PET Imaging of Sphingosine-1-Phosphate Receptor 1 with [18F]TZ4877 in Nonhuman Primates.

Purpose: The sphingosine-1-phosphate receptor-1 (S1PR1) is involved in regulating responses to neuroimmune stimuli. There is a need for S1PR1-specific radioligands with clinically suitable brain pharmcokinetic properties to complement existing radiotracers. This work evaluated a promising S1PR1 radiotracer, [18F]TZ4877, in nonhuman primates. Procedures: [18F]TZ4877 was produced via nucleophilic substitution of tosylate precursor with K[18F]/F- followed by deprotection. Brain PET imaging data were acquired with a Focus220 scanner in two Macaca mulatta (6, 13 years old) for 120-180 min following bolus injection of 118-163 MBq [18F]TZ4877, with arterial blood sampling and metabolite analysis to measure the parent input function and plasma free fraction (f P). Each animal was scanned at baseline, 15-18 min after 0.047-0.063 mg/kg of the S1PR1 inhibitor ponesimod, 33 min after 0.4-0.8 mg/kg of the S1PR1-specific compound TZ82112, and 167-195 min after 1 ng/kg of the immune stimulus endotoxin. Kinetic analysis with metabolite-corrected input function was performed to estimate the free fraction corrected total distribution volume (V T/f P). Whole-body dosimetry scans were acquired in 2 animals (1M, 1F) with a Biograph Vision PET/CT System, and absorbed radiation dose estimates were calculated with OLINDA. Results: [18F]TZ4877 exhibited fast kinetics that were described by the reversible 2-tissue compartment model. Baseline [18F]TZ4877 f P was low (< 1%), and [18F]TZ4877 V T/f P values were 233-866 mL/cm3. TZ82112 dose-dependently reduced [18F]TZ4877 V T/f P, while ponesimod and endotoxin exhibited negligible effects on V T/f P, possibly due to scan timing relative to dosing. Dosimetry studies identified the critical organs of gallbladder (0.42 (M) and 0.31 (F) mSv/MBq) for anesthetized nonhuman primate. Conclusions: [18F]TZ4877 exhibits reversible kinetic properties, but the low f P value limits quantification with this radiotracer. S1PR1 is a compelling PET imaging target, and these data support pursuing alternative F-18 labeled radiotracers for potential future human studies.

Design, synthesis, and biological evaluation of multiple F-18 S1PR1 radiotracers in rodent and nonhuman primate.

Here we report our design and synthesis of 28 new fluorine-containing compounds as potential F-18 radiotracers for CNS imaging of sphingosine-1-phosphate receptor 1 (S1PR1), and determination of their in vitro binding potency and selectivity toward S1PR1 over other S1PR subtypes. Nine potent and selective compounds, 7c&d, 9a&c, 12b, 15b, and 18a-c with IC50 values ranging from 0.6-12.3 nM for S1PR1 and weak binding toward S1PR2, 3, 4, and 5, were further 18F-radiolabeled to produce [18F]7c&d, [18F]9a&c, [18F]12b, [18F]15b, and [18F]18a-c. Multi-step F-18 radiochemistry procedures were investigated for radiosynthesis of [18F]7c&d and [18F]9a&c, and the presumed intermediates were synthesized and authenticated by analytic HPLC. We then performed nonhuman primate (NHP) PET brain imaging studies for eight radiotracers: [18F]7c&d, [18F]9a, [18F]12b, [18F]15b, and [18F]18a-c. Three radiotracers, [18F]7c, [18F]7d, and [18F]15b, had high NHP brain uptake with standardized uptake values (SUVs) at 2 h post-injection of 2.42, 2.84, and 2.00, respectively, and good brain retention. Our ex vivo biodistribution study in rats confirmed [18F]7d had a high brain uptake with no in vivo defluorination. Radiometabolic analysis of [18F]7c and [18F]7d in rat plasma and brain samples found that [18F]7c has a more favorable metabolic profile than [18F]7d. However, the trend of increased brain uptake precludes [18F]7c as a suitable PET radiotracer for imaging S1PR1 in the brain. Further structural optmization is warranted to identify a highly S1PR1-specific radiotracer with rapid brain uptake kinetics.

Metabolite Study and Structural Authentication for the First-in-Human Use Sphingosine-1-phosphate Receptor 1 Radiotracer.

