JP2016193870A - Therapeutic agent for chronic myelogenous leukemia - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a safe and effective CML stem cell remover, TKI-resistant suppressant, and multi-kinase inhibitor-resistant suppressant by clarifying a novel signal transduction pathway involving in maintaining chronic myelogenous leukemia stem cells, and inhibiting the signal transduction pathway selectively, and thereby to provide therapeutic agents for recurrent CML and PhALL and prophylactic agent for recurrent of CML and PhALL.SOLUTION: A chronic myelogenous leukemia therapeutic agent contains a selective inhibitor of p38 MAPK signal. The above therapeutic agent is combined with a tyrosine kinase inhibitor or a multi-kinase inhibitor.SELECTED DRAWING: None

Description

The present invention relates to a therapeutic agent for chronic myelogenous leukemia (hereinafter also referred to as “CML”). More specifically, the present invention is also referred to as a CML therapeutic agent containing a p38MAPK (hereinafter also referred to as “p38”) inhibitor, particularly a CML stem cell removing agent, a tyrosine kinase inhibitor (hereinafter also referred to as “TKI”). ) Resistance inhibitors and multikinase inhibitors Resistance inhibitors; and CML treatments combining p38 inhibitors with TKI or multikinase inhibitors, especially recurrent CML treatments, CML and Philadelphia chromosome positive acute lymph The present invention relates to a prophylactic agent for recurrent leukemia (hereinafter also referred to as “Ph ⁺ ALL”).

  Chronic myelogenous leukemia (CML) is a myeloproliferative disease whose origin originates from hematopoietic stem cells, and progresses from a chronic phase for several years to an acute transition phase that exhibits a serious pathological condition through a transitional phase. In the treatment of CML, it is important that thorough treatment is performed in the chronic phase to prevent transition to the acute phase. CML patients have a chromosomal translocation t (9; 22) (q34; q22) called the Philadelphia chromosome, and this translocation expresses a constitutively activated tyrosine kinase BCR-ABL fusion protein However, it is known as the cause of the development of CML. BCR-ABL tyrosine kinase is deeply involved in the development of CML by phosphorylating intracellular substrates and self, and activating various intracellular signaling related to cell proliferation, transformation, and apoptosis suppression.

A tyrosine kinase inhibitor (TKI) imatinib (marketed drug is mesylate) for ABL has been developed as a treatment for CML, which has significantly improved the outcome of CML patients. Imatinib competitively binds to the ATP binding site of tyrosine kinase and inhibits signal transduction following substrate phosphorylation, thereby suppressing cell proliferation and inducing apoptosis to selectively damage CML cells. Imatinib and the second-generation TKIs nilotinib, dasatinib, bosutinib, and radotinib (IY5511), which are more effective, are used to treat CML patients.
However, in patients with CML after TKI treatment such as imatinib, CML (treatment-resistant recurrent CML) in which TKI has become ineffective has become a serious clinical problem. Recently, as a mechanism of this recurrence, TKI-resistant point mutation of BCR-ABL represented by T315I (imatinib binding site 315th threonine residue is replaced with isoleucine) appears. It became. The multikinase inhibitor ponatinib has been developed as a treatment for CML patients expressing TKI-resistant BCR-ABL1, and has been approved in the US and EU for CML that has become resistant or intolerant to the previous treatment TKI . However, CML stem cells, which are the source of proliferation of TKI-resistant CML cells, cannot be completely eliminated, and there is a concern of recurrence when ponatinib treatment is discontinued. As a result, the patient must continue to take ponatinib therapy. In addition, when CML stem cells having a plurality of gene mutations (compound mutations) are generated from TKI-resistant CML stem cells as a developmental origin, it may cause further ponatinib resistance. Therefore, in order to cure CML, it is necessary to develop a therapeutic method for eradicating CML stem cells.

  Several drugs that suppress CML stem cells have been reported, and CML therapy in combination with TKI has been proposed. For example, the present inventor has previously been able to efficiently remove CML stem cells by inhibiting the TGF-β-FOXO signal (Patent Document 1) and the TGF-β-Smad signal (Patent Document 2). New CML with reduced risk of relapse after remission and acquisition of TKI resistance. Therapeutic means were developed.

  By the way, it is known that the p38 MAPK (mitogen-activated protein kinase) signal transduction pathway is involved in environmental stress, ultraviolet light, apoptosis, and inflammatory reaction. In recent years, activation of p38 has been reported in various cancers (Non-patent Document 1), and a selective inhibitor of p38, LY2228820, has been developed in several cancer models (melanoma, non-small cell lung cancer, ovarian cancer, glia). There is also a report that the growth of the tumor was delayed in the tumor (myeloma, breast cancer) (non-patent document 2). However, the action of p38 in cancer is closely related to the interaction between the tumor and its microenvironment, and is thought to vary greatly depending on the cancer type. In particular, in the context of CML, p38 activation induces apoptosis in CML cells (Non-Patent Documents 3 and 4), and inhibition of p38 in CML cell line K562 cells suppresses apoptosis (Non-Patent Documents 5 and 6). ) Has been reported. There is also a report that activation of p38 is essential for the therapeutic effect of dasatinib on leukemia (Non-patent Document 7).

JP 2010-281656 JP JP 2013-253065

Moller, G.M. et al., FEBS Lett., 581: 1329-1334 (2007) Junttila, M.R. et al., FASEB J., 22: 954-65 (2008) Nakamura, S. et al., Carcinogenesis, 32: 1758-1772 (2011) doi: 10.1093 / carcin / bgr205. Epub 2011 Sep 1. Wu, X-P. Et al., Acta Pharmacol. Sinica, 2015.01.26, doi: 10.1038 / aps. 2014.132 Huang, Y.W. et al., Am. J. Physiol. Cell. Physiol., 306: C37-44 (2014) doi: 10.1152 / ajpcell.00130.2013. Epub 2013 Oct 2. Lee, Y.L. et al., PLoS ONE, 8: e61939 doi: 10.1371 / journal.pone.0061939. Print 2013. Dumka, D. et al., Leukemia & Lymphoma, 50: 2017-2029 (2009) doi: 10.3109 / 10428190903147637

An object of the present invention is to clarify a novel signal transduction pathway involved in maintenance of CML stem cells, and to provide a safe and effective CML stem cell removal agent and TKI resistance inhibitor by selectively inhibiting the signal. is there. Another object of the present invention is to provide a treatment method for CML with reduced risk of recurrence after remission and acquisition of TKI resistance, which can simultaneously eliminate differentiated CML cells and CML stem cells in combination with TKI or the like. In addition, for CML patients expressing mutant BCR-ABL in which TKI typified by T315I has become ineffective, CML and Ph ⁺ ALL of CML and Ph ⁺ ALL can be obtained by using a combination of a third-generation multikinase inhibitor and a CML stem cell depleting agent. To provide a means of preventing and treating recurrence.

  The present inventor has found that the p38 signaling pathway is remarkably activated in CML stem cells in the intensive study to achieve the above object. Therefore, the present inventor co-cultured mouse CML stem cells with feeder cells in the presence of various selective inhibitors of p38 in order to examine the effect of p38 inhibition (this co-culture was performed in vivo in cancer microscopically). Imitating the environment). As a result, all p38 inhibitors suppressed the proliferation of mouse CML stem cells.

  Furthermore, when the combined effect of imatinib and a p38 inhibitor was examined, all of the p38 inhibitors further suppressed the proliferation of mouse CML stem cells in vitro as compared to imatinib alone. In particular, the combined effect was remarkable in LY2228820.

  For human CML stem cells collected from CML patients, the effect of p38 inhibitor alone and in combination with dasatinib was examined in the same manner. Similarly, selective inhibition of p38 was also observed in human CML in vitro. Stem cell proliferation was significantly suppressed.

  Furthermore, experiments using mice transplanted with mouse CML stem cells showed that p38 inhibitors alone had an excellent suppressive effect on CML stem cells in vivo.

