Title of Invention

A PROCESS FOR THE PREPARATION OF DRUG ENCAPSULATED MICROSPHERES

Abstract

A process for the preparation of drug encapsulated microspheres comprising the steps of mixing 1-10ml of the drug is mixed with 5-250gms of the drug absorption enhancer and allowed to react for 1-3 hrs to form the complexed drug, followed by mixing said complexed drug with a biopolymer gel such as herein described to form a gel mixture, subjecting said gel mixture to extrusion into a 0.2-0.5% crosslinker solution to obtain the microspheres containing the drug, allowing the microspheres to harden, followed by filtering and drying to provide the drug encapsulated microspheres.

Full Text

This invention relates to a process for the preparation of drug encapsulated microspheres and the drug encapsulated microspheres produced thereby. The number of people afflicted with diabetes is increasing day be day and many reach 300 million in another 25 years. Majority of them belong to developing countries. However, treatments are by insulin inj ecti ons. Traditional medicines in India show the use of several plant derived material to reduce diabetes. Ayurvedic oral formulations are being investigated by some groups. Despite its high costs and inconvenience, some patients are preferring external insulin pumps. Many private societies in the world have devoted millions of dollars to find an alternative to the injection therapy. Injection requires a strong discipline from the patients and is linked to an important cost. Several research groups are pursui ng permanent solution by transplantation or regeneration of pancreatic islet cells. A number of companies are involved in this research. The findings may lead to a therapy that can regenerate the pancreas but it will not be available at large scale before a long time. Some ongoing developments of novel ways of delivering insulin are descri bed in the literature, like oral, nasal, ophthalmic, rectal, etc.. All these developments are either in early-stage of development or at preclinical testing stages The delivery of therapeutic peptides drugs like insulin is carried out by subcutaneous injecti ons. Even though this ensures high percentage of bioavailability, a few unfavourable but crucia J factors still remain unsolved. The presence of very high and very 1ow amount of the drug in the vascular system is one among them. Inconvenience i n application, pain, requi rement of assistance of a second person for administration, appliances required for delivery, cost, etc. are other disadvantages. Hence, efforts are on for seeking possibilities of alternative routes and methods of administration. Prominent among them are aimed at the various other routes of administration connected with diffe-rent absorption sites present in the human body vis. nasal, enteral, ocular, rectal, bucca1 and so on. Of these, oral route has its merits since it is the norma 1 phys iological route, and also due to the convenience in controlling the dose, and ease of adm i n istr t i on. A number of factors limit the scope of oral administration of peptide drugs. Enzymatic degradation of the peptides in the gastro intestinal tract, pH in the gastric region, solubility limitations of drugs, hydrophilic nature of the peptides, etc. are a few among them. Agai n, maintenance of therapeutic level the concentration required to execute a therapeutic effect - a 1 so i s a necess i ty. Many drugs have been incorporated into biocompatib 2 e matr ices and their in vitro release in both gastric and intestinal pH has been estabiished. Matrices which are pH sensitive so that they remain intact in the hostile gastric region and release the drug in the favourable intestinal region have ako heen exolored. it is easy for lipophilic molecules to get into the vascular system through the cell walls. The hydrophi 1ic nature of many drugs limit their biological absorption. Hence, attempts have been made to entrap peptide mo 1 ecu 1es i nto 1iposomes or othe absorption enhancers for the easier absorption of the molecules into the circulatory system. It is therefore an object of this invention to propose an oral drug delivery system which is capable of encapsu1 ati ng pepti de drugs which