The sphingosine-1-phosphate receptor 1 (S1PR1) radiotracer [11C]CS1P1 has shown promise in proof-of-concept PET imaging of neuroinflammation in multiple sclerosis (MS). Our HPLC radiometabolite analysis of human plasma samples collected during PET scans with [11C]CS1P1 detected a radiometabolite peak that is more lipophilic than [11C]CS1P1. Radiolabeled metabolites that cross the blood-brain barrier complicate quantitative modeling of neuroimaging tracers; thus, characterizing such radiometabolites is important. Here, we report our detailed investigation of the metabolite profile of [11C]CS1P1 in rats, nonhuman primates, and humans. CS1P1 is a fluorine-containing ligand that we labeled with C-11 or F-18 for preclinical studies; the brain uptake was similar for both radiotracers. The same lipophilic radiometabolite found in human studies also was observed in plasma samples of rats and NHPs for CS1P1 labeled with either C-11 or F-18. We characterized the metabolite in detail using rats after injection of the nonradioactive CS1P1. To authenticate the molecular structure of this radiometabolite, we injected rats with 8 mg/kg of CS1P1 to collect plasma for solvent extraction and HPLC injection, followed by LC/MS analysis of the same metabolite. The LC/MS data indicated in vivo mono-oxidation of CS1P1 produces the metabolite. Subsequently, we synthesized three different mono-oxidized derivatives of CS1P1 for further investigation. Comparing the retention times of the mono-oxidized derivatives with the metabolite observed in rats injected with CS1P1 identified the metabolite as N-oxide 1, also named TZ82121. The MS fragmentation pattern of N-oxide 1 also matched that of the major metabolite in rat plasma. To confirm that metabolite TZ82121 does not enter the brain, we radiosynthesized [18F]TZ82121 by the oxidation of [18F]FS1P1. Radio-HPLC analysis confirmed that [18F]TZ82121 matched the radiometabolite observed in rat plasma post injection of [18F]FS1P1. Furthermore, the acute biodistribution study in SD rats and PET brain imaging in a nonhuman primate showed that [18F]TZ82121 does not enter the rat or nonhuman primate brain. Consequently, we concluded that the major lipophilic radiometabolite N-oxide [11C]TZ82121, detected in human plasma post injection of [11C]CS1P1, does not enter the brain to confound quantitative PET data analysis. [11C]CS1P1 is a promising S1PR1 radiotracer for detecting S1PR1 expression in the CNS.

Development of a CD163-Targeted PET Radiotracer That Images Resident Macrophages in Atherosclerosis.

Tissue-resident macrophages are complementary to proinflammatory macrophages to promote the progression of atherosclerosis. The noninvasive detection of their presence and dynamic variation will be important to the understanding of their role in the pathogenesis of atherosclerosis. The goal of this study was to develop a targeted PET radiotracer for imaging CD163-positive (CD163+) macrophages in multiple mouse atherosclerosis models and assess the potential of CD163 as a biomarker for atherosclerosis in humans. Methods: CD163-binding peptide was identified using phage display and conjugated with a NODAGA chelator for 64Cu radiolabeling ([64Cu]Cu-ICT-01). CD163-overexpressing U87 cells were used to measure the binding affinity of [64Cu]Cu-ICT-01. Biodistribution studies were performed on wild-type C57BL/6 mice at multiple time points after tail vein injection. The sensitivity and specificity of [64Cu]Cu-ICT-01 in imaging CD163+ macrophages upregulated on the surface of atherosclerotic plaques were assessed in multiple mouse atherosclerosis models. Immunostaining, flow cytometry, and single-cell RNA sequencing were performed to characterize the expression of CD163 on tissue-resident macrophages. Human carotid atherosclerotic plaques were used to measure the expression of CD163+ resident macrophages and test the binding specificity of [64Cu]Cu-ICT-01. Results: [64Cu]Cu-ICT-01 showed high binding affinity to U87 cells. The biodistribution study showed rapid blood and renal clearance with low retention in all major organs at 1, 2, and 4 h after injection. In an ApoE-/- mouse model, [64Cu]Cu-ICT-01 demonstrated sensitive and specific detection of CD163+ macrophages and capability for tracking the progression of atherosclerotic lesions; these findings were further confirmed in Ldlr-/- and PCSK9 mouse models. Immunostaining showed elevated expression of CD163+ macrophages across the plaques. Flow cytometry and single-cell RNA sequencing confirmed the specific expression of CD163 on tissue-resident macrophages. Human tissue characterization demonstrated high expression of CD163+ macrophages on atherosclerotic lesions, and ex vivo autoradiography revealed specific binding of [64Cu]Cu-ICT-01 to human CD163. Conclusion: This work reported the development of a PET radiotracer binding CD163+ macrophages. The elevated expression of CD163+ resident macrophages on human plaques indicated the potential of CD163 as a biomarker for vulnerable plaques. The sensitivity and specificity of [64Cu]Cu-ICT-01 in imaging CD163+ macrophages warrant further investigation in translational settings.

