Peripheral Vascular Effects of Sulfhydryl-containing Antihypertensive Pharmacotherapy on Microvascular Function and Vessel Remodeling in Hypertensive Humans
Phase: PHASE1
Conditions: Hypertension,Essential
Sponsor: Penn State University

 
 
 
 
 
 
 
 
 
 
Peripheral Vascular Effects of Sulfhydryl-containing Antihypertensive Pharmacotherapy 
on Microvessels in Humans 
NCT03179163 
10/25/2021 
Study Protocol and Statistical Analysis Plan 

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Protocol for
Human Subject Research
with Use of Test Article(s)
Protocol Title: Peripheral vascular effects of sulfhydryl-containing antihypertensive 
pharmacotherapy on microvascular function and vessel remodeling in hypertensive humans 
(IRB#3224)
Principal Investigator:
Name:  Lacy M. Alexander, Ph.D.
Department: Kinesiology
Telephone: 814-863-8557
E-mail Address: lma191@psu.edu
Version Date: 10/25/2021
Clinicaltrials.gov Registration #: 03179163
1.0
Objectives
1.1
Study Objectives
Essential hypertension is a complex pathology and clinical guidelines (JNC8) strongly support angiotensin 
converting enzyme inhibitors (ACEi) for the treatment of essential hypertension. It is increasingly recognized 
that secondary peripheral vascular effects of systemic ACEi significantly contribute to reduced mortality in 
hypertension-associated disease. A specific class of ACEi containing a sulfhydryl group is purported to exert a 
beneficial peripheral effect through their highly lipophilic targeting to the endothelium where they act through 
hydrogen sulfide (H2S)-dependent mechanisms. With the emergence of the importance of H2S as a modulator 
in hypertension, there is an immediate need to explore the therapeutic potential related to these signaling 
mechanisms in humans. The proposed work has the potential to uncover novel vascular signaling mechanisms 
related to the gasotransmitter H2S in humans and better understand target-based therapeutics in hypertension 
associated vascular dysfunction.
Note:  This project covers Specific Aim 2 of the grant.
Specific Aim: To determine the peripheral vascular effects of sulfhydryl (SH)-containing antihypertensive 
pharmacotherapy on in vivo and in vitro indices of microvascular function and vessel remodeling (longitudinal 
intervention). We will utilize a 16-wk randomized intervention comparing and contrasting the effects of (1) a
SH-ACEi, (2) a non-SH containing ACEi, and (3) a thiazide-type diuretic (nonvascular therapeutic control).
Hypothesis a. Both SH-ACEi and ACEi treatments will improve endothelial-dependent vasodilation in response 
to physiological and pharmacological stimuli (in vivo) and improve in vitro markers of vasodilator 
responsiveness (pVASP/VASP) compared to diuretic treatment.
Hypothesis b. SH-ACEi will be more effective at reducing end-organ vasoconstrictor responsiveness to 
adrenergic stimuli than ACEi and diuretic treatments.
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Hypothesis c SH-ACEi will improve in vivo and in vitro indices of microvessel remodeling compared to ACEi 
and diuretic treatment.
Hypothesis d. SH-ACEi will improve exogenous H2S mediated-vasodilation compared to ACEi.
Hypothesis e. SH-ACEi will [synergistically] improve endothelium-dependent vasodilation through both H2S 
and NOS-dependent mechanisms (cross talk).
1.2
Primary Study Endpoints
The study ends when enough subjects have completed the protocol to adequately test the above hypotheses.  
Outcomes include blood pressure (BP), cutaneous vascular conductance (DVD), flow mediated vasodilation 
(FMD), rate of reactive oxygen species (ROS) production, H2S production, protein concentration, and hydrogen 
sulfide (H2S), acetylcholine (ACh), norepinephrine (NE) dose responses.
1.3
Secondary Study Endpoints
NA
2.0
Background 
2.1
Scientific Background and Gaps
Essential hypertension is a complex pathology characterized by systemic inflammation, altered neurovascular 
control, including microvascular dysfunction characterized by a loss of endothelium-dependent signaling 
pathways), and vessel remodeling. Clinical outcomes data forming the basis for the Eight Joint National
Committee (JNC8) guidelines strongly support angiotensin converting enzyme inhibitors (ACEi) for the first line 
treatment of essential hypertension. Importantly, reduced mortality attributable to ACEi therapy is strongly 
related to their secondary peripheral vascular effects.
Emerging data indicate that ACEi containing a sulfhydryl group (SH-ACEi) exert beneficial peripheral vascular 
effects via lipophilic targeting to the endothelium where they act through hydrogen sulfide (H2S)-dependent 
mechanisms. H2S is a gasotransmitter that acts as an endothelium-derived hyperpolarizing factor (EDHF) and 
is synthesized in the vasculature enzymatically by cystathionine-γ-lyase (CSE) and mercaptopyruvate 
sulfurtransferase, (MPST). Because of the convergence of H2S on multiple endothelial signaling pathways 
including NO, examining H2S signaling is critical to elucidating (1) the mechanisms of vascular dysfunction in 
hypertension, and (2) its viability as a therapeutic target for SH-based pharmacologics.
We propose to perform a comprehensive examination of the impact of different classes of antihypertensive 
pharmacotherapy on indices of eutrophic microvessel remodeling. The degree of vessel remodeling in 
hypertension is negatively associated with clinical outcomes. The proposed studies will provide insight into the 
potential benefits of utilizing available SH-containing antihypertensive agents on validated clinical outcomes. The 
research team is well-positioned to translate laboratory findings into [an in vivo human model and determine the 
effects of target-based antihypertensive treatments.
In summary, the proposal represents an integrative and innovative effort to (1) provide mechanistic evidence 
about the putative benefits of specific classes of antihypertensive agents, and (2) identify possible target-based 
treatment strategies in humans.
2.2
Previous Data
This study continues the research and focus of the study, “Cutaneous vascular effects of Hydrogen Sulfide,” 
(IRB# 43891). 
Published data from our laboratory20, 22 demonstrate that essential hypertensive humans have microvascular 
dysfunction characterized by attenuated endothelium-dependent vasodilation to both pharmacological and 
physiological stimuli, increased adrenergic responsiveness, and evidence of eutrophic microvessel 
remodeling1, 2. These data are further supported by biochemical data obtained from the analysis of human skin 
tissue showing reduced pro-vasodilator markers including phosphorylated vasoactive simulator protein 
(pVASP/VASP).
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We have developed and validated in vivo methodology to examine H2S-mediated vasodilation in human 
subjects (N=11). Our exogenous H2S dose-response protocol, in which two different commonly utilized H2S 
donors were delivered locally to the cutaneous microcirculation to elicit vasodilation, demonstrated that there 
was no difference in functional responsiveness or measured concentration of H2S generated from the NaHS or 
Na2S (R2=0.97)3. 
In addition, we have published data determining the downstream mechanisms by which exogenous H2S 
induces vasodilation in the human cutaneous vasculature3. We tested the efficacy of KCa channel inhibitors to 
elucidate the specific mechanisms underlying H2S-mediated vasodilation. The data suggest that KCa channels, 
and not KATP channels, mediate exogenous H2S-induced cutaneous vasodilation. Our published in vivo data 
are novel with respect to mechanistic investigation of H2S-mediated vasodilation in humans3. [Note, while more 
specific K channel inhibitors are available for use in animal studies, to date, the specific KCa and KATP inhibitors 
used in that study are the only available inhibitors available for use in humans. If others become available, we 
would test the efficacy upon IRB and FDA approval.] 
We have also published studies with appropriate NOS and COX blockade to examine potential cross-talk 
mechanisms involved in H2S-mediated vasodilation3. The cutaneous vasodilation response to exogenous H2S 
during both single and double blockade protocols showed that, similar to animal models4, 5, there is cross talk 
between H2S, NOS, and COX pathways in humans (N=8).
Pilot data have been collected on hypertensive subjects naïve to pharmacotherapy (resting MAP=109±5 
mmHg). In support of hypothesis 1a, these data show decreased H2S-mediated vasodilation, illustrating 
decreased peripheral vascular responsiveness specific to H2S [that occurs, in part, through crosstalk with NOS 
systems.
In further support of hypothesis 1a, we have performed dose-response experiments to the endothelium-
dependent agonist acetylcholine in the presence of the H2S enzymatic inhibitor aminooxyacetic acid (AOAA). 
NOTE: AOAA is the only available CSE inhibitor available for human use. We first verified the efficacy of this 
inhibitor by utilizing an in vitro assay. In vivo data demonstrate that H2S inhibition with AOAA attenuated 
acetylcholine-induced endothelium dependent cutaneous vasodilation in healthy adults (83±9), indicating that 
H2S contributes to agonist mediated endothelium-dependent vasodilation healthy adults. In contrast, H2S-
mediated endothelium dependent vasodilation was reduced in 3 hypertensive adults (MAP 105±5) as the 
enzymatic H2S inhibitor AOAA did not alter the response to Ach. These pilot data demonstrate that 
endogenous H2S-mediated vasodilation is reduced in hypertensive humans. 
In support of our in vivo data, in vitro approaches for examining H2S-dependent mechanisms have been 
refined and optimized. Protein expression data for the main enzymatic sources of H2S in our target tissue, CSE 
and MPST (N=7) demonstrate the presence of H2S-generating enzymes in the human cutaneous 
microcirculation. The inhibition of CSE by AOAA was verified in these experiments.
Pilot data in support of hypothesis 2b data show CSE and MPST are expressed in human skin samples. We 
also show that enzymatic production of H2S is increased after treatment with the non-SH donating ACEi 
Lisinopril to normalize blood pressure. The pilot data demonstrate the beneficial peripheral vascular effects of 
systemic ACE-inhibition on these indices.
We have also conducted a preliminary cross-sectional study to examine the peripheral vascular effects of 
different classes of antihypertensive pharmacotherapy (N=10, MAP=96±4mmHg). These pilot data 
demonstrate moderate effectiveness of systemic ACEi to improve cutaneous endothelial measures. These 
improvements are due, in part, to an increase in both EDHF- and NOS-dependent signaling. Consistent (with 
regard to these classes of antihypertensive agents) with hypotheses 2a and 2b, we observe improvements in 
the function of endothelial mediators in subjects prescribed ACEi but do not observe any change in indices of 
eutrophic vessel remodeling. These data highlight the need to conduct a well-controlled interventional study 
testing the efficacy of antihypertensive pharmacotherapy -- including SH-ACEi, ACEi, and diuretics -- on 
indices of eutrophic vessel remodeling.
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In addition to the pilot and preliminary data shown here, our research team has extensive experience 
conducting pharmacological interventional studies in human subjects. We have published extensively on the 
pleiotropic effects of systemic statin therapy6, 7, 8 and systemic supplementation of essential cofactors and 
signaling intermediates to improve vascular function in adults with pre-clinical CVD9, 10. Importantly, these 
studies, published in high impact clinical and physiology journals, utilize similar methodologies to those 
proposed herein.
Our fellow investigator, Dr. Gail Thomas, has utilized a similar experimental design to take advantage of 
between (irbesartan vs chlorthalidone) and within (nebivolol vs. metoprolol) class differences in 
antihypertensive agents to gain insight into the central and peripheral mechanisms causing enhanced 
vasoconstriction in human subjects with essential hypertension11, 12. Consistent with hypothesis 2b 
antihypertensive agents with greater peripheral vasodilatory potential are more effective at reducing 
vasoconstrictor responses114.
2.3
Study Rationale
We developed and validated the human cutaneous microcirculation as a powerful model for the in vivo 
examination of novel signaling mechanisms mediating microvascular dysfunction in hypertensive adults. The 
emerging importance of H2S as an endothelial signaling modulator and inhibitor of eutrophic vessel remodeling 
in hypertension highlights the need to examine target-based intervention strategies related to these 
mechanisms. SH-ACEi have been extensively prescribed for the secondary treatment of cardiovascular 
disease with highly effective clinical outcomes; however, the precise mechanisms that underlie the therapeutic 
benefit of SH-ACEi in primary hypertension are unknown. We propose to elucidate the mechanisms underlying 
attenuated H2S signaling and the mechanistic effects of lipophilic SH-ACEi in hypertensive humans in two 
specific aims. We will utilize in vivo and in vitro approaches to address the following aims to determine the 
peripheral vascular effects of SH-containing antihypertensive pharmacotherapy on indices of microvascular 
function and vessel remodeling in an interventional study with SH-based antihypertensive therapies.
3.0
Inclusion and Exclusion Criteria
3.1
Inclusion Criteria

women and men

40-65 years

blood pressure: 
Normotensive
<120/80 mmHg
Hypertensive
≥140/90 mmHg and <160/110 mmHg

HbA1C of <6.5%

Women are post-menopausal and not taking hormone replacement therapy, or have normal 
cycles and are tested in the early follicular phase. Subjects may or may not be taking one doctor-
prescribed drug to lower blood pressure (e.g. diuretic, ACE inhibitor).
o
Must be able to stop physician-prescribed antihypertensive drug for the duration of the 
subject’s participation in the study (with the approval of their personal physician).
3.2
Exclusion Criteria
Relevant to all subjects:

current medications which could conceivably alter the cardiovascular or thermoregulatory control or 
responses (e.g. beta blockers, calcium channel blockers, angiotensin receptor blockers)

allergy to test substances

allergy to latex

nicotine use (smoking, chewing tobacco, etc.)

illegal/recreational drug use

pregnancy or breastfeeding

diabetes

men taking medication for erectile dysfunction
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Relevant to all hypertensive subjects:

contraindication for all three pharmocotherapy drugs used in this study
o
Note:  Subjects who have a contraindication (e.g. a condition, medication with a known 
interaction, known allergy) to only one or two of the three pharmacotherapy drugs, may be 
assigned one of the pharmocotherapy drugs that is not contraindicated.  

kidney problems

liver problems

history of heart disease or failure

history of blood clots or stroke

angioedema

electrolyte imbalance

planned surgery requiring general anesthesia during the pharmacotherapy period

peripheral vascular disease
Relevant only to hypertensive subjects who are currently taking a physician-prescribed antihypertensive 
drug (e.g. diuretic, ACE inhibitor).

Taking more than one physician-prescribed drug to lower blood pressure

Unable or unwilling to stop the physician-prescribed antihypertensive drug for the duration of the 
subject’s participation in the study (with the approval of their personal physician).

