Title of Invention

AN IMPROVED PROCESS FOR THE MICROENCAPSULATION OF ACTIVE INGREDIENTS IN POLYMERS

Abstract

This invention relates to a process for microencapsulation of active ingredients. More particularly it relates to the micro-encapsulation of hydrophilic macromolecular entities in polymers. The invention specifically relates to an emulsification-solvent removal technique that uses a mixed-solvent system for the polymer and involves a combination of solvent extraction solvent evaporation processes fro solvent removal carries out in a non-aqueous medium to achieve entrapment of the active ingredient, particularly the hydrophilic macromolecular entities. Microencapsulation technology is an important means of designing controlled release devices, immobilization and for imparting protective coatings. Miroencapsulated products prepared by the following process can be used for controlled release applications, enzyme or cell immobilization and protective coating for storage of sensitive active ingredients.

Full Text

This invention relates to a process for microencapsulation of active ingredients. More particularly it relates to the micro- encapsulation of hydrophilic macromolecular entities in polymers. The invention specifically relates to an emulsification-solvent removal technique that uses a mixed-solvent system for the polymer and involves a combination of solvent extraction-solvent evaporation processes for solvent removal carried out in a non¬aqueous medium to achieve entrapment of the active ingredient, particularly the hydrophilic macromolecular entities. Microencapsulation technology is an important means of designing controlled release devices, immobilization and for imparting protective coatings. Microencapsulated products prepared by the following process can be used for controlled release appli-ca-tions, enzyme or cell immobilization and protective coating for storage of sensitive active ingredients. Many active ingredients are encapsulated in polymers; viz. pharmaceuticals, enzymes, food additives, perfumes, fertilizers, insecticides etc. for the above mentioned purposes. Conventional method most widely used for encapsulation utilizes an aqueous processing medium where the hydrophilic active ingredient preferentially partitions out of the polymeric phase during the emulsification stage resulting in low entrapment efficiencies (Bodmeier R. and McGrinity G. W., Int. J. Pharm. 43 (1988) 179- 186). In another method, where oil is used as the external processing medium, the sensitive macromolecular drug ingredient looses its activity, probably due to deleterious interactions of the organic solvents with the macromolecular drug (Wada R, Hyon S.H. andlkada Y., J. Pharm. Sci. 79 (1990) 919-924). It is the object of this invention to provide an improved micro- encapsulation process to entrap active ingredients, especially the macromolecular entities like peptides and proteins, in polymers, which overcomes the problems noted above and results in high efficiencies of entrapment while still retaining its biological activity. In particular, the object is to develop an improved process for encapsulating active ingredients in polymers used in drug delivery, immobilization and protective coatings, namely, poly(lactic- co-glycolic) acid, polyanhydrides, poly(ortho esters), nylon, cellulose derivatives and acrylates. The microencapsulated products prepared by such a process can be used for formulating controlled release delivery devices, designing techniques of immobilization for enzymes or cells and preparing protective coatings of sensitive materials. It is also one of the objects of the present invention to overcome the above deficiencies by using an emulsification-solvent removal technique with a mixed-solvent system for polymers and involving a combination of solvent extraction-solvent evaporation techniques carried out in a non-aqueous medium. A most outstanding feature of the invention is the use of a specially formulated mixed-solvent system for the polymer and the use of a non-aqueous processing medium in which the active ingredient has low solubility. It is most important to select the constituents of the mixed-solvent system and the non-aqueous processing medium appropriately in accordance with the properties of the active ingredient and the polymer. In the description that follows, it will be clear to a person skilled in the art to arrive at a suitable process parameters for successful encapsulation. The term "mixed-solvent system" refers to a mixture of two parts of organic sorvent(s) in which the polymer material fully dissolves, and which has been carefully selected, keeping in view all the physico-chemical properties of the active ingredient, polymer and non¬aqueous processing medium used, from any member of the commonly available organic liquids. The said mixed-solvent