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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 306-314
Efflux of Rhodamine From CD56+ Cells as a Surrogate
Marker for Reversal of P-Glycoprotein-Mediated Drug Efflux by PSC 833
By
Robert Robey,
Susan Bakke,
Wilfred Stein,
Beverly Meadows,
Thomas Litman,
Sheela Patil,
Tom Smith,
Tito Fojo, and
Susan Bates
From the Medicine Branch, National Cancer Institute, National
Institutes of Health, Bethesda, MD; the Hebrew University of Jerusalem,
Jerusalem, Israel; and Novartis Pharmaceutical Corporation, East
Hanover, NJ.
 |
ABSTRACT |
The expression of high levels of P-glycoprotein (Pgp) in circulating
mononuclear cells allowed us to use an ex vivo assay as a surrogate
measure of Pgp antagonism. Efflux of rhodamine from CD56+
cells was measured before the start of PSC 833 and at varying times
thereafter. Patients receiving PSC 833 had decreased rhodamine efflux
from their circulating CD56+ cells. Time course studies
showed that following a single oral dose of PSC 833, decreased
rhodamine efflux was found in some patients within 15 minutes of
treatment. Maximal inhibition was observed at times ranging from 45 minutes to 60 minutes. A dose-response relationship was shown between
the concentration of PSC 833 in the blood and the inhibition of
rhodamine efflux, with an apparent plateau of the inhibition of
rhodamine efflux at approximately 1,000 ng/mL. The Ki,
defined as the concentration required for half-maximal inhibition of
Pgp-mediated rhodamine efflux, was determined to be in the range of 29 to 181 ng/mL; although results in two patients were distinctly
different, with Ki values of 914 and 916 ng/mL. MRK-16 staining was similar among all patients. We conclude that
measurement of rhodamine efflux from CD56+ cells
provides a surrogate assay with the potential for monitoring Pgp
antagonism in clinical trials.
This is a US government work. There are no restrictions on its use.
| |
INTRODUCTION |
EARLY STUDIES AIMED at reversal of
P-glycoprotein (Pgp)-mediated resistance used compounds already
available and in use in clinical medicine for indications unrelated to
oncology. These studies, referred to as `first generation' reversal
studies, provided several insights into the obstacles that must be
surmounted to prove that reversal of Pgp-mediated resistance could be
of clinical benefit. A variety of agents, including verapamil,
quinidine, quinine, cyclosporin A, tamoxifen, and megestrol acetate
were investigated with response rates of 0% to 73%, depending on the tumor type studied, the reversal agent chosen, and the anticancer regimen used.1,2 The failure of these studies to
convincingly show a role for Pgp antagonism in the clinic was likewise
related to the use of antagonists in patients with tumors in whom
resistance was not clearly Pgp-mediated, inadequate blood
concentrations of reversal agents, and the use of drugs or schedules
for which resistance was not clearly present.2 The
observation that blood concentrations of the reversal agents were often
below that needed in the in vitro setting for the reversal of
resistance in cell lines expressing high levels of P-glycoprotein led
to the development of `second generation' antagonists. These second
generation agents, including S9788, GF910128, dexniguldipine, PSC 833, and VX 710, reverse high levels of resistance at concentrations that
are clinically attainable.3 In phase I studies, the maximum
tolerated dose of PSC 833 has been shown to achieve concentrations of
3,000 to 4,000 ng/mL.4 This is 3- to 10-fold higher than
that shown to be effective in vitro.5
In addition to the difficulty in achieving sufficient
concentrations of the reversal agents, in vitro laboratory
studies with a variety of antagonists raised concerns that protein
binding in serum impaired the Pgp antagonism.6-9 PSC 833 was reported by Ludescher et al to be the antagonist least
affected by performing the drug accumulation assay in
serum.6 The extent to which these in vitro studies reflect
decreased antagonist potency in patients has not yet been confirmed,
because direct assays measuring intratumoral drug concentrations
are rare. Although the first-generation antagonists are
effectively used in the clinic for their primary indications, protein
binding could be a greater problem for Pgp antagonism if the affinity
of the antagonist was less for Pgp than for serum proteins or for the
drugs' principal target.
Thus, several attempts have been made to develop surrogate assays for
Pgp antagonism. We used an approach based on the observation that
hematopoietic cells, including circulating mononuclear cells, express
Pgp and show Pgp-mediated efflux of rhodamine, which can be inhibited
by systemic administration of a reversal agent before blood
collection.10 Peripheral mononuclear cells including
CD3+, CD4+, and CD8+ T cells, as
well as the CD56+ natural killer (NK) subset, have been
shown to overexpress P-glycoprotein, with highest levels observed in
CD8+ and CD56+ cells.11-14 The role
that Pgp plays in these cells is largely unexplored.