on administration releases the drug into the gastro intestinal tract, retaining its therapeutic effect. It is a further object of this invention to propose an oral drug delivery system which del'vers the drug in a controlled manner. It is yet another object of this invention to propose an oral drug delivery system for the encapsulation of uncomplexed insulin, like peptides. Thus, accord ing to this invention is provided a process for the preparation of drug encapsu1 ated microspheres comprising mixing the drug with an absorption enhancer to form the complexed dru# foil owed by mixing said complexed drug with a biopolymer gel to form a gel mixture, subjecting said gel mixture to extrusion into a crosslinker solution to obtain the microspheres containing the drug, al1 owing the microspheres to harden, followed by filtering and drying to provide the drug encapsulated microspheres. I n accordance with this invention, a solution of about 0.5 to 5 gms. of a salt of a copolymer of mannuronic acid and guluronic acid (alginate) is prepared in a 100ml salt solution. This solution which forms the anionic biopolymer gel contains mannuronic acid and guluronic acid in a proportion of 30 :40 to 50:60, preferably 39:61. 1 to 10ml of the drug of suitable strength is added to 5 to 250gms of the absorption enhancer and mixed well and allowed to react for about 1-3 hrs.» preferably 2 hrs. to obtain the drug compiexed absorption enhancer. The drug complexed absorption enhancer is mixed with the alginate solution and extruded through a hypodermic needle of suitable diameter into a crosslinking medium. The crosslinker solution or crosslinking medium referred to hereinabove consists of about 0.2 to 5% by weight of calcium chloride. A combination of calcium chloride and about 0.1-3% by weight of chitosan dissolved in dilute hydrochloric acid or acetic acid is another choice. The microspheres thus formed are all owed to harden for about 10-90 mins., the hardened microspheres are separated and dried at a o temperature in the range of 3-15 C. The drug used for the purpose in general are peptide drugs such as insulin are used in particular, either in their solid or solution state. In a preferred embodiment, 40-100 IU of insulin is used as the drug for forming the complex. Various molecules are used for the encapsulation of drugs. Such molecu Ies are chi tosan, alginic acid and its salts, polylysine, polyv iny1 alcohol, hydroxyapatite, etc. of which alginate, chitosan, etc are generally used since they are proven natural polymers. The alginate salt may be a sodium or potassium salt and the salt solution is likewise selected from sodi um chloride or potassium chloride solution. Apart from using the polymeric mo 1 ecu 1es, such as alginate and its salts alone, they have also been used in combination for specific drug release patterns. Such a system is poly anionic alginate and poly cationic chitosan in combination. It optimises the release of the drug to have a more uniform release pattern. Other mo 1 ecules like eye 1odex tr i ns have also been applied as absorption enhancers. All three forms of cyclodextrin such as alpha, beta and gamma cyclodextrin and their derivatives have been used. Accord ing to this invention is further provided a met mixing a drug with an absorption enhancer to form the compiexed drug. foil owed by mixing the compiexed drug with the polysaccharide ge1 to obtain the drug containing gel and stirring, introducing the drug containing gei into an emulsifying medium with stirring followed by adding a crosslinker to form the microspheres, washing and dry ing the microspheres to obtain the drug encapsulated microspheres. In accordance with the further embodiment, a vegetable oil, such as sunflower oil, groundnut oil, coconut oil, singly or in combi- nation is used as the emulsifying agent. About 0.25-5 gms. of a sodium or potassium salt of alginic acid is dissolved in 50-100ml water or a 0.5-1.5% solution of sodium chloride in water to obtain the alginate solution. About 5-10ml of the drug, preferably 40 IU insulin is taken separately and to it is added 5-50mg of an absorption enhancer and the mixture is allowed to react for 1-3 hrs.. This solution is added to the alginate