Transcript Kein Folientitel

Physicochemical Properties at Bayer
HealthCare (Wuppertal) and Their Use
in Medicinal Chemistry
Jörg Keldenich Nov. 2006
2. PhysChem Forum
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Contents
 Measured physico-chemical parameters
 Introduction of our laboratory
 Model systems for lipophilicity
 Solubility
 Use in medicinal chemistry
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Physicochemical Properties Measured
Lipophilicity
Membrane Affinity
MA
Plasma binding
human serum albumin binding
rat serum albumin binding
HSA
RSA
pKa
Solubility
pKa
screening in various buffers
equilibrium in buffer
equilibrium in galenic formulations
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SOL
Logistics for HT Physicochemistry
BLJ input for sample
identification
Vial collecting rack
Bar-coded vial for sample registration
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Data Handling by Laboratory Information and
Management System (LIMS)
Chemistry
Laboratories
Sample
BAYNO/Prepno.,
Lab.Barcode, Scale of
Journal
Tests, Weight,
Molecular Weight, Principle
investigator, Projekt, Comparison
Sampleregistration
Rack
PDH
cMA, cHSA
flag for
bases or
acids
PIX
Solubility
HSA-binding
Membrane affinity
PILO-LIMS
Results,
Calibration Data
Rackno.
Position
Barcode
Area,
Time
Methods
Roboter
Sequences
LISSY Samplepreparation
Bar-code scan
Archive
LC/MS/MS
Waters
Quattro-Micro
Rack
MTP
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Reports
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Our model system
Solid-supported lipid membranes (TRANSIL)
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Why Use Membrane Affinity?
1. comparison with other lipophilicity descriptors
mlogP vs mlogMA
6.0
5.0
mlogMA (Österberg) vs logMA Bayer
4.0
logP
5.0
4.0
y = 0.9977x - 0.4014
R2 = 0.8543
2.0
1.0
3.0
logMA Österberg et al.
3.0
0.0
0.0
2.0
1.0
2.0
3.0
4.0
5.0
6.0
mlogP (Österberg)
mlogP (Österberg) acid
mlogD7.4 vs mlogMA
1.0
4.0
0.0
2.0
mlogP (Österberg) base
3.0
1.0
0.0
mlogD
-1.0
-4.0
-2.0
-1.0
0.0
2.0
-2.0
-2.0
-2.0
-3.0
-1.0
0.0
1.0
2.0
3.0
4.0
5.0
-4.0
logMA Bayer
-5.0
-6.0
logMA
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4.0
6.0
Why use membrane affinity?
2. comparison with physiological membranes
lipiophilicity vs MAerythrocytes
10000000
y = 0.1404x1.4994
R2 = 0.8551
1000000
y = 0.2121x1.3531
R2 = 0.7327
lipophilicity
100000
10000
1000
MA measured
cPow
100
ACD D7.4
cMA
10
100
1000
Potentiell (MA
measured) 100000
Potentiell
(cMA)
10000
MAery
Influence of cholesterol content has to be considered
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Why use membrane affinity?
Influence of cholesterol
MA vs Molratio Lipid/Cholesterol
1000000
MA
100000
10000
1000
100
C. Tradum et al.:Biophysical J. 78 2496-2492
0
0.2
0.4
0.6
0.8
1
Molration
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Why use membrane affinity?
Influence of cholesterol
MA vs MA ERY
MA measured with pure egg lecithin
MA measured with cholesterol/egg lecithin ratio of 0.8
8000
600000
y = 0.6707x
R2 = 0.9892
y = 0.1404x1.4994
R2 = 0.8551
500000
6000
400000
4000
300000
200000
2000
100000
0
0
0
5000
10000
15000
20000
25000
0
5000
MA Ery
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10000
MA Ery
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15000
Why use membrane affinity?
