8
TJn; Jou iL'iAL OF DERM.ATOLOOY Copy righL C IU71 by Tb c Wil liallll! & Willdn8 Co. Vol. 56, No.2 Printd in U.S. A. INTRA VAGINAL A:t\'D INTRARECT AL SCREENING OF ANTIMITOTIC DRUGS FOR TOPI CAL EFFECTIVEl\TE S* E. J. Y. \X COIT, M.D. AND Rl CBARD R. BOXDER. M.D. Drugs with knowu antimitotic properties were screened for these properties on mouse vaginal and rectal ruucosn by i ntravaginal and int rarectal testing. IntrnYaginal screen - ing seems preferablr to intrarcctal screening. T he system may h uw \lsefuluess in identifying t.lrug canJidatcs for topical usage in mau. Data on ilre two daunom_,·cin :wd BCNV rh<.'.>c c!wgs may selectircl.'' affect, \":u:iunl mut•o:>al c·<'lls mrd not·rerml muco.:m cells. Effectiveness of urug;: as canrer chemothera- peutic agents has been rorrelntcd generally with the of thcsr dru!!" to interfere 1>ith the mitotic cycle. Recently we hu1·e sbo"·n that the pr openy of mitotic cyclr interference by drugs can be readily confirmed by measuring effects on m.itosi;c: of llle nor m:1l ant.! rt'Cto.l epithe- lia of mire r c•cei,·in!! "'"ch drug,; im r:•rwritonealJy (IP) (1). T he 1\ '0rkability 01 th:lt sug- ge,:;ts that it br exploit able for screening dru11:::- of unknown phnrmnrologic properliies fo r their possible potent.inl as c·auc·er chemothera- peutic drug cmrdiclatr.;;. SyFtemic ndmini:>t rution of crll :trtiYe dru!! , b:ts been used in the trratmellt of sclectrd dermutolog:tc llowc,•er. cPr1nin disad- ''Dl1ta!!:f'-- of :;:y::lcmic of some drugs thcntpy nf nonmnlie:.nant dermatose; exi•t (2, 3). lt seems important thcrt•forr to ;;cMrh for drug<: thnt would hr dleNi,·e on the cell whl.'u applir>J tojlir-alh· ;:ince •mch mi!!ht be useful in toprcal 1 reatment of dernunol ogic B('t':lll"'e initial 1opicaJ dru!! .-•crrenin!! in man j,. grncraU,· or impractical, a toprcnl .syFtem in lab- animal,: is necded . ._kin is unsatis- 10 mcn'>Ur{' mitotic inl1ihition by a drug brcnn, .;e of 1 hi' normally low miwtir rate of cpi- Supporlr•d in l•:ll'L by N .T .H . Prol!.rllm Proje<·l Grunt 1 PO 1 CA 11ii3G and a grnnt from DorJI( ' L:tboratories, Didl!ion of l\filcs ies. Tn". H N·civrd Aul!ul"t 14. 1970; acc·eplcd for rublirn- rion Nl'pl<'mber 15, 1970. Presented nl. th<, Annual of lhP for l nn>slil!a.lh•p Drrm:uoloa:.v. lrll' .. June 20-21, I 97 0. Ilhnoi.;. From the kin and Cancer H ospitnl. Depart- ment of Temp!P Univt>rsity School of :\l cdir·mc. P hi l ndrl)lhia. P a. 19140. dermal cell.,. Celb of and rectal mucosae on 1 he ot lrc·r !rand are charact.crizcd by compar- r·:tpid rPplicntion :mt.l then•fore are ble for me:.rsu r inl! drug inhibi1 ion of the mitollc cyc·Jp :'llon'cn·er, theFe lattPr ti<:<;ucs farilitate tP•tmg of po.;;.:iblr cffectivcnL""" of topically a p- plied materials bccau-e of their permeability. Epidermi,- j,. characteriZed barrier properties whirh mlrilon or ru·c·\'C'Dt prnrtr:llion oi many mntrriab but 1 hr Yagina and rectum are not kilOII"O ro Jra \'(' :mcl1 lmrr icr properties. T he :;tudy rrpor tc·d lwrPin demonstrates thai the mou;:c Yll, !dna nnd l 'C'C'tllm nrc exploitable for c1·aluation of for I'ITl·c·tivcness on the mi- tot ie c·yt"l<· wh<-n topir·ally administer ed intrang- uwll_,. (J\':11rl or int r;trt•c · Utlly 1 l R). )f ITElllAl.. A1"0 ;\fETRnDS Yirginnl f• ·malr• CBA. C5i or ,.;wJso; random fin lo scn :n wcc k:, old Wt'l'f' iouod to be satisfac- lOJ>' for thPde sludiPs. T<·sl drue:st were given n ·ue: or IH n blunL I e:auge needle. The iodudcd blcomycin, colcentid. ,., rosint urnhrno,:ulc l.n·dror·hlonde (XSC63Si8) 5-Jiuoroumcil. ( )."SC32065), sodium r nl'!loot rc·x:ll e. m r rof.!CO mustard (mC'Chlorethamint· ),,·drocloloridc). vinblnslinc sulfnlc. dncrisfine sul- lnpacltOI (X. C l1905). pscudourca dtloritlr· (XSCiiGOii1 ). hydrochlorid" nnd ncxu <XSC-109962). Onu.: ... ITa!! n.od JR were prepared i11 I lir· fullowin)! wnr er I _,·t osine nrnbl- 5-fluorouraril, rnC'lhorrt>xale. nirrogt>n mw:- raro!, \'inlll:lsLillc); prop)·lcm• f.!:)yc:ol (pseurouren) . prop_ ,It•n .. nJroho!:cli;;li!led walrt 1:1:1 (c·okt'rnid. BC!\U. and t>lll\'l rtllt'r : olin• oi l 1:5 t l\lost drui!S lll'l'd in Ur is sturh· were suppliN h_,. Ca.nrcr Clumothcmp_ ,. :Kational .=:>t>rvie• Cl'nlr•r. !\ationnl Cancrr Institute, Bethesda :\lnr>•lnnd.

