Clinical trial NCT02552888
Conditions: Chronic Kidney Disease
Keywords: Endothelial Dysfunction Inflammation Oxidative Stress eGFR Urine Albumin
Sponsor: Tulane University
Start date: 2016-03-01; Completion date: 2017-06-01
Enrollment: 70 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The proposed randomized controlled trial will test the safety and efficacy of combination therapy with sodium nitrite and isoquercetin on endothelial function and inflammation among patients with chronic kidney disease.

Detailed description:
The proposed randomized controlled trial will test the safety and efficacy of combination therapy with sodium nitrite and isoquercetin on endothelial function and inflammation among patients with chronic kidney disease. Investigators will recruit 70 albuminuric CKD patients and randomly assign participants to combination therapy with sodium nitrite and isoquercetin or placebo for three months.

Arms: arm 1: Immediate release sodium nitrite 40 mg by mouth twice per day and Isoquercetin 225 mg by mouth once per day. arm 2: identical placebos.
Interventions: intervention 1: Immediate release sodium nitrite intervention 2: Isoquercetin intervention 3: placebos
Intervention descriptions: intervention 1: Immediate release sodium nitrite 40 mg by mouth twice per day intervention 2: Isoquercetin 225 mg by mouth once per day intervention 3: placebos

Protocol documents:
Prot_SAP_000.pdf: 
August 30, 2016 
 
 
 
Protocol Version 7.1 
 
Nitrite, Isoquercetin and Endothelial Dysfunction (NICE) Trial 
 
 
PI: Jing Chen, M.D., M.Sc., M.M.Sc 
 
Funding: Louisiana Clinical & Translational Science Center/National Institutes of Health 
 
 
Tulane University School of Medicine 
August 30, 2016 
IND #127397 
 
 
 
 
 
 


August 30, 2016 
Abbreviations 
 
 
AE 
 
Adverse event 
ADMA  
Asymmetrical dimethylarginine 
BP 
 
Blood pressure 
BUN  
Blood Urea Nitrogen 
CBC  
Complete blood count 
CKD  
Chronic kidney disease 
CrCl  
Creatinine clearance 
CRF  
Case Report Forms 
CRP  
C reactive protein 
CVD  
Cardiovascular disease 
DSMB  
Data and Safety Monitoring Board 
eGFR  
Estimate glomerular filtration rate 
ET-1  
Endothlin-1 
FDA  
Food and Drug Administration 
FMD  
Flow-mediated vasodilation 
G6PD  
Glucose-6-phosphate dehydrogenase 
GRAS  
Generally Recognized as Safe 
HbA1c 
Hemoglobin A1c 
HDL-C 
High density lipoprotein cholesterol 
ICAM-1 
intercellular adhesion molecule-1 
ICF 
 
Informed Consent Form 
IL-1 
 
Interleukin-1 
IL1ra  
Interleukin-1 receptor antagonist 
IL-6 
 
Interleukin-6 
IRB 
 
Institutional review board 
IR-nitrite 
Immediate Release Sodium Nitrite 
LDL-C  
Low density lipoprotein cholesterol 
MCP-1 
Monocyte chemoattractant protein-1 
NICE  
Nitrite Isoquercetin Endothelial dysfunction 
NO 
 
Nitric Oxide 
PAD  
Peripheral Arterial Disease 
SAE  
Serious Adverse Event 
TNF- α 
Tumor necrosis factor- α 
VCAM-1 
Vascular adhesion molecule-1 
vWF  
Von Willebrand factor 
WBC  
White blood cell count 
 
 
 
 
 
 
 


August 30, 2016 
 
 
 
 
1.0. TRIAL OVERVIEW .................................................................................................. 4 
2.0. SPECIFIC AIMS ....................................................................................................... 4 
2.1. Primary Objective ................................................. Error! Bookmark not defined. 
2.2. Secondary Objective ............................................. Error! Bookmark not defined. 
2.3. Safety Objective .................................................... Error! Bookmark not defined. 
3.0. BACKGROUND AND SIGNIFICANCE .................................................................... 5 
3.1. Summary of Relevant and Significant Findings from Nonclinical Studies ..... Error! 
Bookmark not defined.  
3.2. Description of and Justification for the Route of Administration, Dosage, Dosage 
Regimen, and Treatment Period .................................................................................. 7 
3.3. Pharmacology of Description of the Investigational Products ............................... 7 
3.4. Safety of Investigational Products ......................................................................... 8 
4.0. INVESTIGATIONAL PLAN .................................................................................. 910 
4.1. Study Design - Overview ...................................................................................... 9 
4.2. Study Population ................................................................................................... 9 
4.3. Inclusion Criteria ................................................................................................. 10 
4.4. Exclusion Criteria ................................................................................................ 10 
4.5. Clinical Screening Visit ....................................................................................... 11 
4.6. Randomization Procedures................................................................................. 11 
4.7. Cohort Follow up and Clinic Visits ...................................................................... 11 
4.8. Study Treatment ................................................................................................. 14 
4.9. Monitoring Participant Symptoms and Laboratory Values .................................. 15 
4.10. Endpoint Definitions and Documentation .......................................................... 15 
4.11. Drug Compliance Monitoring ............................................................................ 16 
4.12. Central Bio-bank and Data Storage .................................................................. 16 
4.13. Adverse Events (AEs) and Serious Adverse Events (SAEs) ............................ 16 
4.14. Data Coordination ............................................................................................. 17 
4.15.Quality Control and Quality Assurance .............................................................. 17 
4.16. Assessment of Efficacy ..................................................................................... 18 
5.0. DATA ANALYSIS PLAN ....................................................................................... 18 
5.1. Statistical Analysis .............................................................................................. 18 
5.2. Sample Size and Power ..................................................................................... 19 
5.3. Missing and Spurious Data ................................................................................. 19 
5.4. DSMB Analyses .................................................................................................. 19 
6.0. HUMAN SUBJECTS CONSEDERATION ............................................................. 20 
6.1. Informed Consent ............................................................................................... 20 
6.2. Risks ................................................................................................................... 21 
6.2. Benefits ............................................................................................................... 21 
6.4. Payments and Costs ........................................................................................... 22 


August 30, 2016 
7.0. REFERENCES ....................................................................................................... 23 
 
1.0. TRIAL OVERVIEW 
The proposed study is a randomized, double-blind, placebo-controlled, parallel-
design clinical trial with a treatment and a control group. The overall objectives of the 
trial are to test the efficacy and safety of combination therapies. The treatment arm 
includes Immediate release sodium nitrite (IR-nitrite formula, 40 mg twice daily) and 
isoquercetin (225 mg, once daily). The control (placebo) arm tests placebo versions of 
IR-nitrite and isoquercetin. Effects will be measured based on endothelial dysfunction, 
inflammation, and oxidative stress among patients with albuminuric and non-albuminuric 
chronic kidney disease (CKD).  
Sodium nitrite at 40 mg and 80 mg doses by mouth twice per day are considered 
safe and well tolerated based on previously conducted animal and phase 1 and 2 
clinical trials.4-7 The FDA has documented that the normal dietary dose of isoquercetin 
(as proposed in this trial) is considered Generally Recognized as Safe.  
Eligible participants will be required to complete a screening visit, 
baseline/randomization visit, and follow-up visits conducted at day 3 (phone only), and 
in-person clinic visits on weeks 1, 6 and 12 following randomization. During the 
intervention period, Flow-mediated dilation (FMD), serum creatinine, urine albumin-to-
creatinine ratio, biomarkers of endothelial dysfunction, inflammation, and oxidative 
stress will be measured at follow-up visits marked by 6 and 12 weeks post 
randomization (introduction to drug therapy). 
The primary trial endpoint is the measurement of net change in endothelium-
dependent FMD between the active treatment group and the placebo-control group over 
the 3-month intervention period. Secondary and exploratory endpoints include the 
expected differences in endothelial dysfunction biomarkers, estimated-glomerular 
filtration rate (eGFR) and urinary albumin-to-creatinine ratios between the intervention 
and control groups. 
 
