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

Siponimod (1 g/kg; i.v.; single dose) exhibits low to moderate levels in monkeys but high levels in rats in metabolism studies with liver microsomes. The absolute bioavailability is 50 and 71% in the rat and monkey, respectively, indicating no major presystemic first pass metabolism[1]. Siponimod (0.3, 3 mg/kg; p.o.; once daily; 23 d) internalizes S1P1 receptors to suppress experimental autoimmune encephalomyelitis (EAE) in rats[2].

Centrifuging the cells at 26900 × g for 30 minutes at 4°C follows the homogenization of the cells. To re-suspend the membranes, mix 20 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, and 0.1% fat-free BSA at a protein concentration of 2-3 mg/mL. The membranes used in the GTPγ[35S] binding assay are 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. With the addition of 200 pM GTPγ[35S], the GTPγ[35S]-binding reaction is initiate. Once the plates have been at room temperature for 120 minutes, they are centrifuged at 300 × g for 10 minutes and then 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
The time (Tmax) to attain maximum plasma concentrations (Cmax) after oral administration of immediate-release oral doses of siponimod was found to be approximately 4 hours ( with a range 3 - 8 hours). Siponimod is heavily absorbed (at a rate greater than or equal to 70%). The absolute oral bioavailability of siponimod is about 84%. Steady-state concentrations were attained after approximately 6 days of daily administration of a single dose of siponimod. **Effects of food on absorption** Food ingestion leads to delayed siponimod absorption (the median Tmax increased by approximately 2-3 hours). Food intake has no effect on the systemic exposure of siponimod (Cmax and AUC). Therefore, siponimod may be taken without regard to food.
Siponimod is eliminated from the systemic circulation mainly due to metabolism, and subsequent biliary/fecal excretion. Unchanged siponimod was not detected in urine.
Siponimod distributes to body tissues with an average volume of distribution of 124 L. Siponimod fraction mesaured in plasma is 68% in humans. Animal studies demonstrate that siponimod readily crosses the blood-brain-barrier.
Apparent systemic clearance of 3.11 L/h has been estimated in MS patients.

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Metabolism / Metabolites
Siponimod is extensively metabolized, mainly by CYP2C9 enzyme (79.3%), and subsequently by CYP3A4 enzyme (18.5%). The pharmacological activity of the main metabolites M3 and M17 is not expected to be responsible for the clinical effect and the safety of siponimod in humans.

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Biological Half-Life
The apparent elimination half-life is approximately 30 hours.

Hepatotoxicity
In large controlled trials of siponimod in patients with multiple sclerosis, serum ALT elevations were common, typically arising during the first 3 months of treatment. The elevations were generally mild and asymptomatic, and they often returned to baseline values even with continuation of treatment or within 3 months of stopping. Aminotransferase elevations above 3 times upper limit of normal (ULN) were reported in 6% to 8% of siponimod recipients compared to less than 2% of placebo recipients. 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 1% if subjects. While siponimod 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 siponimod 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 siponimod is 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 siponimod 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. However, the manufacturer's labeling does not recommend against the use of siponimod 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.
◈ What is siponimod?
Siponimod (Mayzent®) is a medication approved to treat relapsing forms of multiple sclerosis (MS), including clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease. For information on multiple sclerosis, please see the MotherToBaby fact sheet at: https://mothertobaby.org/fact-sheets/multiple-sclerosis/.Sometimes when people find out they are pregnant, they think about changing how they take their medication, or stopping their medication altogether. However, it is important to talk with your healthcare providers before making any changes to how you take this medication. Your healthcare providers can talk with you about the benefits of treating your condition and the risks of untreated illness during pregnancy.
◈ I am taking siponimod, but I would like to stop taking it before becoming pregnant. How long does the drug stay in my body?
People eliminate medication at different rates. In healthy adults it takes up to 10 days, on average, for most of the siponimod to be gone from the body.
◈ I take siponimod. Can it make it harder for me to get pregnant?
It is not known if siponimod can make it harder to become pregnant.
◈ Does taking siponimod increase the chance for miscarriage?
Miscarriage is common and can occur in any pregnancy for many different reasons. According to the product label, experimental animal studies reported an increase in pregnancy loss. Studies have not been done in human pregnancy to see if siponimod increases the chance for miscarriage.
◈ Does taking siponimod increase the chance of birth defects?
Every pregnancy starts out with a 3-5% chance of having a birth defect. This is called the background risk. According to the product label, experimental animal studies reported an increased chance for birth defects. Studies have not been done in human pregnancy to see if siponimod increases the chance for birth defects above the background risk.
◈ Does taking siponimod in pregnancy cause other pregnancy-related problems?
According to the product label, experimental animal studies reported a chance of low birth weight. Studies have not been done in human pregnancy to see if siponimod increases the chance for pregnancy-related problems such as preterm delivery (birth before week 37) or low birth weight (weighing less than 5 pounds, 8 ounces [2500 grams] at birth).
◈ Does taking siponimod in pregnancy affect future behavior or learning for the child?
Studies have not been done to see if siponimod can cause behavior or learning issues for the child.
◈ Breastfeeding while taking siponimod:
Siponimod has not been studied for use while breastfeeding. It is not known if it can enter human breastmilk or how it might affect a nursing child. If you are taking siponimod while breastfeeding and you suspect the baby has any symptoms contact the child’s healthcare provider. Be sure to talk to your healthcare provider about all of your breastfeeding questions
◈ If a male takes siponimod, could it affect fertility (ability to get partner pregnant) or increase the chance of birth defects?
Studies have not been done to see if siponimod could affect male fertility or increase the chance of birth defects. In general, exposures that fathers or sperm donors have are unlikely to increase the risks to a pregnancy. For more information, please see the MotherToBaby fact sheet Paternal Exposures at https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

