Chitosan-N-acetyl cysteine conjugates: in vitro evaluation of permeation enhancing and P-glycoprotein inhibiting properties.

This study evaluated three chitosan-N-acetyl cysteine (CAC) conjugates of increasing molecular mass as a valuable tool to improve the absorption of drugs by assessing its permeation enhancing effect regarding the active P-gp substrate rhodamine-123 in comparison to the trans- and paracellular marker FD 4 both in rat intestine and Caco 2 monolayers. Additional LDH and MTT cytotoxicity tests have attested a non-toxic profile to CAC, which can consequently be seen as a safe and promising novel drug carrier with the ability to enhance drug absorption and to inhibit P-gp efflux transporters.

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.

Papain: an effective permeation enhancer for orally administered low molecular weight heparin.

PURPOSE: The purpose of this study was to evaluate an effect of the proteolytic enzyme papain on permeation of low molecular weight heparin (LMWH) in vitro and in vivo. MATERIALS AND METHODS: In vitro permeation studies were performed using rat small intestine as permeation barrier. In order to determine the ratio of papain to heparin resulting in the highest heparin permeation rate, molar ratios 1:1, 1:2 and 2:1 of papain to heparin were tested. Interactions of heparin with papain were investigated spectro-photometrically. For in vivo studies, 15 mg tablets containing heparin (13%), papain (64%) and hydroxyethylcellulose (22%) were orally administered to rats. RESULTS: Since molar ratio papain to heparin 1:1 resulted in the highest permeation rate, it was used for in vivo studies. The results of binding studies of papain with heparin indicated a strong interaction between papain and heparin. Oral administration of tablets containing LMWH/papain/HEC resulted in sevenfold improvement of plasma anti-Xa activity in comparison to control. For tablets based on heparin/papain/HEC, a relative bioavailability of 9.1% vs. subcutaneous injection was obtained, whereas the relative bioavailability of control was 2.4%. CONCLUSION: The co-administration of papain with heparin represents a new approach in improvement of absorption and bioavailability of orally administered heparin.

Development and in vitro evaluation of a thiomer-based nanoparticulate gene delivery system.

Chitosan-thiobutylamidine was developed and evaluated as a novel tool for gene delivery. The conjugate, displaying 299.1+/-11.5 micromol free thiol groups per gram polymer, formed coacervates with pDNA at a mean size of 125 nm and a zeta potential of +9 mV. Thiol groups, being susceptible for oxidation, were immobilised on the polymeric backbone of chitosan in order to introduce the property of extracellular stability and intracellular pDNA release by forming reversible disulfide bonds. The integrity of the new particles was compared to unmodified chitosan under simulated physiological conditions. Within 10h, pDNA was completely released from chitosan-DNA particles while only 12% were released from the thiomer-based particles. At pH 7, the amount of thiol groups significantly (p<0.05) decreased by more than 25% within 6h. In contrast, in a reducing environment as found intracellularly, chitosan-thiobutylamidine-DNA nanoparticles dissociated continuously, liberating approximately 50% of pDNA within 3h. Transfection studies performed in a Caco2 cell culture evinced the highest efficiency for chitosan-thiobutylamidine-DNA nanoparticles in combination with a glycerol shock solution. The combination of improved stability, enhanced pDNA release under reducing conditions, and higher transfection efficiency identifies chitosan-thiobutylamidine as a promising new vector for gene delivery.

Oral peptide delivery: are there remarkable effects on drugs through sulfhydryl conjugation?

In oral peptide delivery, the gap between convenient administration and low blood concentration has to be minimized. We found that oral peptide drugs have not only to pass the various commonly known barriers encountered with the gastrointestinal tract but that these drugs, under certain conditions, have also to be seen as redox partners for thiol bearing substrates. The interaction of glutathione (GSH) with peptides via thiol-disulfide exchange reactions was investigated for three peptides, vasotocin, oxytocin and octreotide. The extent of thiol-disulfide exchange reactions was investigated by liquid chromatography (LC) and further confirmed by hyphenation to electrospray ionization (ESI) and MALDI-TOF mass spectrometry (MS). We found that the presence of aromatic amino acid residues in the neighbourhood of the disulfide bond minimizes the thiol-disulfide interaction: oxytocin was degraded more than 80% with 1% reduced glutathione at pH 3.0 and vasotocin more than 40% under the same conditions. In the case of octreotide no interaction with GSH was observed. The obtained results revealed that thiol-disulfide exchange reactions have an important impact on the alteration of peptide drugs and proteins in the gastrointestinal tract.

The potential of cystine-knot microproteins as novel pharmacophoric scaffolds in oral peptide drug delivery.