D INTRARECT AL SCREENING OF - COnnecting …TJn; Jou iL'iAL OF l~tV&S'I'IOATfYE DERM.ATOLOOY Cop y righL C IU7 1 b y Tbc Wil liallll! & Willdn8 Co. Vol. 56 , No.2 Printd in U.S.A

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Page 1: D INTRARECT AL SCREENING OF - COnnecting …TJn; Jou iL'iAL OF l~tV&S'I'IOATfYE DERM.ATOLOOY Cop y righL C IU7 1 b y Tbc Wil liallll! & Willdn8 Co. Vol. 56 , No.2 Printd in U.S.A

TJn; Jou iL'iAL OF l~tV&S'I'IOATfYE DERM.ATOLOOY

CopyrighL C IU71 by Tbc Wil liallll! & Willdn8 Co. Vol. 56, No.2

Printd in U.S.A.

INTRA VAGINAL A:t\'D INTRARECT AL SCREENING OF ANTIMITOTIC DRUGS FOR TOPICAL

EFFECTIVEl\TE S*

E. J . Y.\X COIT, M.D. AND RlCBARD R. BOXDER. M.D.

AB~TRACT

Drugs with knowu antimitotic p roperties were screened for these properties on mouse vaginal and rectal ruucosn by intravaginal and int ra rectal testing. IntrnYaginal screen­ing seems preferablr to intrarcctal screening. T he system may huw \lsefuluess in identifying t.lrug canJidatcs for topical usage in mau.

Data on ilre two dru~r,; daunom_,·cin :wd BCNV oU!!~e~t rh<.'.>c c!wgs may selectircl.'' affect, \":u:iunl mut•o:>al c·<'lls mrd not·rerml muco.:m cells.

Effectiveness of urug;: as canrer chemothera­peutic agents has been rorrelntcd generally with the abilit~· of thcsr dru!!" to interfere 1>ith the mitotic cycle. Recently we hu1·e sbo"·n that the propeny of mitotic cyclr interference by drugs can be readily confirmed by measuring effects on m.itosi;c: of llle norm:1l vo~rinal ant.! rt'Cto.l epithe­lia of mire rc•cei,·in!! "'"ch drug,; im r:•rwritonealJy (IP) (1). The 1\'0rkability 01 th:lt ~·,:trm sug­ge,:;ts that it m:~y br exploit able for screening dru11:::- of unknown phnrmnrologic properliies for thei r possible potent.inl as c·auc·er chemothera­peutic drug cmrdiclatr.;;.

SyFtemic ndmini:>trution of crll c~-rlr :trtiYe dru!!, b:ts been used in the trratmellt of sclectrd dermutolog:tc cli~ca::cs. llowc,•er. cPr1nin disad­''Dl1ta!!:f'-- of :;:y::lcmic ndmini.~r!ltion of some drugs a~ thcntpy nf nonmnlie:.nant dermatose; rna~· exi•t (2, 3). lt seems important thcrt•forr to ;;cMrh for drug<: thnt would hr dleNi,·e on the cell (·~-ell' whl.'u applir>J tojlir-alh· ;:ince •mch drug~ mi!!ht be useful in toprcal 1 reatment of dernunologic di~e.'\~cs. B('t':lll"'e initial 1opicaJ dru!! .-•crrenin!! in man j,. grncraU,· un:~.rcC'ptabfP or impractical, a toprcnl ~rn•1·nin~ .syFtem in lab­orator~· animal,: is necded . ._kin it~elf is unsatis­fartor~- 10 mcn'>Ur{' mitotic inl1ihition by a drug brcnn,.;e of 1 hi' normally low miwtir rate of cpi-

Supporlr•d in l•:ll'L by N .T.H. Prol!.rllm Proje<·l Grunt ~o. 1 PO 1 CA 11ii3G and a grnnt from DorJI(' L:tboratories, Didl!ion of l\filcs T~aborator­ies. Tn".

H N·civrd Aul!ul"t 14. 1970; acc·eplcd for rublirn­rion Nl'pl<'mber 15, 1970.

Presented nl. th<, Thirt,,·-flr~l Annual mPetin~ of lhP • ocier~· for l nn>slil!a.lh•p Drrm:uoloa:.v. lrll' .. June 20-21, I 970. Chicu~o. Ilhnoi.;.

• From the kin and Cancer H ospitnl. Depart­ment of Dermatolo~Y. Temp!P Univt>rsity School of :\lcdir·mc. P hilndrl)lhia. P a. 19140.

dermal cell.,. Celb of Ya~rina.l and rectal mucosae on 1 he ot lrc·r !rand are charact.crizcd by compar­ath·cl~ r·:tpid rPplicntion :mt.l then•fore are suit:~­ble for me:.rsu rinl! drug inhibi1 ion of the mitollc cyc·Jp :'llon'cn·er, theFe lattPr ti<:<;ucs farilitate t P•tmg of po.;;.:iblr cffectivcnL""" of topically a p­plied materials bccau-e of their permeability. Epidermi,- j,. characteriZed b~· barrier properties whirh mlrilon or ru·c·\'C'Dt prnrtr:llion oi many mntrriab but 1 hr Yagina and rectum are not kilOII"O ro Jra \'(' :mcl1 lmrricr properties.

The :;tudy rrportc·d lwrPin demonstrates thai the mou;:c Yll,!dna nnd l'C'C'tllm nrc exploitable for c1·aluation of drtl!!~ for I'ITl·c·tivcness on the mi­tot ie c·yt"l<· wh<-n topir·ally administered intrang­uwll_,. (J\':11rl or int r;trt•c·Utlly 1 l R).

