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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Inhibition of Epidermal Growth Factor Receptor Activity by Two Pyrimidopyrimidine Derivatives

Departments of New Chemical Entities Pharmacology (F.S., J.C.A.v.M.) and New Biological Entities Pharmacology (A.B., K.-H.H.), Boehringer Ingelheim, Vienna, Austria; and Department of Chemical Research (E.L., G.D., F.H.), Boehringer Ingelheim, Ingelheim, Germany

Received March 30, 2004; accepted June 15, 2004.

	Abstract

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Overexpression of the epidermal growth factor receptors (EGFRs) and human epidermal growth factor receptor 2 occurs frequently in human cancers and is associated with aggressive tumor behavior and poor patient prognosis. We have investigated the effects in vitro and in vivo of a new class of inhibitor molecules on the growth of several human cancer cell lines. BIBX1382 [N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine] and BIBU1361 [(3-chloro-4-fluoro-phenyl)-[6-(4-diethylaminomethyl-piperidin-1yl)-pyrimido[5,4-d]pyrimidin-4-yl]-amine] are two new selective EGFR kinase inhibitors that do not block the activity of other tyrosine kinases. BIBU1361 blocked epidermal growth factor-induced phosphorylation of EGFR and also prevented downstream responses such as mitogen-activated protein kinase kinase (MAPK/extracellular signal-regulated kinase kinase) and MAPK activation in cells. In accordance with these observations thymidine incorporation into EGFR-expressing KB cells was selectively and potently inhibited by BIBX1382 and BIBU1361 with half-maximally effective doses in the nanomolar range. Oral administration of these compounds inhibited the growth of established human xenografts in athymic mice, including vulval and head and neck squamous cell carcinomas. Tumor growth inhibition by BIBX1382 coincided with reduced pEGFR and Ki-67 levels in vivo, which is in accordance with the expected effect of EGFR inhibitors. Collectively, these results show that the structural class of pyrimidopyrimidines, exemplified here by BIBX1382 and BIBU1361, represents an interesting scaffold for the design of EGFR inhibitors.

The observation that the class I receptor pathway is often implicated in the development and progression of many common human epithelial cancers makes the blockade of this growth pathway an attractive target for anticancer therapy (Sainsbury et al., 1987; Gullick, 1991; Helmout and Dean, 1994; Klapper et al., 2000; Grunwald and Hidalgo, 2003; Mendelsohn and Baselga, 2003; Ross et al., 2003). Several approaches have been used to block class I receptor function in cancer cells. Among these, monoclonal antibodies directed to the EGFR (e.g., IMC-C225) or HER2 receptor (Herceptin), as well as small molecules inhibiting the tyrosine kinase activity show promising activity as anticancer agents in preclinical models and clinical trials (Sridhar et al., 2003). In recent years, several classes of small molecules, inhibiting the function of EGFR or HER2 molecules, have been identified (Traxler, 2003). All these small molecules bind competitively to the ATP binding pocket of the receptor kinase moiety and thus inhibit class I receptor activation. Although similar in their inhibition mode, these compounds differ with regard to potency and selectivity. The quinazolines Iressa and erlotinib, which are the most advanced compounds, are selective and reversible EGFR inhibitors. Irreversible inhibitors such the quinazoline canertinib (Smaill et al., 2000; Slichenmyer et al., 2001) and the cyanoquinoline EKB-569 (Wissner et al., 2003) covalently bind into the catalytic cleft of the EGFR molecule and display long-lasting inhibition that is only overcome by new receptor synthesis (Fry et al., 1998). Some inhibitors such as lapatinib and canertinib are dual kinase inhibitors that simultaneously inhibit the EGFR and HER2 kinase activities. Here, we report about two new EGFR inhibitors belonging to the structural class of pyrimido [5,4-d]-pyrimidines comprising a number of molecules displaying strong inhibitory activity against EGFR-dependent processes in vitro and showing good antitumor efficacy in vivo. Whereas BIBX1382 failed to achieve plasma exposure in humans due to a first path metabolism (Dittrich et al., 2002) BIBU1361 is expected to be unaffected by this metabolism.

	Materials and Methods

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Materials
BIBX1382, BIBU1361, and Iressa. BIBX1382 and BIBU1361 were synthesized at Boehringer Ingelheim GmbH (Ingelheim, Germany) (Himmelsbach et al., 1997), and Iressa [4-(3-chloro-4-fluoro-anilino-7-methoxy-6-(3-morpholinopropoxy) quinazoline] was synthesized as described previously (Gibson, 1996).

