Partition Coefficients in Drug Design - η-Τάξη ΕΚΠΑ Partition Coefficients in Drug Design: How to measure? How to calculate? Anna Tsantili-Kakoulidou Department of Pharmaceutical
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1
Partition Coefficients in Drug Design:
How to measure?
How to calculate?
Anna Tsantili-Kakoulidou
Department of Pharmaceutical Chemistry, School of Pharmacy,
University of Athens
2
Partition Coefficients in Drug Design
Important in :
• Passive diffusion transcellular permeability
• Hydrophobic binding to proteins plasma and tissue proteins,
active transport
metabolizing enzymes
receptors
3
Partition Coefficients in Drug Design
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Partition Coefficients in Drug Design
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Partition Coefficients in Drug Design:
How to measure?
How to calculate?
• Reference System:
logPbio=alogPorg+b
octanol/water
cyclohexane/water
chloroform/ water
di-butyl-ether/water
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Partition Coefficients in Drug Design:
How to measure?
Direct measurement:
Shaking flask
Centrifugal Partition Chromatography (CPC)
Potentiometric titration
Indirect determination
Partition chromatography
HPLC
RP-TLC
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Partition Coefficients in Drug Design:
How to measure?
Shaking flask method: What to consider?
Partition conditions
• Mutual saturation of the two phases
• purity of solvents and solute
• solute concentration ~ 10-5 M
• pH control if necessary
• constant temperarure
• suitable volume ratio
• time for the achievement of equilibration
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Partition Coefficients in Drug Design:
How to measure?
Shaking flask method: What to consider?
Substance characteristics
• Is the substance pure?
• Is the substance stable?
• Is the substance volatile?
• Is the substances absorbed in glass?
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Partition Coefficients in Drug Design:
How to measure?
Shaking flask method: What to consider?
Quantitative substance analysis
• Suitable analytical technique
UV/Vis Spectrophotometry
HPLC
• Analysis of the two phases measurement of substance
concentration by means of calibration equations
• Analysis of aqueous phase only measurement of
absorbance (peak height, peak area) before and after
equilibration
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Partition Coefficients in Drug Design:
How to measure?
Analysis of aqueous phase only:
org
aqu
V
V
A
AAoP *
1
1
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Partition Coefficients in Drug Design:
How to measure?
Shaking flask method: Limitations:
logP range -2.5 to 4
tedious and time consuming
not suitable for all types of substances
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Partition Coefficients in Drug Design:
How to measure?
ALogP - A new automated log P direct partitioning workstation
• Chait, A. and Zaslavsky, B.
Analiza, Inc., 408 Glen Park Drive, Bay Village, OH 44140, USA
• The system performs direct partitioning, with sampling of both phases,
and as such enables the quantification of ionic and non-ionic
compounds alike. The total partitioning system volume is ca. 1 mL,
and the total dissolved sample volume is ca. 200 mL. This miniaturized
partitioning system is based on standardized 96-deepwell plates,
enabling complete automation with user-handling limited to the plate
level….
Lipohilicity in Drug Disposition, Lausanne 2000
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Partition Coefficients in Drug Design:
How to measure?
Centrifugal Partition Chromatography (CPC)
• Unique form of liquid-liquid chromatography free of a
solid support no problem of adsorption
• Two poorly miscible liquids are used as the stationary
and mobile phase
• Stationary phase is maintained by centrifugal and or
Archimedian screw forces - mobile phase is pumped
through the system
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Partition Coefficients in Drug Design:
How to measure?
Centrifugal Partition Chromatography (CPC)
• Hydrostatic Equilibrium Systems
high pressure
low retention of the stationary phase (50%)
• Hydrodynamic Equilibrium Systems
continuous mixing of the two phases
high retention proportion of the stationary phase
high partition efficiency
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Partition Coefficients in Drug Design:
How to measure?
Centrifugal Partition Chromatography (CPC):
What we measure?
aqueous solutionas mobile phase
organic solventas mobile phase
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Partition Coefficients in Drug Design:
How to measure?
Centrifugal Partition Chromatography (CPC):
Some system characteristics
• vertical coiled column
• horizontal flow-through multilayer CPC
• length : 50-60 m, id 2,6, o.d. 3,4 Vt ~300-320 ml
• high quality of tubing
• stable flow rate (0.5-10 ml/min)
• speed of rotation: 800-1000 r.p.m.
• precise determination of dead volume or to
• temperature control
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Partition Coefficients in Drug Design:
How to measure?
• Centrifugal Partition Chromatography (CPC):
Chromatographic conditions
• ‘Normal mode’ process Vt ~300ml
sample volume 20 μl containing 0.1-5mM
For logP> 0 the organic phase is the mobile phase
For logP< 0 the aqueous phase is the mobile phase
• ‘Reversed phase’ process Vt ~30ml
For logP> 0 the aqueous phase is the mobile phase
logP range -2 to 2
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Partition Coefficients in Drug Design:
How to measure?
