Interactions Between Organic Nitrates and Thiol Groups JONATHAN ABRAMS, M.D., Albuquerque,New Mexico
Nitroglycerin and the organic nitrates (RONO2) can be considered prodrugs that require conversion to an active intracellular moiety that initiates vascular smooth muscle relaxation. Vasodilation of veins and arteries occurs when the enzyme guanylate cyclase (GC) is activated, initiating the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP); this is the final pathway for vascular dilation caused by the nitrovasodilators (organic nitrates, sodium nitroprusside, and molsidomine) as well as endothelium-derived relaxing factor (EDRF). The common denominator appears to be the intracellular production of nitric oxide (NO), which is the activated product of organic nitrate denitration. Nitrate tolerance has been associated with a relative depletion or unavailability of thiol groups that are involved in the initial step of denitration of RONO2. Sulfhydryl groups (SH) are oxidized during this process; with continuous nitrate exposure, decreased nitrate metabolism within the vascular smooth muscle cell occurs as a direct result of the depletion of reduced SH groups. Thus, less NO is formed and cGMP production is diminished, with a subsequent decrease or absence of vasodilation. In addition, SH groups or thiols are required for the production of S-nitrosothiols (RSNO). These short-lived compounds have been identified as an end product of organic nitrate metabolism and as possibly obligatory for the induction of GC. It is unclear, however, as to whether S-nitrosothiols are a necessary byproduct of NO production from organic nitrates. It appears that RSNO can be formed outside the cell membrane and may be able to induce vasorelaxation after penetrating the cell and initiating GC activation.
From the Department of Medicine, School of Medicine, The University of New Mexico, Albuquerque, New Mexico. Requests for reprints should be addressedto Jonathan Abrams, M.D., Department of Medicine, School of Medicine,The Universityof New Mexico,Albuquerque, New Mexico 87131-5271, U.S.A.
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Exogenous SH donors, particularly N-acetylcysteine (NAC), have been employed to provide intracellular thiols in efforts to prevent or reverse nitrate tolerance. Nitrate physiologic actions are accentuated following NAC administration in the absence of tolerance. Although controversial, the concept that NAC or other thiols might be able to prevent the development of nitrate tolerance is being actively studied in laboratories around the world. Methionine has also been utilized as an SH donor with some success. Not all data are consistent, however, and the ultimate role of thiol donors for the prevention or reversal of nitrate tolerance remains uncertain. Finally, there has been considerable interest in supplying thiols by use of the SH-containing angiotensin converting enzyme inhibitors, such as captopril. This approach does not seem promising, probably because insufficient thiol can be supplied by therapeutic dosages of these drugs. between organic nitrates and thiols Itionsnteractions have been widely reported after the observaof Needleman and Johnson [1,2] showing that nitrate intracellular action is dependent on sulfhydryl groups (SH). Studies have been carried out using thiol donors in isolated vascular strips, in anireals, and in humans to understand the complexities of the attenuation of nitrate action (nitrate tolerance). This review discusses nitrate metabolism and actions in the nontolerant and tolerant states with a focus on the role of thiols. Nonetheless, many issues remain unresolved. Before describing the role of thiols in intracellular nitrate metabolism, as well as discussing potential agents for prevention of tolerance, a brief review of nitrate actions and clinical indications is in order.
ORGANIC NITRATES These compounds have been a mainstay of cardiovascular medicine since the recognition [3] that nitroglycerin (NTG) is a useful vasodilator in relieving angina pectoris attacks. Nitroglycerin, isosorbide dinitrate (ISDN), and isosorbide-5-mononitrate (5-ISMN), the major metabolite of ISDN, have physiologic potency in humans. The nitrates
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are versatile compounds and are available in a large number of formulations or delivery systems (Table I). Nitroglycerin has been the major formulation that has been studied in in vitro and in vivo investigations of thiol-nitrate interactions. Sodium nitroprusside and molsidomine are also vasodilators. They cause smooth muscle relaxation, but differ from the organic nitrates in their metabolism and that there is no tolerance problem with these compounds.
MECHANISMS OF ACTION Veins and arteries dilate after nitrate administration (Table II). The smaller resistance vessels or arterioles do not relax to conventional doses of hitrates, but do so at high concentrations. Venodilation is pronounced, even with low doses, and is a unique and significant component of nitrovasodilator action in humans. The combination of venous and arterial dilation results in a decrease in cardiac output. The heart becomes smaller as systolic and intraventricular filling pressures diminish after nitrate administration. The circulating blood volume is redistributed, with pooling or sequestration of blood in the splanchnic and mesenteric circulations and less blood returning to the heart. In the presence of depressed left ventricular function and/or congestive heart failure, the arterial dilating effects of the nitrates result in an increase in stroke volume and cardiac output (unloading effect), making these drugs useful as adjunctive therapy in heart failure [4]. While the peripheral circulatory actions of the nitrates, venous and arterial vasodilation, are important, it is clear that the central or coronary arterial actions of the nitrates are also critical to many patients with coronary artery disease. Nitrates can enhance global or regional nutrient coronary blood flow by a number of mechanisms, and these actions are important in the major myocardial ischemic syndromes. Prevention or reversal of coronary vasoconstriction is useful in patients with mixed angina. Prevention of coronary stenosis constriction and/or overt enlargement of a critical coronary narrowing may be an important mechanism for the relief or prevention of chest pain in some angina subjects [5]. It is not known how much the peripheral vs. central actions of the organic nitrates contribute to the relief or prevention of myocardial ischemia in a patient. ANTIPLATELET ACTIVITY OF NITRATES It is known that nitrates have antiaggregatory effects on platelet function in vitro; however, in
TABLEI AvailableNitrateFormulations Nitroglycerin /sosorbide dinitrate 5-1sosorbidemononitrate
SL, spray, orat-SR,buccal, topical, iV SL, spray, chewable, oral, oraI-SR,topical, IV Oral, oraI-SR
SL = sublingual; SR = sustained release; IV = intravenous; topical = ointment, patch, impregnated synthetic membrane.
TABLE II Beneficial Effects of Nitrates: Potential Mechanisms of Action Peripheralor SystemicActions Venous dilation Arterial dilation Arterial dilation (high doses) Central coronary actions Coronary artery vasodilation Prevention/reversal of coronary artery vasoconstriction Coronary stenosis dilation Enhanced collateral flow Endothelial function Nitrates converted to nitric oxide within vascular smooth muscle cell Antiplatelet actions Decreased platelet aggregation and adhesion (controversial)
Effects Smaller right and left cardiac volumes; lower right and left heart filling pressures Decreased afterload; reduced arterial reflectance wave; decreased systolic blood pressure Decreased systemic vascular resistance; decreased afterload All: Increased global and/or regional coronary blood flow, especially during ischemia
Vasodilation and antiplatelet action enhanced in the absence of normal endothelial function Potential antithrombotic benefit in unstable angina and possibly useful for circadian increase in platelet activation in stable angina
early studies pharmacologic doses were used to achieve these effects. A number of recent experiments [6,7] suggest that nitrates have potent antiplatelet activity in animals and humans at conventional doses and that these actions may be important in acute myocardial ischemic syndromes, an area that remains controversial. Nitrate tolerance decreases [8] the platelet response to administered nitrate. Thiol donors have been used with some success [9,10] in enhancing the platelet antiaggregatory action of the organic nitrates. A SH donor-nitrate interaction appears to be a promising approach for future studies into the platelet effects of the organic nitrates.
CLINICAL INDICATIONS FOR THE ORGANIC NITRATES NTG and the other nitrate compounds are effective in a variety of important cardiovascular conditions, with nitrates playing a significant role in the coronary artery syndromes. Use of these drugs in patients with congestive heart failure or impaired left ventricular function in the asymptomatic postinfarction subject is another emerging indication.
