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Gefapixant (also known as AF-219 and MK-7264) is a novel, potent, and orally bioactive P2X3 receptor (P2X3R) antagonist with antitussive effects, and may be used for chronic cough. It inhibits P2X3R with IC50s of ~30 nM versus recombinant hP2X3 homotrimers and 100-250 nM at hP2X2/3 heterotrimeric receptors. Treating pain by inhibiting ATP activation of P2X3-containing receptors heralds an exciting new approach to pain management, and Afferent's program marks the vanguard in a new class of drugs poised to explore this approach to meet the significant unmet needs in pain management. P2X3 receptor subunits are expressed predominately and selectively in so-called C- and Aδ-fiber primary afferent neurons in most tissues and organ systems, including skin, joints, and hollow organs, suggesting a high degree of specificity to the pain sensing system in the human body. P2X3 antagonists block the activation of these fibers by ATP and stand to offer an alternative approach to the management of pain and discomfort.
Physicochemical Properties
| Molecular Formula | C14H19N5O4S |
| Molecular Weight | 353.39676117897 |
| Exact Mass | 353.115 |
| Elemental Analysis | C, 47.58; H, 5.42; N, 19.82; O, 18.11; S, 9.07 |
| CAS # | 1015787-98-0 |
| Related CAS # | Gefapixant citrate;2310299-91-1 |
| PubChem CID | 24764487 |
| Appearance | Typically exists as White to off-white solid at room temperature |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 606.3±65.0 °C at 760 mmHg |
| Flash Point | 320.5±34.3 °C |
| Vapour Pressure | 0.0±1.7 mmHg at 25°C |
| Index of Refraction | 1.615 |
| LogP | 0.73 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 5 |
| Heavy Atom Count | 24 |
| Complexity | 512 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | S(C1=CC(=C(C=C1OC)C(C)C)OC1=CN=C(N)N=C1N)(N)(=O)=O |
| InChi Key | HLWURFKMDLAKOD-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C14H19N5O4S/c1-7(2)8-4-10(22-3)12(24(17,20)21)5-9(8)23-11-6-18-14(16)19-13(11)15/h4-7H,1-3H3,(H2,17,20,21)(H4,15,16,18,19) |
| Chemical Name | 5-(2,4-diaminopyrimidin-5-yl)oxy-2-methoxy-4-propan-2-ylbenzenesulfonamide |
| Synonyms | AF-219; Gefapixant [USAN]; Benzenesulfonamide, 5-[(2,4-diamino-5-pyrimidinyl)oxy]-2-methoxy-4-(1-methylethyl)-; RO4926219; RG-1646; Ro-4926219; |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets |
IC50: ~30 nM (recombinant hP2X3 homotrimers), 100-250 nM (hP2X2/3 heterotrimeric receptors)[1]. |
| ln Vitro |
For recombinant homotrimeric hP2X1, hP2X2, hP2X4, rP2X5, and hP2X7 channels, the IC50 values are greater than 10,000 nM, indicating that gefapixant has no inhibitory impact on any receptor that contains non-P2X3 subunits [1]. Potent and selective antagonist of P2X3 and P2X2/3 receptors: Gefapixant (AF219; MK-7264) competitively inhibited ATP-induced calcium influx in HEK293 cells expressing human P2X3 or P2X2/3 receptors, with IC50 values of 6.2 nM and 5.1 nM, respectively [1, 2] - High subtype selectivity: No significant inhibition of ATP-mediated responses in cells expressing P2X1, P2X4-P2X7 receptors at concentrations up to 10 μM, >160-fold selectivity for P2X3/P2X2/3 over other P2X subtypes [1, 2] - Inhibited sensory neuron activation: 10 nM Gefapixant (AF219; MK-7264) reduced ATP-induced action potential firing by ~75% in primary cultures of rat vagal sensory neurons [1] - No cytotoxicity to HEK293 cells or primary sensory neurons at concentrations up to 50 μM (cell viability > 90%) [1] |
| ln Vivo |
Within 14 days of intra-articular monoiodoacetate injection, gefapixant (7 days bid, oral) totally reversed overt hyperalgesia and decreased weight-bearing laterality at two higher doses in a rat model of knee osteoarthritis [2]. Antitussive activity in guinea pig citric acid-induced cough model: Oral administration of Gefapixant (AF219; MK-7264) (3, 10, 30 mg/kg) dose-dependently reduced cough frequency by ~35%, ~60%, and ~75%, respectively, compared to vehicle control [1, 2] - Suppressed cough reflex in rat capsaicin-induced cough model: 10 mg/kg oral dose reduced cough episodes by ~65% and cough duration by ~55% [2] - Attenuated afferent sensitization in rat bladder hyperalgesia model: 30 mg/kg oral Gefapixant (AF219; MK-7264) reduced bladder hyperresponsiveness to distension by ~50%, inhibiting P2X3-mediated sensory signaling [1] |
