Biodistribution and Radiation Dosimetry of the Integrin Marker 64Cu-BaBaSar-RGD2 Determined from Whole-Body PET/CT in a Non-human Primate

Introduction: ⁶⁴Cu-BaBaSar-RGD₂ is a positron emission radiotracer taken up by integrin α_vβ₃, which is overexpressed in many malignancies. The aim of this study was to evaluate the biodistribution of ⁶⁴Cu-BaBaSar-RGD₂ in a non-human primate with positron emission tomography (PET) and to estimate the absorbed doses in major organs for human.

Materials and Methods: Whole-body PET imaging was done on a Siemens Biograph scanner with a male macaque monkey. After an i.v. injection of 13.1–19.7 MBq/kg of ⁶⁴Cu-BaBaSar-RGD₂, whole body scan was collected for a total duration of 180 min. Attenuation and scatter corrections were applied to reconstructions of the whole-body emission scan. After image reconstruction, three-dimensional volumes of interest (VOI) were hand-drawn on the PET transaxial or coronal slices of the frame where the organ was most conspicuous. Time-activity curves (TACs) for each VOI were obtained, and residence times of each organ were calculated by integration of the time-activity curves. Human absorbed doses were estimated using the standard human model in OLINDA/EXM software.

Results: Injection of ⁶⁴Cu-BaBaSar-RGD₂ was well-tolerated in the macaque monkey, with no serious tracer-related adverse events observed. ⁶⁴Cu-BaBaSar-RGD₂ was cleared rapidly from the blood pool, with a 12.1-min biological half-life. Increased ⁶⁴Cu-BaBaSar-RGD₂ uptake was observed in the kidneys, and bladder, with mean percentage injected dose (ID%) values at 1 h after injection ~35.50 ± 6.47 and 36.89 ± 5.48, respectively. The calculated effective dose was 15.30 ± 2.21 μSv/MBq, and the kidneys had the highest absorbed dose at 108.43 ± 16.41 μGy/MBq using the non-voiding model. For an injected activity of 925 MBq ⁶⁴Cu for human, the effective dose would be 14.2 ± 2.1 mSv.

Discussion: Due to the limited availability of the primates, we evaluated ⁶⁴Cu-BaBaSar-RGD₂ in the same monkey using three imaging sessions. Measured absorbed doses and effective doses of ⁶⁴Cu-BaBaSar-RGD₂ are comparable to other reported RGD-derived radiopharmaceuticals labeled with ⁶⁴Cu and ¹⁸F. Therefore, ⁶⁴Cu-BaBaSar-RGD₂ can be safely injected into humans for studying integrin α_vβ₃ expression non-invasively.

Introduction

Tumor-induced angiogenesis plays a critical role in tumor progression and metastasis. Without new vasculature and blood circulation, tumor stops growing at the size of 1–2 mm³ and may become necrotic or even apoptotic since diffusion is already insufficient to supply the tissue with oxygen and nutrients [1, 2]. Great efforts have been made to develop therapeutic strategies that interrupt the angiogenic process to stop the tumor growth [3]. Integrin α_vβ₃ is a vital component for the angiogenic process by mediating endothelial cell (EC) migration and survival during angiogenesis [4]. For neovasculature formation, ECs need to migrate into an avascular region and to extensively remodel the extracellular matrix (ECM). In this process, integrins α_vβ₃, an immunoglobulin superfamily molecule has proved to be one of the most important cell adhesion receptors for various ECM proteins. In normal tissues, expression of integrin α_vβ₃ is much lower, making integrin α_vβ₃ an ideal target for diagnosis and therapy in cancer study. A protocol to non-invasively quantify its expression levels will provide a method to document integrin levels, which can support the anti-integrin α_vβ₃ treatment for the patients, and effectively monitor treatment progress for the integrin α_vβ₃-positive patients. Non-invasive detection and quantification of integrin α_vβ₃ is also leading to the diagnosis of many types of cancer at their earliest stages [5].

