18F-flurpiridaz has demonstrated favorable imaging characteristics for MPI in preclinical studies: As a derivative of the pyridazinone insecticide pyridaben, it has a high binding affinity towards mitochondrial complex I, with a considerable high first-pass extraction of > 90% as measured in an isolated perfused heart setup [
27,
28]. Comparing
18F-flurpiridaz with the established SPECT agent
99mTc sestamibi in a biodistribution rat study, the cardiac uptake of the
18F-labeled agent was significantly higher at both early (15 min) and late time points (120 min). This experiment was followed by an isolated rabbit heart perfusion study and net
18F-flurpiridaz cardiac uptake increased to a greater extent than that of
201TI or
99mTc sestamibi at physiologically relevant flow rates. Moreover, an in vivo PET study demonstrated almost no lung uptake and rapid liver clearance in rats, rabbits, and primates (pronounced washout between 5 and 15 min). In addition, a rat model of coronary occlusion also showed an excellent correlation with
18F-flurpiridaz uptake and histopathological findings [
29]. These findings were further corroborated in a chronic myocardial infarction (MI) model in rabbits (left coronary artery occlusion, followed by recovery phase over 1 month): compared to controls, a clear defect could be appreciated in the left ventricular wall. The promising safety profile of this imaging agent was further confirmed by electrocardiogram assessments in both controls and MI rabbits [
30]. Huisman et al. also used the Langendorff method and investigated the first-pass extraction of
18F-flurpiridaz in isolated perfused rat hearts, on which the radiotracer demonstrated a high and flow-independent myocardial first-pass extraction fraction. Thus,
18F-flurpiridaz may hold the promise of a linear correlation between radiotracer uptake and cardiac blood flow [
28]. Higuchi and coworkers tested
18F-flurpiridaz in rats in vivo. Normal healthy control rats were found to have a homogoneous delineation of the myocardium up to 2 h after tracer injection. However, for the permanent occlusion model, the defect size remained stable over the entire imaging protocol (15–115 min). This was in contradistinction to the transient ischemia model: reperfusion after short, transient ischemia of 3 min showed radiotracer redistribution to the induced defect (i.e. tracer redistribution after reperfusion). Radiotracer reinjection further enhanced the normalization process. The concept of redistribution is based on underperfused but viable myocardium, which retains the radiotracer while it washes out of normal myocardial areas, i.e. initial defects appear to normalize [
31]. The clinical application are diagnosis of CAD and most importantly, for the assessment of tissue viability, e.g. by radiotracer injection under physical stress with early and delayed imaging protocols, which allows to monitor such redistribution closely over time. Figure
2 shows the superior imaging characteristics of
18F-flurpiridaz PET compared to a common PET MPI agent,
13N-ammonia, in (A) healthy rats and (B) in a rat model after coronary artery occlusion. The
18F-labeled radiotracer demonstrated improved contrast and higher resolution, resulting in better delineation of induced lesions, despite a higher injected dose of
13N-ammonia relative to
18F-flurpiridaz. For the
18F-labeled imaging agent, the inferior/left ventricular wall can be better distinguished from the liver [
32]. Figure
3 displays a head-to-head comparison of
18F-flurpiridaz and
18F-FBnTP in a rat model of short-term occlusion and reperfusion. For the latter radiotracer, retention stability over time was confirmed, while
18F-flurpiridaz showed slow restoration over time. Differences may be explained by the underlying uptake mechanisms:
18F-flurpiridaz targets mitochondrial complex I, while
18F-FBnTP localizes to mitochondria due to membrane potential [
33]. The observed kinetics (redistribution after reperfusion) may allow for the use of
18F-flurpiridaz in a similar way to clinical protocols for the diagnosis of CAD with conventional stress/rest
201TI perfusion protocols or for the assessment of myocardial viability [
32,
34]. In a permanent and transient occlusion model of the left coronary artery, uptake defect assessed by
18F-flurpiridaz closely correlated with histological measured scar sizes confirmed by 2,3,5-triphenyltetrazolium chloride staining [
35]. In a pig model, Guehl et al. demonstrated that accurate rest and stress blood flow estimations with
18F-flurpiridaz are feasible, even in less than 15 min of PET acquisition time by using a single-scan rest-stress method, which further emphasizes the practicality of this radiotracer in clinical routine [
36]. Also in a pig model, Sherif et al. showed that
18F-flurpiridaz retention and standardized uptake values (SUVs) correlated with absolute MBF values at rest and pharmacological stress. As such, SUVs may be used as a substitute for absolute blood flow. As SUV does not require determination of radiotracer input function, tracer injection and exercise treadmill or bicycle stress test protocols could be performed outside the scanner. From a practical standpoint, such an approach may facilitate flow estimation in clinical routine [
37]. By comparison with radioactive microsphere-derived blood flow in a pig model, a high agreement rate with regional MBF using
18F-flurpiridaz was achieved, even over a wide flow range [
38].