E. coli nitroreductase NfsA is a reporter gene for non-invasive PET imaging in cancer gene therapy applications.

The use of reporter genes to non-invasively image molecular processes inside cells has significant translational potential, particularly in the context of systemically administered gene therapy vectors and adoptively administered cells such as immune or stem cell based therapies. Bacterial nitroreductase enzymes possess ideal properties for reporter gene imaging applications, being of non-human origin and possessing the ability to metabolize a range of clinically relevant nitro(hetero)cyclic substrates. Methods: A library of eleven Escherichia coli nitroreductase candidates were screened for the ability to efficiently metabolize 2-nitroimidazole based positron emission tomography (PET) probes originally developed as radiotracers for hypoxic cell imaging. Several complementary methods were utilized to detect formation of cell-entrapped metabolites, including various in vitro and in vivo models to establish the capacity of the 2-nitroimidazole PET agent EF5 to quantify expression of a nitroreductase candidate. Proof-of-principle PET imaging studies were successfully conducted using 18F-HX4. Results: Recombinant enzyme kinetics, bacterial SOS reporter assays, anti-proliferative assays and flow cytometry approaches collectively identified the major oxygen-insensitive nitroreductase NfsA from E. coli (NfsA_Ec) as the most promising nitroreductase reporter gene. Cells expressing NfsA_Ec were demonstrably labelled with the imaging agent EF5 in a manner that was quantitatively superior to hypoxia, in monolayers (2D), multicellular layers (3D), and in human tumor xenograft models. EF5 retention correlated with NfsA_Ec positive cell density over a range of EF5 concentrations in 3D in vitro models and in xenografts in vivo and was predictive of in vivo anti-tumor activity of the cytotoxic prodrug PR-104. Following PET imaging with 18F-HX4, a significantly higher tumor-to-blood ratio was observed in two xenograft models for NfsA_Ec expressing tumors compared to the parental tumors thereof, providing verification of this reporter gene imaging approach. Conclusion: This study establishes that the bacterial nitroreductase NfsA_Ec can be utilized as an imaging capable reporter gene, with the ability to metabolize and trap 2-nitroimidazole PET imaging agents for non-invasive imaging of gene expression.

Intra-articular etanercept attenuates pain and hypoxia from TMJ loading in the rat.

Mechanical overloading of the temporomandibular joint (TMJ) and biochemical changes, like inflammation and hypoxia, contribute to cartilage degeneration and pain associated with osteoarthritis (OA). Yet, how overloading contributes to early dysregulation of chondrocytes is not understood, limiting the development of diagnostics and treatments for TMJ OA. Hypoxia-inducible factors (HIF)-1α/2α in chondrocytes were evaluated at Days 8 and 15 in a rat TMJ pain model induced by jaw loading (1 h/day for 7 days) using immunohistochemistry and compared between cases that induce persistent (3.5 N), acute (2 N), or no (0 N) sensitivity. Hypoxia was measured on Day 8 by immunolabeling of the tracer EF5 and 18 F-EF5 PET imaging. To assess the role of tumor necrosis factor (TNF) in painful TMJ loading, intra-articular etanercept was given before loading. Orofacial sensitivity was evaluated during and after loading. Facial grimace, TNF-α, HIF-2α, and hypoxia levels in the TMJ were measured after loading. HIF-2α was elevated (P = .03) after 3.5 N loading at Day 8, but HIF-1α was unchanged. EF5 uptake increased on Day 8 in the 3.5 N group (P < .048) by tissue assay and 18 F-EF5 PET. At Day 8, both HIF-2α (P = .01) and EF5 uptake (P = .005) were correlated with loading magnitude. Etanercept attenuated sensitivity (P < .01) and the facial grimace on Day 7 (P = .01). It also reduced (P < .01) HIF-2α and EF5 uptake on Day 8; but TNF-α levels were not different from controls at that time. Findings suggest that TMJ loading that induces persistent sensitivity upregulates the catabolic factor HIF-2α and reduces oxygen levels in the cartilage, which may be TNF-driven.

Engineering Escherichia coli NfsB To Activate a Hypoxia-Resistant Analogue of the PET Probe EF5 To Enable Non-Invasive Imaging during Enzyme Prodrug Therapy.

