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Circulation. 1996;93:817-825

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(Circulation. 1996;93:817-825.)
© 1996 American Heart Association, Inc.

Articles

Mechanism of Action of OPC-8490 in Human Ventricular Myocardium

Amelia Focaccio, MD; George Peeters, MD; Matthew Movsesian, MD; Robert Roden, MS; Yutaka Eki, MD; Judith Krall, BS; Michael R. Bristow, MD, PhD

From the University of Naples (Italy), Department of Cardiology (A.F.); the University of Utah Medical Center, Division of Cardiology, Salt Lake City (G.P., J.K.); the Departments of Internal Medicine (Cardiology) and Pharmacology, Salt Lake City (Utah) Veterans Affairs Medical Center and University of Utah School of Medicine, Salt Lake City (M.M.); the University of Colorado Health Sciences Center, Division of Cardiology, Denver (R.R., M.R.B.); and the National Defence Medical College, First Department, Tokaimura, Japan (Y.E.).

Correspondence to Michael R. Bristow, University of Colorado Health Sciences Center, Division of Cardiology, 4200 E 9th Ave, Denver, CO 80262. E-mail bristow_M@defiance.uchsc.edu.


*    Abstract
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 *Abstract
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Background The quinolinone compounds OPC-8212 (vesnarinone), OPC-18790, and OPC-8490 are members of a family of unique positive inotropic compounds that have no positive chronotropic effects. In subjects with heart failure, the prototypic compound OPC-8212 may reduce morbidity and mortality at low doses but increase mortality at high doses.

Methods and Results To further characterize the inotropic mechanism(s) of action of these compounds, we investigated the effects of OPC-8490, a water-soluble quinolinone, on the inotropic response, inhibition of phosphodiesterase (PDE), and action potential in human ventricular myocardial preparations. In isolated right ventricular trabeculae and membranes prepared from left ventricular myocardium, OPC-8490 produced dose-related positive inotropic effects, inhibited type III PDE activity, and prolonged action potential. Comparative experiments with other PDE inhibitors, sodium channel agonists, and potassium channel antagonists indicated that the positive inotropic effects are due to PDE inhibition, whereas the action potential effects of OPC-8490 are due to effects on ion channels.

Conclusions We conclude that OPC-8490 produces selective positive inotropic effects because of type III PDE inhibition combined with ion channel effects, with the latter property inhibiting the positive chronotropic response usually associated with agents that increase intracellular cAMP concentrations.


Key Words: inotropic agents • heart failure • drugs


*    Introduction
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 up arrowAbstract
 *Introduction
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down arrowDiscussion
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The quinolinone derivatives OPC-8212 (vesnarinone), OPC-18790, and OPC-8490 (Fig 1Down) are recently developed inotropic agents with positive inotropic actions that are not accompanied by positive chronotropic effects.1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 In model systems and human subjects with heart failure, these quinolinone compounds produce positive inotropic effects, neutral to negative chronotropic effects, and mild (OPC-8212) or moderate (OPC-18790 and OPC-8490) degrees of vasodilation.1 2 3 4 6 10 11 12 13 14 15 16 17 All three quinolinone compounds prolong the cardiac action potential in several mammalian species,1 2 4 5 6 7 8 9 12 which theoretically could produce a positive inotropic response in the absence of a positive chronotropic effect. Small and medium-sized clinical trials with OPC-8212, a hydrophobic compound available only as an oral agent, have demonstrated hemodynamic and clinical benefits in the short- and long-term treatment of heart failure.10 11 16 17 Recent studies with intravenous OPC-18790 have demonstrated favorable hemodynamic effects in subjects with advanced heart failure, with a myocardial energetic profile superior to that of dobutamine.14 Moreover, a recent large-scale multicenter clinical trial of OPC-8212 in advanced heart failure revealed a 62% reduction in mortality in subjects treated with low-dose OPC-8212 versus placebo.18 This trial,18 however, also reported an increase in mortality at high doses of OPC-8212, suggesting that the mechanism of action of this class of compounds is complex.



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  		Figure 1. Schematic showing the structure of OPC-8212, OPC-8490, and OPC-18790.