Peptides containing Arg-Glu-Asp (RGD) amino acid sequence have a high binding affinity and selectivity for integrin α_vβ₃ [6]. In the last two decades, a number of peptides containing RGD sequences have been developed to target tumors overexpressing α_vβ₃ receptors [7]. RGD peptides have been modified and radiolabeled for positron emission tomography (PET) probe development. ¹⁸F-galacto-RGD is the first RGD probe tested in human subjects for detecting α_vβ₃ expression. With conjugation of a sugar moiety for reducing the liver uptake, ¹⁸F-galacto-RGD is still specifically binding to integrin α_vβ₃, shows a more desirable biodistribution in humans, and provides a better visualization of α_vβ₃ expression in tumors with high contrast [8]. However, a major disadvantage for ¹⁸F-galacto-RGD is the long and sophisticated preparation, including multiple synthetic steps that complicate routine production [9]. Due to the importance of RGD peptides, continued efforts have been made to achieve desirable PET probes for easy production, optimal pharmacokinetics, and higher tumor uptake, such as ¹⁸F-AH111585 [10–12], ¹⁸F-alfatide [13, 14], ¹⁸F-RGD-K5 [15, 16], ¹⁸F-FPPRGD₂ [17, 18], ¹⁸F-fluciclatide [12, 19], and ⁶⁸Ga-NOTA-PRGD₂ [20].

⁶⁴Cu [T_1/2 = 12.7 h; β⁺ 0.653 MeV (17.8%)] has been widely used for radiolabeling proteins, antibodies and peptides for PET probe development. The low β⁺ energy of ⁶⁴Cu gives a resolution down to 1 mm in PET images and is important to achieve lower radiation doses for the patients [21]. Cage-like hexaazamacrobicyclic sarcophagine chelator completely encapsulates the coordinated Cu²⁺ ions. Their complexes exhibit enhanced thermodynamic and kinetic stability to copper-binding proteins in vivo [22]. Starting from hexaazamacrobicyclic sarcophagine, we developed the BaBaSar chelator for conjugation with RGD peptide (BaBaSar-RGD₂). The ⁶⁴Cu labeling chemistry for BaBaSar-RGD₂ was achieved at room temperature to give a quantitative yield. The resulted ⁶⁴Cu-BaBaSar-RGD₂ probe shows great stability both in vitro and in vivo, providing high tumor uptake and low normal organ uptake in U87MG glioblastoma tumor bearing mice [23]. Due to the wide application of RGD peptide in diagnostic and therapeutic applications, a novel PET radiotracer for integrin imaging with easy availability would be of great interest to both radiochemists and physicians. To promote the clinical application of ⁶⁴Cu-BaBaSar-RGD₂ in human, we studied its biodistribution in a non-human primate after intravenous (i.v.) administration of ⁶⁴Cu-BaBaSar-RGD₂ and estimated the radiation exposure for humans.

Materials and Methods

General

All chemicals obtained commercially were of analytic grade and used without further purification. ⁶⁴Cu in hydrochloric acid was obtained from Washington University at St. Louis, MO. Reversed phase extraction C₁₈ Sep-Pak cartridges (Waters, Milford, MA) were pretreated with ethanol and water before use. Analytic reversed-phase high-performance liquid chromatography (RP-HPLC) using a Phenomenex Luna column (5 μ, C₁₈, 250 × 4.6 mm) were performed on a Dionex U3000 chromatography system with a diode array detector and radioactivity flow-count (Eckert & Ziegler, Valencia, CA). The recorded data were processed using Chromeleon version 7.20 software. The flow rate of analytical HPLC was 1.0 mL/min. The mobile phase starts from 95% solvent A [0.1% trifluoroacetic acid (TFA) in water] and 5% solvent B [0.1% TFA in acetonitrile (MeCN)]. From 2 to 32 min, the mobile phase ramped to 35% solvent A and 65% solvent B. The ultraviolet (UV) detector of HPLC was set at 254 nm. The endotoxin analysis was performed on a portable Endosafe^®-PTS™ system consisting of LAL reagent and endotoxin controls applied to a single use, polystyrene cartridge.

Radiopharmaceutical Preparation

Production of ⁶⁴Cu-BaBaSar-RGD₂ suitable for monkey injection was accomplished as previous reports [23]. The final solution was passed through a 0.22 μm sterile filter (Pall Corp.). This pyrogen-free ⁶⁴Cu-BaBaSar-RGD₂ possessed an acceptable pH profile (pH 5.0–7.5).