More importantly, from experiments using doxycycline-induced CML model mice, the combination of p38 inhibitors and dasatinib significantly improved the survival rate of mice compared to dasatinib alone.
From the above findings, the present inventors can efficiently eliminate CML stem cells by inhibiting the p38 MAPK signal transduction pathway, and can suppress TKI-resistant CML stem cells, and in combination with TKI, The inventors have found that the CML therapeutic effect is extremely excellent, and have completed the present invention.

That is, the present invention is as follows.
[1] A therapeutic agent for chronic myelogenous leukemia, comprising a selective inhibitor of p38 MAPK signal.
[2] The agent described in [1] above, wherein the inhibitor inhibits p38α and p38β.
[3] The agent described in [1] or [2] above, wherein the inhibitor is selected from the group consisting of LY2228820, VX-702, BIRB 796 and salts thereof.
[4] The agent described in [3] above, wherein the inhibitor is LY2228820 or a salt thereof.
[5] The agent according to any one of [1] to [4] above, which is a remover of chronic myeloid leukemia stem cells.
[6] The agent according to any one of [1] to [4] above, which is an inhibitor of chronic myeloid leukemia stem cells resistant to tyrosine kinase inhibitors.
[7] The agent according to any one of [1] to [6] above, which is combined with a tyrosine kinase inhibitor or a multikinase inhibitor.
[8] The agent according to [7] above, wherein the tyrosine kinase inhibitor is imatinib, dasatinib, bosutinib, radotinib or nilotinib, and the multikinase inhibitor is KW2449 or ponatinib.

According to the present invention, administration of a p38 inhibitor effectively eliminates CML stem cells that may be eradicated by TKI treatment, particularly CML stem cells that are resistant to any conventional TKI. can do. In addition, the combined use of a p38 inhibitor and a TKI or multikinase inhibitor can further improve the therapeutic efficacy of CML. Furthermore, by using in combination with a p38 inhibitor, the dose of TKI or multikinase inhibitor can be reduced and / or the administration period can be shortened, and side effects can be reduced. It can also prevent progression to more malignant conditions such as Blast crisis and Ph ⁺ ALL, for which no effective treatment is currently available.

FIG. 5 shows the structural formulas of LY2228820 (dimethanesulfonate), VX-702, and BIRB 796. It is a figure which shows that the p38 MAPK signaling pathway is activated in a mouse CML stem cell. (Upper) The phosphorylation states of the threonine residue at position 180 and the tyrosine residue at position 182 of the p38 protein were analyzed in mouse CML stem cells (left) and mouse normal hematopoietic stem cells (right). The dots in the figure indicate that p38 is phosphorylated (activated). (Lower) For comparison, the phosphorylation of serine residues at positions 423 and 425 of Smad3 in mouse CML stem cells (left) and mouse normal hematopoietic stem cells (right) have been reported. It is a figure which shows the inhibitory effect of various p38 inhibitors with respect to the mouse | mouth CML stem cell in vitro. (A) A p38 inhibitor alone was added to the medium. (B) A p38 inhibitor and imatinib were added to the medium. It is a figure which shows the therapeutic effect of single administration of the p38 inhibitor in a mouse | mouth CML stem cell transplant model. It is a figure which shows the combination effect (simultaneous administration start, LY2228820 daily administration) of a dasatinib and a p38 inhibitor in an induction | guidance | derivation type CML mouse model. It is a figure which shows the combined use effect of dasatinib and a p38 inhibitor (Initiation of LY2228820 one week after the start of dasatinib administration, and administration of LY2228820 once every two days or three days) in an induced CML mouse model. It is a figure which shows the inhibitory effect of the p38 inhibitor with respect to the human CML stem cell in vitro. (Control) LY2228820 alone was added to the medium. “Cont” indicates that no drug is added. (Dasatinib) LY2228820 and dasatinib were added to the medium. “Cont” indicates the addition of dasatinib alone.

The present invention provides a therapeutic agent for chronic myelogenous leukemia (CML) comprising a selective inhibitor of p38 MAPK signal (herein, also simply referred to as “p38 inhibitor”).
Here, “CML therapeutic agent” refers not only to the treatment of CML in the chronic phase, but also to the treatment of CML in the acute phase or transition phase, prevention of acute transformation, prevention of recurrence of CML and Ph ⁺ ALL, relapse CML and Ph ⁺ It is also used for the purpose of treating ALL.
In addition, “p38 MAPK signal” here refers to MAPKKK (MEKK1-4, MLK2 / 3, ASK1, TAO1 / 2, TAK1) activated by stimulation of DNA damage, oxidative stress, ultraviolet light, inflammatory cytokines, etc. Activates MAPKK (MKK3 / 6, MKK4), activated MAPKK activates p38, and activated p38 is a variety of transcription factors (Ets1, NFAT, Sap1, Stat1, Max, Myc, ELK1, p53 , CHOP, MEF2, ATF2, etc.) and MAPKAPK (MK2 / 3, MSK1, MNK1 / 2) are activated.

Therefore, “a selective inhibitor of p38 MAPK signal (p38 inhibitor)” inhibits any step of the above signal transduction pathway, or inhibits the expression itself of a molecule mobilized in the signal transduction pathway. As a result, it is a drug that selectively inhibits the signal transduction pathway, and further upstream of MAPKKK (for example, TGF-β receptor, IL-1 receptor, receptor tyrosine kinase (RTK), etc.) And a p38 MAPK signal other than those downstream signals (for example, Tmad-β signal, Smad pathway, Ras pathway, PI3K-Akt pathway, ROCK pathway, etc.) Here, “substantially does not inhibit” means not only inhibiting at all, but also inhibiting to the extent that it does not even adversely affect the administration subject in a therapeutically effective amount of CML, for example, The IC ₅₀ value for the target molecule of p38 MAPK signal is 1/10 or less of the IC ₅₀ value for other biomolecules, preferably 1/20 or less, more preferably 1/50 or less, particularly preferably 1/100 or less. is there.

  As a selective inhibitor of p38 MAPK signal (p38 inhibitor), for example, as a drug that inhibits any step of the above signal transduction pathway, it directly acts on p38 protein to inhibit its activation (phosphorylation) Substance, substance that acts on MAPKK upstream of p38 (MKK3 / 6 or MKK4, preferably MKK3 / 6) to inhibit p38 activation (phosphorylation), or substrate of p38 (transcription factor, MAPKAPK) Examples include, but are not limited to, substances that inhibit activation (phosphorylation). There are four isoforms of α, β, γ and δ in p38, but the p38 inhibitor of the present invention may be any one that inhibits at least one of these isoforms, for example, at least Examples include drugs that inhibit p38α. Preferably, it is an agent that inhibits at least p38α and p38β. In a preferred embodiment, the p38 inhibitor of the present invention is an agent that selectively inhibits p38α and p38β.

  As the p38 inhibitor of the present invention, for example, the following formula (I) disclosed as a p38 inhibitor acting as an ATP competitive inhibitor in US Pat. No. 7,582,652:

(Wherein, W is represented by any of the following formulas (i) to (vii);

X is a nitrogen atom or C—R ¹ ;
R _is, C 1 -C _{7 alkyl,} C 3 -C _{7 cycloalkyl, (C} 1 -C ₇ alkylene) - _(C 3 ~C _{7 cycloalkyl), - SO 2 - (C} 1 ~C 7 alkyl), Or —SO ₂ —NR ⁵ R ⁶ ;
R ¹ is a hydrogen atom, amino, methyl, or —N═CHN (CH ₃ ) ₂ ;
R ² is phenyl optionally substituted by 1 or 2 independently selected halogen atoms;
R ³ is a hydrogen atom, C ₁ -C ₇ alkyl, C ₃ -C ₇ cycloalkyl, or phenyl optionally substituted with 1 or 2 substituents independently selected from a halogen atom and trifluoromethyl. Is;
R ⁴ is a hydrogen atom or C ₁ -C ₇ alkyl;
R ⁵ and R ⁶ are each independently C ₁ -C ₇ alkyl. )
Or a salt thereof,
(Wherein “C ₁ -C ₇ alkyl” includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, and “C ₁ -C ₇ alkylene” means methylene. , ethylene, propylene, isopropylene, butylene, isobutylene, sec- butylene, tert- butylene, pentylene, hexylene, includes a heptylene, _"C 3 -C ₇ cycloalkyl" include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl Including, “(C ₁ -C ₇ alkylene)-(C ₃ -C ₇ cycloalkyl)” means a C ₃ -C ₇ cycloalkyl attached via a C ₁ -C ₇ alkylene linker, Is fluorine atom, chlorine atom, bromine atom and iodine atom Including the.)

