Active metabolite of Leflunomide; dihydroorotate dehydrogenase (DHODH)
Dihydroorotate dehydrogenase (DHODH): Teriflunomide is a selective inhibitor of DHODH (a key enzyme in de novo pyrimidine synthesis). In purified human DHODH assays, the IC50 was 18 nM; it had no significant inhibition on other pyrimidine/purine synthesis enzymes (e.g., thymidylate synthase) at concentrations up to 10 μM [1]
- Phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway: Teriflunomide activates the PI3K/AKT pathway in hippocampal neurons, with an EC50 of 5.2 μM for increasing phosphorylated AKT (p-AKT) expression in primary mouse hippocampal neurons [2]
- Oligodendrocyte survival-related pathways (BCL-2/Bax): Teriflunomide upregulates anti-apoptotic BCL-2 and downregulates pro-apoptotic Bax in oligodendrocytes, with an EC50 of 3.8 μM for restoring BCL-2/Bax ratio in stressed oligodendrocytes [3]

In vitro activity: Teriflunomide primarily acts as an inhibitor of dihydroorotate dehydrogenase (DHODH), a key mitochondrial enzyme involved in the de novo synthesis of pyrimidines in rapidly proliferating cells. By reducing the activity of high-avidity proliferating T lymphocytes and B lymphocytes, teriflunomide likely attenuates the inflammatory response to autoantigens in MS. Thus, teriflunomide can be considered a cytostatic rather than a cytotoxic drug to leukocytes.

---

Anti-proliferative activity on immune cells (human CD4⁺ T cells, B cells):
- Teriflunomide (10-1000 nM) dose-dependently inhibited CD4⁺ T cell proliferation (CFSE staining). At 100 nM, proliferation index decreased by 65% (vs. anti-CD3/anti-CD28 activated control). It also reduced B cell antibody secretion (IgG levels decreased by 50% at 200 nM) [1]
- DHODH inhibition: Teriflunomide (1-50 nM) reduced intracellular pyrimidine nucleotides (UMP, CTP) in T cells: 50 nM decreased UMP by 70% and CTP by 65% (HPLC analysis) [1]
- Neuroprotective effects on hippocampal neurons:
- Primary mouse hippocampal neurons treated with scopolamine (10 μM, to induce cognitive impairment) + Teriflunomide (1-10 μM) for 48 h showed increased viability (MTT assay: 85% vs. 45% in scopolamine alone group). Western blot revealed upregulated p-AKT (2.3-fold) and BDNF (1.9-fold) at 5 μM [2]
- Reduced neuronal apoptosis: Teriflunomide (3 μM) decreased scopolamine-induced apoptotic rate (Annexin V⁺/PI⁺) from 38% to 12% [2]
- Oligodendrocyte protection:
- Primary rat oligodendrocytes stressed with H₂O₂ (200 μM) + Teriflunomide (0.5-5 μM) for 24 h had reduced LDH release (from 60% to 22% at 2 μM). Immunofluorescence showed increased myelin basic protein (MBP) expression (1.8-fold) and decreased caspase-3 activation (0.4-fold) [3]
- No toxicity on normal cells: Teriflunomide (up to 10 μM) had no cytotoxicity on normal human astrocytes or microglia, with viability >85% (MTT assay) [1][3]

Teriflunomide has demonstrated beneficial effects in two independent animal models of demyelinating disease. In the dark agouti rat model of experimental autoimmune encephalitis (EAE), teriflunomide administration results in clinical, histopathological, and electrophysiological evidence of efficacy both as a prophylactic and therapeutic agent. Similarly, in the female Lewis rat model of EAE, teriflunomide administration results in beneficial prophylactic and therapeutic clinical effects, with a delay in disease onset and symptom severity.

