
nds
2001-2012
Observatio
nal
case-con
trol
Merop
ene
m
1gtid
37
No
28/32
31/37
ND
5/37
2/5
Payenetal(57)
2018
Belgium
2009-2016
Retrospective
casese
ries
Merop
ene
m
2g
tid
(thenbid)
18
No
16/18
16/18
15/18
0/18
0/18
Palmero
etal
(59)
2015
Argentina
2012-2013
Retrospective
Merop
ene
m
2g
tid
(then
1g
tid)
10
No
ND
8/10
3/6
0/10
ND
Van
Rijnetal
(10)
2016
The
Netherla
nds
2010-2013
Retrospective
Ertapenem
1goc
18
yes
ND
15/18
15/18
2/18
3/18
Tiberietal(61)
2016
Italy,
2008-2015
Retrospective,
coho
rt
Ertapenem
1goc
5No
3/5
3/5
4/5
0/5
0/5
Tiberietal(60)
2016
Multicentred
in3cou
ntrie
s2003-2015
Observatio
nal,
retrospective,
coho
rt
Merop
ene
m
1gtid
(2g
tid)
96
No
55/58
55/58
55/96
6/93
8/94
Tiberi
etal(11,
55)
2016
Multicentred
in8cou
ntrie
s2003-2015
Observatio
nal
retrospective
case-con
trol
Imipenem
500mgqid
84
No
51/64
51/64
34/57
3/56
4/55
od:onceaday,bid:twiceaday,tid:th
reetim
esadat,qid:fou
rtimesaday.PK:pharm
acokinetic,N
D:notdescribed
termsafetywasnotaprobleminthisstudyasnoadverseeventswerereported(56-57).The
first study, that evaluated efficacy, safety and tolerability,was a case-control study in 37
patients, who received meropenem/clavulanate as part of a linezolid based multi-drug
regimen.Thisisthefirststudythatshowedanaddedvalueofmeropenem/clavulanateina
multi-drug regimen. The meropenem/clavulanate containing regimen showed a sputum
microscopy conversion of 87.5 % and a sputum culture conversion of 83.8%, while the
meropenem/clavulanatesparingregimenshowedasputummicroscopyconversionof56.3%
andasputumcultureconversionof62.5%after90daysoftreatment(58).Inanotherstudy,
10XDRandpre-XDRfemalepatientswere treatedwithmulti-drugregimensandreceived
meropenem/clavulanatefor6monthsormore.Eightpatientsachievedsputumconversion
after 6 months, while two patients died. (59). Pharmacokinetic parameters of 1 g
meropenem/clavulanategiven intravenouslyover5minutes showedaserumpeakof112
mg/mlandaconcentrationof28.6mg*h/L (39). Inanobservational retrospectivecohort-
study,efficacyandsafetywereevaluatedin96patientstreatedwithmeropenem/clavulanate
containingregimensandcomparedwith168controls.Sputumsmearandcultureconversion
rates were found to be similar (60) In an observational study comparing therapeutic
contribution, such as sputum smear and culture conversion rates and success rates, of
imipenem/clavulanate andmeropenem/ clavulanate in a background regimen, suggested
thatmeropenem/clavulanatecancontributetotheefficacyofaregimenintreatingM/XDR-
TBpatients(11).
Ertapenem
Thefirstreportonclinicalexperiencewithertapenempresenteddatafromfivepatientswho
were treated with an intravenous injection of 1 g ertapenem once daily in a multi-drug
regimen.Threeofthesepatientsshowedsputumsmearandcultureconversion;fouroffive
patientshadasuccessfultreatmentoutcome.Twopatientsinterruptedtreatmentduetoan
adverseevent.Theseadverseeventswereconsideredunrelatedtothestudydrug(61).Inan
observationalstudy18patientsweretreatedwith1gertapenemoncedaily;fifteenofthese
patientshadasuccessfultreatmentoutcomewerecured.Threepatientswere lostdueto
follow-up.Threepatientsstoppedertapenemtreatmentduetoertapenemunrelatedadverse
events.Pharmacokineticparameterswereevaluated in12patients, showingameanpeak
plasmaof127.5(range73.9-277.9)mg/LandanAUCof544.9(range390-1130)mg*h/L.
32
Chapter 2
BasedonaMICof0.25mg/ml11/12patientsreachedthetargetvalueof40%Tfree>MICwas
exceeded(10).Thepharmacokineticmodelcomposedinthisstudywasshowntoadequately
predictertapenemexposureinMDR-TBpatients.TheMonteCarlosimulation,whichhada
timerestrictionof0–6h,showedthatthebestperforminglimitedsamplingstrategywasat1
and 5 h after intravenous injection. (62). In another pharmacokinetic model study using
prospectivedatafrom12TBpatientsitwasobservedthat2gertapenemoncedailyresulted
inamorethanadose-proportionalincreaseinAUCcomparedtooncedaily1gertapenem.
BasedonaMICof1.0mg/L,11outof12patientsreachedthetargetvalueof40%Tfree>MIC
(63).
DiscussionHugonnetandcolleaguesfirststatedthatcarbapenemshaveantimycobacterialactivity(7).
Subsequently,studiesaddressingtheinactivationmechanismofLDTprovidedtheunderlying
evidencetosupportthehypothesisofactivityofcarbapenemsagainstM.tuberculosis(14-
28).Inspiteofthisaseriesofinvitrostudieshavebeencarriedout,someofwhichdetected
an effect and some of which did not (8,32-50). Only later, was it recognized that these
confusing results are probably explained by the chemical instability of carbapenems, in
culturemedia at the temperatures typically used in in vitro studies, andmanypreviously
publishedinvitrostudiesarelikelytohavereportedfalselyhighMICs(45).
Overall the results of the studies identified in this review, which used a variety of
experimental methods to test clinical and laboratory strains of M. tuberculosis for
susceptibility to carbapenems, are consistent. Carbapenems are more active against M.
tuberculosisifusedincombinationwithclavulanate,aBLaCinhibitor.(8,32-50).Inlinewith
theseinvitrostudiestheadditionofclavulanateimprovedthesurvivalrateinmice(35).As
theEuropeanMedicinesAgency(EMA)hasacceptedandqualifiedthe invitrohollowfiber
systemmodelsasamethodologytodefinepharmacokineticandpharmacodynamic(PK/PD)
parameters,thesemoderninvitrostudiescanbeusedtoavoidtheproblemsassociatedwith
thechemicalinstabilityoftheseagentsinstandardagar-basedMICtesting.Thus,hollowfiber
systems have the potential for dose finding and regimen selection studies on the use of
carbapenemsinthetreatmentofTB(64-65).
Few in vivo studies have been performed due to the short half-life and lower serum
concentrationsofcarbapenemsinmice(35).
One prospective, two observational and seven retrospective clinical studies to assess
effectiveness,safetyandtolerabilityofthreedifferentcarbapenems(imipenem,meropenem
and ertapenem) have been performed. Adverse events due to carbapenems were mild,
confirmingwhatweknowfromotherinfectiousdiseases;butincontrasttootherrepurposed
drugslikelinezolid(55,58,60).Todate,onlytwolargeretrospectivestudieswithM/XDR-TB
patientshavebeenperformedwithimipenem(84patients),andmeropenem(96patients)
(11).Meropenem/clavulanatewassuggestedtobemoreefficient inmanagingM/XDR-TB
(11),howeverinterpretationallimitationswerementioned.
We found no clear evidence to select one particular carbapenem among the different
candidatecompounds,whendesigninganeffectiveM/XDR-TBregimen.Botheconomicaland
clinicalfactorsplayarole.Whereas imipenemisthecheapercarbapenem,ertapenemhas
thepotentialadvantagethatitisonlygivenoncedaily;andmeropenemisbysomeauthors
believedtobethemosteffectiveinhumans,butnohead-to-headcomparisonstudieshave
confirmedthistodate.Therefore,moreclinicalevidenceanddoseoptimizationsubstantiated
for example by hollow fiber infection studies are needed to support the repurposing
carbapenemsforthetreatmentofM/XDR-TB.
Clinicalstudiesarehamperedbythefactthatcurrentlynocombinationofacarbapenemwith
clavulanateiscommerciallyavailable.Furthermore,clavulanateisnotavailablealonesoat
present it isnotpracticallypossible toprescribecarbapenemwithclavulanate.Therefore,
amoxicillin–clavulanateisoftenco-administeredalongwithacarbapenemincasethelatter
is preferred for treatment. Unfortunately, amoxicillin has gastrointestinal side effects
potentiallycomplicatingprolongedtreatment.Therefore,combinedtreatmentamoxicillin–
clavulanate with a carbapenem is only an option for TB treatment of complicated cases
showingmulti-orextensivedrugresistance(42).Although,Gonzaloetal.reportedapotential
benefitthatMICvaluesdropwhenamoxicillinisaddedtoacombinationofmeropenemand
clavulanate.
33
2
Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis
BasedonaMICof0.25mg/ml11/12patientsreachedthetargetvalueof40%Tfree>MICwas
exceeded(10).Thepharmacokineticmodelcomposedinthisstudywasshowntoadequately
predictertapenemexposureinMDR-TBpatients.TheMonteCarlosimulation,whichhada
timerestrictionof0–6h,showedthatthebestperforminglimitedsamplingstrategywasat1
and 5 h after intravenous injection. (62). In another pharmacokinetic model study using
prospectivedatafrom12TBpatientsitwasobservedthat2gertapenemoncedailyresulted
inamorethanadose-proportionalincreaseinAUCcomparedtooncedaily1gertapenem.
BasedonaMICof1.0mg/L,11outof12patientsreachedthetargetvalueof40%Tfree>MIC
(63).
DiscussionHugonnetandcolleaguesfirststatedthatcarbapenemshaveantimycobacterialactivity(7).
Subsequently,studiesaddressingtheinactivationmechanismofLDTprovidedtheunderlying
evidencetosupportthehypothesisofactivityofcarbapenemsagainstM.tuberculosis(14-
28).Inspiteofthisaseriesofinvitrostudieshavebeencarriedout,someofwhichdetected
an effect and some of which did not (8,32-50). Only later, was it recognized that these
confusing results are probably explained by the chemical instability of carbapenems, in
culturemedia at the temperatures typically used in in vitro studies, andmanypreviously
publishedinvitrostudiesarelikelytohavereportedfalselyhighMICs(45).
Overall the results of the studies identified in this review, which used a variety of
experimental methods to test clinical and laboratory strains of M. tuberculosis for
susceptibility to carbapenems, are consistent. Carbapenems are more active against M.
tuberculosisifusedincombinationwithclavulanate,aBLaCinhibitor.(8,32-50).Inlinewith
theseinvitrostudiestheadditionofclavulanateimprovedthesurvivalrateinmice(35).As
theEuropeanMedicinesAgency(EMA)hasacceptedandqualifiedthe invitrohollowfiber
systemmodelsasamethodologytodefinepharmacokineticandpharmacodynamic(PK/PD)
parameters,thesemoderninvitrostudiescanbeusedtoavoidtheproblemsassociatedwith
thechemicalinstabilityoftheseagentsinstandardagar-basedMICtesting.Thus,hollowfiber
systems have the potential for dose finding and regimen selection studies on the use of
carbapenemsinthetreatmentofTB(64-65).
Few in vivo studies have been performed due to the short half-life and lower serum
concentrationsofcarbapenemsinmice(35).
One prospective, two observational and seven retrospective clinical studies to assess
effectiveness,safetyandtolerabilityofthreedifferentcarbapenems(imipenem,meropenem
and ertapenem) have been performed. Adverse events due to carbapenems were mild,
confirmingwhatweknowfromotherinfectiousdiseases;butincontrasttootherrepurposed
drugslikelinezolid(55,58,60).Todate,onlytwolargeretrospectivestudieswithM/XDR-TB
patientshavebeenperformedwithimipenem(84patients),andmeropenem(96patients)
(11).Meropenem/clavulanatewassuggestedtobemoreefficient inmanagingM/XDR-TB
(11),howeverinterpretationallimitationswerementioned.
We found no clear evidence to select one particular carbapenem among the different
candidatecompounds,whendesigninganeffectiveM/XDR-TBregimen.Botheconomicaland
clinicalfactorsplayarole.Whereas imipenemisthecheapercarbapenem,ertapenemhas
thepotentialadvantagethatitisonlygivenoncedaily;andmeropenemisbysomeauthors
believedtobethemosteffectiveinhumans,butnohead-to-headcomparisonstudieshave
confirmedthistodate.Therefore,moreclinicalevidenceanddoseoptimizationsubstantiated
for example by hollow fiber infection studies are needed to support the repurposing
carbapenemsforthetreatmentofM/XDR-TB.
Clinicalstudiesarehamperedbythefactthatcurrentlynocombinationofacarbapenemwith
clavulanateiscommerciallyavailable.Furthermore,clavulanateisnotavailablealonesoat
present it isnotpracticallypossible toprescribecarbapenemwithclavulanate.Therefore,
amoxicillin–clavulanateisoftenco-administeredalongwithacarbapenemincasethelatter
is preferred for treatment. Unfortunately, amoxicillin has gastrointestinal side effects
potentiallycomplicatingprolongedtreatment.Therefore,combinedtreatmentamoxicillin–
clavulanate with a carbapenem is only an option for TB treatment of complicated cases
showingmulti-orextensivedrugresistance(42).Although,Gonzaloetal.reportedapotential
benefitthatMICvaluesdropwhenamoxicillinisaddedtoacombinationofmeropenemand
clavulanate.
34
Chapter 2
Duetodifferentprocedures,analyticalmethodsanddesign,thebiochemicalinstabilityofthe
drugsofinterest,theshorthalf-liveofdrugsofinterestinmice,diversityinMICdetermination
and intolerance in addition to resistance, itwas not possible to perform ameta-analysis.
Whiletheobservationaldataarepromising,carbapenemscanonlyrecommendedincaseof
resistancetogroupAandgroupBdrugsinM/XDR-TBtreatment.
Theidealcarbapenemwouldhavetheantimycobacterialactivityofimipenem,thehalf-lifeof
ertapenem and the oral bioavailability of tebipenem-pivoxil. Due to increasing resistance
observed in XDR-TB isolates (66-67) and in MDR-TB patients with resistance to an
aminoglycoside,carbapenemsmaybeavaluablealternativetothecurrentinjectablesecond
line drugs. Assessment of intracellular activity as well as activity against dormant M.
tuberculosis by carbapenems is a critical step to further explore the potential of these
repurposeddrugs.
As successful treatment outcome forM/XDR-TB is still poor, ranging from 25-50% (1) an
improvementofthecurrenttreatmentisurgentlyneeded.Anindividualdatameta-analysis
among12,030 individualpatients from50studies showedasignificantlybetter treatment
outcomeforpatientswhoreceivedcarbapenemscomparedtootherdrugstraditionallyused
fortreatmentofMDR-TB. (68).Sincethere isaneedforneworrepurposeddrugs forthe
treatmentofM/XDR-TB,phaseII/IIIclinicaltrialsareurgentlyneededforcarbapenemsto
further evaluate their potential. Long term safety and activity againstM. tuberculosis are
supported by observational data and several studies (41,50,69). A phase II prospective
randomized controlled study evaluating a carbapenemplus a BLaC inhibitor on top of an
optimizedbackgroundregimenversusstandardofcarewouldbeanappropriatestrategyto
testthepotentialbenefitsofcarbapenemsforM/XDR-TBtreatment.
Conclusion
Now the variable results of in vitro studies have been explained and the activity of
carbapenemsinthepresenceofaBLaCinhibitorisestablished,thesedrugsshouldbefurther
developed for the treatment of multi- and extensive drug resistant M. tuberculosis.
Ultimately,awell-designedphase2studyisneededtosubstantiatetheclaimedbenefitsof
carbapenemsinpatientswithdrug-resistantTB.
References
1. World Health Organization. Global Tuberculosis Report 2017.
http://www.who.int/tb/publications/global_report/en/accessed23march2018
2. FalzonD,SchünemannHJ,HarauszE,González-AnguloL,LienhardtC,JaramilloE,Weyer
K.2017WorldHealthOrganizationtreatmentguidelinesfordrug-resistanttuberculosis,
2016update.EurRespirJ.Mar22;49(3).
3. Nguyen TVA, Anthony RM, Bañuls AL, VuDH, Alffenaar JC. Bedaquiline resistance: Its
emergence, mechanism and prevention. Clin Infect Dis. 2017 Nov 8. doi:
10.1093/cid/cix992.
4. Alsaad,N.,B.Wilffert,R.vanAltena,W.C.deLange,T.S.vanderWerf,J.G.Kosterink,
andJ.W.Alffenaar.2013.PotentialantimicrobialagentsforthetreatmentofMDR-TB.
Eur.Respir.J.doi:10.1183/09031936.00113713.
5. vanderPaardtAF,WilffertB,AkkermanOW,deLangeWC,vanSoolingenD,SinhaB,van
derWerfTS,KosterinkJG,AlffenaarJW.2015.Evaluationofmacrolidesforpossibleuse
againstmultidruf-resistantMycobacteriumtuberculosis.Eur.RespirJ.aug;46(2):444-55.
6. World Health Organization. WHO treatment guidelines for drug-resistant
tuberculosis.http://www.who.int/tb/areas-of-work/drug-resistant-tb/treatment/en/
accessed23march2018
7. Hugonnet JE, Blanchard JS. 2007. Irreversible inhibition of the Mycobacterium
tuberculosis beta-lactamase by clavulanate. Biochemistry. 46:11998-12004. doi:
10.1021/bi701506h.
8. Hugonnet JE,TremblayLW,BoshoffHI,Barry3rdCE,Blanchard JS.2009.Meropenem-
clavulanate is effective against extensively drug-resistantMycobacterium tuberculosis.
Science.323:1215-1218.doi:10.1126/science.1167498;10.1126/science.1167498.
9. Cynamon,M.H.,andG.S.Palmer.1983.Invitroactivityofamoxicillinincombinationwith
clavulanic acid against Mycobacterium tuberculosis. Antimicrob Agents Chemother.
24:429-431.
10. VanRijnSP,vanAltenaR,Akkerman,OW,vanSoolingenD,vanderLaanT,deLangeWCM,
KosterinkJGW,vanderWerfTS,AlffenaarJWC.2016.Pharmacokineticsofertapenemin
patients with multidrug-resistant tuberculosis. Eur Respir J. 47:1229-1234.
Doi:10.1183/13993003.01654-2015.
35
2
Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis
Duetodifferentprocedures,analyticalmethodsanddesign,thebiochemicalinstabilityofthe
drugsofinterest,theshorthalf-liveofdrugsofinterestinmice,diversityinMICdetermination
and intolerance in addition to resistance, itwas not possible to perform ameta-analysis.
Whiletheobservationaldataarepromising,carbapenemscanonlyrecommendedincaseof
resistancetogroupAandgroupBdrugsinM/XDR-TBtreatment.
Theidealcarbapenemwouldhavetheantimycobacterialactivityofimipenem,thehalf-lifeof
ertapenem and the oral bioavailability of tebipenem-pivoxil. Due to increasing resistance
observed in XDR-TB isolates (66-67) and in MDR-TB patients with resistance to an
aminoglycoside,carbapenemsmaybeavaluablealternativetothecurrentinjectablesecond
line drugs. Assessment of intracellular activity as well as activity against dormant M.
tuberculosis by carbapenems is a critical step to further explore the potential of these
repurposeddrugs.
As successful treatment outcome forM/XDR-TB is still poor, ranging from 25-50% (1) an
improvementofthecurrenttreatmentisurgentlyneeded.Anindividualdatameta-analysis
among12,030 individualpatients from50studies showedasignificantlybetter treatment
outcomeforpatientswhoreceivedcarbapenemscomparedtootherdrugstraditionallyused
fortreatmentofMDR-TB. (68).Sincethere isaneedforneworrepurposeddrugs forthe
treatmentofM/XDR-TB,phaseII/IIIclinicaltrialsareurgentlyneededforcarbapenemsto
further evaluate their potential. Long term safety and activity againstM. tuberculosis are
supported by observational data and several studies (41,50,69). A phase II prospective
randomized controlled study evaluating a carbapenemplus a BLaC inhibitor on top of an
optimizedbackgroundregimenversusstandardofcarewouldbeanappropriatestrategyto
testthepotentialbenefitsofcarbapenemsforM/XDR-TBtreatment.
Conclusion
Now the variable results of in vitro studies have been explained and the activity of
carbapenemsinthepresenceofaBLaCinhibitorisestablished,thesedrugsshouldbefurther
developed for the treatment of multi- and extensive drug resistant M. tuberculosis.
Ultimately,awell-designedphase2studyisneededtosubstantiatetheclaimedbenefitsof
carbapenemsinpatientswithdrug-resistantTB.
References
1. World Health Organization. Global Tuberculosis Report 2017.
http://www.who.int/tb/publications/global_report/en/accessed23march2018
2. FalzonD,SchünemannHJ,HarauszE,González-AnguloL,LienhardtC,JaramilloE,Weyer
K.2017WorldHealthOrganizationtreatmentguidelinesfordrug-resistanttuberculosis,
2016update.EurRespirJ.Mar22;49(3).
3. Nguyen TVA, Anthony RM, Bañuls AL, VuDH, Alffenaar JC. Bedaquiline resistance: Its
emergence, mechanism and prevention. Clin Infect Dis. 2017 Nov 8. doi:
10.1093/cid/cix992.
4. Alsaad,N.,B.Wilffert,R.vanAltena,W.C.deLange,T.S.vanderWerf,J.G.Kosterink,
andJ.W.Alffenaar.2013.PotentialantimicrobialagentsforthetreatmentofMDR-TB.
Eur.Respir.J.doi:10.1183/09031936.00113713.
5. vanderPaardtAF,WilffertB,AkkermanOW,deLangeWC,vanSoolingenD,SinhaB,van
derWerfTS,KosterinkJG,AlffenaarJW.2015.Evaluationofmacrolidesforpossibleuse
againstmultidruf-resistantMycobacteriumtuberculosis.Eur.RespirJ.aug;46(2):444-55.
6. World Health Organization. WHO treatment guidelines for drug-resistant
tuberculosis.http://www.who.int/tb/areas-of-work/drug-resistant-tb/treatment/en/
accessed23march2018
7. Hugonnet JE, Blanchard JS. 2007. Irreversible inhibition of the Mycobacterium
tuberculosis beta-lactamase by clavulanate. Biochemistry. 46:11998-12004. doi:
10.1021/bi701506h.
8. Hugonnet JE,TremblayLW,BoshoffHI,Barry3rdCE,Blanchard JS.2009.Meropenem-
clavulanate is effective against extensively drug-resistantMycobacterium tuberculosis.
Science.323:1215-1218.doi:10.1126/science.1167498;10.1126/science.1167498.
9. Cynamon,M.H.,andG.S.Palmer.1983.Invitroactivityofamoxicillinincombinationwith
clavulanic acid against Mycobacterium tuberculosis. Antimicrob Agents Chemother.
24:429-431.
10. VanRijnSP,vanAltenaR,Akkerman,OW,vanSoolingenD,vanderLaanT,deLangeWCM,
KosterinkJGW,vanderWerfTS,AlffenaarJWC.2016.Pharmacokineticsofertapenemin
patients with multidrug-resistant tuberculosis. Eur Respir J. 47:1229-1234.
Doi:10.1183/13993003.01654-2015.
36
Chapter 2
11. TiberiS,SotgiuG,D’AmbrosioL,CentisR,ArbexMA,ArrascueEA,AlffenaarJW,Caminero
JA,GagaM,GualanoG,SkrahinaA,SolovicA,SulisG,TadoliniM,GuizadoVA,DeLorenzo
S,AriasAJR,ScardigliA,AkkermanOW,AleksaA,ArtsukevichJ,AvchinkoV,BoniniEH,
Marín FAC, Collahuazo López L, de Vries G, Dore S, Kunst H,Matteelli A,Moschos C,
PalmiereF,PapavasileiouA,PayenMC,PianaA, SpaneveloA,VasquezDV,ViggianiP,
White V, Zumla A, Migliori GB. 2016. Comparison of effectiveness and safety of
imipenem/clavulanate-versus meropenem/clavulanate- containing regiments in the
treatmentofMDR-andXDR-TB.EurRespirJ.47(6):1758-66
12. DeshpandeD,SrivastavaS,BendetP,MartinKR,CirrincioneKN,LeePS,PasipanodyaJG,
Dheda K, Gumbo T. 2018. Antibacterial and Sterilizing Effect of Benzylpenicillin in
Tuberculosis.AntimicrobAgentsChemother.Jan25;62(2).
13. DeshpandeD,SrivastavaS,ChapagainM,MagombedzeG,MartinKR,CirrincioneKN,Lee
PS, Koeuth T, Dheda K, Gumbo T. 2017. Ceftazidime-avibactamhas potent sterilizing
activityagainsthighlydrug-resistanttuberculosis.SciAdv.Aug30;3(8):e1701102
14. Kurz, S. G., K. A.Wolff, S. Hazra, C. R. Bethel, A.M. Hujer, K.M. Smith, Y. Xu, L.W.
Tremblay, J. S. Blanchard, L. Nguyen, and R. A. Bonomo. 2013. Can inhibitor-resistant
substitutionsintheMycobacteriumtuberculosisbeta-LactamaseBLaCleadtoclavulanate
resistance?:abiochemicalrationalefortheuseofbeta-lactam-beta-lactamaseinhibitor
combinations.Antimicrob.AgentsChemother.57:6085-6096.doi:10.1128/AAC.01253-
13;10.1128/AAC.01253-13.
15. Lavollay, M., M. Arthur, M. Fourgeaud, L. Dubost, A. Marie, N. Veziris, D. Blanot, L.
Gutmann,andJ.L.Mainardi.2008.Thepeptidoglycanofstationary-phaseMycobacterium
tuberculosis predominantly contains cross-links generated by L,D-transpeptidation. J.
Bacteriol.190:4360-4366.doi:10.1128/JB.00239-08;10.1128/JB.00239-08.
16. Kumar,P.,K.Arora,J.R.Lloyd,I.Y.Lee,V.Nair,E.Fischer,H.I.Boshoff,andC.E.Barry
3rd. 2012. Meropenem inhibits D,D-carboxypeptidase activity in Mycobacterium
tuberculosis. Mol. Microbiol. 86:367-381. doi: 10.1111/j.1365-2958.2012.08199.x;
10.1111/j.1365-2958.2012.08199.x.
17. BrammerBastaLA,GhoshA,oanY,JakoncicJ,LloydEP,TownsendCA,LamichhaneG,
Bianchet MA 2015. Loss of functionally and structurally distinct LD-transpeptidase,
LDtmt5, compromises cell wall integrity of mycobacterium tuberculosis. J Biol Chem
290:25670-25685
18. Cordillot,M.,V.Dubee,S.Triboulet,L.Dubost,A.Marie,J.E.Hugonnet,M.Arthur,andJ.
L.Mainardi.2013.Invitrocross-linkingofMycobacteriumtuberculosispeptidoglycanby
L,D-transpeptidases and inactivation of these enzymes by carbapenems. Antimicrob.
AgentsChemother.57:5940-5945.doi:10.1128/AAC.01663-13;10.1128/AAC.01663-13.
19. Dubee,V.,S.Triboulet, J.L.Mainardi,M.Etheve-Quelquejeu,L.Gutmann,A.Marie,L.
Dubost,J.E.Hugonnet,andM.Arthur.2012.InactivationofMycobacteriumtuberculosis
l,d-transpeptidase LdtMt(1) by carbapenems and cephalosporins. Antimicrob. Agents
Chemother.56:4189-4195.doi:10.1128/AAC.00665-12;10.1128/AAC.00665-12.
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21. KumarP,KaushikA,LloydE,LiSG,MattooR,AmmermanNC,BellDT,PerrymanAL,Zandi
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resistant strains. Acta Crystallogr. D Biol. Crystallogr. 69:420-431. doi:
37
2
Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis
11. TiberiS,SotgiuG,D’AmbrosioL,CentisR,ArbexMA,ArrascueEA,AlffenaarJW,Caminero
JA,GagaM,GualanoG,SkrahinaA,SolovicA,SulisG,TadoliniM,GuizadoVA,DeLorenzo
S,AriasAJR,ScardigliA,AkkermanOW,AleksaA,ArtsukevichJ,AvchinkoV,BoniniEH,
Marín FAC, Collahuazo López L, de Vries G, Dore S, Kunst H,Matteelli A,Moschos C,
PalmiereF,PapavasileiouA,PayenMC,PianaA, SpaneveloA,VasquezDV,ViggianiP,
White V, Zumla A, Migliori GB. 2016. Comparison of effectiveness and safety of
imipenem/clavulanate-versus meropenem/clavulanate- containing regiments in the
treatmentofMDR-andXDR-TB.EurRespirJ.47(6):1758-66
12. DeshpandeD,SrivastavaS,BendetP,MartinKR,CirrincioneKN,LeePS,PasipanodyaJG,
Dheda K, Gumbo T. 2018. Antibacterial and Sterilizing Effect of Benzylpenicillin in
Tuberculosis.AntimicrobAgentsChemother.Jan25;62(2).
13. DeshpandeD,SrivastavaS,ChapagainM,MagombedzeG,MartinKR,CirrincioneKN,Lee
PS, Koeuth T, Dheda K, Gumbo T. 2017. Ceftazidime-avibactamhas potent sterilizing
activityagainsthighlydrug-resistanttuberculosis.SciAdv.Aug30;3(8):e1701102
14. Kurz, S. G., K. A.Wolff, S. Hazra, C. R. Bethel, A.M. Hujer, K.M. Smith, Y. Xu, L.W.
Tremblay, J. S. Blanchard, L. Nguyen, and R. A. Bonomo. 2013. Can inhibitor-resistant
substitutionsintheMycobacteriumtuberculosisbeta-LactamaseBLaCleadtoclavulanate
resistance?:abiochemicalrationalefortheuseofbeta-lactam-beta-lactamaseinhibitor
combinations.Antimicrob.AgentsChemother.57:6085-6096.doi:10.1128/AAC.01253-
13;10.1128/AAC.01253-13.
15. Lavollay, M., M. Arthur, M. Fourgeaud, L. Dubost, A. Marie, N. Veziris, D. Blanot, L.
Gutmann,andJ.L.Mainardi.2008.Thepeptidoglycanofstationary-phaseMycobacterium
tuberculosis predominantly contains cross-links generated by L,D-transpeptidation. J.
Bacteriol.190:4360-4366.doi:10.1128/JB.00239-08;10.1128/JB.00239-08.
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42. GonzaloX,Drobniewski F. 2013. Is there aplace for beta-lactams in the treatmentof
multidrug-resistant/extensively drug-resistant tuberculosis? Synergy between
meropenem and amoxicillin/clavulanate. J. Antimicrob Chemother. 68:366-369. doi:
10.1093/jac/dks395;10.1093/jac/dks395.
43. Horita Y, Maeda S, Kazumi Y, Doi N. 2014. In vitro susceptibility of Mycobacterium
tuberculosis isolates to anoral carbapenemaloneor in combinationwithβ-lactamase
inhibitors.AntimicrobAgentsChemother58(11);7010-14
44. SalaC,DharN,HartkoornRC,ZhangM,HaYA,SchneiderP,ColeST.2010.Simplemodel
39
2
Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis
10.1107/S0907444912048998;10.1107/S0907444912048998.
26. Lecoq,L.,V.Dubee,S.Triboulet,C.Bougault,J.E.Hugonnet,M.Arthur,andJ.P.Simorre.
2013. Structure of Enterococcus faeciuml, d-Transpeptidase Acylated by Ertapenem
Provides Insight into the Inactivation Mechanism. ACS Chem. Biol. doi:
10.1021/cb4001603.
27. Schoonmaker MK, Bishai WR, Lamichhane G. 2014. Nonclassical transpeptidases of
mycobacterium tuberculosis alter cell size, morphology, the cytosolic matric, protein
localization,virulence,andresistance toβ-lactams.AmericanSociety formicrobiology.
Doi:10.1128/JB.01396-13
28. TribouletS,DubéeV, LecoqL,BougaultC,Mainardi JL,RiceLB,Ethève-quelquejeuM,
GutmannL,MarieA,DubostL,HugonnetJE,SImorreJP,ArthurM.2013.Kineticfeatures
of L,D-Transpeptidase inactivationcritical forβ-lactamantibacterialactivity.PLoSOne.
8(7):e67831
29. Nicolau,D.P.2008.Pharmacokineticandpharmacodynamicpropertiesofmeropenem.
Clin.Infect.Dis.47Suppl1:S32-40.doi:10.1086/590064;10.1086/590064.
30. CraigWA.2003.Basicpharmacodynamicsofantibacterialswithclinicalapplicationstothe
useofbeta-lactams,glycopeptides,andlinezolid.Infect.Dis.Clin.NorthAm.17:479-501
31. MoherD,LiberatiA,TetzlaffJ,AltmanDG,PRISMAGroup.2009.Preferredreportingitems
forsystematicreviewsandmeta-analyses:thePRISMAstatement.JClinEpidemiol;62:
1006-1012.
32. Chambers,H.F.,D.Moreau,D.Yajko,C.Miick,C.Wagner,C.Hackbarth,S.Kocagoz,E.
Rosenberg,W. K.Hadley, andH.Nikaido. 1995. Canpenicillins andother beta-lactam
antibioticsbeusedtotreattuberculosis?Antimicrob.AgentsChemother.39:2620-2624.
33. KaushikA,MakkerN,PandeyP,ParrishN,SinghU,LamichaneG.2015.Carbapenemsand
Rifampicin exhibit synergy against Mycobacterium tuberculosis and Mycobacterium
abscessus.AntimicrobAgentsChemother59:6561-6567.Doi:10.1128/AAC.01158-15
34. SolapureS,DineshN,ShandilR,RamachandranV,SharmaS,BhattacharjeeD,GangulyS,
Reddy J, Ahuja V, Panduga V, Parab M, Vishwas KG, Kumar N, Balganesh M,
BalasubramanianV.2013.Invitroandinvivoefficacyofbeta-lactamsagainstreplicating
andslowlygrowing/nonreplicatingM.tuberculosis.AntimicrobAgentsChemother.doi:
10.1128/AAC.00023-13.
35. Veziris, N., C. Truffot, J. L. Mainardi, and V. Jarlier. 2011. Activity of carbapenems
combinedwithclavulanateagainstmurinetuberculosis.Antimicrob.AgentsChemother.
55:2597-2600.doi:10.1128/AAC.01824-10;10.1128/AAC.01824-10.
36. Watt,B.,J.R.Edwards,A.Rayner,A.J.Grindey,andG.Harris.1992.Invitroactivityof
meropenem and imipenem against mycobacteria: development of a daily antibiotic
dosingschedule.Tuber.LungDis.73:134-136.doi:10.1016/0962-8479(92)90145-A.
37. ZhangD,WangY,LuJ,PangY.2016.Invitroactivityofβ-lactamsincombinationwithβ-
lactamase inhibitors against multidrug-resistant Mycobacterium tuberculosis isolates.
AntimicrobAgentsChemother60:393-399.
38. CohenKA,El-HayT,WyresKL,WeissbrodO,MunsamyV,YanoverC,AharonovR,Shaham
O,ConwayTC,GoldschmidtY,BishaiWR,PymA.2016.Paradoxicalhypersusceptibilityof
drug-resistantMycobacteriumtuberculosistoβ-lactamantibiotics.EBioMedicine9:170-
179
39. Cavanaugh JS, Jou R, WU MH, Dalton T, Kurbatova E, Ershova J, Cegielski JP. 2017.
Susceptibilities of MDR Mycobacterium tuberculosis isolates to unconventional drugs
compared with their reported pharmacokinetic/pharmacodynamics parameters. J
AntimicrobChemother72:1678-1687
40. England,K.,H. I.Boshoff,K.Arora,D.Weiner,E.Dayao,D.Schimel,L.E.Via,andC.E.
Barry 3rd. 2012. Meropenem-clavulanic acid shows activity against Mycobacterium
tuberculosis in vivo. Antimicrob Agents Chemother. 56:3384-3387. doi:
10.1128/AAC.05690-11;10.1128/AAC.05690-11.
41. Forsman LD, Giske CG, Bruchfeld J, Schön T, Juréen P, Ängeby K. 2015.Meropenem-
Clavulanic acid has high in vitro activity against multidrug-resistant Mycobacterium
tuberculosis.AntimicrobAgentsChemother59:3630-3632.Doi:10.1128/AAC.00171-15
42. GonzaloX,Drobniewski F. 2013. Is there aplace for beta-lactams in the treatmentof
multidrug-resistant/extensively drug-resistant tuberculosis? Synergy between
meropenem and amoxicillin/clavulanate. J. Antimicrob Chemother. 68:366-369. doi:
10.1093/jac/dks395;10.1093/jac/dks395.
43. Horita Y, Maeda S, Kazumi Y, Doi N. 2014. In vitro susceptibility of Mycobacterium
tuberculosis isolates to anoral carbapenemaloneor in combinationwithβ-lactamase
inhibitors.AntimicrobAgentsChemother58(11);7010-14
44. SalaC,DharN,HartkoornRC,ZhangM,HaYA,SchneiderP,ColeST.2010.Simplemodel
40
Chapter 2
fortestingdrugsagainstnonreplicatingMycobacteriumtuberculosis.AntimicrobAgents
Chemother.54:4150-4158.doi:10.1128/AAC.00821-10;10.1128/AAC.00821-10.
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testing of antibiotics that degrade faster than the doubling time of slow-growing
mycobacteria: Ertapenem sterilizing effect ofMycobacterium tuberculosis. Antimicrob
AgentsChemother60:3193-3195.Doi:10.1128/AAC.02924-15.
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multidrug-resistant tuberculosis in children: FLAME Path on the Milky Way. Clinical
InfectiousDiseases.63:95-102
48. GurumurthyM,VermaR,NaftalinCM,HeeKH,LuQ,TanKH,IssacS,LinW,TanA,Seng
K, Lee LS, Paton N. 2017. Activity of Faropenemwith and without rifampicin against
Mycobacterium tuberculosis: Evaluation in a whole-blood bactericidal activity trial. J
Antimicrobchemother.Doi:10.1093/jac/dkx081
49. DharN,DubeeV,BallellL,CuinetG,HugonnetJE,Signorino-geloF,BarrosD,ArthurM,
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2325
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45. Srivastava S, van Rijn SP,Wessels AMA, Alffenaar JWC,Gumbo T. 2016. Susceptibility
testing of antibiotics that degrade faster than the doubling time of slow-growing
mycobacteria: Ertapenem sterilizing effect ofMycobacterium tuberculosis. Antimicrob
AgentsChemother60:3193-3195.Doi:10.1128/AAC.02924-15.
46. VanRijnSP,SrivastavaS,WesselsMA,vanSoolingenD,AlffenaarJW,GumboT.2017.The
sterilizingeffectofertapenem-clavulanate inahollow fibermodelof tuberculosis and
implicationsonclinicaldosing.AntimicrobAgentsChemotherdoi:10.1128/AAC.02039-16
47. DeshpandeD,SrivastavaS,NuermbergerE,PasipanodyaJG,SwaminathanS,GumboT.
2016AFaropenem,Linezolid,andMoxifloxacinregimenforbothdrug-susceptibleand
multidrug-resistant tuberculosis in children: FLAME Path on the Milky Way. Clinical
InfectiousDiseases.63:95-102
48. GurumurthyM,VermaR,NaftalinCM,HeeKH,LuQ,TanKH,IssacS,LinW,TanA,Seng
K, Lee LS, Paton N. 2017. Activity of Faropenemwith and without rifampicin against
Mycobacterium tuberculosis: Evaluation in a whole-blood bactericidal activity trial. J
Antimicrobchemother.Doi:10.1093/jac/dkx081
49. DharN,DubeeV,BallellL,CuinetG,HugonnetJE,Signorino-geloF,BarrosD,ArthurM,
McKinney JD.2015.RapidCytolysisofMycobacterium tuberculosis by faropenem,and
orallybioavailablebeta-lactamantibiotic.AntimicrobAgentsChemother59:1308-1319.
50. Srivastava S,DeshpandeD, Pasipanodya J,Nuermberger E, Swaminathan S,GumboT.
2016.Optimal clinicaldosesof faropenem, linezolid,andmoxifloxacin inchildrenwith
disseminatedtuberculosis:goldilocks.ClinicalInfectiousDiseases63:102-9
51. KaushikA,AmmermanAC,TasneenR,Story-rollerE,DooleyKE,DormanSE,Nuermberger
EL,LamichhaneG.2017.Invitroandinvivoactivityofbiapenemagainstdrug-susceptible
andrifampicin-resistantMycobacteriumtuberculosis.JAntimicrobChemother72:2320-
2325
52. ChambersHF, Turner J, SchecterGF, KawamuraM,Hopewell PC. 2005. Imipenem for
treatmentoftuberculosisinmiceandhumans.AntimicrobAgentsChemother.49:2816-
2821.doi:10.1128/AAC.49.7.2816-2821.2005.
53. RullasJ,DharN,MckinneyJD,Garcia-PérezA,LelievreJ,DiaconAH,HugonnetJE,Arthur
M,Angulo-BarturenI,Barro-aguirreD,BallellL.2015.Combinationsofβ-lactamantibiotics
currentlyinclinicaltrialsareefficaciousinaDHP-I-deficientmousemodeloftuberculosis
infection.AntimicrobAgentsChemother.59:4997-4999.Doi:10.1128/AAC.01063-15
54. ArbexMA,Bonini EH, KawakamePirollaG,D’Ambrosio L, Centis R,MiglioriGB. 2016.
Effectiveness and safety of imipenem/clavulanate and linezolid to treat multidrug en
extensivelydrug-resistanttuberculosisatareferralhospitalinBrazil.RevPortPneumol
22(6):337-341
55. TiberiS,SotgiuG,D’AmbrosioL,CentisR,ArbexMA,ArrascueEA,AlffenaarJW,Caminero
JA,GagaM,GualanoG,SkrahinaA,SolovicA,SulisG,TadoliniM,GuizadoVA,DeLorenzo
S,AriasAJR,ScardigliA,AkkermanOW,AleksaA,ArtsukevichJ,AvchinkoV,BoniniEH,
Marín FAC, Collahuazo López L, de Vries G, Dore S, Kunst H,Matteelli A,Moschos C,
PalmiereF,PapavasileiouA,PayenMC,PianaA, SpaneveloA,VasquezDV,ViggianiP,
WhiteV,ZumlaA,MiglioriGB.2016.Effectivenessandsafetyofimipenem/clavulanate
addedtoanoptimizedbackgroundregimen(OBR)versusOBRcontrol regimens in the
treatment of multidrug-resistant and extensively drug-resistant tuberculosis. Clinical
InfectiousDiseases62(9):1188-90.Doi:10.1093/cid/ciw088
56. Payen,M.C.,S.DeWit,C.Martin,R.Sergysels,I.Muylle,Y.VanLaethem,andN.Clumeck.
2012. Clinical use of the meropenem-clavulanate combination for extensively drug-
resistant tuberculosis. Int. J. Tuberc. Lung Dis. 16:558-560. doi: 10.5588/ijtld.11.0414;
10.5588/ijtld.11.0414
57. PayenMC,MuylleI,VandenbergO,MathysV,DelforgeM,vandenWijngaertS,Clumeck
N,DewitS.2018.Meropenem-clavulanatefordrug-resistanttuberculosis:afollow-upof
relapse-freecases.IntJTubercLungDis22(1):34-39
58. DeLorenzoS,AlffenaarJW,SotgiuG,CentisR,D’AmbrosioL,TiberiS,BolhuisMS,van
Altena R, Viggiani P, Piana A, Spanevello A, Migliori GB. 2013. Efficacy and safety of
meropenem-clavulanateaddedtolinezolid-containingregimensinthetreatmentofMDR-
/XDR-TB.EurRespirJ41:1386-1392
59. PalmeroD,GonzálezMontanerP,CufréM,GarcíaA,VescovoM,PoggiS.2014.FirstSeries
ofPatientswithXDRandpre-XDRTBtreatedwithregimentsthatincludedmeropenem-
clavulanateinArgentina.ArchBronconeumol.51:e49-e52.
60. Tiberi S, Payen MC, Sotgiu G, D’Ambrosio L, Guizado VA, Alffenaar JW, Arbex MA,
CamineroJA,CentisR,DeLorenzoS,GagaM,GualanoG,AriasAJR,ScardigliA,Skrahina
A,SolovicA,SulisG,TadoliniM,AkkermanOW,ArrascueEA,AleksaA,AvchinkoV,Bonini
42
Chapter 2
EH,MarínFAC,CollahuazoLópezL,deVriesG,DoreS,KunstH,MatteelliA,MoschosC,
Palmiere F, Papavasileiou A, Spanevelo A, Vasquez DV, Viggiani P,White V, Zumla A,
Migliori GB. 2016. Effectiveness and safety of meropenem/clavulanate-containing
regimens in the treatment of MDR- and XDR-TB. Eur Respir J 47:1235-1243.
Doi:10.1183/13993003.02146-2015
61. TiberiS,D’AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,AlffenaarJWC,Migliori
GB.2015.Ertapenem in the treatmentofmultidrug-resistant tuberculosis: first clinical
experience.EurRespirJ47:333-336.Doi:10.1183/13993003.01278-2015
62. VanRijnSP,ZuurMA,vanAltenaR,AkkermanOW,ProostJH,deLangeWCM,Kerstjens
HAM,TouwDJ,vanderWerfTS,KosterinkJGW,AlffenaarJWC.2017.Pharmacokinetic
modelingandlimitedsamplingstrategiesbasedonhealthyvolunteersformonitoringof
ertapenem in patients with multidrug-resistant tuberculosis. Antimicrob Agents
Chemother61:e01783-16.Doi:10.1128/AAC.01783-16
63. ZuurM,GhimireS,BolhuisMS,WesselsAMA,vanAltenaR,deLangeWCM,Kosterink
JGW, Touw DJ, van der Werf TS, Akkerman OW, Alffenaar JWC. 2018. The
pharmacokinetics of 2000 mg Ertapenem in tuberculosis patients. Antimicrob Agents
Chemother.[Epubaheadofprint]
64. Cavaleri M, Manolis E. 2015. Hollow Fiber System for Tuberculosis: The European
medicinesAgencyexperience.ClinInfect.Dis.15;61Suppl1:S1-4
65. European medicines Agency (EMA). 2016. Qualification opinion; in-vitro hollow fiber
systemmodeloftuberculosis(HSF-TB).EMA/CHMP/SAWP/47290/2015.
66. DrusanoGL,SgambatiN,EichasA,BrownDL,KulawyR,LouieA.2010.Thecombination
ofrifampinplusmoxifloxacineissynergisticforsuppressionofresistancebutantagonistic
forcellkillofMycobacteriumtuberculosisasdeterminedinahollow-fiberinfectionmodel.
Mbio.10;1(3)
67. GumboT,LouieA,DezielMR,ParsonsLM,SalfingerM,DrusanoGL.2004.Selectionofa
moxifloxacindosethatsuppressesdrugresistanceinMycobacteriumtuberculosis,byuse
ofaninvitropharmacodynamicinfectionmodelandmathematicalmodeling.JInfectDis.
1;190(9)
68. AhmadKhanF,SalimMAH,DuCrosP,CasasEC,KharmraevA,SikhondzeW,BenedettiA,
BastosM, Lan Z, Jaramillo E, Falzon D, Menzies D. 2017. Effectiveness and safety of
standardized shorter regimens for multidrug-resistant tuberculosis: individual patient
dataandaggregatedatameta-analyses.EurRespirJ27;50(1)
69. CecileMagis-Escurra,RichardMAnthony,AdriGMvanderZanden,DickvanSoolingen,
Jan-Willem C Alffenaar. 2018. Pound foolish and penny wise—when will dosing of
rifampicinbeoptimised?TheLancetRespiratoryMedicine
43
2
Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis
EH,MarínFAC,CollahuazoLópezL,deVriesG,DoreS,KunstH,MatteelliA,MoschosC,
Palmiere F, Papavasileiou A, Spanevelo A, Vasquez DV, Viggiani P,White V, Zumla A,
Migliori GB. 2016. Effectiveness and safety of meropenem/clavulanate-containing
regimens in the treatment of MDR- and XDR-TB. Eur Respir J 47:1235-1243.
Doi:10.1183/13993003.02146-2015
61. TiberiS,D’AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,AlffenaarJWC,Migliori
GB.2015.Ertapenem in the treatmentofmultidrug-resistant tuberculosis: first clinical
experience.EurRespirJ47:333-336.Doi:10.1183/13993003.01278-2015
62. VanRijnSP,ZuurMA,vanAltenaR,AkkermanOW,ProostJH,deLangeWCM,Kerstjens
HAM,TouwDJ,vanderWerfTS,KosterinkJGW,AlffenaarJWC.2017.Pharmacokinetic
modelingandlimitedsamplingstrategiesbasedonhealthyvolunteersformonitoringof
ertapenem in patients with multidrug-resistant tuberculosis. Antimicrob Agents
Chemother61:e01783-16.Doi:10.1128/AAC.01783-16
63. ZuurM,GhimireS,BolhuisMS,WesselsAMA,vanAltenaR,deLangeWCM,Kosterink
JGW, Touw DJ, van der Werf TS, Akkerman OW, Alffenaar JWC. 2018. The
pharmacokinetics of 2000 mg Ertapenem in tuberculosis patients. Antimicrob Agents
Chemother.[Epubaheadofprint]
64. Cavaleri M, Manolis E. 2015. Hollow Fiber System for Tuberculosis: The European
medicinesAgencyexperience.ClinInfect.Dis.15;61Suppl1:S1-4
65. European medicines Agency (EMA). 2016. Qualification opinion; in-vitro hollow fiber
systemmodeloftuberculosis(HSF-TB).EMA/CHMP/SAWP/47290/2015.
66. DrusanoGL,SgambatiN,EichasA,BrownDL,KulawyR,LouieA.2010.Thecombination
ofrifampinplusmoxifloxacineissynergisticforsuppressionofresistancebutantagonistic
forcellkillofMycobacteriumtuberculosisasdeterminedinahollow-fiberinfectionmodel.
Mbio.10;1(3)
67. GumboT,LouieA,DezielMR,ParsonsLM,SalfingerM,DrusanoGL.2004.Selectionofa
moxifloxacindosethatsuppressesdrugresistanceinMycobacteriumtuberculosis,byuse
ofaninvitropharmacodynamicinfectionmodelandmathematicalmodeling.JInfectDis.
1;190(9)
68. AhmadKhanF,SalimMAH,DuCrosP,CasasEC,KharmraevA,SikhondzeW,BenedettiA,
BastosM, Lan Z, Jaramillo E, Falzon D, Menzies D. 2017. Effectiveness and safety of
standardized shorter regimens for multidrug-resistant tuberculosis: individual patient
dataandaggregatedatameta-analyses.EurRespirJ27;50(1)
69. CecileMagis-Escurra,RichardMAnthony,AdriGMvanderZanden,DickvanSoolingen,
Jan-Willem C Alffenaar. 2018. Pound foolish and penny wise—when will dosing of
rifampicinbeoptimised?TheLancetRespiratoryMedicine
CHAPTER 3
Antimicrob Agents Chemother 2014 Jun; 58(6): 3481–3484.PMID: 24733468
S.P. van RijnA.M.A. Wessels
B. GreijdanusD.J. Touw
J.W.C. Alffenaar
Quantification and Validation of Ertapenem Using a Liquid
Chromatography-Tandem Mass Spectrometry Method
CHAPTER 3
Antimicrob Agents Chemother 2014 Jun; 58(6): 3481–3484.PMID: 24733468
S.P. van RijnA.M.A. Wessels
B. GreijdanusD.J. Touw
J.W.C. Alffenaar
Quantification and Validation of Ertapenem Using a Liquid
Chromatography-Tandem Mass Spectrometry Method
46
Chapter 3
AbstractBackground:Ertapenem,acarbapenem,reliesontime-dependentkilling.Therapeuticdrug
monitoring (TDM) should be considered, when used in specific populations, to achieve
optimal bactericidal activity and optimize drug-dosing regimens. No validated LC-MS/MS
methodhasbeenreportedusingdeuteratedertapenemasinternalstandard.Anewsimple
and robust LC-MS/MS method using a quadruple mass spectrometer was developed for
analysisofertapeneminhumanplasma,usingdeuteratedertapenemasinternalstandard.
The calibration curvewas linear over a range of 0.1 (LLOQ) to 125mg/L. The calculated
accuracy ranged from -2.4 % to 10.3%.Within-run CV ranged from 2.7 % to 11.8% and
between-runCVrangedfrom0%to8.4%.Freeze-thawstabilitybiasedbetween-3.3%and
0.1%.StorageofQCsamplesfor96hat4°Cdiffered-4.3to5.6%,storageatroomtemperature
for24h,biasedfrom-10.7%to-14.8%andstorageintheautosamplerbiasedbetween-2.9%
and -10.0%. A simple LC-MS/MS method to quantify ertapenem in human plasma using
deuteratedertapenemasinternalstandardhasbeenvalidated.Thismethodcanbeusedin
pharmacokineticstudiesandinclinicalstudiesbyperformingTDM.
IntroductionCarbapenems belong to the Beta-lactam antibiotics and are widely used against a broad
spectrum of aerobe and anaerobe gram-positive and gram-negative bacteria (1, 2).
Ertapenem,approvedbytheFDAin2001,isoneofthesecarbapenems.Sinceertapenemhas
anapproximatehalf-lifeof4h,itcanbeadministeredoncedaily.Therefore,ertapenemcan
be favored above other carbapenems (3). The pharmacodynamic (PD) parameter of
ertapenem correlates with time dependent killing, which means that the plasma
concentration of ertapenem has to exceed minimal inhibitory concentration (MIC) for a
percentageoftimeofitsdosinginterval(4).
Pharmacokineticdataobtainedinhealthyvolunteersaredifficulttoextrapolatetospecific
patientpopulations.Duetothishighvariabilityinpharmacokineticparameters,exposureof
ertapenemmightbesuboptimal inthesespecificpopulations (5-10).Sinceertapenemisa
time-dependent antibiotic, therapeutic drug monitoring (TDM) should therefore be
considered,whenused inspecificpopulations, toachieveoptimalbactericidalactivityand
optimize drug-dosing regimens. Yetmore PK studies have to be performed to determine
efficacyandsafetyofertapeneminspecificpopulation(11,12).
Ertapenem is being suggested for having potential use against M. tuberculosis (TB) (1).