Phytohemagglutinin (PHA) activation of mononuclear cells
has been shown to increase Pgp expression.15 One report showed that Pgp antagonists were able to inhibit interleukin-2 (IL-2)
transport in T cells.16 The Pgp present in the
CD56+ population of cells appears functionally identical to
that observed in multidrug resistant cells, with highest levels of RNA,
protein, as well as drug efflux observed in the CD56+
population. For this reason, we examined Pgp function and expression in
the CD56+ population.
The results presented here show that rhodamine efflux in the
CD56+ population provides a surrogate marker for Pgp
antagonism and that inhibition of rhodamine efflux occurs at clinically
achievable concentrations of PSC 833.
| |
MATERIALS AND METHODS |
Clinical studies.
Blood samples were obtained from patients enrolled on two separate
multidrug resistance reversal trials at the Clinical Center, National
Cancer Institute. The 20 patients examined for this study ranged in age
from 20 to 76 years old (mean, 54 years). Diagnoses for these patients
are presented in Table 1. In these trials, patients received PSC 833 formulated in a soft gelatin capsule, which
has 30% to 50% bioavailability, in combination with either vinblastine or paclitaxel.17 Patients on the PSC 833 + vinblastine study were receiving PSC 833 at doses of 3, 4, and 5 mg/kg
every 6 hours, whereas patients on the PSC 833 + paclitaxel study were receiving PSC 833 at 4 or 5 mg/kg every 6 hours. Samples were typically obtained as "pre," before initial exposure to
chemotherapy or to PSC 833; at indicated time points following the
first dose of PSC 833 administered (0, 15, 30, 45, 60, and 120 minutes); as "peak," 2 hours following ingestion of PSC 833; and
as "trough," 6 hours following PSC 833 administration. The peak
and trough samples were obtained after at least 6 days of PSC 833 administration on the every-6-hour schedule. It should be noted that
pharmacokinetic studies have suggested that peak PSC 833 concentration may occur from 2 to 4 hours following ingestion of PSC
833.17,18 The time course studies were obtained over the
initial 2 hours following the first administration of PSC 833.
Materials.
PSC 833 was provided by Novartis Research Institute (Hanover, NJ).
Cell lines.
SW620 Ad2, Ad5, Ad10, Ad20, and Ad300 are multidrug resistant human
colon carcinoma sublines, selected from parental SW620 cells and
maintained in 2, 5, 10, 20, and 300 ng/mL of doxorubicin, respectively.19 Parental cells and sublines are carried in
RPMI, 10% fetal calf serum (FCS), penicillin, streptomycin, and
glutamine. SW620 Ad2, Ad5, Ad10, Ad20, and Ad300 cells possess a
multidrug resistant phenotype and do not appear to have mechanisms of
resistance other than overexpression of Pgp.19
Isolation of peripheral blood mononuclear cells (PBMCs).
Whole blood (15 to 30 mL) was drawn into a heparinized
syringe from patients enrolled on the PSC 833 protocols. The blood was
used directly in the rhodamine efflux assay, as described below, or was
diluted 1:1 with Dulbecco's phosphate-buffered saline (DPBS), layered
onto Lymphocyte Separation Medium (Organon Teknika, Durham, NC) and
centrifuged at 2,000 rpm for 30 minutes. The mononuclear layer was
removed, washed with cold DPBS, and then frozen in 10% DMSO/90% FCS
and stored at  80°C for later use.
Rhodamine 123 efflux.
Assays were performed as previously described in cultured
cells20 and modified as described below for mononuclear
cells. Whole blood was obtained from each patient in a heparinized
syringe. Rhodamine 123 (Sigma, St Louis, MO) with or without PSC 833 was added to aliquots of whole blood to achieve a final rhodamine concentration of 0.5 µg/mL and PSC 833 concentration of 3 µg/mL. The blood was incubated for 30 minutes at 37°C in 5%
CO2. After the accumulation period, the blood was layered
onto Lymphocyte Separation Medium and centrifuged at 2,000 rpm for 5 minutes. The mononuclear cell layer from each tube was transferred to a separate tube, washed with cold DPBS, resuspended in 200 µL cold DPBS
with 2% FCS (DPBS/FCS), and held at 4°C for later staining. Cells
that were to be subjected to an efflux period were then resuspended in
rhodamine-free complete media (phenol red-free improved modified
Eagle's medium [IMEM] with 10% FCS) with or without 3 µg/mL PSC 833 and incubated another 60 minutes at 37°C in 5%
CO2. After the efflux period, cells were sedimented and washed twice with cold DPBS/FCS. After the washings, the cells were
resuspended in 200 µL cold DPBS/FCS. The cells were then stained with
phycoerythrin (PE)-labeled CD56 antibody (Becton Dickinson, San
José, CA) or PE-labeled mouse IgG1 (Becton Dickinson) as a negative control. After staining, the cells were washed twice and
then resuspended in DPBS and kept on ice in the dark until analyzed. A
FACSort flow cytometer (Becton Dickinson) with a 488-nm argon laser was
used to analyze the samples. Rhodamine fluorescence was collected after
a 520 nm bandpass filter and PE fluorescence was collected after
a 585 nm bandpass filter. A minimum of 5,000 events were collected per
sample, and the samples were gated on forward scatter versus side
scatter to exclude clumps and debris. Dead cells were excluded based on
propidium iodide staining.