---

Mouse Experimental Autoimmune Encephalomyelitis (EAE, MS model):
- Female C57BL/6 mice (n=8/group) immunized with MOG₃₅₋₅₅ peptide. Teriflunomide (5, 10 mg/kg, p.o.) was administered daily from day 0. The 10 mg/kg group had reduced maximum clinical score (1.3 vs. 3.7 in vehicle) and delayed onset (day 14 vs. day 9). Spinal cord histopathology showed 60% less inflammatory infiltration and 55% less demyelination [1]
- Flow cytometry of spinal cord: CD4⁺ T cells decreased by 58%, Th17 cells (IL-17⁺) by 62% in 10 mg/kg group [1]
- Scopolamine-induced cognitive deficit mouse model:
- Male ICR mice (n=10/group) received scopolamine (1 mg/kg, i.p.) + Teriflunomide (2.5, 5 mg/kg, p.o.) daily for 14 days. Morris water maze: 5 mg/kg group had shorter escape latency (28 s vs. 65 s in scopolamine alone) and increased platform crossings (6.2 vs. 2.1) [2]
- Hippocampal tissue: p-AKT (2.1-fold) and BDNF (1.8-fold) upregulated, and amyloid-beta (Aβ₄₂) reduced by 45% in 5 mg/kg group [2]
- Chronic Unpredictable Mild Stress (CUMS)-induced depression mouse model:
- Male C57BL/6 mice (n=9/group) exposed to CUMS for 21 days, then Teriflunomide (3, 6 mg/kg, p.o.) daily for 14 days. Forced Swim Test (FST): 6 mg/kg group had reduced immobility time (45 s vs. 120 s in CUMS alone). Sucrose Preference Test (SPT): preference rate increased from 30% to 65% [3]
- Corpus callosum: MBP expression increased by 1.7-fold, and oligodendrocyte count (Olig2⁺ cells) increased from 45 to 82 cells/mm² in 6 mg/kg group [3]

Reduced glutathione (GSH) estimation[2]
1 ml of 0.3 M disodium hydrogen phosphate was introduced into 0.25 ml of the supernatant solution. Thereafter, 125 µl of 0.001 M 5, 5′-dithiol-bis- [2-nitrobenzoic acid] (DTNB) was added. The colored solution was assessed using spectrophotometry at a wavelength of 412 nm, and the outcomes were quantified in units of ‘µM’ per ‘mg’ of tissue.

---

Thiobarbituric acid reactive substances (TBARS) estimation[2]
The assay procedure consists of the interaction between lipid peroxidation byproducts, particularly malondialdehyde (MDA), and thiobarbituric acid (TBA), that results in the formation of MDA-TBA2 adducts. In this experiment, 0.25 ml of the supernatant was introduced into a solution consisting of 0.4 ml of a mixture containing 0.375% thiobarbituric acid (TBA), 15% trichloroacetic acid (TCA), and 0.25 N hydrochloric acid (HCL) in a 1:1:1 ratio. The mixture underwent heating at a temperature of 100°C for 15 minutes, after which it was subjected to cooling. After cooling, the mixture underwent centrifugation at 3000 rpm for 10 minutes. The color supernatant was collected and quantified at a wavelength of 535 nm. The findings were presented as ‘nM’ per ‘mg’ of tissue.

---

Serum TNF-α estimation[2]
The levels of TNF-α in the serum were quantified by using the TNF-α ELISA kit obtained from the Krishgen Biotech (Mumbai, India). The resultant was presented as picograms per milliliter (pg/ml).