)f ITElllAl.. A1"0 ;\fETRnDS

Yirginnl f• ·malr• CBA. C5i or ,.;wJso; random mit~<' fin lo scn:n wcc k:, old Wt'l'f' iouod to be satisfac­lOJ>' for thPde sludiPs. T<·sl drue:st were given n ·ue: or IH tlarmu~h n blunL I e:auge needle. The tln1~" iodudcd podoph~·lho. blcomycin, colcentid. ,., rosint urnhrno,:ulc l.n·dror·hlonde (XSC63Si 8 ) 5 -Jiuoroumcil. h\drox~'l.Jrca ( )."SC32065), sodium rnl'!loot rc·x:ll e. m r rof.!CO mustard (mC'Chlorethamint· ),,·drocloloridc). vinblnslinc sulfnlc. dncrisfine sul­ft~le. lnpacltOI (X. C l1905). pscudourca dih~·drco­dtloritlr· (XSCiiGOii1 ). d:nmom~·rin hydrochlorid" (X~Cq215 l l. nnd ncxu <XSC-109962).

Onu.: ... nclrnini~lr•rr-d ITa!! n.od JR were prepared i11 I lir· fullowin)! \'Phiclc~: wnr er I c·_,·t osine nrnbl­no~rdr. 5-fluorouraril, rnC'lhorrt>xale. nirrogt>n mw:­raro!, \'inlll:lsLillc); prop)·lcm• f.!:)yc:ol (pseurouren) . prop_,It•n .. l!:ly<•o!:elh~ l nJroho!:cli;;li!led walrt 1: 1 : 1 (c·okt'rnid. podoplt~·!lin. BC!\U. I:~r:wl•ul and dnrri~rinr): t>lll\'l rtllt'r:olin• oil 1 :5 (hlr~o-

t l\lost drui!S lll'l'd in Uris sturh· were suppliN h_,. fh<~ Ca.nrcr Clumothcmp_,. :Kational .=:>t>rvie• Cl'nlr•r. !\ationnl Cancrr I nstitute, Bethesda :\lnr>•lnnd.

1~2

Page 2: D INTRARECT AL SCREENING OF - COnnecting …TJn; Jou iL'iAL OF l~tV&S'I'IOATfYE DERM.ATOLOOY Cop y righL C IU7 1 b y Tbc Wil liallll! & Willdn8 Co. Vol. 56 , No.2 Printd in U.S.A

VAGINAL AND RECTAL SCREEN FOR TOPICAL DRUGS 133

myciu). Podophyllin administered IP was pre­pared in distilled water:propyleue glycol:ethyl alcohol4 .5:4.5:l.

T rsl drugs were instilled IVag or IR at time

zero in a volume of 0.05 ml. This dose was re­prated two flours later a~ wLich ~ime 2 mg of potlophylliu were inicc·tE·d IP in a volume of 0.1 ml. All mice were kill ed by cen ·ical d.i.slocaLion 6

Fu;. I. Ynginal mucosa of untreated mou~e during t.he estrogenic pl1ase of lhe cst.rus eyde. x 17~) .

FIG. 2. Yaginnl muc:osn of untreated mouse during the progestational phase of the estrus cycle . X 179.

Fro. 3. Estrogcrtic mu<·osa 6 hours af~cr IP podophyllin. Bigl1 r!JLe of cell rt>plir·aliou is apparen~ by the m1my C'ells that have been arrested il\ me\nphnse by podophyllin. X 179.

FJO. 4. Progestational mucosa 6 hours after lP podophyllin. Rate of cell rcplieation is lower thuo .in est rogeujc mucosa and is reflected by lower numbers of cells arrested in metaphase. X 179.

Page 3: D INTRARECT AL SCREENING OF - COnnecting …TJn; Jou iL'iAL OF l~tV&S'I'IOATfYE DERM.ATOLOOY Cop y righL C IU7 1 b y Tbc Wil liallll! & Willdn8 Co. Vol. 56 , No.2 Printd in U.S.A

134 T HE J O'GRNAL OF 11\\'ESTlGATIVE DERMATOLOGY

FIG. 5. R ectal mm·osR of untreated mouse. X 179 FlG. 6. H.eeLnl murosll of mouse 6 hours after IP t)Odopliyllin . X 179

hours lnt('r, i.e. eight hours after ulili.al instillation oi t.est druc:. Boil• rectum and vugimt were re­mon•u (·n bloc :wd fixed in !Oro buffl'rl'd formalin. TrnU!'\'l'f~(: scrtion.s were t'Ul at 61-1 and stained with hl:mnw:wltn aDtl eo"tn .

. \ o o('ular 'micromett·r permltt(•tl cotuHwg UJC nu~t~lx·r of mitori(' <·ell,- l'l'r length of mut"osn ns m .. nsured 1uong tht> bn~ul •·ell ln.n•r of the vagina and Lhe musrulnris mur·o.<!\ uf the rectum. cat­lr>ted ficin.. of mucosn 1\'(•n • o£>1cr trd aud the numbPr of mtto-I'S in thrr" sepnnllr- 3 millimeter muro~nl lcnt!lhs was del crnunPd and expr£>.ssed as miros··~ em. The rnne:e of \':tn:thtlity of repealed t·ouor,. 1,~ thi& tl'clmiqur· dtd tJOt r"t't·rd 15% ex­ecpt in tho,c inslnnct:s wlu·re nlf·dtan eount;: werP ,·cry lotc