Definition of mean channel shift.
Rhodamine efflux experiments were performed on samples from patients
before and following the clinical administration of PSC 833. Fluorescence histograms for CD56+ cells were generated and
the mean channel numbers for the histograms were compared following
rhodamine accumulation with and without exogenous PSC 833 (PSC Control) and following the efflux period with and without exogenous PSC
833 (PSC/Efflux Efflux). The PSC Control
value is calculated from the mean channel numbers after the 30-minute
period of accumulation in which rhodamine (Control) or rhodamine and
PSC 833 (PSC) are added directly to whole blood. The value is
calculated from the mean channel numbers after the 30-minute
accumulation period, lymphocyte separation, wash, and 60-minute efflux
period occurring in rhodamine-free medium with (PSC/Efflux) or without
(Efflux) added PSC 833. A small value or zero value for PSC Control would indicate complete antagonism of Pgp in the cells by the
PSC 833 present in the patient's blood. Similarly, a low or zero value
for PSC/Efflux Efflux would indicate complete antagonism of Pgp
in the cells by the PSC 833 present in the patient's blood, whereas
higher mean channel shifts would indicate less antagonism.
MRK-16 staining and quantitation.
Cultured cells were trypsinized, washed, and incubated in MRK-16
antibody (Kamiya, Seattle, WA) or the IgG2a negative
isotype control (Becton Dickinson) for 30 minutes, washed twice with
DPBS/FCS, and incubated for 30 minutes with fluorescein isothiocyanate
(FITC)-conjugated horse anti-mouse antibody (Vector Laboratories,
Burlingame, CA). In the case of blood samples, PBMCs were stained again
with PE-conjugated CD56 antibody or PE-conjugated
IgG1 isotype control, washed 2 times in DPBS/FCS, and
analyzed. A minimum of 5,000 events was collected. MRK-16 staining was
expressed as the difference of the mean channel value of the isotype
control histogram from the mean channel value of the MRK-16 histogram
or, in the case of the mononuclear cells, the histogram for cells
stained with both MRK-16 and the desired T-cell marker.21
Determination of PSC 833 levels.
The assay is based on the competition of PSC 833 and
[G-3H]Dihydro-PSC 833 tracer in whole blood and binding
to a monoclonal antibody to Cyclosporine A. PSC 833 concentrations in
human blood were determined using PSC 833 radioimmunoassay kits
provided by ANAWA Laboratorien AG (Zurich, Switzerland). Briefly,
appropriate volumes of blood standards, unknowns, and quality control
samples were dispensed into glass tubes. To each sample, 1 mL methanol was dispensed, vortex mixed, and centrifuged at 3,000 rpm at
approximately 4°C for 10 minutes. The supernatant from each tube
was decanted into a clean tube and evaporated to dryness using a vortex
evaporator at approximately 54°C. To each tube, appropriate volumes
of buffer and human plasma were added and vortex mixed to redissolve
all the sample residue. Subsequently, antibody (specific Cyclosporine A
monoclonal antibody) and tracer (3H-Dihydro-PSC 833) were
added, vortex mixed, and incubated overnight at approximately 4°C.
The following morning, prechilled charcoal suspension was added to each
sample. The sample was vortex mixed and incubated for 10 minutes at
approximately 4°C and then centrifuged for 7 minutes at 4°C and
3,000 rpm. The supernatant was decanted into a scintillation vial
containing 10 mL of Picofluor. Each vial was thoroughly mixed and
counted for 5 minutes in a beta counter.
Estimations of the human blood PSC 833 concentrations in samples
were obtained using the computer program RIAPROG
(Mercer Computer Systems, New York, NY). The program converts the raw counts obtained for the standards to the response curve, (Y). The
Y-response versus standard concentration (standard curve) is linearized
by the logit-log method, and the unknown concentrations are
interpolated form the linearized curve.22
Determination of kinetic parameters.
