Permeability: transporting drugs through (lipid) membranes

1st Physical Chemistry Symposium, November 30, 2005

Paula Garcia

Efflux (or counter-transport)

Metabolic (in)stability: microflora

intestines

liver

Factors Determining Oral Bioavailability

Physicochemical factors:

Biochemical factors

Dissolution (solid to solution)

Aqueous Solubility

Membrane PermeabilityMembrane Permeability

Di, L., Kerns, E., Fan, O.J., Carter, G.T., Eur. J. Med. Chem. 2003, 38, 223.

Permeation Mechanisms

Cellular

Barrier

Passive Diffusion

Active Transport EffluxParacellular

Endocytosis

PAMPA StructureMotifs

MWPolarity

Caco-2MDCK

Passive diffusion: major absorption pathway

Can any of these methods be adapted for high throughput measurements?

*2000, www.fda.gov.cder/guidance/index.html

In-vivo intestinal perfusion studies in humans or animals In-vitro permeation experiments using excised human or animal intestinal tissues In-vitro permeation experiments across a monolayer of cultured human intestinal

cells (e.g Caco-2 cells)

• Methods approved for the Biopharmaceutical Classification System*

Permeability Measurements

Rule of five - Lipinski

Lipophilicity H-bond capacity Molecular size Polar Surface Area (PSA) Quantum properties

• In-silico models based on:

• Because they are made from cultured cells, Caco-2 membranes express all mechanisms of transport;

• Therefore, if a drug goes through a Caco-2 membrane, it will probably be absorbed by the GI tract

Can Caco-2 assay be used for permeability screening?

Yes, but…

It takes several days to create membranes* and requires cell culture skills

* normally 21-25 days4-day culture was recently reported: Int. J. Pharm. 2000, 200, 41.

Drug

basolateral

apical

human colonic cell line

Caco-2 Permeability Assay

Protein

Phospholipid Head

Lipophilic Tail

Structure of the cell membrane

Permeability through lipid membranes ?!

Lipid membranes are quickly and easily made by robots; easily automated for high throughput permeability assays.

a) Artursson P., Book of Abstracts, PAMPA 2002 Conference, 2002, San Francisco; Mandagere A.K., Thompson T.N., J. Med. Chem. 2002, 45, 304.

Transcellular Absorption

80-95% of commercial drugs (a)80-95% of commercial drugs (a)

1998 - PAMPA was initially introduced by Kansy from Hoffmann-La Roche 2005 – 95 hits in Pubmed

Kansy, M.; Senner, F.; Gubernator, K., J. Med. Chem. 1998, 41, 1007.

Membrane: egg lecithin in hydrophobic filter

Good correlation between PAMPA flux

and % HIA

Active transport of polar compounds with

low Mw

PAMPA: Permeability through lipid membranes

Donor

Acceptor

Membrane(20% (W/V) phospholipid mixture in dodecane)

PAMPA: Parallel Artificial Membrane Permeability Assay

Dru

gPassive Diffusion- Pe (cm/s)

Donor

Acceptor(pH=7.4)

Membrane

GastroIntestinal Tract (GIT)

Double-Sink Conditions

SDS micelles

(pH= 5-8)

PAMPA: Parallel Artificial Membrane Permeability Assay

Dru

gPassive Diffusion- Pe (cm/s)

Unstirred Water Layer

PAMPA: workstation

phospholipidcocktail

sink (acceptor at top)

stirrer (donor at bottom)

PAMPA: Sandwich plates

Solubility-Diffusion Model / pH-Partition Theory

Passive diffusion: pKa, solubility and lipophilicity are important!

LogP

The effect of the pH on Permeability

O

O

N

N

O

O

HCl

-8.0

-7.0

-6.0

-5.0

-4.0

-3.0

-2.0

2.0 4.0 6.0 8.0 10.0

pH

log

Pe

Verapamil pKa=9.07Base

Ketoprofen

-6.5

-5.5

-4.5

-3.5

-2.5

2.0 4.0 6.0 8.0 10.0pH

log

Pe

pKa=3.98

O

O

HO

Acid

Faller, B., Wohnsland, F., J.Med.Chem., 2001, 44, 923; Ruell, J.A., Tsinaman, K.L., Avdeef, A., Eur.J.Pharm.Sci., 2003, 20, 39; Kerns, E.H., Di, L., Jupp, P., Pharm.Sci., 2004, 93, 6, 1440;

The effect of the pH on Permeability

Permeability of ionisable compounds is pH dependent!

