Abstract

Introduction

The number of publications devoted to ⁶⁸Ga-radiopharmaceutical basic and clinical research has increased drastically during last two years. Rough estimation demonstrates that the number of ⁶⁸Ga-related scientific articles published during 2011-2012 stands for over 45% of all publications since 1956 (Figure ​(Figure1).1). Rather crucial changes and progress occurred during this short period and they influenced the basic perceptions and thus speculation about the nearest 5-10 year future of the field. Nuclear medicine applications and in particular positron emission tomography (PET) have experienced accelerated development.

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Fig 1

The fraction (%) of ⁶⁸Ga-related publications per two successive years (except for the periods of 1956-1966 and 1967-1978) appeared from 1956 through the end of 2012. PubMed, Scopus, SciFinder Scholar, Web of Science, and Beilstein databases as well as references from published articles were used for the literature search and assay.

The major advantage of PET is that it is not only enables in vivo visualization of physiological processes on molecular level in real time, but it also quantifies them by measuring regional concentration of the radiation source. PET employs imaging agents comprising positron-emitting radionuclides (Figure ​(Figure2A),2A), and scanners detecting radiation (Figure ​(Figure2B).2B). Positron scan registration is based on the 180±0.25° correlation of the 511 keV photons arising from the annihilation of positrons with electrons and detection by means of two opposing counters recording only coincident events ¹. The registered events are reconstructed into images representing spatial distribution of the radioactivity in a subject. Positron emission is an attribute of neutron deficient nuclides requiring artificial production generally by cyclotrons. However ⁶⁸Ga is obtained from a ⁶⁸Ge/⁶⁸Ga generator system which is simple in use and relatively inexpensive.

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Fig 2

Schematic representation of PET principle. (A) After travelling in tissue (positron range) positron looses energy and annihilates with electron resulting in two 511 keV annihilation photons travelling in opposite directions; (B) Annihilation photons are registered externally by radiodetectors consisting of scintillation crystals and photomultiplier tubes and assembled in a ring. Only photons that are registered in coincidence are used for image reconstruction.

PET has become an established method for medical research and clinical routine diagnostics. Development and availability of new radiopharmaceuticals specific for particular diseases is one of the driving forces of the expansion of clinical nuclear medicine providing early personalized diagnosis and efficient therapy. The future development of ⁶⁸Ga-radiopharmaceuticals must be put in the context of several aspects such as role of PET in nuclear medicine, unmet medical needs, identification of new biomarkers, targets and corresponding ligands, production and availability of ⁶⁸Ga, automation of the radiopharmaceutical production, progress of PET technologies and image analysis methodologies for improved quantitation accuracy, PET radiopharmaceutical regulations as well as radiopharmaceutical chemistry advances. This review presents the prospects of the ⁶⁸Ga-based radiopharmaceutical development on the basis of the current status of these aspects. ⁶⁸Ga has demonstrated its applicability for the labelling of small compounds, biological macromolecules as well as nano- and micro-particles promoting the growth of PET field ². The major application domain is oncology; however, potential has been demonstrated for imaging of myocardial perfusion, pulmonary perfusion and ventilation as well as inflammation and infection. Imaging of general biologic properties and processes such as proliferation, apoptosis, hypoxia, glycolysis, and angiogenesis have also been investigated. These prerequisites may trigger an explosive progress and introduction of new ⁶⁸Ga-radiopharmaceuticals into clinics in the nearest future of 5-10 years.

The focus of this critical review resides on the publications from year 2008-2012 reflecting latest achievements as well as selected earlier references that give the background and support the foundation for the future development of basic research and clinical applications. The achievements in gallium radiopharmaceutical chemistry and imaging agent development from 1956 through January 2011 were addressed in a comprehensive and exhaustive review published earlier ² and references therein. The explosive growth of publications reflecting the success of ⁶⁸Ga applications is remarkable. Another strong indication of the worldwide growth of ⁶⁸Ga studies is the “First World Congress on Ga-68 and peptide receptor radionuclide therapy (PRRT)” that took place in 2011 and attracted participants from all continents ³.

Role of PET in nuclear medicine

Nuclear medicine is a quickly expanding field for the diagnostics and therapy on cellular and molecular level. Molecular imaging techniques such as PET and Single Photon Emission Computed Tomography (SPECT) utilize, respectively, positron and gamma emitting radionuclides for the generation of the signal that results in a whole-body scan in a single examination. They provide fast and non-invasive evaluation of physiology and pathology, and together with external and internal radiotherapy merge into theranostics resulting in personalized medicine ^4-7. Imaging diagnostics enables early detection, staging, therapy selection, planning, and monitoring treatment response thus considerably improving cancer therapy. The most pronounced example is the selection of oncological patients for PRRT.

Radionuclidic properties and detection techniques determine the advantages of PET over SPECT in terms of 100-1000 fold higher sensitivity, higher speed, accurate quantitation, and dynamic image reconstruction. However gamma and SPECT scanners are more accessible and have lower cost. Moreover, a wider range of registered gamma emitting radiopharmaceuticals is available, though their price has enhanced currently due to the reduction of public subsidy ⁸. On the other hand, the shorter scanning time and thus higher throughput of PET decreases the cost per patient examination. Nuclear medicine practice is dominated by ^99mTc/SPECT standing for 85% of all clinical examinations in the USA with 50% in cardiology ⁹. Dual-tracer imaging is one more advantage of SPECT, however recent research has demonstrated possibility of multiple PET tracer imaging based on the signal separation according to the differences in radionuclide half-life as well as tracer kinetics and distribution ¹.

Although PET was introduced as early as in the 1970s, only recently it has been recognized clinically relevant, to some extent due to the introduction of hybrid PET-CT scanners that are spreading worldwide with acceleration and have become the gold standard of PET imaging. Rather radical question has been raised if there is a role in oncology for SPECT considering current advantages of PET and respective radiopharmaceuticals ⁸. The authors suggest that PET provides more accurate and quantitative diagnosis enabling individualized cancer therapy planning resulting in the efficient and cost saving treatment. The propagation of PET technique is greatly stimulated by the widespread use of [¹⁸F]-fluorodeoxyglucose ([¹⁸F]FDG/PET-CT) for many indications in oncology as well as centralized production and distribution of [¹⁸F]FDG to satellite clinical centres. Another factor influencing the expansion of PET is the need for imaging agents with disease specific action, and ⁶⁸Ga has a significant contribution to make. The reports on such category of radiopharmaceuticals from molecular imaging and contrast agent database (MICAD) state that PET and SPECT imaging agents stand respectively for 42% and 31% ¹⁰. However, PET still has to overcome the major hurdles such as regulatory barriers for introducing new imaging agents and restriction of reimbursement. The substitution of SPECT with PET might be just a part of evolutionary process until another more advanced technology appears. Just like rectilinear scanners were substituted with gamma cameras.

PET provides more accurate staging than other conventional diagnostic means and thus it is rational to use it independently and as the first diagnostic choice ¹¹. Dramatic impact of PET-CT on patient management has been recognized by Medicare and Medicaid Services ¹². This can be illustrated by the evidence that the course of considerable number of patient treatments (50-60 %) was changed or adjusted on the basis of ⁶⁸Ga/PET-CT examinations using somatostatin (SST) ligand analogues in diagnosis and staging of neuroendocrine tumours (NETs) ^{2, 13}. Somatostatin receptor scintigraphy will most likely be replaced with ⁶⁸Ga/PET-CT in the nearest future ¹⁴.

The widespread opinion is that PET is a costly technique, but in fact the total cost of the otherwise required examinations, hospitalization and the risk associated with biopsy as well as false diagnosis leading to futile surgery cost and patient distress greatly overweighs. In particular, the accuracy of diagnostic methods related to the heterogeneity of primary tumour and between a primary tumour and metastases should be addressed. The biopsy results might be misleading since the tumour diagnosis and staging is conducted ex vivo on invasively collected tissue. The sampled tissue is commonly heterogenic and thus might not be representative, while PET may provide more detailed and accurate information on heterogeneity of the whole tumour non-invasively in a single examination. The cost of [⁶⁸Ga]Ga-DOTA-TOC/PET-CT including material and personnel was found lower as compared to that of ¹¹¹In-DTPA-octreotide/SPECT ¹⁵. Moreover, when using the former fewer additional examinations were required. In addition, the cost-effectiveness and cost-saving of PET-CT was demonstrated by [¹⁸F]FDG/PET-CT used for staging of lung cancer ^{8, 16, 17}. Currently PET stands for only ~1.5% of Medicare (USA) cancer care expenses, nevertheless the total imaging costs are increasing fast ¹⁸, and the reimbursement of [¹⁸F]FDG/PET-CT of lung cancer by public health care system might become reality in the nearest future in industrialized countries, and also will propagate into other cancer groups. This in turn will stimulate the investments and development of new imaging agents. The dramatic growth in PET-CT during the last decade has already been accompanied with reducing costs and introduction of new specific PET tracers.

Unmet medical needs

Early diagnosis is important in order to identify functional abnormalities which precede morphological changes, and in particular for cancer, molecular imaging may contribute to the reduction of morbidity and mortality ¹⁹. Over 90% of clinical PET investigations are nowadays performed with [¹⁸F]FDG, however interest in specific molecular probes for, e.g. cancer and inflammation/infection is getting stronger among scientists and clinicians ^{20, 21}. The reason for this is that although [¹⁸F]FDG has been successfully used in many cancers as a biomarker of glucose transport, it fails in diagnosis of slowly growing tumours and in differentiation between tumour and such processes as inflammation, infection, reactive lymph nodes, tuberculosis and sarcoidosis ^{22, 23}. Moreover, the high uptake in normal organs, particularly in brain and gut, results in poor contrast in those areas and potential failure in lesion detection. Thus alternative imaging agents with specific binding capability to e.g. receptors, antigens, enzymes are of strong interest. Specific imaging agents providing information on the molecular and cellular background of various diseases and in particular cancer would allow improvement in patient management and outcome. Generator produced ⁶⁸Ga may not only enable PET examinations at remote hospitals distant from [¹⁸F]FDG distribution, but would also enrich the radiopharmaceutical arsenal at the medical centers both with and without accelerators.

Various ⁶⁸Ga-based imaging agents have already been tested in humans ⁷. The basic research and development of new ⁶⁸Ga-based agents for targeted imaging of specific protein expression products, pre-targeted imaging as well as non-targeted imaging of pulmonary and myocardial perfusion and pulmonary ventilation is expanding steadily. Imaging of cell proliferation, hypoxia, glycolysis, angiogenes as well as inflammation and infection has also been considered ². Radiochemistry plays a central role in the development and ⁶⁸Ga in particular has demonstrated powerful potential. The prevalent area of ⁶⁸Ga clinical applications in the nearest future will most probably be oncology. More research is required for the development of agents for cardiology. On the other hand, neurology will still be dominated by ¹¹C- and ¹⁸F-based imaging agents since common requirements to specific binding, small size, lipophilicity and charge might be difficult to meet when using bulky radiometal-chelator complex to be attached to a small biologically active molecule.

