Tissue acidosis does not mediate the hypoxia selectivity of [64Cu][Cu(ATSM)] in the isolated perfused rat heart.

Copper-64-Diacetyl-bis(N4-methylthiosemicarbazone) [64Cu][Cu(ATSM)] is a hypoxia-targeting PET tracer with applications in oncology and cardiology. Upon entering a hypoxic cell, [64Cu][Cu(II)(ATSM)] is reduced to a putative [64Cu][Cu(I)(ATSM)]- species which dissociates to deposit radiocopper, thereby providing hypoxic contrast. This process may be dependent upon protonation arising from intracellular acidosis. Since acidosis is a hallmark of ischemic tissue and tumors, the hypoxia specificity of [64Cu][Cu(ATSM)] may be confounded by changes in intracellular pH. We have therefore determined the influence of intracellular pH on [64Cu][Cu(ATSM)] pharmacokinetics. Using isolated perfused rat hearts, acidosis was induced using an ammonium pre-pulse method, with and without hypoxic buffer perfusion. Cardiac [64Cu][Cu(ATSM)] pharmacokinetics were determined using NaI detectors, with intracellular pH and cardiac energetics monitored in parallel by 31P NMR. To distinguish direct acidotic effects on tracer pharmacokinetics from acidosis-induced hypocontractility, parallel studies used lidocaine perfusion to abolish cardiac contraction. Hypoxic myocardium trapped [64Cu][Cu(ATSM)] despite no evidence of it being acidotic when characterised by 31P NMR. Independent induction of tissue acidosis had no direct effect on [64Cu][Cu(ATSM)] pharmacokinetics in either normoxic or hypoxic hearts, beyond decreasing cardiac oxygen consumption to alleviate hypoxia and decrease tracer retention, leading us to conclude that tissue acidosis does not mediate the hypoxia selectivity of [64Cu][Cu(ATSM)].

Modification of intracellular glutathione status does not change the cardiac trapping of (64)Cu(ATSM).

BACKGROUND: The trapping mechanisms of the PET hypoxia imaging agent copper(II)-diacetyl-bis(N (4)-methylthiosemicarbazone) ((64)Cu(ATSM)) remain unresolved, although its reduction prior to dissociation may be mediated by intracellular thiols. Glutathione (GSH) is the most abundant intracellular thiol, and its redox status changes in cancer cells and ischaemic myocardium (two prime applications for (64)Cu(ATSM) PET). We therefore investigated whether modification of intracellular GSH content affects the hypoxia selectivity of (64)Cu(ATSM). METHODS: Isolated rat hearts (n = five per group) were perfused with aerobic buffer (equilibrated with 95%O2/5%CO2) for 15 min, then hypoxic buffer (95%N2/5%CO2) for 20 min. Cardiac glutathione was depleted by buthionine sulphoximine (BSO, 4 mmol/kg/ 48 h intraperitoneal), or augmented by N-acetyl cysteine (NAC, 4 mmol/L) in the perfusion buffer. Cardiac (64)Cu retention from three 2-MBq bolus injections of (64)Cu(ATSM) before and during hypoxia was then monitored by NaI detectors. RESULTS: Cardiac GSH content was elevated by NAC and depleted by BSO (from 7.9 ± 2.0 to 59.3 ± 8.3 nmol/mg and 3.7 ± 1.0 nmol/mg protein, respectively; p < 0.05). Hypoxia did not affect cardiac GSH content in any group. During normoxia, tracer washed out bi-exponentially, with 13.1% ± 1.7% injected dose being retained; this was not affected by GSH augmentation or depletion. Hypoxia significantly increased tracer retention (to 59.1% ± 6.3%, p < 0.05); this effect was not modified by GSH augmentation or depletion. CONCLUSION: Modification of GSH levels had no impact upon the pharmacokinetics or hypoxia selectivity of (64)Cu(ATSM). While thiols may yet prove essential for the intracellular trapping of (64)Cu(ATSM), they are not the determinants of its hypoxia selectivity.