Rationale and design of the RAPID-WATER-FLOW trial: Radiolabeled perfusion to identify coronary artery disease using water to evaluate responses of myocardial FLOW.

OBJECTIVES: The objective of this study was to determine the diagnostic performance of 15O-water positron emission tomography (PET) myocardial perfusion imaging to detect coronary artery disease (CAD) using the truth-standard of invasive coronary angiography (ICA) with fractional flow reserve (FFR) or instantaneous wave-Free Ratio (iFR) or coronary computed tomography angiogram (CCTA). BACKGROUND: 15O-water has a very high first-pass extraction that allows accurate quantification of myocardial blood flow and detection of flow-limiting CAD. However, the need for an on-site cyclotron and lack of automated production at the point of care and relatively complex image analysis protocol has limited its clinical use to date. METHODS: The RAPID WATER FLOW study is an open-label, multicenter, prospective investigation of the accuracy of 15O-water PET to detect obstructive angiographic and physiologically significant stenosis in patients with suspected CAD. The study will include the use of an automated system for producing, dosing, and injecting 15O-water and enrolling approximately 215 individuals with suspected CAD at approximately 10 study sites in North America and Europe. The primary endpoint of the study is the diagnostic sensitivity and specificity of the 15O-water PET study using the truth-standard of ICA with FFR or iFR to determine flow-limiting stenosis, or CCTA to rule out CAD and incorporating a quantitative analytic platform developed for the 15O-water PET acquisitions. Sensitivity and specificity are to be considered positive if the lower bound of the 95% confidence interval is superior to the threshold of 60% for both, consistent with prior registration studies. Subgroup analyses include assessments of diagnostic sensitivity, specificity, and accuracy in female, obese, and diabetic individuals, as well as in those with multivessel disease. All enrolled individuals will be followed for adverse and serious adverse events for up to 32 hours after the index PET scan. The study will have >90% power (one-sided test, α = 0.025) to test the hypothesis that sensitivity and specificity of 15O-water PET are both >60%. CONCLUSIONS: The RAPID WATER FLOW study is a prospective, multicenter study to determine the diagnostic sensitivity and specificity of 15O-water PET as compared to ICA with FFR/iFR or CCTA. This study will introduce several novel aspects to imaging registration studies, including a more relevant truth standard incorporating invasive physiologic indexes, coronary CTA to qualify normal individuals for eligibility, and a more quantitative approach to image analysis than has been done in prior pivotal studies. CLINICAL TRIAL REGISTRATION INFORMATION: Clinical-Trials.gov (#NCT05134012).

Toward Quantitative Multisite Preclinical Imaging Studies in Acute Myocardial Infarction: Evaluation of the Immune-Fibrosis Axis.

The immune-fibrosis axis plays a critical role in cardiac remodeling after acute myocardial infarction. Imaging approaches to monitor temporal inflammation and fibroblast activation in mice have seen wide application in recent years. However, the repeatability of quantitative measurements remains challenging, particularly across multiple imaging centers. We aimed to determine reproducibility of quantitative inflammation and fibroblast activation images acquired at 2 facilities after myocardial infarction in mice. Methods: Mice underwent coronary artery ligation and sequential imaging with 68Ga-DOTA-ECL1i to assess chemokine receptor type 2 expression at 3 d after myocardial infarction and 68Ga-FAPI-46 to assess fibroblast activation protein expression at 7 d after myocardial infarction. Images were acquired at 1 center using either a local or a consensus protocol developed with the second center; the protocols differed in the duration of isoflurane anesthesia and the injected tracer dose. A second group of animals were scanned at the second site using the consensus protocol. Image analyses performed by each site and just by 1 site were also compared. Results: The uptake of 68Ga-DOTA-ECL1i in the infarct territory tended to be higher when the consensus protocol was used (P = 0.03). No difference was observed between protocol acquisitions for 68Ga-FAPI-46. Compared with the local protocol, the consensus protocol decreased variability between individual animals. When a matched consensus protocol was used, the 68Ga-DOTA-ECL1i infarct territory percentage injected dose per gram of tissue was higher on images acquired at site B than on those acquired at site A (P = 0.006). When normalized to body weight as SUV, this difference was mitigated. Both the percentage injected dose per gram of tissue and the SUV were comparable between sites for 68Ga-FAPI-46. Image analyses at the sites differed significantly, but this difference was mitigated when all images were analyzed at site A. Conclusion: The application of a standardized acquisition protocol may lower variability within datasets and facilitate comparison of molecular radiotracer distribution between preclinical imaging centers. Like clinical studies, multicenter preclinical studies should use centralized core-based image analysis to maximize reproducibility across sites.

