WO2011052748A1 - 抗がん剤の感受性判定方法 - Google Patents
抗がん剤の感受性判定方法 Download PDFInfo
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- WO2011052748A1 WO2011052748A1 PCT/JP2010/069362 JP2010069362W WO2011052748A1 WO 2011052748 A1 WO2011052748 A1 WO 2011052748A1 JP 2010069362 W JP2010069362 W JP 2010069362W WO 2011052748 A1 WO2011052748 A1 WO 2011052748A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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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
- A61K31/435—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
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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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
- A61K31/495—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
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to an anticancer drug sensitivity determination marker used for determining whether or not a cancer of a target patient has therapeutic reactivity to an anticancer drug to be used, and its application.
- anticancer agents such as alkylating agents, platinum preparations, antimetabolites, anticancer antibiotics, and anticancer plant alkaloids. These anticancer agents may or may not be effective depending on the type of cancer. However, it is known that some types of cancer that are recognized as effective may or may not be effective depending on the individual patient. Whether or not an anticancer drug shows an effect on such individual patient's cancer is called anticancer drug sensitivity.
- CPT-11 Irinotecan hydrochloride
- CPT-11 is an anticancer agent developed in Japan and having an action mechanism of topoisomerase I inhibition.
- CPT-11 was approved in January 1994 as an effective drug for non-small cell lung cancer, small cell lung cancer, cervical cancer and ovarian cancer, and in July 1995, gastric cancer, colorectal cancer, breast cancer Indications for squamous cell carcinoma and malignant lymphoma are recognized.
- CPT-11 occupies the position of a global standard therapeutic agent as a first-line drug or second-line drug for multi-drug combination therapy particularly in the region of colorectal cancer, and its usefulness has been recognized (Non-patent Documents 1 to 6). .
- Chemotherapy for advanced and metastatic colorectal cancer is centered on key drugs such as CPT-11 and oxaliplatin, which appeared in the 1990s, and fluorouracil (5-FU), which has been a central treatment for colorectal cancer.
- the combined use with the fluorinated pyrimidine preparation dramatically improved clinical outcomes including survival.
- the response rate is still around 50%, and in the current situation, half of the patients who received anticancer drugs at the high risk of serious side effects have not been effective.
- CPT-11 itself has antitumor activity, it is activated by carboxyesterase in the body and has an antitumor activity of about 100 to several thousand times stronger than CPT-11 (7-ethyl-10-hydroxycamptothecin (SN-38)). ). It is considered that CPT-11 and SN-38 are present in the body at the same time to exhibit an antitumor effect.
- SN-38 is glucuronidated by UDP-glucuronosyltransferase (UGT) in hepatocytes to become a non-cytotoxic SN-38 glucuronide conjugate (SN-38G), mainly in bile It is excreted and transferred to the intestinal tract, and then excreted in the stool.
- UDP-glucuronosyltransferase UDP-glucuronosyltransferase
- SN-38G non-cytotoxic SN-38 glucuronide conjugate
- a part of SN-38G excreted in the intestinal tract is deconjugated by ⁇ -glucuronidase contained in the intestinal bacterium to become active SN-38 again, and is reabsorbed via the intestinal epithelial transporter, and enterohepatic circulation, intestinal epithelium. It undergoes metabolism and excretion through steps such as glucuronidation by UGT in cells (Non-patent Document 7).
- SN-38 is thought to damage the intestinal mucosa and induce diarrhea. It also affects the bone marrow where cell division is active and has been shown to cause erythrocyte depletion, leukopenia and thrombocytopenia.
- Tumor-side factors involved in CPT-11 sensitivity or resistance include the presence or absence of the mutation and expression level of SN-38 target topoisomerase I, and carboxylesterase activity involved in the conversion of CPT-11 to SN-38 (non- (Patent Document 9), reports on the involvement of transporters (multidrug resistance protein (MRP) -1, MRP-2, breast cancer resistant protein (BCRP) / ABCG2) affecting the amount of intracellular accumulation of CPT-11 and SN-38
- MRP multidrug resistance protein
- BCRP breast cancer resistant protein
- / ABCG2 cell proliferation antigen Ki-67, tumor suppressor gene p53, and the like have also been investigated for their association with the responsiveness to treatment with CPT-11.
- Non-patent Document 10 plasma levels of Tissue inhibitor of metalloproteinase-1 (TIMP-1), which has anti-apoptotic effects, may be significantly correlated with the clinical prognosis of CPT-11 + 5-FU combination therapy for metastatic colorectal cancer Recently reported (Non-Patent Document 11).
- Tissue inhibitor of metalloproteinase-1 Tissue inhibitor of metalloproteinase-1
- 5-FU is a fluorinated pyrimidine anticancer drug developed in 1957 and is still a basic drug for digestive cancer chemotherapy.
- 5-FU taken up by cancer cells has the main mechanism of action of inhibition of thymidylate synthase (TS) caused by active metabolite fluorodeoxyuridine-5'-monophosphate (FdUMP), and also has an active metabolism. It exhibits a cell-killing effect due to RNA dysfunction caused by 5-fluorouridine triphosphate (FUTP).
- TS thymidylate synthase
- FdUMP active metabolite fluorodeoxyuridine-5'-monophosphate
- FUTP 5-fluorouridine triphosphate
- DPD dihydropyrimidine dehydrogenase
- 5-FU fluorinated pyrimidine anticancer agents
- thymidylate synthase which is a target enzyme of active metabolites
- TS tumors that highly express DPD, which is the rate-determining enzyme of 5-FU catabolism, have been reported to be 5-FU resistant (Non-patent Document 12), but there are many verification reports using clinical specimens. Absent.
- TS expression it has been reported that the amount of TS expression can be a therapeutic effect determinant of fluorinated pyrimidine anticancer agents regardless of measurement methods such as enzyme activity, protein and RNA level (Non-patent Documents 13 and 14).
- the results are not necessarily consistent in all reports, and there is no known clear biomarker that predicts treatment responsiveness to 5-FU at an early stage of treatment.
- An object of the present invention is to provide an anticancer drug sensitivity determination marker capable of discriminating treatment responsiveness of individual patients and a new cancer treatment means using the same.
- the present inventors cultured human cancer cell lines and measured changes in intracellular protein over time after drug exposure using a surface enhanced laser desorption / ionization time-of-flight mass spectrometer (SELDI-TOF MS).
- SELDI-TOF MS surface enhanced laser desorption / ionization time-of-flight mass spectrometer
- a peak was found in which the intracellular expression level in highly sensitive cells increased after 5-FU exposure, and the peak was determined as a peak of m / z 16,450 to 16,620 in the main body or a fragment thereof in the mass spectrometer. The protein was found to be detected.
