Do We Need to Optimize Protein Binding in
Drug Discovery?
NEDMDG Summary Meeting
June 4, 2013
Xingrong Liu, Ph.D.
Genentech
Free drug interacts with drug targets: Plasma
Total IC50 = 0.3- 300x Ki
Free IC50 = 0.1-10x Ki
Liu et al., Drug Metab Dispos 37:1548–1556, 2009
100
100
80
80
SERT Occ (%)
SERT Occ (%)
Brain binding does not contribute to the
activity: free drug interacts with drug targets
60
40
20
0
-20
0.01
60
40
20
0
0.1
1
10
100
1000 10000
-20
0.01
0.1
1
10
100
1000 10000
Total Brain Concentration/KI
Unbound Brain Concentration/KI
Total IC50 = 1-1000x Ki
Free IC50 = 0.3-3x Ki
Liu et al., Drug Metab Dispos 37:1548–1556, 2009
Different effects of protein binding on in vitro
and in vivo free concentrations
In vitro
In vivo
Scenario 1
fu=0.1
fu=0.5
fu ↑
fu=0.1
fu ↑ Clin↔ fu=0.5
Cu↔
Cbound
fu=0.5
Cbound
Cbound
Cbound
Cu ↑
Cu
Cu
Scenario 2
fu ↑ Clin↓
Cu ↑
Increase of free fraction by 15X increases
IV free AUC by 3X
R2
1
1
G-378895 IV 3 mg/kg
R1
Cl
Unbound Conc. (ug/mL)
Total Conc. (ug/mL)
10
G-378242 IV 3 mg/kg
fup=0.01
0.1
R2
0.01
fup=0.15
N
G-378895 IV 3 mg/kg
G-378242 IV 3 mg/kg
0.1
0.01
0.001
R1
0.001
0.0001
0
6
12
Time (h)
18
24
0
6
12
Time (h)
• 3X increase was due to reduced clearance
18
24
Increase of free fraction by 15X increases
oral free AUC 2X
1
G-378895 PO 10 mg/kg
R2
Unbound Conc. (ug/mL)
Total Conc. (ug/mL)
10
G-378242 PO 10 mg/kg
R1
1
Cl
fup=0.01
R2
0.1
N
R1
fup=0.15
G-378242 PO 10 mg/kg
0.1
0.01
0.001
0.01
0
G-378895 PO 10 mg/kg
6
12
Time (h)
18
24
0
6
12
Time (h)
• 2X increase was due to reduced clearance
18
24
Protein binding vs. intrinsic clearance
H
N
O
O
C2H5
HN
R
O
R = -(CH2)nCH3
n = 0-8
Data form Blakey et al., J Pharmacokinet Biopharm 25:277–312, 1997
Protein binding vs. Vdss
H
N
O
 fu plasma 

Vd  Vc  Vt 
 fu tissue 
O
C2H5
HN
R
O
R = -(CH2)nCH3
n = 0-8
Data form Blakey et al., J Pharmacokinet Biopharm 25:277–312, 1997
Protein binding vs. t1/2
H
N
O
O
C2H5
HN
R
O
R = -(CH2)nCH3
n = 0-8
Data form Blakey et al., J Pharmacokinet Biopharm 25:277–312, 1997
Protein binding vs. intrinsic clearance
100000
Acids
10000
Clin (ml/min/kg)
Clin (ml/min/kg)
100000
1000
100
10
Bases
10000
1000
100
10
1
1
0.1
0.0001
0.001
0.01
0.1
0.1
0.0001
1
0.001
fu
0.1
1
fu
Neutrals
1000
10000
Clin (ml/min/kg)
Clin (ml/min/kg)
100000
0.01
1000
100
10
Zwitterions
100
10
1
0.1
0.001
0.01
0.1
fu
1
1
0.01
0.1
1
fu
Data form Obach et al., Drug Metab Dispos 36:1385–1405, 2008
Protein binding vs. Vdss
 fu plasma 

Vd  Vc  Vt 
 fu tissue 
Data form Obach et al., Drug Metab Dispos 36:1385–1405, 2008
Protein binding not associate with t1/2
Acids
10000
100
t1/2 (hr)
1000
t1/2 (hr)
Bases
1000
100
10
1
10
1
0.1
0.1
0.0001
0.001
0.01
0.1
0.01
0.0001
1
0.001
fu
1000
0.01
0.1
1
fu
Neutrals
100
Zwitterions
100
t1/2 (hr)
t1/2 (hr)
10
10
1
1
0.1
0.001
0.01
0.1
fu
1
0.1
0.01
0.1
1
fu
Data form Obach et al., Drug Metab Dispos 36:1385–1405, 2008
Brain unbound conc. is governed by the BBB and
plasma unbound conc.
Cu ,brain 
1
1
 Cu , plasma
Clefflux
PS
Clefflux: efflux transport activity at the BBB
PS: diffusion permeability at the BBB
Protein binding does not associate with
brain penetration
R2=0.077
R2=0.087
Modified from Maurer et al, Drug Metab Dispos 33: 165, 2005
Significant effects of P-gp on brain
concentration
Compound
Efflux
Compound
18
CsA
Desloratadine
14
Verapamil
8
Efflux
Digoxin
16-28
Ivermectin
14-27
Chen et al, Curr Drug Metab, 4: 272, 2003
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.
Acknowledgements
•
Marcel Hop
•
•
Bruce Roth
Joe Lyssikatos
•
•
•
R. Scott Obach
Franco Lombardo
Nigel J. Waters
•
Dennis A. Smith
Protein binding of marketed drugs
30%
n = 267
25%
Drugs (%)
20%
15%
10%
5%
0%
<0.001
0.001-0.01 0.01-0.05
0.05-0.1
0.1-0.3
0.3-0.6
0.6-1
Unbound Fraction
n=14, fu<0.1
Data from Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 11th
0.0001
Modified from Maurer et al, Drug Metab Dispos 33: 165, 2005
Meprobamate
Sulpiride
Hydrocodone
Caffeine
Morphine
Metocloperamide
Methylphenidate
Lamotrigine
Buspirone
Venlafaxinec
Carisoprodol
Zolpidema
Thiopentalc
Carbamazepine
9-OH-Risperidone
Phenytoin
Risperidone
Selegiline
Diazepam
Trazodone
Propoxyphene
Citalopram
Midazolam
Hydroxyzine
Clozapine
Fluvoxamine
Cyclobenzaprine
Haloperidol
Nortriptylinea
Paroxetine
Fluoxetine
Chlorpromazine
Sertraline
Brain fu
Many successful CNS drugs show high
brain binding
1
0.1
0.01
0.001
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34x
AUCu,brain/AUCu,plasma (0-5h)
Plasma and brain binding has a significant
impact on the B/P ratio
25
20
Total Brain/Plasma Ratio
15
10
5
Acid: 0.5 (0.4-0.6)
Base: 6 ± 7 (0.1-24)
Neutral: 0.9 ± 0.7 (0.2-0.8)
0
25
20
Unbound Brain/Plasma Ratio
15
Acid: 0.2 (0.2-0.3)
Base: 1 ± 0.7 (0.1-3.4)
Neutral: 0.5 ± 0.3 (0.1-1)
10
5
0
Modified from Maurer et al, Drug Metab Dispos 33: 165, 20
Protein binding vs. clearance
Data form Obach et al., Drug Metab Dispos 36:1385–1405, 2008
Protein binding vs. free Vdss
Data form Obach et al., Drug Metab Dispos 36:1385–1405, 2008
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? Nonwell 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?