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Small Molecule PET Tracers in Drug Discovery
Small Molecule PET Tracers in Drug Discovery David J. Donnelly, PhD The process of discovering and developing a new pharmaceutical is a long, difficult, and risky process that requires numerous resources. Molecular imaging techniques such as PET have recently become a useful tool for making decisions along a drug candidate’s development timeline. PET is a translational, noninvasive imaging technique that provides quantitative information about a potential drug candidate and its target at the molecular level. Using this technique provides decisional information to ensure that the right drug candidate is being chosen, for the right target, at the right dose within the right patient population. This review will focus on small molecule PET tracers and how they are used within the drug discovery process. PET provides key information about a drug candidate’s pharmacokinetic and pharmacodynamic properties in both preclinical and clinical studies. PET is being used in all phases of the drug discovery and development process, and the goal of these studies are to accelerate the process in which drugs are developed. Semin Nucl Med 47:454–460 © 2017 Published by Elsevier Inc.
Introduction
O
ver the past decade, remarkable progress in drug discovery and development has resulted in novel lifesaving drugs that have provided relief and hope to millions of patients with varying disease conditions. However, the process of discovering and developing new drugs remains a risky and costly endeavor. The cost of developing a drug to market approval is now estimated to be between $2.6-2.9 billion, with development timelines between 5 and 10 years.1 With these costs steadily rising over recent years, a recent trend within the field of drug discovery and development has been focused on translation techniques that can provide decisional information to reduce both the cycle times and costs associated with this process. Molecular imaging techniques such as PET can significantly impact productivity and lower development costs by ensuring a new drug has target engagement, proper receptor occupancy, and guides dose selection in a more rapid timeframe.2-5 PET imaging is a translational medicine approach that provides quantitative information along multiple time points of a new drugs development timeline. PET imaging is an attractive tool because the same tracer and imaging technique can be used in both preclinical and clinical settings. PET allows researchers to bridge important information gaps between the preclinical and clinical reBristol-Myers Squibb Pharmaceutical Research and Development, Princeton, NJ. Address reprint requests to David J. Donnelly, PhD, Discovery Chemical Platforms-PET Radiochemical Synthesis, Bristol-Myers Squibb Pharmaceutical Research and Development, P.O. Box 4000, Princeton, NJ 08543. E-mail: [email protected]
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search. Very promising studies using PET tracers based on antibody frameworks have provided insight into the drug development process.6 However small, synthetic molecules remain prevalent in many companies’ development pipelines and as marketed drugs. These small molecules remain an important part of the modern drug development pipeline and consistently deliver results. This review will focus on an overview of the drug discovery and development process with a focus on how small molecule PET tracers can provide information focused on target identification, lead optimization, and clinical proof of concept of a new drug. This review is not intended to be a comprehensive overview of the use of molecular imaging techniques in drug trails but rather provide some examples of how PET can be used in various places within the drug discovery and development process. Also, many great reviews have covered the topic of [18F]FDG and its impact on drug development; however, this review will focus on small molecule PET tracers outside of [18F]FDG.7-10
The Drug Discovery and Development Process The drug discovery and development process can be characterized by several stages as shown in Figure 1. The goal of this process is to produce the right drug, at the right dose, chosen for the right target within the proper patient population. The discovery of a new drug typically begins with identifying a target that is expressed within a disease condition. For every disease condition there are a large range of known and emerging biochemical hypotheses based on the
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Figure 1 Phases of drug discovery and development.
known biology of a target. From these hypotheses, targets are identified using knowledge of the underlying pathophysiological processes of a disease condition and how a potential drug can interact with this target to reverse the disease conditions effects. The sequencing of the human genome has broadened the number of potential drug targets within drug pipelines.11 Validation of a new drug target is a challenging process, as the development of cellular- or tissue-based assays and animal models that mimic human disease condition are difficult to develop. Once a proper assay or animal model has been identified, libraries of compounds are synthesized and tested, with the goal of generating a lead series of molecules that bind effectively to target. Once a primary lead compound or series of compounds have been identified for this target, the next phase of the process is lead discovery and optimization. Lead optimization itself can be a lengthy, complex nonlinear process. This phase includes refining a series of chemical structures that interact with the target to improve a molecule’s characteristics as a potential drug. As molecules progress through the process from early discovery to preclinical discovery, they are prioritized as function of their binding profiles, pharmaceutical properties, and efficacy in animal models. Thousands of potential drug candidates are narrowed down during this process until the optimal candidate is chosen to move forward into the translational space. Early development studies include safety and tolerability (phase 1), proof of concept, and dose finding studies (phase 2). The drug candidate then enters full development and efficacy studies in phase 3, followed by compound registration, drug approval, and product launch. As part of life cycle management, the drug is monitored in postmarketing or postapproval studies. As a drug candidate progresses from early to late drug development, the costs associated with these clinical studies increases steadily as the number of patients and safety monitoring requirements also increase. Because the costs of clinical drug development increase from phase 1 to 3 so dramatically, it is important that biomarkers that allow companies to make informed decisions early in the process are in place. By discontinuing programs that do not have the characteristics for a successful drug and being able to reinvest funds into programs that have higher potential for success can result in a large research and development cost saving. In a retrospective review of several of its drug candidates, a group of Pfizer scientists looked at ways success rates can be improved for drugs in phase 2 of clinical development.2 In this retrospective review of both successful and failed drug candidates, the authors suggest that “The highest level of
confidence and direct evidence at the site of action that required levels of target binding were being achieved is most probably obtained from pharmacokinetics (PK)/ pharmacodynamic (PD) studies of in vivo occupancy measurements with positron emission tomography (PET) or radiolabeled ligands.”
