FORM 2
THEPATENTS ACT, 1970
&
THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
ORAL INSULIN DELIVERY SYSTEMS FOR CONTROLLING DIABETES
RELIANCE LIFE SCIENCES PVT.LTD
an Indian Company having its Registered Office at
Dhirubhai Ambani Life Sciences Centre,
R-282, TTC Area of MIDC,
Thane Belapur Road, Rabale, Navi Mumbai - 400 701 Maharashtra India.
The following specification particularly describes and ascertains the nature of this invention and the banner in which it is performed:-

TECHNICAL FIELD
The present invention relates to the development of CR formulations containing insulin and description of its methods. More specifically, it relates to the development of a oral CR preparation of insulin encapsulated hydrogel microparticles and/or microparticles produced by microencapsulation of insulin using the newly developed copolymers, polymers, blend polymers and its method.
BACKGROUND ART
Oral administration of therapeutic agents like proteins and peptides is the most preferred means of delivering drugs compared to other routes because of ease of administration, low-cost production and high patient compliance. However, formulating such drug(s) for oral delivery is a complicated process due to the poor intrinsic protein permeability as a result of high. molecular weight, ease of degradation by proteolytic- enzymes in the stomach and in the small intestine as well as chemical instability; major hurdles in developing effective oral formulations for delivering peptides and proteins (Ramesh et al, Expert Opinion, Drug Delivery, 5, 2008, 403-415; Mundargi et al., J. Controlled Release, 125, 2008, 193-209, Emisphere Technologies, Inc., Tarrytown, NY, USA. Diabetes Technology & Therapeutics, 6, 2004, 510-517). Especially Protein/peptide denaturation/degradation can be overcome by designing suitable carriers, which would protect insulin from harsh environments of the stomach before releasing the cargo into more favorable regions of gastrointestinal (GI) tract, specifically lower region of the intestine. In order to control the release of the drug in vivo over specified period of time in order for the drug to safely and effectively exhibit its pharmacological efficacy, a sustained or controlled release of drug can be achieved by the retardation of drug diffusion by graduaf disintegration of the polymer matrix /blowing its app/ication. In case of small peptides, even if decomposition scarcely Occurs, the stability becomes an important issue. Therefore, microencapsulation process of protein-based pharmaceutical products must be free from excessive heat and shear stress, sharp changes in pH, organic solvent, excessive freezing and drying. It is possible that the microencapsulated proteins may be hydrated even during storage and proteins are prone to denaturation and aggregation under these circumstances. The polymer degrades after being administered,

thereby creating highly concentrated acidic microenvironment inside and around the polymer due to the possible decomposed acidic monomer. Under these circumstances, proteins are prone to aggregation, hydrolysis and chemical change, thereby affecting the delivery rate, leading to denaturation, aggregation or inactivation of the proteins. Particularly, insulin becomes the target of a protease and is prone to chemical, physical denaturation in a solution or a suspension (Brange et al., J. Pharm. Sci., 86, 1997, pp 517-525).
Some types of micro or nanotechnoiogy revolves around the use of hydrogels as carriers for drugs, particularly the peptide, insulin as envisaged in the work of Peppas et al., (WO 99/43615. Oct.8, 1998. ''method for oral delivery of proteins"). The principle behind this technology is to use a suitable polymer, which traps the drug such as insulin and then releases it by swelling or de-swelling mechanisms in a controlled manner within the specific tissues, thus allowing higher concentration of the peptide drug in a chosen biodegradable format. These hydrogels are very specialized systems that are generally formulated to meet the specific needs for the delivery of individual drugs. Particularly, drug release from such systems is triggered by changes in pH of the medium or temperature or magnetic field as the case may be. One example of the complexity of such systems is the glucose-sensing hydrogel used to deliver insulin to diabetic patients using an internal pH trigger. Another approach is to use pH-sensitive nature of the delivery device, to mediate the changes in swelling of the hydrogel, since a pH-sensitive hydrogel undergoes large and reversible changes in volume in response to pH changes within a biological environment.
Therefore, from what has been available in the prior art, most efforts have been concentrated to develop therapeutic strategies for optimizing oral insulin absorption and delivery using pH-sensitive hydrogels, micro or nanoparticles prepared from biodegradable polymers or a combination of biodegradable polymers with synthetic polymers, etc. Insulin has been encapsulated in these carriers to be released at specified time intervals over the required period of time. Specifically engineered delivery carriers that would transport across the biological cell membranes, including G1 tract are useful in these applications and many such systems have been proposed in the prior art, yet none of

