Carvedilol, a novel, multiple-action neurohormonal antagonist (Fig. 1), has been shown to have greater cardioprotective efficacy over other β-blockers in animal models of cardiac ischemia (1). Carvedilol also has been shown to scavenge oxygen free radicals and inhibit lipid peroxidation in swine heart and rat brain membranes, and we have found a potency two orders of magnitude greater than that seen for other β-blockers (2,3). The capacity of carvedilol to scavenge oxygen free radicals in both lipid and aqueous environments has been confirmed using electron spin traps to generate EPR spectra (3,4). Carvedilol produced a concentration-dependent decrease in the intensity of the EPR signals formed in the presence of a cell-free oxygen radical-generating system; other β-blockers were ineffective. Similarly, carvedilol has prevented Fe2+/vitamin C-induced depletion of α-to-copherol from brain homogenates, whereas other β-blockers did not (3). A solid correlation between median inhibitory concentration (IC50) values for lipid peroxidation and their oxidation potentials showed that these compounds are effective reducing agents, with the tendency to donate electrons and nullify toxic oxygen free radicals.
Metoprolol (Fig. 1) is a β1-selective adrenoceptor antagonist, whereas carvedilol is an α1 and nonselective β-adrenoceptor antagonist. We had noted superior cardioprotection by carvedilol in comparison with metoprolol in rabbit ischemia and reperfusion models (5). In that study, at doses giving comparable decreases in pressure-rate indices, carvedilol provided superior cardioprotection (smaller infarct size), and reduced neutrophil accumulation into ischemic myocardium, which metoprolol did not (5). Similarly, in isolated perfused rabbit heart, carvedilol was significantly more effective than metoprolol in protecting systolic function after ischemia, and in reducing postischemic contracture (6). In examinations of hypertensive patients given either metoprolol or carvedilol as monotherapy for 4 weeks, carvedilol induced a more favorable hemodynamic profile, not altering cardiac output but decreasing systemic vascular resistance (7). Furthermore, in patients with dilated cardiomyopathy that was unresponsive to metoprolol, carvedilol decreased left ventricular (LV) dimensions, improved LV ejection fraction, and decreased ventricular arrhythmias after 12 months (8).
When carvedilol and metoprolol were recently compared in clinical trials for heart failure, each showed beneficial β-blocker effects such as improved symptoms, quality of life, exercise tolerance, and LV ejection fraction, with no between-group differences (9,10). When thiobarbituric acid-reactive substance (TBARS) levels were measured in plasma as an indirect marker of free radical activity, there also were no between-group differences (10). However, as little as a week's dosing with 3.125 mg b.i.d. of carvedilol was recently found to decrease the generation of reactive oxygen species significantly by polymorphonuclear leukocytes and mononuclear cells (11). Because many previous studies had indicated additional benefits of carvedilol such as savings in myocardial oxygen consumption, or antioxidant and antineutrophil effects, we decided to explore this differentiation further. We therefore examined the antioxidant activity of carvedilol directly in cells and tissues exposed to oxidative stress. The data showed that carvedilol was a potent antioxidant, whereas metoprolol was devoid of similar activity.
Neuron culture and toxicity assays
Primary cultures of rat cerebellar neurons were prepared from 8-day-old Sprague-Dawley rat pups (Taconic Farms, Germantown, NY, U.S.A.) and used after 8 or 9 days in culture (12,13) by washing and incubating in a buffer composed of (mM): NaCl, 154; KCl, 5.6; CaCl2, 2.3; MgCl2, 1.0; glucose, 5.6; and HEPES, 8.6; adjusted to pH 7.4 with NaOH. For free radical-mediated toxicity, granule cell death is dependent on the cellular energy state and occurs in buffer deprived of glucose (12,13). For free radical-mediated toxicity, neurons grown in 35-mm dishes were deprived of glucose for 40 min and exposed for 20 min to the hydroxyl radical (OH•)-generating system, DHF-Fe3+/ADP, with final concentrations of 0.83 mM dihydroxyfumarate, 0.025 mM FeCl3, and 0.25 mM adenosine diphosphate (12,13). After challenge, the OH•-generating system was replaced with fresh, glucose-free buffer, and cell death was quantified after 50-60 min by visually counting neurons stained with the vital dye, fluorescein diacetate. Results are expressed as percentage viability of untreated neurons. To prevent free radical-mediated toxicity, compounds were added to neurons from concentrated stocks in dimethylsulfoxide (DMSO), 15 min before the oxidative stress. Equivalent amounts of the DMSO vehicle had no protective effect in this system.
