Indian Journal of Experimental Biology Vol. 39, Jan uary 200 1, pp. 11 -24

Review Article

Permeation through cornea Manjusha Malh otra & D K M aj umdar* De partment of Pharmace uti cs, College of Pharmacy (U ni versit y of Delhi) , Pushp Vihar, Secto r-Ill , New Delhi 11 00 17 , Indi a The permeability of the cornea to drugs is clinically important because it is the major factor determining the crticacy of topically ap plied ophthalmic preparations. With this perspective, the present art icle gives a bri ef update and overview of corneal structure and proposed mec hanisms of permeation . Physiological, physicochemical and formulation factors affecting drug permeation th rough cornea arc hi ghlighted . lnllucncc of ocular penetration enhancers on drug permeation is also discussed.

The use of medical therapeutics in the treatment of eye di seases is as old as medicine itself. Ocular therapeusis has progressed inexorably but by a circuitous path. The earliest account of ophthalmic treatment on record dates back to the Mesopotamian era, when Imhotep (2,800 BC), an Egyptian physician used malachite topicall y, in the treatment of trachoma . In seventh century BC, powders were blown into the eye through reeds and tubes as a means of topical admini stration . The Greek physicia n Pedacius Dioscorides (40-90 AD) compiled five-vo lume materia medica De Universa Medicina that gave fi rst th erapeutic delivery system, Coll yrium, fo r op hthalmic medication . However, improvement in ocul ar deli very preparations and topical treatments did not occur for many centuries with ocular therapeutics onl y finding mean ingful va lue in the seventeenth century until when the understandin g of transcorneal permeat ion was sketch/. The permeability of th e co rnea to drugs is clinica ll y import an t because it is th e major factor determining the efficacy of topica ll y app lied preparati ons. Therefore, an integrated kn ow ledge of the relevant anatomical and physiological constraints that impede or modify ocular drug and vehi cle disposit ion through co rnea is important.

Corneal structure The cornea is a transparent, avascular tissue, approximately 0.5-0.7 mm thick and abou t 11 .5 mm in diameter. Broadly, cornea is divided into 3 layers: epithelium, stroma and endothelium 2 . Epithelium is a tight junction tissue that is about six cell layers thick . *Correspondent author

Physiologically, the epithelium is relatively impervious to polar or hydrophilic compounds with relative molecular weight greater tha n 60-100 Da. Glucose (Mo l. Wt. =180 Da), for exa mpl e, does not pass 1 th rough the epithelium . Lipophi lic compounds pass the ep itheli um du e to solubilization in the lipid cel l membranes"'·:~, while it provides major resistance to the movement of ions 6 or wate r7 . The anterio r corneal stroma is condensed into a thin membrane known as Bowman's membrane. The stroma constitutes 85-90% of the cornea and consists of fine col lagen fibr il s and mu copolysaccharides ab le to hold a substant ial quanti ty of water. Fibril s are spaced so that the cornea is transparent when at normal thi ck ness. Loosely attached to the posterior surface of the stroma is another interfacia l layer known as Descemet's membrane. The stroma is read il y transversed by wate r soluble, polar compounds and less so by non-polar co mpou nd s. Even high molecul ar weight substances diffu se with ease 8•9 . The innermost layer of the cornea, the endothelium, consists of a single cell layer whic h houses an active water pump that regulates co rn eal thickn ess through hydration 10 - 12 . It is a ve ry porous ti ssue wi th an open intercel lul ar network and thu s large molec ul es (up to 70,000 Da) can trave rse this membrane eas il / 1_1:;.

Mechanisms of permeation through cornea There are two major pathways fo r th e movement of compounds through the corneal tissue : transcellular and paracellular. Tra nscellular drug movement involves cell/tissue partitioning/diffusion, channel diffusion and carrier mediated transport. In contrast, paracellular represents diffusive and co nvecti ve

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INDIAN J EX P BIOL, JANUARY 200 1

transport occurring through intercellul ar spaces and ti gh t junctions. Diffusive transport is a di ssipative process that depends upon the difference in th e so lute concentration and the permeability-surface area properti es of the membrane whi le, convecti ve transport is dominated by a balance of hydrostat ic and osmoti c grad ients, so lute concentrati on and hydrauli c and refl ecti on coefficient of the res tri cti ve barrier 16 • Patti ti onin g of a compound across cel lul ar membranes is especially important for hydrop hob ic molec ul es and is rep resented by a relati vely hi gh activati on energy for diffusion. While aq ueous diffusional channels are important for cornea l transport of hydrophil ic compounds and are characterized by low ac tivation energyl7. The cellul ar arrange ment of epithelium of cornea precludes parace llul ar transport of most ophth almic drugs and limits lateral movement wit hin the anterior ep ithelium 18 . Cornea l surface ep itheli al intrace llul ar poresize has been estimated to be about 60 A (ref. 19). Small ioni c and hydrop hilic molecu les appear to gain access to the anterior chamber through these pores 20 , however, for most dru gs, paracellu lar transport is prec luded by the tight intetjunct ional comp lexes. Stroma exerts a diffusional barrier to highly lipophilic drugs owing to its hydrophi lic nature. There are no tight junction comp lexes in the stroma and parace llular transport through this ti ssue is possible. The endothelial permeability depends on molecular weight and not on the charge or hyd ro?? p h .,. t tc nature o f t I1e compoun d?- 1·--. 23 26 The genera l mechanisms ' of drug movement through cornea are given below: A. Organ level • Rate-limiting membrane for most drugs is the corneal epithelium which ac ts both as a barrier to penetrati on and as a reservo ir for dru g. • The rate-limiting barrier for most drugs appear to reside in th e top two cell layers of th e ep ithel iu m. & Stroma is rate limit ing for very lipid-soluble drugs . B. Cellular level • Small molecules, for example, water, methanol , ethanol, propanol, and butanol, readily traverse the cornea through assumed aqueous pores . Theit permeabi I ity constants are very large. • vVater-soluble compounds traverse the cornea by the paracellular route. The smaller the partition

• • •

coefficient, the smaller is the permeabi I ity constant. Peptides, ions and other charged compounds appear to penetrate th e co rnea by parace llul ar route. Substances th at possess biphasic solubility traverse the cornea more eas il y. Lipid soluble substances pass read il y throu gh the limiting cellul ar membranes.They do not penetrate in proportion to thei r concen tration.

Factors affecting drug permeation through cornea Majority of ocu lar preparations are fo rmul ated in aqueous vehicle. The factors th at large ly determine ocular bioavai lability of drug from aqueous formulation, may be divided into 3 categori es: A. Physiological factors B. Physicochemical fac tors C. Formulation factors A. Physiological factors The loss of dru g from th e precorneal area is a net effect of drainage, tear secretion, non-corneal abso rption and corneal absorption rate processes . Collecti ve ly these processes lead to typ ical co rneal contact time of about 2-4 min in humans, fo r an in st illed soluti on and an ocul ar bi oava il ab ility that is co mmon ly less than I 0% (ref. 27). I . Precorn eal factors- Various precorneal factors causing loss of drug are: (a) Tear turnover-Tears wash out at a rate of 16% per min, except during periods of sleep or during anaesthesia. Normal tear vo lume is only 7 f..!l, so dru g loss is substantial. (b) Insti ll ed solution drainage- The precorneal area can hold approximately 30 111 , including resident tears when the eye is not blinking. T he volume reduces to 10 111 in blin king eye. Therefore, excess of instilled volume either spills or rapidly drains from the eye into the nasolacrimal duct with subseque:1t absorption into the systemic circul ation . Drainage cf an instilled drug sol ution away from the eye is respo nsible fo r a considerable loss of drug and hen ce affec ts the biological activity of drugs in the eye. The rate of th is drainage is related to the volume of dru g sol ution instilled and increases with increasing volume. The drainage rate of an instilled volume increases at a rate proportional to the volume of th e flui d in the eye more than the normal lacrimal vol ume. The drainage rate is about I 00 times faster than . the cornea I absorptton rate-?R . L ee an d Rob'tnson2x gave following equation to predict drainage :