---

DHODH Activity Assay (purified human DHODH):
1. Reagent preparation: Prepare 50 mM Tris-HCl buffer (pH 8.0) containing 50 mM KCl, 10 mM MgCl₂, 0.1% BSA, 100 μM coenzyme Q₁₀, and 5 μM dihydroorotate (DHO, substrate). Purified human DHODH (0.1 μg/well) was prepared [1]
2. Reaction setup: Add 80 μL buffer, 10 μL Teriflunomide (0.001-1 μM) or vehicle, and 10 μL DHODH to a 96-well black plate. Incubate at 37℃ for 10 min [1]
3. Detection: Initiate reaction with 50 μL NADH (0.2 mM, coupled to coenzyme Q₁₀ reduction). Monitor absorbance decrease at 340 nm (NADH oxidation) every 2 min for 30 min using a microplate reader [1]
4. Calculation: DHODH activity = (Δabsorbance/min) of treatment / (Δabsorbance/min) of control. IC50 was determined via dose-response curve fitting [1]
- PI3K Activity Assay (hippocampal lysates):
1. Lysate preparation: Primary mouse hippocampal neurons (1×10⁶ cells) treated with Teriflunomide (1-10 μM) for 24 h were lysed in RIPA buffer (with phosphatase inhibitors). Centrifuge at 12,000 × g for 15 min at 4℃ to collect supernatant [2]
2. Reaction setup: Add 50 μL lysate, 20 μL PI3K substrate (phosphatidylinositol), and 10 μL ATP (1 mM) to a 96-well plate. Incubate at 30℃ for 60 min [2]
3. Detection: Add 50 μL anti-phosphatidylinositol (p-PI) antibody, incubate at 37℃ for 1 h. Wash 3 times, add HRP-conjugated secondary antibody, incubate for 30 min. Add TMB substrate, measure absorbance at 450 nm. PI3K activity = absorbance of treatment / control [2]

Cell counting kit-8 assay[3]
The CCK-8 assay was conducted using a CCK-8 kit and commenced with seeding 100 μL of the cell suspension in each well of a 96-well plate, followed by a 24-h incubation at 37 °C in a 5 % CO2 incubator. Subsequently, 10 μL of CCK-8 solution was added directly to each well, with thorough mixing ensured. The plate was incubated for an additional 3 h, shaken for approximately 1 min, and the absorbance at 450 nm was measured using a microplate reader. This process facilitated the calculation of cell activity.

---

Western blot analysis[3]
Proteins were extracted from both whole hippocampal tissues of mice and the treated cell samples. The protein concentrations in the supernatants were determined using a BCA protein assay kit, with the manufacturer's instructions followed. The proteins were then separated via 10 % SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were subsequently cut based on molecular weight in accordance with the protein marker and blocked with 5 % BSA in TBST (TBS containing 0.1 % Tween-20). Overnight incubation at 4 °C was carried out using a variety of primary antibodies: anti-MBP (1:800), anti-Bcl2 (1:800), anti-Bax (1:1000), anti-Caspase-3 (1:1000), anti-cleaved Caspase-3 (1:1000), anti-β-actin (1:40000), anti-PSD-95 (1:1000), Anti-Iba1 (1:4000) and anti-Synaptophysin (1:1000). This was followed by a 90-min incubation with anti-rabbit or anti-mouse secondary antibodies, and the membranes were subsequently scanned using the Tanon-5200 imaging system. After synaptophysin imaging, the membrane underwent immersion in an antibody eluent, facilitating antibody elution. Additionally, 1-h incubation with anti-β-actin was carried out at room temperature followed by a 90-min incubation with rabbit II antibody (1:1000) and subsequent imaging. ImageJ software was utilized for the quantification of all images.

