Transcript Permeability measurements PAMPA

Permeability: transporting drugs
through (lipid) membranes
Paula Garcia
1st Physical Chemistry Symposium, November 30, 2005
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
Permeation Mechanisms
Passive
Diffusion
Active
Transport
Paracellular
Efflux
Endocytosis
Cellular
Barrier
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.
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)
• In-silico models based on:





Lipophilicity
H-bond capacity
Molecular size
Polar Surface Area (PSA)
Quantum properties
Rule of five - Lipinski
Can any of these methods be adapted for high throughput measurements?
*2000, www.fda.gov.cder/guidance/index.html
Caco-2 Permeability Assay
human colonic cell line
apical
• Because they are made from cultured cells,
Caco-2 membranes express all mechanisms
of transport;
Drug
basolateral
• 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 days
4-day culture was recently reported: Int. J. Pharm. 2000, 200, 41.
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.
PAMPA: Permeability through lipid membranes
1998 - 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.
PAMPA: Parallel Artificial Membrane Permeability Assay
Passive Diffusion- Pe (cm/s)
Acceptor
Donor
Drug
Membrane
(20% (W/V) phospholipid mixture in dodecane)
PAMPA: Parallel Artificial Membrane Permeability Assay
Passive Diffusion- Pe (cm/s)
Acceptor
(pH=7.4)
SDS micelles
GastroIntestinal Tract (GIT)
Double-Sink Conditions
Unstirred Water Layer
Donor
(pH= 5-8)
Drug
Membrane
PAMPA: workstation
sink
stirrer
PAMPA: Sandwich plates
(acceptor at top)
(donor at bottom)
phospholipid
cocktail
Solubility-Diffusion Model / pH-Partition Theory
LogP
Passive diffusion: pKa, solubility and lipophilicity are important!
The effect of the pH on Permeability
-2.5
Ketoprofen
Acid
log P e
-3.5
O
-4.5
HO
pKa=3.98
-5.5
O
-6.5
4.0
2.0
8.0
pH 6.0
-2.0
Base
N
O
O
log Pe
O
HCl
pKa=9.07
Verapamil
-3.0
O
N
10.0
-4.0
-5.0
-6.0
-7.0
-8.0
2.0
4.0
6.0
pH
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;
8.0
10.0
The effect of the pH on Permeability
unionisable
Carbamazepine
-3.0
O
logPe
-3.5
-4.0
NH2
N
-4.5
-5.0
2.0
4.0
pH
6.0
8.0
10.0
Permeability of ionisable compounds is pH dependent!
Unstirred Water Layer and Ionisation
BBB
UWL:
In GIT: 40 mm
In BBB: no UWL
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
PAMPA Assay: DOUBLE-SINK and Stirring
1400,0
without stirring
with stirring
Pex10 -6 (cm/s)
1200,0
1000,0
800,0
Pe is UWL limited!
600,0
400,0
Log P ≥ 2
pH
Ranitidine
Antipyrine
Metoprolol.Tar
Ketoprofen
Carbamazepine
Propranolol
0,0
Verapamil
200,0
Log P < 2
Compounds with a log P ≥ 2, follow the protocol with stirring.
The Effect of lipophilicity on Permeability
-2
-2,5
-3
LogPemax
-3,5
-4
-4,5
-5
-5,5
-6
-6,5
-7
-0,3
0,2
0,7
1,2
1,7
2,2
2,7
3,2
3,7
4,2
4,7
5,2
5,7
6,2
logP
Permeability improves with increase in lipophilicity
The Effect of lipophilicity on Permeability
0
Caco-2 Assay
-1
-2
Pe is UWL limited
Paracellular transport
LogPapp
-3
-4
-5
-6
-7
-8
-0,10
0,40
0,90
1,40
1,90
2,40
2,90
3,40
logP
3,90
4,40
4,90
5,40
5,90
6,40
Caco-2 versus CLogP/D
AstraZeneca database
Organon database
2
800
2
y = -0.2183x + 0.8639x + 0.4508
R 2 = 0.5362
700
Caco-2 (nm/s)
logPapp
1.5
600
500
Membrane Retention
1
0.5
400
300
200
100
0
-1
0
1
2
3
4
0
5
-2 -1
-0.5
cLogD7.4
0
1
2
3
4
5
6
cLogP
What is the Permeability hurdle, lipophilicity or solubility?