However, according to Caminero et al. carbapenems are used as fifth line drugs in the
treatment of TB and can only be used in severe cases only, since carbapenems are
administeredintravenously,costsarehighandclinicalexperienceisrestricted(13).Clinical
studiesassessingefficacyorsafetyprofilesforcarbapenemsarescarce,butshowedfavorable
preliminaryresults (14-16).Beforeclinicalefficacy inTBcanbedeterminedadose-finding
studyshouldbeperformedtoevaluatepharmacokineticparametersinthisspecificpatient
population. Therefore, an analytical method to measure ertapenem concentrations is
mandatory.
There are a few methods published about LC-MS/MS quantification and validation of
ertapenem in human plasma (17, 18). Since LC-MS/MS is easy to use frequently in daily
routine,morepharmacokineticstudiesarebeingperformedtoquantifydrugs.Itistherefore
47
3
Quantification and Validation of Ertapenem Using a LC-MS/MS
AbstractBackground:Ertapenem,acarbapenem,reliesontime-dependentkilling.Therapeuticdrug
monitoring (TDM) should be considered, when used in specific populations, to achieve
optimal bactericidal activity and optimize drug-dosing regimens. No validated LC-MS/MS
methodhasbeenreportedusingdeuteratedertapenemasinternalstandard.Anewsimple
and robust LC-MS/MS method using a quadruple mass spectrometer was developed for
analysisofertapeneminhumanplasma,usingdeuteratedertapenemasinternalstandard.
The calibration curvewas linear over a range of 0.1 (LLOQ) to 125mg/L. The calculated
accuracy ranged from -2.4 % to 10.3%.Within-run CV ranged from 2.7 % to 11.8% and
between-runCVrangedfrom0%to8.4%.Freeze-thawstabilitybiasedbetween-3.3%and
0.1%.StorageofQCsamplesfor96hat4°Cdiffered-4.3to5.6%,storageatroomtemperature
for24h,biasedfrom-10.7%to-14.8%andstorageintheautosamplerbiasedbetween-2.9%
and -10.0%. A simple LC-MS/MS method to quantify ertapenem in human plasma using
deuteratedertapenemasinternalstandardhasbeenvalidated.Thismethodcanbeusedin
pharmacokineticstudiesandinclinicalstudiesbyperformingTDM.
IntroductionCarbapenems belong to the Beta-lactam antibiotics and are widely used against a broad
spectrum of aerobe and anaerobe gram-positive and gram-negative bacteria (1, 2).
Ertapenem,approvedbytheFDAin2001,isoneofthesecarbapenems.Sinceertapenemhas
anapproximatehalf-lifeof4h,itcanbeadministeredoncedaily.Therefore,ertapenemcan
be favored above other carbapenems (3). The pharmacodynamic (PD) parameter of
ertapenem correlates with time dependent killing, which means that the plasma
concentration of ertapenem has to exceed minimal inhibitory concentration (MIC) for a
percentageoftimeofitsdosinginterval(4).
Pharmacokineticdataobtainedinhealthyvolunteersaredifficulttoextrapolatetospecific
patientpopulations.Duetothishighvariabilityinpharmacokineticparameters,exposureof
ertapenemmightbesuboptimal inthesespecificpopulations (5-10).Sinceertapenemisa
time-dependent antibiotic, therapeutic drug monitoring (TDM) should therefore be
considered,whenused inspecificpopulations, toachieveoptimalbactericidalactivityand
optimize drug-dosing regimens. Yetmore PK studies have to be performed to determine
efficacyandsafetyofertapeneminspecificpopulation(11,12).
Ertapenem is being suggested for having potential use against M. tuberculosis (TB) (1).
However, according to Caminero et al. carbapenems are used as fifth line drugs in the
treatment of TB and can only be used in severe cases only, since carbapenems are
administeredintravenously,costsarehighandclinicalexperienceisrestricted(13).Clinical
studiesassessingefficacyorsafetyprofilesforcarbapenemsarescarce,butshowedfavorable
preliminaryresults (14-16).Beforeclinicalefficacy inTBcanbedeterminedadose-finding
studyshouldbeperformedtoevaluatepharmacokineticparametersinthisspecificpatient
population. Therefore, an analytical method to measure ertapenem concentrations is
mandatory.
There are a few methods published about LC-MS/MS quantification and validation of
ertapenem in human plasma (17, 18). Since LC-MS/MS is easy to use frequently in daily
routine,morepharmacokineticstudiesarebeingperformedtoquantifydrugs.Itistherefore
48
Chapter 3
importanttohaveestablishedstandardsinordertocompareresultsofPKstudiesbetween
laboratories.PresentvalidatedLC-MS/MSmethodsforertapenemareusingextensivesample
preparation,e.g.liquid-liquidextraction(LLE),solidphaseextraction(SPE)andnitrogengas
drying(17,18).Thesemethodsaretimeconsumingandlesscost-effective.Thechoiceofan
internal standard for LC-MS/MS is important as it corrects for extraction, injection and
ionizationvariability.Particularlythelatterone,ionsuppressionandionenhancement,isa
sourceofvariability.Onlyadeuteratedinternalstandardissuitabletocompensateforthis
andassurearobusthighthroughputbioanalyticalmethod(19).SincenoLC-MS/MSmethod
has been validated using a deuterated internal standard, the purpose of this study is to
developanewsimpleandrobustLC-MS/MSmethodusingaquadruplemassspectrometer
withoutextensivesampleprocessing,usingdeuteratedertapenemas internal standard to
quantifyconcentrationsofertapeneminhumanplasmaforpharmacokineticstudies.
MaterialandmethodsAnalysis
Ertapenem (Fig 1) and the internal standard used, ertapenem-D4, were purchased from
Alsachim(Illkirch,Graffenstaden,France).
Figure1.Chemicalstructureofertapenem
AcetonitrileforLC-MS/MSwaspurchasedfromBioSolve(Valkenswaard,TheNetherlands).
Thechemicalsused,includingmethanolLichrosolvandtrifluoroaceticanhydrideareofHPLC
oranalyticalgradeandwerepurchasedfromVWR(Amsterdam,TheNetherlands).Purified
waterwasobtainedfromaMilli-Qwaterpurifyingsystem(MilliporeCorporation,Billerica,
MA, USA). The precipitation reagent consisted of amixture ofmethanol and acetonitrile
(4:21,v/v).PooledhumanplasmasampleswithEDTAasanticoagulantandpooledhuman
serum samples were made available according to the standard operating procedures of
UniversityMedicalCenterGroningen.
Thecalibrationstandards,blankandQCsampleswerefullythawedatroomtemperature.To
100µLofeachsampleavolumeof500µLofprecipitationreagentsand10µLofertapenem-
D4(250mg/L)wereaddedinavial.Thesampleswerevortexedfor1minute.Topromote
proteinprecipitation,thevialswerestoredat-20°Cfor30minutes.Thevialswerecentrifuged
for5minutesat11000rpm.FiveµLoftheupperlayerwereinjectedintotheLC-MS/MS.QC
samplesandcalibrationstandardswerestoredat-20°C.
TheanalysiswasperformedonatriplequadrupoleLC-MS/MS(ThermoScientific,SanJose,
CAUSA)withaMSpumpPlus(Finnigan,surveyor)andautosampler(Finnigan,surveyor).The
massspectrometerwasaTSQQuantumAccessMaxmassspectrometer.Theautosampler
temperature was set at 10°C. Liquid chromatographic separation was performed by a
HyPURITYC18,analyticalcolumn,50*2.1mm,3-μm(ThermoScientific,Interscience,Breda,
TheNetherlands)andtemperaturewassetat20°C.Themobilephasehadaflowof300μL/
min and consisted of purified water, acetonitrile and an aqueous buffer (containing
ammoniumacetate10g/L,aceticacid35mg/Landtrifluoroaceticanhydride2mg/Lwater.
The method had a runtime of 4 minutes and the elution gradient is shown in Table 1.
Table1.Eluentgradient
Time(minutes) A(%) B(%) C(%)0.00 5 95 0
0.50 5 35 60
1.30 5 35 60
1.40 5 0 95
2.80 5 0 95
3.00 5 95 0
4.00 5 95 0
A:aqueousbuffer;B:purifiedwater,C:acetonitrile
49
3
Quantification and Validation of Ertapenem Using a LC-MS/MS
importanttohaveestablishedstandardsinordertocompareresultsofPKstudiesbetween
laboratories.PresentvalidatedLC-MS/MSmethodsforertapenemareusingextensivesample
preparation,e.g.liquid-liquidextraction(LLE),solidphaseextraction(SPE)andnitrogengas
drying(17,18).Thesemethodsaretimeconsumingandlesscost-effective.Thechoiceofan
internal standard for LC-MS/MS is important as it corrects for extraction, injection and
ionizationvariability.Particularlythelatterone,ionsuppressionandionenhancement,isa
sourceofvariability.Onlyadeuteratedinternalstandardissuitabletocompensateforthis
andassurearobusthighthroughputbioanalyticalmethod(19).SincenoLC-MS/MSmethod
has been validated using a deuterated internal standard, the purpose of this study is to
developanewsimpleandrobustLC-MS/MSmethodusingaquadruplemassspectrometer
withoutextensivesampleprocessing,usingdeuteratedertapenemas internal standard to
quantifyconcentrationsofertapeneminhumanplasmaforpharmacokineticstudies.
MaterialandmethodsAnalysis
Ertapenem (Fig 1) and the internal standard used, ertapenem-D4, were purchased from
Alsachim(Illkirch,Graffenstaden,France).
Figure1.Chemicalstructureofertapenem
AcetonitrileforLC-MS/MSwaspurchasedfromBioSolve(Valkenswaard,TheNetherlands).
Thechemicalsused,includingmethanolLichrosolvandtrifluoroaceticanhydrideareofHPLC
oranalyticalgradeandwerepurchasedfromVWR(Amsterdam,TheNetherlands).Purified
waterwasobtainedfromaMilli-Qwaterpurifyingsystem(MilliporeCorporation,Billerica,
MA, USA). The precipitation reagent consisted of amixture ofmethanol and acetonitrile
(4:21,v/v).PooledhumanplasmasampleswithEDTAasanticoagulantandpooledhuman
serum samples were made available according to the standard operating procedures of
UniversityMedicalCenterGroningen.
Thecalibrationstandards,blankandQCsampleswerefullythawedatroomtemperature.To
100µLofeachsampleavolumeof500µLofprecipitationreagentsand10µLofertapenem-
D4(250mg/L)wereaddedinavial.Thesampleswerevortexedfor1minute.Topromote
proteinprecipitation,thevialswerestoredat-20°Cfor30minutes.Thevialswerecentrifuged
for5minutesat11000rpm.FiveµLoftheupperlayerwereinjectedintotheLC-MS/MS.QC
samplesandcalibrationstandardswerestoredat-20°C.
TheanalysiswasperformedonatriplequadrupoleLC-MS/MS(ThermoScientific,SanJose,
CAUSA)withaMSpumpPlus(Finnigan,surveyor)andautosampler(Finnigan,surveyor).The
massspectrometerwasaTSQQuantumAccessMaxmassspectrometer.Theautosampler
temperature was set at 10°C. Liquid chromatographic separation was performed by a
HyPURITYC18,analyticalcolumn,50*2.1mm,3-μm(ThermoScientific,Interscience,Breda,
TheNetherlands)andtemperaturewassetat20°C.Themobilephasehadaflowof300μL/
min and consisted of purified water, acetonitrile and an aqueous buffer (containing
ammoniumacetate10g/L,aceticacid35mg/Landtrifluoroaceticanhydride2mg/Lwater.
The method had a runtime of 4 minutes and the elution gradient is shown in Table 1.
Table1.Eluentgradient
Time(minutes) A(%) B(%) C(%)0.00 5 95 0
0.50 5 35 60
1.30 5 35 60
1.40 5 0 95
2.80 5 0 95
3.00 5 95 0
4.00 5 95 0
A:aqueousbuffer;B:purifiedwater,C:acetonitrile
50
Chapter 3
TheMSwasconfiguredontopositiveelectrospray ionizationmodeandSelectedReaction
Monitoring (SRM)witha sprayvoltageof3500V,a capillary temperatureof350°Canda
sheathgaspressureandauxiliarypressure35and5arbitraryunitsrespectively.
Masstransitionsforertapenemwere476.1m/z→432.1m/zandforertapenem-D4480.1
m/z→436.1m/z,usingascanwidthof0.5m/z.Collisionenergywasdeterminedon10eV
forbothtransitions.Peakheight integrationforallcomponentswascalculatedbyXcalibur
softwareversion2.0.7(ThermoFisher,SanJose,CA,USA).
Analyticalmethodvalidation
Validationofthemethodincludedselectivityandsensitivity,linearity,accuracyandprecision,
recovery and dilution integrity conform the guidance for Industry of the Food and Drug
Administration.SincetheFDAdidnotpostulateamaximumCVrequirementforstability,a
maximumCVof15%wasemployedaccordingtoEMAguidelines(20,21).
Insomecases,forexampleinpharmacokineticstudies,humanserumisbeingcollectedfor
the quantification of ertapenem. To check if there’s a difference between the analysis of
ertapeneminhumanplasmaandinhumanserum,amatrixcomparisonhasbeenperformed.
Thecalibrationcurveofertapenemconsistedof8sampleswithconcentrationsof0.1,0.5,
2.0,7.5,20,50,90and125mg/L.QualityControl(QC)sampleswith4differentconcentrations
ofertapenemwereused,whereasLLOQis0.1mg/L,LOWis2.5mg/L,MEDis40mg/Land
HIGH is 120 mg/L. For selectivity 6 pooled human plasma samples were examined for
interferenceandcomparedwithresponseofthelowerlimitofquantificationsamples(LLOQ).
Duringthreedays,eachdayasinglecalibrationcurveinplasmaandinserumwasanalyzed
andaccuracywasmeasuredbyevaluationof fivedeterminationsperQCsampleonthree
consecutive days. Precision is divided into within-run and between-run using the same
methodasaccuracy.ThecoefficientofvariationforLLOQmaynotexceed20%andforother
QClevelsnotexceed15%.
Therecoverywasdeterminedonthreelevels(LOW,MEDandHIGH)infivefold.Asprotein
precipitationisusedastheonlywayofsamplepreparationinthismethod,relativerecovery
wasmeasured by comparing the ratios of integrated peak heights of ertapenem and the
internalstandardoftheprocessedQCsampleswiththeaveragepeakheightsoftherecovery
samples.Recoverysamples(LOW,MEDandHIGH)werepost-extractionblanksamplesspiked
atthesameconcentrationsastheQCsamples.
Stabilityofertapenemwastestedindifferenttestconditions,includingstoragestabilityand
freeze-thawstability.StoragestabilityofertapenemwasexaminedbystoringQCsamplesat
roomtemperature(20°C-25°C)inarefrigeratorat4°Candaftersamplepreparationinthe
autosamplerat10°C. Stabilitywasalso testedusing three freeze-thawcyclesat -20°C.All
stability tests were done using three different QC levels (LOW, MED, HIGH) in five
determinations per concentration. Stability is defined as a change in concentration and
shouldbe≤15%.SinceFDAguidelineshavenorequirementsforthecoefficientofvariation
ofeachQCsample(LOW,MED,HIGH),EMAguidelinerequirementsaretakenintoaccount,
statingthatCVofeachQCsampleshouldnotexceed15%(20,21).
Todeterminethedilutionintegrity,onthreeconsecutivedays,asamplewithaconcentration
of500mgertapenem/Lplasmawasdiluted10timesandthenpreparedinfivefold.
Statistics
Results were analyzed using one-way ANOVA and validated Excel sheets (Microsoft,
Redmond,WA).
ResultsMeanretentiontimesofertapenemandertapenem-D4wereequalto1.5minutes.
Examiningtheselectivityofthisanalyticalmethod,therewerenointerferingpeaksobserved
attheretentiontimeofertapenemorertapenem-D4inanyofthesixlotsofpooledhuman
plasma(Fig.2).
Thecalibrationcurveinplasmawaslinearoverarangeof0.1(LLOQ)to125mg/Landthe
correlationcoefficient(R2)was0.9988.Thecalibrationcurveparametersareasfollows;slope,
0.487 ± 0.00519 (average ± standard deviation); intercept, 0.0149 ± 0.0257; average
regressioncoefficient,0.99762;correlationcoefficient,0.9981.
51
3
Quantification and Validation of Ertapenem Using a LC-MS/MS
TheMSwasconfiguredontopositiveelectrospray ionizationmodeandSelectedReaction
Monitoring (SRM)witha sprayvoltageof3500V,a capillary temperatureof350°Canda
sheathgaspressureandauxiliarypressure35and5arbitraryunitsrespectively.
Masstransitionsforertapenemwere476.1m/z→432.1m/zandforertapenem-D4480.1
m/z→436.1m/z,usingascanwidthof0.5m/z.Collisionenergywasdeterminedon10eV
forbothtransitions.Peakheight integrationforallcomponentswascalculatedbyXcalibur
softwareversion2.0.7(ThermoFisher,SanJose,CA,USA).
Analyticalmethodvalidation
Validationofthemethodincludedselectivityandsensitivity,linearity,accuracyandprecision,
recovery and dilution integrity conform the guidance for Industry of the Food and Drug
Administration.SincetheFDAdidnotpostulateamaximumCVrequirementforstability,a
maximumCVof15%wasemployedaccordingtoEMAguidelines(20,21).
Insomecases,forexampleinpharmacokineticstudies,humanserumisbeingcollectedfor
the quantification of ertapenem. To check if there’s a difference between the analysis of
ertapeneminhumanplasmaandinhumanserum,amatrixcomparisonhasbeenperformed.
Thecalibrationcurveofertapenemconsistedof8sampleswithconcentrationsof0.1,0.5,
2.0,7.5,20,50,90and125mg/L.QualityControl(QC)sampleswith4differentconcentrations
ofertapenemwereused,whereasLLOQis0.1mg/L,LOWis2.5mg/L,MEDis40mg/Land
HIGH is 120 mg/L. For selectivity 6 pooled human plasma samples were examined for
interferenceandcomparedwithresponseofthelowerlimitofquantificationsamples(LLOQ).
Duringthreedays,eachdayasinglecalibrationcurveinplasmaandinserumwasanalyzed
andaccuracywasmeasuredbyevaluationof fivedeterminationsperQCsampleonthree
consecutive days. Precision is divided into within-run and between-run using the same
methodasaccuracy.ThecoefficientofvariationforLLOQmaynotexceed20%andforother
QClevelsnotexceed15%.
Therecoverywasdeterminedonthreelevels(LOW,MEDandHIGH)infivefold.Asprotein
precipitationisusedastheonlywayofsamplepreparationinthismethod,relativerecovery
wasmeasured by comparing the ratios of integrated peak heights of ertapenem and the
internalstandardoftheprocessedQCsampleswiththeaveragepeakheightsoftherecovery
samples.Recoverysamples(LOW,MEDandHIGH)werepost-extractionblanksamplesspiked
atthesameconcentrationsastheQCsamples.
Stabilityofertapenemwastestedindifferenttestconditions,includingstoragestabilityand
freeze-thawstability.StoragestabilityofertapenemwasexaminedbystoringQCsamplesat
roomtemperature(20°C-25°C)inarefrigeratorat4°Candaftersamplepreparationinthe
autosamplerat10°C. Stabilitywasalso testedusing three freeze-thawcyclesat -20°C.All
stability tests were done using three different QC levels (LOW, MED, HIGH) in five
determinations per concentration. Stability is defined as a change in concentration and
shouldbe≤15%.SinceFDAguidelineshavenorequirementsforthecoefficientofvariation
ofeachQCsample(LOW,MED,HIGH),EMAguidelinerequirementsaretakenintoaccount,
statingthatCVofeachQCsampleshouldnotexceed15%(20,21).
Todeterminethedilutionintegrity,onthreeconsecutivedays,asamplewithaconcentration
of500mgertapenem/Lplasmawasdiluted10timesandthenpreparedinfivefold.
Statistics
Results were analyzed using one-way ANOVA and validated Excel sheets (Microsoft,
Redmond,WA).
ResultsMeanretentiontimesofertapenemandertapenem-D4wereequalto1.5minutes.
Examiningtheselectivityofthisanalyticalmethod,therewerenointerferingpeaksobserved
attheretentiontimeofertapenemorertapenem-D4inanyofthesixlotsofpooledhuman
plasma(Fig.2).
Thecalibrationcurveinplasmawaslinearoverarangeof0.1(LLOQ)to125mg/Landthe
correlationcoefficient(R2)was0.9988.Thecalibrationcurveparametersareasfollows;slope,
0.487 ± 0.00519 (average ± standard deviation); intercept, 0.0149 ± 0.0257; average
regressioncoefficient,0.99762;correlationcoefficient,0.9981.
52
Chapter 3
Forcomparisonoftheanalysisinplasmaandinserum,thepeakheightratiosofertapenem
andtheinternalstandardinplasmawerecomparedtothoseinserum.Analyzingbothdata
setsusingPassing-Bablokregression,showednostatisticallysignificantdifferencebetween
thetwomatrices:y=1.01(0.95-1.02)x+0.00(-0.01-0.04)at95%confidencelevel.
Figure2.Chromatogram
(a)ErtapenemD4atLLQ,(b)ertapenematLLQand(c)blankplasma
Accuracyandprecision,dividedinwithinrunandbetweenrun,werecalculatedusingspiked
samples for 5 determinations per concentration on 3 consecutive days. The calculated
accuracyrangedfrom-2.4%to10.3%.Within-runprecisionrangedfrom2.7%to11.8%and
between-runprecisionrangedfrom0%to8.4%.Theresultsofaccuracyandprecisionforall
QClevelsareshowninTable2.
Table2.ConcentrationsofcalibrationstandardsandQCsamples.QCsamples LLOQ LOW MED HIGH
Concentration(mg/L) 0.1 2.5 40 120
Accuracy(%bias) -2.4 9.3 7.3 10.3
Within-runprecision(%CV) 11.8 5.6 3.1 2.7
Between-runprecision(%CV) 8.4 0 1.5 1.6
QCsamplesLOW(2.5mg/L),MED(40mg/L)andHIGH(120mg/L)wereusedtodetermine
recovery; relative recovery was 101.7%, 97.9% and 95.1% for these three samples,
respectively.Dilutionintegritywasdeterminedinfive-foldon3consecutivedays.Accuracy
was 1% and within-run and between-run precisions were 3.2% and 2.3%, respectively.
Stabilityofertapenemusingdifferenttestconditionsisshownintable3.
Table3.Stabilitytestingresultsforertapenem.Testcondition LOW MED HIGHBenchtop,RT*,24h(%bias) -11.9 -10.7 -14.8
Refrigerator,4°C,96h(%bias) 5.6 -0.1 -4.3
Freeze-thaw,-20°C,3cycles(%bias) -0.2 0.1 -3.3
Autosampler,10°C,24h(%bias) -10.0 -2.9 -4.8
*RTroomtemperature
Measured concentrations of QC samples (LOW,MED, HIGH) for the freeze-thaw stability
biased between -3.3% and 0.1% and therefore comply with the guidelines. Stability was
determinedbymeasuringQCsamplesstoredfor96hat4°Canddiffered-4.3to5.6%from
thenominalconcentrations.Afterstorageatroomtemperaturefor24h,theconcentrationof
ertapenem biased from -10.7% to -14.8%, compared to the initial concentrations. After
sample preparation the concentration of ertapenem stored in the autosampler biased
between-2.9%and-10.0%fromthenominalconcentrations.
53
3
Quantification and Validation of Ertapenem Using a LC-MS/MS
Forcomparisonoftheanalysisinplasmaandinserum,thepeakheightratiosofertapenem
andtheinternalstandardinplasmawerecomparedtothoseinserum.Analyzingbothdata
setsusingPassing-Bablokregression,showednostatisticallysignificantdifferencebetween
thetwomatrices:y=1.01(0.95-1.02)x+0.00(-0.01-0.04)at95%confidencelevel.
Figure2.Chromatogram
(a)ErtapenemD4atLLQ,(b)ertapenematLLQand(c)blankplasma
Accuracyandprecision,dividedinwithinrunandbetweenrun,werecalculatedusingspiked
samples for 5 determinations per concentration on 3 consecutive days. The calculated
accuracyrangedfrom-2.4%to10.3%.Within-runprecisionrangedfrom2.7%to11.8%and
between-runprecisionrangedfrom0%to8.4%.Theresultsofaccuracyandprecisionforall
QClevelsareshowninTable2.
Table2.ConcentrationsofcalibrationstandardsandQCsamples.QCsamples LLOQ LOW MED HIGH
Concentration(mg/L) 0.1 2.5 40 120
Accuracy(%bias) -2.4 9.3 7.3 10.3
Within-runprecision(%CV) 11.8 5.6 3.1 2.7
Between-runprecision(%CV) 8.4 0 1.5 1.6
QCsamplesLOW(2.5mg/L),MED(40mg/L)andHIGH(120mg/L)wereusedtodetermine
recovery; relative recovery was 101.7%, 97.9% and 95.1% for these three samples,
respectively.Dilutionintegritywasdeterminedinfive-foldon3consecutivedays.Accuracy
was 1% and within-run and between-run precisions were 3.2% and 2.3%, respectively.
Stabilityofertapenemusingdifferenttestconditionsisshownintable3.
Table3.Stabilitytestingresultsforertapenem.Testcondition LOW MED HIGHBenchtop,RT*,24h(%bias) -11.9 -10.7 -14.8
Refrigerator,4°C,96h(%bias) 5.6 -0.1 -4.3
Freeze-thaw,-20°C,3cycles(%bias) -0.2 0.1 -3.3
Autosampler,10°C,24h(%bias) -10.0 -2.9 -4.8
*RTroomtemperature
Measured concentrations of QC samples (LOW,MED, HIGH) for the freeze-thaw stability
biased between -3.3% and 0.1% and therefore comply with the guidelines. Stability was
determinedbymeasuringQCsamplesstoredfor96hat4°Canddiffered-4.3to5.6%from
thenominalconcentrations.Afterstorageatroomtemperaturefor24h,theconcentrationof
ertapenem biased from -10.7% to -14.8%, compared to the initial concentrations. After
sample preparation the concentration of ertapenem stored in the autosampler biased
between-2.9%and-10.0%fromthenominalconcentrations.
54
Chapter 3
Discussion
Thisisthefirstdesignandvalidationofanew,simpleandrapidanalysismethodusingatriple
quadrupleLC-MS/MSforthequantificationofertapeneminhumanplasmaanddeuterated
ertapenemasinternalstandard.
ThisLC-MS/MSmethodwasvalidatedforaccuracyandprecisionaccordingtoFDAguidelines,
havingbiases<20%forLLOQand<15%forotherQClevels(20).Thecalibrationcurvewas
linearwithin a rangeof 0.1 (LLOQ) – 125µg/mL, compared toother studies,whichwere
validatedup to50µg/mLandhadaLLOQof respectively0.5and1.0µg/mL (17,18)This
methoduseddeuteratedertapenemasinternalstandard,resultinginbetterinter-day,intra-
dayvariationandaccuracy,comparedtomethodsofPickeringetal.andKoaletal,which
usedBeta-lactammonologuesasinternalstandard(17,18).
Matrix comparison showed no difference between the analysis of ertapenem in human
plasmaandinhumanserum.However,becauseofthepoorstabilityofertapenematroom
temperatureit’srecommendedtodrawwholeblood(withEDTAastheanticoagulant)asit
canbeplacedoniceforashorttimeduringtransportfromthenursingwardtotheanalyzing
laboratory.
AsmentionedintheintroductionamajoradvantageofthisLC-MS/MSmethodisthatasimple
proteinprecipitationisusedinsteadofLLE,SPEornitrogengasdrying,resultinginalesstime
consumingandalessexpensivemethod,comparedtootherLC-MS/MSmethods(17,18).The
runtimeisveryshortsincetheretentiontimeofertapenemis1.5minutes.Thisfacilitatesa
highsampletroughput.ThisisagreatadvantageforlaboratorieshavingonlyoneLCMSMS
tosupporttheirclinicalTDMservice.
Ertapeneminplasmastoredatroomtemperaturedecreaseswithinashortperiodtimewith
10to15%.Therefore,itiscrucialtostoresamplesinthefreezeruntilanalysisandtoprocess
samplescontainingertapenemwithinthevalidatedtimeframeofstabilitytoassureaccurate
andpreciseresults.Reinjectionofprocessedsamplesstoredat10°C intheautosampler is
toleratedwithin24hours.
Sinceertapenem isa timedependentantibiotic, it isnecessary thatplasmaconcentration
exceedsMICatleast40%ofitsdosinginterval.Toattainthistargetinpatientssuspectedto
have altered pharmacokinetics due to renal function, high variability in plasma proteins
ertapenemconcentrationmeasuringmaybeofhelp,especiallyifmoreresistantpathogens
are targetedwith higherMIC values. Thismethodmeets the criteria for TDMbut is also
suitableforclinicalpharmacokineticstudiesorclinicaltrialstofurtherinvestigatetheuseof
ertapeneminotherinfectiousdiseasesorotherspecificpatientpopulations.
ConclusionA simple LC-MS/MS method to quantify ertapenem in human plasma using deuterated
ertapenemasinternalstandardhasbeendevelopedandvalidated.Thismethodcanbeused
inpharmacokineticstudiesandinclinicalstudiesbyperformingTDM.
55
3
Quantification and Validation of Ertapenem Using a LC-MS/MS
Discussion
Thisisthefirstdesignandvalidationofanew,simpleandrapidanalysismethodusingatriple
quadrupleLC-MS/MSforthequantificationofertapeneminhumanplasmaanddeuterated
ertapenemasinternalstandard.
ThisLC-MS/MSmethodwasvalidatedforaccuracyandprecisionaccordingtoFDAguidelines,
havingbiases<20%forLLOQand<15%forotherQClevels(20).Thecalibrationcurvewas
linearwithin a rangeof 0.1 (LLOQ) – 125µg/mL, compared toother studies,whichwere
validatedup to50µg/mLandhadaLLOQof respectively0.5and1.0µg/mL (17,18)This
methoduseddeuteratedertapenemasinternalstandard,resultinginbetterinter-day,intra-
dayvariationandaccuracy,comparedtomethodsofPickeringetal.andKoaletal,which
usedBeta-lactammonologuesasinternalstandard(17,18).
Matrix comparison showed no difference between the analysis of ertapenem in human
plasmaandinhumanserum.However,becauseofthepoorstabilityofertapenematroom
temperatureit’srecommendedtodrawwholeblood(withEDTAastheanticoagulant)asit
canbeplacedoniceforashorttimeduringtransportfromthenursingwardtotheanalyzing
laboratory.
AsmentionedintheintroductionamajoradvantageofthisLC-MS/MSmethodisthatasimple
proteinprecipitationisusedinsteadofLLE,SPEornitrogengasdrying,resultinginalesstime
consumingandalessexpensivemethod,comparedtootherLC-MS/MSmethods(17,18).The
runtimeisveryshortsincetheretentiontimeofertapenemis1.5minutes.Thisfacilitatesa
highsampletroughput.ThisisagreatadvantageforlaboratorieshavingonlyoneLCMSMS
tosupporttheirclinicalTDMservice.
Ertapeneminplasmastoredatroomtemperaturedecreaseswithinashortperiodtimewith
10to15%.Therefore,itiscrucialtostoresamplesinthefreezeruntilanalysisandtoprocess
samplescontainingertapenemwithinthevalidatedtimeframeofstabilitytoassureaccurate
andpreciseresults.Reinjectionofprocessedsamplesstoredat10°C intheautosampler is
toleratedwithin24hours.
Sinceertapenem isa timedependentantibiotic, it isnecessary thatplasmaconcentration
exceedsMICatleast40%ofitsdosinginterval.Toattainthistargetinpatientssuspectedto
have altered pharmacokinetics due to renal function, high variability in plasma proteins
ertapenemconcentrationmeasuringmaybeofhelp,especiallyifmoreresistantpathogens
are targetedwith higherMIC values. Thismethodmeets the criteria for TDMbut is also
suitableforclinicalpharmacokineticstudiesorclinicaltrialstofurtherinvestigatetheuseof
ertapeneminotherinfectiousdiseasesorotherspecificpatientpopulations.
ConclusionA simple LC-MS/MS method to quantify ertapenem in human plasma using deuterated
ertapenemasinternalstandardhasbeendevelopedandvalidated.Thismethodcanbeused
inpharmacokineticstudiesandinclinicalstudiesbyperformingTDM.
56
Chapter 3
References1. Hugonnet,J.E.,L.W.Tremblay,H.I.Boshoff,C.E.Barry3rd,andJ.S.Blanchard.2009.
Meropenem-clavulanate is effective against extensively drug-resistantMycobacterium
tuberculosis. Science. 323:1215-1218. doi: 10.1126/science.1167498;
10.1126/science.1167498.
2. Gupta,R.,M.Lavollay,J.L.Mainardi,M.Arthur,W.R.Bishai,andG.Lamichhane.2010.
TheMycobacteriumtuberculosisproteinLdtMt2isanonclassicaltranspeptidaserequired
forvirulenceandresistancetoamoxicillin.Nat.Med.16:466-469.doi:10.1038/nm.2120;
10.1038/nm.2120.
3. Majumdar,A.K.,D.G.Musson,K.L.Birk,C.J.Kitchen,S.Holland,J.McCrea,G.Mistry,M.
Hesney, L. Xi, S. X. Li, R.Haesen, R.A. Blum,R. L. Lins,H.Greenberg, S.Waldman, P.
Deutsch, and J. D. Rogers. 2002. Pharmacokinetics of ertapenem in healthy young
volunteers.Antimicrob.AgentsChemother.46:3506-3511.
4. Nicolau,D.P.2008.Pharmacokineticandpharmacodynamicpropertiesofmeropenem.
Clin.Infect.Dis.47Suppl1:S32-40.doi:10.1086/590064;10.1086/590064.
5. Burkhardt,O., V. Kumar, S. Schmidt, J. T. Kielstein, T.Welte, andH.Derendorf. 2010.
UnderdosingofertapenemincriticallyillpatientswithpneumoniaconfirmedbyMonte
Carlo simulations. Int. J. Antimicrob. Agents. 35:96-97. doi:
10.1016/j.ijantimicag.2009.09.007;10.1016/j.ijantimicag.2009.09.007.
6. Brink,A.J.,G.A.Richards,V.Schillack,S.Kiem,andJ.Schentag.2009.Pharmacokinetics
of once-daily dosing of ertapenem in critically ill patients with severe sepsis. Int. J.
Antimicrob. Agents. 33:432-436. doi: 10.1016/j.ijantimicag.2008.10.005;
10.1016/j.ijantimicag.2008.10.005.
7. Dailly,E.,J.F.Arnould,F.Fraissinet,E.Naux,M.A.LetarddelaBouraliere,R.Bouquie,G.
Deslandes, P. Jolliet, and R. Le Floch. 2013. Pharmacokinetics of ertapenem in burns
patients. Int. J. Antimicrob. Agents. 42:48-52. doi: 10.1016/j.ijantimicag.2013.02.021;
10.1016/j.ijantimicag.2013.02.021.
8. Chen,M.,A.N.Nafziger,G. L.Drusano,L.Ma,and J. S.Bertino Jr.2006.Comparative
pharmacokinetics and pharmacodynamic target attainment of ertapenem in normal-
weight, obese, and extremely obese adults. Antimicrob. Agents Chemother. 50:1222-
1227.doi:10.1128/AAC.50.4.1222-1227.2006.
9. Mistry,G.C.,A.K.Majumdar,S.Swan,D.Sica,A.Fisher,Y.Xu,M.Hesney, L.Xi, J.A.
Wagner,andP.J.Deutsch.2006.Pharmacokineticsofertapeneminpatientswithvarying
degreesofrenalinsufficiencyandinpatientsonhemodialysis.J.Clin.Pharmacol.46:1128-
1138.doi:10.1177/0091270006291839.
10. Abdel-Rahman,S.M.,G.L.Kearns,S.Topelberg,R.F.Jacobs,G.C.Mistry,A.Majumdar,
Y. Xu, J. A. Wagner, C. J. Kitchen, M. Groff, G. Herman, and J. L. Blumer. 2010.
Pharmacokinetics and tolerability of single-dose intravenous ertapenem in infants,
children, and adolescents. Pediatr. Infect. Dis. J. 29:1072-1076. doi:
10.1097/INF.0b013e3181e82608;10.1097/INF.0b013e3181e82608.
11. Wiskirchen, D. E., S. T. Housman, R. Quintiliani, D. P. Nicolau, and J. L. Kuti. 2013.
Comparative pharmacokinetics, pharmacodynamics, and tolerability of ertapenem 1
gram/day administered as a rapid 5-minute infusion versus the standard 30-minute
infusion in healthy adult volunteers. Pharmacotherapy. 33:266-274. doi:
10.1002/phar.1197;10.1002/phar.1197
12. Breilh,D.,C.Fleureau,J.B.Gordien,O.Joanes-Boyau,J.Texier-Maugein,S.Rapaport,E.
Boselli,G.Janvier,andM.C.Saux.2011.Pharmacokineticsoffreeertapenemincritically
illsepticpatients:intermittentversuscontinuousinfusion.MinervaAnestesiol.77:1058-
1062.
13. Caminero, J.A.,G. Sotgiu,A. Zumla, andG.B.Migliori. 2010.Bestdrug treatment for
multidrug-resistantandextensivelydrug-resistanttuberculosis.LancetInfect.Dis.10:621-
629.doi:10.1016/S1473-3099(10)70139-0;10.1016/S1473-3099(10)70139-0.
14. DeLorenzo,S.,J.W.Alffenaar,G.Sotgiu,R.Centis,L.D'Ambrosio,S.Tiberi,M.S.Bolhuis,
R.vanAltena,P.Viggiani,A.Piana,A.Spanevello,andG.B.Migliori.2012.Efficacyand
safetyofmeropenem/clavunateaddedtolinezolidcontainingregimensinthetreatment
ofM/XDR-TB.Eur.Respir.J.doi:10.1183/09031936.00124312.
15. Dauby, N., I. Muylle, F. Mouchet, R. Sergysels, and M. C. Payen. 2011.
Meropenem/clavulanate and linezolid treatment for extensively drug-resistant
tuberculosis. Pediatr. Infect. Dis. J. 30:812-813. doi: 10.1097/INF.0b013e3182154b05;
10.1097/INF.0b013e3182154b05.
16. Payen,M.C.,S.DeWit,C.Martin,R.Sergysels,I.Muylle,Y.VanLaethem,andN.Clumeck.
2012. Clinical use of the meropenem-clavulanate combination for extensively drug-
resistant tuberculosis. Int. J. Tuberc. Lung Dis. 16:558-560. doi: 10.5588/ijtld.11.0414;
57
3
Quantification and Validation of Ertapenem Using a LC-MS/MS
References1. Hugonnet,J.E.,L.W.Tremblay,H.I.Boshoff,C.E.Barry3rd,andJ.S.Blanchard.2009.
Meropenem-clavulanate is effective against extensively drug-resistantMycobacterium
tuberculosis. Science. 323:1215-1218. doi: 10.1126/science.1167498;
10.1126/science.1167498.
2. Gupta,R.,M.Lavollay,J.L.Mainardi,M.Arthur,W.R.Bishai,andG.Lamichhane.2010.
TheMycobacteriumtuberculosisproteinLdtMt2isanonclassicaltranspeptidaserequired
forvirulenceandresistancetoamoxicillin.Nat.Med.16:466-469.doi:10.1038/nm.2120;
10.1038/nm.2120.
3. Majumdar,A.K.,D.G.Musson,K.L.Birk,C.J.Kitchen,S.Holland,J.McCrea,G.Mistry,M.
Hesney, L. Xi, S. X. Li, R.Haesen, R.A. Blum,R. L. Lins,H.Greenberg, S.Waldman, P.
Deutsch, and J. D. Rogers. 2002. Pharmacokinetics of ertapenem in healthy young
volunteers.Antimicrob.AgentsChemother.46:3506-3511.
4. Nicolau,D.P.2008.Pharmacokineticandpharmacodynamicpropertiesofmeropenem.
Clin.Infect.Dis.47Suppl1:S32-40.doi:10.1086/590064;10.1086/590064.
5. Burkhardt,O., V. Kumar, S. Schmidt, J. T. Kielstein, T.Welte, andH.Derendorf. 2010.
UnderdosingofertapenemincriticallyillpatientswithpneumoniaconfirmedbyMonte
Carlo simulations. Int. J. Antimicrob. Agents. 35:96-97. doi:
10.1016/j.ijantimicag.2009.09.007;10.1016/j.ijantimicag.2009.09.007.
6. Brink,A.J.,G.A.Richards,V.Schillack,S.Kiem,andJ.Schentag.2009.Pharmacokinetics
of once-daily dosing of ertapenem in critically ill patients with severe sepsis. Int. J.
Antimicrob. Agents. 33:432-436. doi: 10.1016/j.ijantimicag.2008.10.005;
10.1016/j.ijantimicag.2008.10.005.
7. Dailly,E.,J.F.Arnould,F.Fraissinet,E.Naux,M.A.LetarddelaBouraliere,R.Bouquie,G.
Deslandes, P. Jolliet, and R. Le Floch. 2013. Pharmacokinetics of ertapenem in burns
patients. Int. J. Antimicrob. Agents. 42:48-52. doi: 10.1016/j.ijantimicag.2013.02.021;
10.1016/j.ijantimicag.2013.02.021.
8. Chen,M.,A.N.Nafziger,G. L.Drusano,L.Ma,and J. S.Bertino Jr.2006.Comparative
pharmacokinetics and pharmacodynamic target attainment of ertapenem in normal-
weight, obese, and extremely obese adults. Antimicrob. Agents Chemother. 50:1222-
1227.doi:10.1128/AAC.50.4.1222-1227.2006.
9. Mistry,G.C.,A.K.Majumdar,S.Swan,D.Sica,A.Fisher,Y.Xu,M.Hesney, L.Xi, J.A.
Wagner,andP.J.Deutsch.2006.Pharmacokineticsofertapeneminpatientswithvarying
degreesofrenalinsufficiencyandinpatientsonhemodialysis.J.Clin.Pharmacol.46:1128-
1138.doi:10.1177/0091270006291839.
10. Abdel-Rahman,S.M.,G.L.Kearns,S.Topelberg,R.F.Jacobs,G.C.Mistry,A.Majumdar,
Y. Xu, J. A. Wagner, C. J. Kitchen, M. Groff, G. Herman, and J. L. Blumer. 2010.
Pharmacokinetics and tolerability of single-dose intravenous ertapenem in infants,
children, and adolescents. Pediatr. Infect. Dis. J. 29:1072-1076. doi:
10.1097/INF.0b013e3181e82608;10.1097/INF.0b013e3181e82608.
11. Wiskirchen, D. E., S. T. Housman, R. Quintiliani, D. P. Nicolau, and J. L. Kuti. 2013.
Comparative pharmacokinetics, pharmacodynamics, and tolerability of ertapenem 1
gram/day administered as a rapid 5-minute infusion versus the standard 30-minute
infusion in healthy adult volunteers. Pharmacotherapy. 33:266-274. doi:
10.1002/phar.1197;10.1002/phar.1197
12. Breilh,D.,C.Fleureau,J.B.Gordien,O.Joanes-Boyau,J.Texier-Maugein,S.Rapaport,E.
Boselli,G.Janvier,andM.C.Saux.2011.Pharmacokineticsoffreeertapenemincritically
illsepticpatients:intermittentversuscontinuousinfusion.MinervaAnestesiol.77:1058-
1062.
13. Caminero, J.A.,G. Sotgiu,A. Zumla, andG.B.Migliori. 2010.Bestdrug treatment for
multidrug-resistantandextensivelydrug-resistanttuberculosis.LancetInfect.Dis.10:621-
629.doi:10.1016/S1473-3099(10)70139-0;10.1016/S1473-3099(10)70139-0.
14. DeLorenzo,S.,J.W.Alffenaar,G.Sotgiu,R.Centis,L.D'Ambrosio,S.Tiberi,M.S.Bolhuis,
R.vanAltena,P.Viggiani,A.Piana,A.Spanevello,andG.B.Migliori.2012.Efficacyand
safetyofmeropenem/clavunateaddedtolinezolidcontainingregimensinthetreatment
ofM/XDR-TB.Eur.Respir.J.doi:10.1183/09031936.00124312.
15. Dauby, N., I. Muylle, F. Mouchet, R. Sergysels, and M. C. Payen. 2011.
Meropenem/clavulanate and linezolid treatment for extensively drug-resistant
tuberculosis. Pediatr. Infect. Dis. J. 30:812-813. doi: 10.1097/INF.0b013e3182154b05;
10.1097/INF.0b013e3182154b05.
16. Payen,M.C.,S.DeWit,C.Martin,R.Sergysels,I.Muylle,Y.VanLaethem,andN.Clumeck.
2012. Clinical use of the meropenem-clavulanate combination for extensively drug-
resistant tuberculosis. Int. J. Tuberc. Lung Dis. 16:558-560. doi: 10.5588/ijtld.11.0414;
10.5588/ijtld.11.0414.
17. Koal, T.,M. Deters, K. Resch, and V. Kaever. 2006. Quantification of the carbapenem
antibiotic ertapenem in human plasma by a validated liquid chromatography-mass
spectrometrymethod.Clin.Chim.Acta.364:239-245.doi:10.1016/j.cccn.2005.07.004.
18. Pickering,M.,andS.Brown.2013.QuantificationandvalidationofHPLC-UVandLC-MS
assays for therapeutic drug monitoring of ertapenem in human plasma. Biomed.
Chromatogr.27:568-574.doi:10.1002/bmc.2829;10.1002/bmc.2829.
19. Lowes,S.,J.Jersey,R.Shoup,F.Garofolo,N.Savoie,E.Mortz,S.Needham,M.C.Caturla,
R.Steffen,C.Sheldon,R.Hayes,T.Samuels,L.DiDonato,J.Kamerud,S.Michael,Z.J.Lin,
J.Hillier,M.Moussallie,L.deSouzaTeixeira,M.Rocci,M.Buonarati,J.Truog,S.Hussain,
R. Lundberg,A.Breau,T. Zhang, J. Jonker,N.Berger, S.Gagnon-Carignan,C.Nehls,R.
Nicholson,M.Hilhorst,S.Karnik,T.deBoer,R.Houghton,K.Smith,L.Cojocaru,M.Allen,
T. Harter, S. Fatmi, F. Sayyarpour, J. Vija, M. Malone, and D. Heller. 2011.
Recommendationson:internalstandardcriteria,stability,incurredsamplereanalysisand
recent 483s by the Global CRO Council for Bioanalysis. Bioanalysis. 3:1323-1332. doi:
10.4155/bio.11.135;10.4155/bio.11.135.
20. U.S.DepartmentofHealthandHumanServices,FoodandDrugAdministration.Guidance
for industry,bioanalyticalmethodvalidation.Rockville:CenterforDrugEvaluationand
Research,2001.
21. European Medicines Agency, Committee for Medicinal Products for Human Use.
Guideline on bioanalytical validation (EMEA/CHMP/EWP/192217/2009). London:
EuropeanMedicinesAgency,2011.
59
3
Quantification and Validation of Ertapenem Using a LC-MS/MS
10.5588/ijtld.11.0414.
17. Koal, T.,M. Deters, K. Resch, and V. Kaever. 2006. Quantification of the carbapenem
antibiotic ertapenem in human plasma by a validated liquid chromatography-mass
spectrometrymethod.Clin.Chim.Acta.364:239-245.doi:10.1016/j.cccn.2005.07.004.
18. Pickering,M.,andS.Brown.2013.QuantificationandvalidationofHPLC-UVandLC-MS
assays for therapeutic drug monitoring of ertapenem in human plasma. Biomed.
Chromatogr.27:568-574.doi:10.1002/bmc.2829;10.1002/bmc.2829.
19. Lowes,S.,J.Jersey,R.Shoup,F.Garofolo,N.Savoie,E.Mortz,S.Needham,M.C.Caturla,
R.Steffen,C.Sheldon,R.Hayes,T.Samuels,L.DiDonato,J.Kamerud,S.Michael,Z.J.Lin,
J.Hillier,M.Moussallie,L.deSouzaTeixeira,M.Rocci,M.Buonarati,J.Truog,S.Hussain,
R. Lundberg,A.Breau,T. Zhang, J. Jonker,N.Berger, S.Gagnon-Carignan,C.Nehls,R.
Nicholson,M.Hilhorst,S.Karnik,T.deBoer,R.Houghton,K.Smith,L.Cojocaru,M.Allen,
T. Harter, S. Fatmi, F. Sayyarpour, J. Vija, M. Malone, and D. Heller. 2011.
Recommendationson:internalstandardcriteria,stability,incurredsamplereanalysisand
recent 483s by the Global CRO Council for Bioanalysis. Bioanalysis. 3:1323-1332. doi:
10.4155/bio.11.135;10.4155/bio.11.135.
20. U.S.DepartmentofHealthandHumanServices,FoodandDrugAdministration.Guidance
for industry,bioanalyticalmethodvalidation.Rockville:CenterforDrugEvaluationand
Research,2001.
21. European Medicines Agency, Committee for Medicinal Products for Human Use.
Guideline on bioanalytical validation (EMEA/CHMP/EWP/192217/2009). London:
EuropeanMedicinesAgency,2011.
CHAPTER 4
S.P. van Rijn*R. van Altena*O.W. Akkerman
D. van SoolingenT. van der LaanW.C.M de LangeJ.G.W. KosterinkT.S. van der WerfJ.W.C. Alffenaar
Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant
Tuberculosis
Eur Respir J. 2016 Apr; 47(4): 1229-34.PMID: 26743484
*Both authors contributed equally
CHAPTER 4
S.P. van Rijn*R. van Altena*O.W. Akkerman
D. van SoolingenT. van der LaanW.C.M de LangeJ.G.W. KosterinkT.S. van der WerfJ.W.C. Alffenaar
Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant
Tuberculosis
Eur Respir J. 2016 Apr; 47(4): 1229-34.PMID: 26743484
*Both authors contributed equally
62
Chapter 4
AbstractTreatmentofmultidrugresistant(MDR)andextensivelydrugresistant(XDR)tuberculosis(TB)
isbecomingmorechallengingbecauseofincreasedlevelofdrugresistanceagainstsecond
line tuberculosis drugs. One promising group of antimicrobial drugs are carbapenems.
ErtapenemisanattractivecarbapenemforthetreatmentofMDRandXDR-TBbecause its
relativelonghalf-lifeenablesoncedailydosing.Aretrospectivestudywasperformedforall
MDR-TB suspected patients at the Tuberculosis Center Beatrixoord of UniversityMedical
Center Groningen (Haren, The Netherlands) who received ertapenem as part of their
treatmentregimenbetweenthefirstofDecember2010andthefirstofMarch2013.Safety
and pharmacokinetics were evaluated. Eighteen patients were treated with 1000 mg
ertapenemforameanof77days(range5-210).Sputumsmearandculturewereconverted
inallpatients.Drugexposurewasevaluated in12patients.ThemeanAUC0-24was544,9
(range 309 – 1130)mg*h/L. Themean Cmaxwas 127.5 (73.9 – 277.9)mg/L. In general,
ertapenemtreatmentwaswelltoleratedduringMDR-TBtreatmentandshowedafavourable
PK/PDprofileinMDR-TBpatients.Weconcludethatertapenemisahighlypromisingdrugfor
thetreatmentofMDR-TBthatwarrantsfurtherinvestigation.
IntroductionTreatmentofmultidrugresistant(MDR)andextensivelydrugresistant(XDR)tuberculosis(TB)
isbecomingmorechallengingbecauseofincreasedlevelofdrugresistanceagainstsecond
linetuberculosisdrugs.Newdrugsarebeingevaluatedinclinicaltrials,butonlybedaquiline
anddelamanidhaveenteredthemarkettodate.Therefore,antimicrobialdrugs,whichhave
beendeveloped and labeled for other bacterial infectionsmaybeof potential use in the
treatmentofMDR-TB.
One promising group of antimicrobial drugs are carbapenems [1, 2]. An early in vitro
experimentshowedthatimipenemandmeropenemwereactiveagainstM.tuberculosis[3].
Chambersandco-workersshowedthatimipenemhasanti-mycobacterialactivityinmiceand
humans [4]. Imipenem and meropenem are currently listed as group 5 drugs for the
treatment of MDR-TB [5]. More recently, clinical experience of carbapenems inMDR-TB
patientsshowedpromisingresults[6-7].
CarbapenemsarepoorsubstratesforbetalactamaseC(BLaC)duetorapidacylationandslow
deacylation. Therefore, unlike beta-lactams, they are not rapidly hydrolyzed by BLaC and
therefore maintain their potential activity against M. tuberculosis [8]. The binding of
carbapenems to the LD transpeptidases results in inhibition of the peptidoglycan
polymerization of the cell wall [9]. Combined with a beta lactamase inhibitor, such as
clavulanate,activityagainstM.tuberculosisishigher[10].
Efficacy of carbapenems is correlated with the percentage of time the free plasma drug
concentrationtranscendstheMIC(Tfree>MIC).Maximalbactericidalactivityisreachedifthe
time aboveMIC is at least 40% of dosing interval [11,12]. To reach this target for gram
positive,gramnegativeandanaerobicbacterialinfectionsertapenemisgivenintravenously
inadoseof1000mgoncedaily[13].Ertapenemhastheadvantageoverothercarbapenems
becauseofalonghalf-lifeof4henablingoncedailydosing[12],whichisattractiveforMDR-
TBtreatment.Anotheradvantageisthatertapenemisnotaffectedbydrug-druginteractions
asitisneithermetabolizedbycytochromeP450norasubstrateforP-glycoprotein[14].
Toincludeertapenemamongtheothercarbapenemsasagroup5drugforthetreatmentof
MDR-TBadditionalpharmacokineticandsafetydataareurgentlyneeded[15].Therefore,the
63
Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant Tuberculosis
4
AbstractTreatmentofmultidrugresistant(MDR)andextensivelydrugresistant(XDR)tuberculosis(TB)
isbecomingmorechallengingbecauseofincreasedlevelofdrugresistanceagainstsecond
line tuberculosis drugs. One promising group of antimicrobial drugs are carbapenems.
ErtapenemisanattractivecarbapenemforthetreatmentofMDRandXDR-TBbecause its
relativelonghalf-lifeenablesoncedailydosing.Aretrospectivestudywasperformedforall
MDR-TB suspected patients at the Tuberculosis Center Beatrixoord of UniversityMedical
Center Groningen (Haren, The Netherlands) who received ertapenem as part of their
treatmentregimenbetweenthefirstofDecember2010andthefirstofMarch2013.Safety
and pharmacokinetics were evaluated. Eighteen patients were treated with 1000 mg
ertapenemforameanof77days(range5-210).Sputumsmearandculturewereconverted
inallpatients.Drugexposurewasevaluated in12patients.ThemeanAUC0-24was544,9
(range 309 – 1130)mg*h/L. Themean Cmaxwas 127.5 (73.9 – 277.9)mg/L. In general,
ertapenemtreatmentwaswelltoleratedduringMDR-TBtreatmentandshowedafavourable
PK/PDprofileinMDR-TBpatients.Weconcludethatertapenemisahighlypromisingdrugfor
thetreatmentofMDR-TBthatwarrantsfurtherinvestigation.
