Clinical trial NCT03364270
Conditions: Carotid Arteries
Keywords: atherosclerosis
Sponsor: Balaji Tamarappoo
Start date: 2018-10-10; Completion date: 2020-05-31
Enrollment: 6 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this study is to investigate the ability of a new investigational agent compound \[F-18\] labeled RGD-K5 to detect unstable atherosclerotic plaque in the carotid artery of subjects being considered for carotid endarterectomy (CEA), and to confirm this ability through tissue analysis of samples of carotid artery plaques that will be collected during the planned carotid surgery. \[F-18\] RGD-K5 is a radioactive tracer used in imaging to detect active growth of new blood vessels and presence of macrophages. Patients with unstable plaque may be prone to rupture of the plaque due to increase in macrophage activity and growth of new blood vessels. \[F-18\] RGD-K5 is an investigational agent, which means that it has not yet been approved by the US Food and Drug Administration (FDA).

Unstable atherosclerotic plaque that is prone to rupture is characterized by an increase in the number of macrophages and enhanced angiogenesis. Both neovascular endothelium and macrophages exhibit increased Alpha-v beta3 integrin expression. PET (Positron Emission Tomography) imaging of \[F-18\] RGD-K5 uptake may identify carotid plaque with increased inflammation and neovascularization and may therefore detect unstable plaque in participants with carotid artery stenosis.

Prior to Dr. Tamarappoo's relocation to Cedars Sinai Medical Center (CSMC), 5 subjects were enrolled at the Cleveland Clinic where PET-CT (Positron Emission Tomography - Computed Tomography) was performed. 6 subjects will be scanned at Cedars using PET-MRI (Positron emission tomography-magnetic resonance imaging). Based on preliminary data with PET-CTA, the investigator strongly believes the study will be able to reproducibly detect significant \[F-18\] RGD-K5 uptake in plaque from symptomatic patients. Ultimately, demonstrating preferential \[F-18\] RGD-K5 uptake in symptomatic patients will significantly impact the way in which patients with carotid plaque (at risk for stroke) are treated and it may prevent unnecessary surgical and endovascular procedures in this population

Detailed description:
The primary objectives of this exploratory study are:

* To assess the uptake of \[F-18\] RGD-K5 by carotid plaque with PET/MRI imaging in participants prior to carotid endarterectomy and thereby determine if carotid plaque that causes significant stenosis \[in participants being considered for carotid endarterectomy (CEA)\] is characterized by increased integrin expression and enhanced angiogenesis.
* To collect safety data of \[F-18\] RGD-K5 in participants with carotid atherosclerosis.

Potential subjects will be identified and approached during an inpatient or outpatient clinical visit by a member of the research team. Potential subjects will be identified by their treating physicians and referred to the researchers. Patients' private and identifiable information will not be shared prior to receiving permission from the patient to do so

Summary of research procedures:

Physical exam and vital Signs:

Height, weight, temperature, heart rate and blood pressure will be checked before beginning the imaging tests. Subjects' heart rate and blood pressure will be checked again after each of the two PET/MRI images, and as needed.

Patients will also be asked about the presence of ICD, pacemaker or any metallic implants that would not be compatible with the MRI magnet (presence of MRI non-compatible devices will be an exclusion criteria)

Blood Test:

If labs have been done and the lab values meet the criteria to participate in this study, the investigators will not need to collect these labs again on the day of testing. If these labs have not been collected within 30 days prior to the research imaging tests, the investigators will draw a tube of blood to check kidney function and other necessary blood values prior to the research testing. Patients with a glomerular filtration rate (GFR) \<40ml/min will be excluded from the study.

Urine Pregnancy test:

This will only need to be done in female subjects of childbearing potential. This will need to be checked prior to scheduling subjects' research imaging tests. If subjects' tests are schedules more than 2 days (48 hours) since first checked, this test will need to be rechecked on the day of subjects' research imaging testing.

ECG:

An ECG (electrocardiogram) will be done to assess subjects' heart rhythm before the testing begins, and after each of the PET scans.

IV insertion:

A catheter will be inserted into subjects' arm. It is through this IV catheter that the dose of \[F-18\] RGD-K5 will be given prior to imaging.

Research PET/MRI Imaging of the Carotid Arteries:

2 hours after receiving the dose of \[F-18\] RGD-K5 PET/MRI imaging of subjects' neck (carotid arteries) will be performed. Subjects will be lying on subjects' back with a head fixation device supporting subjects' head to prevent Subjects from moving subjects' head while the images are taken. This scan will take about 30-45 minutes. Subjects will be permitted to get up and move around between receiving the radiotracer and imaging. Subjects will be asked to drink a glass of water before receiving the dose of \[F-18\] RGD-K5.

Histology and Immunohistochemistry:

During Subjects carotid endarterectomy(CEA) which is a clinically indicated procedure, atherosclerotic plaque will be removed. A sample of this plaque will be collected and taken to the histology laboratory at Cedars Sinai for analysis.

Follow Up phone call:

Subjects will be contacted by phone the day after subjects' PET scans to ask if Subjects have had any medical problems and what medications Subjects have taken since the PET scan. If Subjects were seen by a doctor to be evaluated for a medical problem, the investigators may ask permission to contact the doctor or hospital to obtain copies of subjects' medical records

Research poses greater than minimal risk

Anticipated benefits:

There is no personal benefit to participants by participating in this research study.

The knowledge to be gained from this research may be beneficial for other patients, society or science.

The detection by the imaging protocol of unstable atherosclerotic plaque in the carotid artery will not result in any change in their standard of care.

The potential benefit of the study to the patient is that it will allow the physician treating the patient to know the entire extent of the carotid plaque, plaque composition and will inform them of the anatomy of the carotid artery in question.

Furthermore, it will provide information about plaque in the contralateral carotid artery and if that plaque has a high degree of inflammation. If a high inflammatory burden is noted in the contralateral carotid artery, there will be more vigilance in monitoring these patients.

Summary of process by which confidentiality will be maintained:

Research records will be stored in a locked cabinet in a secure location Electronic research records will be stored in a shared network folder on the CSMC network. The list linking the assigned code number to the individual subject will be maintained separately from the other research data. Only certified research personnel who are listed on the approved Institutional Review Board (IRB) application will be given access to identifiable subject information. Research related data and patient/subject information will never be stored on a researcher's personal (private) computer or laptop. Study data will be coded upon abstraction so that Direct Identifiers\*\* are maintained separately from the data set.

Summary of data and safety monitoring plan:

The study will be monitored only by the study investigators and/or sponsor.

How does participation in the research differ from the receipt of standard care:

RISKS OF THE STUDY

As with any research study, there may be adverse events or side effects that are currently unknown and it is possible that certain of these unknown risks could be permanent, serious or life threatening. The study doctor will inform subjects of any significant new information that is discovered during this trial.

Risks of the Study Drug Based upon the toxicity profile to be administered as a single dose, the purity and impurity profile of \[F-18\] RGD-K5, no risk of adverse events is anticipated for this study.

Radiation Risk:

* It is very unlikely that participants will see any harmful effects because of the radiation exposure participants will receive from taking part in this study.
* At high levels of exposure, scientists agree that radiation can cause cancer. This research study involves exposure to radiation from the RGDK5-PET scan. The total amount of radiation exposure participants will receive is equivalent to a uniform whole-body exposure of 0.8 rem. This is comparable to approximately three years' worth of radiation exposure from typical environmental sources (about 0.3 rem per year, which includes radon, natural background radiation, and cosmic rays). This is also comparable to 16% of the maximum annual exposure for radiation workers (5 rem) allowed by the Nuclear Regulatory Commission.

MRI is not associated with any radiation risk.

MRI non-compatible devices such as pacemakers, ICD (implantable cardioverter defibrillator) and metallic implants may cause artifacts in imaging and device malfunction as well as local tissue injury.

Contrast used for MRI is associated with minimal risk among patients who are free of significant renal dysfunction.

When injected into the body, gadolinium contrast medium makes certain tissues, abnormalities or disease processes more clearly visible on a magnetic resonance imaging scan. They are used to improve the clarity of the scanned images or pictures of the body's internal structures.

Gadolinium contrast medium is given by intravenous injection.

The risk of gadolinium contrast is minimal in patients without severe renal disease.

The most common adverse effects of gadolinium agents are

* Itching of the skin (5%)
* Headache (4%)
* Nausea (4%)
* Very small risk of local pain
* Hypersensitivity (allergic reaction)
* Rarely (\<1% of the time) it can cause low blood pressure and lightheadedness. This can be treated immediately with IV fluids.

More serious allergic reactions that are life threatening are rare. Gadolinium is not safe for people with late stage kidney disease or who have had a liver transplant. Gadolinium-based contrast agents increase the risk for a rare, but serious adverse reaction, called nephrogenic systemic fibrosis (NSF) among patients with impaired elimination of the drugs. Patients with kidney disease are at increased risk of developing NSF. NSF may cause skin thickening, joint pain and/or swelling, causing feelings of burning, itching and pain that can be severe. In very rare cases, NSF can lead to lung and heart problems and may be life threatening. Participants will have a blood test to measure kidney function and if the blood test is abnormal, they will not be permitted to receive gadolinium.

There is a very small risk of mild headache and local pain. Rarely (\<1% of the time) it can cause low blood pressure and lightheadedness. This can be treated immediately with IV fluids. More serious allergic reactions that are life threatening are rare. Gadolinium is not safe for people with late stage kidney disease or who have had a liver transplant.

As with all research procedures and administration of study drugs, subjects will be monitored at all times.

Arms: arm 1: PET/CT Imaging with administration of \[F-18\] RGD-K5
Interventions: intervention 1: PET/CT Imaging with [F-18] RGD-K5
Intervention descriptions: intervention 1: PET Imaging/scan with \[F-18\] RGD-K5, PET Imaging/scan with RGD-K5, PET Imaging/scan with K5

Protocol documents:
Prot_SAP_000.pdf: 
 
 
1 
K5-C200 
BALAJI K TAMARAPPOO, MD, PHD 
 
11NOV2019 
 
Section 1.0 
 
General Information 
  
TITLE:   
AN EXPLORATORY, PHASE II, OPEN LABEL, SINGLE-CENTER, NON-RANDOMIZED 
STUDY OF [F-18] RGD-K5 POSITRON EMISSION TOMOGRAPHY (PET) IN 
PARTICIPANTS WITH CAROTID ARTERY STENOSIS; K5-C200 
 
PI:   
BALAJI K TAMARAPPOO, MD, PHD 
 
Study Support Provided By:  
NHLBI-NIH  
 
(NATIONAL INSTITUTES OF HEALTH) 
 
