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Chemically modified chitosans as enzyme inhibitors
Advanced Drug Delivery Reviews 52 (2001) 127–137 www.elsevier.com / locate / drugdeliv
Chemically modified chitosans as enzyme inhibitors ¨ *, Constantia E. Kast Andreas Bernkop-Schnurch Institute of Pharmaceutical Technology and Biopharmaceutics, Center of Pharmacy, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria Received 13 June 2001
Abstract Because of its permeation enhancing effect (I), mucoadhesive properties (II) and the capability to provide a controlled release of incorporated drugs (III), chitosan represents an advantageous excipient in non-invasive peptide delivery. The use of chitosan for such delivery systems, however, is limited by the lack of inhibitory properties towards secreted and membrane bound enzymes. Due to the covalent attachment of enzyme inhibitors and / or complexing agents at the 2-position of this poly(b1-4-D-glucosamine), chitosans can be transformed into polymers that exhibit inhibitory properties. The immobilization of inhibitors such as antipain, chymostatin, elastatinal and Bowman–Birk inhibitor provide a protective effect towards pancreatic serine proteases, whereas covalently attached complexing agents such as EDTA guarantee the inactivation of membrane bound Zn-dependent peptidases as well as carboxypeptidase A and B. As the inhibition of these enzymes strongly improves the bioavailability of non-invasively administered peptide drugs, chemically modified chitosans represent promising auxiliary polymers. 2001 Elsevier Science B.V. All rights reserved. Keywords: Chitosan; Chitosan derivatives; Chitosan–inhibitor conjugates; Chitosan-complexing agent conjugates; Enzyme inhibition; Non-invasive (poly)peptide delivery
Contents 1. Introduction ............................................................................................................................................................................ 2. Advantages of chitosan in (poly)peptide delivery....................................................................................................................... 2.1. Permeation enhancing effect ............................................................................................................................................. 2.2. Mucoadhesive properties .................................................................................................................................................. 2.3. Controlled drug release..................................................................................................................................................... 3. The enzymatic barrier .............................................................................................................................................................. 4. Chemical modifications of chitosan .......................................................................................................................................... 4.1. Immobilization of protease inhibitors................................................................................................................................. 4.2. Immobilization of complexing agents ................................................................................................................................ 4.2.1. Chitosan–nitrilotriacetic acid .................................................................................................................................. 4.2.2. Chitosan–ethylenediaminetetraacetic acid ................................................................................................................
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Abbreviations: DTPA, diethylenetriaminepentaacetic acid; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol bis(b-aminoethyl ether)-N,N9-tetraacetic acid; NTA, nitrilotriacetic acid *Corresponding author. Tel.: 143-1-4277-55413; fax: 143-1-4277-9554. ¨ E-mail address: [email protected] (A. Bernkop-Schnurch). 0169-409X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 01 )00196-X
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4.2.3. Chitosan–diethylenetriaminepentaacetic acid ........................................................................................................... 4.3. Immobilization of protease inhibitors and complexing agents .............................................................................................. 5. Future trends........................................................................................................................................................................... 6. Conclusion ............................................................................................................................................................................. References ..................................................................................................................................................................................
1. Introduction Within the last decade the aminopolysaccharide chitosan has proved to be a useful excipient in various drug delivery systems, as it exhibits favourable features such as non-toxicity, high cohesive properties and a polycationic character. To date, the polymer has been used in direct tablet compression, for the production of controlled release solid dosage forms or for the improvement of drug dissolution [1]. In addition, chitosan proved successful in the production of controlled release implant systems for a sustained delivery of chemotherapeutic agents over extended periods of time [2]. More recently the mucoadhesive properties and the permeation enhancing effect of chitosan have been exploited for noninvasive peptide and protein application [3,4]. In contrast to other polymers, it can also be used as carrier matrix for the non-invasive administration of therapeutic (poly)peptides, however, chitosan does not exhibit enzyme inhibitory properties. For example, polycarbophil proved to be a potent inhibitor towards trypsin and carboxypeptidase B, whereas chitosan even tended to accelerate the activity of these enzymes [5]. The rapid presystemic metabolism of (poly)peptide drugs being unprotected in polymeric carrier systems without inhibitory properties, however, results in a very poor bioavailability of these therapeutic agents. Although this degradation can be minimized by the co-administration of enzyme inhibitors, their use is more of scientific than of practical relevance. Enzyme inhibitors do not remain concentrated on polymer drug carrier systems. They are diluted in various body fluids and subsequently absorbed from mucosal tissues leading to systemic toxic side effects [6]. The covalent immobilization of enzyme inhibitors to chitosan keeps these auxiliary agents concentrated on the un-absorbable drug carrier matrix, thus providing a strong protective effect for incorporated (poly)peptide drugs [7,8]. In particular at the primary amino
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group at the 2-position of each polymer-subunit, enzyme inhibitors and complexing agents can easily be attached covalently to chitosan, rendering the polymer useful for non-invasive (poly)peptide delivery. The objective of this review is to give an overview of chemically modified chitosans displaying enzyme inhibitory properties. It should provide a good starting point for scientists who are looking forward to making use of such chitosan derivatives for their (poly)peptide delivery systems or inspire them for the development of further chitosan derivatives with enzyme inhibitory properties.
2. Advantages of chitosan in (poly)peptide delivery
2.1. Permeation enhancing effect In the 1990s, Illum et al. [4] showed for the first time that chitosan has a permeation enhancing effect for the paracellular route of absorption, which is important for the transport of hydrophilic compounds such as (poly)peptides across the membrane. Their observation was confirmed by following studies carried out on Caco-2 cell monolayers showing a significant decrease in the transepithelial electrical resistance in the presence of chitosan [9]. So far a strongly increased transport of buserelin, insulin and a vasopressin derivative has been achieved by the addition of chitosan on Caco-2 cell monolayers [5,10]. In the presence of the mucus layer, however, this enhanced transport across the membrane was comparatively lower, as the polymer cannot reach the epithelium because of size limited diffusion and / or competitive charge interactions with mucins [11]. Meanwhile, the permeation enhancing effect of chitosan was verified also in vivo. Lueßen et al. [3], for instance, demonstrated an enhanced intestinal
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absorption of buserelin in rats by the co-administration of chitosan.
