JP2016141669A - Chronic myeloid leukemia treating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a safe and effective chronic myeloid leukemia (CML) treating agent.SOLUTION: The CML treating agent is obtained by combining N-((4-([1,2,4]triazolo[1,5-α]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2- yl)methyl)-2-fluoroaniline (TEW-7197) or a salt thereof with 3-(imidazo[1,2-b]pyridazin-3-ylethynyl)-4-methyl-N-{4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide (ponatinib) or a salt thereof.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 relates to a therapeutic agent for CML, particularly a tyrosine kinase inhibitor resistant CML, comprising a combination of EW-7197 and ponatinib.

  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.

As a therapeutic agent for CML, a tyrosine kinase inhibitor for ABL (hereinafter also referred to as “TKI”) imatinib mesylate (hereinafter also referred to as “imatinib”) has been developed, which has significantly improved the treatment 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 and dasatinib, which have higher therapeutic effects, 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. (Non-Patent Document 1).

The multikinase inhibitor ponatinib was developed as a therapeutic agent for CML patients expressing TKI-resistant BCR-ABL1 (Non-patent Document 2). In the US and EU, CML that has become resistant or intolerant to TKI in the previous treatment Approved by the subject (trade name: Iclusig). 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. Moreover, when CML stem cells having a plurality of gene mutations (compound mutations) are generated from TKI-resistant CML stem cells as a developmental origin, this may cause further ponatinib resistance (Non-patent Document 3). Therefore, in order to cure CML, it is necessary to develop a therapeutic method for eradicating CML stem cells.
In addition, in a phase III trial in newly diagnosed CML patients, ponatinib showed higher efficacy earlier than imatinib, but severe arteriovenous blood coagulation in patients receiving ponatinib The clinical trial was discontinued due to the accumulation of cases. Therefore, in order to improve the success rate of treatment with ponatinib, there is a demand for a method for reducing the side effects while maintaining the effectiveness of ponatinib.

TEW-7197 (N-((4-([1,2,4] triazolo [1,5-α] pyridin-6-yl) -5- (6-methylpyridin-2-yl) -1H-imidazol- 2-yl) methyl) -2-fluoroaniline (also called “EW-7197” or “EW7197”) inhibits the serine / threonine kinase activity of TGF-β type I receptor protein (ALK5) Inhibits signal transduction mechanisms that depend on the expression (Patent Documents 1 and 2). TGFβ works protectively in early stages of tumor formation, but as cancer progresses, cancer exacerbation factors such as promoting epithelial-mesenchymal transition (EMT), interfering with immune surveillance, and promoting cancer cell growth Acts as Therefore, TEW-7197, which is an ALK5 inhibitor, has an effect of suppressing tumor cell proliferation and metastasis (see, for example, Patent Documents 1 and 2 and Non-Patent Document 4), and is approved by the National Institutes of Health (NIH). As a result, a phase I clinical trial for patients with solid cancer has already started.
However, there has been no specific report on the therapeutic effect of TEW-7197 against CML and other blood cancers, and the combined effect of TEW-7197 and ponatinib is not known at all.

International Publication No. 2012/002680 Pamphlet US Patent Application Publication No. 2011/0319408

Gorre, M.E. et al., Science, 293 (5531): 876-880 (2001) O'Hare, T. et al., Cancer Cell, 16 (5): 401-412 (2009) Zabriskie, M.S. et al., Cancer Cell, 26 (3): 428 (2014) Son, J.Y. et al., Mol. Cancer Ther., 13 (7): 1704-1716 (2014)

  An object of the present invention is to provide a novel CML therapeutic agent capable of suppressing CML stem cells. Another object of the present invention is to provide a novel CML therapeutic agent, particularly a TKI-resistant CML therapeutic agent, which can improve the therapeutic effect of ponatinib when used in combination with ponatinib.

  As a result of intensive studies to achieve the above object, the present inventors have found that TEW-7197 can remarkably suppress TKI-resistant CML stem cells by single administration, and that TEW-7197 alone can be administered in vivo. Although the therapeutic effect of CML is limited, it is possible to further enhance the therapeutic effect of ponatinib by combining TEW-7197 and ponatinib, and the administration of ponatinib can reduce the dose of ponatinib. The inventors have found that it can be reduced, and have completed the present invention.

