JP6267619B2 - Composition for the treatment of chronic myeloid leukemia - Google Patents

Description

  The present invention relates to a therapeutic composition for chronic myelogenous leukemia, and particularly provides a therapeutic composition that is effective even for patients who are resistant to conventional therapeutic agents.

  Chronic myelogenous leukemia (CML) is a myeloproliferative disease that originates from hematopoietic stem cells. It is caused by an acquired abnormality of hematopoietic stem cell genes due to a chromosomal translocation known as the Philadelphia chromosome. Hematopoietic stem cells proliferate autonomously while maintaining differentiation and maturation ability, and are blood tumors in which white blood cells and platelets increase. This disease is said to account for about 15% of adult leukemia.

  As a therapeutic agent for chronic myelogenous leukemia, imatinib mesylate (hereinafter also simply referred to as “imatinib”), a tyrosine kinase inhibitor (TKI) for Bcr-Abl, which is a gene product of the Philadelphia chromosome, was developed. As a result, treatment outcomes for patients with myeloproliferative disorders have improved significantly. This drug is called a molecular target drug because it targets Bcr-Abl.

  However, when treated with imatinib, it has become a serious clinical problem for the patient to acquire resistance to imatinib. For this reason, second generation Abl tyrosine kinase inhibitors such as dasatinib, nilotinib, and bosutinib have been developed that are effective even for patients who have acquired tolerance.

  On the other hand, the occurrence of molecular target drug resistance in cancer therapeutics is also known in other diseases. For example, an EGFR tyrosine kinase inhibitor (EGFR-TKI) for lung cancer having an epidermal growth factor receptor (EGFR) gene mutation and an ALK tyrosine kinase inhibitor (ALK-TKI) for lung cancer having an EML4-ALK fusion gene are extremely Highly effective. However, with almost no exception, resistance is acquired and recurs in 1 to several years (Non-Patent Document 1).

  Non-Patent Document 1 discloses causes such as secondary mutation of the target itself and activation of the collateral pathway as the cause of acquiring molecular target drug resistance. In particular, as a collateral pathway, it has been introduced that resistance is induced by the activation of a Met protein whose ligand is hepatocyte growth factor (HGF).

  Patent Document 1 discloses a novel c-Met inhibitor for cancer treatment. Patent Document 1 describes a pharmaceutical composition in which a second chemotherapeutic agent is further combined with this c-Met inhibitor. Imatinib is mentioned as one of the group of second chemotherapeutic agents.

JP 2013-241458 A

"Special Feature: Next-Generation Signal Transduction Research-Pioneering Basic Analysis and Development for Clinical and Drug Discovery-Molecular Targeted Treatment and Resistance Signals for Cancer" Seiji Yano, Biochemistry, Vol. 85, No. 6, pp. 475-485, 2013

  Although Patent Document 1 describes a pharmaceutical composition in which imatinib and a c-Met inhibitor are combined, there is no description about what kind of case it can be used and its effect.

  In addition, Non-Patent Document 1 does not mention imatinib resistance acquisition against chronic myelogenous leukemia, although there is disclosure of general knowledge about acquisition of resistance of molecular target drugs. In other words, there are currently no drugs other than second-generation Abl tyrosine kinase inhibitors for the problem of acquiring resistance to imatinib against chronic myelogenous leukemia.

  As a result of intensive studies to solve the above problems, the inventors of the present invention have found that a pharmaceutical composition having an HGF-Met receptor system inhibitor or a pharmaceutical composition containing a MEK / ERK pathway inhibitor is a molecular target. The inventors have found that the sensitivity to imatinib, a therapeutic agent for chronic myeloid leukemia that is resistant to the drug, is enhanced to overcome the resistance, and the present invention has been completed.

More specifically, a composition for treating chronic myeloid leukemia having resistance to a pharmaceutical composition that inhibits Bcr-Abl tyrosine kinase according to the present invention,
A first pharmaceutical composition that inhibits Bcr-Abl tyrosine kinase;
It comprises a second pharmaceutical composition that inhibits Met tyrosine kinase.