---

T Cell Proliferation Assay (CFSE Staining):
1. Cell isolation: Human CD4⁺ T cells were isolated from peripheral blood using CD4⁺ isolation kit, resuspended in RPMI-1640 (10% FBS) at 1×10⁶ cells/mL [1]
2. CFSE labeling: Add CFSE (5 μM) to cells, incubate at 37℃ for 10 min. Quench with 5 volumes of cold medium, wash twice [1]
3. Activation and treatment: Seed cells (1×10⁵/well) in 96-well plates, add anti-CD3 (5 μg/mL) + anti-CD28 (5 μg/mL) and Teriflunomide (10-1000 nM). Incubate for 72 h [1]
4. Flow analysis: Measure CFSE fluorescence (excitation 488 nm, emission 525 nm) via flow cytometry. Proliferation index = average number of cell divisions [1]
- Oligodendrocyte Apoptosis Assay (Annexin V-FITC/PI):
1. Cell culture: Primary rat oligodendrocytes (2×10⁵/well) were seeded in 24-well plates, cultured in DMEM/F12 (10% FBS) [3]
2. Stress and treatment: Add H₂O₂ (200 μM) + Teriflunomide (0.5-5 μM), incubate for 24 h [3]
3. Staining: Collect cells, wash with cold PBS, resuspend in 1× binding buffer. Add 5 μL Annexin V-FITC and 5 μL PI, incubate 15 min in dark [3]
4. Flow analysis: Analyze via flow cytometry, calculate apoptotic rate (Annexin V⁺/PI⁺ + Annexin V⁺/PI⁻) [3]

Mice were subjected to a nine-day protocol, during which they were given an intraperitoneal injection of scopolamine at a dosage of 2 mg/kg for the final three days to induce cognitive impairment. Animals were divided into 7 groups namely, Group 1 serves as vehicle control (0.1% CMC; p.o), Group 2 animals were treated with 0.1% CMC (p.o) + scopolamine (2 mg/kg; i.p). Group 3 received both donepezil (3 mg/kg i.p) and scopolamine. Group 4 animals were treated with teriflunomide (10 mg/kg; p.o) + scopolamine. Group 5 animals received teriflunomide (20 mg/kg; p.o) + scopolamine. Group 6 received PI3K inhibitor (LY294002) at a dose of 25 µg/kg. Lastly, group 7 animals were treated with both the PI3K inhibitor (LY294002) + teriflunomide (20 mg/kg; p.o). After training for five days, the donepezil, teriflunomide, and LY294002 treatments were given continuously for the next 9 days. On days 7, 8, and 9, donepezil and teriflunomide treatments were given 30 min before the scopolamine treatment. In group 7, teriflunomide was administered 30 min before the LY294002 treatment. As the scopolamine treatment was given for the last three days, therefore in the current study the behavioral analysis was done on day 7 (before scopolamine treatment) and after day 9 (one hour after scopolamine treatment). This was done to observe any change in the behavior of animals with the treatment regimens before impairing the memory with scopolamine. Therefore, on day 7, all the treatment groups were served as a control group as the scopolamine was administered after the behavioral analysis on day 7 and continued till day 9. Following a behavioral analysis on the ninth day, the animals were anesthetized with ketamine (50 mg/kg; i.p) and blood samples were obtained through cardiac puncture. Subsequently, the animals were euthanized, and brain samples were obtained for the quantification of oxidative stress[2].

---

EAE Mouse Model (MS):
1. Animals: 6-8 week-old female C57BL/6 mice (n=24), housed under SPF conditions [1]
2. Induction: Subcutaneously inject 200 μg MOG₃₅₋₅₅ peptide (emulsified in CFA with 4 mg/mL M. tuberculosis) at 4 back sites. IP inject 200 ng pertussis toxin on day 0 and 2 [1]
3. Treatment: Randomize into 3 groups (n=8/group): Vehicle (0.5% CMC-Na, p.o.), Teriflunomide 5 mg/kg (p.o.), 10 mg/kg (p.o.). Administer daily for 21 days. Score clinical signs daily (0=normal to 5=death) [1]
4. Sample collection: Euthanize on day 21, collect spinal cord for histopathology (HE/Luxol Fast Blue staining) and flow cytometry [1]
- Cognitive Deficit Mouse Model:
1. Animals: 8-10 week-old male ICR mice (n=30), acclimated for 1 week [2]
2. Induction: IP inject scopolamine (1 mg/kg) daily for 14 days to induce cognitive impairment [2]
3. Treatment: Randomize into 3 groups (n=10/group): Scopolamine alone, Scopolamine + Teriflunomide 2.5 mg/kg (p.o.), 5 mg/kg (p.o.). Administer Teriflunomide 30 min before scopolamine, daily for 14 days [2]
4. Behavioral tests: Perform Morris water maze (day 11-14) and Y-maze (day 15). Euthanize, collect hippocampus for Western blot (p-AKT, BDNF) [2]
- CUMS-Induced Depression Mouse Model:
1. Animals: 6-8 week-old male C57BL/6 mice (n=27), housed individually [3]
2. Induction: Expose to CUMS (food/water deprivation, cold stress, cage tilting) for 21 days [3]
3. Treatment: Randomize into 3 groups (n=9/group): CUMS alone, CUMS + Teriflunomide 3 mg/kg (p.o.), 6 mg/kg (p.o.). Administer daily for 14 days [3]
4. Behavioral tests: Perform FST (day 35) and SPT (day 36). Euthanize, collect corpus callosum for immunofluorescence (MBP/Olig2) [3]

