Imaging in Drug Development

Imaging in Drug Development

ARTICLE IN PRESS Imaging in Drug Development James Nairne*, Peter B. Iveson*, Andreas Meijer† *GE Healthcare, The Grove Centre, Amersham, Buckinghams...

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ARTICLE IN PRESS

Imaging in Drug Development James Nairne*, Peter B. Iveson*, Andreas Meijer† *GE Healthcare, The Grove Centre, Amersham, Buckinghamshire, United Kingdom † GE Healthcare AS, Nydalen, Oslo, Norway

Contents 1. Introduction 2. Magnetic Resonance Imaging 2.1 Use of MRI in the Clinic 2.2 MRI in Drug Development 3. Nuclear Medicine Imaging 3.1 Single-Photon Emission Computed Tomography 3.2 Positron Emission Tomography 4. Summary and Future Prospects References

1 3 4 6 9 9 16 41 42

Keywords: Imaging, Radiochemistry, MRI, PET, SPECT, Fluorine-18, Clinical trials

1. INTRODUCTION In the past 50 years, imaging in medicine has become a staple in the diagnosis of disease. The technology for detection and processing of images has increased in complexity leading to an increase in sophistication in the information that can be derived from these images. Several different underlying technologies and techniques allow various aspects of the human body to be examined. Five basic modalities are used to provide non-invasive imaging for uncovering anatomical and functional detail in the clinic. These are X-ray, ultrasound, magnetic resonance imaging (MRI), nuclear medicine and optical imaging. X-ray was the earliest of the techniques and was able to show only hard tissue through attenuation of the signal from an X-ray source. Subsequent innovations included addition of contrast agents that absorb X-rays and allow complicated cardiac and pulmonary vasculature to be visualised. Abnormalities could then be identified prior to any surgical intervention. Progress in Medicinal Chemistry ISSN 0079-6468 http://dx.doi.org/10.1016/bs.pmch.2014.10.002

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2014 Elsevier B.V. All rights reserved.

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The early 1970s saw the development of computed tomography (CT) in which a 3D image of the anatomy is generated from exposure of the patient to X-rays. The information acquired from X-ray and CT is exclusively anatomical in detail. Its use within drug development has been limited to gross anatomical changes, for example, in tumour mass, and in conjugation with nuclear medicine techniques, in which it provides the anatomical context for the signal obtained from the radioactivity in dual modality techniques such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET) (vide infra). Ultrasound imaging is another important method used in the diagnostic imaging field. In this technique, an ultrasound wave is produced by a transducer and guided into the body, where it is reflected from boundaries at which there are changes in tissue density. Its strength is in the investigation of soft tissue, and it benefits from the lack of use of ionising radiation, portability of equipment and the provision of real-time images. These features make ultrasound very useful in the field of antenatal care. Like X-ray, however, the major limitation of ultrasound is that it can provide information only on an anatomical level and as such has little role to play in drug development outside its diagnostic applications. MRI is an ionising radiation free technology that relies on the detection of a radiofrequency (RF) signal emitted by protons in a magnetic field. As with CT and ultrasound, MRI is capable of visualising anatomical detail; however, the MR technology is also capable of generating images based on the properties of endogenous molecules such as water and haemoglobin. For example, it is possible to assess the macroscopic movements of water to measure blood velocity. It is also possible to measure the microscopic movements of water molecules to assess cellular density. Another example is the possibility of generating images that are influenced by local concentration of haemoglobin and deoxyhaemoglobin, in order to assess the oxygenation of blood. The great capacity of MRI to visualise anatomical detail, without ionising radiation, and to simultaneously provide additional functional information on blood flow, cellular density and oxygenation renders it a very powerful imaging modality in drug development. MR is discussed in more detail in Section 2. Optical imaging is another modality that requires no ionising radiation. In general, the technique uses a fluorescence signal as the imaging output. Fluorescence is the excitation of a chromophore and dissipation of the energy absorbed in the form of light of longer wavelength. The human body is opaque to light in the visible portion of the electromagnetic spectrum and

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CH3 CH3 N

H3C H3C N+

O S O O

OO S O

Figure 1 Structure of indocyanine green.

has absorption minima in the near infrared. Any reporter therefore needs to be able to absorb and emit light in the near infrared portion of the spectrum (750–900 nm) [1]. There are dyes that meet this criterion, the most widely used of which are the cyanine dyes. An example is the heptamethine cyanine, indocyanine green (Figure 1). The reporter is a large molecule and, as such, it is only really applicable for labelling of peptides and proteins. In addition, the absorption of light by tissue and photon scatter limits useful detection to a few millimetres [2]. Although some optical imaging agents have been used diagnostically in the clinic and in preclinical imaging, there are no examples of the modality being used in translational medicine. The last of the major modalities is nuclear medicine imaging. Early imaging agents tracked accumulation in target organs, for example, calcium-45 for bone and iodide-123 for thyroid. The development of radiochemistry has led to an increasingly sophisticated arsenal of chemical techniques that allow the production of radiotracers that are able to reveal the underlying biochemistry of disease with good spatial resolution. The description of these techniques and illustrations of their applications in the clinic forms the bulk of this chapter.

2. MAGNETIC RESONANCE IMAGING MRI is a non-invasive modality that uses strong magnetic fields and RF pulses to produce images of organs and internal structures in the body. Unlike CT, PET and SPECT imaging modalities, no ionising radiation is utilised. The main advantage with MRI over other imaging modalities is the contrast resolution that facilitates anatomical imaging with great soft tissue detail. CT is also capable of anatomical imaging; however, soft tissue detail is not as good as for MRI.

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The MR modality is very flexible with a great number of possibilities for adjusting imaging parameters, in order to further enhance the contrast to background ratio for a certain pathology. It is also possible to inject a contrast agent to further enhance contrast between pathological and normal tissue; however, most MR procedures are performed without the use of a contrast agent. In a simplified view, an MRI scanner consists of three main components: a powerful magnet, an RF transmitter and an RF receiver. When a patient is inserted into an MRI scanner, the powerful magnet will magnetise hydrogen nuclei of molecules present in the patient (principally water and fat) so that they align, either parallel or anti-parallel, with the magnetic field giving rise to a net magnetisation (sum of magnetised hydrogen nuclei). This net magnetisation is excited by an RF pulse to give an unstable state that eventually will return to its initial state, emitting a detectable RF signal, in a process called relaxation. The relaxation proceeds via two independent processes (T1 and T2) which happen simultaneously. These two processes are called intrinsic contrast parameters as they are inherent to various types of tissue and cannot be changed. It is possible to weight the contrast of an image towards one of the two processes by adjusting imaging parameters. The relaxation process can be accelerated by introducing a contrast agent. Several MR contrast agents are available in the clinic and all incorporate a paramagnetic gadolinium ion [3]. The gadolinium ion will accelerate the relaxation process of protons that are in close proximity to the contrast agent, leading to a dramatically altered relaxation process that can be visualised by weighted imaging. The gadolinium ion is toxic and must be entrapped in a chelate to render it safe to use. There are several types of gadolinium chelates (Figure 2) in clinical use and they are all based on polyamino polycarboxylate structures that entrap the gadolinium ion, rendering it safe to use while maintaining the relaxation-enhancing properties.

2.1. Use of MRI in the Clinic MRI is used to diagnose or monitor treatment for a variety of medical conditions by visualisation of anatomical details, detection of pathological tissue or assessment of organ functionality. MR is frequently used to image the central nervous system (CNS) where different compartments and structures can be visualised in high detail in order to detect defects such as aneurysms, tumours, infections or stroke (Figure 3).

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O

N O O

O

O

N

O

O

Gd N

H N

O

O

H3C

N

OO

Gadoterate meglumine Dotarem Guerbet

N H

N

O

O

N

O

N

O N

O

OO

O

Gadodiamide Omniscan GE

O

N

Gd

O N

O

O-

O

Gd

O

O

CH3

Gadopentetate dimeglumine Magnevist Bayer Schering

Figure 2 Examples of gadolinium chelates in use as MR contrast agents.

Figure 3 Anatomical head scan. Adapted with permission from GE Healthcare.

Another example is cardiovascular MR where the vascular anatomy, function, blood perfusion and tissue characteristics can be imaged and quantitated [4]. For some diagnostic situations, it is preferred to inject a contrast agent that will distribute in the blood and the extracellular space before excretion through the kidneys or the liver. Contrast agents are frequently used for angiography applications, where structural or flow related abnormalities are identified and characterised. Contrast agents are also used for oncology applications where the increased perfusion and permeability of tumours result in a high

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concentration of contrast agent. The lesions are then characterised based on structural and morphological criteria (size and shape). MRI is especially powerful for the diagnosis of brain lesions, as the injected agent will leak into the brain only if the blood–brain barrier is damaged, such as when a tumour has disrupted the barrier.

