Effects of Nebivolol Versus Metoprolol on Blood Flow Responses to Exercise and Angiotensin II in Hypertensive Patients
Phase: PHASE4
Conditions: Hypertension
Sponsor: University of Texas Southwestern Medical Center

1 
July 25, 2008 
Protocol NCT01502787 
 
Title:  Attenuation of Angiotensin II-Mediated Vasoconstriction in Hypertension with 
Nebivolol  
 
Investigators 
 
 
Name (printed) 
Deg 
Rank 
Phone 
Mail 
Code 
E-mail 
Wanpen Vongpatanasin 
(Principal Investigator and 
Faculty Sponsor) 
M.D. Associat
e Prof 
(214) 
648-7950 
8586 
Wanpen.Vongpatanasin@ 
utsouthwestern.edu 
Gail Thomas 
(Sub-Investigator) 
PhD Associat
e Prof 
(214) 
648-7950 
8586 
Gail.Thomas@UTSouthwestern.edu 
Debbie Arbique 
(Sub-Investigator) 
R.N. Senior 
R.N. 
(214) 
648-2968 
8586 
Debbie.Arbique@utsouthwestern.edu 
Ronald Victor 
(Sub-Investigator) 
MD Prof 
(214) 
648-7950 
8586 
Ronald.Victor@utsouthwestern.edu 
 
Participating Institutions: 
Univ. of Texas Southwestern Medical Center 
 
 
 
 
 
  
 
Funding:  
 
Forest Research Institute  
 
Table of Contents: 
 
Protocol Section 
 
 
 
 
Pages 
 
1) Schema  
 
 
 
 
2-3  
2) Objectives 
 
 
 
 
2 
3) Background 
 
 
 
 
4  
4) Agent/Device 
 
 
 
 
4-5 
5) Eligibility criteria  
 
 
 
6  
6) Material and data to be accessioned  
6-8 
7) Treatment 
 
 
 
 
8-9  
8) Evaluation criteria 
 
 
 
10 
9) Off-study criteria  
 
 
 
10 
10) Registration and study monitoring 
 
10-11 
11) Statistical considerations  
 
 
12 
12) Gender/minority analysis 
 
 
13  
13) References 
 
 
 
 
14-15  
 
 

2 
July 25, 2008 
Objectives: 
 
The overall objective of this study is to determine if Nebivolol a) attenuates angiotensin II-
induced increase in oxidative stress, thereby attenuating angiotensin II-induced 
vasoconstriction and blood pressure elevation and b) attenuates sympathetic mediated 
vasoconstriction during exercise, thereby reducing functional skeletal muscle ischemia in 
hypertensive patients.  
 
Schema 
In 25 untreated hypertensive subjects, we will measure sympathetic nerve activity (SNA) with 
direct intraneural recording of sympathetic action potential (microneurography), skeletal 
muscle oxygenation (Near Infrared spectroscopy), plasma F2-isoprostanes, BP, forearm blood 
flow (high-resolution ultrasonography), forearm vascular resistance (FVR, the ratio of mean 
arterial pressure to forearm blood flow), cardiac output (noninvasive impedance 
plethysmography), total peripheral resistance (TPR, the ratio of mean arterial pressure to 
cardiac output) in untreated stage I hypertensive subjects at baseline, during handgrip 
exercise, and during infusion of intravenous Ang II. Then, we will randomize our subjects to 
receive either 3 months of Nebivolol or Metoprolol, using a crossover design. There will be a 2-
week washout period between the two treatments. We will assess SNA and skeletal muscle 
oxygenation during handgrip exercise and compare the dose response curve of Ang II-induced 
increase in plasma F2-isoprostanes, FVR, TPR, and BP at baseline, after 3 months of 
Nebivolol, and after 3 months of Metoprolol treatment in the same subjects. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Wk 12
Blood draw; Measure
SNA, muscle O2
during exercise; 
Test BP responses 
to i.v.Ang II
Wk 0
Blood draw; Measure
SNA, muscle O2
during exercise; 
Test BP responses 
to i.v.Ang II
Nebivolol
Metoprolol Succinate
12 weeks
Wk 4
Check BP
Wk 8
Check BP
Wk 26
Blood draw; Measure
SNA, muscle O2
during exercise; 
Test BP responses 
to i.v.Ang II
Metoprolol Succinate
Nebivolol
12 weeks
Wk 4
Check BP
Wk 8
Check BP
Wk 14
Check BP
Blood draw
2 wk
washout
3 wk
washout
Wk -3

