KR101853540B1 - Pharmaceutical composition for preventing or treating blood cancer comprising amphotericin B - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating hematological cancer, which comprises amphotericin B as an active ingredient. According to the present invention, amphotericin B selected by natural killer cell activity screening method is a natural killer As a result, it has been confirmed that the composition containing amphotericin B as an active ingredient activates natural killer cells, and thus, And can be used as a therapeutic agent for cancer diseases and various immune diseases related to natural killer cells or immunoadjuvants for increasing immunity.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating blood cancer, which comprises amphotericin B as an active ingredient,

The present invention relates to a natural killer cell treatment aid, a composition for enhancing immunity and an anticancer activity, which comprises amphotericin B selected through natural killer cell activity screening as an active ingredient.

Of the cells that make up the immune system, in particular natural killer cells (NK cells), function to remove host cells, bacteria, intracellular parasites or virus-infected host cells without pre-sensitization by the antigen , Rejection of inadequate bone marrow transplantation, and regulation of immune responses in T cells, acting on the first line of the in vivo immune system defense mechanism.

As a result, the number and activity of NK cells in the peripheral blood were decreased compared with those in normal subjects. In animal experiments, NK cell activity The risk of developing cancer and cancer is increasing.

In addition, NK cells play an important role in the innate immune response against host-infected pathogens or cancer cells and the acquired immune response through cytokine secretion. The main mechanism that NK cells use to kill target cells is perforin ) And granzyme B are secreted into the target cells through the immune synapses. The secreted perforin makes holes in the target cell wall, and granzyme, which enters the target cell through the hole, Induce apoptosis.

Thus, NK cells have been shown to play a crucial role in cancer progression, in which cells are transformed into malignant cells, and in the early stages of viral infection. Based on these studies, NK cells using NK cells With the emergence of cell therapy, it is necessary to find a substance that increases the cytotoxic ability of NK cells.

Japanese Patent Laid-Open No. 2000-256212 (Sep. 19, 2000)

The present invention provides a pharmaceutical composition for treating cancer and immune diseases in which natural killer cells are involved, by providing a composition containing amphotericin B as an active ingredient as a therapeutic adjuvant for natural killer cells, thereby activating cytotoxicity of natural killer cells .

The present invention is to provide a pharmaceutical composition for preventing or treating blood cancer, which comprises amphotericin B as an active ingredient.

The present invention provides a natural killer cell therapeutic adjuvant containing amphotericin B as an active ingredient.

The present invention provides a T cell therapy adjuvant containing amphotericin B as an active ingredient.

The present invention provides an adjuvant containing amphotericin B as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating immunological diseases containing amphotericin B as an active ingredient.

According to the present invention, it has been confirmed that amphotericin B selected through natural killer cell activity screening effectively increases the degranulation and cytotoxicity of natural killer cells, and it has been confirmed that the cancer cells are effectively killed through the natural killer cell screening method , The present invention can be used as a therapeutic agent for cancer diseases and various immune diseases related to natural killer cells or immunoadjuvants which increase the immunity by activating natural killer cells as a composition containing amphotericin B as an active ingredient.

FIG. 1 shows that the cytotoxic activity of amphotericin B-treated NK cells was increased. 1A shows the effect of amphotericin B -activated natural killer cells treated with 1, 5, 10 and 20 μM FIG. 1B is a graph showing the increase rate of cytotoxic activity per concentration. FIG.
FIG. 2 shows the results of confirming the degree of apoptosis of amoctericin B-treated blood cancer cell line 721.221.
FIG. 3 shows the results of confirming an increase in the degranulation activity of amonoterin B-treated super early NK cells.
FIG. 4 shows the results of confirming the ability of cancer cells to kill cancer cells treated with amphotericin B at 1, 5, 10, and 20 μM concentrations.

The present invention can provide a pharmaceutical composition for preventing or treating blood cancer containing amphotericin B as an active ingredient.

The amphotericin B may increase cytotoxicity and degranulation of natural killer cells (NK cells).

More specifically, amphotericin B is a kind of polyenic antibiotic produced by Streptomyces nodosus. It is associated with sterols existing in the cell membrane of fungi or animal cells, To form a pore, which causes the leakage of intracellular substances and the influx of extracellular fluid to kill the fungus.

