Therapeutic agent for myelofibrosis

Abstract

To provide a therapeutic agent for myelofibrosis that enables inhibition or amelioration of myelofibrosis.SOLUTION: A therapeutic agent for myelofibrosis for administration to a patient with myelofibrosis, the agent containing as an active constituent an HMG-CoA reductase inhibitor. The patient may carry a JAK2 mutation. The myelofibrosis may result from abnormal proliferation of fibrocytes derived from blood. The therapeutic agent may be administered together with a JAK1 / 2 inhibitor. The HMG-CoA reductase inhibitor may be at least one selected from pitavastatin, atorvastatin, cerivastatin, rosuvastatin, fluvastatin, simvastatin, pravastatin, lovastatin, and mevastatin, and pharmaceutically acceptable salts thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for myelofibrosis, which comprises an HMGCoA reductase inhibitor. [Background technology]

[0002] Myelofibrosis (primary myelofibrosis, post-polycythemia vera myelofibrosis, post-essential thrombocythemia myelofibrosis) is a myeloproliferative neoplasm that develops when genetic mutations such as JAK2 mutations occur in hematopoietic stem cells. According to Western statistics, the incidence rate of primary myelofibrosis is 0.3-0.52 per 100,000 population per year, and the number of patients in Japan is estimated to be between 1,000 and 2,000. Patients experience systemic symptoms such as bone marrow fibrosis, hematopoietic failure (anemia, thrombocytopenia), massive splenomegaly, fever, night sweats, and weight loss, resulting in a reduced quality of life.

[0003] Hematopoietic stem cell transplantation is a curative treatment, but due to its prevalence in elderly patients, only 10% of patients are eligible for transplant. The JAK2 inhibitor ruxolitinib improves splenomegaly and systemic symptoms and extends median survival from four to five years. Currently, ruxolitinib is administered as standard treatment for many patients with myelofibrosis, but it does not sufficiently improve the underlying cause of the disease, bone marrow fibrosis. Even in patients who take ruxolitinib for a long period of time, the improvement rate of fibrosis remains at 36%. Furthermore, patients receiving ruxolitinib often experience worsening anemia and thrombocytopenia, often necessitating discontinuation or dose reduction. This is due to ruxolitinib's inhibition of erythropoietin and thrombopoietin signaling.

[0004] In Japan, ruxolitinib is the only drug approved for the treatment of myelofibrosis. In the United States, the JAK2 inhibitors fedratinib, pacritinib, and momelotinib are also approved. However, like ruxolitinib, they are poor at improving bone marrow fibrosis.

[0005] Bone marrow fibrosis is classified into four stages, from Grade 0 to Grade 3. Grade 2 or higher fibrosis is a prognostic factor independent of the International Prognostic Classification System (IPSS) and is a biomarker reflecting disease activity. By improving bone marrow fibrosis, the bone marrow microenvironment is normalized, normal hematopoiesis is restored, and anemia is improved, which is expected to improve the prognosis of myelofibrosis. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Shide K, Shimoda HK, Kumano T, Karube K, Kameda T, Takenaka K, et al. Development of ET, primary myelofibrosis and PV in mice expressing JAK2 V617F. Leukemia. 2008;22(1):87-95. [Non-patent document 2] Ozono Y, Shide K, Kameda T, Kamiunten A, Tahira Y, Sekine M, et al. Neoplastic fibrocytes play an essential role in bone marrow fibrosis in Jak2V617F-induced primary myelofibrosis mice. Leukemia. 2021;35(2):454-67. Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have created Jak2 mutant mice that develop bone marrow fibrosis, anemia, and splenomegaly as a mouse model reflecting the pathology of human myelofibrosis, and have studied the mechanism of fibrosis (Non-Patent Document 1). Recent studies have revealed that bone marrow fibrosis is not caused by the reactive proliferation of fibroblasts, which are mesenchymal cells as previously thought, but by neoplastic fibrocytes that differentiate from Jak2 mutant monocytes (Non-Patent Document 2). Because ruxolinitib does not inhibit the proliferation of neoplastic fibrocytes in vitro, new therapeutic agents for myelofibrosis that use drugs that inhibit the proliferation of neoplastic fibrocytes are needed.