PET Within Drug Discovery and Development Generally, there are three main approaches for the use of PET tracers in the drug discovery and development process. The first is using a radiolabeled candidate to evaluate the distribution and pharmacokinetics of the drug candidate. For small molecules PET tracers, direct labeling with short-lived PET radionuclides such as replacing a native carbon (C-12) with carbon-11 (20 minute half-life) or a native fluorine (F19) with fluorine-18 (109.5 minute half-life) is important to keep the physiochemical properties of the molecule the same. Recent advancements in PET radiochemistry have helped make this process more feasible.12-20 Figure 2 highlights some recent advancements in carbon-11 and fluorine-18 chemistry that have generated several PET-labeled drug candidates from the literature.21-26 The second approach to using PET tracers in drug development is to use a radioligand with known affinity for the molecular target of interest to evaluate the PD properties of the drug candidate. In this case, the PET tracer is no longer the drug candidate and is either a derivate of the main drug or a molecule that is known to bind to the target. The tracer database initiative that is sponsored by the National Institute of Mental Health and the Society for NonInvasive Imaging in Drug Development and the Molecular Imaging and Contrast Agent Database are excellent resources to search for specific PET tracers for a given drug target. Finally, a small molecule PET tracer is used as an imaging biomarker of a known biochemical, metabolic, or physiological process (such as FDG) to evaluate efficacy of a candidate drug.
PET Within Early Discovery PET tracers can provide useful information at many points within the drug discovery and development process. PET tracers can confirm the newly identified drug targets within
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Figure 2 Examples of 11C- and 18F-labeled drug candidates.
cells and tissues of known expression.27 As shown in Figure 3, techniques such as in vitro and ex vivo autoradiography can be used to detect the presence of drug targets in cells and determine their spatial and temporal distribution within tissues. Figure 3A shows a generic example of a PET tracer that has been incubated with cells that express (+) and do not express (−) a new drug target. A successful new drug target will be one that has retained the PET ligand in this assay within the cell line that expresses the target and does not retain radioactivity in the cell lines lacking the target. To further validate this new drug target, these cells can be preincubated with a compound that is known to bind to this target in a dosedependent manner. Figure 3A shows that incubating a blocking compound in increasing concentration can reduce the amount a PET tracer can bind in each cell chamber. One of the major questions posed at this stage of the process is if a target is expressed in the human disease state. Figure 3B shows how using human biopsy tissues and tissue-based autoradiography approach for a PET tracer can provide important information for a drug target. In this generic example, a fluorine-18-labeled PET tracer is incubated with a human tumor tissue slice and then exposed to an autoradiography film to generate the image seen in Figure 3B. In this example, the PET tracer displays significant binding to this tissue, confirming the target is expressed in this tissue. Given the short 109.5 minute half-life of fluorine-18, this same tissue was used to confirm target expression using immunohistochemistry several days later. The power of this technique is by using the same human tissues, we can confirm radioligand and correlate this binding to known histology stains. The expression of the drug target can also be confirmed in the disease condition using these human tissues. This can be used to validate the PET tracer for future translational imaging studies as well. This technique is by no means a replacement for high throughput assays in this phase of the drug discovery process, as they
are also needed to validate a new drug target.28 PET tracers or tritium-labeled radioligands are used to confirm that cellular assays and animal models developed for target indeed express the target of interest. PET tracers can help medicinal chemistry teams by prioritizing lead chemical series (chemotypes) and provide important decisional information regarding which of these series are to progress along the process. A generic example of this is shown in Figure 4. In this hypothetical example, a drug program needs to obtain >90% receptor occupancy for drug efficacy to be achieved in a non-human primate. Images A-C represent longitudinal studies in a non-human primate. In this example, a baseline image is obtained in a nonhuman primate, followed by that same animal being scanned again after administration of increasing concentrations of a lead compound of series A, B, or C. From the chart below, the medicinal chemist would receive important information regarding which of these series to advance. It would be clear in this hypothetical example that chemotype C would differentiate itself against the other series of compounds.
