S1PR1 ( EC50 = 0.39 nM ); S1PR5 ( EC50 = 0.98 nM ); S1PR4 ( EC50 = 750 nM ); S1PR3 ( EC50 > 1000 nM ); S1PR2 ( EC50 > 10000 nM )

BAF312 suppresses encephalomyelitis (EAE) in rats effectively by internalizing S1P1 receptors and making them immune to the lymph node egress signal. When administered as a preventive or therapeutic dose in mice at 0.3 mg/kg, BAF312 significantly lowers clinical scores.

After the cells are homogenized, they are centrifuged for 30 minutes at 4°C at 26900 × g. In 20 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, and 0.1% fat-free BSA, membranes are re-suspended at a protein concentration of 2-3 mg/mL. Membranes (75 mg protein/mL in 50 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 20 μg/mL saponin and 0.1% fat-free BSA; pH 7.4), 5 mg/mL with a wheat-germ agglutinin-coated scintillation proximity assay-bead, and 10 μM GDP for 10–15 min) are used in the GTPγ[35S] binding technique. 200 pM GTPγ[35S] is added to initiate the GTPγ[35S]-binding reaction. Following 120 minutes at room temperature, the plates undergo a 10-minute 300 × g centrifugation before being tallied.

Analysis of CHO cells using flow cytometry reveals antagonist-mediated internalization of S1P1 receptors. An agonist is added to standard culture medium and Myc-tag hS1P1 cells are incubated for 1 hour at 37°C. The cells are then washed with PBS. While one aliquot is left in culture medium (without an agonist) at 37°C for three hours, the other is kept on ice for three hours (or twelve hours). First, the cells are incubated for 60 minutes at 4°C with either 4 μg/mL monoclonal mouse anti-C-myc IgG1 antibody or isotype control mouse IgG1. Next, they are incubated with 1 μg/mL of goat anti-mouse secondary conjugates that have been labeled with Alexa488, which is a fluorescent dye. Using 10,000 viable cells per sample, the cells are measured using flow cytometry.

BAF312 was tested in a rat experimental autoimmune encephalomyelitis (EAE) model. Electrophysiological recordings of G-protein-coupled inwardly rectifying potassium (GIRK) channels were carried out in human atrial myocytes. A Phase I multiple-dose trial studied the pharmacokinetics, pharmacodynamics and safety of BAF312 in 48 healthy subjects.[1]

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Suspended in 1% aqueous carboxy-methylcellulose; 0.03, 0.3 and 3 mg/kg; oral givage

Encephalomyelitis (EAE) model rat

Absorption, Distribution and Excretion
Following oral administration of immediate-release sinimod, the time to reach maximum plasma concentration (Cmax) (Tmax) is approximately 4 hours (range 3–8 hours). Sinimod is highly absorbed (≥70%). The absolute oral bioavailability of sinimod is approximately 84%. Steady-state plasma concentrations are reached approximately 6 days after a single daily dose of sinimod. Effect of Food on Absorption Food intake delays sinimod absorption (median Tmax increases by approximately 2–3 hours). Food intake has no effect on systemic exposure (Cmax and AUC) of sinimod. Therefore, sinimod administration is not affected by food intake. Sinimod is primarily metabolized and subsequently eliminated from systemic circulation via bile/feces. Unmetabolized sinimod is not detected in urine. Sinimod is distributed throughout the body with a mean volume of distribution of 124 liters. In humans, the plasma concentration of sinimod is 68%. Animal studies have shown that sinimod readily crosses the blood-brain barrier. In patients with multiple sclerosis, the estimated epigenetic systemic clearance is 3.11 L/h. Metabolites/Metabolites: Sinimod is primarily metabolized by CYP2C9 enzymes (79.3%), followed by CYP3A4 enzymes (18.5%). The pharmacological activities of the major metabolites M3 and M17 are not expected to affect the clinical efficacy and safety of sinimod in humans. Biological Half-Life: The epigenetic elimination half-life is approximately 30 hours.