The mechanisms of action of these quinolinone derivatives have not been completely clarified. PDE inhibitory activity has been demonstrated for OPC-82121 9 19 and OPC-1879012 but not for OPC-8490. Although a cAMP-dependent action of OPC-8490 has been reported in isolated, blood-perfused canine heart preparations,4 this action was absent in guinea pig dissociated ventricular cells.5 PDE inhibition, however, cannot account for the negative or neutral chronotropic effects of quinolinone derivatives. It has thus been suggested that multiple modes of action allow quinolinones to act as positive inotropic agents with negative or neutral chronotropic effects. A cAMP-dependent or cAMP-independent increase in inward calcium current activity,5 8 20 21 22 an increase of Na+ channel open time,7 9 and a modification of K+ current actions5 8 23 have all been proposed as possible additional mechanisms of action that could contribute to the effects of these drugs. OPC-8490 and OPC-18790 are water-soluble quinolinone derivatives that differ from OPC-8212 in that they can be administered intravenously and have more potent vasodilatory effects.1 4 10 12 14 15 24 Because it is relatively hydrophilic, OPC-8490 can be evaluated in isolated tissue preparations without the use of organic solvent vehicles, which may confound the measurements of PDE inhibition, electrophysiological effects, and mechanical response to OPC-8212.1 6 16 17 18 20 21

The present study was undertaken to evaluate the inotropic effects of OPC-8490 as a model quinolinone inotropic agent to elucidate the mechanism of action of this unique class of agents in the human heart. Specifically, we wanted to determine whether the positive inotropic effects of the quinolinones as represented by OPC-8490 were due to PDE inhibition or to ion channel effects. The effects of OPC-8490 on myocardial type III cAMP PDE activity, inotropic response in RV trabeculae, QT interval, and action potential duration were assessed in preparations obtained at the time of cardiac transplantation from ventricles of human subjects with and without end-stage heart failure. For comparative purposes, the effects of enoximone and milrinone, two well-characterized PDE type III inhibitors,25 were examined in the same preparations. In addition, the inotropic and action potential effects of OPC-8490 were compared with the effects of the Na+ channel activators BDF-914826 27 28 and veratridine and the potassium channel antagonist d-sotalol.29


*    Methods
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 *Methods
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Tissue Acquisition
Sixteen human hearts obtained from transplant recipients or organ donors were used in this study and were classified as either failing or nonfailing on the basis of myocardial function assessment made before explantation. The hearts were rapidly removed, placed in ice-cold oxygenated Tyrode's solution, and transported immediately to the laboratory as previously described.30 31 Of the 12 failing hearts used in these studies, 10 were obtained from patients with end-stage heart failure (NYHA class IV) undergoing cardiac transplantation. Diagnoses included idiopathic dilated cardiomyopathy in 2 patients and ischemic cardiomyopathy, ie, heart failure resulting from coronary artery disease, in 8 patients. All chronically failing hearts had biventricular dilatation on gross examination, and all had increased systolic and diastolic volumes measured by radionuclide and echocardiographic techniques before explantation. Additionally, 2 donor hearts with acute myocardial dysfunction (echocardiographically measured shortening fraction <15%) after brain injury also were used in the failing group. The average age of the subjects with heart failure was 52±2.2 years (range, 41 to 64 years). In the chronically failing hearts, ejection fraction measured by radionuclide ventriculography and cardiac catheterization data obtained within 8 weeks of transplant were as follows: ejection fraction, 17.9±1.6%; mean pulmonary artery pressure, 33.8±39 mm Hg; and cardiac index, 2.1±0.1 L·m-1·m-2. Medications being administered at the time of transplantation included digitalis (n=10), diuretics (n=10), and angiotensin-converting enzyme inhibitors (n=10) in the chronically failing group and dopamine in the acutely failing group. In the 2 acutely failing hearts exposed to dopamine, the baseline systolic tension was 440 mg compared with 595±80 mg in the 10 chronically failing hearts (P=NS).

In addition, RV trabeculae were taken from 3 organ donors and 1 patient with coronary artery disease whose right ventricles were considered to have normal function on the basis of echocardiographic, radionuclide, and RV catheterization data. Nonfailing donor hearts could not be used for transplantation because of size or ABO blood type incompatibility.

