Role of DMPK Platform. in Helping Early. Andy Harrell. Property of GlaxoSmithKline

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1 Role of DMPK Platform Technology & Knowledge in Helping Early Discovery Research Andy Harrell Property of GlaxoSmithKline

2 Discovery Preclinical Clinical Non Clinical Development Clinical

3 Discovery Preclinical Clinical R dl f i ti t f DMPK b t Regardless of organisation, some aspects of DMPK are best deeply embedded within the discovery program, whilst others are best operating as platforms across multiples programs.

4 A collection of Short Stories which demonstrate how platform science and knowledge can be used to help drug discovery 5

5 Definition of a Platform Science and Knowledge An expensive piece of equipment or suite of equipment......or......specialist scientific knowledge/expertise associated with only a few highly trained individuals possibly applied across the whole organisation on most projects at some stage but not necessarily routinely applied within a single project......often contributing through bespoke support to answer a specific question. Presentation title in footer 00 Month

6 Drive Through Metabolism by Steve Thomas & Nigel Deeks Platform: Nuclear Magnetic Resonance Spectroscopy Specialist Knowledge: Metabolite Identification

7 Drive Through Metabolism Promising chemical scaffold. (1) Bulk Microsome Incubation Clearance issue which could not be resolved by mass spectrometry alone (2) Fractionation Response Time (3) Identification NMR +16 % (4) Quantification DRUG METABOLITE 70 30

8 Drive Through Metabolism Major routes of metabolism identified in very short time-frame commensurate with drug discovery. Metabolite amounts estimated by NMR integral of a common proton. OH Methods also applicable to plasma, bile and urine from animals and man. Project team able to address pharmacokinetics through structural modification. OH Evaluation of preparative high performance liquid chromatography and cryoprobe-nuclear magnetic resonance spectroscopy for the early quantitative estimation of drug metabolites in human plasma GJ G.J. Dear, AD A.D. Roberts,C. Beaumont,S.E. SE North

9 Seeing is Believing By Steve Castellino, Bianca Squillaci, i William Hardesty, Reid Groseclose, Peter Marshall and Josie Morrell Platform: MALDI MS Imaging Specialist Knowledge: Drug Disposition

10 Seeing is Believing Extraction of analyte into matrix droplet Matrix Application Co-crystallisation of analyte(s) with matrix Laser MS Spectra acquired across tissue surface Ion distribution map can be generated for any ion of interest Matrix applied evenly across surface using automated device ~ 12µm sections thaw-mounted onto surface e.g. glass slide Frozen tissue Sliced using a cryo-microtome Adjacent slide retained for staining H&E stained tissue MS and H&E data overlaid in software

11 Seeing is Believing MALDI-MS imaging has been used to help the GSK effort to discover new drugs for skin indications by......comparing the skin penetration of various drugs & formulations..... and visualising target engagement for a sweat gland indication.

12 Seeing is Believing MALDI-MS imaging has also been used to help in the discovery of new drugs for respiratory indications. e.g. by correlating drug disposition with histology or pharmacology...or investigating accumulation in the animal lungs Day 1 Day 28

13 Too Hot to Handle by Alison Churchill Ian Baines & Rob Chambers Platform: Radioisotopes Specialist Knowledge: Mechanisms of Metabolism

14 Short Story 2: Too Hot to Handle Targeted covalent binding via Michael addition of αβ unsaturated carbonyl group. Unprecedented mechanism within GSK Program collecting as much information as possible on specificity of binding. Early ADME using [ 14 C] material conducted within DMPK prior to candidate selection Covalent interaction with target. Concerns about non-specific interactions resulting in direct, genetic or immunogenic toxicity. SH

15 Too Hot to Handle % Radioactive Dose (0-24 hours) Urine 25 Faeces 57 Wash 2 Total C

16 Too Hot to Handle Alerts Metabolism Covalent Binding Body Burden Trapping In Vivo CYP In Vitro In Vivo QWBA Tissue Dose Overall Risk deemed moderate based on integrated weight of evidence assessment. Need for low dose emphasised to project team. First Time in Human included a dose level stopping criteria regardless of systemic exposure.

