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Vol. 291, Issue 2, 739-748, November 1999
Department of Genomics, Targets and Cancer Research, Pfizer Central Research, Groton, Connecticut
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Phosphorylation of tyrosine residues on the epidermal growth factor (EGF) receptor (EGFr) is an important early event in signal transduction, leading to cell replication for major human carcinomas. CP-358,774 is a potent and selective inhibitor of the EGFr tyrosine kinase and produces selective inhibition of EGF-mediated tumor cell mitogenesis. To assess the pharmacodynamic aspects of EGFr inhibition, we devised an ex vivo enzyme-linked immunosorbent assay for quantification of EGFr-specific tyrosine phosphorylation in human tumor tissue specimens obtained from xenografts growing s.c. in athymic mice. When coupled with pharmacokinetic analyses, this measurement can be used to describe the extent and duration of kinase inhibition in vivo. CP-358,774 is an effective, orally active inhibitor of EGFr-specific tyrosine phosphorylation (ED50 = 10 mg/kg, single dose). It has a significant duration of action, producing, on average, a 70% reduction in EGFr-associated phosphotyrosine over a 24-h period after a single 100 mg/kg dose. Inhibition of EGFr phosphotyrosine in an ex vivo assay format effectively estimates the potency and degree of inhibition of EGFr-dependent human LICR-LON-HN5 head and neck carcinoma tumor growth. Substantial growth inhibition of human tumor xenografts was achieved with p.o. doses of the compound (ED50 = 10 mg/kg q.d. for 20 days). Combination chemotherapy with cisplatin produced a significant response above that of cisplatin alone with no detectable effects on body weight or lethal toxicity. Taken together, these observations suggest that CP-358,774 may be useful for the treatment of EGFr-driven human carcinomas.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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For
the majority of human carcinomas, growth factor receptors play an
important role in tumorigenesis and progression to terminal disease
states. The epidermal growth factor (EGF) receptor (EGFr) has been
implicated in many human squamous cell carcinomas (Ozanne et al.,
1986
), such as non-small cell lung carcinoma and brain, bladder,
breast, and ovarian carcinomas (Gullick, 1991). EGF at picomolar
concentrations is mitogenic for cells overexpressing the receptor, and
antibodies to EGFr abolish EGF-stimulated mitogenesis in LICR-LON-HN5
head and neck carcinoma (HN5; Modjtahedi et al., 1993b,c) and other
tumor cells (Aboud-Pirak et al., 1988; Yoneda et al., 1991a). As an
early event in the signal transduction process, the ligand transforming
growth factor-
or EGF binds to EGFr on the surface of tumor cells
and stimulates: 1) heterodimerization and homodimerization of EGFr
molecules; 2) intermolecular cross-phosphorylation of intracytoplasmic
tyrosine residues (EGFr autophosphorylation; Honegger et al., 1989);
and 3) activation of the tyrosine kinase activity of EGFr. Apart from
binding to the cognate ligand, all known functions of EGFr depend on
tyrosine kinase activity. Point mutations in the kinase domain that
abrogate ATP binding also abolish ligand-dependent kinase activity and
abrogate EGF/transforming growth factor--induced mitogenesis
(Moolenaar et al., 1988). An intact kinase domain is essential for
activation of numerous downstream effectors, including phospholipase
C-
(Margolis et al., 1990; Nishibe et al., 1990; Wahl et al., 1990)
phosphatidylinositol 3-kinase (Bjorge et al., 1990), and
mitogen-activated protein kinase (Ahn et al., 1990), with the ultimate
cellular response being DNA synthesis and cell division (Honegger et
al., 1987). Transfection experiments have shown that EGFr
overexpression alone may lead to constitutive activation of signal
transduction, leading to uncontrolled mitosis (Di Fiore et al., 1987;
Velu et al., 1987). The degree of EGFr overexpression has been shown to
be related to tumorigenicity in some tumor systems (Santon et al.,
1986; Velu, 1990). Recent studies of biopsy specimens suggest that
overexpression of EGFr is associated with a poor prognosis in bladder
(Neal et al., 1985) and breast (Sainsbury et al., 1985) carcinomas.
Despite homology with other tyrosine kinases, selective inhibitors have
been identified (for a review, see Traxler, 1998). The EGFr tyrosine
kinase therefore represents an attractive molecular target for
pharmacological intervention. To monitor the effects of kinase
inhibition, the degree of EGFr autophosphorylation was examined,
because: 1) autophosphorylation of effector-specific tyrosine residues
increases the velocity of the kinase reaction (Bertics and Gill, 1985);
2) autophosphorylation increases the affinity of the EGFr for its
substrates, such as phospholipase C- (Magni et al., 1991), allowing
these substrates to bind the activated receptor (docking site) and
thereby become tyrosine phosphorylated; and 3) EGFr phosphotyrosine
represents the last known biochemical event before committed steps
toward cellular division are mediated by downstream effector
mechanisms. For these reasons, we believe quantification of EGFr
autophosphorylation is related to, and characterizes, inhibition of the
kinase functionality.