TltP number of miroric- ccfls prc>cnt ufTI'r sr­qtH:nltnl ndministrution of 1 !JP le::.t dntg. followed b,· podophyllin e:in•s thr d"'sirr·d informal ion on rhr if"'i dntg for the followinl!: r~'ll$011. Podop!,yllin nrrt ~t- cf'll.• in mrtnphlll-f· ~~~ dtcy pn$s through mitu~l~. If the· tr;:t rlruu: U:t\'1'11 bl'forc mtrttperito­nl'nl podoph,dltn inlullll· 1lu uttf'rmilolit· phn~e of the· ,., .11 1'\I']P (G,. S. or (; l. fc·w 1'1'11~ will rf'ach rnitu~i~ tr• hP arrr.:::tpcf in mC'tapktJ.P h~· podophylltn and l~t•nl'f I hr numhPr of milotit· l'f·ll• r·ounterl will '" -;lunll. If. on tl1r otht'r l,aml. the tP.-<1 drug is a mt:tuplut-r J,Jot'l.;inc: !ll!rnl ilsr>lf or hn- no efi!'«'f on tlrf' t·t II r\·•·1< lhr· uumlwr of 1ni toti1· N·ll~ ronntf'd will b, !!l:l'ttlt r thrill or Nillltl to thl· numhrr founrl whrn J•IHJoph\'Jiit, j.;, gi, Pll ulom . in 11 hir·h r·a.,r· tit• TC>;;t •lrue: "' llot·n rc!1dntini~IPn·t.l wilhom podo­nhdlitl In r·oofinn 011t or rJ,p n1hcr of thN•P t•o,~i­J,ihli•·~

\II ,·tu.nuol "prc·imens \\'frP •·ln.,,tfic·d a~ heine: in ri1lu r thr· {'"'lro!!"ni<' or proe:c•lational phnsP of the ntnt~ t',Yr)p on 1 he· b:t"'i"' of hisloloj!;ic rhnrnt'lrris­rir·~. Thr l'll.l!innl muros11 of nntrrnlf'd mire dur­in!! 1 hr> phn"P of 1 he ,~tm• f'\ rlr primnril~· du(' to estrog(·n iF r hnrarlcrized In- sl rr11ifirn1ion, krmtini-

zution. and a thick strH.lum t'orucuru (Fig. 1). The \'ltginul mucosa oi an untreaLcd mouse- durin~ Ute pha,e of the estrus cycle in \1 hi ell progesterone ef­ft•ct,. rrrdoruinate i~ muelt tlnnnl'r ttnd i;: muCU!> Qerrr·t to~ rntht::r than kc·ratiuizc·d fFic:. 2) . F ic:urc ;3 ,:hows nn estrns:wrur t>pttlu•lturu six- hour~ :tflet intrnpr>rilon<•lll pudophylltn hns hr·en giyeu. lt i~ dl~rt·mihlf' LhRt. podophYl lin !IITf'~ls r·ells in mctn­phnSf' by tliC! ioc1. lhal rnrtnpbnr;:p li~ur"~ pr~>domt­

nnlc :tnd few if any nnnph!tso• or telophn•c fi~un·­nn· found .. \ m· rnNaphRF• llrre>st inj:: rlrug "til !lh'c• thl' ~nnw rc·sul1. Ji'ig~.m· 4 show~ n T•t·oc:<:slnliunnl r ptlhPlium !<ix l•our< nfrrr intrapr•ttll>ncal porl,)­phdliu Thr numhPr of miH•,... 1• ow:-thinl to on~·-hnlf tll!ll of tlw r.;;trop:rnir Ppithr·lium Uf'~nu~\ proc:r•st:tlton~d epithr·limn rrplir·ntt'" oul\' •ln.--tlmd to Oil\ -half n,; rtt pidly s;:- c>f rogcnic C'pirhr•lium ( 4)

S1 \'en l!roups of trn mir·c each wPrr utilizrd Tfl U{'tr·rmiiH' the medtau rnnJ!r of miloric rou11l~ in ,·np:innl nnrl rertul mucosn.· followinc: inlrnpr·ri io­nP:d podophyUin. The m<'Ciinn counts in estro,rrmi<' l':u!tnlll murosn followiu~ podophyllin :dotH rnnl!:l'd from 130 to 245 mito•P~ 1rm with a mPLlhn <II

lhr•l" mf'dinu~ of 190 nuw~t, rm. ThP ;,nn lo!.!nu­nwdt~tn t'O\tnl~ in pro!!c•«tntiounl mu~n"'l mu!!Pd irom 32 10 120 mi10sr~/rm "ith n mrollnn of tl11 mr>d1an' hf'in~ R2 mitCl~r~ 'rm. The mrrlian mt1ot11· N>tmls m rl'ctnl murosn mnp:C>d irom 27:! w lSi witl1 a mr•linn of lhe nlNli:1n• of 375. Tlw<, ,·nlur­wc·r•· ll~f'tl lnTf'r to rlf'Terminc· whc•t lw1 n 1r·<t rlm~ Qie:uif1,.an l h· :lfiN·(pd I],,. mitolir l'\'1'11'.

P11s<il olr ·, rao:<umrnsal nJ,i>orpl ion of tlru~~ gh•rt• 1\':tl! ror JR \\'ll~ mnoilnr('(] h~· milolir rouut~ ir Li11· ttlll rn'dr oru::rn to nsr•rrlnin \I luther ,.n·c·<·l~ nt mitn,.j, \\'l' rr drtcrtnl·l••. If rfT<•<·r;, on milo~i;. \I'PI•

prr·-r·ut it wa!'> n.•stunr-rl tlor'"" n·<nltcd from ~-~ trmit· nh~nrpliou of fl11· rlrtllot (It nntwo~n.lh· frm the• primnr;t• t!'st orj!nn, For r>xnmpl!', if a mrl:t ph:l" arrr·•tiu!! dntl! ndmtniSI('J'('rJ n ·a!! i< nhRnriJ(•,' •ufli<•i,.ntl.'· to prndtt<'!' ::1 ~,\'~IPmi<· pfff'f'l. tli t> oum

Page 4: D INTRARECT AL SCREENING OF - COnnecting …TJn; Jou iL'iAL OF l~tV&S'I'IOATfYE DERM.ATOLOOY Cop y righL C IU7 1 b y Tbc Wil liallll! & Willdn8 Co. Vol. 56 , No.2 Printd in U.S.A

YAGlNAL Ml> RECTAL SCREE>'\' J·'OR TOPJCAL URl Gf'. ] ::1.5

her of rutlotic <·ells eounlf'd m ilie reclum is ele­' nwd; if tltc drug f:inn !Vag inhibits tloc inter­miLO lie phn.sc of Lhe c·ell cvcle and is :.tbsorbcd sufficiently t.o produce a syet.emi<· f'ff<·cl, the nunl­ber of nuloLir cdls counted w Uoe rccLum is di­minislwd .• \ <·omparablf' 51"1 or c·ir(•umsrances is Dl•11l ic:.~lole fo r IR 1\dU\lUl,t<•rt•d drugs and their d1c•rts on ,·nginal muc•osnl n·lls. Cri1ena for grud­iug tloe SJslemir effect~ of such c.lru~ were :lrui­lra rily eslt~bl U.l11~d as indiettted iu T11ble 1.

