Coronary Flow Reserve to Assess Cardiovascular Inflammation (CIRT-CFR)
Phase: NA
Conditions: Coronary Heart Disease, Metabolic Syndrome, Diabetes Mellitus
Sponsor: Brigham and Women's Hospital

 
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Coronary Flow Reserve to Assess Cardiovascular Inflammation (CIRT-CFR) 
 
A multi-center, double-blind, placebo-controlled, randomized ancillary imaging trial  
of the Cardiovascular Inflammation Reduction Trial  
 
 
ClinicalTrials.gov Identifier: NCT02786134  
 
 
Investigators: 
Viviany R. Taqueti, MD, MPH, Brendan M. Everett, MD, MPH, Scott D. Solomon, MD, Paul 
M. Ridker, MD, MPH, Rob S. Beanlands, MD, Fadi Hage, MD, Marcelo F. Di Carli, MD 
 
 
 
 
Institution: 
Brigham and Women’s Hospital (BWH) 
Boston, MA 
 
 
 
Sponsor: 
National Heart, Lung and Blood Institute,  
National Institutes of Health,  
Bethesda, MD 
 
 
 
24 May 2013  
Revised 24 September 2013 
Revised 28 February 2016 
Revised 4 March 2016 
 
 
 
 
 
Revised 9 March 2016 
Revised 22 March 2016 
Revised May 19, 2016 
Revised June 28, 2016 
Revised October 25, 2017 
Revised 4/5/2018 

 
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I. BACKGROUND AND SIGNIFICANCE 
 
Diabetes mellitus (DM) and prediabetic states are enormous public health burdens in the US and 
worldwide. Recent statistics approximate that 25.8 million children and adults in the US—8.3% 
of the population have DM1. Alarmingly, an additional 79 million people are estimated to have 
prediabetes, and this is likely to increase with the rising prevalence of obesity in the population1. 
Meta-analyses2,3 of large prospective studies suggest that, after accounting for other risk factors, 
DM more than doubles the risk of coronary artery disease (CAD) and ischemic stroke, and that 
this DM-related risk is higher in women than in men. In contrast with the strong associations 
observed between type 2 DM and related cardiometabolic disorders and cardiovascular outcomes, 
a recent meta-analysis of 102 prospective studies found a very modest association of fasting 
glucose level with CAD and stroke risk3. Moreover, large intervention trials4-7 have shown no 
beneficial effect of intensive glucose control on primary cardiovascular endpoints. While glucose 
level itself may play some role in modulating cardiovascular risk, the mechanisms mediating 
increased risk of cardiovascular mortality and morbidity in diabetics are not well understood. 
Although a greater extent of CAD8, as well as abnormalities in cardiac structure and function9 have 
been implicated in diabetics, these pathophysiologic features provide only a partial explanation to 
the excess risk of MI, heart failure and death among diabetics and patients with metabolic 
syndrome. Indeed, the extent of luminal angiographic CAD with or without left ventricular (LV) 
dysfunction does not fully account for the excess clinical risk among subjects with10 or without11 
DM. In our work, the presence of impaired coronary flow reserve (CFR) in patients without overt 
obstructive CAD identifies patients at increased risk of adverse cardiac events, and diabetic 
patients without known CAD with impaired CFR show a risk of cardiac death comparable to, and 
possibly higher than, that for nondiabetic patients with known CAD12. This suggests that diffuse 
atherosclerosis and coronary microvascular dysfunction play a more important role than previously 
appreciated in the pathophysiologic abnormalities leading to increased risk.  
Interestingly, this excess risk may be linked to an enhanced pro-inflammatory state, 
consistently demonstrated in patients with type 2 DM and metabolic syndrome13, and following 
acute MI14. Exactly how the enhanced pro-inflammatory response in DM and metabolic syndrome 
is associated with increased risk of adverse outcomes is not well understood. There is ample 
laboratory evidence that inflammation plays a major role in all stages of atherothrombosis15,16 
including the early functional abnormalities in vascular endothelial and smooth muscle cell 
function. There is also growing in vivo human evidence that increased systemic inflammation is 
associated with coronary vascular dysfunction17-22. Moreover, there is preliminary evidence that 
anti-inflammatory therapies may lead to improved coronary vasoreactivity23-25.  
To date, however, no trial has yet proven that directly reducing inflammation lowers 
cardiovascular event rates, and the Cardiovascular Inflammation Reduction Trial (CIRT)26 has 
been launched to address this issue. A possible mechanism for doing so may involve systemic 
improvement in global coronary vascular function. As such, CIRT provides a unique opportunity 
to assess whether CFR might provide a simple noninvasive method to predict which patients best 
respond to a systemic anti-inflammatory intervention. By testing the fundamental concept of 
whether reducing systemic inflammation can lead to improved myocardial blood flow, tissue 
perfusion and LV function, CIRT-CFR, our proposed randomized placebo-controlled ancillary 
clinical trial, would provide important mechanistic insights of the capabilities of an anti-
inflammatory therapy to improve key clinical determinants of clinical risk. Equally important for 

 
3 
 
continued research development programs and eventual future clinical decision-making is the 
validation of a physiologic imaging biomarker that could also be used to evaluate safety and 
efficacy of novel therapeutic agents during drug development programs, and/or identify patients 
at particularly high risk in need of aggressive therapeutic intervention27. 
 