That is, the present invention is as follows.
[1] N-((4-([1,2,4] triazolo [1,5-α] pyridin-6-yl) -5- (6-methylpyridin-2-yl) -1H-imidazol-2 -Yl) methyl) -2-fluoroaniline (TEW-7197) or a salt thereof and 3- (imidazo [1,2-b] pyridazin-3-ylethynyl) -4-methyl-N- {4-[(4 -Methylpiperazin-1-yl) methyl] -3- (trifluoromethyl) phenyl} benzamide (ponatinib) or a salt thereof, a therapeutic agent for chronic myelogenous leukemia (CML).
[2] The agent described in [1] above, wherein CML is resistant to a tyrosine kinase inhibitor (TKI).
[3] The agent described in [2] above, wherein the resistance to TKI is based on the T315I mutation of BCR-ABL.
[4] The agent according to any one of [1] to [3], which is for oral administration or intravenous administration.
[5] The agent according to [4] above, wherein the oral dose of ponatinib or a salt thereof is 15 to 30 mg / day.
[6] The agent according to [4] or [5] above, wherein the oral dose of TEW-7197 or a salt thereof is 30 to 200 mg / 2 to 4 days.
[7] The agent according to any one of [1] to [6] above, wherein the administration of ponatinib is started prior to the start of administration of TEW-7197 or a salt thereof.

  According to the present invention, CML stem cells that can escape eradication according to TKI treatment, particularly CML stem cells that are resistant to any conventional TKI, can be efficiently eliminated. Moreover, the combined use of TEW-7197 and ponatinib can prevent recurrence of CML, reduce the dose of ponatinib, and reduce side effects.

It is a figure which shows the examination result of the combined administration method of imatinib and TEW-7197 with respect to a CML mouse model. Imatinib and TEW-7197 were started simultaneously on day 8 after CML stem cell transplantation, and both imatinib and EW-7197 were administered daily. It is a figure which shows the examination result of the combined administration method of imatinib and TEW-7197 with respect to a CML mouse model. Imatinib administration was started on the 8th day after CML stem cell transplantation, TEW-7197 administration was started on the 15th day, and both imatinib and EW-7197 were administered every day. It is a figure which shows the examination result of the combined administration method of imatinib and TEW-7197 with respect to a CML mouse model. Imatinib administration was started on day 8 after CML stem cell transplantation, administration of TEW-7197 was started on day 15, imatinib was administered every day, and TEW-7197 was administered once every 3 days. It is a figure which shows the inhibitory effect of (A) TEW-7197 with respect to the mouse | mouth TKI resistant CML stem cell in vitro, and the combined use effect of (B) TEW-7197 and ponatinib. It is a figure which shows the combined use effect of TEW-7197 and ponatinib on the survival rate of the mouse | mouth which transplanted the TKI resistant CML stem cell. It is a figure which shows the effect of TEW-7197 on the survival rate of the CML stem cell in the mouse | mouth transplanted with the TKI resistant CML stem cell, and the combined use effect of TEW-7197 and ponatinib. It is a figure which shows the dose reduction effect of ponatinib by combined use of TEW-7197 and ponatinib, using the survival rate of the mouse transplanted with TKI-resistant CML stem cells as an index.

The present invention relates to a therapeutic agent for chronic myelogenous leukemia (CML) (hereinafter referred to as “the CML therapeutic agent of the present invention” or “the combination agent of the present invention”) comprising a combination of TEW-7197 or a salt thereof and ponatinib or a salt thereof. Say).
The term “CML therapeutic agent” is intended not only for the treatment of CML in the chronic phase, but also for the treatment of CML in the acute phase or transition phase, prevention of acute transformation, prevention of recurrence of CML, treatment of recurrent CML. Used in meaning.

