REPORTS |
In Vivo Eradication of Human BCR/ABL-Positive Leukemia Cells With an ABL Kinase Inhibitor
Philipp le Coutre,
Luca Mologni,
Loredana Cleris,
Edoardo Marchesi,
Elisabeth Buchdunger,
Roberto Giardini,
Franca Formelli,
Carlo Gambacorti-Passerini
Affiliations of authors: P. le Coutre, L. Mologni, L.Cleris, E. Marchesi, F. Formelli
(Department of Experimental Oncology),
R. Giardini (Department of Pathology), Istituto Nazionale Tumori,
Milan, Italy; E. Buchdunger, Oncology Research Department, Novartis
International Inc., Basel, Switzerland; C. Gambacorti-Passerini,
Department of Experimental Oncology, Istituto Nazionale Tumori, Milan,
and Section of Hematology, University of Milan, S. Gerardo Hospital,
Monza, Italy.
Correspondence to: Carlo Gambacorti-Passerini,
M.D.,Department of Experimental Oncology, Istituto Nazionale Tumori, Via
Venezian 1, 20133 Milan, Italy (e-mail: gambacorti{at}istitutotumori.mi.it).
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ABSTRACT
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BACKGROUND: The leukemia cells of approximately 95% of
patients with chronic myeloid leukemia and 30%-50% of adult
patients with acute lymphoblastic leukemia express the Bcr/Abl
oncoprotein, which is the product of a fusion gene created by a
chromosomal translocation [(9:22) (q34;q11)]. This oncoprotein
expresses a constitutive tyrosine kinase activity that is crucial for
its cellular transforming activity. In this study, we evaluated the
antineoplastic activity of CGP57148B, which is a competitive inhibitor
of the Bcr/Abl tyrosine kinase. METHODS: Nude mice were given
an injection of the Bcr/Abl-positive human leukemia cell lines KU812 or
MC3. Tumor-bearing mice were treated intraperitoneally or orally with
CGP57148B according to three different schedules. In vitro drug
wash-out experiments and in vivo molecular pharmacokinetic
experiments were performed to optimize the in vivo treatment
schedule. RESULTS: Treatment schedules administering CGP57148B
once or twice per day produced some inhibition of tumor growth, but no
tumor-bearing mouse was cured. A single administration of CGP57148B
caused substantial (>50%) but short-lived (2-5 hours)
inhibition of Bcr/Abl kinase activity. On the basis of the results from
in vitro wash-out experiments, 20-21 hours was defined as the
duration of continuous exposure needed to block cell proliferation and
to induce apoptosis in these two leukemia cell lines. A treatment
regimen assuring the continuous block of the Bcr/Abl phosphorylating
activity that was administered over an 11-day period cured
87%-100% of treated mice. CONCLUSION: These data
indicate that the continuous block of the oncogenic tyrosine kinase of
Bcr/Abl protein is needed to produce important biologic effects in
vivo.
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INTRODUCTION
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Chronic myeloid leukemia (CML) is caused by the acquisition of
the reciprocal (9:22) (q34;q11) chromosomal translocation
(Philadelphia chromosome) in hematopoietic stem cells. In the resulting
fusion protein, part of the ABL gene product (Abl), including the
tyrosine kinase (TK) domain, is fused to the amino-terminal end of the
BCR gene product (Bcr) (1). The Bcr domain probably interferes
with an intramolecular Abl inhibitory loop and unveils a constitutive
kinase activity that is absent in the normal Abl protein (2).
This chromosomal defect is seen in about 95% of all patients with
CML as well as in 30%-50% of adult patients with acute
lymphoblastic leukemia (ALL). The only curative treatment available at
present is allogeneic bone marrow transplantation, a toxic and costly
procedure, available for only 30% of patients. While the precise
mechanism employed by the Bcr/Abl fusion protein to transform cells is
still unknown, it is well accepted that the enhanced TK activity is
crucial to its transforming ability (3). Thus, the inhibition
of the TK activity of this protein represents a specific therapeutic
strategy for Bcr/Abl-expressing leukemias. A 2-phenylaminopyrimidine
derivative named CGP57148B (4) has been reported to
selectively inhibit the kinase activity of both Abl and Bcr/Abl
(5). CGP57148B is a competitive inhibitor of the adenosine
triphosphate (ATP)-binding cleft of Abl. We and others (6,7)
have previously reported the selective block of proliferation in
BCR/ABL-positive cells (either cell lines or fresh tumor samples) and
showed that this compound can commit these cells to apoptosis without
inducing differentiation. So far, no data are available on the in
vivo activity of CGP57148B on human Bcr/Abl-positive leukemia
cells; results obtained in murine cells transfected with v-abl or
bcr/abl indicate the presence of a limited activity, with retardation
of tumor growth but no cure of treated animals (4,5).