Animal Subjects

The macaque monkey studies were approved by the University of Southern California (USC) Institutional Animal Care and Use Committee (IACUC). A single male macaque monkey was used for 3 imaging sessions. The monkey (M. fascicularis, 8 years old) was initially anesthetized with ketamine (10 mg/kg intramuscularly) to allow preparation and handling of the animals. The animal was intubated, and connected to anesthesia equipment, and i.v. catheters were placed into right and left cephalic veins. Anesthesia was maintained by 1–3% sevoflurane during the study, and the vital signs (heart rate, respiration, and body temperature) were closely monitored and recorded for the duration of the study. ⁶⁴Cu-BaBaSar-RGD₂ was administered intravenously in a 3-mL dose injected with a 10- to 15-s bolus duration.

Once in the PET facility, the animal was placed on the scanner bed in a supine position during the whole scan. The animal was covered with blankets to maintain body temperature. Two intravenous lines were inserted into both cephalic veins, for the administration of the radiotracer and fluids, and for obtaining blood samples during the study, respectively.

PET/CT Acquisition

The primate underwent PET/CT imaging (Biograph Duo LSO; Siemens) immediately after intravenous administration of 111–185 MBq (3–5 mCi) of ⁶⁴Cu-BaBaSar-RGD₂. Low-dose unenhanced CT (pitch, 1.0; 90–130 mAs; 130 kVp) was performed before each scan. Raw CT data were reconstructed using 5-mm slice thickness transverse, and sagittal and coronal CT images were reformatted and generated to coregister to PET, accordingly. A series of 26 whole body PET images was acquired in 2-dimensional mode for a total duration of 180 min. The monkey was placed across the gantry so that one bed position of PET could cover the whole body. A 30-min static scan was acquired 22 h post injection of ⁶⁴Cu-BaBaSar-RGD₂. Venous blood samples were weighed right after they were withdrawn from the monkey. Decay-corrected radioactivity for each blood sample was measured in a NaI(Tl) gamma counter.

PET Reconstruction/Image Analysis

All PET/CT images were recorded in Digital Imaging and Communications in Medicine (DICOM) format and image analysis was performed using a Medical Image Data Examiner (AMIDE 1.0.4). The 26 whole-body images were analyzed individually to obtain the time-activity curves. After careful visual inspection of the images, three dimensional volumes of interests (VOIs) of each major organ were drawn on the emission frame where the organs were most clearly seen, using CT images anatomical reference. The source organs analyzed were including lungs, brain, heart, kidneys, stomach, urinary bladder, liver, and whole body. Time-activity curves (TAC) expressed as the percentage of the injected dose (% ID) at each time point were obtained by the ratio of the VOI activity to the total administered activity of ⁶⁴Cu-BaBaSar-RGD₂. The resulting TACs were fitted to exponential equations, and the curves were extrapolated to the infinity using the fitted equations.

Pharmacokinetics and Metabolic Stability in Blood

After a bolus dose of ⁶⁴Cu-BaBaSar-RGD₂ was administered i.v. into the monkey, 100–300 μL of blood samples were collected at 1, 3, 5, 10, 20, 30, 60, 120, and 180 min after injection. The sample weights were recorded immediately and the activity of each sample was measured by the gamma-counter (Wizard 2, PerkinElmer, Waltham, MA). The pharmacokinetic parameters were calculated using non-compartmental analysis, where half-life was determined using the trapezoidal rule.

Approximately 1 mL blood samples were collected at 30 and 60 min post injection of ⁶⁴Cu-BaBaSar-RGD₂. Blood samples were centrifuged at 3,000 rpm for 5 min. The supernatant was treated with 100 μL trifluoroacetic acid (TFA) and centrifuged at 3,000 rpm for 3 min. The supernatant was passed through Sep-Pak C₁₈ cartridges and the cartridge was washed with 2 mL of water and the fixed activity on C₁₈ was eluted with 2 mL of acetonitrile containing 0.1% TFA. After removal of acetonitrile under reduced pressure, the residue activity was reconstituted in 1.0 mL of water and injected onto an analytic HPLC using the same gradient program with ⁶⁴Cu-BaBaSar-RGD₂ standard. The eluent was collected using a fraction collector (0.5 min/fraction) and the activity of each fraction was measured by a gamma-counter.

