5UN-29-1998 10: 56 FROM MACROCHEI.~ CORPORAT [ OH
`
`TO I/ALTERS, K.
`
`P. 07 .": :2
`
`1997 L Pharm, Pha..-mac~l.
`
`In-vitro Permeability of the Human Nail and of a Keratin
`Membrane from Bovine Hooves: Influence of the Partition
`Coefficient Octanol/Water and the Water Solubility of Drugs on
`their Permeability and Maximum Flux
`
`~RK MEgTIN AND ItER.NHARD C. LIFFOI,.I~
`
`Department of Pharmaceat;cai Technology, Helnrich-Heine-Uni~ersity, Universit£tssrr. I,
`D.40225 Diisseldt~rf, Germany
`
`Abstract
`
`PerLetration of homolt~goug rdcednic acid esters through the hum~ halt and a keratin mcmb,~¢ from bovine
`hooves was investigated by modified Frartz diffusion cells in.vitxo to study ~(cid:128) transport mechanism,
`The partition co:fficien~ octanol/wat~r PCc~/w of the esters was ever the r~,-~ge 7 ~o > 51 000. The
`I~’tn~bility coefficient P of rite (cid:128):Lit plate ~s well as the hoof rasmbranc did not increase with in,easing
`~[fl~ibn eo~fieidmt Ot lipophilicity of ~ pen.e.trafing.guhstance..’I’Ms £n~eams tkat tm~.b~a’iers behave ILkc
`h~idrophilie gel It’~mb.rta.tm ratl~, r *.ham llpophih¢ ]:~tlon rltemomes ~ ram, cpe oi.rdae su’aulm ct2meum.
`penetration s~di¢s wi~ me moael ¢ompotmag paraeetamot ano pnenacenn snoweu mat me maximum nag was
`tint..a timer]on of the Crag solubility m water or in the ~w011en kerati,~ maRSx. Diss~tafi?e hindered the
`diff-a~ion of bertzok: acid and pyridine taro~h the hgOf me rabrane. Since keralSn, a protein with ar isoelcetric
`point of about $, is also eha.rged~ d’fis redueuon can be attri~,uted to an exeausicn or the diss.~iating sur~suancE
`due to the Donnan ¢quitibrlttm. Nevertheless, tlt* sirtlultan~ous erthancEmea~t of the water solubility makes a
`distinct increase of the maximum flux po~fi’e!e.
`In eeder to sersma dongs for potential to1~ic~d al:~lisa~on to ~.aail plat~, a.t~atior, has to be rdd mainly to
`the vra~er t~l~!L~ of the compound- The bo~oe hoof menal~tafie raay iE~e aS an app~pfiSt¢ model for the
`nail,
`
`However, the maximum amount of ~a~g, ~aehin8 the side
`~gat di~a.~s of" ~= null plate a~ount for up to 30-45% of
`al.l..ii~pi..~,"
`of action per lime firmer appl]catbn to an a.,x~ A, is mm,~
`m~ses ~ the prevalence among the population
`b..ter~dag than the pm-m,.~bility. ~ib in the ease of a lilxr.
`~(~al nations is about 5% ~was I990), Serious
`phitle pardfiota membrane the nmximum flux depends on the
`inf~rbm are treated with ~i~ternie ant~mycetics owr several
`drag ~olubiliry in the vehicle a.,’,d the pac:ifsn coefficient
`months, which exposes the organism to a considerable anaoum
`(’Hagedorn.Leweke & Lippold 1995), Ir Irmre~ses with grow.
`of drag, $itto* the introduction of nail laequen, mpiea! treat.
`it~g water solubiliD’ L’~ t,h¢ ease of hydrophilic gel raembna~e~.
`meet hu ~ome nao~ and ram-¢ successful for light and
`The dependency of the maximum flax through the nail plate
`int.~’m~a~ mycoses (Qadripttr et al I9~3; Polak & Zaug
`and the h~of membrane was investigat~ wi~ the model
`19.~:;,~.~. I992; Noking & *cebacher 1993; Herin &
`s~Gn 1095).
`eom1~oun~ psa’aee~tmol ted phenaeetln who~e solubilities ~n
`water differ from r-a~h other by a factor of 17 (C,w~- 1~ 800
`Until now ther~ has been no commcmly accepted in-vitro
`and 950 m$ L-I, Pff.o=iw ----- 5.7 and 70.L respectively).
`model for testing new, tople~dly applicabl~ drags, B¢~aus~
`The solubility of a¢idd¢ or basic s~bsta~ces in water can be
`human nails are not available ia a sufficient number, a model
`bksed an membranes from the bovine hoof was deve~pe, d,’:~he
`increased greatly by dissociation. Eabaacemeu; of the max.
`Strtt~Rtl~ and l~’meabilky cf which e..quatss with those of the
`imam fl~ through the nail plate is possible o_aly wh¢~ the
`d=crea~¢ of the p~rmsabili .ty eo¢fft¢’:er~ due to dissociation is
`h~’xiiGl. Up to now the m~hanism of nail penetration is
`nDt me gramS. Ions cannot pence’ate h~id membranes due to
`largely tmcertain. From i~vestigation~ of the penetrat~o~ of
`low partitioning into the mcrnbran¢. However, they a.m ~ble to
`homologous dvohol~ ~gh the naiI plate, Waiters et a!
`overcome hydrophili~ gel membranes (Zaikov et al 1988). In
`(1983, 1955b) concluded that it is a hydrophtlie gel membrane.