Of the data that we had collected for the 20 patients in the study, 12 were sufficiently complete as to allow a quantitative analysis of the
effect of plasma PSC 833 levels on the reversal of P-glycoprotein. For
these 12 data sets, we plotted the difference between rhodamine
fluorescence in CD56+ cells in the presence of PSC 833 and
in its absence against the plasma concentration of PSC 833. For each
data set, we fitted the points to the equation:
where  is the difference between the rhodamine
fluorescence signal in CD56+ cells obtained during the
accumulation period with and without exogenously added PSC 833 (PSC Control), S is the concentration of PSC 833 in the plasma of
the patient, and Ki is a parameter that determines the
concentration at which a half-maximal effect of the reverser is found.
This equation assumes that the interaction between the reverser and
P-glycoprotein obeys a simple ligand-binding isotherm.23,24
We also fitted the data to the more complex equation:
where n is an additional parameter that accounts for a possible
cooperativity between molecules of the reverser and determines the
slope of the inhibition curve.23,24
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RESULTS |
Rhodamine efflux and MRK-16 staining in mononuclear cell populations.
Figure 1A presents the results of rhodamine
efflux in three different subsets of peripheral mononuclear cells
derived from a normal volunteer. MRK-16 staining of these subsets is
shown in Fig 1B. As noted by other investigators, both MRK-16 cell
surface staining and rhodamine efflux are detectable in the three
subsets: the CD3+ cells, which encompass both the CD4
+ and the CD8+ populations, have the lowest
level of staining, and the least rhodamine efflux of the three;
intermediate levels are observed in the CD8+ population;
and highest levels are observed in CD56+ cells. The
accumulation of rhodamine in the presence of PSC 833 without an efflux
period (PSC) yields a histogram that is identical to that following
efflux in the presence of PSC alone (PSC/Efflux) and thus is not
depicted in the figure.
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| Fig 1.
Rhodamine efflux and MRK-16 staining in T-cell subsets.
(A) Rhodamine efflux in isolated mononuclear cells from a normal
patient. Blank histogram (solid line), cells incubated 30 minutes in
media alone. Control histogram (dotted line), cells incubated 30 minutes in media with 0.5 µg/mL rhodamine 123. PSC/Efflux histogram
(dashed line), cells incubated 30 minutes in media with 0.5 µg/mL
rhodamine 123 and 3 µg/mL PSC 833, washed, and incubated in
rhodamine-free media with 3 µg/mL PSC 833 for 60 minutes. Efflux
histogram (heavy solid line), cells incubated 30 minutes in media with
0.5 µg/mL rhodamine 123, washed, and incubated in rhodamine-free
medium for 60 minutes. Staining with the appropriate T-cell marker was
performed with each sample. (B) MRK-16 staining of normal mononuclear
cells. Blank histogram (solid line), unstained mononuclear cells. IgG
Control histogram (dotted line), cells incubated with mouse
IgG1, stained with FITC-labeled horse anti-mouse
antibody, then stained with the appropriate PE-labeled T-cell
antibody. MRK-16 histogram (dashed line), cells were incubated in
unlabeled MRK-16 antibody for 30 minutes, stained with
FITC-labeled horse anti-mouse, and subsequently labeled with the
desired PE-labeled T-cell marker.
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Rhodamine efflux and MRK-16 staining in multidrug resistant SW620
sublines and patient samples acquired before treatment.
Rhodamine efflux is readily observed in multidrug resistant sublines
selected from SW 620 human colon carcinoma cells. In Fig 2A, increased rhodamine efflux (top
row) and increased MRK-16 staining (bottom row) are observed in the
SW620 Ad10, Ad20, and Ad300 sublines. By quantitative polymerase chain
reaction (PCR), the Ad20 and Ad300 sublines have been shown to
overexpress MDR-1 at levels 100- and 500-fold above the level
found in parental SW620 cells.25 Adriamycin resistance in
the SW620 Ad20 cells can be fully reversed by 10 µg/mL verapamil,
whereas 25 µg/mL verapamil cannot fully reverse resistance in the
Ad300 cells.19 Results from two patients (T19 and T2)
representing the range seen in rhodamine efflux (upper row) and MRK-16
staining (lower row) are shown in Fig 2B. The MRK-16 staining in the
CD56+ cells was highest for patient T2 and appears to be
comparable to that found in the resistant SW620 Ad20 subline, whereas
that of T19 was lowest and appears closer to that found in parental SW620 cells. Figure 2C summarizes the results in 20 patients (empty circles), and the SW620 parental and resistant cell lines (filled squares). MRK-16 staining was correlated with the difference in mean
channel shift values obtained for rhodamine accumulation with and
without added PSC 833, designated PSC Control (right graph of Fig 2C, r = .751), and also with the value for
PSC/Efflux Efflux (left graph of panel C, r = .854).
