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Experimental Therapeutics |
Xenova Limited, Slough, Berkshire SL1 4EF [P. M., A. J. S., W. D., S. O., C. L., D. B., D. T., P. C.], and CRC Department of Oncology, Glasgow G61 1BD [J. A. P.], United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A broad range of compounds that interact with P-gp and block drug efflux have been reported to reverse the MDR phenotype. The first generation modulators consisted of calcium channel blockers, calmodulin inhibitors, hormonal/steroidal derivatives, antibiotics, cardiovascular drugs, the cyclosporins, and other miscellaneous compounds (13) . These compounds were developed for pharmacological uses other than reversal of MDR and were relatively nonspecific and weak inhibitors that were also substrates for P-gp. With the majority of these compounds, deleterious toxicities associated with their use at the required concentrations to inhibit P-gp function have precluded their widespread clinical use (14, 15, 16) . The requirement for more selective and potent agents as resistance modifiers has led to the development of several "second-generation" modulators such as the nonimmunosuppressive cyclosporin D analogue, PSC 833 (17) , VX-710 (18) , the acridone carboxamide derivative GF120918 (GG918; Ref. 19 ), the substituted dibenzosuberane molecule LY335979 (20) , and the diketopiperazine derivative XR9051 (21 , 22) . Clinical trials with some of the second-generation modulators are currently in progress, and initial studies have demonstrated some clinical benefit from the use of modulators such as PSC833 (23) . However, to date, the nonspecific modulators dexverapamil, VX-710, and PSC833 have shown significant enhancement of pharmacokinetics and toxicity of cytotoxics such as paclitaxel, which has necessitated the reduction of the cytotoxic drug dose when administered with these modulators (24 , 25) . In contrast the more potent and specific modulators (GG918, LY335979) have exhibited no significant pharmacokinetic interaction with doxorubicin, etoposide, and paclitaxel in animal studies (19 , 20) . These studies clearly indicate that the development of potent and selective P-gp inhibitors is an important approach to reversing MDR in the clinic.
A chemical program aimed at improving the potency and physicochemical
properties of our MDR modulator XR9051, resulted in the discovery of
XR9576 (Fig. 1)
, a potent and specific inhibitor of P-gp (26)
. The
present studies were undertaken to characterize this novel
anthranilamide derivative. The ability of XR9576 to sensitize a panel
of MDR cell lines in vitro and in vivo to various
cytotoxic drugs was evaluated along with its ability to inhibit drug
efflux and to interact with P-gp. The pharmacokinetic interaction
between XR9576 and paclitaxel was also examined because this commonly
used drug has shown significant interaction with other modulators
including dexverapamil (27)
, VX-710 (24)
, and
PSC833 (25)
. The characteristics of XR9576 indicate that
it holds great promise for the treatment P-gp-mediated MDR cancers.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytotoxics and Reagents
For in vitro use, daunorubicin, doxorubicin,
vincristine, paclitaxel, etoposide, colchicine, and actinomycin D were
obtained from Sigma. Stock solutions (5–100 mM)
of all drugs were prepared in DMSO with the exception of daunorubicin
(1 mM) and doxorubicin (500 µg/ml), which were
prepared in sterile deionized H2O; the stock
solutions were stored as aliquots at -20°C. For cell
treatments, cytotoxics were further diluted in culture medium with the
final DMSO concentration <1%. The resistance modifying agents
XR9576 (free base and mesylate salt) and GG918 (free base and
hydrochloride salt) were synthesized at Xenova. Stock solutions (5
mM) of these resistance modifying agents were
prepared in DMSO. Vpm (Sigma, Poole, United Kingdom) and CsA
(Calbiochem, Nottingham, United Kingdom) were dissolved in
ethanol to give a stock concentration of 10 mM.
For in vivo use epirubicin and doxorubicin were obtained from Pharmatalia (St Albans, United Kingdom); etoposide from Bristol-Myers Squibb Pharmaceuticals Ltd (Middlesex, United Kingdom) and vincristine from David Bull Laboratories (Warwick, United Kingdom). XR9576 (mesylate salt) was dissolved in 5% (w/v) D-(+)-glucose (dextrose) solution at appropriate concentrations for i.v. and p.o. administration. GG918 (hydrochloride salt) was prepared in propylene-glycol:5% dextrose (3:2 v/v) at 10 mg/ml and diluted appropriately in 5% dextrose immediately prior to use.
