PREPARATION AND CHARACTERIZATION OF TOLTERODINE TARTRATE PRONIOSOMES

The present work deals with the preparation of Tolterodine tartrate proniosome formulations by coacervation phase separation method by using different surfactants in different ratios. The prepared proniosomal formulations were evaluated for vesicle size, rate of spontaneity, encapsulation efficiency, drug content etc. In vitro release study was conducted and it indicated that, increases in liphophilicity of surfactants decreases release of Tolterodine tartrate from proniosomal formulations. Stability studies were performed at optimized formulation PG4, indicated that, the prepared formulations remain stable at room and refrigeration temperature.


INTRODUCTION
At present scenario vesicular systems have been receiving a lot of interest as a carrier for advanced drug delivery 1 . Encapsulation of the drug in vesicular structures is one such system, which can be expected to prolong the duration of the drug in systemic circulation 2 . Proniosomes are water soluble carrier particles that are coated with surfactants and can be hydrated to form niosomal dispersion immediately before use in hot aqueous media. Proniosome is a dry free flowing, granular product that could be hydrated immediately before use and would avoid many of the problems associated with aqueous noisome dispersions and problem of physical stability 3 . Proniosome technology offers novel solution for poorly soluble drugs. Proniosomes avoid many of the problems associated with aqueous niosome dispersions, and problems of physical stability (aggregation, fusion, leaking) could be minimized. The additional convenience of the transportation, distribution, storage, and dosing would make 'dry niosomes' a promising industrial product 4 .
Tolterodine acts on M2 and M3 subtypes of muscarinic receptors. Tolterodine tartrate is used for the treatment of overactive bladder with symptoms of urge urinary in continence, urgency and frequency 5 . Use of Tolterodine tartrate is associated with side effects like dry mouth and other side effects like constipation, headache, stomach pain and blurred vision, often leading to discontinuation of therapy 6 . The aim of present study includes development of proniosomes of Tolterodine tartrate to reduce dosing frequency and avoid side effects.

MATERIALS AND METHODS
Tolterodine tartrate was obtained as gift sample from Churchbells Pharma Nigeria Limited. Span 60, Tween 40 and cholesterol were procured from Drugfield Pharmaceuticals Limited, Nigeria. Ethyl alcohol and lecithin was procured from Interpharma Industries Nigeria Limited. All other reagents used were of analytical grades.

Preparation of proniosomal gel
Tolterodine tartrate proniosomal gel formulations were prepared by coacervation phase separation method. Precisely weighed amounts of surfactant, lipid phase and drug were taken in a clean and dry wide mouthed glass vial of 5.0 ml capacity and alcohol was added to it. All the ingredients were mixed well with a glass rod; the open end of the glass vial was covered with a lid to prevent the loss of solvent from it and warmed over water bath at 50-60°C for about 5 minutes until the drug is dissolved completely in surfactant mixture. Then the aqueous phase 1.6ml phosphate buffer (pH 7.4) was added and warmed on a water bath until a clear solution was formed. Preliminary the composition of these formulations is reported in Table 1 and they are referred as PG1 to PG4 6 .

Evaluation of proniosome formulations Vesicle size analysis
Hydration of Tolterodine tartrate proniosomal gel (100 mg) was done by adding saline solution (0.9% solution) in a small glass vial with occasional shaking for 10 min. The dispersion was observed under optical microscope at 45x magnification. The sizes of 200-300 vesicles were measured using a calibrated ocular and stage micrometer (Erma, Tokyo) fitted in the optical microscope 8 .

Drug content
In a 100 ml volumetric flask, 20 mg of proniosomal gel formulations were taken, and volume was made up to mark with pH 7.4. The flask was shaken for 12 hours using an orbital shaker incubator (Finlab, Nigeria). Then the solution was filtered and from the filtrate appropriate dilutions were made and absorbance was measured at 261 nm 8 .

Encapsulation efficiency
To evaluate the loading capacity of proniosomal systems for Tolterodine tartrate gel (100 mg) was dispersed in distilled water and warmed a little for the formation of niosomes. Then the dispersion was centrifuged at 18000 rpm for 40 min the clear fraction was used for the determination of free drug at 281 nm spectrophotometrically. The percentage encapsulation efficiency was calculated from following equation 9, 10 .

pH and Viscosity
Accurately weighed gel was taken and then diluted with the pH 7.4 phosphate buffer and checked the pH by using pH meter (Finlab, Nigeria) and Brook field viscometer is used to determine the viscosity of the gel 11 .

In vitro release study
In vitro release studies on proniosomal gel of Tolterodine tartrate were performed using locally manufactured Franz-diffusion cell. The capacity of receptor compartment was 15 ml. The area of donor compartment exposed to receptor compartment was 1.41cm 2 . The dialysis cellophane membrane (MMCO 14KDC) was mounted be Tween the donor and receptor compartment 12 . A weighed amount of proniosomal gel was placed on one side of the dialysis membrane. The receptor medium was phosphate saline buffer pH 7.4. The receptor compartment was surrounded by a water jacket to maintain the temperature at 37±1 o C. Samples were withdrawn and were replaced by equal volumes of fresh receptor fluid on each occasion. Samples withdrawn were analyzed spectrophotometrically (Finlab, Nigeria) at 281 nm.

Stability Studies
The ability of vesicles to retain the drug was assessed by keeping the proniosomal gel at three different temperature conditions, i.e., refrigeration temperature (4-8 0 C), room temperature (25±2 0 C) and oven (45±2 0 C) for 12 weeks (60% relative humidity). Throughout the study, proniosomal formulations of Tolterodine tartrate were stored in aluminium foilsealed glass vials. The samples were withdrawn at different time intervals and drug leakage from the formulations was analyzed for drug content spectrophotometrically at 281 nm 8 .  minimum effective concentration and it is also used to estimate the drug release profile. The percent drug content was higher for PG4 that is 99±0.47% and lower for PG2 (88±0.12%).   (Figure 2). Thus the room temperature is the favorable storage condition for storage of proniosomes.

CONCLUSION
The results of investigation demonstrated that proniosomes offers an alternative colloidal carrier approach. The results obtained from the present study clearly revealed that Tolterodine tartrate proniosome formulations prepared by using coacervation phase separation method are capable of releasing drug for the extended period of time. Results of the present work have shown that surfactant type affect the encapsulation efficiency and drug release rate from proniosomes. Based on different parameters formulation of batch PG4 was considered as an optimum formulation.

AUTHOR'S CONTRIBUTION
The manuscript was carried out, written, and approved in collaboration with all authors.