IntroductionTreatmentofmultidrugresistant(MDR)andextensivelydrugresistant(XDR)tuberculosis(TB)
isbecomingmorechallengingbecauseofincreasedlevelofdrugresistanceagainstsecond
linetuberculosisdrugs.Newdrugsarebeingevaluatedinclinicaltrials,butonlybedaquiline
anddelamanidhaveenteredthemarkettodate.Therefore,antimicrobialdrugs,whichhave
beendeveloped and labeled for other bacterial infectionsmaybeof potential use in the
treatmentofMDR-TB.
One promising group of antimicrobial drugs are carbapenems [1, 2]. An early in vitro
experimentshowedthatimipenemandmeropenemwereactiveagainstM.tuberculosis[3].
Chambersandco-workersshowedthatimipenemhasanti-mycobacterialactivityinmiceand
humans [4]. Imipenem and meropenem are currently listed as group 5 drugs for the
treatment of MDR-TB [5]. More recently, clinical experience of carbapenems inMDR-TB
patientsshowedpromisingresults[6-7].
CarbapenemsarepoorsubstratesforbetalactamaseC(BLaC)duetorapidacylationandslow
deacylation. Therefore, unlike beta-lactams, they are not rapidly hydrolyzed by BLaC and
therefore maintain their potential activity against M. tuberculosis [8]. The binding of
carbapenems to the LD transpeptidases results in inhibition of the peptidoglycan
polymerization of the cell wall [9]. Combined with a beta lactamase inhibitor, such as
clavulanate,activityagainstM.tuberculosisishigher[10].
Efficacy of carbapenems is correlated with the percentage of time the free plasma drug
concentrationtranscendstheMIC(Tfree>MIC).Maximalbactericidalactivityisreachedifthe
time aboveMIC is at least 40% of dosing interval [11,12]. To reach this target for gram
positive,gramnegativeandanaerobicbacterialinfectionsertapenemisgivenintravenously
inadoseof1000mgoncedaily[13].Ertapenemhastheadvantageoverothercarbapenems
becauseofalonghalf-lifeof4henablingoncedailydosing[12],whichisattractiveforMDR-
TBtreatment.Anotheradvantageisthatertapenemisnotaffectedbydrug-druginteractions
asitisneithermetabolizedbycytochromeP450norasubstrateforP-glycoprotein[14].
Toincludeertapenemamongtheothercarbapenemsasagroup5drugforthetreatmentof
MDR-TBadditionalpharmacokineticandsafetydataareurgentlyneeded[15].Therefore,the
64
Chapter 4
objectiveofthisstudywastoevaluatepharmacokineticsandsafetyinpatientsthatreceived
ertapenemaspartoftheirtreatmentMDR-TBregimen.
Patientsandmethods
Patients
AllpatientssuspectedtoMDR-TBattheTuberculosisCenterBeatrixoordoftheUniversity
MedicalCenterGroningen(Haren,TheNetherlands)whoreceivedertapenemaspartoftheir
treatment regimen between first of December 2010 and the first of March 2013 were
includedinthisretrospectivestudy.ThestudywasevaluatedbytheMedicalEthicalReview
Boardof theUniversityMedicalCenterGroningen (metc2013-492). Theneed forwritten
informedconsentwaswaivedfortheretrospectivecollectionandanalysisofanonymousdata
becauseitwasnotrequiredunderDutchLaw(WMO).ForeachMDR-TBsuspect,age,gender,
weight, length, ethnicity, drug susceptibility pattern, localization of tuberculosis,
antiretroviraltherapy,sputumconversion,adverseeffectsinducedbyertapenem,dose,total
exposuretoertapenem,anddurationoftreatmentwerecollected.
DrugsusceptibilitytoErtapenem
Drugsusceptibilitytesting(DST)ofertapenemwasperformedwithandwithoutclavulanic
acidusingthemiddlebrook7H10agardilutionmethodattheDutchNationalTuberculosis
Reference Laboratory (National Institute for Public Health and the Environment RIVM),
Bilthoven,TheNetherlands)[16].
Pharmacokineticsandpharmacodynamics
All patients receivedertapenem in a dosageof 1000mgoncedaily, given as intravenous
infusionin30min.InallMDR-TBpatientsroutineplasmaconcentrationswerecollectedat
steadystatetoassessdrugexposuretoenableindividualizeddosing.Forplasmasamplinga
peripheral intravenous catheter was inserted. Patency of the peripheral catheter was
maintainedbyasalinedrip.Beforeabloodsamplewastaken,thedripwasstoppedandthe
first4mlsofbloodwerediscarded.Thesampleswerecollectedbeforeadministrationandat
t = 1, 2, 3, 4, 5, 6, 8, 12, hrs post-dosage and stored at –80 °C until analysis. Plasma
concentrationswereassessedandvalidatedusingavalidatedliquidchromatography-tandem
massspectrometry(LC-MS/MS)inthelaboratoryofClinicaltoxicologyandDrugsAnalysisof
the department of Clinical Pharmacy and Pharmacology at theUniversityMedical Center
Groningen[17].PopulationpharmacokineticparameterswerecalculatedusingtheKinPOP
module. Both KinFIT and KinPOP were part of the software package MWPharm 3.82
(Mediware,TheNetherlands).TheTfree>MICwascalculatedasthishasbeenproposedasthe
best pharmacokinetic/pharmacodynamic parameter to predict in vivo efficacy of
carbapenems[10].Freedrugconcentrationwasassumedtobe5%[12,18].Eucastminimal
inhibitoryconcentrationsforertapenem(non-speciesrelated)of0.5and1.0mg/Lwereused
tocalculateTfree>MIC.
Safetyandtolerability
Reported adverse effects (AE) in medical charts were used to evaluate the safety of
ertapenem.SpecificattentionwaspaidtoAE’smentionedinearlierstudies:i.e.diarrheaand
vomiting.TheNaranjoalgorithmwasusedtoevaluateforcausalitybetweenadverseeffects
thatoccurredandertapenem[19].
Statisticsandpharmacokineticevaluation
SPSS 20 was used as statistical software (SPSS, Virgina, IL). Correlation between
pharmacokineticparametersandpatientcharacteristicswereanalyzedusingtheSpearman
correlationcoefficient.MICdatawerestatisticalanalyzedusingamethodologyforcensored
MICdata[20].
Results
Patients
Eighteenpatientstreatedwithertapenem,meanage29(range13-66years),wereretrieved.
Ertapenem was part of the treatment regimen because of suspected extensive drug
resistance,intolerancetosecondlinedrugsorcombinationofboth.Basedontheresultsof
thedrugsusceptibilitytestertapenemwasdiscontinuedinthreepatientswhoappearedto
havedrugsusceptibleTB.Genderwasunequallydistributedbetweenpatientsas8weremale
(44.4%)and10patientswerefemale(55.5%).Themeanbodymassindexwas21.3(range
65
Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant Tuberculosis
4
objectiveofthisstudywastoevaluatepharmacokineticsandsafetyinpatientsthatreceived
ertapenemaspartoftheirtreatmentMDR-TBregimen.
Patientsandmethods
Patients
AllpatientssuspectedtoMDR-TBattheTuberculosisCenterBeatrixoordoftheUniversity
MedicalCenterGroningen(Haren,TheNetherlands)whoreceivedertapenemaspartoftheir
treatment regimen between first of December 2010 and the first of March 2013 were
includedinthisretrospectivestudy.ThestudywasevaluatedbytheMedicalEthicalReview
Boardof theUniversityMedicalCenterGroningen (metc2013-492). Theneed forwritten
informedconsentwaswaivedfortheretrospectivecollectionandanalysisofanonymousdata
becauseitwasnotrequiredunderDutchLaw(WMO).ForeachMDR-TBsuspect,age,gender,
weight, length, ethnicity, drug susceptibility pattern, localization of tuberculosis,
antiretroviraltherapy,sputumconversion,adverseeffectsinducedbyertapenem,dose,total
exposuretoertapenem,anddurationoftreatmentwerecollected.
DrugsusceptibilitytoErtapenem
Drugsusceptibilitytesting(DST)ofertapenemwasperformedwithandwithoutclavulanic
acidusingthemiddlebrook7H10agardilutionmethodattheDutchNationalTuberculosis
Reference Laboratory (National Institute for Public Health and the Environment RIVM),
Bilthoven,TheNetherlands)[16].
Pharmacokineticsandpharmacodynamics
All patients receivedertapenem in a dosageof 1000mgoncedaily, given as intravenous
infusionin30min.InallMDR-TBpatientsroutineplasmaconcentrationswerecollectedat
steadystatetoassessdrugexposuretoenableindividualizeddosing.Forplasmasamplinga
peripheral intravenous catheter was inserted. Patency of the peripheral catheter was
maintainedbyasalinedrip.Beforeabloodsamplewastaken,thedripwasstoppedandthe
first4mlsofbloodwerediscarded.Thesampleswerecollectedbeforeadministrationandat
t = 1, 2, 3, 4, 5, 6, 8, 12, hrs post-dosage and stored at –80 °C until analysis. Plasma
concentrationswereassessedandvalidatedusingavalidatedliquidchromatography-tandem
massspectrometry(LC-MS/MS)inthelaboratoryofClinicaltoxicologyandDrugsAnalysisof
the department of Clinical Pharmacy and Pharmacology at theUniversityMedical Center
Groningen[17].PopulationpharmacokineticparameterswerecalculatedusingtheKinPOP
module. Both KinFIT and KinPOP were part of the software package MWPharm 3.82
(Mediware,TheNetherlands).TheTfree>MICwascalculatedasthishasbeenproposedasthe
best pharmacokinetic/pharmacodynamic parameter to predict in vivo efficacy of
carbapenems[10].Freedrugconcentrationwasassumedtobe5%[12,18].Eucastminimal
inhibitoryconcentrationsforertapenem(non-speciesrelated)of0.5and1.0mg/Lwereused
tocalculateTfree>MIC.
Safetyandtolerability
Reported adverse effects (AE) in medical charts were used to evaluate the safety of
ertapenem.SpecificattentionwaspaidtoAE’smentionedinearlierstudies:i.e.diarrheaand
vomiting.TheNaranjoalgorithmwasusedtoevaluateforcausalitybetweenadverseeffects
thatoccurredandertapenem[19].
Statisticsandpharmacokineticevaluation
SPSS 20 was used as statistical software (SPSS, Virgina, IL). Correlation between
pharmacokineticparametersandpatientcharacteristicswereanalyzedusingtheSpearman
correlationcoefficient.MICdatawerestatisticalanalyzedusingamethodologyforcensored
MICdata[20].
Results
Patients
Eighteenpatientstreatedwithertapenem,meanage29(range13-66years),wereretrieved.
Ertapenem was part of the treatment regimen because of suspected extensive drug
resistance,intolerancetosecondlinedrugsorcombinationofboth.Basedontheresultsof
thedrugsusceptibilitytestertapenemwasdiscontinuedinthreepatientswhoappearedto
havedrugsusceptibleTB.Genderwasunequallydistributedbetweenpatientsas8weremale
(44.4%)and10patientswerefemale(55.5%).Themeanbodymassindexwas21.3(range
66
Chapter 4
13.7-32.6)kg/m2.PatientsoriginatedpredominantlyfromAfrica(11/18)andEurope(5/18).
PatientswereprimarilydiagnosedwithpulmonaryTB(13/18),extrapulmonarysiteswere
involvedin7patients.
Prescribeddosageofertapenemwas1000mgoncedailyinallpatients.Meantotaltreatment
duration of ertapenem was 77 days (range 5-210 days). Drug resistance pattern of the
patientstoanti-tuberculosisagentsareshownintable1.Mostprescribedanti-TBdrugswere:
moxifloxacin(17/18),injectable(16/18),linezolid(15/18),clofazimine(8/18),clarithromycin
(6/18)andpyrazinamide(5/18).
Intotal15patientscompletedtreatmentandwerecured.Threepatientswerelosttofollow
up.Allpatientswithpositivesputum-smearconvertedwithinameanperiodof17days(range
0-97days).CulturesremainednegativeaftercultureconversionandnorelapseofMDR-TB
wasobserved.
Table1.Resistancepattern
WHO ResistantN(%)
SensitiveN(%)
Group1 First-lineoraldrugs Isoniazid 17(94,4) 1(5,55) Rifampicin 15(83,3) 3(16,6) Pyrazinamide 8(44,4) 8(44,4) Ethambutol 11(61,1) 6(33,3) Rifabutin 13(72,2) 4(22,2)Group2 Injectableagents streptomycin 14(77,7) 4(22,2) Amikacin 3(16,6) 14(77,7) kanamycin 3(16,6) 14(77,7)
capreomycin 5(27,7) 12(66,6)
Group3 Fluorquinolones Moxifloxacin 2(11,1) 15(83,3)
Group4 OralbacteriostaticSecond-lineagents protionamide 7(38,8) 10(55,5)Group5 Agentswithunclear Linezolid 17(94,4) Roleintreatmentof Clarithromycin 3(16,6) 7(38,8) drugresistant-TB Clofamizine 8(44,4)Other Ertapenem 18(100)
DataarepresentedasN(%),N=18
DrugsusceptibilityofM.tuberculosistoertapenem
All the M. tuberculosis strains appeared susceptible to ertapenem. However, actual
determinationof theMICwascomplicatedby the fact thatertapenem itself appearedan
instablecompoundat37°C[17].Thiswasconfirmedbythefactthatafter7daysMICvalues
were lower than after 14 days. In addition, freshly prepared plates showed lowerMIC’s
comparedtoplatesstoredat4°C.Therefrigerator-storedplatesshowedlowerMIC’sthan
platesstoredatroomtemperature.IfertapenemwascombinedwithclavulanicacidallMIC’s
wereevenlower.
Figure1.Ertapenemplasmaconcentration-timecurvesin12patients
Pharmacokineticsandpharmacodynamics
Theplasma concentration time curveswere obtained in 12patientswithMDR-TB. In the
remaining6patientsroutineplasmaconcentrationswerecollectedatatimepointatwhich
theydidnotyetreceiveertapenemorthisdrugwasnolongeradministered.Threepatients
hadmultipleplasmaconcentrationtimecurvesandthesewereconsistent.Themeancurve
isshowninfigure1.ThemeanAUC0-24was544,9(range309–1130)mg*h/L.Thesteady
statepharmacokineticparametersareshownintable2.
67
Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant Tuberculosis
4
13.7-32.6)kg/m2.PatientsoriginatedpredominantlyfromAfrica(11/18)andEurope(5/18).
PatientswereprimarilydiagnosedwithpulmonaryTB(13/18),extrapulmonarysiteswere
involvedin7patients.
Prescribeddosageofertapenemwas1000mgoncedailyinallpatients.Meantotaltreatment
duration of ertapenem was 77 days (range 5-210 days). Drug resistance pattern of the
patientstoanti-tuberculosisagentsareshownintable1.Mostprescribedanti-TBdrugswere:
moxifloxacin(17/18),injectable(16/18),linezolid(15/18),clofazimine(8/18),clarithromycin
(6/18)andpyrazinamide(5/18).
Intotal15patientscompletedtreatmentandwerecured.Threepatientswerelosttofollow
up.Allpatientswithpositivesputum-smearconvertedwithinameanperiodof17days(range
0-97days).CulturesremainednegativeaftercultureconversionandnorelapseofMDR-TB
wasobserved.
Table1.Resistancepattern
WHO ResistantN(%)
SensitiveN(%)
Group1 First-lineoraldrugs Isoniazid 17(94,4) 1(5,55) Rifampicin 15(83,3) 3(16,6) Pyrazinamide 8(44,4) 8(44,4) Ethambutol 11(61,1) 6(33,3) Rifabutin 13(72,2) 4(22,2)Group2 Injectableagents streptomycin 14(77,7) 4(22,2) Amikacin 3(16,6) 14(77,7) kanamycin 3(16,6) 14(77,7)
capreomycin 5(27,7) 12(66,6)
Group3 Fluorquinolones Moxifloxacin 2(11,1) 15(83,3)
Group4 OralbacteriostaticSecond-lineagents protionamide 7(38,8) 10(55,5)Group5 Agentswithunclear Linezolid 17(94,4) Roleintreatmentof Clarithromycin 3(16,6) 7(38,8) drugresistant-TB Clofamizine 8(44,4)Other Ertapenem 18(100)
DataarepresentedasN(%),N=18
DrugsusceptibilityofM.tuberculosistoertapenem
All the M. tuberculosis strains appeared susceptible to ertapenem. However, actual
determinationof theMICwascomplicatedby the fact thatertapenem itself appearedan
instablecompoundat37°C[17].Thiswasconfirmedbythefactthatafter7daysMICvalues
were lower than after 14 days. In addition, freshly prepared plates showed lowerMIC’s
comparedtoplatesstoredat4°C.Therefrigerator-storedplatesshowedlowerMIC’sthan
platesstoredatroomtemperature.IfertapenemwascombinedwithclavulanicacidallMIC’s
wereevenlower.
Figure1.Ertapenemplasmaconcentration-timecurvesin12patients
Pharmacokineticsandpharmacodynamics
Theplasma concentration time curveswere obtained in 12patientswithMDR-TB. In the
remaining6patientsroutineplasmaconcentrationswerecollectedatatimepointatwhich
theydidnotyetreceiveertapenemorthisdrugwasnolongeradministered.Threepatients
hadmultipleplasmaconcentrationtimecurvesandthesewereconsistent.Themeancurve
isshowninfigure1.ThemeanAUC0-24was544,9(range309–1130)mg*h/L.Thesteady
statepharmacokineticparametersareshownintable2.
68
Chapter 4
BasedonaMICof0.25mg/L,11outof12patientsexceededaminimumof40%-timeabove
MIC. In9patients theMDR-TBremainedsusceptiblewithaMICof0.5mg/L.Except for2
patients, none exceeded a minimum of 40%-time interval with a MIC of 1 mg/L. The
pharmacokineticpopulationmodel(KinPOP)ofertapenemshowedaclearanceof2.26(range
0.86-3,19)L/h/1.73m2andavolumeofdistributionof8.79(range4.76-13,57)L.
Table2.pharmacokineticparametersofErtapenem
StudyAUC0-24(h*mg/L) Cmax(mg/L) T1/2(h) Vd(L) CL(L/h)
1gIVinMDR-TBpatients
544.9(309–1130)
127.5(73.9–277.9)
2.4(2.047–3.528)
7.3(2.612–11.1)
2.1(0.0884–3.231)
1gIVinhealthyvolunteers[16]
572.1(572–672)
154.9(145–175)
4(3.8-4.4) 8.2 1.8
Dataarepresentedasmean(range).AUC0-24:Areaunderthecurve-timecurveupto24h.Cmax:highestobservedplasmaconcentration.Cl:clearance.T1/2:half-life.Vd:volumeofdistribution.
Safetyandtolerability
Ingeneral,ertapenemwastoleratedverywell.Inthreepatients,treatmentwithertapenem
wasstopped.OneofthesepatientssufferedfromCrohn’sdiseaseanddevelopedMDR-TB
aftermultipledosagesof infliximab,aTNFalpha-blocker [21-22].Thispatientexperienced
allergicfever,shortlyafteradministrationofertapenemandethambutol.Afterreintroduction
thishappenedagain(Naranjoscore=4).Bothadverseeventssubsidedafterwithdrawalof
theoffendingdrug.Inthesecondpatientertapenemwasstoppedafteranincreaseinliver
enzymes(ASAT:109/ALAT:255)after13daysoftreatmentwithertapenem.However,after
twomonths, while this patient was still on treatmentwithout ertapenem, liver enzymes
remainedelevated(Naranjoscore=1).Inonepatientkanamycin,linezolidandertapenem
werestoppedduetolinesepsis.Thiswasconsiderednot-to-berelatedtoertapenem.After
removal of the venous access port, the patient recovered. Ertapenem and a new venous
accessportwerenotreintroduced,sinceitwasnotindicatedanymore,duetolowbacillary
loadatthattimeandtheseIVdrugscouldbesubstitutedwithoralantimycobacterialdrugs.
Noneofthepatientsexperienceddiarrhea,vomitingordizziness.
DiscussionThisisthefirststudy,followingaclinicalreportofertapenem[6],presentingpharmacokinetic
andsafetydata inpatientswithMDR-TB. Incomparisonwithhealthyvolunteers,MDR-TB
patientsshowedlowerAUC0-24values.Meanvaluesofvolumeofdistributionandclearance
of MDR-TB patients were higher compared to healthy volunteers. Our observation is
consistentwithotherstudiesthatshowedalowerdrugexposureofertapeneminpatients
with infectiousdiseases [23-25].Moresurprisingwas the inter-variability inAUCbetween
patientswithMDR-TB.Otherantimycobacterialdrugsalsoshowhighlyvariabilityandlower
drug exposure in TB patients as well [2, 26-27]. It is not yet completely clear why drug
exposureislowerinTBpatients.Apparently,stageofdiseaseandalteredbodycomposition
maypotentiallyhelptoexplainthisobservation.
Since ertapenem belongs to the class of beta-lactams, ertapenem has a time-dependent
bactericidal activity. The Tfree>MIC is therefore important to evaluate the efficacy of
ertapenemagainstM.tuberculosis.Nicolauandcolleaguesindicatedthatincasemeropenem
showed40%T>MICbactericidalactivityisobservedwhereas20%T>MICappearedtohave
bacteriostaticactivity.OtherstudieshavementionedthisT>MICaswell[11-12,14].Protein
binding of ertapenem was assumed to be 5%, since ertapenem shows concentration-
dependent plasma protein binding. Healthy volunteers, whom average a peak plasma
concentrationof150mg/Laftertheendofinfusionof1gofertapenem,haveapercentage
of8%unboundprotein.Whentotaldrugplasmaconcentrationdeclinesbelow50mg/Lpeak
plasma concentration, the percentage of unbound ertapenem is circa 5% [18]. It is very
promisingtonoticethatthenon-speciesrelatedbreakpointofertapenemof0.5mg/Lwas
exceededformorethan40%ofthedayinthemajorityofpatientsassumingthatpatients
haveaproteinbindingof5%.AtahigherMICvalueof1mg/Lbacteriostaticactivitycouldbe
expected.Therefore,ertapenemseemsaveryattractivedrugforMDR-TBtreatment.Itseems
warrantedthatdosesofertapenemhigherthan1g/dayshouldbeusedinthetreatmentof
MDR-TB.However,invitroexperimentsevaluatingPK/PDtargetsforertapenemagainstM.
tuberculosishaveyettobeperformed.ThehollowfiberinfectionmodelissuitableforPK/PD
experimentsandhasalreadybeenusedsuccessfullybefore[28].
69
Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant Tuberculosis
4
BasedonaMICof0.25mg/L,11outof12patientsexceededaminimumof40%-timeabove
MIC. In9patients theMDR-TBremainedsusceptiblewithaMICof0.5mg/L.Except for2
patients, none exceeded a minimum of 40%-time interval with a MIC of 1 mg/L. The
pharmacokineticpopulationmodel(KinPOP)ofertapenemshowedaclearanceof2.26(range
0.86-3,19)L/h/1.73m2andavolumeofdistributionof8.79(range4.76-13,57)L.
Table2.pharmacokineticparametersofErtapenem
StudyAUC0-24(h*mg/L) Cmax(mg/L) T1/2(h) Vd(L) CL(L/h)
1gIVinMDR-TBpatients
544.9(309–1130)
127.5(73.9–277.9)
2.4(2.047–3.528)
7.3(2.612–11.1)
2.1(0.0884–3.231)
1gIVinhealthyvolunteers[16]
572.1(572–672)
154.9(145–175)
4(3.8-4.4) 8.2 1.8
Dataarepresentedasmean(range).AUC0-24:Areaunderthecurve-timecurveupto24h.Cmax:highestobservedplasmaconcentration.Cl:clearance.T1/2:half-life.Vd:volumeofdistribution.
Safetyandtolerability
Ingeneral,ertapenemwastoleratedverywell.Inthreepatients,treatmentwithertapenem
wasstopped.OneofthesepatientssufferedfromCrohn’sdiseaseanddevelopedMDR-TB
aftermultipledosagesof infliximab,aTNFalpha-blocker [21-22].Thispatientexperienced
allergicfever,shortlyafteradministrationofertapenemandethambutol.Afterreintroduction
thishappenedagain(Naranjoscore=4).Bothadverseeventssubsidedafterwithdrawalof
theoffendingdrug.Inthesecondpatientertapenemwasstoppedafteranincreaseinliver
enzymes(ASAT:109/ALAT:255)after13daysoftreatmentwithertapenem.However,after
twomonths, while this patient was still on treatmentwithout ertapenem, liver enzymes
remainedelevated(Naranjoscore=1).Inonepatientkanamycin,linezolidandertapenem
werestoppedduetolinesepsis.Thiswasconsiderednot-to-berelatedtoertapenem.After
removal of the venous access port, the patient recovered. Ertapenem and a new venous
accessportwerenotreintroduced,sinceitwasnotindicatedanymore,duetolowbacillary
loadatthattimeandtheseIVdrugscouldbesubstitutedwithoralantimycobacterialdrugs.
Noneofthepatientsexperienceddiarrhea,vomitingordizziness.
DiscussionThisisthefirststudy,followingaclinicalreportofertapenem[6],presentingpharmacokinetic
andsafetydata inpatientswithMDR-TB. Incomparisonwithhealthyvolunteers,MDR-TB
patientsshowedlowerAUC0-24values.Meanvaluesofvolumeofdistributionandclearance
of MDR-TB patients were higher compared to healthy volunteers. Our observation is
consistentwithotherstudiesthatshowedalowerdrugexposureofertapeneminpatients
with infectiousdiseases [23-25].Moresurprisingwas the inter-variability inAUCbetween
patientswithMDR-TB.Otherantimycobacterialdrugsalsoshowhighlyvariabilityandlower
drug exposure in TB patients as well [2, 26-27]. It is not yet completely clear why drug
exposureislowerinTBpatients.Apparently,stageofdiseaseandalteredbodycomposition
maypotentiallyhelptoexplainthisobservation.
Since ertapenem belongs to the class of beta-lactams, ertapenem has a time-dependent
bactericidal activity. The Tfree>MIC is therefore important to evaluate the efficacy of
ertapenemagainstM.tuberculosis.Nicolauandcolleaguesindicatedthatincasemeropenem
showed40%T>MICbactericidalactivityisobservedwhereas20%T>MICappearedtohave
bacteriostaticactivity.OtherstudieshavementionedthisT>MICaswell[11-12,14].Protein
binding of ertapenem was assumed to be 5%, since ertapenem shows concentration-
dependent plasma protein binding. Healthy volunteers, whom average a peak plasma
concentrationof150mg/Laftertheendofinfusionof1gofertapenem,haveapercentage
of8%unboundprotein.Whentotaldrugplasmaconcentrationdeclinesbelow50mg/Lpeak
plasma concentration, the percentage of unbound ertapenem is circa 5% [18]. It is very
promisingtonoticethatthenon-speciesrelatedbreakpointofertapenemof0.5mg/Lwas
exceededformorethan40%ofthedayinthemajorityofpatientsassumingthatpatients
haveaproteinbindingof5%.AtahigherMICvalueof1mg/Lbacteriostaticactivitycouldbe
expected.Therefore,ertapenemseemsaveryattractivedrugforMDR-TBtreatment.Itseems
warrantedthatdosesofertapenemhigherthan1g/dayshouldbeusedinthetreatmentof
MDR-TB.However,invitroexperimentsevaluatingPK/PDtargetsforertapenemagainstM.
tuberculosishaveyettobeperformed.ThehollowfiberinfectionmodelissuitableforPK/PD
experimentsandhasalreadybeenusedsuccessfullybefore[28].
70
Chapter 4
BesidespharmacokineticsofertapeneminpatientswithMDR-TB,additionalsafetydataare
describedforthefirsttimeaswell.Onlyonepatient,withCrohn’sdisease,experiencedAE,
whichmightbepotentiallyrelatedtoertapenem.Onecanspeculatethismayberelatedto
aninfusionrelatedAE,duetoadevelopedimmunedisorderandeventuallyaconsequence
ofaninfliximabtreatment.DruginducedfeverisacommonAEofinfliximabinthetreatment
of Crohn’s Disease [29]. AE’s of other carbapenems, such as diarrhea, nausea, vomiting,
headache and rash arewell documented and found to bemild [1, 30]. According to the
product leaflet, ertapenem is given for a maximum of two consecutive weeks. Previous
studies explored the safety and tolerability of ertapenem for this period of time and
concludedthatadversesideeffectsweremildtomoderate[13].Inourstudyweshowedthat
AEdidnotincreaseduringprolongedtreatment.
ThemeasurementofactualMICvalueswascomplicatedby the fact thatertapenem isan
instablecompoundat37°C.AsdrugsusceptibilitytestingforM.tuberculosis takesat least
twoweeksat37°C,itishighlylikelythattheinitialdrugconcentrationdecreasesrapidlyin
time.Unfortunately,with the current drug susceptibility systems, e.g.MGITor plate, this
problemcannotbeovercome,asthedrugconcentrationinthemediumcannotbecorrected
foradecrease inconcentrationdue todegradationof thedrug.Recently thehollow fiber
infectionmodelsolvedthisproblemasdrugconcentrationscanbeincreasedtocorrectfor
degradation[31].Assystemsareexpensiveanddifficulttomanageitsnotlikelythatroutine
DSTwillbeperformedusinghollowfibersystems.AnotheralternativemaybetheuseofE-
tests[32].Thisismuchcheaperandeasiertoemploybutitisunclearifitcanhelptoovercome
theinstabilityofertapenem.
Themost important limitation of our study is the retrospective character and its limited
samplesize,andabsenceofcontrolgroup,therebypreventingameaningfulconclusionon
efficacyofertapenem.SecondlytheinabilitytodefineMICforertapeneminclinicalisolates
isanotherlimitation.Nevertheless,allpatientswerecuredandnorelapsewasnoticedafter
being treatedwith combination regimen including ertapenem. Likely the combination of
drugscontributedtosputumcultureconversionandfavorabletreatmentoutcome.Thisisin
line with recently published data on tolerability and outcomes in 5 patients receiving
ertapenem[6].
A recent editorial proposed a new classification of anti-tuberculosis drugs. Itmarked the
potentialofcarbapenemsasgroup5drugs,howevercarbapenemsarestillinneedofproper
evaluationandclinicalevidence[33].Beforeertapenemcanbelabeledasagroup5drugand
usedaspartofMDR-TB treatment,avalidprocedure to testdrugsusceptibilityhas tobe
madeavailable.Ideally,theuseofertapenemwouldbesupportedbytheresultsofaclinical
trial.
Inconclusionthisstudyprovidesnewknowledgeontheuseofertapeneminpatientswith
MDR-TB,presentingpharmacokineticandadditionalsafetydata.
71
Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant Tuberculosis
4
BesidespharmacokineticsofertapeneminpatientswithMDR-TB,additionalsafetydataare
describedforthefirsttimeaswell.Onlyonepatient,withCrohn’sdisease,experiencedAE,
whichmightbepotentiallyrelatedtoertapenem.Onecanspeculatethismayberelatedto
aninfusionrelatedAE,duetoadevelopedimmunedisorderandeventuallyaconsequence
ofaninfliximabtreatment.DruginducedfeverisacommonAEofinfliximabinthetreatment
of Crohn’s Disease [29]. AE’s of other carbapenems, such as diarrhea, nausea, vomiting,
headache and rash arewell documented and found to bemild [1, 30]. According to the
product leaflet, ertapenem is given for a maximum of two consecutive weeks. Previous
studies explored the safety and tolerability of ertapenem for this period of time and
concludedthatadversesideeffectsweremildtomoderate[13].Inourstudyweshowedthat
AEdidnotincreaseduringprolongedtreatment.
ThemeasurementofactualMICvalueswascomplicatedby the fact thatertapenem isan
instablecompoundat37°C.AsdrugsusceptibilitytestingforM.tuberculosis takesat least
twoweeksat37°C,itishighlylikelythattheinitialdrugconcentrationdecreasesrapidlyin
time.Unfortunately,with the current drug susceptibility systems, e.g.MGITor plate, this
problemcannotbeovercome,asthedrugconcentrationinthemediumcannotbecorrected
foradecrease inconcentrationdue todegradationof thedrug.Recently thehollow fiber
infectionmodelsolvedthisproblemasdrugconcentrationscanbeincreasedtocorrectfor
degradation[31].Assystemsareexpensiveanddifficulttomanageitsnotlikelythatroutine
DSTwillbeperformedusinghollowfibersystems.AnotheralternativemaybetheuseofE-
tests[32].Thisismuchcheaperandeasiertoemploybutitisunclearifitcanhelptoovercome
theinstabilityofertapenem.
Themost important limitation of our study is the retrospective character and its limited
samplesize,andabsenceofcontrolgroup,therebypreventingameaningfulconclusionon
efficacyofertapenem.SecondlytheinabilitytodefineMICforertapeneminclinicalisolates
isanotherlimitation.Nevertheless,allpatientswerecuredandnorelapsewasnoticedafter
being treatedwith combination regimen including ertapenem. Likely the combination of
drugscontributedtosputumcultureconversionandfavorabletreatmentoutcome.Thisisin
line with recently published data on tolerability and outcomes in 5 patients receiving
ertapenem[6].
A recent editorial proposed a new classification of anti-tuberculosis drugs. Itmarked the
potentialofcarbapenemsasgroup5drugs,howevercarbapenemsarestillinneedofproper
evaluationandclinicalevidence[33].Beforeertapenemcanbelabeledasagroup5drugand
usedaspartofMDR-TB treatment,avalidprocedure to testdrugsusceptibilityhas tobe
madeavailable.Ideally,theuseofertapenemwouldbesupportedbytheresultsofaclinical
trial.
Inconclusionthisstudyprovidesnewknowledgeontheuseofertapeneminpatientswith
MDR-TB,presentingpharmacokineticandadditionalsafetydata.
72
Chapter 4
References
1. De Lorenzo S, Alffenaar JW, SotgiuG,et al. Efficacy and safety ofmeropenem/clavunate
addedtolinezolidcontainingregimensinthetreatmentofM/XDR-TB.EurRespirJ2012Sep
20.
2. PrangerAD,vanAltenaR,AarnoutseRE,etal.Evaluationofmoxifloxacinforthetreatment
oftuberculosis:3yearsofexperience.EurrespirJ2011;38(4):888-94.
3. WattB,EdwardsJR,RaynerA,etal. Invitroactivityofmeropenemandimipenemagainst
mycobacteria:Developmentofadailyantibioticdosingschedule.Tubercleandlungdisease.
1992;73(3):134-6.
4. ChambersHF,TurnerJ,SchecterGF,etal.Imipenemfortreatmentoftuberculosisinmiceand
humans.AntimicrobAgentsChemother2005;49(7):2816-21.
5. Caminero, J. A., G. Sotgiu, A. Zumla, and G. B. Migliori. 2010. Best drug treatment for
multidrug-resistant andextensively drug-resistant tuberculosis. Lancet Infect.Dis. 10:621-
629.doi:10.1016/S1473-3099(10)70139-0;10.1016/S1473-3099(10)70139-0.5.
6. Tiberi S,D’Ambrosio L,De Lorenzo S, et al. Ertapenem in the treatmentofMDR-TB: first
clinicalexperience.EurRespirJ2015inpress.
7. WintersN,Butler-LaporteG,MenziesD.EfficacyandsafetyofWorldHealthOrganization
group5drugsformultidrug-resistanttuberculosis treatment.EurRespir J.2015sept17. [
Epubadheadofprint]
8. Hugonnet J, Blanchard JS. Irreversible inhibition of the mycobacterium tuberculosis β-
lactamasebyclavulanate.Biochemistry2007;46(43):11998-2004.
9. CordillotM,DubeeV,TribouletS,etal.Invitrocross-linkingofMycobacteriumtuberculosis
peptidoglycanbyL,D-transpeptidasesand inactivationof theseenzymesbycarbapenems.
AntimicrobAgentsChemother201357:5940-45.
10. VezirisN,TruffotC,MainardiJL,etal.Activityofcarbapenemscombinedwithclavulanate
againstmurinetuberculosis.AntimicrobAgentsChemother2011Jun;55(6):2597-600.
11. NicolauDP.Pharmacokineticandpharmacodynamicpropertiesofmeropenem.Clin Infect
Dis.2008Sep15;47Suppl1:S32-40.
12. ZhanelGG,WiebeR,Dilay L,etal. Comparative reviewof the carbapenems.Drugs 2007;
67(7):1027-52.
13. TepplerH,GesserRM,FriedlandIR,etal.SafetyandtolerabilityofErtapenem.JAntimicrob
Chemother2004Jun;53Suppl2:ii75-81.
14. NixDE,MajumdarAK,DiNubileMJ.PharmacokineticsandpharmacodynamicsofErtapenem:
Anoverviewforclinicians.JAntimicrobChemother.2004Jun;53Suppl2:ii23-8.
15. DooleyKE,ObukuEA,DurakovicN,etal.WorldHealthOrganizationgroup5drugsforthe
treatmentofdrug-resistanttuberculosis:unclearefficacyoruntappedpotential?JInfectDis.
2013May1:207(9):1352-8
16. Van Klingeren B, Dessens-Kroon M, van der Laan T, et al. Drug susceptibility testing of
mycobacterium tuberculosis complex by use of a high-throughput, reproducible, absolute
concentrationmethod.JClinMicrobiol.2007;45:2662-8
17. vanRijnSP,WesselsAM,GreijdanusB,etal.QuantificationandvalidationofErtapenemusing
aliquidchromatography-tandemmassspectrometrymethod.AntimicrobAgentsChemother.
2014Jun:58(6):3481-4.
18. MajumdarAK,MussonDG,BirkKL,etal.PharmacokineticsofErtapeneminhealthyyoung
volunteers.AntimicrobAgentsChemother2002Nov;46(11):3506-11.
19. NaranjoCA,BustoU,SellersEM,etal.Amethodforestimatingtheprobabilityofadverse
drugreactions.ClinPharmacolTher1981Aug;30(2):239-45.
20. VandeKassteeleJ,vanSanten-VerheuvelMG,KoedijkFD,etal.Newstatisticaltechniquefor
analyzingMIC-based susceptibility data. Antimicrob Agents Chemother. 2012Mar; 56(3):
1557-63
21. CantiniF,NiccoliL,GolettiD.Adalimub,etanercept,infliximab,andtheriskoftuberculosis:
data from clinical trials, national registries, and postmarketing surveillance. J Rheumatol
Suppl.2014May;91:47-55
73
Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant Tuberculosis
4
References
1. De Lorenzo S, Alffenaar JW, SotgiuG,et al. Efficacy and safety ofmeropenem/clavunate
addedtolinezolidcontainingregimensinthetreatmentofM/XDR-TB.EurRespirJ2012Sep
20.
2. PrangerAD,vanAltenaR,AarnoutseRE,etal.Evaluationofmoxifloxacinforthetreatment
oftuberculosis:3yearsofexperience.EurrespirJ2011;38(4):888-94.
3. WattB,EdwardsJR,RaynerA,etal. Invitroactivityofmeropenemandimipenemagainst
mycobacteria:Developmentofadailyantibioticdosingschedule.Tubercleandlungdisease.
1992;73(3):134-6.
4. ChambersHF,TurnerJ,SchecterGF,etal.Imipenemfortreatmentoftuberculosisinmiceand
humans.AntimicrobAgentsChemother2005;49(7):2816-21.
5. Caminero, J. A., G. Sotgiu, A. Zumla, and G. B. Migliori. 2010. Best drug treatment for
multidrug-resistant andextensively drug-resistant tuberculosis. Lancet Infect.Dis. 10:621-
629.doi:10.1016/S1473-3099(10)70139-0;10.1016/S1473-3099(10)70139-0.5.
6. Tiberi S,D’Ambrosio L,De Lorenzo S, et al. Ertapenem in the treatment ofMDR-TB: first
clinicalexperience.EurRespirJ2015inpress.
7. WintersN,Butler-LaporteG,MenziesD.EfficacyandsafetyofWorldHealthOrganization
group5drugsformultidrug-resistanttuberculosistreatment.EurRespir J.2015sept17. [
Epubadheadofprint]
8. Hugonnet J, Blanchard JS. Irreversible inhibition of the mycobacterium tuberculosis β-
lactamasebyclavulanate.Biochemistry2007;46(43):11998-2004.
9. CordillotM,DubeeV,TribouletS,etal.Invitrocross-linkingofMycobacteriumtuberculosis
peptidoglycanbyL,D-transpeptidasesand inactivationof theseenzymesbycarbapenems.
AntimicrobAgentsChemother201357:5940-45.
10. VezirisN,TruffotC,MainardiJL,etal.Activityofcarbapenemscombinedwithclavulanate
againstmurinetuberculosis.AntimicrobAgentsChemother2011Jun;55(6):2597-600.
11. NicolauDP.Pharmacokineticandpharmacodynamicpropertiesofmeropenem.Clin Infect
Dis.2008Sep15;47Suppl1:S32-40.
12. ZhanelGG,WiebeR,Dilay L,etal. Comparative reviewof the carbapenems.Drugs 2007;
67(7):1027-52.
13. TepplerH,GesserRM,FriedlandIR,etal.SafetyandtolerabilityofErtapenem.JAntimicrob
Chemother2004Jun;53Suppl2:ii75-81.
14. NixDE,MajumdarAK,DiNubileMJ.PharmacokineticsandpharmacodynamicsofErtapenem:
Anoverviewforclinicians.JAntimicrobChemother.2004Jun;53Suppl2:ii23-8.
15. DooleyKE,ObukuEA,DurakovicN,etal.WorldHealthOrganizationgroup5drugsforthe
treatmentofdrug-resistanttuberculosis:unclearefficacyoruntappedpotential?JInfectDis.
2013May1:207(9):1352-8
16. Van Klingeren B, Dessens-Kroon M, van der Laan T, et al. Drug susceptibility testing of
mycobacterium tuberculosis complex by use of a high-throughput, reproducible, absolute
concentrationmethod.JClinMicrobiol.2007;45:2662-8
17. vanRijnSP,WesselsAM,GreijdanusB,etal.QuantificationandvalidationofErtapenemusing
aliquidchromatography-tandemmassspectrometrymethod.AntimicrobAgentsChemother.
2014Jun:58(6):3481-4.
18. MajumdarAK,MussonDG,BirkKL,etal.PharmacokineticsofErtapeneminhealthyyoung
volunteers.AntimicrobAgentsChemother2002Nov;46(11):3506-11.
19. NaranjoCA,BustoU,SellersEM,etal.Amethodforestimatingtheprobabilityofadverse
drugreactions.ClinPharmacolTher1981Aug;30(2):239-45.
20. VandeKassteeleJ,vanSanten-VerheuvelMG,KoedijkFD,etal.Newstatisticaltechniquefor
analyzingMIC-based susceptibility data. Antimicrob Agents Chemother. 2012Mar; 56(3):
1557-63
21. CantiniF,NiccoliL,GolettiD.Adalimub,etanercept,infliximab,andtheriskoftuberculosis:
data from clinical trials, national registries, and postmarketing surveillance. J Rheumatol
Suppl.2014May;91:47-55
74
Chapter 4
22. BumbaceaD,ArendSM,EyubogluF,etal.Theriskoftuberculosisintransplantcandidates
andrecipients:aTBNETconsensusstatement.EurRespirJ.2012Oct:40(4):990-1013.
23. BrinkAJ,RichardsGA,SchillackV,etal.Pharmacokineticsofonce-dailydosingofErtapenem
incriticallyillpatientswithseveresepsis.IntJAntimicrobAgents2009May;33(5):432-6.
24. BurkhardtO,DerendorfH,WelteT.Ertapenem:Thenewcarbapenem5yearsafterfirstFDA
licensingforclinicalpractice.ExpertOpinPharmacother.2007Feb;8(2):237-56.
25. SauermannR,BurianB,BurianA,etal.TissuepharmacokineticsofErtapenematsteady-state
indiabeticpatientswithleginfections.JAntimicrobChemother.2012Dec4.
26. Alsaad N, van Altena R, Pranger AD, et al. Evaluation of co-trimoxazole in treatment of
multidrug-resistanttuberculosis.EurRespirJ.2012Oct25.
27. DongH,WangX,DongY,etal.Clinicalpharmacokinetic/pharmacodynamicprofileoflinezolid
inseverelyillintensivecareunitpatients.IntJAntimicrobAgents2011Oct;38(4):296-300.21.
Hess S, Hospach T, Nossal R, et al. Immune reconstitution after discontinuation of TNF
blockade.EurJPediatr.2011Oct;170(10):1337-42
28. GumboT,LouieA,DezielMR,etal.Selectionofamoxifloxacindosethatsuppressesdrug
resistanceinmycobacteriumtuberculosis,byuseofaninvitropharmacodynamicinfection
modelandmathematicalmodeling.JInfectDis.2004Nov1;190(9):1642-51.
29. HessS,HospachT,NossalR,etal.ImmunereconstitutionafterdiscontinuationofTNF
blockade.EurJPediatr.Oct;170(10):1337-42
30. ZhanelGG,SimorAE,VercaigneL,etal.CanadianCarbapenemDiscussionGroup.Imipenem
andmeropenem:Comparisonofinvitroactivity,pharmacokinetics,clinicaltrialsandadverse
effects.CanJInfectDis.1998Jul;9(4):215-28
31. Srivastava S., VanRijn SP, ShermanC, et al. SterilizingActivityof Ertapenem (ETP) agains
Mycobacterium tuberculosis (Mtb). Poster session presented at: ICAAC 2015; 66th annual
conferenceofInterscienceConferenceofAntimicrobialAgentsandChemotherapy;2015Sep
17-21;SanDiego,CA.
32. Esteban J, Ortiz A, Jiménez MS. Usefulness of E-test strips for testing susceptibility of
Mycobacterium tuberculosis complex strains. Eur J Clin Microbiol Infect Dis. 2005 Dec;
24(12):856-7
33. CamineroJA,ScardigliA.Classificationofantituberculosisdrugs:anewproposalbasedonthe
mostrecentevidence.EurRespirJ2015;46:887-893
75
Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant Tuberculosis
4
22. BumbaceaD,ArendSM,EyubogluF,etal.Theriskoftuberculosisintransplantcandidates
andrecipients:aTBNETconsensusstatement.EurRespirJ.2012Oct:40(4):990-1013.
23. BrinkAJ,RichardsGA,SchillackV,etal.Pharmacokineticsofonce-dailydosingofErtapenem
incriticallyillpatientswithseveresepsis.IntJAntimicrobAgents2009May;33(5):432-6.
24. BurkhardtO,DerendorfH,WelteT.Ertapenem:Thenewcarbapenem5yearsafterfirstFDA
licensingforclinicalpractice.ExpertOpinPharmacother.2007Feb;8(2):237-56.
25. SauermannR,BurianB,BurianA,etal.TissuepharmacokineticsofErtapenematsteady-state
indiabeticpatientswithleginfections.JAntimicrobChemother.2012Dec4.
26. Alsaad N, van Altena R, Pranger AD, et al. Evaluation of co-trimoxazole in treatment of
multidrug-resistanttuberculosis.EurRespirJ.2012Oct25.
27. DongH,WangX,DongY,etal.Clinicalpharmacokinetic/pharmacodynamicprofileoflinezolid
inseverelyillintensivecareunitpatients.IntJAntimicrobAgents2011Oct;38(4):296-300.21.
Hess S, Hospach T, Nossal R, et al. Immune reconstitution after discontinuation of TNF
blockade.EurJPediatr.2011Oct;170(10):1337-42
28. GumboT,LouieA,DezielMR,etal.Selectionofamoxifloxacindosethatsuppressesdrug
resistanceinmycobacteriumtuberculosis,byuseofaninvitropharmacodynamicinfection
modelandmathematicalmodeling.JInfectDis.2004Nov1;190(9):1642-51.
29. HessS,HospachT,NossalR,etal.ImmunereconstitutionafterdiscontinuationofTNF
blockade.EurJPediatr.Oct;170(10):1337-42
30. ZhanelGG,SimorAE,VercaigneL,etal.CanadianCarbapenemDiscussionGroup.Imipenem
andmeropenem:Comparisonofinvitroactivity,pharmacokinetics,clinicaltrialsandadverse
effects.CanJInfectDis.1998Jul;9(4):215-28
31. Srivastava S., VanRijn SP, ShermanC, et al. SterilizingActivityof Ertapenem (ETP) agains
Mycobacterium tuberculosis (Mtb). Poster session presented at: ICAAC 2015; 66th annual
conferenceofInterscienceConferenceofAntimicrobialAgentsandChemotherapy;2015Sep
17-21;SanDiego,CA.
32. Esteban J, Ortiz A, Jiménez MS. Usefulness of E-test strips for testing susceptibility of
Mycobacterium tuberculosis complex strains. Eur J Clin Microbiol Infect Dis. 2005 Dec;
24(12):856-7
33. CamineroJA,ScardigliA.Classificationofantituberculosisdrugs:anewproposalbasedonthe
mostrecentevidence.EurRespirJ2015;46:887-893
CHAPTER 5
S. SrivastavaS.P. van Rijn
A.M.A. WesselsJ.W.C. Alffenaar
T. Gumbo
Susceptibility Testing of Antibiotics That Degrade Faster than the DoublingTime of Slow-Growing Mycobacteria: Ertapenem Sterilizing Effect Versus
Mycobacterium Tuberculosis
Antimicrob Agents Chemother. 2016 Apr 22; 60(5):3193-5.PMID: 26926650
CHAPTER 5
S. SrivastavaS.P. van Rijn
A.M.A. WesselsJ.W.C. Alffenaar
T. Gumbo
Susceptibility Testing of Antibiotics That Degrade Faster than the DoublingTime of Slow-Growing Mycobacteria: Ertapenem Sterilizing Effect Versus
Mycobacterium Tuberculosis
Antimicrob Agents Chemother. 2016 Apr 22; 60(5):3193-5.PMID: 26926650
78
Chapter 5
AbstractDrug susceptibility tests (DST) for Mycobacterium tuberculosis require at least 7 days of
incubation. Forunstable at 37°Cdrugs suchas ertapenem, it is likely that this drug couldbe
degraded before killing or inhibiting slow growing bacteria. This would alter the minimum
inhibitory concentrations (MICs) of the ertapenem, leading to falsely high MICs. Here, we
developedanewstrategytoperformDSTandMICsforsuchunstablecompounds.
Ertapenem,aβ-lactamagentofthecarbapenemclass,hasshownpromisingclinicalresultsand
favourable pharmacokinetics againstMycobacterium tuberculosis (Mtb) (1,2). The scourge of
multidrug-resistanttuberculosis(MDR-TB)andextensivelydrug-resistanttuberculosis(XDR-TB),
aglobalproblem,has increased theurgency for theuseof carbapenemssuchasertapenem,
meropenem,andfaropenem(2-4).RecentlythefirstphaseIIstudy(NCT02349841)evaluating
earlybactericidalactivityofmeropenemand faropenemhasbeencompletedand resultsare
expectedsoon.
Carbapenemsinhibitthepeptidasedomainofpenicillinbindingproteins,leadingtoautolysisand
peptidoglycanweakeningof thecellwall (5).Degradationofertapenemonstorage following
reconstitutionanddilutionistemperaturedependentandtheproposedin-useshelf-lifeis6hr
atroomtemperatureor24hoursat2to8°C(6).Mtbhasadoublingtimeofatleast24hours
under the best of circumstances (7,8). Mtb cell division is particularly slow; FtsZ, a protein
responsible for initiatingcelldivisionand recruitingproteins for formationofnewcellwall is
known tohavea rateofpolymerization that is at least 20 times slower inMtb compared to
Escherichiacoliforexample(9).InMtbatlowpH,thereplicationrateisupto10-20timesslower,
killofsuchsemidormantbacteriaisdefinedassterilizingeffect(7,8,10).Thus,microbialkilland
inhibitionofgrowthbythemosteffectiveofantibioticsisslowandtakesplaceoverseveraldays,
especiallybyβ-lactamsthatdependoncellwallturnover.
Drugsusceptibilitytests(DST)forMtbusingClinicalandLaboratoryStandardsInstitute(CLSI)and
theEuropeanCommitteeonAntimicrobial SusceptibilityTesting (EUCAST)approvedmethods
require at least 7 days of incubation (11). For drugs such as ertapenem, already appearing
unstableat37°C(2),itishighlylikelythatthisdrugcouldbedegradedbeforekillingorinhibiting
slow growing bacteria, especially semi dormant Mtb. This would be expected to alter the
minimuminhibitoryconcentrations(MICs)ofertapenem,leadingtofalselyhighMICsandfalse
resistance.Here,weshowtherapiddeclineofertapenemduringDSTandtherebywedeveloped
anewstrategytoperformDSTandMICsforsuchunstablecompounds.
79
5
Susceptibility testing of Antibiotics: Ertapenem Sterilizing Effect
AbstractDrug susceptibility tests (DST) for Mycobacterium tuberculosis require at least 7 days of
incubation. Forunstable at 37°Cdrugs suchas ertapenem, it is likely that thisdrug couldbe
degraded before killing or inhibiting slow growing bacteria. This would alter the minimum
inhibitory concentrations (MICs) of the ertapenem, leading to falsely high MICs. Here, we
developedanewstrategytoperformDSTandMICsforsuchunstablecompounds.
Ertapenem,aβ-lactamagentofthecarbapenemclass,hasshownpromisingclinicalresultsand
favourable pharmacokinetics againstMycobacterium tuberculosis (Mtb) (1,2). The scourge of
multidrug-resistanttuberculosis(MDR-TB)andextensivelydrug-resistanttuberculosis(XDR-TB),
aglobalproblem,has increased theurgency for theuseof carbapenemssuchasertapenem,
meropenem,andfaropenem(2-4).RecentlythefirstphaseIIstudy(NCT02349841)evaluating
earlybactericidal activityofmeropenemand faropenemhasbeencompletedand resultsare
expectedsoon.