Section 2.0 
 
Background information 
 
Atherosclerotic vascular disease is the primary cause of heart attacks and stroke. The rupture 
and downstream embolization of unstable plaque in the carotid artery can result in stroke or 
transient ischemic attack (TIA). Stroke is the fourth leading cause of mortality in the United 
States. In addition, rupture of unstable atherosclerotic plaque in the coronary artery results in 
myocardial infarction (MI), the major cause of death in the United States.  A noninvasive 
imaging test that can detect unstable plaque is critical for identification of high-risk patients who 
would benefit from aggressive medical therapy, carotid artery stenting or percutaneous coronary 
intervention.  Among patients with carotid artery stenosis, a significant number of them undergo 
carotid endarterectomy (CEA) or stenting purely based on the presence of plaque causing ≥70% 
stenosis even though they may have never had a stroke or TIA [1, 2].  A non-invasive imaging 
test that can accurately identify unstable plaque may prevent unnecessary surgical and 
endovascular procedures and could potentially reduce cardiovascular mortality. 
Current State of the Art for Noninvasive Detection of Unstable Plaque 
CTA:  Carotid plaque is usually detected by Doppler ultrasound and alternatively by CTA and 
magnetic resonance imaging (MRI) [3].  CTA can identify high-risk plaque features such as 
spotty calcification, plaque ulceration and enriched lipid composition [4].  However, dense 
calcification can mask high-risk plaque features on CTA. 
MRI:  Morphologic features associated with unstable plaque such as a necrotic lipid core and 
intraplaque hemorrhage can be imaged with MRI [5-10].  Even still, MRI is not widely used for 
detection of unstable plaque due to the technical challenges associated with obtaining high 
resolution images in smaller arteries [11]. Ultrasmall paramagnetic nanoparticles (USPIO) have 
been used in clinical studies to bind to vulnerable plaque based on the high density of 
macrophages; however, there have been no clinical trials of USPIO for routine identification of 
vulnerable plaque in the United States [12, 13]. 
18F-Sodium fluoride (NaF): NaF has been shown to bind to culprit lesions in the coronary 
artery of patients who have suffered a myocardial infarction and in carotid artery plaque in 
patients who have suffered a stroke or TIA [14, 15].  However, there are no prospective studies 
demonstrating the ability of NaF to predict plaque rupture at this time. 
18F-labeled deoxyglucose (FDG):  FDG is the only molecular imaging agent that has been used 
for positron emission tomography (PET) of atherosclerotic plaque in large clinical studies [16, 
17]. In 2002, Rudd et al. showed that FDG uptake was preferentially increased in carotid artery 
plaque of patients with symptoms of stroke or TIA compared to asymptomatic patients. Plaque 
removed from patients with high carotid FDG uptake was enriched in macrophages and was 


 
 
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BALAJI K TAMARAPPOO, MD, PHD 
 
11NOV2019 
characterized by a large necrotic core. This study concluded that FDG uptake may be a marker 
of plaque vulnerability; however, there are no studies that have tested the benefit of FDG PET 
imaging of plaque for clinical decision-making.  FDG is extracted by a number of metabolically 
active cellular targets cells including macrophages, lymphocytes and smooth muscle cells found 
in plaque.  A plaque imaging agent with selective affinity for macrophages and neovascular 
endothelial cells might significantly improve our ability to identify vulnerable plaque compared 
to FDG.  Molecular imaging agents that target proteins that are presumably enriched in plaque 
such as matrix metalloproteases and annexinVa have been used in animal models of 
atherosclerosis; however, clinical translation of these imaging tracers is still in progress [18]. 
 
Most importantly, there is lack of data in support of clinical decision making based on the 
presence of high-risk plaque-features-using CTA, MRI, FDG-PET and NaF-PET in individuals 
with plaque that causes <70% luminal stenosis. 
Need for Alternate Tracers for Plaque Imaging 
There is an unmet clinical need for a plaque imaging agent that preferentially binds to cells and 
proteins that are present in unstable plaque and will thereby identify patients at risk for stroke or 
MI.  Vulnerable plaque is characterized by high inflammatory activity and an overabundance of 
macrophages and neovessels.  An imaging agent that selectively binds to macrophages and (or) 
neovascular endothelial cells may allow identification of inflamed vulnerable plaque.  Such a-
specific-tracer will provide higher lesion-to-background ratio and will improve clinical accuracy 
in identifying vulnerable plaque. 
18F-flotegatide:  18F-flotegatide, a PET imaging agent with high specificity for αVβIII and 
α5β1 integrin present on macrophages and neovascular endothelial cells may offer the 
opportunity to detect vulnerable plaque.   
The objective of this proposal is to evaluate the use of 18F-flotegatide to noninvasively 
detect unstable plaque in clinical subjects. I propose to accomplish this objective by first 
testing the hypothesis that 18F-flotegatide can bind to atherosclerotic plaque with morphologic, 
histologic and clinical features of vulnerability using PET-MR imaging in patients with carotid 
atherosclerosis. 
 
INNOVATION 
Advantages of using 18F-Flotegatide for the identification of vulnerable plaque 
Integrins are heterodimeric glycoproteins composed of α and β subunits.  They are key 
molecules involved in angiogenesis and mediate cell locomotion through the extracellular matrix 
[19, 20]. The integrin αVβIII plays an important role in the development and progression of 
atherosclerotic plaque and is considered to facilitate inflammatory cell recruitment to the 
damaged vessel wall and angiogenesis [20-23]. The α5β1 integrin is known to be involved in 
macrophage recruitment and angiogenesis in atherosclerotic plaque [24-26]. There is prominent 
expression of αVβIII on the endothelial cell surface of adventitial vasa vasorum and plaque 
neovessels [27].  Integrin αVβIII and α5β1 are also expressed by macrophages in atherosclerotic 
plaque [26, 28, 29].  Until recently, the most promising tracer for non-invasive detection of 
αVβIII expression in humans using PET has been 18F-Galacto-RGD [30, 31]. On the basis of 
the αVβIII receptor binding, the glycosylated cyclic tripeptide, 18F-Galacto-RGD demonstrates 
high affinity and selectivity for the receptor in vivo [32, 33]. Recently, 18F-Galacto-RGD was 
shown to bind to carotid artery plaque in a small group of patients with atherosclerosis [34].  
However, the long labeling time (4h) and complex chemistry required for the synthesis of 
18F-Galacto-RGD makes it less amenable for routine clinical applications [30].  Our 


 
 
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BALAJI K TAMARAPPOO, MD, PHD 
 
11NOV2019 
proposal to use 18F-Flotegatide (18F-RGD) for clinical plaque imaging in patients with carotid 
atherosclerosis is innovative for the following reasons: 
1. The relatively short labeling time (75min) achieved through the use of click chemistry for its 
synthesis in comparison to the synthesis of 18F-GalactoRGD makes 18F-flotegatide an 
attractive agent for large scale clinical investigation. 
2. Blood and background clearance is faster due to the triazole, a by-product of the click 
reaction. The plasma clearance half time is 12min. This in turn provides higher target to 
background ratio (TBR). 
3. The synthesis of 18F-flotegatide is automated (unlike other RGD-tracers), which makes 
production of this tracer manufacturing-friendly and adaptable to various synthesis platforms 
at different clinical sites. 
4. The high affinity of 18F-flotegatide for both αVβIII and α5β1 (Kd of 7.9nM for αVβIII and 
26.5nM for α5β1 measured by inverse surface plasmon resonance) increases the number of 
potential cell surface targets for binding of 18F-flotegatide in atherosclerotic plaque. This 
translates to an improved signal to background ratio of 18F-flotegatide for in-vivo PET 
imaging relative to other integrin targeted agents. 
5. We propose to use PET-MR instead of PET-CTA for the following reasons: 
(a)  MR offers superior tissue characterization for assessment of plaque vulnerability 
compared to CTA. 
(b)  PET-CT and CTA are often performed independently and accurate co-registration 
can be limited. The use of PET-MR allows simultaneous MR and PET imaging and 
allows accurate co-registration of plaque by MRI with the PET uptake of 18F-
flotegatide. 
6. Carotid plaque is removed surgically by CEA in patients with stroke, TIA or significant 
luminal obstruction. This offers a unique opportunity to directly compare observations from 
noninvasive imaging to histologic and immunohistochemical characterization of the same 
plaque after endarterectomy. 
 
7. A significant number of patients with carotid atheroma are sent for carotid stenting with 
optical coherence tomography (OCT) to assess plaque composition at the time of stenting. 
In-vivo information from OCT can be correlated to the tracer uptake and MRI plaque 
features.  
 
8. Our study is conducted by an interdisciplinary, highly synergistic team comprised of a 
cardiologist (PI), neurologist, vascular surgeon, PET-radiochemist, PET and MR physicists 
and radiologists. 
 
Based on our preliminary data with PET-CTA, we strongly believe we will be able to 
reproducibly detect significant 18F-flotegatide uptake in plaque from symptomatic patients.  
Ultimately, demonstrating preferential 18F-flotegatide uptake in symptomatic patients will 
significantly impact the way in which patients with carotid plaque (at risk for stroke) are 
treated and it may prevent unnecessary surgical and endovascular procedures in this 
population. 
 


 
 
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K5-C200 
BALAJI K TAMARAPPOO, MD, PHD 
 
11NOV2019 
Overall Study Design Background: The rupture of unstable plaque in the carotid artery is one of 
the most common causes of stroke. Plaque rupture is followed by activation of the thrombotic 
cascade and formation of an intravascular thrombus. Downstream embolization of all or part of 
the thrombus causes ischemia of brain tissue thus precipitating a stroke.  Smaller emboli that 
originate from unstable plaque may result in TIA. 
 
Currently Accepted Model for Testing New Tracers for Imaging Vulnerable Plaque:  PET 
imaging studies that investigated the use of FDG, and NaF for detection of unstable carotid 
plaque have used stroke as the clinical surrogate marker for presence of unstable plaque [15, 17].  
Although, patients were imaged with FDG or NaF several days after plaque rupture had 
occurred, these studies still showed tracer uptake in the remaining carotid plaque.  We propose to 
use a similar approach to test our hypothesis that 18F-flotegatide is extracted by atherosclerotic 
plaque (Specific Aim I). We also propose to determine if plaque removed from patients who 
exhibit 18F-flotegatide uptake by PET exhibits MRI features and histologic markers of 
vulnerability (Specific Aim II). 
Rationale:  Carotid artery plaque serves as an ideal model system for testing molecular 
imaging agents designed to bind to biological targets in unstable plaque. The carotid artery 
wall is perfused by vasa vasorum similar to the aorta and coronary arteries making 
observations made in the carotid artery broadly applicable to clinically important vascular beds 
in the human body [35-37].  Carotid artery plaque can be localized and imaged by multiple 
noninvasive techniques including ultrasound, CTA and MRI. The relatively large size of 
carotid plaque lends itself to better visualization of intraplaque targets using imaging 
techniques including PET. 
 
Section 3.0 
 
Trial objectives and purpose 
 
Primary Objectives:  
 
Aim 1:  To validate that 18F-flotegatide may be extracted and differentially retained by carotid 
artery plaque in patients with clinical symptoms of stroke or TIA (vulnerable plaque) compared 
to asymptomatic control patients with stable plaque using PET-MR imaging. Our hypothesis is 
that noninvasive PET imaging of 18F-flotegatide uptake can clinically distinguish unstable 
plaque from stable plaque. 
• We propose to validate our preliminary results (that were obtained using PET-CT and 
CTA) by demonstrating that atherosclerotic plaque can be detected by PET-MRI using 18F-
flotegatide. 
• We propose to accomplish this aim by performing in-vivo PET imaging of patients with 
unstable carotid plaque (within 96h of a stroke or TIA). We propose to quantify plaque uptake 
of 18F-flotegatide relative to 18F-flotegatide uptake in segments of the carotid vessel wall that 
are free of atherosclerosis. 
• We also propose to test our hypothesis that 18F-flotegatide is preferentially retained by 
unstable plaque and may be used to distinguish unstable plaque from stable plaque. 
• We propose to achieve this by comparing 18F-flotegatide uptake in patients who have 
been free of antecedent stroke or TIA (asymptomatic-control group) to 18F-flotegatide uptake 
of patients with unstable plaque with recent stroke or TIA.   


 
 
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K5-C200 
BALAJI K TAMARAPPOO, MD, PHD 
 
11NOV2019 
• In the section “Aim 1. Preliminary results”, we demonstrate the relative increase in 18F-
flotegatide uptake by vulnerable atherosclerotic plaque in the carotid artery in 3 patients with 
stroke compared to 2 asymptomatic patients with similar degree of carotid artery stenosis. 
 
Aim 2a and 2b: 
To test our hypothesis that 18F-flotegatide uptake is reflected by histologic markers of plaque 
vulnerability and mediated by an integrin-targeted mechanism. To achieve this goal, we 
propose to characterize the extent of macrophage infiltration and neovascularization in 
atherosclerotic plaque removed during endarterectomy and its association with 18F-flotegatide 
uptake. 
 
