Combined Dietary Nitrate and Exercise Intervention in Peripheral Artery Disease: Protocol Rationale and Design

Background Peripheral artery disease (PAD) is caused by atherosclerotic occlusions in the legs. It affects approximately 8-12 million people in the United States alone, one-third of whom suffer from intermittent claudication (IC), defined as ischemic leg pain that occurs with walking and improves with rest. Patients with IC suffer a markedly impaired quality of life and a high perception of disability. Improving pain-free walking time is a primary goal of rehabilitation in this population. Objective The nitric oxide (NO)-PAD trial is designed to compare the effects that 12 weeks of supervised exercise training, in combination with a high inorganic nitrate-content (beetroot [BR] juice) beverage or placebo (PL) beverage, has on clinical outcomes of exercise and functional capacity in two groups of PAD+IC patients: exercise training plus beetroot (EX+BR) and exercise training plus placebo (EX+PL). The primary aims of this randomized controlled, double-blind pilot study are to determine group differences following 12 weeks of EX+BR versus EX+PL in the changes for (1) exercise capacity: pain-free walking time (claudication onset time, COT), peak walk time (PWT), and maximal exercise capacity (peak oxygen uptake, VO2peak) during a maximal-graded cardiopulmonary exercise test (max CPX) and (2) functional capacity: 6-minute walk (6MW) distance. The secondary aims will provide mechanistic insights into the exercise outcome measures and will include (1) gastrocnemius muscle oxygenation during exercise via near-infrared spectroscopy (NIRS); (2) gastrocnemius muscle angiogenesis: capillaries per unit area and per muscle fiber, and relative fraction of type I, IIa, IIb, and IId/x fibers; and (3) vascular health/function via brachial artery flow-mediated dilation, lower-limb blood flow via plethysmography, and pulse wave velocity and reflection. Methods A total of 30 subjects between 40 and 80 years of age with PAD who are limited by IC will undergo exercise training 3 days per week for 12 weeks (ie, 36 sessions). They will be randomized to either the EX+BR or EX+PL group where participants will consume a beverage high in inorganic nitrate (4.2 mmol) or a low-nitrate placebo, respectively, 3 hours prior to each training session. Results Data collection from this study has been completed and is in the process of analysis and write-up. While the study is too underpowered—EX+BR, n=11; EX+PL, n=13—to determine between-group differences in the primary outcomes of COT, PWT, and 6MW, preliminary observations are promising with Cohen d effect sizes of medium to large. Conclusions Exercise training is currently the most effective therapy to increase functional capacity in PAD+IC. If the addition of inorganic nitrate to an exercise regimen elicits greater benefits, it may redefine the current standard of care for PAD+IC. Trial Registration ClinicalTrials.gov NCT01684930; https://clinicaltrials.gov/ct2/show/NCT01684930 (Archived by WebCite at http://www.webcitation.org/6raXFyEcP)


DESCRIPTION (provided by applicant):
Peripheral artery disease (PAD) is caused by atherosclerotic occlusions in the legs. It affects 5% of the US population over 50 yrs, one third of which suffer from intermittent claudication (IC), defined as ischemic leg pain that occurs with walking and improves with rest. An intervention that could (A) acutely improve oxygenation to areas of ischemia and (B) chronically increase vessel growth to these ischemic areas would allow for greater exercise tolerance and compliance and facilitate greater improvements in function and quality of life. Plasma nitrite was once considered a biologically inert byproduct and marker of endothelial NO production. Recently, several studies have demonstrated an endocrine role for NO equivalents, including nitrite. These equivalents may be transported in the blood to peripheral tissue beds, where under hypoxic conditions they can be converted to NO and increase blood flow and O2 delivery. This may be pertinent in an arterial occlusive disease (PAD), especially during exercise when tissue ischemia is the limiting factor. The hypothesis of this proposal is that in subjects with PAD and IC, regular consumption of a high nitrate supplement which raises plasma nitrite, in conjunction with 8 weeks of supervised exercise training at the limb ischemic threshold (SET) will produce a greater clinical benefit (increases in COT and PWT) than placebo plus supervised exercise at the limb ischemic threshold (PET). In order to adequately develop, power and execute a larger study, the following specific aims will explore our hypothesis using 24 individuals (12 per group) with PAD and IC: Specific Aim 1a: To determine the segment of PAD patients that can achieve the desired level of plasma nitrite (>500nM at 180min post beverage consumption) at a tolerable volume for an 8 week study. Specific Aim 1b: In these subjects, determine differences in COT, PWT and VO2peak during a graded exercise test, between randomization to 8 weeks of SET or PET. Specific Aim 2: To determine differences in (a) tissue oxygenation (by NIRS) and (b) NO-derived species (plasma and RBC nitrite, nitrate, nitrosothiol), cGMP, oxidative and nitrosative stress (plasma nitrotyrosine and F2-isoprostane) during treadmill testing following SET or PET Specific Aim 3: To determine differences in gastrocenemius muscle (via biopsy) (a) angiogenesis and arteriogenesis (capillary density with surrounding pericytes, proliferating cell nuclear antigen, and apoptosis) and oxidative capacity (fiber type composition, citrate synthase activity), and (b) endothelial function (brachial artery FMD) after SET or PET.

