The AAPS Journal, Vol. 11, No. 2, June 2009 ( # 2009)
`DOI: 10.1208/s12248-009-9104-5
`
`Theme: Imaging in Drug Development and Therapeutics
`Guest Editor: Murali Ramanathan
`
`Review Article
`
`A Review of Imaging Agent Development
`
`Eric D. Agdeppa1 and Mary E. Spilker2,3
`
`Received 12 January 2009; accepted 1 April 2009; published online 5 May 2009
`Abstract. This educational review highlights the processes, opportunities, and challenges encountered in
`the discovery and development of imaging agents, mainly positron emission tomography and single-
`photon emission computed tomography tracers. While the development of imaging agents parallels the
`drug development process, unique criteria are needed to identify opportunities for new agents. Imaging
`agent development has the flexibility to pursue functional or nonfunctional targets as long as they play a
`role in the specific disease or mechanism of interest and meet imageability requirements. However, their
`innovation is tempered by relatively small markets for diagnostic imaging agents, intellectual property
`challenges, radiolabeling constraints, and adequate target concentrations for imaging. At the same time,
`preclinical imaging is becoming a key translational tool for proof of mechanism and concept studies.
`Pharmaceutical and imaging industries face a common bottleneck in the form of the limited number of
`trials one company can possibly perform. However, microdosing and theranostics are evidence that
`partnerships between pharmaceutical and imaging companies can accelerate clinical translation of tracers
`and therapeutic interventions. This manuscript will comment on these aspects to provide an educational
`review of the discovery and development processes for imaging agents.
`
`KEY WORDS: drug development; imaging agent development; PET; SPECT.
`
`INTRODUCTION
`
`Imaging biomarkers hold much promise for diagnosing
`disease, monitoring disease progression, tracking therapeutic
`response, and enhancing our knowledge of physiology and
`pathophysiology. With the continued momentum towards
`specialized therapies and personalized medicine, there will
`be an increasing need to monitor the physical state of an
`individual in a noninvasive manner with increased specificity.
`Medical
`imaging has been put forth as one of the major
`players in such assessments, with an emphasis on molecular
`and functional imaging in addition to anatomical imaging.
`Molecular imaging is also expected to play an increas-
`ingly important role in drug discovery and development
`
`1 Medical, Science, and Technology Office, GE Healthcare, 101
`Carnegie Center, Princeton, New Jersey 08540, USA.
`2 Pfizer Global Research and Development, 10646 Science Center
`Drive, San Diego, California 92121, USA.
`3 To whom correspondence should be addressed. (e-mail: Mary.
`Spilker@pfizer.com)
`ABBREVIATIONS: CMS, Centers for Medicare and Medicaid
`Services; CT, computed tomography; IP, intellectual property; MRI,
`magnetic resonance imaging; NOPR, National Oncologic PET
`Registry; PD, pharmacodynamics; PET, positron emission tomography;
`PK, pharmacokinetics; PPP, public–private partnership; RDRC,
`Radioactive Drug Research Committee; SNM, Society of Nuclear
`Medicine; SPECT, single-photon emission computed tomography.
`
`(1–5). A seminal review by Frank and Hargreaves describes
`the type 0 biomarkers along the continuum of the natural
`history of disease, type I biomarkers for detecting a thera-
`peutic drug’s mechanism of action, and type II biomarkers
`that are equivalent to surrogate end points (6). Molecular
`imaging probes have been designed to target all three types of
`biomarkers. To better understand their utility, imaging probes
`also can be grouped according to the markers they are
`target, mechanism, efficacy, and surrogate
`interrogating:
`markers. Probes can interact with target biomarkers to
`temporally determine the presence, quantitative level of
`expression, and spatial localization of specific targets for a
`therapeutic drug. A mechanism biomarker may be interrogated
`by specific molecular imaging probes to assess the therapeutic’s
`modulation of the drug target. Visualization of efficacy markers
`involves the use of molecular imaging to monitor drug action.
`Surrogate markers can be imaging biomarkers if the related
`probe concentration predicts the effect of a therapeutic drug
`in lieu of a clinical end point for regulatory decisions. It can
`be appreciated from these groupings that
`imaging bio-
`markers can provide valuable information in preclinical and
`clinical stages of drug development.
