NOVEL PENETRATION ENHANCERS

VI. NOVEL PENETRATION ENHANCERS

A. a -Amino Acid Most of classical penetration enhancers improve the paracellular transport

across a biological membrane by damaging the tight junctions to varying degrees by their nonspecific actions. As a result, few of them are approved by the FDA because of safety concern. Emisphere Technologies synthesized

a series of small molecular weight a-amino acids, which are used to promote

Ocular Penetration Enhancers 299 oral delivery (Fig. 6) (40,41). These delivery agents successfully increased

absorption of several macromolecules in vivo in rats and primates, including humans, such as salmon calcitonin (42), interferon-a (43), heparin (44), and human growth hormone (hgH) (45). Wu (41) showed that these carriers can increase the permeability coefficient of human growth hormone across Caco-2 monolayers by 10-fold. Although it did have some effects on para- cellular transport, the major pathway was observed to be transcellular. Failure of these carriers to improve the transport of hydrocortisone, a transcellular marker, in Caco-2 monolayers showed that these carriers have a specific interaction with hGH, which makes the hGH more trans- portable, and such an interaction does not exist in the case of hydrocorti- sone. It was clearly established that these carriers do not damage cell membranes and thus are not classical penetration enhancers. Moreover, the carrier-drug complex is not absorbed by an active transport process.

Figure 6 Chemical structures of various amino acid derivative carriers. (From Ref. 41.)

300 Lee and Robinson The working mechanistic assumption is that the carrier shields those hydro-

philic groups on the molecule that restrain absorption. Previous work in our laboratory showed that these carriers increase the permeability coefficient of hGH across the cornea of a rabbit by 10-fold (46). Further study is ongoing in our laboratory to confirm the efficacy and toxicity of these carriers as delivery agents/carriers for ocular drug delivery.

B. Pz-Peptide Pz-peptide (4-phenylazobenzoxycarbonyl-Pro-Leu-Gly-Pro- D -Arg) is a

hydrophilic collagenase-labile pentapeptide with a molecular weight of 777 daltons, which is capable of triggering opening of tight junctions in a transient, reversible manner. As a result, it can facilitate paracellular trans- port in rabbit intestinal segments and Caco-2 monolayers (47,48). Interestingly, it also facilitates its own transport.

The enhancement effect of Pz-peptide on permeability of drug across the cornea and conjunctiva was studied by Chung et al. (49). Pz-peptide increases penetration across the cornea and conjunctiva for a wide range of compounds such as atenolol, propranolol, mannitol, fluorescein, FITC- Dextran 4000, etc. Compared with other traditional penetration enhancers such as a cytochalasin B and EDTA, Pz-peptide is less potent in facilitating paracellular transport since it fails to improve the penetration of FITC- Dextran 10000 across the cornea.

The mechanism of enhancement is believed to involve stimulation of transepithelial Na + flux at the level of the amiloride-sensitive Na + channel and then triggering biochemical changes, which result in opening of tight junctions. This was demonstrated in colonic segments of rabbits and Caco-2 cell monolayers (50). However, this may not be the case in ocular tissues. Amiloride (a Na + channel blocker), hexamethylene amiloride (Na + /H + exchange blocker), ouabain (a Na + /K + ATPase inhibitor), and replace- ment of Na + with choline chloride fails to inhibit Pz-peptide penetration (49). In addition, Pz-peptide can unexpectedly enhance the penetration of propranolol, which transports across a biological membrane solely via a transcellular pathway. This may be due to inhibition of P gp 170 drug efflux pump, which was found in the conjunctiva (51), since propranolol is a substrate for this efflux system. Further investigation has to be carried out to clarify its exact mechanism of enhancement.

Although the enhancement effect of Pz-peptide is promising in vitro and propranolol), its effect is much less pronounced in vivo. Pz-peptide fails

to enhance ocular absorption of propranolol and only improves the absorp- tion of atenolol by 1.4–2.0 times. This may be due to the dilution effect of

Ocular Penetration Enhancers 301 resident tears on Pz-peptide and the applied dose or binding of Pz-peptide to

mucin in other tears proteins.