system essentially consists of two parts - one part is miscible with the processing medium while the other is immiscible, and each part is present in a proportion that could range from 5 to 95% of total volume of the mixed-solvent. At least one of the solvents of the mixed solvent system is immiscible with water containing active ingredient but it is preferable that both parts are immiscible. The mixed-solvent system, as a whole, is required to be immiscible with the said water containing active ingredient. If, however, one of the parts of the mixed-solvent system is miscible with the water containing active ingredient, it is preferable that this part is also miscible with the processing medium. An example of a mixed-solvent system is described as consisting of a 1:1 solvent mixture of tetrahydrofuran-dichloromethane as a solvent for a polymer, poly(D,L) lactic acid while the processing medium is glycerol. In this example, the 1:1 solvent mixture is immiscible with water containing a protein as the substance to be encapsulated, the tetrahydrofuran is miscible with glycerol, while the dichloromethane is not miscible with both water containing the active ingredient and glycerol, processing medium. Solvents for the mixed-solvent system can be any organic liquid like Acetone, Ethyl alcohol, isopropanol, acetonitrile, butanol, halogenated hydrocarbons like dichloromethane, chloroform, dichloroethane, carbontetrachloride, tetrafluoroethane, hexafluoroisopropanol etc., dimethylformamide, dimethylsulphoxide, dioxane, ethyl acetate, benzyl alcohol, toluene, benzene, tetrahydrofuran and their likes. It is extremely important to that the solvent mixture be selected according to the choice of the processing medium used, so as to enable solvent removal by both solvent-extraction and solvent-evaporation. Another equally outstanding feature of this invention is the selection and use of a non¬aqueous processing medium containing a suitable surfactant or stabilizer. A "non¬aqueous" processing medium according to this invention is a liquid that has no water present in it except as may be found as impurities under normal conditions of commercial availability or a liquid mixture of an organic liquid and water in which the active ingredient has less than 50% solubihty as compared to the solubihty in water alone. Such processing media can be any one or more of the commonly available oils such as liquid paraffin, silicone oil, biocompatible vegetable oils like arachis, cottonseed, sunflower, corn, olive, sesame, castor oil etc. or polymers like polyethylene glycols or organic liquids like diethers of alcohols (cellosoh/es), benzyl alcohol, propylene or ethylene glycol, glycerol, sorbitol, dimethylformamide, dioxane, isopropyl alcohol, isopro-pyl myristate, ethyl oleate, cyclohexane, petroleum ether and their likes, or mixtures of water and water-miscible organic liquids like glycerol, polyethylene glycol, ethanoL actonitrile, ethanoL methanol, propylene glycol, ethylene glycol, acetone, and their likes, used in different grades and/or mixtures to achieve the desired physical properties like viscosity, polarity, boiling point etc. suitable for different polymers used as the encapsulating agent and active ingredient to be encapsulated. It is important to choose the processing medium such that the solubihty of the active ingredient being encapsulated is low, preferably negligible, in the processing medium. It is also one of the important features of the invention that the mixed-solvent system and the non-aqueous processing medium is carefully selected for a particular set of active ingredients and polymer, keeping in mind all the physico-chemical parameters of the active ingredient(s) and the polymer such that the combination enables formation of emulsion with the water containing active ingredient phase as well as the non-aqueous processing medium, and also enables solvent removal by a combination of solvent-extraction and solvent-evaporation techniques. In the example cited above, the 1:1 mixture of tetrahydrofuran and dichloromethane containing the polymer dissolved in it, forms a water-in-oil emulsion with the water containing protein as the active ingredient and the resulting emulsion forms a further [(water-in-oil)-in-oil] emulsion with the processing medium glycerol due to immiscibility of dichloromethane. Solvent removal of this mixed-solvent system in this case is done by extraction of tetrahydrofuran into glycerol while dichloromethane is removed by evaporation under ambient or reduced pressure.. It is also another important feature of this invention to carefully select the composition of the mixed-solvent system to enable solvent removal by both solvent extraction as well as solvent evaporation to ensure high efficiencies of entrapment. When the emulsified into the non-aqueous processing medium, the