17 Predictable Pathways by Gary Collins. Platform: Metabolic Prediction Software Specialist Knowledge: Drug Metabolism; Computational Chemistry

18 Predictable Pathways A suite of complementary software packages which, in the hands of an experienced user, can predict metabolic routes and advise on structural modifications which may modify safety, efficacy and pharmacokinetics. M t MetaSite CYP-mediated prediction

19 Predictable Pathways Metabolite Browser and Meteor were used to demonstrate risks associated with benzodiaxoles (autoinhibition and reactive quinones as a result of ring opening)...as a result direction of chemistry was diverted toward active ethoxy structures......metasite predicted O-dealkylation as most likely route of metabolism. DEREK raised a genotoxicity alert for corresponding quinone-imine. Risk mitigated because no extractable proton on tertiary nitrogen. Yet to establish whether O- dealkylation is problematic from a PK view.

20 Predictable Pathways Predicting Increasing Metabolic Stability In this example MetaSite was used to help a discovery programme optimise on metabolic stability Additional advice provided on aldehyde oxidase (see later on).

21 Gone with the Wind by Helen Tracey, Stephanie North. Platform: DMPK WIKI, In vitro assays. Specialist Knowledge: Corporate History, Drug Metabolism.

22 Gone with the wind N N N H Molecules looked progressable based on rat, dog in vivo clearance & microsomal clearance (all species). Moderate - high clearance in cynomolgus monkey. > >

23 Gone with the wind N N N H Aldehyde Oxidase (AO) Previous experience with AO recalled. AO is a non-microsomal, non-cyp enzyme prevalent in man. Substrates usually have good bioavailability in rat & dog and are stable in microsomes. AO substrates, however, often have poor PK in man. AO screen applied to help progress molecules without AO liability.

24 Gone with the wind A considerable amount of information was available on DMPK WIKI (a DMPK knowledge depository similar to wikipedia) which covered both past experiences and scientific information. Examples of regio-selective oxidation by aldehyde oxidase. Electron deficiencies correlate with regioselective metabolism - numbers in red highlight position(s) of oxidation.

25 Challenging Journeys by Nainesh Patel, Donna Fraser, David Kenworthy, Richard Snell Platforms: Chromatography and Mass Spectrometry Specialist Knowledge: Oligonucleotide and peptide analysis in biological matrices

26 Challenging g Journeys Delivery to target tissue remains a key problem for oligonucleotide discovery programs across a variety of disease indications U A G C U A G C U A G C U A G C U A G C U A G C U A G C U A G C U A G C U A G C U A G C U A G C DMPK was asked to help investigate metabolic stability and distribution of base, linker and conjugate modifications for an anti-sense therapy.

27 Challenging g Journeys DPM Tissue Concen ntration / [ng/g g] Target tissue extract mins Oligonucleotide & Metabolites Target Concentration vs. Time 6E+4 5E+4 4E+4 3E+4 2E+4 1E+4 0E Time / Hours Group 1 Group 2 Group 3 Target tissue extract Using radiolabel and cold approaches concentrations were measured & compared concentrations for each separate analogue in target tissues in order to assess the effectiveness of the structural modification. This example draws upon people with deep-seated analytical expertise able to rise to the challenge of many different types of molecules.

28 Challenging g Journeys For a different discovery programme looking at short interfering i RNA( (sirna), the activity it seen in cell lines could not be replicated in vivo. We were able to compare metabolic vulnerability of different anti-sense strands in the relevant biofluid. Doubtful (also) whether the sirna was ever present, in vivo, in the duplex form required for activity. it 70% Turnover of various sirna oligonucleotides Sense Anti-sense OG1 OG2 OG3 OG4 OG5 OG6

29 Challenging Journeys Serum stability was an import consideration for a discovery unit trying to engineer proteolytically resistant peptides that can enter cells and modulate protein interactions Drawing on DMPK experience of peptide analysis, DMPK developed MS assays which compared serum stability for 33 different peptide analogues. Some peptide sequences and conformations were clearly more stable than others providing a clear structural steer for the discovery unit to follow Time Time

30 Conclusions Platforms Metabolite Identification Metabolite Predictions MALDI-MS MS imaging Radioactivity WIKI Chromatography/MS Clearance, Safety and Efficacy Drug Disposition Reactive Metabolites Corporate Knowledge Analytical Knowledge Bioanalysis & Pharmacokinetics (including modelling, PK/PD and dose predictions) can be regarded as central to the drug discovery effort. Many other platforms, however, can & should be fully used during drug discovery to ensure the best candidates are selected for drug development.

31 Key References

32 Additional Information All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals. All human biological samples were sourced ethically and their research use was in accord with the terms of the informed consent.