The use of Tris-lipidation to modify drug cytotoxicity in multidrug resistant cells expressing P-glycoprotein or MRP1

Article abstract of DOI:10.1038/sj.bjp.0704983

1. Increasing the lipophilicity is a strategy often used to improve a compound's cellular uptake and retention but this may also convert it into a substrate for an ATP-dependent transporter such as P-glycoprotein or the multidrug resistance-associated pro

Full text of DOI:10.1038/sj.bjp.0704983

British Journal of Pharmacology (2002) 137, 1280 ± 1286
ã
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The use of Tris-Lipidation to modify drug cytotoxicity in multidrug
resistant cells expressing P-glycoprotein or MRP1
2
2
3
4
*,1Ross A. Davey, Mary W. Davey, Karen V. Cullen, Xanthe E. Wells, Craig L. Francis,
3Hua-Ming Williams, Qi Yang, Minoo J. Moghaddam, Fred Widmer & Robert G. Whittaker
4
3
3
3
2
1Bill Walsh Cancer Research Laboratories, Royal North Shore Hospital, St Leonards, NSW, 2065, Australia; Cell and
Molecular Biology Department, University of Technology, Sydney, Gore Hill, NSW, 2065, Australia; CSIRO Molecular
3
4
Science, Delhi Rd, North Ryde, NSW, 1670 Australia and CSIRO Molecular Science, Bayview Ave, Clayton, VIC, 3169,
Australia
1
Increasing the lipophilicity is a strategy often used to improve a compound's cellular uptake and
retention but this may also convert it into a substrate for an ATP-dependent transporter such as P-
glycoprotein or the multidrug resistance-associated protein (MRP1), which are involved in cellular
e‚ux of drugs. Tris-Lipidation of compounds is a convenient way of modifying drug lipophilicity
and generating an array of derivatives with diverse properties.
2
To determine the e€ect of Tris-Lipidation on a drug's cytoxicity in multidrug resistant cells,
various glycyl-Tris-mono- (GTP1), di- (GTP2) and tri-palmitate (GTP3) derivatives were prepared
of the cancer chemotherapeutic drugs chlorambucil and methotrexate, and of the anti-HIV drug
AZT. The cytotoxicity of these derivatives and their parent compounds was determined in the CEM/
VLB100 cells with increased P-glycoprotein expression, the CEM/E1000 cells that overexpress MRP1
and the parent, drug-sensitive CCRF-CEM cells.
3
Increasing the lipophilicity of AZT increased its cytotoxicity in the sensitive CCRF-CEM
parental cell line while decreased cytotoxicity was observed for the methotrexate derivatives. For the
chlorambucil derivatives, both increased (GTP1) and decreased (GTP2) cytotoxicity occurred in the
CCRF-CEM cells. With the exception of AZT-GTP1, all GTP1 and GTP2 derivatives of
chlorambucil, methotrexate and AZT had decreased cytotoxicity in the P-glycoprotein-expressing
CEM/VLB100 cells while chlorambucil-GTP1, methotrexate-GTP2 and methotrexate-GTP3 were the
only compounds with decreased cytotoxicity in the MRP1-overexpressing CEM/E1000 cells.
4
The number of palmitate residues, the position of derivatisation and the type of linkage all may
a€ect the P-glycoprotein and MRP1 substrate properties.
Tris-Lipidation may therefore provide a useful way of manipulating the pharmacokinetic
5
properties of drugs.
