The Effects of Assay Conditions on PAMPA Results · PDF fileThe Effects of Assay Conditions on...
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The Effects of Assay Conditions on PAMPA Results Kevin Chen, Ph.D. BD Biosciences August 12, 2009
Transcript of The Effects of Assay Conditions on PAMPA Results · PDF fileThe Effects of Assay Conditions on...
The Effects of Assay Conditions on PAMPA ResultsKevin Chen, Ph.D.
BD Biosciences
August 12, 2009
High Throughput Permeability Assay
• Drug candidates are screened for their oral-absorption potential early in the discovery and development phase, as a filter to remove poor performers and identify candidates that need to be modified.
• Most drugs are absorbed through the intestines without using cellular pumps. Therefore, passive permeability assays are useful for screening oral-absorption potential of drug candidates.
• Two permeability assays have become prevalent in recent years:– Cell-based (especially Caco-2) permeability assay– Parallel artificial membrane
permeability assay (PAMPA)
Caco-2 PAMPA
Type of model Cell monolayer Artificial membrane
Type of Permeability Measures the sum of passive and active permeability
Measures passive permeability in absence of transporters or efflux systems
Assay preparation Requires cell culture (up to 21 days)
Easy, fast preparation
Cost High Low
PAMPA and Caco-2 tests can be complementary tests to determine both passive and active permeabilities. Caco-2 tests alone measure the sum of passive and active permeabilities which can not be decoupled without the information obtained from PAMPA tests.
Caco-2 vs. PAMPA
Buffer Compound solutionMultiwell filter plate
Multiwell receiver plate
(Incubation for several hours)
Compounds permeate through membrane barrier
Compound concentration in acceptor well: CA (t)
Compound concentration in donor well: CD (t)
Permeability is calculated from CA (t) and CD (t)
PAMPA Experimental Setup
Existing PAMPA Methods
• Original PAMPA– The lipid solution consists of 10% lecithin in dodecane– Reference: Kansy, M. et al. (1998) J. Med. Chem. 41:1007-1010
• DOPC-PAMPA– The lipid solution consists of 2% DOPC in dodecane– Reference: Avdeef, A. et al. (2001) Eur. J. Pharm. Sci. 14:271-280
• HDM-PAMPA– The lipid solution is 100% hexadecane– Reference: Wohnsland, F. and Faller, B. (2001) J. Med. Chem. 44:923-930
• Bio-mimetic PAMPA (BM-PAMPA)– The lipid solution consists of a mixture of PC, PE, PS, PI and cholesterol in an organic solvent– Reference: Sugano, K. et al. (2001) Int. J. Pharm. 228:181-188
• Double-Sink™ PAMPA (DS-PAMPA), available from pION Inc– The lipid solution consists of 20% dodecane solution of a phospholipid mixture– The acceptor solution contains a surfactant mixture– Reference: Avdeef, A. et al. (2005) Eur. J. Pharm. Sci. 24:333-349
• BD Gentest™ Pre-coated PAMPA Plate, available from BD Biosciences– A lipid-oil-lipid tri-layer structure is constructed in the pores of the porous filter– Solvent contents are reduced by using volatile solvents in membrane preparation– Improves correlation with human absorption and Caco-2, and reduces mass retention– Reference: Chen, X. et al., (2008) Pharm. Res. 25:1511-1520
Lipids solution:Red: lipidsGreen: solvent
Filter plate
Pores
Traditional PAMPA Membrane
• A porous filter is soaked with a solution of lipids• The lipids are likely dispersed in the solvent• The PAMPA membrane is dominated by solvent, instead of lipids• The excess amount of solvent may become an extra barrier for the
compounds to permeate through
Filter plate
PoresRed: lipid layer
Green: oil layer
Structure of the BD Gentest PAMPA Membrane
• A lipid-oil-lipid tri-layer structure is constructed in the pores of the porous filter using three consecutive coating steps
• The oil layer mimics the hydrophobic interior of the biological membrane; and the amphiphilic lipids anchoring on the oil / water interface mimic the exterior of the biological membrane
• The oil layer is crucial for maintaining a robust and stable PAMPA membrane
• The oil layer is ultra thin to minimize compound retention and interference with the compound permeation
• Excessive solvents
• No structured layers
• Long permeation pathway
Traditional PAMPA membrane construction
New PAMPA membrane construction
Porous filter
• No excessive solvents
• Structured layers
• Short permeation pathway (closer to biological membrane)
Comparison of Traditional and BD Gentest PAMPA membrane
Traditional PAMPA membrane construction
New PAMPA membrane construction
Porous filter
Excessive solvents
No excessive solvents
Comparison of Traditional and BD Gentest PAMPA membrane
Type of drug Human absorption values (Fa%)