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Protein Binding
Protein binding of siponimod is higher than 99.9% in healthy patients as well as hepatic and renal impaired patients. Because of the high plasma protein binding of siponimod, hemodialysis is not likely to change the total and unbound siponimod concentration and no dose adjustments are expected based on this.

[1]. The selective sphingosine 1-phosphate receptor modulator BAF312 redirects lymphocyte distribution and has species-specific effects on heart rate. Br J Pharmacol. 2012 Nov;167(5):1035-47.

[2]. Discovery of BAF312 (Siponimod), a Potent and Selective S1P Receptor Modulator. ACS Med Chem Lett. 2013 Jan 4;4(3):333-7.

[3]. Prospects of siponimod in secondary progressive multiple sclerosis. Ther Adv Neurol Disord. 2018 Jul 17;11:1756286418788013.

[4]. Mechanism of Siponimod: Anti-Inflammatory and Neuroprotective Mode of Action. Cells. 2019 Jan 7;8(1):24.

Pharmacodynamics
**Immune system effects** Siponimod causes a dose-dependent decrease of the peripheral blood lymphocyte count within 6 hours of the first dose, caused by the reversible accumulation of lymphocytes in lymphoid tissues, due to lack of lymphocyte release. This results in a decrease in the inflammation that is involved in multiple sclerosis. Lymphocyte counts return to normal in 90% of patients within 10 days after the cessation of therapy. **Effects on heart rate and rhythm** Siponimod causes a temporary decrease in heart rate and atrioventricular conduction upon beginning treatment. The maximum fall in heart rate is observed in the first 6 hours post ingestion. Autonomic heart responses, including diurnal variation of heart rate and response to exercise activities, are not altered by siponimod treatment. **Effects on pulmonary function** Dose-dependent decreases in absolute forced expiratory volume over a time frame of 1 second were noted in siponimod-treated patients and were higher than in patients taking placebo.

C29H35F3N2O3

516.6

516.259

C, 67.42; H, 6.83; F, 11.03; N, 5.42; O, 9.29

1230487-00-9

Siponimod hemifumarate; 1234627-85-0

44599207

White to off-white solid powder

1.2±0.1 g/cm3

602.0±65.0 °C at 760 mmHg

111-112

317.9±34.3 °C

0.0±1.8 mmHg at 25°C

1.571

6.9

1

8

9

37

777

0

FC(C1C([H])=C(C([H])([H])O/N=C(\C([H])([H])[H])/C2C([H])=C([H])C(=C(C=2[H])C([H])([H])C([H])([H])[H])C([H])([H])N2C([H])([H])C([H])(C(=O)O[H])C2([H])[H])C([H])=C([H])C=1C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])(F)F

KIHYPELVXPAIDH-HNSNBQBZSA-N

InChI=1S/C29H35F3N2O3/c1-3-21-14-23(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/h9-14,22,25H,3-8,15-18H2,1-2H3,(H,35,36)/b33-19+

1-[[4-[(E)-N-[[4-cyclohexyl-3-(trifluoromethyl)phenyl]methoxy]-C-methylcarbonimidoyl]-2-ethylphenyl]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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.84 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 (4.84 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 25.0 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: ≥ 2.5 mg/mL (4.84 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 4: 1.67 mg/mL (3.23 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 5: 1.67 mg/mL (3.23 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 6: 0.33 mg/mL (0.64 mM) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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