Recently, several polymers have been reported to modulate drug absorption by inhibition of intestinal efflux pumps such as multidrug resistance proteins (MRPs) and P-glycoprotein (P-gp). The aim of the present study was to evaluate the efficiency of thiolated poly(acrylic acid)(PAA-Cys) to act as a drug absorption modulator for MRP2 efflux pump substrates in vivo, using sulforhodamine 101 as representative MRP2 substrate. In vitro, the permeation enhancing effect of unmodified PAA and PAA (250)-Cys, displaying 580 mumol free thiol groups per gram polymer, was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to buffer control, the sulforhodamine 101 transport in presence of 0.5% unmodified PAA (250) and 0.5%(w/v) PAA (250)-Cys was 1.3-fold and 4.0-fold improved, respectively. In vivo, sulforhodamine 101 solutions containing 4%(w/v) unmodified PAA (250) or 4%(w/v) thiolated PAA (250) were orally given to rats. The PAA (250)-Cys solution increased the area under the plasma concentration time curve (AUC (0-12)) of sulforhodamine 101 3.8-fold in comparison to control and 2.2-fold in comparison to unmodified PAA (250). This in vivo study revealed that PAA (250)-Cys significantly increased the oral bioavailability of MRP2 substrate sulforhodamine 101.

The anticancer agent paclitaxel is currently commercially available only as an infusion due to its low oral bioavailability. An oral formulation would be highly beneficial for patients. Besides the low solubility, the main reason for the limited oral bioavailability of paclitaxel is that it is a substrate of the efflux pump P-glycoprotein (P-gp). Recently, it has been demonstrated that P-gp can be inhibited by thiolated polymers. In this study, an oral paclitaxel formulation based on thiolated polycarbophil was evaluated in vivo in wild-type rats and in mammary cancer-induced rats. The paclitaxel plasma level after a single administration of paclitaxel was observed for 12 h in healthy rats. Moreover, cancer-induced rats were treated weekly for 5 weeks with the novel formulation. It was demonstrated that (1) co-administration of thiolated polycarbophil significantly improved paclitaxel plasma levels,(2) a more constant pharmacokinetic profile could be achieved and (3) the tumor growth was reduced. These effects can most likely be attributed to P-gp inhibition. According to the achieved results, thiolated polymers are believed to be interesting tools for the delivery of P-gp substrates such as paclitaxel.

Within this study, a novel gastrointestinal patch system was developed and investigated regarding water-absorbing capacity, adhesive properties, in vitro release, unidirectional release and permeation enhancing effect. Water uptake studies revealed that the weight of patch systems with Ch-GSH increased about 44.5 +/- 2.3 mg (127%) after 90 min. This patch system remained even after 180 h on the mucosa and released 49.7 +/- 0.7% of FD(4) within 8 h. A 2.5-fold higher transport of FD(4) can be obtained in contrast to control. In conclusion this patch system could be an interesting possibility for the transport through the intestinal mucosa of macromolecules which will normally be degraded in the intestinal tract.

The aim of this study was to evaluate two in vitro models, Caco-2 monolayer and rat intestinal mucosa, regarding their linear correlation with in vivo bioavailability data of therapeutic peptide drugs after oral administration in rat and human. Furthermore the impact of molecular mass (Mm) of the according peptides on their permeability was evaluated.Transport experiments with commercially available water soluble peptide drugs were conducted using Caco-2 cell monolayer grown on transwell filter membranes and with freshly excised rat intestinal mucosa mounted in Using type chambers. Apparent permeability coefficients (P (app)) were calculated and compared with in vivo data derived from the literature.It was shown that, besides a few exceptions, the Mm of peptides linearly correlates with permeability across rat intestinal mucosa (R (2)= 0.86; y =-196.22x + 1354.24), with rat oral bioavailability (R (2)= 0.64; y =-401.90x + 1268.86) as well as with human oral bioavailability (R (2)= 0.91; y =-359.43x + 1103.83). Furthermore it was shown that P (app) values of investigated hydrophilic peptides across Caco-2 monolayer displayed lower permeability than across rat intestinal mucosa. A correlation between P (app) values across rat intestinal mucosa and in vivo oral bioavailability in human (R (2)= 0.98; y = 2.11x + 0.34) attests the rat in vitro model to be a very useful prediction model for human oral bioavailability of hydrophilic peptide drugs.Presented correlations encourage the use of the rat in vitro model for the prediction of human oral bioavailabilities of hydrophilic peptide drugs.

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((R)). 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.

