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WO2008011799A1 - Nouvelle utilisation d'inhibiteurs spécifiques de la tyrosine kinase c-ab1 non réceptrice - Google Patents

Nouvelle utilisation d'inhibiteurs spécifiques de la tyrosine kinase c-ab1 non réceptrice Download PDF

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WO2008011799A1
WO2008011799A1 PCT/CN2007/002113 CN2007002113W WO2008011799A1 WO 2008011799 A1 WO2008011799 A1 WO 2008011799A1 CN 2007002113 W CN2007002113 W CN 2007002113W WO 2008011799 A1 WO2008011799 A1 WO 2008011799A1
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cancer
abl
tumor
cells
gal3
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Xiaoming Li
Cheng Cao
Qingjun Ma
Xuan Liu
Yanwen Jin
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Institute of Bioengineering Chinese Academy of Military Medical Sciences
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Institute of Bioengineering Chinese Academy of Military Medical Sciences
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/541—Non-condensed thiazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to novel uses of non-receptor tyrosine kinase c-Abl specific inhibitors, particularly to the use of non-receptor tyrosine kinase c-Abl specific inhibitors in the preparation of antitumor drugs.
  • Galectin-3 is a galactosidase-binding protein that has a variety of biological functions and is involved in tumor cell adhesion, proliferation, differentiation, angiogenesis, deterioration and metastasis as a cancer-associated protein.
  • Clinical studies have shown that in many types of tissue cells, Gal3 expression is up-regulated when cells undergo malignant transformation (eg cancer in the colon, thyroid, central nervous system, pancreas, bladder, kidney, stomach, etc.), and Up-regulation of Gal3 expression levels in tumor cells increases the malignancy and metastatic invasiveness of tumors.
  • Gal3 is considered to be a cancer-associated protein and plays an important role in carcinogenesis and tumor progression.
  • the anti-apoptotic activity of Gal3 is manifested by the ability to inhibit drug-induced apoptosis and anchorage-dependent apoptosis to allow cell survival.
  • Gal3 as an indicator of clinical detection of immune cytochemistry, has certain value for the diagnosis of benign and malignant tumor cells and the diagnosis of cancer prognosis. Therefore, Gal3 has a very good application prospect as a new target in the diagnosis and treatment of various types of cancer.
  • Protein tyrosine kinases are a class of proteins with tyrosine kinase activity and can be divided into receptor and non-receptor types, which catalyze the transfer of phosphates on ATP to tyrosine residues of many important proteins. On, make it phosphorylated. Protein tyrosine kinases play an important role in the signal transduction pathways in cells, regulating a series of physiological processes such as cell growth, differentiation and apoptosis.
  • the ABL family of non-receptor protein tyrosine kinases includes two members: Abl and Arg.
  • the Abl gene is located on the long arm of chromosome 9 (9q34.1) and encodes the 145kD Abl protein.
  • c-Abl protein Has tyrosine kinase activity, including SH3, SH2, PTK, DNA binding domain, actin binding domain, etc. (Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature, 2001, 411 (6835): 355-65 ).
  • the non-receptor tyrosine kinase c-Abl specific inhibitor can inactivate the ubiquitous Abelson tyrosine kinase (Abl).
  • the application of the present invention is to prepare an antitumor drug or a tumor therapeutic auxiliary drug by using a non-receptor tyrosine kinase c-Abl specific inhibitor as an active ingredient.
  • the non-receptor tyrosine kinase c-Abl specific inhibitors are selected in a variety of ways, specifically STI571, Sunitinib, PKC412, vandetanib Sorafenib, erlotinib, gefitinib, dasatinib and the like.
  • chemotherapeutic drugs such as cisplatin and CTX may be added to the anti-tumor drugs or tumor therapeutic auxiliary drugs prepared by using the non-receptor tyrosine kinase c-Abl specific inhibitor.
