Special Issue Review Received 22 April 2012,
Revised 27 July 2012,
Accepted 6 November 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2997
Positron emission tomography radiotracers for imaging hypoxia† Melinda Wuest and Frank Wuest* Localized hypoxia, the physiological hallmark of many clinical pathologies, is the consequence of acute or chronic ischemia in the affected region or tissue. The versatility, sensitivity, quantitative nature, and increasing availability of positron emission tomography (PET) make it the preclinical and clinical method of choice for functional imaging of tissue hypoxia at the molecular level. The progress and current status of radiotracers for hypoxia-specific PET imaging are reviewed in this article including references mainly focused on radiochemistry and also relevant to molecular imaging of hypoxia in preclinical and clinical studies. Keywords: tumor hypoxia; radiotracers; fluorine-18; radiochemistry
Introduction Tissue hypoxia can develop in the microenvironment of solid tumors or after acute or chronic ischemic events in the brain or the heart. Ischemia is induced by a number of vascular, lymphatic, and membrane changes that prevent delivery of sufficient amounts of oxygen to the hypoxic area.1–3 Tissue hypoxia can be detected with various molecular probes, especially with radiotracers.4,5 Hypoxic cells undergo complex transcriptional and posttranslational cellular adaptations including upregulation of drug resistance transporters and energy metabolism, modulation of apoptotic processes, and induction of gene expression encoding growth factors that promote metastases and compromise chemotherapy outcomes.6–8 In oncology, tumor hypoxia is associated with poor response to treatment protocols and poor clinical outcome. This strongly supports the need to identify tumors with high hypoxic fractions to implement hypoxia-directed treatments and to optimize tumor treatments that are oxygen sensitive.9–11 Molecular probes designed to measure tissue hypoxia have to compete directly with intracellular O2. Hypoxia-sensitive molecular probes should not undergo metabolic trapping when O2 supply within the cells is sufficient but should retain within the cells when oxygen supply is decreased.12 This review summarizes synthesis and application of various positron emission tomography (PET) radiotracers designed and used for hypoxia imaging. On the basis of the topic of this special theme issue dealing with 11C and 18F radiochemistry, special focus will be on 18F-labeled radiotraces for imaging tissue hypoxia, whereas alternative PET radiotracers labeled with 68Ga, 64Cu, and 124 I will only be discussed briefly. 18
F-labeled 2-nitroimidazoles as PET radiotracers for hypoxia imaging
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2-Nitroimidazole-containing compounds were among the first molecular probes for PET imaging of hypoxia. The mechanism of reduction and intracellular trapping of 2-nitroimidazoles
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involves formation of a radical anion, which reacts preferentially with O2 if it is present to re-oxidize the radiotracer into its original form. Under hypoxic conditions when O2 supply is reduced, the radical anion accepts another electron yielding a nitroso intermediate that is further reduced to a primary amine (R0 –NH2) as highly efficient alkylating agent. Covalent bonding of alkylating agent R0 –NH2 results in intracellular retention of the radiolabel (Figure 1).12 Early analysis of tumor hypoxia with 14C-labeled misonidazole revealed selective binding of the radiolabel to hypoxic cells within a tumor.13 On the basis of their lipophilicity, 2-nitroimidazoles enter the cell via passive diffusion.14 This early work stimulated the development of various 2nitroimidazole-containing compounds labeled with 18F for PET imaging of hypoxia. A summary of 18F-labeled 2-nitroimidazoles (except for [18F]3-NTR, which is a 3-nitro-1,2,4-triazole analog of 1-[18F]fluoro-3-(2-nitro-imidazol-1-yl)-propan-2-ol ([18F]FMISO)) is given in Figure 2. One of the most frequently applied 18F-labeled 2-nitroimidazoles is [18F]FMISO. Several different synthesis routes have been described. A two-step synthesis was developed involving [18F] epifluorohydrin as intermediate, which was further reacted with 2-nitroimidazole to provide [18F]FMISO.15,16 HPLC purification was necessary to obtain high radiochemical purity. A more prominent approach relies on nucleophilic substitution reaction of
Department of Oncology, University of Alberta, Edmonton, AB, T6G 1Z2, Canada *Correspondence to: Frank Wuest, PhD, Department of Oncology, University of Alberta, 11560 University Ave, Edmonton, Alberta, T6G 1Z2, Canada. E-mail: [email protected] †
This article is published in Journal of Labelled Compounds and Radiopharmaceuticals as a special issue on Carbon-11 and fluorine-18 chemistry devoted to molecular probes for imaging the brain with PET, edited by Frédéric DOLLÉ, Service Hospitalier Frédéric Joliot, Institut d’Imagerie BioMédicale – CEA, 4 Place du Général Leclerc – F-91406 Orsay – France. {
Dedicated to Prof. Dr. Leonard I. Wiebe on the occasion of his 70th birthday.
Copyright © 2013 John Wiley & Sons, Ltd.
M. Wuest and F. Wuest Biography Melinda Wuest was born in 1972 in Dresden, Germany. She obtained her PhD in Inorganic Chemistry in 1999 from Dresden University of Technology, Germany. She spend one year as postdoc at the School of Medicine, University of St. Louis (U.S.A.) in the group of Dr. Michael J. Welch working on the characterization of novel bifunctional chelators for complexing Cu-64. After returning to Germany she started research activities in Pharmacology, first for one year at the Department of Pharmacy at University of Leipzig, Germany. In 2001 she started a similar position at the Department of Pharmacology (School of Medicine, Dresden University of Technology, Germany) focusing on research of pharmacology of the lower urinary tract. In 2009 she finished a PhD equivalent degree in Pharmacology from the German Society of Experimental and Clinical Pharmacology and Toxicology). In 2008 she started a new research position at the Department of Oncology at the University of Alberta and the Cross Cancer Institute, Canada. Her current research is focused on PET and the radiopharmacological evaluation of novel radiotracers in vivo as well as analysis of therapeutical drug effects in different preclinical in vivo models by utilizing PET.
Biography Frank Wuest was born in 1969 in Erfurt, Germany. He earned his PhD in chemistry in 1999 from the Technical University of Dresden, Germany. He spent one year as a postdoctoral fellow at the School of Medicine in St. Louis working on molecular probe development in the laboratory of Dr. Michael J. Welch. After his return to Germany he became head of the PET-tracer group of the Research Center DresdenRossendorf. He earned his habilitation thesis in biochemistry from the Technical University of Dresden in 2006. In 2008 he started a new position as the Dianne and Irving Kipnes Chair in Radiopharmaceutical Sciences at the University of Alberta in Edmonton (Canada) where he currently holds an appointment as Associate Professor at the Department of Oncology of the University of Alberta. His research activities are aimed at the evaluation and translation of the diagnostic and therapeutic potential of novel molecular targets and specific biochemical signatures associated with the development and progression of cancer. This especially involves the development of innovative PET radiopharmaceuticals for non-invasive assessment of cancer-related metabolic pathways and biochemical processes at the cellular and molecular level.