C. Multifunctional Approach (Polymeric Penetration Enhancers)

1. Colloidal Systems Colloidal systems have been extensively studied as carriers for ocular drug

delivery (52). The mechanism of enhancement is generally believed to be related to prolonged residence time in the cul-de-sac. However, enhanced penetration may also be one of the explanations for improved ocular deliv- ery. Poly-e-caprolactone nanoparticles, nanocapsules, and submicron emul- sions improved ocular bioavailability of indomethacin when compared with aqueous solutions and with a suspension of microparticles (53). It is believed that the colloidal nature, rather than the inner structure or the specific composition of the colloidal carriers, plays a key role in the enhancement since all three colloidal carriers improve the ocular bioavailability of indo- methacin to a similar extent. Confocal microscopy showed that the colloidal carriers penetrate into the epithelial cells of the cornea without causing damage to the cell membrane. This suggests that these carriers enter the epithelium via endocytosis. Therefore, these carriers act as a penetration enhancer or an endocytotic stimulator.

2. Bioadhesives

A mentioned earlier, in order to improve ocular bioavailability, either k eli or k abs have to be increased two- to threefold. Various means have been attempted to prolong residence time (1–4). Obviously, it is desirable to have a delivery system that can stay in the precorneal area for an extended period of time but at the same time enhance corneal penetration. A number of bioadhesive polymers have such properties. Typically, these are macro- molecules that have already been approved by FDA for other purposes. Therefore, safety should not be a big problem.

3. Polyacrylates Poly(acrylic acid) derivatives such as Carbomer and Polycarbol are used

extensively as bioadhesives (54). They do have a membrane-penetrating enhancing effect, although the exact mechanism is not well understood. It was demonstrated that polyacrylic acid gel significantly increased the influx of water in the rat rectum (55). It was speculated that this solvent drag was responsible for the enhanced absorption of low molecular weight com-

302 Lee and Robinson pounds. Reduction of mucus on the microvillus and dilatation of the inter-

cellular space was also observed 5–10 minutes after administration of poly- acrylic acid gel into the rat rectum. However, these changes were reversible and returned to normal relatively soon. It was not likely that polyacrylic acid gel enhanced penetration solely by its detergent action since it inhibited rather than induced hemolysis, which is commonly observed with surfac- tants. Another possible mechanism is related to its chelating activity. Polycarbopol and other polyacrylic acid–based polymers are able to chelate calcium (56), which is an essential component for proper functioning of tight junctions. In addition, chelation of cations that are essential for normal activity of enzymes further improves bioavailability. However, this inhibi- tory effect may be too weak to account for the improved bioavailability (57). In the case of ocular drug delivery, there is reported to be only a minimum amount of metabolizing enzymes in the precorneal area. As a result, there is not likely to be a drastic improvement in ocular bioavailability because of this enzyme inhibitory effect.

4. Chitosan and Derivatives Chitosan (poly[b-(1-4)-2-amino-2-deoxy- D -glucopyranose]) (58) is a hydro-

philic, biocompatible, biodegradable polymer of low toxicity. It is widely used as a pharmaceutical excipient for direct compression of tablets, con- trolled release rate of drugs from a dosage form, enhanced dissolution, etc. (58). It also shows strong mucoadhesive properties (59). Chitosan was eval- uated as a delivery system to increase precorneal drug residence times (60). The positively charged chitosan can reduce the elimination rate from the precorneal area by increasing viscosity and by its interaction with negatively charged mucus (mucoadhesive). The presence of chitosan with tobramycin can bring an improvement in AUC and t 1=2 in the precorneal area. Moreover, this preparation is well tolerated with minimal toxicity.

Besides increasing residence time of a drug in the precorneal area, chitosan can be used as a potential penetration enhancer to improve delivery across the cornea. Dodane et al. (61) showed that chitosan caused a rever- sible, time and dose-dependent decrease in TEER in Caco-2 cell monolayers. The increase in permeability was further confirmed by increased mannitol permeability. They suggested that the above effects might be due to partial alteration of the cytoskeleton, but the exact mechanism is not known. The slight perturbation of the plasma membrane was evident by the rise in extracellular LDH release. However, complete recovery was observed 24 hours after exposure to a low concentration of chitosan (0.005%) for a short period of time (<60 min). Moreover, the chitosan did not affect cell viability as shown by the trypan blue exclusion test.