  • non-receptor tyrosine kinase c-Abl-specific inhibitors can cause Gal3 to undergo degeneration and aggregation and lysosome degradation by inhibiting the kinase activity of C-Abl, resulting in a significant decrease in Gal3 protein levels in tumor cells, resulting in tumors.
  • the cells die of autophagic cell apoptosis.
  • non-receptor tyrosine kinase c-Abl specific inhibitors have a broad spectrum of anti-tumor effects, including breast cancer, prostate cancer, cervical cancer, lung adenocarcinoma, liver cancer, Colon cancer, thyroid cancer, pancreatic cancer, neuroendocrine tumor, bladder cancer, kidney cancer, stomach cancer, and the like.
  • one or more pharmaceutically acceptable excipients may be added, and the excipients include conventional diluents, excipients, and the like in the pharmaceutical field.
  • a filler, a binder, a wetting agent, an absorption enhancer, a surfactant, a lubricant, a stabilizer, etc., and if necessary, a flavoring agent, a sweetener, a coloring matter, and the like may be added.
  • the antitumor drug or the tumor therapeutic auxiliary drug prepared by the invention can be prepared into a plurality of drug forms such as capsules, tablets, powders, granules, and injections, in addition to the oral solution.
  • the above various dosage forms of the drug can be prepared according to a conventional method in the pharmaceutical field.
  • the oral dose of the drug is generally 400rag/day or 5.55rag/kg. body weight/day, which can be used once or in multiple times for 20 days to 30 days.
  • DRAWINGS Figure 1 shows the results of immunoprecipitation and immunoblotting of Gal3-specific phosphorylation by non-receptor tyrosine kinase c-Abl.
  • Figure 2 shows the results of immunoprecipitation and immunoblotting of C -Abl-specific phosphorylation by non-receptor tyrosine kinase c-Abl specific inhibitor STI571.
  • Figure 3 shows the results of immunostaining for the distribution of Gal3 protein in MCF-7 cells treated with lOuM STI571.
  • Figure 4 shows the results of immunostaining assay for changes in Gal3 protein content in MCF 7 cells treated with 5 uM STI571, 0.5 uM apoptosis-inducing agent STS alone and co-treated.
  • Figure 5 shows the results of immunoblotting for the change of Gal3 protein in MCF-7 cells treated with 5uM STI571, 0.5 uM apoptosis inducer STS alone and co-treated.
  • Figure 6 shows the results of TUNEL assay for apoptosis of MCF-7 cells treated with lOuM STI57K 0. 5uM STS alone and in combination.
  • Figure 7 shows the results of Annexin-V-FLU0S assay for apoptosis of MCF-7 cells treated with lOuM STI571, 0.5 uM STS alone and both.
  • Figure 8 shows the Sub G1 assay for apoptosis of MCF-7 cells treated with lOuM STI571, 4 uM CDDP alone and co-treated.
  • Figure 9 shows the treatment of tumors in nude mice. '
  • Figure 10 shows the body weight statistics of nude mice.
  • Figure 11 shows the results of HE staining of tumor sections.
  • the flag tag will be encoded (a peptide consisting of the following 8 amino acids:
  • the cDNA obtained by reverse transcription of total RNA extracted from normal human breast tissue cells carrying the Gal3 gene was used as a template in primer P1 (upstream primer): 5 '-CGGATCCATGGCAGACAATTTTTCGCTCCA-3 ' (underlined Bases are restriction endonucleases ⁇ 3 ⁇ 4/ ⁇ 1 recognition site) and P2 (downstream primers):
  • the 5'-CGGAATTCTTATATCATGGTATATGAAGCA-3 (underlined base is a restriction endonuclease recognition site), the Gal3 gene was amplified by PCR and the restriction enzymes BainH I and EcoR I were added to both ends of the gene fragment. After the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis, and a 753 bp DNA fragment was obtained, which was consistent with the expected result. The target fragment was recovered and purified, sequenced, and the sequencing result was obtained.
