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

Review Article

Permeation through cornea

Manjusha Malhotra & D K Maj umdar*

Department of Pharmaceuti cs, College of Pharmacy (U ni versit y of Delhi) , Pushp Vihar, Sector-Ill , New Delhi 11 00 17 , India

The permeability of the cornea to drugs is clinically important because it is the major factor determining the crticacy of topically applied ophthalmic preparations. With this perspective, the present art icle gives a bri ef update and overview of corneal structure and proposed mechanisms 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 physician Pedacius Dioscorides (40-90 AD) compiled five-vo lume materia medica De Universa Medicina that gave fi rst therapeutic delivery system, Coll yrium, fo r ophthalmic medication . How­ever, improvement in ocular 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 un­derstanding of transcorneal permeat ion was sketch/.

The permeability of the cornea to drugs is clini­ca ll y importan t because it is the major factor deter­mining the efficacy of topica ll y app lied preparations. Therefore, an integrated knowledge of the relevant anatomical and physiological constraints that impede or modify ocular drug and vehicle disposit ion through cornea is important.

Corneal structure The cornea is a transparent, avascular tissue, ap­

proximately 0.5-0.7 mm thick and about 11 .5 mm in diameter. Broadly, cornea is divided into 3 layers: epithelium, stroma and endothelium2

. 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 than 60-100 Da. Glucose (Mo l. Wt. = 180 Da), for example, does not pass th rough the epithelium1

.

Lipophi lic compounds pass the ep itheli um due to solubilization in the lipid cel l membranes"'· :~, while it provides major resistance to the movement of ions6 or water7

. The anterior corneal st roma 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 mucopolysaccha­rides ab le to hold a substant ial quanti ty of water. Fi­bril s are spaced so that the cornea is transparent when at normal thickness. Loosely attached to the posterior surface of the stroma is another interfacial layer known as Descemet's membrane. The stroma is read il y transversed by water soluble, polar com­pounds and less so by non-polar compou nd s. Even high molecular weight substances diffu se with ease8

•9

.

The innermost layer of the cornea, the endothelium, consists of a single cell layer whic h houses an active water pump that regulates corneal thickness through hydration 10

-12

. It is a very porous ti ssue wi th an open intercel lul ar network and thus large molecul es (up to 70,000 Da) can traverse this membrane eas il / 1

_1:;.

Mechanisms of permeation through cornea There are two major pathways fo r the movement of

compounds through the corneal tissue : transcellular and paracellular. Transcellular drug movement in­volves cell/tissue partitioning/diffusion, channel dif­fusion and carrier mediated transport. In contrast, paracellular represents diffusive and convecti ve

12 INDIAN J EXP BIOL, JANUARY 200 1

transport occurring through intercellular spaces and tigh t junctions. Diffusive transport is a di ssipative process that depends upon the difference in the so lute concentration and the permeability-surface area prop­erties of the membrane whi le, convecti ve transport is dominated by a balance of hydrostat ic and osmotic grad ients, so lute concentration and hydraulic and re­fl ecti on coefficient of the res tricti ve barrier 16

• Patti ­ti oning of a compound across cel lul ar membranes is especially important for hydrophob ic molecules and is represented by a relati vely high activati on energy for diffusion. While aqueous diffusional channels are important for cornea l transport of hydrophil ic com­pounds and are characterized by low ac tivation en­ergyl7.

The cellular arrangement of epithelium of cornea precludes parace llular transport of most ophthalmic drugs and limits lateral movement within the anterior ep ithelium 18

. Cornea l surface ep itheli al intrace llular poresize has been estimated to be about 60 A (ref. 19). Small ionic and hydrophilic molecu les ap­pear to gain access to the anterior chamber through these pores20

, however, for most drugs, paracellu lar transport is prec luded by the tight intetjunct ional complexes. Stroma exerts a diffusional barrier to highly lipophilic drugs owing to its hydrophi lic na­ture. There are no tight junction complexes in the stroma and paracellular transport through this ti ssue is possible. The endothelial permeability depends on molecular weight and not on the charge or hyd ro-

h.,. f I d?1 ?? p t tc nature o t 1e compoun - ·--. The genera l mechanisms23

'26 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 penetration and as a reservo ir for drug.

• The rate-limiting barrier for most drugs appear to reside in the top two cell layers of the 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 the cornea by parace llular route.

• Substances that possess biphasic solubility trav­erse the cornea more eas il y.

