Clinical trial NCT04329806
Conditions: Obesity-Associated Insulin Resistance
Sponsor: Italo Biaggioni
Start date: 2021-02-23; Completion date: 2022-07-27
Enrollment: 10 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to study the role of sympathetic mechanisms involved in chronic regulation of cardiovascular and metabolic abnormalities seen in obesity. The investigators will study the effects chronic sympathetic inhibition on insulin sensitivity, inflammation and endothelial function in obese hypertensive human subjects.

Detailed description:
Continuing Review (CR, 2021/08/04) Update: Removal of the angiotensin receptor blockade arm of the study

The presence of obesity increases the risk for hypertension and diabetes, in part due to the development of insulin resistance. Obesity is also associated with sympathetic activation and the overarching hypothesis is that sympathetic activation contributes to insulin resistance with impairment of its vascular and metabolic actions. Preliminary studies suggest that 1) Blood pressure can be normalized by autonomic blockade in obese hypertensives, 2) Sympathetic activation provides no metabolic benefit because the increase in resting energy expenditure associated with obesity is due to an increase in fat free mass rather than sympathetic activation. On the contrary, autonomic blockade: 3) Improves insulin sensitivity in obese hypertensives, 4) Reverses their impaired NO-mediated dilation, and 5) Reduces plasma isoprostanes, a measure of oxidative stress. Furthermore, these abnormalities are interrelated in negative feedback loops, whereby inflammation/oxidative stress impairs nitric oxide mechanisms, which in turn reduces insulin-mediated vasodilation important for substrate delivery, thus contributing to insulin resistance; insulin resistance leads to compensatory increases in insulin levels, which contributes to further sympathetic activation. Current treatment guidelines do not specifically address the treatment of obesity hypertension, and do not target sympathetic activation as a first line approach. It is important, therefore, to determine whether or not targeting sympathetic activation offers unique advantages in the treatment of obesity hypertension over current approaches. The investigators propose a proof-of-concept mechanistic study comparing the metabolic, vascular, and anti-inflammatory effects of sympathetic inhibition, calcium channel blockade and angiotensin receptor blockade in obesity hypertension. The investigators will test the hypotheses that sympathetic activation contributes to 1) metabolic insulin resistance, which impairs the suppression of endogenous glucose production and the stimulation of glucose uptake normally provided by insulin, 2) vascular insulin resistance, which impairs insulin-mediated vasodilation and microvascular recruitment that normally promote glucose uptake, and 3) inflammation and oxidative stress, which contribute to insulin resistance and hypertension. The proposed studies will gauge the contribution of sympathetic activation to the cardiovascular and metabolic complications of obesity and provide the mechanistic insight to determine whether or not it should foster the efforts currently under way to develop novel therapies targeting sympathetic activation for hypertension.

Arms: arm 1: Moxonidine to be administered at a dose to produce a decrease in BP of at least 20% of baseline for 9 weeks arm 2: Amlodipine to be administered at a dose to produce a decrease in BP of at least 20% of baseline for 9 weeks
Interventions: intervention 1: Moxonidine 0.2 MG intervention 2: Amlodipine 5 MG
Intervention descriptions: intervention 1: Moxonidine 0.2 MG twice daily intervention 2: Amlodipine 5 MG twice daily

Protocol documents:
Prot_SAP_000.pdf: 
 
 
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Sympathetic Mechanisms in the Cardiovascular 
and Metabolic Alterations of Obesity 
 
 
Principal Investigator:  
Italo Biaggioni, M.D., Professor 
 
 
Co-Investigators: 
 
Amr Wahba, M.D., 
Research Fellow 
Division of Clinical Pharmacology 
Department of Medicine 
 
 
Alfredo Gamboa, M.D., M.S.C.I., 
Research Associate Professor 
Division of Clinical Pharmacology 
Department of Medicine 
 
William D. Dupont, PhD., 
Research Professor and Vice-Chair 
Department of Biostatistics 
 
 
 
Vanderbilt University School of Medicine 
 
 
 
 


 
 
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Table of Contents: 
 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Previous Human Studies 
4.0 
Inclusion/Exclusion Criteria 
5.0 
Enrollment/Randomization 
6.0 
Study Procedures 
7.0 
Risks of Investigational Agents/Devices (side effects) 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 
9.0 
Study Withdrawal/Discontinuation 
10.0 Statistical Considerations 
11.0 Privacy/Confidentiality Issues 
12.0 Follow-up and Record Retention 
13.0 Reference List 
 
 
 


 
 
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1.0 
Background 
 
As the prevalence of obesity continues to increase, its associated conditions 
hypertension and diabetes) will contribute to greater health care costs and shortened life 
expectancy. More than 78 million adults in the U.S. are obese. Medical costs associated 
with obesity are estimated to be $210 billion annually. Current hypertension treatment 
guidelines emphasize the need to lose weight, but do not otherwise address the 
treatment of obesity hypertension. Given the low success rate of long-term weight loss 
we cannot currently rely on this approach to treat obesity hypertension. Evidence from 
the literature and our studies indicate that sympathetic activation accompanies obesity 
and contributes to insulin resistance and endothelial dysfunction. However, first line 
treatments recommended for hypertension often result in further increases in 
sympathetic activity and worsening of insulin resistance. 
 