Unable or unwilling to obtain the personal physician’s approval to stop the physician-prescribed 
antihypertensive drug for the duration of the subject’s participation in the study.

patients who are taking clonidine

African Americans who are taking a calcium channel blocker (Norvasc)

Potential subjects who are taking physician-prescribed drugs for the control of hypertension will be 
evaluated and excluded when necessary by the principal investigator and/or Dr. Fragin on a case by 
case basis.
3.3
Early Withdrawal of Subjects
3.3.1
Criteria for removal from study
Participants may withdraw at any time.  We may end the participant’s role in the study without her/his consent if 
the researcher deems that her/his health or behavior adversely affects the study or increases the risks beyond 
those approved by the Institutional Review Board and agreed upon by her/him in the informed consent, or we 
develop new exclusion criteria. 
Relevant only to hypertensive subjects who are currently taking a physician-prescribed antihypertensive drug.
Subjects discontinue the physician-prescribed antihypertensive drug for a 14-day washout period prior to 
participating in the first experiment.  During the 14 days, they self-check their blood pressure with a blood 
pressure monitor (supplied by us) every 6-8 hours.  If the subject’s blood pressure exceeds 140 mmHg systolic 
or 90 mmHg diastolic during the 14 days, the subject notifies Susan Slimak, RN (the research nurse), resumes 
taking the physician-prescribed antihypertensive drug, and withdraws from the study.
3.3.2
Follow-up for withdrawn subjects
We follow up with subjects that withdraw from the study within one week of removal or withdrawal. The research 
nurse and/or project-affiliated physician follows up on withdrawals that are related to medical concerns.  We 
contact them by phone or e-mail. Subjects are replaced by recruitment of new subjects. 
  Relevant only to hypertensive subjects who are currently taking a physician-prescribed antihypertensive drug.
We instruct subjects who withdraw from the study due to excessive blood pressure during the 14-day 
washout (see 3.3.1) to notify their personal physicians.  The research nurse or study physician follows up with 
the subject within 24 hours.  The research nurse contacts the subject 10 days later for additional follow-up.  We 
advise subject to follow up with their personal physicians.
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4.0
Recruitment Methods
4.1
Identification of subjects
We advertise for subjects. Interested persons contact us. We also avail ourselves of lists of potential participants 
maintained by the CRC and our lab. The people on these lists have screened for other studies and indicated that 
they wished to be maintained on a list of potential participants to be contacted in the event that they may qualify 
for addition studies.   
4.2
Recruitment process
Research Match sends out emails to those who meet our eligibility criteria.  If the potential subject chooses to 
click “yes” in the email, we receive the contact info for that subject allowing us to contact the potential subject.  
Also, we advertise for subjects (see below).  Interested persons contact us.  Using the IRB-approved “phone-
script” or “ad,” we telephone or email subjects, respectively, from the lists of potential subjects (see above). We 
obtain permission before using a listserv that we determine may aid us in recruitment.  We discuss the study’s 
purpose and protocol as well as the qualifications for the study with potential subjects.  We tell them about the 
study and, if they are interested in participating, give them a chance to come to lab for a screening.
4.3
Recruitment materials
Research Match
Studykik
Newspaper/magazine ads (print and/or online)
Letters/Emails to potential participants  
Flyers/posters (permission to post obtained when needed)
Script - Verbal (i.e., telephone, face-to-face, classroom)
Brochures (describes the general research foci of the lab group)
Web Sites    (website and initial screening form collect the same basic pre-screening information)
Listserv  (on and off campus that target under-represented groups (e.g. FOBA) as well as listservs belonging 
groups (e.g. Elks) and others as we become aware of them; permission obtained)
Note:  We have a recruiting website through the Kinesiology department that we have used for most of our 
studies.  The uploaded phone script and/or ad are used for additional recruiting purposes such as classroom 
announcements and emails.
4.4
Eligibility/screening of subjects
We use the phone questionnaire and the website documents to determine eligibility before obtaining informed 
consent. We inform subjects that the information they provide is kept confidential.  The pre-screening information 
is provided by the subject either verbally over the phone or via electronically via REDCap. If this occurs via 
phone, the investigator records this information on the telephone interview form. The data from people who 
qualify or do not qualify for the study is stored until end of study, then destroyed.  We request permission from 
participants who qualify or do not qualify for the study to maintain the information and to contact for future studies  
Relevant to hypertensive subjects who are currently taking a physician-prescribed antihypertensive drug.
We fax a letter and completed HIPAA form (see uploaded documents) to the subject’s physician 
requesting the physician’s approval for the subject to stop taking the physician-prescribed drug for the duration 
of the subject’s participation in the study.  We only include a subject in the study when her/his personal physician 
returns the letter indicating the physician’s permission.
5.0
Consent Process and Documentation 
5.1
Consent Process 
5.1.1
Obtaining Informed Consent
5.1.1.1 Timing and Location of Consent
When a potential participant reports to Noll Lab or CRC, she/he signs the informed consent before screening 
procedures begin.   
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5.1.1.2 Coercion or Undue Influence during Consent
If subjects are not able to understand the protocol and instructions for any reason, written or verbal, they are not 
included in the study. Subjects are informed throughout the consenting, screening, and conduction of the study 
that their participation is voluntary, and they may discontinue their participation at any time. It is possible that a 
person enrolled in the study could be a student or employee.  We tell the person that participation in the study is 
voluntary and no aspect of their participation or non-participation has an effect on their class or grade, 
employment or salary, respectively.  The person conducting the screening and consenting is not a professor 
from any of the student’s classes or the employee’s supervisor.
Relevant to hypertensive subjects who are currently taking a physician-prescribed antihypertensive drug.
The HIPAA form informs subjects that their treatment, payment, or enrollment in any health plans or 
eligibility for benefits is not affected by their declining to sign the HIPAA form.
5.1.2
Waiver or alteration of the informed consent requirement
NA
5.2
Consent Documentation
5.2.1
Written Documentation of Consent
All participants sign the informed consent document. After the participant signs the consent, we photocopy it and 
give it to the participant.
5.2.2
Waiver of Documentation of Consent
We are requesting a waiver of documentation of consent for the screening procedures only. It will be a verbal 
consent on the phone, or an implied consent if the screening is done in Redcap. These procedures present no 
more than minimal risk. Due to COVID-19, one subject who has her own blood pressure monitor at home will 
provide verbal consent to: (1) monitor her blood pressure at home, (2) report it to the investigators by telephone, 
and (3) will have her medications mailed to her residence directly from the pharmacy.  This participant is 
confirmed post-menopausal and there for a pregnancy test is not required to refill her medication.  
5.3
Consent – Other Considerations 
5.3.1
Non-English Speaking Subjects
All participants will be able to understand written and verbal English and give consent in English.
5.3.2
Cognitively Impaired Adults
This research does not include cognitively impaired adults.
5.3.3
Subjects who are not yet adults (infants, children, teenagers) 
NA
6.0
HIPAA Research Authorization and/or Waiver or Alteration of 
Authorization
NA
6.1
Authorization and/or Waiver or Alteration of Authorization for the Uses and Disclosures of 
PHI
Check all that apply:
X
Authorization will be obtained and documented as part of the consent process.
If a potential subject is taking a personal physician-prescribed antihypertensive drug, the subject may participate if 
her/his personal physician gives permission for the subject to discontinue the antihypertensive drug for the duration of the 
subject’s participation in the study.  In this case, the subject signs a HIPAA form (see related uploaded documents).
Partial waiver is requested for recruitment purposes only (Check this box if patients’ 
medical records will be accessed to determine eligibility before consent/authorization has 
been obtained)
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Full waiver is requested for entire research study (e.g., medical record review studies) 
Alteration is requested to waive requirement for written documentation of authorization
6.2
Waiver or Alteration of Authorization for the Uses and Disclosures of PHI
6.2.1
Access, use or disclosure of PHI representing no more than a minimal risk to the 
privacy of the individual
6.2.1.1 Plan to protect PHI from improper use or disclosure
6.2.1.2 Plan to destroy identifiers or a justification for retaining identifiers 
6.2.2
Explanation for why the research could not be practicably be conducted without 
access to and use of PHI
6.2.3
Explanation for why the research could not practicably be conducted without the 
waiver or alteration of authorization
6.3
Waiver or alteration of authorization statements of agreement
7.0
Study Design and Procedures
7.1
Study Design 
We recruit subjects from central Pennsylvania using resources including the Penn State Center for Healthy 
Aging and our clinical translational research center (CTRC). We over-sample minority populations using 
currently active strategies approved by NIH. Because JNC8 has different treatment recommendations for Black 
hypertensive patients, concerted efforts are made to recruit all races, and secondary analyses are conducted 
to examine race differences.
Upon initial screening and again within a week of testing, all subjects have an assessment of 24-hour 
ambulatory BP. The research nurse and the Penn State CTRC medical staff perform the screening which 
includes anthropometry, and a chemical and lipid profile, liver and renal function. We analyze the blood 
samples of enrolled subjects for ACE activity, Ang II, Ang 1-7, oxLDL subfraction particles, ApoB, and 
assymetrical dimethyl L-arginine (ADMA) concentrations (ELISA), and plasma inflammatory cytokine panel (IL-
6, sIL-6α receptors, CRP, TNFα, and gp130). Subjects also undergo an assessment of conduit vessel 
endothelial and vascular smooth muscle function with brachial artery flow-mediated vasodilation (FMD) and 
sublingual nitroglycerin, respectively.
We include normo- and hypertensive subjects.  We recruit hypertensive subjects who may or may not be 
taking a physician-prescribed antihypertensive drug (e.g. diuretic, ACE inhibitor).  Subjects taking a physician-
prescribed antihypertensive drug cease taking the drug (with their physician’s documented permission) for 14 
days prior to the first experiment.  They do not take their physician-prescribed antihypertensive drug for the 
duration of their participation in the study.
The study is a single blind randomized control trial consisting of 16 weeks of antihypertensive 
pharmacotherapy. (Pharmacokinetic and dynamic data from the literature suggest that blood pressure lowering 
and peripheral vascular effects are maximized by 12 weeks of antihypertensive therapy.  Investigators are 
blinded to the condition, but the physician and nursing oversight are not. Captopril (SH-ACEi), enalapril (ACEi), 
or hydrochlorothiazide (HCTZ) are used for hypertensive therapy as required by the subject to target BP of less 
than 135/85 mmHg. Titration and doses are performed in accordance with FDA-approved labeling. If blood 
pressure is not within the target range after 12 weeks, we recruit additional subjects. A group of age- and sex-
matched normotensive time control subjects are included (no pharmacotherapy).
Subjects undergo initial microdialysis (MD) experiments, and biopsy sampling. Then we randomly assign 
hypertensive subjects to a treatment group. Our nurse coordinator monitors blood pressure every 4 weeks and 
performs weekly compliance checks (telephone and/or email). The nurse performs 24-hour ambulatory blood 
pressure monitoring at week 8 to determine the efficacy of antihypertensive treatment and to inform dosing 
titration.  Pharmacokinetic and dynamic data from the literature indicate that blood pressure lowing and 
peripheral vascular effects are maximized by 12 weeks of antihypertensive therapy and maintained
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thereafter. After 16 weeks of the assigned intervention, subjects repeat the 24-hour BP monitoring, 
FMD/sublingual nitroglycerin trials, and MD experiments. We obtain post-pharmacotherapy cutaneous biopsies 
and repeat the venous blood sampling/analysis.
The normotensive time-control subjects undergo the series of experiments and then repeat the experiments 
about 16 weeks later.
7.2
Study Procedures
Screening:  The subjects drink only water and do not eat after 10 PM the evening before the visit.
The research nurse and/or Clinical Research Center staff perform the screening procedures. The screening includes blood 
pressure, heart rate, height, waist circumference, and weight. If the research nurse is unavailable for an extended period of 
time, the CRC performs the full screening. The CRC clinician reviews the medical history.  The CRC uses their admission 
form to admit potential participants for screening.  Women who are not postmenopausal submit urine samples for pregnancy 
tests. The nurse draws about 30 ml (2 Tbsp) of blood from a vein in the subject’s arm.  We send the blood to labs for some 
analyses.  If the subject takes thyroid hormone, we draw an extra 3.5 ml (0.2 Tbsp) to check the level of thyroid hormone. 
Screening tests performed on blood:  CBC, Lipid panel, and blood chemistry, Interleukin Panel (IL), folic acid 
and B12, homocysteine, oxidized LDL, total antioxidant capacity. We do not perform genetic analyses on the 
blood nor look for presence of disease (e.g. HIV). 
Subjects with hypertension or suspected of being hypertensive undergo ambulatory 24 hour blood pressure 
monitoring.
If a potential subject is taking a single, personal physician-prescribed antihypertensive drug (e.g. diuretic, ACE 
inhibitor), the subject may participate if her/his personal physician gives permission for the subject to discontinue 
the drug for the duration of the subject’s participation in the study.  In this case, the subject signs a HIPAA form 
and the physician signs a permission form (see related uploaded documents).  Upon obtaining permission from 
her/his physician, the subject ceases the physician-prescribed antihypertensive drug for a 14-day washout period 
prior to participating in the first experiment.  During the 14 days, she/he self-checks her/his blood pressure with 
a blood pressure monitor (supplied by us) every 6-8 hours.   During the first week, the study nurse contacts the 
subject daily to check the subject’s progress and discuss concerns the subject may have.  During the second 
week, the study nurse contacts the subject every other day to check the subject’s progress and discuss concerns 
the subject may have.  If the subject’s blood pressure exceeds 168 mmHg systolic or 96 mmHg diastolic during 
the washout, the subject resumes taking the physician-prescribed antihypertensive drug, and withdraws from 
the study.  We will consult with Dr. Fragin concerning those potential subjects taking a beta blocker because the 
length of the washout period could be longer than 2 weeks depending upon the dose of beta blocker the subject 