[0008] The present invention provides a therapeutic agent for myelofibrosis that can suppress or ameliorate myelofibrosis. [Means for solving the problem]

[0009] The present invention includes the following aspects. [1] A drug for treating myelofibrosis that contains an HMGCoA reductase inhibitor as its active ingredient and is administered to patients with myelofibrosis. [2] The myelofibrosis therapeutic agent according to [1], wherein the myelofibrosis is primary myelofibrosis, post-polycythemia vera myelofibrosis, or post-essential thrombocythemia myelofibrosis. [3] The myelofibrosis therapeutic agent described in [1] or [2], wherein the patient has a JAK2 mutation. [4] The therapeutic agent for myelofibrosis according to any one of [1] to [3], wherein the myelofibrosis is caused by proliferation of blood-derived fibrocytes. [5] The therapeutic agent for myelofibrosis according to any one of [1] to [4], which is used in combination with at least one of a JAK1 inhibitor and a JAK2 inhibitor. [6] The myelofibrosis therapeutic agent according to any one of [1] to [5], wherein the HMG-CoA reductase inhibitor is one or more selected from pitavastatin, atorvastatin, cerivastatin, rosuvastatin, fluvastatin, simvastatin, pravastatin, lovastatin, and mevastatin, and pharmaceutically acceptable salts thereof.

[0010] The therapeutic agent for myelofibrosis of the present invention may be used not only for the purpose of treating myelofibrosis but also for the purpose of preventing myelofibrosis. A therapeutic method related to the present invention includes a therapeutic method in which an effective amount of the therapeutic agent for myelofibrosis of the present invention is administered to a patient suffering from myelofibrosis. The use invention related to the present invention includes the use of an HMGCoA reductase inhibitor for the manufacture of a therapeutic agent for myelofibrosis. The invention of the present invention relates to an HMGCoA reductase inhibitor for use in the treatment of myelofibrosis. [Effects of the Invention]

[0011] The therapeutic agent for myelofibrosis of the present invention can suppress or improve myelofibrosis. [Brief explanation of the drawings]

[0012] [Figure 1] The specific effects of six statins on the proliferation of fibrocytes differentiated from hematopoietic cells derived from a myelofibrosis model mouse were investigated in vitro. [Figure 2] In vitro study of the effects of three statins on nonspecific inhibitory activity of hematopoietic cells. [Figure 3] Results of pitavastatin administration on bone marrow fibrosis in a myelofibrosis model mouse. [Figure 4] Results of pitavastatin administration on peripheral blood Hb concentration and bone marrow nucleated cell count (NCC) in myelofibrosis model mice. [Figure 5] Results of pitavastatin administration on organ weight in myelofibrosis model mice. [Figure 6] Results of pitavastatin administration on weight changes over time in myelofibrosis model mice. [Figure 7] In vitro results show that pitavastatin inhibits the proliferation of fibrocytes derived from human myelofibrosis patients. [Figure 8]In vitro results show that pitavastatin inhibits the proliferation of fibrocytes differentiated from hematopoietic cells derived from myelofibrosis model mice by inhibiting the synthesis of mevalonate. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present inventors searched for drugs that inhibit the proliferation of neoplastic fibrocytes differentiated from Jak2 (Janus kinase 2) mutant monocytes. Among 1,630 existing drugs in the existing drug library held by the Drug Discovery Institute, Graduate School of Pharmaceutical Sciences, The University of Tokyo, we identified nine drugs that inhibited the proliferation of Jak2 mutant fibrocytes by 80% or more. These nine drugs included atorvastatin, cerivastatin, and pitavastatin. These statins are HMG-CoA reductase inhibitors, known to lower blood cholesterol levels by inhibiting the activity of HMG-CoA reductase (hydroxymethylglutaryl-CoA reductase).