Hepatotoxicity
Elevated serum ALT levels were common in large controlled trials of sinimod in patients with multiple sclerosis, typically occurring within the first 3 months of treatment. These elevations were usually mild and asymptomatic, and ALT levels often returned to baseline within 3 months of continued treatment or discontinuation. 6% to 8% of patients in the sinimod treatment group reported transaminase elevations exceeding three times the upper limit of normal (ULN), compared to less than 2% in the placebo group. No cases of acute hepatitis or clinically significant liver injury occurred in these premarket clinical trials, but 1% of subjects discontinued treatment due to abnormal liver function tests. While sinimod is associated with lymphopenia and long-term treatment is associated with a risk of relapse of herpes simplex and herpes zoster virus infections, it has not been found to be associated with hepatitis B virus relapse, although one case of hepatitis B virus relapse has been reported with fingolimod. Therefore, mild to moderate transient elevations of serum enzymes during treatment are not uncommon, but there have been no reports of clinically significant liver injury with jaundice caused by sinimod, despite limited clinical experience.
Probability Score: E (Suspected but not confirmed cause of clinically significant liver injury).

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Effects during Pregnancy and Lactation
◉ Overview of Use During Lactation
Although sinimod has a high binding rate in maternal plasma and is unlikely to enter breast milk in large quantities, it still poses potential toxicity to breastfed infants. Since there is currently no published experience regarding the use of sinimod during lactation, expert opinion generally recommends avoiding the use of fingolimod, a drug closely related to it, during lactation, especially when breastfeeding newborns or premature infants. However, the manufacturer's label does not recommend that sinimod be contraindicated in breastfeeding women.
◉ Effects on Breastfed Infants
No relevant published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No relevant published information found as of the revision date.
◈ What is Sinimod?
Sinimod (Mayzent®) is a medication approved for the treatment of relapsing-remitting multiple sclerosis (MS), including clinically isolated syndrome, relapsing-remitting multiple sclerosis, and active secondary progressive multiple sclerosis. For information about multiple sclerosis, please see the MotherToBaby case sheet: https://mothertobaby.org/fact-sheets/multiple-sclerosis/. Sometimes, when people find out they are pregnant, they may consider changing how they take the medication or even stopping it altogether. However, it is essential to talk to your healthcare provider before changing how you take this medication. Your healthcare provider can discuss with you the benefits of treating your condition and the risks of not treating the condition during pregnancy.
◈ I am taking sinimod, but I want to stop taking it before I get pregnant. How long will this medication stay in my body?
Everyone clears the medication at a different rate. For healthy adults, it takes an average of about 10 days to clear most of the sinimod from the body.
◈ I am taking sinimod. Will it make it harder for me to get pregnant?
It is currently unclear whether sinimod makes it harder to get pregnant.
◈ Does taking sinimod increase the risk of miscarriage?
Miscarriage is common and can occur in any pregnancy for many reasons. According to the product label, animal studies report that taking sinimod increases the risk of pregnancy loss. There are currently no studies in human pregnant women to determine whether sinimod increases the risk of miscarriage.
◈ Does taking sinimod increase the risk of birth defects?
There is a 3-5% risk of birth defects in each pregnancy, known as background risk. According to the product label, animal studies report that taking sinimod increases the risk of birth defects. There are currently no studies in human pregnant women to determine whether sinimod further increases the risk of birth defects on top of the background risk.
◈ Will taking sinimod during pregnancy cause other pregnancy-related problems?
According to the product label, animal studies report that taking sinimod may lead to low birth weight in newborns. Currently, there are no studies on human pregnant women to determine whether sinimod increases the risk of pregnancy-related problems, such as preterm birth (delivery before 37 weeks of gestation) or low birth weight (birth weight less than 5 pounds 8 ounces [2500 grams]).
◈ Will taking sinimod during pregnancy affect a child's future behavior or learning abilities?
There are currently no studies to determine whether sinimod causes behavioral or learning problems in children.
◈ Breastfeeding while taking sinimod:
There are currently no studies on taking sinimod while breastfeeding. It is unclear whether sinimod passes into breast milk and what effects it has on breastfed infants. If you are taking sinimod while breastfeeding and suspect your infant has any symptoms, contact your child's healthcare provider. Be sure to consult your healthcare provider about all breastfeeding-related questions.
◈ If men take sinimod, will it affect fertility (the ability to impregnate a partner) or increase the risk of birth defects?
There are currently no studies to explore whether sinimod affects male fertility or increases the risk of birth defects. Generally, exposure to sinimod by the father or sperm donor is unlikely to increase the risk of pregnancy. For more information, please see the “Paternal Exposure to Sinimod” fact sheet on the MotherToBaby website: https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