Centrifugal Partition Chromatography (CPC):
Proposed operational flow rates in different logP ranges
For:
* logP > 2,5 or logP< -2,5 U=0,5 ml/min
* 1,5 <logP <2, or –2,5 < logP < -1,5 U=1 ml/min
* 0,5 < logP < 1 or -1,5 < logP < -0,5 U=3 ml/min
* 0 < logP <0,5 or –0,5 < logP <0 U=6 ml/min
Vt-VM: 88%, 86%, 83%, 77% for octanol/water system
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Partition Coefficients in Drug Design:
How to measure?
Centrifugal Partition Chromatography (CPC):
Advantages
• Precise and accurate
• No problems for adsorption, instability impurity
• and volatility of solutes
• Relatively less time consuming
• Small amount of sample
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Partition Coefficients in Drug Design:
How to measure?
• Centrifugal Partition Chromatography (CPC):
Limitations
• Low number of theoretical plates
• logP range -3 to 3
• Large amount of organic solvent , when using the
organic phase as mobile phase at a flow rate 3-6 ml/min
• a mechanical expertise needed
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Partition Coefficients in Drug Design:
How to measure?
Dual-phase Potentiometric Titration
Two linked titrations:
• alkalimetrical titration of a preacidified solution of a weak
acid to appropriate high pH;
• acidimetrical back titration after addition of octanol (or any
organic solvent immiscible with water)
Simultaneous measurement of pKa and logP
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Partition Coefficients in Drug Design:
How to measure?
Dual-phase Potentiometric Titration
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Partition Coefficients in Drug Design:
How to measure?
Dual-phase Potentiometric Titration
For a monoprotic weak acid:
P= (10 poKa-pKa -1) * Vaqu/Vorg
For a monoprotic weak base
P= [10 -(poKa-pKa) -1) * Vaqu/Vorg
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Partition Coefficients in Drug Design:
How to measure?
Dual-phase Potentiometric Titration
• especially standarized electrode
• use of background electrolyte (0.15 M KCl)
logP range -2 to 7
pKa range 0.6 to 13.3
Limitations: applicable to ionizable compounds;
special, rather expensive instrumentation
(Sirius instruments)
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Partition Coefficients in Drug Design:
How to measure?
Reversed phase chromatographic techniques
RP-TLC HPLC
Indirect determination of octanol water logP
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Partition Coefficients in Drug Design:
How to measure?
• Reversed phase chromatographic techniques
Chromatographic methods combine the possibility
of automation, high dynamic range, low
sensitivity to impurities and are compound
sparing
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Partition Coefficients in Drug Design:
How to measure?
Reversed phase Thin Layer Chromatography
1Rf RM= log -1
Rf
logP= aRM+b Isocratic values
} (Collander type equation)
logP= aRMw+bExtrapolated values
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Partition Coefficients in Drug Design:
How to measure?
Reversed phase Thin Layer Chromatography:
Conditions
Stationary phase:
Silica gel impregnated with a strong hydrophobic agent
(paraffin oil, silicone oil, usually 5%)
Silanized Silica gel, C2, C8, C18
low wettability
Mobile phase:
water + organic modifier (methanol, acetonitrile, THF)
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Partition Coefficients in Drug Design:
How to measure?
Isocratic RM values are converted to logP values by means
of a
Calibration equation
logP= aRM+b
Recommended to measure more than one isocratic RM
values, construct the corresonding calibration equations
and calcultae the average logP
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Partition Coefficients in Drug Design:
How to measure?
Reversed phase Thin Layer Chromatography
Extrapolated RMw
• RM= Aφ2 - Bφ+ C
• RM=-Sφ + RMw logP= a RMw + b
φ %
RMw
RM
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Partition Coefficients in Drug Design:
How to measure?
Reversed phase Thin Layer Chromatography
Isocratic versus extrapolated RM values
less experiments more general indices
possible lipophilicity inversion values in the same order of logP
avoid lipophilicity inversion
Do they contain the same information content?
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Partition Coefficients in Drug Design:
How to measure?
logP/RMw correlation
Reversed phase Thin Layer Chromatography
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Partition Coefficients in Drug Design:
How to measure?
High Performace Liquid Chromatography
retention times tr converted to capacity factors
tr-totr logk’= log( )
to
logP=a logk’ +b Isocratic capacity factors
} (Collander type equation)
logP=alogk’w+b Extrapolated capacity factors
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Partition Coefficients in Drug Design:
How to measure?
