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Vol. 304, Issue 3, 1085-1092, March 2003
Department of Pharmaceutics, University of Minnesota, Minneapolis, Minnesota (H.D., W.F.E.); Novartis Pharma AG, Preclinical Safety, Basel, Switzerland (P.M., M.L.); and Novartis Pharma, East Hanover, New Jersey (M.H.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The adequate distribution of STI-571 (Gleevec) to the central nervous
system (CNS) is critical for its effective use in CNS tumors.
P-glycoprotein-mediated efflux in the blood-brain barrier may play a
role in the CNS delivery of this drug. Whether STI-571 is a substrate
of P-glycoprotein was determined by examining the directional flux of
[14C]STI-571 in parental and MDR1-transfected Madin-Darby
canine kidney (MDCK) II epithelial cell monolayers. The
basolateral-to-apical flux of STI-571 was 39-fold greater than the
apical-to-basolateral flux in the MDR1-transfected cells and
8-fold greater in the parental cell monolayers. This difference in
directional flux was significantly reduced by a specific P-glycoprotein
inhibitor
(2R)-anti-5-{3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy}quinoline trihydrochloride (LY335979). The role of P-glycoprotein in the CNS distribution of STI-571 was examined in vivo, using wild-type and
mdr1a/b (
/) knockout mice that were orally administered 25 mg/kg
[14C]STI-571. In the wild-type mice, the brain-to-plasma
STI-571 concentration ratio at all time points was low (1-3%);
however, there was an 11-fold greater brain partitioning of STI-571 at 1 h postdose in the mdr1a/b (/) mice compared with the
wild-type mice. When 12.5 mg/kg STI-571 was given intravenously, the
brain-to-plasma ratio of STI-571 in the mdr1a/b (/) mice was
approximately 7-fold greater than that of wild-type mice up to 120 min
postdose. These data indicate that STI-571 is a substrate of
P-glycoprotein, and that the inhibition of P-glycoprotein affects the
transport of STI-571 across MDCKII monolayers. Moreover, P-glycoprotein
plays an important role in limiting the distribution of STI-571 to the CNS.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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STI-571
(Gleevec) is an inhibitor of BCR/ABL tyrosine kinase (Buchdunger et
al., 1996
), stem cell factor receptor (c-kit) kinase (Buchdunger et
al., 2000; Heinrich et al., 2000), and platelet-derived growth factor
receptor (PDGFR) kinase (Buchdunger et al., 2000). BCR/ABL tyrosine
kinase is overexpressed in chronic myelogenous leukemia (CML) patients
because of a chromosomal translocation (Philadelphia chromosome) and is
responsible for the oncogenesis of CML (Rowley, 1973; Daley et al.,
1990; Melo, 1996). STI-571 has been shown to be effective for CML
patients, including those who are refractory to interferon-
treatment (Druker et al., 2001). In one study, 53 of 54 patients
examined showed complete and lasting hematological remission (Druker et
al., 2001). The success of this compound in the treatment of CML has
led to the broader examination of its application in the treatment of
other tumors, such as glioblastoma (Kilic et al., 2000; Uhrbom et al.,
2000), small cell lung cancer (Krystal et al., 2000; Wang et al.,
2000), and gastrointestinal stroma (Joensuu et al., 2001; Tuveson et
al., 2001; van Oosterom et al., 2001), in which c-kit or PDGFR is
expressed and participates in the autocrine loop (Uhrbom et al., 2000;
Wang et al., 2000; Tuveson et al., 2001). Animal experiments with nude
mice suggest that STI-571 may be effective against glioblastoma
xenograft (Kilic et al., 2000). Currently, several clinical trials of
glioblastoma patients treated with STI-571 are under way [e.g.,
National Cancer Institute protocol 01-C-0243, a phase I/II trial of
STI571 (NSC716051) in patients with recurrent malignant gliomas].
However, the treatment of CNS tumors is often limited by low
distribution of the antitumor agents into brain because of blood-brain
barrier. Various efflux transporters expressed in the blood-brain
barrier, including P-glycoprotein, can eliminate xenobiotics from the
brain and further limit their CNS distribution (Kusuhara and Sugiyama,
2002). A limited distribution of STI-571 to the cerebrospinal
fluid in humans has been reported (Leis et al., 2001; Petzer et
al., 2002; Takayama et al., 2002), but factors responsible for this low
distribution have not been characterized. This is an important issue
not only for treatment of the primary CNS tumor such as glioblastoma,
but also it may be critical for CML patients, considering that CNS
relapses have been observed in CML patients even though they have
showed a complete hematological response to STI-571 (Leis et al., 2001;
Petzer et al., 2002; Takayama et al., 2002). The objective of this
study was to determine whether STI-571 is a substrate of P-glycoprotein and to examine the role of P-glycoprotein in the distribution of
STI-571 into the brain.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals. STI-571 and [14C]STI-571 (both as the mesylate salt) were provided by Novartis Pharma AG (Basel, Switzerland). (2R)-Anti-5-{3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy}quinoline trihydrochloride (LY335979) (Zosuquidar.3 HCl) was kindly provided by Eli Lilly Cancer Research Laboratory (Indianapolis, IN). Tritiated inulin was purchased from Moravek Biochemicals (Brea, CA). All other chemicals were reagent grade or better.
