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Dyslipidemia alone does not fully explain the increase in cardiovascular events in patients receiving HIV protease inhibitor (PI)-based treatment. Some PIs, such as indinavir, directly induce endothelial dysfunction, an effect that may mediate that portion of the increase in cardiovascular events that is not attributable to dyslipidemia.
Endothelium-dependent vasodilation (EDV), insulin-mediated vasodilation, and whole body and leg glucose uptake during a euglycemic hyperinsulinemic clamp (40 mU/m2/min) were measured in healthy men before and after four weeks of placebo (N=12), atazanavir 400 mg daily (N=9), or lopinavir-ritonavir 400 mg-100mg twice daily (N=9).
Median age (36 years) and mean body mass index (23.4 ± 2.6 kg/m2) did not differ between groups. EDV, the leg blood flow response to intrafemoral artery infusion of 15 μg/min of the endothelium-dependent vasodilator methacholine, did not change after 4 weeks of treatment in any group: 154 ± 102% above basal at baseline and 242 ± 254% at week 4 with atazanavir (p=0.36), 82 ± 63% and 76 ± 79% at week 4 with lopinavir-ritonavir (p=0.68), and 109 ± 84% and 127 ± 153% at week 4 with placebo (p=0.63; between groups p=0.55). The response to the endothelium-independent vasodilator nitroprusside was not different at week 4 in any group, nor was insulin-mediated vasodilation, leg or whole-body insulin-mediated glucose uptake (all within-group p-values >0.1).
Unlike the dramatic impairment seen with indinavir, the newer PIs atazanavir and lopinavir-ritonavir do not induce endothelial dysfunction in healthy subjects. Thus, endothelial dysfunction does not appear to be a PI class effect. The cause of the non-lipid-mediated increase in cardiovascular events reported with PIs remains unclear.
Combination antiretroviral therapy for HIV infection is associated with an increased risk of myocardial infarction, particularly with the use of HIV-1 protease inhibitors (PIs) [1, 2]. Prospective data suggest that antiretroviral therapy-associated lipid disorders alone do not explain all of this increased risk . Endothelial dysfunction is a critical initial step of atherogenesis which contributes to the progression and clinical manifestations of atherosclerosis [3, 4]. PI-based antiretroviral regimens have been associated with endothelial dysfunction . Thus, PI-related endothelial dysfunction may be responsible for increased cardiovascular events under PI therapy that is not merely due to PI-associated lipid changes.
Four weeks of administration of the HIV-1 PI indinavir significantly impaired endothelial function in healthy, non-obese HIV-uninfected subjects in three different studies [6-8]. The magnitude of indinavir-induced endothelial dysfunction in these studies was great, comparable to the degree seen with type 2 diabetes mellitus. However, indinavir is now seldom used in clinical practice. In the United States, the newer PIs atazanavir and the fixed-dose combination of lopinavir-ritonavir account for nearly 70% of the total PI use as of August, 2007 (IMS National Prescription Audit, 8/24/2007). We hypothesized that lopinavir-ritonavir would have an adverse effect on endothelial function, due to its adverse effects on serum lipid parameters [9, 10] or, in some studies , insulin resistance. We further hypothesized that atazanavir, which lacks adverse effects on lipids and insulin resistance [10, 11], would not affect endothelial function. We measured the effect of standard doses of atazanavir, lopinavir-ritonavir, or placebo, each given for four weeks on endothelium-dependent vasodilation (EDV), insulin sensitivity at the level of the whole body and skeletal muscle, and insulin effects on the vasculature in a group of healthy non-obese subjects without HIV infection in a randomized trial.
Demographic characteristics are shown in Table I. All subjects (N=30) were men (mean age 37 ± 1 years) who were HIV seronegative, healthy, not obese (total body fat ≤ 27% by dual x-ray absorptiometry scan, Lunar DPX-L; Lunar Corp., Madison, WI, system software 4.6b), normotensive by cuff measurements per JNC VI criteria , had normal 75 g oral glucose tolerance tests per American Diabetes Association criteria  and normal lipid profiles per National Cholesterol Education Program III criteria  and were not taking any over-the-counter or prescription medications including herbal supplements. None of the women who were screened for the study met the total body fat entry criterion. Studies were approved by the Indiana University-Purdue University and Clarian Health Partners Institutional Review Board, and all volunteers gave written informed consent. Subjects were instructed to maintain their usual dietary and physical activity habits during study.