  Preferably, the following formula (I ′):

Wherein R ′ is 2,2-dimethylpropyl or 1,2,2-trimethylpropyl;
R ² ′ is phenyl, 4-fluorophenyl or 2,4-difluorophenyl;
R ³ ′ is tert-butyl, 2-chloro-6-fluorophenyl, 2-fluoro-6-trifluoromethylphenyl, 2,6-dichlorophenyl or 2,6-difluorophenyl. )
Or a salt thereof,

More preferably, in the above formula (1 ′):
(a) R ′ is 2,2-dimethylpropyl, R ² ′ is 4-fluorophenyl and R ³ ′ is 2-fluoro-6-trifluoromethylphenyl;
(b) R ′ is 2,2-dimethylpropyl, R ² ′ is 4-fluorophenyl and R ³ ′ is 2,6-dichlorophenyl;
(c) R ′ is 2,2-dimethylpropyl, R ² ′ is 4-fluorophenyl and R ³ ′ is tert-butyl;
(d) R ′ is 2,2-dimethylpropyl, R ² ′ is phenyl and R ³ ′ is 2-chloro-6-fluorophenyl;
(e) R ′ is 2,2-dimethylpropyl, R ² ′ is 2,4-difluorophenyl;
(f) R ′ is 1,2,2-trimethylpropyl, R ² ′ is 4-fluorophenyl and R ³ ′ is tert-butyl; or
(g) a compound or a salt thereof, wherein R ′ is 1,2,2-trimethylpropyl, R ² ′ is 4-fluorophenyl and R ³ ′ is 2,6-difluorophenyl;

Particularly preferably, in the above formula (1 ′), R ′ is 2,2-dimethylpropyl, R ² ′ is 4-fluorophenyl and R ³ ′ is tert-butyl [5- (2-tert-butyl -5- (4-Fluorophenyl) -1H-imidazol-4-yl) -3-neopentyl-3H-imidazo [4,5-b] pyridin-2-amine (ralimetinib (LY2228820); FIG. 1); In the book, it may be referred to as “LY2228820”. Or a salt thereof. LY2228820 is a p38 inhibitor selective for p38α and p38β.
The salt of the compound represented by the above formula (I) or (I ′) is not particularly limited as long as it is a pharmaceutically acceptable salt. For example, trifluoroacetic acid, acetic acid, lactic acid, succinic acid, maleic acid , Tartaric acid, citric acid, gluconic acid, ascorbic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, cinnamic acid, fumaric acid, phosphonic acid, hydrochloric acid, nitric acid, hydrobromic acid, hydroiodic acid, sulfamine Acid addition salts with acids such as sulfuric acid, preferably methanesulfonate, succinate, fumarate, dimaleate, dihydrochloride, dimethanesulfonate, etc., more preferably dimethanesulfonic acid Salt.

  Further, as another p38 inhibitor of the present invention, for example, AMG-548, BIRB-796 (FIG. 1), SClO— disclosed in Nikas et al. (Curr Opin Drug Discov Devel. 8, 421-430 (2005)). 469, SCIO-323, VX-702 (FIG. 1), peximetinib (ARRY-614) and its derivatives disclosed in WO 2011/087795, SB203580 acting as an ATP competitive inhibitor disclosed in WO 99/42592, SB202190 and derivatives thereof, disclosed in Underwood et al. (J Pharmacol Exp Ther. 293, 281-288 (2000)), SB239063 and derivatives thereof, Jackson et al. (J Pharmacol Exp Ther. 284, 687-692 (1998)) SB220025 and derivatives thereof disclosed in PD169316, Mclay et al. (Bioorg Med Chem. 9, 537-554 (2001)) disclosed in Gallagher et al. (Bioorg. Med. Chem. 5, 49 (1997)). RPR200765A, FR167653 disclosed in Yamamoto et al. (Eur. J. Pharmacol. 314, 137-142 (1996)), Rosmapimod (GW856553X disclosed in Aston et al. (J. Med. Chem. 52, 6257 (2009)) ), Xing et al. (Bioche m. 48, 6402-6411 (2009)), Haddad (Curr. Opin. Investig. Drugs 2, 1070-1076 (2001)), VX-745, Miwatashi et al. (J. Med. Chem. 48, 5966) -5979 (2005)), Koeberle et al. (Nat. Chem. Biol. 8, 141-143 (2012)), and skepinone-L, and salts thereof.

  The salt of the above compound is not particularly limited as long as it is a pharmaceutically acceptable salt. For example, trifluoroacetic acid, acetic acid, lactic acid, succinic acid, maleic acid, tartaric acid, citric acid, gluconic acid, ascorbic acid, Acid addition salts with acids such as benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, cinnamic acid, fumaric acid, phosphonic acid, hydrochloric acid, nitric acid, hydrobromic acid, hydroiodic acid, sulfamic acid, sulfuric acid; For example, metal salts such as sodium, potassium, magnesium and calcium; for example, salts with organic bases such as trimethylamine, triethylamine, pyridine, picoline, N-methylpyrrolidine, N-methylpiperidine and N-methylmorpholine.

When the above-described low-molecular-weight p38 inhibitor of the present invention has an isomer such as an optical isomer, a stereoisomer, a positional isomer, a rotational isomer, etc., either one isomer or a mixture of isomers As long as it has inhibitory activity, it is included in the p38 inhibitor of the present invention. For example, when an optical isomer exists in the p38 inhibitor of the present invention, the optical isomer resolved from the racemate is also encompassed in the p38 inhibitor of the present invention. Each of these isomers can be obtained as a single product by a known synthesis method or separation method (concentration, solvent extraction, column chromatography, recrystallization, etc.).
The above-described low molecular weight p38 inhibitor of the present invention may be crystalline or amorphous. When the p38 inhibitor is a crystal, a single crystal form or a mixture of crystal forms is included in the p38 inhibitor of the present invention. The crystal can be produced by crystallization by applying a crystallization method known per se.
The low molecular weight p38 inhibitor of the present invention may be a solvate (for example, hydrate etc.) or a solvate, and both are included in the p38 inhibitor of the present invention.
The low molecular weight p38 inhibitor of the present invention may be labeled with an isotope (eg, ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I, etc.).
In addition, when the p38 small molecule inhibitor of the present invention is unstable to acid, it can be provided in the form of an acid-stable prodrug by a method known per se to prevent degradation by gastric acid when administered orally.

  The above-described low molecular weight p38 inhibitors of the present invention are all known compounds and can be produced by methods known per se. In addition, many of these p38 inhibitors are commercially available.

