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WO2010061862A1 - Agent antitumoral contenant un dérivé de thalidomide en tant que substance active - Google Patents

Agent antitumoral contenant un dérivé de thalidomide en tant que substance active Download PDF

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Publication number
WO2010061862A1
WO2010061862A1 PCT/JP2009/069890 JP2009069890W WO2010061862A1 WO 2010061862 A1 WO2010061862 A1 WO 2010061862A1 JP 2009069890 W JP2009069890 W JP 2009069890W WO 2010061862 A1 WO2010061862 A1 WO 2010061862A1
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thalidomide
cells
kms
myeloma
drug
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弘志 柳川
いちご 早川
弘和 始平堂
典子 田畠
豊 服部
健人 山田
剛巳 大槻
佳代子 田原
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Keio University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
    • C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
    • 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
    • A61K31/4035—Isoindoles, e.g. phthalimide
    • 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 a drug that inhibits the growth of various cancer cells including human myeloma containing a thalidomide derivative as an active ingredient, a method for inhibiting the same, and the use of such a drug in human cancer treatment.
  • myeloma is a hematopoietic tumor in which plasma cells grow neoplasticly. Although some chemotherapy and autologous hematopoietic stem cell transplant combined with high-dose therapy have improved the survival of patients with multiple myeloma, they still have no effective treatment. Currently, most patients with multiple myeloma become resistant to treatment and eventually die. Therefore, the development of new therapeutic drugs is widely demanded.
  • Thalidomide was released as a safe hypnotic by Grunenthal in Germany in 1957. In 1958, Dainippon Pharmaceutical launched a thalidomide agent in 1958. However, in 1961, Dr. W. Lenz revealed that when thalidomide was taken by a pregnant woman in early pregnancy, the fetus was malformed. The sale of thalidomide was stopped in Japan in 1962, but many thalidomide children were born. In this way, the drug caused tremendous damage worldwide. In 1994, it was announced by the United States that thalidomide has an anti-angiogenic activity (D'Amato, R.J., ⁇ (1994) Proc. Natl. Acad. Sci. USA 91,4082-4085).
  • Non-patent Document 1 thalidomide is effective for multiple myeloma.
  • the drug has antimyeloma effects through a variety of mechanisms. (1) directly induces myeloma cell death (apoptosis), (2) suppresses mutual use of myeloma cells and stromal cells, (3) suppresses tumor angiogenesis, (4) myeloma cells It has been considered to suppress the production of growth factors, and (5) activate anti-myeloma immunity.
  • thalidomide itself has low biological activity, it is activated in the body, such as TNF ⁇ (tumorcronecrosis factor : ⁇ : tumor necrosis factor ⁇ ), interferon ⁇ (interferon ⁇ ), IL-10 (interleukin-10), Direct bone marrow by acting on cyclooxygenase (cyclooxygenase [COX-2]), nuclear factor- ⁇ B (NF- ⁇ B: nuclear transcription factor ⁇ B), related adhesion focal tyrosine FTkinase (RAFTK: related adhesion focal tyrosine kinase) It is presumed to induce cell cycle G1 arrest and apoptosis of tumor cells (Non-patent document 2; Non-patent document 3; Non-patent document 4).
  • TNF ⁇ tumor necrosis factor ⁇
  • interferon ⁇ interferon ⁇
  • IL-10 interleukin-10
  • COX-2 cyclooxygenase [COX-2]
  • nuclear factor- ⁇ B nuclear
  • lenalidomide a thalidomide derivative
  • Phase III study a therapeutic result surpassing thalidomide was reported, while it was reported that peripheral neuropathy was low.
  • lenalidomide causes proliferation inhibition and apoptosis induction of myeloma cells even in vitro (Non-patent Document 3).
  • Non-Patent Document 6 Non-patent document 7; Non-patent document 8
  • the therapeutic effect with respect to multiple myeloma with a poor prognosis called the high-risk group which caused No.
  • Non-patent literature 9 Non-patent document 10; Regarding thalidomide derivatives, it has been known that TC11 acts as an aminopeptidase N enzyme inhibitor, an angiogenesis inhibitor (Patent Document 1) and a tubulin polymerization inhibitor (Non-Patent Document 12; Non-Patent Document 13). ing. Therefore, it is expected to develop a derivative that enhances the antitumor effect of thalidomide and has few teratogenicity and side effects.
  • Non-patent Document 14 Non-patent Document 14
  • Non-Patent Document 15 Non-Patent Document 15; Reference 16.
  • a drug that has a high antitumor effect against myeloma and does not have teratogenicity or resistance.
  • the object of the present invention is to provide a drug having an effect superior to that of conventional therapeutic drugs.
  • the present inventors screened drugs that show growth inhibition against various myeloma cell lines derived from Japanese myeloma patients from a variety of uniquely designed and synthesized thalidomide derivatives.
  • the present invention was completed by successfully obtaining a drug having a strong antimyeloma action in cancer mice and an effect superior to that of conventional therapeutic drugs.
  • the present invention provides an anticancer agent comprising a thalidomide derivative selected from the following compounds as an active ingredient.
  • the anticancer agent of the present invention is preferably for myeloma.
  • the compound is preferably TC11.
  • Compound TC11 can be used as an apoptosis-inducing agent, tubulin fragmentation-inducing agent, caspase-dependent apoptosis-inducing agent, or therapeutic agent for high-risk myeloma having chromosome 17 deletion.
  • the present invention also provides a method for treating cancer comprising administering the thalidomide derivative to a subject in need of anticancer treatment.
  • anticancer and “antitumor” are used as synonymous terms.
  • a drug exhibiting strong growth inhibition against various myeloma cell lines derived from Japanese myeloma patients were confirmed to have a strong antimyeloma action in invitro and myeloma-bearing mice and to have superior effects compared to conventional therapeutic agents. It is known that side effects such as teratogenesis are observed only in the S-form of optical isomers of thalidomide, an existing pharmaceutical product. Since the thalidomide derivative of the present invention is not an optically active substance, there is a high possibility that there is no side effect such as teratogenesis.