All vascular infusion drugs were diluted in normal saline, achieving concentrations of 25 μg/ml of methacholine chloride (Clinalfa, CH-4448 Läufelfingen, Switzerland) and 7 μg/ml of sodium nitroprusside (SNP; Roche Laboratories, Division of Hoffman-La Roche, Nutley, NJ). Atazanavir 200 mg capsules (Reyataz®, Bristol-Myers-Squibb Co., Princeton NJ) and lopinavir-ritonavir 133/33.3 mg soft gel capsules (Kaletra®, Abbott Laboratories, Abbott Park IL) were kindly provided by their respective manufacturers. Subjects were randomly assigned in a double-blind fashion to receive atazanavir 400 mg (two 200 mg capsules) daily plus lopinavir-ritonavir placebo (N=9), lopinavir-ritonavir 400 mg/100 mg (three soft gel capsules, each containing 133.3 mg lopinavir and 33.3 mg ritonavir) twice daily with food plus atazanavir placebo (N=9), or placebos for both drugs (N=12). Randomization was stratified by body mass index (BMI) of >24 or <24 kg/m2. Subject compliance was assessed by self-report and manual pill counts.
Thirty healthy males were studied between November 2003 and September 2006. Subjects were admitted to the Indiana University General Clinical Research Center one day before study and were fed a weight-maintaining diet. All vascular and metabolic studies (Figure 1) were performed after an overnight fast and abstinence from smoking. The final dose of study drugs were administered at 7 AM on the last day of the study, at ~1 hour prior to methacholine infusion and ~5 hours prior to the final hour of the clamp study. Subjects had body weight, height, basal heart rates, systolic and diastolic blood pressure measurements done at baseline and after 4 weeks of study drug administration.
Hemodynamic measurements (see Figure 1) were obtained with the subjects in the supine position as previously described . Leg blood flow was measured using a modified thermodilution technique. Basal leg blood flow measurements were done at least 30 minutes following catheter placement, then leg blood flow was measured in response to graded intrafemoral artery infusions of 5, 10 and 15 μg/min of the endothelium-dependent vasodilator methacholine. After a washout period following the methacholine infusion where a return of leg blood flow values to basal levels was documented, leg blood flow was then measured in response to graded infusions of 1.75, 3.5 and 7 μg/min of the endothelium-independent vasodilator sodium nitroprusside. Subsequently, a euglycemic hyperinsulinemic clamp study (described below) was initiated and continued for a minimum of 240 minutes. During the fourth hour of the insulin infusion, insulin-mediated vasodilation was assessed by measurement of leg blood flow and compared to basal leg blood flow values. The first 14 subjects received an infusion of NG-Mono-Methyl-L-Arginine (L-NMMA) at the end of the clamp study (data not shown).
Immediately following baseline assessments of leg blood flow, a euglycemic hyperinsulinemic clamp study using an insulin infusion of 40 mU/m2/min was initiated, as in . Subjects were studied in the supine position with a catheter inserted in an antecubital vein for infusion of glucose, insulin, and potassium. The femoral arterial and venous lines described above were used for drawing blood samples. The clamp technique was a modification of the Andres method . Arterial plasma glucose values were determined at 5-minute intervals and the glucose infusion rate adjusted to maintain normoglycemia (target plasma glucose value 90 mg/dL). Venous plasma glucose values were measured at 20-minute intervals for the first three hours of the clamp study, and at 5-minute intervals for the last hour of the clamp study. The clamp procedure was continued for at least 240 minutes after initiation of the insulin and glucose infusions. To prevent hypokalemia, potassium was infused during the studies at a rate of 0.0038 meq/kg/min.
Glucose was assayed by the glucokinase method at the bedside using a YSI apparatus (Yellow Springs Instruments, Yellow Springs, OH). Plasma adiponectin, resistin, and total plasminogen activator inhibitor-1 levels were measured by immunoassay (LINCOPlex assay, Millipore, Billerica MA). Serum lipids were measured by standard enzymatic techniques. Low density lipoprotein cholesterol levels were calculated using the Friedewald formula . Atazanavir, lopinavir, and ritonavir levels were measured by high-performance liquid chromatography at the University of Alabama at Birmingham Antiviral Pharmacology Laboratory.