Gene-directed enzyme prodrug therapy (GDEPT) uses tumor-tropic vectors to deliver prodrug-converting enzymes such as nitroreductases specifically to the tumor environment. The nitroreductase NfsB from Escherichia coli (NfsB_Ec) has been a particular focal point for GDEPT and over the past 25 years has been the subject of several engineering studies seeking to improve catalysis of prodrug substrates. To facilitate clinical development, there is also a need to enable effective non-invasive imaging capabilities. SN33623, a 5-nitroimidazole analogue of 2-nitroimidazole hypoxia probe EF5, has potential for PET imaging exogenously delivered nitroreductases without generating confounding background due to tumor hypoxia. However, we show here that SN33623 is a poor substrate for NfsB_Ec. To address this, we used assay-guided sequence and structure analysis to identify two conserved residues that block SN33623 activation in NfsB_Ec and close homologues. Introduction of the rational substitutions F70A and F108Y into NfsB_Ec conferred high levels of SN33623 activity and enabled specific labeling of E. coli expressing the engineered enzyme. Serendipitously, the F70A and F108Y substitutions also substantially improved activity with the anticancer prodrug CB1954 and the 5-nitroimidazole antibiotic prodrug metronidazole, which is a potential biosafety agent for targeted ablation of nitroreductase-expressing vectors.

18F-EF5 PET-based Imageable Hypoxia Predicts Local Recurrence in Tumors Treated With Highly Conformal Radiation Therapy.

PURPOSE: Tumor hypoxia contributes to radiation resistance. A noninvasive assessment of tumor hypoxia would be valuable for prognostication and possibly selection for hypoxia-targeted therapies. 18F-pentafluorinated etanidazole (18F-EF5) is a nitroimidazole derivative that has demonstrated promise as a positron emission tomography (PET) hypoxia imaging agent in preclinical and clinical studies. However, correlation of imageable hypoxia by 18F-EF5 PET with clinical outcomes after radiation therapy remains limited. METHODS AND MATERIALS: Our study prospectively enrolled 28 patients undergoing radiation therapy for localized lung or other tumors to receive pretreatment 18F-EF5 PET imaging. Depending on the level of 18F-EF5 tumor uptake, patients underwent functional manipulation of tumor oxygenation with either carbogen breathing or oral dichloroacetate followed by repeated 18F-EF5 PET. The hypoxic subvolume of tumor was defined as the proportion of tumor voxels exhibiting higher 18F-EF5 uptake than the 95th percentile of 18F-EF5 uptake in the blood pool. Tumors with a hypoxic subvolume ≥ 10% on baseline 18F-EF5 PET imaging were classified as hypoxic by imaging. A Cox model was used to assess the correlation between imageable hypoxia and clinical outcomes after treatment. RESULTS: At baseline, imageable hypoxia was demonstrated in 43% of all patients (12 of 28), including 6 of 16 patients with early-stage non-small cell lung cancer treated with stereotactic ablative radiation therapy and 6 of 12 patients with other cancers. Carbogen breathing was significantly associated with decreased imageable hypoxia, while dichloroacetate did not result in a significant change under our protocol conditions. Tumors with imageable hypoxia had a higher incidence of local recurrence at 12 months (30%) than those without (0%) (P < .01). CONCLUSIONS: Noninvasive hypoxia imaging by 18F-EF5 PET identified imageable hypoxia in about 40% of tumors in our study population. Local tumor recurrence after highly conformal radiation therapy was higher in tumors with imageable hypoxia.

Repeatability of tumour hypoxia imaging using [18F]EF5 PET/CT in head and neck cancer.

PURPOSE: Hypoxia contributes to radiotherapy resistance and more aggressive behaviour of several types of cancer. This study was designed to evaluate the repeatability of intratumour uptake of the hypoxia tracer [18F]EF5 in paired PET/CT scans. METHODS: Ten patients with newly diagnosed head and neck cancer (HNC) received three static PET/CT scans before chemoradiotherapy: two with [18F]EF5 a median of 7 days apart and one with [18F]FDG. Metabolically active primary tumour volumes were defined in [18F]FDG images and transferred to co-registered [18F]EF5 images for repeatability analysis. A tumour-to-muscle uptake ratio (TMR) of 1.5 at 3 h from injection of [18F]EF5 was used as a threshold representing hypoxic tissue. RESULTS: In 10 paired [18F]EF5 PET/CT image sets, SUVmean, SUVmax, and TMR showed a good correlation with the intraclass correlation coefficients of 0.81, 0.85, and 0.87, respectively. The relative coefficients of repeatability for these parameters were 15%, 17%, and 10%, respectively. Fractional hypoxic volumes of the tumours in the repeated scans had a high correlation using the Spearman rank correlation test (r = 0.94). In a voxel-by-voxel TMR analysis between the repeated scans, the mean of Pearson correlation coefficients of individual patients was 0.65. The mean (± SD) difference of TMR in the pooled data set was 0.03 ± 0.20. CONCLUSION: Pretreatment [18F]EF5 PET/CT within one week shows high repeatability and is feasible for the guiding of hypoxia-targeted treatment interventions in HNC.

