KR20210017823A - Pharmaceutical composition for preventing or treating myeloid leukemia - Google Patents

Abstract

A compound of chemical formula 1 or a pharmaceutically acceptable salt thereof inhibits myosin ATPase of myeloid leukemia cells to inhibit the binding between actin and myosin, inhibits cell proliferation, and induces apoptosis, thereby being able to be used for preventing, alleviating or treating myeloid leukemia. In addition, the present invention can be administered in combination with an existing myeloid leukemia therapeutic agent, and among the same, blebbistatin, which has been used for other medicinal purposes, can be developed more efficiently, thereby being able to shorten a novel drug development process.

Description

Pharmaceutical composition for the prevention or treatment of myeloid leukemia {PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING MYELOID LEUKEMIA}

It relates to a pharmaceutical composition for the prevention or treatment of myeloid leukemia.

Leukemia is a generic term for a disease in which leukocytes proliferate in a neoplastic form. The type of leukemia is divided into myeloid leukemia and lymphocytic leukemia according to the leukocytes from which leukemia originates, and is divided into acute leukemia and chronic leukemia depending on the progression rate. The clinical manifestations of leukemia vary depending on the type of disease and the nature of the cells involved. Lymphocytic leukemia is caused by lymphatic leukemia, myeloid leukemia by myeloid blood cells, and chronic myelogenous leukemia is caused by mutation of cells in maturity, and acute myelogenous leukemia is a myeloid system that starts differentiation in a relatively early hematopoietic process. It is caused by disorders of the hair cells.

Among them, acute myeloid leukemia (AML) is known as acute myeloid leukemia or acute nonlymphatic leukemia (ANLL), and is characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. Loss is a cancer of the blood cells of the bone marrow system. AML is the most common acute leukemia affecting adults, and its incidence increases with age. AML is a relatively rare disease and accounts for about 1.2% of cancer deaths in the United States, but its incidence is expected to increase as the population ages.

Symptoms of AML are caused by the replacement of leukemia cells in the normal bone marrow, which leads to a decrease in red blood cells and platelets and normal white blood cells. Such symptoms include fatigue, shortness of breath, frequent bruising and bleeding, and an increased risk of infection. Some risk factors and chromosomal abnormalities have been identified, but the specific cause is not clear. AML is an acute leukemia that progresses rapidly and is usually fatal within weeks or months if left untreated.

AML has several subtypes: Treatment and prognosis depend on the subtype. The 5-year survival rate varies from 15 to 70%, and the recurrence rate varies from 33 to 78% depending on the subtype. AML is initially treated with chemotherapy aimed at inducing remission. Patients may continue to receive additional chemotherapy or hematopoietic stem cell transplants.

Until now, anticancer drugs targeting myosin were mostly anticancer drugs that acted as targets for kinase that induces myosin phosphorylation. Until now, myeloid leukemia treatment strategies do not directly inhibit the function of myosin, but transmit higher signals. By changing the system, there is a risk of cell resistance to drugs through side effects and signal rewiring. Therefore, there is a need for a treatment strategy that can be applied more directly and widely through the regulation of the activity of a new target.

On the other hand, the main cytoskeletal motor protein that causes cell tension is non-muscular myosin II (referred to as myosin II). Straight et al. (2003) identified specific small molecule inhibitors of non-muscular myosin II in an attempt to analyze cell division. A small molecule with high activity was called blebbistatin, which inhibits cell blebbing. This compound is cell-permeable, positive, and importantly, it is easily reversible.

The IUPAC name of blebbistatin is 3a-Hydroxy-6-methyl-1-phenyl-2,3-dihydropyrrolo[2,3-b]quinolin-4-one, and is used for in vitro function studies of existing myosin. Since it is a formulation with proven efficacy that has been widely used, it is possible to develop a therapeutic agent more efficiently if it is used for a new pharmaceutical use.

One aspect is to provide a pharmaceutical composition for the prevention or treatment of myeloid leukemia comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof:

[Formula 1]

R is selected from the group consisting of hydrogen, deuterium, halogen, cyano group, nitro group, -NR' ₃ ⁺ , carbonyl group, carboxyl group, aldehyde group, ester group, -SO ₃ R'.

Another aspect is to provide a pharmaceutical composition for the prophylaxis or treatment of myeloid leukemia comprising a compound of Formula 2 or a pharmaceutically acceptable salt thereof:

[Formula 2]

R is selected from the group consisting of hydrogen, deuterium, halogen, cyano group, nitro group, -NR' ₃ ⁺ , carbonyl group, carboxyl group, aldehyde group, ester group, -SO ₃ R'.

Another aspect is to provide a pharmaceutical composition for the prevention or treatment of Myeloid Leukemia comprising a compound of Formula 3 or a pharmaceutically acceptable salt thereof:

[Formula 3]

.

.

Another aspect is to provide a health functional food for the prevention or improvement of Myeloid Leukemia, comprising the compound of Formula 1 or a food pharmaceutically acceptable salt thereof:

[Formula 1]

R is selected from the group consisting of hydrogen, deuterium, halogen, cyano group, nitro group, -NR' ₃ ⁺ , carbonyl group, carboxyl group, aldehyde group, ester group, -SO ₃ R'.

Another aspect is to provide a method for preventing or treating Myeloid Leukemia comprising administering the pharmaceutical composition to a subject.

One aspect provides a pharmaceutical composition for the prophylaxis or treatment of myeloid leukemia comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof:

[Formula 1]

R is selected from the group consisting of hydrogen, deuterium, halogen, cyano group, nitro group, -NR' ₃ ⁺ , carbonyl group, carboxyl group, aldehyde group, ester group, -SO ₃ R'.

The R'is, for example, hydrogen, deuterium, -F, -Cl, -Br, -I, a hydroxyl group, a cyano group, a nitro group, an amidino group, a hydrazino group, a hydrazono group, a substituted or unsubstituted C ₁ -C ₅ alkyl group, substituted or unsubstituted C ₂ -C ₅ alkenyl group, substituted or unsubstituted C ₂ -C ₅ alkynyl group, substituted or unsubstituted C ₁ -C ₅ alkoxy group, substituted or unsubstituted A substituted C ₃ -C ₅ cycloalkyl group, a substituted or unsubstituted C ₁ -C ₅ heterocycloalkyl group, a substituted or unsubstituted C ₃ -C ₅ cycloalkenyl group, a substituted or unsubstituted C ₁ -C ₅ heterocyclo Alkenyl group, substituted or unsubstituted aryl group, substituted or unsubstituted aryloxy group, substituted or unsubstituted arylthio group, substituted or unsubstituted heteroaryl group, substituted or unsubstituted monovalent non-aromatic condensed polycyclic It may be selected from a group, a substituted or unsubstituted monovalent non-aromatic condensed heteropolycyclic group.

