Dalton
`Transactions
`Cite this: DOI: 10.1039/c0dt01595d
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`PERSPECTIVE
`A practical guide to the construction of radiometallated bioconjugates for
`positron emission tomography
`
`Brian M. Zeglis and Jason S. Lewis*
`
`Received 16th November 2010, Accepted 7th February 2011
`DOI: 10.1039/c0dt01595d
`
`Positron emission tomography (PET) has become a vital imaging modality in the diagnosis and
`treatment of disease, most notably cancer. A wide array of small molecule PET radiotracers have been
`developed that employ the short half-life radionuclides 11C, 13N, 15O, and 18F. However, PET
`radiopharmaceuticals based on biomolecular targeting vectors have been the subject of dramatically
`increased research in both the laboratory and the clinic. Typically based on antibodies, oligopeptides, or
`oligonucleotides, these tracers have longer biological half-lives than their small molecule counterparts
`and thus require labeling with radionuclides with longer, complementary radioactive half-lives, such as
`the metallic isotopes 64Cu, 68Ga, 86Y, and 89Zr. Each bioconjugate radiopharmaceutical has four
`component parts: biomolecular vector, radiometal, chelator, and covalent link between chelator and
`biomolecule. With the exception of the radiometal, a tremendous variety of choices exists for each of
`these pieces, and a plethora of different chelation, conjugation, and radiometallation strategies have
`been utilized to create agents ranging from 68Ga-labeled pentapeptides to 89Zr-labeled monoclonal
`antibodies. Herein, the authors present a practical guide to the construction of radiometal-based PET
`bioconjugates, in which the design choices and synthetic details of a wide range of biomolecular tracers
`from the literature are collected in a single reference. In assembling this information, the authors hope
`both to illuminate the diverse methods employed in the synthesis of these agents and also to create a
`useful reference for molecular imaging researchers both experienced and new to the field.
`Introduction
`
`Department of Radiology and Program in Molecular Pharmacology and
`Chemistry Memorial Sloan-Kettering Cancer Center, New York, NY 10021,
`USA. E-mail: lewisj2@mskcc.org; Fax: (646)-888-3039; Tel: (646)-888-
`3038
`
`Over the course of the past fifty years, advances in medical imaging
`have revolutionized clinical practice, with a wide variety of imaging
`modalities playing critical roles in the diagnosis and treatment
`
`Dr. Brian Zeglis received his B.S.
`in chemistry summa cum laude
`from Yale University (2004),
`where he worked under
`the
`guidance of Professor Robert
`H. Crabtree. For his graduate
`studies, he attended the Cali-
`fornia Institute of Technology
`as an NSF pre-doctoral fellow.
`At Caltech, Brian worked un-
`der the mentorship of Professor
`Jacqueline K. Barton, studying
`the synthesis and development of
`Brian M. Zeglis, Ph.D.
`DNA-binding octahedral metal
`complexes. After receiving his Ph.D. (2009), Brian moved to
`Memorial Sloan-Kettering Cancer Center, where he works as an
`NIH post-doctoral fellow in the laboratory of Professor Jason
`S. Lewis. Currently, his research is focused on the design, synthesis,
`and evaluation of 64Cu- and 89Zr-based PET radiopharmaceuticals.
`
`Professor Lewis earned a B.Sc.
`Hons (1992) and an M.Sc.
`(1993) in Chemistry from the
`University of Essex. He received
`a Ph.D. in Biochemistry (1996)
`from the University of Kent at
`Canterbury. Following his post-
`doctoral study at the Washington
`University School of Medicine,
`he joined the Radiology faculty
`(2003). In 2008, he moved to
`Memorial Sloan-Kettering Can-
`cer Center (New York) where
`Jason S. Lewis, Ph.D.
`he is currently a Member (with
`tenure) and Vice Chairman for Basic Research, Chief Attending
`Radiochemist, and Director of the Cyclotron Core. He holds
`joint appointments at the Sloan-Kettering Institute, Weill Cornell
`Medical College and Gerstner Sloan-Kettering Graduate School.
`Professor Lewis has co-authored over 110 peer-reviewed journal
`articles, reviews and book chapters.
`
`This journal is © The Royal Society of Chemistry 2011
`
`Dalton Trans.