3 
July 25, 2008 
 
 
Visit 2 (1-4 wks later): Draw 30 cc of blood for plasma isoprostanes
renin, and angiotensin II. Measure SNA, skeletal muscle
oxygenation at rest, during LBNP, and during exercise plus LBNP.
Measure  FVR, TPR, BP at baseline and after intravenous Ang II
infusion. Then, randomize to Nebivolol 5 mg once a day, or
Metoprolol succinate 100 mg once daily.
Visit 4 (4 weeks later): obtain BP (sitting / standing), If SBP < 90
mmHg or DBP < 60 mmHg, reduce the dose of medications by 50%.
If SBP is still ≥ 130 or DBP is ≥ 80 mmHg, increase nebivolol to 20
mg/day or Metoprolol succinate to 300 mg once daily.
Visit 3 (4 wks later): obtain BP (sitting / standing), If SBP < 90
mmHg or DBP < 60 mmHg, reduce the dose of medications by 50%.
If SBP is still ≥ 130 or DBP is ≥ 80 mmHg, increase nebivolol to 10
mg/day or Metoprolol succinate to 200 mg once daily.
Visit 5 (4 wks later): Draw 30 cc of blood for plasma isoprostanes
renin, and angiotensin II. Measure SNA, skeletal muscle
oxygenation at rest, during LBNP, and during exercise plus LBNP.
Measure  FVR, TPR, BP at baseline and after intravenous Ang II
infusion. Then, stop the first medications.
PROTOCOL FLOWCHART
Visit 6 (2 weeks later): obtain BP (sitting / standing). Draw 30 cc of
blood for plasma isoprostanes renin, and angiotensin II. Obtain
FVR, TPR, BP at baseline. Start drug treatment in the remaining
arm.
Visit 1: Inform consent described and signed by subject, check BP, perform physical
examination, obtain ECG, and draw 10 cc of blood for Na, K, glucose and creatinine
(Cr).
Visit 7 (4wks later): Repeat procedure in visit 3
Visit 8 (4wks later): Repeat procedure in visit 4
Visit 9 (4wks later): Repeat procedure in visit 5. End of study.
 

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July 25, 2008 
Background 
 
In normal physiologic states, muscle contraction during exercise blunts alpha-adrenergic mediated 
vasoconstriction in order to maintain blood flow to the exercising muscles, a concept known as 
“functional sympatholysis” 1,2. Studies in animals 3,4 and normal volunteers 5 by Drs. Thomas and Victor 
from our institution indicated a major role of nitric oxide (NO) derived from neuronal nitric oxide 
synthase (nNOS) in the skeletal muscle in the prevention of sympathetic vasoconstriction.  Recent 
study in hypertensive rats indicates presence of impaired functional sympatholysis related to 
Angiotensin II-induced increase in oxidative stress, which inactivates NO 6. This adverse effect of Ang II 
can be reversed by superoxide scavenger, tempol 6. Studies in hypertensive patients in our laboratory 
also recently confirm presence of impaired functional sympatholysis during rhythmic handgrip exercise, 
resulting in augmented skeletal muscle hypoxia, as evidenced by Near Infrared (NIR) Spectroscopy. 
Whether Nebivolol restores increase in forearm blood flow during exercise and improves skeletal 
muscle oxygenation by inhibiting Ang II-induced superoxide formation remains unknown.  
 
Angiotensin II has also been shown to promote peripheral vasoconstriction and elevated blood 
pressure (BP) in both hypertensive and normotensive subjects by increasing circulating levels of 
oxidative stress 7,8. This effect of angiotensin II (Ang II) was reversed by concomitant infusion of 
antioxidant Vitamin C in healthy volunteers 8. Previous study also demonstrated that Ang II-induced BP 
elevation was attenuated by oral administration of carvedilol, but not by metoprolol in patients with 
congestive heart failure 9. Mechanisms underlying this observation is unknown but could be mediated 
by antioxidant effects of carvedilol. Nebivolol has been shown to inhibit Ang II-induced superoxide 
formation by inhibiting NADPH-oxidase in rats 10 and decrease markers of oxidative stress in 
hypertensive patients 11. However, efficacy of Nebivolol in attenuating increase in oxidative stress, 
vasoconstriction, and hypertension during Ang II infusion has not been previously assessed in 
hypertensive patients. 
 
