Imaging Systems

Preclinical Imaging and its Importance in Drug Discovery and Development

By Alexandra De Lille Advances in non-invasive, in vivo imaging techniques have raised the use at Caliper Life Sciences of animal models in preclinical R&D to a new level, enabling the underlying biological processes of disease to be revealed and the effects of therapeutic intervention to be monitored.

During the preclinical stage of Molecular imaging utilises highly settings, both bioluminescence and research in drug discovery and specific probes and gene reporters fluorescencein vivo imaging are the development, in vitro and in vivo tests to monitor gene expression, most established modalities in the are conducted to ensure safe testing tumour growth and metastasis, preclinical environment. They are in humans prior to the initiation of bacterial and viral infections, relatively inexpensive, yet highly clinical trials. Preclinical research homing, and biomarker and sensitive technologies with a flexible employs animal models to facilitate compound biodistribution. The toolbox of reporters and disease- the investigation of human disease hand-in-hand development of specific, targeting probes. and to aid in the development of molecular imaging instrumentation, treatment. The biological relevance transgenic animal models, cells Non-invasive imaging enables the of an animal model is critical to the and probes facilitate this dynamic same animal to be followed over predictive value of clinical outcome. interrogation of molecular pathways time. This principle of longitudinal The recent development of non- in vivo. Therefore, the application studies increases the statistical invasive, in vivo imaging techniques of molecular imaging is key in power of the model significantly, has vastly advanced the sophistication revealing the underlying biological since each animal serves as its of animal models. Non-invasive processes of disease and monitoring own control. Subsequently, this animal imaging delivers fast, therapeutic intervention. practice reduces the number of longitudinal, accurate, real-time and animals needed while providing quantitative assessments of treatment In the clinic, positron emission accurate, whole body, life animal efficacy. The implementation of tomography (PET) and single- and physiologically relevant preclinical imaging enables photon emission computed information. Nonetheless, a single translational medicine, de-risks tomography (SPECT) scanners are imaging modality may not address compound candidates, significantly the primary technologies utilised all biological questions asked shortens ‘time to the clinic’, and to visualise molecular processes in of the model. Multiplexing with lowers costs while maximising humans by means of radioisotopes. various reporters or probes, or the biological understanding. While optical imaging is an emerging combination of molecular and imaging modality in clinical anatomical imaging modalities Molecular Imaging Beyond traditional anatomical imaging modalities, the field of molecular imaging (MI) has developed exponentially over the past decade. Keywords Molecular imaging is a highly sensitive, Preclinical imaging quantitative technique, capable Molecular imaging (MI) of visualising Positron emission biological processes tomography (PET) Figure 1: Brain tumour model expressing non-invasively, a bioluminescent report to track tumour Computed tomography (CT) in living animals. burden and therapeutic effect Images: Caliper Life Sciences

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within a single animal study is Coronal (z=6.5) Sagittal (x=6.3) Figure 2: Co-registered image

increasingly prevalent, with each of 5.0 showing accumulation these parameters offering an added of 2-DG750 at MDA-MB231 dimension to the validation of a metastasis sites specific drug or treatment regime. 4.0

Multimodality Imaging 3.0

With MI, we can detect signal with x 106 an extremely high sensitivity but we inherently lack anatomical context. 2.0 MI is commonly complemented Transaxial (= -11.1)

by anatomical imaging. Computed 1.0 2-DG750 targeting tomography (CT) or magnetic

resonance imaging (MRI) modalities Subject height: 19.8mm Perspective offer excellent spatial resolution Bioluminescence and signal-to-noise characteristics; they also help provide anatomical and organ identification. Many localisation and staging of disease, pre-clinical imaging modalities while MI provides specificity and have cardiac and respiratory gating differentiation of diseased from methods integrated in order to normal tissue. While MRI provides reduce motion artifacts and enhance non-ionising 3D imaging and is image resolution. Lastly, multi- the best technique for soft tissue modality co-registration and data personalised medicine, tailoring resolution, longer acquisition times integration can be challenging if diagnosis and treatment to the and the high Tesla requirement animals are repositioned in between individual patient’s genetic make-up. for high resolution of small modalities or imaging sessions. animals, organs and structures Various software and hardware Imaging can be implemented are relatively costly. Dedicated solutions are available. The mouse at various stages of the drug preclinical microCT instrumentation imaging shuttle from Caliper has development process, and it is may be more readily accessible. built-in fiducial markers compatible important to design an imaging Since CT scanners utilise ionising with the IVIS® Spectrum, Quantum master plan in the early stages. radiation, the cumulative effect FX µCT and many other microCT, In vivo imaging as a whole is a of radiation exposure must be MRI, PET and SPECT scanners from biology-driven practice, in which the carefully considered, especially in diverse vendors. Moreover, the Living disease dictates the animal model longitudinal studies where the total Image® software allows for seamless and the imaging modality. Today, dose may be lethal – the mouse co-registration between the IVIS® it is possible to carry the same

