Optimised production of technetium-94m for PET imaging by proton-irradiation of phosphomolybdic acid in cyclotron liquid target.

Natural-abundance phosphomolybdic acid (H3(Mo12PO40) ‧12H2O, 0.181-0.552 g Mo/mL) solutions were irradiated with 12.9 MeV protons on a GE PETtrace cyclotron using an adapted standard liquid target. Technetium-94m (94mTc) was produced through the 94Mo(p,n)94mTc nuclear reaction with saturation yields of up to 53 ± 6 MBq/µA. End of bombardment activities of 161 ± 17 MBq and 157 ± 7 MBq were achieved for the 0.552 g Mo/mL solution (10 µA for 30 min) and 0.181 g Mo/mL solution (15 µA for 60 min), respectively. No visible degradation of the niobium target body and foil were seen during the irradiations of up to 15 µA for 60 min. The produced 94mTc was separated from the target phosphomolybdic acid solution with >98% recovery using an aqueous biphasic extraction resin. Compared to previous reported liquid target methods for 94mTc production, the better production yield, in-target solution stability during irradiation and 94mTc separation recovery of phosphomolybdic acid makes it a very promising target material for routine clinical 94mTc production at medical facilities with liquid targets already installed for 18F production.

Elevated Na is a dynamic and reversible modulator of mitochondrial metabolism in the heart.

Elevated intracellular sodium Nai adversely affects mitochondrial metabolism and is a common feature of heart failure. The reversibility of acute Na induced metabolic changes is evaluated in Langendorff perfused rat hearts using the Na/K ATPase inhibitor ouabain and the myosin-uncoupler para-aminoblebbistatin to maintain constant energetic demand. Elevated Nai decreases Gibb's free energy of ATP hydrolysis, increases the TCA cycle intermediates succinate and fumarate, decreases ETC activity at Complexes I, II and III, and causes a redox shift of CoQ to CoQH2, which are all reversed on lowering Nai to baseline levels. Pseudo hypoxia and stabilization of HIF-1α is observed despite normal tissue oxygenation. Inhibition of mitochondrial Na/Ca-exchange with CGP-37517 or treatment with the mitochondrial ROS scavenger MitoQ prevents the metabolic alterations during Nai elevation. Elevated Nai plays a reversible role in the metabolic and functional changes and is a novel therapeutic target to correct metabolic dysfunction in heart failure.

Disrupting Na+ ion homeostasis and Na+/K+ ATPase activity in breast cancer cells directly modulates glycolysis in vitro and in vivo.

BACKGROUND: Glycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine triphosphate (ATP), however it is unknown what this extra ATP turnover is used for. We hypothesise that an important contribution to the increased glycolytic flux in cancer cells results from the ATP demand of Na+/K+-ATPase (NKA) due to altered sodium ion homeostasis in cancer cells. METHODS: Live whole-cell measurements of intracellular sodium [Na+]i were performed in three human breast cancer cells (MDA-MB-231, HCC1954, MCF-7), in murine breast cancer cells (4T1), and control human epithelial cells MCF-10A using triple quantum filtered 23Na nuclear magnetic resonance (NMR) spectroscopy. Glycolytic flux was measured by 2H NMR to monitor conversion of [6,6-2H2]D-glucose to [2H]-labelled L-lactate at baseline and in response to NKA inhibition with ouabain. Intracellular [Na+]i was titrated using isotonic buffers with varying [Na+] and [K+] and introducing an artificial Na+ plasma membrane leak using the ionophore gramicidin-A. Experiments were carried out in parallel with cell viability assays, 1H NMR metabolomics of intracellular and extracellular metabolites, extracellular flux analyses and in vivo measurements in a MDA-MB-231 human-xenograft mouse model using 2-deoxy-2-[18F]fluoroglucose (18F-FDG) positron emission tomography (PET). RESULTS: Intracellular [Na+]i was elevated in human and murine breast cancer cells compared to control MCF-10A cells. Acute inhibition of NKA by ouabain resulted in elevated [Na+]i and inhibition of glycolytic flux in all three human cancer cells which are ouabain sensitive, but not in the murine cells which are ouabain resistant. Permeabilization of cell membranes with gramicidin-A led to a titratable increase of [Na+]i in MDA-MB-231 and 4T1 cells and a Na+-dependent increase in glycolytic flux. This was attenuated with ouabain in the human cells but not in the murine cells. 18FDG PET imaging in an MDA-MB-231 human-xenograft mouse model recorded lower 18FDG tumour uptake when treated with ouabain while murine tissue uptake was unaffected. CONCLUSIONS: Glycolytic flux correlates with Na+-driven NKA activity in breast cancer cells, providing evidence for the 'centrality of the [Na+]i-NKA nexus' in the mechanistic basis of the Warburg effect.