The R may be H, and the IUPAC name of the compound in which R of the compound of Formula 1 is H is 3a-Hydroxy-6-methyl-1-phenyl-2,3-dihydropyrrolo[2,3-b]quinolin-4 -one, also called blebbistatin, is an ATPase inhibitor of myosin.

The compound of Formula 1 may be derived from nature or may be synthesized using a known organic synthesis method, and non-protein compounds, peptides, extracts of plant-derived tissues or cells, microorganisms (for example, bacteria or fungi, and In particular, it may be a product obtained by cultivation of yeast).

The term "pharmaceutically acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition.

The term "pharmaceutically acceptable salt" means a salt prepared using a specific compound according to an aspect and a relatively non-toxic acid or base. When the compound contains a relatively acidic functional group, base addition salts can be obtained by contacting a sufficient amount of a base with the neutral form of such compound in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include salts or similar salts of sodium, potassium, calcium, ammonium, organic amine, or magnesium. When the compound contains a relatively basic functional group, acid addition salts can be obtained by contacting a sufficient amount of acid with the neutral form of such compound in a pure solution or a suitable inert solvent. Pharmaceutically acceptable acid addition salts are salts of inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate ion, phosphoric acid, monohydrogen phosphate ion, dihydrogen phosphate ion, sulfuric acid, hydrogen sulfate ion, hydroiodic acid or phosphorous acid, And salts of organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. And salts of amino acids (eg, arginine) and salts of organic acids such as glucuronic acid.

The pharmaceutically acceptable salt can be synthesized by conventional chemical methods from a parent compound containing an acidic or basic moiety. In general, these salts are prepared by reacting the form of the free acid or base of these compounds with a stoichiometrically appropriate amount of a base or acid in water or in an organic solvent or in a mixture of the two. In general, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred.

According to one aspect, the compound of Formula 1 or a pharmaceutically acceptable salt thereof inhibits myosin ATPase in myeloid leukemia cells, inhibits the binding between actin and myosin, inhibits cell proliferation, and causes apoptosis. It may be, and through this may exhibit a preventive or therapeutic effect of myeloid leukemia.

The pharmaceutical composition may contain an active ingredient alone, or may be provided as a pharmaceutical composition including one or more pharmaceutically acceptable carriers, excipients, or diluents.

Specifically, the carrier may be, for example, a colloidal suspension, powder, saline, lipid, liposome, microspheres, or nano spherical particles. They can be complexed or associated with a vehicle and are known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. It can be delivered in vivo using known delivery systems.

When the pharmaceutical composition is formulated, it can be prepared using a diluent or excipient such as a lubricant, sweetener, flavoring agent, emulsifier, suspending agent, preservative, filler, extender, binder, wetting agent, disintegrant, surfactant, etc. I can. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient in the composition, such as starch, calcium carbonate, and sucrose. ) Or lactose (lactose), gelatin, etc. can be prepared by mixing. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc., and various excipients, such as humectants, sweeteners, fragrances, preservatives, etc., may be included in addition to water and liquid paraffin, which are commonly used simple diluents. . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycero gelatin, etc. may be used, and a known diluent or excipient may be used in the preparation of eye drops. have.

In addition, the pharmaceutical composition may be administered in combination with an existing myelogenous leukemia therapeutic agent, that is, mixed with a conventionally known myelogenous leukemia prevention or treatment composition. For example, it may be provided by mixing with radotinib or imatinib.

The pharmaceutical composition may be administered in parallel with a known composition having an effect of preventing or treating myeloid leukemia, and may be administered simultaneously, separately, or sequentially, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

The term "administration" means introducing a predetermined substance to an individual by an appropriate method, and "individual" means all organisms such as rats, mice, livestock, including humans capable of carrying myeloid leukemia. As a specific example, it may be a mammal including a human.

In one aspect, the route of administration of the pharmaceutical composition is, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, This includes topical, sublingual or rectal.

The composition may be administered orally or parenterally, and when administered parenterally, external use of the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection injection method may be selected.

The pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, drug Sensitivity to, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. Specifically, the pharmaceutical composition may be administered at 0.01 to 1000 mg/kg/day, more specifically 0.1 to 500 mg/kg/day. The administration may be administered once a day, or may be divided several times.

Specifically, the effective amount of the pharmaceutical composition may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and drugs used in combination, administration route, obesity It can be increased or decreased depending on the severity, sex, weight, age, etc.

The myeloid leukemia (Myeloid Leukemia) may be specifically acute myeloid leukemia (AML).

The term "prevention" may mean any action of inhibiting or delaying the onset of myeloid leukemia in an individual by administration of the pharmaceutical composition according to an aspect.

The term "treatment" may refer to any action in which symptoms of an individual's myeloid leukemia are improved or advantageously changed by administration of the pharmaceutical composition according to one aspect.

Another aspect provides a pharmaceutical composition for the prevention or treatment of myeloid leukemia comprising a compound of Formula 2 or a pharmaceutically acceptable salt thereof:

[Formula 2]

R is selected from the group consisting of hydrogen, deuterium, halogen, cyano group, nitro group, -NR' ₃ ⁺ , carbonyl group, carboxyl group, aldehyde group, ester group, -SO ₃ R'.

The compound of Formula 2 is one in which the carbon of 3a, which is a chiral center in the compound of Formula 1, has a (S) configuration, and the "pharmaceutically acceptable salt", "myelogenous leukemia", "prevention" and "treatment", etc. May be within the aforementioned range.

Another aspect provides a pharmaceutical composition for the prevention or treatment of Myeloid Leukemia comprising a compound of Formula 3 or a pharmaceutically acceptable salt thereof:

[Formula 3]

.

.

In the compound of Formula 3, in the compound of Formula 1, R is a para-nitro group, and the IUPAC name is 3a-Hydroxy-6-methyl-1-(4-nitrophenyl)-2,3-dihydropyrrolo[2,3- b ]quinolin-4-one.

