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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2019 May 11;176(13):2279–2291. doi: 10.1111/bph.14677

Action of MK‐7264 (gefapixant) at human P2X3 and P2X2/3 receptors and in vivo efficacy in models of sensitisation

David Richards 1, Joel R Gever 2, Anthony P Ford 2, Samuel J Fountain 1,✉
PMCID: PMC6555852  PMID: 30927255

Abstract

Background and Purpose

The P2X3 receptor is an ATP‐gated ion channel expressed by sensory afferent neurons and is used as a target to treat chronic sensitisation conditions. The first‐in‐class, selective P2X3 and P2X2/3 receptor antagonist, the diaminopyrimidine MK‐7264 (gefapixant), has progressed to Phase III trials for refractory or unexplained chronic cough. We used patch clamp to elucidate the pharmacology and kinetics of MK‐7264 and rat models of hypersensitivity and hyperalgesia to test its efficacy on these conditions.

Experimental Approach

Whole‐cell patch clamp of 1321N1 cells expressing human P2X3 and P2X2/3 receptors was used to determine mode of MK‐7264 action, potency, and kinetics. The analgesic efficacy was assessed using paw withdrawal threshold and limb weight distribution in rat models of inflammatory, osteoarthritic, and neuropathic sensitisation.

Key Results

MK‐7264 is a reversible allosteric antagonist at human P2X3 and P2X2/3 receptors. Experiments with the slowly desensitising P2X2/3 heteromer revealed concentration‐ and state‐dependency to wash‐on, with faster rates and greater inhibition when applied before agonist compared to during agonist application. The wash‐on rate (τ value) for MK‐7264 at maximal concentrations was much lower when applied before compared to during agonist application. In vivo, MK‐7264 displayed efficacy comparable to naproxen in inflammatory and osteoarthritic sensitisation models and gabapentin in neuropathic sensitisation models, increasing paw withdrawal threshold and decreasing weight‐bearing discomfort.

Conclusions and Implications

MK‐7264 is a reversible and selective P2X3 and P2X2/3 antagonist, exerting allosteric antagonism via preferential activity at closed channels. Its efficacy in rat models supports its clinical investigation for chronic sensitisation conditions.

Abbreviation

α,β‐meATP

α,β‐methylene ATP.

What is already known

  • P2X3 is an ATP‐gated ion channel expressed on sensory neurons. The antagonist MK‐7264 has shown efficacy in a Phase 2b clinical trial for unexplained or refractory chronic cough.

What this study adds

  • This study reveals the mechanism of action of MK‐7264 at human P2X3 and P2X2/3 receptors and in vivo efficacy in preclinical models of sensitisation.

What is the clinical significance

  • MK‐7264 has progressed to a Phase 3 trial for unexplained or refractory chronic cough and has the potential to be clinically useful for conditions involving sensitisation.

1. INTRODUCTION

P2X receptors are a family of trimeric, ATP‐gated ion channels (North, 2002). The human genome encodes seven pore‐forming subunits (P2X1–7) that are capable of assembling as homomeric and heteromeric receptors in a subunit‐dependent fashion (Surprenant & North, 2009). Each subunit has a double transmembrane topology and large extracellular domain which forms the orthosteric ATP binding site with an adjacent subunit (Kawate, Michel, Birdsong, & Gouaux, 2009; Mansoor et al., 2016; Wang et al., 2018). Several P2X receptor subtypes are implicated in pain, irritation, and hypersensitivity and have been proposed as drug targets, including the P2X3 receptor and P2X2/3 heteromeric receptor (Gever et al., 2010; Jarvis et al., 2002; Pijacka et al., 2016; Stokes, Layhadi, Bibic, Dhuna, & Fountain, 2017). P2X3 receptor tissue expression is very limited with protein and mRNA transcript detected in small diameter C‐fibre sensory neurons (Chen et al., 1995; Lewis et al., 1995; Xiang, Bo, & Burnstock, 1998), particularly those innervating the skin and viscera (Bradbury, Burnstock, & McMahon, 1998), petrosal neurons, and the carotid body afferents (Pijacka et al., 2016). P2X3 and P2X2/P2X3 double knockout mice display reduced nocifensive responses to ATP and formalin injection, as well as bladder hyporeflexia (Cockayne et al., 2000, 2005), and both dorsal root and nodose ganglia neurons lose sensitivity to the selective agonist α,β‐methylene ATP (α,β‐meATP; Zhong et al., 2001). P2X3 expression increases in rat models of inferior alveolar nerve injury (Eriksson, Bongenhielm, Kidd, Matthews, & Fried, 1998), complete Freund's adjuvant (CFA)‐induced monoarthritis (Shinoda, Ozaki, Asai, Nagamine, & Sugiura, 2005), and cast immobilisation (Sekino et al., 2014). In rat models, the number of P2X3‐positive small diameter L4 and L5 dorsal root ganglion neurons increases after chronic constriction of the sciatic nerve (Novakovic et al., 1999) but decreases following axotomy (Bradbury et al., 1998). In humans, P2X3 expression is increased in bladder urothelium during interstitial cystitis (Tempest et al., 2004), endometriosis endometrium, and endometriosis lesions (Ding et al., 2017).

Studies in knockout mice (Cockayne et al., 2000, 2005) as well as with RNAi (Barclay et al., 2002; Honore et al., 2002), small molecule antagonists (Jarvis et al., 2002; Kaan et al., 2010; McGaraughty et al., 2003), the spider venom peptide purotoxin‐1 (Grishin et al., 2010), and blocking monoclonal antibodies (Shcherbatko et al., 2016) have all demonstrated the efficacy of P2X3 and P2X2/3 antagonism to reduce nocifensive responses and neuropathic, inflammatory, arthritic, and visceral pain. Such studies validate P2X3 as a therapeutic target for chronic sensitisation conditions.

