'15 Sep - Scholz Presentation2. Cells incubated on growth media overnight 3. Day 2 –Begin 1:2...

NATURALANTIMICROBIALSTheAbilitytoMaintainSkin’sMicrofloraBalancevs.theDamagingEffectsof

TraditionalSyntheticPreservatives

OUTLINE

I. ImportanceofPreservation

II. TraditionalPreservatives

III. AlternativeAntimicrobials

IV. BacterialSymbiosis:Resistant,Transient,andCommensal

V. IdealBalance:Eliminating“Bad”&Promoting“Good”Bacteria

VI. AlternativesinAction

ImportanceofPreservation

Step1- Theusualsuspects

1.BacteriaandFungus(YeastandMold)I.Gram-Positive– crystalviolet– StaphII.GramNegative– fuchsine– PseudomonasIII.Pathogens

2.HouseOrganisms

3.UsualsourcesofcontaminationI.AirborneII.RawMaterialsIII.Lines/Valves/Pumps/Tanks

ImportanceofPreservation

Step2- Produceacleanproduct

1.Goodmanufacturingpractice(GMP)I.ColdorhotasepticfillingII.Clean-roomtechnology

2.Hygiene-compliantpackaging3.Emulsionform4.Wateractivity5.pHcontrol6.Multifunctionalingredientswithbiostaticeffect7.Remediation– Thermal– Gammaradiation

SchuelkeMeyer- Useofmultifunctionalstoachieveself-preservingsystems

PreservationBasics

1. APC(AerobicPlateCounts)

2. PET(PreservativeEfficacyTest)aka“ChallengeTest

3. PreservationChallengesI. CompatibilityII. NaturalGuidelinesIII. GlobalRegulationsIV. EfficacyV. Change

APC(AerobicPlateCounts)

1. PicksupBacteria,Yeast,andMold

2. TrypticSoyAgarwithlecithinandpolysorbate80temperedat45°C(TSA)SabouraudDextroseAgartemperedat45°C(SDA)

3. 2daysforbacteria5daysforfungus

PET(PreservativeEfficacyTest)aka“ChallengeTest”

1.PCPCmethodM-1.2.PCPCmethodM-3orUSP<51>ChallengeS.aureus,E.coli,P.aeruginosa,C.albicans,andA.brasiliensis

3.PassCriteria>99.999%%at7daysforbacteria>90.0%at7daysforfungus

4.AcceptableLevels

•Babyproducts- nomorethan10²CFU/gram•Eyeareaproducts- nomorethan10²CFU/gram•Allotherproducts- nomorethan10³CFU/gram

TraditionalPreservatives

I. US– EPApesticideregistrationII. Europe– AnnexVoftheCosmeticActIII. Asia– countrybycountryadministeredbytheequivalentoftheFDA

AlternativeAntimicrobials

I. Botanicals–WillowBark/Honeysuckle/WoodExtractsII. GlycolsIII. Organicacids/EstersIV. PeptidesV. Packaging

IMPORTANCEOFPRESERVATION

TheSkin

• Thebody’slargestorganandonethatisconstantlyexposedtotheenvironment

• Itprotectsthebodyfrompathogenswhilesustainingmicroorganismsthatinfluencehumanhealthanddisease.

• Anideallocationforthecontrolledgrowthofbacteria

• Consistsofvariouscutaneousantimicrobialdefencemechanisms:

• StratumCorneum• StratumCorneumLipids• ProductionofLysozyme• Acidity• Defensins• CommensalBacteria

BACTERIALSYMBIOSIS:RESISTANT,TRANSIENT&COMMENSAL

SkinMicrobiome

• Theskinisanecosystem,harboringmicrobialcommunitiesthatliveinarangeofphysiologicallyandtopographicallydistinctniches

• Bacterialgrowthontheskinincludes:• Resident&TransientPathogenicBacteria- capableofinvadingandcausingharm• CommensalBacteria- protectsthehostfromthesepathogens

