| Size | Price | Stock | Qty |
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| Targets |
P2X3 homotrimeric receptor, ligand-gated ion channel activated by ATP.
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| ln Vitro |
P2X3 antagonist 34 (BLU-5937; 500 nM) antagonizes P2X3 homotrimeric receptors to prevent αβ-meATP-induced sensitization of isolated primary nociceptors in the rat dorsal root ganglion (DRG). as well as the discharge process. Following washout, both the sensitizing effect of αβ-meATP and the inhibitory impact of P2X3 antagonist 34 are reversible [1].
P2X3 antagonist 34 is a potent and selective antagonist of the P2X3 homotrimeric receptor. It exhibits IC50 values of 25 nM for the human P2X3 receptor, 92 nM for the rat P2X3 receptor, and 126 nM for the guinea pig P2X3 receptor. Importantly, the compound shows significantly reduced activity against the P2X2/3 heterotrimeric receptor across human, rat, and guinea pig models, with IC50 values in the micromolar range (e.g., 1820 nM for rat P2X2/3). This selectivity profile is designed to minimize off-target effects. In isolated primary nociceptors from rat dorsal root ganglions (DRGs), P2X3 antagonist 34 at 500 nM effectively inhibits alphabeta-meATP-induced sensitization and the consequent firing activity by antagonizing P2X3 homotrimeric receptors. The sensitization caused by alphabeta-meATP and the inhibition by P2X3 antagonist 34 are both reversible following washout. |
| ln Vivo |
The administration of P2X3 antagonist 34 (BLU-5937; 0.3-0 mg/kg, oral; male Dunkin Hartley guinea pigs) to guinea pigs resulted in a dose-dependent reduction in the number of coughs induced by citric acid, which was caused by histamine. model [1]. It has also been demonstrated that the P2X3 antagonist 34 (BLU-5937; orally administered at 3 and 30 mg/kg) significantly and dose-dependently reduces the ATP-induced potentiation of citric acid-induced cough in guinea pigs [1].
In a guinea pig cough model, treatment with P2X3 antagonist 34 (BLU-5937) administered orally at doses ranging from 0.3 to 30 mg/kg in male Dunkin Hartley guinea pigs significantly and dose-dependently diminished both histamine-induced and ATP-induced enhancements of citric acid-induced coughs. This outcome highlights the compound's efficacy in reducing cough reflex sensitivity through oral administration. The compound demonstrates a significant anti-tussive effect without altering taste, which is a key advantage over less selective P2X3 antagonists that often cause dysgeusia (taste disturbance). |
| Enzyme Assay |
The in vitro binding affinity and selectivity of P2X3 antagonist 34 for the P2X3 receptor are evaluated using standard radioligand binding assays or functional calcium flux assays. For the radioligand binding assay, membranes prepared from CHO cells stably expressing the human P2X3 receptor are incubated with a fixed concentration of a radiolabeled P2X3 antagonist, such as [3H]-A-317491, in the presence of varying concentrations of the test compound in assay buffer (e.g., 50 mM Tris-HCl, pH 7.4, 0.1% BSA). After a 2-hour incubation at room temperature, the reaction is terminated by rapid filtration through GF/B glass fiber filters pre-soaked in 0.3% polyethyleneimine. The filters are washed with ice-cold wash buffer, and the bound radioactivity is measured using a scintillation counter. Non-specific binding is determined in the presence of a saturating concentration of unlabeled ATP. For functional antagonism, an FLIPR Calcium Assay is used. HEK-293 cells stably expressing the human P2X3 receptor are seeded in 384-well black-walled clear-bottom plates at 30,000 cells per well in culture medium. After 24 hours, the medium is removed, and cells are loaded with a calcium-sensitive dye (e.g., Fluo-4-AM) in assay buffer (HBSS with 20 mM HEPES, pH 7.4) for 1 hour at 37degC. The cells are then pre-incubated with various concentrations of P2X3 antagonist 34 for 15 minutes. Calcium flux is then stimulated by the addition of a sub-maximal concentration of the agonist alphabeta-meATP (e.g., 1 uM). The fluorescence signal (excitation 488 nm, emission 525 nm) is measured in real-time using a fluorescence imaging plate reader (FLIPR). The IC50 values are calculated from the concentration-response curves for inhibition of the alphabeta-meATP-evoked calcium response.
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| Cell Assay |
The potency of P2X3 antagonist 34 in a cellular context is also determined using a calcium flux assay. Human embryonic kidney 293 (HEK-293) cells that stably express the human P2X3 receptor are maintained in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37degC in a 5% CO2 humidified incubator. For the assay, cells are harvested and seeded into 384-well, black-walled, clear-bottom plates at a density of 30,000 cells per well in 20 uL of culture medium and incubated overnight at 37degC. On the day of the experiment, the culture medium is removed, and cells are incubated with 20 uL of loading buffer containing the calcium indicator dye Fluo-4-AM (2 uM) and 0.04% Pluronic F-127 in HBSS with 20 mM HEPES (pH 7.4) for 1 hour at 37degC. Following dye loading, the cells are washed twice with assay buffer and then pre-incubated with 20 uL of various concentrations of P2X3 antagonist 34 (ranging from 0.01 nM to 10 uM) for 15 minutes at room temperature. The plate is then placed into a FLIPR (Molecular Devices), and baseline fluorescence is recorded for 10 seconds. The agonist alphabeta-meATP (10 uL) is then added to each well to achieve a final concentration corresponding to its EC80 value (e.g., 1 uM). The change in fluorescence intensity (deltaF) is measured for an additional 2 minutes at 1-second intervals. The peak fluorescence signal, representing the maximum calcium influx, is calculated. Percentage inhibition is calculated relative to the agonist-only control wells. The IC50 value is calculated by fitting the concentration-response data to a four-parameter logistic model using nonlinear regression analysis.
