Chitosan-thioglycolic Acid Conjugate Research Articles

Influence of Chitosan Thioglycolic Acid Conjugate in improving bioavailability of an Antiparkinson drug; Rasagiline Mesylate from transdermal patch

: Objective Parkinson Disease (PD) is a chronic disorder of central nervous system mainly affecting the motor systems. The drug of choice to treat PD is Rasagiline Mesylate (RM) and it belongs to BCS class III drug. The objective of the present study was preparation of transdermal drug delivery system for RM. Several permeation enhancers were screened to be included in the formulation. To achieve desired flux a new strategy was developed by including in-house prepared CTC to enhance the permeation of RM. Methods The CTC was prepared by reaction between Chitosan and Thioglycolicacid, characterized by determining physical properties and applying analytical tools. Seven permeation enhancers with different mechanisms were screened. The transdermal patches were prepared with chitosan along with permeation enhancer IPM, various proportions of CTC and evaluated for physical and permeation studies. The optimized transdermal patch was obtained by two factors and three responses to obtain the design space and further evaluated for pharmacokinetic studies. Results The results of the present study confirmed the formation of CTC, IPM was best permeation enhancer among all. The presence of CTC in the formulations significantly improved the permeation of RM to achieve desired steady state flux. The relative bioavailability of optimized transdermal patch was determined and it was observed that improved bioavailability as compared to marketed conventional tablets. Conclusion The study was concluded that CTC has significant influence on permeation enhancing ability of IPM.

Synthesis of conjugated chitosan and its effect on drug permeation from transdermal patches.

The aim of this study was to synthesis the conjugated chitosan by covalent attachment of thiol moieties to the cationic polymer, mediated by a carbodiimide to improve permeation properties of chitosan. Thioglycolic acid was covalently attached to chitosan by the formation of amide bonds between the primary amino groups of the polymer and the carboxylic acid groups of thioglycolic acid. Hence, these polymers are called as thiomers or thiolated polymers. Conjugation of chitosan was confirmed by Fourier transform-infrared and differential scanning calorimetric analysis. Matrix type transdermal patches of carvedilol were prepared using the different proportions of chitosan and chitosan-thioglycolic acid conjugates (2:0, 1.7:0.3, 1.4:0.6, 1:1, 0.6:1.4 and 0.3:1.7) by solvent casting technique. Prepared matrix type patches were evaluated for their physicochemical characterization followed by in vitro evaluation. Selected formulations were subjected for their ex vivo studies on Wistar albino rat skin and human cadaver skin using the modified Franz diffusion cell. As the proportion of conjugated chitosan increased, the transdermal patches showed increased drug permeation. The mechanism of drug release was found to be nonFickian profiles. The present study concludes that the transdermal patches of carvedilol using conjugated chitosan with different proportions of chitosan were successfully developed to provide improved drug permeation. The transdermal patches can be a good approach to improve drug bioavailability by bypassing the extensive hepatic first-pass metabolism of the drug.

Design and evaluation of an intravesical delivery system for superficial bladder cancer: preparation of gemcitabine HCl-loaded chitosan-thioglycolic acid nanoparticles and comparison of chitosan/poloxamer gels as carriers.

This study aimed to develop an intravesical delivery system of gemcitabine HCl for superficial bladder cancer in order to provide a controlled release profile, to prolong the residence time, and to avoid drug elimination via urination. For this aim, bioadhesive nanoparticles were prepared with thiolated chitosan (chitosan–thioglycolic acid conjugate) and were dispersed in bioadhesive chitosan gel or in an in situ gelling poloxamer formulation in order to improve intravesical residence time. In addition, nanoparticle-loaded gels were diluted with artificial urine to mimic in vivo conditions in the bladder and were characterized regarding changes in gel structure. The obtained results showed that chitosanthioglycolic acid nanoparticles with a mean diameter of 174.5±3.762 nm and zeta potential of 32.100±0.575 mV were successfully developed via ionotropic gelation and that the encapsulation efficiency of gemcitabine HCl was nearly 20%. In vitro/ex vivo characterization studies demonstrated that both nanoparticles and nanoparticle-loaded chitosan and poloxamer gels might be alternative carriers for intravesical administration of gemcitabine HCl, prolonging its residence time in the bladder and hence improving treatment efficacy. However, when the gel formulations were diluted with artificial urine, poloxamer gels lost their in situ gelling properties at body temperature, which is in conflict with the aimed formulation property. Therefore, 2% chitosan gel formulation was found to be a more promising carrier system for intravesical administration of nanoparticles.

