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/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 (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% ± 7.2% for (64)Cu-ATS and 70.7% ± 14.5% for (64)Cu-CTS, compared with 63.9% ± 11.7% for (64)Cu-ATSM. Cardiac retention of (18)F-fluoromisonidazole increased from 0.44% ± 0.17% during normoxia to 2.24% ± 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% ± 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. (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.

Keywords: 18FMISO; 64Cu-ATSM; PET; bis(thiosemicarbazones); cardiac ischemia; hypoxia.

Figure 1

Structure and physicochemical properties of ⁶⁴Cu-BTSC complexes. ITLC = instant thin-layer liquid chromatography; ATSE = diacetyl bis(N4-ethylthiosemicarbazone); CTSM = 2,3-pentanedione bis (N4-methylthiosemicarbazone); DTS = 3,4-hexanedione bis(thiosemicarbazone); DTSM = 3,4-hexanedione bis(N4-methylthiosemicarbazone).

Figure 2

(A) Triple-γ-detector system for monitoring radiotracer passage through Langendorff heart apparatus. (B) Representative time–activity curves from ⁶⁴Cu-ATSM displaying input function in arterial line (detector 1), retention/washout through heart (detector 2), and washout (detector 3).

Figure 3

Hemodynamic data from isolated rat hearts during 20 min of normoxic perfusion followed by 45 min of hypoxic perfusion (marked by arrow), showing changes in perfusate partial pressure of O₂ (A), lactate washout (B), and left ventricular developed pressure (LVDP) and left ventricular end diastolic pressure (LVEDP) (C). Data are mean (n = 5) ± SD.

Figure 4

Representative time–activity curves showing myocardial clearance and accumulation of ⁶⁴Cu-ATS, ⁶⁴Cu-ATSM, ¹⁸F-fluoromisonidazole (FMISO), and ⁶⁴CuCl₂ during normoxia (white background) and then hypoxia (gray background). Each spike represents 1-MBq bolus.

Figure 5

(A) Tissue retention of ⁶⁴Cu-BTSC complexes, ¹⁸F-fluoromisonidazole, and ⁶⁴CuCl₂ during normoxia and hypoxia. Asterisk indicates significant difference from corresponding normoxic control (P < 0.05). (B) Normoxic-to-hypoxic contrast ratios as calculated from A. Asterisk indicates significant difference from corresponding ⁶⁴Cu-ATSM values (P < 0.05). Data are mean (n = 5) ± SD. ATSE = diacetyl bis(N4-ethylthiosemicarbazone); CTSM = 2,3-pentanedione bis (N4-methylthiosemicarbazone); DTS = 3,4-hexanedione bis(thiosemicarbazone); DTSM = 3,4-hexanedione bis(N4-methylthiosemicarbazone); FMISO = fluoromisonidazole.

Figure 6

Fast (A) and slow (B) rates of tracer clearance from myocardium (and their respective weights, C and D). Data are mean (n = 5) ± SD. a.u. = arbitrary units. ATSE = diacetyl bis(N4-ethylthiosemicarbazone); CTSM = 2,3-pentanedione bis(N4-methylthiosemicarbazone); DTS = 3,4-hexanedione bis(thiosemicarbazone); DTSM = 3,4-hexanedione bis(N4-methylthiosemicarbazone).

Figure 7

(A) PET/CT images of healthy rat 30 min after injection with ⁶⁴Cu-CTS (5 MBq in 200 μL). (B–D) Time-activity curves showing tracer pharmacokinetics of ⁶⁴Cu-ATS, ⁶⁴Cu-ATSM, and ⁶⁴Cu-CTS in heart, liver, and kidneys, respectively. (E) Tissue biodistribution of each tracer 90 min after injection. Data are mean (n = 3) ± SD. Asterisk indicates significant difference from ⁶⁴Cu-ATSM (P < 0.05).