said processing medium immediately extracts a part of the mixed- solvent system which is miscible with it (as tetrahydrofuran is extracted into glycerol in the above example) which leaves the polymer to be sob/ated only by the other part of the mixed solvent. Such a sudden reduction in amount of solvent results in increase in the viscosity of the polymer, especially if it is a polymer, and thus inhibits diffusion of active ingredient or solution thereof into the outer processing medium and thus helps in increasing entrapment efficiency. Such a process besides ensuring high efficiencies of entrapment, also offers a method to prevent deleterious interaction between organic solvent and sensitive active ingredients like proteins, peptides, enzymes etc. that would alter their biological activity significantly. Accordingly, the present invention provides an improved process for microencapsulation of active ingredients in polymers which comprises; a) dissolving polymer selected from polylactic acid/poly glycolic acid and their copolymer in water miscible/water immiscible mixed - solvent system, b) adding conventional active ingredients (to be encapsulated) in above polymer solution to form emulsion or dispersion, c) emulsifying the said emulsion solution or dispersion obtained in (b) in a non¬aqueous processing medium selected from commonly available oil optionally with conventional surfactants or stabilizers, d) adding diluents used as non-aqueous processing medium, e) removing the solvents by a combination of known methods such as solvent-extraction and solvent-evaporation, under agitation at a temperature 1-50°C below and above the boiling point of solvents, f) isolating the product by conventional methods like decanting, filtering, washing, drying and sieving to obtain desired product. In one of the embodiments of the present invention the microencapsulated product obtained by the above process, has a particle-size range between 0.5 micron to 750 microns as desired, and an entrapment efficiency greater than 90 percent. In another embodiment of the present invention the encapsulated active ingredient, namely lysozyme, was allowed to diffuse out. The released lysozyme retained greater than 80 percent of its biological activity when sampled from the extraction study medium (a pH 7.4 phosphate buffer) 30 days after the start of the study. Such products are useful in controlled release applications immobilization and protective coatings. In one another embodiment of the present invention surfactants used for the emulsion are selected from sorbitan esters (Span series), polysorbates (Tween series), Fatty acid esters, phospholipid derivatives, bile salts or their derivatives, other natural surfactants, Pluronics, Brij series and alikes. According to a feature of the invention, the substances that can be encapsulated are particularly the hydrophilic macromolecular active ingredients such as peptides, proteins, oligonucleotides etc. According to yet another feature of the invention, various oil- soluble polymers such as polymers of lactic and glycolic acids, polyanhydrides, poly(ortho esters), cellulose derivatives, acrylates and their likes can be used as the encapsulating material. According to yet another feature of the invention, the oil used as the external processing medium containing a suitable surfactant can be any one or more of the commonly available oils such as liquid paraffin, silicone oil etc. or can be chosen from any one or more of the biologically compatible oils such as cotton seed oil, sesame oil, olive oil, castor oil etc. used in various proportions and/or grades to achieve the desired viscosities. According to yet another feature of the invention, the (w/oj) emulsion is made by high¬speed stirring and/or sonication or homogenrzation while the final emulsion is made under controlled stirring between 25 to 3,000 rpm. According to yet another feature of the invention, the solvent removal is done under controlled gas-sweep and/or controlled rate of vacuum application and/or at a suitable temperature below, at or slightly above the boiling point of the solvent(s). According to yet another feature of the invention, the particle size of the microencapsulated product can be varied between 0.5 micron to 750 microns diameter by varying the viscosity of the non-aqueous processing medium between 0.02 to 1,000 centistokes and/or by varying the stirring speed from 25 to 3,000 rpm and/or by varying the concentration of polymer material such as a polymer in mixed-solvent sytem from 2 to 75 percent w/v and/or by varying the concentration of surfactant from 0.01 to 10 percent w/v in the processing medium. According to yet another feature of the invention, the porosity of the microencapsulated product can be altered by varying the rate of gas-sweep and/or rate of vacuum application and/or the temperature during processing from 2 to 70%. The invention is described herein below with reference to the examples which should not, however, be construed to limit the scope of this invention. Example 1: 0.75 grams of poly (DL) lactic acid of molecular weight 3,500 (Mw) was dissolved in 2.5 ml of 1:1 Dichloromethane-Acetonitrile mixture. 