`While the outlook for medical and molecular imaging is
`quite promising, the commercial development of imaging
`agents can be as challenging as the development of thera-
`peutics. In fact, current imaging agent development shares
`much in common with standard drug discovery and develop-
`ment practices (Fig. 1) (7). This is especially true for
`molecular imaging agents that bind to a specific target in
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`A Review of Imaging Agent Development
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`vivo. For example, target validation, identification of suitable
`candidate compounds with high affinity and uptake at the
`target site, adequate clearance, and low potential toxicity are
`key considerations for therapeutic and imaging compounds.
`In addition, there are similar stages such as hit identification
`and lead generation as well as multiple phases of clinical trials
`before approval. Despite these similarities, there are differ-
`ences in imaging agent discovery and development processes
`that can be critical to the ultimate success of an imaging
`agent, some of which will be discussed in this review.
`The role of academia and industry in the development of
`therapeutics and diagnostics is one aspect that can differ. The
`main players in the development of diagnostic imaging agents
`include an active and far-reaching community of academic
`investigators as well as a handful of companies involved in
`their commercialization. The academic community is invalu-
`able for exploring new high-risk areas and initially showing
`the feasibility of new imaging approaches. For example, early
`feasibility studies around targeted magnetic resonance imag-
`ing (MRI) (8,9) are opening the door for MRI to be used as a
`molecular imaging modality. Additionally,
`the academic
`community directly develops new agents and is credited with
`the majority of positron emission tomography (PET) and
`single-photon emission computed tomography (SPECT) trac-
`ers discovered to date. These efforts provide significant in-
`licensing opportunities for diagnostic companies, which in
`turn provide a route of commercialization for the tracers
`discovered in academia, although academics also can drive
`the development process from basic research to limited
`clinical studies without industry sponsorship.
`
`Beyond the academic community, there are companies
`which focus solely on the commercial development of diagnostic
`imaging agents, while, in other cases, these efforts are part of
`larger therapeutic or technology companies. In general, the size
`of these companies/divisions is on par with smaller biotechnol-
`ogy companies. This is likely a reflection of the smaller market
`potentials and profits from diagnostic imaging agents compared
`to those associated with pharmaceuticals. Based on 2004
`numbers, it was estimated that the total imaging agent market
`was only 1% of the total therapeutic market (10). This means
`that the majority of imaging agents would likely fall into the
`small/specialty market size. While reliable estimates of time and
`costs for the development of diagnostic agents are not often
`reported, Nunn calculated that a diagnostic imaging agent takes
`approximately 10 years to develop at a cost of $150 million; yet
`generates only $200–$400 million in sales annually, even for a
`highly used diagnostic agent, such as Omnipaque™ (iohexol,N,
`N’-bis(2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl)-acet-
`amido]-2,4,6-triiodoisophthalamide, GE Healthcare) or Cardi-
`olite™ ([Tc-99m]N-(2-methoxy-2-methyl-propyl)methanimine,
`Lantheus). As a comparison, he reported that a therapeutic
`costs approximately $850 million to develop over 14 years but
`could attain as much as $3.4 billion in sales for a blockbuster
`drug (Fig. 1) (10). It is evident from these numbers that the
`commercialization of diagnostic imaging agents will not gener-
`ate the same return on investment achieved in the pharmaceu-
`tical industry. Therefore, diagnostic companies are exploring
`partnerships with academia and Pharma to improve the viability
`of agent development in light of the increasingly personalized
`treatment of patients.
`
`Fig. 1. Discovery and development of therapeutics and diagnostics. a Therapeutic process
`modified from Fig. 4 of the FDA’s Critical Path Initiative; $3.4 billion annual revenue is
`representative of a typical top ten selling drug. b The best selling diagnostic imaging agent
`has an annual revenue of $400 million. Cost and timeline numbers taken from Nunn (10)
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`These partnerships will likely continue to be important in
`the creation of new imaging agents. Yet, the development of
`imaging agents has unique aspects that are critical to its
`success that may not be well known outside of the imaging
`community. Therefore, this review outlines the processes,
`opportunities, and challenges encountered in the discovery
`and development of imaging agents.