LD50/30 (kills 50 per cent within Spectrum and Quantum FX µCT biomarker from in vitro testing all 30 days) is 5-7.6 Gy (1)). DICOM data. Additionally, any the way through to clinical trials, DICOM, TIFF, RAW and VOX and potentially use this same High speed (17 seconds), low dose volumetric data files can be biomarker as a campaign diagnostic (14mGy per scan), high resolution imported to co-register data from or prognostic test. Translation from (10μm) microCT scanners are other 3D modalities. Ultimately, bench to bedside and subsequent now commercially available (for the practice of biology-driven back-translation to the bench is a example, the Quantum µCT and multimodality imaging requires powerful cycle of information- IVIS® SpectrumCT from Caliper access to core imaging facilities. gathering in drug development. Life Sciences). These features Ensuring a smart choice of animal enable longitudinal imaging Translational Imaging models, reporters and imaging for anatomical reference and The primary goal of translational modalities at the earliest preclinical quantification in sensitive disease imaging is to improve the odds time point can reduce time in each models such as tumour models of clinical success and reduce phase of development. Careful and bone remodelling. Short development time and costs. quantitative modelling and acquisition times also permit the Currently, only eight per cent of new de-risking of each NME throughout use of (clinical) contrast agents, molecular entities (NMEs) make it development optimises pipeline facilitating affordable contrast agent to the market at an estimated R&D management and facilitates imaging with uCT for vascularisation, cost of $1.8 billion per NME (2). As a decision-making towards perfusion and cardiac hypertrophy secondary goal, imaging can facilitate abrogating or accelerating trials.

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of the isotype. However, it is equally (unbound) signals in the important to assess the stability of blood pool. Alternative targeting the molecule in serum, its primary agents – such as wavelength intended in vivo environment. Serum responsive smart probes that shift proteins affect the physicochemical wavelength upon binding – facilitate properties of the molecule, lowering the differentiation of bound versus their potency. The ability to rapidly unbound probe. screen directly after circulation in blood is an important The IVIS® Spectrum CT is uniquely step in the therapeutic antibody equipped with a transillumination development decision-making imaging module process. Caliper’s LabChip GXII and advanced 3D reconstruction offers a high throughput analytical software algorithms facilitating in technique with very good precision situ tomographic reconstruction and sensitivity. Fluorescently and absolute quantification in labelled mAbs can easily be picomolar concentrations of the analysed directly from blood, bound marker signal. Anatomical providing real-time information on context is quickly rendered by the behaviour of candidate mAbs co-registration with a built-in in blood. There is no need for gel microCT scanner. slabs – electropherograms will give

Figure 3: A Hypothetical Example a reading in just seconds from a The hypothetical animal model 2D Cerenkov A good example would be the microfluidic chip. comprises immunocompromised dynamic contrast enhancement development of a new therapeutic mice inoculated with a human (DyCE) imaging of antibody. It would be ideal to Biodistribution of fluorescently tumour cell line (human epitopes) 90Y biodistribution develop an in vivo test plan to labelled antibodies in lab animals can expressing, firstly, a constitutive relative abundance is in agreement test the following parameters: readily be monitored in real time by renilla luciferase for tumour growth with ex vivo beta stability, PK/PD, biodistribution, means of an in vivo optical imaging and metastasis tracking, and counter data tumour targeting and efficacy. system. Traditionally, the animal is secondly, a firefly luciferase inducible Quantitative modelling will be imaged at various time points post reporter to assess the efficacy of the greatly facilitated if the antibody injection; the early time points post therapeutic antibody (for example, is tagged with an optical or injection should be particularly hvegfr2-luc for angiogenesis or an radio-isotopic moiety. Although closely monitored. Whole body apoptosis sensitive split luciferase). labelling with a radioisotope imaging offers the advantage of not These sophisticated animal models renders molecule imaging directly just focusing on the target tissue; allow evaluation of antibody translational to humans (PET, SPECT), unexpected accumulation may be biodistribution, pharmacokinetics, the convenience, cost savings and observed in the heart, for example, tumour targeting, efficacy and opportunity to easily multiplex indicating potential cardiotoxicity. pharmacodynamics (for example, various reporters and imaging Furthermore, as opposed to apoptosis), and tumour metastasis modalities from in vitro to in vivo is traditional tumour uptake and all in one animal, longitudinally, enabled by a fluorescent marker. wash-out over time, pharmacokinetic non-invasively and in high-resolution data can be generated from anatomical context. Targeted probes such as antibodies temporal imaging to determine the can easily be labelled with a amount of a marker in various body After the last in vivo image, organs fluorescent dye prior to injection. compartments of the animal. This is are dissected and quickly imaged Various user-friendly antibody commonly practised in PET imaging ex vivo in the in vivo imaging system. labelling kits are available. For in but is only nascent in fluorescence Further ex vivo tissue molecular vivo fluorescence imaging, a dye imaging. With dynamic contrast validation is done by histopathology with a near infrared (NIR) emission enhancement (DyCE) kinetic analysis and/or immunohistochemistry on spectrum is recommended for software, organ-specific accumulation paraffin-embedded fixed tissue. higher sensitivity based on tissue of antibody can be detected. The Fluorescence microscopy validates optical properties. rate of change in concentration, that the fluorescently labelled or the ‘slope’ for an organ, can be antibody not only accumulates Therapeutic antibody selection calculated and specific (bound) in the tumour, but also binds to is largely determined by the antibody signal accumulation can be its antigen. The Nuance liquid understanding of the basic biology spectrally unmixed from non-specific crystal tunable filter microscopy