  According to the present invention, there is provided a pharmaceutical composition capable of enhancing the sensitivity of a myelogenous chronic leukemia showing resistance to molecular target drugs such as imatinib, dasatinib, bafetinib, ponatinib, etc., or overcoming the resistance. Can do.

  In addition, an effective cancer therapeutic agent can be provided for chronic myelogenous leukemia that is resistant to molecular target drugs such as imatinib, dasatinib, bafetinib, and ponatinib. The present invention proposes a method for treating fatal TKI resistance, and its social significance is extremely great.

It is a graph which shows the relationship between the addition amount of imatinib and a cell viability in normal cells (K562) and imatinib resistant cells. It is a graph which shows the relationship between the addition amount of nilotinib, and a cell survival rate in a normal cell (K562) and an imatinib resistant cell. It is a graph which shows the relationship between the addition amount of dasatinib and a cell viability in normal cells (K562) and imatinib resistant cells. It is a graph which shows the relationship between the addition amount of ponatinib and a cell viability in a normal cell (K562) and imatinib resistant cell. It is a graph which shows the relationship between the addition amount of a bafetinib, and a cell survival rate with a normal cell (K562) and an imatinib resistant cell. It is a photograph showing the results of immunoblotting for anti-phosphorylated Met antibody, anti-Met antibody and anti-β-actin antibody in normal cells (K562) and imatinib resistant cells. It is a graph which shows the relationship between the addition amount at the time of using together imatinib and PHA-665752 (met tyrosine kinase inhibitor) and a cell viability in a normal cell (K562) and an imatinib resistant cell. Photograph showing immunoblotting results for anti-phosphorylated ERK1 / 2 antibody, anti-ERK1 / 2 antibody, anti-phosphorylated Akt antibody, anti-Akt antibody, phosphorylated STAT3 antibody, and STAT3 antibody in normal cells (K562) and imatinib resistant cells It is. It is a graph which shows the relationship between the addition amount and cell viability when imatinib and U0126 (MEK inhibitor) are used in combination in normal cells (K562) and imatinib resistant cells. It is a graph which shows the relationship between the addition amount and cell viability when imatinib and LY294002 (PI3K inhibitor) are used in combination in normal cells (K562) and imatinib resistant cells. It is a graph which shows the relationship between the addition amount at the time of using together imatinib and AG490 (JAK2 inhibitor) and a cell viability in a normal cell (K562) and an imatinib resistant cell. In normal cells (K562) and imatinib resistant cells, anti-phosphorylated Met (Tyr1234 / 1235) antibody, anti-phosphorylated Met (Tyr1349) antibody, anti-Met antibody, anti-phosphorylated ERK1 / 2 antibody, anti-ERK1 / 2 antibody, anti-ERK1 / 2 antibody It is a photograph which shows the result of the immunoblotting with respect to (beta) -actin antibody.

  The therapeutic agent for chronic myeloid leukemia according to the present invention will be described below. In addition, the following description is the illustration about one embodiment and one Example of this invention, Comprising: This invention is not limited to the following description. The following embodiments can be modified without departing from the spirit of the present invention.

  The therapeutic agent for chronic myelogenous leukemia according to the present invention comprises a first pharmaceutical composition that selectively inhibits Bcr-Abl tyrosine kinase activity, which is said to be a cause of chronic myelogenous leukemia, and hepatocyte growth factor (HGF) as a ligand. And a second pharmaceutical composition that inhibits Met of the receptor tyrosine kinase.

  Moreover, as a 2nd pharmaceutical composition, you may inhibit the signal pathway of Met / MEK / ERK.

  Imatinib is known as a therapeutic agent for chronic myelogenous leukemia. However, taking imatinib eventually leads to drug resistance. This is thought to be because imatinib becomes difficult to bind due to displacement of the tyrosine kinase domain to which imatinib was bound. Therefore, nilotinib developed as a therapeutic agent for second-generation chronic myelogenous leukemia binds strongly and selectively through Bcr-Abl tyrosine kinase and inhibits its activity.