The chicken chorioallantoic membrane as a low-cost, high-throughput model for cancer imaging.

Mouse models are invaluable tools for radiotracer development and validation. They are, however, expensive, low throughput, and are constrained by animal welfare considerations. Here, we assessed the chicken chorioallantoic membrane (CAM) as an alternative to mice for preclinical cancer imaging studies. NCI-H460 FLuc cells grown in Matrigel on the CAM formed vascularized tumors of reproducible size without compromising embryo viability. By designing a simple method for vessel cannulation it was possible to perform dynamic PET imaging in ovo, producing high tumor-to-background signal for both 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) and (4S)-4-(3-18F-fluoropropyl)-L-glutamate (18F-FSPG). The pattern of 18F-FDG tumor uptake were similar in ovo and in vivo, although tumor-associated radioactivity was higher in the CAM-grown tumors over the 60 min imaging time course. Additionally, 18F-FSPG provided an early marker of both treatment response to external beam radiotherapy and target inhibition in ovo. Overall, the CAM provided a low-cost alternative to tumor xenograft mouse models which may broaden access to PET and SPECT imaging and have utility across multiple applications.

A Reactivity-Based 18F-Labeled Probe for PET Imaging of Oxidative Stress in Chemotherapy-Induced Cardiotoxicity.

Oxidative stress underlies the pathology of many human diseases, including the doxorubicin-induced off-target cardiotoxicity in cancer chemotherapies. Since current diagnostic procedures are only capable of monitoring cardiac function, a noninvasive means of detecting biochemical changes in redox status prior to irreversible functional changes is highly desirable for both early diagnosis and prognosis. We designed a novel 18F-labeled molecular probe, 18F-FPBT, for the direct detection of superoxide in vivo using positron emission tomography (PET). 18F-FPBT was radiosynthesized in one step by nucleophilic radiofluorination. In vitro, 18F-FPBT showed rapid and selective oxidation by superoxide (around 60% in 5 min) compared to other physiological ROS. In healthy mice and rats, 18F-FBPT is distributed to all major organs in the first few minutes post injection and is rapidly cleared via both renal and hepatobiliary routes with minimal background retention in the heart. In a rat model of doxorubicin-induced cardiotoxicity, 18F-FBPT showed significantly higher (P < 0.05) uptake in the hearts of treated animals compared to healthy controls. These results warrant further optimization of 18F-FBPT for clinical translation.

Detecting Validated Intracellular ROS Generation with 18F-dihydroethidine-Based PET.

PURPOSE: To determine the sensitivity of the 18F-radiolabelled dihydroethidine analogue ([18F]DHE) to ROS in a validated ex vivo model of tissue oxidative stress. PROCEDURES: The sensitivity of [18F]DHE to various ROS-generating systems was first established in vitro. Then, isolated rat hearts were perfused under constant flow, with contractile function monitored by intraventricular balloon. Cardiac uptake of infused [18F]DHE (50-150 kBq.min-1) was monitored by γ-detection, while ROS generation was invoked by menadione infusion (0, 10, or 50 µm), validated by parallel measures of cardiac oxidative stress. RESULTS: [18F]DHE was most sensitive to oxidation by superoxide and hydroxyl radicals. Normalised [18F]DHE uptake was significantly greater in menadione-treated hearts (1.44 ± 0.27) versus control (0.81 ± 0.07) (p < 0.05, n = 4/group), associated with concomitant cardiac contractile dysfunction, glutathione depletion, and PKG1α dimerisation. CONCLUSION: [18F]DHE reports on ROS in a validated model of oxidative stress where perfusion (and tracer delivery) is unlikely to impact its pharmacokinetics.

