Title of Invention

A PROCESS FOR THE PREPARATION OF ORAL INSULIN MICROCAPSULES

Abstract

WE CLAIM: 1. A process for the preparation of oral insulin microcapsules comprising emulsifying 1 to 10% solution of chitosan in a vegetable oil; treating with a solidifying agent such as a solution of tripolyphosphate or sodium hydroxide to obtain the chitosan microparticles; filtering and washing the chitosan microparticles; encapsulating the microparticles in alginate solution comprising mixing 100-750 mg chitosan microparticles and 20% slurry of calcium citrate with a 2-6% solution of aliginic acid to obtain a mixture, emulsifying the mixture in a vegetable oil to form capsules, partioning the capsules in a solution of calcium chloride, filtering, washing and drying to obtain the encapsulated microspheres. loading the encapsulated microspheres with 40 to 2000 IU/ml of insulin for a period of 4-16 hrs. at a temperature in the range of 2-6°C, by diffusion filling method to obtain the oral insulin microcapsules.

Full Text

FIELD OF THE INVENTION This invention relates to oral insulin microcapsules and a process for the preparation thereof. This invention further relates to oral insulin microcapsules based on chitosan, alginate and liposome and a process for the preparation thereof. BACKGROUND OF THE INVENTION Diabetes mellitus is a serious disease characterised by the body's inability to produce or respond properly to insulin) a hormone used to convert blood glucose (blood sugar) into energy. The diminished rate of Insulin sec retion by the beta cells of the islets of Langerhans leads to an elevation of blood glucose (hyoerglycemi a) . Each yea.ri diabetes contributes to considerable deaths through its complications such as heart disease, kidney failure, and stroke. Diabetes is also a leading contributor of new cases of blindness and amputat ions and is considered a costly disease . Appro* imately 4L'i*/. of the 1.35 mi 11 ion peopl e diagnosed with diabetes worldwide are rece iv ing insulin as a component of their therapy. Despite its widespread use, conventional subcu¬taneous insulin injection is more—or—less painful and incon¬venient, with poor patient acceptability. Significant inter patient and intra patient variability in absorpt ion, saw-tooth like insulin availab i1i ty in serum that leads to alternative hyperglye emia and hyperinsulinemia, and the risks of blood-borne infect ion assoc iated with repeated inject ions are some of the drawbacks. Other clinically available insulin delivery devices presently usedi such as pen injectors or external insulin pumps are probably under utilised, as they are inconvenient. Several research groups are pursuing permanent solution by transplanta¬tion or regeneration of pancreatic islet cells. 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 nove1 ways of delivering insulin are orali nasal, ophthalmic and rectal. All these developments are either in early-stage of development or at pre-clinical testing stages. In India, also number of people afflicted with diabetes is in an increasing trend. However , treatments are mastly by insulin injections. Traditional medicines in India show the use of several plant-derived materials to reduce diabetes. Some groups are investigating ayurvedic oral formulations. These are mainly targeted to the regeneration of pancreatic cells in type II diabetes. An alternate method of insulin delivery has been the focus of research for past few decades. Oral administration has always been desirable for a patient perspecti ve as witnessed by the frequent need to explain to patients the difference between insulin and oral hypoglycemic agents. Oral de1i very appears as a simple, comfortable and physiological way of administration. However, like dietary proteins, peptide and protein drugs get digested by the proteolytic enzymes in the gastrointestinal tract inhibiting its absorption. It has been reported that oral bioavailabilities of most free peptide and protein drugs are less than 1'/.. Therefore, an insulin formulation that can escape the harsh env ironment of the stomach intact and can deliver insulin in the neutral env i, ran merit of the intestine or colon for optimum bioavailability should be developed and optimized making oral