P2X3 and P2X2/3 receptors are antagonised by a range of molecules with poor selectivity and potency, including suramin, pyridoxal phosphate‐6‐azo(benzene‐2,4‐disulphonic acid) (PPADS), and reactive blue 2, in addition to 2′,3′‐O‐(2,4,6‐trinitrophenyl) ATP (TNP‐ATP) which has low metabolic stability (North, 2002). These molecules are therefore compromised for in vivo investigation (Jarvis et al., 2001; Ueno et al., 2003). A‐317491 (Jarvis et al., 2002), a more potent and selective P2X3 and P2X2/3 antagonist, has been developed; however, this molecule suffers from several undesirable characteristics including low oral bioavailability and very high protein binding (>99.9%). MK‐7264 (formerly AF‐219) is diaminopyrimidine antagonist of human P2X3 and P2X2/3 receptors. The compound has reached Phase 2 proof of concept trials for pain in osteoarthritis of the knee and interstitial cystitis/bladder pain (Ford & Undem, 2013) and Phase 2b trials for refractory chronic cough (Abdulqawi et al., 2015), whereby daytime cough frequency was significantly reduced compared to placebo. MK‐7264 is the first‐in‐class selective P2X3 antagonist, named gefapixant, and has progressed to a Phase 3 trial for unexplained or refractory chronic cough (NCT03449134). This drug is peripherally restricted, but the close structural analogue, AF‐353, is able to traverse the blood–brain barrier (Gever et al., 2010). Although the mechanisms underlying chronic cough are not fully understood, it is widely accepted that heightened sensitivity of afferent limb of the cough reflex plays an important role in the disease (Higenbottam, 2002). The efficacy of P2X3 antagonists as anti‐tussive agents in humans is likely due to the blockade of P2X3 receptors expressed on the sensory neurons within the afferent limb of the cough reflex (Ford, 2012). Mice and rats lack a cough reflex and are therefore not useful in studying the efficacy of P2X3 antagonists as anti‐tussive agents in vivo (Mazzone, 2005). However, these preclinical models are useful in studying the in vivo effects of P2X3 receptors in other processes that involve afferent sensitisation and hyperalgesia. Here, we electrophysiologically characterised MK‐7264 at human P2X3 and P2X2/3 receptors and determined its in vivo efficacy in rat models of sensitisation.

2. METHODS

2.1. Study ethics

All animal experiments were performed at Pharmaron (Beijing, China). Experiments were performed according to the guidelines approved by the Institutional Animal Care and Use Committee of Pharmaron following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care International.

2.2. Cells

1321N1 astrocytoma lines (RRID:CVCL_0110) stably transfected with human P2X2, P2X3, or co‐expressing P2X2 and P2X3 receptors were used in all experiments and provided by Afferent Pharmaceuticals Ltd. Cells were cultured in DMEM with 4.5‐g·L−1 glucose, 4‐mM L‐glutamine, 50‐IU·ml−1 penicillin, 50‐μg·ml−1 streptomycin, and 10% (v/v) FBS. Cells were cultured in the presence of a selection of antibiotics, G418 (P2X2), puromycin (P2X3), or G418 plus puromycin (P2X2/3). Cells were maintained at 37°C and 5% CO2.

2.3. Patch‐clamp electrophysiology

Cells were resuspended at 1 × 106 cells·ml−1 in solution containing (mM) NaCl, 140; KCl, 4; MgCl2, 1; CaCl2, 2; D‐glucose, 5; and HEPES, 10; pH 7.4. Whole‐cell planar patch clamp was conducted using a Nanion Port‐a‐Patch system fitted with an eight‐valve gravity‐fed perfusion panel. The time for total solution exchange was <150 ms. Internal solution contained (mM) CsF, 120; NaCl, 10; MgCl2, 2; and HEPES, 10; pH 7.2. Following gigaseal formation, all recordings were performed in external solution containing (mM) NaCl, 155; KCl, 3; MgCl2, 1; CaCl2, 1.5; and HEPES, 10; pH 7.4. Acquisition protocols were run using HEKA PatchMaster software (RRID:SCR_000034) and a HEKA EPC 10 amplifier. Data were sampled at 1 kHz and filtered at 10 kHz. Cells were held at −80 mV in all experiments. All recordings were performed at room temperature (21–23°C). The values for τ were calculated by fitting a single exponential decay curve to the desensitisation phase of current traces using OriginPro software (version 2016; Originlab).

2.4. Animals

Five‐ to seven‐week‐old female Sprague–Dawley rats (150–180 g; RRID:RGID_10395233) were obtained from the Beijing Vital River Company. Animals were housed at 22 (±3°C) with 12 hr of fluorescent lighting per day and food and water available ad libitum. Animals were kept for 1 week prior to the start of experiments, health checked, and randomly assigned to groups. Experiments began when animals were 6–8 weeks old (180–220 g). MK‐7264, naproxen, and gabapentin were dissolved in 0.5% (w/v) sodium carboxymethyl cellulose (Sigma) and administered by oral gavage at 5 ml·kg−1. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010) and with the recommendations made by the British Journal of Pharmacology.

2.5. CFA inflammatory hyperalgesia

The CFA model of inflammation leads to oedema as well as mechanical allodynia of the injected paw (Stein, Millan, & Herz, 1988). Fifty microlitres of CFA (Chondrex) was injected into the left hind paw subplantar 7 days prior to experimentation.

2.6. Mono‐iodoacetate‐induced osteoarthritis

Mono‐iodoacetic acid is an inhibitor of GAPDH causing the death of chondrocytes and similar histological and pathophysiological changes to those observed in osteoarthritis (Bove et al., 2003). Fifty microlitres of 0.9% normal saline containing 3‐mg mono‐iodoacetate (MIA; Sigma) was injected through the patellar ligament into the right hind knee. The injection was conducted under isoflurane anaesthesia 13 days prior to experimentation.