2.2. MRI in Drug Development 2.2.1 Oncology The efficacy of an oncological treatment is often assessed by morphological criteria such as tumour size and shape [5]. Unfortunately, the progression or regression of a tumour following a specific treatment is not always reflected in a morphological change in a predictable and timely manner, potentially leading to unnecessarily long treatment regimes with expensive and ineffective therapeutic agents. There is, thus, a great need to assess therapy response on tumour function at an earlier stage, to allow for a change in treatment plan if ineffective. Currently there are two main MRI techniques, dynamic contrast enhancement (DCE) and diffusion weighted imaging (DWI) that are in clinical use to assess therapy response. A search of www.clinicaltrials.gov indicates that there are 52 active clinical trials using DCE or DWI as a diagnostic tool in evaluating therapeutic response. 2.2.1.1 Dynamic Contrast Enhancement

Imaging tumour vascularisation has received a lot of interest over the past few years as it gives a better understanding of tumour function, compared to morphological criteria alone. DCE is used to study the flow of contrast agent through the tumour to give data on blood volume, perfusion and permeability. There are two types of DCE in clinical use, qualitative and quantitative. Qualitative DCE studies the wash in and wash out pattern in a tumour to give crude information on its vascularisation that is used in conjunction with morphological data [6]. Quantitative DCE utilises pharmacokinetic modelling of blood flow through various compartments to give parameters that numerically describe perfusion, blood volume and permeability [7]. Although quantitative DCE holds great promise for the detailed study of tumour physiology, it is technically challenging and requires strict control of the imaging setup. The parameters obtained from quantitative DCE are very sensitive towards the choice of contrast agent, scanner type, pulse sequence and pharmacokinetic model [8].

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2.2.1.2 Diffusion Weighted Imaging

DWI is a non-contrast technique that measures the microscopic mobility of water molecules, reported as ADC (Apparent Diffusion Coefficient) values. As the mobility of water molecules is hindered by various membranes in vivo, it is possible to assess the cellular density (Figure 4) [9]. In general, tumours have a higher cellular density than normal cells, whereas necrotic areas have a lower density [10]. DWI has been used in monitoring therapy response as a method of detecting early changes in the tumour microenvironment and much effort is in progress to establish it as a general response biomarker [11,12]. In some studies both DCE and DWI are used to improve the assessment of therapy response [13].

Distribution

2.2.2 Central Nervous System Drug discovery for CNS disorders is particularly challenging due to the often poorly defined pathophysiology and the subjective assessment of treatment efficacy [14]. Novel CNS drugs that enter clinical trials have a very high failure rate, and there is a clear need for biomarkers to objectively and quantifiably study the biological processes in the brain in order to better study the effect of treatments. Functional MRI (fMRI) has emerged as a very

Solid tumour

Water mobility or ⬙ADC⬙

Treatmentinduced cell death

Distribution

Effective therapy

Water mobility or ⬙ADC⬙

Figure 4 Cellular density and DWI. Adapted with permission from Wiley Publishers Ltd.: Ref. [9], © 2010.

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promising tool in understanding how the various regions of the brain are involved in various diseases and it is providing a new way of screening patients and assessing treatment responses [15]. A search of www. clinicaltrials.gov indicates that there are 172 active clinical trials using fMRI as a diagnostic tool in evaluating therapeutic response. 2.2.2.1 Functional MRI

fMRI is a non-contrast technique that detects areas of the brain that are haemodynamically activated. Although it is mainly used to measure brain activity upon a sensory stimulus, it can also be used to assess the effect of pharmaceutical challenges and is sometimes called phMRI (pharmacologic MRI) [16]. There are two main techniques that are in use, bloodoxygenation-level dependent (BOLD) and arterial spin labelling (ASL). The BOLD effect utilises the different magnetic moments of oxygenated and de-oxygenated blood to indirectly measure the level of brain activity. A BOLD signal is a result of a complex interaction between changes in blood flow and blood oxygenation following an increase in neural activity (Figure 5). ASL is a non-contrast technique that can be used to measure cerebral blood flow (CBF) and, indirectly, neural activity. It is based on the magnetic labelling of blood water molecules that are flowing into a region of interest. Synapse

Astrocyte

Glutamate Glutamate

Glucose

Capillary O2

?

Glucose Glucose

· Less Hb · Slower dephasing · Stronger MRI signal

O2

· More Hb · Faster dephasing · Weaker MRI signal

Figure 5 BOLD effect. Adapted with permission from Macmillan Publishers Ltd.: Ref. [17], © 2002.

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The magnetic labelling will alter the signal compared to a non-labelled background and the amount of arterial blood delivered to the region can be measured. Recently, fMRI has been shown to be a promising tool for translational drug discovery as certain spatial and functional characteristics of brain network architectures across species have been mapped. These intrinsic networks could potentially be used as translational biomarkers in early screening for CNS drug discovery [18].

3. NUCLEAR MEDICINE IMAGING The use of radiotracers is a particularly powerful tool for the development of therapeutics. This is mainly due to the high sensitivity of the signal. Typically, radiolabelled compounds are administered intravenously in very low quantities (around 10 μg), levels below that which induces a pharmaceutical response. The radionuclides of use in nuclear medicine imaging have to fulfil certain specific criteria to minimise the exposure of the patient to the deleterious effects of ionising radiation. This is achieved by using nuclides of low energy, those with short half-lives and γ or positronemitting isotopes. Isotopes that emit α-particles are inappropriate for imaging as the α-particles are absorbed by tissue in the immediate vicinity, thus causing damage. The mode of emitting detectable radiation dictates the mode of detection of the signal. The radiation from γ emitting radionuclides is detected directly in a technique called SPECT, whereas the radiation detected from positron-emitting nuclides is detected indirectly; this technique is known as positron emission tomography (PET).

3.1. Single-Photon Emission Computed Tomography Images generated using SPECT are built up from a series of planar images. The γ-rays are captured by a camera that is rotated about the patient. The camera consists of up to three heads, each consisting of a collimator in front of a sodium iodide crystal backed with an array of photomultiplier tubes, which are used to pick up an image of the scanned area. This is reconstructed to give a 3D image of the accumulated radiotracer or radiopharmaceutical. There are three main radionuclides used in SPECT imaging (Table 1); two of these, technetium-99m and indium-111, are radiometals and the third is iodine-123. Good metal-ion chelate stability under physiological conditions is required for imaging applications. If this is not achieved,

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Table 1 Common Radioisotopes Used in SPECT Isotope Production Emissions 99m

Tc

Generator

90% IsT, 10% InT

Half-life

Daughter

6h

99

Tc

111

In

Cyclotron

100% EC

2.8 days

111

123

I

Cyclotron

100% EC

13.2 h

123

Cd Te

EC, electron capture; IsT, isomeric transition (γ emission); InT, internal transition. In internal transition, the nucleus decays by transferring energy to an orbital electron which is ejected instead of the γ-ray.

imaging artefacts can be observed that can complicate image interpretation; for example, free technetium will accumulate in the thyroid gland. 3.1.1 Technetium-99m Tracers Technetium-99m is produced relatively inexpensively using a generator. Molybdenum-99 suspended on an alumina column decays (t½ ¼ 66 h) to form technetium-99m. The singly charged 99mTcO 4 is eluted in preference  to the doubly charged 99 MoO4 2 using saline. Commercially available technetium-99m radiotracers are generally prepared by the simple addition of technetium-99m eluted from the generator to a kit vial containing a freeze-dried formulation of the active ingredient. The technetium-99m half-life of 6 h allows time for preparation of the radiotracer, distribution and patient imaging. The energy of the γ-ray emission (140 keV) is ideal for imaging using gamma cameras. Technetium has a rich coordination chemistry with several potential oxidation states [19]. Most nuclear imaging agents contain technetium99m in the +5, +3 or +1 oxidation states, although it is also present in the +7 oxidation state in the thyroid imaging agent 99m TcO4  , as formed in the generator. Technetium has good affinity for nitrogen, oxygen, phosphorus and sulphur in the most common oxidation states. The preparation of technetium-99m imaging agents is relatively straightforward (Scheme 1); a kit comprising a reducing agent, usually stannous chloride, a weak chelating agent and the cheland is treated with the generator eluate and the mixture incubated for a short time, often at room temperature, giving a preparation that is ready for injection without purification. Approximately 85% of nuclear medicine diagnostic imaging procedures are still done with technetium-99m. The three most widely used technetium-99m tracers in the USA are 99mTc-Sestamibi (Cardiolite) and 99m Tc-Tetrofosmin (Myoview) (Figure 6) primarily for imaging myocardial perfusion and 99mTc-MDP (methylenediphosphonate) for imaging bone

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Sn4+

Sn2+

HO

O

O

99mTc

HO

[99mTcVOx] −

[99mTcVIIO4] −

O

O

O

O

O

OH CO2H

HO

OH OH CO2H

CH3 H3C NO N H3C CH3 Tc NO N H3C CH3 OH OH

Scheme 1 Formation of 99mTc-HMPAO (hexamethylpropyleneamine oxime).