Engineering a Multifunctional Nitroreductase for Improved Activation of Prodrugs and PET Probes for Cancer Gene Therapy.

Gene-directed enzyme-prodrug therapy (GDEPT) is a promising anti-cancer strategy. However, inadequate prodrugs, inefficient prodrug activation, and a lack of non-invasive imaging capabilities have hindered clinical progression. To address these issues, we used a high-throughput Escherichia coli platform to evolve the multifunctional nitroreductase E. coli NfsA for improved activation of a promising next-generation prodrug, PR-104A, as well as clinically relevant nitro-masked positron emission tomography-imaging probes EF5 and HX4, thereby addressing a critical and unmet need for non-invasive bioimaging in nitroreductase GDEPT. The evolved variant performed better in E. coli than in human cells, suggesting optimal usefulness in bacterial rather than viral GDEPT vectors, and highlighting the influence of intracellular environs on enzyme function and the shaping of promiscuous enzyme activities within the "black box" of in vivo evolution. We provide evidence that the dominant contribution to improved PR-104A activity was enhanced affinity for the prodrug over-competing intracellular substrates.

18F-EF5 PET Is Predictive of Response to Fractionated Radiotherapy in Preclinical Tumor Models.

We evaluated the relationship between pre-treatment positron emission tomography (PET) using the hypoxic tracer 18F-[2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl) acetamide] (18F-EF5) and the response of preclinical tumor models to a range of fractionated radiotherapies. Subcutaneous HT29, A549 and RKO tumors grown in nude mice were imaged using 18F-EF5 positron emission tomography (PET) in order to characterize the extent and heterogeneity of hypoxia in these systems. Based on these results, 80 A549 tumors were subsequently grown and imaged using 18F-EF5 PET, and then treated with one, two, or four fraction radiation treatments to a total dose of 10-40 Gy. Response was monitored by serial caliper measurements of tumor volume. Longitudinal post-treatment 18F-EF5 PET imaging was performed on a subset of tumors. Terminal histologic analysis was performed to validate 18F-EF5 PET measures of hypoxia. EF5-positive tumors responded more poorly to low dose single fraction irradiation relative to EF5-negative tumors, however both groups responded similarly to larger single fraction doses. Irradiated tumors exhibited reduced 18F-EF5 uptake one month after treatment compared to control tumors. These findings indicate that pre- treatment 18F-EF5 PET can predict the response of tumors to single fraction radiation treatment. However, increasing the number of fractions delivered abrogates the difference in response between tumors with high and low EF5 uptake pre-treatment, in agreement with traditional radiobiology.

Prognostic value of tumour blood flow, [¹8F]EF5 and [¹8F]FDG PET/CT imaging in patients with head and neck cancer treated with radiochemotherapy.