Agent/Device 
 
1. Nebivolol  
Trade Name: Bystolic 
Source: Forest Pharmaceuticals, Inc 
Pharmacology: Nebivolol is a beta1 selective adrenergic receptor blocker approved by the FDA for 
treatment of hypertension (2.5-40 mg/day). Nebovolol lacks intrinsic sympathomimetic activity at clinical 
relevant doses with no alpha-1 adrenergic blocking activity. Mechanisms of antihypertensive effects of 
beta blockers in general include 1) peripheral inhibition of beta 1 receptors leading to decreased 
cardiac output, 2) a central effect leading to reduced sympathetic outflow to the periphery, and 3) 
suppression of renin activity.  Nebivolol may also reduce BP by increasing endothelium-dependent 
release of nitric oxide in hypertensive patients, which was not observed with other beta blockers 12.  
 
Toxicity: In placebo-controlled clinical trials comparing nebivolol with placebo, discontinuation of 
therapy due to adverse events was reported in 2.8% of patients treated with nebivolol and 2.2% of 
patients given placebo. The most common adverse events that led to discontinuation of nebivolol were 
headache (0.4%), nausea (0.2%) and bradycardia (0.2%). Other adverse events associated with higher 
dose than recommended by the FDA include bronchospasm and heart block. 
 
2. Metoprolol Succinate  
Trade name: Toprol XL 
Source: Astra Zeneca 

5 
July 25, 2008 
Pharmacology: Toprol XL is a beta1 selective adrenergic receptor blocker approved by the FDA for 
treatment of hypertension (25-400 mg/day).  Mechanisms of antihypertensive effects of beta blockers in 
general include 1) peripheral inhibition of beta 1 receptors leading to decreased cardiac output, 2) a 
central effect leading to reduced sympathetic outflow to the periphery, and 3) suppression of renin 
activity.   
 
Toxicity: Tiredness and dizziness have occurred in about 10 of 100 patients. Depression has been 
reported in about 5 of 100 patients. Mental confusion and short-term memory loss have been reported. 
Headache, somnolence, nightmares, and insomnia have also been reported. Shortness of breath and 
bradycardia have occurred in approximately 3 of 100 patients. Cold extremities; arterial insufficiency, 
usually of the Raynaud type; palpitations; congestive heart failure; peripheral edema; syncope; chest 
pain; and hypotension have been reported in about 1 of 100 patients. Wheezing (bronchospasm) and 
dyspnea have been reported in about 1 of 100 patients. Musculoskeletal pain, blurred vision, decreased 
libido, and tinnitus have also been reported. 
 
3. Angiotensin II 
Tradename: N/A 
Source: Merck Biosciences, Switzerland 
Pharmacology: Angiotensin II causes direct vasoconstriction via angiotensin receptor subtype I, 
resulting in elevated BP. Angiotensin II also stimulates release of aldosterone from adrenal gland, 
resulting in increased sodium absorption and potassium secretion from the renal tubules. Angiotensin II 
will be administered intravenously at the dose of 1, 2, and 3 ng/kg/min for 15 minutes at each dose to 
test vascular sensitivity in hypertensive patients.  
 
IND # 53,558 
Toxicity: Potential side effects are hypertension and hypokalemia. However, the dose of 3 ng/kg/min of 
Angiotensin II has been administered to more than 400 untreated essential hypertensive patients and 
was shown to increase mean arterial pressure by 3-20 mmHg 13-15. None of these subjects experienced 
any complication from the drug. A higher dose of Angiotensin II of 5 ng/kg/min has been administered 
to patients with hypertension from aldosterone producing adenoma on 3 separate days without 
complications 16. The increase in mean arterial pressure in that study was between 10-20 mmHg 16. 
 