[0014] (1) Administration of these statins inhibited the proliferation of Jak2 mutation-positive fibrocytes, and (2) administration of mevalonate, a product of HMG-CoA reductase, together with these statins did not inhibit the proliferation of these fibrocytes. Therefore, HMG-CoA reductase inhibitors function as inhibitors of the proliferation of Jak2 mutation-positive fibrocytes and serve as therapeutic agents for myelofibrosis. That is, one aspect of the present invention is a therapeutic agent for myelofibrosis that contains an HMG-CoA reductase inhibitor as an active ingredient and is administered to patients with myelofibrosis.

[0015] As shown in the test example below, pitavastatin had the greatest inhibitory activity against Jak2 mutation-positive fibrocytes among the six statins (see Figure 1). Furthermore, pitavastatin had the smallest nonspecific inhibitory activity against hematopoietic cells in vitro among the three statins (see Figure 2). For these reasons, pitavastatin was selected as a candidate drug for clinical development.

[0016] Administration of pitavastatin to Jak2 mutant mice improved bone marrow fibrosis (see Figure 3), anemia (see Figure 4, left), and splenomegaly (see Figure 5), without affecting body weight (see Figure 6). Administration of ruxolinitinib to Jak2 mutant mice did not affect the suppression of bone marrow fibrosis (see Figure 3). Administration of pitavastatin and ruxolinitinib together to Jak2 mutant mice did not affect the suppression of bone marrow fibrosis by pitavastatin (see Figure 3 and Figure 4, right).

[0017] Pitavastatin also inhibited the proliferation of fibrocytes derived from human myelofibrosis patients in vitro (see FIG. 7).

[0018] When mevalonate, a product of HMG-CoA reductase, was administered together with pitavastatin, the growth inhibition of Jak2 mutation-positive fibrocytes disappeared (see FIG. 8).

[0019] Pitavastatin is a drug widely used for dyslipidemia, and its safety when used in combination with ruxolitinib is expected to be similar to that of ruxolitinib alone. Pitavastatin has the potential to be a curative treatment, eliminating bone marrow fibrosis. It can also improve anemia and reduce transfusions, potentially improving quality of life. It also allows for the administration of sufficient doses of ruxolitinib, which is difficult to achieve with sufficient therapeutic intensity due to bone marrow failure. This may reduce tumor clones and improve survival.

[0020] An example of a method for administering pitavastatin and ruxolitinib in combination is to orally administer pitavastatin once a day and ruxolitinib twice a day.

[0021] From the above, the therapeutic agent for myelofibrosis of the present invention is considered to be an effective drug for treating all of primary myelofibrosis, post-polycythemia vera myelofibrosis, and post-essential thrombocythemia myelofibrosis, because these are diseases in which differentiated blood cells proliferate clonally due to abnormalities in hematopoietic stem cells and exhibit similar clinical symptoms. As for driver mutations, JAK2 mutations are seen in almost all cases of post-polycythemia vera myelofibrosis and approximately 50% of cases of post-essential thrombocythemia myelofibrosis and primary myelofibrosis. CALR mutations are seen in 20-25% of cases of post-essential thrombocythemia myelofibrosis and primary myelofibrosis, and MPL mutations are seen in approximately 5% of cases of post-essential thrombocythemia myelofibrosis and primary myelofibrosis.

[0022] Primary myelofibrosis (PMF) In the early stages of the disease, the bone marrow shows an increase in blood cells, mainly megakaryocytes. As the disease progresses, the bone marrow becomes fibrotic, causing hematopoietic failure (anemia, thrombocytopenia) and massive splenomegaly. The median survival time is approximately 4 years.

[0023] polycythemia vera (PV) It causes pancytosis, mainly of red blood cells. The prognosis is relatively good. Approximately 5% of patients progress to post-polycythemia vera myelofibrosis (post-PV MF).

[0024] Essential thrombocythemia (ET) It is a disease in which platelets increase, and the prognosis is relatively good. Approximately 5% of patients progress to post-essential thrombocythemia myelofibrosis (post-ETMF).

[0025] The therapeutic agent for myelofibrosis of the present invention is considered to be highly effective in patients with JAK2 mutations, because abnormalities in the JAK-STAT pathway involving JAK (Janus kinase) are known to be one of the causes of myelofibrosis, and as shown in the test examples described below, administration of the agent to Jak2 mutant mice showed a therapeutic effect on myelofibrosis.

