1. INTRODUCTION:

The concept of mucoadhesion has been pioneered in the 1980s, numerous attempts have been taken in order to improve the adhesive properties of polymers. These attempts include approaches such as the use of linear poly(ethylene glycol) as adhesion promoter for hydrogels,¹ the neutralization of ionic polymers,² mucoadhesion by a sustained hydration process³ and the development of polymer–adhesin conjugates^4,5 providing a specific binding to epithelium. However, all these systems are based on the formation of non-covalent bonds such as hydrogen bonds, Van der Waal’s forces, and ionic interactions. Accordingly, they provide only relative weak mucoadhesion, in many cases insufficient to guarantee the localization of a drug delivery system at a given target site. Mucoadhesive polymers have therefore in many cases not proven to be effective as a pharmaceutical glue.^{6, 7} A presumptive new generation of mucoadhesive polymers are thiolated polymers or designated thiomers⁸. In contrast to well-established mucoadhesive polymers these novel polymers are capable of forming covalent bonds.

The bridging structure most commonly encountered in biological systems–the disulfide bond–has been discovered for the covalent adhesion of polymers to the mucus gel layer of the mucosa.

Thiomers are mucoadhesive basis polymers, which display thiol bearing side chains Fig 2. Based on thiol/disulfide exchange reactions and/or a simple oxidation process as illustrated in Fig 1, disulfide bonds are formed between such polymers and cysteine-rich subdomains of mucus glycoproteins.⁹ Hence, thiomers mimic the natural mechanism of secreted mucus glycoproteins, which are also covalently anchored in the mucus layer by the formation of disulfide bonds. Within this review the so far gained knowledge on the mucoadhesive properties of thiomers is summarized and discussed.

2. Synthesis of thiomers:

2.1. Cationic thiomers:

Cationic thiomers are mainly based on chitosan. The primary amino group at the 2-position of the glucosamine subunit of this polymer is the main target for the immobilization of thiol groups. As outlined in Fig. 3 sulfhydryl bearing agents can be covalently attached to this primary amino group via the formation of amide or amidine bonds. In case of the formation of amide bonds the carboxylic acid group of the ligands cysteine and thioglycolic acid reacts with the primary amino group of chitosan mediated for instance by carbodiimides.^10-12 An unintended oxidation of thiol groups during synthesis can be avoided by performing the reaction under inert conditions. Alternatively the synthesis can be performed at a pH 5. At this pH-range the concentration of thiolate-anions, representing the reactive form for oxidation of thiol groups, is low, and the formation of disulfide bonds can almost be excluded. Furthermore, disulfide bonds can be reduced after the synthesis process by the addition of reducing agents such as dithiotreithol or borohydride. In case of the formation of amidine bonds 2-iminothiolane is used as coupling reagent.^13,14 It offers the advantage of a simple one step coupling reaction. In addition, the thiol group of the reagent is protected towards oxidation due to its chemical structure. The amount of immobilized thiol groups in reduced and oxidized form can be determined via Ellman’s reagent⁸ with and without previous quantitative reduction of disulfide bonds with borohydride.²²

Fig 1: Mechanism of disulfide bond formation between thiomers and mucus glycoproteins (mucins).⁹

2.2. Anionic thiomers:

So far generated anionic thiolated polymers exhibit all carboxylic acid groups as anionic substructures. These carboxylic acid groups offer also the advantage that sulfhydryl moieties can be easily attached to such polymers via the formation of amide bonds. Appropriate ligands are cysteine, homocysteine and cysteamine.^15-21

The formation of amide bonds can be mediated by carbodiimides. The chemical structure of so far generated anionic thiolated polymers is shown in Fig 3. Thiol oxidation during synthesis can be avoided as described above. The total amount of immobilized reduced and oxidized thiol groups can be determined in the same way as described for cationic thiomers.

3. Mechanisms being responsible for improved mucoadhesion:

3.1. Formation of disulfide bonds with the mucus gel layer:

The formation of disulfide bonds between the thiomer and the mucus gel layer takes place either via thiol/disulfide exchange reactions or via a simple oxidation process of free thiol groups. The different types of mucus glycolproteins or designated mucins exhibiting cysteine-rich subdomains have been reviewed previously.²³ Generally there are no mucosal surfaces in which mucins with cysteine rich subdomains are not present. In contrast to noncovalent bonds disulfide bonds are not influenced by factors such as ionic strength and pH. Velocity and extent of disulfide bond formation depends on the concentration of thiolate anions representing the reactive form for thiol/disulfide exchange reactions and oxidation processes. The concentration of thiolate anions inturn depends on:

Fig 2: Thiolated polymers-thiomers.

