v.
`Galderma Laboratories, Inc.
`IPR2015-__
`Exhibit 1019
`
`Exh. 1019
`
`

`
`·~022-202Xi78/0070-0001$02.00/0
`1
`HE JouRNAL
`Copyr·ight © OF NV~~TIGATIVE DERMATOLOGY, 70:51- 55, 1978
`1978 by lhe W!lhams & Wi lkins Co.
`
`Vol. 70, No. 1
`Printed in U.S.A.
`
`THE EFFECT OF ANTIMICROBIAL AGENTS ON LEUKOCYTE
`CHEMOTAXIS
`NANCY B. EsTERLY , M.D., NANCY L . FuREY, M.D., AND LrLLIAN E . FLANAGAN, B.S.
`Division of Dermatology, Department o(Pediatrics, Michael Reese Hospital and Medical Center, Chicagu, lllinois, and
`Department oj"Derm.atology, Northwestem Uniuersity Seiwal o/ Medicine,
`Chicago, lllinois, U .S .A.
`
`.
`'l'h
`e effects of several chemothcmpeutic agents on thc
`h
`c emoh ·
`fh
`• XIS o
`uman leukocytes were studied in an in vitro
`S
`system u ·
`t , ·h
`.
`smg a ykes-Moo•·c chamber and a doublc-filtcr
`. ect. mque. Chemotactic factor was genemted by the inter-
`1
`ac •on of
`t'
`norma human scrum and zymosan. At concentra-
`t iotns comparablc to and bclow therapeutic blood Ieveis,
`e •·acy r
`H
`Were alc 1.nc . ~1, e~·ythr~mycin base and clin~amycin HCI
`I lflhibJtory, causmg marked suppresswn of leuko-
`d 1· h
`cyte chemotax· ·
`d
`·
`·
`·
`t'
`IS an s 1g t re uctwn of random m1g1·a 1011.
`. . .
`p
`w~~:cllhn G-Na, dapsone, and sulfapyridine did not alter
`. . 1 e cell motility at thc concentrations of dmg tested. 1t
`;s :o~tulated that the Capacity of somc of these agents to
`thn ~bJtf ~eukocyte chemotaxis may account, in part, for
`e1r e flca
`·
`· 1
`.
`cy lfl mf ammatory skin diseases such as acnc
`vulgans.
`
`an in vitro system, using concentrations of a ntibiotics below,
`within, and a bove the range oftherapeutic semm Ievels. Also
`included in these assays are two chemotherapeutic agents
`(dapsone a nd sulfapyridine) that are prescribed frequently für
`a variety of inflammatory skin diseases.
`METHODS
`
`Gell Preparations
`On the day of assay, venous blood was coll ected from hea lthy
`human vol unteers in a hepa rinized syringe. One ml of P lasmagel
`(HTI Corpora t ion , Buffalo, N . Y.) was added für each 10 ml of blood
`collected , and th e syringe was incubated a t 37oC in a n inverted
`position for 2 hr to permit Sedimenta tion of ce lls . The leukocyte- rich
`plasma was separa ted from the erythrocytes a nd centrifuged at 900
`rpm tor 10 min . The cell pell et was suspended in minima l essentia l
`media (MEM) (Gra nd Is la nd Biologica l Company, Gra nd Isla nd ,
`N. Y.) with 10% feta l ca lf ,'erum rFCS) rReheis Chemica l Corpora-
`tion, Phoenix, Ariz.), washed twice with MEM a nd resuspended in
`5 ml of MEM-FCS für counting. Th e fin a l leukocyte concentration
`was adjusted to 2.5 x 10"/ml by di lution with MEM-FCS, a nd a cell
`vi a bility of at least 99% was esta blished , us ing th e trypan blu e dye
`exclusion method.
`
`Chemotactic Factor
`Chemotactic factor was genera ted by in cuba tion of fresh or frozen
`( - 70°C) norma l huma n serum with zymosa n !Sigma Chemi ca l Co.,
`St. Loui s , Mo. ) in a concen tration of 2 mg/ml für 1 hr at 37°C. The
`zymosan was removed by centrifugation, a nd th eserum was hea ted
`to 56°C for 30 min to terminate the reaction I17J.
`
`ce~\al administration of certa in antibiotics a nd, more re-
`. ·~' .topJcal apphcatwn of these antibiotics have been
`c
`01181 eted effective therapeutic rneasures for pa tients with
`acne vulg·a · s
`.
`.
`·
`n s. uccess w1th these agents has been attnbuted,
`ln part t
`th
`.
`.