MALHOTRA & MAJUMDAR: PERMEATION THROUGH CORNEA

Y1 =Y 0 + Y; e-K '11 where, Y, = volume remaining in the conjunctival sac at time t, Yo= normal lacrimal tea r vo lume, Y; = vo lume of instill ed drop, and K,1 =drainage rate consta nt. Drainage rate constant is directly proportional to the vo lume of instilled drop i.e. smaller the drop instill ed, the lower the drainage rate and greater the ex tent of ophthalmic absorption. Instilled volume - In the rabbits, 90% of the dose is cleared within 2 min for an in still ed volume of 50 J..ll , 4 min for an instilled volume of 25 111 , 6min for an instilled volume of 10 111 and 7.5 min for an instill ed vo lume of 5 !11 29 . This vo lume dependency of so luti on drainage rate has been found to exert its ex pected effec t on the percent of dose absorbed into the eye and on the pharmaco logical effect th at follows. The effect of reducing the instill ed volume but keeping the dose constant was evaluated from the mi otic effects of pilocarpine nitrate in rabbits by Chrai et al. 29 . As the vo lume of the instilled drop was reduced from 50 to 25, I 0 or 5 111 , the area under the miosis-time curve increased 1.2, 1.8 and 3-fold, respecti ve ly . In the same study , it was shown that instill ation of 10111 of a 2% epinephrine solution to rabb its caused the same pupillary respo nse as 50 111 of a I 0% soluti on. The same principle was demonstrated by Patton and Francoeur'0, who found equal bi oava il ability for pilocarpine nitrate after instillation of 25 111 of 0.01 M so lution (67.8 !lg) or 5 111 of 0.092 M so lution (26 !lg). Thi s resulted in a 2.6-fold improvement in bi oavail ability from a 5-fold reducti on in dropsize. Brown and coworkers'u 2 ex tended the benefits of thi s phenomenon to the clini cal use of phenylephrine, a hi ghl y effecti ve mydriatic, but wit h significant ca rdiovasc ul ar ri sk in certain pati ents. Eleven neo nates were administered 8 ~LI of 2.5 % phenylephrine hydrochloride in one eye and 30 111 in th e other eye. The mean pupillary diameters (4.86 and 4 .57 mm respecti ve ly) were the sa me. However, when the said vo lumes were administered to two different groups of infants, the plasma concentrati on of phenylep hrin e was 0.9 ng/ml for 8 111 dose and 1.9 ng/ml for the 2 30 111 dose' . Clinical app licati on of the benefi ts of small-volume dosing has also been reported" ·' 5 for dosing mydriatic so lutions to ch ild ren and ad ult s. Tn their studi es, these au th ors found eq ual dil ati ons of the pupil in patients rece iving 0.005 ml ointment or drop dosage form of phenylephrine, cyc lopentolate or tropicamide hydroc hl oride sa lts and in pati ents re-

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ceiving 50-75 111 of repeated aq ueous drops of these medications. These studi es suggest that there is an advantage to small -volume dosing ow ing to an improvement in ocul ar bi oavai labi lity. Thi s al lows a red ucti on in dose and a reduction in the potenti al for 36 systemic side effects. Keister et a!. reported that reducing th e instilled vo lume would increase only the ocular bioavailability of drugs with low permeability and would not effec t th e oc ul ar bioavailability of drugs with hi gh corneal permeability. Therefo re, the clinical implication is that appropriate reduct ion of instill ed volume and the simultaneous increase in insti lied drug co ncentrati on permit sub stanti al dosage reductions without red ucti on in absolute dose of th e dru g37 . Chrai et a/. 38 studi ed the effects of dilution and drainage on the absorpt ion of pilocarpine ni trate and epinep hrine hydroc hl oride in rabbits when dosed topically in different co ncentrati ons and volume and at different do sing interval s. The res ults indicated that two drops instilled immedi ately after one another showed a reduced bioavail abilit y compared to separating the doses by 3-5 min . This implies th at multipl e dosing is more efficient when sufficient time is allowed to elapse between instillations. The vo lu me dependency of the drainage rate has also been observed with suspensions 39 but not with liposomes. Lee et a/. 40 reported th at liposomes were c leared from the co njuncti val sac of albino rabbits with approx imate ly the sa me first order rate constant (0.45 min. 1) over the instill ed volume ran ge of 10-50 111. The size and number of liposomes are more important factors than in still ed volume influencing the ex tent of ocul ar dru g abso rpti on from liposo mes. (c) Protein binding-Tears normally, co ntain about 41 0.7 % protein and the protein level increases durin g infection or infl ammati on. Un like the blood, where the drug-protein co mpl ex co ntinues to circul ate, tears are replaced qui ckly thu s remov ing both free and 41 bound form s of the dru g. Mikkelson et a/. showed that the mi oti c response to topically applied pilocarpine was reduced about 2 times as the albumin co ncentrati on in the preco rnea l fluid was increased from 0-3 %. (d) Non-productive drug absorpti on-Upon inst illati on, dru g is absorbed into the cornea and conjunctiva. The surface area of the conj uncti va is about 17times that of the co rnea 42 with 2-30 times greater 43 permeability to many dru gs . All ti ss ue absorptio n other than the cornea is perceived as non-product ive loss when the target ti ssue is the interi or of the eye.

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I DIAN J EXP BIOL, JANUARY 2001

Thi s loss can be minimized in two ways: Varying drug lipophili c it/~or chan ging the drug formulation. Formulation changes that are most effect ive in minimizing th e rati o of conjunctival to corneal drug absorption are increasing soluti on pH, lowering soluti on tonicity and lowering the concentration of EDT A and 44 benzalkonium chloride in the formulation . The chan ges in lipophilicity and drug formulations have a greater effect on corneal than conjunctival penetration. Chast et a/.~ 5 reported the pharmacokinetics of a single dose of morphine ocularly applied in rabbits before and after lacrimal canalculi ligature and results suggested a great capacity of conjunctiva for drug reabsorption . It is important to mention here, th e assumption that drug absorbed by th e conju nctiva is swept into systemic circulati on is incorrect. Series of experiments conducted by Doane et a/.46 and Ahmed et a I.~ 1-~ 9 proved the same. They postulated that th e drug abso rbed by th e co njunctiva could lead to direct entry into the uvea l tract, bypass ing the cornea and thi s route of dru g ent ry into the eye is cal led non-cornea l route. 2. Membran e jc1ctors-Membrane factors include area available for absorption, thickness, porosity and tortuosity of the cornea and lipophilicit y/hyd rophili ci ty ba lance. The cornea consists of three layers, wi th respect to barrier resistance, namely the epi thelium, stroma and endothelium. Permeability studies on excised corneas have indicated the superficial layers or epithelium as th e primary rat edetermining barrier for penetration of both water soluble and lipid soluble dru gs 2550-52 . Because the epithelium is lipophi lic, low in porosity and relati ve ly high in tortuosity and thickness, a rapidly penetrating drug mu st possess log partition coeffic ient greater th an I in order to achieve a suffic ient penetrati on rat e :~o.:. ~ . Although both epithelium and endothel ium are considered lipophilic, measurements of the water permeability of each laye r indi cate that endothe lium 5 is 2.7 times more permeable than th e ep ithe lium ~. 1 Studies on exc ised corneas by Kim et a/. ~ and Maurice 55 indicate that non-e lectrol yte penetration through the endothelium occ urs primarily through the intercellular spaces. The stroma is basically acellular, hydrophilic in nature, hi gh in porosity and low in tortuosit y but because it represents 90% or the thickness of the cornea , the stro ma is significant in overa ll . . H.uan g et a.I ·' 6 d eterrnme . d co ntn.b ut1on to reststance. the resistance to permeability of each corneal laye r

for a group of P-blocking agents. The results ind icated epithelium as th e rate determ ining barrier for hydroph ilic compounds and stroma for lipophi li c compounds. When absolute values were compared, the lipophi lic compounds were found to have greater permeability coefficients. Authors suggest that penetration through stroma occurs when drug diffuses th rough an aqueous media of gel-like mucopolysaccharide interspersed by a matrix of coll agen fibrils. Kedem and Katchalsk/ 75 8 ana lysed the permeab ilit y characteristics of biological membranes using thermodynamics and concluded that three independent coefficients, e.g. pe rmeability of th e so lute, hydraulic conductivity and reflection coefficient, were necessary to describe the permeability properties of a membrane. Hydraulic conductivity involved a net volume flow of water driven by either hydrostati c or osmotic press ure grad ien ts. The reflection coefficient was dependent on the interaction s of the so lute and water with the membrane and gave a measure of th e relative semipermeabi lity of a membrane. Mishima and Hedbys59 estimated the hydrau lic onductivity of the epithelium and endothelium of the rabbit cornea and the reflection coefficients of th ese layers with various solutes. The hydraulic conducti vity of the epithelium and endothelium was found to be approximately l .Ox Io-~ and 2.3x I o·~ mm/m in msOm/1 respectively . The reflecti on coefficient of the epithelium was found to be I and that of endothelium between 0.6-1. . k u I an d Ro b.mso n60 mvesll . . gate d cornea I Ropnasa permse lectivity by measuring membrane electrokinetic potentials generated either by ionic concentration gradi ent (diffusion potential ) or hydrostati c pressure gradient (streamin g potenti al) . Studies indicated dual-selective character to passage of ions across the cornea. The magnitude and polarity of th e selectivity are control led by the degree~ of protonation of ioni zab le si tes within the cornea. The cornea possesses an isoelectric poi nt of 3.2. At physiologic pH and pH above the isoelectri c point, the corn ea behaves as a negatively charged membrane and allows preferential passage of positive ion s in compari son to negative ions. Below isoelectric point, th e reverse is tru e. A study of th e in vitro flux of lys ine (MW 146, positively charged) and glutamic acid (MW 147 , negatively charged) indicated an app roximately two to three-fo ld difference in the permeabil ity of the two 61 . be1ng . compound s w1t. I1 Iys 1ne more absorpt .tvc . Lo werin g the ionic st rength of the bathing solution

MALHOTR A & M AJUMDAR: PERM EATION THRO UGH COR NEA

caused an increase in membrane effecti ve charge density due to lesser degree of electrostati c shielding which resulted in a significant increase in both membrane resistance and selectivity60 . Due to its dua l capability to termin ate the pharmacological acti viti es of inherentl y ac ti ve dru gs and to transform inact ive dru gs to th eir ac ti ve moieti es, dru g metaboli sm in the eye is an important as pect of dru g acti on. Drugs th at are degraded by oxidati on or reducti on are less likely to be metaboli zed in th e eye than those th at are degraded by hydrolys is62 . Corneal epit helium and th e iris-cilli ary body are metabolica ll y acti ve due to presence of esterases 6 ', aminopepti· dases M and ketone reductase M· . Drugs th at co nta1n ester linkage are likely to be hydrolysed by th e esterases. After the topical applicati on of pilocarpine, to the rabbit eye, when the mioti c effect is at its peak, up to 50% of the drug is hydrol ysed to pil ocarpi c acid62 by the esterases present in the intercellul ar spaces of cornea and aqu eous humor. Esterases play a very im. the act1.vat10n . o f' ester prodru gs 66 . s usportant ro1e 111 ceptibility of th ese prodrugs to esterase-medi ated hydro lys is is an important fac tor th at affects not onl y the onset of dru g acti on but also the ex tent of cornea l penetration . The lipid so luble dru g, dipi vefrin , is transported fas ter ac ross the cornea th an the parent molecul e, epinephrine. Subsequent to the transport, the inacti ve prodrug is presumab ly hydro lysed to acti ve epinephrine in the anteri or chamber by esterases67 .