Theranostics in nuclear medicine has manifested in pre-therapeutic diagnosis for PRRT. In this context the diagnosis and radiotherapy are conducted with the same ligand labelled respectively with imaging radionuclide, e.g. ⁶⁸Ga and therapeutic one, e.g. ¹⁷⁷Lu (Figure ​(Figure3B).3B). The radiopharmaceuticals bind to tumour-type specific receptors enabling personalized approach of accurate quantitative diagnosis and staging for subsequent selection and planning of therapeutic means as well as monitoring response to the treatment. The dosimetry estimation prior to PRRT is one of the most important prerequisites of successful treatment, and the higher spatial resolution and inherent quantitative accuracy of ⁶⁸Ga/PET are of paramount importance for the dosimetry precision and treatment response evaluation. The common perception is that ⁶⁸Ga is not applicable for the pre-therapeutic dosimetry due to the short half-life of ⁶⁸Ga (68 min) mismatching that of therapeutic radionuclides ⁹⁰Y (64 h) and ¹¹⁷Lu (6.71 d), and consequently resulting in different time windows of the pharmacokinetics. However, e.g. in the case of somatostatin analogues their fast pharmacokinetics may enable the utilization of kinetic analysis of ⁶⁸Ga-labelled peptide for the prediction of radiation doses during PRRT with the ¹⁷⁷Lu-labelled analogue. This might be accomplished by the combination of the respective agents, namely by either co-injection or separate use of ⁶⁸Ga- and ¹⁷⁷Lu-labelled peptides. The high accuracy at early time points could be achieved by ⁶⁸Ga/PET quantitation with an error of less than 5% since the tracer accumulation in the organs at risk reaches its maximum within 2-3 half-lives of ⁶⁸Ga ²⁴. The kinetics at later time points could be obtained by either blood sampling during the radiotherapy with ¹⁷⁷Lu-labelled analogue or by imaging gammas emitted by ¹⁷⁷Lu. The dual-tracer examination is possible since the gamma energy of ¹⁷⁷Lu is outside the PET detection window and thus would not interfere with ⁶⁸Ga/PET-CT quantitation. Such combination of the tracers would provide highly improved internal dosimetry.

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Fig 3

A) Depiction of the basic components of an imaging agent comprising a vector for specific binding, pharmacokinetic modifier, and the complex of the chelator moiety with a radiometal; B) Drawing of the interaction of the agent, either imaging if labelled with ⁶⁸Ga (left) or radiotherapetic if labelled with ¹⁷⁷Lu (right), with the cell receptor.

Specific radioactivity (SRA) of an imaging agent is another parameter influencing the organ distribution of the agent and subsequent dosimetry. In the case of macromolecular agents, e.g. based on peptides, SRA is calculated as radioactivity per total amount of the agent. The targeted and pre-targeted imaging might require relatively high SRA in order to avoid saturation of binding sites by non-labeled counterpart and possible pharmacological side effects. In this context the total amount of the injected agent, e.g. peptide, should be optimized prior to the radiotherapy providing the highest possible uptake in the lesions and lowest possible uptake in the healthy tissue and organs with physiological expression of the target ^{2, 24, 25}.

Development of scanner hardware and software technologies

The technology developed from PET scanners to hybrid PET-CT and PET-MRI ^{26, 27}. The morphologic information from CT and MRI is crucial for the accurate determination of location of the lesions. New hardware, detector, and image reconstruction algorithm technologies (e.g. time-of-flight (ToF-PET), point-spread-function (PSF-PET)) continuously improve image contrast, spatial and temporal resolution as well as quantitation accuracy ^{1, 28}. Moreover, dual-tracer imaging might be possible distinguishing signals on the basis of differences in radionuclide half-lives, tracer kinetics and distribution, however dynamic scanning and multivariate software tools such as principle component analysis would be required ¹. PET-MRI combining the high soft tissue contrast of PET and high spatial resolution of MRI has become reality although it is used at present mainly for research in neurology, cardiology, and oncology. It considerably decreases the radiation dose to the patient as compared to PET-CT. The special resolution of PET might also be improved since the positron range can be shortened by the influence of strong magnetic field. PET-MRI with [⁶⁸Ga]Ga-DOTA-TOC was found superior to MRI alone or PET-CT in terms of detection rate and specificity of liver lesions in patients with NETs ²⁹. It has also been found to improve the accuracy of target volume delineation in intensity modulated radiotherapy (IMRT) treatment planning and delivery ³⁰. Moreover, it reduced the number of pretreatment imaging sessions for meningioma patients.

The spatial and temporal resolution of instrumentation, radionuclide decay mode and energy as well as organ movement and size influence the accuracy of PET quantitation which is especially crucial when monitoring the treatment response with marginal changes. Scanning technology and data reconstruction technique have been improved during last 10-15 years in order to meet the requirements. These advances will definitely stimulate development of ⁶⁸Ga-based imaging agents, empowered by the relation to the therapy selection and planning. It should be stressed that the full potential of the imaging technology can only be realized if new imaging agents for specific indications emerge. Improved clinical outcomes of molecular imaging-guided radiation therapy have already been demonstrated ²³. PET radiopharmaceuticals induce adverse reactions extremely seldom with no serious or life-threatening events ³¹. While hybrid imaging, PET-CT and PET-MRI, may require high dose of CT and MRI contrast agents thus enhancing the probability of adverse events.

Overview of common clinical imaging radionuclides and ⁶⁸Ga

The most common radionuclides relevant for the labelling synthesis of radiopharmaceuticals for PET, SPECT, and radiotherapy are presented in Table ​Table1.1. The majority are metals typically involved in coordination labelling chemistry. Only few of the radionuclides are produced in generator systems. Such factors as production mode as well as physical and chemical characteristics determine the choice of a radionuclide.

Two gallium isotopes, ⁶⁶Ga (t_1/2 = 9.5 h) and ⁶⁸Ga (t_1/2 = 68 min), decay by β⁺-emission and are therefore suitable for PET imaging, ⁶⁷Ga (t_1/2 = 78 h) decays by electron capture with concomitant γ-emission and is used for SPECT imaging, examples of clinically used radionuclides include ¹¹C, ¹⁸F, ⁶⁴Cu, ⁸⁹Zr, ^99mTc, ¹¹¹In, ¹²⁴I (Table ​(Table1).1). MICAD has reported that ~41%, 31% and 28% of PET agents is labelled, respectively with ¹⁸F, ¹¹C, and ⁶⁴Cu, ⁶⁸Ga, ⁸⁹Zr, ¹²⁴I taken together ¹⁰. Amongst SPECT agents the leading position belongs to ^99mTc (42%) followed by ¹¹¹In (29%). The major advantage of ^99mTc and ⁶⁸Ga is that they are obtained from generators that are cost-effective alternative to reactors and cyclotrons. They are also cheaper as compared to commercial radionuclides, for example ¹¹¹In and ¹²⁴I. ⁶⁸Ga/PET-CT provides advantage over ^99mTc/SPECT not only due to the inherent benefits of the technology, but also due to the resemblance of coordination chemistry of ⁶⁸Ga, ⁹⁰Y and ¹⁷⁷Lu and, thus possibility to use the same vector molecule for the subsequent radiotherapy. As a result, the diagnostic agent is directed to the same molecular target as radiotherapeutic one allowing the prediction of the treatment efficacy and selection of patients within the frame of theranostics.

With regard to the decay mode, the advantage of positron emitting radionuclides over gamma emitting ones is inherent and related to the benefits of PET technique such as higher sensitivity, resolution, quantitation, and dynamic scanning. The energy and the content of the emitted particles influence the resolution. ⁶⁸Ga with rather high positron energy could be expected to perform with lower resolution as compared to ¹⁸F, however both computational analysis and experimental measurements demonstrated equally high quality images for these two radionuclides assuming the scanner detector resolution of 3 mm ². The disadvantage with ⁶⁴Cu, ⁸⁹Zr, and ¹²⁴I as compared to ⁶⁸Ga include low positron abundance (⁶⁴Cu, ⁸⁹Zr), higher positron energy (¹²⁴I), as well as simultaneous gamma emission (¹²⁴I) resulting in poorer image quality, longer scanning duration, and additional radiation dose. However, their longer half-lives allow development of agents based on vectors with slow pharmacokinetics such as, for example antibodies that require 2-4 days post injection for blood clearance and optimal image contrast. Though, it is worth mentioning that the relatively short half-life of ⁶⁸Ga allows repetitive examinations on the same day ²⁴.

One of the most rapidly expanding areas is the development of peptide-based agents for targeted imaging. Metal and halogen radionuclides such as ⁶⁴Cu, ⁸⁹Zr, ^99mTc, ¹¹¹In, ¹⁸F, and ¹²⁴I are most commonly used for the design of the agents. Typically, the metal ion coordination chemistry is straightforward and mild. However, it requires attachment of a rather bulky chelator to the vector molecule what may deteriorate the biological activity. Simple direct iodination has been used for decades introducing minor modification at tyrosine amino acid residue though demonstrating poor residualizing of the radioactive iodine catabolites in the cell. On the contrary hydrophilic complexes of radiometals stay trapped in the cell after the degradation of the internalized imaging agent. The incorporation of ¹⁸F can be accomplished by the conventional nucleophilic or electrophilic addition, or via prosthetic groups ³². However, the synthesis might be rather complex with harsh conditions and high macromolecule concentration. A novel method utilizing similarity of Al¹⁸F²⁺ with metal cations and thus possibility of coordination labelling chemistry has potential for kit type production like radiometals ³³. The relatively long half-life (110 min) of ¹⁸F enables centralized production and distribution of either the radionuclide or prepared imaging agents to the satellite clinical centres. Most of the PET agents used in clinical studies are ¹⁸F-based. Nevertheless, the simpler labelling chemistry of ⁶⁸Ga and its availability from the generator system which can be used even at distant and isolated medical centres make ⁶⁸Ga more preferable.

Only ¹¹C amongst over mentioned radionuclides provides true tracers since it is an endogenous element and the radioactive compounds are chemically identical to the stable counterparts. It is perfect radionuclide for the labelling of small bioactive organic molecules. This field is rather challenging for metal radionuclides including ⁶⁸Ga, and ¹¹C together with ¹⁸F will most probably dominate in the field of neurology and metabolic tracers in oncology employing small molecules ³⁴.

As mentioned earlier the advantages of ⁶⁸Ga over ¹¹¹In are multiple and have already been demonstrated in patients affected by NETs in terms of diagnostic output and logistics, superior sensitivity and accuracy, higher detection rate and specificity, faster acquisition and shorter examination time, lower radiation exposure as well as cost, and ⁶⁸Ga-labelled somatostatin analogues are predicted to replace [¹¹¹In]In -DTPA-octreotide (Octreoscan®) in the nearest future ^{14, 15, 35, 36}.

Another advantage of ⁶⁸Ga over ¹⁸F, ^99mTc, and ¹¹¹In is comparable or lower level of effective dose (Table ​(Table2)2) ^{37, 38}. The latter was calculated considering administration of commonly used levels of radioactivity of 100, 200, 370, and 570 MBq respectively for [⁶⁸Ga]Ga-DOTA-TOC, [¹¹¹In]In-DTPA-octreotide, [¹⁸F]FDG, and [^99mTc]-MDP. It is worth mentioning that the examination time is considerably shorter for ⁶⁸Ga-agents as compared to SPECT agents and somewhat shorter compared to [¹⁸F] FDG.