`0
`e reductwn m numbers of n01·mal cutaneous
`f1
`'
`w~;~·tharticularly Cor:ynebacterium acnes , and to interference
`3J. Thu e actwn of bacteria l lipases on sebum triglycerides /l -
`and b s, by. reducmg the populations of specific orga msrns
`th Y partJally suppressmg the formation of free fatty ac1ds,
`P e nlatura] Progression of acne lesions frorn comedones and
`apu es to p
`t I
`d
`.
`.
`b
`.
`ru t d
`. us u es an cysts theorebcal ly rn1ght e mter-
`thp e · Although nurnerous investigators have documented
`e effects of a t.b . t.
`·
`t '
`h
`n I 10 1cs on cutaneous flora and on quantita-
`d~e c anges in suJ·face Iipids, not a ll of these studies have
`d monstrated a direct relationsh ip between administration of
`rug and r d
`t·
`·
`.
`.
`.
`·
`t .
`~ uc IOn 1n bacten a l coun ts a lteratwn 111 cornposi-
`Jon of suda
`1· ·d
`. .
`.
`'
`.
`.
`14-13 /. So ~e .1P1 s, a nd chmcaiimprovement of skm l esJ.~ns
`e
`.
`. me of the d1screpancws may be explamed by dlfler-
`u~~es, In rnet~odology; however, the participation of additional
`T~scnbed factors may account für the therapeutic eftect.
`. ere lsevJdence in the lite1·ature to suggest that certain
`.
`dntJmJcrobJ· 1 .
`.
`.
`. a
`agents may suppress leukocyte chemotax1s
`114 15
`J .. Smce mflamrnatory acne lesions which are most
`'
`usceptibl t
`.
`··b·
`.
`.
`,
`.
`S
`e 0 anti IOtJc therapy, are the resu lt ofan wflux of
`ol
`~ Ymorphonuclear leukocytes /16 1 it seemed z·easonable to
`cons1der th h
`·h
`·
`·
`'
`.
`.
`ta .
`.
`e ypot es1s that suppress1on ot leukocyte chemo-
`Thx~s ~ght account, in pa rt, for the effi cacy of these agents.
`
`Prepara.tion o/ Antibiolies
`The füllowin g drugs were tested in chemotax is assays: erythro-
`mycin base lAbbott Laboratori es, North Chi cago, 11 1. ), tetracycline
`HC I ILederl e Laboratories, Pea rl R iver, N . Y.), clinda mycin HCI
`(Upjohn Compa ny , Ka lamazoo, Mich. ), penicill in G-Na rUpjohn
`Compa ny, Ka lamazoo, Mi ch .), da psone IAyerst La boratori es, New
`York, N. Y .), a nd su lfapyridine rLilly La boratories, lndi a napo li s,
`l nd. ). Stock solutions of each a ntibiotic were prepa rcd in MEM a nd
`appropriate a mounts added to ach ieve the desircd concen tra tion in
`the chamber. High concentra tion stock solutions of sulbpyridine
`a nd da psone were prepa red in absolute ethanol a nd diluted with
`MEM. Tetracycline HC I a nd erythromycin basc were assayed 111
`concentra tions of .01 J.Lg/ml, 1 J.Lg/ml , 10 J.Lg/mi, a nd 300 J.Lglm i.
`Clinda mycin H CI was added in concentrations of .2 J.Lg/ml , 2 J.Lg(m i,
`20 J.Lg/ml, and 200 J.Lglm l; peni cill in G-Na in w ncentratwns of .01
`J.tg/m l, .1 J.tg/ml, 10 J.tg/ml, a nd 100 J.tg/ml. Sulfapyndme was tcsted
`at 50 J.tg/ml a nd 100 J.tg/m l a nd dapsone a t 25 J.tg/ml a nd 50 J.tg/ml.
`Antibioti cs were added to the lower (attracta nt) compa rtm e ~1t ofthe
`cha mber in the first set of experirnents. In il second set of ex pen -
`ments
`th e antibiotic was added to the leukocyte Suspension in the
`upper 'compa rtment. Chemotactic assays with each concentra tion of
`drug were perfünned in duplicate or tripli ca te.
`
`Chemotaxis Assays /1 8/
`The cha mbers used for the assays were of the Sy kes-Moore type
`lßellco B iologica l Glasswa re and Equiprnent, Vineland , N.J. ) I lHI .
`Two Millipore filters IMi ll ipore Corporation , Bedfürd, Mass.) 25
`mm in di a met.er a nd 3 1-' porc size were inserted between the t.wo
`compa r tments a nd held in place by a rubber gasket 1201 . The
`cha mber was sea led at both ends by a 25-mm circul a r glass cover
`slip macbester Scientifi c Co., Rochcster, N. Y .). lnjcction of materi -
`
`ec s of several of these agents on leukocyte chemotaxis in
`
`eft' ,t sent study represents an atternpt to measure the
`29.~~~~script received May 5, 1977; accepted for publication Ju ly
`MS hupported in pa rt by the Medi ca l Resea rch Institute Counci l
`Ic aei Reese Hospita l a nd Medi ca l Center.