The "pharmaceutically acceptable salt", "myelogenous leukemia", "prevention" and "treatment" and the like may be within the above-described range.

Another aspect provides a health functional food for the prevention or improvement of Myeloid Leukemia, comprising the compound of Formula 1 or a food pharmaceutically acceptable salt thereof:

[Formula 1]

R is selected from the group consisting of hydrogen, deuterium, halogen, cyano group, nitro group, -NR' ₃ ⁺ , carbonyl group, carboxyl group, aldehyde group, ester group, -SO ₃ R'.

In the health functional food, the "compound of formula 1", "R, R'", "myelogenous leukemia", "prevention" and "treatment" may be within the above-described range.

The term “food wise acceptable” means exhibiting properties that are not toxic to cells or humans exposed to the compound.

The term “food wise acceptable salt” means a salt prepared using a specific compound according to an aspect and a relatively non-toxic acid or base, and may be within the above-described range for “salt”.

The term “improvement” may refer to any action that at least reduces the severity of a parameter related to the condition being treated, eg, symptom. In this case, the health functional food may be used before or after the onset stage of the disease in order to prevent or improve myelogenous leukemia, at the same time as or separately from the drug for treatment.

In the health functional food, the active ingredient may be added to the food as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (for prevention or improvement). In general, when preparing food or beverage, the health functional food may be added in an amount of specifically about 15% by weight or less, and more specifically about 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range.

The health functional food may be formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further including at least one of carriers, diluents, excipients and additives. Foods to which the compound according to one aspect can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

Specific examples of the carrier, excipient, diluent and additive include lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose. , Polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil It may be at least one selected from.

The health functional food may contain other ingredients as essential ingredients without particular limitation in addition to containing the active ingredient. For example, as in ordinary beverages, various flavoring agents or natural carbohydrates may be included as additional ingredients. Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) are advantageously used. I can. The proportion of the natural carbohydrate can be appropriately determined by the choice of a person skilled in the art.

In addition to the above, health functional foods according to one aspect include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof. , Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. These components may be used independently or in combination, and the proportion of these additives may also be appropriately selected by those skilled in the art.

Another aspect provides a method for preventing or treating Myeloid Leukemia comprising administering the pharmaceutical composition to a subject.

In the method for preventing or treating myeloid leukemia, “individual”, “administration”, “pharmaceutical composition”, “myelogenous leukemia” and the like may be within the above-described range.

In addition, the method of preventing or treating myeloid leukemia may further include administering an existing myelogenous leukemia therapeutic agent to the individual.

The compound of formula 1 or a pharmaceutically acceptable salt thereof is used for the prevention or treatment of myeloid leukemia by inhibiting the binding between actin and myosin by inhibiting myosin ATPase in myeloid leukemia cells, inhibiting cell proliferation, and causing apoptosis. Can be used as In addition, it can be administered in combination with existing myelogenous leukemia treatments. Among them, blevisstatin, in particular, has been used for other medicinal purposes, so that the development of myeloid leukemia treatments can be more efficiently progressed, thereby shortening the process of developing new drugs. I can.

1 is a diagram showing the role of actomyosin contractility in human AML cells.
2 is a diagram showing an increase in MYH9 in human AML.
3 is a diagram showing that disturbance of actomyosin contractility controls apoptosis of AML cells.
4 is a diagram showing a cell growth pattern in HL-60 cells deficient in myosin phosphorylation.
5 is a diagram showing the effect of blevisstatin on cells derived from AML patients.
6 is a diagram showing that disturbance of actomyosin contractility increases differentiation of HL-60 cells and blocks mobility.
7A and 7B are diagrams showing that actomyosin contractility modulates transcriptional activity of HL-60 cells.
8 is a diagram showing that actomyosin contractility causes oncogene changes in human AML cells.
9 is a diagram showing the tumor suppression effect in the xenograft model when blevisstatin is administered.
10 is a diagram showing the inhibitory effect of para-nitro blebbistatin, a blevisstatin derivative, on AML cell proliferation.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Reference example

Reference Example 1. Antibodies and reagents

B p65 (C22B4) 항체(4764), YAP (D8H1X) XP^® 항체(14074) 및 TAZ (D3I6D) 항체(70148)는 Cell Signaling Technology로부터 구입하였다. Alexa Fluor^® 488 항-인간 CD11b (561687), Alexa Fluor^® 488 항-인간 CD14 (561706), PE 아넥신(Annexin) V 항체(559763) 및 CD34 APC 항체(345804)는 BD Biosciences에서 구입하였다. 쥐의 아릴 하이드로카본 수용체(aryl hydrocarbon receptor: AhR) 항체(sc-133088)는 Santa Cruz Biotechology에서 구입하였다.Rabbit's purified non-muscular myosin heavy chain II-A antibody (909801) and purified non-muscular myosin heavy chain II-B antibody (909901) were purchased from Biolegend. Rabbit phosphorylated myosin light chain 2 (ser19) antibody (3671), truncated caspase-3 (Asp175) (5A1E) antibody (9664), caspase-7 antibody (9492), NF-

B p65 (C22B4) antibody (4764), YAP (D8H1X) XP ® antibody (14 074), and TAZ (D3I6D) antibody (70 148) were purchased from Cell Signaling Technology. Alexa Fluor ^® 488 anti-human ^{CD11b (561687), Alexa Fluor ®} 488 anti-human CD14 (561706), PE annexin (Annexin) V antibody (559 763) and CD34 APC antibody (345 804) were purchased from BD Biosciences. The murine aryl hydrocarbon receptor (AhR) antibody (sc-133088) was purchased from Santa Cruz Biotechology.

Blebbistatin (B0560) and ML-7 (I2764) were purchased from Sigma. StemRegenin 1 (SR1) (C7710-1) was purchased from Cellagen Technology. CH229131 (3858) and Y27632 (1254) were purchased from Tocris Bioscience. Recombinant human TNF-α (300-01A) was purchased from Peprotech.

