Michael J A Walker

Cardiovascular actions of the κ-agonist, U-50,488H, in the absence and presence of opioid receptor blockade

1 The cardiovascular actions of U‐50,488H, a κ‐receptor agonist, were studied in rat isolated perfused hearts, and in anaesthetized rats, over concentrations or doses generally above those required to produce κ‐receptor‐mediated effects. 2 U‐50,488H dose‐dependently decreased left‐ventricular peak systolic pressure and beating rate in vitro and reduced blood pressure and heart rate in vivo. 3 Over the concentration range of 1–30 μm in vitro, and the dose‐range of 0.5–32 μmol kg−1 in vivo, U‐50,488H prolonged the P—R, QRS and Q—T intervals of the ECG. 4 The effects of U‐50,488H were not antagonized by an opioid receptor antagonist, naloxone (1 μm or 8 μmol kg−1). Similarly, the opioid receptor antagonist, MR 2266, at 8 μmol kg−1 did not significantly reduce the cardiovascular actions of U‐50,488H in vivo. 5 The actions of U‐50,488H on responses to electrical stimulation were also studied. Over the dose range of 0.5–32 μmol kg−1, U‐50,488H altered thresholds and effective refractory period. It had a biphasic action on thresholds for induction of ventricular fibrillation. Thresholds were decreased at lower doses (0.5–4 μmol kg−1) but increased at higher doses (8–32 μmol kg−1). The effects of lower doses were blocked by naloxone. Effective refractory period and threshold pulse width only increased with dose. 6 In conclusion, U‐50,488H at high concentration, had direct depressant actions on cardiac contractility, electrical excitability and the ECG. These depressant effects were not antagonized by the opioid receptor antagonists, naloxone and MR 2266, and probably do not involve opioid receptors. Furthermore, some of the observed effects were those expected to result from sodium channel blockade.

Efficacy and safety of RPL554, a dual PDE3 and PDE4 inhibitor, in healthy volunteers and in patients with asthma or chronic obstructive pulmonary disease: findings from four clinical trials.

BACKGROUND Many patients with asthma or chronic obstructive pulmonary disease (COPD) routinely receive a combination of an inhaled bronchodilator and anti-inflammatory glucocorticosteroid, but those with severe disease often respond poorly to these classes of drug. We assessed the efficacy and safety of a novel inhaled dual phosphodiesterase 3 (PDE3) and PDE4 inhibitor, RPL554 for its ability to act as a bronchodilator and anti-inflammatory drug. METHODS Between February, 2009, and January, 2013, we undertook four proof-of-concept clinical trials in the Netherlands, Italy, and the UK. Nebulised RPL554 was examined in study 1 for safety in 18 healthy men who were randomly assigned (1:1:1) to receive an inhaled dose of RPL554 (0·003 mg/kg or 0·009 mg/kg) or placebo by a computer-generated randomisation table. Subsequently, six non-smoking men with mild allergic asthma received single doses of RPL554 (three received 0·009 mg/kg and three received 0·018 mg/kg) in an open-label, adaptive study, and then ten men with mild allergic asthma were randomly assigned to receive placebo or RPL554 (0·018 mg/kg) by a computer-generated randomisation table for an assessment of safety, bronchodilation, and bronchoprotection. Study 2 examined the reproducibility of the bronchodilator response to a daily dose of nebulised RPL554 (0·018 mg/kg) for 6 consecutive days in a single-blind (patients masked), placebo-controlled study in 12 men with clinically stable asthma. The safety and bronchodilator effect of RPL554 (0·018 mg/kg) was assessed in study 3, an open-label, placebo-controlled crossover trial, in 12 men with mild-to-moderate COPD. In study 4, a placebo-controlled crossover trial, the effect of RPL554 (0·018 mg/kg) on lipopolysaccharide-induced inflammatory cell infiltration in induced sputum was investigated in 21 healthy men. In studies 3 and 4, randomisation was done by computer-generated permutation with a block size of two for study 3 and four for study 4. Unless otherwise stated, participants and clinicians were masked to treatment assignment. Analyses were by intention to treat. All trials were registered with EudraCT, numbers 2008-005048-17, 2011-001698-22, 2010-023573-18, and 2012-000742-34. FINDINGS Safety was a primary endpoint of studies 1 and 3 and a secondary endpoint of studies 2 and 4. Overall, RPL554 was well tolerated, and adverse events were generally mild and of equal frequency between placebo and active treatment groups. Efficacy was a primary endpoint of study 2 and a secondary endpoint of studies 1 and 3. Study 1 measured change in forced expiratory volume in 1 s (FEV1) and provocative concentration of methacholine causing a 20% fall in FEV1 (PC20MCh) in participants with asthma. RPL554 produced rapid bronchodilation in patients with asthma with an FEV1 increase at 1 h of 520 mL (95% CI 320-720; p<0·0001), which was a 14% increase from placebo, and increased the PC20MCh by 1·5 doubling doses (95% CI 0·63-2·28; p=0·004) compared with placebo. The primary endpoint of study 2 was maximum FEV1 reached during 6 h after dosing with RPL554 in patients with asthma. RPL554 produced a similar maximum mean increase in FEV1 from placebo on day 1 (555 mL, 95% CI 442-668), day 3 (505 mL, 392-618), and day 6 (485 mL, 371-598; overall p<0·0001). A secondary endpoint of study 3 (patients with COPD) was the increase from baseline in FEV1. RPL554 produced bronchodilation with a mean maximum FEV1 increase of 17·2% (SE 5·2). In healthy individuals (study 4), the primary endpoint was percentage change in neutrophil counts in induced sputum 6 h after lipopolysaccharide challenge. RPL554 (0·018 mg/kg) did not significantly reduce the percentage of neutrophils in sputum (80·3% in the RPL554 group vs 84·2% in the placebo group; difference -3·9%, 95% CI -9·4 to 1·6, p=0·15), since RPL554 significantly reduced neutrophils (p=0·002) and total cells (p=0·002) to a similar degree. INTERPRETATION In four exploratory studies, inhaled RPL554 is an effective and well tolerated bronchodilator, bronchoprotector, and anti-inflammatory drug and further studies will establish the full potential of this new drug for the treatment of patients with COPD or asthma. FUNDING Verona Pharma.