admin istrat ion feasible. OBJECTS OF THE INVENTION It is therefore an abject of this invention to propose oral insulin microcapsules for an alternative treatment of diabetes me 11itus. It is a further object of this invention to propose oral insulin microcapsules for an alternative treatment of diabeteg mellitus which is cheap and easily sealable, Another object of this invention to propose oral insulin microcapsules for an alternative treatment of diabetes mellitus which is non-invasive and painless. BRIEF DESCRIPTION OF THE INVENTION Thus according to this invention is provided a process for the preparation of oral insulin microcapsules comprising emulsi f y ing a solution of chitosan in a vegetable oil; treat ing with a solidifying agent such as a solution of tripoly¬phosphate or sodium hydroxide to obtain the microparticlesj filtering and washing the microparticles) encapsulating the microspheres in alginate solution to obtain microcapsules; lasd ing the microcapsules wi th insul in to obtain the oral insul in microcapsules. According to this invention is further provided the oral insulin microcapsules . In accordance with this invention, chitosan micro particles are prepared by emu), si f ying an ac id solution of chitosan in a vegetable oil medium, followed t>y solidification to obtain micro particles. After a predetermined period, the micro particles are washed with an organic sol vent and water and dried. A slurry of the micro particles and calcium citrate is mixed with alginic acid solut ion and emulsified in vegetable oil medium. The capsules formed are partitioned into a solution of calcium chloride, f i1tered, washed and dried. These microcapsules are loaded with insulin by diffusion filling method in a solution of o 4i?i"20J0i?i IU/ml of insulin for 4-16 hours at a temperature of 2-6 C. The solut ion of ch i tosan is prepared as a 1--HSV4 solut ion in a 1-5*/. acid i selected from acetic, maleic or citric acids. The step of solidification is effected with agents such as tripolyphos¬phate, sodium hydroxide or the like. In accordance with an alternative embodiment, the microparticles are prepared by spraying a t-l&Y, solution of chitosan into an alkali bath, preferably sodlum hydroxide bath, filtered and d r i a d . In accordance with an embodiment of the invent ion, the drug loaded microcapsules are given a secondary coating with alg inate » by suspending them in a minimum volume of the alginate solution) o fallowed by filtering and drying at a temperature of 2-6 C. In accordance with another embodiment! 50-~200mg of L- phospho- tidyl choline and 25-100mg of cholesterol is dissolved in about 5ml of chloroform in a round bottom flask. The flask is swirled constantly to evaporate the solvent till a thin lipid film is farmed on the walls of the flask. The flask is kept under vacuum for about 30 mins. to remove any traces of chloroform. 2 to Sml of insulin sol Lit ion (40-2000 lU/ml) is added into the lipid coated f1 ask and p1aced on a rotary shaker at 200 rpm for 1 hour to obtain neutral 1ipoinsulin. The drug loaded microcapsules are given a secondary coating with alginate containing the neutral lipainsLilin, by suspending them in l--4ml of the alginate solution containing l--4ml of neutral lipoinsulin, followed by filtering o and drying at a temperature of 2-6 C. According to a preferred embodiment, 50~200mg of L- phospho-tidyl choline, 25-100mg of cholesterol and 35-140mg of stearylamine is dissolved in 5ml of chloroform in a round bottom flask. The flask is swi rled constantly to evapore the solvent till a thin lipid film is farmed an the walls of the flask. The f1 ask is kept under vacuum for about 30 mins. to Remove any traces of chloroform. 2 to Sml of insulin solution (40-2000 lU/ml) is added into the lipid coated flask and placed on a rotary shaker at 200 rpm for i hr. to obtain positively charged 1ipainsulin. The drug loaded microcapsules are given a secondary coating with alginate containing the positively charged lipainsulin, by suspend ing them in i-4ml of the alginate solution containing l-4ml of positively charged lipainsulin, followed by o filtering and drying at a temperature of 2-6 C. According to yet another preferred embod iment, 50~5t?'