2.0. SPECIFIC AIMS 
 
Chronic kidney disease (CKD) is highly prevalent in Louisiana and around the 
world.1,2 Cardiovascular disease (CVD) and end-stage renal disease (ESRD) are the 
major causes of premature death in patients with CKD.3,4 Randomized controlled trials 
have documented that renin-angiotensin system antagonists, blood pressure reduction, 
glycemic control, and lipid lowering can slow CKD progression and reduce CVD risk 
among CKD patients.5,6 However, multiple risk factors contribute to the progression of 
CKD and development of CVD among CKD patients.7 Previous works from our group 
and others indicate that endothelial dysfunction and inflammation may be the common 
etiologic pathway for the rapid progression of CKD and excess risk of CVD independent 
of traditional risk factors.8-12 Nitric oxide (NO) deficiency may be a key component of 


August 30, 2016 
endothelial dysfunction in CKD patients and contribute to accelerate both CKD and CVD 
progression.13-21 Until now, there has been no specific or effective pharmacological 
treatment for endothelial dysfunction and inflammation among CKD patients. Previous 
phase 2 trials suggest that sodium nitrite provides exogenous NO and improves 
endothelial function.22-26 Dietary isoquercetin supplement (a natural flavonoid found in 
fruits and vegetables) has also been shown to reduce inflammation and oxidative 
stress.27-33  
The overall objective of this proposed randomized placebo-controlled trial is to test 
the efficacy and safety of combination therapy with sodium nitrite (immediate-release 
sodium nitrite formula, 40 mg twice daily) and the dietary isoquercetin supplement (225 
mg, once daily) on endothelial function, inflammation and oxidative stress among CKD 
patients, as compared to the placebo group.  
The study plans to recruit 70 patients with albuminuric and non-albuminuric CKD 
from the greater New Orleans area in Louisiana. Participants will be randomly assigned 
to receive combination therapy with sodium nitrite and isoquercetin, or receive placebos 
of IR-nitrite and Isoquercetin for 3 months. The primary study outcome is endothelial 
function assessed by brachial artery flow-mediated dilatation (FMD). Secondary 
outcomes include biomarkers of endothelial dysfunction, inflammation, and oxidative 
stress. This trial is designed to have 80% statistical power to detect a 2.0% difference in 
FMD with a 2-sided significance level of 0.05.  
2.1. Primary objective: To test the efficacy of a combination therapy with sodium nitrite 
(IR-nitrite) and dietary isoquercetin supplement on endothelial function assessed by 
FMD among patients with CKD. 
Primary hypothesis: Compared to CKD patients receiving placebo IR-nitrite and 
Isoquercetin, FMD will be increased among CKD patients receiving the combination 
therapy with IR-nitrite and isoquercetin. 
2.2. Secondary objectives: To test the efficacy of the combination therapy with sodium 
nitrite and dietary isoquercetin supplement on biomarkers of endothelial function, 
inflammation, and oxidative stress among patients with CKD. 
Secondary hypothesis 1: Compared to CKD patients receiving placebo, the plasma 
levels of asymmetric dimethylarginine (ADMA), intercellular adhesion molecule 1 
(ICAM-1), vascular adhesion molecule 1 (VCAM-1),  E-selectin, endothlin-1 (ET-1), von 
Willebrand factorv (vWF), endothlin-1, and endostatin will be reduced among CKD 
patients receiving IR-nitrite and isoquercetin. 
Secondary hypothesis 2: Compared to CKD patients receiving placebo, the plasma 
levels of C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), interleukin-1β 
(IL-1β), interleukin 6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) will be 
reduced among CKD patients receiving IR-nitrite and isoquercetin. 
Secondary hypothesis 3: Compared to CKD patients receiving placebo, the plasma 
levels of oxidized LDL and nitrotyrosines will be reduced among CKD patients receiving 
IR-nitrite and isoquercetin. 
2.3. Safety objective: To test the safety of the combination therapy with IR-nitrite and 
dietary isoquercetin supplement among patients with CKD. 


August 30, 2016 
Safety hypothesis 1: The combination treatment is safe and well-tolerated. Blood levels 
of methemoglobin, nitrite, nitrate, and isoquercetin are within acceptable range in the 
treatment group. 
Safety hypothesis 2: Compared to CKD patients receiving placebo control, eGFR will be 
increased and urinary albumin will be decreased among CKD patients receiving SR-
nitrite and isoquercetin. 
Impact: The proposed research is the first clinical trial to investigate the efficacy and 
safety of combination therapy with IR-nitrite and isoquercetin on endothelial function 
and inflammation among CKD patients. The findings from this study will help in the 
development of novel treatments targeting endothelial dysfunction and inflammation, 
primarily aiming to slow the progression of CKD and development of CVD among 
patients with CKD. This study will generate the necessary pilot data for an NIH-
supported large phase-III clinical trial to test the efficacy of IR-nitrite and isoquercetin 
combined treatment and its effect on CKD progression. 
3.0. BACKGROUND AND SIGNIFICANCE 
 
3.1. Summary of Relevant and Significant Findings from Nonclinical Studies 
 
Sodium Nitrite: Previous studies suggest that sodium nitrite supplement provides 
exogenous nitric oxide and improves endothelial function.1-7 For instance, systemic 
intravenous administration of sodium nitrite (8.7 μmol/min) dilated the radial artery by 
10.7% (95% confidence interval, 6.8-14.7%) under normal oxygenated conditions at 
supraphysiological and near-physiological concentrations in healthy male volunteers.2 
Nitrite enhanced cGMP production in a dose-dependent manner, suggesting nitric 
oxide-mediated vasodilatory effects.2 Nitrite-induced radial artery dilation was maximal 
under conditions of normoxia and inhibited by hypoxia and hyperoxia.2 A phase 2 
clinical trial suggests that diabetic patients with peripheral artery disease receiving 
sodium nitrite (40 mg and 80 mg) by mouth had significantly higher flow-mediated 
vasodilation compared with those receiving the placebo. Furthermore, dietary sodium 
nitrite attenuates brain death-induced renal injury by regulating responses to ischemia 
and inflammation, ultimately leading to improvement in post-transplant kidney function 
in rats.28 
 
Isoquercetin: Isoquercetin has been demonstrated to have a dose-dependent protective 
effects against oxidative endothelial injury in cultured human endothelial cells.13 It has 
also been shown to protect venular endothelium from inflammatory products released 
by activated blood platelets and polymorphonuclear granulocytes in vitro.14 Isoquercetin 
increases antioxidant activities and reduces oxidative stress in combating cadmium-
induced toxicity in animals.15 A small randomized, double-blind, placebo-controlled 
crossover trial reported that quercetin-3-glucoside (160 mg/d) reduced  E-selectin by 
27.7 ng/mL, interleukin -1β by 20.23 pg/mL, and the z score for inflammation by 20.33 in 
37 healthy hypertensive or pre-hypertensive men and women.16 Quercetin (500 mg four 
times per day) also reduced plasma malondialdehyde (a marker of lipid peroxidation) 
and tumor necrosis factor-α levels in a clinical trial among patients with sarcoidosis.17 In 
addition, quercetin 500 mg + vitamin C 250 mg reduced interleukin-6 by 38% compared 


August 30, 2016 
to placebo in a randomized double-blind clinical trial among 60 male physical education 
students. A similar effect was not observed in the subjects who received quercetin 500 
mg/day alone.18 The mechanism of anti-inflammatory effects of quercetin/isoquercetin 
may be via down-regulation of the nuclear factor kappa B (NF-kB) pathway.19-21 It has 
also been reported that quercetin is capable of inhibiting tumor necrosis factor-α 
production as well as tumor necrosis factor-α gene expression via modulation of NF-kB 
in human peripheral blood mononuclear cells.22 Moreover, since NF-kB activation 
induces oxidative stress, the direct inhibition of NF-kB pathway by quercetin therefore 
reduces oxidative stress. A laboratory study also suggested that quercetin decreased 
lipid peroxidation and increased nitric oxide production and antioxidant enzyme 
activity.23 
 