The radiolabeled compounds [3H]daunorubicin (1–5 Ci/mmol) and [3H]azidopine (48 Ci/mmol) were purchased from DuPont and Amersham Life Sciences, respectively.
Drug Potentiation Assays
The ability of modulators to potentiate the cytotoxicity of
various drugs was evaluated in several cell lines as outlined
previously (21)
with minor modifications. Briefly, cells
were seeded into 96-well plates (Falcon) at 6 x 102
to 2 x 104/well, depending on the cell line, in 100 µl
of medium and incubated for 4 h at 37°C. Varying
concentrations of modulator or solvent control (50 µl/well) were
subsequently added and incubated for an additional 1 h before the
addition of the cytotoxic drug. The cytotoxic drug (50 µl) was added
to give a range of final concentrations in quadruplicate wells. After
incubation for an additional 4–6 days, cell proliferation of adherent
cells was assessed using the sulforhodamine B assay and the
proliferation of suspension cells by AlamarBlue.
IC50 values for cytotoxic drugs (concentration
resulting in 50% inhibition of cell growth) were calculated from
plotted results using untreated cells as 100%.
EC50 values for modulators (concentration
required to give 50% of full reversal) were obtained from graphs of
potentiation index (ratio of IC50 of cytotoxic
drug alone:IC50 of cytotoxic drug in the
presence of modulator) plotted against concentration of modulator. Full
reversal was defined as the potentiation index obtained in the presence
of 100 nM GG918.
Intrinsic Cytotoxicity
Cells (EMT6 AR1.0 8 x 102
/well; A2780 5 x 103/well; 2780AD 6 x 103/well) were seeded into 96-well plates. After
4 h, varying concentrations of XR9576 were added, and cells were
incubated for an additional 4 days (EMT6 AR1.0) or 6 days (2780AD)
before quantification of cell growth and calculation of
IC10 values (concentration resulting in 10%
inhibition of cell growth) as described above.
Accumulation of [3H]Daunorubicin in the EMT6/AR1.0
Cells
The ability of modulators to reverse the P-gp-dependent
accumulation deficit in the resistant EMT6 AR1.0 cells was investigated
as described previously (21)
.
Inhibition of P-gp-mediated Efflux
The ability of modulators to maintain inhibition of
P-gp-mediated efflux after their removal from the incubation medium was
assessed using two P-gp substrates, Rh123 and
[3H]daunorubicin. For the studies with Rh123,
A2780 and 2780AD human ovarian cells were trypsinized and resuspended
at a density of 106/ml of culture medium.
Modulators were added at fixed concentrations immediately before the
addition of Rh-123 (0.8 µg/ml), and the cells were incubated for
1 h at 37°C (substrate-loading phase). The cells were then
washed and resuspended in normal growth medium without modulator or
Rh123. During the following "efflux phase," samples were incubated
at 37°C, and aliquots (106 cells) removed at
various time intervals, centrifuged, and washed in ice-cold PBS. The
cells were then resuspended in 1 ml of ice-cold PBS and analyzed by
flow cytometry. Fluorescence was measured from
104 cells and cell-associated fluorescence as a
percentage of T0 was plotted against
time. Similar experiments extended over a 22-h period using
[3H]daunorubicin as the P-gp substrate, and
EMT6/AR1.0 cells were performed as described previously
(21)
.
Persistence of Activity after Removal of Modulator
EMT6 AR1.0 cells were plated as for the accumulation assay and
allowed to attach for 48 h. Cells were then incubated with
modulators for 1 h at 37°C, washed with culture medium, and
further incubated in normal growth medium. At subsequent time points
(up to 22 h) as indicated, the ability of the cells to accumulate
[3H]daunorubicin was assessed as described for
the accumulation assay. Graphs were plotted of cell-associated
radioactivity/105 cells against time, where
T0 represents the end of the modulator
incubation phase.