Changes in leg blood flow are expressed as absolute change (Δ) and percent change (%Δ) to adjust for differences at baseline. Whole body glucose uptake (M) was calculated as the mean glucose infusion rate normalized for body weight and corrected for changes in the glucose pool sizes, according to methods described by DeFronzo  using data from a one hour period late in the clamp (160-220 minutes) before any vasoactive agents were infused. This time period was used to avoid the effect of any L-NMMA infusion on glucose uptake results. Arterio-venous (A-V) glucose differences were calculated [(arterial glucose)-(venous glucose)] every 5 minutes during the last hour of the clamp study. Leg glucose uptake was calculated as the product of leg blood flow * A-V glucose difference during the last hour of the clamp study.
The pre-specified primary study endpoint was EDV, expressed as the increment in leg blood flow from basal (Δ% leg blood flow) in response to maximal methacholine (15 μg/min) . The study was powered to show a within-arm change of 1 standard deviation by a paired t test with 80% power to detect a difference with a nominal level of significance of 0.05. In our prior data with indinavir , the change in the percent increase from basal in leg blood flow during maximal doses of methacholine was 145 ± 80% from baseline values. In the current study, we had power to detect within-arm changes of 80% from baseline, a change that was considerably smaller than what was observed with indinavir. Results are shown as the mean ± SD. Paired t-test was used to compare within-arm differences in metabolic and vascular variables before and after indinavir. To compare results across study arms, analysis of variance (ANOVA) was used for continuous variables and Fisher's exact test was used for categorical variables. Statistical significance was defined as p< 0.05. Analyses were performed with the SAS version 9.1 (SAS Institute, Cary, NC).
Treatment had no effect on BMI or fasting plasma glucose (Table 1). Plasma levels of adiponectin, resistin, and plasminogen activator inhibitor-1 did not change in any group (Table 1). Minor, but statistically significant, increases in serum aminotransferase levels occurred only in the placebo group. A doubling of total bilirubin levels occurred with atazanavir treatment, and a slight but statistically significant increase in total bilirubin also occurred with placebo. Total cholesterol increased significantly only with placebo, but the overall ANOVA between groups for change in cholesterol was not significant. Triglycerides increased significantly only with lopinavir-ritonavir (77 ± 29 mg/dL to 128 ± 48 mg/dL, p=0.002; p-value for between-groups difference=0.002). Low density lipoprotein cholesterol and non-high density lipoprotein cholesterol levels did not change in any group, whilst an increase in high density lipoprotein cholesterol occurred only in the placebo group (Table 1).
Mean glucose concentrations during the clamp (Table 2) were similar before and after study treatment. A-V glucose difference, leg glucose uptake, and whole-body glucose uptake (M) were not affected by any study treatment. Subjects had mean pre-dose and post-dose plasma drug levels as follows: Atazanavir 0.49 ± 0.69 and 1.23 ± 1.19 μg/mL, respectively; lopinavir 5.20 ± 5.17 and 7.61 ± 1.91 μg/mL, respectively; ritonavir 0.33 ± 0.35 and 0.56 ± 0.34 μg/mL, respectively.
Mean arterial pressure decreased slightly with lopinavir-ritonavir, from 94 ± 6 mm Hg to 91 ± 6 mm Hg (p=0.03, Table 3). Leg blood flow increased in a dose-dependent fashion in response to the endothelium dependent vasodilator methacholine at baseline and with both study drugs and with placebo (Table 3, Figure 2). The primary study endpoint of EDV, the increment in leg blood flow (Δ% leg blood flow) in response to maximal methacholine (15 μg/min) did not change significantly after any of the study treatments. With lopinavir-ritonavir (Figure 2B) and with placebo (Figure 2C), the dose-response curves were very similar pre and post study drug. With atazanavir (Figure 2A), the greater increase in leg blood flow post study drug was not statistically different from baseline (p=0.36). Leg blood flow in response to insulin during the final hour of the clamp increased similarly in all arms, before and after study treatment (Table 3).
The newer PIs atazanavir and lopinavir-ritonavir do not induce endothelial dysfunction in healthy subjects, in contrast to the dramatic impairment of EDV in healthy subjects seen with four weeks of the older HIV-1 PI indinavir [6-8]. These observations suggest that endothelial dysfunction is not a PI drug class effect. Additionally, there was no change in insulin's effects on blood flow or insulin-mediated whole-body or leg glucose uptake with either study treatment, indicating no impairment of insulin sensitivity.