[0026] Myelofibrosis patients with abnormalities in the JAK-STAT pathway may be administered JAK1 / 2 inhibitors (such as ruxolinitinib), which are the current standard of care. The therapeutic agent for myelofibrosis of the present invention may be used in combination with at least one of a JAK1 inhibitor and a JAK2 inhibitor, because the therapeutic agent for myelofibrosis of the present invention suppresses myelofibrosis without being affected by a JAK1 / 2 inhibitor, as shown in the test examples described below (the collagen I-positive cell area, which reflects myelofibrosis, is maintained low, and the number of nucleated cells (NCC) in the bone marrow is maintained high).

[0027] The therapeutic agent for myelofibrosis of the present invention is considered to be particularly effective as a therapeutic agent for myelofibrosis caused by the abnormal proliferation of fibrocytes derived from hematopoietic cells, because the abnormal proliferation of fibrocytes derived from hematopoietic cells is considered to be the true nature of myelofibrosis (Non-Patent Document 2), and as shown in the test examples described below, it has been confirmed that an HMG-CoA reductase inhibitor inhibited the proliferation of fibrocytes derived from blood cancer in vitro.

[0028] The HMG-CoA reductase inhibitor contained in the therapeutic agent for myelofibrosis of the present invention may be one or more selected from pitavastatin, atorvastatin, cerivastatin, rosuvastatin, fluvastatin, simvastatin, pravastatin, lovastatin, and mevastatin. In this case, an acid group such as a carboxy group present in the molecule of these statins may form a pharmaceutically acceptable salt.

[0029] The therapeutic agent for myelofibrosis of the present invention may be a pharmaceutical composition containing, in addition to the active ingredient, other auxiliary ingredients contained in conventional common therapeutic agents. For example, it can be formulated using additives such as fillers, lubricants, flavoring agents, colorants, coating agents, disintegrants, and glidants. The therapeutic agent for myelofibrosis of the present invention can be administered by various routes, and the dosage form may be selected appropriately depending on the mode of administration. Dosage forms include, for example, oral preparations such as tablets, granules, and capsules, as well as injections, transdermal preparations, and eye drops.

[0030] The therapeutic agent for myelofibrosis of the present invention can be administered at a dose determined appropriately depending on the condition and stage of the patient's myelofibrosis, other concomitant diseases, and physical conditions such as age, sex, weight, etc. In one embodiment, for example, about 1 to 10,000 mg / day of a therapeutic agent containing an HMG-CoA reductase inhibitor can be administered according to the patient's condition. [Example]

[0031] The present invention will be further described below with reference to examples, but the present invention should not be construed as being limited to these examples.

[0032] [Test Example 1] The test method and results shown in Figure 1 are described below. Jak2 suspended in FibroLife Basal Medium (Lifeline, LM-0001) V617F Bone marrow cells (2.0 × 10) from GFP double transgenic mice (female, 12-16 weeks old) 6100 μL of 100 μM of 100 μM Fibrocyte Growth Regulator (FGF) was dispensed into a flat-bottom 96-well plate (Biolamo 1-1601-06) per well and cultured at 37°C in a 5% CO2 environment for 7 days. After half of the medium was exchanged twice, each drug was added to the indicated final concentration. Culture was continued for an additional 5 days. Cell counts were measured using a hybrid cell count / image cytometer on an all-in-one optical microscope (BZ-X810, KEYENCE). The average cell counts from 5 wells are expressed as a percentage of the drug-free group (mean ± SD). Fibrocyte proliferation was inhibited by 0.1 μM concentrations of pitavastatin, cerivastatin, simvastatin, fluvastatin, and rosuvastatin, and by 1 μM concentration of atorvastatin.

