GluN2B-enriched N-methyl-D-aspartate receptors (NMDARs) are implicated in several neurodegenerative and psychiatric diseases, such as Alzheimer’s disease. No clinically valid GluN1/2B therapeutic exists due to a lack of selective GluN2B imaging tools, and the state-of-the-art [3H]ifenprodil shows poor selectivity in drug screening. To this end, we developed a tritium-labeled form of OF-NB1, a recently reported selective GluN1/2B positron emission tomography imaging (PET) agent, with a molar activity of 1.79 GBq/µmol. The performance of [3H]OF-NB1 and [3H]ifenprodil was compared through head-to-head competitive binding experiments, using the GluN1/2B ligand CP-101,606 and the sigma-1 receptor (σ1R) ligand SA-4503. Contrary to [3H]ifenprodil, the usage of [3H]OF-NB1 differentiated between GluN1/2B and σ1R binding components. These results were corroborated by observations from PET imaging experiments in Wistar rats using the σ1R radioligand [18F]fluspidine. To unravel the binding modes of OF-NB1 and ifenprodil in GluN1/2B and σ1Rs, we performed a retrospective in silico study using a molecular operating environment. OF-NB1 maintained similar interactions to GluN1/2B as ifenprodil, but only ifenprodil successfully fitted in the σ1R pocket, thereby explaining the high GluN1/2B selectivity of OF-NB1 compared to ifenprodil. We successfully showed in a proof-of-concept study the superiority of [3H]OF-NB1 over the gold standard [3H]ifenprodil in the screening of potential GluN1/2B drug candidates.

Background A growing body of evidence highlights sex differences in the diagnostic accuracy of cardiovascular imaging modalities. Nonetheless, the role of sex hormones in modulating myocardial perfusion and coronary flow reserve (CFR) is currently unclear. The aim of our study was to assess the impact of female and male sex hormones on myocardial perfusion and CFR. Methods Rest and stress myocardial perfusion imaging (MPI) was conducted by small animal positron emission tomography (PET) with [18F]flurpiridaz in a total of 56 mice (7–8 months old) including gonadectomized (Gx) and sham-operated males and females, respectively. Myocardial [18F]flurpiridaz uptake (% injected dose per mL, % ID/mL) was used as a surrogate for myocardial perfusion at rest and following intravenous regadenoson injection, as previously reported. Apparent coronary flow reserve (CFRApp) was calculated as the ratio of stress and rest myocardial perfusion. Left ventricular (LV) morphology and function were assessed by cardiac magnetic resonance (CMR) imaging. Results Orchiectomy resulted in a significant decrease of resting myocardial perfusion (Gx vs. sham, 19.4 ± 1.0 vs. 22.2 ± 0.7 % ID/mL, p = 0.034), while myocardial perfusion at stress remained unchanged (Gx vs. sham, 27.5 ± 1.2 vs. 27.3 ± 1.2 % ID/mL, p = 0.896). Accordingly, CFRApp was substantially higher in orchiectomized males (Gx vs. sham, 1.43 ± 0.04 vs. 1.23 ± 0.05, p = 0.004), and low serum testosterone levels were linked to a blunted resting myocardial perfusion (r = 0.438, p = 0.020) as well as an enhanced CFRApp (r = −0.500, p = 0.007). In contrast, oophorectomy did not affect myocardial perfusion in females. Of note, orchiectomized males showed a reduced LV mass, stroke volume, and left ventricular ejection fraction (LVEF) on CMR, while no such effects were observed in oophorectomized females. Conclusion Our experimental data in mice indicate that sex differences in myocardial perfusion are primarily driven by testosterone. Given the diagnostic importance of PET-MPI in clinical routine, further studies are warranted to determine whether testosterone levels affect the interpretation of myocardial perfusion findings in patients.