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`Downloaded by Columbia University on 28 March 2011
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`

`of disease. Today, clinicians have at their disposal a remarkable
`range of medical imaging techniques, from more conventional
`modalities like ultrasound, conventional radiography (X-rays),
`X-ray computed tomography (CT scans), and magnetic resonance
`imaging (MRI) to more specialized methodologies such as single-
`photon emission computed tomography (SPECT) and positron
`emission tomography (PET).
`imaging research has experienced
`In recent years, medical
`a paradigm shift from its foundations in anatomical imaging
`towards techniques aimed at probing tissue phenotype and
`function.1 Indeed, both the cellular expression of disease biomark-
`ers and fluctuations in tissue metabolism and microenvironment
`have emerged as extremely promising targets for imaging.2 With-
`out question, the unique properties of radiopharmaceuticals have
`given nuclear imaging a leading role in this movement. The
`remarkable sensitivity of PET and SPECT combines with their
`ability to provide information complementary to the anatomical
`images produced by other modalities to make these techniques
`ideal for imaging biomarker- and microenvironment-targeted
`tracers.3,4 Both relatively young modalities, SPECT and PET have
`had an impact on medicine (and oncology in particular), which
`belies their novelty, and both have been the topic of numerous
`thorough and well-reasoned reviews.5–9 Both modalities have be-
`come extremely important in the clinic, and while PET is generally
`more expensive on both the clinical and pre-clinical levels, it also
`undoubtedly possesses a number of significant advantages over its
`single-photon cousin, most notably the ability to quantify images,
`higher sensitivity (PET requires tracer concentrations of ~10-8 to
`10-10 M, while SPECT requires concentrations approaching 10-6
`M), and higher resolution (typically 6–8 mm for SPECT, compared
`to 2–3 mm or lower for PET). Therefore, in the interest of scope, the
`article at hand will limit itself to the younger and higher resolution
`of the techniques: positron emission tomography.
`Regardless of the broader perspective, any discussion of PET
`benefits from a brief description of the underlying physical
`phenomena. Starting from the beginning, a positron released by a
`decaying radionuclide will travel in a tissue until it has exhausted
`its kinetic energy. At this point, it will encounter its antiparticle,
`an electron, and the two will mutually annihilate, completely
`
`converting their mass into two 511 keV g-rays that must, due to
`◦
`conservation of momentum, have equal energies and travel 180
`relative to one another. These g-rays will then leave the tissue
`and strike waiting coincidence detectors; importantly, only when
`signals from two coincidence detectors simultaneously trigger the
`circuit is an output generated. The two principal advantages
`of PET thus lie in the physics: the short initial range of the
`positrons results in high resolution, and the coincidence detection
`methodology allows for tremendous sensitivity.
`In the early 1950s, Brownell10 and Sweet11 developed the first
`devices for creating images using the coincident detection of g-rays
`emitted from positron-electron annihilation events. At the same
`time, these researchers and others were pioneering the oncologic
`applications of positron imaging, specifically the imaging of brain
`tumors.10–14 Not until the 1970s, however, did the field take
`the next important practical step forward: tomographic systems
`and computer analysis were first applied to positron imaging,
`innovations which paved the way for the widespread clinical use
`of the modality.
