Constantia E. Kast

Thiolated polymers — thiomers: development and in vitro evaluation of chitosan–thioglycolic acid conjugates

The aim of this study was to improve mucoadhesive properties of chitosan by the covalent attachment of thiol moieties to this cationic polymer. Mediated by a carbodiimide, thioglycolic acid (TGA) was covalently attached to chitosan. This was achieved by the formation of amide bonds between the primary amino groups of the polymer and the carboxylic acid group of TGA. Dependent on the pH-value and the weight ratio of polymer to TGA during the coupling reaction the resulting thiolated polymers, the so-called thiomers, displayed 6.58, 9.88, 27.44, and 38.23 micromole thiol groups per gram polymer. Tensile studies carried out with these chitosan-TGA conjugates on freshly excised porcine intestinal mucosa demonstrated a 6.3-, 8.6-, 8.9-, and 10.3-fold increase in the total work of adhesion (TWA) compared to the unmodified polymer, respectively. In contrast, the combination of chitosan and free unconjugated TGA showed almost no mucoadhesion. These data were in good correlation with further results obtained by another mucoadhesion test demonstrating a prolonged residence time of thiolated chitosan on porcine mucosa. The swelling behavior of all conjugates was thereby exactly in the same range as for an unmodified polymer pretreated in the same way. Furthermore, it could be shown that chitosan-TGA conjugates are still biodegradable by the glycosidase lysozyme. According to these results. chitosan-TGA conjugates represent a promising tool for the development of mucoadhesive drug delivery systems.

Improvement in the mucoadhesive properties of alginate by the covalent attachment of cysteine

The purpose of the present study was to improve the mucoadhesive properties of alginate by the covalent attachment of cysteine. Mediated by a carbodiimide, L-cysteine was covalently linked to the polymer. The resulting thiolated alginate displayed 340.4+/-74.9 micromol thiol groups per g conjugate (means+/-S.D.; n=4). Within 2 h the viscosity of an aqueous mucus/alginate-cysteine conjugate mixture pH 7.0 increased at 37 degrees C by more than 50% compared to a mucus/alginate mixture, indicating enlarged interactions between the mucus and the thiolated polymer. Tensile studies carried out on freshly excised porcine intestinal mucosa demonstrated a total work of adhesion (TWA) of 25.8+/-0.6 and 101.6+/-36.1 microJ for alginate and the alginate-cysteine conjugate, respectively (means+/-S.D.; n=5). The maximum detachment force (MDF) was thereby in good correlation with the TWA. Due to the immobilization of cysteine, the swelling velocity of the polymer was significantly accelerated (P<0.05). In aqueous media the alginate-cysteine conjugate was capable of forming inter- and/or intramolecular disulfide bonds. Because of this crosslinking process within the polymeric network, the cohesive properties of the conjugate were also improved. Tablets comprising the unmodified polymer disintegrated within 49+/-14.5 min, whereas tablets of thiolated alginate remained stable for 148.8+/-39.1 min (means+/-S.D.; n=3). These features should render thiolated alginate useful as excipient for various drug delivery systems providing an improved stability and a prolonged residence time on certain mucosal epithelia.

The role of glutathione in the permeation enhancing effect of thiolated polymers.

AbstractPurpose. To verify or refute the mechanism of permeation enhancement with thiolated polymers via GSH by the use of NaFlu as marker for the paracellular permeation. Methods. The capability of 0.5% polycarbophil cysteine conjugate (PCP-Cys) to reduce 0.02% oxidized glutathione (GSSG) was evaluated via iodometric titration in aqueous solution. Glutathione in its reduced form (GSH; 0.1%-0.4%) and in combination with 0.5% PCP-Cys were tested for their permeation enhancement of sodium fluorescein (NaFlu) and fluorescence labeled bacitracin (bac-FITC) used as paracellular markers. Permeation studies across guinea pig duodenum were carried out in Ussing-type chambers. Opening of the tight junctions was additionally monitored by transepithelial electrical resistance (TEER) measurements. Results. PCP-Cys (0.5%) was shown to reduce 22.0% ± 8.2% of GSSG (0.02%) to GSH in aqueous solution at pH 7.0 and 37°C within 3 h. Permeation of NaFlu was shown to depend on the concentration of GSH. The apparent permeability coefficient (Papp) of NaFlu in buffer only was 4.98 ± 0.5*10-6, while in the presence of 0.4% GSH a Papp of 9.31 ± 0.92*10-6 was achieved, representing an enhancement ratio (R = Papp enhancer system/Papp control) of 1.86. The combination of GSH (0.4%) with PCP-Cys (0.5%) led to a significant (p < 0.001) improvement of R for NaFlu up to 2.93 accompanied by a decrease in TEER of 20.3% ± 1.4%. Incubation of bac-FITC with the same GSH / PCP-Cys combination led to an enhancement ratio of 2.06 within 3 h. Conclusion. GSH plays an important role in the opening of tight junctions of intestinal epithelia. It would appear that PCP-Cys is able to reduce GSSG, prolonging the concentration of GSH at the apical membrane, resulting in significantly enhanced paracellular transport.