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TABLE III Dosing Factors FavoringDevelopmentof Nitrate Tolerance Frequentdoses Largedoses Sustained-releaseformulations Continuousdeliverysystems(IV,patch,ointment) Absentor briefnitrate-freeinterval
Myocardial Ischemia ACUTE EPISODE OF ANGINA PECTORIS:
Rapid-
acting nitrates remain the 'gold standard' for treatment of acute myocardial ischemia (MI). Sublingual (NTG, ISDN), chewable (ISDN), oral spray (NTG, ISDN), and buccal or transmucosal NTG are available as rapidly acting formulations. PROPHYLAXIS
FOR STABLE ANGINA PECTORIS:
Long-acting nitrates decrease angina attacks and improve exercise performance in patients with chronic stable angina. Many of these patients have a variable angina threshold and/or occasional episodes of rest pain (mixed angina pectoris) and these clinical clues demand consideration of nitrate therapy. The nitrates are equally as effective as calcium channel blockers or/3-adrenergic antagonists in the therapy of chronic angina pectoris, and are used in double and triple therapy in more symptomatic or refractory patients. UNSTABLE ANGINA PECTORIS: Intravenous NTG is an extremely useful antianginal agent in unstable angina therapy, and is commonly followed by oral or topical nitrate treatment after the acute syndrome subsides. Combination therapy with calcium channel blockers and/or/3-blockers is useful in this unpredictable syndrome of acute myocardial ischemia. ACUTE MYOCARDIAL INFARCTION: A number of trials suggest a benefit when intravenous NTG is infused for 24-72 hours in subjects with acute myocardial infarction. A decrease in morbidity has been observed, and data from these studies [11] suggest a reduction in hospital mortality. A study [12] of more than 300 patients has shown a decrease in mortality and the incidence of major complications in transmural myocardial infarction (anterior) when an intravenous NTG infusion was given for 48 hours. The NTG-treated patients showed a decrease in adverse left ventricular remodeling in the postinfarction period. This work has been replicated by the same author [13] in an animal model and in a clinical study [14] using buccal NTG for 6 weeks, after the hospitalization period, in patients initially treated with intravenous NTG. Nitrates may be protective against remodeling after transmural myocardial infarction. Two major trials currently under way, GISSI-3 and ISIS-4, will test 3C-108S
this hypothesis. The angiotensin converting enzyme (ACE) inhibitors have been widely used in the prevention of post-MI ventricular remodeling [15] and the data are promising. Congestive Heart Failure Nitrates have been widely used as vasodilator therapy for heart failure. Short-term data [4,16,17] show improvement in cardiac hemodynamics, with a fall in right and left heart filling pressures and an increase in cardiac output. Some data [16,17] suggest an improvement in exercise performance in chronic failure. In the Veterans Administration study [18], the combination of oral ISDN and hydralazine gave a 20-25% reduction in mortality in class I I - I I ! heart failure patients compared with placebo. Data in the literature suggest that nitrate therapy in heart failure reduces signs and symptoms of this clinical syndrome and may be cardioprotective. Nitrates have a similar hemodynamic profile as the ACE inhibitors in congestive heart failure. These drugs are a useful adjunctive modality in the treatmerit of heart failure not easily controlled with diuretics and digitalis. It is likely that the combination of a nitrate and an ACE inhibitor would be more beneficial. NITRATE TOLERANCE The major limitation for the effective utilization of nitrates is in its attenuation or tolerance. When nitrates are administered in a protolerant fashion (Table III), their effects decrease in effect over time. In some studies, complete tolerance to the desired nitrate action is evident. Despite the history of nitrate tolerance being long and controversial, all experts now agree that tolerance is the critical factor that adversely affects nitrate efficacy. Attenuation of nitrate action may be Seen after the first administered dose, especially with long-acting formulations, such as the NTG patch or sustainedrelease compounds of ISDN and 5-ISMN. Tolerance Mechanisms Two hypotheses are believed to be causally related to nitrate tolerance: (a) the thiol or SH depletion theory and (b) the neurohormonal activation/ salt and water retention theory. Both probably play a role in clinical nitrate tolerance and are being actively studied. Activation of neurohumoral systems with subsequent plasma expansion results in stimulation of the renin-angiotensin system and catecholamine release after nitrate administration. There is evidence [19,20] that these phenomena may be clinically important. Captopril has been used recently in
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ENOOTHELIUM organic nitrate (RONOz)
VASCULAR SMOOTHMUSCLE RONOz
Nitroprusside (NP}
X
-NP
.R' SH LR'SSR'
NO
S-nitrosot hiol. (RSNO}Figure1. Intracellular nitrate metabolism in relation to thiols. NO = nitric oxide, GC = guanylate cyclase, EDRF = endothelium-derived relaxing factor, GTP = guanine-5'-triphosphate, cGMP = cyclic guanosine-3',5'-monophosphate. Reproduced with permission from [67].
RSNO
\
-.~..~
GTP
Endothe[iurn -dependent-- --~EDRF vosodi[otors
IRELAXATION]
tolerance studies [21]. This potential thiol donor may function via two modes of action (ACE inhibition and SH donor) and may be effective in preventing tolerance.
The Thiol Depletion Hypothesis It has long been known [1,2] that the first step in organic nitrate denitration within the vascular smooth muscle cells requires reduced thiol groups in the form of cysteine (Figure 1). During nitrate biotransformation, there is an obligatory role for the oxidation of SH donors, as the parent nitrate molecule is converted to nitric acid and subsequently to nitric oxide (NO). Continuous nitrate administration results in the loss of reduced SH groups in the cytoplasm, and inadequate repletion of these reduced thiols decreases nitrate action, and slows vascular nitrate uptake. This is the basic rationale underlying efforts to supply thiol donors that provide reduced SH or cysteine moieties to the smooth muscle cells. In a subsequent step of nitrate metabolism, SH groups also help to form S-nitrosothiols (SNO), short-lived intermediate compounds activating guanylate cyclase [22,23] (Figure 1). It was believed [23] that the nitrosothiols were formed after NO production, but evidence suggests that organic nitrates can be directly converted to NO or SNO, both capable of activating guanylate cyclase [23,24] (Figure 1). Nitroprusside and molsidomine directly form NO and are not reliant on reduced SH availability; tolerance is not found with these compounds.
Nitrate Metabolism The organic nitrates act at the level of the plasma membrane in vascular smooth muscle cells, where they are converted to NO and SN0 [23,24], which activate guanylate cyclase. This enzyme, present in soluble cytoplasm, catalyzes the conversion of guanosine triphosphate to cyclic guanosine-3',5'monophosphate (cGMP), the final metabolic step leading to vascular smooth muscle relaxation, and is associated with a decrease in intracellular calcium and phosphorylation of protein kinase [25]. It is believed [19,20,22,24,26] that the production of SNO may also occur outside the cell when exogenous thiol is provided (Figure 1). The nitrosothiol may penetrate the cell membrane and activate guanylate cyclase with simultaneous intracellular production of NO from denitration of the parent nitrate. Both compounds stimulate guanylate cyclase. Since tolerance predictably appears when sufficient nitrate exposure has occurred, it is presumed that intracellular metabolic conversion of R SN02 to NO is the critical step in the production of nitrate tolerance (Figure 1). Studies [22,27] attempting to show that guanylate cyclase or guanylate cyclase-cGMP interaction is the actual locus of tolerance have been unsuccessful. Guanylate cyclase can respond to NO or nitroprusside in the presence of established nitrate tolerance [28], indicating that the site of tolerance is "proximal" to NO production [22,29].
Nitrate.Free Interval In vitro animal and human studies [30,31] have demonstrated that a sufficient duration of freedom
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from organic nitrate exposure allows vascular smooth muscle to remain fully responsive to administered nitrate. A nitrate-free interval of variable duration restores the desired nitrate response. The duration of this interval depends on the nitrate concentration and the length of pre-exposure to the administered drug. The current clinical strategy is to administer organic nitrates to provide intermittent dosing of nitrate with a predetermined nitratefree period. These dosing regimens vary with the type of formulation; 8-12 hours from the last nitrate dose is necessary to maintain vascular responsiveness.