2.2. Mucoadhesive properties The adhesion of drug delivery systems at the site of drug absorption offers various advantages for an improved uptake of therapeutic (poly)peptides. (I) In the case of peroral peptide delivery, the released peptide will be degraded by luminally secreted proteases on the way between the dosage form and the absorption membrane. Such a presystemic metabolism is strongly reduced by mucoadhesive formulations providing an intimate contact with the intestinal mucosa. (II) The permeation enhancing effect of chitosan can only take place, if the polymer interacts with the absorption membrane. Hence, the adhesion of the delivery system represents a prerequisite for an enhanced drug uptake mediated by the cationic polymer. (III) Moreover, the adhesion of the delivery system on mucosal membranes provides a high concentration gradient of the drug towards the absorption membrane representing the driving force for the passive paracellular uptake. (IV) A prolonged residence time of the dosage form on mucosal tissues such as the buccal or nasal mucosa leads to an extended time period of drug absorption and subsequently to an improved bioavailability. In the last decade the mucoadhesive properties of chitosan were shown by Hassan and Gallo [12], who explained this phenomenon by electrostatic interactions of the cationic polymer with anionic substructures such as sialic acid moieties of the mucus gel layer. Due to the mucoadhesive properties of chitosan-coated liposomes, for instance, the bioavailability of perorally administered insulin could be strongly improved [13].
2.3. Controlled drug release A further advantage of chitosan in (poly)peptide delivery is a controlled drug release which can be guaranteed by the polymer used as carrier matrix.
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For example, a controlled release of insulin over a period of 6 h was provided by incorporating the drug in chitosan–HCl [14]. The retention of therapeutic peptides in the three-dimensional network of the cationic polymer is thereby achieved by a simple time-dependent hydration and diffusion process and / or ionic interactions between the polymer and anionic substructures of the drug. In contrast to many anionic polymers such as carbomer, in most cases a complete drug release can be guaranteed from chitosan. Aydin and Akbuga [15], for instance, demonstrated a complete release of calcitonin from chitosan beads, whereas the peptide remains adsorptively bound on delivery systems based on carbomer.
3. The enzymatic barrier Although chitosan exhibits all these advantages for non-invasive application of (poly)peptides, its efficacy as carrier matrix is in many cases insufficient as the embedded drugs are subject of a severe presystemic metabolism by proteolytic enzymes. In order to give the audience a brief overview of this substantial limiting factor in (poly)peptide delivery, the enzymatic barrier, which can be divided into secreted proteases (I) mainly found in gastric and intestinal fluids, and membrane bound peptidases (II) localized on the surface of various mucosal tissues will be characterized within this chapter. Luminally secreted proteases include the pepsins being secreted into the stomach lumen via the gastric pits and the pancreatic enzymes trypsin, chymotrypsin, elastase, and carboxypeptidase A and B. The pepsins, however, do not represent a threat to orally administered peptide and protein drugs. Coating materials such as polymethacrylates or celluloseacetate phthalate guarantee stability in the prevailing acid conditions. The compositions seem to be in particular useful for the oral administration of therapeutic peptides and proteins. On the contrary, a protective effect towards pancreatic proteases cannot be provided by a coating material as the therapeutic (poly)peptide has to be released in the intestine which would be excluded by the coating. Pancreatic proteases are highly active in the small intestine and are able to survive transit through the entire human digestive tract. These
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peptidases are some of the most studied of all enzymes. Their substrate specificity and mode of action is well-documented. Based on the knowledge of their specificities and activity in intestinal fluids being described in detail in various reviews, for example, Refs. [6,16], the way and extent of enzymatic degradation of therapeutic polypeptides can easily be predicted. In contrast to secreted proteases which are mainly confined to the digestive tract, membrane bound peptidases are present on the surface of all mucosal tissues. They are all large glycoproteins facing outward from the membrane to hydrolyze peptides that come into contact with the cell surface. Generally, membrane bound peptidases can be divided into endopeptidases, aminopeptidases and carboxypeptidases. Most of these enzymes are metallo-peptidases, i.e. they need metal ions such as zinc in order to maintain their conformation and / or enzymatic activity. Membrane bound peptidases are specialized on the cleavage of peptides displaying a molecular mass especially below approximately 3 kDa [17]. The distribution, specificity and enzymatic activity of membrane bound enzymes has also been documented in various reviews [6,16].
4. Chemical modifications of chitosan
4.1. Immobilization of protease inhibitors The covalent attachment of protease inhibitors to chitosan can be achieved via the primary amino groups or the hydroxyl groups of chitosan. Although the number of hydroxyl groups on the polymer is 2-fold higher and the covalent attachment of protease inhibitors via this functional group would offer the advantage of leaving the cationic character of the polymer uninfluenced, up to now all chemical modifications have been focused on the primary amino group of the polymer. A reason for it are the drastic reaction conditions known from the derivatization of cellulose such as high temperature and pressure [18] causing an inactivation of many enzyme inhibitors and / or an oxidative depolymerisation of the polymer. As many protease inhibitors offer at least one carbonic acid moiety being not located in the active
site of the inhibitor, these auxiliary agents can directly be bound to the primary amino group of chitosan-forming amide bonds. The reaction can be mediated by carbodiimides in aqueous solutions excluding remaining traces of otherwise harmful organic solvents. After the coupling reaction the carbodiimide is transformed to a non-toxic urea derivative which can easily be removed from the polymer. Up to now generated and evaluated chitosan–protease inhibitor conjugates are shown in Fig. 1. In contrast to numerous other polymer– inhibitor conjugates none of them exhibits a spacer between the polymer and the inhibitor, as an unhindered access of proteases to immobilized inhibitors seems to be guaranteed even without it. Generally, the extent of the protective effect can be controlled by the amount of covalently attached inhibitors and / or by the share of the chitosan–inhibitor conjugate in the dosage form. The development of drug delivery systems based on chitosan–inhibitor conjugates, however, revealed that only very low concentrations of the modified polymer are necessary to achieve a strong protective effect. The tryptic degradation of insulin embedded in chitosan as drug carrier matrix, for instance, was significantly reduced by the addition of 5% chitosan–inhibitor conjugate to the delivery system. The result of this study as shown in Fig. 2 is representative for many similar peptide delivery systems containing polymer–inhibitor conjugates [14]. Meanwhile the efficacy of an anionic polymer–inhibitor conjugate was verified in vivo as well [24].
4.2. Immobilization of complexing agents Most brushborder membrane bound enzymes as well as the intestinal secreted proteases carboxypeptidase A and B are zinc-dependent peptidases. Apart from competitive inhibitors their inhibition can also be achieved by complexing agents which are able to deprive the essential divalent cation out of the enzyme structure. The most abundant brushborder membrane bound peptidase aminopeptidase N, for instance, can be strongly inhibited by EDTA, EGTA and 1,10-phenantroline [25]. In vivo, however, the inhibitory effect of complexing agents is strongly reduced by their dilution in body fluids and absorption from the mucosa. In order to keep the complex-
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Fig. 1. Chemical structure of chitosan derivatives displaying enzyme inhibitory properties. (numbers in brackets indicate the cited reference).