  

6 
July 25, 2008 
Eligibility Criteria 
 
 
Experiments will be performed in stage I (140-159/90-99 mmHg) untreated hypertensive subjects, age 
18-65, without target organ disease.  
 
Criteria for inclusion of subjects 
 
 
 (1) Patients with stage 1 primary hypertension (BP between 140-159/90-99 mmHg) without 
treatment. 
 
 
Criteria for exclusion of subjects 
 
 
 
(1) Congestive heart failure or coronary artery disease.  
 
 
(2) Blood pressure averaging >159/99 mmHg or resting heart rate < 55 bpm 
 
 
(3) Serum creatinine > 1.4 mg/dL 
 
 
 
(4) Asthma or chronic obstructive pulmonary diseases   
 
 
 
   
 
(5) Left ventricular hypertrophy by echocardiography or ECG  
 
 
 
(6) Pregnancy 
  
(7) Hypersensitivity to beta blockers or angiotensin   
 
 
(8) Any history of substance abuse (other than tobacco) 
 
 
(9) Concomitant drug treatment which raise endogenous nitric oxide levels such as nitrates or 
phosphodiesterase V inhibitors (Viagra, Levitra) 
 
 
(10) History of symptomatic bradycardia or heart block 
 
Material and Data to be accessioned: 
 
Visit 1 (week # –3 to -1): Complete history will be taken and a physical examination will be done. 
Laboratory safety screen, height, weight, sitting blood pressure, vital signs, urine pregnancy tests, and 
urinalysis will be obtained by the investigators in all subjects prior to entering the study. Laboratory 
safety screen will include electrocardiogram, random blood glucose, BUN, creatinine, total bilirubin, 
electrolyte, alkaline phophatase, SGOT, and urinalysis. The presence of inclusion criteria and absence 
of exclusion criteria will be verified and documented on the case report form. Subjects who receive 
pharmacologic treatment of hypertension will be required to all stop antihypertensive medications for at 
least 3 weeks prior to the study. After medications are stopped for 2 weeks, these subjects will be 
asked to return to laboratory for BP check. Subjects with BP of ≥ 160/100 mmHg will be excluded from 
the study and they will be instructed to resume their medications. Subjects who are not on hypertension 
treatment during visit 1 can proceed to visit 2 without delay. 
 
Visit#2 (week #0): Subjects who meet inclusion/exclusion criteria of the study will undergo baseline 
evaluation during this visit. During visit 2, each subject will undergo the following procedures: 
 
1) Microneurographic Recording of Muscle Sympathetic Nerve Activity. Multiunit recordings of 
postganglionic SNA will be obtained with unipolar tungsten microelectrodes inserted selectively into 
muscle nerve fascicles of the peroneal nerves using the microneurographic technique of Valbo et al 17. 
The nerve signals are amplified, filtered (bandwidth 700-2000 Hz), rectified and integrated to obtain a 
mean voltage display of SNA. The criteria for an acceptable recording of muscle SNA are: 1) electrical 
stimulation through the microelectrode evokes twitch contractions of the peroneal muscles without 
paresthesias; 2) stretching the muscles or tendons elicits mechanoreceptor discharge; 3) the inter-burst 
interval is equal to or a multiple of the cardiac cycle length, indicating baroreceptor modulation; and 4) 
the activity increases during the decrease in arterial pressure produced by phases II and III of the 

7 
July 25, 2008 
Valsalva maneuver and decreases during the overshoot in pressure produced by release of the 
maneuver (phase IV). Sympathetic bursts will be detected by inspection of the filtered and mean 
voltage neurograms. A deflection on the mean voltage display is counted as a "burst" if it has a minimal 
signal to noise ratio of 2:1. The interobserver and intraobserver variations in identifying bursts are 
<10% and < 5% 18. When a given subject is studied on repeated occasions, the intra-subject variability 
is less than 15% 19. 
 
