Clinical trial NCT01405456
Conditions: HIV
Keywords: HIV-infection eplerenone aldosterone visceral fat lipodystrophy
Sponsor: Massachusetts General Hospital
Start date: 2012-01-01; Completion date: 2017-11-01
Enrollment: 46 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to test the effects of a drug, eplerenone, along with lifestyle modification to affect sugar metabolism, body fat distribution, and cardiovascular health in HIV-infected individuals. In non-HIV-infected individuals, recent data has shown that aldosterone, a hormone that regulates salt and water balance, is increased in association with increased belly fat and decreased insulin sensitivity. In HIV-infected individuals, aldosterone appears to be higher in individuals with increased belly fat, and increased aldosterone appears to be strongly associated with impaired sugar metabolism. In this study, the investigators will test the effects of eplerenone, which is a medication that blocks the actions of aldosterone, along with lifestyle modification. The investigators hypothesize that eplerenone may improve sugar metabolism, improve markers of cardiovascular health, and reduce fat accumulation in liver and muscle.

Detailed description:
The study is 12 months long, with two phases. In the initial, 6-month phase, volunteers are randomly assigned to receive either eplerenone or placebo (an inactive pill). In addition, all volunteers will receive counseling about healthy diet and lifestyle, and will be asked to follow guidelines for a healthy level of physical activity. In the second 6-months of the study, all volunteers will continue to receive lifestyle modification and all will receive eplerenone.

Arms: arm 1: First 6 months: eplerenone 50mg daily along with lifestyle modification (counseling regarding lifestyle and diet, as well as following healthy activity guidelines) Second 6 months: same (eplerenone open label during second 6 months) arm 2: First 6 months: placebo pill daily along with lifestyle modification (counseling regarding lifestyle and diet, as well as following healthy activity guidelines) Second 6 months: eplerenone (open label) 50mg daily along with continued lifestyle modification
Interventions: intervention 1: Eplerenone and lifestyle intervention 2: placebo and lifestyle
Intervention descriptions: intervention 1: eplerenone 50mg by mouth daily as well as lifestyle counseling intervention 2: placebo pill daily and lifestyle counseling

Protocol documents:
Prot_SAP_001.pdf: 
Randomized placebo-controlled trial to investigate the effects of eplerenone in 
patients with HIV-associated abdominal fat accumulation 
 
Detailed Protocol Version 14 
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Official Title of the Study:  Eplerenone in HIV Associated Abdominal Fat Accumulation 
 
PI: Steven K. Grinspoon, MD 
 
NCT Registration: NCT01405456 
 
Date of Document: 12/1/2016 


Randomized placebo-controlled trial to investigate the effects of eplerenone in 
patients with HIV-associated abdominal fat accumulation 
 
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I.  Background and Significance 
 
 
Metabolic abnormalities and body composition changes are associated with 
combination antiretroviral therapy (cART) in HIV-infection.  Recent studies have shown 
that hypertension [1], impaired glucose tolerance [2], Type 2 diabetes [3, 4], increased 
carotid intima-media thickness (cIMT) [5] as well as coronary atherosclerosis [6] and 
myocardial infarction [7] are more prevalent in HIV-infected individuals than in non-
infected controls.  Visceral adipose tissue (VAT) is increased in HIV-infected patients 
compared to non-infected controls [8] and is associated with cardiac risk factors such as 
decreased insulin sensitivity [9, 10], hypertriglyceridemia and low HDL [11], increased 
systemic inflammatory markers [12-14], and increased subclinical atherosclerosis [15].  
At this time there is no standard of care to adequately treat these abnormalities.   
 
 
The etiology of body composition changes, metabolic abnormalities, and 
increased cardiovascular risk is multifactorial and likely includes perturbation of multiple 
endocrine axes.  In non-HIV-infected individuals, recent data has shown that 
aldosterone, a mineralocorticoid hormone, is increased in association with increased 
VAT [16] and decreased insulin sensitivity [17].  We have demonstrated increased 24-
hour urine aldosterone in HIV-infected women with increased visceral fat accumulation, 
compared to age and BMI-matched healthy controls [18]. Urinary aldosterone secretion 
was strongly related to VAT in this study. In addition, we also observed an independent 
relationship between increased urinary aldosterone and hemoglobin A1c, which was 
independent of BMI or visceral adiposity.  Furthermore, emerging animal and human 
studies have suggested effects of mineralocorticoid blockade on insulin sensitivity, 
inflammation, and hepatosteatosis [19, 20].   
 
Physiology of aldosterone and the mineralocorticoid receptor 
 
Recent data suggest that aldosterone and mineralocorticoid receptor (MR) 
activation may play a role in cardiovascular and metabolic disease [21, 22].  In vitro 
studies demonstrate effects of aldosterone on adipocyte differentiation, adipocytokine 
production, lipid metabolism, and insulin signaling.  The physiologic relevance of these in 
vitro effects is unclear based on our current understanding of aldosterone’s mechanism 
of action.  The mineralocorticoid receptor, which is the only identified receptor for 
aldosterone to date, has equal affinity for aldosterone and cortisol.  Certain tissues, 
including kidney, colonic epithelium, vascular smooth muscle [23], and endothelium [24], 
highly express 11β hydroxysteroid dehydrogenase 2 (11βHSD2), which inactivates 
cortisol and facilitates aldosterone’s specificity of action at the MR.  In other tissues, 
including cardiac and adipose tissue, MR expression is present, but significant 
expression of 11βHSD2 has not been identified [25].  As cortisol circulates in 
concentrations approximately 1000-fold higher than aldosterone under normal 
physiologic conditions, cortisol is thought to be the primary agonist for the MR in these 
tissues, with changes in aldosterone concentration presumably of little physiologic 
significance.  However, emerging evidence challenges this paradigm.  First, numerous 
effects of aldosterone occur too rapidly to attribute to the classic genomic model of 
transcription regulation [26, 27].  These actions, which include stimulating proliferation of 
vascular smooth muscle cells [28] and inducing arterial vasoconstriction [29], may be 
mediated by non-genomic signaling through the MR or may involve a separate receptor 
for aldosterone that has yet to be identified.  Second, animal studies suggest that 


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glucocorticoid may act as an antagonist rather than an agonist of the MR in some 
tissues [30-32].  In a rodent model, infusion of aldosterone alone caused hypertension 
and cardiac fibrosis, whereas concomitant infusion of 30-fold corticosterone significantly 
attenuated the development of hypertension and fibrosis [31].  Moreover, transgenic 
mice that overexpress 11βHSD2 in cardiomyocytes, causing decreased glucocorticoid 
occupancy of the MR and increased signaling by aldosterone, developed cardiac 
fibrosis, dilated cardiac hypertrophy, and heart failure [32].  Taken together, these data 
suggest that aldosterone may have physiological relevance even in tissues without 
significant 11βHSD2 activity.  Increasing evidence suggests that aldosterone may be 
increased in individuals with increased VAT and may play a significant role in lipid 
metabolism, glucose homeostasis, and inflammation.   
 