Normalized Number of Disintegrations

The %ID for each organ at all time points was fitted to an exponential or sum-of-exponentials function in OLINDA/EXM (version 1.0) software to calculate the total accumulated disintegrations per unit administered activity, which was the normalized number of disintegrations used for the dosimetry calculation. Activity in the remainder of the body at each time point was the difference between the injected activity and the activity in all the source organs. The non-voiding models for urinal bladder in OLINDA/EXM software were used to determine the normalized number of disintegrations for the bladder. Absorbed doses for each organ were calculated using the normalized number of disintegrations of all source organs for each subject. The standardized adult male and female models in OLINDA/EXM were used for the absorbed dose calculation. We assumed that the biodistribution of monkey and human are the same, therefore no adjustment of the macaque monkey biodistribution data was done for calculating human absorbed radiation doses.

Results

Radiochemistry

The ⁶⁴Cu-labeling yield was >95%, and the radiochemical purity of ⁶⁴Cu-BaBaSar-RGD₂ was >99% without the need for purification (Figure 1). The retention times for free ⁶⁴CuCl₂ and ⁶⁴Cu-BaBaSar-RGD₂ on HPLC were 2.5 and 13.9 min, respectively. The reaction crude without purifications did not show free ⁶⁴Cu in HPLC chromatograms. All the quality control results met the pre-specified limits. These included half-life, appearance, pH value, identity, endotoxin amount, etc. (Table 1). The specific activity determined by HPLC analysis was between 14.4 and 22.4 GBq/μmol (average 17.5 ± 4.3 GBq/μmol). Therefore, a human dose (<925 MBq) of ⁶⁴Cu-BaBaSar-RGD₂ contained <125 μg of RGD peptide.

FIGURE 1

Figure 1. Structure of RGD and the synthetic scheme for ⁶⁴Cu-BaBaSar-RGD₂.

TABLE 1

Table 1. Quality control data from 3 synthesis runs.

Residence-Time Calculations

Integrations of time-activity curves were used for the residence time calculation for each organ. The residence time for blood was calculated from the direct venous sample at each time point. The residence times for the spleen and red marrow were indirectly obtained from the blood samples. The spleen residence time was calculated based on its blood fraction (1.5%) of the spleen [24]. The red marrow residence time is calculated using the following equation as reported [24, 25].

${\text{A}}_{\text{rm}}=0.19/(1-0.39){\text{A}}_{\text{Blood}}$

Figure 2 shows the time-activity curve of a few major organs including kidneys, liver, heart, and intestines. Calculated from the time-activity curves, Table 2 shows the residence times for 9 major organs.

FIGURE 2

Figure 2. TACs for ⁶⁴Cu-BaBaSar-RGD₂ in kidneys, liver, heart, and intestines. There was no voiding because monkey was anesthetized.

TABLE 2

Table 2. Normalized number of disintegrations of source organs for macaque monkey injected with ⁶⁴Cu-BaBaSar-RGD₂.

Absorbed Dose Estimates from Macaque Imaging

The injection of 13.1–19.7 MBq/kg of ⁶⁴Cu-BaBaSar-RGD₂ in the macaque monkey produced no observable effects on vital signs (blood pressure, pulse, and electrocardiogram) during and 24-h after PET scan. The PET images at 1, 5, 10, 20, 40, 60, 120, and 180 min after injection are shown in Figure 3. At 1 min, rapid uptake of ⁶⁴Cu-BaBaSar-RGD₂ was observed in the heart, and liver. The bladder content was visualized at 10 min after injection and more and more activity was accumulated in urine bladder content. The bladder did not void because the macaque monkey was under anesthesia. Gallbladder uptake was not observed during the whole scan. Rapid clearance of activity in the liver was observed in the images at time points after 1 min. The urinary bladder had the highest uptake, with 51.37 ± 8.73% of injected activity at 1 h post injection. The maximum uptake for the liver, and kidneys were 37.40 ± 6.63%ID (9 min) and 26.79 ± 4.35%ID (0.5 min) respectively. At 3 h of post injection, 8.62 ± 1.41% of injected activity was found in the gallbladder, small intestine, and upper and lower portions of the large intestine.

FIGURE 3

Figure 3. Decay-corrected anterior maximum-intensity projections of PET/CT at 1, 5, 10, 20, 40, 60, 120, and 180 min after injection of ⁶⁴Cu-BaBaSar-RGD₂ in macaque monkey.