 
 
3.2 Description of and Justification for the Route of Administration, Dosage, 
Dosage Regimen, and Treatment Period 
 
Sodium nitrite: Oral administration of IR- nitrite will be used due to its increased safety 
and practicality for routine clinical use, when compared to intravenous therapy. Patients 
will receive IR-nitrite for three months, as empirical evidence suggests that this time 
frame is sufficient to show that changes in endothelial dysfunction (measured by FMD) 
can be detected. During the treatment portion of the trial, the following factors will be 
monitored with these goals in mind: methemoglobin, nitrite, and nitrate (aiming to keep 
methemoglobin <3%, plasma nitrite within the normal range of 1.3-13 µmol/L, and 
plasma nitrate within the normal range of 4.0-45.3 µmol/L).50  
 
Isoquercetin: The effective oral dose of 225 mg isoquercetin (supplied by Quercegen 
Pharmaceuticals), will be administered to participants daily. This dose is below the 
threshold of the FDA’s acceptable daily intake of 293 mg/person/day and the dose for 
no-observed-adverse-effect level (539 mg/kg/day).24 Isoquercetin has been selected 
over quercetin due to the fact that the absorption rate of isoquercetin is roughly 2 times 
higher in comparison to quercetin11,12 and the effective dose of isoquercetin is half that 
of quercetin in combination with vitamin C.18. It is also less likely that participants would 
have any side effects with isoquercetin. Expected clinical symptoms including 
headaches and plasma isoquercetin levels will be closely monitored throughout patient 
participation.   
 
 
 
3.3. Pharmacology and Description of the Investigational Products 
 
Immediate Release Sodium Nitrite (IR-nitrite): The chemical formula appears as NaNO2. 
IR-nitrite is also referred to as TV1001, a product of TheraVasc Inc. (pharmaceutical 
company). IR-Nitrite is absorbed from the stomach. The half-life elimination is 
approximately one hour, with peak effect occurring at roughly 25 minutes, while duration 
of action is close to 10 hours. More detailed information on sodium nitrite, including the 


August 30, 2016 
investigator’s brochure, the compound’s chemistry, manufacturing and control data, and 
pharmacology/toxicology data is described in the FDA approved IND Application (IND 
#108526, Protocol TheraVasc-TV1001-002, submitted by TheraVasc, Inc. on March 14, 
2011, serial #0001, ClinicalTrials.gov identifier: NCT01401517). Please note, the IND# 
specific to the sodium nitrite for packaging label given by TheraVasc is #108526 and 
appears on the Investigator’s Brochure as such. The IND associated with the NICE trial 
by the FDA is #127397. 
Nitrite is a major intravascular storage pool for nitric oxide. Sodium nitrite has been used 
as a nitric oxide-donor to improve endothelial dysfunction in the treatment of 
cardiovascular disease (CVD).1-7  
In brief, sodium nitrite at 40 mg and 80 mg doses by mouth twice per day are safe and 
well tolerated in animal as well as human phase 1 and 2 clinical trials.4-7 Its biological 
effect in improving endothelial dysfunction has been observed in both animal studies 
and phase 2 clinical trials.4-6 The major adverse effects associated with sodium nitrite 
are methemoglobinemia and hypotension. However, these potential adverse effects 
were not observed in patients with peripheral artery disease who received 40 mg or 80 
mg immediate release sodium nitrite by mouth twice per day, or in patients with 
diabetes mellitus and active or healed foot ulcers who received a single dose of 80 mg 
IR-nitrite in clinical trials.4,7  
 
Isoquercetin: Isoquercetin (quercetin 3-glucoside or quercetin-3-O-glucoside) is a 
natural dietary flavonoid of fruits and vegetables. In a human study, isoquercetin was 
shown to peak at 30 minutes after ingestion of 156 mg, with a peak plasma 
concentration of 5 µmol/L. The half-life elimination is reported as 18.5 hours, and urinary 
excretion is measured as 3.6%.8 Isoquercetin has the same therapeutic effects in vivo 
as quercetin, but with better bioavailability, resulting in potentially increased efficacy 
compared to quercetin.9 In a study of male participants, the absorption rate was noted 
to be 52% for quercetin 3-glucosides and 24% for quercetin.10 Rats given quercetin and 
isoquercetin achieved plasma concentration 2.5 to 3 times higher with isoquercetin 
compared to quercetin.11,12  
The FDA has documented isoquercetin to be Generally Recognized as Safe (GRAS) in 
2007 with an acceptable daily intake of 293 mg/person/day. Detailed information on 
isoquercetin and quercetin, such as their chemical and pharmacological properties, as 
well as safety and toxicology data is described in the GRAS application (please see 
attached letter on GRAS for isoquercetin and quercetin). Isoquercetin and quercetin 
have been shown to reduce inflammation and oxidative stress in animal studies and 
phase 2 clinical trials.13-23 No-observed-adverse-effect level of enzymatically 
decomposed rutin (containing 95% isoquercitrin) in Wistar rats was estimated to be 1% 
(539 mg/kg/day, i.e. 38 g for a 70 kg individual) for acute or chronic use. Adverse effects 
of higher doses in rats included mostly (benign) chromaturia.24 This trial will administer a 
225 mg daily dose as this is below the FDA’s threshold of the acceptable daily intake of 
293 mg/person/day as well as the previously discussed dose of539 mg/kg/day, which 
showed no-observed-adverse-effect level. 
 
Vitamin C:  Vitamin C is also referred to asL-ascorbic acid or ascorbate. Participants will 
receive vitamin C 55.8mg by mouth daily, as a stabilizing, inactive ingredient in the 


August 30, 2016 
Isoquercetin capsules, to increase the absorption of isoquercetin and reduce 
methemoglobin, oxidative end products of nitric oxide, and nitrosamine resulting from 
sodium nitrite treatment.25-27 
 
3.4. Safety of Investigational Products 
 
Sodium nitrite: Sodium nitrite has been used as a nitric oxide-donor to improve 
endothelial dysfunction in the treatment of cardiovascular disease (CVD).1-7 Nitric Oxide 
(NO) deficiency may be a key component of endothelial dysfunction in patients with 
chronic kidney disease (CKD) and contribute to accelerate both CKD and CVD 
progression.29-38 Therefore, sodium nitrite may correct nitric oxide deficiency and 
improve clinical CKD and CVD outcomes in CKD patients. However, the increased NO 
production associated with sodium nitrite supplementation also potentially dilates 
vessels and causes side effects such as hypotension and dizziness. Headaches, when 
occurring as a side effect, are transient and reversible. In addition, nitrite can react with 
oxyhemoglobin to form methemoglobin, which may cause tissue hypoxia. The reaction 
of nitrite with secondary amines under acidic conditions may form carcinogenic 
nitrosamines. Increased oxidation of NO may also induce formation of nitrosamines. 
Vitamin C (an antioxidant) can reduce methemoglobin to hemoglobin as well as prevent 
nitrosamine formation.25-27 According to 9 Code of Federal Regulations (CFR) 424.22, 
550 ppm (550ppm = 550mg/kg) of Vitamin C has been used in cured meats (containing 
nitrite) to reduce nitrosamine formation within the food industry, according to the 
regulation of U.S. Department of Agriculture. Therefore, only 0.044 mg of vitamin C is 
needed for 80 mg of sodium nitrite (used in this study, sodium nitrite 40 mg BID) to 
prevent nitrosamine formation.  
 