SR-Associated cAMP PDE Activity
Microsomes were prepared from left ventricular free wall myocardial tissue obtained from the explanted failing hearts of 3 heart transplant recipients with NYHA class IV heart failure resulting from idiopathic dilated cardiomyopathy, as previously described.32 cAMP PDE activity was measured as previously described.33 Microsomes were suspended at 0.012 mg/mL in a reaction mixture made up of 0.1 mmol/L EGTA, 8.3 mmol/L MgCl2, and 50 mmol/L HEPES (pH 7.5, 30°C). cAMP hydrolysis was inhibited by addition of 0.2 µmol/L [3H]AMP (New England Nuclear); each assay was performed in duplicate. The reaction was stopped by the addition of 10.1 mmol/L unlabeled cAMP and 5 mmol/L unlabeled 5'AMP in 0.25 N HCl. The reacting mixture was neutralized with NaOH, and [3H]5'AMP was converted to [3H]adenosine by incubation with Crotalus atrox venom (30 mg/dL) at 30°C for 30 minutes. The reaction mixture was then applied to QAE-Sephadex columns, and adenosine was eluted with H2O at neutral pH. cAMP hydrolysis was determined by measuring [3H]adenosine in the eluent by scintillation spectrometry.

Tissue Contractile Response
The contractile response of isolated ventricular tissue was determined as previously described.30 31 Briefly, hearts were placed in ice-cold Tyrode's buffer31 immediately after cardiectomy. Individual trabeculae of uniform size (1 to 2 by 6 to 8 mm) were isolated from the free wall of the right ventricle and placed in muscle bath chambers containing Tyrode's buffer equilibrated with 95% O2/5% CO2 and kept at 37°C. Trabeculae were suspended between plastic mounting clips and allowed to equilibrate for 2 hours, and bath volumes were exchanged with fresh buffer every 30 minutes. After the first 30 minutes of equilibration, resting tension was set at the length at which maximal isometric tension developed (usually {approx}1 g), and a field current (square-wave pulse) was passed through the bath at a frequency of 1 Hz and a pulse duration of 5 ms at a voltage just above threshold (usually 15 to 20 V). BSA (0.1%) was added to the plastic muscle bath chambers to reduce adherence of the drugs to the plastic. The response to OPC-8490, enoximone, and BDF-9148 was evaluated in the absence and presence of forskolin, which acts by elevating intracellular cAMP levels through activation of adenylyl cyclase.34 Forskolin was delivered at threefold increasing concentrations (starting at 1x10-7 mol/L) with 6-minute intervals between two consecutive doses until a small (15% to 25% of the maximal response) increase in isometric tension was observed. After a 10-minute equilibration period, cumulative dose-response curves were generated for isoproterenol, OPC-8490, enoximone, or BDF-9148 in the presence and absence of forskolin. Drugs were delivered throughout several log doses and at half-log-unit dose intervals (1x10-9 to 1x10-4 mol/L for isoproterenol and 1x10-8 to 1x10-4 mol/L for OPC-8490, enoximone, and BDF-9148). The time between two consecutive dose intervals was 2 minutes for isoproterenol and 4 minutes for all other compounds, reflecting differences in the time necessary to reach maximum tension for each drug. Systolic tension was measured by individual Kistler-Morse deflection sensor cartridges, with signal amplification by Accudata 105 DC amplifiers, and recorded on a 16-channel recorder at a paper speed of 10 and 50 mm/s. One hour after completion of the dose-response curves and washout of drugs, calcium chloride at final concentrations of 2.5, 5.0, and 10 mmol/L was administered to measure the maximal response to calcium. Tension responses were measured as the net gain in amplitude from baseline in 10–3 newtons.

Electrogram Recording
In four experiments, RV trabeculae were placed in muscle bath chambers equipped to simultaneously measure tension and electrogram response to OPC-8490. Trabeculae were stimulated to contract at a frequency of 1 Hz through a bipunctate electrode with use of voltage that was 10% above threshold and a pulse duration of 5 ms. Electrogram and developed isometric tension were then recorded simultaneously during the administration of OPC-8490 (10-4 mol/L). QAT rather than QT interval was measured as the beginning of the Q wave to the peak of the T wave. The QAT interval was used because it may be a more drug-sensitive interval that reflects repolarization changes.35 Recordings were made at slow and fast paper speeds (10 and 100 mm/s) to facilitate measurement of intervals.

