Regadenoson Blood Flow in Type 1 Diabetes (RABIT1D)

The CACTI cohort is being followed for progression of coronary artery calcification (CAC)
for development of clinical CVD and participants have been characterized during the baseline
examination (4/00-3/02) and two follow-up re-examinations 3 and 6 years later. We are
proposing assess a subset of this population to determine vasodilatory reserve as it relates
to early coronary atherosclerosis in T1D. In the CACTI Study sought to determine the
relationship between risk factors for CAD and coronary arterial calcification in T1D
subjects compared to an age and gender matched control cohort. The trial identified CAC as a
high risk marker of CAD and was more extensive in diabetic patients.

T1D patients have more CAC than nondiabetic controls but they also appear to have more
impaired coronary vasoreactive function. Impaired response to acetylcholine testing and
adenosine vasodilation are reported in diabetic subjects. The relationship between coronary
microvascular dysfunction and diabetic cardiomyopathy is supported by observation of similar
microvascular abnormalities. Endothelial dysfunction not only precedes and predicts clinical
macrovascular disease, but also is an independent prognostic marker of adverse long-term
cardiovascular outcomes. Significantly greater CAC in T1D was also noted than in non-DM
subjects. There is also a greater rate of increase noted in the CACTI cohort.

Myocardial Flow Reserve by MRI: Myocardial perfusion distribution and myocardial blood flow
reserve can be simultaneously assessed using a MRI first-pass Gd contrast injection
approach. Rapid MR imaging (MRI) during the first pass injection of Gd compounds is used to
assess myocardial perfusion with a spatial resolution of 2-3 mm, and to detect any regional
impairments of myocardial blood flow (MBF) that may lead to ischemia. The myocardial
perfusion reserve (MPR) is a useful concept for quantifying the vasodilator response. The
perfusion reserve can be estimated from the ratio of MBFs during vasodilation and at
baseline, similar to that described by labeled microspheres. Assessment of myocardial blood
flow and flow reserve by cardiac MR is a robust technique. There is a relationship between
CAC and hyperemic flow reserve, although it may be more or less strong depending upon the
methodology employed. However, CAC is still a marker of the atherosclerotic process and this
process appears to be more aggressive in T1D. The investigation of MBF reserve as a marker
of early or more aggressive vasculopathy is important, especially in a disease with
significant morbidity and mortality from cardiovascular events.

Regadenoson Vasodilation: Regadenoson is a selective A2A adenosine receptor agonist FDA
released as a pharmacologic vasodilator in nuclear stress myocardial perfusion imaging
(MPI). It has a higher affinity for A2A receptors than adenosine and is a more potent
coronary vasodilator. Adenosine infusion is the gold standard for both invasive and
non-invasive assessment of myocardial blood flow reserve, yet regadenoson with potentially
greater coronary arterial vasodilation and a simple bolus injection is an ideal agent with
which to study differences in CFR between different disease states. Limited data is
published on the utility of regadenoson in invasive assessment of CFR and no data are
published regarding the utility of this agent in assessing the hyperemic myocardial blood
flow response with respect to cardiac MRI.

Research Design and Methods POPULATION CACTI Diabetes Study: We propose to recruit subjects
from the CACTI cohort into this sub-study by focusing on individuals completing 6-yr
follow-up.

Imaging Studies Non-DM CAC>100 T1D CAC< 100 T1D CAC> 100 MPI 5 10 10 CMR 5 10 10 InvCBF 5
5-10 5-10 Subject will be recruited to complete all three parts of the protocol myocardial
perfusion imaging (MPI); cardiac MR assessment of myocardial blood flow reserve (MBF); and
only those subject with significant symptoms or clinical indications proceeding to invasive
"Catheterization Laboratory" assessment of CBF reserve (Invasive CBF) Aim 1. Determine the
relationship between myocardial perfusion index, regional CBF and invasively measured
coronary perfusion reserve The first approach will be to assess the differences in
myocardial perfusion between T1D and non-DM subjects employing vasodilatory stress with
regadenoson. The myocardial perfusion index as described previously will be calculated for
each individual. The relative perfusion index will be directly compared to the invasively
obtained CBF reserve in each vascular territory assessed.