e N
N R NO2
e N R
N
NO2
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N R
no-carrier-added (n.c.a.) [18F]fluoride with 1-(20 -nitro-10 -imidazolyl)2-O-tetrahydropyranyl-3-O-toluenesulfonyl-propanediol as labeling precursor followed by acidic hydrolysis of the tetrahydropyranyl (THP) ether protecting group.17–20 Radiosynthesis of [18F]FMISO illustrating commonly employed nucleophilic aliphatic substitution reaction is depicted in Figure 3. Automation of the synthesis including either HPLC or purification by solid phase extraction provided [18F]FMISO in 40% radiochemical yield and 97% radiochemical purity at a specific activity of about 34 GBq/mmol. The total synthesis time was 50 min.21 More recent developments employing electrochemically concentrated anhydrous [18F]fluoride for microfluidic radiosynthesis afforded [18F]FMISO in higher radiochemical yields of 60–80% depending on selected reaction conditions and parameters.22 However, the latter development is not used for routine clinical production of [18F]FMISO. To date, [18F]FMISO represents the most widely used and extensively studied PET radiotracer for quantitative assessment of tumor hypoxia in lung, brain and head, and neck cancer patients as well as in hearts of patients with myocardial ischemia.12 On the basis of its relatively high lipophilicity, [18F]FMISO readily crosses cell membranes by passive diffusion. [18F]FMISO displays slow clearance from normal tissue, which results in longer time (≥3 h) to achieve acceptable tumor/ background ratios for optimal PET imaging.12 [18F]FMISO has found numerous applications in oncology, but its ability to freely diffuse across the blood–brain barrier (BBB) makes [18F]FMISO also an important radiotracer to assess cerebral hypoxia after an ischemic stroke.23 [18F]FMISO targets hypoxic tissue, which reflects the amount of brain area amenable for treatment, and therefore visualizes the target area for early interventions after a stroke.24,25 [18F]FMISO uptake is increased in surrounding tissue of the infarcted core during the acute phase of a stroke. [18F]FMISO is not suitable for hypoxia imaging in the sub-acute phase.26 A more hydrophilic 18F-labeled 2-nitroimidazole is [18F]fluoroazomycin-arabinofuranoside ([18F]FAZA). Synthesis of [18F]FAZA is carried out by the reaction of n.c.a. [18F]fluoride with 1-(2,3di-O-acetyl-5-O-tosyl-a-D-arabinofuranosyl)-2-nitroimidazole as labeling precursor employing standard nucleophilic substitution conditions followed by basic hydrolysis of the acetyl protection groups.27 The synthesis of FAZA was originally developed from its iodinated derivative iodoazomycinarabinofuranoside (IAZA).28,29 Optimal labeling conditions included reaction of n.c.a. [18F]fluoride with 5 mg of labeling precursor for 5 min at elevated temperature of 100 C. This procedure provided up to 60% radiochemical yield for the radiofluoride incorporation step prior to deprotection. Deprotection was performed using 1 mL of 0.1 N NaOH at 20 C for 2 min. Transfer of the radiosynthesis to an automated synthesis module resulted in 20% radiochemical yield. Typical clinical batch productions were reported to provide 9.87 2.3 GBq and 1.0 GBq of [18F]FAZA, respectively.27,30 The automated synthesis was further modified by utilizing a single column purification method as typical for the synthesis of 2-deoxy-2-(18F)fluoro-D-glucose ([18F]FDG). This procedure afforded [18F]FAZA in radiochemical yields of 20%.31 Radiosynthesis of [18F]FAZA in a microfluidic
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Figure 1. Multistep reduction pathway and intracellular trapping for 2-nitroimidazoles in cells.
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M. Wuest and F. Wuest OH N
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[18F]NEFA Figure 2. Structures of
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Figure 3. Radiosynthesis of 1-[18F]fluoro-3-(2-nitro-imidazol-1-yl)-propan-2-ol.
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system provided [18F]FAZA in 40% radiochemical yield within 60 min including HPLC purification.32 This procedure gave sufficient amounts of [18F]FAZA for a single patient dose and for preclinical studies. More recently, further optimization of reaction conditions allowed the synthesis of [18F]FAZA in good radiochemical yields of 50% at high specific activity of >300 GBq/mmol.33 [18F]FAZA is exclusively used for imaging tumor hypoxia in a variety of different human cancers. Interestingly, uptake of [18F]FAZA was also observed in a group of glioblastoma patients, which most likely can be attributed to a disruption of the BBB. [18F] FAZA is not able to cross the intact BBB.30 In comparison with [18F]FMISO and [18F]FAZA, a significantly more lipophilic 18F-labeled 2-nitroimidazole is 2-(2-nitro-1Himidazol-1-yl)-N-(2,2,3,3,3-[18F]pentafluoropropyl)-acetamide ([18F] EF5). Other examples of lipophilic fluoroalkyl acetamide derivatives are 2-(2-nitroimidazol-1-yl)-N-3-[18F]fluoropropyl acetamide [18F]EF1 and 2-(2-nitroimidazol-1-yl)-N-(3,3,3-[18F]trifluoro-propyl) acetamide [18F]EF3.12 The high lipophilicity of [18F]EF5 favors the compound for PET imaging of hypoxia because of its facile
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penetration through cell membranes, which results in easy access of the radiotracer to most tissues including the central nervous system.34 The synthesis of [18F]EF5 was first performed by electrophilic radiofluorination with [18F]F2 gas in trifluoroacetic acid (TFA) as the solvent and 2-(2-nitro-1H-imidazol-1-yl)-N(2,3,3-trifluoroallyl)-acetamide as the labeling precursor.35 However, the synthesis of [18F]EF5 is challenging. The use of [18F]F2 gas implies inherent problems such as limited amounts of starting radioactivity.34 A further challenge is the evaporation of TFA, which can contribute to significant product decomposition resulting in low overall radiochemical yields. [18F]EF5 was prepared in 3% radiochemical yield at moderately high specific activity of 7 GBq/mmol when 1 mg of labeling precursor was used.34 However, specific activity is not a major concern for hypoxia imaging radiotracers. Recently, a simplified synthesis procedure for [18F]EF5 was described involving a modified production of [18F]F2 gas and the implementation of solid phase extraction into the purification process. However, the required use of more labeling precursor (10 mg)
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Microfluidic technology was used for the synthesis of [18F]3NTR, a 3-nitro-1,2,4-triazole analog of [18F]FMISO. The synthesis was performed in a microreactor by nucleophilic radiofluorination of 3-(3-nitro-1,2,4-triazol-2-yl)-2-O-tetrahydropyran-1-yloxy) propyl tosylate. After HPLC purification, [18F]3-NTR was obtained in 36% 10% radiochemical yield at high radiochemical purity >97% and specific activity of 90–150 GBq/mmol at the end of synthesis. Under hypoxic conditions, [18F]3-NTR is reduced faster than [18F]FMISO, but final binding to intracellular marcomolecules is too slow for irreversible trapping. This finding makes [18F]3-NTR not suitable to identify and quantify hypoxic tumor areas.50 The authors of the study concluded that for the development of more optimal PET radiotracers for hypoxia imaging, binding kinetics of reduced intermediate to macromolecules is crucial for desired irreversibility of intracellular trapping.
Novel classes of 18F-labeled radiotracers for imaging hypoxia Recently, various sulfoxides were proposed as novel structural scaffold for the development of 18F-labeled radiotracers for hypoxia imaging (Figure 4). Compounds 1 and 2 were prepared through nucleophilic displacement of corresponding chloride precursors.51 The proposed mechanism of metabolic trapping involves bioreduction of the sulfoxide group followed by the formation of a highly reactive aziridinium ion, which readily alkylates DNA or other intracellular macromolecules.52 [18F]Sulfoxide 1 showed great potential for imaging hypoxia in a rat stroke model.51 However, a major concern with compounds 1 and 2 is their molecular structure being closely related to highly toxic nitrogen mustards.52 Recently, an alternative molecular structure consisting of a propargyl group instead of a chloroethyl group was proposed.52 This structural modification led to the preparation of N-(2-[18F] fluoroethyl)-4-(4-nitrophenylsulfinyl)-N-(prop-2-ynyl)aniline 3 and N-(2-chloroethyl)-N-((1-(2-[18F]fluoroethyl)-1H-1,2,3-triazol-4-yl) methyl)-4-(4-nitrophenylsulfinyl)aniline 4 through direct nucleophilic radiofluorination with n.c.a. [18F]fluoride or click chemistry with 18F]fluoroethyl azide, respectively.52 Initial radiopharmacological studies revealed compound 3 as a promising lead structure for the further development of sulfoxide-based radiotracers for hypoxia imaging.