British Journal of Pharmacology (2002) 137, 1280 ± 1286. doi:10.1038/sj.bjp.0704983
Keywords: Tris-Lipidation; P-glycoprotein; MRP1; drug modi®cation; chlorambucil; methotrexate; AZT; leukaemia cells;
multidrug resistance
Abbreviations: AZT, 3'-azido-3'-deoxythymidine (Zidovudine); BSO, buthionine sulphoximine; GT, glycine-Tris; GTPn,
glycine-Tris-palmitate (1, 2 or 3); HPLC, High Performance Liquid Chromatography; MRP1, multidrug
resistance-associated protein 1; MTT, 3[-4,5 dimethylthiazo]-2,5 diphenyl tetrazolium bromide; SDZ-PSC-833,
Cyclosporin-D derivative produced by Novartis; Tris, 2-amino-2-hydroxymethyl-1,3-propanediol; Z, benzyloxy-
carbonyl
Introduction
The rates of tissue uptake and e‚ux of a drug are important
factors in determining both its speci®city and its e€ective
dose. At the cellular level, uptake and e‚ux are often
in¯uenced by the ABC super-family members of ATP-
dependent transport proteins. The clinical importance of
these transporters is very evident from the role of two family
members, P-glycoprotein (Endicott & Ling, 1989) and the
multidrug resistance-associated protein (MRP1, Cole et al.,
1992) in causing resistance to a wide variety of chemother-
apeutic drugs used to treat cancer (Goldstein et al., 1992;
Borst et al., 2000). It is therefore important in designing new
drugs, to establish whether they are substrates for these
transporters. As more becomes known about the substrate
speci®city and the tissue and cellular location of the ABC
super-family of transporters, it may be possible to engineer
the transportability of a drug to enhance its pharmacokinetic
properties.
Increasing the lipophilicity of a drug is a common strategy
to enhance cellular uptake. However, this approach can be
unsuccessful because it also can produce a more e€ective
substrate for the e‚ux transporters such as P-glycoprotein
and MRP1. The balance between the rate of cellular uptake
and e‚ux is probably dictated by the properties of the
lipophilic groups. However, because there is little under-
standing of which of these properties are important, it is
*Author for correspondence; E-mail: rdavey@med.usyd.edu.au

R.A. Davey et al
P-glycoprotein and MRP1 substrates and Tris-Lipidation
1281
advantageous to use a chemistry capable of generating and
conjugating a diverse array of lipophilic structures.
modi®ed by the addition of fatty acyl groups via Tris to
engineer the desired lipophilicity, solubility, stability, shape
and molecular weight. In addition, pro-drugs or new
chemical entities can be produced by varying the linker.
Despite the advantages of this approach, little is known
about the e€ect that Tris-Lipidation has on the capacity to be
transported by members of the ABC super-family. P-
glycoprotein and MRP1 are the best characterized members
of this family and they are individually overexpressed in the
CEM/VLB100 (Beck et al., 1979) and CEM/E1000 sublines
respectively, relative to the parental CCRF-CEM human T-
cell leukaemia cell line which does not express detectable
levels of P-glycoprotein and expresses a low constitutive level
of MRP1 typical of the majority of human cell lines (Davey
et al., 1995). The drug accumulation characteristics of these
cells and the e€ects of the P-glycoprotein inhibitor, SDZ-
PSC-833 and the MRP1 inhibitor, buthionine sulphoximine
(BSO) have also been well characterized (Davey et al., 1996).
The CEM/VLB100 and CEM/E1000 sublines have been used
previously to investigate the multidrug resistance character-
istics of drugs (Davey et al., 1997, E€erth et al., 2002). They
are ideally suited for this as they have similar levels of
resistance to many lipophilic and natural product drugs, their
multidrug resistance is stable in the absence of continual drug
treatment, they are the same size, they have similar growth
rates and they are derived from the same parental cell line by
selection rather than transfection (Beck et al., 1979; Davey et
al., 1995).
Liposomal formulations and chemical modi®cations, such
as the addition of fatty acyl groups, are commonly used to
increase the lipophilicity of a drug. Fatty acyl groups have
usually been introduced through the addition of di- and
mono-glycerides. Whilst these conjugates have enhanced
biological properties, the chemistry to prepare them is often
dicult and problems arise in scale-up because glycerol is an
unsymmetrical molecule.
