Development of an automated production process of [64Cu][Cu (ATSM)] for positron emission tomography imaging and theranostic applications

Cyclotron-produced copper-64 radioisotope tracers offer the possibility to perform both diagnostic investigation by positron emission tomography (PET) and radiotherapy by a theranostic approach with bifunctional chelators. The versatile chemical properties of copper add to the importance of this isotope in medicinal investigation. [⁶⁴Cu][Cu (ATSM)] has shown to be a viable candidate for imaging of tumor hypoxia; a critical tumor microenvironment characteristic that typically signifies tumor progression and resistance to chemo-radiotherapy. Various production and radiosynthesis methods of [⁶⁴Cu][Cu (ATSM)] exist in labs, but usually involved non-standardized equipment with varying production qualities and may not be easily implemented in wider hospital settings. [⁶⁴Cu][Cu (ATSM)] was synthesized on a modified GE TRACERlab FXN automated synthesis module. End-of-synthesis (EOS) molar activity of [⁶⁴Cu][Cu (ATSM)] was 2.2–5.5 Ci/μmol (HPLC), 2.2–2.6 Ci/μmol (ATSM-titration), and 3.0–4.4 Ci/μmol (ICP-MS). Radiochemical purity was determined to be >99% based on radio-HPLC. The final product maintained radiochemical purity after 20 h. We demonstrated a simple and feasible process development and quality control protocols for automated cyclotron production and synthesis of [⁶⁴Cu][Cu (ATSM)] based on commercially distributed standardized synthesis modules suitable for PET imaging and theranostic studies.     

Validation of 64Cu-ATSM damaging DNA via high-LET Auger electron emission

Radioactive copper (II) (diacetyl-bis N4-methylthiosemicarbazone) (Cu-ATSM) isotopes were originally developed for the imaging of hypoxia in tumors. Because the decay of a ⁶⁴Cu atom is emitting not only positrons but also Auger electrons, this radionuclide has great potential as a theranostic agent. However, the success of ⁶⁴Cu-ATSM internal radiation therapy would depend on the contribution of Auger electrons to tumor cell killing. Therefore, we designed a cell culture system to define the contributions to cell death from Auger electrons to support or refute our hypothesis that the majority of cell death from ⁶⁴Cu-ATSM is a result of high-LET Auger electrons and not positrons or other low-LET radiation. Chinese hamster ovary (CHO) wild type and DNA repair–deficient xrs5 cells were exposed to ⁶⁴Cu-ATSM during hypoxic conditions. Surviving fractions were compared with those surviving gamma-radiation, low-LET hadron radiation, and high-LET heavy ion exposure. The ratio of the D₁₀ values (doses required to achieve 10% cell survival) between CHO wild type and xrs5 cells suggested that ⁶⁴Cu-ATSM toxicity is similar to that of high-LET Carbon ion radiation (70 keV/μm). γH2AX foci assays confirmed DNA double-strand breaks and cluster damage by high-LET Auger electrons from ⁶⁴Cu decay, and complex types of chromosomal aberrations typical of high-LET radiation were observed after ⁶⁴Cu-ATSM exposure. The majority of cell death was caused by high-LET radiation. This work provides strong evidence that ⁶⁴Cu-ATSM damages DNA via high-LET Auger electrons, supporting further study and consideration of ⁶⁴Cu-ATSM as a cancer treatment modality for hypoxic tumors.     