Transmembrane Electrical Activity
To evaluate the effects of increasing concentrations of OPC-8490 (10–6 to 10–4 mol/L), enoximone (10–6 to 10–4 mol/L), and BDF-9148 (10–8 to 10–5 mol/L) on the action potential and peak developed tension, RV trabeculae measuring 1 to 1.5 by 6 to 8 mm were mounted in a horizontal muscle bath warmed at 37°C. The bath was continuously superfused with oxygenated (5% CO2/95% O2) Tyrode's solution. Pacing rate was 0.3 Hz (n=4) or 1 Hz (n=4) in experiments with OPC-8490 and 1 Hz in all the other experiments. Resting tension was set at 2 g during control superfusion. Transmembrane action potentials were recorded with 3 mol/L KCl-filled glass micropipettes with a tip resistance of {approx}10 M{Omega}. Drugs were added rapidly to the muscle bath by switching the superfusate from the control to drug by a stopcock manifold. Drug concentrations were increased in a stepwise fashion at 10-minute intervals. Data were recorded on magnetic tape and monitored on a digital storage oscilloscope. Peak developed tension and APd90 were measured for each drug concentration and normalized to their control values. Resting membrane potentials in this preparation varied from -72 to -90 mV.

Drugs and Reagents
Forskolin was purchased from Sigma Chemical Co. Enoximone was a gift from Marion Merrell Dow Pharmaceuticals Inc. OPC-8490 was a gift from Otsuka America. Stock solutions of forskolin were prepared by dissolving the drug in 100% ethanol; in the amounts used in these experiments, the diluent had no effect on force development. Stock solutions of enoximone and BDF-9148 were obtained by dissolving the drug in nine parts dimethylacetamide (Sigma Chemical Co) and one part 1 N NaOH. OPC-8490 was dissolved in distilled water. Serial dilutions of the drugs were made up in distilled water containing 1 mmol/L ascorbate. OPC-8490 was protected from light. All concentrations refer to the final bath concentration.

Statistical Analysis
Differences in dose-response curves were determined by repeated-measures ANOVA. Inotropic responses to each dose of drug in the absence and presence of forskolin were compared by paired Student's t test. A value of P<.05 in a two-tailed distribution was considered significant. ED50 and IC50 values were obtained by computer modeling of full dose-response curves with Inplot (GraphPad).


*    Results
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 up arrowMethods
*Results
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cAMP PDE Type III Inhibitory Activity
At low concentrations, the cAMP PDE activity associated with the SR of human ventricular myocardium has been shown to be catalytically homogeneous PDE type III.33 For this reason, we examined the effects of OPC-8490 on SR-associated PDE activity in microsomes prepared from failing human left ventricles. For comparative purposes, the effects of enoximone and milrinone, two well-characterized PDE type III inhibitors,25 36 37 were examined in the same preparations. As Fig 2Down shows, OPC-8490, enoximone, and milrinone exhibited type III cAMP PDE inhibitory activity, with IC50 values of 1x10-5, 2x10-6, and 3x10-7 mol/L, respectively.



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  		Figure 2. Plot showing the inhibition of cAMP PDE activity by OPC-8490, enoximone, and milrinone. Measurements were made at 0.2 µmol/L cAMP. Each point represents the mean±SEM of three to five determinations.

Inotropic Effects in Isolated RV Trabeculae
The maximal inotropic response to OPC-8490 in isolated RV trabeculae removed from failing and nonfailing human hearts was compared with the maximal inotropic response to the sodium channel activator BDF-9148 and enoximone, the inotropic efficacy of which is derived exclusively from inhibition of PDE type III activity.25 36 Fig 3Down compares the maximal tension response for OPC-8490, BDF-9148, and enoximone in RV trabeculae from failing and nonfailing human hearts expressed as net gain in force from baseline.



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  		Figure 3. Bar graph showing the effects of OPC-8490 (10 hearts, 23 trabeculae), BDF-9148 (4 hearts, 12 trabeculae), and enoximone (16 hearts, 30 trabeculae) on isometric systolic tension in isolated RV trabeculae in failing and nonfailing tissues. Values are mean±SEM.