`'
`0 ~epnnt request to: Dr. Nancy 8 . Esterly, D irector, Division of
`. e~ %atology, Depa r tment of Pediatrics, Michael Reese Hospita l
`~~616 . edr ca l Center, 29th Street a nd E ll is Avenue, Chicago, Illinois
`Abbrevia t ions:
`FCS: feta l calf seru m
`MEM: minima l essen tia l medi a
`
`51
`
`Exh. 1019
`
`

`
`52
`
`ESTERLY, FUREY, AND FLAN AG A N
`
`20
`
`19
`
`18
`
`17
`
`16
`
`15
`
`14
`
`13
`~
`~ 12
`V)
`-~ II
`Cl:
`
`6
`
`5
`
`4
`
`3
`
`2
`
`I
`
`r
`
`!!Ii
`
`~
`
`1
`
`1
`
`1111
`
`NONE
`
`I
`
`I
`
`I
`
`III
`
`~
`
`300)Jg
`
`I
`I
`~ ilj
`
`rl l
`
`.OI)Jg
`
`... ~
`
`I)Jg
`IO)Jg
`CONCENTRATIONS OF TETRACYCLINE HCL
`I' h
`F1c 1 Chemotact1. · . t. ·t
`.
`c ac IV I y o
`·
`.
`uman leukocytes with dif'l·, ., •
`cuncentrat 1ons of letr 1 .
`e1 enc
`. 1. ,
`of the chamber
`.
`c tyt tne HCI added to the lower compa rtment
`: C!eUI bars represent thc rnean of 8 assays r + 1 SO)
`I. . ..
`01 1 andom m1grat 10n of 1, k . t
`. D
`-
`v· I
`. I + 1 SO
`. .
`.
`eu ocy es.
`arh bars represent mean
`) l01 neutroph d chemotaxis.
`a ues -
`
`<~I ~ ~as, madc. w.i t~ a 25_gauge needle through the chamhcr ports.
`I I.tcement ol .1 second ~aga uge ncedl e 1n the opposite po1·t al lowed
`n1r to e~ca pc dunng III IIng, resulting in a hubble-free charnbcr.
`One-ha ll ml ol lcukocytc suspension 11.25 x 10n cclls) was addcd to
`the uppcr compa rtment, a nd a n 0.8 ml aliquot of fl uid 11 part
`human scrurn w1th or w1thout zy rnosa n plus 1 part MEM-FCS with
`or without drugJ wus added to thc lower chamher. In the second set
`of expcriments, appropriate concentrations of dru g in MEM were
`added to thc lcukocyte suspension in the upper compartment . Thc
`chambers were incubated a t :l7°C for :l hr. The lilters were removccl ,
`lixed and sta ined
`in an eth a no l, hcmatoxyli n, ethanol, xylene
`sequence, a nd mounted with Permount on slides for counting. The
`cells on each hottom li lter were counted in :10 ra ndom fi elds using a
`IOx ocu lnr and 40 x ohjective.
`in a steri le fashion, and th e
`All suspensions werc prcpa red
`cha mhers werc nutoclavcd prior to use. No prohlems with bactcrial
`" rowth were cncountered. Cell viahi lity was checket! aga in with
`Lrypan bluc dye a t t.hc tcrrn in ation of each ex periment and rern ained
`.
`,
`grea tcr thnn ~)9'/r in nll charnhcrs.
`Statisticu l a na lysiswas perform ed us1ng Stud ent s I = lest.
`
`RESU LTS
`T he mca n v;d ues :!:: 1 S O für thc che motaxis assays w i ~h
`te tracycli nc-HC I are depicted in F ig 1. The mcan va lue for
`random migration of' cclls f'rom 8 huma n vo lunteers 18 sepa-
`ra te assaysJ was 2.9 :!:: 0. 8 cc ll s/hpf', whereas the mea n Wiih
`chemotactic factor was 10.8:!:: 2.5 cc lls/hpf' lp =< .0005l . Whe n
`tetJ·acycline was added in concentrntions of' .01 fLg/ ml , 1 fLg/
`rn l a nd 10 fLg/ml , thc1·e was strik ing supp ression of'che mota x1s
`lp =< .0005), most ma rkcd a t conce ntrations of 1 (Lg and 10
`':"[!;/ml. Minima l inhibition of nt ndom migration was ohserved
`at the sa me concentrations lp =< .025 with I (Lg/m l of dru g;
`
`Vol. 70, No . l
`
`other values not sig nifi cant). However, a t a concentration of
`300 fLg/m l, there was no effect on chemotax is (mean of 11 .7 ::!:
`2.1 cells/hpf) or ra ndom migra tion lm ean ~f 3.3 :!:: 0.7 cells/
`hpf) .