PUBLIC HEALTH RELEVANCE:
Peripheral artery disease (PAD) is a form of cardiovascular disease (CVD) caused by atherosclerotic occlusions in the legs. It affects approximately 5% of the US population over 50 yrs of age, one third of which suffer from intermittent claudication (IC), defined as ischemic leg pain that occurs with walking and improves with rest. We propose by supplementing plasma nitrite via a beverage we can (A) acutely improve oxygenation to areas of ischemia and (B) chronically increase vessel growth to these ischemic areas, would allow for greater acute exercise tolerance, ease the burden of exercise compliance and facilitate greater improvements in function and quality of life Overall Impact: There are currently very few treatment options for patients with intermittent claudication. The applicants propose a novel treatment with a high nitrite diet. They have preliminary data that support this concept, and a strong research team to address this topic in a multifaceted way. This has significant potential public health benefit if functional improvements are noted.

Strengths
• Intermittent claudication is an increasingly common disorder that will continue to increase in prevalence in an aging population. There are currently very few medical therapies for intermittent claudication. Given the inevitable increase in prevalence, an effective medical therapy would be a significant advancement. • If successful, this small trial would have a very good chance of leading to a broader trial to more thoroughly address the effects of a high nitrite diet on claudication.

Weaknesses
• Other dietary factors have been addressed in the past, such as folate and homocysteine levels, with respect to their effect on PAD. While they have been shown to correlate with PAD disease severity, dietary supplements have not been shown to influence the course of the disease. Thus, it is difficult to say whether additional attempts at dietary modification will substantially change the course of disease.

Strengths
• Investigator is young but with extensive background in this field of research • Co-investigators and research team provide the expertise needed to perform the research. The skills of the various investigators seem to complement each other well.

Weaknesses
• No specific weaknesses. Seems to be a very well-rounded team.

Strengths
• The proposed pharmacologic intervention utilizing beet juice extract is a novel idea in the treatment of peripheral vascular disease. The use of a low nitrite, identical tasting placebo was an essential component that the investigators seem to have very successfully addressed. • Peripheral vascular disease is a multi-factorial disorder with a number of factors influencing its course, including smoking, diabetes, hyperlipidemia, etc. There is evidence that the symptoms are not solely related to oxygen delivery, and the relationship between tissue oxygenation and physical performance is not linear, so it is difficult to know whether influencing one factor with the proposed intervention will result in measureable differences, especially in such a small group over such a short time period.

Strengths
• The project is in its early stages so the scope of the proposal is relatively small scale. It is set up to answer a number of questions about intermittent claudication including clinical outcomes (walking distance) and biochemical parameters. I think there is a nice combination of clinical and physiologic questions.

Weaknesses
• Most claudication studies are carried out for at least three months, and the investigators have previously done three month studies. The rationale for making this a two month study is not explained. It is questionable whether a two month study with such a small patient cohort (12 patients each group) will be adequate to see a difference in the test parameters.
• Muscle biopsy is an invasive procedure in patients with limb ischemia. While the authors have extensive experience with this, I question how accepting patients will be of this procedure. Perhaps a little more description of how this is done would be helpful. For example, many patients with intermittent claudication are taking antiplatelet agents such as aspirin and clopidogrel. How will medications be managed around the time of the biopsy?
• It is not specifically stated how patients will be recruited except to say that they will be recruited from Durham NC and the surrounding communities. More detail on how patients will be recruited would be helpful.

Strengths
• Scientific work environment appears strong with three separate facilities (Duke, Wake Forest, Virginia) involved utilizing different areas of expertise at the three facilities. There seems to be a good collaborative environment shared by the groups involved.
• Institutional support, equipment and physical resources appear adequate for the proposed research.

Protections for Human Subjects:
• Protection of human subjects adequately addressed. There appears to be minimal potential harm to recruited patients. One potential area of harm is the muscle biopsy, although the investigators report extensive experience with this procedure.