`¯ i~ reelect tl’,¢ peroration of lhe model compounds benzoic
`¯ TIwy ~osmlatcd an additiorsal llpophilic route for the diffusion
`acid (pK-t 4-19) and pyrid~ne (pK, 5.19) through the b~f
`of lipophiEe gubst~ee~. Therefore, the present ~cr~ examtn~
`membrane was investigated at pH 2.0 and 7-4, In these mili¢~
`the dct~mdcncy of the pcrm¢¢bili~ of both lh¢ n~.il plate and
`they ~re ne~ly ~’¢mplet¢ly dissociated or undi~sooiated, The
`the hoof rn~mbcanc on the paxution coefficient between 1-
`nevtraI bcnzyl aleot~ol served lu~ a ~tandard i~ order to eoucider
`octanoI tad water (l:’Coc,/w). Homologous nicotin(cid:128)c acid ~sters
`the l~ssible influence of the pH value on the kgratln sw¢lling,
`were ascd as model compound~ with PCo, v’w vah:es over the
`range 7 to > 51000 (Table t),
`
`Contspoad~tme: 18. C. Lil~p~ld, I~lmrtmeat of l~harma¢¢atical
`T~tmg]ol~_, l.hinrich-l.leine-Uglveriity, Ueivcniaust:- I, D.4027.5
`D~l~Morf. Q~n~a~y,
`
`Materials and Metht~d.~
`
`Chemicals
`Pgosphatn buffer solution with so4it~rn clx’~nde pH 7~ (D/d~
`1996), hydrochloric acid buffer pH 2.0 at ~mmonium chlot~.d¢
`
`ACRUX DDS PTY LTD. et al
`EXHIBIT 1021
`IPR Petition for
`U.S. Patent No. 7,214,506
`
`1 of 5
`
`

`
`JUN-29-1998 18:56 FROM MACROCHEM CORPORAT:ON
`
`TO WALTERS, K,
`
`P. C~,8,- = 2
`
`(rag rnL- )
`
`(r~ mL-’)
`
`Methyl ni¢oti~te
`Ill,y1 me..otina:e
`Bu~: n R;ot~na.W,
`Heayl nicotina~
`Oetyl nicodnam
`
`13"~-1
`] S lot
`179,~
`207,)
`235.3
`
`1 I06
`47
`2-45
`0-17
`0,01
`
`")’624
`1034
`7]S
`5/-,8
`527
`
`6-9
`.-m
`292
`2233
`5 ] IB2
`
`MW = moleculLr weight, C,w= solubility in wattt*, C,t~ =~lt:bility i,~ l,ottanol. PCo,;vw=
`partition (cid:128)odficient between !-ocumol and wa~er.
`
`buff~ pH 10.0 (DAB 1;O6) wen used ~ vekicles. All solutions
`were svm~’di~ to an ionic s~ns~ of 0’158. M~thyi. ethyl.
`butyl and hexyl nie~finate were ob~n~ from Aldrieh-C’nemie
`(Stci~=im), octV! mooti~ato fio~m the Dcpmmi~nt of Phorma-
`ceut~cal L’-’hemi~t~ Of the University of DOsseHorf, paracetamol
`from Bo~hrlngcr Ingelh¢im KG gagelhelm), phenacetin from
`Bayer AG (Leverkusen), benzoic acid from Caesar & Lorentz
`([,Ziid~n), botmyl td=oho] from J~’~ase.n Chiw~aa (~e~l, k3olgit~)
`m~d pwldine from Kra_et OmbH 03ui:~ta’~)_ HPLC grade
`methanol mad acetcmerlie see produzta of Ri~el-d¢ He~
`(Seehe, Gtrm~my). Water was useA. freshly di~tilMd.
`
`Oiff=ion cd/¢
`Two diff,~m¢ diffusion celia wen: used for the investigations
`of the n~t plates md hoof membranes (Menu t995). Both
`galls were raodlllcadom of ~e Fnmz diffusion ohm-abet (~ranz
`1975). Th~ v~rdeal!y ofien~:d donor and accepter OZmp~-
`meat w~ joia~ :ogath=r by =lamps and contained 100 or
`50 mL, respectively. As curves nails mu~t be fixed und~
`p:cssun: to seal ~= cell, the r~’o-part donor comparanent w~
`built from smi.~egs steel ~ nail plats wag fixed between
`these ~’o pm-.~ semwing them tog’~thcr.
`
`Arvalyzieal eoadiaor,~
`In the case of h~cf membranes the ~etta’rainadon of ~e
`accepter eo~nte~tioa wa, carried out specwophotometrically
`(Zr.lss DMR 10, Ob~rkc~h:u), The houfly-¢o~e,,~.d sample,
`were measm’ed a: the long-wave mudmum end thee(cid:128)ariel
`returnS. In the case af the nail plates ~e samples had to k
`analysed by I-IPLC due to the smaller fluxag, The concea~arion
`wa~ asssyed b)’ a modulm" I’I’PL~ unit ~LC=6A, $1",imm~u,
`D~isburg) equi$~ with ~ auloma~ ~ar~le inj,otion mod-
`ule (SIL-SB). system oonu’ollar ($CU-6B), LrV-VIS, spec~o.