`
`DISCOVERY
`
`Identification of Diagnostic Opportunities
`
`Early in the commercial development of an imaging
`agent, several
`factors must be considered beyond the
`scientific challenges. One of the first considerations is to
`identify how the agent will be used. This will then define
`performance metrics required for success. For agents used to
`diagnose disease, their performance criteria include sensitiv-
`ity and specificity requirements. For example, Cardiolite™
`([Tc-99m]-sestamibi), which is used to detect coronary artery
`disease (11), has similar sensitivity and specificity compared
`to Thallium-201, the gold standard at the time of its approval
`(12). Yet the advantageous properties of the Tc-99m label
`improved image quality and allowed for the use of cardiac
`gating, which resulted in significant improvements in speci-
`ficity for female patients (13,14).
`Agents also can be used as efficacy markers to monitor
`therapy, as exemplified by the use of 2-[F-18]fluoro-2-deoxy-
`D-glucose ([F-18]FDG) PET to monitor the reduction of
`tumor metabolism resulting from the cytostatic drug imatinib
`mesylate (Gleevec, Novartis) (15). When considering probe
`development for therapeutic monitoring, the noise level of
`the modality as well as the expected target modulation due to
`therapy and disease progression should be considered in early
`feasibility assessments.
`Preclinical molecular imaging of targets and mechanisms
`is increasingly used in drug discovery and development.
`Imaging probes (e.g.,
`[Tc-99m]scVEGF/Tc) for vascular
`endothelial growth factor receptor have been used for
`determining target expression in tumor-bearing mice (16). In
`addition, bioluminescence of live mice was recently used to
`monitor the inhibition of lymphocyte proliferation by rapa-
`mycin, specifically the interaction between rapamycin-binding
`domain and cytosolic protein FK506-binding protein 12 (17).
`Development of such imaging agents needs to provide
`convincing evidence that they are specifically marking the
`targets and mechanisms they are designed to measure.
`In addition to defining the use of the imaging agent,
`other factors to assess include the market size, optimal
`modality, intellectual property (IP), competing diagnostics,
`and the existence of disease-modifying therapies. With
`relatively small revenues for even large market segments,
`market size is an important factor when prioritizing projects.
`This may lead to technically feasible projects being depriori-
`tized due to considerations of return on investment (10).
`Market size also may be a factor in the selection of the
`imaging modality, which can be driven by the current/future
`install bases as well as factors associated with clinical usage
`(e.g., repeated dosing) and modality constraints (resolution
`and sensitivity). For example, SPECT and SPECT/computed
`tomography (SPECT/CT) equipment has a larger installed
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`Agdeppa and Spilker
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`base (∼12,530 stationary multihead SPECT scanners in the
`US in 2008) compared with PET and PET/CT (∼1,000
`stationary PET and PET/CT in 2008) in the US (18,19).
`Therefore, if the performance of SPECT imaging is adequate,
`then the development of a SPECT tracer may be preferred
`over a PET tracer. This is especially true as advancements in
`SPECT technology are likely to shorten the current resolu-
`tion and sensitivity gaps between the two modalities (20,21).
`To reach users of both modalities, there are examples of PET
`and SPECT tracers under development for the same biolog-
`targets, such as amyloid deposits (22–24) or αvβ3
`ical
`expression (25,26).
`As with therapeutics, knowing the IP landscape before
`project inception helps to identify opportunities and compe-
`tition. For diagnostic companies, competition can come from
`technology advancements, nonimaging biomarkers, and other
`applicable imaging agents, regardless of modality. Given the
`long development times for new imaging agents and the
`continuous technology improvements in the various imaging
`modalities, it is important to consider the emerging technol-
`ogies that could displace the agent’s market potential. For
`example, MRI researchers are ever-expanding opportunities
`to leverage endogenous signals to gain information without a
`contrast agent (27–29). Blood biomarkers and nonimaging
`diagnostics are another form of competition that could limit
`the use of an imaging agent. These diagnostics are typically
`cheaper than imaging tests but lack the spatial information
`that can be gained from imaging.