Enzymatic degradation of thiolated chitosan

The objective of this study was to evaluate the biodegradability of thiolated chitosans in comparison to unmodified chitosan. Mediated by carbodiimide, thioglycolic acid (TGA) and mercaptonicotinic acid (MNA) were covalently attached to chitosan via formation an amide bond. Applying two different concentrations of carbodiimide 50 and 100 mM, two chitosan TGA conjugates (TGA A and TGA B) were obtained. According to chitosan solution (3% m/v) thiomer solutions were prepared and chitosanolytic enzyme solutions were added. Lysozyme, pectinase and cellulase were examined in chitosan degrading activity. The enzymatic degradability of these thiomers was investigated by viscosity measurements with a plate–plate viscometer. The obtained chitosan TGA conjugate A displayed 267.7 µmol and conjugate B displayed 116.3 µmol of immobilized thiol groups. With 325.4 µmol immobilized thiol groups, chitosan MNA conjugate displayed the most content of thiol groups. In rheological studies subsequently the modification proved that chitosan TGA conjugates with a higher coupling rate of thiol groups were not only degraded to a lesser extent by 20.9–26.4% but also more slowly. Chitosan mercaptonicotinic acid was degraded by 31.4–50.1% depending the investigated enzyme and even faster than unmodified chitosan. According to these results the biodegradability can be influenced by various modifications of the polymer which showed in particular that the rate of biodegradation is increased when MNA is the ligand, whereas the degradation is hampered when TGA is used as ligand for chitosan.

<i>In Situ</i> Crosslinkable Thiolated Chitosan as Scaffold Material for Tissue Engineering

The scope of the study was to investigate the potential of thiolated chitosan as scaffold material for tissue engineering. Porous scaffolds were fabricated by freeze-drying method and stailized via disulfide bond formation. Characterizations were focused on morphological structure and degree of disulfide cross-linking. Renal proximal tubule epithelial cells (RPTECs) were used as model cell line. Chitosan-thioglycolic acid conjugate (chitosan-TGA) displayed 624.8 ± 21.2 and 1195.9 ± 61.0 µmol thiol groups/g polymer (chitosan-TGA 625 and chitosan-TGA 1196, respectively). After 4 days of incubation, the attachment of cells on the chitosan-TGA 625 and chitosan-TGA 1196 scaffolds were 101 ± 6.36 and 157.5 ± 2.82 cells/mm2, respectively. However, RPTECs did not grow on the unmodified chitosan scaffolds. These results confirm the potential utility of chitosan-TGA as a novel candidate being used as scaffold material in tissue engineering.

In situ gelling properties of chitosan-thioglycolic acid conjugate in the presence of oxidizing agents

The rheological behaviour of chitosan-thioglycolic acid conjugate (chitosan-TGA) in the presence of four oxidizing agents was investigated. Chitosan-TGA was synthesized via amide bond formation between the primary amino group of chitosan and the carboxylic acid group of thioglycolic acid. The sol–gel phase transition of the polymer was determined by rheological measurements. Moreover, cytotoxicity of the gel in combination with each oxidizing agent was evaluated utilizing LDH and MTT assay. The modified chitosan displayed 1053 ± 44 μmol/g thiol groups. Results of rheological studies showed that 1% (m/v) chitosan-TGA without any oxidizing agents became gel within 40 min. In contrast, when the oxidizing agents hydrogen peroxide, sodium periodate, ammonium persulfate and sodium hypochlorite were added, respectively, gelation took place within a few minutes. Within 20 min, hydrogen peroxide having been added in a final concentration of 25.2 nmol/L increased dynamic viscosity of 1% (m/v) chitosan-TGA up to 16,500-fold. This can be explained by the formation of inter- and/or intramolecular disulfide bonds which were indirectly verified via the decrease in thiol groups. Additionally, evidence of an increase in cross-linking of thiolated chitosan as a function of time was provided by frequency sweep measurements. Furthermore, viability of Caco-2 cells having been incubated with chitosan-TGA/oxidizing agent systems assessed by MTT assay was 70–85% and the percentage of LDH release was only in case of the chitosan-TGA/ammonium persulfate system significantly ( p < 0.05) raising compared to the negative control. According to these results, chitosan-TGA/oxidizing agent combinations might be a promising novel in situ gelling system for various pharmaceutical applications such as a controlled drug release carrier or for tissue engineering.