`photometer ($PD-6AV) and Im integrator (CR 4AX
`Cltmmatopac). The malyses "~ carri~ om at ambicm
`temremnms ~th a t25 x 4ram column packed with
`I.JChrospher I00 RP-lg, 5 gm particle diamemr (’i~. Memh
`Darmstadt), The mobile phase, (cid:128)onslsting of methanol/water
`or a~tord~ie/wa~er mix,tea was puml:~l a~ flow rai, s ran-
`ging from l m 2 re.t, ruin- ~. Injection volumes varied b~tween
`5 ~nd 150 pL. The concentration of the sampl~s was (cid:128)flculated
`from peak z~a$ by ~e external standard met,hod.
`
`Solubilities
`After a tough cstim,!¢ i~ a p~lirnin~’y cxp=dmzat~ the amount
`of substance eort~e;ponding to trace ~e gOlt~bi]ity W&* mix~:l
`Pr*pamtio^ of ~F.a ,all plates and hoof met~ran~
`with 20-50 mL solvent iv, n 100 ~ gb.ss butfl~ by rotating at
`Healthy n~dls of the big tot: were ral~n from dead raea and
`32=C. ARer 24 b the sample~ were filtere~ first by paper
`women, aged 19.-61 yo~a~. 24 h post morton at the lot=st,
`[$:hlck:h~r & Schtmll, Dzasel) and a.ftcr ~.t by cellulose
`Ad, hw:ing tissue of the nail bed or nail fold was removed by the
`method of KIigraan and Cht~stophars (Kligm~n & (~h,’iSto-
`aeemt= membrane ~lter (0.45 pm, 25 mm Z) with a filter
`device of stainless steel (Sanoriua, fi~tmgen). All ma~fials
`phe.rs 1963). With the ~’ene-al side ~wnwards the nails w=r~
`were equilibrated a: ~2°C. Sp~tropho~om=~ai ~say of the
`placed on a cotton pad which w~ soaked with 0.5% trypsin iu
`phosphate buffer pH 7-4 and tem~¢M at 37~C m~er a period of
`eoneeutration followed immedia~ly to pt¢~ent precipitation.
`IO h. A£-mrwards the tissue war ramave.d wi~ t~ccz~rs, the
`nails ~n.q~ wi:h distilled water ~d put into w~ter for 24 h to
`remove remaining trypsin, Because k~radn is hardly d~om-
`pored by pmteina~e~ fit]be & Regi~ 1989), axis method
`|mahled t~eipt of intact nail plates.
`Hoove~ were taken from fTcshly daughtcr¢4 eattle~ rid of
`adberh~g eoem~tiv¢ md aar~il~qnous dssue and pu: ~.nto water
`for 24 h. ~ter~ar~ abo~t 100 pm thick memSrane~ wen
`taken f’:om the distal ~ Of th~ bail hem with a miter.one
`(R~ichert & I’ang, Nu$1och). The hoof pieces w¢t"t strong
`enough ~ot ne, ed embedding, even in the swoll*.a state, The
`membranes wen punch~ with a diaraeter of 25 mm ~d dri~
`at room tcm~ratum, ~.zoful inslx~:tlo- of me tool mem-
`branes by electron microscopy did t~ot show any por~s through
`the membnm~s but only cavi:ies. The thickness of the gwolt~n
`nails and rr, cmbean~ were meaautod with a mierommer cal-
`filer (Teen Mieromasmr, Reo=ns, Swi~erland), in the case of
`~he naib with ~ help of a mrtd ball (5.5 mm rile’act=r).
`
`Penetration studies
`After filling the accepter comparmaem~ the ~woll¢,[,, n~i ~]ate
`m hoof membrane was inserted in the ~ffusicn ceil. The eel]s
`were ~c1~libramd at 32*C in a water bath ~thermost,a~ Julalz3
`Parath¢.rm IM, /uchheim Labo~cck.-fik. Seelbacb), then
`100 rift_ donsr liq~d was t~kled an~l samples wen take~ :~m
`¯ ~ accepter ~odi~y. The meanudng :’mervai was de:~r-
`mined by the punctuation rate If it was kith eaough 01¢~’f
`mcmbrttne) t.h: samplra wc:(cid:128) taken htyarly. In the case of the
`nail plam samples we~ ¢ollec~d daily or weekly because of
`oh© !ow l~rmcabilRy and therefore long teat period. Since all
`investigations with nail plates were antlys~ by l-]~LC, the
`saml:les (I,7 mL) (cid:128)ould not Im rammed ~ut had to be replaced
`by buffer solution_ The a:eeptor Oilution w~ ¢orrtate.d arith-
`metically (eomputee-~idcd). The m~ia were pmserv~ with
`0-02% sodium azldu in the cn_~ of a !oi~g test .~riod. Th~
`accepter was mixed b)’ a magntfic stirrer ~ll~’aag EOA 9 warn
`
`&
`
`b
`
`m
`
`2 of 5
`
`

`
`.1UN-29-1998 18:58 FROM MACROCHEN E:ORPORQTiON
`
`TO [.,;ALTERS,K,
`
`P, !0/!2
`
`DIRK MERTIN ~ BEIL,NRARD C. 1.191~k.,~
`¢oa~ller 1/~ 5, Janke & Kunk~l, Staufen i. Br,. Gormuny)
`during the whole course.