is taking.  We extend the monitoring-protocol used after the first week of washout for washout durations that are 
longer than two weeks.  We advise the subject to follow up with her/his personal physician.
Experiments:
Blood Sample Experiment:  When the subject comes to the lab for one of the first experiments, we obtain a 30 ml (2 Tbsp) 
blood sample to analyze for substances pertinent to the specific aims of this study (e.g. ACE activity, Ang II, Ang 1-7, 
oxLDL subfraction particles, ApoB, and assymetrical dimethyl L-arginine (ADMA) concentrations (ELISA), and plasma 
inflammatory cytokine panel (IL-6, sIL-6α receptors, CRP, TNFα, and gp130)).  We conduct some of the analyses at the 
Noll Lab.  We send some of the sample to an outside lab for the other analyses (see letter from Joseph Cannon, Ph.D.).
Microdialysis Experiments:  (conducted on separate days)
Microdialysis Overview:  Microdialysis (MD) is a procedure in which a thin tube of membrane that mimics the 
capillary blood vessel is implanted in tissue.  As a physiological saline perfuses the membrane, there is bi-
directional exchange of molecules between the perfusing saline and the fluid bathing the tissue surrounding 
the membrane.  However, the membrane prohibits the exchange of large molecules such as proteins.  
Substances of interest may be added to the perfusing saline and thus delivered into the surrounding tissues 
with no systemic effects.  
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Preparation for all MD experiments:  We give printed and verbal instructions outlining what the subjects need to do 
before they come to the lab.  Subjects do not eat or drink anything containing caffeine (ex. coffee, tea, Coca Cola, chocolate) 
for 12 hours before the experiment.  
We measure HR and BP. Women who are not postmenopausal undergo a urine pregnancy test if they have 
not had one within 2 weeks.  The subject washes the ventral forearm with antimicrobial soap.  
Microdialysis probe insertion:  We place a tight band around the arm so we can visualize veins.  For each MD 
site, we make pairs of pen-marks on the arm 2.5 cm (1 inch) apart and away from veins.  We remove the tight 
band.  The MD tubing enters and exits the skin at the marks.  We clean the arm with povidone iodine and alcohol.  
We place an ice bag on the arm for 5 minutes to numb the skin.  Then we insert a thin needle into the skin at 
each entry mark.  The needle’s tip travels between the layers of skin for 2.5 cm (1 inch) and leaves the skin at 
the matching exit mark. We thread the MD tubing through the needle.  Next, we withdraw the needle leaving the 
tubing in the skin.  Any redness of the skin subsides in about 60 – 120 minutes.
We place a cuff on the upper arm sans the MD probes to measure blood pressure.  We place a local skin heater 
and laser Doppler probe over each MD site.  We measure local temperature, and SkBF throughout the MD 
experiments.  We also measure blood pressure every 5-7 minutes (brachial osculation and/or Cardiocap).
MD Experiment 1:  Na2S Dose Response and Local Heating
We prepare 4 MD sites. We clamp the temperatures of the local heaters at 33°C (91.4°F).  We perform the Na2S 
Dose Response and Local Heating procedure concurrently.
Na2S Dose Response
Probe 1. Lactated Ringer’s only (control)
Probe 2. Lactated Ringer’s + LNAME
When the experiment begins, the subject rests for a 20-minute baseline.  When skin blood flow readings 
are stable, we add the test substances to the respective probes as indicated above for 60-90 minutes.  
We obtain a second baseline for about 20 minutes.  When the SkBF is stable, we add the first 
concentration of Na2S to the perfusate of Probes 1-2.  We increase the concentration of Na2S 
approximately every 5 minutes.  The nine concentrations of Na2S range from 10^-6M to 10^-1M.  After 
the perfusion with the last concentration of Na2S, we perfuse the sites with Ringer’s + SNP for about 30 
minutes.  Heating and SNP perfusion causes maximum vasodilation.
Local Heating
Probe 3. Lactated Ringer’s + Cysteine 
Probe 4. Lactated Ringer’s + Cysteine + AOAA
We collect baseline data for 20 minutes, then we add the investigative substances to the probes.  After 
a second 20-minute baseline, we increase the temperature of the local heaters 0.5°C/5 sec to a target 
local skin temperature of 42°C (107.6°F).  We clamp the local temperature at 42°C (107.6°F). After the 
SkBF stabilizes (~40 minutes), we switch perfusates at all MD sites to Ringer’s + sodium nitroprusside 
(SNP) and increase the local skin temperature to 43°C (109.4°F) for about 30 minutes.  Heating and SNP 
perfusion causes maximum vasodilation.
Then the experiment is over, and we remove the MD tubing from the skin and place sterile bandages over the sites.  If the 
subject desires, we can also place a bag of ice on the site for 10 minutes to reduce any bruising that may occur.  We 
measure blood pressure and heart rate before the subject departs.
MD Experiment 2:  Acetylcholine (ACh) and Norepinephrine (NE) Dose Responses
We prepare 4 MD sites.  We clamp the temperatures of the local heaters at 33°C (91.4°F).  We perform the 
ACh and NE Dose Responses concurrently.
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ACh Dose Response
Probe 1. Lactated Ringer’s only (control)
Probe 2. Lactated Ringer’s + LNAME
Probe 3. Lactated Ringer’s + AOAA 
When the experiment begins, the subject rests for a 20-minute baseline.  When skin blood flow readings 
are stable, we add the test substances to the respective probes as indicated above for 60-90 minutes.  
We obtain a second baseline for about 20 minutes.  When the SkBF is stable, we add the first 
concentration of ACh to the perfusate at each probe.  Each site receives 10 increasing concentrations of 
ACh (10^-10 to 10^-1) over 70 minutes.  As SkBF stabilizes with the addition of each concentration of 
ACh, we proceed to the next concentration.  After the last concentration of ACh, we switch perfusates at 
all sites to Ringer’s + SNP and increase the local skin temperature to 43°C (109.4°F) for about 30 
minutes.  Heating and SNP perfusion causes maximum vasodilation.
NE Dose Response
Probe 4. Lactated Ringer’s only
We collect baseline data for about 20 minutes.  We add the first concentration of NE.  We perform the dose response 
of NE with serial log concentration doses from 10^-12M to 10^-2M for a total of 11 doses. The participant receives 
11 different concentrations of NE.  After the perfusion with the last concentration of NE, we perfuse the site with 
Lactated Ringer’s only.  After the effects of the final concentration of norepinephrine subsides (about 30 minutes), 
we switch the perfusate to Ringer’s + SNP and increase the local skin temperature to 43°C (109.4°F) for 
about 30 minutes.  Heating and SNP perfusion causes maximum vasodilation.
Then the experiment is over, and we remove the MD tubing from the skin and place sterile bandages over the sites.  If the 
subject desires, we can also place a bag of ice on the site for 10 minutes to reduce any bruising that may occur.  We 
measure blood pressure and heart rate before the subject departs.
Experiment:  Flow Mediated Dilation (FMD) and sublingual nitroglycerin
FMD:  FMD is an assessment of conduit vessel endothelial function.  The researchers place a BP cuff on a 
forearm, gel on the upper arm just above the elbow, and a Doppler ultrasound probe on the gel.  The ultrasound 
measures vessel size and blood velocity.  After a 3-minute “resting” measurement, they inflate the cuff for 5 
minutes to occlude forearm BF.  After deflating the cuff, they perform a second reading.  
Sublingual nitroglycerin:  This procedure assesses vascular smooth muscle function.  A nurse administers the 
sublingual nitroglycerine and is present throughout the procedure.  The subject is supine on a medical bed or 
recliner.  We apply a blood pressure / heart rate monitor (Cardiocap).  We may take manual blood pressures.  
Also, we perform ultrasonography at the brachial artery at the elbow during the procedure.  We place a 0.4 mg 
nitroglycerin tablet under the subject’s tongue.  The subject closes the mouth immediately after we insert the 
tablet.  The tablet dissolves in 15-90 seconds.  The subject refrains from swallowing until the tablet dissolves.  
Nitroglycerin causes blood vessels to dilate.  The effects last for 5-10 minutes.  The subject remains supine for 
20 minutes following the nitroglycerin administration.  The subject remains in the lab until 20 minutes after 
administration of the nitroglycerin.  If the subject has an exceptional reaction to the nitroglycerin (e.g. drop in 
blood pressure with longer duration), we monitor the subject for up to 60 minutes following nitroglycerin 
administration.
Experiment:  Biopsy:
We give printed and verbal instructions listing what the subjects need to do before they come to the lab.  We take two 
small pieces of skin from the arm (skin biopsy) using standard techniques.  First, the subjects wash the site with soap and 
warm water.  Then the subject sits or lies on a bed.  We clean the top of the lidocaine-vial with alcohol.  An approved 
clinician cleans the skin with alcohol and injects lidocaine into the skin at the biopsy sites to numb them.  We wait a few 
minutes after injecting the lidocaine to give the drug time to work.  We clean the biopsy site 3 times with povidone iodine 
and an alcohol pad.  If the subject is allergic to iodine, we use only alcohol.  We gently touch the site with the tip of a 
needle to see if the subject can feel anything.  The participant may feel the slight pain of the pin-prick or only pressure.  If 
the subject can feel pain, we wait a little longer or the approved clinician adds more lidocaine into the skin.  We use a 
punch-tool that looks like a screwdriver that has a round, hollow tip.  The tip is 3mm (0.12 in) in diameter.  The hollow tip 
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acts like a cookie cutter.  We place the tip of the punch against the skin at the biopsy site and apply mild pressure.  The 
subject feels the pressure.  The tip of the punch goes about 3 mm (0.12 in) into the skin.  The punch collects a small piece 
of skin about 3mm x 2mm (0.12 in x 0.08 in).  We may apply pressure with sterile dressing to the biopsy site to stop 
bleeding, if necessary. We place the piece of skin into a small container.  We use the punch to remove the second piece of 
skin in the same way.  The researcher applies a sterile bandage to the site.  We give instructions concerning care of the 
biopsy site.
Pharmacotherapy Protocol 1:  (Hypertensive subjects only)
Note:  All medications will be double-checked by two people to ensure correctness.  No generic substitutions are allowed
1)    Subjects take their assigned pharmacotherapy (ACEi, SH-ACEi, or diuretic).  
a)  They take the drug for 16 weeks + the time necessary to repeat the experiments.  
b)  Subjects receive enough pills for 4 weeks of therapy at a time.  
c)  Women who are not postmenopausal undergo a pregnancy test every 2 weeks during 
pharmacotherapy. 
Special Note  The women have a pregnancy test during the monthly visits to the lab to pick up 
pills and conduct a pregnancy test at home two weeks after the visit.  We supply the pregnancy 
test for subjects to use in at home. 
2)     The research nurse contacts subjects weekly to check compliance.
3)     After starting the treatment, subjects either (1) come to the lab every 4 weeks to pick up pills or (2) 
if they are male or post-menopausal female, then pills can be mailed directly to the subjects’ residence by the 
pharmacy.
a)  During the monthly visit, we check blood pressure: either (1) we will directly take their blood 
pressure or (2) if participants own an automatic blood pressure cuff, they will take their own blood 
pressure and report to us the results via telephone.
b)  Women who are not postmenopausal, have a urine pregnancy test, and receive test supplies 
to take home.
4)     At week 4 of the treatment, subjects also have a blood draw (about 8.5 ml, 0.6 Tbsp) to check 
potassium, BUN, and creatinine.
5)     At week 8 of the treatment, subjects repeat the 24-hour blood pressure monitoring.
a)  If subjects who are taking HCTZ have not reached the target blood pressure (140/90 mmHg) 
the dose is Increased to 50 mg/day.
6)     At week 12 of treatment (Subjects taking HCTZ only)
a)  If subjects who are taking HCTZ have not reached the target blood pressure (140/90 
mmHg), the subjects are given the opportunity to switch to one of the ACEi pharmacotherapies 
for 16 weeks.  The ACEi pharmacotherapy chosen is randomized.  The subjects washout for 
two weeks before starting the ACEi or SH-ACEi pharmacotherapy.
b)  The nonresponders are replaced to achieve the necessary number of participants who 
respond to HCTZ.
4)     At week 16 of the treatment
a)  Subjects repeat the 24-hour blood pressure monitoring.
b)  Subjects repeat all experiments.
c)   We take another blood sample (about 8.5 ml, 0.6 Tbsp) to check potassium, BUN, and 
creatinine.
Pharmacotherapy Protocol 2:  (Hypertensive subjects who stopped physician-prescribed drugs only)
Note:  All medications will be double-checked by two people to ensure correctness.  No generic substitutions are allowed
1)    Subjects take their assigned pharmacotherapy (ACEi, SH-ACEi, or diuretic).  A participant who has a 
history of being a non-responder to one of the research blood pressure drugs before they enter the 
study will be randomly assigned to one of the other study drugs.
a)  They take the drug for 16 weeks + the time necessary to repeat the experiments.  
b)  During the first month, subjects receive enough pills for 2 weeks of therapy at a time.
c)  Women who are not postmenopausal undergo a pregnancy test every 2 weeks during 
pharmacotherapy. 
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Special Note  The women have a pregnancy test during the monthly visits to the lab to pick up 
pills and conduct a pregnancy test at home two weeks after the visit.  We supply the pregnancy 
test for subjects to use in at home. 
2)     The research nurse contacts subjects weekly to check compliance.
3)     After starting the treatment, subjects come to the lab every 2 weeks for the first month for blood 
pressure check and to pick up pills.
a)  If a subject’s blood pressure is > 140/90 after 2 weeks of pharmacotherapy, we titrate the 
dose of the medication upward to reduce blood pressure below 140/90.
b)  Women who are not postmenopausal, have a urine pregnancy test, and receive test supplies 
to take home.
4)
After the first month on the pharmacotherapy, subjects will either (1) come to the lab every 4 
weeks thereafter to pick up enough pills for 4 weeks of therapy at a time or (2) if they are a male 
or a post-menopausal female, pills can be mailed directly to the subjects’ residence by the 
pharmacy.
a)  During the monthly visit, we check blood pressure: either (1) we will directly take their blood 
pressure or (2) if participants own an automatic blood pressure cuff, they will take their own blood 
pressure and report to us the results via telephone.
i)  Any subjects whose blood pressure is > 140/90 at 4 or more weeks after starting the 
pharmacotherapy end participation in the study.
b)  Postmenopausal women who are continuing in the study, have a urine pregnancy test, and 
receive test supplies to take home.
4)     At week 4 of the treatment, subjects also have a blood draw (about 8.5 ml, 0.6 Tbsp) to check 
potassium, BUN, and creatinine.
5)     At week 8 of the treatment, subjects repeat the 24-hour blood pressure monitoring.
4)     At week 16 of the treatment
a)  Subjects repeat the 24-hour blood pressure monitoring.
b)  Subjects repeat all experiments.
c)   We take another blood sample (about 8.5 ml, 0.6 Tbsp) to check potassium, BUN, and 
creatinine.
Equipment:
Laser Doppler Flowmetry:  The Laser Doppler Flowmeter (Moor Instruments, Inc.) non-invasively provides a qualitative 
measure of skin blood flow to a depth of about 1 mm in the skin using a weak laser light.  This measure is a dimensionless 
value called “flux” that reflects the speed and number of blood cells moving through the microvasculature in an area of 
skin.  The flowmeter continuously measures skin blood flow using a fiber optic probes that fit into holders taped to the skin.  
approved by the FDA
  
