Thiomers: Influence of molecular mass and thiol group content of poly(acrylic acid) on efflux pump inhibition.

The aim of the present study was to investigate the influence of molecular mass and thiol group content of poly(acrylic acid)-cysteine conjugates on the permeation of sulforhodamine 101 and penicillin G. acting as substrates for multidrug resistance-associated protein 2 efflux pump. Poly(acrylic acids) of 2 kDa, 100 kDa, 250 kDa, 450 kDa and 3000 kDa were conjugated with cysteine. The thiol group content of all these polymers was in the range from 343.3 ± 48.4 µmol/g to 450.3 ± 76.1 µmol/g. Transport studies were performed on rat small intestine mounted in Ussing-type chambers. Since 250 kDa poly(acrylic acid) showed the highest permeation enhancing effect, additionally thiolated 250 kDa polyacrylates displaying 157.2 µmol/g, 223.0 ± 18.1 and 355.9 µmol/g thiol groups were synthesized in order to investigate the influence of thiol group content on the permeation enhancement. The permeation of sulforhodamine was 3.93- and 3.85-fold improved using 250 kDa poly(acrylic acid)-cysteine conjugate exhibiting 355.9 ± 39.5 µmol/g and 223.0 ± 18.1 µmol/g thiol groups. Using the same conjugates the permeation of penicillin G was 1.70- and 1.59-fold improved, respectively. The study demonstrates that thiolated poly(acrylic acid) inhibits Mrp2 mediated transport and that the extent of inhibition depends on the molecular mass and degree of thiolation of the polymer.

Permeation studies on freshly excised rat gastric mucosa: influence of pH.

The objective of this study was to evaluate the influence of pH on the permeation of model drugs through freshly excised rat stomach. Additionally, the capability of excised gastric mucosa to maintain an acidic pH was assessed. In vitro permeation studies were performed in Ussing-type diffusion chambers with rat stomach using fluorescence-labeled bacitracin (bac-FITC), sodium fluorescein (NaFlu), propranolol HCl, and cimetidine as model drugs. The pH was adjusted to pH 1, 2, and 6.8 in the donor chamber and pH 7.4 in the acceptor chamber. The study demonstrated that both, the fore stomach and the glandular gastric mucosa, are capable of maintaining an acidic pH of 1-1.2 in the donor chamber. P(app) (permeation coefficients) were found to be 1.4 ± 0.6 ×·10(-7) and 7.6 ± 0.7 ×·10(-7) for bac-FITC and 3.3 ± 1.5 ×·10(-7) and 2.4 ± 0.6 ×·10(-6) cm/sec for NaFlu at pH 2 and 6.8, respectively, in the glandular stomach. In order to evaluate the effect of pH on the integrity of paracellular space, propranolol as high-permeability drug and cimetidine as low-permeability drug were chosen. The P(app) of propranolol HCl was determined to be 5.9 ± 0.3 ×·10(-7) and 1.1 ± 0.7 ×·10(-6) cm/sec at pH 2 and 6.8, respectively, in the glandular stomach. Cimetidine showed a permeability of 1.4 ± 0.4 ×·10(-5) and 9.6 ± 2.3 ×·10(-6) cm/sec at pH 2 and 6.8. Results provide essential basic information for the development of gastric drug delivery systems.

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.

Design and in vivo evaluation of a patch delivery system for insulin based on thiolated polymers.

PURPOSE: The aim of this study was to develop and evaluate a novel three-layered oral delivery system for insulin in vivo. METHODS: The patch system consisted of a mucoadhesive layer, a water insoluble backing layer made of ethylcellulose and an enteric coating made of Eudragit. Drug release studies were performed in media mimicking stomach and intestinal fluids. For in vivo studies patch systems were administered orally to conscious non-diabetic rats. Orally administered insulin in aqueous solution was used as control. After the oral administration of the patch systems a decrease of glucose and increase of insulin blood levels were measured. RESULTS: The mucoadhesive layer, exhibiting a diameter of 2.5mm and a weight of 5mg, comprised polycarbophil-cysteine conjugate (49%), bovine insulin (26%), gluthatione (5%) and mannitol (20%). 74.8+/-4.8% of insulin was released from the delivery system over 6h. Six hours after administration of the patch system mean maximum decrease of blood glucose level of 31.6% of the initial value could be observed. Maximum insulin concentration in blood was 11.3+/-6.2ng/ml and was reached 6h after administration. The relative bioavailability of orally administered patch system versus subcutaneous injection was 2.2%. CONCLUSION: The results indicate that the patch system provides enhancement of intestinal absorption and thereby offers a promising strategy for peroral peptide delivery.

