(T1430-06-40) Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method Development and Pharmacokinetics of PTH-IA in Mice

Purpose: PTH-IA (parathyroid hormone receptor-inverse agonist) is a drug candidate under investigation for the treatment of Jansen’s Metaphyseal Chondrodysplasia (JMC) which is a rare disease of abnormalities in bone development and mineral ion homeostasis. The objective of the present study was to develop a rapid, accurate, and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method to quantify PTH-IA in plasma samples and to assess PTH-IA pharmacokinetic (PK) properties in mice.

Methods: Mass spectrometric analysis was performed on a Waters Xevo TQ-Absolute triple quadrupole instrument using electrospray ionization in positive ionization mode. The selected reaction monitoring (SRM) was used for mass spectrometric detection. The optimized SRM transitions were m/z 636 → 726.9 (quantifier) and 763 → 759.45 (qualifier) for PTH-IA, and m/z 637.6 → 728.9 for the stable isotope labeled internal standard (SILIS: [¹³C₉¹⁵N]-PTH-IA). The analyte was retained and separated on an Atlantis™ PREMIER BEH C₁₈ AX (1.7 µm, 2.1 x 50 mm) column using a Waters Acquity UPLC system. A 2-min gradient method was developed and optimized. The mobile phases were 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Aliquots of 40 µL plasma samples were mixed with 200 µL SILIS in 80% acetonitrile /1% formic acid solution to precipitate proteins in a 96-well plate. A 2 µL supernatant aliquot was injected for UPLC-MS/MS analysis. A single 1.52 mg/kg subcutaneous (SC) or 1.44 mg/kg intravenous (IV) injection of PTH-IA was administrated to wild-type male C57BL6 mice for the PK studies. Plasma samples were collected up to 7 h post dosing and stored at -80^oC until analysis. The pharmacokinetic parameters were calculated with the mean plasma concentration of 3 - 4 mice at each time point, using the non-compartmental method (Model 200) of Phoenix WinNonlin, version 8.3.

Results: Using the developed UPLC-MS/MS method, PTH-IA was analyzed in mouse plasma. The linear calibration range for PTH-IA was 0.5 - 1000 ng/mL and the lower limit of quantitation (LLOQ) was 0.5 ng/mL. Six replicates of QC samples at four concentration levels were prepared in control blank mouse plasma. The precision of the QC samples (CV%, n=6) ranged from 1.4 -12% and the accuracy was between 91% and 112% of the nominal values. No endogenous interference was found at the retention time for PTH-IA and SILIS, as verified by testing control blank plasma. PTH-IA was stable at room temperature and 4^oC for at least 1 hr and 20 hr, respectively, in mouse plasma. The developed UPLC-MS/MS method was successfully applied in PK studies of PTH-IA in mice. Following a single SC administration of 1.52 mg/kg to male C57Bl6 mice, the mean maximum plasma concentration (C_max) was 152 ng/mL at the first sample collection time of 0.083 hr. The terminal half-life (t_1/2) was 2.3 hr. The corresponding AUC_0-∞ was 101 ng∙hr/mL. Following a single IV administration of 1.44 mg/kg, the mean plasma clearance (CL_p) was relatively low, in the range of 10 - 15 mL/min/kg. The volume of distribution at steady-state (Vd_ss) was in the range of 0.21 - 0.45 L/kg. Based on the dose normalized AUC_0-∞ values between the SC and the IV studies, the estimated SC bioavailability for PHT-IA was 4 - 6% in mice.

Conclusion: An accurate, selective, rapid, and robust UPLC-MS/MS method to quantify PTH-IA in mouse plasma was developed. This method was applied successfully to the mouse PK studies. The PK results suggested that PTH-IA has relatively low plasma clearance and steady-state volume distribution in mice.

References: Noda H, Guo J, Khatri A, Dean T, Reyes M, Armanini M, Brooks DJ, Martins JS, Schipani E, Bouxsein ML, Demay MB, Potts JT Jr, Jüppner H, Gardella TJ. An Inverse Agonist Ligand of the PTH Receptor Partially Rescues Skeletal Defects in a Mouse Model of Jansen's Metaphyseal Chondrodysplasia. J Bone Miner Res. 2020 Mar;35(3):540-549. doi: 10.1002/jbmr.3913. Epub 2019 Dec 4.PMID: 31693237
Chambers EE, Lame ME, Bardsley J, Hannam S, Legido-Quigley C, Smith N, Fountain KJ, Collins E, Thomas E. High sensitivity LC-MS/MS method for direct quantification of human parathyroid 1-34 (teriparatide) in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Nov 1;938:96-104. doi: 10.1016/j.jchromb.2013.08.027. Epub 2013 Sep 3. PMID: 24076523.

Acknowledgements: We thank the contributions from NCATS JMC Project Team, NCATS CoMa, NIH DVR to this study. This research was supported in part by the Intramural Research Program of the National Center for Advancing Translational Sciences, National Institutes of Health. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.