Stress Myocardial Perfusion Protocols. Subjects will undergo regadenoson stress MPI with 400
mcg administered as a 10 second bolus followed by injection of 30 mCi of Tc-99m-sestamibi.
Gated SPECT perfusion imaging is then performed within 30 minutes. A rest study is performed
on a separate day with a similar sestamibi dose administered.

Acquisition Protocol. All SPECT studies are performed with parallel hole collimators and
gated acquisitions protocol (10% RR window) acquired into 16 time bins. Data Processing of
Gated data sets are assessed for mean counts, corrected for dose, decay and time of imaging,
are measured in each segment utilizing 4D-MSPECT (INVIA, Ann Arbor, Michigan). MPI is
defined as the corrected stress counts divided by corrected rest counts.

Statistical Analysis: Based upon our prior experience approximately a third of the T1D
subjects will have a MP index of less that 1.5 to direct compare to the invasive measured
flow reserve. This maybe treated as a binary variable or as a continuous variable by
combining all T1D subject compared to the non-DM control subjects. Data will be analyzed
using unpaired t-test, linear and logistic regression. Continuous variables are expressed as
mean ± standard deviation (SD) and are compared across groups by use of unpaired t-tests.
All statistical analysis will be performed using two-sided tests with p < 0.05 considered
statistically significant.

Aim 2. Measure coronary blood flow using Regadenoson stress CMR to determine the MBF reserve
in type 1 DM subjects compared to non-diabetic controls. Using the method of a two
compartment model with K trans function analysis, we will determine the myocardial blood
flow reserve by cardiac MR. This approach will use regadenoson bolus as the vasodilator for
the hyperemic response of the coronary arterial bed. We will assess the relationship of MBF
reserve or lack of reserve to disease state i.e T1D to non-DM subjects. In addition we will
regionally compare MBF reserve to the MP index obtained from radionuclide techniques by
coronary vascular territories. Similarly CAC values obtained at the 6-yr CACTI follow-up
study will be directly compared to blood flow measurement within coronary vascular
distributions.

CMR Protocol: An IV injection into the antecubital vein and connected to a power injector
will be performed in the MR scanner for functional and perfusion imaging (Symphony 1.5 T,
Siemens Medical). All images are acquired with a 4 channel phased array surface coil. Blood
flow imaging is performed in three short-axis simultaneous slices (basal, mid and apical)
acquired during an initial bolus injection of gadolinium (0.04 mmol/kg at a rate of 5
ml/sec) followed by a 10-ml saline flush administered using the power injector. ProHance is
used due to its lower viscosity as the contrast agent. The short-axis slices are acquired
using a SSFP imaging sequence with, TR/TE/flip 185ms/1.2ms/50 degrees dynamically acquired
and inserted into a 330x 380 matrix). Images are acquired using a field of view ranging from
280 to 400 mm. A first perfusion scan will be performed at rest, followed by a second scan
during maximal vasodilation about 15 min later. Hyperemia will be initiated with
administration of 400 mcg bolus injection of regadenoson and acquisition started 1 minute
post regadenoson administration. Subjects will be monitored using MRI compatible blood
pressure monitoring and single lead ECG monitoring.

Data analysis Quantitative analysis of the MR blood flow data will be performed on a
Leonardo Workstation using Argus perfusion analysis software. Images were registered
manually by adjusting the horizontal and vertical registration of each frame. The left and
right ventricular blood pool and six myocardial regions will be analyzed to extract the
linear slope during the initial signal increase as well as the starting and peak signal
intensities. To extract the slope, base, and peak signals, a line was manually fitted to the
upslope signal (typically 4-6 time points), and the base and peak positions of the upslope
data were manually identified. Normalized slope (SlopeN) was calculated by division by the
left ventricular blood pool slope, and the net signal gain (SG) determined by subtracting
the initial from the peak signal intensity. The myocardial flow reserve index was calculated
for each myocardial region as the ratio of the normalized hyperemic time-intensity slope to
the normalized baseline time-intensity slope.