The Cardiovascular Inflammation Reduction Trial (CIRT) is an NHLBI funded multicenter 
clinical trial (U01 HL 101422) that will randomly allocate 7,000 patients with stable CAD and 
either type 2 DM or metabolic syndrome to low-dose methotrexate (LDM; target dose 15 to 20 mg 
per week) plus usual care, or placebo plus usual care over a follow-up period of 2 to 4 years 
(average 3 years)26. CIRT is an event-driven trial such that in the absence of extreme effects, the 
trial will conclude after accrual of at least 530 primary endpoints, estimated to provide 90% power 
to detect a 25% relative risk reduction. The CIRT primary endpoint is a composite of nonfatal 
myocardial infarction (MI), stroke, and cardiovascular death. Secondary endpoints include total 
mortality, coronary revascularization, unstable angina requiring unplanned revascularization, and 
hospitalization for heart failure, with additional pre-specified endpoints such as incident venous 
thrombosis, atrial fibrillation, peripheral artery disease, and severe aortic stenosis. In addition, 
effects on measures of quality of life will be assessed using the standardized SF-36 Health Survey. 
Participants will be recruited from roughly 400 clinical sites in the United States and Canada.  
In addition to LDM or matching placebo, all study participants (including those on placebo) 
will receive folic acid 1.0 mg 6 days per week, a therapy known to reduce side effects that can be 
associated with LDM. To further ensure safety and improve long-term compliance, the trial design 
incorporates a 5- to 6-week open-label, active-therapy run-in (maximum 8 weeks) for all potentially 
eligible participants so that those unable to initially tolerate LDM are excluded prior to 
randomization. Additional details of the CIRT parent trial are included in the appended CIRT IRB 
application, approved on 6/13/2012 (Appendix). 
  
The proposed ancillary imaging trial, CIRT-CFR, is a randomized placebo-controlled clinical trial 
that will test directly whether reducing systemic inflammation in stable CAD patients improves 
myocardial perfusion reserve, as measured by CFR, a novel quantitative imaging marker of clinical 
risk. Coronary flow reserve (CFR) refers to the ratio of global myocardial blood flow quantified 
at stress over that at rest. CFR provides a robust measure of the integrated hemodynamic effects 
of epicardial CAD, diffuse atherosclerosis, vessel remodeling, and microvascular dysfunction on 
myocardial perfusion. CFR quantified using vasodilator PET can identify patients at high risk for 
serious cardiac adverse events, including cardiac death, even in the absence of overt obstructive 
disease (i.e. independently of perfusion score)28-31, and these associations are especially evident 
among high, but heterogeneous risk cohorts including patients with DM12. The demonstration of 
improved CFR and myocardial perfusion after low-dose methotrexate would provide further 
validation of this imaging biomarker as a potential indicator of the complex sequelae of active 
inflammation in the heart. Doing so may provide a direct, quantifiable target that could be useful 
in drug discovery programs, and perhaps for initiation and monitoring of therapeutic efficacy. 
Specifically, this trial aims to investigate a mechanism by which LDM may modulate 
cardiovascular outcomes in a high-risk population enriched for chronic, low-level inflammation. 
Increasing data suggest that LDM, a generic therapy that is widely used among patients with 
rheumatoid arthritis and psoriasis, mediates its beneficial effects not by direct inhibition of cellular 
proliferation, but by promoting release of the robust anti-inflammatory autacoid, adenosine, at 
sites of inflammation32. Increased extracellular concentrations of adenosine modulate recruitment 

 
4 
 
and function of leukocytes, including neutrophils, macrophages and regulatory T lymphocytes, 
leading to local and systemic reduction of inflammatory cytokines such as IL-12, IL-6 and TNF, 
with far-reaching effects that include facilitated cholesterol efflux and reverse cholesterol transport 
from arterial wall foam cells33. Moreover, adenosine mediates these effects while acting as a potent 
vasodilator. As such, treatment with LDM is poised to affect coronary vasoreactivity in diabetics 
and prediabetics in complex ways that are ripe for study using PET measures of CFR. 
Another fundamental physiologic principle of our study is that, by decreasing inflammation 
and enhancing myocardial tissue perfusion to the diabetic or prediabetic heart, we may improve 
cardiac mechanics and, in so doing, prevent or slow the progression to clinical heart failure and 
possibly death. Although some of these pathophysiologic mechanisms have been explored in 
experimental animal models34, the gaps between these experiments in animals and applications in 
humans are just beginning to be addressed35. 
 
Thus, we believe that the use of physiologic imaging techniques, integrated into the 
continuum of clinical care to understand the mechanisms linking inflammation to cardiovascular 
complications, is not only innovative, but will also facilitate needed clinical translation. 
 