CP-358,774 is a potent inhibitor of the EGFr tyrosine kinase with
an IC50 value of 2 nM; CP-358,774 and its analogs
have been shown to be direct-acting, reversible, ATP-competitive
inhibitors of EGFr tyrosine phosphorylation (Moyer et al., 1997;
Pustilnik et al., 1997). Specificity analysis has indicated >1000-fold
selectivity against other tyrosine kinases, such as
pp60v-src, pp145c-abl,
the tyrosine kinase activities of the insulin and the
insulin-like growth factor-1 receptors; selectivity has been shown
against isolated kinases as well as in intact cells (Moyer et al.,
1997). CP-358,774 inhibits autophosphorylation of the EGFr in a variety of EGFr-overexpressing tumor cells (IC50 = 20 nM)
and produces inhibition of mitogenesis, inhibition of tumor cell
division, and cell cycle arrest. In some cell types, such as DiFi,
CP-358,774 induces concentration-dependent apoptosis in vitro.
Here, we report that CP-358,774 is an effective, orally active
inhibitor of EGFr tyrosine autophosphorylation. CP-358,774 can
effectively inhibit EGFr tyrosine phosphorylation in human tumors
growing s.c. in athymic mice with an ED50 value
of 10 mg/kg p.o. It has significant duration of action and produces
substantial inhibition of human EGFr-overexpressing tumors growing s.c.
in athymic mice. Moreover, the degree of inhibition of EGFr
phosphotyrosine shows good agreement with the degree of tumor growth
inhibition in treated animals. The results of these experiments were
previously reported at the American Association for Cancer Research
annual meeting (Pollack et al., 1997). The data suggest that CP-358,774 may be a useful new compound for therapy of human neoplastic diseases.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mice. Three- to 4-week-old female athymic mice (CD-1 nu/nu) were used for human tumor xenografts. Mice were obtained from Charles River Laboratories (Wilmington, MA) and were housed in specific pathogen-free conditions, according to the guidelines of the American Association for Laboratory Animal Care; all studies were carried out with approved institutional experimental animal care and use protocols. During these studies, animals were provided pelleted food and water ad libitum and kept in a room conditioned at 70-75°C and 50 to 60% relative humidity with >15 fresh air changes per hour. Sentinel heterozygous littermates of the athymic animals were monitored routinely (3-week intervals) by serological assays and were found to be free of exposure to the following agents: murine hepatitis virus, ectromelia virus, and Sendai virus. For all studies, the mice were allowed to acclimate for 1 to 3 days after receipt of shipment; test animals were randomized before commencement of treatments and examined twice daily thereafter for compound-induced or tumor-related deaths. Moribund animals were sacrificed to reduce suffering.
Tumor Cell Lines. The HN5 cells were obtained from Dr. M. J. O'Hare (Haddow Labs., Institute of Cancer Research, Sutton, Surrey, UK). All other cells were purchased from the American Type Culture Collection (Rockland, MD). All cell lines were free of reovirus type 3, pneumonia virus of mice, K-virus, Theiler's virus, Sendai virus, ectromelia virus, and lactate dehydrogenase virus (Microbiological Associates, Bethesda, MD).
Cell Culture. Cell lines were passaged by monolayer culture in 175-cm2 tissue culture flasks (Nunclon; Marsh Biomedical Products, Rochester, NY) in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated FBS (Hazelton Research Products, Inc., Lenexa, KS), 300 µg/ml glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 10 µg/ml gentamycin at 37°C in a humidified 95% air/5% CO2 atmosphere. Routine periodic samples of cell culture broths tested negative for Mycoplasma contamination (Microbiological Associates). For implantation in vivo, the tumor cells were harvested from exponentially growing cultures (60-80% confluence), detached by light trypsinization (0.25% trypsin and 0.02% EDTA, 1 min), washed in Hanks' balanced salt solution (HBSS), resuspended in HBSS, mixed with the basement membrane preparation Matrigel (40234; Collaborative Biomedical Products, Bedford, MA), and held in an ice bath <1 h before injection.
Chemotherapeutants.
CP-358,774
[6,7-bis(2-methoxy-ethoxy)quinazoline-4-yl]-(3-ethynylphenyl)amine;
MF = C22H23N3O4),
a colorless, crystalline, anhydrous compound, was synthesized in our
laboratories (Arnold and Schnur, 1998). In these studies, the
hydrochloride salt (molecular weight = 429.9) was used in all
cases, except for that represented in Fig. 7, which used the free base
(molecular weight = 393.4), and the dosage levels shown represent
the quantity of free base administered, excluding the contribution of
the salt. The compound was formulated for i.p. or p.o. administration
by dissolution of the dry powder in a small amount (10% of final
volume) of dimethyl sulfoxide (DMSO), mixed by vortexing until
dissolved; during vortexing, sufficient sterile, pyrogen-free
physiological saline (0.15 N NaCl), containing 0.10% (w/v) Pluronic
P105 (BASF Wyandotte, Parsippany, NJ), was added to produce a
homogeneous fine suspension. The prepared dosage forms did not produce
microbial colonies after incubation on brain-heart infusion agar and
did not contain endotoxin detectable by the Limulus
amoebocyte lysate assay (Associates of Cape Cod, Inc., Woods Hole, MA).
