PURPOSE The influence of various sulfhydryl ligands on permeation-enhancing and P-glycoprotein (P-gp) inhibitory properties of the six established thiolated chitosan conjugates was investigated using Rhodamine-123 (Rho-123) and fluorescein isothiocyanate-dextran 4 (FD4) as model compounds. METHODS Permeation of these compounds was tested on freshly excised rat intestine in Ussing-type chambers. Apparent permeability coefficients (Papp) were calculated and compared to values obtained from the buffer only control. RESULTS The lyophilized polymers had a thiol group content in the range of 230-520 μmol/g. Results of this study led to the following rank order in permeation enhancement: chitosan-6-mercaptonicotinic acid (chitosan-6MNA)> chitosan-cysteine (chitosan-Cys)> chitosan-glutathione (chitosan-GSH)> chitosan-4-thiobutylamidine (chitosan-TBA)> chitosan-thioglycolic acid (chitosan-TGA)> chitosan-N-acetyl cysteine (chitosan-NAC). In P-gp inhibition studies, 0.5%(m/v) chitosan-NAC showed the highest inhibitory effect on P-gp, where the Papp was determined to be 3.78-fold increased compared with the buffer control. Among these thiolated chitosans, chitosan-NAC and chitosan-6MNA are the most effective polymers being responsible for P-gp inhibition and permeation enhancement, respectively. CONCLUSION These thiolated chitosans would therefore be advantageous tools for enhancing the noninvasive bioavailability of active pharmaceutical ingredients.

Drug carriers tailored to fit the physicochemical properties of anticancer agents and the therapeutic peculiarities of tumor management are envisioned for improving the effectiveness/toxicity ratio of the current treatments. Polymeric micelles are attracting much attention owing to their unique beneficial features: i) core-shell structure capable to host hydrophobic drugs, raising the apparent solubility in aqueous medium; ii) size adequate for a preferential accumulation (passive targeting) within the tumor, exhibiting enhanced permeability and retention (EPR effect), and iii) unimers that modulate the activity of efflux pumps involved in multidrug resistance (MDR). This review focuses on amphiphilic poly(ethylene oxide)(PEO) and poly(propylene oxide)(PPO) block copolymers, namely the linear poloxamers (Pluronic® or Lutrol®) and the X-shaped poloxamines (Tetronic®), as components of polymeric micelles able to play these three roles. Specific facets of poloxamers have been highlighted some years ago, but recently their wide range of possibilities is beginning to be fully elucidated and understood. Poloxamines are new excipients in the cancer arena and the comparison of their performance with that of poloxamers may enable to identify aspects of their architecture relevant for the optimization of micellar carriers. Clinical trials in progress indicate that drug-loaded polymeric micelles are beneficial regarding efficiency, safety, and compliance of the treatment and quality of life of the patients. The fact that some copolymers are already approved for internal use and several chemotherapy agents will be off patent soon may help to bring the clinical use of poloxamer- or poloxamine-based micelles into a reality in the coming years.

The aim of the present study was to investigate the potential of different thiolated polymers (thiomers) on the catalytic activity of CYP450s on one hand and to explore new inhibitors for CYP activity on the other hand. Several thiolated polymers including poly(acrylic acid)-cysteine (PAA-cysteine), chitosan-thioglycolic acid (chitosan-TGA), and thiolated PEG-g-PEI copolymer along with brij® 35, myrj® 52 and the well-established CYPP450 inhibitor verapamil were screened for their CYP3A4 and CYP2A6 inhibitory activity, and their IC(50) values were determined. Both enzyme inhibition assays were performed in 96-well microtiter plates. 7-Benzyloxy-4-(trifluoromethyl)-coumarin (BFC) and 7-hydroxycoumarin (7-HC) were used as fluorescent substrates in order to determine CYP3A4 and CYP2A6 catalytic activity, respectively. All investigated compounds inhibited CYP3A4 as well as CYP2A6 activity. All tested (thiolated) polymers were found to be more potent inhibitors of CYP3A4 than of CYP2A6 catalytic activity. Apart from verapamil that is a known CYP3A4 inhibitor, brij® 35 and myrj® 52 were explored as potent inhibitors of CYP3A4 and CYP2A6 catalytic activity. Among the tested polymers, the rank order for CYP3A4 inhibition was PAA-cysteine (100kDa)>brij® 35>thiolated PEG-g-PEI copolymer (16kDa)>myrj® 52>PAA (100kDa)>PAA-cysteine (450kDa)>verapamil>PAA (450kDa)>chitosan-TGA (150kDa)>chitosan (150kDa). On the other hand, the rank order of CYP2A6 inhibition was brij® 35>PAA-cysteine (100kDa)>chitosan-TGA (150kDa)>PAA (100kDa)>thiolated PEG-g-PEI copolymer (16kDa)>PAA-cysteine (450kDa)>chitosan (150kDa)>verapamil>PAA (450kDa)>myrj® 52. Thus, this study suggests that (thiolated) polymers display a promising potential to inhibit cytochrome P450s activity and might turn out to be potentially valuable tools for improving the oral bioavailability of actively secreted compounds by avoiding intestinal metabolism.