2) Measurement of Skeletal Muscle Oxygenation during exercise by Near Infrared (NIR) Spectroscopy: 
The NIR method is based on the principle that laser light with wavelengths in the 700–900 nm range 
penetrates tissues with relative ease, and is absorbed by the iron-porphyrin moieties in hemoglobin and 
myoglobin. Changes in NIR light absorption are proportional to changes in the relative concentrations of 
oxygenated hemoglobin and myoglobin (HbO2+MbO2). To monitor the tissue absorption of NIR light, 
two fiber optical bundles will be placed on the skin over the left flexor digitorum profundus muscle, 
which is the main muscle recruited during handgrip exercise. NIR signals at four different wavelengths 
will be sequentially sampled at a rate of 1 Hz, converted to optical densities by using known algorithms, 
and stored digitally for analysis (NIRO 500, Hamamatsu Photonics, Hamamatsu City, Japan).  
 
Skeletal muscle oxygenation will be measured at rest and during lower body negative pressure 
application to evoke reflex sympathetic activation. The subject’s lower body will be enclosed in a 
negative pressure chamber to the level of the iliac crest. Pressure inside the chamber will be measured 
by a Statham transducer (Gould, Oxnard, CA). Lower body negative pressure (LBNP) at -20 mmHg 
simulates mild orthostatic stress (e.g., the transition from the supine to the seated position). Then, 
subjects will be asked to perform 5 minutes of rhythmic handgrip exercise at 30% of maximal voluntary 
contraction. During the last 2 minutes of exercise, we will apply lower body negative pressure (LBNP) to 
activate sympathetic nerve activity. 
 
Blood pressure, SNA, heart rate, respiration, handgrip force, and NIR signals will be recorded in 
response to 2 min of LBNP applied at rest and during the 3rd and 4th min of each 5-min exercise 
period. Each exercise period will be followed by at least 2 min of forearm circulatory arrest during 
inflation of a pneumatic cuff on the upper arm to 250 mmHg to establish the maximal decrease in 
muscle tissue oxygenation. The total labile signal will be defined as the difference in tissue oxygenation 
in the forearm at rest and during sustained circulatory arrest. Changes in the NIR signals in response to 
LBNP alone and LBNP plus handgrip exercise will be expressed as a percentage of the total labile 
signal. 
 
3) Assessment of vascular sensitivity to angiotensin II:  To avoid confounding influence of sodium 
intake on BP, each subject will be given diet containing 200 mmol sodium, 100 mmol potassium, 800 
mg calcium, and 200–300 g carbohydrate for 7 days prior to the study. On the day of the study, two 
intravenous catheters will be inserted in antecubital veins (one in each arm) for infusion of angiotensin 
II and measurement of levels of isoprostanes, renin, and angiotensin II. Forearm blood flow (FBF) will 
be measured with high resolution Doppler Ultrasonography (Phillips HDI 5000, Bothell, WA, USA) with 
a 12 MHz linear array transducer. All 2D imaging and Doppler data will be digitized into our computer 
and analyzed off-line, using an automatic wall-tracking system (Vascular Analysis Tools, 5.0, Medical 
Imaging Analysis, Coralville IA). Beat-to-beat MBV will be calculated by integrating the total area under 
the Doppler waveform divided by RR interval. Forearm vascular resistance (FVR) will be calculated as 
mean arterial pressure (MAP) divided by forearm blood flow. BP will be measured noninvasively with 
oscillometric method (Welch Allyn, Vital Signs moitor). Cardiac output (CO) will be measured 
noninvasively by thoracic electrical bioimpedance (Bioz, CardioDynamics International Corporation). 
Stroke volume will be derived from change in impedance/time measured during electrical systole. 
Cardiac output will be determined as the product of stroke volume and heart rate. Total peripheral 

8 
July 25, 2008 
resistance (TPR) will be calculated by multiplying mean arterial pressure (MAP) by 80 and dividing the 
product by CO. 
 
Changes in MAP, FVR, TPR, renin, plasma F2-isoprostanes, and angiotensin II will be assessed at 
baseline and in response to intravenous infusion angiotensin II of at the dose of 1, 2, and 3 ng/kg/min 
for 15 minutes at each dose. 
 
Treatment 
After visit#2, each subject will be randomized to receive Nebivolol at the dose of 5 mg once daily or 
Metoprolol succinate (Toprol XL) 100 mg once daily. 
 
Visit#3 (4 weeks later): Subjects will return for BP check. If BP is < 90/60 mmHg or resting heart rate < 
50 bpm, the dose of drug will be reduced by 50%. If BP > 130/80, the dose of Nebivolol will be 
increased to 10 mg and Toprol XL will be increased to 200 mg once daily. 
 