Background: 
 
Aim 1:  Macrophages and Neovessels as Markers for Vulnerable Plaque 
Rupture of the fibrous cap on the plaque surface and accelerated intravascular thrombus 
formation in response to plaque rupture is the primary event that precedes stroke, TIA and 
myocardial infarction.  Atherosclerotic plaque develops in response to entry of LDL into the 
intimal layer of the vessel wall and endothelial injury.  In response to this injury, monocytes are 
recruited into the vessel wall and differentiate into macrophages, whose primary role is the 
phagocytosis of modified LDL [38-40]. The inflammatory cascade initiated by the phagocytosis 
of oxidized LDL by macrophages stimulates angiogenesis and the formation of intraplaque 
neovessels allows the entry of red blood cells (RBCs), leukocytes, lipids and oxidized 
lipoproteins [35, 41].  Phagocytosis of RBCs further stimulates recruitment of new macrophages.  
Macrophage apoptosis and the release of matrix metalloproteases destabilizes the extracellular 
matrix and is thought to cause plaque rupture [39, 42].  Intuitively, a noninvasive imaging 
technique that can detect plaque hemorrhage, neovascularization, and macrophage 
infiltration would enable identification of plaque that is vulnerable and may provide a 
means of distinguishing unstable plaque from stable plaque. 
Imaging of 
Neovessels and 
Macrophages with 
Integrin Targeted 
Imaging Agents 
Due to the relative 
enrichment of 
macrophages and 
neovessels in 
unstable 
atherosclerotic 
plaque, they have 
been used as 
surrogates for 
noninvasive 
detection of 
atherosclerotic 
plaque. The 
expression of 


 
 
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K5-C200 
BALAJI K TAMARAPPOO, MD, PHD 
 
11NOV2019 
αVβIII integrin on the cell surface of macrophages, smooth muscle cells and neovascular 
endothelial cells has offered a convenient target for designing molecular imaging agents with 
improved specificity for unstable atherosclerotic plaque. Multiple ligands can bind to αVβIII 
integrin in an Arg-Gly-Asp (RGD)- dependent manner and the high affinity binding of this 
tripeptide sequence has been exploited for MRI and for PET imaging.  PET tracers designed for 
in vivo imaging of integrin expression utilize the R-G-D peptide motif, usually within a cyclic 
peptide structure. Examples of current tracers include 18F-Galactosyl-RGD [43] a cyclic 
pentapeptide, which enables integrin imaging [34, 44, 45].  Scientists at Siemens Medical 
Solutions, Culver City, CA have synthesized 18F-flotegatide which has a number of advantages 
over other RGD-based imaging agents [46]. 18F-flotegatide is easily labeled through click 
chemistry, without requiring protecting groups, by the reaction of 18F-fluoropentyne and an 
azide precursor in the presence of in situ generated Cu(I) (Figure 2(A)) with a synthesis time of 
75min [47]. 18F- flotegatide is metabolically stable for 2 hours and exhibits favorable 
biodistribution and clearance properties characterized by fast blood washout (plasma clearance 
half time of 12 min) and favorable renal excretion [48] (Figure 2(B)).  In oncologic studies, 
intra-tumor tracer distribution of 18F-flotegatide matches the patterns of integrin and CD31 
expression [47]. We hypothesized that 18F-flotegatide may function as an ideal molecular 
imaging agent for noninvasive detection of unstable plaque since macrophages and 
neovascular endothelial cells express integrin αVβIII and α5β1 and vulnerable plaque is 
characterized by neovascularization and macrophage infiltration. 
MRI for anatomic localization of plaque and PET-MR of 18F-flotegatide uptake 
In order to anatomically localize the plaque, we propose to perform MRI for visualization of 
vulnerable plaque characteristics and to localize the plaque along with time of flight (TOF) 
Magnetic resonance angiography (MRA) of the carotid artery for quantification of stenosis 
severity.  PET images will be co-registered with MRI and 18F-flotegatide uptake will be 
compared to plaque features suggestive of vulnerability including presence of necrotic lipid 
core and intraplaque hemorrhage.  MRI is especially robust for visualization of inflammation 
and neovascularization with the use of specialized pulse sequences and contrast agents.  
Enhancement of atherosclerotic plaque with Gadolinium (Gd) based contrast agent, Gd-
diethylenetriaminepentaacetic acid (DTPA) has been used to differentiate between the fibrous 
cap and the lipid core [49, 50]. Gd enhancement has been shown to correspond to zones of 
neovascularization and inflammation [9, 51]. The extent of plaque neovascularization can also 
be measured by dynamic contrast enhancement (DCE) MRI [57]. The ability to detect 
intraplaque hemorrhage, lipid rich core and neovascularization illustrate the superiority of MRI 
based plaque imaging over CTA.  Furthermore, MRI is not associated with ionizing radiation 
and is therefore safer than CTA and thus lends itself to serial imaging in high risk patients. We 
propose to use MRI in our plaque imaging studies to both localize atherosclerotic plaque in the 
carotid artery and to identify high risk morphologic plaque features such as presence of a lipid 
rich core and a thin fibrous cap. 
Aim 2a and 2b: 
Macrophage density and neovascularization as surrogate markers of unstable plaque 
In clinical studies of atherosclerotic plaque imaging with radiolabeled molecular imaging 
agents, macrophage infiltration and neovascularization have been used as the gold standard 
among histologic biomarkers to adjudicate plaque vulnerability [14, 15, 17, 34]. In an early 
plaque imaging study with FDG, Rudd et al. used ex-vivo incubation of plaque to demonstrate 
that carotid plaque uptake of FDG was confined to macrophage- rich areas of plaque [17]. 


 
 
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BALAJI K TAMARAPPOO, MD, PHD 
 
11NOV2019 
This has been used as evidence that FDG uptake is specific for plaque with histologic features 
consistent with vulnerability [17]. The important role played by macrophages in causing 
plaque rupture has been documented in histologic examination of carotid artery plaque. In 
plaque removed during endarterectomy, macrophage density was increased in patients with 
symptoms of stroke or TIA compared to presumably stable plaque removed from 
asymptomatic patients [55, 56]. The strong association between an increase in plaque 
microvessel density, plaque progression and plaque vulnerability has been demonstrated in ex-
vivo studies [39].  Plaque collected during endarterectomy from patients with antecedent 
stroke contained more staining for CD31, an endothelial cell marker when compared to plaque 
from asymptomatic patients [57] Histologic studies show that dysmorphic neovessels are seen 
more often in patients with symptomatic carotid artery disease [58]. We propose to use a 
similar approach to demonstrate that plaque with increased 18F-flotegatide uptake is 
highly enriched for one or several histologic biomarkers of plaque vulnerability 
including inflammation and angiogenesis. 
A positive correlation between 18F-flotegatide extent and enrichment for necrotic lipid-rich 
zones, increased macrophage density and (or) neovascularization would support to our 
hypothesis that uptake of this radiotracer reflects plaque vulnerability at the histologic level.   
Most importantly, we will perform ex-vivo imaging with a portion of plaque from 3 
symptomatic patients to evaluate 18F-flotegatide binding in the presence or absence of 
unlabeled RGD peptide. The goal of this experiment is to demonstrate that 18F-flotegatide 
uptake occurs through an integrin mediated mechanism.  Absence of ex-vivo 18F-flotegatide 
binding to plaque in the presence of unlabeled RGD peptide would suggest that 18F-flotegatide 
uptake is mediated by a specific integrin-RGD interaction. 
 
Experimental Approach  
 
Aim 1.   
18F-flotegatide will be synthesized as described previously and PET imaging of the carotid 
arteries will be performed with MR based attenuation correction.  18F-flotegatide uptake will 
be quantified using commercially available software and plaque uptake of 18F-flotegatide by 
the carotid artery plaque implicated in the stroke will be compared to the contralateral carotid 
artery or other healthy reference vessel.  18F-flotegatide uptake of carotid plaque will also be 
compared between patients with stroke and asymptomatic patients. 18F-flotegatide uptake of 
carotid plaque will be compared to MRI based plaque features of vulnerability. 
Chemical formulation and synthesis of 18F-Flotegatide and dosage for PET imaging 
Pent-4-yn-1-yl 4-methylbenzenesulfonate (pentyne tosylate) will be reacted with anhydrous 
18F-fluoride in the presence of Kryptofix® 2.2.2 and K2CO3 in MeCN at 110°C with the 
resulting 18F-fluoropentyne distilling into a collection vial for the click reaction [52]. The 
collection vial contains Flotegatide azide precursor, Cu(I) (generated by the in situ reduction 
of CuSO4 with sodium ascorbate), and tris[(1-benzyl-1H-1,2,3-triazol-4- yl)methyl]amine 
(TBTA) in aqueous ethanol/acetonitrile [53]. TBTA will be removed during the semi- 
preparative RP-HPLC step and is not detected in the final dose.  After reacting for 10 to 30 
minutes at room temperature, the crude reaction mixture will be transferred to an intermediate 
HPLC loading vial containing water for dilution prior to semi-preparative HPLC purification.  
After purification of the crude reaction mixture by semi-preparative RP-HPLC 
(acetonitrile:aqueous TFA), the product will be reformulated via C18 cartridge reconstitution 
as a solution in a maximum of 10% EtOH:water.  The total process time is 75 minutes, i.e. 


 
 
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less than one half-life. The final drug product formulated in a hydroalcoholic vehicle 
containing up to 10% ethanol will be administered intravenously at a target imaging dose of 
10mCi per patient. Even at the lowest specific activity allowed-under the IND-for injection 
(0.4Ci/μmol), the mass is only 27μg. 
 
Aim 2a.  
Collection of plaque and processing for histologic studies 
Carotid artery containing the plaque (specimen) from 3 symptomatic and 3 asymptomatic 
patients will be obtained at the time of endarterectomy and labeled with sutures to identify 
cranio-caudal orientation. The specimen will be divided into three segments (cranial, middle 
and caudal). This will allow us to identify any particular slice on axial PET images with high 
18F-flotegatide uptake (based on SUVmax) as located in a specific segment of the specimen. We 
propose to pick the slice corresponding to the segment (cranial, middle or caudal) with the 
highest SUVmax. This segment of the specimen containing the plaque will be excised and placed 
in formalin for processing prior to immunohistochemical studies (Aim 2a. Preliminary Results 
Figure 8). 
Histological Evaluation of Plaque Composition 
Plaque will be placed into 10% Neutral Buffered Formalin for 24 hours after which the samples 
will be decalcified (Cal-Rite, Richard-Allen Scientific, Kalamazoo, MI). After fixation and 
decalcification transferred to 70% Ethanol until processing.  5 μm thick sections will be taken 
at 1 mm steps.  Plaque samples will be processed using the Leica Peloris with a xylene free 
processing protocol and embedded in paraffin.  Sections, 5 microns thick, will be made with a 
Leica microtome and placed on positively charged slides.  Sections will be stained using H&E 
and Movat’s pentachrome staining. H&E staining will identify nuclei (black), calcium (purple), 
and hemorrhage(magenta) in contrast to the general pink staining of other tissues. The Movat 
pentachrome can distinguish fibrous regions (dark yellow-green) from lipid rich zones 
(turquoise). The prior presence of cholesterol appears as “cleft” voids after processing. 
Immunohistochemistry (IHC) will be performed on a Benchmark XT (Ventana Medical 
Systems Inc, Tucson, AZ) automated stainer.  Slides will be deparaffinized and blocked with a 
hydrogen peroxide solution.  Antigen retrieval will be performed using Cell Conditioner 1 
(Ventana, 950-124) and a Tris/borate/EDTA buffer, pH 8.0-8.5 @ 95C for 60 minutes.  Slides 
will be incubated with primary antibody for approximately 32 minutes @ 37C followed by 
incubation with secondary antibody, a biotinylated Mouse/Rabbit mixture. The secondary 
antibody and the HRP-based detection are part of the i-View DAB detection kit (Ventana, 790-
091). The slides will be counterstained with Hematoxylin II (Ventana, 790-2208) and Bluing 
Solution (Ventana, 760-2037). Tissue sections will be imaged in their entirety at 20X 
magnification (0.5um/pixel) using a Leica SCN 400FL Slide Scanner (Leica Microsystems, 
GmbH, Wetzlar, Germany).  Antibody staining will be measured using ImagePro Plus Analysis 
software (Media Cybernetics, Inc., Rockville, MD).  The program’s Color Segmentation tool 
will identify regions of HRP staining (brown) for IHC and fibrous regions (dark yellow-green) 
or lipid rich zones (turquoise) for MOVATS in each tissue section. The area of stained tissue 
will be compared to a measurement of the total tissue area to give the “percent area stained.”  
CD31 (JC70), ventana, 760-4178 and CD68 (KP-1), Ventana, 790-2931 will be used to detect 
and quantify macrophage infiltration and endothelial cell density respectively.  Smooth muscle 
proliferation will be identified and quantified using αSMA antibodyDako, M0851.  In order to 


