OVERVIEW OF CLINICAL TRIALS

Vascular Bi o l o g y in Clinical Pr a c t i c e Vol. 1, No. 2 A CME Monograph Series OVERVIEW OF CLINICAL TRIALS TO IMPROVE ENDOTHELIAL FUNCTION GUEST EDITOR Arshed A. Quyyumi, MD, MRCP National Institutes of He a l t h Bethesda, Ma ry l a n d Published under the auspices of the Vascular Bi o l o gy Wo rking Gro u p Sponsored by College of Medicine

THE ENDOTHELIUM: A NE EDUCATIONAL GOALS Upon successful completion of this continuing education program, you should be able to: Describe the roles of A II and NO in maintaining vascular tone Explain the role of ACE in regulating the components of the RAS Outline the pathway by which CV risk factors lead to endothelial dysfunction and clinical sequelae Discuss the beneficial effect of HMG-CoA reductase inhibitors and ACE inhibitors on endothelial function Summarize recent clinical trials focusing on treatment of CVD and endothelial dysfunction The University of Florida College of Medicine is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to sponsor continuing medical education programs for physicians. The University of Florida College of Medicine designates this educational activity for 1 credit hour in Category 1 of the Physician s Recognition Award of the American Medical Association. Each physician should claim only those hours of credit that he/she actually spent on this educational activity. This continuing medical education activity was planned and produced in accordance with ACCME essentials. As of July 1, 1999, this monograph is approved for 24 months (until July 1, 2001). Fundamental views on the progression of cardiovascular disease are evolving toward a clearer understanding that the endothelium plays a central role in health and disease. The endothelial lining of the vasculature sustains damage in the presence of several conditions including hypertension, dyslipidemia, diabetes and smoking. 1 Each of these conditions causes oxidative stress that leads to abnormal functioning of the endothelium. One of the primary alterations is an imbalance between vasodilator compounds, such as nitric oxide (NO), and vasoconstrictor compounds, such as angiotensin II (A II) and endothelin-1. These vasoactive compounds are part of auto-, para-, and endocrine systems that interact at the vessel wallblood interface. Within the vessel wall A II upregulates endothelin-1 production while NO downregulates it, thus maintaining a delicate balance in blood vessel tone. Disclaimer The editorial content of this monograph expresses the views of the individual contributors and does not necessarily reflect the views or recommendations of the University of Florida College of Medicine, Parke-Davis, or the publisher. The reader is advised to consult the full prescribing information for all medications prior to use. Supported by an unrestricted educational grant from Parke-Davis, A Division of Warner-Lambert Company. Copyright 1999. Medical Education Consultants, Inc. (MEDCON). All rights reserved. Figure 1: Influences on endothelial tone 2 When this delicate balance within the endothelium is disturbed, pathophysiologic changes can occur that often lead to the development or progression of atherosclerosis. 3

HERAPEUTIC TARGET IN CARDIOVASCULAR DISEASE F i g u re 2: Consequences of abnormal tissue ACE pathway THE IMPORTANCE OF THE RENIN- ANGIOTENSIN SYSTEM AND ACE As research reveals more of the complex interaction occurring within the endothelium, approaches are emerging that are beneficial in improving endothelial function. One area of study focuses on inhibitors of angiotensin converting enzyme (ACE). Historically, ACE inhibitors are known to restore the balance within the reninangiotensin system (RAS) once it is activated. It is now known that there are actually two RASs on which ACE inhibitors can act. One system, the classic RAS, is responsible for acute blood pressure regulation. 4 A second system, the tissue RAS, regulates vascular and cardiac function and structure over the long term. 5-7 ACE inhibitors suppress production of A II thereby reducing vasoconstriction and, c o n s e q u e n t l y, blood pre s s u re in hypert e n s i ves. However, it is becoming clear that inhibition of ACE does more than lower blood pressure by reducing A II formation. ACE inhibition also increases production of NO via its effect on bradykinin, as shown in Figure 2, leading to vasodilation. Use of these agents also appears to reduce proliferation and migration of smooth muscle cells, neutrophils and mononuclear cells in the subintimal layer. ACE inhibitors seem to have antiplatelet effects and enhance endogenous fibrinolysis by increasing tpa and reducing PAI-1 levels. Research suggests that the sum effect of ACE inhibitors is to improve arterial compliance and tone, ameliorate endothelial dysfunction, exert an antiatherogenic effect and thereby decrease probability of plaque rupture. 8 Figure 3: Endothelial regulation: Understanding RAS When this delicate balance is disturbed, pathophysiologic changes can occur that often lead to clinical outcomes 3