Interactions between aldosterone, mineralocorticoid receptor, and adipose tissue 
 
Abundant evidence suggests a bidirectional relationship between adipose tissue 
and aldosterone, with adipose tissue potentially increasing aldosterone levels, and 
aldosterone increasing adipogenesis and lipid storage in adipocytes.  Regarding the 
effect of adipose tissue on aldosterone production, angiotensinogen production has 
been demonstrated in both differentiated 3T3-L1 adipocytes [33] and in human 
abdominal subcutaneous (SC) adipose tissue [34].  Angiotensin converting enzyme 
(ACE) mRNA is also present in human SC adipose tissue [34].  Further, Ehrhart-
Bornstein et al. have demonstrated that secretory products from human adipocytes can 
stimulate mineralocorticoid steroidogenesis in human adrenocortical cells even in the 
presence of angiotensin receptor blockade with valsartan, suggesting that other 
“mineralocorticoid releasing factors” in addition to angiotensinogen may be produced by 
adipocyte tissue [35]. Extending on this work, this group has subsequently demonstrated 
that adipocyte-derived factors induce Wnt signaling and increase StAR promoter activity, 
increasing production of cortisol and aldosterone [36].  Further, adipocyte-derived factors 
also appear to sensitize human adrenocortical cells to angiotensin II, also contributing to 
increased aldosterone production [37].  These mechanisms may underlie the numerous 
reports of increased systemic aldosterone levels with increasing body fat.  Twenty-four 
hour urine aldosterone/creatinine is higher in animal models of obesity (db/db mice) 
compared to lean (db/+) mice [19], and, in non-HIV populations, both 24-hour urine 
aldosterone and angiotensin II-stimulated aldosterone are higher in overweight and 
obese normotensive adults compared to lean controls [38].  Some reports suggest that 
the relationship of aldosterone and body fat may be largely mediated by increased VAT.  
Goodfriend et al. demonstrated that plasma aldosterone correlates with VAT in 
normotensive women [16], and, as above, we have demonstrated a positive relationship 
of 24-hour urinary aldosterone with VAT in HIV-infected women [18].   
 
In addition to evidence that adipose tissue increases aldosterone production, 
recent studies suggest that MR activation may play an important role in adipogenesis 
and lipid metabolism.  MRs have been identified in adipocytes from several fat depots, 
including both VAT and SAT from humans [25] and murine brown adipocytes [39].  
Furthermore, several groups have shown that aldosterone stimulates adipocyte 
differentiation and increases triglyceride accumulation [40-42]. In adrenalectomized male 
rats, aldosterone infusion over 12 days resulted in body weight gain correlating to 
epididymal fat pad mass, but not to percent tissue water, suggesting that aldosterone 
stimulates increase in adipose tissue [43].  Notably, as 11HSD2 is not highly expressed 
in adipocytes [25], MR activation, by either cortisol or aldosterone, may be a more 
relevant focus in adipose tissue than aldosterone levels per se.  In this respect, MR 


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appears to play a crucial role in adipocyte differentiation and lipid accumulation.  In 3T3-
L1 cells, MR but not glucocorticoid receptor (GR) downregulation by siRNAs inhibited 
adipocyte differentiation and decreased cell triglyceride content [44].  Furthermore, 
whereas murine adipocytes lacking GR show mild impairment in lipid accumulation 
during early differentiation, adipocytes lacking MR demonstrate failure to accumulate 
lipid throughout differentiation [39]. These observations are supported by in vivo models 
of ob/ob and db/db mice, in which MR blockade with eplerenone significantly decreased 
the number of hypertrophic adipocytes in epididymal fat [45]. Blockade of MR, which we 
propose to study in this grant application, would block MR activation by cortisol as well 
as aldosterone in adipose tissue, and may decrease lipid accumulation and ultimately 
affect body composition.  There is also some evidence to suggest that MR blockade may 
affect hepatosteatosis, which is prevalent in HIV-infected individuals in association with 
increased abdominal adiposity [46, 47].  In a murine model of diet induced obesity, 
Wada et al. demonstrate that spironolactone significantly decreases hepatic triglyceride 
accumulation as well as decreasing serum triglyceride, total cholesterol, and free fatty 
acid [20].  In humans, Fallo et al. demonstrate an increased prevalence of 
hepatosteatosis in patients with primary aldosteronism (PA) compared to normotensive 
controls, but the prevalence in PA (57.5%) was not different from the prevalence found 
in a matched cohort with low-renin essential hypertension (55.8%) [48].  Further studies 
are clearly needed to determine whether mineralocorticoid activity affects hepatic 
triglyceride accumulation.  Aim I of the current proposal will investigate relationships 
between aldosterone level and body fat distribution, including ectopic fat storage in liver. 
 
Aldosterone, Mineralocorticoid Receptor, and Insulin Resistance 
 
             Multiple epidemiologic studies have found positive associations between 
aldosterone and markers of insulin resistance in overweight and obese patients [16, 17, 
38, 49], hypertensive patients [49], and patients with heart failure [50]. Moreover, a high 
prevalence of impaired glucose tolerance has been recognized in primary aldosteronism 
(PA) for several decades [51].  In recent series, patients with PA are generally reported 
to have impaired glucose homeostasis in comparison to both controls and patients with 
essential hypertension [52, 53], although one recent study refutes this association [54].  
Further, treatment of PA with either surgery or aldosterone antagonism improves 
glucose homeostasis in this population [52].  Genetic polymorphisms in aldosterone 
synthase have also demonstrated an association with both fasting glucose and glucose 
following OGTT, which further suggests a possible relationship between aldosterone and 
glucose homeostasis [55].   
 
