Site specific delivery of anti-arthritic drug by gelatin surface modified bovine serum albumin microspheres

Non-steroidal anti-inflammatory drugs are the most commonly used and widely prescribed drugs all over the world. With the wide advantages they are also associated with severe Gastro-Intestinal side effects. Developments of novel drug delivery systems have always been a challenge to formulation scientists because of their high instability and economic factor compared to the conventional dosage forms. Thus the main objective of the present investigation was to develop gelatin surface modified bovine serum albumin microspheres containing anti-arthritic drug and to evaluate its potential as targeted drug delivery system. Hence there is a prolonged release of the drug along with minimized side effects. A brief overview of the methods developed for the preparation of albumin microspheres and the most suitable techniques for optimum entrapment of drug is emphasized. The invitro evaluations are also explained. In order to appreciate the medical application possibilities of albumin microspheres in novel drug delivery, some fundamental aspects are also briefly discussed.


Introduction
Development of new drug molecule is expensive and time consuming. Improving safety efficacy ratio of "old" drugs has been attempted using different methods such as individualizing drug therapy and therapeutic drug monitoring. Delivering drug at controlled rate, slow delivery, and targeted delivery are other very attractive methods that have been pursued very vigorously (Patel et al., 2009, Jain et al., 2001. For drugs with short half-lives and with a clear relationship between concentration and response, it will be necessary to dose at regular, frequent intervals in order to maintain the concentration within the therapeutic range. Higher doses at less frequent intervals will result in higher peak concentrations with the possibility of toxicity. For some drugs with wide margins of safety, this approach may be satisfactory (Tripathi et al., 2003).
A trend in NSAID development has been to improve therapeutic efficacy and reduce the severity of upper GI side effects through altering dosage forms of NSAIDs by modifying release of the formulations to optimize drug delivery.
These formulations are designed to increase patient compliance through a prolonged effect and to reduce adverse effects through lowered peak plasma concentrations. Many controlled-release dosage forms are designed to release the drug at a predetermined rate, thus maintaining relatively constant drug levels in the plasma for an extended period of time.
Several benefits may result from the use of such formulations. Reduction of frequency of dosing, lowered adverse effects, and improved patient compliance are considered the primary advantages of controlled-release dosage forms (Sam et al., 2008).
Further, currently available slow release oral dosage forms, such as enteric coated/ double-layer tablets which release the drug for 12-24 hours still result in inefficient systemic delivery of the drug and potential gastrointestinal irritation. Therefore, currently available slow release oral dosage forms of NSAIDs induces systemic effects and the drug is not efficiently used at the site of inflammation (Lewis et al., 1992) . Formulations can affect the safety of preparations by controlling the rate of release of the drug at sensitive sites, by delivering drug to specific sites to minimize systemic exposure, or delivering drug in such a way so as to change the rate or extent of the formation of toxic metabolite (Lachman et al., 1991).
One such formulation uses polymeric microspheres as carriers of drugs. Many authors have reported that nanoparticles and micro particles have a tendency to accumulate in the inflamed areas of the body. It has been reported that microspheres of NSAIDs reduce the GI toxic effects and exhibit sustained action, thus increasing patient and therapeutic compliance. ISSN 2250-3153 www.ijsrp.org Microspheres can be described as small particles (in 1-1000 micrometer size range) for use as carriers of drugs and other therapeutic agents. The term microspheres describe a monolithic spherical structure with the drug or therapeutic agent distributed throughout the matrix either as a molecular dispersion or as a dispersion of particles.

Microspheres
Microspheres are used as targeted drug delivery system due to; small size and relatively narrow size distribution which provide biological opportunities for site-specific drug delivery, controlled release of active drug over a long period can be achieved and surface modification can easily be accomplished and hence can be used for site specific drug delivery system (Liggins et al., 2004, Vyas et al., 2002.
The present study is an attempt to develop extended release formulation of Aceclofenac to addresses the above issues.
The objectives for the present study are -1. To identify formulation excipients based on compatibility studies. 2. To Optimize formulation and processing parameters (Stirring speed, Viscosity of oil phase and Percentage of emulsifying agent, etc.) using optimization technique (Response Surface Methodology) to get the desired response (particle size, entrapment efficiency and drug release). To predict the optimized formulation based on the desired response obtained. 3. To prepare the Aceclofenac microspheres based on predicted optimized formulation. To evaluate the product through various in-vitro (entrapment efficiency, surface characteristics, particle size, drug release & stability) studies.
A number of novel drug delivery systems have emerged encompassing various routes of administration, to achieve controlled and targeted drug delivery, micro-carriers being one of them. These micro-carriers include microspheres, liposomes, nanoparticles, resealed erythrocytes, and micro emulsion etc.
Microspheres have large surface area on which ligands can be attached for delivering drugs to a specific local area. Modification of microsphere surface with specific ligands have been attempted to use these microsphere as targeted drug delivery system.
Gelatin has a specific interaction with fibronectin. Gelatin microspheres have been investigated and demonstrated to target S-180 mouse sarcoma cells (pathological tissue), which are known to express extra fibronectin on their surface. This results in the cell line's extra fibronectin creating an affinity between cancer cells and gelatin microspheres. Excess fibronectin in the local tissues is associated with a number of disease states, such as inflammatory disorders, cardiovascular disease, rheumatoid arthritis, and cancer. Consequently, gelatin surface modified microspheres may be useful as a targetable controlled-release microsphere system. These microspheres are intended for localized delivery to fibronectin enriched pathological tissues.