[0033] [Test Example 2] The test method and results shown in Figure 2 are described below. Bone marrow cells were collected from the femur and tibia of a Jak2V617F transgenic mouse (female, 8 weeks old). Red blood cells were lysed in 100 mM NH4Cl / 17 mM Tris-HCl solution [pH 7.2] for 8 minutes at 4°C, and then washed once with PBS. 2 x 10 cells were placed in a 35 mm dish (150460, Thermo Scientific) per well for CFU-GM measurement. 4 2 x 10 bone marrow cells per well and 1 mL of MethoCult M3434 (ST-03434, STEMCELL Technologies) were added for CFU-E measurement. 5To each well, 1 mL of MethoCult M3234 (ST-03234, STEMCELL Technologies) and erythropoietin (final concentration 3.0 IU / mL) were added. Pitavastatin, cerivastatin, and atorvastatin were added to the indicated final concentrations. After culturing at 37°C in a 5% CO2 environment for 7 days (CFU-GM) and 3 days (CFU-E), the number of colonies formed was counted using an inverted microscope (Olympus IX70, OLYMPUS). The mean number of colonies in two wells is expressed as a percentage of the control group (mean ± SD). The inhibitory effect on CFU-GM and CFU-E proliferation was smallest when pitavastatin was added.

[0034] [Test Example 3] The test method and results of Figure 3 are described below. V617F Transgenic mice (female, 4 weeks old) were orally administered vehicle (PBS) alone (8 mice), pitavastatin (3 mg / kg, once daily) (4 mice), ruxolitinib (90 mg / kg, twice daily) (3 mice), or both pitavastatin (3 mg / kg, once daily) and ruxolitinib (90 mg / kg, twice daily) (3 mice) for 12 weeks. After dissection, femurs were collected and fixed in formalin for 24 hours, then infiltrated in Morse's solution for 5 days for decalcification. After embedding in paraffin, pathological sections were prepared. After retrieval using a microwave oven, the specimens were stained with a primary antibody (Anti-Collagen I antibody (Abcam ab6308), diluted 1 / 200, left at 4°C for 24 hours) and a secondary antibody (Gout Anti-Mouse IgG H&L (Abcam 150116), diluted 1 / 200, left at 4°C for 2 hours). The specimens were observed with a laser confocal microscope (TCS-SP8, Leica), and the collagen I-positive area was calculated using ImageJ. The percentage of collagen I-positive area in the bone marrow is shown (mean ± SD). Pitavastatin administration reduced the collagen I-positive area in the bone marrow. Ruxolitinib administration had no effect on the collagen I-positive area.

[0035] [Test Example 4] The test method and results of Figure 4 (peripheral blood Hb concentration and bone marrow nucleated cell count (NCC) after administration of pitavastatin) are described below. (Left) Jak2 in wild-type (WT) mice orally treated with vehicle alone (PBS) or pitavastatin (3 mg / kg, once daily) for 12 weeks. V617F Blood was collected from the tail vein of transgenic mice (female, 4 weeks old) every 4 weeks. Hb concentrations were measured using an automated hemocytometer (MEK-6558 Celltac α, Nihon Kohden) (mean ± SD). V617F Transgenic mice exhibited anemia due to decreased Hb levels compared with WT mice, but this anemia was partially improved by administration of pitavastatin. (Right) Jak2 V617F Transgenic mice (female, 4 weeks old) were orally administered vehicle alone (PBS), pitavastatin (3 mg / kg, once daily), ruxolitinib (90 mg / kg, twice daily), or both pitavastatin (3 mg / kg, once daily) and ruxolitinib (90 mg / kg, twice daily) for 12 weeks. After dissection, bone marrow cells were collected from one femur and two tibiae. They were suspended in RPMI 1640 Medium (Invitrogen, 44288-55) and centrifuged at 1500 rpm at 4°C for 5 min, and the supernatant was removed. After incubation with 10 mL of hemolytic agent (100 mM NH4Cl, 17 mM Tris-HCl, pH 7.2) for 8 min, the cells were suspended in PBS (2% FBS), and the cell count was determined (mean ± SD). Pitavastatin administration significantly increased Jak2 expression. V617F The number of bone marrow cells increased in transgenic mice, but ruxolitinib administration did not affect the increase in bone marrow cells.

