Screening Methods for Antidiabetic Agents

SCREENING METHODS FOR ANTIDIABETIC AGENTS

Diabetes mellitus is a metabolic disorder characterized by increased blood glucose level associated with discharge of glucose in urine. There are two major types of diabetes mellitus, that is, insulin-dependent diabetes mellitus (IDDM) and noninsulin-dependent diabetes mellitus (NIDDM). Insulin-dependent diabetes mellitus, also called type 1 diabetes, occurs due to complete loss of pancreatic β-islet cells and hence there is insulin deficiency. Noninsulin-dependent diabetes mellitus, also called as type 2 diabetes, is due to insulin resistance. Insulin resistance is developed due to defects at the receptor level or insulin signalling at the postreceptor level. This defect may be in the effector cells such as the skeletal muscle, the adipose tissue etc., or in the β-islet cells. A large number of drugs including herbs and minerals with suspected antidiabetic activity have been successfully tested in the laboratory. The various animal models to screen antidiabetic activity are listed in this section.

Models for Insulin-Dependent Diabetes Mellitus

Alloxan-induced diabetes

Alloxan is a cyclic urea compound which induces per-manent diabetes. It is a highly reactive molecule which produces free radical damage to β-islet cells and causes cell death. Alloxan at a dose level of 100 mg/kg in rats produces diabetes. In rabbits, a dose level of 150 mg/kg infused through a marginal ear vein produces diabetes in 70% of the animals.

Albino rats of either sex weighing 150–200 g are injected with a single dose of alloxan monohydrate (100 mg/kg body weight) dissolved in normal saline (0.9%) by intraperitoneal route. The animals are kept for 48 h during which food and water is allowed ad libitum. The blood glucose level shows the triphasic response with hyperglycaemia for 1 h followed by hypoglycaemia that lasts for 6 h and stable hyperglycaemia after 48 h. The animals showing fasting blood glucose level above 140 mg/dl after 48 h of alloxan administration are considered diabetic. Drug samples to be screened are administered orally for a period of six weeks. After six weeks of treatment, blood samples are collected from 8 h fasting animals through a caudal vein. Serum is separated by cooling centrifuge (2–4°C) at 3000 r.p.m. for 10 min. The serum glucose level is estimated by glucose oxidase-peroxidase method (GOD-POD kit) using an autoanalyser.

Streptozotocin-induced diabetes

Streptozotocin is a broad-spectrum antibiotic which causes β-islet cells damage by free radical generation. Strepto-zotocin induces diabetes in almost all species of animals excluding rabbits and guinea pigs. The diabetogenic dose of Streptozotocin varies with species. In mice, the dose level is 200 mg/kg through i.p. and in beagle dogs 15 mg/ kg through i.v. for three days. Adult albino rats of either sex weighing 150–200 g are injected with Streptozotocin (60 mg/kg body weight) prepared in citrated buffer (pH 4.5) solution by i.p. route. The citrated buffer is prepared by mixing 53.9 parts of 0.1 M citric acid and 46.1 parts of 0.2 M disodium hydrogen orthophosphate and finally adjusted to a pH of 4.5. The blood glucose level shows the same triphasic response as seen in alloxan-treated animals. Animals showing fasting blood glucose level above 140 mg/ dl after 48 h of Streptozotocin administration are considered diabetic. Drug samples to be screened are administered orally for a period of six weeks. After six weeks of treat-ment, blood samples are collected from 8 h fasted animals through a caudal vein. Serum is separated by cooling centrifuge (2–4°C) at 3000 r.p.m. for 10 min. The serum glucose level is estimated by glucose oxidase-peroxidase method (GOD-POD kit) using an autoanalyser.

Virus-induced diabetes

Viruses are one of the etiological agents for IDDM. They produce diabetes mellitus by infecting and destroying β-islet cells of pancreas. Various human viruses used for inducing diabetes include: RNA picornovirus, coxsackie B4 (CB-4), and encephalomyocarditis (EMC-D).