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[27]. However, the capability of unmodified chitosan to bind divalent metal ions turned out to be not strong enough in order to inhibit metallo-peptidases. In contrast, the complexing properties of the chitosan derivatives: chitosan–nitrilotriacetic acid (–NTA) [19], chitosan–ethylenediaminetetraacetic acid (–EDTA) [20] and chitosan–diethylenetriaminepentaacetic acid (–DTPA) [21] are much higher. By the immobilization of these complexing agents the cationic polymer becomes anionic displaying an excellent swelling behaviour in neutral and alkaline aqueous solutions.
Fig. 2. Amount of degraded insulin (%) in inner (black bars) and lateral parts (white bars) of matrix tablets consisting of chitosan and a chitosan–antipain conjugate. Tablets were incubated for 1.5 h in 20 mM Tris–HCl pH 7.8 containing 0.2 mg of trypsin (180 BAEE units) per ml at 3760.58C (adapted from Bernkop¨ Schnurch et al. [14]).
ing agent therefore concentrated in the area, where an enzyme inhibition is required, the immobilization of these auxiliary agents on the drug carrier matrix seems to be essential. In contrast to competitive inhibitors, complexing agents can provide an inhibition without a direct interaction with the corresponding enzyme. Polymers with strong complexing properties seem therefore especially suitable for the inhibition of membrane bound enzymes, where the covering mucus gel layer hinders polymer immobilized inhibitors on direct interactions. As illustrated in Fig. 3, polymers with complexing properties can provide an inhibition even through the separating mucus. Previous studies revealed that chitosan per se has excellent metal adsorbing features with much higher selectivity than usual commercial chelating resins and a high loading capacity [26]. This is attributed to the large number of primary amino groups of chitosan with high activity as adsorption sites, high hydrophilicity, and the flexible structure of the polymer chains enabling it to take suitable configuration for the complexation with metal ions
4.2.1. Chitosan–nitrilotriacetic acid In the 1990s, Tikhonov et al. [19] immobilized the complexing agent nitrilotriacetic acid to chitosan. The coupling reaction was mediated by a water soluble carbodiimide leading to chitosan derivatives displaying iminodiacetate moieties. The chemical structure of chitosan–nitrilotriacetic acid is shown in Fig. 1. Raising the amount of carbodiimide during the coupling reaction caused a crosslinking of the polymer chains, as nitrilotriacetic acid was covalently attached to more than only one primary amino group of chitosan. Because of this crosslinking process, the viscosity of the polymer increased tremendously. Iminodiacetate as well as iminomonoacetate residues were able to form 1:1-type complexes with divalent cations. 4.2.2. Chitosan–ethylenediaminetetraacetic acid Chitosan–ethylenediaminetetraacetic acid conjugates were the first chitosan derivatives with complexing properties which have already been tested for pharmaceutical use [28]. The immobilization of ethylenediaminetetraacetic acid (EDTA) on chitosan was achieved by the formation of amide bonds between amino groups of the polymer and carboxyl groups of EDTA. Mediated by a carbodiimide EDTA was linked to almost each primary amino group of chitosan [20]. The ratio of chitosan to EDTA at the reaction had thereby a great impact on the resulting conjugate. Comparatively low shares of EDTA during the coupling reaction led to a crosslinking of the chitosan chains by the complexing agent as illustrated in Fig. 4. Because of this crosslinking process, the viscosity of the polymer was strongly improved. The impact of the amount of EDTA on viscosity and
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Fig. 3. Schematic presentation of the drug uptake from a mucoadhesive polymeric delivery system without inhibitory properties (left hand side) and the uptake from the same delivery system but displaying an immobilized complexing agent (right hand side). The inhibition is provided by the deprivation of zinc ions of membrane bound peptidases through the separating mucus gel layer.
Fig. 4. Chemical structure of a crosslinked chitosan–EDTA conjugate.
mucoadhesive properties of the resulting conjugates is listed in Table 1. Zinc binding studies of these conjugates revealed a significantly lower binding capacity than carbomer or polycarbophil (Fig. 5), whereas the binding affinity as shown in Table 2 was much higher. Furthermore, in comparison to carbomer and polycarbophil chitosan–EDTA conjugates are to a higher extent compatible with divalent cations, e.g. leading only to a coagulation with calcium ions in concentrations higher than 5 mM [29]. Generally, an interference of the complexation of zinc ions by calcium ions with a determined concentration of 0.4–0.7 mM in the intestinal fluid [30] should be negligible, as the association constant of calcium towards EDTA, determined to be 10 10.7 is approximately 10 6 times lower than that of zinc. Due to its strong binding affinity for zinc ions, chitosan– EDTA is a very potent inhibitor of zinc-dependent peptidases. The degradation of the peptide drug leucine enkephalin on porcine mucosa, for instance, could be reduced 3-fold in the presence of chitosan–
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Table 1 Comparison of the degree of modification, viscosity and mucoadhesive properties of different chitosan–EDTA conjugates resulting out of an ¨ increasing share of EDTA during the coupling reaction (adapted from Bernkop-Schnurch and Krajicek [20]) Amount of EDTA in the reaction mixture a (g)
Resulting polymer
Remaining free amino groups (%, 6S.D., n53)
Viscosity (polymer concentration: 0.25%) (mPas / s, 6S.D., n550)
Mucoadhesion: maximum detachment force (mN, 6S.D., n54–9)
– 0.454 0.908 1.815 3.63 7.26
Chitosan HCl Conjugate 1:2.5 Conjugate 1:5 Conjugate 1:10 Conjugate 1:20 Conjugate 1:40
100 6.560.67 5.860.75 3.560.31 0.160.03 0.160.06
ND ND ND 1042674 234673 221676
33.1624.4 9.363.3 21.5611.2 21.662.8 43.7614.9 43.567.2
a
Reaction mixture: 10 ml of 1% chitosan HCl pH 3.0, 0.1 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and increasing amounts of EDTA (in g) as listed above. ND, not determined.
EDTA [31]. Moreover, its inhibitory activity towards the zinc-dependent luminally secreted proteases carboxypeptidase A and B was demonstrated [32]. Apart from its enzyme inhibitory properties, chitosan–EDTA displays improved mucoadhesive properties as shown in Table 1 as well as the possibility of a controlled release of polymer embedded drugs [33].
