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many diabetics use insulin to control their glucose level, and the most available dosage form is subcutaneous injections of insulin. This dosage form is painful, therefore has decreased compliance by these patients(1,2). This problem can be solved by developing oral dosage form of insulin which is more convenient(3). But there are mainly two barriers that stand against the development of such dosage form. The first one is the degradation of insulin by digestive enzymes found in the stomach and the upper part of small intestine(3-10). The second barrier is that insulin is absorbed very slowly from the colon(4,7).
,In order to protect the insulin from inactivation by using a carrier that is responsive to the PH. This dosage form is made of insulin-containing microparticles of cross-linked copolymers of poly(methacrylic acid) which is grafted by ethylene (P(MMA-g-EG)). This system show swelling properties according to PH changes(11). In the stomach the PH is low, so the gel is complexed , and the insulin can not be released from the tiny pores of this network and therefore can remain stable. while in the intestine as the PH increases, the complex begins to disintegrate and the insulin can be released through the pores with the increased size(12).
Microparticles of P(MAA-g-EG) were prepared(13) by a free-radical bulk, suspension polymerization of methacrylic acid (MAA) and poly(ethylene glycol) (PEG) monomethacrylate (PEGMA) with PEG of molecular weight 1000.
Drug loading was accomplished by equilibrium partitioning of insulin into the P(MAA-g-EG) microparticles. Crystalline porcine insulin (10 mg, 26.9 IU/mg, Shimizu Pharmaceutical Co., Ltd., Shizuoka, Japan) was dissolved in 100 ÃL of 0.1 N HCl. The insulin solution was diluted with 19.8 mL of phosphate buffer solution (pH 7.4) and normalized with 100 ÃL of 0.1 N NaOH. The final pH of the loading solution was 7.4. Loading was accomplished by soaking 140 mg of dried P(MAA-g- EG) microparticles for 24 h in the insulin solution. The concentration of insulin in the solution was monitored over time using HPLC. The particles were then filtered using filter paper with 1 Ãm pores and washed with 100 mL of 0.1 N HCl solution to collapse the microparticles and exude the remaining buffer solution. The insulin-loaded microspheres were dried under vacuum and stored at 4 Â°C. The degree of loading was determined from HPLC analysis of the insulin concentrations of the initial solutions and the filtrate from the washings. Using this loading technique, 94 ( 9% of the insulin in the initial solution was entrapped within the polymer.19 The activity of the insulin loaded in the gels was verified using an Insulin EIA kit (Abbot Laboratories, Chicago, IL)(14).
In Vivo Studies:
Male wistar rats (200 g) were used in this study. Diabetes is made in these rats by injecting streptozotocin intraperitoneally (40 mg/kg body weight once daily for three days in a row(15).The rats with fasting glucose level of more than 250 mg/dl are regarded as diabetic. While rats with average blood glucose level of 80 mg/dl are considered healthy. These rats were fasted for 2 days before the administration of the new dosage form. The insulin loaded P(MMA-g-EG) microparticles and a solution of insulin as a control were given by the mouth using a gelatin capsule. These capsules dissolved rapidly in the stomach .Meanwhile, a 0.2 blood samples were withdrawn of time intervals 0.25, 0.5,1, 2, 4, 6, 8 hours after dosing . these blood samples were centrifuged in order to isolate the serum. Then by using an insulin EIA kit, serum insulin levels were measured by an enzyme immunoassay. Also, glucose levels in the serum were measured by using a glucose B-test kit by the glucose oxidase method. To measure the relative efficacy of these formulations, healthy and diabetic rats were injected subcutaneously and their blood glucose levels were measured in the same manner. The healthy rats were injected with 0.5, 1, and 3 IU/Kg of insulin, while the diabetic rats were injected with 0.25, 0.5 ,and 1 IU/Kg of insulin. The area under the curve was measured for each dose, and a dose dependent AUC relationship were made:
AUC=219.29 log (SC dose) +145.96 (1a)
AUC=512.64 log (SC dose) +319.76 (1b) (14).
25 IU/Kg and 50 IU/Kg of insulin were given in this dosage form in addition to a control solution of insulin(50 IU/Kg). In Figure1,First the glucose level were risen due to the stress caused by the method of administration. Then, this rise were followed by a decrease in blood glucose levels for 8 hours. This decrease proves that insulin was successfully delivered to the intestine across which it is absorbed to give its hypoglycemic effect. This reduction was greater in rats that received 50 IU/Kg of insulin which indicate that the reduction in blood glucose levels is highly dependent on the dose of insulin. Because of the PH sensitive swelling properties of the gels , it managed to keep the insulin safe from the acidity of the stomach and its digestive enzymes, and managed to finally release the insulin from the polymer due to its dissociation in response to the increased PH in the intestine. Another useful property of these gels is their relatively selective adhesion to the intestinal wall more than to the stomach lining. this selectivity is due to the difference in PH between these two regions of the GIT. This property increases the contact time of insulin with the intestinal wall to enhance its absorption(16-18). Relative efficacy of P(MMA- g- EG) carriers to S.C injection for each dosage form the AUC was obtained , and the bioavailability was expressed as the ratio of the AUC of the oral dosage to the AUC of the SC injection and these data appear in Table 1. The control solution efficacy in comparison to SC injection was less than 1% , while when the polymer microparticles were used, the efficacy has reached up to 4.22% for the gel containing 50 IU/Kg does(14).
Since the main problems in developing oral insulin dosage form is to protect it from the acidic environment of the stomach and to keep the insulin biologically active long enough to be absorbed successfully through the intestinal mucosa , so complexing P(MMA-g-EG) hydrogels are convenient for his purpose. After administration of these insulin-loaded microparticles , the blood glucose levels of both healthy and diabetic rats were decreased for at least 8 hours because insulin was absorbed from the GIT. This effect was highly dependent on the given dose, without the need for additives such as absorption enhancers or protease inhibitors(14).