Synthesis and ex vivo biological evaluation of gallium-68 labelled NODAGA chelates assessing cardiac uptake and retention.

Radiolabelled lipophilic cations can be used to non-invasively report on mitochondrial dysfunction in diseases such as cardiovascular disease, cardiotoxicity and cancer. Several such lipophilic cations are currently used clinically to map myocardial perfusion using SPECT imaging. Since PET offers significant advantages over SPECT in terms of sensitivity, resolution and the capacity for dynamic imaging to allow pharmacokinetic modelling, we have synthesised and radiolabelled a series of NODAGA-based radiotracers, with triarylphosphonium-functionalisation, with gallium-68 to develop PET-compatible cationic complexes. To evaluate their capacity to report upon mitochondrial membrane potential, we assessed their pharmacokinetic profiles in isolated perfused rat hearts before and after mitochondrial depolarisation with the ionophore CCCP. All three tracers radiolabel with over 96% RCY, with log D7.4 values above -0.4 observed for the most lipophilic example of this family of radiotracers. The candidate tracer [68Ga]Ga4c exhibited non-preferential uptake in healthy cardiac tissue over CCCP-infused cardiac tissue. While this approach does show promise, the lipophilicity of this family of probes needs improving in order for them to be effective cardiac imaging agents.

DO2A-based ligands for gallium-68 chelation: synthesis, radiochemistry and ex vivo cardiac uptake.

Radiolabelled lipophilic cations could potentially be used to non-invasively image mitochondrial dysfunction in cardiovascular disease, building on their current role as perfusion imaging agents. We have synthesised and radiolabelled two series of DO2A-based radiotracers, with bistriarylphosphonium- and bisaryl-functionalisation respectively, with gallium-68 to form lipophilic cations. Both sets of tracers radiolabel with over 90% RCP, although the tracers form kinetic/thermodynamic pairs of species upon gallium chelation that can be visualised and separated by radioHPLC. Log D7.4 values above -0.3 are observed for the most lipophilic examples of each series of radiotracers. Both tracers show significant preferential uptake in healthy cardiac tissue over cardiac tissue depolarised by CCCP.

Imaging of Chemotherapy-Induced Acute Cardiotoxicity with 18F-Labeled Lipophilic Cations.

Many chemotherapy agents are toxic to the heart, such that increasing numbers of cancer survivors are now living with the potentially lethal cardiovascular consequences of their treatment. Earlier and more sensitive detection of chemotherapy-induced cardiotoxicity may allow improved treatment strategies and increase long-term survival. Lipophilic cation PET tracers may be suitable for early detection of cardiotoxicity. This study aimed to evaluate an 18F-labeled lipophilic phosphonium cation, [1-(2-18F-fluoroethyl),1H[1,2,3]triazole-4-ethylene]triphenylphosphonium bromide (18F-MitoPhos), as a cardiac imaging agent, comparing it with leading PET and SPECT lipophilic cationic tracers before further assessing its potential for imaging cardiotoxicity in an acute doxorubicin model. Methods: Cardiac uptake and response to decreased mitochondrial membrane potential of 18F-MitoPhos and 99mTc-sestamibi were tested in isolated perfused rat hearts. Baseline pharmacokinetic profiles of 18F-MitoPhos and 18F-fluorobenzyltriphenylphosphonium and their response to acute doxorubicin-induced cardiotoxicity were assessed in rats in vivo (10, 15, or 20 mg of doxorubicin per kilogram, intravenously, 48 h beforehand). Results: Cardiac retention of 18F-MitoPhos was more than double that of 99mTc-sestamibi in isolated perfused rat hearts. A favorable biodistribution of 18F-MitoPhos in vivo was observed, with heart-to-tissue ratios of 304 ± 186, 11.2 ± 1.2, and 3.8 ± 0.6 for plasma, liver, and lung, respectively (60 min). A significant dose-dependent loss of cardiac retention of 18F-MitoPhos was observed on doxorubicin treatment, with average cardiac SUV from 30 to 60 min (mean ± SD) decreasing from 3.5 ± 0.5 (control) to 1.8 ± 0.1 (doxorubicin, 20 mg/kg). Other assessed biomarkers showed no alterations. Conclusion:18F-MitoPhos showed pharmacokinetic parameters suitable for cardiac imaging. A significant dose response of cardiac uptake to doxorubicin treatment was observed before detectable biomarker alterations. 18F-MitoPhos is therefore a promising tracer for imaging chemotherapy-induced cardiotoxicity. To our knowledge, this is the first demonstration of radiolabeled lipophilic cations being used for the PET imaging of chemotherapy-induced cardiotoxicity and indicates the potential application of these compounds in this area.