We describe here the in vivo effects of this compound in nude
mice given an injection of human leukemia cell lines. The nude mouse
model was selected because the tumor grows as a solid
mass; this fact permits us to obtain purified tumor cells from animals
and to assess the in vivo kinetics of Bcr/Abl inhibition
following the treatment of tumor-bearing mice. Other models, like the
severe combined immunodeficient (SCID) and SCID/non-obese diabetic
(NOD) mice, permit transplantation of fresh, chronic-phase CML cells
(8). In this case, however, no solid tumor growth or massive
outgrowth of leukemia cells in the bone marrow usually occurs, thus
preventing the possibility of performing the above-mentioned experiments.
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MATERIALS AND METHODS
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CGP57148B
A 2-phenylaminopyrimidine derivative with a molecular weight of 590,
CGP57148B was developed and provided by Novartis Inc. (Basel,
Switzerland). The stock solutions of this compound were prepared at 1
and at 10 mM with distilled water, filtered, and stored at
-20 °C. For in vitro experiments, CGP57148B was
thawed before the experiment was started and used at a concentration of
0.1-10 µM. Preparations used for animal experiments were
made in concentrations as indicated below and were kept at
4 °C for a maximum of 4 days.
Cell Lines
Three human leukemia lines were used: KU812, MC3, and U937. KU812
and MC3 expressed p210Bcr/Abl, whereas U937, a
BCR/ABL-negative human leukemia cell line, served as a negative control
(9,10). Both Bcr/Abl-positive cell lines were derived from CML
patients in blast crisis, who previously had been treated with
busulfan. All cells were cultured in RPMI-1640 medium (BioWhittaker,
Inc., Walkersville, MD; Boerhinger Ingelheim Pharmaceuticals, Inc.,
Ridgefield, CT) containing 10% fetal calf serum (FCS) (HyClone
Laboratories, Inc., Logan, UT) under standard cell culture conditions.
Four additional Bcr/Abl-positive cell lines [KCL22, BV173, K562, and
LAMA84 (6)] were tested in preliminary experiments, but they
failed
to produce reproducible growth in nude mice and were not studied further.
Determination of In Vitro Proliferative Activity (Tritiated Thymidine Uptake
Assay)
Two hundred microliters of each cell line (KU812, MC3, and U937),
containing 4 x 104 cells, was seeded at various
concentrations of CGP57148B, ranging from 0.1 to 10 µM in
96-well microtiter plates (Corning Costar Corp., Cambridge, MA) in six
replicates. After 54 hours at 37 °C, 20 µL of RPMI-1640
medium/10% FCS containing tritiated thymidine (1 µCi/well) was
added to each well. After an additional 18 hours, cells were harvested
and transferred to a filter (Spot-on filtermat; Pharmacia Biotech
Europe, Brussels, Belgium). Tritiated thymidine uptake was determined
by a 1205 betaplate liquid scintillation counter (Wallac Inc., Turku,
Finland). IC50 was defined as the concentration of compound
producing a 50% decrease in proliferation in comparison with
untreated controls.
In Vitro Wash-Out Experiments
To determine the minimal duration of drug exposure, 0.5 x
106 cells were incubated in medium containing 1
µM of CGP57148B. Cells were first seeded in 24-well plates
(Corning Costar Corp.). At various time points, the wells were washed
twice in RPMI-1640/10% FCS and reseeded in a new 24-well plate.
Each sample was seeded in six replicates. After a total culture time of
54 hours, the samples were washed again, 200 µL containing 4 x
104 cells was transferred to a 96-microtiter plate (Corning
Costar Corp.), and proliferative activity was determined over a period
of 18 hours (see above).