We have previously reported that Tris (2-amino-2-(hydro-
xymethyl)-1,3-propanediol) is easily linked to peptides and
other compounds (Whittaker et al., 1993). Tris is accepted for
use by the pharmaceutical industry and is relatively
inexpensive, non-toxic and readily available. Tris has
structural similarities to glycerol but has the advantage of
being symmetrical around the central carbon atom. This
allows for the easy preparation and puri®cation of mono-, di-
and tri-glyceride mimics because structural isomers are
avoided. Compounds can be readily attached to the amine
group, with or without the use of linkers, to give conjugates
of the general structure shown in Figure 1. Tri-fatty acyl
esters can also be formed using Tris. This is not possible with
glycerol. The chemical properties of a drug can be easily
To determine the e€ect that Tris-Lipidation has on a
drug's potential to be transported by either P-glycoprotein
or MRP1, we have chosen three drugs that are considered
to have little interaction with P-glycoprotein or MRP1, the
anti-cancer alkylating drug chlorambucil, the anti-cancer
folate pathway inhibitor, methotrexate and the nucleoside
reverse transcriptase inhibitor, AZT (3'-azido-3'-deoxythymi-
dine; Zidovudine). These were conjugated to various Tris-
palmitate adducts (Figure 1) and their cytotoxicity against
the P-glycoprotein-expressing CEM/VLB100 and the MRP1-
overexpressing CEM/E1000 multidrug resistant sublines was
determined relative to the drug sensitive parental CCRF-
CEM cells. The e€ects on cytotoxicity of the P-glycoprotein
inhibitor SDZ-PSC-833 and the MRP1 inhibitor BSO
(Davey et al., 1995; Grech et al., 1998) were also
determined.
Methods
Figure 1 presents the general structure of the drug-glycine-
Tris-palmitate conjugates and the structural formulae for
conjugates of chlorambucil, methotrexate and AZT.
Benzyloxycarbonyl-GTPn derivatives were synthesized as
previously described (Whittaker & Bender, 1992; Whittaker
et al., 1993; 1999). This was then conjugated to the various
drugs as described below.
Chlorambucil-Tris conjugates
Chlorambucil-Tris conjugates were prepared as previously
described (Whittaker et al., 1995). Brie¯y, the N-hydro-
xysuccinimide-derived active ester of chlorambucil (Sigma
Chemical Co., U.S.A.) was prepared and reacted with GTP1
Figure 1 Structure of Tris-Lipid drug conjugates. The general
structure (R=fatty acyl group or OH) and the structure of the
Tris-Lipid conjugates of chlorambucil, AZT and methotrexate are
presented.
British Journal of Pharmacology vol 137 (8)

1282
R.A. Davey et al
P-glycoprotein and MRP1 substrates and Tris-Lipidation
or GTP2. Products were puri®ed from the reaction mixture
by column chromatography and/or HPLC.
Results
The GTP1 and GTP2 moieties themselves showed little
cytotoxicity to the CCRF-CEM cells with an IC50 in excess
of 20 mM for both compounds (not shown). However, the
conjugation of these compounds to the three drugs, chlor-
ambucil, methotrexate or AZT, dramatically altered the
cytotoxicity of these drugs in the CCRF-CEM cells (Figure 2
and Table 1). GTP1 conjugation increased the cytotoxicity of
chlorambucil by 10 fold and AZT by 50 fold and decreased the
cytotoxicity of methotrexate by greater than 10 fold. Conjuga-
tion to GTP2 decreased the cytotoxicity of chlorambucil by
twofold, methotrexate by 20 fold and it increased the
cytotoxicity of AZT by 50 fold. For methotrexate, the parent
drug was more cytotoxic than all the derivatives tested while for
AZT the parent drug was less cytotoxic than the GTP1 and
GTP2 conjugates.
Methotrexate-Tris conjugates
Methotrexate was a gift from FH Faulding & Co. Limited,
Australia and the Tris conjugates were prepared as previously
described (Whittaker et al., 1999). Brie¯y, modi®ed glutamic
acid (carboxyl groups were selectively esteri®ed-a-t-butyl and
g-methyl) was attached to 4-amino-4-deoxy-N10-methyl
pteroic acid (Sigma Chemical Co.) followed by selective
methyl ester hydrolysis (Rosowsky et al., 1981). This
liberated the g-COOH site for the attachment of GT,
GTP1, GTP2 or GTP3 using a DCC-mediated coupling
reaction. The a-ester was subsequently cleaved. This
produced conjugates with an amide linkage on the g-COOH.
Conjugates linked through the a-COOH were produced
through the addition of GT or GTP1 to the a-COOH
following selective removal of the a-t-butyl group. Products
were puri®ed by chromatography.
