KR102228406B1 - Pharmaceutical composition for treating chronic myeloid leukemia comprising CXCR2 inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment of chronic myelogenous leukemia comprising a CXCR2 (C-X-C motif chemokine receptor 2) inhibitor as an active ingredient.

Description

Pharmaceutical composition for treating chronic myeloid leukemia comprising CXCR2 inhibitor as an active ingredient

The present invention relates to a pharmaceutical composition for the treatment of chronic myelogenous leukemia comprising a CXCR2 (C-X-C motif chemokine receptor 2) inhibitor as an active ingredient.

Chronic myeloid leukemia (CML) is a malignant blood disease in which various blood cells, which are abnormally increased by massive proliferation of hematopoietic stem cells in the bone marrow, invade peripheral blood or other organs.It is a malignant blood cancer that occurs mainly in adults. The etiology of chronic myelogenous leukemia is known as overexpression of tyrosine kinase due to an abnormality in the BCR-ABL gene, which is caused by the chromosomal translocation of the BCR gene and the ABL gene. It is a well-known fact that the therapeutic effect of myeloid leukemia was greatly increased.

However, the use of a BCR-ABL tyrosine phosphorylase inhibitor (hereinafter, a BCR-ABL inhibitor) causes various point mutations in the ABL kinase domain and is difficult to treat in the case of mutant chronic myelogenous leukemia. In addition, patients who fail drug treatment in chronic myelogenous leukemia without mutations are expected to have a mechanism independent of BCR-ABL. Accordingly, there is an urgent need to discover new therapeutic targets capable of inhibiting intracellular signaling pathways other than BCR-ABL inhibitors, and to develop therapeutics therefor.

Recently, interest in CXC motif chemokine receptor 2 (CXCR2), a cell receptor related to inflammation and apoptosis, is increasing, and several CXCR2s are used for the treatment of chronic obstructive pulmonary disease, asthma and cystic fibrosis. Inhibitors are being developed. In addition, when CXCR2 was inhibited in solid cancer cells, the effect of inhibiting the survival, invasion and proliferation of cancer cells and inhibiting signaling such as AKT and MAPK was confirmed, suggesting that CXCR2 can function as a new therapeutic target. do.

On the other hand, in the case of hematologic cancer, it has been found that CXCR2 overexpression in acute myeloid leukemia cells and a poor prognosis have a relationship, but the effectiveness of CXCR2 as a therapeutic target for chronic myelogenous leukemia has not yet been reported.

Under this background, the present inventors have confirmed that CXCR2 can be an effective therapeutic target in chronic myelogenous leukemia cells, and has a therapeutic effect on cells with resistance as well as chronic myelogenous leukemia cells susceptible to tyrosine phosphorylation enzyme inhibitors. Was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the treatment of chronic myelogenous leukemia comprising a CXCR2 (C-X-C motif chemokine receptor 2) inhibitor as an active ingredient.

However, the problem to be solved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

According to an embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of chronic myelogenous leukemia comprising a CXCR2 (C-X-C motif chemokine receptor 2) inhibitor as an active ingredient.

According to one side, the CXCR2 inhibitor may be a CXCR2 expression inhibitor or a CXCR2 antagonist.

According to one side, the CXCR2 antagonist may be SB225002.

According to one aspect, the composition may promote the death of cells sensitive to a tyrosine phosphorylase inhibitor.

According to one aspect, the composition may promote the death of cells resistant to tyrosine phosphorylation enzyme inhibitors.

According to one side, the tyrosine phosphorylation enzyme inhibitor may be imatinib.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of chronic myelogenous leukemia comprising a CXCR2 inhibitor and a tyrosine phosphorylation enzyme inhibitor as active ingredients.

The pharmaceutical composition for the treatment of chronic myelogenous leukemia comprising a CXC motif chemokine receptor 2 (CXCR2) inhibitor of the present invention as an active ingredient presents CXCR2 receptor as a new therapeutic target, and chronic myelogenous leukemia cells sensitive to tyrosine kinase inhibitors In addition, it can effectively induce the death of resistant cells.

