IC50: ~30 nM (recombinant hP2X3 homotrimers), 100-250 nM (hP2X2/3 heterotrimeric receptors)[1].
Gefapixant citrate targets the P2X3 receptor, a ligand-gated ion channel activated by ATP that is involved in pain and sensory signaling. It acts as a potent antagonist, blocking the activation of P2X3 homotrimers and P2X2/3 heterotrimers. By inhibiting these receptors, Gefapixant reduces sensory nerve activation, which is thought to underlie its potential therapeutic effects in chronic cough and other conditions involving sensory hyperreactivity.

Gemfapic citrate does not inhibit any receptor that contains non-P2X3 subunits (recombinant homotrimeric hP2X1, hP2X2, hP2X4, rP2X5, and hP2X7 channels have IC50 values >10,000 nM) [1].
In vitro, Gefapixant citrate demonstrates potent antagonism of P2X3 receptors. It has an IC₅₀ of ~30 nM for recombinant human P2X3 homotrimers and 100-250 nM for human P2X2/3 heterotrimeric receptors. These values indicate its high potency and selectivity for the P2X3 receptor. The compound's activity has been characterized in cell-based functional assays, such as calcium flux or electrophysiological recordings.

In a rat model of knee osteoarthritis (14 days after intra-articular treatment of monoiodoacetate), gemfapic citrate (7 days b.i.d., orally) reduced weight-bearing laterality at both higher dosages. Complete reversal of severe hyperalgesia [2].
Specific in vivo activity data for Gefapixant citrate are not detailed in the available sources. As an orally active P2X3 receptor antagonist, it would be expected to have effects in animal models of chronic cough, pain, or other sensory disorders. The compound has been evaluated in clinical trials for chronic cough, indicating it has favorable in vivo activity and pharmacokinetic properties in humans. However, specific preclinical in vivo data are not provided.

The aryloxy-pyrimidinediamine, 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]
A cell-free assay for Gefapixant citrate is not directly applicable as it targets a ligand-gated ion channel. Its activity is typically assessed in cell-based functional assays. However, binding affinity to the P2X3 receptor could be measured using radioligand binding assays with membrane preparations expressing the receptor. Specific binding data are not provided in the available sources.

Cellular assays for Gefapixant citrate typically involve measuring its ability to inhibit P2X3 receptor-mediated responses. Cells expressing recombinant human P2X3 or P2X2/3 receptors are stimulated with ATP or a P2X3 agonist, and the resulting calcium influx or electrophysiological response is measured. Gefapixant is added at varying concentrations to determine its IC₅₀ for inhibition. For Gefapixant, IC₅₀ values of ~30 nM (P2X3) and 100-250 nM (P2X2/3) have been reported.

In vivo animal experiments for Gefapixant citrate are not described in the available sources. As a compound being developed for chronic cough, it would likely have been evaluated in animal models of cough, such as guinea pig or mouse models. In such studies, cough frequency would be measured after administration of the compound. However, specific protocols and data are not provided.

Absorption
The absolute bioavailability of gefaspirant has not been evaluated, but is estimated to be ≥78%. The recommended dose is 45 mg twice daily. Steady state is reached within 2 days, with steady-state mean plasma AUC and Cmax of 4,144 ng∙hr/mL and 531 ng/mL, respectively. The time to peak plasma concentration (Tmax) after oral administration is 1 to 4 hours. Concomitant administration of gefaspirant with high-fat, high-calorie foods has no effect on its AUC or Cmax.
Elimination Route
Gefaspirant is primarily excreted via the kidneys. In a healthy male subject, approximately 76.4% of the administered radiolabeled drug was recovered in the urine and 22.6% in the feces after a single oral dose. Of the drug recovered in the feces, 64% was unchanged drug, and of the drug recovered in the urine, 20% was unchanged drug.
Volume of Distribution
Based on population pharmacokinetic analysis, the estimated steady-state apparent volume of distribution for gefapitant 45 mg twice daily is 133.8 L (Vc 101 L, Vp 32.8 L).
Clearance
Population pharmacokinetic analysis integrating Phase I, II, and III 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, with renal clearance of approximately 8.7 L/h.
Metabolisms/Metabolites
Gefapitant is relatively poorly metabolized. Following oral administration, only 14% of the administered dose is excreted as metabolites in urine and feces. The predominant drug-related component in plasma is the unchanged parent drug (87%), with circulating metabolites each accounting for less than 10%. The major biotransformation pathways observed in the ADME study of gefapitant included hydroxylation, O-demethylation, dehydrogenation, oxidation, and direct glucuronidation. Secondary biotransformation pathways included glucuronidation of the O-demethylated metabolite and the formation of O-demethylated and hydrogenated metabolites. The three most abundant circulating metabolites were M1 (O-demethylated gefapitant glucuronide), M5 (direct glucuronidation of the parent drug), and M13 (hydroxylated metabolite), accounting for 1.0%, 6.3%, and 5.8% of the total drug-related components in plasma, respectively.

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Biological Half-Life
The terminal half-life of gefapitant is 6–10 hours.
Gefapixant citrate is an orally active compound. Its citrate salt form is used to enhance oral bioavailability. Specific pharmacokinetic parameters, such as half-life, Cmax, and AUC, are not provided in the available sources. The compound has been evaluated in clinical trials, indicating it has favorable PK properties for oral administration.

Protein binding
Gefapitant exhibits a relatively low protein binding rate (55%) in vitro, therefore drug interactions due to protein substitution are not expected.
Toxicity data for Gefapixant citrate are not detailed in the available sources. As a compound that has been evaluated in clinical trials, its safety profile is known, but specific toxicological information is not provided in the references cited. Common adverse effects may include taste disturbances, which are expected due to the role of P2X3 receptors in taste perception. The compound is intended for therapeutic use under medical supervision.

[1]. 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]. Validation of a visual analog scale for assessing cough severity in patients with chronic cough. Ther Adv Respir Dis. 2021 Jan-Dec;15:17534666211049743.

Drug Indication
Treatment of cough of unknown origin or chronic refractory disease.
Gefapixant citrate (CAS: 2310299-91-1) is an orally active P2X3 receptor antagonist being developed for the treatment of chronic cough. It is also known as MK-7264 citrate and AF-219 citrate. The compound is highly potent, with IC₅₀ values of ~30 nM for human P2X3 homotrimers and 100-250 nM for human P2X2/3 heterotrimers. Its mechanism of action involves blocking ATP-activated P2X3 receptors on sensory nerves, thereby reducing cough reflex sensitivity. It has been investigated in clinical trials for chronic cough and other sensory disorders.

545.52

545.14

C, 44.04; H, 4.99; N, 12.84; O, 32.26; S, 5.88

2310299-91-1

Gefapixant;1015787-98-0

145720531

Solid powder

7

16

10

37

739

0

AIJVJYUOMCRFOE-UHFFFAOYSA-N

InChI=1S/C14H19N5O4S.C6H8O7/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;7-3(8)1-6(13,5(11)12)2-4(9)10/h4-7H,1-3H3,(H2,17,20,21)(H4,15,16,18,19);13H,1-2H2,(H,7,8)(H,9,10)(H,11,12)

5-(2,4-diaminopyrimidin-5-yl)oxy-2-methoxy-4-propan-2-ylbenzenesulfonamide;2-hydroxypropane-1,2,3-tricarboxylic acid

Gefapixant citrate; MK-7264; MK 7264; Gefapixant citrate; DFK0FC2VVV; Gefapixant citrate [USAN]; 2310299-91-1; MK-7264; Gefapixant (citrate); UNII-DFK0FC2VVV; MK7264

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)