`The effects of erythr omycin base on leukocyte chemotaxis
`as shown in Fig 2 were s imi lar to th ose of te tracycli n e . T he
`mca n value for ra ndom migra tion of cell s in 8 assays w as 3.0
`:!:: 0.4 cells/hpf and that for ch e motax is 14.0 :!:: 1.0 cell s /hpf
`lp =<: .005). Concentrations of .01 fLg/ml , 1 (Lg/ml, and 10 p..g/
`m l of et·ythromycin cau sed significa nt suppt·ession of chemo-
`ta~ Js lp = < .0005 for a ll concentrations) a nd b ad a minimal
`~flect on nmdom migration lp = < .01 für .01 fLg/m l; p = < .ü05
`for l (Lg/ml a nd 10 fLg/m l). Erythromycin at 300 fLg/m l fa iled
`to m h1b1t chemotaxis or ra ndom m igration, a nd the v a ]ues
`obta med w1th th is concentration of drug were not sign ifica ntly
`dtfferent fi·om thc mean contml values 13.0 :!:: 0.4 cells/hpffor
`random m igration a nd 14.0 :!:: 1. 1 cells/hpf für chemotax is).
`Cli ndamycin HC I a lso caused inhibition of' chernotaxis a t
`concentrations of .2 fLg/m l, 2 fLg/ rnl, 20 fLg/ ml, a nd 200 fl.gjrnl
`as demonstrated in Fig 3. The mea n value für t·andom migra-
`tiOn was 3.2 :!:: 0.5 cell s/hpf and th a t für hemotaxis 12.9 ::!: 1.9
`cells/hpf (p = < .0005). There was significant suppression of
`ch?motaxis a t a ll concentrations (p = < .0005) a nd a min imal
`eflect on ra ndorn m igration (p = .01 at .2 and 2 fLg/m l; p = < .l
`at 20 [Lg/ m]) with the lower concentrations of drug .
`In contJ·ast, pe n icillin G-Na in concentrations of' .01 f.Lg/ml ,
`.1 [Lg/ml , 10 fLg/m l, a nd 100 fLg/rn l fai led to inhibit leukocyte
`moti li ty IFig 4). Neithe r sulfa pyridine in concentrations of 50
`fLg/ml a nd 100 fLg/m l nor dapsone in concentrations of 25 fLgl
`ml a nd 50 (Lg/ml had a ny perceptible eflect on leukocyte
`move ment unde t· the condit ions of t he assay IFig 5).
`
`20
`
`18
`
`16
`
`14
`
`Q..
`<>::
`
`10
`
`"-Q..
`'1::
`" 12
`"' "' ~
`"' "' ~ :::;, "'
`"' "" "' ~
`
`8
`
`6
`
`I I
`
`I
`
`I
`
`4
`
`2
`
`0
`
`~
`
`rt
`
`~ I
`
`~I
`
`NONE
`
`.O IJJg
`
`(JJQ
`
`I
`I
`
`111
`I'
`dll
`II II!
`
`~ I
`I i
`
`300)Jg
`
`CONCENTRATIONS OF ERYTHROMYCIN BASE
`F'tG 2. Chemotactic activity of huma n leukocytes with dif!'erent
`concentrations of'erythromycin hase ndded to thc lower compa ,·tment
`of th e charnber. Cleur hurs represent the mean of 8 assays I:!:: 1 SD)
`fo r ra ndom migrntion of leukocytes. Durh bars represent mean
`va lues 1::':: 1 SOl for neutrophi l chemotaxis.
`
`Exh. 1019
`
`

`
`~-
`
`Jan.1978
`
`20
`
`18
`
`16
`
`14
`
`~
`
`"-~
`" 12
`"' ~
`"' Q:)
`~ ::::.
`~ 8
`~
`~
`~
`
`!);:
`
`10
`
`6
`
`EFFECT OF A NTIMICROBIAL AGENTS ON LEUKOCYTE C HEMOTAXIS
`
`53
`
`been utilized in previous studies to dernonstra te the effect of
`drugs on the migration of polymorphonuclear leukocytes . The
`antiinllarnmatory agents, chloroquine, hydrocortisone, a nd
`rnethyl prednisolone have been shown to suppress chemotaxis
`by direct action on the neutrophil 122 1. Mafenide aceta te and
`sulfadiazine compounds, widely used for the treatment of
`burns, a lso appear to cause ma rked inhibition of leukocyte
`rnotility 1191.