AZT-Tris conjugates
AZT conjugates were prepared as previously described
(Whittaker et al., 1995) from AZT produced according to
the method of Czernecki & Valery (1991) AZT was treated
with succinic anhydride to form AZT-succinate. The active
ester was prepared and reacted with either GTP1 or GTP2.
Final compounds were isolated by column chromatogra-
phy.
Cell culture
The CCRF-CEM human leukaemia cell line (Foley et al.,
1965), the P-glycoprotein expressing CEM/VLB100 subline
(Beck et al., 1979) and the MRP1 overexpressing CEM/
E1000 subline (Davey et al., 1995) were grown in RPMI-1640
medium (Trace, Sydney, Australia) supplemented with 10%
foetal calf serum (Trace), 20 mM HEPES (Trace) and 10 mM
NaHCO3 at 378C in a humidi®ed atmosphere containing 5%
CO2. The sublines were maintained in the absence of the
selecting drug except for treatment for 3 days every 8 weeks.
All cultures were free of Mycoplasma as determined by
Mycoplasma-speci®c culture (Westmead Hospital Microbiol-
ogy Department, Sydney, Australia).
Cytotoxicity assay
To determine the cytotoxicity of the drugs and their
derivatives, cells were plated out in triplicate in 96-well
plates at a density of 2.56105/ml and cultured in the
presence of the test compound for 4 days after which cell
viability was measured by the MTT assay as previously
described (Marks et al., 1992). The IC50 was determined as
that concentration that caused a 50% decrease in cell
viability. The relative resistance was calculated by dividing
the IC50 of a compound in the resistant subline by the IC50 in
the sensitive CCRF-CEM cell line. Relative reversal was
determined by dividing the IC50 of a compound by the IC50
obtained in the presence of the reversing agent, either 50 mM
BSO (Sigma) or 10 mM SDZ-PSC-833 (gift from Novartis,
Sydney, Australia).
Figure 2 E€ect of drug modi®cation on cytotoxicity in the CCRF-
CEM cells. Cells were incubated continuously in drug for 4 days after
which the cell viability was determined using the MTT assay as
described in the Methods. Assays were performed in triplicate and
the mean and standard deviation of each determination are shown. P,
Parent drug; a-GT, a-glycine-Tris; g-GT, g-glycine-Tris; GTP1,
glycine-Tris-monopalmitate; GTP2, glycine-Tris-dipalmitate; GTP3,
glycine-Tris-tripalmitate.
British Journal of Pharmacology vol 137 (8)

R.A. Davey et al
E€ect of derivitization on the cytotoxicity of
chlorambucil, methotrexate and AZT in multidrug resistant
cells
P-glycoprotein and MRP1 substrates and Tris-Lipidation
1283
Table
1
methotrexate resistance was not detected with shorter drug
exposure times of 1, 4 or 24 h (data not shown).
IC50 mM (fold-resistance)
CCRF-CEM CEM/VLB100 CEM/E1000
Drug
Discussion
Chlorambucil
Chlorambucil-GTP1
Chlorambucil-GTP2
3.4
0.35
6.8
5.2
14.6 (42)
4200 (425)
2.2
1.7 (5)
9.3
Tris-Lipidation allows the preparation of a range of lipophilic
derivatives of a wide range of therapeutic agents. The method
is simple and very versatile. Varying the number and/or type
of fatty acyl group attached, as well as the nature of the
linkage to the drug, can produce a vast array of di€erent
properties. Tris-Lipidation has already been applied to a
range of applications including the delivery of genes
(Cameron et al., 1999), vaccines (Reilly et al., 1991; Walker
et al., 2000) and therapeutic agents (Whittaker et al., 1995).
The versatility of Tris-Lipidation was also evident in our
study. Compared to chlorambucil, the GTP1 derivative was
10 fold more cytotoxic in the parental CCRF-CEM cells
while the GTP2 derivative had half the cytotoxicity of
chlorambucil. Derivatization also had a dramatic e€ect on
chlorambucil's cytotoxicity in the P-glycoprotein-expressing
CEM/VLB100 cells which were 42 fold resistant to the GTP1
derivative and 425 fold resistant to the GTP2 derivative.