PURPOSE: In order to improve the treatment of squamous cell carcinoma of the head and neck, precise information on the treated tumour's biology is required and the prognostic importance of different biological parameters needs to be determined. The aim of our study was to determine the predictive value of pretreatment PET/CT imaging using [(18)F]FDG, a new hypoxia tracer [(18)F]EF5 and the perfusion tracer [(15)O]H2O in patients with squamous cell cancer of the head and neck treated with radiochemotherapy. METHODS: The study group comprised 22 patients with confirmed squamous cell carcinoma of the head and neck who underwent a PET/CT scan using the above tracers before any treatment. Patients were later treated with a combination of radiochemotherapy and surgery. Parametric blood flow was calculated from dynamic [(15)O]H2O PET images using a one-tissue compartment model. [(18)F]FDG images were analysed by calculating standardized uptake values (SUV) and metabolically active tumour volumes (MATV). [(18)F]EF5 images were analysed by calculating tumour-to-muscle uptake ratios (T/M ratio). A T/M ratio of 1.5 was considered a significant threshold and used to determine tumour hypoxic subvolumes (HS) and hypoxic fraction area. The findings were finally correlated with the pretreatment clinical findings (overall stage and TNM stage) as well as the outcome following radiochemotherapy in terms of local control and overall patient survival. RESULTS: Tumour stage and T-classification did not show any significant differences in comparison to the patients' metabolic and functional characteristics measured on PET. Using the Cox proportional hazards model, a shorter overall survival was associated with MATV (p = 0.008, HR = 1.108), maximum [(18)F]EF5 T/M ratio (p = 0.0145, HR = 4.084) and tumour HS (p = 0.0047, HR = 1.112). None of the PET parameters showed a significant effect on patient survival in the log-rank test, although [(18)F]EF5 maximum T/M ratio was the closest (p = 0.109). By contrast, tumour blood flow was not correlated with any of the clinical endpoints. There were no statistically significant correlations among [(18)F]FDG SUVmax, [(18)F]EF5 T/M ratio and blood flow. CONCLUSION: Our study in a limited number of patients confirmed the importance of MATV in the prognosis of locally advanced squamous cell carcinoma of the head and neck. It is of interest that high uptake of the hypoxia tracer [(18)F]EF5 showed a stronger correlation with a poor clinical outcome than [(18)F]FDG uptake. This confirms the importance of hypoxia in treatment outcome and suggests that [(18)F]EF5 may act as a surrogate marker of radioresistance.

Comparison of the Hypoxia PET Tracer (18)F-EF5 to Immunohistochemical Marker EF5 in 3 Different Human Tumor Xenograft Models.

UNLABELLED: The availability of (18)F-labeled and unlabeled 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5) allows for a comparative assessment of tumor hypoxia by PET and immunohistochemistry; however, the combined use of these 2 approaches has not been fully assessed in vivo. The aim of this study was to evaluate (18)F-EF5 tumor uptake versus EF5 binding and hypoxia as determined from immunohistochemistry at both macroscopic and microregional levels. METHODS: Three tumor models-PC3, HCT116, and H460-were evaluated. Tumor-bearing animals were coinjected with (18)F-EF5 and EF5 (30 mg/kg), and PET imaging was performed at 2.5 h after injection. After PET imaging and 2 min after Hoechst 33342 injection, the tumors were excised and evaluated for (18)F-EF5 distribution by autoradiography and EF5 binding by immunohistochemistry. Additionally, the effects of nonradioactive EF5 (30 mg/kg) on the hypoxia-imaging characteristics of (18)F-EF5 were evaluated by comparing the PET data for H460 tumors with those from animals injected with (18)F-EF5 alone. RESULTS: The uptake of (18)F-EF5 in hypoxic tumor regions and the spatial relationship between (18)F-EF5 uptake and EF5 binding varied among tumors. H460 tumors showed higher tumor-to-muscle contrast in PET imaging; however, the distribution and uptake of the tracer was less specific for hypoxia in H460 than in HCT116 and PC3 tumors. Correlation analyses revealed that the highest spatial correlation between (18)F-EF5 uptake and EF5 binding was in PC3 tumors (r = 0.73 ± 0.02) followed by HCT116 (r = 0.60 ± 0.06) and H460 (r = 0.53 ± 0.10). Uptake and binding of (18)F-EF5 and EF5 correlated negatively with Hoechst 33342 perfusion marker distribution in the 3 tumor models. Image contrast and heterogeneous uptake of (18)F-EF5 in H460 tumors was significantly higher when the radiotracer was used alone versus in combination with unlabeled EF5 (tumor-to-muscle ratio of 2.51 ± 0.33 vs. 1.71 ± 0.17, P < 0.001). CONCLUSION: The uptake and hypoxia selectivity of (18)F-EF5 varied among tumor models when animals also received nonradioactive EF5. Combined use of radioactive and nonradioactive EF5 for independent assessment of tumor hypoxia by PET and immunohistochemistry methods is promising; however, the EF5 drug concentrations that are required for immunohistochemistry assays may affect the uptake of (18)F-EF5 in hypoxic cells in certain tumor types as observed in H460 in this study.

Hypoxia in melanoma: using optical spectroscopy and EF5 to assess tumor oxygenation before and during regional chemotherapy for melanoma.

