BiochemicalVerificationofTobaccoUseandCessation:AnUpdate

NealLBenowitzMDUniversityofCalifornia,SanFrancisco

SRNTWebinarOctober13,2017

Disclosures

• Consultanttopharmaceuticalcompaniesthatmarketsmokingcessationproducts,includingPfizer,GlaxoSmithKlineandMcNeil.

• Paidexpertinlitigationagainsttobaccocompanies.

Overview

• Majorbiomarkers:cotinine,CO,NNAL,minortobaccoalkaloids

• Optimalcutpoints• Windowsofdetection• Individualfactorsinfluencingbiomarkerlevels• Overthecountertests• DistinguishingVapingfromSmoking

ConstituentsandBiomarkersofTobacco

Use

ChemicalConstituentsofTobaccoSmoke

Nicotine Formaldehyde*CarbonMonoxide 1,3-butadiene*Acrolein Benzo(a)pyrene*Ammonia Variousnitrosamines*NitrogenOxides Hydrazine*Benzene* Nickel*2-naphylamine* Polonium-210*4-aminobiphenyl* Cadmium

* Known or suspected human carcinogen*ns

Benzene

Carbon Monoxide

1,3-Butadiene

Formaldehyde

AcroleinNitrogen Dioxide

Nicotine

NicotinePolycyclic

AromaticHydrocarbons

PAH

Solanesol

Benzo(a)pyreneMetals

Courtesy of Brett Singer, LBNL

TobaccoSmokeisaMixtureofChemicalsIncludingGasesandParticles

Challenges in Assessing Exposure:Extent of Sorption and Stability Varies

Fraction in air at 2 h (furnished room)

0.0

0.2

0.4

0.6

0.8

1.0

Courtesy of Brett Singer, LBNLMultiple tracers/biomarkers are desirable

TobaccoSpecificBiomarkers

• Nicotineandmetabolites– Cotinine– Urinetotalnicotineequivalents

• Minortobaccoalkaloids– Anabasine,anatabine– Nicotelline

• Tobaccospecificnitrosamines– NNAL

OtherTobaccoSmokeBiomarkers

• Carbonmonoxide• Thiocyanate(cyanide)• Polycyclicaromatichydrocarbons

– 1hydroxypyreneandothers

• Volatileorganiccompounds– Acrylonitrile,acrolein,butadieneandothers

OptimalCotinineLevelstoDistinguishSmokingand

Nonsmoking

PlasmaCotinineLevelsThroughouttheDay

SRNTSubcommitteeonBiochemicalVerification(NTR2002)

Optimalcut-offpointstodistinguishtobaccousevs.notobaccouse.

• Plasmaorserumcotinine15ng/mL• Urinarycotinine 50ng/mL• ExpiredCO 8-10ppm• PlasmaSCN 78-84mmol/L

NHANESParticipants• 1999-2000,2001-2002,2003-2004• NationallyrepresentativesampleofU.S.civilianpopulation

• Adolescents(12-19y.o.)andadults(mean45y.o.)

• 13,078nonsmokersand3,078smokers• NHW(6,896);NHB(3,671);MA(4,472)

All Adults

Fixed: 14.0 ng/mL Specificity = 98.5%Sensitivity = 92.4%

0

2

4

6

8

10

12

14

16

0.035 0.1 1 10 100 1000

Actual % = 57%(cropped at 16%)

ROC: 3.08 ng/mL Specificity = 97.4%Sensitivity = 96.3%

%Po

pula

tion

Overall

Serum Cotinine, ng/mL

RecommendedCotinineCutpointsbyRace/Ethnicity

Adults Adolescents*

NHW- 5ng/ml 3ng/ml

NWB- 6ng/ml 3ng/ml

MA- 1ng/ml 1ng/ml

* sensitivity of cut-point lower in adolescents –likely due to higher prevalence of occasional smoking and more under-reporting of smoking

Caveatsregardingcutpoints

• Theoptimalcutpointisinfluencedbytheprevalenceandintensityofactivetobaccouseandsecondhandsmokeexposure.

• Higherprevalenceandintensityoftobaccoexposurewillresultsinahigheroptimalcutpoint

• Race- andphysiology-relatedmetabolicdifferencescaninfluencecutpoints

Comparisonofbiofluidsforcotininemeasurement

• Blood(serum,plasma)isgoldstandard• Saliva/plasmaratio=1.04(0.95– 1.14)

– Ratioinfluencedbysalivaflowrate• Urine/plasmaratio=4.6(4.0– 5.3)

– RatioinfluencedbyurineflowrateandpH

()– 95%C.I.

