Bryan F. Cox

A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat

P2X3 and P2X2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X3 and P2X2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X3 and P2X2/3 receptor-mediated calcium flux (Ki = 22–92 nM) and was highly selective (IC50 >10 μM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X3 and P2X2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X3 containing channels was stereospecific because the R-enantiomer (A-317344) of A-317491 was significantly less active at P2X3 and P2X2/3 receptors. A-317491 dose-dependently (ED50 = 30 μmol/kg s.c.) reduced complete Freunds adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED50 = 10–15 μmol/kg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R-enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED50 >100 μmol/kg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X3 and P2X2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X3 and P2X2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.

The Canine Purkinje Fiber: An In Vitro Model System for Acquired Long Qt Syndrome and Drug-induced Arrhythmogenesis

Torsade de pointes is a rare but potentially fatal ventricular arrhythmia associated with drug-induced delayed repolarization and prolongation of the QT interval. To determine if the arrhythmogenic potential of noncardiac drugs can be assessed in vitro, we evaluated the effects of 12 drugs on the action potential duration (APD) of cardiac Purkinje fibers and compared results with clinical observations. APD changes in canine and porcine fibers were evaluated under physiologic conditions (37°C, [K+]0 = 4 m M) using standard microelectrode techniques. Six of seven drugs associated with QT prolongation or torsade de pointes in man (cisapride, erythromycin, grepafloxacin, moxifloxacin, sertindole, and sotalol) affected concentration-dependent prolongation of the APD in canine fibers during slow stimulation (2-s basic cycle length), attaining greater than 15% prolongation at high concentrations (≥10-fold clinically encountered plasma levels). Each of five drugs not linked clinically to QT prolongation and torsade de pointes (azithromycin, enalaprilat, fluoxetine, indomethacin, and pinacidil) failed to attain 15% prolongation, with fluoxetine, indomethacin, and pinacidil abbreviating the APD. Drugs eliciting the greatest prolongation also demonstrated prominent reverse rate-dependent effects. The antihistamine terfenadine (linked to dose-dependent QT prolongation and torsade de pointes clinically) only minimally prolonged the APD in canine and porcine fibers (and exerted no effect on midmyocardial fibers from left ventricular free wall) at supratherapeutic concentrations. On the basis of concentration-dependent APD prolongation and reverse rate-dependent effects, this Purkinje fiber model detects six of seven drugs linked clinically to acquired long QT syndrome and torsade de pointes, and clears each of five drugs not associated with repolarization abnormalities (overall 92% accuracy), validating the utility of this Purkinje fiber model in the preclinical evaluation of QT prolongation and proarrhythmic risk by noncardiac drugs.

Effect of bradykinin metabolism inhibitors on evoked hypotension in rats: rank efficacy of enzymes associated with bradykinin-mediated angioedema

Inhibition of bradykinin metabolizing enzymes (BMEs) can cause acute angioedema, as demonstrated in a recent clinical trial in patients administered the antihypertensive, omapatrilat. However, the relative contribution of specific BMEs to this effect is unclear and confounded by the lack of a predictive pre‐clinical model of angioedema.

Analgesic and anti-inflammatory effects of A-286501, a novel orally active adenosine kinase inhibitor

&NA; Adenosine (ADO) is an inhibitory neuromodulator that can increase nociceptive thresholds in response to noxious stimulation. Inhibition of the ADO‐metabolizing enzyme, adenosine kinase (AK) increases extracellular ADO concentrations at sites of tissue trauma and AK inhibitors may have therapeutic potential as analgesic and anti‐inflammatory agents. N7‐((1′R,2′S,3′R,4′S)‐2′,3′‐dihydroxy‐4′‐amino‐cyclopentyl)‐4‐amino‐5‐bromo‐pyrrolo[2,3‐a]pyrimidine (A‐286501) is a novel and potent (IC50=0.47 nM) carbocyclic nucleoside AK inhibitor that has no significant activity (IC50>100 &mgr;M) at other sites of ADO interaction (A1, A2A, A3 receptors, ADO transporter, and ADO deaminase) or other (IC50 values>10 &mgr;M) neurotransmitter and peptide receptors, ion channel proteins, neurotransmitter reuptake sites and enzymes, including cyclooxygenases‐1 and ‐2. A‐286501 showed equivalent potency to inhibit AK from several mammalian species and kinetic studies revealed that A‐286501 was a reversible and competitive inhibitor with respect to ADO and non‐competitive with respect to MgATP2−. A‐286501 was orally effective to reduce nociception in animal models of acute (thermal), inflammatory (formalin and carrageenan), and neuropathic (L5/L6 nerve ligation and streptozotocin‐induced diabetic) pain. A‐286501 was particularly potent (ED50=1 &mgr;mol/kg, p.o.) to reduce carrageenan‐induced inflammatory thermal hyperalgesia as compared to its analgesic actions in other pain models (acute and neuropathic) and its ability to alter hemodynamic function and motor performance. A‐286501 was also effective to reduce carrageenan‐induced paw edema and myeloperoxidase activity, a measure of neutrophil influx (ED50=10 &mgr;mol/kg, p.o.), in the injured paw. The anti‐nociceptive effects of A‐286501 in the L5/L6 nerve injury model of neuropathic pain (ED50=20 &mgr;mol/kg, p.o.) were not blocked by the opioid antagonist naloxone, but were blocked by the ADO receptor antagonist, theophylline. Following repeated administration, A‐286501 showed less potential to produce tolerance as compared to morphine. Thus, A‐286501 is a structurally novel AK inhibitor that effectively attenuates nociception by a non‐opioid, non‐non‐steroidal anti‐inflammatory drug ADO, receptor mediated mechanism.