H3C

CH3

H3CO CH3

H3CO

CH3 H3 CO

N N

N

OEt O

EtO P

CH3

99m

Tc

H3C

OCH3 H3C

P

EtO

N N

OEt

P Tc P

99m

H3C N

OEt

CH3

OCH3 CH3

OEt

O OEt

OEt

OCH3 H3C CH3

99m

99m

Tc-Sestamibi

Figure 6 Structures of

99m

Tc-Sestamibi and

99m

Tc-Tetrofosmin

Tc-Tetrofosmin.

metastases. The delivery of these tracers to the organ of interest is based on a non-target specific localisation mechanism. 99m Tc-Sestamibi and 99mTc-Tetrofosmin are also both transport substrates recognised by the multidrug resistance (MDR) P-glycoprotein (Pgp). Pgp can confer resistance to many cytotoxic cancer therapeutics. 99m Tc-Sestamibi has been used in cancer clinical trials to predict the response to chemotherapy. In a recent meta-analysis, lung cancer patients who had less 99mTc-Sestamibi initial uptake in tumours were found to be less likely to respond to chemotherapy. The sensitivity, specificity and

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accuracy of 99mTc-Sestamibi in identifying chemotherapy responders were 94%, 90% and 92%, respectively [20]. 3.1.2 Indium-111 Tracers Indium-111 is produced in a cyclotron using a cadmium-112 target which is bombarded with protons to produce indium-111 by the (p,2n) reaction. The energies of the γ-ray emissions (171 and 245 keV) are higher than that of technetium-99m. Indium-111 forms complexes in which indium is present in the +3 oxidation state. Either open-chain DTPA or macrocyclic DOTA chelating agents are generally used in indium-111 nuclear medicine applications (Figure 7). In contrast to indium-DTPA which can be formed at room temperature, heat is required to form indium-DOTA complexes [21]. As a result, indium-DOTA complexes may not be suitable for applications in which the peptide or antibody is sensitive to heat. The half-life of indium-111 (2.8 days) is especially suited to imaging antibodies which tend to have longer biological half-lives. 3.1.3 Iodine-123 Tracers Iodine-123 is produced in a cyclotron by bombardment of enriched xenon124 by the (p,2n) reaction via the shorter lived caesium-123 and xenon-123. The energy of the γ-ray emission (159 keV) is close to the ideal for imaging using gamma cameras. Several different iodination methods are employed to synthesise radiolabelled iodine-123 compounds. The most commonly used involve either O O O N O O

N

111In

O

N

N O O

O O

N

N

N

O O

O

111In

O O O

111In-DTPA

111In-DOTA

Figure 7 Structures of indium-DTPA and -DOTA complexes.

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direct electrophilic substitution on activated aromatic systems, electrophilic radio-iododestannylation reactions or nucleophilic exchange [22]. Direct electrophilic substitution of protein or peptide is carried out using iodogen, an oxidant that converts [123I]iodide to [123I]iodonium, which then reacts with an electron-rich amino acid residue such as tyrosine [23]. 123 I-FP-CIT (DaTSCAN, 123I-2-β-carbomethoxy-3β-(4-iodophenyl)N-(3-fluoropropyl)nortropane) is used in patients with clinically uncertain Parkinsonian syndromes and in patients to help differentiate probable dementia with Lewy bodies from Alzheimer’s disease. It is one of the few commercially available iodine-123 radiopharmaceuticals and is prepared by radio-iododestannylation (Scheme 2). The combination of [123I]iodide, part labelled elemental iodine and the hydrogen peroxide oxidising agent, produces the electrophilic species HO*I and H2O*I which then react rapidly with the trimethylstannyl precursor at room temperature. The 13.2 h half-life of iodine-123 allows central manufacturing and a relatively wide distribution area. 123 I-BMIPP ([123I]-beta-methyl-p-iodophenylpentadecanoic acid (Cardiodine/Japan)) is another commercially available radioiodinated tracer which is currently being used as a metabolic imaging agent in drug therapy clinical trials. Long-chain fatty acids such as BMIPP are the main energy source for normal oxidised myocardium and are rapidly metabolised by β-oxidation. With ischaemia, energy metabolism moves to anaerobic metabolism and the main energy substrate changes from fatty acids to glucose. Reduced uptake of 123I-BMIPP can indicate a history of previous ischaemia. This is due to the fact that the recovery of oxidative metabolism of fatty acids within cardiomyocytes lags behind the recovery of perfusion. 123 I-BMIPP is synthesised by nucleophilic exchange of iodine-127 in the presence of a catalytic amount of copper and ascorbic acid as reductant (Scheme 3).

F

F

O

O N

OCH3

N SnMe3

Scheme 2 Preparation of

OCH3

[123I]-iodide

123

I-FP-CIT.

123I

H2O2 / H2SO4

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I OH CH3 O

[123I]-iodide Cu(NO3)2 / ascorbic acid

123I

OH CH3 O Scheme 3 Preparation of

123

I-BMIPP.

3.1.4 Applications of SPECT to Translational Medicine The number of applications of technetium-99m radiotracers in clinical drug development is likely to increase in the future as more target-specific technetium radiotracers are developed. One recent example involved the use of a 99m Tc-labelled folate imaging agent 99mTc-Etarfolatide in the clinical trials of the related drug Vintafolide [24] (Figure 8). In 99mTc-Etarfolatide, 99mTc is stabilised by chelation to a peptide-based N3S chelate which forms a negatively charged complex in which 99mTc is present in the +5 oxidation state. The formation of this hydrophilic complex facilitates the desired rapid clearance through the kidneys. Vintafolide is under development for the treatment of cancers of the ovary, lung and breast among others. 99mTc-Etarfolatide imaging has primarily been used in clinical trials to identify patients expressing high levels of the folate receptor who may be suitable for Vintafolide therapy. The longer half-life of indium-111 makes it suitable for use with antibody-based therapies. 111In-ibritumomab tiuxetan has been used to test the mechanistic hypothesis behind the combination of radiotherapy using 90 Y-iritumomab tiuxetan (Zevalin) and oligonucleotide therapy using CpG 7909 in patients with relapsed indolent non-Hodgkin lymphoma [25]. It had been proposed that CpG 7909 therapy could enhance the delivery of the Zevalin as in vitro experiments had shown increased CD20 expression by malignant B cells. However, no increase in uptake of the indium-111

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O HN

O N NH

N H

HO2C O

N

NH2

N

HN O NH N HO2C 99mTc=O

N

O

S HO2C 99m

Tc-Etarfolatide

O HN

O N

HO2C

N H

NH

O NH2

N

N

O HN NH O HO2C HN

CO2H H2N

NH NH

O NH

CO2H

O

O HN

S S

NH

O

CO2H

HN

O OH

HO

CH3 N

H3C OCH3 HN O H3CO H3C

Vintafolide

Figure 8 Structures of

99m

Tc-Etarfolatide and Vintafolide.

H N HO

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analogue of Zevalin was observed, indicating that there was no enhanced delivery of the radiotherapeutic. 123 I-FP-CIT imaging has been used in a number of clinical trials of disease modifying drugs. A recent study used 123I-FP-CIT imaging to show that the dopamine agonist pramipexole had no modifying properties in patients with early Parkinson’s disease [26]. Baseline 123I-FP-CIT scans were carried out before therapy and at 15 months following treatment with pramipexole. A decrease in striatal dopamine transporter binding observed by imaging at 15 months indicated that pramipexole was not effective in slowing disease progression. A trial in Japan has been set up to determine the efficacy of the Na–H exchange (NHE) inhibitor TY-51924 as an adjunctive therapy to primary percutaneous coronary intervention (pPCI) for patients with ST-elevation myocardial infarction (STEMI) [27]. The trial illustrates the important role radiotracers can play in the clinic. In the acute phase (3–5 days after pPCI), thallium-201 SPECT imaging will be used to determine infarct size, and left ventricular (LV) volume and function, while 123IBMIPP will be used to determine the area at risk. In the follow-up, 3 months after pPCI, 99mTc-Tetrofosmin will be used to determine infarct size and LV volume and function. 201Tl chloride and 99mTc-Tetrofosmin are both myocardial perfusion imaging agents. GlaxoSmithKline (GSK) have used [123I]iodobenzamide and [123I]R91150 123 ( I-4-amino-N-[1-[3-(4-fluorophenoxy)propyl]-4-methyl-4-piperidinyl]5iodo-2-methoxybenzamide) in the study of SB-773812, a putative antipsychotic treatment. The two SPECT tracers bind D2 receptors and [123I] R91150 also binds to 5HT2A[28]. The clinical study was able to show that not only did SB-773812 cross the blood–brain barrier, but it also bound the two receptors in humans. The study contributed towards the decision to proceed with clinical development into Phase III.