 
 
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correlate 18F- flotegatide uptake with integrin expression, we will use an Integrin αVβIII 
antibody from R&D Systems, MAB3050 and α5β1 integrin antibody from Bioss, bs-2016R. 
Comparison of Immunohistochemical and Movat Pentachrome Staining with 18F-flotegatide 
Uptake 
IHC staining will be quantified as the percent area that is stained with the particular antibody 
for the marker of interest (CD68, macrophage; CD31, neovessel endothelium; αSMA, smooth 
muscle and Integrin αVβIII and α5β1).  Percent area of lipid rich necrotic core and fibrous 
zones will be quantified from Movats staining. Correlation between the TBR of the segment 
with the highest 18F-flotegatide uptake on PET imaging for each individual patient to the 
percent area with lipid, fibrous elements, macrophage staining, endothelial staining and smooth 
muscle staining of the tissue sections from this segment will be evaluated in all patients (Aim 2. 
Preliminary Results Figure 8 and 9). We will also quantify and compare staining in patients 
with symptoms (unstable plaque) vs asymptomatic patients (stable plaque) (Aim 2a. 
Preliminary Results Figure 10). 
Aim 2b.  
Ex-vivo Competition Study to Demonstrate Integrin Mediated Binding of 18F-flotegatide 
A 5mm segment of plaque from 3 symptomatic patients will be collected for assessment of 18F-
flotegatide uptake ex-vivo.  This plaque segment will be embedded in optimum cutting 
temperature compound (Tissue- Plus, Fisher Scientific), snap-frozen in liquid nitrogen and 
stored at –70°C. Samples will be sectioned using a cryostat (Leica Microsystems, Wetzlar, 
Germany) and placed on electrically charged glass slides.  20 µm sections will be warmed to 
room temperature (RT), preincubated for 5 min in PBS buffer, and incubated with   10 nM 18F-
flotegatide (specific activity 2.5Ci/μmol) in PBS buffer for 30 min at RT. Every alternate section 
from each plaque will be pre-treated with 10 mmol of unlabeled cyclic RGD (Siemens) prior to 
incubation with 18F- flotegatide. After incubation, the slides will be washed twice for 5 min 
with PBS, rinsed in cold water, air dried, and placed on an autoradiography imaging plate (Fuji 
Imaging Plate BAS-MS2040, Fuji Photo Film Co., Ltd., Japan). After overnight exposure, 18F 
activity in the imaging plates will be measured with Fuji Analyzer FLA- 2000. After co-
registration of autoradiography and H&E-stained histological images of the sections, count 
density (PSL/mm2) of plaques will be quantified. 18F activity (PSL/mm2) in the absence of 
unlabeled competing tracer will be compared to 18F activity in the presence of competing tracer. 
 
Expected Results 
 
Aim 1.  
1. A detectable increase in the (uptake) TBR of 18F-flotegatide in atherosclerotic plaque 
compared to the unaffected arterial wall. 
2. An increase in TBR (18F-flotegatide uptake) in plaque that is implicated in the stroke or TIA 
compared to plaque that may be present in the contralateral carotid artery in symptomatic 
patients. 
3. Most importantly, a detectable increase in atherosclerotic plaque uptake (TBR) of 18F-
flotegatide in patients with stroke or TIA compared to 18F-flotegatide uptake in plaque in 
patients without symptoms of stroke or TIA. 
4. A positive correlation between volume of lipid rich necrotic core and 18F-flotegatide 
uptake and a negative or lack of correlation between thickness of fibrous cap and 18F-
flotegatide uptake. 


 
 
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Aim 2a and 2b.  
1. Percent area of lipid rich necrotic core quantified from MOVATS staining may be 
positively correlated with TBR of the 18F-flotegatide uptake on PET imaging whereas, 
percent area stained for fibrous elements is expected to exhibit a negative correlation with 
18F-flotegatide uptake. 
2. We expect % area of plaque with CD68 (macrophage infiltration) and (or) CD31 staining 
(endothelial cell) to be higher among patients with increased 18F-flotegatide uptake 
relative to plaque from patients with lower levels of 18F-flotegatide uptake detected by 
PET imaging (Figure 9).  Staining for α-smooth muscle actin may be increased in stable 
plaque with reduced 18F-flotegatide uptake. We also expect to find a strong correlation 
between area of staining with integrins αVβIII, α5β1 and 18F-flotegatide uptake. 
2. We expect to find % area with macrophage staining and neovascular endothelial cell staining 
to be greater in plaque from patients with symptoms of stroke or TIA compared to plaque 
from those who are asymptomatic (Figure 10). 
3. Ex-vivo binding studies:  we expect 18F activity (PSL/mm2) of plaque sections measured 
by (autoradiography) in the absence of unlabeled RGD to be significantly greater than 18F 
activity in the presence of competing unlabeled RGD (Similar to Figure 11). 
 
Feasibility 
 
Aim 1.  
Tracer Synthesis and PET Imaging: The Cleveland Clinic Department of Nuclear Medicine 
has a radiochemistry laboratory for the synthesis of novel PET tracers for research.  The 
radiochemistry laboratory has synthesized several batches of 18F-flotegatide for imaging over 
the last 3 years (see Aim 1. Preliminary Results). In our preliminary studies, the average 
isolated yield of 18F-Flotegatide was 26.9% ± 10.3% with specific activity of 2.6 +/- 1.4 
Ci/umol. We have injected this tracer in 5 patients without any adverse effects.  Siemens has 
allowed us to use the tracer and has expressed support for this project (see letter from Siemens 
in the original RO1 application).  We have injected 18F-flotegatide in 5 patients without any 
adverse effects.  Siemens has allowed us to use the tracer and has expressed support for this 
project (see letter from Siemens).  Furthermore, the PI has previously held an IND from the 
FDA for use of this tracer in human subjects for carotid plaque imaging and will be reapplying 
for an IND from Cedars Sinai Medical Center. Our preliminary results in Table 1, show an 
increased uptake of the radiotracer by carotid plaque that is associated with a recent stroke. In 
these same patients, plaque in the contralateral carotid artery which was not associated with 
stroke (and may therefore represent a relatively stable plaque) did not exhibit significant 18F-
flotegatide uptake (Table 1). We also observed that plaque associated with a stroke (unstable 
plaque) exhibited a higher uptake of 18F-flotegatide relative to stable plaque (asymptomatic 
patients) (Aim 1. Preliminary Results Table 1 and Figure 5). 
PET-MR:  PET-MR is routinely performed in our imaging laboratory (Aim 1. Preliminary 
Results Figure 4). We have the technical expertise of Dr. Zhaoyang Fan at Cedars Sinai 
Medical Center (see letter attached in original RO1 application) and he will be able to help us 
with pulse sequences and quantitative image analysis. 
Aim 2a and 2b.  
We have been able to stain carotid artery sections successfully with CD68 and CD31 (Figure 
8 and 10). We have been able to detect prominent staining with CD68 and CD31 in plaque 


 
 
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from a patient who underwent CEA 8 weeks following a stroke (Figure 8). We are currently 
optimizing staining conditions for α-SMA, αVβIII and α5β1 antibodies.   
Furthermore, we show an association between uptake of 18F-flotegatide and macrophage density 
in 5 patients who underwent plaque imaging with 18F-flotegatide (Figure 9). We have also 
demonstrated that atherosclerotic plaque uptake of 18F-flotegatide occurs via an RGD-specific 
mechanism in a mouse model of atherosclerosis (Figure 11). We expect to find similar 
specificity of 18F-flotegatide binding in human plaque samples. 
 
Limitations and Alternative Strategies  
 
Aim 1.  
Radiotracer Synthesis, PET-MR and Plaque Uptake: We have synthesized and labeled several 
batches of 18F-flotegatide and we do not expect to encounter any technical difficulties in the 
synthesis of 18F-flotegatide.  We have technical support from Siemens for trouble shooting if 
any issues relating to the synthesis of 18F-flotegatide were to arise.  High affinity binding of 
purified 18F-flotegatide to αVβIII and α5β1 has been confirmed in-vitro with inverse surface 
plasmon resonance [53, 54].  Documentation of radiotracer purity from our synthesis will also be 
verified by the FDA (IND to be obtained). In the unlikely event of difficulties in performing 
PET-MR, we will be able to seamlessly switch to PET-CT imaging for detection of 18F-
flotegatide uptake. We have 3 PET/CT scanners located in the Department of Nuclear Medicine.  
Our preliminary data in Figure 5 and 6 and Table 1 indicates that we are likely to be successful 
in demonstrating that atherosclerotic plaque extracts 18F-flotegatide and that uptake of 18F-
flotegatide is increased in unstable plaque compared to stable plaque.  Since we are using 
clinical criteria to define plaque vulnerability, there is the possibility that we may be 
misclassifying some patients. We may be able to include LDL and C-reactive protein (CRP) 
levels in our analysis and consider matching symptomatic and asymptomatic patients based on 
these serum markers (classified as normal vs abnormal). This will allow for comparisons to be 
made between symptomatic and asymptomatic patients with a similar inflammatory and lipid 
profile and minimize potential confounding effects. 
 
Aim 2a and 2b.  
Macrophages and neovascular endothelial cells are considered gold standard histologic 
biomarkers for adjudication of unstable plaque in clinical studies [12, 13, 17, 50, 56, 59]. Based 
on our preclinical study of 18F-flotegatide uptake in a mouse model of atherosclerosis and our 
preliminary data from 5 patients with carotid atheroma, there is a high likelihood of detecting an 
association between macrophage density and 18F- flotegatide uptake. We also plan to compare 
18F-flotegatide uptake to histologic plaque features such as area of fibrous elements and lipid 
core. This will serve as an alternate method by which we can confirm that 18F- flotegatide 
uptake is associated with features of plaque vulnerability.  To maximize sampling of 
macrophages, endothelial cells and smooth muscle cells, we propose to obtain multiple sections 
from the plaque segment with the highest 18F-flotegatide uptake. We have optimized the 
conditions for immunohistochemical staining of CD68 and CD31.  For our proposed studies, we 
will use commercially available antibodies to α-SMA and αVβIII that have been used by other 
investigators with formalin fixed, paraffin embedded plaque samples. The histology core is well 
equipped and has expertise for optimization of staining conditions and has access to other 
vendors of antibodies if this becomes necessary. 