• A2009NIHmicrobiomestudyanalyzedribosomalRNAgenesequencesobtainedfrom20distinctskinsitesofhealthyhumans

• Revealedthatphysiologicallycomparablesitesharborsimilarbacterialcommunities

• Thecomplexityandstabilityofthemicrobialcommunityaredependentonthespecificcharacteristicsoftheskinsiteandextrinsicenvironmentalfactors

• Examinedthemicrobialinterdependenciesrequiredtomaintainhealthyskin


SkinMicrobiome

SkinMicrobiome

• Recentresearchhasbeguntodocumenthowskincommensalsinteractwithoneanother,withpathogenicmicrobes,andwithhumancells.Examples:

• Staphylococcusepidermidissecretesantimicrobialsubstancesthathelpfightpathogenicinvaders

• P.acnesusestheskin’slipidstogenerateshort-chainfattyacidsthatcansimilarlywardoffmicrobialthreats

• “Ithinkanythingwecandotorestoremorebalanceormoreappropriatemicrobecompositionintheskin,asinallthedifferenttissues,isextremelyimportant.”

- YasmineBelkaidoftheNationalInstituteofAllergyandInfectiousDisease

• Theseandotherskinmicrobescanimpactthelocalmolecularenvironment,andmaybeabletoalterthebehaviorofhumanimmunecells


IMPORTANCEOFPRESERVATION

CosmeticPreservatives

• Theproperuseofpreservativesandbiocidesaretopreventmicrobialcontaminationwithoutalteringtheskinmicrobiome

• GramnegativeandGrampositivebacteria,yeasts,andmoldshaveallbeenfoundtogrowinvariouscosmeticproducts

• Yet,someofthesemicrobesarealsoresidentcommensalbacteriaontheskin

• Activebiocidesmusteliminatethebad,yetpromotethegoodbacteria

• Sometraditionalpreservativeseffectiveatreducingcosmeticcontamination:• Parabens• Formaldehydedonors• Halogenatedcompounds

• Butaretheysafe?

RoleoftheHDAC

• HistoneDeacetylaces(HDAC’s)areaclassofenzymesexpressedinskincells

• Theseenzymeshelpmaintainhealthyskin bybalancingacetylationactivitiesofhistoneacetyltransferasesonchromatinremodelingandalsoplayessentialrolesinregulatinggenetranscription

• Canberesponsivetomanydifferentenvironmentalsignals

• Specifically,HDAC3whichisthemostprominentlyexpressedhistonedeacetylaseinN-TERThumankeratinocytecells,hasvariousinflammatoryandmetabolicroles


HDAC3• Researchhasfound thatHDAC3isakey

mediatorinmaintainingtheoverallintegrityandfunctionofhumanorganssuchastheintestinesandskin,whileinthepresenceofcommensal“friendly”bacteria

• HDAC3alsoplaysakeyroleintherelationshipbetweenmicrobiotaandinflammation

• Arecentstudyfound thatthereweremajordifferencesbetweenthemicrobialpopulationdiversitywithinnormalmicecomparedtomicedeficientinHDAC3

• Somebacterialspecieswereactuallyover-populatedwhenHDAC3wasreduced


HDAC3

• ResearchprovedthatthereisafundamentalchangeintherelationshipbetweencommensalbacteriaandtheirmammalianhostsfollowingdeletionofHDAC3

• HDAC3notonlyinfluencesthebacterialpopulation,butthebacteriainturninfluencecells’behaviourasaresult,whichincludesgeneexpression


RoleofHDAC

• A2012studyfromtheJournalofCellScienceinvestigatedHDACproteinlevelsandtotalactivitydependenceonArylHydrocarbonReceptorNuclearTranslocator(ARNT)inN-TERTepidermalkeratinocytescells