  TEW-7197 (N-((4-([1,2,4] triazolo [1,5-α] pyridin-6-yl) -5- (6-methylpyridin-2-yl)) used in the present invention -1H-imidazol-2-yl) methyl) -2-fluoroaniline) inhibits the serine / threonine kinase activity of TGF-β type I receptor protein (ALK5) and has a signaling mechanism that depends on TGF-β. Inhibit. The salt of TEW-7197 is not particularly limited as long as it is a pharmaceutically acceptable salt, but an acid addition salt such as hydrochloride is preferable. TEW-7197 can be manufactured by the method described in Patent Document 1.

When TEW-7197 is used as a pharmaceutical, it can be prepared as a pharmaceutical composition as it is or by mixing with a known pharmaceutically acceptable carrier. 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. Preferably, it is oral administration or intravenous administration, more preferably oral administration.
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 dose of TEW-7197 in the concomitant drug of the present invention varies depending on the route of administration, the subject of administration or the age, weight, symptoms, etc. of the patient and cannot be generally specified. The amount of TEW-7197 is about 5 to 1500 mg, preferably about 10 to 500 mg, more preferably about 30 to 200 mg, once a day to once every 5 days, preferably once every 2 days to 4 days. It can be administered once, more preferably once every 3 days. From the results of Examples and Reference Examples described later, TEW-7197 suppresses CML stem cells by promoting differentiation of CML stem cells, but may not suppress differentiated mature CML cells. On the other hand, CML may lead to exacerbation of CML. Since the concomitant drug of the present invention has a remarkable concomitant effect compared with the single administration of TEW-7197, the dose of TEW-7197 used in the concomitant drug is the amount usually used in single administration (No. I In the phase study, the dose can be reduced more than 30/60/100/140/200 mg / day (2 days drug withdrawal cycle after 5 consecutive days administration).

  Ponatinib (3- (imidazo [1,2-b] pyridazin-3-ylethynyl) -4-methyl-N- {4-[(4-methylpiperazin-1-yl) methyl] -3-] used in the present invention (Trifluoromethyl) phenyl} benzamide) is a multikinase inhibitor that inhibits multiple kinase activities including BCR-ABL (Cancer Cell, 16: 401-412 (2009)), such as imatinib, nilotinib, dasatinib It is also effective against T315I mutant BCR-ABL-expressing CML cells in which TKI is ineffective. The salt of ponatinib is not particularly limited as long as it is a pharmaceutically acceptable salt, but an acid addition salt such as hydrochloride is preferable.

When ponatinib is used as a pharmaceutical product, it can be prepared as a pharmaceutical composition as it is or mixed with a known pharmaceutically acceptable carrier. 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. Preferably, it is oral administration or intravenous administration, more preferably oral administration.
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 those similar to TEW-7197.

  The dose of ponatinib in the concomitant drug of the present invention differs depending on the route of administration, the subject of administration or the age, weight, symptoms, etc. of the patient, but cannot generally be defined. 5 to 100 mg, preferably about 10 to 50 mg, more preferably about 15 to 30 mg can be administered once a day or several times a day. The concomitant drug of the present invention has a remarkable concomitant effect as compared to ponatinib alone, so the dose of ponatinib used in the concomitant is the amount normally used in single administration (45 mg / day as the starting dose). ) Can be reduced. Ponatinib inhibits not only BCR-ABL but also signaling by other kinases such as Aurora kinase and FLT3, thus potentially increasing the risk of side effects, and in fact because of the accumulation of thrombotic events, the newly diagnosed chronic phase CML Phase III trial for was canceled. According to the combined use with TEW-7197, the dose of ponatinib can be reduced, and a safer CML therapeutic agent with reduced side effects can be provided.

  In the concomitant drug of the present invention, TEW-7197 and ponatinib may be administered as a combination or as separate preparations simultaneously or over time. However, from the results of Examples and Reference Examples described later, TEW-7197 suppresses CML stem cells by promoting differentiation of CML stem cells, but may not suppress differentiated mature CML cells. Alternatively, if TEW-7197 is started prior to ponatinib, CML may be exacerbated. Therefore, it is preferable to start administration of ponatinib first, after suppressing the mature CML cells to some extent to improve the disease state, and then start administration of TEW-7197. For example, administration of TEW-7197 can be started 3 to 14 days, preferably 5 to 10 days, more preferably about 7 days after the start of ponatinib administration.