Cardiovascular pharmacology of the adenosine A1/A2-receptor agonist AMP 579: coronary hemodynamic and cardioprotective effects in the canine myocardium.

The hemodynamic and cardioprotective properties of the novel adenosine A1/A2 receptor agonist AMP 579 (IS-[1a,2b,3b,4a(S*)]-4-[7-[[1-[(3-chloro-2-thienyl)methyl]propylamino]- 3H-imidazo[4,5-b]pyridin-3-yl]-N-ethyl-2,3-dihydroxy cyclopentanecarboxamide) were studied in two canine models designed to simulate (a) mild single-vessel coronary artery disease, and (b) myocardial ischemia/reperfusion injury. In the first model, a moderate stenosis was placed on the left circumflex coronary artery (LCCA), and the effects of AMP 579 on regional myocardial blood flow were assessed. AMP 579, 10 micrograms/kg/min, i.v., for 10 min, induced coronary dilation without causing endocardial steal. In the model of ischemia/reperfusion injury (60 min LCCA occlusion/5 h reperfusion), AMP 579, 10 micrograms/kg/min, i.v., administered for 15 min before ischemia significantly decreased myocardial infarct size. Control infarct size to area at risk (IS/AAR) equaled 34 +/- 3% (n = 9); IS/AAR for AMP 579-treated dogs equaled 16 +/- 4% (n = 9). Preconditioning (5 min LCCA occlusion + 10 min reperfusion) immediately before the 60-min LCCA occlusion also resulted in a marked decrease in IS/AAR: 9 +/- 3% (n = 6). The selective A1 agonist CPA reduced infarct size when administered at 3 micrograms/kg/min, i.v., for 15 min before LCCA occlusion: IS/AAR = 11 +/- 3% (n = 5). Pretreatment of animals with the adenosine-receptor antagonist 8-SPT, 10 mg/kg, i.v., attenuated the myocardial protective effects associated with preconditioning, CPA, and AMP 579, resulting in IS/AAR values of 28 +/- 7% (n = 7), 28 +/- 4% (n = 8), and 26 +/- 3% (n = 8), respectively. The ability of 8-SPT to block the cardioprotective effects suggests that these effects were mediated through an interaction with adenosine receptors. These experimental results indicate that AMP 579 is an effective coronary vasodilator, which also can protect the heart from ischemic injury. Thus AMP 579 has the potential to be useful in cardiovascular therapeutics.

Cardiovascular and Antilipolytic Effects of the Adenosine Agonist GR79236

Adenosine is known to produce cardiovascular effects such as bradycardia and hypotension via activation of myocardial (A1) and vascular (A2) receptors and antilipolytic effects through activation of adipocyte (A1) receptors. We established the cardiovascular and antilipolytic profile of the adenosine A1 agonist GR79236 (N6-[(1S,trans)-2-hydroxycyclopentyl]-adenosine) and compared it with CPA (N6-cyclopentyl-adenosine). GR79236 was approximately 3-fold less potent than CPA in inhibiting in vitro lipolysis. In conscious rats, both agents were shown to have antilipolytic and glucose-lowering properties. In rats instrumented with telemetry transmitters, orally administered CPA was one log unit more potent than GR79236 as a hypotensive and bradycardiac agent. In summary, GR79236 is an A1-selective adenosine agonist which reduces heart rate and mean arterial pressure and produces decreased plasma lipids and glucose levels.

Magnetic resonance imaging detection and time course of cerebral microhemorrhages during passive immunotherapy in living amyloid precursor protein transgenic mice.