The benefits of ⁶⁸Ga can be summarized as follows. It is produced from a long shelf-life and cost-effective generator. The half-life of ⁶⁸Ga permits production and application of resultant agents, and the labelling synthesis is amenable to automation and kit type preparation. It provides sufficient levels of radioactivity for high quality images, short scanning time (fast patient examination) while minimizing the radiation dose to the patient and personnel, and allows fast dischargement of the patient. It also allows repetitive examinations within the same day ²⁴. The majority of the therapeutic radionuclides is also metals and might allow for the theranostic development.

In comparison with the other radionuclides mentioned above there are a number of advantages and disadvantages, and they all complement each other to make PET imaging applicable to a broad range of diseases. ^99mTc/SPECT has been holding the leading position in nuclear medicine for decades due to such factors as favorable gamma energy, availability from a generator system and kit type radiopharmaceutical production. The future of ⁶⁸Ga is predicted as PET analogue of ^99mTc/SPECT with added value of higher sensitivity, resolution as well as accurate quantitation and personalized medicine. However, even if the vision of ⁶⁸Ga becoming as immense as ^99mTc will not get fulfilled, ⁶⁸Ga has definitely strong contribution to make in the improvement of personalized patient management and there is a niche for ⁶⁸Ga-based agents in nuclear medicine. Moreover, ⁶⁸Ga-agents have the prerequisites to substitute also ¹¹¹In-based radiopharmaceuticals.

Biomarkers, targets, and ligands

Development of agents for imaging and radiotherapy involves identification of biological process and target underlying the pathology as well as respective lead compound. Then the radioactive lead compound counterpart must be designed, chemically characterized as well as preclinically and clinically validated. In particular, the specific targeting and pre-targeted imaging require information on biomarkers and is dependent on their discovery. Thus, advances in biological research and biotechnology are crucial. Proteomics and genomics considerably contribute to the expansion due to the increasing knowledge and access to the vectors and targets such as receptors, enzymes, antigens as well as their ligands and substrates. Proteins demonstrate remarkable capability of molecular recognition. Advances in genetic and biochemical techniques resulted in a large number of antibody radioimmunotherapeutics. That in turn triggered further development with the objective to overcome the drawbacks related to antibody high molecular weight, slow pharmacokinetics and clearance that cause high radiation dose to normal tissue and poor image contrast. Thus, large libraries of high affinity small proteins have been created using combinatorial engineering and phage display techniques that provide efficient screening of ligands as well as identification and selection of antibodies and receptors for drug discovery and therapy. A number of non-immunoglobulin scaffolds has also been studied that resulted in libraries as, for example Affibody^® molecules originating from Z domain scaffold of staphylococcal protein A. Thirteen surface-exposed residues of the scaffold were randomized resulting in a library (3x10⁹ members) of high-affinity binders to various targets ³⁹. Radiolabelled Affibody molecules have extensively been investigated preclinically, and ⁶⁸Ga- and ¹¹¹In-labelled analogues with high affinity to HER2 receptors up-regulated in breast cancer has also been studied in patients ⁴⁰. This is a strong evidence for the future fruitful development of engineered high affinity proteins and abundant source of ligands to various receptors expressed in diseased tissues.

G-protein and G-protein coupled receptor discoveries, both awarded with Nobel Prize, respectively in 1994 and 2012, provided basis for the important and most explored class of imaging agents comprising small regulatory peptides that are involved in many metabolic processes in almost all organs ^41-43. It should be mentioned that one of the factors that tremendously contributed to the progress of ⁶⁸Ga applications was the advent of agonist somatostatin ligand analogue (DOTA-D-Phe¹-Tyr³-Octreotide, (DOTA-TOC)). The current development is directed to pansomatostatin and antagonist analogues ⁴⁴. Other examples of peptide receptor targets in various cancer types are: gastrin-releasing peptide (GRP) receptors in prostate, ovarian and urinary tract cancers; neuropeptide Y receptors in breast cancers; glucagon-like peptide 1 receptors (GLP-1) in benign insulinomas and islet cells; vasoactive intestinal peptide receptor (VPAC-1) in breast, prostate cancer; cholecystokinin 2 receptors (CCK) in medullary thyroid cancers; neurokinin-1 receptor in glioblastoma, breast cancer; melanocortin-1 receptor in melanomas; chemokine receptor 4 (CXCR4) in lung, breast, prostate cancer; neurotensin (NT) receptor in SCLC, colon, breast, prostate cancer. Corresponding peptide ligands such as bombesin, CCK/gastrin, exendin, α-MSH, neurotensin, substance P labelled with ¹⁸F, ⁶⁸Ga, ^99mTc or ¹¹¹In have been studied clinically ^45-50. Even though just few of them were labelled with ⁶⁸Ga, it is a solid background and springboard for ⁶⁸Ga-based analogues to come in the nearest future. Many more studies have been conducted preclinically and it is worth mentioning that the total critical mass of basic knowledge and experience gained until now will lead to the explosively growing clinical studies with ⁶⁸Ga. The design of an imaging agent is a complex process, and although the major factors that influence the biological function of a probe such as peptide/protein sequence and 3D structure, pharmacokinetic modifiers, chelators, metal cations are known, it is not that straightforward to predict the pharmacokinetics and pharmacodynamics of the candidate agent ⁵¹.

Thus fundamental research in biology of surface receptors and antigens, enzyme activity, transport systems, proliferation, apoptosis, hypoxia, glycolysis, and angiogenesis provides invaluable information and lays a basis for imaging agent development.

PET radiopharmaceutical regulations

The absence of legislation and regulations specific to PET radiopharmaceuticals made it difficult to conduct clinical trials and introduce new imaging agents into clinical routine however the situation has improved during last years. It required considerable effort from academic, clinical and patient communities and societies, and the hard work has started giving results. Recent developments indicate possibility of regulatory specific solutions that may allow the clinical use of small scale preparation radiopharmaceuticals without obligation to apply for manufacturing authorization or clinical trial ⁵².

One of the major difficulties was that regulatory bodies evaluated therapeutic and imaging agents by the same process and put forward the same requirements, however there has been an improvement such as the recognition of the microdosing concept ^53-55 by EMEA and FDA, and introduction of the Exploratory Investigational New Drug (eIND) guidelines that reduces the demand on toxicity studies and respective cost burden ^{56, 57}. This is possible because of the high sensitivity of PET and consequently use of nonpharmacological radiopharmaceutical doses of picomoles (nanograms-micrograms). It should also be mentioned that commonly the adverse reactions to PET radiopharmaceuticals are extremely rare and with no serious or life-threatening events ³¹.

The recommendations on compliance with regulatory requirements for radiopharmaceutical production in clinical trials have been discussed for Europe and the USA ^{58, 59}. A comprehensive overview of relevant current EU documentation concerning requirements to the quality of starting materials and final drug products/radiopharmaceuticals as well as to the preparation procedure has been published with references to specific directives and regulations ^{52, 58, 60}. Another guidance is more specific and covers Part B of the EANM “Guidelines on Good Radiopharmacy Practice (GRPP)” for small-scale “in house” production of radiopharmaceuticals that are not intended for industrial production, sale or distribution ⁶¹. European pharmacopoeia monograph on compounding of radiopharmaceuticals that includes chapter dealing with standards of the extemporaneous preparation of radiopharmaceuticals has been prepared and submitted to the authorities.

Recommendations on the patient examination protocols, interpretation and reporting of the results have been summarized as guidelines for the assistance to nuclear medicine physicians ^{62, 63}. Two European Pharmacopeia monographs are in the preparation and publication, “Gallium (⁶⁸Ga) edotreotide injection” ⁶⁴ and “Gallium (⁶⁸Ga) chloride solution for radiolabelling”. One of the critical quality parameters is the breakthrough from the ⁶⁸Ge/⁶⁸Ga generator of long-lived parent radionuclide, ⁶⁸Ge (t_½=270.8 d) and its content in the radiopharmaceutical preparation of not more than 0.001% may restricts the use of the generators and limit clinical applications of ⁶⁸Ga specifically with regard to kit type preparation. The limit defined in the European Pharmacopeia monographs was based on a hypothetical assumption of total accumulation of ⁶⁸Ge(IV) radioactivity in the bone marrow with an infinite retention. However, ⁶⁸Ge(IV) biodistribution studies conducted currently in rats with extrapolation to the human organ and whole-body radiation dosimetry demonstrated that the limit defined in the monograph could be increased at least 100 times without compromising patient safety ⁶⁵. If the ⁶⁸Ge limit is increased, then together with the labelling methods of high selectivity towards ⁶⁸Ga at room temperature, it will open possibility for kit type production at radiopharmacy practice and consequently even wider use of ⁶⁸Ga-based agents. There are no labelling kits for the preparation of ⁶⁸Ga-based radiopharmaceuticals with marketing authorization available currently, and it is a matter of years before they appear on the market.

One more crucial reason postponing the clinical introduction of ⁶⁸Ga-agents was the scarcity of generators, their quality, and absence of corresponding regulations. However currently at least four different generators are commercially available and more are under development. Some of the manufacturers (Eckert & Ziegler; iThemba Labs) hold license for good manufacturing practice (GMP) production and the generators of pharmaceutical grade are on the way (Eckert & Ziegler). The draft monograph, “Gallium (⁶⁸Ga) chloride solution for radiolabelling”, regulates the requirements to the quality and safety.

In order to accelerate the use of PET for assessing treatment responses, standardization and consolidation of image acquisition, interpretation, and quantitation criteria that have been considered in Europe ^{52, 66, 67} and the USA ⁶⁸ are required to be introduced into practice ¹⁸. International Atomic Energy Agency conducted survey of the status of nuclear medicine in developing countries and localized the areas requiring international cooperation and help in order to improve the level of use and the quality as well as promote global standardization, growth, and dissemination ⁶⁹.

There is an ongoing dispute on the benefits of PET-CT in patient management conducted by national authorities. The validity of such review judgments of health technology assessment (HTA) agencies was questioned ⁷⁰. The authors investigated HTA reviews (1996-2010) and found them misleading and that in many countries they unfortunately discredited evidence of PET and PET-CT capability to improve patient outcomes. However, original conclusions of clinicians have proven the opposite, and access to this technology though was delayed it could not be prevented.

In summary, the regulation and legislation of the manufacturing of PET radiopharmaceuticals and in particularly ⁶⁸Ga-related documentation has been defined during current years reflecting the acceptance of obvious benefits of ⁶⁸Ga/PET-CT to the patient management. Interestingly the analysis of various reasons causing performance inefficiency of nuclear medicine centres in developing countries revealed that regulatory hinder was the major problem only at 5% of the centres ⁶⁹. The USA had a 10 y delay in implementing human studies with ⁶⁸Ga-labelled somatostatin analogues as compared to Europe due to mainly poor availability of generators, uncertainty about intellectual property of the ligands and regulation of manufacturing; however regulatory uncertainty and generators are less of a problem currently in the USA ⁷¹. There are at least three sites, University of Iowa, Vanderbilt University, and Excel Diagnostics & Nuclear Oncology Center holding approval from FDA for the use of ⁶⁸Ga-labelled somatostatin analogues in the management of patients with NETs. Peptide based precursors of GMP grade for labelling with ⁶⁸Ga are becoming available commercially which will most likely accelerate the introduction of respective imaging agents into clinical environment. Nevertheless currently the facilitation of the entry of novel radiopharmaceuticals into clinical practice relies mostly on magisterial preparation/prescription and compassionate use under responsibility of the prescribing physician.