High Performace Liquid Chromatography: Conditions
Stationary phase:
Silanized Silica gel:
• chemical bonded octadecyl silica, ODS
silanophilic interactions,
pH limitations 2-7.4
• end-capped BDS, ABZ
reduced silanophilic interactions
pH limitations 2-7.4
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Partition Coefficients in Drug Design:
How to measure?
High Performace Liquid Chromatography: Conditions
Stationary phase:
• octadecyl-polyvinylalcohol copolymer gel, ODP
no silanophilic interactions
no pH limitations
large retentions times
longer equlibration time
• octanol coated ODS column
instability, column bleeding
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Partition Coefficients in Drug Design:
How to measure?
High Performace Liquid Chromatography: Conditions
Stationary phase:
• C18 derivatized Polystyrene-divivylbenzene
C18-PS-DVB suitable for alkane/water simulation?
• Immobilized artifficial membranes (IAM) suitable
for membrane simulation
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Partition Coefficients in Drug Design:
How to measure?
High Performace Liquid Chromatography: Conditions
Mobile Phase
• water (buffer) + organic modifier (not for octanol coated )
phosphate buffer methanol
MOPS acetonitrile (not suitable for ODP)
THF
+ masking agent (hydrophobic amine)
+ octanol (0.25%)
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Partition Coefficients in Drug Design:
How to measure?
High Performace Liquid Chromatography
Isocratic logk’ values are converted to logP values by means
of a
Calibration equation
logP= alogk’+b
Recommended to measure more than one isocratic logk’
values, construct the corresonding calibration equations
and calcultae the average logP
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Partition Coefficients in Drug Design:
How to measure?
High Performace Liquid Chromatography
• logk’=Aφ2-Bφ+C
• logk’= - Sφ+logk’w logP= a logk’w +b
φ %
logk
logkwlogk
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Partition Coefficients in Drug Design:
How to measure?
High Performace Liquid Chromatography
Linearity depends on:
Chromatographic conditions
organic modifier, pH
solutes (presence of strong hydrogen dond acceptors)
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Partition Coefficients in Drug Design:
How to measure?
High Performace Liquid Chromatography
Isocratic versus extrapolated logk’ values
less experiments more general indices
possible lipophilicity inversion values in the same order of logP
avoid lipophilicity inversion
They do not contain the same information content!
Results of LFER analysis (Abraham 1994)
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Partition Coefficients in Drug Design:
How to measure?
High Performace Liquid Chromatography
Isocratic versus extrapolated logk’ values
Under suitable chromatographic conditions and depending
on the stucture of the solutes 1:1 correlations between logP
and logk’w are found
logP= alogk’w +b
a ~1 b ~ 0
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Partition Coefficients in Drug Design:
How to measure?
High Performace Liquid Chromatography
Plot of logk versus φ - Lipophilicity inversion
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Partition Coefficients in Drug Design:
How to measure?
No octanol added octanol added
Lombardo et al. Lipohilicity in Drug Disposition, Lausanne 2000
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Partition Coefficients in Drug Design:
How to measure?
logPoct = 1.0890 ( 0.0969)* logk’w- 0.5435 ( 0.2768)
N = 27, R2 = 0.835, R = 0.914, s = 0.556, F = 126, q2 = 0.808
(No octanol added, 1 mL/min)
logPoct = 1.1014 ( 0.0389)logk’w - 0.0045 ( 0.0941)
N = 27, R2 = 0.970, R = 0.985, s = 0.238, F = 803, q2 =0.965
(Octanol added, 1 mL/min)
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Χρωματογραφία Ακινητοποιημένων Τετνητών Μεμβρανών
Immobilized Artificial Membrane Chromatography
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Partition Coefficients in Drug Design:
How to calculate
• Substitution system Direct Calculation • Fragmental Systems ( based on the
• Atomic Contribution Systems additive/constitutivecharacter)
• Model based equations Indirect calculation
• Molecular Lipophilicity Potential
Lipophilcity=Hydrophobicity +Polarity
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Partition Coefficients in Drug Design:
How to calculate
Hansch’s Hydrophobic substituent constant π
Based on the additivity principle:
logP=logPH +Σπ
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Partition Coefficients in Drug Design:
How to calculate
Fragmental Systems
logP = anfn+ Q
Rekker’s fragmental system
Leo-Hansch’s fragmental system
Meylan-Howard fragmental system
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Partition Coefficients in Drug Design:
How to calculate
Rekker’s fragmental systemreductionnist approach
logP = anfn+ knCM
for octanol- water system: CM =0.289 original system
CM =0.219 revised system
Softwares