Cell Lines. The human cancer cell lines KB (oral epidermoid carcinoma), A431 (vulval squamous cell carcinoma), and FaDu (hypopharyngeal squamous cell carcinoma) were obtained from the American Type Tissue Culture Collection (Manassas, VA). The HN5 cell line (head and neck squamous cell carcinoma) was kindly provided by Michael J. O'Hare (Ludwig Institute for Cancer Research, London, UK). All cell lines were maintained at 37°C in a 5% CO₂ humidified atmosphere in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma Chemie, Deisenhofen, Germany), 1 mM Na-pyruvate, 1% nonessential amino acids, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Methods
Kinase Assays. The cytoplasmic tyrosine kinase domains of EGFR, HER2, VEGFR2, HGFR, IGF1R, -InsRK, and c-src were cloned as such or as fusion proteins into pFastBac (Invitrogen, Renfrew, Renfrewshire, UK). The protein was expressed in Sf9 insect cells using the baculovirus expression system according to the manufacturer's protocol. Sf9 cells were harvested 72 h postinfection, washed twice with PBS, and extracted with HEPEX (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM -glycerophosphate, 10 mM para-nitro-phenylphosphate, 30 mM NaF, 5 mM EDTA, 5% glycerol, 1% Triton X-100, 1 mM Na₃VO₄, 0.1% SDS, 0.5 µg/ml pepstatin A, 2.5 µg/ml 3,4-dichloroisocoumarin, 2.5 µg/ml trans-epoxysuccinyl-L-leucyl-L-amido butane, 20 KIU/ml aprotinin, 2 µg/ml leupeptin, 1 mM benzamidine, and 0.002% phenylmethylsulfonyl fluoride). The extracts were used for the determination of IC₅₀ values. The dilution of enzymes was set so that incorporation of phosphate was linear with respect to time and amount of enzyme. Enzyme activities were assayed in the presence or absence of serial dilutions of the inhibitor performed in 50% dimethyl sulfoxide. Each microtiter plate contained internal controls such as blank, maximum reaction (no inhibitor added), and historical reference compound (substance with known IC₅₀ value).

EGFR kinase activity was assessed in a 50-µl reaction containing 5% dimethyl sulfoxide, 40 mM HEPES, pH 7.4, 10 mM Mg-acetate, 0.5 mg/ml poly (Glu-Tyr), 0.05% Triton X-100, 50 µM ATP, 1 µCi of [-³³P]ATP, and 10 µl of enzyme preparation. Assays were carried out at room temperature for 30 min and terminated by the addition of 10 µl of 5% H₃PO₄. The entire reaction was trapped onto GF/B filters, and the incorporated radioactivity was determined by scintillation counting using a Microbeta counter.

The reaction conditions for the radioactive HER2 kinase assay have been described previously (Stratowa et al., 1999). The radioactive assays for VEGFR2 and HGFR were performed as described above with the following modifications: VEGFR2 assays contained 5% Me₂SO, 40 mM HEPES, pH 7.4, 5 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml poly(EY), 0.05% Triton X-100, 100 µM ATP, and 1 µCi of [-³³P]ATP, and the reaction was carried out at room temperature for 20 min. The HGFR assays were performed like the VEGFR2 assays but in the absence of MnCl₂.

The kinases assays for c-src, -InsRK, and IGF1R were adapted to a nonradioactive setup. For the c-src kinase assays, a 100-µl reaction contained 10 µl of inhibitor in 50% Me₂SO, 20 µl of substrate solution [200 mM HEPES, pH 7.4, 50 mM Mg-acetate, 2.5 mg/ml poly(EY), 5 µg/ml bio-poly(EY), and 1000 µM Na₃VO₄] and 20 µl of enzyme preparation. The enzymatic reaction was started by addition of 50 µl of a 1 mM ATP solution made in 10 mM Mg-acetate. For the -InsR kinase assays, the 20-µl substrate solution contained 250 mM Tris, pH 7.4, 10 mM dithiothreitol, 2.5 mg/ml poly(EY), and 5 µg/ml bio-poly(EY), and the 2 mM ATP solution was made in 8 mM MnCl₂ and 20 mM Mg-acetate. For the IGF1R kinase assays, the 20-µl substrate solution contained 200 mM HEPES, pH 7.4, 2.5 mg/ml poly(EY), and 5 µg/ml bio-poly(EY), and the 2 mM ATP solution was made in 10 mM MgCl₂ and 20 mM MnCl₂. The nonradioactive kinase reactions were carried out at room temperature for 30 min (20 min for IGF1R) and terminated by the addition of 50 µl of stop solution (250 mM EDTA in 20 mM HEPES, pH 7.4). Then, 100 µl was transferred to a streptavidin-coated microtiter plate, after an incubation time of 60 min at room temperature the plate was washed with 200 µl of wash solution (50 mM Tris and 0.05% Tween 20). Then, 100 µl of a horseradish peroxidase-labeled anti-PY antibody (PY20H anti-PY:horseradish peroxidase supplied by BD Transduction Laboratories, Lexington, KY) (250 ng/ml) was added. After a 60-min incubation the plate was washed three times with a 200-µl wash solution. The samples were then developed with a 100-µl substrate solution [1:1 (v/v), supplied by Bender MedSystems, Vienna, Austria; catalog BMS402 and BMS403]. The reaction was stopped after 10 min with 100 µl of 1 M phosphoric acid. The plate was transferred to an ELISA reader, and extinction was measured at OD_{450 nm}.

The data generated with these assays were analyzed with the program Prism version 3.0 (GraphPad Software Inc., San Diego, CA). The inhibitor concentrations were transformed to logarithmic values, and the raw data were normalized. These normalized values were used to calculate the IC₅₀ by a nonlinear regression curve fit (sigmoidal dose response, variable slope). All iteration data had a correlation coefficient above 0.9, and top and bottom values of the curves displayed a window of at least 5.