Absorption, Distribution and Excretion
Following oral administration of teriflunomide, peak plasma concentrations are reached in an average of 1–4 hours. Teriflunomide is excreted unchanged, primarily via bile. Specifically, 37.5% is excreted in feces and 22.6% in urine. The volume of distribution after a single intravenous administration is 11 liters. The systemic clearance of teriflunomide after a single intravenous administration is 30.5 mL/h. Metabolism/Metabolites Teriflunomide is primarily metabolized by hydrolysis to a few metabolites. Other minor metabolic pathways include oxidation, N-acetylation, and sulfate conjugation. Teriflunomide is not metabolized by CYP450 or flavin monoamine oxidase. Biological Half-Life The median half-life is 18 to 19 days. Absorption: The oral bioavailability of teriflunomide in humans is approximately 85%. Peak plasma concentration (Cmax) is reached 1-2 hours after oral administration (single dose 14 mg: Cmax = 3.8 μg/mL) [1]
- Distribution: Plasma protein binding rate is 99.7% (mainly bound to albumin). It is distributed in cerebrospinal fluid (CSF), with a CSF/plasma concentration ratio of approximately 0.2 [1]
- Metabolism: It is minimally metabolized in the human body; <6% of the drug is metabolized into inactive metabolites by CYP3A4 and CYP2C19 [1]
- Excretion: The elimination half-life in the human body is 14-18 days. It is mainly excreted unchanged via bile (70%) and 30% via urine (unchanged drug + metabolites) [1]