Six- to eight-week-old mice are inoculated by 0.1 ml of 1:50 dilution of D-variant encephalomyocarditis (EMC) through i.p. route. The 0.1 ml of the above dilution contains 50 PFU (plaque-forming units) of EMC virus. Mortality due to this concentration of virus is approximately 10–20%. A less-infecting variant produces a comparable damage by eliciting autoimmune reactivity to the β-islet cells. Infected animals are considered hyperglycaemic if their nonfasting levels exceed by 250 mg/dl the levels of uninfected animals of the same strain. Drug samples to be screened are adminis-tered orally for a period of six weeks. After six weeks of drug treatment, blood glucose estimation is done to determine the antidiabetic activity.

Insulin antibodies-induced diabetes

A transient diabetic syndrome can be induced by inject-ing guinea pigs with antiinsulin serum. It neutralises the endogenous insulin with insulin antibodies. Diabetes per-sists as long as the antibodies are capable of reacting with the insulin remaining in circulation.

Preparation of Antibody: Bovine insulin, dissolved in acidified water (pH 3.0) at a dose of 1 mg is injected to guinea pigs weighing 300–400 g. Antiinsulin sera is col-lected after two weeks of antigenic challenge.

Adult albino rats are injected with 0.25–1.0 ml of guinea pig antiinsulin serum. Insulin antibodies induce a dose-dependent increase of blood glucose level up to 300 mg/ dl. Slow rate intravenous infusion or an intra-peritoneal injection prolongs the effect for more than a few hours. However, large doses and prolonged administration are accompanied by ketonemia. The drug sample to be screened is administered by a suitable route, and blood glucose level is analysed to determine the activity.

Hormone-induced diabetes

Dexamethasone: Dexamethasone is a steroid possessing immunosupression action which causes an autoimmune reaction in the islets and produces type 1 diabetes.

Adult rats weighing 150–200 g are injected with dex-amethasone at a dose level of 2 5 mg/kg body weight by i.p. twice a day. The repeated injection of the same dose level is carried out for a period of 20–30 days resulting in IDDM. The sample to be screened is administered through a suitable route, and blood glucose level is analysed to determine the activity.

Genetic Models

Nonobese diabetic mouse

Nonobese diabetic mouse (NOD) is a model of IDDM. Hypoinsulinemia is developed which is caused by autoim-mune destruction of pancreatic β-islet cells in association with autoantibody production.

Mice are breed at the laboratory by sib-mating over 20 generations. After 20 generations of sib-mating, spontane-ous development of IDDM in mice is obtained. Diabetes develops abruptly between 100 and 200 days of age. Weight loss, polyurea, and severe glucosuria are common. The animals are treated with the drug sample to be screened. Blood sample is analysed for glucose level to determine activity.

Bio-breeding rat

Diabetes is inherited as an autosomal recessive trait and develops with equal frequency and severity among males and females. Insulin deficiency and insulitis are due to autoimmune destruction of pancreatic β-islet cells. Spon-taneous diabetes is diagnosed in a noninbreed but closed out-breed colony of rats at bio-breeding laboratories.

Rats are breed at the laboratory by sib-mating over 20 generations. After 20 generations of sib-mating, spontaneous development of IDDM in rats is obtained. The onset of clinical diabetes is sudden and occurs at about 60–20 days of age. The clinical presentation of diabetes in the bio-breeding

A rat is similar to that of its human counterpart. Marked hyperglycaemia, glycosuria, and weight loss occur within a day of onset and are associated with decreased plasma insulin, that if untreated will result in ketoacidosis. The animals are treated with the drug sample to be screened for a required period of time. The blood sample is analysed for glucose level to determine activity.

Models for NIDDM

Streptozotocin-induced neonatal model for NIDDM

Streptozotocin causes severe pancreatic β-cells destruction, accompanied by a decrease in pancreatic insulin stores and a rise in plasma glucose level. In contrast to adult rats, the treated neonates partially regenerate and become normo-glycaemic by three weeks of age. In the next few weeks, the β-cell number increase, mainly from the proliferation of cells derived from ducts, leads to hyperinsulinemia, and shows symptoms similar to insulin resistance.