IMPROVING ENDOTHELIAL FUNCTION IN PATIENTS WITH CAD: THE TREND STUDY (TRIAL ON REVERSING ENDOTHELIAL DYSFUNCTION) The evidence for these beneficial effects of ACE inhibitors on endothelial function comes from a number of recently completed clinical studies. One of these studies is TREND. 9 TREND was a randomized, double-blind, placebo-controlled study that followed up 105 adults with coronary artery disease (CAD) for 6 months. Subjects randomized to the therapy group received quinapril (40 mg/day). The primary study end point was the net change in coronary artery diameter in response to acetylcholine, with responses at baseline compared to those seen after treatment. THE BANFF STUDY (BRACHIAL ARTERY NORMALIZATION OF FOREARM FUNCTION) The BANFF trial also examined the effectiveness of ACE inhibition at improving endothelial function. In this study, the effectiveness of 4 anti-hypertensive agents was compared. 10 Study subjects included patients with documented CAD but without previous coronary artery bypass graft (CABG) surgery, lipid-lowering therapy, uncontrolled hypertension, a left ventricular fraction of <40%, or a total cholesterol level >6.0 mmol/l. Two ACE inhibitors were administered in the study: quinapril (20 mg/day) and enalapril (10 mg/day). The angiotensin receptor antagonist losartan was given (50 mg/day). The fourth treatment was the calcium channel antagonist, amlodipine (5 mg/day). Endothelial function was assessed in these patients by measuring flow-mediated dilation (FMD) of the brachial artery during reactive hyperemia. The only treatment to show significant improvement in FMD was quinapril (P<0.02). A nonsignificant trend toward improvement was observed with losartan and amlodipine. The study authors concluded that some antihypertensive agents are more effective than others at improving endothelial function. Figure 4: Improvement of endothelial function with ACE inhibition: TREND results After 6 months of treatment, the group receiving quinapril demonstrated significant improvement in arterial function compared with the placebo group (P<0.0003). Out of all the clinical and angiographic variables included at baseline (such as smoking status, stenosis severity, blood pressure, gender, initial response to acetylcholine, and lipid values), assignment to the quinapril group was the only independent predictor of improved endothelial function (P=0.022). 4 Figure 5: BANFF trial: Improvement in flow-mediated vasodilation

IN PATIENTS WITH CHF IN PATIENTS UNDERGOING CABG While the TREND and the BANFF studies focused on patients with CAD, a recent investigation by Hornig et al examined the effect of ACE inhibition in patients with congestive heart failure (CHF). 11 FMD was used as a measure of endothelial function. High-resolution ultrasound and Doppler were used to measure radial artery diameter and blood flow in patients given intra-arterial infusions of either quinaprilat 1.6 µg/min or enalaprilat 5 µg/min. Measurements were made at rest and during reactive hyperemia both before and after administration of N- monomethyl-l-arginine (L-NMMA), a substance that inhibits endothelial synthesis of NO. The investigators found that quinaprilat improved overall FMD by >40% (P<0.01) while enalaprilat had no detectable effect. Through use of L-NMMA, the i n vestigators we re able to isolate the portion of F M D attributable to NO. They found that quinaprilat increased NO-dependent FMD by >100% (P<0.01). Quo Vadis study (Effects of Quinapril on Vascular ACE and Determinants of Ischemia) The effect of ACE inhibition on endothelial function via reduction of A II was the focus of one portion of the Quo Vadis study. 12 Figure 7: Quo Vadis: The benefits of increased tissue ACE inhibition In this part of the study, 187 patients scheduled for coronary bypass surgery were randomized at least 7 days prior to surgery. Patients were treated with quinapril (40 mg/day), captopril (150 mg/day) or placebo prior to surgery. Portions of the internal mammary artery not used for CABG we re exposed to increasing doses of A I. A I to A II conversion was measured using 2 methods: the difference between pec50 of the dose-response curves to A I and A II and as the area between the curves (ABC) of A I and A II. The data indicated that quinapril was more effective than captopril or placebo at inhibiting endothelial A II production. Figure 6: Positive effect of some ACE inhibitors on NO-mediated vasodilation These results of the Quo Vadis study support those of the BANFF and Hornig investigations which suggest that ACE inhibitors work by differing mechanisms and that not all are equally effective at altering endothelial function. 5