Both calculations show a significant correlation between rhodamine
efflux and MRK measurement of Pgp.
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| Fig 2.
Correlation of rhodamine efflux with MRK-16 staining. (A)
Rhodamine efflux and MRK-16 staining was performed on SW 620 parental
cells and cells selected in 10, 20, and 300 ng/mL Adriamycin, and
designated as described in Fig 1, except that cells were not subjected
to the CD56 labeling step. (B) Whole blood from each patient was
obtained before initial treatment. Rhodamine efflux and MRK-16 staining
were performed on mononuclear cells, and then stained with PE-labeled
CD56 antibody. Designations as described in Fig 1. (C) The difference
in rhodamine accumulation with and without exogenous PSC 833 (PSC Control) and rhodamine efflux with and without exogenous PSC 833 (PSC/Efflux Efflux) was plotted versus the difference between the
MRK-16 histogram and the IgG1 negative control histogram
for patients before receiving treatment (empty circles) and for the
SW620 parental, Ad2, Ad5, Ad10, Ad20, and Ad300 cell lines (filled
squares).
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Pgp antagonism following vinblastine, paclitaxel, and PSC 833 administration in patients on protocol.
Peripheral mononuclear cells were obtained from patients enrolled on
phase I trials with PSC 833. These patients were treated with a first
cycle of either infusional vinblastine or paclitaxel alone, and then
PSC 833 was administered separately. In this way, the individual
pharmacokinetic parameters of PSC 833 and the chemotherapeutic agent
(taxol or vinblastine) could be obtained. To determine whether the
initial infusion of vinblastine or paclitaxel would affect rhodamine
efflux, peripheral mononuclear cells were obtained before and after the
infusion. No significant difference could be observed in PSC/Efflux Efflux values between the two samples, with the average of
PSC/Efflux Efflux values before chemotherapy being 319 ± 71 and after chemotherapy 338 ± 75. A Student's t-test
yielded a value of P > .5, indicating no difference between
the samples as a result of the initial vinblastine or paclitaxel
treatment.
In contrast, treatment with a single oral dose of PSC 833 resulted in
marked inhibition of rhodamine efflux from the CD56+ cells.
Time course studies were performed on the CD56+ cells, and
serum levels of PSC 833 were obtained.
Figure 3 depicts two time course
studies representing two extremes, one patient with antagonism of
rhodamine efflux and one patient without. Patient T7 (Fig 3A) showed
almost complete antagonism of rhodamine efflux from the
CD56+ cells 2 hours following the first dose of PSC 833. In
contrast, in Patient T12 (Fig 3B), significant reversal was not
observed within the first 2 hours following ingestion of PSC 833. These differences, observed despite the administration of identical PSC 833 doses in the two patients, were correlated with markedly different PSC
833 levels, 3,130 ng/mL and 249 ng/mL for patients T7 and T12,
respectively, 2 hours following PSC 833 administration. This may
represent differences in drug bioavailability or metabolism.
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| Fig 3.
Inhibition of rhodamine efflux from CD56+
cells after initial exposure to PSC 833. Whole blood was obtained 0, 15, 30, 45, 60, and 120 minutes after an initial dose of PSC 833. Rhodamine 123 was added to whole blood and incubated for 30 minutes and
the mononuclear cells were separated and incubated for an additional 60 minutes in rhodamine-free media before staining with PE-labeled CD56
antibody to generate the Efflux histogram (dotted line), or rhodamine
123 and PSC 833 were both added to the blood during the 30-minute
incubation period and the mononuclear cells were separated and
incubated in rhodamine-free media with PSC 833 before PE-labeled CD56
staining to yield the PSC/Efflux histogram (solid line). A small
difference between the Efflux and PSC/Efflux peak signifies greater
blocking of Pgp by PSC 833 in patient blood; a larger difference
indicates poor reversal. (A) Patient T7 shows near complete reversal of
Pgp-mediated rhodamine efflux in CD56+ cells. Reversal
can be seen 45 minutes after a single oral dose of PSC 833. (B) Patient
T12 shows incomplete reversal of Pgp in CD56+ cells.
Virtually no effect is seen 2 hours after administration of PSC 833. The two patients shown received identical doses of PSC 833. The PSC 833 levels in the two patients after two hours are 3130 ng/mL (T7) and 249 ng/mL (T12).