rate guanylate cyclase with subsequent cGMP production [2,8,24,32-40,43-52]. Methionine, a thiol that is converted to cysteine within the cell [20], has been effective in preventing or reversing tolerance in preliminary experiments [35-37]. New data [38,39] suggest that other thiol donors may be identified that are more potent than NAC itself. These experiments are not conclusive with respect to a useful clinical role for other thiols, although preliminary data suggest enhanced biological effects for some thiol compounds in the presence of nitrates [38,39]
Negative Studies Concept of the Sulfhydryl Donor Providing thiol donors in the form of exogenous SH groups for tolerance prevention was first carried out by Needleman et al [1,2]. Recently, N-acetylcysteine (NAC), a reduced SH compound, has been administered with organic nitrates. Studies have focused on the reversal of established nitrate tolerance with NAC administration, or using simultaneous nitrate and NAC to prevent the development of tolerance or enhancement of NTG action with coadministration of NAC or other thiols [9,10,32-36]. The first published study [32] showed heightened NTG hemodynamic action in subjects given intravenous NTG before and after an infusion of NAC. NAC has since been used as a thiol donor in the presence of nitrate tolerance. Other work [33,34] suggests that NAC potentiates the vasodilatory activity of nitrates in nontolerant blood vessels. Preliminary studies [9,10] indicate that NAC enhances the antiplatelet activity of NTG through the formation of NAC-nitrosothiols. NTG effects are increased severalfold with co-administered NAC [26,32-34], suggesting that additional guanylate cyclase is activated by thiol donors [26] although some authors [20] suggest that this is a nonspecific effect. This interaction may not be due to increased intracellular denitration of R-SN02, through the availability of more reduced SH groups, resulting in NO production, but rather due to enhanced extracellular NAC-nitrosothiol formation and subsequent direct stimulation of guanylate cyclase and enhanced cGMP production [20,24,26] (Figure 1). Reversal of Nitrate Tolerance with Thiol Donors Many studies have shown that established nitrate tolerance can be partially reversed by the addition of NAC in oral or in intravenous (IV) formulation. A number of positive studies have been published showing that exogenous thiol donors may provide additional NO and/or SNOs that can acti3C-110S
Many thiol donor studies have been unsuccessful in overcoming established nitrate tolerance [19,20,26,39,40,53-62]. It is unclear why some studies produce negative results while others are promising [36,37]. Differences in experimental design, variable vascular tissue sensitivity to thiols in different vascular beds, differences among animal species, differing NAC and methionine doses and concentrations, different duration of exposure to NTG (and other nitrates), and differing degrees of induced nitrate tolerance may explain the discordant results. Tolerance in patients with angina pectoris may involve different (possibly less complex) mechanisms than tolerance in congestive heart failure. NAC or methionine may have a limited capacity to overcome established tolerance, particularly when it is marked [20,40]. Hutter et al [39] suggest that different thiols have differing actions on potentiation of NTG action as well as reversal of tolerance.
Other Thiol Donors METHIONINE: Methionine is converted to cysteine within smooth muscle cells and is a possible thiol donor [35-37,40]. It prevented or reversed nitrate tolerance in human volunteers when the venous vasodilation response to NTG was evaluated [35,36,40]. Since methionine does not form an extravascular nitrosothiol, it is unclear if this agent will be as effective as NAC [20,40]. CAPTOPRIL: There has been interest in the SH group on the captopril molecule. While preliminary results [21] suggested a positive effect of captopril in preventing nitrate tolerance when compared with a nonsulfhydryl ACE inhibitor, a subsequent report from the same group [41] has been unable to demonstrate a difference between captopril and enalapril. Both drugs seem to have some antitolerance activity, perhaps by interfering with activation of the renin-angiotensin system. In an in vitro model, using aortic vascular rings, captopril prevented tolerance whereas enalapril did not [42].
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Discrepancies in these studies may be due to the differences in experimental design, amount of ACE inhibitor administered, and possible direct coron a g vasodilatory actions of captopril. Chong and Fung [38] have shown that captopril is a poor thiol donor when compared with NAC. Other reports [41,42,63-66] support a physiologic rationale for ACE inhibitors, diuretics, or hydralazine in enhancing nitrate action and preventing or reversing tolerance. It is unlikely that the clinical doses of captopril or other ACE inhibitors with a SH group will provide sufficient quantities of SH groups to be useful as thiol donors in tolerance prevention Or reversal. However, the role of ACE inhibition in preventing or reversing tolerance is controversial and needs further study.
Unresolved Issues Recent reports using thiol donors and the organic nitrates support the potential of this drug interaction. Data are conflicting as to the efficacy of NAC and methionine. Many positive [2,8,24,32-40,4352] as well as negative [19,26,39,40,53-62] studies appear in the literature. In some studies, thiol donors are both beneficial and ineffective, tt may be that NAC or methionine as thiol donors have insufficient potency to overcome marked or complete tolerance. A number of experimental methods have been used in tolerance studies and may account for some of the discrepancy. Furthermore, tolerance is multi-factorial in origin and other mechanisms could be relevant to experiments that result in renin-angiotensin activation, increased catecholamine release, or plasma volume expansion [19,20,63-66]. It remains unclear if NAC and other thiols directly reverse or prevent nitrate tolerance, or whether these compounds primarily enhance residual vasodilatory capacity in partially tolerant veins and arteries. Some studies [26] have demonstrated an increased vasodilatory capacity in partially tolerant vessels exposed to NAC. Thiols enhance the vasodilator response in the nontolerant state by severalfold, and incomplete degrees of nitrate vascular attenuation could result in important responses to administered NAC or methionine. THE FUTURE More basic and human investigative work is likely in this area. The thiol donor theory provides new insights into the mechanisms of nitrate action and the causation of nitrate tolerance itself. The possibility that tolerance can be prevented or limited with a thiol donor has yet to be proven, although some studies are promising. The goals of
enhancing nitrate activity with respect to smooth muscle responses (e.g., angina or congestive heart failure) or antiplatelet action (e.g., unstable angina and acute myocardial infarction) with a thiol donor hold promise for continuing clinical investigation.
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SYMPOSIUMON OXIDANTSAND ANTIOXIDANTS/ABRAMS 23. Ignarro LJ, Lippton H, Edwards JC, et al. Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiots as active intermediates, J Pharmacot Exp Ther 198]; 218: 739-49. 24. Fung HL, Chong S, Kowaluk E, Hough K, Kakemi M. Mechanisms for the pharmacologic interaction of organic nitrates with thiols. Existence of an extraceliular pathway for the reversal of nitrate vascular tolerance by N-acetylcysteine. J Pharmacol Exp Ther 1989; 245: 524-30, 25. Ahlner J, Axetsson KL Nitrates, Mode of action at a cellular level. Drugs 1987; 33(suppl 4): 32-8. 26. Munzel T, Holtz J, Mulsch A, Stewart DJ, Bassenge E. Nitrate tolerance in epicardial arteries or in the venous system is not reversed by acetylcysteine in vivo, but tolerance-independent interactions exist, Circulation 1989; 79: 188-97. 27, Holtz J, Munzel T, Stewart DJ, Bassenge E. Nitrate action on epicardial coronary arteries and tolerance: new aspects based on long term glyceryi trinitrate infusion in dogs. Eur Heart J 1989; 10 (suppl F): 127-33, 28. Mulsch A, Busse R, Bassenge E. Desensitization of guanylate cyclase in nitrate tolerance does not impair endothelium-dependent responses. Eur J Pharmacol 1988; 158: 191-8. 29. Feelisch M, Noack E, Schrocker H. Explanation of the discrepancy between the degree of organic nitrate decomposition, nitrite formation and guanylate cyclase stimulation. Eur Heart J 1988; 9(suppl A): 57-62. 30. DeMots H, Glasser SP. Intermittent transdermal nitroglycerin therapy in the treatment of chronic stable angina. J Am Coil Cardiol 1989; 113: 786-93. 31. Abrams J. Interval therapy to avoid nitrate tolerance: paradise regained? Am J Cardiot 1989; 64: 93i-4. 32. Horowitz LD, Antman EM, Lorell BH, Barry WH, Smith TW. Potentiation of the cardiovascular effects of nitroglycerin by N-aeetylcysteine. Circulation 1983; 687: 1247-53. 33. Bertel O, Noll G. Effects of N-acetylcysteine on nitroglycerin responsiveness before and during nitrate therapy of congestive heart failure. Eur HearL J 1987; 8(suppl i): 44. 34. Winniford MD, Kennedy PL, Wells PJ, Hillis LD. Potentiation of nitroglycerininduced coronary dilatation by N-acefylcysteine. Circulation 1986; 73: 138-42. 35. Levy WS, Katz RJ, Wasserman AG. Methionine reverses tolei'ance to transderreal nitroglyceryn, J Am Coil Cardiol 1989; 13: 230A. 36. Levy WS, Katz RJ, Ruffalo RL, Leiboff RH, Wasserman AG. Potentiation of the hemodynamic effects of administered nitroglycerin by methionine. Circulation 1988; 78: 640-5. 37. Neuberg GW, Packer M, Medina N, Yushak M, Kukin ML. Reversal of nitroglycerin tolerance in patients with chronic heart failure by oral methionine. Circulation 1989; 80 (suppl II): 11-213. 38; Chong S, [-ung HL. Biochemical and pharmacological interactions between nitroglycerin and thiols: effects of thiol structure on nitric oxide generation and tolerance reversal. Bioi:hem Pharmacol (in press). 39. Hutter J, Schmidt M, Rittler J. Effects of sulphydryl-containing compounds on nitrogtycerin-inc[uced coronary dilatation in isolated working rat hearts. Eur J Pharmacol 1988; 156: 215-22. 40. Penn J, Neuberg GW, Kukin ML, Medina N, Yushak M, Packer M. Methionine fails to restore the initial effects of nitroglycerin in tolerant patients: evidence that sulphydryl induced reversal of tolerance is mediated extracellulary. Circulation 1990; 82 (suppl Ill): 111-199. 41. Katz RJ, Levy WS, Buffalo RL, Wassermann AG. Prevention of nitrate tolerance with angiotensin-converting enzyme inhibifors. Circulation 1991; 83: 1271-77. 42. Lawson DL, Nichols WW, Mehta F, Mebta JL. Prevention of vascular tolerance to nitroglycerin by converting enzyme inhibitor (CEI) captoprih Role of CEI activity vs sulphydryl (SH) group. Circulation 1990; 82 (suppl III): 111-265. 43. Bauer JA, Fung HL Differential hemodynamic effects and tolerance properties of nitroglyceirn and an S-nitrosothiol in experimental heart failure. J Phatmacol Exp Ther 1991; 256: 249-54.