Design and in vitro evaluation of a novel bioadhesive vaginal drug delivery system for clotrimazole.

It was the purpose of this study to design and evaluate a new bioadhesive vaginal drug delivery system for clotrimazole. Chitosan, a cationic biopolymer derived by deacetylation of chitin, was modified by the introduction of thioglycolic acid (TGA). The modification was achieved by utilising a carbodiimide to link the carboxylic acid moieties of TGA covalently to the primary amino groups of chitosan. The amount of added carbodiimide was thereby varied, resulting in chitosan-TGA conjugates A and B with 160 microM (=micromol) and 280 microM thiol groups per gram polymer, respectively. In order to characterise the new polymers the water uptake, the disintegration behaviour, the bioadhesive properties utilising the rotating cylinder method, as well as the release of clotrimazole from tablets based on these derivatives were studied. The water uptake and cohesive properties of vaginal tablets consisting of these new conjugates could be significantly (p<0.05) improved. By adding clotrimazole the disintegration time of the conjugates was prolonged 1.6-fold for conjugate A and even 100-fold for conjugate B. Furthermore, the adhesion on vaginal mucosal tissue could be significantly improved. The addition of clotrimazole had also an impact on the adhesion time of chitosan-TGA conjugate B, which remained 26-times longer on vaginal mucosa than the corresponding unmodified polymer. The immobilisation of thiol groups guarantees a controlled drug release. Results of this study demonstrate that these new chitosan-TGA conjugates are very promising vehicles for the vaginal application of clotrimazole in treatment of mycotic infections.

In Vivo Evaluation of an Oral Salmon Calcitonin-Delivery System Based on a Thiolated Chitosan Carrier Matrix

AbstractPurpose. To develop and evaluate an oral delivery system for salmon calcitonin. Methods. 2-Iminothiolane was covalently bound to chitosan in order to improve the mucoadhesive and cohesive properties of the polymer. The resulting chitosan-TBA conjugate (chitosan-4-thiobutylamidine conjugate) was homogenized with salmon calcitonin, mannitol, and a chitosan-Bowman-Birk inhibitor conjugate and a chitosan-elastatinal conjugate (6.75 + 0.25 + 1 + 1 + 1). Optionally 0.5% (m/m) reduced glutathione, used as permeation mediator, was added. Each mixture was compressed to 2 mg microtablets and enteric coated with a polymethacrylate. Biofeedback studies were performed in rats by oral administration of the delivery system and determination of the decrease in plasma calcium level as a function of time. Results. Test formulations led to a significant (p < 0.005) decrease in the plasma calcium level of the dosed animals in comparison to control tablets being based on unmodified chitosan. The addition of glutathione in the tablets led to a further improvement in the oral bioavailability of salmon calcitonin with an earlier onset of action and a decrease in the calcium level of about 10% for at least 10 h. Conclusions. The combination of mucoadhesive thiolated chitosan, chitosan-enzyme-inhibitor conjugates and the permeation mediator glutathione seems to represent a promising strategy for the oral delivery of salmon calcitonin.

Development and in Vivo Evaluation of an Oral Delivery System for Low Molecular Weight Heparin Based on Thiolated Polycarbophil

AbstractPurpose. It was the purpose of this study to develop a new oral drug delivery system for low molecular weight heparin (LMWH) providing an improved bioavailability and a prolonged therapeutic effect. Methods. The permeation enhancing polycarbophil-cysteine conjugate (PCP-Cys) used in this study displayed 111.4 ± 6.4 μM thiol groups per gram polymer. Permeation studies on freshly excised intestinal mucosa were performed in Ussing chambers demonstrating a 2-fold improved uptake of heparin as a result of the addition of 0.5% (w/v) PCP-Cys and the permeation mediator glutathione (GSH). Results. Tablets containing PCP-Cys, GSH, and 279 IU of LMWH showed a sustained drug release over 4 h. To guarantee the swelling of the polymeric carrier matrix in the small intestine tablets were enteric coated. They were orally given to rats. For tablets being based on the thiomer/GSH system an absolute bioavailability of 19.9 ± 9.3% (means ± SD; n = 5) vs. intravenous injection could be achieved, whereas tablets comprising unmodified PCP did not lead to a significant (p < 0.01) heparin concentration in plasma. The permeation enhancing effect and subsequently a therapeutic heparin level was maintained for 24 h after a single dose. Conclusions. Because of the strong and prolonged lasting permeation enhancing effect of the thiomer/GSH system, the oral bioavailability of LMWH could be significantly improved. This new delivery system represents therefore a promising tool for the oral administration of heparin.

