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

Ozanimod, recently approved for treating relapsing multiple sclerosis, 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 mitochondria! fractions revealed K-mapp, V-max, and intrinsic clearance (Cl-int) for CC112273 formation to be 4.8 mu M, 50.3 pmol/min/mg protein, and 12 mu l/min/mg, respectively, whereas Michaelis-Menten constant (K-M) with human recombinant MAO B was 1.1 mu 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 mu M, 80.6, 114, 37.3 pmol/min/mg, and 27.2, 3.25, and 1.14 mu 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 similar to 2-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 (RP1 01074). This showed marked selectivity toward catalysis of the S-isomer (RP1 01075) only. Docking into MAO B crystal structure suggested that although both the isomers occupied its active site, only the orientation of RP101075 presented the C-H on the alpha-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.SIGNIFICANCE STATEMENTThis study evaluates the enzymology and the species differences of the major circulating metabolite of ozanimod, CC112273. Additionally, the study also explores the influence of stereochemistry on MAO B-catalyzed reactions. The study is of significance to the DMD readers given that this oxidation is catalyzed by a non-cytochrome P450 enzyme, and that marked species difference and notable stereospecificity was observed in MAO B-catalyzed biotransformation when the indaneamine enantiomers were used as substrates.