Translational Biomarkers and Bioanalysis Glen Hawthorne and Emilyanne

Translational Biomarkers and Bioanalysis Glen Hawthorne and Emilyanne Leonard 25 10 17

Astra. Zeneca Cambridge Small molecule and biologics R&D • Global pharmaceutical company with a historic presence in UK • 6, 700 employees Avlon, Bristol • 7 sites in the UK Manufacturing home of two Active Pharmaceutical Ingredients (APIs) • Support up to 35, 000 jobs • Cambridge focus is oncology 2 Alderley Park R&D and support from the beginning of the drug discovery process to late stage development Macclesfield Luton Manufacturing, packing, and distribution of medicines to 130 global markets UK Marketing Company (UKMC) handles the marketing of AZ medicines in the UK

3 -6 years Preclinical Studies 4 -7 years Review and Approval Target Identification Compound Screening Lead Identification In vitro studies In vivo studies Investigational New Drug application 1 year Phase I: healthy humans Phase II: patients Phase III: many patients Clinical Trials New Drug Application and FDA review Drug is approved and manufactured Post Release Monitoring 1 -2 years Research & Development

Pre Clinical In Vitro Studies • Major points to explore: – Drug behaviour– absorption and metabolism – Toxicity– are there any safety concerns – Dosing – what is an ideal amount for treatment • Investigations use these technologies: – Cell Culture – Organoids – Microphysiological Systems • Measured in cells or the media: – Biomarkers – Drug 4

Pre Clinical In Vivo Studies • Each study measures: – Efficacy – how well the medicine works – Toxicokinetics – how the medicine moves through the body – Safety – are there any unexpected side effects – Dosing – what is an ideal amount for treatment • More than one study per drug: – Short-term ‘mini’ studies – Month-long studies • Biomarkers and Bioanalysis – Tissues: liver, brain, lung, etc. – Body fluids: plasma, whole blood, urine, etc. 5

Sample Collection and Types • Collected by live animal sciences colleagues • Usually through the tail vein 6 Plasma separated Whole blood

Bone Marrow Sampling Centrifuge LC/MS Snip Flush Filter Dilute 7 Cell Count

Measuring drug in biological samples - bioanalysis

LC-MS: Liquid Chromatography - Mass Spectrometry Signal 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 Time, min

LC-MS: Quantitation Signal 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 Time, min

What is Clinical Pathology and Biomarker Analysis? • Measuring the toxicity of new drugs by testing for specific biomarkers in body fluids • Biomarkers can be cells, proteins, molecules like DNA, etc. • Each marker gives information about the functioning of the body – White blood cells: an low number can mean bone marrow effects – Troponin: raised levels of this protein can indicate heart injury – mi. R-122: changes in levels of this RNA molecule can indicate liver damage • Categories of testing: – haematology – clinical chemistry – molecular pathology 11

What does our instrumentation look like? BD FACSCanto II 12

What are the steps in flow cytometry? 13 Fluorophore labelled antibodies attach to molecule of interest Lasers with specific wavelengths are directed at particle stream Fluorophore is excited by a certain wavelength laser Electrons in the fluorescent molecules move to a higher energy state When electrons return to ground state, photons are emitted at a higher wavelength Photons pass through specific filters to be measured Signal from the filters reaches the detector Signal is converted into a voltage and recorded by the computer

Data Example – Rat Bone Marrow Differential Single Gate TN Ga C te L/M Gate 14

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