 
II. SPECIFIC AIMS   
 
Coronary flow reserve (CFR, calculated as the ratio of hyperemic over rest myocardial blood flow) 
is emerging as a powerful quantitative prognostic imaging marker of clinical cardiovascular risk. 
CFR provides a robust and reproducible clinical measure of the integrated hemodynamic effects 
of epicardial CAD, diffuse atherosclerosis, and microvascular dysfunction on myocardial tissue 
perfusion. Inflammation is a key mediator of this constellation of abnormalities, affecting the entire 
coronary vasculature, but no clinical trial to date has shown that directly reducing inflammation 
lowers cardiovascular event rates. As such, the recently launched Cardiovascular Inflammation 
Reduction Trial (CIRT) provides a unique opportunity for mechanistic investigation of the impact 
of anti-inflammatory therapy on changes in CFR as a reflection of coronary vascular dysfunction, 
which may precede clinical outcomes, particularly in patients at high-risk of events. We are ideally 
positioned to examine the impact of inflammation on CFR, having extensive experience in both 
the quantitation of CFR using clinically-integrated dynamic positron emission tomography (PET) 
and the ability to assess its association with cardiovascular outcomes. 
 
The central hypothesis of this ancillary proposal, CIRT-CFR, is that reducing systemic 
inflammation using low-dose methotrexate (LDM) will, compared to placebo, quantitatively 
improve myocardial blood flow and coronary flow reserve as measured by PET over 1-12 months, 
in stable CAD patients with type 2 diabetes or metabolic syndrome enrolled in CIRT. In so doing, 
improvement in coronary vasoreactivity, endothelial function, and tissue perfusion may have 
beneficial effects on myocardial mechanics, left ventricular deformation and function and, 
ultimately, symptoms and prognosis. 
 
AIM 1. To test the hypothesis that systemic inflammation is a critical determinant of coronary 
flow reserve and myocardial tissue perfusion, as assessed by quantitative PET imaging, in stable 
CAD patients with type 2 diabetes or metabolic syndrome. Specifically, we propose that treatment 
with LDM at a target dose of 15 to 20 mg orally weekly for 1-12 months will, compared to placebo, 
result in: 

 
5 
 
Hypothesis #1: Improved global coronary flow reserve in response to dipyridamole, reflecting 
primarily endothelium-independent vasodilation. 
Hypothesis #2: Reduced total stress perfusion deficit, a semi-quantitative score that combines scar 
and ischemia. 
 
AIM 2. To test the hypothesis that LDM at a target dose of 15 to 20 mg orally weekly for 1-12 
months will, compared to placebo, result in improved left ventricular (LV) myocardial mechanics, 
as assessed by transthoracic echocardiography, and that this functional improvement will be 
correlated with the change in coronary flow reserve after 1-12 months of treatment. In this 
population of patients without severe heart failure, we propose to test the following hypotheses:  
Hypothesis #3: LDM for 1-12 months will result in improved LV systolic function, as assessed by 
measures of LV peak global longitudinal strain, and diastolic function, as assessed by measures of 
tissue Doppler mitral annular early diastolic relaxation velocity (E’). 
Hypothesis #4: Changes in LV systolic and diastolic function after 1-12 months of LDM therapy 
will be correlated with changes in coronary flow reserve.  
 
 
III. SUBJECT SELECTION 
 
Inclusion and exclusion criteria were selected to reflect those of the parent CIRT study. 
Specifically:  
 
Inclusion criteria: 
a. Age ≥18 years at screening; 
b. Documented past history of MI OR past evidence of multivessel CAD by angiography, 
completed any planned coronary revascularization associated with a qualifying event at 
least 60 days prior to enrollment, and clinically stable for ≥60 days prior to enrollment; 
qualifying prior MI must be documented either by hospital records, evidence on current 
ECG of Q waves in 2 contiguous leads, and/or an imaging test demonstrating wall motion 
abnormality or scar; qualifying evidence of multivessel CAD by angiography must be 
documented by CAD in at least two major epicardial vessels defined either as the 
presence of a stent, a coronary artery bypass graft, or an angiographic lesion of 60% or 
greater (left main CAD that has been revascularized with a stent or bypass graft will 
qualify as multivessel disease, as will the presence of a 50% or greater isolated left main 
stenosis); 
c. History of type 2 DM or metabolic syndrome (meeting 2004 AHA/NHLBI definition*) at 
time of study enrollment; *includes any 3 of the following 5 diagnostic criteria: waist 
circumference ≥ 102 cm in men or 88 cm in women; triglycerides ≥ 150 mg/dl or on drug 
treatment for elevated triglycerides; high-density lipoprotein cholesterol (HDL-C)< 40 
mg/dL in men or < 50 mg/dL in women or on drug treatment for reduced HDL-C; 
systolic blood pressure ≥ 130 mm Hg or diastolic blood pressure ≥ 85 mm Hg or on drug 
treatment for hypertension; and elevated fasting glucose ≥ 100 mg/dL or on drug 
treatment for elevated glucose.  
d. Willingness to participate as evidenced by signing the CIRT and CIRT-CFR informed 
consent. 
  

