Locus Coeruleus and Noradrenergic Pharmacology in Neurodegenerative Disease.

Adrenoceptors (ARs) throughout the brain are stimulated by noradrenaline originating mostly from neurons of the locus coeruleus, a brainstem nucleus that is ostensibly the earliest to show detectable pathology in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. The α1-AR, α2-AR, and ß-AR subtypes expressed in target brain regions and on a range of cell populations define the physiological responses to noradrenaline, which includes activation of cognitive function in addition to modulation of neurometabolism, cerebral blood flow, and neuroinflammation. As these heterocellular functions are critical for maintaining brain homeostasis and neuronal health, combating the loss of noradrenergic tone from locus coeruleus degeneration may therefore be an effective treatment for both cognitive symptoms and disease modification in neurodegenerative indications. Two pharmacologic approaches are receiving attention in recent clinical studies: preserving noradrenaline levels (e.g., via reuptake inhibition) and direct activation of target adrenoceptors. Here, we review the expression and role of adrenoceptors in the brain, the preclinical studies which demonstrate that adrenergic stimulation can support cognitive function and cerebral health by reversing the effects of noradrenaline depletion, and the human data provided by pharmacoepidemiologic analyses and clinical trials which together identify adrenoceptors as promising targets for the treatment of neurodegenerative disease.

Gefapixant in two randomised dose-escalation studies in chronic cough.

BACKGROUND AND OBJECTIVES: Gefapixant has previously demonstrated efficacy in the treatment of refractory chronic cough at a high daily dose. The current investigations explore efficacy and tolerability of gefapixant, a P2X3 receptor antagonist, for the treatment of chronic cough using a dose-escalation approach. MATERIALS AND METHODS: Two randomised, double-blind, placebo-controlled, crossover, dose-escalation studies recruited participants with refractory chronic cough. Patients were assigned to receive ascending doses of gefapixant (study 1: 50-200 mg, study 2: 7.5-50 mg) or placebo for 16 days, then crossed-over after washout. The primary end-point was awake cough frequency assessed using a 24-h ambulatory cough monitor at baseline and on day 4 of each dose. Patient-reported outcomes included a cough severity visual analogue scale and the cough severity diary. RESULTS: In clinical studies, gefapixant doses ≥30 mg produced maximal improvements in cough frequency compared with placebo (p<0.05); reported cough severity measures improved at similar doses. Taste disturbance exhibited a different relationship with dose, apparently maximal at doses ≥150 mg. CONCLUSIONS: P2X3 antagonism with gefapixant demonstrates anti-tussive efficacy and improved tolerability at lower doses than previously investigated. Studies of longer duration are warranted.

The effect of gefapixant, a P2X3 antagonist, on cough reflex sensitivity: a randomised placebo-controlled study.

We evaluated the effect of gefapixant on cough reflex sensitivity to evoked tussive challenge.In this phase 2, double-blind, two-period study, patients with chronic cough (CC) and healthy volunteers (HV) were randomised to single-dose gefapixant 100 mg or placebo in a crossover fashion. Sequential inhalational challenges with ATP, citric acid, capsaicin and distilled water were performed 1, 3 and 5 h after dosing. Mean concentrations evoking ≥2 coughs (C2) and ≥5 coughs (C5) post dose versus baseline were co-primary endpoints. Objective cough frequency (coughs·h-1) over 24 h and a cough severity visual analogue scale (VAS) were assessed in CC patients. Adverse events were monitored.24 CC patients and 12 HV were randomised (mean age 61 and 38 years, respectively). The cough challenge threshold increased for ATP by 4.7-fold (C2, p≤0.001) and 3.7-fold (C5, p=0.007) for gefapixant versus placebo in CC patients; in HV, C2 and C5 increased 2.4-fold (C2, p=0.113; C5, p=0.003). The distilled water C2 and C5 thresholds increased significantly (p<0.001) by a factor of 1.4 and 1.3, respectively, in CC patients. Gefapixant had no effect on capsaicin or citric acid challenge. Median cough frequency was reduced by 42% and the least squares mean cough severity VAS was 18.0 mm lower for gefapixant versus placebo in CC patients. Dysgeusia was the most frequent adverse event (75% of HV and 67% of CC patients).ATP-evoked cough was significantly inhibited by gefapixant 100 mg, demonstrating peripheral target engagement. Cough count and severity were reduced in CC patients. Distilled water may also evoke cough through a purinergic pathway.

