Erika Mariotti

Cardiac Hypoxia Imaging: Second-Generation Analogues of 64Cu-ATSM

Myocardial hypoxia is an attractive target for diagnostic and prognostic imaging, but current approaches are insufficiently sensitive for clinical use. The PET tracer copper(II)-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) has promise, but its selectivity and sensitivity could be improved by structural modification. We have therefore evaluated a range of 64Cu-ATSM analogs for imaging hypoxic myocardium. Methods: Isolated rat hearts (n = 5/group) were perfused with normoxic buffer for 30 min and then hypoxic buffer for 45 min within a custom-built triple-γ-detector system to quantify radiotracer infusion, hypoxia-dependent cardiac uptake, and washout. A 1-MBq bolus of each candidate tracer (and 18F-fluoromisonidazole for comparative purposes) was injected into the arterial line during normoxia, and during early and late hypoxia, and their hypoxia selectivity and pharmacokinetics were evaluated. The in vivo pharmacokinetics of promising candidates in healthy rats were then assessed by PET imaging and biodistribution. Results: All tested analogs exhibited hypoxia sensitivity within 5 min. Complexes less lipophilic than 64Cu-ATSM provided significant gains in hypoxic-to-normoxic contrast (14:1 for 64Cu-2,3-butanedione bis(thiosemicarbazone) (ATS), 17:1 for 64Cu-2,3-pentanedione bis(thiosemicarbazone) (CTS), 8:1 for 64Cu-ATSM, P < 0.05). Hypoxic first-pass uptake was 78.2% ± 7.2% for 64Cu-ATS and 70.7% ± 14.5% for 64Cu-CTS, compared with 63.9% ± 11.7% for 64Cu-ATSM. Cardiac retention of 18F-fluoromisonidazole increased from 0.44% ± 0.17% during normoxia to 2.24% ± 0.08% during hypoxia. In vivo, normoxic cardiac retention of 64Cu-CTS was significantly lower than that of 64Cu-ATSM and 64Cu-ATS (0.13% ± 0.02% vs. 0.25% ± 0.04% and 0.24% ± 0.03% injected dose, P < 0.05), with retention of all 3 tracers falling to less than 0.7% injected dose within 6 min. 64Cu-CTS also exhibited lower uptake in liver and lung. Conclusion: 64Cu-ATS and 64Cu-CTS exhibit better cardiac hypoxia selectivity and imaging characteristics than the current lead hypoxia tracers, 64Cu-ATSM and 18F-fluoromisonidazole.

64Cu-CTS: A Promising Radiopharmaceutical for the Identification of Low-Grade Cardiac Hypoxia by PET.

The subtle hypoxia underlying chronic cardiovascular disease is an attractive target for PET imaging, but the lead hypoxia imaging agents 64Cu-2,3-butanedione bis(N4-methylthiosemicarbazone) (ATSM) and 18F-fluoromisonidazole are trapped only at extreme levels of hypoxia and hence are insufficiently sensitive for this purpose. We have therefore sought an analog of 64Cu-ATSM better suited to identify compromised but salvageable myocardium, and we validated it using parallel biomarkers of cardiac energetics comparable to those observed in chronic cardiac ischemic syndromes. Methods: Rat hearts were perfused with aerobic buffer for 20 min, followed by a range of hypoxic buffers (using a computer-controlled gas mixer) for 45 min. Contractility was monitored by intraventricular balloon, energetics by 31P nuclear MR spectroscopy, lactate and creatine kinase release spectrophotometrically, and hypoxia-inducible factor 1-α by Western blotting. Results: We identified a key hypoxia threshold at a 30% buffer O2 saturation that induces a stable and potentially survivable functional and energetic compromise: left ventricular developed pressure was depressed by 20%, and cardiac phosphocreatine was depleted by 65.5% ± 14% (P < 0.05 vs. control), but adenosine triphosphate levels were maintained. Lactate release was elevated (0.21 ± 0.067 mmol/L/min vs. 0.056 ± 0.01 mmol/L/min, P < 0.05) but not maximal (0.46 ± 0.117 mmol/L/min), indicating residual oxidative metabolic capacity. Hypoxia-inducible factor 1-α was elevated but not maximal. At this key threshold, 64Cu-2,3-pentanedione bis(thiosemicarbazone) (CTS) selectively deposited significantly more 64Cu than any other tracer we examined (61.8% ± 9.6% injected dose vs. 29.4% ± 9.5% for 64Cu-ATSM, P < 0.05). Conclusion: The hypoxic threshold that induced survivable metabolic and functional compromise was 30% O2. At this threshold, only 64Cu-CTS delivered a hypoxic-to-normoxic contrast of 3:1, and it therefore warrants in vivo evaluation for imaging chronic cardiac ischemic syndromes.