0.75 ml of a 50 mg/ml Lysozyme in fresh distilled water was added to the polymer solution. The two liquid phases were homogenized by sonicating for 45 seconds with a probe microtip and introduced quickly into 100 gms of liquid paraffin (-65 centistokes) containing 4% Span 80 held in a jacketed round bottomed flask at ambient temperature while stirring at 210 rpm to form the [(w/01/02] emulsion. After stabilizing the emulsion for 10 minutes, a gentle filtered air-sweep was applied over the liquid paraffin while reducing the pressure to 600 and 400 mm Hg in stages of 10 minutes each. Pressure was reduced to about 15 mm Hg without air-sweep for the next 90 minutes. The liquid paraffin was diluted with petroleum ether and product isolated and washed by decantation and finally air-dried. The product was sieved through a 210 micron mesh to remove large irregular aggregates, if any. Most of the particles of particles were of size between 50 to 150 microns and an efficiency of loading was above 90% (Batch No. P-ll). The lysozyme released into pH 7.4 phosphate buffer from this product, one month after incubation in the buffer, were tested for its biological cell-lysis activity on Micrococcus lysodeiktikus cell suspensions. The cell-lysis study showed that atleast 80 percent of the released protein retained its biological activity. Example 2: 1.75 grams of poly[ (D,L) lactic acid] of molecular weight (Mw) 90,000 was dissolved in 7 ml of 1:1 mixture of Dichloromethane and Acetonitrile. 1.75 ml of a 20 mg/ml model protein, Bovine Serum Albumin, in fresh double distilled water was added while stirring the polymer solution to form a coarse w/o1 emulsion. An additional 7 ml of solvent mixture was added and the coarse emulsion was sonicated for 45 seconds with a microtip probe and uitroduced quickly into 100 gms of liquid paraffin (~ 65 centistokes) containing 4% Span 80 held in a jacketed round bottomed flask at ambient temperature while stirring at 210 rpm to form the [(w/01/02] emulsion. After stabilizing the emulsion for 10 minutes, a gentle filtered air-sweep was applied over the liquid paraffin while reducing the pressure to 600 and 400 mm Hg in stages of 10 minutes each. Pressure was reduced to about 15 mm Hg without air-sweep for the next 90 minutes. The liquid paraffin was diluted with petroleum ether and product isolated and washed by decantation and finally air-dried. The product was sieved through a 210 micron mesh to remove large irregular aggregates, if any. Most of the particles were of size between 50 to 150 microns and an efficiency of loading above 70%. The yield ( # 210/37 microns) in triplicate batches were 1.22, 1.18, and 1.31 grams (Batch codes P-4, P-5, and P-6). Example 3: 1.25 grams of poly [ lactic-co-glycolic acid] (80:20 molar ratio of monomers) of molecular weight 50,000 (Mw) was dissolved in 5 ml of 1:1 Dichloromethane-Acetonitrile mixture. 1.25 ml of a 20 mg/ml Lysozyme in fresh distilled water was added with stirring to the polymer solution. An additional 5 ml of the solvent mixture was added and the coarse emulsion homogenized by sonicating for 45 seconds with a probe microtip and introduced quickly into 100 gms of liquid paraffin (~ 65 centistokes) containing 4% Span 80 held in a jacketed round bottomed flask at ambient temperature while stirring at 210 rpm to form the [(w/01/02] emulsion. After stabilizing the emulsion for 10 minutes, a gentle filtered air-sweep was applied over the liquid paraffin while reducing the pressure to 600 and 400 mm Hg in stages of 10 minutes each. Pressure was reduced to about 15 mm Hg without air-sweep for the next 90 minutes. The liquid paraffin was diluted with petroleum ether and product isolated and washed by decantation and finally air-dried. The product was sieved through a 210 micron mesh to remove large irregular aggregates, if any. Most of the particles were of size between 50 to 150 microns and an efficiency of loading above 80%. The yield was 1.11 grams (Batch code G-5). Example 4: 0.5 grams of poly [ lactic-co-glycolic acid] (80:20 molar ratio of monomers) of molecular weight 50,000 (Mw) was dissolved in 2ml of 1:1 Dichloromethane-Acetonitrile mixture. 