Lipophilicity by HPLC retention

www.bio-mimetic-chromatography.com

Lipophilicity in drug discovery

Klara Valko

Bio-Mimetic ChromatographyConsultancy for Successful Drug Discovery

What is lipophilicity?IUPAC definition Lipophilicity represents the affinity of a

molecule or a moiety for a lipophilic environment.

Hydrophobicity measures the association of non-polar groups or molecules in an aqueous environment which arises from the tendency of water to exclude non-polar molecules.


Why do we need to measure lipophilicity?

A compound partitioning between aqueous and organic phase can model compound partitioning in vivo

Compound partitions between two immiscible solvent


Lipophilicity is measured by partition coefficient Partition coefficient is the quotient of the compound

concentration in an aqueous and a non-miscible solvent under equilibrium condition.

It is expressed by the quotient of the compound concentrations in the two phases under equilibrium condition.

It depends on the nature of the two phases, the compound properties, pH, and the temperature.


Measuring lipophilicity by HPLC

Cs. Horváth, W. Melander, I. Molnár, J. Chromatogr. 125 (1976) 129.

Measuring bio-relevant association constants has great impact on drug discovery!

Based on the solvophobic theory the interaction between the solute and the stationary phase is considered as a reversible association of

the solute molecules with the stationary phase moiety (hydrocarboneous, membrane, or protein). Accordingly solute

retention is governed by the dynamic equilibrium constant.

Vs = volume of the stationary phase

Vm = volume of the mobile phase


Gradient retention time for the determination of Chromatographic Hydrophobicity Index (CHI)

The isocratic chromatographic hydrophobicity index (φ0) is linearly related to gradient retention times.

Measure log k values of a set of compounds using 3 to 5 different mobile phase composition. Plot the log k values in the function of the organic phase concentration.

Calculate the organic phase concentration that relates to log k = 0. This is the φ0 value.

Reversed phase gradient retention times can be calibrated by isocratically determined φ0 values.

Use the slope and the intercept of the obtained straight line to convert the gradient retention time of any compound obtained under the same conditions as the calibration set of compounds to CHI.



Chromatographic Hydrophobicity Index (CHI) Approximates the organic phase concentration (%) when the compound elutes from a reversed

phase column using linear gradient. CHI gives a straight line with the gradient retention time.

LunaC18(2) 50 x 3 mm; 1.00 ml/min; Mobile phase A 50 mM ammonium acetate pH 7.4 and B is 100%

acetonitrile. Gradient: 0 - 2.5 min 0 - to 100% B; 2.5 - 2.7 min 100% B.

Calibration of CHI at pH7.4

y = 54.329x - 71.702R 2 = 0.9972

0.00

20.00

40.00

60.00

80.00

100.00

120.00

1.4 1.9 2.4 2.9 3.4

Compound CHI7.4 at pH 7.4

CHI2 at pH 2

CHI10.5 at pH 10.5

Theophylline 18.4 17.9 5.0 Phenyltetrazole 23.6 42.2 16.0 Benzimidazole 34.3 6.3 30.6 Colchicine 43.9 43.9 43.9 Phenyltheophylline 51.7 51.7 51.7 Acetophenone 64.1 64.1 64.1 Indole 72.1 72.1 72.1 Propiophenone 77.4 77.4 77.4 Butyrophenone 87.3 87.3 87.3 Valerophenone 96.4 96.4 96.4


Ultrahigh Performance Liquid Chromatography (uPLC) for CHI

CHI TM1

Time0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80

AU

0.0

5.0e-2

1.0e-1

1.5e-1

2.0e-1

2.5e-1

3.0e-1

CHI TM1_LunapH74 Diode Array 254

Range: 3.969e-11.02

0.86

0.64

0.590.72

0.92

1.27

1.16

1.10

1.37

The CHI test mix is separated in less than 90 secNow we can determine a compound’s lipophilicity in 90 sec

using various starting mobile phase pHCourtesy of Shenaz Bunally at GSK


Parallel measurement of a compound’s retention at various pH to reveal acid/base character

Typical 4-way chromatograms of a base

pH2

pH7.4

pH 10.5

IAM 7.4


CHIs measured at 3 pHs provide an automatic way of grouping molecules according to acid/base character without structural information.

