Objective:
To explore the human in vivo metabolism of SEP-227900 (4H-furo[3, 2-b]pyrrole-carboxylic acid, m.w 151.03), a D-amino-acid oxidase (DAAO) inhibitor by using plasma and urine samples from first-in-human study
Methods:
The human plasma and urine samples were from a single dose cohort that consisted of 9 healthy male volunteers each received 80-mg dose of SEP-227900 orally. The pooled pre-dose urine and the pooled 0-24 h urine sample were created across 9 subjects by equal volume. Plasma samples were pooled by equal volume across 9 subjects to obtain 0-12 h plasma for metabolite searching, and also pooled by timepoints across 9 subjects to obtain 0.5-, 5-, and 12-h plasma for semi-quantitation. The plasma was de-proteinized by acetonitrile (1:3 v/v plasma-acetonitrile) then the supernatant was dried down, reconstituted and injected for LC-HRMS/UV analysis. The urine sample was just simply centrifuged before analysis. LC-HRMS/UV was utilized to search predictable and unknown metabolites and estimate their relative abundances. Accurate mass measurement by Orbitrap-MS and MS/MS were used for metabolite identification. Chromatographic separation was achieved on a MACMOD AQ C8 column (250 × 4.6 mm, 5-µm) with a gradient mobile phase (A: 10 mM NH4Ac; B: acetonitrile; flowrate: 0.700 ml/min) for a total run-time of 65 min. The definite position in the molecule for the glucuronidation metabolism was characterized by detected migration phenomenon, methylation with diazomethane (CH2N2), and NMR.
Results:
Unchanged parent drug and four metabolite peaks were detected in humans: M1 was a mono-oxidative metabolite of SEP-227900; M2 was a glucuronide conjugate of SEP-227900; M3 was a glycine conjugate of SEP-227900; and M4 was a glycine conjugate of M1. The specific position of the oxidation in M1 solely based on the mass spectral (MS and MS/MS) data was not identified. However, for the major metabolite M2, the acyl glucuronidation was unambiguously determined through multiple pieces of experimental evidence such as the observation of a migration pattern, mono-methylation by diazomethane, and NMR measurement. This determination is of significance related to the safety evaluation of an investigational new drug development. The glycine conjugate of SEP-227900, i.e. M3 was found to be the most abundant metabolite in human urine (approximately 3-fold higher level as the glucuronide level). All together (mainly glycine-conjugate and glucuronide), it resulted in greater than 80% of the dosed amount in urine excretion (a separate measurement showed 23% of the dosed amount in urine excretion as the glucuronide).
Conclusion:
Four metabolites were found in humans: SEP-227900-glycine conjugate, SEP227900-glucuronide, mono-oxidative metabolite and its consequent glycine conjugate. The glucuronide metabolite was identified as the acyl glucuronide. Greater than 80% of the dosed amount of SEP-227900 was excreted in urine mainly in the forms of glycine- and glucuronide- conjugates.