Low Permeability CompoundsAcyclovir guanosine analogue antiviral drug 20%Sulpiride anti-psychotic drug 35%Amiloride potassium-sparing diuretic 50%Ranitidine histamine H2-receptor antagonist 50%Terbutaline beta2-adrenergic receptor agonist 60%Furosemide loop diuretic 61%High Permeability CompoundsAtropine anticholinergic drug 90%Metoprolol selective beta1 receptor blocker 95%Verapamil antiarrhythmic drug 95%Propranolol non-selective beta blocker 99%Diltiazem anti-anginal drug 99%Imipramine antidepressant drug 99%Naproxen non-steroidal anti-inflammatory drug (NSAID) 99%Ketoprofen non-steroidal anti-inflammatory drug (NSAID) 100%Rifampicin bactericidal antibiotic drug 90%Antipyrine analgesic 100%Caffeine central nervous system (CNS) stimulant 100%
Drug Compounds Used in This Study
NaproxenpKa = 4.15
KetoprofenpKa = 4.45
Structure of the Acidic High Permeability Compounds used in This Study
Atropine MetoprololpKa = 9.6
VerapamilpKa = 8.92
Propranolol Diltiazem ImipraminepKa = 9.4
Structure of the Basic High Permeability Compounds Used in This Study
Rifampicin Antipyrine Caffeine
Structure of the Neutral High Permeability Compounds Used in This Study
Acyclovir Sulpiride Amiloride
Ranitidine Terbutaline Furosemide
Structure of the Low Permeability Compounds Used in This Study
Assay Conditions
• pH– Donor pH 7.4 (PBS buffer) / Acceptor pH 7.4 (PBS buffer)
– Donor pH 4.0 (acetate buffer) / Acceptor pH 7.4 (PBS buffer)
• Temperature– 26.5°C
– 37°C
Low permeabilityCompounds
Basic Compounds
Acidic Compounds
Neutral compounds
0
5
10
15
20
25
Acyc
lovi
r
Sulp
iride
Amilo
ride
Ran
itidi
ne
Terb
utal
ine
Furo
sem
ide
Atro
pine
Met
opro
lol
Vera
pam
il
Prop
rano
lol
Dilt
iaze
m
Imip
ram
ine
Nap
roxe
n
Keto
prof
en
Rifa
mpi
cin
Antip
yrin
e
Caf
fein
e
Donor pH7.4 / Acceptor pH7.4Donor pH4.0 / Acceptor pH7.4
Perm
eabi
lity
(10-6
cm/s
)
Permeability measured using BD Gentest™ Pre-Coated PAMPA Plate, at 26.5 °C
pH Dependence of Permeability (1)
0
5
10
15
20
25
Acyc
lovi
r
Sulp
iride
Amilo
ride
Ran
itidi
ne
Terb
utal
ine
Furo
sem
ide
Atro
pine
Met
opro
lol
Vera
pam
il
Prop
rano
lol
Dilt
iaze
m
Imip
ram
ine
Nap
roxe
n
Keto
prof
en
Rifa
mpi
cin
Antip
yrin
e
Caf
fein
e
Donor pH7.4 / Acceptor pH7.4Donor pH4.0 / Acceptor pH7.4
Perm
eabi
lity
(10-6
cm/s
)
Permeability measured using BD Gentest™ Pre-Coated PAMPA Plate, at 37.0 °C
Low permeabilityCompounds
Basic Compounds
Acidic Compounds
Neutral compounds
pH Dependence of Permeability (2)
0
5
10
15
20
25
Acyc
lovi
r
Sulp
iride
Amilo
ride
Ran
itidi
ne
Terb
utal
ine
Furo
sem
ide
Atro
pine
Met
opro
lol
Vera
pam
il
Prop
rano
lol
Dilt
iaze
m
Imip
ram
ine
Nap
roxe
n
Keto
prof
en
Rifa
mpi
cin
Antip
yrin
e
Caf
fein
e
26.5 C37.0 C
Low permeabilityCompounds
Perm
eabi
lity
(10-6
cm/s
)
Permeability measured using BD Gentest™ Pre-Coated PAMPA Plate, at pH 7.4
High permeabilityCompounds
Temperature Dependence of Permeability
0
20
40
60
80
100
0.0 5.0 10.0 15.0 20.0
acyclovir
sulpiride
amiloride, ranitidineterbutalinefurosemide
ketoprofen
metoprololrifampicin
naproxenantipyrine
verapamilcaffeine
propranolol atropinediltiazem
imipramine
0
20
40
60
80
100
0.0 10.0 20.0 30.0 40.0 50.0 60.0
Double-Sink PAMPA (1)
DOPC PAMPA (2)acyclovir
sulpiride
amiloride, ranitidineterbutaline
furosemide
atropinerifampicinketoprofennaproxenantipyrinecaffeine
propranolol
imipramine verapamil
diltiazemmetoprolol
Hum
an A
bsor
ptio
n (%
Fa)
Hum
an A
bsor
ptio
n (%
Fa)
Permeability (10-6 cm/s)
Compound Permeability Obtained Using BD Gentest Pre-Coated PAMPA Plate
Compound Permeability Obtained Using Other PAMPA Methods
Permeability (10-6 cm/s)
Correlation of Permeability with Human Absorption Data
Summary
• The permeability of the basic compounds decreases as the donor pH decreases from 7 to 4
• The permeability of the acidic compounds increases as the donor pH decreases from 7 to 4
• The permeability of the neutral compounds is relatively unchanged as the donor pH decreases from 7 to 4
• The permeability of most compounds increases as the incubation temperature increases
• The permeability of compounds with low human absorption (low Fa%) remains small in different conditions, indicating a good correlation between PAMPA and human absorption
• BD Gentest Pre-coated PAMPA Plate improves correlation with human absorption, as compared with other PAMPA methods
BD Gentest Pre-coated PAMPA Plate System
Contact Us
Questions?Contact information:Kevin Chene-mail: [email protected]
Technical Support:In the U.S.tel: 877.232.8995e-mail: [email protected] the U.S.Contact your local distributor or visitbdbiosciences.com/offices to locate your nearest BD Biosciences office.For research use only. Not intended for use in diagnostic or therapeutic procedures. BD, BD Logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2009 BD
