Download 4. Mechanisms of cellular drug uptake

4. Mechanisms of cellular drug uptake
Drug movements by simple diffusion
W.D. Stein. Institute of Life Sciences, Hebrew University, Jerusalem
91904, Israel
It is clear that a large number of drugs and chemotherapeutic substances get
into the target cell after crossing the cell's plasma membrane by simple
diffusion. Two factors determine the permeability of a cell membrane for a
particular drug: (i) the ability of the drug to dissolve in the cell membrane (i.e.,
its partition coefficient) and (ii) the rate at which the drug moves within the
membrane (i.e. its diffusion coefficient). Studies of the basal permeability of
the human red blood cell membrane enable one to sort out these two effects.
By comparing a set of permeability coefficients with the partition coefficients
for model solvents, one can determine which solvent is the best model for the
partitioning properties of the rate-limiting step for movement across the cell
membrane. It turns out that mono- and di-unsaturated hexadecanes model
best this barrier. Having ascertained the best model for the partitioning
behaviour of the membrane, one can attempt to calculate diffusion coefficients
within the membrane. These show a steep size-dependence, suggesting that
the membrane behaves as a thin polymeric sheet rather than as a simple
solvent phase. Applying this model to the movement of cytotoxins as large as
daunomycin suggests that the model applies also to this size range. But the
data on transport rates for such large molecules are also consistent with
movement by a flip-flop mechanism.Transport within the membrane can be
considered in terms of Eyring's activation energy model. The flip-flop model is
consistent with a scheme whereby one high energy barrier separates the two
half-bilayers. The continuum model posits that a number of smaller activation
energy barriers exist in series across the membrane. Partitioning into and out
of the membrane has consequences also for an activation energy model.
Binding of drugs to proteins in the plasma (such as serum albumen) can
drastically lower the concentration of free drug and hence greatly reduce
transport rates into the cell.
The multidrug resistance pump, P-glycoprotein appears to be able to reduce
the rate of influx of drugs into the cell, i.e. to decrease the membrane's
permeability to the drug. The 'vacuum cleaner' model for P-glycoprotein seems
to explain this behaviour.
Passive and active transport of drugs across the plasma
membrane of cells with active drug transport by ABC
transporters
A. Garnier-Suillerot. Laboratoire de Physicochimie Biomoleculaire et Cellulaire, Universite Paris Nord, 74 rue Marcel Cachin, 93017 Bobigny, France
Membrane proteins belonging to the ATP-binding cassette (ABC) family of
transport proteins play a central role in the defence of cells against toxic
compounds. Two human members of the ABC family have been identified that
can render mammalian tumor cells multidrug resistant: the MDR1 P-glycoprotein (Pgp) and the multidrug resistance protein (MRP1). Both proteins confer
drug resistance by active, ATP-dependent extrusion of a range of cytotoxic
drugs from the cell. Drug accumulation is the result of a balance between
passive permeability of the membrane and the effect of an efflux pump (Pgp or
MRP). In the search for new compounds able to overcome MDR, it is of prime
importance to determine the molecular parameters whose modifications would
lead to an increase in the influx and/or to a decrease in the P-glycoproteinmediated efflux. Several anthracyclines were used to analyse the respective
contribution of the influx and the transporter-mediated efflux in their impaired
accumulation in MDR cells (K562/Adr overexpressing P-gp and GLC4/Adr overexpressing MRP). For this purpose, a simple method based on a continuous
spectrofluorometric monitoring of the decrease of the fluorescence signal of
anthracyclines during incubation with living cells, was used. The influx of these
drugs varies over a very large range (for instance the influx of idarubicin is 400
times higher than that of doxorubicin). As can be expected, the influx increases
as the lipophilicity increases. In contrast, the efficiency of their P-gp (or by
MRP1) mediated transport, determined as the ratio V M (maximum rate)/K m
(Michaelis constant), is comparable. With this ratio being almost the same for
the different drugs, it follows that the intracellular free cytosolic drug concentration depends mainly on their passive influx. Therefore, the different resistance factors obtained for these drugs are mainly due to differences in passive
influx and not in P-gp-mediated or MRP-mediated efflux of these drugs.
