The Development And Application Of The Methods Of Screening For The Anti-diabetic Drugs

OBJECTIVE To establish drug screening methods for finding natural small molecular compounds with anti-diabetic activity, in the programs of discovering new drugs with novel mechanism or novel structure. First, a cell-based glucose uptake screening model was developed by using adipocyte cells for finding small molecular compounds stimulating glucose uptake as insulin. Second, another drug screening model based on reporter gene and the signal transduction of PPARγ system was established for finding small molecular compounds with activity of improving insulin sensitivity. Lastly, a diabetic mice model induced by ALX was prepared for confirming the hypoglycemic efffects of "hit" compounds.METHODS (1) In vitro glucose uptake screening model in the microtitre plate. The 3T3-L1 preadipocytes were induced by hormones and differentiated to mature adipocyte cells, the adipocyte cells were treated with different concentrations of insulin or 230 compounds for 48 hours, and then the glucose concentration of cells culture supernatants was detected. The activity of stimulating glucose uptake of 230 compounds was evaluated, compared with insulin; (2) Three recombinant reporter gene vectors contained different PPRE fragments were transiently co-transfected with a PPARy expression vector pCMX- PPARγ into cells respectively. The effect of rosiglitazone on the signal transduction of PPARγ was determined by examing the SEAP or Luc activity. The cell line transfected with recombinant reporter vector with the strongest induction rate was selected for further experiments. The speciality of induction was examined by the ligands of other nuclear receptors. The 63 "hit" compounds having been found in the glucose uptake experiment were further evaluated in this model; (3) T he diabetic mice model was established with different dose of ALX for 48h, the diabetic mice were treated i.g. with positive drug MET at dose of 250mg.Kg-1.d-1 for 7 consecutive days, the effect of MET on the bloodsugar in the diabetic mice was observed, to optimize the dosing scheme of ALX for further experiments. On the basis of the results of the two above models, the hypoglycemic effects of NO.51 , NO.60 were selected to further evaluate in the diabetic mice. The diabetic mice were administrated i.g. with MET and NO.51 at doses of 25> 50, lOOmg ? Kg'1 ? d'1, NO.60 at doses of 12.5> 25, 50mg ? Kg"1 ? d'1 for 5 consecutive days respectively, the blood sugar of the diabetic mice was examined. RESULTS (1) Glucose uptake were stimulated by Insulin at 0.01 ~lOOmg ? L"1 in a good dose-dependent manner. Among the 63 compounds which showed different effects on glucose uptake, NO.33, 51 > 96> 200 > 202^ 21K 212, 213 at concentration of 50mg ? L"1, and NO. 170 at concentration of 5mg ? L'1 showed strongest effect; (2) The highest of expression levels of reporter gene induced by rosiglitazone in 293T cells co-tranfected with pTK-PPRE-Luc and PPARy plasmids was up to 4.9 fold, and in a dose-dependent manner. The Z'factor was 0.72. The induction fold of Luc expression in 293T cells induced by the ligands of other nuclear receptors were about 1.0 fold. No effect of rosiglitazone on the proliferation of 293T was observed. The induction fold of Luc expression in 293T cells induced by NO.60 at dose of lmg ? L"1 was up to 2.0 fold, there was no significant difference compared with rosiglitazone at dose of lOumol ? L"1 (p>0.05); (3) Mice were treated i.v. with ALX 90mg ■ Kg"1, the increase in blood sugar were observed. The diabetic mice were treated i.g. with NO.51 sample at dose of lOOmg ■ Kg'1 ? d"1, or NO.60 sample at dose of 50mg ? Kg"1 ? d"1 for 5 consecutive days. The blood sugar of diabetic mice was significantly decreased, compared with vehicle-treated control group (p<0.01 or p<0.05); There were no significant difference compared with MET group (p>0.05).Two CONCLUTION (1) Two cellular models as the primary screening methods and one diabetic animal model were developed successfully. The glucose uptake model was characterized as the lower cost, the lower quantity of sample, the higher screening efficiency etc, except that poor stability and speciality; Another drug screening model was characterized as the single target, the preferable speciality and stability; Intravenous injection of ALX resulted in significant increase in blood sugar of up to...