  However, when examining imatinib-resistant cells in more detail, it was found that non-activated Met tyrosine kinase is in an active state in cells not having imatinib resistance (hereinafter referred to as “normal cells”). That is, in imatinib-resistant cells, the result that the cells survived by the signal pathway by Met in addition to the signal pathway by Bcr-Abl was obtained.

  Therefore, the present invention is intended to overcome the resistance of a Bcr-Abl tyrosine kinase inhibitor by using a Bcr-Abl tyrosine kinase inhibitor in combination with a Met inhibitor as a therapeutic agent for chronic myeloid leukemia. is there.

  More specifically, as a Bcr-Abl tyrosine kinase inhibitor (first pharmaceutical composition), imatinib, nilotinib, dasatinib, ponatinib, bafetinib and the like are preferably used.

  In addition, as a Met tyrosine kinase inhibitor (second pharmaceutical composition), cabozantinib (1), crizotinib (2), foretinib (3), BMS777607 (4), golbatinib (: Golvatinib) (5), MK-2461 (6), MGCD-265 (7), MK-8033 (8), Ambatinib (9), TAS-115 (10), S49076 (11), A multi-kinase inhibitor such as BMS-754807 (12), BMS-794833 (13), LY2801653 (14), CKI27 / RG7304 / RO5126766 / CH5126766 (15) can be used. Each molecular structure is shown below. A multi-kinase inhibitor called BI847325 can also be used.

  In addition, PHA-666552 (16), EMD1214063 (17), JNJ-38877605 (18), PF-4219793 (19), SGX523 (20), INCB-028060 (21), Tivantinib (22), SAR125844 Met selective inhibitors such as (23), voritinib (24), AMG-458 (25), NVP-BVU972 (26), SU11274 (27), AMG208 (28) can be used. Each molecular structure is shown below. A Met selection inhibitor called AMG337 can also be used.

  Moreover, the 2nd pharmaceutical composition should just inhibit the signal pathway of Met / MEK / ERK, and may be a MEK inhibitor. More specifically, selumetinib (: selumetinib) (29), refametinib (: refametinib) (30), pimasertiv (31), MEK162 / ARRY-162 (32), AZD8330 / ARRY-424704 (33) , Cobimetinib (34), GDC-0623 / RG7421 / XL518 (35), CIF / RG7167 / RO497655 (36), E6201 (37), TAK-733 (38), PD-0325901 (39), CI -1040 / PD184352 (40), AS703026 (41), MEK inhibitor (42), PD318088 (43), PD98059 (44), SL327 (45), trametinib : Trametinib) (46), can be used MEK selection inhibitors such as U0126 (47). Each molecular structure is shown below. In addition, a MEK selective inhibitor called AS703888 / MSC2015103B, WX-554, RRY-300 may be used.

  Moreover, you may inhibit a more downstream signal pathway. Specifically, ERK1 / 2 selective inhibitors such as BVD-523 (48), SCH772984 (49), VTX11e (50), AEZS-131 / AEZS-134 (51), and FR180204 (52) can be used. Each molecular structure is shown below. An ERK1 / 2 selective inhibitor called MK-8353 / SCH9000353 can also be used.

Any of these pharmaceutical compositions (first pharmaceutical composition and second pharmaceutical composition) may contain a pharmaceutically acceptable salt. More specifically, hydrochloride, hydrobromide, phosphoric acid, sulfate, hydrogen sulfate, alkyl sulfonate, aryl sulfonate, acetate, benzoate, citrate, maleate, Acid addition salts including fumarate, succinate, lactate and tartrate; alkali metal cations such as Na ⁺ , K ⁺ , Li ⁺ , alkaline earth metal salts such as Mg or Ca, or organic amine salts Can do.

  The therapeutic composition for chronic myeloid leukemia according to the present invention will be described below with reference to examples.

(Example 1) <Examination of imatinib resistance acquisition factor>
K562 cells (leukemia cell line, cell number in JCRB cell bank: JCRB0019) and imatinib-resistant cells (Imatinib-resistant K562) were seeded on 96-well plates and pre-cultured for 24 hours. Thereafter, imatinib was added to a final concentration of 0, 0.05, 0.1, 0.5, 1, 5, 10, 20 μM. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and this measured value was evaluated as the cell viability against control (0 μM imatinib).