- a peak was found in which the intracellular expression level in highly sensitive cells increased, and this peak was the peak of m / z 16,450-16,620 in the main body or a fragment thereof in the mass spectrometer.
- the main body or a fragment thereof was found to be a protein detected as a peak at m / z 22,080-22,310.
- a peak was found in which the intracellular expression level increased in highly sensitive cells, and the peak was found to be m / z 16,450-16 in the mass spectrometer in the main body or a fragment thereof. , 620, and a peak in which the intracellular expression level in a low-sensitivity cell increases, and the peak is m / z 17,100 in the mass spectrometer. It was found that the protein was detected as a peak at ⁇ 17,270. In addition, a peak having a different intracellular expression level before drug exposure was found in high-sensitivity cells and low-sensitivity cells, and the peak was determined to be m / z 16,450 to 16,620 in the mass spectrometer.
- the protein was detected as a peak, the protein was detected as a peak at m / z 17,100-17,270, and the protein was detected as a peak at m / z 22,080-22,310.
- the cancer of the cancer patient can be identified as 5-FU, SN-38, or 5-FU / Whether or not it has sensitivity to SN-38 combination therapy can be determined, and if suppression of the expression of this substance is used as an index, screening for an anticancer drug sensitivity-enhancing agent can be performed.
- the present inventors have found that the therapeutic effect of the anticancer agent can be drastically improved by using a cancer drug sensitivity enhancing agent in combination with the anticancer agent that is the subject of sensitivity enhancement, and the present invention has been completed.
- the main body or a fragment thereof is detected as a peak at m / z 16,450-16,620 (hereinafter referred to as protein A), the main body or a fragment thereof is m / z 22.
- protein A Proteins detected as peaks at 080 to 22,310
- protein C proteins whose main body or fragments thereof are detected as peaks at m / z 17,100 to 17,270
- An anticancer drug sensitivity determination marker comprising a protein selected from The present invention also provides a method for determining sensitivity to an anticancer agent, comprising measuring protein A, protein B, and protein C in a specimen.
- the present invention also provides a kit for carrying out a method for determining sensitivity to an anticancer drug, comprising a protocol for measuring protein A, protein B, and protein C in a specimen. . Furthermore, the present invention provides a screening method for an anticancer drug sensitivity enhancer using the expression variation of protein A, protein B, and protein C as an index. Furthermore, this invention provides the anticancer agent sensitivity enhancer obtained by said screening method. Furthermore, this invention provides the composition for cancer treatment which combines said anticancer agent sensitivity enhancer and the anticancer agent used as the object of sensitivity enhancement. Furthermore, this invention provides the protein A, protein B, and protein C for determining anticancer agent sensitivity.
- the anticancer drug sensitivity determination marker of the present invention it is possible to accurately determine the anticancer drug sensitivity of an individual patient before the start of treatment or early after the start of the treatment. Is possible. Furthermore, unnecessary side effects can be avoided because it is possible to avoid the use of anticancer agents that are not effective. In addition, because treatment schedules using anticancer drugs are long-term, the sensitivity of anticancer drugs to cancer over time can be determined by determining anticancer drug sensitivity for each treatment cycle even during treatment. Evaluation is possible and it is possible to determine whether or not to continue treatment.
- the measurement reagent of the anticancer drug sensitivity determination marker of the present invention is useful as an anticancer drug sensitivity determination reagent.
- Anticancer drug sensitivity determination markers in the present invention are proteins A to C, and more specifically, using a cation exchange chip in a surface enhanced laser desorption / ionization time-of-flight mass spectrometer (SELDI-TOF MS).
- a cation exchange chip in a surface enhanced laser desorption / ionization time-of-flight mass spectrometer SELDI-TOF MS.
- the main body or a fragment thereof is detected as a peak of m / z 16,450-16,620 (protein A), and the main body or a fragment thereof is m / z 22 .
- Protein detected as a peak at 080 to 22,310 protein B
- protein C a protein whose main body or fragment thereof is detected as a peak at m / z 17,100 to 17,270
- protein A was obtained by examining intracellular protein expression of cultured cancer cells using SELDI-TOF MS. As a result, 5-FU, SN-38, and 5-FU / SN-38 were combined. In HCT116, which is highly sensitive to any of the above, an increase in intracellular level was observed after 5-FU, SN-38, and 5-FU / SN-38 combined exposure. On the other hand, no significant change was observed in the intracellular level of DLD-1, which is hyposensitive to any of 5-FU, SN-38, and 5-FU / SN-38 combination. Therefore, protein A is useful as an anticancer drug sensitivity determination marker, particularly as an anticancer drug sensitivity determination marker when 5-FU, SN-38, and 5-FU / SN-38 are used in combination.
- Protein B is highly sensitive to SN-38 after exposure to SN-38 as a result of examining intracellular protein expression in cultured cancer cells using SELDI-TOF MS, as shown in the Examples below. An increase in intracellular levels was observed with HCT116. On the other hand, DLD-1, which is less sensitive to SN-38, showed no significant change or showed a downward trend. Therefore, protein B is useful as an anticancer agent sensitivity determination marker, particularly as an anticancer agent sensitivity determination marker for SN-38.
- protein C was obtained by examining intracellular protein expression of cultured cancer cells using SELDI-TOF MS. As a result, 5-FU / SN-38 was exposed to 5-FU / SN after exposure with 5-FU / SN-38. Increased intracellular levels were observed with DLD-1, which is less sensitive to the -38 combination. On the other hand, no significant change was observed in HCT116, which is highly sensitive to the 5-FU / SN-38 combination. Therefore, protein C is useful as an anticancer agent sensitivity determination marker, particularly as an anticancer agent sensitivity determination marker when 5-FU / SN-38 is used in combination.
- proteins A to C are highly sensitive to any of 5-FU, SN-38, and 5-FU / SN-38 combination before drug exposure in HCT116 (when not exposed).
- the intracellular level of was significantly higher than that of DLD-1, which is less sensitive to any of the above drugs. Therefore, proteins A to C are useful as anticancer agent sensitivity determination markers, particularly as anticancer agent sensitivity determination markers in combination with 5-FU, SN-38, and 5-FU / SN-38.