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)].

Detection of anthracycline-induced cardiotoxicity using perfusion-corrected 99mTc sestamibi SPECT.

By the time cardiotoxicity-associated cardiac dysfunction is detectable by echocardiography it is often beyond meaningful intervention. 99mTc-sestamibi is used clinically to image cardiac perfusion by single photon emission computed tomography (SPECT) imaging, but as a lipophilic cation its distribution is also governed by mitochondrial membrane potential (ΔΨm). Correcting scans for variations in perfusion (using a ΔΨm-independent perfusion tracer such as (bis(N-ethoxy-N-ethyldithiocarbamato)nitrido 99mTc(V)) (99mTc-NOET) could allow 99mTc-sestamibi to be repurposed to specifically report on ΔΨm as a readout of evolving cardiotoxicity. Isolated rat hearts were perfused within a γ-detection apparatus to characterize the pharmacokinetics of 99mTc-sestamibi and 99mTc-NOET in response to mitochondrial perturbation by hypoxia, ionophore (CCCP) or doxorubicin. All interventions induced 99mTc-sestamibi washout; hypoxia from 24.9 ± 2.6% ID to 0.4 ± 6.2%, CCCP from 22.8 ± 2.5% ID to -3.5 ± 3.1%, and doxorubicin from 23.0 ± 2.2% ID to 17.8 ± 0.7, p < 0.05. Cardiac 99mTc-NOET retention (34.0 ± 8.0% ID) was unaffected in all cases. Translating to an in vivo rat model, 2 weeks after bolus doxorubicin injection, there was a dose-dependent loss of cardiac 99mTc-sestamibi retention (from 2.3 ± 0.3 to 0.9 ± 0.2 ID/g with 10 mg/kg (p < 0.05)), while 99mTc-NOET retention (0.93 ± 0.16 ID/g) was unaffected. 99mTc-NOET therefore traps in myocardium independently of the mitochondrial perturbations that induce 99mTc-sestamibi washout, demonstrating proof-of-concept for an imaging approach to detect evolving cardiotoxicity.

Synthesis, gallium-68 radiolabelling and biological evaluation of a series of triarylphosphonium-functionalized DO3A chelators.

Radiolabelled lipophilic cations that accumulate in mitochondria according to the magnitude of the mitochondrial membrane potential can be used to report non-invasively on mitochondrial dysfunction in cardiovascular disease, cardiotoxicity, and cancer. While several such cations are already commercially available for SPECT imaging, PET offers greater promise in terms of sensitivity, resolution, and capacity for dynamic imaging and pharmacokinetic modelling. We have therefore synthesised a series of three triarylphosphonium-functionalised DO3A chelators for positron emitter gallium-68, with differing alkyl-functionalisation motifs to provide opportunities for tunable lipophilicity as a means of optimising their pharmacokinetics. To assess their capacity to report on mitochondrial membrane potential, we assessed their pharmacokinetic profiles in isolated tumour cells and isolated perfused rat hearts before and after mitochondrial depolarisation with the ionophore CCCP. All three compounds radiolabelled with over 97% RCY and exhibited log D values of between -3.12 and -1.81. In vitro assessment of the uptake of the radiotracers in cultured tumour cells showed a three-fold increase in uptake compared to unchelated [68Ga]Ga(iii). However, each complex exhibited less than 1% retention in healthy hearts, which was not significantly diminished by mitochondrial depolarisation with CCCP. This preliminary work suggests that while this approach is promising, the lipophilicity of this class of tracers must be increased in order for them to be useful as cardiac or cancer imaging agents.

