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. 2019 Sep 26;2(6):453–467. doi: 10.1021/acsptsci.9b00065

Figure 6.

Figure 6

Ralaniten is a substrate for O-glucuronidation by UGT2B enzymes. (A) Chemical structures of ralaniten, EPI-045, and major glucuronide of ralaniten M15 (EPI-002053). Potential sites of glucuronidation are shown in red. (B) HPLC chromatograms show glucuronidated products following incubation in the presence of UDPGA cofactor. While some minor peaks are observed in samples incubated with EPI-045 (labeled 045-G), the intensity is substantially lower than that seen in those incubated with ralaniten. Cotreatment with β-glucuronidase is sufficient to remove glucuronides from substrate, and thus only peaks indicating unbound drugs are seen. (C) Detection of ralaniten metabolites using LC/MS following incubation with human hepatocytes. Direct glucuronidation of ralaniten represents the most prevalent metabolite (M15). Another notable metabolite, M4, represents dechlorination followed by cysteine conjugation. (D) LC–MS chromatographs showing ralaniten metabolites isolated from pooled human plasma samples (ESSA Pharma, Inc.). Patients were treated with 3600 mg of ralaniten acetate daily. M15 represents direct glucuronidation, M19 is an oxidized metabolite. (E) Dose response curves measuring androgen-dependent growth of LNCaP cells treated with escalating concentrations of ralaniten or EPI-002053 (synthetic ralaniten glucuronide structurally equivalent to M15) and stimulated with 0.1 nM R1881. IC50 values were calculated using a linear regression and interpolation/extrapolation where the line crossed 50% growth. RAL, 14.78 μM; EPI-002053, >35 μM; p = 0.007. Data presented as mean ± SEM and analyzed by two-way ANOVA with Sidak’s test applied post hoc, n = 4 independent experiments. (F) Transcript levels of AR, PSA, FKBP5, and RHOU normalized to SDHA harvested from LNCaP cells treated with RAL (35 μM), EPI-002053 (35 μM) or v/v DMSO vehicle and stimulated with 1 nM R1881. Data presented as mean ± SEM and analyzed by one-way ANOVA with Dunnett’s test applied post hoc, n = 4 independent experiments. (G) Protein levels of AR and AR regulated genes from whole cell lysates (LNCaP) treated as in part F. *p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.0001; n.s., not significant. RAL, ralaniten.