Obesity and insulin resistance. Obesity 
is 
associated 
with 
decreased 
responsiveness 
to 
insulin 
(insulin 
resistance) in the liver, skeletal muscle and 
the vasculature. The liver is a major source 
of endogenous glucose production (EGP). 
Suppression of EGP by insulin reduces 
blood glucose, but this process is impaired 
in obesity (hepatic insulin resistance). In 
the muscle, insulin promotes glucose 
uptake and this process is also impaired in obesity. In addition, impaired insulin-induced 
vasodilation, can contribute to insulin resistance by reducing the delivery of substrate 
(glucose) available for tissue uptake.  
 
Obesity and sympathetic activation. Obesity is widely accepted to be associated 
with increased sympathetic nerve activity, and this sympathetic activation is preferential 
to vascular beds that regulate metabolism and blood pressure. For example, muscle 
sympathetic nerve activity (MSNA), which is tightly coupled to baroreflex regulation of 
blood pressure, is not only increased in obesity but this increase correlates with body 
mass index (BMI), body fat, and particularly with visceral fat. Multiple mechanisms 
explain obesity-associated sympathetic activation, including increased leptin and 
decreased adiponectin; insulin itself can induce sympathetic activation directly (by acting 
in CNS centers that increase sympathetic outflow), and reflexively (by inducing 
peripheral vasodilation with unloading of baroreflexes). (Figure 1) 
 
 


 
 
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Sympathetic activation and insulin resistance. Evidence from the literature and 
our laboratory indicates that sympathetic activation contributes to metabolic and 
vascular insulin resistance. Even physiological sympathetic activation (induced by lower 
body negative pressure, LBNP) acutely impairs glucose uptake. Conversely, we have 
shown that autonomic withdrawal improves insulin sensitivity of glucose uptake. 
Sympathetic activation of hepatic nerves promotes hepatic glucose production and can 
contribute to resistance to the normal suppression by insulin of hepatic glucose 
production. We will test this hypothesis in Specific Aim 1. 
 
Sympathetic activation with LBNP also acutely 
impairs flow-mediated dilation (1) and blunts 
vasodilation to acetylcholine. Conversely, our 
preliminary results suggest that autonomic 
withdrawal reverses “endothelial dysfunction” in 
obese hypertensives. The relevance of this 
finding is that this impaired insulin vasodilation 
can contribute to metabolic resistance (impaired 
glucose uptake) by reducing glucose availability. 
Current evidence suggests that the increase in 
vascular glucose delivery by insulin is mediated 
mostly 
by 
opening 
new 
vascular 
beds 
(microvascular recruitment). These observations 
raise the possibility that removal of sympathetic 
vasoconstriction can result in improvement of 
insulin-mediated microvascular recruitment and 
subsequently of sensitivity of insulin-mediated 
glucose uptake. We will test this hypothesis in 
Specific Aim 2. 
 
 
 
Significance and Scientific Premise. 
The evidence presented above supports the 
existence of a negative feedback loop in 
obesity (insulin resistance, increased levels 
of insulin, leading to sympathetic activation 
and promoting further insulin resistance) 
that can be intervened therapeutically. We 
propose a mechanistic study that will test 
the hypothesis that sympathetic inhibition 
provides a unique benefit compared to 
calcium channel blockade by improving 
vascular and metabolic insulin sensitivity 
and reducing inflammation. We hope our 
results will challenge current guidelines in the treatment of hypertension. 
 
2.0 
Rationale and Specific Aims 
 


 
 
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Current treatment guidelines for hypertension are based on outcome trials that enrolled 
patients likely to have cardiovascular events during the duration of the trial. They 
recruited elderly subjects (average age 65 years) who were not obese (average BMI 28). 
Thus, the data obtained from these studies may not be applicable or reflect the 
cardiovascular disease evolution in the case of young (15-20 years younger) obese (5 
BMI points higher) Our preliminary data suggest that sympathetic activation contributes 
to obesity hypertension, which is not directly targeted by drugs commonly used to treat 
hypertension. This is the impetus for this proof-of-concept study comparing the 
metabolic effects of the central sympatholytic moxonidine and the calcium channel 
blocker amlodipine, at doses titrated to produce equivalent decreases in blood pressure.  
 
Moxonidine was chosen because it is an imidazoline agonist devoid of the CNS side 
effects characteristic of older sympatholytic drugs. Amlodipine is one of the most widely 
used antihypertensives with mostly neutral metabolic effects (“negative” control). A 
more detailed discussion of drug selection is included below. We hypothesize that, at 
equipotent antihypertensive doses, moxonidine will be superior to amlodipine in 
improving insulin resistance (Specific Aim 1) insulin-mediated vasodilation and 
microvascular recruitment (Specific Aim 2), and obesity associated inflammation and 
oxidative stress (Specific Aim 3). These three specific aims will be explored within a 
single clinical study (Figure 6). 
 