Other positron emission tomography radiotracers for imaging of tissue hypoxia To the best of our knowledge, to date, no 11C-labeled radiotracer for hypoxia imaging has been described. The rather short halflife of carbon-11 (t1/2 = 20.4 min) seems to be not suitable for molecular imaging of tissue hypoxia, which usually requires longer imaging protocols to obtain a favorable target-to-nontarget contrast. However, besides the plethora of 18F-labeled radiotracers for imaging tissue hypoxia, there are several alternative PET radiotraces labeled with 124I, 64Cu, and 68Ga. Among those radiotracers, especially [64Cu]diacetyl-bis(N4-methylthiosemicarbazone) (ATSM) has become a frequently used PET radiotracer for hypoxia imaging. Prominent examples for 124I-labeled, 64Cu-labeled, and 68 Galabeled hypoxia imaging agents are depicted in Figure 5.
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resulted in the formation of [18F]EF5 at relatively low specific activity (330 MBq/(mmol).36 The high lipophilicity of [18F]EF5 allows for an even and rapid distribution of the radiotracer throughout soft tissues including crossing of the BBB. A clinical trial study with glioblastoma patients demonstrated that [18F]EF5 can be utilized for PET imaging of hypoxic malignant brain lesions compared with the uniform distribution of the radiotracer within the healthy brain.37 Discussions concerning the feasibility of various 18F-labeled 2-nitroimidazoles for imaging glioblastomas tend to favor the use of [18F]EF5 for its ability to cross the BBB, whereas [18F]FAZA is only suitable in cases where the integrity of the BBB is disrupted. Additional examples for 18F-labeled 2-nitroimidazoles are [18F] fluoroetanidazole ([18F]FETA) and [18F]fluoroerythronitroimidazole ([18F]FETNIM). Both compounds showed reduced metabolic rates in vivo and less retention in the liver compared with the first generation 2-nitroimidazole [18F]FMISO.9,12 [18F]FETA was prepared via an active ester coupling reaction between 2,3,5,6tetrafluorophenyl ester of 2-nitroimidazole acetic acid and [18F] fluoroethylamine. [18F]Fluoroethylamine was prepared starting from N-[2-(toluene-4-sulfonyloxy)-ethyl]-phthalimide and n.c.a. [18F]fluoride followed by purification by distillation.38 On the basis of its fairly high metabolic stability and the capability to image tumor hypoxia at earlier time points (~1 h post-injection (p.i.) compared with ~3 h p.i. with [18F]FMISO, [18F]FAZA, and [18F]EF5), [18F]FETA was also proposed for imaging hypoxia after ischemic stroke.39,40 However, to date, [18F]FETA has not been used in clinical trials yet. [18F]FETNIM was synthesized by nucleophilic radiofluorination of corresponding tosylate precursor with n.c.a. [18F]fluoride followed by acidic hydrolysis of the protecting group.41,42 Radiosynthesis was accomplished within 50 min, and [18F]FETNIM could be prepared in 13–20% radiochemical yield at high specific activity of 330 GBq/mmol.43 [18F]FETNIM was used in some early clinical trials demonstrating radiotracer uptake in head and neck tumors as well as in locally advanced non-small-cell lung carcinoma patients.44,45 [18F]HX4 represents a novel, click chemistry-based generation of hypoxia imaging agents. Structure–activity relationships, focused on the incorporation of a 1,2,3-triazole moiety, were used to design an imaging agent with a favorable pharmacokinetic and clearance profile.46 Incorporation of metabolically stable 1,2,3-triazole moiety was achieved through Cu-catalyzed Huisgen [3 + 2] cycloaddition. [18F]HX4 was synthesized via nucleophilic displacement of the nitrobenzenesulfonate group in 3-(formyloxy)-2-{4-[(2-nitro-1H-imidazol-1-yl)methyl]-1H-1,2,3triazol-1-yl}propyl 2-nitrobenzenesulfonate with n.c.a. [18F]fluoride. The purification, radiochemical yield, and the time required to synthesize radiotracer [18F]HX4 were not reported.46,47 Phase I clinical trial showed that [18F]HX4 is suitable for imaging tumor hypoxia. However, final evaluation of usefulness and optimal imaging parameters of [18F]HX4 still remain to be determined.48 More recent developments for the synthesis of 18F-labeled 2-nitroimidazoles involve compounds 2-[18F]fluoro-N-(2-(2-nitro1H-imidazol-1-yl)ethyl)acetamide ([18F]NEFA) and 2-(2-nitro-1Himidazol-1-yl)ethyl 2-[18F]fluoroacetate ([18F]NEFT). 2-Nitroimidazole derivatives [18F]NEFA and [18F]NEFT were prepared by nucleophilic radiofluorination with n.c.a. [18F]fluoride using 2-bromo-N-(2-(2nitro-1H-imidazol-1-yl)ethyl)acetamide and 2-(2-nitro-1H-imidazol-1-yl)ethyl 2-bromo-acetate as labeling precursors to afford radiotracers in radiochemical yields of 6–10%, respectively. Both compounds proved to be metabolically unstable in vivo, and [18F] NEFT showed substantial radiodefluorination in vivo.49
M. Wuest and F. Wuest Cl O S
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[18F]Sulfoxide 3 Figure 4. Structure of sulfoxide-based compounds as hypoxia imaging agents.