3.2. Positron Emission Tomography Positrons are particles that have the same mass as an electron, but are positively charged and as such are very unstable. They have a very short half-life in tissue as they interact with electrons in a process called annihilation, in which the mass of the two particles is converted into energy in the form of two photons, with an energy of 511 keV, emitted in the opposite directions. It is these two photons that are detected in a PET camera. The major advantage of the use of PET over SPECT is that the technique can be used to quantify the uptake of a particular tracer. This is

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possible due to the simultaneous emission of the two photons during annihilation. The detectors in the camera are arranged in a ring around the patient and only signals that arrive at the same time are counted in image accumulation. It is, therefore, certain that the signal is accrued from single emission events and that each event has occurred on a straight line between the two detectors that have detected the signal Most of the common PET radioisotopes have shorter half-lives that those used in SPECT (Table 2). Nitrogen-13 and oxygen-15 are used clinically in the form of [13N]ammonia and [15O]water as blood perfusion agents. However, the most widely used nuclides are carbon-11 and fluorine-18. These are attractive isotopes in drug development as they can form covalent bonds to carbon, allowing them to be incorporated into analogues of drug-like molecules which then to behave in the same way as the drug when introduced into the body. 3.2.1 Carbon-11 Tracers The half-life of carbon-11 is only 20 min. This, therefore, necessitates two prerequisites for the radiochemist. First is in the design of the radiotracer; the radiolabel should be designed to be introduced at the latest possible stage in a synthesis. The second is that the chemistry used needs to be rapid and efficient. Carbon-11 is generated in a cyclotron by proton bombardment of nitrogen gas with ejection of an α-particle from the nucleus (14N2 (p,a)11C). Incorporation of a small amount of oxygen in the target gives rise to carbon-11 as carbon dioxide, while if around 4% hydrogen is mixed with the nitrogen, carbon-11 methane is obtained. These two compounds Table 2 Common PET Radionuclides and Their Properties Isotope Production Emissions Half-life 11 13

C N

Cyclotron

100% β +

Cyclotron

100% β +

Daughter

20.3 min

11

B

10 min

13

C N

15

O

Cyclotron

100% β +

2.1 min

15

18

F

Cyclotron

100% β +

109.8 min

18

O

Generator

89% β +, 11% EC

68 min

68

Zn

68

Ga

89

Zr

Cyclotron

76.6% EC, 23% β +

78.4 h

89m

64

Cu

Cyclotron

44% EC, 38% β , 18% β +

12.7 h

64

Y,

Ni,

89 64

Y

Zn

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provide the basic feedstocks for introduction of carbon-11 atoms into compounds. [11C]Carbon dioxide can be used directly in reactions to incorporate the radiolabel. The radiosynthesis of [11C]WAY100635 (Scheme 4), a 5HT1A receptor ligand [29], and the dopamine D2/D3 binder [11C] PHNO [30] (Scheme 5) was prepared by trapping Grignard reagents with [11C]carbon dioxide, conversion to the corresponding acid chlorides and reaction with amines. Over the years, radiochemists have been able to develop a number of chemical approaches that fulfil the criterion of rapid incorporation of the label. [11C]Carbon dioxide has been converted to [11C]methanol by treatment with lithium aluminium hydride [31] and subsequent treatment with hydriodic acid [32] to form [11C]methyl iodide. This synthon is one of the most widely used methylating agents for introducing labelled methyl groups in nucleophilic methylation reactions (Scheme 6). It has been used in the preparation of methyl ethers, as in the adenosine A2A ligand [11C]KW6002 [33], N-methylated compounds such as the benzodiazepine receptor binding compound, [11C]flumazenil [34], and S-methylated compounds

MgCl [11CO2]

O11 OMgCl C SOCl2

O11 Cl C

11C

O

N

N

OCH3

N

N

[11C]WAY100635 11

Scheme 4 Radiosynthesis of [ C]WAY100635.

CH3CH2MgBr

[11CO2]

CH3CH211CO2MgBr

(i) PDC (ii) DTBP

CH3CH211COCl

O N

11CH CH CH 2 2 3

O [11C]PHNO PDC–phthaloyl dichloride; DTBP–2,6-di-tert-butylpyridine

Scheme 5 Radiosynthesis of [11C]PHNO.

N O

11C

CH2CH3

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O Et O

N N Et

CH3 N

OCH3

N

N

O 11CH 3

[11C]KW-6002

N

O OEt

N

F O

11CH 3

[11C]flumazenil

[11C]CO2

[11C]CH3I

[11C]CH3OH

CO2H H311C

NH2

[11C]methionine

O H311C

S

NH N

O

HO O F OH [11C]FMAU

Scheme 6 Some applications of [11C]methyl iodide in the radiosyntheses of important carbon-11 labelled radiotracers.

in the preparation of [11C]methionine [35], used for imaging tumours. [11C] Methyl iodide has also been used as a substrate in Stille [36], for the preparation of 1-(20 -deoxy-20 -β-D-arabinofuranosyl)-[methyl-11C]thymine ([11C]FMAU), and Suzuki [37] coupling reactions and as a substrate for the preparation of [11C]methylmagnesium iodide [38]. [11C]Methyl triflate has also been used as a methylating agent. This is prepared by treatment [11C]methyl iodide with silver triflate. [11C]Methyl triflate is then used as a very reactive electrophile [39] in similar reactions to [11C]methyl iodide. For example, Langer et al. have prepared [11C] raclopride (Scheme 7) using this approach [40]. [11C]Carbon dioxide can be reduced to [11C]carbon monoxide by reduction over zinc [41] or molybdenum [42] and the resultant gas used in carbonylation reactions using transition metals such as palladium and rhodium. These synthetic routes have been used to prepare malonates [43], ureas [44], carbamates [45], ketones [46,47] and amides [48] among other compound classes.

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H311C OH Cl

O

Et N

N H OH

O

O

Cl

11CH OSO CF 2 3 3

aq NaOH acetone, RT

Et N

N H OH Cl

Cl

[11C]raclopride 11

11

Scheme 7 Radiosynthesis of [ C]raclopride using [ C]methyl triflate.

F

Br N

F

11CN

K11CN

N

N N O

F

Pd(PPh3)4 DMSO

N N O

F

11

Scheme 8 Radiosynthesis of [ C]AZD9272.

Cu11CN 11

I

CN

DMF N

N

Scheme 9 Radiosynthesis of [11C]LY223645.

[11C]Methane is also a valuable cyclotron product used for introducing a carbon-11 label into compounds. It has been utilised to prepare [11C]methyl iodide by treatment with iodine [49]; [11C]hydrogen cyanide via a platinum-catalysed reaction with ammonia [50]; and [11C]phosgene via perchlorination then hydrolysis of the resultant [11C]carbon tetrachloride [51,52]. The latter two carbon-11 reagents have been used to make a variety of tracers. [11C]Hydrogen cyanide is used to introduce a [11C]cyano group using a number of different chemistries, including transition metal-catalysed cyanations, such as used by Andersson et al. in the radiosynthesis of the mGluR5 radioligand [11C]AZD9272 [53] (Scheme 8), and copper cyanide reactions such as that used by Mathews et al. to prepare [11C]LY2232645 (Scheme 9), also an mGluR5 antagonist [54]. 3.2.2 Fluorine-18 Tracers Fluorine-18 has a half-life of 109.8 min, some five-and-a-half times that of carbon-11. This provides the isotope with several advantages over the use of carbon-11 tracers; the greater shelf-life of the tracer once prepared can

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translate as the ability either to image more subjects from a single radiosynthesis or to allow imaging of patients to occur at sites remote from the production of the tracer. Fluorine-18 is also produced by a reaction in a cyclotron; in this case, proton bombardment of oxygen-18. In the case of [18F]fluorine gas, the target is [18O]oxygen gas and for [18F]fluoride the target is [18O]water. The methods for introducing fluorine-18 into a molecule are more limited than is the case for carbon-11, but the same principle for introducing the isotope as late as possible still applies. Much of the early work with fluorine18 used electrophilic reactions. These involved the use of [18F]fluorine gas directly. As fluorine gas is very reactive, the substrates that could be used were limited and the chemoselectivity of the introduction of the label is low. An example of this is the synthesis of [18F]6-F-dopa, a tracer for the quantification of dopamine receptors. The reaction is only 59% regioselective [55], the remaining 41% of radiofluorinated product being a mixture of the 2- and 5-fluoro analogues (Scheme 10). Other electrophilic reagents have been developed are [18F]acetyl hypofluorite, which has been used to prepare [18F]fluorodeoxyglucose (FDG) in its early radiosyntheses [56] (Scheme 11), and N-fluorobenzenesulfonamide [57]. The nuclear chemistry to produce [18F]fluoride is much more straightforward—bombardment of [18O] water with protons gives [18F] fluoride that can be used as a nucleophile in radiofluoridation reactions. HO

CO2H 18F

HO

NH2

[18F]6-F-dopa HO HO

CO2H NH2

[18F]F2/HF

HO

CO2H NH2

HO 18F

[18F]-5F-dopa 18F

HO HO

CO2H NH2 [18F]-2F-dopa

Scheme 10 Electrophilic radiosynthesis of [18F]6-F-dopa.