Assessing radiotracer kinetics in the Langendorff perfused heart

BackgroundThe Langendorff perfused heart is a physiologically relevant and controllable model with potential for assessing the pharmacokinetics of new radiotracers under a range of pathophysiological conditions.. We assess the feasibility of extending the methods validated for in vivo PET data analysis to the characterisation of PET tracer kinetics applied to Langendorff perfused hearts.MethodsMonte Carlo simulations were used to study the accuracy and reproducibility of linear and non-linear spectral analysis (SA/NLSA), the Patlak graphical method and normalised tissue activity (NA). The methods were used to analyse time-activity curves of two widely used PET tracers, [18 F]-FDG and [18 F]-FMISO, acquired ex vivo from Langendorff perfused rat hearts under normoxic and hypoxic conditions.ResultsMonte Carlo simulations showed NLSA to be superior to SA in identifying and quantifying the presence of irreversible trapping component (αo), for low values of αo. The performance of NLSA and SA for high values of trapping was comparable. NLSA was also more precise than SA in determining the absence of trapping over the range of simulated kinetics and SNR. Simulations also suggest that the semi-quantitative method NA is adequate for the evaluation of trapping, and it was found to be more accurate than Patlak. The values of α0 estimated with NLSA from the time series of both [18 F]-FDG and [18 F]-FMISO increased significantly from normoxia to hypoxia in agreement with previous studies. The values of trapping derived using SA increased but not significantly, reflecting the larger error associate with this method. Patlak estimated from the experimental datasets increased from normoxia to hypoxia but was not significant. NA estimated from the [18 F]-FDG data increased from normoxia to hypoxia, but was not significant, whilst NA calculated for [18 F]-FMISO time-activity curves increased significantly.ConclusionsMonte Carlo simulations suggested that spectral-based quantitative analysis methods are adequate for the kinetic characterisation of time-activity curves acquired ex vivo from perfused hearts. The uptake rate Patlak and the index NA also represent a good alternative to the SA and NLSA algorithms when the aim of the kinetic analysis is to measure changes in the amount of tracer trapped in the irreversible compartment in response to external stimuli. For low levels of trapping, NLSA and NA were subject to lower errors than SA and Patlak, respectively.

Modeling non-linear kinetics of hyperpolarized [1-(13)C] pyruvate in the crystalloid-perfused rat heart

Hyperpolarized 13C MR measurements have the potential to display non‐linear kinetics. We have developed an approach to describe possible non‐first‐order kinetics of hyperpolarized [1‐13C] pyruvate employing a system of differential equations that agrees with the principle of conservation of mass of the hyperpolarized signal. Simultaneous fitting to a second‐order model for conversion of [1‐13C] pyruvate to bicarbonate, lactate and alanine was well described in the isolated rat heart perfused with Krebs buffer containing glucose as sole energy substrate, or glucose supplemented with pyruvate. Second‐order modeling yielded significantly improved fits of pyruvate–bicarbonate kinetics compared with the more traditionally used first‐order model and suggested time‐dependent decreases in pyruvate–bicarbonate flux. Second‐order modeling gave time‐dependent changes in forward and reverse reaction kinetics of pyruvate–lactate exchange and pyruvate–alanine exchange in both groups of hearts during the infusion of pyruvate; however, the fits were not significantly improved with respect to a traditional first‐order model. The mechanism giving rise to second‐order pyruvate dehydrogenase (PDH) kinetics was explored experimentally using surface fluorescence measurements of nicotinamide adenine dinucleotide reduced form (NADH) performed under the same conditions, demonstrating a significant increase of NADH during pyruvate infusion. This suggests a simultaneous depletion of available mitochondrial NAD+ (the cofactor for PDH), consistent with the non‐linear nature of the kinetics. NADH levels returned to baseline following cessation of the pyruvate infusion, suggesting this to be a transient effect.

Kinetic analysis of hyperpolarized data with minimum a priori knowledge: Hybrid maximum entropy and nonlinear least squares method (MEM/NLS).

To assess the feasibility of using a hybrid Maximum‐Entropy/Nonlinear Least Squares (MEM/NLS) method for analyzing the kinetics of hyperpolarized dynamic data with minimum a priori knowledge.

Kinetic analysis of hyperpolarized data with minimum a priori knowledge

To assess the feasibility of using a hybrid Maximum‐Entropy/Nonlinear Least Squares (MEM/NLS) method for analyzing the kinetics of hyperpolarized dynamic data with minimum a priori knowledge.

Kinetic analysis of hyperpolarized data with minimum a priori knowledge: Hybrid maximum entropy and nonlinear least squares method (MEM/NLS): MEM/NLS Method for Kinetics of Hyperpolarized Data

To assess the feasibility of using a hybrid Maximum‐Entropy/Nonlinear Least Squares (MEM/NLS) method for analyzing the kinetics of hyperpolarized dynamic data with minimum a priori knowledge.