Kinase experiment:

The purified PPAR_ LBD protein is diluted to 12 mg/mL and 1mM of Indeglitazar and 2x molar excess of steroid receptor coactivator-1 (SRC-1) peptide are added before crystallization by mixing equal volumes of a protein/compound sample with reservoir solution containing 27% polyethylene glycol (PEG) 4000, 0.1 M 2-(bis-(2-hydroxy-ethyl)-amino)-2-hydroxymethyl- propane-1,3-diol (BisTris) buffer at pH 6.5, 0.2 M ammonium acetate, and 5% glycerol. The crystals are soaked in cryo-protective buffer (30% PEG 4000, 0.1 M BisTris buffer at pH 6.5, 0.2 M ammonium acetate, and 5% glycerol) before flash-freezing in liquid nitrogen for data collection[1].

Animal experiment:

Rats[1]Indeglitazar is administered once daily i.v. as a solution (10% SolutolHS15,10%ethanol,80%saline) to ZDF/GmiCrl-fa/fa rats. Treatment is initiated at age 7-8 weeks, and blood samples are analyzed before the treatment and 21 days after the treatment.Mice[1]Ob/ob mouse study. Indeglitazar (10 mg/kg) or Pioglitazone (30 mg/kg) are orally administered to 9-week-old B6.V-Lepob mice for 14 days. Compounds are suspected in 0.5% methylcellulose and 2% Tween 80 before dosing. On the last day, blood is collected for insulin, triglyceride, free fatty acid, and adiponectin measurements.

Indeglitazar is an orally available peroxisome proliferator-activated receptor (PPAR) pan-agonist for all three PPAR subtypes alpha (α), delta (δ) and gamma (γ).

In an assay of preadipocyte differentiation, measuring in part functional insulin sensitization capability of the cells, Indeglitazar shows an EC50 of 0.32 μM compared with Rosiglitazone, which shows an EC50 of 13 nM, although the maximal response obtained from the 2 compounds is comparable[1].

An initial assessment of in vivo activity is carried out using the Zucker rat model of diabetes. The significant lowering of glucose, HbA1C, triglycerides, and total cholesterol are observed after i.v. treatment with 10 mg/kg Indeglitazar once per day for 3 weeks. Notably, the level of Adiponectin (on day 21) is essentially unchanged in treated vs. untreated animals (4.8 mcg/mL vs. 4.9 mcg/mL), thus the observed reductions in glucose and HbA1C are achieved in an adiponectin-independent fashion. These differences in the effects of Indeglitazar in vivo may be a consequence of synergy between the 3 PPAR activities or because of the SPPARM profile of the compound, or a combination of these factors. The oral activity of Indeglitazar is assessed in the ob/ob model of diabetes and insulin resistance. Indeglitazar significantly decreases glucose, insulin, triglycerides, and free fatty acid levels[1].

[1]. Artis DR, et al. Scaffold-based discovery of Indeglitazar, a PPAR pan-active anti-diabetic agent. Proc Natl Acad Sci U S A. 2009 Jan 6;106(1):262-7.