Cellular Flux of STI-571 across the MDR1-Transfected and Wild-Type Madin-Darby Canine Kidney (MDCK) II Monolayers. MDCKII (wild-type and MDR1-transfected) were kindly provided by Dr. Piet Borst (Netherlands Cancer Institute, Amsterdam, The Netherlands) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2.5% penicillin and streptomycin at 37°C in a humidified incubator with 5% CO2. MDCKII cells (1 × 106, wild-type and MDR1-transfected) were seeded in six-well polyester membrane inserts (Transwell, Costar Brand Tissue Culture Products; Fisher Scientific Co., Pittsburgh, PA) with a pore size of 0.4 µm and a diameter of 24.0 mm. The media were changed every 24 h, and a polarized monolayer was established in 3 days. After the monolayers were established, the directional transport of a tracer concentration (1.1 µg/ml) of [14C]STI-571 across the cellular monolayer was examined. Briefly, media were removed from the chambers and the monolayers were washed with phosphate buffer (pH 7.2). The stock solution of [14C]STI-571 was prepared in ethanol and diluted 100-fold with the assay buffer containing122 mM sodium chloride, 25 mM sodium bicarbonate, 10 mM glucose, 10 mM HEPES, 3 mM potassium chloride, 1.2 mM magnesium sulfate, 1.4 mM calcium chloride, and 0.4 mM potassium phosphate dibasic (pH 7.4). Then, 0.01 µCi of [14C]STI-571 in 2 ml of assay buffer was applied to the donor chamber (either the apical side or the basolateral side). The receiver chamber was filled with 2 ml of blank assay buffer. At 0, 15, 30, 60, 90, 120, and 180 min, 200 µl of assay buffer was sampled with replacement from the receiver chamber. The samples were mixed with 4 ml of scintillation cocktail (ScintiSafe Econo 1 cocktail; Fisher Scientific Co.) and counted for beta radioactivity using a Packard Tri-Carb 2500 liquid scintillation counter. The measured radioactivity over the initial radioactivity of the donor chamber is calculated as percentage of the radioactivity transported across the monolayer at different time points (% transported). When the apical side (upper chamber) serves as the donor chamber, the flux from apical-to-basolateral (A-to-B) side of the monolayer was measured and vice versa the basolateral-to-apical (B-to-A) flux was measured. The percentage of STI-571 transported was plotted as a function of time, the slope of which is related to the first order rate constant (K) for the steady-state flux and effective permeability (Peff) as follows: K = slope/100 and Peff = K · V/Area, where V and Area are the volume of the donor chamber and effective cellular surface area of the insert, respectively.
When a specific inhibitor of P-glycoprotein, LY335979, was used, the MDR1-transfected MDCKII monolayer was first preincubated with 1 µM LY335979 in assay buffer for 30 min and then the A-to-B flux and the B-to-A flux were measured with 1 µM LY335979 present in both chambers.CNS Distribution of STI-571 in mdr1a/b (/) Knockout Mice and
Wild-Type Mice.
The mdr1a/b (/) knockout and wild-type mice
(129/Ola × FVB) used for this study were purchased from Taconic
Farms (Germantown, NY). In oral dosing experiments, the mice received
25 mg/kg [14C]STI-571 [approximately 3 µCi
of tracer in a 200-µl saline solution (6.7 ml/kg)] as a single oral
dose via gavage. At different times postdose (30, 60, and 120 min,
n = 4 for each time point), the mice were euthanized
and the plasma and total brain tissue were collected. The brain tissue
was homogenized in 3 volumes of saline phosphate buffer with 5%
albumin using a manual tissue homogenizer (Schinkel et al., 1996). Then
0.2 ml of plasma or brain homogenate was mixed with 4 ml of
scintillation fluid and counted for radioactivity using liquid
scintillation counting. In addition, the plasma and brain tissue
homogenate collected at 60 min postdose were analyzed by HPLC to
separate the parent drug from its metabolites. Briefly, the sample was
deproteinized with 2 volumes of acetonitrile at room temperature. After
centrifugation (6000g), evaporation, and reconstitution with
mobile phase, 30 µl of the solution was injected onto the HPLC column
(base-deactivated C18; Thermo Hypersil, Keystone Scientific Operations, Bellefonte, PA). The mobile phase was 5% acetonitrile, 4% tetrahydrofuran, and 0.5% triethylamine (w/w) in 25 mM phosphate buffer (pH 3), isocratic, at a rate of 0.25 ml/min. The
column eluant was collected every 15 s for 2 min around the peak
of interest (the retention time of STI-571, approximately 18 min) and
every 2 min otherwise for a total of 22 min. The collected eluant was
measured for radioactivity using liquid scintillation counting. The
brain-to-plasma ratio is the ratio of disintegrations per minute per
gram of brain tissue over disintegrations per minute per milliliter of
plasma. The radiopurity of [14C]STI-571
administered was 98.3%, as stated in the certificate of analysis
(Novartis Pharma AG).