Blood Pressure, ECG (Heart rate):  The CardioCap 5 (General Electric – GE) critical care monitor measures blood pressure 
via a cuff inflated every 5-7 minutes on the upper arm and heart rate via 3-lead ECG probes taped to the skin of the chest.  
approved by the FDA
Flow Mediated Dilation (FMD):  FMD measures the health of blood vessels.  The researchers place a blood pressure cuff 
around the forearm.  They put gel on the upper arm just above the elbow.  Then they place a Doppler ultrasound probe on 
the gel.  The ultrasound makes sound waves to measure the size of blood vessels and the speed of the blood.  They make a 
“resting” measurement before they inflate the cuff.  Then they inflate the cuff for 5 minutes to stop blood flowing to and 
from the forearm.  After they deflate the cuff, they perform a second reading for 3 minutes.  ATL HDI 5000 SonoCT 
(Ultrasound) FDA 510 (k) approved by the FDA
7.3
Duration of Participation
Screening (1 Visit) 
less than 1.5 hour
MD Experiments (4 Visits) 
5 hours each
FMD/sublingual Nitroglycerin (2 visits)
1.5 hours
Biopsy (2 visits)
less than 1 hour
Obtain pills (3 Visits, Hypertensives only)
about 15 minutes each
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Total: 27.25 Hours (7-12 visits; It could take a minimum of 18 weeks to complete the study.)Note:  The biopsy 
may occur on the same day as one of the other visits.
Hypertensive subjects taking physician-prescribed drugs:
In addition to the 27.25 hours described above, these subjects undergo a 2-week washout period during which 
they monitor their blood pressure prior to the first experiment.
HCTZ non-responders only
Screening (1 Visit) 
less than 1.5 hour
MD Experiments (4 Visits) 
5 hours each
FMD/sublingual Nitroglycerin (2 visits)
1.5 hours
Biopsy (2 visits)
less than 1 hour
Obtain pills (6 Visits)
about 15 minutes each
Total: 28 Hours (10-15 visits; It could take a minimum of 32 weeks to complete the study.)
Note:  The biopsy may occur on the same day as one of the other visits.
7.4
Test Article(s) (Study Drug(s) and/or Study Device(s))
7.4.1
Description
 Devices:  See “7.2 Study Procedures”
Used with Microdialysis:
Acetylcholine
ACh
Form: Powder
Role in Current Study:  Local heating combined with the local perfusion of endothelial agonist, acetylcholine, 
specifically examines the attenuated endothelium-dependent vasodilation (eNOS-derived NO) associated with 
aging or vascular pathologies.
Concentration:  10^-10 to 10^-1M
Aminooxyacetic acid
AOAA
Form: Powder
Role in current study:  Aminooxyacetic acid is a general inhibitor of pyridoxal phosphate (PLP)-dependent 
enzymes and an inhibitor of cystathionine Y-lyase (CSE), an enzyme that aids in endogenous production of 
hydrogen sulfide from the substrate, l-cysteine.
Concentration:  5mM
Vitamin C
L-Ascorbate
Form: Powder
Role in Current Study:  L-ascorbate stabilizes norepinephrine in solution.  A small amount will be delivered by 
MD to a nickel-sized are of skin.
Concentration:  3mM
Cysteine
L-Cysteine
Form: Powder
Role in Current Study:  L-cysteine is a substrate for the enzyme, cystathionine Y-lyase (CSE) that aids in the 
production of endogenous production of hydrogen sulfide in the body.
Concentration:  20 mM
LNAME 
NG-nitro-L-arginine methyl ester
Form: Powder
Role in current study:  A nitric oxide synthase inhibitor.  L-NAME is an analog to the amino acid, L-arginine.  L-
NAME is a non-specific inhibitor for nitric oxide synthases, thereby inhibiting the production of nitric oxide that 
causes vasodilation.  We deliver small doses of LNAME to a nickel-sized area of the skin.
Concentration:  10mM
Norepinephrine
Noradrenaline
Form: Powder
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Role in Current Study:  Norepinephrine acts on both alpha-1 and alpha-2 adrenoreceptors to cause 
vasoconstriction.  A small amount will be delivered by MD to a nickel-sized are of skin.
Concentration:  10^-12 to 10^-1 M
Sodium Nitroprusside     
SNP
Form: Powder
Role in Current Study:  SNP, acting an NO donor, dilates blood vessels maximally thereby achieving maximal 
skin blood flow (SkBF).  Maximal SkBF is a reference point for other measures of skin blood flow.
Concentration:  28mM
Sodium sulfide 
Na2S
Form: Powder
Role in current study: Once in the human body, sodium sulfide breaks down to produce hydrogen sulfide.  The 
hydrogen sulfide causes vasodilation.  Sodium sulfide solution is strongly alkaline; therefore the solution is 
titrated with HCl is added to correct the pH to 7 thereby rendering the solution suitable for perfusion through a 
MD probe.
Concentration:  10^-6 to 10^-1 M
Lactated Ringer’s
Ringer’s
Form: Liquid
Role in current study:  Lactated Ringer’s acts as the vehicle for the other research substances and as a flush.
Drugs not used with microdialysis
Lidocaine (1%)
Form: Liquid
Role in Current Study:  We use Lidocaine (without epinephrine) to numb the skin for biopsy in an FDA-
approved manner.
Nitroglycerin
Form:  Tablets
Role in Current Study:  Sublingual nitroglycerin coupled with ultrasonography of the brachial artery assesses 
the function of vascular smooth muscle.   This is not an on-label, FDA-approved use for nitroglycerin.  
However, researchers at Noll Lab and elsewhere have successfully used sublingual nitroglycerin in this 
manner without problem.  We included this use of nitroglycerin in the IND application for this study.
Used as Pharmacotherapy:
Enalapril maleate
Vasotec
Form: Tablets
Role in Current Study:  Enalapril is an Angiotensin Converting Enzyme (ACE) inhibitor.  Clinically, oral 
administration of Enalapril is used in the management of hypertension.  Vasotec is FDA-approved for the 
management of hypertension.  Titration and doses are performed in accordance with the FDA-approved 
labeling.
Hydrochlorothiazide
Form: Tablets
Role in Current Study:  Hydrochlorothiazide USP is a diuretic and antihypertensive.  Clinically, oral 
administration of hydrochlorothiazide is used in the management of hypertension either as the sole therapeutic 
agent or to enhance the effectiveness of other antihypertensive drugs in the more severe forms of 
hypertension.  Hydrocholothiazide is sold under various brand names (e.g. Aquazide H, HydroDIURIL, 
Microzide).  Titration and doses are performed in accordance with the FDA-approved labeling.
Captopril
Form:  Tablets
Role in Current Study:  Captopril is an Angiotensin Converting Enzyme (ACE) inhibitor.  Captopril contains a 
sulfhydryl moiety, which has been associated with the capacity to scavenge oxygen-free radicals. The 
antioxidant activity of Captopril has been shown to be present at cardiac and vascular levels both in vitro and in 
vivo.  Captopril is approved by the FDA for clinical use in the United States.    Titration and doses are 
performed in accordance with FDA-approved labeling.
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7.4.2
Treatment Regimen
Investigative Substances used with Microdialysis
The following is a table of the research agents used with intradermal microdialysis.  Based upon research 
literature, we use 14% for calculating maximum delivery of the research agents for all IND applications for our 
MD studies.  
Research Agent
14% delivery (mg)
Acetylcholine (USP)
     
0.051
Aminooxyacetic acid
0.033
Vitamin C
0.002
Cysteine
0.092
L-NAME
0.100
Norepinephrine
0.005
Sodium Nitroprusside
0.070
Sodium Sulfide
          0.067
Drugs not used with microdialysis
Lidocaine (1%):  The dose is less than 0.5mg / biopsy site.
Nitroglycerine:  0.4 mg under the tongue during FMD/sublingual nitroglycerin experiment
Used as Pharmacotherapy:
Enalapril:  The recommended initial dose is 5 mg orally once/day. Dosage is adjusted according to blood 
pressure response. The typical maintenance dosage range is 10 to 40 mg per day administered in a single dose 
or two divided doses.  Titration and doses are performed in accordance with the FDA-approved labeling.
Hydrochlorothiazide:  The recommended initial dose is 25 mg orally once daily.  The maintenance dose can 
increase to 50 mg orally daily, as a single or 2 divided doses.  Titration and doses are performed in accordance 
with the FDA-approved labeling.
Captopril:  Typical initial dose is 25mg orally given 2-3 times daily.  Dosage is adjusted according to blood 
pressure response.  Target dose usually achieved within 4 weeks.  Maintenance dose range is 25-150mg given 
2-3 times daily (usually not necessary to exceed 150mg/day).  The typical maintenance dose of Captopril Tablets 
in hypertension usually does not exceed 50 mg three times per day.  The maximum dose of 450mg/day is not to 
be exceeded.  Titration and doses are performed in accordance with the FDA-approved labeling.
Also see “7.2 Study Procedures”
7.4.3
Method for Assigning Subject to Treatment Groups
The hypertensive subjects are randomly assigned to a treatment group.  If a hypertensive subject has 
contraindications for one or two of the treatments, the subject is assigned to one of the remaining treatments.
7.4.4
Subject Compliance Monitoring
We contact subject weekly to ask if they have been compliant during their treatment period.
7.4.5
Blinding of the Test Article
The members of the research team conducting experiments and data analysis are blinded to the 
pharmacotherapy treatment and do not handle the treatment drugs during the study.  The research nurse and 
research physicians know which treatment the subject receives. Subjects are not blinded to their assigned 
treatment.  We caution the subjects not to reveal the identity of their treatment to the research team conducting 
experiments and data analysis.
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7.4.6
Receiving, Storage, Dispensing and Return 
See uploaded Physician Oversight SOP.
7.4.6.1 Receipt of Test Article 
Used with Microdialysis:  Detailed records are kept in laboratory log books.  The containers are dated upon 
receipt and when opened.    
Acetylcholine
source:  USP
Aminooxyacetic acid
source:  MPBiochemicals, Sigma
Vitamin C
source:  Sigma
Cysteine
source:  MP Biomedicals
LNAME 
source:  EMD, Tocris
Norepinephrine
source:  USP
Sodium Nitroprusside  
source:  USP
Sodium sulfide 
source:  EMD, Sigma
Lactated Ringer’s
source:  VWR, Owens and Minor, McKesson
From uploaded Physician Oversight SOP:  
The investigative agents are purchased by the lab members from reputable sources as indicated in the IRB 
and the FDA IND applications and in accordance with the standards and guidelines imposed by the FDA.
A license from the CRC physician, the nurse manager, or the overseeing physician is filed with vendors (e.g. 
VWR , Owens and Minor) requiring the documentation for the purchase of some investigative agents (e.g. 
Lactated Ringer's) .
The investigative agents are shipped to the Noll Lab or picked up from the pharmacy by lab personnel.
Copies of the prescriptions and orders are maintained in the laboratory's files.
Not used with Microdialysis:  Detailed records are kept in laboratory log books and in individual subjects ' 
charts.  The containers are dated upon receipt and when opened.    
Lidocaine (1%)
source:  University Health Services Pharmacy
Nitroglycerin:  
source:  University Health Services Pharmacy.
From uploaded Physician Oversight SOP:  
Some of the investigative agents are prescription drugs (e.g. lidocaine for skin biopsy). The lab personnel 
obtain prescriptions from a CRC physician, the research nurse manager, or the overseeing physician for the 
investigative agents that are prescription drugs and forward the prescriptions to the pharmacy (usually UHS 
Pharmacy).  The investigative agents are shipped to the Noll Lab or picked up by lab personnel.
A license from the CRC physician, the research nurse manager, or the overseeing physician is filed with 
reputable vendors requiring the documentation for the purchase of some investigative agents.
Copies of the prescriptions and orders are maintained in the laboratory's files.
Used as Pharmacotherapy:  Detailed records are kept in laboratory log books and in individual subjects ' 
charts.  The containers are dated upon receipt and when opened.   All medications will be double-checked by 
two people to ensure correctness.  No generic substitutions are allowed
Vasotec, Captopril, and HCTZ:  
source: Boalsburg Apothecary
 