In addition, examples of substances that inhibit p38 phosphorylation include MKK3 / 6 that mimics p38, MKK4 substrate peptide, dominant negative form of MAPKK, antibodies and aptamers that sterically hinder p38 phosphorylation, etc. . Examples of the substance that inhibits phosphorylation of a downstream factor (transcription factor, MAPAPK) by p38 include a substrate peptide of p38 that mimics the downstream factor, a dominant negative form of p38, and the like. A substrate peptide that mimics p38 or its downstream factor can be produced by chemically synthesizing a peptide consisting of a partial amino acid sequence including the phosphorylation site of each protein by a method known per se. Antibodies and aptamers against p38 can also be obtained by methods known per se.
The dominant negative form of p38 may be any substance as long as it has a substrate binding ability but acts competitively with the wild-type p38 protein and inhibits its function without phosphorylating the substrate. Examples include p38T180A in which threonine at position 180 located in the DNA binding region of p38 in humans and mice is point-mutated to alanine, p38Y182F in which tyrosine at position 182 of p38 in humans and mice is point-mutated to phenylalanine, and the like ( Raingeaud J., J Biol Chem. 270, 7420-7426. (1995)).
A dominant negative form of p38 can be obtained, for example, by the following method. In human p38, for p38α, synthesize an appropriate oligonucleotide as a probe or primer based on the sequence registered as NM_001315 in the NCBI database for p38α, and NM_002751 for p38β. Alternatively, mouse or human p38 cDNA is cloned from a cDNA library using a hybridization method or (RT-) PCR method and subcloned into an appropriate plasmid. Inverse PCR using the plasmid containing the inserted p38 cDNA as a template by synthesizing a primer containing the site in which the codon at the site where the mutation is to be introduced is replaced with a codon encoding another desired amino acid. By performing, a nucleic acid encoding the target dominant negative body is obtained. In the case of a deletion mutant such as p38DD, a primer may be designed outside the site to be deleted, and inverse PCR may be performed in the same manner.
The same applies to the dominant negative form of MAPKK.

  Examples of selective inhibitors of the p38 MAPK signal that inhibit the expression of p38 include antisense nucleic acids, siRNA (shRNA), ribozymes, and the like against p38. Preferably it is siRNA (shRNA). The siRNA for p38 is designed according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)) based on the mouse or human p38 cDNA sequence information shown in SEQ ID NOs: 1-4. can do. For the selected target sequence candidate group, whether or not there is homology in the 16-17 base sequence in the non-target mRNA is determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) And the like, and the specificity of the selected target sequence is confirmed. About the target sequence whose specificity has been confirmed, a sense strand having a 3 'end overhang of TT or UU at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 bases and TT or A double-stranded RNA consisting of an antisense strand having a 3 'end overhang of UU is designed as an siRNA. The shRNA is designed by appropriately selecting an arbitrary linker sequence (for example, about 8-25 bases) that can form a loop structure, and linking the sense strand and the antisense strand via the linker sequence. be able to.

The sequence of siRNA and / or shRNA can be searched using search software provided free of charge on various websites. Examples of such sites include siRNA Target Finder (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) and pSilencer ^™ Expression Vector insert design tool (http: / /www.ambion.com/techlib/misc/psilencer_converter.html), GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) is not limited to these, QIAGEN, Searches are also possible on websites such as Takara Bio, SiSearch, Dharmacon, Whitehead Institute, Invitrogen, and Promega.

  The siRNA for p38 was synthesized by using a DNA / RNA automatic synthesizer with the sense strand and antisense strand oligonucleotides designed as described above, for example, at about 90 to about 95 ° C. in a suitable annealing buffer. It can be prepared by denaturing for about 1 minute and then annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. ShRNA against p38 can be prepared by synthesizing an oligonucleotide having the shRNA sequence designed as described above with a DNA / RNA automatic synthesizer and self-annealing in the same manner as described above.

The nucleotide molecules that make up siRNA and shRNA may be natural RNA, but various chemical modifications may be applied to improve stability (chemical and / or enzyme) and specific activity (affinity with mRNA). Can be included. For example, in order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue. In addition, the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R is, for example, CH ₃ (2′-O-Me), CH ₂ CH ₂ OCH ₃ (2′-O-MOE), CH ₂ CH ₂ NHC (NH) NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN and the like may be substituted). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned.

  The conformation of the sugar part of RNA is dominated by C2'-endo (S type) and C3'-endo (N type). In single-stranded RNA, it exists as an equilibrium between the two, but double-stranded Is fixed to the N type. Therefore, in order to give strong binding ability to the target RNA, BNA (LNA) (Imanishi) is an RNA derivative in which the conformation of the sugar moiety is fixed to N-type by cross-linking 2 'oxygen and 4' carbon. , T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides Nucleotides Nucleicides Nucleic Acids , 22, 1619-21, 2003) can also be preferably used.

  However, if all ribonucleoside molecules in the natural RNA are replaced with a modified form, RNAi activity may be lost, and therefore it is necessary to introduce a minimal modified nucleoside that allows the RISC complex to function.

  The siRNA for p38 can also be purchased from, for example, Santa Cruz (eg, Santa Cruz Cat # sc-29433, sc-29434, sc-44216), SigmaAldrich (eg, SHGLY-NM_011951), and the like.

Since the p38 inhibitor of the present invention can efficiently eliminate CML stem cells, particularly CML stem cells that are resistant to TKI, treatment of CML and Blast crisis, Ph ⁺ ALL, etc. for which there is currently no effective treatment It is effective in suppressing the progression to higher-grade disease states.

  In recent years, p38 has been activated in various cancers, and p38 inhibitors suppress tumor growth in multiple tumor models including melanoma, non-small cell lung cancer, ovarian cancer, glioma, myeloma, and breast cancer A phase I or phase II trial is being conducted in the US and France for the three drugs LY2228820, LY3007113, and ARRY-614. However, for CML, activation of p38 induces apoptosis in CML cells, and conversely, inhibition of p38 in CML cell line K562 cells suppresses apoptosis, and the effect of p38 on the therapeutic effect of dasatinib on CML It has been reported that activation is essential, and it has been suggested that inhibition of p38 may worsen CML. Therefore, in the present invention, it is a surprising discovery that CML therapeutic effect was recognized in vivo by inhibiting p38.

  Small molecule p38 inhibitor of the present invention, MAPKK (MKK3 / 6, MKK4) substrate peptide, dominant negative form of the MAPKK, antibody that sterically interferes with phosphorylation of p38, substrate peptide of p38, dominant negative form of p38 Can be prepared as a pharmaceutical composition as it is or by mixing it with a known pharmacologically acceptable carrier or the like. The pharmaceutical composition is prepared in an orally administered form such as tablets, pills, capsules, powders, granules, syrups, emulsions and suspensions; injections, drops, external preparations, suppositories, etc. Depending on the parenteral administration agent, etc., it can be systemically or locally administered orally or parenterally. In the case of parenteral administration, intravenous administration, intradermal administration, subcutaneous administration, rectal administration, transdermal administration, and the like are possible.

  The content of the p38 inhibitor in the CML therapeutic agent of the present invention is about 0.01% to 100% by weight of the total composition.

  The above-mentioned appropriate dosage form can be produced by blending the active ingredient with a pharmacologically acceptable carrier or the like. Examples of the pharmacologically acceptable carrier include various organic or inorganic carrier substances that are commonly used as pharmaceutical materials, such as excipients, lubricants, binders, disintegrants, water-soluble polymers in solid formulations, Basic inorganic salts; solvents, solubilizers, suspending agents, isotonic agents, buffers, soothing agents and the like in liquid preparations. Further, if necessary, additives such as ordinary preservatives, antioxidants, colorants, sweeteners, sour agents, foaming agents, and fragrances can be used.

  Examples of the “excipient” include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, titanium oxide and the like.

  Examples of the “lubricant” include magnesium stearate, sucrose fatty acid ester, polyethylene glycol, talc, stearic acid and the like.

  Examples of the “binder” include hydroxypropylcellulose, hydroxypropylmethylcellulose, crystalline cellulose, starch, polyvinylpyrrolidone, gum arabic powder, gelatin, pullulan, low-substituted hydroxypropylcellulose, and the like.

  Examples of the “disintegrant” include (1) crospovidone, (2) disintegrant called super disintegrant such as croscarmellose sodium (FMC-Asahi Kasei), carmellose calcium (Gotoku Pharmaceutical), (3) carboxymethyl Examples include starch sodium (eg, Matsutani Chemical Co., Ltd.), (4) low-substituted hydroxypropylcellulose (eg, Shin-Etsu Chemical Co., Ltd.), (5) corn starch and the like. The “crospovidone” is crosslinked with a chemical name of 1-ethenyl-2-pyrrolidinone homopolymer, including polyvinyl polypyrrolidone (PVPP) and 1-vinyl-2-pyrrolidinone homopolymer. Specific examples include Kollidon CL (manufactured by BASF), Polyplaston XL (manufactured by ISP), Polyplaston XL-10 (manufactured by ISP), and Polyplastidone. INF-10 (manufactured by ISP) or the like.