Determination of the Brain Vascular Space by Dual-Labeling Using [3H]Inulin and [14C]STI-571. The brain vascular space was determined by [3H]inulin administered via tail vein together with oral dosing of [14C]STI-571. Briefly, the mice were given an oral dose of [14C]STI-571, and 10 min before sacrificing the mice, [3H]inulin (1 µCi in 0.9% saline) (molecular weight 5000-5500) was administered via the tail vein. After euthanasia by CO2 inhalation, plasma and total brain tissue homogenate were collected and counted for both 3H and 14C radioactivity using spectrum-based dual label counting.
Intravenous Administration of STI-571 to the Wild-Type and
mdr1a/b (/) Mice.
The mice received 12.5 mg/kg STI-571 [in
100 µl of 0.9% saline (3.3 ml/kg)] via tail vein injection. The
plasma and total brain tissue were collected at different times (30, 60, and 120 min postdose, n = 4 for each time point) as
described above. STI-571 was measured using LC-MS as described in
Bakhtiar et al. (2002).
Statistical Analysis. Groups were compared using simple one-way analysis of variance analysis with a significance level of p < 0.05 (Microsoft Excel, 1997). To compare the brain-to-plasma ratios in the wild-type and knockout mice at 1 h postoral dose, where the variance was not equal between groups, the nonparametric alternative of a two-sample t test, the Mann-Whitney test, was used.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Directional Flux across the MDCKII Monolayer.
The directional
flux study measures the percentage of compound in the donor compartment
transported from the A-to-B side and from the B-to-A side of MDCKII
monolayers at different time points. P-glycoprotein is located in the
apical side of the monolayer and it transports substrates from the
basolateral to apical side, thereby decreasing the A-to-B flux and
increasing the B-to-A flux of a substrate, while not affecting the flux
of the nonsubstrates. As shown in Fig. 1,
the B-to-A flux of STI-571 was significantly greater (p < 0.001) and the A-to-B flux was significant lower (p < 0.001) in P-glycoprotein-transfected cell monolayers compared with
wild-type cell monolayers. Also, there was a significant difference
between A-to-B flux and B-to-A flux in both P-glycoprotein-transfected (p < 0.001) and wild-type cell lines
(p < 0.001). However, this difference is significantly
greater in P-glycoprotein-transfected cell monolayer than wild-type
cell monolayer (p < 0.001). The ratio of B-to-A flux
over A-to-B flux is 39 in MDR1-transfected cells and about 8 in
wild-type cells (Fig. 1). These data indicate that the expression of
P-glycoprotein influences the transcellular transport of STI-571 across
this model barrier. The difference between A-to-B and B-to-A flux in
wild-type cells suggests that an endogenous active transporter that
facilitates B-to-A transport, possibly P-glycoprotein, is expressed in
these cells. As shown in Fig. 2, a
specific inhibitor of P-glycoprotein (Starling et al., 1997; Dantzig et
al., 1999), LY335979, significantly reduced the difference between
A-to-B and B-to-A fluxes in the MDR1-transfected MDCKII monolayer,
strongly suggesting the endogenous transporter is P-glycoprotein (Fig.
2). These in vitro cellular experiments lead to the conclusion that
STI-571 is a substrate of the active efflux transporter P-glycoprotein.
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Brain Penetration of STI-571 in mdr1a/b (/) Knockout Mice and
Wild-Type Mice.
When using whole brain homogenates to determine
the distribution of a drug into the brain, the drug remaining in the
brain vascular space needs to be excluded from the drug in the brain tissue to accurately determine the penetration of drug across the
blood-brain barrier. This is especially important when the drug of
interest has a relatively low CNS distribution. To accomplish this in
the current study, the volume of brain vascular space was measured
using [3H]inulin, which is assumed to not
penetrate the blood-brain barrier (Smith et al., 1988). Our results
from total brain homogenates show that the volume of brain vascular
space in mice accounts for 1.4% of the whole brain volume (Fig.
3). This value is comparable with those
of previous studies using inulin and in situ perfusion in the rat and
mouse, where the brain distribution volume of inulin was calculated to
be about 1.2% (Abbruscato et al., 1997; Murakami et al., 2000). Upon
oral administration of radiolabeled STI-571, only 2.7 to 3.3% the
total radioactivity is distributed into the brain, as depicted by the
brain-to-plasma ratio (Fig. 3). Of this percentage of radiolabeled
material, when considering the radioactivity in the brain vascular
space using the inulin distribution data, it can be seen that about 40 to 50% of the total radiolabeled material in the brain is localized
within the brain vascular space. Therefore, all the brain distribution
values of STI-571 radioactivity reported hereafter are corrected for
the brain vascular space using the value measured by
[3H]inulin (1.4%).