Fig. 5. Zinc binding capacity of neutralized carbomer (NaC934P), polycarbophil (NaPCP) and different chitosan–EDTA conjugates according to Table 1 at pH 6.5 and 9.0. Each bar represents the mean6S.D. of at least three experiments. (adapted from Bernkop¨ Schnurch and Krajicek [20]).
4.2.3. Chitosan–diethylenetriaminepentaacetic acid As diethylenetriaminepentaacetic acid (DTPA) displays an association constant of 10 18.6 for zinc, which is even higher than that of EDTA determined to be 10 16.5 , it was also covalently bound to chitosan. Although the concentration of DTPA was 2-fold higher than that of EDTA during the coupling reaction, the amount of covalently attached DTPA was comparatively lower. Because of a possible steric hindrance caused by already covalently bound DTPA which might restrict the access for further DTPA molecules to the vicinally located primary amino groups on the polymer, merely 63.865.8%
Table 2 Binding affinity of 0.25% chitosan–EDTA, carbomer (C934P) and polycarbophil towards calcium and zinc (0.05 mg / ml) at pH 6.5 (adapted ¨ from Bernkop-Schnurch and Krajicek [20]) Tested polymer
Polymer bound calcium (%, mean6S.D.; n53–6)
Polymer bound zinc (%, mean6S.D.; n53–6)
Chitosan–EDTA 1:20 Carbomer Polycarbophil
10060 84.962.5 77.663.3
10060 78.062.3 69.466.2
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(n 5 3; 6S.D.) of the primary amino groups of chitosan were modified by DTPA. Hence, chitosan– DTPA exhibits ambiphilic features because of its cationic and anionic sub-structures making it easily swellable in the acid and alkaline milieu. Although DTPA displays a higher association constant towards zinc than EDTA, the inhibitory effect of the chitosan–EDTA conjugate towards carboxypeptidase A and aminopeptidase N was more pronounced than that of the chitosan–DTPA conjugate [21].
4.3. Immobilization of protease inhibitors and complexing agents The inhibition of serine proteases and zinc-dependent peptidases at the same time can be achieved by the combination of protease inhibitors and complexing agents. To the remaining primary amino groups of chitosan–protease inhibitor conjugates, for instance, EDTA can be attached covalently leading to polymers displaying the demanded features. The polymer immobilized inhibitors provide a protective effect towards pancreatic serine proteases, whereas the immobilized complexing agent guarantees an inhibition of zinc-dependent peptidases rendering it useful for the oral administration of (poly)peptide drugs [32]. In another approach the protease inhibitor Bowman–Birk inhibitor [34] was covalently attached to the carboxylic acid groups of a chitosan–EDTA conjugate via its primary amino groups. The resulting chitosan–EDTA BBI conjugate exhibited a strong protective effect towards trypsin and chymotrypsin [35,36]. However, the protective effect towards elastase was markedly lower. Due to the high binding affinity of EDTA towards zinc, the zincdependent exopeptidases carboxypeptidase A and aminopeptidase N were inhibited by this polymerconjugate as well [36]. The incorporation of the protein drug bromelain in chitosan–EDTA BBI used as carrier matrix, for instance, guaranteed a strong protective effect for the therapeutic agent towards a presystemic metabolism by intestinal proteases [37].
5. Future trends Promising new chitosan derivatives represent thiolated chitosans such as chitosan–cysteine and chitosan–thioglycolic acid conjugates. Although
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their enzyme inhibitory capacities have not been evaluated yet, the inhibition of zinc-dependent proteases seems likely. Thiol moieties are capable of binding zinc ions, and therefore they are strong inhibitors for zinc dependent proteases. For example, carboxypeptidase A and B were strongly inhibited by cysteine irrespectively of whether the sulfhydryl compound was immobilized on a polymer or not [38]. According to these results, similar effects might be observed by thiolated chitosans. Apart from this presumptive enzyme inhibitory effect of thiolated chitosans, also other features of these derivatives might be useful for peptide and protein delivery systems. The chitosan–cysteine conjugate displayed a significantly improved permeation enhancing effect compared to the unmodified polymer [22]. This improved permeation enhancing effect by the thiolation of chitosan is in good agreement with other polymers such as polycarbophil also leading to an enhanced drug uptake by the covalent attachment of cysteine [24]. Furthermore, the mucoadhesive properties of chitosan are improved by the immobilization of thiol moieties. Chitosan–thioglycolic acid conjugates, for instance, showed an even 10-fold higher mucoadhesion on porcine mucosa compared to unmodified chitosan [23]. The possible enzyme inhibitory effect of the sulfhydryl moieties in combination with an improved permeation enhancing effect and extended mucoadhesive properties might render such chitosan derivatives useful in non-invasive (poly)peptide delivery.
6. Conclusion Because of a permeation enhancing effect, mucoadhesive properties and a controlled release, which can be provided by incorporating peptide drugs into the polymer, chitosans represent a promising tool for various peptide delivery systems. In contrast to other polymers such as poly(acrylates) displaying similar advantages for peptide delivery, chitosans do not exhibit any enzyme inhibitory properties. In recent years, however, it has been shown that this drawback can be eliminated by simple chemical modifications of the polymer. Although additionally toxicological studies are required for derivatives displaying strong enzyme inhibitory
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properties, the great potential and the high patent strength of modified chitosans should justify this effort in the future.
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¨ , C.E. Kast / Advanced Drug Delivery Reviews 52 (2001) 127 – 137 A. Bernkop-Schnurch ¨ Charac[30] A. Lindahl, A.-L. Ungell, L. Knutson, H. Lennernas, terization of fluids from the stomach and proximal jejunum in men and women, Pharm. Res. 14 (1997) 497–502. ¨ [31] A. Bernkop-Schnurch, Ch. Paikl, C. Valenta, Novel bioadhesive chitosan–EDTA conjugate protects leucine enkephalin from degradation by aminopeptidase N, Pharm. Res. 14 (1997) 917–922. ¨ [32] A. Bernkop-Schnurch, A. Scerbe-Saiko, Synthesis and in vitro evaluation of chitosan–EDTA–protease inhibitor conjugates which might be useful in oral delivery of peptides and proteins, Pharm. Res. 15 (1998) 263–269. ¨ [33] A. Bernkop-Schnurch, C. Humenberger, C. Valenta, Basic studies on bioadhesive delivery systems for peptide and protein drugs, Int. J. Pharm. 165 (1998) 217–225. [34] Y. Birk, The Bowman–Birk inhibitor, Int. J. Pept. Protein Res. 25 (1985) 113–131.
