KR102273445B1 - A pharmaceutical composition comprising selegiline for preventing or treating acute myeloid leukemia - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating refractory or recurrent acute myeloid leukemia comprising selegiline as an active ingredient. Unlike conventional anticancer drugs, selegiline has excellent apoptosis effect not only on acute myeloid leukemia cells but also on refractory or recurrent acute myeloid leukemia cells, so that acute myeloid leukemia, in particular, refractory or recurrent acute myeloid leukemia. It can be usefully used as a pharmaceutical composition for preventing or treating leukemia.

Description

A pharmaceutical composition comprising selegiline for preventing or treating acute myeloid leukemia comprising selegiline as an active ingredient

The present invention relates to a pharmaceutical composition for preventing or treating acute myeloid leukemia comprising selegiline as an active ingredient.

Acute leukemia is a disease in which abnormal leukocyte progenitor cells or platelet progenitor cells are excessively produced due to a bone marrow abnormality. When cells of the lymphatic system proliferate, acute lymphocytic leukemia (ALL), cells of the myeloid system proliferate This is called acute myeloid leukemia (AML). Since abnormal white blood cells increase and occupy the place of hematopoiesis, normal white blood cells, red blood cells, platelets, etc. are not formed, so infection or bleeding occurs easily, and if not treated, death occurs within several months. Although the survival rate of acute leukemia in infants has improved significantly with recent advances in chemotherapy, the survival rate in adults is still low.

Acute myeloid leukemia is a malignant tumor arising from stem cells of non-lymphocytes or myeloid leukocytes produced in the bone marrow. Genetic mutations in hematopoietic stem cells cause myeloid progenitors to stop differentiation at various stages, and immature myeloblasts become single cells. It is a hematopoietic tumor that proliferates in a group (monoclonal). Symptoms of bone marrow dysfunction such as anemia, fever, increased infectivity, and bleeding tendency, and long-term infiltration of tumor cells such as splenomegaly and lymph node swelling may also appear.

Refractory/relapsed acute myeloid leukemia (refractory/relapsed acute myeloid leukemia) is characterized by no treatment effect at the first treatment after diagnosis, two or more relapses, or 1 year after the first treatment is determined to be cured. case of recurrence soon. The prognosis is poor with an average survival rate of 3 to 4 months, and the main prognostic factors include cytogenetic characteristics, age, treatment response rate and morphological response during remission therapy (Kyunghee Yoon, Master's thesis, 2001). Treatment for sexual or recurrent acute myeloid leukemia is very limited, and there is an urgent need for a new treatment method that overcomes drug resistance, acts specifically on leukemia cells, and has low side effects.

As a standard method for treating leukemia, chemotherapy, hematopoietic stem cell transplantation, radiation therapy, etc. are included, and in the case of chemotherapy, a method of using two or more anticancer agents in combination is usually included. The ideal chemotherapy is that the anti-leukemia drug should show a selective effect only on leukemia cells without inhibiting normal hematopoiesis or causing other harmful side effects. However, although most anti-leukemia drugs can kill leukemia cells close to the ideal state, they also inhibit normal hematopoiesis and cause other harmful side effects, so there is a limit to the treatment of leukemia. In addition, antitumor effects are weak in drug-resistant leukemia cells, side effects may occur, and sufficient chemotherapy may not be administered in some cases.

Hematopoietic stem cell transplantation (HSCT) has gone beyond the area of bone marrow transplantation (BMT) that used bone marrow in the past, and is now used in peripheral blood (PB) and cord blood (CB). It refers to transplantation by using all types of hematopoietic stem cells that exist as a transplant source. Hematopoietic stem cell transplantation is a treatment that removes cancer cells and the patient's own hematopoietic stem cells using chemotherapy and radiotherapy in hematological tumor patients and then transplants new hematopoietic stem cells. It is being established as an effective and promising means of treatment in various fields such as leukemia, aplastic anemia, malignant lymphoma, refractory autoimmune disease, and solid cancer. However, until now, it is a treatment method with high complications due to high-dose chemotherapy and graft-versus-host disease that occurs after allogeneic transplantation. Therefore, it is an important task to develop new therapeutic drugs for the treatment of all leukemia forms.

As a prior art related to a composition for treating leukemia containing selegiline, International Patent Publication No. 2016-088111 discloses a composition for preventing or treating cancer of an arylalkylamine compound, and a prior paper [ThyagaRajan, S et al, J Neuroimmunol 109(2), 95-104, 2000] disclosed the antitumor effect of selegiline, and a previous paper [Lajko, E et al, J Neural Transm, 119(5), 545-556, 2012] reported Mono The basic physiological activity induced by selegiline and its derivatives in Mac 6 human monocytes was disclosed. However, there is no prior literature disclosing that selegiline is effective in overcoming drug resistance as well as in acute leukemia and therapeutic effect in refractory or relapsed acute myeloid leukemia.

International Patent Publication No. 2016-088111, ARYLALKYLAMINE COMPOUNDS FOR USE IN THE PREVENTION OR TREATMENT OF CANCER, published on June 09, 2016.