-5.0

-4.5

-4.0

-3.5

-3.0

2.0 4.0 6.0 8.0 10.0pH

log

Pe

Carbamazepine

N

NH2O

unionisable

Unstirred Water Layer and Ionisation

pKaflux

pH where 50% of the resistance to transport comes from the UWL

and 50% from the membrane

UWL:

In GIT: 40 mIn BBB: no UWL

BBB

GIT

UWL can be reduced by stirring the donor solution

PAMPA Assay: DOUBLE-SINK and Stirring

Pe is UWL limited!

Compounds with a log P ≥ 2, follow the protocol with stirring.

Log P ≥ 2 Log P < 2

The Effect of lipophilicity on Permeability

Permeability improves with increase in lipophilicity

The Effect of lipophilicity on Permeability

Caco-2 Assay

Pe is UWL limitedParacellular transport

AstraZeneca database

0

100

200

300

400

500

600

700

800

-2 -1 0 1 2 3 4 5 6 7 8 9 10 11

cLogPC

aco

-2 (

nm

/s)

What is the Permeability hurdle, lipophilicity or solubility?

Caco-2 versus CLogP/D

y = -0.2183x 2 + 0.8639x + 0.4508R 2 = 0.5362

-0.5

0

0.5

1

1.5

2

-1 0 1 2 3 4 5

cLogD7.4

log

Pap

p

Riley et al, Curr. Drug Metab., 2002, 3, 527

Organon database

Membrane Retention

The Effect of Solubility on Permeability: Co-Solvent PAMPA Assay (b)

Compound logP Solubility Solubility pH

pH 5 (mg/ml) pH 7.4 (mg/ml) H2O H2O: ACN (8:2)v/v

Antipyrine 5 2,1 1,2

0,56 > 197 > 194 6,2 2,1 1,6

7,4 2,3 1,6

Caffeine 5 2,6 0,8

-0,07 > 20000 > 20000 6,2 2,6 0,7

7,4 2,5 0

Theophylline 5 1,2 0,4

0 > 189 > 186 6,2 1,4 0,5

7,4 1,1 0,3

Verapamil.HCl 5 189 12

4,33 > 500 > 500 6,2 763 96

7,4 1707 739

Ketoprofen 5 264 123

3,16 > 259 > 262 6,2 20 14

7,4 4 3

Ketoconazole 5 496 68

4,34 43 11 6,2 und 1254

7,4 0 equ

Danazol 5 0 1754

3,9 0,09 0,06 6,2 0 819

7,4 64 419

Pe x 10-6 (cm/sec)

SOl (ketoconazole)ACN:H2O=53 (pH 5); 37(pH 7.4); SOl (danazol)ACN:H2O=1 (pH 5); 3(pH 7.4)

0

200

400

600

800

1000

1200

1400

1600

1800

AntipyrineCaffeineTheophyllineVerapamil.HClKetoprofenKetoconazoleDanazol

H2O

H2O

H2O:ACN (8:2 v/v)

The Effect of Solubility on Permeability-Co-Solvent PAMPA AssayP

ex1

0-6 (

cm/s

)

Pe is solubility limited(Low Sol., but high Pe)

(High Sol., high Pe)

(High Sol., Low Pe)

Membrane keeps its integrity

Sugano, K., Hamada, H., Machida, M., Ushio, H, Int. J. Pharm. 2001, 228, 181; Ruell, A.J., Tsinman, O., Avdeef, A., Chem. Pharm. Bull. 2004, 52, 561

The Effect of Solubility on Permeability-Co-Solvent PAMPA Assay (b)

Compound MWElogD (7.4)

Sol (pH 5) Sol (pH 7.4) 

Pe x10-6 (cm/sec) 

      (µg/ml) (µg/ml) pH H2O H2O/ACN (8:2 v/v)

