Thomas F. Palmberger

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.

Preparation of Thiomer Microparticles and In Vitro Evaluation of Parameters Influencing Their Mucoadhesive Properties

ABSTRACT It was the aim of this study to develop mucoadhesive microparticulate delivery systems based on thiomers and to investigate parameters influencing their mucoadhesive properties. Microparticles were prepared via coazervation of thiolated or unmodified polycarbophil with fluorescein-diacetate as marker. The protective effect of the polymers toward enzymatic hydrolysis by intestinal enzymes was investigated. Mucoadhesion studies with microparticles, applied in dry and prehydrated form, were performed by ascertaining their residence time on intestinal mucosa. Furthermore, the influence of the amount of thiol groups on mucoadhesion was studied in vitro. Results showed that in comparison to unmodified polycarbophil, thiolated polycarbophil provided a more than 3-fold higher protective effect for the incorporated marker fluorescein-diacetate toward hydrolysis. When being applied in dry form 23.4 ± 4.8% of the fluorescence marker being embedded in thiomer microparticles remained adhering to the intestinal mucosa within 3 h. In contrast, only 11.6 ± 2.0% of the marker remained on the mucosa, when the thiomer microparticles were applied in prehydrated form. In addition, tests performed to assess the impact of the amount of thiol groups pointed out that a high amount of thiol groups is advantageous in order to further improve mucoadhesive properties. This knowledge should contribute to the design of highly efficient drug delivery systems being based on thiomer microparticles.

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, α-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.

In vitro evaluation of the potential of thiomers for the nasal administration of Leu-enkephalin.

Summary.It was the aim of this study to evaluate the potential of thiolated polycarbophil for the nasal administration of Leucine-enkephalin (Leu-enkephalin). The enzymatic degradation of Leu-enkephalin on freshly excised bovine nasal mucosa was analysed qualitatively via thin layer chromatography and quantitatively via high performance liquid chromatography (HPLC). The potential of thiolated polycarbophil gels to provide a sustained release for the therapeutic peptide was investigated via diffusion studies. Permeation studies were performed in Ussing-type diffusion chambers with freshly excised bovine nasal mucosa. Results demonstrated that Leu-enkephalin is mainly degraded by the cleavage of tyrosine from the N-terminus of the peptide. Within one hour more than 63.5 ± 2% of this therapeutic peptide are degraded on the nasal mucosa. In the presence of 0.25% thiolated polycarbophil, this degradation process, however, could be significantly lowered. Diffusion studies demonstrated that Leu-enkephalin being incorporated in a 0.5% thiolated polycarbophil gel is sustained released out of it. The appearent permeability coefficient (Papp) for Leu-enkephalin on the nasal mucosa was determined to be 1.9 ± 1.2 × 10−7 cm/sec. Furthermore, in the presence of 0.5% thiolated polycarbophil and 1% glutathione, which is used as permeation mediator for the thiomer, the uptake of Leu-enkephalin from the nasal mucosa was even 82-fold improved. According to these results thiolated polycarbophil might be a promising excipient for nasal administration of Leu-enkephalin.

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.

In vitro characterization of insulin containing thiomeric microparticles as nasal drug delivery system.

This study focused on a novel two step preparation method for the generation of insulin containing thiomer microparticles. The first step utilized the interpolymer complexation between poly(vinyl pyrrolidone) (PVP) and poly(acrylic acid) (PAA) or poly(acrylic acid)-cysteine (PAA-Cys), respectively, in the presence of insulin. Thereafter lyophilized coprecipitates were micronized via air jet mill. Particles were evaluated regarding size, morphology, insulin release and the effect on ciliary beat frequency of human nasal epithelial cells in vitro. Results displayed mean particle sizes of 2.6±1.6μm and 2.8±1.7μm for PAA/PVP/insulin and PAA-Cys/PVP/insulin microparticles, respectively, in a range where volitional impaction of particles on nasal epithelium takes place. Multi unit dosage forms showed in addition release for the incorporated insulin and nasal safety as to results of ciliary beat frequency studies (CBF). The introduced jet milled microparticles might in conclusion display a safe nasal insulin drug delivery system leading to improved absorption.

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.