I. The pKa value of the thiol group:

In dependence on the polymer backbone and the chemical structure of the ligand, more or less reactive thiomers can be designed. Thiol groups of the chitosan–thiobutylamidine conjugate Fig 3, for instance, exhibit a pKa value of 9.9,¹³ whereas the pKa value of the thiol groups of poly(acrylate)–cysteine conjugates is 8.35. ²⁴

II. The pH of the thiomer:

As only ionic thiomers are used, they all display a high buffer capacity. The buffer capacity of a sodium poly(acrylate) matrix tablet, for instance, can be compared with that of an at least 25 M acetate buffer. As all charged groups remain concentrated on the polymeric network a kind of microclimate can be established.²⁵ The reactivity of thiol groups can consequently be controlled by adjusting the pH of the polymer to a certain level. The higher the pH is adjusted, the more reactive are the thiol groups and vice versa.

III. The pH of the surrounding medium:

The reactivity of thiol groups inside the polymeric network is mainly controlled by the pH of the thiomer, whereas the reactivity on the surface of the polymer is more controlled by the pH of the surrounding medium. As the mucus gel layer being close to the epithelium has a pH around 7, thiol groups penetrating into the mucus are always sufficiently reactive. Evidence for the formation of covalent bonds between thiomers and the mucus gel layer has been provided recently. Leitner et al. could show by four different methods including rheological, diffusion, gel permeation and certain mucoadhesion studies the formation of disulfide bonds between thiolated polymers and mucus glycoproteins.⁹ It was also shown that mucin can be effectively bound to thiolated polyacrylate, while it is not at all bound to unmodified polyacrylate. Due to the addition of the disulfide bond breaker dithiothreitol already immobilized mucin could be completely removed from the thiolated polymer.⁸

Fig 3: Structure of thiolated polymers.^{10, 11, 12,
15}

IV. In situ cross-linking process:

Another likely mechanism being responsible for the improved mucoadhesive properties of thiomers is based on their in situ cross-linking properties. During and after the interpenetration process, which could be verified for mucoadhesive polymers such as poly(acrylic acid) recently,²⁶ disulfide bonds are formed within the thiomer itself leading to additional anchors via chaining up with the mucus gel layer. It is similar to the mechanism on which the adhesive properties of most adhesive are based on, i.e. a penetration of the adhesive into a certain surface structure followed by a stabilization process of the adhesive. In case of superglues, for instance, monomeric cyanoacrylates penetrate into raw surfaces followed by a polymerization process. Thiolated polymers display in situ gelling properties due to the oxidation of thiol groups at physiological pH-values, which results in the formation of inter- and intramolecular disulfide bonds. The in situ gelling behavior of thiomers was characterized in vitro by rheological measurements. The sol–gel transition of thiolated chitosans, for instance, was completed at pH 5.5 after 2 h, when highly crosslinked gels were formed. In parallel, a significant decrease in the thiol group content of the polymers was observed, indicating the formation of disulfide bonds.^13,27 The rheological properties of unmodified chitosan remained constant over the whole observation period. Rheological investigation of thiolated chitosans furthermore demonstrated a clear correlation between the total amount of polymer-linked thiol groups and the increase in elasticity of the formed gel. The more thiol groups were immobilized on chitosan, the higher was the increase in elastic modulus G’ in solutions of thiolated chitosan.^13,27 These in situ gelling properties are in particular of interest for liquid or semisolid vaginal, nasal and ocular formulations, which should stabilize themselves once applied on the site of drug delivery.