PET Within Lead Discovery and Early Clinical Development Once a drug candidate reaches the lead optimization phase of this process, the discovery team begins planning biomarker studies in both preclinical and clinical studies to understand the drug’s characteristics, such as absorption, biodistribution, metabolism, delivery, and doses for first use in humans. It is at this stage where PET tracers are the most valuable. PET tracers can be used in both preclinical animal models to confirm a drug’s target engagement, receptor occupancy, dose selection, and dose ranges to provide information to plan focused human studies in early development. These
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Figure 3 Examples of [18F]-labeled PET tracers in target identification and early discovery phases of drug discovery. (A) Represents an example of cell-based autoradiography and (B) represents an example of tissue-based autoradiography.
same studies with the same tracer can be planned in human studies with high confidence of their success. PET tracers can also provide biodistribution in higher species animals such as non-human primates and provide important metabolism data for a lead molecule at this stage of the process. PET tracers used with the genetically engineered mice or disease stateinduced rodents that mimic many aspects of human diseases can provide a valuable physiologically based platform for deciding which molecules are most likely to succeed and just as important, drugs that are likely not to succeed. As shown in Figure 1, the information obtained from using a PET tracer within lead optimization provides an import feedback loop to the early discovery portion of the process. PET imaging can feedback information to back up programs to deliver a higher quality drug candidate for these programs. In early clinical development, because PET tracers are generated in highly specific activities (>1 mCi/nmol), microdosing
studies in humans can generate a noninvasive image of the molecule’s biodistribution, delivering only nano- to picomoles of the drug molecule to the patient. This makes it ideal for phase 0 clinical microdosing studies. Valuable information regarding PK, bioavailability, biodistribution, and targeting properties of new drugs, in a limited number of human volunteers, is obtained. A recent study has shown that up to 40% of drug candidates fail in phase 1 or 2 clinical trials due to poor pharmacokinetics and pharmacodynamics.29 PET tracers, using a microdose approach, allows for a direct assessment of biodistribution, metabolism, and dose selection both preclinically and clinically to help improve this success rate. Target engagement and establishing a drug candidate that can localize within a tissue of known target expression with confidence is a critical point for a potential drug at this phase of development. A dynamic PET biodistribution study using a PET-labeled drug candidate can provide concentration-
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Figure 4 Examples of determining receptor occupancy to prioritize three chemical series as a function of receptor occupancy in a non-human primate. The dotted red line represents the occupancy needed for efficacy. PET image A represents a single slice image of a non-human primate brain at baseline, image B represents the same non-human primate upon pharmacologic challenge of a lead molecule at 25% occupancy, and image C represents the same nonhuman primate upon pharmacologic challenge of a lead molecule at 90% occupancy.
time course information in the tissue of interest as well as non–target expressing tissues. Additionally, tissues of known toxicity can be examined for accumulation of the radiolabeled drug candidate as a measure of safety. This information can be decisional, as only the most promising candidates that localize in tissues of known expression are carried forward to reduce lengthy and costly clinical phases. Also, in these clinical phases, PET tracers can be used as imaging biomarkers that can, often quantitatively, assess the clinical efficacy of the drugs being tested and thus provide important tools for the assessment of clinical end points. The most common use for this PET biodistribution or target engagement study has been the development of central nervous system drugs.30-33 Recently, PET tracers have been used to optimize oncology, immunology, fibrosis, and heart failure drugs.3,34-42 A good example of using a small molecule PET tracers to guide drug development is [18F] substance-P antagonist-receptor quantifier ([18F] SPA-RQ). [18F] SPA-RQ is a small molecule PET tracer that binds to the neurokinin-1 (NK1) receptor.43 In this study, [18F] SPA-RQ was used in human imaging studies to determine the NK1 receptor in the normal human brain and the receptor occupancy of the drug candidate aprepitant. The researchers examined the relationship between the plasma concentration of aprepitant and the NK1 receptor occupancy using [18F] SPA-RQ. Also, the distribution of NK1 receptors in the central nervous system of healthy male volunteers was evaluated using [18F]SPA-RQ before and after treatment with placebo or increasing doses of aprepitant. A clear concentration vs receptor occupancy relationship was observed using this small molecule PET tracer confirming target engagement. The researchers used this PET imaging data to show that high levels of NK1 receptor occupancy (>90%) were required to achieve optimal antiemetic effects for the drug candidate. This study also guided dose selection for this drug candidate in an an-
tidepressant trail, which led to a no-go decision for that indication.44 By using this approach, the right drug, the right target in the right patient population was used, and further clinical studies for the flawed indication were also avoided. Table provides several examples of small molecule PET tracers that are used for evaluating the metabolic or physiological processes, binding of a drug candidate to key receptors, and assessment of PK profiles within early clinical drug development.
