Synthesis and characterization of a chitosan-N-acetyl cysteine conjugate.

The aim of the present study was to synthesize and characterize a novel thiolated polymer by covalent attachment of N-acetyl cysteine to chitosan. The obtained conjugate was characterized in vitro by quantification of immobilized thiol groups and their pH dependent oxidation, swelling behaviour in artificial intestinal fluid at pH 6.8, rheological properties and evaluation of its mucoadhesive properties on freshly excised porcine mucosa. The chitosan-N-acetyl cysteine conjugate was synthesized via a carbodiimide mediated coupling reaction displaying up to 325.5+/-41.8 micromol of immobilized thiol groups per gram polymer. 79% of the total amount of thiol groups was oxidized to disulfide groups during the coupling reaction. Adhesion studies on the mucosa indicate that the resulting polymer shows a 50-fold longer residence time on the mucosa and 8.3-fold higher total work of adhesion necessary to detach a flat-faced polymeric tablet from the mucosa in comparison to unmodified chitosan. Swelling properties at pH 6.8 were rather limited displaying only 5% of increment in weight after 2h of experiment. Within 1h the viscosity of an aqueous chitosan-N-acetyl cysteine conjugate mixture at 37 degrees C, pH 5.0 decreased by 35% after addition of hen white egg lysozyme demonstrating its biodegradability. Because of these features chitosan-N-acetyl cysteine seems to represent a promising novel tool, which might be useful in particular for the development of mucoadhesive and biodegradable formulations.

Viscoelastic properties of a new in situ gelling thiolated chitosan conjugate.

The aim of this study was the synthesis of a new thiolated chitosan conjugate and the evaluation of its viscoelastic properties in vitro. The modification of chitosan was achieved by covalent attachment of isopropyl-S-acetylthioacetimidate to chitosan. The resulting conjugate (chitosan-TEA; chitosan-thioethylamidine) exhibited 300.7+/-27.4 micromol thiol groups per gram polymer and no disulfide bond. For rheological studies, the pH of 0.5% and 1% polymer solutions was adjusted to 6.5 in order to simulate a physiological pH-level. Both, 0.5% and 1% chitosan-TEA solutions showed the transition from sol to gel within 30 min. Within 6 h of incubation, the storage modulus of 0.5% and 1% chitosan-TEA increased 3354-fold and 6199-fold, whereas the loss modulus increased 11-fold and 38-fold, respectively. Frequency sweep measurements demonstrated an increase in crosslinking of the thiolated polymer as a function of time. The formation of inter- and/or intramolecular disulfide bonds was monitored indirectly via determining the decrease of thiol groups. Unmodified chitosan did not exhibit in situ gelling properties. The release of a fluorescent marker being incorporated in a 0.5% chitosan-TEA solution was significantly (p<0.001) slower, when the formulation was preincubated for one hour and consequently already highly crosslinked. The polymer generated within this study represents a promising novel tool for various drug delivery systems, where in situ gelling properties are advantageous.

Synthesis and in vitro evaluation of a novel thiolated chitosan.

In order to achieve the same properties as chitosan-4-thio-butyl-amidine and to overcome at the same time its insufficient stability, the aim of this study was to evaluate the imidoester reaction of isopropyl-S-acetylthioacetimidate for the chemical modification of chitosan and to study the properties of the resulting chitosan-thioethylamidine (TEA) derivative. The thioalkylamidine substitute was introduced without the formation of N-substituted non-thiol products. The resulting conjugates exhibited 1.05+/-0.17% or 139.68+/-17.13 micromol immobilized free thiol groups per gram polymer and a total amount of reduced and oxidized thiol groups of 1.81+/-0.65% or 179.46+/-67.95 micromol/g polymer. By the immobilization of thiol groups mucoadhesion was strongly improved due to the formation of disulfide bonds with mucus glycoproteins. Chitosan-TEA was investigated regarding to its mucoadhesive properties via tensile studies and the rotating cylinder method. In tensile studies the total work of adhesion of chitosan-TEA was increased 3.3-fold in comparison to unmodified chitosan. Results from the rotating cylinder method showed an improvement ratio of 8.9 for chitosan-TEA compared with unmodified chitosan. In spite of the immobilization of thiol groups onto chitosan its swelling behavior in aqueous solutions was not significantly altered. Cumulative release studies out of matrix tablets comprising the chitosan-TEA and the model compound fluorescence labeled dextrane (FD(4)) demonstrated a controlled release over 3h with a trend toward a pseudo-zero-order kinetic. Because of these features the new chitosan thioamidine conjugate might represent a promising new polymeric excipient for various drug delivery systems.