EGFR and pEGFR ELISA Assays. EGF receptor phosphorylation was assessed in A431 cells using a commercially available ELISA kit (QIA95; Oncogene Research Products, San Diego, CA) according to the manufacturer's protocol with minor modifications. In brief, 1 x 10⁴ A431 cells were transferred into each well of a 96-well microtiter plate in 90 µl of serum-free culture medium. Cells were incubated at 37°C and 5% CO₂ in a humidified atmosphere overnight. The next day, 10 µl of each test compound concentration was added to duplicate wells. Each microtiter plate contained internal controls, such as blank, maximum reaction, and historical reference compound, for the qualification of the results. The microtiter plates were then incubated at 37°C and 5% CO₂ in a humidified atmosphere for 1 h. EGF-stimulation was done at a final concentration of 100 ng/ml for 10 min at room temperature. Cells were washed two times with 200 µl/well ice-cold PBS before addition of 120 µl/well receptor extraction buffer provided in the ELISA kit and shaking for 1 h at room temperature. After extraction, 100 µl of cell extract was transferred to the assay plate provided in the kit. After 1-h incubation at room temperature, the plate was washed three times with water before addition of the horseradish peroxidase-conjugate. The bound antibody was then detected with TMB substrate solution. Coloring was stopped with 100 µl of stop solution. The extinction was measured at 450 nm. All incubation steps were performed on a microtiter plate shaker.

Expression of the EGF receptor was determined with a commercially available assay (QIA35; Oncogene Research Products), and the A431 extracts were diluted with the sample diluent included in the kit and processed as described in the manufacturer's protocol. Each dilution was measured in duplicate.

The data generated with both assays were analyzed with Prism as described above.

Thymidine Incorporation Assays. Subconfluent KB cultures were used for the assays. Cells were trypsinized, washed twice in serum-free culture medium, and seeded in 200 µl of medium at a density of 1500 cells/well in a flat bottom 96-well culture dish. A 10-µl aliquot of either 200 or 60 µM compound dilution made in PBS was added to the respective first well and serially transferred between the wells. All samples were prepared in triplicate. Compounds were tested between 3 and 10,000 nM. EGF-stimulated cells received 30 ng/ml EGF, which was determined to be the optimal dose in these assays; serum-stimulated cells received 0.5% FCS; unstimulated controls received neither EGF nor serum. After addition of the inhibitors and growth factors, the cells were incubated for 76 h, and then [³H]thymidine (0.1 µCi/well) was added. After an additional 16 h, the cells were harvested on glass fiber filters, and the incorporated radioactivity was determined by scintillation counting using a TopCount (PerkinElmer Life and Analytical Sciences, Boston, MA). To determine inhibition of thymidine incorporation by EGFR inhibitors, the difference between the mean counts of stimulated and unstimulated control wells was set to a 100%. Counts incorporated by unstimulated samples were set to 0%. The stimulation of compound-treated samples was expressed in percentage of the full stimulation. The inhibitor concentrations were transformed to logarithmic values. The percentage of stimulation values were used to calculate the IC₅₀ by iteration using a nonlinear regression curve fit program (Prism; sigmoidal dose response with variable slope).

In Vivo Xenograft Experiments. Five- to six-week-old athymic NMRI-nu/nu female mice (21–31 g) were purchased from Harlan Winkelmann (Borchen, Germany) and maintained under specific pathogen-free conditions. All experiments complied with the Declaration of Helsinki and European Policy Legislations (FELASA and GV-SOLAS) on the Care and Use of Laboratory Animals. After acclimatization mice were inoculated s.c. with 1 x 10⁶ (in 100 µl) A431, FaDu, or HN5 cells into the right flank of the animal. After 7 to 11 days, tumors reached a average volume of 40 to 130 mm³. The mice were randomized and treated daily p.o. with BIBX1382, BIBU1361, or vehicle control on the basis of individual weights. Tumors were measured three times a week with calipers, and tumor volumes were calculated by the formula /6 x length x (width)². Experimental compounds were prepared in 25% aqueous hydroxypropyl--cyclodextrin (33259-3; Sigma-Aldrich, St. Louis, MO) and administered by intragastral gavage. The administration volume was 10 ml/kg b.wt.

Determination of pEGFR Level in Tumor Samples. Excised A431 tumors were snap-frozen in liquid nitrogen immediately after dissection. The tumors were lyophilized over night and extracted with HEPEX (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM -glycerophosphate, 10 mM para-nitro-phenylphosphate, 30 mM NaF, 5 mM EDTA, 5% glycerol, 1% Triton X-100, 1 mM Na₃VO₄, 0.1% SDS, 0.5 µg/ml pepstatin A, 2.5 µg/ml 3,4-dichloroisocoumarin, 2.5 µg/ml trans-epoxysuccinyl-L-leucyl-L-amido butane, 20 KIU/ml aprotinin, 2 µg/ml leupeptin, 1 mM benzamidine, and 0.002% phenylmethylsulfonyl fluoride). The EGFR and pEGFR levels were determined using the ELISA procedures described above [QIA95 (pEGFR) and QIA35 (EGFR); Oncogene Research Products]. To asses the PY-EGFR ratio (PY-EGFR units/EGFR molecule), an in-house standard of EGF-stimulated HN5 cells was used. The EGFR concentration of this standard was 8560 fmol/ml as determined by QIA35. The PY-EGFR concentration was arbitrarily assigned a concentration of 2.8 x 10¹⁵ PY units/ml. The PY-EGFR /EFGR ratio was calculated from the results of the PY-EGFR QIA95 ELISA (expressed in units per milliliter), and the EGFR QIA35 ELISA (expressed in femtomoles per milliliter) and expressed as arbitrary PY-units/EGFR molecule.