Doxorubicin (Adriamycin; Rapid Dissolution Formula) was purchased from
Adria Labs. (Columbus, OH). Cisplatin was obtained as a powder from
Sigma Chemical Co. (St. Louis, MO). All dosage forms were freshly
prepared for each day's treatment. CP-358,774 and the reference agents
were dosed according to the optimum formulation, route, and regimens,
as empirically derived in previous studies; aggressive dosing
parameters (single bolus treatments at maximum tolerated dosage levels)
were used for maximum antitumor efficacy of the cytoreductive agents.
Test animals were treated between 7 and 9 AM, immediately after a 12-h
dark photoperiod (active phase), to control for variability introduced
by circadian physiological cycles, according to the methods of Halberg
et al. (1973).
EGFr Phosphotyrosine Determinations by Enzyme-Linked Immunosorbent Assay (ELISA). To determine compound-induced inhibition of EGFr-associated tyrosine phosphorylation in human tumor explants from athymic mice, an ELISA specific for EGFr phosphotyrosine was developed. Tumor tissue was harvested at various times after dosing (usually 1 h) by careful dissection, immediately flash frozen in liquid nitrogen, and then homogenized in buffer formulated to prevent further tyrosine phosphorylation as well as enzymatic phosphatase activity. A double-antibody ELISA provided quantitative determinations of the degree of EGFr tyrosine phosphorylation after specific capture of EGFr protein.
Briefly, athymic mice with s.c. tumors (5-10 mm in diameter) were euthanized humanely, and tumors were excised with the use of small dissecting scissors and mosquito forceps, after which the tumor tissue was immediately flash frozen in liquid nitrogen and stored at
70°C
before homogenization and immunoassay. Tumors were weighed, and for
each 100 mg of tumor tissue, 1 ml of ice-cold, sterile lysis buffer was
added. Lysis buffer contained (per liter) 50 ml of 1 M HEPES, pH 7.4, buffer, 37.5 ml of 4 M sodium chloride, 0.75 ml of 2 M magnesium
chloride, 10 ml of 100 mM EDTA, 10 ml of glycerol, 10 ml of Triton
X-100, 8 ml of 200 mM sodium orthovanadate, 4.2 g of sodium
fluoride, 50 µg/ml phenylmethylsulfonyl fluoride, 25 mg of soybean
trypsin inhibitor, 10 µg/ml leupeptin, and 10 µg/ml aprotinin.
Tumors were homogenized with a Thomas Teflon pestle homogenizer
attached to a power drill (or equivalent) and then clarified by
centrifugation; the resulting supernatant liquid (800 µl) was
transferred to microtiter plates in 200-µl aliquots and maintained at
70°C before assay.
Appropriate dilutions of tumor homogenates (1:20-1:40 dilutions) were
made in blocking buffer containing (per liter) 50 g of bovine
serum albumin, 10 g of ovalbumin, 0.90% NaCl, and 10 mM
Tris · HCl buffer, pH 7.4. After dilution, 100-µl aliquots were
transferred to microtiter wells containing adsorbed monoclonal antibody
to EGFr protein (QIA08; Oncogene Science, Uniondale, NY). The plates
were then incubated for 30 min at 30°C (or 3 h at room
temperature) to allow efficient capture of the EGFr protein from the
tumor homogenates. Microtiter wells were washed six times in a 1:10
dilution of Plate Wash Concentrate (PN 77 0550; DuPont NEN, Boston,
MA). To detect phosphotyrosine residues, 100 µl of horseradish
peroxidase-conjugated monoclonal antibody specific for phosphotyrosine
(diluted 1:1000 in blocking buffer) was added to each well (PY54
conjugate, PT03; Oncogene Science), and plates were incubated for
1 h at 30°C. Microtiter wells were then washed six times in a
1:10 dilution of Plate Wash Concentrate, after which 100 µl/well of
3,3',5,5'-tetramethylbenzidine substrate was added (50-76-04;
Kirkegaard and Perry Laboratories, Gaithersburg, MD); color development
was monitored over 30 min, after which all reactions were stopped with
100 µl/well of 0.09 M sulfuric acid. For quantification, absorbance
was determined at 450 nm with a Bio-Rad (Hercules, CA) model 3550 microplate reader. EGFr phosphotyrosine content was calculated after
normalization of each sample for total protein with a commercial kit
(BCA Protein; Pierce, Rockford, IL).