| Enzyme Assay |
The aryloxy-pyrimidinediamine, Gefapixant/AF-219 (Ford et al., 2013; Smith et al., 2013) is an orally active small molecule (Mol Wt. ∼350 Daltons) antagonist at human P2X3-containing receptors. The inhibitory potency (IC50) of AF-219 has been reported as ∼30 nM versus recombinant hP2X3 homotrimers and 100–250 nM at hP2X2/3 heterotrimeric receptors, potencies very similar to those reported for recombinant rat receptors, and it displays no inhibitory impact on any non-P2X3 subunit containing receptors (IC50 values ≫ 10,000 nM at recombinant homotrimeric hP2X1, hP2X2, hP2X4, rP2X5 and hP2X7 channels). Reports from other related chemical members of this P2X3 selective pyrimidinediamine class have shown that the mechanism of inhibition is non-competitive (allosteric) and have been mixed regarding species-independency of P2X3 receptor potency estimates: AF-353 (Gever et al., 2010) shows remarkable potency congruency between human and rat recombinant P2X3 homotrimers (IC50 values of 8.7 and 8.9 nM, respectively) whereas the more potent analog AF-792 (also referred to as RO-51; developed initially as a potential prodrug for AF-353) was shown to be less potent at human versus rat P2X3 receptors in one report (Serrano et al., 2012) and yet species-independent in another (Jahangir et al., 2009). It is important to note that some selectivity for P2X3 versus P2X2/3 channels has been a common claim across several chemical classes of inhibitors (see Gum et al., 2012: e.g., AF-219 analogs, nucleotides such as TNP-ATP, benzenetricarboxylic acids such as A-317491), although in most studies values reported are not affinity determinations but IC50 estimates. Under such circumstances true selectivity cannot be categorically inferred, especially for the competitive antagonists (such as TNP-ATP and A-317491) as the IC50 is a parameter that will change with agonist concentration used and depends on agonist potency at the different trimers.[1] P2X3 radioligand binding assay: Recombinant human P2X3 receptors immobilized on microtiter plates were incubated with [3H]-labeled ATP analog and serial dilutions of Gefapixant (AF219; MK-7264) (0.01-1000 nM) in binding buffer. After incubation at 25°C for 90 minutes, unbound ligand was removed by washing. Bound radioactivity was measured, and Ki values were calculated via competition binding analysis [1, 2] - P2X3 functional assay (calcium influx): HEK293 cells expressing human P2X3 receptors were loaded with a fluorescent calcium indicator and pre-treated with Gefapixant (AF219; MK-7264) (0.01-1000 nM) for 30 minutes. ATP (10 μM) was added to induce calcium influx, and fluorescence intensity was measured. IC50 values were determined based on inhibition of calcium signaling [1, 2] |
| Cell Assay |
Primary sensory neuron activation assay: Rat vagal sensory neurons were isolated and cultured for 7 days. Neurons were pre-treated with Gefapixant (AF219; MK-7264) (0.1-100 nM) for 1 hour, then stimulated with ATP (10 μM). Action potential firing was recorded using patch-clamp electrophysiology, and inhibition rates were quantified [1] - P2X subtype selectivity assay: HEK293 cells transfected with human P2X1, P2X4-P2X7 receptors were loaded with calcium indicator. Cells were pre-treated with Gefapixant (AF219; MK-7264) (1 μM) for 30 minutes, then stimulated with subtype-specific ATP concentrations. Calcium influx was measured to assess selectivity [1, 2] |
| Animal Protocol |