`Finally, imaging companies are beginning to carry out
`health economic analyses at the front end of development for
`molecular imaging agents and technology. Health economics
`is the science of value and, when applied to imaging agents,
`determines if there is additional benefit from a new agent
`worth the cost to the health care system. Health economics
`and outcomes-based research are expected to assist in the
`identification and commercialization of new medical technologies
`in light of the reimbursement issues.
`Once these concerns have been addressed, the project is
`ready to begin the discovery and development process.
`
`Target Identification/Validation
`
`Standard drug discovery often begins with target identi-
`fication and validation by providing evidence that the drug
`target plays an important mechanistic role in the disease
`process such that inhibiting the target may modify the disease
`(30). Since the drug will have a functional impact, a validated
`target then serves as the basis for early-stage screening of
`potential drug compounds (31). A key difference between
`screening for drugs and imaging agents is that imaging agents
`can be aimed at targets that play critical roles in disease but
`do not need to be functional targets that modify the disease.
`There are several instances where nonfunctional targets are
`markers of the disease process, such as extracellular matrix
`proteins, membrane lipids, structural proteins, or extracellu-
`lar deposited peptides (32–35). Target validation for imaging
`agents also requires determining the amount of
`target
`accessible for high signal-to-noise images. Therefore, the
`absolute amount of accessible target in vivo is important to
`imaging agent performance.
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`While PET displacement studies have helped receptor
`binding determinations of drugs in vivo, there is growing
`evidence that modeling and simulation of molecular imaging
`help characterize nonreceptor nonenzymatic ligand binding.
`For example, saturation binding of a highly specific imaging
`agent can be used to calculate the receptor density (Bmax)
`empirically and has been particularly useful for well-under-
`stood receptor–ligand interactions, which pharmaceutical
`companies have leveraged in receptor occupancy studies
`(36,37). However, interest in complex nonreceptor imaging
`targets related to disease processes is growing. For example,
`imaging beta-amyloid plaques requires ligand binding to a
`complex heterogeneous target with multiple independent
`binding sites (33,38,39). The complexity of some imaging
`targets, such as beta-amyloid, also arises from a target’s
`trafficking pattern in vivo (40,41). In such cases, computa-
`tional and systems biology may provide useful guidance. This
`approach was recently applied to understand the relationship
`between the heterogeneous microenvironment of plaque and
`imaging kinetics (42,43). Further, recent data indicates that
`combining models of pharmacokinetics with physiological
`models of beta-amyloid production and clearance has value in
`understanding the relationship between target concentrations,
`affinity, and image quality (44). The use of mathematical
`models and quantitative assessments early in a project’s
`development can play an important role in assessing the
`feasibility and risk of the imaging approach (45,46). As with
`the pharmaceutical industry, the use of in silico models and
`computation will be important
`tools to help diagnostic
`companies become more efficient in bringing new imaging
`agents to market.
`
`Iterative Discovery Processes
`
`The discovery process for traditional small-molecule
`drugs has typically been represented as a fairly linear process,
`progressing along the major milestones outlined in Fig. 1.
`This is not to say that therapeutic discovery is not iterative, as
`certainly there are times when iterations are necessary to
`achieve the required in vivo therapeutic performance. In
`major pharmaceutical companies focused on small-molecule
`drugs, a large number of chemical compounds can be
`generated and then screened for performance against specific
`assays to arrive at a reasonable number of compounds to take
`forward into in vivo testing. Diagnostic companies often do
`not have a large library of chemical compounds to select
`from. Furthermore, in vitro results do not always translate
`well to in vivo performance (47–49). For example, in the
`search for a SPECT agent equivalent to [F-18]FDG, an
`iodinated mannose analog was investigated. While the
`stability of the tracer looked good in vitro, in vivo perfor-
`mance suggested that the label was rapidly deiodinated (47)
`and therefore would not satisfy imaging requirements. Since
`the pharmacokinetic behavior and signal generation are
`major contributors to performance, chemical synthesis, and
`screening, preclinical studies and lead selection can be quite
`iterative for diagnostics (Fig. 2). This is especially true for
`novel imaging approaches that involve complex mechanisms
`and targets assessed by nontraditional (nonsmall molecule)
`compounds. In these cases, the chemical and in vitro screening
`assays may not fully capture in vivo performance. Therefore,
`
`Fig. 2. The iterative discovery process allows for the early in vivo
`assessment of novel imaging compounds and targeting mechanisms
`
`early studies in simple animal models may be used to
`understand and optimize the performance of the new agents.