The aim of the present study was to improve the inhibitory properties of poly(ethylene glycol)(PEG) as excipient in drug delivery systems by covalent attachment of thiol moieties. This was achieved by grafting PEG to polyethylenimine (PEI) and finally thiolation with γ-thiobutyrolatone. Furthermore, the potential of this novel thiolated PEG-g-PEI co-polymer on the transport of rhodamine-123 (Rho-123) as P-gp substrate across freshly excised rat intestinal mucosa was evaluated in Ussing-type chambers. Apparent permeability coefficients (P(app)) were calculated and compared with values gained from experiments with the well-established P-gp inhibitors verapamil, reduced GSH, 6-mercatopurine and vitamin E-TPGS and the structurally similar compounds, myrj 52 and brij 35. The thiolated co-polymer displayed 145.07 ± 1.64 μmol/g of remaining primary amino groups, 84.30 ± 5.43 μmol/g of immobilized thiol groups and 12.74 ± 1.57 μmol/g of disulfide bonds. The approximate molecular mass of the thiolated co-polymer was 16,000 Da. The (1)H-NMR spectrum of PEG-g-PEI co-polymer was characterized by the presence of signal groups of PEG, hexamethylene diisocyanate (HMDI) and PEI substructures. Studies with Caco-2 cells revealed that the thiolated co-polymer shows 6.69 ± 0.27% of cytotoxicity by LDH assay and 93.33 ± 0.07% of cell viability by MTT assay. The thiolated co-polymer in a concentration of 0.5%(w/v) displayed a more pronounced effect on the absorptive transport of Rho-123 (P(app)=15.2 ± 1.0 × 10(-)(6)cm/s) in comparison to reduced GSH, 6-mercatopurine, vitamin E-TPGS, myrj 52 and brij 35. The thiolated co-polymer increased the transport of Rho-123 up to 3.3-fold in comparison to Rho-123 without any inhibitor used as control (P(app)=4.7 ± 0.1 × 10(-)(6)cm/s). The thiolated co-polymer applied in a concentration of 0.1%, 0.25% and 0. 5%(w/v) did not only enhance the absorption but also decreased the secretory transport of Rho-123 resulting in efflux ratios (secretory P(app)/absorptive P(app)) of 1.0, 1.4 and 2.0, respectively. Because of these features the novel thiolated PEG-g-PEI co-polymer seems to exhibit promising properties as novel P-gp inhibitor.

Multidrug resistance (MDR) is a major impediment to the success of cancer chemotherapy. P-glycoprotein is an important and the best-known membrane transporter involved in MDR. Several strategies have been used to address MDR, especially P-glycoprotein-mediated drug resistance in tumors. However, clinical success has been limited, largely due to issues regarding lack of efficacy and/or safety. Nanoparticles have shown the ability to target tumors based on their unique physical and biological properties. To date, nanoparticles have been investigated primarily to address P-glycoprotein and the observed improved anticancer efficacy suggests that nanomedicinal strategies provide a new opportunity to overcome MDR. This article focuses on nanotechnology-based formulations and current nanomedicine approaches to address MDR in tumors and discusses the proposed mechanisms of action.

