Florian Föger

Oral gene delivery: Strategies to improve stability of pDNA towards intestinal digestion

Purpose: Gastrointestinal (GI) nucleases are responsible for a rapid presystemic degradation of orally administered transgenes. Within the current study, the activity of these degrading enzymes as well as the effect of various nuclease inhibitors on the degradation process were evaluated in order to assess their potential as auxiliary agents in oral gene delivery. Methods: Digestion assays of pDNA with DNaseI and in GI juices were performed in absence and presence of inhibitors. Consequently, a chitosan conjugate with covalently bound ethylendiaminetetraacetic acid disodium salt dihydrat (EDTA) was synthesized and its nuclease inhibitory properties were evaluated. Results: Small intestinal juice was shown to possess a nuclease activity per millilitre corresponding to 0.02 Kunitz units of DNaseI. Inhibition studies revealed that inhibitory activity followed the ranking: EDTA > sodium dodecyl sulfate (SDS) > aurintricarboxylic acid (ATA) > poly (acrylic acid) > cysteine. The chitosan–EDTA conjugate offered good nuclease inhibiting properties. Conclusion: This study determined the nuclease activity of native porcine small intestinal juice as well as enterocytes homogenate. Moreover, several promising strategies to overcome this enzymatic barrier were identified.

Correlation of in vitro and in vivo models for the oral absorption of peptide drugs

Summary.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 (Papp) 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 (R2 = 0.86; y = −196.22x + 1354.24), with rat oral bioavailability (R2 = 0.64; y = −401.90x + 1268.86) as well as with human oral bioavailability (R2 = 0.91; y = −359.43x + 1103.83). Furthermore it was shown that Papp values of investigated hydrophilic peptides across Caco-2 monolayer displayed lower permeability than across rat intestinal mucosa. A correlation between Papp values across rat intestinal mucosa and in vivo oral bioavailability in human (R2 = 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.

Enhanced transport of P-glycoprotein substrate saquinavir in presence of thiolated chitosan

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.

In vivo evaluation of thiolated poly(acrylic acid) as a drug absorption modulator for MRP2 efflux pump substrates.

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 micromol free thiol groups per gram polymer, was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to that of the buffer control, the sulforhodamine 101 transport in the presence of 0.5% unmodified PAA(250) and 0.5% (w/v) PAA(250)-Cys was 1.3- 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.

Design and evaluation of a chitosan-aprotinin conjugate for the peroral delivery of therapeutic peptides and proteins susceptible to enzymatic degradation.

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.

Evaluation of in vitro enzymatic degradation of various thiomers and cross-linked thiomers.

ABSTRACT 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.

Effect of a thiolated polymer on oral paclitaxel absorption and tumor growth in rats

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.

Design and evaluation of a new gastrointestinal mucoadhesive patch system containing chitosan-glutathione.

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 FD4 within 8 h. A 2.5-fold higher transport of FD4 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.