Specific Aim 1. To test the hypothesis that sympathetic activation contributes 
to insulin resistance in obesity hypertension. We propose a two-step 
hyperinsulinemic euglycemic clamp with stables isotopes to determine the effect of 
sympathetic activation on insulin-mediated: 
 
1a. Suppression of Hepatic Glucose Production 
1b. Increase in Glucose Uptake 
Our preliminary studies showed that acute autonomic blockade improved insulin 
resistance (Figure 3). In those studies, we used an acute dose of the ganglionic blocker 
trimethaphan, and a dose of insulin that suppresses endogenous glucose production 
(EGP) so we could selectively measure insulin resistance to glucose uptake. Animal 
studies suggest that sympathetic tone plays an even a greater role in regulating EGP by 
the liver (2). However, to our knowledge the magnitude of this effect in humans, and in 
particular in obesity hypertension, has not been assessed. Therefore, we now propose to 
determine the role of the autonomic nervous system in insulin resistance at baseline, 
and after central sympathetic inhibition with moxonidine vs. treatment with amlodipine 
(Figure 6). Subjects will be studied at baseline and during treatment using a two-step 
hyperinsulinemic euglycemic clamp (Figure 7): first using a low dose insulin infusion, 
coupled with stable isotopes (glucose tracer) to assess insulin sensitivity of suppression 
of endogenous glucose production (EGP, Specific Aim 1a), followed by a higher dose of 
insulin that suppresses EGP, to electively assess glucose uptake (Specific Aim 1b).  
 
Specific aim 2. To test the hypothesis that sympathetic activation contributes 
to impaired vasodilation in obesity hypertension. 
There is significant evidence that obese hypertensive subjects have impaired NO-
mediated dilation, and our group confirmed this abnormality in resistance vessels 
(brachial artery dilation to acetylcholine). In addition, we showed that autonomic 


 
 
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blockade reverses this impairment, indicating that a significant component of impaired 
NO-dilation is secondary to sympathetic activation, at least in initial stages of obesity 
hypertension (3).Considering that insulin vasodilation is NO-mediated, sympathetic 
blockade may improve this mechanism blunted in insulin resistant states. Current 
evidence suggests that the ability of insulin to increase the number of perfused 
capillaries (microvascular recruitment) is important for stimulating glucose uptake, by 
enhancing its delivery to muscle cells (4-6). Therefore, insulin ability to stimulate 
microvascular recruitment, via NO-mediated dilation, is an important component of 
insulin sensitivity of glucose uptake (7).  
 
Our previous findings suggest that sympathetic activation contributes to impairments in 
both NO-mediated dilation (Figure 2) and insulin mediated glucose uptake (Figure 3). 
Therefore, we propose to test the hypothesis that sympathetic inhibition with 
moxonidine improves insulin-stimulated microvascular recruitment. To assess this 
objective, we will use the gold-standard hyperinsulinemic euglycemic clamp combined 
with contrast-enhanced ultrasonography to address insulin sensitivity and insulin-
stimulated microvascular recruitment. To compare the effect of this trial with “selective” 
blunting of sympathetic activity, we propose to use the vasodilator amlodipine as a 
control due to its neutral effect on insulin sensitivity. 
 
Specific Aim 3. To test the hypothesis that sympathetic activation contributes to 
inflammation and oxidative stress in obesity hypertension 
Inflammation is increasingly recognized as an important pathophysiological mechanism 
in hypertension (8). Of relevance to this project, these inflammatory processes are 
driven by an increase in sympathetic outflow (8, 9). In animal models CNS lesions that 
interrupt sympathetic pathways prevent angiotensin II-induced inflammation (10), and 
conversely, activation of sympathetic outflow by a stress paradigm promotes vascular 
inflammation (11). Furthermore, bilateral renal denervation significantly reduces 
inflammation and blunts angiotensin II-induced hypertension in animal models (12). 
Unilateral renal denervation selectively reduces inflammation in the denervated kidney, 
suggesting a link between sympathetic nerves and organ inflammation even 
independent of blood pressure (12). Several studies suggest a role for inflammation and 
immunity in human hypertension. Work from our group showed an increase in the 
percentage of circulating inflammatory CD4+ and CD8+/ CD45RO+ T cells (Figure 9) in 
hypertensive subjects (13). These cells have upregulated production of inflammatory 
cytokines. However, the link between sympathetic activation and inflammatory/immune 
pathways has not been extensively explored in humans; uncontrolled studies in 
hypertensive patients found a correlation between sympathetic activity and inflammation. 
Available data from human studies is in agreement with this postulate, and this provides 
the rationale for the premise that sympathetic activity contributes to inflammation and 
oxidative stress in obesity hypertension. 
 
3.0 
Previous Human Studies 
 
Moxonidine is a newer generation central sympatholytic that activates imidazoline 
receptors in the brain, producing less sedation than classic α2 partial agonists. It also has a 
longer half-life and has equivalent antihypertensive effect as amlodipine at the doses 
proposed in this application (14). Furthermore, uncontrolled studies suggest that 
moxonidine has a positive metabolic profile (15-17). The obvious limitation is that it is 


 
 
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not available in the US (it is approved in Europe and Australia). We should emphasize 
that this is not a drug treatment clinical trial, but a proof of concept study to test the 
hypothesis that sympathetic activation contributes to the cardiovascular and metabolic 
alterations of obesity, and we thought important to use the best pharmacologic probe 
available. We have an IND for the use of moxonidine and have obtained it through the 
Hannover Medical Center Pharmacy (Hannover, Germany).  
 
We selected amlodipine because it is one of the most extensively used 
antihypertensives. Most studies have found amlodipine to be metabolically neutral (18) 
although a few have shown improvement of insulin sensitivity (19, 20). On the other 
hand, it may produce a modest sympathetic activation (21). An alternative comparator 
would have been a thiazide because they are widely used and recommended as first line 
therapy. However, thiazides are known to worsen insulin sensitivity and it would be seen 
as stacking the deck in our favor. Thus, we thought important to include a head-to-head 
comparison between a treatment that specifically targets sympathetic activation 
(moxonidine) with one that is largely metabolically neutral (amlodipine, “negative” 
control). We believe this design will help determine if specifically targeting sympathetic 
activity has a role in the treatment of obesity hypertension compared to medications 
currently considered standards of care. 
 
