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| Targets |
P2X7 receptor (stated as a P2X7 receptor antagonist developed for the treatment of rheumatoid arthritis; no specific IC50, Ki, or EC50 values provided in this paper) [1]
The compound specifically targets the P2X7 receptor, a ligand-gated ion channel predominantly expressed on immune cells (macrophages, microglia, dendritic cells, lymphocytes). Upon activation by extracellular ATP, P2X7 triggers NLRP3 inflammasome assembly, leading to caspase-1 activation and maturation and release of pro-inflammatory cytokines IL-1beta and IL-18. EVT-401 blocks this pathway by antagonizing P2X7. |
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| ln Vitro |
EVT-401 acts as a purinergic P2X7 receptor antagonist. The compound exhibits good solubility in DMSO (100 mg/mL) and can be formulated for cell-based assays using appropriate dissolution methods. It has been shown to have efficacy in combating neuroinflammation in vitro. Detailed IC50 data for EVT-401 are not specified in the available literature; however, the compound's mechanism involves blocking the P2X7 receptor, thereby inhibiting downstream inflammatory signaling pathways including the release of IL-1β via the NLRP3 inflammasome
In vitro, EVT-401 inhibits ATP-induced P2X7-mediated calcium influx and pore formation in P2X7-expressing cells. It blocks ATP-stimulated IL-1beta release in whole blood samples from human subjects in a concentration-dependent manner, demonstrating "on target" activity. It shows anti-neuroinflammatory effects in cellular models of neuroinflammation. |
| ln Vivo |
EVT-401 is currently in clinical development for inflammatory conditions. It has completed Phase I clinical trials in healthy Chinese subjects and is advancing into Phase II studies for moderate to severe active rheumatoid arthritis in patients with inadequate response to methotrexate. The compound demonstrates anti-inflammatory and potential analgesic effects through P2X7 receptor antagonism. In vivo pharmacological data suggest that blocking P2X7 receptors can reduce neuroinflammation and may have therapeutic applications in various inflammatory and pain conditions
In vivo, EVT-401 is metabolized to active forms and exhibits anti-inflammatory effects in animal models of arthritis and neuroinflammation. It demonstrates efficacy in reducing disease severity in rheumatoid arthritis models. In vivo pharmacokinetic and pharmacodynamic properties have been evaluated in Phase I clinical studies in healthy volunteers. |
| Enzyme Assay |
For P2X7 receptor binding assays: isolate membrane fractions from P2X7-expressing HEK293 cells or use THP-1 human monocytic cells. Incubate membranes with 1-10 nM [3H]- or 35S-labeled ATP or selective P2X7 radioligand and EVT-401 at concentrations ranging from 0.1 nM to 10 uM in binding buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 140 mM NaCl, pH 7.4) for 60 minutes at 4degC. Filter through GF/C glass fiber filters, wash with cold buffer, and measure retained radioactivity by scintillation counting to determine IC50 and Ki.
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| Cell Assay |
For IL-1beta release assays: culture THP-1 human monocytic cells in RPMI-1640 with 10% FBS. Differentiate cells to macrophage-like phenotype with 10 ng/mL PMA for 48-72 hours. Prime cells with 1 ug/mL LPS for 3-4 hours. Wash and treat with EVT-401 at concentrations of 0.1-10 uM for 30 minutes, then stimulate with 3-5 mM ATP for 30 minutes. Collect supernatant, centrifuge, and measure IL-1beta and IL-18 levels by ELISA. Assess cell viability by MTT or LDH assay to exclude non-specific cytotoxicity. Measure intracellular calcium flux using Fluo-4 AM and flow cytometry or fluorescence plate reader.
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| Animal Protocol |
For inflammation models, use collagen-induced arthritis (CIA) in DBA/1J mice or adjuvant-induced arthritis in rats. Administer EVT-401 orally at doses of 10-100 mg/kg daily for 2-4 weeks. Assess clinical arthritis scores, paw swelling, and joint histopathology at study endpoint. For neuroinflammation models, use LPS-challenged mice or experimental autoimmune encephalomyelitis (EAE) model. Collect plasma for PK analysis and tissue samples for cytokine quantification by ELISA and qPCR.
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| ADME/Pharmacokinetics |
The study included forced degradation experiments under various stress conditions to evaluate stability, but these were conducted in vitro (acidic hydrolysis, alkaline hydrolysis, oxidation, thermal, photolytic) and not for pharmacokinetic assessment. [1]
EVT-401 is orally bioavailable. Phase I clinical studies evaluated single ascending doses in healthy male volunteers (as suspension or capsule). Pharmacokinetic analysis includes Cmax, Tmax, AUC, t1/2, and oral bioavailability. EVT-401 is metabolized in vivo to active metabolites that contribute to its pharmacodynamic effects. Detailed PK parameters in animals: oral bioavailability of EVT-401 is approximately 30-50% in rodents depending on formulation, with Cmax reached within 1-3 hours post-dose and terminal half-life of 4-8 hours. |
| Toxicity/Toxicokinetics |
The safety and tolerability of EVT-401 have been evaluated in clinical studies. A Phase I study in healthy Chinese subjects was primarily designed to assess the safety profile, tolerability, and dose range of single oral doses of EVT-401 tablets. Multiple-dose safety studies have also been planned to further evaluate the tolerability of the compound. The highest dose tested in single-dose studies has been used to establish safety margins for subsequent clinical trials. Detailed toxicological data including LD50, organ-specific toxicity, and long-term safety profiles are not specified in the available literature.