It was the aim of this study to synthesize and characterize a novel hyaluronic acid-cysteine ethyl ester (HA-Cys) conjugate providing improved mucoadhesive properties and a significantly lowered biodegradation rate. Mediated by carbodiimide and N-hydroxysuccinimide, l-cysteine ethyl ester hydrochloride was covalently attached to hyaluronic acid (HA, hyaluronan) via the formation of an amide bond. The adhesive properties of HA-Cys conjugates were evaluated in vitro on a freshly excised porcine mucosa via the rotating cylinder method. The cohesive properties of the resulting conjugates were evaluated by oxidation experiments. Biodegradability studies were carried out by viscosity measurements and spectrophotometric assays. Release studies were performed with fluorescein isothiocyanate-dextrans (FD) as model compounds. The obtained conjugate displayed 201.3+/-18.7mumol immobilized free thiol groups and 85.7+/-22.3mumol disulfide bonds per gram polymer. Results from the rotating cylinder method showed more than 6.5-fold increase in the adhesion time of HA-Cys versus unmodified HA. In aqueous solutions, the obtained conjugate demonstrated improved cohesive properties. The hydrolysis degree of HA-Cys was lower compared with the corresponding unmodified HA in the framework of viscosity experiments. In addition, the cross-linking process via disulfide bonds additionally reduced the rate of degradation of the new derivative. Cumulative release studies out of matrix tablets comprising HA-Cys and the model compound FD demonstrated a sustained drug release for more than 12h due to in situ formation of inter- and intramolecular disulfide bonds in the thiomer matrix. According to the results of the present study, this novel thiolated polymer seems to represent a promising multifunctional excipient for the development of various drug delivery systems.

One main barrier for the peroral administration of therapeutic peptides and proteins is the enzymatic barrier, that is mediated by luminally secreted and membrane bound proteolytic enzymes. It was the aim of the study to synthesise, characterise and evaluate a novel polymer-inhibitor conjugate in order to improve the bioavailability of orally-administered peptides and proteins. The trypsin/chymotrypsin inhibitor aprotinin was covalently bound to chitosan. The percentage of the inhibitor in the polymer-inhibitor conjugate (m/m) was determined to be between 1.11 +/- 0.36 and 1.92 +/- 0.05%. In vitro enzyme assays clearly demonstrated the potential of the novel conjugate to inhibit trypsin and chymotrypsin. Moreover, studies in rats were performed to evaluate the efficacy of the conjugate in vivo. Eight hours after oral administration of tablets containing insulin and the novel chitosan-aprotinin conjugate, the mean blood glucose level decreased to 84 +/- 6%. In contrast, the mean blood glucose level in the control group increased to 121 +/- 8% of the initial measured blood glucose level. In conclusion it was demonstrated that chitosan-aprotinin conjugate represents a novel and promising tool for the oral administration of therapeutic peptides and proteins susceptible to enzymatic degradation caused by trypsin and chymotrypsin.

The aim of this study was to examine the biodegradability of thiomers and cross-linked thiomers in comparison with unmodified polymers. Disulfide-cross-linked conjugates were prepared by air oxidation at room temperature. Thiomers were investigated by viscosity measurements and spectrophotometric assays. The influence of different factors on the hydrolysis rate, such as the degree of modification of thiomers, structure of the conjugates, pH value of the reaction medium, and the impact of the process of cross-linking were evaluated. Due to the modification, thiolated chitosans degraded 12.9-24.7% less than unmodified chitosan in the framework of viscosity measurements. In addition, the hydrolysis degree of thiolated alginates and modified carboxymethylcelluloses was 25.6-32.4% and 18.4-27.0% lower, respectively, in comparison to the corresponding unmodified polymers. Conjugates with higher coupling rate of thiol groups were degraded even more slowly. Moreover, the cross-linking process via disulfide bonds additionally reduced the rate of thiomer degradation. The range of degradation rates achieved in vitro could be modified by alterations of the contents of thiol and disulfide groups, as well as by suitable design of the polymer structure and ligands used. These results represent helpful basic information for the development of mucoadhesive drug delivery systems, implantable delivery systems and tissue engineering constructs.

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.

It was the aim of this study to investigate the effect of chitosan-4-thiobutylamidine (Ch-TBA) and reduced glutathione (GSH) on the absorption of P-glycoprotein (P-gp) and multidrug resistance protein (MRP) substrate saquinavir in vitro and in vivo. Bidirectional transport studies were performed with Caco-2 cell monolayers and additionally with freshly excised rat small intestinal mucosa mounted in Ussing type chambers. Furthermore, a delivery system based on Ch-TBA and GSH was evaluated in vivo in rats. The functional activity of the efflux pumps in Caco-2 cells and rat intestinal mucosa during the experiment was proven by the efflux ratio of saquinavir, which was 6.4 for Caco-2 cells and 2.1 for rat intestinal mucosa, respectively. Ch-TBA and particularly the combination of Ch-TBA with GSH enhanced apical (AP) absorption and decreased the secretory transport of saquinavir. In presence of 0.5% Ch-TBA and 0.5% GSH, the uptake of saquinavir was 1.6-fold improved in Caco-2 monolayer and 2.1-fold improved in rat intestinal mucosa. In vivo, the area under the plasma concentration time curve (AUC) of saquinavir was 1.4-fold and Cmax 1.6-fold increased, in comparison with control. Results of this study showed that Ch-TBA in combination with GSH can be an interesting tool for increasing the oral bioavailability of actively secreted compounds.