  • a Gal3 gene fragment (CDS sequence of the Gal3 gene) having a 25'-777 deoxyribonucleotide at the 5' end of GenBank Accession Number: BC001120.
  • the Gal3 gene fragment cloned in step 2 was digested with restriction endonucleases BamH I and EcoR I, and the restriction fragment was ligated with the vector pcDNA 3-flag constructed in step 1 which was digested with the same enzyme, and ligated.
  • the product was transformed into E. coli DH5 ci competent cells by calcium chloride method, positive transformants were screened, plasmids were extracted, and restriction endonucleases/ and I were used for restriction enzyme digestion, and 5400 bp and 753 bp fragments were positively digested.
  • the positive clones were further identified by PCR.
  • the primers were P1 and P2.
  • the PCR products were sequenced.
  • the Gal3 gene can express a fusion protein with a Flag tag under the action of a CMV promoter.
  • a human fetal liver cDNA library (clontech product) carrying the wild type c-Abl gene (GenBank Accession Number: ⁇ -007313) was used as a template in the primer Myc-Abl-1 (upstream primer): 5 '-CGAATTCGGATGG6GCAGCAGCCTGGAA-3 ' (underlined bases are restriction endonuclease I recognition sites) and Myc- (downstream primers):
  • the c-Abl gene was amplified by PCR under the guidance of the ⁇ recognition site, and the restriction endonucleases EcoR I and Bgl II were added to the ends of the gene fragment. After the reaction, the PCR amplification products were subjected to the PCR amplification products. After 1% agarose gel electrophoresis, a 3450 bp DNA fragment was obtained, which was consistent with the expected results. The target fragment was recovered and purified, and sequenced. The sequencing result showed that the 5' end was obtained from GenBank Accession Number: ⁇ -007313.
  • the c-Abl gene fragment of the 440th to 3889 deoxyribonucleotides (the CDS sequence of the c-Abl gene).
  • the c-Abl gene fragment cloned in step 1) was digested with restriction endonucleases EcoR I and ⁇ 3 ⁇ 4 ⁇ _ ⁇ , and the restriction fragment was digested with the vector pCMV-Myc (Clontech) which was digested with the same enzyme. Ligation, the ligation product was transformed into E. coli DH5 C1 competent cells by calcium chloride method, positive transformants were screened, plasmids were extracted, and restriction endonucleases were used to identify the digestive enzymes ⁇ 1 and / / II, and obtained by enzyme digestion. 3800 bp (vector fragment) and 3450 bp fragment were positive clones, and the positive clone plasmid was further identified by PCR.
  • the primers used were Myc-Abl-1 and Myc-Abl-2, and the PCR products were sequenced.
  • the c-Abl gene fragment was inserted between the EcoR I and Bgl II restriction sites of pCMV-Myc, and the sequence was correct.
  • the recombinant expression vector was named pCMV_Myc-c-Abl.
  • the Abl gene can express a fusion protein with a Myc tag under the action of the CMV promoter.
  • the 896 bp Ml fragment and the 2609 bp M2 fragment were obtained, which were consistent with the expected results, recovered and purified.
  • Two target fragments then, 1 ⁇ each of M1 and M2, and mixed, using this as a template, PCR-amplified the full sequence of the c-Abl mutant gene under the guidance of primers Myc-Abl-1 and Myc-Abl-2, After the reaction, the PCR amplification product was subjected to 1% agarose gel electrophoresis, and a DNA fragment of (3450 + 17) bp was obtained, which was consistent with the expected result, and the target fragment was recovered and purified.
  • the c_Abl point mutation gene fragment cloned in step 1) was digested with restriction endonucleases EcoR I and Bgl II, and the restriction fragment was ligated with the vector pCMV-Myc digested with the same enzyme, and the ligation product was used.