Other antioxidants, such as vitamin E and erythorbic acid, have also been used for the 
same purpose.  
For the purposes of this trial, IR-nitrite will be given orally, which was demonstrated as 
safe in a phase 2 clinical trial, without any additional vitamin C supplement.4 Patients in 
both arms of the trial will receive a dose of 55.8 mg of vitamin C supplied (more than 
550 ppm vitamin C for sodium nitrite) as a filler ingredient within the isoquercetin 
capsule. The combination of vitamin C and isoquercetin used in this study may further 
increase the effect of antioxidants, thereby reducing nitrosamine formation. In addition, 
as absorption time of IR-nitrite is roughly 30 minutes, participants will be asked to take 
their dosage of sodium nitrite 30 minutes before meals to avoid a reaction with potential 
amines in food. In order to report and treat any potential side effects, we will closely 
monitor methemoglobin and nitrite related oxidation product such as nitrotyrosines 
through blood draws at visits.   
A dose of 40 mg of IR-nitrite twice per day will be used for the treatment arm patients, 
which is a 50% reduction of the FDA-approved dosage of 80 mg twice per day. The 
80mg BID dose was well-tolerated in a clinical trial studying patients with peripheral 
artery disease (PAD).4 The reduced dosage is selected due to increased safety 
considerations amid patients with declined kidney function, as the proposed cohort of 
this clinical trial. 
 


August 30, 2016 
Isoquercetin: Isoquercetin/quercetin have anti-inflammatory and antioxidant effects, 
which have been documented in both phase 2 clinical trials and animal studies.13-23 
Inflammation and oxidative stress are increased in CKD patients and predict CKD and 
CVD progression.39-49 Therefore, CKD patients may benefit from isoquercetin treatment 
through the reduction of inflammation and oxidative stress and ultimately improved CKD 
clinical outcomes. The Isoquercetin capsule additionally contains 55.8 mg of vitamin C 
and 4.5mg of vitamin B3 to increase absorption and stability of isoquercetin. 
 
4.0. INVESTIGATIONAL PLAN 
4.1. Study Design - Overview 
The proposed study is a randomized, double-blind, placebo-controlled, parallel-
design trial. Seventy (70) patients 
with chronic kidney disease who meet 
eligibility criteria will be recruited in 
the greater New Orleans area. 
Eligible participants will be randomly 
assigned to the intervention 
(combination therapy with IR-nitrite 
and isoquercetin) and placebo 
(placebo IR-nitrite and Isoquercetin) 
groups. The intervention will last for 3 
months. The primary, secondary, and 
safety outcomes will be measured 
throughout the proposed time frame 
(Figure 1). 
 
 
 
 
4.2. Study Population 
The study plans to recruit 70 patients with albuminuric and non-albuminuric 
chronic kidney disease (CKD) in the greater New Orleans area including Tulane 
hospital, Louisiana State University Medical Center, and Ochsner Health System. 
Thirty-five (35) patients will be randomly assigned to the active combination treatment 
group and 35 to the placebo control group. 
 
 
4.3. Inclusion Criteria 
a. Men and women aged 21-74 years old of all race/ethnicity backgrounds 
b. eGFR<60 ml/min/1.73m2 or urinary albumin-to-creatinine ratio ≥30 mg/g or 
protein to creatinine ratio ≥150 mg/g 
c. Systolic BP ≥120 and <180 mmHg and/or diastolic BP ≥70 and <110 mmHg 
Screening for patients with albuminuric chronic 
kidney disease in the great New Orleans area 
70 eligible study participants
Placebo control
(35 participants)
Combination therapy with 
sodium nitrite and quercetin 
(35 participants)
Figure 1. Overview of the Study Design
3 month treatment
and follow-up
Net changes in endothelium-dependent flow-mediated vasodilation, 
biomarkers of endothelial function, inflammation, and oxidative stress 
as well as safety measures


August 30, 2016 
4.4. Exclusion Criteria 
a. Allergic to organic nitrite, isoquercetin, and/or vitamin C 
b. Institutionalized (e.g., prisoner, nursing home or skilled nursing facility resident) 
c. Unable or unwilling to give consent  
d. Known HIV infection and/or AIDS 
e. Pregnant or lactating women 
f. Currently on dialysis  
g. Previous or current organ or bone marrow transplant 
h. Receiving immunosuppressive treatment or other immunotherapy 
i. Receiving chemotherapy or alkylating agents for systemic cancer 
j. Recent acute myocardial infarction, cerebrovascular accidence or transient 
ischemic attack, or hospitalization in 3 months 
k. Acute kidney injury within 3 month 
l. Currently taking a phosphodiesterase-5 enzyme inhibitor, such as Viagra 
m. Chronic headaches 
n. Chronically receiving fluoroquinolones, cyclosporin (neural, sandimmune),  
nitrate drug, NSAIDS ( except aspirin ≤ 81 mg daily) , allopurinol or Uloric 
(febuxostat),   meperidine and related central nervous system (CNS) 
depressants, oral glucocorticoids,   and not willing or able to stop during study 
period. 
o. Active infection (i.e. systemic or osteomyelitis) 
p. Class III or IV heart failure 
q. History of hemolytic anemia including sickle cell disease  
r. Hemoglobin <10 
s. History of chronic obstructive pulmonary disease (COPD)  
t. Have a positive screen for glucose-6-phosphate dehydrogenase (G6PD) 
deficiency at screening 
u. Involvement in other clinical trials  
v. Current  alcohol or other substance abuse 
w. Current smokers 
x. Unwillingness to stop flavonoid or nitrite/nitrate supplementation 
 
4.5. Clinical Screening visit 
All subjects will undergo screening visit to assess their eligibility. 
The following information will be obtained at the clinical visit:  
• Informed consent for the screening visit  
• Detailed medical history and use of medication and dietary supplement 
• Blood pressure and pulse (measured using a standardized automatic blood 
pressure monitor); height and weight 


August 30, 2016 
• CBC; BMP (Serum creatinine (eGFR)), urine albumin, and plasma aglucose-6-
phosphate dehydrogenase measured via serum samples- roughly 15mL of blood. 
Exception: should CBC, BMP and UA results be available within three months, 
these labs will not be drawn. 
• Spot urine pregnancy test for women at child-bearing age without surgical 
sterilization (such as tubal ligation).  
• Information about heart function including review of medical records, assessment 
of clinical symptoms, and a full physical exam as needed. 
 
 
4.6. Randomization Procedures 
Eligible participants will be assigned, using a block stratified randomization 
method, to the active treatment or placebo control groups. The randomization will be 
stratified by sex and use random block sizes of 4 or 6 in order to maintain balance 
among groups over time. A statistician, who is not involved in the clinical operation of 
the trial, will prepare the randomization allocation using the SAS program. The 
randomization allocation will be concealed in a sealed envelope, and stored securely 
with the statistician. The sealed envelope will be opened only by the investigational 
(research) pharmacist who is not involved in the clinical operation of the trial. After 
eligibility and consent have been ascertained, the study nurse/coordinator will order the 
study drugs from Tulane pharmacy. The research pharmacist will match the patient 
identifier to the subsequent randomization allocation, and dispense the study drugs 
according to the assignment. Drug packs will be labeled with random pack numbers, 
and each patient will receive one bottle at randomization with a 45-day supply, and one 
bottle at their week-6 visit. Periodic quality control checks will be run between the 
clinical research nurse and the investigational pharmacist to ensure the same number of 
patient identifiers have been placed into allocated positions. Once the assignment has 
been made, trial participants are considered to be officially randomized, and every effort 
will be made to obtain complete follow-up information. 
Study participants, coordinators and investigators will be blinded to treatment 
assignments. In addition, ultrasound technicians and clinical laboratory technicians will 
be blinded to the intervention assignments. Separate rooms at the study clinic will be 
utilized for ultrasound measures, blood draws, and study intervention. All biochemical 
measures will be conducted at Tulane University Medical Center Clinical Laboratory. 
Ultrasound readings will be performed at the University of Pennsylvania Hospital 
Cardiovascular Center, where all technicians will be unaware of the study participants’ 
study arm assignments. 
Placebo and active medications will be stored in the Research Pharmacy at 
Tulane University Medical Center. Research pharmacists will receive written orders for 
the coordinating drug pack(s) to be dispensed to the patient based on their 
randomization assignment. Once dispensed by designated research pharmacists, the 
drug pack(s) will be given to the patients by study staff. Drug Accountability Logs will be 
kept by the research pharmacists, and coordination of inventory will be managed by the 
pharmacists and study nurse.   
 


