S1PR1 1.03 nM (EC50) S1PR5 8.6 nM (EC50)

Ozanimod (RPC-1063) hydrochloride exhibits intrinsic activity and potency as S1P receptor modulators for S1P5 across species with [35S]-GTPgS binding. The EC50 values for Human S1P1, Cynomolgus monkey S1P1, Mouse S1P1, Rat S1P1, and Canine S1P1 are 1.03 nM, 1.29 nM, 0.90 nM, 1.02 nM, and 0.61 nM, respectively; for Cynomolgus monkey S1P5, Mouse S1P5, Rat S1P5, and Canine S1P5 are 8.6 nM, 15.9 nM, 957.5 nM, 2032.7 nM, and 1662.0 nM, respectively[1]. By altering the alanine in the mouse sequence, ozanimod hydrochloride recovers the potency with EC50 from 958 nM for mS1P5 to 6.7 nM for mS1P5_A120T, nearly mirroring the EC50 for hS1P5 of 8.6 nM[1]. The binding affinities of ozanimod hydrochloride for hS1P5, mS1P5, and mS1P5 _A120T are 2.0 nM, 59.9 nM, and 5.6 nM, respectively[1]. In addition to having saturation binding for [3H]-A971432 to S1P5D value of 8.75 nM, ozanimod hydrochloride possesses saturation binding of [3H]-ozanimod to hS1P5, and mS1P5_A120T with KD values of 6.56 nM and 7.35 nM, respectively[1].

Reduced circulating lymphocytes, disease scores, and body weight loss were the outcomes of ozanimod (RPC-1063) hydrochloride (oral gavage; 0.05, 0.2, or 1 mg/kg; once daily; for 14 consecutive days) exposures sufficient to engage S1P1, but not S1P5. Additionally, the spinal cord's levels of inflammation, demyelination, and apoptotic cells were reduced, as was the circulating level of neurofilament light, a marker of neuronal degeneration[1]. During a toxin challenge, ozanimod hydrochloride (oral gavage; 5 mg/kg; once daily) reduced myelin loss and axonal degradation, but it did not promote increased remyelination following intoxication[1]. Mice treated orally with ozanimod hydrochloride (1 or 5 mg/kg for 7 days) exhibit excellent pharmacokinetics[1].

The cAMP (S1P1R) or β-arrestin (S1P4R) signaling was detected by cell signaling assays using the LiveBLAzer-FRET B/G assay. As directed by the manufacturer, assays are carried out in triplicate in 384-well plates. Twenty percent (2-hydoxypropyl)-β-cyclodextrin is used to dilute compound stocks 1:10 at first, and they are kept at 10 mM in 100% DMSO at -50°C. For every 40 times the final assay concentration in 10 mM, a 10-point dose response curve is generated. Hepes 7.4 pH with 0.1% Pluronic F-127. 80 μM forskolin is added to the diluent for the S1P1R assay. To put it briefly, 104 cells per well are cultured for 4 hours at 37°C with a dose response of ligand available. Probenecid and CC4-AM substrate are added, and after an additional two hours of incubation at 37°C, the sample is examined using a Spectramax M5. Data for S1P1R cAMP assays is normalized to the highest fluorescence produced by 2 μM forskolin. In GTPγS binding assays, 1–5 μg/well of membrane protein is incubated for 15 minutes in 96-well plates with 10 μM GDP, 100–500 μg/well Wheat Germ Agglutinin PVT SPA beads in 50 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 20 μg/ml saponin, and 0.1% fatty acid free BSA. The plates are incubated for 120 minutes after the addition of compound and 200 pM GTP [35S] 1250Ci/mmol, and then centrifuged for 5 minutes at 300g. A TopCount Instrument is used to detect radioactivity. A four-parameter variable called slope is used to fit all of the data. Radioactivity is detected with a TopCount Instrument. GraphPad Prism's four parameter variable slope non-linear regression is used to fit all the data in order to determine the maximum efficacy and half-maximum effective concentration (EC50) in relation to S1P.

Ozanimod (RPC1063) was a particular agonist for S1P1 and S1P5 receptors. The suppression of cAMP generation and [35S]-GTPγS binding by S1P1 receptors had EC50 values of 160 ± 60 pM and 410 ± 160 pM, respectively. In relation to the S1P5 receptor, ozanimod's 83% Emax value was 11 ± 4.3 nM. After one hour of incubation, RPC1063 virtually completely and persistently reduced S1P1 receptor re-expression on the cell surface in S1P1 receptor-HEK293T cells. This was observed at concentrations greater than 10 nM.