• Lipid soluble substances pass read il y through the limiting cellular 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 that large ly determine ocular bioavai lability of drug from aqueous formula­tion, may be divided into 3 categories: A. Physiological factors B. Physicochemical fac tors C. Formulation factors

A. Physiological factors The loss of drug from the precorneal area is a net

effect of drainage, tear secret ion, non-corneal absorp­tion and corneal absorption rate processes . Collec­ti ve ly these processes lead to typ ical corneal contact time of about 2-4 min in humans, fo r an in st illed so­lution and an ocul ar bioava il ability that is commonly less than I 0% (ref. 27). I . Precorneal 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 drug 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. The 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 circulation . Drainage cf an instilled drug solution away from the eye is re­sponsible fo r a considerable loss of drug and hence affec ts the biological activity of drugs in the eye. The rate of th is drainage is related to the volume of drug solution instilled and increases with increasing vo­lume. The drainage rate of an instilled volume in­creases at a rate proportional to the volume of the flui d in the eye more than the normal lacrimal vol ­ume. The drainage rate is about I 00 times faster than

I b . ?R L d R b' 2x the cornea a sorptton rate- . ee an o tnson gave following equation to predict drainage:

MALHOTRA & MAJUMDAR: PERMEATION THROUGH CORNEA 13

Y1

=Y0

+ Y; e-K '11

where, Y, = volume remaining in the conjunctival sac at time t, Yo= normal lacrimal tear vo lume, Y; = vo l­ume of instilled drop, and K,1 =drainage rate constant. Drainage rate constant is directly proportional to the vo lume of inst illed drop i.e. smaller the drop instilled, the lower the drainage rate and greater the extent of ophthalmic absorption.

Instilled volume - In the rabbits, 90% of the dose is cleared within 2 min for an instilled volume of 50 J..ll , 4 min for an instilled volume of 25 111 , 6min for an inst illed volume of 10 111 and 7.5 min for an instill ed volume of 5 !11 29

. This vo lume dependency of so lution drainage rate has been found to exert its ex pected ef­fect on the percent of dose absorbed into the eye and on the pharmacological effect that follows. The effect of reducing the instilled 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 rabbits caused the same pupillary response as 50 111 of a I 0% solution. The same principle was demonstrated by Patton and Fran­coeur'0, who found equal bioava 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). This resulted in a 2.6-fold improvement in bioavail ­ability from a 5-fold reduction in dropsize. Brown and coworkers'u2 extended the benefits of thi s phe­nomenon to the clini cal use of phenylephrine, a highl y effecti ve mydriatic, but with significant car­diovascul ar ri sk in certain pati ents. Eleven neonates were administered 8 ~LI of 2.5 % phenylephrine hydro­chloride in one eye and 30 111 in the other eye. The mean pupillary diameters (4.86 and 4 .57 mm respec­ti vely) were the same. However, when the said vo l­umes were administered to two different groups of infants, the plasma concentrati on of phenylephrine was 0.9 ng/ml for 8 111 dose and 1.9 ng/ml for the 30 111 dose' 2

. Clinical application of the benefi ts of small-volume dosing has also been reported" ·'5 for dosing mydriatic so lutions to ch ildren and adult s. Tn their studies, these au thors found equal dil ations of the pupil in patients rece iving 0.005 ml ointment or drop dosage form of phenylephrine, cyc lopentolate or tropicamide hydrochloride sa lts and in patients re-

ceiving 50-75 111 of repeated aqueous drops of these medications. These studies suggest that there is an advantage to small -volume dosing owing to an im­provement in ocul ar bioavai labi lity. This al lows a reducti on in dose and a reduction in the potential for systemic side effects. Keister et a!. 36 reported that reducing the instilled volume would increase only the ocular bioavailability of drugs with low permeability and would not effect the ocul ar bioavailability of drugs with high corneal permeability. Therefore, the clinical implication is that appropriate reduct ion of instilled volume and the simultaneous increase in in­sti lied drug concentrati on permit substanti al dosage reductions without red ucti on in absolute dose of the drug37

. Chrai et a/. 38 studi ed the effects of dilution and drainage on the absorpt ion of pilocarpine ni trate and epinephrine hydrochl oride in rabbits when dosed topically in different concentrations and volume and at different dosing interval s. The results indicated that two drops instilled immedi ately after one another showed a reduced bioavail ability 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 suspensions39 but not with liposomes. Lee et a/. 40 reported that liposomes were c leared from the conjuncti val sac of albino rabbits with approx imately the same first order rate constant (0.45 min.1

) over the instilled volume range of 10-50 111. The size and number of liposomes are more important factors than in still ed volume influencing the extent of ocul ar drug absorption from liposomes.

(c) Protein binding-Tears normally, contain about 0.7 % protein41 and the protein level increases during infection or infl ammati on. Un like the blood, where the drug-protein complex continues to circulate, tears are replaced quickly thus remov ing both free and bound forms of the drug. Mikkelson et a/.41 showed that the miotic response to topically applied pilocar­pine was reduced about 2 times as the albumin con­centrati on in the precornea l fluid was increased from 0-3%.

(d) Non-productive drug absorption-Upon inst il­lati on, drug is absorbed into the cornea and conjunc­tiva. The surface area of the conjuncti va is about 17-times that of the cornea42 with 2-30 times greater permeability to many drugs43

. All ti ssue absorption other than the cornea is perceived as non-product ive loss when the target ti ssue is the interi or of the eye.