  • the active ingredient of the anticancer agent of the present invention is a thalidomide derivative selected from the compounds TC10, TC11, TC14, TC15, TC16, TC19, TC24 and TC25. These thalidomide derivatives also include pharmacologically acceptable salts of TC10, TC11, TC14, TC15, TC16, TC19, TC24 and TC25.
  • thalidomide derivatives are known compounds per se, as described below, or can be synthesized from known compounds by the method described in the present specification as shown in Examples described later.
  • TC10 EP 051563 A TC11: Inatsuki, S., et al. (2005) Bioorganic. Med. Chem. Lett., 15, 321-325; Inatsuki, S., et al. (2008) Int. J. Mol. Med., 21, 163-168; WO 98/07421 TC14: See examples below TC15: See examples below TC16: See examples below TC19: See examples below TC24: See examples below TC25: See examples below
  • the thalidomide derivative which is an active ingredient, can be made into a preparation (pharmaceutical composition) using a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include an excipient or a base.
  • the formulation may contain the additive used normally.
  • the dosage form is appropriately selected depending on the administration route. For example, tablets, capsules, granules, powders, syrups and the like may be administered orally, or injections, suppositories, etc., and parenteral by intraperitoneal or intravenous injection. It can also be administered.
  • the preparation includes a thalidomide derivative of the active ingredient and another anti-cancer agent separately packaged and integrated.
  • the anticancer agent of the present invention can be administered to a patient who is receiving or intending to receive anticancer drug treatment. These thalidomide derivatives may be used alone or in combination with other drugs (for example, other anticancer drugs).
  • the dose of the thalidomide derivative is appropriately selected depending on the anticancer drug treatment targeted, the patient's condition, and the like.
  • the above thalidomide derivative is administered to humans, it is administered once at a dose of about 0.1 to 20 mg / kg (body weight) per day, preferably about 0.1 to 0.5 mg / kg (body weight) per day.
  • it can be administered orally in several divided doses, but the dose and frequency of administration can be appropriately changed depending on symptoms, age, administration method and the like.
  • the above thalidomide derivatives include hematopoietic tumors such as myeloma, malignant lymphoma, leukemia, myelodysplastic syndrome, colon cancer, lung cancer, renal cell cancer, breast cancer, brain tumor, ovarian cancer, melanoma, stomach cancer, prostate cancer, etc. It can be used for treatment of cancer such as solid cancer.
  • the side amide derivative used in the present invention is selected as follows and is considered to have the advantages described below.
  • combichem combinatorial chemistry
  • one of the problems in synthesis is to efficiently introduce an affinity carrier immobilization linker for each drug when constructing the library.
  • Thalidomide was originally used as a hypnotic and is notorious for its teratogenicity. However, in recent years, it has been reported that thalidomide and its derivatives show good drug efficacy in various diseases such as cancer, leprosy, and AIDS. Therefore, the thalidomide skeleton is considered to be a super template in drug development. Thalidomide has the structure shown in FIG. 1, and can be roughly divided into two units, a phthalimide ring and a glutarimide ring. Therefore, it was decided to construct a thalidomide derivative library based on the policy of fixing one unit and converting the other unit (FIG. 1).
  • R-NH 2 was selected mainly for aromatic amines and aromatic alkyl amines. There are many reports of thalidomite derivatives having these substituents, such as angiogenesis inhibition ability, TNF- ⁇ regulation ability, and microtubule polymerization inhibition ability at the cellular level, and it is considered that they are likely to become anticancer agents.
  • R was a saturated ring and thalidomide itself was designed.
  • thalidomide with various activities was designed as one of the active bodies in vivo, with the glutarimide ring opened, and R-NH 2 is an amino acid. .
  • the thalidomide library was synthesized as follows.
  • thalidomide having a benzene ring as a naphthalene ring was synthesized in the same manner.
  • the glutarimide ring was reacted with commercially available modified phthalic anhydride in acetic acid under microwave irradiation. Initially, the imide nitrogen atom was modified with ⁇ -chloroacetic acid ester. In that case, decomposition into a plurality of compounds proceeded during the subsequent conversion to carboxylic acid by hydrolysis. Therefore, the imide nitrogen atom was modified with ⁇ -bromoacetic acid benzyl ester, and the carboxylic acid compound was synthesized by hydrogenolysis. In the tetrafluorophthalimide body and the pyridylphthalimide body, alkylation of the glutarimide ring imide nitrogen atom itself did not proceed.
  • TC11 caused strong growth inhibition (IC50 value of 3-7 ⁇ M) and apoptosis induction for all myeloma cell lines. Induction of apoptosis was also confirmed by DNA and PARP cleavage by gel electrophoresis and flow cytometer detection by Annexin V staining.
  • the seven thalidomide derivatives (TC10, TC14, TC15, TC16, TC19, TC24, TC25) other than TC11 also caused growth inhibition and apoptosis in myeloma cell lines, although there were differences in strength.
  • TC11 irreversibly caused destruction of the Golgi apparatus and microtubule structure in the KMS-34 cell line.
  • the Golgi body and microtubule structure were also destroyed in HeLa cells, but the action is reversible, and when the drug is washed and removed, it quickly returns to its original state. High nature.
  • thalidomide and dexamethasone which are existing myeloma drugs (key drug), inhibit the growth of only the KMS-11 cell line, and other cell lines (KMS-34, KMM-1, KMS-27, RPMI- The growth of 8226) was not suppressed at all, and a clear difference in action specificity with TC11 was observed.
  • KMS-34 tumor-bearing SCID mice were intraperitoneally injected with 20 mg / kg TC11 suspended in 5% carboxymethylcellulose. In the administration for 2 weeks, significant growth inhibition was observed compared with the control, but no systemic toxicity such as weight loss or significant organ damage was observed.
  • thalidomide derivatives originally designed and synthesized by the present inventors could be screened for drugs showing strong growth inhibition against various myeloma cell lines derived from Japanese myeloma patients.
  • the obtained drug has a strong antimyeloma action in invitro and myeloma-bearing mice and has an excellent effect compared with conventional therapeutic agents.
  • side effects such as teratogenesis are observed only in the S-form of optical isomers of thalidomide, an existing pharmaceutical product.