 
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Exclusion criteria: 
a. Prior history of chronic infectious disease, tuberculosis, or severe fungal disease; chronic 
hepatitis B or C infection; renal insufficiency; interstitial pneumonitis, bronchiectasis, or 
pulmonary fibrosis; known chronic pericardial effusion, pleural effusion, or ascites; 
chronic liver disease; myeloproliferative disorders in the past 5 years; non-basal cell 
malignancy or treated lymphoproliferative disease within the past 5 years; known HIV 
positive; life expectancy of <3 years;  
b. Chronic inflammatory condition such as lupus or rheumatoid arthritis, ulcerative colitis or 
Crohn’s disease  
c. White blood cell count <3,500/ul, hematocrit < 32 percent, or platelet count < 75,000/ul  
d. Liver transaminase levels (AST or ALT) >upper limit of normal (ULN) or albumin < the 
lower limit of normal (LLN);  
e. Creatinine clearance < 40 ml/min as estimated with the Cockroft-Gault equation;  
f. History of alcohol abuse or unwillingness to limit alcohol consumption to less than 4 
drinks per week  
g. Women of child bearing potential, even if they are currently using contraception, and 
women intending to breastfeed.  
h. Men who plan to father children during the study period or who are unwilling to use 
effective forms of contraception.  
i. Requirement for use of drugs that alter folate metabolism 
(trimethoprim/sulfamethoxazol) or reduce tubular excretion (probenecid) or known 
allergies to antibiotics making avoidance of trimethoprim impossible;  
j. Current indication for methotrexate therapy;  
k. Chronic use of oral steroid therapy or other immunosuppressive or biologic response 
modifiers (see Exclusionary Medication List in Manual of Operations). Eligible study 
participants will be encouraged to have up to date pneumococcal and influenza 
vaccinations as recommended based on their age and underlying medical conditions.  
l. Chest X-ray evidence in the past 12 months of interstitial pneumonitis, bronchiectasis, or 
pulmonary fibrosis. For participants who do not have a chest X-ray in the prior 12 
months, a chest X-ray will be obtained at baseline as part of the study protocol.  
m. New York Heart Association Class IV congestive heart failure.  
 
 
IV. SUBJECT ENROLLMENT 
 
We propose to recruit 150 patients for CIRT-CFR. This will be a collaborative effort between 6  
clinical sites, with 4 sites in the Boston area: (1) Brigham and Women’s Hospital, BWH (PI: 
Samuel Goldhaber, MD), (2) Joslin Diabetes Center (PI: Allison Goldfine, MD), (3) 
Massachusetts General Hospital (PIs: Linda C. Hemphill, MD, and Laura Benzaquen, MD), (4) 
North Shore Medical Center (PI: Lawrence S. Block, MD), and two additional sites in the 
Ottawa area: (5) Ottawa Heart Institute, OHI (PI: Rob Beanlands, MD), and in Birmingham, 
Alabama: (6) University of Alabama, UAB (PI: Fadi Hage, MD). We will have 3 imaging sites 
(BWH, PI: Marcelo Di Carli, MD; OHI, PI: Robert Beanlands, MD; and UAB, PI: Fadi Hage, 
MD), with BWH serving as the Core Imaging Lab, and all 4 Boston area sites referring 
patients for imaging to BWH. The subjects of the trial will be patients with CAD and type 2 DM 
or metabolic syndrome enrolled in the parent study (CIRT) and recruited from participating 

 
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CIRT sites local to BWH, OHI, and UAB. All patients identified at the 6 participating clinical 
sites as being potentially eligible for CIRT are automatically eligible for participation in this 
ancillary study.  
 
 
V. STUDY PROCEDURES 
 
Randomization and double-blind study treatment period to either placebo or LDM (1:1) of willing 
and eligible patients will occur at the end of the open label run-in phase per the parent CIRT 
protocol, and will be stratified by time since the qualifying event (< 6 or ≥ 6 months from the date 
of MI or most recent angiogram), type of event (MI or multivessel CAD), presence of either type 
2 DM or metabolic syndrome, and site, which will ensure balance in the proposed study. Patients 
willing to participate in CIRT will be asked to enroll into the sub-study and may sign the CIRT-
CFR informed consent at any point between signing the parent CIRT informed consent and 
completing the parent CIRT randomization visit (Visit 4). After giving informed consent for the 
ancillary CIRT-CFR, patients will undergo the baseline PET scan along with echocardiography at 
any point between the parent CIRT post run-in visit (Visit 3) and up to 4 weeks after randomization 
(Visit 4). Per the parent CIRT protocol: i) blood will be collected and stored for biomarkers at 
baseline (Visit 3), ii) patients will complete quality of life (SF-36) questionnaires at enrollment 
(Visit 2) and 1-12 months (Visit 7) after randomization, and iii) patients will be called at 3, 6, and 
10 months after randomization to assure compliance. Final PET scan and echocardiogram will 
occur at approximately 1-12 months after randomization or within 4 weeks (+/- 2 weeks) of Visit 
7. At that time, blood will be collected and stored for biomarkers (using the same protocol as in 
the parent CIRT) to coincide with follow-up imaging in CIRT-CFR. The data collection schedule 
for CIRT and CIRT-CFR is summarized in the Appendix. Trial schema is as follows: 
 
 
 