4.0 
Inclusion/Exclusion Criteria 
 
• 
Inclusion criteria 
o Adults aged between 18 and 65 years old 
o Able to give informed consent 
o Non-smokers 
o Obese (defined as BMI ≥30 kg/m2 and 35% of body fat) 
o Hypertensive (at least 2 measurements of 130<SBP<160mm Hg OR 
85<DBP<100mmHg while off medications or currently being treated with 
anti-hypertensives) 
 
• 
Exclusion criteria 
o Past medical history of long-term diabetes or treatment with insulin, 
pulmonary, renal or hematopoietic disease , cardiovascular disease other 
than hypertension (i.e., stroke, myocardial infarction), or other conditions 
that the study team judges could affect the volunteer’s participation in 
the study (e.g., chronic back pain) 
o Morbid obesity (BMI > 43 kg/m2) 
o Unable to withdraw from medications that the study team judge to have 
effects on energy metabolism or autonomic function 
o Highly trained athletes 
o Pregnancy or breastfeeding 
o Patients with Stage 2 hypertension ≥ 160/100 mmHg 
 
*Of note: chronic medications that can interfere with the autonomic nervous system 
(i.e., antihypertensives, metformin) will be withheld for at least five half-lives before the 
first study visit. 
 
5.0 
Enrollment/Randomization 


 
 
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For all Specific Aims, subjects will be recruited from the community. A study 
investigator or trained research assistant will seek eligible subjects and provide 
them with information regarding the proposed research, including procedures 
involved, and potential risks and benefits. A written informed consent form will 
be obtained from all subjects prior to any study procedures. We have extensive 
experience in conducting acute and longitudinal studies in these patient 
populations. Nevertheless, several strategies will be used to reduce the dropout 
rate and increase adherence to treatment. Compliance with the study will be 
discussed with potential participants before enrollment. Participants will get a 
schedule of the visits at the beginning of the study, and a research nurse will 
make regular contact with them by phone calls, text messages and/or emails to 
send instructions and reminders of upcoming visits and to maximize compliance. 
 
6.0 
Study Procedures / Study timeline 
 
As explained before, we propose a double-blind, randomized, 9-week, parallel 
group study. The clinical trial will consist of 9 visits. 
 
 
 
 
 
 
Visit 1 (Screening and clinical characterization): We will enroll obese 
hypertensive subjects off medications. Antihypertensives and medications that 
can affect autonomic function will be stopped at least for 5 half-lives (~2-3 
weeks). They will be asked to monitor their blood pressure at home and will be 
provided with blood pressure monitors if necessary. Patients with pre-


 
 
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hypertension are included if they have insulin resistance and sympathetic 
activation. Stage II hypertension is excluded because of the likelihood that more 
than one antihypertensive is needed to control BP. 
 
Visit 2 and 3 (Baseline measurements): After 3 weeks off medications (or at 
least 5 half-lives), if the patients still meet the inclusion/exclusion criteria, we will 
perform the following baseline measurements the day before performing the first 
hyperinsulinemic-euglycemic clamp:  
o Sympathetic activity (MSNA) (visit 2 only) 
▪ 
We may perform some autonomic nervous system function tests 
during MSNA such as Valsalva maneuvers 
o Heart rate 
o Blood pressure (supine and 1, 3, 5, 15, 30 minutes standing)  
o Blood samples collected in supine position and after 30 minutes of 
standing for catecholamines, renin activity, angiotensin II and 
aldosterone levels) (visit 2 only) 
o Blood for FAC analysis of immune cells (visit 2 only) 
o Flow mediated dilation and 24-hr blood pressure (visit 2 only) 
o FAC analysis of harvested endothelial cells (visit 2 only) 
o Non-invasive determination of arterial stiffness using tonometric 
(Sphygmocor) method (visit 2 only) 
o Hyperinsulinemic clamp (visit 3 only) 
o Body impedance (visit 2 or 3) 
o Blood volume measurement (visit 2 only) 
o DEXA scan (visit 2 only) 
 
 
Visit 4-7 (Randomization): Randomization, blood pressure adjustments and 
monitoring will be held from visit 4 to visit 7. Patients will be randomized with a 
permuted block double-blinded scheme. Moxonidine and amlodipine will be 
started at 0.2mg bid and 5mg, respectively. Blood pressure will be measured 
using the Sprint protocol with home monitors provided by us, and at weekly 
visits. A blinded study physician will increase medication dose once if needed. 
The dose titration period can be extended if necessary, to reach a stable BP.  
 
Visit 8-9: We will repeat the baseline measurements mentioned above after 9 
weeks of treatment during these visits. Hyperinsulinemic-euglycemic clamp will 
be performed in visit 9 (treatment). No DEXA scan will be performed at visit 8. 
 
Study procedures during the hyperinsulinemic-euglycemic clamps: 
 


 
 
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1. We will draw a blood sample to measure baseline levels of [6,6-2H2]-
dideuterated glucose. We will then start the continuous infusion of [6,6-
2H2]-dideuterated glucose. After at least 90 minutes, we will do baseline 
measurements of HP and BP. We will take blood samples every 15 
minutes for 30 minutes (3 total) to measure plasma glucose, insulin, 
glucagon, C-peptide, and [6,6-2H2]-dideuterated glucose. 
 