• AnHDACactivityassaywasemployed

• HDAC3appearedtobethemostprominentlyexpressedhistonedeacetylaseinthesecells

• Attheproteinlevel,depletionofARNTleadtoasignificantincreaseofHDAC3

• InARNT-depletedHaCaTcells,therewasalsoaprominentincreaseinHDAC3proteinlevels


RoleofHDAC

• TheJournalofBiologicalChemistrypublishedastudy investigatingHDAC3mediationofskininflammation

• TheroleofHDAC3inallergicskininflammationprimarilyremainsunknown

• TheirresultsindicatethatHDAC3interactswithFcεRIandregulatesexpressionofMCP1throughSp1andc-Juntomediateallergicskininflammation

• ConcludedthatHDAC3mediatesallergicskininflammationinrelationwithangiogenesisbyregulatingMCP1

• HDAC3servesasatargetfordevelopmentofallergytherapeutics


RoleofHDAC

• HDACexpressionwithinmultipletissuesystemssuchasthedigestivetractandskin,isanintegralfactorinorganhealthandfunction

• Thisenzymeisextremelysensitivetoenvironmentalandintrinsicfactorssuchaspreservativesandbiocides,i.e.:

• Triclosan• Parabens

• ExpressionofHDACisanessentialcomponentofhowweregulatetherelationshipbetweencommensalbacteriaandcellfunction tomaintainhealthyskin


RoleofHDAC

• WhenHDACisalteredorreducedthentheskin’scommensalbacterial,whichprotectsagainstunwantedmicrobes,isnolongeraseffective

• OurresearchhasshownmosttraditionalbiocidesdecreaseHDACexpressioninhumankeratinocytes

• Thisleadstoacompromisedimmunedefencesystemandreducedskinhealth

• Whatifbiocidescouldonlyeliminatethe“bad”withoutalteringthe“good”commensalmicrobes,whilestillnotaffectingHDACactivity?

• Couldtraditionalpreservativesdothisordonaturalantimicrobialshold thekey?


TRADITIONALBIOCIDES

Triclosan

• Triclosanisabactericidalbroad-spectrumagentdevelopedover40yearsagoandfirstintroducedasasurgicalscrub

• Primarilyusedasatopicalbiocidemoresothanacosmeticproductpreservative,suchasparabens

• Bothbiocidesandpreservativesaffecttheskinmicrobiome

• Overthelast20yearsitsusehasgrownrapidlyinpersonalcareproductsincludingsoap,handsanitizer,cosmetics,andtoothpaste,aswellashouseholdproductssuchasodor-fighting socksandgerm-resistantsponges,kitchenware,andbedding

TRADITIONALBIOCIDES

Triclosan

• Cancausedisruptionofbacterialcellwallsinnonspecifictargets

• DecreasesHDACexpressioninskinkeratinocytes

• Resultsindisturbanceoftheskin’smicroflorabalance• Pathogenicandcommensalbacteriaarekilled• Skinleftdefenselessagainstnewdestructivemicroorganisms

• Canalsocausedangerousantimicrobialresistancetovitalmedicines• Growingthreattohealthcareasawhole

• Mayenhancetheproductionofchloroform,whichisclassifiedbytheEPAasaprobablehumancarcinogen.• A2007studyillustratedthat,undersomecircumstances,triclosantriggered

productionofchloroforminamountsupto40%higherthanbackgroundlevelsinchlorine-treatedtapwater

NATURALANTIMICROBIALS

NewFormulationApproach

• Somenaturalantimicrobialsareequipped tokillpathogenicbacteriawhilestillmaintainingavigorouscommensalmicrofloraontheskin

• OthernaturalantimicrobialswillnothaveanegativeorinhibitoryeffectonHDACexpression

• Thereforenotaffectingthecommensalmicrobiotawhichprotectsandstrengthensoverallskinhealth

• Helpachievethegoalofdevelopingnaturalantimicrobialcosmeticproductswhicharenotharmfulundernormalorforeseeableconditionsofuse


AntimicrobialPeptides

• Exampleofanaturalantimicrobialequippedtokillpathogenicbacteriawhilestillmaintainingavigorouscommensalmicrofloraontheskinareantimicrobialpeptides– Nisin,Natamycin