Transient receptor potential cation channel, subfamily V, member 4 and airway sensory afferent activation: Role of adenosine triphosphate.

BACKGROUND: Sensory nerves innervating the airways play an important role in regulating various cardiopulmonary functions, maintaining homeostasis under healthy conditions and contributing to pathophysiology in disease states. Hypo-osmotic solutions elicit sensory reflexes, including cough, and are a potent stimulus for airway narrowing in asthmatic patients, but the mechanisms involved are not known. Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is widely expressed in the respiratory tract, but its role as a peripheral nociceptor has not been explored. OBJECTIVE: We hypothesized that TRPV4 is expressed on airway afferents and is a key osmosensor initiating reflex events in the lung. METHODS: We used guinea pig primary cells, tissue bioassay, in vivo electrophysiology, and a guinea pig conscious cough model to investigate a role for TRPV4 in mediating sensory nerve activation in vagal afferents and the possible downstream signaling mechanisms. Human vagus nerve was used to confirm key observations in animal tissues. RESULTS: Here we show TRPV4-induced activation of guinea pig airway-specific primary nodose ganglion cells. TRPV4 ligands and hypo-osmotic solutions caused depolarization of murine, guinea pig, and human vagus and firing of Aδ-fibers (not C-fibers), which was inhibited by TRPV4 and P2X3 receptor antagonists. Both antagonists blocked TRPV4-induced cough. CONCLUSION: This study identifies the TRPV4-ATP-P2X3 interaction as a key osmosensing pathway involved in airway sensory nerve reflexes. The absence of TRPV4-ATP-mediated effects on C-fibers indicates a distinct neurobiology for this ion channel and implicates TRPV4 as a novel therapeutic target for neuronal hyperresponsiveness in the airways and symptoms, such as cough.

P2X3 receptor antagonist (AF-219) in refractory chronic cough: a randomised, double-blind, placebo-controlled phase 2 study.

BACKGROUND: Preclinical studies suggest that P2X3 receptors are expressed by airway vagal afferent nerves and contribute to the hypersensitisation of sensory neurons. P2X3 receptors could mediate sensitisation of the cough reflex, leading to chronic cough. We aimed to investigate the efficacy of a first-in-class oral P2X3 antagonist, AF-219, to reduce cough frequency in patients with refractory chronic cough. METHODS: We did a double-blind, placebo-controlled, two-period, crossover study at one UK centre. With a computer-generated sequence, we randomly assigned patients with refractory chronic cough to AF-219, 600 mg twice a day, or to placebo (1:1), and then, after a 2 week washout, assigned patients to receive the other treatment. Patients, health-care providers, and investigators were masked to sequence assignment. We assessed daytime cough frequency (primary endpoint) at baseline and after 2 weeks of treatment using 24 h ambulatory cough recordings. The primary analysis used a mixed effects model with the intention-to-treat population. This study was registered at ClinicalTrials.gov, number NCT01432730. FINDINGS: Of 34 individuals assessed between Sept 22, 2011, and Nov 29, 2012, we randomly assigned 24 patients (mean age 54·5 years; SD 11·1). In the observed case analysis, cough frequency was reduced by 75% when patients were allocated to AF-219 compared when allocated to placebo (p=0·0003). Daytime cough frequency fell from a mean 37 coughs per h (SD 32) to 11 (8) coughs per h after AF-219 treatment versus 65 (163) coughs per h to 44 (51) coughs per h after placebo. Six patients withdrew before the end of the study because of taste disturbances, which were reported by all patients taking AF-219. INTERPRETATION: P2X3 receptors seem to have a key role in mediation of cough neuronal hypersensitivity. Antagonists of P2X3 receptors such as AF-219 are a promising new group of antitussives. FUNDING: Afferent Pharmaceuticals.

Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice.

Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca(2+) transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 µm or 100 µm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities.

Inhibition of urothelial P2X3 receptors prevents desensitization of purinergic detrusor contractions in the rat bladder.

OBJECTIVES: To evaluate whether P2X3 receptors (P2X3R) are expressed in the bladder urothelium and to determine their possible function in modulating purinergic detrusor contractions in the rat urinary bladder. MATERIALS AND METHODS: The expression of urothelial receptors was determined using conventional immunohistochemistry in bladders from normal Sprague-Dawley rats. The urothelial layer was removed by incubation with protamine, and disruption of the urothelium was confirmed using haematoxylin and eosin staining on bladder sections. Open cystometry was used to determine the effects of both urothelial removal as well as intravesical application of a specific P2X3R antagonist on bladder properties from intact and protamine-treated rats. Isometric contractile responses to potassium chloride (KCl) depolarization, electrical field stimulation (EFS) or chemical P2X activation were determined in normal and urothelium-denuded bladder strips, with and without application of the P2X3R antagonist. RESULTS: Immunohistochemical staining showed high expression of P2X3R in the medial and basal layers of the urothelium. Removal of the urothelial layer disturbed normal bladder performance in vivo and eliminated the effects of the P2X3R antagonist on increasing the contractile interval and reducing the amplitude of voiding contractions. Removal of the urothelium did not affect bladder strip contractile responses to KCl depolarization or EFS. Pharmacological inhibition of P2X3R prevented desensitization to P2X-mediated detrusor muscle contractions during EFS only in the strips with an intact urothelium. A concentration-dependent, specific inhibition of P2X3R also prevented desensitization of purinergic contractile responses in intact bladder strips. CONCLUSIONS: In the rat bladder, medial and basal urothelial cells express P2X3R, and specific inhibition of the receptor leads to a more hyporeflexive bladder condition. This pathway may involve P2X3R driving a paracrine amplification of ATP released from umbrella cells to increase afferent transmission in the sub-urothelial sensory plexus and desensitization of P2X1-mediated purinergic detrusor contractions.

Single- and Multiple-Dose Pharmacokinetics of Gefapixant (MK-7264), a P2X3 Receptor Antagonist, in Healthy Adults.

Gefapixant (MK-7264, RO4926219, AF-219) is a first-in-class P2X3 antagonists being developed to treat refractory or unexplained chronic cough. The initial single- and multiple-dose safety, tolerability, and pharmacokinetics of gefapixant at doses ranging from 7.5 to 1800 mg were assessed in four clinical trials. Following single-dose administration of 10-450 mg, the pharmacokinetic (PK) profile of gefapixant in plasma and urine demonstrated low inter-subject variability and a dose-proportional exposure. Following administration of multiple doses twice daily, the plasma exposures were dose-proportional at doses ranging from 7.5 to 50 mg and less than dose-proportional at doses ranging from 100 to 1800 mg. The time to mean peak drug concentration ranged from 2 to 3 h post-dose, and steady state was achieved by 7 days after dosing, with an accumulation ratio of approximately 2, comparing data from day 1 to steady state. The mean apparent terminal half-life ranged from 8.2 to 9.6 h. Gefapixant was primarily excreted unmodified in urine. Gefapixant was well tolerated following single-dose administration up to 1800 mg and multiple doses up to 1800 mg twice daily; there were no serious adverse events (AEs) reported. The most common AE reported was dysgeusia. The PK profile supports a twice-daily dosing regimen.

Cognitive Effects of Three ß-Adrenoceptor Acting Drugs in Healthy Volunteers and Patients with Parkinson's Disease.