Comparative evaluation of cytotoxicity of a glucosamine-TBA conjugate and a chitosan-TBA conjugate.

D-glucosamine and chitosan were modified by the immobilization of thiol groups utilizing 2-iminothiolane. The toxicity profile of the resulting D-glucosamine-TBA (4-thiobutylamidine) conjugate, of chitosan-TBA conjugate and of the corresponding unmodified controls was evaluated in vitro. On the one hand, the cell membrane damaging effect of 0.025% solutions of the test compounds was investigated via red blood cell lysis test. On the other hand, the cytotoxity of 0.025, 0.25 and 0.5% solutions of the test compounds was evaluated on L-929 mouse fibroblast cells utilizing two different bioassays: the MTT assay (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide), which assess the mitochondrial metabolic activity of the cells, and the BrdU-based enzyme-linked immunosorbent assay, which measures the incorporation in the DNA of 5-bromo-2'-deoxyuridine and consequently the cell proliferation. Results of the red blood cell lysis test showed that both thiolated compounds displayed a lower membrane damaging effect causing a significantly lower haemoglobine release than the unmodified compounds. Data obtained by the MTT assay and the BrdU assay revealed a concentration dependent relative cytotoxicity for all tested compounds. The covalent linkage of the TBA-substructure to D-glucosamine did not cause a significant increase in cytotoxicity, whereas at higher concentrations a slightly enhanced cytotoxic effect was caused by the derivatisation of chitosan. In conclusion, the -TBA derivatives show a comparable toxicity profile to the corresponding unmodified compounds, which should not compromise their future use as save pharmaceutical excipients.

Thiomers for oral delivery of hydrophilic macromolecular drugs.

In recent years thiolated polymers (thiomers) have appeared as a promising new tool in oral drug delivery. Thiomers are obtained by the immobilisation of thio-bearing ligands to mucoadhesive polymeric excipients. By the formation of disulfide bonds with mucus glycoproteins, the mucoadhesive properties of thiomers are up to 130-fold improved compared with the corresponding unmodified polymers. Owing to the formation of inter- and intramolecular disulfide bonds within the thiomer itself, matrix tablets and particulate delivery systems show strong cohesive properties, resulting in comparatively higher stability, prolonged disintegration times and a more controlled drug release. The permeation of hydrophilic macromolecular drugs through the gastrointestinal (GI) mucosa can be improved by the use of thiomers. Furthermore, some thiomers exhibit improved inhibitory properties towards GI peptidases. The efficacy of thiomers in oral drug delivery has been demonstrated by various in vivo studies. A pharmacological efficacy of 1%, for example, was achieved in rats by oral administration of calcitonin tablets comprising a thiomer. Furthermore, tablets comprising a thiomer and pegylated insulin resulted in a pharmacological efficacy of 7% after oral application to diabetic mice. Low-molecular-weight heparin embedded in thiolated polycarbophil led to an absolute bioavailability of > or = 20% after oral administration to rats. In these studies, formulations comprising the corresponding unmodified polymer had only a marginal or no effect. These results indicate drug carrier systems based on thiomers appear to be a promising tool for oral delivery of hydrophilic macromolecular drugs.