J. Soc. Cosmet. Chem., 33, 27-37(January/February 1982)

Antimicrobial effectof zincpyrithione GENJI IMOKAWA, HARUO SHIMIZU and KIKUHIKO OKAMOTO, KaoSoapCo.Ltd., TochigiResearch Laboratories, 2606Akabane,Ichikai-Machi Haga-Gun,Tochigi, Japan.

Received April 27, 1981.Presented at theSCCAnnualScientific Seminar,IVashington, D.C., May 21-22, 1981.

Synopsis

The antimicrobial effectof zinc pyrithionehas beenstudiedwith healthyscalpsin relationto its ANTIDANDRUFF effect. Use of zinc pyrithione-containing shampoo sharply reduced the CORNEOCYTECOUNTS,whilethesimilarly activeantimicrobial agent,IrgasanDP-300 © hadno effect on the corneocyte counts.Quantitative estimationof scalpmicroorganisms revealedthat corresponding to thedecrease in dandruff, Pityrospbrum ovalewerespecifically loweredbyzincpyrithione treatments, but not by IrgasanDP-300©. Subsequent observations withoutshampooing indicatedthat the corneocyte countrecovered to controllevelafter6 daysdespitethe continued suppression of Pityrosporum ovale.In contrast,non-medicated shampooing, afterthe suppression of dandruffby zincpyrithione,showeda continuedreducedcorneocyte countdespiterecovered growthof Pityrosporum ovale.

INTRODUCTION

In theirstudyon the pathogenesis of dandruff,Leydenet al (1) indicatedthat after suppression of dandruffandyeastwith selenium sulfide,dandruffreturnsdespite the continuous suppression of Pityrosporum ovaleby amphotericin B and suggested that scalpmicroorganisms do notplaya primaryrolein thepathogenesis of dandruff and that the modeof antidandruffactionof seleniumsulfideis cytostatic.A viewhasbeen

expressed by Kligmanet al (2)thatzincpyrithione couldhavea similarantimetabolic action on the epidermis.Althoughzinc pyrithione,a derivativeof the naturally

occurring antibiotic aspergillic acid(3,4),isa potentantimicrobial agent(5-8),thereare few papersdescribing quantitatively the antimicrobial effectof ZPT on the scalps in vivorelativeto itsantidandruff mechanism. In thepresent study,antimicrobial effectof zincpyrithione hasbeenstudiedon healthyscalpsto testthe relationship between microorganisms anddandruff. MATERIALS

AND

METHODS

SHAMPOOING

Usinghealthyvolunteers, testshampoos containing 1.0%Zinc pyrithione (ZPT) or 0.2%IrgasanDP-300© (DP-300)weretreated3 timesweeklyfor 2 weeksfollowing3 27

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

times weekly pretreatments for 1 week with a non-medicated shampoo.This non-medicated shampoowas composedof the samematerials(mainly1556lauryl

sulfatetriethanolamine salt)excludingZPT or DP-300.Gradingand countswere obtainedon days0, 7, 14 and21,always3 daysafterwashing with eachshampoo. Subjectsweregradedon a ! to 4 scaleseparately for dandruffand itching.The descriptive equivalents of various gradesareasfollows:In dandruff, grade1 showsno or minimalscaling,grade2 mild dandruff,grade3 moderatedandruff,andgrade4 severedandruff.For itching,grade! showsno or minimalitching,grade2 mild itching,grade3 moderateitching,andgrade4 severeitching. SAMPLING

AND

CORNEOCYTE

COUNTS

Samplingof corneocytes, bacteriaand yeastswasperformedat two differentscalp areasfor the samepersonaccordingto the methodof Leydenet al (1), usingtwo 1-minute scrubs with! ml of 0.2%TritonX-100in a 2.0cm2areadelineated bya sterile glasscylinder.Corneocyte countsweredoneaccordingto themethodof McGinleyet

al (9)anddensity expressed asthegeometric meanpercm? MICROBIAL

COUNTS

Aerobicbacteria whichweremostlycomposed of S.epidermidis in oursubject's scalps werecountedby pourplatemethod.In brief,102,103and104-fold dilutions of the scrubbed sample weremade.Oneml of thedilutionand15to 20ml of Tripticase Soy Agar(BBL)with0.5%yeastextractsterilized andthencooledto 45to 50øCwaspoured intostrilepetridish.After theagarsolidified, theplateswereinvertedandincubated at 35øCfor 3 daysto countthe colonies.