In preparations obtained from failing hearts, OPC-8490 produced a marginal increase in isometric systolic tension (0.62±0.49 mN with the maximal concentration used, 10-4 mol/L). Slightly greater inotropic responses were also observed with 3x10-5 (0.62±0.22 mN) and 10-4 mol/L (2.0±0.52 mN) enoximone in failing preparations. In nonfailing tissues, OPC-8490 also exhibited a more moderate tension response relative to both enoximone and BDF-9148. Unlike BDF-9148, however, both OPC-8490 and enoximone exhibited greater tension responses in nonfailing than in failing tissues.

In the presence of a minimally effective dose of forskolin (ie, a dose that produced and sustained a barely detectable increase [{approx}20%] in systolic tension development), the inotropic responses to enoximone (n=6 hearts, 10 trabeculae; P<.01 by ANOVA) and OPC-8490 (n=6 hearts, 24 trabeculae; P<.01 by ANOVA) were markedly potentiated (Fig 4ADown and 4BDown). Fig 4ADown and 4BDown also shows that the dose-response curve slopes for both enoximone (P<.01) and OPC-8490 (P<.01) are increased by forskolin, indicating synergism and implying a cAMP-dependent mechanism for the positive inotropic effect. In the presence of forskolin, the ED50 that elicited an increase in force of contraction was 3x10-5 and 8x10-6 mol/L for enoximone and OPC-8490, respectively.



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  		Figure 4. Plots showing the effects of enoximone (A; 10 hearts, 33 trabeculae), OPC-8490 (B; 8 hearts, 16 trabeculae), BDF-9148 (C; 12 hearts, 11 trabeculae), and d-sotalol (D; 10 hearts, 11 trabeculae) on isometric systolic tension in isolated RV trabeculae that had or had not been potentiated with the adenylyl cyclase activator forskolin. Values are mean±SEM.

In contrast to OPC-8490 and enoximone, the inotropic responses to the Na+ channel activators BDF-9148 (Fig 4CUp) and veratradine (data not shown) and the K+ channel antagonist d-sotalol were not potentiated by forskolin. Fig 4CUp gives the cumulative dose-response curves to BDF-9148 in the presence and absence of forskolin; the maximal increase in isometric systolic tension was 6.2±2.3 mN at 3x10-5 mol/L in the absence of forskolin and 5.1±1.9 mN at 1x10-4 mol/L in the presence of forskolin (P=NS). The EC50 values for BDF-9148 with and without forskolin were 2x10-8 and 2x10-7 mol/L, respectively. Unlike BDF-9148, d-sotalol did not increase systolic tension in either the presence or absence of forskolin (Fig 4DUp).

QAT Interval and Action Potential
QAT interval increased after the addition of OPC-8490 (from a mean basal value of 221±49 to 351±105 ms at 10-4 mol/L; Fig 5ADown). In contrast, enoximone had no effect on QAT (data not shown). In action potential measurements performed at 1.0 and 0.3 Hz (n=4 for each), OPC-8490 induced a dose-dependent increase in APd90, from a mean basal value of 340±38 to 626±115 ms with the maximal dose used (1x10-4 mol/L, P=.01; Fig 5BDown). In four experiments performed at a stimulation rate of 0.3 Hz, OPC-8490 induced APd90 prolongation from a mean basal value of 405±60 to 809±199 ms (P=.07; Fig 6ADown). In four different experiments performed at a pacing rate of 1 Hz, OPC-8490 increased APd90 from a mean control value of 275±19 to 443±13 ms (P=.01; Fig 6BDown). As Fig 6ADown and 6BDown shows, the effects on action potential appeared to occur at lower doses than the tension increase at the slower (0.3 Hz) pacing rate but over the same dose range as the tension responses at 1.0 Hz. Fig 6CDown is a representative trace illustrating prolongation of action potentials recorded from RV trabeculae of an explanted failing heart in which recordings made before and after exposure to 1 mmol/L OPC-8490 are superimposed.



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  		Figure 5. Bar graphs showing the effects of 1x10-4 mol/L OPC-8490 on the QT interval (A; 8 trabeculae) and APd90 (B; 4 trabeculae; stimulation rate, 3 Hz in four experiments, 1 Hz in four experiments), QT interval measured from the beginning of the Q wave to the peak of the T wave (QAT; stimulation rate, 1 Hz), and isometric systolic tension. Values are mean±SEM.