An improved perfusion apparatus for small animal hearts.

A perfusion apparatus for mechanical and electrophysiological studies in small animal (e.g., rat, guinea pig) hearts is described. The apparatus consists of a number of individual perfusion chambers connected to the aortic perfusion cannula of a Langendorff perfused heart. The aortic root perfusion pressure is controlled by the oxygenating gas (usually 5% CO2 in O2) at a range of 0-200 mmHg. Different solutions can be placed in different individual chambers. Pressure in the left ventricle is monitored by means of a special compliant, but nonelastic, balloon while special atraumatic electrodes allow high-voltage, noise-free ECG recordings to be made from all parts of the epicardium. The apparatus keeps hearts beating for hours while the multichamber allows multiple drug applications to be made rapidly. Thus, the apparatus can be used for all types of pharmacodynamic analysis.

RSD931, a novel anti-tussive agent acting on airway sensory nerves

The anti‐tussive effects, of the local anaesthetic, lidocaine and carcainium chloride (RSD931) have been investigated in guinea‐pigs and rabbits. Pre‐treatment of guinea‐pigs with aerosols of lidocaine or RSD931 at 0.1, 1.0 and 10 mg ml−1 reduced the number of citric acid‐induced coughs by 9.3, 32.6 and 40.9% (P>0.05) for lidocaine and by 25.3% (P>0.05), 40.4% (P>0.05) and 97.6% (P<0.01) for RSD931, respectively and increased the latency to onset of cough at 10.0 mg ml−1 only. In addition, RSD931 at 10 mg ml−1 reduced citric acid‐evoked cough responses in rabbits (with prior exposure to ozone at 3 p.p.m. for 1 h) from 22.1±5.1 to 2.7±0.9 coughs (P<0.01). Acute pre‐treatment of guinea‐pigs with aerosols of lidocaine or RSD931 at 10.0 and 30.0 mg ml−1 reduced the number of capsaicin‐evoked coughs by 42.2 and 10.3% (P>0.05) (lidocaine) and by 25% (P>0.05) and 76.9% (P<0.01) (RSD931), respectively. Lidocaine had little effect on the latency of cough onset at either 10.0 or 30.0 mg ml−1, however, RSD at 30.0 mg ml−1 significantly (P<0.05) prolonged the latency of cough onset. RSD931 (10.0 mg ml−1) significantly (P<0.05–<0.01) reduced the spontaneous and histamine‐evoked discharges in Aδ‐fibres originating from airway, rapidly adapting stretch receptors (RARs) without affecting histamine‐evoked bronchoconstriction. Lidocaine at 10.0 mg ml−1 also significantly (P<0.05) inhibited the spontaneous and histamine‐induced discharges of RARs without affecting histamine‐evoked bronchoconstriction. Aerosols of RSD931 (10.0 mg ml−1) caused a transient, but significant (P<0.05), activation of pulmonary C‐fibre endings 2.5 min after administration started. RSD931 had no significant (P>0.05) effects on discharges in bronchial C‐fibres originating from bronchial C‐fibre endings, capsaicin‐evoked discharges of either pulmonary or bronchial C‐fibre endings or on capsaicin‐evoked bronchoconstriction. In contrast, lidocaine (10.0 mg ml−1) significantly (P<0.05) inhibited spontaneous and capsaicin‐induced discharges in both pulmonary and bronchial C‐fibres respectively. Lidocaine also significantly (P<0.05) reduced capsaicin‐evoked bronchoconstriction. These studies suggest that the anti‐tussive actions of RSD931 are mediated via inhibition of discharges in Aδ‐fibres originating from airway RARs. The mechanism of action of RSD931 is distinct from that of the local anaesthetic lidocaine and RSD931 may represent a novel class of anti‐tussive agent.