Histology and Immunohistochemistry. The proliferation of tumor cells in samples was assessed by Ki-67 immunostaining. Frozen tumor tissues were paraffin embedded, sectioned to 3 to 5 µmin thickness, deparaffinized, and subjected to immunohistochemistry protocol as described previously (Heider et al., 1993a,b). For Ki-67 detection an affinity-purified rabbit anti-human Ki-67 serum was applied (DakoCytomation Denmark A/S, Glostrup, Denmark). As secondary antibody either a biotinylated swine anti-rabbit or rabbit anti-mouse antibody was used, followed by incubation with the avidin-biotin-peroxidase complex (DakoCytomation Denmark A/S). After color development in aminoethylcarbazole, the slides were counterstained with hematoxylin and mounted with aqueous mounting medium. For evaluation of histology, H&E-stained serial sections were used.

	Results

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Potency and Selectivity of BIBX1382 and BIBU1361 in Molecular Assays. The data presented in Table 1 demonstrate that BIBX1382 and BIBU1361 are both potent and selective submicromolar inhibitors of the EGFR kinase activity. An IC₅₀ value of 3 nM was determined for both compounds. The potency of these two compounds compares with the one obtained with Iressa, which is a leading EGFR inhibitor in the field. Inhibition of the closest family member, HER2, was 100- to 1000-fold less potent. Furthermore, BIBX1382 and BIBU1361 did not inhibit a number of other related tyrosine kinases.

Inhibition of EGFR Autophosphorylation in Cells. The inhibitory activity of these two compounds was confirmed at the cellular level. A431 cells, which highly express the EGF receptor, were used to examine the effect of BIBU1361 and BIBX1382 on EGF-induced EGFR phosphorylation. A 1-h pretreatment of the cells with either compound inhibited EGF-induced phosphorylation of EGFR in a dose-dependent manner. For BIBU1361, the EC₅₀ was 122 nM. As a control, the EGF receptor expression was monitored and showed no variation in this experimental setup (Fig. 1). Under the same conditions, the drug concentrations required for half-maximal inhibition by BIBX1382 and Iressa were 141 and 86 nM, respectively (data not shown). Thus, these data demonstrate that both pyrimidopyrimidines reach their target in the cell and that the potency compares to that of Iressa.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Inhibition of EGF-induced EGFR tyrosine autophosphorylation by BIBU1361. A, A431 cells were serum-starved overnight to induce EGFR dephosphorylation. On the next day, cells were treated for 1 h with BIBU1361 at indicated concentrations before stimulation with EGF (100 ng/ml). After a 10-min stimulation, the amount of tyrosine-phosphorylated EGFR was determined as described under Materials and Methods. Three experiments were performed that confirmed the nanomolar potency of BIBU1361 in this assay. B, data in the inset show the concentration of EGFR and demonstrate that EGFR expression is not altered during the experiment.

Effects of BIBU1361 on Downstream Signaling. After showing that BIBU1361 is capable of inhibiting the activation of the EGF receptor, we further evaluated whether this compound would also abrogate downstream signaling events. Several signaling pathways that control the G₁/S transition are activated in response to EGF in A431 cells. Therefore, the effect of BIBU1361 on one such pathway (activation of the MAPK signaling) was analyzed in more details by means of electrophoretic analyses. An aliquot of the extracts used for the autophosphorylation assays was loaded on SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes for Western blot analyses. In line with its ability to prevent EGFR activation, BIBU1361 also suppressed the activation of MEK, a central molecule in the MAPK cascade (Fig. 2). Pretreatment of A431 cells with 313 nM resulted in nearly complete inhibition of phospho-MEK signal. Although MAPK is still activated in the starved A431 cells, pretreatment for 1 h with 1250 nM BIBU1361 induced complete dephosphorylation of this molecule.

View larger version (51K): [in this window] [in a new window]   Fig. 2. Inhibition of EGF-induced signaling by BIBU1361 in A431 cells. A431 cells were incubated for 1 h with BIBU1361 at various concentrations and subsequently stimulated with EGF for 10 min. After treatment, the cells were extracted, and aliquots were subjected to SDS-polyacrylamide gel electrophoresis before blotting onto polyvinylidene difluoride membranes. A Western blot analysis revealing MEK (middle), phosphorylated MEK (pMEK), and MAPK (pMAPK) is shown. The dot in the upper left corner of the pMEK and MEK panels is due to nonspecific staining.

In line with the previous results, treatment of cells with either BIBX1382 or BIBU1361 suppresses EGF-induced thymidine incorporation in KB cells in a dose-dependent manner. For BIBX1382, the EC₅₀ value is 150 nM. Pretreatment of KB cells with 400 nM resulted in almost complete inhibition of new DNA synthesis. Similarly, the EC₅₀ value for BIBU1361 was 225 nM (data not shown). In comparison, the FCS-dependent incorporation was inhibited with an EC₅₀ value of 3195 nM (Fig. 3). Thymidine incorporation can also be stimulated by hepatocyte growth factor in this cell line. The EC₅₀ value was 1815 nM (data not shown). Together, the cellular data show that BIBX1382 and BIBU1361 are potent and selective inhibitors of EGF-induced DNA synthesis.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Inhibition of thymidine incorporation into KB cells. KB cells were seeded at a density of 1500 cells/well in a flat bottom 96-well culture dish, supplemented with BIBU1361 at indicated concentrations, and maintained for 3 days at 37°C. After adding 0.1 µCi of [3H]thymidine to each well, the cells were stimulated either with EGF (30 ng/ml) or 0.5% FCS. Thymidine incorporation was determined by scintillation counting, whereby the difference between the mean counts of stimulated and unstimulated wells was set to 100%.