RE~L'L'l'S

Effects on mitosis of di'U(JS administered /Vag (Table If). Podophyllin, Yinblus\ine, vincristine, ~d rolccmid were tdrntificd lis agents blocking the rcll eyrie in mito~i.~ w!Jen topically f.ldminis­rerPd lYa!!;. Their meraphnse nrresLiug property was rorrclnted ~·ith l1ic:h number,; of mctnpha.-;E> figure-'> in tht' Yatrinal rpith<·linm. The steps by whtch these drug~ might be 1dent\fi<'d if ;;crt'<'n£•u u;; unknowns are illustrated by the example of ,;nbla4ine. Tlte drug w M ti r:;t admini;;tered and followl'Cl two hour:; Inter with podopbyUm JP. The use of both dn1gs yieldrd a rugh ,·arrinnl mitotic count, reprc~enting the combined mi\osi:< arrcFtiiHr effe-ct:. of both Yinbla~>Une and podo­phyllin. The rrutosic; arresting property of Yin­bh;tiot' itFrlf \YU~ then a::<~rrlllincd bY admini"­t crimr it IYtlg without !'lllFE>quent Jl>. podoph):l­lin: in t hiti in"-lanee a high mit out• coum wns ll!!ain obtained. The ~nunt wa" somewhat lower than tlte f'OUnl obtuin<'d whc·n b.,th drug:; wt'rl' tN-d huL was \'a:;ll~· 11i!!:ltc·r than in :m unt re:tted animal.

Podophyllm, YiJtbl:..;.tint' :md colcemid gjYcn n ·ag in the· do•e:- indicatPd 311f'ear to ha,·e· been llb~orbcd trarHllucos:tlly to the r'\tent that e'i~ deuce of somt• ::y::tpm•r rffect wa:: detecwhle bv elentf'd mitotic· <'ountc; in tht· rrrrnl mu('os~. The Plf'v:.tions of rN'1nl roi1ntic count;: a<'so­CJ:tted wit.h podoph~llin nnd Ymbb;line IYag wrre srnnll, hut wrrr la rgl' with TYnrr colcemid. ~o i'uc·h rectal <·,·idrnf'E> of ,;,·st<'mir ~ff(>('t from 1 ran~<,·;. ginn! ahsorpt icm "a:; dc•tect<·d wnb J\' ag vi.nc·ri line in the c·oncentrn1ion used.

'Xitroc;cn mustnru IYag effectively blocked nlginnl mitoses, ns indiC!Ilecl hy n mi1mir count similnr to that of an untreated animal. The low rount obi fl incd indicut r~ fe"' cells entered meta­ph"~c to be arrested rhcrf' by pndophyllin. No <>vidl'ncc of systemic died from transYag:im\l ab­sorpl ion was detected by the mitotic count of recwl muco'>:t. SimiJarly, p~cudourca and 5-fluo­rouracil IVng were found to inhibit the intermi­totic phase of vaginal mucoeal cells without d&-

TABLE I Crilrriu j11r .•y.~f• llti•· uL~<orpltnn l(f •lr11g.~

'Rctt•l mitu-..~·cm

---<75

75-125 l:!r; 200 :wr aoo >300

Mitosts/cm in c~tn:genic

muc:csa

<2{1 21- .j(J 51 100

101 130 > 130

1\ ag ~dministertd lcsl drug.

( , rading of systemic Grading cf >ntemic tfftl t (~om m~t.nphasc cil~tlrom in\t!1'1'0l\otic

•rrcstmg \t:Sl drug inhibiting lt:5l gh tn "tlhuut rl!llf; iollowcd

ro<fupbyllin by P<>dopbyUin

~11!1(' 3-;-± 'l .J..

1+ 1+ H :± 3+ 1\; <lire

j Gradwg of sy«emic Gr~ding of synemir cllt-cl from mcl4ph.ar.e clJ«tlnJm in\ermit.o\ic:

orrosung ~e<t drug it>bibitin~ test dru; !:"·en ~ ttb~ut toUo" ed by

podOJJhylhn pudopby Uin

.:\ llltP 3..!.. ± 2+

1+ 1+ 2-!- ± 3+ X <J!IG

rc·c·r:thlr. e,·idcuc·c (1( "~·-tNUir f•ft(:('·- nn rectal llllf('(J~fl.

EJirc·ti1·rnr,;• of r.\'tw,ine :lrabinosiJe. h\'droxY­un•:i :11Hl met hot rex:ti I' iu blorkin~ the int.er~i­to1!t' pha . ..;c· (Jf 1lw cf:'ll c~·rlr whrn :tdminic;tered IYac; i- idcnltfic·d h~· low mi1nnr CC>Illlf- in ngi­n·d rpir helmm. ] r i- nnt rlcfll bowr·1·~r whether r hi ... utl1tlli1 ion i- due to the•e r!rU!!" act.intr lo­f't\11~· or ~~·-temt<'ullr -ffrmd3r_,. rn tran"'Ya~inal ah-orpuou ;;inrl' "0111(' inhibtrton, ~bru!!h small rm rPrtal mil<l"<':'o i- dl.'ti.'Ct:\hlc. I\-'il! 1~-ti:ng of Juwer ronrrmmtion- of thr-<' rtru~r-< "·nu!d ascer~ l:~in 1dwthr r mhillltJon of 1'1!ttn:Jl mito~i" is nt· t·riu:~lok 'lnthnut t·\·idmrc· ot' ")""TrmiP effet't.