these devices are available in the market. Insulin delivery is one of the major targets for the CR preparation that is being investigated most vigorously.
Research inventions from the various groups have proposed many CR formulations containing insulin that are capable of reducing the concentration of serum glucose levels continuously for a long time after in vivo administration. However, most insulin delivery carriers currently being used are based on the reaction between glucose-oxidase tied up into a polymer in a drug delivery system and glucose present in the blood. If the reaction between glucose and glucose-oxidase results in a decrease of pH of the microenvironment, the polymer system is swollen such that the amount of released insulin increases. The polymer system used includes that of A^'-dimethyiaminoethyl methacrylate and polyacrylamide.
Further the bioavailability of insulin after oral administration is normally low, due to its instability in the GIT, low partition coefficient and physical barrier of the intestinal epithelium. Innumerable attempts in the prior art to develop the oral insulin formulations have met with two hurdles that are to be overcome: (i) insulin transport across the mucosal barrier is restricted and (ii) insulin degradation by proteolytic enzymes of the stomach as well as intestinal lumen. Such formulations are described in detail in US patent numbers 70}$980, 5824638, 6258377, 636S6)9, PCT patent application 97/34581 and 99/43615
Wood et al., (AIChE Annual Meeting, Conference Proceedings, San Francisco, CA, United States, Nov. 12-17, 2006, American Institute of Chemical Engineers, New York) suggested that functionalizing complexation hydrogels with wheat germ agglutinin (WGA) improved the mucoadhesive properties. Katsuma. et al (Inter. J. Pharmaceutics 307, 2006, pp 156-162) demonstrated colon-specific delivery of insulin using sodium glycocholate (GC) to increase hypoglycemic effects after oral administration of insulin. Nakamura and Yakuzaigaku (Nakamura and Yakuzaigaku, 64, 2004, pp 350-353. Publisher: Nippon Yakuzai Gakkai) developed insulin base oral delivery device using

copolymer of methacrylic acid and ethylene glycol. The polymer size and ionic strength of the medium influenced the release of insulin.
Singh et al., (Indian Pat. Appl. 2008, 35 pp. IN 2006DE01437 A 20080104, Application: IN 2006-DE1437 20060616) invented hydrogel copolymer microparticles of P(MAA-co-PEGDMA), P(MAA-co-PEGDA), P(AA-co-PEGDA) and P(AA-co-PEGDMA) by copolymerization of poly(ethylene glycol) dimethacrylate (PEGDMA) and poly{ethylene glycol) aery late (PEGDA) of various molecular weights with methacrylic acid (MAA)/acrylic acid (AA), respectively.
Hassan et al in Egyptian patent (EG 2002-827 20020720) developed the CR insulin oral capsule formulations containing chitosan, hydroxypropyl cellulose, methyl cellulose, and methacrylic acid copolymers providing optimum pH for insulin release.
Development and characterization of new insulin containing polysaccharide nanoparticles is the subject of a recent invention (Sarmento et al., Colloids and Surfaces B: Biointerfaces, 53, 2006, pp 193-202). Other studies in the prior art on P(MAA-g-EG) hydrogels have dealt with their potential to bind calcium (Nakamura et. al., J. Control. Rel., 95, 2004, pp 589-599; Aragoa et. al., Eur. J. Pharm. Sci., 11, 2000, pp 333-341) to affect the proteolytic activity of calcium-dependent enzymes, such as trypsin Insulin-loaded P(MAA-g-EG) hydrogels for oral insulin delivery were studied by Morishita (J. Control. Rel., 97, 2004, pp 115-124). Other oral insulin delivery devices based on (i) biodegradable poly(glycolic acid), (PGA), poly(lactic acid), (PLA), poly(lactic acid-co-glycolic acid), (PLGA), poly(lactic acid-co-po)y(ethylene glycol), (PLA-PEG), dextran-PEG; (ii) pH-sensitive polymers like poly(acrylic acid) i.e., PAA and poly(methacrylic acid), PMAA; and (iii) complexing hydrogel graft polymers like P(MAA-g-EG) i.e., P(PAA-g-EG) in addition to biopolymers like chitosan, cyclodextrin, etc., in various combinations with methacrylic or acrylic-polymers have been reported. According to the most recent prior art (Teply, Biomaterials, 29, 2008, pp 1216-1223), a new formulation strategy was developed for prolonging the intestinal retention of protein drug giving substantial absorption. This method involved using the negatively charged PLGA

