Comparison of P-gp surface densities and P-gp elementary kinetic rate
constants for binding and transport in Caco-2 and MDCKII-hMDR1-NKI
cells to extend in vitro/in vivo extrapolations (IVIVE).
Zhou Meng & Joe Bentz, Department of Biology, College of Arts & Sciences, Drexel University, Philadelphia, PA
Abstract
Results
Cells
[P-gp]
k1
2
AMP
QND
LPM
-1
kr
-1
k2
-1
kA
-1
P-gp
Pgp k1
Cinner monolayer
kr
Cim
KPC
Ccytosol
PBC
Passive
Passive
permeability
CB
-1
(Pgp / µm )
(M s )
(s )
(s )
(s )
Caco-2
180±40
(1.7±0.5)E+08
(4±1)E+04
12±2
0
0
MDCK
800±200
(1±0.4)E+08
(7±3)E+04
30±8
0
0
Caco-2
Same as AMP
Same as AMP
(7±2)E+03
1.2±0.2
0
0
MDCK
Same as AMP
Same as AMP
(4±2)E+03
3±0.4
0
0
Caco-2
Same as AMP
Same as AMP
(1±0.4)E+04
1.2±0.02
0
CBasolateral chamber
P-gp expressing cells
T0 + Cim
k1
DGX
MDCK
Same as AMP
Same as AMP
(2±1)E+04
0.4±0.08
0
kr
T1
B:B>A
1
A:A>B
B:A>B
Caco-2
Same as AMP
Same as AMP
(5.3±2.5)E+04
7.6±1.5
0+
MDCK
Same as AMP
Same as AMP
(3±1)E+04
3±1
0
2
4
6
Time (h)
37±3
40±20
40±3
1B
Table 1: Comparison of fitted consensus kinetic values between MDCKIIhMDR1 and Caco-2 cell monolayers. +Needed a time course >12hrs
1.6
1.4
Symbols: measured data
Dotted Lines: fits using the
only P-gp fitted kinetic
parameters.
Solid Lines: Fits using P-gp and
basolateral uptake transporter
fitted kinetic parameters
1 µM Digoxin
1.2
Efflux active
P-gp surface
density
Relative Pgp
expression by
western blot
mRNA
Absolute Pgp
expression by
LCMS
Predicted
number of efflux
active Pgp
P-gp/ µm
2
Copies of P-gp
mRNA /ng
Pgp expression
level by
densitometry
pmol Pgp/well
pmol Pgp/well
1
0.6
0.4
800±200
6748±?
25.1
0.65±0.25
0.5
Caco-2
180±40
918±?
26.2
0.8±0.3
0.1
0.2
Ratio of
MDCK/Caco-2
~4.6
~7
~1
~1
~5
0
0
+Same amount of protein was loaded for each cell line in gel electrophoresis.
Table 2: Correlation of active P-gp and total P-gp in MDCK-hMDR1 and
Caco-2 cell lines.
2
Time (h)
4
6
Figure 1: Transport data of amprenavir and digoxin over 6 hours and their
fitted curves across Caoco-2 cell monolayers.
For Caco-2 cells, only the
Pgp near the top of the
microvilli is efflux active.
Acharya et al.(2006) predicted that the efflux active P-gp would be less than the total
P-gp (LCMS data in Table 2) due to re-adsorption of previously effluxed P-gp
substrates by microvilli before they can diffuse to the aqueous apical chamber, since
substrates are amphipathic. The extent of re-adsorption would increase with increased
height and/or closer spacing between adjacent microvilli. For the MDCKII-hMDR1-NKI
cells, the reduction is a modest 30%. For the Caco-2 cells, the reduction is a whopping
8-fold. Following the same hypothesis, we would predict that the microvilli of the Caco2 cells are substantially taller and/or more closely spaced than those of the MDCKhMDR1-NKI cells.
Best fits with basolateral
uptake transporter
0.8
MDCKII-hMDR1
Conclusions
•
The similarity in P-gp elementary rate constants in two different cell lines shows these
can be extrapolated between systems.
•
In vitro to in vivo extrapolation (IVIVE) is typically based on the hybrid parameters Km,
Vmax and total Pgp present in plasma membrane. For P-gp present in micro-villous
membranes, only Pgp at the micro-villous tip succeeds in terminal removal of substrate
from cell, i.e. contributes to Vmax.
•
With the current emphasis on mechanistic PBPK modeling, the availability of the
elementary kinetic parameters and a measure of efflux active transporter level (which
constitute the hybrid Km and Vmax values) will support robust model building and
validation.
T0 + CA
Mass action
reaction
2
100±7
CA
k2
Only P-gp kr &k2 value
are required for fitting
23±3
PAC
CC
3
0
Structural Mass Action Kinetic Model (SMAKM)
CApical chamber
A:B>A
2µm Amprenavir
kB
+
k2
1A
Conc. (µM)
Drug
Efflux Active
Dissociation Efflux to
Other
P-gp
Association
from P-gp to
Apical Bidirectiona
Surface
to P-gp
Apical Bilayer Chamber l transporter
Density
Conc. (µM)
Purpose
Can elementary on-, off- and efflux rate constants (k1, kr and k2) to Pgp be extrapolated
between cell lines of different origin and for in vitro/in vivo extrapolation (IVIVE)?
Methods
Caco-2 cells were seeded on 12 well transwell plates and used for experiments 21 days after
seeding. Transport of amprenavir, quinidine, loperamide and digoxin was measured in the
basolateral-to-apical as well as apical-to-basolateral direction as a function of time and drug
concentration. P-gp surface density and elementary rate constants were fitted as described
in Agnani et al., PLoS-One, 2011. P-gp mRNA levels were determined by RT-PCR and P-gp
protein level was quantified by LC/MS/MS in MDCKII-hMDR1 and Caco-2 cells.
Results
The results show that the elementary rate constant for the above P-gp substrates obtained in
Caco-2 cells are very similar in magnitude (within 4 fold difference) to those obtained
previously in MDCKII-hMDR1 cells. Similarly, the total P-gp protein level is almost the same
between two cell lines. On the contrary, the fitted efflux active P-gp surface densities and
measured mRNA levels are substantially different between the cell lines. Efflux active P-gp
surface density was 4.5 fold and mRNA levels 7 fold lower in Caco-2 compared to the
MDCKII-hMDR1 cells.
Conclusion
The similarity in P-gp elementary rate constants in two different cell lines suggests that these
elementary rate constants can be extrapolated to in vivo. In vitro to in vivo extrapolation
(IVIVE) is typically based on the hybrid parameters developed from Michaelis-Menten theory
published over 100 years ago from the accepted mass action kinetic model for enzymology
(Vmax & Km), and appended to membrane transport without a clear idea of assumptions.
With the current FDA emphasis on mechanistic PBPK modeling, the availability of the
elementary kinetic parameters provide the ability to upgrade PBPK modellng.
4
References
Acharya, P., T. T. Tran, J. W. Polli, A. Ayrton, H. Ellens and J. Bentz.
Biochemistry. 45:15505-15519 (2006).
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