  • the thalidomide derivative of the present invention is not an optically active substance, there is a high possibility that there is no side effect such as teratogenesis. From now on, by further examination, it can be expected that these thalidomide derivatives will be applied to antimyeloma therapeutic agents without side effects.
  • TC11 is considered to have an antitumor effect that is different from conventional anticancer agents or an antitumor effect superior to conventional anticancer agents.
  • Nucleophosmin and ⁇ -tubulin were identified as target proteins for the drug TC11. This indicates that it is associated with phenotypes such as apoptosis and tubulin fragmentation.
  • TC11 causes apoptosis, but further cleavage of procaspases 2, 3, 8, and 9 was observed, and apoptosis signals were detected between DeathDereceptor and mitochondria. I found it coming from both pathways. Due to the difference in the mechanism of action, TC11 is considered to exhibit an antitumor effect that is different from conventional anticancer agents.
  • compound TC11 can be used as an apoptosis inducer, a tubulin fragmentation inducer, a caspase-dependent apoptosis inducer, or a therapeutic agent for high-risk myeloma having chromosome 17 deletion. it is conceivable that.
  • Example 1 Synthesis of thalidomide derivatives (TC11) TC11 was synthesized according to the following scheme.
  • Thalidomide derivatives other than TC11 are TC10, TC14, TC15, TC16, TC19, TC24, TC25, 4-nitrophthalic anhydride and cyclohexylamine, p-benzylaniline, o-phenylaniline, p-phenoxyaniline, phenylbutylamine, naphthyl, respectively.
  • Ethylamine and phenylmethylamine were synthesized by reacting in acetic acid according to the same method as TC11, and then catalytic reduction.
  • a normal overheating method may be used instead of using microwaves.
  • the microwave heating portion may be set to “several hours heating reflux”.
  • KMM-1, KMS-11, KMS-27, KMS-34, RPMI-8226, and MUM24 were established by Takeshi Ohtsuki and others at Kawasaki Medical School.
  • RPMI-8226 was obtained from a cell bank (Moore, GE, Kitamura, H., 1968 NY State., J. Med. 2054-2060; IFO 50013; JCRB0034).
  • MUM24 was independently established by Yutaka Hattori from bone marrow tumor patients.
  • chromosomes 4 and 14 are translocated. In both cell lines, the expression of fibroblast growth factor FGF3 (Fibroblast factor 3) is excessive. This translocation is clinically regarded as a poor prognostic factor.
  • FGF3 fibroblast growth factor 3
  • MUM24 is a cell line whose hat was originally established from bone marrow blood of a 64-year-old IgG (k) type myeloma patient. Grows in RPMI 1640 + 10% fetal calf serum medium, no cytokines required. The cell lacks one of chromosome 13.
  • Chromosome 13 deletion is considered in the treatment of myeloma, chemotherapy, autologous hematopoietic stem cell transplantation (Chang, H., ⁇ (2005) Bone Marrow transplantation 36,793-796), thalidomide therapy (Hattori, Y., ⁇ (2008) Cancer Science, 99,1243) is regarded as an independent poor prognostic factor.
  • cases with chromosome 13 deletion have a very poor prognosis with existing therapies and are referred to as high-risk groups.
  • MUM24 cells can be an important tool. For example, by using the same cells in screening for a myeloma therapeutic drug, it is possible to selectively select an effective drug even in cases having chromosome 13 deletion. At present, there are very few commonly available myeloma cell lines with chromosome 13 deletion, and MUM24 is extremely useful for elucidating the pathology of high-risk myeloma and developing effective therapeutic agents.
  • thalidomide derivatives (0.5% DMSO solution) were added to each 96-well plate.
  • the plate was placed in RPMI1640 medium (Invitrogen) containing 10% (v / v) FBS (Gibco), 1% (v / v) penicillin / streptomycin (Gibco) at 37 ° C. and 5% CO 2.
  • RPMI1640 medium Invitrogen
  • FBS FBS
  • 1% (v / v) penicillin / streptomycin Gibco
  • Six cultured myeloma cells were seeded at a density of 1-2.5 ⁇ 10 4 cells per well. After culturing the cells for 72 hours, Cell Proliferation Reagent WST-1 (Roche) was added and further cultured for 1 hour.
  • the absorbance of the cells was measured with a plate reader to determine the number of viable cells. With respect to this absorbance, the cell viability was calculated with 100% non-drug added (0.5% DMSO added) and 0% when no cells were seeded.
  • thalidomide derivatives (TC10, TC11, TC14, TC15, TC16, TC19, TC24, TC25) are divided into six types of human cultured myeloma cell lines (KMM-1, KMS-11, KMS-27, KMS-34, It showed cytostatic activity against RPMI-8226 and MUM24).
  • TC11 showed strong growth inhibitory activity (IC50 value of 3-7 ⁇ M) against any myeloma cell line.
  • thalidomide and dexamethasone which are existing myeloma drugs (key drug)
  • KMS-34, KMM-1, KMS-27, RPMI- 8226 and KMU24 were not inhibited at all, and a clear difference in action specificity from TC11 was observed.
  • thalidomide derivatives Five concentrations (50, 20, 10, 5, 0 ⁇ M final concentration) of thalidomide derivatives and each of thalidomide and staurosporine (0.5% DMSO) were added to 12-well plates, respectively.
  • the plate was placed in RPMI1640 medium (Invitrogen) containing 10% (v / v) FBS (Gibco), 1% (v / v) penicillin / streptomycin (Gibco) at 37 ° C. and 5% CO 2.
  • Cultured myeloma cells were seeded at a density of 2.5 ⁇ 10 5 cells per well. After culturing for 6 hours, the mixture was centrifuged at 1,000 rpm for 5 minutes, and the supernatant was removed and suspended in D-PBS.
  • the obtained cell suspension was subjected to ultrasonic disruption and then heated at 96 ° C. for 10 minutes for Western blotting.
  • 12% polyacrylamide gel was used for SDS-PAGE, anti-PARP antibody (Cell Signaling) was used as the primary antibody, and HRP-labeled antibody was used as the secondary antibody.