 
 
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Preliminary results  
 
Aim 1.   
Carotid MRI for plaque characterization (Figure 4) and 18F-flotegatide PET-CT in 5 patients (3, 
with stroke due to carotid plaque rupture) with. Preliminary results confirm uptake of 18F-
flotegatide by atherosclerotic plaque in the carotid artery (Figures 5 and 6) and increased uptake 
of 18F-flotegatide by plaque in patients with stroke compared to asymptomatic patients (Figure 
5 and Table 1).  
 
 
 
 
 


 
 
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Aim 2a and 2b.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TIMELINE (ALL AIMS) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
FUTURE DIRECTIONS 
Our long-term goal is to translate the use of 18F-flotegatide for identification of vulnerable 
plaque in individuals with carotid atherosclerosis. The R56 bridge award will enable us to 
optimize PET-MRI of carotid plaque with 18F-flotegatide, demonstrate that 18F-flotegatide 
uptake in carotid plaque can be imaged with PET-MRI and will establish that flotegatide uptake 
in carotid plaque is integrin mediated.  Results from the studies proposed in this R56 application 
will be used as preliminary data (demonstrating the feasibility of using 18F-flotegatide for 
imaging unstable plaque with PET-MRI) in an RO1 application.  In the future RO1 application 


 
 
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we propose to test our hypothesis that 18F-flotegatide may be preferentially extracted and 
retained by carotid artery plaque in patients with clinical symptoms of stroke or TIA (vulnerable 
plaque) compared to asymptomatic control patients (stable plaque) in a larger cohort of 20 
symptomatic patients and 20 asymptomatic controls.  
 
Section 4.0 
 
Trial Design 
  
APPROACH 
PET-MR of Carotid Plaque 
PET-Protocol:  
A total of 6 patients (3 with stroke or TIA and 3 asymptomatic) will undergo noninvasive PET-
MR imaging (Imaging protocol is described below) after injection of 18F-Flotegatide. Our 
preliminary studies show that there is maximal plaque to blood pool contrast at 2h after injection 
of 18F-flotegatide. A dose of 10-12.5 milliCurie (370MBq-462.5MBq) of 18F-flotegatide will 
be injected intravenously and PET-MR imaging will be performed 2 hours (+/- 30 mins) after 
injection on a Siemens Biograph mMR (Siemens Healthcare Erlangen, Germany) using a 
dedicated head and neck coil (Siemens Healthcare). Scout images will be obtained for 
localization of the carotid arteries. An mMR standard Dixon water-fat MR sequence will be 
obtained and segmented (into air, lung tissue, soft tissue and fat) for MR-based PET attenuation 
correction (MR-AC) [54]. PET acquisition will begin with the start of the Dixon MR sequence 
in the same bed position. PET acquisition time will be 15 min in three-dimensional list mode. 
The PET data from the PET-MR scan will be reconstructed using ordered-subsets expectation 
maximization iterative reconstruction algorithm (OSEM 3D) (6 iterations, 21 subsets, zoom 2.0) 
with MR based attenuation correction yielding 512 x 512 image slices (voxel size: 0.70 x 0.70 x 
2.03 mm). Attenuation maps for correction of the PET data from the PET-MR scanner will be 
generated on the basis of the Dixon water-fat MR sequence using the postprocessing software of 
the scanner. Once plaque is localized on the MR images, the corresponding coregistered PET 
image will be visually evaluated for 18F-flotegatide uptake. The region corresponding to the 
plaque on the PET image will be analyzed with commercially available software (MIM) to 
quantify standard uptake value (SUV) and TBR of 18F-flotegatide. MRI of plaque will be 
examined for markers of vulnerability including, fibrous cap, lipid rich necrotic core and 
intraplaque hemorrhage. 
 
MRI-MRA for visualization and co-registration of atherosclerotic plaque:  
The MRI protocol will consist of high- spatial resolution 3D imaging sequences in order to 
detect major vulnerable plaque features and minimize partial volume effects due to tortuous 
carotid arteries. The sequences will be: a) 3D MP-RAGE -a heavily T1- weighted imaging 
sequence for detection of intraplaque hemorrhage; b) 3D time-of-flight (TOF) for definition of 
luminal stenosis; and c) pre- and post-contrast T1-weighted 3D turbo spin echo (SPACE) to 
detect and quantify the lipid-rich necrotic core and fibrous cap. This will be followed by 
contrast injection (Gadolinium- DTPA, single dose 0.1 mmol/kg bodyweight, injection rate 2-3 
cc/sec). Post-contrast SPACE will be acquired 5 minutes post injection. In addition, contrast-
enhanced MRA may also be added into the protocol to obtain high- quality MRA.  Typical 
imaging parameters are:  3D MP-RAGE axial acquisition, TR/TE = 15/3.5 msec, FOV = 
160x160 mm2, resolution = 0.6x0.6 mm2, slice = 2 mm interpolated to 1 mm, flip angle = 15º, 
inversion time = 500 msec, turbo factor = 30.  3D TOF axial acquisition, TR/TE = 20/4.6 msec, 


 
 
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FOV = 160x160 mm2, resolution = 0.6x0.6 mm2, slice = 2 mm interpolated to 1 mm, flip angle 
= 20º.  3D SPACE coronal acquisition, TR/TE = 100/15 msec, FOV = 160x160 mm2, resolution 
= 0.6x0.6 mm2, slice = 0.6 mm, echo train = 50.  Image quality review, processing (multi-planar 
reconstruction, MPR), and tissue characterization (outlining and size quantification) will be 
conducted on an image workstation with FDA-approved carotid plaque analysis software, MRI-
Plaqueview (VPDiagnostics, Seattle, WA). From each of 3D sequences, contiguous cross- 
sectional slices that are perpendicular to the common carotid artery will be reconstructed. 
Intraplaque hemorrhage will be identified on 3D MP-RAGE as a hyper-intense area. Lipid rich 
necrotic core and fibrous cap will be identified on post-contrast 3D SPACE as hypo-intense and 
hyper-intense areas, respectively, with pre- contrast SPACE as reference. Vessel wall, lumen, 
and individual components will be outlined in a semi- automatic fashion. Their volumes will 
then be reported by the software. 
 
Figure 1. Overall Experimental Scheme 
 
 
Patients will be enrolled when they present to the neurologist for evaluation of stroke or TIA or 
when they are seen by vascular surgeon for CEA or an interventional cardiologist for carotid 
stent (Figure 1).  All patients will have fasting lipid levels, C- Reactive protein and HgBA1C 
measured as routine standard of care.  Symptomatic patients would be required to have plaque 
≥70% in at least one carotid artery that would be implicated as the source of embolus responsible 
for the stroke. We propose to enroll 3 patients with stroke or TIA for PET imaging. They will 
undergo PET-MR of the neck (to image the bilateral carotid arteries) with 18F-Flotegatide 
within 96h of stroke or TIA and a simultaneous MRI and magnetic resonance angiography 
(MRA) of the carotid arteries for anatomic localization of the plaque (Specific Aim 1).  In 
addition to providing an anatomic map for coregistration of the images obtained by PET, MRI 


 
 
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will provide morphologic information about the carotid plaque.  Plaque removed during CEA or 
carotid stenting in all patients (within 7d of carotid PET-MR) will be used for histologic 
examination (Specific Aim 2a).  A small portion of the plaque from 3 symptomatic patients will 
be used for ex-vivo 18F-flotegatide uptake-competition studies to confirm integrin specific 
binding (Specific Aim 2b). We propose to also enroll 3 asymptomatic patients with a luminal 
stenosis of ≥70% in at least one carotid artery (stable plaque) referred for CEA or carotid 
stenting. These patients will be age-matched by deciles to symptomatic patients.  These 
patients will undergo PET-MR of the neck (to image the bilateral carotid arteries) with 18F-
flotegatide along with an MRI and MRA of their carotid arteries.  Plaque removed from 
asymptomatic patients at the time of CEA or carotid stenting (within 7d of PET-MR) will be 
used for histologic examination. Exclusion criteria include, stroke due to atrial fibrillation, 
preexisting carotid stents in the artery of interest, renal dysfunction defined as glomerular 
filtration rate <40ml/min, allergy to gadolinium based contrast agents, metal implants 
incompatible with MRI, pregnancy, inability to provide informed consent and age ≤18 years. 
We propose to enroll a total of 6 patients over a period of 1 year. 
 
Section 5.0. 
 
Selection of Subjects 
 
Inclusion Criteria: 
1.   Patients presenting  
a. to the neurologist for evaluation of stroke or TIA (Symptomatic) with: 
i. Symptomatic patients with plaque ≥70% in at least one carotid artery that would be 
implicated as the source of embolus responsible for the stroke/TIA. 
b. or to the vascular surgeon for CEA (Asymptomatic) with: 
i. asymptomatic patients with a luminal stenosis of ≥70% in at least one carotid artery 
(stable plaque) referred for CEA 
ii. age-matched by deciles to symptomatic patients 
c. or to the interventional cardiologist for evaluation and possible carotid stenting with:  
i. asymptomatic patients with a luminal stenosis of ≥50% in at least one carotid artery 
(stable plaque) referred for carotid stenting 
ii. age-matched by deciles to symptomatic patients 
 
Exclusion criteria: 
1. stroke due to atrial fibrillation,  
2. preexisting carotid stents in the artery of interest,  
3. renal dysfunction defined as glomerular filtration rate <40ml/min,  
4. allergy to gadolinium based contrast agents,  
5. Volunteers who have had four or more prior previous gadolinium contrast scans 
6. metal implants incompatible with MRI or other condition that prohibits MRI,  
7. pregnancy,  
8. allergy to animal dander or animal-instigated asthma 
9. inability to provide informed consent and  
10. age ≤18 years.  
 
We propose to enroll a total of 6 patients over a period of 1 year. 
 


 
 
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Section   6.0 
 
Treatment of Subjects 
 
All eligible subjects will undergo PET-MR of the neck (to image the bilateral carotid arteries) 
with 18F-Flotegatide within 96h of stroke or TIA and a simultaneous MRI and magnetic 
resonance angiography (MRA) of the carotid arteries for anatomic localization of the plaque 
 
Plaque removed from asymptomatic patients at the time of CEA or carotid stenting (within 7d of 
PET-MR) will be used for histologic examination. 
 
Section 7.0  
 
Assessment of Efficacy 
 
Validation of the atherosclerotic plaque detected by PET-MRI using 18F-flotegatide will 
be completed via in-vivo PET imaging of patients with unstable carotid plaque. Plaque will 
be quantified by measuring the plaque uptake of 18F-flotegatide relative to 18F-flotegatide 
uptake in segments of the carotid vessel wall that are free of atherosclerosis. 
 
Section 9.0. 
   
Statistics 
 
Image Analysis of plaque using MRI and MRA: Hemorrhage appears bright on MP-RAGE 
(Figure 4, panel A, yellow arrow).  On contrast enhanced T1W images, fibrous portions of 
atherosclerotic plaque appear bright (Figure 4 Panel B, arrowhead) and necrotic core in the 
plaque will be identified on the postcontrast T1W image as the area with no or slight contrast 
enhancement (Figure 4 panel B yellow arrow).  The percent stenosis will be quantified from 
TOF images (panel C, bright lumen is shown by the yellow arrow).  Thickness of the fibrous cap 
and volume of lipid rich necrotic core will be quantified and plaque features including 
hemorrhage, and intact vs ruptured fibrous cap will be recorded. 
 