Background: Noradrenergic signaling declines in Parkinson's disease (PD) following locus coeruleus neurodegeneration. Epidemiologic studies demonstrate that ß-acting drugs slow PD progression. Objective: The primary objective was to compare the safety and effects of 3 ß-adrenoceptor (ß-AR) acting drugs on central nervous system (CNS) function after a single dose in healthy volunteers (HVs) and evaluate the effects of multiple doses of ß-AR acting drugs in HVs and PD-patients. Methods: In Part A, HVs received single doses of 32 mg salbutamol, 160µg clenbuterol, 60 mg pindolol and placebo administered in a randomized, 4-way cross-over study. In Part B (randomized cross-over) and Part C (parallel, 2:1 randomized), placebo and/or clenbuterol (20µg on Day 1, 40µg on Day 2, 80µg on Days 3-7) were administered. CNS functions were assessed using the NeuroCart test battery, including pupillometry, adaptive tracking and recall tests. Results: Twenty-seven HVs and 12 PD-patients completed the study. Clenbuterol improved and pindolol reduced the adaptive tracking and immediate verbal recall performance. Clenbuterol and salbutamol increased and pindolol decreased pupil-to-iris ratios. Clenbuterol was selected for Parts B and C. In Part B, clenbuterol significantly increased performance in adaptive tracking with a tendency toward improved performance in immediate and delayed verbal recall. In Part C trends toward improved performance in immediate and delayed verbal recall were observed in PD-patients. Typical cardiovascular peripheral ß2-AR effects were observed with clenbuterol. Conclusions: This study demonstrates the pro-cognitive effects of clenbuterol in HVs with similar trends in PD-patients. The mechanism of action is likely activation of ß2-ARs in the CNS.

Aims and Purpose of the Research:This research aimed to explore how three different drugs affect brain function. These drugs are salbutamol, clenbuterol, and pindolol and work in the brain by stimulating specific brain cells that can improve aspects like memory and coordination. The main question was to see how safe these drugs were and how they impact the brain function after one dose in healthy people, and after multiple doses in both healthy people and those with Parkinson's disease.Background of the Research:Parkinson's disease is a condition where brain cells start to die, which affects different areas of the brain, including movement function, as well as memory and attention. This research matters because finding drugs that affect the brain function could improve the lives of people with Parkinson's disease.Methods and Research Design:The study was conducted in three parts. In the first part, healthy volunteers took one dose of each of the three drugs­ salbutamol, clenbuterol, and pindolol­ as well as a placebo (a harmless pill that has no effect). The researchers tested the participants' brain functions using various tasks including memory tests and eye response measurements. In the second and third part, healthy people and people with Parkinson's disease took the drug that performed best in healthy volunteers for seven days.Results and Importance:In the first part, a single dose of clenbuterol was safe and improved memory and attentions tasks in healthy people, and therefore was chosen for further testing in the second and third part. In these parts, multiple doses of clenbuterol were safe and helped improve memory and attention tasks in healthy people, with similar positive trends seen in people with Parkinson's disease. The study suggests that clenbuterol might help improve brain function by activating specific receptors in the brain.These results are important because they suggest that clenbuterol could be a potential treatment to help improve brain function in people with Parkinson's disease. However, more research is needed to fully understand its effects and to confirm these findings.

Fingerprinting heterocellular ß-adrenoceptor functional expression in the brain using agonist activity profiles.

Noradrenergic projections from the brainstem locus coeruleus drive arousal, attentiveness, mood, and memory, but specific adrenoceptor (AR) function across the varied brain cell types has not been extensively characterized, especially with agonists. This study reports a pharmacological analysis of brain AR function, offering insights for innovative therapeutic interventions that might serve to compensate for locus coeruleus decline, known to develop in the earliest phases of neurodegenerative diseases. First, ß-AR agonist activities were measured in recombinant cell systems and compared with those of isoprenaline to generate Δlog(Emax/EC50) values, system-independent metrics of agonist activity, that, in turn, provide receptor subtype fingerprints. These fingerprints were then used to assess receptor subtype expression across human brain cell systems and compared with Δlog(Emax/EC50) values arising from ß-arrestin activation or measurements of cAMP response desensitization to assess the possibility of ligand bias among ß-AR agonists. Agonist activity profiles were confirmed to be system-independent and, in particular, revealed ß2-AR functional expression across several human brain cell types. Broad ß2-AR function observed is consistent with noradrenergic tone arising from the locus coeruleus exerting heterocellular neuroexcitatory and homeostatic influence. Notably, Δlog(Emax/EC50) measurements suggest that tested ß-AR agonists do not show ligand bias as it pertains to homologous receptor desensitization in the system examined. Δlog(Emax/EC50) agonist fingerprinting is a powerful means of assessing receptor subtype expression regardless of receptor expression levels or assay readout, and the method may be applicable to future use for novel ligands and tissues expressing any receptor with available reference agonists.