Photoaffinity Labeling of P-gp
The photoaffinity labeling of P-gp was evaluated using a
modified method of Ferry et al. (30)
. Cell
membrane-enriched fractions of H69/P and H69/LX4 cells were adjusted to
1.0 mg/ml in "predilution buffer" [final concentration, 50
mM Tris-HCl (pH 7.4), 0.1
mM AEBSF, 0.25 mM
sucrose, and 5 mM MgCl]. Samples of membrane (30
µl) were incubated in wells of a V-well polyvinyl chloride microtiter
plate, with increasing concentrations of modulator (10 µl) prediluted
in labeling buffer [50 mM Tris-HCl (pH 7.4), 0.1
mM p-aminoethylbenzenesulfonyl
fluoride, 0.25 mM sucrose, and 5
mM MgCl] and 10 µl of
[3H]azidopine prediluted in labeling buffer.
The final concentration of [3H]azidopine in the
incubation mixture was 1 µM. Incubation was for
1 h in darkness on ice. Samples were then exposed to UV light (366
nm) for 20 min on ice. Each incubation mixture was then diluted 1:1
with sample buffer [100 mM Tris-HCl (pH 6.8),
200 mM DTT, 4% SDS, 0.2% bromphenol blue, and
20% glycerol] and electrophoresed on a 7.5% SDS-PAGE gel. Gels were
then fixed in 5% glacial acetic acid and 5% isopropyl alcohol and
rinsed in distilled water, and the signal was amplified by bathing in
Autofluor (National Diagnostics) for 1 h. Dried gels were then
exposed to XAR-5 film with an intensifying screen for 7 days at
-70°C. After development of film, bands corresponding to P-gp at
Mr 170,000 were quantified by
densitometry (Pharmacia Gel Scanner).
In Vivo Efficacy Evaluation
MC26 Murine Colon Carcinoma Model.
All of the animal experimentation was performed to United Kingdom Home
Office regulations and the United Kingdom CCCR guidelines were
adhered to at all times. The in vivo efficacy of XR9576 was
evaluated using the intrinsically resistant MC26 colon carcinoma tumors
that exhibited low levels of P-gp-mediated drug resistance as outlined
previously (22)
. MC26 tumor slurry was implanted s.c. in
BALB/c mice (day 0). The animals were then randomized, 24 h later,
into groups of 15–18 and treated once with various regimens. XR9576 or
vehicle was administered either i.v. via a lateral tail vein or p.o.
with doxorubicin (5 mg/kg) or vehicle i.v. The modulator was
administered either i.v. at 2–4 mg/kg (10 ml/kg) at the same time as
doxorubicin or p.o. at 2–8 mg/kg (10 ml/kg) 1 h before the
cytotoxic drug. GG918 was administered p.o. 1 h before
doxorubicin. All of the animals were weighed twice weekly. The animals
were killed by cervical dislocation on day 14, and the tumors were
excised and weighed. The data were analyzed by Student’s t
test.
Human Carcinoma Xenografts.
The efficacy of XR9576 was also evaluated using MDR human carcinoma
xenografts. Studies in parental (A2780) and resistant (2780AD) human
ovarian carcinoma xenografts were performed using a modification of the
method of Plumb et al. (31)
. Briefly, A2780 and
2780AD cells grown in vitro were harvested by exposure to
trypsin-EDTA, washed three times in PBS, and implanted s.c. in female
nude mice (2 x 106 cells in 0.1
ml). When the tumors had reached a mean diameter of 0.5–1.0 cm, the
animals were randomized into groups of 6 and treated with various
regimens on days 0 (start of treatment), 2, and 4. XR9576 or vehicle
was administered i.v 1 h before or p.o. 2 h before paclitaxel
(15 mg/ml; formulated in 5% cremophor EL and 5% ethanol in (5% w/v)
dextrose) or vehicle i.v. Administration of all of the i.v. compounds
was via a lateral tail vein. The efficacy of GG918 was also evaluated
after i.v. administration. Tumor volume and body weights were recorded
as described previously (31)
, and an ANOVA model was used
to assess significance between treatment groups.
The ability of XR9576 to potentiate the antitumor activity of etoposide and vincristine was evaluated using the H69/LX4 SCLC xenografts. The xenografts were established in female nude mice by s.c. implantation of 7–8 x 106 cells resuspended in PBS (0.1 ml) after harvesting from in vitro culture. When the tumors had reached a mean diameter of about 0.6 cm, the animals were randomized into groups and treated on days 0, 5, and 10 with XR9576 (i.v. or p.o.) and etoposide (30 mg/kg) or vincristine (0.5 mg/kg) i.v. The modulator and the cytotoxic drug were mixed together immediately prior to i.v. administration by the caudal vein. Control animals were treated with vehicle. Tumor volumes and body weights were recorded as described previously (22) .