  • Blocking was performed at room temperature for 30 minutes using Blocking buffer [PBS containing 1% BSA (Nacalai), 0.05% Sodium Azide (Nacalai)].
  • the cells were rinsed with PBS, and anti-cleaved PARP antibody, anti-GM130 antibody, and anti-tubulin antibody (Cell Signaling), which are primary antibodies at any concentration, were diluted with Blocking Buffer and added to the cells. After 1 hour, the cells were rinsed with PBS, and Blocking buffer containing 1/600 vol secondary antibody was added to the cells.
  • TC11 caused cleavage of PMS in KMS-34 cells, but did not cause cleavage of PARP in HeLa cells.
  • Staurosporine which is known to cause strong apoptosis, caused the cleavage of PARP in both KMS-34 and HeLa cells.
  • thalidomide did not cause PARP cleavage in either cell. This indicates that TC11 strongly induces apoptosis in KMS-34 cells.
  • TC11 irreversibly caused Golgi structural destruction in the KMS-34 cell line. At the same time, the microtubule structure was destroyed. On the other hand, TC11 also caused destruction of the Golgi apparatus and microtubule structure on HeLa cells, but the action was reversible immediately after washing and removal of TC11. It is likely that this is a selective phenomenon.
  • thalidomide derivative and staurosporine (0.5% DMSO) were added to 6 cm dishes, respectively.
  • the plate was placed in RPMI1640 medium (Invitrogen) containing 10% (v / v) FBS (Gibco), 1% (v / v) penicillin / streptomycin (Gibco) at 37 ° C. and 5% CO 2.
  • Cultured myeloma cells were seeded at a density of 1 ⁇ 10 6 cells per well.
  • the cells were cultured for 6 hours and then centrifuged at 1,000 rpm for 5 minutes, and the precipitate was suspended in a lysis buffer (0.2% Triton X-100, 10 mM Tris-HCl buffer (pH 7.4), 10 mM EDTA).
  • the cells were lysed by allowing them to stand for 15 minutes on ice, and collected by centrifugation at 10,000 ⁇ g for 20 minutes.
  • RNase A was added to the collected supernatant and reacted at 37 ° C. for 1 hour to degrade RNA. From this cell suspension, DNA was recovered using MaXtract low density gel (Qiagen). The experimental procedure was performed according to the protocol attached to the kit, and elution was performed with TE buffer.
  • the obtained eluate was subjected to electrophoresis using 2% agarose gel (containing EtBr), and the band was detected with a fluorescence imager.
  • TC11 caused DNA cleavage of KMS-34 cells.
  • Positive control staurosporine also caused DNA breaks in KMS-34 cells.
  • Tumor size was measured over time using calipers, and tumor body diameter was calculated as major axis ⁇ minor axis 2 ⁇ 0.52 (Tomioka, D., et al. (2001) Cancer Res., 61, 7518-7524) . Evaluation was made by t-test on day 7, day 10, and day 14, and P ⁇ 0.05 was considered significant. This research was conducted with the approval of Keio University Animal Experiment Committee (Approval No. 09118- (0)).
  • TC11 TC11 (20 ⁇ g when tumor size exceeded 50 mm 3 for the first time. / g mouse) was started.
  • the change in tumor size over time is shown in FIG.
  • t-tests were performed on day 7, day 10, and day 14.
  • the TC11 treatment group showed significant growth inhibition compared to controls, but no systemic toxicity such as weight loss or significant organ damage was observed.
  • mice were intraperitoneally injected with 5% carboxymethylcellulose (control) and 3 mice with TC11 (20 ⁇ g / g mouse) on the 3rd. Mice were killed by ether anesthesia 14 days after the start of TC11 or control injection (day 14), and the tumors were isolated by skin incision and fixed with 10% formalin to prepare paraffin-embedded specimens. Apoptosis was examined by hematoxylin and eosin staining.
  • ssDNA anti-single-stranded DNA
  • ssDNA rabbit polyclonal antibody
  • 1/100 diluted anti-MIB-1 mouse monoclonal was used to assess cell proliferation.
  • an antibody DakoCytomation, Carpinteria, CA
  • a peroxidase-labeled secondary antibody was added, and DAB color development was performed.
  • the TC11 treatment group had significantly more apoptotic images such as nuclear condensation and fragmentation than the control group. Also, according to immunohistochemistry using anti-ssDNA (single strand DNA) antibody, the TC11 group had significantly more apoptotic cells that caused DNA cleavage and became single strand DNA than the control group. . Thus, it was found that TC11 strongly induces not only in vitro but also apoptosis of in vivo myeloma cells.
  • Example 2 Identification of target protein that binds to drug TC11
  • the following experiment was performed. As a result, it was revealed that nucleophosmin and ⁇ -tubulin bind to TC11.
  • the primers used in this example are as follows.
  • PCR was carried out at 94 ° C. for 5 minutes, followed by 24 cycles of 94 ° C., 30 seconds, 58 ° C. for 30 seconds, 68 ° C. for 2 minutes, and then reacted at 68 ° C. for 5 minutes.
  • the cDNA library was purified by Wizard Plus Minipreps DNA Purification System (Promega) and recovered as a 60 ⁇ l DNA solution, and then electrophoresed on a 1% low melting point agarose gel (Sigma), using molecular weight markers as a guideline for 200-400 bp, 400-750 bp, Bands of 750-1400 bp and 1400-3000 bp were cut out.
  • the gel was heated at 70 ° C for 20 minutes, thawed, purified by Wizard Plus Minipreps DNA Purification System (Promega), recovered as 40 ⁇ l of DNA solution, and cDNA libraries KMS-XS (200-400bp), KMS-S (400-750bp) KMS-L (750-1400 bp) and KMS-XL (1400-3000 bp).
  • Immobilization of target drug on sensor chip Biacore used the Biacore 3000 system to fix the drug on sensor chip SA.
  • the flow was performed with a buffer HBS-EP (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20) at 10 ⁇ l / min.
  • Pretreatment for immobilization was performed by repeatedly injecting 20 ⁇ l of a solution containing 50 mM NaOH and 1M NaCl into the flow cells 1 to 4 five times.