Cpd 1 539 4.6 55 7 5.0 1137.6 57.4

          6.2 1875.1 331.3

          7.4 0.0 916.7

Cpd 2 555 4.4 > 58 8.9 5.0 891.4 78

          6.2 1361.1 364.5

          7.4 0.0 862.5

Cpd 3 422 4,1 0.5 0.3 5.0 78.5 89.6

          6.2 391.1 166.9

          7.4 0.0 168.2

Cpd 4 529 5.2 < 0.1 0.2 5.0 0.0 879.5

          6.2 0.0 173.3

          7.4 0.0 216.4

Cpd 5 650 4.4 1 1.1 5.0 256.2 109.5

          6.2 347.9 129.4

          7.4 270.1 201.5 

Cpd 6 543 6 0.5 0.7 5.0 0.0 0.0

          6.2 98.0 4.89

          7.4 918.0 143.9

PAMPA Assay in Organon

Ionisable compounds display Permeability- pH profile

a)- Kern, E. et al., Pharm. Sci., 2004, 93, 6, 1440;

Lipophilicity ↔ Permeability

• Reducing the UWL is important for lipophilic compounds.

• Highly lipophilic compounds display a high membrane retention.

b)- Bermejo, M. et al., Eur. J. Pharm. Sci., 2004, 21, 429;

Low aqueous solubility might be a limiting factor in Permeability measurements:

• Use of co-solvent method allows to differentiate compounds from classes III and IV in the BCS system.

• 20% of ACN doesn’t interfere with the integrity of the membrane.

d) Avdeef, A., et al., Chem. Pharm. Bull., 2004, 52, 561; Sugano, K., et al., Int. J. Pharm., 2001, 181.

30

40

50

60

70

80

90

100

110

-7,00 -6,50 -6,00 -5,50 -5,00 -4,50 -4,00 -3,50 -3,00 -2,50 -2,00

log PeMax (pH 5.0, 6.2, 7.4)

% H

IA

Theophiline

Caffeine

Antipyrine

Atenolol

Furosemide

Tertbutaline

Hydrocholorthiazide

Ranitidine

Dexamethasone

Metoprolol

PiroxicamCarbamazepin

Quinine

Ketoprofen TestosteronePropanolol

VerapamilDesipramine

Max-Pe PAMPA Model for Prediction of Human Intestinal Absorption

high permeabilitylow permeability

a)- Avdeef, A., Absorption and Drug Development, 2003, Hoboken, NJ: Wiley-Interscience,b)- Avdeef, A. Curr. Top. Med. Chem., 2001, 1, 277.

Classification Pe

High >20x10-6 cm/s

Medium 10- 20x10-6 cm/s

Low <10x10-6 cm/s

Factors Determining Intestinal Drug Absorption

Fraction of drug absorbed (Fa) is governed by several processes:

Dose/Dissolution ratio,

Chemical degradation and/or metabolism in the lumen,

Complex binding in the lumen,

Intestinal Transit,

Effective Permeability across the Intestinal Mucosa (HJP)Effective Permeability across the Intestinal Mucosa (HJP)

Winimater, S., Bonham, N. M., Lernnernas, H., J. Med. Chem., 1998, 41, 4939.

PAMPA Model for prediction the Human Jejunal Permeability (HJP)(a)

Double–Sink (pH=5.0/7.4)

y = 0,4731x - 1,4404

R2 = 0,9408

-6,0

-5,5

-5,0

-4,5

-4,0

-3,5

-3,0

-2,5

-2,0

-8,5 -7,5 -6,5 -5,5 -4,5 -3,5 -2,5

log Pe 5.0/7.4

Lo

g P

eH

JP

Hydrochlorotiazide

Tertbutaline

Ranitidine

Atenolol

Furosemide

Antipirine

Metoprolol

Carbamazepine

Piroxicam

Propanolol

Ketoprofen

Naproxen

Verapamil

Desipramine

a)- Avdeef, A., Absorption and Drug Development, 2003, Hoboken, NJ: Wiley-Interscience, b)- Karlsson, J. P., Artursson, P., Int. J. Pharm., 1991, 7, 55; Karlsson, J. P., Artursson, P., Eur. J. Pharm. Sci., 1999, 9, 47.

