In vivo evaluation of anionic thiolated polymers as oral delivery systems for efflux pump inhibition.

Recently, the cationic polymer thiolated chitosan has been reported to modulate drug absorption by inhibition of intestinal efflux pumps. The objective of this study was to evaluate in vitro and in vivo whether thiolated anionic biopolymers also show an efflux pump inhibitory effect in order to improve intestinal transcellular drug uptake. Therefore, three thiomers have been synthesized due covalent attachment of cysteine to various polymer backbones: pectin-cysteine (pect-cys), carboxymethylcellulose-cysteine (CMC-cys) and alginate-cysteine (alg-cys). In vitro, the permeation enhancing properties of these thiomers and their corresponding unmodified polymers have been evaluated on rat small intestine in Ussing-type chambers, using sulforhodamine 101 (SR-101) as MRP2 model substrate. In comparison to buffer only, SR-101 transport in presence of pect-cys, CMC-cys and alg-cys was improved 1.5-fold, 1.8-fold and 3.0-fold, respectively. Due to the comparatively best in vitro performance of thiolated alginate, it has been chosen for in vivo studies: a SR-101 solution containing 4% (w/v) alg-cys led to an AUC0 ≥ 12 of SR-101 of 109 ng ml(-1)h in rats representing a 3.8-fold improvement in comparison to a SR-101 buffer solution. Unmodified alginate improved the AUC0 ≥ 12 of SR-101 by a factor of 1.9. These findings suggest thiolated alginate as promising auxiliary agent for drugs being anionic efflux pump substrates, since the oral bioavailability of a MRP2 substrate could be significantly improved.

Safety assessment of thiolated polymers: effect on ciliary beat frequency in human nasal epithelial cells.

OBJECTIVE: The aim of this study was to investigate the nasal safety of gel formulations of thiolated polymers (thiomers) by assessing their effect on ciliary beat frequency (CBF) in human nasal epithelial cells. METHODS: Poly(acrylic acid) 450 kDa-cysteine (PAA-cys) and alginate-cysteine (alg-cys) were synthesized by covalent attachment of L-cysteine to the polymeric backbone. The cationic polymer chitosan-thiobutylamidine (chito-TBA) was synthesized by attaching iminothiolane to chitosan. CBF using was measured by a photometric system. CBF was measured before incubating the cells with test gels, during incubation and after washing out the polymeric test gels to evaluate reversibility of cilio-inhibition. The influence of viscosity on CBF was determined by using hydroxyethylcellulose (HEC)-gels of various concentrations. RESULTS: Ciliary beating was observed to be affected by viscosity, but cilia were still beating in the presence of a HEC-gel displaying an apparent viscosity of 25 Pa.s. In case of thiolated polymers and their unmodified control, a concentration-dependent decrease in CBF could be observed. PAA-cys, alg-cys, chito-TBA and their corresponding unmodified controls exhibited a moderate cilio-inhibitory effect, followed by a partial recovery of CBF when used at a concentration of 1%. Alg-cys 2% and chito-TBA 2% (m/v) gels exhibited severe cilio-inhibition, which was partially reversible. L-cysteine and reduced glutathione led to mild cilio-inhibition at concentrations of 3% (m/v). CONCLUSIONS: Taking into account that dilution after application and cilio-modifying effects is usually more pronounced under in vitro conditions, thiomers can be considered as suitable excipients for nasal drug delivery systems.

Insulin-loaded poly(acrylic acid)-cysteine nanoparticles: stability studies towards digestive enzymes of the intestine.

This study evaluated thiolated poly(acrylic acid) nanoparticles as a valuable tool to protect insulin from degradation by serinproteases of the intestine. Nanaoparticles were characterized concerning particle size, zeta potential, and drug load. Furthermore, in vitro release studies were performed. Within in vitro degradation studies with trypsin, alpha-chymotrypsin, and elastase it could be demonstrated that the obtained nanoparticles are capable of protecting 44.47 +/- 0.89% of the initial insulin amount from tryptic degradation, 21.33 +/- 5.34% from chymotryptic degradation, and 45.01 +/- 1.40% from degradation by elastase compared to insulin solutions.