Carbapenemsinhibitthepeptidasedomainofpenicillinbindingproteins,leadingtoautolysisand
peptidoglycanweakeningof thecellwall (5).Degradationofertapenemonstorage following
reconstitutionanddilutionistemperaturedependentandtheproposedin-useshelf-lifeis6hr
atroomtemperatureor24hoursat2to8°C(6).Mtbhasadoublingtimeofatleast24hours
under the best of circumstances (7,8). Mtb cell division is particularly slow; FtsZ, a protein
responsible for initiating celldivisionand recruitingproteins for formationofnewcellwall is
known tohavea rateofpolymerization that is at least 20 times slower inMtb compared to
Escherichiacoliforexample(9).InMtbatlowpH,thereplicationrateisupto10-20timesslower,
killofsuchsemidormantbacteriaisdefinedassterilizingeffect(7,8,10).Thus,microbialkilland
inhibitionofgrowthbythemosteffectiveofantibioticsisslowandtakesplaceoverseveraldays,
especiallybyβ-lactamsthatdependoncellwallturnover.
Drugsusceptibilitytests(DST)forMtbusingClinicalandLaboratoryStandardsInstitute(CLSI)and
theEuropeanCommitteeonAntimicrobial SusceptibilityTesting (EUCAST)approvedmethods
require at least 7 days of incubation (11). For drugs such as ertapenem, already appearing
unstableat37°C(2),itishighlylikelythatthisdrugcouldbedegradedbeforekillingorinhibiting
slow growing bacteria, especially semi dormant Mtb. This would be expected to alter the
minimuminhibitoryconcentrations(MICs)ofertapenem,leadingtofalselyhighMICsandfalse
resistance.Here,weshowtherapiddeclineofertapenemduringDSTandtherebywedeveloped
anewstrategytoperformDSTandMICsforsuchunstablecompounds.
80
Chapter 5
Ertapenem (purchased from SIGMA) was first dissolved in purified water and subsequently
diluted in Middlebrook 7H9 broth to the desired drug concentrations of 5.0 and 50 mg/L
respectively.Thetwosolutionswereincubatedat37ºC.After0,5,8,24,32and48hours,three
sampleswerecollectedfromeachsolutionandimmediatelystoredat-80ºCuntilfurtheranalysis.
Allsampleswerethenfullythawedatroomtemperatureandanalysedinduplicateusingafully
validatedassay(12).Thecalibrationcurveofertapenemwaslinearoverarangeof0.1to125
mg/L and the correlation coefficient was 0.999. The % coefficient of variation between the
replicatesforeachconcentrationateachtimepointwas2.7-11.2%.Figure1showsthedecrease
in ertapenem concentration in the solution at 37°C. After 5 hours of incubation, ertapenem
concentrationwasreducedby45.3%and40.7%incomparisonwiththeinitialconcentrationsof
5and50mg/L,respectively.After48hours,theconcentrationswere20.1%and26.8%ofthe
timezeroconcentrations.
Figure1.LC-MS/MSanalysisofertapenemtodeterminetherateofdegradationat370C.
Panel A and B, 5 and 50 mg/L initial concentration, respectively show the percentage of
ertapenemmeasuredafterincubationat370C.M.tuberculosisgrows“inslowmotion”compared
totheseotherbacteria(72timesslowerthanE.coliwheninlog-phasegrowthand720times
slower for semi dormantM. tuberculosis) (5). Whereas, as shown in the figure, longer the
incubationperiodmore isthedegradationofertapenem.Thiscouldeventually leadtofalsely
highertapenemMIC.
Mtb H37Ra (ATCC 25177) was used in the MIC and dose-response experiments. For each
experiment, one stock vialwas thawedandbacteria grown to logarithmic growthphase (log
phase growth) inMiddlebrook7H9broth enrichedwith 10%OADC for 4 days at 37°Cunder
shakingconditionsand5%CO2.Forsemidormantbacteriaunderacidicconditions, theday4
culturewas inoculated inMiddlebrook 7H9 acidified to pH of 5.8 bymeans of citric acid as
describedearlier (7).SterilizingeffectMICswere identifiedusingboth thebrothdilutionand
resazurincolorimetricassay(11,13).MtbinacidifiedMiddlebrook7H9brothwasexposedtothe
followingertapenemconcentrations,intriplicate:0,0.075,0.15,0.3,0.6,1,1.25,2,2.5,4,5,8,
16,32,and64mg/L.Thecultureswereincubatedat370Cwith5%CO2for7days.Inoneset,each
replicatereceivedadaily50%supplementationofertapenemconcentrationatvolumesof<1%.
Fortheresazurinassay,analiquotoftheday7cultureshadresazurin(0.001%v/v)addedtothe
samplesandplates,whichwerethenfurtherincubatedfor24hoursat37°Cunder5%CO2.The
ertapenem MIC without daily ertapenem supplementation was 64 mg/L, while that with
supplementationwas0.6mg/L.
Day7culturesdescribedabovethatwerenotusedforresazurinassayswerewashedtwicein
normalsalinetopreventdrugcarryover,andweresubsequentlyspreadonMiddlebrook7H10
agar,andincubatedfor3weeksat37°Cforenumerationofcolonyformingunits(CFU)counts.
InhibitorysigmoidEmaxcurvesforconcentrationversusCFU/mLundersterilizingeffectconditions
areshown inFigure2. Comparisonof the tworegressions,with thenullhypothesis that the
maximalkill(Emax)orefficacyandconcentrationmediating50%ofEmax(EC50)orpotencyrevealed
aratioofprobabilitiesof7.46andadifferenceincorrectedAkaikeInformationcriteriascoresof
4.02,whichmeansthattheefficacyandpotencydifferedwithsupplementation.TheEC50was
1.41 mg/L without ertapenem supplementation compared to 0.19 mg/L with daily
supplementation. Theefficacywas0.751 log10 CFU/mLwithoutdaily supplementation versus
2.38log10CFU/mLwithsupplementation.Thus,ertapenemdisplayspotentialforsterilizingeffect
thatwouldotherwisebemaskedbynotaccountingforthedegradation.
81
5
Susceptibility testing of Antibiotics: Ertapenem Sterilizing Effect
Ertapenem (purchased from SIGMA) was first dissolved in purified water and subsequently
diluted in Middlebrook 7H9 broth to the desired drug concentrations of 5.0 and 50 mg/L
respectively.Thetwosolutionswereincubatedat37ºC.After0,5,8,24,32and48hours,three
sampleswerecollectedfromeachsolutionandimmediatelystoredat-80ºCuntilfurtheranalysis.
Allsampleswerethenfullythawedatroomtemperatureandanalysedinduplicateusingafully
validatedassay(12).Thecalibrationcurveofertapenemwaslinearoverarangeof0.1to125
mg/L and the correlation coefficient was 0.999. The % coefficient of variation between the
replicatesforeachconcentrationateachtimepointwas2.7-11.2%.Figure1showsthedecrease
in ertapenem concentration in the solution at 37°C. After 5 hours of incubation, ertapenem
concentrationwasreducedby45.3%and40.7%incomparisonwiththeinitialconcentrationsof
5and50mg/L,respectively.After48hours,theconcentrationswere20.1%and26.8%ofthe
timezeroconcentrations.
Figure1.LC-MS/MSanalysisofertapenemtodeterminetherateofdegradationat370C.
Panel A and B, 5 and 50 mg/L initial concentration, respectively show the percentage of
ertapenemmeasuredafterincubationat370C.M.tuberculosisgrows“inslowmotion”compared
totheseotherbacteria(72timesslowerthanE.coliwheninlog-phasegrowthand720times
slower for semi dormantM. tuberculosis) (5). Whereas, as shown in the figure, longer the
incubationperiodmore isthedegradationofertapenem.Thiscouldeventually leadtofalsely
highertapenemMIC.
Mtb H37Ra (ATCC 25177) was used in the MIC and dose-response experiments. For each
experiment, one stock vialwas thawedandbacteria grown to logarithmic growthphase (log
phase growth) inMiddlebrook7H9broth enrichedwith 10%OADC for 4 days at 37°Cunder
shakingconditionsand5%CO2.Forsemidormantbacteriaunderacidicconditions, theday4
culturewas inoculated inMiddlebrook 7H9 acidified to pH of 5.8 bymeans of citric acid as
describedearlier (7).SterilizingeffectMICswere identifiedusingboth thebrothdilutionand
resazurincolorimetricassay(11,13).MtbinacidifiedMiddlebrook7H9brothwasexposedtothe
followingertapenemconcentrations,intriplicate:0,0.075,0.15,0.3,0.6,1,1.25,2,2.5,4,5,8,
16,32,and64mg/L.Thecultureswereincubatedat370Cwith5%CO2for7days.Inoneset,each
replicatereceivedadaily50%supplementationofertapenemconcentrationatvolumesof<1%.
Fortheresazurinassay,analiquotoftheday7cultureshadresazurin(0.001%v/v)addedtothe
samplesandplates,whichwerethenfurtherincubatedfor24hoursat37°Cunder5%CO2.The
ertapenem MIC without daily ertapenem supplementation was 64 mg/L, while that with
supplementationwas0.6mg/L.
Day7culturesdescribedabovethatwerenotusedforresazurinassayswerewashedtwicein
normalsalinetopreventdrugcarryover,andweresubsequentlyspreadonMiddlebrook7H10
agar,andincubatedfor3weeksat37°Cforenumerationofcolonyformingunits(CFU)counts.
InhibitorysigmoidEmaxcurvesforconcentrationversusCFU/mLundersterilizingeffectconditions
areshown inFigure2. Comparisonof the tworegressions,with thenullhypothesis that the
maximalkill(Emax)orefficacyandconcentrationmediating50%ofEmax(EC50)orpotencyrevealed
aratioofprobabilitiesof7.46andadifferenceincorrectedAkaikeInformationcriteriascoresof
4.02,whichmeansthattheefficacyandpotencydifferedwithsupplementation.TheEC50was
1.41 mg/L without ertapenem supplementation compared to 0.19 mg/L with daily
supplementation. Theefficacywas0.751 log10 CFU/mLwithoutdaily supplementation versus
2.38log10CFU/mLwithsupplementation.Thus,ertapenemdisplayspotentialforsterilizingeffect
thatwouldotherwisebemaskedbynotaccountingforthedegradation.
82
Chapter 5
Figure2.ErtapenemdoseresponseagainstslowlyreplicatingM.tuberculosis.
Asshowninthefigure,whenthedegradationratewastakenintoaccountbysupplementingthe
drugdaily,therewasbetterkillofsemi-dormantM.tuberculosisbyertapenem.
Here,firstweshowthatertapenemdegradesconsiderably,atrates>20-foldthedoublingtimes
ofMtbunderacidicconditions.Thiseffect is strikingwhenonecompared theMICswithand
withoutsupplementation.TheMICsintheabsenceofsupplementationwouldbeconsideredin
the resistance range by EUCAST breakpoints (14). Second, we show that ertapenem
supplementationbrings itwellwithin the susceptibility range. This suggests thatmostof the
published MICs for this drug are likely falsely high and rates of resistance are likely falsely
elevated.Inaddition,sincemanypeoplehaveusedMICstochoosewhichofthecarbapenems
wouldbebettersuitedfortreatmentofXDR-TB,gooddrugsmayhavebeendiscardedbecause
oftheartefactualmannerinwhichcurrentMICsareperformedforunstablemolecules.Third,
weshowthatertapenemislikelytohavegoodsterilizingeffectintuberculosis.Followuphollow
fiberstudiesforsterilizingeffecthavenowbeencompletedinordertoidentifytheertapenem
dose, which can be used against both drug resistant and sensitive Mtb (manuscript in
preparation).
ACKNOWLDGEMENTS
FUNDING
This work was supported by National Institutes of Health (NIH) via DP2 OD001886 and
R01AI079497toTawandaGumbo.
POTENTIALCONFLICTOFINTEREST
TawandaGumboisaconsultantforAstellasPharmaUSAandLuminaCaresolutions;andfounded
Jacaranda Biomed Inc. Jan-Willem Alffenaar received financial support from Merck, Pfizer,
AstellasandGileadforinvestigator-initiatedstudiesand/oracademicsymposia.
83
5
Susceptibility testing of Antibiotics: Ertapenem Sterilizing Effect
Figure2.ErtapenemdoseresponseagainstslowlyreplicatingM.tuberculosis.
Asshowninthefigure,whenthedegradationratewastakenintoaccountbysupplementingthe
drugdaily,therewasbetterkillofsemi-dormantM.tuberculosisbyertapenem.
Here,firstweshowthatertapenemdegradesconsiderably,atrates>20-foldthedoublingtimes
ofMtbunderacidicconditions.Thiseffect is strikingwhenonecompared theMICswithand
withoutsupplementation.TheMICsintheabsenceofsupplementationwouldbeconsideredin
the resistance range by EUCAST breakpoints (14). Second, we show that ertapenem
supplementationbrings itwellwithin the susceptibility range. This suggests thatmostof the
published MICs for this drug are likely falsely high and rates of resistance are likely falsely
elevated.Inaddition,sincemanypeoplehaveusedMICstochoosewhichofthecarbapenems
wouldbebettersuitedfortreatmentofXDR-TB,gooddrugsmayhavebeendiscardedbecause
oftheartefactualmannerinwhichcurrentMICsareperformedforunstablemolecules.Third,
weshowthatertapenemislikelytohavegoodsterilizingeffectintuberculosis.Followuphollow
fiberstudiesforsterilizingeffecthavenowbeencompletedinordertoidentifytheertapenem
dose, which can be used against both drug resistant and sensitive Mtb (manuscript in
preparation).
ACKNOWLDGEMENTS
FUNDING
This work was supported by National Institutes of Health (NIH) via DP2 OD001886 and
R01AI079497toTawandaGumbo.
POTENTIALCONFLICTOFINTEREST
TawandaGumboisaconsultantforAstellasPharmaUSAandLuminaCaresolutions;andfounded
Jacaranda Biomed Inc. Jan-Willem Alffenaar received financial support from Merck, Pfizer,
AstellasandGileadforinvestigator-initiatedstudiesand/oracademicsymposia.
84
Chapter 5
References1. TiberiS,D’AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,AlffenaarJW,MiglioriGB.
2015. Ertapenem in the treatment of multidrug-resistant tuberculosis: First clinical
experience.EurRespirJ.doi:10.1183/13993003.01278-2015
2. Van Rijn SP, Altena R, AkkermanOW, van Soolingen D, van der Laan T, de LangeWCM,
Kosterink JGW, vanderWerf TS,Alffenaar JWC. 2015. Pharmacokinetics of ertapenem in
patientswitgmultidrug-resistanttuberculosis.EurRespir J.doi:10.1183/13993003.01654-
2015
3. DeLorenzoS,AlffenaarJW,SotgiuG,CentisR,D'AmbrosioL,TiberiS,BolhuisMS,vanAltena
R,ViggianiP,PianaA,SpanevelloA,MiglioriGB.2013.Efficacyandsafetyofmeropenem-
clavulanate added to linezolid-containing regimens in the treatment of MDR-/XDR-TB.
Eur.Respir.J.41:1386-1392.
4. DhedaK,GumboT,GandhiNR,MurrayM,TheronG,UdwadiaZ,MiglioriGB,WarrenR.2014.
Globalcontroloftuberculosis: fromextensively-drugresistanttountreatabletuberculosis.
LancetRespirMed.2:321-338.
5. CordillotM,DubeeV,TribouletS,DubostL,MarieA,HugonnetJE,ArthurM,MainardiJL.
2013. In vitro cross-linking of Mycobacterium tuberculosis peptidoglycan by L,D-
transpeptidasesandinactivationoftheseenzymesbycarbapenems.AntimicrobAgentsand
Chemother.57:5940-45
6. Scientific discussion for the approval of Invanz.
http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-
_Scientific_Discussion/human/000389/WC500033918.pdf.
7. Gumbo T, Dona CS, Meek C, Leff R. 2009. Pharmacokinetics-pharmacodynamics of
pyrazinamideinanovel invitromodeloftuberculosisforsterilizingeffect:aparadigmfor
fasterassessmentofnewantituberculosisdrugs.AntimicrobialAgentsandChemotherapy.
53:3197-3204.
8. MusukaS,SrivastavaS,SiyambalapitiyageDonaCW,MeekC,LeffR,PasipanodyaJ,Gumbo
T. 2013. Thioridazine pharmacokinetic-pharmacodynamic parameters "wobble" during
treatmentoftuberculosis:atheoreticalbasisforshorter-durationcurativemonotherapywith
congeners.AntimicrobialAgentsandChemotherapy.57:5870-5877.
9. Hett EC, Rubin EJ. 2008. Bacterial growth and cell division: a mycobacterial perspective.
Microbiol.Mol.Biol.Rev.72:126-56,table.
10. MitchisonDA.1979.Basicmechanismsofchemotherapy.Chest.76:771-781.
11. Clinical and Laboratory Standards Institute. 2003. Susceptibility testing ofMycobacteria,
Nocardiae,andotheraerobicActinomycetes;ApprovedStandard.M24-A2
12. Van Rijn SP,Wessels AM,Greijdanus B, TouwDJ, Alffenaar JW. 2014.Quantification and
validationofertapenemusingaliquidchromatography-tandemmassspectrometrymethod.
AntimicrobialAgentsandChemotherapy.58:3481-3484.
13. Srivastava S, Pasipanodya J, Sherman CM, Meek C, Leff R, Gumbo T. 2015. Rapid drug
toleranceanddramaticsterilizingeffectofmoxifloxacinmonotherapyinanovelhollow-fiber
model of intracellular Mycobacterium kansasii disease.Antimicrobial Agents and
Chemotherapy.59:2273-2279.
14. VezirisN, Truffot C,Mainardi JL, Jarlier, V. 2011. Activity of carbapenems combinedwith
clavulanateagainstmurinetuberculosis.AntimicrobialAgentsandChemotherapy.55:2597-
2600.
85
5
Susceptibility testing of Antibiotics: Ertapenem Sterilizing Effect
References1. TiberiS,D’AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,AlffenaarJW,MiglioriGB.
2015. Ertapenem in the treatment of multidrug-resistant tuberculosis: First clinical
experience.EurRespirJ.doi:10.1183/13993003.01278-2015
2. Van Rijn SP, Altena R, AkkermanOW, van Soolingen D, van der Laan T, de LangeWCM,
Kosterink JGW, vanderWerf TS,Alffenaar JWC. 2015. Pharmacokinetics of ertapenem in
patientswitgmultidrug-resistanttuberculosis.EurRespir J.doi:10.1183/13993003.01654-
2015
3. DeLorenzoS,AlffenaarJW,SotgiuG,CentisR,D'AmbrosioL,TiberiS,BolhuisMS,vanAltena
R,ViggianiP,PianaA,SpanevelloA,MiglioriGB.2013.Efficacyandsafetyofmeropenem-
clavulanate added to linezolid-containing regimens in the treatment of MDR-/XDR-TB.
Eur.Respir.J.41:1386-1392.
4. DhedaK,GumboT,GandhiNR,MurrayM,TheronG,UdwadiaZ,MiglioriGB,WarrenR.2014.
Globalcontroloftuberculosis: fromextensively-drugresistanttountreatabletuberculosis.
LancetRespirMed.2:321-338.
5. CordillotM,DubeeV,TribouletS,DubostL,MarieA,HugonnetJE,ArthurM,MainardiJL.
2013. In vitro cross-linking of Mycobacterium tuberculosis peptidoglycan by L,D-
transpeptidasesandinactivationoftheseenzymesbycarbapenems.AntimicrobAgentsand
Chemother.57:5940-45
6. Scientific discussion for the approval of Invanz.
http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-
_Scientific_Discussion/human/000389/WC500033918.pdf.
7. Gumbo T, Dona CS, Meek C, Leff R. 2009. Pharmacokinetics-pharmacodynamics of
pyrazinamideinanovel invitromodeloftuberculosisforsterilizingeffect:aparadigmfor
fasterassessmentofnewantituberculosisdrugs.AntimicrobialAgentsandChemotherapy.
53:3197-3204.
8. MusukaS,SrivastavaS,SiyambalapitiyageDonaCW,MeekC,LeffR,PasipanodyaJ,Gumbo
T. 2013. Thioridazine pharmacokinetic-pharmacodynamic parameters "wobble" during
treatmentoftuberculosis:atheoreticalbasisforshorter-durationcurativemonotherapywith
congeners.AntimicrobialAgentsandChemotherapy.57:5870-5877.
9. Hett EC, Rubin EJ. 2008. Bacterial growth and cell division: a mycobacterial perspective.
Microbiol.Mol.Biol.Rev.72:126-56,table.
10. MitchisonDA.1979.Basicmechanismsofchemotherapy.Chest.76:771-781.
11. Clinical and Laboratory Standards Institute. 2003. Susceptibility testing ofMycobacteria,
Nocardiae,andotheraerobicActinomycetes;ApprovedStandard.M24-A2
12. Van Rijn SP,Wessels AM,Greijdanus B, TouwDJ, Alffenaar JW. 2014.Quantification and
validationofertapenemusingaliquidchromatography-tandemmassspectrometrymethod.
AntimicrobialAgentsandChemotherapy.58:3481-3484.
13. Srivastava S, Pasipanodya J, Sherman CM, Meek C, Leff R, Gumbo T. 2015. Rapid drug
toleranceanddramaticsterilizingeffectofmoxifloxacinmonotherapyinanovelhollow-fiber
model of intracellular Mycobacterium kansasii disease.Antimicrobial Agents and
Chemotherapy.59:2273-2279.
14. VezirisN, Truffot C,Mainardi JL, Jarlier, V. 2011. Activity of carbapenems combinedwith
clavulanateagainstmurinetuberculosis.AntimicrobialAgentsandChemotherapy.55:2597-
2600.
CHAPTER 6
S.P. van Rijn*S. Srivastava*
A.M.A. WesselsD. Soolingen
J.W.C. AlffenaarT. Gumbo
Sterilizing Effect of Ertapenem-Clavulanate in a
Hollow-Fiber Model ofTuberculosis and Implications on
Clinical Dosing
Antimicrob Agents Chemother. 2017 Aug 24; 61(9).PMID: 28696238
*Both authors contributed equally
CHAPTER 6
S.P. van Rijn*S. Srivastava*
A.M.A. WesselsD. Soolingen
J.W.C. AlffenaarT. Gumbo
Sterilizing Effect of Ertapenem-Clavulanate in a
Hollow-Fiber Model ofTuberculosis and Implications on
Clinical Dosing
Antimicrob Agents Chemother. 2017 Aug 24; 61(9).PMID: 28696238
*Both authors contributed equally
88
Chapter 6
AbstractCarbapenems are now being explored for treatment of multi-drug resistant tuberculosis
(MDR-TB),especiallyinconjunctionwithclavulanate.Clinicaluseisconstrainedbytheneed
formultipleparenteraldosesperday,andlackofknowledgeoftheoptimaldoseforsterilizing
effect. Our objective was to identify the ertapenem exposure associated with optimal
sterilizingeffectandthendesignaonceadaydoseforclinicaluse.Weutilizedthehollow
fiber system model of tuberculosis in a 28-day exposure-response study of 8 different
ertapenem doses in combination with clavulanate. The systems were sampled at
predetermined time-points to verify the concentration-time profile and identify the total
bacterial burden. Inhibitory sigmoid Emax modelling was used to identify the relationship
between total bacterial burden and the drug exposure, and identify optimal exposures.
Contrarytotheliterature,ertapenem-clavulanatecombinationdemonstratedgoodmicrobial
kill and sterilizingeffect. In adose-fractionationhollow fiber study,efficacywas linked to
percentageofthe24-hourdosingintervalofertapenemconcentrationpersistingaboveMIC
(%TMIC). We performed 10,000MDR-TB patient computer-aided clinical trial simulations,
basedonMonteCarlomethods,to identifythedosesandschedulethatwouldachieveor
exceed%TMIC³40%.Weidentifiedanintravenousdoseof2gramsonceperdayasachieving
the target in 96% of patients. An ertapenem susceptibility breakpoint MIC 2 mg/L was
identifiedforthatdose.Anertapenemdoseof2goncedailyisthemostsuitabletobetested
inaphaseIIstudyofsterilizingeffectinMDR-TBpatients.
IntroductionTheemergenceofdrugresistanttuberculosis(TB),especiallymultidrug-resistantTB(MDR-
TB), extensively drug resistant (XDR-TB), and virtually incurable TB (termed totally drug-
resistant TB by some), is a global emergency that threatens to underminemany gains of
chemotherapy(1-4).Asaresult,thereiscurrentlyafourprongedeffort;(i)identificationof
new smallmolecules to kill drug resistantMycobacterium tuberculosis, (ii) repurposingof
antimicrobial drugs not currently used to treat TB into TB therapeutics, (iii) host-directed
therapy, and (iv) use of pharmacokinetic/pharmacodynamics science to optimize efficacy
while suppressing emergenceof acquireddrug resistance (5-8).Carbapenems, extensively
used to treatGram-negativebacteriaover the last30 years,havealsobeen shown tobe
effectiveagainstM.tuberculosisinvitroandinvivowheninthepresenceofaβ-lactamase
inhibitor(6,9).
Severalinitiativesareongoingtoexploretheaddedvalueofcarbapenemsgivenaspartofa
multidrugregimenforM/XDR-TB(10,11).InmurineTB,efficacyhasbeendemonstratedfor
meropenemandimipenemwithclavulanate;howeverertapenemwasnobetterthannon-
treated controls (9). In addition, ertapenemdemonstratedhighMICs, suggestingpossible
natural resistance. However, ertapenem degrades rapidly in in vitro growth media at
incubationtemperaturesusedtomeasureMICswithconventionalmethods(12).Wehave
sincedevelopedaMICassaythatcorrectsforthisdegradation,whichhasdemonstratedmuch
lowerMICs(12).Themainadvantageofertapenemtopatientscouldbeitshalf-lifeof4hours,
which could allow a once a day schedule, as opposed to 0.6-0.7 hrs formeropenemand
imipenemwhichnecessitatesmultipleandprolongedintravenousinfusionsperday(13).The
multiple infusions per day with meropenem and imipenem make it rather difficult to
administer long duration therapy in M/XDR-TB. Recently the first TB clinical data with
ertapenem showed that it was well tolerated as part of a salvage regimen for MDR-TB
patients(13,14).Unfortunately,theefficacyofthedrugcouldnotbeassessedasitwasused
inamultidrugregimen;moreover,itssterilizingeffectisunknown.
ThehollowfibersystemmodelofTB(HFS-TB)hasbeenusedtoexaminethesterilizingeffect
of anti-TBagents,definedas theability to kill either semi-dormantM. tuberculosis under
89
6
Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of Tuberculosis
AbstractCarbapenems are now being explored for treatment of multi-drug resistant tuberculosis
(MDR-TB),especiallyinconjunctionwithclavulanate.Clinicaluseisconstrainedbytheneed
formultipleparenteraldosesperday,andlackofknowledgeoftheoptimaldoseforsterilizing
effect. Our objective was to identify the ertapenem exposure associated with optimal
sterilizingeffectandthendesignaonceadaydoseforclinicaluse.Weutilizedthehollow
fiber system model of tuberculosis in a 28-day exposure-response study of 8 different
ertapenem doses in combination with clavulanate. The systems were sampled at
predetermined time-points to verify the concentration-time profile and identify the total
bacterial burden. Inhibitory sigmoid Emax modelling was used to identify the relationship
between total bacterial burden and the drug exposure, and identify optimal exposures.
Contrarytotheliterature,ertapenem-clavulanatecombinationdemonstratedgoodmicrobial
kill and sterilizingeffect. In adose-fractionationhollow fiber study,efficacywas linked to
percentageofthe24-hourdosingintervalofertapenemconcentrationpersistingaboveMIC
(%TMIC). We performed 10,000MDR-TB patient computer-aided clinical trial simulations,
basedonMonteCarlomethods,to identifythedosesandschedulethatwouldachieveor
exceed%TMIC³40%.Weidentifiedanintravenousdoseof2gramsonceperdayasachieving
the target in 96% of patients. An ertapenem susceptibility breakpoint MIC 2 mg/L was
identifiedforthatdose.Anertapenemdoseof2goncedailyisthemostsuitabletobetested
inaphaseIIstudyofsterilizingeffectinMDR-TBpatients.
IntroductionTheemergenceofdrugresistanttuberculosis(TB),especiallymultidrug-resistantTB(MDR-
TB), extensively drug resistant (XDR-TB), and virtually incurable TB (termed totally drug-
resistant TB by some), is a global emergency that threatens to underminemany gains of
chemotherapy(1-4).Asaresult,thereiscurrentlyafourprongedeffort;(i)identificationof
new smallmolecules to kill drug resistantMycobacterium tuberculosis, (ii) repurposingof
antimicrobial drugs not currently used to treat TB into TB therapeutics, (iii) host-directed
therapy, and (iv) use of pharmacokinetic/pharmacodynamics science to optimize efficacy
while suppressing emergenceof acquireddrug resistance (5-8).Carbapenems, extensively
used to treatGram-negativebacteriaover the last30 years,havealsobeen shown tobe
effectiveagainstM.tuberculosisinvitroandinvivowheninthepresenceofaβ-lactamase
inhibitor(6,9).
Severalinitiativesareongoingtoexploretheaddedvalueofcarbapenemsgivenaspartofa
multidrugregimenforM/XDR-TB(10,11).InmurineTB,efficacyhasbeendemonstratedfor
meropenemandimipenemwithclavulanate;howeverertapenemwasnobetterthannon-
treated controls (9). In addition, ertapenemdemonstratedhighMICs, suggestingpossible
natural resistance. However, ertapenem degrades rapidly in in vitro growth media at
incubationtemperaturesusedtomeasureMICswithconventionalmethods(12).Wehave
sincedevelopedaMICassaythatcorrectsforthisdegradation,whichhasdemonstratedmuch
lowerMICs(12).Themainadvantageofertapenemtopatientscouldbeitshalf-lifeof4hours,
which could allow a once a day schedule, as opposed to 0.6-0.7 hrs formeropenemand
imipenemwhichnecessitatesmultipleandprolongedintravenousinfusionsperday(13).The
multiple infusions per day with meropenem and imipenem make it rather difficult to
administer long duration therapy in M/XDR-TB. Recently the first TB clinical data with
ertapenem showed that it was well tolerated as part of a salvage regimen for MDR-TB
patients(13,14).Unfortunately,theefficacyofthedrugcouldnotbeassessedasitwasused
inamultidrugregimen;moreover,itssterilizingeffectisunknown.
ThehollowfibersystemmodelofTB(HFS-TB)hasbeenusedtoexaminethesterilizingeffect
of anti-TBagents,definedas theability to kill either semi-dormantM. tuberculosis under
90
Chapter 6
acidicconditionsorofnon-replicatingpersistersunderhypoxia(15-17). Itwasqualifiedby
the European Medicines Agency and editorially endorsed by the US Food and Drug
Administration (http://www.ema.europa.eu/docs/en_GB/document_library/Regulatory_an
d_procedural_guideline/2015/02/WC50018199.pdf).TheHFS-TBintandemwithcomputer-
aidedclinicaltrialsimulationswasfoundtohaveaforecastingaccuracyof>94%ofobserved
optimalexposuresanddosesinTBpatientsintheclinic(18-20).Thismakesthismodelideal
toidentifyoptimaldosesfortreatmentofM/XDR-TB,whichcandirectlybetranslatedinto
clinicaluse.Ourobjectivewastousethesemodelstoidentifytheoptimalsterilizingeffect
doseofertapenemfortreatmentofMDR-TB.
ResultsDose-effectHFS-TBstudyforsterilizingeffect
InthefirstHFS-TB,whichwasmainlyadoserangingstudy,weculturedM.tuberculosisH37Ra
underacidicconditionstoasemidormantstate,andtheinoculatedintoHFS-TBunitswith
circulating Middlebrook 7H9 acidified to a pH of 5.8, as described in the past (15, 21).
Differentertapenemexposures,basedonhumanequivalentdosesof0.25,0.5,1.0,2.0,3.0,
5.0,and10.0gramswereadministeredintothecentralcompartmentofduplicateHFS-TBvia
a computer-controlled syringepumpover30minutes, as inpatients;drug concentrations
achievedineachofthe16HFS-TBweremeasuredat8differenttimepointsoverthefirst24
hours.Clavulanatewasalsodispensedviasyringepumptoachieveapeakof3mg/Latthe
endof30minutesinfusion.Pharmacokineticmodellingofthemeasureddrugconcentrations,
revealedthatthelowestAkaikeInformationCriteriascores(22)wereforaonecompartment
model. The ertapenem total clearance (± standard deviation) was 4.11±1.83L, and the
volume of 22.55±4.0 L, which translates to a half-life of 3.80 hours. The regression for
observed concentrations versus pharmacokinetic model predicted concentrations had an
r2=0.997andtheslopewas0.996±0.006,whichisclosetounity.Thus,theonecompartment
modeldescribedthedatawell,withnobias.
Figure1showsthatertapenemachievedgoodsterilizingeffect.Thebacterialburdenatthe
start of therapywas 4.0 log10 CFU/mL. The data are presented as inhibitory sigmoid Emax
modelsbetween“nominal”humanequivalentdoseandmicrobialburden.
Figure1.Ertapenem-clavulanatedose-effectsterilizingeffectinthehollowfibermodel.
Drug treatmentsaredepictedas “nominal”humanequivalentdoses.Onday3, inhibitorysigmoidEmaxmodellingdemonstratednomodelconverge,andtherewasverylittlekill,thusregressionsforday3wereleftoutofthefigure.However,byday7therewasalreadygoodmicrobialkill,characterizedbymaximalkill(Emax)of1.13±0.34log10CFU/mL.Byday28,allertapenemtreatedHFS-TBcompletelysterilizedthebacteria.
InFigure1,therewasnomodelconvergenceonday3,whileonday28,attheendofthe
experiment, all ertapenem treated systems now had bacterial burden below limits of
detection.Allsystemsachieved%oftimeaboveMIC(%TMIC)for100%ofthedosinginterval;
thetroughat23.5hrswas>4mg/Linallsystems,andallachievedthesamemicrobialkillon
day28.
Ertapenemdose-fractionationstudyintheHFS-TB
Next, we performed a new HFS-TB, this time using M. tuberculosis H37Rv and a dose-
fractionationdesign, fora treatmentdurationof14days.Onmeasurementofertapenem
concentrations,similartothefirststudy,theconcentrationswerealsobestdescribedusinga
onecompartmentmodel;theobservedversuspredictedconcentrationsrevealedaslopeof
0.995±0.002(r2>0.999).Theconcentration-timeprofilesachievedwitheachdoseareshown
bydosingscheduleinFigures2A-C.,togetherwiththeertapenem(plusclavulanate2.5mg/L)
MICof4mg/L.InhibitorysigmoidEmaxmodelfittingbyexposureexpressedaseitherCmax/MIC
orAUC0-24/MICor%TMICrevealedAkaikeInformationCriteriascoresshowninTable1.
91
6
Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of Tuberculosis
acidicconditionsorofnon-replicatingpersistersunderhypoxia(15-17). Itwasqualifiedby
the European Medicines Agency and editorially endorsed by the US Food and Drug
Administration (http://www.ema.europa.eu/docs/en_GB/document_library/Regulatory_an
d_procedural_guideline/2015/02/WC50018199.pdf).TheHFS-TBintandemwithcomputer-
aidedclinicaltrialsimulationswasfoundtohaveaforecastingaccuracyof>94%ofobserved
optimalexposuresanddosesinTBpatientsintheclinic(18-20).Thismakesthismodelideal
toidentifyoptimaldosesfortreatmentofM/XDR-TB,whichcandirectlybetranslatedinto
clinicaluse.Ourobjectivewastousethesemodelstoidentifytheoptimalsterilizingeffect
doseofertapenemfortreatmentofMDR-TB.
ResultsDose-effectHFS-TBstudyforsterilizingeffect
InthefirstHFS-TB,whichwasmainlyadoserangingstudy,weculturedM.tuberculosisH37Ra
underacidicconditionstoasemidormantstate,andtheinoculatedintoHFS-TBunitswith
circulating Middlebrook 7H9 acidified to a pH of 5.8, as described in the past (15, 21).
Differentertapenemexposures,basedonhumanequivalentdosesof0.25,0.5,1.0,2.0,3.0,
5.0,and10.0gramswereadministeredintothecentralcompartmentofduplicateHFS-TBvia
a computer-controlled syringepumpover30minutes, as inpatients;drug concentrations
achievedineachofthe16HFS-TBweremeasuredat8differenttimepointsoverthefirst24
hours.Clavulanatewasalsodispensedviasyringepumptoachieveapeakof3mg/Latthe
endof30minutesinfusion.Pharmacokineticmodellingofthemeasureddrugconcentrations,
revealedthatthelowestAkaikeInformationCriteriascores(22)wereforaonecompartment
model. The ertapenem total clearance (± standard deviation) was 4.11±1.83L, and the
volume of 22.55±4.0 L, which translates to a half-life of 3.80 hours. The regression for
observed concentrations versus pharmacokinetic model predicted concentrations had an
r2=0.997andtheslopewas0.996±0.006,whichisclosetounity.Thus,theonecompartment
modeldescribedthedatawell,withnobias.
Figure1showsthatertapenemachievedgoodsterilizingeffect.Thebacterialburdenatthe
start of therapywas 4.0 log10 CFU/mL. The data are presented as inhibitory sigmoid Emax
modelsbetween“nominal”humanequivalentdoseandmicrobialburden.
Figure1.Ertapenem-clavulanatedose-effectsterilizingeffectinthehollowfibermodel.
Drug treatmentsaredepictedas “nominal”humanequivalentdoses.Onday3, inhibitorysigmoidEmaxmodellingdemonstratednomodelconverge,andtherewasverylittlekill,thusregressionsforday3wereleftoutofthefigure.However,byday7therewasalreadygoodmicrobialkill,characterizedbymaximalkill(Emax)of1.13±0.34log10CFU/mL.Byday28,allertapenemtreatedHFS-TBcompletelysterilizedthebacteria.
InFigure1,therewasnomodelconvergenceonday3,whileonday28,attheendofthe
experiment, all ertapenem treated systems now had bacterial burden below limits of
detection.Allsystemsachieved%oftimeaboveMIC(%TMIC)for100%ofthedosinginterval;
thetroughat23.5hrswas>4mg/Linallsystems,andallachievedthesamemicrobialkillon
day28.
Ertapenemdose-fractionationstudyintheHFS-TB
Next, we performed a new HFS-TB, this time using M. tuberculosis H37Rv and a dose-
fractionationdesign, fora treatmentdurationof14days.Onmeasurementofertapenem
concentrations,similartothefirststudy,theconcentrationswerealsobestdescribedusinga
onecompartmentmodel;theobservedversuspredictedconcentrationsrevealedaslopeof
0.995±0.002(r2>0.999).Theconcentration-timeprofilesachievedwitheachdoseareshown
bydosingscheduleinFigures2A-C.,togetherwiththeertapenem(plusclavulanate2.5mg/L)
MICof4mg/L.InhibitorysigmoidEmaxmodelfittingbyexposureexpressedaseitherCmax/MIC
orAUC0-24/MICor%TMICrevealedAkaikeInformationCriteriascoresshowninTable1.
92
Chapter 6
Figure2.DosefractionationstudytoPK/PDindexlinkedtoertapenemefficacy
TheconcentrationtimeprofilesareshownrelativetotheMIC.Symbolsindicatemeasuredconcentrations,andthelinesmodeledprofile.A.Concentration-timeprofilesofertapenemidentified in theHFS-TBwithevery8hrdosingschedule.B.Concentration-timeprofilesofertapenemidentifiedintheHFS-TBwithevery12hrdosingschedule.C.Concentration-timeprofilesofertapenemidentifiedintheHFS-TBwithonceadaydosingschedule.Giventheconcentrationrange,thescaleobscuresthetimethatconcentrationspersistedaboveMICforsomeofdoses.Fortheblueopencircles,thelowestconcentration,thetimeaboveMICwas0hrs.Forthedoseshownbycayennetrinagles,thetimewas3hr,fortheblackopendiamondsitwas8.32hr,whilefortheopenmagentasquaresitwas11.7hr.Therestcanbereadoffthegraph. D. Inhibitory sigmoidEmaxmodel for%TMIC versusbacterialburden.Onday7, themaximalkill(Emax)was1.14log10CFU/mL,consistentwithfindingsinthefirstHFS-TBdose-effectstudy.Thestudywasforonly14days.Examinationofthecurvesoneachdayshowsthat80%ofmaximalkilloccursarounda%TMICof40%onallsamplingdays,exceptday3whenitoccurswithlowerexposures.
Table1.AkaikeInformationCriteriaScoresforPK/PDindexversusertapenemsterilizingeffect.
Day3 Day7 Day10 Day14AUC0-24/MIC -30.26 -18.99 -31.41 -5.112Cmax/MIC -30.63 -17.19 -31.55 -2.144%TMIC -60.39 -62.96 -49.39 -45.06
Thelowestscoreswerefor%TMIC,whichmeansthisisthePK/PDindexlinkedtomicrobialkill.
Figure2DshowstheinhibitorysigmoidEmaxMICcurvesforeachsamplingdaybasedon%TMIC.
Based on day 10, which had the highest r2 of 0.94, the relationship between %TMIC and
bacterialburdenwas:
log10CFU/mL=5.68-%TMIC2.56/[23.522.56+%TMIC
2.56]
Fromthisrelationship,wecalculatedtheEC80asa%TMICof40.41%ofthedosinginterval.
Indeed,thiscanbereadoffFigure2Daswell,whichshowsthatonegetsthesameexposure
foroptimalkillwhicheversamplingdayisexamined.
MonteCarlosimulationstoidentifyoptimalertapenemdose
InTBpatients,pharmacokineticvariabilityisoneofthemostimportantdriversofsterilizing
effect(23-29).Therefore,inordertoidentifytheoptimalertapenemdoseforpulmonaryTB,
we performedMonte Carlo simulations of 10,000 patientswith pulmonary TB, using the
pharmacokineticparameterestimatesandbetween-patientvariabilityindicesshowninTable
2basedonBurkhardtetal(30-32).Wealsoaccountedfortheertapenempenetrationinto
epithelialliningfluid(ELF)of7.48±8.17%(whichmirrorsthenon-proteinboundconcentration
of5-15%),andthatinlungtissueof23.6±12.3%(33).Weperformedsimulationstodetermine
howmuch1.0gonceaday,or1.0gtwiceaday,or2.0gonceaday,or2.0gtwiceaday,or
3.0g once a daywould achieve or exceed the target exposure, which is %TMIC of 40.41%
associated with optimal sterilizing effect in ELF of patients. For internal validation, we
compared the pharmacokinetic parameters in the 10,000 simulated patients to those of
Burkhardt et al in Table 2, which shows that the simulations faithfully recapitulated the
pharmacokinetic parameters and variability. As an extra external validation step, we
comparedthepharmacokineticparametersinthesimulationstothoseweactuallyobserved
inourMDR-TBpatientsintheNetherlands,asshowninTable2(13).Table2showsthatthe
pharmacokineticparametersandvarianceinoursimulationswerevirtuallyidenticaltothose
weobserved inpatients.Therefore, the simulationswereaccurate in reproducingwhat is
identifiedintheclinic.
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Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of Tuberculosis
Figure2.DosefractionationstudytoPK/PDindexlinkedtoertapenemefficacy
TheconcentrationtimeprofilesareshownrelativetotheMIC.Symbolsindicatemeasuredconcentrations,andthelinesmodeledprofile.A.Concentration-timeprofilesofertapenemidentified in theHFS-TBwithevery8hrdosingschedule.B.Concentration-timeprofilesofertapenemidentifiedintheHFS-TBwithevery12hrdosingschedule.C.Concentration-timeprofilesofertapenemidentifiedintheHFS-TBwithonceadaydosingschedule.Giventheconcentrationrange,thescaleobscuresthetimethatconcentrationspersistedaboveMICforsomeofdoses.Fortheblueopencircles,thelowestconcentration,thetimeaboveMICwas0hrs.Forthedoseshownbycayennetrinagles,thetimewas3hr,fortheblackopendiamondsitwas8.32hr,whilefortheopenmagentasquaresitwas11.7hr.Therestcanbereadoffthegraph. D. Inhibitory sigmoidEmaxmodel for%TMIC versusbacterialburden.Onday7, themaximalkill(Emax)was1.14log10CFU/mL,consistentwithfindingsinthefirstHFS-TBdose-effectstudy.Thestudywasforonly14days.Examinationofthecurvesoneachdayshowsthat80%ofmaximalkilloccursarounda%TMICof40%onallsamplingdays,exceptday3whenitoccurswithlowerexposures.
Table1.AkaikeInformationCriteriaScoresforPK/PDindexversusertapenemsterilizingeffect.
Day3 Day7 Day10 Day14AUC0-24/MIC -30.26 -18.99 -31.41 -5.112Cmax/MIC -30.63 -17.19 -31.55 -2.144%TMIC -60.39 -62.96 -49.39 -45.06
Thelowestscoreswerefor%TMIC,whichmeansthisisthePK/PDindexlinkedtomicrobialkill.
Figure2DshowstheinhibitorysigmoidEmaxMICcurvesforeachsamplingdaybasedon%TMIC.
Based on day 10, which had the highest r2 of 0.94, the relationship between %TMIC and
bacterialburdenwas:
log10CFU/mL=5.68-%TMIC2.56/[23.522.56+%TMIC
2.56]
Fromthisrelationship,wecalculatedtheEC80asa%TMICof40.41%ofthedosinginterval.
Indeed,thiscanbereadoffFigure2Daswell,whichshowsthatonegetsthesameexposure
foroptimalkillwhicheversamplingdayisexamined.
MonteCarlosimulationstoidentifyoptimalertapenemdose
InTBpatients,pharmacokineticvariabilityisoneofthemostimportantdriversofsterilizing
effect(23-29).Therefore,inordertoidentifytheoptimalertapenemdoseforpulmonaryTB,
we performedMonte Carlo simulations of 10,000 patientswith pulmonary TB, using the
pharmacokineticparameterestimatesandbetween-patientvariabilityindicesshowninTable
2basedonBurkhardtetal(30-32).Wealsoaccountedfortheertapenempenetrationinto
epithelialliningfluid(ELF)of7.48±8.17%(whichmirrorsthenon-proteinboundconcentration
of5-15%),andthatinlungtissueof23.6±12.3%(33).Weperformedsimulationstodetermine
howmuch1.0gonceaday,or1.0gtwiceaday,or2.0gonceaday,or2.0gtwiceaday,or
3.0g once a daywould achieve or exceed the target exposure, which is %TMIC of 40.41%
associated with optimal sterilizing effect in ELF of patients. For internal validation, we
compared the pharmacokinetic parameters in the 10,000 simulated patients to those of
Burkhardt et al in Table 2, which shows that the simulations faithfully recapitulated the
pharmacokinetic parameters and variability. As an extra external validation step, we
comparedthepharmacokineticparametersinthesimulationstothoseweactuallyobserved
inourMDR-TBpatientsintheNetherlands,asshowninTable2(13).Table2showsthatthe
pharmacokineticparametersandvarianceinoursimulationswerevirtuallyidenticaltothose
weobserved inpatients.Therefore, the simulationswereaccurate in reproducingwhat is
identifiedintheclinic.
94
Chapter 6
Table2.Comparisonofpharmacokineticparameterandconcentrationestimatesandrangesin10,000simulatedpatientstothoseactuallyobservedinpatientstreatedwith1G.
SubroutinePRIORbasedonliterature(±SD)
10,000simulatedTBpatients
ObservedinMDR-TBpatients
TotalclearanceinL/hr 2.63±0.83 2.6(0.02-6.00) 2.1(0.09–3.23)VolumeinL 10.6±2.51 11(1.2-19) 7.3(2.61–11.10)Half-lifeinhours - 2.8(2.20-3.70) 2.4(2.05–3.53)AUC0-24inmg*h/L - 448(166-4255) 545(309–1130)
Figure3Ashowsthetargetattainmentprobability(TAP)foreachdoseanddosingschedule
astheMICchanges.Ononeextreme,the1gmonceadaydosehadaTAPlessthan90%once
theMICwas1mg/L,whilethedoseof3gmtwiceadayachievedahighTAPuntil8mg/L,then
fellprecipitouslyat16mg/L.For2gmaday,theTAPfellatanMICof2mg/L.Thismeansthat
thesusceptibilitybreakpointforertapenemplusclavulanatewillfallbetweenMICof1and
16mg/L,andwilldependonthefinaldosechosen.
SinceMICvariabilityisalsoanimportantdeterminantoftherapyresponseinTBpatients(25,
34-36),wealsotookintoaccounttheMICdistribution.Figure3BshowstheertapenemMIC
distribution from 33 MDR-TB patients isolates in the Netherlands, in the presence of
clavulanate.Figure3BshowsthatallisolateswouldhaveMICsbetweengreaterthan1mg/L
andbelow128mg/L.However,inthepastwehaveshownthatertapenemdegradesduring
theMICtesting,andifonceaccountsforthedegradation,thereisa4-tubedilutiondecrease
inMICs; ifclavulanate isaddedandertapenemissupplementedthere isa7-tubedilution
difference(12).Thus,wetransformedtheMICsforthe33clinical isolatesdownby4-tube
dilutionsaswell,asshowninFigure3B.Inthatscenario,only6.5%of isolateshadanMIC
greaterthan1mg/L.
SummationofallTAPstoaccountfordistributionofMICsgivestheproportionof10,000TB
patientswhowouldachieve theargett exposureof%TMICof 40%, termed the cumulative
fractionofresponse(CFR).Figure3CshowstheCFRsfortheonceadaydosingschedulefor
bothobservedMICsandtransformedMICs.ForthetransformedMICsthedoseof2gmaday
hadaCFRof96%.
Figure 3. Target attainment probability and cumulative fraction of response for variousertapenemdoses.
A.Targetattainmentprobabilityfor%TMICof40%asM.tuberculosisMICchanges.NodoseordosingscheduleiseffectiveonceMICsare16mg/L.B.ErtapenemMICdistributioninisolatesfrom the Netherlands, with and without transformation to account for ertapenemdegradation.C.Proportionof10,000patientswhoachievedorexceeded%TMICof40%withonceadaydosing.TheproportionishighlysensitivetotheMIC,andfellonsensitivityanalysis,aworst-casescenario.D.Proportionof10,000patientswhoachievedorexceeded%TMICof40%withtwiceadaydosing.Thetwiceadaydosingscheduleachievedthetargetinhigherproportionsofpatients,evenonsensitivityanalysis.However,giventhehardshipoftwiceaday administration of therapy in TB, we chose the 2gm once a day dose as being mostpractical. WealsoperformedertapenemplusclavalunateMICsin4clinical isolatesincubatedat4°C
versus37°Ctotryandslowdowndrugdegradation:MICswerelowerat4°Cby4,2,3,and
2-tube dilutions. Therefore, we performed sensitivity testing by examining CFR is MIC
transformationwasonly2-tubedilution(worstcasescenario).Figure3Cshowsthatthedose
of2gmonceadaywouldnotachievethetarget in90%ofpatients;nevertheless itwould
achievethisin63%ofpatients,whichisstillreasonable.Figure3Dshowstheresultsoftwice
aweekdosingschedule;aswouldbeexpectedfroma%TMICdrivendrug,thisdosingschedule
95
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Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of Tuberculosis
Table2.Comparisonofpharmacokineticparameterandconcentrationestimatesandrangesin10,000simulatedpatientstothoseactuallyobservedinpatientstreatedwith1G.
SubroutinePRIORbasedonliterature(±SD)
10,000simulatedTBpatients
ObservedinMDR-TBpatients
TotalclearanceinL/hr 2.63±0.83 2.6(0.02-6.00) 2.1(0.09–3.23)VolumeinL 10.6±2.51 11(1.2-19) 7.3(2.61–11.10)Half-lifeinhours - 2.8(2.20-3.70) 2.4(2.05–3.53)AUC0-24inmg*h/L - 448(166-4255) 545(309–1130)
Figure3Ashowsthetargetattainmentprobability(TAP)foreachdoseanddosingschedule
astheMICchanges.Ononeextreme,the1gmonceadaydosehadaTAPlessthan90%once
theMICwas1mg/L,whilethedoseof3gmtwiceadayachievedahighTAPuntil8mg/L,then
fellprecipitouslyat16mg/L.For2gmaday,theTAPfellatanMICof2mg/L.Thismeansthat
thesusceptibilitybreakpointforertapenemplusclavulanatewillfallbetweenMICof1and
16mg/L,andwilldependonthefinaldosechosen.
SinceMICvariabilityisalsoanimportantdeterminantoftherapyresponseinTBpatients(25,
34-36),wealsotookintoaccounttheMICdistribution.Figure3BshowstheertapenemMIC
distribution from 33 MDR-TB patients isolates in the Netherlands, in the presence of
clavulanate.Figure3BshowsthatallisolateswouldhaveMICsbetweengreaterthan1mg/L
andbelow128mg/L.However,inthepastwehaveshownthatertapenemdegradesduring
theMICtesting,andifonceaccountsforthedegradation,thereisa4-tubedilutiondecrease
inMICs; ifclavulanate isaddedandertapenemissupplementedthere isa7-tubedilution
difference(12).Thus,wetransformedtheMICsforthe33clinical isolatesdownby4-tube
dilutionsaswell,asshowninFigure3B.Inthatscenario,only6.5%of isolateshadanMIC
greaterthan1mg/L.
SummationofallTAPstoaccountfordistributionofMICsgivestheproportionof10,000TB
patientswhowouldachieve theargett exposureof%TMICof 40%, termed the cumulative
fractionofresponse(CFR).Figure3CshowstheCFRsfortheonceadaydosingschedulefor
bothobservedMICsandtransformedMICs.ForthetransformedMICsthedoseof2gmaday
hadaCFRof96%.
Figure 3. Target attainment probability and cumulative fraction of response for variousertapenemdoses.
A.Targetattainmentprobabilityfor%TMICof40%asM.tuberculosisMICchanges.NodoseordosingscheduleiseffectiveonceMICsare16mg/L.B.ErtapenemMICdistributioninisolatesfrom the Netherlands, with and without transformation to account for ertapenemdegradation.C.Proportionof10,000patientswhoachievedorexceeded%TMICof40%withonceadaydosing.TheproportionishighlysensitivetotheMIC,andfellonsensitivityanalysis,aworst-casescenario.D.Proportionof10,000patientswhoachievedorexceeded%TMICof40%withtwiceadaydosing.Thetwiceadaydosingscheduleachievedthetargetinhigherproportionsofpatients,evenonsensitivityanalysis.However,giventhehardshipoftwiceaday administration of therapy in TB, we chose the 2gm once a day dose as being mostpractical. WealsoperformedertapenemplusclavalunateMICsin4clinical isolatesincubatedat4°C
versus37°Ctotryandslowdowndrugdegradation:MICswerelowerat4°Cby4,2,3,and
2-tube dilutions. Therefore, we performed sensitivity testing by examining CFR is MIC
transformationwasonly2-tubedilution(worstcasescenario).Figure3Cshowsthatthedose
of2gmonceadaywouldnotachievethetarget in90%ofpatients;nevertheless itwould
achievethisin63%ofpatients,whichisstillreasonable.Figure3Dshowstheresultsoftwice
aweekdosingschedule;aswouldbeexpectedfroma%TMICdrivendrug,thisdosingschedule
96
Chapter 6
performedbetter.Thedoseof1.0gmtwiceadaywouldachievetargetexposurein99%of
patients,andonsensitivitytestingwouldstillachievethis in70%ofpatients. Thedoseof
2gmtwiceadaywouldachieve>90%evenonsensitivitytesting.
