Title of Invention

''A PROCEES FOR THE PREPARATION OF HARDENED POLYLACTIDE CO-GLYCOLIDE MICROPARTICLES''

Abstract

This invention relates to A process for the preparation of hardened polylactide co-glycolide microparticles having anti-tubercular drugs encapsulated therein comprising:- (i) preparing an aqueous solution of the drug in phosphate buffered saline, (ii) preparing a polymer solution such as polylactide co-glycolide in an organic solvent such as dichlorio methane, (ii) mixing equal volumes of the above solutions to make an emulsion, (iv) sonicating said emulsion at a temperature of 4-8°C for 0.5 to 1 minute in cycles (20 sec/cycle) and putting the same under stirring, (v) adding polyvinyl alcohol there to drop-wise such that to form hardened microparticles, (vi) stirring said emulsion mixture and centrifuging the same, (vii) washing said particles with said phosphate buffer saline followed by reconstituting the particles.

Full Text

FIELD OF INVENTION This invention relates to a process for the preparation of hardened polylactide co-glycolide microparticles having antitubercular drugs encapsulated therein and which can be administered orally or subcutaneously. In distinction to porous or non porous particles, the present invention relates only to hardened microparticles. BACKGROUND OF INVENTION Tuberculosis is usually treated with oral administration of drugs for 6-9 months, depending upon the severity of the disease. For the administration of drugs, the dosage and time intervals between doses must be carefully regulated. Free drug administration normally results into inadequate drug at the target site, low permeability into cells, toxic side effects and most importantly patient non-compliance, which is the most serious threat to success of therapeutic process. Tuberculosis drugs are known to have side effects and cause hepatoxicity, and primarily due to this reason the daily higher dosage is orally provided so as to maintain MIC levels (Minimum Inhibited Concentration). Microsphere drug delivery systems using various kinds of biodegradable polymers have been extensively studied during the past two decades. Several processes for the synthesis of microparticles have been described in literature. The preparation procedures described in literature make use of either spray drying, single or double emulsification or solvent evaporation methods. However, no single method proved to be perfect in terms of sizing of microparticles, drug encapsulation efficiency and drug release kinetics. Most of the previously described procedures for the preparation of poly-lactide-co-glycolide (PLG) microparticles employs large drug:polymer ratio and use ethyl acetate, polyvinyl alcohol (PVA) etc. as emulsifying agents. Solvent evaporation is the most commonly used preparation process for the PLG microparticles. In this process, microsphere formation is a phase separation process in which polymer solution is transformed into precipitated solid spheres. Polymer precipitation induced by solvent evaporation depends to a great extent on evaporation conditions. However, a distinct disadvantage of this method is the poor encapsulation efficiencies of water soluble drugs. The drug usually diffuses out from the dispersed oil phase into the aqueous continuous phase and microcrystalline fragments of hydrophilic drugs are dispersed in microsphere surface and in the matrix. This results in poor trapping of the hydrophilic drugs and initial rapid release of drugs called the burst effect particularly with respect to porous, and non-porous microparticles formed from emulsion. The preparation of emulsifying agent, which is PVA controls the stability and drug release kinetics of PLG microparticles, and is employed in a range of 0.3 to 1 w/w of PVA. An object of this invention is to propose a process for the preparation of hardened polylactide co-glycolide microparticles having antitubercular drugs encapsulated and obviates the disadvantages associated with the known art. Another object of this invention is to propose a process for the preparation of hardened polylactide co-glycolide microparticles having antitubercular drugs encapsulated which provides a prolonged and sustained release of the drug. Yet another object of this invention is to propose a process for the preparation of hardened polylactide co-glycolide microparticles having antitubercular drugs encapsulated capable of being modulated to entrap maximum drug. Still another object of this invention is to propose a process for the preparation of hardened polyactide co-glycolide microparticles having antitubercular drugs encapsulated capable of ditributing the drugs evenly to different sites in which tubercle bacteria resides. A further object of this invention is to propose a process for the preparation of ahrdened polylactide co-glycolide microparticles having antitubercular drugs