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Determination of mean channel shifts following PSC 833.
The fluorescence-activated cell sorter (FACS) results were quantitated
by determining the difference in rhodamine fluorescence in
CD56+ cells measured in mean channel numbers, termed the
mean channel shift, as a result of adding PSC 833 in the laboratory.
The two values quantitated, PSC Control and PSC/Efflux Efflux, have been previously shown to reflect the degree of inhibition
of P-glycoprotein-mediated drug efflux.20,26 In this
assay, the cells incubated with exogenous PSC 833 will provide a
"maximum" fluorescence value, whereas the fluorescence measured
in the Control and Efflux histograms will depend on the level of PSC
833 present in the patient's plasma. The PSC Control value
compares the increased rhodamine fluorescence in CD56+
cells due to PSC 833 in the patient's plasma to the "maximum" value obtained by the addition of PSC 833 in the laboratory. The second
value, PSC/Efflux Efflux, compares the ability of PSC 833 in
the patient's plasma to increase rhodamine fluorescence in
CD56+ cells during the accumulation period and prevent
rhodamine efflux during the efflux phase to the "maximum"
fluorescence value. This measurement is highest in cells obtained from
patients before PSC 833 administration and has the widest range among
the patients. Because the PBMCs must be allowed efflux in
rhodamine-free media during the efflux assay, the cells are subjected
to a washing step which effectively removes the PSC 833 as well as the
rhodamine. The PSC/Efflux Efflux value, therefore, measures the
effective inhibition of Pgp-mediated rhodamine efflux by PSC 833 remaining inside the cell. Although PSC 833 is poorly transported by
Pgp, it may diffuse from the cell or be effluxed at a low level in the
PSC 833-free, rhodamine-free media used for the efflux assay. Thus, the
channel shift observed in the patient samples following the efflux
period could underestimate the extent of Pgp inhibition occurring in
the patient during continued exposure to PSC 833. As the PSC Control value is not affected by this washing step, the difference
between the PSC and the Control histograms is smaller and provides a
narrower range in which to evaluate differences among patients. As a
result, the data were quantitated in both ways. As seen in
Fig 4, a wide range in basal efflux levels
was observed in patients before administration of PSC 833, at the pre-
and 0-minute time points. Complete inhibition of efflux is seen in the
samples obtained 2 hours following administration of oral PSC 833 as
evidenced by the PSC Control value, with the mean channel
shifts decreasing to an insignificant difference of 0 to 12 channel
numbers in the majority of samples. The differences following the
efflux period, as represented by the PSC/Efflux Efflux values
are markedly reduced, but more variable than the PSC Control
samples. This is most likely due to the wash step mentioned previously.
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| Fig 4.
Effect of PSC 833 in 12 patients studied. (A) Difference
in rhodamine fluorescence in CD56+ cells with and without
exogenous PSC 833 (PSC Control) or following the efflux period with
and without exogenous PSC 833 (PSC/Efflux Efflux) were compared at
the "pre" time point, before initial chemotherapy treatment; the
"peak" time point, 2 hours following ingestion of PSC 833; and
the "trough" time point, 6 hours following PSC 833 administration for 12 patients on study. The peak and trough samples
were obtained after at least 6 days of PSC 833 administration. (B)
PSC Control and PSC/Efflux Efflux values were compared at
0, 15, 30, 45, 60, and 120 minutes after a single dose of PSC 833.
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Correlation of Pgp antagonism with PSC 833 blood level.
For each time point, PSC 833 levels in the blood were measured by
radioimmunoassay. For 10 of the 12 data sets, complete inhibition of
rhodamine efflux was observed
(Fig 5A), with a
Ki value in the range 29 to 181 ng/mL (mean 108 ± 16 SE, n = 10). For two sets (open squares [T5] and open diamonds [T6]
in Fig 5A), higher Ki values, 914 and 916 ng/mL, were
calculated, suggesting that in these two patients, complete inhibition
of rhodamine efflux was not achieved at the PSC 833 levels attained.
However, one aberrant value in patient T6 may have contributed to the
higher Ki. For a number of data sets (in particular for
those where numerous data points were available) the dose dependence of
the reversal effect was steeper than a simple binding isotherm would
predict. Figure 5B depicts two of these data sets, replotted from Fig
5A on a larger scale. In this graph, the dashed lines represent the best-fit predictions modeled on a simple binding isotherm. The solid
lines are the predictions of the more complex model in which a number
of PSC 833 molecules cooperate together to block P-glycoprotein. The
fact that the solid lines visually provide a better fit suggests a
possible interaction between several PSC 833 molecules that results in
greater inhibition of rhodamine efflux. Such a phenomenon has been
documented for several Pgp antagonists in in vitro
experiments.9,27,28
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| Fig 5.