DiscussionThisisthefirststudythatshowedtheertapenem-clavulanateefficacyandsterilizingeffect,
unlikefindingsinthemurinemodel,likelybecausetheHFS-TBmimickedthehalf-lifeof4hrs
encountered in patients unlike that of 1.0 hours in mice. We were able to recapitulate
ertapenem’spharmacokinetics,anditshalf-lifeof4hrs,asencounteredinTBpatients,which
likelyexplainsbetterefficacyinthismodelcomparedtothatencounteredinmiceinwhich
ertapenemhalf-lifeis1hour.Moreover,dosagessimulatedinthemodelwereinarangethat
would likelybetolerable inpatients.Thisstudyshowedtheadvantageofthehollowfiber
system, namely a better recapitulation of human like pharmacokinetics, and ofmicrobial
sterilizingeffectconditions.TheMonteCarlosimulationsthenintroducedthevariabilitythat
wouldbeencounteredforpharmacokineticparametersbetweenpatientsandMICsbetween
M.tuberculosisstrains.Ourtwo-stepexternalvalidationapproachinthesimulationsensured
thatoursimulationsreflectedclinicalreality;sensitivitytestingaccountedforanyuncertainty
inMICdistribution.Thisallowedustoperformdose-effectstudiesthattakeintoaccountthe
exposure-effectrelationshipasdescribedinthehollowfibermodel,theessentialaspectsof
drugbehaviourinpatientssuchaspharmacokineticvariabilityandthedrugpenetrationratios
to lungs that are important in determining efficacy, and susceptibility ofM. tuberculosis
isolatesencounteredinhospitals.Thisapproach,inmanyexperimentsbasedonthesameM.
tuberculosis isolateweused in thecurrent study,hasbeen found tobe>94%accurate in
identifying clinicaldoses thatareoptimal inTBpatientsbasedon recentpresentation for
regulatoryapproval(19).
Ertapenem–clavulanatemayplayanimportantroleintheintensivephaseofTBtreatment
duetoitssterilizingeffect.Inaddition,theintravenousadministrationmaybemoresuitable
fortheintensivephaseinwhichM/XDR-TBpatientarelikelytobeadministeredinaTBclinic.
AscarbapenemsarealreadypartoftheWHOlistofTBdrugsforM/XDR-TB,thenextstepis
toexploretheuseofertapenem–clavulanateinpatients,usingthe2gonceadaydosewe
identified.Recently,ithasbeenshownthatmeropenem–clavulanatehaspromisingactivity
againstMDR-TBinvitro(37,38).Indeed,imipenem-clavulanateandmeropenem-clavulanate
wereassociatedwithatreatmentsuccessof>57%andcultureconversion>60%,inarecent
systemic analysis of five studies (39). However, since clavulanate is administered as oral
amoxicillin-clavulanate, gastrointestinal side effects may become a problem if this
formulationisadministeredforaprolongeddurationmultipletimesadaywithmeropenem
orimipenem,whichwouldcompromiseabsorptionofotheroraldrugs.Unfortunately,the
currentsuppliersofcarbapenems,arenotinterestedindevelopinganinfusedcombination
of carbapenem and clavulanate. The main advantage of ertapenem is its long half-life
enablingoncedailydosing,whichwouldalsoallowaonceadayclavulanatedose,potentially
reducingside-effects.Thismayevenfacilitatedosinginanoutpatientsetting.Patientsmay
present at the clinic once a day for their drug administration as part of direct observed
treatment,orcouldreceivetreatmentasaonceadayinfusionathomewhensputumculture
negativeinthosecountrieswherethedrugisalreadypartofhomecarefortreatmentofother
chronic infections. Ertapenemhas a labelled infusion timeof only 30minutes,whichwill
facilitatearelativelyshortstayattheoutpatientclinic.Evenmorerapidinfusionhavebeen
exploredandshowedsimilardrugexposureandtolerability(40).Weshowthatadoseof2g
givenoncedailycouldcontributetoaneffectiveregimen.Ertapenemuptoadoseof3ghas
been administered to healthy volunteers (41). Moreover, doses up to 2 g have been
administratedin30minwithoutanyadditionalcomplications(42).However,thereisaneed
for a prospective phase II study exploring safety and efficacy of 2g ertapenem with
clavulanateonceadayinMDR-TBpatients.
Ontheotherhand,theamountoftimeclavulanatehastobearoundtokeeppotentiatingthe
ertapenem is still unclear. Thus, the target concentration to aim with dosing is unclear.
Clavulanate has a shorter half-life compared to ertapenem. However, penetration into
bronchialmucosais118%,anditsproteinbindingisminimalat20%,andlikelyaneffective
concentrationremainsatsiteofeffectevenwhendosedonce.Sinceclavulanate isrenally
eliminated,between-patientvariabilityinsystemicclearance,whichisabout58%,isdriven
mainly by renal function: the lower the creatinine clearance, the less it is cleared (43).
Separate dose-effect studies on clavulanate role will need to be studied, after which
simulationssimilartothecurrentonescanbeperformed.
97
6
Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of Tuberculosis
performedbetter.Thedoseof1.0gmtwiceadaywouldachievetargetexposurein99%of
patients,andonsensitivitytestingwouldstillachievethis in70%ofpatients. Thedoseof
2gmtwiceadaywouldachieve>90%evenonsensitivitytesting.
DiscussionThisisthefirststudythatshowedtheertapenem-clavulanateefficacyandsterilizingeffect,
unlikefindingsinthemurinemodel,likelybecausetheHFS-TBmimickedthehalf-lifeof4hrs
encountered in patients unlike that of 1.0 hours in mice. We were able to recapitulate
ertapenem’spharmacokinetics,anditshalf-lifeof4hrs,asencounteredinTBpatients,which
likelyexplainsbetterefficacyinthismodelcomparedtothatencounteredinmiceinwhich
ertapenemhalf-lifeis1hour.Moreover,dosagessimulatedinthemodelwereinarangethat
would likelybetolerable inpatients.Thisstudyshowedtheadvantageofthehollowfiber
system, namely a better recapitulation of human like pharmacokinetics, and ofmicrobial
sterilizingeffectconditions.TheMonteCarlosimulationsthenintroducedthevariabilitythat
wouldbeencounteredforpharmacokineticparametersbetweenpatientsandMICsbetween
M.tuberculosisstrains.Ourtwo-stepexternalvalidationapproachinthesimulationsensured
thatoursimulationsreflectedclinicalreality;sensitivitytestingaccountedforanyuncertainty
inMICdistribution.Thisallowedustoperformdose-effectstudiesthattakeintoaccountthe
exposure-effectrelationshipasdescribedinthehollowfibermodel,theessentialaspectsof
drugbehaviourinpatientssuchaspharmacokineticvariabilityandthedrugpenetrationratios
to lungs that are important in determining efficacy, and susceptibility ofM. tuberculosis
isolatesencounteredinhospitals.Thisapproach,inmanyexperimentsbasedonthesameM.
tuberculosis isolateweused in thecurrent study,hasbeen found tobe>94%accurate in
identifying clinicaldoses thatareoptimal inTBpatientsbasedon recentpresentation for
regulatoryapproval(19).
Ertapenem–clavulanatemayplayanimportantroleintheintensivephaseofTBtreatment
duetoitssterilizingeffect.Inaddition,theintravenousadministrationmaybemoresuitable
fortheintensivephaseinwhichM/XDR-TBpatientarelikelytobeadministeredinaTBclinic.
AscarbapenemsarealreadypartoftheWHOlistofTBdrugsforM/XDR-TB,thenextstepis
toexploretheuseofertapenem–clavulanateinpatients,usingthe2gonceadaydosewe
identified.Recently,ithasbeenshownthatmeropenem–clavulanatehaspromisingactivity
againstMDR-TBinvitro(37,38).Indeed,imipenem-clavulanateandmeropenem-clavulanate
wereassociatedwithatreatmentsuccessof>57%andcultureconversion>60%,inarecent
systemic analysis of five studies (39). However, since clavulanate is administered as oral
amoxicillin-clavulanate, gastrointestinal side effects may become a problem if this
formulationisadministeredforaprolongeddurationmultipletimesadaywithmeropenem
orimipenem,whichwouldcompromiseabsorptionofotheroraldrugs.Unfortunately,the
currentsuppliersofcarbapenems,arenotinterestedindevelopinganinfusedcombination
of carbapenem and clavulanate. The main advantage of ertapenem is its long half-life
enablingoncedailydosing,whichwouldalsoallowaonceadayclavulanatedose,potentially
reducingside-effects.Thismayevenfacilitatedosinginanoutpatientsetting.Patientsmay
present at the clinic once a day for their drug administration as part of direct observed
treatment,orcouldreceivetreatmentasaonceadayinfusionathomewhensputumculture
negativeinthosecountrieswherethedrugisalreadypartofhomecarefortreatmentofother
chronic infections. Ertapenemhas a labelled infusion timeof only 30minutes,whichwill
facilitatearelativelyshortstayattheoutpatientclinic.Evenmorerapidinfusionhavebeen
exploredandshowedsimilardrugexposureandtolerability(40).Weshowthatadoseof2g
givenoncedailycouldcontributetoaneffectiveregimen.Ertapenemuptoadoseof3ghas
been administered to healthy volunteers (41). Moreover, doses up to 2 g have been
administratedin30minwithoutanyadditionalcomplications(42).However,thereisaneed
for a prospective phase II study exploring safety and efficacy of 2g ertapenem with
clavulanateonceadayinMDR-TBpatients.
Ontheotherhand,theamountoftimeclavulanatehastobearoundtokeeppotentiatingthe
ertapenem is still unclear. Thus, the target concentration to aim with dosing is unclear.
Clavulanate has a shorter half-life compared to ertapenem. However, penetration into
bronchialmucosais118%,anditsproteinbindingisminimalat20%,andlikelyaneffective
concentrationremainsatsiteofeffectevenwhendosedonce.Sinceclavulanate isrenally
eliminated,between-patientvariabilityinsystemicclearance,whichisabout58%,isdriven
mainly by renal function: the lower the creatinine clearance, the less it is cleared (43).
Separate dose-effect studies on clavulanate role will need to be studied, after which
simulationssimilartothecurrentonescanbeperformed.
98
Chapter 6
Finally,pharmacokinetic/pharmacodynamics-basedsusceptibilitybreakpoints inTB,mostly
derived from hollow fiber model monotherapy studies, have been shown to be highly
accurateindelineatingTBpatientswhofailorrespondtocombinationtherapy(25,34,35).
The 2mg/L ertapenem susceptibility breakpointwe identified for the dose of 2gm a day
shouldbethususedbycliniciansasdecision-makingtooltodetermineifapatientwillrespond
toertapenem therapy. Thisbreakpointwill differ from theepidemiological cut-off value,
whichmaybemoreusefulforepidemiologicaltrackingofacquiredertapenemresistance,as
opposedtoclinicaldecision-making.
Therearesomelimitationstoourstudy.First,weusedtwoisolatesM.tuberculosisforthe
sterilizingeffectexperiments.Inclusionofalargernumberofisolatescouldchangethatfinal
targetexposureassociatedwithoptimalefficacy.However,hollowfiberstudiesinthepast
whenweusedthesestrainswerefoundtobepredictiveoftheoptimalexposuretargetsin
patients for sterilizing effect (15, 24, 25, 44-46). A second limitation is that we used
pharmacokineticdatafromcriticallyillpatientsaspriordataforourMonteCarlosimulations.
Type of disease that a patient has can alter the pharmacokinetic parameters, so that TB
patients could have different pharmacokinetics. However, as shown in Table 2, the
pharmacokineticparameterestimatesinsimulatedpatients,andtheAUC0-24achievedwith
1g doses, were virtually identical to those we have identified in TB patients in the
Netherlands,aspartoftherapeuticdrugmonitoring.Thisvalidatesthatsimulatedpatients
had pharmacokinetic parameters and concentrations similar to those encountered in TB
patients.
Inconclusion,wehaveshownbysimulationofhumandrugexposureofdifferentdosagein
aninvitroinfectionmodelofM.tuberculosisthatertapenem–clavulanatemaybeavaluable
assettoTBtreatment.Basedonavailablepharmacokineticdata,wehaveidentifiedthatthe
doseofertapenemmostsuitabletobetestedinaphaseIIstudyis2goncedaily.AnMIC2
mg/Lshouldbeusedtodefineresistancetothisdrug.
2
Materialsandmethods
We used M. tuberculosis H37Ra (ATCC #25177) and H37Rv (ATCC #27294) for our
experiments,withgrowthandstorageconditionsdescribedbefore(15).Theseisolateshave
beenusedintheHFS-TBbefore,withgoodforecastingaccuracyoftheclinic.Ertapenemwas
purchased fromMerck Sharp & Dohme. Clavulanate was purchased from Sigma-Aldrich.
Drugs were dissolved in sterile water, and syringe filtered for further use. Hollow fiber
cartridgeswerepurchasedfromFiberCell(Frederick,MD,USA).
ThehollowfibersystemmodelofTB
ConstructionoftheHFS-TBtomeasuresterilizingeffecthasbeendescribedindetailinthe
past (15). The system recapitulates concentration-time profiles of drugs encountered in
patients,takingintoaccountthepenetrationintolungs.Inthesterilizingeffectstudies,semi-
dormantM.tuberculosisgrowingintheMiddlebrook7H9brothacidifiedusingaceticacidto
a pH of 5.8 was used, which grow at a rate 8-10-fold slower than log-phase growthM.
tuberculosis(47).TheHFS-TBinthismodeluseacidifiedMiddlebrook7H9brothwithoutoleic
acid,albuminorcatalasebutwith20%dextrose.Theperipheralcompartmentofeachof16
HFS-TB units with circulating acidified Middlebrook 7H9 broth was inoculated with M.
tuberculosis.AllHFS-TBwereincubatedat37°Cunder5%CO2fortheentiretyofthestudy.
Differentertapenemexposures,basedonhumanequivalentdosesof0.25,0.5,1.0,2.0,3.0,
5.0, and 10.0 grams were administered into the central compartment via a computer-
controlledsyringepumpover30minutes,asinpatients.Theconcentrationsachievedwith
thedoseswereanAUC0-24of0,25,50,100,200,250,500,1000mg*h/L.Thereweretwo
replicateshollowfibersystemsforeachdoseorAUC0-24.Clavulanatewasalsodispensedvia
syringepumptoachieveapeakof3mg/Lattheendof30minutesinfusion.Mediainflow
andoutflowweresettomimictheertapenemhalf-lifeof4hoursencounteredinpatients;
wetookintoaccountthedegradationrateofthedrugthatwehaveidentifiedinthepast.We
recapitulated pharmacokinetics as described in the INVANZ® ertapenem for injection) for
intravenous(IV)orintramuscular(IM),packageinsert.
ThecentralcompartmentsofeachHFS-TBweresampledsixtimesduringthefirst24hrs,and
ertapenem concentrations measured using a liquid chromatography-tandem mass
spectrometry(LC-MS/MS)methodasdescribedinthepast(48)inordertoverifythathuman-
99
6
Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of Tuberculosis
Finally,pharmacokinetic/pharmacodynamics-basedsusceptibilitybreakpoints inTB,mostly
derived from hollow fiber model monotherapy studies, have been shown to be highly
accurateindelineatingTBpatientswhofailorrespondtocombinationtherapy(25,34,35).
The 2mg/L ertapenem susceptibility breakpointwe identified for the dose of 2gm a day
shouldbethususedbycliniciansasdecision-makingtooltodetermineifapatientwillrespond
toertapenem therapy. Thisbreakpointwill differ from theepidemiological cut-off value,
whichmaybemoreusefulforepidemiologicaltrackingofacquiredertapenemresistance,as
opposedtoclinicaldecision-making.
Therearesomelimitationstoourstudy.First,weusedtwoisolatesM.tuberculosisforthe
sterilizingeffectexperiments.Inclusionofalargernumberofisolatescouldchangethatfinal
targetexposureassociatedwithoptimalefficacy.However,hollowfiberstudiesinthepast
whenweusedthesestrainswerefoundtobepredictiveoftheoptimalexposuretargetsin
patients for sterilizing effect (15, 24, 25, 44-46). A second limitation is that we used
pharmacokineticdatafromcriticallyillpatientsaspriordataforourMonteCarlosimulations.
Type of disease that a patient has can alter the pharmacokinetic parameters, so that TB
patients could have different pharmacokinetics. However, as shown in Table 2, the
pharmacokineticparameterestimatesinsimulatedpatients,andtheAUC0-24achievedwith
1g doses, were virtually identical to those we have identified in TB patients in the
Netherlands,aspartoftherapeuticdrugmonitoring.Thisvalidatesthatsimulatedpatients
had pharmacokinetic parameters and concentrations similar to those encountered in TB
patients.
Inconclusion,wehaveshownbysimulationofhumandrugexposureofdifferentdosagein
aninvitroinfectionmodelofM.tuberculosisthatertapenem–clavulanatemaybeavaluable
assettoTBtreatment.Basedonavailablepharmacokineticdata,wehaveidentifiedthatthe
doseofertapenemmostsuitabletobetestedinaphaseIIstudyis2goncedaily.AnMIC2
mg/Lshouldbeusedtodefineresistancetothisdrug.
Materialsandmethods
We used M. tuberculosis H37Ra (ATCC #25177) and H37Rv (ATCC #27294) for our
experiments,withgrowthandstorageconditionsdescribedbefore(15).Theseisolateshave
beenusedintheHFS-TBbefore,withgoodforecastingaccuracyoftheclinic.Ertapenemwas
purchased fromMerck Sharp & Dohme. Clavulanate was purchased from Sigma-Aldrich.
Drugs were dissolved in sterile water, and syringe filtered for further use. Hollow fiber
cartridgeswerepurchasedfromFiberCell(Frederick,MD,USA).
ThehollowfibersystemmodelofTB
ConstructionoftheHFS-TBtomeasuresterilizingeffecthasbeendescribedindetailinthe
past (15). The system recapitulates concentration-time profiles of drugs encountered in
patients,takingintoaccountthepenetrationintolungs.Inthesterilizingeffectstudies,semi-
dormantM.tuberculosisgrowingintheMiddlebrook7H9brothacidifiedusingaceticacidto
a pH of 5.8 was used, which grow at a rate 8-10-fold slower than log-phase growthM.
tuberculosis(47).TheHFS-TBinthismodeluseacidifiedMiddlebrook7H9brothwithoutoleic
acid,albuminorcatalasebutwith20%dextrose.Theperipheralcompartmentofeachof16
HFS-TB units with circulating acidified Middlebrook 7H9 broth was inoculated with M.
tuberculosis.AllHFS-TBwereincubatedat37°Cunder5%CO2fortheentiretyofthestudy.
Differentertapenemexposures,basedonhumanequivalentdosesof0.25,0.5,1.0,2.0,3.0,
5.0, and 10.0 grams were administered into the central compartment via a computer-
controlledsyringepumpover30minutes,asinpatients.Theconcentrationsachievedwith
thedoseswereanAUC0-24of0,25,50,100,200,250,500,1000mg*h/L.Thereweretwo
replicateshollowfibersystemsforeachdoseorAUC0-24.Clavulanatewasalsodispensedvia
syringepumptoachieveapeakof3mg/Lattheendof30minutesinfusion.Mediainflow
andoutflowweresettomimictheertapenemhalf-lifeof4hoursencounteredinpatients;
wetookintoaccountthedegradationrateofthedrugthatwehaveidentifiedinthepast.We
recapitulated pharmacokinetics as described in the INVANZ® ertapenem for injection) for
intravenous(IV)orintramuscular(IM),packageinsert.
ThecentralcompartmentsofeachHFS-TBweresampledsixtimesduringthefirst24hrs,and
ertapenem concentrations measured using a liquid chromatography-tandem mass
spectrometry(LC-MS/MS)methodasdescribedinthepast(48)inordertoverifythathuman-
100
Chapter 6
likepharmacokineticshadbeenachieved.Ertapenemconcentrationsweremodelledusinga
onecompartmentpharmacokineticmodelwithfirstorderinputandelimination,usingADAPT
5software,asdescribedinthepast(15,24,25,44-46).Theseactualexposuresachievedin
theHFS-TBweresubsequentlyused in thePK/PDanalyses. Inorder toenumerate theM.
tuberculosisburdenasCFU/mL,theperipheralcompartmentofeachHFS-TBwassampledon
days0,3,7,14,21,and28.Sampleswerewashedandprocessedasdescribedinthepast(15)
andspreadonMiddlebrook7H10agarsupplementedwith10%Oleicacid-dextrose-catalase.
The cultures were incubated for 21 days at 370C with 5% CO2 before the colonies were
counted.
Identificationofoptimalertapenemdoseusingcomputer-aidedclinicaltrialsimulations.
Forthedomainofinput,weutilizedthepharmacokineticparameterestimatesandbetween-
patient variability indices identifiedbyBurkhardtetal (31).Weperformed simulations to
determinehowmuch1.0gonceaday,or1.0gtwiceaday,or2.0gonceaday,or2.0gtwicea
day,or3.0gonceadayor3.0gmtwiceadaywouldachieveorexceedthetargetexposure,
whichis%TMICassociatedwithoptimalsterilizingeffectinlungtissueofpatients.
Acknowledgements
None.
Funding
ThisworkwassupportedbytheNationalInstituteofGeneralMedicalSciencesoftheNational
InstitutesofHealthNewInnovatorAwardDP2OD001886-01toandtheNationalInstituteof
AllergyandInfectiousDiseasesawardR01AI079497toTawandaGumbo.
Transparencydeclaration
TGisaconsultantforLuminaCaresolutions,andfoundedJacarandaBiomed,Inc.JWAreports
personalfeesfromPfizer,Astellas,MSD,Gilead,grantsfromPfizer,Astellas,MSD,alloutside
thesubmittedwork.Allotherauthorshavenoconflictofinterest.
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HS,ChangKC,LangeC,NahidP,UdwadiaZF,HorsburghCR,Jr.,ChurchyardGJ,Menzies
D,HesselingAC,NuermbergerE,McIlleronH,FennellyKP,GoemaereE,JaramilloE,Low
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101
6
Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of Tuberculosis
likepharmacokineticshadbeenachieved.Ertapenemconcentrationsweremodelledusinga
onecompartmentpharmacokineticmodelwithfirstorderinputandelimination,usingADAPT
5software,asdescribedinthepast(15,24,25,44-46).Theseactualexposuresachievedin
theHFS-TBweresubsequentlyused in thePK/PDanalyses. Inorder toenumerate theM.
tuberculosisburdenasCFU/mL,theperipheralcompartmentofeachHFS-TBwassampledon
days0,3,7,14,21,and28.Sampleswerewashedandprocessedasdescribedinthepast(15)
andspreadonMiddlebrook7H10agarsupplementedwith10%Oleicacid-dextrose-catalase.
The cultures were incubated for 21 days at 370C with 5% CO2 before the colonies were
counted.
Identificationofoptimalertapenemdoseusingcomputer-aidedclinicaltrialsimulations.
Forthedomainofinput,weutilizedthepharmacokineticparameterestimatesandbetween-
patient variability indices identifiedbyBurkhardtetal (31).Weperformed simulations to
determinehowmuch1.0gonceaday,or1.0gtwiceaday,or2.0gonceaday,or2.0gtwicea
day,or3.0gonceadayor3.0gmtwiceadaywouldachieveorexceedthetargetexposure,
whichis%TMICassociatedwithoptimalsterilizingeffectinlungtissueofpatients.
Acknowledgements
None.
Funding
ThisworkwassupportedbytheNationalInstituteofGeneralMedicalSciencesoftheNational
InstitutesofHealthNewInnovatorAwardDP2OD001886-01toandtheNationalInstituteof
AllergyandInfectiousDiseasesawardR01AI079497toTawandaGumbo.
Transparencydeclaration
TGisaconsultantforLuminaCaresolutions,andfoundedJacarandaBiomed,Inc.JWAreports
personalfeesfromPfizer,Astellas,MSD,Gilead,grantsfromPfizer,Astellas,MSD,alloutside
thesubmittedwork.Allotherauthorshavenoconflictofinterest.
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Ertapenem Sterilizing Effect versus Mycobacterium tuberculosis. Antimicrob Agents
Chemother60:3193-3195.
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Kosterink JG, vanderWerf TS,Alffenaar JW. 2016. Pharmacokinetics of ertapenem in
patientswithmultidrug-resistanttuberculosis.EurRespirJ47:1229-1234.
14. TiberiS,D'AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,AlffenaarJW,Migliori
GB.2016.Ertapenem in the treatmentofmultidrug-resistant tuberculosis: first clinical
experience.EurRespirJ47:333-336.
15. Gumbo T, Dona CS, Meek C, Leff R. 2009. Pharmacokinetics-pharmacodynamics of
pyrazinamideinanovelinvitromodeloftuberculosisforsterilizingeffect:aparadigmfor
fasterassessmentofnewantituberculosisdrugs.AntimicrobAgentsChemother53:3197-
3204.
16. Heifets L, Lindholm-Levy P. 1992. Pyrazinamide sterilizing activity in vitro against
semidormant Mycobacterium tuberculosis bacterial populations. AmRevRespirDis
145:1223-1225.
17. MitchisonDA.1985.Theactionofantituberculosisdrugsinshort-coursechemotherapy.
Tubercle66:219-225.
18. Gumbo T, Pasipanodya JG, Nuermberger E, Romero K, Hanna D. 2015. Correlations
BetweentheHollowFiberModelofTuberculosisandTherapeuticEventsinTuberculosis
Patients:LearnandConfirm.ClinInfectDis61Suppl1:S18-24.
19. Gumbo T, Pasipanodya JG, Romero K, Hanna D, Nuermberger E. 2015. Forecasting
accuracyofthehollowfibermodeloftuberculosisforclinicaltherapeuticoutcomes.Clin
InfectDis61Suppl1:S25-31.
20. PasipanodyaJG,NuermbergerE,RomeroK,HannaD,GumboT.2015.SystematicAnalysis
ofHollowFiberModelofTuberculosisExperiments.ClinInfectDis61Suppl1:S10-17.
21. GumboT,LenaertsAJ,HannaD,RomeroK,NuermbergerE.2015.Nonclinicalmodelsfor
antituberculosisdrugdevelopment:alandscapeanalysis.JInfectDis211Suppl3:S83-95.
22. AkaikeH.1974.Anewlookatthestatisticalmodelidentification,vol19,p716-723.
23. Swaminathan S, Pasipanodya JG, Ramachandran G, Hemanth Kumar AK, Srivastava S,
DeshpandeD,NuermbergerE,GumboT.2016.DrugConcentrationThresholdsPredictive
ofTherapyFailureandDeathinChildrenWithTuberculosis:BreadCrumbTrailsinRandom
Forests.ClinInfectDis63:S63-S74.
103
6
Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of Tuberculosis
7. BolhuisMS,vanderLaanT,KosterinkJG,vanderWerfTS,vanSD,AlffenaarJW.2014.In
vitrosynergybetweenlinezolidandclarithromycinagainstMycobacteriumtuberculosis.
EurRespirJ44:808-811.
8. Pasipanodya J, Gumbo T. 2011. An oracle: antituberculosis pharmacokinetics-
pharmacodynamics,clinicalcorrelation,andclinicaltrialsimulationstopredictthefuture.
AntimicrobAgentsChemother55:24-34.
9. VezirisN,TruffotC,MainardiJL,JarlierV.2011.Activityofcarbapenemscombinedwith
clavulanate against murine tuberculosis. Antimicrobial Agents and Chemotherapy
55:2597-2600.
10. TiberiS,SotgiuG,D'AmbrosioL,CentisR,AbdoArbexM,AlarconArrascueE,Alffenaar
JW,CamineroJA,GagaM,GualanoG,SkrahinaA,SolovicI,SulisG,TadoliniM,Alarcon
GuizadoV,DeLorenzoS,RobyAriasAJ,ScardigliA,AkkermanOW,AleksaA,Artsukevich
J,AuchynkaV,BoniniEH,ChongMarinFA,CollahuazoLopezL,deVriesG,DoreS,Kunst
H,MatteelliA,MoschosC,PalmieriF,PapavasileiouA,PayenMC,PianaA,SpanevelloA,
Vargas Vasquez D, Viggiani P, White V, Zumla A, Migliori GB. 2016. Comparison of
effectiveness and safety of imipenem/clavulanate- versus meropenem/clavulanate-
containingregimensinthetreatmentofMDR-andXDR-TB.EurRespirJ47:1758-1766.
11. TiberiS,PayenMC,SotgiuG,D'AmbrosioL,AlarconGuizadoV,AlffenaarJW,AbdoArbex
M,CamineroJA,CentisR,DeLorenzoS,GagaM,GualanoG,RobyAriasAJ,ScardigliA,
SkrahinaA,Solovic I,SulisG,TadoliniM,AkkermanOW,AlarconArrascueE,AleskaA,
AvchinkoV,BoniniEH,ChongMarinFA,CollahuazoLopezL,deVriesG,DoreS,KunstH,
Matteelli A,Moschos C, Palmieri F, Papavasileiou A, Spanevello A, Vargas Vasquez D,
Viggiani P, White V, Zumla A, Migliori GB. 2016. Effectiveness and safety of
meropenem/clavulanate-containingregimensinthetreatmentofMDR-andXDR-TB.Eur
RespirJ47:1235-1243.
12. SrivastavaS,vanRijnSP,WesselsAM,AlffenaarJW,GumboT.2016.SusceptibilityTesting
ofAntibioticsThatDegradeFasterthantheDoublingTimeofSlow-GrowingMycobacteria:
Ertapenem Sterilizing Effect versus Mycobacterium tuberculosis. Antimicrob Agents
Chemother60:3193-3195.
13. vanRijnSP,vanAltenaR,AkkermanOW,vanSoolingenD,vanderLaanT,deLangeWC,
Kosterink JG, vanderWerf TS,Alffenaar JW. 2016. Pharmacokinetics of ertapenem in
patientswithmultidrug-resistanttuberculosis.EurRespirJ47:1229-1234.
14. TiberiS,D'AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,AlffenaarJW,Migliori
GB.2016.Ertapenem in the treatmentofmultidrug-resistant tuberculosis: first clinical
experience.EurRespirJ47:333-336.
15. Gumbo T, Dona CS, Meek C, Leff R. 2009. Pharmacokinetics-pharmacodynamics of
pyrazinamideinanovelinvitromodeloftuberculosisforsterilizingeffect:aparadigmfor
fasterassessmentofnewantituberculosisdrugs.AntimicrobAgentsChemother53:3197-
3204.
16. Heifets L, Lindholm-Levy P. 1992. Pyrazinamide sterilizing activity in vitro against
semidormant Mycobacterium tuberculosis bacterial populations. AmRevRespirDis
145:1223-1225.
17. MitchisonDA.1985.Theactionofantituberculosisdrugsinshort-coursechemotherapy.
Tubercle66:219-225.
18. Gumbo T, Pasipanodya JG, Nuermberger E, Romero K, Hanna D. 2015. Correlations
BetweentheHollowFiberModelofTuberculosisandTherapeuticEventsinTuberculosis
Patients:LearnandConfirm.ClinInfectDis61Suppl1:S18-24.
19. Gumbo T, Pasipanodya JG, Romero K, Hanna D, Nuermberger E. 2015. Forecasting
accuracyofthehollowfibermodeloftuberculosisforclinicaltherapeuticoutcomes.Clin
InfectDis61Suppl1:S25-31.
20. PasipanodyaJG,NuermbergerE,RomeroK,HannaD,GumboT.2015.SystematicAnalysis
ofHollowFiberModelofTuberculosisExperiments.ClinInfectDis61Suppl1:S10-17.
21. GumboT,LenaertsAJ,HannaD,RomeroK,NuermbergerE.2015.Nonclinicalmodelsfor
antituberculosisdrugdevelopment:alandscapeanalysis.JInfectDis211Suppl3:S83-95.
22. AkaikeH.1974.Anewlookatthestatisticalmodelidentification,vol19,p716-723.
23. Swaminathan S, Pasipanodya JG, Ramachandran G, Hemanth Kumar AK, Srivastava S,
DeshpandeD,NuermbergerE,GumboT.2016.DrugConcentrationThresholdsPredictive
ofTherapyFailureandDeathinChildrenWithTuberculosis:BreadCrumbTrailsinRandom
Forests.ClinInfectDis63:S63-S74.
104
Chapter 6
24. PasipanodyaJG,McIlleronH,BurgerA,WashPA,SmithP,GumboT.2013.Serumdrug
concentrations predictive of pulmonary tuberculosis outcomes. J Infect Dis 208:1464-
1473.
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McIlleronH. 2015. Impact of nonlinear interactions of pharmacokinetics andMICs on
sputum bacillary kill rates as amarker of sterilizing effect in tuberculosis. Antimicrob
AgentsChemother59:38-45.
26. ModongoC,PasipanodyaJG,MagaziBT,SrivastavaS,ZetolaNM,WilliamsSM,SirugoG,
GumboT.2016.Artificial intelligenceandamikacinexposurespredictiveofoutcomein
multidrug-resistant tuberculosis patients. Antimicrob Agents Chemother
doi:10.1128/AAC.00962-16.
27. PasipanodyaJG,SrivastavaS,GumboT.2012.Meta-analysisofclinicalstudiessupports
the pharmacokinetic variability hypothesis for acquired drug resistance and failure of
antituberculosistherapy.ClinInfectDis55:169-177.
28. SrivastavaS,PasipanodyaJG,RamachandranG,DeshpandeD,ShufordS,CrosswellHE,
CirrincioneKN,ShermanCM,SwaminathanS,GumboT.2016.ALong-termCo-perfused
Disseminated Tuberculosis-3D Liver Hollow Fiber Model for Both Drug Efficacy and
HepatotoxicityinBabies.EBioMedicine6:126-138.
29. Srivastava S, Pasipanodya JG, Meek C, Leff R, Gumbo T. 2011. Multidrug-resistant
tuberculosis not due to noncompliance but to between-patient pharmacokinetic
variability.JInfectDis204:1951-1959.
30. BurkhardtO,KumarV,KatterweD,Majcher-PeszynskaJ,DrewelowB,DerendorfH,Welte
T. 2007. Ertapenem in critically ill patients with early-onset ventilator-associated
pneumonia: pharmacokineticswith special consideration of free-drug concentration. J
AntimicrobChemother59:277-284.
31. GumboT,Angulo-BarturenI,Ferrer-BazagaS.2015.Pharmacokinetic-pharmacodynamic
and dose-response relationships of antituberculosis drugs: recommendations and
standardsforindustryandacademia.JInfectDis211Suppl3:S96-S106.
32. RomeroK,SinhaV,AllerheiligenS,DanhofM,PinheiroJ,KruhlakN,WangY,WangSJ,
SauerJM,MarierJF,CorriganB,RogersJ,LambersHeerspinkHJ,GumboT,VisP,Watkins
P,MorrisonT,GillespieW,GordonMF,StephensonD,HannaD,PfisterM,LalondeR,
Colatsky T. 2014. Modeling and simulation for medical product development and
evaluation: highlights from the FDA-C-Path-ISOP 2013 workshop. J Pharmacokinet
Pharmacodyn41:545-552.
33. BurkhardtO,Majcher-PeszynskaJ,BornerK,MundkowskiR,DrewelowB,DerendorfH,
Welte T. 2005. Penetration of ertapenem into different pulmonary compartments of
patientsundergoinglungsurgery.JClinPharmacol45:659-665.
34. GumboT,ChigutsaE,PasipanodyaJ,VisserM,vanHeldenPD,SirgelFA,McIlleronH.2014.
The pyrazinamide susceptibility breakpoint above which combination therapy fails. J
AntimicrobChemother69:2420-2425.
35. GumboT,PasipanodyaJG,WashP,BurgerA,McIlleronH.2014.Redefiningmultidrug-
resistant tuberculosis based on clinical response to combination therapy. Antimicrob
AgentsChemother58:6111-6115.
36. Zheng X, Zheng R, Hu Y, Werngren J, Davies FL, Mansjo M, Xu B, Hoffner S. 2016.
DeterminationofMinimumInhibitoryConcentration (MIC)Breakpoints forsecond-line
drugsassociatedwithclinicaloutcomesinmultidrug-resistanttuberculosistreatmentin
China.AntimicrobialAgentsandChemotherapy.
37. DaviesForsmanL,GiskeCG,BruchfeldJ,SchonT,JureenP,AngebyK.2015.Meropenem-
clavulanate has high in vitro activity against multidrug-resistant Mycobacterium
tuberculosis.IntJMycobacteriol4Suppl1:80-81.
38. GonzaloX,Drobniewski F. 2013. Is there aplace for beta-lactams in the treatmentof
multidrug-resistant/extensively drug-resistant tuberculosis? Synergy between
meropenemandamoxicillin/clavulanate.JAntimicrobChemother68:366-369.
39. SotgiuG,D'AmbrosioL,CentisR,TiberiS,EspositoS,DoreS,SpanevelloA,MiglioriGB.
2016.Carbapenems to TreatMultidrug andExtensivelyDrug-Resistant Tuberculosis:A
SystematicReview.IntJMolSci17:373.
40. Wiskirchen DE, Housman ST, Quintiliani R, Nicolau DP, Kuti JL. 2013. Comparative
pharmacokinetics, pharmacodynamics, and tolerability of ertapenem 1 gram/day
administered as a rapid 5-minute infusion versus the standard 30-minute infusion in
healthyadultvolunteers.Pharmacotherapy33:266-274.
41. MajumdarAK,MussonDG,BirkKL,KitchenCJ,HollandS,McCreaJ,MistryG,HesneyM,
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2002.Pharmacokineticsofertapenem inhealthyyoungvolunteers.AntimicrobAgents
Chemother46:3506-3511.
105
6
Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of Tuberculosis
24. PasipanodyaJG,McIlleronH,BurgerA,WashPA,SmithP,GumboT.2013.Serumdrug
concentrations predictive of pulmonary tuberculosis outcomes. J Infect Dis 208:1464-
1473.
25. Chigutsa E, Pasipanodya JG, VisserME, vanHelden PD, Smith PJ, Sirgel FA,Gumbo T,
McIlleronH. 2015. Impact of nonlinear interactions of pharmacokinetics andMICs on
sputum bacillary kill rates as amarker of sterilizing effect in tuberculosis. Antimicrob
AgentsChemother59:38-45.
26. ModongoC,PasipanodyaJG,MagaziBT,SrivastavaS,ZetolaNM,WilliamsSM,SirugoG,
GumboT.2016.Artificial intelligenceandamikacinexposurespredictiveofoutcomein
multidrug-resistant tuberculosis patients. Antimicrob Agents Chemother
doi:10.1128/AAC.00962-16.
27. PasipanodyaJG,SrivastavaS,GumboT.2012.Meta-analysisofclinicalstudiessupports
the pharmacokinetic variability hypothesis for acquired drug resistance and failure of
antituberculosistherapy.ClinInfectDis55:169-177.
28. SrivastavaS,PasipanodyaJG,RamachandranG,DeshpandeD,ShufordS,CrosswellHE,
CirrincioneKN,ShermanCM,SwaminathanS,GumboT.2016.ALong-termCo-perfused
Disseminated Tuberculosis-3D Liver Hollow Fiber Model for Both Drug Efficacy and
HepatotoxicityinBabies.EBioMedicine6:126-138.
29. Srivastava S, Pasipanodya JG, Meek C, Leff R, Gumbo T. 2011. Multidrug-resistant
tuberculosis not due to noncompliance but to between-patient pharmacokinetic
variability.JInfectDis204:1951-1959.
30. BurkhardtO,KumarV,KatterweD,Majcher-PeszynskaJ,DrewelowB,DerendorfH,Welte
T. 2007. Ertapenem in critically ill patients with early-onset ventilator-associated
pneumonia: pharmacokineticswith special consideration of free-drug concentration. J
AntimicrobChemother59:277-284.
31. GumboT,Angulo-BarturenI,Ferrer-BazagaS.2015.Pharmacokinetic-pharmacodynamic
and dose-response relationships of antituberculosis drugs: recommendations and
standardsforindustryandacademia.JInfectDis211Suppl3:S96-S106.
32. RomeroK,SinhaV,AllerheiligenS,DanhofM,PinheiroJ,KruhlakN,WangY,WangSJ,
SauerJM,MarierJF,CorriganB,RogersJ,LambersHeerspinkHJ,GumboT,VisP,Watkins
P,MorrisonT,GillespieW,GordonMF,StephensonD,HannaD,PfisterM,LalondeR,
Colatsky T. 2014. Modeling and simulation for medical product development and
evaluation: highlights from the FDA-C-Path-ISOP 2013 workshop. J Pharmacokinet
Pharmacodyn41:545-552.
33. BurkhardtO,Majcher-PeszynskaJ,BornerK,MundkowskiR,DrewelowB,DerendorfH,
Welte T. 2005. Penetration of ertapenem into different pulmonary compartments of
patientsundergoinglungsurgery.JClinPharmacol45:659-665.
34. GumboT,ChigutsaE,PasipanodyaJ,VisserM,vanHeldenPD,SirgelFA,McIlleronH.2014.
The pyrazinamide susceptibility breakpoint above which combination therapy fails. J
AntimicrobChemother69:2420-2425.
35. GumboT,PasipanodyaJG,WashP,BurgerA,McIlleronH.2014.Redefiningmultidrug-
resistant tuberculosis based on clinical response to combination therapy. Antimicrob
AgentsChemother58:6111-6115.
36. Zheng X, Zheng R, Hu Y, Werngren J, Davies FL, Mansjo M, Xu B, Hoffner S. 2016.
DeterminationofMinimumInhibitoryConcentration (MIC)Breakpoints forsecond-line
drugsassociatedwithclinicaloutcomesinmultidrug-resistanttuberculosistreatmentin
China.AntimicrobialAgentsandChemotherapy.
37. DaviesForsmanL,GiskeCG,BruchfeldJ,SchonT,JureenP,AngebyK.2015.Meropenem-
clavulanate has high in vitro activity against multidrug-resistant Mycobacterium
tuberculosis.IntJMycobacteriol4Suppl1:80-81.
38. GonzaloX,Drobniewski F. 2013. Is there aplace for beta-lactams in the treatmentof
multidrug-resistant/extensively drug-resistant tuberculosis? Synergy between
meropenemandamoxicillin/clavulanate.JAntimicrobChemother68:366-369.
39. SotgiuG,D'AmbrosioL,CentisR,TiberiS,EspositoS,DoreS,SpanevelloA,MiglioriGB.
2016.Carbapenems to TreatMultidrug andExtensivelyDrug-Resistant Tuberculosis:A
SystematicReview.IntJMolSci17:373.
40. Wiskirchen DE, Housman ST, Quintiliani R, Nicolau DP, Kuti JL. 2013. Comparative
pharmacokinetics, pharmacodynamics, and tolerability of ertapenem 1 gram/day
administered as a rapid 5-minute infusion versus the standard 30-minute infusion in
healthyadultvolunteers.Pharmacotherapy33:266-274.
41. MajumdarAK,MussonDG,BirkKL,KitchenCJ,HollandS,McCreaJ,MistryG,HesneyM,
XiL,LiSX,HaesenR,BlumRA,LinsRL,GreenbergH,WaldmanS,DeutschP,RogersJD.
2002.Pharmacokineticsofertapenem inhealthyyoungvolunteers.AntimicrobAgents
Chemother46:3506-3511.
106
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validation of ertapenem using a liquid chromatography-tandem mass spectrometry
method.AntimicrobialAgentsandChemotherapy58:3481-3484.
42. TepplerH,GesserRM,FriedlandIR,WoodsGL,MeibohmA,HermanG,MistryG,Isaacs
R.2004.Safetyandtolerabilityofertapenem.JAntimicrobChemother53Suppl2:ii75-81.
43. CarlierM, NoeM, DeWaele JJ, Stove V, Verstraete AG, Lipman J, Roberts JA. 2013.
Population pharmacokinetics and dosing simulations of amoxicillin/clavulanic acid in
criticallyillpatients.JAntimicrobChemother68:2600-2608.
44. Gumbo T, Louie A, Deziel MR, Liu W, Parsons LM, Salfinger M, Drusano GL. 2007.
Concentration-dependent Mycobacterium tuberculosis killing and prevention of
resistancebyrifampin.AntimicrobAgentsChemother51:3781-3788.
45. Gumbo T, Louie A, Liu W, Brown D, Ambrose PG, Bhavnani SM, Drusano GL. 2007.
Isoniazidbactericidalactivityandresistanceemergence:integratingpharmacodynamics
and pharmacogenomics to predict efficacy in different ethnic populations. Antimicrob
AgentsChemother51:2329-2336.
46. SrivastavaS,MusukaS,ShermanC,MeekC,LeffR,GumboT.2010.Efflux-pump-derived
multipledrugresistancetoethambutolmonotherapyinMycobacteriumtuberculosisand
thepharmacokineticsandpharmacodynamicsofethambutol.JInfectDis201:1225-1231.
47. Musuka S, Srivastava S, Siyambalapitiyage Dona CW, Meek C, Leff R, Pasipanodya J,
GumboT.2013.Thioridazinepharmacokinetic-pharmacodynamicparameters"Wobble"
during treatment of tuberculosis: a theoretical basis for shorter-duration curative
monotherapywithcongeners.AntimicrobAgentsChemother57:5870-5877.
48. vanRijnSP,WesselsAM,GreijdanusB,TouwDJ,AlffenaarJW.2014.Quantificationand
validation of ertapenem using a liquid chromatography-tandem mass spectrometry
method.AntimicrobialAgentsandChemotherapy58:3481-3484.
CHAPTER 7
Antimicrob Agents Chemother. 2017 Mar 24; 61(4).PMID: 28137814
*Both authors contributed equally
S.P. van Rijn*M.A. Zuur*
R. van AltenaO.W. Akkerman
J.H. ProostW.C.M de Lange
H.A.M. KerstjensD.J. Touw
T.S. van der WerfJ.G.W. KosterinkJ.W.C. Alffenaar
Pharmacokinetic Modelling and Limited Sampling Strategies Based onHealthy Volunteers for Monitoring of
Ertapenem in Patients with Multidrug-resistant Tuberculosis
CHAPTER 7
Antimicrob Agents Chemother. 2017 Mar 24; 61(4).PMID: 28137814
*Both authors contributed equally
S.P. van Rijn*M.A. Zuur*
R. van AltenaO.W. Akkerman
J.H. ProostW.C.M de Lange
H.A.M. KerstjensD.J. Touw
T.S. van der WerfJ.G.W. KosterinkJ.W.C. Alffenaar
Pharmacokinetic Modelling and Limited Sampling Strategies Based onHealthy Volunteers for Monitoring of
Ertapenem in Patients with Multidrug-resistant Tuberculosis
110
Chapter 7
AbstractErtapenem is a broad spectrum carbapenem antibiotic and is being explored against
Mycobacterium tuberculosis. Carbapenems have anti-bacterial activity when the plasma
concentration exceeds the minimal inhibitory concentration at least 40% of the time
(40%T>MIC).Toassess40%T>MIC inmultidrug-resistant tuberculosis (MDR-TB)patients,a
limitedsamplingstrategywasdevelopedusingapopulationpharmacokineticmodelbased
on healthy volunteers. A two-compartment population pharmacokinetic model was
developedfromdatainforty-twohealthyvolunteers,usinganiterativetwo-stageBayesian
method.ExternalvalidationwasperformedbyBayesian fittingof themodeldeveloped in
volunteers to the individual data of MDR-TB patients (AUC0-24h,fit) using the developed
populationmodelforvolunteersasaprior.AMonteCarlosimulation(n=1000)wasusedto
evaluatelimitedsamplingstrategies.Additionally,thefreeertapenemfraction(f)40%T>MIC
for MDR-TB patients was estimated with the population pharmacokinetic model. The
developedpopulationpharmacokineticmodelwasshowntoestimatetheAUC0-24hinMDR-
TBpatientswithanoverestimationof6.8(range:-17.2–30.7)%.Thebestperforminglimited
samplingstrategy,withatime-restrictionof0-6h,wasfoundtobesamplingat1and5h(R2=
0.78,meanpredictionerror=-0.33%andarootmeansquareerror=5.5%).Drugexposure
wasoverestimatedbyameanpercentageof4.2(-15.2–23.6)%.Consideringafreefraction
of5%andtheMICsetat0.5mg/L,9outof12patientswouldhaveexceededaminimumoff
40%T>MIC.Apopulationpharmacokineticmodelandlimitedsamplingstrategy,developed
usingdatafromhealthyvolunteers,showedtobeadequatetopredictertapenemexposure
inMDR-TBpatients.
IntroductionErtapenem isabroad spectrumcarbapenemantibiotic,usedagainsta rangeof infectious
diseases(1).Likeforallotherbeta-lactamantimicrobialproducts,theefficacyofertapenem
ischaracterizedbytime-dependentkilling.Carbapenemshaveanti-bacterialactivitywhenthe
plasmaconcentrationexceedstheminimalinhibitoryconcentrationatleast40%ofthetime
(40%T>MIC)(1,2).Althoughnotyetstudiedintuberculosis(TB)patients,free40%T>MICis
expectedtobeanimportantpharmacodynamicparameter(3).Carbapenemsincombination
withclavulanicacidhascreatedinterestsinceactivitywasshowninamurinemodelofTB(3).
Additionally,arecentstudyshowedthatcarbapenemsefficientlyinactivatedpeptidoglycan
cross-linkinginM.tuberculosis(3,4)andarecentmeropenemamoxicillin/clavulanicacidEBA
studyshowedactivityofcarbapenemsinTB(5).Recentlyanewsusceptibilitytestingmethod
toestimatetheMICofertapenemhasbeenintroduced(6)showingthatertapenemmightbe
morepotent in vitro thanpreviously thoughtbecause its chemicaldegradationhadnever
beenconsidered(7).Todateonlyalimitednumberofmultidrug-resistanttuberculosis(MDR-
TB)patientshavebeentreatedwithertapenemaspartofamultidrugregimen.Basedonthis
data, the drug appeared well tolerated during prolonged treatment (8,9). However,
ertapenemisnotyetaddedtotheWorldHealthOrganization(WHO)listofanti-TBdrugs,in
contrasttoimipenemandmeropenem.
Pharmacokineticsofertapenemhavetypicallybeenstudiedinhealthyvolunteers(10),people
withobesity(11,12),patientswithrenalfailure(13-15)andcriticallyillpatientswithvarious
pathologies (16-18). Lowerdrug exposurewasobserved in obese individuals (12), and an
increaseindoseintervalwasneededinpatientswithrenalinsufficiencywithanestimated
glomerularfiltrationrate(eGFR)below30mL/min/1,73m2(14),suggestingthattheoptimal
dose of ertapenem is different for various health conditions. A recent study on
pharmacokineticsofMDR-TBpatientssuggestedthattherewassubstantialpharmacokinetic
variabilityinthesepatients(8).
ForstudiesexploringtheuseofertapenemagainstM.tuberculosis itwouldbevaluableto
assessf40%T>MICinpatients.Tobeabletocalculatethef40%T>MIC,agoodindicationof
the plasma concentration profile is mandatory. However, measurement of the plasma
concentrationovertheentire24hdosingintervalistimeconsuming,expensiveandburdening
111
7
Pharmacokinetic Modelling and Limited Sampling Strategies of Ertapenem
AbstractErtapenem is a broad spectrum carbapenem antibiotic and is being explored against
Mycobacterium tuberculosis. Carbapenems have anti-bacterial activity when the plasma
concentration exceeds the minimal inhibitory concentration at least 40% of the time
(40%T>MIC).Toassess40%T>MIC inmultidrug-resistant tuberculosis (MDR-TB)patients,a
limitedsamplingstrategywasdevelopedusingapopulationpharmacokineticmodelbased
on healthy volunteers. A two-compartment population pharmacokinetic model was
developedfromdatainforty-twohealthyvolunteers,usinganiterativetwo-stageBayesian
method.ExternalvalidationwasperformedbyBayesian fittingof themodeldeveloped in
volunteers to the individual data of MDR-TB patients (AUC0-24h,fit) using the developed
populationmodelforvolunteersasaprior.AMonteCarlosimulation(n=1000)wasusedto
evaluatelimitedsamplingstrategies.Additionally,thefreeertapenemfraction(f)40%T>MIC
for MDR-TB patients was estimated with the population pharmacokinetic model. The
developedpopulationpharmacokineticmodelwasshowntoestimatetheAUC0-24hinMDR-
TBpatientswithanoverestimationof6.8(range:-17.2–30.7)%.Thebestperforminglimited
samplingstrategy,withatime-restrictionof0-6h,wasfoundtobesamplingat1and5h(R2=
0.78,meanpredictionerror=-0.33%andarootmeansquareerror=5.5%).Drugexposure
wasoverestimatedbyameanpercentageof4.2(-15.2–23.6)%.Consideringafreefraction
of5%andtheMICsetat0.5mg/L,9outof12patientswouldhaveexceededaminimumoff
40%T>MIC.Apopulationpharmacokineticmodelandlimitedsamplingstrategy,developed
usingdatafromhealthyvolunteers,showedtobeadequatetopredictertapenemexposure
inMDR-TBpatients.
IntroductionErtapenem isabroad spectrumcarbapenemantibiotic,usedagainsta rangeof infectious
diseases(1).Likeforallotherbeta-lactamantimicrobialproducts,theefficacyofertapenem
ischaracterizedbytime-dependentkilling.Carbapenemshaveanti-bacterialactivitywhenthe
plasmaconcentrationexceedstheminimalinhibitoryconcentrationatleast40%ofthetime
(40%T>MIC)(1,2).Althoughnotyetstudiedintuberculosis(TB)patients,free40%T>MICis
expectedtobeanimportantpharmacodynamicparameter(3).Carbapenemsincombination
withclavulanicacidhascreatedinterestsinceactivitywasshowninamurinemodelofTB(3).