solution mentioned above and mixed gently to obtain the drug containing alginate. In a separate vessel, 250-1000m1 of a vegetable oil is stirred at a speed of 500-2000 rpm for 10-60 roins., depending on the required size of the microspheres. The drug-cyc1odextrin-a1ginate mixture is added to it and stirring is conti nued for another 20 - 45 mins.. A super saturated solution of calcium chloride in the same vegetable oil is added to it and stirring is again continued for another 15-25 mins.. The microspheres formed are separated, washed with a suitable solvent like petroleum ether and dried at Still further according to this invention is provided a process for the preparation of drug encapsu1 ated microspheres compr is i ng preparing a biopolymer gel and adding said gel to an emulsifying medium with stirring followed by adding a crosslinker solution thereto and stirring for a further period to obtain the microspheres, followed by separating, washing and drying the microspheres so formed, mixing a drug with an absorption enhancer to form the complexed drug, subjecting it to dilution with ca1cium chloride solution and adjusting the pHf adding said microspheres to the solution of drug complexed enhancer and calcium chloride to obtain the drug loaded microspheres, separating and drying the same to the drug encapsulated microspheres. In accordance with this further embodiment, about 250-1000ml of vegetable oil like sunflower oil, coconut oil, etc. is taken in a vessel. It is stirred at 500-2000 rpm for 10-60 mins.. The alginate solution alone mentioned under Example 2 (Soln, A) is added to it and stirring is continued for 20-45 mins.. A super saturated solution of calcium chloride in the same oil is added to it and the stirring is continued for another 15-25 mins- The microspheres formed are separated and washed in a solvent like o petroleum ether and dried at 8-10 C. In another beaker, 5ml of insulin 5-50 mg. of alpha, beta or gamma cyclodextrin or their derivative is added. After 1-3 hrs.t it is diluted with 0.5-3.5 gm% calcium chloride solution and pH adjusted to pH 3-7.0 (SoJn, C). The dried alginate microspheres are added to the above mentioned solution of insulin containing cyclodextrin and calcium chloride ie. (Soln. C). After 3-8 hrs., the microspheres are separated, o dried at 8-15 C and stored According to this invention is further provided drug encapsulated microspheres for oral delivery of therapeutic peptides, compri sing a drug complexed with a drug absorption enhancer eneapsulated in a natural biopolymer. The invention will now be explained in greater detail with the help of the foil owing non-1imiting examp 1es. EXAMPLE i Three grams of the sodium salt of a copolymer of Mannuronic acid and guluronic acid in the ratio 39:61 (alginate), is weighed out. It is introduced into 100ml of 0.9% sodium chloride solution and sti rred (Soln. I ) . In another beaker, 5ml of insulin of suitable concentration (40-100 JU) is taken. 50mg of beta cyclodextrin is added to it and giantly shaken. It is all owed to react for 2 hrs. , This is mixed with 45ml of Soln. 1 (the copolymer solution of mannuronic and guluronic acids). This mixture of insulin, beta cyclodextrin and alginate solution will be known as Soln. II. In a thi rd beaker, a crosslinking med ium cons isting of 0. 3gm of chitosan in 100ml of 01 N HCl and 1.0gm of calcium chloride is taken (Soln. 111). The alginate-drug-beta cyclodextrin (Soln. 1 I) solution is extruded through a hypodermic needle into the crosslinking medium consisting of chitosan, HCl and calcium chloride (Soln.lll) with gentle stirring. The microspheres formed are allowed to harden for 30 mins. in the crosslinking medium. They are separated and o dried at 8-10 C. EXAMPLE 2 One gram of sodium salt of alginic acid (alginate) is weighed out in a beaker and is dissolved in 100ml 0,996 sod ium ch1oride. In another beaker, 5ml insulin (40 1U per ml) is taken, 50mg of beta cyclodextrin is added to it and gently mixed. After two hours, it is added to the alginate solution and mixed gently. About 250ml coconut oil is taken in a round bottomed reactor. The oil is stirred at 1000 rpm for about 20 mins.. The insulin-beta cyclodextrin-alginate mixture