Kyunghee Yoon, Effect of mitoxantrone combination therapy for remission in the treatment of refractory acute myeloid leukemia, Master's thesis, 2001. Lajko, E et al, Basic cell physiological activities (cell adhesion, chemotaxis and proliferation) induced by selegiline and its derivatives in Mono Mac 6 human monocytes, J Neural Transm, 119(5), 545-556, 2012. ThyagaRajan, S et al, Anti-tumor effect of Ldeprenyl is associated with enhanced central and peripheral neurotransmission and immune reactivity in rats with carcinogen-induced mammary tumors, J Neuroimmunol 109(2), 95-104, 2000.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating acute myeloid leukemia, particularly refractory or recurrent acute myeloid leukemia, comprising selegiline as an active ingredient.

The present invention relates to a pharmaceutical composition for preventing or treating acute myeloid leukemia comprising selegiline as an active ingredient, preferably, the acute myeloid leukemia is refractory or relapsed acute myeloid leukemia).

The pharmaceutical composition according to the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. In addition, the composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl paraoxybenzoate, propyl paraoxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto. In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, stabilizers, binders, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one or more excipients, for example, starch, microcrystalline cellulose, sucrose or lactose in selegiline of the present invention. , low-substituted hydroxypropyl cellulose, hypromellose, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used. In order to formulate a formulation for parenteral administration, the compound or a pharmaceutically acceptable salt thereof is sterilized and/or adjuvants such as preservatives, stabilizers, wetting agents or emulsification accelerators, salts and/or buffers for regulating osmotic pressure, and other treatments It can be prepared as a solution or suspension by mixing it with water with useful substances, and it can be prepared in ampoules or vial unit dosage form.

The pharmaceutical composition comprising selegiline of the present invention as an active ingredient may be administered to mammals such as mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection. The dosage may vary depending on the age, sex, weight, specific disease or pathology to be treated, the severity of the disease or pathology, administration time, administration route, absorption, distribution and excretion rate of the drug, the type of other drugs used, and the prescriber's It will depend on judgment, etc. Dosage determination based on these factors is within the level of one of ordinary skill in the art, and generally dosages range from 001 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

The present invention relates to a pharmaceutical composition for preventing or treating refractory or recurrent acute myeloid leukemia comprising selegiline as an active ingredient. Unlike conventional anticancer drugs, selegiline has excellent apoptosis effect not only on acute myeloid leukemia cells but also on refractory or recurrent acute myeloid leukemia cells, so that acute myeloid leukemia, in particular, refractory or recurrent acute myeloid leukemia. It can be usefully used as a pharmaceutical composition for preventing or treating leukemia.

1 is a result of confirming the cell viability according to the concentration (0, 05, 1, 2, 4 mM) in the treatment of selegiline (FIG. 1A) or cytarabine (FIG. 1B) in KG-1α cells.
2 is a result of confirming the oxygen consumption rate according to the concentration (0, 05, 1, 2, 4 mM) when selegiline is treated in KG-1α cells.
3 is a result of confirming the amount of ATP produced according to the concentration (0, 4 mM) when selegiline is treated in KG-1α cells.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

< Example 1 Preparation of acute and chronic myeloid leukemia cell lines>

The acute myeloid leukemia cell line used in the present invention is U937 cells, and the refractory or relapsed acute myeloid leukemia cell line is KG-1α cells. U937 cells (Acute myeloid leukemia cells) use RPMI medium containing 10% FBS, KG-1α cells (Refractory Acute myeloid leukemia cells) using IMDM medium containing 20% FBS (fetal bovine serum), It was cultured in an incubator at 37° C., 5% CO 2 conditions, and subcultured once every 2 days was used for the experiment.

The chronic myeloid leukemia cell line used K562 cells (Chronic myeloid leukemia cells), and this cell line was cultured in an incubator at 37 ° C., 5% CO2 condition using RPMI medium containing 10% fetal bovine serum (FBS), It was used for the experiment by subculture once every 2 days.

< Example 2 Measurement of cell viability>

In order to measure the cell viability of acute myeloid leukemia cells, refractory or relapsed acute myeloid leukemia cells and chronic myeloid leukemia cells, the cells of the present invention were added to U937 cells, KG-1α cells and K562 cells cultured in Example 1 above. By treatment with ziline, the change in cell viability according to the treatment with selegiline was confirmed.

First, the U937 cells, KG-1α cells, and K562 cells cultured in Example 1 were inoculated in a 96-well plate at 3×10 ⁴ cells/well, and selegiline (M003, Sigma Aldrich) dissolved in PBS (Phosphate buffered saline). , St Louis, MO, USA) and the comparative compound cytarabine (1162002-250MG, Sigma Aldrich, St Louis, MO, USA) were treated with each concentration (0.5, 1, 2 and 4 mM), and then for 24 hours. Incubated at 37°C. In addition, PBS was used as a control.

