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Discovery PhysChem at UCB: AKAS and Beyond

Discovery PhysChem at UCB: AKAS and Beyond. Christine Prosser UCB, Slough, UK PhysChem Forum 30 th November 2005. Target Validation. Lead Optimisation. Target ID. HTS. Hit 2 Lead.

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Discovery PhysChem at UCB: AKAS and Beyond

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  1. Discovery PhysChem at UCB:AKAS and Beyond Christine ProsserUCB, Slough, UKPhysChem Forum30th November 2005

  2. Target Validation Lead Optimisation Target ID HTS Hit 2 Lead Discovery PhysChem work in collaboration with DMPK and CADD to provide full data package on all novel compounds. Later stage research supported by colleagues in Cambridge, UK and Braine, BE. Gold standard data generated on selected compounds. Discovery PhysChem within UCB

  3. Discovery PhysChem within UCB • Highlight potential issues such as poor solubility, permeability and instability to allow accurate interpretation of biological results • Give greater confidence in-vitro and in-vivo data • Set up a cascade of assays to aid decision making in project teams

  4. 10 mM DMSO Stock QC Check Solubility Permeability Others AKAS (all NCEs) BioSol (all NCEs) SnapSol (request) GI PAMPA (all NCEs) BBB PAMPA (all NCEs) HT Log D7.4 (all NCEs) QuickSol (request) TSol (request) Caco-2 (request) DMPK (Slh) PPB (request) Log P (request) pKa (request) Discovery PhysChem (Slh) PhysChem (Brne + Cam)

  5. 10 mM DMSO Stock QC Check Solubility Permeability Others AKAS (all NCEs) BioSol (all NCEs) SnapSol (request) GI PAMPA (all NCEs) BBB PAMPA (all NCEs) HT Log D7.4 (all NCEs) QuickSol (request) TSol (request) Caco-2 (request) DMPK (Slh) PPB (request) Log P (request) pKa (request) Discovery PhysChem (Slh) PhysChem (Brne + Cam)

  6. 10 mM DMSO Stock QC Check Solubility Permeability Others AKAS (all NCEs) BioSol (all NCEs) SnapSol (request) GI PAMPA (all NCEs) BBB PAMPA (all NCEs) HT Log D7.4 (all NCEs) QuickSol (request) TSol (request) Caco-2 (request) DMPK (Slh) PPB (request) Log P (request) pKa (request) Discovery PhysChem (Slh) PhysChem (Brne + Cam)

  7. AKASAutomated Kinetic Aqueous Solubility • All novel compounds pass through the assay (~100/week) • Kinetic solubility measured across a pH range • Allows estimate of pKa • Stability issues can be identified

  8. AKAS - Experimental • 10mM DMSO stock buffered at 4 pHs • 5% final DMSO concentration • Samples shaken for 90mins then filtered • Concentration of sample in filtrate determined by UV analysis against calibration curve • 240-400nm • λmax / isosbestic point • Solubility of all compounds measured in duplicate

  9. AKAS – Results (pKa from Solubility Profile) Calibration Solubility Chlorpromazine • No spectral change • Basic ionisation event between pH7.4 and pH9 • pKa ~9

  10. AKAS – Results (Spectral Changes) Calibration Solubility Labetolol • Spectral change upon ionisation • Isobestic point used instead of λmax • Acidic ionisation event between pH5 and pH7.4 • Spectral change indicates ionisation event – not apparent from solubility profile

  11. AKAS– Results (Stability Issues) Calibration Solubility Compound X • Calibration shows no UV spectral change • Solubility curve at pH9 shows spectral change • Possible stability issue at high pH • ND result returned

  12. 10 mM DMSO Stock QC Check Solubility Permeability Others AKAS (all NCEs) BioSol (all NCEs) SnapSol (request) GI PAMPA (all NCEs) BBB PAMPA (all NCEs) HT Log D7.4 (all NCEs) QuickSol (request) TSol (request) Caco-2 (request) DMPK (Slh) PPB (request) Log P (request) pKa (request) Discovery PhysChem (Slh) PhysChem (Brne + Cam)