microparticles, which were subsequently mixed with the positively charged micromagnets to form stable complexes through the electrostatic interactions.
Paul and Sharma, (J. Pharma. Sci., 97, 2008, pp 875-882) attempted to load insulin in tricaicium phosphate (TCP) microspheres coated with a pH-sensitive polymer of methacrylate derivative to study the stability and conformational variations of insulin as well as their biological activity in diabetic rats. The microspheres were coated with Eudragit SI00, a pH-dependent anionic copolymer of methacrylic acid and methyl methacrylate, solubilizing above the pH of 7.4 for target delivery to large intestine.
Dave et al., (J Chromatography A, 1177, 2008, pp 282-286) developed a conjugated insulin product (IN-105) exhibiting high bioavailability wherein the recombinant human insulin was conjugated covalently with a monodisperse short chain methoxy polyethylene glycol derivative.
In another invention (Agarwal, et. al., Inter. J. Pharmaceutics, 225, 2001, pp 31-39), a coprecipitation technique was used to prepare microparticles of insulin using Eudragit L100 (polymethacrylate). The effect of variables like the addition of salts in the precipitating medium and ratio of polymeric solution to volume of precipitating medium on dissolution and encapsulation efficiency of insulin microparticles was tested. The pH-sensitive graft copolymers of poly(methacrylic acid) and poly(ethylene glycol) i.e.. P(MAA-g-EG) were invented for insulin delivery (Lowman and Peppas, Macromolecules 30, 1997, pp 4959-4965). Tuesca et al., (J. Pharma.Sci.,, 97, 2008, pp 2607-2618) developed hydrogels of poly(methacrylic acid) grafted onto poly(ethylene glycol) P(MAA-g-EG) for oral insulin release. Insulin absorption of this study was dependent on the amount of polymer as well as the concentration of insulin, giving a maximum bioavailability of 8.0 %. Polyelectrolyte complexes of cationic chitosan (CS) with anionic polymers like sodium carboxy methylcellulose, sodium alginate, PAA/PMAA, etc., have been developed for drug delivery (Hari et.aL, J. Appl. Polym. Sci., 59, 1996, pp 1795-1801; Yoshioka et. al., Biotechnol. Bioeng., 35, 1990, pp 66-72; Du et. al., J. Biomed. Mater. Res., B: Appl. Biomater. 72B, 2005, pp 299-304; Hu et.aL, Biomaterials, 23, 2002, pp 3193-3201), under mild conditions without the surfactants, organic solvents or

steric stabilizers. MAA can be polymerized in the presence of CS and PEG by optimizing polymer compositions. Damge et. al., (J. Control. Rel., 117, 2007, pp 163-170) developed nanoparticles of poly(e-caprolactone) (biodegradable) and a non-biodegradable Eudragit® RS blends that gave encapsulation efficiency of 96 % applicable for oral insulin administration wherein Damge et.al have prepared nanoparticles using poly(e-caprolactone) and Eudragit RSI00 polymers using polyvinyl alcohol) as a surfactant. In this study, poIy(e-caprolactone) is biodegradable and it takes longer time to degrade, which is not useful for the short-acting oral insulin delivery.
Thus, formulation and delivery are the most important factors in developing protein/peptide-loaded pharmaceuticals. In general, proteins or peptides have high molecular weights and a tertiary structure, on which their activity with physical property are greatly dependent. Hence, these biomolecules are easily prone to denaturation and thus, their formulation becomes important. As noted in the prior art, insulin is easily denatured during microencapsulation generating the deaminated products with approximately 50 % of loss of activity occurring in the overall protein. However, the initial burst occurring due to the partition on the surface of protein during drying of microparticles may cause hypoglycemic effects.
Since, careful consideration must be taken into the stability of the formulated products, the present invention aims to provide microparticle-based encapsulation to attain the stability of insulin contained in the formulations. Therefore, the present invention focuses on developing CR formulation of insulin that will minimize the denaturation of insulin to increase its stability during microencapsulation. In general, emulsifying a polymer matrix with a drug or protein produces microparticles. The size of microparticle, which is an important parameter to determine the internal behavior of the drug or protein, can be adjusted by selecting appropriate formulation conditions. In the present invention, different types of biodegradable or biocompatible polymers were used to develop insulin-ioaded formulations by microencapsulation. Examples of these biodegradable polymers are Eudragit L100, Eudragit RS100, and derivatives.

