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Xenova, Ltd., Slough, SL1 4EF, United Kingdom
ABSTRACT
XR9576 is a
novel inhibitor of P-glycoprotein (P-gp) that has been shown to reverse
P-gp-dependent multidrug-resistance in tumor cell lines and
tumor-bearing animals. Here we report the first i.v. and p.o.
administration to healthy volunteers of XR9576 in dose-escalating
studies with the aim of investigating its effects on safety, its
pharmacokinetics, and a surrogate marker of efficacy. XR9576 was
administered as a single dose-upward titration of 0.1, 0.2, 0.5, 1.0,
and 2 mg/kg XR9576 i.v. or 50, 100, 200, 500, and 750 mg/volunteer p.o.
The surrogate marker for in vivo efficacy examined the
accumulation of the P-gp substrate Rhodamine-123 (Rh-123) in
P-gp-expressing CD56+ lymphocytes by flow cytometry.
Addition of Rh-123 to blood samples from subjects given XR9576 or a
placebo demonstrated drug-dependent modulation of P-gp activity. Even
at the lowest doses, significant effects were observed on Rh-123
accumulation in CD56+ cells. Maximal effects were seen
during the i.v. infusion or 4–6 h after oral administration. As the
dose was increased, a concomitant rise in the level and duration of
P-gp blockade was observed. A dose of 2.0 mg/kg i.v. and 
200
mg/volunteer p.o. gave 
100% inhibition of P-gp for in excess of
24 h. All doses of XR9576 were well tolerated. Inhibition
increased with XR9576 plasma concentration, and maximal activity was
achieved at 150–200 ng/ml XR9576. In conclusion, XR9576 has
demonstrated sustained inhibition of P-gp after i.v. and oral
administration and, supported by the elimination half-life of about
24 h, XR9576 is being taken into Phase II as a once-daily
agent.
INTRODUCTION
MDR2 attributable to the overexpression of P-gp is known to develop in a wide variety of tumor types and significantly attenuates the efficacy of many commonly used chemotherapeutic agents (e.g., doxorubicin and paclitaxel; Ref. 1 ). Expression of P-gp has been linked to MDR in both intrinsically resistant tumors and in those that acquire the resistant phenotype during treatment (2) . P-gp is a member of the ATP-binding cassette superfamily and acts as an efflux pump to reduce the intracellular concentrations of cytotoxics (2) .
Significant improvement has been made since it was discovered that first-generation inhibitors, originally designed for targets other than P-gp (e.g., the calcium channel blocker verapamil) had unacceptable toxicities in clinical studies (3 , 4) . More recently, molecules have been designed to interact specifically with P-gp, and a number of these, including LY79553 (5) and PSC833 (6) , have advanced to the clinic. PSC833 is at the most advanced stage of clinical development (Phase III) and is reported as sensitizing refractory ovarian tumors to paclitaxel (7) .
XR9576, also designed as a specific P-gp modulator (Fig. 1)
, is a highly potent novel anthranilic
acid derivative that potentiates the activity of a number of
MDR-associated cytotoxics in cell lines overexpressing P-gp (8
, 9)
. Typically it has half-maximal effects at approximately 30
nM. This in vitro activity translates to
tumor-bearing animals where XR9576 has been shown to reverse the
resistance of multidrug-resistant human xenografts at well-tolerated
doses (9)
. XR9576 has a high affinity for P-gp
(Kd = 2.5 ± 0.7
nM; Ref. 10
) and, importantly, has
no effect on the related transporter multidrug-resistance-associated
protein (8)
.
|
MATERIALS AND METHODS
Clinical Study Design.
