THR-β (EC50 = 0.21 μM)

Compared to THR-α (EC50=3.74 μM), Resmetirom (MGL-3196) exhibits a 28-fold increase in selectivity for THR-β (EC50=0.21 μM). Resmetirom (MGL-3196) is used to inhibit hERG channels and has an IC20 of about 30 μM. The inhibitory effect on CYP2C9 is relatively mild (about 22 μM), whereas the IC50 of CYP3A4/5 and CYP2C19 is >50 μM [1].

In rats, resmetirom (MGL-3196) showed reasonable oral bioavailability and excellent exposure. There is little distribution volume and little clearing. A dose-proportional increase in exposure was seen in DIO mice after oral administration of Resmetirom (MGL-3196) solution [1]. Due to hepatic TG, cholesterol and liver size were decreased in rats given Resmetirom (MGL-3196). Animals treated with Resmetirom (MGL-3196) showed no change in heart or kidney size, or bone mineral density (BMD) [1].

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Treatment with resmetirom did not influence body weight but led to significant reduction in liver weight, hepatic steatosis, plasma alanine aminotransferase activity, liver and plasma cholesterol, and blood glucose. These metabolic effects translated into significant improvement in NAFLD activity score. Moreover, a lower content of α-smooth muscle actin and down-regulation of genes involved in fibrogenesis indicated a decrease in hepatic fibrosis.[2]

THR/RXR/GRIP1 Assay[1]
The ligand binding domain (amino acids 148–410) of THR-β (H6-THR-β) and the ligand binding domain (amino acids 202–461) of THR-α (H6-THR-α) were cloned into an E. coli expression vector pET28a that contained a N-terminal hexa His sequence. The resulting recombinant hexa His tagged proteins were produced in E. coli BL21(DE3) cells. Cells were grown in Terrific Broth (in-house prepared medium of Bacto tryptone (3.3%, w/v), Difico yeast extract (2.0%, w/v), and NaCl (0.5%, w/v)) using shake flasks with a 24 h induction in 0.2 mM IPTG at 25 °C, harvested, and lysed with five volumes of buffer A (0.05 M Tris, 0.3 M NaCL, 1%W/V betaine, 0.01 M imidazole, 0.02 M β-mercaptoethanol, pH 8.0). Lysozyme (1.0 mg/mL) and Complete protease inhibitor cocktail were added to slurry, and the solution was sonicated for 1 min five times at 4 °C. The suspension was centrifuged in a Ti45 Beckmann rotor for 2 h at 127 300 RCF, and the supernatant was loaded onto NI_ NTA agarose (Quigen 30210) column. After a washing with buffer A, H6-TRβ or H6-TRα was eluted with buffer A containing 0.25 M imidazole.[1]
The ligand binding domain of human retinoid X receptor (amino acids 225–462) (RxRα) was engineered with N-terminal His6 and EE (EFMPME) tags, a thrombin cleavage site between the His6 and EE sequences, and cloned into pACYC vector. The resulting His6-EE-tagged protein was produced in E. coli cells. Cells were grown using shake flasks with an 18 h induction in 0.1 mM IPTG at 18 °C, harvested, and suspended with five volumes of buffer B (0.025 M Tris, 0.3 M NaCl, 0.02 M imidazole, 0.01 M β-mercaptoethanol, pH 8.0). Lysozyme (0.2 mg/mL,) and Complete protease inhibitor cocktail were added and stirred for 30 min at 4 °C. The suspension was sonicated for 30 s, five times, at 4 °C. The suspension was centrifuged for 20 min at 12 000 RCF. The supernatant was filtered by 0.45 μm pore size membrane, and 0.5% NP-40 was added. The His6-tagged protein was bound to and eluted from NiNTA metal-affinity resin. The protein was concentrated and dialyzed. The His6 tag was removed from EE-RxRα by thrombin digestion, using 10 units of thrombin (Pharmacia, Piscataway, NJ) per milligram of protein and incubating for 2 h at 25 °C. Removal of thrombin was done batchwise using benzamidine-Sepharose 6B. The protein was concentrated and dialyzed. This protein was used in the coactivator peptide recruitment assay. [1]