Inclusion of Women, Minorities and Children:
• Inclusion of women and minorities is addressed. It is stated that the targeted enrollment is 50% women and 50% minorities, but the specifics of how this targeted enrollment is to be achieved are not addressed Overall Impact: This pilot study will test an oral high nitrate supplement that raises plasma nitrite in addition to 8 weeks of supervised exercise training in patients with claudication. The hypothesis is that the plasma nitrite may be transported to peripheral tissue and converted to nitric oxide under hypoxic 1 R21 HL111972-01 6 CICS ALLEN, J conditions. Exercise training in PAD induces ischemia repeatedly during each session, thus underscoring the potential of such an agent in this disease state. Thus those consuming the nitrate supplement could have greater improvement in pain claudication onset time and peak walking time than those undergoing exercise training alone. They also intend to evaluate the changes in tissue oxygenation during exercise between groups via NIRS and changes in gastrocnemius muscle tissue obtained through skeletal muscle biopsy. The protocol is very well designed and presented. The investigative team has the collective expertise to successfully complete the study and if effective, could add an additional treatment option for patients with claudication, for whom therapeutic options are limited.

Strengths
• Oral agents that are effective in claudication have been very elusive despite considerable effort to develop such agents. Should this supplement prove to be effective, it would be very significant for patients with claudication.

Weaknesses Weaknesses
• Minimal

Strengths
• This is a strong investigative team with the collective expertise recruit the subjects, perform the measurements proposed, deliver the interventions, and analyze the data.

Weaknesses
• None identified

Strengths
• The mechanism of this supplement triggered by the ischemic state seen with exertion in PAD is novel • Examining changes in tissue oxygenation in the dynamic state while exercising as well as changes in skeletal muscle tissue in response to the intervention is also novel.

Weaknesses
• Treadmill exercise training is not novel, but appropriate for this protocol.

Strengths
• This study is well designed and described in the application.  Overall Impact: Peripheral artery disease (PAD) is common. Other than supervised exercise, there is a paucity of useful treatments for intermittent claudication. The present study, supported by animal data and by preliminary human data, suggests that dietary nitrates may improve exercise performance in PAD, by raising blood nitrite levels which may serve as a source to nitric oxide at sites of ischemia.

Recommend as Requested
The applicants wish to conduct a study to investigate dietary nitrates for the treatment PAD/intermittent claudication. In a pilot placebo controlled parallel design study, they will look for changes in exercise performance, nitric oxide bioavailability and for evidence of enhanced angiogenesis in skeletal muscle biopsies.
This study is innovative and the pilot data generated by this R21 grant would serve to prepare a more definitive clinical trial. The investigators are expert in the proposed measurements and in a good position to complete the proposed aims. The study is significant as new treatments for intermittent claudication are much needed.

Strengths
• Careful attention is paid to most experimental details. Collaborations are in place, including the analysis of muscle biopsies, to accomplish the aims.

Weaknesses
• Why assume that improvements in exercise with nitrate/nitrite are due to more efficient distribution of blood flow and not due to altered skeletal muscle and/or mitochondrial bioenergetics related to increases in nitric oxide (e.g. Journal of Cell Science 119, 2855-2862)? This single focus on local blood flow to the exclusion of skeletal muscle and mitochondrial function is one potential weakness in this grant.
• Given the variability of stress testing in PAD, is this rather small study adequate to inform about sample sizes for a larger study?
• The use of plethysmography to measure lower extremity blood flow is noted in the grant application. Will plethysmographic blood flow be assessed during reactive hyperemia (it is not stated)? The peak and particularly the duration of reactive hyperemia reflect NO bioavailability (e.g. Am J Physiol 1996; 270:H1435). This ischemic stimulus seems particularly relevant as it would create the conditions (i.e. ischemia) that facilitate conversion of nitrite to NO.

Protections for Human Subjects:
Acceptable Risks and/or Adequate Protections

Inclusion of Women, Minorities and Children:
G1A -Both Genders, Acceptable M1A -Minority and Non-minority, Acceptable

Not Applicable (No Vertebrate Animals)
Biohazards:

Budget and Period of Support:
Recommended budget modifications or possible overlap identified: • Dr. Gary Miller's request for 3.0 month effort is not well justified. 1.0 month seems more appropriate to the stated contribution. + Derived from the range of percentile values calculated for the study section that reviewed this application.

THE FOLLOWING RESUME SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW OFFICER TO SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE ON
NIH has modified its policy regarding the receipt of resubmissions (amended applications). See Guide Notice NOT-OD-10-080 at http://grants.nih.gov/grants/guide/notice-files/NOT-OD-10-080.html. The impact/priority score is calculated after discussion of an application by averaging the overall scores (1-9) given by all voting reviewers on the committee and multiplying by 10. The 1 R21 HL111972-01 10 CICS ALLEN, J criterion scores are submitted prior to the meeting by the individual reviewers assigned to an application, and are not discussed specifically at the review meeting or calculated into the overall impact score. For details on the review process, see http://grants.nih.gov/grants/peer_review_process.htm#scoring.