The effect of plasma membrane transport on the intracellular free drug concentration
J. Lankelma. Free University Hospital, Dept. of Medical Oncology, Room BR
230, P.O. Box 7057,1007 MB, Amsterdam, The Netherlands
When the free drug concentration in the extracellular fluid (Co) is raised
stepwise, inward drug transport across the plasma membrane begins and the
cytosolic free drug concentration (C,) rises. C, is assumed to rapidly equilibrate
with the target concentration. If the molecules inside the cell distribute relatively
rapidly so that the local intracellular free drug concentration (C,) is the same,
everywhere in the cell, the cell can be described by a one-compartment model.
The time it takes to reach the steady state depends on the turnover time for
efflux. This turnover time (n) is proportional to the apparent cellular distribution
volume (Vd). Vd depends on binding and sequestration in endosomes/lysosomes. Assuming that C, and C o are the concentrations of the transportable
form of the molecule, only in the case of active drug pumping against a
concentration gradient (e.g. by P-glycoprotein or multidrug resistance protein)
will C, be less than Co at the steady state. When C , « KM and Michaelis-Menten
kinetics for drug pumping is followed, C|/Co can be calculated from the
dimensionless factor Vj^x/fKm.k) (in which k = permeation coefficient for
passive transport, expresssed in / (106 cells min)" 1 ). In addition to the available
methodology for measuring transport parameters we have developed a flowthrough-system and an in-cuvette transport assay for monolayer cells. With the
first method we can measure C, and P-glycoprotein mediated pumping rate
simultaneously. The latter method allows us to measure cellular efflux of a
broad range of fluorescence probes. Examples will be presented. A drug pump
can pump as a cytosolic drug pump and/or as a vacuum cleaner. The latter
manifests as decreased cellular influx, in which case the drug is pumped out of
a 'membrane-compartment'. At steady state, the contribution of lowering of Q
by both modes cannot be distinguished. However, in a dynamic situation, when
the extracellular drug concentration varies in time, the C, versus time profiles
are different for the two modes, and it appears that a vacuum cleaner is
especially effective in keeping Ci low.
C, can be increased by drug pump inhibitors (reversers). Studies on inhibition
of P-glycoprotein and multidrug resistance protein by reversers has indicated
competitive and non-competitive inhibition. Distinguishing between these two
mechanisms has not (yet) shown to be important for chemotherapy with blood
concentrations, which are most of the time under the KM for drug pumping.
More studies on the mechanisms of inhibition could lead to selective inhibitors
with interesting therapeutic applications. It could be of advantage to selectively
inhibit P-glycoprotein mediated drug transport, e.g. of the blood-brain-barrier.
Carrier mediated transport of anti-neoplasties and the
multiplicity of transport systems in mammalian cells
I.D. Goldman. Albert Einstein College of Medicine, Department of Medicine
and Molecular Pharmacology Comprehensive Cancer Center, Chanin Two,
1300 Morris Park Avenue, Bronx, NY 10461, U.S.A.
A variety of antineoplastics utilize carrier transport mechanisms to traverse
cells membranes. These agents are predominantly structural analogs of naturally occurring substrates that parasitize transport routes normally utilized by
these substrates. Important examples are transporters that mediate translocation of nucleosides and antifolates.
Transport carriers can be equilibrating or uphill. The former facilitate the
rapid translocation of substrates across the cell membrane but do not directly
utilize free energy to generate transmembrane gradients. Carriers that are
intrinsically equilibrating can, however, generate gradients when the flow of
one substrate is linked to energy-dependent flow of other substrates concentrated by a different transport mechanism. There are, in addition, transport
processes that are directly linked to the release of free energy in the hydrolysis
of ATP.
Transport of most antineoplastics is usually mediated by several transport
routes. Hence, unidirectional fluxes and steady-state levels achieved are
determined by the net effect of these processes. For antifolates, in particular,
uphill transport into the cell mediated by the reduced folate carrier is opposed
by a high capacity anion exporter.
The recent cloning of the reduced folate carrier has now permitted a very
careful analysis of mutations that occur under antifolate selective pressure that
result in impaired drug transport and consequent drug resistance. These
analyses have provided very important structure-function insights and clarify
how cells can lose the ability to transport a drug while preserving transport
capacity for the natural species required to sustain growth.
Finally, tranport systems control the rates of drug entry and exit from cells
along with the steady-state concentrations achieved. Each of these parameters
may play a critical role in determining drug efficacy based upon the relationship
between transport and the interaction between the drug and its target or an
intermediary catabolic or anabolic enzyme.