August 30, 2016 
4.7. Cohort Follow up and Clinic Visits 
Study participants will be recruited from hospitals and clinics in the greater New 
Orleans area. Following the pre-screening visit, interested patients meeting the eligibility 
criteria will be invited for a clinical screening visit. 
 
Baseline/randomization visit  
After eligibility is confirmed, patients will be invited to attend a 
baseline/randomization visit. At this time, study participants will receive their 
randomization assignments and begin treatment or placebo therapy. Patients will 
receive 50 tablets of all medications prescribed based on their randomization 
assignment to facilitate compliance and follow-up.  
As a safety precaution, given the immediate absorption rate of the IR-Sodium 
Nitrate, all patients (regardless of treatment or placebo randomization assignment) will 
be asked to take both pills in clinic for the first time, and will be monitored for 30 minutes 
following their dose for potential side effects (see section 4.9 for further detail).  
 
The following information will be obtained at the clinical visit: 
• Contact Information confirmed 
Table 1.  Study visits and data collection 
 
Screening 
Baseline/ 
Randomization 
Follow-up visits 
Day 7 Week 6 Week 12 
Consent form 
X 
 
 
 
 
Medical history and medications 
X 
 
X 
X 
X 
Blood pressure, pulse, weight 
X 
X 
 
X 
X 
Height 
X 
 
 
 
 
eGFR and/or urine albumin-Cr 
ratio 
X 
X 
X 
X 
X 
Glucose-6-phosphate 
dehydrogenase (G6PD) 
X 
 
 
 
 
CBC, BMP 
X 
 
X 
X 
X 
Methemoglobin, nitrite, nitrate, 
isoquercetin, renal function and 
chemistry panels 
 
X 
X 
X 
X 
Lipid Panel 
 
X 
 
 
X 
Flow-mediated dilation 
 
X 
 
X 
X 
Biomarkers for endothelial 
dysfunction, inflammation, and 
oxidative stress 
 
X 
 
X 
X 
Adherence and adverse events 
 
 
X 
X 
X 
CBC: complete blood count; eGFR: estimated-glomerular filtration rate 


August 30, 2016 
• Questions regarding medications and dietary supplement(s) that the subject is 
currently taking, and discussion of other health related events.  
• Vital Signs (blood pressure and pulse), and Anthropometric measure (height, 
weight). 
• Symptoms or health problems since the previous clinic visit. 
• Blood Samples (approximately 2-4 tablespoons) for:  
o Routine tests, including chemistry and renal function panel, lipid panel, 
complete blood counts. 
o Methemoglobin, nitrite, nitrate, isoquercetin. 
o Biomarkers related to endothelial dysfunction, inflammation, oxidative 
stress, and other related biomarkers. 
• Urine sample to test urine protein, creatinine, and other related biomarkers. 
• Forearm ultrasound to measure vascular function (see below for details.) 
 
Forearm Ultrasound Test (Brachial Artery Reactivity Test) 
 
 
Endothelium-dependent flow-mediated vasodilation (FMD) of the brachial artery 
is induced by the elaboration of endothelium-derived NO.  Endothelium-dependent 
FMD in response to reactive hyperemia will be evaluated non-invasively by use of 
high-resolution ultrasound (113-115).  Ultrasound measurements will be performed 
according to a standard method.  Briefly, with subjects in a supine position, at rest, 
and in a quiet temperature-controlled room (22°C to 25°C), B-mode scans of the 
right brachial artery will be obtained in longitudinal sections 2 to 8 cm above the 
elbow by use of a 7.5-MHz linear array transducer and a Hewlett Packard 5500 
duplex ultrasound machine. To ensure consistency of the image with serial scans, 
the transducer position will be marked on the skin. The end-diastolic arterial 
diameter will be measured in ECG-gated end-diastolic frames from 1 media-
adventitia interface to the other at the clearest section. Reactive hyperemia will be 
induced by inflating a pneumatic cuff to suprasystolic pressures at the level of the 
forearm, distal to the brachial artery. After 5 minutes, the cuff will be deflated, 
resulting in a brief episode of reactive hyperemia. Measurements of brachial artery 
diameter will be made 1 minute before application of the cuff and every 60 seconds 
after cuff deflation for 5 minutes. Images will be recorded on a MOD (Magneto 
Optical Disk) for quantitative analysis at the University Of Pennsylvania Vascular 
Research Center, where there is a reading center for several large national studies 
on endothelial dysfunction.  Endothelium-dependent FMD will be calculated as the 
maximal percentage change in vessel size during hyperemia.   
 
Patients will be asked a series of questions by study staff to confirm eligibility in 
continuing the BART exam process. Should a patient report he has taken Viagra 
within 8 hours of the BART exam, the exam will be postponed to a time that can be 
>8 hours from their last dose of the medication so as to avoid potential increased 
vasoactive side effects. Patients will also be reminded that Viagra is an exclusionary 
medication during their participation in the study.  


August 30, 2016 
 
Follow-up visits 
At weeks 6 and 12 after randomization, study participants will be assessed for 
the following information at clinical visits: 
• Detailed medical history and use of medication and dietary supplement(s) 
• Blood pressure, pulse, anthropometric measures (weight)  
• Flow-mediated dilation 
• Urine albumin/cr ratio 
• Blood Samples (approximately 2-4 tablespoons) for:  
o Routine tests, including chemistry and renal function panel, lipid panel, 
complete blood counts. 
o Methemoglobin, nitrite, nitrate, isoquercetin. 
o Biomarkers related to endothelial dysfunction, inflammation, oxidative 
stress, and other related biomarkers. 
Patients will also receive 50 additional tablets of all required medications to last until the 
week 12 visit.  
Safety monitoring visits 
Study staff will contact the participants by phone or email three days after the first dose 
of therapy to confirm that no adverse events have occurred. At day 7 after starting 
treatment, medical history, medication, blood pressure, pulse, blood methemoglobin, 
nitrite, nitrate, isoquercetin, blood chemistry panels, and CBC will be assessed during 
the in-person visit for safety monitoring. Clinical adverse events will also be evaluated. 
A one-week window will be allowed for patients experiencing difficulty with scheduling 
their follow-up visits for weeks 6, and 12. This will allow the patients to complete their 
visits one week prior to or following their scheduled visit date to ensure compliance. 
 
4.8. Study Treatment 
The treatment group will receive combination therapy with IR- nitrite and 
isoquercetin. An immediate-release formulation of sodium nitrite (IR-nitrite or TV1001IR) 
will be administrated. The minimal effective dose of IR-nitrite (40 mg twice daily) will be 
administered.  
As a naturally occurring compound, a small amount of isoquercetin exists in the 
human diet. Isoquercetin will be given at a dose of 225 mg daily, which is below the 
threshold of the FDA’s acceptable daily intake. This dose falls within the normal dietary 
intake range.  The Isoquercetin capsule additionally contains 55.8mg of vitamin C and 
4.5mg vitamin B3 to increase absorption and stability of isoquercetin. 
Patients randomized to the placebo arm will receive IR-nitrite in its placebo form 
(with no notable difference in appearance of the tablet). A placebo dose of Isoquercetin 


August 30, 2016 
(225mg per day) will also be administered to placebo patients. All therapy for both 
selected arms will be taken orally.  
 
