1. Modern Tools for Drug Discovery NIMBUS Biotechnology Modern Tools for Drug Discovery www.nimbus-biotechnology.com

2. Modern Tools for Drug Discovery Innovative tools in “ready to go plates” Modern Tools for Drug Discovery NIMBUS Biotechnology

3. Modern Tools for Drug Discovery Bioavailability - an important issue Absorption Distribution Metabolism Excretion Pharmacokinetic Bioavailability

4. Modern Tools for Drug Discovery Absorption on a cellular level getting into the blood system Cross section of the gut wall TRANSPORT: passive paracellular passive transcellular active transcellular efflux blood stream

5. Modern Tools for Drug Discovery Distribution bloodstream Plasma/Tissue partitioning tissue e.g. brain Balance of interactions: lipophilicity vs. serum binding

6. Modern Tools for Drug Discovery Absorption on a cellular level getting into the blood system Cross section of the gut wall TRANSPORT: passive paracellular passive transcellular active transcellular efflux blood stream

7. Modern Tools for Drug Discovery K water/lipid K lipid/water Passive Transport in search of a good descriptor lipid bilayersmimicking cellular systems Passive Transport In search of a good descriptor

8. Modern Tools for Drug Discovery Passive Transport in search of a good descriptor “only absorption”additional information “hidden” K lipid/water Passive Transport In search of a good descriptor

9. Modern Tools for Drug Discovery Lipids and Proteins on Solid Supports Innovative tools in “ready to go plates” Prediction of absorption processes Unspecific binding to HSA Prediction of distribution

10. Modern Tools for Drug Discovery Determination of LIPOPHILICITY = Membrane Affinity (logMA) Non-covalently attached SINGLE LIPID BILAYER Different LIPID COMPOSITIONS Mimicking natural membranes High LONG TERM STABILITY 9 months DMSO ASSAY CONCENTRATION up to 5 % FAST SEPARATION due to the solid support TRANSIL ®

11. Modern Tools for Drug Discovery Validation log MA (charged and uncharged compounds) Comparison to liposome partitioning Good correlation with liposome approach

12. Modern Tools for Drug Discovery Validation fraction absorbed logMA correlates with fraction absorbed -8 -4 0 4 -2 0 24

13. Modern Tools for Drug Discovery High reproducibility from lot to lot Validation III Lot to Lot and Reproducibility High reproducibility from measurement to measurement

14. Modern Tools for Drug Discovery Validation IV 01234 0 1 2 3 4 logMA_NIMBUS 1:1 correlation logMA NIMBUS in-house data logMA Bayer Health Care Good lab to lab reproducibility Lab to Lab * Introduced by Seiffert at NIMBUS Meeting 2005

15. Modern Tools for Drug Discovery TRANSIL  -HSA Properties of the material random orientation immobilized on a soft and inert surface binding sites freely accessible stable in presence of organic modifiers (e.g. 5% DMSO)

16. Modern Tools for Drug Discovery good correlation with classical equilibrium dialysis TRANSIL  -HSA Validation TRANSIL ® -HSA vs. equilibrium dialysis

17. Modern Tools for Drug Discovery TRANSIL  - HSA Lot-to-lot reproducibility good lot-to-lot reproducibility

18. Modern Tools for Drug Discovery TRANSIL  -Assays Principles

19. Modern Tools for Drug Discovery TRANSIL ® Assays Available Formats TypeCompounds per 96-well plate Compounds per 384-well plate High-Throughput 2393 High-Precision 1045 High-Throughput High-Precision

20. Modern Tools for Drug Discovery ??? Lipophilicity/ Membrane Affinity ADME- Optimised Compounds HSA Binding logMA Log K d,HSA PK-Map TM

21. Modern Tools for Drug Discovery PK-Map TM...via Physiology-based ADME Models PHYSIOLOGICAL PROPERTIES organ volume and composition, blood flow rates, pH, effective accessible surface area, gastric emptying and intestinal transit time, feeding status,... Physico-chemical-/ in vitro properties membrane affinity HSA binding solubility molecular weight... Pharmacokinetic-/ADME- in vivo properties fraction dose absorbed organ/plasma PC free serum concentration volume of distribution...

22. Modern Tools for Drug Discovery Validation of the Absorption Model Human F abs calculated only from MA and Mw compared to published in vivo data Willmann et al., J. Med. Chem. 2004,47, pp 4022-4032 Excellent agreement All outliers known as substrates for active transporters. Compound set (126 marketed drugs)

23. Modern Tools for Drug Discovery Validation of the Distribution Model organ/plasma partition coefficients K org/water

24. Modern Tools for Drug Discovery Combination of TRANSIL  and PK-MAP ™ * as the most reliable tool for ADME Prediction PK-MAP ™ * Lipophilicity/ Membrane Affinity ADME- Optimised Compounds HSA Binding logMA Additional Input Solubility, MW Log K d,HSA Conclusions

25. Modern Tools for Drug Discovery TRANSIL  and TRANSIL  -HSA Benefits and Summary Determination of lipophilicity and serum binding in real High Throughput Processing time is less than one minute per drug in the 384 well format First 384 well assay for both parameters Small amount of compound needed Easy handling: Compound addition, separation, quantification Correlation with established approaches and to ADME parameters High throughput assays for high quality data