Reference Example 2. Cell Culture and Human Patient Sample

및 5% CO₂ 조건 하에서 10% FBS (SH30084.03, Hyclone), 1% 피루브산나트륨(11360-070, Gibco) 및 1% 페니실린/스트렙토마이신(15140-122, Gibco)이 보충된 RPMI-1640(SH30027.01, Hyclone)에서 배양되었다. 32D 세포들에는 10 ng/mL 쥐의 IL-3(213-13, Peprotech)이 보충되었다. HL-60 세포들은 1.3% DMSO가 포함된 배양 배지에서 분화되었고, 세포들은 분화 테스트를 위해 3일 간 배양되었다. 세포들은 모든 실험에서 3 내지 15 계대에서 사용되었다. 유전자 변형 세포주 HL-60-eGFP 및 HL-60 mMYL9-eGFP(MRLC-AA) 세포들은 Sirion biotechnology로부터 구입하였고, 0.5 μg/mL 퓨로마이신을 포함하는 배지에서 배양되어 형질도입된 세포들을 선별하였다. 1차 PBMC들은 한국 국립 암센터의 혈액학과에서 서면 동의를 얻은 후 진단받은 AML 환자들로부터 얻었다. 환자들의 특성은 하기 표 1에 나타내었다. 혈액 샘플들의 분석은 한국 국립암센터의 지침에 따라 승인되었다. 냉동된 정상 PBMC들은 stem cell technology(70025.2)로부터 구입하였다. 1차 세포들은 표시된 처리 하루 전에 20% FBS가 포함된 RPMI-1640 배지에서 성장되었다. CD34⁺ 세포들은 형광 활성 세포 분리 분석(fluorescence-activated cell sorting: FACS)에 의해 분석되었다. 제조사의 지침에 따라 MACSxpress 호중구 분리 키트(130-104-434)를 사용하여 1차 인간 호중구들이 농축되었다.Human leukemia cell lines HL-60 (10240), THP1 (40202), U937 (21593.1), Jurkat (clone E6-1, 40152), and K562 (10243) were purchased from Korean cell line bank. The murine myeloid 32D cell line was purchased from ATCC (CRL-11346). All cells 37

And RPMI-1640 supplemented with 10% FBS (SH30084.03, Hyclone), 1% sodium pyruvate (11360-070, Gibco) and 1% penicillin/streptomycin (15140-122, Gibco) under 5% CO ₂ conditions ( SH30027.01, Hyclone). The 32D cells were supplemented with 10 ng/mL murine IL-3 (213-13, Peprotech). HL-60 cells were differentiated in a culture medium containing 1.3% DMSO, and cells were cultured for 3 days for differentiation testing. Cells were used at passages 3-15 in all experiments. Genetically modified cell lines HL-60-eGFP and HL-60 mMYL9-eGFP (MRLC-AA) cells were purchased from Sirion biotechnology, cultured in a medium containing 0.5 μg/mL puromycin, and the transduced cells were selected. Primary PBMCs were obtained from AML patients diagnosed after obtaining written consent from the Department of Hematology at the National Cancer Center in Korea. The characteristics of the patients are shown in Table 1 below. Analysis of blood samples was approved according to the guidelines of the National Cancer Center of Korea. Frozen normal PBMCs were purchased from stem cell technology (70025.2). Primary cells were grown in RPMI-1640 medium containing 20% FBS before the indicated treatment day. CD34 ⁺ cells were analyzed by fluorescence-activated cell sorting (FACS). Primary human neutrophils were concentrated using the MACSxpress Neutrophil Isolation Kit (130-104-434) according to the manufacturer's instructions.

Reference Example 3. Cell Growth Test

The cell numbers of various leukemia cells and normal bone marrow cells were evaluated after 24, 48, 72 hours in the presence of various concentrations of blevisstatin. The number of viable cells was determined by trypan blue dye exclusion (TBDE). Cell viability was also determined with Cell Counting Kit-8 (CCK-8, CK04, Dojindo Molecular Technologies). The absorbance (450 nm) of WST-8 with reduced cell count was measured in three wells.

Reference Example 4. Colony formation analysis

및 5% CO₂ 조건 하에서 10 내지 50 μM 블레비스타틴이 첨가된 MethoCult H4230(STEMCELL Technologies)에 시딩되었다. 콜로니들은 14일 후에 계수되었다.HL-60 cells (2000 cells/well) are 37

And MethoCult H4230 (STEMCELL Technologies) to which 10-50 μM blevisstatin was added under 5% CO ₂ conditions. Colonies were counted after 14 days.

Colony analysis of FACS-sorted CD34 ⁺ stem/progenitor cells from AML patients and normal healthy donors was performed as described above.

Reference Example 5. Immunofluorescence

HL-60, THP1, U937, Jurkat, K562 cells and 32D cells (1Х10 ⁵ cells/well) were plated on a 96 well glass bottom plate (0611129L2L, Matrical Bioscience) coated with cell-tak (354240, Corning). . Samples were washed with PBS containing 0.1% Tween-20, fixed, and permeated with PBS containing 0.1% Triton X-100 for 15 minutes. Then, a blocking solution containing 10% FBS and 0.1% BSA was added for 1 hour at room temperature. Thereafter, cells were incubated overnight at 4° C. with anti-non-muscular myosin heavy chain II-A and B antibodies, phosphorylated lukewarm light chain 2 (Ser19) antibody and cleaved caspase-3 antibody (1:200), Incubated with Alexa Fluor 488 goat anti-rabbit IgG antibody (1:500; A11034, Invitrogen) for 1 hour. DAPI (Vector Laboratories) and phalloidin (T7471, Invitrogen) were used for nuclear staining and cytoskeleton staining. Images were captured with a laser scanning confocal microscope (LSM 700, Carl Zeiss).

Reference Example 6. Western Blot

B p65와 함게 배양되었다. 세척 후, 막들은 항토끼 IgG-HRP (1:5000; sc-2030, Santa Cruz Biotech)와 배양되었다. 궁극적으로 면역반응성 밴드를 ECL 시약(32106, Thermo Scientific)을 이용하여 시각화하였고, Chemidoc XRS+ 시스템(Bio-Rad Laboratories)으로 검출하였다.Whole cell lysates of HL-60 cells were collected in CRB buffer (FNN0011, Invitrogen) supplemented with a mixture of protease and phosphatase inhibitors (78443, Thermo Scientific) for 30 minutes. Samples were centrifuged at 12000 rpm at 4° C., and the supernatant was used for immunoblotting. Separation of the nucleus and cytoplasm of HL-60 cells was performed by nuclear and cytoplasmic extraction reagent (78833, Pierce). Protein concentration was measured with the BCA protein assay kit (23227, Pierce). Protein samples were analyzed on an SDS-PAGE gel (Bio-Rad Laboratories). After electrophoretic separation using an iBLOT 2 dry blotting system (IB21001, Thermo Scientific), proteins were transferred to a nitrocellulose membrane. Thereafter, the membranes were blocked with 10% fat-free milk, phosphorylated myosin light chain 2 (Ser19) at a concentration of 1:500, cleaved caspase-3, caspase-7, AhR, YAP, TAZ and NF-

Incubated with B p65. After washing, the membranes were incubated with anti-rabbit IgG-HRP (1:5000; sc-2030, Santa Cruz Biotech). Ultimately, the immunoreactive band was visualized using ECL reagent (32106, Thermo Scientific) and detected with a Chemidoc XRS+ system (Bio-Rad Laboratories).

