Reversible oxidation/reduction steps in the metabolic degradation of the glycerol side chain of the S1P1 modulator ponesimod.

1. Ponesimod is a selective modulator of the sphingosine 1-phosphate receptor 1 (S1P1) approved for the treatment of active relapsing forms of multiple sclerosis. The chemical structure of ponesimod contains a glycerol side chain which is the major target of drug metabolism in humans.2. The two major metabolic pathways give the acids M12 (-OCH2CH(OH)COOH) and M13 (-OCH2COOH). While the former results from oxidation of the terminal alcohol, the mechanism yielding the chain-shortened acid M13 is less obvious. A detailed mechanistic study with human liver microsomes and hepatocytes using ponesimod, M12 and some of the suspected intermediates revealed an unexpectedly complex pattern of enzyme-mediated and chemical reactions.3. Metabolic pathways for both acids were not independent and several of the transformations were reversible, depending on reaction conditions. Formation of M13 occurred either via initial oxidation of the secondary alcohol, or as a downstream process starting from M12.4. The phenol metabolite M32 was produced as part of several pathways. Control experiments at various pH values and in the absence of metabolising enzymes support the conclusion that its formation resulted from chemical degradation rather than from metabolic processes.

Deconstructing the Pharmacological Contribution of Sphingosine-1 Phosphate Receptors to Mouse Models of Multiple Sclerosis Using the Species Selectivity of Ozanimod, a Dual Modulator of Human Sphingosine 1-Phosphate Receptor Subtypes 1 and 5.

Ozanimod, a sphingosine 1-phosphate (S1P) receptor modulator that binds with high affinity selectively to S1P receptor subtypes 1 (S1P1) and 5 (S1P5), is approved for the treatment of relapsing multiple sclerosis (MS) in multiple countries. Ozanimod profiling revealed a species difference in its potency for S1P5 in mouse, rat, and canine compared with that for human and monkey. Site-directed mutagenesis identified amino acid alanine at position 120 to be responsible for loss of activity for mouse, rat, and canine S1P5, and mutation back to threonine as in human/monkey S1P5 restored activity. Radioligand binding analysis performed with mouse S1P5 confirmed the potency loss is a consequence of a loss of affinity of ozanimod for mouse S1P5 and was restored with mutation of alanine 120 to threonine. Study of ozanimod in preclinical mouse models of MS can now determine the S1P receptor(s) responsible for observed efficacies with receptor engagement as measured using pharmacokinetic exposures of free drug. Hence, in the experimental autoimmune encephalomyelitis model, ozanimod exposures sufficient to engage S1P1, but not S1P5, resulted in reduced circulating lymphocytes, disease scores, and body weight loss; reduced inflammation, demyelination, and apoptotic cell counts in the spinal cord; and reduced circulating levels of the neuronal degeneration marker, neurofilament light. In the demyelinating cuprizone model, ozanimod prevented axonal degradation and myelin loss during toxin challenge but did not facilitate enhanced remyelination after intoxication. Since free drug levels in this model only engaged S1P1, we concluded that S1P1 activation is neuroprotective but does not appear to affect remyelination. SIGNIFICANCE STATEMENT: Ozanimod, a selective modulator of human sphingisone 1-phosphate receptor subtypes 1 and 5 (S1P1/5), displays reduced potency for rodent and dog S1P5 compared with human, which results from mutation of threonine to alanine at position 120. Ozanimod can thus be used as a selective S1P1 agonist in mouse models of multiple sclerosis to define efficacies driven by S1P1 but not S1P5. Based on readouts for experimental autoimmune encephalomyelitis and cuprizone intoxication, S1P1 modulation is neuroprotective, but S1P5 activity may be required for remyelination.

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.

Structure-function analysis of lipid substrates and inhibitors of sphingosine kinases.