- an anticancer agent used as the target of the anticancer agent sensitivity determination marker of this invention for example, oxaliplatin, cyclophosphamide (ifosfamide), thiotepa (thiotepa), melphalan ( melphalan, busulfan, nimustine, ranimustine, dacarbazine (da), procarbazine, templatin, cisplatin, cisplatin, cisplatin, cisplatin, cisplatin, cisplatin methotrexate , Pemetrexed, fluorouracil, tegafur / uracil (tegaful erac), doxyfluridine, tegafur / gimeracil / citabin, tegafur / citabin enocitabine, gemcitabine, 6-mercaptopurine, fludarabine, pentostatin, cladribine, hydroxyurea, hydroxyurea
- fluorinated pyrimidine-based anticancer agents and plant alkaloid-based anticancer agents are preferable, and fluorouracil, irinotecan, SN-38 or salts thereof are particularly preferable. Furthermore, it can be suitably used in a combination agent of fluorouracil or a salt thereof and irinotecan, SN-38 or a salt thereof.
- the proteins A to C in the sample may be measured.
- a biological sample derived from a subject having cancer such as blood, serum, plasma, a cancer tissue biopsy specimen, a cancer excision surgical specimen, stool, urine, ascites, pleural effusion, Although cerebrospinal fluid, sputum, etc. are mentioned, serum is particularly preferable.
- lip, oral and pharyngeal cancer typified by pharyngeal cancer
- digestive organ cancer typified by esophageal cancer
- stomach cancer colon / rectal cancer
- lung cancer Respiratory and intrathoracic organ cancer, bone and joint cartilage cancer, cutaneous malignant melanoma, squamous cell carcinoma and other skin cancers, mesothelioma and mesothelioma Tissue cancer, breast cancer, uterine cancer, female genital cancer represented by ovarian cancer, male genital cancer represented by prostate cancer, urinary tract cancer represented by bladder cancer, brain tumor Representative eye, brain and central nervous system cancer, thyroid and other endocrine adenocarcinoma, non-Hodgkin lymphoma, lymphoid leukemia, lymphoid tissue, hematopoietic tissue and related tissue cancer, and these as the primary focus Cancers of metastatic tissues, etc., especially gastric cancer and colorec
- the measuring means for the proteins A to C in the specimen can be measured by, for example, SELDI-TOF MS, immunological measurement method, or the like.
- an immunoassay using an anti-protein A to C antibody is preferable.
- the anti-protein A to C antibody used may be a monoclonal antibody or a polyclonal antibody. More specifically, for example, radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, luminescence immunoassay, immunoprecipitation method, immunoturbidimetric method, Western blot, immunostaining, immunodiffusion method, etc. can be mentioned, preferably Western blot
- an enzyme immunoassay is particularly preferred, such as Western blotting and enzyme-linked immunosorbent assay (ELISA) (for example, sandwich ELISA).
- the concentration of protein A and protein B in a biological sample derived from a cancer patient before and after administration of the anticancer agent is measured.
- the concentrations of protein A and protein B before and after administration of the anticancer agent show a tendency to increase, the cancer is sensitive to the anticancer agent, and protein A after administration compared to before administration of the anticancer agent.
- the concentration of protein B does not change or decreases, it can be determined that the cancer is not sensitive to an anticancer agent. That is, when the concentrations of protein A and protein B have a concentration that is determined to be higher than the predetermined standard concentration at an early stage after administration, the cancer is sensitive to the target anticancer agent.
- the cancer Since it can be determined, it can be used as a marker for actively continuing treatment for a patient who can expect a therapeutic effect.
- concentrations of protein A and protein B are determined to be lower than a predetermined standard concentration, the cancer is not sensitive to the anticancer drug of interest. Can be determined. If there is no sensitivity to the targeted anticancer drug, its efficacy cannot be expected, and if the administration of an anticancer drug that cannot be expected to have such efficacy is continued, There is concern about progression and increased side effects.
- the anticancer drug sensitivity determination marker in the present invention is not only a determination of anticancer drug treatment responsiveness, but also an increase in cancer progression and side effects associated with continuous administration of an anticancer drug that cannot be expected to have a medicinal effect. It can also be used as a marker for avoiding.
- the target anticancer agent is a combination of 5-FU / SN-38
- cancer patients before and after administration of the anticancer agent
- the concentration of protein C after administration does not change or decreases compared to before, it can be determined that the cancer is sensitive to an anticancer agent. That is, if the concentration of protein C has a concentration that is determined to be lower than the predetermined standard concentration at an early stage after administration, it can be determined that the cancer is sensitive to the target anticancer agent.
- the anticancer drug sensitivity determination marker in the present invention is not only a determination of anticancer drug treatment responsiveness, but also an increase in cancer progression and side effects associated with continuous administration of an anticancer drug that cannot be expected to have a medicinal effect. It can also be used as a marker for avoiding.
- the target anticancer agent is a combination of 5-FU, SN-38, and 5-FU / SN-38
- the target anticancer agent is a combination of 5-FU, SN-38, and 5-FU / SN-38
- the concentration of these proteins is determined to be lower than the prescribed standard concentration, the cancer is the target. It can be determined that it is not sensitive to anticancer drugs. When there is no sensitivity to the target anticancer agent, it is not possible to expect its medicinal effect, and it is considered that only side effects due to the anticancer agent are expressed.
- the determination marker can also be used as a marker for avoiding the development of unnecessary side effects or the progression of cancer and the increase in side effects associated with continued invalid treatment.
- concentration of these proteins is determined to be higher than a predetermined standard concentration, it can be determined that the cancer is sensitive to the target anticancer agent, and thus a therapeutic effect is expected. It can also be used as a marker to actively select patients who can do it.
- protein C as a marker during administration of an anticancer agent, if the protein concentration after administration does not change or decreases compared to before administration of the anticancer agent, the cancer is anticancer. If it has a concentration that is judged to be lower than a predetermined standard concentration as a marker before administration of an anticancer drug, it can be determined that the cancer is not anticancer drug sensitive . Since the positioning as a marker is different during the administration of the anticancer agent or before the administration of the anticancer agent, it is preferable to use it with attention to that point.
- kits including a protocol for measuring proteins A to C in a specimen.
- the kit includes a measurement reagent for proteins A to C, a method for using the measurement reagent, a standard for determining the presence or absence of anticancer drug sensitivity, and the like.
- the standards include standard concentrations of proteins A to C, concentrations judged to be high, concentrations judged to be low, factors affecting the measurement results, and the degree of their influence, etc. It is possible to set for each anticancer drug. Using the reference, it can be determined as described above.
- anti-cancer drug sensitivity-enhancing agents can be screened using the expression fluctuation of these proteins, specifically, the increase in expression as an index. That is, a substance that increases the expression of protein A and protein B in vitro or in vivo enhances anticancer drug sensitivity.
- a substance that increases the expression of protein A and protein B in vitro or in vivo enhances anticancer drug sensitivity.
