Affiliations of authors: Preclinical Chemotherapy and Pharmacology Unit (G. Cuccuru, CL, G. Cassinelli, G. Pratesi, MT, G. Petrangolini, DL, CZ, FZ), and Nuclear Medicine Unit (ES, AM), Istituto Nazionale Tumori, Milan, Italy.
Correspondence to: Cinzia Lanzi, PhD, Department of Experimental Oncology, Preclinical Chemotherapy and Pharmacology Unit, Istituto Nazionale Tumori, via Venezian 1, 20133 Milan, Italy (e-mail: cinzia.lanzi{at}istitutotumori.mi.it)
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ABSTRACT |
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INTRODUCTION |
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Ret receptor tyrosine kinase is frequently aberrantly activated in thyroid tumors. Several lines of evidence suggest that it might represent a useful target for specific therapeutic approaches. Oncogenic activation of the RET gene is recognized as an early pathogenic event in papillary and medullary thyroid carcinomas. In papillary carcinoma, the most frequent thyroid malignancy, somatic rearrangements of the RET gene that generate oncogenic fusion proteins are found in approximately 30% of the cases (5,6). Somatically arising RET point mutations are present in about 50% of sporadic medullary thyroid carcinoma cases. The finding that germline missense mutations are present in virtually all families with MEN2 syndromes led to the implementation of early genetic screening as the standard for diagnosing MEN2 syndromes (7). The clinical relevance of RET in thyroid cancer is further strengthened by evidence that genetically engineered mice expressing thyroid-targeted RET oncogenes develop thyroid tumors that mimic human tumor phenotypes (8,9).
The RET proto-oncogene is characterized by the presence of cadherin-related motifs and a cysteine-rich domain in the extracellular domain. Two major Ret isoforms of 1072 (short) or 1114 (long) amino acids are generated by alternative splicing (10) and posttranslational glycosylation (11). Partially and fully glycosylated forms of Ret, the precursors and mature forms of the receptor, are expressed in the endoplasmic reticulum and at the cell surface, respectively (12). Activation of the wild-type Ret receptor requires interaction with a ligand belonging to the glial cell linederived neurotrophic factor (GDNF) family, which induces the subsequent autophosphorylation of Ret. As in the case of other receptor tyrosine kinases, specific phosphorylated tyrosine residues represent the docking sites for molecules containing SH2 or phosphotyrosine-binding (PTB) domains. The recruitment of multiprotein complexes, in turn, elicits downstream signaling events involving Ras/ERK (extracellular signal-regulated kinase), PI3K/AKT, and phospholipase C (PLC
)dependent pathways (6,10).
Dominant gain-of-function mutations of the RET gene found in human tumors generate ligand-independent, constitutively active tyrosine kinase oncoproteins that are able to transform NIH3T3 fibroblasts (5,1315). The mechanism of oncogenic activation varies among different RET mutants. In MEN2A patients, Ret mutations occur in the extracellular, cysteine-rich domain at cysteine residues 609, 611, 618, 620, 630, or 634 (C634 mutants are found in approximately 87% of MEN2A patients) and result in the substitution of another amino acid in place of the cysteine. In the wild-type Ret protein, these cysteines are connected by intramolecular disulfide bonds. Their loss by mutation may result in the formation of intermolecular bonds through partner cysteine residues of other Ret mutants, thereby inducing Ret activation by covalent dimerization (7,16).
The constitutive tyrosine kinase activity of Ret oncoproteins suggests the possibility of using small-molecule inhibitors to block their transforming signaling (17). In previous studies, we showed that the small molecule inhibitor arylidene-2-indolinone RPI-1 can inhibit the product of RET/papillary thyroid carcinoma-1 (PTC1), the most frequent RET oncogene found in sporadic papillary thyroid carcinomas (6). RPI-1 abolished the constitutive tyrosine phosphorylation of the Ret/ptc1 oncoprotein and its signaling in NIH3T3 mouse fibroblasts transfected with RET/PTC1 and in human papillary thyroid carcinoma cells spontaneously expressing the RET/PTC1 rearrangement (18,19). These findings prompted us to investigate the effects of RPI-1 in cells carrying medullary thyroid carcinoma-associated RET mutations. In this study, we examined the cellular and biochemical effects of RPI-1 treatment on cells containing constitutively active Ret via mutation of C634. Specifically, our cellular models included NIH3T3 fibroblasts transfected with a RET mutant carrying the C634R substitution (RETC634R), which is found in more than 50% of MEN2A patients, and the human medullary thyroid carcinoma cell line TT, which spontaneously expresses another MEN2A-associated RET mutant, RETC634W. In addition, we investigated the ability of RPI-1 to inhibit the in vivo growth of TT tumor xenografts.