Antitumor Activity of BIBX1382 and BIBU1361. The antitumor activity of BIBX1382 and BIBU1361 was evaluated in vivo in various xenograft models, including A431, HN5, and FaDu. In nude mice, oral once daily dosing at 10 mg/kg with either BIBX1382 or BIBU1361 completely suppressed tumor growth of human A431 xenografts with respective T/C values of 15 and 6% after 2 weeks of treatment (Fig. 4). The average tumor volume of the control group had reached at least 1000 mm³. On the last treatment day, plasma samples were obtained 4, 8, and 24 h after application. Both compounds reached their maximum 4 h after administration. The C_4h values were 2222 (BIBX1382) and 755 nM for BIBU1361. The C_24h with 244 and 356 nM, respectively, were in the range of the EC₅₀ values in the different cellular assays. No weight loss was observed in treated animals over the course of the 2-week treatment period. Hence, these data demonstrate that both compounds at a daily dose of 10 mg/kg are well tolerated in mice and display antitumor activity as single agents.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Inhibition of A431 tumor growth in nude mice by BIBX1382 and BIBU1361. After establishment of tumors, the animals were treated daily p.o. for 2 weeks with either 10 mg/kg BIBX1382 (), 10 mg/kg BIBU1361 (), or vehicle alone (). Tumor size was measured at indicated time points using electronic calipers. In the graph, the average tumor volume ± standard deviation is plotted over time. A p value <0.01 was determined by statistical analysis of the tumor volumes in each group using one-way analysis of variance (Dunnett's multiple test) (see also Table 2).

The antineoplastic potential of BIBX1382 was further explored in a long-term oral administration schedule. A431-tumor bearing mice were treated once daily for 170 days at a dose of 50 mg/kg. The body weights were stable, and no adverse clinical signs could be observed in the treated animals for the whole treatment period. Tumor regressions were obtained in all treated individuals (n = 7) after 90 days. Although two of the animals were tumor free at the end of the experiment, and this even after the 100-day recovery period, two of the tumors started to regrow after cessation of treatment and three tumors did not significantly progress in size during the experiment (Fig. 5). Thus, these data demonstrate the feasibility of long-term treatment schedules with BIBX1382.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Inhibition of A431 tumor growth in nude mice by BIBX1382. After establishment of tumors, the animals were treated daily p.o. for 170 days with 50 mg/kg BIBX1382 () or vehicle alone (). Tumor size was measured at indicated time points using electronic calipers. The relative tumor volume of each individual mouse is plotted over time. The blue curves describe tumors that regrow after cessation of treatment. The green curves show tumors that remain constant, whereas the red curves depict tumors that disappeared.

Effects of BIBX1382 on Tumor Cells in Vivo. To correlate the antitumor activity of these compounds with biomarker modulation, we investigated in vivo, the ability of BIBX1382 to inhibit EGFR phosphorylation in tumors derived from A431 xenograft-bearing mice treated daily p.o. for 14 days, using biochemical and immunohistochemical approaches. The data in Fig. 6A show that the EGF receptor is constitutively phosphorylated in control A431 tumors. Treatment of mice, carrying established A431 tumors, at a dose of 50 mg/kg/day results in dephosphorylation of the EGF receptor (Fig. 6A). Thus, the antitumor activity of BIBX1382 coincides with inhibition of the constitutive tyrosine phosphorylation of the EGF receptor molecule in A431 xenografts.

View larger version (98K): [in this window] [in a new window]   Fig. 6. In vivo modulation of phosphorylated EGFR and Ki-67 nuclear antigen by BIBX1382 in A431 xenografts. Animals carrying established A431 tumors were treated daily p.o. with either 50 mg/kg (A) or 30 mg/kg (C) BIBX1382 for 2 weeks. At the end of the experiment, tumors were excised 6 h after the last application and subjected to biochemical analysis for activated EGFR (A) or immunohistochemical analysis for Ki-67 nuclear antigen (B, control; C, treated). The PY-EGFR/EGFR ratio was determined as described under Materials and Methods. The data were gathered from four vehicle-treated animals (control) and 4 BIBX1382-treated animals. The reduction in pEGFR level was highly significant (p = 0.0011; unpaired t test). Reduced pEGFR staining is paralleled by reduced Ki-67 stain on tumor cells (C, arrows). In addition, occasional Ki-67 reactivity with host stromal cells can be observed. Original magnification, 200x (B and C). The structural elements were revealed by hematoxylin counterstain.

The antiproliferative effect of BIBX1382 was confirmed in vivo by assessing tumor cell proliferation as determined by Ki-67 immunostaining of tumor tissue. A431 xenografts were treated daily p.o. with vehicle control or 30 mg/kg BIBX1382 for 2 weeks. The formalin-fixed and paraffin-embedded tumor tissues were then subjected to Ki-67 immunohistochemistry (see Materials and Methods). The counts of proliferative tumor cells in treated tissues were significantly decreased compared with vehicle-treated control tumors. The microphotographs of Ki-67 immunostaining illustrating a reduction in proliferation are shown in Fig. 6, B and C. A similar reduction in Ki-67 staining was observed with BIBU1361 (data not shown). These data demonstrate that BIBX1382 and BIBU1361 inhibit tumor cell proliferation in vivo.