Delivery of biologically active agents to animals is often perceived to be the poor relation of human drug delivery. Yet this field has a long and successful history of species-specific device and formulation development, ranging from simple approaches and devices used in production animals to more sophisticated formulations and approaches for a wide range of species. While several technologies using biodegradable polymers have been successfully marketed in a range of veterinary and human products, the transfer of delivery technologies has not been similarly applied across species. This may be due to a combination of specific technical requirements for use of devices in different species, inter-species pharmacokinetic, pharmacodynamic and physiological differences, and distinct market drivers for drug classes used in companion and food-producing animals. This chapter reviews selected commercialised and research-based parenteral and non-parenteral veterinary drug delivery technologies in selected domestic species. Emphasis is also placed on the impact of endogenous drug transporters on drug distribution characteristics in different species. In vitro models used to investigate carrier-dependent transport are reviewed. Species-specific expression of transporters in several tissues can account for inter-animal or inter-species pharmacokinetic variability, lack of predictability of drug efficacy, and potential drug-drug interactions.

This study proposes a new concept of double coated nanocapsules to improve the oral bioavailability of a P-glycoprotein (P-gp) substrate drug, tacrolimus, without modulating the physiological activity of the P-gp pump. Tacrolimus was incorporated in nanocapsules with different ratios of two polymethacrylate polymers followed by microencapsulation of these nanocapsules within hydroxypropylmethylcellulose using a spray drying technique. The influence of different formulations of tacrolimus administered orally to rats and pigs on the drug's absorption was investigated. Histopathological studies were performed on rats to follow the nanocapsule path in enterocytes. The novel formulations that released mostly drug loaded nanocapsules in the intestine were shown to enhance markedly the oral absorption of tacrolimus. The relative oral bioavailability of tacrolimus was 4.9 and 2.45 fold compared to the commercial product in rats and pigs respectively. Although there is no direct evidence that intact nanocapsules internalized in the enterocytes, numerous small oil cores were detected within the enterocytes showing the potential of P-gp substrates incorporated in such nanocarriers to escape the efflux pump.

The cationic polysaccharide chitosan has been extensively studied for oral drug delivery. In recent years, chemically modified chitosans developed in order to improve the properties of chitosan for oral drug delivery have gained increasing attention. Representatives of these novel polymers are trimethyl-chitosans, thiolated chitosans, carboxymethyl chitosan and derivatives, hydrophobic chitosans, chitosan succinate and phthalate, PEGylated chitosans and chitosan-enzyme inhibitor conjugates. Besides their use for oral delivery of therapeutic peptides and proteins, they have recently been evaluated regarding their potential for the delivery of other substance classes, including genes and efflux pump substrates. Within the current review, various modified chitosan derivatives, their properties and synthesis are discussed.(c) 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci.

A new oral patch delivery system has been designed to increase the overall oral bioavailability of drugs within the gastrointestinal tract. The patch system consists of four layered films: a mucoadhesive matrix layer, a water insoluble backing layer, a middle layer and an enteric surface layer. The separation layer between the two matrix layers contained lactose, starch and confectioners' sugar. The matrix layer, exhibiting a diameter of 2.5 mm and a weight of 5 mg, comprised Polycarbophil-cysteine conjugate (49%), fluoresceine isothiocyanate-dextran (26%), glutathione (5%), and mannitol (20%). A standard tablet formulation consisting of the same matrix served as control. Entire fluoresceine isothiocyanate-dextran (FD(4)) was released from the delivery system within 2 h. For in vivo studies patch systems were administered orally to Male Sprague-Dawley rats. Maximum FD(4) concentration in blood of the patch system was 46.1 +/- 8.9 ng/mL and was reached 3 h after administration. In contrast c(max) of control tablets displayed 50.5 +/- 14.9 ng/mL after 2 h and the absorption of FD(4) after administration in oral solution was negligible. The absolute bioavailability of orally administered patch systems and control tablets was 0.54% and 0.32% respectively. Results of this study indicate that a prolonged and higher oral bioavailability of FD(4) is obtained with patches thou with tablets.(c) 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci.

Microparticles were formulated by incorporation of the model protein horseradish peroxidase in (thiolated) chitosan and (thiolated) poly(acrylic acid) via co-precipitation. Dried protein/polymer complexes were ground with an air jet mill and resulting particles were evaluated regarding size distribution, shape, zeta potential, drug load, protein activity, release pattern, swelling behaviour and cytotoxicity. The mean particle size distribution was 0.5-12mum. Non-porous microparticles with a smooth surface were prepared. Microparticles from (thiolated) chitosan had a positive charge whereas microparticles from (thiolated) poly(acrylic acid) were negatively charged. The maximum protein load for microparticles based on chitosan, chitosan-glutathione (Ch-GSH), poly(acrylic acid)(PAA) and for poly(acrylic acid)-glutathione (PAA-GSH) was 7+/-1%, 11+/-2%, 4+/-0.2% and 7+/-2%, respectively. The release profile of all microparticles followed a first order release kinetic. Chitosan (0.5mg), Ch-GSH, PAA and PAA-GSH particles showed a 31.4-, 13.8-, 54.2- and a 42.2-fold increase in weight, respectively. No significant cytotoxicity could be found. Thiolated microparticles prepared by jet milling technique were shown to be stable and to have controlled drug release characteristics. After further optimizations the preparation method described here might be a useful tool for the production of protein loaded drug delivery systems.