Rat ventricular membrane preparations were prepared as reported previously (2). In brief, rat ventricular tissues were homogenized with a Polytron homogenizer in cold saline, filtered through four layers of cheesecloth, centrifuged at 1,000 g for 15 min at 4°C, and the pellet was discarded. The supernatant was recentrifuged at 48,000 g, and the pellet was collected, resuspended in saline, and used. The rat brain homogenate was made in saline containing 10 mg tissue/ml, as previously described (3).
Measurement of lipid peroxidation
Lipid peroxidation was measured by the formation of TBARS. Rat ventricular membrane preparations or rat brain homogenates (1 ml) were preincubated at 15°C with 10 μl of vehicle or a test compound at the indicated concentrations. Lipid peroxidation was initiated by the addition of 0.1 ml of 25 mM FeCl2 and 1 mM ascorbic acid (14). After 30 min of incubation at 37°C, the reaction was stopped by adding 0.1 ml of 0.2% butylated hydroxytoluene. TBA reagent was then added, and the mixture was heated for 30 min in a boiling water bath. The TBARS was extracted by n-butanol, and absorbance was measured at 532 nm. Basal peroxidation of tissues was between 6% (brain) and 12% (heart) of that stimulated by Fe2+/ascorbate. Basal TBARS was subtracted from that generated by Fe2+/ascorbate, and the data presented as percentage inhibition of TBA formation.
The determination of a Calculated logP value for a compound allows an estimation of the octanol/water partition co-efficient to determine the degree of lipophilicity. Therefore, it can determine the extent to which a molecule will intercalate or interact with biologic membranes or model lipid bilayers. We determined ClogP values using the ClogP Program, version 4.51, distributed by Daylight Chemical Information Systems, Inc. (Irvine, CA, U.S.A.) and developed by the Pomona College Medicinal Chemistry Project, which has validated this approach for thousands of compounds.
Statistical analysis and curve fitting
Data from all protection assays were analyzed by nonlinear regression analysis and curve-fitting, using GraphPad Prism, version 3.0 (GraphPad Software, San Diego, CA, U.S.A.). Previously obtained carvedilol data from refs. 12 and 13 were combined with additional carvedilol data from experiments run alongside those with metoprolol, and reanalyzed with GraphPad Prism.
Free radical toxicity
Cultured cerebellar granule cell neurons were very sensitive to the toxic action of the free radical-generating system, DHF-Fe3+/ADP, which generates superoxide anion (•O2−) and OH•(15). Carvedilol was a potent inhibitor with a PC50 (protective concentration giving 50% viability) of 5.2 μM. As shown in Fig. 2, carvedilol was much more protective than the antioxidant, vitamin E, which gave a PC50 of 72 μM. However, there was no protection noted for metoprolol (Fig. 2) at concentrations as high as 200 μM.
Free radical-mediated lipid peroxidation
We also examined the production of free radical-induced TBARS from rat heart and brain membranes, determined in the presence and absence of carvedilol or metoprolol (Fig. 3A and B). The addition of FeCl2/ascorbate to membrane preparations caused an eight-fold (heart) and 16-fold (brain) increase in TBARS. As shown, we found that only carvedilol protected from this lipid peroxidation, with an IC50 of 8 μM in heart and 7.7 μM in brain. However, metoprolol was ineffective at concentrations as high as 300 μM.