Development and in vitro evaluation of surface modified poly(lactide-co-glycolide) nanoparticles with chitosan-4-thiobutylamidine.

The objective of this study was to develop a nanoparticulate drug delivery system based on the surface modification of poly(lactide-co-glycolide) (PLGA) nanoparticles with a thiolated chitosan. PLGA nanoparticles were prepared by the emulsification-solvent evaporation method. Immobilization of chitosan to the surface of PLGA nanoparticles via amide bonds was mediated by a carbodiimide. Thiol groups were covalently bound to the chitosan surface of particles by reaction with 2-iminothiolane. Obtained nanoparticles were characterized in vitro regarding size, zeta potential, thiol group content, stability at different pH values, mucoadhesion, and drug release. Results demonstrated that the surface modification of PLGA nanoparticles with thiolated chitosan (chitosan-TBA) leads to nanoparticles of a mean diameter of 889.5 +/- 72 nm and positive zeta potential of + 24.74 mV. The modified nanoparticles contained 7.32 +/- 0.24 micromol thiol groups per gram nanoparticles. The size of nanoparticles was strongly influenced by the pH of the surrounding medium, being 925.0 +/- 76.3 nm at pH 2 and 577.8 +/- 66.7 nm at pH 7.4. Thiolated nanoparticles showed a 3.3-fold prolonged residence time on the mucosa and an unchanged release profile in comparison to unmodified PLGA nanoparticles. These data suggest that surface modified chitosan-TBA conjugate PLGA nanoparticles have the potential to be used as mucoadhesive drug delivery system.

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.

In vivo evaluation of a nasal insulin delivery system based on thiolated chitosan.

The aim of this study was the preparation and in vivo evaluation of a nasal insulin delivery system based on thiolated chitosan. 2-Iminothiolane was covalently attached to chitosan. The resulting conjugate (chitosan-TBA) exhibited 304.9 +/- 63.5 micromol thiol groups per gram polymer. Microparticles were prepared via a new precipitation-micronization technique. The microparticulate delivery system comprised insulin, reduced glutathione and chitosan-TBA (Chito-TBA/Ins) or unmodified chitosan (Chito/Ins) and control microparticles were composed of insulin and mannitol (Mannitol/Ins). Due to a hydration process the size of Chito-TBA/Ins and Chito/Ins microparticles increased in phosphate buffer pH 6.8 2.6- and 2.2-fold, respectively. Fluorescent-labeled insulin-loaded chitosan-TBA microparticles showed a controlled release over 4 h. Chito-TBA/Ins administered nasally to rats led to an absolute bioavailability of 6.9 +/- 1.5%. The blood glucose level decreased for more than 2 h and the calculated absolute pharmacological efficacy was 4.9 +/- 1.4%. Chito/Ins, in comparison, displayed a bioavailability of 4.2 +/- 1.8% and a pharmacological efficacy of 0.7 +/- 0.6%. Mannitol/Ins showed a bioavailability of 1.6 +/- 0.4% and no reduction of the blood glucose level at all. According to these findings microparticles comprising chitosan-TBA seem to have substantial higher potential for nasal insulin administration than unmodified chitosan.

Comparison of the mucoadhesive properties of various polymers.

In this study the mucoadhesive potential of nineteen different, most often referred mucoadhesive polymers was evaluated and characterized by adhesion time and total work of adhesion (TWA) of the polymer to porcine small intestinal mucosa. In addition, the influence of pH of the polymer and of method of drying on adhesion was evaluated. Aqueous polymer solutions were therefore adjusted to pH 3.0 and 7.0. Solutions were either dried by lyophilization (lyo.) or precipitated (pr.) in organic solvent and air-dried. Results of this study led to the following rank order of adhesion time: chitosan-4-thiobuthylamidine pH 3 lyo. >chitosan-4-thiobuthylamidine pH 6.5 pr.>polycarbophil-cysteine pH 3 lyo.>chitosan-4-thiobuthylamidine pH 6.5 lyo.>PAA450-cysteine pH 3 lyo.>pH 7 pr.>Carbopol 980 pH 7 pr.>Carbopol 974P pH 7 pr.>polycarbophil pH 7 pr.>980 pH 3 lyo. The rank order obtained for adhesion time was in agreement with the rank order obtained for total work of adhesion. The highest mucoadhesion was shown by thiolated polymers at pH 3.0, dried by lyophilization. In contrary, polyacrylates were most mucoadhesive in form of precipitated neutral sodium salts. Other tested polymers like natural polysaccharides, cellulose derivatives, polyvinylpirrolidone and polyethylenglycole, although previously reported as good mucoadhesives, showed low to almost no mucoadhesion. The pH of polymer and drying method were found to be important factors influencing the mucoadhesive potential of polymers.