Windowofcotininedetection

• Detection~5half-lives,dependingoninitiallevelandbiofluidtested(longerdetectioninurine)

• Cotininehalf-liferange10to30hr• Detectionrange50to150hours

LimitationsofCotinineasBiomarkerofNicotineandTobacco

SmokeExposure

NICOTINE

COTININE TRANS-3’-HYDROXYCOTININE

TRANS-3’-HYDROXYCOTININE

NICOTINE-1’-N-OXIDE NORNICOTINENICOTINE

NICOTINEGLUCURONIDE

COTININE

COTININEGLUCURONIDE COTININE-N-OXIDE NORCOTININE

TRANS-3’HYDROXYCOTININE

GLUCURONIDE

-80%

4.4%9.8%

4.2%

13.0%

12.6% 2.4% 2% 7.4%

0.4%

33.6%

NicotineMetabolicProfile

Cotinine as a Quantitative Measure of Nicotine Intake from Tobacco Use

Daily Generation of Cotinine =

Daily Nicotine Dose (D) × % Conversion of Nicotine to Cotinine (f)

Elimination rate for Cotinine =

Clearance of Cotinine (ClCOT) × Blood Cotinine Concentration (COT)

Cotinine as a Quantitative Measure of Nicotine Intake from Tobacco Use

• The conversion factor K = ClCOT / f

• Cl cot is determined by activity of CYP2A6, UGT2B10 and the renal clearance of cotinine

• f is determined by activity of CYP2A6, UGT2B10, FM03 and the renal clearance of nicotine

CotinineasaQuantitativeMeasureofNicotineIntakefromTobaccoUse

• TypicalK=0.08mg/24h/ng/ml

• A20cigaretteperdaysmokershasabloodcotininelevelof300ng/ml

D=(0.08)(300)=24mg/day=1.2mg/cigarette

FactorsPotentiallyInfluencingCotinineasaBiomarkerofNicotine

Intake

Genetics KidneyfailureSex Oralcontraceptive/HormoneRace replacementCigarette Anticonvulsantdrugs/rifampinsmoking Menthol

SexDifferencesinRateofNicotineMetabolism

15.617.6

22.5

27.0

0

10

20

30

Nico

tine

Clea

ranc

e (m

l/min

/kg)

Men Women(no OCP)

Women(+ OCP)

PregnantWomen

All Groups Significantly Different from One Another

JPET 2002; 301:594CPT 2006; 79:480

Kvaluesindifferentpopulationsofsmokers

Units:mgnicotine/24hr/ng/ml cotinineAverageK=0.08LowerinmenHigherinwomenVeryhighinpregnancyLowerinAfricanAmericansandAsians

OvertheCounterCotinineTests

OTCCotinineTests

• Fastest,easiestandleastexpensivetest• Immunoanalysisusingcotinineantibodies• Urineandsalivatests• Semi-quantitativewithrelativelyhighcotininethreshold– highspecificity,lowsensitivity

• InadequatesensitivityforSHSandoccasionaltobacco/nicotineuse

• PossiblebetterclassificationwhencombinedwithexpiredCO

COT iScreen OFD NicCheck ISalimetricsELISA Kit

SafeCare

Saliva Saliva Urine UrineSaliva

99% 99% 98% 99%99%

4 ng/ml 30 ng/ml 200 ng/ml 200 ng/ml20 ng/ml50 ng/ml

Active Use Cutoff

Sensitivity

Specimen

NicAlertStrips

SalivaUrine

99%

10 ng/ml (S)100 ng/ml (U)

Marrone, Addiction, 2011

Marrone, Addiction 2011; 106:1325-1334

UrineandSalivaNicAlert andSmokingStatus

Urine

6 (>1000)

5 (500-1000)

4 (200-500)

3 (100-200)

2 (30-100)

1 (10-30)

0 (0-10) Heavy

SmokersLight

SmokersNot ExposedNonsmokers

ExposedNonsmokers

Nic

Ale

rt Z

ones

, Cot

inin

e C

once

ntra

tions

(ng/

ml)

Saliva

Heavy Smokers

LightSmokers

Not ExposedNonsmokers

ExposedNonsmokers

OptimalCOLevelstoDistinguishSmokingandNonsmoking

Marrone, Addiction, 2011

Marrone, Addiction 2011; 106:1325-1334

BreathCOandSmokingStatus

Heavy Smokers

LightSmokers

Not ExposedNonsmokers

Exposed Nonsmokers

Bre

ath

CO

(ppm

)70

60

50

40

30

20

10

0

ExpiredCOandNicAlertPerformance

Measures Cut Point Sensitivity (%) Specificity (%)Expired CO ≥ 5 ppm 94.4 97.6

Urine NicAlert ≥ 3 92.2 97.6

Saliva NicAlert ≥ 1 76.7 85.4

CO / Urine NicAlert ≥ 5 / ≥ 3 97.6 95.1

WindowofCOdetection• Detection~5half-lives• COhalf-life:

– Average4hr– Exercise2hr– Sleep8hrDetectionrange10to20hr (COlevelscanstillbeelevatedafterovernightabstinence)ConsiderothersourcesofCO(vehicleexhaust;marijuanasmoking)

OptimalNNALLevelstoDistinguishSmokersfromNonsmokers

TobaccoSpecificNitrosamines:NNK,NNNandNNAL

• NNKandNNNarepotentlung,esophagealandoralcavitycarcinogensthatarederivedbynicotineinthetobaccocuringandcombustionprocess.

