Characterization of three diaminopyrimidines as potent and selective antagonists of P2X3 and P2X2/3 receptors with in vivo efficacy in a pain model

BACKGROUND AND PURPOSE

P2X3 and P2X2/3 receptors are highly localized on the peripheral and central pathways of nociceptive signal transmission. The discovery of A-317491 allowed their validation as chronic inflammatory and neuropathic pain targets, but this molecule has a very limited oral bioavailability and CNS penetration. Recently, potent P2X3 and P2X2/3 blockers with a diaminopyrimidine core group and better bioavailability were synthesized and represent a new opportunity for the validation of P2X3-containing receptors as targets for pain. Here we present a characterization of three representative diaminopyrimidines.

EXPERIMENTAL APPROACH

The activity of compounds was evaluated in intracellular calcium flux and electrophysiological recordings from P2X receptors expressed in mammalian cells and in a in vivo model of inflammatory pain (complete Freund''s adjuvant (CFA) in rat paws).

KEY RESULTS

Compound A potently blocked P2X3 (pIC₅₀ = 7.39) and P2X2/3 (pIC₅₀ = 6.68) and showed no detectable activity at P2X1, P2X2, P2X4 and P2X7 receptors (pIC₅₀ < 4.7). Whole-cell voltage clamp electrophysiology confirmed these results. Compounds showed good selectivities when tested against a panel of different classes of target. In the CFA model, compound B showed significant anti-nociceptive effects (57% reversal at 3 mg·kg⁻¹).

CONCLUSIONS AND IMPLICATIONS

The diaminopyrimidines were potent and selective P2X3 and P2X2/3 receptor antagonists, showing efficacy in vivo and represent useful tools to validate these receptors as targets for inflammatory and neuropathic pain and provide promising progress in the identification of therapeutic tools for the treatment of pain-related disorders.     

A-317491, a selective P2X3/P2X2/3 receptor antagonist, reverses inflammatory mechanical hyperalgesia through action at peripheral receptors in rats

The effect of A-317491 (5-([(3-Phenoxybenzyl)[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl)-1,2,4-benzenetricarboxylic acid), a recently described selective P2X3 and P2X(2/3) receptor antagonist, on inflammatory mechanical hyperalgesia was examined. In the rat Freund's complete adjuvant model of inflammatory pain, s.c. administration of A-317491 dose-dependently reversed mechanical hyperalgesia. Maximum percent reversal (72%) was seen 3 h after administration at 10 mg/kg. Substantial plasma concentrations were measured for A-317491 after s.c. dosing 3, 10 and 30 mg/kg. However, the brain-to-plasma concentration ratio, determined 1 h after a 10 mg/kg s.c. dose, indicated limited penetration of A-317491 into the central nervous system. As revealed by neural activity recorded from single C-fiber nociceptive afferent in a Freund's complete adjuvant-inflamed rat skin-nerve preparation, topical application of A-317491 completely blocked afferent activation and mechanical sensitization induced by alpha,beta-methylene ATP, a P2X agonist. These results suggest that A-317491 is a peripherally acting P2X blocker. Its efficacy demonstrates the importance of peripheral P2X3/P2X(2/3) receptors in mediating ATP-associated mechanical hyperalgesia following inflammation, confirming previous suggestions of a significant role for P2X(2/3).     

Isolated porcine bronchi provide a reliable model for development of bronchodilator anti-muscarinic agents for human use

BACKGROUND AND PURPOSE: In human airways, muscarinic acetylcholine receptors (mAChRs) exert a predominant role in the control of airways resistance and anti-muscarinic agents are currently included in the pharmacological treatment of chronic obstructive pulmonary disease (COPD). However, the development of more effective mAChR antagonists is hampered by considerable species variability in the ultrastrucural and functional control of airway smooth muscle, making extrapolation of any particular animal model questionable. This study was designed to characterize the mAChRs in a bronchial preparation from pigs, animals considered to provide close models of human biology. EXPERIMENTAL APPROACH: Smooth muscle bronchial strips were examined by electron microscopy in order to compare their neuromuscular structure with that of human bronchi and used to study the affinity of a series of selective mAChR antagonists, estimated as pKis in competition binding assays with NMS and pA2, by Schild analysis, in contractile experiments. KEY RESULTS: Pharmacodynamic binding parameters and affinity profiles of a series of antagonists were consistent with the presence of a majority of M2 mAChRs along with a minor population of M3 mAChRs. Functionally, the highly significant correlation between postjunctional pA2 affinities and corresponding affinity constants at human recombinant M1-M5 subtypes indicated that smooth muscle contraction in porcine bronchi, as in human bronchi, was dependent on the M3 subtype. CONCLUSION AND IMPLICATIONS: Based on the characterization of mAChRs, isolated porcine bronchi provide an additional experimental model for development of mAChR antagonists for the treatment of human airway dysfunctions.     