BACKGROUND: There is increasing evidence that tumor hypoxia plays a significant role in the chemoresistance of melanoma, but to our knowledge, real-time tumor oxygenation during isolated limb infusion (ILI) has not been studied. We sought to demonstrate the feasibility of measuring real-time alterations in tissue oxygenation. METHODS: Consecutive patients with histologically confirmed in-transit melanoma were enrolled onto a prospective single-arm pilot study and administered the hypoxia marker drug EF5. All patients were treated with ILI. Optical spectroscopy readings were obtained at three locations: two discrete target lesions and one normal skin control. Measurements were taken at 11 predefined time points during ILI. RESULTS: A total of six patients were enrolled onto this pilot study. Intratumor and normal skin optical spectroscopy readings were found to have discrete inflection points throughout the duration of therapy, corresponding with established time points. Baseline hypoxia as measured by both optical spectroscopy and EF5 immunofluorescence was variable, but on the basis of optical spectra, tumors appeared to become more hypoxic compared to normal skin after tourniquet application. The optical hypoxia signature was variable between patients while hemoglobin absorption increased. CONCLUSIONS: To our knowledge, this is the first use of real-time optical spectroscopy to evaluate oxygenation and perfusion within melanoma lesions during regional chemotherapy. We report our development of this new noninvasive means of assessing tumor vascular function, which has the potential to be a powerful tool for noninvasive examination of the melanoma tumor microenvironment.

18F-EF5 PET imaging as an early response biomarker for the hypoxia-activated prodrug SN30000 combined with radiation treatment in a non-small cell lung cancer xenograft model.

UNLABELLED: Hypoxia is a significant therapeutic problem for solid tumors because hypoxic cells are treatment-resistant and more aggressive. Hypoxia-activated prodrugs such as SN30000 use a mechanism of activation in hypoxic cells similar to that of 2-nitroimidazole hypoxia PET tracers. Therefore, we have evaluated the usefulness of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-(18)F-pentafluoropropyl)-acetamide ((18)F-EF5) PET to monitor and predict tumor response to SN30000 plus radiation treatment (RT). METHODS: Human non-small cell lung cancer xenografts (H460) in athymic rats were imaged with (18)F-EF5 PET before and after treatment with SN30000 (90 mg/kg), with or without 15-Gy RT. The feasibility of imaging early changes in hypoxia in response to SN30000 was examined 24 h after treatment, followed by ex vivo γ-counting and immunohistochemical examination to study drug-induced apoptosis. Subsequently, the therapeutic effects of SN30000 with or without RT were evaluated in tumor growth delay studies and compared with early treatment-induced changes observed by (18)F-EF5 PET. Changes in tumor hemoglobin oxygen saturation as a function of time after treatment measured by optical spectroscopy were compared with PET data. RESULTS: The uptake of (18)F-EF5 was significantly lower in SN30000-treated tumors than in saline controls 24 h after treatment (mean standardized uptake value, 0.44 ± 0.08 vs. 0.56 ± 0.08 for control group; P < 0.05). Apoptosis was significantly higher in SN30000-treated tumors than in controls. Early treatment-induced changes in (18)F-EF5 uptake were indicative of tumor response in growth delay studies at the group level. SN30000 plus RT significantly decreased (18)F-EF5 uptake relative to baseline and resulted in complete tumor remission in 5 of 7 animals. SN30000 alone decreased (18)F-EF5 uptake, generally in tumors with high initial standardized uptake values, and showed a minor tumor growth delay effect. The changes induced by SN30000 with or without RT in (18)F-EF5 uptake correlated with baseline hypoxia levels. RT caused significant increases in tumor oxygen concentration and hemoglobin oxygen saturation. CONCLUSION: A hypoxia PET imaging agent can measure changes in tumor hypoxic fraction in response to SN30000. These results suggest the utility of (18)F-EF5 PET for monitoring early response to tumor treatment with SN30000 plus RT in the clinical development of this novel hypoxia-activated prodrug.

Electrophilic addition of chlorine monofluoride for PET tracers.