P2X3 receptor antagonism attenuates the progression of heart failure.

Despite advances in the treatment of heart failure, prognosis is poor, mortality high and there remains no cure. Heart failure is associated with reduced cardiac pump function, autonomic dysregulation, systemic inflammation and sleep-disordered breathing; these morbidities are exacerbated by peripheral chemoreceptor dysfunction. We reveal that in heart failure the carotid body generates spontaneous, episodic burst discharges coincident with the onset of disordered breathing in male rats. Purinergic (P2X3) receptors were upregulated two-fold in peripheral chemosensory afferents in heart failure, and when antagonized abolished these episodic discharges, normalized both peripheral chemoreceptor sensitivity and the breathing pattern, reinstated autonomic balance, improved cardiac function, and reduced both inflammation and biomarkers of cardiac failure. Aberrant ATP transmission in the carotid body triggers episodic discharges that via P2X3 receptors play a crucial role in the progression of heart failure and as such offer a distinct therapeutic angle to reverse multiple components of its pathogenesis.

A role for ATP in bronchoconstriction-induced activation of guinea pig vagal intrapulmonary C-fibres.

Activation of vagal afferent sensory C-fibres in the lungs leads to reflex responses that produce many of the symptoms associated with airway allergy. There are two subtypes of respiratory C-fibres whose cell bodies reside within two distinct ganglia, the nodose and jugular, and whose properties allow for differing responses to stimuli. We here used extracellular recording of action potentials in an ex vivo isolated, perfused lung-nerve preparation to study the electrical activity of nodose C-fibres in response to bronchoconstriction. We found that treatment with both histamine and methacholine caused strong increases in tracheal perfusion pressure that were accompanied by action potential discharge in nodose, but not in jugular C-fibres. Both the increase in tracheal perfusion pressure and action potential discharge in response to histamine were significantly reduced by functionally antagonizing the smooth muscle contraction with isoproterenol, or by blocking myosin light chain kinase with ML-7. We further found that pretreatment with AF-353 or 2',3'-O-(2,4,6-Trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), structurally distinct P2X3 and P2X2/3 purinoceptor antagonists, blocked the bronchoconstriction-induced nodose C-fibre discharge. Likewise, treatment with the ATPase apyrase, in the presence of the adenosine A1 and A2 receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and SCH 58261, blocked the C-fibre response to histamine, without inhibiting the bronchoconstriction. These results suggest that ATP released within the tissues in response to bronchoconstriction plays a pivotal role in the mechanical activation of nodose C-fibres.

Modulation of bladder afferent signals in normal and spinal cord-injured rats by purinergic P2X3 and P2X2/3 receptors.

OBJECTIVE: To evaluate the role of bladder sensory purinergic P2X3 and P2X2/3 receptors on modulating the activity of lumbosacral neurones and urinary bladder contractions in vivo in normal or spinal cord-injured (SCI) rats with neurogenic bladder overactivity. MATERIALS AND METHODS: SCI was induced in female rats by complete transection at T8-T9 and experiments were performed 4 weeks later, when bladder overactivity developed. Non-transected rats were used as controls (normal rats). Neural activity was recorded in the dorsal horn of the spinal cord and field potentials were acquired in response to intravesical pressure steps via a suprapubic catheter. Field potentials were recorded under control conditions, after stimulation of bladder mucosal purinergic receptors with intravesical ATP (1 mm), and after intravenous injection of the P2X3/P2X2/3 antagonist AF-353 (10 mg/kg and 20 mg/kg). Cystometry was performed in urethane-anaesthetised rats intravesically infused with saline. AF-353 (10 mg/kg) was systemically applied after baseline recordings; the rats also received a second dose of AF-353 (20 mg/kg). Changes in the frequency of voiding (VC) and non-voiding (NVC) contractions were evaluated. RESULTS: SCI rats had significantly higher frequencies for field potentials and NVC than NL rats. Intravesical ATP increased field potential frequency in control but not SCI rats, while systemic AF-353 significantly reduced this parameter in both groups. AF-353 also reduced the inter-contractile interval in control but not in SCI rats; however, the frequency of NVC in SCI rats was significantly reduced. CONCLUSION: The P2X3/P2X2/3 receptors on bladder afferent nerves positively regulate sensory activity and NVCs in overactive bladders.