`The donor (cid:128)oneant~t~oa of methyl, ethyt and butyl nieod-
`natc was about 1000 mg L-k while hexy] and octyl nicotinate
`werB used ~ ~turated solutions at 32°C. Ottyl nicotinate was
`investigated only with the hoof membram due to i~ low
`solubility m~d low flux. P~ac¢~,nol and phenacetin were used
`u saturated solutions at 52°C. In the ease of the inwstigations
`of ch~ge influence t,~e donor eoncantraUolts were between
`1000 q~yridine) and 5000 mg L-~ 00enzoi¢ acid),
`
`-7’
`
`l~/fut~¢e of rh¢ partlrion cae.~eienr ¢¢tanol/wxer on the
`perraea~iliry ¢o~fficlent
`evOlUtion of the data is b~¢d an Fick~ lawi
`dM Da " A
`
`(1)
`
`F~=I.. Rda~iomMp between per~.eability g~ffidtat P me. partXtion
`¢oemm~nt oetalte, l/water PCe~.. of h~ ai~e~Me .~-;a .., ........
`
`m~an:~-s.d,), MN=me~yl nie~nate, .~q=e~hyl nico6ast~i
`BN ~b~lTl ~c~na~, I-rlqmhexy! nicotinate ON _-oe I nicotiaale.
`P in era" s" ty
`
`-9’
`
`in w]~ieh ~/dt is the ~aetratiog a-’tenure per time, D) the
`effective di.f~sio~ coefficient in the barn(cid:128)z, h~ the thiegness of
`the barrier, ~d C~a and C~ the eonegnzr~fion in the border
`on the donor and the accepmr ~iae, rcspc, edvely. As ~e (cid:128)on-
`eantratlon in the barrier is ,~ormally unknown, Cnt~ Js replaced
`lay PCniv-Cv, in which PC~rv i~ th~ pa.rtki¢l~ coefficient bgr-
`ri~r/vehicb and Cv is the vehicle (cid:128)on:narration of the pene-
`trating compound in the (cid:128)lonor. The te~. DnPCs/v is cail,d
`permeability (cid:128)odficiem P. On rl~ condition that not more that
`10% of the drug araO~nt in the donor panotrato~, P g~ be
`eabula:¢d from the ~lap~ of a plm M vs t:
`
`ease of the hoof membrane: eomptzed with the nail plate th,
`ke~r~n mat"ix in th~ hoof membrane Js probabb’ wider-
`meshed, so that the irmma,ing molecular weight reda~’a~ the
`diffusion co¢~¢icm only insignific~tly. The enlargement of a
`molecule o~y L’a one dire(cid:128)tion, as is the ca~ wi~ hnmolugs.
`m~rely leads m a small decrease of D, because the cmss-
`seetjott r~mains constant assuming ofienta~n in the direction
`of diffusion (’F!yna et .at 1974).
`Although the t~rrnea~,ilities presented here are m the sam:
`¢r~= of magnitude as the m¢ults of W~Iten e~ al (19~2,
`lg$5b), there 18 a~3 ¢ssenfi~d dlffmence in that ~e decrease of
`hhe permeabilities from the C:- tO ~h: Cvalcohol [from
`M -- D~, PCn/v ¯ A- Cv P. A Cv. t
`1.6 x 10me to %5 x 10-s cm s-k) is much s~-onger but to#
`ha -, t - h~ (2)
`diffusion coef~ciant~ am nearly the ~gme, The savors justified
`IS P is dependent on the lipopl’dliclty, then it grows udth
`the assumption of a de,musing I~at’ddon ccefficiem ~il/~ausr
`increaxing PC~/v, T~S term is norma!ly nltknown and its
`with an endesgoni¢ transfer of a methyl group from water into
`dete, rminarion is diffieuh, So PCa/v is approached by the
`¯ e nail (cid:128)ompartment. This t:xpl~atio:l is only conelusivt
`pardtion .~oeftieient octanoliwater (PCc~Vw) in the case of
`when the affinity of a subs~nee to the nail plate decma~s
`biologi¢~J membrane~. Consklea~ng the equation of Collander
`faxter than it, affinity to water with increasing lipophilimty.
`(1947}, who qLlantifi¢,’l the reladonsblp tx:tween PC~,/’w and
`Harrison & Si~akman (1958) also reported a decreasing dif.
`l~-~,.’v, log P ca~ be ¢aleuJat~l from:
`fu,*ian of n-alcanoles in ke~arln with izcreasing chain length.
`AS pat~ aicohol~ wer(cid:128) use~ for these inuesfigations, the ~.x-
`meabilities decreased probably because of the r~uced -~wel.
`Ling ~ kemtirt or iacw,:asiag molecular volume, Our resulu
`with homologous meotinic acid esters ccr’r~:spond with the
`i~vestigations of Wdt©m nt al (1983, 1985b) as far ~ the nai2
`plate is eharaeteei~d a,¢ a hydrophili¢ gel membrane rath¢~
`than a lipophJli¢ partition membrane. In contm~t to the stta~m
`gomeum, the lipid content of the nail plat~ i* m~ch lower (0,1-
`1% t%VMt~s & Flyrm I983); awamm comeum’ I5% of dry
`weight ff3ynn 19~5)), The high water contain Of the swollen
`nail plate (about 27% (Me~n 1995)2 indicates the oresenee of
`a hydrophilic gel membrane~ t~. "/’he be¢.ff membrau¢ behaves
`sin’&l~ly’, th¢.~ is nO deI~ndcnr:y of the 1~?~ 0:" the Iipo-
`philieity on ~e permeabili~/of the penetrating drag.