Anaerobic bacteria whichpredominantly consisted of a fewspecies of Propionibacterium werecountedby surfaceculturemethod.0.1 ml eachof 102,103and 104-fold dilutions of thescrubbed sample werespread on PUK-Agar.Thecolonies whichwere judgedas strictanaerobewere countedafter incubationat 35øCfor 7 daysin an atmosphere of 90%N2 and 1056CO2.Yeastwhichmainlyconsisted of Pityrosporum ovaleand Pityrosporum orbiculare with averageratio of 10:1weredirectlycounted according to the methodof McGinley(10).0.2 ml of eachscrubbed samplewas pouredin a flat hollow of 1.4-cm-dia. equippedon a slideglass.After drying, triple-strength Giemsastainwasaddedfor 1 hr,theslideglasses rinsedwithtapwater, thendriedandcountedlight microscopically underoil immersion. Microbialcounts wereexpressed asthegeometric meanpercm•. The significance of the difference wascalculated by the Student's t-test.

RESULTS COMPARISON

OF ANTIDANDRUFF

EFFECT BETWEEN

ZPT AND

DP-300 BY SHAMPOOING

Antidandruff effectof zincpyrithione (ZPT) hasbeenstudied in comparison withthe similarlyactiveantimicrobialagent,IrgasanDP-300 (DP-300).The use of ZPTcontainingshampoosharplyreducesthe amountof dandruff,as revealedby both clinicalgrade(Figurela) andcorneocyte count(Figurelb) (p < 0.01)2 weeksafter initiationof testshampooing, whereas thesimilarly activeantimicrobial agent,DP-300

ANTIMICROBIAL

EFFECT

OF ZPT

29

..4

ß ZPT

1.0 %

o DP,-300

Before

1

2

Control Shampoo

3

0.20/0

Week

Test Shampoo

Figure la. Antidandruffeffectof ZPT by shampootreatmentas expressed by clinicalgrade,in comparison withthe similarlyactiveantimicrobial agent,DP-300. 6.0-

o ß

0.2% 1.0%

DP-300 ZPT

ß

o

ß

o )

o o

o

ß o

5.0 P•O.01

i

I

I

Before

1

2

Control Shampoo

3

Week

Test Shampoo

Figure lb. Antidandruffeffectof ZPT by shampootreatmentas expressed by corneocyte count,in comparison with the similarlyactiveantimicrobial agent,DP-300.

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

•

o

o

o

ßZPT!.0%

O D P-300 0.2O/o

Before

1 ,L

;

Control

2

3

Test

Shampoo

I

Wee k I

Shampoo

Figure lc. Antidandruffeffectof ZPT by shampootreatmentas expressed by itching,in comparison with the similarlyactiveantimicrobial agent,DP-300.

has no effect.As for itching,ZPT indicatedreducedeffectcorresponding to the decrease of dandruff,in contrastto no effectby DP-300(Figurelc).

Quantitative estimation of scalpmicroorganisms (Figure2) revealed thatcorresponding to the decrease in dandruff,Pityrosporum ovaleare specifically suppressed by ZPT-containing shampoo (p < 0.001),butnotbyDP-300.Despitea greatreduction of clinicalgradesor corneocytecountsinduced by ZPT, no substantialeffect on anaerobic bacteriacouldbe demonstrated. Thus,theonlynoteworthydifference in the microbialchangesbetweenZPT and DP-300is the specificdecrease in Pityrosporum ovalebyZPT. Althoughonemayconsider thatthissuppression of Pityrosporum ovaleis a resultof decreased nutrientsdueto the decrease in corneocytes, suchan environment

must be appliedequallyto other microorganisms and shouldresult in similar suppression of anaerobic bacteria.However,ourresultsdo not coincidewiththisview. At firstsight,theyseemto suggest thatthe decrease in Pityrosporum ovalecontributes to the reduction of dandruff.

EFFECT INDUCED

OF

NON-MEDICATED BY

SHAMPOOING

ON

SUPPRESSED

DANDRUFF

SITUATIONS

ZPT

In orderto furtherclarifythe relationship betweenPityrosporum ovaleanddandruff, subsequent observations with non-medicated shampooing followingthe suppression of bothdandruffandPityrosporum ovaleby useof ZPT havebeencarriedout.Thus, discontinuance of ZPT by threeweeklynon-medicated shampooing indicatedrecoveryof thenumberof Pityrosporum ovaleup to thepretreatment levelbya week(Figure 3a)evenwith the continuedly reducedcorneocyte counts(Figure3b).Thesefindings suggestthat the amountsof dandruffdo not alwaysparallel the number of

ANTIMICROBIALEFFECTOF ZPT

31

1 06

t

o o

•10 $ o

I 04

1 03

©o

Ye

ßoAnaerobic bacteria ,,

•e

st

Shampoo

Figure 2. Quantitative microbiology ofthescalp observed before and after ZPTshampoo treatment in

comparison withthatofDP-300. I ß ZPTshampoo; O[] DP-300 shampoo.

