Possible Mechanisms of Nitrate Tolerance Uri Elkayam,
Prolonged exposure to organic nitrates has been !zihomtoleadtotherapkldevekpmentoftolerante to the peripheral and -WV-W efbctsofthesedrugs.AsaresuItofthlsphenamenon, the hemodynamk and anti-lsdwmk effect8ofnltratesmayberapldlyattenuatedIn patlentswtthlschemkheartdlsease,cangesthre heart faibre, or both. lids nitrate tokrance appeatstobebothdose-andthne-dependent. Ukelymechanlsmsproposedforltsdevelopment are multlfactobial and inch& depktk~ of suIfhymedlabdhweaseinblood W imw, a nbb+ volume, and nau robmmml stlmulatkn wlth actlvatlonofv~hfemechanlsms. (Am J Cawllol1992;7&49SS4G)
From the Department of Medicine, Division of Cardiology, University of Southern California (USC) Los Angeles, California. Address for reprints: Uri Elkayam, MD, Division of Cardiology, USC School of Medicine, 2025 Zonal Avenue, Los Angeles, California 90033.
he rapid development of tolerance with prolonged exposure to organic nitrates is well documented in the scientific literature. Nitroglycerin tolerance was described as early as 1905 by Steward,’ who expressed frustration at an inability to overcome tolerance to the drug, despite a 160-fold increase in the nitrate dose level. The development of immunity to the undesirable side effects of nitroglycerin, particularly headache, within 3-4 days by workers engaged in its manufacture further suggested nitrate tolerance.2 This immunity was lost rapidly, as indicated by the reappearance of symptoms of nitroglycerin toxicity after an absence from work of only a few days. For this reason, it became common practice for nitroglycerin workers to place some of the product in their hatbands during periods of absence from the factory so as to maintain their immunity. Thus as early as 1914 the importance of intermittent exposure to nitrates for the maintenance of the drug effect was being recognized.2 A recent review of data from 22 studies conducted between 1980 and 1989 provided strong evidence for the development of nitrate tolerance in patients with angina pectoris.3 These patients were treated with various types of nitrate preparations, including standard and sustained-release oral formulations, transdermal systems, and intravenous formulations, for periods ranging from 1 day to 2 months. Despite considerable data supporting the occurrence of nitrate tolerance, its clinical importance has been questioned in the past. This earlier skepticism was attributable to findings such as those of Danahy and Aronow.4 These investigators conducted treadmill exercise tests in 21 men with typical effort angina 1, 3, and 5 hours after an initial oral dose of isosorbide dinitrate (ISDN) or placebo. Exercise performance at all 3 evaluation periods was enhanced in the ISDN-treated patients compared with placebo. When the researchers repeated exercise testing after a mean of 5.6 months of treatment with ISDN, they found that the antianginal effect of the drug was maintained. Careful examination of the protocol followed in this study, however, revealed that patients were A SYMPOSIUM:
allowed a 16-hour nitrate-free interval prior to the reevaluation of ISDN effect. Similarly, a period of nitrate withdrawal was also used by Franciosa and Cohn5 who reported persistence of the hemodynamic effect of ISDN in patients with congestive heart failure. Nevertheless, the information available in the early 1980s led to the conclusion that prolonged therapy with long-acting nitrates was not associated with clinically important tolerance, even when these agents were used in large doses.