Background: Myocardial perfusion imaging by positron emission tomography (PET-MPI) is the current gold standard for quantification of myocardial blood flow. 18F-flurpiridaz was recently introduced as a valid alternative to currently used PET-MPI probes. Nonetheless, optimum scan duration and time interval for image analysis are currently unknown. Further, it is unclear whether rest/stress PET-MPI with 18F-flurpiridaz is feasible in mice. Methods: Rest/stress PET-MPI was performed with 18F-flurpiridaz (0.6-3.0 MBq) in 29 mice aged 7-8 months. Regadenoson (0.1 μg/g) was used for induction of vasodilator stress. Kinetic modeling was performed using a metabolite-corrected arterial input function. Image-derived myocardial 18F-flurpiridaz uptake was assessed for different time intervals by placing a volume of interest in the left ventricular myocardium. Results: Tracer kinetics were best described by a two-tissue compartment model. K1 ranged from 6.7-20.0 mL/cm3/min, while myocardial volumes of distribution (VT) were between 34.6 and 83.6 mL/cm3. Of note, myocardial 18F-flurpiridaz uptake (%ID/g) was significantly correlated with K1 at rest and following pharmacological stress testing for all time intervals assessed. However, while Spearman coefficients (rs) ranged between 0.478 and 0.672, R2 values were generally low. In contrast, an excellent correlation of myocardial 18F-flurpiridaz uptake with VT was obtained, particularly when employing the averaged myocardial uptake from 20-40 min post tracer injection (R2 ≥0.98). Notably, K1 and VT were similarly sensitive to pharmacological stress induction. Further, mean stress-to-rest ratios of K1, VT, and %ID/g 18F-flurpiridaz were virtually identical, suggesting that %ID/g 18F-flurpiridaz can be used to estimate CFR in mice. Conclusion: Our findings suggest that a simplified assessment of relative myocardial perfusion and coronary flow reserve (CFR), based on image-derived tracer uptake, is feasible with 18F-flurpiridaz in mice, enabling high-throughput mechanistic CFR studies in rodents.

Abstract[18F]Flurpiridaz is a recently developed positron emission tomography tracer that is currently being investigated in phase III clinical trials to measure myocardial blood flow. The relatively long physical half‐life of fluorine‐18 alongside the high spatial resolution and outstanding myocardium‐to‐background ratio fuels its potential to be the next gold standard for the early detection of coronary artery disease. Notwithstanding the expected widespread use of [18F]flurpiridaz, the reported multistep synthesis of its precursor for radiofluorination involves a hazardous alkylation step using carcinogenic ethylene oxide, and a low overall chemical yield of 7 %. In this work, we have improved the overall yield more than fivefold and concurrently replaced the hazardous step. Specificity of binding of [18F]flurpiridaz to mitochondrial complex 1 was demonstrated by in vitro autoradiography on mouse heart tissue sections. These results thus pave the way for assessing myocardial blood flow and coronary flow reserve in mouse models of cardiovascular disease.

Background Myocardial perfusion imaging by positron emission tomography (PET-MPI) is the current gold standard for quantification of myocardial blood flow. 18F-flurpiridaz was recently introduced as a valid alternative to currently used PET-MPI probes. Nonetheless, optimum scan duration and time interval for image analysis are currently unknown. Further, it is unclear whether rest/stress PET-MPI with 18F-flurpiridaz is feasible in mice. Methods Rest/stress PET-MPI was performed with 18F-flurpiridaz (0.6-3.0 MBq) in 27 mice aged 7–8 months. Regadenoson (0.1 µg/g) was used for induction of vasodilator stress. Kinetic modeling was performed using a metabolite-corrected arterial input function. Image-derived myocardial 18F-flurpiridaz uptake was assessed for different time intervals by placing a volume of interest in the left ventricular myocardium. Results Tracer kinetics were best described by a two-tissue compartment model. K1 ranged from 6.7 to 20.0 mL·cm−3·min−1, while myocardial volumes of distribution (VT) were between 34.6 and 83.6 mL·cm−3. Of note, myocardial 18F-flurpiridaz uptake (%ID/g) was significantly correlated with K1 at rest and following pharmacological vasodilation for all time intervals assessed. However, while Spearman’s coefficients (rs) ranged between 0.478 and 0.681, R2 values were generally low. In contrast, an excellent correlation of myocardial 18F-flurpiridaz uptake with VT was obtained, particularly when employing the averaged myocardial uptake from 20 to 40 min post tracer injection (R2 ≥ 0.98). Notably, K1 and VT were similarly sensitive to pharmacological vasodilation induction. Further, mean stress-to-rest ratios of K1, VT, and %ID/g 18F-flurpiridaz were virtually identical, suggesting that %ID/g 18F-flurpiridaz can be used to estimate coronary flow reserve (CFR) in mice. Conclusion Our findings suggest that a simplified assessment of relative myocardial perfusion and CFR, based on image-derived tracer uptake, is feasible with 18F-flurpiridaz in mice, enabling high-throughput mechanistic CFR studies in rodents.