Although impaired insulin secretion secondary to hypokalemia is one likely cause 
of impaired glucose homeostasis in hyperaldosteronism, in vitro and animal studies have 
demonstrated additional mechanisms linking aldosterone directly to glucose 
homeostasis.  Hitomi et al., using vascular smooth muscle cells from rat aorta, 
demonstrate that aldosterone dose-dependently decreases insulin receptor substrate-1 
(IRS-1), prevents insulin-induced Akt phosphorylation, and reduces insulin-stimulated 
glucose uptake [56].  Notably, all of these effects are reversed with addition of 
eplerenone to achieve MR blockade [56].  Wada et al. demonstrate similar effects of 
aldosterone to reduce IRS-1 and IRS-2, decrease Akt phosphorylation, and decrease 
glucose uptake in 3T3-L1 adipocytes [57].  In their model, however, glucocorticoid 
antagonism with RU486 but not MR antagonism with eplerenone reversed the effects of 
aldosterone, suggesting possible action through the GR rather than the MR [57].  
Another potential mechanism has been demonstrated in vivo using Wistar rats, in which 


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aldosterone treatment significantly decreased glucose uptake in muscle and liver in 
conjunction with decreased GLUT4 protein expression in muscle and decreased GLUT2 
protein expression in liver [58], suggesting that aldosterone may directly contribute to 
insulin resistance at the level of the liver and the muscle.  In vivo rodent models also 
demonstrate beneficial effects of mineralocorticoid blockade on glucose homeostasis.  
Hirata et al. demonstrate that eplerenone treatment of ob/ob and db/db mice significantly 
reduced glucose and HOMA-IR [45], and Guo et al. also describe decrease in HOMA-IR 
in db/db mice treated with eplerenone [19].  Wada et al. demonstrate similar results in a 
murine model of diet induced obesity, with improved response to both glucose tolerance 
test and insulin tolerance test following treatment with spironolactone [20].  Taken 
together, these data strongly support a relationship between mineralocorticoid activity 
and glucose homeostasis and suggest that mineralocorticoid blockade may be a useful 
strategy to improve glucose homeostasis in models of increased adiposity. 
 
Mineralocorticoid receptor and subclinical inflammation 
 
HIV-infection is accompanied by increased markers of systemic inflammation 
[12-14], and levels of inflammatory cytokines predict atherosclerosis [6, 59] and 
myocardial infarction [60] in HIV-infected individuals.  In addition to the recognized role 
of angiotensin II to stimulate production of reactive oxidant species [61, 62], recent 
studies suggest that MR activation may play a distinct role in both cardiovascular and 
systemic inflammation.  In Sprague-Dawley rats, aldosterone administration for 4 weeks 
produced significant medial thickening of coronary arteries as well as perivascular 
leukocyte infiltration and, in some animals, significant leukocyte infiltration into cardiac 
tissue with subsequent cardiomyocyte degeneration and necrosis [63].  These changes 
were accompanied by increased gene expression of cyclooxygenase-2 (COX-2), 
macrophage chemoattractant protein-1 (MCP-1), osteopontin, and ICAM-1.  
Concomitant eplerenone treatment significantly ameliorated inflammatory changes and 
attenuated alterations in gene expression [63].  Although a role of hypertension cannot 
be excluded in this model, a model of atherosclerosis-prone apoE deficient mice 
supports the association between MR activation and vascular inflammation, independent 
of blood pressure.  Six weeks of high-cholesterol diet in these mice led to formation of 
atherosclerotic lesions in the aorta, with increased superoxide production and increased 
vascular expression of NADPH oxidase, TNFα, MCP-1 [64].  Eplerenone treatment did 
not alter total cholesterol levels or blood pressure, but significantly reduced 
atherosclerotic lesion area and decreased superoxide production as well as expression 
of NADPH oxidase, TNFα and MCP-1 [64].  Moreover, although treatment with valsartan 
alone also had beneficial effects to decrease lesion area and reduce oxidative stress, 
the combination of valsartan and eplerenone was significantly more effective than 
valsartan alone to reduce atherosclerotic area and oxidative stress, suggesting an effect 
of MR blockade distinct from that of ATII blockade [64].  Similar results have been 
reported in a rabbit model, with eplerenone treatment significantly improving endothelial 
function and decreasing superoxide production and NADPH oxidase activity in aorta of 
animals fed a high-lipid diet [65].  Taken together, these animal data suggest a direct 
effect of MR activation on oxidative stress, endothelial function, and atherosclerotic 
changes in the vasculature. 
 
Effects of aldosterone on adipocyte inflammation and on systemic levels of 
inflammation have also been demonstrated.  Aldosterone has also been shown to 
contribute to angiotensin-II induced increase in C-reactive protein (CRP) [66], and 
aldosterone stimulation of adipose tissue increases mRNA expression of IL-6 and PAI-1 


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[39].  Further, aldosterone increases LDL-oxidation in human vascular smooth muscle 
cells [67].  Evidence that MR blockade ameliorates inflammation confirms that 
inflammatory actions of aldosterone occur via the MR.  Eplerenone treatment in 
angiotensin II-infused, hypertensive rats decreases both serum and urinary CRP [66].  
As described further in our preliminary data, MR blockade by treatment with eplerenone 
in obese db/db mice markedly reversed obesity related increases in mRNA expression 
of TNF- and other inflammatory markers locally in retroperitoneal adipose tissue and 
increased adiponectin mRNA expression [19].  Similarly, Hirata et al. describe numerous 
effects of eplerenone in adipose tissue of ob/ob and db/db mice [45].  Significantly, they 
report that MR mRNA levels in adipose tissue were significantly increased in ob/ob and 
db/db mice compared to lean controls, supporting a key role for MR in adipocyte biology.  
Eplerenone treatment in these mice significantly reduced reactive oxygen species and 
decreased mRNA expression of NADPH oxidase subunits [45].  Further, in ob/ob mice, 
eplerenone reduced mRNA expression of MCP-1, IL-6, and TNFα [45].  Similar effects 
were reported in a diet-induced obesity mouse model, in which spironolactone resulted 
in significant decreases in hepatic expression of TNFα, IL-6, and MCP-1 [20].  Given this 
abundant animal data, research is greatly needed into the effects of aldosterone and MR 
blockade on systemic and local inflammation in humans.  In this study we will carefully 
study the effects of MR blockade on systemic inflammation and endothelial function in 
HIV-infected individuals, with the hypothesis that MR blockade will have beneficial 
effects to reduce systemic inflammatory markers and improve endothelial function in this 
population with increased risk of atherosclerosis and CV morbidity. 
 