2.7. Spared nerve injury neuropathic pain model

The spared nerve injury model induces increased mechanical and thermal sensitivity of the treated paw, mimicking clinical neuropathic pain (Decosterd & Woolf, 2000). The sciatic nerve and its three terminal branches (sural, common peroneal, and tibial nerves) were exposed by skin incision on the lateral surface of the thigh and by sectioning the biceps femoris muscle of the left lateral leg. The peroneal and tibial nerves were ligated, and 4 ± 2 mm of the distal nerve stump was axotomised followed by muscle and skin closure. The sural nerve was not ligated and remained intact. Surgery was conducted under anaesthesia 10 days prior to experimentation. Anaesthesia was induced using 5% isoflurane and maintained at 3% isoflurane with a gas flow of 1 L·min−1 using a precision vaporiser. The level of anaesthesia was monitored by heart rate, respiratory rate, temperature, and pedal reflex. Animals received post‐operative analgesia via s.c. injection of carprofen (5 mg·kg−1) for up to 72 hr. For sham animals, the sciatic nerve was exposed via a skin incision on the lateral surface of the thigh followed by sectioning of the biceps femoris muscle, but the nerve was not axotomised. Post‐operative analgesia was provided for sham animals as for animals undergoing sciatic nerve axotomy.

2.8. Von Frey filaments testing for mechanical allodynia

The paw withdrawal threshold was measured by applying Von Frey filaments (Bioseb) of increasing force, perpendicular to the plantar surface of the hind paw of the treated limb. Following acclimatisation, rats were allowed to stand on mesh, and filaments were applied from beneath. The average of two to three repeated stimuli was taken as the withdrawal threshold. Experimenters were blinded to drug treatments.

2.9. Static weight‐bearing test for joint discomfort

Weight redistribution between hind paws was measured using a weight balance changing instrument (Bio‐Medical, USA). Weight distribution (g) is calculated as contralateral limb minus ipsilateral limb weight bearing; hence, the difference diminishes as hyperalgesia is reduced. Average weight bearing was measured over a 10‐s period. Experimenters were blinded to drug treatments.

2.10. Pharmacokinetic profiling

Rats received a single p.o. dose of MK‐7264, and a single blood sample was taken from each animal by the retro‐orbital route for a given time point. Retro‐orbital sampling was performed under general anaesthesia. General anaesthesia was induced by i.p. injection of ketamine (80 mg·kg−1) and xylazine (10 mg·kg−1). The level of anaesthesia was monitored by heart rate, respiratory rate, temperature, and pedal reflex. Following blood sampling, animals were not allowed to recovered from general anaesthesia and were killed by CO2 asphyxiation. MK‐7264 was quantified by LC‐MS (API 4000 QTrap, Applied Biosystems).

2.11. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology. All statistical analysis was performed using OriginPro (version 2016; OriginLab), unless otherwise stated. The threshold for statistical significance was P < .05 throughout. Data were first tested with a Shapiro–Wilk test for normality and Levene's test for equality of variance. For antagonist IC50 data sets, the normalised response was plotted against Log10 antagonist concentration and fitted with a modified Hill equation as below:

Y=Start+End−StartXnkn+Xn,

where k = Michealis constant and n = number of cooperative sites. The data sets were then compared with an F test. Pairwise comparison of EC50 values generated in curve shift experiments was conducted using a paired sample t test. Wash‐on rate in the presence and absence of agonist was compared with a one‐way ANOVA followed by Tukey's post hoc test. τ on and τ off rates were estimated by fitting a single exponential decay curve to the desensitisation phase of current traces:

Y=A×exp−xt+Y0,

where Y0 = offset, A = amplitude, and t = time constant. τ of desensitisation and residual current data was compared with a Kruskal–Wallis ANOVA followed by Dunn's post hoc test. Analyses of paw withdrawal (Von Frey filaments) and static weight bearing were conducted with a two‐way repeated measures ANOVA. The two factors were (a) time of measurement after drug treatment (within groups measure) and (b) drug treatment (between groups measure). Where significant interactions were detected, a subsequent Fisher least significant difference post hoc test was used to compare the effect of drug treatments at individual time points.

2.12. Materials

All basic salts, ATP, sodium carboxymethyl cellulose, gabapentin, naproxen, and mono‐iodoacetic acid were purchased from Sigma. CFA was purchased from Chondrex. α,β‐meATP was purchased from Tocris. For electrophysiological studies, all drugs were dissolved in DMSO, with the final DMSO concentration of 0.1% (v/v) in all experiments. For in vivo studies, all drugs were dissolved in 0.5% (w/v) sodium carboxymethyl cellulose and administered p.o. at a volume of 5 ml·kg−1.

2.13. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017).