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Figure 5. Selection of alternative hypoxia imaging agents labeled with 124I,
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The special mechanism of accumulation of copper complexes such as [64Cu]ATSM has been extensively discussed in the literature.12
Hybrid compounds for positron emission tomography imaging of hypoxia
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All radiotracers discussed so far rely on passive diffusion to enter hypoxic cells. This usually leads to extended uptake and retention times, which results in late PET imaging times to achieve a favorable target-to-non-target signal. Especially 2-nitroimidazolecontaining compounds often require more than a 2 h p.i. waiting time in patients prior to the PET scan. Therefore, recent research activities were directed towards the development of hybrid compounds to combine a hypoxiasensitive part (e.g., 2-nitroimidazole) with active transport molecules such as glucose or tyrosine to facilitate and enhance cell uptake.56–58 Figure 6 illustrates examples of recently prepared hybrid compounds. The concept of hybrid compounds for targeting hypoxic cells dates back to the early 1990s when 2-nitroimidazole was conjugated to L-phenylalanine methyl ester to exploit amino acid transport to enhance drug transport into cells.59 Patt and co-workers have used [18F]FDG to prepare a hybrid compound ([18F]FDG-NIm) with 2-nitroimidazole.56 Cold FDG-NIm was able to block cellular uptake of [18F]FDG as indicative of an interaction with GLUT1, but the used concentration range had to be
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HO2 C
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fairly high to observe an effect. Similar results were found when 2-nitroimidazole was coupled via a linker to position 6 of the glucose molecule.57 18F was introduced via nucleophilic substitution using a tetra-acetylated nosylate precursor to afford N-(2-[18F]fluoro-3-(6-O-glucosyl)propyl-azomycin ([18F]F-GAZ). The synthesis proved to be challenging, and a racemate was formed. In vitro cellular uptake of [18F]F-GAZ in EMT6 cells suggested interaction with GLUT1. However, in vivo studies showed continuous washout of radioactivity from the EMT6 tumor. In contrast, [18F]FAZA showed uptake and retention in EMT6 tumors suggesting no sufficient trapping of hybrid compound [18F]F-GAZ in hypoxic tissue.57 More recently, the efficient radiosynthesis of the tyrosine-2-nitroimidazole hybrid ([18F]FNT]) was reported. [18F]FNT] was prepared via nucleophilic displacement of tosyl group in N-[(benzyloxy)carbonyl]-O-[2-{[(4-methylphenyl)sulfonyl] oxy}-3-(2-nitro-1H-imidazol-1-yl)propyl]-tyrosine methyl ester as precursor with n.c.a. [18F]fluoride.58 No data on radiopharmacological evaluation were reported. Another approach included conjugation of diacetyl-bis(N-4ethylthiosemicarbazone) (ATSE) and ATSM to either glucose or 2-nitro-imidazole.60,61 In the case of glucose conjugation, the copper complex was attached via a linker to the glycosidic bond of the glucose molecule. For complexation with 64Cu via transmetalation to yield [64Cu]ATSE-glucose, Zn-complex Zn-ATSE-glucose was synthesized.62 Compound [64Cu]ATSE-glucose retained a hypoxiatargeting profile in vitro, but in vivo studies showed no glucosespecific transport. [64Cu]ATSE-glucose seems to enter hypoxic tumor
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Figure 6. Hybrid compounds for facilitated and enhanced cell uptake.
cells via passive diffusion. Conjugation of [64Cu]ATSM to 2-nitroimidazoles derived from either the bifunctional bis(thiosemicarbazone) H2ATSM/A or diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3(4-N-ethylamino-3-thiosemi-carbazone) H2ATSM/en resulted in no higher retention of radioactivity in hypoxic cells and tissue.61
Conclusion
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Acknowledgement F. W. would like to thank the Dianne and Irving Kipnes Foundation for supporting the radiopharmaceutical chemistry program at the Cross Cancer Institute.
Conflict of Interest The authors have declared that there is no conflict of interest.
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Research on PET radiotracers for imaging hypoxia has witnessed a stormy development since the original introduction of [18F] FMISO in 1986. On the basis of the pioneering work involving radioiodine-labeled 2-nitroimidazoles, numerous 18F-labeled compounds have been used in preclinical and clinical studies, most notably [18F]FMISO, [18F]FAZA, and [18F]EF5. Fluorine-18 has served as the radionuclide of choice for the development of 2-nitroimidazole-based hypoxia imaging agents. This also includes recent developments with sulfoxides as alternative bioreductively active structural motifs. As a result of the favorable chemical and physical properties of short-lived positron emitter 18 F, the majority of hypoxia imaging agents is based on radiotracers labeled with 18F. A parallel major development on hypoxia imaging agents was directed to application of coordination chemistry with radiocopper isotopes. These research activities resulted in the introduction of [64Cu]ATSM for preclinical and clinical imaging of hypoxia. All approaches involving 2-nitroimidazoles and neutral Cu(II)complexes rely on passive diffusion of the respective radiotracers into hypoxic cells. This usually implies long waiting times prior to the PET scan to achieve a favorable clearance profile from normal tissue and sufficient trapping of the radiotracer in hypoxic tissue. In light of this intrinsic problem of classical hypoxia imaging agents, a new generation of hypoxia imaging agents has recently been proposed. Hybrid compounds combining intracellular trapping of 2-nitroimidazoles or neutral Cu(II)-complexes with active transport via hexose or amino acid transporters are aimed at a faster and enhanced cell uptake of the radiotracer.
This interesting and innovative approach has the potential to provide hypoxia imaging agents with significantly improved radiopharmacological profile for molecular imaging. This eventually can lead to the development of radiotracers needed for a broader and more efficient application of hypoxia imaging in preclinical and clinical research, and clinical monitoring of hypoxia upon therapeutic interventions.
M. Wuest and F. Wuest [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40]
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[41] D. J. Yang, S. Wallace, A. Cherif, L. Chun, M. B. Gretzer, E. E. Kim, D. A. Podoloff, Radiology 1995, 194, 795. [42] T. Gronross, L. Berentzen, P. Marjamaki, R. Murata, M. R. Horsman, S. Keiding, O. Eskola, M. Haaparanta, H. Minn, O. Solin, Eur. J. Nucl. Med. Mol. Imaging 2004, 31, 513. [43] T. Gronross, O. Eskola, K. Lehtio, H. Minn, P. Marjamaki, J. Bergman, M. Haaparanta, S. Forsback, O. Solin, J. Nucl. Med. 2001, 42, 1397. [44] K. Lehtio, V. Oikonen, S. Nyman, T. Gronroos, A. Roivainen, O. Eskola, H. Minn, Eur. J. Nucl. Med. Mol. 2003, 30, 101. [45] L. Li, M. Hu, H. Zhu, W. Zhao, G. Yang, J. Yu, Clin. Lung Cancer 2010, 11, 335. [46] L. J. Dubois, N. G. Lieuwes, M. H. Janssen, W. J. Peeters, A. D. Windhorst, J. C. Walsh, H. C. Kolb, M. C. Ollers, J. Bussink, G. A. van Dongen, A. van der Kogel, P. Lambin, Proc. Natl. Acad. Sci. U. S.A. 2011, 108, 14620. [47] M. Doss, J. J. Zhang, M. J. Belanger, J. B. Stubbs, E. D. Hostetler, K. Alpaugh, H. C. Kolb, J. Q. Yu, Nucl. Med. Commun. 2010, 12, 1016. [48] J. Van Loon, M. H. Janssen, M. C. Ollers, H. J. Aerts, L. J. Dubois, M. Hoechstenbag, A. M. Dingemans, R. Lalisang, B. Brans, B. Windhorst, G. A. van Dongen, H. Kolb, J. Ahng, D. De Ruyssher, P. Lambin, Eur. J. Nucl. Med. Mol. Imaging 2010, 37, 1663. [49] Z. Zha, L. Zhu, Y. Liu, F. Du, H. Gan, J. Quia, H. F. Kung, Nucl. Med. Biol. 2011, 38, 501. [50] R. Bejot, V. Kersemans, C. Kelly, L. Carroll, R. C. King, V. Gouverneur, Nucl. Med. Biol. 2010, 37, 565. [51] C. L. Falzon, U. Ackermann, N. Spratt, H. J. Tochon-Danguy, J. White, D. Howells, A. M. Scott, J. Label. Compd. Radiopharm. 2006, 49, 1089. [52] E. Laurens, S. D. Yeoh, A. Rigopoulus, D. Cao, G. A. Cartwright, G. J. O’Keefe, H. J. Tochon-Danguy, J. M. White, A. M. Scott, U. Ackermann, Nucl. Med. Biol. 2012, 39, 871–882. [53] G. Reischl, D. S. Dorow, C. Cullinane, A. Katsifis, P. Roselt, D. Binns, R. J. Hicks, J. Pharm. Pharm. Sci. 2007, 10, 203. [54] C. J. Anderson, R. Ferdani, Cancer Biother. Radiopharm. 2009, 24, 379. [55] L. Hoigebazar, J. M. Jeong, S. Y. Choi, J. Y. Choi, D. Shetty, Y.-S. Lee, D. S. Lee, J.-K. Chung, M. C. Lee, Y. K. Chung, J. Med. Chem. 2010, 53, 6378. [56] M. Patt, D. Sorger, M. Scheunemann, G. Stoecklin, Appl. Radiat. Isotopes. 2002, 57, 705. [57] M. Wuest, P. Kumar, H.-S. Jans, D. Murray, W. Roa, L. I. Wiebe, Radiother. Oncol. 2010, 96(S2), S61. [58] N. Malik, X. Lin, D. Loffler, B. Shen, C. Solbach, G. Reischl, W. Voelter, H.-J. Machulla, J. Radioanal. Nucl. Chem. 2012. DOI: 10.1007/s10967– 012–1683–4. [59] P. K. Garg, S. Garg, W. G. Degraff, M. R. Zalutsky, J. B. Mitchell, Int. J. Radiat. Oncol. Biol. Phys. 1992, 22, 593. [60] S. R. Bayly, R. C. King, D. J. Honess, P. J. Barnard, H. M. Betts, J. P. Holland, R. Hueting, P. D. Bonnitcha, J. R. Dilworth, F. I. Aigbirhio, M. Christlieb, J. Nucl. Med. 2008, 49, 1862. [61] P. D. Bonnitcha, S. R. Bayly, M. B. M. Theobald, H. M. Betts, J. S. Lewis, J. R. Dilworth, J. Inorg. Biochem. 2010, 104, 126. [62] J. P. Holland, F. I. Aigbirhio, H. M. Betts, P. D. Bonnitcha, P. Burke, M. Christlieb, G. C. Churchill, A. R. Cowley, J. R. Dilworth, P. S. Donnelly, J. C. Green, J. M. Peach, S. R. Vasudevan, J. E. Warren Inorg. Chem. 2007, 46, 465.