Imaging will be performed at the 3 imaging centers (BWH, OHI, and UAB). To minimize 
participant and site burden, only a baseline and single follow-up imaging time point will be 
pursued. The CIRT-CFR Screening Visit has been incorporated into the Consent Visit in an 
attempt to further reduce participant and site burden. Imaging tests (PET and echo) will be 
scheduled on the same day for patient convenience if possible, and no more than one week apart. 
“Baseline” study visit imaging will follow the open label run-in period of the parent trial to 
enhance long-term compliance and eliminate risk of radiation exposure for any individuals with 
immediate intolerance to the LDM study protocol. The imaging tests proposed are non-invasive, 
routinely performed, and historically well tolerated by patients. The radiation incurred through 
PET scanning is small (~2.8 mSv per study), and comparable to that incurred by an average 
American in a given year from background radiation. Clinical coordinators will be encouraged to 
enroll all CIRT-eligible patients at the 5 clinical sites participating in this ancillary study, so as to 
remove the possibility of selection bias. To minimize and understand potential selection bias, we 
will maintain an evaluation log of all patients who were screened and enrolled at the 6 sites. 

 
8 
 
 
Timing of baseline imaging assessments. Baseline PET scanning and echocardiography will 
follow CIRT’s post run-in (open label, 6-8 week run-in phase) visit. The justification for this 
decision is based on the pharmacodynamics of LDM in rheumatoid arthritis, as well as additional 
biologic and trial logistic reasons. There are no data on the timing of changes in myocardial blood 
flow following LDM. Methotrexate is a prodrug that becomes active only when polyglutamated 
within cells, a slow iterative process that can take as long as ~27 weeks to reach steady state36. In 
patients with rheumatoid arthritis, a clinical response to methotrexate usually begins within the 
first 6–12 weeks after initiation of therapy, with a maximal response observed at 6 months37,38. 
This suggests that the longer-chain polyglutamates, which take longer to become detectable and 
reach steady state, have a more important role in the clinical response to methotrexate36. 
 
Timing of follow-up imaging assessments.  Follow-up PET scanning and echocardiography will 
occur between 1 and 12 months post randomization.   
 
Positron Emission Tomography (PET): Regional and global myocardial blood flow (MBF) will 
be assessed using PET imaging in accordance with the study-specific acquisition protocol. The 
investigative teams at BWH, OHI, and UAB have extensive experience in performing these 
procedures, and quantifying myocardial blood flow. PET scans will be performed using a whole 
body PET/CT scanner. Anti-hypertensives and beta-blockers will be withheld on the morning of 
the study. Patients will be allowed to continue using sublingual nitroglycerin as needed. Studies 
will be performed after 4 hours of fasting and 24 hours of abstinence from caffeine. The visit will 
take approximately 5 hours, including patient preparation. MBF will be measured at rest and 
during dipyridamole, adenosine, or regadenoson infusion, using 13N-ammonia or 82Rubidium, as 
the flow tracer. Importantly, the same radiopharmaceutical should always be used for the baseline 
and follow-up scans. After transmission imaging and beginning with the intravenous (IV) bolus 
administration of 13N-ammonia (~10-20 mCi, adjusted based on whether 2D- or 3D-imaging is 
used) or 82Rubidium (~10-20 mCi for 3D imaging), list mode images will be acquired for 20 or 7 
minutes, respectively. Thirty (13N-ammonia) or 10 (82Rubidium) minutes later, patients will 
undergo a standard infusion of dipyridamole (0.56 mg/kg over 4 minutes), adenosine (0.14 
mg/kg/min for 4 minutes), or regadenoson (0.4 mg bolus over 10 seconds). At peak hyperemia, a 
second dose of 13N-ammonia (~10-20 mCi, adjusted based on whether 2D- or 3D-imaging is used) 
or 82Rubidium (~10-20 mCi for 3D-, and 30-50 mCi for 2D imaging) will be injected IV, and 
images recorded in the same manner. The heart rate, blood pressure, and 12-lead ECG will be 
recorded at baseline and throughout the infusion of dipyridamole, and at recovery.  All studies will 
be reviewed at the sites by site investigators for clinically important findings. All PET scans will 
be done for research (non-clinical) purposes only. For safety reasons, all studies will be reviewed 
at the sites by site investigators for clinically important findings. No reports or analyses will be 
provided to sites from the PET core laboratory and studies will not be assessed in real-time.   
 
Two-dimensional echocardiography: Echocardiograms will be performed by site sonographers 
trained and certified by the echo core laboratory in the study-specific acquisition protocol39. The 
noninvasive core laboratory at BWH has extensive experience in quantitative echocardiography in 
the context of therapeutic clinical trials40,41. Dr. Solomon also directs the core laboratory in the 
analysis of echocardiographic images in the NIH-funded ARIC study42.  Images will be acquired 
as recommended by the American Society of Echocardiography (ASE) and in a manner consistent 

 
9 
 
with standard practices for patient comfort and position. In addition, the echo protocol includes 
specific views to ensure optimal imaging for specific views to: (1) assess LV diastolic function, 
and (2) ensure optimal imaging for off-line LV deformation analysis with speckle-tracking 
software (i.e., images of the LV will be captured at high frame rates of 50-70 fps). The protocol 
will take approximately 35 minutes, including patient preparation. A superset of a standard echo 
exam will be performed, emphasizing endocardial border optimization and on-axis imaging.  All 
studies will be acquired and stored in digital DICOM format, with CD/DVD copy for site backup.  
All studies will be reviewed at the sites by site investigators for clinically important findings. All 
echos will be done for research (non-clinical) purposes only. No reports or analyses will be 
provided to the sites from the echo core laboratory and studies will not be assessed in real-time.   
 