PROLOGP-cdr original system, modified
SANALOGP_EO original system
SANALOGP_ER revised system
Σf –Sybyl revised system
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Partition Coefficients in Drug Design:
How to calculate
Leo-Hansch’s fragmental system
constructionnist approach
logP = anfn+ bmFm
tested for 8000 compounds (Starlist)
r= 0,970 και s=0.398
Software: CLOGP
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Partition Coefficients in Drug Design:
How to calculate
Meylan-Howard fragmental system
• logP=(finI) +(cjnj) +0,229 regression
• fragments 130 analysis
• correction factors 235
• derived/tested for 2351 compounds
• n=2351 r =0,991 s=0,216
• Software : KOWWIN, LOGKOW
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Partition Coefficients in Drug Design:
How to calculate
Atomic Contribution Systems
logP=Σaiαi
• Broto
• Ghose-Crippen
• Klopman and Wang regression methods
• Suzuki-Kudo
• Dubost
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Partition Coefficients in Drug Design:
How to calculate
Atomic Contribution Systems
Broto’s system
• test series: 1868 compound (no possibility of internal
hydrogen bonds)• descriptors 222 (up to 4 atoms with specific bonding
pathways up to 4 )
Software : PROLOGP-atomic, MOLCAD
presision : 0.4 log units
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Partition Coefficients in Drug Design:
How to calculate
Atomic Contribution Systems
Ghose-Crippen system
test series: 830 compounds
descriptors: 110
precision : r=0,964 s=0,47
Softwares:
MOLCAD,Tsar 2.2, PROLOGP- atomic5 (modified),
HyperChem/Chemplus
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Partition Coefficients in Drug Design:
How to calculate
Atomic Contribution Systems
• Klopman -Wang system (Computer-automated structure
evaluation, CASE)test series: 935 compounds
descriptors: 10 atomic parameters + 76 ‘star centered’ fragemens + 2 Indicator variables 39 statistical
significant
Precision : r=0.965 s=0.385
Software: KLOGP
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Partition Coefficients in Drug Design:
How to calculate
Atomic Contribution Systems
Suzuki-Kudo system
test series: 1465 compounds
descriptors (Increments): 415+ 9 atom fragments
Mean absolute error: 0.35
Software: CHEMICALC-2
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Octanol-Water Partition Coefficients:
How to estimate
• Construction of Artificial Neural Networks
based on Electrotopological State Indices
(Interactive Analysis, AlogP)
http://146.107.217.178/lab/alogps/index.html
http://amorgos.pharm.auth.gr/HelSocMedChem/home.html
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Partition Coefficients in Drug Design:
How to calculate
Model Equations based on Molecular
properties Conformation dependent
Size parameters, charge density, dipole moment
Sovent accassible surface area (with 1.4A
solvent ‘coat’)
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Partition Coefficients in Drug Design:
How to calculate
• Charge density method
(Klopman and Iroff ,1981)
Data set: 61 compounds
Huckel-type charge density calculation
logP=0.258+0.197NH_0.122NC-2.235NN-2.264NO+21.39Σq2C-
9.335Σq2N-5.452Σq2O+1.325NA+0.715NT+0.701NM
n=61 r2=0.988 s=0.13
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Partition Coefficients in Drug Design:
How to calculate
(Bodor and Huang, 1990))
charhe density, van der Waals radii, molecular surface area,
volume , ovality
logP=9.55-0.057D+1.039 Ialkane-17.377Qn4+32.243Qn2-
8.514Qn-5.419Qo4+20.346Qo2-4.625Qo-
5.004Qon+0.0052861MW-1.1414x10-4S2+0.059838S-
7.666O-5.596O2+0.083249nc-0.27406ABSQ+2.1050O4
n=302 r=0.978 s=0.305
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Limitations of Calculation Systems:
• positional isomers
• conformation effects
• zwitterions /charged molecules
• complex structures
Partition Coefficients in Drug Design:
How to calculate
63
Partition Coefficients in Drug Design:
How to calculate
• 3-D description of lipophilicity based on a fragmental
system and s distance function
)(1
ij
N
i
i dfctfMLPj
)exp(1
)exp(1
1 bad
abfMLP
ij
N
i
ij
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Partition Coefficients in Drug Design:
How to calculate
• Molecular Lipophilicity Potential (Testa et al)
• Software CLIP
• logP=2.86x10-3 ΣMLP+ +1.52x10-3 ΣMLP- -0.10
n=114 r2=0.94 s=0.37
• logP=2.35x10-3 ΣMLP+ +1.78x10-3 ΣMLP- -0.39
• n=114 r2=0.89 s=0.53
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Εσρος της λιποπιφιλίας διαφορετικών διαμορφωμερών
66
Partition Coefficients in Drug Design:
How to calculate
Abraham-Kellog
• Hydrophobic INTeractions
based on
Solvent Accessible Surface
Area (s),
fragment constants (α)
and a distance function (R)
• Software :HINT
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Prediction of logD
Software PROLOGD
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Partition Coefficients in Drug Design:
• How to measure?
• How to calculate?
Still a lot to be done!
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