The pharmaceutical composition of the present invention can significantly increase the death of chronic myelogenous leukemia cells when administered in combination with a tyrosine phosphorylation enzyme inhibitor.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1a is a hematopoietic stem cell (CD34+) and a chronic myelogenous leukemia cell line CXCR2 gene expression level was measured by real-time polymerase chain reaction, Figure 1b is the CXCR2 protein expression level measured by Western blot analysis.
Figure 2a is a DAPI fluorescence staining confirms whether or not nuclear fragments are formed when a CXCR2 inhibitor is treated in a chronic myelogenous leukemia cell line. FIG. 2B shows whether or not apoptosis protein was increased when CXCR2 inhibitors were treated in hematopoietic stem cells (CD34+) and chronic myelogenous leukemia cell lines.
FIG. 3A is an observation of whether or not nuclear fragments are generated by DAPI fluorescence staining to confirm whether apoptosis is induced when a CXCR2 inhibitor is treated with an imatinib-resistant chronic myelogenous leukemia cell line. It was observed whether the expression was increased.
Figure 4 shows the apoptosis effect in the case of treatment with a CXCR2 inhibitor and imatinib alone and in combination in a chronic myelogenous leukemia cell line susceptible to a tyrosine kinase inhibitor, Figure 4a shows the cell viability, Figure 4b is It shows the drug combination index (combination index) based on the results of Figure 4a.
FIG. 5 shows the apoptosis effect when treated with a CXCR2 inhibitor and imatinib alone and in combination in a chronic myelogenous leukemia cell line resistant to a tyrosine phosphorylase inhibitor. FIG. 5A shows the cell viability, and FIG. 5B is It shows the drug combination index (combination index) based on the results of Figure 4a.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various changes may be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It is to be understood that all changes, equivalents, or substitutes to the embodiments are included in the scope of the rights.

The terms used in the examples are used for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present specification, terms such as "comprise" or "have" are intended to designate the presence of features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features. It is to be understood that the presence or addition of elements or numbers, steps, actions, components, parts, or combinations thereof does not preclude in advance.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms as defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in the present application. Does not.

In addition, in the description with reference to the accompanying drawings, the same reference numerals are assigned to the same components regardless of the reference numerals, and redundant descriptions thereof will be omitted. In describing the embodiments, when it is determined that a detailed description of related known technologies may unnecessarily obscure the subject matter of the embodiments, the detailed description thereof will be omitted.

The present inventors found that CXCR2 is effective as a new therapeutic target by confirming that apoptosis is promoted when CXCR (C-X-C motif chemokine receptor 2) is inhibited in chronic myelogenous leukemia cells. In particular, the present inventors have completed the present invention for a novel CXCR2 target therapeutic agent by demonstrating that the CXCR2 inhibitor can induce the death of chronic myelogenous leukemia cells that are resistant to imatinib, a first-generation tyrosine phosphorylation enzyme inhibitor.

Accordingly, according to an embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of chronic myelogenous leukemia comprising a CXCR2 (C-X-C motif chemokine receptor 2) inhibitor as an active ingredient.

In the present invention, the term "chronic myelogenous leukemia (CML)" refers to a hematologic stem cell disease caused by uncontrolled growth and excessive accumulation of leukocytes due to increased bone marrow cells in the bone marrow, and refers to the Philadelphia chromosome. Includes diseases caused by abnormal proliferation of hematopoietic stem cells in the bone marrow.

In the present invention, the term "CXCR2 (CXC motif chemokine receptor 2)" refers to a cytokine receptor that exists on the surface of leukocytes and its naturally occurring ligand is Gro-α, βγIL-8, ENA-78 or GCP-2. .

In the present invention, the term "treatment" refers to any action in which symptoms caused by chronic myelogenous leukemia are improved or advantageously changed by administration of the pharmaceutical composition of the present invention.

As the CXCR2 inhibitor included in the pharmaceutical composition of the present invention, an expression inhibitor of CXCR2 or an antagonist of CXCR2 may be used.

As the CXCR2 expression inhibitor, siRNA or shRNA specific for CXCR2 mRNA can be used, and RNAi has a sequence complementary to a part of the CXCR2 gene, and can degrade the mRNA of the CXCR2 gene or inhibit translation.