14 I DIAN J EXP BIOL, JANUARY 2001

This loss can be minimized in two ways: Varying drug lipophilicit/~or changing the drug formulation. Formulation changes that are most effect ive in mini­mizing the rati o of conjunctival to corneal drug ab­sorption are increasing solution pH, lowering solution tonicity and lowering the concentration of EDT A and benzalkonium chloride in the formulation44

. The changes 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, the assumption that drug absorbed by the conjunctiva is swept into sys­temic circulation is incorrect. Series of experiments

46 I ~ 1-~9 conducted by Doane et a/. and Ahmed et a . proved the same. They postulated that the drug ab­sorbed by the conjunctiva could lead to direct entry into the uvea l tract, bypass ing the cornea and thi s route of drug entry 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 lipophilic­ity/hydrophilici 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 the primary rate­determining barrier for penetration of both water soluble and lipid soluble dru gs2550

-52

. Because the epithelium is lipophi lic, low in porosity and relati vely high in tortuosity and thickness, a rapidly penetrating drug must possess log partition coeffic ient greater than 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 layer indicate that endothelium is 2.7 times more permeable than the ep ithe lium5~. Studies on exc ised corneas by Kim et a/. 1 ~ and Maurice55 indicate that non-e lectrol yte penetration through the endothelium occurs primarily through the intercellular spaces. The stroma is basically acellular, hydrophilic in nature, hi gh in porosity and low in tortuosity but because it represents 90% or the thick­ness of the cornea , the stroma is significant in overa ll

.b . . H I ' 6 d . d contn ut1on to reststance. .uang et a. · eterrnme the resistance to permeability of each corneal layer

for a group of P-blocking agents. The results ind i­cated epithelium as the rate determining 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 ility characteristics of biological membranes using thermodynamics and concluded that three independ­ent coefficients, e.g. permeability of the 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 pressure grad ien ts. The reflection coefficient was dependent on the interactions of the so lute and water with the membrane and gave a measure of the 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 these layers with various solutes. The hydraulic conducti vity of the epithelium and endothelium was found to be ap­proximately l .Ox I o-~ 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 I d R b. 60 . . d I Ropnasa u an o mson mvesll gate cornea permselectivity by measuring membrane electro­kinetic potentials generated either by ionic concen­tration gradient (diffusion potential ) or hydrostati c pressure gradient (streaming potenti al) . Studies indi­cated dual-selective character to passage of ions across the cornea. The magnitude and polarity of the 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 isoelectric point, the cornea be­haves as a negatively charged membrane and allows preferential passage of positive ions in compari son to negative ions. Below isoelectric point, the reverse is true. A study of the in vitro flux of lys ine (MW 146, positively charged) and glutamic acid (MW 147, negatively charged) indicated an approximately two to three-fo ld difference in the permeabil ity of the two

d . I I . b . b . 61 compoun s w1t 1 ys 1ne e1ng more a sorpt tvc . Lowering the ionic st rength of the bathing solution

MALHOTRA & MAJUMDAR: PERMEATION THROUGH CORNEA 15

caused an increase in membrane effecti ve charge den­sity due to lesser degree of electrostatic shielding which resulted in a significant increase in both mem­brane resistance and selectivity60

.

Due to its dual capability to terminate the pharma­cological acti viti es of inherentl y ac ti ve drugs and to transform inact ive drugs to their ac ti ve moieties, drug metaboli sm in the eye is an important aspect of drug action. Drugs th at are degraded by oxidati on or re­duction are less likely to be metaboli zed in the eye than those that are degraded by hydrolys is62

. Corneal epithelium and the iris-cilli ary body are metabolica ll y acti ve due to presence of esterases6

', aminopepti-M M · dases and ketone reductase · . Drugs that conta1n

ester linkage are likely to be hydrolysed by the ester­ases. After the topical application of pilocarpine, to the rabbit eye, when the miotic effect is at its peak, up to 50% of the drug is hydrol ysed to pilocarpic acid62

by the esterases present in the intercellular spaces of cornea and aqueous humor. Esterases play a very im-

1 . h . . f' d 66 s portant ro e 111 t e act1 vat10n o ester pro rugs . us-ceptibility of these prodrugs to esterase-medi ated hy­drolys is is an important fac tor that affects not onl y the onset of drug acti on but also the extent of cornea l penetration . The lipid so luble drug, dipi vefrin , is transported fas ter across the cornea than the parent molecule, epinephrine. Subsequent to the transport , the inacti ve prodrug is presumably hydro lysed to ac­ti ve epinephrine in the anteri or chamber by ester­ases67 .

B. Physicochemical factors

Phys icochemical fac tors are the major determi­nants to pass ive di ffu sion across the cornea.

I . Partition coefficient - Partit ion coefficient (between octanol/water) is a parameter for quick as­sessing of penetration potentials of drugs into differ­ent biological membranes. Corwin Hansch desc ri bed I. 68 b ,. 69 I . h. b d f' mear or para o 1c re at1ons 1p etween rug e -fect ivencss and the part iti on coefficient (lipoph ilic character) of the drug. A parabo lic graph identi fies an optimal partiti on coefficient at its apex . Partiti on co­efficient-penetrability correlati on is helpful in the des ign of optimall y permeable ophthalmic drugs. Schoenwald and Ward51 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. Narurkar and Mitra70 observed parabolic relationsh ip between

