(M1330-03-19) Development and Validation of a UPLC Assay for Quantification of a Novel Anti-CRPC Agent PC257 for Preformulation and In Vitro Metabolism Studies

Purpose: PC257 is a first-in-class novel compound that specifically targets the FKBP52 cochaperone to treat castration-resistant prostate cancer (CRPC). ^{(Cox et al.)} The promise of treatment capability by PC257 is exhibited through the inhibition behaviors observed towards androgen, glucocorticoid, and progesterone receptors (AR, GR, PR). ^{(Cox et al.)} Preliminary studies showed that PC257 was insoluble in aqueous solution and showed sparse solubility in organic solvents. The purpose of this study is to develop an analytical method for PC257 quantification.

Methods: A Waters Acquity UPLC system was used for this analysis. The compound was isolated using a Waters BEH C₁₈ column (2.1 x 50 mm, 1.7 µm) with water (A) and acetonitrile (B) as the mobile phases. Prednisone was used as an internal standard (IS). The method was validated according to U.S. Food and Drug Administration (FDA) Bioanalytical Guidelines for Industry. The method was applied in solubility, stability, in vitro metabolism studies, as well as permeability determination using Caco-2 cell culture model.

Results: The results showed that the assay was linear from 0.5208 μg/mL to 200 μg/mL (R² = 0.999). Intra-day and inter-day accuracy and precision fell within the ±15% allowance outlined by the FDA guidance. pH solubility study showed ≤ 250 µg/mL solubility of PC257 across the entire range of buffers. The most stable environments were pH 4.0 and 5.0 buffers, where ≤10% degradation occurred over 120 hours at 7.6 ug/mL. Stability was maintained in acidic and neutral environments for at least 48 hours. The basic pH buffer solutions (9.0,10.0,11.0) showed approximately 40% degradation after 24 hours. The phase one metabolism study parent peak for PC257 elutes at 2.320 mins and starting concentration at 0.5 hours was 20 µg/mL and decreased to 0.5208 µg/mL at hour six in the preliminary phase on metabolism study. Three metabolite peaks were observed in the 0.5 and 6-hour time point samples with the metabolite concentrations at ≤ 2.5 µg/mL. Retention times were 2.087, 2.133, 2.244 mins, respectively. Caco-2 study showed that efflux ratio of PC257 is higher than 2, suggesting efflux transporter(s) may be involved. Further studies using specific chemical inhibitors showed that PC257 is a substrate of Pgp, suggesting that efflux might be a factor affecting its oral bioavailability.

Conclusion: Validation studies show that the method developed for PC257 quantitation to be robust and reliable. Validation stability studies reveal the compound to be stable in various environments. pH solubility studies were on trend with early preformulation studies. Oral bioavailability of PC257 might present challenges due to efflux and metabolism as evidenced in the in vitro studies.

References: 1. Marc B. Cox, Artem Cherkasov, Jaideep Chaudhary. Direct Targeting of the FKBP52 Cochaperone for the Treatment of Castration-Resistant Prostate Cancer. 2020 August.
2. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine. United States Food and Drug Administration - Bioanalytical Method Validation Guidance for Industry. www.fda.gov.; 2024 December 27. Available from: https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf.

Acknowledgements: This study was funded by NIH/NIMHD grant U54MD007605 (Xie, Liang) and the Cancer Prevention & Research Institute of Texas (CPRIT) Core Facilities Support Awards (RP240430, Liang) and the. We thank Dr. Marc Cox at the University of Texas El Paso for providing compound PC257.