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, 173 (11), 1778-92

Ozanimod (RPC1063) Is a Potent sphingosine-1-phosphate receptor-1 (S1P1 ) and receptor-5 (S1P5 ) Agonist With Autoimmune Disease-Modifying Activity

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Ozanimod (RPC1063) Is a Potent sphingosine-1-phosphate receptor-1 (S1P1 ) and receptor-5 (S1P5 ) Agonist With Autoimmune Disease-Modifying Activity

F L Scott et al. Br J Pharmacol.

Abstract

Background and purpose: Sphingosine1-phosphate (S1P) receptors mediate multiple events including lymphocyte trafficking, cardiac function, and endothelial barrier integrity. Stimulation of S1P1 receptors sequesters lymphocyte subsets in peripheral lymphoid organs, preventing their trafficking to inflamed tissue sites, modulating immunity. Targeting S1P receptors for treating autoimmune disease has been established in clinical studies with the non-selective S1P modulator, FTY720 (fingolimod, Gilenya™). The purpose of this study was to assess RPC1063 for its therapeutic utility in autoimmune diseases.

Experimental approach: The specificity and potency of RPC1063 (ozanimod) was evaluated for all five S1P receptors, and its effect on cell surface S1P1 receptor expression, was characterized in vitro. The oral pharmacokinetic (PK) parameters and pharmacodynamic effects were established in rodents, and its activity in three models of autoimmune disease (experimental autoimmune encephalitis, 2,4,6-trinitrobenzenesulfonic acid colitis and CD4(+) CD45RB(hi) T cell adoptive transfer colitis) was assessed.

Key results: RPC1063 was specific for S1P1 and S1P5 receptors, induced S1P1 receptor internalization and induced a reversible reduction in circulating B and CCR7(+) T lymphocytes in vivo. RPC1063 showed high oral bioavailability and volume of distribution, and a circulatory half-life that supports once daily dosing. Oral RPC1063 reduced inflammation and disease parameters in all three autoimmune disease models.

Conclusions and implications: S1P receptor selectivity, favourable PK properties and efficacy in three distinct disease models supports the clinical development of RPC1063 for the treatment of relapsing multiple sclerosis and inflammatory bowel disease, differentiates RPC1063 from other S1P receptor agonists, and could result in improved safety outcomes in the clinic.

Figures

Figure 1
Figure 1
Chemical structure of RPC1063.
Figure 2
Figure 2
RPC1063 is a potent S1P1 receptor (S1P1R) agonist and induces sustained receptor internalization. (A) S1P1 receptor CREbla CHO‐K1 cells were incubated with 2 μM forskolin in the presence of increasing doses of compound for 4 h. Inhibition of forskolin‐induced cAMP generation is indicative of agonist activity on Gi/o coupled S1P1 receptors. (B) Representative histograms of transfected HEK293T cells expressing S1P1 receptors incubated with vehicle control or 1 μM RPC1063. HEK293T cells were incubated with increasing doses of compound for 1 h (C), or for 1 h followed by extensive washing to remove compound, then a 24 h recovery period (D) and cell surface receptor expression was monitored. Error bars represent SEM of triplicate assays.
Figure 3
Figure 3
RPC1063 induces dose‐dependent, selective lymphopenia and rapid lymphocyte repopulation kinetics. (A) C57BL/6 mice (n = 3 per time‐point) were dosed 1 mg·kg−1 RPC1063 p.o. and blood samples collected at 1, 3, 6, 12 and 24 h post dose. (B) Sprague Dawley rats (n = 6) were dosed 0.5 mg kg−1 RPC1063 p.o. and blood samples collected at 1, 2, 4, 7, 12 and 24 h post dose. (C) C57BL/6 mice (n = 3) were dosed 1 mg·kg−1 RPC1063 p.o. and blood samples were collected at 6 h post dose. Lymphocyte subsets were then evaluated by flow cytometry. (D) Sprague Dawley rats (n = 3) were administered 0.2 mg·kg−1 RPC1063 p.o. or 0.1 mg·kg−1 FTY720 p.o. daily for 5 days. Blood samples were taken 0, 6 and 24 h after the final dose. RPC1063 was measured in plasma and circulating T lymphocytes quantified by flow cytometry. Error bars represent SEM.
Figure 4
Figure 4
RPC1063 treatment reduces ongoing disease symptoms in EAE. EAE was induced in mice by immunization with MOG35–55 peptide and at the first signs of clinical disease (limp tail) individual mice were randomized and dosed with test compound for 14 days. Clinical disease scores were assessed daily (A) and at the end of the study (B). Body weights (C) were assessed at the end of the study. The number of circulating lymphocytes was assessed by differential count 24 h after the final dose (D). n = 9–10 per group. Error bars represent SEM; *P < 0.05 by One‐way ANOVA.
Figure 5
Figure 5
RPC1063 treatment reduces disease activity in TNBS‐induced colitis. Colitis was induced by rectal instillation of TNBS followed 2 h later by daily oral drug treatment. Body weight was assessed daily (A) and *P < 0.05 (determined using Student's t‐test). Colon weight to length ratio (B) and macroscopic colon disease score (C) were determined at the end of the study. The percentage of CD4+ T cells in circulation relative to vehicle treated healthy animals was assessed 24 h after the final dose (D). n = 6–8 per group. Error bars represent SEM. (B‐D) *P < 0.05 by one‐way ANOVA.
Figure 6
Figure 6
Therapeutic administration of RPC1063 preserves body weight and reduces inflammation in a naïve T‐cell adoptive transfer model of colitis. (A) Lymphopenia induced by RPC1063 and KRP203 in C57Bl/6 mice after 5 days of oral dosing, as assessed by measuring circulating CD4+ and CD8+ T cells, 6 and 24 h after the final dose and compared to vehicle treated animals. (B‐D) Colitis was induced by adoptive transfer of CD4+CD45Rbhi T cells into SCID mice. Daily drug treatment began 21 days post‐transfer, when the first signs of weight loss were observed. (B and D) Body weight was assessed every other day and *P < 0.05 (determined using Student's t‐test). Data are from the same study but displayed on different graphs for clarity. (C) Colon weight to length ratio was determined at the end of the study. n = 10 per group. Error bars represent SEM; *P < 0.05 by one‐way ANOVA.
Figure 7
Figure 7
Therapeutic administration of RPC1063 reduces histological disease scores in a naïve T‐cell adoptive transfer model of colitis. (A) End of study histopathological assessment of disease measuring multiple parameters. Scoring system is described in Supplemental Information. (B) Representative images of H&E‐stained distal colon tissue sections demonstrating disruption of gut architecture and mononuclear cell infiltration into the lamina propria of diseased animals, but absent in RPC1063‐treated animals. (C) Mucosal thickness was measured at the end of the study. n = 10 per group. Error bars represent SEM; *P < 0.05 by one‐way ANOVA.
Figure 8
Figure 8
RPC1063 dose‐dependently reduces inflammatory cytokine expression in a naïve T‐cell adoptive transfer model of colitis. At the end of the study, protein was extracted from a 200 mg section of the distal colon and analysed in duplicate for cytokines using Meso Scale Discoveries Electrochemiluminescence system. n = 10 per group. Error bars represent SEM; *P < 0.05 by one‐way ANOVA.

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