The CML patient to which the CML therapeutic agent 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 phase or transition phase. Since TEW-7197 is particularly useful for the removal of CML stem cells, the CML therapeutic agent of the present invention containing TEW-7197 is particularly resistant to TKI such as imatinib, or an initially refractory CML patient, This is effective for patients with relapse who have CML stem cells remaining after TKI treatment. 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 BCR-ABL dependent and independent. The former includes amplification of BCR-ABL gene, point mutation (eg, phosphate binding loop (p-loop) in kinase domain (eg, G250E, Q252H, Y253F, Y253H, E255K, E255V, etc.), imatinib binding site ( For example, T315I, T315A, F317L, F317V, etc.), activation loop, catalyst loop). On the other hand, the latter includes increased activity downstream of the BCR-ABL signal, increased activity of signal transduction pathways not involved in BCR-ABL, enhanced discharge of imatinib by multidrug resistance (MRD) protein, imatinib binding protein (α1-acid glycoprotein ) And the like. Of these, it has been reported that the kinase domain point mutation is the main one (Cancer Cell, 2: 117-125 (2002)), but TEW-7197 can suppress CML stem cells and thus has a mutant BCR. -It may be effective not only for ABL-expressing CML but also for 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.
Ponatinib has been shown to be effective against T315I mutant BCR-ABL expressing cells in which the threonine residue at position 315, which is the imatinib binding site of BCR-ABL, is replaced with isoleucine. The number of cases leading to complete remission is limited, and the development of a more powerful therapeutic agent for T315I mutant BCR-ABL-expressing CML cells, a concomitant drug that can complement the therapeutic effect of ponatinib, has been desired. TEW-7197, on the other hand, has a significant antiproliferative effect on imatinib-resistant CML stem cells that express the T315I mutant BCR-ABL, but it is effective in treating CML in vivo using improved survival as an index. May be limited. However, by using TEW-7197 together with ponatinib, it is possible to further suppress the growth of CML stem cells than when ponatinib is administered alone, so that the appearance of ponatinib-resistant CML stem cells can be significantly suppressed, Thereby, the therapeutic effect of ponatinib can be further enhanced. Therefore, the concomitant drug of the present invention is particularly effective against T315I mutant BCR-ABL-expressing CML, which has been extremely refractory.
In addition, TEW-7197 exhibits an excellent CML stem cell growth inhibitory effect both in the single administration and in combination with ponatinib under culture conditions that mimic the cancer microenvironment. Since TGF-β produced from a cancer microenvironment (niche cell) is thought to contribute to the survival of CML stem cells and acquisition of imatinib resistance, the concomitant drug of the present invention provides BCR- It can be a particularly effective therapeutic agent for imatinib-resistant CML based on increased activity of a signal transduction pathway not involved in ABL.

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

Reference example 1

Examination of administration method of TEW-7197 in combination therapy with TKI
In the combined treatment of TKI and TEW-7197 for CML-onset mice, the most effective method of combined administration of TEW-7197 was examined.
For mice that developed CML by transplanting hematopoietic stem cells introduced with the BCR-ABL1-GFP gene,
1) Combined treatment by daily administration of imatinib (manufactured by Novartis, 100 mg / kg / day) and TEW-7197 (5 mg / kg / day) 8 days after transplantation (Fig. 1),
2) Imatinib (100 mg / kg / day) from 8 days after transplantation, plus combined treatment by daily administration of TEW-7197 (2.5 mg / kg / day) from 15 days after transplantation (Figure 2), and
3) Imatinib (100 mg / kg / day) 8 days after transplantation, plus 15 days after transplantation, TEW-7197 (2.5 mg / kg / 3 days) once every 3 days (Fig. 3)
The therapeutic effect of was analyzed.
As a result, compared to treatment with imatinib and TEW-7197 on the same day (Fig. 1), delayed administration of TEW-7197 7 days after imatinib administration had an effect of improving CML recurrence (Fig. 2). ). Furthermore, in TEW-7197 delayed administration 7 days after the start of imatinib administration, the recurrence of CML was caused by administration of TEW-7197 once every 3 days (FIG. 3), compared with daily administration of TEW-7197 (FIG. 2). It was revealed that it can be effectively suppressed.
The delay in the start of administration of TEW-7197 in the combination therapy with TKI and the improvement in the effect of the combination therapy due to the decrease in the number of administrations are unexpected results. As a reason why such combined treatment effect was obtained, TEW-7197 is assumed to have an effect of promoting differentiation from CML stem cells to mature CML cells, and by performing TKI treatment prior to TEW-7197 treatment, It is possible to synergistically enhance the combined effect of TKI and TEW-7197. Furthermore, even during the combined treatment with TKI and TEW-7197, the suppressive effect of CML stem cells (ie, the effect of promoting differentiation into mature CML cells) can be sufficiently treated with administration once every 3 days. it is conceivable that.
Based on the above results, in the following in vivo combination administration test of ponatinib and TEW-7197, except for replacing imatinib (100 mg / kg / day) with ponatinib (100 mg / kg / day), the administration protocol of 3) above It was decided to adopt.