`Martin et al. observed reduced migra tion of leukocytes
`obtained fi·om human volunteers with Mycopla.sma. pnew rw-
`nia.e infection who were receiving tetracycline 1231. Subse-
`quent studies demonstra ted tha t concentrations oftetracycline
`ranging from 0.01 to 10 J.Lg/ml depressed both random migra-
`tion a nd chemotaxis of leukocytes toward Mycopla.smu a nti-
`gen 1141. In contrast, concentra tions of tetracyclinc nmging
`from 30-300 J.Lg/ml caused Stimula tion of neutrophil move-
`ment. The mecha nisms by which the drug influences white
`cell movement are unknown, but it has been suggested that
`these effects may rela te to altera tions in white cell metabo-
`lism.
`Although we used zymosa n-induced chemotactic factor
`ra ther tha n Mycoplasm.a a ntigen as a chemoa ttractant, our
`studies confirm the findings of Ma rtin a nd co-workers. Gon-
`centrations of tetracycline in the ra nge of therapeutic blood
`Ievels !0.1- 10 J.Lg/m]) appeared to inhibit neutrophil chemo-
`taxis whereas tetracycline in very high concentm ti on 1300
`J.Lg"/ml) fai led to suppress chernotaxis. The effect of tetracycli ne
`on random migra tionwas less pronounced in ow· assay system
`tha n in the study of Ma rtin et al. Thi s disparity in t·esults
`rnay be a ttributable to severa l differences in methodology . lt
`is uncl ear whether the tetracycline was added to the upper
`
`20
`
`18
`
`)6
`
`14
`
`~
`
`"-~
`" 12
`"' ~
`~
`~ 10
`!);: "' Q:)
`~
`::::.
`""
`~
`"<(
`.....
`~
`
`8
`
`6
`
`4
`
`2
`
`0
`
`!
`I
`I
`
`I
`
`I
`rt l
`
`I
`
`I
`
`r±
`
`rr
`
`rf
`
`~
`
`NONE
`
`./)J g
`
`IOJ.Jg
`
`/00)Jg
`
`CONCENTRATIONS OF PENI CI LLIN G Na
`F1c 4. Chemotactic activ ity of huma n leu kocytes with diflerent
`concentrations of penicillin G-Na added to the lower compa rtment
`of the cha mber. C/ea r ba rs represent the mea n of 8 assays I± I SOl
`for ra ndom migration of leukocy tes. Dark hars represen t rn ean
`va lues ( ± I SD) for neutrophil chemotnx is.
`
`4
`
`2
`
`0
`
`rf
`
`~
`
`~
`
`tt
`
`NONE
`
`.2)Jg
`
`&
`
`200)Jg
`
`CONCENTRATIONS OF CLINDAMYCIN-HCL
`FIG 3. Chemotactic activity of huma n leukocytes with different
`concentra tions of clindamycin H CI added to the lower compa rtment
`of the chamber. Clear ba.rs represent the mean of 8 assays ( ± 1 SDl
`for ra ndom migra tion of leukocytes. Dark bars represent mean
`valu es ( ± 1 SDl for neutrophil chemotax is.
`
`In the second set of experirnents when the drugs were
`added directly to the leukocyte suspensions prior to incuba-
`tion , the results obtained were cornpa rable to those depicted
`in F ig 1- 5 (Ta ble). Tetracycline HCI, erythromycin base a nd
`clinda mycin-HCI a t the s~ rne concentra tions indica ted in the
`fi gures ca used ma rked suppression of chernotaxis a nd minimal
`suppression of m ndom rnig ra tion. The rnean values for ran-
`dom rnigration fell slightly below the control v~ lues , a nd.
`were the same order of magnitude as in the ftrst set of
`expe rime nts . The only significa nt difl"erence noted between
`this set of experirnents and those described above was a
`fa ilure of clinda rnycin HCI to inhibit chemotaxis when added
`in a conce nt1 c~tion of 200 J.Lg/ml (p = < .0005). Penicillin G- Na,
`sulfa pyridine a nd dapsone had no efl"ect on leukocyte chemo-
`t axis when added to the upper compa rtment of the cha mber.