Reference Example 7. Caspase-3/7 apoptosis test

Apoptosis was detected by caspase 3/7 green detection reagent (C10423, Invitrogen). Cells were plated in assay 96-well plates at a density of 1 10 ⁵ cells per well and cultured in complete medium. 2 μM Caspase 3/7 reagent was treated for 30 minutes. Cell activity on the 3 wells was measured by fluorescence (stimulation/emission = 502/530 nm).

Reference Example 8. Flow cytometric analysis

Cell death and differentiation were confirmed by flow cytometry. HL-60 cells were washed twice with PBS, and then phycoerythrin-conjugated annexin V (phycoerythrin-conjugated annexin V) and 7-amino-actinomycin (7-) for 15 minutes at room temperature for apoptosis assay. Amino-Actinomycin: 7-AAD) or incubated with conjugated CD11b and CD14 antibodies at 4° C. for 30 minutes for differentiation test. HL-60 eGFP and HL-60 MRLC-AA-eGFP were stained with propidium iodide (PI, P4864, Sigma) immediately before cell death assay. All PBMNCs were stained with stem and liver cell surface markers CD34 ⁺ and PE Annexin V for 30 minutes at 4° C. and in the dark. Stained cells were transferred to 5 mL FACS tubes and analyzed by flow cytometry using a BD-Aria ² instrument.

Reference Example 8. Real-time PCR and transcript sequencing

Leukemia cells were harvested for RNA extraction using the RNeasy Mini kit (74140, Qiagen). SuperScript?? Reverse transcriptase was used to prepare cDNA from 1 μg of total RNA in conjunction with the IV4 First-Strand Synthesis System (18091050, Invitrogen). Quantitative real-time PCR was performed using cDNA and SYBR PCR master mix (4309155, Applied BioSystems). Samples were analyzed with an Applied Biosystems 7300 instrument. The following primers were used: TNF: forward 5'-AGACGCCACATCCCCTGACAA-3' (SEQ ID NO: 1) and reverse 5'-GACGGCGATGCGGCTGATG-3' (SEQ ID NO: 2); KIT: forward 5'-TCATGGTCGGATCACAAAGA-3' (SEQ ID NO: 3) and reverse 5'-AGGGGCTGCTTCCTAAAGAG-3' (SEQ ID NO: 4); Ankrd1: forward 5'-AGC CCA GAT CGA ATT CCG TG-3' (SEQ ID NO: 5) and reverse 5'- CTC CTT CTC TGT CTT TGG CGT-3' (SEQ ID NO: 6); Ctgf: forward, 5'-ACC GAC TGG AAG ACA CGT TTG-3' (SEQ ID NO: 7) and reverse 5'-CCA GGT CAG CTT CGC AAG G-3' (SEQ ID NO: 8); Cyr61: forward 5'-TGA AGC GGC TCC CTG TTT T-3' (SEQ ID NO: 9) and reverse 5'-CGG GTT TCT TTC ACA AGG CG-3' (SEQ ID NO: 10); GAPDH: forward 5'-CAA AGT TGT CAT GGA TGA CC-3' (SEQ ID NO: 11) and reverse 5'-CCA TGG AGA AGG CTG GGG-3' (SEQ ID NO: 12). For transcript sequence analysis, RNA was isolated from HL-60 cells 16 hours after treatment with 50 μM of blevisstatin or vehicle. For mRNA-Seq samples, Illumina TruSeq?? Obtained using an RNA sample preparation kit. Gene expression data pretreatment and normalization were performed using the unit of fragments per kilobase of exon per million fragments mapped (FPKM) method. The Illumina platform was run on Macrogen.

Reference Example 9. Small interfering RNA (siRNA)

To temporarily knock down the expression of AhR, YAP and TAZ, HL-60 cells were transfected with ON-TARGETplus SMARTpool siRNAs (Dharmacon) through electroporation using Neon transfection system (MPK5000, Invitrogen). Made it. AhR siRNA sequence is as follows: GCAAGUUAAUGGCAUGUUU (SEQ ID NO: 13), GAACUCAAGCUGUAUGGUA (SEQ ID NO: 14), GCACGAGAGGCUCAGGUUA (SEQ ID NO: 15), GCAACAAGAUGAGUCUAUU (SEQ ID NO: 16). The YAP1 siRNA sequence is as follows: ACAGGUGGCUCAAUUCUUG (SEQ ID NO: 17), GUAAGGAUAUGGCAGCAGU (SEQ ID NO: 18), GAUCGAAGCCAGUGAAGGU (SEQ ID NO: 19), GCCGAGAAGUGCAGUCCAA (SEQ ID NO: 20). The TAZ1 siRNA sequence is as follows: GGCCAGAGAUACUUCCUUA (SEQ ID NO: 21), CCACAGGGCUCAUGAGUGU (SEQ ID NO: 22), GGAUUAGGAUGCGUCAAGA (SEQ ID NO: 23), CGAGAUGGAUACAGGUGAA (SEQ ID NO: 24). Non-target siRNA sequences are as follows: UGGUUUACAUGUCGACUAA (SEQ ID NO: 25), UGGUUUACAUGUUGUGUGA (SEQ ID NO: 26), UGGUUUACAUGUUUUCUGA (SEQ ID NO: 27), UGGUUUACAUGUUUUCCUA (SEQ ID NO: 28). siRNAs were used at a final concentration of 100 nM. Successful knockdown of genes was determined by Western blot 48 hours after transfection.