Influence of cholesterol
 Influence on passive permeation expected to be
strong
 flexibility of plasma membrane is strongly
influence by cholesterol, content usually
about 80 mol% of phospholipids content
 Influence on distribution expected to be low
 80% of all membranes are intracellular with
a cholesterol content about 4 mol% of
phospholipids content
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Comparison of solubility methods
 precipitation
 from powder
 all compounds
 selected compounds
 small amounts
 large amounts (two samples)
 fast analytics
 specific analytics
 compound dissolved in
 sensitive to morphology
organic solvent
 sensitive to purity
 oversaturated solutions
possible
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 sensitive to solvent
impurities
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Comparison of solubility methods
 precipitation
 from powder
 Dissolve compound in DMSO
(2mg/40µl)
 Weight an appropriate amount
of compound as solid
 Add 10µl of this solution to
1000µl buffer (1% DMSO)
 Shake for 24h at room
temperature
 Add 1000µl buffer
 Shake for 24h at room
temperature
 Centrifuge to get supernatant
 Centrifuge to get supernatant
 Establish LC/MS/MS method
 Measure calibration standards
 Establish LC/MS/MS method
 Measure calibration standards
and probe
and probe
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Comparison of solubility methods
EXAMPLES:
Compound
Cpd 5
Cpd 6
Cpd 7
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Cpd 1
Cpd 2
Cpd 3
Cpd 4
mod I
mod II
mod B
amorphous
mod I
mod II
SOL (precipitation) [mg/l]
SOL (from powder) [mg/l]
0.5 ± 0.2
7.9 ± 1.2
8.8 ± 3.8
3.9 ± 0.7
0.8 ± 0.08
1.5 ± 0.4
<0.1
<0.1
350 ± 18
330 ± 26
0.5 ± 0.3
1.3 ± 0.2
4.2 ± 1.1
0.6 ± 0.1
0.4 ± 0.08
1.1 ± 0.08
<0.1
<0.1
420 ± 17
380 ± 19
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Comparison of solubility methods
Name
MOLSTRUCTURE
OH
Warfarin
Yalkowsky charged neutral
compounds
(water)
40
O
235
O O
Solubility [mg/l]: comparison PILO vs literatue (Yalkowsky)
O
HN
N
H O
Primidone
10000
H
N
Cl
O
H2NSO
Metolazone
N
O
N+O
O
O
O
O
O
Nifedipine
500
88
6
9
N
H
1000
O
O
OH
Ketoprofen
Cl
PILO (buffer 6.5)
60
Glyburide
O
N
O H
H
N
H
N
S OO
O
100
140
275
4
1.1
8.6
240
O
HO
O
Indomethacin
N
O
Cl
HN N S
Cimetidine
10
neutral compounds
1
Phenytoin
1
10
100
1000
10000
literature (water)
H
N
O
800
O
Cl
Haloperidol
11000
O
Phenacetin
charged compounds
H
H N
N
N
N
HO
O
NH
N
H O
HO
Ibuprofen
Acetanilide
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F
N
O
N
H
14
1080
180
26
O
36
6300
23
290
Lessons learned from solubility comparisons
 method differences not really critical
 physical form very important
differences between research and development result from:
 morphology differences
 impurities
• solvent content
 counter-ions and buffers are important when compound is
charged in solution
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Case Histories: The Use of Physicochemical Properties
Two different projects as examples:
1. Reducing lipophilicity and HSA binding to
increase fraction unbound: erectile disfunction
2. Influence of solubility on in vivo efficacy:
the HSV project
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Reducing Lipophilicity and Protein
Binding to Increase Fraction Unbound
Starting point: initial compound
moderate effective IC50 PDE-5: 530nM
DP1 compound: Vardenafil
highly effective IC50 PDE-5: 2nM
O
O
O
N
HN
N
HN
N
N
N
N
O S
O
N
N
Insufficient physicochemical properties:
improved physicochemical properties:
high membrane affinity:
16500
reduced membrane affinity:
580
high protein binding:
1.7e-5 mol/l
reduced protein binding: 1.2e-4 mol/l
solubility:
below detection limit solubility:
220 mg/l
fraction unbound:
<1%
fraction unbound:
no in vivo efficacy
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14%
excellent in vivo efficacy
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Reducing lipophilicity and protein
binding to increase fraction unbound
Even the prediction of the in vivo effect from IC50 and fraction
unbound (calculated from MA and HSA) was possible
1.E+02
Dose measured in vivo
initial compound
1.E+01
1.E+00
1.E-01
Vardenafil
1.E-02
1.E-02
1.E-01
1.E+00
1.E+01
1.E+02
Dose calculated from IC50 and physicochemical
properties
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1.E+03
Influence of solubility on in vivo efficacy
survival % of HSV infected mice at 60mg/kg vs
free serum normalized with IC50
Starting point: Example 1
moderate activity IC50: 750nM
N
N
H
Physicochemical properties:
Membrane affinity: 1430
protein binding: 2e-4 mol/l
fraction unbound: 10%
Solubility: 17mg/l
0.8
survival % at 60mg/kg
S
N
O
0.9
O
N
S
O
N
O
1.0
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.00
optimization of physicochemistry not
necessary, activity has to be improved
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0.01
0.10
1.00
free serum/IC50
20
10.00
100.00
Influence of solubility on in vivo efficacy
survival % of HSV infected mice at 60mg/kg vs
free serum normalized with IC50
Development candidate
in vitro activity IC50: 20 nM
1.0
Example 2: brilliant
compound in vitro IC50:
<1 nM
N
O
N
N
S
O
S NH
2
O
Physicochemical
properties:
Membrane affinity: 1590
protein binding: 1e-5 mol/l
fraction unbound: 1%
Solubility: 2.7mg/l
good in vivo efficacy
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survival % at 60mg/kg
0.9
0.8
O
N
N
0.7
S
O
H
S N
O
0.6
Physicochemical
properties:
0.5
0.4
Solubility: <0.1 mg/l
0.3
0.2
0.1
0.0
0.00
0.01
0.10
1.00
10.00
100.00
free serum/IC50
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excellent in vivo
efficacy when
administered as
solution, no in vivo
efficacy even as
micronized powder
Conclusion
 Impact of Physicochemistry Proven
 Physicochemistry/ADME Implemented in
Medicinal Chemistry
 Properties Routinely Measured for Every
Strategic Project
 Use in Lead Optimization and Exploratory
Research Established
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