0

10

20

30

40

50

60

70

80

90

100

Neutral(Zwitterionic)

Strong acid Weak acid Strong base Weak base Amphoteric

pH2

pH7.4pH10.5

The change of CHI values by changing the pHCHI

CHI values at pH 2, pH 7.4 and pH 10.5 reveal acid/base character of compounds


We can use biological macromolecules as stationary phase to obtain bio-mimetic properties Reversed phase retention reveals how compounds

interact/partition/bind to hydrophobic environment such as aliphatic hydrocarbons. This environment is common in the inner part of phospholipid bi-layers.

Applying protein stationary phases can be used to reveal compounds’ interaction/affinity to biologically important proteins, such as plasma proteins

Immobilized artificial membrane (IAM stationary phases were developed to emulate the phospholipid membrane on solid surfaces.


Bio-mimetic HPLC measurement of Human Serum Albumin (HSA), α-1acidglycoprotein (AGP) and Immobilized Artificial Membrane (IAM) partition

HSA AGP IAM

pH 7.4 aqueous mobile phases

IPA IPA ACN

Bio-Mimetic Stationary phases


Measuring bio-mimetic properties The retention times obtained on bio-mimetic

stationary phase should be calibrated

Measure a set of compounds retention for which the bio-mimetic binding properties are available using other methods, such as equilibrium dialysis or ultra filtration


Biomimetic lipophilicity measurements (Membrane partition) using Immobilised Artificial Membrane stationary phase

Stationary phaseIAM calibration y = 26.647x - 37.653

R2 = 0.9966

0

10

20

30

40

50

60

1 1.5 2 2.5 3 3.5gtR

CHI

Typical calibration

Compound gtR CHI IAMOctanophenone 3.269 49.4Heptanophenone 3.145 45.7Hexanophenone 3.001 41.8Valerophenone 2.822 37.3Butyrophenone 2.601 32Propiophenone 2.341 25.9Acetophenone 2.013 17.2Acetanilide 1.83 11.5Paracetamol 1.591 2.9

Column: IAM PC2 (CH2)12 150 x 4.6

Mobile Phase flow rate: 2 ml/min

Gradient: 0 to 3 min 0 to 80% acetonitrile

3 to 3.5 min 80% acetonitrile

3.5 to 3.7 min 0% acetonitrile

Cycle time: 5 min

K. Valko et al. J. Pharm. Sci.89 (2000) 1085-1096


Calibration ploty = 2.177x + 0.1304

R2 = 0.9612

-0.50

0.00

0.50

1.00

1.50

2.00

2.50

-0.40 -0.20 0.00 0.20 0.40 0.60 0.80 1.00

logtR

logK

lite

ratu

re

Calculate %Binding

logK = slope * log(tR) + intK = %B / (101-%B)

y = 0.9309x - 0.3329R2 = 0.879

0

20

40

60

80

100

0 20 40 60 80 100

HSA Column

Lite

ratu

re %

bin

ding n=71

K. Valko et al. J. Pharm. Sci. 92 (2003) 2236

Column: HSA 50 x 3 mm (Chrom Tech, Chiral Technologies)Flow rate: 1.8 ml/min at 300CMobile phase: 50 mM ammonium acetate pH7.4Gradient: 0 - 3 min 0 to 30% 2-propanol;

3 to 10 min 30% 2-propanol; 10 to 10.5 min 0% 2-propanolCycle time: 15 min

Serum albumin binding measurement using chemically bonded serum albumin stationary phases


Retention time of compounds can be converted to % binding or K association constant

)(logexplog HSAKHSAkHSAke

2 min


Accuracy and reproducibility The reproducibility of the retention time

measurements are very good. Variations can be expected only in the third

decimal places in the minutes. The accuracy increase as the binding

increases. We have still several minutes left after the

peak of 99% bound compound, that we can accurately measure.