#mg of B~ cyclodextrin or its derivatives is mi: o (40-2000 lU/ml) and kept at 2-a C for 2,0 to 120 mins. for the complexation of insulin with B-cycladextrin. This B-cyclodextrin complexed insulin is used instead of plain insulin, both far drug loading and the preparation of neutral and positively charged eyelodextrin camplexed lipainsulin. The invention will now be explained in greater detail with the help of the following non~limi ting examp 1es. EXAMPLE 1 About 1 to 10 grams of chitosan is dissolved in 100 ml of distilled water containing 1 to 5 ml of glacial acetic acid 50 ml of well-mixed and filtered solution of chitosan is dispersed in 500 ml of groundnut oil by stirring at 400 rpm using a three bladed stirrer. After 15 minutes, 50 ml of 1 to 10 % aqueous tripolyphosphate solution is added slowly to solidify chitosan particles. These particles are filtered and washed with petroleum ether to remove oil and dried to constant weight. Chitosan microparticles (200 to 1500 mg) are dispersed in 20 ml of 2 to 10 % solution of alginic acid containing 0.9 % sodium chloride. The pH of the mixture is adjusted to 8 by adding 0.1N NaOH solution. To this 1 ml of a 20% slurry of calcium citrate is added and mixed well with a magnetic stirrer. This mixture is emulsified in 100 ml of groundnut oil. After about 15 minutes 1 ml of glacial acetic acid, dissolved in 2 mi of groundnut oil, is added to effect gelation of alginate. The microcapsules formed are solidified and separated by adding 100 ml of 50 mM calcium chloride solution The separated microcapsules are dried at room temperature. These microcapsules are loaded with insulin by diffusion filling method. For that microcapsules are suspended, in a solution of 40-2000 lU/ml of insulin for 16 hours at a temperature of 2-6°C and dried. EXAMPLE 2 About 1 to 10 grams of chitosan is dissolved in 100 ml of distilled water containing 1 to 5 mi of glacial acetic acid Well-mixed and filtered solution of chitosan is sprayed into a bath of 1N NaOH solution by gentle shaking. Microparticles formed are allowed to settle after about 1 hour The particles are filtered; washed thoroughly in distilled water to remove traces of NaOH and boiled in distilled water for 30 min. Particles are dried at room temperature. Chitosan microparticles (200 to 1500 mg) are dispersed in 20 ml of 2 to 10 % solution of alginic acid containing 0.9 % sodium chloride. The pH of the mixture is adjusted to 8 by adding 0.1N NaOH solution. To this 1 ml of a 20% slurry of calcium citrate is added and mixed well with a magnetic stirrer. This mixture is emulsified in 100 ml of groundnut oil. After about 15 minutes 1 ml of glacial acetic acid, dissolved in 2 ml of groundnut oil, is added to effect gelation of alginate. The microcapsules formed are solidified and separated by adding 100 ml of 50 mM calcium chloride solution. The separated microcapsules are dried at room temperature. These microcapsules are loaded with insulin by diffusion filling method. For that microcapsules are suspended in a solution of 40-2000 lU/ml of insulin for 16 hours at a temperature of 2-6°C and dried. EXAMPLE 3 Examp\e 1 is repeated. The drug loaded microcapsules are given a secondary coating with 4% alginate, by suspending them in a minimum volume of the alginate solution, followed by filtering and drying at a temperature of 2-6°C. EXAMPLE 4 50-200 mg of L-a phosphatidyl choline and 25-100 mg of cholesterol is dissolved in 5 ml of chloroform in a round bottom flask. The flask is swirled constantly to evaporate the solvent till a thin lipid film is formed on the walls of the flask. The flask is kept under vacuum for 30 min to remove any traces of chloroform. 