Ocular Penetration Enhancers 303 Conjugation of an enzyme inhibitor to chitosan is another strategy to

improve drug delivery (62,63). However, due to the limited amounts of enzymes in the precorneal area, this strategy may not be beneficial in ocular drug delivery.

It was shown that mucus may inhibit the binding of chitosan to an epithelia surface and hence decreases its absorption-enhancing effect in intestinal epithelia. The absorption-enhancing effect of chitosan has not been evaluated in the eye. The extent of inhibition of chitosan binding to mucus in the precorneal area has yet to be determined.

VII. CONCLUSIONS The use of penetration enhancers in ocular drug delivery has been studied

for more than a decade, but none has been approved by the FDA mainly due to their nonspecific actions, which often give an unfavorable safety profile. In order to design a more specific penetration enhancer, it is neces- sary to have a better understanding of membrane transport, physiology of tight junctions, etc. Another alternative is to reversibly modify physico- chemical properties of a drug so that it becomes more transportable (e.g., prodrugs or carriers).

Obviously, penetration enhancement has its limit. It is not possible to increase drug bioavailability indefinitely by use of penetration enhancement alone. Other approaches such as increased residence time and inhibition of metabolizing enzymes should be used in conjunction with penetration enhancement. We hope that the coming biomaterial era will bring us such

a drug delivery system.

REFERENCES 1. Sieg, J. W., and Robinson, J. R. Vehicle effects on ocular drug bioavailability.

1. Evaluation of fluorometholone. J. Pharm. Sci. 64:931–936, 1975. 2. Patton, T. F., and Robinson, J. R., Ocular evaluation of polyvinyl alcohol vehicle in rabbits. J. Pharm. Sci. 64:1312–1316, 1975. 3. Robinson, J. R., and Mlynek, G. M. Bioadhesive and phase-change polymers for ocular drug delivery. Adv. Drug. Delivery Rev. 16:45–50, 1995. 4. Salminen, L., and Urtti, H. Prolonged pulse-entry of pilocarpine with a solu- ble drug insert. Graefes Arch. Clin. Exp. Ophthalmol. 221:96, 1983. 5. Worakul, N., and Robinson, J. R. Ocular pharmacokinetics/pharmacody- namics. Eur. J. Pharm. Biopharm. 44:71–83, 1997.