The absorbance values for samples from each of the tumor-bearing
animals (sample size, four mice/treatment group) were entered into a
custom Microsoft Excel spreadsheet, where the endpoints (i.e., protein
concentrations and phosphotyrosine levels) were calculated. In all
cases, the EGFr-associated tyrosine phosphorylation was expressed as
absorbance units/mg total protein. For statistical inferences, the
relationships between groups (i.e., test versus control group) were
identified using a computer program for the one-way ANOVA, where the
significance level was assigned at 0.05. P values were
determined using Dunnett's t statistic. A set of internal
laboratory standards (i.e., aliquots from previously frozen tissue for
both treated and control groups) was used to assess the quality and
reproducibility of the immunoassay; in the course of 5 years' routine
testing, the results were highly reproducible (i.e., the coefficient of
variation was <6.0%).
HPLC Determinations of CP-358,774 in Plasma and Tumor Tissue. Determination of drug concentration was made by organic extraction (acetonitrile) of plasma and tumor samples, followed by HPLC. CP-358,774 in plasma and tumor tissues was extracted from 200-µl samples spiked with 100 µl of internal standard (CP-292,597; 0.8 ng/µl in acetonitrile) with 5 ml of methyl t-butyl ether using an Oberbach reciprocating shaker for 10 min. Before extraction, tumor tissue was homogenized in 4 parts deionized water to 1 part tumor specimen (v/m) using an Omni 2000 (Omni International, Gainesville, VA) tissue homogenizer. Samples were centrifuged at 3000 rpm for 5 min at 22°C using a Jouan centrifuge. The organic layer of each sample was transferred to a clean tube, and the methyl t-butyl ether was evaporated to dryness in a Zymark Turbo-Vap at 60°C. All samples were reconstituted in 200 µl of mobile phase consisting of 70% water and 30% acetonitrile (v/v) brought to pH 2.4 with trifluoroacetic anhydride (Acros Organics). A 2-liter volume of mobile phase consisted of 1400 ml of Milli-Q deionized water, 600 ml of acetonitrile, and 550 µl of trifluoroacetic anhydride. The analytical column was a YMC Basic C-18 (4.6 mm × 150 mm, 3 µm). A pump (Thermo Separation Products Constametric 4100) was used to establish a 1.5 ml/min flow rate through the column. CP-358,774 was detected at 345 nm (AUFS 0.001) using an ultraviolet detector (Milton Roy Spectro Monitor 3100 variable wavelength detector). The retention time for CP-358,774 was 6.5. The lower limit of quantification of the assay was 10 ng/ml for plasma and 50 ng/g for tumor tissue.
Tumor Growth Inhibition Studies In Vivo.
The tumor
growth inhibitory effects of CP-358,774 were measured in young athymic
mice bearing established, palpable (2-4-mm diameter) human HN5 or A431
tumors. Tumors were induced in the left flank of 3- to 4-week old
athymic mice by s.c. injection of 1 × 106 cultured,
log phase HN5 or A431 cells in 0.20 ml of HBSS containing 50%
Matrigel. Tumor size was measured in millimeters with Vernier calipers
across two diameters three times/week, and the tumor volume
(mm3) was calculated using the formula: tumor volume = (length × [width]2)/2, according to standard
methods (Geran et al., 1972); results are expressed as tumor volume
(TuV) in mm3. To calculate tumor growth inhibition,
the following formula was used: inhibition (%) = (TuGcontrol TuGtest)/TuGcontrol × 100%, where tumor
growth (TuG) equals the final tumor size minus the pretreatment tumor
size for individual treatment groups. This method of tumor implantation
provided reproducible growth in athymic mice, enabling the
determination of dose-response effects for a variety of
chemotherapeutic agents. For each experiment, athymic mice were
randomized on receipt of shipment and again after tumor implantation
(i.e., before commencement of treatment). Data collected from the
antitumor studies (e.g., tumor volume) were evaluated for statistical
significance using one-way ANOVA (for significant antitumor activity,
P < .05).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Inhibition of EGFr Phosphotyrosine in HN5 Xenografts.
HN5
possesses many of the characteristics of EGFr-dependent squamous cell
carcinomas both in vitro (Modjtahedi et al., 1993b,c) and in vivo
(Modjtahedi et al., 1993a,b). In particular, monoclonal antibodies
directed at the EGFr can completely block cellular proliferation in
vitro, and for this reason, the tumor cell line was selected to
evaluate a large series of EGFr tyrosine kinase inhibitors. When
administered orally (by gavage) or parenterally (i.p.), CP-358,774
consistently produced significant, dose-related inhibition of HN5 EGFr
tyrosine phosphorylation 1 h after dosing (Fig.
1). Compared with vehicle-treated
controls, a maximum of 80% reduction in phosphotyrosine was observed
after dosing by p.o. or i.p. routes. In several preliminary
experiments, the vehicle (10% DMSO, 0.85% NaCl, and 0.10% Pluronic
P105) produced no inhibition of EGFr phosphotyrosine compared with
water or saline treatments.
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Relationship of Plasma Concentration to EGFr Phosphotyrosine
Inhibition.