2. We will then perform a two-hour glucose clamp. In ancillary studies 
(Luther M, unpublished) new found that an autonomic blockade enhances 
insulin suppression of EGP, we decided to use the lower dose of 15 
mU/m2/min insulin produced a >50% suppression of EGP (Bock et al. 
2007). Plasma glucose will be monitored every 5 minutes and euglycemia 
(90-100 mg/dl) will be maintained with variable infusion of 20% glucose. 
Potassium chloride 10 mmol/hr will be infused to prevent hypokalemia.  
 
3. Once the glucose clamp is started, the glucose tracer infusion will be 
gradually reduced to 1.0 mg/m2 (half the starting dose), to account for 
the expected decrease in hepatic glucose output. To minimize changes in 
glucose isotopic enrichment, tracer will be added to the 20% Dextrose 
solution. We will alter tracer delivery commensurate with changes in 
glucose flux to maintain a constant plasma specific activity of glucose, as 
done previously (28-31).  
 
4. Within the last 30 minutes after achieving stable glucose levels, samples 
will be drawn for every 15 minutes for 30 minutes (3 total) to measure 
glucose and [6,6- 2 H2]-glucose levels. At the third blood draw, we will 
also obtain samples for insulin, glucagon, C-peptide, catecholamines, and 
free fatty acids. In particular, glucagon and free fatty acids regulate EGP, 
but 
we have 
previously shown that their suppression during 
hyperinsulinemia is not altered by sympathetic withdrawal (32).  
 
5. We will then increase the dose of insulin to 60 mU/m2 /min for an 
additional two hours. At this dose, insulin produces complete suppression 
of hepatic glucose production even in insulin resistant patients. Glucose 
utilization under these conditions, therefore, will reflect glucose uptake. 
The glucose infusion rate during the last 30 minutes of the clamp will be 
used as a measure of insulin sensitivity of glucose uptake. Within the last 


 
 
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30 minutes after achieving stable glucose levels, samples will be drawn 
every 15 minutes for 30 minutes (3 total) to measure glucose and [6,6-
2H2]-dideuterated glucose levels. At the third blood draw, we will also 
obtain samples for insulin glucagon, C-peptide, catecholamines, and free 
fatty acids. 
 
7.0 
Potential risks 
 
Hyperinsulinemic-euglycemic clamp: Of note, we have performed more than 40 
hyperinsulinemic euglycemic clamps and at least 20 of them during autonomic 
blockade. We have been able to both maintain glycemia at 5% of fasting levels 
and clamp systolic blood pressure to ~140 mm Hg. We therefore do not foresee 
either hypoglycemia or hypotension. 
 
Stopping medications may increase blood pressure: The symptoms of having 
elevated blood pressure vary among individuals but may include headache, 
dizziness, blurred vision, nausea and edema. Blood pressure and symptoms will 
be followed closely and regularly, and a physician will supervise all subjects with 
medications restarted if necessary.  
 
Moxonidine: Treatment with moxonidine should result in changes in blood 
pressure. This medication is considered an investigational drug because it is not 
approved by the FDA in the US. It is approved for use in humans in Europe, Asia, 
and Australia. Side effects of moxonidine are those expected of sympathetic 
withdrawal, and include dry mouth, headache, fatigue, dizziness, intermittent 
facial edema, nausea, sleep disturbances (rarely sedation), vasodilatation, and 
rarely, skin reactions. Moxonidine will be used under IND 106975, sponsored by 
Dr. Biaggioni, and approved by the FDA.  
 
Amlodipine : treatment with amlodipine should also result in changes in blood 
pressure. Common side effects of amlodipine include peripheral edema, fatigue, 
dizziness, palpitations, headache, somnolence and nausea. For the measurement 
of glucose turn-over we will use the stable isotope [6,6- 2 H2]-glucose as the 
tracer.  
 
Peripheral venous catheterization: risks associated with this procedure are mostly 
related to pain and bruising on insertion site and will be explained in the consent 
form.  
 
Infusion of the contrast agent (Definity) to measure microvascular blood flow can 
result in dizziness, chest pain, shortness of breath, or back pain may occur within 
minutes (1-15 minutes) of being given the drug. A very serious allergic reaction 
to this drug is extremely rare, but could include rash, itching/swelling (especially 
of the face/tongue/throat.  
 
Cardiac output: The inert gas rebreathing technique for the assessment of 
cardiac output carries minimal risk.  
 


 
 
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Microneurography (MSNA): During this procedure, the nerve is first localized by 
external electrical stimulation (between 10-15 volts, 1-5 mA through an electrical 
isolated unit specifically designed for this purpose so the amount of current 
reaching the volunteer is limited. After the nerve is localized, an ultrafine 
tungsten electrode is introduced into the nerve to directly record MSNA. A 
second electrode is placed subcutaneously to serve as a reference. Due to the 
small size of the electrode, no anesthesia is required for the procedure. There 
should be no pain or discomfort during MSNA recordings while the electrode is in 
place. However, during placement of the electrode, paresthesias and/or pain 
running to the foot can occur if pain fibers are encountered. The electrode is 
then relocated to avoid this uncomfortable side effect. We have employed this 
technique at Vanderbilt for over twenty years. The method has very few, if any, 
side effects. Pain at the site of placement of the electrode may occur some hours 
following the procedure. Paresthesias distal to the recording site may persist for 
up to a week after the procedure, and in very rare instances, for longer. Subjects 
are advised against strenuous leg exercise for at least 24 hours after the 
procedure to avoid this side effect. Bruising and infection are other possible but 
very unlikely side effects. All intravenous infusions will be performed in a hospital 
inpatient setting by trained nurses who are experts in this task. Sterile technique 
will be used to minimize the risk of a catheter infection. 
 