In recent years immunotherapy-based approaches for treating Alzheimers disease have become the subject of intensive research. However, an important mechanistic-related safety concern is exacerbation of the risk of microhemorrhage that may be associated with fast removal of amyloid-β (Aβ) deposits found in blood vessels or brain parenchyma. Rapid in vivo detection of microhemorrhages in living amyloid precursor protein transgenic mice has not been described, and histological analysis can take several months before this risk is assessed. Aged transgenic mice were divided into two groups that would undergo longitudinal passive immunotherapy for 12 or 18 weeks. 6G1, a nonselective anti-Aβ monoclonal antibody, and 8F5, a more selective antioligomeric Aβ monoclonal antibody, were examined in both longitudinal studies. High-resolution T2*-weighted magnetic resonance microscopy (100 × 100 × 400 μm) was used for microhemorrhage detection in vivo. Cerebral microhemorrhages by magnetic resonance imaging were compared with histological hemosiderin staining in each animal; results showed that T2*-weighted magnetic resonance microscopy can reliably detect microhemorrhages of ≥60 μm in diameter at baseline and after 12 to 18 weeks of treatment in the same animals in vivo. This correlated significantly with histological readings. This new imaging safety biomarker can be readily applied to preclinical antibody screening in a longitudinal manner. 6G1 and 8F5, however, both increased microhemorrhage incidence in aged amyloid precursor protein transgenic mice compared with their baseline and vehicle treatment. A highly selective antibody for soluble Aβ is needed to address the question of whether antibodies that do not bind to deposited Aβ have microhemorrhage liability.

Bimoclomol elevates heat shock protein 70 and cytoprotects rat neonatal cardiomyocytes.

Bimoclomol is a new compound that improves cell survival under experimental stress conditions partly by increasing intracellular heat shock proteins (HSPs). HSPs, especially HSP70, play a cytoprotective role in the rat heart. Rat neonatal cardiomyocytes were used to determine the ability of bimoclomol to induce HSP70 and affect cell survival across a broad concentration range (0.01-100 microM). Bimoclomol significantly elevated HSP70 levels at concentrations ranging from 0.01 to 10 microM, depending on the time of exposure. Pretreatment with bimoclomol for 24 h significantly increased survival of cells. There was a significant correlation between the increased levels of HSP70 and the increase in cell survival as a result of the treatment with bimoclomol. In conclusion, bimoclomol improved cell survival in rat neonatal cardiomyocytes, in part, by increasing the levels of HSP70. This cytoprotection began at the relatively low concentration of 0.1 microM, which is a concentration that can be achieved clinically.

Importance of Species Selection in Arrythmogenic Models of Q-T Interval Prolongation