The mean organ doses for the male human phantom were calculated with Olinda/EXM using ⁶⁴Cu-BaBaSar-RGD₂ biodistribution in monkey (Table 3). The kidneys had the highest radiation-absorbed doses (108.43 μGy/MBq), followed by the urinary bladder wall (87.07 μGy/MBq). The mean effective dose of ⁶⁴Cu-BaBaSar-RGD₂ was 15.30 ± 2.21 μSv/MBq. When 925-MBq of ⁶⁴Cu-BaBaSar-RGD₂ is administrated into human subject, the effective dose for the non-voiding model is estimated to be 14.2 mSv, which is comparable to the estimated 6.23 mSv dose in a whole-body PET scan with 2-deoxy-2-[¹⁸F]fluoro-D-glucose (¹⁸F-FDG) [26]. The estimated doses for the female human were higher by 18% because body and organ sizes of women are smaller than those men (data not shown).

TABLE 3

Table 3. Estimated human absorbed doses of ⁶⁴Cu-BaBaSar-RGD₂ to normal organs using biodistribution data from macaque monkey.

Venous blood samples were withdrawn from monkey during the PET scan. Based on the decay corrected activity per unit of blood sample, we found that ⁶⁴Cu-BaBaSar-RGD₂ was cleared rapidly from the blood. By 3 h after injection, 2.88 ± 0.88%ID remained (range, 2.07–3.82%ID). At 22 h after injection, the activity in the blood decreased to 0.79 ± 0.52%ID. Based on the percentage of injected dose in blood sample, the half life of ⁶⁴Cu-BaBaSar-RGD₂ in the blood pool was calculated as 12.1 ± 4.0 min (n = 3).

Metabolite Analysis

Extraction efficiency of the blood samples for metabolite analysis was ~75%. Due to the low radioactivity in blood samples, 30-s fractions from HPLC eluents were collected and radioactivity in each fraction was measured using a gamma counter. Then, the radio-trace chromatograms of each blood sample were re-plotted using the activity in each fraction against the fraction time. No metabolites or free ⁶⁴Cu were detected by HPLC analysis of the blood samples obtained at 30 and 60 min after ⁶⁴Cu-BaBaSar-RGD₂ administration (Figure 4).

FIGURE 4

Figure 4. (A) Standard radiotrace chromatogram of ⁶⁴Cu-BaBaSar-RGD₂. Analysis of the blood samples drawn at 30 min (B) and 60 min (C) after injection of ⁶⁴Cu-BaBaSar-RGD₂ in the monkey.

Discussion

The aims of this work were to estimate the human absorbed dose in a non-human primate model before clinical use of ⁶⁴Cu-BaBaSar-RGD₂ for integrin α_vβ₃ studies. Non human primates are genetically closer to humans than any other animals, so they are considered to be the optimal substitution to study radiopharmaceuticals for human use. The results from this study demonstrate that ⁶⁴Cu-BaBaSar-RGD₂ is promising as a PET agent, based on the biodistribution and safety profile in monkey. With the information of the uptake levels in the monkey normal organs, the estimated radiation exposure for humans through WB PET of ⁶⁴Cu-BaBaSar-RGD₂ was obtained.

In the macaque monkey, the biodistribution of ⁶⁴Cu-BaBaSar-RGD₂ shows high uptake in the kidney and bladder and steady renal clearance. At 1 h after injection of ⁶⁴Cu-BaBaSar-RGD₂, 72.39 ± 10.57% of injected activity was accumulated in kidney and bladder. To our delight, ⁶⁴Cu-BaBaSar-RGD₂ was cleared rapidly from all the organs. The intestinal elimination of ⁶⁴Cu-BaBaSar-RGD₂ was indicated by gut activity loss. The liver and heart had high activity accumulation in the early stages after injection, which cleared very rapidly as time progressed.

