(W0930-02-09) Identification of New Novel Biotransformations of Fasiligfam Using cIMS and an Integrated Informatics Workflow

Purpose: Identify the metabolic biotransformation of the GPR40 agonist Fasiglifam and evaluate any of the resulting drug metabolites as potential cause of liver injury

Methods: Male Sprague-Dawley rats were dosed orally with 50 mg/kg fasiglifam as a suspension in 0.5% carboxymethylcellulose in water. Blood was collected from the tail vein immediately prior to dosing and at regular intervals over the following 96 hours. Plasma obtained from blood samples was subjected to protein precipitation via mixing with acetonitrile at a ratio of 3:1 (acetonitrile:sample). Following vortex mixing the samples were centrifuged at 13,000 rcf for 10 minutes and transferred to an autosampler vial for analysis. Authentic fasiglifam standard was dissolved in methanol and diluted with 50:50 acetonitrile:water to give a final drug concentration of 100 ng/mL. This sample was used to obtain representative retention time, MS, MS/MS, CCS and neutral loss outcomes for the parent compound.The authentic standard and extracted rat plasma samples were analyzed using reverse phase liquid chromatography coupled with negative ion electrospray ion mobility mass spectrometry detection. Briefly, a 1 mL aliquot of the sample was loaded onto a 2.1 x 100 mm ACQUITYTM HSS T3 1.7 m C18 column, the column was maintained at 40 oC and eluted with a linear solvent gradient of 40 – 70% 0.1% aqueous formic acid: acetonitrile over 10 min at a flow rate of 600 mL/min. Mass spectrometry data was collected in negative ion mode over the range of 60 – 1200 m/z, using a collision energy ramp of 20 – 40 eV and a single pass of the ion mobility cell.

Results: Previous Bioanalytical analysis on these samples allowed for the pharmacokinetics of the IV and PO samples to be accurately determined. The Cmax occurred 3hr post dose for the PO samples, fasiglifam was eliminated with a half life of 4 - 7hr dependent upon dose with a Cl of 1.7 to 1.0 mcg.hr/mL. 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 tR = 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, in which the biotransformations appear to be associated with the substituted dihydrobenzofuran moiety. The structure of these new metabolites is currently under further investigation. fasiglifam and the major metabolites follow a similar elimination profile with a maximum concentration observed at 9 h and a gradual reduction in concentration of the metabolites 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. The metabolites were confirmed by a combination of the exact mass m/z value, fragment ion analysis and comparison of the collision cross section of the structure with predicted values

Conclusion: A total of 15 drug-related metabolites were detected across the time course with an average m/z error of –0.77 ppm.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. Complete analysis of these samples resulted in the detection of the previously reported metabolites together with several new, and previously unreported metabolites, in which the biotransformations appear to be associated with the substituted dihydrobenzofuran moiety.


Fasiglifam and metabolite elimination profile


Summary of fasiglifam related biotransfoirmations