Development and in vitro evaluation of a mucoadhesive vaginal delivery system for progesterone.

The purpose of the present study was to design a novel carrier system based on a mucoadhesive polymer exhibiting improved properties concerning drug delivery to the vaginal mucosa. This was reached by the covalent attachment of L-cysteine to commercially available polyacrylic acid (Carbopol 974P). Mediated by a carbodiimide, increasing amounts of L-cysteine were covalently linked to the polymer. The resulting thiolated polyacrylic acid conjugates (NaC974P-Cys) displayed between 24.8 and 45.8 micromol thiol groups per gram of polymer. Because of the formation of intra- and/or intermolecular disulfide bonds, the viscosity of an aqueous thiolated polymer gel (3%) increased about 50% at pH 7.0 within 1 h. In oscillatory rheological measurements, it was shown that this increase in viscosity is mainly due to the increase in elasticity. Tensile studies carried out on freshly excised cow vagina demonstrated a significant (P<0.05) increase in the total work of adhesion (TWA) compared to the unmodified polymer. An amount of 24.8 micromol thiol groups per gram of polymer resulted in a 1.45-fold increase in the TWA, whereas an amount of 45.8 micromol showed an even 2.28-fold increase. These improved mucoadhesive properties can be explained by the formation of disulfide bonds between the thiolated polymer and cysteine rich subdomaines of the mucus layer. The release rate of the model drug progesterone from tablets based on microcrystalline cellulose serving as the reference was approximately 1% per hour, whereas it was 0.58% per hour for the unmodified polymer (NaC974P) and 0.12% per hour for the thiolated polymer (NaC974P-Cys). Therefore, this thiolated polymer is a promising carrier for progesterone providing a prolonged residence time and a controlled drug release.

Chitosan-thioglycolic acid conjugate: a new scaffold material for tissue engineering?

It was the aim of this study to evaluate chitosan-thioglycolic acid (chitosan-TGA) conjugate as scaffold material in tissue engineering. Chitosan was modified by the introduction of thiol groups. Briefly, TGA was introduced to chitosan via amide bond formation mediated by a carbodiimide. The properties of the resulting polymer were thereby altered in regard to water solubility, mucoadhesion, biodegradability and in situ gelling compared to the original polymer. Due to the immobilised thiol groups (240+/-30 micromol thiol groups per gram polymer), the viscosity of a 1.5% chitosan-TGA solution was improved 4.3-fold. This can be explained by the formation of disulphide bonds within this polymeric network. The conjugate was tested as scaffold material in form of a gel and sheets. Furthermore, the influence of the thiol groups on the viability of L-929 mouse fibroblasts was evaluated. It was shown that the L-929 mouse fibroblasts grew on both scaffolds despite the thiol groups, although the different surface conditions seemed to have an influence on the growing rate. Chitosan-TGA sheets seemed to be the more preferred layer. The improved in situ gelling may be important for ongoing developments. Direct injectable matrices at the site of tissue damage mimicking the tissue being restored may be a future trend on this topic. Hence, chitosan-TGA is a promising candidate as scaffold material in tissue engineering.

Chemically modified chitosans as enzyme inhibitors.

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(beta 1-4-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.

Polymer–cysteamine conjugates: new mucoadhesive excipients for drug delivery?

In the present study, the features of two new thiolated polymers--the so-called thiomers--were investigated. Mediated by a carbodiimide cysteamine was covalently attached to sodium carboxymethylcellulose (Na-CMC) and neutralised polycarbophil (Na-PCP). Depending on the weight-ratio polymer to cysteamine during the coupling reaction, the resulting CMC-cysteamine conjugate and PCP-cysteamine conjugate showed in maximum 43 +/- 15 and 138 +/- 22 micromole thiol groups per g polymer (mean +/- S.D.; n=3), respectively, which were used for further characterisation. Tensile studies carried out with the CMC-cysteamine conjugate on freshly excised porcine intestinal mucosa displayed no significantly (P<0.01) improved mucoadhesion, whereas, the mucoadhesive properties of the PCP-cysteamine conjugate were increased 2.5-fold compared with the unmodified polymer. The swelling behaviour of the CMC-cysteamine conjugate was uninfluenced by the covalent attachment of the sulfhydryl compound. In contrast the swelling behaviour of the PCP-cysteamine conjugate was improved significantly (P<0.01) versus unmodified PCP. Furthermore, in aqueous solutions the disintegration time of tablets based on the CMC- and PCP-cysteamine conjugates was prolonged 1.5 and 3.2-fold, respectively, in comparison to tablets containing the corresponding unmodified polymers. According to these results, especially the PCP-cysteamine conjugate represents a promising new pharmaceutical excipient for various drug delivery systems.