Pityrosporum ovale, although there isapossiblity thatcorneocyte counts have acertain

lagperiod torecover tothecontrol level even after theincrease inPityrosporum ovale. NON-SHAMPOOING EFFECT ON SUPPRESSED DANDRUFFSITUATIONS

Theeffect ofno-shampooing ondandruff andmicrobial counts suppressed byZPT

wassubsequently studied. Noshampooing forsixsuccessive days disclosed recovered

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

1 07

•: 06

o

ß

o

o

o

o

o o

o

o

ß

o

•-,.

0

o-•-........ -O4= =1

05 o

o

ß

P-•0.001

o 04

Aerobe

Anaero

be

Yeast

I

I

I

I

I

2 -*-Test

I

I

3

&

4

Week

Period -•

Figure3a. Effectof non-medicated shampooing for 1 weekonthemicrobial countof thescalp,where dandruffsuppression hasbeeninducedby ZPT.

ANTIMICROBIAL

EFFECT

OF

ZPT

33

6.0

1.0%ZPT

ß

5.0

I

•

Control --•

2

•-Tre

at

3

• •-Control

4

Week

-•

Figure 3b. Effectof non-medicated shampooing for ! weekonthecorneocyte countof thescalp,where dandruffsuppression hasbeeninducedby ZPT.

amountsof dandruff(Figures4a & 4b) despitethe continuedly significant suppression of Pityrosporum ovale(Figure5). In thisexperiment, an apparenteffecton aerobicand anaerobicbacteriawashardlydemonstrated althougha slightincrease in theirnumbers occurredthrough6 daysof no shampooing. DISCUSSION

The questionof microbialinvolvementin the antidandruffmechanism of ZPT still remains to befurtherclarified.Thus,sinceMalassez (! 1)pointedout Pityrosporum ovale aschiefsuspect in dandruffetiology,therehavebeena largenumberof reportstrying to confirmthe link betweenthe two. Most investigators (2,10,12-14)haveconcluded that thereis no connection.However,thesestudieshavenot includedmicrobiological analysis followingZPT treatments. SinceZPT is knownto be a uniqueantidandruff agentavailablein the cosmeticfield(15) whichhasa wide rangeof antibacterial and antifungal properties (5-8),it oughtto be utilizedfor quantitative analysis withrespect to the questionof microbialinvolvementin the antidandruffmechanism.These factorsled us to investigate relationship betweenmicroorganisms and dandruffin varietyof ZPT application system. Our microbialstudiesdemonstrated the following:1) the only noteworthydifference in the microorganisms whichoccurscorresponding to the reductionof dandruffisthe

34

JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

Dandruff Itching

ß

ß

ß

WO

SO

i

I

I

I

I

i

0

3

4

5

6

7

Day

Figure4a. Effectoœ noshampooing forsixsuccessive daysondandruff suppressed byZPT,asexpressed by clinicalgradeanditching.

6.0

P*•O.001

5.0

i

7 Day Figure 4b. Effectof no shampooing for six successive dayson dandruffsuppressed by ZPT, as expressed by corneocyte count.

ANTIMICROBIAL

EFFECT

OF ZPT

35

P•0.01

P•0.01

•

O

oo

I 06

0

P•O.01 o

•1 05

o

o

P•0•5

I 04

o

Aerobe

0

Anaerobe

ß Yeast

3

3

4

$

6

7

Day Figure 5. Effectof no subsequent shampooing for six successive dayson microbialcount,following treatments with 1.0%ZPT-containing shampoo for 3 weeks.

36

JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

specificdecreasein Pityrosporum ovale;2) aerobicand anaerobicbacteriaare not influencedby the useof ZPT evenunderthe suppressed dandruffsituation;3) no shampooing aftersuppression of dandruffandPityrosporum ovaleinduces recovery of dandruffupto controllevel,butdoesnot increase thenumberof Pityrosporum ovalein 6 days.Fromthesefindings,it is clearthat the presence of ZPT molecules on scalpin concentrations capableof maintainingthe continuoussuppression of Pityrosporum ovaledoesnot alwaysresultin reductionof dandruff.Thissuggests thattheremaybe greatdifferences betweenthe effectiveconcentrations of ZPT requiredto suppress dandruffandPityrosporum ovale.Our microbialstudiesfrom someviewpoints fail to revealdirectevidencethat the decreased numberof Pityrosporum ovalecontributeto the reductionof dandruffinducedby the useof ZPT. It is verylikelythat ZPT may havean influenceon other sitesof the skin in a differentmannerfrom the way it suppresses Pityrosporum ovaleandreduces theamountsof dandruff. Leydenet al (1) demonstrated similarsuppression of Pityrosporum ovalewithouta decreasein the amountsof dandruffby use of AmphotericinB and emphasized significanceof antimetabolicaction by antidandruffagentsin improvementof dandruff.Kligmanet al (2) hypothesized thatthe modeof antidandruff actionby both ZPT and SeSis cytostatic,althoughno data were given regardingZPT's effectin decreasing epidermalproliferation.SeSdoesdecrease the corneocytecount,with a corresponding declinein the labelingindex. Concerning themodeof antidandruff actionof ZPT, littleisknownaboutthepossible biologicalfunctionof ZPT-dependent reductionof dandruffexceptfor a few actions associated withitshighlyantimicrobial effect.Priestly(16)speculated, basedon acute toxicityof ZPT to culturedcells,that ZPT's actionagainstdandruffis a resultof non-specific toxicityfor epidermal cells.Glooret al (17)alsohavereported antimitotic effect of omadineMDS, which is chemicallysimilarto zinc pyrithione.Since pyrithioneis a generalinhibitorof membranetransportprocessin fungi(7), it is possible that the suppression in dandruffby ZPT is dueto a markeddecrease in the activitiesof a varietyof independently regulated transportsystems in epidermal cells. In conclusion, the questionof whetherthe mode of antidandruffactionof ZPT is involvedwith antimetabolic effecton epidermisor not may requirefurtheranalysis, especially of its cellulareffects.