Visit#4 (4 weeks later): Subjects will return for BP check. If BP is < 90/60 mmHg or resting heart rate < 
50 bpm, the dose of drug will be reduced by 50%. If BP > 130/80, the dose of Nebivolol will be 
increased to 20 mg and Toprol XL will be increased to 300 mg once daily. 
 
Visit#5 (4 weeks later): Subjects will return for measurement of SNA and skeletal muscle oxygenation 
at rest, during LBNP, and during exercise plus LBNP. Measurement of MAP, FVR, TPR, renin, plasma 
isoprostanes, and angiotensin II at baseline and in response to intravenous infusion angiotensin II of at 
the dose of 1, 2, and 3 ng/kg/min each for 15 minutes at each dose will be repeated as outlined in visit 
#2. Then the medications will be stopped for 2 weeks. This duration of washout period was chosen 
because previous crossover studies have demonstrated it to be adequate to allow detection of the 
difference in endothelial function between Nebivolol and other beta blockers without carryover effect. 
12,20 
 
Visit#6 (2 weeks later): Subjects will return for measurement of MAP, FVR, TPR, renin, plasma 
isoprostanes, and angiotensin II at baseline only. Then subjects will be given BP medications in the 
remaining arm (either Toprol XL 100 mg once daily or Nebivolol at the dose of 5 mg once daily). 
 
Visit#7 (4 weeks later): Subjects will return for BP check. If BP is < 90/60 mmHg or resting heart rate < 
50 bpm, the dose of drug will be reduced by 50%. If BP > 130/80, the dose of Nebivolol will be 
increased to 10 mg and Toprol XL will be increased to 200 mg once daily. 
 
Visit#8 (4 weeks later): Subjects will return for BP check. If BP is < 90/60 mmHg or resting heart rate < 
50 bpm, the dose of drug will be reduced by 50%. If BP > 130/80, the dose of Nebivolol will be 
increased to 20 mg and Toprol XL will be increased to 300 mg once daily. 
 
Visit#9 (4 weeks later): Subjects will return for measurement of SNA and skeletal muscle oxygenation 
at rest, during LBNP, and during exercise plus LBNP. Measurement of MAP, FVR, TPR, renin, plasma 
isoprostanes, and angiotensin II at baseline and in response to intravenous infusion angiotensin II of at 
the dose of 0.5, 1, and 3 ng/kg/min for 30 minutes at each dose will be repeated as outlined in visit #2 
and 5. End of study. 
 
Potential Risks: 
The potential risks are related to: a) microneurography, b) venous cannulation, c) intravenous infusion 
of angiotensin II, d) administration of nebivolol and metoprolol, e) lower body negative pressure, f) 
handgrip exercise and forearm circulatory arrest during inflation of a pneumatic cuff. 
 

9 
July 25, 2008 
a) The potential risks of microneurography: 10% of subjects may experience transient leg tiredness, or 
increase sensitivity to touch (hyperesthesia), motor weakness in the leg lasting less than 1 week. Since 
1979, microneurography has been performed on more than several thousand subjects without 
permanent complications related to procedure. 
 
b) The risks of intravenous catheters are infection, bleeding, and vasovagal reaction. 
 
 
c) The potential risks of infusing i.v. angiotensin II (IND #53,558) are hypertension and hypokalemia 
with chronic infusion. 
 
d) The potential risks of nebivolol and metoprolol are hypotension, bradycardia, fatigue, dizziness, and 
depression.  
 
e) Potential risks of lower body negative pressure application is vasovagal syncope if negative pressure 
is applied at -40 mmHg or lower for a prolonged period of time. However, in our study we will applied 
pressure at the lowest level of –20 mmHg for less than 5 minutes. Therefore, vasovagal syncope is 
unlikely to occur. 
 
f) Potential risks of handgrip exercise and forearm circulatory arrest during inflation of a pneumatic cuff 
are muscle ache or cramp. 
 