partitiOn coeffi cient and cornea l permeability of 5' aliphatic esters of 5 iodo-2' -deoxyuridine. In vitro corneal permeability was optimum at log part ition coeffici ent of 0.88 . Likewise, Mosher and Mikkel­son71 determined the i11. vit ro corneal transport of n­alkyl-p-aminobenzoate ester homologues and ob­served a parabolic relationship with an optimal per­meability at a log partition coeffi cient of 2.5-2.6. Similar parabolic relationship between corneal per­meability and partition coefficient has been reported for ~ -blocker72 whereas Wang et a/. 43 descri bed a sigmoidal relati onship fo r the same. However the guiding criteri a for des ign of prodrugs 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 cornea l penetration efficiency of drugs is the partit ion coefficient. The percentage contribution of each cornea l barrier layer against transport of ~-bl ockin g agents and a few other drugs across exci sed rabbit cornea has been stud­ied56·73. The results show that for hydrophil ic drugs (log partition coeffi cient < 0), the epithelium provides a large percentage of the res istance to corneal pene­tration. For lipophilic drugs with log partiti on coeffi­cient between 1.6-2.5, the stroma contributes a sig­nificant percentage of the res istance. And fo r drugs with log partiti on coeffi cients between 0- 1 .6, the sum of the stromal and endothelial res istances equals the epitheli al res istance. Ki shida74 observed in vit ro rela­ti onship between transcorneal permeability of drugs and their phys icochemical properti es and fo und that the transcorneal permeabilities of hydrophobic and biphasic soluble substances were dependent on their lipid solubility.

Thus an optimal lipophilic/hydroph ilic balance in the molecul ar structure of the penetrant must be achieved to affect rapid penetration through the lipo­philic and hydrophilic barri ers of the cornea.

2. Solubility- The maxi mum penetration rate at­tainable by a drug permeating the cornea is a multi­plicati ve factor of permeability coefficient and tear solubility. If a drug is poorl y so luble, its concentra­tion in the precornea l tear fil m may be li mited and therefore its rate of absorpt ion may not be high enough to achieve adequate concent rati on for thera­peutic ac ti vity and reverse is also true.

3. Ionization constant - The pKa of ionizab le drugs is an important factor in corneal penetration . The degree of ionization influences the ex tent of dif-

16 INDIAN J EX P BIOL, JANUAR Y 200 1

fusion across a membrane. M any drugs are weak ac­ids or weak bases and the refore are partial ly ioni zed at physiological pH . The average pH of the tears is 7.2 and the pKa of the drug if within I o r 2 unit s o f this va lue, the cornea l penetration would be more be­cause maj or proporti on of the admini ste red dose would be in the unionized form . The ionized form o f

the drug is minimally lipid soluble and if thi s frac tion is too large, the rate of corneal penetration may not be suffi cient to produce the rapeutic levels of the drug in the eye . Henderson-Hasse lbalc h equat ions re late the degree of ioni zat ion of a weak acid or weak base to pH of the medium and pKa of the drug as fo ll ows:

pKa- pH= log 1_ (for an acidic drug ) f;

pKa- pH= log _!j_ ( for a basic drug) fll

From these equations one can determine the amoun t of uni o nized drug availabl e for transcorneal movement. The greater the fraction of drug present in the unionized form, the greater is the extent of pas­sive diffusion. Chyi-Fu et a/. 75 proved the assu mpti on that only uni oni zed drugs cross the membrane inco r­rect. The ir s tudies showed that ketoro lac (an anion) could permeate through exci sed rabb it cornea both in unionized and ionized forms. Permeati on studi es us­ing exc ised goat cornea also showed si mil ar res ult s for ketoro lac76

, ibupro fen and flurbiprofen77.

4. Molecular weight - Molecular weight is re lated to the diffu sio nal forces active during corneal pe r­meation. For small molecules, the diffusion coeffi­cients a re in versely re lated to the square roots of the ir molecu lar weight, while for la rge mo lecules, the dif­fus ion coeffic ients in water are in ve rse ly re lated to the cube roots of the ir mo lecular we ight s. The changes in mo lecular weight show an inverse re la­tionship to pe rmeabi lity. Compounds with a mo lecu­lar weight greate r than 500 Da offe r poor corneal penetration because pass ive diffu sion no longer re­mains the predominant mode of drug transfe r. Com­pounds of mo lecul ar weights less than 500 Da gener­ally diffuse through both biol ogica l and synthe ti c membranesn. Molecular weight is a less criti cal fac­tor s ince most ophtha lmi c drugs have a re latively small and narrow range of mo lecul ar weight. Excep­tions to thi s may be the hi gher mo lecula r weight anti­biotics such as bac itrac in (MW 14 11 ), coli st imethate sod ium (M W 1250), coli stin sulphate (MW 1250), and polymixin (M W 1200) wh ich pene trate the cor-

nea of di sease mode ls but not those of no rmal rabb it eyes4

. The transcorneal pe rmeabilities of hyd rophobic su bstances and biphasic-so lubl e substances are not governed significant ly by the ir molecula r wei ght but by the ir li pid so lubilit/~. while that of hydrophili c subs tances is governed by the ir molecular weight7

'>.

Liaw et a /. 80 measured the transport c haracte ri stics of series of diffe rent mo lecul ar we ight polyeth yle ne g ly­cols (PEG) and found that corneal absorpti on of PEG 's showed a dependence on molecu lar weight with cut-off between PEG 400 and PEG 600.