Automation of the labelling procedures

Automated synthesis reduces the radiation exposure to the operator, improves robustness of the production as well as provides on-line documentation of the manufacturing process thus improving GMP compliance. Considerable number of semi-automated and fully automated devices with either stationary tubing system or disposable cassettes for the automated production of radioactive probes is available on the market as well as built “in house” ^72-85. They offer application of three most common methods: fractionation and preconcentration/prepurification of the generator eluate using either cation or anion exchange resins. Synthesizers based on the disposable sterile cassettes and devoted to the production of ⁶⁸Ga-comprising imaging agents are most preferred in routine clinical setup even though the cost in higher. They exclude the risk of cross-contamination, improve the reproducibility and reduce the radiation burden upon the operator. Both hardware and software should comply with GMP and good automated manufacturing practice 5 (GAMP5). The automation may provide possibility for the harmonized and standardized multicentre clinical studies that in turn would accelerate the introduction of new radiopharmaceuticals as well as their regulatory approval. This in turn will motivate investments into the research and development of novel ⁶⁸Ga-based radiopharmaceuticals.

Chemistry of ⁶⁸Ga-based imaging agents

Above mentioned interrelated aspects are critical and prepare soil for the justification and stimulation of the development of new imaging agents which is an expensive and time consuming process. However, the influence is two way and the development of these aspects is in turn motivated by the advent of new agents. Chemistry is a driving force of the development of nuclear medicine and hundreds of various imaging agents have been synthesized and preclinically evaluated.

The labelling with radiometals can be direct or chelator-mediated (tagged). The direct ⁶⁸Ga-labelling of macromolecules is limited and applies to proteins (e.g. lactoferrin, transferrin, ferritin) designed by nature for iron binding thus utilizing chemistry similarity of Ga(III) and Fe(III) ⁸⁶. The direct ⁶⁸Ga-labelling and formation of low molecular weight complexes is commonly employed for the development of imaging agents for perfusion or for imaging of biological processes where the agent uptake is defined by its charge, lipophilicity, and size. Particulate agents can also be produced by the direct ⁶⁸Ga-labelling either by co-precipitation (e.g. macroaggregated albumin) or by co-condensation (e.g. ⁶⁸Ga-carbon nanoparticles) ^87-92. The chelator mediated ⁶⁸Ga-labelling, requiring first synthesis of a bioconjugate comprising vector molecule and chelate moiety for the coordination of the radiometal ion, is the most common pathway of imaging agent design. The principle components of such agents are targeting vector, chelator, and radionuclide (Figure ​(Figure3A).3A). The modulation of pharmacokinetics, biodistribution, and stability can be achieved by the introduction of pharmacokinetic modifiers (PKM) such as hydrocarbon chain, polyethylene glycol (PEG), carbohydrate, and polypeptide chain. PKM may also serve as linker/spacer between the bulky chelate moiety and the active site of the vector molecule. Thermodynamic and kinetic stability, geometry and lipophilicity of a chelator-metal ion complex are important parameters in the development of radiometal based radiopharmaceuticals.

These design principles are valid for the ⁶⁸Ga-labelling of small biologically active organic molecules, biological macromolecules, complexes of variable charge and lipophilicity as well as particles. Thus the chemistry considered in the development of ⁶⁸Ga-based imaging agents has several cornerstones: aqueous chemistry of ⁶⁸Ga, coordination chemistry of Ga(III), design of mono- and bifunctional chelators, and bioconjugate chemistry. As mentioned above most of ⁶⁸Ga imaging agents known by now are tagged which means that their biodistribution is defined by the vector molecule and ⁶⁸Ga may or may not influence biodistribution or affinity to the target. Such are agents for targeting and pre-targeted imaging of receptors, enzymes, antigens, and transporters.

Coordination chemistry and design of chelators

The hard acid Ga(III) can form four-, five-, and six-coordinated complexes. The latter are the most stable with octahedral coordination sphere. Oxygen, nitrogen, and sulfur donor atoms form stable coordinate bonds with Ga(III). The examples of the most common functional groups are amine, carboxylate, hydroxamate, phenolate ². The coordination reaction requires buffering for two reasons: first, to ensure right pH for the deprotonation of electron donor atoms; second, to weekly coordinate and maintain Ga(III) in solution that might otherwise form Ga(OH)₃ and precipitate at pH 3-7 ². Various buffers such as sodium acetate, 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) buffer, succinate, formate, tris, glutamate, lactate, oxalate and tartrate have been investigated ^{75, 99}. Although HEPES is biologically compatible buffer ^{24, 25} and provides high radioactivity incorporation (>95%) and specific radioactivity ^{75, 99, 111}, it is not listed in pharmacopoeia and thus prior to clinical use the radiopharmaceutical should be purified from it and additional quality control must be conducted to ensure that HEPES does not exceed the limit. The use of sodium acetate has the advantage of being eligible for human use possibly simplifying the regulatory approval of the radiopharmaceutical as well as feasibility to omit additional quality control tests ⁷⁵.

As mentioned above coordination chemistry is one of the cornerstones of the development of ⁶⁸Ga-based imaging agents and thus advances in mono- and bifunctional chelator design is of paramount importance. The topic has recently been reviewed elsewhere ^{2, 112} and here only the fundamental and recent advances that shape the future of the field are discussed. The basic structures that have been most thoroughly studied are polyaminopolycarboxylate, hydroxyaromatic, macrocyclic and amine-thiol type ligands. The principle requirements are that they should form stable complexes with Ga(III) favourably of octahedral geometry in order to yield stable complexes. The association kinetics must be fast and reaction desirably taking place at room temperature while dissociation kinetics must be very slow ². Charge and lipophilicity can be adjusted dependent of the application. Two classes of chelators namely open chain and macrocyclic have been considered. Although the former commonly formed insufficiently stable complexes the interest towards them still remains with the desire for fast complexation kinetics at room temperature that would like NOTA ¹¹³ provide possibility for kit type preparation of radioparmaceuticals and labelling of temperature labile molecules.

Several open-chain chelators such as hydroxyaromatic diamines like N,N'-di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED), pyridoxylethylenediamines such as N,N'-dipyridoxyethylenediamine-N,N'-diacetic acid (PLED), mono- and bifunctional open chain chelators based on [tris(aminomethyl)ethane] (TAME) formed complexes with improved stability ². However, neither of them or four-, five- and six-coordinate amino-thiol (N₂S₂, S₃N, 4SS, 5SS, 6SS, N₃S₃) based open chain chelators ² have found further application. There have been just few publications reporting on open-chain chelators and not all of them demonstrated desired properties. ⁶⁷Ga-labelled tripodal 3-hydroxy-4-pyridinone (1, NTP (PrHP)₃, Figure ​Figure4)4) showed in vivo stability and fast renal excretion in healthy rats ¹¹⁴. Similar chelator, tripodaltris(hydroxypyridinone) (YM103), was functionalized with maleimide for the conjugation to proteins via Cys ¹¹⁵. The efficient and stable ⁶⁸Ga-labelling occurred at room temperature. H₂dedpa (N₄O₂), and its bifunctional derivatives containing amine, pyridine and carboxyl moieties (H₂dedpa (3), dedpa-1 (4), dedpa-2 (5), Figure ​Figure4)4) were stably labelled with ^67/68Ga at room temperature (SRA~360 MBq/nmol) using 0.1 µM chelate ¹¹⁶. While HBED possessing hydroxybenzyl and amine groups ¹¹⁷ showed low labelling efficiency and slow blood clearance. The isothiocyanato derivatives of the H₂dedpa (H₂dp-bb-NCS (6), H₂dp-N-NCS (7)) have been synthesized and conjugated to c(RGDyK) resulting in monomer and dimer ¹¹⁸. The uptake of monomer was higher than that of dimer in RAG2M xenografts. However, very slow clearance from blood requires further improvement of pharmacokinetic properties.

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Fig 4

Basic structures of open chain mono- and bifunctional chelators.

An exhaustive number of triazacyclononane (TACN (8)) and tetraazacyclododecane (TACD (9)) (Figure ​(Figure5)5) derivatives have been synthesized. The backbone and pendant arms were functionalized for the conjugation to vector molecules and in order to modulate complexation kinetics, charge, lipophilicity and stability of the complex as well as biodistribution, pharmacokinetics, excretion pathways and blood clearance rate. The pendant arm modifications include such functional groups as carboxylic acid, phosphinic acid, α-haloacetyl, alkoxy, alkyl- and arylamine, alkyl- and aryl sulphide, phenol, hydroxamate. A number of TACN based molecules functionalized with 3-hydroxy-4-pyrone pendant arms (H₃NOKA (10)), with carboxylic pendant arms (NOTA (11)) and its various derivatives, namely, 1,4,7-tris(2-mercaptoethyl)-1,4,7-triazacyclononane (TACN-TM (12)), 1,4,7-triazacyclononane-1-succinic acid-4,7-deacetic acid (NODASA (13)), 1,4,7-triazacyclononane-N,N′,N′′-tris(methylenephosphonic) acid (NOTP (14)), 1,4,7-triazacyclononane-N,N′,N′′-tris(methylenephosphonate-monoethylester) (NOTPME (15)) (Figure ​(Figure5)5) demonstrated similar plasma and in vivo stability. NOTA and its bioconjugates showed efficient chelation (>95%) of ⁶⁸Ga at pH 3.5 and room temperature within 10 min ^{113, 119, 120}. Mechanistic studies of the unexpectedly fast complexation kinetics at such low pH suggested that the transchelation step from the buffer to NOTA involved protonation of the buffer and decoordination that lead to the final Ga-NOTA product ¹²¹. The room temperature is advantageous for the labelling of fragile molecules as well as “shake and shoot” type kit production. Triazacyclononane with either hydroxybenzyl or hydroxypyridyl pendant arms at the nitrogens (TACN-meHP (16), TACN-TX (17), TACN-HP (18), TACN-HB (19), TACN-TM-Bn (20) Figure ​Figure5)5) were synthesized in order to increase the lipophilicity of gallium complexes and enable the blood brain barrier penetration ². The resultant complexes were stable however did not serve the purpose.

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Fig 5

Examples of TACN and TACD based mono- and bifunctional chelators.