 
VI. BIOSTATISTICAL ANALYSIS 
 
CIRT-CFR sites participating in the proposed ancillary study. Based on our power analysis and 
sample size calculations, we estimate that each of the 6 clinical sites will be required to enroll 
approximately 25 patients over approximately 3 years. This is equivalent to 1 patient every 6 weeks 
per site, which we believe is reasonable and attainable. All sites have received IRB approval for 
the parent study and are actively enrolling patients in the parent CIRT trial. The proposed imaging 
studies (PET scans and echos) are routinely performed at the 3 imaging sites (BWH, OHI, and 
UAB). Drs. Di Carli (PI) and Solomon (both at BWH), Beanlands (OHI), and Hage (UAB) are 
recognized experts in quantitative PET imaging and echocardiography, have published widely on 
these topics, and have extensive experience in conducting clinical trials using imaging endpoints40, 
43, 44-48. 
 
Aim 1. To test the hypothesis that systemic inflammation is a critical determinant of coronary 
flow reserve (CFR) and myocardial tissue perfusion, as assessed by quantitative PET imaging, 
in stable CAD patients with type 2 DM or metabolic syndrome. Specifically, we propose that 
treatment with LDM at a target dose of 15 to 20 mg orally weekly for 1-12 months will, 
compared to placebo, result in: 
Hypothesis #1: Improved global CFR in response to dipyridamole, reflecting primarily 
endotheliumindependent vasodilation. 
Hypothesis #2: Reduced total stress perfusion deficit, a semi-quantitative score combining scar 
and ischemia. 
 
Methods: PET scans will be performed at the 3 imaging sites at randomization and at 1-12-
month follow-up using a standardized study protocol. As described in section V, we have 
previously demonstrated feasibility of measuring myocardial blood flow with high 
reproducibility49,50. In addition to CFR and coronary hemodynamics, we will also measure the 
total perfusion deficit (TPD) during stress and at rest, and calculate the difference TPD between 
stress and rest (reflecting total ischemic burden) using previously validated methods51,52. The 
primary endpoint for the efficacy portion of Aim 1 is the change in global CFR response to 
dipyridamole, reflecting primarily endothelium-independent coronary vasodilatation, over 1-12 
months. A key secondary endpoint is the change in dipyridamole-stimulated TPDstress, reflecting 
the combined extent and severity of ischemia and scar, over 1-12 months. Tertiary endpoints 
include the change in hyperemic global and regional (i.e., non-infarcted myocardium) 

 
10 
 
myocardial blood flow (MBF), and the change in global and regional TPDstress-rest (reflecting the 
ischemia score) over 1-12 months. 
 
Statistical analyses: For the analyses of both primary and secondary endpoints included in 
Hypotheses 1 and 2, we will use separate linear regression models with the respective change in 
global CFR (in response to dipyridamole) and TPDstress over 1-12 months as the outcome 
variables. Each of these regression models will control for baseline variables of stratum (DM or 
metabolic syndrome), time since index MI (< 6 months or ≥6 months), imaging site (BWH or 
OHI), the corresponding baseline global CFR score, as well as treatment assignment. Although 
we expect the distribution of change scores to be approximately normal, preliminary analyses 
will evaluate whether a transformation from the Box-Cox family will improve that assumption. 
From our experience in prior clinical trials using a similar design to CIRT-CFR, we expect a 10- 
15% attrition rate of patients due to death or loss to follow-up. Because it is not expected that site 
randomization assignment will influence these events, and because the available information is 
unlikely to allow for accurate imputation of missing values for CFR, subjects who do not have a 
repeat 1-12-month PET scan will not be included in the primary or secondary analyses. 
 
Power calculations: For Aim 1, we used prior publications from our group to characterize the 
reproducibility of the global, dipyridamole mediated CFR measurements in patients with DM or 
metabolic syndrome43, 53. We used data from the patients enrolled in the placebo arm of these 
trials to estimate the standard deviation in the change in global CFR (Time 2 CFR – Time 1 
CFR), and found a mean standard deviation of 0.45. Table 1 shows the sample size needed to 
reach 80-90% power for an expected effect size of 20% relative improvement in CFR in the 
active treatment group compared to placebo. Using a total sample size of 130 evaluable patients 
and a two-tailed alpha of 0.05, power will be 85% for this analysis. Estimated power by this 
approach should be considered conservative since the control for baseline CFR is expected to 
enhance power. We expect to randomize approximately 150 patients to have at least 130 
evaluable subjects completing the study (estimating a 10-15% attrition rate between time of 
randomization and final PET).  
 
 
 
Aim 2. To test the hypothesis that LDM at a target dose of 15 to 20 mg orally weekly for 1-12 
months will, compared to placebo, result in improved LV myocardial mechanics, as assessed 
by transthoracic echocardiography, and that this functional improvement will correlate with 
the change in CFR after 1-12 months of treatment. Specifically: 
Hypothesis #3: LDM for 1-12 months will result in improved LV systolic function, as assessed 
by measures of LV peak global longitudinal strain, and diastolic function, as assessed by 
measures of tissue Doppler mitral annular early diastolic relaxation velocity (E’). 