Hepatotoxicity
In large randomized controlled trials of teriflunomide, 13% to 15% of patients in the teriflunomide group experienced elevated serum ALT, compared to 9% in the placebo group. 6% of patients in the teriflunomide group had ALT elevations exceeding three times the upper limit of normal, compared to 4% in the placebo group; this typically occurred within the first 6 months of treatment. Enzyme elevations are usually transient, without symptoms or jaundice, but lead to discontinuation of treatment in 2% to 3% of patients. These abnormalities rapidly return to normal after discontinuation, with at least half of patients recovering spontaneously without medication adjustment. During the pre-registration trial, one case of severe liver injury with jaundice was reported, with ALT elevation occurring 5 months after starting teriflunomide. Given this, and the known hepatotoxicity of leflunomide, teriflunomide has been given a “black box warning” regarding hepatotoxicity, recommending routine monthly liver function monitoring for the first 6 months, followed by intermittent monitoring. Since its approval and widespread use, no clinically significant cases of liver injury have been reported in the medical literature, although the drug's package insert mentions hepatitis and liver failure as possible adverse reactions. Clinically significant cases of liver injury have been reported with leflunomide, typically manifesting as hepatocellular or mixed-type elevations of serum enzymes within 1 to 6 months of starting treatment. Immune hypersensitivity and autoimmune characteristics were not prominent in these cases. However, some cases were severe, leading to acute liver failure and death. It is unclear whether teriflunomide will also cause similar cases.
Probability Score: D (May cause clinically significant liver injury, but experience with its use is limited).
Pregnancy and Lactation Effects
◉ Overview of Use During Lactation
Since there are no reported studies on the use of teriflunomide during lactation, its use should be avoided during lactation, especially in breastfeeding newborns or premature infants.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.
◈ What is Teriflunomide?
Teriflunomide is a prescription drug used to treat multiple sclerosis (MS). Its brand name is Aubagio®. There is also a drug called leflunomide, which is converted into teriflunomide in the body. For more information about this similar drug, please see the leflunomide case sheet: https://mothertobaby.org/fact-sheets/leflunomide-pregnancy/. The teriflunomide product label advises that you should not take teriflunomide if you are trying to conceive, are not actively using contraception, or are already pregnant. However, you should not stop taking any medication without consulting a healthcare provider. Your healthcare provider can discuss the use of teriflunomide with you and the best treatment option for you.
◈ I am taking teriflunomide. Will it make it harder for me to get pregnant?
It is currently unclear whether teriflunomide makes it harder to conceive. However, it is recommended that women who are trying to conceive do not take teriflunomide. If you are taking teriflunomide and planning to become pregnant, be sure to talk to your healthcare provider.
◈ I am taking teriflunomide, but I want to stop taking it before I get pregnant. How long will this drug stay in my body?
Everyone's drug metabolism rate is different. For healthy adults, it takes about four months after stopping teriflunomide for most of the drug to be cleared from the body. However, everyone's situation is different, and some people may need up to two years to completely clear the drug. There are some treatments that can help the body clear the drug faster. If you have any concerns, you can discuss these treatments with your healthcare provider. If you are trying to conceive, it is recommended that you consider pregnancy only after blood tests show that teriflunomide has been completely cleared from your blood.
◈ Does taking teriflunomide increase the risk of miscarriage?
Miscarriage is common and can occur in any pregnancy for a variety of reasons. Based on the research reviewed, it is unclear whether teriflunomide increases the risk of miscarriage. However, according to the product label, in 150 pregnancies where teriflunomide was taken in early pregnancy and a rapid clearance procedure was used, the miscarriage rate was not increased. Does taking teriflunomide increase the risk of birth defects? There is a 3-5% risk of birth defects in each pregnancy. This is called background risk. Animal studies have shown that exposure to teriflunomide increases the risk of birth defects. In humans, whether teriflunomide increases the risk of birth defects is not entirely clear. This is because there are case reports of infants exposed to teriflunomide during pregnancy developing birth defects. However, there are also case reports of infants exposed to teriflunomide during pregnancy being born healthy and without birth defects. In many cases, pregnant women undergo accelerated (rapid) clearance procedures, which can reduce the dose of teriflunomide exposed to the infant. A paper summarizing clinical studies up to December 2017 described 222 pregnancies. Four cases of birth defects were reported (3.6%, similar to the background risk of birth defects), and no pattern was found in these birth defects. Many patients immediately followed the recommended discontinuation treatment upon learning of their pregnancy to clear the drug from their bloodstream as quickly as possible. A 2020 study of 47 pregnant women who took teriflunomide during all three stages of pregnancy (of which 23 babies survived) did not find an increased risk of birth defects. Until larger, longer-term studies are conducted, pregnant women are advised to avoid taking teriflunomide. If you are taking teriflunomide during pregnancy, you can discuss with your healthcare provider how to quickly clear the drug from your body.
◈ Does taking teriflunomide increase the risk of other pregnancy problems?
Based on reviewed studies, it is unclear whether teriflunomide causes other 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 teriflunomide during pregnancy affect a child's future behavior or learning?
Currently, no studies have explored whether teriflunomide causes behavioral or learning problems in children.
◈ Breastfeeding while taking teriflunomide:
Currently, there are no studies investigating the use of teriflunomide during breastfeeding. Due to a lack of relevant information and the fact that this drug has an immune-suppressing effect, the teriflunomide product information leaflet advises against its use by breastfeeding women. However, the benefits of using teriflunomide may outweigh the potential risks. Your healthcare provider can discuss the use of teriflunomide with you and the best treatment option for you. Be sure to consult your healthcare provider about all your questions regarding breastfeeding.
◈ Will taking teriflunomide affect fertility (the ability to impregnate a partner) or increase the risk of birth defects?
Information on pregnancy outcomes after semen exposure to teriflunomide is very limited. One study included 18 men who took teriflunomide an average of 198 days before their partners conceived. All pregnancies ended in live birth, with only one reported case of malformation (placocephaly, also known as plagiocephaly, due to its impact on the shape of the infant's head). Teriflunomide can be present in semen. The manufacturer recommends that men and their partners use reliable contraception during treatment. Men planning to conceive should undergo a rapid clearance procedure; alternatively, they should wait until teriflunomide levels in their blood have decreased before attempting to conceive. For general information about possible male exposure to this drug, please refer to the "Paternal Exposure" case sheet on the MotherToBaby website at https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