‘j MANIFESTA~ONS OF NITRATE TOLERANCE There are other data, however, to support the rapid and marked attenuation of all aspects of nitrate activity, including effects on the peripheral and coronary circulation and on platelet activity, with continuous exposure or frequent dosing. Effect on peripheral circulation: A 1985 study by Manyari et al7 demonstrated a significant increase in regional blood volume, measured by the radionuclide blood pool method, in patients with stable angina after an initial dose of 0.6 mg of sublingual glyceryl trinitrate. After 4 weeks of treatment with ISDN, the response to glyceryl trinitrate was markedly attenuated (Figure 1). The earlier changes in blood pressure and heart rate achieved with glyceryl trinitrate were also signifi-
m.=.9 Stage A
Time (min) FWURE l. Changes In regional blood volume In msponse -7---------
(IBDN, stage B). Patients received 0.6 mg of subllmal nHto&cedn attlnm0. *p
THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 70
cantly diminished at this repeat evaluation. Thus the study not only showed attenuation of the peripheral effect of nitrates with long-term therapy but also established the existence of cross-tolerance between ISDN and sublingual nitroglycerin. Attenuation of the effect of nitroglycerin on pulmonary arterial wedge pressure (PAWP) in patients with congestive heart failure treated with continuous intravenous infusion of the drug has also been reported. In 1 study by our group,8 patients whose PAWP fell by at least 30% or 10 mm Hg in response to intravenous nitroglycerin were randomly assigned to receive placebo or to continue on nitroglycerin therapy. An immediate increase in PAWP occurred in the placebo group. In contrast, PAWP in patients who continued to receive nitroglycerin was significantly lower than at baseline for the first 8 hours of the infusion period. After that time, however, PAWP in the nitroglycerin treatment group began to rise, and values recorded at 12, 20, and 24 hours were no different from those noted in the placebo group. Attenuation of efFect on coronary circulation:There is also evidence for the attenuation of the effect of nitrates on the coronary circulation. May et al9 demonstrated a reproducible, doserelated increase in coronary sinus blood flow in a group of 19 subjects (17 with coronary artery disease) after administration of graded doses of lo-100 kg of intracoronary nitroglycerin. The patients then received a 24-hour intravenous infusion of nitroglycerin (n = 12) or saline (n = 7). Repeat intracoronary instillation of the same doses of intracoronary nitroglycerin after the intravenous nitroglycerin infusion indicated a substantial reduction in coronary response: the percent increase in coronary sinus blood flow, which had been 3052%, dropped to only 16-27%. In contrast, similar percent increases in coronary flow were noted before and after the intravenous saline infusion. Attenuation of antiplatelet effect: Information obtained primarily from in vitro studies shows that nitrates affect platelet activity, although the clinical significance of these effects has not yet been determined. Nitrates prevent platelet aggregation,‘O-l3 disperse already formed platelet clumps,r1J4 and prevent platelet adhesion to damaged intimal linings. l5 Interestingly, an in vitro study by Loscalzo and Amarantel’j indicated that antiplatelet effects are also attenuated after prolonged exposure to nitroglycerin. When these investigators preincubated platelet-rich plasma in the absence of nitroglycerin, the median inhibitory concentration (IC50) of nitroglycerin required for NOVEMBER 27, 1992
inhibition of adenosine diphosphate-induced platelet aggregation was approximately 40 $‘kf. When platelet-rich plasma was preincubated with nitroglycerin, the IC5a increased 9-fold, to 360 u&f.