4. Mucoadhesive properties:

The mucoadhesive properties of thiomers in comparison to well-established polymers are discussed in detail by Grabovac et al. Due to the immobilization of thiol groups on all so far tested polymers, their mucoadhesive properties were significantly improved irrespectively from the evaluation method. In case of anionic mucoadhesive polymers the poly(acrylic acid)–cysteine conjugate seems to be a good example for this observation. Marschu¨tz et al. could shows that the viscosity of poly(acrylic acid)/ mucin mixtures–directly correlating with the interactions of the polymer with the mucus and consequently indicating the mucoadhesive properties can be more than 10-fold improved.²⁸ The same thiolated polymer showed in comparison to the corresponding unmodified polymer more than 2-fold and 20-fold improved mucoadhesive properties in tensile studies and by using the rotating cylinder method, respectively.²⁸ In addition, it could be shown that the residence time of poly(acrylic acid) microparticles on the small intestinal mucosa can be more than 3-fold prolonged by the immobilization of thiol groups.²⁹ In case of cationic thiomers, on the other hand, the chitosan–thiobutylamidine conjugate seems to be a good example, as it has been evaluated by various mucoadhesion test system. It was demonstrated that a more than 100-fold increased viscosity of chitosan–thiobutylamidine conjugate in comparison to unmodified chitosan.¹³ Moreover in tensile studies and rotating cylinder studies the mucoadhesive properties of the thiolated version were 100-fold and 140-fold improved.^13,14 In case of both mentioned thiomers the molecular mass of the polymer chains had a great impact on their mucoadhesive properties. For the anionic as well as for the cationic thiomer the highest mucoadhesive properties were achieved when they exhibited a medium molecular mass. In case of poly(acrylic acid)–cysteine, polymer conjugates exhibiting a molecular mass of 450 kDa were more mucoadhesive than once of a molecular mass of 2 kDa, 45 kDa and 1000–3000 kDa.³⁰ On the other hand, tensile studies performed with thiolated chitosan exhibiting a molecular mass of 150 kDa, 400 kDa and 600 kDa showed the relatively highest mucoadhesive properties for the medium molecular mass thiomer.¹⁴ Utilizing a medium molecular mass chitosan–thiobutylamidine conjugate displaying 264 A⁰ thiol groups per gram polymer consequently led to a more than 100-fold improvement in mucoadhesion in comparison to unmodified chitosan.¹⁴ Generally, it could be observed in most performed mucoadhesion studies with thiomers, that the higher the amount of immobilized thiol groups was, the higher were the mucoadhesive properties. Furthermore, the mucoadhesive properties of thiomers exhibiting a relative low pH are always higher.^31,32

5. Dosage forms based on thiomers:

5.1. Microparticles and nanoparticles:

As microparticles and nanoparticles have small particle size, they show a prolonged gastrointestinal residence time even without any mucoadhesive properties by diffusing into the mucus gel layer. Coupe etal., for instance, could demonstrate that particulate delivery systems display a more prolonged gastrointestinal transit time compared to single-unit dosage forms.³³ In order to further improve the residence time of drug delivery systems on mucosal membranes, both approaches: mucoadhesive polymers and micro-/nanoparticles were consequently combined. Micro- and nanoparticles based on anionic or cationic mucoadhesive polymers, however, disintegrate very rapidly, unless multivalent cationic or anionic compounds such as Ca²⁺-ions or sulfate ions are added, respectively, leading to stabilization via an ionic cross-linking process.^34-35 Due to the addition of such ionic cross-linkers, on the other hand, the mucoadhesive properties of these polymers are strongly reduced. On the contrary, due to the immobilization of thiol groups on well-established polymers their mucoadhesive properties are even further improved, although micro- and nanoparticles being based on thiolated polymers do not disintegrate. Because of the formation of disulfide bonds within the polymeric network, the particles are stabilized.^29,
36 consequently, also a controlled drug release out of thiomer micro- and nanoparticles can be provided. Recently, microparticles comprising poly(acrylate)–cysteine were generated via the solvent evaporation emulsification method. Particles were of spherical shape and partially porous structure and had a main size in the range of 20–60 A⁰ with a center at 35 A⁰. Because of the formation of disulfide bonds within the particles they did not disintegrate under physiological conditions within 48 h. In addition, a controlled drug release of a model peptide drug was achieved. Due to the immobilization of thiol groups on poly(acrylic acid) the mucoadhesive properties of the corresponding microparticles were 3-fold improved.³⁶