B. Physicochemical factors Phys icochem ical fac tors are the major determ inants to pass ive di ffu sion across the cornea. I . Partition coefficient Partit ion coefficient (between octanol/water) is a parameter for qui ck assessing of penetration potentials of drugs into different biological membranes. Corwin Hansch desc ri bed I .mear68 or para bo ,.1c69 re Iat1.ons h.1p between dru g e f'fect ivencss an d the part iti on coefficient (lipoph ilic character) of th e drug. A parabo lic graph identi fies an optimal partiti on coefficient at its apex . Partiti on coefficient-penetra bility correlati on is helpful in th e des ign of optimall y permeable ophthalmi c drugs. Schoenwa ld and Ward 51 determined rates of permeabi lity across exc ised rabbi t corneas fo r I I steroids and a plot of permeabi lity vs. log part ition coefficient resulted in a parabol ic relati onshi p with optimal permeability at log partition coefficient of 2.9. Narurka r and Mitra70 observed parabolic relationsh ip between

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partitiOn coeffi cient and cornea l permeability of 5' aliphatic esters of 5 iodo-2' -deoxy uridine. In vitro corneal permeability was optimum at log part ition coeffici ent of 0.88 . Likewi se, Mosher and Mikkelson71 determined the i11. vit ro corneal transport of nalkyl-p-aminobenzoate ester homologues and observed a parabolic relati onship with an optimal permeability at a log partiti on coeffi cient of 2.5-2.6. Similar parabolic relation ship between corneal permeability and partiti on coe fficient has bee n reported for ~ -bl ocker72 whereas Wang et a/. 43 desc ri bed a sigmoidal relati onship fo r the same. However the guidin g criteri a for des ign of prodru gs fo r enhanced corneal permeati on have been the parabo li c re lat ion. 66 s(11p. In any study, a measure of the co rnea l penetration efficiency of drugs is th e partit ion coefficient. The percentage contributi on of each cornea l barri er layer again st transport of ~-bl oc kin g agents and a few other drugs across exci sed rabbit co rnea has been stud ied56·73. The results show th at for hydrophil ic drugs (log partition coeffi cient < 0), th e epithelium provides a large percentage of the res istance to corn eal penetration. For lipophilic dru gs with log partiti on coefficient between 1.6-2.5, th e stroma co ntributes a significant percentage of the res istance. And fo r drugs with log partiti on coeffi cients betwee n 0- 1.6, the sum of the stromal and end othelial res istances equals th e epitheli al res istance. Ki shid a74 observed in vit ro relati onship between transcorn eal permeab ility of drugs and their phys icochemical properti es and fo un d that the transcorn eal permea bilities of hyd rophobic and biphas ic solu ble substances were dependent on their lipid solubility. Thu s an optimal lipophilic/hyd roph ilic balance in the molecul ar structure of the penetrant must be ac hieved to affect rapid penetration through the lipophilic and hydrophili c barri ers of th e co rn ea. 2. Solubility- The maxi mum penetration rate attainable by a dru g permeatin g the co rnea is a multiplicati ve factor of permeability coefficient and tear solubility. If a dru g is poo rl y so lu ble, its concentrati on in the precornea l tear fil m may be li mited and therefore its rate of absorpt ion may not be hi gh enough to achieve adeq uate concent rati on for th erapeuti c ac ti vity and reverse is also tru e. 3. Io nization constant - The pKa of ionizab le drugs is an important factor in corneal penetration . The degree of ionization influences the ex tent of dif-

INDIAN J EX P BIOL, JAN UAR Y 200 1

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fusion across a me mbran e. M any dru gs are weak acids o r weak bases and the refore are partial ly ioni zed at physiological pH . Th e ave rage pH of th e tears is 7.2 and the pKa of the drug if within I o r 2 unit s o f this va lue, the co rn ea l pe netration would be more because maj o r propo rti o n of the ad mini ste red dose would be in the unionized form . The ionized form o f the dru g is minima ll y lipid soluble and if thi s frac ti o n is too large, th e rate of cornea l penetration may not be suffi cient to produce th e ra peuti c leve ls of the dru g in the eye . Henderson -Hasse lbalc h equat ions re late th e degree of ioni zat ion of a weak acid or weak base to pH of th e medium and pKa of the drug as fo ll ows: p Ka- pH= log

1_ (for an acidic drug ) f;

pKa- pH= log

_!j_ ( for a basic drug) fll

From these equations o ne can determine th e amoun t of uni o nized drug availabl e for transcorneal moveme nt. The g reater the fraction of drug present in the unionized form , the greater is the extent of pas75 sive diffusion. Chyi-Fu et a/. proved the assu mpti on that o nly uni o ni zed drugs cross the me mbrane inco rrect. The ir studies showed that ketoro lac (an anion) could pe rmeate throug h exci sed rabb it co rn ea both in unionized and ionized form s. Permeati on studi es using exc ised goat corn ea also s howed si mil ar res ult s 77 76 for ketoro lac , ibupro fe n and flurbiprofe n . 4. Molecular weight - Mol ec ular we ight is re lated to th e diffu sio na l forces active durin g co rnea l pe rmeation. For sma ll molecul es, th e diffusion coefficients a re in versely re lated to th e square roots of the ir molecu lar weight, while for la rge mo lec ul es, th e diffus ion coeffic ie nts in water are in ve rse ly re lated to the cube roots of the ir mo lec ular we ight s. The changes in mo lecular we ig ht s how an inve rse re lationship to pe rmeabi lit y. Compo und s wit h a mo lec ular weight greate r th an 500 Da offe r poor corneal penetration because pass ive diffu sio n no longer rema in s the predominant mode of dru g transfe r. Compounds of mo lec ul ar weights less th an 500 Da generally diffuse through both biol og ica l and sy nthe ti c membranesn. Molecular we ig ht is a less criti ca l factor s ince most ophth a lmi c dru gs have a re latively small a nd narrow range of mo lec ul ar weight. Exce ptions to thi s may be the hi g he r mo lec ul a r we ig ht antibiotics such as bac itrac in (MW 14 11 ), co li st imethate sod ium (M W 1250), co li stin s ulphate (MW 1250), and polymixin (M W 1200) wh ic h pe ne trate the co r-

nea of di sease mode ls but no t those of no rmal rabb it 4 eyes . Th e tran sco rneal pe rmeabilities of hyd rophobic su bstances a nd biphasic-so lubl e s ubstances are not governed signifi cant ly by the ir molec ul a r wei g ht but by the ir li pid so lubilit /~. while th at of hydro phili c subs tances is gove rned by the ir mol ec ular weight 7 '>. Liaw et a /. 80 meas ured th e tran s port c ha racte ri stics of series of diffe rent mo lec ul ar we ig ht polyeth yle ne g lycols (PEG) and found that corn ea l absorpti o n of PEG 's showed a de pe nde nce on molecu lar weight with cut-o ff between PE G 400 and PEG 600.