Effects of A-317491, a novel and selective P2X3/P2X2/3 receptor antagonist, on neuropathic, inflammatory and chemogenic nociception following intrathecal and intraplantar administration

We have recently reported that systemic delivery of A-317491, the first non-nucleotide antagonist that has high affinity and selectivity for blocking P2X3 homomeric and P2X2/3 heteromeric channels, is antinociceptive in rat models of chronic inflammatory and neuropathic pain. In an effort to further evaluate the role of P2X3/P2X2/3 receptors in nociceptive transmission, A-317491 was administered either intrathecally or into the hindpaw of a rat in several models of acute and chronic nociception. Intraplantar (ED50=300 nmol) and intrathecal (ED50=30 nmol) injections of A-317491 produced dose-related antinociception in the CFA model of chronic thermal hyperalgesia. Administration of A-317491 by either route was much less effective to reduce thermal hyperalgesia in the carrageenan model of acute inflammatory hyperalgesia. Intrathecal, but not intraplantar, delivery of A-317491 attenuated mechanical allodynia in both the chronic constriction injury and L5-L6 nerve ligation models of neuropathy (ED50=10 nmol for both models). Intrathecal injections of A-317491 did not impede locomotor performance. Both routes of injection were effective in reducing the number of nocifensive events triggered by the injection of formalin into a hindpaw. Nocifensive behaviors were significantly reduced in both the first and second phases of the formalin assay (intrathecal ED50=10 nmol, intraplantar ED50>300 nmol). Nocifensive behaviors induced by the P2X receptor agonist alpha,beta-meATP were also significantly reduced by intraplantar injection of A-317491. These data indicate that both spinal and peripheral P2X3/P2X2/3 receptors have significant contributions to nociception in several animal models of nerve or tissue injury. Intrathecal administration of A-317491 appears to be more effective than intraplantar administration to reduce tactile allodynia following peripheral nerve injury.     

P2X3受体在糖尿病神经病理性疼痛模型中的作用

目的观察P2X3受体在糖尿病神经病理性疼痛大鼠模型中的作用和意义。方法将40只SD大鼠随机分为造模组（n=30）和对照组（n=10）。按照60 mg/kg一次性腹腔注射链脲佐菌素（STZ）建立糖尿病大鼠模型。于STZ注射前和注射后第7、14、28天分别测定大鼠血糖、体重以及机械性痛阈的变化。造模组又均分为造模1周组、造模2周组、造模4周组三个亚组。在造模前及造模后1、2、4周利用von Frey hairs法测定大鼠机械痛阈,并通过Real-time法测定每个时间点大鼠脊髓背根神经节（DRG）P2X3受体的表达水平。结果肉眼观察糖尿病大鼠毛色暗淡,尾静脉采血点愈合缓慢,每日饮水,饮食,尿量显著增加,表现符合糖尿病大鼠的临床特征。各模型组血糖浓度均〉16.7 mmol/L,各模型组与对照组比较,差异有统计学意义（P〈0.01）。糖尿病大鼠体重较对照组同期大鼠增长缓慢,差异有统计学意义（P〈0.01）。造模组机械痛阈在造模1周后明显下降（P〈0.01）,并持续至4周（P〈0.01）;在造模1周后脊髓DRG内P2X3受体表达水平出现显著上调（P〈0.01）。大鼠P2X3受体表达水平变化与机械痛阈的下降存在相关性。结论糖尿病早期大鼠脊髓DRG P2X3受体表达即出现上调,并与疼痛相关,说明P2X3受体可能在糖尿病大鼠周围神经疼痛的发生及痛敏的维持阶段起重要作用。     

Structure-activity relationship studies of spinorphin as a potent and selective human P2X(3) receptor antagonist

Spinorphin, an endogenous antinociceptive peptide (LVVYPWT), showed potent and non-competitive antagonism at the ATP-activated human P2X3 receptor (IC50 = 8.3 pM) in a two-electrode voltage clamp assay with recombinant human P2X3 receptors expressed in Xenopus oocytes. Single alanine substitutions from 1st to 4th amino acids and the cyclic form of LVVYPWT sustained antagonistic properties at the human P2X3 receptors, whereas the threonine to alanine substitution resulted in an enhancing effect of the agonistic activity.     