Within this study, the potential of three clinically relevant microproteins (SE-AG-AZ, SE-EM and SE-EP) with cystine-knot architecture as pharmacophoric scaffolds for oral peptide delivery was investigated. Cystine-knot microproteins (CKM) were analysed regarding their stability towards the most important gastrointestinal secreted and membrane bound proteases in physiological concentrations. In addition, their permeation behaviour through freshly excised rat intestinal mucosa as well as important parameters such as aggregation behaviour, stability in rat plasma and isoelectric point were evaluated and compared to the properties of the model peptide drugs bacitracin and insulin. Aggregation studies indicate that under physiological conditions between 25 and 70% of the CKMs occur as monomers, whereas the rest forms di- and trimers. Pepsin and elastase cause no or only minor degradation to CKMs, whereas trypsin and chymotrypsin degrade CKMs extensively. Removing the theoretical chymotrypsin cleavage site from a CKM, however, led to stabilization towards this protease. Two of the three evaluated CKMs are stable against membrane bound proteases. P(app) values were determined to be 5.96 +/- 0.98 x 10(-6) and 6.63 +/- 0.47 x 10(-6) cm/s. In conclusion, this study indicates that CKM are promising novel pharmacophoric scaffolds for oral peptide delivery.

In vivo evaluation of an oral delivery system for P-gp substrates based on thiolated chitosan.

Recently, thiolated polymers, so called thiomers, have been reported to modulate drug absorption by inhibition of intestinal P-glycoprotein (P-gp). The aim of the present study was to provide a proof-of-principle for a delivery system based on thiolated chitosan in vivo in rats, using rhodamine-123 (Rho-123) as representative P-gp substrate. In vitro, the permeation enhancing effect of unmodified chitosan, chitosan-4 thiobutylamidine (Ch-TBA) and the combination of Ch-TBA with reduced glutathione (GSH) was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to buffer only, Rho-123 transport in presence of 0.5% (w/v) chitosan, 0.5% (w/v) Ch-TBA and the combination of 0.5% (w/v) Ch-TBA/0.5% (w/v) GSH, was 1.8-fold, 2.6-fold, 3.8-fold improved, respectively. Furthermore, enteric-coated tablets based on unmodified chitosan or Ch-TBA/GSH, were investigated in vivo. In rats, the Ch-TBA/GSH tablets increased the area under the plasma concentration time curve (AUC0-12) of Rho-123 by 217% in comparison to buffer control and by 58% in comparison to unmodified chitosan. This in vivo study showed that a delivery system based on thiolated chitosan significantly increased the oral bioavailability of P-gp substrate Rho-123.

Elaboration and characterization of thiolated chitosan-coated acrylic nanoparticles.

The aim of the present work was to investigate the use of thiolated chitosan in the development of polysaccharide-coated nanoparticles in order to confer specific functionality to the system. After chemical modification of commercial and hydrolysed chitosan (400,000 and 9400 g/mol respectively), thiolated chitosans were used to elaborate particles in the nano-range. They were characterized in terms of size and surface charge measurement. Both analysis showed nanoparticles of mean hydrodynamic diameter around 200 nm and positive zeta potential values, indicating the presence of the cationic polysaccharide at the nanoparticle surface. Moreover, the Ellman's reaction was used to demonstrate the presence of thiol groups at the particle surface. The observation of nanoparticles by scanning electronic microscopy (SEM) showed spherical nanoparticles for all formulations. This new system, combining both the advantages of thiolated polymers and colloidal particles can be proposed as an original drug carrier system for mucosal delivery of biotechnology products.

Oral heparin delivery: design and in vivo evaluation of a stomach-targeted mucoadhesive delivery system.

Low molecular weight heparin (LMWH) is an agent of choice in the anti-coagulant therapy and prophylaxis of thrombosis and coronary syndromes. However, the therapeutic use is partially limited due to a poor oral bioavailability. It was therefore the aim of this study to design and evaluate a highly efficient stomach-targeted oral delivery system for LMWH. In order to appraise the influence of the molecular weight on the oral bioavailability, mini-tablets comprising 3 kDa (279 IU) and 6 kDa (300 IU) LMWH, respectively, were generated and tested in vivo in rats. The potential of the test formulations based on thiolated polycarbophil, was evaluated in comparison to hydroxyethylcellulose (HEC) as control carrier matrix. The plasma levels of LMWH after oral versus subcutaneous administration were determined in order to calculate the relative bioavailability. With the delivery system containing 3 kDa LMWH (279 IU) a relative bioavailability of 19.1% was achieved, offering a significantly (p < 0.05) better bioavailability than the control system displaying a relative bioavailability of 8.1% The 6 kDa LMWH (300 IU) formulation displayed a relative bioavailability of 10.7% in contrast to the control displaying a relative bioavailability of 2.1%. In conclusion, these results suggest that mucoadhesive thiolated polymers are a promising tool for the non-invasive stomach-targeted systemic delivery of LMWH as model for a hydrophilic macromolecular polysaccharide.