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MATERIALS AND METHODS |
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The synthesis and chemical structure of RPI-1 (1,3-dihydro-5,6-dimethoxy-3-[(4-hydrophenyl)methylene]1-H-indol-2-one) have been reported previously (18). For in vitro experiments, an RPI-1 stock solution was prepared in 100% dimethylsulfoxide (DMSO). RPI-1 and DMSO (control) were diluted in culture medium for use (final concentration of solvent = 0.5% DMSO, vol/vol). For in vivo studies, solid RPI-1 was dissolved in absolute ethanol and Cremophor EL (each 5% of the final volume). The solution was stirred at 4 °C until clear and maintained at 4 °C for 1224 hours. Just before use, it was diluted by slowly adding cold 0.9% NaCl solution (90% of the final volume) while stirring. During and after dilution, the RPI-1 solution remained on ice. The final RPI-1 concentration was 3.3 mg/mL (maximum concentration allowed before precipitation).
NIH3T3MEN2A and NIH3T3H-RAS cells are NIH3T3 mouse fibroblasts transfected with the C634R mutant of RET (RETC634R short isoform) or H-RAS oncogenes, respectively (20). Both transfected cell lines were routinely maintained at 37 °C in a 10% CO2 atmosphere in Dulbeccos modified Eagle Medium (DMEM; BioWhittaker, Verviers, Belgium) supplemented with 5% calf serum (Colorado Serum Company, Denver, CO). Untransfected NIH3T3 cells were cultured in DMEM containing 10% calf serum. The human TT cell line was derived from a metastatic medullary thyroid carcinoma harboring a MEN2A-type RET mutation, specifically, a cysteine-to-tryptophan substitution at codon 634 (C634W) (21,22). TT cells were subcultured in Hams F12 medium (BioWhittaker) supplemented with 15% fetal bovine serum (Life Technologies, Gaithersburg, MD) and were maintained at 37 °C in a 5% CO2 atmosphere.
Cell Growth Assays
Cell sensitivity to RPI-1 was assessed by cell growth and anchorage-independent growth inhibition assays. To measure cell proliferation, exponentially growing cells were seeded in duplicate on six-well plates at 250010 000 cells per square centimeter and treated with RPI-1 or DMSO alone, after at least one round of cell division (96 hours for TT cells and 24 hours for the NIH3T3 transfectants). After 3 or 7 days of drug exposure, cells were harvested by trypsin treatment and counted directly with a Coulter counter (Coulter Electronics, Luton, U.K.). Alternatively, TT cells were seeded in octuplicate on 96-well plates at 10 000 cells per square centimeter, treated with RPI-1 or DMSO, and then subjected to the sulforhodamine B colorimetric assay (23) at different time points over a period of 21 days. The anchorage-independent growth assay was performed as previously described (18). Briefly, a cell suspension (15 000 cells per milliliter) in medium containing 0.33% agarose (Sigma Chemical Company, St. Louis, MO) and the appropriate concentration of RPI-1 or vehicle (DMSO) was layered onto semisolid agarose (0.5% agarose in medium) in duplicate on 9.6-cm2 dishes. After 810 days of incubation at 37 °C, cell colonies were stained with p-iodonitrotetrazolium violet (Sigma), visualized with a magnifying projector, and counted. In both assays, the RPI-1 IC50 values (drug concentrations producing 50% inhibition) were calculated from the doseresponse curves obtained with Microcal Origin, version 4.10 (Microcal Software, Northampton, MA). Each experiment was performed 34 times.
Immunoprecipitation and Western Blot Analysis
For immunoblots performed with whole-cell extracts, samples were prepared after treatment by lysing cells in sodium dodecyl sulfate (SDS) sample buffer (62.5 mM TrisHCl [pH 6.8], 2% SDS) containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL pepstatin, 12.5 µg/mL leupeptin, 2 µg/mL aprotinin, 1 mM sodium orthovanadate, and 1 mM sodium molybdate. Protein concentration was determined using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL). Samples were then prepared for electrophoresis by adding 10% glycerol, 5% -mercaptoethanol, and 0.001% bromophenol blue.
