Webinar - Imaging technologies to visualise drug discovery
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Imaging allows a non-invasive assessment of biochemical and biological processes in a living subject. Monitoring, assessing, and characterising novel therapeutics in pre-clinical models is an essential part of drug development. In this webinar Dr Juliana Maynard, Lead Scientist in Pre-clinical Imaging, and Dr Philippa Hart, Lead Scientist in Mass Spectrometry Imaging, explore available imaging technologies and techniques and explain how they can help at different stages of the drug development process.
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Webinar - Imaging technologies to visualise drug discovery
- 1. Imaging Technologies to Visualise Drug Discovery Alderley Park September 2019 Juliana Maynard & Philippa Hart
- 2. © 2019 Medicines Discovery Catapult. All rights reserved. Intro: MDC Science Focus Building the link between compound - target - biological effect in translatable systems to reduce pre-clinical attrition of drug discovery projects Translating clinical information back into the pre-clinical phase to ensure better models, better biomarkers and a patient selection strategy Pre-clinical imagingComplex Cell Models Target Engagement & Validation Bioanalytical Technologies Biomarkers
- 3. © 2019 Medicines Discovery Catapult. All rights reserved. What can imaging do?At the Medicines Discovery Catapult: Imaging expertise in multi-parametric imaging modalities: Positron Emission Tomography (PET) Gamma counting biodistribution analysis Computed Tomography (CT) High Frequency Ultrasound (HFUS) Bioluminescence/ NIR imaging (IVIS) Mass Spectrometry Imaging (DESI, MALDI) In-Vivo/Ex-vivoanatomical andfunctionalimaging Home Office PPL and radiological licence Access to novel radiochemistry &commercial radiotracers Access to animal models across disease areas Access to pre-commercial technologies Multi-modal enhanced interpretation of functional and molecular data MolecularPathology Advanced Microscopy (super- resolution/confocal/multiphoton) Digital Spatial Profiler (Nanostring DSP)
- 4. © 2019 Medicines Discovery Catapult. All rights reserved. Imaging in drug discovery Imaging technologies provide visualization of biological functions to accelerate disease understanding and drug development Organ accumulation (PK) Target expression & engagement (biomarkers, stratification) Biological activity (PD, optimal biological dose) Toxicities/Drug interactions (safety) Surrogacy for clinical endpoint (predictive efficacy) PK,PK/PDandbiologicalmodelling Multimodal and multiscale imaging solutions enable quantification and understanding of:
- 5. © 2019 Medicines Discovery Catapult. All rights reserved. In-vivo Imaging
- 6. © 2019 Medicines Discovery Catapult. All rights reserved. Positron Emission Tomography & Near Infra-Red imaging NIR fluorescence and bioluminescent imaging NIR Imaging • Deep whole body penetration • Good signal to noise allows reproducible quantification Bioluminescence imaging • Ability to perform lentiviral transduction of luciferase expressing cell lines and commercially buy 89Zr PET distribution • Distribution of radiolabelled candidate • Distribution and accumulation of the compound across time • Allows for determination of PK and distribution of large molecules • Fully-quantitative method
- 7. © 2019 Medicines Discovery Catapult. All rights reserved. Target engagement– Binding of Drug to Target Maynard et al; 2016: https://DOI: 10.1186/s13550-016-0220-9 AZD8186 : PI3Kβ/δ inhibitor Hypothesis: Tumours with a PTEN deficiency will benefit from AZD8186 therapy and 18F- FDG is a good biomarker Deliverable: A significant reduction in 18F- FDG uptake was seen in 786-0 and U87-MG cell lines (both PTEN null) and not BT474C (PTEN avid)
- 8. © 2019 Medicines Discovery Catapult. All rights reserved. Dynamic 18F-FDG PET Mechanism 18F-FDG uptake complicated: GLUT activity, intracellular 18F-FDG phosphorylation, tumour oxygenation, blood flow and vessel permeability Mechanism 18F-FDG uptake complicated: GLUT activity, intracellular 18F-FDG phosphorylation, tumour oxygenation, blood flow and vessel permeability Compartment 1: Delivery of 18F-FDG in interstitial space Compartment 2: 18F-FDG trapped in intracellular space Mechanism 18F-FDG uptake complicated: GLUT activity, intracellular 18F-FDG phosphorylation, tumour oxygenation, blood flow and vessel permeability
- 9. © 2019 Medicines Discovery Catapult. All rights reserved. Biological activity – is the drug efficacious? Maynard et al; 2017: https://DOI: 10.1371/journal.pone.0183048 AZD8835 : PI3Kα inhibitor Hypothesis: 18F-FDG is a good biomarker to determine the minimal effective dose of AZD8835 Deliverable: Decrease in 18F-FDG uptake was dose dependant with excellent PK/PD correlation observed
- 10. © 2019 Medicines Discovery Catapult. All rights reserved. Toxicities– is the drug safe to be used? Mechanism 18F-FDG uptake complicated: GLUT activity, intracellular 18F-FDG phosphorylation, tumour oxygenation, blood flow and vessel permeability Mechanism 18F-FDG uptake complicated: GLUT activity, intracellular 18F-FDG phosphorylation, tumour oxygenation, blood flow and vessel permeability Cardiac Hypertrophy Compensatory enlargement of the heart in cardiac disease Clinical Cardiology : Echocardiography Gold Standard Ability to track the pathological process and obtain information on functional changes (often preceding anatomical change) Longitudinal pre-clinical characterisation reduces the need to sacrifice animals at each time point. Combining in-vivo imaging with histopathology gives a complete assessment of toxicological effects Ability to monitor disease progression Allow for interventional therapy in a toxicity trial Sysa-Shah et al
- 11. © 2019 Medicines Discovery Catapult. All rights reserved. Surrogate– Can we use imaging to predict response? Mechanism 18F-FDG uptake complicated: GLUT activity, intracellular 18F-FDG phosphorylation, tumour oxygenation, blood flow and vessel permeability Maynard et al; 2013: https:// DOI: 10.1007/s11307-013-0613-3 AZD5363 : AKT inhibitor Hypothesis: 18F-FDG PET is a promising pharmacodynamic biomarker of AKT pathway inhibition. Deliverable: Decrease in 18F-FDG PET only in models responsive to treatment Outcomes: 18F-FDG may have potential as a biomarker that may stop ineffective drug schedules, helping to make early stop decisions and identify responding subsets of patients, resulting in improved clinical decision making both during drug development and patient management.