Animal/Disease Models: Experimental Autoimmune Encephalomyelitis Model[1]
Doses: 0.05, 0.2, or 1 mg/kg
Route of Administration: oral gavage; 0.05, 0.2, or 1 mg/kg; one time/day; for 14 days
Experimental Results: Attenuated body weight loss, terminal disease scores were Dramatically attenuated with the 0.2 and 1 mg/kg doses and ALCs were Dramatically decreased in all dose groups. decreased spinal cord inflammation and demyelination, as well as attenuated the number of spinal cord apoptotic cells, and Dramatically decreased the levels of circulating neurofilament light at the top dose of 1 mg/kg.

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Animal/Disease Models: Cuprizone/Rapamycin Demyelination Model[1]
Doses: 5 mg/kg
Route of Administration: oral gavage; 5 mg/kg; once-daily
Experimental Results: Protected neuronal axons, preventing breakage and ovoid formation in the corpus callosum of CPZ/Rapa treated mice. Dramatically attenuated the extent to which the corpus callosum demonstrated decreased myelin content as visualized by MRI. Did not result in enhanced myelin content.

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MOG35–55 Experimental Autoimmune Encephalomyelitis Model[1]
Experimental autoimmune encephalomyelitis (EAE) was induced in 10-week-old female C57BL/6 mice (Taconic Biosciences, Rensselaer, NY) by subcutaneous immunization with an emulsion of myelin oligodendrocyte glycoprotein 35–55 (MOG35–55) in complete Freund’s adjuvant (CFA) followed by intraperitoneal injections of pertussis toxin 2 and 24 hours later. Mice received two subcutaneous injections, one in the upper and one in the lower back, of 0.1 ml MOG35–55/CFA emulsion per site, and both intraperitoneal injections of pertussis toxin were 100 ng per dose at a volume of 0.1 ml per dose. The study was performed at Hooke Laboratories (Lawrence, MA) using Hooke Kit MOG35–55/CFA Emulsion PTX number EK-2110. Female mice were selected for EAE experimentation since more females than males suffer clinically with MS as well as other autoimmune diseases (Voskuhl, 2011). EAE is an immune-driven preclinical model of MS, and female mice are reported to experience greater severity of disease (Papenfuss et al., 2004; Rahn et al., 2014).
Mice were assessed daily and upon the first emergence of signs of disease, randomized into treatment groups (n = 12) on the basis of comparable group average values for time of EAE onset and disease score at the onset of treatment. Dosing was initiated on the first day of EAE disease via once daily oral gavage of vehicle (5% v/v DMSO, 5% v/v Tween20, 90% v/v Milli-Q water; 5 ml/kg) or ozanimod at doses of 0.05, 0.2, or 1 mg/kg for 14 consecutive days. Efficacy was evaluated by recording daily visual EAE disease scores, as described previously by Scott et al. (2016), as well as body weight measurement three times per week. Approximately 24 hours after the final dose, a blood sample was collected in EDTA coagulant for the assessment of absolute numbers of circulating lymphocytes by differential count, and a separate plasma sample was processed and stored at −80°C for subsequent analysis of neurofilament light by Quanterix (Lexington, MA) using the Simoa NF-light Advantage kit (102258). Mice were anesthetized and perfused with phosphate buffered saline, and the spinal cords collected and stored in 10% buffered formalin for imaging analysis. For each mouse spinal cord, three hematoxylin and eosin sections were prepared and analyzed for the number of inflammatory foci (approximately 20 cells per foci), estimation of demyelinated area (scores of 0–5 representing <5%, 5 to 20%, 20 to 40%, 40 to 60%, 60 to 80%, and 80 to 100% demyelinated area, respectively, and as defined by interruption of normal structure such as pallor and vacuolation consistent with edema and demyelination, as well as dilated axons) and apoptotic cell counts. Histologic analysis was performed by a pathologist blinded to the experimental design and readouts.