3. RESULTS

3.1. MK‐7264 antagonises P2X3‐containing receptors selectively and reversibly

Human P2X3 expressed in 1321N1 cells displayed rapidly desensitising (millisecond) agonist evoked inward currents that recover slowly (Figure 1a). Complete recovery for responses to α,β‐meATP was achieved using an interval of 4 min between agonist application (Figure 1a). P2X2/3‐mediated currents were isolated in 1321N1 co‐expressing P2X2 and P2X3 by using α,β‐meATP which does not activate P2X2 homomers. In some cells, initial responses in 1321N1 co‐expressing P2X2 and P2X3 to α,β‐meATP evoked mixed P2X3 (transient, rapidly desensitising) and P2X2/3 (slowly desensitising) responses; however, the contribution of P2X3 homomer responses was lost after several α,β‐meATP applications at 30‐s intervals leaving P2X2/3 currents for further investigation (Figure 1b). MK‐7264 could potently inhibit P2X3 and P2X2/3 currents in a reversible and concentration‐dependent fashion (Figure 1a,b). No effect of 10‐μM MK‐7264 on human P2X2 currents was observed (N = 5; Figure 1c). The half‐maximal concentration of MK‐7264 antagonism at P2X3 and P2X2/3 receptors was 153 ± 59 nM (N = 5) and 220 ± 47 nM (N = 5; Figure 1d), respectively. These were found to be significantly different by an F test (F = 11.74, P < .05). Measurements were made for reduction in peak and steady‐state currents for P2X3 and P2X2/3, respectively. The Hill coefficient for MK‐7264 concentration inhibition was 1.0 ± 0.4 for P2X3 and 1.0 ± 0.2 for P2X2/3, indicating that MK‐7264 acts in a non‐cooperative manner. Antagonism of human P2X3 receptors using a submaximal MK‐7264 concentration (300 nM; approximate IC70) was not surmounted by increasing the concentration of α,β‐meATP agonist (Figure 1e), indicating that MK‐7264 acts independently of agonist concentration to block P2X3. In addition to suppression of response maxima, MK‐7264 also significantly reduced α,β‐meATP potency (EC50 1.0 ± 0.1 μM vehicle vs. 6.0 ± 1.5 μM with 300‐nM MK‐7264; N = 5, paired sample t test P < .05). These data suggest that MK‐7264 is a negative allosteric modulator of the human P2X3 receptor.

Figure 1.

Figure 1

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MK‐7264 activity at human P2X2, P2X3, and P2X2/3 receptors. (a–c) Representative concatenated traces of whole‐cell patch‐clamp recordings for human P2X3 (a), P2X2/3 (b), and P2X2 (c) receptor currents in 1312N1 stably transfected cells. Fully recovered P2X3 currents were evoked by 10‐μM α,β‐meATP applied for 1 s every 4 min and 10‐μM α,β‐meATP applied for 2 s at 30‐s intervals for P2X2/3 currents. P2X2 currents were evoked by 10‐μM ATP every 4 min. MK‐7264 was applied 4 min, 30 s, and 4 min prior to agonist application for P2X3 (1 μM), P2X2/3 (1 μM), and P2X2 (10 μM) receptors, respectively. Agonist application is indicated by the perfusion bar. Currents shaded in grey are in the presence of MK‐7264. (d) MK‐7264 concentration–dependent inhibition curve at human P2X3 (closed circles) and P2X2/3 receptors (open circles; N = 5). Data sets were compared with an F test and found to be significantly different from one another (F = 11.74, P = .018). (e) Effect of MK‐7264 on α,β‐meATP concentration–response curve at P2X3 receptors. Currents are in the presence of 300‐nM MK‐7264 (open circles) or vehicle control (closed circles; N = 6). MK‐7264 was applied for 4 min prior to agonist application. EC50 values were compared with a paired sample t test and found to be significantly different (P = .018). The holding potential was −80 mV for all experiments

3.2. Onset and offset of MK‐7264 action

Due to the prolonged recovery time required and rapid intrinsic desensitisation of P2X3 receptors, the slowly desensitising and rapidly recovering P2X2/3 receptor was used to quantify wash‐on and wash‐off rates of MK‐7264. Agonist applied for 2 s every 30 s allowed for complete recovery of the P2X2/3 response; we therefore applied α,β‐meATP every 30 s whilst bath perfusing a maximal concentration of MK‐7264 until steady‐state inhibition of the P2X2/3 current was achieved (Figure 2a,b). To quantify wash‐off, the bath was perfused with vehicle following a period of steady‐state inhibition and recovery monitored by agonist application every 30 s until steady‐state response was obtained (Figure 2b). For all wash‐on and wash‐off experiments, the mean current (1–2 s from onset) was then normalised against the vehicle control response. Exponential decay curves were fitted to individual data sets and τ on and τ off derived from curve time constants (Figure 2b). The concentration‐dependence of the effects of MK‐7264 on τ on and τ off is given in Table 1. The τ on rate decreased with increasing concentrations of MK‐7264 ranging between 12 and 360 s for the concentration range 100 nM to 3 μM. The τ off value increased with decreasing concentrations of MK‐7264.

Figure 2.

Figure 2

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MK‐7264 antagonism has different on rates when applied before or during agonist application at the human P2X2/3 receptor. (a) Representative trace showing effect of a 30 s pre‐incubation with 3‐μM MK‐7264 on currents evoked by 10‐μM α,β‐meATP at human P2X2/3 receptors. Agonist application is indicated by the perfusion bar. Currents shaded in grey are in the presence of MK‐7264. (b) The effect of varying the pre‐incubation time of MK‐7264 (3 μM) on its antagonism of P2X2/3 responses (10‐μM α,β‐meATP, 2 s; closed circles; N = 7), and the effect of wash‐off time on response recovery following maximal antagonism (open circles). Time constants (τ values) were calculated from single exponential curve fits. Data were normalised to the control response prior to MK‐7264 application. (c) Representative trace showing effect of rapid co‐application of agonist and MK‐7264. Responses were evoked with 10‐μM α,β‐meATP for 2 s followed by co‐application with 3‐μM MK‐7264 or vehicle. (d) Relationship between magnitude of P2X2/3 antagonism and wash‐on time during α,β‐meATP application (open circles) and before application (closed circles). Data were normalised to response prior to MK‐7264 application (closed circles) or the magnitude of response at the same time point in the presence of vehicle (open circles; N = 5). Data sets were compared with a one‐way ANOVA (F = 45.95, P < .05) followed by Tukey's post hoc test

Table 1.

Mean τ on and τ off times for MK‐7264 at the human P2X2/3 receptor

MK‐7264 concentration (nM) Mean τ on ± SEM (s) Mean τ off ± SEM (s)
3,000 11.82 ± 0.66 113.43 ± 14.42
1,000 38.04 ± 2.34 152.95 ± 14.12
300 176.04 ± 27.59 183.04 ± 24.14
100 359.78 ± 141.98 258.11 ± 45.96
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Note. Rates were estimated by fitting a single exponential decay to individual data sets for mean 1‐ to 2‐s current during onset and offset of antagonist effect. Data shown are the mean ± SEM of six cells. Cells were held at −80 mV.