	Discussion

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Excess EGFR signaling is implicated in the development of a significant proportion of human carcinomas and glioblastomas. Therefore, inhibition of EGFR-signaling has been viewed as a promising approach for the treatment of EGFR-positive human tumors. Both, anti-EGFR antibodies and small-molecule EGFR tyrosine kinase inhibitors are being actively tested in clinical trials (Grunwald and Hidalgo, 2003).

Here, we report a new structural class of small-molecule EGFR tyrosine kinase inhibitors, namely, pyrimido-[5,4-d]-pyrimidines. BIBX1382 and BIBU1361 are two examples of this class and inhibit the EGFR tyrosine kinase activity in vitro in the low nanomolar range. This potency is comparable with other small-molecule inhibitors derived from other structural classes (Table 1; Traxler, 2003). Relative to four distinct members of the tyrosine kinase family, both compounds seem to be selective for the class I receptors. Furthermore, at a concentration of 10 µM BIBX1382 and BIBU1361 failed to inhibit the kinase activities of a number of serine/threonine kinases comprising MEK, phosphorylase kinase, protein kinase A, and stress-activated protein kinase 2 (data not shown), thus confirming the selectivity of BIBX1382 and BIBU1361 for the EGFR kinase. Because all the kinase assays were performed at saturating ATP concentrations, cellular activity for these compounds could be anticipated.

Indeed, BIBX1382 and BIBU1361 in the nanomolar range could interfere with the EGF-induced EGFR autophosphorylation in intact A431 cells (Fig. 1). In accordance with this observation, BIBU1361 also blocked downstream EGFR signaling events. Many intracellular proteins get phosphorylated in response to mitogenic signals such as EGF. Some examples are phospholipase C, phosphoinositide 3-kinase, Src homology 2 domain-containing transforming protein, or MEK. In some cells, constitutive activation (phosphorylation) of these proteins has been linked to the transformed phenotype. Thus, a reduction in the phosphorylation status in one of these signal transducers induced by BIBU1361 could suggest an antiproliferative effect of this compound. Therefore, we analyzed the effect of BIBU1361, 10 min after EGF addition, on the activation of MEK and MAPK. As shown in Fig. 2, BIBU1361 at concentrations of 313 nM completely blocked activation of MEK. In mitogenic assays performed with KB cells (Fig. 3), BIBU1361 as well as BIBX1382 demonstrated antiproliferative activity. The EC₅₀ values for the two compounds were in the nanomolar range when EGF was used as a mitogenic agent. The maintained capacity of KB cells for proliferation in response to hepatocyte growth factor or FCS in the presence of micromolar concentrations of either BIBU1361 or BIBX1382 confirms the selectivity of these compounds. Together, the cellular data show that BIBX1382 and BIBU1361 are potent and selective inhibitors of the EGFR pathway.

Both BIBX1382 and BIBU1361 in mice and rats show serum concentrations of the compounds that are sufficient to achieve therapeutic efficacy. The ability of BIBX1382 and BIBU1361 to inhibit either A431 or HN5 tumor growth is similar to that reported for other EGFR inhibitors currently in clinical trials (Fan et al., 1993; Modjtahedi et al., 1998). Treatment for 14 days, at doses ranging between 10 and 30 mg/kg/day, resulted in complete tumor growth suppression in these models (Fig. 4; Table 2).

To further explore the potential of these compounds as anticancer agents, a long-term treatment study with BIBX1382 was initiated. Mice bearing A431 tumors were treated daily for a period of 170 days. Tumor regressions were observed in all treated individuals after 90 days (Fig. 5). Therefore, these data could suggest that sustained long-term inhibition of EGFR signaling in A431 tumors is required for a curative effect. Indeed, in 5 of 7 individuals, tumors had either disappeared or remained growth suppressed even during the 100-day treatment-free period. It has to be acknowledged that two of the tumors did regrow after termination of treatment. Interestingly, one of these tumors when passaged into mice responded again to BIBX1382 treatment (data not shown). These data also clearly demonstrate that, in mice, BIBX1382 can be given for an extended period of time with good tolerability.

In the 14-day A431 xenograft experiments, determination of the plasma concentrations of BIBX1382 and BIBU1361 on the last day of treatment, 24 h postapplication, revealed that the minimum plasma levels were 244 and 356 nM, respectively. It is noteworthy that in the cellular assays described herein, such concentrations resulted in nearly complete inhibition of EGFR-dependent processes. Therefore, this observation suggests that in vivo the EGF-dependent processes are completely and permanently inhibited by BIBX1382 and BIBU1361 over a 24-h period. The biochemical data shown in Fig. 6A is consistent with this observation. Indeed, a 2-week treatment of mice carrying established A431 tumors with BIBX1382-induced dephosphorylation of constitutively activated EGFR. Because of its ability to block early EGFR signaling in vivo, it was anticipated that BIBX1382 would also inhibit tumor cell proliferation in the tumor. Ki-67 immunostaining was used to visualize proliferating cells in vivo. In the treated samples (Fig. 6C), a reduction of stained cells was evident.

