Discovery of p1736, a novel antidiabetic compound that improves peripheral insulin sensitivity in mice models

Abstract

Insulin resistance is a characteristic feature of Type 2 diabetes. Insulin resistance has also been implicated in the pathogenesis of cardiovascular disease. Currently used thiazolidinedione (TZD) insulin sensitizers although effective, have adverse side effects of weight gain, fluid retention and heart failure. Using fat cell-based phenotypic drug discovery approach we identified P1736, a novel antidiabetic molecule that has completed Phase II clinical trials. The present study evaluated the in vitro and in vivo pharmacological properties of P1736. P1736 is a non-TZD and it did not activate human PPAR(Peroxisome Proliferator Activated Receptor Gamma )receptors. P1736 caused dose dependent increase in glucose uptake (EC50-400 nM) in the insulin resistant 3T3 adipocytes. The compound (10 µM) induced translocation of GLUT-4 (Glucose Transporter type 4) transporters in these adipocytes while metformin (1.0mM) was inactive. In diabetic db/db mice, P1736 (150 mg/kg) was more efficacious than metformin in lowering plasma glucose (35% vs 25%) and triglyceride levels (38% vs 31%). P1736 tested at 5mg/kg, twice daily doses, reduced glucose by 41% and triglycerides by 32%, in db/db mice. These effects were not associated with adverse effects on body weight or liver function. Rosiglitazone (5mg/kg, twice daily) caused 60% and 40 % decreases in glucose and triglyceride levels, respectively. However, rosiglitazone induced 13% weight gain (p<0.05) in db/db mice. P1736 was also efficacious in ob/ob mice wherein 30-35% decrease in glucose and significant improvement in hyperinsulinemia were observed. Administration of P1736 to ob/ob mice resulted in 70% increase in glucose uptake in soleus muscles while metformin caused 38% increase. P1736 exhibited excellent safety profile and was weight neutral in all preclinical models of diabetes. Thus, P1736 with its unique pharmacology coupled with PPAR- independent mode of action could represent an alternative option in the management of insulin resistant Type 2 diabetic patients.

Figure 1. P1736 acts as a potent in vitro insulin sensitizer.

a, Chemical structure of P1736. b, Insulin stimulated glucose uptake in normal adipocytes(empty bar), insulin resistant adipocytes(solid bar) and insulin resistant adipocytes treated with rosiglitazone (bar with stripes). c, Glucose uptake in the dexamethasone - induced insulin resistant adipocytes exposed to vehicle(empty bar) or P1736 (10 µM, solid bar). d, Glucose uptake in the dexamethasone-induced insulin resistant adipocytes exposed to vehicle (empty bar) or metformin (1.0mM, bar with stripes). e, Dose response of P1736 on glucose uptake in dexamethasone - induced insulin resistant adipocytes. Data are expressed as Mean ± SEM. Difference between vehicle and treatment groups were statistically evaluated by t-test., ^#p<0.01 compared to normal adipocytes, **p<0.01 compared to insulin resistant adipocytes (vehicle).

Figure 2. P1736 does not activate human Peroxisome Proliferator Activated Receptors (PPAR).

a, P1736 was added to HEK-293 cells expressing ligand binding domain of human PPARα receptor and the transactivation ability was assayed using renilla luciferase activity. Fenofibrate was used as the positive control. b, P1736 was added to HEK-293 cells expressing ligand binding domain of human PPARγ receptor and the transactivation ability was assayed using rosiglitazone as the positive control.

Figure 3. P1736 reduces circulating glucose and triglyceride levels in diabetic db/dbmice.

a, Plasma glucose levels in male db/db mice that received once daily oral doses of vehicle(empty bar) or P1736 (150 mg/kg, solid bar) or metformin (150 mg/kg, bar with stripes) for 15 days. b, Plasma triglyceride levels of db/db mice that received vehicle(empty bar) or P1736 (150 mg/kg, solid bar) or metformin (150 mg/kg, bar with stripes) for 15 days. c, Plasma glucose and d, Plasma triglyceride levels of male db/db mice that received vehicle(empty bar) or P1736 (5 mg/kg twice daily, solid bars) or equal dose of rosiglitazone (bar with dots). e, Plasma glucose levels of male db/db mice with high initial plasma glucose, that received vehicle(empty bar) orP1736 (150 mg/kg once daily, solid bar) or metformin (150 mg/kg once daily, bar with stripes) for 10 days. Data are expressed as Mean ± SEM for 7 - 10 animals per treatment group. Differences between vehicle and compound treated groups were statistically evaluated by t-test. *p<0.05, **p<0.01, ***p<0.001 vs.vehicle.

Figure 4. P1736 lowers plasma glucose and insulin levels in insulin resistant and obese ob/ob mice.

a, Plasma glucose levels of male ob/ob mice that received oral doses of either vehicle (empty bar) or P1736 (100 mg/kg, 200 mg/kg, solid bars) measured after 10 days. b, Change in plasma insulin levels of ob/ob mice which received vehicle(empty bar) or P1736 (5 mg/kg, 100 mg/kg, solid bars) or rosiglitazone (5 mg/kg, bar with stripes) or metformin (150 mg/kg, dotted bars) for 10 days. Data are expressed in Mean ± SEM for 8 -12 animals per treatment group. Differences between vehicle and treatment groups were statistically evaluated by t-test *p<0.05, **p<0.01, ***p<0.001 vs. vehicle.

Figure 5. P1736 promotes glucose uptake by modulating insulin signaling pathways.

a, Effect of P1736 on PI3 - Kinase mediated glucose uptake in insulin resistant 3T3 adipocytes. Insulin resistant adipocytes were treated with P1736 (10 μM) or rosiglitazone (0.1 μM) as mentioned in methods. At the end of 4 days, cells were treated with vehicle (empty bar) or 100 nM wortmannin (solid bar) for 30 minutes. Later 200nmol/l insulin stimulated 2-deoxyglucose uptake was determined. b, Effect of P1736 on GLUT-4 translocation in insulin resistant adipocytes. Insulin resistant 3T3 adipocytes were treated with vehicle or P1736 (10 μM) or Metformin (1 mM) or rosiglitazone (0.1 μM) for 4 days. Later the cells were stimulated with 200nmol/l insulin for 25 minutes. Whole cell immunofluorescence assay for GLUT-4 translocation was performed using rabbit antibody against GLUT - 4 followed by incubation with FITC labeled secondary antibody. Confocal images were obtained using Bio-Rad Laser Scanning Microscope.

Figure 6. P1736 did not adversely affect body weight in preclinical diabetic models.

a, Body weights of male db/db mice treated with vehicle (empty diamonds) or P1736 (150mg/kg, solid square) or metformin (solid triangle) for 15 days. Body weight was measured daily and recorded. b, Change in the weights of db/db mice treated with vehicle(empty diamonds) or P1736 (5 mg/kg twice daily, solid squares) or rosiglitazone (5 mg/kg, solid triangles) for 15 days. Data are expressed as Mean ± SEM for 7 - 10 animals per treatment group. * p< 0.05 compared to vehicle.