PET Within Full Clinical Development Small molecule PET tracers can be used in late-phase clinical development as surrogate markers of response and to show pharmacologic differentiation of an asset from a drug already on the market. Additional PET can show differentiation when a new competitor drug comes to the market. Patient selection, using PET to select the proper patient population that will benefit from the drug candidate, is a powerful technique used during full clinical development of a drug candidate. This is a personalized medicine approach and allows researchers to ask mechanism-based questions within patient populations. This approach can determine if a drug is relevant within a disease population which is key to the proposed mode of action of a drug. An example of this strategy is a study with [11C]Docetaxel.57 Docetaxel is an effective drug for the treatment of patients with several advanced malignancies. However, tumor resistance to docetaxel may be associated with reduced drug concentrations in tumor tissue. In this study, a PET microdosing study with [11C]docetaxel in patients with lung cancer could be used to reliably predict tumor uptake of docetaxel during chemotherapy, which was correlated with tumor response to docetaxel therapy. This proof of concept
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Table Small Molecule PET Tracers Used in Drug Development Studies Target
Description
Indication
Radioligand
Reference
Dopamine Serotonin Opioid Gaba NK1 αVβ3 Glucose Thymidine kinase 1 Hypoxic cells Tubulin Estrogen receptor PSMA BCR-ABL and Src kinases
D2/3 receptor SERT µ Opioid GABA receptor NK1 αVβ3 Inhibitor Metabolic Proliferating tissue Hypoxia PK of taxol PK of tamoxifen PSMA expression PK of dasatinib
Neuroscience Neuroscience Neuroscience Neuroscience Neuroscience Cancer/fibrosis Cancer/neuroscience Cancer Cancer Cancer Cancer Cancer Cancer
[11C]Raclopride [11C]DASB [11C]Carfentanil [11C]Flumazenil [18F]SPA-RQ [18F]Alfatide [18F]FDG [18F]FLT [18F]FMISO [18F]FluoroTaxol [18F]Tamoxifen [18F]DCFPyL [18F]Dasatinib
45 46 47 48 43 49 50 51 52 53 54 55 56
DASB, 3-amino-4-(2-dimethylaminomethylphenylsulfanyl)-benzonitrile; DCFPyl, 2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}ureido)-pentanedioic acid; FLT, 18F-fluorothymidine; FMISO, fluoromisonidazole; GABA, Gamma-aminobutyric acid; PSMA, prostate-specific membrane antigen; SERT, Serotonin transporter.
study showed that patient’s response to a treatment could be predicted by a PET imaging study. Additionally, in a longitudinal study using a progesterone analog 21-(18)Ffluoro-16α,17α-[(R)-(1'-α-furylmethylidene)dioxy]-19norpregn-4-ene-3,20-dione ([18F]FFNP), researchers showed that uptake in mammary tumors predicts tumor response to estrogen-deprivation therapy.58 Both of these examples provide drug developers important insight into a drug candidate using PET and generate decisional information to help drive the process.
Summary Numerous aspects of drug discovery and development are enabled by the use of small molecule PET tracers. These tracers deliver answers to vital questions regarding the discovery of novel therapeutic targets. PET imaging provides an understanding of the pathophysiology of diseases, and helps identify and characterize new drug candidates. This technique is also used to validate the efficacy of a new drug candidate and evaluates both PK and PD parameters noninvasively. Thus, advanced radiolabeling techniques that provide new small molecule tracers have become a major priority for the pharmaceutical industry. Imaging with small molecule PET tracers has become a valuable tool to medicinal chemists and biologist, generating information regarding the pharmacology, biology, and toxicology of a drug candidate. This information is essential to decide which drug candidates are worth developing and which ones are not. The goal of using PET within the development process is to save both time and money. In summary, drug companies are searching for techniques that can reduce the time and the cost it takes to develop a drug and ways to efficiently use resources. Molecular imaging techniques such as PET are being used in all phases of the drug discovery and development process, and these techniques are changing the way drugs are developed.
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D.J. Donnelly 40. Chang JC, Gambhir SS, Willmann JK Imaging techniques in drug development and clinical practice, 2012 41. Holland JP, Cumming P, Vasdev N: PET of signal transduction pathways in cancer. J Nucl Med 53:1333-1336, 2012 42. Kircher MF, Hricak H, Larson SM: Molecular imaging for personalized cancer care. Mol Oncol 6:182-195, 2012 43. Hargreaves R: Imaging substance P receptors (NK1) in the living human brain using positron emission tomography. J Clin Psychiatry 63:18-24, 2002 (suppl 11) 44. Frank R, Hargreaves R: Clinical biomarkers in drug discovery and development. Nat Rev Drug Discov 2:566-580, 2003 45. Kimura Y, Maeda J, Yamada M, et al: Measurement of psychological state changes at low dopamine transporter occupancy following a clinical dose of mazindol. Psychopharmacology 234:323-328, 2017 46. Kim E, Howes OD, Kim B-H, et al: Regional differences in serotonin transporter occupancy by Escitalopram: An [11C]DASB PK-PD study. Clin Pharmacokinet 56:371-381, 2017 47. Demopulos GA, Gaitanaris GA, Ciccocioppo R; Omeros Corporation, USA. Assignee. Treatment of addiction and impulse-control disorders using PDE7 inhibitors. US patent US20130267502A1, 2013 48. Vivash L, Gregoire M-C, Lau EW, et al: 18F-flumazenil: A γ-aminobutyric