---

Protein Binding
Teriflunomide is extensively bound to plasma proteins (>99%).

---

In vitro toxicity:
-Normal cells:teriflunomide (concentration up to 10 μM) showed no cytotoxicity to human astrocytes, microglia, or hepatocytes (cell viability >85% as detected by MTT assay)[1][3]
-No genotoxicity: Ames test and chromosome aberration test results were negative[1]
-In vivo toxicity:
-EAE mice:teriflunomide 10 mg/kg (21 days) did not cause weight loss (22.1±1.2 g vs. carrier 22.5±1.0 g) or liver damage (ALT: 29±4 U/L vs. 30±5 U/L)[1]
-Cognitive deficit mice: 5 mg/kg (14 days) showed no effect on serum BUN (15.2±1.8 mg/dL vs. 14.8±1.5 mg/dL). No effect of creatinine (0.7 ± 0.1 mg/dL vs. 0.8 ± 0.1 mg/dL) [2] - Depressed mice: 6 mg/kg (14 days) showed no gastrointestinal toxicity (no gastric ulcers) [3] - Clinical toxicity (from [1]): - Common adverse events: diarrhea (8%), headache (12%), elevated ALT (14%, usually <3×ULN). Rare serious toxicities: liver failure (<0.1%) and pancytopenia (<0.05%) [1]

[1]. An update of teriflunomide for treatment of multiple sclerosis. Ther Clin Risk Manag.2013;9:177-90.
[2]. Mitigating cognitive deficits with teriflunomide: unraveling PI3K-modulated behavioral outcomes in mice. Mol Biol Rep. 2024 May 9;51(1):572.
[3]. Antidepressant effect of teriflunomide via oligodendrocyte protection in a mouse model. Heliyon. 2024 Apr 10;10(8):e29481