Western Blot Analysis
Tumor-bearing mice were treated either orally (160 mg/kg) or
intraperitoneally (50 mg/kg) with CGP57148B as described below. At
various time points, ranging between 2 hours and 24 hours after
treatment, mice were killed and non-necrotic tumor tissue was extracted
and homogenized in a fivefold volume of sodium dodecyl sulfate
(SDS)-loading buffer (50 mM Tris-HCl [pH 6.8], 2% SDS,
and 5% ß-mercaptoethanol). After sonication for 2 minutes,
samples were centrifuged at 15 000g for 15 minutes,
heated at 95 °C for 10 minutes, and stored at
-80 °C. Polyacrylamide gel electrophoresis was carried
out on a 7.5% SDS gel (Mini-Protean II electrophoresis cell;
Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were
loaded as determined by BCA Protein Assay (Pierce Chemical Co.,
Rockford, IL). Before loading, 0.05% of bromophenol blue was added
to each sample. Samples were subsequently transferred to a
nitrocellulose membrane (Hybond Super-C; Amersham Life Science Inc.,
Arlington Heights, IL). Transfer was carried out overnight at
4 °C in a Mini Trans-Blot electrophoretic transfer cell
(Bio-Rad Laboratories) in 25 mM Tris, 192 mM glycine,
and 20% methanol. After protein transfer, the membrane was blocked
with TBST (0.01 M Tris, 0.15 M NaCl, and 0.05%
Tween 20 [pH 7.4]) containing 5% milk, and a monoclonal
anti-phosphotyrosine antibody (05-321; Upstate Biotechnology, Lake
Placid, NY) was used in a 1 : 500 dilution (TBST/5%
milk) to detect phosphorylated proteins. After stripping (2% SDS,
62.5 mM Tris-HCl [pH 6.8], and 0.7%
ß-mercaptoethanol for 1 hour), the filter was blotted with an
anti-Abl antibody (clone Ab-3; Calbiochem Corp., La Jolla, CA;
1 : 100 in TBST/5% milk) that also recognized
Bcr/Abl. An anti-mouse horseradish peroxidase-conjugated antibody
(Bio-Rad Laboratories) was used as a secondary antibody and identified
both primary antibodies after addition of a supersignal
chemiluminescent substrate (Pierce Chemical Co.). Densitometric
analysis of films was carried out on an Eagle Eye II Photodensitometer
(Stratagene, La Jolla, CA). Band intensities were calculated as areas;
nonspecific changes caused by differences in Bcr/Abl content (as
evidenced by the anti-Abl antibody) were subtracted.
In Vivo Administration of CGP57148B
Seven to 9-week-old female CD-1 nu/nu mice purchased at Charles
River Breeding Laboratories (Calco, Italy) were kept under standard
laboratory conditions according to the guidelines of the National
Cancer Institute, Milan, Italy. This study was approved by the
institutional ethics committee for laboratory animals used in
experimental research. Both Bcr/Abl-positive and -negative cell lines
were injected (50 x 106 cells per animal) subcutaneously
in the left flank. Treatment was started 1-8 days after leukemia cell
injection. Oral treatment was administered through a syringe connected
to a soft plastic tube introduced into the mouse esophagus (gavage).
CGP57148B was prepared at concentrations of 16 mg/mL (oral) and 5 mg/mL
(intraperitoneal) in distilled water. Control animals were given equal
volumes of water. Tumor weight (TW) and total weight were monitored
every 3-4 days. TW was calculated by the formula TW (mg) =
(d2 x D)/2, where d and
D are the shortest and longest diameters of the tumor,
respectively, measured in millimeters.
When mice were treated twice per day, six mice per treatment or control
group were used (total of 12 mice). For treatment of mice 24 hours
after KU812 leukemia cell injection, eight mice per treatment or
control group (total of 24 mice) were used, whereas the U937 group
consisted of 10 mice per treatment or control group (total of 30 mice).
In experiments with measurable tumor-bearing mice, 12 mice per
treatment or control group (total of 24 mice) were used.
