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

Abstract

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)].

Figure 1

(left) Structure of [Cu(ATSM)], (right) Generalised schematic of the proposed trapping mechanisms for [⁶⁴Cu][Cu(BTSC)] PET tracers. [⁶⁴Cu][Cu(II)(BTSCs)] passively diffuse into cells where they can be reduced to a charged Cu(I) complex which is unable to leave the cell. In the presence of oxygen this Cu(I) complex is rapidly reoxidised back to Cu(II) which is again able to diffuse out of the cell. If oxygen is insufficient, the Cu(I) complex can become further reduced and dissociate. The Cu(I) then becomes sequestered by copper chelating proteins and trapped inside the cell (Adapted with permission from Pell et al. 2018 under a CC BY open access licence).

Figure 2

Langendorff hemodynamic data showing changes in perfusion pressure, left ventricular developed pressure and left ventricular end-diastolic pressure from all treatment groups. Data are expressed as mean +/− SD (n = 5).

Figure 3

(A) Representative stacked plot of ³¹P NMR spectra acquired from a hypoxic control (20% O₂) heart along with single spectra stacked plot and myocardial pH_i. (B) Representative stacked plot of ³¹P NMR spectra acquired from a hypoxic (20% O₂) acidotic heart along with single spectra stacked plot and myocardial pH_i (data are expressed as mean ± standard deviation, n = 5).

Figure 4

Cardiac lactate release. Lactate concentration was measured in perfusate samples collected from isolated perfused hearts. Data are expressed as means ± SD (n = 5).

Figure 5

Representative time-activity curves demonstrating Copper-64 retention from [⁶⁴Cu][Cu(ATSM)] across all treatment groups.

Figure 6

Tissue retention of [⁶⁴Cu][Cu(ATSM)] (% of injected dose) across all treatment groups. Data are expressed as mean ± SD. (n = 5).

Figure 7

Perfusion protocols for hearts from all treatment groups. (A) Normoxic control, (B) Normoxia + acidosis (C) Hypoxic control (D) Hypoxia + acidosis (E) Normoxia + lidocaine infusion (F) Hypoxia + lidocaine infusion.