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.

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.

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.

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.

PURPOSE: It was the aim of this study to synthesize and characterize a novel chitosan-glutathione (GSH) conjugate providing improved mucoadhesive and permeation-enhancing properties. METHODS: Mediated by carbodiimide and N-hydroxysuccinimide, glutathione was covalently attached to chitosan via the formation of an amide bond. The adhesive properties of chitosan-GSH conjugate were evaluated in vitro on freshly excised porcine mucosa via tensile studies and the rotating cylinder method. The cohesive properties and stability of the resulting conjugate were evaluated by disintegration test and by oxidation experiments, respectively. The permeation-enhancing effect of the chitosan-GSH/GSH system was evaluated in Ussing chambers by using rhodamine 123 as model compound. RESULTS: The obtained conjugate displayed 265.5 mumol immobilized free thiol groups and 397.9 micromol disulfide bonds per gram polymer. Because of the formation of disulfide bonds within the polymer, the stability of matrix tablets could be strongly improved. In tensile studies, the total work of adhesion of the conjugate was determined to be 9.9-fold increased in comparison to unmodified chitosan. Results from the rotating cylinder method showed more than 55-fold increase in the adhesion time of thiolated chitosan vs. unmodified chitosan. In addition, the conjugate in combination with GSH displayed a 4.9-fold higher permeation-enhancing effect compared with unmodified chitosan. CONCLUSIONS: Because of the improved mucoadhesive and cohesive properties, and the strong permeation-enhancing effect of the chitosan-GSH conjugate/GSH system, the novel thiolated chitosan seems to represent a promising multifunctional excipient for various drug delivery systems.

The aim of this study was to establish and evaluate a high pressure homogenization method for the preparation of thiomer nanoparticles. Particles were formulated by incorporation of the model protein horseradish peroxidase in chitosan-glutathione (Ch-GSH) and poly(acrylic acid)-glutathione (PAA-GSH) via co-precipitation followed by air jet milling. The resulting microparticles were suspended in distilled water using an Ultraturax and subsequently micronized by high pressure homogenization. Finally, resulting particles were evaluated regarding size distribution, shape, zeta potential, drug load, protein activity and release behaviour. The mean particle size after 30 cycles with a pressure of 1500 bar was 538 +/- 94 nm for particles consisting of Ch-GSH and 638 +/- 94 nm for particles consisting of PAA-GSH. Nanoparticles of Ch-GSH had a positive zeta-potential of +1.03 mv, whereas nanoparticles from PAA-GSH had a negative zeta potential of -6.21 mv. The maximum protein load for nanoparticles based on Ch-GSH and based on PAA-GSH was 45 +/- 2% and 37 +/-%, respectively. The release profile of nanoparticles followed a first order release kinetic. Thiolated nanoparticles prepared by a high pressure homogenization technique were shown to be stable and provide controlled drug release characteristics. The preparation method described here might be a useful tool for a more upscaled production of nanoparticulate drug delivery systems.

Objective: The aim of this review is to provide the reader general and inspiring prospects in various attempts to make noninvasive delivery systems of calcitonin and teriparatide feasible and as convenient as possible. Background: Calcitonin and teriparatide play an important role in both calcium homeostasis and bone remodelling. Currently calcitonin is available as a subcutaneous injection and as a nasal spray whereas teriparatide is administered subcutaneously. In the past few years, an increasing number of articles about drug delivery systems for calcitonin and teriparatide have been published. These delivery systems have been developed to overcome the inherent barriers for the uptake across the diverse membranes on the various routes for protein and peptide delivery. Results: Co-administration of permeation enhancers, mucoadhesive agents, viscosity modifying agents, multifunctional polymers, protease inhibitors as well as encapsulation and chemical modification are utilized in order to improve calcitonin and teriparatide absorption after oral, nasal, pulmonal, or buccal administration. Conclusion: The majority of research groups have been working on the development of formulations based on the encapsulation of molecules in biodegradable and biocompatible polymeric nanoparticles. However these observations are based on data obtained under different experimental conditions. Hence, it is difficult to compare the obtained results in order to draw general conclusions about the most promising characteristics required for oral and nasal formulations for these peptides.