Conclusion 
Despite the critical need to reduce CVD risk in HIV-infected patients, a 
comprehensive and effective strategy has not yet been developed to accomplish this 
purpose. Indeed, numerous strategies have been tried with varying degrees of success 
to improve the metabolic and cardiovascular abnormalities in HIV-infected patients.  
Lifestyle modification (LSM) strategies have demonstrated a substantial benefit to 
reduce CVD risk factors among HIV-infected patients. We have previously demonstrated 
positive effects of a well designed, achievable LSM program on HDL, waist 
circumference and HgbA1c [68]. Based on our data, we maintain that LSM should be the 
foundation of any treatment strategy.  As shown in our preliminary data and in other 
studies using this strategy, however, LSM is unlikely to reverse completely the metabolic 
abnormalities associated with HIV [68], [69], [70], [71]. 
 
In summary, epidemiologic studies have established that aldosterone is 
increased in association with increased BMI, and some evidence suggests that VAT in 
particular may be related to increased aldosterone.   In vitro and animal data 
demonstrate a cross-talk between aldosterone/MR and adipose tissue, such that 
increased adiposity may increase aldosterone, and MR activation may, in turn, increase 
adipocyte differentiation and triglyceride accumulation.  Further, increasing research 
demonstrates a role for MR activation in glucose homeostasis, inflammation, and 
atherosclerosis.  Lo et al. [18] demonstrated increased aldosterone levels in association 
with increased VAT in HIV-infected women.  MR activation may be a mediator of some 
of the detrimental effects of excess visceral adiposity in HIV, and MR blockade may 
provide to be an important element of a larger strategy to reverse endocrine 
abnormalities, improve insulin sensitivity, and decrease inflammation in HIV.  
Evidence suggests that MR blockade may have beneficial effects on 
cardiovascular and metabolic parameters, and the selective MR blocker eplerenone has 


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proven safe and effective in non-HIV infected populations with cardiovascular disease.  
Our preliminary data suggest increased aldosterone in HIV-infected individuals with 
increased abdominal adiposity, a population with significant risk of insulin resistance and 
cardiovascular morbidity and for whom no specific treatment is currently available.  MR 
blockade with eplerenone may be one beneficial strategy to address impaired glucose 
homeostasis and cardiovascular risk in this group, but the effects of eplerenone in HIV-
infected individuals with abdominal fat accumulation and impaired glucose tolerance are 
not known.  We hypothesize that by blocking the effects of increased aldosterone 
secretion, eplerenone treatment will significantly improve insulin sensitivity in this 
population and may also decrease visceral adipose tissue, and improve dyslipidemia 
and inflammation, thereby improving vascular function.  In a 6-month, randomized, 
placebo-controlled study followed by 6-month open-label extension, we will test for the 
first time the effects of the selective mineralocorticoid blocker, eplerenone, on markers of 
insulin sensitivity, dyslipidemia, body composition, inflammation and vascular function in 
this population. 
Eplerenone was chosen for this study instead of an ACE-I or Angiotensin II 
Receptor Blocker (ARB) because we want to specifically test the effects of MR blockade, 
including in tissues in which cortisol may active the MR due to lack of significant 
11βHSD2 expression.  Evidence suggests that MR activation in adipose tissue may 
affect adipocyte differentiation and triglyceride accumulation, and cortisol may act 
similarly to aldosterone at the MR in this regard.  As we aim to determine the effects of 
MR blockade on cardiometabolic parameters and body composition, specifically VAT 
accumulation, we have chosen eplerenone in order to block the effects of both 
aldosterone and potentially cortisol at the MR.  Eplerenone was chosen rather than 
spironolactone because eplerenone is more selective for the MR, does not affect adrenal 
steroid synthesis as spironolactone does, and has a better side effect profile.   
 
II.  Specific Aims 
 
To determine if mineralocorticoid receptor blockade with eplerenone among HIV-infected 
patients with increased waist circumference and impaired glucose tolerance will 
ameliorate cardiovascular risk parameters, we will perform a randomized double-blind 
study of eplerenone vs. placebo for 6 months, with a 6 month safety extension.  We will 
provide all subjects with lifestyle modification throughout the study.  We hypothesize that 
addition of eplerenone to lifestyle modification will significantly: 
A. Improve insulin sensitivity as measured by euglycemic hyperinsulinemic 
clamp. 
B. Improve endothelial function and adipokines. 
C. Reduce critical measures of adiposity, including liver fat and IMCL 
 
III. Subject Selection 
 
Inclusion/Exclusion: 
Inclusion criteria:  
1. Increased waist circumference based on NCEP guidelines (>102cm in men and 
>88cm in women) and impaired glucose tolerance (either IFG > 100 mg/dL but < 
126 mg/dL or 2hr glucose > 140 mg/dl but < 200 mg/dL, or fasting insulin >12 
uIU/mL)   
2.   HIV positive for 5 years and on a stable ART regimen for at least 12 months 
3. Age ≥ 30 and ≤ 65 years of age 


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4. Stable CD4 count and HIV viral load in the past year 
 
Exclusion criteria: 
1. ACE Inhibitor, ARB, verapamil, or spironolactone  
2. Potassium supplementation 
3. Estimated GFR<60, creatinine > 1.5 mg/dL 
4. Serum K > 5.5 mEq/L, ALT > 2.5 times the upper limit of normal, Hgb < 11g/dL 
5. Uncontrolled hypertension (SBP ≥ 160 or DBP ≥ 100) 
6. Current or prior steroid use within past 6 months 
7. Known history of diabetes mellitus or current use of anti-diabetic medications 
8. Concomitant use of  full dose ritonavir, nelfinavir, clarithromycin and other strong 
inhibitors of CYP34A 
9. Use of St. John’s Wart (CYP3A4 inducer) 
10. For women: Pregnant or actively seeking pregnancy, breastfeeding, failure to use 
an acceptable non-hormonal form of birth control, including abstinence, barrier 
contraceptives, or non-hormonal IUD.   
11. Estrogen or progestational derivative use within 3 months 
12. Testosterone use for non-physiologic purposes, or physiologic testosterone 
replacement for < 3 months. 
13. Current growth hormone or growth hormone releasing hormone use 
14. Current viral, bacterial or other infections (excluding HIV) 
15. Current active substance abuse 
16. Patients with a significant history of cardiovascular disease, including prior MI or 
stroke 
 