In conclusion, we have shown that pyrimidopyrimidines, exemplified here by BIBX1382 and BIBU1361, are potent inhibitors of EGFR-mediated signaling in vitro. In vivo, both compounds demonstrate antitumor activity in xenograft models such as A431, FaDu, and HN5. All these models have proven to be reliable predictors for class I receptor-targeted therapeutics such as erlotinib (Pollack et al., 1999), Iressa (Sirotnak et al., 2000), canertinib (Nelson and Fry, 2001), or lapatinib (Rusnak et al., 2001), which all have advanced to clinical trials. The results described above also show that BIBX1382 is well tolerated in long-term treatment regimens in animal models with sufficient exposure. Unfortunately, BIBX1382, which was tested in clinical phase I studies, did not show acceptable plasma exposure in humans. The reason for this behavior has been attributed to a first path metabolism (Dittrich et al., 2002), and the enzymes involved have been resolved and are the subject of another publication. Unlike BIBX1382, BIBU1361 is not a substrate for this enzyme and therefore plasma exposure in humans is expected. Thus, pyrimidopyrimidines, in principle, can be viewed as an attractive structural class for the development anticancer agents for patients whose cancers display aberrant EGFR signaling. Attention should be paid to first path metabolism effects as demonstrated for BIBX1382.

	Acknowledgements

 
We thank L. Süly, T. Troels, U. Strobl, M. Leber, R. Meyer, and I. Kothbauer for excellent technical assistance. We also thank A. Greischel and S. Blech for providing preclinical pharmacokinetic data. The unequivocal support of T. Metz (Oncotest, Freiburg, Germany) for this program is gratefully acknowledged.

	Footnotes

 
doi:10.1124/jpet.104.069138.

ABBREVIATIONS: EGF, epidermal growth factor; HER, human epidermal growth factor receptor; MAPK, mitogen-activated protein kinase; EGFR, epidermal growth factor receptor; BIBX1382, N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine; BIBU1361, (3-chloro-4-fluoro-phenyl)-[6-(4-diethylaminomethyl-piperidin-1-yl)-pyrimido[5,4-d]pyrimidin-4-yl]-amine; VEGFR2, vascular endothelial growth factor receptor 2; HGFR, hepatocyte growth factor receptor; IGF1R, insulin-like growth factor 1 receptor; InsRK, insulin receptor kinase; PBS, phosphate-buffered saline; FCS, fetal calf serum; pEGFR, phosphorylated epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; MEK, mitogen-activated protein kinase kinase.

Address correspondence to: Dr. Flavio Solca, Department of NCE Pharmacology, Boehringer-Ingelheim, Austria, Dr. Boehringer-Gasse 5-11, A-1120 Vienna, Austria. E-mail: flavio.solca{at}vie.boehringer-ingelheim.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Arteaga CL (2001) The epidermal growth factor receptor: from mutant oncogene in nonhuman cancers to therapeutic target in human neoplasia. J Clin Oncol 19 (Suppl 18): 32S–40S.

Carpenter G (2000) The EGF receptor: a nexus for trafficking and signaling. Bioessays 22: 697–707.[CrossRef][Medline]

Carraway KL 3rd and Cantley LC (1994) A neu acquaintance for erbB3 and erbB4: a role for receptor heterodimerization in growth signaling. Cell 78: 5–8.[CrossRef][Medline]

Dittrich C, Greim G, Borner M, Weigang-Köhler K, Huisman H, Amelsberg A, Ehret A, Wanders J, Hanauske A, and Fumoleau P (2002) Phase I and pharmacokinetic study of BIBX1382 BS, an epidermal growth factor receptor (EGFR) inhibitor, given in a continuous daily oral administration. Eur J Cancer 38: 1072–1080.

Fan Z, Baselga J, Masui H, and Mendelsohn J (1993) Antitumor effect of antiepidermal growth factor receptor monoclonal antibodies plus cis-diamminedichloroplatinum on well established A431 cell xenografts. Cancer Res 53: 4637–4642.[Abstract/Free Full Text]

Fernandes A, Hamburger AW, and Gerwin BI (1999) ErbB-2 kinase is required for constitutive stat 3 activation in malignant human lung epithelial cells. Int J Cancer 83: 564–570.[CrossRef][Medline]

Fry DW, Bridges AJ, Denny WA, Doherty A, Greis KD, Hicks JL, Hook KE, Keller PR, Leopold WR, Loo JA, et al. (1998) Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor. Proc Natl Acad Sci USA 95: 12022–12027.[Abstract/Free Full Text]

Gibson KH (1996) inventor, Zeneca Limited, UK, assignee. Quinazoline derivatives. Worldwide patent WO9633980. 1996 Oct 31.

Grunwald V and Hidalgo M (2003) Developing inhibitors of the epidermal growth factor receptor for cancer treatment. J Natl Cancer Inst 95: 851–867.[Abstract/Free Full Text]

Gullick WJ (1991) Prevalence of aberrant expression of the epidermal growth factor receptor in human cancers. Br Med Bull 47: 87–98.[Abstract/Free Full Text]

Heider KH, Dammrich J, Skroch-Angel P, Muller-Hermelink HK, Vollmers HP, Herrlich P, and Ponta H (1993a) Differential expression of CD44 splice variants in intestinal- and diffuse-type human gastric carcinomas and normal gastric mucosa. Cancer Res 53: 4197–4203.[Abstract/Free Full Text]

Heider KH, Hofmann M, Hors E, Van den BF, Ponta H, Herrlich P, and Pals ST (1993b) A human homologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps. J Cell Biol 120: 227–233.[Abstract/Free Full Text]

Himmelsbach F, Von Rueden T, Dahmann G, and Metz T (1997) inventors, Boehringer Ingelheim Pharma KG, assignee. Pyrimido[5,4-d]pyrimidines, medicaments containing these compounds, their use and process for production. Worldwide patent WO9732880. 1997 Sep 12.