Reversal of Pgp activity by PSC 833 in plasma. Ordinates,
(PSC Control). Abscissas, concentration of PSC 833 in plasma in
ng/mL. (A) Data for 12 different patients. Open squares aberrant
patient T5, open diamonds aberrant patient T6. (B) Data for patients T2
(left panel) and T4 (right panel) replotted from (A). Dashed lines,
best-fit predictions using the simple binding isotherm model; solid
lines, best fits for model that includes cooperative interactions
between molecules of PSC 833. The lines drawn are obtained by using the
Marquardt-Levenberg algorithm.
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| |
DISCUSSION |
As our knowledge of the molecular mechanisms of drug action and
resistance increases, measurement of a drug's effects will enhance
clinical trial designs. For studies designed to overcome Pgp-mediated
resistance, measurement of the extent of in vivo inhibition of
Pgp-mediated drug efflux is deemed essential. To that end, this
manuscript presents the results of an ex vivo assay developed to detect
reversal of multidrug resistance in patients receiving a Pgp
antagonist. Blood samples were obtained from patients on either of two
multidrug resistance reversal studies using PSC 833. Following
incubation of cells with rhodamine 123, peripheral mononuclear cells
were separated by centrifugation, and rhodamine fluorescence in
CD56+ cells was determined by FACS analysis.
Rhodamine efflux was measured in two ways: (1) by comparing rhodamine
fluorescence in cells after an accumulation period with or without
exogenous PSC 833 (PSC Control) or (2) by comparing rhodamine
fluorescence in cells following an efflux period during which
exogenous PSC 833 was or was not present (PSC/Efflux Efflux). Both measurements show inhibition of rhodamine efflux from
CD56+ cells in patients treated with PSC 833.
Efflux of rhodamine from CD56+ cells as a surrogate marker
requires the assumption that Pgp in CD56+ cells has an
identical substrate and antagonist profile to Pgp in cancer cells.
Support for this assumption includes several studies that describe
increased uptake of either a chemotherapeutic agent or a drug surrogate
in the normal liver and kidney, following the administration of Pgp
antagonists in both humans and in animal models.29-34 These
studies support the thesis that Pgp in normal tissues has the
same substrate and antagonist specificities as cancer cells and provide
the basis for the development of a surrogate assay to measure
inhibition of Pgp-mediated drug efflux.
The need for a surrogate marker to establish the extent of inhibition
of Pgp-mediated efflux is clear; the results of clinical trials cannot
be interpreted without knowledge of the extent of Pgp inhibition. The
ideal measurement would be actual drug accumulation in malignant cells.
However, because this ideal is not possible, alternative approaches
have been evaluated, usually with PSC 833, the first "second
generation" antagonist to be evaluated in patients. One such
approach is the inhibition of Tc-99m sestamibi efflux from tumors.
Sestamibi is a radionuclide imaging agent that is a substrate for
Pgp-mediated efflux.35 Imaging studies with sestamibi have
shown efflux from Pgp-expressing tumors in both animals and
man.36-39 Following PSC 833 administration, these studies showed marked increases in sestamibi uptake in the liver and kidneys as
well as an increase in a subset of tumors.36,37,40 However, a dose-response relationship between sestamibi uptake and PSC 833 level
was not shown.37 This may be related to interpatient variability in tumor or normal tissue uptake that may or may not be due
to Pgp expression. Additional studies are needed to establish the
sensitivity of sestamibi imaging to determine its adequacy as a
surrogate marker and to evaluate how well the pharmacodynamics of
sestamibi reflect the pharmacodynamics of the anticancer agents.
A second approach to evaluate the extent of Pgp inhibition has been to
add serum or plasma from patients treated with Pgp modulators to an in
vitro assay comprised of multidrug-resistant cells and a detectable Pgp
substrate.9 Rhodamine or radiolabeled daunomycin or
mitoxantrone have been used as substrates in this assay. Lehnert et al
showed a threefold to eightfold increase in rhodamine retention in
8226/Dox6 human myeloma cells, using serum from patients treated with
dexverapamil.41 Solary et al showed a 20% to 80% increase
in mitoxantrone uptake in CEM/VBL cells exposed to serum from patients
with quinine levels above 4 µg/mL.42 As with the results
described herein, these methods do not provide information about
antagonist levels or availability in tumor tissue itself.