Source of subjects and recruitment methods: 
 
Participants will be recruited from community flyers and announcements at HIV focused 
community centers, community newspaper advertising, and referrals from infectious 
disease physicians and primary care physicians familiar with the study.  Recruitment 
may also be done via RSVP for Health and/or internet announcements such as Partners 
“All User Broadcast.”  Potential participants, from all referral sources, will contact the 
study investigator directly by phone for a pre-study evaluation via telephone to determine 
eligibility.  
 
IV.  Subject Enrollment 
 
Informed consent will be obtained at the screening visit by a licensed physician or nurse 
practitioner, prior to any procedures or interventions.  Potential research subjects will 
have the entire protocol explained in detail and study investigators will review the 
consent form item by item, answering any and all questions.  Once risks and benefits 
have been reviewed and subjects have agreed to participate by signing the consent, the 
screening visit will continue as described in the subject selection section.  Once final 
eligibility has been determined subjects will be scheduled for a baseline visit.   
 
Treatment assignment and randomization: 
 
During the baseline visit randomization will be done.  All subjects will receive lifestyle 
modification as described below.  Subjects will be randomized to receive eplerenone 
50mg po qd or identical placebo for 6 months.  Doses will start at 25mg for the first week 
to avoid hypotension.  The study will be double-blind.  Randomization will be stratified by 


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gender, age (>45 y), and BP (SBP>140 or DBP>90 vs. normotensive).  After the first 6 
months, open-label eplerenone will be given for 6 months in all patients, and lifestyle 
modification will continue.  During the open-label period all subjects will start eplerenone 
25 mg a day for 1 week and dose will be increased to 50 mg a day at the 25 week safety 
visit to maintain blinding.   
 
Prior to randomization in all cases, the subject’s primary care physician will be consulted 
regarding the appropriateness of the protocol including the lifestyle modification 
program.  Subjects are not eligible if they do not meet all of the enrollment criteria 
 
 
V.  Study Procedures 
Data Collection/Study Procedures 
Screen Visit (to determine eligibility)  
1. Informed consent; history and PE, vital signs including blood pressure, and urine 
pregnancy test for all female subjects 
2. Bionutrition: Height, metabolic weight, and anthropometric measurements, 
including waist and hip circumferences, distribution of a 4-day food record 
3. Fasting blood: potassium, creatinine, CBC, ALT, FSH (women only) 
4. 2 hour oral glucose tolerance test 
 
Prior to the baseline, 3, 6 and 12 month visits, subjects will go on a uniform 200 meq/day 
sodium diet.   
Baseline, 6 months, and 12 months: 
1. Urine pregnancy test of all female subjects 
2. MRI/MRS for abdominal visceral adipose tissue, subcutaneous adipose tissue, 
liver fat, intramyocellular lipid content 
3. Flow mediated vasodilation 
4. Bionutrition: DEXA, REE, anthropometric measurements and review of a session 
in the lifestyle modification workbook 
5. Fasting blood will be drawn for CBC, lipid profile, HgbA1c, chem 7, ALT, CD4/8, 
HIV viral load, aldosterone, plasma renin activity, inflammatory markers and 
adipokines: MCP-1, CRP, adiponectin, PAI-1, resistin, TNFα, TNFR2, IL-6. 
SHBG, total and free testosterone, LH, FSH, estradiol (females only). 
6. 24 hour urine will be collected for aldosterone, potassium, sodium, cortisol and 
creatinine 
7. Euglycemic hyperinsulinemic clamp  
8. Interval medical history and physical exam including blood pressure check 
9. Modifiable activity questionnaire 
10. Overnight q 20 minute sampling for GH pulsatility analysis. 
 
Interim Safety Visits 1 wk, 2 wks, 4 wks, 2 months and same schedule post 6 month 
crossover with an additional visit at 10 months 
1. Physical exam and interval medical history including blood pressure check 
2. Blood: creatinine, K, CBC, ALT 
3. Urine pregnancy test of all female subjects 
 
 
3 Month Visit 
1. Urine pregnancy test for all female subjects 
2. Physical exam, including blood pressure check, and medical history 


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3. Euglycemic hyperinsulinemic clamp  
4. Bionutrition:  DEXA, anthropometric measurements, and review of a session from 
the lifestyle modification workbook. 
5. Fasting blood: lipid profile, HgbA1c, chem. 7, CBC, potassium, creatinine, ALT, 
CD4/8, viral load, aldosterone, plasma renin activity, inflammatory markers and 
adipokines: MCP-1, CRP, adiponectin, PAI-1, resistin, TNFα, TNFR2, IL-6.  
SHBG, total and free testosterone, LH, FSH, estradiol (females only).   
6. 24 hour urine will be collected for aldosterone, potassium, sodium, cortisol, and 
creatinine 
7. Optional overnight q 20 minute sampling for growth hormone pulsatility analysis 
8. Modifiable activity questionnaire 
 
Oral Glucose Tolerance Test:  A standard 75g OGTT will be performed on the morning 
of the screen visit following a 12 hour fast, with interval measurements of insulin and 
glucose at 0 and 120 min. 
Euglycemic-Hyperinsulinemic Clamp:  After 12-h overnight fast, patients will receive a 
primed infusion of regular insulin mixed with albumin, 80 mU/m2, for 2 hours, with a 
variable rate of dextrose administered to maintain blood glucose at 5.0 mmol/L (90 
mg/dL).  Blood samples will be collected for glucose at time 0 and every 5 min during the 
2-hour clamp, and samples for insulin will be collected every 20 min from 0-120 minutes.  
Insulin sensitivity will be calculated. 
Optional GH Pulsatility Analysis: Starting at 2000 until 0740 the next morning, fasting 
blood will be sampled every 20 minutes. Approximately 1.5cc of blood will be drawn at 
each time point for a total of 54cc. Mean overnight GH concentration, basal 
concentration, pulse frequency, pulse amplitude and overall pattern regularity will be 
determined using the deconvolution analysis and cluster computerized pulsatility 
program. 
 