The blood cancers of the present invention may be, but are not limited to, myeloid blood cancers.

According to one embodiment of the present invention, when amphotericin B was directly treated with 721.221 cells of hematological cancer cells, the death of cancer cells was found to be about 5% as shown in Fig. 2, while amphotericin B , Cytotoxicity of cancer cells treated with amphotericin B-activated natural killer cells at concentrations of 1, 5, 10 and 20 μM was 1.25, 1.41, and 1.38, respectively, as compared to those of untreated natural killer cells And 1.19 times, respectively.

In addition, according to another embodiment of the present invention, it was confirmed that degranulation was increased in the natural killer cells treated with amphotericin B as shown in FIG.

Accordingly, the present invention can provide a natural killer cell therapeutic adjuvant containing amphotericin B as an active ingredient.

In addition, the present invention can provide a T cell therapy adjuvant containing amphotericin B as an active ingredient.

More specifically, the T cells may be selected from the group consisting of cytotoxic T cells and chimeric antigen receptor T cells (CAR-T).

According to another embodiment of the present invention, an NK cell activator In order to confirm that the candidate substance amphotericin B promotes the cancer cell killing ability of NK cells for cell therapy, amphotericin B at a concentration of 1, 5, 10 and 20 μM was treated with NK cells for cell therapy, As shown in FIG. 4, when cancer cells were treated with NK cells treated with amphotericin B at a concentration of 1, 5, 10 and 20 μM, The toxicity was 1.76, 1.70, 1.57 and 1.44 times higher, respectively, as compared with the cancer cell control treated with NK cells for cell therapy without treatment with amphotericin B, respectively.

From the above results, amphotericin B can effectively increase the cytotoxicity of NK cells for cell therapy, and NK cells for cell therapy activated by amphotericin B effectively kill cancer cells. Considering that the cancer cell killing mechanism of NK cells and cytotoxic T cells is common, amphotericin B inhibits cytotoxic T cells and chimeric antigen receptor-T cells (CAR-T) as well as NK cells for cell therapy, Can also be effectively used for increasing the cytotoxicity of < RTI ID = 0.0 >

In addition, the present invention can provide an adjuvant containing amphotericin B as an active ingredient.

The adjuvants of the present invention are agents that promote specific immune responses to vaccines or inoculations. An adjuvant is any substance or agent that, when incorporated into a vaccine or inoculum, generally or specifically acts to accelerate, prolong or enhance its specific immunoreactivity to the immunogenic material in its preparation . ≪ / RTI >

The adjuvants of the present invention may further comprise suitable excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, the adjuvant containing amphotericin B according to the present invention can be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, And can be used as formulations.

Examples of carriers, excipients and diluents that can be included in the adjuvant including amphotericin B include lactose, textol, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. As the liquid preparation for oral administration, suspensions, solutions, emulsions, syrups and the like may be used. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, . Formulations for non-oral administration include sterilized aqueous solutions, non-aqueous agents, suspensions, emulsions, and freeze-drying agents.

As the non-aqueous preparation and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.

The dosage of the adjuvant containing amphotericin B of the present invention may vary depending on the age, sex, body weight, etc. of the subject to be administered and the dose of the vaccine may be increased or decreased depending on the administration route, degree of disease, sex, .

In addition, the present invention can provide a pharmaceutical composition for preventing or treating immunological diseases containing amphotericin B as an active ingredient.

The immunological diseases of the present invention can be selected from the group consisting of autoimmune diseases, viral infection diseases, graft rejection diseases and inflammatory diseases.

More specifically, the autoimmune disease may be selected from the group consisting of multiple sclerosis, rheumatoid arthritis, rheumatoid arthritis, Crohn's disease and type 1 diabetes, wherein the viral infectious disease is selected from the group consisting of acquired immunodeficiency syndrome, hepatitis B, hepatitis C, measles , Herpes zoster, and papilloma virus. The inflammatory disease may be selected from the group consisting of asthma, sinusitis, atopy, periodontitis and Behcet's disease, but is not limited thereto.