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¨ [35] A. Bernkop-Schnurch, A. Krauland, C. Valenta, Development and in vitro evaluation of a drug delivery system based on chitosan–EDTA BBI conjugate, J. Drug Target. 6 (1998) 207–214. ¨ [36] A. Bernkop-Schnurch, M. Pasta, Intestinal peptide and protein delivery: Novel bioadhesive drug carrier matrix shielding from enzymatic attack, J. Pharm. Sci. 87 (1998) 430–434. ¨ [37] A. Bernkop-Schnurch, R. Giovanelli, C. Valenta, Peroral administration of enzymes: Strategies to improve the galenic of dosage forms for trypsin and bromelain, Drug Dev. Ind. Pharm. 26 (2000) 115–121. ¨ [38] A. Bernkop-Schnurch, S. Thaler, Polycarbophil–cysteine conjugates as platforms for oral (poly)peptide delivery systems, J. Pharm. Sci. 89 (2000) 901–909.
Chemically modified chitosans as enzyme inhibitors ¨ *, Constantia E. Kast Andreas Bernkop-Schnurch Institute of Pharmaceutical Technology and Biopharmaceutics, Center of Pharmacy, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria Received 13 June 2001
Abstract Because of its permeation enhancing effect (I), mucoadhesive properties (II) and the capability to provide a controlled release of incorporated drugs (III), chitosan represents an advantageous excipient in non-invasive peptide delivery. The use of chitosan for such delivery systems, however, is limited by the lack of inhibitory properties towards secreted and membrane bound enzymes. Due to the covalent attachment of enzyme inhibitors and / or complexing agents at the 2-position of this poly(b1-4-D-glucosamine), chitosans can be transformed into polymers that exhibit inhibitory properties. The immobilization of inhibitors such as antipain, chymostatin, elastatinal and Bowman–Birk inhibitor provide a protective effect towards pancreatic serine proteases, whereas covalently attached complexing agents such as EDTA guarantee the inactivation of membrane bound Zn-dependent peptidases as well as carboxypeptidase A and B. As the inhibition of these enzymes strongly improves the bioavailability of non-invasively administered peptide drugs, chemically modified chitosans represent promising auxiliary polymers. 2001 Elsevier Science B.V. All rights reserved. Keywords: Chitosan; Chitosan derivatives; Chitosan–inhibitor conjugates; Chitosan-complexing agent conjugates; Enzyme inhibition; Non-invasive (poly)peptide delivery
Contents 1. Introduction ............................................................................................................................................................................ 2. Advantages of chitosan in (poly)peptide delivery....................................................................................................................... 2.1. Permeation enhancing effect ............................................................................................................................................. 2.2. Mucoadhesive properties .................................................................................................................................................. 2.3. Controlled drug release..................................................................................................................................................... 3. The enzymatic barrier .............................................................................................................................................................. 4. Chemical modifications of chitosan .......................................................................................................................................... 4.1. Immobilization of protease inhibitors................................................................................................................................. 4.2. Immobilization of complexing agents ................................................................................................................................ 4.2.1. Chitosan–nitrilotriacetic acid .................................................................................................................................. 4.2.2. Chitosan–ethylenediaminetetraacetic acid ................................................................................................................
128 128 128 129 129 129 130 130 130 132 132
Abbreviations: DTPA, diethylenetriaminepentaacetic acid; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol bis(b-aminoethyl ether)-N,N9-tetraacetic acid; NTA, nitrilotriacetic acid *Corresponding author. Tel.: 143-1-4277-55413; fax: 143-1-4277-9554. ¨ E-mail address: [email protected] (A. Bernkop-Schnurch). 0169-409X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 01 )00196-X
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4.2.3. Chitosan–diethylenetriaminepentaacetic acid ........................................................................................................... 4.3. Immobilization of protease inhibitors and complexing agents .............................................................................................. 5. Future trends........................................................................................................................................................................... 6. Conclusion ............................................................................................................................................................................. References ..................................................................................................................................................................................
1. Introduction Within the last decade the aminopolysaccharide chitosan has proved to be a useful excipient in various drug delivery systems, as it exhibits favourable features such as non-toxicity, high cohesive properties and a polycationic character. To date, the polymer has been used in direct tablet compression, for the production of controlled release solid dosage forms or for the improvement of drug dissolution [1]. In addition, chitosan proved successful in the production of controlled release implant systems for a sustained delivery of chemotherapeutic agents over extended periods of time [2]. More recently the mucoadhesive properties and the permeation enhancing effect of chitosan have been exploited for noninvasive peptide and protein application [3,4]. In contrast to other polymers, it can also be used as carrier matrix for the non-invasive administration of therapeutic (poly)peptides, however, chitosan does not exhibit enzyme inhibitory properties. For example, polycarbophil proved to be a potent inhibitor towards trypsin and carboxypeptidase B, whereas chitosan even tended to accelerate the activity of these enzymes [5]. The rapid presystemic metabolism of (poly)peptide drugs being unprotected in polymeric carrier systems without inhibitory properties, however, results in a very poor bioavailability of these therapeutic agents. Although this degradation can be minimized by the co-administration of enzyme inhibitors, their use is more of scientific than of practical relevance. Enzyme inhibitors do not remain concentrated on polymer drug carrier systems. They are diluted in various body fluids and subsequently absorbed from mucosal tissues leading to systemic toxic side effects [6]. The covalent immobilization of enzyme inhibitors to chitosan keeps these auxiliary agents concentrated on the un-absorbable drug carrier matrix, thus providing a strong protective effect for incorporated (poly)peptide drugs [7,8]. In particular at the primary amino
134 135 135 135 136
group at the 2-position of each polymer-subunit, enzyme inhibitors and complexing agents can easily be attached covalently to chitosan, rendering the polymer useful for non-invasive (poly)peptide delivery. The objective of this review is to give an overview of chemically modified chitosans displaying enzyme inhibitory properties. It should provide a good starting point for scientists who are looking forward to making use of such chitosan derivatives for their (poly)peptide delivery systems or inspire them for the development of further chitosan derivatives with enzyme inhibitory properties.
