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
Phase: PHASE2
Conditions: Carotid Arteries
Sponsor: Balaji Tamarappoo

 
 
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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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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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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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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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• 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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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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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 

 
 
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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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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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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.