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44. Kowaluk EA, Poliszczuk R, Fung HL. Tolerance to relaxation in rat aorta: comparison of an S-nitrosothiol with nitroglycerin. Eur J Pharmacol 1987; 144: 379-83. 45. Axelssson KL, Anderson RGG, Wikberg JES. Acta Pharmacol Toxicol 1982; 50: 350-57. 46. Boesgaard S, Adeshvile J, Pederson F, Pieterson A, Madsen JK, Grande P. Circulation 1990; 82 (suppl III): 199A. 47. May DC, Popoma JJ, Black WH, Schaffer S, Lee HR, Levine BD, Hillis LD. N Engl J Med 1987; 317: 805-09. 48. Newman CM, Wassen JB, Taylor GW, Boobis AR, Davies DS. Rapid tolerance to the hypotensive effects of glyceryl trinitrate in the rat: prevention by N-acetyI-L-, but not -D-cysteine. Br J Pharmacol 1990; 99: 825. 49. Packer et al. Arch Int Pharmacol Ther 1987; 317: 799. 50. Rosen R, Konig E, Klaus W. Different sensitivities of arteries and veins to glyceryl trinitrate-induced relaxa{ion and tolerance: an 'in-vitro' study on isolated vessels from rabbits. Arch Int Pharmacol Thei 1987; 285: 777-83. 51. Torresi J, Horowitz JD, Dusting GJ. Prevention and reversal of tolerance to nitroglycerin with N-acetylcysteine. J Cardiovasc Pharmacoi 1985; 7: 777-83. 52. Tsuneyoshi H, Akatsuka N, Ohno M, Hara K, Ochiai M, Moroi M. Inhibition of development of tolerance to nitroglycerin by preventive administration of N-acetylcysteine in rats. Jpn Heart J 1989; 30: 733-41. 53. Abdallah A, Moffat JA, Armstrong PW. N-Acetylcysteine does not modify nitroglycerin-induced tolerance in canine vascular rings. J Cardiovasc Pharmaco11987; 9: 445-50. 5& Gruetter CA, Lemke SM. Dissociation of cysteine and glutathione levels from nitroglycerin-induced relaxation. Eur J Pharmacol 1985; 111: 85-95. 55. Henry PJ, Horowitz JD, Louis WJ. Determinants of in vitro nitroglycerin tolerance induction and reversal: Influence of dose regimen, nitrate-free period, and sulfhydryl supplementation. J Cardiovasc Pharmacol 1989; 14: 31-37. 56. Hogan JC, Lewis MJ, Henderson AH. N-Acetylcysteine fails to attenuate hemodynamic tolerance to glyceryl trinitrate in healthy volunteers. Br J Clin Pharrnacol 1989; 28: 421-26. 57. Kawamoto JH, Brian JF, Marks GS, Nakatsu K. Mechanism of glyceryl trinitrateinduced vasodilation. I1. Lack of evidence for specific binding of GTN to bovine pulmonary vein. J Pharmacol Exp Ther 1988; 244: 328-34. 58. Keith RA, Burkman AM. In vitro induction of nitroglycerin (NGG) tolerance in vascular tissue at pH 7.4 and 9.0 occurs prior to the decrease in sulfhydi'yl (SH) content. Fed Proc Am Soc Exp Biol 1981; 40:729 (A2849). 59. Moffat JA, Armstrong PW Marks GS. Investigations into the role of sulfhydryi groups in the mechanism of action of the nitrates. Can J Physiol Pharmacol 1982; 60: 1261-66. 60. Munzel TH, Holtz J, Mulsch A, Stewart DJ, Bassenger E. Z Kardiol 1989; 78 (suppl 2): 26-28. 61. Munzel TH, Mulsch A, Just H, Bassenger E. Cardiovascular pharmacology: basic mechanisms. J Am Coil Cardiol 1990; 15: 159A. 62. Parker JO, Farrell B, Lahey KA, Rose BE Nitrate tolerance: the lack of effect of N-acetyicysteine. Circulation 1987; 76: 572-76. 63. Muiesan ML, Boni E, Ceils G, et aL Efficacy of transdermal nitroglycerin in combination with an ACE inhibitor in patients with chronic stable angina pectoris. J Am Coil Cardiol 1991; 17 (suppl A): 337A. 64. Parker JD, Farrell B, Parker AC, Cohanim MA, Parker JO. Neurohumoral responses and the hemodynamic adaptation to nitrates. Circulation 1990; 82 (suppl III): 111-200. 65. Sussex BA, Campbell NRC, Raju MK. Nitrate tolerance is modified by diuretic treatment. Circulation 1990; 82 (suppl III): 111-200. 66. Bauer JA, Fung HL. Concurrent hydralazine administration prevents nitroglycerin-induced hemodynamic tolerance in experimental heart failure. Circulation (in press). 67. Kowaluk E, Fung HL Pharmacology and pharmacokinetics of organic nitrates. In: Abrams J, Pepine C, Thadani V, eds. Medical therapy of ischemic hear disease. Boston: Little, Brown and Co., 1991 (in press).
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Nitroglycerin and the organic nitrates (RONO2) can be considered prodrugs that require conversion to an active intracellular moiety that initiates vascular smooth muscle relaxation. Vasodilation of veins and arteries occurs when the enzyme guanylate cyclase (GC) is activated, initiating the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP); this is the final pathway for vascular dilation caused by the nitrovasodilators (organic nitrates, sodium nitroprusside, and molsidomine) as well as endothelium-derived relaxing factor (EDRF). The common denominator appears to be the intracellular production of nitric oxide (NO), which is the activated product of organic nitrate denitration. Nitrate tolerance has been associated with a relative depletion or unavailability of thiol groups that are involved in the initial step of denitration of RONO2. Sulfhydryl groups (SH) are oxidized during this process; with continuous nitrate exposure, decreased nitrate metabolism within the vascular smooth muscle cell occurs as a direct result of the depletion of reduced SH groups. Thus, less NO is formed and cGMP production is diminished, with a subsequent decrease or absence of vasodilation. In addition, SH groups or thiols are required for the production of S-nitrosothiols (RSNO). These short-lived compounds have been identified as an end product of organic nitrate metabolism and as possibly obligatory for the induction of GC. It is unclear, however, as to whether S-nitrosothiols are a necessary byproduct of NO production from organic nitrates. It appears that RSNO can be formed outside the cell membrane and may be able to induce vasorelaxation after penetrating the cell and initiating GC activation.
From the Department of Medicine, School of Medicine, The University of New Mexico, Albuquerque, New Mexico. Requests for reprints should be addressedto Jonathan Abrams, M.D., Department of Medicine, School of Medicine,The Universityof New Mexico,Albuquerque, New Mexico 87131-5271, U.S.A.