  • TC11-biotin was adjusted to 1 ⁇ M (H 2 O, 0.05% DMSO), and TC11-biotin was immobilized on the flow cells 1 to 4.
  • TC11-biotin was bonded to Flow Cell 1 at 203.2 RU, Flow Cell 2 at 205.0 RU, Flow Cell 3 at 206.2 RU, and Flow Cell 4 at 206.8 RU.
  • the injection of 30 ⁇ l / min with buffer HBS-EP, 30 ⁇ l of the same buffer, and 15 ⁇ l of Glycin HCl pH 2.0 was performed twice.
  • the gel was washed 3 times with 500 ⁇ l of TBST and eluted with 100 ⁇ l of FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Lys) (SEQ ID NO: 9) 100 ⁇ g / ml.
  • Fluorescence was detected with a Multi-detection Microplate Reader POWERSCAN HT at an excitation wavelength of 485 nm and a fluorescence wavelength of 528 nm, and IVV fractions eluted from 4 to 7 wells were collected.
  • the IVV fraction was diluted with about 100 ⁇ l of HBS-EP in about 200 ⁇ l and injected into Biacore.
  • Selection at Biacore uses buffer HBS-EP at a flow rate of 20 ⁇ l / min, after 750 seconds of binding and after 1000 seconds of dissociation, using Biacore recovery method, washing part is buffer HBS-EP 600 seconds, elution part is TC11 200 ⁇ M (H 2 O, 1% DMSO) was performed for 600 seconds, and 7 ⁇ l was collected each time.
  • 1 round of selection experiment KMS-L ⁇ KMS-L1) 1 wash, 2 rounds (KMS-L1 ⁇ KMS-L2) 2 washings, 3 rounds (KMS-L2 ⁇ KMS-L3) washing 3 times and 4 rounds (KMS-L3 ⁇ KMS-L4) washing was performed 4 times, and the selection pressure was increased for each round (FIG. 5).
  • Reaction solution 2.5 ⁇ l subjected to reverse transcription reaction, 10 ⁇ KOD plus buffer (TOYOBO) 2.5 ⁇ l, 2 mM dNTPs (TOYOBO) 2.5 ⁇ l, 25 mM MgSO 4 1 ⁇ l, forward primer: GSP6omega F (10 pmol / ⁇ l) 0.75 ⁇ l, Reverse primer: 0.75 ⁇ l of 3RV30 (10 pmol / ⁇ l), 0.5 ⁇ l of KOD plus polymerase (TOYOBO), and RNase-free water were added to carry out PCR reaction with a total volume of 25 ⁇ l. PCR was performed at 94 ° C.
  • the cDNA library was purified with Wizard Plus Minipreps DNA Purification System (Promega) and recovered as a 60 ⁇ l DNA solution, then electrophoresed with 1% low-melting point agarose gel (Sigma), and the molecular weight marker as a guideline for KMS-L (750-1400 bp) A band was cut out. The gel was heated at 70 ° C. for 10 minutes and thawed, then purified by Wizard Plus Minipreps DNA Purification System (Promega) and recovered as a 42 ⁇ l DNA solution to obtain DNA libraries KMS-L1 to KMS-L4 (750-1400 bp).
  • the product was purified by Wizard Plus Minipreps DNA Purification System (Promega) and recovered as 20 ⁇ l of DNA solution, and then mixed with 4 ⁇ l of DNA, 1 ⁇ l of Topo vector (Invitorogen) and 1 ⁇ l of Salt solution (Invitorogen) and left at room temperature for 20 minutes. .
  • 5.5 ⁇ l of the mixed solution was placed in a competent cell dissolved on ice and allowed to stand on ice for 25 minutes, followed by heat shock at 43 ° C. for 32 seconds. Place 250 ⁇ l of SOC (Invitorogen) in the cell and incubate on a shaking incubator for 1 hour at 37 ° C.
  • Two agar plates (5 g tryptone, 2.5 g yeast extract, 5 g NaCl, 7.5 g agar, carbenicillin in 500 ml) 25 mg) (9 cm petri dish) and cultured overnight at 37 ° C.
  • PCR was conducted at 96 ° C for 5 minutes, followed by 30 cycles of 96 ° C, 30 seconds, 58 ° C for 30 seconds, 72 ° C for 1 minute, and then reacted at 72 ° C for 5 minutes.
  • the amplified gene was confirmed by 1% agarose gel electrophoresis and then purified by Wizard Plus Minipreps DNA Purification System (Promega) and recovered as a 25 ⁇ l DNA solution.
  • the obtained PCR reaction product was transferred to a 1.5 ml tube, and (3M) NaOAc 1 ⁇ l, (0.1M) EDTA 1 ⁇ l, (20 mg / ml) glycogen solution (Nacalai Tesque) 1 ⁇ l was mixed well and cooled (100% ) 60 ⁇ l of ethanol was added and mixed well. The supernatant was removed by centrifugation at 4 ° C., 14000 rpm for 15 minutes, and the supernatant was removed (70%). The pellet was washed with 200 ⁇ l of ethanol, and then centrifuged again at 14000 rpm for 2 minutes to remove the supernatant twice. It was. Subsequently, after centrifugal drying for 30-40 minutes, 40 ⁇ l of deionized formamide (Beckman® coulter) was added and mixed well. Sequence analysis was performed with CEQ 2000 DNA Analysis System (Beckman coulter).
  • nucleophosmin is a candidate protein binding protein TC11.
  • Amino acid sequences of KMS-L31-35 and NPM1 KMS-L31-35 obtained in the selection experiment was 1-183 amino acids including the amino terminus of the full-length 294 amino acid residues of nucleophosmin. .
  • FIG. 7 shows the domain structure of nucleophosmin.
  • the via core 3000 system was used as the via core, and the drug was immobilized on the sensor chip SA.
  • the flow was carried out with buffer HBS-EP at 10 ⁇ l / min.
  • the flow cells 1 to 4 were pretreated for immobilization by repeatedly injecting 20 ⁇ l of a solution containing 50 mM NaOH and 1M NaCl five times.