C. Formulation factors I . Concentration - Cornea l penetrati on enhance­

ment can be achieved by increasing the solut ion con­centrati o n of a drug, resulting in improved the rapy. Davi s et a/. 50 studied the e ffect of in creas ing con­centration of tobramycin on Pseudomonas kera riris. The resu lts showed that a I 0-fold reduct ion in num­ber of corneal co loni es could be ac hi eved by in­c reasing the concentrati on of topica ll y applied to bramycin from 4 to 40%. T he driv ing force behind this enh ancement is the concentrat io n grad ient de­sc ribed by Fick's first law of diffusion ~ 1 • However inc reas ing the app lieJ concentration to overcome poor c linica l effi cacy works onl y till the drug has reached a pl ateau of its doze-effect curve. Drance and Nash 82 s tudied the effec t of instill ations of increasing concentrati on ( I, 2, 4 and 8%) of p ilocarpine hydro­ch loride on intraocular pressure. Maximum reducti on in pressure was obtained with 4% so lution . The 8% so luti on th ough showed the inc rease in duration , but not an increase in pressure reduction.

Also, inc reas ing concentratio n may result in hy­pertoni c solutions, wh ich are potenti a ll y uncomfort­abl e and can induce increased lacrimat io n which can accele rate the dra inage rate and reduce percent ab-

8' sorption . Ramer and Gassett ·' showed that the ex tent of ocul ar absorption fo r 10% (hyperto nic) pilocarpine hydrochloride was on ly 3-fold greater than that of the 2% so luti on. In a s imila r study, Asseff et al. R~ showed that in comparison with I% pilocarpine hy­drochloride, a 2-fold inc rease in cornea l penetration occurred with 4 % drops, whereas only a 2.5-fold in­crease was produced with an 8% (hypertonic) prepa­ration.

2. Particle shape, size and dissolution rate-Sus­pension s a re wide ly used in ocul ar drug therapy to admini ste r sparing ly soluble drugs or complexes of so luble drugs or to obtain a s low di sso lution and a

MALHOTRA & MAJUMDAR: PERMEATION THROUGH CORNEA 17

prolonged release of the drug. Ophthalmic suspen­sions are generally gentl y deposi ted in the cul-de-sac. The drug particles deposited on the eye surface are moved by the eyelids at each blink , which cou ld cause an abrasion of the outer ep ithelium layers85. These may al so cause irritation of sensory nerves in the epithelium86

. The irritation cou ld elicit refl ex blinks and reflex lacrimation. Concentrati on, shape and particle-size together interact to determine the irritat ion potential of the suspended partic les. Forms with sharp angles 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 drug particle size influences bioavailability inversel / 7

. Three suspensions of 0.1 % triti ated dexamethasone were evaluated with varying mean particle size of 5.75, 11.5 and 22.0 Jlm. As the parti­cle size increased, the in vivo disso lution rate de­creased to the point that the part icles were removed from the conjuncti va l sac before dissolution was complete . Both the rate and extent of dexamethasone penetration into the anterior chamber of the rabbit eye decreased .

In another study, with fluorometholone8R (a spar­

ingly so luble compou nd), it was fou nd that 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 property. The dissolution rate of the drug is an important parameter which de­termines the amount of drug actually in solution and thus available for transport through the cornea during the short res idence time in the eye89. Although the range of particle-size that could be used for ocul ar suspensions 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 lution rate. Though the surface specifi c dissolution rate of a sparingly soluble dru g increases with a decreasing particle­size90, the particle-s ize has no significant influence on the ocular permeati on and that permeability rate rather than the dissolution rate represents the rate-1. . . 'JJ tmttmg step .

3. pH and tonic ity- The pH for human tears ranges between 7.14-7.28. Tears possess a relatively weak buffer capac it y of approximately 3.6x I o·5 (refs 92, 93) . The osmolality of the lac rimal fluid is mainly dependent on the number of di ssolved ions and crys-

talloids . During sleep, the osmolality of the tears varies 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 ion, the permeability of the epithelium is increased con­siderabl y and reverse is true for hypertonic solution95. The inst illati on of a hypotonic drug solution creates an osmotic gradient between the tear film and the sur­rounding ti ssues. The corneal epithelium is extremely tolerant to large variations in pH and tonici ty96

. The sod ium permeability of the epi thelium is unchanged from pH 4- 10 (ref. 63) . Outside thi s range, epithelial permeability increases especia lly when bathed with more alkaline so lutions. Depending on the drop size, solutions with an osmolality lower than I 00 or 266 mOsm/kg and higher than 480 or 640 mOsm/kg are irritant97