DEXA scan: The DEXA scan may provide up to the equivalent of 9 days of natural 
radiation. 
 
Harvesting of endothelial cells for FAC analysis has the potential to increase the 
risk of bruising. For this procedure a J-tipped wire is advanced 10 cm through 
the vein catheter. Volunteers may find this sensation unpleasant but not painful. 
 
Non-invasive determination of arterial stiffness will be done using a Sphygmocor 
device. There are no known risks associated with this measurement. With this 
method a blood pressure cuff is placed on the arm as with regular blood 
pressure determinations. This may cause transient discomfort. The measurement 
takes only a few minutes,  as with a regular blood pressure determination.  
 
Body impedance: Measurements of body impedance (electrical resistance) are 
made by recording the changes in the transmission of a very weak electrical 
current at the patches placed on the skin. It delivers a very small electrical 
current (0.6 mA). There are no known risks associated with this procedure. 
 
Blood volume measurement (CO rebreathing): Subjects may experience mild 
headache and reduction of exercise capacity. It should clear from the body 
within 12-14 hours. 
 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 
 
The PI will be responsible for data integrity, the safety of study participants, and 
ensuring that all adverse events are documented, followed and reported to the 


 
 
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IRB and the Data Safety Monitor (DSM) as appropriate. The PI will review the 
records of the study subjects following the participation of each subject to 
ensure patient safety.  The adverse event will be described with the 
following information: description of the event, outcome of the event, 
duration of the event, relationship to study procedure, requirement if any 
for treatment or intervention and overall outcome. Adverse events will be 
graded according the following scale: 
 
0 no adverse event or within normal limits 
1 mild adverse event (transient and mild in nature with no treatment 
necessary) 
2 moderate adverse event (some intervention and treatment necessary, 
but patient completely recovers) 
3 severe adverse event (the event results in hospitalization, disability, 
death, or is life-threatening).  
 
Any adverse events (AE) will be rated on a 0-5 scale (0-no AE; 1-mild AE, no 
treatment required; 2- moderate AE that responds to treatment; 3-severe AE 
that would limit normal activities and require a prolongation of hospitalization; 4-
life-threatening or disabling AE; 5-Fatal AE).  
 
Any AE rated 3 or greater will be immediately reported to the IRB and the DSM. 
All AEs will be reported to the IRB on an annual basis. In the case of serious AE, 
the IRB makes an immediate determination about the necessity to modify the 
protocol, including additional information in the consent form, informing previous 
participants, temporarily holding enrollment of patients, or terminating the study.  
 
All study procedures and cumulative adverse events are subject to full committee 
review at least yearly. The DSM will provide objective review of treatment results 
as they relate to human safety and data quality. Dr. Katherine T. Murray, 
Professor of Medicine, and Pharmacology, Division of Clinical Pharmacology, with 
extensive experience in human research and clinical trials, has agreed to take on 
this role. The PI will provide at least yearly reports to the DSM, or more often if 
needed. The information produced includes recruiting, safety reporting, data 
quality, and efficacy. Instances of non-compliance with the protocol will be 
reported annually. 
 
The DSM will assess safety data including common and serious adverse events. 
The DSM will have the authority to modify the protocol or to terminate the study 
if it deems such actions to be warranted. The DSM will also receive quarterly 
reports of enrollment, protocol adherence, data quality and adverse events via e-
mail. The DSM will review all serious adverse events. Any serious adverse event 
will be reported within 24 hours of the PI’s notification of the event.. During 
regularly scheduled meetings, the PI will provide the DSM with a list of non-
serious adverse events. The DSM may choose to perform an interim analysis; 
however, it is expected that this would not occur without reasonable concern 
related to either patient safety or data validity. 
 


 
 
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9.0 
Study Withdrawal/Discontinuation 
 
The study investigators or Vanderbilt University Medical Center may withdraw subjects 
from this study at any time after they have provided informed consent, but before study 
completion. Study withdrawal may occur for several reasons including but not limited to: 
non-compliance with the protocol, concern for patient safety, the availability of new 
information that may affect continued participation, or if the study is stopped. Subjects 
are free to withdraw from this study at any time. We will cease to collect study 
information at the time of withdrawal of consent, but data already collected will be 
retained. Withdrawal of consent or refusal to participate will not prejudice subject health 
care. 
 
10.0 Statistical Considerations 
 
In this proposal, we will be testing the general hypothesis that nine weeks of treatment 
with the sympathetic blocker, moxonidine, will improve insulin sensitivity (Specific aim 
1), microvascular blood flow (Specific aim 2), and inflammation (Specific aim 3), in 
obese hypertensive human subjects. We will be comparing the changes before and after 
treatment with the central sympathetic inhibitor moxonidine (MOX), versus the changes 
produced with treatment with the calcium channel blocker amlodipine (AML), an 
antihypertensive medication considered to have neutral metabolic and anti-inflammatory 
effects. Our main scientific goal is to demonstrate the efficacy of MOX in a superiority 
trial, and for that reason the study is powered to perform the main statistical analysis 
between the MOX and AML arms. The clinical comparison between the two responses, 
not previously done, will provide important new information. Nonetheless, as a 
secondary analysis, the differences in primary outcomes between the treatment and 
baseline study days will be compared across the two study arms using a linear 
regression model. Given the fact that the degree of obesity, baseline sympathetic 
activity and blood pressure may affect EGP these variables as well as gender and age 
will be included as covariates in the model to take into account potential confounding 
effects. We make no a priori assumptions about the outcome of these studies. Any 
result, therefore will provide important new information. 
 
