Ceralifimod (also known as ONO-4641) is a novel sphingosine-1-phosphate (S1P) receptor agonist selective for S1P1 and S1P5. While it has been demonstrated that treating autoimmune diseases like relapsing-remitting multiple sclerosis (RRMS) with modulation of the S1P1 receptor is effective, activation of the S1P3 receptor has also been linked to certain negative effects. The clinical candidate ceralifimod was found to be potent in a peripheral lymphocyte lowering (PLL) test in mice (ED50 = 0.029 mg/kg, 24 hours after oral dosing) and to have >30,000-fold selectivity for S1P1 over S1P3.

Physicochemical Properties

Molecular Formula	C27H33NO4
Molecular Weight	435.56
Exact Mass	435.241
CAS #	891859-12-4
Related CAS #	891859-12-4
PubChem CID	11502996
Appearance	White to off-white solid powder
LogP	4.9
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	9
Heavy Atom Count	32
Complexity	671
Defined Atom Stereocenter Count	0
SMILES	O=C(C1CN(CC2CCC3C(=CC=C(C=3)OCC3C(OC)=CC(CCC)=CC=3)C=2C)C1)O
InChi Key	QDDQIPUKAXBMBX-UHFFFAOYSA-N
InChi Code	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)
Chemical Name	1-[[6-[(2-methoxy-4-propylphenyl)methoxy]-1-methyl-3,4-dihydronaphthalen-2-yl]methyl]azetidine-3-carboxylic acid
Synonyms	ONO-4641; ONO 4641; ONO4641
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	hS1P1 ( EC50 = 27.3 pM ); hS1P5 ( EC50 = 334 pM )
ln Vitro	In vitro activity: Ceralifimod (ONO-4641) has an agonistic action for both S1P1 and S1P5, and its agonistic action for S1P1 is the same in rats and humans. Ceralifimod (ONO-4641) also down-regulates S1P1 in a concentration-dependent way, with a 90% effect at 25 nM[1].
ln Vivo	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].
Enzyme Assay	Ceralifimod (also known as ONO-4641) is a novel selective agonist of the sphingosine-1-phosphate (S1P) receptor that targets only S1P1 and S1P5.
Animal Protocol	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].
References	[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.
Additional Infomation	Ceralifimod is under investigation in clinical trial NCT01226745 (Phase 2 Extension Trial in Patients With Relapsing-remitting Multiple Sclerosis (RRMS)).

Solubility Data

Solubility (In Vitro)

DMSO: ~2.9 (~6.6 mM) Water: N/A Ethanol: N/A

Solubility (In Vivo)

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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2959 mL	11.4795 mL	22.9589 mL
	5 mM	0.4592 mL	2.2959 mL	4.5918 mL
	10 mM	0.2296 mL	1.1479 mL	2.2959 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.