  The results of cell viability with imatinib addition are shown in FIG. The horizontal axis indicates the amount of imatinib added (μM), and the vertical axis indicates the cell viability (Cell viability:%). The white bar represents K562 cells, and the black bar represents imatinib resistant cells.

  As the amount of imatinib added increased, the survival rate of K562 cells decreased. When imatinib was added at 10 μM or more, the cell survival rate of K562 cells was zero. However, in imatinib resistant cells, the cell viability was almost 100% even when the amount of imatinib added increased. In addition, including the subsequent graphs, “P” in the graph indicates a significance probability.

  From the above, it was confirmed that cell death was not induced in imatinib-resistant cells at a concentration that induced cell death by imatinib in K562 cells. From this result, it was revealed that the prepared imatinib resistant cells showed imatinib resistance.

(Example 2) <Confirmation of nilotinib resistance in imatinib resistant cells>
K562 cells and imatinib-resistant cells (Imatinib-resistant K562) were seeded on 96-well plates and pre-cultured for 24 hours. Nilotinib was then added to a final concentration of 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2 μM. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and this measured value was evaluated as the cell viability with respect to control (0 μM nilotinib).

  The results of cell viability by adding nilotinib are shown in FIG. The abscissa indicates the amount of nilotinib added (μM), and the ordinate indicates the cell viability (Cell viability:%). The white bar represents K562 cells, and the black bar represents imatinib resistant cells.

  As the amount of nilotinib added increased, the survival rate of K562 cells decreased. When nilotinib was added in an amount of 0.2 μM or more, the cell survival rate of K562 cells was zero. However, in imatinib-resistant cells, the cell viability was almost 100% even when the amount of nilotinib added increased. The cell viability of imatinib-resistant cells decreased to about 60% when the amount of nilotinib added was increased to 2 μM.

  From the above, it was confirmed that cell death was not induced in imatinib-resistant cells even at a concentration at which cell death was induced by nilotinib in K562 cells. From this result, it was clarified that the prepared imatinib resistant cells also showed nilotinib resistance.

(Example 3) <Confirmation of dasatinib resistance in imatinib resistant cells>
K562 cells and imatinib-resistant cells (Imatinib-resistant K562) were seeded on 96-well plates and pre-cultured for 24 hours. Thereafter, dasatinib was added to a final concentration of 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2 μM. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and this measured value was evaluated as the cell viability against control (0 μM dasatinib).

  The result of cell viability by adding dasatinib is shown in FIG. The horizontal axis represents the amount of dasatinib (Dasatinib) added (μM), and the vertical axis represents the cell viability (Cell viability:%). The white bar represents K562 cells, and the black bar represents imatinib resistant cells.

  As the amount of dasatinib added increased, the survival rate of K562 cells decreased. When dasatinib was added in an amount of 0.2 μM or more, the cell viability of K562 cells was zero. However, in imatinib resistant cells, the cell viability was almost 100% even when the amount of dasatinib added increased. The cell viability of imatinib resistant cells decreased to about 80% when the amount of dasatinib added was increased to 2 μM.

  From the above, it was confirmed that cell death was not induced in imatinib-resistant cells at a concentration that induced cell death by dasatinib in K562 cells. From this result, it was revealed that the prepared imatinib resistant cells also show dasatinib resistance.

(Example 4) <Confirmation of ponatinib resistance in imatinib resistant cells>
K562 cells and imatinib-resistant cells (Imatinib-resistant K562) were seeded on 96-well plates and pre-cultured for 24 hours. Thereafter, ponatinib was added to a final concentration of 0, 5, 10, 20, 100, 250, 500, 1000 nM. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and this measured value was evaluated as the cell viability with respect to control (0 μM ponatinib).

  The results of cell viability by adding ponatinib are shown in FIG. The horizontal axis represents the amount of ponatinib (Ponatinib) added (μM), and the vertical axis represents the cell viability (Cell viability:%). The white bar represents K562 cells, and the black bar represents imatinib resistant cells.