Characterization of a selective and potent antagonist of human P2X(7) receptors, AZ11645373

BACKGROUND AND PURPOSE: The ATP-gated P2X(7) receptor has been shown to play a role in several inflammatory processes, making it an attractive target for anti-inflammatory drug discovery. We have recently identified a novel set of cyclic imide compounds that inhibited P2X(7) receptor-mediated dye uptake in human macrophage THP-1 cells. In this study the actions and selectivity of one of these compounds, AZ11645373, were characterized. EXPERIMENTAL APPROACH: We measured membrane currents, calcium influx, and YOPRO-1 uptake from HEK cells expressing individual P2X receptors, and YOPRO1 uptake and interleukin-1beta release from THP-1 cells in response to ATP and the ATP analogue benzoylbenzoyl ATP (BzATP). KEY RESULTS: AZ11645373 up to 10 microM, had no agonist or antagonist actions on membrane currents due to P2X receptor activation at human P2X(1), rat P2X(2), human P2X(3), rat P2X(2/3), human P2X(4), or human P2X(5) receptors expressed in HEK cells. AZ11645373 inhibited human P2X(7) receptor responses in HEK cells in a non-surmountable manner with K (B) values ranging from 5 - 20 nM, with mean values not significantly different between assays. K (B) values were not altered by removing extracellular calcium and magnesium. ATP-evoked IL-1beta release from lipopolysaccharide-activated THP-1 cells was inhibited by AZ11645373, IC(50) = 90 nM. AZ11645373 was > 500-fold less effective at inhibiting rat P2X(7) receptor-mediated currents with less than 50% inhibition occurring at 10 microM. CONCLUSIONS AND IMPLICATIONS: AZ11645373 is a highly selective and potent antagonist at human but not rat P2X(7) receptors and will have much practical value in studies of human cells.     

Evidence for the expression of multiple uracil nucleotide-stimulated P2 receptors coupled to smooth muscle contraction in porcine isolated arteries

BACKGROUND AND PURPOSE: The uracil nucleotides UDP and UTP have been reported to activate P2Y2, P2Y4 and P2Y6 receptors to cause vasoconstriction. We have performed a comparative analysis of these receptors in endothelium-denuded smooth muscle from porcine isolated coronary and ear arteries, using pharmacological and molecular tools. EXPERIMENTAL APPROACH: Tissue segments were used to construct non-cumulative concentration response curves for UTP and UDP, in the absence and presence of the P2 receptor antagonists PPADS or suramin. RT-PCR and immunoblot analyses were employed to define gene expression and immunoreactivity for P2Y2, P2Y4 and P2Y6 receptors. KEY RESULTS: In the coronary artery, UTP-evoked contractile responses were reduced in the presence of suramin, but not PPADS, while the smaller responses to UDP were unaffected by either antagonist. In the ear artery, contractile responses to UDP were much smaller than those to UTP; responses to UTP were inhibited by both PPADS and suramin. RT-PCR suggested predominant expression of P2Y2 receptors in the coronary artery, while P2Y4 and P2Y6 receptor gene expression appeared equivalent in both tissues. Immunoblot analyses provided evidence for P2Y6 receptors in both tissues, with equivocal evidence of P2Y2 and P2Y4 receptor immunoreactivities. CONCLUSIONS AND IMPLICATIONS: We conclude that UTP-evoked contraction of porcine coronary artery smooth muscle appears to be predominantly P2Y2-mediated, while the ear artery appears to express a uracil nucleotide-sensitive P2 receptor(s) which fails to fit readily into the current classification.     

3H]A-804598 ([3H]2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine) is a novel, potent, and selective antagonist radioligand for P2X7 receptors

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1β, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC₅₀ = 10 nM), mouse (IC₅₀ = 9 nM) and human (IC₅₀ = 11 nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1β and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated ([³H]A-804598; 8.1 Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. [³H]A-804598 labeled a single class of high affinity binding sites (K_d = 2.4 nM and apparent B_max = 0.56 pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit [³H]A-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r = 0.95, P < 0.05). These data demonstrate that A-804598 is one of the most potent and selective antagonists for mammalian P2X7 receptors described to date and [³H]A-804598 is a high affinity antagonist radioligand that specifically labels rat P2X7 receptors.     

Myocardial pharmacokinetics of ebastine, a substrate for cytochrome P450 2J, in rat isolated heart

BACKGROUND AND PURPOSE

It is well established that cytochrome P450 2J (CYP2J) enzymes are expressed preferentially in the heart, and that ebastine is a substrate for CYP2J, but it is not known whether ebastine is metabolized in myocardium. Therefore, we investigated its pharmacokinetics in the rat isolated perfused heart.

EXPERIMENTAL APPROACH

Rat isolated hearts were perfused in the recirculating mode with ebastine for 130 min. The concentrations of ebastine and its metabolites, hydroxyebastine and carebastine, were measured using liquid chromatography with a tandem mass spectrometry. The data were analysed by a compartmental model. The time course of negative inotropic response was linked to ebastine concentration to determine the concentration–effect relationship.

KEY RESULTS

Ebastine was metabolized to an intermediate compound, hydroxyebastine, which was subsequently further metabolized to carebastine. No desalkylebastine was found. The kinetics of the sequential metabolism of ebastine was well described by the compartmental model. The EC₅₀ of the negative inotropic effect of ebastine in rat isolated heart was much higher than free plasma concentrations in humans after clinical doses.