After 24 hours of incubation, 5 μl of CCK-8 (cell counting kit-8) solution was added to each well, _{and incubated for 2 hours in an incubator at 37° C. and 5% CO 2} condition, and then 450 nm using a multi-scan machine. The cell viability was confirmed from the absorbance, and it is shown in Table 1 and FIG. 1 .

cell line compound Compound treatment concentration control
(PBS) 05mM 1 mM 2 mM 4 mM refractory or relapse
sexual acute myeloid bag
blood clot cells (KG-1α) selegiline 93.1% 86.0% 63.0% 51.8% 100% Comparative compound 1
(Cytarabine) 95.2% 96.9% 97.7% 95.3% 100% acute myeloid leukemia
disease cell (U937) Comparative compound 1
(Cytarabine) 45.3% 43.9% 40.9% 40.7% 100%

Referring to Table 1 and FIG. 1 , it was found that treatment of the present invention selegiline to refractory or recurrent acute myeloid leukemia cells (KG-1α) exhibited excellent apoptosis effect. In particular, in the case of cytarabine, an existing anticancer drug, the apoptosis effect on acute leukemia cells was excellent, but it did not show an apoptosis effect on refractory or recurrent acute myeloid leukemia cells, whereas selegiline of the present invention showed acute leukemia cells and refractory cells. It was confirmed that all of the apoptotic effects on the sexual or recurrent acute myeloid leukemia cells were shown.

In addition, although not shown in Table 1 above, in the case of selegiline of the present invention, the apoptosis effect on chronic myeloid leukemia cells (K562) rather than acute myeloid leukemia cells (U937) or refractory or recurrent acute myeloid leukemia cells (KG-1α) The apoptosis effect was excellent.

Therefore, unlike conventional anticancer drugs, selegiline has an excellent apoptosis effect not only on acute myeloid leukemia cells but also on refractory or relapsed acute myeloid leukemia cells, so it can overcome drug resistance and be used in the treatment of refractory or relapsed acute myeloid leukemia. It was confirmed that the composition was effective.

< Example 3 Check the change in the rate of mitochondrial oxygen consumption>

By treating refractory or recurrent acute myeloid leukemia cells with the selegiline of the present invention and measuring the change in the rate of intracellular mitochondrial oxygen consumption, changes in energy metabolism ability for leukemia cells were confirmed.

The KG-1α cells cultured in Example 1 were stained with Trypan blue to measure the number of cells, and then ^{inoculated into an XF-plate at 3×10 4} cells/well. Thereafter, selegiline was injected and the change in oxygen consumption rate occurring for 20 minutes was measured and shown in FIG. 2 .

Referring to FIG. 2 , it was confirmed that the oxygen consumption rate decreased in a concentration-dependent manner when selegiline was treated in refractory or recurrent acute myeloid leukemia cells (KG-1α).

< Example 4 Check the change in ATP production>

The KG-1α cells cultured in Example 1 were inoculated into a 96-well plate at 3×10 ⁴ cells/well, treated with 4mM selezilin dissolved in PBS (Phosphate buffered saline), and then cultured at 37° C. for 20 minutes. did. As a control, PBS was used, and after 20 minutes, the total amount of energy in the cell was measured with ATPlite 1step kit (PerkinElmer) and shown in FIG. 3 .

Referring to FIG. 3 , it was confirmed that ATP production was reduced when selegiline was treated with refractory or recurrent acute myeloid leukemia cells (KG-1α).

Therefore, from the results of FIGS. 2 and 3, selegiline reduces the oxygen consumption rate and ATP production of refractory or relapsed acute myeloid leukemia cells, thereby reducing the mitochondrial function of leukemia cells, that is, lowering energy metabolism ability, so refractory or relapse It can be usefully used as a composition for the prevention or treatment of acute myeloid leukemia.

< Formulation example 1 Pharmaceutical preparations>

Formulation example 1-1 powdery Produce

A powder was prepared by mixing 2 g of selegiline of the present invention and 1 g of lactose and filling it in an airtight bag.

Formulation example Preparation of 1-2 tablets

100 mg of selegiline of the present invention, 100 mg of microcrystalline cellulose, 60 mg of lactose hydrate, 20 mg of low-substituted hydroxypropyl cellulose, and 2 mg of magnesium stearate are mixed, and then tableted according to a conventional tablet manufacturing method to obtain tablets. prepared.

Formulation example 1-3 Preparation of capsules

After mixing 100 mg of selegiline of the present invention, 100 mg of microcrystalline cellulose, 60 mg of lactose hydrate, 20 mg of low-substituted hydroxypropyl cellulose and 2 mg of magnesium stearate, the above components were prepared according to a conventional capsule preparation method. The mixture was mixed and filled into gelatin capsules to prepare capsules.

Formulation example 1-4 Preparation of injections

After mixing 10 mg of the selegiline of the present invention, an appropriate amount of sterile distilled water for injection, and an appropriate amount of a pH adjusting agent, the content of the above components per 1 ampoule (2 ml) was prepared according to a conventional method for preparing an injection.

Claims (2)

A pharmaceutical composition for inhibiting KG-1α cancer cells which is a human-derived acute myeloid leukemia cell line and is refractory to cytarabine, comprising selegiline as an active ingredient. delete

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