Image Analysis of PET-MR:  PET-MR images will be fused with the simultaneously acquired 
MR images. Coregistration of PET images with MRI will be performed using anatomic 
landmarks.  Plaque is normally present in the carotid bifurcation and the proximal internal 
carotid artery. Plaque localized on the axial MR images will be correlated with PET signal.  
Uptake of 18F-flotegatide will be quantified in regions of interest (ROI) including both the 
vessel wall and the vessel lumen. The standard uptake value (SUV) in each slice position on 
the axial images will be quantified as SUVmean and SUVmax using commercially available 
software (MIM software Inc, Cleveland, OH). These are derived from the injected dose, time 
to acquisition to account for decay and patient weight. The SUVmax and SUVmean of 
unaffected segment of the carotid artery as well as the uptake of the tracer in the same level in 
the contralateral carotid artery will be quantified.  SUVmean of the blood pool in the aortic 
arch will be quantified to derive background uptake.  SUVmax of the target ROI is divided by 
the background SUVmean to derive a target to background ratio (TBR). The TBR of 18F-
flotegatide in the plaque will be compared to TBR of unaffected vessel wall. 
Comparison of morphologic markers of vulnerability with 18F-flotegatide uptake:  MRI based 
plaque features including, volume of lipid core and thickness of fibrous cap will be directly 
compared to plaque uptake of 18F-flotegatide expressed as TBR from PET-MR images. 


 
 
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Figure 3.  Power Calculations 
 
Statistical Analysis 
Aim 1.  
The statistical analysis is provided to demonstrate how we propose to analyze data when we are 
able to test our hypothesis in a larger group of patients as part of our future RO1.  18F-
flotegatide uptake will be analyzed as a continuous variable.  The thickness of fibrous cap and 
volume of lipid rich necrotic core will also be treated as continuous variables. We will evaluate 
the use of a parametric test or a transformation of the data (e.g. log transform) prior to using a 
parametric test.   However, if this is not appropriate due to distributional assumptions etc., we 
will use a nonparametric Mann Whitney U test with the following null hypotheses: 
1. 18F-flotegatide uptake by carotid plaque (TBR of plaque) in patients with symptoms 
of stroke or TIA is similar to 18F-flotegatide uptake by the unaffected vessel wall. 
2. 18F-flotegatide uptake by carotid plaque (TBR of plaque) implicated in stroke or TIA in 
symptomatic patients is similar to 18F-flotegatide uptake by stable plaque in asymptomatic 
patients. 
3. There is no correlation between 18F-flotegatide uptake and area of the fibrous cap or 
volume of necrotic lipid core quantified by MRI. 
With a sample size of 17 per group, we will have 90% power to detect a 1.2-fold difference in 
the TBR of the 
plaque responsible for causing stroke/TIA compared to TBR of unaffected vessel wall (based on 
level of significance of 0.05 and the standard deviation of 0.3 for TBR measurements).  For our 
correlation studies 
between TBR of 18F-flotegatide uptake and MRI based characteristics of vulnerability, a sample 
size of 19 will 
provide 80% power to detect at least moderate association (correlation coefficient of 0.6) at a 
level of significance of 0.05. 
The goal of our proposed studies in this R56 application is to demonstrate that detection of 
18F-flotegatide by PET-MRI is feasible and that there is a detectable difference in 18F-
flotegatide uptake between unstable and stable atherosclerotic plaque.  Even though it may not 
be statistically significant due to the limited number of patients who will be enrolled during the 


 
 
19 
K5-C200 
BALAJI K TAMARAPPOO, MD, PHD 
 
11NOV2019 
1-year time-frame, we will be able to observe qualitative differences in plaque uptake of 18F-
flotegatide between symptomatic and asymptomatic patients. 
Aim 2a and 2b.  
Figure 7.  Power Calculations for Estimating Sample Size for Histologic 
Comparison Studies 
 
 
We propose to use a Spearman 
correlation (a nonparametric test of 
association) that does not assume 
linearity or normal distribution of the 
values of 18F-flotegatide uptake and 
staining density for macrophages, 
smooth muscle cells and endothelial 
cells. An overall sample size of 19, 
will provide 80% power to detect 
moderate to strong associations 
(correlation coefficient of 0.6 or 
greater) at a level of significance of 
0.05 (Figure 7). Our plan to enroll 40 patients is significantly greater than the sample size 
needed for 80% power.   
 
The goals of Aims 2a and 2b in this R56 application are to demonstrate that 18F-flotegatide 
uptake in carotid plaque is associated with markers of vulnerability and is mediated by 
integrin-flotegatide interaction.  Even though our planned enrollment will not be powered to 
reach statistical significance for Aim 2a, we will be able to demonstrate qualitative differences 
in histologic markers of plaque vulnerability between symptomatic and asymptomatic patients.  
We will also be able to unequivocally demonstrate that 18F-flotegatide uptake in 
atherosclerotic plaque is mediated by integrin binding with our ex-vivo assay (Aim 2b).  
 
Section 10.0.   
 
Quality Control and Quality Assurance 
 
Descriptive summaries will be provided for all primary and secondary end-points. All 
analyses and reports will be developed per Good Clinical Practices. 
 
Section 11.0    
 
Ethical considerations relating to the trial 
This study will be conducted in accordance with Good Clinical Practice (GCP) 
requirements described in the current revision of International Conference on 
Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH) 
Guidelines and all applicable regulations, including current United States Code of 
Federal Regulations (CFR), Title 21, Parts 11, 50, 54, 56, and 312 and Title 45, 
Part 164. Compliance with these regulations and guidelines also constitutes 
compliance with the ethical principles described in the current revision of the 
Declaration of Helsinki. This study will also be carried out in accordance with local 
legal requirements. 


 
 
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BALAJI K TAMARAPPOO, MD, PHD 
 
11NOV2019 
Section 12.0.  
 
Data Handling and Recordkeeping 
 
Patients’ names will remain confidential and will not be included in the database. Only patient 
number, patient initials, and birth date will be recorded in the data system. If the patient name 
appears on any other document collected (e.g., hospital discharge summary), the name will be 
obliterated before the document is transmitted. All study findings will be stored in paper CRFs. 
The patients will give explicit permission for representatives of the Sponsor, regulatory 
authorities, and the IRB/IEC to inspect their medical records to verify the information collected. 
Patients will be informed that all personal information made available for inspection will be 
handled in the strictest confidence and in accordance with all state, local, and federal data 
protection/privacy laws, including, without limitation, the HIPAA. All participants in the study 
will provide written authorization to disclose private health information either as a part of the 
written ICF or as a separate authorization form. The authorization will contain all required 
elements specified by 45 CFR 164 and ICH E6 as applicable, and will contain a waiver of 
patient access to study-related private health information until the conclusion of the clinical 
study. The authorization will remain valid and in full force and effect until the first to occur of 
(1) the expiration of 2 years after the study therapy is approved for the indication being studied, 
or (2) the expiration of 2 years after the research program is discontinued. 
 
Individual patient medical information obtained during this study is confidential and its 
disclosure to third parties (other than those mentioned in this section) is strictly prohibited. In 
addition, medical information obtained during this study may be provided to the patient's 
personal physician or to other appropriate medical personnel when required in connection with 
the patient's continued health and welfare. 
 
The study site will maintain a personal patient identification list (patient and treatment numbers 
with the corresponding patient names) to enable records to be identified. 
 
In accordance with 21 CFR 312.62, an Investigator participating in this study shall retain 
records, including the CRFs and supporting data including signed and dated consent forms, and 
medical records, progress notes of the physician, the individual's hospital chart(s), and the 
nurses' and staff notes. 
 


 
 
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BALAJI K TAMARAPPOO, MD, PHD 
 
11NOV2019 
Section 13.0 
FLOWCHART OF PROCEDURES  
 
Procedures 
 Visit 1 3 
Baseline 
Visit 2  
(Pre-Operative 
PET Scan) 
Follow-up 
Phone Call 
Visit 3 
(Surgical Day) 
 
(-28 days) 
(-7 to 0 days) 
(-4 - +3 Days) 
Day 0 
Standard of Care Procedures: Items, drugs and services that are part of regular care and 
would be done even if you did not take part in this research study.  These will be billed to you 
and/or your insurance company. 
Medical History 
Review 
X 
 
 
 
Medication Review 
X 
X 
X 
Vital Signs x 3 
  
X 
 
Physical Exam 
  
X 
  
 
Electrocardiogram x 
3 
  
X 
  
 
Pre-op blood tests  
including Fasting 
lipid panels, C-
reactive protein and 
HgBA1C1 
 X1 
X1 
  
 
Pregnancy test, if 
applicable 
  
X 
 
 
IV (intravenous) line 
inserted 
  
X 
 
 
Carotid 
Endarterectomy or 
Stenting of Carotid 
Artery 
 
 
 
X 
MRI/MRA Imaging2 
 
X 
 
Research Related Procedures: Items and services done for research purposes only.  These will 
NOT be billed to your insurance company. 
Subject Informed 
Consent 
X 
  
  
  
Inclusion/Exclusion 
Criteria 
X 
  
  
  
PET/MR2 Imaging 
with 18-flotegatilde 
 
X 
 
 
Adverse Events 
As they occur
Sample of carotid 
artery plaque 
removed 
  
  
  
X 
1If labs been collected within 30 days prior to the research imaging tests, no blood will be drawn to 
check your kidney function. 
2 Within 96 hours of stroke or TIA (if applicable; “Symptomatic” subjects only) 
3 Baseline visit (Visit 1) may be combined with Visit 2.
 

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CONSENT FORM FOR RESEARCH
TITLE: 
K5-C200: An Exploratory, Phase II, Open Label, Single-Center, Non-Randomized 
Study Of [F-18] RGD-K5 Positron Emission Tomography (PET) In Participants With 
Carotid Artery Stenosis
STUDY SUPPORT PROVIDED BY: 
NHLBI-NIH (National Institutes of Health) 
PARTICIPATING RESEARCHERS:
Balaji K Tamarappoo, MD, PhD 
Daniel Berman, MD
Louise Thomson, MD
Patrick Lyden, MD
Shlee Song, MD
Suhail Dohad, MD
STUDY CONTACT PHONE NUMBER AT CSMC:   
(310) 423-3095
AFTER HOURS CONTACT (24 HOURS):  
(310) 423-3300
This research study is sponsored by the NIH as part of a grant provided to Dr. Tamarappoo as 
Principal investigator.  NIH only reimburses Cedars-Sinai Medical Center for the costs associated with 
running the study. The NIH is not providing additional compensation to Cedars Sinai Medical Center 
or the Principal Investigator for their participation in the study.
1.
WHAT IS THE PURPOSE OF THIS RESEARCH STUDY?
We are doing this study to examine a new investigational agent compound [F- 18] labeled RGD-K5. 
This agent will be used to help identify areas of plaque in your carotid arteries that may be “unstable” 
or at risk of breaking loose.  This atherosclerotic plaque (a disease of the arteries characterized by the 
deposition of plaques of fatty material on their inner walls) in the carotid artery (major blood vessels in 
the neck that supply blood to the brain, neck, and face) of subjects being considered for carotid 
revascularization [endarterectomy (CEA, i.e. surgical repair of a blocked Carotid artery) or stenting of 
your carotid artery (procedure where a stent is deployed within the lumen of the carotid artery to treat 
narrowing)].  We want to know if this new agent can detect regions of unstable atherosclerotic plaque 
in the carotid artery.
You are being asked to participate in this research study because you have carotid artery stenosis 
(narrowing of your artery due to plaque buildup) and you may undergo a carotid endarterectomy 
(CEA) or stenting of your carotid artery. 
The study may enroll up to 6 people in total at Cedars-Sinai.  
This research study is designed to test the investigational use of compound [F- 18] labeled RGD-K5.  
This drug has not been approved by the U.S. Food and Drug Administration (FDA).
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In this study, we want to learn what effects, good or bad, of compound [F- 18] labeled RGD-K5 has on 
people with your condition. We will give the compound [F- 18] labeled RGD-K5 during the research 
PET/MRI scans to research participants, assess whether it can detect regions of unstable 
atherosclerotic plaque in the carotid artery and watch carefully for any side effects.
2.
WHAT WILL HAPPEN DURING THE STUDY?
This section provides a general overview of what will happen during your participation in this study.  
Information included below should be considered along with the flowchart of procedures attached as 
Appendix A.  
The flowchart shows a timeline for research-related or standard of care procedures that are involved in 
your participation in this study.  Research-related procedures are those performed solely for the 
research.  They would not be performed if you did not take part in the study. Standard of care 
(routine) procedures would be performed both as part of this study and if you do not participate in this 
study.   
    