- substances that increase the concentrations of protein A and protein B in the presence of anticancer drugs in various cancer cell lines are substances that enhance the sensitivity of the anticancer drug (anticancer drug sensitivity).
- Enhancer substances that increase the concentration of protein A and protein B before and after administration of an anticancer drug in cancer-bearing animals are substances that increase the sensitivity of the anticancer drug (enhanced anticancer drug sensitivity).
- Agent for example, in vitro, substances that increase the concentrations of protein A and protein B in the presence of anticancer drugs in various cancer cell lines are substances that enhance the sensitivity of the anticancer drug (antican
- the anticancer agent sensitivity enhancer Can be screened. That is, a substance that suppresses the expression of protein C in vitro or in vivo enhances anticancer drug sensitivity.
- a substance that decreases the concentration of protein C in the presence of an anticancer agent in various cancer cell lines is a substance that enhances the sensitivity of the anticancer agent (anticancer agent sensitivity enhancer). It is.
- a substance that enhances the decrease in protein C concentration before and after administration of an anticancer agent in a cancer-bearing animal is a substance that enhances the sensitivity of the anticancer agent (anticancer agent sensitivity enhancer). is there.
- anticancer drug sensitivity-enhancing agents can be screened. That is, a substance that increases protein expression in vitro or in vivo enhances anticancer drug sensitivity. For example, in vitro, a substance that increases the protein concentration in the absence of an anticancer drug in various cancer cell lines is a substance that enhances the sensitivity of the anticancer drug (anticancer drug sensitivity enhancer). It is.
- the substance that enhances the increase in protein concentration is a substance that enhances the sensitivity of the anticancer agent (anticancer agent sensitivity enhancer).
- anticancer agent sensitivity enhancer since the behavior of protein C varies depending on whether the anticancer agent is exposed or not exposed to the anticancer agent, it is preferable to screen for an anticancer agent sensitivity-enhancing agent with this point in mind.
- the combined form of the anticancer agent sensitivity-enhancing agent and the anticancer agent targeted for sensitivity enhancement may be a single composition containing both of these components. There may be. In addition, these components may be different administration routes.
- Anticancer agents to be used here are the same as described above, and are oxaliplatin, cyclophosphamide, ifosfamide, thiotepa, melphalan, busulfan.
- fluorinated pyrimidine-based anticancer agents and plant alkaloid-based anticancer agents are preferable, and fluorouracil, irinotecan, SN-38 or salts thereof are particularly preferable.
- a concomitant drug of fluorouracil or a salt thereof and irinotecan, SN-38 or a salt thereof can be suitably used.
- Example 1 (1) Method (a) Cells used Two types of human colorectal cancer cell lines (HCT-116, DLD-1) were obtained from ECACC. The culture was performed under conditions of ⁇ 100 mm / Tissue Culture Dish (IWAKI), medium (D-MEM, 2 mM Glutamine, 10% Fetal Bovine Serum), 37 ° C., 5% CO 2 .
- IWAKI Tissue Culture Dish
- D-MEM 2 mM Glutamine, 10% Fetal Bovine Serum
- the drug exposure conditions are as follows: Control (0 ⁇ M) as 5-FU single agent, 11 conditions of 0.001 ⁇ M, 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 3 ⁇ M, 10 ⁇ M, 30 ⁇ M, 100 ⁇ M, 1000 ⁇ M, and 10,000 ⁇ M, as SN-38 single agent 11 conditions of Control (0 nM), 0.001 nM, 0.01 nM, 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, and 1000 nM were used.
- 5-FU 2 ⁇ M, 10 ⁇ M, 30 ⁇ M, and 100 ⁇ M were used as a 5-FU single agent at the same concentration as each combination condition.
- Sensitivity was evaluated by measuring three samples for each cell line, drug exposure time, and drug exposure condition in one test, and performing three times with cells of different passage numbers. The analysis was performed based on the survival rate calculated from the result of MTS assay.
- the survival rate at the time of combination was compared with the survival rate at the time of single agent exposure of each drug at the same concentration as used at the time of combination. If it decreased, it was determined that there was a combined effect.
- (D) Drug exposure experiment Based on the results of (c) above, the drug concentration used in the exposure experiment is the same as that used in the combination exposure of 5-FU + SN-38: 2 ⁇ M + 10 nM, 2 ⁇ M + 100 nM, 100 ⁇ M + 10 nM, 100 ⁇ M + 100 nM.
- the exposure experiment was conducted under the conditions of 5-FU for 2 ⁇ M and 100 ⁇ M, SN-38 for 10 nM and 100 nM, and all of these conditions including drug-free control added to each concentration of each drug alone.
- the drug exposure time was set to four types immediately before exposure (0 hour), 4 hours, 24 hours, and 48 hours, and the number of cells at the end of the exposure was counted to extract intracellular proteins.
- HCT116 was determined to be a highly sensitive cell for 5-FU / SN-38 combination, and DLD-1 was determined to be a low sensitive cell (FIG. 1).
- Example 2 In Example 1, as a main body or a fragment thereof, a protein (protein A) detected as m / z 16,450-16,620, a protein (protein B) detected as m / z 22,080-22,310 , M / z 17,100 to 17,270, a standard substance having a known molecular weight, Cytochrome c (equine) (m / z 123600.96 + 1H), Apomyoglobin (equine) (m / z 16952.27 + 1H), Aldorase (reabbit muscle) (m / z 39212.28 + 1H), and molecular weight correction (internal calibration) using two molecular weights of two kinds of standard substances sandwiching each target peak.
- Cytochrome c equine
- Apomyoglobin equine
- Aldorase reabbit muscle
- m z 39212.28 + 1H Aldorase
- Example 1 the detected protein A, protein B, and protein C were m / z 16, 450 to 16,620, m / z 22,080 to 22,310, m / z 17, respectively. It was confirmed that it was detected between 100 and 17,270.
- Example 3 In Example 1, m / z 16,450-16,620 (protein A), m / z 22,080-22,310 (protein B), m / z 17,100-17,270 (protein C) In order to investigate the property of the protein detected as a peak in more detail, changes in peak intensity accompanying changes in pH were examined.
- the pH at which the decrease in peak intensity is recognized is near the isoelectric point (pI) at which the protein ionization is lost.
- the isoelectric point (pI) of the peak detected in Example 1 is pH 4.5 to 7.5 and m / z 22,080 to 22 for m / z 16,450 to 16,620 (protein A).
- 310 (protein B), pH 4.0 to 7.0, and m / z 17, 100 to 17, 270 (protein C) were estimated to correspond to pH 5.0 to 8.0 in the vicinity of pI.