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Exogenous SH donors, particularly N-acetylcysteine (NAC), have been employed to provide intracellular thiols in efforts to prevent or reverse nitrate tolerance. Nitrate physiologic actions are accentuated following NAC administration in the absence of tolerance. Although controversial, the concept that NAC or other thiols might be able to prevent the development of nitrate tolerance is being actively studied in laboratories around the world. Methionine has also been utilized as an SH donor with some success. Not all data are consistent, however, and the ultimate role of thiol donors for the prevention or reversal of nitrate tolerance remains uncertain. Finally, there has been considerable interest in supplying thiols by use of the SH-containing angiotensin converting enzyme inhibitors, such as captopril. This approach does not seem promising, probably because insufficient thiol can be supplied by therapeutic dosages of these drugs. between organic nitrates and thiols Itionsnteractions have been widely reported after the observaof Needleman and Johnson [1,2] showing that nitrate intracellular action is dependent on sulfhydryl groups (SH). Studies have been carried out using thiol donors in isolated vascular strips, in anireals, and in humans to understand the complexities of the attenuation of nitrate action (nitrate tolerance). This review discusses nitrate metabolism and actions in the nontolerant and tolerant states with a focus on the role of thiols. Nonetheless, many issues remain unresolved. Before describing the role of thiols in intracellular nitrate metabolism, as well as discussing potential agents for prevention of tolerance, a brief review of nitrate actions and clinical indications is in order.
ORGANIC NITRATES These compounds have been a mainstay of cardiovascular medicine since the recognition [3] that nitroglycerin (NTG) is a useful vasodilator in relieving angina pectoris attacks. Nitroglycerin, isosorbide dinitrate (ISDN), and isosorbide-5-mononitrate (5-ISMN), the major metabolite of ISDN, have physiologic potency in humans. The nitrates
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are versatile compounds and are available in a large number of formulations or delivery systems (Table I). Nitroglycerin has been the major formulation that has been studied in in vitro and in vivo investigations of thiol-nitrate interactions. Sodium nitroprusside and molsidomine are also vasodilators. They cause smooth muscle relaxation, but differ from the organic nitrates in their metabolism and that there is no tolerance problem with these compounds.
MECHANISMS OF ACTION Veins and arteries dilate after nitrate administration (Table II). The smaller resistance vessels or arterioles do not relax to conventional doses of hitrates, but do so at high concentrations. Venodilation is pronounced, even with low doses, and is a unique and significant component of nitrovasodilator action in humans. The combination of venous and arterial dilation results in a decrease in cardiac output. The heart becomes smaller as systolic and intraventricular filling pressures diminish after nitrate administration. The circulating blood volume is redistributed, with pooling or sequestration of blood in the splanchnic and mesenteric circulations and less blood returning to the heart. In the presence of depressed left ventricular function and/or congestive heart failure, the arterial dilating effects of the nitrates result in an increase in stroke volume and cardiac output (unloading effect), making these drugs useful as adjunctive therapy in heart failure [4]. While the peripheral circulatory actions of the nitrates, venous and arterial vasodilation, are important, it is clear that the central or coronary arterial actions of the nitrates are also critical to many patients with coronary artery disease. Nitrates can enhance global or regional nutrient coronary blood flow by a number of mechanisms, and these actions are important in the major myocardial ischemic syndromes. Prevention or reversal of coronary vasoconstriction is useful in patients with mixed angina. Prevention of coronary stenosis constriction and/or overt enlargement of a critical coronary narrowing may be an important mechanism for the relief or prevention of chest pain in some angina subjects [5]. It is not known how much the peripheral vs. central actions of the organic nitrates contribute to the relief or prevention of myocardial ischemia in a patient. ANTIPLATELET ACTIVITY OF NITRATES It is known that nitrates have antiaggregatory effects on platelet function in vitro; however, in
TABLEI AvailableNitrateFormulations Nitroglycerin /sosorbide dinitrate 5-1sosorbidemononitrate
SL, spray, orat-SR,buccal, topical, iV SL, spray, chewable, oral, oraI-SR,topical, IV Oral, oraI-SR
SL = sublingual; SR = sustained release; IV = intravenous; topical = ointment, patch, impregnated synthetic membrane.
TABLE II Beneficial Effects of Nitrates: Potential Mechanisms of Action Peripheralor SystemicActions Venous dilation Arterial dilation Arterial dilation (high doses) Central coronary actions Coronary artery vasodilation Prevention/reversal of coronary artery vasoconstriction Coronary stenosis dilation Enhanced collateral flow Endothelial function Nitrates converted to nitric oxide within vascular smooth muscle cell Antiplatelet actions Decreased platelet aggregation and adhesion (controversial)
Effects Smaller right and left cardiac volumes; lower right and left heart filling pressures Decreased afterload; reduced arterial reflectance wave; decreased systolic blood pressure Decreased systemic vascular resistance; decreased afterload All: Increased global and/or regional coronary blood flow, especially during ischemia
Vasodilation and antiplatelet action enhanced in the absence of normal endothelial function Potential antithrombotic benefit in unstable angina and possibly useful for circadian increase in platelet activation in stable angina
early studies pharmacologic doses were used to achieve these effects. A number of recent experiments [6,7] suggest that nitrates have potent antiplatelet activity in animals and humans at conventional doses and that these actions may be important in acute myocardial ischemic syndromes, an area that remains controversial. Nitrate tolerance decreases [8] the platelet response to administered nitrate. Thiol donors have been used with some success [9,10] in enhancing the platelet antiaggregatory action of the organic nitrates. A SH donor-nitrate interaction appears to be a promising approach for future studies into the platelet effects of the organic nitrates.
CLINICAL INDICATIONS FOR THE ORGANIC NITRATES NTG and the other nitrate compounds are effective in a variety of important cardiovascular conditions, with nitrates playing a significant role in the coronary artery syndromes. Use of these drugs in patients with congestive heart failure or impaired left ventricular function in the asymptomatic postinfarction subject is another emerging indication.
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TABLE III Dosing Factors FavoringDevelopmentof Nitrate Tolerance Frequentdoses Largedoses Sustained-releaseformulations Continuousdeliverysystems(IV,patch,ointment) Absentor briefnitrate-freeinterval
Myocardial Ischemia ACUTE EPISODE OF ANGINA PECTORIS:
Rapid-
acting nitrates remain the 'gold standard' for treatment of acute myocardial ischemia (MI). Sublingual (NTG, ISDN), chewable (ISDN), oral spray (NTG, ISDN), and buccal or transmucosal NTG are available as rapidly acting formulations. PROPHYLAXIS
FOR STABLE ANGINA PECTORIS:
Long-acting nitrates decrease angina attacks and improve exercise performance in patients with chronic stable angina. Many of these patients have a variable angina threshold and/or occasional episodes of rest pain (mixed angina pectoris) and these clinical clues demand consideration of nitrate therapy. The nitrates are equally as effective as calcium channel blockers or/3-adrenergic antagonists in the therapy of chronic angina pectoris, and are used in double and triple therapy in more symptomatic or refractory patients. UNSTABLE ANGINA PECTORIS: Intravenous NTG is an extremely useful antianginal agent in unstable angina therapy, and is commonly followed by oral or topical nitrate treatment after the acute syndrome subsides. Combination therapy with calcium channel blockers and/or/3-blockers is useful in this unpredictable syndrome of acute myocardial ischemia. ACUTE MYOCARDIAL INFARCTION: A number of trials suggest a benefit when intravenous NTG is infused for 24-72 hours in subjects with acute myocardial infarction. A decrease in morbidity has been observed, and data from these studies [11] suggest a reduction in hospital mortality. A study [12] of more than 300 patients has shown a decrease in mortality and the incidence of major complications in transmural myocardial infarction (anterior) when an intravenous NTG infusion was given for 48 hours. The NTG-treated patients showed a decrease in adverse left ventricular remodeling in the postinfarction period. This work has been replicated by the same author [13] in an animal model and in a clinical study [14] using buccal NTG for 6 weeks, after the hospitalization period, in patients initially treated with intravenous NTG. Nitrates may be protective against remodeling after transmural myocardial infarction. Two major trials currently under way, GISSI-3 and ISIS-4, will test 3C-108S
this hypothesis. The angiotensin converting enzyme (ACE) inhibitors have been widely used in the prevention of post-MI ventricular remodeling [15] and the data are promising. Congestive Heart Failure Nitrates have been widely used as vasodilator therapy for heart failure. Short-term data [4,16,17] show improvement in cardiac hemodynamics, with a fall in right and left heart filling pressures and an increase in cardiac output. Some data [16,17] suggest an improvement in exercise performance in chronic failure. In the Veterans Administration study [18], the combination of oral ISDN and hydralazine gave a 20-25% reduction in mortality in class I I - I I ! heart failure patients compared with placebo. Data in the literature suggest that nitrate therapy in heart failure reduces signs and symptoms of this clinical syndrome and may be cardioprotective. Nitrates have a similar hemodynamic profile as the ACE inhibitors in congestive heart failure. These drugs are a useful adjunctive modality in the treatmerit of heart failure not easily controlled with diuretics and digitalis. It is likely that the combination of a nitrate and an ACE inhibitor would be more beneficial. NITRATE TOLERANCE The major limitation for the effective utilization of nitrates is in its attenuation or tolerance. When nitrates are administered in a protolerant fashion (Table III), their effects decrease in effect over time. In some studies, complete tolerance to the desired nitrate action is evident. Despite the history of nitrate tolerance being long and controversial, all experts now agree that tolerance is the critical factor that adversely affects nitrate efficacy. Attenuation of nitrate action may be Seen after the first administered dose, especially with long-acting formulations, such as the NTG patch or sustainedrelease compounds of ISDN and 5-ISMN. Tolerance Mechanisms Two hypotheses are believed to be causally related to nitrate tolerance: (a) the thiol or SH depletion theory and (b) the neurohormonal activation/ salt and water retention theory. Both probably play a role in clinical nitrate tolerance and are being actively studied. Activation of neurohumoral systems with subsequent plasma expansion results in stimulation of the renin-angiotensin system and catecholamine release after nitrate administration. There is evidence [19,20] that these phenomena may be clinically important. Captopril has been used recently in
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ENOOTHELIUM organic nitrate (RONOz)
VASCULAR SMOOTHMUSCLE RONOz
Nitroprusside (NP}
X
-NP
.R' SH LR'SSR'
NO
S-nitrosot hiol. (RSNO}Figure1. Intracellular nitrate metabolism in relation to thiols. NO = nitric oxide, GC = guanylate cyclase, EDRF = endothelium-derived relaxing factor, GTP = guanine-5'-triphosphate, cGMP = cyclic guanosine-3',5'-monophosphate. Reproduced with permission from [67].