A table describing common medical procedures done solely for the purposes of monitoring and 
assessing your condition during this study is attached as Appendix B to the end of this consent form.  
Standard of care procedures that will be repeated or done at greater frequency are listed in the flow 
chart, attached Appendix A. 
 
Overview of study:
Visit
What procedures/tests that will be 
done at this visit?
What will need to be brought 
and/or done for this visit?
Baseline Visit 1  
(May happen in your 
doctor’s office or 
hospital and may be 
combined with Visit 2)

Informed Consent reviewed and 
signed*
* Must occur 1st, prior to any other 
research activities
•   Medical History reviewed 
Eligibility Labs, if needed
The purpose of this visit will 
be to gather information to 
ensure you qualify for the 
study
Visit 2 – PET Scans

Vital Signs obtained (2)

Physical exam 

ECG obtained (1)

Blood drawn, if needed

Pregnancy test, if applicable

IV (intravenous) line inserted 

A dose of [F-18]RGD-K5 will be 
given through the IV

PET imaging 
Bring list of medications.
You will be asked how you 
are feeling during your visit
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Follow up Phone call

Phone call day after PET scans 
List of medications taken 
since PET scans
You will be asked how you 
have felt since your PET/CT 
Scans.
Visit 3 - Day of Surgery

After the surgeons complete the 
surgery to remove the plaque in 
your carotid artery, a portion of 
the removed plaque will be 
collected for this research.
Please notify us if your 
surgery is rescheduled or 
cancelled.
 
Research PET/MRI Imaging of the Carotid Arteries:  2 hours (+/- 30 minutes) after receiving the 
dose of [F-18] RGD-K5, PET/MRI imaging of your neck (carotid arteries) will be performed. You will 
be lying on your back with a head fixation device supporting your head to prevent you from moving 
your head while the images are taken. This scan will take about 30-45min minutes. You will be 
permitted to get up and move around between injection of the PET agent and testing. You will be 
asked to drink a glass of water before receiving the dose of [F-18] RGD-K5.
How long will you be in the study:
You will be asked to come to the site approximately for 1 to 2 visits.  The day of your scans you will 
have a PET/MRI it will last approximately 2 ½ to 4 hours. You will be contacted by phone the day 
after the PET/MRI scans for follow up. On the day that you return for your scheduled surgery, a 
portion of the tissue samples from the plaque that is removed by your surgeons will be picked up and 
sent to the lab for this research study.  It is expected that your participation in the study will be less 
than 2 weeks.
3.
WHAT ARE THE POSSIBLE RISKS?
Risks of common medical procedures performed solely for research purposes are described in a table 
attached to the end of this consent form as Appendix B.  Side effects and risks of standard of care 
procedures are not described in this consent form. 
This section describes the possible risks and/or discomforts we anticipate you could experience that 
are related to the study procedures: 
Unknown Risks
There may be risks or side effects related to the study drug that are unknown at this time. Many side 
effects may go away shortly after the study medication or procedure is stopped.  However, in some 
cases they can be serious, long-lasting, permanent, and/or fatal.  You will be notified of any significant 
new findings that become known that may affect your willingness to continue in the study.
Risks Associated with [F-18] RGD-K5: 
Based upon the information from a prior study of 5 subjects, the administration of a single dose of the 
study medication ([F-18] RGD-K5) presented no risks in humans and no adverse events were reported.  
The results of studies in human for compounds that were similar in structure to RDG-K5, reported no 
adverse events.
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Radiation Risk:
This research study involves exposure to radiation from the RGDK5-PET/MRI scan. This radiation 
exposure is not necessary for your medical care and is for research purposes only. The total amount of 
radiation exposure you will receive is equivalent to a uniform whole body exposure of 0.8 rem. This 
amount is comparable to 16% of the maximum annual exposure for radiation workers (5 rem) allowed 
by the Nuclear Regulatory Commission. This use involves minimal risk and is necessary for the 
research information desired. 
Pregnant women, fertile females:
Pregnancy tests will be performed on all women of child-bearing potential before enrolling in the 
study. If the test is positive, you will not be permitted to participate in this study.
Reproductive and Lactation Risks 
Taking part in this research study can affect an unborn baby.  Therefore, you should not become 
pregnant or father a baby while on this study.  If you or your partner is capable of becoming pregnant 
you will need to use birth control.   Check with the researcher about approved birth control methods to 
use while participating in this study.   
Women should not breastfeed a baby while on this study.  
Unknown Risks to the Developing Embryo or Fetus (an unborn baby)
If you are pregnant, or become pregnant during participation in this research, the study drug, 
procedures and radiation might involve risks to an embryo or fetus, which are currently unknown.  It is 
important that you contact the researcher immediately if you believe you might be pregnant.
MRI Related Risks:
The physical procedure of MRI and administration of intravenous contrast are generally considered 
low-risk in patients without significant renal dysfunction and other contraindications to intravenous 
contrast. Since we will exclude patients with significantly elevated creatinine, the risk of renal 
dysfunction is minimal.
Contrast- We will use Gadovist® which is a gadolinium-based contrast agent routinely used in clinical 
magnetic resonance imaging. When injected into the body, gadolinium contrast medium makes certain 
tissues, abnormalities or disease processes more clearly visible on a magnetic resonance imaging scan. 
They are used to improve the clarity of the scanned images or pictures of the body’s internal 
structures. 
Gadolinium contrast medium is given by intravenous injection. 
The risk of gadolinium contrast is minimal in patients without severe renal disease. 
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The most common adverse effects of gadolinium agents (such as Gadovist®) are: 
Common, some may be 
serious (occurs in 
greater than 20% of 
people)
Occasional, some may 
be serious (occurs in 4-
20% of people)
Possible, some may be 
serious (occurs in 1-
3% of people)
Rare, and serious 
(occurs in less than 
1% of people)
None

Itching of the skin 
(5%) 

Headache (4%) 

Nausea (4%)

Very small risk of 
local pain

Hypersensitivity 
(allergic reaction)

low blood pressure 
and 
lightheadedness. 
This can be treated 
immediately with 
IV fluids.
More serious allergic reactions that are life threatening are rare. Gadolinium is not safe for people with 
late stage kidney disease or who have had a liver transplant. Gadolinium-based contrast agents 
increase the risk for a rare, but serious adverse reaction, called nephrogenic systemic fibrosis (NSF) 
among patients with impaired elimination of the drugs. Patients with kidney disease are at increased 
risk of developing NSF. NSF may cause skin thickening, joint pain and/or swelling, causing feelings 
of burning, itching and pain that can be severe. In very rare cases, NSF can lead to lung and heart 
problems and may be life threatening. Participants will have a blood test to measure kidney function 
and if the blood test is abnormal, they will not be permitted to receive gadolinium. 
There is a very small risk of mild headache and local pain. Rarely (<1% of the time) it can cause low 
blood pressure and lightheadedness. This can be treated immediately with IV fluids. More serious 
allergic reactions that are life threatening are rare. Gadolinium is not safe for people with late stage 
kidney disease or who have had a liver transplant. 
As with all research procedures and administration of study drugs, subjects will be monitored at all 
times.
Risks Associated with Contrast Agents:  The U.S. Food and Drug Administration (FDA) is 
investigating the risk of brain deposits following repeated use of gadolinium-based contrast agents 
(GBCAs) for magnetic resonance imaging (MRI). MRIs help detect abnormalities of body organs, 
blood vessels, and other tissues. Recent publications in the medical literature have reported that 
deposits of GBCAs remain in the brains of some patients who undergo four or more contrast MRI 
scans, long after the last administration.  It is unknown whether these gadolinium deposits are harmful 
or can lead to adverse health effects.  FDA, including its National Center for Toxicological Research 
(NCTR), will study this possible safety risk further.  The FDA is working with the research 
community and industry to understand the mechanism of gadolinium retention and to determine if 
there are any potential adverse health effects.  To reduce the potential for gadolinium accumulation,  
FDA has cautioned health care professionals to consider limiting GBCA use to clinical circumstances 
in which the additional information provided by the contrast is necessary. Health care professionals are 
also urged to reassess the necessity of repetitive GBCA MRIs in established treatment protocols.  
Healthy control participants should understand your exposure to GBCAs is not clinically indicated.
Incidental Findings and Duplicate Tests
The procedures in this study are for research purposes and no clinical care or information will be 
provided to you as part of this study. It is possible that the study procedures could detect a medical 
problem that was not the focus of the research and about which you may not already be aware.  If we 
learn that the results of research procedures could suggest information relevant in an important way to 
your health, we will notify you. We will not provide any specific diagnosis about the information seen 
during research scans performed at the Research Imaging Core facility, although we may suggest your 
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physician order a particular test or procedure to further investigate the finding. Your primary care 
physician will determine if it is in your best interest to obtain this test for you. There may be added 
risks of having further diagnostic tests, and we suggest that you discuss this with your primary care 
physician.
You will not be provided with information from the screening blood test to check your kidney function 
other than noting whether your screen met our standards.  If not, you will be told and encouraged to 
discuss this with your primary physician.  We will not provide the blood test results to your primary 
physician.
4.
ARE THERE BENEFITS IN TAKING PART IN THE STUDY?
If you agree to take part in this research study, there may or may not be direct medical benefit to you.  
The possible benefits of taking part in the research study are:  it will allow the study doctor to know 
the entire extent of the carotid plaque, plaque composition and will inform them of the anatomy of the 
carotid artery. Furthermore, if a high inflammatory signal is noted in the carotid artery, there will be a 
greater degree of monitoring from the doctors. However, no benefit is guaranteed.  It is possible that 
your condition may remain unchanged or even get worse.
The knowledge to be gained from this research may be beneficial for other patients, society or science.
5.
WHY WOULD MY PARTICIPATION BE STOPPED?
Your participation in this study may be stopped at any time by the researcher or the sponsor without 
your consent for any reason, including:
•    The study is stopped or suspended;
• 
Funding for the study is reduced, stopped or withdrawn;
•
If it is in your best interest;
• 
You do not consent to continue in the study after being told of changes in the research that may 
affect you;

You do not follow the study procedures.
 