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Cuprizone/Rapamycin Demyelination Model: Neuroprotection and Remyelination[1]
Cuprizone/rapamycin-induced demyelination was initiated in 8-week-old male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) by ad libitum access to normal rodent diet (Harlan Teklad, Madison, WI) containing cuprizone (0.3% w/w) for a period of 6 weeks with once daily intraperitoneal injection of rapamycin. Rapamycin was prepared fresh daily at 10 mg/kg at a volume of 5 ml/kg in 5% v/v pure ethanol/5% v/v Tween 80/5% PEG1000, aqueous. Age-matched control mice had ad libitum access to the same diet not containing cuprizone and received daily intraperitoneal injection with vehicle. Mice were group housed 4 to 5 per cage, and fresh food was provided three times weekly. All mice had ad libitum access to reverse osmosis filtered, acidified palatable drinking water at a pH level of 2.5 to 3.0. The study was performed at Renovo Neural, Inc. (Cleveland, OH). Male mice were chosen for the demyelination model since a number of studies have reported that females are more resistant to the toxin and hence more robust demyelination is observed in males (MacArthur and Papanikolaou, 2014).

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After 2 weeks of acclimation, mice were randomly assigned to dose groups and received once-daily oral gavage administration of vehicle (5% v/v DMSO, 5% v/v Tween20, 90% v/v Milli-Q water; 5 ml/kg) or ozanimod 5 mg/kg after the dosing and sample collection/testing regimen depicted in Fig. 1. For the assessment of ozanimod on neuroprotection and demyelination, dosing was initiated on day 1 concurrent with cuprizone/rapamycin and continued daily for 6 weeks. For the assessment of ozanimod’s effect on remyelination, daily dosing was also initiated on day 1 but continued beyond the 6-week cuprizone/rapamycin challenge for a further 12-week period (weeks 7–18 of the study). Mice in the remyelination arms of assessment were discontinued from cuprizone diet and daily intraperitoneal rapamycin injection at the end of the 6-week challenge period and returned to normal rodent diet.
In vivo brain magnetic resonance imaging (MRI) was used to monitor the effects of the 6-week cuprizone/rapamycin treatment and after a further 12 weeks after the demyelination challenge (study weeks 6 and 18). Mice were imaged on a 7T/20 Bruker-Biospec system to acquire high quality three-dimensional MRI longitudinally in the same animals. Mice were sedated with 1 to 3% isoflurane with adjusted respiration rate of approximately 50 to 80 breaths per minute. Level of induction was constantly monitored during the MRI. The heated bed of the system-maintained animals at 35°C for the duration of the experiment. At the end of the scan, isoflurane was discontinued, and the mouse was returned to its cage to recover. To quantify changes in myelin loss sensitive magnetization transfer ratio, magnetization transfer-weighted MRI images were acquired. After outlier removal based on image quality and animal stability in the MRI machine, group sizes were 6 to 9 mice.
Mice were not treated on the day of termination. Twelve animals per group (six for age-matched controls) were euthanized after 6 weeks of cuprizone/rapamycin treatment, whereas the remaining animals continued on treatment until study weeks 9, 12, and 18, at which point these animals were sacrificed and samples collected (n = 6 per group for study weeks 9 and 12, n = 12 per group for study week 18). Animals were perfused with phosphate buffered saline, and the brains were removed and fixed in 4% paraformaldehyde overnight at 4°C. The brains were dissected using a custom brain-slicing mold and further trimmed to isolate the corpus callosum, which was then fixed in a 2.5% glutaraldehyde/4% paraformaldehyde mix for at least 12 hours. A small piece of corpus callosum was identified by specific morphologic landmarks, then cut and embedded in Epon resin. The rostral and caudal part of the brain (either side of the slice) was placed in a cryoprotection solution at 4°C overnight. The rostral section was sectioned with a microtome to generate 30 μm thick free-floating sections; two sections per animal were stained with either SMI-32 (nonphosphorylated neurofilament H) or myelin proteolipid protein (PLP) antibodies and visualized by 3,3′-diaminobenzidine. The SMI-32–stained sections were evaluated to assess axonal ovoids in the white matter (corpus callosum), and the PLP-stained sections were evaluated to assess the extent of remyelination in the hippocampus and cortex.