The sphingosine kinases, SK1 and SK2, catalyse the formation of the bioactive signalling lipid, sphingosine 1-phosphate (S1P), from sphingosine. SK1 and SK2 differ in their subcellular localisation, trafficking and regulation, but the isoforms are also distinct in their selectivity toward naturally occurring and synthetic ligands as substrates and inhibitors. To date, only the structure of SK1 has been determined, and a structural basis for selectivity differences in substrate handling by SK2 has yet to be established. Here we present a structural rationale, based on homology modelling and ligand docking, to account for the capacity of SK2, but not SK1, to efficiently process the pharmacologically active substances, fingolimod (FTY720) and safingol, as substrates. We propose that two key residue differences in hSK2 (Ser305/Thr584 in place of Ala175/Ala339 in hSK1) facilitate conformational switching in the lipid head group anchor residue, Asp308 (corresponding to Asp178 in hSK1), to accommodate substrate diversity for SK2. Our analysis accounts for the contrasting behaviour of fingolimod and safingol as non-turnover inhibitors of SK1, but substrates for SK2, and the observed stereoselectivity for phosphorylation of the pro-S hydroxymethyl group of fingolimod to generate (S)-FTY720-P in vivo. We also rationalise why methylation of the pro-R hydroxymethyl of FTY720 switches the behaviour of the resulting compound, (R)-FTY720 methyl ether (ROMe), to SK2-selective inhibition. Whilst the pharmacological significance of (S)-FTY720-P is firmly established, as the active principle of fingolimod in treating relapsing-remitting multiple sclerosis, the potential importance of SK-mediated phosphorylation of other substrates, such as safingol and non-canonical naturally occuring substrates such as (4E,nZ)-sphingadienes, is less widely appreciated. Thus, the contribution of SK2-derived safingol 1-phosphate to the anti-cancer activity of safingol should be considered. Similarly, the biological role of sphingadiene 1-phosphates derived from plant-based dietary sphingadienes, which we also show here are substrates for both SK1 and SK2, merits investigation.

Does Siponimod Exert Direct Effects in the Central Nervous System?

The modulation of the sphingosine 1-phosphate receptor is an approved treatment for relapsing multiple sclerosis because of its anti-inflammatory effect of retaining lymphocytes in lymph nodes. Different sphingosine 1-phosphate receptor subtypes are expressed in the brain and spinal cord, and their pharmacological effects may improve disease development and neuropathology. Siponimod (BAF312) is a novel sphingosine 1-phosphate receptor modulator that has recently been approved for the treatment of active secondary progressive multiple sclerosis (MS). In this review article, we summarize recent evidence suggesting that the active role of siponimod in patients with progressive MS may be due to direct interaction with central nervous system cells. Additionally, we tried to summarize our current understanding of the function of siponimod and discuss the effects observed in the case of MS.

Bioequivalence Study of 2 Capsule Formulations of Fingolimod 0.5 mg Assessing Both Parent Drug and Active Metabolite in New Zealand Healthy Subjects (Truncated Design).

Fingolimod is indicated for the treatment of patients with the relapsing-remitting form of multiple sclerosis. The primary study objective was to evaluate the bioequivalence of a test formulation, 0.5 mg fingolimod HCl capsule (Lebrina, Asofarma Sociedad Anónima Industrial y Comercial, Argentina) relative to a reference formulation, 0.5 mg fingolimod capsule (Gilenya, Novartis Pharmaceutical, Australia). In a single-center, randomized, single-dose, single-blinded, 2-way crossover study, 33 New Zealand healthy subjects of both sexes were enrolled to receive a 0.5-mg dose of 3 capsules of each fingolimod formulation under fasting conditions, with a 42-day washout period between administrations. Additional pharmacokinetic information regarding its main active metabolite, fingolimod phosphate, was also provided. The point estimate and 90% confidence intervals of the ratios of maximum concentration and area under the plasma concentration-time curve from time 0 to 72 hours were 99.07 (95.83-102.41) and 97.64 (95.33-100.00) for fingolimod, and 95.60 (90.95-100.49) and 98.54 (96.19-100.96), for fingolimod phosphate. Primary parameters, maximum concentration and area under the plasma concentration-time curve from time 0 to 72 hours for fingolimod and fingolimod phosphate were found to have no significant difference when test and reference formulations were compared. Fingolimod and fingolimod phosphate of both formulations were within the accepted 90% confidence interval limits of 80.00% and 125.00%. No significant differences between the test and reference drug products were detected in any of the pharmacokinetic parameters estimated. Notwithstanding the primary conclusion of bioequivalence is focused on the measurement of the parent compound, compliance with the same criteria by the active metabolite reinforces the comparability between the pharmacokinetic profiles of both formulations (ClinicalTrials.gov Identifier: NCT03757338).