The most promising and thoroughly investigated group of chelators is based on TACN and functionalized with phosphinic acid pendant arms. In particular, chelates with basic structure of N,N′,N′′-trisubstitutedtriazacyclononane with methyl(2-carboxyethyl)phosphinic acid pendant arms (PrP9 or TRAP-Pr (21)) were synthesized for the fast complexation with ⁶⁸Ga ¹²². The labelling was possible at ambient temperature and as extreme pH as 1. The complexes were thermodynamically stable (logK=26.24) and kinetically inert. TRAP-Pr was activated with propargylamine for the further conjugation with cyclo(RGDfK) at each pendant arm of the chelate resulting in RGD-trimer ¹²³. The in vivo performance of [⁶⁸Ga]Ga-TRAP-(RGD)₃ was evaluated in athymic nude mice bearing M21 (α_vβ₃-positive) and M21L (low expression of α_vβ₃) human melanoma xenografts. The uptake could be fully blocked by excess of unlabelled precursor. Another derivative of TRAP possessing just one site for bioconjugation (NOPO) was conjugated with cyclo(RGDfK) and NOC and labelled with ⁶⁸Ga with as high SRA as 5.6 GBq/nmol ¹²⁴. The uptake of [⁶⁸Ga]Ga-NOPO-RGD and [⁶⁸Ga]Ga-NOPO-NOC respectively in M21 and AR42J mouse xenografts was specific. NOPO demonstrated highly selective complexation with Ga(III) as compared to Fe(III) and Zn(II) ¹²⁴. The higher complexation selectivity of TACD derivatives for Ga(III) as compared to In(III) and Al(III) has also been demonstrated using DOTA-TOC ²⁵. A comprehensive and thorough investigation has been conducted on the influence of Zn(II), Cu(II), Fe(III), Al(II), Ti(IV), and Sn(IV) on the incorporation of ⁶⁸Ga(III) into NOTA, DOTA (22), TRAP, TRAP-Pr, and NOPO as well as their peptide conjugates ¹²⁵. The selectivity of TRAP, TRAP-Pr, and NOPO towards Ga(III) was considerably improved as compared to NOTA and DOTA. This is particularly very critical with regard to Fe(III) and Zn(II). The structural investigation of the underlying mechanism of the coordination revealed formation of various complex diastereoisomers ¹²⁶. The presence of phosphinic acid pendant arms improved the ligand coordination ability and resulted in fast complexation in acidic media. The complexes were kinetically inert in acidic as well as basic solutions. The broad range of pH at which the complexation reaction can take place would allow avoiding the pH 3-7 when the Ga(III) is insoluble and thus omit the use of buffers that contribute to cation contamination. This might improve the complexation efficiency and thus SRA. In addition the stability constant for [⁶⁸Ga]Ga-DOTA was recently revised and demonstrated to be even higher (logK = 26) than previously known (logK = 21.33) ¹²⁷.

Four bifunctional macrocyclic chelators have been investigated with the objective to compare their labelling chemistry and in vivo stability and clearance ¹²⁸. Nine and twelve member rings were considered, namely 1,4,7-triazacyclononane-1,4,7-triacetic acid (p-NO₂-Bn-NOTA (23)), 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (p-NO₂-Bn-Oxo (24)), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-NO₂-Bn-DOTA (25)) and 3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15), 11,13-triene-3,6,9-triacetic acid (p-NO₂-Bn-PCTA (26)) (Figure ​(Figure5).5). The labelling was more efficient for p-NO₂-Bn-NOTA, p-NO₂-Bn-PCTA, and p-NO₂-Bn-Oxo as compared to p-NO₂-Bn-DOTA. However, p-NO₂-Bn-Oxo was unstable in vivo. p-NO₂-Bn-NOTA cleared rapidly from the blood and muscle but had 5-fold higher uptake in kidneys.

A novel approach providing a chelator, protoporphyrinIX (PPIX), that can be used both for in vitro fluorescence microscopy and in vivo PET or SPECT imaging has an advantage of using exactly the same molecule ¹²⁹. PPIX was conjugated to RGD peptide sequence and ⁶⁸Ga-labelled under microwave heating. The resulting probe was evaluated in MDA-MB-435 cancer cell line expressing integrin receptors, and demonstrated binding specificity. Porphyrins specifically accumulate in tumour tissue and five various ⁶⁸Ga-labelled analogues were suggested as imaging agents ¹³⁰. Preliminary results indicated accumulation in DS sarcoma tumour in rat, however the mechanism of the uptake requires further investigation ¹³⁰.

Another class of macrocyclic chelators is bridged or macrobicyclic ¹³¹. The structure of [Ga-(1-NH₃-8-NH₂-sar)]⁴⁺ (where sar =3,6,10,13,16,19-hexaazabicyclo[6.6.6.]icosane) was determined by X-ray crystallography as distorted octahedral with six nitrogen atoms. The chelator was coupled to two RGDfK moieties. The resulting conjugate was labelled with ⁶⁸Ga at elevated temperature (85 °C). The specific uptake was observed in vivo in 66c14β3 xenografts in mice.

In summary, even though open chain chelators serve the aim of rapid chelation at room temperature they have it difficult to compete with macrocyclic chelators based on TACN and TACD that provide stability, selectivity, fast association kinetics and extremely slow dissociation kinetics as well as high thermodynamic stability. It should also be mentioned that the advent of macrocyclic chelators was one of the critical factors influencing the broader demand for ⁶⁸Ga and its versatile applications. The current improvement in macrocyclic design is substantial, and mechanistic as well as structural investigation of coordination chemistry provides knowledge for more efficient and diverse imaging agent development and radiopharmaceutical routine production. The development of mono- and bifunctional chelators will direct towards fast complexation and room temperature, broad working pH range and high SRA. Although TACN derived chelators offer an advantage over TACD with respect to fast complexation kinetics at room temperature and higher selectivity there are more aspects to take into consideration such as theranostics applications where it is desirable that both diagnostic and therapeutic radionuclides can be coordinated by the same chelator moiety.

Current status and directions of ⁶⁸Ga-based imaging agent development

⁶⁸Ga-based imaging agents comprising small molecules, large biomolecules, and particles have been explored for the imaging and quantitation of various physiological disorders and biological functions. The molecular imaging of oncological diseases have been investigated most extensively targeting receptors (e.g. G-protein coupled receptor family and human epidermal growth factor receptor (HER) family, folate and urokinase receptors), enzymes, antigenes as well as visualizing downstream biological processes such as angiogenesis, hypoxia, proliferation, apoptosis, glycolysis. Probes for non-targeted imaging of pulmonary and myocardial perfusion and ventilation as well as targeting imaging of inflammation, infection, and mRNA have also been considered and fundamental exploration is ongoing.

Oncological application

Various approaches have been developed for the imaging of certain biological processes involved in cancer diseases with receptor imaging being most thoroughly investigated. There are currently few ⁶⁸Ga-based imaging agents in routine clinical practice and clinical studies however the number of applications is increasing with acceleration. Somatostatin analogues are already established in clinical routine. Imaging feasibility of GRPR, GLP-1R, MSH, HER2 as well as prostate-specific membrane antigen, osteoblastic bone metastases, amino acid uptake, glucose transport, angiogenesis has been clinically demonstrated in patients ^{7, 40, 141-145}. Extensive basic research is conducted on the development of ⁶⁸Ga-based probes for the imaging of CCK, CXCR, MSH, gonadotropin releasing hormone (GnRH), NT, folate, integrin, vascular endothelial growth factor (VEGF), urokinase receptors as well as enzymes, antigens, and multidrug resistance (MDR1) P-glycoprotein (Pgp). Imaging probes for visualizing such processes in tumours as proliferation, apoptosis, hypoxia, glycolysis, and angiogenesis as well as bone metastases have also been investigated. A number of particulate agents have been developed for the delivery of imaging reporters or therapeutic agents to the tumour target.

Imaging of G-protein coupled receptor family

Somatostatin receptor imaging

The expression of SSTRs has been found in neuroendocrine tumours, small cell lung cancer, renal cell carcinoma, malignant lymphoma, breast cancer, and prostate cancer. PET-CT using somatostatin ligand analogues labelled with ⁶⁸Ga has become a new golden standard in imaging of NETs with specificity and sensitivity well above 90% and advantages over conventional radiologic and scintigraphic imaging ^{14, 146-149}. It is the most pronounced example of theranostics ^{6, 7, 150}.

Extensive basic research has been conducted on the development and biological validation of analogues varied in peptide sequence, size and number of peptide rings, chelator (DFO, DTPA, DOTA, NOTA, and their derivatives) and radiometal (Ga, Y, Tc, In, Lu) type. Their receptor binding affinity, internalization and biodistribution have been shown to be dependent on the chemical modifications ². Fast tumour localization, blood clearance, and renal excretion are typical characteristics of clinically used [⁶⁸Ga]Ga-DOTA-TATE, [⁶⁸Ga]Ga-DOTA-TOC, [⁶⁸Ga]Ga-DOTA-1-Nal³-octreotide ([⁶⁸Ga]Ga-DOTA-NOC). Structure activity relation studies allowed fine tuning for the agent properties such as receptor affinity, in vivo stability, biodistribution, pharmacokinetics, excretion pathway, and kidney uptake, and pharmacological activity ^{138, 151-155}.

Consistently higher uptake of antagonist SSTR ligand as compared to agonist counterpart was found in forty-eight SST2-positive human tumour frozen sections in vitro ¹⁵⁶. Similar peptide sequences, p-Cl-Phe-cyclo(D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys)D-Tyr-NH₂ (where d-Aph(Cbm) is d-4-amino-carbamoyl-phenylalanine) (LM3), p-NO₂-Phe-cyclo(D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys)D-Tyr-NH₂ (JR10), and Cpa-cyclo(D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys)D-Tyr-NH₂ were conjugated to DOTA and NODAGA ^{157, 158}. The agents demonstrated antagonistic properties with higher affinity of NODAGA counterparts. Clinical case report with ¹¹¹In-lablled SST analogues demonstrated higher detection rate for the antagonist counterpart ¹⁵⁹. This fact opens possibility for the use of SST analogues not only for the diagnosis of neuroendocrine tumours but also breast carcinomas, renal cell carcinomas, non-Hodgkin lymphomas that express SSTR2 to lesser extent.

The feasibility of quantitation of SSTR density has been demonstrated both preclinically ^{25, 160} and clinically ²⁴ using [⁶⁸Ga]Ga-DOTA-TOC. Although a ten-fold higher affinity for the SSTR₂ had been demonstrated for DOTA-TATE as compared to DOTA-TOC in vitro in transfected cell cultures ¹⁶¹, no statistically significant difference between [⁶⁸Ga]Ga-DOTA-TOC and [⁶⁸Ga]Ga-DOTA-TATE uptake could be observed in vitro in monkey brain tissue sections or in vivo in rat organs expressing SSTRs ( pituitary, adrenal, pancreas) ¹⁰³. Moreover, clinical study involving 40 patients did not verify the 10-fold higher affinity for the SSTR₂ of [⁶⁸Ga]Ga-DOTA-TATE, on the contrary, standardized uptake value (SUV_max) of [⁶⁸Ga]Ga-DOTA-TOC tented to be higher ¹⁶². Another aspect investigated preclinically is the influence of treatment with octreotide and interferon-a (IFNa) used for biotherapy of NETs on the uptake of [⁶⁸Ga]Ga-DOTA-TATE. The exposure of the animals to the cold octreotide did not enhance the uptake of the tracer while IFNa did, however the mechanism of the observation was not clear ^{163, 164}. Gene therapy protocols were optimized on the basis of in vivo imaging ([⁶⁸Ga]Ga-DOTA-TATE) of gene expression and quantitative monitoring of gene transfer ¹⁶⁵.

