Investigation of Human Metabolism of SEP-227900 Using the Samples from First-in-Human Study by LC-HRMS/UV and NMR.

OBJECTIVE

The study aims 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 an 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 was used for metabolite identification. Chromatographic separation was achieved on a MACMOD AQ C column (250 × 4.6 mm, 5-μm) with a gradient mobile phase (A: 10 mM NHAc; 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 the detected migration phenomenon, methylation with diazomethane (CHN), 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; 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 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 than 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, SEP- 227900-glucuronide, mono-oxidative metabolite, and its consequent glycine conjugate. The glucuronide metabolite was identified as acyl glucuronide. Greater than 80% of the dosed amount of SEP-227900 was excreted in the urine, mainly in the forms of glycine- and glucuronide- conjugates.