The studies were rising-single-dose, randomized, double-blind, and
placebo-controlled in healthy male volunteers to assess the
tolerability, pharmacokinetics, and effect on a surrogate marker of
efficacy of single doses of i.v.- and p.o.-administered XR9576. All
volunteers gave informed consent, and study entry requirements
included an age range of 18–55 years, being within 15% of the normal
height for weight according to Metropolitan Life Assurance
tables, and having no clinically significant abnormalities at
screening. All volunteers underwent a complete medical history and
examination that included the cardiovascular, respiratory, hemopoietic,
central and peripheral nervous, gastrointestinal, endocrine, and
renal systems. Laboratory tests included a 12-lead ECG, full blood
count with differential white cell and platelet counts, urea and
electrolytes, liver function tests, and measurement of serum proteins,
calcium, phosphate glucose, and creatinine. Panels of five
subjects (four on XR9576 and one on a placebo) received single doses of
the drug at the given dose-level on only one occasion. XR9576 was
administered as a 250-ml i.v. infusion over 30 min (mesylate salt) or
in hard gelatin capsules (free-base; 50 or 100 mg base/capsule). Blood
samples were taken into heparinized Vacutainers (Becton Dickinson) at
the time points shown in the results section for measurement of XR9576
plasma levels and surrogate marker activity
(CD56+ cells).
Assay of Plasma Drug Levels.
XR9576 was assayed in human plasma by a validated procedure involving
SPE followed by liquid chromatography with tandem mass-spectrometric
detection. Aliquots of the plasma samples (100 µl) and an
internal standard (structurally related to XR9576) were mixed and
centrifuged. The supernatants, containing XR9576 and the internal
standard, were extracted using OASIS HLB SPE cartridges. The eluates
from the SPE cartridges, containing the analytes, were subjected to
tandem mass-spectrometric detection analysis. The lower limit of
quantification for XR9576 in human plasma was 2.5ng/ml with linearity
demonstrable to 1000 ng/ml. Full details of the analytical procedure
will be published elsewhere.
Surrogate Marker Assay.
The assay for inhibition of P-gp in CD56+
lymphocytes was carried out using modifications of the method published
by Witherspoon et al. (11)
Duplicate blood
samples were aliquoted into polypropylene tubes (0.5 ml/tube) and
processed in parallel. Predose samples were split into 4 x 0.5 ml
aliquots, two of which (duplicates) were spiked with XR9576 to a final
concentration of 3.0 µM from a
30-µM stock in RPMI. This was incubated
at 37°C for 1 h prior to addition of 55 µl of Rh-123 (Sigma)
from a 10-fold concentrated stock in RPMI/25 mM
HEPES to give a final concentration of 150 ng/ml. For all other samples
that were not spiked, 50 µl of Rh-123 were added. Samples were
incubated at 37°C for 1 h then chilled on ice and stored at
4°C with agitation until preparation of WBCs. Preliminary
studies had confirmed that overnight storage of blood samples did not
disrupt the assay (data not shown).
WBCs were prepared by dilution of the chilled blood samples with 20 volumes of erythrocyte lysis buffer [0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA (pH 7.2–7.4), containing 0.04% sodium azide; all reagents from Sigma) at 4°C. Samples were then agitated gently for 1 h at 4°C before being centrifuged at 4°C. The cell pellet contained principally WBCs, with only minor contamination with RBCs. Cells were transferred to a 4-ml flow cytometry tube and washed twice with PBS/0.1% BSA/0.04% sodium azide (Sigma) before staining with an anti-CD56+ monoclonal antibody or an IgG1 isotype control (both Becton Dickinson) using conditions recommended by the supplier. After 30 min incubation on ice, cells were washed before being resuspended in 0.5 ml PBS/BSA/azide containing 1 µg/ml PI (Sigma) to stain for dead cells.
Flow cytometry analysis was carried out using an Ortho Diagnostics flow cytometer (Cytoron Absolute) using three color detection on 65,000 total events as follows: (a) green, Rh-123; (b) orange, anti-CD56-PE; and (c) red, PI. Using a gate on the PI-negative population of lymphocytes, it was possible to measure the orange and green fluorescence only in live cells. IgG1 isotype controls were used to set a cutoff whereby Rh-123 accumulation was measured only in CD56+ cells. Analysis was carried out using Ortho Diagnostics Immunocount software.