`Since the advent of PET in both the clinic and medical research
`laboratories, a number of positron-emitting isotopes have been
`developed for use in radiopharmaceuticals. For years, the field
`was dominated by small molecule tracers, radiopharmaceuticals
`whose short biological half-lives favor the use of non-metallic
`radionuclides with correspondingly short radioactive half-lives,
`such as 18F, 15O, 13N, and 11C (Table 1). In many ways, this is still
`true: [18F]-fluoride and the ubiquitous [18F]-fluorodeoxyglucose
`([18F]-FDG) are the only FDA-approved PET radiopharmaceu-
`ticals commonly employed in oncology ([13N]-NH3 and [82Rb]-
`RbCl are FDA-approved but are used principally for myocardial
`perfusion scans). Further still, an examination of the list of PET
`radiotracers currently in NIH-sponsored clinical trials reveals an
`overwhelming majority of agents with non-metallic radionuclides,
`including among others the promising agents [18F]-FLT, [18F]-FES,
`[18F]-FDHT, [18F]-FMISO, [18F]-FACBC, [18F]-fluoroethylcholine,
`[18F]-deshydroxycholine, [18F]-FMAU, [11C]-acetate, [11C]-choline,
`[11C]-MeAIB, [11C]-MET, [124I]-IAZGP, and [124I]-FIAU.15
`Yet despite the significant successes of small molecule probes
`labeled with non-metallic isotopes, these radionuclides possess
`
`Table 1 Physical decay characteristics of conventional PET radionuclides158,159
`
`Radionuclide
`
`Half-life
`
`Decay mode
`(% branching ratio)
`
`Production route
`
`E(b+)/keV
`
`b+ end-point
`energy/keV
`
`Abundance,
`Ib+/%
`
`Eg/keV
`(intensity, Ig/%)
`
`11C
`
`13N
`
`15O
`
`18F
`
`124I
`
`1223.1 (12) s
`
`9.965 (4) m
`
`122.24 (16) s
`
`b+ (100)
`
`b+ (100)
`
`b+ (100)
`
`109.77 (5) m
`
`b+ (100)
`
`4.1760 (3) d
`
`e + b+ (100)
`b+ (22.7 [13])
`
`14N(p,a)11C
`
`16O(p,a)13N
`
`15N(p,n)15O
`14N(d,n)15O
`
`18O(p,n)18F
`20Ne(d,a)18F
`
`124Te(p,n)124I
`
`385.6 (4)
`
`960.2 (9)
`
`99.759 (15)
`
`511.0 (199.5)
`
`491.82 (12)
`
`1198.5 (3)
`
`99.8036 (20)
`
`511.0 (199.6)
`
`735.28 (23)
`
`1732.0 (5)
`
`99.9003 (10)
`
`511.0 (199.8)
`
`249.8 (3)
`
`633.5 (6)
`
`96.73 (4)
`
`511.0 (193.5)
`
`687.04 (85)
`974.74 (85)
`
`1,534.9 (19)
`2,137.6 (19)
`
`11.7 (10)
`10.7 (9)
`
`511.0 (45)
`602.7 (62.9)
`722.8 (10.4)
`1,691.0 (11.2)
`
`e = electron capture; m = minutes; d = days; s = seconds. Where positrons or g-rays of different energies are emitted, only those with abundances of greater
`than 10% are listed. Unless otherwise stated, standard deviations are given in parentheses.
`
`Dalton Trans.
`
`This journal is © The Royal Society of Chemistry 2011
`
`Downloaded by Columbia University on 28 March 2011
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`Published on 25 March 2011 on http://pubs.rsc.org | doi:10.1039/C0DT01595D
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`Petitioner GE Healthcare – Ex. 1010, p. 2
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`

`

`a few critical limitations. First, the short half-lives of the most
`common non-metallic radionuclides - approximately 20 min for
`11C, 10 min for 13N, 2 min for 15O, and 110 min for 18F - allow
`only for investigations of biological processes on the order of
`minutes or a few hours using tracers with rapid pharmacokinetic
`profiles. Second, both the short half-lives of the radionuclides and
`the frequent necessity of incorporating the radioisotopes into the
`core structure of the tracer (rather than in an appended chelator
`or prosthetic group) often necessitate demanding and complex
`syntheses. Third, the clinical and pre-clinical use of short half-life,
`non-metallic radionuclides often requires a local cyclotron facility;
`in its absence, the radionuclide in question will undergo many half-
`lives of decay while in transit. Given the resources required for the
`construction and operation of medical cyclotrons, this is simply
`not an option in many locations.
