Use Of Immobilised Artificial Membrane (IAM)

Chromatography with Aqueous and simulated intestinal fluid as mobile

phase

David Sherwood

NOV 2006

IAM Alternative to LogD

• Uses Phosphatidycholine (PC) embedded in silica as stationary phase

• PC analogs closely mimic the surface of biological cell membranes

•IAM more closely mimic the interactions of analytes with biological membranes, where hydrophobic, ion pairing and hydrogen bonding interactions are possible

•IAM pH7.4 being developed in Cambridge based on method developed by colleagues in Braine

IAM stationary phase

IAM pH7.4 General ProcedureAT UCB

• 1mg of sample dissolved in 1ml MeOH and filtered • Hydrophilic compounds 1 isocratic run in 100%PBS pH7.4• Lipophilic 5 isocratic HPLC runs per sample using pH7.4 PBS as mobile phase

in 5 different %MeCN (15%, 20%, 25%, 30%, 35%)• extrapolate back to 0% MeCN Take the Log value of the intercept

• Log K’IAM is therefore quoted as being in 100% pH7.4 PBS

• Calculate Capacity factor K’IAM Tr.-To

k’IAM = ------------- To

• Tr.: retention time of the compound• To: void volume , the retention time of a product that is not retained on

the column, NaNO2 in our case

Extrapolation

Log k’IAM 1.91

y = -0.08x + 1.91R2 = 0.99

-2.0-1.00.01.02.03.0

0 20 40

%MeCN

Log

K'I

AM

Correlation Of K’IAM with other Data

• Jean-Philippe Starck, Patrice Talaga, Luc Que´re´et.al, ‘Potent anti-muscarinic activity in a novel series of quinuclidine derivatives’ Bioorganic & Medicinal’ Chemistry Letters 16 (2006) 373–377

• Suggest that IAM can be an effective tool in predicting metabolic clearance providing structure is taken into consideration

• Ferenc Hollosy, Klara Valko, Anne Hersey et. al ‘Estimation of Volume of Distribution in Humans from High Throughput HPLC-Based Measurements of Human Serum Albumin Binding and Immobilized Artificial MembranePartitioning’ J. Med. Chem. 2006, 49, 6958-6971

•  Have suggested a way of estimating Volume distribution using IAM and HAS for Acids Bases and zwitterions

Log K’IAM Vs Metabolic Clearance

(series of compounds with similar structure)

N=8

Clearance R2 = 0.9678

0

20

40

60

80

100

120

140

0 0.5 1 1.5 2 2.5

Log k'IAM

ml/

min

Note that this correlation suggests the reverse of whatWe would expect

IAM Vs Clearance

(Same series more data points)

clearanceR2 = 0.329

0

50

100

150

200

250

0 0.5 1 1.5 2 2.5

LogK'IAM

ml/

min

N=12Posses the question how many data points required to prove correlation

IAM v Target Cell potency

N=6

Log K'IAM vs Log Potency Cell Assay

R2 = 0.9753

-1

-0.5

0

0.5

1

1.5

2

2.5

0 0.5 1 1.5 2 2.5

Log K'IAM

Lo

g P

ote

ncy

Target protein potency

N=6

Log K'IAM vs Target protien potency

R2 = 0.8819

-6-4-202468

1012

0 0.5 1 1.5 2 2.5

Log K'IAM

Pro

tien

po

ten

cy (

nM

)

IAM Fessif

• Fessif mobile phase          Fed State Simulated Intestinal Fluid composition according to

Dressman et al.,1998. Dissolution testing as a prognostic tool for oral absorption: immediate release dosage forms. Pharm. Res. 15, 11-22.

• pH 5.0 Buffer: Dissolve 4.04 g of NaOH, 8.65 g of CH3COOH 100% and

15.15 g of KCl and sufficient water to make 1000 ml. Adjust to pH 5.0 with HCl or NaOH 1M if necessary.

• Fessif: Dissolve 6.6 g of Taurocholic Acid in 50 ml of buffer pH 5.0. Add 2.36g lecithin add stirrer bar place on stirrer over night. add sufficient pH5.0 buffer to make 800 ml.

