Biomedical Engineering Reference
In-Depth Information
10.9. P-GLYCOPROTEIN-MEDIATED DRUG TRANSPORT
The transport activity of Pgp can be studied in intact cells or in simpler subcellular
systems such as plasma membrane vesicles and reconstituted proteoliposomes. In
general, it has proved difficult to characterize the transport properties of Pgp in com-
plex intact cell systems. However, one exception to this has been the use of polarized
epithelial cells (such as MDCK, LLC-PK1, or Caco-2 cells) grown as monolayers on
permeable filters that allow separate access to the basal and apical compartments.
92
Transfection of Pgp results in expression of the protein at the apical surface, and
quantitative measurements of basal-to-apical and apical-to-basal fluxes of a drug can
be made.
93-95
This approach can be very useful for direct determination of whether
a drug is transported by Pgp, and showed that many MDR modulators are them-
selves transport substrates.
94
,
96-98
However, these cell lines may also show endoge-
nous expression of drug transporters, although at low levels, which may complicate
interpretation of experimental data.
Plasma membrane vesicles from MDR cells have been used extensively for mea-
surements of Pgp-mediated drug transport. Inside-out vesicles (present in variable
amounts in plasma membrane preparations) transport drug into the lumen when sup-
plied with ATP and an ATP-regenerating system.
54
,
56
,
99-103
Radiolabeled drugs such
as [
3
H]vinblastine, [
3
H]daunorubicin, or [
3
H]colchicine are usually employed. Some
early work purporting to measure drug-binding to membrane vesicles did not differ-
entiate between binding and transport, since ATP was included in the samples (at the
time it was not known if ATP was required for drug-binding). These studies probably
measured drug transport rather than ATP-dependent drug-binding. Osmotic sensitiv-
ity is a useful test to differentiate between transport and binding, and has been used for
both plasma membrane vesicles
54
,
99
,
100
and reconstituted systems.
54
One additional
difficulty is the high background levels of drug often observed for hydrophobic drugs
such as vinblastine.
54
In general, drug transport into plasma membrane vesicles or proteoliposomes is
saturable at high drug concentrations and requires ATP hydrolysis; nonhydrolyzable
analogs do not support transport. A drug concentration gradient is generated across the
membrane, which can usually only be estimated indirectly.
54
Drugs and modulators
block transport of other drugs with varying degrees of effectiveness. Reconstituted
proteoliposomes containing fully or partially purified Pgp have also been used to
characterize drug transport. An ATP-regenerating system is often not required, since
other membrane-bound enzymes do not deplete ATP. Similar approaches using radi-
olabeled substrates have been used to monitor transport in proteoliposomes. In this
more defined system, the magnitude of the drug concentration gradient was esti-
mated more precisely; Pgp built up a five- to six-fold gradient of colchicine
25
and
NAc-LLY-amide.
26
Real-time fluorescence assays can monitor the Pgp-mediated transport of fluores-
cent substrates continuously. H33342 is highly fluorescent when partitioned into the
membrane but loses fluorescence after export into the aqueous solution, allowing the
initial rate of movement of the dye out of plasma membrane vesicle to be quanti-
tated in real time.
104
The same system was used to demonstrate H33342 transport by
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