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(i) [18F]CH3COOF (ii) HCl

O

HO

O

HO

F

HO

HO

OH

OH

OH

Scheme 11 Electrophilic radiosynthesis of [18F]FDG.

AcO

O

AcO

18

OAc

(i) [ F]KF, K222

OTf

(ii) HCl

OAc

HO

O

OH 18

HO

F

OH

Scheme 12 Nucleophilic radiosynthesis of [18F]FDG.

As fluoride is a relatively poor nucleophile, it needs to be processed to improve its ability to participate in SN2 reactions. [18F]Fluoride is trapped on an ion exchange cartridge, the [18O]water recovered for reuse and the [18F]fluoride eluted with a mixture of potassium carbonate and kryptofix222 (K222). This gives a solution that, once dried, gives anhydrous fluoride that is rendered more reactive by the sequestration of the potassium ion by the kryptofix-222 [58]. In this form, the fluoride can react in both SN2 and SNAr reactions used in the preparation of [18F]fluoroalkanes and [18F] fluoroarenes, respectively. 3.2.2.1 Preparation of [18F]Fluoroalkanes

[18F]Fluoroalkanes can be prepared from precursors such as alkyl tosylates, mesylates and triflates in solvents such as acetonitrile, DMF and DMSO. The basicity of fluoride means that use of protecting groups is often required to prevent side reactions or suppress [18F]hydrogen fluoride formation by proton abstraction. This chemistry has been exploited widely in the preparation of radiotracers for applications in neurology and oncology. The most widely used PET radiopharmaceutical is [18F]FDG, now prepared via the nucleophilic substitution of a triflate group in mannose triflate tetraacetate [59] (Scheme 12). [18F]FDG is a marker for glucose hypometabolism—it is taken into cells via the glucose transporter mechanism and then undergoes phosphorylation on the 6-hydroxyl function, but cannot undergo the second step of glucose metabolism—phosphorylation at the 2-position—as this is blocked by the fluorine-18 atom (Scheme 13). The phosphorylated derivative is then trapped in the cell, leading to an accumulation of the monophosphorylated

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HO

-

18

HO

O P O O

O

OH

O

hexokinase

F

OH

O

18

HO

OH

F

OH

Scheme 13 Metabolism of [18F]FDG. 18F

O

O 18F

N

N H OCH3

N

N

N

OCH3 N

OCH3 [18F]Fallypride

H3C

[18F]MeFWAY

H N 18F

N

O

O

O

18F

[18F]Florbetapir 18

CO2H NH2

[18F]Fluciclovine 18

18

Figure 9 Structures of [ F]fallypride, [ F]MeFWAY, [ F]florbetapir and [18F] fluciclovine.

[18F]FDG that is detected by the PET camera [60]. The greater the glucose demand of the cells in question, the greater the radioactive signal. This tracer has, therefore, found particular use in oncology, as well as cardiology and neurology [61]. Many tracers have been developed using this type of chemistry via direct labelling methods. These include analogues of carbon-11 tracers such as the dopamine D2/D3 binder fallypride [62], an analogue of raclopride, and the 5HT1A tracer MeFWAY [63], an analogue of WAY100635; and tracers that have been approved for clinical use. This latter group includes florbetapir [64], a radiopharmaceutical for the detection of amyloid plaques, and others undergoing development, for example, fluciclovine (FACBC) [65], an L-amino acid transporter marker that has potential applications in prostate cancer diagnosis (Figure 9). In all of the above cases, the fluorine is introduced by a direct method, that is, one in which a pharmacophore containing a leaving group is treated

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with [18F]fluoride. Some compounds contain functionality that is sensitive to the conditions for reaction with fluoride or contain functionality that prevent fluoride acting as a nucleophile. In these cases, a so-called indirect method of fluorination can be used. The fluorine is introduced to a small molecule that undergoes a reaction with the pharmacophore under mild conditions. Examples of this approach are demonstrated by the synthesis of [18F]fluorocholine [66] and [18F]fluoroethyltyrosine [67], both used in oncology, and [18F]GE-179 [68], a marker for activated NMDA receptors. Radiosynthesis of [18F]fluorocholine is carried out via the synthesis of [18F] fluorobromomethane from dibromomethane (Scheme 14) and the radiosyntheses of [18F]fluoroethyltyrosine and [18F]GE-179 via the synthesis of [18F]fluoroethyl tosylate from ethylene ditosylate (Scheme 15). 3.2.2.2 Preparation of [18F]Fluoroarenes

Fluorination of aromatic rings with [18F]fluoride follows the same rules as those for preparative SNAr reactions in that, generally, there is a requirement CH3 H3 C Br

18F

Br

N

OH 18F

Br

CH3 N

H 3C

OH

Scheme 14 Radiosynthesis of [18F]fluorocholine using [18F]bromofluoromethane.

TsO

OTs

CH3 N

H N

18

NH Cl

F

CO2H

OTs HO

SH H N

NH2

CH3 N CO2H

NH Cl

18

S 18

F

O

NH2

F [18F] fluoroethyltyrosine

[18F] GE-179 18

18

Scheme 15 Radiosyntheses of [ F]GE-179 and [ F]fluoroethyltyrosine.

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for an electron-withdrawing group capable of delocalising the incoming negative charge. For simple direct labelling reactions, therefore, there needs to be suitable functionality in the ortho- or para-position to the [18F]fluoride label. Leaving groups are typical for the types of reaction—nitro, trimethylammonium and halides have all been used, though nitro and trimethylammonium are preferred due to ease of separation of the product from the excess of precursor. The two dopamine tracers [18F]haloperidol [69] (Scheme 16) and [18F]spiperone [70] (Scheme 17) have been prepared in one step from their nitro precursors. There are some examples where the standard SNAr-activating groups are not present, so particularly stringent reaction conditions are required to effect the radiosyntheses; [18F]flutemetamol [71] (the fluorinated equivalent of the carbon-11 amyloid tracer [11C]PiB [72]) and [18F]flumazenil (a GABA tracer) [73] have been prepared from [18F]fluoride (Figure 10).

OH

OH

O

O Cl

Cl

O2N

18

F

[18F]Haloperidol 18

Scheme 16 Radiosynthesis of [ F]haloperidol.

O O

H N

O O

N

H N N

N

N 18F

O2N

18

[ F]Spiperone

Scheme 17 Radiosynthesis of [18F]spiperone. N

18F

HO

HO

S N

NH CH3

S N

18F

N O

[18F]Flutemetamol

[11C]PiB

CO2Et

N

NH 11CH 3

CH3

[18F]Flumazenil

Figure 10 Structures of [18F]flutemetamol, [11C]PiB and [18F]flumazenil.

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The constraints placed on the electronic requirements of SNAr reactions have led to a number of different approaches to introduce fluorine-18 into aromatic systems. Initial attempts were to redesign syntheses so that the radiochemical precursor was activated to nucleophilic attack, and then carry out subsequent chemistry to provide the tracer required. Much of this effort was aimed at radiosyntheses of the dopamine transporter tracer [18F] 6-F-dopa. Lemaire et al. reported a four-step radiosynthesis of this tracer in which the fluoride was introduced via nucleophilic displacement of a trimethylammonium salt derived from veratraldehyde. Subsequent reduction, iodination, asymmetric alkylation and deprotection gave [18F]6-F-dopa [74] (Scheme 18). The Machulla group developed a similar nucleophilic method using a veratraldehyde [75]. Although it was not used for the synthesis of [18F] 6-F-dopa, the same group developed a method for the introduction of [18F] fluoride by incorporation of a formyl group acting as a transient activating group that was removed using Wilkinson’s catalyst [76] (Scheme 19). A further strategy towards [18F]6-F-dopa was reported by Coenen who demonstrated incorporation of fluoride in the 6-position via activation through a ketone in the 3-position. The ketone was subsequently converted to the required phenol via a Baeyer–Villiger oxidation reaction [77]. Each of these syntheses has the disadvantage of a large number of steps with the fluorine-18 in the molecule leading to low radiochemical yields of tracer. A more direct approach has been shown in the use of iodonium salts by Carroll and DiMagno. Carroll showed that it was possible to access 3-fluoropyridine using this technology in 55–63% radiochemical yields (Scheme 20) [78]. Hitherto, the yields of this compound using fluoride were significantly worse than for the activated 2- and 4-fluoro analogues [79].

H3CO

CHO

H3CO

H3CO

R

H3CO

CHO

HO

18F

HO

CO2H 18

F

NH2

Scheme 18 Outline of radiosynthesis of [18F]F-dopa [73]. H3CO

CH3

H3CO

H3CO

F

H3CO

CHO

CH3 18

F

H3CO H3CO

CH3 18

F

CHO

Scheme 19 Use of a formyl group to activate an aromatic ring towards SNAr using [18F] fluoride [75].

