[64Cu]diacetyl-bis(N4-methyl-thiosemicarbazone) — a radiotracer for tumor hypoxia

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Abstract

Positron emission tomography scanning using the radiotracer-labeled copper (II)-diacetyl-bis(N4-methylthiosemicarbazone) has been proposed as a noninvasive method for evaluating tumor hypoxia. Tumor hypoxia results in a more aggressive tumor phenotype together with resistance to both radiotherapy and chemotherapy. A noninvasive technique for evaluation of tumor hypoxia is not currently available. Validation of this technique would provide clinicians with a tool for determining the most appropriate cancer therapy, prognostic information, and subvolume delineation for the radiotherapy dose escalation to the radioresistant hypoxic regions within a tumor. This review article describes the background to the development of this tracer, its proposed retention mechanism, biodistribution dosimetry and the preclinical and clinical studies to date. It outlines the potential use of this radiotracer for imaging in the field of oncology.

Introduction

For many years, tumor hypoxia has been recognized as an indicator of an unfavorable prognosis, a cause of radiation resistance and treatment failure. This is most marked when the partial pressure of oxygen (pO2) in a tumor is <10 mmHg, although radiation sensitivity declines with a pO2 of <30 mmHg (mean arterial pO2 is 70–100 mmHg). The technological advances in radiotherapy mean that it is now possible to tailor treatment according to the biological phenotype of individual tumors. For example, with intensity-modulated radiotherapy (IMRT), knowledge of the distribution of hypoxia will allow escalation of radiotherapy dose to relatively radioresistant hypoxic areas. Such directed treatment will potentially improve the chance of tumor control and cure. In addition, detailed information of hypoxia within tumors may provide prognostic information [1], [2], [3]. However, despite extensive research, there is currently no feasible and widely available noninvasive method for determining a hypoxia map of an entire tumor.

Positron emission tomography (PET) is an imaging technique that allows us to look for specific biological tissue functions using radiotracers. The most widely available and well-characterized tracer currently available is 18F-fluorodexoyglucose (FDG). This radiotracer seeks out tissues that are metabolically active, providing images of its distribution that can be used for the purposes of diagnosis, tumor staging and treatment response. The ability to image tissues with a specific biological characteristic has resulted in an explosion of activity to find tracers that can be used to define a range of tissue and cellular functions for potential use in a wide range of medical and surgical specialities including oncology, endocrinology and cardiology. The ability to define regions within a tissue with a particular biological behavior that may be different among neighboring cells has resulted in the search for a PET radiotracer for tumor hypoxia. Labeled nitroimidazole compounds were the obvious first choice for evaluation as PET tracers of tumor hypoxia, and 18F-misonidazole has been the most clinically evaluated agent. The nitroimidazoles have a long history in the field of tumor hypoxia. They are retained in hypoxic tumor cells and can be used for the immunohistochemical detection of hypoxia at a cellular level (pimonidazole, EF-5); in addition, they have been given therapeutically as radiosensitizers (nimorazole) to improve the efficacy of radiotherapy in poorly oxygenated tumors. However, despite their initial promise, tumor uptake of these compounds as PET tracers is poor and requires a minimum period of scanning of 2 h in order to visualize tumors with a good tumor-to-background ratio [4]. As a result of the poor image quality obtained with the nitroimidazoles, alternative tumor hypoxia tracers have been sought.

The cellular retention mechanism of the non-tissue-selective blood perfusion tracer Cu(II)-pyruvaldehyde-bis(N4-methyl-thiosemicarbazone) (Cu-PTSM) was used as a design model for developing a hypoxia radiotracer. Cu-PTSM is a low-molecular-weight, planar, lipophilic compound that can move rapidly in to cells by passive diffusion [5], [6], [7]. Once inside, it becomes trapped, probably as a result of mitochondrial reduction of Cu(II) to Cu(I) [8], [9]. It was thought that if a tracer could be designed that had a lower redox potential such that it was only retained in cells with a more reducing environment, that is, hypoxic cells, this could lead to generation of a hypoxic radiotracer. Cu(II)-diacetyl-bis(N4-methyl-thiosemicarbazone) (Cu-ATSM) with its a high membrane permeability, and low redox potential was one of the first bis(thiosemicarbazone) complexes to be evaluated for its role as an agent to identify cellular hypoxia. In 1997, Fujibayashi et al. [10] published the results of their investigation in to a new hypoxia imaging agent, [62Cu]diacetyl-bis(N4-methylthiosemicarbazone) ([62Cu]ATSM). This study evaluated the effect of hypoxia on cellular retention of [62Cu]ATSM in a rat heart model. It showed that [62Cu]ATSM was retained in hypoxic myocardium, however, under conditions of normoxia or reoxygenation after a period of hypoxia [62Cu]ATSM washed out. A subsequent study in the mouse mammary tumor cell line EMT6 demonstrated that [64Cu]ATSM uptake increased as oxygen concentration decreased and uptake was rapid peaking at 10 min postinjection [11]. This was also seen in the 9L gliosarcoma rodent tumor model whereby tumor oxygenation was modified using hydralazine to decrease tumor oxygenation and inhalation of 100% oxygen to increase tumor oxygenation. Again, Cu-ATSM uptake was rapid (with a peak uptake at 80 s postinjection) and greater in the tumors treated with hydralazine [12].