. Ramselaar et a!.'JX found that human corneal fluorescein permeability was unaffected in a pH range from 4.5 to 7 and tonicity rangi ng from isotonic (270 mOsm) to hypertonic (620 mOsm). Conrad et a/.99

reported that alkaline pH induced greater lacrimation than ac idic pH in the albino rabbit. Thi s is consistent with the lower buffer capac ity of tears in the basic h . I .d. '(){} 101 s· I t an Ill t 1e act tc range · . mce tears are poor y

buffe red 100, induced lacr imation can be minimized by reducing the tonicity of the solution. Hind and Goyan 102 discussed in qualitative terms, sign ificant aspects of the use of buffers in ophthalmic solutions and reported buffer concentration or buffer capacity (index) as an important factor in formulation of oph­thalmic so lutions containing ionizable drugs. The concentration of the buffer in an instilled so lu tion upon mixing wi th precornea l fluid dictates the time course of pH, drug ioni zation and therefore drug ab­sorption . By progressively reducing the buffe r con­centration of a pH 4.5 citrate buffe r from 0.11 to 0 M, Mitra and Mikkelson103 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 from so lutions contai ning different phosphate bu ffer concentrations at pH 4.5. Seig and Robinson88

· 105

studied the effect of vehicle pH 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 solution was ad­j usted to va lues away from physiological pH. How-

e d I')') .

ever, onra et a . suggested that the mechamsm responsible for the decreased ocular availability of pilocarpine from inst illed so lutions 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 concentration 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 drugs . Phosphate buffer has a hi gh res idual buffer capac ity and yie lds a lower bio­ava ilability of pilocarpine than does an acetate buffer101 . Bioavailability from solutions containing no buffer was max imum and those containing citrate buffer was minimum whi le acetate and phosphate

. d" 106 p f h gave mterme 1ate response . retreatment o t e eye with a sterile drop of isotonic buffer (pH 9.2), totem­porarily alter the tear pH reduces the necessary dose approximately I 0-fold for tropicamide, homatropine hyclrobromide, phenylephrine hydrochloride and cy-1 I I d I I "d 107-109 c open to ate 1y roc 1 on e .

The corneal end othelium is far more sensitive to changes in both pH and tonicity . The optimum endo­theli al pH is 7.4 to 7.5. Below pH 6.7 and above pH 8.5, the electrical potential difference across the en­dothelium reaches zero 10 and both structural and functional alterations occur to the endothelia1 10. The end othelium can withstand vari ati ons in the tonicity from 200-400 m0sm1 11.

4. Viscosity- It is generally beli eved that inclu­sion of a visco li zing agent in an ophthalmic so lution will increase ocul ar bioavailability of the drug due to prolonged res idence time of an inst illed dose in the conj uncti val sac. The more commonly used viscoliz­ing agents include water soluble polymers like poly­vinyl alcohol (PV A), polyvinylpyrrolidone (PVP) and cellulosic polymers. Methylcellulose was first used in ophthalmology to increase the viscosity of aqueous ophthalmic solu tions 112

. In 1965, PYA was introduced to ophthalmology 1

1.1 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 Robinson 11 6 reported that the rate of so lution drai nage decreased with increas ing viscosity and over a range of 1-15 cps viscosity, 3-fold change in the drainage rate constan t was ob­tained . A further 3-fold change over the viscosity range of 15-100 cps was also observed . The drug bio­availabil ity however, was not proportional to contact time. Even with as much as I 00-fold increase in vis­L-Osity and a I 0-fold decrease in drainage rate, the maximum improvement in drug activity, be it mio-

. I 17.118 . h"b" . f . f . I 19 I SIS , 111 1 1t1on o 111 ect1 on or aqueous mmor levels1 16 is about twice that of an aqueous solution. The decline in precorneal drug concentration is of the first order and rate of dec I ine is proportional to the viscosi ty of the instill ed solution. A linear relation­ship between the first order drainage rate constant and both the miotic activity and aqueous humor drug lev­els was obtained over a range of 1- 15 cps solution viscosity in studi es conducted by Chrai and Robin­son1 16. The optimum viscos ity to use is in the range of I 2-1 5 cps120 beyond which the gain in ocul 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 drugs 122

.

A large number of drug studies in humans and animals have been conducted with the use of said polymer vehicles and the results have been ex pressed . f d . . . . I I 7 I I 8. I 2.\ I 24 . I . b. . Ill terms 0 my naSIS Or mi OS IS . . , IIlli 1t1 011

f · f · I I 9 I 25 · d · I 26- I 29 o 111 ect1on · , mcrease contact ti me , aque-1 I I f d J>o-J'' d . I ous 1umor eve s o rug · · · an mtraocu ar pres-J\4 115 S d. . I . I sure · · · · . tu 1es companng t 1e vanous po ymer

vehicles have attempted to emphasize the superi or ity of one vehicle over the other.

The instillation of water-soluble polymers either change the physiological processes, such as drainage or alters the physicochemical parameters that govern the tear film stability. Some viscos ity enhancers, 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 consequent ly the eliminat ion of the drug instil led. In genera l, these polymer vehicles impart a slight lowering of surface tension and an increase in visco­sity to the tear film1

.16. 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 precorneal tea:· film was not strictly because of viscosity of the vehi­cle or its surface wetti ng properties but because of surface spread ing characteristics and water draggin g capac ity of the visco li zi ng agent. 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 phy.-icochemical characteristics as well as concentration of the poly­mer used affected ocu lar retention 1 w_

When a viscous po lymer solution i~ instilled into the cu l-de-sac, seve1al processes reduce its viscosity.