304 Lee and Robinson 6. Keister, J. C., Cooper, E. R., Missel, P. J., Lang, J. C., and Hager, D. F.

Limits on optimizing ocular drug delivery. J. Pharm. Sci. 80:50–53, 1990. 7. Harris, D., and Robinson, J. R. Bioadhesive polymers in peptide drug deliv- ery. Biomaterials 11:652–658, 1990. 8. Grass, G. M. Mechanisms of Corneal Drug Penetration. Ph.D. thesis, School of Pharmacy, University of Wisconsin–Madison, 1985. 9. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D. Molecular Biology of the Cell, 3rd ed., Garland Publishing, Inc., New York, 1994. 10. Liaw, J., and Robinson, J. R. Ocular penetration enhancers. In: Ophthalmic Drug Delivery Systems (Mitra A., ed.), Marcel Dekker, New York, 1993, pp. 369–381. 11. Rojanasakul, Y., and Robinson, J. R. Transport mechanisms of the cornea: Characterization of barrier permselectivity. Int. J. Pharm. 55:237–246, 1989. 12. Lee, V. H. L., and Robinson, J. R. Review: Topical ocular drug delivery: Recent developments and future challenges. J. Ocular Pharmacol. 2:67–108, 1986. 13. Robinson, J. R., and Yang, X. Absorption enhancers. In: Encyclopedia of Pharmaceutical Technology, Vol. 18 (Swarbrick J. and Boylan J. C. (eds.), Marcel Dekker, New York, 1999, pp. 1–27. 14. Sasaki, H., Yamamura, K., Mukai, Nishida, K., Nakamura, J., Nakashima, M., and Ichikawa, M. Enhancement of ocular drug penetration, Crit. Rev. Drug Carrier Syst. 16:85–146, 1999. 15. Junginger, H. E., and Verhoef, J. C. Macromolecules as safe penetration enhancers for hydrophilic drugs—a fiction? Pharmaceut. Sci. Technol. Today 1:370–376, 1998. 16. Grass, G. M., Wood, R. W., and Robinson, J. R. Effects of calcium chelating agents on corneal permeability. Invest. Ophthalmol. Vis. Sci. 26:110–113, 1985. 17. Rojanasakul, Y., Liaw, J. and Robinson, J. R. Mechanisms of action of some penetration enhancers in the cornea: laser scanning confocal microscopic and electrophysiological studies. Int. J. Pharm. 66:131–142, 1990. 18. Nishihata, T., Tomida, H., Frederick, G., Rytting, J. H., and Higuchi, T. Comparison of the effects of sodium salicylate, disodium ethylenediamine tetraacetic acid and polyoxylene-23-lauryl ether as adjuvants for the rectal absorption of sodium cefoxitin. J. Pharm. Sci. 37:159, 1985. 19. Frederiksen, O., and Leyssac, P. P. Effects of cytochalasin B. and dimethyl- sulsulfoxide on isoosmotic transport by rabbit gall-bladder in vitro. J. Facial (Lund.) 265:103–118, 1977. 20. Madara, J. L., et al. Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: Further evidence that the cytoskeleton may influ- ence paracellular permeability and junctional charge selectivity. J. Cell Biol. 102:2125, 1986. 21. Martinez-Palomo, A. Structure of tight junctions in epithelia with different permeability. Proc. Natl. Acad. Sci. USA 72:4487–4491, 1975.

Ocular Penetration Enhancers 305 22. Lee, V. H. L., ed. Peptides and Proteins Drug Delivery. Marcel Dekker, New

York, 1990. 23. Murakami, M., et al. Intestinal absorption enhanced by unsaturated fatty acids: Inhibitory effect of sulfhydryl modifiers. Biochim. Biophys. Acta 293:238, 1988. 24. Cyclodextrins in Pharmacy (Fromming, K.-H., and Szejtli, J., eds.), Kluwer Academic Publishers, Boston, 1994. 25. Davies, N. M., Wang, G., and Tucker, I. G. Evaluation of a hydrocortisone/ hydroxypropyl-beta-cyclodextrin solution for ocular drug delivery. Int. J. Pharm. 156:201–209, 1997. 26. Jarho, P., Jarvinen, K., Urtti, A., Stella, V. J., and Jarvinen, T. Use of cyclo- dextrins in ophthalmic formulations of dipivefrin. Int. J. Pharm. 153:225–233, 1997. 27. Siefert, B., and Keipert, S. Influence of alpha-cyclodextrin and hydroxyalky- lated b-cyclodextrin derivatives on the in vitro corneal uptake and permeation of aqueous pilocarpine-HCl solutions. J. Pharm. Sci. 86:716–720, 1997. 28. Michniak, B. B., Player, M. R., Godwin, D. A., Lockhart, C. C., and Sowell, J. W. In vitro evaluation of azone analogs as dermal penetration enhancers. Part 5. Miscellaneous compounds. Int. J. Pharm. 161:169–178, 1998. 29. Valenta, C., and Wedenig, S. Effects of penetration enhancer on the in vitro percutaneous absorption of progesterone. J. Pharm. Pharmacol. 49:955–959, 1997. 30. Chatterjee, D. J., Li, W. Y., and Koda, R. T. Effect of vehicles and penetra- tion enhancers on the in vitro and in vivo percutaneous absorption of metho- trexate and edatrexate through hairless mouse skin. Pharm. Res. 14:1058– 1065, 1997. 31. Tang-Liu, D. D-S., Richman, J. B., Weinkam, R. J., and Takruri, H. Effects of four penetration enhancers on corneal permeability of drugs in vitro. J. Pharm. Sci. 83:85–90, 1994.