In one embodiment of the present invention, the pharmaceutical composition containing amphotericin B as an active ingredient may be administered orally, parenterally, orally in the form of injections, granules, powders, tablets, pills, capsules, suppositories, gels, Or a liquid preparation can be used.

In another embodiment of the present invention, the pharmaceutical composition containing amphotericin B as an active ingredient may be formulated with a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, lubricant, And may further comprise at least one additive selected from the group consisting of flavor agents, antioxidants, buffers, bacteriostats, diluents, dispersants, surfactants, binders and lubricants.

Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

According to one embodiment of the present invention, the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, ≪ / RTI > can be administered to the subject in a conventional manner.

The preferred dosage of amphotericin B may vary depending on the condition and body weight of the subject, the type and degree of disease, the type of drug, the route of administration and the period of time, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dose may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Cell culture

Human NKL cells were obtained from M. Robertson of the University of Indiana, USA and 721.221 cells of blood cancer cells were obtained from J. Gumperz and P. Parham of Stanford University in USA and K562 cells of blood cancer cell line were obtained from USA ATCC (American Type Culture Collection). Each cell was cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, 1% sodium pyruvate or 10% FBS, 1% penicillin / streptomycin And cultured in an IMDM (Iscove's Mod. Of DMEM) medium containing the mycine at 37 ° C and 5% CO ₂ . Materials and reagents necessary for cell culture were purchased from Gibco / BRL, Cellgro / MediaTech, Inc., Nalgene-Nunc International, and Sigma-Aldrich Co.

< Example  2> Natural killer cells ( NK  Cell) Active substance screening

One. NK  Cell cytotoxicity ( cytotoxicity ) Confirm

Cytotoxicity, the ability of target cells to kill NK cells, was confirmed by Europium fluorescence assay.

Human NKL cells were treated with 200 U / ml IL-2 for 2 days. 5 × 10 ⁵ 721.221 cells per well of a 96-well plate were dispensed and the BATDA reagent (Perkin Elmer), which is an improved fluorescence ligand, was added to the final 40 μM and reacted at 37 ° C. for 30 minutes. Then, 5% fetal bovine serum (Iscove's Mod. Of DMEM) medium containing 100 [mu] L / well was added and stored.

NK cells and 721.221 cells were mixed at a cell number ratio of 10: 1, 5: 1, 2.5: 1 and 1.25: 1, respectively, in order to confirm the number of NK cells suitable for inducing cytotoxicity in the target cells. NK cells and 721.221 cells were mixed at the above ratios, and cultured at 37 ° C for 1 hour and 30 minutes. After centrifugation, the supernatant was reacted with Eu ^{3 +} solution, and a DELPIA Europium cytotoxicity assay (Perkin Elmer) The degree of fluorescence expression was measured using the manufacturer's recommendation.

As a result, it was confirmed that a ratio of 5: 1, which killed 40 ~ 50% of 721.221 cell line, was suitable for NK cell activation factor screening.

2. High-speed bulk screening  Natural killer cells using the method ( NK  Cell) Active substance screening

Candidates that increase the activity of NK cells were selected from LOPAC (Sigma, 1280 species) and PRESTWICK (Prestwick, 1200 species) using the high throughput screening (HTS) method.

Human NKL cells were plated at a concentration of 2.5 x 10 &lt; ⁴ &gt; / 50 [mu] l per well of a 96-well V bottom plate and the compounds contained in LOPAC and PRESTWICK were incubated with 1 [mu] M and 5 [ Lt; 0 &gt; C for 1 hour. NKL cells after the completion of the pre-mixed with 5 × 10 ³ / 50㎕ concentration of 721.221 cells were incubated for 1 hour and 30 minutes at 37 ℃. After the incubation, the supernatant was centrifuged to react with the Eu ^{3 +} solution, and TRF (time resolved fluorescence) fluorescence was measured with VICTOR (Perkin Elmer) to select candidates that increase NK cell activity.

As a result, amphotericin B was selected as a substance that increases the ability of NK cells to kill cancer cells.

< Example  3> NK  Identification of cell cytotoxicity increasing effect

NK cell activator selected through primary screening In order to confirm the ability of the candidate substance, amphotericin B, to kill NK cells, NK cells were treated with 1, 5, 10 and 20 μM concentrations of each, and the ability of NK cells treated with each substance to kill cancer cells was confirmed .