3.3. MK‐7264 action before and during agonist application

The slow intrinsic desensitising properties of P2X2/3 currents provided the opportunity to investigate the effects of rapid MK‐7264 application to activated channels. In these experiments, currents were initiated by rapid 2‐s perfusion of α,β‐meATP, quickly followed by combined rapid perfusion of α,β‐meATP and MK‐7264 allowing the effect of MK‐7264 on the steady‐state current to be quantified (Figure 2c). The current magnitude was quantified every 5 s following MK‐7264 co‐application. We observed that the on rate of inhibition by MK‐7264 was significantly greater following application to the closed versus open channel (Figure 2d), with MK‐7264 inhibiting the response by 83 ± 2% compared to 41± 5% following a 30‐s perfusion prior to agonist application and during agonist application, respectively (one‐way ANOVA F = 45.94, P ≤ .05, followed by Tukey's post hoc test; Figure 2a vs. Figure 2d).

3.4. Changes in P2X3 receptor desensitisation

MK‐7264 antagonism of human P2X3 receptors was associated with a slowing of the second phase of desensitisation that was relieved upon agonist wash‐off (Figure 3a). This apparent slowing of desensitisation kinetic of the response was different from control response which decayed to baseline during agonist application (Figure 3a). The desensitisation phase of P2X3 after MK‐7264 exposure consists of a rapid phase (~200 ms) which is characteristic of P2X3 and appears unchanged apart from the reduction in magnitude, followed by a slower phase with shallow gradient dependent upon MK‐7264 concentration. This prolonged desensitisation effect of MK‐7264 washed off at a similar rate to the inhibition of peak response (Figure 3b). The prolonged desensitisation phase is shallow and not well fit by a single exponential curve to calculate a τ value, and therefore, a curve was fitted 250–750 ms after the onset of agonist response to estimate the rate of this phase (Figure 3a,c). MK‐7264 at 1 μM significantly decreased the rate of desensitisation from 206 ± 11 ms to 6.6 ± 3.5 s (N = 6; Kruskal–Wallis ANOVA followed by Dunn's post hoc P < .05 vs. vehicle control), respectively. The residual current at 1 s after onset of agonist response was measured and shown as a fraction of peak current in Figure 3d. The residual current increased with antagonist concentration and was significantly different from the vehicle control at 300 nM and 1 μM (Kruskal–Wallis ANOVA followed by Dunn's post hoc test, P < .05).

Figure 3.

Figure 3

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MK‐7264 antagonism of human P2X3 receptor is associated with an onset of slowed desensitisation. (a) Representative trace showing a diminished peak response and appearance of slow desensitisation are associated with MK‐7264 antagonism. (b) Representative superimposed trace showing time‐dependent recovery of P2X3 fast desensitisation following MK‐7264 (300 nM) wash‐off. Agonist applied every 4 min. (c) Relationship between MK‐7264 concentration and the rate of desensitisation. Time constants (τ values) were derived from single exponential curves fitted to the desensitisation phase 250–750 ms after the peak response. The τ values were compared with a Kruskal–Wallis ANOVA (P < .05) followed by Dunn's post hoc test; N = 6. (d) The change in shape of the current following MK‐7264 antagonism quantified as a fractional response (residual current, 1 s after onset of agonist response over peak current) and compared with a Kruskal–Wallis ANOVA followed by Dunn's post hoc test. *P < 0.05 versus control response; N = 7

3.5. Efficacy of MK‐7264 in rat model of inflammatory hyperalgesia

MK‐7264 and the non‐steroidal anti‐inflammatory naproxen were tested for their ability to reduce mechanical sensitivity and weight‐bearing discomfort in a CFA model of inflammatory hyperalgesia (Figure 4). For the Von Frey measurements, there were significant differences between the time of measurement (F = 17.6, P < .05), the treatments (F = 24.4, P < .05), and the interaction between these factors (F = 5.03, P < .05). For weight‐bearing measurements, there were significant differences between the time of measurement (F = 44.29, P < .05), the treatments (F = 28.8, P < .05), and the interaction between these factors (F = 9.97, P < .05). Fisher least significant difference post hoc test indicated that both naproxen and MK‐7264 at 20 and 30 mg·kg−1, respectively, significantly reduced mechanical sensitivity (increased paw withdrawal) threshold compared to vehicle control, 1 and 3 hr after dosing and to a comparable level (N = 10; P < .05 for both treatments vs. vehicle control; Figure 4a). The maximum reduction for both treatments was observed at 3 hr, where the mean withdrawal threshold was 6.0 ± 0.6 g, 8.2 ± 0.9 g, and 8.1 ± 0.5 g for vehicle, naproxen, and MK‐7264, respectively. Withdrawal threshold was 13.3 ± 0.2 g (N = 20) in naïve control animals. MK‐7264 was also efficacious in reducing weight‐bearing discomfort at 1 and 3 hr after dosing (Figure 4b). The maximum effect of MK‐7264 (30 mg·kg−1) was observed at 3 hr and similar to that of naproxen (20 mg·kg−1), where difference in weight bearing between contralateral and ipsilateral limbs was 55 ± 6 g, 35 ± 4 g, and 34 ± 4 g for vehicle control, MK‐7264, and naproxen (N = 10; P < .05 for all treatments vs. vehicle control), respectively. These values are compared to 5.8 ± 0.8 g (N = 12) for sham animals. Individual differences detected with a Fisher least significant difference post hoc test are shown in Figure 4.

Figure 4.