OBJECTIVES OF THE INVENTION:
It is the aim of the present invention to provide a novel pH sensitive copolymer.
It is the aim of the present invention to provide a pH sensitive copolymer blend of Eudragits.
It is the aim of the present invention to provide oral formulations of pH sensitive active ingredient encapsulated by the copolymer of the present invention.
It is the aim of the present invention to provide hydrogel compositions of the insulin.
It is the aim of the present invention to provide negligible release of the insulin in the acidic pH.
It is the aim of the present invention to avoid burst release of the insulin in neutral pH.
It is therefore the object of the present invention to provide increased encapsulation efficiency and optimization of insulin loading/release.
It is aim of the present invention to provide a method of microparticle preparation of insulin without any denaturation of the protein.
SUMMARY OF INVENTION
The present invention provides a novel pH sensitive biodegradable copolymer blends for microencapsulation of insulin. Examples of these biocompatible polymers Eudragit L100, Eudragit RS100, and derivatives.
The present invention also provides a Eudragit L100 and blend of Eudragit 100 and Eudragit RS100 for oral delivery of pH sensitive active ingredients.

In one embodiment, the present invention provides a method of microparticle preparation of insulin in a liquid phase which is then stored in a solid form after freeze-drying, thus avoiding protein denaturation that may occur during the course of freeze-drying.
In one embodiment, the present invention provides the composition by by using different methods such as emulsion polymerization, dispersion polymerization, solvent evaporation, in situ gel forming, precipitation method, etc. In one preferred embodiment, the present invention provides method of preparation of composition using free radical polymerization.
In one embodiment, the present invention provides compositions for the oral delivery of pH sensitive active ingredient using the copolymer of the present invention. In one embodiment the present invention provides compositions for oral delivery of insulin.
In one embodiment, the present invention provides oral formulations of pH sensitive active ingredient using copolymer of the present invention. In one preferred embodiment, the oral formulation is a hydrogel.
In one embodiment, the present invention provides hydrogel which does not release the active ingredient in acidic pH but swell in neutral pH to release the active ingredient. In one preferred embodiment the hydrogel of insulin shrinks at pH 1.2 and swells in pH 7.4 to release the insulin.
In one embodiment, the present invention provides a novel copolymer which can be used composition which does not alter the stability of the active ingredient.
BRIEF DESCRIPTION OF DRAWINGS
The following drawings form part of the present specification and are included to further
demonstrate certain aspects of the present disclosure, the inventions of which can be
better understood by reference to one or more of these drawings in combination with the
detailed description of specific embodiments presented herein.
FIG. 1 Photographs of scanning electron microscope, illustrating the group of
microparticles of Eudragit L100.
FIG. 2. In vitro release of insulin loaded Eudragit L100 in pH 1.2 and 7.4.

FIG. 3. In vitro release of Eudragit L100 coated insulin loaded Eudragit L100 tablet in pH 1.2 and 7.4.
FIG. 4. In vitro release of insulin loaded Eudragit L100- Eudragit RS100 in pH 1.2 and
7.4
FIG. 5: In vivo animal experiments: symbols: (♦) Control, (■) Insulin loaded Eudragit L100 particles (20 units/200 g) and (A) insulin loaded Eudragit L100/RS100 blend particles (20 units/200g).
FIG. 6: % of inhibition of oral insulin delivery formulations of insulin-loaded Eudragit L100 and Eudragit L100/RS100 blend particles
DESCRIPTION OF EMBODIMENTS DEFINITIONS:
The term "MAA" as used herein refers to methacrylic acid.
The term " pH sensitive copolymer" as used herein refers to EL 100
The term "acidic pH" as used herein refers to 1.2
The term "neutral pH" as used herein refers to range 7.4
The term" insulin" as used herein refers to human insulin, porcine insulin, bovine insulin as well as their analogues, such as recombinants
The term "derivatives" used herein comprises polymers having substitution of chemical groups like alkyl, alkylene, hydroxyhtion, oxidation, addition and other similar modifications made by the one skilled in the art in a conventional manner.
The present invention provides novel types of biodegradable / biocompatible polymers for the controlled release of drugs. Examples of these biodegradable polymers include Eudragit L100, Eudragit RS100.
Biodegradable polymers in general, are degraded in vivo both by enzymatic and non-enzymatic hydrolysis, surface or bulk erosion. Microparticles of the present invention prepared by microencapsulating insulin are used for the CR preparations of insulin by which pharmaceutical efficacy of insulin can be continuously retained in vivo for a long