C. Formulation factors I . Concentration - Cornea l penetrati o n e nh a ncement ca n be achi eved by increasing the solut ion concentrati o n of a dru g, resulting in improved th e rapy. 50 Davi s et a/. studied the e ffect of in creas in g concentration of tobramycin on Pseudomonas kera riris. The resu lts showed that a I 0-fold reduct io n in number of corneal co lo ni es could be ac hi eved by inc reasing the concen trati o n of topica ll y applied to bramycin from 4 to 40 %. T he driv in g force be hin d this e nh ancement is the co nce ntrat io n g rad ie nt de1 sc ribed by Fick's first law of diffusion ~ • However in c reas in g the app li eJ concentratio n to overcome poo r c lini ca l effi cacy wo rk s o nl y till the drug has reached a pl ateau of its doze-effect c urve. Dra nce and 82 Nash studied th e effec t of in still ations of increasing co nce ntrati o n ( I, 2, 4 and 8 %) of p iloca rpine hyd roch loride on intraocular pressure. Maximum red ucti on in pressure was ob tained w ith 4% so lution . Th e 8 % so luti on th oug h showed the inc rease in duration , but not an increase in press ure reduction. Also, inc reas in g concentrati o n may res ult in hype rto ni c so lution s, wh ic h are po te nti a ll y unco mfortabl e and can induce increased lacrimat io n which ca n accel e rate the dra inage rate and red uce pe rce nt ab8' sorption . Rame r and Gassett ·' showed that th e ex te nt of ocul ar absorption fo r 10% (hype rto ni c) pilocarpine hydrochl o ride was on ly 3-fold greater th an th at of the 2 % so luti o n. In a s imil a r study , Asse ff et al. R~ showed th at in comparison with I % pilocarpine hydrochloride, a 2-fold inc rease in corn ea l penetration occurred wit h 4 % drops, w he reas only a 2.5-fold in crease was produced with a n 8 % (hypertonic) preparati o n. 2. Particle shape, size and dissolution rate-S uspe nsion s a re wide ly used in oc ul ar dru g therapy to admini ste r s pa rin g ly so lubl e drugs o r complexes of so lubl e dru gs o r to obtain a s low di sso luti o n and a

MALHOTRA & MAJUMDAR: PERMEATION THROUGH CORNEA

prol onged release of the dru g. Ophthalmic suspensions are generally gentl y deposited in the cul-de-sac. The drug particles deposited on the eye surface are mo ved by the eyelid s at eac h blink , whi ch cou ld cause an abrasion of the outer ep ithelium laye rs85. These may al so cause irritation of sensory nerves in the epithelium 86 . The irritation cou ld elicit refl ex blinks and reflex lacrimation. Co ncentrati on, shape and particle-size together interact to determine the irritat ion potential of the suspended partic les. Forms with sharp an gles and edges are more irritant than isometric particles with obtuse angles and edges. Commercial suspensions are formulated to contain nearly spherical particles less than I 0 11m in size. Increase in dru g particle size influences bioavailability inversel / 7 . Three suspension s of 0.1 % triti ated dexamethasone were evaluated with varying mea n particle size of 5.75, 11.5 and 22.0 Jlm . As the particle size increased, the in vivo disso lution rate decreased to th e poi nt that the part icles we re removed from the conjuncti va l sac before dissolution was complete . Both the rate and extent of dexamethasone penetration into th e anterior chamber of th e rabbit eye decreased . In another stud y, wit h fluorometholone 8R (a sparingly so lubl e co mpou nd ), it was fou nd th at the use of a higher concentration of equivalent particle-size did not improve the aqueous humor drug concentrationtime profile and absorption of the drug was regu lated by its inheren t dissolution propert y. The dissolution rate of the dru g is an important parameter whi ch determines the amount of dru g actually in solution and thus ava ilable for transport through the cornea during the short res idence time in th e eye 89 . Although the range of particle-size that could be used for ocul ar suspension s is limited, it is well known that a decrease in particle-size leads to an increased surface area and thus an increase in di sso luti on rate. Though the surface specifi c dissolution rate of a sparingly soluble dru g increases with a decreasing parti clesize90, the parti cle-s ize has no significant influence on the ocular permeati on and that permea bility rate rather th an the dissolution rate represe nts the rate'JJ . .. 1tmttmg step . 3. pH and tonic ity- The pH for human tears ranges betwee n 7. 14-7.28. Tears possess a relatively weak buffer capac it y of approximately 3.6x I o·5 (refs 92, 93) . The osmolalit y of th e lac rimal fluid is mainl y dependent on the number of di ssolved ions and crys-

17

talloids . During sleep, the osmolality of the tears vari es between 280-293 mOsm/kg and when eyes are open during day, it varies between 302-318 m0sm/kg94 . On instillation of a hypotonic solut io n, the permeability of the epithelium is increased considerabl y and reverse is true for hypertoni c solution 95 . The instillati on of a hypotoni c drug soluti on creates an os moti c gradi ent between the tear film and the surrounding ti ssues. The corneal epithelium is ex tremely tolerant to large variati ons in pH and toni ci ty96 . The sod ium permeability of the epi thelium is unchanged from pH 4- 10 (ref. 63) . Outside thi s ran ge, epithelial permeability increases especia ll y when bathed with more alkaline so lution s. Depending on th e drop size, soluti ons with an osmo lality lower than I 00 or 266 mOs m/kg and higher than 480 or 640 mOsm/kg are irritant 97 . Ramselaar et a!.'JX found that human corneal fluorescein permeab ility was unaffected in a pH ran ge from 4.5 to 7 and tonicity ran gi ng from isotonic (270 mOsm) to hypertonic (620 mOsm). Conrad et a/. 99 repo rted that alka line pH induced greater lacrimati on than ac idi c pH in the albino rabbit. Thi s is co nsistent with th e lower buffer capac ity of tears in the basic . tI1e act.d.tc range '(){}·101 . s·mce tears are poor Iy t han Ill buffe red 100 , induced lacr imation can be minimi zed by red ucin g the tonicity of th e soluti on. Hind and Goyan 102 disc ussed in qualitative terms, sign ificant aspec ts of the use of buffers in ophthalmic solutions and repo rted buffer co ncentration or buffer capacity (index) as an important factor in formulation of op hth almic so lutions containing ioni zable dru gs. The concentration of the buffer in an instilled so lu tion upon mixing wi th precornea l fluid di ctates the time course of pH, dru g ioni zation and therefore drug abso rption . By progressively reduc ing the bu ffe r concentration of a pH 4.5 c itrate buffe r from 0.11 to 0 M, Mitra and Mikke lson 103 observed a 5-fold increase in the ocular bioavailability of pilocarpine. Similar results were reported by Ahmed and Patton 104 for ocular penetrat ion and prccorneal di sposition of pilocarpine fro m so lutions contai nin g different phosphate bu ffer concentrations at pH 4.5. Seig and Robin so n88 · 105 studi ed the effect of vehicle p H on the absorpti on of pilocarpine and reported lower absorption with decrease in pH due to pH-induced lacri mati on which increased as the pH of !he instilled soluti on was adj usted to va lues away from physiological pH. How')') . ever, e onrad et a I . suggested that the mec hamsm res ponsible for the decreased ocular availability of pilocarpine from in st illed so luti ons of lower pH was

18

INDIAN J EXP BIOL, JANUARY 2001

due to both pH-partition effect and pH induced lacr imati on, the magnitude of which depends not necessarily on the pH of an instilled so lution, but on the co ncentration of the buffer contained in an instilled solution. In addition to buffer concentration, the buffer type used also affects the absorption efficiency of topically applied dru gs . Phos phate buffer has a hi gh res idual buffer capac ity and yie lds a lower bioava ilability of pilocarpine than does an acetate buffer 101 . Bioavailability from soluti ons containing no buffer was max imum and th ose containing citrate buffer was minimum whi le acetate and phosphate d"1ate response 106 . p retreatment o f t he eye . gave mterme with a steril e drop of isotonic buffer (pH 9.2), totemporarily alter the tear pH reduces the necessary dose approximately I 0-fold for tropi camide, homatropine hyclrobromide, phenylephrine hydrochloride and cyc1opento Iate I1yd roc I1Ion"d e 107-109 . The corn eal end othelium is far more sensitive to changes in both pH and tonicity . The optimum end otheli al pH is 7.4 to 7.5. Be low pH 6.7 and above pH 8.5, the electrical potential difference ac ross th e endothelium reaches zero 10 and both structural and functional alterations occur to the endoth elia 110 . The end othelium can withstand vari ati ons in the toni city from 200-400 m0sm 111. 4. Viscosity- It is generall y beli eved that inclusion of a visco li zing agent in an ophthalmic so luti on will increase oc ul ar bi oavailability of the dru g du e to prolonged res idence time of an inst illed dose in th e conj un cti val sac. The more commonly used viscolizing agents include wate r soluble polymers like polyvin yl alcohol (PV A), polyvinylpyrrolidone (PVP) and cellul osic polymers. Methylcellulose was first used in op hth almol ogy to increase the viscosity of aqueous op hthalmic solu tions 112 . In 1965, PYA was introdu ced to op hth almology 11.1 and it was proved that PV A does not blur vision 114 . In 1968, studies on hydroxypropyl methylcellulose (HPMC), a derivative of methylcellulose, showed it to be a superior visco li zing agent than PV A 115 . Chrai and Robin son 11 6 repo rted th at the rate of so luti on drai nage decreased with increas ing viscosity and over a range of 1-15 cps viscosity, 3fold change in th e drainage rate constan t was obtained . A further 3-fold change over th e viscosity range of 15-100 cps was also observed . The dru g bi oavailabil ity however, was not proportional to contact time. Even with as much as I 00-fold increase in visL-Osity an d a I0-fold decrease in drainage rate, the maximum improvement in drug activity, be it mio-