Reference Example 10. Cell Mobility Test

For transwell analysis, HL-60 cells (2Х10 ⁵ ) in serum-free medium were added to the upper chamber of a 5 μm pore size transwell membrane filter (3421, Corning). 10% FBS was added to the lower chamber for use as a positive control. Blevistatin was applied to both the upper and lower chambers. Cells were allowed to migrate at 37° C. for 16 hours. Migrated cells collected from the bottom well were quantified by hemocytometer.

For 2D migration analysis, a 96-well glass bottom plate was coated with fibronectin (100 μg/ml in PBS) for 1 hour at 37°C. Plates were washed twice with PBS and then cells were seeded in serum-free medium for 1 hour to set before imaging. Low-speed images were obtained on a JuLi Br live cell analyzer (JULI-BR04, NanoEnTek Inc.) using a 4x objective. For rate quantification and tracking, images were obtained every 3 minutes for 16 hours at 37° C. after blevisstatin was added. Instantaneous speed is expressed as instantaneous distance divided by time. Average cell velocity represents the average of all instantaneous velocities of a cell. Image analysis was performed with Image J software (National Institutes of Health).

Reference Example 11. Mouse xenograft (The Mice xenograft)

6-8 weeks old RAG2 ^-/- gc ^-/- mice were purchased from Jackson Laboratory. For the local xenograft model, 10 ⁷ HL-60 cells were injected subcutaneously into 8-10 week old nude mice. When tumors were established (~500 mm ³ ), rats were prepared to maximize uniformity between groups (n = 5), and blevisstatin was injected intraperitoneally (0, 0.5, 1.5 mg/day) every 2 days. kg). Mice were monitored daily, and tumor growth was measured every 3-4 days with a digital caliper using Equation 1 below:

[Equation 1]

Volume = length Х width Х width Х 0.5.

When the tumor volume exceeded 2,000 mm ³ , animals were sacrificed. All animal experiments were approved according to the guidelines of the Tea Medical School.

Reference Example 12. Statistical Analysis

All experiments were repeated at least 3 times. Data are expressed as mean ± standard error (SEM) or median ± minimum/maximum value. Statistically significant differences between the two groups were evaluated by a two-sided t-test. Multiple comparisons were performed using one-way analysis of variance (ANOVA). Pair differences between groups were determined using a two-sided t-test with Bonferroni correction. Statistical analysis was performed in Microsoft Excel.

Example

Example 1. Confirmation of the inhibitory effect of blevisstatin on proliferation of AML (Acute Myeloid Leukemia) cells

First, the spatial relationship between the location of the non-muscle myosin ±: NMII?) isomer in AML cells and the filamentous actin (F-actin) was analyzed. Hematopoietic stem cells (HSC) differentially express two heavy chain isotype proteins: NMII: A (MYH9) and B (MYH10). In HL-60 cells, a representative acute promyelocytic leukemia cell line, NM±A and -±B have distinct cell location patterns, and actin indicates a distinct cortical location. NM±B is exclusively located in the nucleus, while NM±A is distributed throughout the cell including the cell membrane and cytoplasm. As a result of immunofluorescence staining, the repositional coordination of NM±A on the plasma membrane and cortical actin was significant (Fig. 1A). The BloodSpot database (www.bloodspot.eu) showed that MYH9 mRNA expression was increased in primary human AML compared to normal HSC and precursor controls via cytogenetic subgroups (Fig. 2A). However, BloodSpot analysis did not show a significant increase in MYH10 expression in the AML subset (FIG. 2B ).

Myosin light chain (MLC) phosphorylation is sufficient to support ATPase activity and is essential for the binding of actin filaments by the myosin head. Therefore, phosphorylation of MLC (pMLC) in AML cells was analyzed. As a result, it was found through immunofluorescence that pMLC levels were significantly increased in established AML cell lines (HL-60, THP1, U937) (P <0.001; FIGS. 1B and 1C). These data indicate that overexpression of NM±A and increased phosphorylation of MLC may be common in AML modification.

The development of new compounds with high specificity has been introduced to study myosin activity under various pathological conditions. Blevistatin is a reversible inhibitor of myosin II ATPase that binds to the fissure between actin and the ATP binding site, inhibits Pi release from the MgADP-Pi complex, and separates actin. Therefore, the effect of blevisstatin on AML cells was evaluated. The number of HL-60 cells was dose-dependently decreased by blevisstatin in the culture medium (Fig. 1D). It was confirmed that the number of colonies was decreased in the group treated with blevisstatin through methylcellulose colony formation analysis (FIG. 1E).

Next, it was analyzed how blevisstatin affects normal hematopoietic cells. Bone marrow 32D cells are a non-carcinogenic cell model that is widely used in in vitro studies. As a result, the degree of change in the number of cells to blevisstatin was observed less in 32D cells. At 72 hours 32D cells only showed a decrease of less than 10%, while HL-60 cells showed a significant decrease in cell number (48 h: 54%; 72 h: 73%) (Figure 1F). In addition, the effects of blevisstatin on other leukemia cell lines including Jurkat cells (acute lymphocytic leukemia), K562 cells (chronic myelogenous leukemia) and other AML cells (THP1 and U937) were investigated. Blevistatin was more sensitive to AML cell lines that could be used for therapeutic benefit (Fig. 1G).

Example 2. Confirmation of the effect of blevisstatin inducing AML cell death

Next, the mechanism of blevisstatin inducing cell number reduction was confirmed. As a result, as measured by Annexin V staining, apoptosis of HL-60 cells remarkably increased 24 hours after blevisstatin treatment (Fig. 3A). HL-60 cells also displayed a distinct caspase 3/7 cell death signal in the presence of blevisstatin (FIG. 3B ). Consistently, the caspase-3/7 apoptosis signal of 32D cells increased to a degree similar to that seen with HL-60 at 24 hours (40.72 ± 3.92% (32D) vs 44.53 ± 3.37% (HL-60); P = 0.42; Fig. 3C), this level of cell death was maintained until 72 hours. On the other hand, HL-60 cells showed a sharp increase in apoptosis, and the apoptosis fluorescence increased strongly up to 90% at 72 hours. In addition, the apoptosis effect of blevisstatin on other leukemia cell lines indicates that blevisstatin further induces apoptosis signals of AML cell lines (FIGS. 3D and 3E ).

