1. Do We Need to Optimize Protein Binding in Drug Discovery? NEDMDG Summary Meeting Xingrong Liu, Ph.D. Genentech

2. Free drug interacts with drug targets: Plasma Total IC 50 = 0.3- 300x K i Free IC 50 = 0.1-10x K i Liu et al., Drug Metab Dispos 37:1548–1556, 2009

3. Brain binding does not contribute to the activity: free drug interacts with drug targets Total IC 50 = 1-1000x K i Free IC 50 = 0.3-3x K i Liu et al., Drug Metab Dispos 37:1548–1556, 2009

4. Different effects of protein binding on in vitro and in vivo free concentrations C u ↑ C bound CuCu f u =0.1 C bound f u =0.5 f u =0.1 f u =0.5 C u ↑ f u =0.5 CuCu f u ↑ Cl in ↓ fu ↑ Cl in ↔ C bound Cu↔Cu↔ Scenario 1 Scenario 2 In vitroIn vivo fu ↑

5. Increase of free fraction by 15X increases IV free AUC by 3X 3X increase was due to reduced clearance f up =0.15 f up =0.01

6. Increase of free fraction by 15X increases oral free AUC 2X 2X increase was due to reduced clearance f up =0.01 f up =0.15

7. Protein binding vs. intrinsic clearance Data form Blakey et al., J Pharmacokinet Biopharm 25:277–312, 1997 R = -(CH 2 ) n CH 3 n = 0-8

8. Protein binding vs. Vdss Data form Blakey et al., J Pharmacokinet Biopharm 25:277–312, 1997 R = -(CH 2 ) n CH 3 n = 0-8

9. Protein binding vs. t 1/2 Data form Blakey et al., J Pharmacokinet Biopharm 25:277–312, 1997 R = -(CH 2 ) n CH 3 n = 0-8

10. Protein binding vs. intrinsic clearance Data form Obach et al., Drug Metab Dispos 36:1385–1405, 2008

11. Protein binding vs. Vdss Data form Obach et al., Drug Metab Dispos 36:1385–1405, 2008

12. Protein binding not associate with t 1/2 Data form Obach et al., Drug Metab Dispos 36:1385–1405, 2008

13. Brain unbound conc. is governed by the BBB and plasma unbound conc. Cl efflux : efflux transport activity at the BBB PS: diffusion permeability at the BBB

14. Protein binding does not associate with brain penetration Modified from Maurer et al, Drug Metab Dispos 33: 165, 2005 R 2 =0.077 R 2 =0.087

15. Significant effects of P-gp on brain concentration Chen et al, Curr Drug Metab, 4: 272, 2003 8 14 14-27 16-28 18 CsA Digoxin IvermectinVerapamil Desloratadine CompoundEffluxCompoundEfflux

16. Considerations of protein binding in drug discovery No need to specifically optimize plasma binding –Reduction of plasma protein binding alone does not increase free drug concentration –Reduction of clearance increases free drug concentration –Reduction of plasma protein binding tends to associated with reduction of clearance, as both of the parameters are governed by lipophilicity No need to specifically optimize brain binding Reduction of brain tissue binding alone does not increase free brain drug concentration Reduction of efflux activity enhance free brain drug concentration Need to consider protein binding in … –PK/PD, human PK/dose/DDI prediction, drug transporter/tissue penetration, safety margin calculation, regulatory filing, etc.

17. Acknowledgements Marcel Hop Bruce Roth Joe Lyssikatos R. Scott Obach Franco Lombardo Nigel J. Waters Dennis A. Smith

18. Protein binding of marketed drugs Data from Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 11 th n = 267 n=14, fu<0.1

19. Many successful CNS drugs show high brain binding Modified from Maurer et al, Drug Metab Dispos 33: 165, 2005

20. Plasma and brain binding has a significant impact on the B/P ratio Total Brain/Plasma Ratio Unbound Brain/Plasma Ratio Acid: 0.5 (0.4-0.6) Base: 6 ± 7 (0.1-24) Neutral: 0.9 ± 0.7 (0.2-0.8) Acid: 0.2 (0.2-0.3) Base: 1 ± 0.7 (0.1-3.4) Neutral: 0.5 ± 0.3 (0.1-1) Modified from Maurer et al, Drug Metab Dispos 33: 165, 2005 240x 34x

21. Protein binding vs. clearance Data form Obach et al., Drug Metab Dispos 36:1385–1405, 2008

22. Protein binding vs. free Vdss Data form Obach et al., Drug Metab Dispos 36:1385–1405, 2008

23. Questions for protein binding PPB non-linearity – saturation of PPB as a strategy to increase unbound concentration – is this a viable strategy? Covalent modifiers – does PPB even matter? Is it always total plasma concentration? What about "exceptions to the rule"? - e.g. Active transport? Non- well stirred assumption? Impact on Cmax and Cmin – this could be sensitive to fu – see Derendorf paper I circulated Determination of unbound PK parameters (Clunbound – Vd,ssunbound) – does this simplify or complicate things? Species differences? What should we use PPB data for in drug discovery?