1H Magnetic Resonance Spectroscopy & MRI Scanning:  After 12-hour overnight fast 
subjects will under go 1H -MRS of the calf muscle and of the liver.  For calf muscle 1H -
MRS, voxel measuring 15 x 15 x 15 mm (3.4 ml) will be placed on the axial T1-weighted 
slice with largest muscle cross-sectional area of the tibialis anterior and subsequently 
the soleus, avoiding visible interstitial tissue, fat or vessels.  Single-voxel 1H -MRS data 
were acquired using point-resolved spatially localized spectroscopy pulse sequence.  
For liver 1H-MRS, a breath-hold true fast imaging with steady precession sequence will 
be obtained.  A voxel measuring 20 x 20 x 20 mm (8 ml) will be placed within the right 
lobe of the liver, avoiding vessels or artifact.  Breath-hold single-voxel 1H -MRS data will 
be acquired using point-resolved spatially localized spectroscopy pulse sequence 
without water suppression with the following parameters: TE of 30 msec, TR of 1500 
msec, eight acquisitions, 1024 data points, and receiver bandwidth of 2000 Hz.  For 
abdominal visceral and subcutaneous fat areas, conventional MR images will be 
acquired for anatomic reference of 1H-MRS overlays.  Image series will include Axial T1-
weighted localizers.  A single axial slice of the abdomen and mid thigh will be obtained 
with magnetic resonance imaging. Abdominal visceral and subcutaneous fat areas, as 
well as mid thigh subcutaneous fat area will be determined based on offline analysis of 
tracings obtained utilizing commercial software. No intravenous contrast will be used.  
The total amount of time in the magnet will be approximately 1 hour.   
 


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Flow Mediated Vasodilation:  After a 12 hour overnight fast a blood pressure cuff will be 
inflated to 50 mmHg above systolic blood pressure (or 250 mmHg, whichever is less) for 
five minutes.  The diameter of the brachial artery will be recorded by ultrasound under 
no-flow conditions and over a five minute period after the cuff is deflated (reflow). The 
images will be recorded for analysis of the time-course and extent of dilation of the 
brachial artery. 
 
Whole Body Dual Energy X-ray Absorptiometry (DEXA) 
DEXA (Hologic) will be used to determine whole body and regional fat will be assessed. 
The technique has a precision error (1 SD) of 3% for whole body fat and 1.5% for lean 
mass. It estimates body composition from the attenuation of X-rays pulsed in synchrony 
between 100 and 140 kV with the line frequency for each pixel of the scanned image. 
(As an external standard for analysis of body composition, a step phantom, with six 
fields of acrylic and aluminum of varying thickness and known absorptive properties, was 
scanned periodically for calibration.)  The DEXA scan will be used to determine total 
body fat as well as regional body fat measurements. 
 
Anthropometric Measurements 
Subjects’ height and weight will be obtained and plotted on standard weight charts to 
determine percent of ideal body weight. Body mass index will be calculated. 
Anthropometric measurements of waist to hip ratio, leg circumference, arm 
circumference and neck circumference will be performed using a standardized 
technique. Waist-to-hip ratio will be determined from the circumferential measurements 
of the iliac waist and the hips at the level of the iliac crest taken with the patient in an 
upright standing position. 
 
Bionutritional Analysis:  Protein, carbohydrate, fat, micronutrient, dietary supplements 
and alcohol intake will be determined from 4-day food records (3 weekdays and 1 
weekend day) (Nutrition Data systems).   
 
High Sodium Diet:  A high sodium diet will be followed for 6 days prior to the Baseline, 3, 
6 and 12 month visits and consists of the subject’s normal, ad lib diet supplemented with 
high sodium meals and/or chicken broth, and snacks to equal approximately 200 mEq of 
sodium.  The 7th day (last day) of the high sodium diet will be completed on the first day 
of the Baseline, 3, 6 and 12 month visit.  
 
Lifestyle Modification:  Goals derived from the AACE and NCEP-ATP III guidelines and 
the Diabetes Prevention Program are as follows: <35% calories from fat, < 7% calories 
from saturated fat, up to 10% calories from polyunsaturated fat, reduction of trans fatty 
acid intake, up to 20% calories from monounsaturated fat, and 25-35g of fiber per day.  3 
hrs of physical activity/week at moderate intensity, >10,000 steps in daily activity, 
measured by pedometer.  The curriculum is modeled after the Diabetes Prevention 
Program.  Subjects will complete lifestyle sessions in the offices of the Program in 
Nutritional Metabolism, the Clinical Research Center at MGH or by phone conversation 
with protocol study staff trained to implement the curriculum.  The curriculum will be 
introduced and reinforced with weekly sessions during wks 0-24 (see Table), then 
continued with monthly sessions after open label extension. There will also be $100 per 
patient allowance that will be used in individualized ways to promote lifestyle goals:  For 
example, subsidizing a gym membership, purchasing of relevant cookbooks or purchase 


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of appropriate food items from health food stores, etc. 
 