[⁶⁸Ga]Ga-DOTA-TOC (27), [⁶⁸Ga]Ga-DOTA-TATE (28) and [⁶⁸Ga]Ga-DOTA-NOC (29) (Figure ​(Figure6)6) are the most commonly used analogues in clinical studies ^{2, 166, 167}. Their pharmacokinetics, blood clearance and target localization rate are compatible with half-life of ⁶⁸Ga. Renal excretion, short scanning time, high sensitivity and resolution assure high contrast and quality images over organs of interest as well as accurate quantitation. Relatively low radiation dose is one more advantage that should be mentioned. They served for diagnosis, staging, prognosis, therapy selection and response monitoring of NETs and other types of cancers and diseases. [⁶⁸Ga]Ga-DOTA-TATE was compared with [⁶⁸Ga]Ga-DOTA-NOC in 20 patients in terms of detection rate and SUVs ¹⁶⁸. The agents had comparable diagnostic accuracy with higher SUV_max for the former. One more analogue, [⁶⁸Ga]Ga-DOTA-2-Nal, Tyr³, ThrNH₂⁸-octreotide (DOTA-lanreotide, DOTA-LAN) was successfully used for lung and thyroid tumour detection ¹⁶⁹.

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Fig 6

Structural formulae of the clinically used somatostatin analogue imaging agents. TOC stands for D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr(OH); TATE stands for D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr; and NOC stands for D-Phe-Cys-Nal-D-Trp-Lys-Thr-Cys-Thr(OH). The differences in structures are highlighted.

The individualized diagnosis has been practiced in the selection of patients for PRRT, target definition for fractionated stereotactic radiotherapy (FSRT) planning, target volume delineation for intensity modulated radiotherapy ^170-174. The diagnosis on the cellular and molecular level and determination of the disease associated biomarkers provides basis for the treatment optimization and efficacy for a particular patient ¹⁷⁵. The personalized therapy planning necessity was demonstrated in retrospective study of ten patients examined with [⁶⁸Ga]Ga-DOTA-TATE ¹⁷⁶. It was concluded that the radiotherapeutical dose should be determined by the tumour burden since the latter influences the radioactivity distribution to the healthy organs, and in particular the higher burden decreases the radiation accumulation in the kidney. The starting point of PRRT after preceding cold octreotide therapy in patients with NETs could be determined ¹⁷⁷. These imaging agents improved the detection rate and diagnostic accuracy ^{178, 179}. They were used for therapy planning and monitoring response to treatment ¹⁸⁰, as well as influenced and changed the therapeutic course ^181-184.

The majority of the clinical studies have been conducted in the field of GEP NETs however considerable number of other indications has been reported demonstrating wider application possibilities. [⁶⁸Ga]Ga-DOTA-NOC was successfully used for the assessment of NETs ¹⁸⁵, well-differentiated medullary thyroid carcinoma (MTC) ¹⁸⁶, bronchial carcinoid (BC) and Von-HippelLindau (VHL) disease ¹⁸⁷, alveolar rhabdomyosarcoma with neuroendocrine differentiation ¹⁸⁸, carotid body chemodectomas (CBCs) ¹⁸⁹, idiopathic pulmonary fibrosis ¹⁹⁰. [⁶⁸Ga]Ga-DOTA-TOC was found valuable for therapy planning and accurate diagnosis of multiple endocrine neoplasia ¹⁸⁴, sudden onset of vision problems ¹⁸³, duodenopancreatic NET ¹⁸², autoimmune thyroid disease like Graves' disease and Hashimoto's disease ¹⁹¹. Patients affected by prostate cancer with bone metastases were examined with [⁶⁸Ga]Ga-DOTA-TOC ¹⁹². However, due to the low expression of SSTR₂ and SSTR₅ the uptake in the lesions was low and the signal was weak. Even broader areas were covered by [⁶⁸Ga]Ga-DOTA-TATE. Benign and malignant thrombi were distinguished in a patient affected by pancreatic NET tumour with hepatic and regional lymph node metastases ^{193, 194}. It was found relevant for the diagnosis and therapy planning for meningiomas ¹⁹⁵, recurrent medullary carcinoma ¹⁹⁶, plaque imaging and characterization in the coronary arteries ¹⁹⁷. In addition, the usefulness of ⁶⁸Ga-labelled somatostatin analogues has been discussed in the context of other available imaging techniques for staging of lung neuroendocrine cancer and therapy selection ¹⁴⁷ as well as for the evaluation of Hurthle cell thyroid carcinoma ¹⁹⁸, and neuroectodermaltumours ¹⁶⁷.

The large number of clinical applications of somatostatin ligand analogues triggers investigations that refine the examination protocols and interpretation of results. Somatostatin receptors are expressed also physiologically in healthy organs and the awareness of that is very crucial for correct interpretation of examination results, and possible causes for false negative or positive findings should be kept in mind. Distribution of [⁶⁸Ga]Ga-DOTA-TATE and organ uptake (SUV) in healthy volunteers has been studied and compared with in vitro findings ¹⁹⁹. The organs with physiological expression of SSTR₂ as well as metabolic and excretion sites (pituitary, adrenals, pancreas, spleen, thyroid, stomach wall, liver, salivary glands, bowel, pelvicalyceal system, kidneys, urinary bladder) demonstrated distinguished uptake. Forty-three patients with NETs were examined with [⁶⁸Ga]Ga-DOTA-TOC in order to distinguish pathological and physiological uptake of the head of pancreas ²⁰⁰. The uptake that was similar to that of liver was considered physiological. The findings stressed importance of accurate quantitation of the uptake for the avoidance of false-positive diagnosis. The uptake in the head of pancreas was also considered physiological in the retrospective study of 100 patients ²⁰¹ and specifically designed study of 96 patients ²⁰² since the accumulation was stable over time regardless of the uptake intensity or shape. [⁶⁸Ga]Ga-DOTA-TOC scans of 165 patients with various thyroid pathologies were analyzed ²⁰³ and in 8 cases of normal thyroids with increased uptake, follow-up examination after 6-14 months did not show any thyroid pathology. Another case study using [⁶⁸Ga]Ga-DOTA-TOC reported on the uptake in wandering spleen which could mistakenly be interpreted as malignant process instead of physiological expression of SSTR₂ in spleen in an unusual position ²⁰⁴.

The option to quantify the disease underlying processes and provide personalized diagnosis and therapy is one of the crucial advantages of PET-CT. Various methodologies and approaches are being considered with the aim to assure accuracy and unambiguity of the quantitation. The uptake of [⁶⁸Ga]Ga-DOTA-NOC measured in SUV_max correlated with pathologic features and was suggested as a prognostic tool characterizing stable disease with SUV_max cutoff value ranging from 17.9 to 19.3 ²⁰⁵. The quantitation of diagnostic procedures is getting refined. The correlation between [⁶⁸Ga]Ga-DOTA-TOC (SUV) and SST₂ mRNA was investigated in 120 patients and a novel normative database was created for the accurate quantitative evaluation. It may improve diagnostics, treatment monitoring and therapy of SST expressing tumours or inflammation on a molecular basis ²⁰⁶. In another study the evaluation of the response to the radiotherapeutic treatment was conducted using tumour-to-spleen SUV ratio which was found more accurate measure than solely SUV_max ¹⁸⁰. Treatment with cold somatostatin analogue did not reduce the binding of [⁶⁸Ga]Ga-DOTA-TATE, but improved the tumour-to-background ratio ²⁰⁷. The detection rate of pre-therapeutic [⁶⁸Ga]Ga-DOTA-TATE/PET-CT was found higher as compared to sequential planar scans with [¹⁷⁷Lu]Lu-DOTA-TATE (9% of the lesions not detected) ²⁰⁸. Most importantly the highest detection concordance was observed at later time point (72h) thus indicating the potential of [⁶⁸Ga]Ga-DOTA-TATE for pre-therapeutic dosimetry and dose planning.

The superiority of ⁶⁸Ga-labelled somatostatin analogues in terms of specificity, sensitivity, staging accuracy, detection rate, quantitation, acquisition time to [¹⁸F]-FDG ²¹⁰, [¹⁸F]F-DOPA ²¹¹, ¹²³I-metaiodobenzylguanidine (¹²³I-MIBG) ^{212, 213}, and [¹¹¹In]In-octreotide (Octreoscan®) (Figure ​(Figure7)7) ²⁰⁹, [¹⁸F]-NaF and [^99mTc]-dicarboxypropanediphosphonate ²¹⁴ has been demonstrated. Advantage of ⁶⁸Ga/PET-CT in terms of detection rate over ultrasonography, CT, MRI, and skeletal scintigraphy was confirmed in over 5800 clinical cases ⁷.

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Fig 7

A 69-y-old man with low grade metastatic midgut NET. (A) Arterial-phase CT shows multiple arterially enhancing and low-attenuation liver metastases. (B and C) Anterior and posterior whole body ¹¹¹In-DTPA-octreotide scintigraphy shows low grade (Krenning score), 1) mesenteric metastases (arrow) but no liver metastases. (D and E) Axial ¹¹¹In-DTPA-octreotide SPECT at level of spleen shows heterogeneous liver uptake with no discernable liver deposits. (F) Maximum-intensity-projection [⁶⁸Ga]Ga-DOTA-TATE PET shows multiple deposits in liver and mesentery. (G) Coronal [⁶⁸Ga]Ga-DOTA-TATE PET anterior to kidney shows multiple liver metastases. (H) [⁶⁸Ga]Ga-DOTA-TATE PET of G. (I) Axial [⁶⁸Ga]Ga-DOTATATE PET at level of spleen shows multiple liver metastases. (J) Axial [⁶⁸Ga]Ga-DOTA-TATE PET at level of spleen shows multiple liver metastases. Reproduced by permission of SNMMI from ²⁰⁹.

Further unique application is the use of hand-held gamma probe in combination with either [⁶⁸Ga]Ga-DOTA-TATE or [⁶⁸Ga]Ga-DOTA-NOC intraoperatively in order to localize metastases and primary tumours ²¹⁵. The surgery was conducted 30 min after agent injection. One of the most significant advantages of the radioguided surgery (RGS) was the differentiation of scar tissue from small tumour metastases. RGS resulted in change in the operative procedure in 56%. In addition, overexpression of SSTR has been found in patients with inflammatory bowel diseases, pulmonary diseases, rheumatoid arthritis, Sjogren's syndrome, Grave's disease ^{43, 216}.

Human epidermal growth factor receptor (HER) family

HER family receptors are overexpressed in head-and-neck, lung, breast, colorectal, ovary, and urothelial cancer cells. A number of imaging agents labelled with ⁶⁸Ga has been preclinically evaluated for the visualization of the receptors: natural peptide EGFR ligand (DOTA-hEGF) ²⁴⁶, Affibody^® molecules ^247-249, 2-helix Affibody derivative ²⁵⁰, F(ab')₂ fragment of trastuzumab ²⁵¹, as well as tyrosine kinase inhibitor ²⁵². An innovative approach where various affibody molecules were pre-administered and allowed saturation of physiological EGFR uptake prior to the injection of [^67/68Ga]Ga-DOTA-hEGF was successfully proved with the aim to improve the image contrast ²⁵³. Fast target localization, blood clearance and normal tissue wash out providing high image contrast as well as high kidney uptake are characteristic to various Affibody molecule based agents. A clinical imaging of metastatic breast cancer using [⁶⁸Ga]Ga-ABY-002 proved to be an efficient method for the non-invasive determination of HER2 status of metastases not amenable to biopsy ⁴⁰. Two-helix Affibody derivative ([⁶⁸Ga]Ga-DOTA-MUT-DS) ²⁵⁰, Herceptin fragment ([⁶⁸Ga]Ga-DOTA-F(ab')₂) ²⁵⁴, camelid heavy-chain antibody variable domain based ligand ([⁶⁸Ga]Ga-Df-Bz-NCS-7D12) ²⁵⁵ were developed from a larger molecules in order to accelerate pharmacokinetics and demonstrated compatibility with ⁶⁸Ga half-life time frame.