 
11 
 
Hypothesis #4: Changes in LV systolic and diastolic function after 1-12 months of LDM therapy 
will be correlated with changes in coronary flow reserve. 
Methods: 2-D echocardiograms will be performed at the 3 enrolling imaging sites at baseline 
and 12 months after randomization in accordance with standard clinical practice and utilizing a 
standardized study protocol as outlined in section V. As described in section V, we have 
previously demonstrated feasibility of measuring cardiac mechanics with high reproducibility. 
The primary efficacy endpoints for Aim 2 are the change in LV peak global longitudinal strain 
(GLS) (systolic function) and in tissue Doppler mitral annular early diastolic relaxation velocity 
(E’) (diastolic function). Key secondary endpoints are change in other LV systolic function 
measures (LVEF and tissue Doppler peak systolic mitral annular velocity (S’)), as well as change 
in other LV diastolic function measures (LV early diastolic strain rate by speckle-tracking, and 
mitral inflow Doppler E velocity to E’ ratio (E/E’)). Tertiary endpoints include changes in more 
conventional measures of LV structure (e.g. volumes, wall thickness, mass) over 1-12 months. 
 
Statistical analyses. Hypothesis 3: We will use separate linear regression models with the 
respective change in LV peak GLS and E’ over 1-12 months as the outcome variables. Each of 
these regression models will control for baseline variables of stratum (DM or metabolic 
syndrome), time since index MI (< 6 months or ≥ 6 months), imaging site (BWH, OHI, or UAB), 
the corresponding baseline LV peak GLS and E’ scores, as well as treatment assignment. 
Hypothesis 4: Although temporality cannot be established in correlational analysis of concurrent 
changes, we will also use regression models to clarify the association of changes in CFR and LV 
function. Controlling for treatment assignment, baseline stratification variables, baseline CFR 
and LV function, as well as change in LV function, our outcome will be change in CFR. By 
adding the baseline and change in LV function to our primary model for Aim 2, we will explore 
whether changes in CFR are associated with changes in LV function, or whether independent 
treatment effects remain. 
 
Power calculations: For Aim 2, we used prior publications from our team to characterize the 
reproducibility of the LV measures of systolic and diastolic function40, 54. Power analysis is based 
upon the following assumptions: the expected effect size is 10% relative improvement in each of 
the primary and secondary outcome measures in the active treatment group compared to placebo. 
Table 2 shows the sample size needed to reach 80-90% power for each of the primary and 
secondary efficacy measures. As for Aim 1, estimated power by this approach should be 
considered conservative since the control for baseline scores is expected to enhance power. Of 
the 150 patients we expect to randomize, there will be at least 130 evaluable subjects completing 
the study (estimating a 10-15% attrition rate between time of randomization and final echo).  
 

 
12 
 
 
Exploratory analyses: The proposed ancillary study will not be powered to detect changes in 
clinical outcomes, patient symptoms, or changes in circulating biomarkers. However, we 
recognize the importance of exploring the correlations between the changes in CFR and LV 
function over the 1-12-month observation period and these meaningful outcomes. We propose 
the following exploratory tertiary objectives: (1) Explore an interaction between the change in 
CFR at 1-12 months with composite MACE that includes cardiac and all-cause mortality, 
recurrent MI, stroke, coronary revascularization, and hospitalization for unstable angina 
requiring unplanned revascularization or for congestive heart failure, occurring 3-4 years after 
randomization. We will use a proportional hazards model to explore relationships of CFR and its 
change with time until development of the first clinical event occurring through the end of 
randomization. We recognize that this exploratory analysis, in particular, will be limited because 
we expect only ~25 composite events in total in our study sample. (2) Correlate the change in 
CFR at 1-12 months with the corresponding change in QOL scores, as assessed by the 
standardized SF-36 Health Survey to be administered during enrollment, 12-month, and final 
study visits of the parent CIRT trial. Prior assessments with this tool have shown significant 
differences in baseline and 6-month follow-up scores in a modest-sized (n=120) patient 
population post-MI55. (3) Correlate the change in CFR and LV function with the corresponding 
changes in circulating biomarkers of inflammation (hsCRP, IL-1) and myocardial strain 
(NTproBNP, hsTroponin), as assessed from stored biorepository samples at 8, 12 and 24 months 
after randomization. Analyses of changes in QOL scores and biomarkers will use linear 
regression models, after transforming changes to normality as necessary, and with control for 
baseline trial stratification variables, baseline QOL scores and biomarker levels, and treatment 
assignment. Main independent variables will be baseline and 1-12-month change in CFR. (4) 
Assess correlations among the baseline PET, echo, biomarker, and SF-36 data using linear 
regression models, as described above for continuous variables. 
 
 
VII. RISKS AND DISCOMFORTS   
 
Blood Sampling and Intravenous Catheter Risks 
There are minor risks and discomforts associated with placement of an intravenous line, including 
the possibility of bleeding, pain, inflammation (redness and swelling), and leaking of contrast 
agent outside of the vein at the site of the IV. These problems are usually self-limited or require 
only minor treatments, such as application of an ice-pack or slight pressure for a few minutes. 
 