2. Advantages of chitosan in (poly)peptide delivery
2.1. Permeation enhancing effect In the 1990s, Illum et al. [4] showed for the first time that chitosan has a permeation enhancing effect for the paracellular route of absorption, which is important for the transport of hydrophilic compounds such as (poly)peptides across the membrane. Their observation was confirmed by following studies carried out on Caco-2 cell monolayers showing a significant decrease in the transepithelial electrical resistance in the presence of chitosan [9]. So far a strongly increased transport of buserelin, insulin and a vasopressin derivative has been achieved by the addition of chitosan on Caco-2 cell monolayers [5,10]. In the presence of the mucus layer, however, this enhanced transport across the membrane was comparatively lower, as the polymer cannot reach the epithelium because of size limited diffusion and / or competitive charge interactions with mucins [11]. Meanwhile, the permeation enhancing effect of chitosan was verified also in vivo. Lueßen et al. [3], for instance, demonstrated an enhanced intestinal
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absorption of buserelin in rats by the co-administration of chitosan.
2.2. Mucoadhesive properties The adhesion of drug delivery systems at the site of drug absorption offers various advantages for an improved uptake of therapeutic (poly)peptides. (I) In the case of peroral peptide delivery, the released peptide will be degraded by luminally secreted proteases on the way between the dosage form and the absorption membrane. Such a presystemic metabolism is strongly reduced by mucoadhesive formulations providing an intimate contact with the intestinal mucosa. (II) The permeation enhancing effect of chitosan can only take place, if the polymer interacts with the absorption membrane. Hence, the adhesion of the delivery system represents a prerequisite for an enhanced drug uptake mediated by the cationic polymer. (III) Moreover, the adhesion of the delivery system on mucosal membranes provides a high concentration gradient of the drug towards the absorption membrane representing the driving force for the passive paracellular uptake. (IV) A prolonged residence time of the dosage form on mucosal tissues such as the buccal or nasal mucosa leads to an extended time period of drug absorption and subsequently to an improved bioavailability. In the last decade the mucoadhesive properties of chitosan were shown by Hassan and Gallo [12], who explained this phenomenon by electrostatic interactions of the cationic polymer with anionic substructures such as sialic acid moieties of the mucus gel layer. Due to the mucoadhesive properties of chitosan-coated liposomes, for instance, the bioavailability of perorally administered insulin could be strongly improved [13].
2.3. Controlled drug release A further advantage of chitosan in (poly)peptide delivery is a controlled drug release which can be guaranteed by the polymer used as carrier matrix.
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For example, a controlled release of insulin over a period of 6 h was provided by incorporating the drug in chitosan–HCl [14]. The retention of therapeutic peptides in the three-dimensional network of the cationic polymer is thereby achieved by a simple time-dependent hydration and diffusion process and / or ionic interactions between the polymer and anionic substructures of the drug. In contrast to many anionic polymers such as carbomer, in most cases a complete drug release can be guaranteed from chitosan. Aydin and Akbuga [15], for instance, demonstrated a complete release of calcitonin from chitosan beads, whereas the peptide remains adsorptively bound on delivery systems based on carbomer.
3. The enzymatic barrier Although chitosan exhibits all these advantages for non-invasive application of (poly)peptides, its efficacy as carrier matrix is in many cases insufficient as the embedded drugs are subject of a severe presystemic metabolism by proteolytic enzymes. In order to give the audience a brief overview of this substantial limiting factor in (poly)peptide delivery, the enzymatic barrier, which can be divided into secreted proteases (I) mainly found in gastric and intestinal fluids, and membrane bound peptidases (II) localized on the surface of various mucosal tissues will be characterized within this chapter. Luminally secreted proteases include the pepsins being secreted into the stomach lumen via the gastric pits and the pancreatic enzymes trypsin, chymotrypsin, elastase, and carboxypeptidase A and B. The pepsins, however, do not represent a threat to orally administered peptide and protein drugs. Coating materials such as polymethacrylates or celluloseacetate phthalate guarantee stability in the prevailing acid conditions. The compositions seem to be in particular useful for the oral administration of therapeutic peptides and proteins. On the contrary, a protective effect towards pancreatic proteases cannot be provided by a coating material as the therapeutic (poly)peptide has to be released in the intestine which would be excluded by the coating. Pancreatic proteases are highly active in the small intestine and are able to survive transit through the entire human digestive tract. These
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peptidases are some of the most studied of all enzymes. Their substrate specificity and mode of action is well-documented. Based on the knowledge of their specificities and activity in intestinal fluids being described in detail in various reviews, for example, Refs. [6,16], the way and extent of enzymatic degradation of therapeutic polypeptides can easily be predicted. In contrast to secreted proteases which are mainly confined to the digestive tract, membrane bound peptidases are present on the surface of all mucosal tissues. They are all large glycoproteins facing outward from the membrane to hydrolyze peptides that come into contact with the cell surface. Generally, membrane bound peptidases can be divided into endopeptidases, aminopeptidases and carboxypeptidases. Most of these enzymes are metallo-peptidases, i.e. they need metal ions such as zinc in order to maintain their conformation and / or enzymatic activity. Membrane bound peptidases are specialized on the cleavage of peptides displaying a molecular mass especially below approximately 3 kDa [17]. The distribution, specificity and enzymatic activity of membrane bound enzymes has also been documented in various reviews [6,16].
4. Chemical modifications of chitosan
4.1. Immobilization of protease inhibitors The covalent attachment of protease inhibitors to chitosan can be achieved via the primary amino groups or the hydroxyl groups of chitosan. Although the number of hydroxyl groups on the polymer is 2-fold higher and the covalent attachment of protease inhibitors via this functional group would offer the advantage of leaving the cationic character of the polymer uninfluenced, up to now all chemical modifications have been focused on the primary amino group of the polymer. A reason for it are the drastic reaction conditions known from the derivatization of cellulose such as high temperature and pressure [18] causing an inactivation of many enzyme inhibitors and / or an oxidative depolymerisation of the polymer. As many protease inhibitors offer at least one carbonic acid moiety being not located in the active
site of the inhibitor, these auxiliary agents can directly be bound to the primary amino group of chitosan-forming amide bonds. The reaction can be mediated by carbodiimides in aqueous solutions excluding remaining traces of otherwise harmful organic solvents. After the coupling reaction the carbodiimide is transformed to a non-toxic urea derivative which can easily be removed from the polymer. Up to now generated and evaluated chitosan–protease inhibitor conjugates are shown in Fig. 1. In contrast to numerous other polymer– inhibitor conjugates none of them exhibits a spacer between the polymer and the inhibitor, as an unhindered access of proteases to immobilized inhibitors seems to be guaranteed even without it. Generally, the extent of the protective effect can be controlled by the amount of covalently attached inhibitors and / or by the share of the chitosan–inhibitor conjugate in the dosage form. The development of drug delivery systems based on chitosan–inhibitor conjugates, however, revealed that only very low concentrations of the modified polymer are necessary to achieve a strong protective effect. The tryptic degradation of insulin embedded in chitosan as drug carrier matrix, for instance, was significantly reduced by the addition of 5% chitosan–inhibitor conjugate to the delivery system. The result of this study as shown in Fig. 2 is representative for many similar peptide delivery systems containing polymer–inhibitor conjugates [14]. Meanwhile the efficacy of an anionic polymer–inhibitor conjugate was verified in vivo as well [24].