Figure 4

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MK‐7264 efficacy in a rat model of inflammatory hyperalgesia. CFA hind paw injection model of inflammatory hyperalgesia. (a) Effect of a single p.o. dose of vehicle, naproxen, or MK‐7264 on paw withdrawal threshold determined by Von Frey filament testing (N = 10). (b) Effect of single p.o. dose of vehicle, naproxen, or MK‐7264 on postural equilibrium (weight bearing) of inflamed versus non‐inflamed hindlimb (N = 10). Naproxen servesd as a positive control. Sodium carboxymethyl cellulose is the vehicle control. Drug treatments were compared with a two‐way repeated measures ANOVA followed by Fisher least significant difference post hoc test. *P < .05 versus vehicle control

3.6. Efficacy of MK‐7264 in rat model of osteoarthritis pain

MK‐7264 in vivo efficacy was investigated further to provide relief from mechanical hypersensitivity in a model of MIA‐induced osteoarthritis. In these experiments three doses of MK‐7264 (3, 10, and 30 mg·kg−1) were compared with naproxen (20 mg·kg−1) and vehicle control (Figure 5a). For the Von Frey measurements there were significant differences between the time of measurement (F = 157.27, P < .05), the treatments (F = 87.99, P < .05) and the interaction between these factors (F = 30.37, P < .05). For weight‐bearing measurements, there were significant differences between the time of measurement (F = 44.8, P < .05), the treatments (F = 14.35, P < .05) and the interaction between these factors (F = 4.96, P < 0.05). A Fisher least significant difference post hoc test indicated that naproxen and MK‐7264 at 10 and 30 mg·kg−1 significantly increased the paw withdrawal threshold 1 and 3 hr after administration (Figure 5a). The maximum effect of all drug treatments was observed at 3 hr following administration, where the paw withdrawal threshold for 6.4 ± 0.7 g, 10.9 ± 0.6 g, 9.6 ± 0.8 g and 10.7 ± 0.8 g for vehicle control, naproxen, and MK‐7264 at 10 and 30 mg·kg−1, respectively (N = 10; P < .05 for all treatments vs. vehicle control). Withdrawal threshold was 13.3 ± 0.2 g (N = 20) in control animals. MK‐7264 at 3 mg·kg−1 produced no significant effect over vehicle control (Figure 5a). Similar effects of MK‐7264 dosing were observed in reducing the difference in weight bearing between contralateral and ipsilateral limbs in the osteoarthritis model at 1 and 3 hr following administration (Figure 5b). Naproxen and MK‐7264 (30 mg·kg−1) significantly reduced the difference in weight bearing between contralateral and ipsilateral limbs to 24.2 ± 4.1 g and 24.5 ± 3.1 g, respectively, compared to 35.9 ± 4.1 g in vehicle control (N = 10; P < .05 for treatments vs. vehicle control). These values are compared to 6.8 ± 0.5 g (N = 10) for sham animals. Individual differences detected with a Fisher least significant difference post hoc test are shown in Figure 5.

Figure 5.

Figure 5

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MK‐7264 efficacy in a rat model of osteoarthritic pain. MIA hind knee injection model of osteoarthritic pain. (a) Effect of single p.o. dose of vehicle and naproxen and a range of MK‐7264 doses on paw withdrawal threshold determined by Von Frey filament testing (N = 10). (b) Effect of single p.o. dose of vehicle and naproxen or a range of MK‐7264 doses on postural equilibrium (weight bearing) of injected versus non‐injected hindlimb (N = 10). Drugs were administered 13 days following MIA injection. Naproxen served as a positive control. Sodium carboxymethyl cellulose is the vehicle control. Drug treatments were compared with a two‐way repeated measures ANOVA followed by Fisher least significant difference post hoc test.*P < 0.05 versus vehicle control

3.7. Efficacy of MK‐7264 in rat neuropathic pain model

Finally, we sought further verification for the in vivo efficacy of MK‐7264 in a rat model of neuropathic pain (Figure 6). Hypersensitivity was assayed by measurement of paw withdrawal threshold and weight‐bearing discomfort. In a Phase I experiment to determine the efficacy of MK‐7264 in reducing hypersensitivity, animals were administered with either vehicle control, gabapentin (100 mg·kg−1) as a positive control, or MK‐7264 at either 10 or 30 mg·kg−1. Paw withdrawal threshold and weight‐bearing discomfort were measured 1, 3, and 8 hr following administration. For Von Frey assessment of paw withdrawal threshold, there were significant differences between the time of measurement (F = 22.99, P < .05), the treatments (F = 11.75, P < .05), and the interaction between these factors (F = 2.18, P < .05; Figure 6a). Nerve injury produced hypersensitivity as assessed by paw withdrawal. Thresholds were 7.4 ± 0.5 g (N = 10) for animals that underwent nerve injury, compared to 13.3 ± 0.2 g (N = 20; P < .05) for sham surgery animals. Gabapentin (100 mg·kg−1) and MK‐7264 administered at 10 and 20 mg·kg−1 all significantly increased paw withdrawal threshold compared to vehicle control at 1 and 3 hr post‐administration (Figure 6a). Following Phase I experiments, a second dosing regimen was undertaken to test for drug tolerance. In these experiments (termed Phase II), animals were repeatedly dosed for 12‐hr intervals, and drug efficacy was re‐evaluated at the same time points as Phase I starting at Day 5.5. For the Von Frey measurements, there were significant differences between the time of measurement (F = 22.13, P < .05), the treatments (F = 49.61, P < .05), and the interaction between these factors (F = 6.57, P < .05). After the 5.5‐day repetitive dosing region, administration of gabapentin (100 mg·kg−1) and MK‐7264 (30 mg·kg−1) significantly increased paw withdrawal threshold for 8 hr post‐administration compared to administration of vehicle control (Figure 6b). The efficacy of MK‐7264 at 10 mg·kg−1 diminished significantly after 3 hr post‐administration (Figure 6b).

Figure 6.