  Examples of the “water-soluble polymer” include ethanol-soluble water-soluble polymers [for example, cellulose derivatives such as hydroxypropylcellulose (hereinafter sometimes referred to as HPC), polyvinylpyrrolidone, etc.], ethanol-insoluble water-soluble polymers Molecules [for example, hydroxypropylmethylcellulose (hereinafter sometimes referred to as HPMC), cellulose derivatives such as methylcellulose, sodium carboxymethylcellulose, sodium polyacrylate, polyvinyl alcohol, sodium alginate, guar gum, etc.] and the like.

Examples of the “basic inorganic salt” include basic inorganic salts of sodium, potassium, magnesium and / or calcium. Preferred is a basic inorganic salt of magnesium and / or calcium. More preferred is a basic inorganic salt of magnesium. Examples of the basic inorganic salt of sodium include sodium carbonate, sodium hydrogen carbonate, disodium hydrogen phosphate and the like. Examples of the basic inorganic salt of potassium include potassium carbonate and potassium hydrogen carbonate. Examples of the basic inorganic salt of magnesium include heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide, magnesium metasilicate aluminate, magnesium silicate, magnesium aluminate, synthetic hydrotalcite [Mg ₆ Al ₂ ( OH) ₁₆ · CO ₃ · 4H ₂ O] and alumina / magnesium hydroxide, preferably heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide and the like. Examples of the basic inorganic salt of calcium include precipitated calcium carbonate and calcium hydroxide.

  Examples of the “solvent” include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like.

  Examples of the “dissolution aid” include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

  Examples of the “suspending agent” include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; And hydrophilic polymers such as polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

  Examples of the “isotonic agent” include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol and the like.

  Examples of the “buffering agent” include buffer solutions of phosphate, acetate, carbonate, citrate, and the like.

  Examples of the “soothing agent” include benzyl alcohol and the like.

  Examples of the “preservative” include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.

  Examples of the “antioxidant” include sulfite, ascorbic acid, α-tocopherol and the like.

  Examples of the “colorant” include edible pigments such as edible yellow No. 5, edible red No. 2, and edible blue No. 2; edible lake pigments, bengara and the like.

  Examples of the “sweetening agent” include saccharin sodium, dipotassium glycyrrhizin, aspartame, stevia, thaumatin and the like.

  Examples of the “sour agent” include citric acid (anhydrous citric acid), tartaric acid, malic acid and the like.

  Examples of the “foaming agent” include sodium bicarbonate.

  The “fragrance” may be a synthetic product or a natural product, and examples thereof include lemon, lime, orange, menthol, and strawberry.

  The low molecular weight p38 inhibitor of the present invention is compression-molded according to a method known per se, for example, by adding a carrier such as an excipient, a disintegrant, a binder or a lubricant, and then, if necessary, masking of taste, For enteric or long-lasting purposes, an oral preparation can be obtained by coating by a method known per se. In the case of an enteric preparation, an intermediate layer may be provided between the enteric layer and the drug-containing layer by a method known per se for the purpose of separating both layers.

  When the low molecular p38 inhibitor of the present invention is, for example, an orally disintegrating tablet, for example, a core containing crystalline cellulose and lactose is coated with the low molecular p38 inhibitor of the present invention and, if necessary, a basic inorganic salt, A composition is obtained by coating with a water-soluble polymer-containing coating layer, and the resulting composition is coated with a polyethylene glycol-containing enteric coating layer, followed by a triethyl citrate-containing enteric coating layer, and further polyethylene glycol It can be produced by a method of coating with the containing enteric coating layer and finally coating with mannitol to obtain fine particles, mixing the obtained fine particles and additives, and molding.

  Examples of the “enteric coating layer” include cellulose acetate phthalate (CAP), hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, and a methacrylic acid copolymer [for example, Eudragit L30D-55 (trade name; Rame Co., Ltd.), Kollicoat MAE30DP (trade name; manufactured by BASF Corp.), Polykid PA30 (trade name; manufactured by Sanyo Kasei Co., Ltd.), etc.], carboxymethyl ethyl cellulose, shellac and other water-based enteric polymer bases; Sustained release bases such as coalescent [eg Eudragit NE30D (trade name), Eudragit RL30D (trade name), Eudragit RS30D (trade name), etc.]; water-soluble polymer; triethyl citrate, polyethylene glycol, acetylated monoglyceride And a layer composed of one or a mixture of two or more plasticizers such as triacetin and castor oil.

  Examples of the “additive” include water-soluble sugar alcohols (eg, sorbitol, mannitol, maltitol, reduced starch saccharified product, xylitol, reduced palatinose, erythritol, etc.), crystalline cellulose (eg, Theolas KG 801, Avicel PH 101). , Avicel PH 102, Avicel PH 301, Avicel PH 302, Avicel RC-591 (crystalline cellulose / carmellose sodium), etc., low-substituted hydroxypropyl cellulose (eg, LH-22, LH-32, LH-23, LH) -33 (Shin-Etsu Chemical Co., Ltd.) and mixtures thereof) and the like, and further, binders, acidulants, foaming agents, sweeteners, fragrances, lubricants, colorants, stabilizers, excipients, Disintegrants are also used.

  The dose of the therapeutic agent for CML containing the low-molecular-weight p38 inhibitor of the present invention as an active ingredient varies depending on the type of compound, administration subject, administration route, severity of CML, etc., but for example, LY2228820 is administered orally In this case, the amount of LY2228820 per dose is 0.5 to 10 mg / kg, preferably 1 to 5 mg / kg, 3 times a day to once every 5 days, preferably once a day to once every 3 days. More preferably, it can be administered once every 2 to 3 days. For example, the frequency of “once every two days” includes not only the case of administration every two days but also the case of administration for two consecutive days and drug withdrawal for two days. The maximum tolerated dose (MTD) of LY2228820 in the Phase 1 trial for advanced cancer (solid cancer) is 420 mg, and the recommended dose based on side effects and therapeutic efficacy is 300 mg (14 days off after 14 days of administration twice a day) Cycle (corresponding to 14 kg / kg / day and 10 mg / kg / day, respectively, assuming a body weight of 60 kg), and in animal experiments using various solid cancer transplantation models, 10-30 mg / kg 2 to 3 times a day In view of the fact that it is orally administered, it was surprising that significant increase in survival time was observed by administering LY2228820, 2.5 mg / kg once every 3 days, as shown in the Examples below. At the same time, it can be said to be a very advantageous effect considering the patient's drug tolerance. P38 MAPK may be expressed ubiquitously, and various side effects have been reported in clinical trials of p38 inhibitors (eg, clinical trials of BIRB 796 for rheumatoid arthritis), and safety issues have been pointed out. However, regarding CML, it is possible to avoid the problem of side effects because it is effective at low doses.

On the other hand, when a nucleic acid p38 inhibitor such as an antisense nucleic acid against p38, siRNA (shRNA), ribozyme, aptamer or the like is used as a CML therapeutic agent, it can be formulated and administered according to a method known per se. That is, the nucleic acid is inserted alone or in a functional manner into an appropriate expression vector for mammalian cells such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc., and then formulated according to conventional means. Can do. The nucleic acid can be administered as it is or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
Furthermore, for the purpose of improving the pharmacokinetics, prolonging the half-life, and improving the efficiency of cellular uptake, the nucleic acid may be formulated (injection) alone or with a carrier such as liposome and administered intravenously, subcutaneously, etc. .

  The nucleic acid p38 inhibitor of the present invention may be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for administration may contain the nucleic acid and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.

  As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included. Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared, for example, by dissolving, suspending or emulsifying the nucleic acid of the present invention in a sterile aqueous liquid or oily liquid usually used for injection. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. Suppositories used for rectal administration may be prepared by mixing the nucleic acid with a normal suppository base.

  Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

  The above parenteral or oral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories. The nucleic acid p38 inhibitor of the present invention is preferably contained, for example, usually 5 to 500 mg per dosage unit form, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.

  The dose of the therapeutic agent for CML containing the nucleic acid p38 inhibitor of the present invention varies depending on the subject of administration, symptoms, administration route, etc., but usually about 0.01 to 20 mg / kg body weight of the nucleic acid per dose. Preferably, about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight is administered by intravenous injection about 1 to 3 times a day to about once every 5 days. is there. In the case of other parenteral administration and oral administration, an equivalent amount can be administered.

  The CML patient to which the p38 inhibitor of the present invention is to be administered is not particularly limited, but is desirably a CML patient in the chronic phase before passing the blast crisis phase or transition phase. Since the p38 inhibitor of the present invention is particularly useful for removing CML stem cells, the CML therapeutic agent of the present invention containing the inhibitor is particularly resistant to TKI such as imatinib, or is initially refractory. It is effective for CML patients, relapsed patients with CML stem cells remaining after TKI treatment, and the like. In this specification, unless otherwise specified, “resisting to TKI” includes not only the case of becoming resistant to TKI during treatment but also the case of initial refractory. It will be used in the meaning.

  Imatinib resistance may be dependent on BCR-ABL-dependent and independent mechanisms. The former includes amplification of the BCR-ABL gene, point mutation (eg, phosphate-binding loop (p-loop in the kinase domain) ) (E.g., G250E, Q252H, Y253F, Y253H, E255K, E255V, etc.), imatinib binding sites (e.g., T315I, T315A, F317L, F317V, etc.), activation loop, catalytic loop), while the latter is , Increased activity downstream of the BCR-ABL signal, increased activity of signal transduction pathways not involved in BCR-ABL, increased discharge of imatinib by multidrug resistance (MRD) protein, increased imatinib binding protein (α1-acid glycoprotein), etc. Can be mentioned. Among these, it has been reported that the main cause is a point mutation in the kinase domain (Cancer Cell, 2: 117-125 (2002)), but the p38 inhibitor of the present invention can suppress CML stem cells. In addition to mutant BCR-ABL-expressing CML, it may be effective against treatment-resistant CML by any mechanism.

  Among the BCR-ABL point mutations, mutations in the p-loop, which is the N-terminal side of the kinase domain, are common in patients in the blast crisis and transition phase, and have been reported to worsen the prognosis (Blood , 102 (1): 276-283 (2003)), the CML therapeutic agent of the present invention is a therapeutic agent for BCR-ABL-expressing CML having a mutation in the p-loop, an acute transformation inhibitor, a prognosis improving agent, etc. It is particularly useful.

  Second-generation TKIs such as nilotinib, a multikinase inhibitor with improved affinity for ABL, and dasatinib, which has an ABL tyrosine kinase inhibitory effect approximately 260 times that of imatinib, have proliferated many BCR-ABL point mutation-expressing cells. Although it can be suppressed, it is ineffective for cells expressing T315I mutant BCR-ABL in which the threonine residue at position 315, which is the imatinib binding site, is substituted with isoleucine. Third-generation multikinase inhibitors such as KW2449 (Blood, 114: 1607-1617 (2009)) and ponatinib (Cancer Cell, 16: 401-412 (2009)) are also effective against T315I mutant BCR-ABL expressing cells Phase I trials have shown that only a limited number of cases lead to complete cytogenetic remission, with more potent treatments for T315I mutant BCR-ABL-expressing CML cells, such as KW2449 and ponatinib. Development of concomitant drugs that can complement the therapeutic effect is desired. The p38 inhibitor of the present invention has a remarkable growth inhibitory effect on imatinib-resistant CML stem cells that express T315I mutant BCR-ABL, and when used in combination with KW2449 or ponatinib, these multikinase inhibitors The p38 inhibitor of the present invention is a particularly effective therapeutic agent for T315I mutant BCR-ABL-expressing CML, which has been extremely refractory in the past, because it can further suppress the proliferation of CML stem cells than when administered alone. obtain.

  In addition, the p38 inhibitor of the present invention exhibits an excellent CML stem cell growth inhibitory effect both in the single administration and in combination with TKI under culture conditions that mimic the cancer microenvironment. Various signals produced from the cancer microenvironment (niche cells) are thought to contribute to the survival of CML stem cells and the acquisition of imatinib resistance. Therefore, in combination with TKI and multikinase inhibitors, CML It may also be effective against imatinib-resistant CML based on enhanced signaling pathways not involved in BCR-ABL in stem cells.

  Therefore, the present invention also provides a CML therapeutic agent (combination agent) comprising a combination of the p38 inhibitor of the present invention and a TKI or multikinase inhibitor. Currently approved tyrosine kinase inhibitors include imatinib, dasatinib, nilotinib, bosutinib and radotinib, and second-generation TKIs currently in clinical trials include bafetinib. On the other hand, there are KW2449, ponatinib, AT9283, etc. as multikinase inhibitors, but any TKI and multikinase inhibitor that acts as a molecular target drug for BCR-ABL can be used.

  The dosage form, administration route, dosage and the like of TKI and multikinase inhibitor used in the concomitant drug of the present invention may be those normally used for CML patients to which it is applied. For example, in the case of imatinib, 400-600 mg / day is administered to patients with chronic phase CML and 600-800 mg / day is administered to patients in transitional phase or blast crisis in a single dose or in two divided doses. be able to. In the case of nilotinib, 400 mg per dose can be orally administered twice daily to CML patients in the chronic or transitional phase. In the case of dasatinib, 100-140 mg once a day for CML patients in the chronic phase and 70-90 mg once a day for patients in the transitional phase or blast crisis phase may be orally administered twice a day. it can. In the case of KW2449, 50-500 mg per day can be administered orally in 1 or 2 divided doses. In the case of ponatinib, 45 mg can be orally administered once a day.

  Since the concomitant drug of the present invention has a remarkable concomitant effect compared to single administration of TKI or multikinase inhibitor, the dose of TKI or multikinase inhibitor used in the concomitant drug is usually used in single administration. The amount can be reduced from the amount. Hematological toxicity (platelet, neutrophil, leukocyte reduction, etc.) is frequently observed with imatinib and dasatinib, skin symptoms (rash, etc.), digestive symptoms (nausea, diarrhea, etc.), fluid retention (pleural effusion, eyelids) Side effects such as edema), liver damage, and acute pancreatitis have been reported. Reduction of TKI is expected to reduce these side effects. Multikinase inhibitors also block signaling by other kinases such as Aurora kinase and FLT3 as well as BCR-ABL, potentially posing a high risk of side effects and, indeed, due to the accumulation of thrombotic events The Phase III trial for newly diagnosed chronic phase CML was discontinued. Therefore, if the dose of the multikinase inhibitor can be reduced by the combined use with the p38 inhibitor of the present invention, a safer CML therapeutic agent with reduced side effects can be provided.

  The p38 inhibitor and the TKI or multikinase inhibitor of the present invention may be administered as a combination, separately, simultaneously or over time. However, from the results of Examples described later, if administration of the p38 inhibitor of the present invention is started at the same time as TKI or multikinase inhibitor or prior to administration of TKI or multikinase inhibitor, CML may be worsened on the contrary. . Without being bound by any theory, the p38 inhibitor of the present invention suppresses CML stem cells, but it cannot be ruled out that it does not suppress differentiated mature CML cells, so first administered TKI or multikinase inhibitor It is preferable to start administration of the p38 inhibitor of the present invention after starting maturation and improving the disease state by suppressing mature CML cells to some extent. For example, the administration of the p38 inhibitor of the present invention can be started 3 to 14 days, preferably 5 to 10 days, more preferably about 7 days after the start of TKI or multikinase inhibitor administration.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail, this invention is not limited to these Examples.

Reference example 1

Activation of p38 MAPK signaling pathway in mouse CML stem cells In this reference example, phosphorylation states of p38MAPK threonine 180 residues and tyrosine 182 residues were analyzed using CML stem cells purified from a tetracycline-induced CML mouse model.