acid A-specific PET radiotracer for the localization of drug-resistant temporal lobe epilepsy. J Nucl Med 54:1270-1277, 2013 49. Guo J, Guo N, Lang L, et al: 18F-alfatide II and 18F-FDG dual-tracer dynamic PET for parametric, early prediction of tumor response to therapy. J Nucl Med 55:154-160, 2014 50. Kurland BF, Peterson LM, Lee JH, et al: Estrogen receptor binding (18F-FES PET) and glycolytic activity (18F-FDG PET) predict progression-free survival on endocrine therapy in patients with ER+ breast cancer. Clin Cancer Res 23:407-415, 2017 51. Menda Y, Ponto LLB, Dornfeld KJ, et al: Kinetic analysis of 3'-deoxy-3'18F-fluorothymidine (18F-FLT) in head and neck cancer patients before and early after initiation of chemoradiation therapy. J Nucl Med 50:1028-1035, 2009 52. Hernandez-Agudo E, Mondejar T, Soto-Montenegro ML, et al: Monitoring vascular normalization induced by antiangiogenic treatment with 18F-fluoromisonidazole-PET. Mol Oncol 10:704-718, 2016 53. Hsueh W-A, Kesner AL, Gangloff A, et al: Predicting chemotherapy response to paclitaxel with 18F-fluoropaclitaxel and PET. J Nucl Med 47:1995-1999, 2006 54. Linden HM, Kurland BF, Peterson LM, et al: Fluoroestradiol positron emission tomography reveals differences in pharmacodynamics of aromatase inhibitors, tamoxifen, and fulvestrant in patients with metastatic breast cancer. Clin Cancer Res 17:4799-4805, 2011 55. Szabo Z, Mena E, Rowe SP, et al: Initial evaluation of [(18)F]DCFPyL for Prostate-Specific Membrane Antigen (PSMA)-targeted PET imaging of prostate cancer. Mol Imaging Biol 17:565-574, 2015 56. Dunphy MPS, Zanzonico P, Veach D, et al: Dosimetry of 18F-labeled tyrosine kinase inhibitor SKI-249380, a dasatinib-tracer for PET imaging. Mol Imaging Biol 14:25-31, 2012 57. van der Veldt AAM, Lubberink M, Mathijssen RHJ, et al: Toward prediction of efficacy of chemotherapy: A proof of concept study in lung cancer patients using [11C]docetaxel and positron emission tomography. Clin Cancer Res 19:4163-4173, 2013 58. Chan SR, Fowler AM, Allen JA, et al: Longitudinal noninvasive imaging of progesterone receptor as a predictive biomarker of tumor responsiveness to estrogen deprivation therapy. Clin Cancer Res 21:1063-1070, 2015
Introduction
O
ver the past decade, remarkable progress in drug discovery and development has resulted in novel lifesaving drugs that have provided relief and hope to millions of patients with varying disease conditions. However, the process of discovering and developing new drugs remains a risky and costly endeavor. The cost of developing a drug to market approval is now estimated to be between $2.6-2.9 billion, with development timelines between 5 and 10 years.1 With these costs steadily rising over recent years, a recent trend within the field of drug discovery and development has been focused on translation techniques that can provide decisional information to reduce both the cycle times and costs associated with this process. Molecular imaging techniques such as PET can significantly impact productivity and lower development costs by ensuring a new drug has target engagement, proper receptor occupancy, and guides dose selection in a more rapid timeframe.2-5 PET imaging is a translational medicine approach that provides quantitative information along multiple time points of a new drugs development timeline. PET imaging is an attractive tool because the same tracer and imaging technique can be used in both preclinical and clinical settings. PET allows researchers to bridge important information gaps between the preclinical and clinical reBristol-Myers Squibb Pharmaceutical Research and Development, Princeton, NJ. Address reprint requests to David J. Donnelly, PhD, Discovery Chemical Platforms-PET Radiochemical Synthesis, Bristol-Myers Squibb Pharmaceutical Research and Development, P.O. Box 4000, Princeton, NJ 08543. E-mail: [email protected]
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search. Very promising studies using PET tracers based on antibody frameworks have provided insight into the drug development process.6 However small, synthetic molecules remain prevalent in many companies’ development pipelines and as marketed drugs. These small molecules remain an important part of the modern drug development pipeline and consistently deliver results. This review will focus on an overview of the drug discovery and development process with a focus on how small molecule PET tracers can provide information focused on target identification, lead optimization, and clinical proof of concept of a new drug. This review is not intended to be a comprehensive overview of the use of molecular imaging techniques in drug trails but rather provide some examples of how PET can be used in various places within the drug discovery and development process. Also, many great reviews have covered the topic of [18F]FDG and its impact on drug development; however, this review will focus on small molecule PET tracers outside of [18F]FDG.7-10
The Drug Discovery and Development Process The drug discovery and development process can be characterized by several stages as shown in Figure 1. The goal of this process is to produce the right drug, at the right dose, chosen for the right target within the proper patient population. The discovery of a new drug typically begins with identifying a target that is expressed within a disease condition. For every disease condition there are a large range of known and emerging biochemical hypotheses based on the
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Figure 1 Phases of drug discovery and development.