AGP binding measurement by HPLC

Same principle as HSA binding measurements:AGP column2-propanol gradientpH 7.4 ammonium acetateCalibration with AGP binding data derived from published % AGP bound values

Typical AGP column calibration plot y = 2.7976x - 0.5289R2 = 0.9744

-0.400

-0.200

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

log tR

logK

AG

P

Calibration set of compounds: Nizatidine, Bromazepam, Warfarin, Propranolol, Imipramin, Nicardipine, Chlorpromazine


How can we use the bio-mimetic chromatographic data?

We can build models for in vivo distribution of drug molecules

We can predict Volume of distribution Unbound volume of distribution Drug efficiency max Minimum efficacious dose when the potency data are available

Ask for help to set up these measurements! You need only an HPLC, several HPLC columns and calibration set of compounds.

In three days you can be up and running your bio-mimetic chromatography!

ReferencesC. Hansch, A. Leo, ρ-σ-π analysis. A method for correlation of biological activity and chemical structure. J. Amer. Chem.Soc.

86(1964) 1616-1624 Horvath, Cs., Melander, W., Molnar, I. Solvophobic interactions in liquid chromatography with non-polar stationary phases,

Journal of Chromatography, 125 (1976) 129-156Valko, K.; Snyder, L.R.; Glajch, G.L. Retention in reversed-phase liquid chromatography as a function of mobile-phase

composition. J. Chromatogr. A 656, (1993) 501–520.Giaginis, A. Tsantili-Kakoulidou, Current state of the art in HPLC Methodology for lipophilicity assessment of basic drugs

(Review) Journal of Liquid Chromatography & Related Technologies, 31: (2008) 79–96.Lombardo, F., Shalaeva, M. Y., Tupper, K. A., Gao, F., ElogDoct: A tool for Lipophilicity Determination in Drug Discovery. 2.

Basic and neutral compounds. (2001) 2490-2497Gocan, S., Cimpan, G., Comer, J., Lipophilicity measurements by liquid chromatography in Advances in Chromatography,

Eds: E. Grushka, N. Grinberg, 44 (2005) 79-176, Taylor & Francis Group, 1574447343Valko, K., Bevan, C., Reynolds, D., Chromatographic hydrophobicity index by fast-gradient RP-HPLC: A high throughput

alternative to log P/log D. Analytical Chemistry 69 (1997) 2022-2029 Valko, K. Measurements of lipophilicity and acid/base character using HPLC methods. In “Pharmaceutical profiling in drug

discovery for lead selection” Eds. Borchardt, R., Kerns, E., AAPS (2004)Arlington, VA 127-182

K. Valko, C.M. Du, C.D. Bevan, D.P. Reynolds, M.H. Abraham, Rapid-gradient HPLC method for measuring drug interactions with immobilized artificial membrane: Comparison with other lipophilicity measures, J. Pharm. Sci. 89 (2000)

1085– 1096. K. Valko, C.M. Du, C. Bevan, D.P. Reynolds, M.H. Abraham, Rapid Method for the Estimation of Octanol / Water Partition Coefficient ( Log P oct ) from Gradient RP-HPLC Retention and a Hydrogen Bond Acidity Term, (2001) 1137–1146.K. Valko, S. Nunhuck, C. Bevan, M.H.M.H. Abraham, D.P.D.P. Reynolds, Fast gradient HPLC method to determine compounds binding to human serum albumin. Relationships with octanol/water and immobilized artificial membrane lipophilicity., J. Pharm. Sci. 92 (2003) 2236–48.


Science

Lipophilicity by HPLC retention