Figure 2 illustrates the
relationship of plasma concentration of CP-358,774 to inhibition of
EGFr-associated phosphotyrosine of HN5 xenografts. At 1 h
post-treatment with a single dose, plasma concentrations of 2 to 10 µM CP-358,774 (0.79-3.9 µg/ml) were associated with a significant
reduction in EGFr phosphotyrosine (~40% reduction relative to
vehicle-treated controls). At higher plasma concentrations (i.e.,
10-100 µM, 3.9-39 µg/ml), the reduction in EGFr phosphotyrosine
ranged from 65 to 75%. By interpolation, the effective plasma
concentration for 50% inhibition of the target receptor was estimated
at 8 µM (3.1 µg/ml) and ~12 µM (4.7 µg/ml) for p.o. and i.p.
dosing, respectively. In mouse plasma, 95% of CP-358,774 is bound to
plasma proteins. Taking these data into account, at 1 h after the
dose, 50% inhibition of EGFr-associated phosphotyrosine of HN5 tumors
occurred at free plasma concentrations of 400 nM (160 ng/ml) for p.o.
and 600 nM (240 ng/ml) for i.p. doses of CP-358,774.
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Duration of Action of CP-358,774.
The duration of reduction in
EGFr phosphotyrosine after a single 92 mg/kg dose of CP-358,774 was
evaluated in the HN5 model (Fig. 3).
After p.o. dosing, significant and substantial inhibition of EGFr
phosphotyrosine (75-85%) was observed for 12 h; reduction was
still measurable (25-40%), and statistically significant, after
24 h. To a similar degree of efficacy, parenterally (i.p.) dosed
mice showed substantial inhibition of EGFr phosphotyrosine for 12 h; however, no reduction was observed at 24 h (data not shown).
Calculation of the area under the curve for reduction in EGFr
phosphotyrosine provides an estimation of the overall degree of
inhibition over a 24-h period. Based on the assumption that complete
inhibition (100%) of EGFr autophosphorylation over a 24-h period would
produce inhibition of 2400%-h (100% "coverage"), p.o.
dosing elicited inhibition of 1690%-h (70.4% coverage), whereas parenteral dosing (i.p.) showed an area under the curve of 1420%-h (59.0% coverage).
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Antitumor Effects of CP-358,774 (HN5).
The antitumor effects
of CP-358,774 were determined in the EGFr-overexpressing human HN5 and
human A431 epidermoid carcinomas. Both tumors have been shown to be
inhibited by monospecific anti-EGFr antibodies in cell culture and in
xenograft models (Fan et al., 1993; Modjtahedi et al., 1993a). Oral
administration of CP-358,774 produced significant dose-related
antitumor effects against established HN5 growing s.c. in athymic mice
(Fig. 4). When test animals were dosed
for 20 consecutive days beginning at 4 days after tumor implantation
(tumor diameter, 2-4 mm), the minimal effective dose for significant
antitumor effects was 5.7 mg/kg/day p.o., using one-way ANOVA
(P < .05 with Dunnett's test). Doses of 11 to 92 mg/kg/day p.o. produced substantial antitumor effects (i.e., >50% inhibition). During the course of dosing (days 4-23 after
implantation), tumor-bearing mice treated with vehicle alone showed
progressive enlargement of tumors; spontaneous regressions in
vehicle-treated or untreated animals have not been observed in this
model.
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11 mg/kg, and some tumor growth inhibition,
although not as pronounced, was evident at a dose as low as 5.7 mg/kg
(Fig. 7). Tumors in treated animals (11 mg/kg) appeared to be in stasis during the treatment or to slightly
decrease in size during the dosing period. In addition, the static
tumor profile appeared to extend beyond the treatment period, such that
tumor sizes for the treated animals did not exceed pretreatment levels
until at least day 60, which was 33 days after the cessation of
treatment. Although tumors resumed growth at days 60 to 70, subsequent
growth did not occur at the same rate as the controls. These data,
taken together, suggest that treatment with CP-358,774 at effective
dosage levels produces tumor stasis up to, and beyond, the dosing
period and suggest that effective control of established tumors may be
expected from chronic treatment regimens. In this and several other
experiments, treatment with the maximally tolerated dose of doxorubicin
had little therapeutic effect on large, well-established HN5 xenografts (Fig. 7), although tumor growth inhibition could readily be
demonstrated with small tumor masses (tumor diameter, 2-4 mm; Fig. 8).
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Combinations of CP-358,774 and Cisplatin.
Current chemotherapy
regimens use multiple agents for the treatment of carcinomas to obtain
improved efficacy and avoid emergence of resistance. Combined therapy
of experimental tumors with anti-EGFr antibodies and cytotoxic therapy
can produce greater antitumor effects than that use of either modality
alone (Baselga et al., 1993; Fan et al., 1993). We therefore examined
the effects of CP-358,774 treatment in combination with a conventional
cytotoxic drug (i.e., cisplatin) to characterize both the toxicity and
efficacy of combination regimens.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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CP-358,774 is a potent, selective inhibitor of the tyrosine kinase
of the human EGFr that blocks tumor cell division, produces cell cycle
arrest, and initiates programmed cell death in EGFr-overexpressing human tumor cells (Moyer et al., 1997). To examine the therapeutic effects of this inhibitor, we selected a human tumor system (HN5) that
is dependent on EGFr for mitogenic signals (Modjtahedi et al.,
1993b,c). In parallel, we measured EGFr tyrosine phosphorylation to
assess inhibition of the kinase activity in situ because the functions
of this receptor in signal transduction require kinase activity. EGFr
autophosphorylation represents the last known biochemical event before
committed steps toward cell division are mediated by numerous
downstream effectors.