• Derivedfromfermentationwithprovenefficacy

• Typicallyshortchainsoflessthan50aminoacids

• Synthesizedbyanumberoforganismsasprotectiveorcompetitiveadvantagemechanisms

• Viafermentationtechnology,thesepeptidesaredesigned toprovide:• Superiorantimicrobialefficacy• PromotionofHDACactivityandcommensalbacteriabalance


PeptideFormulaMICResults

PeptideMICresultsdemonstratepotentialeffectivenessatproductadditionratesof1to3%


DoubleChallengeTestResults

ChallengeTestresultsshowsuccessfulprotectionofagenericcreamformulation

PeptideHDACAssayProtocol:HDAC-Glo™AssayandScreeningSystem

• Asingle-reagent-addition,homogeneous,luminescentassaythatmeasurestherelativeactivityofHDACenzymesfromcells

• Usesanacetylated,live-cell-permeant,luminogenicpeptidesubstratethatcanbedeacetylatedbyHDACactivities.

• DeacetylationofthepeptidesubstrateismeasuredusingacoupledenzymaticsysteminwhichaproteaseintheDeveloperReagentcleavesthepeptidefromaminoluciferin

• ThisisquantifiedinareactionusingUltra-Glo™RecombinantLuciferase

• TheHDAC-mediatedluminescentsignalispersistentandproportionaltodeacetlyaseactivity,whichallowsforprocessingofmultiwellplates

ANTIMICROBIALPEPTIDE:HDACDATA

PeptideHDACAssay

Protocol:HDAC-Glo™AssayandScreeningSystem

1. HumanKeratinocytesplatedonDay12. Cellsincubatedongrowthmediaovernight3. Day2– Begin1:2serialdilutions onseparatemultiwellplate4. HDACBufferaddedtoeachwellondilutionplate5. Mediadiscardedfromcellplatetoreplacewithfreshmedia6. Dilutionswithstandardtransferredontocellplate7. Mixforonehourtohomogenize8. Incubatedfor30minutestoallowHDACinhibitortobegin9. Luminescentreagentprepared10. Reagentaddedtocellplate11. Incubatedfor30minutesatroomtemp(23-25C)12. HDACactivitymeasuredoncolorimetricplatereader


PeptideHDACAssayResults




1.56nM

0

500

1000

1500

2000

2500

3000

Luminescence

HDACAssay

Peptide Phenonip Trichostatin A Triclosan



• Demonstratessuccessfulrelationshipbetweenantimicrobialpeptideandskinmicroflora.

• Although theantimicrobialpeptidestillhasslighteffectonHDACactivity,itdoesnotinhibitorreduceHDACnearlyasmuchastriclosanorparabens

• TraditionalpreservativesandbiocidesareshowtodrasticallyinhibitHDACactivity,whichnegativelyaffectsskin’snaturalmicroflorabalance

• Withnaturalantimicrobialpeptide, theskin’scommensalbacterial,whichprotectsagainstunwantedmicrobes,remainseffectiveandintact

TransformingTheFaceOfPreservation:PeptideTechnology

AntimicrobialPeptideSystems

LacticAcidBacteria(LAB)Family

Producenovelantimicrobialpeptides– Bacteriocins

• Proteinaceousmaterials

• Inhibitgrowthofbacterialstrains

• Structurally,functionally,andecologicallydiverse


Leuconostoc/RadishRootFermentFiltrate

Thenextgenerationofproactive,innovativeandefficacious antimicrobialsystems


Leuconostoc/RadishRootFermentFiltrate

Mechanism of ActionLAB family – Leuconostoc kimchii produces lactic acid

Restricts the growth ofmicroorganisms by acidifying their environment

Fermentation of Leuconostoc in a defined growth medium containingRadish creates bacteriocins

