click to enlarge Figure 1. Palladium mediated electrophilic fluorination produces novel PET tracers. (Source: SciFluor)

A number of recently approved cancer drugs have utilized a companion biomarker test for patient selection. Crizotinib was approved for ALK-positive non-small cell lung cancer and vemurafinib for BRAF V600E-positive melanoma. Both of these agents were approved in less than half the average time needed to approve the oncology drugs in the 1990s.¹ The success of these targeted therapies is predicated on the ability to identify patients—by the use of in vitro diagnostic assays—whose tumors harbor a genetic mutation making them highly responsive to the targeted treatment.

These targeted therapies, however, require that a patient’s diseased tissue be readily available for testing. Unfortunately, this is not always the case leaving molecular imaging using single photon emission computed tomography (SPECT) or positron emission tomography (PET) to fill in the gaps. SPECT and PET are increasingly being explored as non-invasive tools to help characterize the status of patients’ disease. The PRECEDENT study² used molecular imaging to identify a sub-population of ovarian cancer patients who are more responsive to EC145, a folate-receptor targeted vinblastine analog.  The imaging agent, EC20, is now being developed as an in vivo imaging companion diagnostic.

PET imaging involves the injection of small quantities of short-lived radiotracers that emit positrons during radioactive decay. The positrons undergo annihilation within a radius of the point of emission and each annihilation event results in the emission of two 511 keV photons at an 180 degree relative orientation that can be readily detected by external imaging cameras specifically designed for either preclinical studies in small animals or human clinical studies. Suitable positron-emitting isotopes include fluorine-18 (half-life: 110 minutes), carbon-11 (half-life: 20 minutes), and nitrogen-13 (half-life: 10 minutes), with fluorine-18 being the most widely used because of its longer half-life and resulting wider availability.

The application of PET imaging to drug discovery and development is finding wide-spread utility in multiple therapeutic areas and encompasses three broad categories:

• Justification for a biological target for therapeutic intervention. This involves measurement of target level or function, or change in level/function in disease or with therapeutic intervention. A classic example is the use of fluorine-18-fluorodopa in elucidating the role of dopamine in schizophrenia.³
• Determining the biodistribution of a new drug, e.g. blood-brain barrier transit, target engagement, and normal tissue localization and excretion. Target engagement refers to the establishment that the drug is interacting with the desired biological target. Establishing a link between target engagement and a biologic change that is expected to give a clinical benefit—termed proof of biology—is a significant milestone in drug development.
• Rational therapeutic dosing and clinical proof of concept. PET imaging is used to determine receptor occupancy and guide clinical-dosing studies, especially for psychotropic drugs. PET imaging can help identify the optimum therapeutic dose in fewer dose cohorts, expediting clinical proof-of-concept when engagement of the target is linked to a clinical-efficacy endpoint. PET imaging can also determine whether the receptor occupancy will allow clinical benefit to be achieved within the maximum tolerated dose. If not, further development can be terminated (i.e. a “quick kill” of a drug that will not be successful). Alternatively, PET imaging that demonstrates substantial receptor occupancy in patients that show no clinical benefit can provide the rationale for rejecting a disease mechanism, as in the role of the neurokinin-1 receptor in depression.⁴

These applications have spurred rapid growth in the use of PET imaging in drug discovery and development. Of the currently recruiting clinical trials listed on ClinTrials.gov, over 700 utilize PET imaging. While a majority of these studies utilize well-established radiotracers, such as fluorine-18-fluorodeoxyglucose (FDG), there are approximately 40 different fluorine-18 radiotracers in use, roughly evenly distributed between oncology and neurology applications.

The clinical studies that are utilizing fluorine-18 tracers fall into four general types: therapy planning, either for pharmaceutical therapy or radiation therapy; predictive- or response-marker studies for a new pharmaceutical using an established tracer; development of a new tracer for clinical-disease diagnosis; and development of a new biomarker for pharmaceutical therapy. The first two involve tracers with previous clinical experience while the latter include a number of Phase 1 studies to determine the safety, dosimetry, and optimum imaging parameters of a new tracer. The clinical application may be to ultimately use the new tracer as an approved diagnostic tool for disease diagnosis or for use as a clinical research and development tool. The latter category represents the most active area, a reflection of the strong interest in the use of fluorine-18 PET imaging in drug discovery and development.

In addition to the increasing use of fluorine-18 tracers in clinical development studies, there is a rapid expansion in the number of reports of preclinical testing of new fluorine-18-labeled molecules. In 2011 alone, the synthesis or preclinical testing of approximately 200 new or recently discovered fluorine-18 tracers were reported.

