During the last 30 years, several dopaminergic agonists have been developed for the treatment of cardiovascular diseases (1). Of these dopaminergic agonists, ibopamine (the prodrug of epinine) was the first to be used on a large scale in heart failure. Ibopamine primarily stimulates both D1- and D2-like receptors, but at higher dosages is also known to stimulate α and β receptors (2).
Initial studies with ibopamine, in patients with mild to moderate heart failure, reported mainly beneficial effects (3,4). However, the PRIME-II mortality study, after the effects of ibopamine in patients with severe heart failure, could not confirm these promising results (5). On the contrary, in this study, ibopamine treatment was unfavorable, as mortality was increased. Although the cause of this undesirable effect has not yet been elucidated, it is tempting to speculate that in severe heart failure, β- and α-adrenergic stimulation may occur. Consequently, alternative dopaminergic agonists devoid of α- and β-receptor stimulation could be more effective than ibopamine. Z1046 is such an alternative mixed dopaminergic agonist. In vitro studies of Z1046 in animal tissues already showed important D1- and D2-like agonistic effects together with α1-antagonistic and absence of β-adrenergic effects (6-9). However, these observations in animals models may not fully reflect the response in humans, or in patients with cardiovascular diseases, because receptor profiles may be totally different. Therefore in addition to these previous studies in animal preparations, we evaluated (in vitro) the α-adrenergic and dopaminergic effects of Z1046 in human arteries obtained from patients undergoing elective coronary bypass surgery. In this study, we compared the responses of Z1046 with those of the established dopaminergic compounds epinine and dopamine.
MATERIALS AND METHODS
Human internal mammary arteries (IMA) were obtained intraoperatively from 28 male patients (mean age, 61.3 ± 1.9 years; age range, 45-81 years) undergoing elective coronary artery bypass grafting. Use of excess IMA tissue was granted by the Human Ethics Committee of the University Hospital Groningen (Groningen, The Netherlands). During operation, the arteries were dissected free, but remained in their original anatomic environment (as previous described by Lüscher et al. (10). Excess IMA material was collected in a physiologic salt solution (NaCl, 0.9%), and immediately transferred from the operating room to the laboratory. The IMA segments were then placed in a Krebs solution (in mM consisting of NaCl, 120.4; KCl, 5.9; CaCl2, 2.5; MgCl2, 1.2; NaH2PO4, 1.2; glucose, 11.5; NaHCO3, 25.0; EDTA 0.01; and aerated with 95% O2/5% CO2). In this buffer, the segments were cleaned of surrounding tissue, and cut in several 2-mm rings with a sharp razor blade. All artery rings were connected to isotonic displacement transducers, and subjected to a 14 mN tonus, while mounted in 15-ml organ baths filled with Krebs solution at 37.5°C (11). Before starting the actual protocols, all IMA rings were left to equilibrate for 60 min, while repeatedly washed with fresh Krebs solution. Thereafter, the rings were primed by evoking initial contraction with 10 μM phenylephrine 3 times to check for viability. Each contraction to phenylephrine was followed by repeated washout and renewed stabilization. The third contraction to phenylephrine served as a control response in the different experiments. At the end of the total protocol, the condition of the preparation was tested by inducing a receptor-independent contraction to a single dose of KCl (60 mM).
α1-Antagonistic effects of Z1046
In the first set of paired experiments, α1-antagonistic effects of the new dopaminergic agonist Z1046 were studied by performing cumulative dose-response curves with the α1-selective agonist phenylephrine (dosages, 0.01 μM-1 mM, half decades) in the presence of increasing concentrations of Z1046 (0, 0.3, 1, or 3 μM). From these dose-response relations, the pA2 value (the concentration inducing 50% of the maximal antagonistic effect) of Z1046 was calculated by means of a Schild plot.
Contractile effects of dopaminergic agonists
In a second set of experiments, the concomitant contractile effects of dopaminergic agonists were studied, by performing cumulative dose-response relations to baseline tension, with dopamine, epinine, and Z1046 (dosages, 0.01 μM-0.1 mM in half decades). A single high dose of Z1046 (0.1 mM) was administered on top of the final concentration of dopamine and epinine.
