Investigation of Human in vivo Metabolism of SEP-227900 Using the Samples
from First-in-Human Study by LC-HRMS/UV and NMR
Authors: Chen Yu-Luan , Skende Estela , Wetie Armand Gatien Ngounou , Wang Peter Li-Quan 
Published: March 2022

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 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 the 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; 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.
(Clinical protocol No. 900-001 under IND No. 102,379).