S-protected thiolated chitosan: synthesis and in vitro characterization.

Purpose of the present study was the generation and evaluation of novel thiolated chitosans, so-named S-protected thiolated chitosans as mucosal drug delivery systems. Stability of all conjugates concerning swelling and disintegration behavior as well as drug release was examined. Mucoadhesive properties were evaluated in vitro on intestinal mucosa. Different thiolated chitosans were generated displaying increasing amounts of attached free thiol groups on the polymer, whereby more than 50% of these thiol groups were linked with 6-mercaptonicotinamide. Based on the implementation of this hydrophobic residue, the swelling behavior was 2-fold decreased, whereas stability was essentially improved. Their mucoadhesive properties were 2- and 14-fold increased compared to corresponding thiolated and unmodified chitosans, respectively. Release studies out of matrix tablets comprising the novel conjugates revealed a controlled release of a model peptide. Accordingly, S-protected thiomers represent a promising type of mucoadhesive polymers for the development of various mucosal drug delivery systems.

In vivo evaluation of an oral drug delivery system for peptides based on S-protected thiolated chitosan.

The aim of the present study was the development and evaluation in vitro as well as in vivo of an oral delivery system based on a novel type of thiolated chitosan, so-called S-protected thiolated chitosan, for the peptide drug antide. The sulfhydryl ligand thioglycolic acid (TGA) was covalently attached to chitosan (CS) in the first step of modification. In the second step, these thiol groups of thiolated chitosan were protected by disulfide bond formation with the thiolated aromatic residue 6-mercaptonicotinamide (6-MNA). Absorptive transport studies of antide were evaluated ex vivo using rat intestinal mucosa. Matrix tablets of each polymer sample were prepared and their effect on the absorption of antide evaluated in vivo in male Sprague-Dawley rats. In addition, tablets were examined in terms of their disintegration, swelling and drug release behavior. The resulting S-protected thiomer (TGA-MNA) exhibited 840µmol of covalently linked 6-MNA per gram thiomer. Based on the implementation of this hydrophobic ligand on the thiolated backbone, the disintegration behavior was reduced greatly and a controlled release of the peptide could be achieved. Furthermore, permeation studies with TGA-MNA on rat intestine revealed a 4.5-fold enhanced absorptive transport of the peptide in comparison to antide in solution. Additional in vivo studies confirmed the potential of this novel conjugate. Oral administration of antide in solution led to only very small detectable quantities in plasma with an absolute and relative bioavailability (BA) of 0.003 and 0.03%, only. In contrast, with antide incorporated in TGA-MNA matrix tablets an absolute and relative BA of 1.4 and 10.9% could be reached, resulting in a 421-fold increased area under the plasma concentration time curve (AUC) compared to the antide solution. According to these results, S-protected thiolated chitosan as oral drug delivery system might be a valuable tool for improving the bioavailability of peptides.

S-protected thiolated chitosan for oral delivery of hydrophilic macromolecules: evaluation of permeation enhancing and efflux pump inhibitory properties.

The objective of this study was the investigation of permeation enhancing and P-glycoprotein (P-gp) inhibition effects of a novel thiolated chitosan, the so-named S-protected thiolated chitosan. Mediated by a carbodiimide, increasing amounts of thioglycolic acid (TGA) were covalently bound to chitosan (CS) in the first step of modification. In the second step, these thiol groups of thiolated chitosan were protected by disulfide bond formation with the thiolated aromatic residue 6-mercaptonicotinamide (6-MNA). Mucoadhesive properties of all conjugates were evaluated in vitro on porcine intestinal mucosa based on tensile strength investigations. Permeation enhancing effects were evaluated ex vivo using rat intestinal mucosa and in vitro via Caco-2 cells using the hydrophilic macromolecule FD(4) as the model drug. Caco-2 cells were further used to show P-gp inhibition effects by using Rho-123 as P-gp substrate. Apparent permeability coefficients (P(app)) were calculated and compared to values obtained from each buffer control. Three different thiolated chitosans were generated in the first step of modification, which displayed increasing amounts of covalently attached free thiol groups on the polymer backbone. In the second modification step, more than 50% of these free thiol groups were covalently linked with 6-MNA. Within 3 h of permeation studies on excised rat intestine, P(app) values of all S-protected chitosans were at least 1.3-fold higher compared to those of corresponding thiomers and more than twice as high as that of unmodified chitosan. Additional permeation studies on Caco-2 cells confirmed these results. Because of the chemical modification and higher amount of reactive thiol groups, all S-protected thiolated chitosans exhibit at least 1.4-fold pronounced P-gp inhibition effects in contrast to their corresponding thiomers. These features approve S-protected thiolated chitosan as a promising excipient for various drug delivery systems providing improved permeation enhancing and efflux inhibition effects.

Modulation of basal and stress-induced amygdaloid substance P release by the potent and selective NK1 receptor antagonist L-822429.

It has been shown that anxiety and stress responses are modulated by substance P (SP) released within the amygdala. However, there is an important gap in our knowledge concerning the mechanisms regulating extracellular SP in this brain region. To study a possible self-regulating role of SP, we used a selective neurokinin-1 (NK1) receptor antagonist to investigate whether blockade of NK1 receptors results in altered basal and/or stress-evoked SP release in the medial amygdala (MeA), a critical brain area for a functional involvement of SP transmission in enhanced anxiety responses induced by stressor exposure. In vitro binding and functional receptor assays revealed that L-822429 represents a potent and selective rat NK1 receptor antagonist. Intra-amygdaloid administration of L-822429 via inverse microdialysis enhanced basal, but attenuated swim stress-induced SP release, while the low-affinity enantiomer of L-822429 had no effect. Using light and electron microscopy, synaptic contacts between SP-containing fibres and dendrites expressing NK1 receptors was demonstrated in the medial amygdala. Our findings suggest self-regulatory capacity of SP-mediated neurotransmission that differs in the effect on basal and stress-induced release of SP. Under basal conditions endogenous SP can serve as a signal that tonically inhibits its own release via a NK1 receptor-mediated negative feedback action, while under stress conditions SP release is further facilitated by activation of NK1 receptors, likely leading to high local levels of SP and activation of receptors to which SP binds with lower affinity.