State-of-the-Art Imaging of Infiltrative Cardiomyopathies: A Scientific Statement From the American Heart Association.

Infiltrative cardiomyopathies comprise a broad spectrum of inherited or acquired conditions caused by deposition of abnormal substances within the myocardium. Increased wall thickness, inflammation, microvascular dysfunction, and fibrosis are the common pathological processes that lead to abnormal myocardial filling, chamber dilation, and disruption of conduction system. Advanced disease presents as heart failure and cardiac arrhythmias conferring poor prognosis. Infiltrative cardiomyopathies are often diagnosed late or misclassified as other more common conditions, such as hypertrophic cardiomyopathy, hypertensive heart disease, ischemic or other forms of nonischemic cardiomyopathies. Accurate diagnosis is also critical because clinical features, testing methodologies, and approach to treatment vary significantly even within the different types of infiltrative cardiomyopathies on the basis of the type of substance deposited. Substantial advances in noninvasive cardiac imaging have enabled accurate and early diagnosis. thereby eliminating the need for endomyocardial biopsy in most cases. This scientific statement discusses the role of contemporary multimodality imaging of infiltrative cardiomyopathies, including echocardiography, nuclear and cardiac magnetic resonance imaging in the diagnosis, prognostication, and assessment of response to treatment.

Chemokine Receptor 2 Is A Theranostic Biomarker for Abdominal Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) is a degenerative vascular disease impacting aging populations with a high mortality upon rupture. There are no effective medical therapies to prevent AAA expansion and rupture. We previously demonstrated the role of the monocyte chemoattractant protein-1 (MCP-1) / C-C chemokine receptor type 2 (CCR2) axis in rodent AAA pathogenesis via positron emission tomography/computed tomography (PET/CT) using CCR2 targeted radiotracer 64 Cu-DOTA-ECL1i. We have since translated this radiotracer into patients with AAA. CCR2 PET showed intense radiotracer uptake along the AAA wall in patients while little signal was observed in healthy volunteers. AAA tissues collected from individuals scanned with 64 Cu-DOTA-ECL1i and underwent open-repair later demonstrated more abundant CCR2+ cells compared to non-diseased aortas. We then used a CCR2 inhibitor (CCR2i) as targeted therapy in our established male and female rat AAA rupture models. We observed that CCR2i completely prevented AAA rupture in male rats and significantly decreased rupture rate in female AAA rats. PET/CT revealed substantial reduction of 64 Cu-DOTA-ECL1i uptake following CCR2i treatment in both rat models. Characterization of AAA tissues demonstrated decreased expression of CCR2+ cells and improved histopathological features. Taken together, our results indicate the potential of CCR2 as a theranostic biomarker for AAA management.

Quantitative Analysis of S1PR1 Expression in the Postmortem Multiple Sclerosis Central Nervous System.

Multiple sclerosis (MS) is an immune-mediated disease that is characterized by demyelination and inflammation in the central nervous system (CNS). Previous studies demonstrated that sphingosine-1-phosphate receptor (S1PR) modulators effectively inhibit S1PR1 in immune cell trafficking and reduce entry of pathogenic cells into the CNS. Studies have also implicated a nonimmune, inflammatory role of S1PR1 within the CNS in MS. In this study, we explored the expression of S1PR1 in the development and progression of demyelinating pathology of MS by quantitative assessment of S1PR1 expression using our S1PR1-specific radioligand, [3H]CS1P1, in the postmortem human CNS tissues including cortex, cerebellum, and spinal cord of MS cases and age- and sex-matched healthy cases. Immunohistochemistry with whole slide scanning for S1PR1 and various myelin proteins was also performed. Autoradiographic analysis using [3H]CS1P1 showed that the expression of S1PR1 was statistically significantly elevated in lesions compared to nonlesion regions in the MS cases, as well as normal healthy controls. The uptake of [3H]CS1P1 in the gray matter and nonlesion white matter did not significantly differ between healthy and MS CNS tissues. Saturation autoradiography analysis showed an increased binding affinity (Kd) of [3H]CS1P1 to S1PR1 in both gray matter and white matter of MS brains compared to healthy brains. Our blocking study using NIBR-0213, a S1PR1 antagonist, indicated [3H]CS1P1 is highly specific to S1PR1. Our findings demonstrated the activation of S1PR1 and an increased uptake of [3H]CS1P1 in the lesions of MS CNS. In summary, our quantitative autoradiography analysis using [3H]CS1P1 on human postmortem tissues shows the feasibility of novel imaging strategies for MS by targeting S1PR1.