• NNKisonlyfoundintobaccoproducts;itslevelsdifferfromproducttoproduct,dependingontypeoftobaccoandnitratecontent.

• NNKismetabolizedinthebodytoNNAL,whichcanbemeasuredintheurineoftobaccousers.

UrinaryNNALinCigaretteSmokersandNon-Smokers

NHANES 2011-2012 • 6705 Participants • Ages ≥ 6 years old• 62% Overall Detection

Rate• 95% percentile urine

NNAL in nonsmokers=11.4 pg/mg creat Urinary NNAL, pg/mg

creatinine

Perc

ent

OptimalurinecotinineandNNALcutpointstodistinguishactivevs

passivesmoking

Goniewiczetal.2011:Cot31.1ng/ml;NNAL47.3pg/ml[337smokers,228heavySHS]

Benowitzetal2017:Cot30ng/ml;NNAL14.4pg/ml [54smokers,303lightSHSornon-exposed]

UrineNNALwithUseofDifferentTobaccoProducts

0

100

200

300

400

Non-Tobacco use

Pipe or Cigar Cigarette Snuff or Chewing Tobacco

NN

AL

(pg/

mg

crea

tinin

e)

AmongNon-SmokersUrineNNALGreatestinChildren

WindowofNNALdetection

• Detection~5half-lives• NNALhalf-liferange10to16days• Detectionrange6to12weeks

MinorTobaccoAlkaloidsasBiomarkers

ofTobaccoUse

MinorNicotineAlkaloids

Anabasine andAnatabine areBiomarkersofTobacco(asOpposedtoNicotine)

Consumption• NotpresentinNRT.NoneorminimalinE-cigaretteliquid

• Urineconcentrationsof2ng/mlhighlyspecificfortobaccousewhileusingNRT

• CanestimatenicotineintakecomingfromtobaccoevenwithconcomitantNRTuse

• Potentialbiomarkertoassesstobaccovse-cigarettevsdualuse

Nicotelline

• Becauseofstabilityandlowvolatility,nicotellinehasbeenproposedasanenvironmentaltracerandbiomarkerforparticulatematterderivedfromtobaccosmoke

Nicotelline HighlyCorrelatedwithPMinCigaretteEmissions

ElectronicCigarettes

ECLiquidandAerosol• Nicotine• Propyleneglycol• Glycerin• Particulates(includingmetals)• Volatileorganicchemicals(acrolein,formaldehyde)

• Flavorants• Miscellaneouscontaminants

ReducedNNKexposureafterSwitching

**

E-CigStudy- Nicotelline inUrine

Changes in Biomarkers after Switching from Cigarettes to E-Cigarettes

TNE

NNAL

NICOTELLINE

Days0 1 2 2 Months

SummaryofBiomarkerstoDistinguishE-cigarettefromTobaccoUse

• Nicotineintakesimilartocigarettesmoking,sonicotinebiomarkersarenotuseful

• Nicotellineisusefulbiomarkertodistinguishsmokingvsnon-combustibletobaccouse.

• Anabasine/abatabinepotentiallyuseful,butsomeE-liquidscontainsubstantialamountsofthesealkaloids

• ForstudiesofE-cigderivedtoxicantbiomarkers,toexcludedualtobaccouseIrecommendurineNNALmeasurement(<1pg/mgcreat)

Summary

Mainpoints

• Cotinine,COandNNALalluseful• OptimalcutpointsdependonprevalenceandheavinessofsmokingandSHSexposure

• Differencesinwindowofdetection:– CO– hours– Cotinine– days– NNAL- weeks

Mainpoints• Cotininelevelsinfluencedbyindividualmetabolicdifferences

• COinfluencedbyenvironmentalsourcesandsmokingmarijuana

• OTCcotininetestseasyandcheap,specificbutlacksensitivityforlight/occasionaluse

• MinortobaccoalkaloidsmostusefulfordistinguishingtobaccofromnicotineorE-cigaretteuse

OptimalBiochemicalCutPointsSeparatingSmokersand

Nonsmokers

Biomarker Biospecimen Concentration

Cotinine Blood/saliva 3-5 ng/ml

Cotinine Urine 30 ng/ml

Carbon Monoxide Breath 5-6 ppm

NNAL Urine 10 pg/mg creat

AssayCost/SensitivityBiomarker Specimen Method LLOQ Cost Per Sample Exhaled CO Breath Portable

Instrument0.5 ppm $200 - 3000

per instrument

Cotinine Urine OTC Kit NA $0.50-5.00 Nicotine and Cotinine

Urine Saliva Blood

GC-NPD 1 &10 ng/ml, respectively

$50-100

Nicotine Urine Saliva Blood

GC-MS/MS 0.2 ng/ml $75-200 nic+cot

Cotinine Urine SalivaBlood

GC-MS/MS 2 ng/ml $75-200nic+cot

Cotinine Urine LC-MS/MS 0.05 ng/ml $100-200cot +3-OH cot

Total Nicotine Equivalents

Urine LC-MS/MS NA $180-300

NNAL Urine LC-MS/MS 0.25 pg/ml $180-300