5 × 10 ⁵ 721.221 cells were dispensed per well of a 96-well plate, and BATDA reagent (Perkin Elmer, 40 μM), which is an improved fluorescent ligand, was added and reacted at 37 ° C. for 30 minutes. Then, 5% fetal bovine serum was included And 100 [mu] L / well of Iscove's Mod. Of DMEM (DMEM) medium was added and stored. Each material was treated with NK cells at concentrations of 1, 5, 10 and 20 μM and mixed with BATDA labeled 721.221 blood cancer cells at a ratio of 5: 1. After incubation at 37 ° C for 1 hour and 30 minutes, the supernatant was reacted with Eu ^{3 +} solution and the fluorescence expression level was measured using a DELPIA Europium cytotoxicity assay (Perkin Elmer).

As a result, as shown in Fig. 1, the cytotoxicity of cancer cells treated with NK cells treated with amphotericin B at concentrations of 1, 5, 10 and 20 μM was significantly lower than that of NK cells treated with amphotericin B 1.41, 1.38, and 1.19 times, respectively, as compared with the control group.

In order to confirm whether the increased NK cell cytotoxicity by the selected amphotericin B was specific to NK cells, 721.221 cell lines of cancer cell line were treated with Amphotericin B at a concentration of 1, 5, 10 and 20 μM Were individually treated to confirm the Specific Lysis%.

As a result, it was confirmed that apoptosis of 721.221 cell line by amphotericin B was about 5% as shown in Fig.

From these results, it was confirmed that NK cell cytotoxicity by amphotericin B specifically acts on NK cells, and that NK cells activated by amphotericin B effectively kill blood cancer cells.

< Example  4> Amphotericin  By B NK  Cell Degranulation ( Degranulation ) Increase confirmation

To confirm the effect of amphotericin B on the activity of NK cells, Degranulation assay, which is closely related to cytotoxicity, was carried out using early NK cells as follows.

Peripheral blood mononuclear cells were separated from normal blood by peripheral blood mononuclear cells treated with heparin-treated peripheral blood using the Ficoll-Hypaque technique. Separated mononuclear cells were washed twice with DPBS (Dulbecco's phosphate buffered saline) and then treated with 1% penicillin / streptomycin, 10% fetal bovine serum (FBS), 1% glutamine, IMDM supplemented with 400 U / ml IL- Iscove's Mod. of DMEM) by using the medium and cultured in a 96-well round bottom plates in each well 4 × 10 ⁵ / seeded to 200㎕ 37 ℃, 5% CO ₂ incubator for 24 hours.

A frequency divider and the cultured mononuclear cells in 96-well V 2 × 10 ⁵ / 100㎕ per well in the bottom plate was then pretreated for one hour amphotericin B in respectively 1, 5, 10 and 20 μM concentration.

After pretreatment, mononuclear cells, monocytes, CD107a-FITC antibody and NK cell-sensitive cell line K562 (blood cancer cells) were mixed at the same concentration as the mononuclear cells and incubated for 2 hours at 37 ° C in a 5% CO ₂ incubator Respectively.

The cultured samples were treated with anti-CD56-PE and anti-CD3-PerCP antibodies at 4 DEG C for 30 minutes. Three color fluorescent stained cells were collected with Accuri C6 (BD) and data were analyzed using FlowJo 7.6.5 program. The lymphocyte compartment was defined as primary gating with FSC (forward-scatter) and SSC (side-scatter). After the second gating of only NK cells (CD56 + / CD3-) with anti-CD3-PerCP and anti-CD56-PE, the degree of NK cell surface expression of CD107a in the CD56 + The pattern of degranulation was analyzed.

As a result, as shown in FIG. 3 and Table 1, it was confirmed that the degranulation activity of NK cells was increased in the experimental group treated with 1 μM, 5 μM and 10 μM of amphotericin B treatment.

The concentration of the compound Control group 1 [mu] M 5 [mu] M 10 μM 20 μM Amphotericin  B 24.8 28.0 28.6 26.6 23.8

From the above results, it was confirmed that amphotericin B increases the degranulation activity of NK cells involved in the target cell killing ability and cytotoxicity of NK cells, and thus amphotericin B is used as various cell therapy adjuvants involved in NK cells .