  • the calcium chloride method was used to transform E. coli DH5 a competent cells, positive transformants were screened, plasmids were extracted, and restriction endonucleases ⁇ ⁇ and ll were used for restriction enzyme digestion, and 3800 bp and 3450 bp fragments were positively cloned by restriction enzyme digestion.
  • the positive clone plasmid was further identified by PCR.
  • the primers used were Myc-Abl-1 and Myc-Abl-2, and the PCR product was sequenced. The result showed that the c-Abl point mutation gene fragment was inserted into pCMV-Myc. Between the EcoRl and ⁇ /11 cleavage sites, and the sequence is correct, the recombinant expression vector was named pCMV-Myc-c-Abl (K290R).
  • the c-Abl point mutation gene fragment was changed from AAG to AGG from the 5' end of the GenBank Accession Number: NM_007313, and the other was from the 5' end of GenBank Accession Number: ⁇ 007313
  • the base sequence of the deoxyribonucleotide at position 440 to position 3889 is the same, and the Abl point mutation gene can express the fusion protein carrying the Myc tag under the action of the CMV promoter.
  • Abl, Arg double knockout mouse fibroblast MEF (Abl/Arg double knockout mouse embryo) was co-transfected with pcDNA 3-Flag-Gal3 and any of the following three expression vectors, respectively.
  • the transfected cells were lysed and immunoprecipitated with anti-Flag antibody (M5, Sigma). All the procedures should be carried out strictly in ice bath or low temperature conditions. The specific method is: Collect cells, 1000g Centrifuge for 3 minutes and discard the medium. The cells were washed with 5 mL of ice-cold lxPBS, centrifuged at 100 g for 2 minutes, and the supernatant was discarded and washed three times in the same manner.
  • ⁇ 100mm per cell requires 600 ⁇ l of single detergent lysate (50 awake ol/L Tris ⁇ CI, 150 mmol/L NaCl, 0.02% sodium azide, 1% NP-40 and protease inhibitors) (Roche, Inc) 1 tablet / 25 mL), 200 ⁇ l of single detergent lysate was added to each cell of 60 mra. Mix the cells with the lysate, ice bath for 5-10 minutes, and centrifuge at 16000 g for 10 minutes. The cell lysis supernatant was carefully aspirated, and 10-15 ⁇ M anti-Fat antibody cross-linked agarose beads (Sigma) were added and incubated at 4 ° C for 2 hours to perform immunoprecipitation.
  • single detergent lysate 50 awake ol/L Tris ⁇ CI, 150 mmol/L NaCl, 0.02% sodium azide, 1% NP-40 and protease inhibitors
  • the protein was transferred to a nitrocellulose membrane (NC membrane) by: cutting two 2. 5 mm thick filter paper (Bio-Rad, Inc.) and a nitrocellulose membrane into gel size. Soak for 30 minutes in lx transfer buffer (39 mmol/L glycine, 8mraol/L Tris, 0.0037% SDS and 20% methanol). Subsequently, it was placed on the semi-dry transfer apparatus (Bio-Rad, Inc.) from top to bottom in the order of filter paper-gel-nitrocellulose membrane-filter paper, and the air bubbles in the interlayer were exhausted as much as possible. Transfer film voltage 15V, 2- 2.
  • HRP-mouse Ig G horseradish peroxidase
  • step 3 After the NC membrane blocked in step 3 was incubated with primary antibody for 1 hour at room temperature, the membrane was washed three times with lxPBST for 5-10 minutes each time; then the enzyme-labeled secondary antibody was added, and after incubation for 1 hour at room temperature, the membrane was washed with lxPBST. Three times, 5-10 minutes each time. The above incubations were all carried out on a horizontal shaker.
  • the chemiluminescence reaction is carried out by: uniformly dropping the sensitizing liquid WESTERN LIGHTNING (PerkinElmer Life Sciences, Inc.) containing the horseradish peroxidase substrate onto the NC film, and then reacting the coloring solution for 1 minute. Dry, exposed with X-ray film (X-ray film and developer, fixing solution were purchased from Kodak Company).
