Kinase experiment:

Ligand binding to bacterially expressed ligand binding domain (LBD) of hPPAR-γ is determined by scintillation proximity assay (SPA). The assay measures the ability of putative ligands to displace receptor bound [3H]BRL 49653. Assays are conducted in 96-well plates. Wells contained varying concentrations of GW1929 or troglitazone; streptavidin-modified SPA beads to which biotinylates PPAR-γ LBD is prebound; and 10 nM of the specific radioligand [3H]BRL 49653 in a volume of 100 μL. The amount of nonspecific binding, as assessed by control wells that contained 50 μM of the corresponding unlabeled ligand, is subtracted from each data point. For each compound tested, plots of ligand concentration versus counts/min of radioligand bound are constructed, and apparent Ki values are estimated from a nonlinear least squares fit of the data, assuming simple competitive binding. The results are expressed as pKi, where pKi = -log10(KI)[1].

Cell experiment:

For the experiments, the cells are plated in 96-well plates at a density 2 × 105 cells per cm2 and cultured in the presence of TBBPA, in a concentrations range from 1 nM to 100 μM TBBPA. TBBPA is dissolved in DMSO, resulting in a final vehicle concentration of 0.1 % (v/v). Control (no vehicle) and DMSO-treated wells are included in the experimental design to determine the effect of DMSO. To study whether PPAR-γ is involved in the neurotoxic effect of TBBPA, cells are co-treated with 10 μM TBBPA and 10 μM GW1929 or GW9662. After 6 or 24 h of culture, 100 μL medium is collected for the LDH analysis, and the cells are collected and frozen at –70℃ for the caspase-3 activity measurements[2].

Animal experiment:

Animals are housed at 72°F and 50% relative humidity with a 12-h light and dark cycle, and fed Formulab Diet 5008. Age- (60-day) and glucose-matched male Zucker diabetic fatty rats are gavaged twice daily for 14 days with vehicle (0.05 M N-methylglucamine), GW1929 (0.5, 1.0, or 5.0 mg/kg), or troglitazone (as the milled extrudate, in a suspension in methylcellulose, 50, 150, and 500 mg/kg). Another group of animals receives a mixture of Humulin N and Humulin R by subcutaneous injection twice daily. On days 7 and 14 of dosing, nonfasted measurements of glucose, lactate, insuline, total cholesterol, TGs, F FAs, and hematocrit are obtained. On day 14 of dosing, samples for serum drug levels (2-h postdose) and glycosylated hemoglobin measurements are also collected. In addition, once weekly, three animals from each group are placed in metabolic chambers for 48 h for quantitation of 24-h food and water consumption. Body weights are recorded throughout the study. At the conclusion of the study, perfused pancreas experiments are performed on 12 animals (n = 4 per group) that have received either GW1929 (1 and 5 mg/kg) or vehicle, to directly evaluate the effects of treatment on basal and glucose-stimulated insuline secretion. The remaining animals are killed, and their pancreases are processed for immunocytochemistry[1].

IC50: 5.0 μM

GW1929 is a nonthiazolidinedione PPARγ agonist. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors with three isoforms: PPARα, PPARγ, and PPARβ/δ. Among these, PPARγ is highly expressed in various brain regions. A growing evidences have suggested that PPARγ play a key role in pathogenesis of cerebral IR injury

In vitro: GW1929 was tested on currents through L-type voltage-dependent calcium channels (VDCC) in freshly isolated smooth muscle cells. Using Ba2+ as the charge carrier through VDCC, the IC50s for GW1929 and pioglitazone were determined to be 5.0 +/- 0.7 and 10.0 +/- 0.8 μM, respectively. GW1929 and pioglitazone were both effective on inhibiting VDCC and relaxing pressurized arteries, indicating the vasodilation of resistance arteries might be explained by the inhibition of calcium entry through VDCC [1].

In vivo: GW1929 treatment attenuated the neurological damage in focal cerebral IR injury significantly. In addition, the neuroprotective effects of GW1929 were found to be associated with significant reduction in the levels of MMP-9, COX-2, iNOS, TNFα and IL-6. Neuroprotective effects of GW1929 related with significant reduction in TUNEL positive cells in IR challenged brain [2].

Clinical trial: N/A

References:
[1] Heppner TJ,Bonev AD,Eckman DM,Gomez MF,Petkov GV,Nelson MT. Novel PPARgamma agonists GI 262570, GW 7845, GW 1929, and pioglitazone decrease calcium channel function and myogenic tone in rat mesenteric arteries. Pharmacology.2005 Jan;73(1):15-22.
[2] Kaundal RK,Sharma SS.  Ameliorative effects of GW1929, a nonthiazolidinedione PPARγ agonist, on inflammation and apoptosis in focal cerebral ischemic-reperfusion injury. Curr Neurovasc Res.2011 Aug 1;8(3):236-45.