(1) Purification of mouse chronic myeloid leukemia (CML) stem cells and normal mouse hematopoietic stem cells using a cell sorter
(1-1) Tetracycline Inducible Mouse CML Model This CML mouse model is a transgenic mouse Scl-tTA mouse that expresses a tetracycline-regulated transcriptional activator (tTA) specifically in hematopoietic stem cells (purchased from Jackson Laboratory: # 6209) and a BCR-ABL1 transgenic mouse tetO-BCR-ABL1 mouse (purchased from Jackson Laboratories: # 6202) whose expression can be regulated by tTA (Blood 113, 1613- 1630 (2009)). Scl-tTA / tetO-BCR-ABL1 mice with two transgenes suppress the expression of BCR-ABL1 by administration of tetracycline derivative doxycycline (Dox) (20 mg / l; manufactured by Sigma) and discontinue Dox administration Can induce the expression of BCR-ABL1. Dox administration of this Scl-tTA · tetO-BCR-ABL1 double transgenic mouse was discontinued to induce BCR-ABL1 expression, and 5 weeks later, a CML-onset mouse was obtained.

(1-2) Purification of mouse hematopoietic stem cells and mouse CML stem cells As described above, Scl-tTA · tetO-BCR-ABL1 double transgenic mice were used as a CML mouse model, while littermate Scl- A tTA-only mouse transgenic was used as a healthy mouse.
Dox administration of these mice was discontinued, and 5 weeks later, bone marrow mononuclear cells were obtained from CML-onset mice and normal mice. These bone marrow mononuclear cells were divided into anti-CD4 (L3T4), anti-CD8 (53-6.7), anti-B220 (RA3-6B2), anti-TER119 (Ly-76), anti-Gr-1 (RB6-8C5), anti-Mac1 ( M1 / 70), anti-Sca-1 (E13-161.7), and anti-c-Kit (2B8) antibody (above, manufactured by BD bioscience), anti-CD150 / SLAM (TC15-12F12.2) -Pacific blue, anti-CD48 (HM48-1) -APC-Cy7 antibody (manufactured by BioLegend), anti-CD135 / Flk2 / Flt3 (A2F10) -biotin antibody (manufactured by eBiosciences), streptavidin-PE-Cy7 (BD Biosciences) And stained. From these stained mouse CML cells and normal bone marrow cells, using a cell sorter (FACS Aria III BD), mouse long-term CML stem cells (CD150 positive, CD135 negative, CD48 negative, cKit positive, differentiation marker, respectively) (CD4, CD8, B220, TER119, Mac1, Gr-1) negative, Sca-1 positive cells (hereinafter referred to as "CD150 ⁺ KLS cells")}, and long-term hematopoietic stem cells (CD150 ⁺ KLS cells) went.

(2) Activation of p38 MAPK signal pathway in CML stem cells (1-2) Using mouse CML stem cells purified with mouse normal hematopoietic stem cells, the phosphorylation state of p38MAPK was analyzed by Duolink PLA method (Olink) . Rabbit anti-phosphorylated p38 MAPK antibody (D13E1 #, manufactured by Cell Signaling) that specifically detects phosphorylation of threonine at position 180 and tyrosine at position 182 of p38 MAPK. 8690) and mouse anti-p38 MAPK antibody (Cell Signaling, 28D10 # 9216), and, as a control, activation of TGF-β signaling pathway, which has been reported to be activated in CML stem cells and normal hematopoietic stem cells Rabbit anti-phosphorylated Smad3 antibody (Abcam, ab51451) that specifically detects serine at position 423 and serine at position 425 of Smad3, and mouse anti-Smad3 antibody (Abcam, ab75512) Each combination was analyzed.
First, phosphorylation of Smad3, which shows activation of TGF-β signal, was confirmed in normal hematopoietic stem cells and CML stem cells (lower panel in FIG. 2). Next, as a result of analyzing the phosphorylation state of p38 MAPK, phosphorylated threonine 180 and tyrosine 182 showing activation of the p38 MAPK signal pathway were detected in CML stem cells (upper left of FIG. 2). Therefore, it was revealed that the p38 MAPK signaling pathway was activated in CML stem cells, unlike normal hematopoietic stem cells.

Inhibitory effect of p38 MAPK inhibitor on mouse CML stem cells in vitro
As described above, since it was revealed that the p38 MAPK pathway was activated in CML stem cells, the suppressive effect of CML stem cells by p38 MAPK inhibitors in vitro was analyzed.
In order to analyze the maintenance of CML stem cells in vitro, a co-culture system with a mouse mesenchymal cell line OP-9 cells that mimics the microenvironment supporting CML stem cells in vivo was used. The co-culture system with OP-9 cells was used to analyze the colony-forming ability of mouse CML stem cells treated with p38 MAPK inhibitors, and the inhibitory effect of p38 MAPK inhibitors on the maintenance of CML stem cells was analyzed.
First, 100,000 cells of mouse mesenchymal cell line OP-9 cells were cultured in a monolayer in a 24-well plate for 1 day. On this cell, 1,000 mouse long-term CML stem cell (CD150 + KLS) cells purified as described above were added. Several known p38 MAPK inhibitors Ly2228820 (purchased from Santacruz, final concentration 5 μM), VX-702 (purchased from Santacruz, final concentration 5 μM), BIRB 796 (purchased from Calbiochem, final concentration) 5 μM) (the structural formula of each compound is shown in FIG. 1) was added, and the cells were cultured at 37 ° C. for 5 days under 3% oxygen concentration conditions.
CML cells treated with these p38 MAPK inhibitors were recovered, and after remaining inhibitors were washed with phosphate buffer, 3% oxygen concentration in methylcellulose semi-solid medium (GFM3434; Stem cell technology) Under the conditions, the cells were cultured at 37 ° C. for 1 week to evaluate the colony forming ability.
As a result, it was clarified that the colony-forming ability of mouse long-term CML stem cells can be suppressed by treatment with a p38 MAPK inhibitor (FIG. 3A).

Combined effect of p38 inhibitor and TKI on mouse CML stem cells in vitro, and in order to verify the therapeutic effect on TKI-resistant CML stem cells, the above-mentioned p38 MAPK inhibitors (Ly2228820, VX-702, and BIRB 796), and The ability of CML stem cells to form colonies after co-treatment with imatinib mesylate was analyzed.
The mouse mesenchymal cell line OP-9 cells, 100,000 cells, were monolayer cultured in 24-well plates for 1 day. On this OP9 mesenchymal cell, 3,000 mouse long-term CML stem cells (CD150 + KLS) cells were added. The above-mentioned p38 MAPK inhibitor Ly2228820 (5 μM), VX-702 (5 μM), and BIRB 796 (5 μM) were added to the culture solution, and cultured at 37 ° C. for 3 days under 3% oxygen concentration conditions. Furthermore, imatinib mesylate (purchased from Axon Medchem, final concentration 1 μM) was added to this culture solution, and cultured at 37 ° C. under 3% oxygen concentration conditions for 4 days (total 5 days).
CML cells co-treated with p38 MAPK inhibitor and imatinib mesylate were collected, and after remaining inhibitors were washed with phosphate buffer, methylcellulose semisolid medium (GFM3434; manufactured by Stem cell technology) Then, the colony forming ability was evaluated by culturing at 37 ° C. for 1 week under the condition of 3% oxygen concentration.
As a result, it was revealed that the p38 MAPK inhibitor has an inhibitory effect on mouse CML stem cells under the condition of imatinib (FIG. 3B). That is, it is considered that the p38 MAPK inhibitor has an inhibitory effect on TKI-resistant CML stem cells.

Therapeutic effects of p38 MAPK inhibitors administered to mouse CML stem cell transplanted mice alone In order to analyze the therapeutic effects of p38 MAPK inhibitors on CML stem cells in vivo, mice transplanted with CML stem cells developed CML. In contrast, the p38 MAPK inhibitor Ly2228820 was administered alone, and the survival period of the mice was analyzed.