RNA interference (RNAi) is a process in which the expression of a target gene is inhibited by the selective inactivation of the corresponding mRNA by double-stranded RNA (dsRNA). RNA interference effect is activated by double-stranded RNA delivered to the intracellular cytoplasm, and this mechanism can induce degradation or translational inhibition of target mRNA through small interfering RNA (siRNA), short hairpin RNA (shRNA), and bi-functional shRNA. I can.

As a substance that suppresses the expression of CXCR2 by RNAi, shRNA or an expression vector containing the DNA, or a cell containing the expression vector can be used, for example, using a viral vector or a gene introduction method using liposomes It is possible. Viruses (or viral vectors) useful for delivering shRNA in the present invention include lentiviruses, adenoviruses, retroviruses, adenoviruses, and the like.

In addition, as the CXCR2 antagonist, known substances may be used without limitation, and for example, SB225002 may be used.

The pharmaceutical composition of the present invention may promote the death of cells susceptible to a tyrosine phosphorylation enzyme inhibitor, or may promote the death of cells resistant to a tyrosine phosphorylation enzyme inhibitor.

Here, the tyrosine phosphorylation enzyme inhibitor may be imatinib.

The pharmaceutical composition of the present invention may contain a CXCR2 inhibitor alone, or may be prepared by a method known in the pharmaceutical field to be used as a drug, and a pharmaceutically acceptable carrier, excipient, diluent, stabilizer, preservative, etc. By mixing, it may be prepared and used in a dosage form such as powder, granule, tablet, capsule, or injection.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery substances used for oral administration of the peptide preparation. In addition, the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizing agents include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl-or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, etc. in addition to the above components.

The diluent is a non-aqueous solvent such as propylene glycol, polyethylene glycol, olive oil, vegetable oil such as peanut oil, or saline (preferably 0.8% saline), water containing a buffer medium (preferably 0.05M phosphate buffer) And an aqueous solvent such as, but is not limited thereto.

The excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene, glycol, water. , Ethanol, and the like, but are not limited thereto.

Examples of the stabilizing agent include carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamate, and glucose, or proteins such as animal, vegetable or microbial proteins such as milk powder, serum albumin, and casein, but are limited thereto. no.

Examples of the preservatives include thimerosal, merthiolate, gentamicin, neomycin, nystatin, amphotericin B, tetracycline, penicillin, streptomycin, and polymyxin B, but are not limited thereto.

The pharmaceutical composition of the present invention may be administered to mammals including humans by any method, for example, orally or parenterally. The parenteral administration method may be intravenous, intramuscular, intraarterial, intramedullary, intrathecal intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration, but is limited thereto. no.

The pharmaceutical composition of the present invention may be formulated as a formulation for oral administration or parenteral administration according to the route of administration as described above.

The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient in a single dose, and may be administered by a divided treatment regimen administered for a long period in multiple doses. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. This may be determined in consideration of various factors such as the formulation method, the route of administration and the number of treatments, as well as the patient's age, weight, health condition, disease symptoms, administration time and method. In view of these points, one of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in the formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.

In addition, according to another embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of chronic myelogenous leukemia comprising a CXCR2 inhibitor and a tyrosine phosphorylation enzyme inhibitor as active ingredients. In the case of co-administration of a CXCR2 inhibitor and a tyrosine kinase inhibitor, side effects can be reduced by using a small amount of a tyrosine kinase inhibitor, and at the same time, the probability of developing drug resistance can be lowered.

Hereinafter, the present invention will be described in more detail through examples. The following examples are described for the purpose of illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: chronic myeloid As a therapeutic target for leukemia Of CXCR2 effectiveness

CXCR2 mRNA expression levels and protein expression levels in hematopoietic stem cells (CD34+) and chronic myelogenous leukemia cell lines (K562, KU812) were measured by real time PCR and Western blot, respectively.