- Example 4 The main body or a fragment thereof found in Example 1 is a protein having m / z 16,450-16,620 (protein A), a protein having m / z 22,080-22,310 (protein B), m / z 17 , 100-17,270 proteins (Protein C), based on the information of Examples 2 and 3, using The ExPASy proteomics server TagIdent tool (http://au.expasy.org/tools/tagient.html). Database search. The results were as follows.
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Abstract
Description
進行性・転移大腸癌に対する化学療法は、1990年代に登場したCPT-11、オキサリプラチンなどのkey drugと、それまで大腸癌治療の中心的薬剤であったフルオロウラシル(5-FU)を中心とするフッ化ピリミジン製剤とを併用することにより、生存率をはじめとする臨床成績が劇的に改善された。しかしそれでもなお奏効率はおよそ50%程度であり、重篤な副作用という高リスクを冒して抗がん剤が投与された患者の半分では効果が得られていないのが現状であり、個々の治療反応性(レスポンダー・ノンレスポンダー)を判別する抗がん剤感受性予測マーカーの確立は急務である。
重篤な下痢や好中球減少症などの副作用は、UGT1A1遺伝多型がもたらすSN-38体内曝露量の変化が一因であることが示されている。しかし治療効果に関しては、プロドラッグであるCPT-11から活性代謝物SN-38への変換とその解毒、さらに腸管循環の過程におけるSN-38の再生成や、CPT-11自体の代謝と代謝物からのSN-38の生成といった体内動態の複雑性により、また、抗腫瘍効果が腫瘍側の要因により規定されることが多いため、薬物動態により治療効果を予測できるとする報告は未だなされていない。末梢血単核球細胞のカルボキシルエステラーゼmRNA発現量がSN-38とSN-38GのAUC比とは相関するものの腫瘍縮小効果とは相関がなかったとする報告もなされている(非特許文献8)。
しかし、すべての報告において必ずしも一致する結果は得られておらず、さらに5-FUに対する治療反応性を治療早期に予測する明確なバイオマーカーは知られていない。
さらに、一般にがん化学療法の治療スケジュールは長期に渡るため、治療継続中における抗がん剤に対する感受性の経時的モニターは治療継続の是非の判定を可能とし、患者負担や副作用軽減につながるのみならず医療経済の観点からも有用であると考えられる。個々の患者における治療反応性を予測、そして早期に診断して適切な薬剤や治療レジメンを選択する「個別化治療」実現のためには、5-FU、CPT-11、及び5-FU/CPT-11併用時の効果予測もしくは治療応答性の早期診断を可能とするバイオマーカーの確立は急務である。
同様に、SN-38曝露後に、高感受性細胞における細胞内発現レベルが上昇するピークを見出し、そのピークが、質量分析計において、本体もしくはそのフラグメントがm/z 16,450~16,620のピークとして検出されるタンパク質、あるいは本体もしくはそのフラグメントがm/z 22,080~22,310のピークとして検出されるタンパク質であることを見出した。
さらに、5-FU/SN-38併用曝露後に、高感受性細胞における細胞内発現レベルが上昇するピークを見出し、そのピークが、質量分析計において、本体もしくはそのフラグメントがm/z 16,450~16,620のピークとして検出されるタンパク質であること、また、低感受性細胞における細胞内発現レベルが上昇するピークを見出し、そのピークが、質量分析計において、本体もしくはそのフラグメントがm/z 17,100~17,270のピークとして検出されるタンパク質であることを見出した。
また、高感受性細胞と低感受性細胞において、薬剤曝露前の細胞内発現レベルが異なるピークを見出し、そのピークが、質量分析計において、本体もしくはそのフラグメントがm/z 16,450~16,620のピークとして検出されるタンパク質、m/z 17,100~17,270のピークとして検出されるタンパク質、m/z 22,080~22,310のピークとして検出されるタンパク質であることを見出した。
かかる知見に基づき、さらに検討した結果、がん患者由来の生体試料中の当該タンパク質の濃度を測定すれば、当該がん患者のがんが、5-FU、SN-38、或いは5-FU/SN-38の併用療法に対して感受性を有するか否かを判定できること、また、この物質の発現抑制を指標とすれば抗がん剤感受性亢進剤のスクリーニングが可能になること、さらに当該抗がん剤感受性亢進剤と感受性亢進の対象となる抗がん剤を併用すれば、当該抗がん剤の治療効果が飛躍的に向上することを見出し、本発明を完成した。
また、本発明は、検体中のタンパク質A、タンパク質B、タンパク質Cを測定することを特徴とする抗がん剤感受性の判定方法を提供するものである。
また、本発明は、検体中のタンパク質A、タンパク質B、タンパク質Cを測定するためのプロトコールを含むことを特徴とする抗がん剤感受性の判定方法を実施するためのキットを提供するものである。
さらに本発明は、タンパク質A、タンパク質B、タンパク質Cの発現変動を指標とする抗がん剤感受性亢進剤のスクリーニング方法を提供するものである。
さらにまた本発明は、上記のスクリーニング方法により得られた抗がん剤感受性亢進剤を提供するものである。
さらに本発明は、上記の抗がん剤感受性亢進剤と感受性亢進の対象となる抗がん剤を組み合わせてなるがん治療用組成物を提供するものである。
さらに本発明は、抗がん剤感受性を判定するためのタンパク質A、タンパク質B、タンパク質Cを提供するものである。
さらに、また、このマーカーを用いれば、抗がん剤感受性を亢進させる薬剤がスクリーニングでき、その対象となった抗がん剤と抗がん剤感受性亢進剤とを併用すれば、がん治療効果が飛躍的に向上する。本発明の抗がん剤感受性判定マーカーの測定試薬は、抗がん剤感受性判定試薬として有用である。
また、投与後早期の段階において、タンパク質A、タンパク質Bの濃度が所定の標準濃度より低いと判断される濃度を有する場合は、そのがんは対象とする抗がん剤に対して感受性ではないと判定できる。対象とする抗がん剤に対して感受性を有さない場合は、その薬効を期待することができず、このような薬効の期待できない抗がん剤の投与が続けられた場合、がんの進行、副作用の増大が危惧される。このように、本発明における抗がん剤感受性判定マーカーは、抗がん剤治療反応性の判定のみならず、薬効の期待できない抗がん剤の継続投与に伴うがんの進行や副作用の増大を回避するためのマーカーとしても使用できる。