Pharmacokinetic Studies.
Male CD rats (3 animals per time point) were dosed i.v. with paclitaxel
alone [15 min infusion at 10 mg/kg in Tween 80:ethanol:5% dextrose
(5:10:85% v/v/v)] or in combination with XR9576 (10 mg/kg).
XR9576 was administered as a bolus (i.v.) dose 15 min before infusion
of paclitaxel. Blood samples were collected by cardiac puncture using
heparinized syringes at various times between 0.083 and 48 h and
were centrifuged to prepare plasma, which was stored at -20°C until
analysis. Paclitaxel concentration in plasma samples was measured by a
LC-MS/MS assay. Paclitaxel and Baccatin III (internal standard) were
extracted from 100 µl of rat plasma using liquid-liquid extraction
procedure with diethyl ether (7 ml). The plasma samples were mixed with
diethyl ether and centrifuged, and the organic layer was evaporated to
dryness under nitrogen. The residues were reconstituted in 100 µl of
mobile phase and aliquots (5–15 µl) were analyzed using an isocratic
LC-MS/MS. The chromatography was carried out using a Hypersil
C1 column [5 µm x 100
mm x 2.1 mm (i.d.)] with acetonitrile:0.1%
aqueous formic acid (50:50 v/v) mobile phase at a flow rate of
0.2 ml/min. Detection was via MS/MS in multiple reaction monitoring
mode. The area under the concentration time curve (AUC) and
half life of paclitaxel in plasma was calculated by noncompartmental
analysis using WinNonlin software (Scientific Consulting Inc.).
Statistical analysis was performed using an ANOVA model.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The four resistant cell lines used exhibited either acquired
(EMT6/AR1.0, 2780AD, and H69/LX4) or intrinsic (MC26) MDR phenotype as
a result of P-gp overexpression and were described previously
(21)
. XR9576 was highly active across the panel of cell
lines and gave significant reversal of resistance to doxorubicin at
concentrations as low as 10 nM and almost complete reversal
at 30 nM. The potency of XR9576 was comparable with that of
GG918 and between 10- and 30-fold greater than that of PSC833 in these
assays. XR9576 at 100 nM, caused a weak sensitization
effect in the EMT6/P parental cell line to doxorubicin, presumably as a
result of the basal level of P-gp expression (21)
. No
effect was observed in the other parental cell lines examined.
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. The activity is expressed as EC50 values, the
concentration of modulator required to reverse resistance to the
various cytotoxic agents by 50% of the maximal level observed in the
presence of 100 nM GG918 (see "Materials and Methods").
The results showed that XR9576 was very potent at fully reversing
resistance to various agents associated with the MDR phenotype in all
three of the cell lines, with EC50 in the range
of 12–38 nM. In these assays, XR9576 was up to 2-fold more
potent than GG918 (EC50 range, 14–63
nM; Table 2
). Moreover, the presence of 100 nM
XR9576 had no effect on the activity of these cytotoxic agents in the
parental cell lines (A2780 and H69/P) lacking P-gp. Importantly, XR9576
did not affect the activity of non-MDR cytotoxics, cisplatin and
methotrexate, in the resistant cells (data not shown). These results
indicate that reversal of resistance was attributable to the
inhibition of P-gp function.
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Effect on Accumulation of Daunorubicin.
The ability of XR9576 to inhibit P-gp-mediated transport was evaluated
by measuring reversal of [3H]daunorubicin
accumulation deficit in the MDR EMT6/AR1.0 cells. XR9576 had a
significant effect on the accumulation of
[3H]daunorubicin in these cells at
concentrations as low as 10 nM (Fig. 2)
. Half-maximal reversal of accumulation deficit was observed at
38 ± 18 nM (n = 4) and near maximal at 300 nM. The potency
of XR9576 was comparable with that observed for GG918
(EC50, 48 ± 26
nM; n = 4) but was
significantly greater than that observed for CsA
(EC50, 440 ± 230
nM) and Vpm (EC50,
580 ± 220 nM; data not shown).