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). It has been shown in our in vitro
directional flux studies that STI-571 is a substrate of P-glycoprotein
and it can therefore be hypothesized that the CNS distribution of
STI-571 will be greater in the mdr1a/b (/) knockout mice. When
measuring the total radioactivity in brain homogenates and plasma after
oral dosing, the penetration of STI-571 radioequivalents into brain
tissue (brain-to-plasma ratio) in mdr1a/b (/) knockout mice was
1.37-fold greater at 30 min, which is not statistically significant
(p > 0.05), and significantly greater at 60 min
(2.73-fold, p < 0.01) and 120 min (2.3-fold, p < 0.001), compared with that of wild-type mice (Fig.
4). Treatment of the wild-type mice with
the P-glycoprotein-specific inhibitor LY335979 increased the brain
penetration of [14C]STI-571 total radioactivity
(p < 0.01) to a similar extent as seen in mdr1a/b
(/) knockout mice (Fig. 4).
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/) knockout mice is 11.2-fold higher than that
of wild-type mice 1 h after oral dosing (p < 0.005) (Fig. 5).
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). The
results indicate that the STI-571 level in the brain is significantly greater in the knockout mice compared with the wild-type mice, even
though the plasma levels of STI-571 were similar in wild-type and the
knockout mice (Fig. 6). The
brain-to-plasma ratio of STI-571 in mdr1a/b (/) knockout mice is 6- to7-fold greater than that of wild-type mice (p < 0.001). These data clearly indicate that P-glycoprotein is an important
factor in limiting the distribution of STI-571 into the CNS.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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STI-571 is the first molecularly targeted antitumor agent,
exerting its antitumor effect by inhibiting signal transduction pathways (Buchdunger et al., 1996; Druker et al., 1996; Deininger et
al., 1997; Heinrich et al., 2000). Approximately 95% of patients with
CML and some patients with acute lymphoid leukemia or acute myeloid
leukemia, exhibit the Philadelphia chromosome, a translocation between
chromosome 9 and 22. This results in the fusion gene BCR/ABL that
encodes a 210-kDa protein that has deregulated tyrosine kinase activity
(Rowley, 1973; Daley et al., 1990; Melo, 1996). This mutant tyrosine
kinase activates downstream signal transduction pathways that lead to
CML (Konopka et al., 1984; Kelliher et al., 1990). STI-571 can inhibit
the BCR/ABL-encoded tyrosine kinase and subsequently the downstream
transduction pathway (Buchdunger et al., 1996; Druker et al., 1996). As
a result, it inhibits the proliferation of the tumor cells (Druker et
al., 1996; Deininger et al., 1997) and induces apoptosis
(Gambacorti-Passerini et al., 1997). Because it attacks a specific
target, STI-571 has so far been shown to have mild side effects in
contrast with conventional nontargeted cytotoxic agents (Druker et al.,
2001). In addition to BCR/ABL, STI-571 has inhibitory effects on c-kit
and PDGFR kinase (Buchdunger et al., 2000; Heinrich et al., 2000).
These two enzymes are involved in the development of glioblastoma,
gastrointestinal stromal tumor, and small cell lung carcinoma (Uhrbom
et al., 2000; Wang et al., 2000; Tuveson et al., 2001). Thus, it is
possible that STI-571 may also have therapeutic effects on these
tumors. However, the therapeutic effect of a drug will depend largely on the targeted bioavailability of the drug at the site of action.
A major challenge in the treatment of brain tumors, including secondary
brain tumors, is the effective delivery of antitumor compounds across
blood-brain barrier into the brain (Lesniak et al., 2001). It is
possible that the blood-brain barrier is disrupted by the disease
process in brain tumor (Davies, 2002), although the role of the
blood-brain barrier in drug delivery to brain tumors has been
controversial (Groothuis, 2000). In some cases, it may be that a drug
such as STI-571 can enter the brain parencyhma through a "leaky"
blood-brain barrier in some areas of the tumor. Nevertheless, it is
important to recognize that the barrier of interest may be the
blood-brain barrier in the brain around tumor, or the growing edge of a
brain tumor, where the blood-brain barrier may have a full complement
of anatomical and physiological features to limit the transport of
drugs (Levin et al., 1975). It has been shown that various efflux
transporters are expressed in blood-brain barrier and
blood-cerebrospinal fluid barrier that eliminate compounds from the
brain or cerebrospinal fluid to the blood (Kusuhara and Sugiyama,
2002). P-glycoprotein is expressed on the apical side of the brain
capillary endothelium (Cordon-Cardo et al., 1989; Thiebaut et al.,
1989; Beaulieu et al., 1997) and can transport substrates from the
basolateral side (brain) to the apical side (blood) of the blood-brain
barrier and thus limit the brain distribution and decrease the specific
brain tissue bioavailability of many therapeutic agents (Kusuhara and
Sugiyama, 2002), including those used for brain tumors (Regina et al.,
2001). Therefore, for rational use of STI-571 in brain tumor, it
is important to know whether STI-571 is a substrate of P-glycoprotein
and whether this active efflux transporter is an effective limiting
determinant of STI-571 distribution to the brain in vivo. Such
information would provide valuable insight for the various ongoing
clinical trials of STI-571 in the treatment of brain tumor.