Thus the objective of the present investigation was to develop gelatin surface modified bovine serum albumin microspheres containing anti-arthritic drug and to evaluate its potential as targeted drug delivery system in vivo.
Drug:-Aceclofenac is one of the well tolerated COX-2 inhibitor and is often the drug of choice in the treatment of osteoarthritis, rheumatic arthritis and other related conditions. However, because of its short half-life (2-4 hrs.) it requires dosing of 100 mg twice daily. Missing of dose, which is often common, would cause inconsistence in drug level in the blood, which would in turn reflect in poor therapeutic outcome. It has been reported that more than 50% of patients fail to take medicine as advised. Extended release formulations are the tools useful in promoting medication adherence and improving therapeutic outcomes. Medication adherence in chronic conditions like arthritis improves the quality of life of the patients.

METHOD OF PREPARATION 3.4.1 Selection of method
Following two methods were investigated for the preparation of BSA microspheres: 3.4.1.1 Emulsion chemical cross-linking 100 mg of bovine serum albumin (BSA) was dissolved in 5 ml of distilled water. Tween-80 was added at a concentration of 2% wt/wt. 20 mg of finely powdered Aceclofenac was added the above solution and sonicated to obtain a uniform dispersion. One milliliter of this dispersion was injected (drop by drop) into a mixture of 20 ml of heavy liquid paraffin and 1.0 ml of span-85, while stirring at 2000 rpm. Stirring was continued for 10 minutes to obtain a water/oil (w/o) emulsion. One milliliter of 25% w/v glutaraldehyde was added into the emulsion to cross-link the albumin present in the internal phase of the emulsion. Microspheres formed were then separated by centrifugation (8000 rpm for 15 minutes) and washed with 30 ml of petroleum ether to remove liquid paraffin. The microspheres were then suspended in 10 ml of 5% wt/vol. sodium bisulphite solution and stirred on a magnetic stirrer for 10 minutes to remove the residual glutaraldehyde. Finally, the microspheres were washed with distllled water until they were free from residual glutaraldehyde. The microspheres were dried at room temperature and stored in a dessicator (Thakkar H, et al., 2005)

Emulsification-heat stabilization technique
10 mg of aceclofenac was added to a 5% w/v solution of BSA containing 0.1% Tween 80 and used as the aqueous phase. The oil phase composed of 30 ml maize oil and 10 ml petroleum ether with 1% Span 80 as emulsifier were stirred for 10 min at 1000 rpm. The aqueous phase was added drop wise to the oil phase and stirred at 1000 rpm for 30 min to form the initial emulsion. This www.ijsrp.org emulsion was then added to 40 ml of maize oil preheated to 120° C and stirred at 1000 rpm for 15 min to allow the formation of microspheres. The microsphere suspension was centrifuged at 3500 rpm for 30 min and the settled microspheres were washed three times with 50 ml ether to remove traces of oil. The microspheres were dried in a desiccator overnight and stored (Tabassi SAS, et al., 2003). In-vitro drug release study of aceclofenac from BSA and gelatin surface modified BSA microspheres was carried out for up to 24 hrs using dissolution test apparatus (paddle method) containing 900 ml of phosphate buffer saline (PBS) pH 6.8 as dissolution medium. Microspheres equivalent to 20 mg aceclofenac were kept in muslin cloth and tied to the paddles, stirred at 100 rpm. At various time periods (0.25-24hrs), 5 ml of the sample solutions were withdrawn and diluted with methanol up to 10 ml. An equivalent volume of fresh buffer was added into the dissolution medium. The quantity of drug was estimated against reagent blank by UV spectroscopy at 277.0 nm (Jayaprakash S, et al., 2009). .