• Provides broadspectrumantimicrobial activity• Proven conditioningbenefits

ModulatedActivityWillowBark Extract is added – facilitates controlledcell lysis

Ensures the release of the antimicrobialpeptidesformaximised activity


Leuconostoc/RadishRootFermentFiltrateChallengeTestinaCreamFormulation

MIC %

E.coli 1.60

S.aureus 0.80

P.aeruginosa 1.60

C.albicans 0.80

A.brasiliensis 2.40

MICData

Table1:ChallengeTestResultsfor2.00%Leuconostoc/RadishRootFermentFiltrateinO/WCreamformulationinoculatedonday0andre-inoculatedonday28


LactobacillusFerment

Mechanism of ActionLAB family – Lactobacillus bulgaricus produces lactic acid

Restricts the growth ofmicroorganisms by acidifying their environment

Fermentationof Lactobacillus creates bacteriocins• Provides broadspectrumantimicrobial activity• Proven conditioningbenefits

ModulatedActivityLysozyme is added to the ferment filtrate – facilitates controlledcell lysis

Ensures the release of the antimicrobialpeptidesformaximised activity


LactobacillusFermentChallengeTestinaCreamFormulation

MIC %

E.coli 0.75

S.aureus 1.00

P.aeruginosa 1.00

C.albicans 0.75

A.brasiliensis 1.00

MICData

Table2:ChallengeTestResultsfor2.0%LactobacillusFermentinO/WCreamformulationinoculatedonday0andre-inoculatedonday28



PCPC Method 3 – Determination of Preservation Adequacy of Water –Miscible Cosmetic and Toiletry FormulationsGenericBase Cream+ 2.0% LactobacillusFerment – pH 3, 4, 5, 6, 7, 8, and 9

PurposeThese studies were initiated to determine the efficacy of a cosmeticingredient with antimicrobial properties in cream formulations againstbioburdenas a function of time.

TestedOrganisms• Escherichia coli• Pseudomonas aeruginosa• Staphylococcus aureus• Aspergillus brasiliensis


LactobacillusFermentChallengeTestinGenericBaseCream–pH3

Table3:ChallengeTestResultsforGenericCreamwith2.0%LactobacillusFermentinoculatedonDay0.Resultsshowpercent(%)reductioninviableorganisms.

The results of this Challenge Testdemonstrate the effectiveness ofLactobacillus Ferment as an antimicrobialagent in a cream formulation pH 3.

The gram negative and gram positivebacteria were reduced by greater that99.9% within 7 days of each challenge.The mould was reduced by greater than90.0%within 7 daysof each challenge.


LactobacillusFermentChallengeTestinGenericBaseCream–pH5


The gram negative and gram positivebacteria were reduced by greater that99.9%within 7 days of each challenge. Themould was reduced by greater than 90.0%within 7 daysof each challenge.





The gram negative and gram positivebacteria were reduced by greater that 99.9%within 7 days of each challenge. The mouldwas reduced by greater than 90.0% within 7days of each challenge.

ChallengeTestinGenericBaseCream–pH7




The results of this Challenge Test demonstratethe effectiveness of Lactobacillus Ferment asan antimicrobial agent in a cream formulationpH 9.

The gram negative and gram positive bacteriawere reduced by greater that 99.9% within 7days of each challenge. The mould wasreduced by greater than 90.0% within 7 daysof each challenge.


ChallengeTestinGenericBaseCream–pH9


• Thehistoryofthecosmeticindustryoverthepast30yearshascreatedincreasingpressureonconventionalpreservativesandbiocides

• Thiswillcontinuetointensifyunlesswefindalternativeapproachestothepreservationofcosmeticsandpersonalcareproducts

• Thereareeffectivenaturalalternativestoconventionalpreservationmethodsthatonlytargetmicrobesbutnottheunderlyingenzymesandcellfunctions theyaffect,suchasHDAC

• Providesasolutiontofindingtheidealbalancebetweeneliminatingbadandpromotinggoodbacteria

Conclusion

'15 Sep - Scholz Presentation2. Cells incubated on growth media overnight 3. Day 2 –Begin 1:2...

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