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camera is a powerful tool for disease models that recapitulate Conclusion multispectral imaging on bright the complexities of human Preclinical imaging not only impacts field and fluorescence microscopes; and metastatic progression. BLI cancer research; infectious disease, its multispectral imaging systems allows quantitative assessment of Alzheimer’s disease, gene therapy, enable the quantification of the antitumour and antimetastatic inflammation, diabetes, stem cell multiple disease and drug response efficacy. BLI is a sensitive, noninvasive therapy and immunotherapy are molecular markers, even when tool for longitudinal assessment all beneficiaries of this technology. they are co-localised in a single of tumorigenesis, metastasis, and Researchers have a vision that tissue section, producing clear and therapeutic intervention in animal molecular imaging will revolutionise accurate images of each individual models. By using optimised BLI our biological understanding of label on a multi-label tissue section. technique, clinically relevant disease disease, facilitating therapeutic The Nuance integrates a unique models can be developed for discovery and personalised treatment. multispectral imaging technology efficient evaluation of drug effects, Aggressive innovation of new with proprietary image analysis thereby allowing scientists to gain technology is key to bridging the gap algorithms to achieve unparalleled deeper knowledge of the underlying between in vitro assays and in vivo accuracy and sensitivity, rapidly biology during disease progression results, enabling the translation of and cost-effectively. Biomarker and to accelerate cancer those results into cures for disease. We multiplexing microscopy of drug discovery” (3). should strive to transform ‘sick care’ to histopathology sections from ‘health care’ through next-generation clinical trials produces quantitative Even though bioluminescence biomarker discovery, patient data facilitating rapid NME decision- and fluorescence imaging (FLI) stratification studies and companion making. In the clinic, biomarker have direct translational limits, it diagnostics for personalised medicine. multiplexing microscopy allows for is clear that their implementation better diagnostics and can be used provides an insightful technology References to guide personalised therapy. for fast, efficacious and cost- 1. Tremoleda et al, Imaging effective use and characterisation technologies for preclinical Prior to clinical trials, the antibody of animal disease models and models of bone and joint can be tagged with a clinical drug discovery validation. BLI and disorders, EJNMMI Research radionuclide. For translational FLI represent a good alternative 1(11), 2011 purposes, the distribution of this and/or complementary validation 2. Paul SM, Mytelkal DS, radionuclide labelled antibody can mechanism for more invasive Dunwiddie CT et al, How to be screened in lab animals using an techniques such as histopathology improve R&D productivity: the IVIS in vivo optical imaging system and biochemical assays. Through pharmaceutical industry’s grand which has the sensitivity to detect the combination of various imaging challenge, Nature Reviews Drug the Cerenkov light emission from b+ modalities, molecular, anatomical Discovery 9, pp203-214, 2010 or b- emitting radiotracers. Cerenkov and functional data can accurately 3. Zhang C et al, Advancing light imaging data has been found and quantitatively de-risk preclinical bioluminescence imaging to give a strong correlation with NME discovery efforts and help technology for the evaluation of immuno-PET and biodistribution ineffective drug candidates to anticancer agents in the MDA- studies and offers the potential fail more quickly. The ability to MB-435-HAL-Luc mammary fat to conduct rapid, low-cost, high- image live animals is one of the pad and subrenal capsule tumor throughput screening of novel most important advantages of models, Clin Cancer Res 15(1), radiotracers in vivo. these technologies. pp238-46, 2009

The translational relevance of preclinical bioluminescence imaging Alexandra De Lille, DVM, PhD, is Director of Technical Applications at Caliper Life (BLI) has been advocated by Dr Cathy Sciences (Hopkinton, MA, US), a PerkinElmer Company. She graduated with honours as a veterinarian at the University of Ghent (Belgium) and subsequently attended Zhang from Pfizer’s Department of a PhD programme in reproductive physiology/molecular biology at the Colorado Cancer Biology: State University (Fort Collins, CO, US) studying angiogenesis and growth factors in the bovine placenta of cloned pregnancies. For her post-doctoral research, Alexandra “Cancer metastasis presents an managed a cancer pharmacology laboratory at the Animal Cancer Center of the Colorado State ongoing challenge for modern University in collaboration with the Department of Pharmacology at the University of Colorado Health drug discovery. Recent advances Sciences Center. This laboratory investigated drug delivery of cancer therapeutics in animal models in optical imaging technology using imaging technologies. She then went on to join Caliper in her present position. provide great opportunities for Email: [email protected] researchers developing animal

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