 
13 
 
Vasodilator Risks 
Vasodilator-stress with Dipyridamole (Persantine®), as will be performed in this study, has been 
used routinely for many years for evaluation of known or suspected coronary artery disease in 
conjunction with myocardial perfusion imaging. During the Dipyridamole infusion, the patient 
may experience flushing, chest pain/pressure, nausea, or lightheadedness. If significant symptoms 
are present, patients will be given aminophylline IV to relieve these symptoms. There will be 
continuous monitoring of heart rate, blood pressure, and 12-lead ECG throughout the infusion and 
recovery. These procedures are routinely performed in patients with CAD, as those in the CIRT 
trial, and are considered safe. 
 
For patients with contraindications to Dipyridamole, the PI or Co-Investigator can decide to use 
Adenosine or Regadenoson for safety purposes.  
 
Regadenoson stress as performed in this study has been used routinely for many years for 
evaluating patients with known or suspected CAD. The most common side effects associated with 
the regadenoson bolus include: flushing, chest pain/pressure, shortness of breath, palpitations, 
headache, mild hypotension and heart block. These side effects are usually mild and self-limiting. 
If they are severe in intensity, aminophylline IV (1 mg/kg) will be given as per standard protocol. 
Subjects with a history of seizures may receive a medicine called adenosine. The side effects 
associated with this product include: facial flushing, headache, sweating, palpitations, chest pain, 
hypotension, shortness of breath/dyspnea, chest pressure, hyperventilation, head pressure, 
lightheadedness, dizziness, tingling in arms, numbness, apprehension, blurred vision, burning 
sensation, heaviness in arms, neck and back pain, nausea, metallic taste, tightness in throat, and 
pressure in groin. Adenosine is administered as a continuous infusion over 4 minutes and 
administered in a dose of 0.14mg/kg/min.  The half life of adenosine is less than 10 seconds.  If 
the patient is having symptoms the infusion can be stopped, no further medication is 
administered, and the symptoms would stop within seconds because the medication would 
already be cleared from the heart. Therefore, no reversal medication is needed.   
 
Aminophyline Infusion 
Rarely, an aminophylline IV injection may be used to reverse side effects of dypiridamole 
infusion. Aminophylline is generally well tolerated; possible side effects of aminophylline may 
include nausea, headache, restlessness, convulsions, rapid breathing, a rapid heart rate, and 
allergic reactions such as rash. 
 
Radiation Risks 
The PET scans will involve exposure to radiation for research purposes. The two radioactive drug 
studies (13N-ammonia) per visit will expose the patient to approximately 2.82 mSv of radiation for 
each visit, or a total of approximately 5.64 mSv for both the baseline and follow-up studies (a mSv 
is a unit of radiation dose).  For sites using 82Rubidium, the two radioactive drug studies per visit 
will expose the patient to approximately 2.6 mSv of radiation for each visit, or a total of 
approximately 5.2 mSv for both the baseline and follow-up studies (a mSv is a unit of radiation 
dose). The total whole body effective dose from the ancillary study is equivalent to approximately 
11% of the 50 mSv annual limit for a person who works with radiation, or 2 times the natural 
environmental radiation an average person receives in the United States annually. This risk from 

 
14 
 
medical imaging is considered low. Women of child-bearing potential are excluded from the parent 
trial. 
 
Potential Risks from 2-D Echocardiograms 
There is minimal discomfort from the echocardiograms including slight discomfort from pressure 
from the transducer while images are collected, or rarely irritation from the gel used to obtain the 
echocardiographic images. These studies are routinely performed in patients with CAD, as those 
in the CIRT trial, and well tolerated. 
 
Reproductive Risks 
Pregnant or breastfeeding patients will be excluded from this study.  
 
Unknown Risks 
There may be other risks and side effects that are not known at this time. 
 
 
VIII. POTENTIAL BENEFITS 
 
There are no direct benefits for the patient from taking part in this study. The proposed ancillary 
study will test the fundamental concept of whether reducing systemic inflammation with LDM can 
lead to improved myocardial blood flow and tissue perfusion and LV function. Thus, this ancillary 
study will provide important and unique mechanistic insights of the capabilities of anti-
inflammatory therapies to improve key clinical determinants of clinical risk (improve coronary 
blood flow, reduce ischemia, and improved LV function) and, in so doing, improve patient 
symptoms and quality of life, as well as outcomes.  
 
 
IX. MONITORING AND QUALITY ASSURANCE 
 
Safety of patients will be monitored through an independent Data and Safety Monitoring Board 
(DSMB) for the main CIRT trial.  
 
Ancillary study investigators will report to the CIRT leadership (Ancillary Studies Committee 
and Steering Committee) on an annual basis regarding the status of study funding, initiation and 
terminations dates, and success of recruitment and data collection.  
 
The PI will monitor the safety of subjects enrolled at BWH.   
 
 
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APPENDIX:  
CIRT Protocol Summary, Data Collection Schedule, Informed Consent Form  
and IRB Approval Letter