Controlled release matrix tablet formulation using synthesized N-acyl Thiolated Chitosan derivatives

2.1 Materials

Chitosan (medium molecular mass: 400 KDa; degree of deacetylation: 83–85%) was purchased from Research Laboratories, Acylchlorides, ammonium thiocyanate, Elman’s Reagent were obtained from Research Lab and aluminium isopropoxide was procured from SIGMA. All chemicals were of analytical grade. NMR analysis was performed at IIT (Powai).

2.2 Preparation of acylthiocyanate reagent

The solution of ammonium thiocyanate (0.011 mol) in dry acetone (25 ml) was prepared. Benzoyl chloride (0.01 mol) was added slowly in above solution with stirring. The reaction mixture was subjected to microwave irradiation for 3 mins at 560 W power to yield an acylthiocyanate reagent (Kumaraswamy et al., 2006).

2.3 Synthesis of acylthiouride of chitosan

A solution of chitosan in 2%AcOH/MeOH (50 ml) was added slowly to the above solution so as to maintain reflux condition. After the addition was complete, the mixture was stirred for 90 min at room temperature, which is separated as solid precipitate on pouring in NaOH solution (pH 10) Zhong et al., 2008; Champagne, 2008.

2.4 Synthesis of Thiolated Chitosan derivative

The above precipitate was washed with acetone thoroughly to remove the traces of acylthiocyanate. The product was then treated with aluminium isopropoxide in Isopropanol which reduces the thioketone group from acylthiouride to form the thiol resulting in the formation of Thiolated Chitosan. The aluminium isopropoxide in isopropanol solution is as specific reducing agent for ketones (Finar, 2006) (Fig. 1).

Close

Figure 1 N-acyl Thiolated Chitosan derivatives.

Export to PPT

2.5 Procedure for quantification of sulfhydryl groups

A dilution buffer (0.1 M Sodium phosphate, 1 mM EDTA, pH 8.0) and DTNB [(5,5′-dithio-bis-(2-nitrobenzoic acid)] working solution were prepared initially. The solutions were stored at +4 °C. 4 mg of DTNB was dissolved in 1 ml dilution buffer. A set of Sulfhydryl standard (Cysteine HCl) with sample dilution buffer (or distilled water) was prepared. Cysteine HCl (26.34 mg) was dissolve in the media to prepare 1.5 mM solution, serial dilutions 1.25 mM, 1.0 mM, 0.75 mM, 0.5 mM and 0.25 mM were prepared and were used immediately. Following components were added subsequently to the test tubes:

• 250 μl Sample/Standard

• 2.5 ml dilution buffer

• 50 μl DTNB reagent

The resulting solution was incubated for 15 min and absorbance was measured at the wavelength of 412 nm and concentration of Sulfhydryl group was determined using Cysteine as standard solution. Molar extinction coefficient at 412 nm of DTNB is 14,150 M⁻¹ cm⁻¹ (Irene and Christine, 2007; Alvear et al., 1989) (Table 1).

2.6 Swelling Index

Swelling index of the synthesized derivative was determined by soaking 150 mg flat faced 8 mm tablet in 6 ml of water in a Petri-dish. Initial and final weights of the tablets were recorded (Table 2).

Swelling index was calculated by using following formula: $$\text{Swelling index}=\text{Final weight}-\text{initial weight}/\text{Final weight}\times 100$$

2.7 Solubility test

The solubility of the copolymers was tested in several organic solvents, distilled water as well as in 0.1 M phosphate buffer (pH 4.0), 0.1 M phosphate buffer (pH 7.0) and DMSO. The samples were soaked in each solvent at the concentration of 5 mg/ml (Gorochovceva and Kulbokaite, 2004) (Table 3).

2.8 Mucoadhesivity assessment

Recently, it has been shown that polymers with thiol groups provide much higher adhesive properties than polymers generally considered to be mucoadhesive. The enhancement of mucoadhesion can be explained by the formation of covalent bonds between the polymer and the mucus layer which are stronger than non-covalent bonds. This theory was supported by the results of assessment of Mucoadhesive strength demonstrated a positive correlation between the degree of modification with thiol bearing moieties and the adhesive properties of the polymer (Sreenivas and Pai, 2008; Deshmane et al., 2009).

For the measurement of the Mucoadhesive strength, the pure polymer was taken and the disc of 150 mg was prepared by using 8 mm die on the rotary compression machine. After that the tablet was removed and used for the measurement of the mucoadhesive strength (Fig. 4).

2.9 Thiol derivative as controlled release matrix

Thiolated Chitosan represents, primarily due to its mucoadhesive properties, a valuable tool for non-invasive drug delivery. The longer residence time of formulations based on mucoadhesive polymers at the absorption site is believed to contribute to an increased absorption rate of the incorporated drug. However, such an enhanced bioavailability can be achieved only if a controlled release of the active agent out of the formulation is provided. Thiolated Chitosans also display, besides their strong mucoadhesive and permeation enhancing properties, excellent cohesive properties. The cohesion and stability of a drug delivery system over the intended duration of drug liberation is often a substantial requirement for a controlled release. The usefulness of Thiolated Chitosans as carrier matrices for controlled drug release was demonstrated with model drug as Amodiaquine (Sreenivas and Pai, 2008) (Table 4).