また、投与後早期の段階において、タンパク質Cの濃度が所定の標準濃度より高いと判断される濃度を有する場合は、そのがんは対象とする抗がん剤に対して感受性ではないと判定できる。対象とする抗がん剤に対して感受性を有さない場合は、その薬効を期待することができず、このような薬効の期待できない抗がん剤の投与が続けられた場合、がんの進行、副作用の増大が危惧される。このように、本発明における抗がん剤感受性判定マーカーは、抗がん剤治療反応性の判定のみならず、薬効の期待できない抗がん剤の継続投与に伴うがんの進行や副作用の増大を回避するためのマーカーとしても使用できる。
また、これらのタンパク質の濃度が所定の標準濃度より高いと判断される濃度を有する場合は、そのがんは対象とする抗がん剤に対して感受性を有すると判定できるため、治療効果を期待できる患者を積極的に選出するためのマーカーとしても使用できる。
ここで用いられる対象となる抗がん剤としては、前記と同様であり、オキサリプラチン、シクロフォスファミド(cyclophosphamide)、イフォスファミド(ifosfamide)、チオテパ(thiotepa)、メルファラン(melphalan)、ブスルファン(busulfan)、ニムスチン(nimustine)、ラニムスチン(ranimustine)、ダカルバジン(dacarbazine)、プロカルバジン(procarbazine)、テモゾロミド(temozolomide)、シスプラチン(cisplatin)、カルボプラチン(carboplatin)、ネダプラチン(nedaplatin)、メトトレキサート(methotrexate)、ペメトレキセド(pemetrexed)、フルオロウラシル(fluorouracil)、テガフール/ウラシル(tegaful・uracil)、ドキシフルリジン(doxifluridine)、テガフール/ギメラシル/オテラシル(tegaful・gimeracil・oteracil)、カペシタビン(capecitabine)、シタラビン(cytarabine)、エノシタビン(enocitabine)、ゲムシタビン(gemcitabine)、6-メルカプトプリン(6-mercaptopurine)、フルダラビン(fuludarabin)、ペントスタチン(pentostatin)、クラドリビン(cladribine)、ヒドロキシウレア(hydroxyurea)、ドキソルビシン(doxorubicin)、エピルビシン(epirubicin)、ダウノルビシン(daunorubicin)、イダルビシン(idarubicine)、ピラルビシン(pirarubicin)、ミトキサントロン(mitoxantrone)、アムルビシン(amurubicin)、アクチノマイシンD(actinomycin D)、ブレオマイシン(bleomycine)、ペプレオマイシン(pepleomycin)、マイトマイシンC(mytomycin C)、アクラルビシン(aclarubicin)、ジノスタチン(zinostatin)、ビンクリスチン(vincristine)、ビンデシン(vindesine)、ビンブラスチン(vinblastine)、ビノレルビン(vinorelbine)、パクリタキセル(paclitaxel)、ドセタキセル(docetaxel)、イリノテカン(irinotecan)、イリノテカン活性代謝物(SN-38)、ノギテカン(nogitecan、topotecan)、エトポシド(etoposide)、プレドニゾロン(prednisolone)、デキサメタゾン(dexamethasone)、タモキシフェン(tamoxifen)、トレミフェン(toremifene)、メドロキシプロゲステロン(medroxyprogesterone)、アナストロゾール(anastrozole)、エキセメスタン(exemestane)、レトロゾール(letrozole)、リツキシマブ(rituximab)、イマチニブ(imatinib)、ゲフィチニブ(gefitinib)、ゲムツズマブ・オゾガマイシン(gemtuzumab ozogamicin)、ボルテゾミブ(bortezomib)、エルロチニブ(erlotinib)、セツキシマブ(cetuximab)、ベバシズマブ(bevacizumab)、スニチニブ(sunitinib)、ソラフェニブ(sorafenib)、ダサチニブ(dasatinib)、パニツムマブ(panitumumab)、アスパラギナーゼ(asparaginase)、トレチノイン(tretinoin)、三酸化ヒ素(arsenic trioxide)、又はそれらの塩、又はそれらの活性代謝物等が挙げられる。このうちフッ化ピリミジン系抗がん剤、植物アルカロイド系抗がん剤が好ましく、特にフルオロウラシル、イリノテカン、SN-38又はそれらの塩が好ましい。さらに、フルオロウラシル又はその塩と、イリノテカン、SN-38又はそれらの塩との併用剤を好適に利用することができる。
(1)方法
(a)使用細胞
ヒト大腸癌細胞株2種類(HCT-116、DLD-1)は、ECACCより入手した。培養は、φ100mm/Tissue Culture Dish(IWAKI)、培地(D-MEM、2mM Glutamine、10% Fetal Bovine Serum)、37℃、5%CO2の条件下にて行なった。
フルオロウラシル(5-FU)はSigma社より購入し、SN-38原末は、株式会社ヤクルト本社より入手した。
2種類の大腸癌細胞株HCT116、DLD-1(いずれもECACCより入手)を用い、薬剤曝露48時間後、72時間後の細胞生存率をMTS assay(CellTiter96TMAQueous One Solution Cell Proliferation Assay、Promega)にて評価した。薬剤曝露条件は5-FU単剤としてControl(0μM)、0.001μM、0.01μM、0.1μM、1μM、3μM、10μM、30μM、100μM、1000μM、10000μMの11条件、SN-38単剤としてControl(0nM)、0.001nM、0.01nM、0.1nM、0.3nM、1nM、3nM、10nM、30nM、100nM、1000nMの11条件を用いた。併用としては5-FUの2μM、10μM、30μM、100μMの4濃度それぞれに、SN-38の0.001nM、0.01nM、0.1nM、0.3nM、1nM、3nM、10nM、30nM、100nM、1000nMの10濃度を加えた40条件、及び各併用条件と同濃度の5-FU単剤として4条件とControl(0μM)を用いた。感受性の評価は、1回の試験においてそれぞれの細胞株、薬剤曝露時間、薬剤曝露条件について3サンプルずつ測定し、異なる継代数の細胞にて3回実施した。
解析は、MTS assayの結果から算出した生存率をもとに行った。併用効果の有無の判定は、併用時における生存率(viability)と、併用時に用いたのと同濃度での各薬剤の単剤曝露時における生存率とを比較し、双方から有意に生存率が低下していれば併用効果があると判定した。