REFERENCES

(1) J. J. Leyden,K.J. McGinley and A.M.

Kligman,Role of microorganisms in dandruff,Arch

Dermatol.,112, 333-338(1976).

(2) A.M. Kligman,K.J. McGinleyandJ.J. Leyden,The natureof dandruff, J. Soc.Cosmet. Chem.,27, 111-139(1976).

(3) E. Shaw,Analogsof aspergillic acid.I. The tautomerism of the hydroxypyridine N-oxide,J. Am. Chem.Soc.,71, 67-70 (1949).

(4) E. Shaw,J. Bernstein,K. Loseeand W. Lott, Analogsof aspergillicacid. IV. Substituted 2-bromopyridine-N-oxides andtheirconversion to cyclicthiohydroxamic acid,J.Am.Chem. Soc., 72, 4362-4364(1950).

(5) F. E. Pansy,H. Slander,W. L. Koerberand R. Donovich,In vitrostudieswith 1-hydroxy-2(1H) pyridinethione, Proc.Soc.Exp.Bid. Med.,82, 122-124(1953). (6) H. Yale, Sulfurand seleniumcompounds of pyridine,Part IV, in Pyridineand its derivatives, E. Klingsberg, Ed.,(Interscience, New York, 1964),p. 345-437.

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EFFECT

OF

ZPT

37

(7) E. W. Brauer,D. L. OpdykeandC. M. Burnett,The anti-seborrheic properties of zincpyrithione in a creamvehicle,J.Invest.Dermatol.,47, 174-178(1966). (8) C. j. Chandlerand I. H. Segel,Mechanismof the antimicrobialactionof pyrithione:Effectson membrane transport, ATP levels,andproteinsynthesis, Antimicrob. Ag.Chemother. 14, 60-68(1978). (9) K.J. McGinley,R. R. Marplesand G. Plewig,A new methodfor visualizing and quantitating the desquamating portionof thehumanstratumcorneum, J. Invest.Dermatol., 53, 107-111(1%9). (10) K. J. McGinley,J. j. Leyden,R. R. Marples,M. R. C. Path and A.M. Kligman,Quantitative microbiologyof the scalpin non-dandruff, dandruff,and seborrheic dermatitis, J. Invest.Dermatol., 64, 401-405(1975).

(11) L. Malassez, Note surle champignon dela pelade,Arch.Physiol. Norm.Pathol.,II, 1,203(1874). (12) R. W. VanderWyk andK. E. Hechemy,A comparison of the bacterialandyeastfloraof the human scalpandtheireffectupondandruffproduction,J. Soc.Cosmet. Chem.,18, 629-632(1%7). (13) A. B. Ackermanand A.M. Kligman,Someobservations on dandruff,J. Soc.Cosmet. Chem.,20, 81-101(1969).

(14) G. PlewigandA.M. Kligman,The effectof selenium sulfideon epidermal turnoverof normaland dandruffscalps,J. Soc.Cosmet. Chem.,20, 767-775(1%9). (15) A.M. Kligman,R. R. Marples,L. R. LantisandK.J. McGinley,Appraisal of efficacyof antidandruff formulations,J. Soc.Cosmet. Chem.,25, 73-91(1974). (16) G. C. Priestley andJ. C. Brown,Acutetoxicityof zincpyrithione to humanskincellsin vitro,Acta Dermato-Venereol., 60, 145-148(1980).

(17) M. Gloor,M. DresselandU. W. Schnyder, The effectof coaltar distillate,cadmiumsulfide,ichtyol sodiumandomadineMDS on the epidermis of theguineapig,Dermatologica, 156, 238-243(1978).