Antiarrhythmic properties of tedisamil (KC8857), a putative transient outward K+ current blocker

1 Rats were used to evaluate the antiarrhythmic properties of tedisamil, a novel agent with the electrophysiological properties of a Class III antiarrhythmic drug. Tedisamil was tested against coronary artery occlusion‐induced arrhythmias in conscious animals. 2 The actions of tedisamil on the ECG, as well as responses to electrical stimulation, were compared with those on the configuration of epicardial intracellular action potentials recorded in vivo. 3 Tedisamil (1–4 mg kg−1, i.v.) caused bradycardia, elevated blood pressure and dose‐dependently reduced ventricular fibrillation (VF) induced by occlusion of the left anterior descending coronary artery. Other ischaemia‐associated arrhythmias were not so well suppressed. Antiarrhythmic activity was greatest when the tedisamil‐induced bradycardia was prevented by electrically‐pacing the left ventricle. 4 Tedisamil dose‐dependently lengthened the effective refractory period and prevented electrically‐induced VF. In vivo, tedisamil (0.5–4 mg kg−1, i.v.) prolonged the duration of epicardial intracellular action potentials by up to 400%. 5 Results showed that tedisamil possessed antifibrillatory actions in rats that were related to Class III electrophysiological actions as revealed by electrical stimulation and electrophysiological analyses.

The mechanism of action of the optical enantiomers of verapamil against ischaemia-induced arrhythmias in the conscious rat

1 The actions of (‐)‐verapamil (0.2–6 mg kg−1) and (+)‐verapamil (0.4–12 mg kg−1) against arrhythmias induced by coronary artery occlusion were studied in conscious rats. 2 Intravenously administered (‐)‐ and (+)‐verapamil dose‐dependently reduced ventricular arrhythmias. (‐)‐Verapamil was consistently 4 times more potent than (+)‐verapamil. 3 In the same animals, (‐)‐verapamil was approximately 4 times more potent than (+)‐verapamil for effects on heart rate and blood pressure. Both enantiomers prolonged P‐R interval, but had no effect on QRS interval. 4 In separate groups of conscious rats, neither enantiomer influenced the threshold voltage and pulse width required to elicit fibrillo‐flutter, or altered the maximum following frequency, during electrical stimulation of the left ventricle. 5 In isolated, paced, Langendorff‐perfused ventricles of the rat, both enantiomers dose‐dependently reduced contractility, (‐)‐verapamil being 8–21 times more potent than (+)‐verapamil; both absolute and relative potencies were dependent on potassium concentration. 6 These results are compatible with the hypothesis that calcium antagonism in the ischaemic ventricular myocardium is antiarrhythmic during acute myocardial ischaemia.

Antiarrhythmic effects of U-50,488H in rats subject to coronary artery occlusion

The antiarrhythmic actions of low and high doses of U-50,488H, a selective kappa-receptor agonist, were examined in pentobarbitone-anaesthetized rats subjected to occlusion of the left anterior descending coronary artery. At a high dose (16 mumol/kg) U-50,488H reduced blood pressure, heart rate and prolonged the P-R and QRS intervals of the electrocardiogram. This dose reduced the incidence of ventricular arrhythmias produced by occlusion. The blood pressure, heart rate, ECG and antiarrhythmic actions of a high dose of U-50,488H were not antagonized by 8 mumol/kg naloxone, a dose which had no cardiovascular or ECG actions. Naloxone alone reduced arrhythmia incidence but to a lesser extent than U-50,488H. A low dose (0.2 mumol/kg) of U50,488H in the absence or presence of naloxone had no effect on arrhythmias. Thus, U-50,488H had antiarrhythmic actions at a high dose which were independent of opioid receptors.