前記(c)の結果にもとづき、曝露実験に用いる薬剤濃度は、5-FU+SN-38の併用として2μM+10nM、2μM+100nM、100μM+10nM、100μM+100nMの4種類、これら併用曝露で用いるのと同じ濃度の各薬剤単独曝露として5-FUは2μM、100μM、SN-38は10nM、100nM、及びこれらに薬剤フリーのControlを加えた全9条件で曝露実験を行った。薬剤曝露時間は曝露直前(0時間)、4時間、24時間、48時間の4通りとし、曝露終了時点における細胞数を計測し、細胞内タンパク質を抽出した。
dishより培地を除き氷冷PBSで3回洗浄後、ラバーポリスマンにて剥がしてかき集め、細胞懸濁液を1.5mLマイクロチューブに移した。4℃にて1,200×g、10分間遠心して細胞を集め、上清を除いた後に、細胞数1000万個あたり200μLの細胞溶解バッファー(9mol/L Urea、2% CHAPS、1mM DTT、プロテアーゼインヒビターカクテル(Sigma))を加え、氷冷下で超音波破砕処理を行なった後、4℃にて16,000×g、20分間遠心し、上清を液体窒素にて急速凍結後、分析するまで-80℃にて保存した。上清の一部を用いてタンパク質定量(DC Protein Assay Kit、Bio-Rad)を行なった。
細胞溶解バッファー(プロテアーゼインヒビターを除く)にて2.5mg/mLに調製し、さらに、pH4.5の希釈/洗浄バッファー(50mM sodium acetate buffer)(以下、バッファー)にて0.5mg/mLに調製したサンプル100μLを、同バッファーにて前処理した陽イオン交換チップアレイ(CM10、Bio-Rad)のスポットにapplyし、1時間インキュベートして反応させた後、バッファーにて3回洗浄、milliQ水にて2回リンスした。風乾後、energy absorbing molecule(EAM:50% ACN/0.5% TFA溶液によるsinapinic acidの飽和溶液)1.0μLを、0.5μLずつ2回に分けて各スポットにapplyし、スポット表面が乾いた後、プロテインチップアレイの分析を行なった。
タンパク質発現解析は、表面エンハンス型レーザー脱離イオン化飛行時間型質量分析計(surface-enhanced laser desorption/ionization time-of-flight mass spectrometry:SELDI-TOF MS)にて行なった。分析機器としては、ProteinChipTM Reader(Model PCS4000 Personal Edition、Bio-Rad)を用い、Mass Range 0~70,000 Daltons、Focus Mass 8,000 Dalton、Energy 4000 nJ、1サンプルあたり合計265shotsの条件にて分析を行なった。
signal-to-noise ratio(S/N比)2以上のピークの抽出とタンパク質発現比較解析は、CiphergenExpressTM Data Manager 3.0を用いて行なった。
SELDI-TOF MSによる分析の結果、S/N比2以上の条件で各サンプルにつき112~147個のタンパク質ピークを抽出した。まず、CiphergenExpressTM Data Manager 3.0によりピーククラスターを作成した。次に、各条件において、薬剤曝露後に経時的に有意にピーク強度が変化しているピークと、各曝露時間(4、24、48時間)において、薬剤曝露条件によってピーク強度が有意に変化しているピークをピックアップした。さらに、両者に共通して検出されるピーク、すなわち曝露時間による発現変化と薬剤条件による発現変化のいずれをも認めるピークを絞り込んだ。
(a)HCT-116及びDLD-1における薬剤感受性の評価
100μMの5-FUにて48時間曝露後の細胞生存率は、HCT116で約24%、DLD-1で約49%となり、DLD-1がHCT116に比べ5-FU低感受性であると判断した。100nMのSN-38にて72時間曝露後の細胞生存率は、HCT116で約24%、DLD-1で約76%となり、DLD-1はHCT116に比べSN-38低感受性であると判断した。5-FU/SN-38併用曝露後においては、5-FU 2μM+SN-38 10nM、及び5-FU 100μM+SN-38 100nMの72時間曝露後、HCT116では各々の単剤曝露後の細胞生存率よりも併用時において有意に低い細胞生存率を示し併用効果を認めたが、DLD1では有意な併用効果は認められなかった。したがって、HCT116は5-FU/SN-38併用に対する高感受性細胞、DLD-1は低感受性細胞と判断した(図1)。
SELDI-TOF MSを用いたプロテオーム解析手法により、5-FU曝露、SN-38曝露、5-FU/SN-38併用曝露に伴う細胞内タンパク質の変動を網羅的に解析した。(1)方法で示した手法により解析した結果、各薬剤曝露後に特徴的な変動を示す以下のタンパク質を見出した。
(1)5-FU曝露後、HCT116で細胞内レベルの上昇が認められたピーク(図2)・m/z 16,450~16,620(タンパク質A)
(2)SN-38曝露後、HCT116で細胞内レベルの上昇が認められたピーク(図2、3)
・m/z 16,450~16,620(タンパク質A)
・m/z 22,080~22,310(タンパク質B)
(3)5-FU/SN-38併用曝露後、HCT116で細胞内レベルの上昇が認められたピーク(図2)
・m/z 16,450~16,620(タンパク質A)
(4)5-FU/SN-38併用曝露後、DLD-1で細胞内レベルの上昇が認められたピーク(図4)
・m/z 17,100~17,270(タンパク質C)
(5)薬剤曝露前(非曝露時)の細胞内レベルが、DLD-1と比較してHCT116で有意に高値を示したピーク
・m/z 16,450~16,620(タンパク質A)
・m/z 22,080~22,310(タンパク質B)
・m/z 17,100~17,270(タンパク質C)
タンパク質Aにおける薬剤曝露前の細胞内レベルは、SELDI-TOF MS分析によるピーク強度(μA)(平均値±S.D.、n=27)として、HCT116では15.0±3.30、DLD-1では8.59±3.49、同様にタンパク質Bでは、HCT116 5.95±1.10、DLD-1 2.99±0.81、タンパク質Cでは、HCT116 26.9±3.94、DLD-1 5.01±2.01であり、いずれもHCT116における薬剤曝露前(非曝露時)細胞内レベルはDLD-1よりも有意に高値を示した。
実施例1において、本体もしくはそのフラグメントとして、m/z 16,450~16,620として検出されたタンパク質(タンパク質A)、m/z 22,080~22,310として検出されたタンパク質(タンパク質B)、m/z 17,100~17,270として検出されたタンパク質(タンパク質C)について、分子量既知の標準物質として、Cytochrome c(equine)(m/z 12360.96+1H)、Apomyoglobin(equine)(m/z 16952.27+1H)、Aldorase(reabbit muscle)(m/z 39212.28+1H)を用い、各目的ピークを挟む2種類の標準物質の分子量2点を用いた分子量補正(internal calibration)によりm/zの確認を行った。その結果、実施例1において、検出されたタンパク質A、タンパク質B及びタンパク質Cは、それぞれ、m/z 16,450~16,620、m/z 22,080~22,310、m/z 17,100~17,270の間に検出されることを確認した。