`These limitations have been brought into focus by the increasing
`study and development of biomolecular targeting agents for
`cancer, including short peptides, antibodies, antibody fragments,
`and natural and non-natural oligonucleotides. Given that Nature
`herself has designed or inspired these agents, they often show
`sensitivities and specificities for cancer cell biomarkers that far
`exceed those of their small molecule counterparts. However,
`these biomolecular tracers typically have biological half-lives that
`are much longer than the radioactive half-lives of the most
`common non-metallic positron-emitting radionuclides; further,
`though less pressing, many of these biomolecules are incompatible
`with the chemistry required for direct labeling with non-metallic
`radionuclides.16–19
`Given the enormous potential of biomolecular imaging agents,
`significant effort has been dedicated to the production, purifica-
`tion, and radiochemistry of positron-emitting radioisotopes of
`the metals Zr, Y, Ga, and Cu. These isotopes, specifically 64Cu,
`68Ga, 86Y, and 89Zr, have radioactive half-lives (roughly 12.7,
`1.1, 14.7, and 78.4 h, respectively) that favorably complement
`the biological half-lives of many biomolecular targeting vectors
`(Table 2). Although all four radiometals emit positrons, each
`
`Table 2 Physical decay characteristics of common PET radiometals40
`
`has a characteristic positron range, which is the principal factor
`in determining imaging resolution. 64Cu and 89Zr emit very low
`energy positrons, producing image resolution comparable to that
`of 18F. 86Y and 68Ga, in contrast, emit higher energy positrons,
`which can result in slightly lower imaging resolutions, though this
`can be corrected through the use of mathematical algorithms.20
`Further still, and equally critical, all four metals form stable
`chelate complexes that may be employed for the radiolabeling
`of biomacromolecules. To be sure, not all biomolecular PET
`tracers are labeled with radiometals, nor are all radiometallated
`PET tracers biomolecules. An 18F-labeled variant of the integrin-
`targeting RGD peptide21 and an 124I-labeled carbonic anhydrase-
`targeting antibody22 have produced very exciting results and
`are currently being employed in human studies. Moreover, a
`few radiometal-based small molecule tracers have also proved
`extremely promising, most notably [64Cu]-Cu(PTSM)23 and [64Cu]-
`Cu(ATSM),24 with the latter currently in a multi-center clinical
`trial as an imaging agent for hypoxia.25–28 Yet, despite these
`exceptions, the single most important application of positron-
`emitting radiometals is the development of tracers based on
`peptides, antibodies, and oligonucleotides.
`Importantly, the basic strategy for the incorporation of a
`radiometal into a biomolecule differs somewhat from the synthesis
`of a small molecule radiotracer containing a non-metallic PET
`radionuclide. In small molecule tracers, the radionuclide most
`often replaces an isotopologue (e.g. [11C]-acetate or [15O]-H2O)
`or is incorporated into the basic structure of a molecule with
`either the intent of strategically altering the behavior of the parent
`molecule (e.g. [18F]-FDG) or, more likely, disturbing the activity
`of the parent molecule as little as possible (e.g. [18F]-FDHT or
`[18F]-FES). In contrast, in biomolecular tracers, the radiometal is
`almost never directly attached to the biomolecule itself. Rather,
`the radionuclide is bound to a chelating moiety (e.g. DOTA29 or
`EDTA30), which is first covalently appended to the biomolecule
`with the intent of altering the vector’s biochemical properties as
`little as possible.31,32
`
`Radionuclide Half-life
`
`Decay mode
`(% branching ratio) Production route E(b+)/keV
`
`b+ end-point
`energy/keV
`
`Abundance,
`Ib+/%
`
`Eg/keV
`(intensity, Ig/%) Ref.
`
`64Cu
`
`68Ga
`
`86Y
`
`12.701(2) h e + b+ (61.5 [3])
`b+ (17.6 [22])
`b- (38.5 [3])
`
`67.71 (9) m e + b+ (100)
`b+ (89.14 [12])
`
`14.74 (2) h
`
`e + b+ (100)
`b+ (31.9 [21])
`
`64Ni(p,n)64Cu
`
`278.21 (9)
`
`653.03 (20)
`
`17.60 (22)
`
`511.0 (35.2)
`
`84, 85
`
`68Ge/68Ga
`
`836.02 (56)
`
`1889.1 (12)
`
`87.94 (12)
`
`511.0 (178.3)
`
`160, 70, 161
`
`86Sr(p,n)86Y
`
`535 (7)
`
`1221 (14)
`
`11.9 (5)
`
`443.1 (16.9)
`511.0 (64)
`627.7 (36.2)
`703.3 (15)
`777.4 (22.4)
`1076.6 (82.5)
`1153.0 (30.5)
`1854.4 (17.2)
`1920.7 (20.8)
`
`511.0 (45.5)
`909.2 (99.0
`
`77
`
`79, 83, 162–164
`
`89Zr
`
`78.4 (12) h
`
`e + b+ (100)
`b+ (22.74 [24])
`
`89Y(p,n)89Zr
`
`395.5 (11)
`
`902 (3)
`
`22.74 (24)
`
`e = electron capture; m = minutes; h = hours; s = seconds. Where positrons or g-rays of different energies are emitted, only those with abundances of
`greater than 10% are listed. Unless otherwise stated, standard deviations are given in parentheses.
`
`This journal is © The Royal Society of Chemistry 2011
`
`Dalton Trans.