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Although the use of iodonium salts shows promise for simple molecule and synthons [80], the chemistry used to prepare them is incompatible with functionality that is sensitive to oxidation or strong acid. Recent years have seen the extension of transition metal chemistry for the introduction of fluoride. Initial work by the Buchwald group showed that the introduction of 19F fluoride was possible using palladium chemistry [81]. The use of palladium-catalysed chemistry to introduce [18F]fluoride into unactivated positions was reported by Ritter [82]. A fluorine-18 palladium(IV) complex is prepared from the 4-methylpyridine precursor (Scheme 21), and it is this that is used to react with palladium(II) complexes of the arene that is to be fluorinated to give the desired [18F]fluoroarene. This chemistry was illustrated by the syntheses of [18F]fluorodeoxyestrone and [18F]AS-252424 (Figure 11). Ritter recognised the need for further simplification of his method and followed up by publishing on the fluorination of nickel(II) complexes in aqueous solutions [83] in the presence of an oxidant. These single-step radiosyntheses also remove the need for the time-consuming drying step. 18

I+ N

OCH3

F

N

18

Scheme 20 Radiosynthesis of [ F]3-fluoropyridine using an iodonium salt. CH3

18

F

N N

N N Pd

N

N

B N N N N N

Pd N

N N N B N N N N

Scheme 21 Synthesis of [18F]-containing palladium(IV) complex for preparation of [18F] fluoroarenes [81]. CH3O

O NH

H H 18F

S

OH H

18F

O O

Figure 11 Structures of [18F]fluorodeoxyestrone and [18F]AS-252424.

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The utility of the method was exemplified by the formal syntheses of [18F]6F-dopa as well as [18F]fluorodeoxyestrone. In a further development in the flexibility of preparation of [18F]fluoroarenes, Tredwell et al. have recently published a paper on the use of copper(II)-mediated chemistry to introduce [18F]fluorine into aromatic systems via boronic esters [84] (Scheme 22). This methodology has the benefits of well-established chemistry for the preparation of the precursors and relatively mild reaction conditions for the fluorination reaction itself. The method was illustrated with the radiosyntheses of [18F]DAA1106, a tracer for translocator protein (TSPO), [18F]6fluorotyrosine and [18F]6-F-dopa. 3.2.2.3 Labelling of Peptides and Proteins with Fluorine-18

All the methods discussed earlier are applicable to the preparation of small molecules, but outside neurology, there is an increasing emphasis on the use of biopharmaceuticals, consisting of peptide or proteins. The requirements of most of the methods are for anhydrous conditions or often the absence of acidic protons to allow fluoride to be introduced. Moreover, site-selective chemistry for peptides and proteins has long been a challenge for molecular biologists. The radiolabelling of radiotracers for in vivo use presents an additional challenge due to the stoichiometry of the radiochemistry reactions. A considerable amount of work has been put into realising methodologies for the introduction of [18F]fluorine to this type of molecule. One of the advantages of dealing with high molecular weight pharmacophores is that the addition of extra functional groups can be done with little effect on the binding properties and pharmacokinetics, provided that the group is added away from the molecule’s binding site. This strategy has been broadly exploited. The groups added are often called “synthons” or “prosthetic groups” and are designed to react with functional groups not found in native amino acid residues. By definition, these strategies are two-step H3CO

H3CO

O H3C

OCH3 N

18

OPh O

B O

[Cu(OTf)2(py)4] [ F]KF/K222 DMF, 110 °C, 20 min

O H3C

OCH3 N OPh

18F

Scheme 22 Radiosynthesis of [18F]DAA1106 using copper(II)-mediated chemistry.

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processes—the synthons must be readily radiolabelled with [18F]fluoride and then undergo rapid chemoselective reactions to give the labelled macromolecule (Scheme 23). The most commonly used synthon is [18F]4-fluorobenzaldehyde. The conjugation chemistry to add this to proteins and peptides is through the reaction of the aldehyde with either an aminoxy or hydrazine group. The aminoxy group can be introduced by the reaction of an amine in the peptide or protein with aminooxyacetic acid, as exemplified by the αvβ3 integrin binding peptide, [18F]fluciclatide [85] (Figure 12), and the hydrazine group can be introduced using the HYNIC group, for example, the LTB4 antagonist, MB67 [86] (Figure 13). This strategy works well for peptides that can be synthesised, as the introduction of the reactive group can be done in a site-specific manner before deprotection of the amino acid side chains. More complex molecules such as the HER-2-binding Affibody ZHER2:2891 can be labelled via cysteine–maleimide conjugates [87] (Figure 14). The 4-fluorobenzaldehyde group is a relatively lipophilic synthon; the O’Hagan group has developed a simple, more hydrophilic equivalent in a synthon derived from ribose, 5-deoxy-5-fluororibofuranose (FDR) (Scheme 24). FDR has an advantage over FDG in that the conjugate is Peptide-ONH 2 CHO

18 -

CHO

F

Me3N

18

TfO-

N 18

F

O

Peptide

F

Scheme 23 Outline scheme for labelling peptides and proteins using [18F]4fluorobenzaldehyde as a synthon. 18

F

N O HN O

NH2

NH NH

H N

HN O

O

O N H S

H N O

O N H

CO2H H N O

Ph

O N H

S

H N O S

Figure 12 Structure of [18F]fluciclatide.

O

O N H

O

O

O

N H

O O

NH2

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N

O

N N N N H3C CH3

O

O

O

O

N H

O

NH

NaO3S HN O O

SO3Na

NH

NaO3S HN O

SO3Na N NH

HN O

18

F

H N

N O

O

NH O

SO3Na

NaO3S HN O

NH O

N

O

SO3Na

NaO3S HN

N N N N

O

H3C CH 3

O N H

O

O

NH

O

18

Figure 13 Structure of [ F]MB67. 18F

H N

O

N O

N S

O O CO2H

ZHER2:2891

NH

Figure 14 Structure of [18F]-labelled HER-2 affibody.

18

F HO

O

OH

O OH OH

O

N

18

F

N OH

glutathione S

O

Scheme 24 Reaction of [18F]FDR with aminoxy-tagged glutathione.

OH

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formed more rapidly than the corresponding FDG equivalent and that it exists predominantly in the open-chain form [88]. Another method for introducing fluorine-18 via a synthon approach has been to use the Huisgen 1,3-dipolar cycloaddition reaction, also known as Click chemistry. Usually, the peptide or protein is derivatised with a terminal acetylene then treated with an [18F]ω-fluoroalkyl azide which reacts to give the target molecule tagged with a [18F]fluoroalkyl-1,2,3-triazole. This has been demonstrated by the Sutcliffe group in the preparation of a labelled version of A20FDMV, a binder to αvβ6-expressing tumours [89]. Ramenda et al. also used click chemistry to label human serum albumin (HSA), but in this case, they prepared a fluorine-18 substituted alkyne and reacted it with azide-functionalised HSA [90] (Figure 15). There have been successful attempts to label peptides and proteins directly with fluoride. Wa¨ngler et al. developed a silicon-based prosthetic group that reacts with [18F]fluoride to give a compound with a hydrolytically stable silicon–fluorine bond [91] (Figure 16). The second recent development has been reported by D’Souza et al. who used a chelated aluminium ion linked to a targeting agent and then carried out a ligand exchange reaction with [18F]fluoride [92] (Scheme 25). This chemistry has recently been reported as being used to prepare the integrin imaging agent [18F]-alfatide (Figure 17) for use in lung cancer patients [93]. 3.2.3 Iodine-124 Tracers Iodine-124 is produced in a cyclotron by bombardment of enriched tellurium-124 either by protons in a (p,n) reaction or by deuterons in a O N N

O

O

S

N N H

N

A20FMDV2 18

F

H3C

18

F

Figure 15 Structures of [18F]-labelled A20FDMV and the [18F]-labelled alkyne synthon developed by Ramenda.

O N t-Bu 18

F

O

N H

Aux--Peptide

Si t-Bu

Figure 16 General structure of peptide labelled with a stable [18F]fluoride–silicon bond.

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O HN

O HO

N

HO

N

D -Lys(HSG)- D -Tyr- D -Lys(HSG)-NH2

N

O

AlCl3

O HN

O O O

D -Lys(HSG)- D -Tyr- D -Lys(HSG)-NH2

N

Al N N

Na18F

OH O

O HN

O O O

D -Lys(HSG)-D -Tyr- D -Lys(HSG)-NH2

N

Al N N 18F

O

Scheme 25 Preparation of [18F]-labelled aluminium conjugate.

