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.

Julie V Selkirk,Yingzhuo Grace Yan,Andrea Bortolato,Carlos Lopez,Iliana Ruiz,Jeffrey M Schkeryantz,Shameek Biswas,Deepak Dalvie,Richard Hargreaves,Nathan Ching,Kevin C Dines

doi:10.1124/jpet.121.000741

Abstract

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.

Highlights

Sphingosine-1 phosphate (S1P) and its cognate family of receptors play a key role in the immunomodulatory and direct central nervous system (CNS) effects associated with multiple sclerosis (MS) (Cohan et al, 2020)
The family of sphingosine-1 phosphate (S1P) receptors is comprised of five G protein-coupled receptors (GPCR) of the class A family of seven transmembrane domain GPCRs, designated S1P subtypes 1-5 (S1P1-5) (Rosen et al, 2013; Kihara et al, 2014), with S1P1 and S1P5 expressed on key cell types that contribute to MS disease pathology (Rothhammer et al, 2017; Groves et al, 2018; Kim et al, 2018;)
Potency and intrinsic activity assessment of ozanimod and RP101075 for S1P1 and S1P5 across human and preclinical species revealed that while the potency for S1P1 was maintained across all species, the potency for S1P5 was significantly reduced for the mouse, rat, and canine homologues compared with that of human and cyno (Table 1)

Summary

Introduction

Sphingosine-1 phosphate (S1P) and its cognate family of receptors play a key role in the immunomodulatory and direct central nervous system (CNS) effects associated with multiple sclerosis (MS) (Cohan et al, 2020). The predominant S1P receptor involved in lymphocyte trafficking in MS is S1P1, which is expressed on B and T cells, and following exposure to agonist is rapidly down modulated from the plasma membrane with internalization to the intracellular compartment (Sanna et al, 2004; Scott et al, 2016). This internalization of S1P1 prevents specific subsets of B and T cells in the peripheral lymphoid tissue from sensing the S1P concentration gradient that exists between tissues and the systemic circulation and blocks traffic out of the lymphoid tissue into the circulation. S1P1 is expressed by astrocyte and microglial cells within the CNS (Choi et al, 2011; Noda et al, 2013; Musella et al, 2020), and down modulation of S1P1 in these cell types attenuates their activation state which may abrogate the local pro-inflammatory environment that exists during an MS relapse

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