PURPOSE: We have studied the utility of [(18)F]ClF electrophilic addition to the carbon-carbon double bond of analogues of a model positron emission tomography (PET) tracer, [(18)F]EF5. The consequence of simultaneous chlorine/fluorine addition on lipophilicity and biological activity of the molecule is evaluated. PROCEDURES: Post-target produced [(18)F]F2 was reacted with Cl2 to produce [(18)F]ClF, which was used in electrophilic addition. RESULTS: [(18)F]ClF was produced and used to label chlorinated analogues of [(18)F]EF5. The chlorinated analogues, [(18)F]EF4Cla and [(18)F]EF4Clb, were synthesized simultaneously. The in vivo uptake of the analogues compared well with [(18)F]EF5 uptake in tumor-bearing mice. CONCLUSION: [(18)F]ClF is a suitable labeling reagent for electrophilic addition to double bonds of PET tracers. The results show that the modification of the pentafluoro group of [(18)F]EF5 by monofluorine-for-chlorine exchange affected the lipophilicity, but the hypoxia avidity of these molecules was not apparently altered.

Radiation dosimetry and biodistribution of the hypoxia tracer (18)F-EF5 in oncologic patients.

UNLABELLED: The primary goals of this study were to determine the biodistribution and excretion of (18)F-EF5 in oncologic patients, to estimate the radiation-absorbed dose and to determine the safety of this drug. METHODS: Sixteen patients with histologically confirmed malignancy received a mean intravenous infusion of 217 MBq (range 107-364 MBq) of (18)F-EF5. Over a 4-6-hour period, four to five serial positron emission tomography (PET) or PET/computed tomography (CT) scans were obtained. To calculate the radiation dosimetry estimates, volumes of interest were drawn over the source organs for each PET scan or on the CT for each PET/CT scan. Serial blood samples were obtained to measure (18)F-EF5 blood clearance. Bladder-wall dose was calculated based on urine activity measurements. RESULTS: The urinary bladder received the largest radiation-absorbed dose, 0.12 ± 0.034 mSv/MBq (mean ± SD). The average effective dose equivalent and the effective dose of (18)F-EF5 were 0.021 ± 0.003 mSv/MBq and 0.018 ± 0.002 mSv/MBq, respectively. (18)F-EF5 was well tolerated in all subjects. CONCLUSIONS: (18)F-EF5 was demonstrated to be safe for patients, and the radiation exposure is clinically acceptable. As with any radiotracer with primary excretion in the urine, the bladder-wall dose can be minimized by active hydration and frequent voiding.

PET of hypoxia: current and future perspectives.

In the past 25 y, a large amount of clinical experience with hypoxia PET tracers has accumulated. This article discusses recent improvements in image acquisition protocols and tracer pharmacology that have resulted in improved understanding of the underlying physiologic processes. The widespread clinical adoption of hypoxia PET tracers will depend largely on their utility in treatment prescription and in outcome monitoring. The establishment and validation of hypoxia-directed treatment protocols are still under development, and it is envisaged that the design and use of future hypoxia PET tracers will develop as part of this process.

A simplified synthesis of the hypoxia imaging agent 2-(2-Nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-[(18)F]pentafluoropropyl)-acetamide ([18F]EF5).

INTRODUCTION: [(18)F]EF5 is a validated marker for PET imaging of tumor hypoxia. It is prepared by reacting a trifluoroallyl precursor with carrier-added [(18)F]F(2) gas in trifluoroacetic acid (TFA) solvent. We report here an improved radiosynthesis and purification of [(18)F]EF5 by utilizing an electroformed nickel (Ni) target for [(18)F]F(2) production, and Oasis® HLB cartridges for on-line solid phase extraction of [(18)F]EF5 prior to HPLC purification. METHODS: [(18)F]F(2) was produced by deuteron bombardment of neon plus F(2) in an Ni target, and bubbled through the radiolabelling precursor solution. Purification was achieved by extracting the contents of the crude reaction mixture onto Oasis HLB cartridges, and subsequently eluted onto a semi-preparative HPLC column for further separation. Purified [(18)F]EF5 was evaluated in small animal PET studies using HCT116 tumor xenografts in nude mice. RESULTS: The electroformed Ni target enabled recovery of >75% of the radioactivity from the cyclotron target, resulting in 16.2 ± 2.2 GBq (438 ± 58 mCi) of [(18)F]F(2) available for the synthesis. Use of Oasis cartridges yielded a less complex mixture for purification. On average, 1140 ± 200 MBq (30.8 ± 5.4 mCi) of [(18)F]EF5 were collected at EOS. Small animal PET imaging studies showed specific retention of [(18)F]EF5 in tumors, with tumor-to-muscle ratios of 2.7 ± 0.3 at about 160 min after injection. CONCLUSION: A simple procedure has been developed for the routine synthesis of [(18)F]EF5 in amounts and purity required for clinical studies. This new method avoids the need for TFA evaporation and also enables facile automation of the synthesis using commercially available radiosynthesis modules.