Pre-targeted imaging

Although the number of antibody based radioimmunotherapeutic agents is considerable, their use is limited by the slow pharmacokinetics and blood clearance that cause high radiation dose to the normal organs ²⁵⁶. In the case of imaging the achievement of optimal image contrast might require as long as 2-4 d. In order to improve the tissue penetration and facilitate clearance and excretion, the size of an antibody was reduced to antibody fragments (F(ab')₂, Fab'), medium-sized proteins and small peptides with high affinity for the same target ^{2, 257}. Another method that has attracted strong interest is pre-targeting. It uses bispecific antibody (bsmAb) with two binding sites for the interaction with a cell target and a hapten molecule carrying radionuclide. First bsmAb is administered and after its localization at the target, clearance from the blood, and washout from the normal tissue, the radiolabelled hapten molecule is administered. The latter has fast pharmacokinetics so that high contrast imaging is possible within short time. Hapten molecules comprising histamine-succinyl-glycine (HSG) motif and chelate moiety (30) (Figure ​(Figure8)8) ^258-261 have been used for the imaging of carcinoembryonic antigen (CEA) pre-targeted with anti-CEA bsmAb ^262-264.

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Fig 8

The principle of pre-targeting: A) An example structure of peptide hapten molecule containing histamine-succinyl-glycine residues and coupled to NOTA chelate moiety complexed with ⁶⁸Ga (***); B) Bispecific antibody with two antigen binding F(ab')2 arms (*) and one anti-hapten binding F(ab) arm (**) interacts with the target antigene (CEA). ⁶⁸Ga-labelled hapten molecule (***) binds to the bispecific antibody (**) on the next step.

Transplantation of islets of Langerhans is a promising treatment for type 1 diabetes mellitus. Avidin-covered agarose resin microbeads were used as a model for islet transplantation in mice ^{265, 266}. After intraportal transplantation they were visualized in hepatic volume by [⁶⁸Ga]Ga-DOTA-(PEG)₂-biotin (31) (Figure ​(Figure99).

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Fig 9

Left) Structure of [⁶⁸Ga]Ga-DOTA-(PEG)₂-biotin (31) analogue used for the imaging of avidin-covered agarose resins transplanted in mice. Right) Coronal views showing total tracer uptake in left kidney (black arrows) and liver (red arrows) in transplanted (top left) and non-transplanted mice (top right). Pretreatment with unlabelled biotin displaced a majority of the tracer uptake in both transplanted (bottom left) and non-transplanted mice (bottom right). The color bar indicates SUV ranging from 0 to 6. All images are summations of the initial 20 minutes following tracer administration. Reproduced from ²⁶⁵.

Imaging hypoxia, bone metastases, proliferation, glycolysis

Small biologically active molecules have also been tagged with ⁶⁸Ga for the visualization of hypoxia, glucose transport, cell proliferation, and bone metastases. Although most of them are currently on preclinical level however these examples demonstrate potential of ⁶⁸Ga for the diversity of imaging agents like ^99mTc-based radiopharmaceuticals. Biphosphonates ^{144, 284-287} and ⁶⁸Ga-labelled ethylene diamino-N,N,N',N'-tetrakis-methylene phosphoric acid ^{288, 289} were found useful for early diagnosis of bone metastases. An encouraging illustration is ⁶⁸Ga-labelled bisphosphonate probe (BPAMD (32), Figure ​Figure10)10) that successfully proved the concept in a clinical examination of a patient with prostate cancer. Osteoblastic bone metastases were visualized with high contrast and detection rate (Figure ​(Figure11)11) ¹⁴⁴. Analogues comprising nitroimidazole, mercaptobenzynamine or 5-nitroimidazole derivatives coupled to DOTA have been investigated for the imaging of hypoxia which is an important parameter in tumour and myocardial ischemia physiology ^290-293. The monitoring of hypoxia could contribute to improved diagnosis, prognosis, treatment planning and validation of response to therapy. DOTA- and NOTA-coupled alanine, lysine and tyrosine analogues targeting transporters for the cell proliferation visualization showed uptake in animal tumour models ^294-296. [⁶⁸Ga]Ga-DOTA-2-deoxy-D-glucosamine demonstrated higher tumour-to-organ ratio as compared to [¹⁸F]FDG ²⁹⁷.

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Fig 10

Low molecular weight vectors for the labelling with ⁶⁸Ga and subsequent imaging of bone metastases (32), folate receptors (33), MDR1 Pgp (34-35), myocardial perfusion (36-39), and necrosis (40).

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Fig 11

[⁶⁸Ga]BPAMD was injected i.v. into a patient with known extensive bone metastases of prostate cancer, and revealed intense accumulation in multiple osteoblastic lesions in the central skeleton, ribs and proximal extremities: a) coronal PET; b) sagittal PET/CT; c) ¹⁸F-fluoride PET (sagittal). Reproduced from ¹⁴⁴.

Miscellaneous imaging targets in oncology

Breast, cervical, ovarian, colorectal, nasopharyngeal, renal, and endometrial cancers express folate receptors (FR). [¹¹¹In]In-DTPA-folate is clinically used and motivates development of an imaging agent comprising ⁶⁸Ga. Folic acid has been conjugated to NOTA and DOTA (Folate (33), Figure ​Figure10)10) based chelators and resultant analogues were validated in mouse tumour models ^{306, 307}. The improvement of tumour-to-kidney ratio and protection of kidneys remains a challenging task.

An agent comprising 9 amino acid residues, DOTA and ⁶⁸Ga was developed for the detection of the receptors of urokinase-type plasminogen activator (uPAR) upregulated in gastric, colorectal, and breast cancers ³⁰⁸. Its uptake in human glioblastoma cancer xenograft mouse model could be observed, though tumour-to-background ratio was low.

Tria- and diabody derivatives of an antibody to epithelial cell adhesion molecule (EpCAM) expressed in tumours were constructed with the goal to accelerate the blood clearance and improve the image contrast ²⁵⁷. The diaboby variant labelled with ⁶⁸Ga via HBED-CC showed faster blood clearance and normal tissue washout as well as rapid tumour uptake in mice A-431 xenograft.

The response to therapy can be evaluated by monitoring expression of multidrug resistance (MDR1) P-glycoprotein (Pgp). ⁶⁸Ga-labelled hexadentate Schiff base ³⁰⁹ (MFL6.MZ (34), Figure ​Figure10)10) and [⁶⁸Ga]Ga-(bis(3-ethoxy-2-hydroxy-benzylidene)-N,N'-bis(2,2-dimethyl-3-propyl)ethylenediamine) [⁶⁸Ga]Ga-ENBDMPI ³¹⁰ (3-ethoxy-ENBDMPI (35), Figure ​Figure10)10) were used for the imaging of Pgp activity dependent on the extracellular acidosis. [⁶⁸Ga]Ga-ENBDMPI demonstrated better balance between uptake and inhibition in Pgp-positive tumour cells ³¹⁰.

Annexin A5 was used for the development of a probe for the imaging and quantitation of apoptosis in the evaluation of therapy response ³¹¹. Regiospecific coupling of the protein to DOTA maleimide was achieved via Cys² and Cys¹⁶⁵ amino acid residues thus maintaining the active binding sites intact. The resultant bioconjugate labelled with ⁶⁸Ga demonstrated statistically higher uptake after cancer therapy in mouse model of hepatic apoptosis.

Rapid progress has taken place in imaging of prostate cancer reaching clinical studies within short period of time. The urea-based inhibitor of the prostate-specific membrane antigen (PSMA) ³¹² was modified in order to adjust all components required for the strong interaction with PSMA. For one of the analogues HBED-CC was added as a lipophilic side chain to the hydrophilic pharmacophore Glu-NH-CO-NH-Lys for the interaction with the active binding site of PSMA. This analogue demonstrated enhanced internalization in LNCaP cells in vitro and high accumulation in mice LNCaP tumour xenografts. Thorough study on structure-activity relation has been performed by ³¹³. [⁶⁸Ga]Ga-HBED-CC-Lys-NH-CO-NH-Glu was taken further to the clinical study and compared with [¹⁸F]FECH ¹⁴⁵. Prostate carcinoma relapses could be clearly visualized only by ⁶⁸Ga-agent. The biodistribution in 37 patients demonstrated uptake in kidneys, salivary glands, lacrimal glands, liver, spleen, and bowel (Figure ​(Figure12)12) ³¹⁴. The lesions were detected with excellent contrast already at 1 h p.i. even at low PSMA levels.

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Fig 12

⁶⁸Ga-PSMA PET/CT of patient 22 who received the lowest dose of radiotracer (52 MBq). Red arrows point to several small lymph nodes with clearly visible pathological tracer uptake. A1 CT, A2 PET, B1 fusion of PET and CT, B2 MIP. Reproduced from ³¹⁴.

⁶⁸Ga-based agents for imaging of thrombosis, atherosclerosis, intracoronary radiation therapy, imaging agent for the diagnosis of hepatobiliary and renal disorders, mitochondrial-targeting imaging of cancer were suggested earlier ². However, to my best knowledge no related studies have been published during last five years.

Myocardial perfusion, necrosis, and lung ventilation

Imaging agents for myocardial perfusion were constructed using either small lipophilic chelators or particles. Lipophilic and cationic gallium complexes with bis- and tris(salicylaldimine) and bisaminothiolate (36, 37) (Figure ​(Figure10)10) analogues have shown myocardial uptake and retention in rodents. However, the optimization of the lipophilicity of the agents so that the higher uptake in the heart is not accompanied by the high uptake in the liver is very challenging. Highly lipophilic [⁶⁷Ga]Ga-bisaminothiolate complex was accumulated during the first-pass extraction with high uptake in the healthy heart, but not in the permanently legated coronary artery of infarct Sprague-Dawley rat heart ³²². Still the imaging quality was deteriorated by high liver uptake. Tris(4,6-dimethoxysalicylaldimin-N,N'-bis(3-aminopropyl) -N,N'-ethylenediamine (BAPEN (38), Figure ​Figure10)10) ³²³, hexadentate bis(salicylaldimine) ligands (BAPDMEN (39), Figure ​Figure10)10) ³²⁴ with variable aromatic ring substituents for lipophilicity modulation have also demonstrated hepatobiliary excretion. The distribution in rat varied dependent on the imaging agent and animal species. Four analogues of BAPEN with varied substituents on the salicyl ring and a number of salicyl rings that were labelled with ⁶⁸Ga and tested in pigs at rest and under adenosine stress did not reveal correlation with myocardial perfusion measured with [¹⁵O]water and their myocardial accumulation was not determined by perfusion ³²⁵.