RSNO
\
-.~..~
GTP
Endothe[iurn -dependent-- --~EDRF vosodi[otors
IRELAXATION]
tolerance studies [21]. This potential thiol donor may function via two modes of action (ACE inhibition and SH donor) and may be effective in preventing tolerance.
The Thiol Depletion Hypothesis It has long been known [1,2] that the first step in organic nitrate denitration within the vascular smooth muscle cells requires reduced thiol groups in the form of cysteine (Figure 1). During nitrate biotransformation, there is an obligatory role for the oxidation of SH donors, as the parent nitrate molecule is converted to nitric acid and subsequently to nitric oxide (NO). Continuous nitrate administration results in the loss of reduced SH groups in the cytoplasm, and inadequate repletion of these reduced thiols decreases nitrate action, and slows vascular nitrate uptake. This is the basic rationale underlying efforts to supply thiol donors that provide reduced SH or cysteine moieties to the smooth muscle cells. In a subsequent step of nitrate metabolism, SH groups also help to form S-nitrosothiols (SNO), short-lived intermediate compounds activating guanylate cyclase [22,23] (Figure 1). It was believed [23] that the nitrosothiols were formed after NO production, but evidence suggests that organic nitrates can be directly converted to NO or SNO, both capable of activating guanylate cyclase [23,24] (Figure 1). Nitroprusside and molsidomine directly form NO and are not reliant on reduced SH availability; tolerance is not found with these compounds.
Nitrate Metabolism The organic nitrates act at the level of the plasma membrane in vascular smooth muscle cells, where they are converted to NO and SN0 [23,24], which activate guanylate cyclase. This enzyme, present in soluble cytoplasm, catalyzes the conversion of guanosine triphosphate to cyclic guanosine-3',5'monophosphate (cGMP), the final metabolic step leading to vascular smooth muscle relaxation, and is associated with a decrease in intracellular calcium and phosphorylation of protein kinase [25]. It is believed [19,20,22,24,26] that the production of SNO may also occur outside the cell when exogenous thiol is provided (Figure 1). The nitrosothiol may penetrate the cell membrane and activate guanylate cyclase with simultaneous intracellular production of NO from denitration of the parent nitrate. Both compounds stimulate guanylate cyclase. Since tolerance predictably appears when sufficient nitrate exposure has occurred, it is presumed that intracellular metabolic conversion of R SN02 to NO is the critical step in the production of nitrate tolerance (Figure 1). Studies [22,27] attempting to show that guanylate cyclase or guanylate cyclase-cGMP interaction is the actual locus of tolerance have been unsuccessful. Guanylate cyclase can respond to NO or nitroprusside in the presence of established nitrate tolerance [28], indicating that the site of tolerance is "proximal" to NO production [22,29].
Nitrate.Free Interval In vitro animal and human studies [30,31] have demonstrated that a sufficient duration of freedom
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from organic nitrate exposure allows vascular smooth muscle to remain fully responsive to administered nitrate. A nitrate-free interval of variable duration restores the desired nitrate response. The duration of this interval depends on the nitrate concentration and the length of pre-exposure to the administered drug. The current clinical strategy is to administer organic nitrates to provide intermittent dosing of nitrate with a predetermined nitratefree period. These dosing regimens vary with the type of formulation; 8-12 hours from the last nitrate dose is necessary to maintain vascular responsiveness.
rate guanylate cyclase with subsequent cGMP production [2,8,24,32-40,43-52]. Methionine, a thiol that is converted to cysteine within the cell [20], has been effective in preventing or reversing tolerance in preliminary experiments [35-37]. New data [38,39] suggest that other thiol donors may be identified that are more potent than NAC itself. These experiments are not conclusive with respect to a useful clinical role for other thiols, although preliminary data suggest enhanced biological effects for some thiol compounds in the presence of nitrates [38,39]
Negative Studies Concept of the Sulfhydryl Donor Providing thiol donors in the form of exogenous SH groups for tolerance prevention was first carried out by Needleman et al [1,2]. Recently, N-acetylcysteine (NAC), a reduced SH compound, has been administered with organic nitrates. Studies have focused on the reversal of established nitrate tolerance with NAC administration, or using simultaneous nitrate and NAC to prevent the development of tolerance or enhancement of NTG action with coadministration of NAC or other thiols [9,10,32-36]. The first published study [32] showed heightened NTG hemodynamic action in subjects given intravenous NTG before and after an infusion of NAC. NAC has since been used as a thiol donor in the presence of nitrate tolerance. Other work [33,34] suggests that NAC potentiates the vasodilatory activity of nitrates in nontolerant blood vessels. Preliminary studies [9,10] indicate that NAC enhances the antiplatelet activity of NTG through the formation of NAC-nitrosothiols. NTG effects are increased severalfold with co-administered NAC [26,32-34], suggesting that additional guanylate cyclase is activated by thiol donors [26] although some authors [20] suggest that this is a nonspecific effect. This interaction may not be due to increased intracellular denitration of R-SN02, through the availability of more reduced SH groups, resulting in NO production, but rather due to enhanced extracellular NAC-nitrosothiol formation and subsequent direct stimulation of guanylate cyclase and enhanced cGMP production [20,24,26] (Figure 1). Reversal of Nitrate Tolerance with Thiol Donors Many studies have shown that established nitrate tolerance can be partially reversed by the addition of NAC in oral or in intravenous (IV) formulation. A number of positive studies have been published showing that exogenous thiol donors may provide additional NO and/or SNOs that can acti3C-110S
Many thiol donor studies have been unsuccessful in overcoming established nitrate tolerance [19,20,26,39,40,53-62]. It is unclear why some studies produce negative results while others are promising [36,37]. Differences in experimental design, variable vascular tissue sensitivity to thiols in different vascular beds, differences among animal species, differing NAC and methionine doses and concentrations, different duration of exposure to NTG (and other nitrates), and differing degrees of induced nitrate tolerance may explain the discordant results. Tolerance in patients with angina pectoris may involve different (possibly less complex) mechanisms than tolerance in congestive heart failure. NAC or methionine may have a limited capacity to overcome established tolerance, particularly when it is marked [20,40]. Hutter et al [39] suggest that different thiols have differing actions on potentiation of NTG action as well as reversal of tolerance.
Other Thiol Donors METHIONINE: Methionine is converted to cysteine within smooth muscle cells and is a possible thiol donor [35-37,40]. It prevented or reversed nitrate tolerance in human volunteers when the venous vasodilation response to NTG was evaluated [35,36,40]. Since methionine does not form an extravascular nitrosothiol, it is unclear if this agent will be as effective as NAC [20,40]. CAPTOPRIL: There has been interest in the SH group on the captopril molecule. While preliminary results [21] suggested a positive effect of captopril in preventing nitrate tolerance when compared with a nonsulfhydryl ACE inhibitor, a subsequent report from the same group [41] has been unable to demonstrate a difference between captopril and enalapril. Both drugs seem to have some antitolerance activity, perhaps by interfering with activation of the renin-angiotensin system. In an in vitro model, using aortic vascular rings, captopril prevented tolerance whereas enalapril did not [42].