period of time. The CR preparation of insulin may therefore include the pharmaceutical ly acceptable diluents, carriers or additives. Preferably, the CR preparation according to the present invention can be made in various forms suitable in oral delivery form, which is the most preferred route.
To this end, the present invention provides the CR formulations containing insulin by which microparticles or even nanoparticles are prepared. The insulin-loaded CR formulations according to the present invention are therefore a release device, which can help to reduce the number of administrations of insulin due to the continuous exhibition of pharmaceutical efficacy. Also, the extent of initial burst of insulin can be controlled, minimized or completely stopped to prevent the sharp decrease in serum glucose concentration.
Biodegradable polymers used for microencapsulation differ in decompositions depending on their physical properties or compositions thereof, so a considerable time is required for their complete decomposition. Thus, if a microencapsulated formulation using a biodegradable polymer as a carrier is continuously administered, the accumulation of polymer in the living body will occur. Hence, understanding of their toxicity aspects is of great importance. This problem becomes important particularly in case of polymers that have not been identified or classified as completely biodegradable.
Generally, micro or nanoparticles of this study can be freeze-dried for storage purpose and dispersed prior to their characterization or actual usage. However, in case of protein-based pharmaceutical products, denaturation may occur due to the surface partition of the microparticles caused possibly during the process of drying. Among peptides for therapeutic purposes, insulin becomes a target of a protease and is prone to chemical, physical denaturation in a solution or suspension (Brange, et al., J. Pharm. Sci., 86, 1997, pp 517-525). Therefore, consideration must be taken in to the stability of pharmaceutical products in view of the formulation as well. Thus, in order to facilitate insulin administration and to prevent the initial burst, the present invention provides a method of preserving the formulation by freeze-drying of the encapsulated product. The other

objects and advantages of the invention will be further described and these will be apparent by the detailed embodiments of the present invention. This invention relates to a microencapsulation method while attaining stability of insulin contained in formulation.
The microparicles of the present invention can be used for targeted delivery to the colon not only for local colonic pathologies to avoid systemic effects of drugs or inconvenient and painful trans-colonic administration of drugs, but also for systemic delivery of drugs like proteins and peptides, which are otherwise degraded and/or poorly absorbed in the stomach and small intestine, but may be better absorbed from the more benign environment of the colon.
As has been discussed before, hydrogels have been generally used to deliver hydrophilic. small-molecular weight drugs, which have high solubilities in both hydrophilic hydrogel matrix and surrounding aqueous media. In the matrices of the type invented herein, it is possible to achieve good encapsulation efficiency into the swollen hydrogel matrix and subsequently release the hydrophilic drug pay load into an aqueous environment
The matrices of the present invention may be potential for the treatment of diseases sensitive to circadian rhythms such as asthma, angina and arthritis. Furthermore, colon delivery of drugs that are absorbable in the colon, such as steroids, which would increase the efficiency and enable reduction of the required effective dose, can be administered using these matrices. The treatment of disorders of the large intestine, such as irritable bowel syndrome, colitis, Crohn's disease and colon disease, where it is necessary to attain a high concentration of the drug, may be efficiently achieved by colon-specific delivery using the type of matrices developed in this art. Overall, the present invention relates to a system or systems for releasing a drug or drugs specifically in the colon of the GI tract. More particularly, it relates to a colon-specific drug release system, which comprises a drug, encapsulated in an organic acid-soluble polymer material and/or polysaccharide (Mundargi et al., Drug Development and Industrial Pharmacy, 33, 2007, 1-10).