Effect of TEW-7197 on mouse TKI resistant CML stem cells in vitro
In order to mimic the microenvironment supporting CML stem cells in vivo and analyze the inhibitory effect of TEW-7197 and the combined effect of TEW-7197 and ponatinib in vitro, the mouse mesenchymal cell line OP- The influence on the maintenance of TKI resistant mouse CML stem cells was analyzed by co-culture experiment with 9 cells.
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. TKI-resistant mouse CML stem cells (T315I mutant BCR-ABL1-GFP ⁺ cKit ⁺ lineage ^- Sca1 ⁺ (KLS) CML-MPPs (Naka K, et al. Nature. 2010; 463: 676-680)) 1,000 cells on this cell Was added. Further, TEW-7197 (provided by MedPacto, final concentration 5 μM) was added to this culture solution, and the cells were cultured at 37 ° C. under 3% oxygen concentration conditions for 3 days. Similarly, imatinib mesylate (purchased from Axon Medchem, final concentration 1 μM) was added as a control, and the cells were cultured for 3 days at 37 ° C. under 3% oxygen concentration conditions.

T315I mutant CML cells treated with TEW-7197 or imatinib mesylate as described above were recovered, and the remaining inhibitor was washed with phosphate buffer, followed by methylcellulose semi-solid medium (GFM3434; manufactured by Stem cell technology) Among them, the colony-forming ability was evaluated by culturing at 37 ° C. for 1 week under 3% oxygen concentration condition.
As a result, as expected, imatinib treatment did not show an inhibitory effect on the colony forming ability of TKI-resistant mouse CML stem cells. On the other hand, TEW-7197 was found to have an inhibitory effect on TKI-resistant mouse CML stem cells (FIG. 4A).

Combined effect of TEW-7197 and ponatinib on mouse CML stem cells in vitro
In order to analyze the combined effect of TEW-7197 and ponatinib on TKI-resistant mouse CML stem cells, mouse CML stem cells were co-cultured on the aforementioned mouse mesenchymal cell line OP-9 cells.
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 cells of TKI resistant mouse CML stem cells (well not treated with ponatinib) or 3,000 cells (well treated with ponatinib) were added. TEW-7197 was added to this culture solution at a final concentration of 5 μM, and cultured at 37 ° C. under 3% oxygen concentration conditions for 1 day. Further, ponatinib (manufactured by Selleck Chemicals) was added to this culture solution at a final concentration of 1 μM, and cultured for 2 days at 37 ° C. under 3% oxygen concentration conditions (3 days in total). After recovering the cells and washing away the remaining inhibitor using phosphate buffer, the cells are cultured in methylcellulose semi-solid medium (GFM3434; manufactured by Stem cell technology) at 37 ° C for 1 week under conditions of 3% oxygen concentration. The colony forming ability was evaluated.
As a result, the combined use of ponatinib and TEW-7197 suppressed colony-forming ability as compared to ponatinib alone (FIG. 4B). That is, TEW-7197 is considered to have an inhibitory effect on TKI-resistant CML stem cells.