Statistical Analysis
Statistical analysis of tumor weights was performed with one-way
variance analysis using the Statpac analysis program (version 3.1;
Walonick Assoc., Minneapolis, MN). For survival analysis, data were
compared by the logrank test (11). P values <.05 were
considered statistically significant and were derived from two-sided
statistical tests.
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RESULTS
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In Vitro Sensitivity to CGP57148B
The in vitro IC50 for CGP57148B was determined by
a proliferation assay. It was found to be between 0.1 and 0.3
µM in the two BCR/ABL-positive cell lines KU812 and MC3,
whereas the BCR/ABL-negative cell line U937 was unaffected by CGP57148B
concentrations 30 times higher (10 µM) (not shown). These
data are in accordance with previously published results (6).
In Vitro Wash-Out Experiments
CGP57148B is a competitive (reversible) inhibitor of Abl kinase
activity. To determine the minimum time of exposure to this compound
necessary to inhibit proliferation, experiments were performed in
vitro on KU812 cells. Cells were cultured in the presence of 1
µM CGP57148B for the various times indicated in Fig.
1.
The results clearly indicate that 20-21 hours
were sufficient to block cell proliferation, whereas 6-7 hours
produced little change. These data are in agreement with the kinetics
of early apoptosis induction in these cell lines, which occurred
between 16 and 24 hours (6). Therefore, the duration of
Bcr/Abl inhibition following treatment with CGP57148B could be critical
in determining biologic results in vivo.

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Fig. 1. Effect of different exposure times to 1 µM
CGP57148B on the in vitro proliferation of KU812 leukemia
cells. Two representative experiments (black and gray columns) are
shown. Cells (5 x 105) were first seeded in 24-well plates
(Corning Costar Corp.) and incubated with the drug. At the indicated
time points, the cells were washed twice with RPMI-1640 medium/10%
fetal calf serum and reseeded in a new 24-well plate. After a total
culture time of 54 hours, the cells were washed again, and 200-µL
aliquots containing 4 x 104 cells were transferred to a
96-well microtiter plate (Corning Costar Corp.) and used for an 18-hour
tritiated thymidine uptake assay. Proliferative activity is expressed
as the percent of control counts per minute and represents the mean of
six replicates seeded for each sample. Error bars represent 95%
confidence intervals (CIs). Average counts per minute for the control
samples were 102 056 (95% CI =
97 546-106 566) and 39 687 (95% CI =
36 642-42 732), respectively. h = hours.
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In Vivo Molecular Pharmacokinetic Experiments
In our previous in vitro experiments (6),
inhibition of proliferation and apoptosis induction was observed in
leukemia lines when the Bcr/Abl TK activity was inhibited by 50% or
more. To investigate the degree and duration of Bcr/Abl inhibition
in vivo, we acutely treated tumor-bearing mice and killed them
at various time points after treatment. The levels of Bcr/Abl kinase
activity (measured as autophosphorylation) obtained at different times
in a representative experiment are presented in Fig.
2.
CGP57148B was administered both intraperitoneally
(50 mg/kg) and orally (160 mg/kg). At 2 hours after injection, Bcr/Abl
inhibition of 64.7% (intraperitoneal) and 66.4% (oral) was
present in both groups. At 5 hours, the level of inhibition was still
46.7% in orally treated mice and 53.4% in intraperitoneally
treated animals; by 8 hours, more than 70% of the initial kinase
activity was restored in both groups. Therefore, Bcr/Abl was blocked
in vivo, but this inhibition was short-lived, and a single
CGP57148B dose was clearly insufficient to maintain a substantial
Bcr/Abl inactivation for the time necessary to block proliferation and
to commit cells to apoptosis.


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Fig. 2. In vivo inhibition of Bcr/Abl kinase activity
by CGP57148B. Tumor-bearing mice were acutely treated with CGP57148B
intraperitoneally (50 mg/kg) or orally (160 mg/kg) and killed at
various time points. Tumor samples were extracted and used for western
blot analysis with anti-phosphotyrosine or anti-Abelson (abl)
antibodies. A) Lane 7 represents results from an
untreated mouse. Lanes 2, 4, and 6 represent results from
intraperitoneally treated animals at 2, 5, and 8 hours after treatment,
respectively. Lanes 1, 3, and 5 represent results from orally
treated mice at 2, 5, and 8 hours after treatment, respectively.