. I 17.118 . h"b" . . fect1.on I 19 or aqueous Immor SIS , 111 1 1t1on o f 111 116 levels is about twice that of an aqueous soluti on. The decline in preco rneal drug co ncentration is of the first order and rate of dec I ine is proportional to the viscosi ty of the instill ed soluti on. A linear relati onship between the first order drainage rate constant and both the miotic activity and aqueous humor dru g levels was obtained over a ran ge of 1- 15 cps solution viscosity in studi es conducted by Chra i and Robinso n1 16. The optimum viscos ity to use is in th e ran ge of I 2-1 5 cps 120 beyond which the gain in oc ul ar absorption would be minimal , while the ri sk of inaccuracy of instill ati on and blurring of vi sion would increase121. Even with a 100-fold increase in viscosity, the ga in in ocul ar drug absorption is modest, being less fo r oi l-soluble than for water- solu ble dru gs 122 . A large number of dru g studi es in humans and animals ha ve been co ndu cted with the use of said polymer vehicles and th e results have been ex pressed . terms 0 f myd naSIS . . Or mi.OS IS . I I 7. I I 8. I 2.\ . I 24 , IIlli . I .b. . Ill 1t1 011 · f · I I 9 I 25 · d · I 26- I 29 o f 111 ect1on · , mcrease co ntact ti me , aqueous 11umor Ieve Is o f d ru g J>o-J'' · · · and .mtraocu Iar pres. . sure J\4 · · 115 · · . S tu d.1es companng tI1e vanous po Iymer vehicles have attempted to emphasize the superi ority of one vehicle over the other. The instillation of water-soluble polymers either chan ge the physiological processes, such as drainage or alters the physicochemical parameters th at govern th e tear film stability. Some viscos ity enhan cers, exhibit surface acti vity, hence interact with the lacrimal film and alter the spreadin g characterist ics of the precorneal tear film, the break-up time, the blinking rate and co nsequent ly th e eliminat ion of the drug instil led. In genera l, these polymer ve hi c les impart a slight lowerin g of surface tension and an increase in viscosity to the tear film 1.1 6. A lowering of surface tension improves drug mixing with the tear fil m and its subsequent penetration 1.\7. Benedetto et a!. 1.16 demonstrated that retention of drug in the preco rneal tea:· film was not strictl y because of viscosity of the vehicle or its su rface wetti ng properti es but because of surface spread ing characteristi cs and wate r draggin g capac ity of the visco li zi ng age nt. Ludwig and Van OoteghemJ.18 reported that vehic les of simi lar viscosity but different surface tension showed no signi fican t differences of AUC values and that ph y.-icochemical characteristics as well as co ncentration of the polymer used affected ocu lar retention 1w_ When a viscous po lymer solution i~ instilled into the cu l-de-sac, seve1al processes reduce its viscosity.

MALH OTR A & M AJUMDAR: PERM EATION THRO UGH COR NEA

Firstl y, the visco us soluti on is diluted by res ident tears and subsequentl y by incomin g tears. Secondl y, the viscous so luti on undergoes shear thinnin g durin g blinking, th ereby increasing contact area and fac ilitating mixing. A compari son of th e redu cti on in so luti on drain age rate by me~hy l cellul ose and polyv in yl alcohol and th e resultin g increase in aqueous humor . . p1.,ocarpme concentrati.on .m tI1e a lb 1no ra bb It. 116·1?1- , suggest that it is the fl ow properti es of the vehi cle in questi on and its viscosity, not the co ncentrati on, th at determines th e effect of polymers on soluti on drainage and oc ul ar drug abso rpti on. In oth er wo rd s, the rheological characteristi cs of a po lymer are important fo r the retention pattern on the oc ul ar surface. Saettone et al. 140 studi ed th e influence of different polymers on the mydri ati c response of tropicamide. They fo und that time profil es of the mydriati c responses indicated a preference fo r pseud opl asti c systems rather th an New toni an systems. Saettone et a /. 140·141 also showed that all polymers yielding same vi scos it y did not effect oc ul ar dru g absorpti on to same ex tent. These in vestigators demonstrated th at equi viscous solutions of CMC, HPMC, PV A and PVP enh ance the ocular abso rpti on of pilocarpine as we ll as tropicamide to different extents in humans. The different acti vi ty of these polymers was att ributed to their influence on spreadin g charac teri sti cs and the thickn ess of th e medi cati on layer on th e precorneal area. NonNewtoni an fluid vehi cles un dergo rh eological changes (thinnin g) when ex posed to shear stress during blinking 142 while Newtoni an vehicles do not. Dilatant fluid vehi cles thi cken with shear while pl asti c flu id vehi cles thin as with pseudop lasti c once the yield value is exceeded 121 . 143 Saettone et a!. showed th at soluble mucoadhesive polyanion ic po lymers, li ke hya luroni c ac id, polygalacturonic acid, mesoglycan, carboxy meth ylchitin and polyac rylic acid, enh anced the ocul ar abso rpti on of pi locarpine more than PV A of eq ui va len t viscosi ty. Si milar favo urab le effects with hya luronic ac id over HPMC and polyac rylic ac id (carbopo l 934 P) over PY A have been reported 144 ' 145 . Des hpande and Shirolkar 146 reported hyd rogels prepared wit h carbopo l 940 to be better than those prepared wi th sodi um CMC and HPMC. Rozier et a!. 147 observed an increased contact time and ocular bioavailabil it y of timc lol from gel rite (ion activated, in situ ge lli ng polymer) formulat ion as compared to formulation containing an eq uiviscous solution of hydroxycthyl cellulose.

19

On the positive side, increas ing viscosity pro longs contact time and th erefore promotes absorpti on. On the negati ve side; it can slow diffusion of dru g in solution and can create mi xing problems of the drug solution with tears. In addition , the polymer may adsorb onto absorpti ve surfaces, creatin g a barri er fo r drug penetration . Further, increas ing di scomfort due to increasing viscosity may induce lacrimati on. Hence optimum selection of viscolizing agent and vi scosity of so lution is necessary for optimum corneal penetrati on.

Penetration enhancers The bi oavail ability of topicall y appli ed ophthalmic dru gs is usuall y very low (< I 0%) (Ref. 27). Attempts have been made to improve ocul ar bioavail abili ty of dru gs through the use of penetrati on enh ancers such as actin cytoskeleton inhib itors, surfactants. bile salts, chelators, preservati ves and ion-pairing salts. Actin cytoskeleton inhibitors - Actin cytoskeleto n inhi bitors like Cytochalas in B, act by disrupti on of actin microfil aments at ti ght juncti ons of corneal epithelium resultin g in increased permeabili ty at these . . . B .mcreases Ill . vuro . JUncti ons 148·149 . Cytoc I1a Ias 1n transco rneal permeati on of PEG 400 and PEG 600 and appears to cause minimum cell membrane damageso_ Surfactants - Benzalkonium chl oride, a cationi c surfac tant, is often added to aqu eo us op hthalmic preparati ons as an anti -bacterial preservati ve in co ncentrati on varying from 0.004 to 0.02%. Benzalkonium chl oride has been fou nd to increase the corneal penetrati on of a number of dru gs. For example, increased co rneal permea bility due to benzalkonium chl oride has been reported for pilocarpi ne 150 , prednisolone130, chl orampheni co l131 , dexameth asone 150, ke. ?'i·, 1' buprof en77 , fl urb'1pro fen 77 , toro 1ac tromet hamme 1 51 timolol , cyc lopentolate 151 ' atenolol 152 , betaxalo l152 . 1s1·. Electron microsco . . f.111 d.mgs 1s1 and fluorescem pic · .. 154 showed th at cati oni c surfac tants prod uce breakdown in the cohes ion of the epi the lium by increasing the width of the interce llul ar space. They also produce so me dis ruption of the cell cytop lasm presumably by their actions on the plasma cell membranes . The cati oni c su rfac tants prod uce an increase in the width of the intercell ular space of the superficial epi thelial layers and also cause disruption of the superficial cells indicating that they effect not only the ce ll membranes but also the intercellular route and hence resulting in increased permeab ilit y. Topically

20

I DIAN J EXP BIOL, JANUARY 2001

applied benzalkonium chloride (0.005 M) , also, produces a loosenin g in adhesion between the corn ea l epithelium and stroma 153 . Fu and Lidgate 75 reported th at increased cornea l permea bilit y of ketorolac, an anioni c dru g, in presence of benza lkonium chloride was not only because of di srupti on of th e ep itheli al membrane but also because of fo rmati on of more lipid soluble ion-pair between ketorolac and benza lkonium chloride. It has been proposed 41 '155 that competitive inhibiti on of dru g-prote in binding by ioni c surfactants, also may be partiall y responsible for th e increased corneal penetration. In vitro eq uilibrium dialysis studies conducted with pilocarpine nitrate, sulfi soxazo le and meth ylpred ni so lone showed that binding of these dru gs to protein components of human tears is inh ibited by ionic surfactants. Smolen et a /. 156·157 hypothesized that benzalkonium chl oride enhanced the ophthalmic bi oavai lab ility of carbachol by inducing th e release of bound dru g from binding sites on th e corneal surface. Sodium Iaury! sulphate, an anionic surfactant, also en I1ances co rnea I permea bt., .tty l ·'i1· ·ISS · . Stu d'tes on most surfactants show a permeability en hance ment of the cornea with non-i oni c, cationic and anionic surfactantsl53, 159. Marsh and Maurice 15 Y determined th e effect of non-ion ic surfac tants on the cornea l penet rati on of fluorescein in human subjects. Spans and Tweens were mixed to ac hi eve hydrophilic-lipophilic balance (HLB) va lues ranging from 2- 18. The res ults showed th at single instill ation of non-i oni c surfac tant with a HLB ran ge of 16-17 increased fluorescein permeation . Tween20 with a HLB value of 16.7 showed th e greatest effec t. While most surfacta nts followed a pattern of increasing permeability when thi s HLB value was achieved , some surfactants did not follow thi s general rul e suggesting th at chemical spec ificity 160 found th at also pl ay a ro le. Tani guchi et a/. Tween80 increased th e rate of dexamethasone penetration across the iso lated rabb it corn ea. Saettone et a/. 151 studi ed the effect of different permeation enhan cers e.g. polyoxyethylene glyco l Iaury! ether (Brij35), polyoxyethylene glyco l stearyl ether (B rij78), polyoxyethylene glycol oley l ether (Brij 98), heteroglycosides (saponin and digitonin) and benza lkonium chloride on in vitro transcorneal permeation of rimolol , levobun olol, and cyc lopcntolate. Among all the Brij, Brij7 8 showed the max imum permeati on of timolol with minimum corneal damage. Brij78 al so increased permeation rates of other two drugs but to a