Session                      Topic                                        Initial Session     Reinforcement 
1 
Welcome to the Lifestyle Balance Program 
Week 1 
Week 13 
2 
How to Read a Nutrition Label 
Week 2 
Week 14 
3 
The Components of a Healthy Diet 
Week 3 
Week 15 
4 
Things to Know Before Becoming Active 
Week 4 
Week 16 
5 
Living on Active Lifestyle 
Week 5 
Week 17 
6 
Focus on Fiber 
Week 6 
Week 18 
7 
Whole Grains 
Week 7 
Week 19 
8 
Fats & Cholesterol 
Week 8 
Week 20 
9 
Fruit, Vegetables & Milk 
Week 9 
Week 21 
10 
Fish Meat, Poultry, Eggs, Peas & Beans 
Week 10 
Week 22 
11 
Tips for Health Eating Out 
Week 11 
Week 23 
12 
Food Safety 
Week 12 
Week 24 
13 
Appendix-printable fact sheets and logs 
 
 
 
 
VI.  Biostatistical Analysis 
 
The primary endpoint will be change in insulin stimulated glucose uptake as measured 
by euglycemic clamp. Secondary endpoints include change in levels of adipokines and 
endothelial function.  In addition, we will assess eplerenone’s effects on hepatic fat and 
IMCL. VAT and SAT will be measured simultaneously and controlled for in the analyses 
of hepatic fat and IMCL. Randomization will be stratified by gender, age (< or > 45), and 
BP (SBP >140 or DBP>90, or normotensive).  Wilk-Shapiro test will be used to assess 
for normality of distribution of all variables, and variables that are not normally distributed 
will be log-transformed.  Baseline variables will be compared between treatment groups 
by t-test for continuous variables and Wilcoxon Rank Sum test for variables that are not 
normally distributed.  Any baseline variable that is statistically different between 
treatment groups will be adjusted for in subsequent analysis.  We will analyze the entire 
set of repeated measurements (baseline, 3 and 6 mos) by using the longitudinal mixed 
effects model, using all available data, including interim data from those who do not 
complete the study, to determine differences in longitudinal mean changes between 
epleronone and placebo over 6 mos of treatment (i.e., test for time x group interaction). 
The analyses will be intention to treat, using all interim data for subjects who do not 
complete the study.  We will control for gender, race, anti-hypertensive and lipid lowering 
medication use, urine free cortisol, dietary intake (caloric, macronutrient, alcohol, 
supplements, salt intake), activity, ARV use and smoking. We will analyze the dietary 
data in terms of overall caloric intake and macronutrient composition, to determine 
whether there are any differences in diet by study treatment, or whether change in 
dietary intake patterns, including vitamins, affects the treatment results. Similar analyses 
will be performed for activity using the Modifiable Activity Questionnaire.  In a sensitivity 
analysis if the dropout rate is higher than expected or differs between groups, we will 


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use a LOCF approach carrying forward the last available data to use in the analysis, to 
determine if the dropouts affected the results.   All patients cross-over to open label 
eplerenone from mos 6 to 12. The primary analysis will be the comparison of placebo 
and eplerenone after 6 months and the purpose of the open label design after 6 mos is 
to encourage recruitment and retention. Safety data will be collected at all time points 
and continued treatment over 12 mos will allow us to determine safety and tolerability 
over one year and whether changes in critical endpoints continue to improve with one 
year of dosing. 
 
Sample Size Calculation 
Assuming 25 evaluable patients in each group, the study will have 80% power to detect 
a between groups difference of 0.8 SD in the percent change over 6 mos, at P=0.05 by 
an independent samples t-test.  Thus the power of the longitudinal mixed models 
approach will be greater than the first-and last pair analysis.  Based on data from our 
group, mean±SD of insulin stimulated glucose uptake in this population using our clamp 
protocol is 7.4±1.7mg/kg/min.  The current study will be powered to detect a change in 
glucose uptake of 1.4mg/kg/min, or approximately 20% change, which would be 
physiologically relevant.   
 
VII.  Risks and Discomforts 
 
Epelerenone: Adverse drug reactions associated with the use of eplerenone include: 
hyperkalaemia, hypotension, dizziness, headache, stomach pain, cough, breast 
enlargement, rash, abnormal vaginal bleeding, erectile dysfunction, and altered renal 
function.  Other rare side effects have been reported include fatigue, chest pain, tingling 
in arms and legs, loss of muscle tone, weakness or heaviness in legs, confusion, lack of 
energy, cold, gray skin or irregular heartbeat, and diarrhea.  These potential side effects 
will be assessed for throughout the study.  Any subject with serum K >5.5 mEq/L at 
screen will be excluded from the study.  During the study, a subject who has an elevated 
K of > 5.5 mEq/L at any study visit will have a recheck; if the K remains elevated after 
recheck they will be excluded from the study.  Additionally, subjects who experience 
symptoms of hyperkalemia and have a K > 5.0 mEq/L will be excluded from continued 
participation in the study.  Subjects who have hypotension (SBP<90) at any study visit 
after titration to full dose eplerenone 50mg/d will be given a dose reduction to 25 mg/d.  
If a subject remains hypotensive (SBP <90) or symptomatic of hypotension at the 
reduced dose they will be dropped from the study.  Since patients with renal insufficiency 
may require reduced dosing based on creatinine clearance, subjects with renal 
insufficiency (serum creatinine > 1.5 mg/dl) will also be excluded from participation.  
Concomitant use of full dose ritonavir, nelfinavir, clarithromycin and other strong 
inhibitors of CYP34A (see Appendix A for complete list) will be an exclusion in the study 
as will the use of St. John’s Wart (CYP3A4 inducer). 
 
  
Oral Glucose Tolerance Test:  Subjects may find the 75g glucose beverage distasteful.  
In addition, there is a possibility of “reactive” hypoglycemia in the latter part of the OGTT 
or after its conclusion.  If subjects develop symptoms of hypoglycemia they will have 
their blood sugar tested and will be treated appropriately.  Subjects will be given a meal 
immediately following the conclusion of the OGTT.   
 


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Radiation  
As a result of participation in this study, subjects will be exposed to 4 DEXA scans over 
12 months. The radiation risk associated with the whole body DEXA scan is 
approximately 0.26 mrem of radiation, for a total of approximately 1 mrem during the 
study.  The total radiation exposure associated with this study does not pose excessive 
risk to subjects.   
 
Magnetic Resonance Imaging:  Subjects will be carefully screened for metal implants 
such as surgical clips or pacemakers prior to MRI scanning.  Subjects will be given 
earplugs due to the loud noises during the test.  Subjects who feel uncomfortable in 
confined spaces may have difficulty in the narrow cylinder of the MRI, and the MRI can 
be stopped at any time at the patient’s request. There are no known hazards for 
pregnant women. 
 