  • TC11-biotin was adjusted to 1 ⁇ M (H 2 O, 0.05% DMSO), and TC11-biotin was immobilized on the flow cell 2.
  • TC11-biotin bound to 276.4.2 RU.
  • the injection of 30 ⁇ l / min with buffer HBS-EP, 30 ⁇ l of the same buffer, and 15 ⁇ l of Glycin HCl pH 2.0 was performed twice.
  • PCR was conducted at 94 ° C. for 5 minutes, followed by 24 cycles of 94 ° C., 30 seconds, 58 ° C. 30 seconds, 68 ° C. 1 minute 20 seconds, and then reacted at 68 ° C. for 5 minutes.
  • the cDNA library was purified by Wizard Plus Minipreps DNA Purification System (Promega) and recovered as a 60 ⁇ l DNA solution.
  • ANTI-FLAG M2 affinity gel 50 ⁇ l (50% slurry) was washed three times with TBST (20 M Tris-HCl buffer, pH 7.5, 138 mMClNaCl, 0.1% Tween-20) 500 ⁇ l 150ml translated protein solution, 150 ⁇ l TBST The mixture was mixed and rotated and stirred with a mini disk rotor (Bio craft) at 4 ° C. for 1 hour. The gel was washed 3 times with 500 ⁇ l of TBST and eluted with FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Lys) (SEQ ID NO: 9) 100 ⁇ g / ml 100 ⁇ l.
  • Biacore was analyzed using HBS-EP at a flow rate of 20 ⁇ l / min. Samples were injected with KINJECT and regeneration was performed with 15 ⁇ l of Glycine-20, 15 ⁇ l of (50 mM) NaCl, and (1 M) NaCl.
  • FIG. 9 shows a sensorgram of KMS-L31-35 (nucleophosmin).
  • the KD was estimated by subtracting and fitting the flow cell to which no drug was added from the flow cell to which TC11-biotin was bound according to a conventional method. A fitting was performed and a KD value of 3.84 ⁇ 10 ⁇ 5 M was calculated. Nucleophosmin and TC11 prepared in a wheat germ cell-free translation system showed a relatively weak interaction.
  • E. coli expressed protein Clone KMS-L31-35 DNA obtained by cloning 0.01 pmol, 10 ⁇ KOD plus buffer (TOYOBO) 10 ⁇ l, 2 mM dNTPs (TOYOBO) 10 ⁇ l, 25 mM MgSO 4 4 ⁇ l , Forward primer: CACC-NPM1-F (10 pmol / ⁇ l) 3 ⁇ l, reverse primer: NPM1-FlagHis (10 pmol / ⁇ l) 3 ⁇ l, KOD plus polymerase (TOYOBO) 2 ⁇ l with RNase-free water added to 100 ⁇ l total volume Were put into one tube, and a total of 4 tubes were subjected to PCR reaction.
  • PCR was conducted at 94 ° C. for 5 minutes, followed by 24 cycles of 94 ° C., 30 seconds, 58 ° C. 30 seconds, 68 ° C. 1 minute 20 seconds, and then reacted at 68 ° C. for 5 minutes.
  • the cDNA library was purified by Wizard Plus Minipreps DNA Purification System (Promega) and recovered as a 60 ⁇ l DNA solution.
  • the obtained PCR product and the resulting product were introduced into pET101 / D-TOPO (Invitrogen) and transformed into One shot top10 (Invitrogen) to obtain a plasmid.
  • the prepared plasmid was introduced into E. coli to express nucleophosmin. Plasmid DNA was transformed into BL21Star (DE3) (Invitrogen), inoculated into LB medium (+100 ⁇ g / mL carbenicillin), and pre-cultured at 37 ° C overnight. The pre-cultured bacterial solution was added to 20 times the amount of TB medium and cultured at 37 ° C. with shaking until the OD600 reached 0.4. IPTG was added to the culture solution so that the final concentration was 0.1 ⁇ mM, and expression induction was performed at 37 ° C. for 2 to 4 hours. The culture was centrifuged at 8,500 rpm for 8 minutes, and the cells were collected.
  • the collected cells were suspended in TBS containing 0.02% (v / v) protease inhibitor cocktail, pulverized with ultrasonic waves, centrifuged at 9,500 rpm for 15 minutes, and the supernatant was collected. After equilibrating 4 mL of TALON CellThru Resin (Clontech) with a TBS buffer containing 10 mM imidazole, the supernatant (soluble fraction) was added to this resin and allowed to bind overnight at 4 ° C. The reaction solution was added to the column, washed with a washing buffer (TBS, 10 mM imidazole), and then the protein was eluted with an elution buffer (TBS, 250 mM imidazole).
  • TBS washing buffer
  • TBS elution buffer
  • Biacore was analyzed using HBS-EP at a flow rate of 20 ⁇ l / min. Samples were injected with KINJECT and regeneration was performed with 15 ⁇ l of Glycine-20, 15 ⁇ l of (50 mM) NaCl, and (1 M) NaCl.
  • FIG. 10 shows a sensorgram of E. coli expressed protein (nucleophosmin).
  • the KD was estimated by subtracting and fitting the flow cell to which no drug was added from the flow cell to which TC11-biotin was bound according to a conventional method. After fitting, the high molecular weight fraction was calculated as the low molecular weight fraction of 1.25 ⁇ 10 ⁇ 4 M and the KD value of 6.64 ⁇ 10 ⁇ 8 M, respectively.
  • Nucleophosmin expressed in E. coli could be separated into a fraction with strong interaction with TC11 and a fraction with weak interaction by size fractionation by gel filtration. The nucleophosmin in the high molecular weight fraction was expected to exist as an aggregate and the interaction was weak, but the low molecular weight fraction was expected to have a monomeric structure. The interaction with TC11 was relatively strong.
  • nucleophosmin is a target protein that binds to the drug TC11.
  • ⁇ -tubulin was 1 out of 23 clones (4%) in 3 rounds, 1 out of 11 clones (9%) in 4 rounds, and 2 out of 34 clones in total. Clones (6%) were included. From this result, it was revealed that ⁇ -tubulin is a candidate for the protein TC11 binding protein.