5.2. Matrix tablets:

Mucoadhesive matrix tablets are useful for intraoral, peroral, ocular and vaginal—local or systemic delivery. Due to the in situ cross-linking properties of thiomers the cohesiveness and subsequently the stability of the swollen carrier matrix can be guaranteed.³⁷ Disintegration studies, for instance, performed with tablets comprising unmodified polycarbophil revealed a stability of less than 2 h, whereas tablets being based on thiolated polycarbophil did not disintegrable at all.¹⁷ Moreover even no erosion of this swollen drug carrier matrix could be observed within an observation period of 24 h. When attached in dry form the mucoadhesion of matrix tablets is additionally improved by an augmented interpenetration process depending on the swelling behavior of the delivery system. In order to make use of this simple adhesion by hydration process also in case of peroral delivery, matrix tablets can be enteric coated. If an adhesion shall be achieved already in the stomach, the coating with a triglyceride seems to be sufficient in order to avoid an unintended adhesion in the oral cavity or oesophagus.³⁸ Additionally, matrix tablets comprising a thiomer offer the advantage that a controlled drug release can be easily achieved out of this type of mucoadhesive dosage forms, which could already be demonstrated for numerous drugs.^37,39,40,41 Hornof et al., for instance, could show that by simple homogenizing the thiomer with the drug of choice and compressing tablets out of it results in many cases in delivery systems, which can guarantee even a zero order release profile for several hours.⁴¹ The drug release rate is thereby predominately controlled by a hydration and diffusion process.

5.3. Gels:

Mucoadhesive gels are useful in case of intraoral, vaginal, nasal and ocular delivery. So far, however, mucoadhesive gel formulations have not reached their full potential, as the adhesive properties of such delivery systems are often insufficient. The great advantage of the use of thiomers in gel formulations has to be seen not only in their mucoadhesive but also in their in situ gelling properties.^13,19 Strong mucoadhesive properties are senseless, if the adhesive bond fails within the gel formulation itself than between the gel and the mucosa. Due to the in situ gelling properties of thiomers, however, this shortcoming can be overcome.

5.4. Liquid formulations:

Thiomers were shown to be stable when stored in dry form.⁴² In aqueous solutions; however, they were shown to form disulfide bonds in a pH-dependent manner. Because of this instability in aqueous solutions thiomers have so far not been used in liquid formulations. Recently, however, Hornof could demonstrate that thiomers can even be stabilized in aqueous solutions when the liquid formulations are produced under inert conditions and the vessels are packed in an aluminium foil containing an oxygen scavenger such as iron-oxides inside.⁴³ Based on this technology first mucoadhesive liquid formulation comprising thiomers were prepared and tested in vivo. In particular in the ophthalmic field thiomers have already shown potential in form of liquid formulations. In case of the dry eye syndrome the most prevalent disease in the eye, for instance, liquid thiomer formulations might be highly beneficial. One of the most important reasons for this disease seems to be a defective mucus layer on the ocular surface. Mucus acts as surfactant, and is therefore important for the wettability of the epithelial surface.⁴⁴ The main treatment for this disease is the use of tear substitutes. Most of these formulations contain mucoadhesive hydrophilic polymers such as carbomer or sodium hyaluronate. The mucoadhesive properties of these polymers, however, are quite insufficient making a frequent instillation necessary. Because of their ability to interact with cysteine-rich subdomains of mucus glycoproteins on the ocular surface, eye drops containing a thiomer should be able to prolong the stability of the precorneal tear film for a comparable longer time period. A parameter to characterize the quality of the tear film is the tear film break-up time, which is defined as the time period after a blink in which the tear film becomes unstable and dry spots evolve on the cornea. Normally the break-up time exceeds the timespan between blinks, but in patients with dry eye syndrome the break-up time is decreased to less than 5s. The comparison of the effect of a well established commercial product containing carbomer and a formulation containing 0.2% (m/v) polyacrylic acid–cysteine and mannitol as tonicity agent on the tear film break-up time is shown in human volunteers. The eye drops containing thiolated polyacrylic acid had a positive effect on the tear film stability, whereas no difference in the tear film break-up time was observed after application of an isotonic mannitol solution or the commercially available formulation.⁴³

CONCLUSION:

The chemical modification of well-established mucoadhesive polymers via derivatisation with various reagents bearing sulfhydryl functions causes a dramatic improvement in the polymer’s properties. Mucoadhesiveness and cohesiveness are strongly improved. Furthermore, thiolated polymers display in situ-gelling features. In future the efficacy of this new generation of mucoadhesive polymers can be demonstrated by various in vivo studies in different species on the gastrointestinal, nasal and ocular mucosa.

REFERENCE:

1. Sahlin JJ, Peppas NA, Enhanced hydrogel adhesion by polymer interdiffusion: use of linear poly(ethylene glycol) as an adhesion promoter. J Biomater Sci 1997; 8:421– 436.

2. Smart JD, Kellaway, Worthington HEC. An in-vitro investigation of mucosa-adhesive materials for use in controlled drug delivery. J Pharm Pharmacol 1984; 36:295– 299.