Human data [5-8] and experimental models [18-22] have implicated PIs as a cause of endothelial dysfunction. The mechanism appears to be impaired nitric oxide bioavailability [8, 19]. Stein and colleagues  documented dyslipidemia and severe endothelial dysfunction in a cross-sectional observational study of subjects who received long-term PI-based antiretroviral therapy (mean 70 total months, which included 31 months on a PI), but not in those treated for HIV without a PI. Importantly, half of their PI-treated subjects received the PI indinavir . More contemporary studies in which few subjects received indinavir have failed to confirm a role for PI-containing antiretroviral regimens in endothelial dysfunction . In fact, there was a nonsignificant trend for better endothelial function measured by brachial flow-mediated dilation among subjects receiving PIs (predominantly nelfinavir and lopinavir-ritonavir) in that study . Similarly, use of the PI lopinavir-ritonavir was the strongest predictor examined of better endothelial function by brachial flow-mediated dilation in a small cross-sectional study .
Although not all PIs have been examined in this manner, in studies in healthy subjects only indinavir has been implicated as a cause of endothelial dysfunction [6-8] and thus this effect may be agent-specific. Consistent with our results, lopinavir-ritonavir administration to six healthy subjects for four weeks actually led to a non-significant percentage increases in forearm blood flow during acetylcholine infusion as compared to baseline values . In experimental models only indinavir and ritonavir have been consistently implicated to cause endothelial dysfunction. In our study, only low-dose ritonavir (total daily dose 200 mg/d) was used, which may have resulted in insufficient drug exposure to lead to endothelial dysfunction. As is the case with glucose and lipid metabolism effects , different PIs appear to have divergent effects on endothelial function.
No effects on glucose metabolism, including insulin stimulated whole-body glucose uptake and leg glucose uptake, were observed with four weeks of study drug administration. This is in contrast to studies that demonstrated reduced whole-body glucose uptake, or insulin resistance, after four weeks of indinavir and using an insulin clamp dose similar to that used in the present study [6, 27]. Similar negative results were seen with lopinavir-ritonavir administered for 4 weeks to healthy subjects , although five days of lopinavir-ritonavir caused insulin resistance in one study . We did not observe increases in adiponectin levels with either study drug, in contrast to previous reports that included indinavir [6, 28] and lopinavir-ritonavir .
We observed the expected increase in indirect bilirubin levels with atazanavir . Triglycerides increased with lopinavir-ritonavir, while total and LDL cholesterol did not change, similar to other studies of healthy subjects [10, 28]. The cause of the increases in total and HDL cholesterol with placebo are not clear.
Limitations to this study include a relatively small number of subjects and a short observation time, which may not detect more subtle long-term effects. However, significant endothelial function was documented with the PI indinavir in several studies that included fewer subjects studied for a similar period of time [6, 8]. It is also possible that delayed endothelial dysfunction may occur with PIs such as lopinavir-ritonavir due to chronic elevations in triglycerides and potentially atherogenic lipoproteins. Because HIV-uninfected subjects were studied, our data cannot address the effect of HIV infection on endothelial function. Similarly, our data cannot address the effects of nucleoside reverse transcriptase inhibitors  on endothelial dysfunction.
We conclude that the PIs atazanavir and lopinavir, when administered for four weeks to healthy nonobese subjects, do not induce endothelial dysfunction or insulin resistance. The mechanisms behind the divergence of these results with the dramatic endothelial dysfunction seen with indinavir [6-8] deserve further study. Because of the great metabolic and vascular diversity of existing PI agents, it will be important to study the effects of all PIs in common clinical use and those in clinical development for their metabolic and vascular properties.
Financial support. This work was supported by grants HL72711 and M01-RR00750 from the NIH, and gifts of drug from Abbott Laboratories and Bristol-Myers-Squibb Co. Employees of Abbott and Bristol-Myers-Squibb reviewed the manuscript prior to submission and provided comments but were not involved in the approval of the final version or the decision for journal submission.
We are indebted to the subjects who volunteered for this study, to the staff of the Indiana University General Clinical Research Center, and to Kathy L. Flynn and Gina-Bob Dubé for managing the references.
Potential conflicts of interest. M.P.D. has served as a consultant or received research support from Abbott, Bristol-Myers Squibb, GlaxoSmithKline, and Merck. C.S., M.G., and K.J.M. - no conflicts.