Additionally,arecentstudyshowedthatcarbapenemsefficientlyinactivatedpeptidoglycan
cross-linkinginM.tuberculosis(3,4)andarecentmeropenemamoxicillin/clavulanicacidEBA
studyshowedactivityofcarbapenemsinTB(5).Recentlyanewsusceptibilitytestingmethod
toestimatetheMICofertapenemhasbeenintroduced(6)showingthatertapenemmightbe
morepotent in vitro thanpreviously thoughtbecause its chemicaldegradationhadnever
beenconsidered(7).Todateonlyalimitednumberofmultidrug-resistanttuberculosis(MDR-
TB)patientshavebeentreatedwithertapenemaspartofamultidrugregimen.Basedonthis
data, the drug appeared well tolerated during prolonged treatment (8,9). However,
ertapenemisnotyetaddedtotheWorldHealthOrganization(WHO)listofanti-TBdrugs,in
contrasttoimipenemandmeropenem.
Pharmacokineticsofertapenemhavetypicallybeenstudiedinhealthyvolunteers(10),people
withobesity(11,12),patientswithrenalfailure(13-15)andcriticallyillpatientswithvarious
pathologies (16-18). Lowerdrug exposurewasobserved in obese individuals (12), and an
increaseindoseintervalwasneededinpatientswithrenalinsufficiencywithanestimated
glomerularfiltrationrate(eGFR)below30mL/min/1,73m2(14),suggestingthattheoptimal
dose of ertapenem is different for various health conditions. A recent study on
pharmacokineticsofMDR-TBpatientssuggestedthattherewassubstantialpharmacokinetic
variabilityinthesepatients(8).
ForstudiesexploringtheuseofertapenemagainstM.tuberculosis itwouldbevaluableto
assessf40%T>MICinpatients.Tobeabletocalculatethef40%T>MIC,agoodindicationof
the plasma concentration profile is mandatory. However, measurement of the plasma
concentrationovertheentire24hdosingintervalistimeconsuming,expensiveandburdening
112
Chapter 7
topatients.Alimitedsamplingstrategythroughapopulationpharmacokineticmodelcanbe
usedtopredictthisplasmaconcentrationprofileashasbeendoneforotheranti-TBdrugs
(19-22).
The aim of this studywas to develop a population pharmacokineticmodel and a limited
samplingstrategybasedondatafromhealthyvolunteers,inordertoestimatedrugexposure
ofertapeneminMDR-TBpatients.
Materialsandmethods
This studywas based on two data sets. The first setwas comprised of forty-two healthy
volunteers fromfiveclinicalstudiesreceiving0.25to2g i.v.dosesofertapenem(10).For
populationpharmacokineticmodelcomparisonwithMDR-TBpatientsweonlyusedthedata
ofhealthyvolunteersreceiving1g.Thesecondsetwascomprisedofaretrospectivedataset
ofpatientswithMDR-TB,receiving1gertapenemadministeredoncedailyviaa30-minute
infusionattheTuberculosisCenterBeatrixoord,UniversityMedicalCenterGroningen,The
NetherlandsbetweenDecember1,2010andMarch1,2013(8).Plasmaconcentrationsof
ertapenemwerecollectedatsteadystatebeforeadministrationandat1,2,3,4,5,6,8and
12 hours after administration. Ertapenem plasma concentrations were analyzed by a
validatedliquidchromatography-tandemmass-spectrometry(LC-MS/MS)method(23).Both
datasetsincludeddemographicandmedicaldata,suchasageatstartoftreatment,height
andbodyweightandserumcreatinineatthetimeofpharmacokineticassessment.
Populationpharmacokineticmodel
All pharmacokinetic calculations were performed using MW\Pharm 3.82 (Mediware,
Zuidhorn,TheNetherlands).Basedonpreviousreportsandrecentpharmacokineticstudies
on ertapenem (11-18, 24-26), concentration time-curves were evaluated in a one-
compartmentalandtwo-compartmentalmodel.Thefinalmodelwasselectedbasedonthe
Akaike information criterion (AIC) (27). The drug plasma-concentrations of the forty-two
healthyvolunteerswereusedtodevelopatwo-compartmentalpopulationpharmacokinetic
modelusinganiterativetwo-stageBayesian(ITSB)procedure(moduleKinPopofMW\Pharm)
(28).TheclearancewascalculatedusingCL=CLm(metabolicclearance(L/h/1.85m2))*BSA
(body surface area (m2)) /1.85 + fr (drug clearance – creatinine clearance ratio) * CLcr
(creatinine clearance (L/h)) (24). Pharmacokinetic parameters were assumed to be log-
normallydistributedandtheresidualerrorwasassumedtobenormallydistributedwithSD
=0.1+0.1*C,inwhichCistheobservedplasmaconcentrationofertapenem.Nonparametric
95%confidence intervalsof thepopulationparametersand their inter-individual standard
deviationwereestimatedusingabootstrapanalysis(n=1000).Theareaundertheplasma
concentration-timecurvefrom0upto24hours(AUC0-24h,ref),usedasareferencevalue,was
calculatedusingthelogtrapezoidalrule(KinFitmoduleofMW\Pharm).
Internal validation was performed by leaving three healthy volunteers out of the
pharmacokinetic model development, creating fourteen n-3 sub models, obtained by
randomizationusingMicrosoftExcel2010.TheestimatedAUC0-24h,(AUC0-24h,n-3)wasobtained
byBayesianfittingusingthedataofthethreeleftoutvolunteersinthecorrespondingn-3
submodels.AgreementbetweenAUC0-24h,n-3andAUC0-24h,refwasassessedbyBland-Altman
analysisandPassingandBablokregressionandsubsequentresidualplot.Externalvalidation
wasperformedbyBayesianfittingofthemodeldevelopedinvolunteerstotheindividualdata
ofMDR-TBpatients(AUC0-24h,fit),usingthedevelopedpopulationmodelforvolunteersasa
prior.ForcomparisonofthepharmacokineticsbetweenMDR-TBpatientsandvolunteers,a
similaranalysiswasperformedwiththedataof18volunteerswhoreceived1gofertapenem.
Bland-Altmananalysiswasalsoused toassess theagreementbetweenAUC0-24h,fitand the
AUC0-24h,refofMDR-TBpatients.
Limitedsamplingstrategies
AMonteCarlosimulationwasusedtocalculatelimitedsamplingstrategiesforestimationof
AUC (AUC0-24h,LSS), as implemented inMW\Pharm. This stochastic simulation consisted of
1000randompatientsdrawnfromthepopulationpharmacokineticmodel.Foreachpatient
limitedsamplingstrategieswerecalculatedat1to3samplingtimepointsbyBayesianMAP
procedure.We evaluated limited sampling strategies based on separate calculationswith
time restrictions of 0-6h, 0-12h, and 0-24h. Performance was considered suitable for
applicationinprospectivestudies iftheadjustedrsquared(R2)was>0.95,therootmean
squareerror(RMSE)was<15%,andthemeanpredictionerror(MPE)was<5%.Theprediction
errorswerecalculatedas(AUC0-24h,LSS-AUC0-24h,ref)/AUC0-24h,ref·100%.
113
7
Pharmacokinetic Modelling and Limited Sampling Strategies of Ertapenem
topatients.Alimitedsamplingstrategythroughapopulationpharmacokineticmodelcanbe
usedtopredictthisplasmaconcentrationprofileashasbeendoneforotheranti-TBdrugs
(19-22).
The aim of this studywas to develop a population pharmacokineticmodel and a limited
samplingstrategybasedondatafromhealthyvolunteers,inordertoestimatedrugexposure
ofertapeneminMDR-TBpatients.
Materialsandmethods
This studywas based on two data sets. The first setwas comprised of forty-two healthy
volunteers fromfiveclinicalstudiesreceiving0.25to2g i.v.dosesofertapenem(10).For
populationpharmacokineticmodelcomparisonwithMDR-TBpatientsweonlyusedthedata
ofhealthyvolunteersreceiving1g.Thesecondsetwascomprisedofaretrospectivedataset
ofpatientswithMDR-TB,receiving1gertapenemadministeredoncedailyviaa30-minute
infusionattheTuberculosisCenterBeatrixoord,UniversityMedicalCenterGroningen,The
NetherlandsbetweenDecember1,2010andMarch1,2013(8).Plasmaconcentrationsof
ertapenemwerecollectedatsteadystatebeforeadministrationandat1,2,3,4,5,6,8and
12 hours after administration. Ertapenem plasma concentrations were analyzed by a
validatedliquidchromatography-tandemmass-spectrometry(LC-MS/MS)method(23).Both
datasetsincludeddemographicandmedicaldata,suchasageatstartoftreatment,height
andbodyweightandserumcreatinineatthetimeofpharmacokineticassessment.
Populationpharmacokineticmodel
All pharmacokinetic calculations were performed using MW\Pharm 3.82 (Mediware,
Zuidhorn,TheNetherlands).Basedonpreviousreportsandrecentpharmacokineticstudies
on ertapenem (11-18, 24-26), concentration time-curves were evaluated in a one-
compartmentalandtwo-compartmentalmodel.Thefinalmodelwasselectedbasedonthe
Akaike information criterion (AIC) (27). The drug plasma-concentrations of the forty-two
healthyvolunteerswereusedtodevelopatwo-compartmentalpopulationpharmacokinetic
modelusinganiterativetwo-stageBayesian(ITSB)procedure(moduleKinPopofMW\Pharm)
(28).TheclearancewascalculatedusingCL=CLm(metabolicclearance(L/h/1.85m2))*BSA
(body surface area (m2)) /1.85 + fr (drug clearance – creatinine clearance ratio) * CLcr
(creatinine clearance (L/h)) (24). Pharmacokinetic parameters were assumed to be log-
normallydistributedandtheresidualerrorwasassumedtobenormallydistributedwithSD
=0.1+0.1*C,inwhichCistheobservedplasmaconcentrationofertapenem.Nonparametric
95%confidence intervalsof thepopulationparametersand their inter-individual standard
deviationwereestimatedusingabootstrapanalysis(n=1000).Theareaundertheplasma
concentration-timecurvefrom0upto24hours(AUC0-24h,ref),usedasareferencevalue,was
calculatedusingthelogtrapezoidalrule(KinFitmoduleofMW\Pharm).
Internal validation was performed by leaving three healthy volunteers out of the
pharmacokinetic model development, creating fourteen n-3 sub models, obtained by
randomizationusingMicrosoftExcel2010.TheestimatedAUC0-24h,(AUC0-24h,n-3)wasobtained
byBayesianfittingusingthedataofthethreeleftoutvolunteersinthecorrespondingn-3
submodels.AgreementbetweenAUC0-24h,n-3andAUC0-24h,refwasassessedbyBland-Altman
analysisandPassingandBablokregressionandsubsequentresidualplot.Externalvalidation
wasperformedbyBayesianfittingofthemodeldevelopedinvolunteerstotheindividualdata
ofMDR-TBpatients(AUC0-24h,fit),usingthedevelopedpopulationmodelforvolunteersasa
prior.ForcomparisonofthepharmacokineticsbetweenMDR-TBpatientsandvolunteers,a
similaranalysiswasperformedwiththedataof18volunteerswhoreceived1gofertapenem.
Bland-Altmananalysiswasalsoused toassess theagreementbetweenAUC0-24h,fitand the
AUC0-24h,refofMDR-TBpatients.
Limitedsamplingstrategies
AMonteCarlosimulationwasusedtocalculatelimitedsamplingstrategiesforestimationof
AUC (AUC0-24h,LSS), as implemented inMW\Pharm. This stochastic simulation consisted of
1000randompatientsdrawnfromthepopulationpharmacokineticmodel.Foreachpatient
limitedsamplingstrategieswerecalculatedat1to3samplingtimepointsbyBayesianMAP
procedure.We evaluated limited sampling strategies based on separate calculationswith
time restrictions of 0-6h, 0-12h, and 0-24h. Performance was considered suitable for
applicationinprospectivestudies iftheadjustedrsquared(R2)was>0.95,therootmean
squareerror(RMSE)was<15%,andthemeanpredictionerror(MPE)was<5%.Theprediction
errorswerecalculatedas(AUC0-24h,LSS-AUC0-24h,ref)/AUC0-24h,ref·100%.
114
Chapter 7
Predictionoffree40%T>MIC
The ertapenem concentration-time curve of each patient was used to establish if the f
40%T>MICwasreached.Forthiscausethetimethattheconcentration-timecurvewasabove
theMICwasassessedinMW/Pharm.Thepercentageproteinunboundertapenemusedfor
the assessment was 5 (4,7). European Committee on Antimicrobial Susceptibility Testing
(EUCAST)MICvalueforertapenem(non-speciesrelated)of0.5mg·L−1wasusedtocalculate
f40%T>MIC(6,7).Exposurewasconsideredadequateiftheconcentrationwas40%oftime
aboveMIC,whichcorrespondswith9.6hofeach24hintervalasshowninfigure1.
Figure1.Ertapenemplasmaconcentrationtimecurve. 40%oftimeiscorrespondingwith9.6hofeach24hinterval.
Statistics
Differences between the population characteristics and pharmacokinetic parameters of
healthy volunteers and TB patients were calculated using the MannWhitney U test. All
statisticswerecalculatedwithAnalyse-it™forMicrosoftExcel(version2.30).
ResultsDataset
Dataofforty-twohealthyvolunteerswasusedtodevelopthepopulationpharmacokinetic
model.SincebloodsamplesfromMDR-TBpatientswerecollectedforanotherpurpose,no
datawasavailablebetween5and8hours.Thebaselinecharacteristicsofhealthyvolunteers
andMDR-TBpatientswereshowntodiffersignificantly(P<0.05),exceptforage(table1).The
medianageofthevolunteerswas31(23-38)yearsandbodymassindexwas24.5(23.6–
26.2)kg/m2.
Table1.Baselinecharacteristicsofhealthyvolunteersversusmultidrug-resistanttuberculosispatients
Healthyvolunteers MDR-TBpatients p-value n=42 n=12 Sex[n(%)] Male 36(86%) 5(42%) 0.0083aFemale 6(14%) 7(58%) Age(years;median,IQR) 31(23-38) 25(18-30) 0.064bWeight(kg;median,IQR) 78.9(72.2-83.8) 55.5(47.3-70.3) 0.000b
Height(cm;median,IQR) 178(172-183) 167(164-174) 0.004b
BodymassIndex(kg/m2;median,IQR) 24.5(23.6-26.2) 19.2(17.9-23.7) 0.002b Ethnicity Black(%) 5(12%) 7(58%) 0.000cCaucasian(%) 36(86%) 3(25%) Asian(%) 1(2%) 1(8%) Other(%) 0(0%) 1(8%) SerumCreatinine(mg/dl;median,IQR)* 0.9(0.8-1.1)** 0.5(0.4-0.7) 0.000b
Creatinineclearance(ml/min/1.73m2;median,IQR) 91(81–97) 181(122–248) <0.0001b
Dose/weight(mg/kg;median;IQR) 18.0(14.2-21.1)** 12.9(6.0-20.0) 0.044b
Samplesperpatient(median;IQR) 28(16-48) 7(6-8)
IQR=Interquartilerange*Onthedaytheplasmaconcentrationsweredetermined**Somepatientsreceivedmorethanonedosewithsubsequentserumcreatininea.Fisherexacttest,b.Mann-WhitneyUtest,c.PearsonChi-Squaredtest
115
7
Pharmacokinetic Modelling and Limited Sampling Strategies of Ertapenem
Predictionoffree40%T>MIC
The ertapenem concentration-time curve of each patient was used to establish if the f
40%T>MICwasreached.Forthiscausethetimethattheconcentration-timecurvewasabove
theMICwasassessedinMW/Pharm.Thepercentageproteinunboundertapenemusedfor
the assessment was 5 (4,7). European Committee on Antimicrobial Susceptibility Testing
(EUCAST)MICvalueforertapenem(non-speciesrelated)of0.5mg·L−1wasusedtocalculate
f40%T>MIC(6,7).Exposurewasconsideredadequateiftheconcentrationwas40%oftime
aboveMIC,whichcorrespondswith9.6hofeach24hintervalasshowninfigure1.
Figure1.Ertapenemplasmaconcentrationtimecurve. 40%oftimeiscorrespondingwith9.6hofeach24hinterval.
Statistics
Differences between the population characteristics and pharmacokinetic parameters of
healthy volunteers and TB patients were calculated using the MannWhitney U test. All
statisticswerecalculatedwithAnalyse-it™forMicrosoftExcel(version2.30).
ResultsDataset
Dataofforty-twohealthyvolunteerswasusedtodevelopthepopulationpharmacokinetic
model.SincebloodsamplesfromMDR-TBpatientswerecollectedforanotherpurpose,no
datawasavailablebetween5and8hours.Thebaselinecharacteristicsofhealthyvolunteers
andMDR-TBpatientswereshowntodiffersignificantly(P<0.05),exceptforage(table1).The
medianageofthevolunteerswas31(23-38)yearsandbodymassindexwas24.5(23.6–
26.2)kg/m2.
Table1.Baselinecharacteristicsofhealthyvolunteersversusmultidrug-resistanttuberculosispatients
Healthyvolunteers MDR-TBpatients p-value n=42 n=12 Sex[n(%)] Male 36(86%) 5(42%) 0.0083aFemale 6(14%) 7(58%) Age(years;median,IQR) 31(23-38) 25(18-30) 0.064bWeight(kg;median,IQR) 78.9(72.2-83.8) 55.5(47.3-70.3) 0.000b
Height(cm;median,IQR) 178(172-183) 167(164-174) 0.004b
BodymassIndex(kg/m2;median,IQR) 24.5(23.6-26.2) 19.2(17.9-23.7) 0.002b Ethnicity Black(%) 5(12%) 7(58%) 0.000cCaucasian(%) 36(86%) 3(25%) Asian(%) 1(2%) 1(8%) Other(%) 0(0%) 1(8%) SerumCreatinine(mg/dl;median,IQR)* 0.9(0.8-1.1)** 0.5(0.4-0.7) 0.000b
Creatinineclearance(ml/min/1.73m2;median,IQR) 91(81–97) 181(122–248) <0.0001b
Dose/weight(mg/kg;median;IQR) 18.0(14.2-21.1)** 12.9(6.0-20.0) 0.044b
Samplesperpatient(median;IQR) 28(16-48) 7(6-8)
IQR=Interquartilerange*Onthedaytheplasmaconcentrationsweredetermined**Somepatientsreceivedmorethanonedosewithsubsequentserumcreatininea.Fisherexacttest,b.Mann-WhitneyUtest,c.PearsonChi-Squaredtest
116
Chapter 7
Pharmacokineticmodel
Developmentofpharmacokineticmodelinvolunteers
Theselectionofthetwo-compartmentalmodelwasbasedontheAICvaluesfortheone-(AIC
=1280)andtwo-(AIC=-1073)compartmentmodels(28).Finalpopulationpharmacokinetic
modelparametersdevelopedfromdataofhealthyvolunteers(N=42)areshownintable2.
AUC0-24h,n-3valuesestimatedintheinternalvalidationwereunderestimatedbyameanvalue
of0.3%(range:-8.1–7.6),whencomparedwithAUC0-24h,refshowninFigure2.Theobserved
AUC0-24h,refandmodelcalculatedertapenemAUC0-24h,n=3wereassessedforagreement,using
PassingandBablokregressioninfigure3.
Figure 2. Internal validation of the population pharmacokineticmodel. Bland-Altman plotshowing theagreementbetween theareaunder the concentration-time curve for 24hofhealthyvolunteersestimatedwiththepopulationpharmacokineticmodel(AUC0-24h,n=3)andtheAUC0-24h,ref
Table2.Pharmacokineticpopulationmodeldevelopedfromdatainvolunteers.
Parameter Mean(95%CI) SD(95%CI)CLm(L/h/1.85m2) 1.06(0.54–1.34) 0.16(0.09–0.23)fr 0.130(0.073–0.237) 0.039(0.016–0.063)V1(L/kg) 0.0824(0.0789–0.0860) 0.0095(0.0073–0.0119)CL12(L/h/1.85m2) 2.56(2.34–2.85) 0.14(0.11–0.28)V2(L/kg) 0.0543(0.0527–0.0559) 0.0016(0.0013–0.0021)
CLm = metabolic clearance; fr =ertapenem clearance/creatinine clearance ratio; V1 = volume ofdistribution of the central compartment; CL12 = inter-compartmental clearance; V2 = volume ofdistributionintheperipheralcompartment;CI=nonparametricconfidenceintervalfrombootstrapanalysisPopulationPKparametersTBpatientscomparedtohealthyvolunteers
Pharmacokinetic parameters of 18 healthy volunteers, who received 1 g of ertapenem,
comparedtoTBpatientsareshownintable3.AUC0-24h,fitvaluesoftheMDR-TBpatientdata
wereunderestimatedbymean6.8%(range:-17.2–30.7),whencomparedwithAUC0-24h,ref.
AUC0-24h,fit correlatedwell with AUC0-24h,ref , as determinedwith a Bland-Altman analysis -
showninFigure4.
Table3.Pharmacokineticparametersinvolunteerswhoreceiveda1gdoseandMDR-TBpatients.
Healthyvolunteers(n=18) Tuberculosispatients(n=12) p-value*CLm(L/h/1.85m2) 1.06±0.08 0.944±0.132 0.0023fr 0.158±0.051 0.104±0.039 0.0017V1(L/kg) 0.0846±0.0094 0.0876±0.0166 0.4717CL12(L/h/1.85m2) 2.52±0.03 2.55±0.04 0.0023V2(L/kg) 0.0543±0.0005 0.0549±0.0008 0.2885
*Mann-WhitneyUtestClm = metabolic clearance; fr =ertapenem clearance/creatinine clearance ratio; V1 = volume ofdistribution of the central compartment; CL12 = inter-compartmental clearance; V2 = volume ofdistributionintheperipheralcompartment
117
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Pharmacokinetic Modelling and Limited Sampling Strategies of Ertapenem
Pharmacokineticmodel
Developmentofpharmacokineticmodelinvolunteers
Theselectionofthetwo-compartmentalmodelwasbasedontheAICvaluesfortheone-(AIC
=1280)andtwo-(AIC=-1073)compartmentmodels(28).Finalpopulationpharmacokinetic
modelparametersdevelopedfromdataofhealthyvolunteers(N=42)areshownintable2.
AUC0-24h,n-3valuesestimatedintheinternalvalidationwereunderestimatedbyameanvalue
of0.3%(range:-8.1–7.6),whencomparedwithAUC0-24h,refshowninFigure2.Theobserved
AUC0-24h,refandmodelcalculatedertapenemAUC0-24h,n=3wereassessedforagreement,using
PassingandBablokregressioninfigure3.
Figure 2. Internal validation of the population pharmacokineticmodel. Bland-Altman plotshowing theagreementbetween theareaunder the concentration-time curve for 24hofhealthyvolunteersestimatedwiththepopulationpharmacokineticmodel(AUC0-24h,n=3)andtheAUC0-24h,ref
Table2.Pharmacokineticpopulationmodeldevelopedfromdatainvolunteers.
Parameter Mean(95%CI) SD(95%CI)CLm(L/h/1.85m2) 1.06(0.54–1.34) 0.16(0.09–0.23)fr 0.130(0.073–0.237) 0.039(0.016–0.063)V1(L/kg) 0.0824(0.0789–0.0860) 0.0095(0.0073–0.0119)CL12(L/h/1.85m2) 2.56(2.34–2.85) 0.14(0.11–0.28)V2(L/kg) 0.0543(0.0527–0.0559) 0.0016(0.0013–0.0021)
CLm = metabolic clearance; fr =ertapenem clearance/creatinine clearance ratio; V1 = volume ofdistribution of the central compartment; CL12 = inter-compartmental clearance; V2 = volume ofdistributionintheperipheralcompartment;CI=nonparametricconfidenceintervalfrombootstrapanalysisPopulationPKparametersTBpatientscomparedtohealthyvolunteers
Pharmacokinetic parameters of 18 healthy volunteers, who received 1 g of ertapenem,
comparedtoTBpatientsareshownintable3.AUC0-24h,fitvaluesoftheMDR-TBpatientdata
wereunderestimatedbymean6.8%(range:-17.2–30.7),whencomparedwithAUC0-24h,ref.
AUC0-24h,fit correlatedwell with AUC0-24h,ref , as determinedwith a Bland-Altman analysis -
showninFigure4.
Table3.Pharmacokineticparametersinvolunteerswhoreceiveda1gdoseandMDR-TBpatients.
Healthyvolunteers(n=18) Tuberculosispatients(n=12) p-value*CLm(L/h/1.85m2) 1.06±0.08 0.944±0.132 0.0023fr 0.158±0.051 0.104±0.039 0.0017V1(L/kg) 0.0846±0.0094 0.0876±0.0166 0.4717CL12(L/h/1.85m2) 2.52±0.03 2.55±0.04 0.0023V2(L/kg) 0.0543±0.0005 0.0549±0.0008 0.2885
*Mann-WhitneyUtestClm = metabolic clearance; fr =ertapenem clearance/creatinine clearance ratio; V1 = volume ofdistribution of the central compartment; CL12 = inter-compartmental clearance; V2 = volume ofdistributionintheperipheralcompartment
118
Chapter 7
Figure3.PassingandBablokregression.Plotshowingtheagreementbetweentheareaunderthe concentration-time curve for 24h (AUC0-24h,ref) and theAUC0-24h,n=3 estimatedwith thepopulationpharmacokineticmodel(dottedlines:95%confidenceinterval(CI)).
Limitedsamplingstrategies
Usingthepopulationpharmacokineticmodel,limitedsamplingstrategieswereevaluatedfor
0-6h, 0-12h and 0-24h restriction of the dosing intervals. The R2, bias and RMSE were
subsequentlydetermined.Foreachdosingintervalandforone,twoorthreesamplingtime
points,thelimitedsamplingstrategieswiththebestperformanceofRMSEandbiasareshown
inTable4.
Figure4. External validationof thepopulationpharmacokineticmodel.BlandAltmanplotshowing the agreement of the area under the concentration-time curve of multidrug-resistanttuberculosispatients(AUC0-24h,ref)andtheAUC0-24h,fitestimatedwiththepopulationpharmacokineticmodel.MeanofAllisthemeanAUCofbothAUC0-24h,fitandAUC0-24h,ref.
Table4.Best-performinglimitedsamplingstrategies
Firstsamplingtimepoint(h)
Secondsamplingtimepoint(h)
Thirdsamplingtimepoint(h)
Coefficientofdetermination(r2)
Meanpredictionerror(%bias) %RMSEa
0-6h 5 0.57 -0.09 7.40-6h 1 5 0.78 -0.33 5.50-6h 1 3 5 0.83 -0.39 4.90-12h 8 0.72 -0.84 7.10-12h 1 11 0.83 -0.83 5.60-12h 1 4 8 0.87 -0.89 4.90-24h 14 0.82 -0.19 6.90-24h 1 14 0.88 -0.44 5.50-24h 1 9 14 0.92 -0.46 4.7
a. Rootmeansquareerror
119
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Pharmacokinetic Modelling and Limited Sampling Strategies of Ertapenem
Figure3.PassingandBablokregression.Plotshowingtheagreementbetweentheareaunderthe concentration-time curve for 24h (AUC0-24h,ref) and theAUC0-24h,n=3 estimatedwith thepopulationpharmacokineticmodel(dottedlines:95%confidenceinterval(CI)).
Limitedsamplingstrategies
Usingthepopulationpharmacokineticmodel,limitedsamplingstrategieswereevaluatedfor
0-6h, 0-12h and 0-24h restriction of the dosing intervals. The R2, bias and RMSE were
subsequentlydetermined.Foreachdosingintervalandforone,twoorthreesamplingtime
points,thelimitedsamplingstrategieswiththebestperformanceofRMSEandbiasareshown
inTable4.
Figure4. External validationof thepopulationpharmacokineticmodel.BlandAltmanplotshowing the agreement of the area under the concentration-time curve of multidrug-resistanttuberculosispatients(AUC0-24h,ref)andtheAUC0-24h,fitestimatedwiththepopulationpharmacokineticmodel.MeanofAllisthemeanAUCofbothAUC0-24h,fitandAUC0-24h,ref.
Table4.Best-performinglimitedsamplingstrategies
Firstsamplingtimepoint(h)
Secondsamplingtimepoint(h)
Thirdsamplingtimepoint(h)
Coefficientofdetermination(r2)
Meanpredictionerror(%bias) %RMSEa
0-6h 5 0.57 -0.09 7.40-6h 1 5 0.78 -0.33 5.50-6h 1 3 5 0.83 -0.39 4.90-12h 8 0.72 -0.84 7.10-12h 1 11 0.83 -0.83 5.60-12h 1 4 8 0.87 -0.89 4.90-24h 14 0.82 -0.19 6.90-24h 1 14 0.88 -0.44 5.50-24h 1 9 14 0.92 -0.46 4.7
a. Rootmeansquareerror
120
Chapter 7
Figure 5. Validation of the limited sampling strategy. The Bland-Altman plot shows theagreementbetweentheareaundertheconcentration-timecurvefor24hfrommultidrug-resistanttuberculosispatientsobtainedfromthepopulationpharmacokineticmodelapplyingthelimitedsamplingstrategyof1and5h(AUC0-24h,LSS)andtheAUC0-24h,refMeanofAllisthemeanAUCofbothAUC0-24h,fitandAUC0-24h,ref.
AlllimitedsamplingstrategiesmetthebiasandRMSEcriteria.Threesamplingtimepoints,at
1, 4 and 9h, enabled the best prediction of ertapenemexposure reflected byAUC0-24h,LSS,
consideringbias,RMSEandR2(R2=0.92,MPE=—0,46%andRMSE=4.7%).However,dueto
thelackofdatawithinthesetimeintervalsandclinicalrelevanceoftimesamplingswithina
certainamountoftime,itwouldbepreferredtouse1,3and5h(R2=0.83,RMSE=4.7%and
MPE = -0.39%). Based on clinical suitability, using two sampling time-pointswith a time-
restrictionof0-6h,alimitedsamplingstrategyat1and5h,showedthelowestRMSE(5.5%)
andlowMPE(-0.33%).TheAUC0-24h,LSS,estimatedbyapplyingthistwosamplingtime-point
limited sampling strategywas comparedwith theAUC0-24h,ref usingBland-Altmananalysis,
showingabiasinAUC0-24h,LSS,of4.2%(-15.2–23.6)(figure5).
%T>MIC
Consideringafreefractionof5%andtheMICsetat0.5mg/L,9outof12patientswouldhave
exceeded a minimum of f 40% T>MIC (range 6.8h – 19.7h) thereby having sufficient
therapeuticeffectinMDR-TBPatientswithoncedailydosing.
DiscussionThisisthefirststudyshowingthatapopulationpharmacokineticmodelofertapenembased
ondataofhealthyvolunteerscanpredictpharmacokineticsofertapeneminpatientswith
MDR-TB,eventhoughthebaselinecharacteristicsofbothhealthyvolunteersandMDR-TB
patientsdifferedsignificantly(table1).WeshowedthattheAUC0-24hofMDR-TBpatientscould
beestimatedwiththispopulationpharmacokineticmodelwithameanoverestimationof6.8
(range:-17.2–30.7%).
Therobustnessofthispopulationpharmacokineticmodelwasvalidatedusingan-3cross-
validation, showing an underestimation of 0.3%. The limited sampling strategy that we
presentherecanbeusedtoassessindividualdrugexposureofertapeneminTBpatientswith
limitedtreatmentoptions.Moreover,themodelandlimitedsamplingstrategycanbeused
toevaluatedrugexposureofertapeneminphaseIIstudiesstudyingearlybactericidalactivity
ofertapeneminTBpatients.Suchastudyisurgentlyneededtoprovidedataonefficacyof
thepotentialattractiveTBdrug.
Inthepopulationpharmacokineticmodel,multipledosesweretreatedassingledosesonday
onetoavoidduplication,asanearlierstudyhadfoundthattherewasnoaccumulationof
ertapenemfollowingdosingovereightdaysandmeanplasmaconcentrationswerefoundto
beverysimilarondayoneaswellasondayeight(9).
Pharmacokineticmodelingofertapenemhasbeenperformedinpreviousstudies(11-18,24-
26),butithasneverbeenperformedforapplicationinMDR-TBtreatment.Comparinghealthy
volunteersandtheTBpatients,we foundthat there isasmallpharmacokineticvariability
betweenthetwogroupsincontrasttootherantimicrobialdrugs,whichshowhighvariability
inpharmacokineticparameters(19,20).Thismightbeexplainedbytheparenteralrouteof
121
7
Pharmacokinetic Modelling and Limited Sampling Strategies of Ertapenem
Figure 5. Validation of the limited sampling strategy. The Bland-Altman plot shows theagreementbetweentheareaundertheconcentration-timecurvefor24hfrommultidrug-resistanttuberculosispatientsobtainedfromthepopulationpharmacokineticmodelapplyingthelimitedsamplingstrategyof1and5h(AUC0-24h,LSS)andtheAUC0-24h,refMeanofAllisthemeanAUCofbothAUC0-24h,fitandAUC0-24h,ref.
AlllimitedsamplingstrategiesmetthebiasandRMSEcriteria.Threesamplingtimepoints,at
1, 4 and 9h, enabled the best prediction of ertapenemexposure reflected byAUC0-24h,LSS,
consideringbias,RMSEandR2(R2=0.92,MPE=—0,46%andRMSE=4.7%).However,dueto
thelackofdatawithinthesetimeintervalsandclinicalrelevanceoftimesamplingswithina
certainamountoftime,itwouldbepreferredtouse1,3and5h(R2=0.83,RMSE=4.7%and
MPE = -0.39%). Based on clinical suitability, using two sampling time-pointswith a time-
restrictionof0-6h,alimitedsamplingstrategyat1and5h,showedthelowestRMSE(5.5%)
andlowMPE(-0.33%).TheAUC0-24h,LSS,estimatedbyapplyingthistwosamplingtime-point
limited sampling strategywas comparedwith theAUC0-24h,ref usingBland-Altmananalysis,
showingabiasinAUC0-24h,LSS,of4.2%(-15.2–23.6)(figure5).
%T>MIC
Consideringafreefractionof5%andtheMICsetat0.5mg/L,9outof12patientswouldhave
exceeded a minimum of f 40% T>MIC (range 6.8h – 19.7h) thereby having sufficient
therapeuticeffectinMDR-TBPatientswithoncedailydosing.
DiscussionThisisthefirststudyshowingthatapopulationpharmacokineticmodelofertapenembased
ondataofhealthyvolunteerscanpredictpharmacokineticsofertapeneminpatientswith
MDR-TB,eventhoughthebaselinecharacteristicsofbothhealthyvolunteersandMDR-TB
patientsdifferedsignificantly(table1).WeshowedthattheAUC0-24hofMDR-TBpatientscould
beestimatedwiththispopulationpharmacokineticmodelwithameanoverestimationof6.8
(range:-17.2–30.7%).
Therobustnessofthispopulationpharmacokineticmodelwasvalidatedusingan-3cross-
validation, showing an underestimation of 0.3%. The limited sampling strategy that we
presentherecanbeusedtoassessindividualdrugexposureofertapeneminTBpatientswith
limitedtreatmentoptions.Moreover,themodelandlimitedsamplingstrategycanbeused
toevaluatedrugexposureofertapeneminphaseIIstudiesstudyingearlybactericidalactivity
ofertapeneminTBpatients.Suchastudyisurgentlyneededtoprovidedataonefficacyof
thepotentialattractiveTBdrug.
Inthepopulationpharmacokineticmodel,multipledosesweretreatedassingledosesonday
onetoavoidduplication,asanearlierstudyhadfoundthattherewasnoaccumulationof
ertapenemfollowingdosingovereightdaysandmeanplasmaconcentrationswerefoundto
beverysimilarondayoneaswellasondayeight(9).
Pharmacokineticmodelingofertapenemhasbeenperformedinpreviousstudies(11-18,24-
26),butithasneverbeenperformedforapplicationinMDR-TBtreatment.Comparinghealthy
volunteersandtheTBpatients,we foundthat there isasmallpharmacokineticvariability
betweenthetwogroupsincontrasttootherantimicrobialdrugs,whichshowhighvariability
inpharmacokineticparameters(19,20).Thismightbeexplainedbytheparenteralrouteof
122
Chapter 7
administrationofertapenemtherebyhavingnolossofertapenemduetoabsorption.Several
studieslookingattheexposureofertapeneminpatientshaveshownthattheexposurein
patients varies greatly (14,15). There is little pharmacokinetic variability in the MDR-TB
patients(table3).
A limitedsamplingstrategyusingtwosamplingtimepoints is favoredas itwouldgivethe
leastburdentopatientsas it isminimally invasiveand leasttime-consuming.Additionally,
less timebetweensamplingtimepoints ismorefeasible inclinicalpractice,since it is less
pronetosamplingmistakes.Moreover, limitationofthisstudyisthatafter6hsparsedata
wasavailable,thereforeresultsafter6hislesssubstantiatedwithclinicaldata.
AstherearelimitedoptionsfortreatingMDR-TBandresistanceofantibioticsisanemerging
problem,wethink it is timetostartassessingefficacyofertapeneminMDR-Bpatients in
Phase II clinical trial testing theearlybactericidalactivity.Thedeveloped limitedsampling
strategycanbeusedtoevaluatedrugexposureandtherebyreducecostsandburdenforthe
studysubjects.
Conclusion
A pharmacokinetic model and limited sampling strategy bases on data from healthy
volunteerswasabletopredicttheAUC0-24handf40%T>MICinMDR-TBpatients.Thismodel
canbeusedinphaseIIstudies.
Conflictofinterest:nonedeclared
Acknowledgement:TheauthorsthankMerck&Co.Inc.USA,forprovidingdatafromearlier
pharmacokineticsstudiesofertapenem
References1. MoutonJW,TouzwDJ,Horrevorts,VinkAA.2000.Comparativepharmacokineticsofthe
carbapenems:Clinicalimplications.ClinPharmacokinet39:185-201.
2. Craig WA. 1998. Pharmacokinetic/pharmacodynamic parameters: Rationale for
antibacterialdosingofmiceandmen.ClinInfectDis26:1,10;quiz11-2.
3. VezirisN,TruffotC,MainardiJL,JarlierV.2011.Activityofcarbapenemscombinedwith
clavulanateagainstmurinetuberculosis.AntimicrobAgentsChemother55:2597-600.
4. CordillotM,DubeeV,TribouletS,DubostL,MarieA,HugonnetJE,ARthurM,Mainardi
JL. 2013. In vitro cross-linking of mycobacterium tuberculosis peptidoglycan by L,D-
transpeptidasesandinactivationoftheseenzymesbycarbapenems.AntimicrobAgents
Chemother57:5940-5.
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ofAntibioticsthatdegradefasterthanthedoublingtimeofslow-growingmycobacteria:
Ertapenem sterilizing effect versus Mycobacterium tuberculosis. Antimicrob Agents
Chemother60(5):3193-5
7. VezirisN,TruffotC,MainardiJL,JarlierV.2011.Activityofcarbapenemscombinedwith
clavulanateagainstmurinetuberculosis.AntimicrobAgentsChemother55(6):2597-600
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Kostering JG, van derWerf TS, Alffenaar JW 2016. Pharmacokinetics of ertapenem in
patientswithmultidrug-resistanttuberculosis.EurRespirJ47:1229-34
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experience.EurRespirJ47:333-6.
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Chemother.46:3506-11.
123
7
Pharmacokinetic Modelling and Limited Sampling Strategies of Ertapenem
administrationofertapenemtherebyhavingnolossofertapenemduetoabsorption.Several
studieslookingattheexposureofertapeneminpatientshaveshownthattheexposurein
patients varies greatly (14,15). There is little pharmacokinetic variability in the MDR-TB
patients(table3).
A limitedsamplingstrategyusingtwosamplingtimepoints is favoredas itwouldgivethe
leastburdentopatientsas it isminimally invasiveand leasttime-consuming.Additionally,
less timebetweensamplingtimepoints ismorefeasible inclinicalpractice,since it is less
pronetosamplingmistakes.Moreover, limitationofthisstudyisthatafter6hsparsedata
wasavailable,thereforeresultsafter6hislesssubstantiatedwithclinicaldata.
AstherearelimitedoptionsfortreatingMDR-TBandresistanceofantibioticsisanemerging
problem,wethink it is timetostartassessingefficacyofertapeneminMDR-Bpatients in
Phase II clinical trial testing theearlybactericidalactivity.Thedeveloped limitedsampling
strategycanbeusedtoevaluatedrugexposureandtherebyreducecostsandburdenforthe
studysubjects.
Conclusion
A pharmacokinetic model and limited sampling strategy bases on data from healthy
volunteerswasabletopredicttheAUC0-24handf40%T>MICinMDR-TBpatients.Thismodel
canbeusedinphaseIIstudies.
Conflictofinterest:nonedeclared
Acknowledgement:TheauthorsthankMerck&Co.Inc.USA,forprovidingdatafromearlier
pharmacokineticsstudiesofertapenem
References1. MoutonJW,TouzwDJ,Horrevorts,VinkAA.2000.Comparativepharmacokineticsofthe
carbapenems:Clinicalimplications.ClinPharmacokinet39:185-201.
2. Craig WA. 1998. Pharmacokinetic/pharmacodynamic parameters: Rationale for
antibacterialdosingofmiceandmen.ClinInfectDis26:1,10;quiz11-2.
3. VezirisN,TruffotC,MainardiJL,JarlierV.2011.Activityofcarbapenemscombinedwith
clavulanateagainstmurinetuberculosis.AntimicrobAgentsChemother55:2597-600.
4. CordillotM,DubeeV,TribouletS,DubostL,MarieA,HugonnetJE,ARthurM,Mainardi
JL. 2013. In vitro cross-linking of mycobacterium tuberculosis peptidoglycan by L,D-
transpeptidasesandinactivationoftheseenzymesbycarbapenems.AntimicrobAgents
Chemother57:5940-5.
5. DiaconAH, vanderMerweL,BarnardM, vonGroote-Bidlingmaier F, LangeC,García-
BasteiroAL,SeveneE,BallellL,Barros-AguirreD.2016.β-LactamsagainstTuberculosis--
New Trick for an Old Dog? N Engl J Med. Jul 28;375(4):393-4. doi:
10.1056/NEJMc1513236.Epub2016Jul13.
6. SrivastaS,vanRijnSP,WesselsAM,AlffenaarJW,GumboT.2016.Susceptibilitytesting
ofAntibioticsthatdegradefasterthanthedoublingtimeofslow-growingmycobacteria:
Ertapenem sterilizing effect versus Mycobacterium tuberculosis. Antimicrob Agents
Chemother60(5):3193-5
7. VezirisN,TruffotC,MainardiJL,JarlierV.2011.Activityofcarbapenemscombinedwith
clavulanateagainstmurinetuberculosis.AntimicrobAgentsChemother55(6):2597-600
8. vanRijnSP,vanAltenaR,AkkermanOW,vanSoolingenD,vanderLaanT,deLangeWC,
Kostering JG, van derWerf TS, Alffenaar JW 2016. Pharmacokinetics of ertapenem in
patientswithmultidrug-resistanttuberculosis.EurRespirJ47:1229-34
9. TiberiS,D'AmbrosioL,DeLorenzoSViggianiP,CentisR,Sothiu,AlfenaarJW,MiglioriGB.
2016. Ertapenem in the treatment of multidrug-resistant tuberculosis: First clinical
experience.EurRespirJ47:333-6.
10. MajumdarAK,MussonDG,BirkKL,KitchenCJ,HollandS,McCreaJ,MistryG,HesneyM,
XiL,LiSX,HaesenR,BlumRA,LinsRL,GreenbergH,WaldmanS,DeutschP,RogersJD.
2002.Pharmacokineticsof ertapenem inhealthy young volunteers.AntimicrobAgents
Chemother.46:3506-11.
124
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Model-Based Drug Development—Part 2: Introduction to Pharmacokinetic Modeling
Methods.CPTPharmacometricsSystPharmacol.Apr;2(4):e38.
23. vanRijnSP,WesselsAM,GreijdanusB,TouwDJ,AlffenaarJW.2014.Quantificationand
validation of ertapenem using a liquid chromatography-tandem mass spectrometry
method.AntimicrobAgentsChemother58(6):3481-4.
24. DaillyE,ArnouldJF,FraissinetF,NauxE,LetarddelaBouraliereMA,BouquieR,Deslandes
G, Jolliet P, Le Floch R. 2013. Pharmacokinetics of ertapenem in burns patients. Int J
AntimicrobAgents42:48-52.
25. LiebchenU,KratzerA,WichaSG,KeesF,KloftC,KeesMG.2014.Unboundfractionof
ertapeneminintensivecareunitpatients.JAntimicrobChemother69(11):3108-11
26. 2Lewis SJ, KaysMB,Mueller BA. 2016. Use ofMonte Carlo simulations to determine
optimalcarbapenemdosingincriticallyillpatientsreceivingprolongedintermittentrenal
replacementtherapy.JClinPharmacol26.doi:10.1002/jcph.727.[Epubaheadofprint]
27. 27.SheinerLB,BealSL.1981.SomeSuggestionsforMeasuringPredictivePerformance.
JournalofPharmacokineticsandBiopharmaceutics9(4):503-512
28. 28.ProostJH,EleveldDJ.2006.Performanceofaniterativetwo-stageBayesiantechnique
for population pharmacokinetic analysis of rich data-sets. Pharm Res 23:2748-2759.
http://dx.doi.org/10.1007/s11095-006-9116-0
125
7
Pharmacokinetic Modelling and Limited Sampling Strategies of Ertapenem
11. BorracciT,AdembriC,AccettaG,BertiJ,CappelliniI,LuccheseM,BiggeriA,DeGaudio
AR,NovelliA.2014.Useoftheparenteralantibioticertapenemasshorttermprophylaxis
inbariatricsurgery:Apharmaco-kinetic-pharmacodynamicstudyinclassIIIobesefemale
patients.MinervaAnestesiol80:1005-11.
12. Chen M, Nafziger AN, Drusano GL, Ma L, Bertino JS Jr. 2006. Comparative
pharmacokinetics and pharmacodynamic target attainment of ertapenem in normal-
weight,obese,andextremelyobeseadults.AntimicrobAgentsChemother50:1222-7.
13. EylerRF,VilayAM,NaderAM,HeungM,PlevaM,SowinskiKM,DePestelDD,SorgelF,
Kinzig M, Mueller BA. 2014. Pharmacokinetics of ertapenem in critically ill patients
receivingcontinuousvenovenoushemodialysisorhemodiafiltration.AntimicrobAgents
Chemother8:1320-6.
14. MistryGC,Majumdar AK, Swan S, SicaD, Fisher A, Xu Y, HesneyM, Xi L,Wagner JA,
Deutsch PJ. 2006. Pharmacokinetics of ertapenem in patientswith varying degrees of
renalinsufficiencyandinpatientsonhemodialysis.JClinPharmacol46:1128-38.
15. BurkhardtO,HaferC,LanghoffA,KaeverV,KumarV,WelteT,HallerH,FliserD,Kielsteijn
JT.2009.Pharmacokineticsofertapenemincriticallyillpatientswithacuterenalfailure
undergoingextendeddailydialysis.NephrolDialTransplant24:267-71.
16. BrinkAJ,RichardsGA,SchillackV,KiemS,SchentagJ.2009.Pharmacokineticsofonce-
daily dosing of ertapenem in critically ill patients with severe sepsis. Int J Antimicrob
Agents33:432-6.
17. BurkhardtO,KumarV,KatterweD,Majcher-PeszynskaJ,DrewelowB,DerendorfH,Welte
T. 2007. Ertapenem in critically ill patients with early-onset ventilator-associated
pneumonia: Pharmacokineticswith special consideration of free-drug concentration. J
AntimicrobChemother59:277-84.
18. CardoneKE,GrabeDW,KulawyRW,KulawyRW,DaouiR,RoglieriJ,MeolaS,DrusanoGL,
LodiseTP2012.Ertapenempharmacokineticsandpharmacodynamicsduringcontinuous
ambulatoryperitonealdialysis.AntimicrobAgentsChemother56:725-30.
19. Alffenaar JW, Kosterink JG, vanAltenaR, van derWerf TS,UgesDR, Proost JH. 2010.
Limitedsamplingstrategiesfortherapeuticdrugmonitoringoflinezolidinpatientswith
multidrug-resistanttuberculosis.TherDrugMonit.32:97-101.
20. PrangerAD,KosterinkJG,vanAltenaR,AarnoutseRE,vanderWerfTS,UgesDR,Alffenaar
JW.2011.Limited-samplingstrategiesfortherapeuticdrugmonitoringofmoxifloxacinin
patientswithtuberculosis.TherDrugMonit.33:350-4.
21. Magis-Escurra C, Alffenaar JW, Hoefnagels I, Dekhuijzen PN, BoereeMR, van Ingen J,
Aarnoutse RE. 2013. Pharmacokinetic studies in patients with nontuberculous
mycobacteriallunginfections.IntJAntimicrobAgents42:256-61.
22. Mould DR, Upton RN. 2013. Basic Concepts in Population Modeling, Simulation, and
Model-Based Drug Development—Part 2: Introduction to Pharmacokinetic Modeling
Methods.CPTPharmacometricsSystPharmacol.Apr;2(4):e38.
23. vanRijnSP,WesselsAM,GreijdanusB,TouwDJ,AlffenaarJW.2014.Quantificationand
validation of ertapenem using a liquid chromatography-tandem mass spectrometry
method.AntimicrobAgentsChemother58(6):3481-4.
24. DaillyE,ArnouldJF,FraissinetF,NauxE,LetarddelaBouraliereMA,BouquieR,Deslandes
G, Jolliet P, Le Floch R. 2013. Pharmacokinetics of ertapenem in burns patients. Int J
AntimicrobAgents42:48-52.
25. LiebchenU,KratzerA,WichaSG,KeesF,KloftC,KeesMG.2014.Unboundfractionof
ertapeneminintensivecareunitpatients.JAntimicrobChemother69(11):3108-11
26. 2Lewis SJ, KaysMB,Mueller BA. 2016. Use ofMonte Carlo simulations to determine
optimalcarbapenemdosingincriticallyillpatientsreceivingprolongedintermittentrenal
replacementtherapy.JClinPharmacol26.doi:10.1002/jcph.727.[Epubaheadofprint]
27. 27.SheinerLB,BealSL.1981.SomeSuggestionsforMeasuringPredictivePerformance.
JournalofPharmacokineticsandBiopharmaceutics9(4):503-512
28. 28.ProostJH,EleveldDJ.2006.Performanceofaniterativetwo-stageBayesiantechnique
for population pharmacokinetic analysis of rich data-sets. Pharm Res 23:2748-2759.
http://dx.doi.org/10.1007/s11095-006-9116-0
CHAPTER 8
General Discussion and Future Perspectives
CHAPTER 8
General Discussion and Future Perspectives
128
Chapter 8
GeneralDiscussionThelandscapeofMulti-andExtensivedrugresistant(M/XDR)treatmentofTBiscontinuously
changing.M/XDR-TBmustbetreatedwithmultipledrugstoachieveefficacyandtoprevent
development of further resistance [1].Multiple trials for different regimenswithminimal
resistanceareongoingtoassessshorter,affordable,fullyoralandoptimizedTBtreatment.
Recently, an individual patient data meta-analysis was performed of observational and
experimental studies, published between January 2009 and April 2016, reporting end of
treatmentoutcomes(completion,failureorrelapse)of12,030patientsfrom25countriesin
50 studies. Treatment success of MDR-TB was positively associated with the use of
bedaquiline,latergenerationfluoroquinolones,linezolid,clofazimineandcarbapenems.This
meta-analysis of predominantly observational studies had a large impact on setting new
standardsofcareforM/XDRtreatment.InthenewguidelinesissuedbyWHO[2],theroleof
2nd line injectable agents changed entirely. These compoundswere no longer considered
primordialtoobtainsuccessfuloutcomes;twooftheseagents-kanamycinandcapreomycin
-wereevenassociatedwithapooroutcome,andarenolongerrecommendedintherevised
treatment[2].FortheinitialphaseofMDR-TBtreatment,atleastfoureffectivedrugs;andin
thecontinuationphase,atleastfiveagentsarerecommended.Bedaquilline,moxifloxacinand
linezolidhavebeenlistedasbackboneandstandardofcareinlongerMDR-TBregimens[1-
2].Imipenem-cilastatinandmeropenemhavebeenlistedasGroupCdrugsforuseinlonger
MDR-TBregimensguidelinestocompleteregimens,whenothermedicinescannotbeused
[2].Whileertapenemhasnotyetbeen included in theWHOregimen, ithasa favourable
advantageovertheothercarbapenemsduetoitsonce-dailydosingitsactivityinMDR-TBhas
been demonstrated. This thesis provides additional data on ertapenem as potentially
advantageouscarbapenemcompoundintreatmentofMDR-TB.
DrugSusceptibilityTesting
Effective management of M/XDR-TB relies on rapid diagnosis and treatment. Currently,
phenotypicDrugSusceptibilityTesting(DST)isthegoldstandardfordrugresistancedetection
[3].ThisDSTmethodologyhasbeeninuse inmedicalmicrobiologybuthas inherentflaws
whenitcomestopredictingwhathappensinvivo.Thedrugthatistobetestedisaddedto
theculturemediumatafixedconcentration;inanimalmodelsandinhumanstreatedforTB,
however,drugconcentrationstypicallyfollowapharmacokineticconcentration-timecurve.
Moreover,with every new drug dosage every day, in vivo systems are not vulnerable to
chemicaldegradationwhichisaninherentflawintheinvitrostandardDSTassay.Oneway
toovercomethesemethodologicalflawswouldbetousethehollowfiberinfectionmodel
(HF).HFisaninvitrosystemthatusesapumpsystemwithculturemediawithfluctuating
drugconcentrationsovertime,mimickingmuchmorecloselythe invivoexposuretodrug
concentrationsovertime,correctingforchemicaldegradationofthecompoundunderstudy
aswellasmanyotherinherentflawsintheclassicalDSTassay.