  • FIG. 11 shows the domain structure of ⁇ -tubulin.
  • the ⁇ -tubulin region (1-163) screened using TC11 as a bait contains a GTP-binding domain, and TC11 competes with GTP to produce ⁇ -tube. It is likely that the polymerization of phosphorus and ⁇ -tubulin is inhibited. This hypothesis is consistent with the phenomenon of tubulin fragmentation in myeloma cells in the presence of TC11.
  • PCR was conducted at 94 ° C. for 5 minutes, followed by 24 cycles of 94 ° C., 30 seconds, 58 ° C. 30 seconds, 68 ° C. 1 minute 20 seconds, and then reacted at 68 ° C. for 5 minutes.
  • the cDNA library was purified by Wizard Plus Minipreps DNA Purification System (Promega) and recovered as a 30 ⁇ l DNA solution.
  • SDS-PAGE was performed with a 4-12% Bis-Tris NuPAGE gel and MES electrophoresis buffer (Invitrogen) at 200 V, 400 mA for 35 minutes, and then transferred to a nitrocellulose membrane with an iBlot dry blotting system.
  • Membrane blocked with Blocking One Buffer: TBST (1: 9) then detected using ERP using HRP-conjugated mouse anti-Flag • tag antibody (Sigma; 2: 3000 / Blocking One Buffer: TBST (1: 9)) I went there.
  • Fig. 12 shows the results of a competitive experiment with KMS-L31-36 (tubulin).
  • KMS-L31-36 ( ⁇ -tubulin) showed a protein band superior to the control with respect to the resin to which TC11 was bound.
  • the competitive drug inhibited binding to the resin predominantly. Therefore, a specific interaction between TC11 and KMS-L31-36 ( ⁇ -tubulin) was confirmed.
  • Example 3 Induction of caspase-dependent apoptosis by TC11
  • treatment of human bone marrow tumor cell KMS-34 with TC11 gives fragments of tubulin and Golgi, which are proteins that form microtubules and centrosomes of cells. Observed.
  • PRP and DNA were cleaved by treating KMS-34 cells with TC11 for a long time, indicating that TC11 induces apoptosis of these cancer cells.
  • which caspase pathway this apoptosis is caused by was examined by Western blot using various caspase antibodies.
  • caspase 3 which is an effector caspase
  • caspases 8 and 9 that act as initiator caspases upstream thereof were cleaved and activated (FIG. 13, A).
  • caspase-8 is activated by a signal pathway via death receptor and caspase-9 by a pathway via mitochondrion (Spek, E., Bloem, AC, Lokhorst, HM, Kessel, B., Bogers-Boer, L., Donk, N. Inhibition of the mevalonate pathway potentiates the effects of lenalidomide in myeloma. Leukemia Res., 33, 100-108 (2009)).
  • Caspase 2 which is activated by disruption of the cytoskeleton, has been reported to induce caspase-9 pathway-dependent apoptosis by activating its substrate Bid (Ho, LH, Read, SH , Dorstyn, L., Lambrusco, L., Kumar, S. Caspase-2 is required for cell death induced by cytoskeletal disruption. Oncogene, 27, 3393-3404 (2008)). Therefore, it was examined whether TC11 affects caspase 2 activity, but caspase 2 activation was not observed (FIG. 13, B).
  • Example 4 Colony assay for verification of hematopoietic disorder effect of TC11
  • the toxicity test of TC11 was performed using a colony assay method for examining colony-forming ability of normal bone marrow cells.
  • Bone marrow cells are collected from the femur of an ICR mouse ( ⁇ , 13 weeks old) using a 26 gauge needle, and become 5 ⁇ 10 5 cells / mL in 2 ⁇ alpha MEM, 10% FCS medium. Adjusted as follows.
  • Cytokine (final concentration) in methyl cellulose medium (containing 10% 2 ⁇ alpha MEM, 30% FCS, 1% BSA, 0.1 mM 2ME, 0.1 mM hemine, 1% P / S, 1% L-Glu, 1.2% methyl cellulose)
  • IL-3 20 ng / mL, IL-6 10 ng / mL, SCF 20 ng / mL, EPO 1U ng / mL) and various concentrations of TC11 or doxorubicin hydrochloride were added to the above bone marrow cells 5 ⁇ 10 4 cells / mL were added.
  • TC11 is prepared by diluting 20 mM DMSO stock solution with 5% DMSO EtOH to make 1 mM, 0.2 mM, and 0.04 mM, and adding 0.025 mL to 1 mL of methyl cellulose medium, respectively, to a final concentration of 25 ⁇ M, 5 ⁇ M, and 1 ⁇ M. Adjusted as follows. In addition, the concentration of DMSO and EtOH was adjusted to 0.125% and 2.5% for specimens not containing TC11, respectively. Note that CTRL (-) does not include DMSO and EtOH.
  • Example 5 Changes in blood concentration after intraperitoneal administration of TC11 to mice
  • the course is such that drugs are less effective at small doses, the effect is stronger when doses are increased, and at the same time, side effects occur, and death occurs when doses are further increased. It has a nature to follow. Therefore, it is necessary to decide how much is appropriate in order to maximize the effect of the drug and minimize the occurrence of side effects. Even if people take the same amount of medicine, the same blood concentration cannot be obtained. It is said that the drug blood concentration obtained by taking the same amount of drug will be 5 times different. Some drugs are 30 times different. When a drug is administered, if the blood concentration during administration is maintained within the effective range, the drug can be used safely and effectively.
  • the drug blood concentration is below the effective range of the person, and the drug effect is not exerted. turn into.
  • the effectiveness of performing drug therapy while measuring blood concentration is recognized and used in practice. This is why it is said that the drug blood concentration is more important than the dose.
  • low and high doses of TC11 were injected into the abdominal cavity of mice, and the change in blood concentration over time was measured.