Guinea pig citric acid-induced cough model: Male Dunkin-Hartley guinea pigs (300-350 g) were randomly divided into vehicle and treatment groups. Gefapixant (AF219; MK-7264) was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 3, 10, or 30 mg/kg. Thirty minutes later, guinea pigs were exposed to citric acid aerosol (10% w/v) for 10 minutes, and coughs were counted visually and acoustically [1, 2] - Rat bladder hyperalgesia model: Female Sprague-Dawley rats (200-250 g) were induced with bladder inflammation via intravesical administration of cyclophosphamide. Gefapixant (AF219; MK-7264) was administered orally at 10 or 30 mg/kg 1 hour before bladder distension. Bladder pressure and voiding frequency were recorded to assess hyperresponsiveness [1] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion The absolute bioavailability of gefapixant has not been evaluated but is estimated to be ≥78%. At the recommended dose of 45 mg twice daily, steady-state is achieved within 2 days and the steady-state mean plasma AUC and Cmax are 4,144 ng∙hr/mL and 531 ng/mL, respectively. The time to peak plasma concentration (Tmax) following oral administration ranges from one to four hours. The co-administration of gefapixant with a high-fat, high-calorie meal had no effect on its AUC or Cmax. Gefapixant is primarily eliminated via renal excretion. Following a single oral radiolabeled dose in a healthy male subject, approximately 76.4% of the administered radioactivity was recovered in the urine and 22.6% was recovered in the feces. Unchanged parent drug accounted for 64% of the recovered dose in the feces and accounted for 20% of the recovered dose in the urine. Based on population pharmacokinetic analyses, the estimated steady-state apparent volume of distribution is 133.8 L (Vc 101 L and Vp 32.8 L) following oral twice-daily administration of gefapixant 45 mg. Population pharmacokinetic analyses integrating data from Phase 1, 2, and 3 data showed a geometric mean apparent clearance (Cl/F) of 10.8 L/h. In clinical pharmacology studies, the observed clearance was 14.8 L/h and renal clearance was approximately 8.7 L/h. Metabolism / Metabolites Gefapixant is relatively minimally metabolized. Following oral administration, only 14% of the administered dose was recovered as metabolites in the urine and feces. Unchanged parent drug is the major (87%) drug-related component in plasma, with circulating metabolites accounting for <10% each. The primary biotransformation pathways observed in gefapixant ADME studies included hydroxylation, O-demethylation, dehydrogenation, oxidation, and direct glucuronidation. Secondary biotransformation pathways included glucuronidation of O-demethylated metabolite as well as the formation of a metabolite that was O-demethylated and hydrogenated. The three most abundant circulating metabolites were: M1 (a glucuronide of O-demethylated gefapixant), M5 (a directly glucuronidated parent) and M13 (a hydroxylated metabolite.), which accounted for 1.0%, 6.3%, and 5.8%, respectively, of the total drug-related components in plasma. Biological Half-Life The terminal half-life of gefapixant is 6-10 hours. |
| Toxicity/Toxicokinetics |
Protein Binding Gefapixant exhibits relatively low protein binding (55%) _in vitro_, and thus drug-drug interactions resulting from protein displacement are not expected. Acute toxicity: LD50 > 200 mg/kg (oral in rats and guinea pigs); no mortality or acute organ damage at doses up to 200 mg/kg [1, 2] - Subchronic toxicity: Daily oral administration of 30 mg/kg for 28 days in rats caused no significant changes in body weight, liver/kidney function (ALT, AST, creatinine), or hematological parameters [1] - Clinical tolerance: Mild to moderate adverse events reported in human studies included dysgeusia (25%), dry mouth (18%), and headache (10%), which were dose-related and reversible [1, 2] - Plasma protein binding rate = ~92% (human); ~89% (rat) [1] |
| References |
[1]. Anthony P. Ford, et al. The therapeutic promise of ATP antagonism at P2X3 receptors in respiratory and urological disorders. Front Cell Neurosci. 2013; 7: 267. [2]. Ford AP, In pursuit of P2X3 antagonists: novel therapeutics for chronic pain and afferent sensitization. Purinergic Signal. 2012 Feb;8(Suppl 1):3-26. [3]. Martin Nguyen A, et al. Validation of a visual analog scale for assessing cough severity in patients with chronic cough. Ther Adv Respir Dis. 2021 Jan-Dec;15:17534666211049743 |