Imaging tumor sensitivity to a bioreductive prodrug: two for the price of one!

Hypoxia is an important characteristic of many solid tumors and has a major negative effect on treatment response. A way to combat this effect is with drugs called "bioreductive prodrugs" or "hypoxic cytotoxins," which are metabolized under hypoxia to toxic species. However, the patients with hypoxic tumors need to be identified.

Tracer level electrophilic synthesis and pharmacokinetics of the hypoxia tracer [(18)F]EF5.

PURPOSE: 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide labeled with [(18)F]-fluorine ([(18)F]EF5), a promising tracer for tumor hypoxia, has previously been synthesized in low yields and low specific radioactivity. In pharmacokinetic evaluations, in the presence of non-radioactive EF5, a uniform and low background uptake and high in vivo stability of [(18)F]EF5 have been demonstrated. Our purpose was to increase the specific radioactivity of [(18)F]EF5 to enable to study the pharmacokinetics at trace level. PROCEDURES: [(18)F]EF5 was synthesized using high specific radioactivity electrophilic [(18)F]F(2) as labelling reagent. Biodistribution of [(18)F]EF5 was determined in a prostate tumor mouse model, and formation of radiolabelled metabolites was studied in mouse, rat and human plasma. RESULTS: On average, 595 ± 153 MBq of [(18)F]EF5 was produced. Specific radioactivity was 6.6 ± 1.9 GBq/µmol and the radiochemical purity exceeded 99.0%. [(18)F]EF5 was distributed uniformly in tissues, with highest uptake in liver, kidney, and intestine. Several radiolabelled metabolites were detected in mouse plasma and tissues, whereas low amounts of metabolites were detected in human and rat plasma. CONCLUSIONS: [(18)F]EF5 was synthesized by electrophilic labelling with high quality and high yields. Pharmacokinetics of [(18)F]EF5 was determined at trace level in several species. Our results suggest that the trace-level approach does not affect the biodistribution of [(18)F]EF5. Extensive metabolism was seen in mouse.

The 2-nitroimidazole EF5 is a biomarker for oxidoreductases that activate the bioreductive prodrug CEN-209 under hypoxia.

PURPOSE: Benzotriazine-N-oxide bioreductive prodrugs such as tirapazamine and its improved analogue CEN-209 (SN30000) have potential for exploiting hypoxia in tumors. Here, we test the hypothesis that the 2-nitroimidazole EF5, in clinical development for both immunohistochemical and positron emission tomography imaging of hypoxia, can detect not only hypoxia but also the one-electron reductases required for activation of these hypoxia-targeted prodrugs. EXPERIMENTAL DESIGN: Aerobic and hypoxic covalent binding of [(14)C]-EF5 was determined in human tumor cell lines, including lines with overexpression of NADPH:cytochrome P450 oxidoreductase (CYPOR), and reductive metabolism of tirapazamine and CEN-209 by mass spectrometry. DNA damage response was measured by γH2AX formation. Bioreductive metabolism was modulated in HCT116 tumor xenografts by overexpression of CYPOR and breathing of hyperbaric oxygen or 10% oxygen. RESULTS: Overexpression of CYPOR induced similar 2- to 4-fold increases in EF5 binding and metabolic reduction of tirapazamine and CEN-209 in SiHa and HCT116 cell lines, and similar enhancement of γH2AX formation. EF5 binding and metabolic reduction of the prodrugs were highly correlated in a panel of 14 hypoxic tumor cell lines. In HCT116 xenografts, CYPOR overexpression also significantly increased EF5 binding and CEN-209 reduction, and modification of tumor hypoxia caused similar changes to the bioreductive activation of both agents, resulting in a strong correlation between EF5 binding and CEN209-induced DNA damage (R(2) = 0.68, P < 0.0001) at the individual tumor level. CONCLUSIONS: EF5 binding is a promising stratification biomarker for benzotriazine-N-oxide bioreductive prodrugs because of its potential for interrogating reductase activity as well as hypoxia in individual tumors.