Species dependence of [64Cu]Cu-Bis(thiosemicarbazone) radiopharmaceutical binding to serum albumins

INTRODUCTION: Interactions of three copper(II) bis(thiosemicarbazone) positron emission tomography radiopharmaceuticals with human serum albumin, and the serum albumins of four additional mammalian species, were evaluated. METHODS: 64Cu-labeled diacetyl bis(N4-methylthiosemicarbazonato)copper(II) (Cu-ATSM), pyruvaldehyde bis(N4-methylthiosemicarbazonato)copper(II) (Cu-PTSM) and ethylglyoxal bis(thiosemicarbazonato)copper(II) (Cu-ETS) were synthesized and their binding to human, canine, rat, baboon and porcine serum albumins quantified by ultrafiltration. Protein binding was also measured for each tracer in human, porcine, rat and mouse serum. RESULTS: The interaction of these neutral, lipophilic copper chelates with serum albumin is highly compound- and species-dependent. Cu-PTSM and Cu-ATSM exhibit particularly high affinity for human serum albumin (HSA), while the albumin binding of Cu-ETS is relatively insensitive to species. At HSA concentrations of 40 mg/ml, "% free" (non-albumin-bound) levels of radiopharmaceutical were 4.0+/-0.1%, 5.3+/-0.2% and 38.6+/-0.8% for Cu-PTSM, Cu-ATSM and Cu-ETS, respectively. CONCLUSIONS: Species-dependent variations in radiopharmaceutical binding to serum albumin may need to be considered when using animal models to predict the distribution and kinetics of these compounds in humans.     

Tumor Hypoxia Detected by Positron Emission Tomography with <Superscript>60</Superscript>Cu-ATSM as a Predictor of Response and Survival in Patients Undergoing Neoadjuvant Chemoradiotherapy for Rectal Carcinoma: A Pilot Study

PURPOSE  The response of rectal cancers to neoadjuvant chemoradiotherapy is variable. Tumor hypoxia reduces the effectiveness of both radiation therapy and chemotherapy and is a well-known risk factor for tumor radioresistence. We hypothesized that imaging with the novel hypoxia-detecting agent, ⁶⁰Cu-diacetyl-bis (N⁴-methylthiosemicarbazone) (⁶⁰Cu-ATSM), previously validated in cervical and lung cancers, would predict the response of rectal cancers to neoadjuvant chemoradiotherapy and prognosis. METHODS  Patients with locally invasive (T2–4) primary or node-positive rectal cancer located <12 cm from the anal verge were recruited for this pilot study. Pretreatment tumor size and stage were determined by endorectal ultrasonography, CT, and magnetic resonance imaging. Eleven patients also underwent clinical positron emission tomography with ¹⁸F-fluorodeoxyglucose at the discretion of the treating clinician. The primary tumor was imaged by positron emission tomography with ⁶⁰Cu-ATSM, and accumulation of the tracer was measured semiquantitatively by determining the tumor-to-muscle activity ratio. Neoadjuvant chemoradiotherapy was then administered (within 2 weeks of ⁶⁰Cu-ATSM-positron emission tomography) and consisted of 45 Gy given in 25 fractions to the pelvis with continuous intravenous infusion of 5-fluorouracil (225 mg/m²/day). Proctectomy was performed six to eight weeks after neoadjuvant chemoradiotherapy and the tumor submitted to pathology for size measurement and staging. Tumor-to-muscle activity ratios were compared with tumor ¹⁸F-fluorodeoxyglucose uptake, tumor response to neoadjuvant chemoradiotherapy, and with patient survival. RESULTS  Nineteen patients were enrolled in the study, two of whom were excluded from final analysis (1 death during neoadjuvant chemoradiotherapy and 1 tumor perforation during neoadjuvant chemoradiotherapy requiring emergent surgery). Of the 17 remaining patients, 14 had a reduction in tumor size and 13 were downstaged. The median tumor-to-muscle activity ratio of 2.6 discriminated those with worse prognosis from those with better prognosis. Both overall and progression-free survivals were worse with hypoxic tumors (tumor-to-muscle activity ratio >2.6) than with nonhypoxic tumors (tumor-to-muscle activity ratio ≤2.6; both P < 0.05). In addition, 2 of the 3 tumors with no change in size had tumor-to-muscle activity ratios >2.6 (positive predictive value 66 percent), whereas 6 of 14 with decreased size had tumor-to-muscle activity ratios >2.6 (negative predictive value 57 percent). Three of the 4 tumors not downstaged had tumor-to-muscle activity ratios >2.6 (positive predictive value 75 percent), whereas 5 of 13 downstaged tumors had tumor-to-muscle activity ratios >2.6 (negative predictive value 62 percent). The mean tumor-to-muscle activity ratio for downstaged tumors (2.2) was significantly lower than that of nondownstaged tumors (3.3) (P = 0.03). The difference in mean tumor-to-muscle activity ratio between downsized (2.3) and nondownsized (2.9) tumors did not reach statistical significance (P = 0.36). Tumor ¹⁸F-fluorodeoxyglucose uptake (n = 11) did not correlate with ⁶⁰Cu-ATSM uptake (r = 0.4; P = 0.9) and there was no significant difference in mean tumor ¹⁸F-fluorodeoxyglucose uptake between patients with hypoxic tumors and those with normoxic tumors (P = 0.3). CONCLUSIONS  The results of this small pilot study suggest that ⁶⁰Cu-ATSM-PET may be predictive of survival and, possibly, tumor response to neoadjuvant chemoradiotherapy in patients with rectal cancer. A larger Phase II study is warranted to validate these results. Poster presentation at the meeting of The American Society of Colon and Rectal Surgeons, St. Louis, Missouri, June 2 to 6, 2007. Supported by an American Cancer Society Institutional Research Grant (ACS-IRG #58–010–46).     