L:qJ:trhol 1 Ya~ w~t" nor Jot met ro hnYe anv dt•lf•t•tahlt> <'ITN'1" on 1bl' cell rHh• -

Effect.• (ln mito.~i.< of riru{l:• 1Jrimini11tercd I R ( Tal,lc Ill). \Itt h ~orne drll!!' re.;uft ~ on rccral nmroo:a J rum T R t <':'I in~r were .;;im il~ r to tllose ol1t~1 im•d nn Hte:in:il muro;;:~t r ront n·:~!! testing. \\iih other drii,I!'S, howe,·cr. TR te~t in!! did not dete<'t am imito1ic propl'rtj~ de,~ctNI bv the f\':t ~ 1 e.:: f. -

Podoph\'llin IR did not :me~t rectal muco~a <'ell~ in mrttt phase 1 o an\' si!!nifirnnr rlrgrrc, e1·en in hi!!h courentra.tion. This result ~~ in contrast to lhr drug's clear effecth·ene35 in arrestine: mi-

Page 5: D INTRARECT AL SCREENING OF - COnnecting …TJn; Jou iL'iAL OF l~tV&S'I'IOATfYE DERM.ATOLOOY Cop y righL C IU7 1 b y Tbc Wil liallll! & Willdn8 Co. Vol. 56 , No.2 Printd in U.S.A

136 THE JOURN.<UJ OF Th'VESTIGATIVJ!J DlffiMATOLOGY

TABLE Il Effect., Ml milo.~i.s of tlrug11 adm1:rn'stcrcJ i n lrlli>OOinally

Drus lonc:cntrn.tion X umber of nnimols of drug

N one (t·unt,rnl ) E• p~

Podophyllin int,rupcr itonen.lly • E (coni rol) p

Podophyllin ?f· E - ( p

Yinblz<siinP + P<'dophyllin 0. 5"; E p

Vin bln.sJinP 0.51 ( E p

I

\ ' incri;;tiue 0 .03!3!(. I E p

Colecmid Jrf E p

Kitrogcn mmnanl + podophyl-O.QJ I(

E lin p

Cytosinf' n.rnhiunside + pocln-

r

E phyllin

J'( I'

llydr<Jxyuren + podophyllin f!l t E

I p

tllf't hot rc~xatc + podoJlhyllin 1'; E p

5-tluorouracil + pudophyllin 5r-;. E p

Pseuuuurea -L podo phyllin (} . . 'J' ( E

I p

Lapuchol + podophyllin 2CO E p

Lapachnl ?<7 E - , c. p

t E = Estrogenic mucn~:~n

2 P = P rogestational mucosa a .1\s monitored by effects on rectal mucosa mitoses • J.P. Dose; 2 mg

(j

9

51 ]!)

1

!I 1

!)

1

!)

1

6 I

:~

ti

H 5

I~

-A

"j

3

8 2

7 3

5 2

.J (i

Mit uses/ em

R!Ulge I .MediiUl

2- 12 7 lh'i 1

117--10!-1

I HlO

25- 14-1 82

58-~2 ' 100 72 72

;{ 75-1 282 lG HiS

2-370 HJCi 3 3

5-3 l ] (),

12 12

5D-3fil 2U7 HO llO

I ll - 21i 20 0- 11 2

J:i-152 l 5.)

2-1 :~" I 5;~

0-120 12 2--18

I 7

2!l-Hi:3 ()

5-Jl 2i

1&- !JO G2 J;-1- 2 2l

2G- 140 fi2 22-li~ a.J

iu- l G!I l 14U 21-28 2:)

JQ-15 13 2-H 5

Systemic absorption•

-

H

-

J+

!\otlP

:H

Non!'

I H

I

I ±

J+

N one

'!\one

Nune

None

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VAGINAL AND RECTAL SCREEN FOR TOPJC.~ DRUGS 137

T.ABJ,E I II Effecls on milosi.~ of clrugs administered intrareclal/y

11-ito~:cm

Drux I Conccntr.ltJon I X umber of ~)·ste~ic of drul( animals I ab5l>rpUonl

l ~ Range Median

None (control) n }!)-~0 311

I ! P odophyllin intruperitcmenll.r (<-oo!rol) 70 l!12--fo3i 3<5

Pt>doph_dlirl 0. I r·t I 10 I;H!l -40 l'·:onP l

Pudoph~·lJiu 2 . 0~( I 10 14- iSl 80 ±

ViJ1bl11stine + podophyllin 0.1"; 10 200- 12 Mi~

Vinblas 1 inc 0.5(1 I() ! 5:!2-9 4 75J ± I

Nitr()geu must a rd + podophy llin (l. 01 ('~ 6 I fo-{iO 24 :\nne

5-f:luoro llrFl{'il + pudophy llirt ~ . (11'1 10 I 2- J II I

10 !).""n<'

Iiyuroxyuren + podophyllin 5.0'( !1 I 2-48 22 :?+

Cytosine urahinos ide 1 podophy llin 1 .0'( 10 144-527 ~ 1 )i' (J!I(!

1\leLbotre:-.atc + podophyllin O. l r(. 11 JOI\-140 2&i ':1::

' Af: monitored by PffN:tl' 011 v11giunl rouco!':n n1i(vses ~ J.P. dm•e; Z mg

toses in \·aginal mucosa when given IVag, and :< simibr effecth ·eness 011 both epithel.i11 when ginm IP. On the other hand, ,·inbJast ine was rlr:uly tdentilied as a mitosis a rresting agent wl1en gi,·en JR, jn keeping with other data on this drug.