Dopaminergic vasodilating effects
In the third set of paired experiments, dopaminergic effects of Z1046, dopamine, and epinine were determined, according to the same protocol that was previously used by Ferlenga et al. (12). This was to demonstrate direct dopaminergic effects in animal preparations. First, levo-propranolol (1 μM) and indomethacin (10 μM) were added to the organ baths to exclude possible interfering β-adrenoceptor and prostanoid mediated effects, and were left to incubate for 45 min (both compounds remained present throughout the protocol). Then α1-adrenergic effects were excluded by treating the artery rings with the irreversible α1-antagonist phenoxybenzamine (30 μM), which was left to incubate for 30 min. This second incubation period was followed by repeated washout (3 times within 15 min). Finally, the artery rings were treated with IBMX (10 μM) to inhibit cyclic adenosine monophosphate (cAMP) breakdown, and 30 min later, a contraction was induced with the stable thromboxane A2 mimetic, U46619, in a submaximal dose (0.1 μM). After obtaining stable precontractions, dose-dependent relaxations were made to dopamine, epinine, and Z1046 (0.01 μM-0.1 mM, half decades). Control artery rings singly treated with U46619 were included to test stability of the contraction.
In the fourth set of experiments, the selectivity of Z1046 for D1-like receptors was checked by performing cumulative dose-response relaxation curves with Z1046 (0.01 μM-0.1 mM, half decades) in the presence of increasing concentrations of SCH23390 (0.01, 0.1, and 1.0 nM). This protocol was preceded by the same incubation protocol described earlier to exclude possible interfering receptor stimulation. The data obtained in this set were used to calculate the pA2 value of SCH23390 by means of a Schild plot.
Both epinine (N-methyldopamine) and Z1046 (i.e., ((S)-6-[[6-[[2-(2-methoxy phenoxy) ethyl]amino]propylamino]-5,6,7,8-tetrahydro-1,2 naphthalenediol] dihydrochloride)) were supplied by the ZAMBON Group SpA, Milan, Italy. Dopamine, phenylephrine, indomethacin, IBMX (3-isobutyl-1-methylxanthine), and U46619 (9,11-dideoxy,9a-11a-epoxymethano-PGF2α) were all purchased from Sigma, and Levo-propranolol was purchased from ICN Biomedicals. Phenoxybenzamine hydrochloride and SCH23390 were purchased from Research Biochemical Incorporated.
All drugs were dissolved in distilled water, except for the following drugs: IBMX (dissolved in 10% dimethylsulfoxide aqueous solution); indomethacin [dissolved with a stoichiometric amount of NaOH (0.1 M)]; and U46619, which was dissolved in freshly prepared sodium bicarbonate solution (2 mg/ml).
Data are presented as mean ± SEM, and are expressed as percentage of phenylephrine control response or U46619 precontraction. Differences between maximal dose of epinine and dopamine with or without a single dose of Z1046, were tested with paired t test. Comparisons between time controls and dopaminergic receptor-mediated relaxation (of a U46619 precontraction) by Z1046, epinine, and dopamine were tested by a one-way analysis of variance for repeated measures. A value of p < 0.05 was considered statistically significant.
All control responses to phenylephrine were comparable between the artery rings (average response, 291 ± 19 μm). All artery rings showed adequate post hoc contraction to KCl (average response, 319 ± 30 μm).
α1-Antagonistic effects of Z1046
Increasing doses of Z1046 induced a rightward shift of the dose-response curve to the selective α1-adrenoceptor agonist phenylephrine (Fig. 1; n = 7, 28-artery rings), implying competitive antagonism between phenylephrine and Z1046 for the α1-adrenoceptor. The Schild plot analysis of Z1046 resulted in a pA2 value of 6.6; and the slope of the plot was 1.4 (Fig. 1, inset).
Contractile effects of dopaminergic agonists
Epinine caused a potent contraction of the artery rings under baseline conditions. Especially at higher dosages, the contractions to epinine were of the same magnitude as those of the control contractions to the selective α1-agonist phenylephrine. Dopamine also induced contraction, although to lesser extent. In contrast, Z1046 had no effect on the artery rings at baseline, either at lower or at higher dosages. The single high-dose Z1046 significantly reduced the contractions to epinine and dopamine (p < 0.05; Fig. 2, n = 6, 18-artery rings).
Dopaminergic vasodilating effects
Thromboxane precontracted artery rings showed significant relaxations to both epinine and Z1046 (to maximal 35% of the thromboxane precontraction; Fig. 3; n = 6, 24-artery rings). Dopamine also induced significant relaxations; however, this was less pronounced (to maximal 24% of the thromboxane precontraction). Contractions in untreated time controls remained stable.