Effect of combined use of TEW-7197 and ponatinib on TKI-resistant mouse CML stem cells in vivo To analyze the therapeutic effect of TEW-7197 on TKI-resistant CML stem cells in vivo The administration of TEW-7197, single administration of ponatinib, and combined administration of TEW-7197 and ponatinib were performed to evaluate the survival time of mice and recurrence after the end of treatment.
Control administration (artificial gastric juice; 900 ml double-distilled water) from 8 days to 60 days after transplantation to mice that developed TKI-resistant CML by transplanting the above-mentioned hematopoietic stem cells introduced with the T315I BCR-ABL1-GFP gene , 2.0 g NaCl, 7 ml conc. HCl, 3.2 g pepsin) or ponatinib (15 mg / kg / day, AP24534, Selleck Chemicals) (dissolved in artificial gastric juice) was orally administered. Divide these ponatinib-treated mice into two groups, one group with 200 μl artificial gastric juice, the other group with 200 μl artificial gastric fluid containing TEW-7197 (0.2 mg / ml), TEW-7197 final concentration 2.5 mg Orally administered every 3 days from the 15th day to the 60th day after transplantation. The survival period of these mice and the recurrence of ponatinib resistance after ponatinib administration were analyzed up to 100 days after transplantation. This analysis examined the therapeutic effect of TEW-7197 administration in maintaining TKI-resistant CML stem cells resistant to ponatinib.
As a result, the recurrence of CML after the end of ponatinib treatment was improved in the combination administration group of TEW-7197 and ponatinib compared to the ponatinib alone administration group (FIG. 5). Therefore, TEW-7197 has an inhibitory effect on ponatinib-resistant CML stem cells in vivo, and is considered to be able to enhance the therapeutic effect of ponatinib.

Effect of TEW-7197 on TKI-resistant CML stem cells in vivo Analysis of the therapeutic effect of TEW-7197 on TKI-resistant CML stem cells and the inhibitory effect on TKI-resistant CML stem cells that are resistant to ponatinib in vivo Therefore, the remaining TKI-resistant CML stem cells were evaluated by administering TEW-7197 alone or in combination with TEW-7197 and ponatinib to mice that developed T315I CML.
Oral administration of ponatinib (15 mg / kg / day, AP24534, manufactured by Selleck Chemicals) was performed from 8 days to 30 days after transplantation in mice with TKI-resistant CML in which the above T315I BCR-ABL1-GFP gene was introduced It was. This ponatinib-treated mouse is divided into 2 groups, 1 group contains 200 μl 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 contains TEW- 200 μl artificial gastric juice containing 7197 (0.2 mg / ml) was orally administered every 3 days from the 15th day to the 30th day after transplantation at a final concentration of 2.5 mg / kg / 3 days of TEW-7197. Bone marrow mononuclear cells were isolated from the bone marrow of these mice, and the frequency of KLS cells in GFP-positive leukemia cells expressing T315I BCR-ABL1 was analyzed.
As a result, in the mice administered with TEW-7197, the frequency of T315I BCR-ABL1-expressing KLS cells was remarkably reduced (second bar graph from the upper right of FIG. 6A and the left from FIG. 6B). In other words, it is considered that TKI-resistant CML stem cells can be treated by TEW-7197 single agent administration. In Example 3, the improvement in the survival rate of mice by TEW-7197 single agent administration was limited, and this result was unexpected considering that it was equivalent to the untreated group after the end of administration. is there. TEW-7197 is not expected to suppress mature CML cells, and factors other than maintenance of TKI-resistant CML stem cells, such as proliferation of CML cells that have already differentiated from TKI-resistant CML stem cells, prevented improvement in survival rate. It is considered a thing.
On the other hand, in the group administered with ponatinib alone, T315I BCR-ABL1-expressing KLS cells remained. Therefore, it is considered that ponatinib-resistant TKI-resistant CML stem cells exist and cannot be excluded (FIG. 6A, lower left, FIG. 6). 6B 3rd bar graph from the left)
However, in the TEW-7197 and ponatinib combination administration group, the remaining ponatinib-resistant TKI-resistant CML stem cells decreased compared to the ponatinib alone administration group (FIG. 6A lower right, FIG. 6B fourth bar from the left). Therefore, TEW-7197 has the effect of suppressing the appearance of ponatinib-resistant TKI-resistant CML stem cells in vivo, and it is considered that the therapeutic effect of ponatinib can be enhanced.