Temporary Stops 
In the interest of maintaining safety and best clinical practices, a temporary stop will be 
available to all enrolled patients should specific circumstances arise. Should a patient 
begin a temporary dose of antibiotics or steroids, which may be considered an 
interaction risk by the study physician, treatment may be stopped on a temporary basis. 
Enrolled participants will be advised to inform study staff any infection, arthritis pain, and 
other conditions requiring systemic steroid treatment. Patients will also be asked to note 
any persistent or intolerable symptoms that could be related to the study drugs pending 
evaluation by the study physician. In addition, participants will have the option to 
temporarily stop the study drugs due to symptoms that are potentially related to study 
drugs, following an evaluation by the study physician.  
In the event of the temporary stop, patients will continue to follow up with their 
protocol visits as scheduled, and side effects and additional medications will be 
assessed at that time. The study participant is to restart the study medications 
immediately upon conclusion of the contraindicated treatment or once related side 
effects subside based on the evaluation of the study physician.   
All study drugs and placebos will be directly shipped to Tulane inpatient 
pharmacy. The designated research pharmacist will fill and dispense the study drugs 
according to written orders from the study physician and nurse.  
 
4.9. Monitoring Participant Symptoms and Laboratory Values  
The following safety parameters will be assessed: medical and medication 
history; concomitant medication usage; physical examination; blood pressure; pulse; 
clinical chemistries (including glucose, urea nitrogen (BUN), creatinine, calcium, 
phosphorus, bicarbonate, cholesterol, triglycerides, high-density lipoprotein cholesterol, 
low-density lipoprotein cholesterol), complete blood count (CBC), side effects, and 
adverse events. The plasma levels of methemoglobin, nitrite, nitrate, and isoquercetin 
will be monitored.   
Side effects will be recorded via a questionnaire, and adverse events will be 
assessed at 1-week, 6-week, and 12-week follow-up visits. Blood methemoglobin, 
nitrite, nitrate, and isoquercetin, chemistry panels, as well as CBC, will be assessed at 
baseline, 1 week, 6 weeks, and 12 weeks. Plasma nitrite and nitrate will be measured 
using the Griess reaction coupled to high-performance liquid chromatography 
separation with the ENO-20 analyzer (Eicom, Kyoto, Japan). Plasma isoquercetin level 
will be determined by HPLC at Dr. David Nieman’s lab at Appalachian State University, 
North Carolina. Blood methemoglobin and G6PD will be measured at Tulane Medical 
Center Clinical Lab, New Orleans, LA.  


August 30, 2016 
As an added precaution, and due to the immediate absorption rate of the IR-
Sodium Nitrate (30 minutes or less), participants will be asked to remain in the clinic for 
up to 30 minutes following their dose administration at the randomization visit. 
According to the published Investigator’s Brochure from drug manufacturer, TheraVasc, 
decreased SBP and DBP have been noted to be caused by the Sodium Nitrate, as well 
as a compensatory increased pulse rate. Patient’s blood pressure will be taken by the 
automatic machine prior to their leaving clinic. Patients will also be asked if they are 
experiencing any dizziness, fatigue, or headache, due to the vasodilating nature of the 
medication. Should patient’s BP measure ≤100/60mmHg, the patient will be asked to 
remain in clinic until BP remains stable, a nursing or physician assessment will be 
performed prior to the patient’s leaving clinic.  
As an addition to the safety monitoring, inclusion criteria has been carefully 
selected to include patients with a BP of ≥120/70mmHg, in order to minimize the risk of 
hypotension resulting from the study medication. Increased vasoactivity effects of 
sodium nitrite have been noted only with intravenous doses of sodium nitrite, and with 
this method, a mean blood pressure decrease was noted to be 7mmHg. This level is not 
anticipated with the proposed study patients, as the dose will be given orally rather than 
intravenously, however, side effects and measurements will be monitored to ensure 
utmost concern for safety.  
Other serious adverse events noted by the Investigator’s Brochure are not 
relevant for monitoring at the dose prescribed for study patients, as those side effects 
resulting from Methemoglobinemia are associated with much larger doses of IV 
administered sodium nitrite. The minimally effective dose (40 mg BID) of IR-Sodium 
Nitrite has been chosen for use in the study in order to minimize vasoactivity associated 
side effects. Per previous report, there was 0% new hypotension and no apparent 
orthostatic change in pulse or blood pressure throughout the study in the 40 mg BID 
group.4 
 
4.10. Endpoint Definitions and Documentation 
Primary endpoints 
The primary outcome of interest is the net change in endothelium-dependent 
flow-mediated vasodilation (FMD) between the active treatment group and the placebo-
control group over the 3-month intervention period. FMD will be measured using high 
resolution ultrasound on the brachial artery at baseline, 6 weeks, and 12 weeks 
according to a standard method, and images will be analyzed quantitatively at the 
vascular laboratory of the University of Pennsylvania. FMD will be calculated as the 
maximal percentage change in vessel size during hyperemia. 
Secondary endpoints 
The secondary outcomes include endothelial dysfunction biomarkers 
(asymmetrical dimethylarginine [ADMA], intercellular adhesion molecule-1 [ICAM-1], 
vascular adhesion molecule-1 [VCAM-1],  E-selectin, endothlin-1 [ET-1], von Willebrand 
factor [vWF], endothlin-1, and endostatin), inflammatory biomarkers (C-reactive protein 


August 30, 2016 
[CRP], tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], interleukin-6 [IL-6], and 
monocyte chemoattractant protein-1 [MCP-1]), and oxidative stress biomarkers 
(oxidized LDL and nitrotyrosines). All biomarkers will be measured using ELISA assays 
according to a standard protocol.  
Exploratory endpoints 
The estimated-glomerular filtration rate (eGFR) and urinary albumin-to-creatinine 
ratio will be measured and compared between the intervention and control groups. 
4.11. Drug Compliance Monitoring 
Participants will be instructed to return unused study medications and empty 
packaging at each study visit. All returned capsules/tablets will be counted and recorded 
to the corresponding case report forms. Compliance will be calculated as the number of 
capsules/tablets taken divided by the number of capsules/tablets expected. If a 
participant has taken fewer capsules/tablets than expected, the study staff will counsel 
the participant on the importance of compliance. The research pharmacy at the 
investigators’ institute will be responsible for receipt and proper storage of study 
medication, as well as for maintaining records of product delivery to the site, dispensing 
of product to each patient, and return of product at the end of the study. 
4.12. Central Bio-bank and Data Storage 
In addition to the safety laboratory testing, blood samples will be collected during 
the trial and stored in a central facility (Tulane National Primate Research Center).  
These samples will be used after the trial is complete to increase the understanding of 
the study results.  These samples will also be used to research how the study drug is 
processed by the body. It is possible that new tests may become available in the future, 
which could be useful in understanding kidney and heart disease. Stored blood and 
urine may be used later to identify new risk factors for kidney disease. 
The key that connects code numbers to the participants’ identifying information will be 
kept by the study staff.  It will be stored in a locked file within a password-protected 
computer and be accessible for study use only. The study records will also be stored in 
a locked area at the Tulane clinical site.  Only the study staff members will have access 
to the identifying information. 
4.13. Adverse Events (AEs) and Serious Adverse Events (SAEs) 
For the purpose of this trial, an Adverse Event (AE) is defined as any untoward medical 
occurrence, including any clinically significant abnormal sign (e.g., abnormal physical 
exam or laboratory finding), symptom, or disease associated with the use of a drug in 
humans, whether or not considered drug-related. Serious adverse events (SAEs) are 
defined as any undesirable experience associated with the use of a medical product 
that meets any of the following criteria: fatal or life-threatening; resulting in significant or 
persistent disability; requiring or prolonging hospitalization (greater than 24-hours); 
resulting in a congenital anomaly/birth defect; important medical events that 
investigators judge to represent significant hazards or harm to research participants. 
Any AE that meets the criteria outlined as an SAE will be documented and reported as 
such.  