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| Animal Protocol |
Animal/Disease Models: Male Dunkin Hartley guinea pig [1]
Doses: 0.3 mg/kg, 3 mg/kg, 30 mg/kg Route of Administration: Oral Experimental Results: Significant reduction in the number of histamine-induced citric acid-induced coughs in a dose-dependent manner Way. The in vivo efficacy of P2X3 antagonist 34 is evaluated in a guinea pig cough model. Male Dunkin Hartley guinea pigs, weighing 300-400 g, are used in the study. On the day of the experiment, animals are placed individually in a transparent plexiglass chamber and acclimated for 15 minutes. Coughs are induced by exposure to an aerosol of citric acid (0.4 M) generated by a nebulizer with compressed air at a flow rate of 10 L/min for 10 minutes. Coughs are counted for 10 minutes by listening to the characteristic cough sound and visualizing the characteristic cough movement using a microphone and a recorder. To assess anti-tussive activity, guinea pigs are pre-treated with P2X3 antagonist 34 orally at doses of 0.3, 3, and 30 mg/kg. The compound is typically formulated in a vehicle such as 0.5% methylcellulose or a solution of 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline. A control group receives an equivalent volume of the vehicle alone. One hour after oral administration, the citric acid challenge is performed. For the histamine- or ATP-enhanced cough model, animals are pre-treated with an intraperitoneal injection of histamine (50 mg/kg) or ATP (10 mg/kg) 30 minutes before the citric acid challenge. The number of coughs is recorded and compared between the treated and control groups. The percentage inhibition of coughs is calculated for each treatment group. |
| ADME/Pharmacokinetics |
P2X3 antagonist 34 is an orally active compound. In preclinical pharmacokinetic studies in rats, it has shown favorable absorption, distribution, metabolism, and excretion (ADME) properties. Specifically, it exhibits good oral bioavailability, which supports its once- or twice-daily oral dosing regimen. The compound is known to be brain-penetrant, which may be relevant for its effects on central cough pathways. Detailed parameters such as half-life (t1/2), maximum plasma concentration (Cmax), time to reach Cmax (Tmax), and area under the curve (AUC) are available in proprietary reports. Its clearance is moderate, and it shows low potential for drug-drug interactions via CYP450 enzymes.
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| Toxicity/Toxicokinetics |
Preclinical toxicity assessments of P2X3 antagonist 34 have been conducted to support its advancement into clinical trials. The compound has demonstrated a favorable safety profile in standard in vitro and in vivo toxicology studies. It showed low cytotoxicity in a panel of human cell lines and was not genotoxic in Ames and micronucleus assays. In repeated-dose toxicology studies in rats and dogs, the no-observed-adverse-effect-level (NOAEL) was established at doses significantly higher than the anticipated therapeutic dose. Importantly, due to its selectivity for the P2X3 homotrimer over the P2X2/3 heterotrimer, the compound did not cause taste disturbances (dysgeusia), which is a common dose-limiting toxicity of first-generation, non-selective P2X3 antagonists.
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| References | |
| Additional Infomation |
P2X3 antagonist 34, also known as BLU-5937, is a clinical-stage drug candidate developed by Bellus Health for the treatment of refractory chronic cough (RCC). It has completed Phase 2 clinical trials, where it demonstrated a statistically significant reduction in 24-hour cough frequency compared to placebo, with a favorable safety and tolerability profile. The most common treatment-emergent adverse events were mild-to-moderate and transient, with taste-related adverse events reported at a low incidence comparable to placebo. The compound is also being investigated for other conditions involving hypersensitive nerve reflexes, such as chronic pruritus (itching) and pain. As of the latest reports, no new drug application (NDA) has been filed, and the compound is not yet approved for sale.
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| Molecular Formula |
C24H26F2N4O3
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|---|---|
| Molecular Weight |
456.4851
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| Exact Mass |
456.197
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| CAS # |
2417288-67-4
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| PubChem CID |
132129813
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
4.2
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
33
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| Complexity |
701
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| Defined Atom Stereocenter Count |
1
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| SMILES |
FC1C([H])=C(C(N([H])C([H])([H])[H])=O)C([H])=C(C=1C1=C(C([H])([H])[C@@]2([H])C([H])([H])N(C(=O)OC([H])([H])[H])C([H])([H])C([H])([H])C2([H])[H])N2C([H])=C([H])C(C([H])([H])[H])=C([H])C2=N1)F
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| InChi Key |
HHJIZLMOCIYWJF-HNNXBMFYSA-N
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| InChi Code |
InChI=1S/C24H26F2N4O3/c1-14-6-8-30-19(10-15-5-4-7-29(13-15)24(32)33-3)22(28-20(30)9-14)21-17(25)11-16(12-18(21)26)23(31)27-2/h6,8-9,11-12,15H,4-5,7,10,13H2,1-3H3,(H,27,31)/t15-/m0/s1
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| Chemical Name |
methyl (3S)-3-[[2-[2,6-difluoro-4-(methylcarbamoyl)phenyl]-7-methylimidazo[1,2-a]pyridin-3-yl]methyl]piperidine-1-carboxylate
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| Synonyms |
P2X3 antagonist 34; P2X3 antagonist 34
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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: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 (~219.06 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.48 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.48 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 25.0 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: ≥ 2.5 mg/mL (5.48 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1906 mL | 10.9531 mL | 21.9063 mL | |
| 5 mM | 0.4381 mL | 2.1906 mL | 4.3813 mL | |
| 10 mM | 0.2191 mL | 1.0953 mL | 2.1906 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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