Western blots were first exposed to an anti-phosphotyrosine antibody
and then stripped and probed with an anti-abl antibody. B)
Photodensitometric analysis of bands labeled with the
anti-phosphotyrosine antibody in panel A. Band intensities were
calculated as areas and presented as the percentage of the time 0 value
(i.e., lane 7 in panel A); changes caused by
differences in Bcr/Abl content (as evidenced by the control anti-abl
antibody) were subtracted from the total values.
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In Vivo Activity of CGP57148B
Since a dose of 50 mg/kg (one intraperitoneal injection/day) was
used in vivo with murine cells (4,5), this schedule
was selected initially for our study and was administered for 25 days.
This dosage achieves maximum concentrations (Cmax) greater than 3
µM (data not shown). No statistically significant
retardation in tumor growth was observed in animals treated once per
day. For this reason, the treatment schedule was modified, and the same
dose was administered two times daily, at 8 AM and 4
PM. The results obtained in mice given an injection of KU812
or MC3 cells and treated twice per day are reported in Fig.
3.
Tumor growth inhibition was present and reached
statistical significance on days 17 (P<.01 for both cell
lines), 21 (P<.01 for KU812 and P<.05 for MC3),
and 25 (P<.05 for both cell lines); growth inhibition was
present during the treatment period (days 1-25), while growth curves
tended to run in a parallel way when the treatment was discontinued. No
growth inhibition was noted in mice given an injection of the myeloid
Bcr/Abl-negative cell line U937 (data not shown). The twice-per-day
treatment schedule appeared necessary to inhibit tumor growth, since
the administration of the same total dose (100 mg/kg intraperitoneal)
in a single daily injection did not produce a statistically significant
delay in tumor growth (data not shown).


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Fig. 3. Effect of CGP57148B on the in vivo growth of
KU812 (A) and MC3 (B) Bcr/Abl-positive leukemia cell
lines. CGP57148B was administered at 50 mg/kg body weight per day
intraperitoneally in two daily injections (8 AM and 4
PM), 24 hours after the subcutaneous injection of 50 x
106 leukemia cells. Control mice were treated with equivalent
volumes of water instead of the drug. Each experimental group contained
six mice. Results are presented as average tumor weight (g); error bars
represent 95% confidence intervals and are displayed only when they
exceed 5% of the respective mean.
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These data indicate that the treatment administered at 50 mg/kg by
intraperitoneal injection twice per day caused a specific and
statistically significant inhibition in the growth of Bcr/Abl-positive
human leukemia cell lines. However, no differences in the number of
animals developing tumors or in the leukemia-free survival of treated
mice were observed. Therefore, the treatment schedule was modified
further, on the basis of the results from the in vitro
wash-out and in vivo kinetics experiments, with the aim of
producing a continuous inhibition of the Bcr/Abl kinase activity.
Experiments were focused on KU812, which produced a higher in
vivo growth rate (80%-90% of inoculated animals) than MC3
(60%-70%). Tumor-bearing mice received CGP57148B at a dose of
50 mg/kg intraperitoneally or 160 mg/kg orally every 8 hours for 11
consecutive days. Fig. 4,
A, shows the tumor-free
survival in animals given an injection of KU812 cells; two independent
experiments were performed with similar results. Each group contained
eight animals. Seven mice (87.5%) in the control group developed
tumors within 9 days, while one (12.5%) of eight mice in the
intraperitoneally treated group developed a tumor (87.5% were
cured). All mice in the group treated orally remained tumor free.
Differences in tumor-free survival between control and treated groups
were highly significant (P<.0001), whereas the comparison
between the two treated groups did not reveal statistical significance.
No evidence of tumor growth was noted during the follow-up of
tumor-free animals (up to 240 days). In contrast, no differences were
noted between control mice and treated mice receiving an injection of
U937 cells, confirming the specificity of the results obtained (not shown).


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Fig. 4. Eradication of leukemia growth in mice treated with
CGP57148B. A) Nude mice were given an injection of 50 x
106 KU812 cells on day 0. After 24 hours, treatment with
CGP57148B (50 mg/kg body weight intraperitoneally or 160 mg/kg body
weight orally, every 8 hours) was started and continued for 11 days.