`Information learned through these studies is then fed back to
`the chemists for modifications to improve performance before
`taking them into more rigorous preclinical studies. Once a lead
`compound has been identified, the compound will then be
`optimized and formulated for use in humans.
`
`Chemical Synthesis
`
`The historical success of neurological tracers for nuclear
`medicine was governed in part by the same principles
`followed in therapeutic drug development. Parameters for
`optimization included affinity for the target, selectivity,
`metabolism, lipophilicity, and molecular weight/size (50–54).
`Equally important is the selection and incorporation of the
`signaling moiety into the chemical construct, which can
`significantly alter the delivery, retention, binding, and clear-
`ance of an imaging agent. Nuclear tracers can be grouped into
`three broad classes according to how the radiolabel
`is
`integrated into the imaging agent. The first class of radio-
`labeled tracer is the radionuclide itself (Fig. 3). For instance,
`sodium [Tc-99m]pertechnetate is simply the oxidized gamma-
`emitting technetium metal used for SPECT blood pool
`imaging. Likewise, sodium [F-18]fluoride is a bone-seeking
`PET agent used in imaging osteosarcoma. The second class
`involves a pendant radiolabel extending from the molecular
`scaffold that imparts targeting of molecular markers. This
`second grouping is traditionally applied to radiometals and
`their chelators for SPECT imaging (e.g., [Tc-99m]TRODAT-
`1; Fig. 3) (55); however, the pendant can be applied to PET
`agents, where the pendant label is a radiometal (e.g., Ga-68
`DOTA in [Ga-68]DOTATOC, Fig. 3) or an [F-18]alkyl
`fluoride, such as in 3-N-(2-[F-18]fluoroethyl)spiperone
`(Fig. 3). The third class of tracers incorporates the radiolabel
`within the structure of the targeting molecule. For example,
`experimental estradiol derivatives for SPECT imaging inte-
`grate the receptor binding sites of the molecule on the
`exterior of the technetium radiometal (Fig. 3). For PET
`imaging, positron-emitting radionuclides C-11, N-13, and O-
`15, which are isotopes to endogenous elements like C, N, and
`O, have been incorporated into molecules without changing
`chemical properties. For example, a comparison between L-
`[C-11]DOPA and L-6-fluoro[F-18]DOPA clearly shows that
`the in vivo decarboxylation rate differs (56). While fluorine is
`not as ubiquitous in endogenous biomolecules, F-19 is a common
`element in many drug pharmacophores. Therefore, substitution
`of F-19 with F-18 may be ideal for the labeling of some small-
`molecule drugs. However, Pharma scientists should note that F-
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`Fig. 3. Three classes of tracers (modified from Dilworth and Parrott (55)). a Class 1 tracers are those
`where the radionuclide is the imaging agent itself. Class 2 tracers have pendent radiolabels. Class 3
`incorporates the radionuclide within the targeting molecule. b (1) [Tc-99m]TRODAT-1, (2) Experimental
`[Tc-99m]estradiol. c (3) [Ga-68]DOTATOC, (4) [C-11]DOPA, (5) 3-N-(2[F-18]Fluoroethyl)spiperone
`
`18-labeled drugs are analogs with different pharmacokinetic and
`pharmacodynamic properties in vivo unless there is F-19 in the
`drug structure that is amenable to F-18 substitution.
`Early probe development in academia has utilized C-11
`chemistry due in part to the synthetic opportunities using a
`radioisotope of carbon relative to the other positron-emitting
`radionuclides. However, fluorine-18 and other relatively
`longer-lived radioisotopes have been used clinically and
`commercially. The 110-min half-life of F-18 (compared with
`
`the 20-min half-life of C-11) allows for longer radiosynthesis
`of tracers and imparts greater potential for regional distribu-
`tion of PET tracers to clinics without cyclotrons. It
`is
`estimated that 90% of all PET scanners do not have facilities
`that can produce C-11 tracers (24), thereby being dependent
`on local distribution of tracers with longer half-lives.