CP-358,774 produces potent reduction in EGFr tyrosine phosphorylation within 1 h of dosing (ED50 = 9.9 mg/kg p.o.). The plasma CP-358,774 concentration associated with this effect was 8 µM (3.1 µg/ml); because 95% of CP-358,774 is bound to mouse plasma proteins, we calculated that 50% inhibition of EGFr phosphotyrosine occurred at free plasma concentrations of 400 nM (160 ng/ml) for p.o. doses of CP-358,774. Substantial reduction in EGFr phosphotyrosine was observed for at least 12 h after the dose (100 mg/kg p.o.), and significant reduction was noted at 24 h. The antitumor effects of CP-358,774 correlated well with inhibition of tumor-associated EGFr phosphotyrosine.
These results are comparable to those of Kunkel et al. (1996), who used
Western blots to show 80 to 90% reduction in EGFr phosphotyrosine in
A431 tumors as early as 15 min after i.p. administration of the EGFr
inhibitor PD153035. Our findings differ in that we observed a longer
duration of action for CP-358,774, whereas in the study of Kunkel et
al. (1996), tyrosine phosphorylation returned to baseline after 3 h. At a dose of 40 mg/kg i.p., PD153035 produced a reduction in EGFr
phosphotyrosine but no measurable changes in tumor growth when
administered b.i.d from days 5 to 13 after s.c. implantation of A431.
The importance of duration of receptor modulation to antitumor effects
is underscored in data on a related compound. PD168393, an irreversible
EGFr/erbB-2 inhibitor, suppressed EGFr phosphotyrosine for at least
24 h, whereas suppression by the reversible inhibitor PD174265 was
short-lived (i.e., <8 h); only PD168393 showed antitumor activity (Fry
et al., 1998). In our work, we were able to show substantial antitumor
effects for the HN5 and A431 carcinomas at doses that can reduce EGFr
autophosphorylation in extracted xenografts. Despite a number of
EGFr-targeted therapeutants, little attention has been paid to
assessment of receptor modulation in the growing tumor until very
recently (Pollack et al., 1997; Vincent et al., 1998). Our data suggest
that inhibition of EGFr, as evidenced by monitoring EGFr
phosphotyrosine in situ, is directly related in this system to
inhibition of established tumor growth (Fig. 6).
Although the benefit of an EGFr inhibitor has yet to be proved
clinically, EGFr-specific therapeutants produce considerable inhibition
of human xenografts in athymic mice. Monoclonal antibodies were shown
by Masui et al. (1984) to prevent tumor growth in vivo. Inhibition by
EGFr-specific monoclonal antibodies has been shown subsequently with a
variety of therapeutic endpoints, including tumor growth inhibition,
regression of tumor implants, reduced metastases, and increased
lifespan (Aboud-Pirak et al., 1988; Mueller et al., 1991; Yoneda et
al., 1991a; Fan et al., 1993a,b; Schnurch et al., 1994; Prewett et al.,
1996). Striking antitumor effects were shown by Modjtahedi et al.
(1993a,b; Dean et al., 1994) with rat monoclonal antibodies ICR62 and
ICR16 and, very recently, by Yang et al. (1999) using humanized
monoclonal antibody E7.6.3. These antibodies induced complete
regressions of human xenografts if treatment was initiated on the day
of tumor implantation and induced significant regressions in large (>1
cm) tumors.
Despite differences in methodologies and endpoints, our results are
similar to those with monoclonal antibodies in the degree and timing of
treatment effectiveness. The responses of EGFr-overexpressing HN5 and
A431 carcinomas to treatment with CP-358,774 involved a marked
cessation of tumor growth detected shortly after commencement of
dosing. This was observed repeatedly in experiments involving 20 doses
or as few as 5 daily doses (data not shown) and is a consistent finding
with EGFr-targeted monoclonal antibodies (Masui et al., 1986; Yoneda et
al., 1991a; Baselga et al., 1993; Modjtahedi et al., 1993a,b; Schnurch
et al., 1994; Ciardiello et al., 1996; Yang et al., 1999). In addition,
the responses are clearly potent (ED50 = 10 mg/kg
p.o.), dose dependent, and substantial (i.e., >50% inhibition).
CP-358,774 shares another pharmacological feature common to monoclonal
antibodies, one that distinguishes these forms of therapies from
conventional cytoreductive agents. When very large tumors were used
(1-cm diameter), CP-358,774 produced stasis of tumor growth and a
noticeable reduction in the size of treated tumors (i.e., partial
regression). This is a confirmed observation for the monoclonal
antibodies (Baselga et al., 1991; Yoneda et al., 1991a; Prewett
et al., 1996); with doxorubicin, however, treatment is very effective
when small tumors are treated (100-200 mm3; Fig.
