(T1230-10-66) Addressing the MicroSampling Sensitivity Challenge in DMPK Studies Using Vacuum Jacketed Column UHPLC-cyclic Ion Mobility MS

Purpose: Microsampling offers an attractive approach to reduce animal requirements whilst maintaining the quality and information in discovery and preclinical studies, thus supporting the 3Rs (reduction, replacement, refinement) principles. However, these reduced sample volumes requires improved sample handling and increased instrument sensitivity to obtain the required DMPK data for confident decision making. Here we report the use of microsampling combined with one step sample preparation, novel high sensitivity vacuum jacketed column (VJC) LC/MS/MS and cyclic ion mobility MS to determine the pharmacokinetics and metabolic biotransformation of the GPR40 agonist fasiglifam in the Sprague-Dawley rat.

Methods: Male Sprague-Dawley rats were dosed intravenously and orally with fasiglifam at 10 and 50 mg/kg respectively. Blood was collected via microsampling (10 µL) from the tail vein at regular intervals over the following 96 hours. Derived plasma was subjected to protein precipitation with acetonitrile, vortex mixed and centrifuged prior to analysis. Fasiglifam pharmacokinetics was obtained via quantitative analysis using a 2 min reversed - phase VJC UHPLC method coupled to a tandem quadrupole mass spectrometer operating in negative ion MRM mode using a SIL internal standard. Metabolite identification was performed on a 10 min reversed – phase UHPLC coupled to a cyclic ion mobility mass spectrometer operating in positive and negative ion mode.

Results: The IV and PO pharmacokinetics and metabolism of fasiglifam in the male and female rat were accurately determined using UHPLC/MS/MS using 10 µL of plasma. Following intravenous dosing, peak observed plasma concentrations of 8.8/9.2 µg/mL (5 mg/kg) were seen in male and female rats respectively. Following oral dosing peak plasma concentrations at 1 h of ca. 16.9/12.9 µg/mL for 10 mg/kg and 76.2/87.5 µg/mL for 50 mg/kg doses were obtained for male and female rats respectively. Drug concentrations then declined in the plasma of both sexes with t_1/2’s of 3.8 and 3.9 h. Oral bioavailability was estimated to be 89-117% in males and females at both dose levels. Urinary excretion was low, but in a significant sex-related difference female rats eliminated ca. 10 fold more drug related material by this route. A total of 15 drug related metabolites were identified in the PO samples. The major and previously reported metabolites of fasiglifam were identified using a combination of targeted screening using library entries, diagnostic fragment ions, common neutral losses and established metabolic cleavage, functionalization and conjugation reactions. For example, the glucuronide metabolite of fasiglifam, eluting with a retention time t_R = 6.26 min with a deprotonated molecular ion m/z = 699.2113, with diagnostic fragment ions at m/z = 523.1786, corresponding to a neutral loss of the glucuronide moiety, and the glucuronide itself as a product ion. Several new, and previously unreported metabolites were detected, in which the bio-transformations appear to be associated with the substituted dihydrobenzofuran moiety. Fasiglifam and major metabolites follow a similar elimination profile with a maximum concentration observed at 9 h and a gradual reduction in concentration over the following 96 h. However, it is evident that even after 96 h the parent drug and metabolites are still not fully eliminated from the systemic circulation.

Conclusion: Metabolic fate of fasiglifam in the rat was determined using Microsampling, high sensitivity VJC UHPLC/MS/MS and ion mobility LC/MS.