physicochemical properties at bayer healthcare (wuppertal) and their use in medicinal chemistry

Jörg Keldenich Nov. 2006 2. PhysChem Forum 1

Physicochemical Properties at Bayer HealthCare (Wuppertal) and Their Use

in Medicinal Chemistry


Contents

Measured physico-chemical parameters

Introduction of our laboratory

Model systems for lipophilicity

Solubility

Use in medicinal chemistry


Physicochemical Properties Measured

Lipophilicity Membrane Affinity MA

Plasma binding human serum albumin binding HSArat serum albumin binding RSA

pKa pKa

Solubility screening in various buffers SOL

equilibrium in bufferequilibrium in galenic formulations


Logistics for HT Physicochemistry

Bar-coded vial for sample registration

BLJ input for sample identificationVial collecting rack


BAYNO/Prepno.,Barcode, Scale ofTests, Weight,Molecular Weight, Principleinvestigator, Projekt, Comparison

Data Handling by Laboratory Information and Management System (LIMS)

ChemistryLaboratories

PILO-LIMS

Lab.-Journal PIXPDH

LISSY Sample-preparation

LC/MS/MSWaters

Quattro-Micro

RoboterBar-code scan

Sample

Rackno.PositionBarcode

Methods

Rack

MTP

Sequences

SolubilityHSA-bindingMembrane affinity

Sample-registration

RackArea,Time

ReportsResults,Calibration Data

cMA, cHSAflag for

bases or acids

Archive


Our model system

Solid-supported lipid membranes (TRANSIL)


Why Use Membrane Affinity?1. comparison with other lipophilicity descriptors

mlogMA (Österberg) vs logMA Bayer

y = 0.9977x - 0.4014R2 = 0.8543

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

-2.0 -1.0 0.0 1.0 2.0 3.0 4.0 5.0

logMA Bayer

logM

A Ö

ster

berg

et a

l.

mlogP vs mlogMA

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0

logMA

log

P

mlogP (Österberg)

mlogP (Österberg) acid

mlogP (Österberg) basemlogD7.4 vs mlogMA

-6.0

-5.0

-4.0

-3.0

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

-4.0 -2.0 0.0 2.0 4.0 6.0

mlo

gD


Why use membrane affinity?2. comparison with physiological membranes

lipiophilicity vs MAerythrocytes

y = 0.1404x1.4994

R2 = 0.8551

y = 0.2121x1.3531

R2 = 0.7327

10

100

1000

10000

100000

1000000

10000000

100 1000 10000 100000

MAery

lip

op

hil

icit

y

MA measured

cPow

ACD D7.4

cMA

Potentiell (MAmeasured)Potentiell(cMA)

Influence of cholesterol content has to be considered


Why use membrane affinity?Influence of cholesterol

C. Tradum et al.:Biophysical J. 78 2496-2492

MA vs Molratio Lipid/Cholesterol

100

1000

10000

100000

1000000

0 0.2 0.4 0.6 0.8 1

Molration

MA



MA vs MA ERY

y = 0.1404x1.4994

R2 = 0.8551

0

100000

200000

300000

400000

500000

600000

0 5000 10000 15000 20000 25000

y = 0.6707x

R2 = 0.9892

0

2000

4000

6000

8000

0 5000 10000 15000

MA measured with pure egg lecithin MA measured with cholesterol/egg lecithin ratio of 0.8

MA Ery MA Ery



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


Comparison of solubility methods

precipitation

all compounds

small amounts

fast analytics

compound dissolved in

organic solvent

oversaturated solutions

possible

from powder

selected compounds

large amounts (two samples)

specific analytics

sensitive to morphology

sensitive to purity

sensitive to solvent

impurities



precipitation

Dissolve compound in DMSO

(2mg/40µl)

Add 10µl of this solution to

1000µl buffer (1% DMSO)

Shake for 24h at room

temperature

Centrifuge to get supernatant

Establish LC/MS/MS method

Measure calibration standards

and probe

from powder

Weight an appropriate amount

of compound as solid

Add 1000µl buffer

Shake for 24h at room

temperature

Centrifuge to get supernatant

Establish LC/MS/MS method

Measure calibration standards

and probe


Compound SOL (precipitation) [mg/l] SOL (from powder) [mg/l]