One outcome of our study is the observation that a plateau in efflux
antagonism occurs, suggesting that the levels of PSC 833 achievable in
patients reach a maximum inhibition of Pgp-mediated efflux. The plateau
begins at approximately 1,000 ng/mL, a concentration well below that
achieved at maximally tolerated doses of PSC 833. Calculations using
experimental models with high levels of P-glycoprotein suggest that a
single modulator cannot completely inhibit Pgp-mediated efflux.43 The plateaus observed in the present study are
consistent with the absence of complete reversal. However, they
indicate substantial (near maximal) reversal, which may be sufficient
to improve drug efficacy. Additionally, we found that the data most fit
a model in which cooperativity between molecules of PSC 833 is assumed,
as shown in Fig 5B. Cooperativity may be beneficial to the patient
because it means that the reversal effect happens over a small
concentration range.
Furthermore, this assay offers the ability to follow reversal of
Pgp-mediated efflux clinically, to ensure that adequate levels are
reached. It could also guide the addition of a second modulator, by
indicating whether further inhibition of efflux can be achieved, and it
could similarly guide the clinical use of Pgp antagonists by helping to
establish the minimum dose of a modulator needed to inhibit drug
efflux. This is particularly exemplified in Fig 3, in which the
histograms of patient T7 suggest that PSC 833 provides complete
antagonism, while little is observed in CD56+ cells of
patient T12. The PSC 833 blood levels of the two patients, 3,130 ng/mL (T7) and 249 ng/mL (T12) at 2 hours are concordant with the
reversal observed.
The possibility that the inhibition of rhodamine efflux from
CD56+ lymphocytes may not accurately reflect the inhibition
of anticancer drug efflux from a solid tumor must be considered. A
number of factors may dictate results that are inferior to those
obtained in the surrogate assay using circulating cells. These factors include (1) decreased drug and modulator uptake in tumor due to poor
vascularity and perfusion, increased interstitial pressure, and binding
to interstitial macromolecules; (2) increased affinity of the
anticancer drug for intracellular targets, such that less compound is
available for efflux, relative to rhodamine, which is compartmentalized
in mitochondria and readily effluxed from the cell; and (3) differences
in affinity of the anticancer drug for Pgp, because not all compounds
are equally well transported. These factors preclude precise
conclusions about Pgp reversal in solid tumors. This assay could also
be used in hematopoietic malignancies to confirm effective inhibition
of drug transport. Because the malignant cells are circulating, the
degree of Pgp antagonism should be comparable to that observed in
CD56+ cells.
Similarly, the potential for variability in Pgp expression in
lymphocytes must also be considered. As shown in Fig 2, increased expression of Pgp as measured by MRK-16 staining correlates with a
decreased PSC 833 effect, and a range in expression can be detected. Furthermore, in one or two patients, antagonism was inferior compared with that achieved in the other patients. An expanded study, with increased numbers of patients would provide information about how
frequently this type of aberration would be observed. Very high levels
of Pgp, or the presence of another transporter, could potentially
explain the failure to antagonize the rhodamine efflux in patient T5.
Several reports suggest interpatient variation in the expression of Pgp
in circulating lymphocytes. An early study provided indirect evidence
that prior chemotherapy (with Adriamycin) may increase Pgp level or
function,44 whereas increased expression was noted in
rheumatoid arthritis patients receiving prednisolone45 and
in patients undergoing a kidney transplant following treatment with
cyclosporin A.46 However, a subset analysis was not
performed in these two studies, precluding a direct extrapolation of
these findings to the CD56+ population. Furthermore, an
increase in Pgp expression in CD4+ and CD8+
lymphocytes obtained from older patients suggests a potential correlation between age and Pgp expression.47 This
variability could impact on the clinical utility of the rhodamine
reversal assay described here.
Clearly, the ideal assay would determine inhibition of drug efflux from
tumor cells. However, with the exception of leukemia or multiple
myeloma, where repetitive sample acquisition is possible, this ideal
cannot be achieved in the clinical setting. Despite its limitations, we
believe the rhodamine assay described here should provide valuable
information. The ability to obtain serial specimens with minimal risk
to patients, its relative simplicity, and its proven accuracy and
reproducibility suggests it can serve as a surrogate marker for
modulation of Pgp in patients.
| |
ACKNOWLEDGMENT |
The authors thank Anne Rutt for coordinating the collection of patient
samples, the nurses of 12W and 13E for collection of patient samples,
and Susan Mertins for advice on statistical calculations.
| |
FOOTNOTES |
Submitted June 1, 1998;
accepted September 4, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Susan Bates, MD, NIH, NCI, 9000 Rockville
Pike, Bldg 10 Room 12N226, Bethesda, MD 20892; e-mail:
sebates{at}helix.nih.gov.
| |
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Top
Abstract
Introduction