`
`1(cid:128),~ P. log Da ÷ b + a. log PCo~/w (~)
`
`If the so!ability proporde~ of oetanol and the lipid barrier ar~
`not the same, the slope a diffm-s more or iess from the ideal
`value of unity bat b di~ncfly grcat~ than O. But if r.~ barrier
`bch~.ves IL~e ~ hyOa’ophilie get membrane, ).hen lhc I~rm0-
`ability is indepcndcm of ~e PCo~!w and the slope becomes 0.
`The ~¢n~eability coeffi¢iemts ~f ~e hoof Itt¢~brfme (’P~
`~xcee~ed that of the hall plate fP~¢) 10- m 30.tbld ~Fig, 1).
`Uulik© the hoof membr~e, whera ~t lag-~’n¢ wa~ unix a few
`rMnutes, s~dy-state Ixmetmtio~ th.rough the nail plate
`~cur~d after 10 (methyl aioofinate) up to 80 (hexyl nbotl-
`note) h. The plot off v,~ PCc~q,v in a Iogadthraic scale
`aeeorddng to tquatlon 3 dc~g ant show 8, positive slope. Rather
`the pe.rmeabil~ty of ~ hoof membnm¢ is independent of the
`tipophi!ieity of the penetrating suhs~ces (PRO.05). How-
`ever, the Oexma~ of the petm~biliry in ~ar~ oftha nail phte is
`highly siBrdFa:a~t (p_ 0,01), but ~ds¢ contradiom the model of
`a partition membrane. It can be expldn~l by rbe decreaginl~
`diffiaslon ¢~fficicnt tiue to the iner¢.asing molecular volume. It
`can only ~ assumed why this f;~-tor has no in~ueaee m the
`
`Mazlmurn fiu.res and water solubilities
`The eqtmtioo for calcul8~ng the maximum fl~ (3mix) felk, ws
`di~cfly from the first Fiek’s law (~.qn I). J,~.~ i~ me a.rnoun: of
`substance that p~ne~te~ ~.~ugh a burner of ~e area A aa~
`the thickness ha lXa uni: of time from a sameat¢.d solution
`
`3 of 5
`
`

`
`JUN-29-1998 10:59 FROM MACROCHEM CORPORATION
`
`TO WALTERS,K.
`
`P. LI.!~!
`
`NAIl. AND KERATIN 33
`that, thi~ investigation with only two m~¢l ¢ompoand~ m~st
`integrated carefully. This agrees wit~ ~e m~l¢! of a
`laydrophilic gel membrane wh=e the solubility in tl~ barrier,
`which de~em’fines the maximum flu~. (cid:128)err=signals to the
`~olubility in the swelling medium ~at is water. [.n (cid:128)ontr~t, the
`maximum flux ffu’ot~gh a lilx~phillc partirdon mambrane is a
`f~ncflon of the laa.nitlon (cid:128)oef’ficien~ and the ~olol:dliry in the
`veh~le. Approaching ~(cid:128) solubility in the barrier by the t~lu.
`bllity in octanol, eomFlex eq~tiens result With their help
`maximum ~uxe~ can be pmclic~d by knowledge of the drug
`solubility in the vehicle and in oetar~l ~Iagndom-Lew©k¢
`& Li~pold 1995). The (cid:128)ons¢,quenee is that one has to pa~
`attention primarily to the highly different wa(er solubili;i~s and
`only secondly to the morn similar permeability eoeffidents in
`or~r to gereen drugs for potential topical ap~liea~on to ~e
`hall phm.
`
`a:a ~2 3:4 3:e " 3’.s ’ ;:n 4:~~-4
`Log ¢,w
`~_ G. 2. Rdadon.ddp bcrw~n ~e ;oiadflan of the ra~immu flux J~,~
`
`000 ~a) ~d ~© ]ogatilhrn of the waP..r so]ubiiit~ of ~h= model
`~ounds p_ayao¢Cgmol ~AR) ~d pkrgacetin (PI-~,) ;~¢rO~s human
`l~late I~11) aaa ~nne ~9oz mem¢~a~¢ (M) at 32"C (n~4,
`mea~s±s,d.}. Ja,~ (1000/~m) in mg cm- s" ; ¢.w in mg L-k
`
`(sir’.k-e0~di~iDas~:
`
`P
`
`C4)
`
`Pet~tmtion of ,~le¢t~ly:~$
`Di~soaiadon of benzoic acid and pyrid.ine led ~o t redo(cid:128)tier ef
`¯ ~" ~e~i~ioa ate tlm~ugh ~(cid:128) h~ membrane. Table 2
`shows that the p~tmeabill~ coefficients of ~) io~(cid:128) forms
`wer~ dgn~ficantly lower than those of the neutral form.
`B¢¢at~ th~ gen,-n-atior~ of b~nzyl alcolml de¢:r¢,,~e5 sip
`nificgntl~ by a half (t-teSt. P ~ 0.05) ~[tcr d~e transltlon from an
`acidic to a ne, umd or basic milieu, ~e neutral oompound fgrves
`as a stand~d. To this end th~ qu0ticnts of tim perm~bility
`¢~ffici~-n~ Pl~,_,..i~ ~¢ia or P~,yr*cia~ and P~yi ,):~ gre bu.;lt
`Negl¢cdag ¢~ccts of the molccu!a.r volanle lind ~sstm:fing a
`~d (cid:128)~mpared with each other ("l’abie 3~.