`
`Downloaded by Columbia University on 28 March 2011
`
`Published on 25 March 2011 on http://pubs.rsc.org | doi:10.1039/C0DT01595D
`
`View Online
`
`Petitioner GE Healthcare – Ex. 1010, p. 3
`
`

`

`As new targets are described and radiometals become more
`available to the wider molecular imaging community, the amount
`of research into radiometal-based PET tracers has exploded in
`recent years. For example, over 60% of all publications describing
`89Zr-PET have been published in the last four years (with well over
`20% in 2010 alone).33 Indeed, the dramatic growth in this area and
`the expansion in the availability of radiometals have had the dual
`effects of broadening the appeal of biomolecular PET imaging
`and opening the field to investigators who previously may have
`left the development of PET probes to dedicated radiochemistry
`and molecular imaging laboratories. However, the frenetic pace of
`the field and the array of choices in chelation, conjugation, and
`metallation strategies may serve as an obstacle to those who are
`interested in the development of radiometallated PET tracers but
`lack significant bioconjugation or radiochemical experience.
`This perspective aims at lowering this barrier. Here, we strive to
`create a practical guide to the synthesis of radiometal-based PET
`tracers. To this end, we have compiled the experimental details
`of chelator choice, conjugation strategy, and radiometallation
`conditions from the syntheses of a wide array of 64Cu-, 68Ga-, 86Y-,
`and 89Zr-labeled PET agents. Typically, reviews discuss the struc-
`ture, behavior, biology, and imaging applications of these agents,
`with the experimental details touched upon only briefly or simply
`referenced.7,16,34–37 All too often, however, the search for a specific
`conjugation or metallation protocol results in an elongated, and in
`some cases circuitous, trek through the literature to find a simple
`incubation time or buffer concentration. Importantly, we do not
`strive for an exhaustive review of the radiochemistry or imaging
`applications of radiometal-based PET tracers. Others - most
`notably Carolyn Anderson and her coworkers at the Washington
`University School of Medicine and Martin Brechbiel and his
`coworkers at the National Cancer Institute - have produced well-
`written and remarkably thorough reviews on these topics.3,30,34,38–45
`The core of this perspective lies not in the text but rather in
`the series of tables containing the practical details of chelator
`conjugation and radiometallation from a diverse collection of
`64Cu-, 68Ga-, 86Y-, and 89Zr-labeled bioconjugates. We have elected
`not to include two types of macromolecular radiopharmaceuticals,
`bispecific antibodies and biomolecule-based nanoparticles, in the
`interest of space and scope, though these have been addressed
`well elsewhere.46–49 Further, it is important to note that some of
`the conjugation strategies described herein are now, for the most
`part, obsolete with respect to their original vector; for example, a
`number of syntheses for DOTATOC will be outlined, though this
`DOTA-modified somatostatin analogue is now widely commer-
`cially available. Yet we believe it is important to detail these con-
`jugation methods nonetheless, for the synthetic routes themselves
`may prove useful in the future for the creation of conjugates with
`different biomolecular vectors. In collecting these techniques in
`one place, we hope not only to shed light upon the diverse methods
`employed in the synthesis of these agents but also, and perhaps
`more importantly, to create a useful reference for both experienced
`molecular imaging scientists and researchers new to the field.
`
`The anatomy of a PET bioconjugate
`
`A radiometallated PET bioconjugate has four component parts,
`each of which must be carefully considered during the design and
`synthesis of the tracer: (1) the biomolecular targeting vector, (2)
`
`the radiometal, (3) the chelator, and (4) the linker connecting the
`chelator and the biomolecule (Fig. 1). A detailed discussion of
`the possible targeting vectors lies outside the scope of this work,
`though biomolecules ranging from cyclic pentapeptides and short
`oligonucleotides to 40-amino acid peptides, antibody fragments,
`and full antibodies have been employed.30 Of course, the most
`important facet of the biomolecule moiety is its specificity for
`its biomarker target. Indeed, a wide array of biomarkers have
`been exploited. Most often, the chosen target is a cell surface
`marker protein or receptor, such as the somatostatin receptor
`integrin family (e.g. avb3),51 gastrin-releasing
`family (SSTr),50
`peptide receptor (GRPR),52 and epidermal growth factor receptor
`(EGFR).53 In more specialized cases, disialogangliosides (e.g.