The cellular accumulation and directional flux of a compound across a polarized cell monolayer, particularly those transfected with a specific transporter, are useful in vitro methods to determine the substrate status of a drug. Compared with cellular accumulation studies, one of the advantages of the directional flux method is that it gives information about whether the compound is transported by a particular active transporter if one knows the orientation of that transporter. The directional flux results shows that the B-to-A flux of STI-571 is significantly greater than the A-to-B flux in the MDCKII monolayer, indicating STI-571 is transported from the basolateral-to-apical side by an active transporter, which is consistent with the localization and orientation of P-glycoprotein. This is more evident in the MDR1-transfected MDCKII cellular monolayer than the wild-type monolayer. Moreover, when a specific inhibitor of P-glycoprotein is used (LY335979), the difference between the B-to-A flux and A-to-B flux in the mdr1-transfected cells is significantly reduced. These data clearly demonstrate that STI-571 is a substrate of P-glycoprotein.
The CNS distribution of antitumor agents can be limited by many
factors, including 1) the physicochemical properties of the compound
(hydrophilicity or size), 2) plasma protein binding, and 3) efflux
transport by CNS efflux transporters. A compound with limited brain
distribution may be a substrate of competing active transport systems,
which would make it difficult to sort out the significance of various
contributing factors that may limit its CNS distribution. The use of
knockout mice, however, will give us an important tool to achieve this
goal. Because the mdr1a/b genes are absent in mdr1a/b (/) knockout
mice, the contribution of P-glycoprotein in limiting the brain
distribution of STI-571 can be clearly demonstrated. In this study, by
using the mdr1a/b (/) knockout mice, it is shown that lacking
P-glycoprotein leads to a severalfold increase in the CNS penetration
of STI-571, indicating P-glycoprotein plays a significant role in the
distribution of STI-571 into the brain. The results in this study are
the first direct evidence to show that P-glycoprotein transports
STI-571 and limits its distribution into the brain.
When [14C]STI-571 was given orally, it is found that only 35 to 40% of the total radioactivity is associated with the STI-571 parent compound in the plasma at 60 min postdose, as indicated by HPLC. Under such a situation, the brain-to-plasma ratio of total radioactivity of [14C]STI-571 will not reflect the true brain-to-plasma ratio of parental STI-571. The increase in the brain-to-plasma ratio of total radioactivity of [14C]STI-571 in the knockout versus the wild-type mice is only about 2.7 at 1 h postdose and it is about 11-fold after correcting for the radioactivity associated with the STI-571 peak. However, when the drug is given intravenously, the brain-to-plasma ratio of STI-571 in knockout mice is about 7-fold greater than that of wild-type. It is possible that this difference may be due to different plasma and brain concentration-time profiles of STI-571 after different routes of administration. However, with both dosing modalities, STI-571 has a greater CNS penetration in the mice deficient in P-glycoprotein than the wild-type mice, indicating the importance of P-glycoprotein in the CNS distribution of STI-571.
It is interesting to note that the concentration-time profile of STI-571 in the brain parallels that of the drug in plasma as seen in Fig. 6. One question that arises is how this rapid equilibrium is established in the face of a relatively low permeability. We have considered this in regard to the present sparse data, and we feel that one explanation may be that for a drug with a small volume of distribution in the CNS, and a rapid efflux, the concentration-time profile in the brain will quickly reflect changes in the concentration-time profile in the plasma because the small volume will quickly respond to a change in driving force as long as the drug can exit easily. Further studies need to be performed to fully describe the distributional kinetics of STI-571 in the brain.
Also of interest, in case of intravenous administration, the brain-to-plasma ratio is higher than that of oral dosing (compare Figs. 4 and 6). There could be several reasons for this difference, and we do not want to speculate too much at this stage of the work. However, one possibility is that the free concentration of STI-571 after i.v. bolus dosing is higher than after oral dosing. Bearing in mind that STI-571 is a highly plasma protein-bound drug, it is possible that a higher concentration of STI-571 could cause saturation of protein binding and lead to a greater free fraction, which would result in a greater driving force for drug in the plasma to enter the brain. Our future experimental studies will be to quantitatively describe the kinetics of unbound STI-571 in both plasma and brain using microdialysis. Also, at this stage we cannot rule out other possibilities such as different concentration-time profiles due to different routes of administration of drug, or saturation of other efflux transporters by the high plasma concentration in case of i.v. administration. No matter what the reason may be, however, the conclusion regarding the P-glycoprotein substrate status of STI-571 is not affected.