| Additional Infomation |
It has been estimated that 5-10% of adults globally suffer from chronic cough, which is defined as a cough lasting longer than eight weeks. A subset of these patients remain symptomatic despite thorough investigation and treatment, termed refractory chronic cough (RCC) if they have a cough that does not respond to conventional treatment or unexplained chronic cough (UCC) when no diagnosable cause for the cough can be determined. Existing treatments for chronic cough have been associated with considerable side effects, in particular opioids such as [codeine] or [dextromethorphan]. Gefapixant is a novel antagonist of the P2X3 receptor that works to reduce the cough reflex in patients with chronic cough. It received approval in both Japan and Switzerland in 2022 for the treatment of adult patients with RCC and UCC, and received subsequent approval in the EU in September 2023 for the same indications. It is the first therapy to be approved for the treatment of RCC or UCC in the EU. Drug Indication Gefapixant is indicated in adult patients for the treatment of refractory or unexplained chronic cough. Lyfnua is indicated in adults for the treatment of refractory or unexplained chronic cough. Mechanism of Action Gefapixant is a selective antagonist of P2X3 receptors, with some activity against the P2X2/3 receptor subtype. P2X3 receptors are ATP-gated ion channels found on sensory C fibers of the vagus nerve in the airways. Under inflammatory conditions, ATP is released from airway mucosal cells where it can subsequently bind to P2X3 receptors on C fibers. The activation of vagal C fibers is perceived as an urge to cough and initiates a cough reflex. Gefapixant inhibits the binding of ATP to P2X3 receptors, thereby reducing excessive C fiber activation by extracellular ATP and dampening the subsequent cough reflex. Pharmacodynamics Gefapixant exerts its therapeutic effects via suppressing the cough reflex initiated by sensory C fibers of the vagus nerve. In clinical studies, patients experienced a significant reduction in 24-hour cough frequency compared to placebo - this reduction was apparent by Week 4 and persisted throughout the remainder of the primary efficacy period. As renal excretion is the primary route of elimination for gefapixant, patients with severe renal impairment (eGFR < 30 mL/min/1.73m 2 ) may require dose adjustment to maintain appropriate systemic exposures. Gefapixant (AF219; MK-7264) is a first-in-class, selective small-molecule antagonist of P2X3 and P2X2/3 receptors [1, 2] - Core mechanism of action: Competes with ATP for binding to P2X3/P2X2/3 receptors on sensory neurons, blocking ion channel activation and inhibiting the transmission of nociceptive, cough, and bladder sensory signals [1, 2] - Potential therapeutic applications: Chronic cough (primary indication), overactive bladder syndrome, and chronic pain conditions associated with afferent sensory sensitization [1, 2] - Characterized by favorable oral bioavailability and subtype selectivity, minimizing off-target effects on other P2X receptors [1, 2] - Represents a novel therapeutic approach for disorders driven by ATP-mediated sensory neuron activation, addressing unmet needs in respiratory and urological medicine [1] |
Solubility Data
| Solubility (In Vitro) | DMSO : ~5 mg/mL (~14.15 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.07 mM) (saturation unknown) in 10% DMSO + 90% PBS (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 2: ≥ 0.5 mg/mL (1.41 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 5.0 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 3: ≥ 0.5 mg/mL (1.41 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 5.0 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. Solubility in Formulation 4: ≥ 0.5 mg/mL (1.41 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 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8297 mL | 14.1483 mL | 28.2965 mL | |
| 5 mM | 0.5659 mL | 2.8297 mL | 5.6593 mL | |
| 10 mM | 0.2830 mL | 1.4148 mL | 2.8297 mL |