X itrogen mustard and 5-fluorouri\cil lR eD'Pc­tively bloc:ked the inteimitotit· phases of rectal mucosal cells as indicat ed by low mitot ic counts similtlr to that of an tmtrea\r.J a.nir.n.il. Xo e\•i­J em·c of systcmir effect from transret'raJ absoi'P­tion was ri£>1C'cted in ,·aginal conuts .

n"hile hydrosyurctt IR was ~ssocilltcd wit h a low recta l count, this result. may J1aYe been due tn .~ systemic effert of Lhe Jrug from transrectaJ absorption since :;ystcmir effects were detect ed by lo1Y mitotir coturia in vaginal mucosa-tl cir­cumstance similar to t.hat t'J1countered with this drug in the !Vag test.

Cy1 osine arabinosicle and mrLhotrexate were not shown to have any significant antimitotic effects when given IR in the doses used. This was in contrast to their antimitotic cff~.>cts dem­onstrated when given IVag, o.nd IP (1).

E.fJects o 11 mitosis of BCXL" und daw.umyciJl ( TalJle n ·) The ntiwuc effects o( theoe t \YO

dnw:s are prr-sc:ni ed separately sincl' \he data ~ugge::t the..-oe drugs selectiYely affect vagmal mu­co:o:t riDJ 110i n·clal mucosa when gi1·en JP s.s well •tS wlren e-in•n lH . ln t fw bttt>r !ClOt proce­durr it may be assumed tlun rc<•tnl mueo:;:a was ndequately exposed to the dmga since only by tnmsrecral absorption can the systenue effects that were identitird in \'Dgmal muco.:::1 be ae­cr>Ullt~d for. The dnti• do not permit dctPmtiu­ing whether or not trau:-·vaginnl ::tbsorptwn oc~ Ct:LrrCU when Lhe drugs were gi\·en I\'a!! since s;y~1 rmi<: c.ffects '''ere monitored only by effects on rect al muco>::t. Insofar a:; rectal muc0£:1 of the morts<• "<·CI11!:> unrr•;:ponsh·c 1o these drugs by any route. t h i.s single mc~'ltl., of monit oriU!!' t ra.ns,·ag­io:J l absorption i- nn1 Yalid in 1bi" panicular circttmsta.n<·e.

The drugs selected from tbis study mcluded rhemical agents from t he categories of spindle poisons (metaphase nrn•stors or stathmokinetic

Page 7: D INTRARECT AL SCREENING OF - COnnecting …TJn; Jou iL'iAL OF l~tV&S'I'IOATfYE DERM.ATOLOOY Cop y righL C IU7 1 b y Tbc Wil liallll! & Willdn8 Co. Vol. 56 , No.2 Printd in U.S.A

T.\Ul~g 1\'

EjJrl'l~> ll/1 mi/11.•i~ of /1('1\' l ' 11111/ "'"'""llllf•·in arlmini.~ll'rt•d !I', ll'oo ond !Hfollowt•d l1y podopllyllin TP ----

l n1tapcritont.1l route

~ l ite t5/Lm

Prus Number - --Total or \ agino do•e :.mima i~ - -

Ccme. of Redum drug

Range Medinn Range 1 Mediun I -----

·~1 :! 27- 130 50 BC:\U O.li mg p s i 1 15 4 74 5llOI 2lili I 0 . 1%

E 7 1- 92 l Daunomycin 0.2 mg p 3 7-12 11

l!J0-6101 -100 I 0.5''

1 E = EslrogE'nic mucosn 1 P ~ Progeslntiouul mucosn 1 As monitored by eiTectB un rett nl mucosu. ~,.,.. t t'xt rnr P.xrcpl i•11t • As monitored by efTPcts ou Vl\~tinal ltiLH'O:Hl

I

I

lutrnvnginn I rou le Tntrarecto.l route

T J\1 itote•/ • m Mitoses/em

Numbr1 of

ttnimaiJ\ Ran~c !Median

Sy•wnk I\ umber --f'unr. of

•b~orp drug or l ion' 3nimals Range :11edinn

--- --- -- ---- --- ---1·: (J Hi-1 10 45 p 'l ~ i 54 51

Nnu<' O. lt, lU ~~ 77:1 3:iO

I ~ /j i -Hi 2~ p 5 0 HI I X

:\uue 0.5', liJ 257 -501 371

-

Syotemic ahsorn-

tion'

----2+

I+

,_.

""

~

~ ~ ~

~ ~

~ I ~ en ...,

Page 8: D INTRARECT AL SCREENING OF - COnnecting …TJn; Jou iL'iAL OF l~tV&S'I'IOATfYE DERM.ATOLOOY Cop y righL C IU7 1 b y Tbc Wil liallll! & Willdn8 Co. Vol. 56 , No.2 Printd in U.S.A

\'t\Gl~ \L AX"D RECT.\L !o.CIU:EN FOB 1'0PJCAL DRL'GS 139

agents), alkyhning ngenls .tnd antimebholite:. -drug3 who.,e modes of act ion are generally un­derstood quite well and all having tile property of interrupting the pa3;;agr oi rcll;; throutrb one or another pba.--e of the tell r<'"plicat ion e~·clc (5-11) . The mode of action of one dnw:, lll­pachol, has not yet hern a~eerrained ( 121.

Mouse vaginal 1111d rectal epithelia provide systems by which to screen tlw nntimir otic prop­erties of drugs admmisterc·r! JJ) (I). The da1.a of tl1e present stud~' indica1 C' that .irltrav11gin:ll sorcr.ning can establish whNher dr11~;: are locally effective as ant.imit.otic:; \Yhen applied topically to this organ. Information received from the in­tra\'ncinal screen cw.n be c>.Jiccted to predict po­tcnt.inl antimitotic effectiveness of dru!!. rundi­dtlt~ on 1 be skin of man. Howf>\·er. I he lower pcrme.'lbiliry of c•ptclrrtnL" :1::: compared to ,.al!i­n:'LI rnurosu dol'~ not a !low immedwJ P or dirert tran~lauon of the :uwnal darn into ib :lpplicabil­ity in regard to human ~km, !tt Jea<=t wil110ui some furthrr rcfin<>rnenl- fn r mterprerma: tlw data prOYJded by the ~trr·en. Further \\·ork is needed to develop tlH-.e refinem~ms inc·luding Eome Ililot e\·u.lu~ttion'> in dw ~km nf man.