PET Imaging of Cardiac Hypoxia: Hitting Hypoxia Where It Hurts.

PURPOSE OF REVIEW: In this review, we outline the potential for hypoxia imaging as a diagnostic and prognostic tool in cardiology. We describe the lead hypoxia PET radiotracers currently in development and propose a rationale for how they should most appropriately be screened and validated. RECENT FINDINGS: While the majority of hypoxia imaging agents has been developed for oncology, the requirements for hypoxia imaging in cardiology are different. Recent work suggests that the bis(thiosemicarbazone) family of compounds may be capable of detecting the subtle degrees of hypoxia associated with cardiovascular syndromes, and that they have the potential to be "tuned" to provide different tracers for different applications. SUMMARY: New tracers currently in development show significant promise for imaging evolving cardiovascular disease. Fundamental to their exploitation is their careful, considered validation and characterization so that the information they provide delivers the greatest prognostic insight achievable.

Opportunities and challenges for metal chemistry in molecular imaging: from gamma camera imaging to PET and multimodality imaging.

The development of medical imaging is a highly multidisciplinary endeavor requiring the close cooperation of clinicians, physicists, engineers, biologists and chemists to identify capabilities, conceive challenges and solutions and apply them in the clinic. The chemistry described in this article illustrates how synergistic advances in these areas drive the technology and its applications forward, with each discipline producing innovations that in turn drive innovations in the others. The main thread running through the article is the shift from single photon radionuclide imaging towards PET, and in turn the emerging shift from PET/CT towards PET/MRI and further, combination of these with optical imaging. Chemistry to support these transitions is exemplified by building on a summary of the status quo, and recent developments, in technetium-99m chemistry for SPECT imaging, followed by a report of recent developments to support clinical application of short lived (Ga-68) and long-lived (Zr-89) positron emitting isotopes, copper isotopes for PET imaging, and combined modality imaging agents based on radiolabelled iron oxide based nanoparticles.

Multiple quantum filtered (23)Na NMR in the Langendorff perfused mouse heart: Ratio of triple/double quantum filtered signals correlates with [Na]i.

We investigate the potential of multiple quantum filtered (MQF) (23)Na NMR to probe intracellular [Na]i in the Langendorff perfused mouse heart. In the presence of Tm(DOTP) shift reagent the triple quantum filtered (TQF) signal originated largely from the intracellular sodium pool with a 32±6% contribution of the total TQF signal arising from extracellular sodium, whilst the rank 2 double-quantum filtered signal (DQF), acquired with a 54.7° flip-angle pulse, originated exclusively from the extracellular sodium pool. Given the different cellular origins of the (23)Na MQF signals we propose that the TQF/DQF ratio can be used as a semi-quantitative measure of [Na]i in the mouse heart. We demonstrate a good correlation of this ratio with [Na]i measured with shift reagent at baseline and under conditions of elevated [Na]i. We compare the measurements of [Na]i using both shift reagent and TQF/DQF ratio in a cohort of wild type mouse hearts and in a transgenic PLM(3SA) mouse expressing a non-phosphorylatable form of phospholemman, showing a modest but measurable elevation of baseline [Na]i. MQF filtered (23)Na NMR is a potentially useful tool for studying normal and pathophysiological changes in [Na]i, particularly in transgenic mouse models with altered Na regulation.