  As the amount of ponatinib added increased, the survival rate of K562 cells decreased. When ponatinib was added at 500 nM or more, the cell viability of K562 cells was zero. However, in imatinib-resistant cells, the cell viability was almost 100% even when the amount of ponatinib added was increased.

  From the above, it was confirmed that cell death was not induced in imatinib-resistant cells at a concentration that induced cell death by ponatinib in K562 cells. From this result, it was clarified that the prepared imatinib resistant cells also exhibited ponatinib resistance.

(Example 5) <Confirmation of resistance to buffetinib in imatinib resistant cells>
K562 cells and imatinib-resistant cells (Imatinib-resistant K562) were seeded on 96-well plates and pre-cultured for 24 hours. Thereafter, buffetinib was added to a final concentration of 0, 5, 10, 50, 100, 500, 1000, 2500 nM. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and the measured value was evaluated as the cell viability against control (0 μM buffetinib).

  FIG. 5 shows the results of cell viability by adding bafetinib. The horizontal axis indicates the amount of addition of bafetinib (Bafetinib) (μM), and the vertical axis indicates the cell viability (Cell viability:%). The white bar represents K562 cells, and the black bar represents imatinib resistant cells.

  As the amount of bafetinib added increased, the survival rate of K562 cells decreased. Further, when vifetinib was added to 2500 nM or more, the cell viability of K562 cells was zero. However, in imatinib-resistant cells, the cell viability was almost 100% even when the amount of buffetinib added increased. In addition, when the addition amount of bafetinib reached 2500 nM, the cell viability of imatinib resistant cells decreased to about 80%.

  From the above, it was confirmed that cell death was not induced in imatinib-resistant cells at a concentration that induced cell death by buffetinib in K562 cells. From this result, it was clarified that the prepared imatinib-resistant cells also showed resistance to buffetinib.

(Example 6) <Examination of imatinib resistance acquisition factor>
Using the prepared imatinib-resistant cells, factors involved in resistance were examined by immunoblotting analysis.

After seeding K562 cells and imatinib-resistant cells (Imatinib-resistant K562) in a 150 cm ² flask, protein was extracted from the cells cultured for 72 hours with a cell lysate. Protein quantification was performed using BCA Protein Assay (Thermo Fisher Scientific; Waltham, MA, USA).

  Each sample was transferred to PVDF membrane after SDS-PAGE, anti-phospho-Met (Tyr1234 / 1235) antibody, anti-phospho-Met (Tyr1349) antibody, anti-Met antibody (Cell Signaling Technology; Beverly, MA, USA), and Phosphorylation of Met was examined using an anti-β-actin antibody (Sigma).

  The result of immunoblotting is shown in FIG. FIG. 6 shows photographs of immunoblotting results of K562 cells and imatinib resistant cells for each antibody. Luminescence appears as a black shadow on the photo. In the anti-phospho-Met (Tyr1234 / 1235) antibody, the shadow of imatinib resistant cells was darker than the shadow of K562 cells.

  In the anti-phospho-Met (Tyr1349) antibody, K562 cells were thin enough to be confirmed, but imatinib resistant cells were clearly darker than the K562 cells. In other words, the results of immunoblotting of imatinib-resistant cells were darker than those of K562 cells in both Tyr1234 / 1235 in the tyrosine kinase domain and Tyr1349 in the C-terminal.

  With anti-met antibody, both K562 cells and imatinib resistant cells were clearly black shadows. However, imatinib-resistant cells had a smaller black shadow. With the anti-β-actin antibody, a clear black shadow was observed in both K562 cells and imatinib resistant cells.

  From the above, it was revealed that phosphorylated Met was significantly increased in imatinib-resistant cells compared to the expression of phosphorylated Met in K562 cells. This suggests that signal transduction by met is activated in imatinib resistant cells. Since β-actin was observed to have the same degree in both K562 cells and imatinib resistant cells, it can be judged that the result of this immunoblotting is correct.