ACE INHIBITION AND VEGF: EFFECTS ON ANGIOGENESIS ACE inhibition may affect more than vascular tone; a recent investigation by Fabre et al suggests that it may induce angiogenesis as well. 13 A rabbit model of chronic hindlimb ischemia was used in the Fabre investigation to test the influence of ACE inhibition on new blood vessel growth. Vascular endothelial growth factor (VEGF) was used as a positive control in the same model. VEGF was first isolated in 1989 and appears to facilitate angiogenesis as well as induce migration and proliferation of endothelial cells, enhance vascular permeability and modulate thrombogenicity. 14 VEGF is produced by endothelial cells, macrophages, smooth muscle cells and tumor cells. 15-17 In the Fabre study, several measurements of angiogenesis, including angiographic score, capillary density and an estimation of endothelial function, were taken 10 days after inducing ischemia to obtain a baseline. Either placebo, VEGF, quinapril or captopril was then administered and the same measurements were taken at day 40. Based on all measurements, both VEGF and quinapril significantly improved endothelial function assessed by the response to acetylcholine and enhanced angiogenesis that was demonstrated as increased blood vessel density (P<0.01). months. The patients in the placebo group experienced little change (+1%) in vasoresponsiveness to acetylcholine whereas patients in the lovastatin group had a mean change of +16%. This difference was significant (P=0.004). Figure 8: Coronary artery responsiveness increased with HMG-CoA reductase inhibition The authors concluded that cholesterol-lowering with a statin significantly improved endothelial function in patients with atherosclerosis. Potentially, this improvement in endothelial activity could influence blood flow to relieve symptoms of ischemia and may reduce progression of atherosclerotic plaques. HMG-COA REDUCTASE INHIBITORS IMPROVE ENDOTHELIAL FUNCTION ACE inhibitors are not the only class of drugs that improve endothelial function. A number of clinical studies have demonstrated positive effects on endothelial activity with HMG-CoA reductase inhibitors or statins. One such investigation was conducted by Treasure et al. 18 In this study 23 patients with CAD were randomized to either lovastatin or placebo, in addition to a lipid-lowering diet. Responsiveness of the coronary arteries to acetylcholine was used as a means of determining endothelial function. The segment demonstrating the greatest degree of constriction was studied at 12 days and at 5 1/2 HMG-COA REDUCTASE INHIBITORS AND ANTIOXIDANTS ENHANCE ENDOTHELIAL ACTIVITY IN HYPERCHOLESTEROLEMIC PATIENTS HMG-CoA reductase inhibitors have been combined with other agents such as antioxidants to reduce endothelial dysfunction. In a study by Anderson et al, 23 patients with hypercholesterolemia were randomized to 1of 3 groups: those consuming an American Heart Association Step 1 diet (7 patients), those taking lovastatin and cholestyramine (7 patients) or those taking lovastatin and probucol (9 patients). 19 They maintained these treatments for 1 year at which time endothelium-dependent vasomotion was studied using acetylcholine and quantitative angiography. 6

Susceptibility of low-density lipoprotein (LDL) to oxidation was determined by measuring the formation of conjugated dienes by Cu 2+ and calculating the lag phase. A prolonged lag phase indicates low susceptibility to oxidation. Taking all of the groups together, the lag time of conjugated diene formation correlated closely with coronary vascular response to acetylcholine (r=0.62, P=0.002). Assignment to a treatment group was also a significant predictor of responsiveness to acetylcholine (P=0.002). The group treated with lovastatin and probucol had a substantially increased lag phase. Figure 9: HMG-CoA reductase inhibitors and antioxidants enhance endothelial activity in hypercholesterolemic patients Based on data from this investigation and related studies by the authors, they conclude that treatment with an HMG-CoA reductase inhibitor combined with an antioxidant provides marked improvement in endothelial function by protecting LDL from oxidation as well as by reducing levels of LDL. Based on all measurements, both VEGF and quinapril significantly improved endothelial function and increased blood vessel density (P<0.01). FUTURE CLINICAL TRIALS: IDENTIFYING TREATMENTS FOR CVD AND CHD BASED ON MECHANISMS OF ACTION The studies highlighted in this monograph are only a few of the clinical trials that have investigated therapies to ameliorate endothelial dysfunction. This monograph has focused on trials of ACE inhibitors as well as 2 trials of HMG-CoA reductase inhibitors that studied the efficacy of these agents at reducing endothelial dysfunction. Overall, the results from the numerous studies conducted so far are varied and may suggest mechanistic differences between ACE inhibitors. This concept was proposed in the study discussed earlier by Hornig; the authors stated, differences exist among ACE inhibitors with regard to their ability to improve endothelial function. However, a clear understanding of the mechanistic and functional differences among ACE inhibitors is lacking. New clinical studies with larger patient populations are needed to elucidate differences in the ways ACE inhibitors reduce hypertension by attacking its underlying endothelial pathology. Some ongoing or recently completed trials such as QUIET, HOPE, ALLHAT and PEACE that have yet to be published may further knowledge in this area. 3 Figure 10: Selected studies of ACE inhibition in endothelial function 7

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