For immunoprecipitation experiments, cells were treated with RPI-1 or DMSO for 24 hours. Cell monolayers were rinsed twice with cold phosphate-buffered saline (137 mM NaCl, 1.76 mM KH2PO4, 2.7 mM KCl, 8.1 mM Na2HPO4 [pH 7.4]) supplemented with 0.1 mM sodium orthovanadate and incubated on ice for 20 minutes in lysis buffer (50 mM HEPES [pH 7.6], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, and four protease inhibitors10 µg/mL antipain, 20 µg/mL chymostatin, 10 µg/mL E64, and 1 mg/mL Pefabloc SC). Cells were collected by scraping, pushed through a 22-gauge needle, and centrifuged at 10 000g, for 20 minutes at 4 °C. Supernatants (cell extracts) were collected, and protein concentration was determined by the BCA reaction. Cell extracts (0.55 mg) were immediately incubated with pre-swelled protein Aagarose resin (Sigma) and the indicated antibody for 2 hours at 4 °C with rotation. For each milligram of cell extract, 18 µg of anti-Ret (24) or 4 µg of anti-PLC (Santa Cruz Biotechnology, Santa Cruz, CA) were used. After washing the resin three times with 20 mM HEPES (pH 7.6), 150 mM NaCl, 10% glycerol, and 0.1% Triton X-100, the immunoprecipitates were eluted with complete sample buffer (SDS sample buffer containing 10% glycerol, 5%
-mercaptoethanol, and 0.001% bromophenol blue).
Immunoprecipitates or whole-cell lysates (3060 µg) were resolved by SDSpolyacrylamide gel electrophoresis (7.5% or 10% acrylamide wt/vol) and electrophoretically transferred to nitrocellulose filters. Nonspecific binding was blocked by incubating membranes in 1% bovine serum albumin, 3% ovalbumin (wt/vol) in TBS-T (25 mM TrisHCl [pH 7.2], 150 mM NaCl, and 0.1% Tween 20) for 1 hour at room temperature. Membranes were then incubated with primary antibodies in TBS-T overnight at 4 °C. Immunoreactive bands were visualized with enhanced chemiluminescence detection systems from Pierce or Amersham Biosciences (Little Chalfont, U.K.) after incubating membranes with horseradish peroxidaseconjugated anti-mouse or anti-rabbit secondary antibodies (Amersham Biosciences). When needed, membranes were stripped with 2% SDS, 62.5 mM TrisHCl [pH 6.8], and 0.7% -mercaptoethanol for 30 minutes at 45 °C and re-probed as described above.
The following primary rabbit polyclonal antibodies were used: anti-Ret, which recognizes a carboxy-terminal sequence (amino acids 10001014) common to the short and long Ret isoforms (24); anti-pY1062-Ret, which recognizes the Y1062-phosphorylated Ret (25); anti-pAkt, which recognizes the phosphorylated S473 of Akt (Cell Signaling Technology, Beverly, MA); anti-p44/42 ERKs (Upstate Biotechnology, Lake Placid, NY); anti-PLC (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-actin (Sigma). The monoclonal antibodies were anti-pTyr antibody clone 4G10 (Upstate Biotechnology), which recognizes phosphorylated tyrosine residues; anti-PKB
/Akt (Transduction Laboratories, Lexington, KY); and anti-phospho-p44/42 ERKs (pERKs) (Thr202/Tyr204), which recognize ERKs that are doubly phosphorylated at these residues (Cell Signaling Technology).
In Vivo Studies
All in vivo experiments were performed using 8- to 11-week-old female athymic nude CD-1 mice (Charles River, Calco, Italy). Mice were maintained in laminar flow rooms with constant temperature and humidity. Experimental protocols were approved by the Ethics Committee for Animal Experimentation of the Istituto Nazionale Tumori (Milan, Italy), according to the United Kingdom Coordinating Committee on Cancer Research Guidelines (26).
Human medullary thyroid carcinoma TT cells (1.6 x 107 cells in suspension) were inoculated subcutaneously into the right flank of mice. Each control or RPI-1-treated group included 810 tumors (one tumor per mouse). TT cells were injected on day 0, and tumor growth was followed by biweekly tumor diameter measurements using a vernier caliper. Tumor weight was calculated, considering tumor density equal to 1, according to the following formula: tumor weight (mg) = tumor volume (mm3) = d2 x D/2, where d and D are the shortest and the longest diameter, respectively. RPI-1 treatment started when tumors were just measurable (mean tumor weight was 50 mg), at approximately 25 days after tumor cell inoculation. RPI-1 was delivered orally twice a day for 10 or 40 days. The drug was administered in a volume of 1530 mL/kg of body weight. Control mice were treated with vehicle (ethanol, Cremophor EL, 0.9% NaCl solution at 5 : 5 : 90) in parallel with mice treated with RPI-1.
