1. Permeability: transporting drugs through (lipid) membranes
Paula Garcia
1st Physical Chemistry Symposium, November 30, 2005

2. Factors Determining Oral Bioavailability
Physicochemical factors:
Dissolution (solid to solution)
Aqueous Solubility
Membrane Permeability
Biochemical factors
Efflux (or counter-transport)
Metabolic (in)stability: microflora
intestines
liver

3. Permeation Mechanisms
Cellular
Barrier
Passive Diffusion
Active Transport
Efflux
Paracellular
Endocytosis
PAMPA
Structure
Motifs MW
Polarity
Caco-2
MDCK
Passive diffusion: major absorption pathway
Di, L., Kerns, E., Fan, O.J., Carter, G.T., Eur. J. Med. Chem. 2003, 38, 223.

4. Permeability Measurements
Methods approved for the Biopharmaceutical Classification System*
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)
Rule of five - Lipinski
Lipophilicity
H-bond capacity
Molecular size
Polar Surface Area (PSA)
Quantum properties
In-silico models based on:
Can any of these methods be adapted for high throughput measurements?
*2000,

5. Caco-2 Permeability Assay
Drug
basolateral
apical
human colonic cell line
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 days
4-day culture was recently reported: Int. J. Pharm. 2000, 200, 41.

6. Permeability through lipid membranes ?!
Transcellular Absorption
Structure of the cell membrane
Phospholipid Head
Lipophilic Tail
Protein
80-95% of commercial drugs (a)
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.

7. PAMPA: Permeability through lipid membranes
PAMPA was initially introduced by Kansy from Hoffmann-La Roche
2005 – 95 hits in Pubmed
Membrane: egg lecithin in hydrophobic filter
Good correlation between PAMPA flux and % HIA
Active transport of polar compounds with low Mw
Kansy, M.; Senner, F.; Gubernator, K., J. Med. Chem. 1998, 41, 1007.

8. PAMPA: Parallel Artificial Membrane Permeability Assay
Passive Diffusion- Pe (cm/s)
Acceptor
Membrane
(20% (W/V) phospholipid mixture in dodecane)
Drug
Donor

9. PAMPA: Parallel Artificial Membrane Permeability Assay
Passive Diffusion- Pe (cm/s)
GastroIntestinal Tract (GIT)
Double-Sink Conditions
SDS micelles
(pH= 5-8)
Acceptor
(pH=7.4)
Membrane
Drug
Unstirred Water Layer
Donor

10. PAMPA: workstation

11. PAMPA: Sandwich plates
sink (acceptor at top)
phospholipid
cocktail
stirrer (donor at bottom)

12. Solubility-Diffusion Model / pH-Partition Theory
LogP
Passive diffusion: pKa, solubility and lipophilicity are important!

13. The effect of the pH on Permeability
Acid
Base
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;

14. The effect of the pH on Permeability
unionisable
Permeability of ionisable compounds is pH dependent!

15. Unstirred Water Layer and Ionisation
UWL:
In GIT: 40 mm
In BBB: no UWL
BBB
GIT
pKaflux
pH where 50% of the resistance to transport comes from the UWL and 50% from the membrane
UWL can be reduced by stirring the donor solution

16. 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

17. The Effect of lipophilicity on Permeability
Permeability improves with increase in lipophilicity

18. The Effect of lipophilicity on Permeability
Caco-2 Assay
Pe is UWL limited
Paracellular transport

19. AstraZeneca database Organon database
Caco-2 versus CLogP/D
AstraZeneca database
Organon database
y = x 2 x R =
-0.5
0.5 1
1.5 -1 3 4 5
cLogD7.4
logPapp
Membrane Retention
What is the Permeability hurdle, lipophilicity or solubility?
Riley et al, Curr. Drug Metab., 2002, 3, 527

20. The Effect of Solubility on Permeability: Co-Solvent PAMPA Assay (b)
SOl (ketoconazole)ACN:H2O=53 (pH 5); 37(pH 7.4); SOl (danazol)ACN:H2O=1 (pH 5); 3(pH 7.4)

21. The Effect of Solubility on Permeability-Co-Solvent PAMPA Assay
Pe is solubility limited
(Low Sol., but high Pe)
(High Sol., high Pe)
(High Sol., Low Pe)
Pex10-6 (cm/s)
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

22. The Effect of Solubility on Permeability-Co-Solvent PAMPA Assay (b)
Compound MW
ElogD (7.4)
Sol (pH 5)
Sol (pH 7.4)
Pe x10-6 (cm/sec)
(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
891.4 78
1361.1
364.5
862.5
Cpd 3
422
4,1
0.5
0.3
78.5
89.6
391.1
166.9
168.2
Cpd 4
529
5.2
< 0.1
0.2
879.5
173.3
216.4
Cpd 5
650 1
1.1
256.2
109.5
347.9
129.4
270.1
201.5 
Cpd 6
543 6
0.7
98.0
4.89
918.0
143.9

23. Ionisable compounds display Permeability- pH profile
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.

24. Max-Pe PAMPA Model for Prediction of Human Intestinal Absorption
high permeability
low permeability
Classification Pe
High
>20x10-6 cm/s
Medium
10- 20x10-6 cm/s
Low
<10x10-6 cm/s
a)- Avdeef, A., Absorption and Drug Development, 2003, Hoboken, NJ: Wiley-Interscience,
b)- Avdeef, A. Curr. Top. Med. Chem., 2001, 1, 277.

25. 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.

26. PAMPA Model for prediction the Human Jejunal Permeability (HJP)(a)
Double–Sink (pH=5.0/7.4) .
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.

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

28. Passive Diffusion Transport
Caco-2 vs. PAMPA
BCS compounds
Passive Diffusion Transport
Absorptive Transport
Secretory transport
Kerns, E.H., Di, L., Petusky, S., J. Pharm. Sci., 2004, 93, 6,1440.

29. Comparison of PAMPA and Caco-2 Permeability Assay Characteristics
Membrane composition
Phospholipid in alkane
Caco-2 cell monolayer
Permeability mechanism
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.

30. Strategy for Combined Use of PAMPA and Caco-2
Passive Diffusion
Passive, active,
influx, efflux
and paracellular
Mechanistic
Information
PAMPA
Caco-2
PAMPA + Caco-2
Exploratory
Discovery
Pre-Development
Development
Kern, E., (Wyeth Research), J. Pharm. Sci. 2004, 93, 6, 1440.

31. Acknowledgements Medicinal Chemistry Maarten Honing Marcel Hermkens
Michiel Scheffer
Department of Medicinal Chemistry

32. PSA Model for Prediction of Human Intestinal Absorption

33. Membrane keeps its integrity
Co-Solvent PAMPA Assay
Pex10-6 (cm/s)
Membrane keeps its integrity

34. Physicochemical properties of 309 NCEs with low and high bioavailability in rats
Bad two or more properties out the preferred range
Moderate one property out the preferred range
Most important properties are logP, Mw and rotatable bonds