PET imaging of cardiac hypoxia: opportunities and challenges

Myocardial hypoxia is a major factor in the pathology of cardiac ischemia and myocardial infarction. Hypoxia also occurs in microvascular disease and cardiac hypertrophy, and is thought to be a prime determinant of the progression to heart failure, as well as the driving force for compensatory angiogenesis. The non-invasive delineation and quantification of hypoxia in cardiac tissue therefore has the potential to be an invaluable experimental, diagnostic and prognostic biomarker for applications in cardiology. However, at this time there are no validated methodologies sufficiently sensitive or reliable for clinical use. PET imaging provides real-time spatial information on the biodistribution of injected radiolabeled tracer molecules. Its inherent high sensitivity allows quantitative imaging of these tracers, even when injected at sub-pharmacological (≥ pM) concentrations, allowing the non-invasive investigation of biological systems without perturbing them. PET is therefore an attractive approach for the delineation and quantification of cardiac hypoxia and ischemia. In this review we discuss the key concepts which must be considered when imaging hypoxia in the heart. We summarize the PET tracers which are currently available, and we look forward to the next generation of hypoxia-specific PET imaging agents currently being developed. We describe their potential advantages and shortcomings compared to existing imaging approaches, and what is needed in terms of validation and characterization before these agents can be exploited clinically.     

Characterization of positron emission tomography hypoxia tracer uptake and tissue oxygenation via electrochemical modeling

Purpose

Unique uptake and retention mechanisms of positron emission tomography (PET) hypoxia tracers make in vivo comparison between them challenging. Differences in imaged uptake of two common hypoxia radiotracers, [⁶¹Cu]Cu-ATSM and [¹⁸F]FMISO, were characterized via computational modeling to address these challenges.

Materials and Methods

An electrochemical formalism describing bioreductive retention mechanisms of these tracers under steady-state conditions was adopted to relate time-averaged activity concentration to tissue partial oxygen tension (PO₂), a common metric of hypoxia. Chemical equilibrium constants of product concentration to reactant concentration ratios were determined from free energy changes and reduction potentials of pertinent reactions reported in the literature. Resulting transformation functions between tracer uptake and PO₂ were compared against measured values in preclinical models. Additionally, calculated PO₂ distributions from imaged Cu-ATSM tracer activity concentrations of 12 head and neck squamous cell carcinoma (HNSCC) patients were validated against microelectrode PO₂ measurements in 69 HNSCC patients.