It is expected that a limited distribution of STI-571 into the CNS
would decrease the efficacy of this peripherally effective compound in
the treatment of CNS tumor, either primary glioblastoma or secondary
CML in the CNS. Indeed, it is reported that isolated CML relapse has
occurred in the brain even after the peripheral leukemia has been
successfully treated (Leis et al., 2001; Petzer et al., 2002; Takayama
et al., 2002). It has also been established that resistance to STI-571
has occurred in some patients due to a point mutation in the
ATP-binding site of the enzyme or multiplication of the BCR/ABL gene
(Gorre et al., 2001), and it has been shown that long-term exposure to
a suboptimal concentration of STI-571 could lead to the above-mentioned
mutation or gene multiplication (Mahon et al., 2000). Thus, it is
likely that low concentrations of STI-571 in CNS may also lead to a
mutation or overexpression of BCR/ABL in the CNS tumor cells, which in
turn would lead to resistance to this drug. Therefore, elevating the
STI-571 concentration in the brain by inhibiting P-glycoprotein will
not only increase the efficacy in primary and secondary brain tumors
but also may possibly reduce the development of resistance. It is worth
noting that P-glycoprotein may also be expressed in the cytoplasmic
membrane of the tumor cells, including glioblastoma (Tews et al., 2000; Demeule et al., 2001) and gastrointestinal stroma (Plaat et al., 2000),
and as such may limit the penetration of the drug into the cell.
Inhibition of P-glycoprotein would further increase the targeted
bioavailability of STI-571 into the tumor cells.
In summary, the results reported here conclusively show that STI-571 is a substrate of P-glycoprotein and that this efflux transporter is an important determinant of distribution of STI-571 to the central nervous system. The functional evidence, both in vitro and in vivo, indicates that inhibition of P-glycoprotein may enhance the CNS delivery of STI-571. Additional studies are needed to quantitatively characterize the role of drug efflux in limiting the targeted bioavailability of this important drug to the brain.
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Acknowledgments |
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We thank Eli Lilly Cancer Research Laboratory and Dr. Piet Borst (Netherlands Cancer Institute) for generously providing the Pgp inhibitor LY335979 and the MDCKII cell lines, respectively.
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Footnotes |
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Accepted for publication November 25, 2002.
Received for publication October 7, 2002.
This project was partially supported by National Institutes of Health Grant CA75466, a grant from Novartis Pharma, and by a fellowship (to H.D.) from the graduate school of University of Nebraska Medical Center.
DOI: 10.1124/jpet.102.045260
Address correspondence to: Dr. William F. Elmquist, Department of Pharmaceutics, University of Minnesota, 308 Harvard St. SE, Minneapolis MN 55455. E-mail: elmqu011{at}umn.edu
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Abbreviations |
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PDGFR, platelet-derived growth factor receptor; CML, chronic myelogenous leukemia; CNS, central nervous system; MDR1, multidrug resistance-1 gene; MDCK, Madin-Darby canine kidney; A-to-B, apical-to-basal; B-to-A, basal-to-apical; HPLC, high-performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry.
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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  Y. Chen, S. Agarwal, N. M. Shaik, C. Chen, Z. Yang, and W. F. Elmquist P-glycoprotein and Breast Cancer Resistance Protein Influence Brain Distribution of Dasatinib J. Pharmacol. Exp. Ther., September 1, 2009; 330(3): 956 - 963. [Abstract] [Full Text] [PDF]
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 J. S. Lagas, R. A.B. van Waterschoot, V. A.C.J. van Tilburg, M. J. Hillebrand, N. Lankheet, H. Rosing, J. H. Beijnen, and A. H. Schinkel Brain Accumulation of Dasatinib Is Restricted by P-Glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2) and Can Be Enhanced by Elacridar Treatment Clin. Cancer Res., April 1, 2009; 15(7): 2344 - 2351. [Abstract] [Full Text] [PDF]
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 N. Giri, S. Agarwal, N. Shaik, G. Pan, Y. Chen, and W. F. Elmquist Substrate-Dependent Breast Cancer Resistance Protein (Bcrp1/Abcg2)-Mediated Interactions: Consideration of Multiple Binding Sites in in Vitro Assay Design Drug Metab. Dispos., March 1, 2009; 37(3): 560 - 570. [Abstract] [Full Text] [PDF]
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O. Benny, L. G. Menon, G. Ariel, E. Goren, S.-K. Kim, C. Stewman, P. M. Black, R. S. Carroll, and M. Machluf Local Delivery of Poly Lactic-co-glycolic Acid Microspheres Containing Imatinib Mesylate Inhibits Intracranial Xenograft Glioma Growth Clin. Cancer Res., February 15, 2009; 15(4): 1222 - 1231. [Abstract] [Full Text] [PDF]