Drug efficacy was assessed either as mean percentage of tumor weight inhibition in drug-treated versus control mice (expressed as tumor weight inhibition percent = 100 [mean tumor weight of treated mice/mean tumor weight of control mice x 100]), evaluated during and after drug treatment, or as "cure," that is, no evidence of tumor at the end of the experiments. Experimental groups were sacrificed by cervical dislocation when mean tumor weight was at a maximum of 2.0 ± 0.5 g.
RPI-1 tolerability was assessed in tumor-bearing mice as either lethal toxicity (i.e., any death in treated mice occurring before any control death) or percent body weight loss (body weight loss percent = 100 [body weight on day x/body weight on day 1 x 100], where x represents 1 day after or during the treatment period). The maximum body weight loss values are reported.
Calcitonin Assay
Control and RPI-1-treated mice were bled under general anesthesia by venous orbital puncture. For anesthesia, a solution of ketamine (Ketavet 50; Farmaceutici Gellini, Aprilia, Italy), xylazine (Rompun; Bayer, Leverkusen, Germany) and saline (at a ratio of 20 : 2.5 : 77.5) was prepared and delivered intraperitoneally at a dose of 10 mL/kg of body weight. Blood samples were centrifuged (4000g for 10 minutes), and the plasma (supernatant) was frozen until analysis. All procedures were performed on ice to prevent the enzymatic degradation of calcitonin. Calcitonin levels in plasma were measured with an immunoradiometric assay (IRMA) kit (Calcitonin Assay; Scantibodies Laboratory, Santee, CA), with two different anti-human calcitonin goat polyclonal antibodies, according to the manufacturers instructions.
Statistical Analysis
Students t test (two-tailed) was used to compare tumor weight and calcitonin levels in control and RPI-1-treated mice. The Spearman rank correlation analysis (27) was used to determine the relationship between tumor weight and calcitonin levels.
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RESULTS |
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We investigated the effects of RPI-1 in NIH3T3 fibroblasts stably transfected to express the RET mutant C634R (RETC634R). The effect of RPI-1 on the proliferation of these cells was examined by counting cells after 72 hours of RPI-1 treatment. As shown in Table 1, NIH3T3 cells transfected with the RETC634R mutant (NIH3T3MEN2A) were nearly four times more sensitive to the growth inhibitory effects of RPI-1 than NIH3T3 cells transfected with H-RAS (NIH3T3H-RAS) or than parental NIH3T3 cells. The selectivity of RPI-1 for RETC634R-expressing cells was higher in the soft agar assay. In this assay, the growth of NIH3T3MEN2A colonies was 10 times more sensitive to RPI-1 than that of NIH3T3H-RAS colonies.
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Effects of RPI-1 on Human TT Medullary Thyroid Carcinoma Cells
The effects of RPI-1 treatment were further investigated in a human medullary thyroid carcinoma cell line, known as TT, that naturally expresses the RETC634W mutant (21,22). Because of their slow doubling time (4 days), TT cells were monitored for up to 21 days in the presence of RPI-1. Dose-dependent inhibition of TT cell proliferation was evident, with a growth arrest at drug concentrations higher than 10 µM (Fig. 2). After 7 days of treatment, the RPI-1 IC50 was 7.2 µM (95% confidence interval = 6.6 to 7.8 µM). Experiments in which we replaced medium and treatment drugs once a week gave similar results to those above (data not shown).
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Next, we examined the effect of RPI-1 on the phosphorylation status of the multifunctional docking site pY1062 of Ret, which is considered crucial for the transforming activity of MEN2A-type C634 mutant Ret receptors and other Ret oncoproteins (29). Whole-cell lysates prepared from TT cells treated with increasing concentrations of RPI-1 (7, 20, and 60 µM) for 24 hours were analyzed by western blotting with a phospho-specific anti-pY1062-Ret antibody. Like overall Ret tyrosine phosphorylation (Fig. 3, A), pY1062 disappeared with increasing RPI-1 dose (Fig. 3, C). The pY1062 docking site has been implicated in the activation of Ras/ERK and PI3K/AKT pathways by either ligand-activated or oncogenic Ret proteins (30,31). Thus, we examined the phosphorylation status of ERKs and AKT in control and RPI-1-treated TT cells by western blot analysis with antibodies that specifically recognize the phosphorylated (i.e., activated) forms of these proteins. Fig. 3, C, shows that phosphorylation, and hence activation, of ERKs and AKT was inhibited by RPI-1 in a dose-dependent manner, similar to that observed for Ret inhibition (Fig. 3, A).