3. Bologna WJ, Levine HL, Cartier PH, Ziegler D. Extended release buccal bioadhesive tablets, WO00/10536(1998).

4. Naisbett B, Woodley J. The potential use of tomato lectin for oral drug delivery. Lectin binding to rat small intestine in vitro. Int J Pharm 1994; 107:223– 230.

5. Bernkop-Schnurch A, Gabor F, Szostak MP, W. Lubitz, An adhesive drug delivery system based on K99-fimbriae. Eur J Pharm Sci 1995; 3:293–299.

6. Khosla R, Davis SS. The effect of polycarbophil on the gastric emptying of pellets. J Pharm Pharmacol 1987; 39:47– 49.

7. Lehr CM. Bioadhesion Technologies for the delivery of peptide and protein drugs to the gastrointestinal tract. Crit Rev Ther Drug 1994; 11:119– 160.

8. Bernkop-Schnurch A, Schwarz V, Steininger S. Polymers with thiol groups: a new generation of mucoadhesive polymers. Pharm Res 1999; 16:876–881.

9. Leitner VM, Walker GF, Bernkop-Schnu¨ rch A. Thiolated polymers: evidence for the formation of disulphide bonds with mucus glycoproteins. Eur J Pharm Biopharm 2003; 56:207– 214.

10. Bernkop-Schnurch A, Brandt UM, Clausen AE. Synthesis and in vitro evaluation of chitosan–cysteine conjugates. Sci Pharm 1999; 67:196– 208.

11. Bernkop-Schnurch A, Hopf TE. Synthesis and in vitro evaluation of chitosan–thioglycolic acid conjugates. Sci Pharm 2001; 69:109– 118.

12. Kast CE, Bernkop-Schnurch A. Thiolated polymers–thiomers: development and in vitro evaluation of chitosan–thioglycolic acid conjugates. Biomaterials 2001; 22:2345– 2352.

13. Bernkop-Schnurch A, Hornof M, Zoidl T. Thiolated polymers–thiomers: synthesis and in vitro evaluation of chitosan-2-iminothiolane conjugates. Int J Pharm 2003; 260:229–237.

14. Roldo M, Hornof M, Caliceti P, Bernkop-Schnu¨rch A. Mucoadhesive thiolated chitosans as platforms for oral controlled drug delivery: synthesis and in vitro evaluation. Eur J Pharm Biopharm 2004; 57:115–121.

15. Bernkop-Schnurch A, Kast CE, Richter MF. Improvement in the mucoadhesive properties of alginate by the covalent attachment of cysteine. J Control Release 2001; 71:277–285.

16. Bernkop-Schnurch A, Steininger S. Synthesis and characterization of mucoadhesive thiolated polymers. Int J Pharm 2000; 194:239–247.

17. Bernkop-Schnurch A, Scholler S, Biebel RG. Development of controlled drug release systems based on polymer–cysteine conjugates. J Control Release 2000; 66:39– 48.

18. A. Bernkop-Schnurch A, Clausen AE, Hnatyszyn M. Thiolated polymers: synthesis and in vitro evaluation of polymer–cysteamine conjugates. Int J Pharm 2001; 226:185– 194.

19. Krauland A, Leitner VM, Bernkop-Schnu¨ rch A. Improvement in the in situ gelling properties of deacetylated gellan gum by the immobilization of thiol groups. J Pharm Sci 1993; 92:1234–1241.

20. Bernkop-Schnurch A, Ko¨ nig V, Leitner V, Krauland A, Brodnik I. The preparation and characterisation of mucoadhesive thiolated poly(methacrylic acid)–starch compositions. Eur J Pharm Biopharm 2004; 57:219– 224.

21. Bernkop-Schnurch A, Leitner V, Moser V. Synthesis and in vitro characterisation of a poly(acrylic acid)–homocysteine conjugate. Drug Dev Ind Pharm 2004; 30:1– 8.

22. Habeeb AF. A sensitive method for localization of disulfide containing peptides in column effluents. Anal Biochem 1973; 56:60–65.

23. Bernkop-Schnurch A. Mucoadhesive polymers, in: Severian Dumitriu (Ed.), Polymeric Biomaterials, 2nd edition, Marcel Dekker, New York, 2002.

24. Roth HJ, Eger K, Troschutz R (Eds.). Pharmazeutische Chemie II: Arzneistoffanalyse, 3rd ed., Thieme Verlag Stuttgart, Germany, 1990, pp. 188–192.