Antiarrhythmic actions of verapamil against ischaemic arrhythmias in the rat

1 The actions of intravenous verapamil against arrhythmias induced by occlusion of a coronary artery were investigated in conscious rats. 2 Verapamil (2–20 mg kg−1, i.v. given pre‐occlusion) dose‐dependently reduced arrhythmias in rats with either large or small occluded zones at an ED50 of 6 mg kg−1. This dose was effective when given immediately post‐occlusion. 3 Severe arrhythmias, as opposed to PVC, were preferentially reduced. 4 In conscious, and pentobarbitone‐anaesthetized rats, verapamil (6 mg kg−1) had different effects on electrically‐induced arrhythmias, and the ECG, from an equi‐effective anti‐arrhythmic dose of quinidine (20 mg kg−1, i.v.). Quinidine decreased following frequency, but increased threshold current and pulse width, whereas verapamil did not. Both drugs increased P‐R interval, but only quinidine increased QRS and Q‐T intervals. 5 Thirty minutes post‐occlusion, the verapamil content of tissue and blood was determined after a 6 mg kg−1 dose given pre‐ or post‐occlusion. Measurable levels of verapamil were found in both normal and ischaemic myocardium. Plasma and plasma water concentrations were 3.6 ± 0.8 μmol l−1 and 0.6 ± 0.1 μmol l−1 (x̄ ± s.e.mean), respectively following post‐occlusion administration vs. 2.7 ± 1.2 and 0.24 ± 0.04 for pre‐occlusion administration. 6 Plasma water concentrations were close to IC50 values for inhibition of contractility in rat atria and ventricles. Similar concentrations depressed slow action potentials induced in rat ventricles by raised K+ 7 We suggest that the ability of verapamil to prevent severe ventricular arrhythmias following myocardial ischaemia in the conscious rat is largely due to the calcium antagonist effects of the drug.

The effect of modification of sympathetic activity on responses to ligation of a coronary artery in the conscious rat

1 Ligation of a coronary artery was performed in conscious rats whose sympathetic system activity had been altered by various treatments. 2 β‐Adrenoceptor blockade with acute (0.2 mg kg−1 plus 0.1 μg kg−1 min−1) or chronic (50–60 mg kg−1 daily for 12 days) propranolol treatment had little effect on arrhythmias, or other responses to ligation. 3 Abrupt withdrawal of chronic propranolol two days before ligation was also without effect. 4 Reduction of sympathetic activity acutely with labetalol (5 mg kg−1), or chronically with adrenomedullectomy and 6‐hydroxydopamine treatment, accentuated the adverse effects of ligation. 5 The results of this study suggest that, while activity of the sympathetic system is not detrimental during ligation in the conscious rat, it may be important for survival.

Electrophysiological and antiarrhythmic actions of the κ agonist PD 129290, and its R,R (+)‐enantiomer, PD 129289

1 The S,S (−)‐enantiomer PD 129290, a κ agonist, and its corresponding inactive R,R (+)‐enantiomer (PD 129289) were studied in rat isolated hearts and in intact rats for cardiovascular and antiarrhythmic actions. The electrophysiological actions of PD 129290 were also studied in rat isolated cardiac myocytes using voltage clamp. 2 Ventricular pressure, heart rate and ECG were studied in isolated hearts while blood pressure, heart rate and ECG were studied in pentobarbitone‐anaesthetized rats. In the latter, responses to electrical stimulation and coronary occlusion were also investigated. 3 In isolated hearts both enantiomers, over the concentration range 2–16 μm, dose‐dependently reduced systolic ventricular pressure and heart rate while prolonging the P–R and QRS intervals of the ECG. 4 At doses of 1–32 μmol kg−1 both enantiomers reduced blood pressure and heart rate in anaesthetized rats. In addition, both enantiomers increased the size of the RSh and increased P–R, QRS, and Q–T intervals of the ECG. The thresholds for premature beats and ventricular fibrillation were dose‐dependently increased by PD 129289. At lower doses PD 129290 decreased thresholds. These decreases were blocked by naloxone to reveal underlying increases similar to those seen with PD 129289. Both enantiomers increased refractory periods. 5 Naloxone (8 μmol kg−1) did not alter any of the actions of PD 129290, except to abolish the initial decreases in thresholds in intact rats seen with lower doses of PD 129290. 6 Both enantiomers (2 and 8 μmol kg−1) equally reduced arrhythmias in anaesthetized rats subject to occlusion of a coronary artery. 7 In rat isolated cardiac myocytes 20 μm PD 129290, in the presence and absence of naloxone decreased the amplitude of the transient sodium current by about 50% without affecting the voltage‐dependence of activation or inactivation of this current. 8 The antiarrhythmic actions of both enantiomers appear to primarily result from their Class I (sodium channel blockade) properties which are independent of κ agonism.