32. Tang-Liu, D. D.-S., and Burke, J. The effect of Azone 1 on ocular levobunolol absorption: Calculating the area under the curve and its standard error using tissue sampling compartments. Pharm. Res. 5:238–241, 1988. 33. Ismail, I. M., Chen, C.-C., Richman, J. B., Andersen, J. S., and Tang-Liu, D. D.-S. Comparison of azone and hexamethylene lauramide in toxicological effects and penetration enhancement of cimetidine in rabbit eyes. Pharm. Res. 9:817–821, 1992. 34. Chiou, G. C. Y., Chang, C. Y., and Chang, M. S. Systemic delivery of insulin through eyes to lower the glucose concentration. J. Ocular Pharmacol. 5:81–

91, 1989. 35. Pillion, D. J., Atchison, J. A., Stott, J., McCracken, D. L., Gargiulo, C., and Meezan, E. Efficacy of insulin eyedrops. J. Ocular Pharmacol. 10:461–470, 1994. 36. Pillion, D. J., McCracken, D. L., Yang, M., and Atchison, J. A. Glucagon administration to the rat via eye drops. J. Ocular Pharmacol. 4:349–358, 1992.

306 Lee and Robinson 37. Pillion, D. J., Amsden, J. A., Kensil, C. R., and Recchia, J. Structure-function

relationship among Quillaja saponins serving as excipients for nasal and ocu- lar delivery of insulin. J. Pharm. Sci. 85:518–524, 1996. 38. Morgan, R. V. Delivery of systemic regular insulin via the ocular route in cats. J. Ocular Pharmacol. 11:565–573, 1995. 39. Morgan, R. V., and Huntzicker, M. A. Delivery of systemic regular insulin via the ocular route in dogs. J. Ocular Pharmacol. 12:515–526, 1996. 40. Steiner, S., and Rosen, R. Delivery System for Pharmacological Agents Encapsulated Proteinoids. U.S. Patent, 1990. 41. Wu, S.-Y. Mechanistic studies on the enhanced mucosal transport of human growth hormone by certain amino acid derivatives. Ph.D. thesis, School of Pharmacy, University of Wisconsin–Madison, 1999. 42. Leone-Bay, A., McInnes, C., Wang, N., DeMorin, F., Achan, D., Lercara, C., Sarubbi, D., Haas, S., Press, J., Barantsevich, E., O’Broin, B., Milstein, S., and Patron, D. Microsphere formation in a series of derivatized a-amino acids: Properties, molecular modeling, and oral delivery of salmon calcitonin. J. Med. Chem. 38:4257–4262, 1995. 43. Leone-Bay, A., Santiago, N., Achan, D., Chaudhary, K., DeMorin, F., Falzarano, L., Haas, S., Kalbag, D., Leipold, H., Lercara, C., O’Toole, D., Rivera, T., Rosado, C., Sarubbi, D., Vuocolo, E., Wang, N., Milstein, S., and Baughman, R. A. N-acylated a-amino acids as novel oral delivery agents for proteins. J. Med. Chem. 38:4263–4269, 1995. 44. Brayden D., Creed E., O’Connell A., Leipold, H., Lercara C., Agarwal R., and Leone-Bay A. Heparin absorption across the intestine: Effects of sodium N-[8(2-hydroxybenzoyl)amino]caprylate in Caco-2 monolayers and in rat in situ intestinal instillations. Pharm. Res. 14:1772–1779, 1997. 45. Leone-Bay, A., Ho, K., Agarwal, R., Baughman, R. A., Chaudhary, K., DeMorin, F., Genoble, L., McInnes, C., Lercara, C., Milstein, S., O’Toole, D.,