`
`DISCUSSION
`A ntimi crobi a l agents are frequently used to treat infla m-
`m a tory disorders of the skin tha t a re cha t·acterized by a n
`infl ux of polyrnorphonuclear leukocytes but a re not due to
`bacterial infection. Although severa l hypotheses have been
`advanced to account for the eflect of these drugs in infl a mma-
`tory conditions, the proposed mecha nisms of action a re still
`controversia l, owing to conflicting da ta . A possible effect on
`]eukocyte migration has received little consideration in the
`development of these concepts; therefore, it seemed of i nterest
`to study the interaction of a ntimicmbia l drugs and polymor-
`phonuclear leukocytes in a n assay systemfür chemotaxis:
`The modjfied micropore cha mber has proved tobe a reltable
`systern in which the chemotactic t·esponse of white bl ood cells
`ca n be assayed in the la bora tory 1211. This technique has
`
`Exh. 1019
`
`

`
`54
`
`ESTERLY, FUREY, AND FLANAGAN
`
`Vol . 70, No.l
`Chemotactic activity oj' hunwn leulwcytes with different concentmtions o{antimicrohia/ agen.ts culded to the upper compartm.ent oj' the
`clwmber. Vulues areexpressedas means o/'8 assays :t:: 1 SD
`----~--~--------~~==~
`~~--~--------~-------
`Erythr omycin
`Tetracycli ne
`Clindamycin
`Pen icill in
`Su lfapyridin e
`Dapsone
`None
`None
`None
`3.0 :t:: .:l
`2.5 :t:: .5
`2.2 :t:: .4
`None
`2.4 :t:: . J
`3.7 :t:: .5
`3.7 :t:: .5
`Zyrnosan
`Zyrno-
`Zyrno-
`12.7 :t:: . 7
`11.2 :t:: .3
`11.0 :t:: 1.0
`Zyrno-
`13.3 :t:: .5
`13.5 :t:: .9
`13.5 :t:: .9
`san
`san
`san
`. 01 jJ.g
`.2 jJ.g
`100 jJ.g
`.2 jJ.g
`. 01 jJ.g
`100 JJ.g
`+ Z
`+Z
`+ Z
`1 jJ.g
`2 jJ.g
`50 JJ.g
`1 jJ.g
`2 jJ.g
`50 JJ.g
`+Z
`+ Z
`+Z
`10 jJ.g
`20 jJ.g
`10 jJ.g
`20 jJ.g
`+Z
`+ Z
`300 jJ.g
`200 JJ.g
`2.2 :t:: .3
`300 jJ.g
`200 jJ.g
`10.8 ± .9
`+Z
`+ Z
`~~- ~- -- ~- . -
`- -~- ---
`
`2.2 ::!::: .:3
`2.2 :t:: .5
`
`1. 9 :t:: .4
`2.4 :t:: .2
`
`2.0 ± .4
`2.6 ± .4
`
`.01 jJ.g
`.01 jJ.g
`+ Z"
`1 JJ.g
`I JJ.g
`+Z
`10 jJ.g
`10 jJ.g
`+Z
`300 jJ.g
`:100 jJ.g
`+Z
`" Z = zymosan
`
`2.5 :t:: .8
`2.2 :t:: .5
`
`2.3 :t:: .2
`2.2 :t:: .1
`
`2.4 ± .2
`2.2 ± .3
`
`2.:J :t:: .4
`12.0 ± .8
`
`2.0 :t:: .1
`2.2 :t:: .2
`
`2.2 :t:: .1
`2.1 :t:: .2
`
`2.2 ± .2
`2.6 ± .6
`
`None
`Zyrno-
`san
`.01 jJ.g
`.01 JJ.g
`+ Z
`.1 jJ.g
`.1 jJ.g
`+ Z
`10 JJ.g
`10 jJ.g
`+ Z
`100 J.Lg
`100 JJ.g
`+Z
`
`2.1 :t:: .1
`13. 1 :t:: .8
`
`2.1 :t:: .2
`12.5 :t:: .8
`
`2.1 ± .2
`13.2 ± 1. 6
`
`2.3 :t:: .2
`13.7 ± .6
`
`None
`Zyrno-
`san
`50 jJ.g
`50 jJ.g
`+Z
`25 jJ.g
`25 jJ.g
`+Z
`
`3.2 :t:: .4
`13.7 :t:: 1.5
`
`3.1 :t:: .2
`13.6 ± 1.3
`
`:L6 :t:: .4
`14.1 :t:: .7
`
`3.7 :t:: .9
`14.4 ± 1.8
`
`2.9 :t:: .4
`12.7 ± 1.7
`
`20
`
`18
`
`16 -
`
`14
`
`12
`
`10
`
`8
`
`6
`
`-
`4
`
`2
`
`0
`
`I
`
`~ 1:
`I I
`I
`
`I
`
`I
`I
`
`I
`
`1
`
`I
`
`I
`
`I
`
`t
`
`I
`I
`I
`
`I I
`
`I
`I
`
`I
`
`I
`I
`I
`I
`I
`
`I
`
`i
`I
`
`I
`
`1111
`
`NONE
`
`S50)Jg
`
`SIOOJ.Jg
`
`CONCENTRATIONS OF SULFAPYR/0/NE (S) ANO OAPSONE (0)
`FIG 5. Chemotactic activity of human leukocytes with di ffe rent
`concentrations of sulfa.pyridin.e and dap::;one added to the low.er
`compartrnent of the charnber. Clear bars represent the mean o! 8
`assays ( ± J SDJ for randorn mig1·ation of leukocytes .. Durh bars
`represent mean values I ± 1 SDJ for neutroph!l chemotax1s.