  13. Kinetic vs. Thermodynamic Solubility? • Kinetic solubility: • Better suited to HT • Doesn’t take into account dissolution rate • Co-solvent present • Thermodynamic solubility: • Expensive with respect to sample quantity and time • Not suited to HT

  14. QuickSolPseudothermodynamic Solubility • Starts from solid material • Single time point • QuickSol measured on requested compounds • solubility >150uM in AKAS at pH5 or pH7.4

  15. QuickSol - Experimental • Buffer added to solid sample • 0.5mg solid per pH • measured in duplicate at 2 pHs • Samples shaken for 90mins then filtered • Concentration of sample in filtrate determined against calibration curve • Analysis by HPLC

  16. QuickSol - Results Ketoconazole • Comparison of AKAS and QuickSol results shows effect of DMSO on solubility

  17. AKAS and QuickSol • AKAS – best case scenario • QuickSol – worst case scenario • Combination of AKAS and QuickSol data used to highlight compounds where solubility may be a serious limitation • interpretation of biological results

  18. 10 mM DMSO Stock QC Check Solubility Permeability Others AKAS (all NCEs) BioSol (all NCEs) SnapSol (request) GI PAMPA (all NCEs) BBB PAMPA (all NCEs) HT Log D7.4 (all NCEs) QuickSol (request) TSol (request) Caco-2 (request) DMPK (Slh) PPB (request) Log P (request) pKa (request) Discovery PhysChem (Slh) PhysChem (Brne + Cam)

  19. BioSolSolubility under Biological Assay Conditions • Primary assay data generated in enzyme or protein based assays • Solubility may be compromised by presence of proteins or incubation media • Cellular assays – DMSO must be kept to minimum • All novel compounds pass through assay • Assay measures solubility at relevant DMSO concentration in incubation media • Same experiment carried out without protein to flag any potential protein binding issues (can then be investigated further by traditional DMPK methods)

  20. BioSol - Experimental • 10mM DMSO stock diluted in D-PBS • 0.2% final DMSO concentration (sample concentration 20uM) • 10% bovine serum albumin • 10% glucose • MgCl2, CaCl2 (1μM) • Control plate without protein • Shaken for 90mins then filtered • Filtrate centrifuged through 10kDa cut off plates • Analysis by HPLC

  21. BioSol - Results Warfarin • Comparison of AKAS and QuickSol results shows no evidence of DMSO effect on solubility • BioSol result much lower than QuickSol • Comparison of BioSol with protein to BioSol blank suggests protein binding

  22. 10 mM DMSO Stock QC Check Solubility Permeability Others AKAS (all NCEs) BioSol (all NCEs) SnapSol (request) GI PAMPA (all NCEs) BBB PAMPA (all NCEs) HT Log D7.4 (all NCEs) QuickSol (request) TSol (request) Caco-2 (request) DMPK (Slh) PPB (request) Log P (request) pKa (request) Discovery PhysChem (Slh) PhysChem (Brne + Cam)

  23. SnapSolHT Single Point Solubility Assay • Developed as an aid to fragment screening to ensure that compounds meet minimum solubility criteria • Can be used to screen a selection of compounds under conditions which mimic closely those of the screening experiment Experimental • 10mM DMSO stock solutions are diluted in the buffer system used for the screening experiment • buffer type • DMSO concentration • Shaken for 90 minutes then filtered • Analysis of the filtrate by UV plate reader at fixed wavelength against a calibration curve

  24. 10 mM DMSO Stock QC Check Solubility Permeability Others AKAS (all NCEs) BioSol (all NCEs) SnapSol (request) GI PAMPA (all NCEs) BBB PAMPA (all NCEs) HT Log D7.4 (all NCEs) QuickSol (request) TSol (request) Caco-2 (request) DMPK (Slh) PPB (request) Log P (request) pKa (request) Discovery PhysChem (Slh) PhysChem (Brne + Cam)