75mg of p-nitrophenol, a model hydrophobic active ingredient was dissolved in 2ml of a similar solvent mixture and added to the polymer solution to give a clear homogenous solution. This solution was quickly introduced into 45 gms of liquid paraffin (~ 65 centis¬tokes) containing 4% Span 80 held in a jacketed round bottomed flask at ambient temperature while stirring at 190 rpm to form the [(01/02] emulsion. After stabilizing the emulsion for 10 minutes, a gentle filtered air-sweep was applied over the liquid paraffin while reducing the pressure to 600 and 400 mm Hg in stages of 10 minutes each. Pressure was reduced to about 15 mm Hg without air-sweep for the next 90 minutes. The liquid paraffin was diluted with petroleum ether and product isolated and washed by decantation and finally air-dried. The product was sieved through a 210 micron mesh to remove large irregular aggregates, if any. Most of the particles were of size between 37 to 75 microns. The yield was 0.37 grams (Batch code GP-3). The process of the present invention has following advantages 1. The process provides a means of encapsulating substances, particularly macromolecules such as peptides and proteins, in polymers with high entrapment efficiency while hydrophobic active ingredients can be encapsulated without surface crystallization. 2. The process provides a means of encapsulating sensitive macromolecular active ingredients, like peptides and proteins, in polymers without seriously altering their biological activity. Claim: 1. An improved process for microencapsulation of active ingredients in polymers which comprises; a) dissolving polymer selected from polylactic acid/poly glycolic acid and their copolymer in water miscible/water immiscible mixed - solvent system, b) adding conventional active ingredients (to be encapsulated) in above polymer solution to form emulsion or dispersion, c) emulsifying the said emulsion solution or dispersion obtained in (b) in a non¬aqueous processing medium selected from commonly available oil optionally with conventional surfactants or stabilizers, d) adding diluents used as non-aqueous processing medium, e) removing the solvents by a combination of known methods such as solvent-extraction and solvent-evaporation, under agitation at a temperature 1-50°C below and above the boiling point of solvents, f) isolating the product by conventional methods like decanting, filtering, washing, drying and sieving to obtain desired product. 2. A process as claimed in claim 1 wherein the substances (active ingredients) to be encapsulated are selected from hydrophilic entities more preferably the macromolecular entities like peptides, proteins, vaccines, antigens, antibodies, vaccine adjuvants, oligonucleotides and mixtures thereof. 3. A process as claimed in claims 1 to 2 wherein the polymers are also selected from polyanhydrides, poly ( ortho esters), cellulose derivatives, acrylates and mixtures thereof. 4. A process as claimed in claim 1 to 3 wherein the water-immiscible oil-miscible solvent system comprises of any one or more of the following: Chloroform, Dichloromethane, Dichloroethane, Ethyl acetate, Carbon tetrachloride, Benzene, Toluene and n-Butanol. 5. A process as claimed in claim 1 to 4 wherein the water - miscible solvent system that is immiscible with the processing oil medium consist of Acetone, Ethyl alcohol, Acetonitrile, Ethyl acetate, N,N-Dimethylformamide, Dimethylsulfoxide, 1,4-Dioxan and Tetrahydrofuran, or mixtures thereof. 6. A process as claimed on claim 1-5 wherein the oil used as non-aqueous processing medium is selected from the commonly available oils like liquid paraffin, silicone oil of different viscosity ranges, more preferably from the biologically compatible oils such as cotton seed oil, sesame oil, olive oil, castor oil or alkies or mixtures thereof. 7. A process as claimed in claim 6 wherein the proportion of the non-aqueous processing media are selected in such a way so as to achieve desired viscosities between 0.02 to 1000 centistokes. 8. A process as claimed in claim 1-7 wherein the emulsification in the polymeric solution phase is carried out by stirring and /or sonication or homogenization. 9. A process as claimed in claim 1-8, wherein the emulsification in the non-aqueous processing medium is done under controlled stirring at 25 to 3,000 rpm. 10. A process as claimed in claim 1-9, wherein the"surfactants used for the emulsion are selected from sorbitan esters (Span series), polysorbates (Tween series), Fatty acid esters, phospholipid derivatives, bile salts or their derivatives, other natural surfactants, Pluronics, Brij series and alikes. 11. A process from the microencapsulation of active ingredients in polymers as substantially described herein before with reference to examples.

Documents:

377-del-1996-abstract.pdf

377-del-1996-claims.pdf

377-del-1996-complete specification granted.pdf

377-del-1996-correspondence-others.pdf

377-del-1996-correspondence-po.pdf

377-del-1996-description (complete).pdf

377-del-1996-form-1.pdf

377-del-1996-form-13.pdf

377-del-1996-form-2.pdf

377-del-1996-form-3.pdf

377-DEL-1996-Form-4.pdf

377-del-1996-form-6.pdf

377-del-1996-petition-others.pdf