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THR-β/RXR/GRIP1 Coactivator Peptide Recruitment Assay[1]
An amount of 30 μL of H6-THR-β (50 nM) in 50 mM Hepes, pH 7.0, 1 mM DTT, 0.05% NP40, and 0.2 mg/mL BSA (binding buffer) was mixed with an equal volume of EE-RxRα (50 nM) in binding buffer. An amount of 6 μL of T3 (0–14.8 μM) or test compound (0–1.2 mM) in DMSO was then added and the solution incubated at 37 °C for 30 min. Then 30 μL of biotin-GRIP1 peptide (biotin-Aca-HGTSLKEKHKILHRLLQDSSSPVDL-CONH2) (100 nM) in 30 μL of binding buffer plus 5% DMSO was added and the solution incubated at 37 °C for 30 min. An amount of 30 μL of solution containing 12 nM europium-conjugated anti hexa His antibody and 160 nM APC-conjugated streptavidin in 50 mM Tris, pH 7.4, 100 mM NaCl, and 0.2 mg/mL BSA was added, and the solution was incubated at 4 °C overnight. An aliquot (35 μL/sample) was transferred to 384-well black microtiter plates. The HTRF signal was read on the Victor 5 reader.[1]

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THR-α/RXR/GRIP1 Coactivator Peptide Recruitment Assay[1]
The assay protocol is essentially the same as that of THR-β/RXR/GRIP1 coactivator peptide recruitment assay as described above except that 125 nM H6-THR-α, 125 nM EE-RxRα, and 250 nM biotin-GRIP1 were used.

Thyroidectomized Rat Cardiac Myocyte Assay[1]
These assays were conducted under the direction of Irwin Klein using the previously described procedures. Compounds 53 [Resmetirom (MGL-3196)], 54, and 55 were formulated in 4% DMSO, 15% PEG-400, and 81% of 30% HPBCD in phosphate buffer and were administered intraperitoneally. For 53 and 54, 4 rats per group were tested at 5, 20, and 37.5 mg/kg. For 55, 3 rats per group were tested at 5 and 15 mg/kg and 4 rats were tested at 50 mg/kg. Compound 21 was formulated in 2% Klucel LF, 0.1% Tween 80, water and dosed orally (n = 3 per group) at 0.0625, 0.25, and 1.25 mg/kg.[1]

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C57Bl/6J-Diet-Induced Obese (DIO) Mice Study[1]
Six week old C57Bl/6J mice were placed on a high fat diet for 34 weeks. At day 0, 9 mice per group were treated daily doses by oral gavage with vehicle (2% Klucel LF, 0.1% Tween 80 in water) or 0.3, 1, 3, or 10 mg/kg 53 [Resmetirom (MGL-3196)] for 23 days. In a parallel study, at day 0, 9 mice per group were treated with daily doses of vehicle (Dulbecco’s phosphate buffered saline, pH adjusted to 9.0 with 1 N NaOH) or 10, 30, or 100 μg/kg T3. Body weight and food intake were monitored during the study. BMD and body composition assessments were made on day 22. On day 23 at necropsy, organ weights were obtained for determination of organ weight and blood samples were assessed for cholesterol and other chemistry parameters.[1]

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C57Bl/6J mice were fed a diet high in fat, fructose, and cholesterol for 34 weeks, and only biopsy-confirmed DIO-NASH mice with fibrosis were included. Resmetirom was administered at a daily dose of 3 mg·kg−1 p.o., for 8 weeks. Systemic and hepatic metabolic parameters, histological non-alcoholic fatty liver disease (NAFLD) activity and fibrosis scores, and liver RNA expression profiles were determined to assess the effect of THR-β activation. Resmetirom was administered once daily, in the morning, by oral gavage at a dose of 3 mg·kg−1. The vehicle used for compound formulation and control injections was 0.6% methyl cellulose with 0.5% Tween 80. The resmetirom concentration in the dosing solution was 0.6 mg·ml−1.[2]

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For CDAHFD induced NASH model construction, after 2 weeks of acclimation, mice were initiated on choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Oral gavage dosing of Resmetirom (10 mg/kg daily) was initiated on week 4 of CDAHFD and continued until termination. [2]
For HFD+CCl4 induced NASH model construction, after 2 weeks of acclimation, mice were initiated on high-fat diet (HFD). Mice were orally gavaged with Resmetirom(10 mg/kg daily) on week 10 of HFD and intraperitoneally injected with CCl4 (0.05 mL/kg) until termination. [2]
For exogenous cholic acid (CA) intervention experiments, normal mice (male C57BL/6J mice) or CDAHFD-fed mice were administered vehicle or 10 mg/kg Resmetirom, together with or without 250 mg/kg CA for 5 consecutive days. CA was administered twice daily with a 12 h interval.[3]

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Dissolved in2% Klucel LF, 0.1% Tween 80 in water; 0.3, 1, 3, or 10 mg/kg 53 for 23 days; p.o.