`hyclrophilio ~el member(cid:128), ~(cid:128) tercrt bg P/ha in e-quatbn 5 is
`Dissociation ~educes me diffiasion rate of benzoic acid ~.o a
`constant and the maximum aax is only del~adcnt on ~e
`tilth end tha~ O~ py’t’idda~ to a quar~r. A~surt~ing the Jso¢Icc~c
`solubility of the d,~g i~ wa~er. Then we expect, for a lfl¢~ of
`p~int of kera~n is ~ou~ 5 ~acshall 1983), it is positively
`logarithmic maximum fluxes vs l~gadthmie water solubilities,
`ch~ged at pH 2.0 and negatively charged at p~ 7-4. However,
`a linear (cid:128)orrelation wi~ the ideal gl~pe of" u~iry. Thi~ was
`nothing is known gbout ~(cid:128) charge d~ns~ty. "flit decrease of
`confirmed by the regults of the nall plat¢ (slop¢ 1.082,~ as well
`penetttl:ion caused by dissociation |g due to ~) Do~ (cid:128)ff~t
`as the hoof mcmbraa¢ (slope 1.003, Fig. 2). Fat bcrtct" oom-
`(,Metres 1968; Hig~ ¢t el 1990, 1991) or the eIectrosmt~c
`pcJ1~bility L~ maximum fluxes were standa~ized to a bar~et
`~lrabion ~:twesa the membrane and th~ diffusing molecuI¢
`thiehaess ~f 10(~ urn. The slight ~itive dgdatio~ of the
`(Kebzy,q,~i et a! 1994), At pH 7.4 ~uzoate is a co-ion to me
`sbp¢ in ~r e~e of ~e nail plat~ ~vas e~xl~lained ID, the ~tmn~r
`nrgativcly t;harged keratin and hence it i~ displaced from the
`influence of abe mol~ular size on rb,,,, permeability. The dcnngr
`m~rl11~°ane: the lower ¢one~aa’ation gradient then leads ~O a
`network of filgmentou~ kar-a~ ¢ora~ed wi~ that of the beef
`reduction of ~e pm"meability. In ~he ea,w. of ~ acidic milieu,
`memb~n¢ exhibited morn raisin.nee to the more voluraiaous
`the pyridiniura-catJon is d.isplar~d from the now posi,ivdy
`phcnac~dn than m pa:-acetamol. Ti~i~ wgs exp~s~cd not only
`chatg©d kcradn leadlng to a decreased p~netra;io~ (cid:128)ompared
`by the lowe1’ ma.x.~rn fll.~x (Fig. 2), bu~ also by the ]awer
`with me neutral term. Nev~laetess, diffusion of ionic sub-
`permeabil~q, eo~cient eaIculatcd from these r~ult~:
`S~e, nces through the h~of membrane ¢onf!r’m the azsumpfi~n of
`I..75 ~- 0.32 × 10-s cmz s- t for ~aeetamo/ and
`a hyd.,T~hilic gel memb.q~u¢ (Zaikov e~ tl I988),
`1.40 4- 0.47 × 10-s emz S- J for pheaaeetJn resp~eti,~ely,
`Some ~u~.hors at~ibum the lowering of the el¢=trol/tc dif.
`It has to be emphasized that the maximum flux of a sub-
`fusion through eha~g~d l~lymnts to sorption =ff~s (Medley
`stance firstly del~nds tm its water solubility and, COnsidering 19571 Msates 1968; Zaikov et ~1 1988). Since only the free
`
`Taking the logarithms result~ iS;
`
`log ~I,~.~ = log/~ + log Csv
`
`~a~ le 2. P~n’t¢~bi1~ (cid:128)~:flicie.a~ ,,~ i~.~zyl alcoboi, beanie a=id and wddine
`gls the bo~ia) haot membr~e.
`
`pH
`
`ia’erme~bili:y (cid:128)~ffi¢ie~tt (I(~-s =ml (cid:128) ~ ~)
`
`Berm~l P.,k;ohol 2.0 7S.24 ± 1~,45
`7.4
`i0,0
`2-0
`7-4
`2-0
`7~,
`
`41,72±9.07
`4S78 ± 8-40
`7~.62 ~- 16-43
`i.29 le 146
`t9-1a~7,05
`~.80± I0,35
`
`Ben.zaie acid
`Pyrldlnc
`
`Tem~ratur~= ~2.C (n-=, 4, raears q- ¢,d,L
`
`)
`
`4 of 5
`
`

`
`JL~-29_-!$98 i i .’ 08 FROM MQ=ROCHEM COPPORAT I ON
`
`TO b]ALTERS, K.
`
`P. L2/!2.
`
`PaAc/PI~,b
`
`Pr~/l~,L
`
`0-21)
`
`{~.2g
`l -O?