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 244–250
Revised 27 July 2012,
Accepted 6 November 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2997
Positron emission tomography radiotracers for imaging hypoxia† Melinda Wuest and Frank Wuest* Localized hypoxia, the physiological hallmark of many clinical pathologies, is the consequence of acute or chronic ischemia in the affected region or tissue. The versatility, sensitivity, quantitative nature, and increasing availability of positron emission tomography (PET) make it the preclinical and clinical method of choice for functional imaging of tissue hypoxia at the molecular level. The progress and current status of radiotracers for hypoxia-specific PET imaging are reviewed in this article including references mainly focused on radiochemistry and also relevant to molecular imaging of hypoxia in preclinical and clinical studies. Keywords: tumor hypoxia; radiotracers; fluorine-18; radiochemistry
Introduction Tissue hypoxia can develop in the microenvironment of solid tumors or after acute or chronic ischemic events in the brain or the heart. Ischemia is induced by a number of vascular, lymphatic, and membrane changes that prevent delivery of sufficient amounts of oxygen to the hypoxic area.1–3 Tissue hypoxia can be detected with various molecular probes, especially with radiotracers.4,5 Hypoxic cells undergo complex transcriptional and posttranslational cellular adaptations including upregulation of drug resistance transporters and energy metabolism, modulation of apoptotic processes, and induction of gene expression encoding growth factors that promote metastases and compromise chemotherapy outcomes.6–8 In oncology, tumor hypoxia is associated with poor response to treatment protocols and poor clinical outcome. This strongly supports the need to identify tumors with high hypoxic fractions to implement hypoxia-directed treatments and to optimize tumor treatments that are oxygen sensitive.9–11 Molecular probes designed to measure tissue hypoxia have to compete directly with intracellular O2. Hypoxia-sensitive molecular probes should not undergo metabolic trapping when O2 supply within the cells is sufficient but should retain within the cells when oxygen supply is decreased.12 This review summarizes synthesis and application of various positron emission tomography (PET) radiotracers designed and used for hypoxia imaging. On the basis of the topic of this special theme issue dealing with 11C and 18F radiochemistry, special focus will be on 18F-labeled radiotraces for imaging tissue hypoxia, whereas alternative PET radiotracers labeled with 68Ga, 64Cu, and 124 I will only be discussed briefly. 18
F-labeled 2-nitroimidazoles as PET radiotracers for hypoxia imaging
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2-Nitroimidazole-containing compounds were among the first molecular probes for PET imaging of hypoxia. The mechanism of reduction and intracellular trapping of 2-nitroimidazoles
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involves formation of a radical anion, which reacts preferentially with O2 if it is present to re-oxidize the radiotracer into its original form. Under hypoxic conditions when O2 supply is reduced, the radical anion accepts another electron yielding a nitroso intermediate that is further reduced to a primary amine (R0 –NH2) as highly efficient alkylating agent. Covalent bonding of alkylating agent R0 –NH2 results in intracellular retention of the radiolabel (Figure 1).12 Early analysis of tumor hypoxia with 14C-labeled misonidazole revealed selective binding of the radiolabel to hypoxic cells within a tumor.13 On the basis of their lipophilicity, 2-nitroimidazoles enter the cell via passive diffusion.14 This early work stimulated the development of various 2nitroimidazole-containing compounds labeled with 18F for PET imaging of hypoxia. A summary of 18F-labeled 2-nitroimidazoles (except for [18F]3-NTR, which is a 3-nitro-1,2,4-triazole analog of 1-[18F]fluoro-3-(2-nitro-imidazol-1-yl)-propan-2-ol ([18F]FMISO)) is given in Figure 2. One of the most frequently applied 18F-labeled 2-nitroimidazoles is [18F]FMISO. Several different synthesis routes have been described. A two-step synthesis was developed involving [18F] epifluorohydrin as intermediate, which was further reacted with 2-nitroimidazole to provide [18F]FMISO.15,16 HPLC purification was necessary to obtain high radiochemical purity. A more prominent approach relies on nucleophilic substitution reaction of
Department of Oncology, University of Alberta, Edmonton, AB, T6G 1Z2, Canada *Correspondence to: Frank Wuest, PhD, Department of Oncology, University of Alberta, 11560 University Ave, Edmonton, Alberta, T6G 1Z2, Canada. E-mail: [email protected] †
This article is published in Journal of Labelled Compounds and Radiopharmaceuticals as a special issue on Carbon-11 and fluorine-18 chemistry devoted to molecular probes for imaging the brain with PET, edited by Frédéric DOLLÉ, Service Hospitalier Frédéric Joliot, Institut d’Imagerie BioMédicale – CEA, 4 Place du Général Leclerc – F-91406 Orsay – France. {
Dedicated to Prof. Dr. Leonard I. Wiebe on the occasion of his 70th birthday.
Copyright © 2013 John Wiley & Sons, Ltd.
M. Wuest and F. Wuest Biography Melinda Wuest was born in 1972 in Dresden, Germany. She obtained her PhD in Inorganic Chemistry in 1999 from Dresden University of Technology, Germany. She spend one year as postdoc at the School of Medicine, University of St. Louis (U.S.A.) in the group of Dr. Michael J. Welch working on the characterization of novel bifunctional chelators for complexing Cu-64. After returning to Germany she started research activities in Pharmacology, first for one year at the Department of Pharmacy at University of Leipzig, Germany. In 2001 she started a similar position at the Department of Pharmacology (School of Medicine, Dresden University of Technology, Germany) focusing on research of pharmacology of the lower urinary tract. In 2009 she finished a PhD equivalent degree in Pharmacology from the German Society of Experimental and Clinical Pharmacology and Toxicology). In 2008 she started a new research position at the Department of Oncology at the University of Alberta and the Cross Cancer Institute, Canada. Her current research is focused on PET and the radiopharmacological evaluation of novel radiotracers in vivo as well as analysis of therapeutical drug effects in different preclinical in vivo models by utilizing PET.