Results

Both Cu-ASTM- and FMISO-modeled PO₂ transformation functions were in agreement with preclinical measured values within single-deviation confidence intervals. High correlation (r²=0.94, P<.05) was achieved between modeled PO₂ distributions and measured distributions in the patient populations. On average, microelectrode hypoxia thresholds (2.5 and 5.0 mmHg) corresponded to higher Cu-ATSM uptake [2.5 and 2.0 standardized uptake value (SUV)] and lower FMISO uptake (2.0 and 1.4 SUV). Uncertainties in the models were dominated by variations in the estimated specific activity and intracellular acidity.

Conclusions

Results indicated that the high dynamic range of Cu-ATSM uptake was representative of a narrow range of low oxygen tension whose values were dependent on microenvironment acidity, while FMISO uptake was representative of a wide range of PO₂ values that were independent of acidity. The models shed light on possible causes of these discrepancies, particularly as it pertains to image contrast, and may prove to be a useful methodology in quantifying relationships between other hypoxia tracers. Comprehensive and robust assessment of tumor hypoxia prior to as well as in response to therapy may be best provided by imaging of multiple hypoxia markers that provide complementary rather than interchangeable information.     

Assessing tumor hypoxia in cervical cancer by positron emission tomography with 60Cu-ATSM: relationship to therapeutic response-a preliminary report

PURPOSE: Tumor hypoxia is associated with poor response to therapy. We have investigated whether pretreatment tumor hypoxia assessed by positron emission tomography (PET) with Cu-60 diacetyl-bis(N(4)-methylthiosemicarbazone) ((60)Cu-ATSM) predicts responsiveness to subsequent therapy in cervical cancer. METHODS AND MATERIALS: Fourteen patients with biopsy-proved cervical cancer were studied by PET with (60)Cu-ATSM before initiation of radiotherapy and chemotherapy. (60)Cu-ATSM uptake was evaluated semiquantitatively by determining the tumor-to-muscle activity ratio (T/M) and peak slope index of tumor tracer uptake. All patients also underwent clinical PET with F-18 fluorodeoxyglucose (FDG) before institution of therapy. The PET results were correlated with follow-up evaluation (14-24 months). RESULTS: Tumor uptake of (60)Cu-ATSM was inversely related to progression-free survival and overall survival (log-rank p = 0.0005 and p = 0.015, respectively). An arbitrarily selected T/M threshold of 3.5 discriminated those likely to develop recurrence; 6 of 9 patients with normoxic tumors (T/M < 3.5) are free of disease at last follow-up, whereas all of 5 patients with hypoxic tumors (T/M > 3.5) have already developed recurrence. Similar discrimination was achieved with the peak slope index. The frequency of locoregional nodal metastasis was greater in hypoxic tumors (p = 0.03). Tumor FDG uptake did not correlate with (60)Cu-ATSM uptake (r = 0.04; p = 0.80), and there was no significant difference in tumor FDG uptake between patients with hypoxic tumors and those with normoxic tumors. CONCLUSION: (60)Cu-ATSM-PET in patients with cervical cancer revealed clinically relevant information about tumor oxygenation that was predictive of tumor behavior and response to therapy in this small study.     

Retention mechanism of hypoxia selective nuclear imaging/radiotherapeutic agent Cu-diacetyl-bis(N 4-methylthiosemicarbazone) (Cu-ATSM) in tumor cells

The retention mechanism of the novel imaging/radiotherapeutic agent, Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) in tumor cells was clarified in comparison with that in normal tissue in vitro. With Cu-ATSM and reversed phase HPLC analysis, the reductive metabolism of Cu-ATSM in subcellular fractions obtained from Ehrlich ascites tumor cells was examined. As a reference, mouse brain was used. To determine the contribution of enzymes in the retention mechanisms, and specific inhibitor studies were performed. In subcellular fractions of tumor cells, Cu-ATSM was reduced mainly in the microsome/cytosol fraction rather than in the mitochondria. This finding was completely different from that found in normal brain cells. The reduction process in the microsome/cytosol was heat-sensitive and enhanced by adding exogenous NAD(P)H, an indication of enzymatic reduction of Cu-ATSM in tumor cells. Among the known bioreductive enzymes, NADH-cytochrome b5 reductase and NADPH-cytochrome P450 reductase in microsome played a major role in the reductive retention of Cu-ATSM in tumors. This enzymatic reduction was enhanced by the induction of hypoxia. Radiocopper labeled Cu-ATSM provides useful information for the detection of hypoxia as well as the microsomal bioreductive enzyme expression in tumor.     

