Active metabolite of Leflunomide; dihydroorotate dehydrogenase (DHODH)

Dihydroorotate dehydrogenase (DHODH), a crucial mitochondrial enzyme involved in the de novo synthesis of pyrimidines in rapidly growing cells, is the primary enzyme that teriflunomide inhibits. It is possible that teriflunomide, which is a cytostatic rather than a cytotoxic medication to leukocytes, lessens the inflammatory response to autoantigens in MS by inhibiting the activity of high-avidity proliferating T lymphocytes and B lymphocytes[1].

In two different animal models of demyelinating illness, teriflunomide has proven to have positive effects. Teriflunomide administration produces clinical, histological, and electrical evidence of efficacy as a preventative and therapeutic drug in the dark agouti rat model of experimental autoimmune encephalitis (EAE). Similar to this, teriflunomide treatment delays the development of the disease and its severity in the female Lewis rat model of EAE[1]. This has positive preventative and therapeutic clinical benefits.

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

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 of their partners' 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 is 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/.

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

[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).

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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 the applicable population for proven efficacy, see Section 5.1).
Treatment of Multiple Sclerosis

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Mechanism of Action
The exact mechanism of action of teriflunomide in MS is not known. 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.

C12H9F3N2O2

270.21

270.061

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

163451-81-8

(E/Z)-Teriflunomide;108605-62-5

54684141

Off-white to yellow solid powder

1.4±0.1 g/cm3

410.8±45.0 °C at 760 mmHg

202.3±28.7 °C

0.0±1.0 mmHg at 25°C

1.552

2.51

2

6

2

19

426

0

C/C(=C(\C#N)/C(=O)NC1=CC=C(C=C1)C(F)(F)F)/O

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

Teriflunomide; 163451-81-8; Flucyamide; HMR 1726; teriflunomida; SU 20; teriflunomidum; A 77-1726;

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)

DMSO: 33.33 mg/mL (123.35 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.25 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 (9.25 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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 (Please use freshly prepared in vivo formulations for optimal results.)