Figure 6

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MK‐7264 efficacy in a rat spared nerve model of neuropathic pain. Sciatic nerve injury model induced by axotomy of peroneal and tibial nerves. Phase I experiments represent single p.o. dosing 10 days after surgery. Following completion of the Phase I dosing regimen, Phase II experiments were undertaken using p.o. dosing every 12 hr for 5.5 days to test for drug tolerance. (a, b) Effect of Phase I dosing (a) and Phase II dosing (b) regimens on paw withdrawal threshold determined by Von Frey filament testing (N = 10). (c, d) Effect of Phase I dosing (c) and Phase II dosing (d) regimens on postural equilibrium (weight bearing) of injected versus non‐injected hindlimb (N = 10). Gabapentin served as a positive control. Sodium carboxymethyl cellulose is the vehicle control. Drug treatments were compared with a two‐way repeated measures ANOVA followed by Fisher least significant difference post hoc test.*P < 0.05 versus vehicle control

For Phase I weight‐bearing discomfort measurements, there were significant differences between the time of measurement (F = 25.64, P < .05) and the treatments (F = 14.35, P < .05) but no significant interaction between these factors (Figure 6c). Nerve injury produced hypersensitivity as assessed by weight‐bearing discomfort. Difference in weight bearing between contralateral and ipsilateral limbs was 51.1 ± 7.8 g (N = 10) for animals that underwent nerve injury, compared to 5.8 ± 0.8 g (N = 12; P < .05) for sham surgery animals. In Phase I experiments, administration of gabapentin (100 mg·kg−1) or MK‐7264 at 10 and 30 mg·kg−1 significantly reduced the difference in weight bearing between contralateral and ipsilateral limbs up to 3 hr post‐administration compared to administration of vehicle control (Figure 6c). In Phase II experiments of weight‐bearing discomfort, there were significant differences between the time of measurement (F = 4.21, P < .05) and drug treatment (F = 12.68, P < .05). Administration of gabapentin (100 mg·kg−1) and MK‐7264 at 30 mg·kg−1 caused a significant reduction in the difference in weight bearing between contralateral and ipsilateral limbs up to 3 hr post‐administration compared to administration of vehicle control (Figure 6d). MK‐7264 at 10 mg·kg−1 reduced weight‐bearing difference up to 1 hr, but its effects had diminished by 3 hr (Figure 6d).

3.8. MK‐7264 blood plasma concentration following dosing in vivo in rats

Rats received a single p.o. dose of MK‐7264, and plasma concentrations were determined for different dosages at 0, 1, and 3 hr after dosing to determine the pharmacokinetic profile (Figure 7). The plasma concentration following a single oral dose of MK‐7264 was determined post‐administration by orbital blood sampling to correlate with exposure times that proved efficacious in inflammatory pain models. After 3 hr, plasma concentrations ranged from 250 to 1,087 nM (mean 605 ± 257 nM; N = 10) for 10 mg·kg−1, from 365 to 1,882 nM (mean 1,027 ± 597 nM; N = 10) for 20 mg·kg−1, and from 1,194 to 3,396 nM (mean 2,077 ± 775 nM; N = 10) for 60 mg·kg−1.

Figure 7.

Figure 7

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Plasma concentration of MK‐7264 following p.o. dosing in rats. Quantification of MK‐7264 plasma concentration following p.o. dosing with 10 (open circles), 20 (closed circles), and 60 (squares) mg·kg−1 at 0, 1, and 3 hr after dosing (N = 5). Sampling from orbital blood

4. DISCUSSION

These studies reveal nanomolar potency of the human P2X3 and P2X2/3 receptor antagonist MK‐7264, with marginal selectivity for P2X3 over recombinant P2X2/3 receptors. Both P2X3 and P2X2/3 are expressed by somatosensory neurons (Bradbury et al., 1998; Chen et al., 1995; Lewis et al., 1995), and selective antagonism of these receptors reduces nocifensive responses in several rat models of pain (Grishin et al., 2010; Jarvis et al., 2002; Kaan et al., 2010; McGaraughty et al., 2003). Our pharmacological analysis demonstrates that the action of MK‐7264 is insurmountable by increasing agonist concentration. Like its structural relative AF‐353 (Gever et al., 2010), MK‐7264 attenuated the response maxima in addition to decreasing agonist potency. This demonstrates that MK‐7264 does not act as a pure non‐competitive antagonist but rather exerts its antagonism through negative allosteric modulation of the P2X3 receptor. The allosteric nature of MK‐7264 is supported by the recent publication of the human P2X3 homomeric receptor with MK‐7264 bound to it (Wang et al., 2018). Radioligand binding studies with AF‐353 (Gever et al., 2010) suggest that this series of compounds does not alter agonist affinity but rather negatively modulates efficacy. The proposed MK‐7264 binding site resides within an inter‐subunit interface created by the lower body and dorsal fin of one P2X3 subunit and the lower body domain of an adjacent P2X3 subunit. This site is distinct from the orthosteric ATP binding site in P2X receptors (Mansoor et al., 2016) and supportive of MK‐7264 acting allosterically. Interestingly, the key residues proposed to form the MK‐7264 binding site by Wang et al. (2018) have a high degree of physicochemical conservation across all human P2X receptors. Our observation that human P2X2 homomeric receptors are insensitive to MK‐7264 suggests that the presence of these residues alone is not sufficient to convey sensitivity, rather the spatial positioning of these residues is important. This hypothesis is supported by in silico measurements of the spatial distribution of these key residues indicating that they are spaced further apart in the zebrafish P2X4 crystal structure (Kawate et al., 2009), which mostly shares the key MK‐7264 binding residues with human P2X3, but they are spatially further apart. The stoichiometry of native P2X2/3 remains undetermined, but at least one P2X3–P2X3 interface may exist and convey MK‐7264 sensitivity to the P2X2/3 heteromer, though with slightly less sensitivity compared to P2X3 homomeric receptors. It is also possible that a P2X2–P2X3 interface creates a suitable binding pocket.