MALHOTRA & MAJUMDAR: PERM EATION THROUGH CORNEA 19

Firstly, the viscous solution is diluted by res ident tears and subsequentl y by incoming tears. Secondl y, the viscous so lution undergoes shear thinning during blinking, thereby increasing contact area and fac ili­tating mixing. A compari son of the reducti on in so lu­tion drainage rate by me~hy l cellul ose and polyvinyl alcohol and the resulting increase in aqueous humor ., . . . I lb . bb . 116 1?1 p1 ocarpme concentrati on m t 1e a 1no ra It · - ,

suggest that it is the fl ow properties of the vehicle in question and its viscosity, not the concentration, that determines the effect of polymers on solution drain­age and ocul ar drug absorpti on. In other words, the rheological characteristics of a po lymer are important fo r the retention pattern on the ocul ar surface. Saet­tone et al. 140 studi ed the influence of different poly­mers on the mydri atic response of tropicamide. They found that time profil es of the mydriatic responses indicated a preference fo r pseudoplasti c systems rather th an Newtoni an systems. Saettone et a /.140·141

also showed that all polymers yielding same vi scos ity did not effect ocul ar drug absorpti on to same ex tent. These in vestigators demonstrated that equi viscous solutions of CMC, HPMC, PV A and PVP enhance the ocular absorption of pilocarpine as well as tro­picamide to different extents in humans. The different acti vi ty of these polymers was att ributed to their in­fluence on spreading charac teri stics and the thickness of the medicati on layer on the precorneal area. Non­Newtoni an fluid vehicles undergo rheological changes (thinning) when exposed to shear stress dur­ing blinking142 while Newtoni an vehicles do not. Di­latant fluid vehicles thicken with shear while pl astic flu id vehicles thin as with pseudop lastic once the yield value is exceeded 121 .

Saettone et a!. 143 showed that soluble mucoadhe­sive polyanion ic po lymers, li ke hyaluronic ac id, polygalacturonic acid, mesoglycan, carboxymethyl­chitin and polyacrylic acid, enhanced the ocul ar ab­sorpti on of pi locarpine more than PV A of equi va len t viscosi ty. Si milar favourab le effects with hyaluronic ac id over HPMC and polyacrylic acid (carbopo l 934 P) over PYA have been reported 144'145. Deshpande and Shirolkar 146 reported hyd rogels prepared wit h carbopol 940 to be better than those prepared wi th sodium CMC and HPMC. Rozier et a!. 147 observed an increased contact time and ocular bioavailabil ity of timclol from gel rite (ion activated, in situ ge lli ng polymer) formulat ion as compared to formulation containing an equiviscous solution of hydroxycthyl cellulose.

On the positive side, increas ing viscosity pro longs contact time and therefore promotes absorption. On the negati ve side; it can slow diffusion of drug in so­lution and can create mi xing problems of the drug solution with tears. In addition , the polymer may ad­sorb onto absorptive surfaces, creating a barrier 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 bioavail ability of topicall y applied ophthalmic

drugs is usuall y very low (< I 0%) (Ref. 27). Attempts have been made to improve ocul ar bioavail abili ty of drugs through the use of penetrati on enhancers such as actin cytoskeleton inhib itors, surfactants. bile salts, chelators, preservati ves and ion-pairing salts.

Actin cytoskeleton inhibitors - Actin cytoskeleton inhi bitors like Cytochalas in B, act by disrupti on of actin microfil aments at tight junctions of corneal epithelium resulting in increased permeabili ty at these . . 148 149 C I I . B . . . JUncti ons · . ytoc 1a as 1n mcreases Ill vuro transcorneal permeation of PEG 400 and PEG 600 and appears to cause minimum cell membrane dam­ageso_

Surfactants - Benzalkonium chl oride, a cationic surfac tant, is often added to aqueo us ophthalmic preparati ons as an anti -bacterial preservati ve in con­centrati on varying from 0.004 to 0.02%. Benzalko­nium chloride has been fou nd to increase the corneal penetrati on of a number of drugs. For example, in­creased corneal permeability due to benzalkonium chl oride has been reported for pilocarpi ne 150, prednis­olone130, chl orampheni co l131 , dexamethasone150, ke-

1 h . ?'i ' b f 77 fl b' f 77 toro ac tromet amme ·, 1 upro en , ur 1pro en , timolol151 , cyc lopentolate 151 ' atenolol152, betaxalo l152

d fl . 1s1 El . . f . d. 1s1 an uorescem ·. ectron microscopic 111 mgs · .. 154 showed that cati onic surfac tants produce break­down in the cohes ion of the epi thelium by increasing the width of the interce llular space. They also pro­duce some dis ruption of the cell cytop lasm presuma­bly by their actions on the plasma cell membranes . The cationic su rfac tants produce 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 permeability. Topically

20 I DIAN J EXP BIOL, JANUARY 2001

applied benzalkonium chloride (0.005 M) , also, pro­duces a loosening in adhesion between the cornea l epithelium and stroma153 . Fu and Lidgate75 reported that increased cornea l permeability of ketorolac, an anionic drug, in presence of benza lkonium chloride was not only because of di srupti on of the 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 proposed41 '155 that com­petitive inhibition of drug-prote in binding by ioni c surfactants, also may be partiall y responsible for the increased corneal penetration. In vitro equilibrium dialysis studies conducted with pilocarpine nitrate, sulfi soxazo le and methylpredni so lone showed that binding of these drugs 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 bioavai lab ility of carbachol by inducing the release of bound drug from binding sites on the corneal surface.