known biology of a target. From these hypotheses, targets are identified using knowledge of the underlying pathophysiological processes of a disease condition and how a potential drug can interact with this target to reverse the disease conditions effects. The sequencing of the human genome has broadened the number of potential drug targets within drug pipelines.11 Validation of a new drug target is a challenging process, as the development of cellular- or tissue-based assays and animal models that mimic human disease condition are difficult to develop. Once a proper assay or animal model has been identified, libraries of compounds are synthesized and tested, with the goal of generating a lead series of molecules that bind effectively to target. Once a primary lead compound or series of compounds have been identified for this target, the next phase of the process is lead discovery and optimization. Lead optimization itself can be a lengthy, complex nonlinear process. This phase includes refining a series of chemical structures that interact with the target to improve a molecule’s characteristics as a potential drug. As molecules progress through the process from early discovery to preclinical discovery, they are prioritized as function of their binding profiles, pharmaceutical properties, and efficacy in animal models. Thousands of potential drug candidates are narrowed down during this process until the optimal candidate is chosen to move forward into the translational space. Early development studies include safety and tolerability (phase 1), proof of concept, and dose finding studies (phase 2). The drug candidate then enters full development and efficacy studies in phase 3, followed by compound registration, drug approval, and product launch. As part of life cycle management, the drug is monitored in postmarketing or postapproval studies. As a drug candidate progresses from early to late drug development, the costs associated with these clinical studies increases steadily as the number of patients and safety monitoring requirements also increase. Because the costs of clinical drug development increase from phase 1 to 3 so dramatically, it is important that biomarkers that allow companies to make informed decisions early in the process are in place. By discontinuing programs that do not have the characteristics for a successful drug and being able to reinvest funds into programs that have higher potential for success can result in a large research and development cost saving. In a retrospective review of several of its drug candidates, a group of Pfizer scientists looked at ways success rates can be improved for drugs in phase 2 of clinical development.2 In this retrospective review of both successful and failed drug candidates, the authors suggest that “The highest level of
confidence and direct evidence at the site of action that required levels of target binding were being achieved is most probably obtained from pharmacokinetics (PK)/ pharmacodynamic (PD) studies of in vivo occupancy measurements with positron emission tomography (PET) or radiolabeled ligands.”
PET Within Drug Discovery and Development Generally, there are three main approaches for the use of PET tracers in the drug discovery and development process. The first is using a radiolabeled candidate to evaluate the distribution and pharmacokinetics of the drug candidate. For small molecules PET tracers, direct labeling with short-lived PET radionuclides such as replacing a native carbon (C-12) with carbon-11 (20 minute half-life) or a native fluorine (F19) with fluorine-18 (109.5 minute half-life) is important to keep the physiochemical properties of the molecule the same. Recent advancements in PET radiochemistry have helped make this process more feasible.12-20 Figure 2 highlights some recent advancements in carbon-11 and fluorine-18 chemistry that have generated several PET-labeled drug candidates from the literature.21-26 The second approach to using PET tracers in drug development is to use a radioligand with known affinity for the molecular target of interest to evaluate the PD properties of the drug candidate. In this case, the PET tracer is no longer the drug candidate and is either a derivate of the main drug or a molecule that is known to bind to the target. The tracer database initiative that is sponsored by the National Institute of Mental Health and the Society for NonInvasive Imaging in Drug Development and the Molecular Imaging and Contrast Agent Database are excellent resources to search for specific PET tracers for a given drug target. Finally, a small molecule PET tracer is used as an imaging biomarker of a known biochemical, metabolic, or physiological process (such as FDG) to evaluate efficacy of a candidate drug.
PET Within Early Discovery PET tracers can provide useful information at many points within the drug discovery and development process. PET tracers can confirm the newly identified drug targets within
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Figure 2 Examples of 11C- and 18F-labeled drug candidates.