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.

A new oral patch delivery system has been designed to increase the overall oral bioavailability of drugs within the gastrointestinal tract. The patch system consists of four layered films: a mucoadhesive matrix layer, a water insoluble backing layer, a middle layer and an enteric surface layer. The separation layer between the two matrix layers contained lactose, starch and confectioners' sugar. The matrix layer, exhibiting a diameter of 2.5 mm and a weight of 5 mg, comprised Polycarbophil-cysteine conjugate (49%), fluoresceine isothiocyanate-dextran (26%), glutathione (5%), and mannitol (20%). A standard tablet formulation consisting of the same matrix served as control. Entire fluoresceine isothiocyanate-dextran (FD(4)) was released from the delivery system within 2 h. For in vivo studies patch systems were administered orally to Male Sprague-Dawley rats. Maximum FD(4) concentration in blood of the patch system was 46.1 +/- 8.9 ng/mL and was reached 3 h after administration. In contrast c(max) of control tablets displayed 50.5 +/- 14.9 ng/mL after 2 h and the absorption of FD(4) after administration in oral solution was negligible. The absolute bioavailability of orally administered patch systems and control tablets was 0.54% and 0.32% respectively. Results of this study indicate that a prolonged and higher oral bioavailability of FD(4) is obtained with patches thou with tablets.(c) 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci.

Microparticles were formulated by incorporation of the model protein horseradish peroxidase in (thiolated) chitosan and (thiolated) poly(acrylic acid) via co-precipitation. Dried protein/polymer complexes were ground with an air jet mill and resulting particles were evaluated regarding size distribution, shape, zeta potential, drug load, protein activity, release pattern, swelling behaviour and cytotoxicity. The mean particle size distribution was 0.5-12mum. Non-porous microparticles with a smooth surface were prepared. Microparticles from (thiolated) chitosan had a positive charge whereas microparticles from (thiolated) poly(acrylic acid) were negatively charged. The maximum protein load for microparticles based on chitosan, chitosan-glutathione (Ch-GSH), poly(acrylic acid)(PAA) and for poly(acrylic acid)-glutathione (PAA-GSH) was 7+/-1%, 11+/-2%, 4+/-0.2% and 7+/-2%, respectively. The release profile of all microparticles followed a first order release kinetic. Chitosan (0.5mg), Ch-GSH, PAA and PAA-GSH particles showed a 31.4-, 13.8-, 54.2- and a 42.2-fold increase in weight, respectively. No significant cytotoxicity could be found. Thiolated microparticles prepared by jet milling technique were shown to be stable and to have controlled drug release characteristics. After further optimizations the preparation method described here might be a useful tool for the production of protein loaded drug delivery systems.

Purpose: The objective of this study was the investigation of permeation enhancing and P-glycoprotein (P-gp) inhibition effects of a novel thiolated chitosan, so-named S-protected thiolated chitosan. Methods: 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 FD4 as 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 (Papp) were calculated and compared to values obtained from each buffer control. Results: 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 permeation studies on excised rat intestine, Papp values of all S-protected chitosans were at least 1.3-fold higher compared to corresponding thiomers and more than twice as high as unmodified chitosan. Additional permeation studies on Caco-2 cells confirmed these results. Due to 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. Conclusion: These features approve S-protected thiolated chitosan as promising excipient for various drug delivery systems providing improved permeation enhancing and efflux inhibition effects.

PURPOSE: To evaluate of the effect of size and surface characteristics of poly(isobutylcyanoacrylate) nanoparticles coated with pluronic F68 and thiolated chitosan on mucoadhesion. METHODS: Nanoparticles were obtained by radical emulsion polymerization in presence of different amounts of F68 (0-4%w/v). Mucoadhesion was ex vivo evaluated by applying nanoparticle suspension on rat intestinal mucosa and quantifying the amount of attached nanoparticles after incubation. RESULTS: F68 unimers added in the polymerization medium allowed decreasing nanoparticle size from 251 to 83 nm, but resulted in nanoparticle surface modification. The amount of thiolated chitosan onto nanoparticle surface was decreased resulting in lower thiol groups and zeta potential. Consequently, the decrease of nanoparticle hydrodynamic diameter resulted in eight-fold-increase of the number of nanoparticles attached to the mucosa but a significant decrease of the weight of attached nanoparticles was observed. This unexpected result was due to a decrease of the amount of chitosan and thiolated chitosan available to interact with mucus upon addition of F68 in the polymerization medium. CONCLUSIONS: Addition of F68 should not be recommended to improve the amount of mucoadherent nanoparticles. Further studies could allow understanding if the low amount of small size nanoparticles could be able to improve oral bioavailability.