25. Bernkop-Schnurch A, Gilge B. Anionic mucoadhesive polymers as auxiliary agents for the peroral administration of (poly) peptide drugs: influence of the gastric fluid. Drug Dev Ind Pharm 2000; 26:107– 113.

26. Imam ME, Hornof M, Valenta C, Bernkop-Schnu¨rch A. Evidence for the interpenetration of mucoadhesive polymers into the mucus gel layer. STP Pharma 2003; 13:171–176.

27. Hornof MD, Kast CE, Bernkop-Schnurch A. In vitro evaluation of the viscoelastic behavior of chitosan–thioglycolic acid conjugates. Eur J Pharm Biopharm 2003; 55:185– 190.

28. Marschu¨ tz MK, Bernkop-Schnurch A. Thiolated polymers: advance in mucoadhesion by use of in-situ crosslinking poly(acrylic acid)–cysteine conjugates. Eur J Pharm Sci 2002; 15:387– 394.

29. Krauland A, Bernkop-Schnurch A. Thiomers: development and in vitro evaluation of an oral microparticulate peptide delivery system. Eur J Pharm Biopharm 2004; 57:181–187.

30. Leitner VM, Marschutz MK, Bernkop-Schnu¨rch A. Mucoadhesive and cohesive properties of poly(acrylic acid)–cysteine conjugates with regard to their molecular mass. Eur J Pharm Sci 2003; 18:89–96.

31. Guggi D, Marschutz MK, Bernkop-Schnu¨rch A. Matrix tablets based on thiolated poly(acrylic acid): pH-dependent variation in disintegration and mucoadhesion. Int J Pharm 2004; 274:97– 105.

32. Bernkop-Schnurch A, Guggi D, Pinter Y. Thiolated chitosans: development and in vivo evaluation of a mucoadhesive permeation enhancing oral drug delivery system. J Control Release 2004; 94:177–186.

33. Coupe AJ, Davis SS, Wilding IR. Variation in gastrointestinal transit of pharmaceutical dosage forms in healthy subjects. Pharm Res 1991; 8:360–364.

34. Ko JA, Park HJ, Park YS, Hwang SJ, Park JB. Chitosan microparticle preparation for controlled drug release by response surface methodology. J Microencapsul 2003; 20:791– 797.

35. Fundueanu G, Nastruzzi C, Carpov A, Desbrieres J, Rinaudo M. Physico-chemical characterization of Ca-alginate microparticles produced with different methods. Biomaterials 1999; 20:1427– 1435.

36. Bernkop-Schnurch A, Egger C, Elhassan IM, Krauland A. Preparation and in vitro characterization of poly(acrylic acid)–cysteine microparticles. J Control Release 2003; 93:29– 38.

37. Clausen AE, Bernkop-Schnu¨rch A. Development and in vitro evaluation of a peptide drug delivery system based on thiolated polycarbophil, Pharm Ind 2001; 63:312– 317.

38. Guggi D, Krauland AH, Bernkop-Schnu¨rch A. Systemic peptide delivery via the stomach: In vivo evaluation of an oral dosage form for salmon calcitonin. J Control Release 2003; 92:125– 135.

39. Bernkop-Schnurch A, Telsnig J, Hornof M. Development and in vitro evaluation of a mucoadhesive oral delivery system for antisense oligonucleotides. Sci Pharm 2003; 71:165– 177.

40. Kast CE, Valenta C, Leopold M, Bernkop-Schnu¨ rch A. Design and in vitro evaluation of a novel bioadhesive vaginal drug delivery system for clotrimazole. J Control Release 2002; 81:347– 354.

41. Hornof MD, Weyenberg W, Ludwig A, Bernkop-Schnu¨rch A. A mucoadhesive ocular insert: development and in vivo evaluation in humans. J. Control Release 2003; 89:419– 428.

42. Bernkop-Schnurch A, Hornof MD, Kast CE, Langoth N. Thiolated polymers: stability of thiol moieties under different storage conditions. Sci. Pharm 2002; 70:331– 339.

43. Hornof M. In vitro and in vivo evaluation of novel polymeric excipients in the ophthalmic field, Thesis. University of Vienna, 2003.

44. Argu¨eso P, Gipson IK. Epithelial mucins on the ocular surface: structure, biosynthesis and function. Exp Eye Res 2001:281– 289.

Received on 03.01.2009 Modified on 12.04.2009

Research J. Pharm. and Tech.2(2): April.-June.2009,;Page 250-255