(1) Establishment of mouse CML stem cell transplantation model by BCRABL1-GFP gene transfer Bone marrow mononuclear cells were obtained from normal mice (C57BL / 6), and anti-CD4 (L3T4), anti-CD8 (53-6.7), anti-B220 (RA3 -6B2), anti-TER119 (Ly-76), anti-Gr-1 (RB6-8C5), anti-Mac1 (M1 / 70), anti-Sca-1 (E13-161.7), and anti-c-Kit (2B8) antibodies Was used for staining. From this mouse bone marrow mononuclear cell, using a cell sorter (FACS Aria III, manufactured by BD), negative differentiation markers (CD4, CD8, B220, TER119, Gr-1, Mac1) including mouse normal hematopoietic stem cells are negative. Kit positive and Sca-1 positive cell populations (hereinafter referred to as “KLS cells”) were purified. This cell population was divided into 100 ng / ml human TPO (Thrombopoietin; manufactured by PeproTech) and 100 ng / ml mouse SCF (Stem cell factor; manufactured by Wako Pure Chemical Industries), serum-free medium S-Clone (SF-O3 And cultured for 1 day. BCRABL1-GFP gene was introduced into the KLS cells using a retroviral vector. Recipient mice (9.0Gy) irradiated (9.0Gy) with bone marrow mononuclear cells (5x10 ⁵ cells per recipient mouse) of mice (C57BL / 6) obtained separately after culturing in cytokine-containing S-Clone medium for 1 day C57BL / 6) was transplanted (injected from the tail vein) (primary transplantation). For radiation irradiation, MBR-1520R-3 manufactured by Hitachi Medical Corporation was used, and the radiation was filtered with a 0.5 mm aluminum / 0.5 mm copper plate and irradiated at 0.45 to 0.55 Gy / min.

(2) Therapeutic effect of p38 MAPK inhibitor on CML stem cell transplanted mice
Control administration (artificial gastric juice: 900 ml double-distilled water, 2.0 g NaCl, 7 days after transplantation from 8 to 50 days after transplantation of hematopoietic stem cells introduced with the BCR-ABL1-GFP gene) HCl, 3.2 g pepsin) or Ly2228820 (prepared by diluting 1/100 of the 25 mg / ml DMSO solution with the above artificial gastric juice. Final concentration 0.25 mg / ml) Was orally administered every 3 days at a final concentration of 2.5 mg / kg. The survival period of these CML mice was analyzed.
As a result, the onset of CML was improved in the Ly2228820 administration group compared to the control group (FIG. 4). Therefore, p38 MAPK inhibitor is considered to have a therapeutic effect on CML stem cells in vivo.

Combined effect of p38 MAPK inhibitor and dasatinib on induced CML mice
(1) Administration protocol 1 (Ly2228820 simultaneous administration, daily administration)
The induced CML mouse model used in Reference Example 1 was used to evaluate the therapeutic effects of dasatinib alone and the combined administration of p38 MAPK inhibitor and dasatinib.
Using the above Scl-tTA / tetO-BCR-ABl1 double transgenic mouse, doxycycline administration was discontinued, and from day 0 to 30 days, dasatinib (trade name Spricel, 5 mg / kg / day, manufactured by Bristol Myers) Was administered orally. The Splicel-administered mice were divided into two groups, one group containing 200 μl of artificial gastric juice (containing 900 ml double-distilled water, 2.0 g NaCl, 7 ml conc. HCl, 3.2 g pepsin), and the other group Ly2228820 ( 200 μl artificial gastric fluid containing 0.25 mg / ml) was orally administered daily from day 0 to day 30 after CML induction at a final concentration of Ly2228820 of 2.5 mg / kg / day.
As a result, as compared with the dasatinib alone administration group, the dasatinib and Ly2228820 combination administration group did not show a clear life-prolonging effect of CML-induced mice (FIG. 5).

(2) Administration protocol 2 (Ly2228820 administration started 1 week later, administration every 2-3 days)
Since p38 MAPK is expressed in ubiquitous, and many side effects have been reported in various clinical trials of p38 MAPK inhibitors, p38 MAPK inhibitors were administered 2 days after daily administration (n = 15) or The administration interval was tried once every 3 days (n = 16). In addition, although the suppressive effect on CML stem cells of p38 inhibitors has been demonstrated, since the response to differentiated mature CML cells is not clear, first kill CML cells to some extent with dasatinib and then act on p38 MAPK inhibitors Administration of Ly2228820 was started one week after the start of dasatinib administration.
As a result, it became clear that the survival rate of CML-induced mice was improved in the dasatinib and Ly2228820 combination administration group compared to the dasatinib alone administration group (FIG. 6). In other words, combined use with a p38 MAPK inhibitor is expected to increase the therapeutic effect of dasatinib-resistant CML stem cells and suppress the onset of CML.

Inhibitory effect of p38 MAPK inhibitor on human CML stem cells in vitro In order to evaluate the therapeutic effect of p38 MAPK inhibitor on dasatinib-resistant human CML stem cells, it was derived from CML patients on mouse mesenchymal cell line OP-9 cells Co-culture of human CML stem cells was performed.
First, 100,000 cells of mouse mesenchymal cell line OP-9 cells were cultured in a monolayer in a 24-well plate for 1 day. On this cell, 6,000 human CML stem cells (well not treated with dasatinib) or 100,000 cells (well treated with dasatinib) were added. Ly2228820 was added to this culture solution at a final concentration of 5 μM, and cultured at 37 ° C. under 5% oxygen concentration conditions for 1 day. Dasatinib was added to this culture solution at a final concentration of 500 nM, and cultured at 37 ° C. under 5% oxygen concentration conditions for 2 days (3 days in total). After recovering the cells and washing away the remaining inhibitor with phosphate buffer, in a methylcellulose semi-solid medium (GF ⁺ H 4435; manufactured by Stem cell technology), the cells were isolated at 37 ° C under 5% oxygen concentration. After culturing for a week, colony forming ability was evaluated.
As a result, the p38 MAPK inhibitor showed an inhibitory effect on CML stem cells derived from human CML patients (FIG. 7 left (control)). Moreover, the combined use of dasatinib and a p38 MAPK inhibitor further suppressed the colony-forming ability as compared to dasatinib alone (FIG. 7, right (dasatinib)). In other words, p38 MAPK inhibitors are thought to have an inhibitory effect on TKI-resistant human CML stem cells. Therefore, p38 MAPK inhibitors may be therapeutic agents for human CML stem cells in CML patients.

According to the TKI treatment, CML stem cells (particularly TKI-resistant CML stem cells including T315I mutant BCR-ABL-expressing CML stem cells) that can escape eradication can be efficiently eliminated. In addition, combined use with TKI and multi-kinase inhibitors can improve the response to treatment, prevent relapse of CML and Ph ⁺ ALL, and reduce the dose of TKI and multi-kinase inhibitors Can be useful in reducing side effects. Furthermore, this is a new treatment method for recurrent CML patients for which no effective treatment method has been established so far.

Claims (8)

  A therapeutic agent for chronic myeloid leukemia, comprising a selective inhibitor of p38 MAPK signal.   The agent according to claim 1, wherein the inhibitor inhibits p38α and p38β.   The agent according to claim 1 or 2, wherein the inhibitor is selected from the group consisting of LY2228820, VX-702, BIRB 796 and salts thereof.   The agent according to claim 3, wherein the inhibitor is LY2228820 or a salt thereof.   The agent of any one of Claims 1-4 which is a removal agent of a chronic myeloid leukemia stem cell.   The agent according to any one of claims 1 to 4, which is an inhibitor of chronic myeloid leukemia stem cells resistant to a tyrosine kinase inhibitor.   The agent of any one of Claims 1-6 formed in combination with a tyrosine kinase inhibitor or a multikinase inhibitor.   The agent according to claim 7, wherein the tyrosine kinase inhibitor is imatinib, dasatinib, bosutinib, radotinib or nilotinib, and the multikinase inhibitor is KW2449 or ponatinib.