  25. PAMPAParallel Artificial Membrane Permeability Assay • Permeability traditionally measured in in-vitro cell based assays • not suitable for high throughput • PAMPA measures passive diffusion through a lipid membrane • no active transport mechanisms • no efflux • All novel compounds pass through assays • Used as a screening tool to select compounds for further permeability studies

  26. GI PAMPA - Experimental • Phosphatidyl choline artificial membrane dispensed onto a solid filter support • 10mM DMSO stocks diluted in buffer (100uM, 5% DMSO) • PAMPA sandwich incubated at 20ºC for 4.5hrs, shaking at 750rpm • Analysis of carried out by UV plate reader • Papp values returned • % membrane retention also calculated using mass balance equations

  27. BBB PAMPABlood Brain Barrier PAMPA • Used to determine whether compounds are CNS+ or CNS- • Important to UCB as we are targeting both CNS and non-CNS disease states • Same methodology as GI PAMPA but using porcine polar brain lipid L.Di, E.Kerns et al Eur. J. Med. Chem 38 (2003)223-232 • Assay validated against known CNS active and inactive drugs

  28. PAMPA – Results (GI vs. BBB) Oxazepam • GI-PAMPA shows poor permeability. • BBB-PAMPA shows good permeability. Consistent with literature, CNS+ drug.

  29. PAMPA – Results (comparison with cell assays) Verapamil • GI PAMPA shows good permeability • Caco-2 shows poor permeability • Efflux mechanisms Caffeine • Both GI and BBB PAMPA show poor permeability. • But caffeine known to cross BBB - actively transported across BBB

  30. 10 mM DMSO Stock QC Check Solubility Permeability Others AKAS (all NCEs) BioSol (all NCEs) SnapSol (request) GI PAMPA (all NCEs) BBB PAMPA (all NCEs) HT Log D7.4 (all NCEs) QuickSol (request) TSol (request) Caco-2 (request) DMPK (Slh) PPB (request) Log P (request) pKa (request) Discovery PhysChem (Slh) PhysChem (Brne + Cam)

  31. HT LogD7.4 • All novel compounds pass through the assay • Robotic based assay to measure partition between octanol and aqueous buffer • Used to rank compounds within a series and flag compounds that have LogD outside desired range.

  32. HT LogD7.4- Experimental • 10mM DMSO stock dissolved in 1:1 mixture of octanol and D-PBS (100uM, 5% DMSO) • Samples shaken for 90mins then centrifuged • Octanol and aqueous phases separated by liquid handling robot • Concentration of sample in each phase determined by HPLC

  33. HT LogD7.4– Results (Std Drugs) Metolazone * A. Avdeef, Absorption and Drug Development, John Wiley & Sons, 2003

  34. HT LogD7.4– Results (UCB Compounds)

  35. 10 mM DMSO Stock QC Check Solubility Permeability Others AKAS (all NCEs) BioSol (all NCEs) SnapSol (request) GI PAMPA (all NCEs) BBB PAMPA (all NCEs) HT Log D7.4 (all NCEs) QuickSol (request) BIO-PAMPA (all NCEs) HT pKa (all NCEs) TSol (request) Caco-2 (request) DMPK (Slh) PPB (request) Log P (request) pKa (request) Discovery PhysChem (Slh) PhysChem (Brne + Cam)

  36. Conclusions • In our opinion, data should be used as a package • no single piece of data should make or break a project • Provide information to allow an informed interpretation of results

  37. PhysChem, Slough, UK Richard Taylor Barbara Mason Emily Freeman PhysChem, Cambridge, UK John Cooper Benedicte Fau Dave Sherwood Acknowledgements • PhysChem, Braine, Belgium • Luc Quere • Liliane Ellens • Geraldine Longfils • DMPK, Slough, UK • Ted Parton • Lloyd King • Hanna Hailu • Mark Baker • Sarah Bartlett • Simon Carter • Judith van Asperen

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