Euglycemic Hyperinsulinemic clamp/insulin: Administration of insulin infusion can cause 
hypoglycemia.  Blood sugar will be monitored every 5 minutes during the insulin clamp, 
and 20% dextrose is infused per protocol to achieve target blood glucose of 90mg/dL.  In 
the event of hypoglycemia, 50% dextrose is available at the bedside, and a physician or 
nurse practitioner is present throughout the insulin clamp procedure. There is a 
theoretical risk of infection associated with the use of albumin, a protein purified from 
donated blood. The risk of transmitting disease is reduced by testing blood donors for 
infections, and by heat treating and purifying the albumin. The albumin that will be used 
for the insulin mixture is the same formulation that is commonly used for patients in the 
hospital and is FDA approved. Because of these measures, the risk is considered to be 
very small; no cases of disease transmission have ever been identified for albumin. 
 
Blood Drawing: The total blood drawn for subjects completing the study is equivalent to 
approximately 2 3/4 cups during 12 month period.  Patients who have the optional 
overnight blood sampling will have an additional ¾ cup of blood drawn.  This quantity of 
blood drawing over 12 months does not pose excessive risk to patients.  Patients with a 
hemoglobin < 11 mg/dL will be excluded from the study.  There will be minimal risk and 
discomfort associated with blood drawing and IV placement.  The risks of these 
procedures are minor bruising or bleeding at the site of the blood draw or IV catheter. 
 
Flow Mediated Vasodilation:  There are no known risks of ultrasound measurements. 
There is the possibility of discomfort from having the blood pressure cuff inflated for 5 
minutes, this includes numbness or tingling of the arm.  
 
VIII.  Potential Benefits 
The potential benefits of this study include improved body composition, cardiovascular 
health, and improved glucose and insulin metabolism associated with HIV associated 
metabolic abnormalities.  These benefits may be associated with eplerenone, lifestyle 
modification or both.  In addition, all subjects will be provided with tools to help them 
meet the prescribed goals, including a core curriculum binder.  All of the participant 
educational handouts have been included as attachments to this document.   
 
IX.  Monitoring and Quality Assurance 
 


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Written informed consent for this protocol will be obtained from all subjects at the first 
screening visit, prior to any procedures or interventions.  In all cases, consent will be 
witnessed by an appropriate health care professional (investigator/co-investigator staff).  
All efforts will be made to insure the privacy rights of the subject.  No written or oral 
communication will be made about any subject with anyone other than the subject, 
unless the subject so requests.  Medical information obtained from the study may 
become part of the subject’s permanent hospital record, subject to the confidentiality and 
privacy regulations of the Massachusetts General Hospital.  Subjects will be closely 
monitored to insure that study participation is not adversely affecting their quality of life, 
and any adverse events will be reported in a timely fashion and in accordance with the 
Partners HRC guidelines for adverse event reporting.  Kidney function and potassium 
levels will be serially monitored.  If a subject experiences an opportunistic infection or 
any acute illness, the lifestyle modification program will be held while the subject is 
acutely ill.   
 
Hyperkalemia is a potential side effect of eplerenone use, and contraindications for 
theuse of eplerenone include serum K > 5.5 mEq/L at initiation.  Therefore, any subject 
with serum K >5.5 mEq/L at screen will be excluded from the study.  During the study, a 
subject who has an elevated K of > 5.5 mEq/L at any study visit will have a recheck; if 
the K remains elevated after recheck they will be excluded from the study.  Additionally, 
subjects who experience symptoms of hyperkalemia and have a K > 5.0 mEq/L will be 
excluded from continued participation in the study.  Subjects who have hypotension 
(SBP<90) at any study visit after titration to full dose eplerenone 50mg/d will be given a 
dose reduction to 25 mg/d.  If a subject remains hypotensive (SBP <90) or symptomatic 
of hypotension at the reduced dose they will be dropped from the study.  Since patients 
with renal insufficiency may require reduced dosing based on creatinine clearance, 
subjects with renal insufficiency (serum creatinine > 1.5 mg/dl) will also be excluded from 
participation.  Other side effects have been reported and include dizziness, fatigue, 
diarrhea, headache, stomach pain, cough, breast enlargement or breast enlargement, 
rash, abnormal vaginal bleeding.  Rare but serious side effects include chest pain, 
tingling in arms and legs, loss of muscle tone, weakness or heaviness in legs, confusion, 
lack of energy, cold, gray skin or irregular heartbeat.  These potential side effects will be 
assessed for throughout the study.  
 
Exclusion criteria during the study include use of diabetic agents; initiation of ACE-I, 
ARB, or spironolactone; Hgb < 11; ALT >2.5 x ULN; persistent hyperkalemia (K >5.5 
mEq/L despite recheck and/or symptoms of hyperkalemia if K >5.0 mEq/L).  If a subject 
remains hypotensive (SBP <90) or symptomatic of hypotension after a dose reduction of 
eplerenone to 25mg for this same reason during the study, they will be dropped from the 
study. 
 
 
Data Safety Monitoring Plan 
Prior to the start of study, a data safety monitoring board will be created and this board 
will consist of a statistician, a clinician not directly related to the research project, but one 
who is familiar with HIV infection and its complications, and a member of the HIV 
community.  This board will meet quarterly to review any potential side effects or 
adverse events in relation to the use of eplerenone or participation in the study. The 
DSMB will meet and review adverse events in accordance with the guidelines of the 
Massachusetts General Hospital Human Research Committee.  Reports generated by 


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the DSMB will be submitted to the Partners Institutional Review Board when otherwise 
indicated or requested. 
 
The principal investigator and/or co-investigator will be responsible for following patients 
during the study.  Patients will be assessed at every visit during their study participation.  
The investigators and study staff will assure that all CRFs, source documents and 
informed consent are accurate and complete after each study visit. 
 
X.  References 
 
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Randomized placebo-controlled trial to investigate the effects of eplerenone in 
patients with HIV-associated abdominal fat accumulation 
 
Detailed Protocol Version 14 
- 20 - 
 
 
 
Appendix A 
 
Strong Inhibitors of CYP3A4 
 
Ketoconazole 
 
Itraconazole 
 
Fluconazole 
 
Ritonavir 
 
Nelfinavir 
 
Indinavir 
 
Clarithromycin 
 
Telithromycin  
 
Erythromycin 
 
Nefazadone 
 
Bergamottin (in grapefruit juice) 
 
Quercetin (nutritional supplement) 
 
Aprepitant 
 
Verapamil 
 
Chloramphenicol 
 
Saquinavir