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Pharmacokinetics[1]
The pharmacokinetic profiles of ozanimod and its primary active rodent metabolite, RP101075, are similar in male and female C57BL/6J mice and so were assessed in plasma and brains of 8-week-old male C57BL/6J mice (Jackson Laboratories) after daily oral dosing with ozanimod for 7 consecutive days. Ozanimod was dosed at either 1 or 5 mg/kg in the same vehicle as used for the MOG35–55 EAE and cuprizone/rapamycin in vivo efficacy studies and terminal plasma, and brain samples were collected 3, 6, and 24 hours after the seventh daily dose of ozanimod. Of note, in the clinical setting, the dosing of ozanimod involves a dose titration to avoid and potential risk of mechanism-based bradycardia, but this is not adopted when assessing efficacy in preclinical studies where dosing is initiated straight away with the dose to be assessed without titration. Brains were homogenized in acetonitrile at a 1:3 (w/v) ratio using a Biospec Bead Beater-16 with 1 mm glass beads and proteins precipitated further with a 1:10 dilution in acetonitrile to 1:30 (w/v) final. Plasma proteins were precipitated with acetonitrile at a 1:3 ratio (v/v). Samples were centrifuged and supernatants were analyzed by liquid chromatography–mass spectrometry (LC-MS/MS). For the tissue analysis, a standard curve was prepared using homogenized brain samples from untreated animals. A 10-point standard curve of ozanimod or RP101075 spanning a range of 0.046 nM to 500 nM was included with each bioanalytical run using a Kinetex C18 2.6μ 30 × 3 mm column (Phenomenex Inc., Torrance, CA), 0.1% formic acid in deionized H2O mobile phase A, and 0.1% formic acid in acetonitrile mobile phase B. Data were collected and analyzed using Analyst software version 1.5.1.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Although ozanimod and its active metabolites are highly bound in maternal plasma and unlikely to reach the breastmilk in large amounts, it is potentially toxic to the breastfed infant. Because there is no published experience with ozanimod during breastfeeding, expert opinion generally recommends that the closely related drug fingolimod should be avoided during breastfeeding, especially while nursing a newborn or preterm infant. Some guidelines recommend avoiding ozanimod during breastfeeding because of a lack of data; however, the manufacturer's labeling does not recommend against the use of ozanimod in breastfeeding.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

[1]. 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 Subtyp.

Ozanimod Hydrochloride is the hydrochloride salt form of ozanimod, an orally bioavailable sphingosine-1-phosphate (S1P) receptors 1 (S1PR1, S1P1) and 5 (S1PR5, S1P5) modulator, with potential anti-inflammatory and immunomodulating activities. Upon oral administration, ozanimod selectively targets and binds to S1PR1 on lymphocytes and induces S1PR1 internalization and degradation. This results in the sequestration of lymphocytes in lymph nodes. By preventing egress of lymphocytes, ozanimod reduces both the amount of circulating peripheral lymphocytes and the infiltration of lymphocytes into target tissues. This prevents a lymphocyte-mediated immune response and may reduce inflammation. S1PR1, a G-protein coupled receptor, plays a key role in lymphocyte migration from lymphoid tissues. Modulation of S1PR5 by ozanimod may be neuroprotective.
See also: Ozanimod (has active moiety).

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Drug Indication
Multiple sclerosisZeposia is indicated for the treatment of adult patients with relapsing remitting multiple sclerosis (RRMS) with active disease as defined by clinical or imaging features. Ulcerative colitisZeposia is indicated for the treatment of adult patients with moderately to severely active ulcerative colitis (UC) who have had an inadequate response, lost response, or were intolerant to either conventional therapy or a biologic agent.
Treatment of multiple sclerosis
Treatment of Crohn's disease
Treatment of ulcerative colitis

C23H25CLN4O3

440.92

440.161

C, 62.65; H, 5.72; Cl, 8.04; N, 12.71; O, 10.89

1618636-37-5

Ozanimod;1306760-87-1

91618104

White to off-white solid powder

3

7

7

31

609

1

Cl[H].O1C(C2C([H])=C([H])C(=C(C#N)C=2[H])OC([H])(C([H])([H])[H])C([H])([H])[H])=NC(C2C([H])=C([H])C([H])=C3C=2C([H])([H])C([H])([H])[C@]3([H])N([H])C([H])([H])C([H])([H])O[H])=N1

KBXLMOYQNDMHQT-QGZVFWFLSA-N

InChI=1S/C23H24N4O3.ClH/c1-14(2)29-21-9-6-15(12-16(21)13-24)23-26-22(27-30-23)19-5-3-4-18-17(19)7-8-20(18)25-10-11-28;/h3-6,9,12,14,20,25,28H,7-8,10-11H2,1-2H3;1H/t20-;/m0./s1

(S)-5-(3-(1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile hydrochloride

RPC-1063 HCl; RPC1063 hydrochloride; RPC-1063 hydrochloride; Zeposia; UNII-3UPR33JAAM; 3UPR33JAAM

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 200 mg/mL (453.60 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (11.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (11.34 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 5 mg/mL (11.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)