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illustrates clearly that XR9576 inhibited the efflux of Rh-123 from the
2780AD cells and that the modulator remained effective even when it was
excluded from the efflux medium. Maximal level of inhibition was
retained up to the end of the efflux period (2 h) when XR9576 was
present at 300 and 200 nM concentration during the loading
phase only. Even at 100 and 50 nM XR9576, 80 and 50% of
the cellular Rh-123 was retained at 2 h. GG918 displayed a similar
concentration-dependent effect on efflux as that observed with XR9576
(data not shown). In contrast, >90% of the Rh-123 was effluxed by
1 h after incubation with CsA at 20 µM, a
supramaximal concentration (Fig. 3)
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. This suggested that the modulator was effective at inhibiting
P-gp-mediated transport for at least 22 h after its removal from
the medium. At lower concentrations (50 and 30 nM), XR9576
still showed inhibitory activity over an extended period of time,
although activity was lost more rapidly as the concentration of XR9576
was decreased (Fig. 4)
. Again, CsA, even after exposure to very high
concentrations (20 µM), displayed only a short duration
of inhibition (<60 min) when omitted from the efflux phase (data not
shown).
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. At 50-nM concentration, XR9576 produced slightly less
initial accumulation of [3H]daunorubicin;
however, 62% of this was retained at 22 h. This long duration of
action of XR9576 was in contrast to that observed with 1
µM PSC833 and 20 µM CsA, which lost the
majority of their activity (70 and 90%, respectively) within 5 h
of removal from the medium (Fig. 5)
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. The identity of this band was confirmed by Western blot analysis
using C219 P-gp antibody (data not shown). Fig. 6
shows that XR9576
inhibited the labeling of P-gp by [3H]azidopine
in a dose-dependent manner. Densitometric evaluation indicated that
XR9576 was more effective than CsA and Vpm at inhibiting
[3H]azidopine binding. The
IC50 values were 0.55, 1.17, and 20
µM for XR9576, CsA, and Vpm, respectively,
which reflects the potency order obtained in accumulation and cytotoxic
potentiation studies.
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5-fold; Table 1
). This confirms that the MC26 cells have low levels
of P-gp-mediated MDR that may be present in many human tumors
(19)
. XR9576 was found to also significantly potentiate
the antitumor activity of doxorubicin against s.c. MC26 tumors in
vivo (Fig. 7)
. Treatment of MC26 tumor-bearing BALB/c mice with doxorubicin (5
mg/kg, i.v.) or XR9576 (p.o. or i.v.) alone had little or no effect on
the growth rate of the tumors (Fig. 7)
. However, coadministration of
XR9576 at 2–8 mg/kg p.o. (Fig. 7, A and B)
with
doxorubicin reduced the growth rate of the tumors significantly
(P < 0.001). The enhancement in antitumor
activity of doxorubicin was related to the dose of the modulator, and
results from two separate experiments showed that maximum modulatory
activity was obtained between a 2- and 4-mg/kg p.o. dose of XR9576. In
comparison, coadministration of 9.5 mg/kg GG918 p.o. was required to
produce a similar reduction in tumor growth (Fig. 7, A and B)
. Importantly, there was no substantial or reproducible
increase in body weight loss in animals treated with doxorubicin plus
XR9576 compared with drug alone groups (Fig. 7, C and D)
. The observed reduction in body weights in all groups on
day 14 may be attributable to tumor-induced cachexia. Similar results
were obtained when XR9576 was coadministered i.v. at 2 and 4 mg/kg with
doxorubicin (data not shown). The similar potency observed after i.v.
and p.o. administration of XR9576 correlates well with the observed
high (80%) p.o. bioavailability of the modulator in BALB/c mice
(32)
.
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demonstrates that i.v. paclitaxel (15 mg/kg) alone significantly
reduces the growth rate of the parental A2780 xenografts and that
coadministration of XR9576 p.o. (12 mg/kg) did not enhance the activity
of paclitaxel. Similar results were obtained after i.v.
coadministration of XR9576 (10 mg/kg) with paclitaxel (data not shown).
In contrast, paclitaxel had no effect on the growth rate of the
resistant 2780AD tumors, and coadministration of XR9576 p.o. or i.v.
restored the antitumor activity of the cytotoxic drug (Fig. 9, A and B)
. Furthermore, this effect was related
to the dose of XR9576. Comparison of growth delay observed in the
parental and resistant tumor models indicated that coadministration of
XR9576 at 12 mg/kg p.o. fully reversed resistance against paclitaxel in
the highly resistant 2780AD tumors. Importantly, the effective
combination schedules were well tolerated as indicated by changes in
body weights compared with those in drug-alone groups. The enhancement
in antitumor activity of paclitaxel by i.v. XR9576 was comparable with
that observed with i.v. GG918, thus confirming the in vitro
results (data not shown).
