Hypha will be exhibiting and presenting a poster on “Accessing Metabolites of the Drug Cenobamate: Challenges and Solutions” at the 2021 ISSX Meeting.

This virtual meeting will bring together individuals who are actively working on drug/xenobiotic research in diverse fields including basic and clinical pharmacology and therapeutics, toxicology, oncology, endocrinology, physiology, biochemistry, medicinal chemistry, molecular and structural biology, and genetics.

Abstract for Poster P07: Accessing Metabolites of the Drug Cenobamate: Challenges and Solutions

Lisbet Kvaerno¹, Chris Drake¹, Liam Evans¹, Lana Gabriel², Renia Gemmell¹, Adriana Gomez¹, Tetsuo Kokubun¹, William Hodds¹, Ravi Manohar¹, Richard Phipps¹, Julia Shanu-Wilson¹, Jonathan Steele¹, Stephen Wrigley¹

¹Hypha Discovery Limited, 154B Brook Drive, Milton Park, Abingdon, OX14 4SD, UK

² SK Life Science, Inc., 461 From Road, 5^th Flr, Paramus, NJ 07652, USA

Cenobamate (Xcopri/ Ontozry), a drug developed by SK Life Science for the treatment of partial-onset seizures, is extensively metabolized, primarily by glucuronidation via UGT2B7 and by oxidation via CYP2E1, CYP2A6 and CYP2B6. Metabolites of cenobamate are formed slowly in the kidney and eliminated in the urine, with an N-glucuronide, M1, comprising the main human metabolite.Other significant excretory metabolites include a phenolic glucuronide M2b and dihydrodiol metabolites M6 and M7.

Objective and methods used: Three metabolites of cenobamate, M1, M2b and a phenolic intermediate M8b, were required by SK Life Science for definitive structure elucidation and various in vitro studies. The metabolites were synthesized and characterized by Hypha using a combination of late-stage chemical synthesis techniques and microbial biotransformation followed by structure elucidation using NMR spectroscopy.

Results: Late-stage chemical glucuronidation using mild deprotection conditions enabled a gram of the N-glucuronide to be prepared, which was identical to the small amount of M1 purified from human urine. Preparation of the other two metabolites, M8b and the secondary glucuronide M2b, provided more of a challenge. In order to do this, and because the position of hydroxylation was unknown, a small amount (0.8 mg) of the M2b had to be purified from a complex human urine background. The structure was then identified using cryoprobe NMR spectroscopy, in order that the correct phenol aglycone could be synthesized, revealing that the aromatic hydroxyl of M2b is para to the aromatic alkyl substituent of cenobamate. The next step was the synthesis of M2b from M8b. M8b was unstable under the chemical glucuronidation conditions attempted, however several microbes were able to make the glucuronide. The best of the microbes was fermented at 8L scale, dosed with 560 mg of M8b, from which 255 mg of M2b was purified from the extract. Attempts to isolate and purify dihydrodiol metabolites M6 and M7 from human urine were unsuccessful due to instability of M6 and M7 in stored urine.

Conclusion: Isolation of M1 and M2b from human urine, comparison of isolated M1 with synthetic M1, and structure elucidation of M2b allowed for the identification of suitable methods for scaling up M1, M8b, and M2b. As a result, the desired major human cenobamate metabolites M1, M2b, and M8b were all obtained in >200 mg quantities and at >95% chemical purity by LC-UV, LC-ELSD, and 1H-NMR. The pure metabolites were shipped to SK Life Science in order to conduct various in vitro tests.