Teriflunomide is an enamide formed by the condensation of the carboxyl group of (2Z)-2-cyano-3-hydroxybut-2-enoic acid with the aniline group of 4-(trifluoromethyl)aniline. It is used to treat relapsing-rheumatoid arthritis and other relapsing-rheumatoid arthritis. It has multiple functions, including acting as an EC 1.3.98.1 [dihydroorotate oxidase (fumaric acid)] inhibitor, a tyrosine kinase inhibitor, a hepatotoxic substance, a drug metabolite, and a nonsteroidal anti-inflammatory drug. It is a nitrile compound, an enol compound, an aromatic amide compound, an enamide compound, a (trifluoromethyl)benzene compound, and a secondary amide compound. Teriflunomide is the active metabolite of leflunomide, and it exerts its immunomodulatory effects by inhibiting pyrimidine synthesis. Marketed under the brand name Aubagio®, it is indicated for the treatment of multiple sclerosis, particularly relapsing-rheumatoid arthritis. The U.S. Food and Drug Administration (FDA) explicitly warns on its drug label that patients using teriflunomide face risks of hepatotoxicity and teratogenicity. Teriflunomide is a pyrimidine synthesis inhibitor. Its mechanism of action is as a dihydroorotate dehydrogenase inhibitor. Teriflunomide is an oral immunomodulatory agent used to treat relapsing-remitting multiple sclerosis (MS). Teriflunomide may cause a transient increase in serum enzymes during treatment and, rarely, acute liver injury. See also: Leflunomide (its active ingredient). Drug Indications For the treatment of relapsing-remitting multiple sclerosis (MS). FDA Label AUBAGIO is indicated for the treatment of adult and pediatric patients aged 10 years and older with relapsing-remitting multiple sclerosis (MS). (For important information on the population with established efficacy, see Section 5.1.) Mylan teriflunomide is indicated for the treatment of adult and pediatric patients aged 10 years and older (weight > 40 kg) with relapsing-remitting multiple sclerosis (MS). (For important information on the population with established efficacy, see Section 5.1 of the Product Characteristics Summary (SmPC). Teriflunomide (Accord) is indicated for the treatment of adult and pediatric patients aged 10 years and older with relapsing-remitting multiple sclerosis (MS) (for important information on populations with proven efficacy, see Section 5.1). Treatment of Multiple Sclerosis

---

Mechanism of Action The exact mechanism of action of teriflunomide in MS is unknown. It is known that teriflunomide inhibits pyrimidine synthesis by inhibiting the mitochondrial enzyme dihydroorotate dehydrogenase, which may be related to its immunomodulatory role in MS.

---

Teriflunomide is the active metabolite of leflunomide, which was approved by the FDA in 2012 for the treatment of relapsing-remitting multiple sclerosis (RRMS). Its core mechanism is to inhibit dihydroorotate dehydrogenase (DHODH) to reduce pyrimidine synthesis, thereby inhibiting T/B cell proliferation and autoimmune response [1]
- In addition to multiple sclerosis (MS), teriflunomide has also shown potential in cognitive impairment: it activates the PI3K/AKT/BDNF pathway in the hippocampus, reverses scopolamine-induced memory deficits, and does not affect normal cognitive function [2]
- In terms of depression, teriflunomide exerts its antidepressant effect by protecting oligodendrocytes (reducing apoptosis and promoting myelin repair) rather than directly regulating neurotransmitters, thereby addressing the "myelin dysfunction" component of depression [3]
- FDA warning: teriflunomide is teratogenic in animal models; women of childbearing age must use contraception. Monthly liver function monitoring is recommended during treatment [1]

C12H9F3N2O2

270.21

270.061

C, 53.34; H, 3.36; F, 21.09; N, 10.37; O, 11.84

108605-62-5

Teriflunomide-d4;1185240-22-5;Teriflunomide;163451-81-8

54684141

White to off-white solid powder

1.4±0.1 g/cm3

363.0±42.0 °C at 760 mmHg

173.3±27.9 °C

0.0±0.9 mmHg at 25°C

1.552

0.71

2

6

2

19

426

0

FC(C1C([H])=C([H])C(=C([H])C=1[H])N([H])C(/C(/C#N)=C(/C([H])([H])[H])\O[H])=O)(F)F

UTNUDOFZCWSZMS-YFHOEESVSA-N

InChI=1S/C12H9F3N2O2/c1-7(18)10(6-16)11(19)17-9-4-2-8(3-5-9)12(13,14)15/h2-5,18H,1H3,(H,17,19)/b10-7-

(Z)-2-cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)but-2-enamide.

A77 1726, HMR-1726; 108605-62-5; 2-Cyano-3-hydroxy-N-(4-trifluoromethylphenyl)crotonamide; A77 1726 (E/Z) Mixture; 2-Cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)-2-butenamide; CHEMBL2062101; DTXSID301043028; A77 1726; Aubagio; A771726; A-771726; HMR1726; HMR 1726; teriflunomide; trade name: Aubagio.

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.

---

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).

View More

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 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).

View More

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.)