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. The potentiation of etoposide antitumor activity was related to the
p.o. dose of XR9576, and comparison with the response of the parental
xenografts to etoposide alone suggested that maximum reversal of
resistance was achieved by the administration of 6 and 12 mg/kg of the
modulator. The efficacious combination schedules were well tolerated as
indicated by changes in body weights compared with those in drug-alone
groups (data not shown). Similarly, the administration of XR9576 p.o.
(12 mg/kg) significantly potentiated the antitumor activity of
vincristine (0.5 mg/kg, i.v.) against the H69/LX4 xenografts (Fig. 11)
, and, again, the combination schedules were well tolerated.
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. The AUC0-t over 48 h for paclitaxel
was similar in the groups treated with or without XR9576 (6500 and 5681
ng•h/ml, respectively). Also, no significant differences were
observed in the elimination half life (13.5 and 13.2 h) or the
Cmax values (8208 and 8642 ng/ml) for
paclitaxel in the two groups, respectively.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several studies were performed to examine the inhibition of
P-gp-mediated transport by XR9576 and to determine the interaction of
the modulator with this protein. XR9576 was very potent at reversing
the accumulation deficit and at blocking the efflux of P-gp substrates,
daunorubicin and Rh123, from P-gp-overexpressing cell lines. The
finding that drug efflux and accumulation was not affected in the
parental cell lines that lacked P-gp, indicated that reversal of drug
resistance by XR9576 was probably attributable to the inhibition of
P-gp-mediated efflux. The inhibition of drug efflux as a result of
direct interaction of XR9576 with P-gp was implied by the potent
displacement of a photoaffinity label,
[3H]azidopine. The direct interaction with P-gp
was confirmed by equilibrium-binding studies with
[3H]XR9576 and membranes isolated from P-gp
overexpressing MDR Chinese hamster ovary cells, which demonstrated that
XR9576 binds to P-gp with a very high affinity,
Kd, 5.1 nM
(34)
. The displacement and binding studies confirmed the
superior potency of XR9576 compared with CsA and Vpm. These results
suggest that XR9576 binds tightly to a site(s) distinct from, but
linked to, those of the cytotoxics and modulators that are P-gp
substrates. The efflux and persistence assays also showed that XR9576
has a long duration of action; the modulator inhibited P-gp function in
cells in excess of 22 h after a short exposure to 
100
nM XR9576 and subsequent removal from the
incubation medium. In contrast, PSC 833 (1 µM),
CsA (20 µM) and Vpm (50
µM) lost the majority of their P-gp inhibition
activity within 1–5 h after removal of the incubation medium. These
data along with the observations that accumulation of
[3H]XR9576 in sensitive (Aux B1) and MDR
(CHrB30) Chinese hamster ovary cells was the same
and was unaffected by coexposure to GG918 (34)
suggest
that XR9576 is not a substrate of P-gp. This property may give XR9576
significant advantage for clinical administration because it should be
able to reverse P-gp-mediated drug resistance in tumors over prolonged
periods after exposure to low nM concentrations.
Studies in mdr1a and mdr1b knockout mice have
suggested that P-gp is not essential for normal function but is
required for protection against xenobiotics because it can influence
their pharmacokinetics/pharmacodynamics (35)
. Therefore,
prolonged inhibition of P-gp by XR9576 may affect the pharmacokinetics
and toxicity of anticancer agents. However, XR9576 had no substantial
effect on plasma pharmacokinetics of paclitaxel or on the toxicity of
various cytotoxic drugs at efficacious doses. Similar results have been
reported for other specific modulators such as GG918 (19)
and LY335979 (20)
, whereas nonspecific modulators such as
VX-710 (24)
, CsA, and PSC 833 (25)
have shown
significant enhancement of pharmacokinetics and toxicity of cytotoxic
drugs. If these results translate to humans, then XR9576 could be used
in combination with a normal dose of cytotoxic agents in front-line
therapy regimen. It has been proposed that such a regimen may improve
the overall response to therapy by preventing the emergence of
drug-resistant cells expressing either P-gp or other mechanisms of
resistance in response to exposure to cytotoxic drugs (reviewed in
Refs. 15
and 36
).