Evaluation of62Cu labeled diacetyl-bis(N 4-methylthiosemicarbazone) as a hypoxic tissue tracer in patients with lung cancer

62Cu labeled diacetyl-bis(N4-methylthiosemicarbazone) (62Cu-ATSM) has been proposed as a generator-produced, positron-emitting tracer for hypoxic tissue imaging. From basic studies, the retention mechanism of 62Cu-ATSM is considered to be closely related to cytosolic/microsomal bioreduction, a possible system for hypoxic bioreductive drug activation. In order to evaluate the characteristics of 62Cu-ATSM, PET studies were performed in 4 normal subjects and 6 patients with lung cancer. 62Cu-ATSM cleared rapidly from the blood with little lung uptake (0.43+/-0.09, uptake ratio; divided by the arterial input function) in normal subjects. Intense tumor uptake of 62Cu-ATSM was observed in all patients with lung cancer (3.00+/-1.50). A negative correlation was observed between blood flow and flow-normalized 62Cu-ATSM uptake in three of four patients. In contrast, 62Cu-ATSM uptake was not related to that of 18F-fluorodeoxyglucose. The negative correlation between blood flow and flow normalized 62Cu-ATSM uptake suggests an enhancement of retention of 62Cu-ATSM by low flow. 62Cu-ATSM is a promising PET tracer for tumor imaging, which might bring new information for chemotherapeutic treatment as well as radiotherapy of hypoxic tumors.     

Examining the relationship between Cu-ATSM hypoxia selectivity and fatty acid synthase expression in human prostate cancer cell lines

INTRODUCTION: Positron emission tomography (PET) imaging with copper (II)-diacetyl-bis(N4-Methylthiosemicarbazone)(Cu-ATSM) for delineating hypoxia has provided valuable clinical information, but investigations in animal models of prostate cancer have shown some inconsistencies. As a defense mechanism in prostate cancer cells, the fatty acid synthesis pathway harnesses its oxidizing power for improving the redox balance despite conditions of extreme hypoxia, potentially altering Cu-ATSM hypoxia selectivity. METHODS: Human prostate tumor-cultured cell lines (PC-3, 22Rv1, LNCaP and LAPC-4), were treated with a fatty acid synthase (FAS) inhibitor (C75, 100 microM) under anoxia. The 64Cu-ATSM uptake in these treated cells and nontreated anoxic cells was then examined. Fatty acid synthase expression level in each cell line was subsequently quantified by ELISA. An additional study was performed in PC-3 cells to examine the relationship between the restoration of 64Cu-ATSM hypoxia selectivity and the concentration of C75 (100, 20, 4 or 0.8 microM) administered to the cells. RESULTS: Inhibition of fatty acid synthesis with C75 resulted in a significant increase in 64Cu-ATSM retention in prostate tumor cells in vitro under anoxia over 60 min. Inhibition studies demonstrated higher uptake values of 20.9+/-3.27%, 103.0+/-32.6%, 144.2+/-32.3% and 200.1+/-79.3% at 15 min over control values for LAPC-4, PC-3, LNCaP and 22Rv1 cells, respectively. A correlation was seen (R2=.911) with FAS expression plotted against percentage change in 64Cu-ATSM uptake with C75 treatment. CONCLUSIONS: Although Cu-ATSM has clinical relevance in the PET imaging of hypoxia in many tumor types, its translation to the imaging of prostate cancer may be limited by the overexpression of FAS associated with prostatic malignancies.