From uploaded Physician Oversight SOP:  
Some of the investigative agents are prescription drugs (e.g. lidocaine for skin biopsy). The lab personnel obtain 
prescriptions from a CRC physician, the research nurse manager, or the overseeing physician for the 
investigative agents that are prescription drugs and forward the prescriptions to the pharmacy (usually UHS 
Pharmacy).  The investigative agents are shipped to the Noll Lab or picked up by lab personnel.
A license from the CRC physician, the research nurse manager, or the overseeing physician is filed with 
reputable vendors requiring the documentation for the purchase of some investigative agents.
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Copies of the prescriptions and orders are maintained in the laboratory's files.
7.4.6.2 Storage
Used with Microdialysis:
The investigational substances are stored under environmental conditions according to manufacturers’ 
instructions in cabinets, refrigerators, or freezers located in Room 228 or 224A Noll Laboratory.  The room is 
locked when unoccupied.  Each location has a thermometer.  The temperatures are recorded daily during 
weekdays.  The temperature of the room is also monitored by the Central Control System of the Environmental 
Systems at PSU.
Not used with Microdialysis:
Lidocaine, Nitroglycerin:  We store the investigational substances under environmental conditions according to 
manufacturers’ instructions in cabinets, refrigerators, or freezers located in Room 228 or 224A Noll Laboratory.  
The room is locked when unoccupied.  Each location has a thermometer.  The temperatures are recorded daily 
during weekdays.  The temperature of the room is also monitored by the Central Control System of the 
Environmental Systems at PSU.
Used as Pharmacotherapy:
Vasotec, HCTZ, Captopril:  We store the drugs in a locked cabinet in Room 228 under environmental conditions 
according to manufacturers’ instructions.  The room is locked when unoccupied.  The location has a 
thermometer.  The temperatures are recorded daily during weekdays.  The temperature of the room is also 
monitored by the Central Control System of the Environmental Systems at PSU.
Used systemically in experiment:
Nitroglycerin:  We store the drug in a locked cabinet in Room 228 under environmental conditions according to 
manufacturers’ instructions.  The room is locked when unoccupied.  The location has a thermometer.  The 
temperatures are recorded daily during weekdays.  The temperature of the room is also monitored by the Central 
Control System of the Environmental Systems at PSU.
7.4.6.3 Preparation and Dispensing
Used with Microdialysis:
None of the microdialysis drugs are dispensed to the participant; rather we use them in the experiment, as 
outlined in the Standard Operating Procedures (Phys Ovrsght SOP). The physician providing oversight approves 
the laboratory standard operating procedure for the obtainment, preparation, and administration of all 
investigational agents for MD and other procedures in experiments. 
The following procedures have been examined and approved by the FDA.
Trained lab personnel prepare the perfusates in accordance with the procedure described in the IRB and FDA 
IND applications. When mixing the perfusates, the experienced technician washes the hands, wears protection 
(e.g. gloves, lab coat), and uses glassware that has been washed with cleaner (designed for use with healthcare 
instruments , pharmaceutical process equipment , tissue culture apparatus , etc.), and rinsed multiple times in 
tap and then doubly- distilled water and air-dried.  Most of the investigational agents are obtained in ultra-pure 
solid form. The solid investigational agents are weighed on a microbalance and then mixed with sterile 
pharmaceutical-grade Lactated Ringer's solution to the desired concentration.  The solution is drawn into the 
syringe through a 0.2 µm filter.  Prior to injecting a solution into or withdrawing solution from a sterile container 
through a sterile hypodermic needle, the stopper of the sterile container is cleaned thoroughly with alcohol.  Most 
perfusates are used within minutes or hours after preparation.  Stock solutions are made for some investigational 
agents.  The stock solutions are drawn into sterile 1-cc syringes through 0.2 µm filters for storage.  The sterile 
stock solutions are labeled accordingly (e.g. content, date), and protected from light.   Stock solutions are not 
used beyond 1 week (refrigerated) and 6 months (frozen) after mixing.  Stock solutions are diluted with sterile 
Lactated Ringer's when preparing the perfusate.  The final dilutions of the perfusates are drawn into sterile 
syringes through 0.2 µm filters within minutes or hours of their use in the experiment.
The protocol that includes the administration of the investigative substances has been examined and approved 
by the FDA.
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The administration of the investigative substances is performed by the trained and approved lab personnel as 
described in the protocol included in the IRB and FDA IND applications.  The overseeing physician has observed 
and approved the microdialysis technique as performed by Dr. Lacy Alexander.  The lab personnel performing 
microdialysis have been trained and approved to perform the technique by Dr. Lacy Alexander, approved by the 
overseeing physician, and approved by the IRB.  Letters of approval from the overseeing physician for each 
member of the lab who performs microdialysis in the research project have been submitted to the IRB. 
Drugs not used with microdialysis
Lidocaine:  The research or CRC nurse administers the drug under the authority and according to the instructions 
provided by the supervising physician.  Detailed records are kept in laboratory log books and in individual 
subjects ' charts.
Nitroglycerin:  The research or CRC nurse administers the sublingual nitroglycerin during the experiment. 
Detailed records are kept in laboratory log books and in individual subjects ' charts.
Used as Pharmacotherapy:
Vasotec, Captopril, and HCTZ:  The Boalsburg Apothecary places the pills into bottles labeled with the subject 
ID number, strength (mg), dosing instructions, date, etc.  Each bottle contains the number of pills as prescribed 
by the CRC clinicians who are working with the subjects. All medications will be double-checked by two people 
to ensure correctness.  No generic substitutions are allowed.  The research nurse gives the bottle of pills to the 
subject along with handouts that contain detailed information and dosing instructions pertinent to their assigned 
drug.  Detailed records are kept in laboratory log books and in individual subjects ' charts.
Also see uploaded Physician Oversight SOP.  
7.4.6.4 Return or Destruction of the Test Article
Subjects are required to return any unused pills to the investigators. The pills are disposed of in accordance with 
the policies of the Penn State’s Environmental Health and Safety Department at University Park.
7.4.6.5 Prior and Concomitant Therapy
Subjects are screened out if they are currently taking chronic medications that may affect vascular function or 
be contraindicated for the pharmacotherapy. Medications that are not specifically mentioned in exclusion criteria 
may be excluded on a case by case basis upon review by the research clinicians or PI.
8.0
Data and Specimen Banking For Future Undetermined Research
NA
9.0
Statistical Plan
9.1
Sample size determination
Based on our previously published data with pharmacological intervention studies, we calculate that 10 subjects 
will be necessary to detect an effect size of 15% difference in microdialysis treatment sites (1 sample t-test (pre 
vs. post intervention), power=0.08, α=0.05). We calculate that 14 subjects per group to detect a meaningful 
physiological differences between groups and microdialysis treatment sites to measure an effect size of at least 
15%; (SA2, 3 group ANOVA, power=0.80, α=0.05). Conservatively, we recommend increasing to 19 subjects 
per group to account for potential dropout along the course of the 16 week intervention and possibility that not 
all subjects will respond to the antihypertensive treatments. In our previous interventional studies we have been 
able to maintain 100% compliance.  We plan to include about 10 time-control normotensive subjects.
9.2
Statistical methods
We will use a within-person experimental design and associated repeated measures ANOVA tests with 
planned contrasts. For all experiments, significance will be accepted at p<0.05. P-values and 95% confidence 
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intervals for the mean difference estimates will be adjusted using Tukey’s multiple comparison procedure to 
account for post-hoc multiple comparison testing and ensure a family-wise type I error rate less than 5%.
Unless indicated otherwise, data will be analyzed using SAS PROC MIXED, a flexible analytic tool that 
accommodates a wide variety of ANOVA and regression models and nested and incomplete data structures.
Microdialysis experiments. For these protocols, the primary outcome variable will be CVC. Separate 3-way 
repeated measures analysis of variance (ANOVA) will be performed to examine systemic treatment difference 
(Captopril, Enalopril, or Hydrochlorothiazide), and localized microdialysis treatment differences across the 
phases or doses of pharmacological or physiological stimuli. Appropriate post-hoc analyses with corrections for 
multiple comparisons will be performed when main effects are identified.
Pharmacological curve modeling. Prior to analysis, H2S, ACh, NE dose concentrations will be log-
transformed.  CVC will be normalized to absolute and as a percentage of maximum or baseline, respectively. 
Vascular responsiveness will be modeled using a sigmoidal dose-response curve with variable slope, as 
previously described by Wenner et al. and will be used to obtain parameters describing potency (logEC50), and 
cooperativity (Hill slope).  Additional growth curve analysis (nonlinear modeling) will be used to determine 
maximal vasoactive responses (Emax) between groups and treatment conditions.
Biopsy experiments. Repeated measures ANOVA will be used to examine group differences, and changes 
that accrue with systemic treatment. Densitometry analysis will be conducted to examine differences between
the groups in enzyme activity, and the protein concentrations obtained from Western blots.
10.0 Confidentiality, Privacy and Data Management 
10.1
Confidentiality.  
The computerized data files are password protected, and automatically and manually backed up.  Only Anna 
Stanhewicz, and the research personnel directly involved in these protocols will have access to these passwords. 
The computerized spreadsheets that are being used are also constructed to ensure the validity and 
confidentiality of the subject’s identity (no names, only subject #), as well as the accuracy of the data itself (i.e., 
automated check for allowable response range).  Once data are entered and completeness is verified, analyses 
proceed with summary results and statistical analyses.  The data collected are used exclusively for research 
purposes. 
The investigators maintain data in the laboratory in locked cabinets and on password-protected computers 
maintained in locked rooms.  Most of the data are coded and do not contain personal identifying information.  
Documents allowing identification of participants do not leave the investigator’s labs and are only available to 
authorized persons.  Coded data shared with unauthorized persons cannot be traced to individuals.  The 
investigators store any list linking the code to participants’ identity in locked cabinets and on password-protected 
computers maintained in a locked room.  Potential participants may submit pre-screening questionnaires via the 
investigator’s website / Qualtrics (commercial survey website).  All web traffic to and from the Qualtrics 
application is done via a Secure Socket Layer (SSL) that encrypts the data in transmission.  Data is deleted from 
the Qualtric’s servers within 24 hours after the user deletes it from the website.  Hard copy data forms containing 
identifiable information are shredded when no longer needed (within 5 years after publication of results).  
Screening data from subjects who are not accepted into the study are shredded when the project ends.  Subjects 
may give permission to have their contact information retained in the investigator’s secured files if they wish to 
be considered for participation in future studies.  After the investigators complete the study, they remove all 
identifiers from the study’s data and store the data indefinitely.  Individual data may be used without identifying 
the subject to illustrate representative responses.
10.1.1 Identifiers associated with data and/or specimens
All of the data and specimens collected in the laboratory are coded according to subject numbers. These subject 
numbers are randomly assigned by the CRC personnel when the subject screens for the study. The investigators 
maintain data in the laboratory in locked cabinets and on password-protected computers maintained in locked 
rooms.  Coded data shared with unauthorized persons cannot be traced to individuals.
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10.1.1.1
Use of Codes, Master List
Documents allowing identification of participants do not leave the investigator’s labs and are only available to 
authorized persons. The investigators store any list linking the code to participants’ identity in locked cabinets 
and on password-protected computers maintained in a locked room.  Only authorized persons have access to 
the code.  The code is destroyed within 5 years of publication of the data.
10.1.2 Storage of Data and/or Specimens
The investigators maintain data in the laboratory in locked cabinets and on password-protected computers 
maintained in locked rooms.  Most of the data are coded and do not contain personal identifying information.  
Documents allowing identification of participants do not leave the investigator’s labs and are only available to 
authorized persons.  Coded data shared with unauthorized persons cannot be traced to individuals.  The 
investigators store any list linking the code to participants’ identity in locked cabinets and on password-protected 
computers maintained in a locked room.  Potential participants may submit pre-screening questionnaires via the 
investigator’s website / Qualtrics (commercial survey website).  All web traffic to and from the Qualtrics 
application is done via a Secure Socket Layer (SSL) that encrypts the data in transmission.  Data is deleted from 
the Qualtric’s servers within 24 hours after the user deletes it from the website.  Hard copy data forms containing 
identifiable information are shredded when no longer needed (within 5 years after publication of results).  
Screening data from subjects who are not accepted into the study are shredded when the project ends.  Subjects 
may give permission to have their contact information retained in the investigator’s secured files if they wish to 
be considered for participation in future studies.  After the investigators complete the study, they remove all 
identifiers from the study’s data and store the data indefinitely.  Individual data may be used without identifying 
the subject to illustrate representative responses.
Biological specimens will be stored at University Park and Quest Lab, Chantilly, VA, Johns Hopkins, Baltimore, 
MD, and Georgia Health Sciences University, Augusta, GA. At University Park the specimens will be stored in a 
-80ºC freezer in Noll first floor hallway. All specimens not exhausted upon analysis are maintained no longer 
than 5 years after publication.
This study will be issued a Certificate of Confidentiality. Researchers will not disclose or provide any identifiable 
information without the subject’s prior consent or where permitted according to NIH’s Policy on Issuing 
Certificates of Confidentiality
10.1.3 Access to Data and/or Specimens
Only IRB approved laboratory personnel will have access to the data. Non-PSU study team members will be 
made aware they are also subject to the disclosure restrictions according to NIH’s Policy on Issuing Certificates 
of Confidentiality.
10.1.4 Transferring Data and/or Specimens
Some specimens may be transferred to Quest Diagnostics (Chantilly, VA), or the laboratories of Dan Berkowitz, 
MD, at Johns Hopkins, Baltimore, MD or that of Dr. Joseph Cannon at the Medical College of Georgia. 
Specimens are labeled with subject numbers only, and the recipients are not given access to the code matching 
the subject number to information that could identify the research participant. All specimens not exhausted upon 
analysis are maintained no longer than 5 years after publication.
The researchers do not plan to release identifiable information collected in the study. However, if researchers 
consider releasing identifiable information in the future – the individual or institution receiving the identifiable 
information will be made aware they are also subject to the requirements of subsection 301(d) of the Public 
Health Service Act.
                                                                                                                                                          
