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

Ozanimod (RPC-1063) (oral gavage; 0.05, 0.2, or 1 mg/kg; once daily; for 14 consecutive days) exposures sufficient to engage S1P1, but not S1P5, lead to decreased levels of circulating lymphocytes, disease scores, and body weight loss; lessen the number of apoptotic cells, inflammation, and demyelination in the spinal cord; and decrease the amount of neurofilament light, a marker of neuronal degeneration, in circulation[1]. Ozanimod (oral gavage; 5 mg/kg; once-daily) does not promote enhanced remyelination following intoxication, but it does stop axonal degradation and myelin loss during toxin challenge[1]. Ozanimod (oral, 1 or 5 mg/kg, for 7 days) exhibits favorable pharmacoKinetics in mice[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.

Dissolved in 5% DMSO, 5% Tween-20, 90% 0.1N HCl; 0.1-3 mg/kg; oral givage; MOG-induced EAE model in C57Bl6 mice, TNBS model of inflammatory bowel disease in male Sprague Dawley rats, and Naive CD4+CD45Rbhi T cell adoptive transfer model in SCID mice

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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.

Absorption, Distribution and Excretion
Ozanimod is absorbed in the gastrointestinal tract after oral administration. The Cmax of ozanimod is 0.244 ng/mL and is achieved at 6 to 8 hours after administration, reaching steady-state at about 102 hours after administration. The AUC is 4.46 ng*h/mL. Its delayed absorption reduces effects that may occur after the first dose, such as heart rate changes. The peak plasma concentration of ozanimod is low due to a high volume of distribution.
The kidneys are not a major source of elimination for ozanimod. After a 0.92 mg dose of radiolabeled ozanimod was administered, about 26% of the labeled drug was accounted for in the urine and 37 % in the feces, mainly in the form of inactive metabolites.
The average volume of distribution of ozanimod is 5590L. Another reference mentions a volume of distribution ranging from 73-101 L/kg. This drug crosses the blood-brain barrier.
The mean apparent oral clearance of ozanimod, according to prescribing information, is 192 L/h. Another reference indicates an oral clearance of 233 L/h.

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Metabolism / Metabolites
Ozanimod has two major active metabolites CC112273 and CC1084037 and minor active metabolites such as RP101988, RP101075, and RP101509, which target the S1P1 and S1P5 receptors. The enzymes involved in the metabolism of ozanimod include ALDH/ADH, NAT-2, Monoamine Oxidase B, and AKR 1C1/1C2. After metabolism, ozanimod (6%), CC112273 (73%), and CC1084037 (15%) are accounted for in the circulation.

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Biological Half-Life
The half-life of ozanimod ranges from 17-21 hours.

Hepatotoxicity
In large controlled trials of ozanimod in patients with multiple sclerosis, serum ALT elevations were common (~5% of recipients) but were typically mild and asymptomatic, and they returned to baseline values within a few months of stopping and often even with continuation of therapy. Aminotransferase elevations above 3 times upper limit of normal (ULN) were reported in 4% of ozanimod recipients compared to less than 1% of placebo recipients and elevations above 5 times ULN in 1%. In these prelicensure clinical trials, there were no cases of acute hepatitis or clinically apparent liver injury but elevations in liver tests led to discontinuation in approximately 1% of subjects. While ozanimod is associated with lymphopenia and long term therapy is associated with risk for reactivation of herpes simplex and zoster infections, it has not been linked to cases of reactivation of hepatitis B although one such instance has been reported with fingolimod. Thus, mild-to-moderate and transient serum enzyme elevations during therapy are not uncommon, but clinically apparent liver injury with jaundice due to ozanimod has not been reported, although the clinical experience with its use has been limited.
Likelihood score: E* (suspected but unproven cause of clinically apparent liver injury).

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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.

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Protein Binding
The plasma protein binding of ozanimod and its metabolites exceeds 98%.

[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 Subtypes 1 and 5. J Pharmacol Exp Ther. 2021 Dec;379(3):386-399.

Pharmacodynamics
Ozanimod reduces circulating lymphocytes that cause the neuroinflammation associated with MS, reducing debilitating symptoms and, possibly, disease progression. During clinical trials, ozanimod reduced MS-associated brain volume loss in several regions. Ozanimod causes the sequestration of peripheral lymphocytes, reducing circulating lymphocytes in the gastrointestinal tract.

C23H24N4O3

404.46

404.184

C, 68.30; H, 5.98; N, 13.85; O, 11.87

1306760-87-1

Ozanimod hydrochloride; 1618636-37-5

52938427

White to off-white solid powder

1.3±0.1 g/cm3

648.3±65.0 °C at 760 mmHg

134-137

345.9±34.3 °C

0.0±2.0 mmHg at 25°C

1.635

4.25

2

7

7

30

609

1

N#CC1=CC(C2=NC(C3=CC=CC4=C3CC[C@@H]4NCCO)=NO2)=CC=C1OC(C)C

XRVDGNKRPOAQTN-FQEVSTJZSA-N

InChI=1S/C23H24N4O3/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/t20-/m0/s1

5-[3-[(1S)-1-(2-hydroxyethylamino)-2,3-dihydro-1H-inden-4-yl]-1,2,4-oxadiazol-5-yl]-2-propan-2-yloxybenzonitrile

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.18 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 25.0 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: ≥ 2.5 mg/mL (6.18 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 5%DMSO + Corn oil: 2.0mg/ml (4.94mM)

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