Karunagaran D, Tzahar E, Beerli RR, Chen X, Graus-Porta D, Ratzkin BJ, Seger R, Hynes NE, and Yarden Y (1996) ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. EMBO J 15: 254–264.[Medline]

Klapper LN, Kirschbaum MH, Sela M, and Yarden Y (2000) Biochemical and clinical implications of the ErbB/HER signaling network of growth factor receptors. Adv Cancer Res 77: 25–79.[Medline]

Mendelsohn J and Baselga J (2003) Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 21: 2787–2799.[Abstract/Free Full Text]

Modjtahedi H, Affleck K, Stubberfield C, and Dean C (1998) EGFR blockade by tyrosine kinase inhibitor or monoclonal antibody inhibits growth, directs terminal differentiation and induces apoptosis in the human squamous cell carcinoma HN5. Int J Oncol 13: 335–342.[Medline]

Modjtahedi H and Dean C (1994) The receptor for EGF and its ligands: expression, prognostic value and target for therapy in cancer (review). Int J Oncol 4: 277–296.

Nelson JM and Fry DW (2001) Akt, MAPK (Erk1/2) and p38 act in concert to promote apoptosis in response to ErbB receptor family inhibition. J Biol Chem 276: 14842–14847.[Abstract/Free Full Text]

Pollack VA, Savage DM, Baker DA, Tsaparikos KE, Sloan DE, Moyer JD, Barbacci EG, Pustilnik LR, Smolarek TA, Davis JA, et al. (1999) Inhibition of epidermal growth factor receptor-associated tyrosine phosphorylation in human carcinomas with CP-358,774: dynamics of receptor inhibition in situ and antitumor effects in athymic mice. J Pharmacol Exp Ther 291: 739–748.[Abstract/Free Full Text]

Ross JS, Fletcher JA, Linette GP, Stec J, Clark E, Ayers M, Symmans WF, Pusztai L, and Bloom KJ (2003) The HER-2/neu gene and protein in breast cancer 2003: biomarker and target of therapy. Oncologist 8: 307–325.[Abstract/Free Full Text]

Rusnak DW, Lackey K, Affleck K, Wood ER, Alligood KJ, Rhodes N, Keith BR, Murray DM, Knight WB, Mullin RJ, et al. (2001) 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 1: 85–94.[Abstract/Free Full Text]

Sainsbury JR, Farndon JR, Needham GK, Malcolm AJ, and Harris AL (1987) Epidermal-growth-factor receptor status as predictor of early recurrence of and death from breast cancer. Lancet 1: 1398–1402.[Medline]

Shawver LK (1999) Tyrosine kinase inhibitors: from the emergence of targets to their clinical development, in American Society of Clinical Oncology 1999 Educational Book (Perry MC ed) pp 29–47, American Society of Clinical Oncology, Alexandria, VA.

Sirotnak FM, Zakowski MF, Miller VA, Scher HI, and Kris MG (2000) Efficacy of cytotoxic agents against human tumor xenografts is markedly enhanced by coad-ministration of ZD1839 (Iressa), an inhibitor of EGFR tyrosine kinase. Clin Cancer Res 6: 4885–4892.[Abstract/Free Full Text]

Slichenmyer WJ, Elliott WL, and Fry DW (2001) CI-1033, a pan-erbB tyrosine kinase inhibitor. Semin Oncol 28: 80–85.[CrossRef][Medline]

Smaill JB, Rewcastle GW, Loo JA, Greis KD, Chan OH, Reyner EL, Lipka E, Showalter HD, Vincent PW, Elliott WL, et al. (2000) Tyrosine kinase inhibitors. 17. Irreversible inhibitors of the epidermal growth factor receptor: 4-(phenylamino)quinazoline- and 4-(phenylamino)pyrido[3,2-d]pyrimidine-6-acrylamides bearing additional solubilizing functions. J Med Chem 43: 1380–1397.[CrossRef][Medline]

Sridhar SS, Seymour L, and Shepherd FA (2003) Inhibitors of epidermal-growth-factor receptors: a review of clinical research with a focus on non-small-cell lung cancer. Lancet Oncol 4: 397–406.[CrossRef][Medline]

Stratowa C, Baum A, Castanon MJ, Dahmann G, Himmelsbach F, Himmler A, Loeber G, Metz T, Schnitzer R, Solca F, et al. (1999) A comparative cell-based high throughput screening strategy for the discovery of selective tyrosine kinase inhibitors with anticancer activity. Anticancer Drug Des 14: 393–402.[Medline]

Traxler P (2003) Tyrosine kinases as targets in cancer therapy–successes and failures. Expert Opin Ther Targets 7: 215–234.[CrossRef][Medline]

Ullrich A and Schlessinger J (1990) Signal transduction by receptors with tyrosine kinase activity. Cell 61: 203–212.[CrossRef][Medline]

van der Geer P, Hunter T, and Lindberg RA (1994) Receptor protein-tyrosine kinases and their signal transduction pathways. Annu Rev Cell Biol 10: 251–337.[CrossRef][Medline]

Wissner A, Overbeek E, Reich MF, Floyd MB, Johnson BD, Mamuya N, Rosfjord EC, Discafani C, Davis R, Shi X, et al. (2003) Synthesis and structure-activity relationships of 6,7-disubstituted 4-anilinoquinoline-3-carbonitriles. The design of an orally active, irreversible inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and the human epidermal growth factor receptor-2 (HER-2). J Med Chem 46: 49–63.[CrossRef][Medline]

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