Previous studies have shown that the aryl hydrocarbon receptor (AHR) is associated with blevisstatin, which induces apoptosis in hematopoietic cells, and thus AHR was expected to be related to the regulation of apoptosis in AML cells. AHR is a ligand-induced transcription factor that affects the major stages of tumorigenesis. Both the antagonists StemRegenin-1 (SR1) and CH223191 partially reduce the blevisstatin-induced cell caspase 3/7 activity when cultured for 72 hours (Fig. 3G). On the other hand, blevisstatin also induced cleavage of caspase 3 and caspase 7 obtained by the two antagonists (Fig. 3H). Inhibition of AHR using RNAi-mediated silencing confirmed the association of AHR signaling in blevisstatin-induced cell death, and the silencing efficiency was confirmed by Western blot (FIGS. 3I and 3J ).

In addition, it is known that blevisstatin can prevent furrow ingression during cell mitosis, and the resulting multinuclear cells undergo various fates including death by apoptosis. It was observed that the level of multinuclear cells increased sharply to 78.2% 48 hours after blevisstatin treatment. Cell multinucleation partially activates the caspase-3/7 apoptosis pathway by increased phosphorylation of histone H2AX (γH2AX) (phosphor-H2A.X data). Taken together, blevisstatin activates the endogenous apoptosis pathway through induction of caspase 3/7 activity through AHR signaling and inhibition of cell division (FIG. 3F).

In addition, HL-60 overexpressing EGFP and MRLC-AA-EGFP was constructed to compare the effects of phosphorylated MLC on cell growth and cell death. The two cell lines showed high transduction efficiency, and the level of phosphorylated MLC changed through transduction was confirmed by Western blot (FIGS. 4A and 4B ). As expected, MRLC-AA-EGFP showed higher cell death rate and lower growth rate compared to control EGFP-HL-60 cells (FIGS. 4C and 4D ).

We confirmed the anti-leukemia effect of blevisstatin in human AML patient samples. Normal donor-derived samples showed low sensitivity to blevisstatin, whereas AML patient-derived cells showed apoptotic response 72 hours after blevisstatin treatment (as analyzed by CD34 ⁺ and Annexin V double staining, FIG. 5). .

Example 3. Confirmation of the effect of inducing differentiation of blevisstatin and inhibiting cell migration

Since AML cells are characterized by uncontrolled survival and misprogrammed hematopoietic cell differentiation, then cell differentiation to blevisstatin treatment was tested. As a result, the surface expression of both CD11b and CD14 (the differentiation markers of bone marrow cells) was clearly amplified in the blevisstatin-treated cells in HL-60 cells in differentiation medium (19.67% increase in CD11b; 17.7% increase in CD14). ; P <0.05; Fig. 6A). Similar results were obtained at the mRNA expression level of CD14 in HL-60 (Fig. 6B).

The ability to spread from blood to peripheral tissues depends on cell mobility. The change in cell mobility according to the blevisstatin treatment was measured. HL-60 cells migrated to the lower chamber of the transwell were reduced when blevisstatin was treated (1.76 ± 0.08% (control) vs 0.65 ± 0.14% (blevisstatin); Figs. 6C and 6D), cell migration pathways The analysis results showed that the cell migration rate was reduced by blevisstatin (285.09 ± 7.29 μm/h (control) vs 92.02 ± 3.42 μm/h (blevisstatin); FIGS. 6E and 6F). These results indicate that blevisstatin has the effect of modulating various physiological functions of AML cells such as survival, differentiation and mobility.

Example 4. Confirmation of the regulation of the transcriptional activity of HL-60 cells by blevisstatin

To better understand the transcriptional changes in AML cells induced by blevisstatin at the molecular level, the gene expression profiles of HL-60 cells were analyzed by total transcriptome analysis using RNA sequencing. 16 hours after treatment, the blevisstatin-treated cells showed 25 differentially expressed genes (DEGs). Among the DEGs, 8 genes increased by 1.5-fold or more while 17 genes decreased by 1.5-fold or more (Fig. 7A). In the volume plot, 6 top-ranked genes were listed (3 upregulated and 3 downregulated; Fig.7AB), and KEGG pathway enrichment analysis to identify the enriched pathway (P<0.05) in the gene set. (KEGG pathway enrichment analysis) was performed (C in Fig. 7A).

B 신호전달 활성의 변화가 확인되었다(도 7b의 E). NF

B의 과활성화는 1차 AML 세포에서 흔히 발견되며, 그 활성화의 차단은 세포 사멸을 유발할 수 있다. In particular, several genes in our DEG list are known to be highly related to leukemia. As the number 1 downregulated gene, TNF is known to play a role in the peripheral blood of AML patients. TNF levels were significantly higher in AML, which promotes survival of leukemia cells. The oncogene c-KIT is expressed in about 80% of AML cases, and high c-KIT expression serves as a reliable molecular marker of poor prognosis of AML. Higher MMP-2 activity is more specific for AML patients who do not respond to chemotherapy. In order to verify the change in the expression pattern of AML-related tumor genes (TNF and c-KIT) shown in the RNA sequence analysis results, we used a quantitative reverse transcription PCR (qRT-PCR) method to determine the gene expression pattern. It was confirmed that they matched (FIG. 7BD). In addition, down-regulation of TNF and c-KIT was found in other AML cells upon treatment with blevisstatin (FIGS. 8A and 8B ). NF, a sub-signaling pathway of TNF after blevisstatin treatment

The change in B signaling activity was confirmed (E in FIG. 7B). NF

Overactivation of B is commonly found in primary AML cells, and blocking its activation can lead to cell death.

Since the important value of these genetic findings in malignant tumors is known, we next focused on the molecular mechanisms of blevisstatin-induced transcriptional changes. Recent studies have shown that cellular contractility can regulate gene expression through signaling pathways including Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1 or TAZ). It was hypothesized that the genetic change according to blevisstatin treatment in AML cells is mediated by YAP/TAZ activity (FIG. 7B). We observed the change in the position of YAP from the cytoplasm to the nucleus, which means the activity of YAP during blevisstatin treatment, and YAP-related genes (Ankrd1, Ctgf) were also upregulated according to the treatment of blevisstatin. This indicates that blevisstatin enhances YAP/TAZ transcriptional activity (G and H in FIG. 7B). Conversely, inhibition of YAP and TAZ using RNAi-mediated silencing enhanced c-KIT and TNF expression, which is the opposite effect of treatment with blevisstatin (Fig. 7A) (Fig. 7B, J). Silence efficiency was confirmed by Western blot (I in Fig. 7B). YAP/TAZ double knockdown rescued blevisstatin-induced c-KIT downregulation, and treatment with verteporfin (YAP-TEAD binding inhibitor) restored TNF expression levels (K and L in FIG. 7B). These data imply that blevisstatin has an inhibitory effect on the expression of tumor-associated genes through induction of YAP/TAZ activity.