  • the expression product of pCMV-Myc-c-Abl (K290R) is phosphorylated by Myc_c-Abl (K290R).
  • K290R Myc_c-Abl
  • the reason is that the K290 site of Abl is a key amino acid of the ATP-binding site in the C-Abl kinase domain.
  • c-Abl loses tyrosine kinase activity.
  • Gal3 can specifically Phosphorylated by c-Abl.
  • STI571 Inhibition of c-Abl phosphorylation by non-receptor tyrosine kinase C -Abl specific inhibitor STI571 was detected by transfecting Flag-Gal3 expression plasmid pcDNA 3_Flag-Gal3 into 293 cells, 24 hours later. 10 mol/L STI571 (product of Novartis, trade name: Gleevec, generic name: imatinib mesylate) was transfected with transfected cells for 12 hours to be transfected with an equivalent amount of DMS0 (solvent of STI571) The cells were used as controls, and the cells were lysed, and immunoprecipitation and immunoblotting were carried out in the same manner as in the third step.
  • the Gal3 protein stained secondary antibody was FITC-rabbit (Beijing Zhongshan Jinqiao Co., Ltd.), diluted at a ratio of 1:200 during use, incubated for 1 h at room temperature, and washed three times with PBS for 5 min each time.
  • the lysosomal staining secondary antibody was TRITC-mouse and was stained with Gal3 using the method. Finally, nuclear staining was performed.
  • the nuclear dye Hochest33342 (purchased from Dingguo Biotechnology Co., Ltd.) was used at a concentration of lu g /mL, incubated at room temperature for 30 min, and washed three times with PBS for 5 min each time. Observe under a fluorescence microscope.
  • Fig. 3 from left to right in Fig. A and Fig. B, green fluorescently labeled Gal3 protein, red Mito-Tracker labeled mitochondria, nuclear dye Hochest33342 labeled nuclei, combined images.
  • Figure C from left to right are the green fluorescently labeled Gal3 protein, the red fluorescently labeled lysosome, the nuclear dye Hochest33342 labeled nuclei, and the combined images.
  • strurosporine (sigma, S4400) was treated for 150 min, and the fourth group was treated with 5 uM STI571 for 18 h and then added with 0.5 uu STS for 150 min.
  • the immunostaining method is the same as step one.
  • the MCF-7 cells were inoculated into four 100-well cell culture dishes. The same treatment as the above immunostaining experiments was divided into four groups. The change of Gal3 protein in the cells was further detected by Western blotting. Gal3 detection Resistance to galectin-3 (B-2) (santa cruz).
  • the primary antibody is ⁇ -actin monoclonal antibody ( santa cruz biotechnology, inc)
  • the secondary antibody is HRP -mouse Ig G (santa cruz biotechnology, inc)
  • the above antibodies are purchased from Friendship Zhonglian Biotechnology Co., Ltd.
  • Example 3 Detection of apoptosis of MCF-7 cells treated with ⁇ STI571, 0.5 ⁇ STS alone and both
  • the experiment was divided into four groups: the first group of MCF-7 cells were not treated; the second group of MCF-7 cells were treated with ⁇ STI571 for 18 h; the third group of MCF-7 cells were treated with 0.5 ⁇ of apoptosis inducer STS for 4-5 h, The fourth group of MCF-7 cells were treated with ⁇ STI571 for 18 h and then added with 0.5 ⁇ M STS for 4-5 h.
  • TUNEL method was used to detect the apoptosis of MCF-7 cells treated with ⁇ STI571, 0.5 ⁇ STS.
  • the apoptosis of the four groups of MCF-7 cells treated with different treatments was detected by TUNEL-FLU0S kit (purchased from Roche) and with reference to the kit instructions.