In pursuit of P2X3 antagonists: novel therapeutics for chronic pain and afferent sensitization.

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. In addition, P2X3 is expressed pre-synaptically at central terminals of C-fiber afferent neurons, where ATP further sensitizes transmission of painful signals. As a result of the selectivity of the expression of P2X3, there is a lower likelihood of adverse effects in the brain, gastrointestinal, or cardiovascular tissues, effects which remain limiting factors for many existing pain therapeutics. In the periphery, ATP (the factor that triggers P2X3 receptor activation) can be released from various cells as a result of tissue inflammation, injury or stress, as well as visceral organ distension, and stimulate these local nociceptors. The P2X3 receptor rationale has aroused a formidable level of investigation producing many reports that clarify the potential role of ATP as a pain mediator, in chronic sensitized states in particular, and has piqued the interest of pharmaceutical companies. P2X receptor-mediated afferent activation has been implicated in inflammatory, visceral, and neuropathic pain states, as well as in airways hyperreactivity, migraine, itch, and cancer pain. It is well appreciated that oftentimes new mechanisms translate poorly from models into clinical efficacy and effectiveness; however, the breadth of activity seen from P2X3 inhibition in models offers a realistic chance that this novel mechanism to inhibit afferent nerve sensitization may find its place in the sun and bring some merciful relief to the torment of persistent discomfort and pain. The development philosophy at Afferent is to conduct proof of concept patient studies and best identify target patient groups that may benefit from this new intervention.

Clinical Formulation Bridging of Gefapixant, a P2X3-Receptor Antagonist, for the Treatment of Chronic Cough.

Gefapixant is a P2X3-receptor antagonist being developed for treatment of refractory or unexplained chronic cough. Four phase 1 studies were conducted in healthy participants that bridged the early-phase gefapixant formulation (F01) to the phase 3 (F04A) and intended commercial (F04B) formulations. In addition, food and proton pump inhibitor (PPI) coadministration effects on gefapixant exposure were assessed. The gefapixant free base formulation (F01) was used in the initial early-phase clinical studies. Adding citric acid to the F01 formulation (to generate F02) enhanced drug solubilization, resulting in similar bioavailability and mitigating food and gastric pH effects. The subsequently developed gefapixant citrate salt formulation (F04) achieved exposures that were comparable to F02 in the fed state (90%CIs of geometric mean ratios for area under the plasma concentration-time curve from time 0 extrapolated to infinity and maximum observed concentration were within 0.80 and 1.25) and were not meaningfully affected by food or PPIs (90%CIs of geometric mean ratios for area under the plasma concentration-time curve from time 0 extrapolated to infinity and maximum observed concentration were within 0.80 and 1.25). Minor compositional changes were made to generate the F04A and F04B formulations. In vitro dissolution studies were used to bridge F04 to F04A, and clinical bioequivalence was then established between F04A and F04B. These data support use of the proposed commercial gefapixant formulation without significant food and PPI effects.

Endogenous purinergic control of bladder activity via presynaptic P2X3 and P2X2/3 receptors in the spinal cord.

