(M1130-03-15) Method Development and Validation of Liquid Chromatography–Tandem Mass Spectrometry for SLP-1A6 in Rat Plasma

Purpose: Prostate cancer (PCa) is a prevalent cancer in men that can progress to metastatic castration-resistant prostate cancer (mCRPC), leading to treatment challenges. While docetaxel chemotherapy can temporarily improve survival, many patients develop resistance. Recent studies reveal that the embryonic ectoderm development (EED) protein contributes to chemoresistance through interactions with enhancer of zeste homolog 2 (EZH2). Targeting EED may improve treatment outcomes and offer new drug development opportunities. Nicardipine, a dihydropyridine calcium-channel blocker, was recently repurposed as an EED inhibitor that disrupts the EED–EZH2 complex, induces EZH2 degradation, and triggers apoptosis in chemoresistant prostate cancer cells. However, its rapid clearance and off-target cardiovascular effects limit its anticancer utility. To address these drawbacks, SLP-1A6, a nicardipine-derived analog featuring a three-carbon alkyl chain modification, was synthesized to enhance EED/EZH2 selectivity, potency, and pharmacokinetic stability while retaining the parent compound’s favorable safety profile. SLP-1A6 has proven to be a specific and potent inhibitor of chemoresistant PCa cells that also reduces bone growth of chemoresistant PCa in mouse models. SLP-1A6 has also demonstrated nanomolar (3.08 nM) IC50 values in representative chemoresistant PCa cells. Herein, the aim of this study was twofold: (1) to develop and rigorously validate a sensitive, selective, and reproducible protein-precipitation LC–MS/MS assay for quantifying SLP-1A6 in rat plasma in accordance with FDA bioanalytical method validation guidelines, and (2) to apply the validated method in a single-dose pharmacokinetic (PK) study following intravenous administration in male Sprague–Dawley rats, thereby enabling informed dose selection for future efficacy and toxicity studies.

Methods: Stock solutions of SLP-1A6 (10 mg/mL in DMSO) and internal standard nicardipine (10 mg/mL in DMSO, diluted to 100 ng/mL) were prepared. Calibration standards (5, 50, 100, 500, 1000, 2500 ng/mL) and quality control (QC) samples at 10, 750, and 2000 ng/mL were spiked into blank rat plasma. Sample preparation involved mixing 20 µL plasma with 200 µL methanol containing IS, vortexing 30 s, incubating on ice for 15 min, then centrifuging at 13,000 × g (4 °C) for 15 min. Supernatants were injected (2 µL) onto an Ascentis Express C18 column (50 × 2.1 mm, 2.7 µm) on a Shimadzu LC-30AD coupled to a SCIEX API 4000. Mobile phases were 5 mM ammonium acetate (pH 4.4) (A) and methanol (B), delivered at 0.4 mL/min with a gradient: 5% B (0.5 min) → 98% B (6.5 min) → hold 0.5 min → re-equilibration (0.1 min), total run time 8 min. Detection used negative ESI MRM transitions m/z 534.2 → 121.6 for SLP-1A6 and m/z 478.1 → 121.6 for IS, with ion spray voltage −4000 V, source temp 500 °C, curtain gas 35 psi, nebulizer 40 psi, and collision energy −35 V. Validation parameters included selectivity (no interference at analyte/IS RT), linearity (r² > 0.997), accuracy and precision (±15%, ±20% at LLOQ), extraction recovery, matrix effect, carry-over ( < 0.1% LLOQ), and stability (autosampler 6 h; freeze–thaw ×3; –80 °C 14 d) .

Results: Calibration curves were linear from 5–2500 ng/mL (r² > 0.997). Intra- and inter-day accuracy ranged from –3.5% to 12.0% (precision ≤19.9% CV) at LLOQ and QC levels. Mean extraction recovery was 94.8% (±3.4%), and matrix effects were within ±12%. Carry-over in blank injections was < 0.08% of the LLOQ signal. SLP-1A6 remained stable under all tested conditions (deviation ≤15%). In the PK study (1 mg/kg IV; n = 3), plasma concentrations were quantified up to 8 h post-dose. Non-compartmental analysis yielded: t₁/₂ = 1.1 ± 0.2 h, CL = 2.4 ± 0.1 L/h/kg, Vss = 5.1 ± 1.2 L/kg, AUC₀–∞ = 411.4 ± 19.7 ng·h/mL, and mean residence time = 2.1 ± 0.4 h—values closely mirroring published nicardipine parameters (t₁/₂ 1.3 h; CL 2.8 L/h/kg; Vss 5.5 L/kg).

Conclusion: The validated LC–MS/MS assay provides a rapid (8 min/run), high-throughput, and low-volume (20 µL) method for SLP-1A6 quantification in rat plasma, fully compliant with regulatory standards. Pharmacokinetic profiling demonstrates that the three-carbon alkyl modification in SLP-1A6 maintains nicardipine-like clearance and distribution characteristics while offering enhanced target specificity, supporting its continued preclinical development as a candidate for overcoming chemoresistance in prostate cancer.

Acknowledgements: This study was funded by the NIMHD grant U54MD007605 (Xie, Liang), Cancer Prevention & Research Institute of Texas (CPRIT) Core Facilities Support Awards RP180748 and renewal RP240430 (Liang), NCI grants R01CA256058 and R42CA217491, NIMHD grant U54MD007590 (Project 1) (Wu).