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 A. Giannoudis, A. Davies, C. M. Lucas, R. J. Harris, M. Pirmohamed, and R. E. Clark Effective dasatinib uptake may occur without human organic cation transporter 1 (hOCT1): implications for the treatment of imatinib-resistant chronic myeloid leukemia Blood, October 15, 2008; 112(8): 3348 - 3354. [Abstract] [Full Text] [PDF]
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 E. Raymond, A. A. Brandes, C. Dittrich, P. Fumoleau, B. Coudert, P. M.J. Clement, M. Frenay, R. Rampling, R. Stupp, J. M. Kros, et al. Phase II Study of Imatinib in Patients With Recurrent Gliomas of Various Histologies: A European Organisation for Research and Treatment of Cancer Brain Tumor Group Study J. Clin. Oncol., October 1, 2008; 26(28): 4659 - 4665. [Abstract] [Full Text] [PDF]
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 G. I. Cancino, E. M. Toledo, N. R. Leal, D. E. Hernandez, L. F. Yevenes, N. C. Inestrosa, and A. R. Alvarez STI571 prevents apoptosis, tau phosphorylation and behavioural impairments induced by Alzheimer's {beta}-amyloid deposits Brain, September 1, 2008; 131(9): 2425 - 2442. [Abstract] [Full Text] [PDF]
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K. Porkka, P. Koskenvesa, T. Lundan, J. Rimpilainen, S. Mustjoki, R. Smykla, R. Wild, R. Luo, M. Arnan, B. Brethon, et al. Dasatinib crosses the blood-brain barrier and is an efficient therapy for central nervous system Philadelphia chromosome-positive leukemia Blood, August 15, 2008; 112(4): 1005 - 1012. [Abstract] [Full Text] [PDF]
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N. Giri, N. Shaik, G. Pan, T. Terasaki, C. Mukai, S. Kitagaki, N. Miyakoshi, and W. F. Elmquist Investigation of the Role of Breast Cancer Resistance Protein (Bcrp/Abcg2) on Pharmacokinetics and Central Nervous System Penetration of Abacavir and Zidovudine in the Mouse Drug Metab. Dispos., August 1, 2008; 36(8): 1476 - 1484. [Abstract] [Full Text] [PDF]
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 V. S. Narang, C. Fraga, N. Kumar, J. Shen, S. Throm, C. F. Stewart, and C. M. Waters Dexamethasone increases expression and activity of multidrug resistance transporters at the rat blood-brain barrier Am J Physiol Cell Physiol, August 1, 2008; 295(2): C440 - C450. [Abstract] [Full Text] [PDF]
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D. K. Hiwase, V. Saunders, D. Hewett, A. Frede, S. Zrim, P. Dang, L. Eadie, L. B. To, J. Melo, S. Kumar, et al. Dasatinib Cellular Uptake and Efflux in Chronic Myeloid Leukemia Cells: Therapeutic Implications Clin. Cancer Res., June 15, 2008; 14(12): 3881 - 3888. [Abstract] [Full Text] [PDF]
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H. H. Helgason, H. A. Mallo, H. Droogendijk, John.G. Haanen, A. A.M. van der Veldt, A. J. van den Eertwegh, and E. Boven Brain Metastases in Patients With Renal Cell Cancer Receiving New Targeted Treatment J. Clin. Oncol., January 1, 2008; 26(1): 152 - 154. [Full Text] [PDF]
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 D. B. Hoelzinger, T. Demuth, and M. E. Berens Autocrine Factors That Sustain Glioma Invasion and Paracrine Biology in the Brain Microenvironment J Natl Cancer Inst, November 7, 2007; 99(21): 1583 - 1593. [Abstract] [Full Text] [PDF]
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N. Shaik, N. Giri, G. Pan, and W. F. Elmquist P-glycoprotein-Mediated Active Efflux of the Anti-HIV1 Nucleoside Abacavir Limits Cellular Accumulation and Brain Distribution Drug Metab. Dispos., November 1, 2007; 35(11): 2076 - 2085. [Abstract] [Full Text] [PDF]
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N. A. de Vries, J. Zhao, E. Kroon, T. Buckle, J. H. Beijnen, and O. van Tellingen P-Glycoprotein and Breast Cancer Resistance Protein: Two Dominant Transporters Working Together in Limiting the Brain Penetration of Topotecan Clin. Cancer Res., November 1, 2007; 13(21): 6440 - 6449. [Abstract] [Full Text] [PDF]
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O. Ottmann, H. Dombret, G. Martinelli, B. Simonsson, F. Guilhot, R. A. Larson, G. Rege-Cambrin, J. Radich, A. Hochhaus, A. M. Apanovitch, et al. Dasatinib induces rapid hematologic and cytogenetic responses in adult patients with Philadelphia chromosome positive acute lymphoblastic leukemia with resistance or intolerance to imatinib: interim results of a phase 2 study Blood, October 1, 2007; 110(7): 2309 - 2315. [Abstract] [Full Text] [PDF]
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 S. Marchetti, R. Mazzanti, J. H. Beijnen, and J. H. M. Schellens Concise Review: Clinical Relevance of Drug Drug and Herb Drug Interactions Mediated by the ABC Transporter ABCB1 (MDR1, P-glycoprotein) Oncologist, August 1, 2007; 12(8): 927 - 941. [Abstract] [Full Text] [PDF]