Biography Frank Wuest was born in 1969 in Erfurt, Germany. He earned his PhD in chemistry in 1999 from the Technical University of Dresden, Germany. He spent one year as a postdoctoral fellow at the School of Medicine in St. Louis working on molecular probe development in the laboratory of Dr. Michael J. Welch. After his return to Germany he became head of the PET-tracer group of the Research Center DresdenRossendorf. He earned his habilitation thesis in biochemistry from the Technical University of Dresden in 2006. In 2008 he started a new position as the Dianne and Irving Kipnes Chair in Radiopharmaceutical Sciences at the University of Alberta in Edmonton (Canada) where he currently holds an appointment as Associate Professor at the Department of Oncology of the University of Alberta. His research activities are aimed at the evaluation and translation of the diagnostic and therapeutic potential of novel molecular targets and specific biochemical signatures associated with the development and progression of cancer. This especially involves the development of innovative PET radiopharmaceuticals for non-invasive assessment of cancer-related metabolic pathways and biochemical processes at the cellular and molecular level.
e N
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e N R
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no-carrier-added (n.c.a.) [18F]fluoride with 1-(20 -nitro-10 -imidazolyl)2-O-tetrahydropyranyl-3-O-toluenesulfonyl-propanediol as labeling precursor followed by acidic hydrolysis of the tetrahydropyranyl (THP) ether protecting group.17–20 Radiosynthesis of [18F]FMISO illustrating commonly employed nucleophilic aliphatic substitution reaction is depicted in Figure 3. Automation of the synthesis including either HPLC or purification by solid phase extraction provided [18F]FMISO in 40% radiochemical yield and 97% radiochemical purity at a specific activity of about 34 GBq/mmol. The total synthesis time was 50 min.21 More recent developments employing electrochemically concentrated anhydrous [18F]fluoride for microfluidic radiosynthesis afforded [18F]FMISO in higher radiochemical yields of 60–80% depending on selected reaction conditions and parameters.22 However, the latter development is not used for routine clinical production of [18F]FMISO. To date, [18F]FMISO represents the most widely used and extensively studied PET radiotracer for quantitative assessment of tumor hypoxia in lung, brain and head, and neck cancer patients as well as in hearts of patients with myocardial ischemia.12 On the basis of its relatively high lipophilicity, [18F]FMISO readily crosses cell membranes by passive diffusion. [18F]FMISO displays slow clearance from normal tissue, which results in longer time (≥3 h) to achieve acceptable tumor/ background ratios for optimal PET imaging.12 [18F]FMISO has found numerous applications in oncology, but its ability to freely diffuse across the blood–brain barrier (BBB) makes [18F]FMISO also an important radiotracer to assess cerebral hypoxia after an ischemic stroke.23 [18F]FMISO targets hypoxic tissue, which reflects the amount of brain area amenable for treatment, and therefore visualizes the target area for early interventions after a stroke.24,25 [18F]FMISO uptake is increased in surrounding tissue of the infarcted core during the acute phase of a stroke. [18F]FMISO is not suitable for hypoxia imaging in the sub-acute phase.26 A more hydrophilic 18F-labeled 2-nitroimidazole is [18F]fluoroazomycin-arabinofuranoside ([18F]FAZA). Synthesis of [18F]FAZA is carried out by the reaction of n.c.a. [18F]fluoride with 1-(2,3di-O-acetyl-5-O-tosyl-a-D-arabinofuranosyl)-2-nitroimidazole as labeling precursor employing standard nucleophilic substitution conditions followed by basic hydrolysis of the acetyl protection groups.27 The synthesis of FAZA was originally developed from its iodinated derivative iodoazomycinarabinofuranoside (IAZA).28,29 Optimal labeling conditions included reaction of n.c.a. [18F]fluoride with 5 mg of labeling precursor for 5 min at elevated temperature of 100 C. This procedure provided up to 60% radiochemical yield for the radiofluoride incorporation step prior to deprotection. Deprotection was performed using 1 mL of 0.1 N NaOH at 20 C for 2 min. Transfer of the radiosynthesis to an automated synthesis module resulted in 20% radiochemical yield. Typical clinical batch productions were reported to provide 9.87 2.3 GBq and 1.0 GBq of [18F]FAZA, respectively.27,30 The automated synthesis was further modified by utilizing a single column purification method as typical for the synthesis of 2-deoxy-2-(18F)fluoro-D-glucose ([18F]FDG). This procedure afforded [18F]FAZA in radiochemical yields of 20%.31 Radiosynthesis of [18F]FAZA in a microfluidic
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Figure 1. Multistep reduction pathway and intracellular trapping for 2-nitroimidazoles in cells.
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M. Wuest and F. Wuest OH N
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[18F]NEFA Figure 2. Structures of
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F-labeled 2-nitroimidazoles.
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Figure 3. Radiosynthesis of 1-[18F]fluoro-3-(2-nitro-imidazol-1-yl)-propan-2-ol.
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system provided [18F]FAZA in 40% radiochemical yield within 60 min including HPLC purification.32 This procedure gave sufficient amounts of [18F]FAZA for a single patient dose and for preclinical studies. More recently, further optimization of reaction conditions allowed the synthesis of [18F]FAZA in good radiochemical yields of 50% at high specific activity of >300 GBq/mmol.33 [18F]FAZA is exclusively used for imaging tumor hypoxia in a variety of different human cancers. Interestingly, uptake of [18F]FAZA was also observed in a group of glioblastoma patients, which most likely can be attributed to a disruption of the BBB. [18F] FAZA is not able to cross the intact BBB.30 In comparison with [18F]FMISO and [18F]FAZA, a significantly more lipophilic 18F-labeled 2-nitroimidazole is 2-(2-nitro-1Himidazol-1-yl)-N-(2,2,3,3,3-[18F]pentafluoropropyl)-acetamide ([18F] EF5). Other examples of lipophilic fluoroalkyl acetamide derivatives are 2-(2-nitroimidazol-1-yl)-N-3-[18F]fluoropropyl acetamide [18F]EF1 and 2-(2-nitroimidazol-1-yl)-N-(3,3,3-[18F]trifluoro-propyl) acetamide [18F]EF3.12 The high lipophilicity of [18F]EF5 favors the compound for PET imaging of hypoxia because of its facile
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penetration through cell membranes, which results in easy access of the radiotracer to most tissues including the central nervous system.34 The synthesis of [18F]EF5 was first performed by electrophilic radiofluorination with [18F]F2 gas in trifluoroacetic acid (TFA) as the solvent and 2-(2-nitro-1H-imidazol-1-yl)-N(2,3,3-trifluoroallyl)-acetamide as the labeling precursor.35 However, the synthesis of [18F]EF5 is challenging. The use of [18F]F2 gas implies inherent problems such as limited amounts of starting radioactivity.34 A further challenge is the evaporation of TFA, which can contribute to significant product decomposition resulting in low overall radiochemical yields. [18F]EF5 was prepared in 3% radiochemical yield at moderately high specific activity of 7 GBq/mmol when 1 mg of labeling precursor was used.34 However, specific activity is not a major concern for hypoxia imaging radiotracers. Recently, a simplified synthesis procedure for [18F]EF5 was described involving a modified production of [18F]F2 gas and the implementation of solid phase extraction into the purification process. However, the required use of more labeling precursor (10 mg)
Copyright © 2013 John Wiley & Sons, Ltd.