Effects on Human TT Medullary Thyroid Carcinoma Xenografts
The antitumor efficacy of RPI-1 was investigated in mice carrying subcutaneous xenograft tumors of human medullary thyroid carcinoma TT cells (Table 2). Two daily RPI-1 dose levels (each fractionated in two oral administrations) were investigated. This schedule was chosen to allow continuous exposure of tumor cells to the drug. A dose-dependent effect on tumor growth was observed, with a dose of 2 x 50 mg/kg/day resulting in less tumor weight inhibition than the 2 x 100 mg/kg/day dose after 10 days of treatment (P = .003 and P<.001, versus controls, respectively, by two-sided Students t test). Moreover, in the group receiving the highest dose, two of eight mice were tumor-free at the end of the experiment (day 53) (Table 2, experiment A). An improved antitumor effect, without evidence of toxicity, was achieved by a more prolonged RPI-1 administration of 2 x 100 mg/kg/day, which was well-tolerated for up to 40 days (maximum time investigated) in terms of body weight loss and lethal toxicity (Table 2, experiment B). The inhibitory effect of the drug was observed immediately, and tumor growth was maintained under control for the treatment period, reaching a tumor weight inhibition of 81% relative to that in controls (P<.001) at the end of treatment (day 66). Approximately 2 months after the interruption of treatment (day 110), treated tumors were still statistically significantly smaller than control tumors (tumor weight inhibition = 52%; P = .01), and two of eight mice were still tumor-free at the end of the experiment (day 140). In these two mice, tumors had disappeared during treatment, at approximately day 50. The growth curves of the two experiments summarized in Table 2 are shown in Fig. 4 (mean values from 810 mice per point). Because of drug solubility and formulation limitations, higher doses were not tested.
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DISCUSSION |
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The clinical relevance of RET in papillary and medullary thyroid cancers has been recognized for many years (6) and has recently been supported by the observation that transgenic mice expressing RET oncogenes develop thyroid tumors (8,9). Thus, the involvement of the RET proto-oncogene in the molecular pathology of thyroid cancers provided a rational basis to investigate the role of Ret tyrosine kinases as therapeutic targets. Our previous studies demonstrated that RPI-1 inhibits the tyrosine kinase activity of the Ret/ptc1 oncoprotein and selectively arrests the growth of either NIH3T3 transfectants carrying the Ret/ptc1 oncoprotein or a papillary thyroid carcinoma cell line spontaneously expressing the RET/PTC1 rearrangement (18,19). Similar results have been obtained by others with structurally different tyrosine kinase inhibitors such as the pyrazolo pyrimidines PP1 and PP2 (33,34) and the quinazoline ZD6474 (35). These studies suggested the possibility of using Ret inhibitors to treat tumors carrying Ret abnormalities including medullary thyroid carcinoma, a more aggressive disease than papillary thyroid carcinoma with a poor responsiveness to conventional chemotherapy. In the present study, the in vitro experiments demonstrated that RPI-1 concentrations that inhibit Ret tyrosine autophosphorylation and signaling have antiproliferative activity on cells expressing ectopic and endogenous MEN2A-type RET mutants (NIH3T3MEN2A transfectants and TT cells), supporting the hypothesis that RPI-1 may have therapeutic potential in the treatment of patients with medullary thyroid carcinomas carrying MEN2A-type mutations.
An interesting feature shared by RPI-1 and the two pyrazolo pyrimidines PP1 and PP2 is the ability to specifically lower the expression of mutant RETC634 receptors [(20) and Figs. 1, A, and 3, A]. The molecular mechanism by which RPI-1 lowers Ret expression is not yet known. The inhibitory effect of PP1 on receptor expression has been ascribed to stimulation of the proteasome-mediated degradative pathway (20). Other tyrosine kinase inhibitors have recently been described as activating a chaperone-mediated pathway that targets ErbB2 for intracellular degradation (36). Together, these observations suggest that the therapeutic efficacy of tyrosine kinase inhibitors might be attributed also to their ability to reduce expression of oncoproteins. A better knowledge of the mechanisms that regulate oncoprotein activity and expression will provide the basis for devising more specific strategies to inhibit them.