Example 7 <Examination of Imatinib Resistance Overcoming Effect by Met Inhibitor>
Imatinib-resistant cells (Imatinib-resistant K562) were seeded in 96-well plates and pre-cultured for 24 hours. Thereafter, imatinib was added to a final concentration of 5 μM. Separately, PHA-6655752 (Met inhibitor) was added in combination with imatinib so that the final concentration was 1, 2, 2.5, or 3 μM.

  What added the solvent which melt | dissolved the chemical | medical agent as Control was cultured similarly. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and this measured value was evaluated as the cell viability with respect to control.

  The results of the Met inhibitor combination are shown in FIG. The horizontal axis represents the difference in the amount of imatinib and PHA-666552, and the vertical axis represents the cell viability (Cell viability:%). The white bar represents control (K562 cells), and the black bar represents imatinib resistant cells. When imatinib and PHA-666552 were added alone, the cell viability was about 70% even when 3 μM PHA-665752 was added.

  On the other hand, when imatinib 5 μM and PHA-665752 were used in combination, the cell viability decreased according to the amount of PHA-665752 added, and the cell viability became 0% with the addition of PHA-665752 3 μM.

  From this, it was recognized that imatinib resistance was overcome in imatinib-resistant cells depending on the concentration of the Met inhibitor. From these results, it was revealed that Met is involved in imatinib resistance.

(Example 8) <Examination of imatinib resistance factor in Met downstream signaling factor>
The activity kinetics of ERK1 / 2, Akt, and STAT3 of downstream signals of Met whose activation was confirmed in imatinib resistant strains were examined by immunoblotting.

After seeding K562 cells and imatinib-resistant cells (Imatinib-resistant K562) in a 150 cm ² flask, the protein was extracted from the cells cultured for 72 hours, and used as a sample. Protein quantification was performed using BCA Protein Assay (Thermo Fisher Scientific; Waltham, MA, USA).

  Each sample was transferred to a PVDF membrane after SDS-PAGE, and anti-phospho-ERK1 / 2 antibody, anti-ERK1 / 2 antibody, anti-phospho-Akt antibody, anti-Akt antibody, anti-phospho-STAT3 antibody, and anti-STAT3 antibody (Cell The phosphorylation of ERK1 / 2, Akt, and STAT3 was examined using Signaling Technology (Beverly, MA, USA).

  The result of immunoblotting is shown in FIG. In FIG. 8, the results of immunoblotting of K562 cells and imatinib resistant cells are shown side by side for each antibody. According to FIG. 8, with the anti-ERK1 / 2 antibody, the anti-Akt antibody, and the anti-STAT3 antibody, K562 cells and imatinib resistant cells have the same level of black shadow. On the other hand, in the anti-phosphorylated ERK1 / 2 antibody, the anti-phosphorylated Akt antibody, and the anti-phosphorylated STAT3 antibody, the dark shadow of imatinib-resistant cells is clearly darker than K562 cells.

  More specifically, it can be confirmed that the anti-ERK1 / 2 antibody has two shadows at the top and bottom. The upper shadow corresponds to ERK1, and the lower shadow corresponds to ERK2. It shows that both K562 and imatinib resistant cells have both ERK1 / 2.

  On the other hand, in the case of the anti-phosphorylated ERK1 / 2 antibody, phosphorylated ERK2 has a light shadow and phosphorylated ERK1 has almost no shadow in K562 cells. Compared to this, in imatinib-resistant cells, the phosphorylated ERK1 has a thin shadow, but phosphorylated ERK2 has a clearly dark shadow.

  Based on the above, phosphorylated ERK1 / 2, phosphorylated Akt, and phosphorylated STAT3 are significantly increased in imatinib-resistant cells compared to the expression of phosphorylated ERK1 / 2, phosphorylated Akt, and phosphorylated STAT3 in K562 cells. It became clear.

  This suggests that imatinib-resistant cells activate not only signal pathways due to Bcr-Abl activity that is mainly inhibited by imatinib but also signal pathways such as ERK-MARK pathway and PI3K-Akt pathway.