The wash‐on rate of MK‐7264 to the P2X2/3 receptor was concentration dependent and significantly attenuated in the presence of agonist, and as with AF‐353, the high compound potency is due to comparatively slow on and off rates (Gever et al., 2010). This is in contrast to rapid rates of other P2X receptor antagonists such as PPADS (Li, 2000), TNP‐ATP, and suramin (Spelta, Jiang, Surprenant, & North, 2002). The disparity in MK‐7264 wash‐on rate in the absence (presumed predominantly closed channel population) and presence (presumed predominantly open/desensitising channel population) indicates that MK‐7264 exerts its effects more rapidly at agonist unbound (closed) channels. This is in contrast to PPADS which was found to bind three times more rapidly to the open over the closed P2X2/3 receptor (Spelta et al., 2002). The crystal structure of human P2X3 in the open and closed confirmation (Mansoor et al., 2016) indicates that the MK‐7264 binding pocket undergoes a dynamic change upon ATP binding, whereby the dorsal fin domain shifts upwards and the left fin moves downwards. This structural transition disrupts the MK‐7264 binding pocket and is the likely molecular basis for the disparity between MK‐7264 on rates in the presence and absence of agonist. A further pharmacological characteristic of MK‐7264 is a slowing of the rate of desensitisation. The reason for this slowing of desensitisation and propagation of a residual current is not known, but it could be that MK‐7264 binding interferes with the transition of open to desensitised channel. An alternative explanation may be that partial antagonist saturation of the receptor leads to a non‐desensitising conductance or sub‐conductance state. The observation that this effect is relieved upon agonist wash‐off indicates that it is an agonist‐dependent process. Although mechanistically interesting, this slowing of desensitisation observed under conditions of voltage clamp does not prevent the therapeutic activity of MK‐7264 in humans (Abdulqawi et al., 2015) or in rat models. MK‐7264 has a relatively slow time course of action in the in vivo rat models employed in this study. The slow on/off rates at the receptor level cannot completely account for this observation. The pharmacokinetics of MK‐7264 following oral dosing are likely to be a major contributor to the slow onset of in vivo efficacy. A sufficiently high concentration of MK‐7264 to achieve complete blockade of P2X3 (low micromolar) is achieved by 1 hr in doses in excess of 20 mg·kg−1 (but not at 10 mg·kg−1) and maintained until at least 3 hr. The time taken for plasma concentrations to peak to a point where P2X3 receptors are maximally blocked in in vitro experiments is likely to account for why the greatest in vivo efficacy is not observed until 3 hr after dosing.

ATP is released by mammalian cells under inflammatory conditions, both passively from cells undergoing lysis and actively via vesicular and channel‐mediated processes (Dosch, Gerber, Jebbawi, & Beldi, 2018). The expression of P2X3 on somatosensory neurons therefore makes it a potential target for a range of peripheral sensory disorders (Ford, 2012). In both the CFA model of inflammatory hyperalgesia and the monosodium iodoacetate injection model of osteoarthritis, 30‐mg·kg−1 MK‐7264 acted with the same efficacy as 20‐mg·kg−1 naproxen, indicating that MK‐7264 provides relief from inflammatory and osteoarthritic pain in these models. In the spared nerve injury model of neuropathic pain, acute MK‐7264 dosing could relieve pain with the same efficacy as 100‐mg·kg−1 gabapentin. After chronic dosing with MK‐7264, the response to acute noxious stimuli (Von Frey) was reduced at all time points tested, but this did not translate to a prolonged reduction in weight‐bearing discomfort. These data indicate that MK‐7264 works efficaciously to reduce pain hypersensitivity in vivo. MK‐7264 was as effective as current treatments and continued to show efficacy after repeated dosing in the neuropathic pain model, suggesting lack of tolerance. Finally, the plasma concentrations of MK‐7264 at 3 hr post‐oral administration suggest high bioavailability, with low micromolar plasma concentrations achieved at 20 and 60 mg·kg−1. These concentrations are sufficient to reduce P2X3 and P2X2/3 activity by 75–95% in vitro and significantly reduce rat nocifensive responses and discomfort in vivo. MK‐7264 is peripherally restricted (Pijacka et al., 2016), and therefore, the in vivo effects are not the result of centrally mediated mechanisms. ATP released from damaged or inflamed tissues can activate P2X receptors expressed on primary afferent neurons. The resulting depolarisation can initiate action potentials that are interpreted centrally as pain (North, 2004). This is also observed in humans where intracutaneous injections of ATP elicited discharge of afferent neurons innervating the peroneal nerve (Hilliges et al., 2002). P2X3 has very limited expression, predominantly located on afferent neurons, including those innervating the skin, viscera, and knee joint (Bradbury et al., 1998; Teixeira, Bobinski, Parada, Sluka, & Tambeli, 2017). Inhibition of P2X3‐dependent action potential of afferent neurons innervating peripheral tissue and nerves is therefore the likely mechanism mediating the in vivo effects of MK‐7264 in pain models.

In summary, MK‐7264 is a first‐in‐class, selective, reversible, and orally bioavailable P2X3 and P2X2/3 receptor antagonist with in vivo efficacy for the relief of inflammatory, osteoarthritic, and neuropathic pain. MK‐7264 likely exerts its antagonism via negative allosteric modulation.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

D.R. undertook the experimental work, analysis, and manuscript writing. A.P.F. undertook experimental analysis and study design. J.R.G. and S.J.F. undertook data analysis, study design, and wrote the manuscript.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, and Animal Experimentation, and as recommended by funding agencies, publishers, and other organisations engaged with supporting research.

ACKNOWLEDGEMENTS

This work was funded by Afferent Pharmaceuticals and Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.

Richards D, Gever JR, Ford AP, Fountain SJ. Action of MK‐7264 (gefapixant) at human P2X3 and P2X2/3 receptors and in vivo efficacy in models of sensitisation. Br J Pharmacol. 2019;176:2279–2291. 10.1111/bph.14677

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