a) Specific Aim 1a - The null hypothesis is that there will be no changes in the 
insulin-mediated suppression of endogenous glucose production in response to 
sympathetic blockade with MOX. We will evaluate this hypothesis at Visit 9 (post 
treatment) by measuring EGP at baseline (EGPBsl) and during the last 30 
minutes of a hyperinsulinemic euglycemic clamp (EGPClamp) in a double-blind 
randomized trial (protocol shown in figure 6). For each study arm, the primary 
endpoint will be DEGP = (EGPBsl ‒ EGPClamp) the difference in the change of 
insulin-mediated suppression of basal EGP between the pre and post treatment 
visit. The primary analysis will be performed between the moxonidine and 
amlodipine arms, at Visit 9 using Welch’s unequal-variance an unpaired t-test to 
test whether the primary end point will be equal in these two treatment groups. 
Note that although we are collecting multiple measures over time, this endpoint 
is based on a single visit and a scalar response feature ΔEGP. Hence, a fixed 
effects analysis is appropriate for this aim. Appropriate non-parametric analysis 
methods will also be used if data are not normally distributed. Since the degree 


 
 
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of obesity, baseline sympathetic activity, blood pressure, age and gender may 
affect EGP, these variables could confound the effect of treatment on the change 
in EGP. For this reason, in a secondary linear regression analysis we will compare 
DEGP in the moxonidine and amlodipine arms adjusted for these variables. We 
will also assess the effects of treatment on EGP over time by conducting a 
repeated-measures GEE regression analysis of EGP against treatment and these 
confounding variables at baseline and the 9 subsequent visit times. These 
analyses will use a normal random component and the identity link function. 
Spaghetti graphs and residual analyses will be used to assess the model fit. 
Restricted cubic splines will be used if needed to account for non-linear effects of 
time on treatment response (33).   
 
Sample size calculation. Power calculations are based on previous studies 
comparing the suppression of EGP between subjects with impaired fasting 
glucose and normal fasting glucose (34). In this study among obese subjects 
with impaired fasting glucose (N=30), the mean and the standard error of mean 
(SEM) for EGP at baseline was 15.2 ± 0.2 μmol×min-1 × kg-1, and during the 
clamp, EGP was 10.2±0.7 μmol×min-1 × kg1. Hence, the standard deviation 
(SD) of EGP at baseline and during clamp was 0.2√30 = 1.1 and 0.7√30 = 3.8, 
respectively. We will power our study to detect a difference in inhibition of 3 
μmol×min-1 ×kg-1. Making the conservative assumption that clamped and 
baseline EGP values are independent, we estimate the SD of this difference to be 
*(1.1, + 3.8,) = 4.0. With this SD, 29 subjects per group would provide 80% 
power to detect a mean difference in the inhibitions of 3 μmol×min-1 × kg-1 
with the type I error of a = 0.05. Assuming a drop–out rate of 20%, which 
includes the subjects that will be enrolled based on HOMA but later shown not to 
have insulin resistance during the clamps (see Section C.2), we will need to 
enroll 36 subjects in each study arm. This sample size will also be used in Aims 
1b, 2 and 3 even though smaller sample sizes would suffice for these aims.  
 
Specific Aim 1b - The null hypothesis is that chronic sympathetic blockade will 
not improve whole body insulin sensitivity. We will measure insulin sensitivity as 
the glucose infusion rate, corrected by body weight (MBW) needed to maintain 
euglycemia during the last 30 minutes of a hyperinsulinemic euglycemic clamp. 
The same three groups of subjects will be studied twice, at the beginning (BSL) 
and the end of 9 weeks of treatment with either the sympathetic blocker 
moxonidine (MOX) or the calcium channel blocker amlodipine (AML). The null 
hypothesis is that we will not see any difference in the change in body-weight-
corrected insulin sensitivity with MOX as compared with AML. The primary 
analysis will assess changes in MBW (MBW_clamp ‒ MBW_bsl) between patients 
receiving MOX and will use a t-test similar to that described for Aim 1a.  
Sample size calculation. We estimated the sample size based on previous 
studies (17) using a similar design to the present study, which found a 21% 
improvement of insulin sensitivity with moxonidine. From our previous studies, 
we expect obese insulin resistant hypertensive subjects to have a MBW of 
3.1±0.99 mg/kg/min (17). An increase of 21% in the moxonidine arm will 
translate as a MBWMOX of 3.75 mg/kg/min (a difference of 0.65 mg/kg/min), 
after 9 weeks of treatment. Assuming that a SD of the difference of 0.75 


 
 
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mg/kg/min, a sample size of of 27 subjects per group will provide 88% power to 
detect this difference with a = 0.05.  
 
