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 Cu-64-2,3-butanedione bis(N4-methylthiosemicarbazone) (ATSM) and F-18-fluoromisonidazole are trapped only at extreme levels of hypoxia and hence are insufficiently sensitive for this purpose. We have therefore sought an analog of Cu-64-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 P-31 nuclear MR spectroscopy, lactate and creatine kinase release spectrophotometrically, and hypoxia-inducible factor 1-alpha by Western blotting. Results: We identified a key hypoxia threshold at a 30% buffer O-2 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-alpha was elevated but not maximal. At this key threshold, Cu-64-2,3-pentanedione bis(thiosemicarbazone) (CTS) selectively deposited significantly more Cu-64 than any other tracer we examined (61.8% +/- 9.6% injected dose vs. 29.4% +/- 9.5% for Cu-64-ATSM, P < 0.05). Conclusion: The hypoxic threshold that induced survivable metabolic and functional compromise was 30% O-2. At this threshold, only Cu-64-CTS delivered a hypoxic-to-normoxic contrast of 3: 1, and it therefore warrants in vivo evaluation for imaging chronic cardiac ischemic syndromes.