Active metabolite of Leflunomide; dihydroorotate dehydrogenase (DHODH)

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.

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.

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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.

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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).

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.

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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.

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].

Absorption, Distribution and Excretion
After oral administration of teriflunomide, maximum plasma concentrations are reached, on average, in 1-4 hours.
Teriflunomide is eliminated unchanged and mainly through bile. Specifically 37.5% is eliminated in the feces and 22.6% in urine.
After a single intravenous dose, the volume of distribution is 11 L.
After a single IV dose, teriflunomide has a total body clearance of 30.5 mL/h.

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Metabolism / Metabolites
Teriflunomide mainly undergoes hydrolyis to minor metabolites. Other minor metabolic pathways include oxidation, N-acetylation and sulfate conjugation. Teriflunomide is not metabolized by CYP450 or flavin monoamine oxidase.

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Biological Half-Life
The median half-life is 18 to 19 days.

Hepatotoxicity
In large randomized controlled trials of teriflunomide, serum ALT elevations occurred in 13% to 15% of teriflunomide compared to 9% of placebo recipients. Elevations above 3 times the upper limit of normal occurred in 6% of teriflunomide versus 4% of placebo recipients, usually within the first 6 months of therapy. The enzyme elevations were usually transient and not associated with symptoms or jaundice, but led to drug discontinuation in 2% to 3% of patients. The abnormalities resolved rapidly with drug discontinuation and resolved spontaneously in at least half of patients without drug modification. During preregistration trials, a single case of severe liver injury with jaundice was described, ALT elevations appearing 5 months after starting teriflunomide. Because of this and the known hepatotoxic potential of leflunomide, teriflunomide was given a "black box" warning regarding hepatotoxicity, and routine monitoring of liver tests is recommended monthly for the first 6 months and intermittently thereafter. Since approval and more wide scale usage, there have been no cases of clinical apparent liver injury published in the medical literature, although the package label mentions hepatitis and hepatic failure as possible adverse events. Clinically apparent liver injury has been reported with leflunomide, generally presenting with a hepatocellular or mixed pattern of serum enzyme elevations within 1 to 6 months of starting therapy. Immunoallergic and autoimmune features have not been prominent in these cases. Some cases, however, have been severe leading to acute liver failure and death. Whether similar cases occur with teriflunomide is not known.
Likelihood score: D (possible cause of clinically apparent liver injury but experience with its use is limited).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Because there is no published experience with teriflunomide during breastfeeding, it should be avoided during breastfeeding, especially while nursing a newborn or preterm infant.
◉ 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 teriflunomide?
Teriflunomide is a prescription medication used to treat multiple sclerosis (MS). It is marketed under the brand name Aubagio®. There is another medication, called leflunomide, which turns into teriflunomide in the body. For more information on this similar medication, please see the fact sheet leflunomide at https://mothertobaby.org/fact-sheets/leflunomide-pregnancy/.The product label for teriflunomide recommends people should NOT take teriflunomide if they are trying to get pregnant, if they are not actively using birth control to prevent a pregnancy, or if they are already pregnant. However, you should not stop taking any medications without first talking with your healthcare provider. Your healthcare providers can talk with you about using teriflunomide and what treatment is best for you.
◈ I take teriflunomide. Can it make it harder for me to get pregnant?
It is not known if teriflunomide can make it more difficult to get pregnant. However, it is recommended that people who are trying to get pregnant not take teriflunomide. If you are trying to become pregnant and are on teriflunomide it is important for you to talk with your healthcare providers.
◈ I am taking teriflunomide, 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 four months after stopping teriflunomide for most of the teriflunomide to be gone from the body. However, not everyone is the same and it could take up to 2 years for some people to clear this medication from their body. There are treatments to help the body clear this medication faster. These can be discussed with your healthcare provider if there is a concern for you. If you are trying to become pregnant, it has been recommended that you wait to get pregnant until AFTER a blood test shows teriflunomide is completely out of your blood.
◈ Does taking teriflunomide increase the chance for miscarriage?
Miscarriage is common and can occur in any pregnancy for many different reasons. Based on the studies reviewed, it is not known if teriflunomide increases the chance for miscarriage. However, according to the product labeling there was no increase in miscarriage in 150 pregnancies with teriflunomide exposure early in the 1st trimester and use of the rapid elimination procedure.
◈ Does taking teriflunomide 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. Experimental animal studies show an increased chance of birth defects with exposure to teriflunomide.In humans it is not fully understood whether teriflunomide increases the chance for birth defects. This is because there are case reports where babies exposed to teriflunomide during pregnancy had birth defects. However, there are also case reports of babies born healthy and without birth defects with exposure to teriflunomide during pregnancy. In many cases the pregnant person underwent the accelerated (rapid) elimination procedure which would have reduced the teriflunomide exposure to the baby.A paper summarizing clinical studies through December of 2017 described 222 pregnancies. Among these, 4 birth defects were reported (3.6%, which is similar to the background risk of birth defects) and no pattern was seen in those birth defects. Many of these people took the recommended elimination treatment once they found out they were pregnant to remove the drug as quickly as possible from their blood.A 2020 study of 47 pregnancies exposed to teriflunomide during all three trimesters (with 23 live-born infants) did not find an increased chance of birth defects. Until larger and longer-term studies are done, it has been recommended to avoid teriflunomide during pregnancy. If teriflunomide is taken during pregnancy, rapid elimination of the medication can be discussed with your healthcare provider.
◈ Does taking teriflunomide increase the chance of other pregnancy problems?
Based on the studies reviewed, it is not known if teriflunomide can cause other 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 teriflunomide in pregnancy affect future behavior or learning for the child?
Studies have not been done to see if teriflunomide can cause behavior or learning issues for the child.
◈ Breastfeeding while taking teriflunomide:
There are no studies looking at the use of teriflunomide in breastfeeding. Because there is a lack of information, and the medication has the ability to suppress the immune system, the product label for teriflunomide recommends people who are breastfeeding not use this medication. But, the benefit of using teriflunomide may outweigh possible risks. Your healthcare providers can talk with you about using teriflunomide and what treatment is best for you. Be sure to talk to your healthcare provider about all of your breastfeeding questions.
◈ If a male takes teriflunomide, could it affect fertility (ability to get partner pregnant) or increase the chance of birth defects?
Very little information is available on the outcomes of pregnancies if semen was exposed to teriflunomide. There is 1 study among 18 males who received teriflunomide an average of 198 days leading up to conception in their partner. All pregnancies in their partners resulted in live births, with only one report of a malformation (plagiocephaly, also called "flat head syndrome" because of the way it affects the shape of a baby's head). Teriflunomide is found in semen. The manufacturer recommends that males and their partners should use reliable contraception during therapy. The rapid elimination procedure should be used in males who are planning to conceive; or they should wait to try to get a partner pregnant until after their blood levels of teriflunomide are low. For general information on exposures that males might have, please see the MotherToBaby fact sheet called Paternal Exposures at https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