8) but is much less effective with large tumor masses (i.e., 500-1000 mm3; Fig. 7). Unlike monoclonal antibodies
(Rodeck et al., 1987; Modjtahedi et al., 1993a,b; Dean et al., 1994),
we have not seen complete regression in any of several xenograft
therapy experiments to date; when treatment is stopped, tumors resume
growth, although the growth rate appears somewhat less than that of the
vehicle-treated controls.
Distinct differences exist between CP-358,774 and monoclonal antibodies
as pharmacological agents, and these may emanate from differences in
their respective mechanisms of action. Although CP-358,774 targets the
intracellular domain of EGFr (Moyer et al., 1997), monoclonal
antibodies act primarily as a blockade of the external EGF-binding
domain. Immunoglobulins, however, may rely in part on recruitment of
host effector mechanisms (immune cytolysis and antibody-dependent
cellular cytotoxicity). Significant antitumor effects have been shown
with Ig F(ab')2 fragments by some investigators
(Aboud-Pirak et al., 1988; Fan et al., 1993b), although not by others
(Mueller et al., 1991), but there are studies that suggest monoclonal
antibodies derive at least some therapeutic effects from immune
recruitment (Herlyn and Koprowski, 1982; Bier et al., 1998). The
cessation of tumor growth (i.e., stasis) is consistent with inhibition
of EGFr signal transduction and with inhibition of subsequent cellular
proliferation in vivo, but the contribution of immune mechanisms to
antibody-induced regressions requires further clarification. In
addition, the incubation of DiFi cells with CP-358,774 induces
apoptotic cell death (Moyer et al., 1997), and the extent to which
CP-358,774 induces apoptosis in vivo in the HN5 is currently under investigation.
The antitumor effects of CP-358,774 in conjunction with conventional
cytoreductive agents (i.e., cisplatin) were similar to those observed
for monoclonal antibody/cytoreductive combinations. Cisplatin and
monoclonal antibodies have been shown to have additive and, in some
cases, synergistic (supra-additive) antitumor effects (Aboud-Pirak et
al., 1988; Fan et al., 1993a,b; Prewett et al., 1996). Additive effects
of monoclonal antibodies have also been demonstrated for the
tyrphostins (Yoneda et al., 1991b) and doxorubicin (Baselga et al.,
1993; Baselga and Mendelsohn, 1994). Our observations with CP-358,774
and cisplatin indicated additive antitumor effects regardless of
whether CP-358,774 was administered before, during, or after treatment
with the cytoreductive; that is, we found significant differences in
tumor growth inhibition for the combination compared with either agent
alone. We did not observe supra-additive effects with this or other
conventional chemotherapeutants, and the experiments were conducted
under conditions that would have allowed synergistic responses to be
detected. The major goal of the drug interaction studies, however, was
to identify incompatibilities, of which there were none detected for
either antitumor effects or toxicity endpoints.
Other small molecules have been reported to inhibit the EGFr. The
tyrphostin RG-13022 inhibits EGFr autophosphorylation and EGFr-dependent tumor cell proliferation. At 400 µg/mouse/day, RG-13022 significantly inhibited MH-85 xenografts and produced increased lifespan (Yoneda et al., 1991b) but had little activity against HN5, albeit with treatment schedules and endpoints that differed somewhat from the original work (McLeod et al., 1996). Naamidine A, an alkaloid purified from a Fijian
Leucetta sp. sponge, showed potent antitumor effects against
the A431 carcinoma with an ED50 value of ~12.5
mg/kg/day when treatment was initiated 1 day after implantation (Copp
et al., 1998). In addition, impressive tumor growth inhibition, stasis,
and, in some models, regressions were achieved with the
anilinoquinazoline ZD1839 against an array of human carcinoma
xenografts; this compound has been advanced to clinical development
(Woodburn et al., 1997). Although the treatment parameters and
endpoints may differ for the EGFr inhibitors, these reports clearly
indicate that the EGFr represents a potentially important molecular
target for cancer therapy.
In conclusion, we found that CP-358,774 is a very potent and selective inhibitor of the EGFr tyrosine kinase and is capable of inhibiting EGFr tyrosine phosphorylation in human tumors in athymic mice after p.o. or i.p. administration. Most importantly, we observed that in tumor growth inhibition studies, the degree of inhibition of EGFr phosphotyrosine is clearly correlated with significant and substantial tumor growth inhibition. For these reasons, we believe that CP-358,774 may be useful for the therapy of EGFr-dependent human carcinomas.
| |
Acknowledgments |
|---|
We gratefully acknowledge the efforts of Drs. D. S. Salsburg and A. C. Swindell in biostatistical analyses and experimental design. In addition, we greatly appreciate the advice and support of Drs. Alan R. Proctor and Kelvin Cooper. Last, we would like to recognize the creativity and commitment of the late Rodney C. Schnur.
| |
Footnotes |
|---|
Accepted for publication August 5, 1999.