  • mice Ten week old male lcr / scid mice were injected intraperitoneally with 240 ⁇ L of TC11 2.5 mg / mL (low dose) or 12.5 mg / mL (high dose). After 0, 1.5, 4, and 8 hours, the animals were fainted by ether anesthesia. EDTA was added in a 1 mL syringe to a final concentration of 4.2 mmol / L, and mice were blood collected using a 21G needle. After blood collection, the mixture was centrifuged at 2500 rpm, 15 min, 4 ° C., and the supernatant (plasma) was collected and stored at ⁇ 30 ° C. until measurement.
  • TC11 solution for preparing a calibration curve
  • 5.8 mg of TC11 was weighed and dissolved in 5.8 mL of ethanol to 1000 ⁇ g / mL (stored in a cold place).
  • 15 mL of distilled water was added to Consera (registered trademark) (Nissui Pharmaceutical) and left to stand for 30 minutes.
  • the TC11 solution was diluted with ethanol and further diluted 10-fold with a consera (0, 0.5, 1.5, 2.5, 3.5, 5 ⁇ g / mL), and used as a standard serum for preparing a calibration curve.
  • Mouse plasma that had been cryopreserved was dissolved at the time of measurement, and ethanol was added to a final concentration of 10%.
  • Sample preparation C18 type solid phase extraction cartridge SepPak (Waters) is conditioned with 500 ⁇ L of methanol and 500 ⁇ L of distilled water, loaded with 200 ⁇ L of measurement sample, washed with 500 ⁇ L of distilled water and 500 ⁇ L of 40% CH 3 CN Then, elution was performed with 1 mL of 100% CH 3 CN. The eluate was evaporated under reduced pressure using an evaporator, redissolved in 1 mL of a mobile phase for HPLC, and centrifuged at 20400 g, 10 min, 25 ° C.
  • HPLC system used was JASCO pump PU-980, autosampler AS-950, column oven CO-2060Plus, UV detector UV-970, and fluorescence detector FP-1520S.
  • the company's ChromNAV was used as the data processor.
  • a calibration curve of TC11 (relationship between TC11 concentration ⁇ g / mL and HPLC peak height) is shown in FIG.
  • FIG. 16 shows the results of comparing the blood concentration (converted to ⁇ M) between the case where TC11 was administered at a low dose and the case where a high dose was administered into the abdominal cavity of a mouse.
  • Low dose administration is 240 ⁇ L of a 2.5 mg / mL solution of TC11, ie 20 mg / mouse kg
  • high dose administration is 240 ⁇ L of a 12.5 mg / mL solution of TC11, ie 100 mg / mouse kg).
  • the blood concentration of TC11 reached 7 ⁇ M 1.5 hours after administration, and almost disappeared after 4 hours.
  • the blood concentration of TC11 was estimated to reach 33 ⁇ M 2 hours after administration, decreased to 4 ⁇ M after 4 hours, and almost disappeared after 9 hours. Since the IC50 value of TC11 for bone marrow tumor cell KMS-34 is 3.5 ⁇ M, an effective blood concentration equal to or higher than the IC50 value was maintained up to about 2 hours after administration even at a low dose. Further, in the high dose administration of 5 times the low dose administration, an effective blood concentration of IC50 value or more was maintained until about 4 hours after administration. From the above results, the blood kinetics of TC11 is quite good, and this result is supported by the fact that the antitumor effect of TC11 using a bone marrow tumor cell KMS34 cancer-bearing mouse was also found to be significant.
  • p53 tumor suppressor gene encodes nuclear protein p53 consisting of 393 amino acids.
  • the main molecular function of p53 is a transcriptional activator, which is activated by post-translational modifications such as phosphorylation and acetylation due to various cellular stresses such as DNA damage and oncogene signals, and then transcription of multiple downstream genes. It binds to a specific base sequence [(5′-PuPuPuC (A / T) (T / A) GPyPyPy-3 ′)] ⁇ 2 existing in the regulatory region to activate transcription.
  • p53 is a core domain (DNA binding domain) that occupies residues 100-300 of the central part of p53 protein important for base sequence-specific DNA binding, its transcriptional activation domain on the N-terminal side and C-terminal side, In addition to the tetramerization domain, there are phosphorylation and acetylation residues and many protein binding sites, which are considered to be important sites for p53 function regulation. To date, p53 mutations have been reported in many human cancers, and it is known that about 50% of all human cancers have p53 mutations depending on the type of tumor (Hussain SP, Harris CC.
  • bortezomib and lenalidomide have been reported to prolong survival in patients with chromosome 13 deletion and t (4; 14) translocation, which have been considered poor in chemotherapy and autologous hematopoietic stem cell transplantation. They should be used from an early stage.
  • the p53 gene is present on chromosome 17, and myeloma having the deletion and mutation cannot be compared with existing therapies. Therefore, with the primary goal of overcoming cases with p53 abnormalities that have been the barriers to current treatment by developing new drugs, the p53 gene deficiency of KMS34, a patient-derived myeloma cell line, I examined mutations.
  • the FISH probes were purchased from Abbott Japan (Tokyo) from VYSIS LSI P53 (17P13.1) SPECTRUMORANGE PROBE (Catalog Number 32-190008) and CEP 17 (D17Z1) SPECTRUMGREEN PROBE (Catalog Number 32-132017).
  • the deletion of p53 gene was analyzed by FISH method.
  • SpectrumRed labeled p53 DNA probe (LSI p53; Vysis, Downers Grove, IL) is located on chromosome 17p13.1 and is p53 locus specific.
  • Spectrum Green-labeled chromosome 17-satellite-DNA-centromere probe CEP17, Vysis
  • Red / green signal ratio of 2R2G (p53 / CEP17 signal is 2/2) is normal, 1R2G (p53 / CEP17 signal is 1/2), or 1R1G (p53 / CEP17 signal is 1/1) is judged to have p53 deletion did. All cells were counted per 1000 cells. The results are shown in Table 1. As a result, it was found that K53, KMS28, KMS26, KMS11, and KMM1 had p53 deletion in one allele, and KMS27 and KMS21 had no p53 allele deletion.
  • a drug exhibiting strong growth inhibition against various myeloma cell lines derived from Japanese myeloma patients.

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Abstract

La présente invention concerne un agent antitumoral qui contient, en tant que substance active, un dérivé de thalidomide choisi parmi les composés décrits ci-dessous.
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