  • the specific method was as follows: using freshly prepared 4% paraformaldehyde at room temperature. The cells were fixed for 30 min, washed with PBS and incubated with blocking agent (3 ⁇ 40 2 methanol solution) for 30 min at room temperature, washed with PBS, and 0.1% Triton X-100 was added and incubated for 2 min in an ice bath.
  • the cells also have apoptosis after being treated with 0.5 uM STS for 4-5 hours, and green fluorescence can be seen in the nucleus.
  • the red Gal3 protein is still distributed in the whole cytoplasm; as shown in Figure 4 in Figure 6, the cells are treated with 10uM STI571 for 18h and then added to 0. 5uM STS for another 4-5h, a strong green fluorescence can be seen. It is expressed in the nucleus, and the nucleus becomes small and shrunken. At this time, the red Gal3 protein is extremely weakly expressed and the apoptosis is remarkable.
  • the apoptosis of the four groups of MCF-7 cells treated with different treatments was detected by using the Annexin-V-FLUOS kit (purchased from Roche) and the kit was as follows: The cells were collected by centrifugation at 500 g for 5 min, and the cells were harvested with PBS. Suspend the cells, wash once, resuspend the cells in a 200 ⁇ l binding buffer (provided with the kit), add ⁇ Annexin-V-FITC and 5 ⁇ 1 ⁇ , mix gently, incubate for 15 min at room temperature, and use flow cytometry. Instrument detection.
  • FIG. 7 top left panel: normal MCF-7 cells, upper right panel: MCF-7 cells treated with 0.5 ⁇ apoptosis inducer STS for 4-5 h, lower left panel: MCF treated with 10 ⁇ STI571 for 18 h- 7 cells, right lower panel: after treatment with 10
  • Apoptosis when treated with STI571 and STS, showed significant apoptosis, further demonstrating that non-receptor tyrosine kinase c-Abl specific inhibitor STI571 can induce tumor cell apoptosis.
  • MCF-7 cells were divided into STI571 (10 ⁇ ) treatment and no treatment. After 18 hours, the two groups of cells were subdivided into two groups treated with the chemotherapy drug cisplatin CDDP (4MM). After 24 hours, the flow was used. Apoptosis analysis was performed using a cytometer.
  • the cells were trypsinized, and the cells were collected with 10-12 mL of PBS into a 15 ml tube, 500 g, and the cells were pelleted by centrifugation for 3 min, the cells were washed with 5 mL of PBS, and the cells were again pelleted by centrifugation. 2 mL of 70% ethanol (70% ethanol in PBS) was added dropwise while shaking. - Incubate overnight at 20 °C. After repeated washing with PBS twice, the pellet was centrifuged, and 2 mL of PC Buffer was added and incubated for 30 min [0. 2 M NaH 2 P0 4 192 mL; 0.2 M Sodium Citrate sodium citrate 8 mL].
  • mice Male, 18-20 g, purchased from the Experimental Animal Center of the Academy of Military Medical Sciences
  • MCF-7 cells in logarithmic growth phase were inoculated into nude mice by pancreatic enzyme digestion at 5 ⁇ 10 6 cells/subcutaneously. After inoculation, nude mice were randomly divided into 5 groups and randomized into groups. 7 only. The first group was the saline group (negative control group), and the nude mice were given saline by intragastric administration twice a day, ⁇ /g body weight twice a day; the second group was STI571 oral administration group.
  • the nude mice were administered by intragastric administration the next day after inoculation of cells, twice a day, 40 mg/kg each time, the solvent was normal saline, and the volume per administration was ⁇ /g body weight; the third group was 1/4 Conventional dose of CTX (cyclophosphamide (product of Jiangsu Hengrui Pharmaceutical Co., Ltd.), intraperitoneal injection group, nude mice were intraperitoneally administered on the 7th day after inoculation of cells, once every two weeks, 25 mg/kg each time, the solvent was Normal saline, each injection volume was ⁇ /g body weight; the fourth group was the combined administration group, and STI571 and CTX were administered in combination according to the second and third groups.