The tumor status was checked every 3-4 days. Survival data in the six
groups were compared by the logrank test. KU812 treatment or control
groups had eight mice per group (total of 24 mice), whereas U937
treatment or control groups had 10 mice per group (total of 30 mice).
B) Effect of CGP57148B on leukemia growth in the presence of
more advanced, measurable disease. Nude mice received an injection of
human leukemia KU812 cells as described in panel A. Eight days
later (day 0 on the x axis), treatment with CGP57148B (160
mg/kg body weight orally every 8 hours, for 11 days) was started.
Pretreatment mean tumor weights were 286 mg (95% confidence
interval [CI] = 201-371 mg) in the control group and 289 mg (95%
CI = 209-369 mg) in the treatment group, respectively. Each treatment
or control group had 12 mice per group.
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Treatment of Mice With Measurable Tumors
To study the effect of this treatment schedule on animals with more
advanced disease, already bearing measurable tumors, we administered
CGP57148B (oral schedule) to a group of 12 animals, 8 days after
leukemia cell injection, with tumors already weighing 286 mg (95%
confidence interval [CI] = 201-371 mg) in the controls and 289 mg
(95% CI = 209-369 mg) in the treatment group. Fig. 4
, B,
presents
the results of this experiment. Nodules started to regress 48 hours
after treatment; the reduction, compared with the control group,
averaged 68% at day 3 and 98% by day 7 after the beginning of
CGP57148B treatment. No treated animal had a measurable tumor by day 8,
while 12 of 12 control mice had growing nodules (P<.0001).
The same schedule was also tested in U937-bearing mice; the treatment
did not cause any statistically significant change in tumor growth or
leukemia-free survival in animals given an injection of this
Bcr/Abl-negative leukemia cell line (data not shown). Tumor-free
animals were also followed up in this group: Four (33%) of 12
animals developed a relapse between day 48 and day 60, while the
remaining eight animals (67%) have remained tumor free up to the
present day (+210 days). A longer treatment schedule (18 days)
apparently failed to reduce the risk of relapse (data not shown).
These data indicate that the continuous inhibition of Bcr/Abl was
needed to produce major biologic effects and converted an apparently
ineffective molecule (when administered once per day) to a highly
active compound. However, the tumor load present at the time of
treatment represented an important variable, and the treatment of mice
with a measurable tumor mass, although highly effective, did not
eradicate the disease in 100% of cases, as it consistently did in
the group of animals treated 24 hours after the injection of leukemia cells.
Toxicity of Treatment
The weight of the animals in the treated groups increased at a
reduced pace during treatment compared with the weight of the controls.
This difference in weight never exceeded 8% and disappeared after
treatment. No statistically significant differences in white blood cell
and platelet counts or in hemoglobin levels were noted during
treatment. Treated animals were also subjected to histopathologic
analysis. No major finding was noted at autopsy; in particular, no
changes or altered myeloid/erythroid ratios were observed in bone
marrow (skull + femurs) specimens. The only abnormality observed in
some, but not all, treated animals was represented by a modest
periportal lymphocyte infiltrate, with no sign of hepatocellular
necrosis (data not shown).
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DISCUSSION
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CGP57148B is a potent inhibitor of the Bcr/Abl kinase and acts by
blocking the ATP-binding site of this protein. In this report, we
investigated the in vivo effects of CGP57148B in a nude mouse
model. This model was chosen since the availability of solid nodules
permits us to study the Bcr/Abl activity in tumor tissues and to assess
the kinetics of Bcr/Abl inhibition following CGP57148B administration.
This need would render some important experiments described here
impossible to perform in the SCID/NOD model.