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Discrepancies in these studies may be due to the differences in experimental design, amount of ACE inhibitor administered, and possible direct coron a g vasodilatory actions of captopril. Chong and Fung [38] have shown that captopril is a poor thiol donor when compared with NAC. Other reports [41,42,63-66] support a physiologic rationale for ACE inhibitors, diuretics, or hydralazine in enhancing nitrate action and preventing or reversing tolerance. It is unlikely that the clinical doses of captopril or other ACE inhibitors with a SH group will provide sufficient quantities of SH groups to be useful as thiol donors in tolerance prevention Or reversal. However, the role of ACE inhibition in preventing or reversing tolerance is controversial and needs further study.
Unresolved Issues Recent reports using thiol donors and the organic nitrates support the potential of this drug interaction. Data are conflicting as to the efficacy of NAC and methionine. Many positive [2,8,24,32-40,4352] as well as negative [19,26,39,40,53-62] studies appear in the literature. In some studies, thiol donors are both beneficial and ineffective, tt may be that NAC or methionine as thiol donors have insufficient potency to overcome marked or complete tolerance. A number of experimental methods have been used in tolerance studies and may account for some of the discrepancy. Furthermore, tolerance is multi-factorial in origin and other mechanisms could be relevant to experiments that result in renin-angiotensin activation, increased catecholamine release, or plasma volume expansion [19,20,63-66]. It remains unclear if NAC and other thiols directly reverse or prevent nitrate tolerance, or whether these compounds primarily enhance residual vasodilatory capacity in partially tolerant veins and arteries. Some studies [26] have demonstrated an increased vasodilatory capacity in partially tolerant vessels exposed to NAC. Thiols enhance the vasodilator response in the nontolerant state by severalfold, and incomplete degrees of nitrate vascular attenuation could result in important responses to administered NAC or methionine. THE FUTURE More basic and human investigative work is likely in this area. The thiol donor theory provides new insights into the mechanisms of nitrate action and the causation of nitrate tolerance itself. The possibility that tolerance can be prevented or limited with a thiol donor has yet to be proven, although some studies are promising. The goals of
enhancing nitrate activity with respect to smooth muscle responses (e.g., angina or congestive heart failure) or antiplatelet action (e.g., unstable angina and acute myocardial infarction) with a thiol donor hold promise for continuing clinical investigation.
REFERENCES 1. Needleman P, Johnson EM, The pharmacological and biochemical interaction of organic nitrates with sulphydryls: possible correlations with the mechanism for tolerance development, vasodilation and mitochondrial and enzyme reactions. In: Needleman P, ed. Organic nitrates. Handbook of experimental pharmacology, Vol. 40. New York: Springer-Verlag, 1975; 97-114. 2. Needleman P. Biotranformation of organic nitrates. In: Needleman P, ed. Organic nitrates. Handbook of experimental pharmacology, VoL 40. New York: SpringerVerlag, 1975: 57-95. 3. Murrell W. Nitro-glycerine as a remedy for angina pectoris, Lancet 1879; 1: 80, 113,225,284,642-6. 4. Abrams J. Pharmacology of nitroglycerin and long-acting nitrates and their Usefulness in the treatment of chronic congestive heart failure. In: Gould L, Reddy CVR, eds. Vasodilator therapy for cardiac disorders. Mount Kisco, NY: Futura Publishing Co., 1979: 129-68. 5, Gage JE, Hess OM, Murakami T, Riher M, Grimm J, Krayenbuehl HP. Vasocontriction of stenotiC coronary arteries during dynamic exercise in patients with classic angina pectoris: reversibility by nitroglycerin. Circulation 1986; 73: 865-76. 6. Diodati J, Theroux P, Latour J-G, et al, Nitroglycerin at therapeutic doses inhibits platelet aggregation in man. J Am Coil Cardiol 1988; 11: 54A. 7. Lam JYT, Chesebro JH, Fuster V, Platelets, vasoconstriction and nitroglycerin during arterial wall injury, Circulation I988; 78: 712-16, 8. Loscalzo J, Amarante P. Nitrate tolerance in piatetets: a model for the process and prevention by reduced thiol. Circulation 1989; 80(suppl II): 11-213. 9, Loscalzo J. N-acetylcysteine potentiates inhibition of platelet aggregation by nitroglycerin. J Clin Invest 1985; 76: 703-8. 10. Folts JD, Stamler J, Loscalzo J. N-Acetylcysteine potentiates intravenous nitroglycerin in inhibiting periodic platelet thrombus formation in stenosed dog coronary arterie, J Am Coil Cardiol 1989; 13: 145A. 11. Yusuf S, Collins R, MacMahon S, Peto R. Effect of intravenous nitrates on mortality in acute myocardial infarction: an overview of the randomized trials. Lancet 1988; 2: 1088-92. 12. Jugdutt BI, Warnica JW. Intravenous nitroglycerin therapy to limit infarct size, expansion, and complications, Effect of timing, dosage, and infarct location. Circulation 1988; 78: 706-14. 13. Jugdutt BI, Michorowski BL, O'Kelly BE Pharmacologic modification of left ventricular remodeling during healing after myocardial infarction. J Am Coil Cardiol 1988; 11: 252A. 14. Humen D, McCornick L, Jugdutt BI. Reduction of left ventricular volumes at rest and exercise in patients treated with nitroglycerin following anterior ML J Am Coil Cardiol 1989; 13: 25A. 15. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation 1990; 81: 1161-72. 16. Franciosa JA, Cohn JN. Sustained hemodynamic effects without tolerance during long-term isosorbide dinitrate treatment in chronic left ventricular failure. Am J Cardiol 1980; 45: 640-54. 17. Leier CV, Huss P, Magorien RD, Unverferth DV. Improved exercise capacity and differing arterial and venous tolerance during chronic isosorbide dinitrate therapy for congestive heart failure. Circulation 1983; 67: 817-22, 18. Cohn JN. Effect of vasodilator therapy on mortality in chronic congestive heart failure. Results of a Veterans Administration Cooperative Study. N Engl J Med 1986; 374: 1547-52. 19, Dupuis J, Lalonde G, Lemieux R, Rouleau JL Tolerance to intravenous nitroglycerin in patients with congestive heart failure: role of increased intravascular volume, neurohumorai activation and lack of prevention with N-acetylcysteine. J Am Coil Cardiol 1990; 16: 923-35. 20. Packer M, What causes tolerance to nitroglyucerin? The 100 year old mystery continues. J Am Coil Cardiol 1990; 16: 932-4. 21. Levy WS, Katz RJ, Buff L, Wasserman AG. Nitroglycerin tolerance is modified by angiotensin converting enzyme inhibitors. Circulation 1989; 80 (suppl II): 11-214. 22. Fung HL, Chung SL, Chong S, Hough K, Kakemi M, Kowaluk E. Cellular mechanisms of nitrate action. Z Kardiol 1989; 78 (suppl): 14-7.