In our recent study of the biophysical interactions of carvedilol with model lipid bilayers (13), we determined that the ClogP values of several dihydropyridines were correlated with relative placement within lipid bilayers, as shown by small angle x-ray diffraction studies, a biophysical technique that has proved useful for interpreting drug/membrane interactions (16). Accordingly, the ClogP value of 3.841 determined for carvedilol may correlate with the antioxidant potencies shown here. In comparison, we calculated metoprolol to have a ClogP value of only 1.346, which is much less lipophilic.
When carvedilol and metoprolol were recently compared in clinical trials for heart failure, each showed beneficial β-blocker effects such as improved symptoms, quality of life, exercise tolerance, and left ventricular ejection fraction, with no between-group differences (9,10). When TBARS levels were measured in plasma as an indirect marker of free radical activity, there also were no between-group differences (10). However, as little as a week's dosing with 3.125 mg b.i.d. of carvedilol was recently found to decrease significantly the generation of reactive oxygen species by polymorphonuclear leukocytes and mononuclear cells (11). Because many previous studies had indicated additional benefits of carvedilol such as savings in myocardial oxygen consumption, or antioxidant and antineutrophil effects, we examined the antioxidant activity of carvedilol and metoprolol directly in cells and tissues exposed to oxidative stress. Comparing the efficacy of carvedilol with that of metoprolol within the three oxidative stress-generating systems studied here, it is apparent that carvedilol is the superior antioxidant. This may be a result of molecular positioning of this compound within the lipid bilayer (13,17), or a result of the lower redox potential of carvedilol, which will give it the tendency to donate electrons more readily to "scavenge" the activities of oxygen free radicals (3). The carbazole ring structure of carvedilol is much different from the phenoxy ring of metoprolol, which seems unlikely to undergo the redox recycling that has been determined for carvedilol (3,17). These findings may be clinically relevant because the carvedilol metabolite, SB 211475, is found in humans (unpublished data) and is a very protective compound in the model systems used here (13,18). In addition, SB 211475 has been shown to be an exceptionally potent antioxidant in a postischemic rat heart model, where its protective action at very low doses was thought to be mediated primarily through a lipid peroxidation chain-breaking mechanism rather than by direct radical scavenging (19). Furthermore, in atrial myocardial preparations, carvedilol and its metabolite effectively inhibited hydroxyl radical-induced contractile dysfunction, whereas metoprolol was ineffective (20). It is possible that the relative efficacy of metoprolol in reducing TBARS in clinical studies of congestive heart failure might be due to further metabolism of this compound, but an antioxidant effect of the major metabolite, α-hxdroxymetoprolol, has not been reported.
Carvedilol is a lipophilic compound that is extensively bound to tissues, achieving a high distribution volume of 132 L/kg in humans (21). It is likely that this lipophilic membrane sequestration results in the protective effects shown here, which may augment the pharmacologic utilities of this class of compounds. It is known that many lipophilic drugs such as probucol, as well as vitamin E, prevent peroxidative damage to membranes through membrane-active physical effects that supplement chemical activities (22,23), as we have recently shown for carvedilol (13). In that study, comparison of carvedilol and SB 211475 with several dihydropyridines showed a correlation between ClogP values (lipophilicity) and the degree of mobilization and localization of compounds within both model lipid and brain membrane bilayers. The antioxidant activity of SB 211475 could be explained by restricted intercalation into the glycerol phosphate/hydrocarbon interface, creating an increase in volume associated with the phospholipid acyl chains, which would then become resistant to lipid peroxidation (13). On the basis of the ClogP determinations here, metoprolol is 300-fold less lipophilic than carvedilol, which would limit its interaction within biologic membranes. A comparison of the volume of distribution within humans shows that metoprolol (24) has only about one-tenth the volume of distribution of carvedilol (20). Therefore, the degree of lipophilicity evidenced by these amphipathic molecules may determine, to a certain extent, their relative effectiveness as antioxidants.
Therefore, in addition to its ability to offer protection from myocardial ischemia by virtue of β-blockade and vasodilation, carvedilol may have additional therapeutic value to reduce risk by free radical scavenging. This benefit may be important for long-term therapy (25).
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