The present invention can be used for insulin delivery but can effectively used for various polypeptides, proteins and derivatives thereof that are easily degraded in the upper part of the GI tract and are absorbed in the lower part of the GI tract to exhibit their pharmacological activities. Examples of such drugs may include insulin, calcitonin, angiotensin, vasopressin, desmopressin, luteinizing hormone-releasing hormone (LH-RH), somatostatin, glucagon, oxytocin, gastrin, cyclosporin, somatomedin, secretin, human artial natriuretic peptide (h-ANP), melanocyte-stimulating hormone, (MSH), adrenocorticotropic hormone (ACTH), [3-endorphin, muramyl dipeptide, enkephalin, neurotensin, bombesin, vasoacive intestinal polypeptide (VIP), parathyroid hormone (PTH), calcitonin gene-related peptide (CGRP), cholecystokinin-8 (CK-8), thyrotropin-releasing hormone (TRH), endocerine, human growth hormone (hGH), cytokines (e.g., interleukin, interferon, colon-stimulating factor, and tumor necrosis factor), as well as derivatives thereof.
The above-mentioned peptides and proteins include not only naturally occurring substances, but pharmacologically active drug derivatives thereof and the analogues thereof (R.C. Mundargi et a!., J. Control. Rel., 125, 2008, 193-209). For example, insulin used in the present art, includes human insulin, porcine insulin, bovine insulin as well as their analogues, such as recombinants.
Drugs effective on diseases of lower part of GI tract, such as Crohn's disease, ulcerative colitis, irritable colitis, amoebiasis and colon cancer are also useful in the present invention. Examples of such drugs include: salazosulfapyridine, 5-aminosalicylic acid, cortisone acetate, triamcinolone, dexamethasone, budesonide, tegafur, budesonide. metronidazole, mesalazine, sulfasalazine, fluorouracil (A.P. Rokhade et al, J Microencap 24 (2007) 274-288) and derivatives thereof.
In addition to the above drugs of interest, the inventions of this patent will also cover physiologically active substances that can be used as the main active ingredient that is absorbed efficiently from lower part of GI tract. These include for instance, antitussive expectorants, such as theophylline (A.P. Rokhade et al, Carbohydrate Polymers 69,

2007, 678-687), vasodilators, such as nicardipine hydrochloride (K..S. Soppimath et al, Drug Dev Ind Pharm, 27, 2001, 507-515) and nifedipine (N.B Shelke and T.M. Aminabhavi, Int. J. Pharma. 345. 2007, 51-58), atenolol and carvedilol (R.C. Mundargi et al., Carbohydrate Polymers, 69, 2007, 130-141, diltiazem hydrochloride (T.M. Aminabhavi et al., Designed Monomers and Polymers, 1, 1998, 347-372), coronary vasodilators, such as iso-sorbide nitrite; antipyretic analgesics, such as acetaminophen, indomethacin (M. Sairam et al., J. App. Polym. Sci., 104, 2007, 1860-1865), hydrocortisone, ibuprofen (R.C Mundargi et al., J. Microencapsulation, 25, 2008, 228-240), salazopyrin, etc.
As confirmed from the above-described invention and examples therein, the developed oral CR formulations of insulin in which insulin was microencapsulated, can reduce the denaturation of insulin that may possibly occur during microencapsulation step as well as reduce the initial burst of insulin in a living body and thereby, preventing the risk of hypoglycemia. According to the present invention, insulin-loaded micron or submicron level formulations are suitably prepared for successful oral delivery of insulin. Further, increased encapsulation efficiency and % inhibition of insulin on fasted as well as diabetic induced rat experiments suggest the success in developing the development of the devices for oral insulin delivery in a living body. The CR formulation according to the present invention exhibits stable pharmaceutical efficacy in a living body continuously for a long period of time, it is thus possible to adjust the serum glucose concentration of a diabetic patient in a more stable and controllable manner, while reducing the number of administrations and avoiding the s.c. route injections. Further, the method developed is so simple that it can easily be scaled-up for large-scale applications.
EXAMPLES
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific

embodiments which are disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention.
EXAMPLE 1:
Insulin-loaded Eudragit L100 particles by solvent evaporation method
Solvent evaporation is a popular, simple and commercially accepted method of producing microparticles of uniform size. . The method as is well known in the prior art (R.C. Mundargi et. al. J. Control. Rel., 125, 2008, pp 193-209) involves selecting of a suitable solvent to dissolve the polymer. In the present invention, methanol was used for dissolving Eudragit L100. In the next step insulin solution prepared in 0.1 M HC1 was added. The insulin containing polymer solution was then homogenized using a high speed homogenizer. This solution was poured into light liquid paraffin oil. The total solution was stirred to evaporate the solvent to obtain microparticles of uniform size. The method was repeated several times to obtain the desired size of the final particles.
In the present invention, 500 mg of Eudragit L100 was dissolved in 5 mL of methanol under constant stirring in 50 mL beaker. To this, 20 mg of human insulin dissolved in 0.5 mL of 0.1 M HC1 was added and homogenized at 11,000 rpm for 2 min. The entire solution was transferred to beaker containing 100 mL of light liquid paraffin oil and 0.5 % of span-80 surfactant. The solution was stirred at 600 rpm for 3 h to evaporate methanol. After 3 h, the particles were filtered and washed with double distilled water to remove excess paraffin oil and span-80. The washed particles were dried at room temperature for 24 h and stored at -20°C before characterization and further analysis.
EXAMPLE 2:
Insulin-loaded Eudragit LlOO/Eudragit RS100 blend particles by solvent
evaporation method
In the present invention, 250 mg of Eudragit L100 was dissolved in 10 mL of methanol and 250 mg of Eudragit RS100 was dissolved in 10 mL of dichloromethane under constant stirring in 50 mL beaker. Both the solutions were mixed with constant stirring. To this, 20 mg of human insulin dissolved in 0.5 mL of 0.1 M HC1 was added and homogenized at 11,000 rpm for 2 min. The entire solution was transferred to a beaker

containing 100 mL of light liquid paraffin oil and 0.5 % of span-80, a surfactant. The solution was stirred at 600rpm speed for 3 h to evaporate methanol and dichloromethane. After 3 h. particles were filtered and washed with milli Q water to remove the excess paraffin oil and span-80. The washed particles were dried at room temperature for 24 h and stored at - 20°C before characterization and further analysis. Similarly insulin loaded Eudragit L 100 blend particles were prepared.
EXAMPLE 3: Characterization of the copolymer: A) Size, shape and morphology analysis
Size distribution analysis was done by laser diffraction spectroscopy using Malvern zeta sizer 3000HS (Malvern Instruments, UK). Mean diameters of the aqueous suspensions were determined in triplicate and the size distribution was represented based on the number.
For scanning electron microscopy (SEM). insulin-loaded micro and nanoparticles were mounted on metal stubs using double-sided adhesive tape, drying in a vacuum chamber, sputter-coating with a gold layer and viewing under the SEM (JSM-840, Jeol Instruments, Tokyo, Japan) to characterize shape and morphology as well as to confirm the particle size. SEM photographs are given in Fig.l. The pictures shown in Fig.6 are of Eudragit LlOO particles of sizes in the range of 1 micron or less. The SEM shows slightly smooth, spherical particles.
EXAMPLE 3: Extent of encapsulation of insulin
Insulin was encapsulated into the hydrogel during the preparation of hydrogel by free radical polymerization technique. Its encapsulation was found to be around 52 %.The encapsulation efficiency of Eudragit LlOO and Eudragit L100/RSI00 was 26 %. The lower encapsulation efficiency was because of the high loss of insulin in paraffin oil. Additionally, insulin solution was homogenized with organic solvent, which reduces the insulin entrapment into the polymeric particles.