6.
ARE THERE ANY OTHER OPTIONS?
Your participation is voluntary so you have the right to decline to participate or to withdraw from this 
research study at any time without any penalty or loss of benefits to which you would be entitled 
outside of the study.  Choosing not to participate will not affect the care you receive at Cedars-Sinai 
Health System.
The alternative is not to participate. You will receive the same standard of clinical care whether you 
participate or not.
7.
WILL MY INFORMATION BE KEPT CONFIDENTIAL?
We will do our best to make sure that the personal information collected as part of this study is kept 
private.  However, we cannot guarantee total privacy.  A copy of your research consent and 
authorization forms may be filed in your electronic medical record at CSMC.  Your personal 
information may be given out if required by law.  If information from this study is published or 
presented at scientific meetings, your name and other identifiable personal information will not be 
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used. Organizations that may look at and/or copy your medical records for research, quality assurance, 
and data analysis include: accrediting agencies, government and regulatory groups (such as Food and 
Drug Administration (FDA), Office for Human Research Protections (OHRP), etc.), safety monitors, 
companies that sponsor the study, and authorized representatives of the sponsor. 
The study team will facilitate any required access to your records by authorized representatives of the 
Sponsor to verify the information collected for the study.  
You may, depending on the circumstances of the study and applicable law, be asked to sign a separate 
“Authorization Form” that outlines with whom your information may be shared for the purposes of 
this research and under what circumstances.  
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required by 
U.S. Law.  This Website will not include information that can identify you.  At most, the Website will 
include a summary of the results.  You can search this Website at any time.
8.
WHAT IF I BECOME ILL OR INJURED BECAUSE OF TAKING PART IN THIS 
STUDY?
Contact your researcher at once if you feel that you are ill or have been injured because of taking part 
in this study.  If it is a medical emergency, call 911 or go to an emergency room.  Promptly notify your 
researcher of your situation at the phone number listed on page 1 of this consent form.
Who pays for my research related illness or injury?
Cedars-Sinai has no plans to pay for costs associated with the treatment of research-related injury or 
illness.  We will make every effort to seek reimbursement from your health plan.  However, you will 
be responsible for any deductibles and co-payments required under your health plan and for any claims 
ultimately denied by your health plan.  Financial assistance may be available under Cedars-Sinai’s 
Charity Care Policy and Procedure.  If you feel that you have had a research-related injury and need 
financial assistance, please contact the IRB Office at 310-423-3783.
9.
FINANCIAL CONSIDERATIONS
Costs of Participation
Please review the attached Appendix A flowchart for a listing of items, drugs and services that will be 
billed to you and/or your insurance and those that will be covered by the study sponsor.
Only items, drugs and services that are reasonable and necessary for your medical care throughout the 
study will be billed to your insurance.  You remain responsible for all deductibles, co-pays, and 
balances under your health benefit plan. If your insurance company does not pay, you will be billed for 
those charges.  You should check with your health benefit plan if you have questions or concerns 
about your insurance coverage.
Compensation for Participating
You will be reimbursed $300.00 at the completion of the PET/CT scans visit for your time and travel 
involved in coming to the study visit.
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You may be required to complete a W-9 Form in order to receive payment.  The W-9 Form will be 
maintained by our accounting department at Cedars-Sinai.  Although any amount of payment may be 
reportable (check with a tax professional if you have questions about your obligations), if total 
payment by Cedars-Sinai  is $600 or more in a calendar year, a 1099 Form will be filed with the IRS 
in accordance with federal tax law.
If you are a Cedars-Sinai employee, you should provide your employee identification number to the 
research team so that your payment can be appropriately processed through Payroll.  For your own 
protection and to comply with tax laws, your payment for participation will be reported to the IRS 
together with other compensation you receive from Cedars-Sinai.
Financial Interest in the Research
The PI and institution have no potential financial conflict of interest with respect to this study.
10.
WHAT IF I HAVE QUESTIONS OR PROBLEMS?
Please contact one of the investigators listed on the first page of this form for questions or concerns 
about the research.  
If you have questions, problems, or concerns that you want to discuss with someone who is not 
associated with this study, or want to offer suggestions or feedback, please contact:
Cedars-Sinai Human Research Protection Program (HRPP) Phone: (310) 423-3783
Email: ResearchConcerns@cshs.org
The Cedars-Sinai HRPP has been established to protect the rights and welfare of research participants.  
You may also contact the Cedars-Sinai HRPP if you want to offer input or obtain information 
regarding the study.
11. CONSENT PROVISIONS 
If you sign this form below, it means that:  
(1) You have taken the time to carefully read and understand the information presented in this 
informed consent form; you should discuss it with others, and if appropriate seek a second opinion to 
make an informed decision;
(2) The information concerning the research study and its involved procedures has been fully 
explained to you and your questions have been answered to your satisfaction; 
(3) You have received and understand all of the information you desire regarding your participation 
in the research study; 
(4) You have considered the potential risks, any anticipated benefits and alternatives (and their 
relative risks and benefits) of participation;
(5) You are voluntarily agreeing to participate in this research study;
(6) You understand that by consenting to participate in the research, you are not giving up any of your 
legal rights; 
(7) You understand that you have the right to be informed of significant new findings related to this 
research study which may affect your willingness to continue participating in this study; and
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(8) You have been provided with a copy of the “Experimental Subject’s Bill of Rights”, if applicable 
to this research study, and have been provided with an opportunity to ask questions regarding the Bill 
of Rights.
We will give you a copy of this signed and dated consent form and a signed copy of the Experimental 
Subject’s Bill of Rights.
SIGNATURE BY THE PARTICIPANT:
Name of Participant (Print)
Signature of Participant
Date of Signature
SIGNATURE BY THE INVESTIGATOR:
I have personally explained the research to the participant in non-technical terms, answered all 
questions, and attest that he/she freely consents to participate. The participant has been provided with 
the Experimental Subject’s Bill of Rights.
Signature of the Investigator Who Obtained Consent
Date of Signature
SIGNATURE BY THE INTERPRETER/WITNESS
(Signature of an interpreter is only required when a non-English speaking subject is consented with 
the assistance of an interpreter and an IRB-approved ‘short form.’  The witness may be any person 
who is conversant in both English and the language of the Non-English speaking subject, such as the 
interpreter (the certified hospital interpreter), study staff, a family member, or other person.  The 
witness signs the consent forms to confirm that the oral interpretation occurred.)
Signature of Interpreter/Witness
Date of Signature
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EXPERIMENTAL SUBJECT’S BILL OF RIGHTS
In accordance with California Health and Safety Code 24172, any person who is required to consent to 
participate as a subject in a research study involving a medical experiment or who is requested to 
consent on behalf of another has the right to:
1.
Be informed of the nature and purpose of the experiment.
2.
Be given an explanation of the procedures to be followed in the medical experiment, and any 
drug or device to be utilized.
3.
Be given a description of any attendant discomforts and risks to the subject reasonably to be 
expected from the experiment.
4.
Be given an explanation of any benefits to the subject reasonably to be expected from the 
experiment, if applicable.
5.
Be given a disclosure of any appropriate alternative procedures, drugs or devices that might be 
advantageous to the subject, and their relative risks and benefits.
6.
Be informed of the avenues of medical treatment, if any, available to the subject after the 
experiment if complications should arise.
7.
Be given an opportunity to ask any questions concerning the experiment or the procedure 
involved.
8.
Be instructed that consent to participate in the medical experiment may be withdrawn at any 
time and the subject may discontinue participation in the medical experiment without 
prejudice.
9.
Be given a copy of any signed and dated written consent form used in relation to the 
experiment.
10.
Be given the opportunity to decide to consent or not to consent to a medical experiment 
without the intervention of any element of force, fraud, deceit, duress, coercion, or undue 
influence on the subject’s decision.
               ___________________________________________                                ___________________________
Signature of Experimental Subject
Date
CSMC IRB No: Pro00047624 / MOD00000746
CSMC Date Effective: 11/15/2019


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Distribution instruction for researchers:
The signed (i) Consent form, (ii) Authorization for Use and Disclosure of Identifiable Health 
Information and (iii) "Experimental Subject’s Bill of Rights" (the latter required if the research study 
involves medical interventions)* should be distributed to:
1) Medical Chart
2) Research Participant
3) Pharmacy (if a drug study)
4) Principal Investigator’s research records (original)
CSMC IRB No: Pro00047624 / MOD00000746
CSMC Date Effective: 11/15/2019


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APPENDIX A:  FLOWCHART OF PROCEDURES 
Procedures
 
Baselin
e
Visit 2 
(Pre-Operative 
PET Scan)
Follow-up 
Phone Call
Visit 3 
(Surgical 
Day)
(-28 
days)
(-7 to 0 days)
(-4 - +3 
Days)
Day 0
Standard of Care Procedures: Items, drugs and services that are part of regular care and 
would be done even if you did not take part in this research study.  These will be billed to 
you and/or your insurance company.
Medical History Review
X
Medication Review
X
X
X
 
Vital Signs x 3
 
X
 
Physical Exam
 
X
 
Electrocardiogram x 3
 
X
 
Pre-op blood tests  including Fasting lipid 
panels, C-reactive protein and HgBA1C1
 X1
X1
 
Pregnancy test, if applicable
 
X
IV (intravenous) line inserted
 
X
Carotid Endarterectomy or Stenting of 
Carotid Artery
X
MRI/MRA Imaging2
X
Research Related Procedures: Items and services done for research purposes only.  
These will NOT be billed to your insurance company.
Subject Informed Consent
X
 
 
 
Inclusion/Exclusion Criteria
X
 
 
 
PET/MR2 Imaging with 18-flotegatilde
X
Adverse Events
As they occur
Sample of carotid artery plaque 
removed
 
 
 
X
1If labs been collected within 30 days prior to the research imaging tests, no blood will be drawn to check 
your kidney function.
2 Within 96 hours of stroke or TIA (if applicable; “Symptomatic” subjects only)
3 Baseline & Screening visits may be combined and performed in a single visit.


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APPENDIX B:  Detailed Description of Common Medical Procedures Performed for Research Purposes 
and Associated Risks
The procedures listed below are often performed as part of routine care for a person with your condition.  
They are being repeated, or performed more frequently as part of this research.  However, the risks 
associated with each procedure should be comparable to what you would experience even if you were 
undergoing the procedure outside this research study.
Study Procedure
Related Risks
Blood draw: A needle is placed in the vein in 
your arm to draw blood
Blood drawing may cause some pain and has a small 
risk of bleeding, bruising, or infection at the puncture 
site.  There is also a small risk of fainting.
Histology and Immunohistochemistry: During 
your carotid endarterectomy(CEA), atherosclerotic 
plaque will be removed. A sample of this plaque 
will be collected and taken to the histology core 
facility at Lerner Research Institute at the 
Cleveland Clinic, Cleveland, OH. 
There’s no pain or risk associated with the removal of 
the plaque from your carotid artery during the CEA.
Urine Pregnancy test: 
This will only need to be done if you are a woman 
of childbearing potential. This will need to be 
checked prior to scheduling your research imaging 
tests. If your tests are schedules more than 2 days 
(48 hours) since first checked, this test will need to 
be rechecked on the day of your research imaging 
testing. 
There’s no pain or risk associated with a urine 
pregnancy test.
Electrocardiogram (ECG): abbreviated as EKG 
or ECG – is a test that measures the electrical 
activity of the heartbeat using electrodes 
(disposable adhesive discs placed on the skin).
There’s no pain or risk associated with having an 
electrocardiogram.  When the disposable adhesive 
discs are removed from your skin, there may be some 
minor skin discomfort or irritation.  You may 
experience temporary discomfort (pulling on the 
skin/skin hair) during removal of the patches.  This 
hair may be shaved for patch placement.
Intravenous (IV) lines: You will receive the 
study drug or other medications or contrast agent 
through an intravenous (IV) line. An IV line is a 
small tube that is attached to a catheter and 
inserted by needle into a vein usually in your hand 
or arm.  Qualified medical professionals will place 
IV lines for use in this study.
IV lines are usually safe and well tolerated and 
complications are rare, but can include phlebitis 
(swelling of the vein) and infection.  The IV may 
come out accidentally or blood may leak around the 
line.  If the IV is not in the vein, medication or fluid 
can enter the surrounding soft tissues, and can be 
associated with swelling, discomfort, bruising and 
irritation.  Rarely, a clot can develop in the IV line 
itself.  If this happens, the staff may remove the old 
IV line and start a new IV line.  There is also a small 
risk of feeling lightheaded and fainting.
Physical Exam: Includes height, weight, vital 
signs (heart rate and blood pressure)
There are no physical risks associated with these 
procedures.
Concomitant Medications: You will be asked 
about your previous and current medications that 
you take. 
There are no physical risks associated with these 
procedures.
CSMC IRB No: Pro00047624 / MOD00000746
CSMC Date Effective: 11/15/2019


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Medical History Review: You will be asked 
about your medical and surgical history 
There are no physical risks associated with this 
procedure.
Demographic Information: You will be asked 
about your age, gender, race, ethnicity
There are no physical risks associated with these 
procedures.
CSMC IRB No: Pro00047624 / MOD00000746
CSMC Date Effective: 11/15/2019