`
`ileukocyte) compartment or the Jower (chemoattractant) com-
`partme nt of the cha mber or whether the leukocytes were.
`preincubated with tetracycline prior to assay m the study oi
`Martin et a l. Other investigators 122,24 1 have shown that
`::;uppression of white cell movement is more pronounced when
`the inhibiting substance is added to the leukocyte rather
`than the chemoattractant side of the micropore ti lter. In
`addition, preincubation of leukocytes with drug prior to injec-
`~o~i~nto the chamber rnay augment the effects on leukocyte
`lty 1.24 1. Poss1bly our fai lure to demoostrate a drarnatic
`
`effect on random rnigration may reside in this latter proce-
`dural difference.
`Erythromycin base and clindamycin HCI, Jike tetracycline,
`also appear to inhibit leukocyte motility in vitro . These data
`support the tindings of Plewig and Schöpf [15J in a study of
`the etl'ects of antimicrobial agents on pustular dermatitis JD
`humans, induced by 40% potassium iodide ointment. Topical
`application of tetracycl ine and erythromycin suppressed the
`pustular reaction whereas penicil lin G-Na and diaminodi-
`phenylsulfone had no effect. Oral administration of tetracy-
`cline and erythrornycin also inhibited the pustular reaction to
`topical potassium iodide, but oral penicillin G-Na fail ed to
`suppress the dermatitis. Systemica lly administered diamino-
`diphenylsulfone exhibited the most pronounced a ntiinflarn-
`matory effect in contrast to its inefl'ectiveness when applied
`topically. The concentrations of diaminodiphenylsulfone used
`in our study are h igher than the blood Ievels that would be
`expected from the oral dosages utilized by P lewig and Schöpf
`125-27 1. Although we detected no suppression of leukocyte
`chemotaxis, it is conceivable that suppression m ight be ob-.
`served a t lower concentrations. Alternatively, the effect of
`diaminodiphenylsulfone on the inflammatory response rnaY
`be mediated through mechan isms other tha n chemotaxis 1.27-
`30 I, however, these data are controversial 1.31].
`Plewig and Schöpf postulated that their data m ight be
`explained by a n effect of specific drugs on leukocyte rnigratioD ·
`Our data support this hypothesis and suggest that inhibit!On
`of leukocyte rnovement may, in some circumstances, be an
`important mechanism by which certain antimicrobial agents
`suppress intlammatory skin disease .
`
`REFERENCES
`1. Kligman AM: An overview of acne. J Invest Dermatol 62:268-
`~87,_ 1974
`.
`.
`.
`.
`. I
`2. Shahta AR: Genes1s of free fatty ac1ds. J Invest Derrnato
`J
`62:332-335, 1974
`3. Marples RR: The microOora of the face and acne lesions.
`Invest Derrnatol 62:326- 331, 1974
`4. Kraus SJ : Reduction in skin surface free fatty acids with topical
`tetracycli ne. J Invest Derrnatol 51:431- 434, 1968
`5. Marples RR, Downing DT, Kligrnan AM: Control of free fattj
`ac1ds m human surface hp1ds by Co,ynebacterium acnes ·
`Invest Derrnatol 56:127- 131, 1971
`6. Hassing GS: Inhibition of Corynebucterium acn.e::; by tetrac.Y-
`cli ne. J lnvest Dermatol 56:189- 192, 1971
`7. Mills OH, Marples RR, Kligman AM: Acne vulgaris oral theraPh
`w1th tetracychne and top1cal therapy with vitarnin A. AJC
`Dermatol 106:200-203, 1972
`8. Cun liffe WJ, Coterill JA, Williamson B: The effect of cii ndalli.I'J
`cm m acne: A chn!cal and Iabaratory investigation. BI
`Dermatol 87:37- 41, 1972
`C
`9. Cunliffe WJ, Forster RA, Greenwood ND, Beatherington
`'
`
`Exh. 1019
`
`

`
`Jan.J978
`
`Holland KT H
`William ,
`, . olmes RL, Khan S, Roberts CD, Williams M,
`Iab
`son B. Tetracyclme and acne vulgans: A chmcal and
`10. Fulto~r~~ry mvestigation._ Br Med J 2:332-335, 1973
`Stud of' :ablo G : Top1cal antibacterial therapy for acne:
`86, 1§74 t e family of erythromycins. Arch Dermatal 110:83-
`11. Baer RL L h
`apy . ' es aw SM, Shalita AR: High-dose tetracycline ther-
`12. Resh :;) s~vere acne. Arch Dermatal 112:479-481, 1976
`vulga ' .. tau?htan RB: Topically applied antibiotics in acne
`riu
`n s. Chmcal response and suppression of Corynebacte-
`m~~i
`n open comedones. Arch De1matal 112:182-184,
`1976
`13. Anderson RL
`topi , 1
`, Cook CH, Smith DH: The effect of oral and
`com ca . tetracyclme on acne severity and on surface Iipid
`14. Marti~~Ihon. J Invest Dermatal 66:172- 177, 1976
`oftetra R, Warr GA, Couch RB, Yeager H, Knight V: Effects
`15. Plewig G c1c!~~e on leukot~txis . J Infect Dis 129:110- 11_6, 1974.