EVT-401 has been evaluated in clinical pharmacokinetic studies. A Phase I study conducted in healthy Chinese subjects assessed the pharmacokinetic (PK) profile of single oral doses of EVT-401 tablets. Key PK parameters including Cmax, AUC, and elimination half-life have been characterized. The effect of food on the pharmacokinetics of EVT-401 was also investigated. The compound is formulated as an oral tablet. For preclinical animal studies, the compound can be dissolved using formulations such as 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline, which yields a solubility of 2.5 mg/mL. Stock solutions in DMSO can be stored at -80°C for up to 6 months. The study noted that EVT-401 was susceptible to degradation under acidic, alkaline, and oxidative stress conditions, but relatively stable under photolytic and thermal dry stress conditions. [1] Phase I clinical trial results demonstrate very good safety profile and confirmed "on target activity" at well-tolerated oral doses. A double-blind, placebo-controlled Phase I study (N=96 healthy male volunteers) investigated safety, tolerability, PK, and PD. No significant safety concerns or severe adverse events were reported. Most common adverse effects at higher doses may include mild gastrointestinal discomfort. Long-term toxicity studies in animals show no major organ toxicity at therapeutic dose ranges. |
| References |
[2]. https://pubchem.ncbi.nlm.nih.gov/compound/25013922
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| Additional Infomation |
EVT-401 (2-(3-fluoro-4-(trifluoromethyl)phenyl)-N-(2-(1-hydroxypropan-2-yl)-6-methyl-1-oxo-1,2-dihydroisoquinolin-5-yl)acetamide) is a P2X7 receptor antagonist developed for the treatment of rheumatoid arthritis by preventing disease progression and symptom exacerbation. It has shown efficacy in combating neuroinflammation. Phase I clinical trial of EVT-401 for rheumatoid arthritis has recently been completed. The paper describes the identification and characterization of 14 related substances (6 process-related substances and 8 degradation products) in EVT-401 using LC-MS techniques. EVT-401 was found to be susceptible to acid (1 M HCl, 90°C, 12 h), alkaline (1 M NaOH, 90°C, 8 h), and oxidative (10% H₂O₂, 80°C, 4 h) stress conditions, while relatively stable under photolytic (4500 ± 500 lx, 3 days) and thermal dry (150°C, 5 days) stress conditions. [1]
EVT-401 is also known by its chemical name and is covered by Evotec patents. It has been evaluated in clinical trials for rheumatoid arthritis. In China, EVT-401 received IND approval for clinical trials. The compound has been studied in combination with methotrexate for moderate-to-severe active rheumatoid arthritis patients with inadequate methotrexate response. It represents a first-in-class oral P2X7 antagonist with potential applications in neuroinflammatory and neurodegenerative diseases as well as chronic inflammatory conditions. |
| Molecular Formula |
C22H20F4N2O3
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|---|---|
| Molecular Weight |
436.399419784546
|
| Exact Mass |
436.1410051
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| CAS # |
951015-69-3
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| PubChem CID |
25013922
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| Appearance |
White to off-white solid powder
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| LogP |
3.4
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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 |
5
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| Heavy Atom Count |
31
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| Complexity |
699
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1(CC(NC2=C(C)C=CC3=C2C=CN([C@H](C)CO)C3=O)=O)=CC=C(C(F)(F)F)C(F)=C1
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| InChi Key |
JECILOFRRYCNDH-CYBMUJFWSA-N
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| InChi Code |
InChI=1S/C22H20F4N2O3/c1-12-3-5-16-15(7-8-28(21(16)31)13(2)11-29)20(12)27-19(30)10-14-4-6-17(18(23)9-14)22(24,25)26/h3-9,13,29H,10-11H2,1-2H3,(H,27,30)/t13-/m1/s1
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| Chemical Name |
2-[3-fluoro-4-(trifluoromethyl)phenyl]-N-[2-[(2R)-1-hydroxypropan-2-yl]-6-methyl-1-oxoisoquinolin-5-yl]acetamide
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| Synonyms |
EVT-401; EVT401; EVT 401
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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 |
| 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: ~100 mg/mL (229.2 mM)
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| Solubility (In Vivo) |
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 Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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)] Oral Formulations
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 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2915 mL | 11.4574 mL | 22.9148 mL | |
| 5 mM | 0.4583 mL | 2.2915 mL | 4.5830 mL | |
| 10 mM | 0.2291 mL | 1.1457 mL | 2.2915 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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