Flow cytometry analysis generated data sets for the amount of green
fluorescence (MCF) in the CD56+ population of
WBCs that directly correlated with the amount of cell-associated
Rh-123. The MCF was converted to percentage inhibition using the
predose and the 3-µM XR9576 (final concentration) spike
controls as follows:
 |
All data represent the mean of duplicate points.
RESULTS
Inhibition of P-gp in CD56+ Cells by XR9576
ex Vivo.
Flow cytometry analysis of WBCs from healthy volunteers allowed
simple separation of the lymphocyte population (Fig. 2
A, Gate A) from
monocytes and granulocytes. Low numbers of residual RBCs were excluded
by setting a threshold on the forward and side-scatter
measurements. This lymphocyte population typically contained
>95% viable cells as judged by exclusion of PI (data not shown). The
CD56+ subpopulation, visualized by addition of a
phycoerythrin-conjugated antibody (orange fluorescence)
represented 5–30% of total viable lymphocytes (Fig. 2
B,
Gate C). Significant variation in this number was seen
between volunteers. Although all lymphocytes accumulated Rh-123
(green fluorescence), the addition of a XR9576 spike to
whole blood before purification of WBCs resulted in a significant
increase in the level of Rh-123 accumulation (Fig. 2
D,
Gate C). This increase was greater in the
CD56+ population than in the remaining
lymphocytes because of the higher level of P-gp in these cells.
|
. Rh-123 fluorescence levels were
converted to a relative measure of P-gp inhibition as described in
"Materials and Methods." This demonstrated clear inhibition of P-gp
activity by XR9576 with half-maximal effect at approximately 30
nM over a number of experiments using blood from different
volunteers.
|
). At this dose level, data
were only gathered on four volunteers (three on XR9576 and one on
a placebo). Subject 1 received a placebo and showed minimal fluctuation
in basal Rh-123 accumulation. Subjects 2–4 were given XR9576 and
significant blockade of P-gp activity was seen in their
CD56+ cells. Maximal inhibition in this cohort
(range, 78–92%; mean, 83% ±6.3 SD) was seen during the infusion
period, after which the inhibition dropped to 40% of maximum at 1 h followed by a decline between 2 and 6 h. P-gp inhibition was
still evident at 6 h post-dose (range, 16–40%; mean, 24.5%
±12.9). Additional time points to 24 h were collected for
subsequent dose levels.
|
greater effects on Rh-123 accumulation
in CD56+ cells were observed. During the infusion
period, 85% of maximal inhibition was seen with 0.5 mg/kg, and this
was increased to complete (100%) inhibition at 1.0 and 2.0 mg/kg. In
addition to this increased effect during the infusion period, the
duration of P-gp blockade was also increased. A dose of 2.0 mg/kg gave
complete inhibition for in-excess-of 24 h. The 1-mg/kg cohort did
not include a placebo, so it cannot be corrected for drift of the
baseline. However, given the level of inhibition seen at the earliest
time points, and from trends seen throughout this and other studies, it
can be inferred that this dose level also gave close to full inhibition
for 24 h.
|
. The inhibition of P-gp at individual
time points in individual subjects was also seen to correlate with
XR9576 plasma concentrations measured in the same samples (Fig. 7)
up to a plasma concentration of
150–200 ng/ml that gave 100% blockade.
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The lowest oral dose of XR9576 (50 mg) gave significant
inhibition of P-gp activity in all four subjects who received the drug
(Fig. 8)
. The level of inhibition varied
between 40 and 80% (mean 65.6% ±17.0) with a
Tmax between 2 and 6 h. Subject
30, who received XR9576 and had the lowest level of inhibition, also
had substantially lower plasma levels of XR9576 than the other subjects
(data not shown). Inhibition of P-gp slowly declined over time, but at
24 h, a mean of approximately 20% inhibition was still apparent.