after supplying sulfhydryl groups in the form of dithiothreitol and N-acetylcysteine (NAC).19,21,22 Interestingly, one in vitro study showed that captopril, a sulfhydryl-containing angiotensinconverting enzyme (ACE) inhibitor, prevented the development of tolerance to the effect of nitroglycerin on aortic vascular rings.23 Enalapril, a nonsulfhydryl-containing ACE inhibitor, failed to prevent development of tolerance. Furthermore, only negligible tolerance has been seen with thiol-independent nitrate vasodilators, such as molsidomine and
PROPOSED MECHANISMS OF NITRATE TOLERANCE The precise cause of nitrate tolerance is still not entirely clear, but a number of mechanisms have been postulated. Among these are pharmacokinetic changes, depletion of sulfhydryl groups, the activation of neurohormonal vasoconstrictive mechanisms, and the expansion of intravascular volume. Pharmacokinetic changes: A reduction in A blood nitrate levels due to pharmacokinetic changes, such as alterations in drug absorption, 24 distribution, or elimination, has been proposed as a mechanism for the diminishing effect of nitrate preparations over time. The feasibility of this explanation, however, is refuted by a recent study by Elkayam et al. l7 The patients enrolled in this study demonstrated attenuation of the ISDNinduced reduction in PAWP within 24 hours after initiation of a 4-hour dosing regimen (Figure 2A). Despite the loss of clinical efficacy, plasma ISDN levels were significantly higher on the second day of dosing than on the first day (Figure 2B). The persistent elevation in blood levels of ISDN was, in 18 fact, considered the probable cause of nitrate 4 6 1 2 tolerance in these patients. Time (hours) Depletion of sulfhydryl groups: Organic niB trates are prodrugs that must undergo biotransformation and activation in order to exert a clinical effect. This activation occurs via an interaction 3 with sulfhydryl groups that is primarily intracelluE 80 lar but also occurs extracellularly (Figure 3). The aI 5 products of this interaction, S-nitrosothiol and nitric oxide, then stimulate guanylate cyclase, the +?I 60 enzyme responsible for the formation of cyclic 5 guanosine monophosphate (GMP). Cyclic GMP g 40 - 27 can, by a variety of mechanisms, lower the cytosolic 3 cl free calcium and make contractile proteins less 2: sensitive to calcium, an effect that eventually leads g202 to vasodilation. Experimental evidence obtained f both in vitro and in vivo lends support to the I I I 1 sulfhydryl depletion hypothesis of nitrate tolerBL 1 2 3 4 ance. Among the in vitro evidence is the decrease Time (hours) in the tissue content of sulfhydryl groups that occurs with prolonged exposure of vascular strips FlGUl?E2. A, Attonwtlon of the dlbot of laowMdo dlnlto nitroglycerin, which was first shown by Needle- trato (ISBN) on pulmonary art&al ww& pressure (PAWP) = lLB,Plasma manls as early as 1970 and then demonstrated in wltha4-hourdoslngmglman(q4h);n ISDN levels achkved wlth a 44our doslng -men; subsequent studies. 19,20In addition, there are sev- n=5.BL=base4lne.(Adaptedwt&hpennlsslanfrom eral reports of the reversal of vascular tolerance Circulation.~ A SYMPOSIUM:
R-ONO, + R-SH +
FIGURE 3. Role of wmyayl @-m&~~f-lrr
R-OH + NO, 1 / NO + R-SH-S-Nitrosothiol
mnylatecydase;-p= cyclk @mosine momphosphateq NO = nttdcoxide.
+ +Cytosolic free Ca*+ Vasodilation
nitroprusside, which do not require interaction with sulfhydryl groups for their effect.24y25The same is true for the vasorelaxation induced by nitric oxide.26 In vivo evidence for the sulfhydryl depletion hypothesis includes the potentiation of the hemodynamic effects of nitroglycerin by the sulfhydryl group donor NAC. This effect has been observed in both rats and human beings.27-30Even more striking is the partial reversal of nitroglycerin tolerance in patients with congestive heart failure and coronary artery disease31,32and in normal subjects33by sulfhydryl groups, supplied by either NAC or methionine. In one study conducted by Packer et aP1 in 35 