(Example 9) <Examination of effect of overcoming imatinib resistance by MEK inhibitor, PI3K inhibitor, and JAK2 inhibitor>
Imatinib-resistant cells (Imatinib-resistant K562) were seeded in 96-well plates and pre-cultured for 24 hours. Thereafter, imatinib was added to a final concentration of 5 μM. Separately, in combination with imatinib, U0126 (MEK inhibitor) has a final concentration of 0.1, 1, 10 μM, and LY294002 (PI3K inhibitor) has a final concentration of 0.1, 1, 10 μM, AG490 (JAK2 inhibition). Agent) was added to a final concentration of 1, 5, 10 μM.

  What added the solvent which melt | dissolved the chemical | medical agent as Control was cultured similarly. The change in the number of cells after culturing for 72 hours was calculated by the trypan blue dye exclusion staining test method, and this measured value was evaluated as the cell viability with respect to control.

  The results of the combined use of various inhibitors are shown in FIGS. 9 to 11, the horizontal axis represents imatinib and U0126 (FIG. 9), imatinib and LY294002 (FIG. 10), imatinib and AG490 (FIG. 11), and the vertical axis represents any graph. The cell viability (Cell viability:%) is shown. The white bar represents control (K562 cells), and the black bar represents imatinib resistant cells.

  Referring to FIG. 9, when imatinib and U0126 (MEK inhibitor) were added alone, the cell viability was about 70% even when 10 μM of U0126 was added. On the other hand, when imatinib 5 μM and U0126 were used in combination, the cell viability was clearly reduced according to the amount of U0126 added. When imatinib 5 μM and U0126 were combined 10 μM, the cell viability decreased to about 35%.

  Referring to FIG. 10, in the case of a combination of imatinib and LY294002 (PI3K inhibitor), there was no significant change in cell viability when used alone or in combination. The cell viability was about 75% when imatinib 5 μM and LY294002 were combined 10 μM.

  Referring to FIG. 11, in the case of a combination of imatinib and AG490 (JAK2 inhibitor), there was no significant change in cell viability when used alone or in combination. The cell viability was about 90% when imatinib 5 μM and LY294002 were combined 10 μM.

  From the above, it was recognized that imatinib resistance was overcome when imatinib-resistant cells were combined with 5 μM imatinib and 10 μM U0126 (MEK inhibitor). From these results, it became clear that the MEK / ERK pathway is involved in imatinib resistance.

(Example 10) <Examination of ERK inhibitory effect by Met inhibitor>
It was suggested that the increase in phosphorylated Met in imatinib-resistant cells is involved in the acquisition of resistance (see Example 6 and FIG. 6). Therefore, the expression changes of Met and downstream signals when Met inhibitor was added were immunoblotted. And examined.

K562 cells, imatinib-resistant cells (Imatinib-resistant K562) were seeded in 150 cm ² flasks, cultured at 37 ° C. under 5% CO ₂ for 48 hours, and imatinib resistant cells were seeded in 150 cm ² flasks for 24 hours. After pre-culture, PHA-665752 was added to imatinib-resistant cells to a final concentration of 1, ₂ , and 2.5 μM, and cultured for 24 hours under conditions of 37 ° C. and 5% CO ₂ . Protein was extracted from this culture solution with a cell lysate to prepare a sample.

  Protein quantification was performed using BCA Protein Assay. Each sample was transferred to a PVDF membrane after SDS-PAGE, anti-phospho-Met (Tyr1234 / 1235) antibody, anti-phospho-Met (Tyr1349) antibody, anti-Met antibody, anti-phospho-ERK1 / 2 antibody, and anti-ERK1 / Two antibodies were used to examine phosphorylation of Met and ERK1 / 2. In addition, anti-β-actin antibody was allowed to act on each cell sample, and the validity of immunoblotting was confirmed.

  The result of immunoblotting is shown in FIG. In the horizontal direction of the photograph, the case of K562 cells and the case of imatinib resistant cells are shown side by side. In the case of imatinib resistant cells, the addition amount (μM) of PHA-665752 is indicated by a number. In the photo vertical direction, the results (photos) for each antibody are shown.