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

[1]. Ther Clin Risk Manag.2013;9:177-90.
[2]. Mol Biol Rep . 2024 May 9;51(1):572. doi: 10.1007/s11033-024-09502-9.
[3]. Heliyon . 2024 Apr 10;10(8):e29481. doi: 10.1016/j.heliyon.2024.e29481.

Teriflunomide is an enamide obtained by formal condensation of the carboxy group of (2Z)-2-cyano-3-hydroxybut-2-enoic acid with the anilino group of 4-(trifluoromethyl)aniline. Used for the treatment of relapsing forms of multiple sclerosis and rheumatoid arthritis. It has a role as an EC 1.3.98.1 [dihydroorotate oxidase (fumarate)] inhibitor, a tyrosine kinase inhibitor, a hepatotoxic agent, a drug metabolite and a non-steroidal anti-inflammatory drug. It is a nitrile, an enol, an aromatic amide, an enamide, a member of (trifluoromethyl)benzenes and a secondary carboxamide.
Teriflunomide is the active metabolite of leflunomide, and it acts as an immunomodulatory agent by inhibiting pyrimidine synthesis. It is marketed under the name Aubagio® and is indicated for the treatment of multiple sclerosis, specifically relapsing forms. The FDA label states an important warning about the risk of hepatoxicity and teratogenicity for patients using teriflunomide.
Teriflunomide is a Pyrimidine Synthesis Inhibitor. The mechanism of action of teriflunomide is as a Dihydroorotate Dehydrogenase Inhibitor.
Teriflunomide is an orally available immunomodulatory agent used to treat relapsing multiple sclerosis. Teriflunomide is associated with transient serum enzyme elevations during therapy and with rare instances of acute liver injury.
See also: Leflunomide (is active moiety of).

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Drug Indication
Used in the treatment of relapsing forms of multiple sclerosis (MS).
FDA Label
AUBAGIO is indicated for the treatment of adult patients and paediatric patients aged 10 years and older with relapsing remitting multiple sclerosis (MS) (please refer to section 5. 1 for important information on the population for which efficacy has been established).
Teriflunomide Mylan is indicated for the treatment of adult patients and paediatric patients aged 10 years and older (body weight > 40 kg) with relapsing remitting multiple sclerosis (MS) (please refer to section 5. 1 of the SmPC for important information on the population for which efficacy has been established).  
Teriflunomide Accord is indicated for the treatment of adult patients and paediatric patients aged 10 years and older with relapsing remitting multiple sclerosis (MS) (please refer to section 5. 1 for important information on the population for which efficacy has been established).
Treatment of multiple sclerosis

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Mechanism of Action
The exact mechanism by which teriflunomide acts in MS is not known. What is known is that teriflunomide prevents pyrimidine synthesis by inhibiting the mitochondrial enzyme dihydroorotate dehydrogenase, and this may be involved in its immunomodulatory effect in MS.

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; 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.

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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.)