CONCLUSIONS AND IMPLICATIONS

The kinetics of ebastine conversion to carebastine via hydroxyebastine resembled that observed in human liver microsomes. The results may be of interest for functional characterization of CYP2J activity in rat heart.     

Structure and immunological properties of thymosin β9 Met,a new analog of thymosin β4 isolated from porcine thymus

During the course for the studies of thymosin β₄ and prothymosin α from porcine thymus, a new analog of thymosin β₄ has been identified. This peptide consists of 41 amino acid residues. The amino terminus is blocked by an acetyl group as revealed by fast atom bombardment mass spectrometric analysis. Amino acid sequence studies disclosed that this peptide is identical to bovine thymosin β₉ except that leucine at position 6 in β₉ is substituted by methionine. Thus, this new peptide has been termed thymosin β₉ Met. The recoveries of β₉Met, β₄, and prothymosin α in porcine tissues have been determined (in μg/g tissue) as follows: thymus (43, 85, 133); spleen (68, 203, 37); liver (10, 31, 27); heart (1.5, 10, 0); kidney (5, 51, 37); brain (0.8, 31, 5). Biologically, thymosin β₉ Met was found to be more active than β₄ in enhancing γ-interferon production in cord blood lymphocytes. However, β₄ appeared to stimulate higher amounts of interleukin 2 and tumor necrotic factor. The significance for the coexistence of two homologous peptides with similar functions in the thymus and a number of other organs is not clear, and deserves further investigation.     

Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening

Background and Purpose

It is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane.

Experimental Approach

A molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP.

Key Results

A series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,β-methylene ATP (α,β-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,β-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression.

Conclusions and Implications

In conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.     

Minodronic acid, a third-generation bisphosphonate, antagonizes purinergic P2X(2/3) receptor function and exerts an analgesic effect in pain models

The P2X(2/3) receptor has an important role in the nociceptive transmission. Minodronic acid is a third third-generation bisphosphonate and a potent inhibitor of bone resorption. We found that minodronic acid inhibited alpha,beta-methylene ATP-induced cation uptake with the potency higher than that of suramin in the P2X(2/3) receptor receptor-expressing cells. Other bisphosphonates did not show such activity. Subcutaneously administered (10-50 mg/kg) minodronic acid significantly inhibited the alpha,beta-methylene ATP-, acetic acid- and formalin-induced nociceptive behaviors in mice. These unique effects of minodronic acid would be beneficial for the treatment of accelerated bone turnover diseases accompanied by bone pain, including bone metastases.     

Exploiting Streptomyces coelicolor A3(2) P450s as a model for application in drug discovery

One of the surprising discoveries about the genomics of the cytochrome P450 (CYP) superfamily is the large number of CYPs in the bacterial class of actinomycetes. It had previously been imagined that bacteria have small numbers of CYPs or none at all. Particularly intriguing is that the bacterial genus Streptomyces, which produce a large number of secondary metabolites with important medical application, has a large CYP complement reflecting the ecological niche that the organism finds itself in. In 2001 the first complete Streptomyces species genome (Streptomyces coelicolor A3[2]) was published, revealing the presence of 18 CYP genes. Subsequently, genomes for Streptomyces avermitilis, with 33 CYPs, and Streptomyces peucetius, with 15 CYPs, have been reported. Although a certain number of these CYPs have known functions in secondary metabolism, as identified biochemically or through gene locus organisation, in the vast majority of Streptomyces species, CYP functions are unknown. The first detailed analysis of the CYP complement from a Streptomyces species genome has begun in the laboratories of Waterman et al. The long-term goal of this effort is to identify orphan CYP function, to establish their high resolution structure and to establish a strategy for producing novel secondary metabolites that have new biomedical function. This chapter provides an overview of CYP systems in Streptomyces species and provides a plan of how new drugs might be generated from streptomycetes by modifying the structure of specific CYPs.     

Alcohol, drugs, and attention-deficit/ hyperactivity disorder: a model for the study of addictions in youth

There has been increasing interest in the developmental origins of substance use disorders (SUDs) in children and adolescents. Because of its early onset, high prevalence and known risk for SUD, attention deficit hyperactivity disorder (ADHD) is a model developmental disorder to evaluate in context to SUDs. A selected review of the literature was undertaken examining ADHD as an antecedent disorder to subsequent SUD. ADHD and its co-occurring comorbid psychopathology increase the risk for cigarette smoking and SUD and is associated with greater SUD severity and chronicity. The treatment of ADHD appears to decrease the risk for cigarette smoking and SUD. ADHD is an important antecedent disorder in children and adolescents worthy of further targeted preventive efforts to diminish the risk for cigarette smoking and SUD.