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CLINICAL IMAGING reason why targeted optical agents for clini- cal use are unavailable, despite the high Drug-Based Optical Agents: expectations set by demonstration of these agents in animals. Yet, at the heart of the Inf ltrating Clinics at Lower Risk translational dif culty are not the addition- al resources required for translation, but Werner Scheuer,1 Gooitzen M. van Dam,2 Michael Dobosz,1 Markus the certainty associated with the imaging Schwaiger,3 Vasilis Ntziachristos4* performance of a given optical agent in hu- mans. In other words, if an agent would be Fluorescent agents with specif city to cellular and subcellular moieties present promise certain to of er suitable imaging accuracy in for enhancing diagnostics and theranostics, yet challenges associated with regulatory humans—if the risks were removed—then approvals of experimental agents stif e the clinical translation. As a result, targeted f uo- it would be straightforward to secure the rescent agents have remained predominantly as preclinical imaging tools. We discuss the resources for clinically translating it from potential of using optically labeled drugs to accelerate the clinical acceptance of optical public funding sources or the industry. and optoacoustic agents, in analogy to nuclear medicine approaches. This strategy, Translation of f uorescent probes to corroborated with microdosing studies, outlines a promising approach for overcoming medical applications can therefore be ex- bottlenecks and advancing photonic clinical imaging. pedited by minimizing the uncertainty of their clinical imaging performance. Here, we discuss how the use of optical agents TR OUBLE WITH TRANSLATION (EMA). T is of en implies high-risk indus- based on approved drugs, or on drugs al- Advances in optical imaging bring diagnos- trial or academic investments owing to the ready being explored therapeutically in tic and theranostic potential that is unavail- time and cost associated with the clinical late-phase clinical trials, may minimize able to conventional visual inspection. Op- translation of a new agent. Consequently, the translational risk compared with novel tical coherence tomography (OCT), narrow there has been little showcased ability to optical agents that are based on molecules

band imaging (NBI), f ber-based confocal bring novel f uorescent agents to use in hu- never administered to humans. We further on May 30, 2012 microscopy, or hyperspectral autof uores- mans. Clinical f uorescence imaging there- discuss the use of optical imaging at micro- cence imaging, for example, can of er sub- fore still relates to the use of ICG or f uores- dosing amounts as a strategy to further ac- surface visualization in vivo and enhance cein—agents approved several decades ago, celerate the clinical translation of f uores- anatomical, functional, and biochemical primarily for resolving anatomical or physi- cence molecular imaging. Lastly, we explain information (1). To improve target detec- ological features. ICG, for example, has why accurate f uorescence imaging tech- tion over intrinsic tissue contrast, contrast been used clinically for eye angiography, nology is required in clinical studies and in enhancement can be further imparted by hepatic clearance studies, and visualization successful regulatory approval.