4.2. Immobilization of complexing agents Most brushborder membrane bound enzymes as well as the intestinal secreted proteases carboxypeptidase A and B are zinc-dependent peptidases. Apart from competitive inhibitors their inhibition can also be achieved by complexing agents which are able to deprive the essential divalent cation out of the enzyme structure. The most abundant brushborder membrane bound peptidase aminopeptidase N, for instance, can be strongly inhibited by EDTA, EGTA and 1,10-phenantroline [25]. In vivo, however, the inhibitory effect of complexing agents is strongly reduced by their dilution in body fluids and absorption from the mucosa. In order to keep the complex-
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Fig. 1. Chemical structure of chitosan derivatives displaying enzyme inhibitory properties. (numbers in brackets indicate the cited reference).
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[27]. However, the capability of unmodified chitosan to bind divalent metal ions turned out to be not strong enough in order to inhibit metallo-peptidases. In contrast, the complexing properties of the chitosan derivatives: chitosan–nitrilotriacetic acid (–NTA) [19], chitosan–ethylenediaminetetraacetic acid (–EDTA) [20] and chitosan–diethylenetriaminepentaacetic acid (–DTPA) [21] are much higher. By the immobilization of these complexing agents the cationic polymer becomes anionic displaying an excellent swelling behaviour in neutral and alkaline aqueous solutions.
Fig. 2. Amount of degraded insulin (%) in inner (black bars) and lateral parts (white bars) of matrix tablets consisting of chitosan and a chitosan–antipain conjugate. Tablets were incubated for 1.5 h in 20 mM Tris–HCl pH 7.8 containing 0.2 mg of trypsin (180 BAEE units) per ml at 3760.58C (adapted from Bernkop¨ Schnurch et al. [14]).
ing agent therefore concentrated in the area, where an enzyme inhibition is required, the immobilization of these auxiliary agents on the drug carrier matrix seems to be essential. In contrast to competitive inhibitors, complexing agents can provide an inhibition without a direct interaction with the corresponding enzyme. Polymers with strong complexing properties seem therefore especially suitable for the inhibition of membrane bound enzymes, where the covering mucus gel layer hinders polymer immobilized inhibitors on direct interactions. As illustrated in Fig. 3, polymers with complexing properties can provide an inhibition even through the separating mucus. Previous studies revealed that chitosan per se has excellent metal adsorbing features with much higher selectivity than usual commercial chelating resins and a high loading capacity [26]. This is attributed to the large number of primary amino groups of chitosan with high activity as adsorption sites, high hydrophilicity, and the flexible structure of the polymer chains enabling it to take suitable configuration for the complexation with metal ions
4.2.1. Chitosan–nitrilotriacetic acid In the 1990s, Tikhonov et al. [19] immobilized the complexing agent nitrilotriacetic acid to chitosan. The coupling reaction was mediated by a water soluble carbodiimide leading to chitosan derivatives displaying iminodiacetate moieties. The chemical structure of chitosan–nitrilotriacetic acid is shown in Fig. 1. Raising the amount of carbodiimide during the coupling reaction caused a crosslinking of the polymer chains, as nitrilotriacetic acid was covalently attached to more than only one primary amino group of chitosan. Because of this crosslinking process, the viscosity of the polymer increased tremendously. Iminodiacetate as well as iminomonoacetate residues were able to form 1:1-type complexes with divalent cations. 4.2.2. Chitosan–ethylenediaminetetraacetic acid Chitosan–ethylenediaminetetraacetic acid conjugates were the first chitosan derivatives with complexing properties which have already been tested for pharmaceutical use [28]. The immobilization of ethylenediaminetetraacetic acid (EDTA) on chitosan was achieved by the formation of amide bonds between amino groups of the polymer and carboxyl groups of EDTA. Mediated by a carbodiimide EDTA was linked to almost each primary amino group of chitosan [20]. The ratio of chitosan to EDTA at the reaction had thereby a great impact on the resulting conjugate. Comparatively low shares of EDTA during the coupling reaction led to a crosslinking of the chitosan chains by the complexing agent as illustrated in Fig. 4. Because of this crosslinking process, the viscosity of the polymer was strongly improved. The impact of the amount of EDTA on viscosity and
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Fig. 3. Schematic presentation of the drug uptake from a mucoadhesive polymeric delivery system without inhibitory properties (left hand side) and the uptake from the same delivery system but displaying an immobilized complexing agent (right hand side). The inhibition is provided by the deprivation of zinc ions of membrane bound peptidases through the separating mucus gel layer.
Fig. 4. Chemical structure of a crosslinked chitosan–EDTA conjugate.
mucoadhesive properties of the resulting conjugates is listed in Table 1. Zinc binding studies of these conjugates revealed a significantly lower binding capacity than carbomer or polycarbophil (Fig. 5), whereas the binding affinity as shown in Table 2 was much higher. Furthermore, in comparison to carbomer and polycarbophil chitosan–EDTA conjugates are to a higher extent compatible with divalent cations, e.g. leading only to a coagulation with calcium ions in concentrations higher than 5 mM [29]. Generally, an interference of the complexation of zinc ions by calcium ions with a determined concentration of 0.4–0.7 mM in the intestinal fluid [30] should be negligible, as the association constant of calcium towards EDTA, determined to be 10 10.7 is approximately 10 6 times lower than that of zinc. Due to its strong binding affinity for zinc ions, chitosan– EDTA is a very potent inhibitor of zinc-dependent peptidases. The degradation of the peptide drug leucine enkephalin on porcine mucosa, for instance, could be reduced 3-fold in the presence of chitosan–
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Table 1 Comparison of the degree of modification, viscosity and mucoadhesive properties of different chitosan–EDTA conjugates resulting out of an ¨ increasing share of EDTA during the coupling reaction (adapted from Bernkop-Schnurch and Krajicek [20]) Amount of EDTA in the reaction mixture a (g)
Resulting polymer
Remaining free amino groups (%, 6S.D., n53)
Viscosity (polymer concentration: 0.25%) (mPas / s, 6S.D., n550)
Mucoadhesion: maximum detachment force (mN, 6S.D., n54–9)
– 0.454 0.908 1.815 3.63 7.26
Chitosan HCl Conjugate 1:2.5 Conjugate 1:5 Conjugate 1:10 Conjugate 1:20 Conjugate 1:40
100 6.560.67 5.860.75 3.560.31 0.160.03 0.160.06
ND ND ND 1042674 234673 221676
33.1624.4 9.363.3 21.5611.2 21.662.8 43.7614.9 43.567.2
a
Reaction mixture: 10 ml of 1% chitosan HCl pH 3.0, 0.1 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and increasing amounts of EDTA (in g) as listed above. ND, not determined.