Chitosan-thioglycolic acid conjugate microspheres: their use for oral delivery of rEPO

Chitosan-thioglycolic acid conjugates were synthesized and their characteristics, including the thiol group content and bioadhesive properties, were evaluated. Chitosan-thioglycolic acid conjugate microspheres (C-TGACMs), containing erythropoietin (rEPO) as a model drug, with or without an enteric coating were prepared, and the entrapment efficiency, the drug release behavior in vitro and the antianaemic effect of rEPO were investigated. It was found that the chitosan-thioglycolic acid conjugate with a 5.56% (w/w) thiol group content exhibited better bioadhesion. Also, the C-TGACMs, with or without an enteric coating, had a good spherical shape and release profiles consisted of a quick-release phase and a stable sustained-release phase. The entrapment efficiencies of C-TGACMs, with and without an enteric coating, were 84.3 and 85.7%, respectively. When rEPO loaded enteric-coated C-TGACMs were given orally to rats, they produced an excellent antianaemic effect, which was evaluated by the Smear and Sysmex methods.

Development of a mucoadhesive and permeation enhancing buccal delivery system for PACAP (pituitary adenylate cyclase-activating polypeptide)

The buccal mucosa providing direct entry into the systemic circulation appears to be a potential site for the delivery of PACAP (pituitary adenylate cyclase-activating polypeptide), a new therapeutic agent in the treatment of type 2 diabetes. In order to reach a sufficient buccal bioavailability a drug delivery system with strong permeation enhancing and mucoadhesive properties is needed. In this study the enhancing effect of a strongly mucoadhesive chitosan–thioglycolic acid (TGA) conjugate in combination with reduced glutathione (GSH) on the permeation of PACAP across the buccal mucosa was investigated. The apparent permeability coefficient ( P app) of PACAP in buffer only was (5.7 ± 3.1) × 10 −8, while in the presence of chitosan–TGA conjugate (1%) a P app of (20.0 ± 3.4) × 10 −8 was achieved. The combination of chitosan–TGA (1%) with GSH (2%) led to an improvement of the P app up to (57.3 ± 31.7) × 10 −8. Release studies of PACAP demonstrated that a controlled release can be provided from tablets consisting of chitosan–TGA at a pH of 5, whereas more than twice as much was released from chitosan–TGA tablets pH 4. According to the combination of permeation enhancing properties, controlled drug release and the mucoadhesive character, chitosan–TGA conjugates represent a promising tool for the buccal administration of PACAP.

Application of chitin and chitosan derivatives in the pharmaceutical field.

Chitin and chitosan derivatives are used as excipients and drug carriers in the pharmaceutical field. Their derivatization contributed to expansion of application and decrease toxicity. Chitosan is used as an excipient in oral dosage form. Chitosan tablet can exhibit a sustained drug release compared to commercial products. Films prepared using chitin or chitosan have been developed as wound dressings, oral mucoadhesive and water-resisting adhesive by virtue of their release characteristics and adhesion. Intratumoral administration of gadopentetic acid-chitosan complex nanoparticles (approximately 430 nm in diameter) has been more effective for gadolinium neutron-capture therapy compared with a group treated with the solution. Compared to intragastrical feeding with diphtheria toxoid (DT) in PBS, a strong enhancement of the systemic (IgG) and local (IgA) immune responses against DT has been observed in mice fed with DT loaded chitosan microparticles (approximately 4.7 microm in size). When DNA-loaded chitosan microspheres (1.15 - 1.28 microm) were intramuscularly administrated into mice, high beta-galactosidase and luciferase productions were obtained even after a long post-transfection period (12 weeks). N-Succinyl-chitosan (Suc-Chi) has been studied for cancer chemotherapy as a drug carrier and the conjugates of mitomycin C with Suc-Chi exhibited good antitumor activities against various tumors. Furthermore, trimethyl-chitosan and monocarboxymethyl-chitosan has been shown to be effective as intestinal absorption enhancers due to their physiological properties. Chitosan-thioglycolic acid conjugates has been found to be a promising candidate as scaffold material in tissue engineering due to their physicochemical properties. This review summarizes the application of chitin and chitosan derivatives for hospital preparations and drug carriers.