< Example  5> for cell therapy NK  Identification of cell cytotoxicity increasing effect

NK cell activator In order to confirm that the candidate substance amphotericin B promotes the cancer cell killing ability of NK cells for cell therapy, amphotericin B at a concentration of 1, 5, 10 and 20 μM was treated with NK cells for cell therapy, And confirmed the ability of cancer cells to kill NK cells treated with B.

NK cells were isolated from normal mononuclear cells using a StemCell kit for NK cell isolation and NK cells for cell therapy were prepared.

First, the separated NK cells were washed twice with SCGM (Stem Cell Growth Medium), and then 100 Gy-irradiated K562-mb-41BBL nutrient cells, 100 U / ml IL-2 and 5 ng / ml IL- SCGM medium to 2 × 10 ⁶ / ml on a 24-well plate and observed at 2-day intervals. When NK cells were about 4 × 10 ⁶ per well, the cells were divided into two wells using a fresh medium for 2 weeks NK cells were recovered and the anticancer activity against the target cell, 721.221 cells, was measured.

In order to measure the anticancer activity, 5 × 10 ⁵ 721.221 cells per well of a 96-well plate were dispensed and the BATDA reagent (Perkin Elmer, 40 μM), which is an improved fluorescent ligand, was added and reacted at 37 ° C. for 30 minutes, IMDM (Iscove's Mod. Of DMEM) medium containing 5% fetal bovine serum was added to each well of 100 μl.

Amphotericin B was pretreated with NK cells at 1, 5, 10, and 20 μM for 1 hour, mixed with 721.221 cells labeled with BATDA at a ratio of 1.25: 1, cultured at 37 ° C for 1 hour, and centrifuged.

After centrifugation, the supernatant was reacted with Eu ^{3 +} solution and the fluorescence expression level was measured using a DELPIA Europium cytotoxicity assay (Perkin Elmer).

As a result, as shown in FIG. 4, in the case of the cancer cell group administered with NK cells treated with amphotericin B at the concentrations of 1, 5, 10 and 20 μM, cytotoxicity was observed in the cells treated with amphotericin B 1.70, 1.57, and 1.44 times, respectively, as compared with the cancer cell control treated with NK cells for 1 hour.

From the above results, amphotericin B can effectively increase the cytotoxicity of NK cells for cell therapy, and NK cells for cell therapy activated by amphotericin B effectively kill cancer cells. Since NK cells and cytotoxic T cells have a common cancer cell death mechanism, amphotericin B is effective not only in increasing NK cells for cell therapy but also in increasing cytotoxicity of cytotoxic T cells and chimeric antigen receptor T cells Can be used.

Amphotericin B according to the present invention can be formulated into various forms according to the purpose. Hereinafter, some formulation methods in which amphotericin B according to the present invention is contained as an active ingredient are exemplified, and the present invention is not limited thereto.

< Formulation example  1 > Prescription example

&Lt; Formulation Example 1-1 > Preparation of injection

10 mg of amphotericin B, 3.0 mg of sodium metabisulfite, 0.8 mg of methylparaben, 0.1 mg of propylparaben, and an appropriate amount of sterile distilled water for injection were mixed, and the mixture was adjusted to a final volume of 2 mL by a conventional method. Ampicillin and sterilized to prepare an injection.

&Lt; Formulation Example 2-1 > Preparation of tablet

10 mg of amphotericin B, 100 mg of lactose, 100 mg of starch and an appropriate amount of magnesium stearate, and tableted according to a conventional tablet preparation method.

&Lt; Formulation Example 3-1 > Preparation of capsules

10 mg of amphotericin B, 50 mg of lactose, 50 mg of starch, 2 mg of talc, and magnesium stearate were mixed and filled in gelatin capsules according to a conventional capsule preparation method to prepare capsules.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (12)

delete delete delete delete delete delete (NK cells) activated by amphotericin B as an active ingredient, and amphotericin B enhances the cytotoxicity of natural killer cells (NK cells) Wherein the pharmaceutical composition is for preventing or treating blood cancer. delete delete delete delete delete

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