encapsulated which can be used both as as injectable as well as oral preparation. Another object of this invention is to propose a process for the preparation of ahrdened polylactide co-glycolide microparticles having antitubercular drugs encapsulated and which no longer exhibits hepatoxicity. STATEMENT OF THE INVENTION According to this invention there is provided a process for the preparation of hardened polylactide co-glycolide microparticles having anti-tubercular drugs encapsulated therein comprises the steps of: (i) preparing an aqueous solution of the drug in buffer, (iiij preparing a polymer solution of polylactide co-glycolide in an organic solvent, (iiij mixing the above solutions to make an emulsion, (iv) sonicating said emulsion followed by stirring, (v) adding polyvinyl alcohol there to drop-wise to form hardened microparticles, (vij stirring and centrifuging the emulsion mixture, (viij washing said particles with said phosphate buffered saline followed by reconstituting the same by suspending in the buffer to obtain the particles characterized by the following: (1) the drug used in step(i) is selected from INH (isoniazid), PV'Z (Pyrazinamidej and RIF (Rifompreinj, the buffer of step (ii) is phosphate buffered saline and the ratio between the drug and buffered saline is 1:10-1200 weight/volume, the solvent used in step (iiij is dichloromethane and the ratio between the polymer solution and solvent is 1:10-150 weight/volume, and the ratio between aqueous solution of drug and polymer solution of step (iiij is 1:1; (2) sonicating said emulsion of step (iiij at a temperature of 4-8°C for 0.5 to 1 minute in cycles (20 sec/cycle). In accordance with this invention an aqueous solution of a drug is prepared in a solvent preferably in phosphate buffered saline in the ratio of 1:10-1200 weight by volume. A solution of polymer is prepared in a solvent preferably dichloromethane in the ratio of 1:10-150 weight by volume. The aqueous solution of the drug and the polymer solution are mixed together in the ratio of 1:1 i.e. equal volume of drug solution and polymer solution are mixed together by vortexing for a period of 15-30 seconds so as to prepare an emulsion. The emulsion so obtained is sonicated at a temperature of 4-8C for a period of 0.5-10 minutes in cycles (20 sec/cycle). The sonicated emulsion is then put immediately under stirring. One ml of 15-25% polyvinyl alcohol solution is added into the sonicated emulsion dropwise till hardened microparticles are formed which are stirred overnight. The stirred mixture is centrifuged at 10,000 Xg for 20-30 minutes for separating the pellets or microparticles. The pellets so obtained are then washed 3-4 times with phosphate buffer saline of a pH of 7.2-7.4 or neutral solution. The washed pellets are then reconstituted by suspending the same into the same buffer in order to get the microparticles of anti-tubercular drugs encapsulated therein. The ratio of the drug and polymer is kept as 0.5-1.5:1. A process for the preparation of hardened polylactide co-glycolide microparticles having antitubercular drugs encapsulated therein is herein described in detail with the help of the following examples. EXAMPLE 1 150 mg IMH (isoniazid) was dissolved in 2 ml phosphate buffered solution. 100 mg polymer PLG (polylactide co-glycolide) was dissolved in 2 ml dichloromethane. Equal amounts of the drug solution and polymer solution were taken and mixed together by vortexing for a period of 30 seconds to obtain and emulsion. The emulsion so obtained was sonicated under cold conditions in cycles of the duration of 20 seconds. The sonicated emulsion was stirred immediately and 1 ml of 18% polyvinyl alcohol solution was added dropwise into the stirred emulsion such that to form hardened microparticles. The emulsion mixture so obtained was stirred overnight and centrifuged at 10,000 Xg for 30 minutes. The pellets/micro particles obtained after centrifugation were washed 4 times with phosphate buffered saline (pH 7.2). The washed pellets were then reconstituted by suspending in the same buffer solution in order to get the INH containing microparticles. EXAMPLE 2 2 mg PYZ (Pyrazinamide) was dissolved in 2 ml phosphate buffered solution. 20 mg polymer PLG (polylactide co-glycolide) was dissolved in 2 ml dichloromethane. Equal amounts of the drug solution and polymer solution were taken and mixed together by vortexing for a period of 30 seconds to obtain an emulsion. The emulsion so obtained was sonicated under cold conditions in cycles of the duration of 20 seconds. The sonicated emulsion was stirred immediately and 1 ml of 18% polyvinyl alcohol solution was added dropwise into the stirred emulsion such that to form hardened micro particles. The emulsion mixture so obtained was stirred overnight and centrifuged at 10,000 Xg for a period of 30 minute. The pellets/micro particles obtained after centrifugation were washed 4 times with phosphate buffered saline (pH 7.2). The washed pellets were then reconstituted by suspending in the same buffer solution in order to get the micro particles containing PYZ. EXAMPLE 3 10 mg RIF (Rifomprcin) was dissolved in 3 ml phosphate buffered solution. 