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SYMPOSIUMON OXIDANTSAND ANTIOXIDANTS/ABRAMS 23. Ignarro LJ, Lippton H, Edwards JC, et al. Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiots as active intermediates, J Pharmacot Exp Ther 198]; 218: 739-49. 24. Fung HL, Chong S, Kowaluk E, Hough K, Kakemi M. Mechanisms for the pharmacologic interaction of organic nitrates with thiols. Existence of an extraceliular pathway for the reversal of nitrate vascular tolerance by N-acetylcysteine. J Pharmacol Exp Ther 1989; 245: 524-30, 25. Ahlner J, Axetsson KL Nitrates, Mode of action at a cellular level. Drugs 1987; 33(suppl 4): 32-8. 26. Munzel T, Holtz J, Mulsch A, Stewart DJ, Bassenge E. Nitrate tolerance in epicardial arteries or in the venous system is not reversed by acetylcysteine in vivo, but tolerance-independent interactions exist, Circulation 1989; 79: 188-97. 27, Holtz J, Munzel T, Stewart DJ, Bassenge E. Nitrate action on epicardial coronary arteries and tolerance: new aspects based on long term glyceryi trinitrate infusion in dogs. Eur Heart J 1989; 10 (suppl F): 127-33, 28. Mulsch A, Busse R, Bassenge E. Desensitization of guanylate cyclase in nitrate tolerance does not impair endothelium-dependent responses. Eur J Pharmacol 1988; 158: 191-8. 29. Feelisch M, Noack E, Schrocker H. Explanation of the discrepancy between the degree of organic nitrate decomposition, nitrite formation and guanylate cyclase stimulation. Eur Heart J 1988; 9(suppl A): 57-62. 30. DeMots H, Glasser SP. Intermittent transdermal nitroglycerin therapy in the treatment of chronic stable angina. J Am Coil Cardiol 1989; 113: 786-93. 31. Abrams J. Interval therapy to avoid nitrate tolerance: paradise regained? Am J Cardiot 1989; 64: 93i-4. 32. Horowitz LD, Antman EM, Lorell BH, Barry WH, Smith TW. Potentiation of the cardiovascular effects of nitroglycerin by N-aeetylcysteine. Circulation 1983; 687: 1247-53. 33. Bertel O, Noll G. Effects of N-acetylcysteine on nitroglycerin responsiveness before and during nitrate therapy of congestive heart failure. Eur HearL J 1987; 8(suppl i): 44. 34. Winniford MD, Kennedy PL, Wells PJ, Hillis LD. Potentiation of nitroglycerininduced coronary dilatation by N-acefylcysteine. Circulation 1986; 73: 138-42. 35. Levy WS, Katz RJ, Wasserman AG. Methionine reverses tolei'ance to transderreal nitroglyceryn, J Am Coil Cardiol 1989; 13: 230A. 36. Levy WS, Katz RJ, Ruffalo RL, Leiboff RH, Wasserman AG. Potentiation of the hemodynamic effects of administered nitroglycerin by methionine. Circulation 1988; 78: 640-5. 37. Neuberg GW, Packer M, Medina N, Yushak M, Kukin ML. Reversal of nitroglycerin tolerance in patients with chronic heart failure by oral methionine. Circulation 1989; 80 (suppl II): 11-213. 38; Chong S, [-ung HL. Biochemical and pharmacological interactions between nitroglycerin and thiols: effects of thiol structure on nitric oxide generation and tolerance reversal. Bioi:hem Pharmacol (in press). 39. Hutter J, Schmidt M, Rittler J. Effects of sulphydryl-containing compounds on nitrogtycerin-inc[uced coronary dilatation in isolated working rat hearts. Eur J Pharmacol 1988; 156: 215-22. 40. Penn J, Neuberg GW, Kukin ML, Medina N, Yushak M, Packer M. Methionine fails to restore the initial effects of nitroglycerin in tolerant patients: evidence that sulphydryl induced reversal of tolerance is mediated extracellulary. Circulation 1990; 82 (suppl Ill): 111-199. 41. Katz RJ, Levy WS, Buffalo RL, Wassermann AG. Prevention of nitrate tolerance with angiotensin-converting enzyme inhibifors. Circulation 1991; 83: 1271-77. 42. Lawson DL, Nichols WW, Mehta F, Mebta JL. Prevention of vascular tolerance to nitroglycerin by converting enzyme inhibitor (CEI) captoprih Role of CEI activity vs sulphydryl (SH) group. Circulation 1990; 82 (suppl III): 111-265. 43. Bauer JA, Fung HL Differential hemodynamic effects and tolerance properties of nitroglyceirn and an S-nitrosothiol in experimental heart failure. J Phatmacol Exp Ther 1991; 256: 249-54.
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44. Kowaluk EA, Poliszczuk R, Fung HL. Tolerance to relaxation in rat aorta: comparison of an S-nitrosothiol with nitroglycerin. Eur J Pharmacol 1987; 144: 379-83. 45. Axelssson KL, Anderson RGG, Wikberg JES. Acta Pharmacol Toxicol 1982; 50: 350-57. 46. Boesgaard S, Adeshvile J, Pederson F, Pieterson A, Madsen JK, Grande P. Circulation 1990; 82 (suppl III): 199A. 47. May DC, Popoma JJ, Black WH, Schaffer S, Lee HR, Levine BD, Hillis LD. N Engl J Med 1987; 317: 805-09. 48. Newman CM, Wassen JB, Taylor GW, Boobis AR, Davies DS. Rapid tolerance to the hypotensive effects of glyceryl trinitrate in the rat: prevention by N-acetyI-L-, but not -D-cysteine. Br J Pharmacol 1990; 99: 825. 49. Packer et al. Arch Int Pharmacol Ther 1987; 317: 799. 50. Rosen R, Konig E, Klaus W. Different sensitivities of arteries and veins to glyceryl trinitrate-induced relaxa{ion and tolerance: an 'in-vitro' study on isolated vessels from rabbits. Arch Int Pharmacol Thei 1987; 285: 777-83. 51. Torresi J, Horowitz JD, Dusting GJ. Prevention and reversal of tolerance to nitroglycerin with N-acetylcysteine. J Cardiovasc Pharmacoi 1985; 7: 777-83. 52. Tsuneyoshi H, Akatsuka N, Ohno M, Hara K, Ochiai M, Moroi M. Inhibition of development of tolerance to nitroglycerin by preventive administration of N-acetylcysteine in rats. Jpn Heart J 1989; 30: 733-41. 53. Abdallah A, Moffat JA, Armstrong PW. N-Acetylcysteine does not modify nitroglycerin-induced tolerance in canine vascular rings. J Cardiovasc Pharmaco11987; 9: 445-50. 5& Gruetter CA, Lemke SM. Dissociation of cysteine and glutathione levels from nitroglycerin-induced relaxation. Eur J Pharmacol 1985; 111: 85-95. 55. Henry PJ, Horowitz JD, Louis WJ. Determinants of in vitro nitroglycerin tolerance induction and reversal: Influence of dose regimen, nitrate-free period, and sulfhydryl supplementation. J Cardiovasc Pharmacol 1989; 14: 31-37. 56. Hogan JC, Lewis MJ, Henderson AH. N-Acetylcysteine fails to attenuate hemodynamic tolerance to glyceryl trinitrate in healthy volunteers. Br J Clin Pharrnacol 1989; 28: 421-26. 57. Kawamoto JH, Brian JF, Marks GS, Nakatsu K. Mechanism of glyceryl trinitrateinduced vasodilation. I1. Lack of evidence for specific binding of GTN to bovine pulmonary vein. J Pharmacol Exp Ther 1988; 244: 328-34. 58. Keith RA, Burkman AM. In vitro induction of nitroglycerin (NGG) tolerance in vascular tissue at pH 7.4 and 9.0 occurs prior to the decrease in sulfhydi'yl (SH) content. Fed Proc Am Soc Exp Biol 1981; 40:729 (A2849). 59. Moffat JA, Armstrong PW Marks GS. Investigations into the role of sulfhydryi groups in the mechanism of action of the nitrates. Can J Physiol Pharmacol 1982; 60: 1261-66. 60. Munzel TH, Holtz J, Mulsch A, Stewart DJ, Bassenger E. Z Kardiol 1989; 78 (suppl 2): 26-28. 61. Munzel TH, Mulsch A, Just H, Bassenger E. Cardiovascular pharmacology: basic mechanisms. J Am Coil Cardiol 1990; 15: 159A. 62. Parker JO, Farrell B, Lahey KA, Rose BE Nitrate tolerance: the lack of effect of N-acetyicysteine. Circulation 1987; 76: 572-76. 63. Muiesan ML, Boni E, Ceils G, et aL Efficacy of transdermal nitroglycerin in combination with an ACE inhibitor in patients with chronic stable angina pectoris. J Am Coil Cardiol 1991; 17 (suppl A): 337A. 64. Parker JD, Farrell B, Parker AC, Cohanim MA, Parker JO. Neurohumoral responses and the hemodynamic adaptation to nitrates. Circulation 1990; 82 (suppl III): 111-200. 65. Sussex BA, Campbell NRC, Raju MK. Nitrate tolerance is modified by diuretic treatment. Circulation 1990; 82 (suppl III): 111-200. 66. Bauer JA, Fung HL. Concurrent hydralazine administration prevents nitroglycerin-induced hemodynamic tolerance in experimental heart failure. Circulation (in press). 67. Kowaluk E, Fung HL Pharmacology and pharmacokinetics of organic nitrates. In: Abrams J, Pepine C, Thadani V, eds. Medical therapy of ischemic hear disease. Boston: Little, Brown and Co., 1991 (in press).
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