Cpd 1 0.5 ± 0.2 0.5 ± 0.3 Cpd 2 7.9 ± 1.2 1.3 ± 0.2 Cpd 3 8.8 ± 3.8 4.2 ± 1.1 Cpd 4 3.9 ± 0.7 0.6 ± 0.1

Cpd 5 mod I mod II

0.8 ± 0.08 1.5 ± 0.4

0.4 ± 0.08 1.1 ± 0.08

Cpd 6 mod B amorphous

<0.1 <0.1

<0.1 <0.1

Cpd 7 mod I mod II

350 ± 18 330 ± 26

420 ± 17 380 ± 19


EXAMPLES:


Solubility [mg/l]: comparison PILO vs literatue (Yalkowsky)

1

10

100

1000

10000

1 10 100 1000 10000

literature (water)

PIL

O (

bu

ffe

r 6

.5)

charged compounds

neutral compounds

Name MOLSTRUCTUREYalkowsky (water)

charged neutral compounds

Warfarin 40 235

Primidone 500

Metolazone 60 88

Nifedipine 6 9

Ketoprofen 140 275

Glyburide 4 1.1

Indomethacin 8.6 240

Cimetidine 11000

Phenacetin 800 1080

Haloperidol 14 180

Phenytoin 26 23

Ibuprofen 36 290

Acetanilide 6300

OH

OOO

NHO

NH O

NH

NO

S

Cl

NH2

O

O

N+

NH

OO

OOO

O

O

OH

O

ONH

OCl

SNH

OO

NH

O

NO

OOH

O

Cl

NNH S NH

NNH

N

NH

O

O

OHN

ClO

F

NHNH

O

O

OHO

NH

O



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


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


Reducing Lipophilicity and Protein Binding to Increase Fraction Unbound

NH

N N

N

ONH

NN

N

O

O

SN OO

N

Starting point: initial compoundmoderate effective IC50 PDE-5: 530nM

DP1 compound: Vardenafilhighly effective IC50 PDE-5: 2nM

Insufficient physicochemical properties:

high membrane affinity: 16500high protein binding: 1.7e-5 mol/lsolubility: below detection limitfraction unbound: <1%

no in vivo efficacy

improved physicochemical properties:

reduced membrane affinity: 580reduced protein binding: 1.2e-4 mol/lsolubility: 220 mg/lfraction unbound: 14%

excellent in vivo efficacy


Even the prediction of the in vivo effect from IC50 and fraction unbound (calculated from MA and HSA) was possible

Reducing lipophilicity and protein binding to increase fraction unbound

1.E-02

1.E-01

1.E+00

1.E+01

1.E+02

1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03

Dose calculated from IC50 and physicochemical

properties

Do

se m

easu

red

in

viv

oinitial compound

Vardenafil


Influence of solubility on in vivo efficacy

survival % of HSV infected mice at 60mg/kg vs free serum normalized with IC50

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0.00 0.01 0.10 1.00 10.00 100.00

free serum/IC50

su

rviv

al

% a

t 6

0m

g/k

g S

N

N

S

NH

O

N

O

O

NO

Starting point: Example 1moderate activity IC50: 750nM

Physicochemical properties:

Membrane affinity: 1430protein binding: 2e-4 mol/l

fraction unbound: 10%Solubility: 17mg/l

optimization of physicochemistry notnecessary, activity has to be improved


S

N

NS

ONH

O

O

Example 2: brilliant compound in vitro IC50:

<1 nM


Solubility: <0.1 mg/l

excellent in vivo efficacy when

administered as solution, no in vivo

efficacy even as micronized powder

S

N

NS

ONH2

O

ON

Development candidatein vitro activity IC50: 20 nM


Membrane affinity: 1590protein binding: 1e-5 mol/l

fraction unbound: 1%Solubility: 2.7mg/l

good in vivo efficacy

Influence of solubility on in vivo efficacy

survival % of HSV infected mice at 60mg/kg vsfree serum normalized with IC50

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0.00 0.01 0.10 1.00 10.00 100.00

free serum/IC50

surv

ival

% a

t 60

mg

/kg


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

physicochemical properties at bayer healthcare (wuppertal) and their use in medicinal chemistry

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