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  		Figure 6. Plots showing the effects of OPC-8490 on APd90 and isometric tension at two different pacing rates: 3 Hz (A; 4 trabeculae) and 1 Hz (B; 4 trabeculae). Values are mean±SEM. C, Prolongation of action potentials recorded from RV trabeculae of explanted failing human heart after OPC-8490 (1-Hz stimulation at 37°C). The action potentials recorded before and after drug infusion are superimposed. The APd90 of control subjects was 0.306 second. After 10 minutes of superfusion with 0.1 mmol/L OPC-8490, APd90 was prolonged to 0.426 second.

As Fig 7ADown shows, BDF-9148 increased APd90 slightly, from a mean control value of 310±79 to 341±69 ms. With enoximone, APd90 did not increase despite an increase in muscle contraction (Fig 7BDown); with d-sotalol, APd90 was slightly increased and muscle contraction tended to decrease (Fig 7CDown). Fig 8Down shows the comparative effects on action potential duration of increasing concentrations of OPC-8490, enoximone, and BDF-9148 at a pacing rate of 1.0 Hz.



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  		Figure 7. Plots showing the effects of BDF-9148 (A), enoximone (B), or d-sotalol (C) on APd90 (stimulation rate, 1 Hz) and force of contraction. Values are mean±SEM.



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  		Figure 8. Plot showing action potential in isolated RV trabeculae before and after increasing concentrations of OPC-8490 (10–6 to 10–4 mol/L), enoximone (10–6 to 10–4 mol/L), or BDF-9148 (10–8 to 10–4 mol/L) at 1-Hz stimulation rate.

Comparative Effects of OPC-8490 on Systolic Tension, PDE, and Action Potential
Fig 9Down plots grouped normalized dose-response data for OPC-8490 for positive inotropic (in the presence of forskolin), PDE type III inhibitory, and action potential prolongation responses, with each response normalized to percentage of maximum effect. As can be seen, the PDE inhibitory effects and action potential effects occur over the same dose range, resulting in similar EC50 values that are within twofold of each other (1.00x10-5 and 1.80x10-5 mol/L, respectively; P=NS). In contrast, the tension effects occurred at a slightly higher EC50 (1.5x10-4 mol/L), which was not significantly different from the PDE inhibitory and action potential EC50 values.



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  		Figure 9. Plot showing the comparative curve analysis of PDE inhibition (5 hearts), systolic tension response (16 hearts), and action potential prolongation (4 hearts) with increasing concentrations of OPC-8490. Mean curves are plotted as a percent of maximal effect.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
The inotropic mechanisms of action of quinolinone derivatives have been previously investigated in animal systems, but no consensus has emerged to explain their unique pharmacological profile. Because species differences exist in at least two potentially important quinolinone mechanisms of action (PDE inhibition25 and effects on action potential22 ), we thought it important to examine the effects of this novel class of compounds in preparations of nonfailing and failing human heart. PDE inhibition has been observed with OPC-8212 in bovine,9 canine,1 20 21 and human19 cardiac muscles, but some of this effect may have been directly attributable to sulfolane, the solvent used for OPC-8212.1 6 A relatively small degree of PDE inhibitory activity was reported for the water-soluble compound OPC-8490 in isolated, blood-perfused canine preparations,4 but the drug did not affect the intracellular cAMP concentration in single guinea pig myocytes.5

Our data unequivocally demonstrate that OPC-8490 has PDE type III inhibitory activity in human ventricular myocardium. OPC-8490 inhibited cardiac SR-associated PDE activity in a concentration-dependent manner, with an IC50 value of 1x10-5 mol/L. In comparison, the classic PDE inhibitors enoximone and milrinone yielded IC50 values of 2x10-6 and 3x10-7 mol/L, respectively, in the same system. The SR-associated PDE in human ventricular myocardium is homogeneous PDE type III,33 and this specific form of PDE has been shown to be directly correlated to the positive inotropic response to PDE inhibitors such as enoximone or milrinone.25 33 37 38