`agents-' Ac _opf E: AntJ-mflammatary effects of antimicrobial
`16. Kunts
`· D n m VIVo study. J Invest Dermatal 65:532-536, 1975
`Th on D: Ultrastructura l observations in acne vulgaris:
`De~m~otorml al sebaceous follicle and acne lesions. J. Invest
`17. c
`a
`62:288- 307 1974
`ornely HP· R
`'
`.
`· eversal of chemotaxis in vitro and chemotactJc
`acti .t
`335 ,v;9~~f leukocyte fractions. Proc Soc Exp Bio! Med 122:831-
`18. Baum J M
`mea
`'
`owat AG, Kirk JA: A simplified method for the
`froms~rement of chemotaxis of polymorphonuclear leukocytes
`19. Ward ~man blood. J Lab Clin Med 77:501- 509, 1971
`cy~nh D, Mason AD Jr, Pruitt BA Jr: Suppression of leuko-
`th
`c emotaxis in vitro by chemotherapeutic agents used in
`19; 5 management of thermal injw·ies. Ann Surg 181:363-369,
`
`22.
`23.
`
`55
`20. Keller HV, Bore! JF, Wilkinson PC, Hess MW, Cottier H:
`Reassessment of Boyden's technique for measuring chemo-
`taxis. J Immunol Meth 1:165- 168, 1972
`Keller HV, Hess MW, Cottier H: Physiology of chemotaxis and
`random motility. Semin Hematal 12:47-57, 1975
`Ward PA: The chemosuppression of chemotaxis. J Exp Med
`124:209-226, 1966
`Martin RR, Warr G, Couch R, Knight V: Chemotaxis of human
`leukocytes: Responsiveness ta Mycoplasma pneumon.iae. J Lab
`Clin Med 81:520-529, 1973
`Goetzl EJ, Wasserman SI, Gigli I, Austen KF: Enhancement of
`random migration and chemotactic response of human leuko-
`cytes by ascorbic acid. J Cl in lnvest 53:813- 818, 1974
`25. Lorincz AL, Pearson RW: Sulfapyridine and sulfone type drugs
`in dermatalogy. Arch Dermatal 85:42-56, 1962
`26. Alexander JO'd, Young E, McFadyen T, Fraser NG, Duguid
`WP, Meredith EM: Absorption and excretion of""S dapsone in
`dermatitis herpetiformis. Br J Dermatal 83:620-631, 1970
`Mier PD, V an Den Hurk JMA: Inhibition of lysosomal enzymes
`by dapsone. Br J Dermatal 93:471-472, 1975
`Barranco VP: Inhibition of lysosomal enzymes by dapsone. Arch
`Dermatal 110:563-566, 1974
`Thompson DM, Souhami R: Suppression of the Arthus reaction
`in the guinea pig by dapsone. Proc R Soc Med 68:273, 1975
`Provost TT, Tomasi Jr TB: Evidence for the activation of
`complement via the a lternate pathway in skin disease: II.
`Dermatitis herpetiformis. Cl in Immunol Immunopathai 3:178-
`186, 1974
`Katz SI, Hertz KG, Crawford PS, Gazze LA, Frank MM, Lawley
`PJ: The effect of sulfones on complement deposition in derma-
`titis herpetif01'mis and on complement-mediated guinea pig
`reactions. J lnvest Dermatal 67:688- 690, 1976
`
`EFFECT OF ANTIMICROBIAL AGENTS ON LEUKOCYTE CHEMOTAXIS
`
`21.
`
`24
`.
`
`27.
`
`28.
`
`29.
`
`30.
`
`31.
`
`Exh. 1019