First, total RNA was extracted from the cells according to the manual using TRIZOL (invitrogen). 1 μg of total RNA was transcribed into cDNA using a cDNA synthesis kit (Intron), and the synthesized cDNA was analyzed by real time PCR using SYBR Green (Bio Rad) to quantitatively analyze the amount of CXCR2, and the primers used Is as follows:

<Real time PCR primer>

Forward direction: 5'-CAATGAATGAATGAATGGCTAAG-3'

Reverse: 3'-AAAGTTTTCAAGGTTCGTCCGTGTT-5'

<GAPDH primer>

Forward direction: 5'-GAGTCCACTGGCGTCTTCAC-3'

Reverse: 3’-TTCACACCCATGACGAACAT-5’

Then, Western blot was performed to measure the amount of CXCR2 protein expression, and then the protein was extracted using protein lysis buffer (Intron), and 20 μg of the protein was hung on the SDS-PAGE gel.

As a result, as shown in FIGS. 1A and 1B, chronic myelogenous leukemia cells had higher levels of mRNA and protein expression of CXCR2 compared to the control group, hematopoietic stem cells, thereby demonstrating the possibility of treating chronic myelogenous leukemia through CXCR2 inhibition. I could confirm.

Example 2: CXCR2 Effect of Inhibitors on Promoting Chronic Myeloid Leukemia Apoptosis

2-1. Tyrosine Phosphorylase Inhibitor Susceptible Cells

Chronic myelogenous leukemia cell lines (K562, KU812) were treated with 1 μg/ml of the CXCR2 inhibitor SB225002 for 24 hours, then the cells were collected and fixed with 4% formaldehyde to perform DAPI fluorescence staining, and then the nuclear fragments were formed through a fluorescence microscope. Was observed. In addition, for quantitative analysis, the total number of cells visible on a spectral microscope was counted, and the number of cells in which the fragment of the nucleus was visible was counted, and then expressed as a percentage and expressed as a graph.

As a result, as shown in Fig. 2a, it was confirmed that the number of nuclear fragments was increased in the cell line treated with the CXCR2 inhibitor compared to the control group not treated with the CXCR2 inhibitor.

Next, the CXCR2 inhibitor SB225002 was treated with 1 μg/ml for 24 hours to the chronic myelogenous leukemia cell line, and then the protein was extracted and 20 μg of the protein was applied to the SDS-PAGE gel. The expression levels of apoptosis proteins were observed using cleavage-PARP, cleavage-caspase 3, and caspase 8 antibody (cell signaling technologies), which are markers for confirming cell death.

As a result, as shown in FIG. 2b, cleaved-PARP, cleaved-caspase-3 and caspase-8 bands were observed in the cell line treated with the CXCR2 inhibitor, indicating that the death of the CML cell line was induced by CXCR2.

2-2. Tyrosine Phosphorylase Inhibitor Resistant Cells

Chronic myelogenous leukemia cell lines (K562, KU812) were first treated with imatinib at a concentration of 10 ng/ml, and then the concentration was increased twice every week, and finally 1 μg/ml of imatinib was treated. /IR, KU812/IR) was produced.

The prepared imatinib-resistant cell line was treated with CXCR2 in the same manner as in Example 2-1, and then the production of nuclear fragments and the amount of apoptosis protein expression were measured to determine whether apoptosis was induced, and are shown in FIGS. 3A and 3B.

As a result, as shown in FIGS. 3A and 3B, it was confirmed that when the imatinib-resistant cell line was treated with a CXCR2 inhibitor at a concentration of 1 μg/ml, the fragmentation of the nucleus was increased, and the expression of cleaved-PARP, a cell death protein, was increased. .

The above results suggest that the CXCR2 inhibitor can induce apoptosis in all chronic myelogenous leukemia cell lines sensitive or resistant to imatinib.

Example 3: CXCR2 Effect of co-administration of inhibitor and tyrosine kinase inhibitor

3-1. Tyrosine Phosphorylase Inhibitor Susceptible Cells

Chronic myelogenous leukemia cells susceptible to tyrosine kinase inhibitors were spread on a 96 well plate at 1x10⁴cells/well, and after 24 hours, 0.1μg/ml, 0.5μg/ml, 1μg/ml, 5μg/ml, 10μg/ml, 50μg/ ml of imatinib and CXCR2 inhibitor were treated, respectively, and mixed in a ratio of 1:1 and treated in combination. 48 hours after treatment with the drug, the effect of co-administration of the drug was confirmed using a CCK-8 solution.