KINETIC MODELING OF DISSOLUTION PROFILES
In vitro dissolution has been recognized as an important element in drug development under certain assessment of Bioequivalence. Several theory kinetic model describe drug dissolution from immediate and modified release dosage forms. There is several models to represent the drug dissolution profile where ft is function of ‛t'(time) related to the amount of drug dissolved form the pharmaceutical dosage system. whenever a new solid dosage form is developed or produced, the drug release/dissolution from solid pharmaceutical dosage form is necessary to ensure that the drug dissolution occurs in an appropriate manner. Several theories/kinetics models describe drug dissolution form immediate and modified release dosage. These represent the drug dissolution profile where ft is a function of ‛t'(time) related to the amount of drug dissolved from the pharmaceutical dosage form. The quantitative interpretation of the value obtained from the dissolution assay by mathematical equation which translates the dissolution curve in function of some parameters related with the pharmaceutical dosage form. In the present study, data of the in vitro release were fitted to different equations and kinetic models to explain the release kinetics of Aceclofenac from the microspheres. The kinetic models used were a Zero order equation, first order, Higuchi model, Hixon crowell and peppas model.

Preparation of assay reagent
The assay reagent was prepared by diluting one volume of dye stock solution with four volumes of distill water. The solution is brown in color with a pH of 1.1. It is stable for weeks in dark bottle at 4 0 C. www.ijsrp.org

Preparation of Standard curve of gelatin
Accurately weighed 100 mg of gelatin was dissolved in 100 ml of PBS pH (7.4) to give a solution of 1000 μg/ml concentration. This solution served as standard stock solution (І). From this solution 10 ml was taken and diluted to 100 ml using PBS pH (7.4) to get 100 μg/ml concentrations. 1 ml, 2 ml, 3 ml and 4 ml samples were taken and diluted up to 10 ml with PBS pH (7.4) to get concentration of 10, 20, 30, and 40µg/ml respectively. 2 ml of the assay reagent was added into each test tube and incubated at 37 0 C for 1 hrs. The resultant color intensity was measured by colorimetry at the wavelength 590 nm.

.2.4.4 Protein Assay Procedure
Accurately weighed 10 mg of gelatin adsorbed BSA microspheres of aceclofenac (GBM-6) were taken and mixed with 10 ml PBS pH (7.4). 2.0 ml assay reagent was added and the solution was shaken for 10 minutes. Gelatin content of the microspheres was determined by measuring the optical density (OD) of the solution at 590 nm (Bradford M, 1976

Drug Release Kinetics
To examine the drug release kinetics, the release data was fitted into models representing zero order, first order, and Higuchi's square root of time kinetics. The coefficient of determination (R 2 ) values were calculated from the plots of Q vs t for zero order, log(Qo-Q) vs t for first order and Q vs √t for Higuchi model, and log cumulative % drug release vs. log time for korsmeyer model, where Q is the amount of drug released at time t, (Q 0 -Q) is the amount of drug remaining after time t.

.3 STABILITY STUDIES
Ability of a formulation to retain its properties in specified limits throughout its shelf life is referred to as stability.
Stability of a pharmaceutical product may be defined as capability of a particular formulation, in a specific container, to retain its physical, chemical, microbiological, therapeutic, and toxicological specification. The objective of stability study is to determine the shelf life, namely the time period of storage at a specified condition within which the drug product still meets its established specifications. The stability of finished pharmaceutical products depends on several factors. On one hand it depends on environmental factors such as ambient temperature, humidity, and light. On the other hand, it depends on the product related factors such as physical and chemical properties of active substance and pharmaceutical excipients, dosage form and its composition, manufacturing process, nature of container, closure system, and properties of packaging materials. The chemical stability of drug is of great importance since it becomes less effective as it undergoes degradation. Also drug decomposition may yield toxic by products that are harmful to the patient. Microbiological instability of a sterile drug product could also be hazardous. Stability of a formulated product on shelf becomes an important factor in successful development of any dosage form. A study of stability of a pharmaceutical product is essential for three main reasons; safety of the patient, legal requirements concerned with the identity, strength, purity and quality of product and to prevent the economic repercussion of marketing an unsuitable product. A well designed stability testing plan is essential and pertinent part of the quality assurance program.

Storage Condition
Prepared formulation (GBM-6) was stored in screw capped glass bottle at refrigerated temperature (2-8 0 C) and room temperature (20-25 0 C). Samples were analyzed for residual drug content after a period of 7, 15, 30 45 and 60 days. Initial drug content was taken as 100% for each formulation.

Results and Discussion of stability
The optimized formulation GBM-6 was stored at two different temperature conditions refrigerated temperature and room temperature. To obtain an optimal storage condition, percent residual drug content and log % residual drug content was calculated at different time interval (0, 7, 15, 30, 45, and 60 days) and graphs were plotted between percent residual drug content vs time, and log % residual drug content vs time. [