optical agents that recognize otherwise- of the lymphatic system (8, 9). Nevertheless, stm.sciencemag.org invisible disease biomarkers (2, 3) . T e ICG and other nonspecif c f uorescent dyes DRUGS DOUBLE AS IMAGING AGENTS development of optical and optoacoustic have not been found particularly useful in Analogous to radioisotopes in nuclear im- agents with molecular specif city has seen many other clinical application areas—for aging, various optical tags, such as f uores- substantial growth in the past decade. Op- example, in cancer detection—owing to the cent dyes, can be used to label drugs and tical agents targeting specif c disease moi- lack of specif city. create agents that retain the targeting prop- eties promise to improve disease detection T e clinical propagation of novel agents erties of the drug while providing optical over the use of intrinsic tissue contrast or engineered to be disease-specif c has two contrast. Compared with optical agents nonspecif c f uorescent organic dyes, such levels of associated challenges. T e f rst based on molecules with undocumented Downloaded from as indocyanine green (ICG) (2). and more straightforward regards the tox- biodistribution and targeting prof les in In animals, f uorescent and optoacoustic icity and stability studies that are required humans, the consideration of known drugs agents have been successfully used to target for obtaining human use approvals. T e labeled with a f uorescent dye comes with cancer, cardiovascular disease, neurode- primary limitation in clinical translation, advantages that can accelerate the clini- generative disease, and bacterial infections, however, comes from the second chal- cal translation of the optical agent. First, a among others (4–7). However, the clinical lenge: the risk associated with the ef cacy wide portfolio of drug molecules is avail- translation of novel optical imaging agents of the agent. Preclinical (animal) studies able for doubling as optical imaging agents. requires overcoming regulatory barriers set do not accurately predict human ef cacy Already, antibody- or antibody fragment– by the U.S. Food and Drug Administration of a new agent. Instead, a stepwise process based agents as well as nonpeptidic small (FDA) or the European Medicines Agency from phase I to phase III clinical studies molecules considered originally for thera- is required to establish the usefulness of a peutics have shown potent imaging perfor- 1Pharma Research and Early Development, Discovery Oncology, Roche Diagnostics GmbH, Penzberg 82377, novel imaging agent. T ese translational mance in animal studies (2, 3). Many ad- Germany. 2University Medical Center Groningen, De- studies constitute a leap in the resources ditional agents can be potentially derived partment of Surgery, 9700 RB Groningen, Netherlands. required in terms of cost, time, skill, and from approved or late-phase clinical trial 3Nuclear Medicine, Klinikum Rechts der Isar, Technische critical mass over the original development drugs (Table 1). Second, each of the drugs Universität München 81675, Germany. 4Biological and Medical Imaging, Technische Universität München and and demonstration of a novel agent in ani- considered comes with knowledge of the Helmholtz Zentrum, München 81675, Germany. mal studies. T e added cost associated with pharmacokinetics and targeting ef ciency *Corresponding author. E-mail: [email protected] clinical translation is typically the primary and specif city in humans—data that were

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Table 1. Examples of clinically approved or investigated drugs that target established can- successfully undergone phase III trials may cer-related biomarkers. These drugs could be considered for use as optical imaging agents for come under these conditions at higher per- microdosing studies. formance certainty (reduced risk) as com- pared with those of new imaging agents, Drug Type Biomarker so as to justify the investment required for Chimeric EGFR clinical translation. Chimeric monoclonal antibody EGFR Chimeric monoclonal antibody EGFR FEATURES OF A LABELED DRUG With the above assumptions, it is clear that Monoclonal antibody EGFR risk management is an important criterion Human monoclonal antibody EGFR in accelerating the clinical propagation of Humanized monoclonal antibody VEGF f uorescence molecular imaging. From sev- eral optical imaging agents described in the Monoclonal antibody fragment VEGF literature (2, 3), a narrowed selection that Pazopanib Small molecule VEGF receptor (VEGFR) will, with high probability, lead to a success- Human monoclonal antibody VEGFR ful outcome is important to help boost the f eld and reach patients faster. Risk manage- Humanized monoclonal antibody αvβ3 integrin ment could be achieved by using not only Chimeric monoclonal antibody α β integrin 5 1 labeled drugs for optical imaging but also Humanized monoclonal antibody HER2 molecules that were discontinued from the Humanized monoclonal antibody HER2 clinical translation pipeline owing to unsat- Recombinant human monoclonal Epithelial cell adhesion isfactory therapeutic ef cacy. Even a change antibody molecule (EpCAM) in a drug’s strategic priority, especially if targeted to disease-specif c moieties, would Monoclonal antibody Folate binding protein

also af ect risk management. on May 30, 2012 Many drugs may not be ideal imaging agents. Besides favorable biodistribution obtained during clinical trials. T ird, each animal data, animal studies do not always to disease, an imaging agent also needs to drug comes with corresponding informa- predict performance in humans. T en, the generate a strong signal and high contrast tion on possible side ef ects to humans, key question of new imaging agents tested within acceptable time frames. Accumu- which can ref ne the selection process only on animal models relates to the overall lation and binding patterns will also play toward minimizing safety risks. When it performance in humans. a vital role in the success of a drug as an