August 30, 2016 
In addition, a select list of important events, regardless of whether they resulted in 
hospitalization, will also be considered SAEs, including: injurious falls, syncope, and 
unexpected events which the investigator believes that the study intervention caused or 
contributed to. Participants will be queried for SAEs and selected AEs at each clinic 
visit.  
Situations that may require temporary reduction or elimination of a study medication 
include side effects such as hypotension, acute kidney injury, methemoglobinemia, and 
other illnesses. All AEs and SAEs are provided to the site clinician for his/her action and 
signature. The study site will be responsible for timely reporting to the Data Safety 
Monitoring Board (DSMB). The study site will provide reports of SAEs for review by the 
DSMB at their meetings. Causality of AEs and SAEs will be evaluated by the PI and site 
physician to determine whether they are probably, possibly, or not related to study 
intervention. Probably or possibly related events will be considered drug-related for 
regulatory reporting purposes, and will be captured as such. 
Expected events: Headaches may be caused by sodium nitrite or isoquercetin. 
Dizziness, lightheadedness, and hypotension may be caused by combination of sodium 
nitrite and isoquercetin. Blood pressure and pulse will be closely monitored, as a 
decrease in these vital signs may cause the aforementioned side effects. Sodium nitrite 
may also cause methemoglobinemia. Participants with history of headaches and with 
systolic blood pressure below 120 mm Hg will be excluded due to safety concerns. 
Clinical safety alerts (including SBP<90, pulse >100 or <45, serum creatinine increase 
by at least 50% to a value >=1.5 mg/dL, or methemoglobin >3%) are to be provided to 
the site clinician for his/her decision of action. Study staff will send safety alerts to the 
participants’ designated physician, noted at the screening visits. The site clinician will 
review all lab results regularly and determine whether results should be sent to the 
participants’ treating physician for further review or treatment. 
The site physician and nurse will track AEs and their progress through the 
resolution, and determine whether post-study follow-up is necessary. SAEs will be 
followed until resolution, stabilization, or until it is determined that study participation is 
not the cause. 
 
4.14. Data Coordination  
All data will be double-entered into a computer database. Subject identifiers and contact 
information will be kept in a separate file and not eligible for data entry. The computer 
database will be protected by a series of passwords available only to study investigators 
and key study personnel. The link between patient identity and a unique study 
identification number will be destroyed after the study is complete. Published reports will 
contain only aggregate and statistical results, with no disclosure of individual results or 
patient identifiers. These safeguards minimize the chances of a breach of confidentiality 
or invasion of privacy. 
We will permit trial-related monitoring, audits, IRB/IEC review, and regulatory 
inspection(s) by providing direct access to source data/documents. 


August 30, 2016 
 
4.15 Quality Control and Quality Assurance 
Personnel training and certification: A comprehensive training session will be conducted 
with all study personnel and encompass all aspects of the study including 
communication, principles of good clinical practice, study implementation and 
procedures, data entry and verification, and specimen testing/handling. All clinical 
technicians have been trained and certified for standard measurements of weight, 
height, and BP in ongoing studies. The ultrasound technologists are also certified in 
their field. Flow-mediated dilation (FMD) results will be read by trained personnel. 
Quality control: All clinical equipment will be checked regularly and standardized by the 
study staff.  Prior to completion of each visit, all questionnaires, forms, and reports of 
laboratory studies will be checked for completion and accuracy.  All lab equipment will 
be checked regularly and standardized by study staff.  
 
4.16 Assessment of Efficacy 
Specification of the efficacy parameters: 
Please see the section “Endpoint Definition and Documentation” of this protocol for 
details. In brief, we will assess the net changes of flow-mediated dilatation (FMD) 
(primary endpoints) between the active treatment group and the placebo-control group 
over the 3-month intervention period. In addition, the secondary outcomes, including 
biomarkers of endothelial dysfunction, inflammation, and oxidative stress, as well as 
exploratory endpoints including eGFR and urinary albumin, will be measured and 
compared between the intervention and control groups. 
Methods and timing for assessing, recording, and analyzing efficacy parameters: 
Flow-mediated dilatation (FMD) will be measured using high resolution ultrasound on 
the brachial artery at baseline, 6 weeks, and 12 weeks according to a standard method 
and images will be analyzed quantitatively at the vascular laboratory of the University of 
Pennsylvania. FMD will be calculated as the maximal percentage change in vessel size 
during hyperemia. All biomarkers will be measured using ELISA assays according to a 
standard protocol using specimens collected at baseline, 6 weeks, and 12 weeks. eGFR 
and urinary albumin will be measured following a standard protocol using specimens 
collected at baseline, 6 weeks, and 12 weeks. 
5.0. DATA ANALYSIS PLAN 
 
5.1. Statistical Analysis 
The primary outcome of interest is the difference in changes of flow-mediated 
dilation between the active intervention and control groups over the 3-month 
intervention period. The changes of FMD during the intervention will be calculated for 
each participant. A mixed-effects model will be used to assess the effects of the active 


August 30, 2016 
treatment on the slope of FMD, in which participants will be assumed to be random 
effects and treatment will be assumed to be an estimable fixed effect. PROC MIXED of 
SAS version 9.2 (SAS Institute Inc, Cary, NC) will be used to obtain point estimates and 
standard errors of the treatment effects and to test for differences between treatment 
arms. An autoregressive correlation matrix will be used to correct within-subject 
correlation for repeated measurements.  
The intention-to-treat (ITT) analysis will be used in all primary analyses. In this 
analysis strategy, all subjects are compared in the treatment groups to which they were 
originally randomized, regardless of any treatment that they subsequently received. In 
addition, the per protocol analysis (PPA) will be used in the secondary analyses. In this 
analysis strategy, subjects who are not fully compliant with the study protocol will be 
excluded. By focusing only on the fully compliant subjects, one can determine the 
maximal efficacy of a treatment. 
The same analysis approaches will be used for the secondary and exploratory 
study outcomes. The level of significance will be 0.05 for a 2-sided test. 
Any deviation(s) from the original statistical plan will be described and justified in 
the updated study protocol and final report. 
 
 
5.2. Sample Size and Power 
The proposed trial has sufficient statistical power to detect a small but clinically 
meaningful effect of combination treatment on FMD. Sample size has been calculated 
based on the following: 
• 80% statistical power; 
• a 2-sided significance level of 0.05; 
• a standard deviation of 4.6%;  
• A detectable difference of 2.8% between intervention and control groups with an 
average of 3 repeated measurements (at baseline, week 6 and week 12); and 
• A correlation between FMD measures over time of 0.55. 
Based on these assumptions, thirty (30) participants are required in each 
treatment arm, for a total of sixty (60) participants. To take into account for withdrawal 
and loss to follow-up, seventy (70) patients will be recruited (35 in each arm of 
treatment). 
 
5.3. Missing and Spurious Data 
All data collection will follow a rigorous quality control process. Spurious and 
missing data will be checked against the original case report forms (CRFs). As a last 
resort, should questionable data not be resolved from the CRFs, such data will be 
coded as missing. When conducting multivariable analyses, multiple imputation for 
missing data will be conducted using the Markov chain Monte Carlo method. 


August 30, 2016 
 
5.4. DSMB Analyses 
Due to the potential risks to study subjects, and the fact that this is a Phase II clinical 
trial, the study will have a formal, and independent Data and Safety Monitoring Board 
(DSMB). A group of experts in Biostatistics, Clinical Nephrology, and Cardiology from 
Tulane University will comprise an internal DSMB. The board will regularly review trial 
progress, including unblinded interim results, and can recommend that the trial 
prematurely end if participants are at risk or if it becomes clear that the trial will not be 
able to yield statistically significant results. Study investigators will prepare accurate and 
timely data tables and reports for the DSMB meetings.  
If methemoglobinemia is shown to be significantly increased in the treatment 
group (p<0.01), the trial will be discontinued. As an extra protective measures, should 
individual patients develop methemoglobinemia, they will be withdrawn from the trial. 
 
