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 mitochondrial fractions revealed K Mapp, V max, and intrinsic clearance (Clint) for CC112273 formation to be 4.8 µM, 50.3 pmol/min/mg protein, and 12 µl/min/mg, respectively, whereas Michaelis-Menten constant (K M) with human recombinant MAO B was 1.1 µM. Studies with liver mitochondrial fractions from preclinical species led to K Mapp, V max, and Clint estimates of 3.0, 35, and 33 µM, 80.6, 114, 37.3 pmol/min/mg, and 27.2, 3.25, and 1.14 µl/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 Clint estimates revealed monkey to be ∼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 (RP101074). This showed marked selectivity toward catalysis of the S-isomer (RP101075) 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 α-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 STATEMENT: This 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.

Absorption, Metabolism, and Excretion, In Vitro Pharmacology, and Clinical Pharmacokinetics of Ozanimod, a Novel Sphingosine 1-Phosphate Receptor Modulator.

Ozanimod is approved for the treatment of relapsing forms of multiple sclerosis. Absorption, metabolism, and excretion of ozanimod were investigated after a single oral dose of 1.0 mg [14C]ozanimod hydrochloride to six healthy subjects. In vitro experiments were conducted to understand the metabolic pathways and enzymes involved in the metabolism of ozanimod and its active metabolites. The total mean recovery of the administered radioactivity was ∼63%, with ∼26% and ∼37% recovered from urine and feces, respectively. Based on exposure, the major circulating components were active metabolite CC112273 and inactive metabolite RP101124, which together accounted for 50% of the circulating total radioactivity exposure, whereas ozanimod accounted for 6.7% of the total radioactive exposure. Ozanimod was extensively metabolized, with 14 metabolites identified, including two major active metabolites (CC112273 and CC1084037) and one major inactive metabolite (RP101124) in circulation. Ozanimod is metabolized by three primary pathways, including aldehyde dehydrogenase and alcohol dehydrogenase, cytochrome P450 isoforms 3A4 and 1A1, and reductive metabolism by gut microflora. The primary metabolite RP101075 is further metabolized to form major active metabolite CC112273 by monoamine oxidase B, which further undergoes reduction by carbonyl reductases to form CC1084037 or CYP2C8-mediated oxidation to form RP101509. CC1084037 is oxidized rapidly to form CC112273 by aldo-keto reductase 1C1/1C2 and/or 3ß- and 11ß-hydroxysteroid dehydrogenase, and this reversible oxidoreduction between two active metabolites favors CC112273. The ozanimod example illustrates the need for conducting timely radiolabeled human absorption, distribution, metabolism, and excretion studies for characterization of disproportionate metabolites and assessment of exposure coverage during drug development. SIGNIFICANCE STATEMENT: Absorption, metabolism, and excretion of ozanimod were characterized in humans, and the enzymes involved in complex metabolism were elucidated. Disproportionate metabolites were identified, and the activity of these metabolites was determined.

Efficiency in Drug Discovery: Liver S9 Fraction Assay As a Screen for Metabolic Stability.

BACKGROUND: A rapid and comprehensive metabolic stability screen at the top of a drug discovery flow chart serves as an effective gate in eliminating low value compounds. This imparts a significant level of efficiency and saves valuable resources. While microsomes are amenable to high throughput automation and are cost effective, their enzymatic make-up is limited to that which is contained in endoplasmic reticulum, thereby informing only on Phase I metabolism. Lack of Phase II metabolism data can become a potential liability later in the process, adversely affecting discovery projects' timelines and budget. Hepatocytes offer a full complement of metabolic enzymes and retain their cellular compartments, better representing liver metabolic function. However, hepatocyte screens are relatively expensive, labor intensive, and not easily automatable. Liver S9 fractions include Phase I and II metabolic enzymes, are relatively inexpensive, easy to use, and amenable to automation, making them a more appropriate screening system. We compare the data from the three systems and present the results. RESULTS: Liver S9 and hepatocyte stability assays binned into the same category 70-84% of the time. Microsome and hepatocyte data were in agreement 73-82% of the time. The true rate for stability versus plasma clearance was 45% for hepatocytes and 43% for S9. CONCLUSION: In our opinion, replacing liver microsome and hepatocyte assays with S9 assay for high throughput metabolic screening purposes provides the combined benefit of comprehensive and high quality data at a reasonable expense for drug discovery programs.

Proposing advancement criteria for efficient DMPK triage of new chemical entities.

With the goal of refining our discovery DMPK workflow, we conducted a retrospective analysis on internal Celgene compounds by calculating the physicochemical properties and gathering data from several assays including solubility, rat and human liver S9 stability, Caco-2 permeability, and rat intravenous (iv.) and oral pharmacokinetics. Our analysis identified plasma clearance to be most statistically relevant for prediction of oral exposure. In rat, compounds with rat S9 stability of ≥70% at 60 min and a plasma clearance of ≤43 ml/min/kg had the greatest chance of achieving oral exposures above 3 µM.h. Compounds with the dual advantage of plasma clearance ≤43 ml/min/kg and Caco-2 permeability ≥8 × 10(-6) cm/s or efflux ratio ≤8 were highly likely to achieve those oral exposures. Implementation of these criteria leads to a significant increase in efficiency, good pharmacokinetic properties, cost savings and a reduction in the use of animals.

Discovery of CC-930, an orally active anti-fibrotic JNK inhibitor.

In this Letter we describe the discovery of potent, selective, and orally active aminopurine JNK inhibitors. Improving the physico-chemical properties as well as increasing the potency and selectivity of a subseries with rat plasma exposure, led to the identification of four structurally diverse inhibitors. Differentiation based on PK profiles in multiple species as well as activity in a chronic efficacy model led to the identification of 1 (CC-930) as a development candidate, which is currently in Phase II clinical trial for IPF.

Application of 1D and 2D 1H-NMR in the structural elucidation of a N-glucuronidated metabolite and oxidized metabolites generated in microsomal incubation.

Metabolite identification can provide tremendous value in identifying metabolic soft-spots on molecules of interest and to evaluate the potential for generating reactive species. This information is useful in designing stable analogs with acceptable drug-like properties. Two key compounds were found to generate major metabolites that could not be elucidated by mass spectrometry. Nuclear Magnetic Resonance (NMR) is a non-destructive method to obtain structural information. It requires milligram quantities of putative metabolites, typically unavailable in early stage discovery projects. Herein, we demonstrated the application of NMR using microgram quantities of samples to identify the structures of the major metabolites of two discovery compounds. In the first case, we studied structural elucidation of a Nglucuronide on a pyrazole moiety using 1H-NMR due to the instability of the glucuronidated metabolite under mass spectrometric conditions. In the second example, we characterized two oxidized metabolites having identical mass fragmentation using 2D-NMR. In both cases, chemists incorporated these findings into designing analogs to improve metabolic stability.

Discovery and optimization of thieno[2,3-d]pyrimidines as B-Raf inhibitors.

The serine/threonine specific protein kinase B-Raf is part of the MAPK pathway and is an interesting oncology target. We have identified thieno[2,3-d]pyrimidines as a core scaffold of small molecule B-Raf inhibitors. The SAR of analogs in this series will be described.