LGD-3303

Kinase experiment:

In vitro binding is determined using recombinant baculovirus expressed human androgen receptor (hAR), human glucocorticoid receptor (hGR), human mineralocorticoid receptor (hMR), or human progesterone receptor (hPR). Tritium-labeled reference ligand is used with varying concentrations of LGD-3303 as a competing ligand. Inhibition constant (Ki) values are calculated by application of the Cheng-Prusoff equation. Reporter assays are performed. Briefly, CV1 cells are cultured in DMEM supplemented with 10% charcoal resin-stripped fetal bovine serum (FBS), and seeded 48 h before transfection in 96-well microtiter plates. Cells are transiently transfected using a nonliposomal formulation, the FuGENE 6 transfection reagent, with luciferase reporter plasmids MMTV-LUC or MTV-ERE5-LUC, a β-galactosidase (β-Gal) expression plasmid coding for the constitutive expression of Escherichia coli β-galactosidase, and hAR, hGR, hMR, hPR, or human estrogen receptor α (pRShERα) expression plasmids. Cells are treated with varying concentrations of LGD-3303 or reference compound for 40 h. The normalized luciferase response is calculated as relative luciferase units/(β-gal O.D415/β-Gal incubation time in minutes). The effective concentration that produces 50% of the maximum response (EC50) is determined, and agonist efficacy is calculated as a percent of normalized luciferase response relative to the maximum response by the reference agonist (i.e., DHT for hAR, dexamethasone for hGR, aldosterone for hMR, progesterone for hPR-B, or 17β-estradiol for hERα)[1].

Animal experiment:

Rats[2]Male Sprague-Dawley rats (7-8 weeks old, 200 g) are used. Rats are sorted by weight, assigned to experimental groups (n=5/group), and surgery is performed. Experimental groups consist of LGD-3303 (doses ranged from 0.1-300 mg/kg/day) or vehicle. LGD-3303 is administered by once daily oral gavage in a volume of 4 mL/kg. On the 14th day, blood is collected into lithium heparin tubes by jugular puncture at 0, 0.5, 1, 2, 4, and 6 h postdosing from the animals in the high-dose groups. Blood is centrifuged, and plasma is stored at -20°C for pharmacokinetic analysis. On the 15th day, rats are killed by decapitation, and trunk blood is collected, allowed to clot in serum separator tubes, and centrifuged, and serum is stored at -80°C for future analysis of serum luteinizing hormone (LH) levels. The wet weights of the ventral prostate, levator ani muscle, and preputial gland are measured at necropsy[2].

References:

[1]. Vajda EG, et al. Combination treatment with a selective androgen receptor modulator q(SARM) and a bisphosphonate has additive effects in osteopenic female rats. J Bone Miner Res. 2009 Feb;24(2):231-40.
[2]. Vajda EG, et al. Pharmacokinetics and pharmacodynamics of LGD-3303 [9-chloro-2-ethyl-1-methyl-3-(2,2,2-trifluoroethyl)-3H-pyrrolo-[3,2-f]quinolin-7(6H)-one], an orally available nonsteroidal-selective androgen receptor modulator. J Pharmacol Exp Ther. 2009 Feb;328(2):663-70.