Protection Against Risks: 
 
a) Microneurography will be performed under the supervision of Dr. Vongpatanasin who has extensive 
experience.  A stimulus isolation unit (special electronic circuit) will be used to prevent electrocution 
from the recording apparatus. Subjects will be given a microneurographic questionnaire to report any 
adverse reactions occurring up to one week after the study.  
 
b) All subjects will have a routine history and physical examination performed by the physician-
investigators. In addition, hospital medical records will be reviewed prior to the initiation of these 
studies.  
 
c) ACLS-certified personnel, will be present during all experiments. Resuscitative equipment and drugs 
will be available in the laboratory. Blood pressure and the subject’s heart rate will be monitored 
continuously during the microneurographic experiments. Infusions of angiotensin II will be discontinued 
if mean arterial pressure decreases or increases by more than 20 mmHg above the baseline level. 
 
d) During treatment with antihypertensive medication with Nebivolol and Metoprolol, patients will be 
seen at the cardiovascular physiology research laboratory for monitoring of BP.   
 
e) Venous cannulation will be performed by an experienced nurse practitioner, Debbie Arbique, RN.  
 
Procedures to Maintain Confidentiality   
1.  Information will be given only to the physician-investigators.   
2.  The nature of the information concerns pertinent medical and social history. 
3. The purpose of the disclosure is solely for research purposes, in particular to determine whether 
potential subjects meet criteria for inclusion or exclusion and to identify specific subgroups of subjects.     
4.  The subject’s right of confidentiality will be given strict priority. No mention of the subjects’ identities 
will be made either directly or indirectly in oral or written presentation of this work. The investigators, 
who are physicians, will be responsible for providing medical care in the event of any adverse effects to 
the subjects. 

10 
July 25, 2008 
5. All medical and biographical information will be held strictly confidential and no disclosures of 
personal identity will be allowed unless specifically requested by the subject. Copies of executed 
consent forms, as well as the experimental log book, will be kept in a locked file cabinet in our research 
laboratory. 
 
Evaluation Criteria 
The study is designed to evaluate SNA, skeletal muscle oxygenation, BP, FVR, and TPR, and 
neurohormonal responses to exercise and angiotensin II during treatment with two antihypertensive 
medications which are already approved by the FDA for treatment of hypertension. The study is not 
designed to evaluate survival, efficacy, or toxicities of a new drug. The maximal dose of each drug used 
in this study is described under treatment section above. 
 
Off-study Criteria 
Stop Points:  The following will constitute stop points indicating permanent discontinuation of study 
drug by participant and scheduling of close out visit.   
 
• 
Withholding of study medication for > 4 weeks for whatever reason. 
• 
Any reason including serious adverse events that results in participant’s inability to continue with 
study protocol and procedures.  
• 
Severe hypertension defined as persistent elevation of systolic BP > 180 mmHg or diastolic BP 
> 110 mmHg on 3 consecutive measurements after randomization 
• 
Refractory hypotension defined as persistent systolic BP < 90 mmHg   
• 
Depression requiring drug treatment or psychiatric evaluation 
• 
Asthma or brochospasm 
• 
Symptomatic bradycardia with heart rate persistently below 50 beats/min 
• 
Pregnancy 
• 
Death  
 
Conditions for temporarily halting study medications:  These include 1) BP < 90/60 mmHg; c) 
Severe uncontrolled hypertension in the outpatient setting defined as elevation of systolic BP > 180 
mmHg or diastolic BP > 110 mmHg on two consecutive occasions measured at least 48 hours apart 
despite effort to lower BP by diet or pharmacologic intervention. 
 
  
Conditions for permanently halting study medication in an individual:  A stop point will be 
defined as any serious adverse event that in the opinion of the Principal investigator could be 
reasonably attributed to the study intervention and could be made worse or recur by continued 
administration of the study medication. At this time the participants study medication will be 
permanently discontinued and a close out visit will be scheduled.   
  
 
 
Conditions for stopping the protocol entirely: Conditions for stopping the entire protocol are 1) 
cumulative incidence of severe uncontrolled hypertension (>180/110 mmHg) or heart rate < 50 bpm of 
> 50% of the study cohort after a minimum of 10 patients have been randomized to study drug, (2) any 
evidence of myocardial ischemia or infarction (chest pain, EKG changes), (2) any new onset of 
sustained cardiac arrhythmia including heart block, atrial fibrillation, supraventricular tachycardia, 
ventricular tachycardia, or ventricular fibrillation; (3) any death. Under such circumstances, the study 
will not resume without IRB approval.   
 