comes to side ef ects, an advantage of an A labeled drug comes with a dif erent imaging agent. For example, trastuzumab stm.sciencemag.org imaging session over a therapeutic proto- question at hand: Does the labeling pro- labels specif c cancer cells and may be ideal col is that a labeled drug considered for op- cess change the performance of the active for margin delineation of a subset of tumors tical imaging will be typically administered molecule, which has already been charac- expressing human epidermal once in quantities that are much smaller terized in vivo in humans? Conf rmation receptor 2 (HER2) (Fig. 1, A and B), where- than the total dose administered over a that active sites of the drug-based agent are as bevacizumab preferentially accumulates treatment protocol. not blocked by optical labeling can be per- in the stroma and may be better as a generic formed easily in the laboratory. Similarly, tumor marker (Fig. 1, C and D). T erefore, REDUCING TRANSLATIONAL RISK animals and pilot human examinations can a careful selection from a long list of thera- Downloaded from Despite these advantages, a labeled drug is quickly reject probes with altered biodistri- peutic molecules, such as those in Table 1, considered a new molecule in regulatory bution or pharmacokinetic prof les result- can lead to optimal disease imaging. approvals for human use. T erefore, even ing from labeling. Nuclear imaging data for pilot clinical trials a labeled drug needs may also be available as part of the drug MICRODOSING to undergo toxicity studies in an identical development process, which could corrob- Microdosing is a tool developed to acceler- manner to new imaging agents—in other orate the biodistribution studies. ate the discovery of new drugs by allowing words, agents based on new molecules In contrast, the clinical translation of a fast regulatory approval of pharmacoki- without previous experience on humans. At new imaging probe requires information on netic studies of a new molecule when ad- f rst impression, it would appear that there broader questions that include the targeting ministered in very low, nonpharmacologi- is no practical benef t when selecting a la- ability, pharmacokinetics, and side ef ects cally active doses. Under the EMA Safety beled drug over a new optical agent. Yet, the of the new molecule in humans, which Working Party Position paper (10) and the major challenge in the clinical translation are typically only conclusively addressed FDA exploratory investigational new drug process is not the investment associated af er the completion of phase III clinical (IND) application guidelines (11), a mi- with the toxicity study itself but the over- trials. Eventually, both labeled drugs and crodose is def ned as the administration of all performance of the agent during phase new imaging agents will undergo phase II 100 µg of a small-molecule ligand or 1% of I to III clinical trials. Although many new and phase III clinical trials for approval in the pharmacological dose determined on optical imaging agents appear promising clinical use. However, a promising imaging animal data to yield a pharmacological ef- for human use on the basis of preclinical agent based on a molecule that has already fect, whichever is the lesser (12). Because of

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A B T e selection of small animals (typically rodents) for toxicity demonstration and the availability of complete pharmacological and dosing prof les of the active molecule, obtained through the drug discovery pro- cess of the active molecule, lead to straight- forward design and implementation of phase 0/I human clinical trials. Conversely, administration of an agent at doses above the microdosing limits typically requires studies on at least two species before trans- lation to humans. One species is selected to be a small animal, such as a rat, and the second species is a larger animal (dog or monkey) (11). T e use of large animals and the corresponding production of the agent at larger doses, to account for the larger ani- C D mal weight over rodents, adds to the cost and time investment required. Microdosing is another strategy to lower the translational barrier of new imaging agents (11), as it can reduce the cost and accelerate early studies investigating the clinical performance of optical agents. T e

combination of labeled drugs with micro- on May 30, 2012 dosing has synergistic ef ects toward risk minimization because it may quickly ad- dress the issue of labeled versus unlabeled drug similarity, providing evidence for fur- ther investing in phase II and III trials. Collecting early clinical imaging ef cacy data, as facilitated by microdosing, is an