Paton, D. R. 4-[4-[(2- Hydroxybenzoyl)amino]phenyl]butyric acid as a novel oral delivery agent for recombinant human growth hormone. J. Med. Chem. 39:2571–2578, 1996. 46. Robinson, J. R., unpublished data, 1998. 47. Yen, W.-C., and Lee, V. H. L. Paracellular transport of a proteolytically labile pentapeptide across the colonic and other intestinal segments of the albino rabbit: Implications for peptide drug design. J. Contr. Rel. 28:97–109, 1994. 48. Yen, W.-C., and Lee, V. H. L. Penetration enhancement effect of Pz-peptide, a paracellularly transported peptide, in rabbit intestinal segments and Caco-2 cell monolayers. J Contr. Rel. 36:25–37, 1995. 49. Chung, Y. B., Han, K., Nishiura, A., and Lee, V. H. L. Ocular absorption of Pz-peptide and its effect on the ocular and systemic pharmacokinetics of topically applied drugs in the rabbit. Pharm. Res. 15:1882–1887, 1998. 50. Yen, W.-C., and Lee, V. H. L. Role of Na + in the asymmetric paracellular transport of 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg across rabbit colonic segments and Caco-2 cell monolayers. J. Pharmacol. Exp. Ther. J. Contr. Rel. 36:25–37, 275:114–119, 1995.

Sarubbi, D.,

Variano, B.,

and

Ocular Penetration Enhancers 307 51. Saha, P., Yang, J., and Lee, V. H. L. Existence of a P-glycoprotein drug efflux

pump in cultured rabbit conjunctival epithelial cells. Invest. Ophthalmol. Vis. Sci. 39:1221–1226, 1998. 52. Zimmer, A., and Kreuter, J. Microcapsules and nanoparticles used in ocular delivery systems. Adv. Drug Delivery Rev. 16:61–73, 1995. 53. Calvo, P., Alonson, M. J., Vila-Jato, J. L., and Robinson, J. R. Improved ocular bioavailability of indomethacin by novel ocular drug carriers. J. Pharm. Pharmacol. 48:1147–1152, 1996. 54. Yang, X., and Robinson, J. R. Bioadhesion in mucosal drug delivery. In: Biomaterials for Drug Delivery (Okano, T., ed.), Elsevier, Amsterdam, 1998. 55. Morimoto, K., Iwamoto, T., and Morisaka, K. Possible mechanisms for the enhancement of rectal absorption of hydrophilic drugs and polypeptides by aqueous polyacrylic acid gel. J. Pharmacobio-Dyn. 10:85–91, 1987. 56. Kriwet, B., and Kissel, T. Interactions between bioadhesive poly(acrylic acid) and calcium ions. Int. J. Pharm. 127:135–145, 1996. 57. Lußen, H. L., Bohner, V., Pe’rard, D., Langguth, P., Verhoef, J. C., de Boer,

A. G., Merkle, H. P., and Junginger, H. E. Mucoadhesive polymers in peroral peptide drug delivery: V. Effect of poly(acrylates) on the enzymatic degrada- tion of peptide drugs by intestinal brush border membrane vesicles. Int. J. Pharm. 141:39, 1996. 58. Illum, L. Chitosan and its use as a pharmaceutical excipient. Pharm. Res. 15:1326–1331, 1998. 59. Yoshiaki, K., Yamamoto, H., Takeuchi, H., and Kuno, Y. Mucoadhesive dl- lactide/glycolide copolymer nanospheres coated with chitosan to improve oral delivery elcatonin. Pharm. Dev. Tech. 5:77–85, 2000. 60. Felt, O., Furrer, P., Mayer, J. M., Plazonnet, B., Buri, P., and Gurny, R. Topical use of chitosan in ophthalmology: tolerance assessment and evalua- tion of precorneal retention. Int. J. Pharm. 180:185–193, 1999. 61. Dodane, V., Khan, A. M., and Merwin, J. R. Effect of chitosan on epithelial permeability and structure. Int. J. Pharm. 182:21–32, 1999. 62. Bernkop-Schnurch, A., Paikl, C., and Pasta, M. Novel bioadhesive chitosan- EDTA conjugate protects leucine enkephalin from degradation by aminopep- tidase N. Pharm. Res. 14:917–922, 1997. 63. Bernkop-Schnurch, A., and Pasta, M. Intestinal peptide and protein delivery: Novel bioadhesive drug-carrier matrix shielding from enzymatic attack. J. Pharm. Sci. 87:430–434, 1998.