hS1P1 ( EC50 = 27.3 pM ); hS1P5 ( EC50 = 334 pM )
Sphingosine 1-phosphate receptor 1 (S1P₁, EC₅₀ = 0.0273 nM); Sphingosine 1-phosphate receptor 5 (S1P₅, EC₅₀ = 0.334 nM) [1]

Ceralifimod (ONO-4641) 0.03 and 0.1 mg/kg groups' clinical scores are still lower than those of the control group. The Ceralifimod (ONO-4641) groups experience a dose-dependent decrease in maximum clinical scores, with scores in the 0.03 and 0.1 mg/kg groups being significantly lower than those in the control group. Particularly, in seven out of eight animals in the Ceralifimod (ONO-4641) 0.1 mg/kg group, paralysis is completely inhibited. Perivascular blood lymphocyte counts in normal NOD mice are reduced by roughly 20, 60, and 80% at 24 hours following a single oral dosage of 0.01 mg/kg, 0.03 mg/kg, and 0.1 mg/kg of Ceralifimod (ONO-4641), respectively. Two of the nine animals in the control group of the NOD mouse model of relapsing-remitting EAE die, and the relapse rate is 90.0%. The control group's cumulative clinical score is 65.4±18.50. On the other hand, no animals in the Ceralifimod (ONO-4641) 0.1 mg/kg group experience a relapse; at 0.1 mg/kg, Ceralifimod totally prevents relapse. Two of the nine animals in the 0.01 mg/kg group in the Ceralifimod (ONO-4641) groups pass away[1]. In female Lewis rats, oral administration of Ceralifimod (doses: 0.01, 0.03, 0.1, 0.3, 1 mg/kg) dose-dependently decreased peripheral blood lymphocyte counts, with the effect observed as early as 2 hours post-administration and persisting for up to 120 hours [1] - In a rat experimental autoimmune encephalomyelitis (EAE) model: Ceralifimod was administered orally once daily at doses of 0.03 and 0.1 mg/kg from day 4 to 21 post-immunization. It suppressed the onset of neurological symptoms (assessed by clinical scoring) and dose-dependently inhibited lymphocyte infiltration into the lumbosacral spinal cord (measured by perivascular cellular infiltrate count in H&E-stained specimens) [1] - In a non-obese diabetic (NOD) mouse model of relapsing-remitting EAE: Ceralifimod was administered orally once daily at doses of 0.01, 0.03, and 0.1 mg/kg for 8 weeks. It significantly prevented disease relapse and reduced the cumulative clinical score compared to the control group [1] - In a rat adoptive transfer experiment: Oral administration of Ceralifimod (0.1 mg/kg) reduced the number of transferred lymphocytes in peripheral blood, while increasing their retention in inguinal, axillary, and mesenteric lymph nodes, confirming inhibition of lymphocyte egress from secondary lymphoid tissues [1]

Ceralifimod (also known as ONO-4641) is a novel selective agonist of the sphingosine-1-phosphate (S1P) receptor that targets only S1P1 and S1P5.

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S1P receptor agonistic activity assay: CHO-K1 cells stably expressing human S1P₁ or S1P₅ were seeded and incubated at 37°C for 60 minutes with serial concentrations of Ceralifimod (0.008–125 nM) or control. Non-transfected CHO-K1 cells were used as negative controls. Agonistic activity was evaluated based on receptor-mediated signaling responses, and EC₅₀ values were calculated from dose-response curves [1]
- S1P₁ receptor down-regulation assay: hS1P₁-expressing CHO-K1 cells were treated with Ceralifimod (0.008–125 nM), S1P (10–10,000 nM), or SEW2871 (0.8–12,500 nM) under standard incubation conditions. After treatment, cells were stained with S1P₁-specific fluorescent antibodies, and receptor expression levels were quantified as the percentage of mean fluorescence intensity relative to the control group (set to 100%) [1]

S1P₁ receptor expression analysis: hS1P₁-expressing CHO-K1 cells were divided into experimental groups (treated with Ceralifimod at different concentrations) and control groups (untreated or treated with reference compounds). Cells were incubated at 37°C for a set period, then harvested and washed. Fluorescently labeled primary antibodies against S1P₁ were added, and the cells were incubated in the dark. Flow cytometry was used to measure fluorescence intensity, and the percentage of S1P₁ receptor expression was calculated to assess down-regulation efficacy [1]

Rats: Inducer injections into the footpad at a volume of 0.1 mL are used to immunize female Lewis rats subcutaneously. Oral administration of ceralifimod (ONO-4641: 0.01, 0.03, and 0.1 mg/kg), prednisolone (3 mg/kg), or 0.5% MC is done once a day for immunization days 4 through 11. The clinical score is specifically determined by grading the degree of paralysis on a scoring scale.
Mouse: The inducer is injected into the left footpad of NOD mice at a volume of 0.05 mL. Forty-eight hours later, another PTX injection is given. Once every day for eight weeks, either 0.5% MC or Ceralifimod (ONO-4641: 0.01, 0.03, or 0.1 mg/kg) is given orally to each group of animals that achieve remission following the initial onset. The degree of paralysis determines how neurological symptoms are rated. Animals that are dead are given a clinical score of 5 until the end of the observation[1].