- 12. © 2019 Medicines Discovery Catapult. All rights reserved. Ex-Vivo Imaging
- 13. © 2019 Medicines Discovery Catapult. All rights reserved. Mass Spectrometry Imaging (MSI) Desorption ElectroSpray Ionisation Matrix Assisted Laser Desorption Ionisation
- 14. © 2019 Medicines Discovery Catapult. All rights reserved. Mass Spectrometry Imaging (MSI) • Unlabelled • Can analyse: Small molecules, Metabolites, lipids, peptides/proteins, glycans etc. • In some cases, different molecular classes can be analysed from a single section • Relative/semi-quantitation
- 15. © 2019 Medicines Discovery Catapult. All rights reserved. Conventional H&E with DESI-MSI and MALDI-MSI Compound penetration: • DESI-MSI for drug distribution • Overlaid with H&E stain • MALDI-MSI for improved resolution and confirmation of localisation 20 µm 50 µm
- 16. © 2019 Medicines Discovery Catapult. All rights reserved. Multimodal imaging of organoids • Human induced pluripotent stem cell (iPSC)-derived kidney organoid containing at least 12 separate kidney cell types • Organoid stained with antibodies to markers of different kidney cell types • Immunofluorescence imaging using confocal microscopy Glomeruli Proximal Tubule Distal Tubule Collecting Duct
- 17. © 2019 Medicines Discovery Catapult. All rights reserved. Multimodal imaging of organoids • MALDI-MSI of endogenous lipid distribution • Segmentation mapping resulting from K-means clustering 20 μm 10 μm
- 18. © 2019 Medicines Discovery Catapult. All rights reserved. Wide-field STORM localisations: 20m 5m 200nm EGF-biotin/ Streptavidin-Alexa647 EGF Receptor Clustering STORM Localisation (stochastic imaging) Advanced Microscopy:
- 19. © 2019 Medicines Discovery Catapult. All rights reserved. • The DSP can quantitatively analyse up to 800 protein or RNA targets with spatial context. • Nanostring have developed this assay based on FFPE tissue We’re participating in the GeoMx Priority Site (GPS) initiative First access to commercial instrument Protein multiplex data from Nanostring; Digital Micromirror adjusts to the shape of the immune compartment: Contour masking around macrophage-boundaries reveals spatial heterogeneity of immune responses Nanostring GeoMx Digital Spatial Profiler – Immuno Oncology Profiling
- 20. © 2019 Medicines Discovery Catapult. All rights reserved. RNA multiplex data from Nanostring 96-plex IO-targets mRNA spatially profiled in many different tumour types Nanostring GeoMx Digital Spatial Profiler – Immuno Oncology Profiling
- 21. © 2019 Medicines Discovery Catapult. All rights reserved. Future opportunities
- 22. © 2019 Medicines Discovery Catapult. All rights reserved. Developing opportunity: 89Zr cell labelling approach for PET Mechanism 18F-FDG uptake complicated: GLUT activity, intracellular 18F-FDG phosphorylation, tumour oxygenation, blood flow and vessel permeability Mechanism 18F-FDG uptake complicated: GLUT activity, intracellular 18F-FDG phosphorylation, tumour oxygenation, blood flow and vessel permeability Discovery and Research Driven; Challenging and optimising the use of imaging and testing Novel 89Zr cell labelling approach for PET-based cell trafficking studies ABILITY TO ACCESS VALIDATED 89Zr VALIDATED RADIOCHEMISTRY FOR USE Bansel et al; 2015: https:// DOI: 10.1186/s13550-015-0098-y Need: Robust cell labelling and imaging methods for in-vivo tracking of living cells urgently needed Deliverable: Robust biostable 89Zr labelling strategy shows promise for wide application PET based non-invasive in-vivo cell trafficking -T cell-specific PET imaging can visualize tumour-infiltrating lymphocytes and monitor the dynamics of T cells in response to chemotherapy, radiotherapy, molecularly targeted therapy, immunotherapy, and adoptive cell transfer.