`GD2), mRNA gene products, and even the low pH environment
`of tumors have been targeted by antibodies,54 oligonucleotides,55
`and short peptides,56 respectively. Targeting cytosolic proteins
`and enzymes with antibodies and oligopeptides is rare due to
`the considerable difficulty of getting large biomolecules into the
`cytoplasm. However, significant progress is being made in the
`development of cell- and nucleus-penetration strategies, and this
`technology may prove productive for intracellular or intranuclear
`PET imaging agents in the near future.
`
`Fig. 1 The anatomy of a PET bioconjugate.
`
`Radiometals: properties and production
`The principal radiometals employed for the labeling of biomolec-
`68Ga,
`86Y, and 89Zr. Of course, these
`ular tracers are 64Cu,
`are not the only positron-emitting radiometals. Some metallic
`radioisotopes, such as 60Cu, 61Cu, 62Cu, 82Rb, 52mMn, and 94mTc,
`have been used in PET studies to varying degrees, but their half-
`lives make them far better suited for small molecule tracers (e.g.
`[60Cu]-Cu(ATSM)).57–60 Other positron-emitting radiometals, in-
`cluding 45Ti ([45Ti]-transferrin61), 52Fe ([52Fe]-citrate/transferrin62),
`55Co ([55Co]-antiCEA F(ab¢)2
`63,64), 66Ga ([66Ga]-octreotate65), 110mIn
`([110mIn]-octreotate66), and 74As ([74As]-bavituximab67,68), have been
`employed in the synthesis of biomolecular radiopharmaceuticals.41
`However, these will not receive more than a brief discussion here,
`due to either the lack of more than one or two radiotracers per
`isotope, the limited availability of the radionuclide in question, or
`decay characteristics that make the isotope sub-optimal for use in
`a clinical PET radiopharmaceutical.40
`The selection of a radiometal from the four main candidates,
`64Cu, 68Ga, 86Y, and 89Zr, is a critical factor in determining the
`ultimate properties of a PET bioconjugate. In this regard, one
`of the most important considerations is matching the radioactive
`
`Dalton Trans.
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`This journal is © The Royal Society of Chemistry 2011
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`Downloaded by Columbia University on 28 March 2011
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`

`

`half-life of the isotope to the biological half-life of the biomolecule.
`For example, 68Ga is an inappropriate choice for labeling fully
`intact IgG molecules, for the radionuclide will decay through a
`number of half-lives before the antibody reaches its fully optimal
`biodistribution within the body. Therefore, the longer lived
`radiometals 64Cu, 86Y, and especially 89Zr are most often employed
`for immunoPET with fully intact mAbs. That said, 68Ga has
`been used successfully in the construction of PET bioconjugates
`based on antibody fragments with shorter biological half-lives.
`Conversely, 89Zr would be an inappropriate choice for a short
`peptide radiotracer; in this case, the multi-day radioactive half-life
`of 89Zr would far exceed what is typically a multi-hour biological
`half-life of the peptide, resulting in poor PET counting statistics
`and unnecessarily increased radiation dose to the patient. Thus,
`64Cu, 86Y, and 68Ga are most often employed for oligopeptide PET
`tracers. It is important to note that 64Cu and 86Y occupy a favorable
`middle ground with respect to radioactive half-life, allowing these
`radionuclides to be utilized advantageously in both antibody- and
`peptide-based based tracers.
`The production of radiometals in high radionuclidic purity
`and specific activity is essential to the development of effective
`bioconjugates for PET imaging, and while an in-depth understand-
`ing of the nuclear reactions and purification chemistry behind
`their production may not be necessary for the biomedical use of
`these isotopes, a brief overview of the processes surely has merit.
`The production methods for radionuclides fall into three general
`categories: generator, cyclotron, and nuclear reactor (Fig. 2). Of
`the positron-emitting radiometals addressed in this perspective,
`68Ga is generator-produced, while 64Cu, 86Y, and 89Zr are produced
`using a medical cyclotron.
`68Ga is produced via the electron capture decay of its parent
`radionuclide, 68Ge. In the laboratory and clinic, 68Ga can be pro-
`duced using a compact, cost-effective, and convenient 68Ge/68Ga
`generator system, which is capable of providing 68Ga for PET
`tracers for 1–2 years before being replaced.69 The 68Ga is eluted
`from the generator in 0.1 M HCl, providing a 68GaCl3 starting
`material for radiolabeling.70 Despite its convenience, the system
`does have some limitations, most notably high eluent volumes that
`often must be pH-adjusted prior to radiolabeling reactions, 68Ge
`break-through from the generator, and metal-based impurities.