patients with severe congestive heart failure, continuous intravenous infusion of nitroglycerin for 48 hours initially caused a statistically significant reduction in left ventricular filling pressure (LVFP) and mean right atria1 pressure (MRAP). By the end of the treatment period, these effects were no longer present. The nitroglycerin-induced decrease in LVFP and MRAP was partially restored by administration of NAC. Parker was unable to repeat the results in patients with angina pectoris who developed tolerance to ISDN, and he concluded that the interaction with NAC is drug-specific and does not occur with ISDN.34 However, in a recently completed study by Mehra et aP5 in patients with congestive heart failure, the addition of NAC to ISDN augmented the nitrate-induced reduction in PAWP, indicating an interaction between ISDN and NAC. Activation of neurohormonal vasoconstrio tlve mechanisms The strong vasodilatory response to nitroprusside causes reflex stimulation of catecholamines and renin, resulting in vasoconstriction and at least a partial attenuation of the 520
THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 70
vasodilatory effect of nitroprusside. With abrupt discontinuation of nitroprusside, rebound vasoconstriction may also occur. It has been suggested that similar activation of neurohormonal vasoconstrictive mechanisms may play a role in the development of tolerance to nitrates. The role of neurohormonal vasoconstrictive factors in the development of nitrate tolerance is supported by a number of reports of increased catecholamine levels or plasma renin activity in patients who develop nitrate tolerance. In the study mentioned above of patients with congestive heart failure conducted by Packer et aL31plasma renin activity rose significantly, from 7.5 + 2.9 ng/mL/hour at baseline to 12.9 2 4.8 ng/mL/hour after a continuous 48-hour infusion of nitroglycerin. In contrast, in patients receiving intermittent nitrate therapy, plasma renin activity showed a much smaller and statistically insignificant increase, from 6.1 + 2.7 ng/mL/hour before infusion to 7.7 + 3.0 ng/mL/hour after infusion. Expansion of intravascular volume: Several studies have shown that nitrate therapy is associated with either an expansion of plasma volume or an increase in body weight. This effect has been demonstrated in patients with myocardial infarction and congestive heart failure.31,36y37 Dupuis et al, for example, reported a significant increase in blood volume of 745 ? 382 mL, primarily during the first hour of nitroglycerin therapy in patients with congestive heart failure.37 These patients showed no weight gain at that time, leading the investigators to propose a shift of fluid from the extravascular to the intravascular compartment as the cause of the augmented blood volume. An increase in blood volume is likely to attenuate the increase in venous capacitance initially observed with nitroglycerin. NOVEMBER 27, 1992
CONCLUSION The mechanisms responsible for the development of nitrate tolerance are not yet completely understood, although several possible hypotheses have been proposed. A combination of these various mechanisms is thought to be the most likely explanation for the development of tolerance to these drugs. REFERENCES L Steward DD. Tolerance to nitroglycerin. J%4A 1905;55:1678. 2. Ebright GE. The effect of nitroglycerin on those engaged in its manufacture. .&&lx 1914;x:20-202. 3. EIkayam U. Tolerance to organic nitrates: evidence, mechanisms, clinical relevance, and strategies for prevention.Ann Inrem Med 1991;114:667-677. 4. Danahy DT, Aronow WS. Hemodynamics and antianginal effects of high dose oral isosorbide dinitrate after chronic use. Circulation 1977;56:205212. S. Franciosa JA, Cohn JN. Sustained hemodynamic effects without tolerance during long-term isosorbide dinitrate treatment of chronic left ventricular faiare. Am J Cam201 1980;45:-54. 6. Elkayam U, Aronow WS. Glyceryl trinitrate (nitroglycerin) ointment and isosorbide dir&rate: a review of their pharmacological properties and therapeutic use. Drugs 1982;23:16>194. 