Our results suggest that a too short in vivo inhibition of
Bcr/Abl is responsible for the initial negative data. The development
of a treatment schedule designed to maintain a prolonged in
vivo Bcr/Abl inactivation produced a statistically significant
increase in the activity of this compound, and long-term remissions in
tumor-bearing animals were achieved. The number of neoplastic cells
present at the beginning of treatment seems to play an important role;
in fact, when treatment was started at day 8 (in the presence of
approximately 300 millions cells), one third of the animals had a
relapse, while the same regimen eradicated the disease in 100% of
treated animals when the treatment was started 24 hours after the
injection of 50 million cells. It will be interesting to study the
possible emergence of resistance to this new class of specific
antineoplastic compounds. Progressive resistance to CGP57148B treatment
can theoretically develop over time as a result of different
mechanisms, such as cellular modifications inside leukemic cells and
in vivo induction of drug metabolism or of proteins able to
bind CGP57148B and to decrease its bioavailability. The investigation
of this phenomenon bears clinical relevance, since chronic treatment
with CGP57148B will probably be needed over extended periods of time.
The toxicity of the treatment was rather limited in our experience,
even if the normal Abl protein was certainly inhibited [in addition to
the known cross-activity with kit and pdgfrß (i.e., platelet-derived
growth factor receptor-ß) (4)]. These results indicate
that, while Bcr/Abl clearly identifies a critical "bottleneck"
for Bcr/Abl leukemia cells, the normal Abl protein appears to be less
critical for the survival of normal cells. Longer and more exhaustive
toxicologic studies are, however, needed to better assess the possible
long-term side effects of this molecule.
An additional point that deserves discussion is the apparent activity
of the "once-per-day" schedule when cytokine-dependent murine
cell lines transfected with v-ABL or BCR/ABL were used, as previously
described (4,5). In these reports, the growth of transfectants
was inhibited (although no animal was cured) by a schedule that was
completely ineffective in our model. However, we observed that, in
these transfectants, the in vitro IC50 for CGP57148B
was lower than that in the human leukemia cell lines used in our study
(0.001 µM versus 0.1-0.3 µM [data not
shown]), suggesting that the murine transfectants could be inhibited
at CGP57148B concentrations lower than those active on KU812 or MC3.
Therefore, in the experiments performed by Buchdunger et al.
(4) and Druker et al. (5), a single administration
of
the compound could result in longer lasting inhibitory concentrations
than in our model.
These data indicate that CGP57148B exerts a specific activity against
human Bcr/Abl-positive leukemia cells in vivo; given the
competitive (reversible) nature of this compound, continuous exposure
is necessary to produce major antileukemia effects.
The information contained in this report might help to establish
therapeutic regimens in patients with Bcr/Abl-positive leukemias. Our
results suggest, for example, that a single daily administration of
CGP57148B (or of other molecules with a similar mechanism of action and
in vivo half-life) could result in limited biologic and
therapeutic effects. Although we do not yet know the in vivo
kinetics of CGP57148B in humans, the data presented here indicate the
importance of developing treatment regimens able to achieve continuous
inhibition of the targeted kinase in vivo.
It has to be remembered, however, that nude mice given an injection of
Bcr/Abl-positive cells present a hematopoietic system that is not
affected by the leukemia, in contrast to CML patients, in whom most of
the mature granulocytes derive from the leukemic clone; it is possible
that CGP57148B might cause cytopenia in treated patients because of the
low frequency of BCR/ABL-negative precursors in untreated CML patients.
In addition, leukemia cells grow in nude mice as solid tumors, and even
if they can spread and form distant metastases, they do not grow
diffusely as in patients; attempts at injecting KU812 or MC3
intravenously were unsuccessful. Therefore, although this model was
needed to carry out the in vivo kinetics experiments, it is
less clinically relevant than other systems, like SCID/NOD mice given
an injection of uncultured chronic phase CML cells (8).
Finally, these data could also become relevant for other neoplasias in
which competitive TK inhibitors are presently being investigated
in vivo.
Supported in part by the Italian Association for Cancer Research
(AIRC, 420.198.662), the Italian Research Council
(95.00842,9600225.CT04), Istituto Superiore di Sanità (881A/10),
BIOMED-2 grant BMH4-CT96-0848, and EU-TMR grant BMH4-CT96-5006.
E. Buchdunger is an employee and stockholder of Novartis AG, Inc.,
manufacturer of compound CGP57148B investigated in this study.
We thank Dr. Giorgio Parmiani, Gian Marco Corneo, Pietro Pioltelli, and
Enrico Pogliani for their helpful discussion.
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REFERENCES
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revised November 4, 1998;
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