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

The purpose of the present study was to design and evaluate a novel vaginal delivery system for nystatin based on mucoadhesive polymers. L-Cysteine and cysteamine, respectively, were covalently attached to poly(acrylic acid), and the two different thiolated polymers were evaluated in vitro regarding their swelling behavior, mucoadhesive properties and release behavior. Tablets comprising these thiolated polymers and nystatin demonstrated a high stability in vaginal fluid simulant pH 4.2 and an increase in weight by swelling whereas control tablets comprising unmodified poly(acrylic acid) disintegrated and dissolved. The mucoadhesion time of tablets on freshly excised bovine vaginal mucosa on a rotating cylinder and the total work of adhesion of gels and tablets increased significantly due to the formation of disulfide bonds between the thiolated polymer and cysteine rich subdomaines of the mucus layer. The drug nystatin was released more slowly out of thiomer tablets and gels than out of PAA control tablets and gels. Therefore these thiolated polymers are promising delivery systems for nystatin providing a prolonged residence time and a sustained drug release in vitro under physiological relevant conditions.

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.

Thiolated chitosan: development and in vitro evaluation of an oral delivery system for acyclovir.

The aim of the study was to develop a novel oral delivery system for the efflux pump substrate acyclovir (ACY) utilizing thiolated chitosan as excipient which is capable of inhibiting P-glycoprotein (P-gp). Three chitosan-4-thiobutylamidine (Chito-TBA) conjugates with increasing molecular mass (Chito-9.4kDa-TBA, Chito-150kDa-TBA and Chito-600kDa-TBA) were synthesized and permeation studies on rat intestinal mucosa and Caco-2 monolayers were performed. Additionally, tablets comprising the conjugates and ACY were tested towards their drug release behaviour. The efflux ratio (secretory P(app)/absorptive P(app)) of ACY across Caco-2 monolayers was determined to be 2.5 and in presence of 100microM verapamil 1.1 which indicates ACY as P-gp substrate. In comparison to buffer only, the transport of ACY in presence of 0.5% (m/v) unmodified chitosan, 0.5% (m/v) Chito-150kDa-TBA and 0.5% (m/v) Chito-150kDa-TBA with 0.5% (m/v) reduced glutathione (GSH), was 1.3-, 1.6- and 2.1-fold improved, respectively. Transport studies across Caco-2 monolayers showed that P-gp inhibition is dependent on the average molecular mass of thiolated chitosan showing following rank order: 0.5% (m/v) Chito-150kDa-TBA/GSH>0.5% (m/v) Chito-9.4kDa-TBA/GSH>0.5% (m/v) Chito-600kDa-TBA/GSH. The higher the molecular mass of Chito-TBA was, the more sustained was the release of ACY. Chito-150kDa-TBA/GSH might be an appropriate sustained release drug delivery system for ACY, which is able to enhance ACY transport due to efflux pump inhibition.

Thiolated polymers: evaluation of the influence of the amount of covalently attached L-cysteine to poly(acrylic acid).

It was the aim of this study to investigate the influence of the amount of thiol groups being covalently attached to poly(acrylic acid) 450 kDa on its properties. Five different PAA(450)-L-cysteine conjugates (PAA(450)-Cys) were synthesized bearing 53.0 (PAA I), 113.4 (PAA II), 288.8 (PAA III), 549.1 (PAA IV) and 767.0 (PAA V) micromol immobilized thiol groups per gram polymer. Mucoadhesion studies utilizing the rotating cylinder method, tensile studies and disintegration studies were performed. Self-crosslinking properties were measured by the increase in viscosity. Permeation studies were performed on rat small intestine and Caco-2 monolayers using sodium fluorescein as model drug. Following residence times on the rotating cylinder could be identified: PAA I 3.1; PAA II 5.2; PAA III 22.0; PAA IV 33.8; PAA V 53.7; control 1.3 [h]. The disintegration time of all PAA(450)-Cys tablets was strongly dependent on the degree of thiolation of the polymer. Self-crosslinking studies showed that the different PAA(450)-Cys conjugates (3% m/v) in phosphate buffer, pH 6.8, formed intramolecular disulfide bonds. In case of Caco-2 monolayer transport studies following P(app)-values could be identified: PAA I 9.8; PAA II 10.1; PAA III 11.1; PAA IV 8.9; PAA V 8.2; control 6.4 [P(app)x10(-6), cms(-1)]. Mucoadhesive and self-crosslinking properties are strongly dependent on the degree of thiolation of the polymer and with respect to transport studies, an optimum amount of covalently attached L-cysteine could be identified.