`
`DmK MEg’rn~ ~ B~ C. LIPPed.I)
`Flyna, O. L. (1985) Meghamsm of pereutaneous at~on fm~,-a
`physi¢.cohemleal evide.(cid:128)~, la: BronaaBh, R. L. Mtibach, H. I,
`(eels) Pt~at~eo~ Absorption: Mtr.hanisms-Mr..tho~olo~-
`Drug DelNcry (I~rmetology. Vol. 6). Marcel l~gker IBc., New
`York. l~ 17,.,41
`Fi~, O. L., Ro~m~, ’r, L (19"rl) Moatb’can¢ difl~io~ T.I: iw3~cn¢e
`of ph)’t|e.all ~iorpuon on ~e mvle~ar flux th~ l’lel~:--Jll~¢ot~.s
`dh~thyltmlysilcx~ barriers. J. Pharm. Scl. 60:1758--1"796
`b"lyan, G. L., Ytllow~7, S. H., gos~man, T. I. (D74) Mm trt~pon
`phemem~a tnd models: dm~’sdc, al anne(cid:128)am L Phm’m, $~. 63;
`+79---51 ~
`Fmnz, T, I, 0975) p~.uumo~as absorption. On the mlevanc~ of in
`~t~ da~. J, In’vaSt. D¢nnatol. 54:190-195
`For.w:, R. D, B,, Maegae, T, P. (1973) Co~ormadoa in fi~rous
`l)fot~its: chapter 1@; ke’rad,’,s, Aoadernle P~. New York. pp
`4{~9-4S~
`Hagedom.I,~wr-.k+, O,, Lipp¢Id, B1 Ci [ 1995) Abf, oi’~tion of ttltascr~eo~
`and other ;omFoua~ ~ro~sB ham~ ski~, in viva" dtwivauoa ol
`mt~od to prr~gt maxim,~m fi~e~, Phstm. l~s. 12:135g-1~60
`Hma, M,, ~ryst:n, H, M, (1995) A_moroifin¢ - e revi¢~ ~f its pbtt.
`macologic~ p.’oNr~rs and th~ralg~ic pct,,ati~ i~ tim tr~atmem ef
`anychomycosis and ohqer s,jp¢ffamal f,.mga] infe~tiorts, Drag; 49:
`
`pR
`
`74
`2,0
`7,4
`
`Ibcidiae
`
`"rem~ratur;: =~2°~ (a -~g
`
`fraction i$ able to pentt[a;(cid:128), sorptioa to ~ctional groups
`oould lead to a ~¢a’tase of the concentration gradi~n:. How-
`ever, investigatloas wi’,h polydlmethylfiloxan¢ films contain-
`ing a silica ~l]~r show that sorp~on oniv causes a prolongation
`of the lag-time but not a reduced steady-senSe flux
`(Flyrm & g~scm~ 19"~a). Due ~o th~ d~smi¢ each~age ofth~
`free mbsta~:e bet-~e~ membrane ~d donor .~olution, the
`resulting concentration of th~ f~e drug in the membrane will
`am almr ~ the adj~straent of the st)rptior~ equilibrium m the
`study state ~d is independ:nt of the degr~ of sartwatlot~ ~f
`¯ e furmdougl groups, eta the coadiLion that the.re is no d:cr¢a~
`of the donor concentrttlon by the sorp6on, Therefore. aa
`iuterlu*tation of the hindare¢ pene~ation oP ~arg~ molecules
`is la’eferrexl on the basis of the Do.,man potential
`reduced pttmaabili~ of the ~ (cid:128)ompeted l~nzyl
`ale6~.oi ~ pH 7-4 ~ ~n~m~I with pH 2-0 is pro~bly OaUWA
`b3’ a ~ of the kerat~ twelling due to the ~hargr In.#erslen
`of the Imra~n g the Wassltlon from an a~i¢li¢ to a mmtrai or basic
`etvironmenL Th~ weak, not significant increase of the pent-
`tradon ofth~ banal ak~h¢l oh~gitg the pH from 7-4 to 10.0
`l¢~s f~ah~t ouF~c~rt to this h~eds, ~ca~ ’,he number of
`negatively charged gro~lps in kmfia (IF.8-5) at pH 10-~ is
`ltOtrly the same aS in s milieu of p]-I 7.g.
`Applying these results to the human w-it, they ¢ontradlc: the
`in,~tigations of Wahers et a~ (IgSSa), wko (cid:128)~uH net find a
`dependency of the nail ~rmcabilJty of micona~Ic On th*
`degree of dissociation. Maybe this contradiction i* oxpltiped
`by the higher ioni~ sa’eng[h of the solutions; in this case the
`i~fluence of l.he Donee equilibrium is lo~d, gin~ t.h¢
`amino a¢i~l composition of keratin is clopendem on th~ species
`(Fras= & Ma~Ra¢ 1973), th.e difftr¢~n~ ~vat~ ~ould ~Iso ba
`pat down to .~arying eh~ge densities or hoof and null keradn_
`The ~du~tion of the hoof ~¢rme.ability 1~ a qua, or oause~ by
`(cid:128)omplett dissociation of ~e subgm~ce is much lower than tim
`possible enhancement of the ~01ubiLit7 of hardly ~oluble a¢i~
`~d banes, So the solub|lity is Lncrrased a hundred fold at S9%
`d~gree of ionization (assuming suffigicnt soh~iliri of the sa)t).
`Sin~ the mpximmn 8ux is primarily a function of ~e solo-
`billed in wamr (.’~(cid:128) ttbove), it is possible to heigh~a t~ per-
`meability of bofiq the beef msmbran¢ a~d the nat1 plum
`dis~,gia~on of ~e penetrating dtu~ ]f ti’,~ ndl~eu in th= ba_,’fier
`¢ausog no pre.¢ipimtie~ of the substance.