Riley et al, Curr. Drug Metab., 2002, 3, 527
7
8
9
10 11
The Effect of Solubility on Permeability: Co-Solvent PAMPA Assay (b)
Compound
logP
Solubility
Solubility
pH
Pe x 10-6 (cm/sec)
H2O H2O: ACN (8:2)v/v
5
6,2
7,4
5
6,2
7,4
5
6,2
7,4
2,1
2,1
2,3
2,6
2,6
2,5
1,2
1,4
1,1
1,2
1,6
1,6
0,8
0,7
0
0,4
0,5
0,3
5
6,2
7,4
5
6,2
7,4
189
763
1707
264
20
4
12
96
739
123
14
3
5
6,2
7,4
5
6,2
7,4
496
und
0
0
0
64
68
1254
equ
1754
819
419
pH 5 (mg/ml) pH 7.4 (mg/ml)
Antipyrine
0,56
> 197
> 194
-0,07
> 20000
> 20000
0
> 189
> 186
Caffeine
Theophylline
Verapamil.HCl
4,33
> 500
> 500
3,16
> 259
> 262
Ketoprofen
Ketoconazole
4,34
43
11
3,9
0,09
0,06
Danazol
SOl (ketoconazole)ACN:H2O=53 (pH 5); 37(pH 7.4); SOl (danazol)ACN:H2O=1 (pH 5); 3(pH 7.4)
The Effect of Solubility on Permeability-Co-Solvent PAMPA Assay
Pe is solubility limited
(Low Sol., but high Pe)
H2O
(High Sol., high Pe)
H2O:ACN (8:2 v/v)
1800
1600
Pex10-6 (cm/s)
1400
1200
(High Sol., Low Pe)
1000
800
600
400
200
H2O
0
Danazol
Ketoconazole
Ketoprofen
Verapamil.HCl
Theophylline
Caffeine
Antipyrine
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
Cpd 1
Cpd 2
Cpd 3
Cpd 4
Cpd 5
Cpd 6
MW
539
555
422
529
650
543
ElogD
(7.4)
4.6
4.4
4,1
5.2
4.4
6
Pe x10-6 (cm/sec)
Sol (pH 5)
Sol (pH 7.4)
(µg/ml)
(µg/ml)
pH
H2O
H2O/ACN (8:2 v/v)
55
7
5.0
1137.6
57.4
6.2
1875.1
331.3
7.4
0.0
916.7
5.0
891.4
78
6.2
1361.1
364.5
7.4
0.0
862.5
5.0
78.5
89.6
6.2
391.1
166.9
7.4
0.0
168.2
5.0
0.0
879.5
6.2
0.0
173.3
7.4
0.0
216.4
5.0
256.2
109.5
6.2
347.9
129.4
7.4
270.1
201.5
5.0
0.0
0.0
6.2
98.0
4.89
7.4
918.0
143.9
> 58
0.5
< 0.1
1
0.5
8.9
0.3
0.2
1.1
0.7
PAMPA Assay in Organon
 Ionisable compounds display Permeability- pH profile
 Lipophilicity ↔ Permeability
• Reducing the UWL is important for lipophilic compounds.
• Highly lipophilic compounds display a high membrane retention.
 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.
a)- Kern, E. et al., Pharm. Sci., 2004, 93, 6, 1440;
b)- Bermejo, M. et al., Eur. J. Pharm. Sci., 2004, 21, 429;
d) Avdeef, A., et al., Chem. Pharm. Bull., 2004, 52, 561; Sugano, K., et al., Int. J. Pharm., 2001, 181.