- 23. © 2019 Medicines Discovery Catapult. All rights reserved. Other Opportunities : What does the future look like? Mechanism 18F-FDG uptake complicated: GLUT activity, intracellular 18F-FDG phosphorylation, tumour oxygenation, blood flow and vessel permeability Mechanism 18F-FDG uptake complicated: GLUT activity, intracellular 18F-FDG phosphorylation, tumour oxygenation, blood flow and vessel permeability Discovery and Research Driven; Challenging and optimising the use of imaging and testing Using Pre-Clinical Imaging to better humanise drug discovery CLINICPRECLINICAL MODEL IMAGING Example Project: ORTHOTOPIC TUMOUR MODELS Challenge: Transplanting cells anatomically into the host organism poses a difficulty in assessing tumour burden Solution: Imaging offers significant potential and use of multimodal techniques and capabilities allow the ability to derive complex biological characterisation Impact: Highest failure rate of new agents – inability to recapitulate the patient situation. PoC pre-clinical data in early pre-clinical models and demonstration of efficacy and effect in humanised model viewed positively by MHRA EXPLOIT AND USE IMAGING TO CHARACTERISE COMPLEX MODELS ACROSS MULTIPLE DISEASE AREAS
- 24. © 2019 Medicines Discovery Catapult. All rights reserved. In-vivo, real time imaging Summary: Multimodal pipelines Ex-vivo imaging In-vitro, live real time imaging Fixed endpoint imaging PET-CT, Ultrasound, NIR Incucyte, confocal microscopy MSI, DSP, Microscopy MSI, DSP, Microscopy
- 25. © 2019 Medicines Discovery Catapult. All rights reserved. Summary: Implementing Multimodal, Multiscale Imaging in Drug Discovery Biomarkers Targeted, real-time in-vivo or in-vitro Untargeted, exploratory/ biomarker discovery, need for high content dataTargeted, known and quantifiable Targeted, known spatial correlation with drug and drug metabolites Choose the right approach to answer your biological question, e.g: Future Challenge: Dynamic integration of imaging data…
- 26. © 2019 Medicines Discovery Catapult. All rights reserved. Acknowledgements Gayle Marshall Isabel Peset Martin Kevin Randall Ekta Patel Lorna Hale Matthew Burnham Benedetta Arno Discovery Science and Technology
Editor's Notes
- Selecting the right drugs for the right patients, using biomarkers to stratify this and improve predictability of drug response.
- Next I will introduce the technologies and show some selected examples before going into multi-modality.
- Looking at samples on different scales is one reason ofor implementing multiple imaging techniques. However as you may know, there is a wealth of other information to be gained.
- As discussed each of the modalities offer a unique offering – PET and NIR imaging are frequently used to measure and assess compound distribution and accumulation – PET is a functional imaging technique radiolabelled distribution studies can be performed either using direct labelling or indirect approaches – they can be used to assess the distribution of the candidate compounds longitudinally – especially with larger molecules and biologics where there are limited ways to gain quantititvate assessment of distribution – PET is a very sensitive methods and can be done in 3D
- Looking at samples on different scales is one reason for implementing multiple imaging techniques. However as you may know, there is a wealth of other information to be gained. In general, the following ex-vivo techniques can be used for assessing biological activity, and toxicity (biomarkers), and in some cases for looking at drug distribution and evidence or target engagement or target validation.
- In this particular case these images were generated by Ekta from FFPE tissue where paraffin removal, antigen retrieval and on-tissue, in-situ digestion was carried out. Generally we find fresh frozen tissue to be best due to the harsh treatment required to handle fFPE tissues.
- For model characterisation, for drug distribution, for biomarker analysis
- For model characterisation, for drug distribution, for biomarker analysis. This work is ongoing and workflows will be applied to other organoid and spheroid models for drug distribution and monitoring of biomarkers for biological activity, evidence of efficacy, toxicity etc.
- When increasing the resolution we see that there are many more receptors present in the cell membrane than there appears to be in the lower res images. This allows for better measurement of distance between molecules and for precise imaging of defined structures, such as mitochondria. This is intended to be implemented for target validation and engagement studies.
- Looking at samples on different scales is one reason ofor implementing multiple imaging techniques. However as you may know, there is a wealth of other information to be gained.
- Can track cells in-vivo, mainly immune, t-cells, b-cells, macrophages. New and noevl ways of labelling them where we don’t currently have any way of doing so at the moment (easily, in-vivo). We are also looking to further this by taking a similar but unlabelled approach with MSI
- In addition to the humanised approach we are aiming for with the organoid and spheroid models in-vitro (mentioned previously), there are a number of ways that we can use preclinical imaging to humanise the drug discovery approach. Using orthotopic models in addition to subcutaneous models
- Summary: there are a variety of different approaches that can be used for in-vivo, ex-vivo and in-vitro imaging.