Reduction effect of ponatinib dose in combination with TEW-7197
In the treatment of mice with TKI-resistant CML, it was investigated whether the dose of ponatinib could be suppressed by administering TEW-7197 and ponatinib in combination compared to ponatinib alone. TKI-resistant CML mice were treated with ponatinib (15 mg / kg / day) alone or with ponatinib (3 mg / kg / day) and TEW-7197 (2.5 mg / kg / 3 day) for survival did.
From mice 8 days to 60 days after transplantation to mice that developed TKI-resistant CML by transplanting the above-mentioned T315I BCR-ABL1-GFP gene-transduced hematopoietic stem cells 1) Control administration (artificial gastric fluid: 900 ml twice Distilled water, 2.0 g NaCl, 7 ml conc. HCl, 3.2 g pepsin), 2) ponatinib (15 mg / kg / day, AP24534, sold by Selleck Chemicals) (dissolved in artificial gastric juice), and 3) ponatinib (3 mg / kg / day) was orally administered. Furthermore, from day 15 to day 60 after transplantation, 1) control-administered mice, and 2) ponatinib (15 mg / kg / day) mice received 200 μl of artificial gastric juice, 3) ponatinib (3 mg / kg / day) In addition, TEW-7197 (2.5 mg / kg / 3 days) was additionally administered orally to the administration mice. The survival period of these mice and the recurrence of ponatinib resistance after ponatinib administration were analyzed up to 100 days after transplantation. Based on this analysis, the effect of reducing the dose of ponatinib by administration of TEW-7197 was examined.
As a result, there was no significant difference in the survival time of the mice of the ponatinib (15 mg / kg / day) single administration group and the combination administration group of TEW-7197 and ponatinib (3 mg / kg / day) (FIG. 7). Thus, TEW-7197 could enhance the therapeutic effect of ponatinib and thereby reduce the dose of ponatinib.

  By administration of TEW-7197, ponatinib-resistant TKI-resistant CML stem cells (TKI-resistant CML stem cells including T315I mutant BCR-ABL1-expressing CML stem cells) that cannot be eradicated by ponatinib treatment can be efficiently eliminated. Therefore, the combined use of TEW-7197 and ponatinib can further improve the response to treatment, provide a preventive effect on the recurrence of CML, reduce the dose of ponatinib, and extremely reduce side effects. Useful. Furthermore, this is a new treatment method for recurrent CML patients for which no effective treatment method has been established so far.

Claims (7)

  N-((4-([1,2,4] triazolo [1,5-α] pyridin-6-yl) -5- (6-methylpyridin-2-yl) -1H-imidazol-2-yl) Methyl) -2-fluoroaniline (TEW-7197) or a salt thereof and 3- (imidazo [1,2-b] pyridazin-3-ylethynyl) -4-methyl-N- {4-[(4-methylpiperazine -1-yl) methyl] -3- (trifluoromethyl) phenyl} benzamide (ponatinib) or a salt thereof, a therapeutic agent for chronic myelogenous leukemia (CML).   The agent according to claim 1, wherein the CML is resistant to a tyrosine kinase inhibitor (TKI).   The agent according to claim 2, wherein the resistance to TKI is based on the T315I mutation of BCR-ABL.   The agent according to any one of claims 1 to 3, which is for oral administration or intravenous administration.   The agent according to claim 4, wherein the oral dose of ponatinib or a salt thereof is 15 to 30 mg / day.   The agent according to claim 4 or 5, wherein the oral dose of TEW-7197 or a salt thereof is 30 to 200 mg / 2 to 4 days.   The agent according to any one of claims 1 to 6, wherein administration of ponatinib is started prior to the start of administration of TEW-7197 or a salt thereof.