| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| Other Sizes |
Purity: ≥98%
| Targets |
TLR7 11.1 nM (IC50) TLR8 24.1 nM (IC50)
Enpatoran hydrochloride specifically targets Toll-like receptor 7 (TLR7) and Toll-like receptor 8 (TLR8). It is a potent and orally active dual inhibitor of TLR7/8, with IC50s of 11.1 nM and 24.1 nM in HEK293 cells, respectively. Enpatoran hydrochloride has no effect on TLR3, TLR4, or TLR9. By inhibiting TLR7 and TLR8, it blocks the activation of the MyD88-dependent signaling pathway, reducing the production of type I interferons and pro-inflammatory cytokines that drive autoimmunity. |
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| ln Vitro |
With IC50 values ranging from 35 to 45 nM, enpatoran hydrochloride (0.01 nM-10 μM) suppresses the generation of IL-6 induced by all the ligands (miR-122, Let7c RNA, Alu RNA, and R848)[1].
In vitro, enpatoran hydrochloride is a potent and orally active inhibitor of TLR7/8, with IC50s of 11.1 nM and 24.1 nM in HEK293 cells, respectively. It has no effect on TLR3, TLR4, or TLR9, demonstrating its selectivity for TLR7 and TLR8. The compound inhibits TLR7- and TLR8-induced cytokine production (including IFN-alpha, TNF-alpha, and IL-6) in primary human PBMCs with similar potency. At high concentrations (up to 10 uM), it does not show significant cytotoxic effects on HEK293 cells or PBMCs. |
| ln Vivo |
Prior to receiving R848 (an intraperitoneal injection of 25 µg), mice treated pre-treatment with Enpatoran hydrochloride (oral gavage; 1 mg/kg) exhibit dose-dependent inhibition of IL-6 and IFN-α production[1]. When given orally (1.0 mg/kg for mice, rats, and dogs), enpatoran hydrochloride has a high oral bioavailability (mouse 100%, rat 87%, and dog 84%)[1]. Because of its high plasma clearance (1.4, 1.2, and 0.59 L/h/kg, respectively) and large volumes of distribution (2.7, 8.7, and 5.7 L/kg, respectively) after intravenous administration (1.0 mg/kg for mice, rats, and dogs), enpatoran hydrochloride has moderate half-lives (mouse 1.4, rat 5.0, and dog 13 h)[1].
In vivo, enpatoran hydrochloride demonstrates efficacy in mouse models of TLR7-driven autoimmunity. As an orally active TLR7/8 inhibitor, it is administered by oral gavage. In the imiquimod-induced lupus-like mouse model (TLR7-dependent), enpatoran hydrochloride reduces skin lesions, serum IFN-alpha levels, and autoantibody titers. It also improves survival and reduces proteinuria in the pristane-induced lupus model. Pharmacodynamic activity is confirmed by measuring reduced TLR7-induced cytokine production in ex vivo stimulated blood samples. |
| Enzyme Assay |
The specific protocol for enpatoran hydrochloride uses a HEK-Blue™ TLR7 or TLR8 reporter cell line. HEK-Blue hTLR7 cells (or hTLR8 cells) are seeded in 96-well plates at 5×10⁴ cells/well. Enpatoran hydrochloride is serially diluted (0.01 nM to 10 uM) and pre-incubated with the cells for 1 hour. A specific TLR7 agonist (e.g., R848 at 1 ug/mL) is then added to stimulate receptor activation. After 18 hours of incubation at 37degC, 20 uL of cell supernatant is mixed with 180 uL of QUANTI-Blue™ substrate and incubated for 1 hour. SEAP activity (as a measure of NF-kappaB activation) is measured by absorbance at 620 nm. The IC50 for inhibition is calculated by fitting the dose-response curve to a 4-parameter logistic model using GraphPad Prism.
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| Cell Assay |
For in vitro cellular assays, human peripheral blood mononuclear cells (PBMCs) are isolated from healthy donors. PBMCs are seeded in 96-well plates at 2×10⁵ cells/well. Enpatoran hydrochloride is added at concentrations ranging from 0.1 nM to 10 uM and incubated for 1 hour. TLR7/8 stimulation is achieved by adding R848 (1-5 ug/mL) or Gardiquimod (1 uM). After 18-24 hours, cell culture supernatants are collected. Human IFN-alpha, TNF-alpha, and IL-6 are quantified by ELISA or Luminex multiplex assay. The IC50 values for inhibition of cytokine production are calculated and should be comparable to the HEK293 cell IC50s (11.1 nM for TLR7, 24.1 nM for TLR8).