The present study aimed to improve the in vivo effectiveness of biochanin A as a P-gp inhibitor by formulation in solid dispersion (SD). SDs were prepared with Solutol HS15 and hydroxypropylmethyl cellulose (HPMC2910) and their inhibition effect on P-gp mediated cellular efflux was examined by using NCI/ADR-RES cells overexpressing P-gp. Compared to the untreated biochanin A, SD formulations enhanced significantly (p < 0.01) the cellular uptake of rhodamine-123, a P-gp substrate by approximately 2-3 folds in NCI/ADR-RES cells. Furthermore, the oral and intravenous pharmacokinetics of diltiazem, a P-gp substrate as well as its active metabolite, desacetyldiltiazem, was determined in rats after pretreatment with biochanin A. Pretreatment with biochanin A in a SD formulation significantly (p < 0.05) increased the AUC of desacetyldiltiazem by 3-fold, although the oral exposure of diltiazem was not altered. In contrast, the intravenous pharmacokinetics of diltiazem and desacetyldiltiazem were not changed by the concurrent use of biochanin A, implying that oral biochanin A affected mainly the intestinal absorption of diltiazem rather than the hepatic extraction. In conclusion, SD formulation improved the in vivo effectiveness of biochanin A as a P-gp inhibitor.

The aim of the present study was to develop an oral delivery system for the peptide drug leuprolide. Gel formulations based on unmodified chitosan/reduced glutathione (GSH) and chitosan-thioglycolic acid (chitosan-TGA)/GSH were prepared, and their effect on the absorption of leuprolide was evaluated in vitro and in vivo in male Sprague Dawley rats. Transport studies were performed with freshly excised rat intestinal mucosa mounted in Ussing-type chambers. Due to the addition of gel formulations comprising 0.5%(m/v) unmodified chitosan/0.5%(m/v) GSH and 0.5%(m/v) chitosan-TGA/0.5%(m/v) GSH, the transport of leuprolide across excised mucosa was improved up to 2.06-fold and 3.79-fold, respectively, in comparison with leuprolide applied in buffer (P(app)=2.87±0.77×10(-6)cm/s). In vivo, the addition of oral gel formulation comprising 8mg of unmodified chitosan, 1mg of GSH and 1mg of leuprolide increased the area under the plasma concentration-time curve (AUC(0-8)) of leuprolide 1.39-fold in comparison with leuprolide having been administered just in saline. Moreover, the administration of oral gel formulation comprising 8mg of chitosan-TGA, 1mg of GSH and 1mg of leuprolide resulted in a further enhanced leuprolide plasma concentration, and the area under the plasma concentration-time curve (AUC(0-8)) of leuprolide was increased 3.72-fold in comparison with the control. With the oral gel formulation comprising 8mg of chitosan-TGA, a relative bioavailability (versus s.c. injection) of 4.5% was achieved in contrast to the control displaying a relative bioavailability of 1.2%. Thus, according to the achieved results, it is suggested that chitosan-TGA in combination with GSH is a valuable tool for improving the oral bioavailability of the peptide drug leuprolide.