A number of copper bis(thiosemicarbazones) have been investigated for the purposes of demonstrating tumor hypoxia; however, Cu-ATSM has so far been shown to be the most effective marker for delineating hypoxic viable tissue. The use of copper radionuclides as part of these complexes promotes great versatility given the array of isotopes available, their half lives and decay schemes (Table 1). There is potential for their use in diagnostic imaging (60Cu t1/2=23.4 min, 61Cu t1/2=204.5 min, 62Cu t1/2=9.7 min, 64Cu t1/2=761.9 min) and therapy (64Cu, 67Cu). These features together with the increasing availability of copper isotopes to the medical community make this copper complex an attractive option for investigation and use in clinical practice [13], [14].

This review aims to provide the readership with the background leading to the current interest in this radiotracer. In addition, the structure, biodistribution, dosimetry and proposed mechanisms of retention of [64Cu]ATSM in hypoxic tumor cells will be described. A summary of the preclinical and clinical investigations thus far will be presented together with the specific applications this radiotracer could have within the field of oncology.

Section snippets

Structure

In 1998, Dearling et al. [15] published details on a series of experiments performed to determine the optimal structure of bis(thiosemicarbazone) for cellular hypoxia selectivity.

Thirteen different complexes were synthesized differing by the combination of methyl (CH3), ethyl (C2H5), phenyl (C6H5) and hydrogen attached to the terminal nitrogen atom (R3, R4) and the diketone backbone (R1, R2) of the bis(thiosemicarbazone) structure (Fig. 1). Hypoxia selectivity was measured using a cell

Biodistribution

Most data on the biodistribution of Cu-ATSM has been done in preclinical models and then extrapolated to humans. Lewis et al. [11] observed the course of [64Cu]ATSM in an EMT6-bearing BALB/c mice up to 40 min postinjection. Early uptake was seen in the heart and brain with a myocardial uptake of 8.75% ID/g at 1 min and a brain uptake of 10.46% ID/g at 1 min. At 40 min, these diminished to 3.81% ID/g and 3.34% ID/g, respectively, consistent with an early vascular phase. The same pattern was seen

Dosimetry

Human absorbed dose estimates for Cu-ATSM have been based on rat data using [61Cu]ATSM and patients with NSCLC using [60Cu]ATSM. The animal data were extrapolated to estimate the human dosimetry of [60Cu]ATSM, whereas the human data were used to measure organ residence times for the final calculation of organ doses using 60Cu, 61Cu, 62Cu and 64Cu [18]. The human radiation dose estimates from the rat biodistribution data indicate that the kidney and liver are the dose-limiting organs; however,

Cellular retention mechanism

The movement of Cu-ATSM in to the cell was initially thought to be as a result of passive diffusion; however, endocytosis has been proposed as further possible mechanism for entry of Cu-ATSM (Pokrovska T, Churchill GC, University of Oxford, May 2007). Once inside the cell, a number of different, but overlapping, mechanisms have been suggested for the retention of the copper radionuclide as a marker of cellular hypoxia:

  • (i)

    Fujibayashi et al. [10] postulated that Cu(II)-ATSM is reduced selectively to

Preclinical studies

In 1997, Fujibayashi et al. published data to show that [62Cu]ATSM uptake in a rat heart model was related to oxygen tension and that it was dependent on the oxic state of the cell and not on the long-term effects of hypoxic insult [10]. In addition, they reported on the effects on [62Cu]ATSM uptake in the rat heart after occlusion of the left anterior descending coronary artery and compared this with 201thallium, providing an index of myocardial perfusion. In regions of very low perfusion, [62

Clinical studies evaluating Cu-ATSM

The vast majority of studies on Cu-ATSM have been preclinical, involving rodent tumor models. Although these are extremely important, as we have seen, the key to validating Cu-ATSM as a tumor hypoxia marker is looking at its behavior in human tumors. There are currently two published studies on the use of Cu-ATSM as a hypoxia marker; however, a number of centers are evaluating this radiotracer in clinical trials (Lewis J, Washington University of Medicine, 2007; Saunders MI, University College

Potential uses of Cu-ATSM PET

A PET radiotracer, such as Cu-ATSM, that delineates tumor hypoxia would have a role in radiotherapy planning, informing treatment decisions, and providing additional information on disease prognosis. PET images can already be incorporated in to radiotherapy planning systems, which would make implementation of Cu-ATSM PET informed planning a relatively straightforward process. The identification of radioresistant hypoxic subvolumes that can be treated with higher radiotherapy doses than they

Conclusion

Cu-ATSM does show promise as a radiotracer for delineating tumor hypoxia, although it is unlikely to be useful across the whole range of tumor types; however, the same can be said for [18F]FDG, currently our most readily available and widely used PET tracer.

It is unclear why preclinical results have been so variable; however, both clinical studies reported have found that Cu-ATSM PET scanning provides a good prediction of treatment response. It would be naive to think that a single radiotracer

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