`There also can be advantages to using SPECT radio-
`labels (e.g., Tc-99m, I-123) for biomolecules and drug labeling
`and there is a long history of various published techniques for
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`radioiodine labeling of proteins, nucleic acids, and small
`molecules (57). The relatively facile labeling with radioiodine,
`its longer half-life (13.2 h for I-123), and gamma emissions ideal
`for sodium-iodide-based SPECT detectors are all reasons why I-
`123-labeled SPECT tracers are still being introduced to the
`clinic (e.g., I-123 MIBG). However, the workhorse radioisotope
`for nuclear medicine continues to be technetium-99m (Tc-99m)
`due to its convenient production, half-life of 6 h, and 140- and
`142-keV gamma emissions which are ideal for NaI detectors. In
`addition, Tc-99m is produced by a molybdenum-99:technetium-
`99m generator which can be conveniently located in small hot
`laboratories in nuclear medicine clinics.
`Radiolabeled molecules for PET and SPECT imaging are
`produced and administered in very low concentrations, which
`allow for detection of ligand–target interactions without prompt-
`ing a pharmacological effect. The high sensitivity of scanners for
`the radioactive emissions from the tracers enables detection of
`picomolar concentrations of C-11-labeled PET tracers, for
`instance (58,59). Therefore, the molecular and biochemical
`system can be interrogated using PET imaging without perturb-
`ing the system. This so-called tracer method is made possible
`with highly specific radioactivity (radioactivity per mass of
`radioactive and nonradioactive molecules) of the cyclotron- or
`generator-produced radionuclide. The challenge of achieving
`and maintaining high specific activity should not be under-
`estimated because of the effects of isotopic dilution (particularly
`for C-11) and the promise of microdosing (discussed below) that
`is incumbent on the high specific activity of a radiotracer. As
`new imaging biomarkers are identified, new radiotracers need
`to be developed with high specific activity in order to target the
`typically low-concentration markers of early disease. This also is
`a consideration in preclinical studies, where the mass of injected
`tracer can lead to much greater receptor occupancy levels than
`anticipated (60).
`
`Preclinical Testing
`
`Preclinical studies are undertaken to assess the pharma-
`cokinetic behavior of the tracer, as well as provide evidence
`for proof of mechanism (POM) and/or proof of concept (POC),
`assess potential toxicity risks, and address translation to humans.
`As with pharmaceuticals, most imaging agents available today
`are small-molecule compounds that rapidly distribute and clear
`from the body. However, this is changing as labeling of antibodies
`and antibody fragments is being explored as well as macro-
`molecules and nanoparticles for various applications (61–65).
`Imaging agents are typically intravenously administered,
`which simplifies their pharmacokinetic characterization. As
`stated earlier, since the pharmacokinetics of an imaging agent
`is particularly important to its performance, the chemical
`compound may go through modifications based on preclinical
`imaging results to improve its pharmacokinetic behavior. In
`addition, prior to human use, the complete pharmacokinetics
`of the tracer is typically evaluated in preclinical studies. Using
`biodistribution or dynamic imaging studies, the time-depen-
`dent concentration of an agent in the major body tissues and
`excrement is recorded. This information is then used to assess
`dosimetry, identify the dose-limiting organ for toxicity and
`radiation exposure, and can be used in the translation to
`humans (66–68). Similar studies in humans are performed in
`
`the first phase of the clinical trials to determine definitive
`human pharmacokinetics since results in preclinical species
`do not always translate well to humans. While nuclear tracers
`usually have limited toxicity issues due to the low mass dose
`administered, MRI and CT contrast agents are dosed at much
`greater amounts and therefore carry a greater risk of toxicity.
`Thus, safety assessment is a critical factor in the development
`of contrast agents for these modalities.
`Since the signal of a nuclear tracer is due to the
`radiolabel, a thorough understanding of the parent/intact
`agent along with any metabolites, biotransformations, and
`free label is important to understand and quantify if possible.
`For new tracers originating in academia, mathematical
`models are often reported to describe the distribution of the
`tracer in select tissues based in preclinical or clinical studies
`(69–74). The mathematical model can then be used to
`quantify specific parameters of interest, such as a binding
`potential or distribution volume for neurotracers (75,76).