Registration and Study Monitoring 
The PI will immediately report all unexpected and any serious adverse events and the 
recommendations derived from data and safety monitoring (such as continuation or conclusion of the 
trial) to the IRB.   

11 
July 25, 2008 
The PI will submit copies of adverse events to the IRB. 
The PI will anonymize all reports of local adverse events submitted to the IRB to protect a subject’s 
privacy. 
The PI will educate all key personnel about the requirements to report unexpected and serious adverse 
events to the IRB. 
 
Statistical Methods   
SAS (SAS Institute, Cary, NC) statistical software will be used for statistical analyses, particularly 
PROC MIXED for linear models.  Model assumptions such as normality and covariance structure will be 
carefully assessed.  Data transformations or nonparametric tests may be performed to meet analysis 
assumptions. Hypothesis tests will be two-sided using the 0.05 significance level.   Bonferroni type 
adjustments for multiple testing will be implemented to control Type I errors as necessary.  
 
Sample size, power estimates, statistical method 
Power calculations were based on the within-person variability of the angiotensin II infusion rate 
required to raise mean arterial pressure by 20 mm Hg (Pd20). These values were previously 
determined by studies in our laboratory and others 21. Normotensive men had intra-individual 
coefficients of variation that averaged 18% for angiotensin II 21. Assuming a greater BP variability in 
hypertensive patients’ of approximately 25%, it was estimated that a crossover study of 20 patients 
would have 80% certainty of demonstrating a 15% difference in cardiovascular reactivity between 
nebovolol and toprol XL at the alpha level of 0.05. 
 
To allow for 25% attrition during the study, total of 25 subjects will be enrolled in the study. Treatment 
order will be randomized using blocked randomization. 
 
Statistical method: Descriptive statistics will be used to summarize the responses over infusion time for 
each phase of this study. To compare responses between the study phases, repeated measure 
analysis of variance models with repeated factors for both study phase and infusion time will be 
constructed. Polynomial contrasts will be constructed to compare the linear trends or slope of the MAP, 
TPR, and FVR responses to infusion of Ang II over time between the study phases.  A between group 
factor will be included in these models to assess treatment order effects.  A mixed linear model 
approach will be used for repeated measures analysis that allows flexibility in the specification of the 
covariance structure over time and can accommodate missing data in most situations.  
 
Gender/Minority Analysis: 
 
Inclusion of Women 
Approximately 50% of our subjects will be women.  
 
Inclusion of Minorities 
We are over-sampling non-Hispanic Blacks because (1) prevalence of hypertension in the U.S. are 
higher in blacks than nonblacks, (2) the class of drugs we are studying is thought in general to be more 
effective in lowering blood pressure in blacks than other ethnic groups. Our aim is to obtain a 50:50 split 
in ethnicity between non-Hispanic blacks (“blacks”) and all other ethnicities (“non-blacks”). The non-
black group will be ~60% white, 35% Hispanic, and 5% other.  
 
Inclusion of Children  

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July 25, 2008 
Children will not be included because: (1) the safety of microneurography in children has not been 
established and children are not likely to undergo successful microneurographic procedure which 
requires staying still in bed while keeping the leg absolutely motionless for 3-4 hours to allow adequate 
nerve recording, (2) the safety of administration of angiotensin II is unknown, and 3) children are much 
more likely to have secondary hypertension than adults.     
 

13 
July 25, 2008 
Targeted/Planned Enrollment Table: 
 
Study Title: Attenuation of Angiotensin II-Mediated Vasoconstriction in Hypertension with 
Nebivolol 
Total Planned Enrollment: 25 
Ethnic Category 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
2 
2 
4 
Not Hispanic or Latino 
11 
10 
21 
Ethnic Category Total of All Subjects* 
13 
12 
25 
Racial Categories 
 
American Indian/Alaska Native 
0 
0 
0 
Asian 
0 
0 
0 
Native Hawaiian or Other Pacific Islander  
0 
0 
0 
Black or African American  
7 
6 
13 
White 
6 
6 
12 
Racial Categories: Total of All Subjects * 
13 
12 
25 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 

14 
July 25, 2008 
 
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