is added to it and stirring is continued for 25 mins.. A super saturated solution of calci urn chloride in 20ml of the same oil is add 1 to it and the stirring is continued for 25 mins.. The microspheres formed are collected, washed with petro- o Ieum ether and dried at 8-10 C, EXAMPLE 3 One gram of sodium salt of alginic acid (alginate) is weighed out in a beaker and is dissolved in 100ml 0.9% sod ium ch1 or ide. About 250ml coconut oil is taken in a round bottomed reactor. The oil is stirred at 1000 rpm for about 20 mins. The a 1ginate solution prepared is added to the oi1 and the stir-ring is continued for about 25 mins.. 20ml of coconut oi1 super saturated with calcium chloride is added to it and the stirring is continued for another 25 mins.. The microspheres formed are separated and washed with petroleum o ether and dried at 10 C. In another beaker, 5ml of insulin (40 IU per ml) is taken and is mixed with 50mg of beta cyclodextrin and gently mixed. After two hours, 2m1 of lgm% calei urn chloride is added to it and the pH of this solution is adjusted to 5 with phthalate buffer- The dried microspheres already prepared are added to it. After five hours, o the drug loaded microspheres are separated and dried at 10 C. EXPERIMENTS 1. In vitro release of the drug in simulated gastric medium (SGM) The in vitro release of the drug was performed in simulated gastric medium (without enzymes) as per the US pharmacopoea at 1.2 pH. A weighed amount of the microspheres were added to a measured quantity of the buffer SGM. 0.5ml of sampe is withdrawn f rom it after 30 mins., 60 mins., 2 hrs., 3 hrs. and 4 hrs.. The drug concentration is estimated using Lowry's method. The drug release in simulated gastric med ium i s shown in Fig.l of the accompany ing drawings. 2. In vitro release of the drug in s imulated intestinal med ium The in vitro release of the drug in SIM was done as per the US pharma copea (without enzymes). A weighed quantity of the (0.2gm) microspheres was introduced into measured quantity of SIM (pH 7.4). Exactly 0.5ml of the sample was withdrawn at the end of 30 mins., 60 mins,, 1 hr., 2 hrs., 3 hrs. and 5 hrs.. The drug concentration was estimated using Lowry's method. Drug re]ease in simulated intestinal medium is shown in Fig.2 of the acccom-panying drawings. 3. Pay 1oad The total amount of drug encapsulated per gram of the microspheres were estimated to be 20-30 IU per gram of microspheres. 4. The therapeutic effect of the microsphere The therapeut ic effect of the microspheres was tested in albino rats. Diabetes was induced on the animals by subcutaneous administration of 150mg alloxan per kg. weight of the animal. The blood glucose level of the animals were monitored using glucose k i ts. The animaIs were maintained in the normal state by intraperitoneal injection of long acting bovine insulin for about. seven days. Afterwards, drug administration was withdrawn and g1ucose 1evel was a 1 1 owed to mount upto 400 mg/dl and above. In two animals, microspheres equivalent to 2 units of insulin was administered through special equipments meant, for it. The third one was maintained as a control. The glucose level was monitored at the end of 1,2,3,6,9 and 24 hrs. using glucose kits. A second dose of 2.0 IU was given at the end of the third hour and a third dose of 2 IU at the end of the ninth hour (Table I J. Results The in vitro release studies suggests that there is less than 0.09 IU per ml release of the drug in 3 hrs. in the acidic pH of the gastric region. The release in the simulated intestinal medium is about 0.2 IU per ml. The total amount of the drug that could be loaded in one gram of the microspheres is about 20-30 1U. The g 3 ucose level of diabetic i nduced animaIs has come down from the third hour onwards to a hypoglycemic state thus ascertaining that this system is capable of reducing g1ucose level in diabetic rats. WE CLAIM: 1. A process for the preparation of drug encapsulated microspheres compr ising the steps of mixing the drug with an absorption enhancer to form the compJexed drug followed by mixing said complexed drug with a biopolymer gel to form a gel mixture, subjecting said gel mixture to extrusion into a crosslinker solution to obtain the microspheres containing the drug, allowing the microspheres to harden, foil owed by filtering and drying to provide the drug encapsulated microspheres. 