PURPOSE The influence of various sulfhydryl ligands on permeation-enhancing and P-glycoprotein (P-gp) inhibitory properties of the six established thiolated chitosan conjugates was investigated using Rhodamine-123 (Rho-123) and fluorescein isothiocyanate-dextran 4 (FD4) as model compounds. METHODS Permeation of these compounds was tested on freshly excised rat intestine in Ussing-type chambers. Apparent permeability coefficients (Papp) were calculated and compared to values obtained from the buffer only control. RESULTS The lyophilized polymers had a thiol group content in the range of 230-520 μmol/g. Results of this study led to the following rank order in permeation enhancement: chitosan-6-mercaptonicotinic acid (chitosan-6MNA)> chitosan-cysteine (chitosan-Cys)> chitosan-glutathione (chitosan-GSH)> chitosan-4-thiobutylamidine (chitosan-TBA)> chitosan-thioglycolic acid (chitosan-TGA)> chitosan-N-acetyl cysteine (chitosan-NAC). In P-gp inhibition studies, 0.5%(m/v) chitosan-NAC showed the highest inhibitory effect on P-gp, where the Papp was determined to be 3.78-fold increased compared with the buffer control. Among these thiolated chitosans, chitosan-NAC and chitosan-6MNA are the most effective polymers being responsible for P-gp inhibition and permeation enhancement, respectively. CONCLUSION These thiolated chitosans would therefore be advantageous tools for enhancing the noninvasive bioavailability of active pharmaceutical ingredients.

Thiolated polyacrylates were shown to be permeation enhancers with notable potential. The aim of this study was to evaluate the permeation enhancing properties of a thiolated polycarbophil/glutathione (PCP-Cys/GSH) system for oral drug application in comparison to a well-established permeation enhancer, namely sodium caprate. In vitro permeation studies were conducted in Ussing-type chambers with sodium fluoresceine (NaFlu) and fluoresceine isothiocyanate labeled dextran (molecular mass 4 kDa; FD4) as model compounds. Bioavailability studies were carried out in Sprague Dawley rats with various formulations. Moreover, cytotoxic effects of both permeation enhancers were compared. Permeation enhancement ratios of 1% sodium caprate were found to be 3.0 (FD4) and 2.3 (NaFlu), whereas 1% PCP-Cys/0.5% GSH displayed enhancement ratios of 2.4 and 2.2. Both excipients performed at a similar level in vivo. Sodium caprate solutions increased oral bioavailability 2.2-fold (FD4) and 2.3-fold (NaFlu), while PCP-Cys hydrogels led to a 3.2-fold and 2.2-fold enhancement. Cell viability experiments revealed a significantly higher tolerance of Caco-2 cells towards 0.5% PCP-Cys (81% survival) compared to 0.5% sodium caprate (5%). As PCP-Cys is not absorbed from mucosal membranes due to its comparatively high molecular mass, systemic side-effects can be excluded. In conclusion, both systems displayed a similar potency for permeation enhancement of hydrophilic compounds. However, PCP-Cys seems to be less harmful to cultured cells.

INTRODUCTION: The ATP-binding cassette superfamily contains membrane transporter proteins that transport a wide range of diverse compounds across cellular membranes. The P-glycoprotein (P-gp) is an important member of this family and a multi-specific drug efflux transporter that plays a significant role in governing the bioavailability of many clinically active drugs. The inhibition of this efflux transporter by various P-gp inhibitors forms a distinctive approach in improving bioavailability and conquering drug resistance. Most P-gp inhibitors exhibit limitations associated with their safety and unwanted pharmacokinetic interactions, thereby restraining their clinical applicability. AREAS COVERED: This review explores the investigations on the feasibility and applicability of various classes of P-gp inhibitors as described in recent patents for enhanced drug delivery. EXPERT OPINION: Several candidates presently under development look promising as P-gp inhibitors, e.g., tariquidar and elacridar. Pharmaceutical excipients currently constitute the most promising class of P-gp inhibitors and are considered safe and pharmaceutically acceptable for use in formulations. In addition, lipid-based excipients and thiolated polymers play an active role in affecting P-gp-mediated transport not only by altering the membrane fluidity or ATPase activity but by down regulating P-gp expression. An additional overture such as the prodrug derivatization of P-gp substrates is a feasible approach to bypass P-gp-mediated efflux.

The purpose of this study was to develop and evaluate an oral oligonucleotide delivery system based on a thiolated polymer/reduced glutathione (GSH) system providing a protective effect toward nucleases and permeation enhancement. A polycarbophil-cysteine conjugate (PCP-Cys) was synthesized. Enzymatic degradation of a model oligonucleotide by DNase I and within freshly collected intestinal fluid was investigated in the absence and presence of PCP-Cys. Permeation studies with PCP-Cys/GSH versus control were performed in vitro on Caco-2 cell monolayers and ex vivo on rat intestinal mucosa. PCP-Cys displayed 223 ± 13.8 μmol thiol groups per gram polymer. After 4h, 61% of the free oligonucleotides were degraded by DNase I and 80% within intestinal fluid. In contrast, less than 41%(DNase I) and 60%(intestinal fluid) were degraded in the presence of 0.02%(m/v) PCP-Cys. Permeation studies revealed an 8-fold (Caco-2) and 10-fold (intestinal mucosa) increase in apparent permeability compared to buffer control. Hence, this PCP-Cys/GSH system might be a promising tool for the oral administration of oligonucleotides as it allows a significant protection toward degrading enzymes and facilitates their transport across intestinal membranes.