Fessif

• K’IAM has been reasonably correlated with many in-vitro permeability studies

• But we make drugs for humans so the earlier we model in-vitro permeability on Human in-vivo the better

• Also most permeability assays are performed in aqueous buffer, but the intestinal juices are more complex than that and may influence permeability

• Aim of K’IAM Fessif was to see if it could be correlated to human permeability and was there a significant difference to using Aq buffer

Peff logPeff logD5.5antipyrine 4.5 -3.35 0.6atenolol 0.2 -4.7 <-2carbamazepine 4.3 -3.37 2.45creatinine 0.3 -4.52 <-2desipramine 4.4 -6.72 1.1furosemide 0.05 -5.3 0.4hydrochlorothiazide 0.04 -5.4 -0.2ketoprofen 8.4 -3.08 1.8L-leucine 6.2 -3.21 -1.6L-dopa 3.4 -3.47 <-2a-methyldopa 0.2 -4.7 <-2metoprolol 1/2 tartate 1.3 -3.89 -0.6naproxen 8.3 -3.08 2.1piroxicam 7.8 -3.11propranolol HCL 2.9 -3.54 0.7terbualine 1/2 sulphate 0.3 -4.52 -1.3verapomil 6.7 -3.17 1.2

S. Winiwarter. et al. 'Correlation of Human Jejunal Permeability (in Vivo ) of Drugs with Experimental and Theoretical Derived Perameters. A multivariate Data Analysis Aproach' J. Med. Chem.1998 Vol41 No25 4939-4949

N=17

Fessif K’IAM

• For low lipophilic compounds use 100% fessif mobile phase similar to that of IAM ph7.4

• Extrapolations in varying degrees of MeCN cannot be used as mobile phase crashes

• MeOH has been tried to no success

• At the moment it is not feasible to analyse compounds above LogK’IAM 0.5 (Fessif) so correlation of 17 broad spectrum standards with intestinal absorption is not possible

• However, differences have been noted between Fessif pH5.0 and Aq pH5.0

LogK’Fessif vs Human Effective permeability (Peff)

N=6

Correlation is non existent however Only the low K’IAM were able to be tested

fessifR2 = 0.5216

-1

-0.5

0

0.5

1

1.5

0.00 1.00 2.00 3.00 4.00 5.00

Peff 10-4cm/s

log

k'IA

M

Peff LogK' IAM Fessifantuipyrine 4.50 0.12atenolol 0.20 -0.53desipramine 4.40 1.29hydrochlorothiazide 0.04 0.12methyldopa 0.20 -0.69metoprolol 1.30 0.21terbutaline 0.30 -0.16

fessifR2 = 0.5216

-1

-0.5

0

0.5

1

1.5

0.00 1.00 2.00 3.00 4.00 5.00

permeabilitylo

g k

'IAM

Log Peff R2 = 0.5216

-1

-0.5

0

0.5

1

1.5

0.00 1.00 2.00 3.00 4.00 5.00

Log Peff

Lo

g K

'IAM

Fes

sif

Log k’IAM pH5 vs Human Effective Permeability (Peff)

N=6

LogK'IAM pH 5.0 vs PeffR2 = 0.2331

-1

-0.5

0

0.5

1

1.5

0.00 1.00 2.00 3.00 4.00 5.00

Peff 10-4cm/s

Lo

gK

'IA

M

Peff LogK' IAM aq pH5.0antuipyrine 4.50 0.23atenolol 0.20 -0.14desipramine 4.40 1.19hydrochlorothiazide 0.04 0.76methyldopa 0.20 -0.58metoprolol 1.30 0.67terbutaline 0.30 0.18

logK’ IAM from Fessif Vs Aq pH5.0 mobile phase

N=6

LogK'IAM pH 5.0 vs FessifR2 = 0.8481

-1

-0.5

0

0.5

1

1.5

-1 -0.5 0 0.5 1 1.5

LogK'IAM Fessif

Lo

gK

'IAM

pH

5.0

Some variation between fessif and aq pH5.0 may suggest Bile surfactants and lipids have an impact on

Compound interactions with PC stationary phase And thus may influence permeability

Summary

• At Cambridge we are only beginning to utilise IAM

• We want K’IAM pH7.4 and K’IAM Fessif to be meaningful not just more data to go unnoticed

• K’IAM pH7.4 has been reported by many sources including colleagues in Braine to have correlation with some compound series and clearance providing structure is taken into concideration.

• K’IAM pH5- pH6.8 has been correlated with in-vitro permeability methods which may not be good enough should be correlated with in-vivo human

• Early data suggest that bile surfactants and lipids may have influence on how compounds interact with biological membranes

• I believe that the use of fessif in K’IAM and in-vitro permeability studies

may be the way forward

Aknowledgements

• Luc Quere, Ariane Descamps, Géraldine Longfils Sandrine Rome, at UCB Braine for their support and assistance