lesser extent. Digitonin , sapon in and benzalkonium chl oride increased permeation rates of timolol but caused substantial cornea l damage. Studi es co nducted earli er indicate that digitonin 80.149 significantl y increases co rneal permeability but may al so cause severe corneal damage. Recently Saettone et a /. 152 have reported 0.05 % w/w co ncentration of polyoxyet hylene alkyl ethers (Brij 35, Brij 78) as sa fe and effecti ve permeation enhan cers for ~-bl oc k e r s like atenolol and timolol. The study confirms th e corneal damaging effect of saponin (0.0 15 % w/w), di gi tonin (0.005 % w/w) and benzalkonium chl oride ( 0 .02cro wlw) . Surfactants are known to impair co rnea l wound healing 158 and increased opaci ty of iso lated bovine cornea is related to anionic and non-ion ic surfac tants but not to cati oni c surfactants 161. Bile sa lts - Bil e salts ha ve been re ported to enhance th e permeabili ty of poorly absorbed dru gs through the small intestin e, rectum and oth er mu cosa l membranes 162"16 ·'. Morimoto et a/. 164 studi ed the promoting effects of th e trih ydroxy bile salt, sodium taurochol ate (TC-Na) and the dih yd rox y bile salt , sodium taurodeoxycholate (TDC-Na) on in vitro co rnea l pe rmeability of hydrophilic compounds and macromolecul ar compounds. The results showed th at TC-Na and TDC-Na increased corn eal permeability of these compounds. The magnitude of the enh ancement by TDC- a was greater than that by TC-Na. The diffe rences in the physicochemica l properti es of bile sa lts like solubi li zing activity, lipoph ili city and calcium ion sequestrati on capac ity, relates to thei r permeability enh ancing effects. The criti ca l mice ll ar concentrati on (CMC) of dih ydroxy bile salts is genera ll y lower and their aggregation numbers larger than th ose of trih ydroxy salts 164 . Thus th e so lub ilizing activ ity of TDC-Na is hi gher than th at of TC-Na. The lipophilicity and calcium ion sequestrati on act ivity of TDC-Na are also hi gher than th at of TC-Na 163 . These higher ph ys icochemica l activities of TDC-Na cause loosening of th e ti ght junctions of cornea l ep itheli al ba rri ers resultin g in increased permeability. A recen t stud y ind icates 0.05 % (w/w) co ncentrati on of bile salts (sod ium taurodeoxycholate and sod ium ursodeoxycholate) as safe and effective ocu lar permeation enhancers for ~­ bl ockers like atenolol and timolol m . Chelators-Di sodium edetate (E DTA) is a chelating age nt that binds divalent cati ons such as calcium and magnesium. As hton 165 showed that EDT A in creased rabbit corneal epithelial permeab ility to sor-

MALHOTRA & MAJUMDAR : PERMEATION THROUGH CORNEA

bitol. Likewise rabbit corneal permeability in vivo to polar compounds was also increased in presence of 0.5% EDTA 166 . In epithelia, calcium maintains the 167 and is therefore, an essential intercellular matrix factor in determining the size of potential paracellular routes for drug transport. EDTA binds to calcium present in the tight junctions of epithelia. This results in a decrease in calcium concentration in these junctions and thus decreasing the transepithelial resistance to water soluble compounds. EDTA may also cause se149 vere corneal damage . Preservatives- Commonly used preservatives in ophthalmic solutions and suspensions are benzalkonium chloride, organomercurials and chlorbutanol. Permeation enhancement effect of benzalkonium chloride has already been discussed. The major disadvantage of the three organomercurials (phenyl mercuric acetate, phenyl mercuric nitrate or thiomersal) are their tendency to deposit mercury in corneal tissues168 and hypersensitivity that is incurred with these 169 agents . Organomercurials react with the membrane sulfhydryl groups and alter membrane permeability 170 and transport systems . Even low concentration of 0.001-0.005 % (w/v) commonly used in ophthalmic 170 171 preparation causes functional changes - . Thiomersal has only a small effect on corneal permeability and does not greatly influence drug penetration at normal concentrations. Chlorbutanol reduces oxygen utilization in the cornea that results in loosened epithelial adhesions 172 . Chlorbutanol when used at concentrations normally found in ophthalmic solution increases corneal 150 173 epithelial permeability to drugs · . Ion pairing salts-Transport of ionized molecules across natural membranes can be greatly facilitated by provision of a suitable counter ion. This enhancement is the result of an association of oppositely charged species giving rise to an ion pair. Since ion pairs possess no net charge they are far more lipid soluble than the constituent ions and hence better able 74 to permeate through membranes . Neubere has summarized the enhancement in transport rates of some common drugs across natural and artificial membranes in presence of ion pairing salts. Ion pair formation between anti-inflammatory agent chromoglycate and a quaternary ammonium compound has been found to increase the corneal transport of the 175 drug . Ion pairing with m-chlorobenzyltrimethylphosphonium chloride increases in vivo cor176 neal uptake of chloramphenicol succinate • Simi-

21

75 larly, increased permeation (in vitro) of ketorolac by ion pairing with quaternary ammonium compound (dodecyltrimethylammonium bromide) has been observed . Enhanced permeability of benzolamide (sulfonamide) in presence of ion pairing salts e.g. tetra-

phenylphosphonium chloride, tetraphenylarsonium chloride, and trimethylphenylammonium chloride 177 through excised rabbit cornea has been reported . Ion pairing salts thus offer means of enhancing transcorneal permeability of ionized drugs provided either the ion pairing salt or the resultant ion pair does not damage the cornea. Thus most of the penetration enhancers while promoting corneal permeation of drugs may also damage the cornea. Recent studies however, indicate nonionic surfactants like Brij, surface-active bile salts (e.g. TDC-Na) and cytoskeletal modulator (cytochalasin B) as promising agents. However, further studies in vivo are needed to know the practical applicability of these penetration enhancers.

Acknowledgement Authors are thankful to CSIR, New Delhi for Senior research fellowship to Manjusha Malhotra .

References I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16

17 18 19 20 21

Denissenko, Klin Mbl Augenheilk, 20 ( 1882) 299. Maurice D M, Int. Ophthalmol Clin, 20 (1980) 7. Bachman W G & Wil son G , In vest Ophthalmol Vis Sci, 26 (1985) 1484. BensonH,ArchOphthalmo/,91 (1974)313. Swan K & WhiteN, Am J Ophthalmol, 25 ( 1942) I 043 . Klyce S D, J Physiol, 226 ( 1972) 407 . Green K, Am J Ophthalmol, 67 ( 1969) II 0. Maurice D M & Watson P G , Exp.Eye Res, 4 ( 1965) 355. Green K, Laughter L & Hull D S, Curr Eye Res, 2 ( 1983) 797. Fishbarg 1 & Lim 11, J Physiol, 241 (1974) 647 . Hodson S & Miller F, J Physiol, 263 ( 1976) 563. Huff 1 W & Green K, Exp.Eye Res, 36 (1983) 607 . Mishima S & Trenberth S M, In vest Ophthalmol, 7 ( 1968) 34. Kim J H, Green K, Martinez M & Paton D, Exp.Eye Res, 12 ( 1971) 231. Hull D S, Green K, Boyd M & Wynn H R, Invest Ophthalmol Vis Sci, 16 (1977) 883 . Liaw 1 & Robinson 1 R, in Ophthalmic drug delivet)' systems, edited by A K Mitra (Marcel Dekker, Inc., New York, Basel, Hongkong), 1993 , 372. Grass G M & Robinson 1 R, J ?harm Sci, 77 ( I), ( 1988) 3. Grass G M & Robinson J R, J ?harm Sci, 77 ( I), ( 1988) 15. Lee V H L, J Controlled Rei, II (1990) 79. Klyce S D & Crosson C E, Curr Eye Res, 4 (1985) 323. Tonjum AM, Acta Ophthalmol, 52 ( 1975) 650 .