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M. Wuest and F. Wuest
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Microfluidic technology was used for the synthesis of [18F]3NTR, a 3-nitro-1,2,4-triazole analog of [18F]FMISO. The synthesis was performed in a microreactor by nucleophilic radiofluorination of 3-(3-nitro-1,2,4-triazol-2-yl)-2-O-tetrahydropyran-1-yloxy) propyl tosylate. After HPLC purification, [18F]3-NTR was obtained in 36% 10% radiochemical yield at high radiochemical purity >97% and specific activity of 90–150 GBq/mmol at the end of synthesis. Under hypoxic conditions, [18F]3-NTR is reduced faster than [18F]FMISO, but final binding to intracellular marcomolecules is too slow for irreversible trapping. This finding makes [18F]3-NTR not suitable to identify and quantify hypoxic tumor areas.50 The authors of the study concluded that for the development of more optimal PET radiotracers for hypoxia imaging, binding kinetics of reduced intermediate to macromolecules is crucial for desired irreversibility of intracellular trapping.
Novel classes of 18F-labeled radiotracers for imaging hypoxia Recently, various sulfoxides were proposed as novel structural scaffold for the development of 18F-labeled radiotracers for hypoxia imaging (Figure 4). Compounds 1 and 2 were prepared through nucleophilic displacement of corresponding chloride precursors.51 The proposed mechanism of metabolic trapping involves bioreduction of the sulfoxide group followed by the formation of a highly reactive aziridinium ion, which readily alkylates DNA or other intracellular macromolecules.52 [18F]Sulfoxide 1 showed great potential for imaging hypoxia in a rat stroke model.51 However, a major concern with compounds 1 and 2 is their molecular structure being closely related to highly toxic nitrogen mustards.52 Recently, an alternative molecular structure consisting of a propargyl group instead of a chloroethyl group was proposed.52 This structural modification led to the preparation of N-(2-[18F] fluoroethyl)-4-(4-nitrophenylsulfinyl)-N-(prop-2-ynyl)aniline 3 and N-(2-chloroethyl)-N-((1-(2-[18F]fluoroethyl)-1H-1,2,3-triazol-4-yl) methyl)-4-(4-nitrophenylsulfinyl)aniline 4 through direct nucleophilic radiofluorination with n.c.a. [18F]fluoride or click chemistry with 18F]fluoroethyl azide, respectively.52 Initial radiopharmacological studies revealed compound 3 as a promising lead structure for the further development of sulfoxide-based radiotracers for hypoxia imaging.
Other positron emission tomography radiotracers for imaging of tissue hypoxia To the best of our knowledge, to date, no 11C-labeled radiotracer for hypoxia imaging has been described. The rather short halflife of carbon-11 (t1/2 = 20.4 min) seems to be not suitable for molecular imaging of tissue hypoxia, which usually requires longer imaging protocols to obtain a favorable target-to-nontarget contrast. However, besides the plethora of 18F-labeled radiotracers for imaging tissue hypoxia, there are several alternative PET radiotraces labeled with 124I, 64Cu, and 68Ga. Among those radiotracers, especially [64Cu]diacetyl-bis(N4-methylthiosemicarbazone) (ATSM) has become a frequently used PET radiotracer for hypoxia imaging. Prominent examples for 124I-labeled, 64Cu-labeled, and 68 Galabeled hypoxia imaging agents are depicted in Figure 5.
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resulted in the formation of [18F]EF5 at relatively low specific activity (330 MBq/(mmol).36 The high lipophilicity of [18F]EF5 allows for an even and rapid distribution of the radiotracer throughout soft tissues including crossing of the BBB. A clinical trial study with glioblastoma patients demonstrated that [18F]EF5 can be utilized for PET imaging of hypoxic malignant brain lesions compared with the uniform distribution of the radiotracer within the healthy brain.37 Discussions concerning the feasibility of various 18F-labeled 2-nitroimidazoles for imaging glioblastomas tend to favor the use of [18F]EF5 for its ability to cross the BBB, whereas [18F]FAZA is only suitable in cases where the integrity of the BBB is disrupted. Additional examples for 18F-labeled 2-nitroimidazoles are [18F] fluoroetanidazole ([18F]FETA) and [18F]fluoroerythronitroimidazole ([18F]FETNIM). Both compounds showed reduced metabolic rates in vivo and less retention in the liver compared with the first generation 2-nitroimidazole [18F]FMISO.9,12 [18F]FETA was prepared via an active ester coupling reaction between 2,3,5,6tetrafluorophenyl ester of 2-nitroimidazole acetic acid and [18F] fluoroethylamine. [18F]Fluoroethylamine was prepared starting from N-[2-(toluene-4-sulfonyloxy)-ethyl]-phthalimide and n.c.a. [18F]fluoride followed by purification by distillation.38 On the basis of its fairly high metabolic stability and the capability to image tumor hypoxia at earlier time points (~1 h post-injection (p.i.) compared with ~3 h p.i. with [18F]FMISO, [18F]FAZA, and [18F]EF5), [18F]FETA was also proposed for imaging hypoxia after ischemic stroke.39,40 However, to date, [18F]FETA has not been used in clinical trials yet. [18F]FETNIM was synthesized by nucleophilic radiofluorination of corresponding tosylate precursor with n.c.a. [18F]fluoride followed by acidic hydrolysis of the protecting group.41,42 Radiosynthesis was accomplished within 50 min, and [18F]FETNIM could be prepared in 13–20% radiochemical yield at high specific activity of 330 GBq/mmol.43 [18F]FETNIM was used in some early clinical trials demonstrating radiotracer uptake in head and neck tumors as well as in locally advanced non-small-cell lung carcinoma patients.44,45 [18F]HX4 represents a novel, click chemistry-based generation of hypoxia imaging agents. Structure–activity relationships, focused on the incorporation of a 1,2,3-triazole moiety, were used to design an imaging agent with a favorable pharmacokinetic and clearance profile.46 Incorporation of metabolically stable 1,2,3-triazole moiety was achieved through Cu-catalyzed Huisgen [3 + 2] cycloaddition. [18F]HX4 was synthesized via nucleophilic displacement of the nitrobenzenesulfonate group in 3-(formyloxy)-2-{4-[(2-nitro-1H-imidazol-1-yl)methyl]-1H-1,2,3triazol-1-yl}propyl 2-nitrobenzenesulfonate with n.c.a. [18F]fluoride. The purification, radiochemical yield, and the time required to synthesize radiotracer [18F]HX4 were not reported.46,47 Phase I clinical trial showed that [18F]HX4 is suitable for imaging tumor hypoxia. However, final evaluation of usefulness and optimal imaging parameters of [18F]HX4 still remain to be determined.48 More recent developments for the synthesis of 18F-labeled 2-nitroimidazoles involve compounds 2-[18F]fluoro-N-(2-(2-nitro1H-imidazol-1-yl)ethyl)acetamide ([18F]NEFA) and 2-(2-nitro-1Himidazol-1-yl)ethyl 2-[18F]fluoroacetate ([18F]NEFT). 2-Nitroimidazole derivatives [18F]NEFA and [18F]NEFT were prepared by nucleophilic radiofluorination with n.c.a. [18F]fluoride using 2-bromo-N-(2-(2nitro-1H-imidazol-1-yl)ethyl)acetamide and 2-(2-nitro-1H-imidazol-1-yl)ethyl 2-bromo-acetate as labeling precursors to afford radiotracers in radiochemical yields of 6–10%, respectively. Both compounds proved to be metabolically unstable in vivo, and [18F] NEFT showed substantial radiodefluorination in vivo.49
M. Wuest and F. Wuest Cl O S
Cl O S Me
N 18
N
F
18
F
O2 N [18F]Sulfoxide 1
O S
[18F]Sulfoxide 2
O S
N 18
N N N
N
Cl
F
18
F
O2 N
O2 N
[18F]Sulfoxide 4
[18F]Sulfoxide 3 Figure 4. Structure of sulfoxide-based compounds as hypoxia imaging agents.