Various human serum albumin (HSA) based nano- and microparticles have been developed for the lung function monitoring by measuring pulmonary and myocardial perfusion as well as vascular permeability ². The first studies on ⁶⁸Ga-labelled HSA microspheres were conducted in the 1970s but nowadays the interest is revitalized. The labelling with ⁶⁸Ga was accomplished either by co-precipitation with the commercially available macroaggregated albumin (MAA) for ^99mTc-labelling ^{88, 89} or by complexation with DOTA chelator attached to HSAM via surface lysine amino groups and p-SCN-Bn-DOTA ^{326, 327}. Simultaneous formation of MMA and its ⁶⁸Ga-labelling under microwave heating resulted in stable agent that was successfully tested in a piglet model of pulmonary embolism ⁸⁷. [⁶⁸Ga]Ga-MAA has been used to quantify spatial differences in pulmonary blood flow in rats in different postures ⁹⁰. The visualization of pulmonary thromboembolism and perfusion was conducted prior selective intra-arterial therapy using [⁶⁸Ga]Ga-MMA ⁷.

N,N'-bis(diethylenetriaminepentaacetic acid)-pamoic acid bis-hydrazide (bis-DTPA-PA (40), Figure ​Figure10)10) was labelled with ⁶⁸Ga for the visualization of necrosis that occurs in acute myocardial infarction, stroke, chronic heart failure, neurodegenerative disorders, allograft rejection, and inflammation ³²⁸.

Acute pulmonary emboli requires fast evaluation of lung ventilation distribution for accurate diagnosis. PET provides necessary prerequisites such as high resolution and quantitation. ⁶⁸Ga-labelled carbon nanoparticles in the form of inhalation aerosol ([⁶⁸Ga]Ga-GallGas) were tested in piglets with lobar obstruction or diffuse airway obstruction ⁹¹. The same animals were examined with [^99mTc]-Technegas aerosol for comparison (Figure ​(Figure13).13). The heterogeneity of the airway response in the case of bronchoconstrictive challenge was more distinguishable in the investigation with [⁶⁸Ga]Ga-GallGas. The distribution of the aerosol in the lung, alveolar space of healthy volunteers was homogeneous and without bronchial deposition ¹⁰⁵. In an intraindividual comparative study ten patients with suspected pulmonary embolism underwent conventional ventilation-perfusion (V/Q) scintigraphy and PET-CT V/Q imaging after inhalation of ⁶⁸Ga-carbon nanoparticles (“Galligas”) and administration of ⁶⁸Ga-macrosggregated albumin ⁹². PET-CT provided images of higher resolution and possibility of lung function quantitation, and thus more accurate diagnosis. The short half-life of ⁶⁸Ga enabled more flexible acquisition protocols.

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Fig 13

Lung ventilation imaging using carbon particles behaving as a “pseudo gas” for diagnosis of acute pulmonary emboli. Dual study using [⁶⁸Ga]Ga-GallGas (left) and [^99mTc]-Technegas (right) in healthy piglets demonstrating similarity in ventilation distribution (A), in piglets with lower lobar obstruction (B), and in the piglets with diffuse airway obstruction induced by infusion of methacholine (C).

Imaging inflammation and infection

The development of ⁶⁸Ga-based imaging agents of various action mechanisms for the differentiation and diagnosis of inflammation and infection started during last five years ^{77, 86, 282, 329-338}. Several peptide analogues were designed for the visualization of vascular adhesion protein-1(VAP-1) expressed on endothelial surface in inflammation and infection. A ligand to VAP-1, nine amino acid residue linear peptide conjugated with DOTA, was labelled and studied in rats with diffuse Staphylococcus aureus tibial osteomyelitis (infection), in rats with healing cortical bone defect (inflammation) demonstrating specific binding and capability to differentiate between inflammation and infection ^{329, 332}. In order to prolong the in vivo stability, peptide was coupled to PEG₄ spacer ³³³. The resulting agent demonstrated higher stability and target-to-background ratio. The renal excretion was modulated also by PEG modification introduced to another ligand to VAP-1 (cyclic 17 amino acid residue peptide; CARLSLSWRGLTLPSK) ^{331, 334}. The accumulation in the site of inflammation induced in rats was clearly observed in the whole body coronal PET image. The important function of such agent must be the differentiation between tumour and inflammation/infection. However, this novel tracer demonstrated also uptake in the melanoma xenografts in mice.

Siderophores that are iron transporters in bacteria, fungi, and some plants were used for the development of imaging agent for invasive pulmonary aspergillosis (IPA) caused by Aspergillusfumigatus ³³⁵. Desferri-triacetylfusarinine C (TAFC) and desferri-ferricrocin (FC) siderophores were labelled with ⁶⁸Ga. [⁶⁸Ga]Ga-TAFC demonstrated specific binding, fast blood clearance and high stability in a rat model of A. fumigatus infection while [⁶⁸Ga]Ga-FC showed prolonged retention in blood and rapid metabolism. TAFC and ferrioxamine E (FOXE) ³³⁶ were selected from a group of 7 various siderophores as best candidates for Aspergillus infection imaging in terms of ⁶⁸Ga complex stability, uptake specificity, rapid clearance and elimination. Both agents demonstrated lung focal uptake in invasive aspergillosis rat model correlated with the severity of disease and high image contrast ³³⁷.

[⁶⁸Ga]Ga-citrate was found effective in clinical examination of bone infection such as osteomyelitis and diskitis. The overall accuracy of diagnosis was of 90% ^{77, 338}. Apo-transferrin was labelled with ⁶⁸Ga and subsequently used for the bacterial infection imaging in rat model with Staphyococcusaureus ⁸⁶. Infection site could be clearly visualized within 1 h after intravenous injection.

Future perspective

First generators were introduced in the late 1950s early 1960s and ⁶⁸Ga-based imaging agents were available already from the 1970s but further progress was precluded by the absence of reliable sources of the generators as well as their properties and quality. During last decade PET has been recognized as a clinically relevant and valuable diagnostic technique providing quantitative information on the physiological status of a disease and possibility to perform a whole-body scan in a single examination. There are many interrelated factors that contributed to the clinical acceptance of PET in general and blossom of ⁶⁸Ga/PET in particular. Technological aspects include achievements in improved sensitivity, resolution, accurate quantitation, and introduction of hybrid systems, PET-CT and PET-MRI; availability and increasing assortment of commercial ⁶⁸Ge/⁶⁸Ga generators; advances in biological research and biotechnology providing identification of biomarkers; accessibility of automated synthesizers for GMP compliant production of imaging agents. The automation may also provide possibility for the harmonized and standardized multicentre clinical studies that in turn will accelerate the introduction of new radiopharmaceuticals as well as their regulatory approval. Steady progress is being observed in PET radiopharmaceutical regulations and multicenter trials. The plausible correction and increase of the parent ⁶⁸Ge breakthrough limit in the radiopharmaceutical preparation (European Pharmacopoeia) may together with the labelling methods of high selectivity towards ⁶⁸Ga(III) at room temperature allow for kit type production at radiopharmacy practice and consequently even wider allocation and use of ⁶⁸Ga-radiopharmaceuticals. The feasibility of PET-CT being cost-effective and cost-saving has been demonstrated, especially in combination with cheaper, as compared to cyclotron, generator produced ⁶⁸Ga radionuclide. Further technological development might render cost reduction and consequently improve profitability and affordability. This in turn may stimulate investments in radiopharmaceutical research and development.

The above mentioned factors have played prominent roles in accelerating the growth of ⁶⁸Ga-radiopharmaceutical field and they will stimulate the investments into development of new imaging agents. The growth has been reflected in the explosive increase of related publication number and more ⁶⁸Ga-agents reported at an ever-increasing rate. However the central role in the expansion of nuclear medicine belongs to identification of unmet medical needs as well as development and introduction of new specific imaging agents and thus achievements in chemistry.

Methods for the enhancement of ⁶⁸Ga quality have been developed and automated however their further simplification is required. Mono- and bifunctional chelators for fast, efficient and selective complexation with ⁶⁸Ga are established and further development will be directed towards radiopharmaceuticals comprising chelators that are highly selective for Ga(III), form efficiently and rapidly complexes at room temperature, broad pH range and low concentration. Conjugation chemistry will further develop towards fast and regioselective coupling reactions at mild conditions and low concentrations.

The ⁶⁸Ga-applications in oncology will continue growing in harmony with the introduction of new receptor specific peptides and high affinity proteins for tumour-type specific diagnosis and treatment that would allow personalized approach of accurate quantitative diagnosis and staging for subsequent selection and planning of therapeutic means as well as monitoring response to the treatment. The imaging of receptors with peptidic agents is the most explored field. The high clinical value of ⁶⁸Ga-labelled SST ligand analogues is recognized and clinical routine practice and regulations are getting established. Imaging feasibility of GRPR, GLP-1R, MSH, HER2 as well as prostate-specific membrane antigen, osteoblastic bone metastases, amino acid uptake, glucose transport, angiogenesis using ⁶⁸Ga-agents has been clinically demonstrated in patients. The experience with a number of ligands specific to G-protein coupled receptors (GRP, CCK, GLP-1, MSH, neuropeptide Y, melanocortin-1, VPAC-1, NT, CXCR4, GnRHR) labelled with ^99mTc, ¹¹¹In, ¹⁸F, ⁶⁴Cu, ⁹⁰Y, ¹⁷⁷Lu and used in clinical studies provide solid basis and will facilitate advent of ⁶⁸Ga-labelled analogues in the nearest future. Obviously, oncology will be the major arena for ⁶⁸Ga-agents, especially those based on regulatory small peptides specific to disease type. This is greatly supported by the robustness, favourable pharmacokinetics and accessibility of the peptides. Immuno-PET with ⁶⁸Ga will also expand due to the pre-targeted imaging technique. The basic research for the development of agents for myocardial perfusion imaging will most probably persist in order to meet the need for quantitative diagnostic means as well as to compensate for ^99mTc shortage. Strong clinical need for the specific and quantitative imaging of inflammation and infection will stimulate the development of the corresponding ⁶⁸Ga-agents, and the vast experience of ^99mTc-radiopharmaceuticals provides valuable basis ²¹⁶.

The future of ⁶⁸Ga is predicted as PET analogue of ^99mTc with added value of higher sensitivity, resolution, quantitation, dynamic scanning, and personalized medicine. The production of ⁶⁸Ga-based imaging agents can be accomplished either under GMP or radiopharmacy practice, and it is a cost-effective complement to cyclotron-based tracers. The kit type preparation similar to that of ^99mTc is feasible. Most importantly it will enable PET-CT investigations globally in remote hospitals without access to accelerators and radiopharmaceutical distribution centres thus promoting worldwide PET technique for earlier better diagnostics and further for individualized medicine. The input of ⁶⁸Ga into oncology and development of theranostics bridging quantitative diagnosis with subsequent therapeutic management has already been proven and will continue to expand. Moreover, it is simply a matter of time when the imaging of bone, sentinel lymph node, lung ventilation/perfusion will be taken over by ⁶⁸Ga/PET-CT. However, even if the vision of ⁶⁸Ga becoming as immense as ^99mTc will not get fulfilled in the nearest future, ⁶⁸Ga definitely has strong contribution to make in the improvement of personalized patient management and there is a niche for ⁶⁸Ga-based agents in nuclear medicine. Moreover, ⁶⁸Ga-agents have the prerequisites to substitute also ¹¹¹In-based radiopharmaceuticals.

References