The promising activity of XR9576 demonstrated in vitro was
confirmed in in vivo efficacy studies. The modulator
exhibited potent activity after i.v. and p.o. administration in mice
bearing MDR murine and human tumor xenografts, restoring antitumor
activity of several drugs without an apparent increase in toxicity.
Against the intrinsically resistant MC26 murine colon tumor, which
exhibits a low level of P-gp-mediated resistance (29)
,
coadministration of XR9576 i.v. or p.o. significantly
(P < 0.001) increased the antitumor activity
of doxorubicin in a dose-related manner without an apparent increase in
toxicity. Moreover, maximum modulatory activity was observed after a
single administration of a low dose of XR9576 (between 2 and 4 mg/kg)
by either route. Oral bioavailability of the modulator in BALB/c mice
is 80% (32)
, thus explaining the result that both
routes of administration appear equipotent. Even in mice bearing human
carcinoma xenografts exhibiting high levels of P-gp-mediated acquired
MDR, coadministration of 6–12 mg/kg XR9576 p.o. was sufficient to
restore the antitumor activity of paclitaxel, etoposide, and
vincristine. All of the efficacious combination schedules with XR9576
appeared to be well tolerated, as indicated by the minimal changes in
body weights of the various treatment groups. Furthermore, XR9576 did
not enhance the activity of paclitaxel against the parental tumor.
These observations along with the lack of effect on plasma
pharmacokinetics of paclitaxel indicate that in vivo
reversal of resistance is attributable to inhibition of P-gp function
and is unlikely to be caused by an increase in the pharmacokinetics of
the cytotoxic drug. The effect of XR9576 on pharmacokinetics of the
other cytotoxic drugs is being investigated and will be reported
separately.
In conclusion, XR9576 is one of the most potent in vitro and in vivo modulators of P-gp-mediated MDR described to date. In addition, it is selective, binds to P-gp with a high affinity, and has a long duration of action. XR9576 also exhibits good oral bioavailability and potent i.v. and p.o. activity, restoring sensitivity of MDR murine and human tumors to a range of chemotherapeutic drugs at well-tolerated doses. Moreover, XR9576 does not alter plasma pharmacokinetics of paclitaxel and hence the enhancement in its activity against MDR tumors by the modulator does not appear to be related to an increase in plasma concentrations of the drug. These data indicate that it may be possible to use XR9576 as part of a first-line therapy with paclitaxel in the clinic. This preclinical profile for XR9576 supported the progression of the modulator into Phase I clinical trials in healthy volunteers. These studies have included measurement of P-gp activity in CD56+ lymphocytes as a surrogate marker of efficacy. Complete inhibition of P-gp activity by XR9576 was observed in excess of 24 h after a single i.v. dose (2.0 mg/kg), which supports a long duration of action for this modulator (37) . Thus, the properties of XR9576 and the Phase I studies indicate that it holds great promise for the treatment of P-gp-mediated MDR cancers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Xenova Limited, Slough, Berkshire SL1 4EF, United
Kingdom. Phone: (44) 1895-866102; Fax: (44) 1895-866130. 
2 The abbreviations used are: MDR, multidrug
resistance; P-gp, P-glycoprotein; MRP, MDR protein; Rh123, rhodamine
123; Vpm, verapamil; CsA, cyclosporin A; ABC, ATP-binding cassette;
SCLC, small cell lung carcinoma; MS/MS, tandem mass spectrometry;
LC-MS/MS, liquid-chromatographic MS/MS.
Received 9/28/99. Accepted 11/14/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), or
100 µM Vpm plus [3H]daunorubicin for 1 h before measuring cell-associated radioactivity as described in
"Materials and Methods." Results are expressed as a percentage of
that seen in the presence of 100 µM Vpm. Data
plotted, the mean ± SD of quadruplicate data
points.
), 100 nM
(
), and 50 nM (
) or CsA at 20 µM (•),
the cells were washed and incubated in fresh medium for the indicated
times (efflux period). At the end of the efflux periods, the
cell-associated Rh123 was measured as described in "Materials and
Methods."
; 10 mg/kg, i.v.). The
modulator was administered 15 min before paclitaxel, and plasma samples
were collected and analyzed by LC-MS/MS as described in the
"Materials and Methods" section. The values represent
mean ± SD of three animals per group.