2. The process as claimed in claim 1 wherein said drug used is a peptide drug* 3. The process as claimed in claim 2 wherein said peptide drug is insulin of strength 40-100 IU. 4. The process as claimed in claim 1 wherein the drug is used in solid or solution state. 5. The process as claimed in claim 1 wherein said absorption enhancer is cyclodextr in and its derivatives such as a 1pha, beta, gamm cyclodextrin and their derivatives. 6. The process as claimed in claim 1 wherein said biopolymer is selected from atleast one of the metal salts of molecules such as chi tosan, alginic acid and its salts, polylysine, polyvinyl alcohol, hydroxyapatite. 7. The process as claimed in claim 6 wherein a preferred bio-polymer system is a combination of polyanionic alginate and poly-cat ionic chitosan. 8. The process as claimed in claim 6 wherein the metal is se1ected f rom sodium and potassi urn. 9. The process as claimed in claim 1 wherein said crosslinker used is selected from calcium chloride or a mixture of calcium chloride and chitosan dissolved in an acid. 10. The process as claimed in claim 9 wherein said acid is an organic or inorganic acid such as acetic acid, hydrochloric acid and the like. 11. The process as cla imed in claim 1 wherein 1-10ml of the drug is mixed with 5-250gms of the drug absorption enhancer and al lowed to react for 1-3 hrs. . 12. The process as claimed in claim 1 wherein the biopolymer gel solution is an alginate acid gel. 13. The process as claimed in claim 12 wherein said alginate acid gel is prepared by mixing about 0.5-5gms of mannuronic acid and guluronic acid in a ratio of 30:40 to 50:80 with 50~100ml sodium chloride solution. 14. The process as claimed in claim 1 wherein said crosslinker solution compr ises 0.2-5% by weight of calci um chloride. 15. The process as claimed in claim 1 wherein said cross!inker solution comprises calcium chloride and 0.1-3% by weight of chitosan dissolved in dilute hydrochloric acid or acet ic acid. 16. The process as claimed in claim 1 wherein said microspheres are allowed to harden for abut 10-90 mins.. 17. The process as claimed in claim 1 wherein said microspheres o are dried at 3-15 C. 18. A process for the preparation of drug encapsulated micro spheres coropr ising preparing a po iysaccharide gel, mixing a drug with an absorption enhancer to form the complexed drug, followed by mixing t ■:> complexed drug with the polysaccharide gel to obtain the drug containing gel and stirring, introducing the drug containing gel into an emuIsifying medium with stirring foil owed by adding a crosslinker to form the microspheres, washing and drying the microspheres to obtain the drug encap- sulated microspheres* 19. The process as claimed in claim 1 wherein said drug used is a peptide drug. 20. The process as claimed in claim 2 wherein said peptide drug is insulin of strength 40-100 IU. 21. The process as claimed in claim 1 wherein the drug is used in solid or solution state. 22. The process as claimed in claim 1 wherein said absorption enhancer is cyclodextrin and its derivatives such as alpha, beta, gamm eyelodextrin and their derivatives. 23. The process as claimed in claim 1 wherein said biopolymer is se1ected f rom atleast one of the metal salts of molecules such as chitosan, alginic acid and its salts, poly lysine, polyvinyl alcohol, hydroxyapat i te. 24. The process as claimed in claim 6 wherein a preferred biopolymer system is a combination of polyanionic alginate and po1y-cationic chitosan. 25. The process as claimed in claim 6 wherein the metal is selected from sodium and potassium. 26. The process as claimed in claim 1 wherein said emulsifying medium compr ises a vegetable oil such as sunflower oil, groundnut oi1 or coconut oi1. 