Fondaparinux is an agent of choice for the prevention and initial treatment of venous thromboembolism (VTE) as well as myocardial infarction. Nevertheless, as a negatively charged molecule fondaparinux can pass the intestinal epithelial barrier after oral administration only partially. It was therefore the aim of this study to design a highly efficient small-intestinal-targeted oral delivery system for fondaparinux based on thiolated polycarbophil (PCP-Cys) and glutathione (GSH) combined with sodium decanoate. The formulations were tested in vitro with regard to their release, cytotoxicity profiles and their permeation-enhancing properties across small-intestinal mucosa. For the in vivo study, rats were treated with a single oral dose of fondaparinux gels or mini-tablets (5mg/kg) and the subcutaneous and intravenous groups with a dose of 200μg/kg fondaparinux. The anti-factor Xa activity in the plasma was measured. In the presence of PCP-Cys/GSH/sodium decanoate the uptake of fondaparinux from the intestinal mucosa was 4.1-fold improved. The area under concentration-time curve in rat plasma from 0 to 24h with PCP-Cys/GSH/sodium decanoate gel was 135.3μgmin/ml and 1.3-fold improved with the tablets. C(max) value of mini-tablets was 0.23μg/ml and the absolute bioavailability of 4.4% was 6.2-fold improved, while the control solution was not absorbed orally. PCP-Cys/GSH/sodium decanoate demonstrated potential for increasing the oral bioavailability of the indirect factor Xa inhibitor as an alternative to currently used subcutaneous delivery.

The aim of the present study was to improve the inhibitory properties of poly(ethylene glycol)(PEG) as excipient in drug delivery systems by covalent attachment of thiol moieties. This was achieved by grafting PEG to polyethylenimine (PEI) and finally thiolation with γ-thiobutyrolatone. Furthermore, the potential of this novel thiolated PEG-g-PEI co-polymer on the transport of rhodamine-123 (Rho-123) as P-gp substrate across freshly excised rat intestinal mucosa was evaluated in Ussing-type chambers. Apparent permeability coefficients (P(app)) were calculated and compared with values gained from experiments with the well-established P-gp inhibitors verapamil, reduced GSH, 6-mercatopurine and vitamin E-TPGS and the structurally similar compounds, myrj 52 and brij 35. The thiolated co-polymer displayed 145.07 ± 1.64 μmol/g of remaining primary amino groups, 84.30 ± 5.43 μmol/g of immobilized thiol groups and 12.74 ± 1.57 μmol/g of disulfide bonds. The approximate molecular mass of the thiolated co-polymer was 16,000 Da. The (1)H-NMR spectrum of PEG-g-PEI co-polymer was characterized by the presence of signal groups of PEG, hexamethylene diisocyanate (HMDI) and PEI substructures. Studies with Caco-2 cells revealed that the thiolated co-polymer shows 6.69 ± 0.27% of cytotoxicity by LDH assay and 93.33 ± 0.07% of cell viability by MTT assay. The thiolated co-polymer in a concentration of 0.5%(w/v) displayed a more pronounced effect on the absorptive transport of Rho-123 (P(app)=15.2 ± 1.0 × 10(-)(6)cm/s) in comparison to reduced GSH, 6-mercatopurine, vitamin E-TPGS, myrj 52 and brij 35. The thiolated co-polymer increased the transport of Rho-123 up to 3.3-fold in comparison to Rho-123 without any inhibitor used as control (P(app)=4.7 ± 0.1 × 10(-)(6)cm/s). The thiolated co-polymer applied in a concentration of 0.1%, 0.25% and 0. 5%(w/v) did not only enhance the absorption but also decreased the secretory transport of Rho-123 resulting in efflux ratios (secretory P(app)/absorptive P(app)) of 1.0, 1.4 and 2.0, respectively. Because of these features the novel thiolated PEG-g-PEI co-polymer seems to exhibit promising properties as novel P-gp inhibitor.