P2X(3) and P2X(2/3) receptors are localized on sensory afferents both peripherally and centrally and have been implicated in various sensory functions. However, the physiological role of these receptors expressed presynaptically in the spinal cord in regulating sensory transmission remains to be elucidated. Here, a novel selective P2X(3) and P2X(2/3) antagonist, AF-792 [5-(5-ethynyl-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine, previously known as RO-5], in addition to less selective purinoceptor ligands, was applied intrathecally in vivo. Cystometry recordings were made to assess changes in the micturition reflex contractions after drug treatments. We found that AF-792 inhibited micturition reflex activity significantly (300 nmol; from baseline contraction intervals of 1.18 +/- 0.07 to 9.33 +/- 2.50 min). Furthermore, inhibition of P2X(3) and P2X(2/3) receptors in the spinal cord significantly attenuated spinal activation of extracellular-signal regulated kinases induced by acute peripheral stimulation of the bladder with 1% acetic acid by 46.4 +/- 12.0% on average. Hence, the data suggest that afferent signals originating from the bladder are regulated by spinal P2X(3) and P2X(2/3) receptors and establish directly an endogenous central presynaptic purinergic mechanism to regulate visceral sensory transmission. Identification of this spinal purinergic control in visceral activities may help the development of P2X(3) and P2X(2/3) antagonist to treat urological dysfunction, such as overactive bladder, and possibly other debilitating sensory disorders, including chronic pain states.

Systemic blockade of P2X3 and P2X2/3 receptors attenuates bone cancer pain behaviour in rats.

Pain remains an area of considerable unmet clinical need, and this is particularly true of pain associated with bone metastases, in part because existing analgesic drugs show only limited efficacy in many patients and in part because of the adverse side effects associated with these agents. An important issue is that the nature and roles of the algogens produced in bone that drive pain-signalling systems remain unknown. Here, we tested the hypothesis that adenosine triphosphate is one such key mediator through actions on P2X3 and P2X2/3 receptors, which are expressed selectively on primary afferent nocioceptors, including those innervating the bone. Using a well-established rat model of bone cancer pain, AF-353, a recently described potent and selective P2X3 and P2X2/3 receptor antagonist, was administered orally to rats and found to produce highly significant prevention and reversal of bone cancer pain behaviour. This attenuation occurred without apparent modification of the disease, since bone destruction induced by rat MRMT-1 carcinoma cells was not significantly altered by AF-353. Using in vivo electrophysiology, evidence for a central site of action was provided by dose-dependent reductions in electrical, mechanical and thermal stimuli-evoked dorsal horn neuronal hyperexcitability following direct AF-353 administration onto the spinal cord of bone cancer animals. A peripheral site of action was also suggested by studies on the extracellular release of adenosine triphosphate from MRMT-1 carcinoma cells. Moreover, elevated phosphorylated-extracellular signal-regulated kinase expression in dorsal root ganglion neurons, induced by co-cultured MRMT-1 carcinoma cells, was significantly reduced in the presence of AF-353. These data suggest that blockade of P2X3 and P2X2/3 receptors on both the peripheral and central terminals of nocioceptors contributes to analgesic efficacy in a model of bone cancer pain. Thus, systemic P2X3 and P2X2/3 receptor antagonists with central nervous system penetration may offer a promising therapeutic tool in treating bone cancer pain.

ATP and P2X purinoceptors in urinary tract disorders.

The pharmacological concept of specifically targeting purinoceptors (receptors for ATP and related nucleotides) has emerged over the last two decades in the quest for novel, differentiated therapeutics. Investigations from many laboratories have established a prominent role for ATP in the functional regulation of most tissue and organ systems, including the urinary tract, under normal and pathophysiological conditions. In the particular case of the urinary tract, ATP signaling via P2X1 receptors participates in the efferent control of detrusor smooth muscle excitability, and this function may be heightened in disease and aging. Perhaps of greater interest, ATP also appears to be involved in bladder sensation, operating via activation of P2X3-containing receptors on sensory afferent neurones, both on peripheral terminals within the urinary tract tissues (e.g., ureters, bladder) and on central synapses in the dorsal horn of the spinal cord. Such findings are based on results from classical pharmacological and localization studies in nonhuman and human tissues, gene knockout mice, and studies using recently identified pharmacological antagonists - some of which have progressed as candidate drug molecules. Based on recent advances in this field, it is apparent that the development of selective antagonists for these receptors will occur that could lead to therapies offering better relief of storage, voiding, and sensory symptoms for patients, while minimizing the systemic side effects that curb the clinical effectiveness of current urologic medicines.