Increasing the lipophilicity of AZT by adding GTP1 and
GTP2 groups caused increased cytotoxicity (greater than 50
fold) and this has previously been reported for other cell lines
(Wells et al., 1999). The strategy of increasing the
lipophilicity is often used to increase the uptake and ecacy
of a compound. Our results show that while increasing the
lipophilicity by conjugation with Tris-palmitates increased the
cytotoxicity of AZT, this was not the case for the other two
drugs (Figure 2). The GTP1 conjugate of chlorambucil was
more cytotoxic, however, chlorambucil-GTP2 was less
cytotoxic than the parent drug. All methotrexate-conjugates
were less cytotoxic.
Processes other than cellular uptake and retention are also
important in determining the cytotoxicity of a compound.
This includes the intracellular modi®cation by conjugation to
compounds including glutathione, glucuronate, sulphonate
and polyglutamate. In the case of methotrexate, the g-
carboxyl group is the site of polyglutamylation, a process
important in cellular drug retention and enzyme binding.
Modi®cation of the g-carboxyl group would therefore reduce
the cytotoxicity and our results are consistent with this as all
methotrexate-g-conjugates were less cytotoxic than the parent
compound (Figure 2). This con®rms the earlier observations
of Wells et al. (1999). Our results also show that modi®cation
to the a-carboxyl group is more disruptive than changes to
the g-carboxyl group since the a-GT conjugate was far less
cytotoxic than the g-GT conjugate of methotrexate (Figure
2). This demonstrates that the site of conjugation is also
important in determining drug cytotoxicity.
Methotrexate
0.017
36
0.57
0.21
0.4
0.045 (3)
164 (5)
1.2 (2)
1.2 (6)
2.0 (5)
99
0.015
48
0.52
0.27
0.7 (2)
Methotrexate-a-GT
Methotrexate-g-GT
Methotrexate-g-GTP1
Methotrexate-g-GTP2
Methotrexate-g-GTP3
94
202 (2)
AZT
AZT-GTP1
AZT-GTP2
5000
93
100
5000
80
227 (2)
6000
80
101
Cells were incubated continuously in drug for 4 days after
which the viability was determined using the MTT assay and
the fold resistance ( ) calculated as described in Methods.
The e€ect of Tris-Lipidation on drug cytotoxicity was
determined in the P-glycoprotein expressing CEM/VLB100
cells relative to that in the CCRF-CEM cells. Figure 3 shows
that the CEM/VLB100 cells were 42 fold more resistant to
chlorambucil-GTP1 than the parental CCRF-CEM cells. The
CEM/VLB100 cells were also greater than 25 fold resistant to
the GTP2 derivative. The P-glycoprotein inhibitor, SDZ-
PSC-833 reversed the resistance 14 fold for chlorambucil-
GTP1 and greater than 20 fold for chlorambucil-GTP2 in the
CEM/VLB100 cells which provided further evidence that these
derivatives were probably interacting with P-glycoprotein.
AZT-GTP2 also appears to interact with P-glycoprotein since
the CEM/VLB100 cells were 2 fold more resistant than the
CCRF-CEM cells and SDZ-PSC-833 reversed this resistance
(Figure 3 and Table 1).
The CEM/VLB100 cells were 6 fold more resistant to
methotrexate-g-GTP1 and 5 fold more resistant to metho-
trexate-g-GTP2 compared to the CCRF-CEM cells while the
CEM/VLB100 cells were not resistant to methotrexate-g-
GTP3 (Table 1). This suggests that if the addition of -g-GTP1
and -g-GTP2 to methotrexate promotes interaction with P-
glycoprotein, the addition of a further palmitate residue
reverses this e€ect. We also examined whether or not the
addition of GT (with no palmitate(s) attached) to the a- or g-
carboxyl groups of methotrexate a€ected cytotoxicity.
Addition of both the -a-GT and -g-GT increased the
resistance in the CEM/VLB100 cells 5 and 2 fold respectively
(Table 1).