Example 5. Confirmation of the tumor suppression effect of blevisstatin in the Xenograft model

Next, it was confirmed that blebbsitatin inhibits the growth of tumor tissue in animal models through the Xenograft technique. Specifically, after injecting HL-60 cells into Rac2 ^-/- gc ^-/- KO immunodeficient mice, tumor growth was induced to about 500 mm ³ for 20 days, and then blebbistatin at a concentration of 0 to 1.5 mg/kg was administered every 2 days. It was injected intraperitoneally (Fig. 9A). As a result, it was confirmed that as the concentration of blebbistatin increased, the growth of tumor tissue was markedly suppressed (FIGS. 9B and 9C).

Example 6. Confirmation of AML cell proliferation inhibitory effect of blevisstatin derivative

Next, para-nitro blebbistatin ((3a S )-3a-Hydroxy-6-methyl-1-phenyl-2,3-dihydropyrrolo[2,3-b]quinolin-4-one) derivative of blebbistatin ((3a S )-3a S ) -3a-Hydroxy-6-methyl-1-(4-nitrophenyl)-2,3-dihydropyrrolo[2,3- b ]quinolin-4-one) in the medium in the presence of HL-60 cells to 24, As a result of measuring under a microscope using a hemocytometer at 48 hour intervals, it was confirmed that HL-60 cells did not grow when treated with para-nitro blebbistatin (FIG. 10). This indicates that blevisstatin derivatives also have a cytostatic effect on AML.

<110> Seoul National University Industry Foundation SUNGKWANG MEDICAL FOUNDATION NATIONAL CANCER CENTER <120> PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING MYELOID LEUKEMIA <130> PN126421KR <160> 28 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TNF-F <400> 1 agacgccaca tcccctgaca a 21 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TNF-R <400> 2 gacggcgatg cggctgatg 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KIT-F <400> 3 tcatggtcgg atcacaaaga 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KIT-R <400> 4 aggggctgct tcctaaagag 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ankrd1-F <400> 5 agcccagatc gaattccgtg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ankrd1-R <400> 6 ctccttctct gtctttggcg t 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ctgf-F <400> 7 accgactgga agacacgttt g 21 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ctgf-R <400> 8 ccaggtcagc ttcgcaagg 19 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cyr61-F <400> 9 tgaagcggct ccctgtttt 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cyr61-R <400> 10 cgggtttctt tcacaaggcg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-F <400> 11 caaagttgtc atggatgacc 20 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-R <400> 12 ccatggagaa ggctgggg 18 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> AhR siRNA <400> 13 gcaaguuaau ggcauguuu 19 <210> 14 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> AhR siRNA <400> 14 gaacucaagc uguauggua 19 <210> 15 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> AhR siRNA <400> 15 gcacgagagg cucagguua 19 <210> 16 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> AhR siRNA <400> 16 gcaacaagau gagucuauu 19 <210> 17 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> YAP1 siRNA <400> 17 acagguggcu caauucuug 19 <210> 18 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> YAP1 siRNA <400> 18 guaaggauau ggcagcagu 19 <210> 19 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> YAP1 siRNA <400> 19 gaucgaagcc agugaaggu 19 <210> 20 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> YAP1 siRNA <400> 20 gccgagaagu gcaguccaa 19 <210> 21 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> TAZ1 siRNA <400> 21 ggccagagau acuuccuua 19 <210> 22 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> TAZ1 siRNA <400> 22 ccacagggcu caugagugu 19 <210> 23 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> TAZ1 siRNA <400> 23 ggauuaggau gcgucaaga 19 <210> 24 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> TAZ1 siRNA <400> 24 cgagauggau acaggugaa 19 <210> 25 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> non-target siRNA <400> 25 ugguuuacau gucgacuaa 19 <210> 26 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> non-target siRNA <400> 26 ugguuuacau guuguguga 19 <210> 27 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> non-target siRNA <400> 27 ugguuuacau guuuucuga 19 <210> 28 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> non-target siRNA <400> 28 ugguuuacau guuuuccua 19

Claims (9)

A pharmaceutical composition for the prevention or treatment of myeloid leukemia (Myeloid Leukemia) comprising a compound of the following formula 1 or a pharmaceutically acceptable salt thereof:
[Formula 1]

R is selected from the group consisting of hydrogen, deuterium, halogen, cyano group, nitro group, -NR' ₃ ⁺ , carbonyl group, carboxyl group, aldehyde group, ester group, -SO ₃ R'.
The pharmaceutical composition of claim 1, wherein R is H.
The pharmaceutical composition according to claim 1, wherein the compound of Formula 1 or a pharmaceutically acceptable salt thereof inhibits myosin ATPase of myeloid leukemia cells, thereby inhibiting cell proliferation and causing apoptosis.
The pharmaceutical composition according to claim 1, wherein the myeloid leukemia is acute myeloid leukemia (AML).
A pharmaceutical composition for the prevention or treatment of myeloid leukemia (Myeloid Leukemia) comprising a compound of the following formula 2 or a pharmaceutically acceptable salt thereof:
[Formula 2]

R is selected from the group consisting of hydrogen, deuterium, halogen, cyano group, nitro group, -NR' ₃ ⁺ , carbonyl group, carboxyl group, aldehyde group, ester group, -SO ₃ R'.
A pharmaceutical composition for the prevention or treatment of myeloid leukemia comprising a compound of the following formula 3 or a pharmaceutically acceptable salt thereof:
[Formula 3]

.
Health functional food for the prevention or improvement of myeloid leukemia (Myeloid Leukemia) comprising a compound of the following formula 1 or a food acceptable salt thereof:
[Formula 1]

R is selected from the group consisting of hydrogen, deuterium, halogen, cyano group, nitro group, -NR' ₃ ⁺ , carbonyl group, carboxyl group, aldehyde group, ester group, -SO ₃ R'.
A method for preventing or treating myeloid leukemia, comprising administering the pharmaceutical composition of claim 1 to an individual.
The method for preventing or treating myeloid leukemia according to claim 8, further comprising administering an existing myeloid leukemia treatment to the individual.

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