Statistical Analysis: Based upon prior experience in the literature approximately a third of
the T1D subjects will have a MP index of less than 1.5 to direct compare to the invasive
measured flow reserve. Data will be analyzed using unpaired t-test, linear and logistic
regression.

Aim 3. Determine the relationship between CBF reserve in vascular distributions to the
degree of coronary arterial calcification.

Coronary artery calcification. All returning patients have previously undergone EBT scan.
These data as described above will be used to compare to the new MPI, CMR myocardial blood
flow reserve, and to the invasively measured CFR. Regional change will determined within
standard coronary vascular distributions.

Flow Reserve Substudy Protocol: High-risk diabetics with CAC> 100 (n=10) and stratified
random sample high risk non-diabetics (n=10) will be studied. The inclusion of subjects with
an abnormal perfusion reserve is important, because this value identified subjects with high
risk in the first phase of the study and therefore is used to select individuals with high
risk of cardiac events that clinically would undergo invasive assessment of coronary
anatomy. Perfusion imaging will then be the test that stratifies subjects into an additional
higher risk than the CAC > 100. Selected patients identified as part of Specific Aim 2 will
undergo a clinically indicated cardiac catheterization and baseline CFR study. Coronary
angiography is the clinically accepted standard for evaluation and diagnosis of coronary
artery disease.

Subsequently, coronary artery vasodilator reserve will be assessed using the regadenoson
intravenous challenge technique measured as the coronary flow reserve and fraction flow
reserve assess by a Doppler flow guide wire.

Methods for Flow Reserve:Selective coronary angiography is performed using standard
technique with 5F diagnostic catheters in the LCA and RCA. The artery or interest determined
by the abnormal area of myocardial perfusion by SPECT or MR imaging will be interrogated.
The artery of interest will be cannulated with a 6F or 7F Guide catheter. A 0.014 in x 300cm
pressure and Doppler-tipped guidewire (CombMap XT, Volcano Corp) will be advanced into the
distal bed of the vessel of interest. After optimization of the pressure and Doppler signal,
velocity and pressure measurements will be obtained at rest and after induction of maximal
hyperemia with an intravenous bolus of regadenoson. Coronary flow reserve (CFR) will be
measured in the target vessel of interest after 1-2 minutes following the IV bolus. Absolute
CFVR will be calculated as the ratio of hyperemic to baseline average peak flow velocity and
the relative CFVR as the ratio of the absolute CFVR in the target vessel to the absolute
CFVR in the reference artery. FFR and CFR values will be categorized according to published
cut-off values of 0.75 and 2.0, respectively.

Inclusion Criteria:

- 1. Inclusion Criteria Nondiabetic controls High-risk (n=5)

1. Completed visit 6yr f/u CACTI Trial

2. No history of previous MI, revascularization or angina

3. CAC > 100

4. Stratified random sample to reflect age-sex- distribution of the high risk
diabetic group T1Diabetic subjects High-risk group (n= 10)

1. Completed visit 6yr f/u CACTI Trial

2. No history of previous MI, revascularization or angina

3. CAC > 100

4. preferably MPR of > 1.5 T1Diabetic subjects Lower-risk group (n= 10)

1. Completed visit 1.A and 1.B CACTI Trial

2. No history of previous MI, revascularization or angina

3. CAC < 100

Exclusion Criteria:

2. Exclusion Criteria:

1. Pregnant or lactating women, women who plan to become pregnant

2. Claustrophobia

3. Moderate or severe congestive heart failure at baseline, Left ventricular
ejection fraction (LVEF) < 25%

4. Uncontrolled hypertension

5. Unwillingness to complete all components of the study

6. Significant CAD or prior revascularization

7. Smoker

8. Subject cannot have >50% reduction in lumen diameter of left main coronary
artery

9. Asthma requiring daily bronchodilators

10. Methylxanthine therapy

11. Moderate to severe renal insufficiency: GFR < 40 mL/min