The e€ect of Tris-Lipidation on drug cytotoxicity in the
MRP1-overexpressing CEM/E1000 cells, relative to that in
the CCRF-CEM cells, was determined. Of the derivatives
tested, chlorambucil-GTP1 had the highest relative resistance
(5 fold) in the CEM/E1000 cells and this was reversed
(7 fold) by BSO treatment (Figure 4). Relative to the
CCRF-CEM cells, methotrexate-GTP2 and -GPT3 were also
less cytotoxic in the CEM/E1000 cells (Table 1).
The GTP2 derivative was less cytotoxic than the GTP1
derivative for all three parent compounds and for metho-
trexate, the GTP3 derivative was less cytotoxic than the
GTP2 derivative. This trend of decreasing cytotoxicity with
increasing number of palmitate groups could be because an
increase in the number of palmitate residues may reduce the
rate of transport into cells. Alternatively the increased
lipophilicity may reduce solubility and consequently decrease
availability to the cells. Using ¯uorescent labelled conjugates,
we have previously observed that the GTP1 conjugates enter
It is also worth noting that the CEM/E1000 cells were not
resistant to the parent drug, methotrexate under these
conditions of 4 day drug exposure (Table 1). Further,
British Journal of Pharmacology vol 137 (8)

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R.A. Davey et al
P-glycoprotein and MRP1 substrates and Tris-Lipidation
The stability of the linkage between the drug and the
adduct is probably important in determining the drug's
cytotoxicity. In the case of the AZT, the Tris-palmitate
moieties are attached via an ester linkage which is more labile
due to cellular esterases than the amide link used for the
chlorambucil and methotrexate derivatives. Once inside the
cell, the AZT derivative would be quickly converted to free
AZT which is not as likely to be actively e‚uxed from the
cell. This is consistent with the dramatic 50 fold increase in
cytotoxicity of the AZT derivatives over the parent
compound compared to that for the chlorambucil and
methotrexate derivatives (Table 1).
P-glycoprotein and MRP1 cause resistance to lipophilic
natural product drugs. Therefore altering the lipophilicity of
a drug may a€ect the interaction with these multidrug
transporters. This has been reported previously for the
anthracyclines (Lampidis et al., 1997). While there is no
clear relationship between the degree of lipophilicity and P-
glycoprotein and MRP1 substrate properties, the addition of
GTPn to the drugs described above did generally reduce their
cytotoxicity, particularly in the P-glycoprotein-expressing
CEM/VLB100 cells. The exception was the addition of
GTP3 to methotrexate. No resistance to this derivative was
observed in the CEM/VLB100 cells. However, this was
associated with a dramatic decrease in cytotoxicity (greater
than 2000 fold increase in IC50) in all cells suggesting either
that methotrexate-GTP3 may have diculty entering the cell
or that its limited solubility reduces its bio-availability in this
cell culture system.
Chlorambucil-GTP1, methotrexate-GTP2 and methotrex-
ate-GTP3 were the only compounds to be less cytotoxic in
the MRP1-overexpressing CEM/E1000 cells and this was
minor compared to their decreased cytotoxicity in the P-
glycoprotein-expressing CEM/VLB100 cells. The increase in
sensitivity to chlorambucil-GTP1 associated with the BSO
treatment of the CCRF-CEM cells (Figure 4) was consistent
with the endogenous level of MRP1 expression in these cells
(Davey et al., 1995).
From these data it is dicult to establish which properties
determine the P-glycoprotein and MRP1 substrate character-
istics of a compound. If it is assumed that a decrease in
cytotoxicity of a compound in the multidrug resistant cell
lines compared to that in the sensitive CCRF-CEM cells
indicates that compound is a substrate for the relevant
multidrug resistant transporter, then there is evidence that the
position of the Tris-Lipidation and the number of palmitate
residues are important in determining the substrate proper-
ties. Attachment of GT alone (no palmitate attached) to the
a-carboxyl group improved the P-glycoprotein substrate
properties of methotrexate (5 fold resistant) while attachment
to the g-carboxyl group had a reduced e€ect (2 fold resistant,
Table 1). This also suggests that interfering with the
polyglutamylation of methotrexate, which occurs on the g-
position, has little e€ect on its P-glycoprotein substrate
properties. The further addition of one or two palmitates to
the methotrexate-g-GT increased the P-glycoprotein substrate
properties while addition of the third palmitate produced
poor substrate properties. Therefore the number of palmitate
residues appears to in¯uence the substrate properties. It is
interesting to note that although methotrexate-GTP3 appears
to be a poor P-glycoprotein substrate, it was a potential
substrate for MRP1. This suggests that changing the degree
Figure 3 E€ect of Tris-Lipidation and SDZ-PSC-833 on cytotoxi-
city. The CCRF-CEM cells and CEM/VLB100 cells were incubated
continuously in the drug derivative for 4 days either in the presence
or absence of 10 mM SDZ-PSC-833 after which the cell viability was
determined by the MTT assay as described in the Methods. Assays
were performed in triplicate and the mean and standard deviation of
each determination are shown.