`
`References
`Co]tt.,a~’, R. (1947) O~ "lipoid so|ub~1]~". Aom Physiol, Scan& 1~;
`
`Evans, E. ft. V, (1990) flat! dcrm~ttopb,_v~is: the aam.’~ and s:~lc of
`~ l~mbltm. J. Dorrt, stof Treat. 1 (SuppL 2): 47-.~$
`Fal.~,.J., Re#m, M. (198S).,...,^.R~pp Otorale-l.axikop, O. Trdome
`
`Hin~on, D., $~a~, L B. (195S) The Imm fi~* of kor~tln. Tern
`Res, J, 28:I005-1005
`HigL M., Ta’tioka, A., Miygglgg, K, (19~) A ~tudy of iot~ ~rme, adon
`ecros~ a cEarg¢~ membrane in raulticomfx3ntnt ion systerr~ as a
`~nction of ra~m~me eh~-ge density, J, MambO. S¢i. 49:145-169
`H~go, M,, Tanioka, A,, Miya~, K, I19gl) An expa~meatal study of
`ion p~’rneitt~on ia mtflticompou~n; ion syite.ra~ ms t f~nction of
`raembt"ane rha~ge d~nslty. L Membr. $~. 64:2~$-262
`K!~g~mtn, A. M., Christoph~n, E. (1962) .z~¢Fmafion o1~ isolate,(cid:128)] sheoW
`of haman stratum ¢orneura. A.’cL l~rmatol. 8g: 702--70~
`Kobaymhl, ’if’.. Nagai. "r., S~’~kL T., blo~ka, Y., Fuji], N, (1994)
`Reslricted l~f’rrt.*aflaa of d~xh"z~s~ILfltt~ by ~e¢lro~ti~ ba~er of
`negatively e1~rged uln’~la’adon m~mb~u~; s~dt (cid:128)ff¢¢t ~. the
`p~m~bon. J. Mambo+ So’. $6:47~55
`Le, V. H., LiEp~ld~ g. C. (1995) [~aence of physicochenuc~d propor-
`t~ of Immologous ester* of Riccrinic acid on ~(cid:128) ~m~tl ~rmeability
`mad mt.ximor~ l%x. Int, J, Mtm-m. 12,(cid:128): 285..292
`Marshall, P-, ~ (t9$3) ~hgrtaeriz~o~ of the proteins of ~tuman hair
`~md ~ai.l by ~l~r~ph,rn’esis, L Imtesl. L-M.=Isw-tel gO: ~ lg..’~A.
`M¢,~w¢, P. (1968) Tnm~port ia ion.¢.xeh~$e Imlymer~. In: Cran.L ~.,
`Park, O, S. (eds) Diffusion in Polymers. Aoad~mi~ Press, New
`York, pp ~’/3.-~28
`MeAley, 1, A, (19~7) Th: dlff~slor, cf acid ions in keratin. T’m.ns.
`pandsy ~(cid:128), ~3:1380-1388
`Meisel, C. W. {,1992) Imm.ma,+nnat symresium ~n am~rotfir~¢. Haul 3;
`64-7O
`Martin, D. (1995) Pe.,.meg~il]ziit d~r me~.~:hiichcn Nagelpl:tte und ~e
`Verlm~agemi~$E,’b.ke~r. L’~tmIs e~a~.r Keea:inmembraa tiefischer
`HtrkaM’t, "-¢erl~t~; Maine, A~hsa
`Nolting, g,, ~=ebeeher, C. (Eig)3) CielopitoxOlzra~.t-Weg~rise."
`t~pischm Mlikes~-Tb.¢rapie, Uai~e~t~tsverla@ Jena. J~n~, pp ~.-6~
`Pol~k, A., Z~ag, M. (t990) A.ma~l~¢, L~: gyley, J. F. (~.) C’ae-
`mothorwy ~t Perusal Dimue (Rm,dbook of ,~perime©tal P~s’m~-
`gvlogT, Vol, 96), SW.m$¢r-V~la$, Berllu. pp ~0~-~21
`¯ ~adripuro S, A,, Horn, G,, Hi$}-A~r~ T. (1 ~ 1) ZAtt Lokglwirk~a.,tsk*it vet
`Odop|m~ola,’ni~ b~l Na~elrayko:~a. Art, aria- Far.b, ~1: :36g-
`1372
`W~ten, K, A,, P1ynn, ~, L. (1~3) l~m~’.abi!i~ ehamgt~rigius .~. th<.
`humaa nail pla+c, ln~ L Cosme~, 8d. 5:2.M.-.246
`Waiters, K. A., Hy1~, G, L., Ma.’~’¢l, L R. 0!)82) Physi¢~bemi~
`nlamagtorizafion of l.~e hum~ mall; left.cation pa~ern for wear an,
`tim horao]egohts alcohols ~d dl~a’zu~¢s with r~s:~:ct to hhe SBW.|t~r
`eomeam. J. Phatm. l~arma¢¢l, 25:28-33
`Waltz-s, K. A., F~yon, O. L, Marvel, I, R, (t985a) F’~n~watlon oft.h,
`h~m~ axi2 #ate: Lhe off.tots of ~,’ehi¢l¢ pH on th~ pomm~on (cid:128)
`~icom~le. J. Phi. I~az’m~o~. 37:49g,~99
`W~te.rt, K. A., F"lymL G, L., Mar#el, J. R_ (19S~b) Phy~icoober~c:
`ohM’golwi~afioll ¢fhhe ~mta’~ a.~il: sol.vent effects on ~ 9¢rmcaUo
`o(cid:128) hr)mologOa~ g¢ohols, 1- I~arm. Phatm~ol, 37: "r’/~-’l’;~
`2aike% G, IL, Iotdanr&.ii, A, 1.., Ma.-kin, V. S. (198$) Diff3.,sion c
`Eh~’,0l~qcs ho Polyme~ s/gP~ U~ehL pp "/3---I:?.6
`TOTAl_ P, !2
`
`5 of 5