[0036] [Test Example 5] The test method and results of Figure 5 are described below. V617FTransgenic mice (female, 4 weeks old) were orally administered vehicle (PBS), pitavastatin (3 mg / kg, once daily), ruxolitinib (90 mg / kg, twice daily), or both pitavastatin (3 mg / kg, once daily) and ruxolitinib (90 mg / kg, twice daily) for 12 weeks. After necropsy, the liver (left) and spleen (right) were removed and weighed (mean ± SD). Pitavastatin administration reduced liver and spleen weights and improved organ enlargement. Ruxolitinib administration reduced liver and spleen weights, but the concomitant administration of the two drugs did not affect the weight loss of the liver and spleen.

[0037] [Test Example 6] The test method and results of Figure 6 (body weight after pitavastatin administration) are described below. Wild-type (WT) mice, Jak2 mice orally administered vehicle (PBS) or pitavastatin (3 mg / kg, once daily) for 12 weeks. V617F The body weight of transgenic mice (female, 4 weeks old) was measured weekly (mean ± SD). Pitavastatin administration did not affect the body weight of the mice.

[0038] [Test Example 7] The test method for Figure 7 (Pitavastatin inhibits proliferation of human peripheral blood-derived fibrocytes) is described below. 10 mL of peripheral blood was collected from the cubital vein of healthy volunteers and myelofibrosis patients using EDTA Na as an anticoagulant. 10 mL of peripheral blood was diluted 2-fold with PBS and layered on 3 mL of Ficoll-Paque Plus (Cytiva, 17144003). After centrifugation at 1200 rpm at room temperature for 30 minutes, the intermediate layer was collected and mononuclear cells were isolated. After washing twice with PBS, 5.0 × 10 cells were placed in FibroLife Basal Medium (Lifeline, LM-0001). 5The cells were suspended at 0.1 cells / mL. 100 μL of the solution was dispensed into a flat-bottom 96-well plate (Biolamo 1-1601-06) per well and cultured at 37°C in a 5% CO2 environment for 4 days. After half of the medium was replaced twice, pitavastatin was added to a final concentration of 0.1 μM and the cells were cultured for an additional 3 days. The number of cells was counted visually using an optical microscope. The average cell count (mean ± SD) from 5 wells is shown.

[0039] [Test Example 8] The test method for Figure 8 (mechanism of fibrocyte proliferation inhibition by pitavastatin) is as follows. Jak2 suspended in FibroLife Basal Medium (Lifeline, LM-0001) was used. V617F Bone marrow cells (2.0 × 10) from GFP double transgenic mice (female, 12-16 weeks old) 6 100 μL of pitavastatin (cells / mL) was dispensed into a flat-bottom 96-well plate (Biolamo 1-1601-06) at each well and cultured for 7 days at 37°C in a 5% CO2 environment. After two half-medium changes, pitavastatin and mevalonic acid were added to the indicated final concentrations. Culture was continued for an additional 5 days. Cell counts were measured using a hybrid cell count / image cytometer on an all-in-one optical microscope (BZ-X810, KEYENCE). The average cell counts for 5 wells are expressed as a percentage of the drug-free group (mean ± SD). Fibrocyte proliferation, inhibited by 1 μM pitavastatin, was restored by the addition of 500 μM mevalonic acid.

Claims

1. A therapeutic agent for myelofibrosis, which is administered to a patient with myelofibrosis and contains an HMGCoA reductase inhibitor as an active ingredient.

2. The myelofibrosis therapeutic agent according to claim 1, wherein the myelofibrosis is primary myelofibrosis, post-polycythemia vera myelofibrosis, or post-essential thrombocythemia myelofibrosis.

3. The myelofibrosis therapeutic agent of claim 1, wherein the patient has a JAK2 mutation.

4. The therapeutic agent for myelofibrosis according to claim 1, wherein the myelofibrosis is caused by proliferation of blood-derived fibrocytes.

5. The myelofibrosis therapeutic agent according to claim 1, which is used in combination with at least one of a JAK1 inhibitor and a JAK2 inhibitor.

6. 2. The method for treating myelofibrosis according to claim 1, wherein the HMG-CoA reductase inhibitor is one or more selected from the group consisting of pitavastatin, atorvastatin, cerivastatin, rosuvastatin, fluvastatin, simvastatin, pravastatin, lovastatin, and mevastatin, and pharmaceutically acceptable salts thereof.

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