Characterization of a S1PR2 specific 11C-labeled radiotracer in streptozotocin-induced diabetic murine model.

BACKGROUND: Diabetes mellitus is a chronic progressive metabolic disorder that affects millions of people worldwide. Emerging evidence suggests the important roles of sphingolipid metabolism in diabetes. In particular, sphingosine-1-phosphate (S1P) and S1P receptor 2 (S1PR2) have important metabolic functions and are involved in several metabolic diseases. In diabetes, S1PR2 can effectively preserve ß cells and improve glucose/insulin tolerance in high-fat diet induced and streptozotocin (STZ)-induced diabetic mouse models. We previously developed a group of potent and selective S1PR2 ligands and radioligands. METHODS: In this study, we continued our efforts and characterized our leading S1PR2 radioligand, [11C]TZ34125, in a STZ-induced diabetic mouse model. [11C]TZ34125 was radiosynthesized in an automated synthesis module and in vitro saturation binding assay was performed using recombinant human S1PR2 membrane. In vitro saturation autoradiography analysis was also performed to determine the binding affinity of [11C]TZ34125 against mouse tissues. Type-1 diabetic mouse model was developed following a single high dose of STZ in C57BL/6 mice. Ex vivo biodistribution was performed to evaluate the distribution and amount of [11C]TZ34125 in tissues. In vitro autoradiography analysis was performed to compare the uptake of [11C]TZ34125 between diabetic and control animals in mouse spleen and pancreas. RESULTS: Our in vitro saturation binding assay using [11C]TZ34125 confirmed [11C]TZ34125 is a potent radioligand to recombinant human S1PR2 membrane with a Kd value of 0.9 nM. Saturation autoradiographic analysis showed [11C]TZ34125 has a Kd of 67.5, 45.9, and 25.0 nM to mouse kidney, spleen, and liver tissues respectively. Biodistribution study in STZ-induced diabetic mice showed the uptake of [11C]TZ34125 was significantly elevated in the spleen (~2 fold higher) and pancreas (~1.4 fold higher) compared to normal controls. The increased uptake of [11C]TZ34125 was further confirmed using autoradiographic analysis in the spleen and pancreases of STZ-induced diabetic mice, indicating S1PR2 can potentially act as a biomarker of diabetes in pancreases and inflammation in spleen. Future mechanistic analysis and in vivo quantitative assessment using non-invasive PET imaging in large animal model of diabetes is worthwhile. CONCLUSIONS: Overall, our data showed an increased uptake of our lead S1PR2-specific radioligand, [11C]TZ34125, in the spleen and pancreases of STZ-induced diabetic mice, and demonstrated [11C]TZ34125 has a great potential for preclinical and clinical usage for assessment of S1PR2 in diabetes and inflammation.

Multiparametric Immunoimaging Maps Inflammatory Signatures in Murine Myocardial Infarction Models.

In the past 2 decades, research on atherosclerotic cardiovascular disease has uncovered inflammation to be a key driver of the pathophysiological process. A pressing need therefore exists to quantitatively and longitudinally probe inflammation, in preclinical models and in cardiovascular disease patients, ideally using non-invasive methods and at multiple levels. Here, we developed and employed in vivo multiparametric imaging approaches to investigate the immune response following myocardial infarction. The myocardial infarction models encompassed either transient or permanent left anterior descending coronary artery occlusion in C57BL/6 and Apoe-/-mice. We performed nanotracer-based fluorine magnetic resonance imaging and positron emission tomography (PET) imaging using a CD11b-specific nanobody and a C-C motif chemokine receptor 2-binding probe. We found that immune cell influx in the infarct was more pronounced in the permanent occlusion model. Further, using 18F-fluorothymidine and 18F-fluorodeoxyglucose PET, we detected increased hematopoietic activity after myocardial infarction, with no difference between the models. Finally, we observed persistent systemic inflammation and exacerbated atherosclerosis in Apoe-/- mice, regardless of which infarction model was used. Taken together, we showed the strengths and capabilities of multiparametric imaging in detecting inflammatory activity in cardiovascular disease, which augments the development of clinical readouts.