As a result, as shown in Fig. 4a, when the two drugs are treated in combination with the chronic myelogenous leukemia cell line, the cell survival rate was lower than that of the case where the drug was used alone, and it was confirmed that apoptosis was more effectively induced.

Next, based on the results obtained using CCK-8, the synergistic effect on the drug combination was confirmed using the Chou-Talalay method. After obtaining values and graphs using Compusyn software, the combination index is defined as additive effect (CI=1), synergy (CI<1), and antagonism (CI>1) in the drug combination. It is shown in Table 1 below.

Total Dose K562 CI Value KU812 CI Value 0.2 0.03 0.14 One 0.10 0.14 2 0.16 0.17 10 0.74 0.60 20 1.36 1.10 100 5.15 3.44

As shown in Table 1 and FIG. 4B, when the imatinib and CXCR2 inhibitors were treated in combination with 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml, and 5 μg/ml, respectively, the CI values of all chronic myelogenous leukemia cell lines were 1 or less. It could be confirmed that there is a synergistic effect, and the combination treatment showed a high effect even at a low concentration.

3-2. Tyrosine kinase inhibitor resistant cells

A chronic myelogenous leukemia-resistant cell line was prepared in the same manner as in Example 2-2, and, as in Example 3-1, imatinib and CXCR2 inhibitor were treated or combined with each of the resistant cell lines by concentration to obtain synergistic effects of the drug combination. Confirmed.

As a result, as shown in FIG. 5A, when the two drugs were treated in combination with the chronic myelogenous leukemia cell line, the cell viability was lower than that of the case where the drug was used alone, and apoptosis was more effectively induced.

In addition, as a result of confirming the synergistic effect based on the above results, the combination index (CI) was shown in Table 2 below.

Total Dose K562 CI Value KU812 CI Value 0.2 10.06 10.06 One 0.31 0.24 2 0.15 0.56 10 0.59 0.56 20 1.18 1.02 100 2.80 4.01

As shown in Table 2 and FIG. 5B, when imatinib and CXCR2 inhibitor were treated in combination at 0.5 μg/ml, 1 μg/ml, and 5 μg/ml, respectively, the CI values of all chronic myelogenous leukemia resistant cell lines were 1 or less, indicating that there was a synergistic effect. It could be confirmed that, at the time of combined treatment, a high effect was exhibited even at a low concentration.

As described above, although the embodiments have been described by the limited drawings, a person of ordinary skill in the art can apply various technical modifications and variations based on the above. For example, even if the described techniques are performed in a different order from the described method, and/or the described components are combined or combined in a form different from the described method, or are replaced or substituted by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and claims and equivalents fall within the scope of the following claims.

Claims (7)

In the pharmaceutical composition for the treatment of tyrosine kinase inhibitor-sensitive chronic myelogenous leukemia, comprising as active ingredients SB225002, a CXC motif chemokine receptor 2 (CXCR2) inhibitor, and imatinib, a tyrosine phosphorylation enzyme inhibitor,
The SB225002 content 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml or 5 μg/ml, and the imatinib content 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml or 5 μg/ml, 1:1 A pharmaceutical composition for the treatment of chronic myelogenous leukemia susceptible to a tyrosine phosphorylase inhibitor, administered in combination by volume.
In the pharmaceutical composition for the treatment of tyrosine kinase inhibitor-resistant chronic myeloid leukemia, comprising as active ingredients SB225002, a CXC motif chemokine receptor 2 (CXCR2) inhibitor, and imatinib, a tyrosine phosphorylation enzyme inhibitor,
The SB225002 content 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml or 5 μg/ml, and the imatinib content 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml or 5 μg/ml, 1:1 A pharmaceutical composition for the treatment of tyrosine kinase inhibitor-resistant chronic myeloid leukemia, which is administered in combination in a volume ratio.

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