27. The process as claimed in claim 1 wherein said crossl inker-comprises a super saturated solution of calcium chloride in vegetable oil. 28. The process as claimed in claim i wherein about 0.25-5gms of sodium or potassium salt of alginic acid is dissolved in 50-100ml of water or 0.5-i.5% sod ium chloride solution in water to obtain the bi opolymer gel. 29. The process as claimed in claim 1 wherein 5- 10m1 insulin is mixed with .5-50mg of the drug absorption enhancer and all owed to react for 1-3 hrs.. 30. The process as claimed in claim i wherein 250-1000ml of the vegetable oi1 stirred at a speed of 500-2000 rpm for about 10-60 mins. is used as the emulsifying medium. 31. The process as claimed in claim 1 wherein the emulsifying med i urn with the drug containing gel is stirred for 20-45 mins.. 32. The process as cla imed in claim 1 wherein said crosslinker is a super saturated solution of calcium chloride in vegetable oil. 33. The process as claimed in claim 1 wherein the microspheres are washed in an organic solvent such as petroleum ether. 34. The process as claimed in claim 1 wherein the microspheres o are dried at 8-15 C. 35. A process for the preparation of drug encapsulated microspheres compri s%ng preparing a biopoi ymer gel and adding said gel to an emuls ifying medium with stirring foil owed by adding a crosslinker solution thereto and stirring for a further period to obtain the microspheres, followed by separating, washing and drying the microspheres so formed, mixing a drug wi th an absorption enhancer to form the complexed drug, subjecting it to dilution with calcium chloride solution and adjusting the pH, adding said microspheres to the solution of drug complexed enhancer and calcium chloride to obtain the drug loaded microspheres, separating and drying the same to the drug encapsulated microspheres. 36. The process as claimed in claim 1 wherein said drug used is a peptide drug. 37. The process as claimed in claim 2 wherein said peptide drug is insulin of strength 40-100 W. 38. The process as claimed in claim 1 wherein the drug is used in solid or solution state. 39. The process as claimed in claim 1 wherein said absorption enhancer is cyclodextrin and its derivatives such as alpha, beta, gamm cyclodextrin and their derivatives. 40. The process as claimed in claim 1 wherein said biopolymer is selected f rom at 1eas4 one of the metal salts of molecules such as chitosan, alginic acid and its salts, polylysine, polyvinyl alcohol, hydroxyapatite. 41. The process as claimed in claim 6 wherein the meta 1 i s selected from sodium and potassium* 42. The process as claimed in claim 1 wherein said emulsifying medium comprises a vegetable oil such as sunflower oil, groundnut oil or coconut oil. 43. The process as claimed in claim 1 wherein said cross!inker solution used is a saturated solution of calci urn chloride in vegetable oi1. 44. The process as claimed in claim 1 wherein 250-1000ml of said emulsifying medi urn is stirred at 500-2000 rpm for 10-60 mins. before addition of the biopolymer gel. 45. The process as claimed in claim 1 wherein the emulsifying medium with the biopolymer gel is stirred for another 20-45 mins.. 46. The process as claimed in claim 1 wherein the emulsifying med i urn with the biopolymer gel is stirred for a further period of 15-25 mins. after addition of the crosslinker solution. 47. The process as claimed in claim 1 wherein said microspheres are washed in an organic solvent like petroleum ether. 48. The process as claimed in claim 1 wherein 5ml of 40 W insulin is mixed with 5-50 ug of the drug absorption enhancer. 49. The process as claimed in claim 1 wherein for dilution, a 0.5-3.5 gm% calcium chloride solution is used. 50. The process as claimed in claim 1 wherein the pH is adjusted to 3-7. 51. The process as claimed in claim 1 wherein the drug loaded microspheres are separated after 3-8 hrs.. 52. The process as claimed in claim 1 wherein said microspheres o are dried at 8-15 C. 53. Drug encapsulated microspheres for oral delivery of thera peutic peptides, comprising a drug complexed with a drug absorp tion enhancer encapsulated in a natural bi opo1ymer.

Documents:

1115-mas-2000 abstract.pdf

1115-mas-2000 claims granted.pdf

1115-mas-2000 description (complete) granted.pdf

1115-mas-2000-claims.pdf

1115-mas-2000-correspondence others.pdf

1115-mas-2000-correspondence po.pdf

1115-mas-2000-description complete.pdf

1115-mas-2000-description provisinol.pdf

1115-mas-2000-drawings.pdf

1115-mas-2000-form 1.pdf

1115-mas-2000-form 3.pdf