(d,2n) reaction. The imaging characteristics of iodine-124 are not ideal as it has a complex decay scheme with many high energy γ emissions and positron emissions: only 23% of its decay leads to positron emissions [94]. However, data correction with modern PET scanners can solve most of the associated imaging problems. The half-life of 4.2 days allows imaging studies at longer timepoints necessary for tracers with slow pharmacokinetics such as antibodies. The strategies for synthesis of iodine-124-labelled compounds are the same as for iodine-123 tracers (vide supra). The utility of iodine-124 has recently been demonstrated in a study of colorectal cancer patients in which an 124I-labelled humanised anti-A33 antibody (hUA33) was used to determine the treatment strategy [95].

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O

18F

N N Al N

O O O

S NH NH O O NH2 HN O OH

NH O

O

HN

O HN

O

HN

NH

NH

O

O

O

NH O HO2C HN

HN O

O HN

HO

HN O

NH

O NH HO2C

NH

HN O

HN NH2

Figure 17 Structure of [18F]-alfatide.

[124I]huA33 is produced by direct electrophilic substitution on the tyrosine residues in the antibody. Na[124I] is added to a tube coated with the oxidant iodogen (1,3,4,6-tetrachloro-3α,6α-diphenylglycouril) followed by addition of a solution containing hUA33. The reaction is terminated by removal of the reaction mixture from the tube. Damage to the antibody is limited due to the poor water solubility of iodogen. PET/CT scans were carried out 1 week after injection and just before surgical removal of tumour. A linear relationship was observed between tissue A33 antigen and uptake of [124I]huA33 in both tumour and normal colon. The amount of [124I]huA33 in A33 antigen-positive colon tumour tissue was three times the amount in the average colon tissue section. As the A33 antigen-positive tumour regions were typically 1–2 mm in dimension, it was concluded that a lower energy/shorter range β emitter would be more suitable for radiotherapy. Due to the persistent tumour retention of [124I]huA33 and uptake in normal gut, a multistep approach to therapy

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was suggested. This could involve initial administration of a modified antibody construct with specificity for a small molecule. Injection of the smallmolecule radiotherapeutic would then take place after clearance of the antibody construct from non-tumour sites. 3.2.4 Zirconium-89 Tracers The most widely used cyclotron production method of zirconium-89 is from yttrium-89 using a (p,n) reaction. The cost of zirconium-89 is relatively low for a cyclotron produced isotope as the 100% naturally abundant yttrium-89 target requires no enrichment and so is commercially available. Although zirconium-89 has a relatively low probability of positron emission (23%), good-quality images can be obtained because of its relatively low energy (397 keV). Zirconium-89 forms complexes in which zirconium is present in the +4 oxidation state. Desferrioxamine (DFO) is the most widely used chelating agent (Figure 18) and the number of studies has increased significantly since bifunctional DFO-chelating agents became commercially available [21]. No zirconium-89 molecular imaging agent is commercially available although there are an increasing number of published preclinical and clinical studies with 89Zr-labelled radiotracers. There is some evidence of undesirable demetallation of the complexes in vivo. Zirconium is a bone seeker and demetallation can lead to increased radiation dose to bone marrow. As a result, several groups have recently H N

O

N

O

O

N

O 89Zr

HN

O

CH3

O O

O

N N H

Figure 18 Structure of 89Zr-desferrioxamine.

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N N

N

O

O

O

O

O O N

O

O

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89Zr

O

N

O

O

O

O

O

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89Zr

O

O

O

NH

N

O

O

N

O

N

Figure 19 Structures of improved 89Zr complexes.

developed new chelating agents for zirconium (Figure 19) that appear to form more stable complexes than those formed by DFO [96,97]. DFO provides only six of eight required donor atoms for zirconium. The improved stability of the new compounds is due to optimisation of the chelate ring size and complete satisfaction of the zirconium coordination sphere. Most clinical studies are with zirconium-89-labelled antibodies as the long half-life (78.4 h) allows imaging after several days. One recent study utilised 89Zr-trastuzumab, 89Zr-bevacizumab and 18F-FDG imaging to assess the ability of an HSP90 inhibitor to degrade client proteins in breast cancer patients [98]. HER-2 and ER oestrogen receptor are two important HSP90 client proteins involved in metastatic breast cancer. Imaging scans were performed at 2 and 4 days post-injections. One injection was carried out prior to therapy with the HSP90 inhibitor NVP-AUY922. A second injection was carried out 15 days later. 89Zr-trastuzumab tumour uptake was found to correlate with change in tumour size as measured by CT. Quantification of 89Zr-trastuzumab uptake in the tumour lesions showed heterogeneity within tumour lesions in the same and in different patients. No correlation was observed between 89Zr-bevacizumab uptake and tumour size. One reason for this could be that most 89Zr-bevacizumab tumour uptake was in bone lesions not measurable by CT. A clinical benefit was observed in 50% of heavily pretreated patients with progressive breast cancer although larger studies are required to confirm this. 3.2.5 Copper-64 Tracers The most widely used cyclotron production method of copper-64 is from nickel-64 using a (p,n) reaction. Copper-64 radiopharmaceuticals almost

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always contain copper-64 in the +2 oxidation state as complexes containing copper in the +1 oxidation state tend to be unstable in biological media. Macrocyclic chelating agents are used to prepare copper-64 radiopharmaceuticals as the resulting copper complexes are kinetically more stable than those formed with acyclic chelating agents. A variety of chelating agents have been developed in recent years to find ones which can form copper-64 complexes at relatively low temperatures, but which will still have sufficient kinetic inertness to be stable in vivo [99]. The focus of most of these efforts is now on cross-bridged macrocycles. A recent example of a cyclam-based chelating agent with pendant carboxylic and phosphonic acid groups (Figure 20) shows that high labelling yields of the peptide-derivatised chelating agents (>90%) can be obtained at close to room temperature after 1 h [100]. It has also been suggested, however, that copper-64 complexes can be formed at room temperature with good in vivo stability with more conventional macrocyclic chelating agents such as TETA (Figure 20), if high purity copper-64 is used [101]. There are still relatively few literature examples of the use of copper-64 radiopharmaceuticals in clinical trials. The relatively short half-life of 12.7 h means that iodine-124 and zirconium-89 tend to be favoured for antibody applications. However, a recent study in HER-2-positive metastatic breast cancer patients using 64Cu-trastuzumab shows that good-quality images can be obtained up to 48 h post-injection if 18F-FDG is used beforehand to determine tumour locations [102]. A trastuzumab dose of 50 mg before the 64Cu-trastuzumab injection was found to improve image quality by increasing blood retention and decreasing liver uptake.

O O

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O

P N

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O

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N N

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O O O

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CB-TE1A1P

Figure 20 Structures of

64

64Cu-TETA

Cu CB-TE1A1P and TETA.

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3.2.6 Gallium-68 Tracers Generator produced gallium-68 is an attractive isotope for PET imaging as the 271-day half-life of the parent germanium-68 means that generators can be used for approximately 1 year [103]. The gallium-68 generator contains germanium-68 absorbed on alumina, SnO2 or TiO2 columns. Dilute HCl solutions are used to elute the decay product gallium-68 from the column. The recent approval of the Eckert and Ziegler pharmaceutical grade gallium-68 generator in Europe is likely to increase the number of future clinical studies. Gallium-68 forms complexes in which gallium is present in the +3 oxidation state. DOTA is the mostly widely used chelating agent as the 68GaDOTA complex (Figure 21) has good stability in vivo [21,103]. Formation of the 68Ga-DOTA complex, however, requires heating to 90  C and as such is not suitable for the labelling of compounds which are sensitive to heat. In addition to the rediscovery of NOTA as a chelating agent for gallium-68 [104], a number of new chelating agents have recently been developed which can coordinate gallium to form more stable complexes than those formed with DOTA. Of particular interest are the TRAP and CP256 chelating agents illustrated in Figure 22[105,106]. 68Ga-TRAP complexes can be formed on heating in acid solution as low as pH 0.5 and TRAP can potentially be used to directly label acidic [68Ga]gallium trichloride solutions eluting from the generator. CP256 was found to complex gallium-68 at room temperature using lower chelating agent amounts than required to form gallium-68 complexes with established chelating agents such as DOTA and NOTA. Radiochemical yields of 68Ga-CP256 of greater than 70% could be obtained at chelating agent concentrations of just 1 μM. O O

N

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O N -

O

68Ga

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Figure 21 Structure of 68Ga-DOTA complex.

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O P

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OH O

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NH

O O

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O O

O

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N H

NH

N

CH3

O

O

O O

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68Ga-TRAP

Ga-NOTA

Figure 22 Structures of

68

68

Ga-CP256

Ga NOTA, TRAP and CP256.