The most common biological targets or processes for the new tracers in oncology include: apoptosis, hypoxia, integrins (angiogenesis), and transporters that are up-regulated to meet the metabolic demand of the tumor cells for glucose, glutamate, and L-type amino acids. In neurological diseases, there are a wide variety of targets for which new tracers are being designed and tested. Particularly active areas of research involve the 18 kDa translocator protein for looking at neurodegenerative processes; glycine tranporter-1 for schizophrenia; metabotropic glutamate receptors-1 and -5 for a variety of neurological and psychiatric conditions. such as epilepsy and addiction; the dopaminergic system for Parkinson’s disease; and the serotonin system for depression. In addition, a number of fluorine-18 tracers are being investigated as markers of β-amyloid deposition—a hallmark of Alzheimer’s disease. This interest is in part driven by the recent FDA approval of florbetapir for clinical diagnostic use, as well as by the use of florbetapir and several other development-stage, amyloid-binding tracers as response markers in clinical trials for anti-amyloid directed therapies. 

Fluorine-18 chemistry methodologies are an active area of research, aimed at improving the selectivity and yield of the fluorination reaction as well as the specific activity [ratio of fluorine-18 atoms to total fluorine (18F plus 19F)] of the resulting tracer. While considerable effort is directed at identifying new methods to incorporate fluorine-18 into peptides and antibody fragments—driven by the expanding interest in biologic drugs for treating a variety of illnesses—there remains a significant need for improved methods for incorporating fluorine-18 into small-molecule drugs.

Nucleophilic substitution methods are predominantly utilized to synthesize fluorine-18 small-molecule radiotracers and most commonly incorporate fluorine-18 into an alkyl or alkoxy substituent on the drug. Nucleophilic substitution methods have limited application for adding fluorine-18 to aromatic rings, a very common feature in small-molecule therapeutics. The aromatic rings need to bear electron-withdrawing substituents in the ortho and/or para positions to the site of substitution. This has led to the development of electrophilic methods of incorporating fluorine-18 into aromatic rings, for example, using fluorine-18-fluorine gas; however, the methods utilized to date result in low yields and the formation of unwanted by-products. 

A versatile method of performing electrophilic substitution of fluorine-18 on aromatic rings in high yield and with high specificity using palladium chemistry has been developed.⁵ The method involves the use of commercially available, cyclotron-generated fluorine-18 -fluoride to form a palladium (IV)-fluorine-18 complex that serves as the electrophilic fluorination reagent. The palladium (IV)-fluorine-18 complex is then reacted with the precursor to the fluorine-18 radiotracer, a palladium (II) aryl complex, in which the position to be fluorinated is bonded to the palladium (II) center, to produce the fluorine-18 radiotracer (see Figure 1).  This chemistry allows the synthesis of fluorine-18 radiotracers that have not been accessible previously. The biological targets of fluorine-18 radiotracers include the serotonin 5HT2c receptor for neurological imaging applications and PI3Kγ for inflammation imaging. The technology has been shown to result in radiotracers that have utility in imaging biological targets in animal models. An extensive effort to synthesize additional fluorine-18-labeled molecules is underway at SciFluor Life Sciences LLC, the licensee of the technology.

SciFluor is attempting to discover small-molecule drugs that have improved pharmacological properties due to the strategic incorporation of fluorine into the molecule. This approach allows registered medicines—compounds in clinical development that have established clinical proof-of-concept—to be optimized to generate new preclinical candidates without an extensive drug discovery effort. The preclinical and clinical development of these new chemical entities are able to leverage the mechanistic and clinical development knowledge of the parent compounds; in many cases, the development of the fluorinated drugs can be expedited by the use of fluorine-18 radiotracers.

The advantages of molecularly targeted therapeutics are being realized with highly efficacious medicines for treating major diseases. This success is driving an expanded effort to discover and develop new technologies to identify and characterize new targets for treatment and to understand the biodistribution, targeting, and pharmacodynamics of new targeted drugs. PET imaging is being applied to everything from characterization of disease models to supporting establishment of proof of biology to use in selection of patients for the treatments. The increasing use of PET imaging has spurred a dramatic increase in the number of novel fluorine-18 tracers studied in preclinical models and advancing into clinical studies to aid drug development. New methodologies for synthesizing fluorine-18 tracers are being developed to support this expansion, including highly innovative chemistry to incorporate fluorine-18 into a wider array of small-molecule drugs.

References
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2. ClinicalTrials.gov. Bethesda (MD): National Library of Medicine (US). Identifier NCT00722592, Platinum resistant ovarian cancer evaluation of Doxil and EC145 combination therapy (PRECEDENT). Available from: http://clinicaltrials.gov/ct2/show/NCT00722592?term=NCT00722592&rank=1.
3. Breier A, et al. Schizophrenia is associated with elevated amphetamine-induced synaptic dopamine concentration: Evidence from a novel positron emission tomography method. Proc Natl Acad Sci USA. 1997; 94: 2569–2574.
4. Yasuno F, et al. PET imaging of neurokinin receptors with [(18)F]SPA-RQ in human subjects: assessment of reference tissue models and their test-retest reproducibility. Synapse. 2007; 61(4):242-251.
5. Lee, S, Kamlet, A, et al. A fluoride-derived electrophilic late-stage fluorination reagent for PET imaging. Science. 2011; 334(4):639-642.