EDTA [31]. Moreover, its inhibitory activity towards the zinc-dependent luminally secreted proteases carboxypeptidase A and B was demonstrated [32]. Apart from its enzyme inhibitory properties, chitosan–EDTA displays improved mucoadhesive properties as shown in Table 1 as well as the possibility of a controlled release of polymer embedded drugs [33].
Fig. 5. Zinc binding capacity of neutralized carbomer (NaC934P), polycarbophil (NaPCP) and different chitosan–EDTA conjugates according to Table 1 at pH 6.5 and 9.0. Each bar represents the mean6S.D. of at least three experiments. (adapted from Bernkop¨ Schnurch and Krajicek [20]).
4.2.3. Chitosan–diethylenetriaminepentaacetic acid As diethylenetriaminepentaacetic acid (DTPA) displays an association constant of 10 18.6 for zinc, which is even higher than that of EDTA determined to be 10 16.5 , it was also covalently bound to chitosan. Although the concentration of DTPA was 2-fold higher than that of EDTA during the coupling reaction, the amount of covalently attached DTPA was comparatively lower. Because of a possible steric hindrance caused by already covalently bound DTPA which might restrict the access for further DTPA molecules to the vicinally located primary amino groups on the polymer, merely 63.865.8%
Table 2 Binding affinity of 0.25% chitosan–EDTA, carbomer (C934P) and polycarbophil towards calcium and zinc (0.05 mg / ml) at pH 6.5 (adapted ¨ from Bernkop-Schnurch and Krajicek [20]) Tested polymer
Polymer bound calcium (%, mean6S.D.; n53–6)
Polymer bound zinc (%, mean6S.D.; n53–6)
Chitosan–EDTA 1:20 Carbomer Polycarbophil
10060 84.962.5 77.663.3
10060 78.062.3 69.466.2
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(n 5 3; 6S.D.) of the primary amino groups of chitosan were modified by DTPA. Hence, chitosan– DTPA exhibits ambiphilic features because of its cationic and anionic sub-structures making it easily swellable in the acid and alkaline milieu. Although DTPA displays a higher association constant towards zinc than EDTA, the inhibitory effect of the chitosan–EDTA conjugate towards carboxypeptidase A and aminopeptidase N was more pronounced than that of the chitosan–DTPA conjugate [21].
4.3. Immobilization of protease inhibitors and complexing agents The inhibition of serine proteases and zinc-dependent peptidases at the same time can be achieved by the combination of protease inhibitors and complexing agents. To the remaining primary amino groups of chitosan–protease inhibitor conjugates, for instance, EDTA can be attached covalently leading to polymers displaying the demanded features. The polymer immobilized inhibitors provide a protective effect towards pancreatic serine proteases, whereas the immobilized complexing agent guarantees an inhibition of zinc-dependent peptidases rendering it useful for the oral administration of (poly)peptide drugs [32]. In another approach the protease inhibitor Bowman–Birk inhibitor [34] was covalently attached to the carboxylic acid groups of a chitosan–EDTA conjugate via its primary amino groups. The resulting chitosan–EDTA BBI conjugate exhibited a strong protective effect towards trypsin and chymotrypsin [35,36]. However, the protective effect towards elastase was markedly lower. Due to the high binding affinity of EDTA towards zinc, the zincdependent exopeptidases carboxypeptidase A and aminopeptidase N were inhibited by this polymerconjugate as well [36]. The incorporation of the protein drug bromelain in chitosan–EDTA BBI used as carrier matrix, for instance, guaranteed a strong protective effect for the therapeutic agent towards a presystemic metabolism by intestinal proteases [37].
5. Future trends Promising new chitosan derivatives represent thiolated chitosans such as chitosan–cysteine and chitosan–thioglycolic acid conjugates. Although
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their enzyme inhibitory capacities have not been evaluated yet, the inhibition of zinc-dependent proteases seems likely. Thiol moieties are capable of binding zinc ions, and therefore they are strong inhibitors for zinc dependent proteases. For example, carboxypeptidase A and B were strongly inhibited by cysteine irrespectively of whether the sulfhydryl compound was immobilized on a polymer or not [38]. According to these results, similar effects might be observed by thiolated chitosans. Apart from this presumptive enzyme inhibitory effect of thiolated chitosans, also other features of these derivatives might be useful for peptide and protein delivery systems. The chitosan–cysteine conjugate displayed a significantly improved permeation enhancing effect compared to the unmodified polymer [22]. This improved permeation enhancing effect by the thiolation of chitosan is in good agreement with other polymers such as polycarbophil also leading to an enhanced drug uptake by the covalent attachment of cysteine [24]. Furthermore, the mucoadhesive properties of chitosan are improved by the immobilization of thiol moieties. Chitosan–thioglycolic acid conjugates, for instance, showed an even 10-fold higher mucoadhesion on porcine mucosa compared to unmodified chitosan [23]. The possible enzyme inhibitory effect of the sulfhydryl moieties in combination with an improved permeation enhancing effect and extended mucoadhesive properties might render such chitosan derivatives useful in non-invasive (poly)peptide delivery.
6. Conclusion Because of a permeation enhancing effect, mucoadhesive properties and a controlled release, which can be provided by incorporating peptide drugs into the polymer, chitosans represent a promising tool for various peptide delivery systems. In contrast to other polymers such as poly(acrylates) displaying similar advantages for peptide delivery, chitosans do not exhibit any enzyme inhibitory properties. In recent years, however, it has been shown that this drawback can be eliminated by simple chemical modifications of the polymer. Although additionally toxicological studies are required for derivatives displaying strong enzyme inhibitory
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properties, the great potential and the high patent strength of modified chitosans should justify this effort in the future.
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