`POM studies should demonstrate the mechanism by
`which the agent is acting, while POC studies should have
`relevance to the clinical disease of interest and may also be
`used to show performance against gold standards or compet-
`itor agents. POM does not necessarily require in vivo studies,
`as long as the mechanism can be convincingly demonstrated
`(77–79). In cases where in vivo studies are used,
`the
`expression of the target may be amplified and modulated to
`provide evidence that the imaging agent is hitting its target.
`Since PET and SPECT imaging agents do not elicit a
`pharmacologic effect, a pharmacodynamic response is not
`measurable. The burden of proof therefore lies in the ability
`to correlate uptake of the imaging agent with the modulated
`target density. Modulation of a target can be accomplished
`therapeutics or genetically modified
`through the use of
`animals. In addition, competition and inhibition studies are
`important to show that the observed uptake is due to specific
`binding to a target and not an alternative mechanism.
`A key step in this process is validation of target modulation.
`The validation of imaging biomarkers is traditionally dependent
`on histologic assays despite the wave of recent genomic and
`proteomic discoveries in medicine. Medical and molecular
`imaging is still very much focused on the precise localization of
`an imaging marker in the context of anatomy. Hence, autora-
`diography and radiologic–pathologic correlates are gold stand-
`ards for validation of in vivo images (24,80–83). Expression
`profiling of RNA and DNA, while valuable for the development
`of in vitro diagnostics and molecular therapies, is not as useful in
`providing a measure of an imaging probe’s likelihood of success.
`The discovery process for molecular imaging probes is
`exemplified by the lack of success with Congo red and
`Thioflavine T derivatives and the eventual recognition of
`benzothiazole analogs as in vivo imaging agents for the
`visualization and detection of beta-amyloid plaques. As
`previously mentioned, beta-amyloid aggregates form plaques
`in the Alzheimer’s disease brain and contain multiple
`independent binding sites for small-molecule probes. Congo
`red and Thioflavine T are gold standard bright-field and
`fluorescent dyes, respectively, used for in vitro histological
`staining of beta-amyloid aggregates in ex vivo brain speci-
`mens. Early attempts to translate these charged in vitro dyes
`into in vivo probes for the beta-amyloid aggregates in plaques
`were not entirely successful or optimal for eventual clinical
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`molecular imaging. Congo red is a charged molecule that
`lacks sufficient hydrophobicity for diffusion through the
`blood–brain barrier (84,85). A lipophilic analog of Congo
`red was synthesized and demonstrated improved blood–brain
`barrier permeation of the probe (86). However, the in vivo
`two-photon confocal microscopy showed unfavorable phar-
`macokinetics for optimal signal to background necessary for
`routine PET imaging in mammals. Similarly, a lipophilic
`radioiodinated analog of Thioflavine T also was synthesized
`and demonstrated ex vivo to enter the brain of normal mice
`(87). But the analog reached maximal uptake at 30 min in the
`mouse brain which suggests less than optimal uptake in the
`brain for in vivo imaging in humans.
`The iterative improvement of Thioflavine T derivatives
`resulted from the recognition of molecular requirements for
`the binding of this family of compounds to beta-amyloid
`plaques and eventually led to successful in vivo PET imaging
`of plaques in Alzheimer’s disease patients using a C-11-
`labeled derivative called 6-OH-BTA-1 (88-91). The develop-
`ment of an F-18 analog of the C-11 derivative continues the
`iterative improvements with the goal being a PET probe with
`the longer 110-min radioactive half-life for F-18 rather than
`the 20-min half-life of C-11 (92). This will result in less
`radioactive decay of the probe when it is commercially
`distributed from regional PET tracer distribution sites to
`imaging clinics without onsite cyclotrons for F-18 radioisotope
`production. In general, the longer half-life of F-18 over C-11
`also allows for the imaging of relatively longer biological
`
`processes or probes with slow distribution and clearance of
`free probe. Thus, it is evident that the typical iterations in the
`imaging agent development process are similar to feedback
`loops in drug development except for the radiolabeling of
`potential probes which includes additional recursive steps in
`order to optimize the