3.2.7 Application of PET to Translational Medicine The chemical methodologies illustrated above demonstrate that there is a broad range of methods for introducing PET isotopes into molecules. The choice of chemistry and isotope is dependent on the application. There are four main areas for the application of PET in the drug development arena. These are to provide information on the biodistribution of a candidate; receptor occupancy studies that can be used to predict efficacy of drug candidates; monitoring of the effectiveness of therapy in early drug development phases; and as a method for pre-screening patient populations for clinical trials. Information on the biodistribution of a drug will require a radioactive analogue of the drug itself. This is most commonly done by the preparation of carbon-11 versions of the drug candidate or, if it has an accessible fluorine atom, fluorine-18 can be considered. Bergstr€ om et al. developed a radiosynthesis of [11C]-zolmitriptan for studying the biodistribution of the drug itself [107]. The study was aimed at demonstrating blood–brain barrier penetration of the drug. As most radiotracers are administered by bolus injection, delivery is usually very different to the drug studies and as such the tracer by itself cannot be used to measure bioavailability. In this study, two imaging measurements were carried out—one before and one 30 min after administration of the unlabelled drug. The difference in the uptake of the tracer was used to infer the biodistribution of the drug itself. Kiesewetter developed a radiosynthesis of racemic [18F]BMS-204352 for use in biodistribution studies to support the development of MaxiPost™ [108]. Radiometals may also be used for studying biodistribution of drugs. The therapeutic is likely to be a biologic as the biodistribution will be affected less by addition of a chelated metal. Roche is currently using a radiolabelled antibody against HER-3 89Zr-RO5479599 to provide information on their trial

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of RO5479599 alone or in combination with erlotinib or cetuximab in patients with HER-3-positive solid tumours [109]. One of the biggest applications of PET in translational medicine is in investigating the receptor occupancy of drugs and drug candidates. This has been used as an end point in a number of studies in neuropsychiatry in particular. The key aspect of use of PET in this arena is that there needs to be a valid biomarker of the receptor to be studied that is not structurally similar to the drug under study. This will ensure that any displacement of the tracer can be attributed to the drug. The antipsychotic drug quetiapine has been shown to have efficacy in a range of psychiatric disorders including schizophrenia, depression and anxiety. The drug has been shown to have high affinity for the 5-HT1A and dopamine D2 receptors, while its human metabolite, norquetiapine, also has high affinity to the norepinephrine transporter (NET). It was this latter property that was shown to be prevalent in a clinical study using the specific NET radiotracer (s,s)-[18F]FMeNER-D2 [110]. PET has also been used to study the pharmacokinetics of drugs. For example, the occupancy of dopamine D2 and D3 receptors by ziprasidone across a 23 h period has been reported. The occupancy was measured by assessing the uptake of [11C]raclopride, which binds to both D2 and D3 receptors. In this case, the occupancy half-life was found to be 8.3 h, data that support the clinical twice-daily treatment [111]. [11C]Raclopride has also been used to assess drug occupancy of the investigational antipsychotic drugs JNJ-37822681 [112], YKP1358 (structure not disclosed) [113] and BL-1020 [114] (Figure 23). More selective tracers for dopamine receptors

F

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N

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CF3

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F

N

Cl

BL-1020 NH2 N

H311C

O

N N

NH2 O

N H JNJ-37822681

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N N O N

[11C]SCH442416

Figure 23 Structures of JNJ-37822681, BL-1020 and [11C]SCH442416.

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can be used to improve the understanding of mechanisms of action; [11C] PHNO has been used to understand the effects of antipsychotics on the D3 receptor [115]. This study showed that the receptor was possibly upregulated after short-term treatment with either risperidone or olanzapine. Dose occupancy studies have been used to justify the continuation of development of compounds. [11C]SCH442416 has been used to assess the binding of vipadenant to the A2A receptor [116]. Applications of PET in translation medicine in oncology have focussed on the effectiveness of treatment. Several tracers that are proliferation markers have been developed, and the wide availability of [18F]FDG makes it a commonly used marker for therapeutic trials [117]. The application of other markers is much less widespread. One of the features of FDG is that it is cleared through the urinary system and so accumulates in the bladder. This makes it suboptimal for imaging bladder and prostate conditions. Imaging agents that are under evaluation in the clinic for monitoring the effectiveness of therapies for such conditions are those derived from choline—[11C]choline [118] and [18F]fluorocholine [119]—and the amino acid analogue [18F] fluciclovine (FACBC) [120]. These three tracers have been used in other oncology applications too. [18F]Fluorothymidine ([18F]FLT) uptake has been shown to change in response to treatment of patients with capecitabine for breast cancer [121], erlotinib for non-small cell lung cancer [122] and bevacizumab for recurrent malignant glioma [123] among others. There are opportunities to show effectiveness earlier in studies by using tracers that probe the mechanism of action of the drug. For example, the integrin binder [18F]fluciclatide is being used in the early determination of the effectiveness of anti-angiogenic therapies in clinical studies [124], following preclinical work showing the ability of the tracer to demonstrate the effectiveness of sunitinib in reducing microvessel density ahead of reduction of tumour volume [125]. A further application of imaging in translational medicine is as a method for selection of patients for therapy groups. This can be particularly useful if the aim of the therapy is to treat diseases in their early phases. An example of this is in the attempts to treat neurodegenerative disorders such as Alzheimer’s disease. It is known that some of the underlying pathology of the disease exists before any symptoms show [126]. This leads to the possibility that healthy volunteers may be on the path to the disease, thus having an adverse effect on the outcome of the drug under study. Merck are using this strategy in assessing the diagnosis of prodromal Alzheimer’s disease by using [18F]flutemetamol in the initial trial of MK-8931 [127]. Lilly used

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[18F]AV-45 ([18F]-florbetapir) as a secondary endpoint in the Phase III assessment of LY450139 [128]. Similar strategies could be employed in the development of treatment of Parkinson’s disease where it has already been shown that a reduction in uptake of [18F]F-dopa precedes observation of symptoms [129]. Clinical trials involving targeted gallium-68 tracers are dominated by the three somatostatin receptor imaging agents 68Ga-DOTA TOC, 68 Ga-DOTA TATE and 68Ga-DOTA NOC. Although not commercially available, all three tracers are now widely used in Europe for the diagnosis of neuroendocrine tumours (NET). The gallium-68 NET imaging agents have been used extensively during the development of the corresponding yttrium-90 and lutetium-177 radiotherapeutics. A recent study concluded that high baseline tracer uptake values obtained with 68Ga DOTA TOC imaging were suggestive of a better therapeutic response to 90Y/177Lu DOTA TOC radiotherapy in patients with hepatic metastases from gastro-entero-pancreatic NETs [130].

4. SUMMARY AND FUTURE PROSPECTS Imaging has the ability to show functional and biochemical changes that can help not only the understanding of disease mechanisms but also the response of the body to treatment. fMRI has the capability to look at these changes in real time, thereby providing a great opportunity to give an early indication of the effect of a putative treatment. The number of clinical trials that already have an fMRI study as part of the protocol indicates that the technique is making big strides in its impact on drug development. This is likely to continue as instrument sensitivity improves and the development of new sequences enables the extraction of more information from endogenous molecules. There is also the prospect of the introduction of molecular imaging techniques; use of hyperpolarised carbon-13 pyruvate has been tried [131] and there is potential of using fluorine-19 [132], but these techniques are some way in the future. Nuclear medicine is a little further back in the scale of its adoption in translational medicine. There are a few reasons for this. Despite the large installed base of SPECT cameras and the ready availability of radionuclides for the modality, there has been less emphasis on the development of SPECT radiotracers for molecular imaging techniques than for PET. This has been due to the reduced accuracy of absolute quantification available for SPECT until the recent development of improved cameras and software. In addition, the

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isotopes available for SPECT are less amenable for application to small molecules, though we have shown some examples of compounds that meet this criterion. As the pharmaceutical industry increases the use of peptides, proteins, antibodies and even cells, these drawbacks become less relevant. PET has long been considered by the nuclear medicine community as the tool that can deliver information at a biochemical level, and as such a great deal of research has gone into the development of tracers that can indicate the mechanism of action of compounds by use of labelled analogues of drugs and observing the effect of such drugs on the biochemistry of the body. Much of this early work has been done using carbon-11 as the reporter. The major challenge in using this radionuclide is the short half-life that has prevented widespread use in large clinical trials. Fluorine-18 has been the main radionuclide of choice for the development of radiotracers due to its longer half-life. This makes the application of PET to large-scale multi-centre clinical trials feasible. As well as the ability to detect changes in the biochemistry of disease early, PET can be used to select clinical trial groups based on the underlying pathology they show. Many neurodegenerative diseases remain asymptomatic until the disease is well advanced; inclusion of patients with early stage in control groups can mask the benefits of a treatment in the treated group. Although a disadvantage in this case, PET could be used in the future to identify asymptomatic patient groups that would benefit from treatment. Recent advances in nuclear medicine imaging highlighted in this review provide the opportunity for the use of radiotracers in the development of small molecule drugs with the use of iodine-123, carbon-11 and fluorine18-labelled molecules and peptide therapies and biopharmaceuticals through labelling with technetium-99m, gallium-68 and zirconium-89. Following the biochemical changes in response to drug treatment gives the possibility for early indications of clinical efficacy; it is anticipated that the application of imaging in this area will continue to grow.

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