The result obtUJUPd from LR :Himini"tration of drul!s that were at ,·ariance with resul ts ob­tained f rom JP and 1\'ag admini::rnllion may be due 1o low J1Cnetrnbilitr of the rlrug;; imo rPctal mucoil:t. On the other hnnd, and in m:~ny ways more likely, expo.:ure time- of rcrr.:d mucosa to the drugs may l1c short bC!<·uu•e of the drua:.; bPm~ e>.'J)elled by the procPA.- of drfcration. Ad~ di\iona.lly, the luminnJ volumP of the rectum is nol a:: sharply dt'lincatro as that oi the Yagina, and dr~s instilled intrarecta.Uy II1lly be distrib­uted retrograde into the colon as well as defe­cated making dosage condition- highly \'ariable.

The s<>eminl!' "clrctivn~· of :tctiou of BCS11 and daunomycin on vnf.!innl epithelium ns op­posed to rPctal epithrlium is nor readilr explain­ablt>. These dru,!!s are dis.~1m1lar to the f'xtent that one, BC~U. is an alkylaung trgent (J J) whilr the other i~ c·n.tegorized ~au nntimetnbo­lite affc.cl.in~; the cell rycle predominant!~· in the S phase (7). This categorization howeYcr ca.rrjes with it no implication of physicochemical prop-

e:rt iel> uf the drul!~ wh1ch ma~ dPtermioe 1 he1r peuctruhihty Hllo difl'crrm cc·ll l,vpe:;. On(' tentn­li,•c cxpi:J.uaricm of their -,elf'<'li\'HY of action as ob.~f'f\'t'd 111 this ,..:t udy, would be that rcrt:tl mu­ro,;a JWl'IDit:S ptu:Sage of tht~ drug" intPrcellul:trly hut that \he mucoS!ll cell membranes ll rr nol perrnP.aiJlc 10 the drugs. Alternate hypothese, muy be constructed, hut would be equally con­jertural. Additional studies hre neceE>.~ary to pro­nc.le f11rlhrr in iortrl!ltion on Lhc absorpti,-e and harrir r prnprrtics of rectal mucosa.

TlJe t(;chuical assislaot·o of }Vl r. Bernol Leitch is gruiPfully llcknowlccl!!(;d

REFI.i:HE!\CEci nondf• r·. R . II. ancl Yan .. ·r·orL. E .• J. : Epilhrli<tl

SI'Tl'NUU~ •. \'Sll'ID!' ior ntilOLJC c ffecf.S of sys-1<•1111t:tlh· admiotS!r>red uru~. Pro<' .. \mer. A~~cw. Cuu~·(;r Hes .. JJ: 10. 1970.

2 l\ lullt·r. ;", A.: }',oria.Qt". methoLrexate, aud f'lrrho.•a~ .• \ r<·h. Durm.,JOJ: 379. 1970.

3. ,-uorhC'f"-. J. J .: Is it sale to trent p;;ona.sr'­with rnethotrr·x:l.lt:'! .\rc:la. Derm .. 101: 3~. 1!)70.

.J . Tlar!t!'lwr . .J. D .. Clark. F l. rtnd Clark£. D. H.: Cluml!c~ in tiH: 'al!in:tl C'pi UJclial tell c.' clc HI n·l11 tion I o n·pn I of rlw c·~rrous c,·clr·. Exp. C<·ll Rr,. .. . ~5: 2:l2. 1967. ·

5 Frf'>l H I . K. Wlonng, J., Scogrrin.-.;;, H 13 . \'an .. coli. .K .L. Hall. D. P. and BC'n. l\1 .: The slutlllllfJkln<·! it· effect of ,·int·ristinc. Cancer Rr!<., 24: Hll . IH<H.

6. Johnson. l. ;:; .. • \'Obocll.l. G. H . and Wrig:J,t, R F : Experimental ha is for clinical e\'alua­tion of lwo new alkaloids irom Vinca rosea linn. Proc .• '\mer .• \.s-oc. Cancer Res., S: 331, 1962.

7. RuSl'Oni. .\ . n.nd DiMarco . .\ : luiJjbition of nurlc1r nrid s.,·n thesis b~- d1lunomycin and Its rel:ll ionship to the uptake oi tbc drug in ltelt~ c~lls Can<'C'T Re~; .. 29: 1507. 1969.

. J\rishun .. \ .: Ttmc-lnp..,c· and ultrastrUcture studit·~ on tlw reversal of rn.itotit· arrest in­duc~d b,· nnblsstine suHate in Earle's L t·ell!'. J. :\' n 1. Cancer Iu.::t .. !,I: 581. 1968.

9. Yarbro .. J. " ·.: Furtiwr !'tudies on the m<•cha­uism of :t('tion o£ h,·droX\1Jr£>a. Canrf'r Rc~ .. g : 10, 2 196". • .

10. l\l rDonuld. C. J .und B Prllno . . T. R.: Parenteral Inethotrcx:tti' in l®ria.~is. Arch. Derm .. 100: 655. 1969.

11. B eirlelb<'rll'er. C.: The need for additional alkylatiuv; agent!' and antimetabol)1es. Can­cer Rt:>s .. &fJ: 2435. 1969.

12. Rao. K V .. M cBride, T . .1. lllld Oleson. J . J .: Recognition Rnd evllhtation or lapachol as nn 1111 1itumor flgt' nt. Cnnccr Res., 28: 1952, 100 .