Investigation into MAO B–Mediated Formation of CC112273, a Major Circulating Metabolite of Ozanimod, in Humans and Preclinical Species: Stereospecific Oxidative Deamination of (S)-Enantiomer of Indaneamine (RP101075) by MAO B

Abstract

Ozanimod, recently approved for treating relapsing MS, produced a disproportionate, active, MAO B-catalyzed metabolite (CC112273) that showed remarkable interspecies differences and led to challenges in safety testing. This study explored the kinetics of CC112273 formation from its precursor RP101075. Incubations with human liver mitochondrial fractions revealed K Mapp , V max and Cl int for CC112273 formation to be 4.8 M, 50.3 pmol/min/mg protein and 12 l/min/mg, respectively, while K M with human recombinant MAO B was 1.1 M. Studies with liver mitochondrial fractions from preclinical species led to K Mapp , V max and Cl int estimates of 3.0, 35 and 33 M, 80.6, 114, 37.3 pmol/min/mg and 27.2, 3.25 and 1.14 l/min/mg in monkey, rat and mouse, respectively, and revealed marked differences between rodents and primates, primarily attributable to differences in the K M . Comparison of Cl int estimates revealed monkey to be ~two-fold more efficient and the mouse and rat to be 11 and 4-fold less efficient than humans in CC112273 formation. The influence of stereochemistry on MAO B-mediated oxidation was also investigated using the R-isomer of RP101075 (RP101074). This showed marked selectivity towards catalysis of the S-isomer (RP101075) only. Docking into MAO B crystal structure suggested that even though both the isomers occupied its active site, only the orientation of RP101075 presented the C-H on the -carbon that was ideal for the C-H bond cleavage, which is a requisite for oxidative deamination. These studies explain the basis for the observed interspecies differences in the metabolism of ozanimod as well as the substrate stereospecificity for formation of CC112273.

The sphingosine‐1‐phosphate receptor 1 modulator ponesimod repairs cuprizone‐induced demyelination and induces oligodendrocyte differentiation

“…Previous research elucidated a species‐based difference of S1P5 selectivity of ozanimod, which shows reduced potency for mouse S1P5 resulting from a mutation of threonine to alanine at position 120 19 . Nonetheless, it was shown that mice are up to 11‐fold less efficient in generating the major ozanimod metabolite (CC112273) 48 . Therefore, ozanimod might require human metabolization for full receptor engagement 49,50 …”

“…19 Nonetheless, it was shown that mice are up to 11-fold less efficient in generating the major ozanimod metabolite (CC112273). 48 Therefore, ozanimod might require human metabolization for full receptor engagement. 49,50 In our study, we confirm that functional antagonism of S1P1 by ponesimod drives OPC differentiation, a key step in initiating (re)myelination, but does not affect axonal wrapping by OPCs in vitro.…”

The Importance of Tracking “Missing” Metabolites: How and Why?

“…The metabolite was minimally observed in vitro due to sequential metabolism through P450 and MAO-B, and it had a much longer half-life (∼10 times that of ozanimod) in vivo . To make it more complex for development advancement, CC112273 was an active metabolite that had equal potency as ozanimod, making it the primary active drug related species in circulation that needed further characterization. , Bridging repeated-dose GLP PK studies in preclinical species had to be conducted to address the exposure and safety multiplier of disproportionate metabolites; additionally, the clinical PK-PD relationship and DDI potential had to be re-assessed before this new drug could gain approval.…”