  In the photo of anti-phosphorylated Met (Tyr1234 / 1235) antibody, the darkest shadow was darkest when PHA-665752 in imatinib resistant cells was 0 μM. In imatinib resistant cells, the black shadow faded with increasing PHA-666552. The black shadow of K562 cells was as thick as when 2 μM of PHA-666552 was added to imatinib resistant cells.

  The result of the anti-phosphorylated Met (Tyr1349) antibody was almost the same. However, the anti-phosphorylated Met (Tyr1349) antibody was generally less shaded than the anti-phosphorylated Met (Tyr1234 / 1235) antibody.

  With anti-Met antibody, imatinib resistant cells were shaded with almost the same density regardless of the amount of PHA-665752 added. The photo of K562 cells was a shadow that was slightly larger than the shadow of imatinib resistant cells.

  With anti-phosphorylated ERK1 / 2 antibody, only a slight shadow was seen in ERK2 in the photo of K562 cells. The thickness was also thin. In imatinib resistant cells, both ERK1 and ERK2 had a clearly darker shadow than K562. In addition, the black shadow became lighter with the addition of PHA-665752.

  With the anti-ERK1 / 2 antibody, the shadow of the K562 cell was slightly smaller than the shadow of imatinib resistant cells, but the darkness was sufficiently black. In imatinib resistant cells, a large and dark shadow was observed regardless of the amount of PHA-665752 added.

  In β-actin, both K562 cells and imatinib resistant cells were almost unchanged, and a large black shadow was observed in both.

  No phosphorylated ERK1 band was observed in K562 cells. On the other hand, the phosphorylated ERK1 band was confirmed in imatinib resistant cells, and the phosphorylated ERK2 band was also stronger compared to K562 cells. That is, the MEK / ERK pathway was not active in K562 cells, whereas the imatinib resistant cells showed that the MEK / ERK pathway was active.

  When a Met inhibitor (PHA-665752) was added to this imatinib-resistant cell, the phosphorylated ERK1 band disappeared and the phosphorylated ERK2 band became thinner, approaching the state of K562 cells.

  From the above, it was confirmed that phosphorylated Met was inhibited by the Met inhibitor PHA-665752. In Example 9, it was clear that the MEK / ERK pathway is involved in imatinib resistance. Thus, it was revealed that imatinib resistance was overcome by inhibition of the MEK / ERK pathway.

  The composition for the treatment of chronic myelogenous leukemia according to the present invention is resistant to Bcr-Abl tyrosine kinase inhibitor, which is said to have a dramatic effect on chronic myelogenous leukemia, and the cause of resistance is Met / MEK / ERK. It is clarified that the pathway is the cause, and the resistance is overcome by using an inhibitor of this pathway in combination with a Bcr-Abl tyrosine kinase inhibitor. Therefore, resistance can be overcome even for patients who have acquired resistance to dasatinib or nilotinib, which has higher binding to Bcr-Abl than imatinib.

Claims (2)

A first pharmaceutical composition that inhibits Bcr-Abl tyrosine kinase;
A composition for treating chronic myeloid leukemia having resistance to the first pharmaceutical composition, comprising a second pharmaceutical composition that inhibits Met tyrosine kinase. The first pharmaceutical composition is at least one pharmaceutical composition selected from imatinib, nilotinib, dasatinib, ponatinib, bafetinib;
Said second pharmaceutical composition is cabozantinib, crizotinib, foatinib, BMS777607, golvatinib, MK-2461, MGCD-265, MK-8033, ambatinib, TAS-115, S49076, BMS-7504807, BMS-779453, LY2801653, LY2801653 RG7304 / RO5126766 / CH5126766, BI847325, PHA-666552, EMD1214063, JNJ-38877605, PF-4217903, SGX523, INCB-028060, Tivantinib, SAR125844, Boritinib, AMG-458A, NVP-BVU972G, S 2. At least one pharmaceutical composition Therapeutic compositions of chronic myelogenous leukemia resistant to the described first pharmaceutical composition.