Six week old C57Bl/6J mice

Hepatotoxicity
Mild, transient serum aminotransferase elevations develop in a high proportion of patients receiving resmetirom, generally within the first 4 weeks of therapy. These elevations are typically mild, self-limited, and not associated with symptoms or jaundice. Furthermore, these early changes were usually followed by a decrease in serum enzymes which were often within normal range 3 to 6 months later. These improvements in liver related enzymes correlated to some extent with the decrease in hepatic fat and histologic evidence of steatohepatitis. After 52 weeks of treatment, liver biopsies demonstrated resolution of NASH in 26% to 30% of patients. Whether these changes are sustained or increase with further therapy is not known. Therapy does not result in weight loss, and the improvements in hepatic histology and fibrosis may be lost once therapy is discontinued.
Analysis of liver tests from more than 1300 adults with NASH treated with resmetirom in doses of 80 or 100 mg daily for up to one year identified 2 patients with liver injury that was considered at least possibly due to resmetirom. The latency to initial onset was 2 and 3 months [ALT 236 U/L and 578 U/L, Alk P unknown and 64 U/L, bilirubin 0.6 and 1.1 mg/dL]. Both patients recovered completely within 1 to 2 months of stopping treatment. One patient was restarted on treatment and redeveloped liver injury within 28 days (ALT 3226 U/L, Alk P 140 U/L, bilirubin 10.9 mg/dL) that was more severe than the initial episode, but that resolved spontaneously within 2 months of stopping. In both cases, other diagnoses remained possible.
Likelihood score: D (possible rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of resmetirom during breastfeeding. Because resmetirom is more than 99% bound to plasma proteins, the amount in milk is likely to be low. If the mother requires resmetirom, it is not a reason to discontinue breastfeeding. Until more data are available, resmetirom should be used with careful infant monitoring 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.

[1]. Discovery of 2-[3,5-dichloro-4-(5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yloxy)phenyl]-3,5-dioxo-2,3,4,5-tetrahydro[1,2,4]triazine-6-carbonitrile (MGL-3196), a Highly Selective Thyroid Hormone Receptor β agonist in clinical trials for the treatment of dyslipidemia. J Med Chem. 2014 May 22;57(10):3912-23.
[2]. Activation of thyroid hormone receptor-β improved disease activity and metabolism independent of body weight in a mouse model of non-alcoholic steatohepatitis and fibrosis. Br J Pharmacol . 2021 Jun;178(12):2412-2423.
[3]. A new mechanism of thyroid hormone receptor β agonists ameliorating nonalcoholic steatohepatitis by inhibiting intestinal lipid absorption via remodeling bile acid profiles. Acta Pharmacologica Sinica (2024).

MGL-3196 has been used in trials studying the treatment of Non-alcoholic steatohepatitis and Heterozygous Familial Hypercholesterolemia.
Resmetirom is a thyroid hormone receptor beta (THR-β) agonist used in conjunction with diet and exercise in the therapy of nonalcoholic steatohepatitis (NASH) with moderate-to-severe fibrosis. Resmetirom therapy is associated with mild and transient serum aminotransferase elevations during the first month of therapy and with rare instances of acute liver injury which can be severe, but which reverses on drug discontinuation.

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Drug Indication
Treatment of non-alcoholic steatohepatitis (NASH)

C17H12CL2N6O4

435.22

434.03

C, 46.92; H, 2.78; Cl, 16.29; N, 19.31; O, 14.70

920509-32-6

15981237

Typically exists as Yellow to orange solids at room temperature

1.65±0.1 g/cm3 (20 °C, 760 mmHg)

2.098

2

7

4

29

878

0

N#CC1C(=O)NC(=O)N(C2C=C(Cl)C(OC3C=C(C(C)C)C(=O)NN=3)=C(Cl)C=2)N=1

FDBYIYFVSAHJLY-UHFFFAOYSA-N

InChI=1S/C17H12Cl2N6O4/c1-7(2)9-5-13(22-23-15(9)26)29-14-10(18)3-8(4-11(14)19)25-17(28)21-16(27)12(6-20)24-25/h3-5,7H,1-2H3,(H,23,26)(H,21,27,28)

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)

Solubility in Formulation 1: ≥ 3.75 mg/mL (8.62 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 37.5 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: ≥ 3.75 mg/mL (8.62 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 37.5 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: ≥ 3.75 mg/mL (8.62 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 37.5 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.)