cells and tissues of known expression.27 As shown in Figure 3, techniques such as in vitro and ex vivo autoradiography can be used to detect the presence of drug targets in cells and determine their spatial and temporal distribution within tissues. Figure 3A shows a generic example of a PET tracer that has been incubated with cells that express (+) and do not express (−) a new drug target. A successful new drug target will be one that has retained the PET ligand in this assay within the cell line that expresses the target and does not retain radioactivity in the cell lines lacking the target. To further validate this new drug target, these cells can be preincubated with a compound that is known to bind to this target in a dosedependent manner. Figure 3A shows that incubating a blocking compound in increasing concentration can reduce the amount a PET tracer can bind in each cell chamber. One of the major questions posed at this stage of the process is if a target is expressed in the human disease state. Figure 3B shows how using human biopsy tissues and tissue-based autoradiography approach for a PET tracer can provide important information for a drug target. In this generic example, a fluorine-18-labeled PET tracer is incubated with a human tumor tissue slice and then exposed to an autoradiography film to generate the image seen in Figure 3B. In this example, the PET tracer displays significant binding to this tissue, confirming the target is expressed in this tissue. Given the short 109.5 minute half-life of fluorine-18, this same tissue was used to confirm target expression using immunohistochemistry several days later. The power of this technique is by using the same human tissues, we can confirm radioligand and correlate this binding to known histology stains. The expression of the drug target can also be confirmed in the disease condition using these human tissues. This can be used to validate the PET tracer for future translational imaging studies as well. This technique is by no means a replacement for high throughput assays in this phase of the drug discovery process, as they
are also needed to validate a new drug target.28 PET tracers or tritium-labeled radioligands are used to confirm that cellular assays and animal models developed for target indeed express the target of interest. PET tracers can help medicinal chemistry teams by prioritizing lead chemical series (chemotypes) and provide important decisional information regarding which of these series are to progress along the process. A generic example of this is shown in Figure 4. In this hypothetical example, a drug program needs to obtain >90% receptor occupancy for drug efficacy to be achieved in a non-human primate. Images A-C represent longitudinal studies in a non-human primate. In this example, a baseline image is obtained in a nonhuman primate, followed by that same animal being scanned again after administration of increasing concentrations of a lead compound of series A, B, or C. From the chart below, the medicinal chemist would receive important information regarding which of these series to advance. It would be clear in this hypothetical example that chemotype C would differentiate itself against the other series of compounds.
PET Within Lead Discovery and Early Clinical Development Once a drug candidate reaches the lead optimization phase of this process, the discovery team begins planning biomarker studies in both preclinical and clinical studies to understand the drug’s characteristics, such as absorption, biodistribution, metabolism, delivery, and doses for first use in humans. It is at this stage where PET tracers are the most valuable. PET tracers can be used in both preclinical animal models to confirm a drug’s target engagement, receptor occupancy, dose selection, and dose ranges to provide information to plan focused human studies in early development. These
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Figure 3 Examples of [18F]-labeled PET tracers in target identification and early discovery phases of drug discovery. (A) Represents an example of cell-based autoradiography and (B) represents an example of tissue-based autoradiography.
same studies with the same tracer can be planned in human studies with high confidence of their success. PET tracers can also provide biodistribution in higher species animals such as non-human primates and provide important metabolism data for a lead molecule at this stage of the process. PET tracers used with the genetically engineered mice or disease stateinduced rodents that mimic many aspects of human diseases can provide a valuable physiologically based platform for deciding which molecules are most likely to succeed and just as important, drugs that are likely not to succeed. As shown in Figure 1, the information obtained from using a PET tracer within lead optimization provides an import feedback loop to the early discovery portion of the process. PET imaging can feedback information to back up programs to deliver a higher quality drug candidate for these programs. In early clinical development, because PET tracers are generated in highly specific activities (>1 mCi/nmol), microdosing
studies in humans can generate a noninvasive image of the molecule’s biodistribution, delivering only nano- to picomoles of the drug molecule to the patient. This makes it ideal for phase 0 clinical microdosing studies. Valuable information regarding PK, bioavailability, biodistribution, and targeting properties of new drugs, in a limited number of human volunteers, is obtained. A recent study has shown that up to 40% of drug candidates fail in phase 1 or 2 clinical trials due to poor pharmacokinetics and pharmacodynamics.29 PET tracers, using a microdose approach, allows for a direct assessment of biodistribution, metabolism, and dose selection both preclinically and clinically to help improve this success rate. Target engagement and establishing a drug candidate that can localize within a tissue of known target expression with confidence is a critical point for a potential drug at this phase of development. A dynamic PET biodistribution study using a PET-labeled drug candidate can provide concentration-
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Figure 4 Examples of determining receptor occupancy to prioritize three chemical series as a function of receptor occupancy in a non-human primate. The dotted red line represents the occupancy needed for efficacy. PET image A represents a single slice image of a non-human primate brain at baseline, image B represents the same non-human primate upon pharmacologic challenge of a lead molecule at 25% occupancy, and image C represents the same nonhuman primate upon pharmacologic challenge of a lead molecule at 90% occupancy.