InChapter2,wediscussinvitrostudiesreportingMICsofimipenemandmeropenemalone
orcombinedwithclavulanicacidatarangebetween0.16–32mg/L[4].Thishighvariability
of results between in vitro studiesmight be best explained by the chemical instability of
carbapenemsingrowthmediaattemperaturestypicallyusedininvitrostudies.Carbapenems
areheat-sensitiveandasM.tuberculosisgrowthneedsanincubationat37°Cfor15daysin
liquidmediaand4weeksinsolidmedia,carbapenemshavetowithstandahightemperature
foralongperiodoftime.Ertapenem,thatisunstableat37°C,islikelytobedegradedbefore
killingorinhibitingslow-growingM.tuberculosis,suggestingthaterroneouslyhighMICsfor
Mtbmighthavebeenreportedforcarbapenems[5].Itiscomprehensiblethatthisstability
issueofertapenemhaslikelynotoccurredbefore,sinceertapenemhasonlybeenprescribed
forawiderangeofbacterialinfections,consistingmostlyofawiderangeofgram-positive,
gram-negativeandanaerobicbacteriawithatypicalgenerationtimeof20min.DSTforthese
micro-organismsareavailablewithindays(maximumof48h),comparedtoslowgrowingM.
tuberculosiswithgenerationtimearound24h-resultinginDSTreportingtimeinweeks.
InChapter5,wedemonstrateanewstrategythatshowshowperformDSTsandmeasure
MICsforsuchunstablecompounds[6].Wefirstshowthatertapenemdegradesconsiderably
and secondly,we show thatertapenemsupplementation - to compensate for lossdue to
chemicaldegradation-bringsitwellwithintheEUCASTsusceptibilityrangeof0.5–1mg/L
[6]. However, only one reference strain, M. tuberculosis H37Ra, was used in our MIC
experiment.TodayantimicrobialDSTisbasedoncriticalconcentration-alsoreferredtoas
thebreakpoint -definedas ‘the lowestconcentrationofdrugthatwill inhibit95%ofwild
strainsofM.tuberculosisthathaveneverbeenexposedtodrugs,whileatthesametimenot
inhibitingclinicalstrainsofM.tuberculosisthatareconsideredtoberesistant’[3].Anextstep
129
8
General Discussion and Future Perspectives
GeneralDiscussionThelandscapeofMulti-andExtensivedrugresistant(M/XDR)treatmentofTBiscontinuously
changing.M/XDR-TBmustbetreatedwithmultipledrugstoachieveefficacyandtoprevent
development of further resistance [1].Multiple trials for different regimenswithminimal
resistanceareongoingtoassessshorter,affordable,fullyoralandoptimizedTBtreatment.
Recently, an individual patient data meta-analysis was performed of observational and
experimental studies, published between January 2009 and April 2016, reporting end of
treatmentoutcomes(completion,failureorrelapse)of12,030patientsfrom25countriesin
50 studies. Treatment success of MDR-TB was positively associated with the use of
bedaquiline,latergenerationfluoroquinolones,linezolid,clofazimineandcarbapenems.This
meta-analysis of predominantly observational studies had a large impact on setting new
standardsofcareforM/XDRtreatment.InthenewguidelinesissuedbyWHO[2],theroleof
2nd line injectable agents changed entirely. These compoundswere no longer considered
primordialtoobtainsuccessfuloutcomes;twooftheseagents-kanamycinandcapreomycin
-wereevenassociatedwithapooroutcome,andarenolongerrecommendedintherevised
treatment[2].FortheinitialphaseofMDR-TBtreatment,atleastfoureffectivedrugs;andin
thecontinuationphase,atleastfiveagentsarerecommended.Bedaquilline,moxifloxacinand
linezolidhavebeenlistedasbackboneandstandardofcareinlongerMDR-TBregimens[1-
2].Imipenem-cilastatinandmeropenemhavebeenlistedasGroupCdrugsforuseinlonger
MDR-TBregimensguidelinestocompleteregimens,whenothermedicinescannotbeused
[2].Whileertapenemhasnotyetbeen included in theWHOregimen, ithasa favourable
advantageovertheothercarbapenemsduetoitsonce-dailydosingitsactivityinMDR-TBhas
been demonstrated. This thesis provides additional data on ertapenem as potentially
advantageouscarbapenemcompoundintreatmentofMDR-TB.
DrugSusceptibilityTesting
Effective management of M/XDR-TB relies on rapid diagnosis and treatment. Currently,
phenotypicDrugSusceptibilityTesting(DST)isthegoldstandardfordrugresistancedetection
[3].ThisDSTmethodologyhasbeeninuse inmedicalmicrobiologybuthas inherentflaws
whenitcomestopredictingwhathappensinvivo.Thedrugthatistobetestedisaddedto
theculturemediumatafixedconcentration;inanimalmodelsandinhumanstreatedforTB,
however,drugconcentrationstypicallyfollowapharmacokineticconcentration-timecurve.
Moreover,with every new drug dosage every day, in vivo systems are not vulnerable to
chemicaldegradationwhichisaninherentflawintheinvitrostandardDSTassay.Oneway
toovercomethesemethodologicalflawswouldbetousethehollowfiberinfectionmodel
(HF).HFisaninvitrosystemthatusesapumpsystemwithculturemediawithfluctuating
drugconcentrationsovertime,mimickingmuchmorecloselythe invivoexposuretodrug
concentrationsovertime,correctingforchemicaldegradationofthecompoundunderstudy
aswellasmanyotherinherentflawsintheclassicalDSTassay.
InChapter2,wediscussinvitrostudiesreportingMICsofimipenemandmeropenemalone
orcombinedwithclavulanicacidatarangebetween0.16–32mg/L[4].Thishighvariability
of results between in vitro studiesmight be best explained by the chemical instability of
carbapenemsingrowthmediaattemperaturestypicallyusedininvitrostudies.Carbapenems
areheat-sensitiveandasM.tuberculosisgrowthneedsanincubationat37°Cfor15daysin
liquidmediaand4weeksinsolidmedia,carbapenemshavetowithstandahightemperature
foralongperiodoftime.Ertapenem,thatisunstableat37°C,islikelytobedegradedbefore
killingorinhibitingslow-growingM.tuberculosis,suggestingthaterroneouslyhighMICsfor
Mtbmighthavebeenreportedforcarbapenems[5].Itiscomprehensiblethatthisstability
issueofertapenemhaslikelynotoccurredbefore,sinceertapenemhasonlybeenprescribed
forawiderangeofbacterialinfections,consistingmostlyofawiderangeofgram-positive,
gram-negativeandanaerobicbacteriawithatypicalgenerationtimeof20min.DSTforthese
micro-organismsareavailablewithindays(maximumof48h),comparedtoslowgrowingM.
tuberculosiswithgenerationtimearound24h-resultinginDSTreportingtimeinweeks.
InChapter5,wedemonstrateanewstrategythatshowshowperformDSTsandmeasure
MICsforsuchunstablecompounds[6].Wefirstshowthatertapenemdegradesconsiderably
and secondly,we show thatertapenemsupplementation - to compensate for lossdue to
chemicaldegradation-bringsitwellwithintheEUCASTsusceptibilityrangeof0.5–1mg/L
[6]. However, only one reference strain, M. tuberculosis H37Ra, was used in our MIC
experiment.TodayantimicrobialDSTisbasedoncriticalconcentration-alsoreferredtoas
thebreakpoint -definedas ‘the lowestconcentrationofdrugthatwill inhibit95%ofwild
strainsofM.tuberculosisthathaveneverbeenexposedtodrugs,whileatthesametimenot
inhibitingclinicalstrainsofM.tuberculosisthatareconsideredtoberesistant’[3].Anextstep
130
Chapter 8
shouldbetostudythewildtypepopulationdistributionMICofcarbapenemswithaminimum
rangeofatleast15putativewild-typeisolatesandwithanoveralltotalofatleast100isolates,
preferably using the HF model. Subsequently, the critical concentration and the
epidemiologicalcutoff(ECOFF)canthenbedefined[7].
TreatmentofMDR-TB
InChapter2,weshowthatthereisapaucityofstudiesontheuseofcarbapenemsinM/XDR
TB.Fewinvivostudieshavebeencarriedouttodate,andonlytwolargeretrospectivestudies
withM/XDRTBpatientsusingimipenemandmeropenemhavebeenperformed;wefound
noclearevidencetoselectoneparticularcarbapenem[4].Noevidenceisavailabletolabel
ertapenemaspotentialGroupCdrugamongthecarbapenemsinthetreatmentofM/XDR
TB. Our focus of this thesis was therefore, to explore ertapenem treatment in order to
performaphaseIIstudyforertapenemtosubstantiateitspotentialasrepurposeddrug.
For studies exploring the clinical efficacy of ertapenem, a pharmacokinetic profile of
ertapenem needs to be established andwe therefore need a procedure to quantify and
validateertapenem inhumanplasmaandhumanserum.Asmentionedabove,stabilityof
ertapenemisanissue,thereforeertapenemneedstobetestedindifferenttestconditions,
including storage stability and at different storage temperatures as samples need to be
movedfromclinicaltrialsitestolaboratories.Wehavethereforedevelopedandvalidateda
liquid chromatography tandem-mass spectrometry (LS-MS/MS) method to quantify
ertapeneminhumanplasma,describedinChapter3ofthisthesis[8].
Inchapter4,weperformedaretrospectivestudytoevaluatesafetyandpharmacokineticsfor
allpatientswithsuspectedMDR-TBwhoreceived1gertapenemonadailybasisaspartof
theirtreatmentregimen[9].Itisinterestingtonotethatpatientsweregivenertapenemfor
aprolongedtime,upto9months;thisisincontrastwiththetreatmentofGram-negativeor
Gram-positivebacterialinfectionswheretheon-labelusetypicallylaststwoweeks.Inastudy
onsafetyandefficacyoflong-termoutpatientertapenemtreatment,ertapenemwasgiven
consecutivelyforaprolongedperiodoftime,8to16weeks,forconsecutivelyskinandsoft
tissueinfections,intra-abdominalinfectionsandosteomyelitis[10].
Ingeneral,ertapenemtreatmentappearswelltoleratedinlong-termtherapyinabroadrange
of bacterial infections. Ertapenem also showed a favorable pharmacokinetic/
pharmacodynamicprofile,withoutanyconstraintsinMDR-TBpatients[9].
Likeallbeta-lactamantimicrobials,ertapenemexhibitstime-dependantkilling.Assessment
of drug exposure of ertapenem should aim at assessing cumulative time above theMIC.
Obviously,andideally,theassessmentshouldaddressthefulldrugcombinationusedtotreat
MDR-TB[11].Usinglimitedsamplingstrategiesmightfacilitatetrialsinlessaffluentsettings.
Inchapter7of this thesis,wedescribea two-compartmentpharmacokineticmodelanda
limitedsamplingstrategythatwedevelopedtosupporttherapeuticdrugmonitoringforan
ertapenemdosageof1gdaily.Weshowthattwowell-timedsamples-at1and5h-was
adequatetopredictertapenemexposureinMDR-TBpatientstoreducetheburdenimposed
onpatientsandthehealthsystembymoreextensivemonitoring[12].Typically,twoorthree
samples are needed to estimate exposure.Other limited sampling strategieswith one to
threesamplingtimepointspost-dosehavealreadybeendevelopedforothersecond-lineanti-
TBdrugs,suchaslinezolid,levofloxacinandamikacin.Resultsshowedthatsamplingpointsat
1and5hpost-dosingwouldbesufficientforamikacin,0and5hforlevofloxacinandat0and
12h for linezolid.Asonlybest limitedsamplingstrategieswereshownfortheseTBdrugs
individually,itwouldbeimportantasanextsteptodeveloponelimitedsamplingstrategyfor
allanti-TBdrugs,usedasbackbonetherapyinthetreatmentofMDR-TB[13-15].
Dosefinding
Correctdosingisacriticalfirststepduringclinicaldevelopment.Thoughourpatientswere
treatedwithadosageof1g,thisdosewasnotclinicallyexploredinaprospectivephase2
(dose-finding)study.Pharmacokinetics/pharmacodynamicmodellingisessentialtooptimise
dosing.DosefractionationisdeemednecessarytodeterminewhichPK/PDparameterismost
importantforclinicalefficacy.Theoptimaltherapeuticdoserangecanbeselectedinadose-
findingstudyusingaHFinfectionmodelstudytomimicpharmacokineticconcentration-time
curvesofantibioticsobservedinTBpatients.ResultsofthisHFsystemcanbeusedinaMonte
Carlosimulationtoidentifytheoptimaldoseofertapenemandthesusceptibilitybreakpoint
basedonMICabovewhichtherapybyertapenemwillfail[16-17].
131
8
General Discussion and Future Perspectives
shouldbetostudythewildtypepopulationdistributionMICofcarbapenemswithaminimum
rangeofatleast15putativewild-typeisolatesandwithanoveralltotalofatleast100isolates,
preferably using the HF model. Subsequently, the critical concentration and the
epidemiologicalcutoff(ECOFF)canthenbedefined[7].
TreatmentofMDR-TB
InChapter2,weshowthatthereisapaucityofstudiesontheuseofcarbapenemsinM/XDR
TB.Fewinvivostudieshavebeencarriedouttodate,andonlytwolargeretrospectivestudies
withM/XDRTBpatientsusingimipenemandmeropenemhavebeenperformed;wefound
noclearevidencetoselectoneparticularcarbapenem[4].Noevidenceisavailabletolabel
ertapenemaspotentialGroupCdrugamongthecarbapenemsinthetreatmentofM/XDR
TB. Our focus of this thesis was therefore, to explore ertapenem treatment in order to
performaphaseIIstudyforertapenemtosubstantiateitspotentialasrepurposeddrug.
For studies exploring the clinical efficacy of ertapenem, a pharmacokinetic profile of
ertapenem needs to be established andwe therefore need a procedure to quantify and
validateertapenem inhumanplasmaandhumanserum.Asmentionedabove,stabilityof
ertapenemisanissue,thereforeertapenemneedstobetestedindifferenttestconditions,
including storage stability and at different storage temperatures as samples need to be
movedfromclinicaltrialsitestolaboratories.Wehavethereforedevelopedandvalidateda
liquid chromatography tandem-mass spectrometry (LS-MS/MS) method to quantify
ertapeneminhumanplasma,describedinChapter3ofthisthesis[8].
Inchapter4,weperformedaretrospectivestudytoevaluatesafetyandpharmacokineticsfor
allpatientswithsuspectedMDR-TBwhoreceived1gertapenemonadailybasisaspartof
theirtreatmentregimen[9].Itisinterestingtonotethatpatientsweregivenertapenemfor
aprolongedtime,upto9months;thisisincontrastwiththetreatmentofGram-negativeor
Gram-positivebacterialinfectionswheretheon-labelusetypicallylaststwoweeks.Inastudy
onsafetyandefficacyoflong-termoutpatientertapenemtreatment,ertapenemwasgiven
consecutivelyforaprolongedperiodoftime,8to16weeks,forconsecutivelyskinandsoft
tissueinfections,intra-abdominalinfectionsandosteomyelitis[10].
Ingeneral,ertapenemtreatmentappearswelltoleratedinlong-termtherapyinabroadrange
of bacterial infections. Ertapenem also showed a favorable pharmacokinetic/
pharmacodynamicprofile,withoutanyconstraintsinMDR-TBpatients[9].
Likeallbeta-lactamantimicrobials,ertapenemexhibitstime-dependantkilling.Assessment
of drug exposure of ertapenem should aim at assessing cumulative time above theMIC.
Obviously,andideally,theassessmentshouldaddressthefulldrugcombinationusedtotreat
MDR-TB[11].Usinglimitedsamplingstrategiesmightfacilitatetrialsinlessaffluentsettings.
Inchapter7of this thesis,wedescribea two-compartmentpharmacokineticmodelanda
limitedsamplingstrategythatwedevelopedtosupporttherapeuticdrugmonitoringforan
ertapenemdosageof1gdaily.Weshowthattwowell-timedsamples-at1and5h-was
adequatetopredictertapenemexposureinMDR-TBpatientstoreducetheburdenimposed
onpatientsandthehealthsystembymoreextensivemonitoring[12].Typically,twoorthree
samples are needed to estimate exposure.Other limited sampling strategieswith one to
threesamplingtimepointspost-dosehavealreadybeendevelopedforothersecond-lineanti-
TBdrugs,suchaslinezolid,levofloxacinandamikacin.Resultsshowedthatsamplingpointsat
1and5hpost-dosingwouldbesufficientforamikacin,0and5hforlevofloxacinandat0and
12h for linezolid.Asonlybest limitedsamplingstrategieswereshownfortheseTBdrugs
individually,itwouldbeimportantasanextsteptodeveloponelimitedsamplingstrategyfor
allanti-TBdrugs,usedasbackbonetherapyinthetreatmentofMDR-TB[13-15].
Dosefinding
Correctdosingisacriticalfirststepduringclinicaldevelopment.Thoughourpatientswere
treatedwithadosageof1g,thisdosewasnotclinicallyexploredinaprospectivephase2
(dose-finding)study.Pharmacokinetics/pharmacodynamicmodellingisessentialtooptimise
dosing.DosefractionationisdeemednecessarytodeterminewhichPK/PDparameterismost
importantforclinicalefficacy.Theoptimaltherapeuticdoserangecanbeselectedinadose-
findingstudyusingaHFinfectionmodelstudytomimicpharmacokineticconcentration-time
curvesofantibioticsobservedinTBpatients.ResultsofthisHFsystemcanbeusedinaMonte
Carlosimulationtoidentifytheoptimaldoseofertapenemandthesusceptibilitybreakpoint
basedonMICabovewhichtherapybyertapenemwillfail[16-17].
132
Chapter 8
Inchapter6,wepresentaHFinfectionmodelfordosefindingontheuseofertapenem.We
testeddifferentertapenemexposures,basedonhumanequivalentdosesinarangeof0.25–
10gertapenem.Dosefractionationshowedthatertapenemwaslinkedtothepercentageof
the 24 h dosing interval of ertapenem concentration persisting aboveMIC (%T/MIC). An
intravenousdosageof2gonceperdaywasidentifiedasmosteffectiveforsterilizingeffect.
This dosage can be used as a once-a-day dose for the treatment of MDR-TB.
AnertapenemsusceptibilitybreakpointMICof2mg/lwasidentifiedforthatdose[18].
The best possibleway to establish efficacy in a proof-of-principle studywould be to test
ertapeneminanearlybactericidalactivity(EBA)studyof2-weeksdurationintreatment-naive
patientswithdrug-susceptibleTB.Therearetwotechniques thatcanbeusedtomeasure
EBA;inliquidandsolidmedia.Insolidmedia,theEBAismeasuredtothefallinlog10CFUof
Mycobacteriumtuberculosispermlsputumperdayoverthefirst14daysoftreatment. In
liquidmediatheEBAisdeterminedbymeasuringthedailyprolongationoftimetopositivity
(TTP) frombaseline. ForTBdrugs,anEBAstudyhasbeenestablishedas thebestway to
establishefficacy[19-20].Datainthisthesiscanbeusedasastartingpointforawell-designed
prospective phase 2 EBA study to substantiate efficacy and safety of 2 g ertapenem in
combinationwithclavulanicacidontopofanoptimizedbackgroundregimenversusstandard
ofcareinpatientswithdrug-resistantTB.Apopulationmodelandlimitedsamplingstrategy
wasdesignedtosupporttherapeuticdrugmonitoringfor2gertapenem[21].
Futureperspectives
CurrentstandardDSTsystemscannotovercomerapiddecreaseofinitialdrugconcentration
over time due to chemical instability of ertapenem in standard-agar based MIC assays.
Althoughconceptuallysuperior,HFinfectionmodelsareexpensiveforroutineDST.Genotypic
testing might be an option to monitor for resistance to carbapenems. In other bacteria
unrelated to M. tuberculosis, it has already been shown that changes in membrane
permeability andpresenceof effluxpumpsmight lead to resistance tobeta lactams [22].
Recently,a singlenucleotidepolymorphism (SNP) indeRv2421c-Rv2422 intergenic region
wasfoundtobecommonamongM.tuberculosismutants,namedascarbapenemresistance
factor A (CrfA) [22].Whole-genome sequencingwas compelling to attribute carbapenem
resistance to thismutation. Incontrast to time-consumingMIC testing,utilizinggenotypic
testingforthesemutationswouldaccelerateknowledgeoncarbapenemresistance,thereby
preventingpoortreatmentoutcomes.Wholegenomesequencing(WGS)hasthepotentialto
rapidly enable insight into resistance profiles of Mycobacterium tuberculosis strains and
improveindividualizedtreatmentonalargescale[23].Therefore,furthergeneticstudiesand
explorationofthiscarbapenemresistancefactorareessentialtounderstanditspotentialfor
detectionofcarbapenemresistancetoM.tuberculosis.
Ahigh-qualityindividualpatientdatameta-analysisreportedalackofbenefitofcommonly
prescribed second-line injectable (kanamycin, capreomycin) andoraldrugs (ethionamide);
indeed,theirusewasassociatedwithpooroutcomes[1].Itwascommunicatedthatitisnot
expedient or desirable to give these second-line injectables. Moreover, FDA black box
warning for aminoglycosides appeared on the package insert due to side effects such as
hearinglossandkidneydamage[24].Wehypothesizethatcarbapenemsmightbepreferred
abovethese injectabledrugs.Afterefficacyandsafetyofertapenemhasbeenshown ina
phase 2 EBA study, a phase 3 study could help for evaluating the different TB injectable
classes. Amikacin could be comparedwith the carbapenem that shows best efficacy in a
comparativeEBAstudy;suchphase3trialmighthelptoestablishwhichinjectableshouldbe
preferredinMDR-TBtreatment.
Beta-LactamaseC(BLaC)inhibitors,forexampleclavulanate,tazobactamandsulbactam,are
currentlynotavailableonthemarketassingleagentorincombinationwithacarbapenem.
Clinicalstudiesareconfinedsincecarbapenemsneedcombinedtreatment incombination
with amoxicillin/clavulanate. Unfortunately, gastrointestinal side effects are common,
complicating prolonged treatment. Addition of amoxicillin in combination showed a
synergisticeffectinvitro,butitsusewasassociatedwithsignificantlylesssuccessandgreat
mortality[1,25].Therefore,amoxicillinisnotrecommendedasaseparateagentagainstMDR-
TB[2].Itwouldbringaddedvaluewhenfiguringouthowmanypatientswouldbenefitfrom
acombinationofacarbapenemwithabeta-lactamaseinhibitortocreateabusinesscasefor
genericpharmaceuticalcompanies.Choosingacombinationofacarbapenemwithabeta-
lactamaseinhibitor,thebestpartnerbeta-lactamantibioticneedstobeidentified.Clavulanic
acid, tazobactam and sulbactam all have a half-life of 1h, however pharmacokinetic
propertiesas invitroactivity incombinationwithcarbapenemsareconsiderablydifferent
133
8
General Discussion and Future Perspectives
Inchapter6,wepresentaHFinfectionmodelfordosefindingontheuseofertapenem.We
testeddifferentertapenemexposures,basedonhumanequivalentdosesinarangeof0.25–
10gertapenem.Dosefractionationshowedthatertapenemwaslinkedtothepercentageof
the 24 h dosing interval of ertapenem concentration persisting aboveMIC (%T/MIC). An
intravenousdosageof2gonceperdaywasidentifiedasmosteffectiveforsterilizingeffect.
This dosage can be used as a once-a-day dose for the treatment of MDR-TB.
AnertapenemsusceptibilitybreakpointMICof2mg/lwasidentifiedforthatdose[18].
The best possibleway to establish efficacy in a proof-of-principle studywould be to test
ertapeneminanearlybactericidalactivity(EBA)studyof2-weeksdurationintreatment-naive
patientswithdrug-susceptibleTB.Therearetwotechniques thatcanbeusedtomeasure
EBA;inliquidandsolidmedia.Insolidmedia,theEBAismeasuredtothefallinlog10CFUof
Mycobacteriumtuberculosispermlsputumperdayoverthefirst14daysoftreatment. In
liquidmediatheEBAisdeterminedbymeasuringthedailyprolongationoftimetopositivity
(TTP) frombaseline. ForTBdrugs,anEBAstudyhasbeenestablishedas thebestway to
establishefficacy[19-20].Datainthisthesiscanbeusedasastartingpointforawell-designed
prospective phase 2 EBA study to substantiate efficacy and safety of 2 g ertapenem in
combinationwithclavulanicacidontopofanoptimizedbackgroundregimenversusstandard
ofcareinpatientswithdrug-resistantTB.Apopulationmodelandlimitedsamplingstrategy
wasdesignedtosupporttherapeuticdrugmonitoringfor2gertapenem[21].
Futureperspectives
CurrentstandardDSTsystemscannotovercomerapiddecreaseofinitialdrugconcentration
over time due to chemical instability of ertapenem in standard-agar based MIC assays.
Althoughconceptuallysuperior,HFinfectionmodelsareexpensiveforroutineDST.Genotypic
testing might be an option to monitor for resistance to carbapenems. In other bacteria
unrelated to M. tuberculosis, it has already been shown that changes in membrane
permeability andpresenceof effluxpumpsmight lead to resistance tobeta lactams [22].
Recently,a singlenucleotidepolymorphism (SNP) indeRv2421c-Rv2422 intergenic region
wasfoundtobecommonamongM.tuberculosismutants,namedascarbapenemresistance
factor A (CrfA) [22].Whole-genome sequencingwas compelling to attribute carbapenem
resistance to thismutation. Incontrast to time-consumingMIC testing,utilizinggenotypic
testingforthesemutationswouldaccelerateknowledgeoncarbapenemresistance,thereby
preventingpoortreatmentoutcomes.Wholegenomesequencing(WGS)hasthepotentialto
rapidly enable insight into resistance profiles of Mycobacterium tuberculosis strains and
improveindividualizedtreatmentonalargescale[23].Therefore,furthergeneticstudiesand
explorationofthiscarbapenemresistancefactorareessentialtounderstanditspotentialfor
detectionofcarbapenemresistancetoM.tuberculosis.
Ahigh-qualityindividualpatientdatameta-analysisreportedalackofbenefitofcommonly
prescribed second-line injectable (kanamycin, capreomycin) andoraldrugs (ethionamide);
indeed,theirusewasassociatedwithpooroutcomes[1].Itwascommunicatedthatitisnot
expedient or desirable to give these second-line injectables. Moreover, FDA black box
warning for aminoglycosides appeared on the package insert due to side effects such as
hearinglossandkidneydamage[24].Wehypothesizethatcarbapenemsmightbepreferred
abovethese injectabledrugs.Afterefficacyandsafetyofertapenemhasbeenshown ina
phase 2 EBA study, a phase 3 study could help for evaluating the different TB injectable
classes. Amikacin could be comparedwith the carbapenem that shows best efficacy in a
comparativeEBAstudy;suchphase3trialmighthelptoestablishwhichinjectableshouldbe
preferredinMDR-TBtreatment.
Beta-LactamaseC(BLaC)inhibitors,forexampleclavulanate,tazobactamandsulbactam,are
currentlynotavailableonthemarketassingleagentorincombinationwithacarbapenem.
Clinicalstudiesareconfinedsincecarbapenemsneedcombinedtreatment incombination
with amoxicillin/clavulanate. Unfortunately, gastrointestinal side effects are common,
complicating prolonged treatment. Addition of amoxicillin in combination showed a
synergisticeffectinvitro,butitsusewasassociatedwithsignificantlylesssuccessandgreat
mortality[1,25].Therefore,amoxicillinisnotrecommendedasaseparateagentagainstMDR-
TB[2].Itwouldbringaddedvaluewhenfiguringouthowmanypatientswouldbenefitfrom
acombinationofacarbapenemwithabeta-lactamaseinhibitortocreateabusinesscasefor
genericpharmaceuticalcompanies.Choosingacombinationofacarbapenemwithabeta-
lactamaseinhibitor,thebestpartnerbeta-lactamantibioticneedstobeidentified.Clavulanic
acid, tazobactam and sulbactam all have a half-life of 1h, however pharmacokinetic
propertiesas invitroactivity incombinationwithcarbapenemsareconsiderablydifferent
134
Chapter 8
between these compounds [26]. Sulbactamwas shown to have themost potent activity
againstMDR-TBisolates[27].Meanwhile,ceftazidime-avibactam,acephalosporinisavailable
onthemarket incombinationwithaBLaC inhibitorasparentalagentandshowedpotent
sterilizingactivityagainstdrug-resistantTB[28].
TheWorldHealthOrganizationhasnow rankedTBmedicationwith apreference fororal
agentsoverinjectabledrugs[2].Therefore,weanticipatethatTBprogramswillstartusing
injectables less frequently. In order for ertapenem to have a place in an oral treatment
regimen,anextstepwouldbetoalterthephysical-chemicalpropertiesofertapeneminsuch
amatterthatitcanbedesignedasanoraldrug.Asoftoday,twocarbapenems,faropenem-
medoxomilandtebipenem-pivoxil,arealreadyavailableasoralpro-drugs,butbothhaveyet
tobeapprovedinEurope(EMA)andtheUnitedstates(FDA).Tebipenem-pivoxil isnowin
clinicaldevelopmentasfirstoralcarbapenemfortreatment inbacterial infections inadult
patients[29].Likeertapenemabetterunderstandingofsafety,tolerability,pharmacokinetics
andadose-findingstudyforbothdrugsinMDR-TBpatientsshouldbeperformedtoexplore
thefeasibilityasanti-TBoralagents.
Recently, inhaled antibiotic therapy has attracted increased attention and is becoming a
promising alternative for parental administration. A formulation of colistin improved the
aerolization of meropenem and showed synergistic bacterial killing against multi-drug
resistant gram-negative pathogens [30]. Some of the potential benefits of inhalation
antibiotics are that the inhaled drug doses are delivered directly to the target areas in
pulmonaryTB;andthattopicaldeliverymightreducesystemicsideeffects.Therefore,dry
powderinhalationantibiotics in low-costgeneric inhalers,whichcandelivermultiplehigh-
dosedrypowdercapsulesinresource-lowsettingsmightbethelifesavereveryoneiswaiting
for.
Conclusion
Inconclusion,ertapenemshouldbefurtherdevelopedandstudiedtoexploreitspotentialas
avaluableassetinthetreatmentofMDR-TB.Itistimetostudyitsmeritsinaphase2EBA
studyandnext,labelertapenemasgroupCdrugamongsttheothercarbapenems.
References1. WHO report: Rapid Communication: Key changes to treatment of multidrug- and
rifampicin-resistant tuberculosis (MDR/RR-TB) 2018. Available at:
http://www.who.int/tb/publications/2018/rapid_communications_MDR/en/
2. Collaborative Group for the Meta-Analysis of Individual Patient Data in MDR-TB
treatment–2017,AhmadN,AhujaSD,AkkermanOW,AlffenaarJC,AndersonLF,Baghaei
P,BangD,BarryPM,BastosML,BeheraD,BenedettiA,BissonGP,BoereeMJ,BonnetM,
BrodeSK,BrustJCM,CaiY,CaumesE,CegielskiJP,CentisR,ChanPC,ChanED,ChangKC,
CharlesM,CiruleA,DalcolmoMP,D'AmbrosioL,deVriesG,DhedaK,EsmailA,FloodJ,
FoxGJ,Fréchet-JachymM,FregonaG,GayosoR,GegiaM,GlerMT,GuS,GuglielmettiL,
HoltzTH,HughesJ,IsaakidisP,JarlsbergL,KempkerRR,KeshavjeeS,KhanFA,KipianiM,
KoenigSP,KohWJ,KritskiA,KuksaL,KvasnovskyCL,KwakN,LanZ,LangeC,Laniado-
LaborínR,LeeM,LeimaneV,LeungCC,LeungEC,LiPZ,LowenthalP,MacielEL,Marks
SM,MaseS,MbuagbawL,MiglioriGB,MilanovV,MillerAC,MitnickCD,ModongoC,
MohrE,MonederoI,NahidP,NdjekaN,O'DonnellMR,PadayatchiN,PalmeroD,Pape
JW,PodewilsLJ,Reynolds I,RiekstinaV,Robert J,RodriguezM,SeaworthB,SeungKJ,
Schnippel K, Shim TS, Singla R, Smith SE, Sotgiu G, Sukhbaatar G, Tabarsi P, Tiberi S,
TrajmanA,TrieuL,UdwadiaZF,vanderWerfTS,VezirisN,ViikleppP,VilbrunSC,Walsh
K,WestenhouseJ,YewWW,YimJJ,ZetolaNM,ZignolM,MenziesD.Treatmentcorrelates
of successful outcomes in pulmonary multidrug-resistant tuberculosis: an individual
patientdatameta-analysis.Lancet.2018Sep8;392(10150):821-834.doi:10.1016/S0140-
6736(18)31644-1.Review.
3. WHO report 2018: Technical report on critical concentrations for drug susceptibility
testingofmedicinesused in the treatmentofdrug-resistant tuberculosis.Availableat:
www.who.int/tb/publications/2018/WHO_technical_report_concentrations_TB_drug_s
usceptibility/en/
4. SanderP.vanRijn,MarlankaA.Zuur,RichardAnthony,BobWilffert,RichardvanAltena,
OnnoW.Akkerman,WielC.M.deLange,TjipS.vanderWerf,JosG.W.Kosterink,Jan-
Willem C. Alffenaar. Evaluation of carbapenems for multi/extensive-drug resistant
Mycobacterium tuberculosis treatment. Antimicrob. Agents Chemother. [To be
published].
135
8
General Discussion and Future Perspectives
between these compounds [26]. Sulbactamwas shown to have themost potent activity
againstMDR-TBisolates[27].Meanwhile,ceftazidime-avibactam,acephalosporinisavailable
onthemarket incombinationwithaBLaC inhibitorasparentalagentandshowedpotent
sterilizingactivityagainstdrug-resistantTB[28].
TheWorldHealthOrganizationhasnow rankedTBmedicationwith apreference fororal
agentsoverinjectabledrugs[2].Therefore,weanticipatethatTBprogramswillstartusing
injectables less frequently. In order for ertapenem to have a place in an oral treatment
regimen,anextstepwouldbetoalterthephysical-chemicalpropertiesofertapeneminsuch
amatterthatitcanbedesignedasanoraldrug.Asoftoday,twocarbapenems,faropenem-
medoxomilandtebipenem-pivoxil,arealreadyavailableasoralpro-drugs,butbothhaveyet
tobeapprovedinEurope(EMA)andtheUnitedstates(FDA).Tebipenem-pivoxil isnowin
clinicaldevelopmentasfirstoralcarbapenemfortreatment inbacterial infections inadult
patients[29].Likeertapenemabetterunderstandingofsafety,tolerability,pharmacokinetics
andadose-findingstudyforbothdrugsinMDR-TBpatientsshouldbeperformedtoexplore
thefeasibilityasanti-TBoralagents.
Recently, inhaled antibiotic therapy has attracted increased attention and is becoming a
promising alternative for parental administration. A formulation of colistin improved the
aerolization of meropenem and showed synergistic bacterial killing against multi-drug
resistant gram-negative pathogens [30]. Some of the potential benefits of inhalation
antibiotics are that the inhaled drug doses are delivered directly to the target areas in
pulmonaryTB;andthattopicaldeliverymightreducesystemicsideeffects.Therefore,dry
powderinhalationantibiotics in low-costgeneric inhalers,whichcandelivermultiplehigh-
dosedrypowdercapsulesinresource-lowsettingsmightbethelifesavereveryoneiswaiting
for.
Conclusion
Inconclusion,ertapenemshouldbefurtherdevelopedandstudiedtoexploreitspotentialas
avaluableassetinthetreatmentofMDR-TB.Itistimetostudyitsmeritsinaphase2EBA
studyandnext,labelertapenemasgroupCdrugamongsttheothercarbapenems.
References1. WHO report: Rapid Communication: Key changes to treatment of multidrug- and
rifampicin-resistant tuberculosis (MDR/RR-TB) 2018. Available at:
http://www.who.int/tb/publications/2018/rapid_communications_MDR/en/
2. Collaborative Group for the Meta-Analysis of Individual Patient Data in MDR-TB
treatment–2017,AhmadN,AhujaSD,AkkermanOW,AlffenaarJC,AndersonLF,Baghaei
P,BangD,BarryPM,BastosML,BeheraD,BenedettiA,BissonGP,BoereeMJ,BonnetM,
BrodeSK,BrustJCM,CaiY,CaumesE,CegielskiJP,CentisR,ChanPC,ChanED,ChangKC,
CharlesM,CiruleA,DalcolmoMP,D'AmbrosioL,deVriesG,DhedaK,EsmailA,FloodJ,
FoxGJ,Fréchet-JachymM,FregonaG,GayosoR,GegiaM,GlerMT,GuS,GuglielmettiL,
HoltzTH,HughesJ,IsaakidisP,JarlsbergL,KempkerRR,KeshavjeeS,KhanFA,KipianiM,
KoenigSP,KohWJ,KritskiA,KuksaL,KvasnovskyCL,KwakN,LanZ,LangeC,Laniado-
LaborínR,LeeM,LeimaneV,LeungCC,LeungEC,LiPZ,LowenthalP,MacielEL,Marks
SM,MaseS,MbuagbawL,MiglioriGB,MilanovV,MillerAC,MitnickCD,ModongoC,
MohrE,MonederoI,NahidP,NdjekaN,O'DonnellMR,PadayatchiN,PalmeroD,Pape
JW,PodewilsLJ,Reynolds I,RiekstinaV,Robert J,RodriguezM,SeaworthB,SeungKJ,
Schnippel K, Shim TS, Singla R, Smith SE, Sotgiu G, Sukhbaatar G, Tabarsi P, Tiberi S,
TrajmanA,TrieuL,UdwadiaZF,vanderWerfTS,VezirisN,ViikleppP,VilbrunSC,Walsh
K,WestenhouseJ,YewWW,YimJJ,ZetolaNM,ZignolM,MenziesD.Treatmentcorrelates
of successful outcomes in pulmonary multidrug-resistant tuberculosis: an individual
patientdatameta-analysis.Lancet.2018Sep8;392(10150):821-834.doi:10.1016/S0140-
6736(18)31644-1.Review.
3. WHO report 2018: Technical report on critical concentrations for drug susceptibility
testingofmedicinesused in the treatmentofdrug-resistant tuberculosis.Availableat:
www.who.int/tb/publications/2018/WHO_technical_report_concentrations_TB_drug_s
usceptibility/en/
4. SanderP.vanRijn,MarlankaA.Zuur,RichardAnthony,BobWilffert,RichardvanAltena,
OnnoW.Akkerman,WielC.M.deLange,TjipS.vanderWerf,JosG.W.Kosterink,Jan-
Willem C. Alffenaar. Evaluation of carbapenems for multi/extensive-drug resistant
Mycobacterium tuberculosis treatment. Antimicrob. Agents Chemother. [To be
published].
136
Chapter 8
5. Veziris, N., C. Truffot, J. L. Mainardi, and V. Jarlier. 2011. Activity of carbapenems
combinedwithclavulanateagainstmurinetuberculosis.Antimicrob.AgentsChemother.
55:2597-2600.doi:10.1128/AAC.01824-10;10.1128/AAC.01824-10.
6. Srivastava S, van Rijn SP,Wessels AMA, Alffenaar JWC,Gumbo T. 2016. Susceptibility
testing of antibiotics that degrade faster than the doubling time of slow-growing
mycobacteria: Ertapenem sterilizing effect ofMycobacterium tuberculosis. Antimicrob
AgentsChemother60:3193-3195.Doi:10.1128/AAC.02924-15.
7. EUCAST: Standard operating procedure MIC distributions and the setting of
epidemiological cut-off (ECOFF) values. 2017. Available at:
http://www.eucast.org/mic_distributions_and_ecoffs/
8. vanRijnSP,WesselsAM,GreijdanusB,etal.Quantificationandvalidationofertapenem
usinga liquidchromatography-tandemmassspectrometrymethod.AntimicrobAgents
Chemother2014;58:3481–3484.
9. VanRijnSP,vanAltenaR,Akkerman,OW,vanSoolingenD,vanderLaanT,deLangeWCM,
KosterinkJGW,vanderWerfTS,AlffenaarJWC.2016.Pharmacokineticsofertapenemin
patients with multidrug-resistant tuberculosis. Eur Respir J. 47:1229-1234.
Doi:10.1183/13993003.01654-2015.
10. QureshiZA,SyedA,DoiY.2014.Safetyandefficacyoflong-termoutpatientertapenem
therapy.AntimicrobAgentsChemother.Jun;58(6)
11. NicolauDP.Pharmacokineticandpharmacodynamicpropertiesofmeropenem.ClinInfect
Dis2008;47:Suppl.1,S32–S40.
12. VanRijnSP,ZuurMA,vanAltenaR,AkkermanOW,ProostJH,deLangeWCM,Kerstjens
HAM,TouwDJ,vanderWerfTS,KosterinkJGW,AlffenaarJWC.2017.Pharmacokinetic
modelingandlimitedsamplingstrategiesbasedonhealthyvolunteersformonitoringof
ertapenem in patients with multidrug-resistant tuberculosis. Antimicrob Agents
Chemother61:e01783-16.Doi:10.1128/AAC.01783-16
13. KampJ,BolhuisMS,TiberiS,AkkermanOW,CentisR,deLangeWC,KosterinkJG,vander
WerfTS,MiglioriGB,AlffenaarJC.Simplestrategytoassesslinezolidexposureinpatients
with multi-drug-resistant and extensively-drug-resistant tuberculosis. Int J Antimicrob
Agents.2017Jun;49(6):688-694.doi:10.1016/j.ijantimicag.2017.01.017.Epub2017Apr4
14. vandenElsenSHJ,SturkenboomMGG,Van'tBoveneind-VrubleuskayaN,SkrahinaA,van
derWerfTS,Heysell SK,MpagamaS,MiglioriGB,PeloquinCA,TouwDJ,Alffenaar JC.
2018. Population Pharmacokinetic Model and Limited Sampling Strategies for
Personalized Dosing of Levofloxacin in Tuberculosis Patients. Antimicrob Agents
Chemother.Nov26;62(12).
15. Dijkstra JA, van Altena R, Akkerman OW, de LangeWC, Proost JH, van derWerf TS,
KosterinkJG,AlffenaarJW.Limitedsamplingstrategiesfortherapeuticdrugmonitoring
of amikacin and kanamycin in patients with multidrug-resistant tuberculosis. Int J
AntimicrobAgents.2015Sep;46(3):332-7.doi:10.1016/j.ijantimicag.2015.06.008.Epub
2015Jul15.
16. GumboT,Angulo-BarturenI,Ferrer-BazagaS.2015.Pharmacokinetic-Pharmacodynamic
and Dose-Response Relationships of Antituberculosis Drugs: Recommendations and
StandardsforIndustryandAcademia.Jinfectdis.211(S3):S96-106
17. EMA Guideline on Hollow fiber system. 2018: Available at:
https://www.ema.europa.eu/documents/regulatory-procedural-guideline/qualification-
opinion-vitro-hollow-fibre-system-model-tuberculosis-hfs-tb_en.pdf
18. VanRijnSP,SrivastavaS,WesselsMA,vanSoolingenD,AlffenaarJW,GumboT.2017.The
sterilizingeffectofertapenem-clavulanate inahollow fibermodelof tuberculosis and
implicationsonclinicaldosing.AntimicrobAgentsChemotherdoi:10.1128/AAC.02039-16
19. DiaconAH,DawsonR,vonGroote-BidlingmaierF,SymonsG,VenterA,DonaldPR,van
Niekerk C, Everitt D, Winter H, Becker P, Mendel CM, Spigelman MK. 2012. 14-day
bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin
combinations:arandomisedtrial.Lancet.Sep15;380(8846):986-93.
20. Diacon AH, Donald PR. 2014. The early bactericidal acitivity of antituberculosis drugs.
ExpertRev.AntiInfect.Ther.12(2):223-37.
21. ZuurM,GhimireS,BolhuisMS,WesselsAMA,vanAltenaR,deLangeWCM,Kosterink
JGW, Touw DJ, van der Werf TS, Akkerman OW, Alffenaar JWC. 2018. The
pharmacokinetics of 2000 mg Ertapenem in tuberculosis patients. Antimicrob Agents
Chemother.
137
8
General Discussion and Future Perspectives
5. Veziris, N., C. Truffot, J. L. Mainardi, and V. Jarlier. 2011. Activity of carbapenems
combinedwithclavulanateagainstmurinetuberculosis.Antimicrob.AgentsChemother.
55:2597-2600.doi:10.1128/AAC.01824-10;10.1128/AAC.01824-10.
6. Srivastava S, van Rijn SP,Wessels AMA, Alffenaar JWC,Gumbo T. 2016. Susceptibility
testing of antibiotics that degrade faster than the doubling time of slow-growing
mycobacteria: Ertapenem sterilizing effect ofMycobacterium tuberculosis. Antimicrob
AgentsChemother60:3193-3195.Doi:10.1128/AAC.02924-15.
7. EUCAST: Standard operating procedure MIC distributions and the setting of
epidemiological cut-off (ECOFF) values. 2017. Available at:
http://www.eucast.org/mic_distributions_and_ecoffs/
8. vanRijnSP,WesselsAM,GreijdanusB,etal.Quantificationandvalidationofertapenem
usinga liquidchromatography-tandemmassspectrometrymethod.AntimicrobAgents
Chemother2014;58:3481–3484.
9. VanRijnSP,vanAltenaR,Akkerman,OW,vanSoolingenD,vanderLaanT,deLangeWCM,
KosterinkJGW,vanderWerfTS,AlffenaarJWC.2016.Pharmacokineticsofertapenemin
patients with multidrug-resistant tuberculosis. Eur Respir J. 47:1229-1234.
Doi:10.1183/13993003.01654-2015.
10. QureshiZA,SyedA,DoiY.2014.Safetyandefficacyoflong-termoutpatientertapenem
therapy.AntimicrobAgentsChemother.Jun;58(6)
11. NicolauDP.Pharmacokineticandpharmacodynamicpropertiesofmeropenem.ClinInfect
Dis2008;47:Suppl.1,S32–S40.
12. VanRijnSP,ZuurMA,vanAltenaR,AkkermanOW,ProostJH,deLangeWCM,Kerstjens
HAM,TouwDJ,vanderWerfTS,KosterinkJGW,AlffenaarJWC.2017.Pharmacokinetic
modelingandlimitedsamplingstrategiesbasedonhealthyvolunteersformonitoringof
ertapenem in patients with multidrug-resistant tuberculosis. Antimicrob Agents
Chemother61:e01783-16.Doi:10.1128/AAC.01783-16
13. KampJ,BolhuisMS,TiberiS,AkkermanOW,CentisR,deLangeWC,KosterinkJG,vander
WerfTS,MiglioriGB,AlffenaarJC.Simplestrategytoassesslinezolidexposureinpatients
with multi-drug-resistant and extensively-drug-resistant tuberculosis. Int J Antimicrob
Agents.2017Jun;49(6):688-694.doi:10.1016/j.ijantimicag.2017.01.017.Epub2017Apr4
14. vandenElsenSHJ,SturkenboomMGG,Van'tBoveneind-VrubleuskayaN,SkrahinaA,van
derWerfTS,Heysell SK,MpagamaS,MiglioriGB,PeloquinCA,TouwDJ,Alffenaar JC.
2018. Population Pharmacokinetic Model and Limited Sampling Strategies for
Personalized Dosing of Levofloxacin in Tuberculosis Patients. Antimicrob Agents
Chemother.Nov26;62(12).
15. Dijkstra JA, van Altena R, Akkerman OW, de LangeWC, Proost JH, van derWerf TS,
KosterinkJG,AlffenaarJW.Limitedsamplingstrategiesfortherapeuticdrugmonitoring
of amikacin and kanamycin in patients with multidrug-resistant tuberculosis. Int J
AntimicrobAgents.2015Sep;46(3):332-7.doi:10.1016/j.ijantimicag.2015.06.008.Epub
2015Jul15.
16. GumboT,Angulo-BarturenI,Ferrer-BazagaS.2015.Pharmacokinetic-Pharmacodynamic
and Dose-Response Relationships of Antituberculosis Drugs: Recommendations and
StandardsforIndustryandAcademia.Jinfectdis.211(S3):S96-106
17. EMA Guideline on Hollow fiber system. 2018: Available at:
https://www.ema.europa.eu/documents/regulatory-procedural-guideline/qualification-
opinion-vitro-hollow-fibre-system-model-tuberculosis-hfs-tb_en.pdf
18. VanRijnSP,SrivastavaS,WesselsMA,vanSoolingenD,AlffenaarJW,GumboT.2017.The
sterilizingeffectofertapenem-clavulanate inahollow fibermodelof tuberculosis and
implicationsonclinicaldosing.AntimicrobAgentsChemotherdoi:10.1128/AAC.02039-16
19. DiaconAH,DawsonR,vonGroote-BidlingmaierF,SymonsG,VenterA,DonaldPR,van
Niekerk C, Everitt D, Winter H, Becker P, Mendel CM, Spigelman MK. 2012. 14-day
bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin
combinations:arandomisedtrial.Lancet.Sep15;380(8846):986-93.
20. Diacon AH, Donald PR. 2014. The early bactericidal acitivity of antituberculosis drugs.
ExpertRev.AntiInfect.Ther.12(2):223-37.
21. ZuurM,GhimireS,BolhuisMS,WesselsAMA,vanAltenaR,deLangeWCM,Kosterink
JGW, Touw DJ, van der Werf TS, Akkerman OW, Alffenaar JWC. 2018. The
pharmacokinetics of 2000 mg Ertapenem in tuberculosis patients. Antimicrob Agents
Chemother.
138
Chapter 8
22. KumarP,KaushikA,BellDT,ChauhanV,XiaF,StevensRL,LamichhaneG.2017.Mutation
inanunannotatedproteinconferscarbapenemresistanceinmycobacteriumtuberculosis.
Antimicrobagentschemother.Feb23;61(3).
23. GröshelMI,WalkerTM,vanderWerfTS,LangeC,NiemannS,MerkerM.2018.Pathogen-
basedprecisionmedicinefordrug-resistanttuberculosis.PLoSPathog12(10):e1007297.
24. vanAltenaR,DijkstraJA,vanderMeerME,BorjasHowardJF,KosterinkJG,vanSoolingen
D, van der Werf TS, Alffenaar JW. Reduced Chance of Hearing Loss Associated with
TherapeuticDrugMonitoringofAminoglycosidesintheTreatmentofMultidrug-Resistant
Tuberculosis. Antimicrob Agents Chemother. 2017 Feb 23;61(3). pii: e01400-16. doi:
10.1128/AAC.01400-16.Print2017Mar.
25. GonzaloX,Drobniewski F. 2013. Is there aplace for beta-lactams in the treatmentof
multidrug-resistant/extensively drug-resistant tuberculosis? Synergy between
meropenem and amoxicillin/clavulanate. J. Antimicrob Chemother. 68:366-369. doi:
10.1093/jac/dks395;10.1093/jac/dks395.
26. delaPenaA,DerendorfH.1999.Pharmacokineticpropertiesofbeta-lactamaseinhibitors.
IntJClinPharmacolTher.Feb;37(2):63-75
27. ZhangD,WangY,LuJ,PangY.2015.InVitroActivityofβ-LactamsinCombinationwithβ-
Lactamase Inhibitors againstMultidrug-ResistantMycobacterium tuberculosis Isolates.
AntimicrobAgentsChemother.Nov2;60(1):393-9.
28. DeshpandeD,SrivastavaS,ChapagainM,MagombedzeG,MartinKR,CirrincioneKN,Lee
PS, Koeuth T, Dheda K, Gumbo T. 2017. Ceftazidime-avibactam has potent sterilizing
activityagainsthighlydrug-resistanttuberculosis.SciAdv.2017Aug30;3(8)
29. JainA,UtleyL,ParrTR,ZabawaT,PucciMJ.2018.Tebipenem,thefirstoralcarbapenem
antibiotic.ExpertRevAntiInfectTher.Jul;16)7:513-522
30. MangalS,ParkH,ZengL,YuHH,LinYW,VelkovT,DenmanJA,ZemlyanovD,LiJ,Zhou
QT.Compositeparticleformulationsofcolistinandmeropenemwithimproved in-vitro
bacterialkillingandaerosolizationforinhalation.IntJPharm.2018Sep5;548(1):443-453.
22. KumarP,KaushikA,BellDT,ChauhanV,XiaF,StevensRL,LamichhaneG.2017.Mutation
inanunannotatedproteinconferscarbapenemresistanceinmycobacteriumtuberculosis.
Antimicrobagentschemother.Feb23;61(3).
23. GröshelMI,WalkerTM,vanderWerfTS,LangeC,NiemannS,MerkerM.2018.Pathogen-
basedprecisionmedicinefordrug-resistanttuberculosis.PLoSPathog12(10):e1007297.
24. vanAltenaR,DijkstraJA,vanderMeerME,BorjasHowardJF,KosterinkJG,vanSoolingen
D, van der Werf TS, Alffenaar JW. Reduced Chance of Hearing Loss Associated with
TherapeuticDrugMonitoringofAminoglycosidesintheTreatmentofMultidrug-Resistant
Tuberculosis. Antimicrob Agents Chemother. 2017 Feb 23;61(3). pii: e01400-16. doi:
10.1128/AAC.01400-16.Print2017Mar.
25. GonzaloX,Drobniewski F. 2013. Is there aplace for beta-lactams in the treatmentof
multidrug-resistant/extensively drug-resistant tuberculosis? Synergy between
meropenem and amoxicillin/clavulanate. J. Antimicrob Chemother. 68:366-369. doi:
10.1093/jac/dks395;10.1093/jac/dks395.
26. delaPenaA,DerendorfH.1999.Pharmacokineticpropertiesofbeta-lactamaseinhibitors.
IntJClinPharmacolTher.Feb;37(2):63-75
27. ZhangD,WangY,LuJ,PangY.2015.InVitroActivityofβ-LactamsinCombinationwithβ-
Lactamase Inhibitors againstMultidrug-ResistantMycobacterium tuberculosis Isolates.
AntimicrobAgentsChemother.Nov2;60(1):393-9.
28. DeshpandeD,SrivastavaS,ChapagainM,MagombedzeG,MartinKR,CirrincioneKN,Lee
PS, Koeuth T, Dheda K, Gumbo T. 2017. Ceftazidime-avibactam has potent sterilizing
activityagainsthighlydrug-resistanttuberculosis.SciAdv.2017Aug30;3(8)
29. JainA,UtleyL,ParrTR,ZabawaT,PucciMJ.2018.Tebipenem,thefirstoralcarbapenem
antibiotic.ExpertRevAntiInfectTher.Jul;16)7:513-522
30. MangalS,ParkH,ZengL,YuHH,LinYW,VelkovT,DenmanJA,ZemlyanovD,LiJ,Zhou
QT.Compositeparticleformulationsofcolistinandmeropenemwithimprovedin-vitro
bacterialkillingandaerosolizationforinhalation.IntJPharm.2018Sep5;548(1):443-453.