Both OPC-8490 and enoximone produced low-level inotropic effects in isolated human RV trabeculae removed from end-stage failing human hearts. Although the response to enoximone was somewhat greater, the response to OPC-8490 was only 7% of that produced by isoproterenol. The effects of both OPC-8490 and enoximone were markedly reduced in failing versus nonfailing preparations. In contrast, the contractile response to the sodium channel agonist BDF-9148 was not decreased in preparations taken from failing hearts and, in agreement with previous reports,39 tended to be increased compared with nonfailing hearts. Diminished effectiveness of PDE inhibitors in the failing human heart has been previously described and is related to a deficient production of cAMP.38 40 A low dose of forskolin, which acts to increase intracellular cAMP levels through direct activation of adenylyl cyclase,30 has been shown to restore the responsiveness of isolated muscle from failing hearts to the PDE inhibitors milrinone and isobutyl methyl xanthine.38 Accordingly, in our study a minimally effective dose of forskolin markedly potentiated the inotropic response of the PDE inhibitor enoximone and OPC-8490. Thus, OPC-8490 exhibits a weak positive inotropic effect in isolated failing human hearts, but its effectiveness is markedly enhanced by increasing intracellular cAMP levels with forskolin. The increase in isometric systolic tension in the presence of forskolin was dose dependent, with an ED50 of 8.4x10-6 and 2.9x10-5 mol/L for enoximone and OPC-8490, respectively. On the other hand, forskolin did not potentiate the inotropic effects of the sodium channel agents BDF-9148 and veratridine. The similarity of the relative potencies of OPC-8490 and enoximone as PDE type III inhibitors and inotropic agents, the enhancement of the latter effects by treatment with forskolin, and the reduced contractile effects of these two agents in failing myocardium indicate that PDE inhibitory activity is at least one component of the mechanism of action of OPC-8490 in the failing human heart.

d-Sotalol, a type III antiarrhythmic agent by virtue of potassium channel antagonist properties,29 41 was used to explore the inotropic effects of action potential prolongation through K+ channel antagonism. Although d-sotalol prolonged action potential, it did not produce a positive inotropic effect in either the absence or presence of forskolin. Thus, of three potential mechanisms offered for the inotropic effect of OPC-8490 and related quinolinones (PDE inhibition, sodium channel agonism, and K+ channel antagonism), the pattern of response to OPC-8490 was identical to the reference PDE inhibitor enoximone and dissimilar to sodium channel agonists and a K+ channel antagonist.

Unlike other PDE inhibitors, OPC-8212, OPC-18790, and OPC-8490 produce positive inotropic effects that are not accompanied by an increase in heart rate. This observation suggests that the mechanism of action of quinolinone derivatives is complex. Because all three quinolinones increase action potential duration,1 2 4 5 12 a direct action on transmembrane ion channel activity in addition to the PDE inhibitory activity is thought to be present. An inhibitory effect on the delayed outward K+ current has been reported for OPC-82128 and OPC-84905 24 in guinea pig ventricular myocytes but not for OPC-8212 in rabbit or a limited number of human cardiac myocytes.9 In addition, a possible direct (cAMP-independent) action on voltage-dependent Ca2+ channels has been proposed for OPC-84905 and possibly for OPC-8212.22 However, studies in chick heart42 and single guinea pig ventricular cells43 have shown that increases in the slow inward calcium current may be produced by an increase in intracellular concentration of cAMP, which increases the number of slow channels available for voltage activation.42 43 44 45 Thus, in model systems an increase in cAMP concentration leads to an increase in contractility and prolongation of the action potential. However, we did not observe any APd90 prolongation by enoximone, which suggests that cAMP increases resulting from PDE inhibition do not prolong the human ventricular myocardial action potential. On the other hand, OPC-8490 produced a marked dose-dependent prolongation of APd90 and QAT, which strongly supports the idea that the quinolinone compounds have both PDE inhibitor and direct ion channel effects in the human heart.