Specific Aim 2 - The null hypothesis is that insulin will not produce any 
different changes in microvascular blood volume (MBV) using contrast enhance 
ultrasonography (CEU) in response to nine weeks of treatment with moxonidine 
as compared to amlodipine. We will measure MBV at baseline (MBVbsl), during 
the last 30 minutes of the lower insulin dose (15 mU/m2 /min) portion of the 
hyperinsulinemic euglycemic clamp (MBVegp), and during the last 30 minutes of 
the higher insulin dose (60 mU/m2/min) portion of the clamp (MBVclamp) on 
two different occasions (double blind, randomized, protocol shown in figure 6). 
Subjects will be randomized to treatment with moxonidine (MOX) or amlodipine 
(AML). The primary endpoint would be the average difference in the change in 
MBV (MBVclamp-MBVbsl) between the MOX and AML arms. The primary analysis 
will be conducted between the moxonidine and amlodipine arms using a t-test as 
described in Aim 1a.  
 
 
Sample size calculation. We estimated the sample size based on our 
preliminary results (Figure 8) and previous studies in obese human subjects (35). 
In our study, on the SAL day, we expect to see a blunted increase in MBV only 
due to insulin among obese subjects (from 20.4±3.6 to 18.8±3.8, as in the 
paper by Haenni and Lithell (17)). We believe that an increase of 3.5 arbitrary 
intensity units (AIU) of MVB (half of the what was seen in healthy subjects) 
during MOX treatment would be a clinically significant difference. Healthy 
subjects showed an increase from 18.7±3.3 to 25.0±4.1 in the same paper, and 
we have observed similar results (see figure 8). With a SD of 3.2 AIU, a sample 
size of 27 subjects per group would provide 97% power to detect a difference of 
3.5 AIU with a = 0.05.  
 
Specific Aim 3 - The null hypothesis is that chronic sympathetic blockade will 
not improve inflammation in obese hypertensive subjects. We will measure the 
percent of circulating T cells that express both CD4 and CD45RO (CD4+ 
/CD45RO+ ), at baseline and after nine weeks of treatment with each of the 
study drugs as in the previous specific aims. Obese hypertensive subjects will be 
studied twice, at the beginning (BSL) and the end of 9 weeks of treatment with 
either the sympathetic blocker moxonidine (MOX) and the calcium channel 
blocker amlodipine (AML). The primary outcome variable will be the change from 
baseline in the CD8+ /CD45RO+ percentage of circulating T cells. The null 
hypothesis will be that there is no difference in this outcome in patients treated 
with moxonidine compared to patients treated with amlodipine. The primary 
analysis will be to conduct an unpaired t-test on the primary outcome that is 
similar to that described for Aim 1.a. Secondary analyses to account for a 
possible skewed distribution of the primary outcome, to adjust for potential 
confounding variables will also be performed in a fashion that is similar to the 
analyses for Aim 1.a.  
Sample size calculation. We estimated the sample size based on previous 
studies ((36), figure 9) that have found an increased percentage of CD4+ 


 
 
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/CD45RO+ circulation T cells in hypertensive human subjects. They found that 
hypertensive subjects have 20% more circulating T cells expressing both CD4 
and CD45RO as compared to healthy controls. We expect to see a reduction of at 
least 15±5 percent after 9 weeks of treatment with moxonidine. A decrease of 
15% in the moxonidine arm will translate as 45% of CD4+ /CD45RO+ (from 
60% at baseline), after 9 weeks of MOX treatment. Assuming that a SD of the 
difference is 15%, a sample size of 27 subjects per arm would provide 95% 
power to detect a difference of this magnitude with a = 0.05. 
 
11.0 Privacy/Confidentiality Issues 
 
All studies will be done by trained personnel in a quiet room, within or adjacent to the 
Clinical Research Center. Women of child-bearing potential will have a serum pregnancy 
test to exclude pregnancy immediately before study participation. In order to minimize 
any potential risk, a physician and a research nurse will be present and monitor all 
procedures involving the administration of investigational medications. Careful and 
continuous monitoring of all vital signs during the studies will help minimize potential 
risks. Blood pressure and heart rate are monitored continuously using non-invasive 
techniques whenever drugs are infused. Confidentiality will be maintained for the 
identity of participants in this study except as necessary for oversight by the Secretary 
of the Department of Health and Human Services or his designated representative. 
Subjects will be instructed to contact the investigator immediately if they experience any 
signs of adverse events during withdrawal of medications. Any adverse events will be 
reported in a timely fashion to the principal investigator. All adverse events will be 
documented on the Adverse Event form and reported to the IRB. All subjects who have 
adverse events will be monitored to determine the outcome. The clinical course of the 
adverse event will be followed according to accepted standards of medical practice, even 
after the end of the period of observation. To date, no subjects have had adverse events 
to the procedures described. 
 
12.0 Follow-up and Record Retention 
 
The project will take 5 years, during the first two quarters of the funding period, we will 
obtain regulatory approvals and develop the database and case report forms. Patient 
recruitment will start on the third quarter and continue through Year 5. We propose to 
enroll a total of 72 subjects. We estimate 30% will be screen failures or dropouts. 
Eligible participants will be studied on the CRC. Since the same patients will participate 
in aims 1 and 2, the studies on all aims will be conducted simultaneously. Final data 
analysis and manuscript preparation will be started the fourth year 
 
The research material obtained in this proposal will be recorded data obtained 
specifically for research purposes. Blood and urine samples will be obtained for 
screening purposes and for analysis of the various parameters under study. A separate 
secure database of patient information will be maintained using the REDCap web-based 
software, a HIPAA compliant secure database. Only investigators listed in this 
application will have access to research information and will follow IRB and institutional 
HIPAA guidelines. Both paper and computer files will be saved under lock/password 
protection. 


 
 
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