  • CTX cyclophosphamide (product of Jiangsu Hengrui Pharmaceutical Co., Ltd.)
  • CTX full-dose intraperitoneal injection group (Positive control group) nude mice were intraperitoneally administered on the 7th day after inoculation of cells, once every two weeks, 100 mg/kg each time, the solvent was physiological saline, and the volume of each injection was ⁇ /g body weight.
  • the tumor growth inhibition rate was calculated according to the following formula:
  • IR (1 - tumor weight of the treatment group / tumor weight of the negative control group) X 100%.
  • STI571 has a certain anti-tumor effect, and it can play a synergistic anti-tumor effect when combined with the tumor chemotherapy drug CTX.
  • the weight results of the five groups of nude mice are shown in Figure 10.
  • the fourth group of STI571 and 1/4 conventional dose CTX combination group (indicated by "STI571 + CTX (25mg/kg)")
  • the fifth group of CTX full-dose administration group (indicated by "CTX (100 mg/kg)”) showed significant differences in the average body weight of nude mice, but the tumor treatment effect did not exist. Significant differences indicate that STI571, as a combination of tumor chemotherapy drugs, can significantly reduce the side effects of cancer chemotherapy drugs.
  • Control is a negative control group.
  • the present invention provides a novel use of a non-receptor tyrosine kinase c-Abl specific inhibitor, i.e., in the preparation of an antitumor drug or a tumor therapeutic adjuvant.
  • non-receptor tyrosine kinase c-Abl specific inhibitors can cause Gal3 to undergo degeneration and aggregation and lysosome degradation by inhibiting the kinase activity of c-Abl, resulting in a significant decrease in Gal3 protein levels in tumor cells, thereby allowing tumors to The cells die of autophagic cell apoptosis.
  • the non-receptor tyrosine kinase c-Abl specific inhibitor can also make tumor cells extremely sensitive to a variety of tumor therapeutic drugs, and a low concentration of apoptosis-inducing agents can cause all cells to die in a short period of time, thus
  • the specific inhibitor of c-Abl, a tyrosine kinase enhances the therapeutic effect of existing cytotoxic chemotherapeutic drugs, and can greatly reduce the side effects by reducing the dose of chemotherapeutic drugs.
  • Preparation of anti-tumor drugs with non-receptor tyrosine kinase c-Abl specific inhibitor as active ingredient has the following advantages: 1) remarkable curative effect, tumor inhibition rate up to 81.4%; 2) high safety; 3) main The route of administration is oral, and the administration is convenient; 4) the inhibitor is inexpensive, and thus the cost of preparing the antitumor drug is low, thereby reducing the economic burden on the patient.
  • the invention provides a new way for the prevention and treatment of tumors, and has broad application prospects in the field of medicine.

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Abstract

L'invention concerne l'utilisation d'inhibiteurs spécifiques de la tyrosine kinase c-Ab1 non réceptrice, seuls ou combinés à la cisplatine ou au cyclodiméthoate dans la préparation de médicaments antitumoraux et/ou de médicaments antitumoraux auxiliaires. Des expériences prouvent que des inhibiteurs spécifiques de la tyrosine kinase c-Ab1 non réceptrice peuvent inhiber l'activité de la kinase c-Ab1 pour déboucher sur l'agrégation de dénaturation et la dégradation de lysosome de Gal3 et pour diminuer le niveau de protéines Gal3 dans les cellules tumorales et, ce de manière à tuer les cellules tumorales dans une apoptose autophagique.
PCT/CN2007/002113 2006-07-20 2007-07-10 Nouvelle utilisation d'inhibiteurs spécifiques de la tyrosine kinase c-ab1 non réceptrice Ceased WO2008011799A1 (fr)

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