`However, a number of purification techniques have been developed
`to circumvent the problems presented by the trace impurities in
`the 68Ga eluent.
`86Y is the first of the three cyclotron-produced radiometals
`to be addressed here. 86Y is most often produced through the
`86Sr(p,n)86Y reaction via bombardment of an isotopically enriched
`
`86SrCO3 or 86SrO target with 8–15 MeV protons.71–74 A range of
`purification methods have been employed, including combinations
`of precipitation, ion exchange chromatography, chromatography
`with a Sr-selective resin, and electrolysis.75–77
`89Zr has been produced via both the 89Y(p,n)89Zr and
`89Y(d,2n)89Zr reactions. In the past, these methods have been used
`to successfully produce the radiometal using 13 MeV protons
`and 16 MeV deuterons, respectively, though both pathways
`have been complicated and limited by problematic purification
`protocols.78–80 A significant improvement upon these methods was
`provided by another production strategy that yielded 89Zr via the
`bombardment of 89Y on a copper target with 14 MeV protons,
`oxidation of Zr0 to Zr4+ with H2O2, and purification via anion
`exchange chromatography and subsequent sublimation steps.81,82
`In the last few years, these methods have been improved upon
`further through the use of an 89Y thin-foil target (99% purity,
`0.1 mm width), the optimization of bombardment conditions
`◦
`(15 MeV, 15 mA, 10
`angle of incidence), and an improved solid
`phase hydroxamate resin purification to produce 89Zr reliably and
`reproducibly in very high specific activity (470–1195 mCi/mmol)
`and radionuclidic purity (>99.99%).83
`Finally, 64Cu can be produced with either a nuclear reactor
`or a cyclotron via a variety of reaction pathways.3 In a nuclear
`reactor, 64Cu can be produced through the 63Cu(n,g )64Cu and
`64Zn(n,p)64Cu pathways. On a biomedical cyclotron, carrier-free
`64Cu can be produced using the 64Ni(p,n)64Cu and 64Ni(d,2n)64Cu
`reactions.84–88 The former pathway has proven more successful
`and is currently used to provide 64Cu to research laboratories
`throughout the United States. In this method, the 64Cu is processed
`and purified via anion exchange chromatography to yield no
`carrier-added 64Cu2+. The expense of the enriched 64Ni target is
`a limitation of this production pathway, though a technique for
`the recycling of 64Ni has ameliorated this issue somewhat. In
`the last few years, a number of groups have worked to develop
`methods for the production of 64Cu using Zn targets through the
`64Zn(d,2p)64Cu,66Zn(d,a)64Cu, and 68Zn(p,an)64Cu reactions.89–92
`These efforts have yielded some promising results but have failed
`to supplant the cyclotron-based 64Ni(p,n)64Cu pathway as the main
`route for 64Cu production.
`
`Radiometal chelation chemistry
`
`With both the targeting vectors and radiometals in hand, the
`spotlight next falls on how to combine these two essential parts of
`the PET bioconjugate. Indeed, both the formation of a kinetically
`inert metal chelate and the stable covalent attachment of the
`
`Fig. 2 Three methods for the production of radionuclides: (A) 68Ga generator, (B) cyclotron, and (C) nuclear reactor. The authors acknowledge David
`Nickolaus of the Missouri University Research Reactor for the photo of the nuclear reactor.
`
`This journal is © The Royal Society of Chemistry 2011
`
`Dalton Trans.
`
`Downloaded by Columbia University on 28 March 2011
`
`Published on 25 March 2011 on http://pubs.rsc.org | doi:10.1039/C0DT01595D
`
`View Online
`
`Petitioner GE Healthcare – Ex. 1010, p. 5
`
`

`

`Fig. 3 Selected chelators and bifunctional chelators for 64Cu, 68Ga, 86Y, and 89Zr.
`
`chelator moiety to the biomolecule are essential to the creation
`of an effective radiopharmaceutical. To this end, a wide variety
`of metal-chelating molecules have been synthesized, studied, and,
`in many cases, made bifunctional to facilitate their conjugation
`
`to a biomolecular vector (Fig. 3 and 4, vide infra). Transition
`metal chelators fall into two broad classes: macrocylic chelators
`and acyclic chelators. Each has its own unique set of advantages:
`while macrocyclic chelators typically offer greater kinetic stability,
`
`Dalton Trans.