This value for inhibition of P-gp may be an underestimate, as the
values obtained from the subject given a placebo (subject 28) showed a
downward drift between 0 and 24 h. Normalization of the
XR9576-treated group data to the placebo values would result in
an increase in the apparent P-gp inhibition. Nevertheless, even in the
absence of normalization of the data it is clear that XR9576 gave
significant inhibition of P-gp after oral administration.
|
. Administration of 200, 500, and 750
mg all gave complete (100%) inhibition of P-gp activity in
CD56+ cells between 4 and 12 h. The highest
dose administered (750 mg) gave complete inhibition for in-excess-of
24 h, whereas 200 and 500 mg gave 80 and 70% inhibition at
24 h, respectively.
|
Although Phase I studies are primarily designed to assess the safety, tolerance and pharmacokinetics of a novel drug, we chose to include a surrogate marker for XR9576 activity and to use this information to guide the selection of the XR9576 dose for Phase II studies. The CD56+/Rh-123 surrogate marker assay (11) has been used previously to show activity of the P-gp modulator, GG918 (12) . This assay, which uses the relatively high expression of P-gp on CD56+ lymphocytes as a surrogate for multidrug resistant tumor cells, can be used to look at P-gp transport by flow cytometry in the presence or absence of a P-gp modulator. We have shown previously that XR9576 is a potent and specific P-gp modulator (8 , 9) , and this system uses the relatively high expression of P-gp on CD56+ lymphocytes. Thus we believe that the XR9576-mediated effect on Rh-123 accumulation reflects modulation of P-gp in this system. The data obtained in this Phase I trial are particularly encouraging when correlated with preclinical studies where XR9576 was seen to accumulate in tumors relative to plasma and where efficacy was observed in solid tumors (9) . This correlation with preclinical data is important because one possible limitation of the surrogate marker assay is that P-gp modulation in a CD56+ lymphocytes may not address all of the issues associated with delivery of a drug to solid tumors.
Using this assay we have shown that an appropriate single dose of XR9576, administered by either the i.v. or the oral route, gives complete inhibition of P-gp for in excess of 24 h. Although the clinical trial design allowed for only one placebo control at each dose level, the data for these samples were consistent over the study and throughout the assay validation before initiation of the clinical trial (data not shown). These data demonstrate that XR9576 is a very effective modulator of P-gp in humans and support the excellent preclinical profile generated for this compound (8 , 9) .
The clear correlation between XR9576 plasma concentrations and
inhibition of P-gp has allowed the definition of a target concentration
of XR9576 for future studies where it may not be practical to run a
surrogate marker assay. The results described here demonstrate that a
XR9576 plasma concentration of approximately 150–200 ng/ml gave
complete inhibition of P-gp. Plasma levels of XR9576 were in excess of
the 200-ng/ml target concentration for in-excess-of 24 h
(13)
, resulting in complete inhibition of P-gp throughout
this period after i.v. (1.0 mg/kg) and oral administration (100
mg/volunteer). This suggests that XR9576 can be administered on a
once-daily dosing regime and is supported by an elimination half-life
of approximately 24 h.3
Importantly,
XR9576 was well tolerated throughout these studies. No drug
abnormalities were noted in any of the hematological or clinical
chemistry parameters measured. These properties of XR9576 should allow
simple incorporation into combination protocols with anticancer agents.
In conclusion, these data confirm the potential of XR9576 in the treatment of multidrug resistant tumors using well-tolerated regimes. The data have also supported the choice of XR9576 dose (150 mg/volunteer i.v. and 500 mg/volunteer p.o.) for Phase IIa clinical trials in a number of centers in the United States and the United Kingdom.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom requests for reprints should be
addressed, at Xenova, Ltd., 240 Bath Road, Slough, SL1 4EF, United
Kingdom. Fax: 44-1753-706607. 
2 The abbreviations used are: MDR,
multidrug-resistance; P-gp, P-glycoprotein; SPE, solid phase
extraction; Rh-123, Rhodamine-123; PI, propidium iodide; MCF, mean
channel fluorescence.
Received 4/10/00; revised 8/28/00; accepted 8/30/00.
REFERENCES
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