In a recent article by Ohtani and colleagues (4), prolongation of the Q-T interval in an anesthetized-rat electrocardiogram (ECG) model was used to determine the arrhythmogenic potential of macrolide antibiotics. Prolongation of the Q-T interval on an ECG has been linked to the rare (but potentially fatal) ventricular arrhythmia known as Torsade de Pointes. In vitro studies suggest that most Q-T-prolonging drugs block the delayed rectifier potassium current (iKr) carried by the pore-forming subunit encoded by hERG (human ether-a-go-go-related gene) in humans (6). While hERG-like mRNA and functional iKr current have been found in many species including dogs, guinea pigs, and rabbits (3), little (if any) functional iKr or hERG-like current is found in the rat ventricle (8). Therefore, we were surprised at the sensitivity of this rat ECG model to the selected macrolide antibiotics, given that these drugs do not elicit iKr blocking until supratherapeutic concentrations are achieved (7). To clarify these issues, we chose to compare the effects of erythromycin and two established iKr-blocking drugs on repolarization of rat ventricular muscle, because Q-T prolongation in vivo is reflective of action potential prolongation. We used standard microelectrode techniques (2) to evaluate the effects of erythromycin (at a concentration achieved by Ohtani et al. in plasma [3.6 μM]) as well as two standard iKr blockers, dofetilide (10 nM) and E-4031 (1.0 μM), at concentrations 2.5-fold greater than those shown to block 50% of native iKr current (1, 5). These concentrations have also been shown to significantly prolong the action potential duration (APD) in other species prominently expressing iKr (5). In brief, male CD rats (300 to 350 g; Charles River Labs) were anesthetized, hearts were removed, and ventricular papillary muscles were excised and placed in a warmed (37°C) superfusion chamber perfused at the rate of 8 to 10 ml/min with Tyrodes solution containing (millimolar concentrations) NaCl, 131; NaHCO3, 18; NaH2PO4, 1.8; MgCl2, 0.5; dextrose, 5.5; KCl, 4; and CaCl2, 2 (aerated with 95% O2-5% CO2 [pH = 7.4] at room temperature). Muscle preparations were field stimulated at a basic cycle length of 180 ms (corresponding to a typical rat heart rate of approximately 330 beats per min) with a biphasic waveform at a twofold threshold using platinum electrodes in the bath floor. Preparations were paced and equilibrated for a minimum of 1 h before recording. Preparations were considered suitable for study if the maximum diastolic potential was more negative than −70 mV and the APD was greater than 20 ms. Records were obtained during a 10-min drug-free control period and ensuing 25-min period of drug exposure. Impalements were maintained throughout each experiment. APD was measured from the upstroke until repolarization reached 10 mV positive to the maximum diastolic potential. All drugs were made up fresh daily as stocks in 100% dimethyl sulfoxide and then diluted in Tyrodes solution to achieve a final dimethyl sulfoxide concentration of 0.1%. Values are presented as means ± standard errors of the means. Erythromycin lactobionate and dofetilide were synthesized in-house; E-4031 was purchased from Wako Chemicals. The typical effects of erythromycin, E-4031, and dofetilide on rat ventricular muscle action potentials are illustrated in Fig. ​Fig.1.1. Rat papillary muscle repolarization is very sensitive to erythromycin (Fig. 1B) but not the iKr-blocking drug E-4031 (Fig. 1A). Figure 1C summarizes results obtained from 15 preparations (10 hearts); drugs are arranged on the abscissa in the order of increasing potency for native-iKr blockade. Whereas erythromycin elicited 76.6% ± 6.4% APD prolongation at concentrations substantially lower than those required for iKr blockade, the iKr blockers dofetilide and E-4031 elicited much smaller prolongation (2.4% ± 2.9% and 1.8% ± 2.1%, respectively) at concentrations 2.5-fold the 50% inhibitory concentration for iKr blockade. The greater APD prolongation of rat papillary muscle with erythromycin compared to potent iKr-blocking drugs suggests that iKr is not the current predominantly affected by erythromycin in the rat papillary muscle. FIG. 1. Effects of select drugs on rat ventricular repolarization. (A and B) Action potential traces from rat papillary muscle preparations. In each panel, pairs of superimposed traces show effects obtained during continuous recordings before and after drug exposure. ... In conclusion, this in vitro study corroborates the findings of Ohtani et al. regarding the sensitivity of the rat in vivo ECG model for erythromycin. However, the lack of effects of high concentrations of known iKr-blocking drugs on the APD corroborates the reported paucity of iKr in the rat ventricular muscle and suggests that currents other than iKr are responsible for delaying repolarization with erythromycin. Thus, the present studies suggest that the rat ECG model is inappropriate for evaluating the proarrhythmic potential of noncardiovascular drugs that prolong the Q-T interval by blocking iKr. In addition, this highlights the need to consider differences in the ionic currents expressed in preparations employed to evaluate cardiac safety. Further studies are necessary to determine the ionic channel(s) responsible for delayed repolarization in the rat myocardium seen with macrolide antibiotics.

Pharmacological characterization of AMP 579, a novel adenosine A1/A2 receptor agonist and cardioprotective

AMP 579 1S‐[1α,2β,3β,4α(S*)]‐4‐[7‐[[1‐[(3‐chloro‐2‐thienyl)methyl]propylamino]‐3H‐imidazo[4,5‐b]pyridin‐3‐yl]‐N‐ethyl‐2,3‐dihydroxy cyclopentanecarboxamide) is a novel cardioprotective adenosine agonist with the following order of affinity at adenosine receptors: A1 > A2A > A3. Agonism at A1 receptors was demonstrated in vitro in three different systems: 1) inhibition of lipolysis in rat and human isolated adipocytes, 2) restoration of insulin‐dependent glucose transport in rat adipocytes, and 3) reduction of heart rate in spontaneously beating rat right atria. Agonism at A2A receptors was reflected in vasorelaxation of porcine coronary arterial rings (IC50 = 0.3 μM); in comparison, agonism at A2B receptors was ∼100‐fold weaker, as reflected in relaxation of guinea pig aorta (IC50 = 28 μM). When given iv to conscious Sprague‐Dawley (SD) rats, AMP 579 dose‐dependently lowered free fatty acids (FFA), heart rate (HR), and mean arterial pressure (MAP), but was 25‐fold more potent at reducing FFA than at decreasing HR and MAP. In anesthetized rats undergoing myocardial ischemia‐reperfusion injury, AMP 579 (3 μg/kg + 0.3 μg/kg/min iv and 10 μg/kg + 1 μg/kg/min iv) was able to reduce infarct size by 55% and 63%, respectively, compared to control animals, when given 10 min prior to and throughout the first hour of reperfusion. These cardioprotective doses of AMP 579 caused no significant change in blood pressure or coronary blood flow. In summary, AMP 579 is a novel adenosine A1/A2A receptor agonist which causes long‐lasting reductions in FFA in vivo and has cardioprotective effects in a rat model of myocardial ischemia‐reperfusion injury at doses which have minimal hemodynamic effects. Thus, AMP 579 has significant potential for the therapy of acute myocardial infarction. Drug Dev. Res. 45:30–43, 1998.© 1998 Wiley‐Liss, Inc.