In Table 4, we compared the radiation doses of ⁶⁴Cu-BaBaSar-RGD₂ with a few other PET probes including ¹⁸F-galacto-RGD [9], ¹⁸F-FDG [27], and ⁶⁴Cu-RGD₄ [28]. The absorbed doses in the heart, and brain were much lower for ⁶⁴Cu-BaBaSar-RGD₂ than those for ¹⁸F-FDG. The low brain uptake could be attributed to the fact that ⁶⁴Cu-BaBaSar-RGD₂ could not cross the blood-brain barrier (BBB). Because the major clearance route was the urinal system, the absorbed doses of ⁶⁴Cu-BaBaSar-RGD₂ in urinary bladder, and kidneys were higher than those for ¹⁸F-FDG. From previous reports, all the RGD-derived PET probes were excreted through the urinary system, so that the urinary bladder doses of RGD-related PET probes such as ¹⁸F-Galacto-RGD, ⁶⁴Cu-RGD₄, and ⁶⁴Cu-BaBaSar-RGD₂ were much higher than that for ¹⁸F-FDG. However, patients can urinate frequently to reduce the bladder dose, which is the second highest among all the organs in this study. Comparing the dosimetry of ⁶⁴Cu-RGD₄ and ⁶⁴Cu-BaBaSar-RGD₂, we find out that ⁶⁴Cu-BaBaSar-RGD₂ has much higher kidney dose than ⁶⁴Cu-RGD₄. BaBaSar bifunctional chelator is a polyamine macrobicyclic ligand, which is more positively charged than the DOTA (tetraazacyclododecane-1,4,7,10-tetraacetic acid) counterpart in ⁶⁴Cu-RGD₄. The electrostatic interaction between more positively charged ⁶⁴Cu-BaBaSar-RGD₂ and negatively charged surface of renal proximal tubular epithelial cells would favor the re-absorption of the filtered ⁶⁴Cu-BaBaSar-RGD₂ by the glomerular apparatus. This re-absorption process possibly increases the retention of ⁶⁴Cu-BaBaSar-RGD₂ in kidneys and their corresponding radiation dose. This result is consistent with previous study for the molecular charges on the renal uptake of ¹¹¹In-labeled octreotide [29]. On the contrary, the liver and pancreas doses of ⁶⁴Cu-RGD₄ are much higher than those of ⁶⁴Cu-BaBaSar-RGD₂, which might be due to the higher lipophilicity of the RGD tetramer than the RGD dimer [28].

TABLE 4

Table 4. Organ doses in μGy/MBq for several pet imaging agents.

Between the monkey and human, there were many similarities on biodistribution and dosimetry data. Despite being similar physiology to humans, human effective dose using monkey data could be either underestimated or overestimated. For example, the effective dose of hypoxia marker ¹⁸F-HX4 estimated from monkey study is significantly higher (42 μSv/MBq) than the results obtained from humans (27 μSv/MBq) [30]. Moreover, although we do not expect that the anesthesia affects the biodistribution and metabolism of ⁶⁴Cu-BaBaSar-RGD₂, it has been found that different anesthetic protocols can result a significant effect on the uptake of many organs [31]. Another issue to be discussed in this study is that we used a single monkey for 3 imaging sessions due to the limitation of monkey number. To make up for the limited number of animals, we performed the following arrangements. (1) The 3 imaging sessions were acquired over 8-month period. It was considerably long interval for the monkey to recover. Over this period, the monkey body weights were changed which would have an impact on the biodistribution of the tracer for statistical purpose. (2) We injected different amounts of tracer (13.1–19.7 MBq/kg) into the animal to test the variations of each scan. Our plan is to test ⁶⁴Cu-BaBaSar-RGD₂ in healthy volunteers and patients for integrin quantification. One injection per human will be applied in the study. The human radiation dose will be calculated and compared with this study after the first human trial with ⁶⁴Cu-BaBaSar-RGD₂.

Conclusion

We have successfully estimated human radiation dosimetry of ⁶⁴Cu-BaBaSar-RGD₂ after intravenous administration in a macaque monkey, by PET imaging and OLINDA/EXM calculations. The critical organs were kidneys and urinary bladder wall. The mean effective dose, determined with the male adult model, was 15.30 ± 2.21 μSv/MBq. This novel PET probe demonstrates an acceptable radiation dose comparable to other reported RGD-derived radiopharmaceuticals. The findings from this investigation show great promise of ⁶⁴Cu-BaBaSar-RGD₂ as an integrin marker, with a desirable biodistribution and safety characteristics in monkey. Therefore, ⁶⁴Cu-BaBaSar-RGD₂ can safely be used for human scans for further evaluation of its performance as an integrin-targeting probe.

Author Contributions

SL designed and performed research, analyzed data and wrote the paper. IV and RP performed research and analyzed data. PC designed the research and wrote the paper.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work was supported by the Whittier Foundation and University of Southern California's Molecular Imaging Center. We thank Kristin Oleary for operating the clinic PET/CT for achieving the raw data.

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