Bioanalysis of the Ex Vivo Labile PACE4 Inhibitory Peptide Ac-[ d- Leu]LLLRVK-Amba in Whole Blood Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Quantification

The calcium-dependent serine endoprotease PACE4 is evaluated as a therapeutic target for prostate cancer. The peptide Ac-[ d -Leu]LLLRVK-amba inhibits PACE4 with high affinity and has shown efficacy in preclinical mice xenograft models of prostate cancer. To support in vivo examinations of the potential therapeutic peptide Ac-[ d -Leu]LLLRVK-amba, we established a highly sensitive assay for its quantification in mouse whole blood microsamples based on UPLC-MS/MS determination. Ac-[ d -Leu]LLLRVK-amba was very labile during sample processing, which was particularly pronounced in plasma. High resolution mass spectrometric investigations of the metabolism/degradation in plasma revealed that no peptide bond hydrolysis generated products were formed, leaving the cause of the observed consumption of the peptide elusive. As a consequence, whole-blood quantification was developed relying on the immediate snap-freezing of blood samples after collection and immediate sample processing after serial thawing to ensure accurate and reliable quantification. The assay was validated according to the applicable recommendations of the FDA and EMA in a range of 10–10,000 ng/mL and applied to determine the pharmacokinetics of Ac-[ d -Leu]LLLRVK-amba after intravenous and intraperitoneal administration to mice. Individual pharmacokinetic profiles were assessed using four microsamplings per animal. Intraperitoneal absorption was found to be efficient, demonstrating that this well-manageable route of administration is feasible for preclinical efficacy experiments with Ac-[ d -Leu]LLLRVK-amba.