important f rst step for the clinical trans- stm.sciencemag.org E F lation of f uorescence molecular imaging. Similarly to nuclear imaging that can detect microdosing amounts from tissues, opti- cal methods of er very sensitive detection of agents when imaging superf cial activ- ity from tissues—for example, in surgical procedures or gastrointestinal endoscopic inspection. Typically, tens of organic f uo- Downloaded from rescent dye femptomoles (or less) can be detected with sensitive charge-coupled de- vice (CCD) cameras in vivo. One clinical study using targeted f uorescent agents for enhancing surgical vision (13) used ~6 to Fig. 1. Anticancer drugs double as imaging agents. (A to D) Macroscopic imaging of a human 7 mg of agent, which is more than the ac- breast cancer xenograft overexpressing HER2 (A, C) and histological confi rmation of excised tumor cepted microdosing amounts of 100 µg in (B, D) confi rms in vivo tagging in mice. Cy5-labeled trastuzumab (yellow) preferentially targets tu- this case. T e f uorescence system used in mor cells (cyan) (B); whereas Cy5-labeled bevacizumab (yellow) accumulated in the tumor stroma the study operated in the visible range (480 (cyan) (D). (E and F) Color (E) and hybrid (F) imaging of an patient imaged intraop- to 530 nm), a spectral region that required α eratively after the injection of a fl uorescence probe targeting the folate- receptor. The images narrow-band optical f ltering leading to re- were obtained with permission from (13). duced detection sensitivity (Fig. 1, E and F). Operation in the near-infrared part of the the larger molecular weight, the maximum that can mitigate the clinical translation spectrum (700 to 900 nm) combined with dose considered for protein products is ≤30 of optical agents. T is is because regula- wider-band f ltering can lead to substantial nanomoles (11). tory guidelines accept the use of a single sensitivity gains to bring imaging of tracer Administration of an optical agent at mammalian species to showcase safety in f uorescent agent amounts (microdosing microdosing (tracer) amounts is a strategy support of single-dose studies in humans. amounts) within reach.

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POTENT TARGETS AND DRUGS 1, C and D. Similarly, labeled trastuzumab of ered for f uorescence imaging operate Successful imaging requires the selection (Herceptin) has shown good binding and with simple photographic principles. Data of specif c molecules against biomarkers signal strength characteristics in the case standardization is a common topic in ra- (physiological or molecular characteristics of HER2-positive tumors (Fig. 1, A and B). diological imaging studies, especially when of the target disease). T e prevalence and Etaracizumab, Mpab, and other nonpep- multicenter clinical trials are considered. Be- specif city of the biomarker in the group of tide-based small molecules can also be con- cause visible and near-infrared photons have patients imaged will play a central role in sidered for targeting and imaging integrins a strong nonlinear dependence on the geo- the selection process. Selection criteria for (Table 1). Despite their overall widespread metrical and optical parameters of tissue, the imaging biomarkers have been suggested re- distribution and varied functions, certain issue of data standardization is a particularly cently (14). Some validated cancer biomark- integrins, such as αvβ3, are closely associ- important topic in optical imaging applica- ers are summarized in Table 1, as well as ated with angiogenesis and and tions. T erefore, the use of optical imaging corresponding drugs considered for clinical have been investigated as imaging targets technology that yields accurate tissue- and use that target them, which can be applied for cancer using positron emission tomog- system-independent measurements is im- as optical or optoacoustic imaging agents. raphy (PET) (20). portant for ensuring data of quality and ac- T e ultimate clinical application will Overall, small molecules may exert bet- curacy standards that are suf cient for regu- also def ne requirements for the imaging ter tumor penetration and nonbinding frac- latory and clinical approvals (13, 21). molecule used. T eranostic applications, tion and are typically cleared faster from the such as surgical guidance, may choose an circulation than are antibody-based agents, MINIMIZING RISK, MAXIMIZING optical agent that can best delineate tumor resulting in a better plasma-to-tumor ratio. TRANSLATION margins or microscopic residual disease. Conversely, the impact of labeling on the A major bottleneck in performing early- For many cancer patients, tissue microar- biodistribution and clearance of an agent phase clinical studies is not the cost or the ray conf rmation of expression markers can should be studied closely as the size of the ef ort involved in the regulatory process, but be performed on tumor biopsies to select active small molecule becomes comparable the risk of long-term clinical failure. With prominent targets present in the individual in size with the f uorescent label. Moreover, costs of preclinical validation and toxicity