200 mg polymer PLG (polylactide co-glycolide) was dissolved in 3 ml dichloromethane. Equal amounts of the drug solution and polymer solution were taken and mixed together by vortexing for a period of 30 seconds to obtain an emulsion. The emulsion so obtained was sonicated under cold conditions in cycles of the duration of 20 seconds. The sonicated emulsion was stirred immediately and 1 ml of 18% polyvinyl alcohol solution was added dropwise into the stirred emulsion such that to form hardened micro particles. The emulsion mixture so obtained was stirred overnight and centrifuged at 10,000 Xg for a period of 30 minute. The pellets/micro particles obtained after centrifugation were washed 4 times with phosphate buffered saline of the pH of 7.2. The washed pellets were then reconstituted by suspending in the same buffer solution in order to get the micro particles containing RIF. The drug particles so obtained were administered to the mice and results are given in Table 1. TABLE 1 Colony Forming Units (CFUs) of Mycobacterium tuberculosis in lungs of mice after high and low dose drug treatment. (Table Removed) Free INH was given daily for 6 weeks whereas PLG-INH was administered as a single subcutaneous injection/dose. ***p Preparation of Poly (DL-lactide-co-glycolide) microparticles and determination of the drug content in PLG microparticles. Different formulations of PLG microparticles (mps) containing entrapped drugs were prepared. Various ratios of the drug and polymer were tried in order to achieve the maximum entrapment of drugs in the microparticles. The drug particles so manufactured were tested for toxicity and the results are given in Table 2. TABLE 2 Plasma levels of alkaline phosphatase, serum glutamate pyruvate transaminase and total, bilirubin post chemotherapy with combination drugs. (Table Removed) All values are mean ±SD of 5-6 animals. Toxicity studies Table 2 depicts the levels of ALP, SGPT and total bilirubin obtained in plasma of mice post chemotherapy with free combination drugs and PLG-combination drugs at high and low doses. As evident from the results, there was not any change in the levels of these parameters post chemotherapy in any of the treatment groups as compared to the controls indicating no hepatotoxicity. WE CLAIM 1. A process for the preparation of hardened polylactide co-glycolide microparticles having anti-tubercular drugs encapsulated therein comprises the steps of: (i) preparing an aqueous solution of the drug in buffer, (ii) preparing a polymer solution of polylactide co-glycolide in an organic solvent, (ii) mixing the above solutions to make an emulsion, (iii) (iv) sonicating said emulsion followed by stirring, (iv) (v) adding polyvinyl alcohol there to drop-wise to form hardened microparticles, (vi) stirring and centrifuging the emulsion mixture, (vii) washing said particles with said phosphate buffered saline followed by reconstituting the same by suspending in the buffer to obtain the particles characterized by the following: (1) the drug used in step (i) is selected from INH (isoniazid), PYZ (Pyrazinamide) and RIF (Rifomprein), the buffer of step («) is phosphate buffered saline and the ratio between the drug and buffered saline is 1:10-1200 weight/volume, the solvent used in step (ii) is dichloromethane and the ratio between the polymer solution and solvent is 1:10-150 weight/volume, and the ratio between aqueous solution of drug and polymer solution of step (iii) is 1:1; (2) sonicating said emulsion of step (iii) at a temperature of 4-86C for 0.5 to 1 minute in cycles (20 sec/cycle). 2. A process as claimed in claim 1 wherein said drug and polymer solution are mixed together by vortexing for 15-30 seconds. 3. A process as claimed in claim 1 wherein said polyvinyl alcohol is of the strength of 15-25%. 4. A process as claimed in claim 1 wherein said step of centrifuging is carried on at 10,000 xg for a period of 20-30 minutes. 5. A process as claimed in claim 1 wherein said microparticles are washed 3-4 times with phosphate buffered solution having the pH of 7.2-7.4. 6. A process for the preparation of hardened polylactide co-gycolide micorparticles having anti-tubercular drugs encapsulated therein substantially as herein described and illustrated in the examples.

Documents:

688-del-2001-abstract.pdf

688-del-2001-claims.pdf

688-del-2001-correspondence-others.pdf

688-del-2001-correspondence-po.pdf

688-del-2001-description (complete).pdf

688-del-2001-form-1.pdf

688-del-2001-form-19.pdf

688-del-2001-form-2.pdf

688-del-2001-form-3.pdf

688-del-2001-form-5.pdf

688-del-2001-gpa.pdf