Chitosan-thioglycolic acid conjugate: a new scaffold material for tissue engineering?

It was the aim of this study to evaluate chitosan-thioglycolic acid (chitosan-TGA) conjugate as scaffold material in tissue engineering. Chitosan was modified by the introduction of thiol groups. Briefly, TGA was introduced to chitosan via amide bond formation mediated by a carbodiimide. The properties of the resulting polymer were thereby altered in regard to water solubility, mucoadhesion, biodegradability and in situ gelling compared to the original polymer. Due to the immobilised thiol groups (240±30 μmol thiol groups per gram polymer), the viscosity of a 1.5% chitosan-TGA solution was improved 4.3-fold. This can be explained by the formation of disulphide bonds within this polymeric network. The conjugate was tested as scaffold material in form of a gel and sheets. Furthermore, the influence of the thiol groups on the viability of L-929 mouse fibroblasts was evaluated. It was shown that the L-929 mouse fibroblasts grew on both scaffolds despite the thiol groups, although the different surface conditions seemed to have an influence on the growing rate. Chitosan-TGA sheets seemed to be the more preferred layer. The improved in situ gelling may be important for ongoing developments. Direct injectable matrices at the site of tissue damage mimicking the tissue being restored may be a future trend on this topic. Hence, chitosan-TGA is a promising candidate as scaffold material in tissue engineering.

In vitro evaluation of the viscoelastic properties of chitosan–thioglycolic acid conjugates

The aim of this study was to evaluate the viscoelastic properties of chitosan–thioglycolic acid conjugates with different amounts of thiol groups immobilized on the polymer. The modification of chitosan was achieved via the covalent attachment of thioglycolic acid mediated by a carbodiimide. Chitosan–thioglycolic acid conjugates displaying 120, 209 and 439 μM thiol groups per gram of polymer were synthesized. The rheological properties of the three different conjugates were investigated. The elastic properties of the gels were found to increase significantly at pH 5.5. After 6 h the elastic modulus G′ of chitosan–TGA 120, chitosan–TGA 209 and chitosan–TGA 439 gels increased 7-, 32- and 168-fold, respectively. In parallel the formation of disulfide bonds was observed. Accordingly, proof of principle that chitosan modified by the introduction of thiol groups has in situ gelling properties due to the formation of inter- and intramolecular disulfide bonds at physiological pH values is provided. Based on their in situ gelling properties, chitosan–thioglycolic acid conjugates seem to be very promising new excipients for liquid or semisolid formulations which should stabilize themselves once applied on the site of drug delivery.

Thiolated polymers: Stability of thiol moieties under different storage conditions

The purpose of this study was to evaluate the stability of thiolated polymers - so-called thiomers. A polycarbophil-cysteine conjugate and a chitosan-thioglycolic acid conjugate were chosen as representative anionic and cationic thiomer. The thiol group bearing compounds L-cysteine and thioglycolic acid were introduced to polycarbophil and chitosan, respectively with a coupling reaction mediated by a carbodiimide. The resulting thiolated polymers were freeze-dried and the amount of thiol groups on the thiomer was determined spectrophotometrically. Each kind of polymer was directly used or compressed into 1 mg matrix-tablets. Polymers were stored for a period of six months at four different storage conditions, namely at -20°C (56% relative humidity; RH), 4°C (53% RH), at 20°C (70% RH), and at 22°C (25% RH). Samples were taken after 6 months to determine the formation of disulfide bonds and the remaining thiol groups on the polymer. When the polycarbophil-cysteine and chitosan-thioglycolic acid conjugate were stored as powder a decrease of free thiol groups was observed only after storage at 20°C and 70% RH. Both polymers were found to be stable under all storage conditions when compressed into matrix tablets. The results provide the base for the use of thiomers as auxiliary agents in commercial products.