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Protein Binding
In healthy patients and patients with impaired liver or kidney function, the protein binding rate of sinimod is greater than 99.9%. Due to the high binding rate of sinimod to plasma proteins, hemodialysis is unlikely to alter the total and free concentrations of sinimod, and therefore, no dose adjustment is expected.

[1]. Br J Pharmacol . 2012 Nov;167(5):1035-47.

[2]. J Immunol . 2013 Apr 1;190(7):3533-40.

Pharmacodynamics
Immune System Effects Sinimod causes a dose-dependent decrease in peripheral blood lymphocyte count within 6 hours of the first dose, due to insufficient lymphocyte release leading to reversible accumulation of lymphocytes in lymphoid tissues. This can reduce inflammation associated with multiple sclerosis. Lymphocyte counts return to normal in 90% of patients within 10 days of discontinuation. Effects on Heart Rate and Rhythm Sinimod causes a transient decrease in heart rate and atrioventricular conduction upon initiation of treatment. The most significant decrease in heart rate occurs within 6 hours of administration. Sinimod treatment does not alter autonomic cardiac responses, including diurnal variations in heart rate and responses to exercise. Effects on Lung Function A dose-dependent decrease in absolute forced expiratory volume within 1 second was observed in patients receiving sinimod, and this was higher than in patients receiving placebo.

C62H74F6N4O10

632.68

1148.53

C, 64.80; H, 6.49; F, 9.92; N, 4.88; O, 13.92

1234627-85-0

Siponimod; 1230487-00-9

44599207

Solid powder

111-112

4.8

1

8

9

37

777

0

FC(C1C=C(CO/N=C(/C)\C2C=CC(=C(C=2)CC)CN2CC(C(=O)O)C2)C=CC=1C1CCCCC1)(F)F.FC(C1C=C(CO/N=C(/C)\C2C=CC(=C(C=2)CC)CN2CC(C(=O)O)C2)C=CC=1C1CCCCC1)(F)F.OC(/C=C/C(=O)O)=O

JNLIKIBISICTMS-PEJBKAKVSA-N

InChI=1S/2C29H35F3N2O3.C4H4O4/c2*1-3-21-14-23(10-11-24(21)15-34-16-25(17-34)28(35)36)19(2)33-37-18-20-9-12-26(22-7-5-4-6-8-22)27(13-20)29(30,31)32;5-3(6)1-2-4(7)8/h2*9-14,22,25H,3-8,15-18H2,1-2H3,(H,35,36);1-2H,(H,5,6)(H,7,8)/b2*33-19+;2-1+

(E)-but-2-enedioic acid;1-[[4-[(E)-N-[[4-cyclohexyl-3-(trifluoromethyl)phenyl]methoxy]-C-methylcarbonimidoyl]-2-ethylphenyl]methyl]azetidine-3-carboxylic acid

NVP-BAF-312; NVP-BAF 312; NVP-BAF312-NX; NVP-BAF312; NVP-BAF312-AEA; Siponimod fumarate; Siponimod; Mayzent; WHO 9491; WHO-9491; WHO9491; BAF-312; BAF 312; Siponimod hemifumarate; NVP-BAF312-AEA; Siponimod hemifumaric acid; Z7G02XZ0M6; Siponimod fumarate (USAN); Siponimod fumarate [USAN];BAF312

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)