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I DIAN J EXP BIOL, JANUARY 200 1

Mi shima S, ln vesl Ophlhalmol Vis Sci, 2 1 ( 198 1) 504. Slovin E M & Robi nson J R, in Biopharmaceulics of ocula r drug deli very, edited by P Edman, (CRC Press, Londo n), 1993, 147 . Mauri ce D M, Oplllhalmol Lil, 7 ( 1953) 3. Seig J W & Rob inson J R, J ?ha rm Sci, 65 ( 12), ( 1976) 18 16. Hu ang A J W, Tseng S C G & Kenyo n K R, lnvesl OphIhalmol Vis Sci, 30 ( 1989) 684. Burstein N I & Anderso n J A, .I Ocula r Pha nnaco l, ( 1985) 309. Lee V H L & Robi nson J R, J Pha rm Sci, 68 ( 1979) 673. Ch rai S S, Patton T F, Meht a A & Robinson J R, J Phw m Sci , 62{7),( 1973) 111 2. Patt on T F, Francoeur M, Am J Opl11halm ol, 85 ( 1978) 225. Brown R H, Wood T S, Lynch M G, Schoenwald R D & Chein D S, Ophlhalmology, 94 ( 1987) 847 . Lynch M G, Brown R H, Goode S M, Schoenwald R D & Chcin D S, A rch Ophlhalmol, l 05 ( 1987) 1364. Hendri ckson R 0 & Hann a C, Arch Opllllw lmol, 96 ( 1977) 333. Cab le M K, Hendri ckso n R 0 & Han na C, Arch Oplllhalmol, 96 ( 1978) 84 . Brow n C & Hann a C, Am .I Ophlhalmol, 86 ( 1978) 820. Keister J C, Cooper E R, Missel P J, Lang J C & Hage r D F, .I Pharm Sci, 80 ( 199 1) 50. Patto n T F, .I ? harm Sci, 66 ( 1977) I 058. Chrai S S, Makoid M C, Eri ksen S P & Robinso n J R, .I Pharm Sci, 63 ( 1974) 333 . Seig J W & Triplett J W, .I Pharm Sci, 69 ( 1980) 863. Lee V 1-1 L, Takemoto K A & limo to D S, Curr Eye Res ,3 ( 1984) 585. Mikkelso n T J, Chrai S S & Robinso n J R, .I Phwm Sci, 62 ( 1973) 1648. Walsky M A, Jab lonski M M & Edelhauser H F, Curr Eye Res, 7 ( 1988) 483. Wa ng W, Sasaki H, Chein D S & Lee V 1-1 L, Curr E\'e Res, 10 ( 199 1) 57 1. As hton P, Podder S K & Lee V 1-1 L, Pha rm Res, 8 ( 199 1) 11 66 . Chas t F, Bardin C, Sauvagao n-manre H, Callaert S & Chaumeil J C, .I Ph a rm Sci, 80 ( I 0), ( 199 1) 19 1 I . Doane M G, Jensc A D & Dohlman C H, Am J Ophlhalmol, 85 ( 1978) 383 . Ahmed I, Gok halc R D, Shah M V & Patton T F, .I Pharm Sci, 76 (8). ( 1987) 583. Ahmed I & Patton T F, !tzvesl Oplllhalmol Vis Sci, 26 ( 1985 ) 584. Ahmed I & Patton T F, In ! .I Ph an u, 48 ( 1987) 9. Davis S D, S:1riT L D & Hyndiuk R A. Arch Ophlhalm ol 46 ( 1978) 123. Kl ein berg J, Dca F J, Anderson J A & Leopold I 1-1 , A rch Oplllhalmol, 97 ( 1979) 933 . Mau rice D M. in Th e Ere, Vo l. I , 2nd cdn. Edited by H Davson (Academic Press, London , cw York), 1969, 489. Schoenwa ld R D & Ward R L, .I Pharm Sci ,67 ( 1978) 786 . Ma urice D M & Ril ey M V, in BiochemislJT of ih e eye, edited by C N Gray more (Academi c Press, Lond on, New York), 1970, 3. Maurice D M, in The slnlcfll re of Ihe ere, edi ted by G K Smelser (Academic Press, London , New York) , 196 1, 38 1.

56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81

82 83 84 85

86 87 88 89 90 91 92 93 94 95

Huang H S, Schoenwald R D & Lach J L, .I Plw rm Sci, 72 ( 1983) 1272. Kadem 0 & Katchalsky A, Biochem Biophys Acia, 27 ( 1958) 229. Kadem 0 & Katchalsky A, J Cen Physiol, 45 ( 196 1) 143. Mi shima S & Hed bys B 0 , Exp Eye Res, 6 ( 1967) I 0. Rojanasaku l Y & Robinson J R, In! .I Pha rm, 55 ( 1989) 237. Li aw J, Rojanasakul Y & Robinson J R, Pharm Res, 6 ( 1989) 90. Lee V 1-1 L, Hui H W & Robinson J R, lnvesl Ophlhalm ol Vis Sci , 9 ( 1980) 2 10. Lee V H L, Morimito K W & Stratford R E, Biopharm Drug Dispos, 3 ( 1982) 29 1. Dodd a Kas hi S & Lee V H L, lnvesl Opl11halmol Vis Sci ,27 ( 1986) 1300. Lee V 1-1 L, Chein D S & Sasak i 1-1 , J Plw nnacol Exp Th er, 246 ( 1988) 871. Lee V H L & Li V H K, Adv Dmg Deliv Re v, 3 ( 1989) I. Neufeld A H & Page E D, lnvesl Oph lhalm ol Vis Sci, 16 ( 1977), 111 8. Hansch C & Dunn I I I W J, .I Ph arm Sci, 62 ( 1972) I . 1-I ansch C & Clayton M, .I Pham z Sci, 62 ( 1973) I . Narurkar M M & Mi tra A K, Pharm Res, 6 ( 1989) 887. Mosher G L & Mikkelson T J, In! J Ph arm, 2 ( 1979) 239 . Schoenwald R D & Huang 1-1 S, J Pharm Sci, 72 ( 1983) 1266. Schoenwald R D, Clin Pha n nacokin el, 18 ( 1990) 255. Kishida K & Otori T, .lpn J Opluhalmol, 24 ( 1980) 251 . Chyi Fu R C & Lidgatc D M, Drug Dev lnd Pharm, 12 ( 14), ( 1986) 2403. Malhotra M & Maj umdar D K, Indian .I Exp Bioi, 35 ( 1997) 94 1. Gu pta M & Maj umdar D K, In dian J Exp Bioi, 35 ( !997) 844. Weld C B, Feindel W H & Davson J, Am .I Physiol, 137 ( 1942) 42 1. Sh in I S, Kim J 1-1 & Rhee S W, J Korean Op/11halmol Soc, 14 ( 1973) 183 . Li aw J & Robinson J R, In! .I Pharm, 88 ( 1992) 125. Ri ggs D S, in Th e malh emalical approach 10 physiological problems, edited by D S Ri ggs (Wi lliams & Wil kins, Baltimore), 1963, 18 1. Drance S M & Nas h P A, Can .I Op/11/w lm ol, 6 ( 197 1) 9. Ramer R M & Gasset A R, Ann Ophlha/mol,6 ( 1974) 1160. AsscfT C F, Weisman R L, Podos S lVI and Becker B, Am J Oplllhalmol, 75 ( 1973) 2 12. Parrish C M & Chandl er B A, in The Corn ea, edi ted by H E Kaufman, B A Barron, M B McDonald & S R Walt man (Churchill Li vingstone, New York ), 1988, 599. Millodot M, Op /11/w lmol Physiol Op!, 4 ( 1984) 305 . Schoenwald R D & Stewart P, .I Ph a rm Sci, 69 ( 1980)391. Seig J W & Robinson J R, .I ? harm Sci, 64 ( 1975 ) 93 1. Hui 1-1 W & Robinso n J R, .I Pha rm Sci, 75 ( 1986) 280. Bis rat M & 1ystro m C, In! .I Pharm , 47 ( 1988) 223. Bi srat M, Nys trom C & Edman P, In ! .I Pha rm , 63 ( 1990) 49. Carney L G & Hill R M, Arch Opluhalmol, 94 ( 1976) 82 1. Carney L G & Hill R M, Arch Ophihalmol. 97 ( 1979) 95 I. Terry J E & Hill R M, A rch Ophlhalmol. 96 ( 1978) 120. Mi shi ma S. A rch Oplllhalmol, 73 ( 1965) 233 .

MALHOTR A & MAJUMD AR: PERMEATION THROUGH CORN EA

Maurice D M, Br J Ophtha lm ol, 39 ( 1955) 463. Maurice D M, Exp Eye Res , II ( 1971 ) 30. Ramsel aar JAM , Boot J P, Van Hae rin gen N J, Van Best J A & Oosterhui s J A, Curr Eye Res, 7 ( 1988) 947 . 99 Conrad J M, Reay W A, Polcyn E & Robinson J R, J Par-

96 97 98

ent Drug Assoc., 32 ( 1978) 149.