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L. L. Muldoon, C. Soussain, K. Jahnke, C. Johanson, T. Siegal, Q. R. Smith, W. A. Hall, K. Hynynen, P. D. Senter, D. M. Peereboom, et al. Chemotherapy Delivery Issues in Central Nervous System Malignancy: A Reality Check J. Clin. Oncol., June 1, 2007; 25(16): 2295 - 2305. [Abstract] [Full Text] [PDF]
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A. Yokota, S. Kimura, S. Masuda, E. Ashihara, J. Kuroda, K. Sato, Y. Kamitsuji, E. Kawata, Y. Deguchi, Y. Urasaki, et al. INNO-406, a novel BCR-ABL/Lyn dual tyrosine kinase inhibitor, suppresses the growth of Ph+ leukemia cells in the central nervous system, and cyclosporine A augments its in vivo activity Blood, January 1, 2007; 109(1): 306 - 314. [Abstract] [Full Text] [PDF]
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S. Bihorel, G. Camenisch, G. Gross, M. Lemaire, and J.-M. Scherrmann Influence of Hydroxyurea On Imatinib Mesylate (Gleevec) Transport at the Mouse Blood-Brain Barrier Drug Metab. Dispos., December 1, 2006; 34(12): 1945 - 1949. [Abstract] [Full Text] [PDF]
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H. J. Kim, C. W. Jung, K. Kim, J. S. Ahn, W. S. Kim, K. Park, Y. H. Ko, W. K. Kang, and K. Park Isolated Blast Crisis in CNS in a Patient With Chronic Myelogenous Leukemia Maintaining Major Cytogenetic Response After Imatinib J. Clin. Oncol., August 20, 2006; 24(24): 4028 - 4029. [Full Text] [PDF]
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P. Y. Wen, W.K. A. Yung, K. R. Lamborn, P. L. Dahia, Y. Wang, B. Peng, L. E. Abrey, J. Raizer, T. F. Cloughesy, K. Fink, et al. Phase I/II Study of Imatinib Mesylate for Recurrent Malignant Gliomas: North American Brain Tumor Consortium Study 99-08. Clin. Cancer Res., August 15, 2006; 12(16): 4899 - 4907. [Abstract] [Full Text] [PDF]
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D. A. Reardon, J. N. Rich, H. S. Friedman, and D. D. Bigner Recent Advances in the Treatment of Malignant Astrocytoma J. Clin. Oncol., March 10, 2006; 24(8): 1253 - 1265. [Abstract] [Full Text] [PDF]
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D. A. Reardon, M. J. Egorin, J. A. Quinn, J. N. Rich Sr, I. Gururangan, J. J. Vredenburgh, A. Desjardins, S. Sathornsumetee, J. M. Provenzale, J. E. Herndon II, et al. Phase II Study of Imatinib Mesylate Plus Hydroxyurea in Adults With Recurrent Glioblastoma Multiforme J. Clin. Oncol., December 20, 2005; 23(36): 9359 - 9368. [Abstract] [Full Text] [PDF]
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L. C. Crossman, B. J. Druker, M. W. N. Deininger, M. Pirmohamed, L. Wang, and R. E. Clark hOCT 1 and resistance to imatinib Blood, August 1, 2005; 106(3): 1133 - 1134. [Full Text] [PDF]
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 P. Breedveld, D. Pluim, G. Cipriani, P. Wielinga, O. van Tellingen, A. H. Schinkel, and J. H.M. Schellens The Effect of Bcrp1 (Abcg2) on the In vivo Pharmacokinetics and Brain Penetration of Imatinib Mesylate (Gleevec): Implications for the Use of Breast Cancer Resistance Protein and P-Glycoprotein Inhibitors to Enable the Brain Penetration of Imatinib in Patients Cancer Res., April 1, 2005; 65(7): 2577 - 2582. [Abstract] [Full Text] [PDF]
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M. Deininger, E. Buchdunger, and B. J. Druker The development of imatinib as a therapeutic agent for chronic myeloid leukemia Blood, April 1, 2005; 105(7): 2640 - 2653. [Abstract] [Full Text] [PDF]
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J. Thomas, L. Wang, R. E. Clark, and M. Pirmohamed Active transport of imatinib into and out of cells: implications for drug resistance Blood, December 1, 2004; 104(12): 3739 - 3745. [Abstract] [Full Text] [PDF]
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N. C. Wolff, D. E. Randle, M. J. Egorin, J. D. Minna, and R. L. Ilaria Jr. Imatinib Mesylate Efficiently Achieves Therapeutic Intratumor Concentrations in Vivo but Has Limited Activity in a Xenograft Model of Small Cell Lung Cancer Clin. Cancer Res., May 15, 2004; 10(10): 3528 - 3534. [Abstract] [Full Text] [PDF]
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A. Hamada, H. Miyano, H. Watanabe, and H. Saito Interaction of Imatinib Mesilate with Human P-Glycoprotein J. Pharmacol. Exp. Ther., November 1, 2003; 307(2): 824 - 828. [Abstract] [Full Text] [PDF]
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H. Pfeifer, B. Wassmann, W.-K. Hofmann, M. Komor, U. Scheuring, P. Bruck, A. Binckebanck, E. Schleyer, N. Gokbuget, T. Wolff, et al. Risk and Prognosis of Central Nervous System Leukemia in Patients with Philadelphia Chromosome-Positive Acute Leukemias Treated with Imatinib Mesylate Clin. Cancer Res., October 15, 2003; 9(13): 4674 - 4681. [Abstract] [Full Text] [PDF]
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