10.2
Privacy
We tell participants that they may decline to answer questions and decline to participate in the study.  The 
researchers obtain only info given to them by the participant.  We perform all procedures and experiments in a 
private room. Only authorized personnel are present during screening and experiments.  On occasion (e.g. 
educational visit, visiting colleague, site visit) participants may give permission for visitors to observe a procedure 
or experiment. All laboratory personnel are aware and trained to protect and respect participants' confidentiality 
and privacy.
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11.0 Data and Safety Monitoring Plan
11.1
Periodic evaluation of data
Dr. Alexander discusses the study with the group at weekly lab meetings.  If necessary, she conducts additional 
special meetings with relevant personnel if there is an immediate, specific issue must be addressed (e.g. 
untoward events).
11.2
Data that are reviewed 
Relevant data safety and management procedures, interim data evaluation, untoward events (rare in this 
research), new developments in related research, and quality control issues are discussed.
11.3
Method of collection of safety information
Lab members keep lab notebooks into which they note relevant safety issues and data that they identify for 
discussion.  In the case of untoward events, those lab members present during the event are included in a 
debriefing and notes of the meeting are taken.  The participant involved in an untoward event is interviewed in 
person or via telephone concerning the event and the responses are included into the notes for that event.
11.4
Frequency of data collection
Data collection (taking notes) concerning safety is ongoing and begins with the beginning of the project.
11.5
Individual’s reviewing the data
The PI and lab group review the data.
11.6
Frequency of review of cumulative data
NA
11.7
Statistical tests
NA
11.8
Suspension of research
In the case of an adverse event, research is suspended while evaluation, reporting, and rectification proceeds.  
Research is restored when the IRB and PI decide that a rectification of the problem is satisfactory.
12.0 Risks
General note: The research group’s members are trained and competent in their duties.  The group, led by Dr. 
Alexander, evaluates the effectiveness and safety of protocols and procedures in an ongoing fashion.  They 
discuss the protocol with candidates, invite questions, and offer tours of the laboratory.  Prior to medical 
screening, candidates read and sign informed consent forms detailing protocols, procedures, risks, sensations, 
compensation, etc.  The researchers give candidates witnessed copies of the signed consent forms.  After 
accepting participants into the study, the researchers discuss and review the procedures and protocols with them 
generally and at each step throughout the project.  They frequently remind participants of the option to withdraw 
from the study at any time.  Restricting access to experiments, data, and coding to authorized personnel 
maintains confidentiality.  The Noll Lab’s electronics technician certifies the electrical devices for human use.  
Lists of emergency numbers remain by lab telephones.  At least one cell phone is present at each experiment.  
A hospital and emergency medical services are within 1-2 miles of the lab.  An AED hangs nearby in the hallway.
Microdialysis:  The researchers insert a 25 g needle horizontally into and then out of the layers of the skin of the 
ventral forearm.  The needle’s entry and exit are about 2.5 cm apart.  They thread the microdialysis “probe,” 
comprising a tube of membrane (320 um OD) with tubing attached at both ends (650 um OD), through the needle.  
Then they withdraw the needle leaving the membrane under the skin.  They perfuse the probe with sterile saline 
using a syringe pump.
Cutaneous microdialysis commonly causes some pain and bruising similar to that experienced during 
venipuncture.  There is usually no pain after the probe is in place.  The participant may experience mild pain 
while the researchers remove probe.  Minor bleeding may occur.  As with routine venipuncture, a participant who 
is nervous about needles could have increased heart rate and blood pressure, become lightheaded or 
nauseated, or could faint.
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As with venipuncture or any event that breaks the skin, infection is possible.  However, no participants in any of 
the researchers’ experiments been reported infection.  The researchers place a sterile bandage on the site after 
the experiment.  Although rare, if the membrane should break in half during removal, they remove the remaining 
half by gently pulling the attached tubing.  This presents no additional risk to the participant. In the unlikely event 
in which the membrane breaks during removal leaving an isolated piece of membrane under the skin, they treat 
the piece of membrane in a manner similar to that for a splinter in the skin.  In this case, they have to make a 
superficial incision for removal.  Such an event has not occurred in projects that have used MD in this lab (over 
1,000 MD probes have been placed by Dr. Alexander alone). 
Perfusate:  Lactated Ringer’s solution flows through the microdialysis probes.  An allergic reaction to this 
physiological saline solution is highly unlikely.
All substances (ACh, AOAA, ascorbate, cysteine, LNAME, norepinephrine, propranolol, sodium sulfide, SNP, 
yohimbine) added to the fluid perfusing the microdialysis probes have been used previously in clinically and/or 
research in humans:
Microdialysis delivers small amounts of the substances to a nickel-sized area of the skin.  The small quantities 
used and the extremely localized administration during microdialysis does not produce systemic effects.  To the 
researchers’ knowledge, there are no reports of short- or long-term side effects of these substances administered 
through microdialysis.  The chance of adverse reactions to these substances is extremely small given the minute 
amount delivered to the a very small area of skin, the lack of adverse reactions to similar amounts delivered via 
microdialysis in many other studies, and the lack of adverse effects in human cell cultures.  There is a slight 
chance of allergic reaction to these substances that could produce redness, itching, rash, and/or swelling.  A 
severe reaction (anaphylactic shock) could also cause fever, difficulty in breathing, changes in pulse, 
convulsions, and/or loss of consciousness.
Pharmacologic Intervention:  Subject screening will help to identify potential subjects who have known 
contraindications.  Subjects who have a contraindication (e.g. a condition, medication with a known interaction, 
known allergy) to one or two of the three pharmacotherapy drugs, may be assigned one of the pharmocotherapy 
drugs that is not contraindicated.  If contraindications do not allow them to be assigned any of the drugs, the 
subjects are excluded.  Subjects receive a handout containing information (e.g. side effects, drug interactions, 
etc.) and instructions relevant to the drug.  The physician associated with the study can decide if the occurrence 
of a side effect warrants medical treatment and/or removal from the study.  The subject is contacted weekly to 
check on compliance.  This also provides an easy weekly opportunity for the subject to ask new questions or 
express new concerns.  Subjects come to the lab monthly for pill-pickup and BP measurement.  During the eighth 
week of therapy the BP measurement comprises 24-hour blood pressure monitoring. Women with normal cycles 
undergo a urine pregnancy test every two weeks during pharmacotherapy.  The subject may choose to stop 
taking the drug and remove her/himself from the study at any time.  
Enalopril:  Subjects could have an allergic or anaphylactic reaction to the drug.  Liver and kidney 
problems, history of heart disease/failure, history of blood clots/stroke, diabetes, and angioedema are 
contraindicative.  Titration and doses are performed in accordance with the FDA-approved labeling.  See 
handout for more information.
   Possible common mild side effects include:
   feeling tired or weak 
   lightheadedness  
        cough 
   blurred vision 
   confusion  
        sweating  
   More severe side effects that include: 
   feeling like you will faint
   pounding heart beats      fluttering-feeling in your chest  
   slow heart rate
   weak pulse
        muscle weakness
   tingly-feeling
   ill feeling
        fever
   chills
   painful mouth sores         painful swallowing
   skin sores
   flu-like symptoms
        trouble breathing
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   reduced or no urine output    painful urination
        swelling of feet or ankles
   feeling tired
   shortness of breath
   Less common side effects include:
   chest pain
   cough producing mucus   vomiting
   runny stool
   fainting
        nausea
   sneezing
   sore throat
        tightness in the chest
Hydrochlorothiazide:  Subjects could have an allergic or anaphylactic reaction to the drug.  Liver and 
kidney problems, glaucoma, asthma, gout, sensitivity to sulfonamide-derived drugs, and diabetes are 
contraindicative.  Titration and doses are performed in accordance with the FDA-approved labeling.  See 
handout for more information.
   Possible milder side effects include: 
   runny stools
mild stomach pain
blurred vision
constipation 
   More severe side effects include 
   eye pain
vision problems
dry mouth/thirst.  
   Nausea
 
vomiting
rash.  
   feeling weak
drowsiness 
restlessness
   light-headedness
fast/uneven heartbeat
muscle pain or weakness
   numbness
tingly feeling.  
Captopril:  Subjects could have an allergic or anaphylactic reaction to the drug.  Known hypersensitivity 
to captopril or another ACE inhibitor is contraindicative.  Titration and doses are performed in accordance 
with the FDA-approved labeling.  See handout for more information
Possible common side effects include:
light-headedness
flushing (warmth, redness, or tingly feeling
loss of taste sensation
mild itching/rash
numbness, tingling, or burning pain in hands/feet
cough
More severe side effects include:
dizziness
reduced or increase urinating           
chest pain or pressure, pounding heartbeats, fluttering in chest
shortness of breath (even with mild exertion), swelling, rapid weight gain
high potassium (slow heart rate, weak pulse, muscle weakness, tingly feeling)
sudden weakness or ill feeling, fever, chills, sore throat, painful mouth sores, pain when swallowing, skin 
sores, cold or flu symptoms
Captopril can cause injury or death to the unborn baby if the medicine is taken during the second or third 
trimester of pregnancy.  Women who are not post-menopausal should use reliable birth control while 
taking Captopril.  If a woman becomes pregnant, she will stop taking Captopril, notify the her personal 
physician and the researcher, and exit the study.
Sublingual Nitroglycerine:  Subjects may experience some of the following reactions to the nitroglycerine 
headache
lightheadedness
dry mouth
flushing
irregular heart beat 
weakness
nausea 
vomiting 
 