100

Carney L G, Mauger T F & Hill R M, In vest Ophthalmo/

Vis Sci, 30 ( 1989) 74 7. 10 1 Ahmed I & Chaudhu ri B, lnt J Pharm, 44 ( 1988) 97. 102 Hind H W & Goyan F W, J Am Phann Ass Sci Ed, 36 ( 1947) 33. 103 Mitra A K & Mikkelson T J, lnt J Pharm, 10 ( 1982) 2 19. 104 Ahmedl&PattonTF, /ntJPha rm, 19( 1984)2 15 . I05 Seig J W & Robinson J R, J Ph a rm Sci, 66 ( 1977) 1222. 106 Mit ra A K & Mikkelson T J, lnt J Pharm, 37 ( 1987) 19. 107 Lee M & Hammerlund E R, J Pha rm Sci, 63 ( 1974) 72 1. I 08 Wang E S N & Hammerlund E R, J Ph a rm Sci, 59 ( 1970) 1559. 109 Bobcrg-Ans J, Grove-Ras mu ssen K F & Ham merlund E R, Br J Oph thalm ol, 43 ( 1959) 670. 11 0 Go nnerin g R, Edelliauscr H F, Va n Horn D L & Duran t W, In vest Oph thalmol Vis Sci , 18 ( 1979) 373.

Ill

Edelhauser H F, Hanneken A M, Pederson H J & Van Horn

D L, Arch Ophtha /m ol, 99 ( 198 1) 128 1. 11 2 Swan K C, A rch Ophth alm ol, 33 ( 1945) 378. 11 3 Kri shna N & Mitchell B, A m J Ophthalmol, 59 ( 1965) 860. 11 4 Kri shn aN & Brow F, Am J Ophthalm ol, 57 ( 1964) 99. 11 5 Linn M L & Jones L D, Am J Oph thalmol, 65 ( 1968) 76. 11 6 Chrai S S & Robinson J R, J Ph arm Sci, 63 ( 1974) 12 18. 117 Bl aug S N & Canada Jr. A T, Am J Hasp Pharm , 22 ( 1965) 662. 11 8 Mueller W H & Deardro iT D L, J Am Pharm Assoc Sci Ed, 45 ( 1956) 334. 11 9 Bach F C, Riddcl G, Mill er C, Mart in J A & Mullins J D, Am J Ophthalmol. 68 ( 1970) 659. 120 Patton T F & Robinson J R, J Phw m Sci, 65 ( 1976) 1295. 12 1 Patton T F & Robinson J R, J Ph arm Sci, 64 ( 1975) 13 12. 122 Grass G M & Robinson J R, J Pha rm Sci, 73 ( 1984) I02 1. 123 Smolen V F & Schoenwald R D, J Pha rm Sci, 63 ( 1974) 1582 . 124 Merrill D L & Hoss J S, Am J Ophthalmol, 54 ( 1962) 2 1. 125 Davis S D, Sarff L D & Hyndiu k R A, Arch Ophthalm ol. 95 ( 1977) 1638. 126 Trueblood J H, Rosso mondo R M, Carlton W H & Wil son LA , Arch OfJhtha lmol, 93 ( 1975) 127. 127 Hardbergc r R, Hann a C & Boyd C M, A rch OfJhthalm ol. 93 ( 1975 ) 4 2. 128 Bac h F C, Adam J B, McWh istcr H C & Johnson J E. A n11 Op htha lmol. 4 ( 1972) I 16. 129 Waltman S R & Patrowicz T C, In vest Oph thalmol. 9 ( 1970) 966. 130 Green K & Down s S J, In vest Ophthalm o/, 13 ( 1974) 3 16. 13 1 Green K & Mac kccn D L, l11 vest Ophthalmol, 15 ( 1976) 220. 132 Green K & Downs S J, A rch Ophthalmol, 93 ( 1975) 11 65 . 133 Hard bergc r R E, Hanna C & Goodart R, Am J Ophthalm ol, 80( 1975 ) 133. 134 Davies D J G, Jones D E P, Meak in B J & Nort on D A, OfJhthalmol Digest, 39 ( 197 7) 13. 135 Kassc m M A, Atti a M A, Habi b F S & Mohamed A A, Dmg Dev l11d Plwrm. l 3 (8), ( 1987) 1447.

136

23

Benedetto D A, Shah D 0 & Kaufman H E, In vest Ophtha/mol, 14 ( 1975 ) 887.

137 138

Marsh R J & Mauri ce D M ,l:.xp Eye Res, II ( 1971 ) 43. Ludwi g A & Van Ooteghem M, Dmg Dev lnd Pharm, 14 ( 1988) 2267.

139 140 14 1 142 143 144

Ludwi g A & Van Ooteghem M, ltll J Pha rm, 54 ( 1989) 95 . Saettone M F, Gi annacini B, Ravecc u S, La Marca F & Tota G, lnt J Pharm, 20 ( 1984) 187. Saettone M F, Gi annacini B, Tcncggi A, Sav igin i P & TelliniN , J Pha rm Pharmaco/,34 ( 1982) 464. Sei g J W & Robinson J R, J Pharm Sci,68 (6), ( 1979) 724. Saettone M F, Monti D, Toracca M T, Chetoni P & Giannacini B, Drug Dev lnd Pha rm, 15 ( 1989) 2475. Camber 0 , Edman P & Gurn y R, Curr Eye Res, 6 ( 1987) 779.

145

Davies N M, Farr S J, Hadgraft J & Kellaway I W, Pharm Res,8 ( 199 1) 1039.

146

Deshpande S G & Shi ro lkar S, J Pharm Pha nnaco/,4 1 ( 1989) 197.

147 148 149 150 151 152 153

Rozier A, Mazuel C, Grove J & Pl azonnet B, lnt J Pharm, 57 ( 1989) 163. Cohen E, £rp Cell Res. 156 ( 1985) I03.

Rojanasakul Y, Li aw J & Robinson J R, l11 t .I Pharm,66 ( 1990) 133 . Camber 0 & Edman P, fill .I Pha nn,39 ( 1987) 229. Saettone M F, Chetoni P, Ccrbai R & Mazza nti G, Proceed Int ern Symp Control Re/Bioact Mater, 2 1 ( 1994) 591. Saettone M F, Chetoni P, Ccrbai R, Gabriela M & Braghiroli L, lnt J Pha rm, 142 ( 1996) I03. Green K & Tonjum A, Am J Ophthalmol, 72 (5) ( 197 1) 897.

154 155 156

Green K & Tonj um A M, Acw Ophtha /m o /,53 ( 1975) 348. Chrai S S & Robinson J R, J Pha rm Sci,65 ( 1976) 437. Smolen V F, Clevenge r J M, Will iams E J & Bergdoll M

157

Smolen V F, Park C S & Willi ams E J, J Ph arm Sci, 64

158

Green K, John so n R E. Chapman J M, Nelson E & Checks

W, J Pharm Sci, 62 ( 1973) 958. ( 1975 ) 520.

159 160 16 1 162 163 164 165 166 167 168 169 170

L, J Toxico l Cut an OCII ! Toxicol, 8 ( 1989) 253. Marsh R J & Mauri ce D M, Exp Eye Res. II ( 1971) 43 . Tani guchi K, ltak ura K. Morisaka K & Hayash i S,J Plwrmacobiodyll, I I ( 1988) 685. Igaras hi H & orthovcr A M. To.ricol Leu,39 ( 1987) 249 . Morimoto K, Mori saka K & Kamada A, J Pha rm Phannaco /,3 7 ( 1985) 134. Murakami T, Sasak i Y, Yamajo R & Yata N, Chem Pharm Bull, 32 ( 1984) 1948 . Morimoto K, Nakai T & Morisaka K, J Pharm Pharmacal, 39 ( 1937) 124. Ashton P, Di epo ld R, Pl atzer A & Lee V H L, J Ocular Pha rmacal, 6 ( 1990) 37. Grass G M, Wood R W & Robinson J R, l11 vest Ophthalmol Vis Sci, 26 ( 1985) I I 0. Rose B, Simpson I & Locnstcin W R, Nat ure, 267 ( 1977) 625. Fraunfelde r F T & Meyer S M .Com ea, 5 ( 1986) 55. Mondino B J, Salamon S M & Zaidman G W, Surv OfJhtlwlm o/, 26 ( 1982) 33 7.

Van Horn D L, Edelhauscr H F, Prodanov ich G, Eilerman R & Pederson H J, flll 'est Ophtha lmol Vis Sci, 16 ( 1977) 273.

24 171 172

173

INDIAN J EXP BIOL, JANUARY 200 1

Burstein N L & Kl yce S D, Invest Ophthalmol Vis Sci, 16 ( 1977) 899. Hu ghes P M & Mitra A K, in Ophthalmic dru g delivery syslems, edited by A K Mitra, (Marcel Dekker, Inc., New York , Base l, Hong Kong), 1993, II. Gobbel s M & Spitznas M, Albrecht Von Graefs Arch Klin Exp Ophthalmol, 227 ( 1989) 139.

174 175 176 177

Neubert R, Pharm Res, 6 ( 1989) 743. Davi s S S, Tomlison E & Wil so n C G, Br 1 Phannacol, 64 ( 1989) 444. Olejnik 0 , Davis S S & Wi lso n C G, Invest Ophthalmol Vis Sci, 275 ( 1986) 347. Co nroy C W & Buck R H, 1 Ocular Pha nnacol, 8 ( 1992) 233.