The increasing concentrations SCH23390 induced a rightward shift of the dose-dependent relaxation to Z1046 (Fig. 4; n = 9, 24-artery rings), thereby implying competitive antagonism between Z1046 and SCH23390 for the D1-like receptor. The Schild plot analysis was calculated from the EC90 values, derived from the fitted curves. This resulted in pA2 value for SCH23390 of 13.1 and a slope of 0.8 (Fig. 4, inset). (A Schild plot on the EC50 values derived from the fitted curves resulted in a comparable pA2).
This study characterizes both the D1-like and α1-receptor mediated effects in human IMA of the relatively new dopaminergic agonist Z1046, and of the known dopaminergic agonists epinine and dopamine. The main conclusions are (a) Z1046 induces a rightward shift of the selective α1-agonist phenylephrine, thereby indicating that it is a competitive antagonist to this receptor; (b) Z1046 did not induce contraction of the artery rings at higher dosages, this while epinine induced a dose-dependent vasoconstriction at higher dosages that was even more pronounced than that of dopamine; (c) Single-dose Z1046 significantly reduced the contraction to epinine and dopamine, thereby indicating that this contraction was mediated by α1-receptor stimulation; (d) Epinine and Z1046 equally effectively induced a significant D1-like receptor-mediated relaxation. This relaxation was more pronounced than that of dopamine.
By itself, direct vasodilation by combined α1-receptor inhibition and D1-like receptor stimulation may eventually have adverse effects, because it is accompanied by reflex sympathetic activation, which is known to induce cardiovascular hypertrophy (13). However, because Z1046 not only induces vasodilation but also inhibits reflex sympathetic activation through stimulation of D2-like receptors [shown by Ferlenga et al. (12,14)], this adverse effect may not occur. In fact, a previous study from our laboratory demonstrated in infarcted rats that prolonged treatment with Z1046 was not accompanied by reflex sympathetic activation (15). In humans, such inhibition of reflex sympathetic activation after longterm treatment with Z1046 should be confirmed; however, because inhibitory presynaptic D2-like receptors are present at the sympathetic nerve endings (16,17), inhibition of reflex norepinephrine outflow can be anticipated.
There is increasing evidence for a functional peripheral dopaminergic system in several species including humans (18-22). The evidence also is still increasing that this system can be applied to reduce progression of heart failure. First, this dopaminergic system can be applied to regulate vascular tone and thereby reduce total peripheral resistance. Although in our study, only large conduit arteries were used, the data presented here further support the concept that the total peripheral resistance can be reduced, because the effects of dopaminergic agonists were found to be even more pronounced in smaller resistance arteries (23,24). Second, as stated before, there is increasing evidence that presynaptic inhibitory receptors regulate the sympathetic output, and third, renal blood flow and natriuresis are improved after dopaminergic receptor stimulation (25,26). Together these effects may reduce the progression of heart failure.
These data once again emphasize that, in contrast to Z1046, epinine induces vasoconstriction at high dosages, mediated by α1-receptors. Although the in vitro dosages that induced this contraction in our study were quite high, it is not unlikely that in patients with severe heart failure, locally these high concentrations are reached, particularly because the metabolism is reduced in these severe heart failure patients. The finding that the increased mortality in the PRIME-II study was clustered in patients with more severe heart failure seems to support this hypothesis. Consequently, the disappointing results of the PRIME II study may relate to α- or β-receptor stimulation. Study of plasma epinine levels in PRIME II patients, together with analysis of possible beneficial effects of concomitant treatment, could verify this hypothesis. The results of the PRIME-II study should generate caution toward the dopaminergic concept, but cannot be used for full rejection of the dopaminergic concept.
In conclusion, the new mixed dopaminergic agonist Z1046 combines direct anti-α-adrenergic effects with direct dopaminergic receptor-mediated vasodilation. Other studies also revealed effective inhibition of reflex sympathetic activation. Therefore Z1046 appears to have a more favorable profile for clinical use than does ibopamine. Because prognosis of patients with heart failure could be further improved, and use of ibopamine in advanced (but not severe) heart failure seems promising, new dopaminergic agonists such as Z1046, with different receptor profiles than ibopamine, deserve further study.
Acknowledgment: We are grateful to the surgeons of the Thorax Centre of the University Hospital Groningen, for supplying tissue for use in the in vitro studies. We thank Drs. Inge Zuhorn for her assistance, and Drs. Claudio Semeraro and Francesco Marchini (Zambon group S.p.A., Milan, Italy) for constructive remarks on the design of the study.
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