Received for publication May 18, 1999.
1 Portions of this work were presented at the annual meeting of the American Association for Cancer Research, April 1997.
2 Present address: Department of Chemistry, BASF Bioresearch Corp., 100 Research Dr., Worcester, MA 01605-4314.
3 Address: OSI Pharmaceuticals, Inc., 106 Charles Lindbergh Blvd., Uniondale, NY 11553.
Send reprint requests to: Dr. Vincent A. Pollack, Department of Genomics, Targets and Cancer Research, Pfizer Central Research, Eastern Point Road, Groton, CT 06340. E-mail: vincent_a_pollack{at}groton.pfizer.com
| |
Abbreviations |
|---|
EGF, epidermal growth factor; EGFr, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; HBSS, Hanks' balanced salt solution; HN5, LICR-LON-HN5 head and neck carcinoma.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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|
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|
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|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
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|
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|
|
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E. G. Barbacci, L. R. Pustilnik, A. M. K. Rossi, E. Emerson, P. E. Miller, B. P. Boscoe, E. D. Cox, K. K. Iwata, J. P. Jani, K. Provoncha, et al. The Biological and Biochemical Effects of CP-654577, a Selective erbB2 Kinase Inhibitor, on Human Breast Cancer Cells Cancer Res., August 1, 2003; 63(15): 4450 - 4459. [Abstract] [Full Text] [PDF]
|
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S. N. Malik, L. L. Siu, E. K. Rowinsky, L. deGraffenried, L. A. Hammond, J. Rizzo, S. Bacus, M. G. Brattain, J. I. Kreisberg, and M. Hidalgo Pharmacodynamic Evaluation of the Epidermal Growth Factor Receptor Inhibitor OSI-774 in Human Epidermis of Cancer Patients Clin. Cancer Res., July 1, 2003; 9(7): 2478 - 2486. [Abstract] [Full Text] [PDF]
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 V. Grunwald and M. Hidalgo Developing Inhibitors of the Epidermal Growth Factor Receptor for Cancer Treatment J Natl Cancer Inst, June 18, 2003; 95(12): 851 - 867. [Abstract] [Full Text] [PDF]
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R. Nahta, G. N. Hortobagyi, and F. J. Esteva Growth Factor Receptors in Breast Cancer: Potential for Therapeutic Intervention Oncologist, February 1, 2003; 8(1): 5 - 17. [Abstract] [Full Text] [PDF]
|
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E. E.W. Cohen and E. E. Vokes Searching for a Standard J. Clin. Oncol., January 15, 2002; 20(2): 359 - 361. [Full Text] [PDF]
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D. W. Rusnak, K. Lackey, K. Affleck, E. R. Wood, K. J. Alligood, N. Rhodes, B. R. Keith, D. M. Murray, W. B. Knight, R. J. Mullin, et al. The Effects of the Novel, Reversible Epidermal Growth Factor Receptor/ErbB-2 Tyrosine Kinase Inhibitor, GW2016, on the Growth of Human Normal and Tumor-derived Cell Lines in Vitro and in Vivo Mol. Cancer Ther., December 1, 2001; 1(2): 85 - 94. [Abstract] [Full Text] [PDF]
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J. G. Christensen, R. E. Schreck, E. Chan, X. Wang, C. Yang, L. Liu, J. Cui, L. Sun, J. Wei, J. M. Cherrington, et al. High Levels of HER-2 Expression Alter the Ability of Epidermal Growth Factor Receptor (EGFR) Family Tyrosine Kinase Inhibitors to Inhibit EGFR Phosphorylation in Vivo Clin. Cancer Res., December 1, 2001; 7(12): 4230 - 4238. [Abstract] [Full Text] [PDF]
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Y. A. Elsayed and E. A. Sausville Selected Novel Anticancer Treatments Targeting Cell Signaling Proteins Oncologist, December 1, 2001; 6(6): 517 - 537. [Abstract] [Full Text] [PDF]
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F. Ciardiello and G. Tortora A Novel Approach in the Treatment of Cancer: Targeting the Epidermal Growth Factor Receptor Clin. Cancer Res., October 1, 2001; 7(10): 2958 - 2970. [Abstract] [Full Text] [PDF]
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M. Hidalgo, L. L. Siu, J. Nemunaitis, J. Rizzo, L. A. Hammond, C. Takimoto, S. G. Eckhardt, A. Tolcher, C. D. Britten, L. Denis, et al. Phase I and Pharmacologic Study of OSI-774, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, in Patients With Advanced Solid Malignancies J. Clin. Oncol., July 1, 2001; 19(13): 3267 - 3279. [Abstract] [Full Text] [PDF]
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L. M. Strawn, F. Kabbinavar, D. P. Schwartz, E. Mann, L. K. Shawver, D. J. Slamon, and J. M. Cherrington Effects of SU101 in Combination with Cytotoxic Agents on the Growth of Subcutaneous Tumor Xenografts Clin. Cancer Res., July 1, 2000; 6(7): 2931 - 2940. [Abstract] [Full Text]
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