There was only a minimal association between APd90 prolongation and the increase in isometric tension development with the sodium channel agonist BDF-9148. With d-sotalol, there was no relation between the relatively modest APd90 prolongation and a positive inotropic effect; in fact, d-sotalol produced a slight negative inotropic effect. Taken together, these data suggest that prolongation of the action potential per se does not produce a positive inotropic effect in human ventricular myocardium and that the marked effects of OPC-8490 on the cardiac action potential are independent of the positive inotropic PDE inhibitory activity of the drug. From the similarity of the OPC-8490 ED50s for PDE inhibition, APd90 prolongation, and systolic tension response, it appears that the action potential properties are manifest over the same dose range as the PDE inhibitory inotropic properties. It therefore seems plausible to propose that the ion channel properties of OPC-8490 may prevent the heart rate increase ordinarily associated with PDE inhibitory properties, which explains the unique inotropic and chronotropic profile of this class of agents.

The specific effect of OPC-8490 on human ventricular myocardial ion channels has not been elucidated by this study. From the observation that OPC-8490 prolonged the cardiac action potential to a much greater degree than maximal doses of sodium channel agonists or d-sotalol, it is possible that the quinolinones act primarily on another channel in the human heart, such as directly on calcium channels. In that regard, Lathrop et al22 recently reported the electrophysiological effects of OPC-8212 on two isolated cardiac myocytes from one failing human right ventricle. In these cells, the prolongation of the action potential appeared to be unrelated to blockade of K+ currents and probably is due to augmentation of the secondary inward calcium current.22 Our data indicate that elevations in cAMP, which presumably increase the secondary inward current by calcium channel phosphorylation, do not cause action potential prolongation in human ventricular myocardium. Therefore, a direct calcium channel effect of the quinolinones as the explanation for their action potential prolongation would have to include a channel-modulating mechanism that is different from that produced by cAMP. Based on these considerations, the magnitude of the effect of OPC-8490 on the cardiac action potential, and the known effect of this and other quinolinones in antagonizing the delayed rectifying potassium current (IK) in isolated guinea pig myocytes,5 8 it is likely that the action potential–prolonging effects of OPC-8490 are due to IK antagonism. This would explain the neutral to inhibitory effects of the quinolinones on heart rate as IK antagonism in humans lowers heart rate,46 which would negate the positive chronotropic consequences of PDE inhibition.

In a previous clinical trial, OPC-8212 (vesnarinone) increased mortality at a high (120 mg/d) dose and lowered mortality at a low (60 mg/d) dose.18 The PDE inhibitor milrinone has also been shown to increase mortality47 at what was a high dose from a hemodynamic standpoint.48 Moreover, the increased mortality from milrinone was from a proarrhythmic effect,49 as may have been the case with high-dose vesnarinone.18 Therefore, it is tempting to speculate that the adverse survival effects of high-dose vesnarinone are due to PDE inhibition and elevated levels of cAMP,44 whereas the favorable effects of a low dose are due to more subtle elevations in cAMP combined with no increase or even a decrease in heart rate50 plus or minus a favorable ancillary property such as cytokine inhibition.51 Alternatively, the increased mortality of high-dose vesnarinone could be due to proarrhythmic effects related to action potential prolongation such as what may occur with type III antiarrhythmic agents.

We conclude that the inotropic mechanism of action of OPC-8490 in failing human myocardium involves inhibition of the SR-associated cAMP PDE type III. The action potential prolongation produced by this agent is not related to the positive inotropic effects of the drug but may explain the neutral or negative chronotropic properties of OPC-8490 and this class of compounds. Thus, OPC-8490 and presumably the closely related compounds OPC-8212 and OPC-18790 should be considered mixed action agents, with the positive inotropic action mediated by PDE type III inhibition and the negative chronotropic action mediated by electrophysiological effects.


*    Selected Abbreviations and Acronyms
 
APd90 = action potential duration measured at 90% of repolarization
NYHA = New York Heart Association
PDE = phosphodiesterase
RV = right ventricular
SR = sarcoplasmic reticulum


*    Acknowledgments
 
We gratefully acknowledge the financial and technical support from Otsuka America Pharmaceuticals, Inc. Dr Movsesian is supported by the Department of Veterans Affairs Medical Research Funds and a Grant-in-Aid from the American Heart Association, Utah Affiliate. He also is the recipient of a Career Development Award from the Department of Veterans Affairs.

Received July 17, 1995; revision received September 27, 1995; accepted October 4, 1995.


*    References
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*References
 

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