Max-Pe PAMPA Model for Prediction of Human Intestinal Absorption
110
Caffeine
100
Theophiline
Classification
Dexamethasone
% HIA
Verapamil
Desipramine
cm/s
Quinine
10- 20x10-6
cm/s
<10x10-6 cm/s
Medium
Furosemide
Ranitidine
Tertbutaline
50
Atenolol
30
-7,00
Testosterone
Propanolol
Hydrocholorthiazide
60
40
Ketoprofen
Pe
>20x10-6
High
80
Piroxicam
Metoprolol
Antipyrine
90
70
Carbamazepin
Low
low permeability
-6,50
-6,00
high permeability
-5,50
-5,00
-4,50
-4,00
-3,50
log Pe Max (pH 5.0, 6.2, 7.4)
a)- Avdeef, A., Absorption and Drug Development, 2003, Hoboken, NJ: Wiley-Interscience,
b)- Avdeef, A. Curr. Top. Med. Chem., 2001, 1, 277.
-3,00
-2,50
-
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)
-2,0
y = 0,4731x - 1,4404
2
R = 0,9408
-2,5
Verapamil
Piroxicam
-3,0
Antipirine
Naproxen
Carbamazepine
-3,5
Desipramine
HJP
Log Pe
Ketoprofen
Propanolol
-4,0
Metoprolol
Tertbutaline
-4,5
Ranitidine
Atenolol
-5,0
-5,5
Furosemide
Hydrochlorotiazide
-6,0
-8,5
-7,5
-6,5
-5,5
log Pe
-4,5
-3,5
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.
-2,5
Permeability and Molecular Properties
Compound
ClogP
MW
Nb Rot
HBD
(-2<X<5)
(X<500)
(X<12)
(X<6)
3,8
314
1
0
HBA
PSA
In-Silico
(X<11) (X<110)
2
31,4
274
0
1
2
38,1
362
2
3
5
78,3
360
2
2
5
77,1
458
3
2
3
55,2
197
3
5
5
108,0
455
14
0
6
61,8
650
13
1
9
114,0
18
26
34
1185
20
0
0
1188
15
Bad
Cpd 2
1600
5.2
Cpd 3
28
Moderate
Verapamil
3.0
35
Moderate
L-Dopa
4,5
422
Moderate
Cpd 1
-2,8
20
Good
Cortisone
5,9
1879
Good
Hydrocortisone
1,3
x10-6
Good
Nandrolone
1,7
x10-6
Good
Progesterone
2,7
PAMPA Caco-2
557
12
0
10
102,0
Bad
500
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
Passive Diffusion Transport
Absorptive Transport
-3.20
-3.70
y = 0.6155x - 2.5577
R2 = 0.8032
Antipyrine
Naproxen
-4.20
Metoprolol
Log Pe Caco-2
Caffeine
Propanolol
Ketoprofen
Verapamil
-4.70
Quinine
Carbamazepine
Desipramine
Theophiline
-5.20
-5.70
-6.20
Secretory transport
Ranitidine
Hydrochlorotiazide
-6.70
Furosemide
-7.20
-7.00
-6.50
-6.00
-5.50
-4.50
-5.00
Log Pe
Max (pH 5.0, 6.2, 7.4)
Kerns, E.H., Di, L., Petusky, S., J. Pharm. Sci., 2004, 93, 6,1440.
-4.00
-3.50
-3.00
-2.50
-2.00
Comparison of PAMPA and Caco-2 Permeability Assay Characteristics
Characteristics
PAMPA
Caco-2
Membrane 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
Passive, active,
influx, efflux
and paracellular
Passive Diffusion
Mechanistic
Information
PAMPA
Caco-2
PAMPA + Caco-2
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
PSA Model for Prediction of Human Intestinal Absorption
110
100
Piroxicam
Cortisone
Dexamethasone
90
% HIA
80
PSA> 62, poorly absorved
Hydrochlorotiazide
70
Tertbutaline
PSA< 62, highly absorved
60
Furosemide
50
Atenolol
RAnitifine
40
30
10
20
30
40
50
60
PSA
70
80
90
100
Co-Solvent PAMPA Assay
700,0
Pex10-6 (cm/s)
600,0
500,0
water
400,0
water:ACN (8:2)
300,0
200,0
100,0
0,0
5,0
6,2
Ketoprofen
7,4
5,0
6,2
Verapamil
7,4
5,0
6,2
7,4
Antipyrine
Membrane keeps its integrity
number of compounds
Physicochemical properties of 309 NCEs with low
and high bioavailability in rats
150
≤30%
125
>30%
100
75
50
25
0
Bad*
Moderate*
Good
Monika prediction
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