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

UNLABELLED: The subtle hypoxia underlying chronic cardiovascular disease is an attractive target for PET imaging, but the lead hypoxia imaging agents (64)Cu-2,3-butanedione bis(N4-methylthiosemicarbazone) (ATSM) and (18)F-fluoromisonidazole are trapped only at extreme levels of hypoxia and hence are insufficiently sensitive for this purpose. We have therefore sought an analog of (64)Cu-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 (31)P nuclear MR spectroscopy, lactate and creatine kinase release spectrophotometrically, and hypoxia-inducible factor 1-α by Western blotting. RESULTS: We identified a key hypoxia threshold at a 30% buffer O2 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-α was elevated but not maximal. At this key threshold, (64)Cu-2,3-pentanedione bis(thiosemicarbazone) (CTS) selectively deposited significantly more (64)Cu than any other tracer we examined (61.8% ± 9.6% injected dose vs. 29.4% ± 9.5% for (64)Cu-ATSM, P < 0.05). CONCLUSION: The hypoxic threshold that induced survivable metabolic and functional compromise was 30% O2. At this threshold, only (64)Cu-CTS delivered a hypoxic-to-normoxic contrast of 3:1, and it therefore warrants in vivo evaluation for imaging chronic cardiac ischemic syndromes.

Kinetic analysis of hyperpolarized data with minimum a priori knowledge: Hybrid maximum entropy and nonlinear least squares method (MEM/NLS).

PURPOSE: To assess the feasibility of using a hybrid Maximum-Entropy/Nonlinear Least Squares (MEM/NLS) method for analyzing the kinetics of hyperpolarized dynamic data with minimum a priori knowledge. THEORY AND METHODS: A continuous distribution of rates obtained through the Laplace inversion of the data is used as a constraint on the NLS fitting to derive a discrete spectrum of rates. Performance of the MEM/NLS algorithm was assessed through Monte Carlo simulations and validated by fitting the longitudinal relaxation time curves of hyperpolarized [1-(13) C] pyruvate acquired at 9.4 Tesla and at three different flip angles. The method was further used to assess the kinetics of hyperpolarized pyruvate-lactate exchange acquired in vitro in whole blood and to re-analyze the previously published in vitro reaction of hyperpolarized (15) N choline with choline kinase. RESULTS: The MEM/NLS method was found to be adequate for the kinetic characterization of hyperpolarized in vitro time-series. Additional insights were obtained from experimental data in blood as well as from previously published (15) N choline experimental data. CONCLUSION: The proposed method informs on the compartmental model that best approximate the biological system observed using hyperpolarized (13) C MR especially when the metabolic pathway assessed is complex or a new hyperpolarized probe is used.

Cardiac hypoxia imaging: second-generation analogues of 64Cu-ATSM.

UNLABELLED: 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.

Targeting hexokinase II to mitochondria to modulate energy metabolism and reduce ischaemia-reperfusion injury in heart.

Mitochondrially bound hexokinase II (mtHKII) has long been known to confer cancer cells with their resilience against cell death. More recently, mtHKII has emerged as a powerful protector against cardiac cell death. mtHKII protects against ischaemia-reperfusion (IR) injury in skeletal muscle and heart, attenuates cardiac hypertrophy and remodelling, and is one of the major end-effectors through which ischaemic preconditioning protects against myocardial IR injury. Mechanisms of mtHKII cardioprotection against reperfusion injury entail the maintenance of regulated outer mitochondrial membrane (OMM) permeability during ischaemia and reperfusion resulting in stabilization of mitochondrial membrane potential, the prevention of OMM breakage and cytochrome C release, and reduced reactive oxygen species production. Increasing mtHK may also have important metabolic consequences, such as improvement of glucose-induced insulin release, prevention of acidosis through enhanced coupling of glycolysis and glucose oxidation, and inhibition of fatty acid oxidation. Deficiencies in expression and distorted cellular signalling of HKII may contribute to the altered sensitivity of diabetes to cardiac ischaemic diseases. The interaction of HKII with the mitochondrion constitutes a powerful endogenous molecular mechanism to protect against cell death in almost all cell types examined (neurons, tumours, kidney, lung, skeletal muscle, heart). The challenge now is to harness mtHKII in the treatment of infarction, stroke, elective surgery and transplantation. Remote ischaemic preconditioning, metformin administration and miR-155/miR-144 manipulations are potential means of doing just that.

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.