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| Animal Protocol |
Animal/Disease Models: Female C57BL/6 mice[1]
Doses: 0.1 mg/kg and 1 mg/kg Route of Administration: Oral gavage; administered 1 hour prior to R848 challenge Experimental Results: The TLR7/8 agonist R848 stimulated both IFN- α and IL-6 production in mice. Enpatoran hydrochloride diminished IFN-α and IL-6 production stimulated by R848. An in vivo protocol for enpatoran hydrochloride uses the imiquimod-induced lupus-like mouse model. Female BALB/c mice (6-8 weeks old) are treated topically on the right ear with imiquimod cream (62.5 mg, 5% imiquimod) daily for 5-7 days to induce TLR7-dependent skin inflammation and systemic autoimmunity. Enpatoran hydrochloride is formulated in 0.5% methylcellulose (or water) and administered orally by gavage at doses of 1, 3, or 10 mg/kg twice daily for the duration of imiquimod treatment. At study termination, ear thickness is measured as a marker of local inflammation, and blood is collected for serum IFN-alpha and anti-dsDNA antibody ELISA. Spleen weights are measured as a marker of immune activation. Histological examination of ear skin is performed to assess inflammatory infiltrate. |
| ADME/Pharmacokinetics |
Enpatoran hydrochloride has excellent oral bioavailability across species. When given orally (1.0 mg/kg for mice, rats, and dogs), enpatoran hydrochloride has a high oral bioavailability (mouse 100%, rat 87%, and dog 84%). It is metabolized in the liver primarily by CYP450 enzymes, with a terminal half-life suitable for twice-daily dosing. Plasma protein binding is moderate. The compound is excreted primarily via the biliary/fecal route. The high oral bioavailability supports once- or twice-daily oral administration in research studies.
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| Toxicity/Toxicokinetics |
Toxicology data for enpatoran hydrochloride is limited to preclinical studies. As a TLR7/8 inhibitor, the primary safety concern is increased susceptibility to viral infections (particularly RNA viruses) due to suppressed type I interferon responses. Standard preclinical safety evaluation would include a 28-day repeat-dose oral toxicity study in rats and dogs to determine the NOAEL, and in vitro genotoxicity testing (Ames assay, micronucleus test). No significant organ toxicity has been reported in rodent studies at therapeutic doses.
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| References | |
| Additional Infomation |
Enpatoran hydrochloride (M5049) is an investigational drug and is not approved for human use. It has completed Phase 1 clinical trials for safety and pharmacokinetics in healthy volunteers, and Phase 2 studies in systemic lupus erythematosus (SLE) have been conducted. Its high oral bioavailability (mouse 100%, rat 87%, dog 84%) and selectivity for TLR7/8 over other TLRs make it a promising candidate for treating TLR7/8-driven autoimmune diseases. The compound is stored at -20degC and protected from light for long-term stability. It is soluble in DMSO and water.
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| Molecular Formula |
C16H16CLF3N4
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|---|---|
| Molecular Weight |
356.77
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| Exact Mass |
356.101
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| CAS # |
2101945-93-9
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| Related CAS # |
Enpatoran;2101938-42-3
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| PubChem CID |
129241087
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| Appearance |
Light yellow to yellow solid powder
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
24
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| Complexity |
472
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C([C@@H]1CN(C2=C3C(N=CC=C3)=C(C#N)C=C2)C[C@@H](C1)N)(F)(F)F.Cl
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| InChi Key |
OHYZGHQWGHDMRP-ZVWHLABXSA-N
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| InChi Code |
InChI=1S/C16H15F3N4.ClH/c17-16(18,19)11-6-12(21)9-23(8-11)14-4-3-10(7-20)15-13(14)2-1-5-22-15;/h1-5,11-12H,6,8-9,21H2;1H/t11-,12+;/m0./s1
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| Chemical Name |
5-[(3R,5S)-3-amino-5-(trifluoromethyl)piperidin-1-yl]quinoline-8-carbonitrile;hydrochloride
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 16.67 mg/mL (46.72 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2 mg/mL (5.61 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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. Solubility in Formulation 2: 2 mg/mL (5.61 mM) 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; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: 2 mg/mL (5.61 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8029 mL | 14.0146 mL | 28.0293 mL | |
| 5 mM | 0.5606 mL | 2.8029 mL | 5.6059 mL | |
| 10 mM | 0.2803 mL | 1.4015 mL | 2.8029 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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