Me
Me
HO2 C
124
I
O HO HO
NO2
N N
N N
S
HN
N
N
N
64Cu
Ga
S
N
N
N N H
N
Me 68Ga-labeled
NOTA complex containing a 2-nitroimidazole moiety [55]
[ 64 Cu]ATSM [54]
Figure 5. Selection of alternative hypoxia imaging agents labeled with 124I,
64
Cu, and
The special mechanism of accumulation of copper complexes such as [64Cu]ATSM has been extensively discussed in the literature.12
Hybrid compounds for positron emission tomography imaging of hypoxia
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All radiotracers discussed so far rely on passive diffusion to enter hypoxic cells. This usually leads to extended uptake and retention times, which results in late PET imaging times to achieve a favorable target-to-non-target signal. Especially 2-nitroimidazolecontaining compounds often require more than a 2 h p.i. waiting time in patients prior to the PET scan. Therefore, recent research activities were directed towards the development of hybrid compounds to combine a hypoxiasensitive part (e.g., 2-nitroimidazole) with active transport molecules such as glucose or tyrosine to facilitate and enhance cell uptake.56–58 Figure 6 illustrates examples of recently prepared hybrid compounds. The concept of hybrid compounds for targeting hypoxic cells dates back to the early 1990s when 2-nitroimidazole was conjugated to L-phenylalanine methyl ester to exploit amino acid transport to enhance drug transport into cells.59 Patt and co-workers have used [18F]FDG to prepare a hybrid compound ([18F]FDG-NIm) with 2-nitroimidazole.56 Cold FDG-NIm was able to block cellular uptake of [18F]FDG as indicative of an interaction with GLUT1, but the used concentration range had to be
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HO2 C
NH
Me [124 I]IAZA[53]
O2 N O
68
68
Ga.
fairly high to observe an effect. Similar results were found when 2-nitroimidazole was coupled via a linker to position 6 of the glucose molecule.57 18F was introduced via nucleophilic substitution using a tetra-acetylated nosylate precursor to afford N-(2-[18F]fluoro-3-(6-O-glucosyl)propyl-azomycin ([18F]F-GAZ). The synthesis proved to be challenging, and a racemate was formed. In vitro cellular uptake of [18F]F-GAZ in EMT6 cells suggested interaction with GLUT1. However, in vivo studies showed continuous washout of radioactivity from the EMT6 tumor. In contrast, [18F]FAZA showed uptake and retention in EMT6 tumors suggesting no sufficient trapping of hybrid compound [18F]F-GAZ in hypoxic tissue.57 More recently, the efficient radiosynthesis of the tyrosine-2-nitroimidazole hybrid ([18F]FNT]) was reported. [18F]FNT] was prepared via nucleophilic displacement of tosyl group in N-[(benzyloxy)carbonyl]-O-[2-{[(4-methylphenyl)sulfonyl] oxy}-3-(2-nitro-1H-imidazol-1-yl)propyl]-tyrosine methyl ester as precursor with n.c.a. [18F]fluoride.58 No data on radiopharmacological evaluation were reported. Another approach included conjugation of diacetyl-bis(N-4ethylthiosemicarbazone) (ATSE) and ATSM to either glucose or 2-nitro-imidazole.60,61 In the case of glucose conjugation, the copper complex was attached via a linker to the glycosidic bond of the glucose molecule. For complexation with 64Cu via transmetalation to yield [64Cu]ATSE-glucose, Zn-complex Zn-ATSE-glucose was synthesized.62 Compound [64Cu]ATSE-glucose retained a hypoxiatargeting profile in vitro, but in vivo studies showed no glucosespecific transport. [64Cu]ATSE-glucose seems to enter hypoxic tumor
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 244–250
M. Wuest and F. Wuest N N NO2 18 F
OH
O O
HO HO
N
N 18
F
N
OH
F
H N
N H
[18F]FNT
Me
Me
N
OH O
18
[18F]F-GAZ
Me
N Cu
S
S
Me
N N
N
HN Et
NH2
O
N
N
OH
[18 F]FDG-NIm
HO HO
OH
NO 2
CO2 H
O2 N
O
HO HO
HN
N Cu
S
S
Me
[64 Cu]ATSE-glucose
N N H
H N
N O
N NO 2
[64Cu]ATSM-NIm
Figure 6. Hybrid compounds for facilitated and enhanced cell uptake.
cells via passive diffusion. Conjugation of [64Cu]ATSM to 2-nitroimidazoles derived from either the bifunctional bis(thiosemicarbazone) H2ATSM/A or diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3(4-N-ethylamino-3-thiosemi-carbazone) H2ATSM/en resulted in no higher retention of radioactivity in hypoxic cells and tissue.61
Conclusion
J. Label Compd. Radiopharm 2013, 56 244–250
Acknowledgement F. W. would like to thank the Dianne and Irving Kipnes Foundation for supporting the radiopharmaceutical chemistry program at the Cross Cancer Institute.
Conflict of Interest The authors have declared that there is no conflict of interest.
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Research on PET radiotracers for imaging hypoxia has witnessed a stormy development since the original introduction of [18F] FMISO in 1986. On the basis of the pioneering work involving radioiodine-labeled 2-nitroimidazoles, numerous 18F-labeled compounds have been used in preclinical and clinical studies, most notably [18F]FMISO, [18F]FAZA, and [18F]EF5. Fluorine-18 has served as the radionuclide of choice for the development of 2-nitroimidazole-based hypoxia imaging agents. This also includes recent developments with sulfoxides as alternative bioreductively active structural motifs. As a result of the favorable chemical and physical properties of short-lived positron emitter 18 F, the majority of hypoxia imaging agents is based on radiotracers labeled with 18F. A parallel major development on hypoxia imaging agents was directed to application of coordination chemistry with radiocopper isotopes. These research activities resulted in the introduction of [64Cu]ATSM for preclinical and clinical imaging of hypoxia. All approaches involving 2-nitroimidazoles and neutral Cu(II)complexes rely on passive diffusion of the respective radiotracers into hypoxic cells. This usually implies long waiting times prior to the PET scan to achieve a favorable clearance profile from normal tissue and sufficient trapping of the radiotracer in hypoxic tissue. In light of this intrinsic problem of classical hypoxia imaging agents, a new generation of hypoxia imaging agents has recently been proposed. Hybrid compounds combining intracellular trapping of 2-nitroimidazoles or neutral Cu(II)-complexes with active transport via hexose or amino acid transporters are aimed at a faster and enhanced cell uptake of the radiotracer.
This interesting and innovative approach has the potential to provide hypoxia imaging agents with significantly improved radiopharmacological profile for molecular imaging. This eventually can lead to the development of radiotracers needed for a broader and more efficient application of hypoxia imaging in preclinical and clinical research, and clinical monitoring of hypoxia upon therapeutic interventions.
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