2 to 8 ml of insulin solution (40 - 2000 lU/ml) is added into the lipid coated flask and placed on a rotary shaker at 200 rpm for 1 hour to obtain neutral lipoinsuiin. EXAMPLE 5 Example 4 and example 1 is repeated. The drug loaded microcapsules are given a secondary coating with 4% alginate containing the neutral lipoinsuiin, by suspending them in 1-4 ml of the alginate solution containing 1-4 ml of neutral lipoinsuiin, followed by filtering and drying at a temperature of 2-6°C. EXAMPLE 6 50-200 mg of L-a phosphatidyl choline, 25-100 mg of cholesterol and 35-140 mg of stearylamine is dissolved in 5 ml of chloroform in a round bottom flask. The flask is swirled constantly to evaporate the solvent till a thin lipid film is formed on the walls of the fusk. The flask is kept under vacuum for 30 min to remove any traces of chloroform 2 to 8 ml of insulin solution (40 - 2000 Ill/ml) is added into the lipid coated flask and placed on a rotary shaker at 200 rpm for 1 hour to obtain positively charged lipoinsulin EXAMPLE 7 Example 6 and example 1 is repeated The drug loaded microcapsules are given a secondary coating with 4% alginate containing the positively charged lipoinsulin, by suspending them in 1-4 ml of the alginate solution containing 1-4 ml of positively charged lipoinsulin, followed by filtering and drying at a temperature of 2-6"C. EXAMPLE 8 Example 4 and example 2 is repeated. The drug loaded microcapsules are given a secondary coating with 4% alginate containing the neutral lipoinsulin, by suspending them in 1-4 ml of the alginate solution containing 1-4 ml of neutral lipoinsulin, followed by filtering and drying at a temperature of 2-6°C. EXAMPLE 9 Example 6 and example 2 is repeated. The drug loaded microcapsules are given a secondary coating with 4% alginate containing the positively charged lipoinsulin, by suspending them in 1-4 ml of the alginate solution containing 1-4 ml of positively charged lipoinsulin, followed by filtering and drying at a temperature of 2-6°C. EXAMPLE 10 50 - 500 mg of fS-cyclodextrin or its derivatives is mixed with 5 ml of insulin solution (40-2000 lU/ml) and kept at 2-8°C for 30 mif> to 120 min for the complexation of insulin with (J-cyclodextrin. Example 4 is repeated by adding complexed insulin instead of plain insulin to obtain neutral cyclodextrin complexed lipoinsulin. Example 1 is repeated by loading cyclodextrin complexed insulin instead of plain insulin The drug loaded microcapsules are given a secondary coating with 4% alginate containing the neutral cyclodextrin complexed lipoinsulin, by suspending them in 1-4 ml of the alginate solution containing 1-4 ml of neutral cyclodextrin complexed lipoinsulin, followed by filtering and drying at a temperature of2-6°C. EXAMPLE 11 50 - 500 mg of p-cyclodextrin or its derivatives is mixed with 5 ml of insulin solution (40-2000 lU/ml) and kept at 2-8°C for 30 min to 120 min for the complexation of insulin with P-cyclodextrin. Example 6 is repeated by adding complexed insulin instead of plain insulin to obtain positively charged cyclodextrin complexed lipoinsulin. Example 1 is repeated by loading cyclodextrin complexed insulin instead of plain insulin. The drug loaded microcapsules are given a secondary coating with 4% alginate containing the positively charged cyclodextrin complexed lipoinsulin, by suspending them in 1-4 ml of the alginate solution containing 1-4 ml of positively charged cyclodextrin complexed lipoinsulin, followed by filtering and drying at a temperature of 2-6°C. WE CLAIM: 1. A process for the preparation of oral insulin microcapsules comprising emulsifying 1 to 10% solution of chitosan in a vegetable oil; treating with a solidifying agent such as a solution of tripolyphosphate or sodium hydroxide to obtain the chitosan microparticles; filtering and washing the chitosan microparticles; encapsulating the microparticles in alginate solution comprising mixing 100-750 mg chitosan microparticles and 20% slurry of calcium citrate with a 2-6% solution of aliginic acid to obtain a mixture, emulsifying the mixture in a vegetable oil to form capsules, partioning the capsules in a solution of calcium chloride, filtering, washing and drying to obtain the encapsulated microspheres. loading the encapsulated microspheres with 40 to 2000 IU/ml of insulin for a period of 4-16 hrs. at a temperature in the range of 2-6°C, by diffusion filling method to obtain the oral insulin microcapsules. 2. The process as claimed in claim 1 wherein said solution of chitosan is an acid solution in acids such as glacial acetic acid, maleic acid, citric acid. 3. The process as claimed in claim 2 wherein a solution of chitosan in 1-5% acid is used for emulsification. 4, The process as claimed in claim 1 wherein said vegetable oil is, for eg. groundnut oil. 5, The process as claimed in claim 1 wherein the solidifying agent is a 1-10% solution of tripolyphosphate or sodium hydroxide. 