5-10 minute drop in blood pressure
fainting 
dizziness 
sweating
Subjects may notice a sweet taste and/or tingling sensation in the mouth while the tablet dissolves.  All these 
effects are usually short-lived.  We minimized the effects by having subjects remain supine for 20 minutes after 
receiving the tablet.  In subjects who experience a drop in blood pressure, values usually return to within 10 
mmHg of baseline levels upon resolution of the testing.  We monitor subjects for up to an hour after they receive 
the nitroglycerin if they have a strong or bad reaction.  Subjects could have a mild or severe allergic response to 
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the drug.  This response could include rash, itching, difficulty breathing, and swelling of the face, lips, tongue, or 
throat.  In the event that blood pressure does not return to baseline, coupled with related symptoms, we refer 
subjects for medical follow-up.  In the event of a severe reaction (e.g. anaphylaxis) we call 911.
The effects of nitroglycerin on pregnant or nursing women are unknown.  We exclude subjects who are pregnant 
or nursing.  The use of nitroglycerin for artery measurements is not an FDA-approved use of this drug.  However, 
nitroglycerin has been used in this way in many research studies nation-wide without problem.  Also, sublingual 
nitroglycerin is often prescribed for and self-administered by heart patients who have, or are at risk for, angina 
(heart pain).
Laser Doppler Flowmetry:  The probe attaches to the skin with double-sided tape and measures skin blood flow 
in a 1mm3 volume of skin.  Weak lasers can hurt the eye if one should stare into the light for a long time. The 
red light seen on the surface of the skin is harmless.  The researchers have used this technique in their lab with 
IRB approval for many years without incident.  The researchers do not turn on the laser until they tape the probes 
to a surface. They remove the tape carefully afterward.
Local Heating and Local Skin Temperature:  The local heating control unit (Moor Instruments) precisely controls 
and monitors the temperature of the heating-rings and heated probe holders used with the Laser Doppler 
Flowmeter.  To determine the maximal skin blood flow, the researchers increase the temperature of the heating 
units slowly (about 0.1°C every 1 second).  The skin feels very warm but not painful. Local heating causes 
temporary redness of the skin that subsides within several hours.  This technique is very unlikely to produce 
long-term ill effects.  The local heating controllers (Moor Instruments) precisely control and monitor the 
temperature of the heating-rings and heated probe holders.  The system has programmed maximum temperature 
limits. This technique is very unlikely to produce long-term ill effects.
Blood pressure (manual; CardioCap 5):  The cuff inflates on the upper arm.  The cuff slowly deflates while the 
researchers listen to pulse-sounds at the inside of the elbow with a stethoscope, or the CardioCap 5 takes a 
measurement.  The inflated cuff may make the arm (manual and CardioCap 5) feel tingly and numb, and the cuff 
may temporarily bruise the arm.  Efficient and competent measurement technique minimizes the duration of cuff 
inflation.
Blood draw:  Blood draws can cause anxiety (with increased heart rate and blood pressure), mild pain, swelling, 
nausea, lightheadedness, fainting, or bleeding.  There is a slight chance of infection.  Subjects may have pain 
or bruising at the site where the catheter is placed in the arm, and there is a small risk of infection. Fainting 
sometimes occurs during or shortly after blood is drawn. A competent nurse performs blood draws using standard 
venipuncture-procedure and techniques that minimize the chance of infection.   Participants may recline for the 
procedure.  To minimize the risk of infection associated with blood collection, only sterile, single use catheters 
are used.
Povidone Iodine:  Researchers and hospitals use this orange-colored fluid to clean the skin.  If the subject informs 
us of a known allergic to iodine, we use only alcohol.  An allergic reaction could cause redness, itching, rash, 
and/or swelling.  A worse reaction could also cause fever, breathing problems, changes in pulse, convulsions, 
and/or fainting.
Tape and adhesive disks:   Participants could be sensitive to the adhesive of the tape and double-sided adhesive 
disks used in the study causing redness, rash, tenderness, and/or itching. The researchers remove the tape and 
adhesive disks carefully.  Ointment is available, if needed.
Medical Screening:  The screening includes blood sample, height, waist circumference, weight, heart rate, blood 
pressure, resting ECG, pregnancy test, and history performed by the competent research staff. Participants may 
be uncomfortable giving medical information or being measured. The participants may decline to answer 
questions or participate in measurements.  The researchers conduct screenings professionally and privately.
Initial Screening form:   The researchers use the form to initially determine a candidate’s suitability for the study.  
The initial interview gathers minimally invasive personal data that is kept confidential.  They used similar 
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interviews in the past without problem.  Candidates may complete the form at the lab group’s web site via the 
Qualtrics commercial survey website. The participant may decline to answer questions.  The researchers 
conduct phone interviews professionally and privately.  Only authorized personnel may access completed forms.
Local Heating:  The local heating control unit (Moor Instruments) precisely controls and monitors the temperature 
of the heated probe holders used with the Laser Doppler Flowmeter.  To determine the maximal SkBF, the 
researchers increase the temperature of the heating units slowly (about 0.1°C every 1 second).   The skin feels 
very warm but not painful.  Local heating causes temporary redness of the skin that subsides within several 
hours.  This technique is very unlikely to produce long-term ill effects.  The local heating controllers (Moor 
Instruments) precisely control and monitor the temperature of the heated probe holders.  The system has 
programmed maximum temperature limits.  This technique is unlikely to cause long-term ill effects.
Skin Biopsy:  Skin biopsy could cause pain, lightheadedness, nausea, and/or fainting.  Subjects who are nervous 
about needles could experience temporary increased heart rate and blood pressure. There is a chance of 
infection.  Subjects may stop the procedure at any time.  Trained staff performs the biopsy.  Subjects may lie on 
a bed or sit in a Trendelenburg chair.  The researcher insures subjects are informed and ready.  Trained staff 
uses proper techniques and sterile supplies to keep risk of infection minimal.  The stopper of the vial of lidocaine 
is cleaned with alcohol before needle insertion.  The skin of the biopsy sites is cleaned with alcohol.  The research 
nurse infiltrates the sites with 1% Lidocaine w/o epinephrine intradermally to numb the sites so the subject feels 
little or no pain.  Pressure with sterile dressing stops bleeding. A sterile bandage is placed on sites.  Scarring 
can be reduced through proper care.  The researcher instructs subjects concerning care of biopsy sites.  Subjects 
may take acetaminophen should they experience minor pain when the lidocaine wears off.  People with a history 
of bleeding problems, excessive scarring, or keloid formation do not participate in the biopsy.
Lidocaine:  Subject’s may feel brief pain from the needle and a mild burning during the infiltration.  Although 
unlikely, a bad reaction to lidocaine cause fever, breathing problems, changes in pulse and/or blood pressure, 
convulsions, shock, and/or loss of consciousness.  We ask subjects if they are allergic to lidocaine.  If so, saline 
infiltration or ice can be used to reduce pain instead.  In the case of a severe reaction, we call 911.  
Thermoregulation Lab Website:  Potential subjects may enter data into the screening form (see above) via the 
Qualtrics website.  Qualtrics is a secure website and survey application designed to support data capture for 
research studies. All web traffic to and from the Qualtrics application website is done via a Secure Socket Layer 
(SSL) that encrypts the data in transmission.  The questionnaire contains statements advising of the limitations 
of technology and that there is no confidentiality guarantee. Data is deleted from the Qualtric’s servers within 24 
hours after the user deletes it from the website.  Participants may choose a personal interview instead.
Flow Mediated Vasodilation (FMD) Test  / Doppler Ultrasound:  There is a small chance the probe could irritate 
the skin.  Placing the probe on the arm’s skin may cause temporary minor redness.  The inflated cuffs may cause 
the participant’s arms and feet to feel numb or tingly, and the skin’s color to change slightly.  The cuffs could 
cause mild bruising.  The gel is the same as that used with medical ultrasound tests.  The gel may feel cool or 
cold on the skin.  A bad reaction to the gel is highly unlikely.  The cuffs inflate for a minimal amount of time.  The 
temporary redness from the probe is unlikely to have lasting ill effects.  The participant may decline the test.
Relevant only to people who stop taking their doctor-prescribed drugs to lower their blood pressure:
Stopping doctor-prescribed blood pressure drugs:  Deleterious effects from abruptly stopping blood pressure 
medications are uncommon1.  However, withdrawal effects can happen with stopping any blood pressure drug   Most 
effects occur when stopping drugs that affect the central nervous system (e.g. propranolol, verapamil, clonidine)1.  
Symptoms could include agitation, headache, sweating and nausea.  Less commonly, blood pressure could 
suddenly spike upwards1,2.  A person who has heart disease who abruptly stops taking beta blockers could be at 
increased risk for chest pain, heart attack, or sudden death.  Any subjects whose blood pressure is > 140/90 at 4 or 
more weeks after starting the pharmacotherapy ends participation in the study.  The risk of symptoms is greater 
when a subject suddenly stops more than one blood pressure drug at the same time1.  Therefore, we do not include 
potential subjects the study if they are taking more than one drug prescribed by doctors to control blood pressure.  
We will exclude patients who are taking clonidine.  We will exclude African Americans who are taking a calcium 
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channel blocker (Norvasc).  Also, we will consult with Dr. Fragin, the cardiologist providing medical oversight for this 
study concerning those potential subjects taking a beta blocker because the length of the washout period could be 
longer than 2 weeks depending upon the dose of beta blocker the subject is taking.
References:
1.
 SB, Weber MA, Priest RT, Brewer DD, Hubbell FA. The abrupt discontinuation of antihypertensive 
treatment. J Clin Pharmacol. 1979 Aug-Sep;19(8-9 Pt 1):476-86.
2.
Reidenberg, MM. Drug discontinuation effects are part of the pharmacology of a drug. J Pharmacology 
Exp Ther. 2011 Nov;339(2):324-8.
13.0 Potential Benefits to Subjects and Others
13.1
Potential Benefits to Subjects
The subjects receive medical screenings providing information about their blood pressure status and physical 
well-being.  We have had occasions when the screening for our projects enabled us to alert a subject to a 
developing medical condition whereupon the subject obtained prompt professional care that prevented the 
condition from progressing into a more serious problem.
13.2
Potential Benefits to Others
Hypertension is one of the most important preventable contributors to cardiovascular disease (CVD) and 
death. It is estimated that 33% of adults are hypertensive. Abundant evidence from clinical trials shows the 
benefit of antihypertensive treatment for improving clinical outcomes and reducing overall morbidity and 
mortality. New JNC8 guidelines strongly recommended the use of ACE inhibitors alone or in combination with 
a thiazide-type diuretic to achieve blood pressure treatment goals. While it is increasingly recognized that the 
secondary peripheral vascular effects of ACEi significantly contribute to reduced mortality in hypertension 
associated-disease83, the precise mechanisms remain unclear.
The present proposal has the potential to advance scientific knowledge of the peripheral vascular effects of 
antihypertensive treatment. The proposed work addresses key questions and research topics identified by 
JNC8 and the American Heart Association:
Which classes of antihypertensive agent should be used for the optimal treatment of hypertension?
Does target-based treatment of hypertension lead to superior outcomes?
Identify and characterize new therapeutic targets for the treatment of hypertension. 
In addition, this project will provide additional training and learning opportunities for post docs and students of 
the Pennsylvania State University.
14.0 Sharing Results with Subjects
The researchers give participants copies of their individual lab results.  When the researchers complete data 
analysis, participants may attend an optional presentation of the study’s general findings at Noll Lab.
15.0 Economic Burden to Subjects
15.1
Costs 
None
15.2
Compensation for research-related injury
It is the policy of the institution to provide neither financial compensation nor free medical treatment for research-
related injury. In the event of injury resulting from this research, medical treatment is available but will be provided 
at the usual charge. Costs for the treatment of research-related injuries will be charged to subjects or their 
insurance carriers. 
16.0 Number of Subjects
Total number of subjects:  70 (about 60 subjects with hypertension, about 10 normal subjects as time-controls)
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17.0 Resources Available
17.1
Facilities and locations
The Noll laboratory is a 4-floor (35,000 sq. ft.) free-standing building devoted to basic, clinical, and applied 
physiological research.  Biochemistry laboratories are available for sample processing and analyses.  Available 
equipment includes centrifuges, osmometer, assay plate reader, shaker,  
washer, NA/K analyzer, 
spectrophotometer, i-Stat analyzer, refractometer, hemocue, hematocrit system, fraction collector, hydrogen 
sulphide analyzer, sonicator, microbalances, laser doppler flowmeters, full-field laser perfusion imager, mini-
syringe pumps/ controllers,  sweat rate monitoring systems, hospital beds, medical infusion chair, ECG analysis 
system, finipres, plethysmograph, refrigerators, pharmaceutical refrigerator,  -20 C and -80 C freezers, 
heated/refrigerated circulators, water-perfused suits, local skin cooling/ heating systems, critical care monitors, 
blood pressure monitors, metabolic cart, gas sterilization equipment, and autoclave facilities.  In addition to ample 
office and laboratory laptop or desktop computers for research and student use, Noll Laboratory has two 
available servers and a trunk line connection to the University’s mainframe computer system.  Internal, office 
and laboratory computers are linked.  The laboratory has four PC-based data acquisition systems equipped with 
Dataq hardware driven by Windaq software, two of which are located in computer controlled environmental 
chambers equipped with treadmills and bikes.   A Clinical Research Center is contiguous to the Noll Laboratory 
in the 14,000 sq. ft. Elmore wing, and available for the proposed studies.  Up to 12 subject beds for overnight (or 
longer) stays, a metabolic kitchen, medical examination rooms, and several specialized procedure rooms are 
available.  Because the CRC is contiguous with existing laboratory space, it is readily available for research 
support, e.g., pre-test subject screening.  Noll Laboratory has exceptional in-house support facilities including 
machine and electronics shops.
17.2
Feasibility of recruiting the required number of subjects
We are using recruiting methods that will reach a large percent of the general population.  We have employed 
these methods successfully for earlier projects that targeted the same groups.
17.3
PI Time devoted to conducting the research
The researchers have been conducting similar experiments for many years and are familiar with the duration of 
the procedures/protocols/projects and the amount of staffing required to complete the project while maintaining 
their other obligations.  
17.4
Availability of medical or psychological resources
The CRC is located within the same building as the Noll Lab.  A hospital and emergency medical services are 
within 1-2 miles of the lab.  An AED hangs near the labs in the hallway.  A CPR mask hangs in each lab.  The 
faculty, staff, grad students, and post docs in the lab group have current BLS training.
17.5
Process for informing Study Team
The PI and senior lab members train new members in the necessary procedures.  The researchers conduct 
weekly lab meetings.  They review any applicable changes or issues.
18.0 Other Approvals
None
19.0 Subject Stipend and/or Travel Reimbursements
MD experiments:   ($15.00 for each MD probe + $40 for completing an MD experiment)
Na2S Dose Response /Local Heating
$200.00
($100.00 x 2, pre and post-treatment)
ACh/Ne Dose Response
$200.00
($100.00 x 2, pre and post-treatment)
$400.00 
total
Biopsy:  
$200.00 
$50.00 each (2 pre + 2 post-treatment)
FMD/Sublingual Nitroglycerin: 
$100.00 
$50.00 each (1 pre + 1 post-treatment)
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Total = $700.00
HCTZ non-responders:  
Receive $60.00 for the 12 weeks on HCTZ
     Switched to an ACEi
$760.00 total = $700.00 (pre and post experiments) + $60.00
     Not switched to an ACEi
$410.00 total = $350.00 (pre experiments only) + $60.00 
For incomplete experiments, the researchers pay an amount of money equal to the part completed.  For instance, 
if a subject completes half of an MD experiment, the subject receives $15.00 for each MD probe inserted + $20 
($20.00 is one-half of $40.00).  The researchers may ask subjects to repeat a trial.  If subjects agree to repeat a 
trial, they receive payment for the repeated trial as stated above. They are reimbursed for gasoline if they live 
more than 20 miles from Noll Lab.
20.0 Multi-Site Research
NA
21.0 Adverse Event Reporting 
21.1
Adverse Event Definitions
For drug studies, incorporate the following definitions into the below 
responses, as written:
Adverse event
Any untoward medical occurrence associated with the use of the drug in 
humans, whether or not considered drug related
Adverse reaction
Any adverse event caused by a drug
Suspected 
adverse reaction
Any adverse event for which there is a reasonable possibility that the drug 
caused the adverse event.  Suspected adverse reaction implies a lesser degree 
of certainty about causality than “adverse reaction”.

Reasonable possibility.  For the purpose of IND safety reporting, 
“reasonable possibility” means there is evidence to suggest a causal 
relationship between the drug and the adverse event.
Serious adverse 
event or Serious 
suspected 
adverse reaction
Serious adverse event or Serious suspected adverse reaction: An adverse 
event or suspected adverse reaction that in the view of either the investigator or 
sponsor, it results in any of the following outcomes: Death, a life-threatening 
adverse event, inpatient hospitalization or prolongation of existing 
hospitalization, a persistent or significant incapacity or substantial disruption of 
the ability to conduct normal life functions, or a congenital anomaly/birth defect. 
Important medical events that may not result in death, be life-threatening, or 
require hospitalization may be considered serious when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and 
may require medical or surgical intervention to prevent one of the outcomes 
listed in this definition. 
Life-threatening 
adverse event or 
life-threatening 
suspected 
adverse reaction
An adverse event or suspected adverse reaction is considered “life-threatening” 
if, in the view of either the Investigator (i.e., the study site principal investigator) 
or Sponsor, its occurrence places the patient or research subject at immediate 
risk of death.  It does not include an adverse event or suspected adverse 
reaction that had it occurred in a more severe form, might have caused death.
Unexpected 
adverse event or 
Unexpected 
suspected 
adverse reaction.  
An adverse event or suspected adverse reaction is considered “unexpected” if it 
is not listed in the investigator brochure, general investigational plan, clinical 
protocol, or elsewhere in the current IND application; or is not listed at the 
specificity or severity that has been previously observed and/or specified.
For device studies, incorporate the following definitions into the below 
responses, as written:
Unanticipated 
adverse device 
Any serious adverse effect on health or safety or any life-threatening problem or 
death caused by, or associated with, a device, if that effect, problem, or death 
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effect
was not previously identified in nature, severity, or degree of incidence in the 
investigational plan or IDE application (including a supplementary plan or 
application), or any other unanticipated serious problem associated with a 
device that relates to the rights, safety, or welfare of subjects.
21.2
Recording of Adverse Events
In the case of untoward events, those lab members present during the event are included in a debriefing and 
notes of the meeting are taken.  The participant involved in an untoward event is interviewed in person or via 
telephone concerning the event and the responses are included into the notes for that event.
All adverse events (serious or non-serious) and abnormal test findings observed or reported to study team 
believed to be associated with the study drug(s) or device(s) will be followed until the event (or its sequelae) or 
the abnormal test finding resolves or stabilizes at a level acceptable to the investigator.
An abnormal test finding will be classified as an adverse event if one or more of the following criteria are met:

The test finding is accompanied by clinical symptoms

The test finding necessitates additional diagnostic evaluation(s) or medical/surgical 
intervention; including significant additional concomitant drug treatment or other therapy
Note: Simply repeating a test finding, in the absence of any of the other listed criteria, does 
not constitute an adverse event.  