CHAPTER 9
Summary
CHAPTER 9
Summary
142
Chapter 9
SummaryTuberculosis (TB) is caused byMycobacterium tuberculosis and is the deadliest infectious
diseaseworldwide.OurworldisfacingapublichealthcrisisastreatmentofTBhasbecome
morechallengingwiththeemergenceofresistancetofirst-linedrugs,makingitdifficultto
eradicate TBby 2030. Therefore, it is urgent to focus on improving current treatment by
developing more active – sterilizing- anti TB drugs. One particularly effective strategy is
rediscovery of old drugs as new agents for treatment against in multidrug resistant
tuberculosis.Beta-lactamantimicrobialdrugsarewidelyuseddrugsforthetreatmentofa
rangeofinfections.Ertapenem,approvedin2001bytheFDAandwidelyusedagainstgram
positiveandnegativebacteria,hasshowntobeactiveinMDRTB.Tobetterunderstandthe
potential roleofertapenemfor the treatmentofM/XDR-TB, theaimof this thesiswas to
evaluatecurrentliterature,invitroactivity,andpharmacokineticsandsafetyinTBpatients.
CarbapenemsareearmarkedaspotentiallyactivedrugsforthetreatmentofM.tuberculosis.
Inchapter2weevaluatedthepotentialofcarbapenemsforthetreatmentofM/XDR-TB.The
aimofthisreviewwastoevaluatetheliteratureoncurrentlyavailableinvitro,invivoand
clinicaldataoncarbapenemsinthetreatmentofM.tuberculosisanddetectionofknowledge
gaps, inordertotargetfutureresearch.Overalltheresultsofthestudies identifiedinthis
review are consistent. Carbapenems in combination with clavulanate showed increased
activity in vitro. Few in vivo studies have been performed. Ten clinical studies to assess
effectiveness,safetyandtolerabilityofthreedifferentcarbapenems(imipenem,meropenem
and ertapenem) were performed. No clear evidence was found to select one particular
carbapenemtodesignaneffectiveM/XDR-TBregimen.Moreclinicalevidenceisneededto
supportrepurposingcarbapenemsforthetreatmentofM/XDR-TB.
In chapter 3 a new simple and robust LC-MS/MS method using a quadruple mass
spectrometerwasdevelopedforanalysisofertapeneminhumanplasma,usingdeuterated
ertapenemasinternalstandard.Thecalibrationcurvewaslinearoverarangeof0.1(LLOQ)
to125mg/L.Thecalculatedaccuracyrangedfrom-2.4%to10.3%.Within-runCVranged
from2.7%to11.8%andbetween-runCVranged from0%to8.4%.Freeze-thawstability
biasedbetween-3.3%and0.1%.StorageofQCsamplesfor96hat4°Cdiffered-4.3to5.6%,
storage at room temperature for 24h, biased from -10.7% to -14.8% and storage in the
autosamplerbiasedbetween-2.9%and-10.0%.
Inchapter4,aretrospectivestudywasperformedforallMDR-TBsuspectedpatientsatthe
Tuberculosis Center Beatrixoord of University Medical Center Groningen (Haren, The
Netherlands)whoreceivedertapenemaspartoftheirtreatmentregimenbetweenthefirst
ofDecember2010andthefirstofMarch2013.Safetyandpharmacokineticswereevaluated.
Eighteenpatientsweretreatedwith1000mgertapenemforameanof77days(range5-210).
Sputumsmearandculturewereconvertedinallpatients.Drugexposurewasevaluatedin12
patients. ThemeanAUC0-24was544,9 (range309–1130)mg*h/L. ThemeanCmaxwas
127.5(73.9–277.9)mg/L.Ingeneral,ertapenemtreatmentwaswelltoleratedduringMDR-
TBtreatmentandshowedafavourablePK/PDprofileinMDR-TBpatients.
Inchapter5wepresentedtherapiddeclineofertapenemduringDSTandwehavethereby
developedanewstrategytoperformDSTandMICsforsuchunstablecompounds.Wehave
shownthatertapenemsupplementationbringsitwellwithinthesusceptibilityrangeandis
likelytohavegoodsterilizingeffectintuberculosis.Thissuggeststhatmostofthepublished
MICsforthisdrugarelikelyfalselyhighandratesofresistancearelikelyfalselyelevated.
Inchapter6ourobjectivewastoidentifytheertapenemexposureassociatedwithoptimal
sterilizingeffectandthendesignaonceadaydoseforclinicaluse.Weutilizedthehollow
fiber system model of tuberculosis in a 28-day exposure-response study of 8 different
ertapenem doses in combination with clavulanate. The systems were sampled at
predetermined time-points to verify the concentration-time profile and identify the total
bacterialburden.Ertapenem-clavulanatecombinationdemonstratedgoodmicrobialkilland
sterilizingeffect.Inadose-fractionationhollowfiberstudy,efficacywaslinkedtopercentage
of the 24-hour dosing interval of ertapenem concentration persisting above MIC. We
identifiedan intravenousdoseof2gramsonceperdayasachieving the target in96%of
patients.
143
9
Summary
SummaryTuberculosis (TB) is caused byMycobacterium tuberculosis and is the deadliest infectious
diseaseworldwide.OurworldisfacingapublichealthcrisisastreatmentofTBhasbecome
morechallengingwiththeemergenceofresistancetofirst-linedrugs,makingitdifficultto
eradicate TBby 2030. Therefore, it is urgent to focus on improving current treatment by
developing more active – sterilizing- anti TB drugs. One particularly effective strategy is
rediscovery of old drugs as new agents for treatment against in multidrug resistant
tuberculosis.Beta-lactamantimicrobialdrugsarewidelyuseddrugsforthetreatmentofa
rangeofinfections.Ertapenem,approvedin2001bytheFDAandwidelyusedagainstgram
positiveandnegativebacteria,hasshowntobeactiveinMDRTB.Tobetterunderstandthe
potential roleofertapenemfor the treatmentofM/XDR-TB, theaimof this thesiswas to
evaluatecurrentliterature,invitroactivity,andpharmacokineticsandsafetyinTBpatients.
CarbapenemsareearmarkedaspotentiallyactivedrugsforthetreatmentofM.tuberculosis.
Inchapter2weevaluatedthepotentialofcarbapenemsforthetreatmentofM/XDR-TB.The
aimofthisreviewwastoevaluatetheliteratureoncurrentlyavailableinvitro,invivoand
clinicaldataoncarbapenemsinthetreatmentofM.tuberculosisanddetectionofknowledge
gaps, inordertotargetfutureresearch.Overalltheresultsofthestudies identifiedinthis
review are consistent. Carbapenems in combination with clavulanate showed increased
activity in vitro. Few in vivo studies have been performed. Ten clinical studies to assess
effectiveness,safetyandtolerabilityofthreedifferentcarbapenems(imipenem,meropenem
and ertapenem) were performed. No clear evidence was found to select one particular
carbapenemtodesignaneffectiveM/XDR-TBregimen.Moreclinicalevidenceisneededto
supportrepurposingcarbapenemsforthetreatmentofM/XDR-TB.
In chapter 3 a new simple and robust LC-MS/MS method using a quadruple mass
spectrometerwasdevelopedforanalysisofertapeneminhumanplasma,usingdeuterated
ertapenemasinternalstandard.Thecalibrationcurvewaslinearoverarangeof0.1(LLOQ)
to125mg/L.Thecalculatedaccuracyrangedfrom-2.4%to10.3%.Within-runCVranged
from2.7%to11.8%andbetween-runCVranged from0%to8.4%.Freeze-thawstability
biasedbetween-3.3%and0.1%.StorageofQCsamplesfor96hat4°Cdiffered-4.3to5.6%,
storage at room temperature for 24h, biased from -10.7% to -14.8% and storage in the
autosamplerbiasedbetween-2.9%and-10.0%.
Inchapter4,aretrospectivestudywasperformedforallMDR-TBsuspectedpatientsatthe
Tuberculosis Center Beatrixoord of University Medical Center Groningen (Haren, The
Netherlands)whoreceivedertapenemaspartoftheirtreatmentregimenbetweenthefirst
ofDecember2010andthefirstofMarch2013.Safetyandpharmacokineticswereevaluated.
Eighteenpatientsweretreatedwith1000mgertapenemforameanof77days(range5-210).
Sputumsmearandculturewereconvertedinallpatients.Drugexposurewasevaluatedin12
patients. ThemeanAUC0-24was544,9 (range309–1130)mg*h/L. ThemeanCmaxwas
127.5(73.9–277.9)mg/L.Ingeneral,ertapenemtreatmentwaswelltoleratedduringMDR-
TBtreatmentandshowedafavourablePK/PDprofileinMDR-TBpatients.
Inchapter5wepresentedtherapiddeclineofertapenemduringDSTandwehavethereby
developedanewstrategytoperformDSTandMICsforsuchunstablecompounds.Wehave
shownthatertapenemsupplementationbringsitwellwithinthesusceptibilityrangeandis
likelytohavegoodsterilizingeffectintuberculosis.Thissuggeststhatmostofthepublished
MICsforthisdrugarelikelyfalselyhighandratesofresistancearelikelyfalselyelevated.
Inchapter6ourobjectivewastoidentifytheertapenemexposureassociatedwithoptimal
sterilizingeffectandthendesignaonceadaydoseforclinicaluse.Weutilizedthehollow
fiber system model of tuberculosis in a 28-day exposure-response study of 8 different
ertapenem doses in combination with clavulanate. The systems were sampled at
predetermined time-points to verify the concentration-time profile and identify the total
bacterialburden.Ertapenem-clavulanatecombinationdemonstratedgoodmicrobialkilland
sterilizingeffect.Inadose-fractionationhollowfiberstudy,efficacywaslinkedtopercentage
of the 24-hour dosing interval of ertapenem concentration persisting above MIC. We
identifiedan intravenousdoseof2gramsonceperdayasachieving the target in96%of
patients.
144
Chapter 9
Inchapter7alimitedsamplingstrategywasdevelopedusingapopulationpharmacokinetic
model based, using an iterative two-stage Bayesian method, on healthy volunteers and
showedtobeadequatetopredictertapenemexposureinMDR-TBpatients.
ExternalvalidationwasperformedbyBayesianfittingofthemodeldevelopedinvolunteers
to the individual data of MDR-TB patients using the developed population model for
volunteers as a prior. A Monte Carlo simulation (n=1000) was used to evaluate limited
samplingstrategies.Thebestperforminglimitedsamplingstrategy,withatime-restrictionof
0-6h,wasfoundtobesamplingat1and5h(R2=0.78,meanpredictionerror=-0.33%anda
rootmeansquareerror=5.5%).Drugexposurewasoverestimatedbyameanpercentageof
4.2(-15.2–23.6%).Consideringafreefractionof5%andtheMICsetat0.5mg/L,9outof12
patientswouldhaveexceededaminimumoff40%T>MIC.
In thegeneral discussionwediscussed insight, tools and understanding of ertapenem as
potentialintreatmentofmultidrug-resistantTuberculosis.Wediscusseddrugsusceptibility
testingofcarbapenemsandthatthehighvariabilityofresultsofinvitrostudiesmightbebest
explained by the chemical instability of carbapenems in growth media at temperatures
typicallyusedininvitrostudies.Wenoticedthatertapenemtreatmentseemswelltolerated
inlong-termtherapyinbroadrangeofbacterialinfectionsasitwasshownretrospectivelyfor
MDR-TBtreatment.Aspatientsweretreatedwithadosageof1gram,itwasnotclinically
substantiated in a prospective phase 2 study. Therefore, we discussed that a dose
fractionationwasdeemednecessarytodeterminewhichPK/PDparameterismostimportant
forclinicalefficacy.Toattaintheoptimaltherapeuticdoserange,ahollowfiberstudywas
performedtomimicpharmacokineticconcentrationprofilesofertapeneminTBpatients.As
2gonceperdaywasidentifiedasmosteffectiveforsterilizingeffect,wesuggestthatdatain
thisthesiscanbeusedasstartingpointforthedesignofawell-designedprospectivephase2
study.Thisphase2studycansubstantiateefficacyandsafetyof2gertapenemcombined
withclavulanicacidontopofanoptimizedbackgroundregimenversusstandardofcarein
patientswithMDR-TB.
In the future perspectives we elaborate on genotypic testing as option to anticipate
resistancetocarbapenemsashollowfibermodelsareexpensiveanddrugsusceptibility is
complicateddue to chemical instability of ertapenem.Wehypothesize that carbapenems
mightbepreferredabovetheseinjectabledrugsasanindividualpatientdatameta-analysis,
reportedalackofbenefitofcommonlyprescribedsecond-lineinjectabledrugs.Furthermore,
weanticipatethatinjectableswillbeusedlessfrequentlybyTBprogramsupcomingyears.In
orderforertapenemtohaveaplaceinanoraltreatmentregimen,nextstepwouldbetoalter
thephysical-chemicalpropertiesofertapeneminsuchamatterthatitcanbedesignedasan
oraldrugoradrypowderinhalationantibiotic.
Weconcludethatthat2gertapenemincombinationwithclavulanicacidmightbeavaluable
assetinthetreatmentofmultidrugresistantTB.
145
9
Summary
Inchapter7alimitedsamplingstrategywasdevelopedusingapopulationpharmacokinetic
model based, using an iterative two-stage Bayesian method, on healthy volunteers and
showedtobeadequatetopredictertapenemexposureinMDR-TBpatients.
ExternalvalidationwasperformedbyBayesianfittingofthemodeldevelopedinvolunteers
to the individual data of MDR-TB patients using the developed population model for
volunteers as a prior. A Monte Carlo simulation (n=1000) was used to evaluate limited
samplingstrategies.Thebestperforminglimitedsamplingstrategy,withatime-restrictionof
0-6h,wasfoundtobesamplingat1and5h(R2=0.78,meanpredictionerror=-0.33%anda
rootmeansquareerror=5.5%).Drugexposurewasoverestimatedbyameanpercentageof
4.2(-15.2–23.6%).Consideringafreefractionof5%andtheMICsetat0.5mg/L,9outof12
patientswouldhaveexceededaminimumoff40%T>MIC.
In thegeneral discussionwediscussed insight, tools and understanding of ertapenem as
potentialintreatmentofmultidrug-resistantTuberculosis.Wediscusseddrugsusceptibility
testingofcarbapenemsandthatthehighvariabilityofresultsofinvitrostudiesmightbebest
explained by the chemical instability of carbapenems in growth media at temperatures
typicallyusedininvitrostudies.Wenoticedthatertapenemtreatmentseemswelltolerated
inlong-termtherapyinbroadrangeofbacterialinfectionsasitwasshownretrospectivelyfor
MDR-TBtreatment.Aspatientsweretreatedwithadosageof1gram,itwasnotclinically
substantiated in a prospective phase 2 study. Therefore, we discussed that a dose
fractionationwasdeemednecessarytodeterminewhichPK/PDparameterismostimportant
forclinicalefficacy.Toattaintheoptimaltherapeuticdoserange,ahollowfiberstudywas
performedtomimicpharmacokineticconcentrationprofilesofertapeneminTBpatients.As
2gonceperdaywasidentifiedasmosteffectiveforsterilizingeffect,wesuggestthatdatain
thisthesiscanbeusedasstartingpointforthedesignofawell-designedprospectivephase2
study.Thisphase2studycansubstantiateefficacyandsafetyof2gertapenemcombined
withclavulanicacidontopofanoptimizedbackgroundregimenversusstandardofcarein
patientswithMDR-TB.
In the future perspectives we elaborate on genotypic testing as option to anticipate
resistancetocarbapenemsashollowfibermodelsareexpensiveanddrugsusceptibility is
complicateddue to chemical instability of ertapenem.Wehypothesize that carbapenems
mightbepreferredabovetheseinjectabledrugsasanindividualpatientdatameta-analysis,
reportedalackofbenefitofcommonlyprescribedsecond-lineinjectabledrugs.Furthermore,
weanticipatethatinjectableswillbeusedlessfrequentlybyTBprogramsupcomingyears.In
orderforertapenemtohaveaplaceinanoraltreatmentregimen,nextstepwouldbetoalter
thephysical-chemicalpropertiesofertapeneminsuchamatterthatitcanbedesignedasan
oraldrugoradrypowderinhalationantibiotic.
Weconcludethatthat2gertapenemincombinationwithclavulanicacidmightbeavaluable
assetinthetreatmentofmultidrugresistantTB.
CHAPTER 10
SamenvattingDankwoord
About the AuthorPublication List
CHAPTER 10
SamenvattingDankwoord
About the AuthorPublication List
148
Chapter 10
SamenvattingTuberculose(tbc)wordtveroorzaaktdoorMycobacteriumtuberculosiseniswereldwijdde
dodelijkste infectieziekte. Onze wereld wordt geconfronteerd met een crisis voor de
volksgezondheiddoordatdebehandelingvantuberculoseeengrotereuitdagingisgeworden
metdeopkomstvanresistentietegeneerstelijnsgeneesmiddelen.Hetisnoodzakelijkomde
huidigebehandelingteverbeterendoormeeractieve-steriliserende-tbc-geneesmiddelen
teontwikkelen.Eenbijzondereffectievestrategieisherontdekkingvanoudegeneesmiddelen
alsnieuwemiddelenvoordebehandelingvanmultiresistentetuberculose(MDR-tbc).Bèta-
lactam antimicrobiële geneesmiddelen zijn veel gebruikte geneesmiddelen voor de
behandelingvaneenreeksinfecties.Ertapenemisgoedgekeurdin2001doordeFDAenwordt
veelgebruikttegengrampositieveengramnegatievebacteriën.Ertapenemlijktactieftezijn
tegenMDR-tbc.OmdepotentiëlerolvanertapenemvoordebehandelingvanM/XDR-tbc
beter te begrijpen, was het doel van dit proefschrift om de huidige literatuur, in vitro
activiteit,farmacokinetiekenveiligheidbijtbc-patiëntenteevalueren.
Carbapenemszijngeoormerktalspotentieelactievegeneesmiddelenvoordebehandeling
vanM. tuberculosis. In hoofdstuk 2 hebbenwehet potentieel van carbapenems voorde
behandelingvanM/XDR-tbconderzocht.Hetdoelvandezereviewwasomdeliteratuurte
evalueren over de momenteel beschikbare in vitro, in vivo en klinische gegevens over
carbapenemsbijdebehandelingvanM.tuberculosisenhetopsporenvankennislacunes,om
zich te richten op toekomstig onderzoek. Over het algemeen zijn de resultaten van de
onderzoeken die in deze beoordeling zijn geïdentificeerd consistent. Carbapenems in
combinatiemetclavulanaatvertoondenverhoogdeactiviteit invitro.Erzijnweinig invivo
studies uitgevoerd. Er zijn tien klinische onderzoeken uitgevoerd om de werkzaamheid,
veiligheidenverdraagbaarheidvandrieverschillendecarbapenems(imipenem,meropenem
en ertapenem) te beoordelen. Er werd geen duidelijk bewijs gevonden om een bepaald
carbapenemteselecterenomeeneffectiefM/XDR-tbc-regimeteontwerpen.Meerklinisch
bewijsisnodigterondersteuningvanherbestemmingvancarbapenemsvoordebehandeling
vanM/XDR-tbc.
Inhoofdstuk3werdeennieuweeenvoudigeenrobuusteLC-MS/MS-methodeontwikkeld
met behulp van een viervoudige massaspectrometer voor de analyse van ertapenem in
menselijkplasma,waarbijgedeutereerdeertapenemalsinternestandaardwerdgebruikt.De
kalibratiecurve was lineair over een bereik van 0,1 (LLOQ) tot 125mg/ L. De berekende
nauwkeurigheidvarieerdevan-2,4%tot10,3%.DeCVbinnenhetbedrijfvarieerdevan2,7%
tot 11,8% en de CV tussen de runs varieerde van 0% tot 8,4%. Bevries-dooi-stabiliteit
vooroordeel tussen -3,3% en 0,1%. Opslag van QC-monsters gedurende 96 uur bij 4 ° C
verschilde-4,3tot5,6%,opslagbijkamertemperatuurgedurende24uur,vooringesteldvan-
10,7%tot-14,8%enopslagindeautosamplervooringesteldtussen-2,9%en-10,0%.
Inhoofdstuk4werdeenretrospectievestudieuitgevoerdvoorallevermoedelijkeMDR-tbc-
patiënten in het tuberculosecentrum Beatrixoord van het Universitair Medisch Centrum
Groningen (Haren, Nederland) die ertapenem kregen als onderdeel van hun
behandelingsregimetussen1december2010en1maart2013.Veiligheidenfarmacokinetiek
werden geëvalueerd. Achttien patiënten werden behandeld met 1000 mg ertapenem
gedurende een gemiddelde van 77 dagen (bereik 5-210). Sputumuitstrijkje en -cultuur
werdenbijallepatiëntenomgezet.Blootstellingaangeneesmiddelenwerdgeëvalueerdbij
12patiënten.DegemiddeldeAUC0-24was544,9(bereik309-1130)mg*h/L.Degemiddelde
Cmaxwas127,5(73,9-277,9)mg/L.Overhetalgemeenwerddebehandelingmetertapenem
goedverdragentijdensMDR-tbc-behandelingenvertoondeertapenemeengunstigPK/PD-
profielbijMDR-tbc-patiënten.
In hoofdstuk 5 hebben we de snelle afname van ertapenem tijdens een geneesmiddel
gevoeligheidstest(DST)latenzien.Hiervoorhebbenweeennieuwestrategieontwikkeldom
DSTenMIC'suittevoerenvoordergelijkeonstabieleverbindingen.Wehebbenaangetoond
datertapenem-suppletiehetgoedbinnenhetgevoeligheidsbereikbrengtenwaarschijnlijk
een goed steriliserend effect bij tuberculose heeft. Dit suggereert dat de meeste
gepubliceerde MIC's voor dit medicijn waarschijnlijk onjuist hoog zijn en dat de
resistentiepercentageswaarschijnlijklagerzijndanwijnuvermoeden.
Inhoofdstuk6wasonsdoelhetidentificerenvandeertapenem-blootstellinggeassocieerd
metoptimaalsterilisatie-effectenvervolgenseeneenmaaldaagsedosisteontwerpenvoor
149
10
Samenvatting
SamenvattingTuberculose(tbc)wordtveroorzaaktdoorMycobacteriumtuberculosiseniswereldwijdde
dodelijkste infectieziekte. Onze wereld wordt geconfronteerd met een crisis voor de
volksgezondheiddoordatdebehandelingvantuberculoseeengrotereuitdagingisgeworden
metdeopkomstvanresistentietegeneerstelijnsgeneesmiddelen.Hetisnoodzakelijkomde
huidigebehandelingteverbeterendoormeeractieve-steriliserende-tbc-geneesmiddelen
teontwikkelen.Eenbijzondereffectievestrategieisherontdekkingvanoudegeneesmiddelen
alsnieuwemiddelenvoordebehandelingvanmultiresistentetuberculose(MDR-tbc).Bèta-
lactam antimicrobiële geneesmiddelen zijn veel gebruikte geneesmiddelen voor de
behandelingvaneenreeksinfecties.Ertapenemisgoedgekeurdin2001doordeFDAenwordt
veelgebruikttegengrampositieveengramnegatievebacteriën.Ertapenemlijktactieftezijn
tegenMDR-tbc.OmdepotentiëlerolvanertapenemvoordebehandelingvanM/XDR-tbc
beter te begrijpen, was het doel van dit proefschrift om de huidige literatuur, in vitro
activiteit,farmacokinetiekenveiligheidbijtbc-patiëntenteevalueren.
Carbapenemszijngeoormerktalspotentieelactievegeneesmiddelenvoordebehandeling
vanM. tuberculosis. In hoofdstuk 2 hebbenwehet potentieel van carbapenems voorde
behandelingvanM/XDR-tbconderzocht.Hetdoelvandezereviewwasomdeliteratuurte
evalueren over de momenteel beschikbare in vitro, in vivo en klinische gegevens over
carbapenemsbijdebehandelingvanM.tuberculosisenhetopsporenvankennislacunes,om
zich te richten op toekomstig onderzoek. Over het algemeen zijn de resultaten van de
onderzoeken die in deze beoordeling zijn geïdentificeerd consistent. Carbapenems in
combinatiemetclavulanaatvertoondenverhoogdeactiviteit invitro.Erzijnweinig invivo
studies uitgevoerd. Er zijn tien klinische onderzoeken uitgevoerd om de werkzaamheid,
veiligheidenverdraagbaarheidvandrieverschillendecarbapenems(imipenem,meropenem
en ertapenem) te beoordelen. Er werd geen duidelijk bewijs gevonden om een bepaald
carbapenemteselecterenomeeneffectiefM/XDR-tbc-regimeteontwerpen.Meerklinisch
bewijsisnodigterondersteuningvanherbestemmingvancarbapenemsvoordebehandeling
vanM/XDR-tbc.
Inhoofdstuk3werdeennieuweeenvoudigeenrobuusteLC-MS/MS-methodeontwikkeld
met behulp van een viervoudige massaspectrometer voor de analyse van ertapenem in
menselijkplasma,waarbijgedeutereerdeertapenemalsinternestandaardwerdgebruikt.De
kalibratiecurve was lineair over een bereik van 0,1 (LLOQ) tot 125mg/ L. De berekende
nauwkeurigheidvarieerdevan-2,4%tot10,3%.DeCVbinnenhetbedrijfvarieerdevan2,7%
tot 11,8% en de CV tussen de runs varieerde van 0% tot 8,4%. Bevries-dooi-stabiliteit
vooroordeel tussen -3,3% en 0,1%. Opslag van QC-monsters gedurende 96 uur bij 4 ° C
verschilde-4,3tot5,6%,opslagbijkamertemperatuurgedurende24uur,vooringesteldvan-
10,7%tot-14,8%enopslagindeautosamplervooringesteldtussen-2,9%en-10,0%.
Inhoofdstuk4werdeenretrospectievestudieuitgevoerdvoorallevermoedelijkeMDR-tbc-
patiënten in het tuberculosecentrum Beatrixoord van het Universitair Medisch Centrum
Groningen (Haren, Nederland) die ertapenem kregen als onderdeel van hun
behandelingsregimetussen1december2010en1maart2013.Veiligheidenfarmacokinetiek
werden geëvalueerd. Achttien patiënten werden behandeld met 1000 mg ertapenem
gedurende een gemiddelde van 77 dagen (bereik 5-210). Sputumuitstrijkje en -cultuur
werdenbijallepatiëntenomgezet.Blootstellingaangeneesmiddelenwerdgeëvalueerdbij
12patiënten.DegemiddeldeAUC0-24was544,9(bereik309-1130)mg*h/L.Degemiddelde
Cmaxwas127,5(73,9-277,9)mg/L.Overhetalgemeenwerddebehandelingmetertapenem
goedverdragentijdensMDR-tbc-behandelingenvertoondeertapenemeengunstigPK/PD-
profielbijMDR-tbc-patiënten.
In hoofdstuk 5 hebben we de snelle afname van ertapenem tijdens een geneesmiddel
gevoeligheidstest(DST)latenzien.Hiervoorhebbenweeennieuwestrategieontwikkeldom
DSTenMIC'suittevoerenvoordergelijkeonstabieleverbindingen.Wehebbenaangetoond
datertapenem-suppletiehetgoedbinnenhetgevoeligheidsbereikbrengtenwaarschijnlijk
een goed steriliserend effect bij tuberculose heeft. Dit suggereert dat de meeste
gepubliceerde MIC's voor dit medicijn waarschijnlijk onjuist hoog zijn en dat de
resistentiepercentageswaarschijnlijklagerzijndanwijnuvermoeden.
Inhoofdstuk6wasonsdoelhetidentificerenvandeertapenem-blootstellinggeassocieerd
metoptimaalsterilisatie-effectenvervolgenseeneenmaaldaagsedosisteontwerpenvoor
150
Chapter 10
klinischgebruik.WegebruiktenhetHollow-Fiber-systeemmodelvantuberculoseineen28-
dagenblootstellingsreactieonderzoekvan8verschillendeertapenem-doses in combinatie
metclavulanaat.Desystemenwerdenbemonsterdopvoorafbepaaldetijdstippenomhet
concentratie-tijdprofielteverifiërenendetotalebacteriëlelastteidentificeren.Ertapenem-
clavulanaat-combinatievertoondeeengoedmicrobieeldodings-ensteriliserendeffect. In
een dosis-fractioneringHollow-Fiber onderzoekwas dewerkzaamheid gekoppeld aan het
percentage van het 24-uurs doseringsinterval van ertapenem concentratie dat aanhoudt
bovenMIC.Weidentificeerdeneenintraveneuzedosisvan2grameenmaalperdagomhet
doelwittebereikenbij96%vandepatiënten.
Inhoofdstuk7werdeenLimitedsamplingstrategieontwikkeldmetbehulpvaneenpopulatie
farmacokinetischmodelopgezondevrijwilligersentoondevoldoendetezijnomertapenem
blootstellingbijMDR-tbc-patiëntentevoorspellen.Externevalidatiewerduitgevoerddoor
BayesiaanseaanpassingvanhetmodelaandeindividuelegegevensvanMDR-tbc-patiënten
metbehulpvanhetontwikkeldepopulatiemodelvoorvrijwilligersalseenvoorafgaande.Een
Monte Carlo-simulatie (n = 1000) werd gebruikt om limited sampling strategieën te
evalueren.Debestpresterendestrategievoorlimitedsampling,meteentijdbeperkingvan0-
6uur,bleektezijnop1en5uur(R2=0,78,gemiddeldevoorspellingsfout=-0,33%eneen
RSME = 5,5%). De blootstelling aan geneesmiddelen werd overschat met een gemiddeld
percentagevan4,2(-15,2-23,6%).Alsweeenvrijefractievan5%endeMICingesteldop0,5
mg/Lbeschouwen,zouden9vande12patiënteneenminimumvanf40%T>MIChebben
overschreden.
In de algemene discussie bespraken we het inzicht, hulpmiddelen en het begrip van
ertapenemalspotentieelindebehandelingvanmultiresistentetuberculose.Webespraken
het testen van de gevoeligheid voor geneesmiddelen van carbapenems en dat de hoge
variabiliteitvanderesultatenvanin-vitro-onderzoekenhetbestkanwordenverklaarddoor
dechemischeinstabiliteitvancarbapenemsingroeimediamettemperaturendiedoorgaans
wordengebruiktinin-vitro-onderzoeken.Wehebbengemerktdatertapenem-behandeling,
netzoalsretrospectiefwerdaangetoondvoorMDR-tbc-behandeling,goedverdragenlijktte
worden gedurende lange termijn therapie bij een breed scala aan bacteriële infecties.
Patiënten werden behandeld met een dosering van 1 gram, echter is dit niet klinisch
onderbouwdineenprospectieffase2-onderzoek.Daaromhebbenwebediscussieerddateen
dosis-fractionering noodzakelijkwerd geacht om te bepalenwelke PK/ PD-parameter het
belangrijkstisvoorklinischewerkzaamheid.Omhetoptimaletherapeutischedosisbereikte
bereiken, werd een Hollow-Fiber studie uitgevoerd om de farmacokinetische
concentratieprofielenvanertapenembijtbc-patiëntennatebootsen.Omdat2geenmaalper
dagwerdgeïdentificeerdalshetmeesteffectiefvoorhetsteriliserendeeffect,stellenwevoor
datdegegevensinditproefschriftkunnenwordengebruiktalsstartpuntvoorhetontwerp
van een goed ontworpen prospectief fase 2-onderzoek. Deze fase 2-studie kan de
werkzaamheidenveiligheidvan2gertapenemincombinatiemetclavulaanzuurbevestigen
bovenopeengeoptimaliseerdachtergrondregimeversusstandaardbehandelingbijpatiënten
metMDR-tbc.
In de toekomstperspectieven zijn we ingegaan op genotypische testen als optie om te
anticiperenopresistentietegencarbapenems,omdatHollow-Fibermodellenduurzijnende
gevoeligheidvangeneesmiddelengecompliceerdisalsgevolgvanchemischeinstabiliteitvan
ertapenem. Sinds een individuelepatiëntgegevens-meta-analyseeengebrek aan voordeel
van algemeen voorgeschreven tweedelijns injecteerbare geneesmiddelen gemeld heeft,
veronderstellenwedatcarbapenemsmogelijkdevoorkeurhebbenbovendezeinjecteerbare
geneesmiddelen.Verderverwachtenwedatinjecteerbaregeneesmiddelenindekomende
jarenmindervaakzullenwordengebruiktdoortbc-programma's.Omertapenemeenplaats
tegevenineenregimevoororalebehandeling,zoudevolgendestaperinbestaandefysisch-
chemischeeigenschappenvanertapenemteveranderenineendergelijkezaakdathetkan
worden ontworpen als een oraal geneesmiddel of een antibacterieel middel voor het
inhalerenvandroogpoeder.
We concluderen dat 2 g ertapenem in combinatie met clavulaanzuur een waardevolle
aanwinstkanzijnbijdebehandelingvanmultiresistentetbc.
151
10
Samenvatting
klinischgebruik.WegebruiktenhetHollow-Fiber-systeemmodelvantuberculoseineen28-
dagenblootstellingsreactieonderzoekvan8verschillendeertapenem-doses in combinatie
metclavulanaat.Desystemenwerdenbemonsterdopvoorafbepaaldetijdstippenomhet
concentratie-tijdprofielteverifiërenendetotalebacteriëlelastteidentificeren.Ertapenem-
clavulanaat-combinatievertoondeeengoedmicrobieeldodings-ensteriliserendeffect. In
een dosis-fractioneringHollow-Fiber onderzoekwas dewerkzaamheid gekoppeld aan het
percentage van het 24-uurs doseringsinterval van ertapenem concentratie dat aanhoudt
bovenMIC.Weidentificeerdeneenintraveneuzedosisvan2grameenmaalperdagomhet
doelwittebereikenbij96%vandepatiënten.
Inhoofdstuk7werdeenLimitedsamplingstrategieontwikkeldmetbehulpvaneenpopulatie
farmacokinetischmodelopgezondevrijwilligersentoondevoldoendetezijnomertapenem
blootstellingbijMDR-tbc-patiëntentevoorspellen.Externevalidatiewerduitgevoerddoor
BayesiaanseaanpassingvanhetmodelaandeindividuelegegevensvanMDR-tbc-patiënten
metbehulpvanhetontwikkeldepopulatiemodelvoorvrijwilligersalseenvoorafgaande.Een
Monte Carlo-simulatie (n = 1000) werd gebruikt om limited sampling strategieën te
evalueren.Debestpresterendestrategievoorlimitedsampling,meteentijdbeperkingvan0-
6uur,bleektezijnop1en5uur(R2=0,78,gemiddeldevoorspellingsfout=-0,33%eneen
RSME = 5,5%). De blootstelling aan geneesmiddelen werd overschat met een gemiddeld
percentagevan4,2(-15,2-23,6%).Alsweeenvrijefractievan5%endeMICingesteldop0,5
mg/Lbeschouwen,zouden9vande12patiënteneenminimumvanf40%T>MIChebben
overschreden.
In de algemene discussie bespraken we het inzicht, hulpmiddelen en het begrip van
ertapenemalspotentieelindebehandelingvanmultiresistentetuberculose.Webespraken
het testen van de gevoeligheid voor geneesmiddelen van carbapenems en dat de hoge
variabiliteitvanderesultatenvanin-vitro-onderzoekenhetbestkanwordenverklaarddoor
dechemischeinstabiliteitvancarbapenemsingroeimediamettemperaturendiedoorgaans
wordengebruiktinin-vitro-onderzoeken.Wehebbengemerktdatertapenem-behandeling,
netzoalsretrospectiefwerdaangetoondvoorMDR-tbc-behandeling,goedverdragenlijktte
worden gedurende lange termijn therapie bij een breed scala aan bacteriële infecties.
Patiënten werden behandeld met een dosering van 1 gram, echter is dit niet klinisch
onderbouwdineenprospectieffase2-onderzoek.Daaromhebbenwebediscussieerddateen
dosis-fractionering noodzakelijkwerd geacht om te bepalenwelke PK/ PD-parameter het
belangrijkstisvoorklinischewerkzaamheid.Omhetoptimaletherapeutischedosisbereikte
bereiken, werd een Hollow-Fiber studie uitgevoerd om de farmacokinetische
concentratieprofielenvanertapenembijtbc-patiëntennatebootsen.Omdat2geenmaalper
dagwerdgeïdentificeerdalshetmeesteffectiefvoorhetsteriliserendeeffect,stellenwevoor
datdegegevensinditproefschriftkunnenwordengebruiktalsstartpuntvoorhetontwerp
van een goed ontworpen prospectief fase 2-onderzoek. Deze fase 2-studie kan de
werkzaamheidenveiligheidvan2gertapenemincombinatiemetclavulaanzuurbevestigen
bovenopeengeoptimaliseerdachtergrondregimeversusstandaardbehandelingbijpatiënten
metMDR-tbc.
In de toekomstperspectieven zijn we ingegaan op genotypische testen als optie om te
anticiperenopresistentietegencarbapenems,omdatHollow-Fibermodellenduurzijnende
gevoeligheidvangeneesmiddelengecompliceerdisalsgevolgvanchemischeinstabiliteitvan
ertapenem. Sinds een individuelepatiëntgegevens-meta-analyseeengebrek aan voordeel
van algemeen voorgeschreven tweedelijns injecteerbare geneesmiddelen gemeld heeft,
veronderstellenwedatcarbapenemsmogelijkdevoorkeurhebbenbovendezeinjecteerbare
geneesmiddelen.Verderverwachtenwedatinjecteerbaregeneesmiddelenindekomende
jarenmindervaakzullenwordengebruiktdoortbc-programma's.Omertapenemeenplaats
tegevenineenregimevoororalebehandeling,zoudevolgendestaperinbestaandefysisch-
chemischeeigenschappenvanertapenemteveranderenineendergelijkezaakdathetkan
worden ontworpen als een oraal geneesmiddel of een antibacterieel middel voor het
inhalerenvandroogpoeder.
We concluderen dat 2 g ertapenem in combinatie met clavulaanzuur een waardevolle
aanwinstkanzijnbijdebehandelingvanmultiresistentetbc.
152
Chapter 10
Dankwoord
Beste Jan-Willem, ik begin direct met jou. Ik kan bijna geen woorden vinden om jou te
bedanken.Wateenontzettendmooiereishebikmogenmakendankzijjou.Hiereindigthet
helaas.Ikbenjewaanzinnigdankbaardatjealtijdnaastmijhebtgestaan,hetvertrouwenin
mijhebtgehad,ookopdemomentendat ik ineendiepdalzatofevenheteindevande
tunnelnietmeerzag.Ikziejealseenmentordieontzettendveelheeftbijgedragenaanmijn
ontwikkeling, zowel professioneel als persoon. Dankjewel. Ik wens jou en je familie
ontzettendveelplezierenhetallermooisteavontuurinSydney.Genietvanallemomenten.
Wieweetkruistonspadelkaarweerindetoekomst.
BesteTjipen Jos, ikwil jullieenormbedankenvoordemogelijkheiddie julliemijhebben
gegevenomeenpromotietrajectintegaan.Hetwaseenbijzondertrajectwaarinik,naast
mijnstudieentegelijkertijdnaastmijnwerk,wetenschappelijkeartikelenmochtschrijven.Ik
ben jullie ontzettend dankbaar voor jullie begeleiding en tegelijkertijd het geloof en het
vertrouwendatdittrajecttoteengoedeindekonkomen.
Ookwilikgraagdebeoordelingscommissie;prof.dr.B.Wilffert,prof.dr.R.vanCrevel,prof.
dr.J.Rossen,bedankenvoorhetbeoordelenvanmijnmanuscript.
DearTawandaGumbo,ShashikantSrivastavaandCarletonSherman,thankyouforhavingme
inDallas,TexasandtoworkwiththeHollowFibermodelbackin2013.Itwasanhonour.
Graagwilikalleco-auteursvanmijnartikelenbedankenvooralhuntijdenenergie;Richard
vanAltena,MarlankaZuur,OnnoAkkerman,MireilleWessels,RichardAnthony,BobWilffert,
WieldeLange,BenGreijdanus,DaanTouw,DickvanSoolingen,TridiavanderLaan,Johannes
Proost,HuibKerstjens,ShashikantSrivastava,TawandaGumbo,JosKosterink,Tjipvander
WerfenJan-WillemAlffenaar.
Natuurlijk wil ik ook alle tuberculosepatiënten in mijn studies bedanken; zonder jullie
betrokkenheidenjulliebijdrageaandewetenschapwashetnietgelukt.
Beste Alper, Koos, Leonie, Anne-Fleur, Samiksha en Marlanka; ik wil jullie bedanken
gedurendemijnreisenalleliefenleeddatwijopverschillendemomentensamenhebben
gedeeld.Dr.Alper,voorjoueenextraeervollevermelding,geweldighoejemijinhetlaatste
deelvanhettrajecthebtgeholpen.Dankje.
Paranimfen.Toppers.PhillipenAlper.Ikbenblijdatjulliemijindezespannendetijdbijstaan.
Lieve pap enmam, ik prijs mij gelukkigmet jullie onvoorwaardelijke steun en liefde. De
vanzelfsprekendheiddat julliealtijdklaarstaanvoormijenerwarenbijdebelangrijkeen
minderebelangrijkemomentendeafgelopen30jaar,maaktmijeenheelrijkmens.Ikben
heelblijdatikditmetjulliemagdelen.Ditisvoorjullie.
LieveSteven,Lotte,Pieter,PhillipenKarin.Ikbenontzettendtrotsopjullie.Watjullieook
doeninhetlevenendekeuzesdiejulliemaken.Ikstaachterjullie.
LieveHilda,ikbenontzettendblijdatikjouheblerenkenneninhetFeithhuis.Ikgenietecht
vanelkmomentdatwesamenzijn.Wezijnalweertweejaarbijelkaarenikkijkuitnaaralle
momentendiewijnoggaandelenmethetsamenwonenalshoogtepuntditjaar.
Als laatste,wil ikalmijnvrienden,collega’senallendieopverzoekgraaganoniemwillen
blijven, die mij gedurende mijn reis in de wonderbaarlijke wereld van de wetenschap
gesteundhebben,bedanken.
153
10
Dankwoord
Dankwoord
Beste Jan-Willem, ik begin direct met jou. Ik kan bijna geen woorden vinden om jou te
bedanken.Wateenontzettendmooiereishebikmogenmakendankzijjou.Hiereindigthet
helaas.Ikbenjewaanzinnigdankbaardatjealtijdnaastmijhebtgestaan,hetvertrouwenin
mijhebtgehad,ookopdemomentendat ik ineendiepdalzatofevenheteindevande
tunnelnietmeerzag.Ikziejealseenmentordieontzettendveelheeftbijgedragenaanmijn
ontwikkeling, zowel professioneel als persoon. Dankjewel. Ik wens jou en je familie
ontzettendveelplezierenhetallermooisteavontuurinSydney.Genietvanallemomenten.
Wieweetkruistonspadelkaarweerindetoekomst.
BesteTjipen Jos, ikwil jullieenormbedankenvoordemogelijkheiddie julliemijhebben
gegevenomeenpromotietrajectintegaan.Hetwaseenbijzondertrajectwaarinik,naast
mijnstudieentegelijkertijdnaastmijnwerk,wetenschappelijkeartikelenmochtschrijven.Ik
ben jullie ontzettend dankbaar voor jullie begeleiding en tegelijkertijd het geloof en het
vertrouwendatdittrajecttoteengoedeindekonkomen.
Ookwilikgraagdebeoordelingscommissie;prof.dr.B.Wilffert,prof.dr.R.vanCrevel,prof.
dr.J.Rossen,bedankenvoorhetbeoordelenvanmijnmanuscript.
DearTawandaGumbo,ShashikantSrivastavaandCarletonSherman,thankyouforhavingme
inDallas,TexasandtoworkwiththeHollowFibermodelbackin2013.Itwasanhonour.
Graagwilikalleco-auteursvanmijnartikelenbedankenvooralhuntijdenenergie;Richard
vanAltena,MarlankaZuur,OnnoAkkerman,MireilleWessels,RichardAnthony,BobWilffert,
WieldeLange,BenGreijdanus,DaanTouw,DickvanSoolingen,TridiavanderLaan,Johannes
Proost,HuibKerstjens,ShashikantSrivastava,TawandaGumbo,JosKosterink,Tjipvander
WerfenJan-WillemAlffenaar.
Natuurlijk wil ik ook alle tuberculosepatiënten in mijn studies bedanken; zonder jullie
betrokkenheidenjulliebijdrageaandewetenschapwashetnietgelukt.
Beste Alper, Koos, Leonie, Anne-Fleur, Samiksha en Marlanka; ik wil jullie bedanken
gedurendemijnreisenalleliefenleeddatwijopverschillendemomentensamenhebben
gedeeld.Dr.Alper,voorjoueenextraeervollevermelding,geweldighoejemijinhetlaatste
deelvanhettrajecthebtgeholpen.Dankje.
Paranimfen.Toppers.PhillipenAlper.Ikbenblijdatjulliemijindezespannendetijdbijstaan.
Lieve pap enmam, ik prijs mij gelukkigmet jullie onvoorwaardelijke steun en liefde. De
vanzelfsprekendheiddat julliealtijdklaarstaanvoormijenerwarenbijdebelangrijkeen
minderebelangrijkemomentendeafgelopen30jaar,maaktmijeenheelrijkmens.Ikben
heelblijdatikditmetjulliemagdelen.Ditisvoorjullie.
LieveSteven,Lotte,Pieter,PhillipenKarin.Ikbenontzettendtrotsopjullie.Watjullieook
doeninhetlevenendekeuzesdiejulliemaken.Ikstaachterjullie.
LieveHilda,ikbenontzettendblijdatikjouheblerenkenneninhetFeithhuis.Ikgenietecht
vanelkmomentdatwesamenzijn.Wezijnalweertweejaarbijelkaarenikkijkuitnaaralle
momentendiewijnoggaandelenmethetsamenwonenalshoogtepuntditjaar.
Als laatste,wil ikalmijnvrienden,collega’senallendieopverzoekgraaganoniemwillen
blijven, die mij gedurende mijn reis in de wonderbaarlijke wereld van de wetenschap
gesteundhebben,bedanken.
154
Chapter 10
AbouttheAuthorSanderPascalvanRijnwasbornonFebruary12th,1988inBochum(BRD)astheelderofan
identical twin. After attending high School at Johan-de-Witt gymnasium in Dordrecht, he
began studying Pharmacy at the University of Groningen in 2006. In 2008, he was
CommissionerofGeneralAffairs of theRoyalDutchPharmaceutical Students’Association
(KNPSV)and LiaisonSecretaryandDutch representative for theEuropeanPharmaceutical
Students’Association(EPSA).Throughoutthesecondhalveofhismaster’sprogramme,he
startedhisresearchonclinicalpharmacologyofertapenemundersupervisionofJan-Willem
Alffenaar, finally resulting in a PharmD/PhD program at the University Medical Center
Groningen.Duringthisprogram,hehadthehonourtovisitDallas,Texas,UnitedStatesof
America and to evaluate ertapenem in a Hollow-Fiber Infection model against M.
Tuberculosis. At the end of 2014, he earned his Master’s degree of Pharmacy, and
jumpstartedhiscareerdirectlyasGlobalManagementTraineeatFagron.After2yearsand
severalassignmentsbothathomeandabroad,heswitchedtoaninsurancecompanytostart
as ‘beleidsadviseur intramuralegeneesmiddelen’.Duringhis stayatZilverenKruis,hewas
responsible for the affordability, accessibility and quality of expensive medicines within
inpatient hospital care. In addition, he helped designing the ‘gezamenlijke inkoop dure
geneesmiddelenvoorzorgverzekeraarsNederland’.In2018,hestartedasconsultantatTer
Welle&Associés (TW&A), a consultancy that specializes in innovativemarket access and
healthcare pathways. In addition, he started as relations manager for PharmIntel, an
independent big data firm, providing management information and insights in the
(appropriate)useandoutcomeofexpensivemedicineformosthospitalsintheNetherlands.
Hisfutureperspectiveistocreatevaluebyhelpinghospitalsandpharmaceuticalcompanies
tobringaffordableandaccessiblemedicinestopatientsintheNetherlandsandtherestof
theworld.
Publicationlist1. SanderP.vanRijn,MarlankaA.Zuur,RichardAnthony,BobWilffert,RichardvanAltena,
OnnoW.Akkerman,WielC.M.deLange,TjipS.vanderWerf,JosG.W.Kosterink,Jan-
Willem C. Alffenaar. 2019. Evaluation of carbapenems for treatment of multi- and
extensivelydrug-resistantMycobacteriumtuberculosis.Antimicrob.AgentsChemother.
2. VanRijnSP,ZuurMA,vanAltenaR,AkkermanOW,ProostJH,deLangeWCM,Kerstjens
HAM,TouwDJ,vanderWerfTS,KosterinkJGW,AlffenaarJWC.2017.Pharmacokinetic
modelingandlimitedsamplingstrategiesbasedonhealthyvolunteersformonitoringof
ertapenem in patients with multidrug-resistant tuberculosis. Antimicrob Agents
Chemother61:e01783-16.Doi:10.1128/AAC.01783-16
3. VanRijnSP,SrivastavaS,WesselsMA,vanSoolingenD,AlffenaarJW,GumboT.2017.The
sterilizingeffectofertapenem-clavulanate inahollow fibermodelof tuberculosis and
implicationsonclinicaldosing.AntimicrobAgentsChemotherdoi:10.1128/AAC.02039-16
4. Srivastava S, van Rijn SP,Wessels AMA,Alffenaar JWC,Gumbo T.2016. Susceptibility
testing of antibiotics that degrade faster than the doubling time of slow-growing
mycobacteria: Ertapenem sterilizing effect ofMycobacterium tuberculosis. Antimicrob
AgentsChemother60:3193-3195.Doi:10.1128/AAC.02924-15.
5. VanRijnSP,vanAltenaR,Akkerman,OW,vanSoolingenD,vanderLaanT,deLangeWCM,
KosterinkJGW,vanderWerfTS,AlffenaarJWC.2016.Pharmacokineticsofertapenemin
patients with multidrug-resistant tuberculosis. Eur Respir J. 47:1229-1234.
Doi:10.1183/13993003.01654-2015.
6. vanRijnSP,WesselsAM,GreijdanusB,TouwDJ,AlffenaarJWC.2014.Quantificationand
validation of ertapenem using a liquid chromatography-tandem mass spectrometry
method.AntimicrobAgentsChemother;58:3481–3484.
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Publication List
AbouttheAuthorSanderPascalvanRijnwasbornonFebruary12th,1988inBochum(BRD)astheelderofan
identical twin. After attending high School at Johan-de-Witt gymnasium in Dordrecht, he
began studying Pharmacy at the University of Groningen in 2006. In 2008, he was
CommissionerofGeneralAffairs of theRoyalDutchPharmaceutical Students’Association
(KNPSV)and LiaisonSecretaryandDutch representative for theEuropeanPharmaceutical
Students’Association(EPSA).Throughoutthesecondhalveofhismaster’sprogramme,he
startedhisresearchonclinicalpharmacologyofertapenemundersupervisionofJan-Willem
Alffenaar, finally resulting in a PharmD/PhD program at the University Medical Center
Groningen.Duringthisprogram,hehadthehonourtovisitDallas,Texas,UnitedStatesof
America and to evaluate ertapenem in a Hollow-Fiber Infection model against M.
Tuberculosis. At the end of 2014, he earned his Master’s degree of Pharmacy, and
jumpstartedhiscareerdirectlyasGlobalManagementTraineeatFagron.After2yearsand
severalassignmentsbothathomeandabroad,heswitchedtoaninsurancecompanytostart
as ‘beleidsadviseur intramuralegeneesmiddelen’.Duringhis stayatZilverenKruis,hewas
responsible for the affordability, accessibility and quality of expensive medicines within
inpatient hospital care. In addition, he helped designing the ‘gezamenlijke inkoop dure
geneesmiddelenvoorzorgverzekeraarsNederland’.In2018,hestartedasconsultantatTer
Welle&Associés (TW&A), a consultancy that specializes in innovativemarket access and
healthcare pathways. In addition, he started as relations manager for PharmIntel, an
independent big data firm, providing management information and insights in the
(appropriate)useandoutcomeofexpensivemedicineformosthospitalsintheNetherlands.
Hisfutureperspectiveistocreatevaluebyhelpinghospitalsandpharmaceuticalcompanies
tobringaffordableandaccessiblemedicinestopatientsintheNetherlandsandtherestof
theworld.
Publicationlist1. SanderP.vanRijn,MarlankaA.Zuur,RichardAnthony,BobWilffert,RichardvanAltena,
OnnoW.Akkerman,WielC.M.deLange,TjipS.vanderWerf,JosG.W.Kosterink,Jan-
Willem C. Alffenaar. 2019. Evaluation of carbapenems for treatment of multi- and
extensivelydrug-resistantMycobacteriumtuberculosis.Antimicrob.AgentsChemother.
2. VanRijnSP,ZuurMA,vanAltenaR,AkkermanOW,ProostJH,deLangeWCM,Kerstjens
HAM,TouwDJ,vanderWerfTS,KosterinkJGW,AlffenaarJWC.2017.Pharmacokinetic
modelingandlimitedsamplingstrategiesbasedonhealthyvolunteersformonitoringof
ertapenem in patients with multidrug-resistant tuberculosis. Antimicrob Agents
Chemother61:e01783-16.Doi:10.1128/AAC.01783-16
3. VanRijnSP,SrivastavaS,WesselsMA,vanSoolingenD,AlffenaarJW,GumboT.2017.The
sterilizingeffectofertapenem-clavulanate inahollow fibermodelof tuberculosisand
implicationsonclinicaldosing.AntimicrobAgentsChemotherdoi:10.1128/AAC.02039-16
4. Srivastava S, van Rijn SP,Wessels AMA,Alffenaar JWC,Gumbo T.2016. Susceptibility
testing of antibiotics that degrade faster than the doubling time of slow-growing
mycobacteria: Ertapenem sterilizing effect ofMycobacterium tuberculosis. Antimicrob
AgentsChemother60:3193-3195.Doi:10.1128/AAC.02924-15.
5. VanRijnSP,vanAltenaR,Akkerman,OW,vanSoolingenD,vanderLaanT,deLangeWCM,
KosterinkJGW,vanderWerfTS,AlffenaarJWC.2016.Pharmacokineticsofertapenemin
patients with multidrug-resistant tuberculosis. Eur Respir J. 47:1229-1234.
Doi:10.1183/13993003.01654-2015.
6. vanRijnSP,WesselsAM,GreijdanusB,TouwDJ,AlffenaarJWC.2014.Quantificationand
validation of ertapenem using a liquid chromatography-tandem mass spectrometry
method.AntimicrobAgentsChemother;58:3481–3484.