Design and in vitro evaluation of a novel bioadhesive vaginal drug delivery system for clotrimazole

It was the purpose of this study to design and evaluate a new bioadhesive vaginal drug delivery system for clotrimazole. Chitosan, a cationic biopolymer derived by deacetylation of chitin, was modified by the introduction of thioglycolic acid (TGA). The modification was achieved by utilising a carbodiimide to link the carboxylic acid moieties of TGA covalently to the primary amino groups of chitosan. The amount of added carbodiimide was thereby varied, resulting in chitosan–TGA conjugates A and B with 160 μM (=μmol) and 280 μM thiol groups per gram polymer, respectively. In order to characterise the new polymers the water uptake, the disintegration behaviour, the bioadhesive properties utilising the rotating cylinder method, as well as the release of clotrimazole from tablets based on these derivatives were studied. The water uptake and cohesive properties of vaginal tablets consisting of these new conjugates could be significantly ( p<0.05) improved. By adding clotrimazole the disintegration time of the conjugates was prolonged 1.6-fold for conjugate A and even 100-fold for conjugate B. Furthermore, the adhesion on vaginal mucosal tissue could be significantly improved. The addition of clotrimazole had also an impact on the adhesion time of chitosan–TGA conjugate B, which remained 26-times longer on vaginal mucosa than the corresponding unmodified polymer. The immobilisation of thiol groups guarantees a controlled drug release. Results of this study demonstrate that these new chitosan–TGA conjugates are very promising vehicles for the vaginal application of clotrimazole in treatment of mycotic infections.

Thiolated polymers — thiomers: development and in vitro evaluation of chitosan–thioglycolic acid conjugates

The aim of this study was to improve mucoadhesive properties of chitosan by the covalent attachment of thiol moieties to this cationic polymer. Mediated by a carbodiimide, thioglycolic acid (TGA) was covalently attached to chitosan. This was achieved by the formation of amide bonds between the primary amino groups of the polymer and the carboxylic acid group of TGA. Dependent on the pH-value and the weight ratio of polymer to TGA during the coupling reaction the resulting thiolated polymers, the so-called thiomers, displayed 6.58, 9.88, 27.44, and 38.23 μmole thiol groups per gram polymer. Tensile studies carried out with these chitosan–TGA conjugates on freshly excised porcine intestinal mucosa demonstrated a 6.3-, 8.6-, 8.9-, and 10.3-fold increase in the total work of adhesion (TWA) compared to the unmodified polymer, respectively. In contrast, the combination of chitosan and free unconjugated TGA showed almost no mucoadhesion. These data were in good correlation with further results obtained by another mucoadhesion test demonstrating a prolonged residence time of thiolated chitosan on porcine mucosa. The swelling behavior of all conjugates was thereby exactly in the same range as for an unmodified polymer pretreated in the same way. Furthermore, it could be shown that chitosan–TGA conjugates are still biodegradable by the glycosidase lysozyme. According to these results, chitosan–TGA conjugates represent a promising tool for the development of mucoadhesive drug delivery systems.

Synthesis and In Vitro Evaluation of Chitosan-Thioglycolic Acid Conjugates

The cationic thiomer chitosan-thioglycolic acid (TGA) shows excellent mucoadhesive features. In order to deepen the knowledge concerning this new excipient the optimization of its synthesis and a detailed characterization of its properties was the objective of this study Mediated by increasing quantities of a carbodiimide, thioglycolic acid was covalently attached to chitosan forming amide bonds with the primary amino groups of the polymer Determined with Ellman's reagent, 38 ± 3, 104 ± 2, 685 ± 43, and 885 ± 7 pmol thiol groups (n=3; ± SD) were bound per gram polymer at carbodiimide concentrations of 50, 75, 100, and 125 mM, respectively. The immobilized thiol groups displayed a comparatively higher reactivity to form disulfide bonds than the thiol groups in a corresponding mixture of chitosan and free unconjugated TGA. In an aqueous 0.5% (rnlv) chitosan-TGA gel 59 ± 5% of the thiol groups formed disulfide bonds within 6 hours at pH 6.0, whereas merely 5 ± 3% were oxidized in the corresponding physical mixture of chitosan and TGA. Diffusion studies showed that the modified polymer was capable of binding cysteine and cysteine methyl ester. The result supports the theory that the improved mucoadhesive properties of thiolated chitosan are based on the formation of disulfide bonds with cysteine moieties of mucus glycoproteins. Because of its availability via an efficient synthetic pathway and its mucoadhesive properties based on the capability to bind cysteine subunits, chitosan-TGA seems to be a promising new excipient for various drug delivery systems.