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Rat peripheral blood lymphocyte count experiment: Female Lewis rats (n=6 per group) were given a single oral dose of Ceralifimod (0.01, 0.03, 0.1, 0.3, 1 mg/kg) or 0.5% methylcellulose (control). Blood samples were collected via the tail vein before administration (0 hour) and at 2, 4, 8, 12, 24, 48, 72, 96, and 120 hours post-administration. Peripheral blood lymphocyte counts were measured at each time point to evaluate dose-dependent effects [1]
- Rat EAE model experiment: Female Lewis rats (n=8 per group) were immunized by subcutaneous injection of inducer into the footpad. From day 4 to 21 post-immunization, rats were administered Ceralifimod (0.01, 0.03, 0.1 mg/kg), prednisolone (3 mg/kg, positive control), or 0.5% methylcellulose (control) orally once daily. Neurological symptoms were assessed from day 9 to 22 post-immunization using a clinical scoring system. On day 16 post-immunization, spinal cords were harvested, fixed, and stained with H&E to count perivascular cellular infiltrates [1]
- NOD mouse relapsing-remitting EAE model experiment: NOD mice were grouped (n=9–10 per group) and administered Ceralifimod (0.01, 0.03, 0.1 mg/kg) or 0.5% methylcellulose (control) orally once daily for 8 weeks starting from the grouping day. Neurological symptoms were observed from day 7 post-immunization to day 56 of treatment, and cumulative clinical scores were calculated [1]
- Rat adoptive transfer experiment: Female Lewis rats were given a single oral dose of Ceralifimod (0.1 mg/kg) or 0.5% methylcellulose (control). After 2.5 hours, adoptive cell suspension was injected intravenously. Four hours post-administration, inguinal, axillary, and mesenteric lymph nodes were removed, and blood was collected. Flow cytometry was used to quantify the number of transferred lymphocytes in each tissue [1]

[1]. Efficacy and immunomodulatory actions of ONO-4641, a novel selective agonist for sphingosine 1-phosphate receptors 1 and 5, in preclinical models of multiple sclerosis. Clin Exp Immunol. 2013 Jan;171(1):54-62.

Ceralifimod is being studied in the clinical trial NCT01226745 (a phase II extension trial in patients with relapsing-remitting multiple sclerosis (RRMS)).

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Ceralifimod (ONO-4641) is a new generation of selective sphingosine-1-phosphate receptor 1 (S1P₁) and 5 (S1P₅) agonists[1]
- Its core mechanism of action is to downregulate S1P₁ receptors on lymphocyte membranes, inhibiting lymphocyte migration from secondary lymphoid tissues, thereby reducing peripheral blood lymphocyte counts and preventing lymphocyte infiltration into the central nervous system[1]
- Preclinical data indicate that Ceralifimod is effective in both transient and relapsing-remitting EAE models, supporting its potential efficacy in the treatment of multiple sclerosis[1]
- Ceralifimod has shown high efficacy and selectivity for… S1P₁ and S1P₅ have very low activity against other S1P receptor subtypes (not explicitly tested in this study)[1]

C27H33NO4

435.56

435.241

891859-12-4

11502996

White to off-white solid powder

4.9

1

5

9

32

671

0

O=C(C1CN(CC2CCC3C(=CC=C(C=3)OCC3C(OC)=CC(CCC)=CC=3)C=2C)C1)O

QDDQIPUKAXBMBX-UHFFFAOYSA-N

InChI=1S/C27H33NO4/c1-4-5-19-6-7-22(26(12-19)31-3)17-32-24-10-11-25-18(2)21(9-8-20(25)13-24)14-28-15-23(16-28)27(29)30/h6-7,10-13,23H,4-5,8-9,14-17H2,1-3H3,(H,29,30)

1-[[6-[(2-methoxy-4-propylphenyl)methoxy]-1-methyl-3,4-dihydronaphthalen-2-yl]methyl]azetidine-3-carboxylic acid

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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