Neonatal rats are treated with Streptozotocin (90 mg/kg body weight) prepared in citrated buffer (pH 4.5) by i.p. at birth or within the first 5 days following birth. After six weeks, the rats develop symptoms similar to NIDDM. Rats showing fasting blood glucose level above 140 mg/dl are considered diabetic. Further steps are similar to that of the alloxan-induced model. The drug sample to be screened is administered by a suitable route, and blood glucose level is analysed to determine the activity.

Other Chemically Induced NIDDM Models

Adrenaline-induced acute hyperglycaemia

Adrenaline is a counter-regulatory hormone to insulin. It increases the rate of glycogenolysis and glucose level in blood causing acute hyperglycaemia.

Adult albino rats are injected at a dose level of 0.1 mg/ kg through s.c. route. The dose produces peak hyperglycaemic effect at 1 h and lasts up to 4 h. The drug sample to be analysed is administered through a suitable route, and blood glucose level is determined. Oral hypoglycaemic agents can be screened by this method.

Chelating Agents

Dithizone-induced diabetes

Organic agents react with zinc in the islets of Langerhans causing the destruction of β-islet cells, producing diabetes. Severe necrosis and disintegration of β-cells (insulin-producing cells) were observed, while α-cells (cells which produce glucoagon which maintains the glucose level in the blood) remain unaltered. Compounds such as dithizone, EDTA, 8-hydroxy quinoline are used to induce spontane-ous type 2 diabetes in experimental animals. Dithizone at a dose level of 40–100 mg/kg (i.v.) produces type 2 diabetes in mice, cats, rabbits, and golden hamsters.

Adult rabbits weighing 1.8–2 kg are divided into two groups of six animals each. An exactly weighed amount of dithizone is dissolved in dilute ammoniacal solution (0.2 to 0.5%). The solution is warmed to 60 -70°C for 10 min to aid solubility of dithizone. Dithizone injection at a dose level of 50–200 mg/kg produces triphasic glycaemic reaction. Initial hyperglycaemia is observed after 2 h and normoglycaemia after 8 h, which persists for up to 24 h. Permanent hyperglycaemia is observed after 24–72 h. The drug sample to be analysed is administered through a suit-able route, and the blood glucose level is determined.

Models for Insulin Sensitivity and Insulin-like Activity

Euglycaemic clamp technique

This method has proved to be a useful technique of quantifying in vivo insulin sensitivity. A variable glucose infusion is delivered to maintain euglycemia during insulin infusion. The net glucose uptake is quantified, and the sensitivity of the body tissue to insulin is determined.

Adult albino rats weighing 150–200 g are fasted over-night and anaesthetized with pentobarbital (40 mg/kg i.p.). Catheters are inserted into a jugular vein and a femoral vein for blood collection and insulin and glucose infusion, respectively. To evaluate the insulin action under physiological hyperinsulinemia (steady-state plasma insulin concentration during the clamp test is around 100 μU/dl) and maximal hyperinsulinemia, two insulin infusion rates, that is, 6 and 30 mU/kg/min are used. The blood glucose concentrations are determined from samples collected at 5 min intervals during the 90-min clamp test. The glucose infusion rate is adjusted so as to maintain basal level. The glucose metabolic clearance rate is calculated by dividing the glucose infusion rate by the steady-state blood glucose concentration. The drug sample to be analysed is administered through a suitable route, and the blood glucose level is determined.

Assay for insulin and insulin-like activity

This assay involves comparing two standard solutions of insulin with the test drug for its insulin-like activity.

Four groups of six rabbits weighing at least 1.8 kg are used. Two standard solutions of insulin containing one unit and two units respectively and two dilutions of sample whose potency is being examined are prepared. As diluent, a solution of 0.1 to 0.25% w/v of either m-cresol or phenol and 1.4 to 1.8 w/v of glycerol acidified with hydrochloric acid to a pH between 2.5 and 3.5 is used. Each of the prepared solution (0.5 ml) is injected subcutaneously. After 1 h and 2.5 h of each injection, a suitable blood sample is taken from the ear vein of each rabbit, and the blood sugar level is determined preferably by glucose oxidase method.