time course information in the tissue of interest as well as non–target expressing tissues. Additionally, tissues of known toxicity can be examined for accumulation of the radiolabeled drug candidate as a measure of safety. This information can be decisional, as only the most promising candidates that localize in tissues of known expression are carried forward to reduce lengthy and costly clinical phases. Also, in these clinical phases, PET tracers can be used as imaging biomarkers that can, often quantitatively, assess the clinical efficacy of the drugs being tested and thus provide important tools for the assessment of clinical end points. The most common use for this PET biodistribution or target engagement study has been the development of central nervous system drugs.30-33 Recently, PET tracers have been used to optimize oncology, immunology, fibrosis, and heart failure drugs.3,34-42 A good example of using a small molecule PET tracers to guide drug development is [18F] substance-P antagonist-receptor quantifier ([18F] SPA-RQ). [18F] SPA-RQ is a small molecule PET tracer that binds to the neurokinin-1 (NK1) receptor.43 In this study, [18F] SPA-RQ was used in human imaging studies to determine the NK1 receptor in the normal human brain and the receptor occupancy of the drug candidate aprepitant. The researchers examined the relationship between the plasma concentration of aprepitant and the NK1 receptor occupancy using [18F] SPA-RQ. Also, the distribution of NK1 receptors in the central nervous system of healthy male volunteers was evaluated using [18F]SPA-RQ before and after treatment with placebo or increasing doses of aprepitant. A clear concentration vs receptor occupancy relationship was observed using this small molecule PET tracer confirming target engagement. The researchers used this PET imaging data to show that high levels of NK1 receptor occupancy (>90%) were required to achieve optimal antiemetic effects for the drug candidate. This study also guided dose selection for this drug candidate in an an-
tidepressant trail, which led to a no-go decision for that indication.44 By using this approach, the right drug, the right target in the right patient population was used, and further clinical studies for the flawed indication were also avoided. Table provides several examples of small molecule PET tracers that are used for evaluating the metabolic or physiological processes, binding of a drug candidate to key receptors, and assessment of PK profiles within early clinical drug development.
PET Within Full Clinical Development Small molecule PET tracers can be used in late-phase clinical development as surrogate markers of response and to show pharmacologic differentiation of an asset from a drug already on the market. Additional PET can show differentiation when a new competitor drug comes to the market. Patient selection, using PET to select the proper patient population that will benefit from the drug candidate, is a powerful technique used during full clinical development of a drug candidate. This is a personalized medicine approach and allows researchers to ask mechanism-based questions within patient populations. This approach can determine if a drug is relevant within a disease population which is key to the proposed mode of action of a drug. An example of this strategy is a study with [11C]Docetaxel.57 Docetaxel is an effective drug for the treatment of patients with several advanced malignancies. However, tumor resistance to docetaxel may be associated with reduced drug concentrations in tumor tissue. In this study, a PET microdosing study with [11C]docetaxel in patients with lung cancer could be used to reliably predict tumor uptake of docetaxel during chemotherapy, which was correlated with tumor response to docetaxel therapy. This proof of concept
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Table Small Molecule PET Tracers Used in Drug Development Studies Target
Description
Indication
Radioligand
Reference
Dopamine Serotonin Opioid Gaba NK1 αVβ3 Glucose Thymidine kinase 1 Hypoxic cells Tubulin Estrogen receptor PSMA BCR-ABL and Src kinases
D2/3 receptor SERT µ Opioid GABA receptor NK1 αVβ3 Inhibitor Metabolic Proliferating tissue Hypoxia PK of taxol PK of tamoxifen PSMA expression PK of dasatinib
Neuroscience Neuroscience Neuroscience Neuroscience Neuroscience Cancer/fibrosis Cancer/neuroscience Cancer Cancer Cancer Cancer Cancer Cancer
[11C]Raclopride [11C]DASB [11C]Carfentanil [11C]Flumazenil [18F]SPA-RQ [18F]Alfatide [18F]FDG [18F]FLT [18F]FMISO [18F]FluoroTaxol [18F]Tamoxifen [18F]DCFPyL [18F]Dasatinib
45 46 47 48 43 49 50 51 52 53 54 55 56
DASB, 3-amino-4-(2-dimethylaminomethylphenylsulfanyl)-benzonitrile; DCFPyl, 2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}ureido)-pentanedioic acid; FLT, 18F-fluorothymidine; FMISO, fluoromisonidazole; GABA, Gamma-aminobutyric acid; PSMA, prostate-specific membrane antigen; SERT, Serotonin transporter.
study showed that patient’s response to a treatment could be predicted by a PET imaging study. Additionally, in a longitudinal study using a progesterone analog 21-(18)Ffluoro-16α,17α-[(R)-(1'-α-furylmethylidene)dioxy]-19norpregn-4-ene-3,20-dione ([18F]FFNP), researchers showed that uptake in mammary tumors predicts tumor response to estrogen-deprivation therapy.58 Both of these examples provide drug developers important insight into a drug candidate using PET and generate decisional information to help drive the process.
Summary Numerous aspects of drug discovery and development are enabled by the use of small molecule PET tracers. These tracers deliver answers to vital questions regarding the discovery of novel therapeutic targets. PET imaging provides an understanding of the pathophysiology of diseases, and helps identify and characterize new drug candidates. This technique is also used to validate the efficacy of a new drug candidate and evaluates both PK and PD parameters noninvasively. Thus, advanced radiolabeling techniques that provide new small molecule tracers have become a major priority for the pharmaceutical industry. Imaging with small molecule PET tracers has become a valuable tool to medicinal chemists and biologist, generating information regarding the pharmacology, biology, and toxicology of a drug candidate. This information is essential to decide which drug candidates are worth developing and which ones are not. The goal of using PET within the development process is to save both time and money. In summary, drug companies are searching for techniques that can reduce the time and the cost it takes to develop a drug and ways to efficiently use resources. Molecular imaging techniques such as PET are being used in all phases of the drug discovery and development process, and these techniques are changing the way drugs are developed.
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