`
`This journal is © The Royal Society of Chemistry 2011
`
`Downloaded by Columbia University on 28 March 2011
`
`Published on 25 March 2011 on http://pubs.rsc.org | doi:10.1039/C0DT01595D
`
`View Online
`
`Petitioner GE Healthcare – Ex. 1010, p. 6
`
`

`

`This journal is © The Royal Society of Chemistry 2011
`
`Dalton Trans.
`
`Fig. 4 Selected chelators and bifunctional chelators for 64Cu, 68Ga, 86Y, and 89Zr.
`
`Downloaded by Columbia University on 28 March 2011
`
`Published on 25 March 2011 on http://pubs.rsc.org | doi:10.1039/C0DT01595D
`
`View Online
`
`Petitioner GE Healthcare – Ex. 1010, p. 7
`
`

`

`Table 3 Coordination number, donor set, and geometrya for selected
`complexes of Cu(II), Ga(III), Y(III), and Zr(IV)30
`
`Metal Chelator
`
`Ligand
`Donor
`Set
`
`Total
`CN Coordination Geometry
`
`N3O3
`Cu(II) DTPA
`N3O3
`NOTA
`N4O2
`DOTA
`N4O2
`TETA
`CB-TE2A N4O2
`N2S2
`EC
`DIAMSAR N6
`
`6
`6
`6
`6
`6
`4
`6
`
`—
`distorted trigonal prism
`distorted octahedron
`distorted octahedron
`distorted octahedron
`distorted square planar
`distorted octahedron or
`trigonal prism
`
`Ga(III) EDTA
`N2O4
`N2O4
`HBED
`N3O3
`DTPA
`N3O3
`NOTA
`N4O2
`DOTA
`N4O2
`TETA
`CB-TE2A N4O2
`DFO
`O6
`
`distorted octahedron
`6
`—
`6
`6? —
`6
`distorted octahedron
`6
`distorted octahedron
`6
`distorted octahedron
`6
`distorted octahedron
`6
`—
`
`Y(III) DTPA
`
`DOTA
`
`N3O5
`
`N4O4
`
`8
`
`8
`
`monocapped square
`antiprism
`square antiprism
`
`TETA
`
`N4O2
`
`8?
`
`distorted dodecahedron(?)
`
`Zr(IV) DFO
`DOTA
`EDTA
`DTPA
`
`O6
`N4O4
`N2O4
`N3O5
`
`7 or 8 —
`8?
`square antiprism?
`8
`distorted dodecahedron
`8
`distorted dodecahedron
`
`Ref.
`
`165
`166, 167
`168–170
`168–170
`101, 171
`172
`173
`
`174, 175
`176, 177
`175
`168, 178
`168
`168
`30, 179
`180
`
`116, 181,
`182
`114, 116,
`182
`183
`
`125
`115
`181, 184
`181, 184
`
`a Question marks denote uncertainty in coordination number or geometry,
`and “—” denotes that the coordination geometry is not known.
`
`acyclic chelators usually have faster rates of metal binding.
`Generally, transition metal chelators offer at least four (and usually
`six or more) coordinating atoms, arrayed in a configuration that
`suits the preferred geometry of the oxidation state and d-orbital
`electron configuration of the metal in question. Yet simply having
`a generic chelator with well-organized and plentiful donor atoms is
`not enough; in every case, an appropriate chelator must be chosen
`to suit the selected radiometal (Table 3). Of course, however,
`some (e.g. DOTA) are more universally applicable than others
`(e.g. DiamSar). The most relevant oxidation states for the metals
`discussed here are Zr(IV), Ga(III), Y(III), and Cu(II); in vivo, only
`Cu(II) is at significant risk for reduction reactions. In terms of the
`commonly-employed ‘hard-soft’ system of classification, Zr(IV) is
`considered a very hard cation, with Y(III) and Ga(III) close behind
`on the spectrum. Cu(II), which is a borderline acid, straddles the
`hard/soft border and is thus easily the softest of the four.
`Cu(II) has a rich chelation chemistry, capable of the forma-
`tion of four-, five-, and six-coordinate complexes, with geome-
`tries ranging from square planar to trigonal bipyrimidal and
`octahedral.3,30,32,36,42,93 Due to its position on the b