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

Abstract

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) ( 64 Cu-ATSM) has promise, but its selectivity and sensitivity could be improved by structural modification. We have therefore evaluated a range of 64 Cu-ATSM analogs for imaging hypoxic myocardium. Methods: Isolated rat hearts (n 5 5/group) were perfused with normoxic buffer for 30 min and then hypoxic buffer for 45 min within a custom-built triple-g-detector system to quantify radiotracer infusion, hypoxiadependent cardiac uptake, and washout. A 1-MBq bolus of each candidate tracer (and 18 F-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 64 Cu-ATSM provided significant gains in hypoxic-to-normoxic contrast (14:1 for 64 Cu-2,3-butanedione bis(thiosemicarbazone) (ATS), 17:1 for 64 Cu-2,3-pentanedione bis(thiosemicarbazone) (CTS), 8:1 for 64 Cu-ATSM, P , 0.05). Hypoxic first-pass uptake was 78.2% 6 7.2% for 64 Cu-ATS and 70.7% 6 14.5% for 64 Cu-CTS, compared with 63.9% 6 11.7% for 64 Cu-ATSM. Cardiac retention of 18 F-fluoromisonidazole increased from 0.44% 6 0.17% during normoxia to 2.24% 6 0.08% during hypoxia. In vivo, normoxic cardiac retention of 64 Cu-CTS was significantly lower than that of 64 Cu-ATSM and 64 Cu-ATS (0.13% 6 0.02% vs. 0.25% 6 0.04% and 0.24% 6 0.03% injected dose, P , 0.05), with retention of all 3 tracers falling to less than 0.7% injected dose within 6 min. 64 Cu-CTS also exhibited lower uptake in liver and lung. Conclusion: 64 Cu-ATS and 64 Cu-CTS exhibit better cardiac hypoxia selectivity and imaging characteristics than the current lead hypoxia tracers, 64 Cu-ATSM and 18 F-fluoromisonidazole.

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

“…Hueting et al have recently suggested in this journal that the biodistribution and pharmacokinetics of 64 Cuacetate in tumor-bearing mice is similar to that of 64 Cu-ATSM when assessed after 2 or 16 h, and that 64 Cu-acetate exhibits hypoxia selectivity in cultured cancer cells, suggesting that 64 Cu-bis(thiosemicarbazone) complexes dissociate quickly in vivo and that the resultant biodistribution observed by PET is that of free copper, questioning the validity of 64 Cu-ATSM as a hypoxia tracer (34). We and other groups have observed no such nonspecific uptake of ionic 64 Cu 21 salts in normoxic or hypoxic myocytes in culture (15), CHO cells (35), or isolated buffer-perfused hearts (17,21). The wide variation in pharmacokinetics and early biodistributions that the different bis(thiosemicarbazones) have been shown to display, with some being hypoxia-selective (ATSM, ATS, CTS) and some not (pyruvaldehyde-bis(N4-methylthiosemicarbazone) [PTSM], glyoxal-bis(N4-methylthiosemicarbazone) [GTSM]), and with some crossing the blood-brain barrier (GTSM, PTSM, ATSM) and some not (ATS) (36,37), would also argue against a common nonselective mechanism of early tissue uptake.…”

“…We recently demonstrated that 64 Cu-CTS and its analog 64 Cu-ATS exhibit significantly greater hypoxia selectivity than the current lead compound 64 Cu-ATSM in hearts perfused with hypoxic (0% O 2 ) buffer (17). Although this finding was a promising first step in the screening process, the key to the progression of this class of compounds is to identify complexes selective for levels of hypoxia that correlate with survivable (and treatable) cardiac disease (9,10).…”

“…64 Cu was produced and used to radiolabel 2,3-butanedione-bis (thiosemicarbazone) (ATS), diacetyl-bis(N4-ethylthiosemicarbazone) (ATSE), 2,3-butanedione bis(N4-methylthiosemicarbazone) (ATSM), and 2,3-pentanedione bis(thiosemicarbazone) (CTS) as previously described (17).…”

“…The hearts were perfused at a constant flow (to simplify pharmacokinetic modeling) of 14 mL/min with a modified Krebs-Henseleit buffer (KHB) described previously (17) and gassed with a 95% O 2 /5% CO 2 mixture, and contractile function was measured with a left ventricular balloon. Perfusion pressure was monitored by a pressure transducer in the arterial line.…”

“…We have therefore performed a series of hypoxia titrations in the isolated heart to determine where this threshold lies using a panel of biochemical and biophysical assays. We then used a recently developed g-detection array (17) to identify analogs of 64 Cu-ATSM exhibiting greater sensitivity to hypoxia at this more relevant threshold.…”