patient. In this case, agent specif city be- antibodies and their fragments generally of an agent, including good manufacturing on May 30, 2012 comes important. Conversely, diagnostic have functional groups available for con- practices for clinical-grade production, be- applications focus on the ability to more jugation with f uorescent dyes, but small- ing relatively low (<0.5 to 1 million euros), generically identify disease, and thus the molecule drugs may not have such groups the main barrier for generating phase I clin- selection of an imaging agent may benef t existing in their structures and thus might ical data is the overall certainty of how well from a broader targeting prof le. require some structural modif cation. A an agent will perform during phase II and Many antibodies, antibody fragments, new molecular structure, however, may III clinical trials—studies that require a larg- and small molecules developed for thera- introduce targeting, biodistribution, and er investment in time and cost (>1 million

peutic purposes have shown good poten- clearance dif erences over the original mol- euros). T erefore, the selection of molecules stm.sciencemag.org tial as imaging agents in nuclear imaging ecule, qualifying the new probe as a new that reduce the risk of clinical failure can in animals and humans or in f uorescence agent and increasing the risk of translation. accelerate the application of f uorescence imaging studies in animals (3, 14). Popular molecular imaging to humans. Molecules targets include proteins involved in tumor IMAGING TECHNOLOGY of known performance in humans of er a growth, extracellular matrix invasion, cell T e optical imaging technology used can potent way to risk minimization. Combined adhesion and migration, and tumor cell also play an important role in the concept of with the possibility of micro-dosing stud- signaling. Cetuximab, nimotuzumab, and risk minimization and the success of clinical ies, such molecules can expedite building panitumumab have been developed for translation. T e use of accurate and sensi- evidence on imaging ef cacy for justifying Downloaded from targeting the epithelial growth factor recep- tive detection systems, in combination with investments into navigating the agent ap- tor (EGFR), which has been implicated in bright optical labels, is crucial for achieving proval process. T is example can follow the cancer growth and migration, with high reliable operation at micro-dosing condi- paradigm of nuclear imaging, which uses prevalence in several dif erent tumor types tions. Unfortunately, the intensity captured microdosing studies to accelerate the de- (Table 1) (14). Such drugs already have in epi-illumination (photographic) f uores- velopment of a clinical agent portfolio, and been considered as nuclear (15, 16) or op- cence imaging does not represent true f uo- potentially benef t from the clinical experi- tical agents (17). T e monoclonal antibody rescence biodistribution because it depends ence using radio-labeled agents (22). Fur- bevacizumab and the derived fragment ra- not only on the underlying f uorochrome thermore, accurate imaging technology and nibizumab have been developed to bind the concentration but also on the local tissue op- the establishment of data standardization vascular epithelial growth factor A (VEGF- tical properties and the f uorochrome’s depth concepts will be benef cial for ensuring the A), which has already been considered as a (1, 21). T e selection of optical imaging tech- generation of high-quality multicenter clini- clinical imaging target in nuclear medicine nology for clinical use should therefore of er cal trial data. Besides camera-based f uores- applications (18, 19). Although VEGF is operating characteristics that yield accurate cence imaging methods, advanced photonic only partly membrane-bound, imaging f uorescent readings that are independent approaches including multispectral opto- studies using radio-labeled drugs have been of tissue discolorations or light illumination acoustic tomography (4) or f ber-based con- able to identify areas of neovascularization. patterns and shadows while of ering real- focal methods can further improve the ther- T e ability to use bevacizumab as an opti- time video operation. Such technology is anostic and diagnostic potential of clinical cal imaging agent is showcased in Figure not generally available because most systems optical molecular imaging.

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