The test finding leads to a change in study drug dosing or discontinuation of subject 
participation in the clinical research study

The test finding is considered an adverse event by the investigator.
21.3
Causality and Severity Assessments
The investigator will promptly review documented adverse events and abnormal test findings to determine 1) if 
the abnormal test finding should be classified as an adverse event; 2) if there is a reasonable possibility that the 
adverse event was caused by the study drug(s) or device(s); and 3) if the adverse event meets the criteria for a 
serious adverse event.
If the investigator’s final determination of causality is “unknown and of questionable relationship to the study 
drug(s) or device(s)”, the adverse event will be classified as associated with the use of the study drug(s) or 
device(s) for reporting purposes.  If the investigator’s final determination of causality is “unknown but not related 
to the study drug(s) or device(s)”, this determination and the rationale for the determination will be documented 
in the respective subject’s case history.
21.4
Reporting of Adverse Reactions and Unanticipated Problems to the FDA  
21.4.1 Written IND Safety Reports
NA
21.4.2 Telephoned IND Safety Reports – Fatal or Life-threatening Suspected Adverse 
Reactions
NA
21.5
Reporting Adverse Reactions and Unanticipated Problems to the Responsible IRB
Problems Not Requiring Prompt Reporting:  The investigators log internal events/problems that are not problems 
that require prompt reporting to the IRB (i.e., expected and related events) in an accumulative tracking log that 
is reported to the IRB with the yearly Continuing Progress Report (CPR) and at the close of the study.
Problems Requiring Prompt Reporting:  Internal problems that require prompt reporting and are fatal or life-
threatening are reported to the IRB in a Problem Report within one weekday of the principal investigator 
becoming aware of the problem.  All other internal problems that require prompt reporting are reported within 
five weekdays of the principal investigator becoming aware of the event or problem.  External problems that 
require prompt reporting are reported within 30 days of their receipt by the principal investigator.  The 
investigators issue problem reports to the IRB in the case of adverse events that are: (1) unexpected and (2) 
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related/likely related to the research as determined by the Penn State University (PSU).  These events include:  
specific protocol-defined events that require prompt reporting to the sponsor, breach of confidentiality, 
incarceration of a participant in a protocol not approved to enroll prisoners, an accidental or unintentional 
deviation to the IRB-approved protocol that involved risks, an emergency protocol deviation take without prior 
IRB review to eliminate an apparent immediate hazard to a research participant, a complaint of a participant that 
indicates an unanticipated risk or any complaint that cannot be resolved by the research staff, information that 
indicates a change to the risks or potential benefits of the research,  change in FDA labeling or withdrawal from 
marketing of the study drug, device or biologic used in this research protocol, or sponsor-imposed suspension 
for risk.  For each problem, the investigator adds the event to an accumulative problem report log included with 
the Problem Report submitted to the IRB.  The investigator submits the log to the IRB with the yearly Continuing 
Progress Report (CPR) and at the close of the study. 
In accordance with applicable policies of The Pennsylvania State University Institutional Review Board (IRB), 
the investigator will report, to the IRB, any observed or reported harm (adverse event) experienced by a subject 
or other individual, which in the opinion of the investigator is determined to be (1) unexpected; and (2) probably 
related to the research procedures. Harms (adverse events) will be submitted to the IRB in accordance with the 
IRB policies and procedures.
21.6
Unblinding Procedures
Participants are not blinded.  The research team members conducting the experiments will be unblinded to a 
particular subject’s treatment if the subject is having a reaction that could be associated with the treatment.  The 
research nurse and physicians are not blinded. If this is associated with an adverse event, the event is reported.
21.7
Stopping Rules
Although such events are extremely unlikely to occur, we are prepared to immediately stop experiments and 
seek medical assistance if the subjects should experience the more serious reactions described in the informed 
consents, and IRB applications such as signs and symptoms of an allergic reaction, anaphylactic shock, chest 
pain, dizziness, and fainting.  As always, we remind our subjects throughout the protocol that they may stop the 
experiment at any time.  Also, the investigators exercise the discretion to end a subject’s participation if the 
subject should engage in behavior that could jeopardize his/her own health and well-being or that of others.  The 
investigators ends the experiments if the subject’s systolic or diastolic blood pressures exceed 180 or 110 mmHg, 
respectively, heart rate exceeds 85% of the age-predicted maximum, or the body's core temperature reaches 
39°C (102 °F) or decreases 1°C (1.8 °F) from baseline.  Human subjects undergoing the pharmacotherapy end 
treatment and cease participation in the study if the research physicians determine that the therapy produces 
undesirable reactions.
22.0 Study Monitoring, Auditing and Inspecting
22.1
Study Monitoring Plan
22.1.1 Quality Assurance and Quality Control
Lacy Alexander, PhD has ultimate responsibility for this study, with oversight from Jason Fragin, D.O. Dr. 
Alexander meets regularly with the study team to discuss the project, procedures, and data.  Dr. Alexander is 
responsible for the conduct of the study being in compliance with this protocol, with institutional and IRB policies, 
with Good Clinical Practice guidelines and any other applicable regulatory requirements.  The monitoring is 
ongoing.
Only trained, experienced persons perform the procedures of this project.  The licensed research nurse (RN) 
performs blood draws.  If the research nurse is unavailable, a CRC nurse or clinician draws blood.  Trained, 
experienced, and IRB-approved research team members perform the MD insertions and skin biopsies.  The 
CFRC clinician reviews the medical history during screening.  Dr. Fragin is the consulting physician overseeing 
the project.  The CRC clinician oversees participants during pharmacotherapy.  The members of the research 
lab (grad students, staff, and select undergrads) have current basic life support training through CentreLife Link 
in State College, PA.
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22.1.2 Safety Monitoring
All adverse events are classified in accordance with the then current policy of the IRB at Pennsylvania State 
University (Adverse Events, Definitions and Reporting Requirements).  A detailed description of each procedure 
posing risk and the assessment of that risk is included in the relevant sections of the application.
Reporting:  The PI evaluates subject eligibility, adherence to protocols and reporting of adverse events at six-
month intervals.  All adverse events are reported by the PI to the IRB, according to the then current policy of the 
IRB at Pennsylvania State University (Adverse Events, Definitions and Reporting Requirements) and copied to 
the Research Safety Advocate.  In addition, all changes in protocol procedures will be submitted for review and 
approval of the IRB and, when approved, will be promptly implemented.  The project receives an extensive 
annual review by the IRB.  The PI submits reportable-changes in protocol and annual reports to the FDA.
23.0 References
“Previous Data” Section
1.
Smith CJ, Santhanam L, Alexander LM. Rho-kinase activity and cutaneous vasoconstriction is upregulated in essential 
hypertensive humans. Microvasc Res. 2013
2.
Bruning RS, Kenney WL, Alexander LM. Altered skin flowmotion in hypertensive humans. Microvasc Res. 2014
3.
Kutz JL, Greaney JL, Santhanam L, Alexander LM. Evidence for a functional vasodilatory role for hydrogen sulfide in 
the human cutaneous microvasculature. J Physiol. 2015
4.
Beltowski J, Jamroz-Wisniewska A, Tokarzewska D. Hydrogen sulfide and its modulation in arterial hypertension and 
atherosclerosis. Cardiovasc Hematol Agents Med Chem. 2010;8:173-186
5.
Coletta C, Papapetropoulos A, Erdelyi K, Olah G, Modis K, Panopoulos P, Asimakopoulou A, Gero D, Sharina I, Martin 
E, Szabo C. Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and 
endothelium-dependent vasorelaxation. Proceedings of the National Academy of Sciences of the United States of 
America. 2012;109:9161-916613
6.
Holowatz LA, Santhanam L, Webb A, Berkowitz DE, Kenney WL. Oral atorvastatin therapy restores cutaneous 
microvascular function by decreasing arginase activity in hypercholesterolaemic humans. J Physiol. 2011;589:2093-
2103
7.
Holowatz LA, Kenney WL. Acute localized administration of tetrahydrobiopterin and chronic systemic atorvastatin 
treatment restore cutaneous microvascular function in hypercholesterolaemic humans. The Journal of physiology. 
2011;589:4787-4797
8.
Holowatz LA, Kenney WL. Oral atorvastatin therapy increases nitric oxide-dependent cutaneous vasodilation in 
humans by decreasing ascorbate-sensitive oxidants. Am J Physiol Regul Integr Comp Physiol. 2011;301:R763-768
9.
Stanhewicz AE, Alexander LM, Kenney WL. Oral sapropterin augments reflex vasoconstriction in aged human skin 
through noradrenergic mechanisms. Journal of applied physiology. 2013;115:1025-1031
10.
Stanhewicz AE, Alexander LM, Kenney WL. Oral sapropterin acutely augments reflex vasodilation in aged human skin 
through nitric oxide-dependent mechanisms. Journal of applied physiology: respiratory, environmental and exercise 
physiology. 2013
11.
Vongpatanasin W, Wang Z, Arbique D, Arbique G, Adams-Huet B, Mitchell JH, Victor RG, Thomas GD. Functional 
sympatholysis is impaired in hypertensive humans. J Physiol. 2011;589:1209-1220
12.
Price A, Raheja P, Wang Z, Arbique D, Adams-Huet B, Mitchell JH, Victor RG, Thomas GD, Vongpatanasin W. 
Differential effects of nebivolol versus metoprolol on functional sympatholysis in hypertensive humans. Hypertension. 
2013;61:1263-1269
Pharmacologic
L-NAME, SNP, ACh
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21. Schulman, S. P., L. C. Becker, D. A. Kass, H. C. Champion, M. L. Terrin, S. Forman, K.  V. Ernst, M. D. Kelemen, S. N. 
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3.
Isbister GK1,2, Downes MA1,2, Mcnamara K2, Berling I1,2, Whyte IM1,2, Page CB1,2,3. A prospective observational 
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2970, 2006.
9. Holowatz, L. A., C. S. Thompson, and W. L. Kenney. L-arginine supplementation or arginase inhibition augments reflex 
cutaneous vasodilatation in aged human skin. J Physiol, 2006.
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13. Kellogg, D. L., Jr., C. G. Crandall, Y. Liu, N. Charkoudian, and J. M. Johnson. Nitric oxide and cutaneous active 
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15. McCord, G. R., J. L. Cracowski, and C. T. Minson. Prostanoids contribute to cutaneous active vasodilation in humans. 
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attenuated in older humans. Am J Physiol Regul Integr Comp Physiol. 288:R1108-1113, 2005.
22. Thompson, C. S. and W. L. Kenney. Altered neurotransmitter control of reflex vasoconstriction in aged human skin. J 
Physiol. 558:697-704, 2004.
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23. Ungerstedt, U. Microdialysis--principles and applications for studies in animals and man. J Intern Med. 230:365-373, 
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vasodilatation in humans. J Physiol. 548:963-969, 2003.
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28. Wong, B. J. and C. T. Minson. Neurokinin-1 Receptor Desensitisation Attenuates Cutaneous Active Vasodilatation in 
Humans. J Physiol, 2006.
29. Wong, B. J., N. J. Tublitz, and C. T. Minson. Neurokinin-1 receptor desensitization to consecutive microdialysis infusions 
of substance P in human skin. J Physiol. 568:1047- 1056, 2005.
30. Wong, B. J., B. W. Wilkins, L. A. Holowatz, and C. T. Minson. Nitric oxide synthase inhibition does not alter the reactive 
hyperemic response in the cutaneous circulation. J Appl Physiol. 95:504-510, 2003.
31. Wong, B. J., B. W. Wilkins, and C. T. Minson. H1 but not H2 histamine receptor activation contributes to the rise in skin 
blood flow during whole body heating in humans. J Physiol. 560:941-948, 2004.
32. Zhao, J. L., P. E. Pergola, L. J. Roman, and D. L. Kellogg, Jr. Bioactive nitric oxide concentration does not increase 
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Pyrogen References
1. Brown, S., and A.C. Fuller, “Depyrogenation of Pharmaceutical Solutions Using Submicron and Ultrafilters,” J. Parenteral 
Sci. & Tech., Vol. 47, No. 6, 1993, pp. 285-288.
The effect of varying the pH and ionic strength on endotoxin removal (depyrogenation) from Water for Injections (WFI) 
was investigated. Studies using submicron filters showed that endotoxin aggregation and filter retention increased with 
increasing molarity and decreasing pH. Using a Sartorius 0.01 micron filter, greater than 98% endotoxin retention could 
be achieved with 10 endotoxin units (EU)/ml bulk solution, and greater than 97% endotoxin retention with the 500 
EU/ml bulk solution. Depyrogenation of active and placebo solutions of the radiopaque, Iohexol (350 mgI/ml), using 
ultrafilters of varying nominal molecular weight limit (NMWL 10,000- 300,000) and a Pall Posidyne 0.2 micron filter was 
also investigated. Results with the ultrafilters showed that it was possible to increase the molecular weight cut-off of 
an ultrafilter from 10,000 to 100,000, without affecting the efficiency of endotoxin removal, thereb increasing flow rate 
and reducing filtration time. The Posidyne filter was able to depyrogenate Iohexol active and placebo product. The use 
of submicron filtration in place of ultrafiltration would provide significant cost benefits in terms of filtration time and 
equipment costs, and they have been shown to be capable of efficient depyrogenation of these pharmaceutical 
products.
2. McCormick J, Pragman A, Stolpa J, Leung D, Schlievert P, “Funtional Characterization of Streptococal Pyrogenic 
Exotoxin J, a Novel Superantigen” Infection and Immunity, Vol. 69, No. 3: p. 1381-1388, 2001.
3. Orwin P, Leung D, Tripp T, Bohach G, Earhart C, Ohlendorf D, Schlievert P. 2002. Characterization of a novel 
staphylococcal enterotoxin-like superantigen, a member of the group V subfamily of pyrogenic toxins. Biochemistry 
41:14033-14040, 2002.
Staphylococcus aureus is an important human pathogen, causing a variety of diseases. Major virulence factors of this 
organism include staphylococcal enterotoxins (SEs) that cause food poisoning and toxic shock syndrome. Our study 
identified a novel enterotoxin-like protein that is a member of the new subfamily (group V) of pyrogenic toxin 
superantigens (PTSAgs) and examined its biochemical and immunobiological properties. The gene encoding the SE-
like protein is directly 5‘ of another recently identified PTSAg, SEK. The SE-like protein had a molecular weight of 
26000 and an experimentally determined isoelectric point between 7.5 and 8.0. We demonstrated that the PTSAg had 
many of the biological activities associated with SEs, including superantigenicity, pyrogenicity, and ability to enhance 
endotoxin shock, but lacked both lethality in rabbits when administered in subcutaneous miniosmotic pumps and 
emetic activity in monkeys. Recombinant protein stimulated human CD4 and CD8 T cells in a T cell receptor variable 
region, β chain (TCRVβ) specific manner. T cells bearing TCRVβ2, 5.1, and 21.3 were significantly stimulated.
4. Orwin P, Fitzgerald JR, Leung D, Gutierrez J, Bohach G, Schlievert P, “Characterization of Staphylococcus aureus 
Entertoxin L, “Infection and Immunity, Vol. 71, No. 5p.: 2916-2919, 2003.
5. Weber C, Linsberger I, Rafiee-Tehrani M, Falkenhagen D: Permeability and adsorption capacity of dialysis membranes 
to lipid A. Int J Artif Organs 20: 144-152, 1997.
Hemodialysis membranes were tested in vitro for possible penetration by low molecular weight endotoxins containing 
lipid A. Using lipid A from Escherichia coli as a model substance for this kind of pyrogen, different dialyzers (F4, E3. 
Acepal 1300, Altraflux, F 40, Polyflux 110, Filtral 12, F 60) were challenged by tangential filtration in aqueous medium. 
All membranes exhibited impermability to lipid A (as well as to LPS from Pseudomonas aeruginosa), which was proved 
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by additional experiments using culture filtrates of Pseudomonas aeruginosa in bicarbonate dialysis fluid, as well as 
by employing miniaturized dialyzers with synthetic lipid A as a contaminant. Furthermore, the highest adsorption 
capacities were found for polysulfone and polyamide membranes.
24.0 Appendix
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