7. Manyari DE, Smith ER, Sprang J. Isosorbide dinitrate and glyceryl trintrate: demonstration of cross tolerance in the capacitance vessels. Am J Cardi 1985;55:927-931. 8. EIkayam U, Kulick D, McIntosh N, Roth A, Hsueh W, Rahiitoola SH. Incidence of early tolerance to hemodynamic effects of continuous infusion of nitroglycerin in patients with coronary artery disease and heart failure. Circuladon 1987;w:577-584. 9. May DC, Popma JJ, Black WH, Schaefer S, Lee HR, Levine BD, Hillis LD. In viva induction and reversal of nitroglycerin tolerance in human coronary arteries. N Engl J Med 1987;317:80%809. lO.Fitzgerald DJ, Roy I, Robertson RM, Fitzgerald GA. The effects of organic nitrates on prostaqclin biosynthesis and platelet function in humans. Cimdion 1984,70:297-302. iL StamIer J, Cunningham M, Los&m J. Reduced thiols and the effect of intravenous nitroglycerin on platelet aggregation. Am J Can&l 1988;62:377380. 12. Diodati J, Theroux P, L&our J-G, Lacoste L, Lam JY, Waters D. Effects of nitroglycerin at therapeutic doses on platelet aggregation in unstable angina pectoris and acote myocardial infarction. Am J Canfiol 1990,6668X88. l.3. Loscalzo J. N-acetylcysteine markedly potentiates the inhibition of platelet aggregation by nitroglycerin. J Clin Invesr 1985;76:70%708. 14. Stamler JS, Mendelsohn ME, Amarante P, Smick D, Andon N, Davies PF, Cooke JP, Loscalzo J. N-acetylcysteine potentiates platelet inhibition by endothlium-derived relaxing factor. Cti Res 1989;65:789-795. 15. Lam JYT, Cbesebm JH, Foster V. Platelets, vasoconstriction, and nitroglycerin during arterial waU injury. A new antithrombotic role for an old drug. Circulation 1988;78:712-716. l6. Loscalzo J, Amarante P. Nitrate tolerance in platelets: a model for the process and prevention by reduced thiol. Circulation 1989;80:11-213. 17. Elkayam U, Roth A, Mehra A, Ostrzega E, Shotan A, Kulick D, Jam&on M, Johnston JV, Rahiitoola SH. Randomized study to evaluate the relation between oral isosorbide dinitrate dosing interval and the development of early tolerance to its effeti in left ventricular wing pressure in patients with chronic heart failure. Circularion 1991;84:[email protected]
&2048. l.8. Needleman P. Tolerance to the vascular effects of glyceryl trinitrate. J Phamacol .&p Ther 1970;171:9%102. lS.Needleman P, Johnson EM. Mechanism of tolerance development to organic nitrate. J Phamcol Eq The? 1973;184:709-715. 20. Ahlner J, Axe&m KL, Bornfeldt K. Biological effects of organic nitroesters and their mechanism of action. Acta Phamcol Toxic01 1986;59 (Suppl VI):17-25. 2L Torres I, Homwitz JD, Dusting GJ. Prevention and reversal of tolerance to nitroglycerin with N-acetylcysteine. J Cardbvasc Phmmacol1985;7:777-783. 22. Rosen R, Konig E, Klaus W. Different sensltwmes of arteries and veins to glyceryl trinitrate-induced relaxation and tolerance: an in vitro study on isolated vessels from rabbits. Arch Int Phmnacorfvn Ther 1987;285:22&237. 23. Lawson DL, Nicols WW, Mebta P, Mehta JL. Captopril-induced reversal
of nitroglycerin tolerance. Role of sulthydryl group vs. ACE inhibitory activity. J Cardiovax Phnmcol1991;17:411~18. 24. Kukovetz WR, Holzman S. Mechanism of vasodilation by molsidomine. Am Hem J 1985;109:637~. 2% Stewart DJ, Elsner D, Sommer 0, Holtz J, Bassenge E. Altered spectrum of nitroglycerin action in long-term treatment: nitroglycerin-specific venous tolerance with maintenance of arterial vasodepressor potency. Circulation 1986; 7457S5-582. 26. Miilsch A, Busse R, Winter I, Bassenge E. Endothelium- and sydnoniminedependent responses of native and cultured aortic smooth muscle cells are not impaired by nitroglycerin intolerance. Namyn Schmiedebe~ Arch Phnmcol 1989;339:56&574. 27. Horowitz JD, Antman EM, Lorell BH, Barry WH, Smith ‘IW. Potentiation of the cardiovascular effects of nitroglycerin by N-acetylqteine. Cirrulation 1983;77:787-794. 28. Winniford MD, Kennedy PL, Wells PJ, Hillis LD. Potentiation of nitroglycerin-induced coronary dilatation by N-acetylcysteine. Cim&ion 1986,73:13& 142. 