6. The process as claimed in claim 1 wherein the treatment with the solidifying agent comprising a step selected from adding al- 10% solution of the solidifying agent to the chitosan solution or spraying a 1-10% solution of chirosan into a NaOH bath. 7, The process as claimed in claim 1 wherein the microcapsules are optionally coated with a secondary coating selected from alginate, alginate-neutral lipoinsulin, alginate-positively charged lipoinsulin. 8. The process as claimed in claim 7 wherein the secondary coating of alginate is provided by suspending the insulin loaded microcapsules in a 2-4% solution of alginic acid followed by filtering and drying to obtain the microcapsules with a secondary alginate coating. 9. The process as claimed in claim 1 wherein for providing the secondary coating of alginate and neutral lipoinsulin, 50-200 mg of L-a-phosphotidyl choline and 25-100 mg of cholesterol are mixed to form a lipid film followed by adding 2-8 ml of insulin thereto to obtain neutral lipoinsulin, suspending the drug loaded microcapsules in a 1-4 ml of the alginate solution containing 1-4 ml of neutral lipoinsulin, followed by filtering and drying to obtain the microcapsules. 10. The process as claimed in claim 1 wherein for providing the secondary coating of alginate and positively charged lipoinsuin 50-200 mg of L-a-phosphotidyl choline, 25-100 mg of cholesterol and 35-140 mg stearylamine are mixed to form a lipid film, followed by adding 2-8 ml of insulin solution thereto to obtain the positively charged lipoinsulin, suspending the drug loaded microcapsules in a 1-4 alginate solution containing 1-4 ml of the positively charged lipoinsulin, followed by filtering and drying to obtain the microcapsules. 11. The process as claimed in claim 1 wherein for the step of loading the microcapsules, preferably cyclodextrin complexed insulin is used. 12. The process as claimed in claim 11 wherein 50-500 mg of [3-cyclodextrin or its derivative is mixed with insulin at 2-8°C for 30-120 mins. to obtain cyclodextrin complexed insulin. 13. The process as claimed in claim 12 wherein a secondary coating of alginate containing neutral cyclodextrin complexed lipoinsulin is provided to the cyclodextrin complexed insulin loaded microcapsules. 14. The process as claimed in claim 13 wherein the cyclodextrin complexed insulin loaded microcapsules are suspended in a 1-4 ml alginate solution containing 1-4 ml of neutral cyclodextrin complexed lipoinsulin to obtain the secondary coating. 15. The process as claimed in claim 12 wherein a secondary coating of positively charged cyclodextrin complexed lipoinsulin is provided to the cyclodextrin cornplexed insulin loaded microcapsules. 16. The process as claimed in claim 13 wherein the cyclodextrin cornplexed insulin loaded microcapsules are suspended in a 1-4 ml alginate solution containing 1-4 ml of positively charged cyclodextrin cornplexed lipoinsulin to obtain the secondary coating. 17. Oral insulin microcapsules comprising chitosan microparticles encapsulated in alginate loaded insulin. 18. The oral insulin capsule as claimed in claim 17 comprising of chitosan microparticles encapsulated in alginate loaded with insulin and having a secondary alginate coat. 19. The oral insulin capsule as claimed in claims 17 and 18 wherein neutral lipoinsulin is present in the secondary coating of alginate. 20. The oral insulin capsule as claimed in claims 17 and 18 wherein positively charged lipoinsulin is present in the secondary coating of alginate. 21. The oral insulin capsule as claimed in claims 17 and 18 wherein p-cyclodextrin or its derivative complexed insulin is loaded into the capsules.

Documents:

0034-mas-2002 abstract -duplicate.pdf

0034-mas-2002 claims-duplicate.pdf

0034-mas-2002 description (complete)-duplicate.pdf

0034-mas-2002 description (provisional)-duplicate.pdf

0034-mas-2002 drawings-duplicate.pdf

034-mas-2002-abstract.pdf

034-mas-2002-correspondnece-others.pdf

034-mas-2002-correspondnece-po.pdf

034-mas-2002-description(provisional).pdf

034-mas-2002-drawings.pdf

034-mas-2002-form 1.pdf

034-mas-2002-form 19.pdf

034-mas-2002-form 3.pdf

034-mas-2002-form 4.pdf

034-mas-2002-form 5.pdf