cells more rapidly and possibly via a di€erent transport route
than GTP2 and GTP3 conjugates (Bender et al., 1994).
Alternatively, it could be speculated that di€erences in
molecular con®guration between the GTP1 and GTP2
conjugates may alter the susceptibility of the drug-GTPn
bond to degradation and this would a€ect cytotoxicity.
British Journal of Pharmacology vol 137 (8)

R.A. Davey et al
P-glycoprotein and MRP1 substrates and Tris-Lipidation
1285
There are, however, reports suggesting that methotrexate is
an MRP1 substrate (Hooijberg et al., 1999; Bakos et al.,
2000) but resistance in MRP1-expressing cells, is only evident
after short drug exposure times. Our data supports the view
that methotrexate is not a substrate for MRP1. Table 1
shows that the MRP1-overexpressing CEM/E1000 cells were
not resistant to methotrexate and we have further shown that
there is no methotrexate resistance detected after shorter drug
exposure times of 1 and 4 h (data not shown). Since little
polyglutamation occurs within 4 h, it is unlikely that
polyglutamation accounts for the di€erence between our
results and the other reports (Hooijberg et al., 1999; Bakos et
al., 2000). One explanation for these di€erences is that our
studies were using whole cells selected for resistance whereas
the evidence supporting methotrexate as a substrate for
MRP1 comes from transport studies using isolated inside-out
membrane vesicles and transfected cells.
Although we have made the assumption that resistance to
a compound in a resistant cell equates to transport of that
compound, transport is but one of several reasons for
resistance. At the very least, our studies have highlighted
the profound e€ects that Tris-Lipidation can have on a
drug's cytoxicity, irrespective of the resistance mechanisms
operating. Thus we have demonstrated the utility of
derivatising drugs by Tris-Lipidation and the diverse e€ects
this has on drug cytotoxicity in sensitive and multidrug
resistant cells. The extent of the diversity was unexpected. As
more becomes known about the substrate speci®city and
tissue distribution of the ABC transporters, it will be this
diversity that may allow us to manipulate the pharmacoki-
netic properties of drugs by Tris-Lipidation.
Figure 4 E€ect of BSO on Chlorambucil-GTP1 cytotoxicity. The
CCRF-CEM cells and CEM/E1000 cells were incubated continuously
in chlorambucil-GTP1 for 4 days either in the presence or absence of
50 mM BSO after which the cell viability was determined by the MTT
assay as described in the Methods. Assays were performed in
triplicate and the mean and standard deviation of each determination
are shown.
of Lipidation may be a way of altering a drug's speci®city for
members of the ABC super-family of transporters.
Based on this assumption that increased resistance in the
CEM/VLB100 cells equates to increased P-glycoprotein
substrate properties, Table 1 shows that methotrexate has
low, but signi®cant P-glycoprotein substrate activity. Metho-
trexate is not usually considered to be a P-glycoprotein
substrate. However, there have been reports (Bebawy et al.,
1999; Gi€ord et al., 1998; de Graaf et al., 1996) that suggest
it is and our data support this.
This work has been partly sponsored by F.H. Faulding & Co
Limited, SA, Australia. We thank Mrs Grace Broadhead, CSIRO
Molecular Science, North Ryde, NSW, Australia, for her
assistance with the preparation of some of the methotrexate
conjugates.
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(Received June 19, 2002
Revised August 30, 2002
Accepted September 13, 2002)
British Journal of Pharmacology vol 137 (8)