29. Fung HL, Chong S, Kowaluk E, Hough K, Kakemi M. Mechanisms for the pharmacologic interaction of organic nitrates with thiols. Existence of an extracellular pathway for the reversal of nitrate vascular tolerance by N-acetylcysteinc. J Phamcol Exp Ther 1988;245:524-530. 30. Munzel T, Holtz J, Mulsch A, Stewart D, Bassenge E. Nitrate tolerance in epicardial arteries in the venous system is not reversed by N-acetylqsteine in viva, but tolerance-independent interactions exist. Cimdarion 1989;79:188-197. 3L Packer M, Lee WH, Kessler PD, Gottlieb SS, Medma N, Yushak M. Prevention and reversal of nitrate tolerance in patients with congestive heart failure. N Engl J Med 1987;317:79%%4. 32. May DG, Pompa JJ, Black WH, Schaefer S, Lee HR, Levine BD, Hillis LD. In vitro induction and reversal of nitroglycerin tolerance in human coronay arteries. N Engl J Med 1987. 33. Levy WS, Katz RJ, Ruffalo RL, Leiboff RH, Wasserman AG. Potentiation of the hemodynamic effects of acutely administered nitroglycerin by [email protected]
nine. Circulation 1988;78:[email protected]
34. Parker JO, Farrel B, Lahey KA, Rose BF. Nitrate tolerance: the lack of effect of N-acetykysteine. Circulation 198776572-576. 35. Mehra A, Ostrzega E, Shotan A, Johnson J, Rahiitoola SK Elkayam U. Potentiation of nitrate effect does occur after sulfhydlyl groups supply in heart failure patients treated with isosorbide dinitrate. (Abstr.) Cim&ti~ 1991;84: IL57. 36.Lis Y, Bennett D, Lambert D, Robson 0. A preliminary double-blind study of intravenous nitroglycerin in acute myocardial infarction. Inrensive Care Med 1984;10:17!+184. 37. Dupuis J, Lalonde G, Lemieux R, Rouleau JL. Tolerance to intravenous nitroglycerin in patients with congestive heart failure: role of increased intravascular volume, neurohumoral activation and lack of prevention with N-acetylqsteine. J Am Coli Car&l 1990;16:923-931.
DISCUSSION Participant: Will diuretics restore sensitivity to nitrates? Dr. Uri Elkayam: A preliminary report presented at a recent meeting indicated that nitrate tolerance might be prevented with diuretics in patients with angina. However, a paper that I reviewed recently from a group that has investigated nitrate tolerance intensively showed diuretits to have no effect on nitrate tolerance in normal volunteers. Thus, at this time, the effect of diuretics remains questionable. Participant: There is a clear consensus that nitrate tolerance does develop. Is there tolerance to the anti-ischemic effect of nitrates, as manifested by the frequency of ischemic events recorded on 24-hour ambulatory monitoring? Dr. Andrew P. Selwyn: Studies have shown that nitrates produce a consistent and modest decrease A SYMPOSIUM:
in the frequency and duration of &hernia seen with 24-hour ambulatory monitoring, due primarily to a preferential decrease in episodes of ischemia occurring at lower heart rates of about 70-80 beats/min. That beneficial effect disappears by 24 hours. There are too few studies available to know whether intermittent application can preserve the effect on ischemic events observed during 24-hour ambulatory monitoring. Dr. William H. Frishman: We have seen evidence of nitrate tolerance in terms of hemodynamic effects and exercise performance but not in terms of angina frequency, although that is not an objective criterion. Most studies suggest the persis-
tence of some anti-ischemic effect, particularly an effect on spontaneous attacks of angina. Dr. Udho Thadani: There are published data showing that angina frequency and tolerance to the anti-ischemic effects develop with continuous transdermal nitroglycerin therapy in patients with a very high frequency of angina1 attacks. Dr. Jay N. Cohn: There is no question that continuous nitrate application and a constant blood level are most likely to produce tolerance. Although the optimal drug-free interval for maintaining efficacy is somewhat controversial, at least some efficacy is certainly maintained with intermittent regimens.