 
6.0. HUMAN SUBJECTS CONSIDERATION 
Recruitment and informed consent: Study participants will be identified by research 
personnel employed by the proposed project, based on screening of patients with 
evidence for albuminuric and non-albuminuric chronic kidney disease. Patients who 
meet eligibility criteria to participate in this project are required to undergo an informed 
consent procedure. The consent outlines all of the participation procedures of the study. 
 
6.1. Informed Consent 
Prior to signing the informed consent (ICF), the Research Coordinator will review the 
details of the consent form orally with the potential participant in a private setting, and 
answer any questions that the participant has concerning participation. The original 
signed ICF is stored in the participant study file at the clinical center, and a copy of the 
signed consent form is given to the participant. Specifically, the following must be 
accomplished during the informed consent process: 
• 
The participant must be informed that participation in the study is voluntary 
and that refusal to participate will involve no penalty or loss of benefits or 
negative impact on their medical care. 
• 
The participant must be informed of the purpose of the study and that it 
involves research. 
• 
The participant must be informed of any alternative procedures, if applicable. 
• 
The participant must be informed of any reasonably foreseeable risks. 
• 
The participant must be informed of any benefits from the research. 
• 
An outline of safeguards to protect participant confidentiality must be 
included, as well as an indication of the participant’s right to withdraw without 
penalty. This should be balanced with a discussion of the effect withdrawals 


August 30, 2016 
have on the study, and the responsibility a participant has, within limits, to 
continue in the study if he or she decides to enroll. 
• 
The participant must be informed whom to contact for information about 
research subjects’ rights, information about the research study, and in the event 
of research-related injury. 
• 
The participant must be informed as to whether or not compensation is offered 
for participation in the study and/or in the event of a medical injury. 
• 
The participant must be informed that he/she will be notified of any significant 
changes in the protocol that might affect their willingness to continue in the 
study. 
 
6.2. Risks 
Taking IR-nitrite and isoquercetin may cause one or more side effects including 
headaches, dizziness, hypotension, and methemoglobinemia. In general, the side 
effects are considered transient and reversible.  The use of the minimally effective dose 
of IR-nitrite and Isoquercetin is expected to decrease the risk of side effects in study 
participants. There may be other unknown risks, and for this reason, participants will be 
closely monitored for side effects. The study physician will be informed about any side 
effects potentially related to intervention, and take action accordingly. 
As a result of venipuncture, there is a risk of bruising (the appearance of a black 
and blue mark) or pain at the site. There is also a small risk of infection, 
lightheadedness, and/or fainting. Trained and certified staff members will draw blood to 
minimize complications. Moreover, a physician will be available to evaluate patients as 
needed.  
For patients with diabetes taking insulin, fasting before specified study visits may 
cause hypoglycemia.  This may result in confusion, weakness, or more serious 
conditions and will be treated by providing the patient with high-fructose food or drinks. 
Participants will be advised to hold their diabetes drugs the day before the visit if fasting 
is required.  
There is no major risk reported for the forearm ultrasound test. Temporary 
decrease of blood flow through the artery in the participant’s arm during the forearm 
ultrasound test may cause temporary numbness and tingling in the forearm and hand.  
The risk of numbness involved is slightly greater than the risk during routine blood 
pressure measurements.   
Additional risks of the study relate to unauthorized disclosure of medical 
information or laboratory data. Subject identifiers and contact information will be kept in 
a separate file and not entered into the computer database to prevent this occurrence.  
 
6.3. Benefits 
Potential benefits for the participants on the active combination treatment group may 
exist, as combination therapy may provide improvement of endothelial dysfunction, 
inflammation, and oxidative stress. Progression of CKD to ESRD is the major cause of 


August 30, 2016 
premature death in patients with CKD. The proposed study will test a novel treatment 
targeting the improvement of endothelial dysfunction and inflammation in CKD patients. 
This study will also provide data to test the effect of combination therapy on the delay of 
CKD progression. There may be substantial benefits to medical knowledge, and since 
there is very little risk to individual subjects, the risk to benefit ratio is favorable. 
 
6.4 Subject Withdrawal Criteria and Procedures 
Subject withdrawal from the trial 
Participants will be withdrawn if there are significant changes in safety 
parameters or significant AEs related to treatment with study medications (i.e. an 
imbalance in the safety profile in subjects receiving the active drug vs. placebo), if there 
is any safety issue raised by the DSMB, or if a participant is unwilling to continue to 
participate in the study.  
 
Data to be collected for withdrawn subjects 
Participants who are withdrawn from the study will be required to sign and date 
the withdrawal form. The data collected before withdrawal will be used in the final data 
analyses. 
Replacement of withdrawn subjects 
Subjects withdrawn from the trial will not be replaced using further recruitment 
methods. In order to ensure adequate numbers of participants to complete the study 
and measure results based on acceptable statistical power, we plan to recruit an 
additional 15% of study participants for the trial. 
Follow-up for withdrawn subjects 
Participants will be allowed to contact study staff/site if they have any concerns 
related to the study. 
 
6.4. Payments and Costs 
Participants will be paid $25 for each completed visit. If a participant withdraws or does 
not complete all protocol study visits, payment will be adjusted accordingly. The study 
will not provide insurance for any study related medical or non-medical issues, which 
will be clearly stated in the participant’s ICF. There will be no costs to the subject for 
participating in this research study 
7.0. REFERENCES 
 
 
 


August 30, 2016 
1. 
Ormerod JO, Arif S, Mukadam M, Evans JD, Beadle R, Fernandez BO, Bonser 
RS, Feelisch M, Madhani M, Frenneaux MP. Short-Term Intravenous Sodium 
Nitrite Infusion Improves Cardiac and Pulmonary Hemodynamics in Heart Failure 
Patients. Circ Heart Fail. 2015 Apr 2. pii: CIRCHEARTFAILURE.114.001716. 
[Epub ahead ofprint]. 
2. 
Omar SA, Fok H, Tilgner KD, Nair A, Hunt J, Jiang B, Taylor P, Chowienczyk P,  
Webb AJ. Paradoxical normoxia-dependent selective actions of inorganic nitrite 
in human muscular conduit arteries and related selective actions on central blood 
pressures. Circulation. 2015 ;131(4):381-9. 
3. 
Ingram TE, Fraser AG, Bleasdale RA, Ellins EA, Margulescu AD, Halcox JP, 
James PE. Low-dose sodium nitrite attenuates myocardial ischemia and vascular 
ischemia-reperfusion injury in human models. J Am Coll Cardiol. 2013 Jun 
25;61(25):2534-41. 
4. 
Mohler ER 3rd, Hiatt WR, Gornik HL, Kevil CG, Quyyumi A, Haynes WG, Annex 
BH.  Sodium nitrite in patients with peripheral artery disease and diabetes 
mellitus:  safety, walking distance and endothelial function. Vasc Med. 2014 
Feb;19(1):9-17. 
5. 
Bradley JM, Islam KN, Polhemus DJ, Donnarumma E, Brewster LP, Tao YX, 
Goodchild TT, Lefer DJ. Sustained release nitrite therapy results in myocardial 
protection in a porcine model of metabolic syndrome with peripheral vascular 
disease. Am J Physiol Heart Circ Physiol. 2015 Jul 15;309(2):H305-17. 
6. 
Polhemus DJ, Bradley JM, Islam KN, Brewster LP, Calvert JW, Tao YX, Chang 
CC,  Pipinos II, Goodchild TT, Lefer DJ. Therapeutic potential of sustained-
release sodium nitrite for critical limb ischemia in the setting of metabolic 
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