EXAMPLE 4;
A) In vitro release of insulin-loaded particles
The in vitro release experiments were done by taking 100 mg of insulin-loaded Eudragit
L100 and Eudragit L100/RS100 blend particles in a flask containing 25 mL of buffer
solution. Particles were placed in buffer solution to observe insulin release. The
dissolution was carried out in an incubator maintained at 37°c under constant stirring at
200 rpm. At regular intervals of time, aliquot samples (2 mL each time) were withdrawn
and analyzed for insulin using the HPLC at the A.max value of 210 nm employing the
gradient method. In order to simulate the stomach and intestinal environments, all release
experiments were performed in buffer solutions of pH of \2 and 7.4. respectively. The
formulations were kept in 1.2 pH media for the first 2 h and later, in pH of 7.4 media to
follow the intestinal environment. The in vitro data of formulation prepared are
graphically displayed in Figures 2, 3 and 4.
B) In vivo efficacy of insulin-loaded particles on diabetic rats
Male Wistar rats (250 g) were housed in a 12-12 h light-dark cycle, constant temperature environment of 22°C, relative humidity of 55 and allowed free access to water and food during acclimatization. To minimize the diurnal variance of blood glucose, all experiments were performed in the morning. Diabetes was induced with intravenous injection of 150 mg/kg alloxan in saline (0.9% NaCl). Ten days after the treatment, rats with frequent urination, loss of weight and blood glucose levels higher than 300 mg/dL were included in experiments. Blood glucose levels Were determined by glucose oxidase/peroxidase method using a glucometer. A 5 % dextrose solution was given in feeding bottle for a day to over come the early hypoglycemic phase. After 72 hours blood glucose was measured by glucometer. The diabetic rats (glucose level > 300 mg/dl) were separated.
In order to investigate the effects of oral insulin-loaded panicles, 12 h fasted diabetic rats were fed with insulin-loaded particles (20 IU) or placebo particles as control. Glucose was measured on a drop of blood collected from the taj] vein before and at different

intervals up to 4 h after oral administration. Results were expressed as means ± standard deviation (SD) or means ± standard errors of means.
Group I received the saline by the intraperitoneal (IP) route. Group 2 rats has received the 20 IU of insulin-loaded particles Eudragit L100 particles dispersed in a mixture of 9 ml of 5 % carboxy methyl cellulose (CMC) and 1 ml of conc.HCI solution through the oral route using oral feeding needle. Yet another Group 3 rats has received the 20 IU of insulin-loaded particles Eudragit L100/RS100 blend particles dispersed in a mixture of 9 ml of 5 % carboxy methyl cellulose (CMC) and 1 ml of conc.HCI solution through the oral route using oral feeding needle.
C) Estimation of blood glucose level:
The pulsatom gluco-strips (stored in refrigerator) were used for estimation. The glucometer is calibrated to 660 units or as according to the specifications mentioned in the strips. The blood removed from the rat is immediately spread on the marked end of the strip. The strip is inserted in the glucometer where two electrodes are situated. After few seconds the glucometer displays the blood glucose level.
After 30 min of the above administrations, rats of all groups were orally treated with 2 g/kg of glucose. Blood samples were collected from the rat tail vein/retro orbital just prior to glucose administration i.e., 0 min and at 30, 60 and 90 min after glucose loading. Blood glucose levels were measured immediately using a glucometer.
Figure 4 represents in vivo efficacy (oral glucose tolerance test) of insulin loaded Eudragit L100 and Eudragit RS100. In this, insulin-loaded Eudragit L100 particles administered by oral route gave glucose drop from 455 to 62 with 86% inhibition where as insulin loaded Eudragit L100 and RS100 blend particles administered by oral route gave glucose drop from 319 to 258. with 42 % inhibition are given in Fig.5.

D) Storage data:
The insulin loaded Eudragit Ll00 and Eudragit L100/RS100 microparticles are prepared are stable up to 3 months. There was no change in the color or anv omer physical characteristics.
E) Release profile with respect to pH
For instance, at pH of 1.2, the hydrogel shrinks, while at PH of 7.4. the hydrogel swells to release the insulin. At pH of 1.2, Eudragit L100 has released 9% of insulin (Fig.2) but to minimize this quantity of insulin release in pH 1.2, we nave made tne insulin loaded particles in to tablet form using polyvinylpyrrolidone magnesium stearate and microcrystalline cellulose. Later on this tablet was coated with 5% of eudragit L100 solution in isopropylaclcohol, where the insulin release in pH 1.2 is totally minimized for
F) Release profile with respect to time.
The release profile of hydrogel changes with time and pH. the in vitro release was carried out both in pH 1.2 and 7.4. The release starts from 3rd hour i.e., in pH 7.4 but not in the first 2 hours i.e., in pH 1.2. The maximum amount of insulin is released in about 6 hours. In case of Eudragit L100, a 9 % of insulin was released at pH 1.2, while burst release was observed at pH 7.4 (see Figure 2). However. blending of Eudragit RS 100 with Eudragit L100 prevented the insulin release at pH 1.2 completely (see Figure 4).

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Dated this 31st day of March, 2009
For Reliance Life Sciences Pvt. Ltd