実施例1において、m/z 16,450~16,620(タンパク質A)、m/z 22,080~22,310(タンパク質B)、m/z 17,100~17,270(タンパク質C)のピークとして検出されたタンパク質について、その性質をさらに詳細に調べるために、pHの変化に伴うピーク強度の変化について検討を行った。
(a)検討に用いたプロテインチップアレイとバッファー条件
陽イオン交換チップアレイ(CM10、Bio-Rad)に対して、pH3.0(50mM Glycine-HCl buffer)、pH3.5(50mM sodium acetate buffer)、pH4.0(50mM sodium acetate buffer)、pH4.5(50mM sodium acetate buffer)、pH5.0(50mM sodium acetate buffer)、pH5.5(50mM sodium acetate buffer)、pH6.0(50mM phosphate buffer)、pH6.5(50mM phosphate buffer)、pH7.0(50mM phosphate buffer)、pH7.5(50mM phosphate buffer)、pH8.0(50mM Tris-HCl buffer)、pH8.5(50mM Tris-HCl buffer)、pH9.0(50mM Glycine-NaOH buffer)、pH9.5(50mM Glycine-NaOH buffer)、pH10.0(50mM Glycine-NaOH buffer)の15種類のバッファーを用いた。
CM10チップアレイを用いた分析のためのサンプル調製及びプロテインチップの作製は、(a)の各バッファーを用いて実施例1の(1)方法前記(f)に準じて実施した。
CM10チップアレイを用いた分析において、ピーク強度の低下を認めるpHがそのタンパク質のイオン化が失われる等電点(pI)付近と判断できる。その結果、実施例1で検出したピークの等電点(pI)は、m/z 16,450~16,620(タンパク質A)ではpH4.5~7.5、m/z 22,080~22,310(タンパク質B)ではpH4.0~7.0、m/z 17,100~17,270(タンパク質C)ではpH5.0~8.0がpI付近に相当すると推定された。
実施例1で見出した、本体もしくはそのフラグメントがm/z 16,450~16,620のタンパク質(タンパク質A)、m/z 22,080~22,310のタンパク質(タンパク質B)、m/z 17,100~17,270のタンパク質(タンパク質C)について、実施例2及び3の情報にもとづき、The ExPASy proteomics serverのTagIdent tool(http://au.expasy.org/tools/tagident.html)を用いてデータベース検索を行った。その結果は以下のとおりであった。
Claims (25)
- 質量分析計において、本体もしくはそのフラグメントがm/z 16,450~16,620のピークとして検出されるタンパク質、本体もしくはそのフラグメントがm/z 22,080~22,310のピークとして検出されるタンパク質、及び本体もしくはそのフラグメントがm/z 17,100~17,270のピークとして検出されるタンパク質から選ばれるタンパク質からなる抗がん剤感受性判定マーカー。
- 抗がん剤が、フッ化ピリミジン系抗がん剤及び植物アルカロイド系抗がん剤から選ばれる抗がん剤である請求項1記載の抗がん剤感受性判定マーカー。
- 抗がん剤が、フルオロウラシル、イリノテカン、SN-38及びそれらの塩から選ばれるものである請求項1又は2記載の抗がん剤感受性判定マーカー。
- 抗がん剤が、フルオロウラシル又はその塩とイリノテカン、SN-38又はそれらの塩との組み合わせである請求項1又は2記載の抗がん剤感受性判定マーカー。
- 検体中の請求項1~4のいずれか1項記載のタンパク質を測定することを特徴とする抗がん剤感受性の判定方法。
- 検体が、がんを有する被験者由来の生体試料である請求項5記載の判定方法。
- 検体が、抗がん剤を投与された、がんを有する被験者由来の生体試料である請求項5又は6記載の判定方法。
- 抗がん剤が、フッ化ピリミジン系抗がん剤及び植物アルカロイド系抗がん剤から選ばれる抗がん剤である請求項5~7のいずれか1項記載の判定方法。
- 抗がん剤が、フルオロウラシル、イリノテカン、SN-38及びそれらの塩から選ばれるものである請求項5~8のいずれか1項記載の判定方法。
- 抗がん剤が、フルオロウラシル又はその塩とイリノテカン、SN-38又はそれらの塩との組み合わせである請求項5~8のいずれか1項記載の判定方法。
- 検体中の請求項1~4のいずれか1項記載のタンパク質を測定するためのプロトコールを含むことを特徴とする請求項5~10のいずれか1項記載の判定方法を実施するためのキット。
- 検体が、がんを有する被験者由来の生体試料である請求項11記載のキット。
- 検体が、抗がん剤を投与された、がんを有する被験者由来の生体試料である請求項11又は12記載のキット。
- 抗がん剤が、フッ化ピリミジン系抗がん剤及び植物アルカロイド系抗がん剤から選ばれる抗がん剤である請求項11~13のいずれか1項記載のキット。
- 抗がん剤が、フルオロウラシル、イリノテカン、SN-38及びそれらの塩から選ばれるものである請求項11~14のいずれか1項記載のキット。
- 抗がん剤が、フルオロウラシル又はその塩とイリノテカン、SN-38又はそれらの塩との組み合わせである請求項11~14のいずれか1項記載のキット。
- 請求項1~4のいずれか1項記載のタンパク質の発現変動を指標とする抗がん剤感受性亢進剤のスクリーニング方法。
- 抗がん剤が、フッ化ピリミジン系抗がん剤及び植物アルカロイド系抗がん剤から選ばれる抗がん剤である請求項17記載のスクリーニング方法。
- 抗がん剤が、フルオロウラシル、イリノテカン、SN-38及びそれらの塩から選ばれるものである請求項17又は18記載のスクリーニング方法。
- 抗がん剤が、フルオロウラシル又はその塩とイリノテカン、SN-38又はそれらの塩との組み合わせである請求項17又は18記載のスクリーニング方法。
- 請求項17~20のいずれか1項記載の方法により得られた抗がん剤感受性亢進剤。
- 請求項21記載の抗がん剤感受性亢進剤と、感受性亢進の対象となる抗がん剤とを組み合せてなるがん治療用組成物。
- 抗がん剤が、フッ化ピリミジン系抗がん剤及び植物アルカロイド系抗がん剤から選ばれる抗がん剤である請求項22記載のがん治療用組成物。
- 抗がん剤が、フルオロウラシル、イリノテカン、SN-38及びそれらの塩から選ばれるものである請求項22又は23記載のがん治療用組成物。
- 抗がん剤が、フルオロウラシル又はその塩とイリノテカン、SN-38又はそれらの塩との組み合わせである請求項22又は23記載のがん治療用組成物。
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