The molecular basis of the inhibitory effects of RPI-1 was supported by the results of experiments designed to examine Ret-dependent signaling in the presence and absence of RPI-1. RPI-1 blocked Ret-dependent signaling in our experimental model. Specifically, Ret did not bind to PLC, and overall, PLC
was inactive in RPI-1-treated TT cells. Similar results were obtained in papillary thyroid carcinoma cells expressing Ret/ptc1 (19). The interaction of Ret/ptc2 or of Ret/ptc1 with PLC
through pY1015 has been shown to be required for full oncogenic activation (24) and for thyroid tumor formation in transgenic mice (37). The precise definition of the role of PLC
in the signaling of receptor Ret oncoproteins is needed to clarify the biologic consequences of its inhibition. Our findings also showed that Ret inactivation by RPI-1 was associated with inhibition of AKT- and ERK-dependent pathways in TT cells, which supports the previous finding that the transforming ability of the MEN2A Ret mutant requires activation of the PI3K/AKT signaling pathway (30). Indeed, our data support the finding that the pY1062 Ret docking site is essential for the activation of both pathways (38), because pY1062 phosphorylation disappeared following the same dose-dependent, drug-induced AKT and ERK dephosphorylation in our experimental model. Although we cannot exclude the possibility that the inhibition of additional receptor tyrosine kinases by RPI-1 might contribute to the inhibition of downstream pathways, as reported for other inhibitors of the indolinone series (39), our results suggest that the inhibition of Ret has a strong effect on cellular signal transduction pathways.
The most relevant result of our study was the strong and reproducible antitumor activity of RPI-1 against the human medullary thyroid carcinoma TT xenograft in nude mice. Tumor growth inhibition was dose- and time-dependent, and optimal effects were observed after prolonged daily treatment. Importantly, the compound was delivered orally, the most suitable delivery route for prolonged treatment. The low solubility of RPI-1 did not allow us to establish a maximum tolerated dose in mice. However, a strong antitumor effect was achieved by a well-tolerated dose (100 mg/kg of body weight). At this RPI-1 dose, 25% of treated mice were tumor-free at the end of the experiments. The antitumor effect was obtained in spite of the relatively low oral bioavailability of the drug in the current formulation, as found in preliminary pharmacokinetic studies. Therefore, detailed in vivo studies will be necessary to define the optimal treatment conditions to fully exploit the therapeutic potential of the drug.
Several clinical studies have addressed the problem of identifying the maximum tolerated dose in the development of "target-oriented drugs," including tyrosine kinase inhibitors (40,41). Data from phase II clinical trials with Gleevec revealed that it was possible to identify a "biologically active dose" lower than the maximum tolerated dose assessed in a typical phase I study (42). For the clinical development of tyrosine kinase inhibitors, determination of pharmacodynamic parameters should allow optimization of the therapy. Our study indicated that plasma calcitonin levels effectively reflect the TT tumor response to RPI-1 treatment. Thus, the plasma calcitonin level, which is recognized as a specific and sensitive medullary thyroid carcinoma marker, might be a useful tool for measuring RPI-1 efficacy in clinical trials.
In conclusion, our studies indicate that Ret oncoproteins represent exploitable therapeutic targets in MEN2A-associated medullary thyroid carcinoma and have the potential to provide a new option for the treatment of RET oncogene-related thyroid carcinomas. Because of its ability to inhibit tumor growth at nontoxic doses and its oral bioavailability, the 2-indolinone RPI-1 is a Ret inhibitor of particular pharmacologic interest.
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NOTES |
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We thank Marco A. Pierotti for encouragement and involvement in this research project, the Medicinal Chemistry Department of Novuspharma S.p.A., now Cell Therapeutics, Inc., Europe (Bresso, Italy) for the synthesis of RPI-1, Italia Bongarzone and Maria G. Borrello for discussion and providing the cell lines and the anti-Ret antibody used in this study, Masahiko Yamamoto for providing the anti-p1062-Ret antibody, and Laura Zanesi for help in editing the text.
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Manuscript received September 29, 2003; revised April 26, 2004; accepted May 14, 2004.
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