Sodium Iaury! sulphate, an anionic surfactant , also I I b., . l 'i1 ISS S d' en 1ances cornea permea t tty · · · · . tu tes on most

surfactants show a permeability enhancement of the cornea with non-i onic, cationic and anionic surfac­tantsl53, 159.

Marsh and Maurice 15Y determined the 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 achi eve hydrophilic-lipophilic balance (HLB) values ranging from 2- 18. The results showed that single instill ation of non-ionic surfac tant with a HLB range of 16-17 increased fluorescein permea­tion . Tween20 with a HLB value of 16.7 showed the greatest effect. While most surfactants followed a pattern of increasing permeability when thi s HLB value was achieved , some surfactants did not follow thi s general rule suggesting th at chemical spec ificity also pl ay a ro le. Taniguchi et a/.

160 found that Tween80 increased the rate of dexamethasone pene­tration across the iso lated rabb it cornea. Saettone et a/. 151 studied the effect of different permeation en­hancers e.g. polyoxyethylene glyco l Iaury! ether (Brij35), polyoxyethylene glycol 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 , levobunolol, and cyc lopcntolate. Among all the Brij, Brij78 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 chloride increased permeation rates of timolol but caused substantial cornea l damage. Studi es conducted earli er indicate that digitonin80.149 significantl y in­creases corneal permeability but may al so cause se­vere corneal damage. Recently Saettone et a /. 152 have reported 0.05 % w/w concentration of polyoxyethyl­ene alkyl ethers (Brij 35, Brij 78) as sa fe and effec­ti ve permeation enhancers for ~-bl oc kers like atenolol and timolol. The study confirms the 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 cornea l wound healing158 and increased opaci ty of iso lated bovine cornea is related to anionic and non-ion ic surfac tants but not to cati onic surfactants161.

Bile salts - Bile salts have been reported to en­hance the permeabili ty of poorly absorbed drugs through the small intestine, rectum and other mucosa l membranes 162"16·' .

Morimoto et a/. 164 studied the promoting effects of the trihydroxy bile salt, sodium taurocholate (TC-Na) and the dihydroxy bile salt , sodium taurodeoxycho­late (TDC-Na) on in vitro cornea l permeability of hydrophilic compounds and macromolecul ar com­pounds. The results showed that TC-Na and TDC-Na increased corneal permeability of these compounds. The magnitude of the enhancement by TDC- a was greater than that by TC-Na. The diffe rences in the physicochemica l properties of bile sa lts like solubi ­li zing activity, lipoph ili city and calcium ion seques­trati on capac ity, relates to thei r permeability enhanc­ing effects. The critica l mice ll ar concentrati on (CMC) of dihydroxy bile salts is genera ll y lower and their aggregation numbers larger than those of trihydroxy salts164 . Thus the so lub ilizing activ ity of TDC-Na is higher than that of TC-Na. The lipophilicity and cal­cium ion sequestrati on act ivity of TDC-Na are also higher than that of TC-Na 163. These higher phys ico­chemica l activities of TDC-Na cause loosening of the tight junctions of cornea l ep itheli al barriers resulting in increased permeability. A recen t study ind icates 0.05 % (w/w) concentrati on of bile salts (sod ium tau­rodeoxycholate and sod ium ursodeoxycholate) as safe and effective ocu lar permeation enhancers for ~­blockers like atenolol and timolol m .

Chelators-Di sodium edetate (EDTA) is a chelat­ing agent that binds divalent cati ons such as calcium and magnesium. Ashton 165 showed that EDT A in ­creased rabbit corneal epithelial permeability to sor-

MALHOTRA & MAJUMDAR: PERMEATION THROUGH CORNEA 21

bitol. Likewise rabbit corneal permeability in vivo to polar compounds was also increased in presence of 0.5% EDTA166. In epithelia, calcium maintains the intercellular matrix 167 and is therefore, an essential 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 se­vere corneal damage 149.

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 agents 169. Organomercurials react with the membrane sulfhydryl groups and alter membrane permeability and transport systems 170. Even low concentration of 0.001-0.005 % (w/v) commonly used in ophthalmic preparation causes functional changes 170-171 . 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 epithelial permeability to drugs 150·173

.

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 to permeate through membranes . Neubere 74 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 drug175

. Ion pairing with m-chlorobenzyl­trimethylphosphonium chloride increases in vivo cor­neal uptake of chloramphenicol succinate176

• Simi-

larly, increased permeation (in vitro) of ketorolac75 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. tetra­phenylphosphonium chloride, tetraphenylarsonium chloride, and trimethylphenylammonium chloride through excised rabbit cornea has been reported 177

.

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 Sen­

ior research fellowship to Manjusha Malhotra .

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