Compound ClogP MW Nb Rot HBD HBA PSA In-Silico PAMPA Caco-2

(-2<X<5) (X<500) (X<12) (X<6) (X<11) (X<110) x10-6 x10-6

Progesterone

3,8 314 1 0 2 31,4 Good

1879 20

Nandrolone

2,7 274 0 1 2 38,1 Good

422 35

Hydrocortisone

1,7 362 2 3 5 78,3 Good

28 18

Cortisone

1,3 360 2 2 5 77,1 Good

26 34

Cpd 1

5,9 458 3 2 3 55,2 Moderate

1185 20

L-Dopa

-2,8 197 3 5 5 108,0 Moderate

0 0

Verapamil

4,5 455 14 0 6 61,8 Moderate

1188 15

Cpd 2

3.0 650 13 1 9 114,0 Bad

1600

Cpd 35.2 557 12 0 10 102,0 Bad

500

Permeability and Molecular Properties

Pe is a physicochemical process that depends on physicochemical properties of a molecule and its interactions with a membrane.

Caco-2 vs. PAMPA

BCS compounds

Kerns, E.H., Di, L., Petusky, S., J. Pharm. Sci., 2004, 93, 6,1440.

Absorptive Transport

Passive Diffusion Transport

Secretory transport

y = 0.6155x - 2.5577

R2 = 0.8032

-7.20

-6.70

-6.20

-5.70

-5.20

-4.70

-4.20

-3.70

-3.20

-7.00 -6.50 -6.00 -5.50 -5.00 -4.50 -4.00 -3.50 -3.00 -2.50 -2.00

Log Pe Max (pH 5.0, 6.2, 7.4)

Lo

g P

e C

ac

o-2

Theophiline

Antipyrine

Caffeine

Ranitidine

Hydrochlorotiazide

Furosemide

Carbamazepine

MetoprololKetoprofen

Naproxen

Quinine Desipramine

Propanolol

Verapamil

Comparison of PAMPA and Caco-2 Permeability Assay Characteristics

Characteristics PAMPA Caco-2Membrane composition Phospholipid in alkane Caco-2 cell monolayer

Permeability mechanism Passive diffusion Passive diffusion

Active transport

Active efflux

Paracellular

Metabolism No Yes

Max. throughput/instrument 690 cpd/week (3 plates/day in duplicate)

50 cps/week (2 plates/day in both A>B and B>A directions)

Resources Robot, plate washer, UV reader, 1 scientist

Cell culture lab., robot, HPLC or LC/MS, 1.5 scientist

Supplies ++ +++

Estimate cost/sample 1X 15-20X

• PAMPA is a good choice for Screening on Permeability.

Strategy for Combined Use of PAMPA and Caco-2

PAMPA

PAMPA + Caco-2

Caco-2

Passive Diffusion

MechanisticInformation

Passive, active, influx, efflux

and paracellular

Exploratory Discovery Pre-Development Development

Kern, E., (Wyeth Research), J. Pharm. Sci. 2004, 93, 6, 1440.

Acknowledgements

Medicinal Chemistry

• Maarten Honing

• Marcel Hermkens

• Michiel Scheffer

• Department of Medicinal Chemistry

30

40

50

60

70

80

90

100

110

10 20 30 40 50 60 70 80 90 100

PSA

% H

IA

Piroxicam

Tertbutaline

Atenolol RAnitifine

Hydrochlorotiazide

Furosemide

PSA< 62, highly absorved

PSA> 62, poorly absorved

CortisoneDexamethasone

PSA Model for Prediction of Human Intestinal Absorption

Co-Solvent PAMPA Assay

Membrane keeps its integrity

Pex1

0-6 (c

m/s

)

Physicochemical properties of 309 NCEs with low and high bioavailability in rats

0

25

50

75

100

125

150

Bad* Moderate* Good

Monika prediction

nu

mb

er

of

co

mp

ou

nd

s

≤30%

>30%

Bad two or more properties out the preferred rangeModerate one property out the preferred rangeMost important properties are logP, Mw and rotatable bonds