KR100267258B1 - Protective antidotes of anthrax toxin-induced cytotoxicity - Google Patents

Abstract

PURPOSE: An antidote for the toxin caused by Bacillus anthracis is provided, thereby the peritoneal macrophages, which are usually injured by the toxin of Bacillus anthracis, in abdominal can be effectively protected. CONSTITUTION: The antidote for the toxin caused by Bacillus anthracis contains dantrolene which inhibits calcium from being mobilized from the calcium storing place in a cell; cyclosporine A which prevents calcium from being released from mitochondria, which is a calcium storing place in a cell, by protecting the mitochondria membrane; and a phospholipase A2 inhibitor which is activated by increasing calcium concentration in a cell. Wherein the phospholipase A2 inhibitor is butacaine, 4-bromophenacyl bromide and quinacrine.

Description

Cytotoxic Antidote Caused by Anthrax Toxin

The present invention relates to an antidote for anthrax toxin. More specifically, the present invention relates to intracellular calcium migration inhibitors, mitochondrial membrane protective agents, and phospholipids, phospholipases, upon cytotoxic expression by anthrax toxin in ICR-based mouse peritoneal macrophage. The present invention relates to an antidote capable of protecting cells from anthrax toxin, comprising at least one component selected from inhibitors of A ₂ (phospholipase A ₂ ) as an active ingredient.

In humans or animals, anthrax is caused by the infiltration of Bacillus anthracis spores into the body through wounded skin, by insect bites , or through the respiratory tract. Spores that penetrate into the body germinate and spread through the lymphocytes to the blood vessels. Symptoms of anthrax are similar to those of shock. In the early stages of the onset, high fever and severe coughing, followed by severe respiratory distress and cyanosis. Usually, people die within 24 or 36 hours after dyspnea. Pathogenesis of anthrax bacilli not Write System for due to the anthrax toxin secreted by the (Bacillus anthracis) anthrax toxin is composed of three protein exotoxin (exotoxin protein). The components include lethal factor (LF), protective antigen (PA), and edema factor (EF). The protective antigen is 83 kDa and the edema factor is 88 kDa. Each of these toxins binds to each other and exhibits two forms of toxic effects on cells or tissues. The first toxic action is the combination of lethal factor and protective antigen to form lethal toxin (LeTx), which causes cell death. Another toxic effect is that the edema factor and the protective antigen combine to form edema toxin (EdTx), causing edema in the skin. The role of these exotoxins in cells is that protective antigens are necessary factors for lethal or edema factors to enter the cell surface by attaching them to receptors on the cell surface, and edema factors are calcium ions (Ca ²⁺ ) or calmodulin ( calmodulin) and acts as adenylate cyclase. However, the role of lethal factors in killing cells is not yet clear. Recent reports have shown that the amino acid sequence of lethal factor is similar to the protease regulated by Zn ²⁺ (Kllmpel et al. (1994) Mol. Micro. 13, 1903). Another team reports that cytotoxicity caused by anthrax toxin causes reactive oxygen to be generated by these factors, leading to cell necrosis (Hanna et al. (1994) Mol. Med. 1, 7).

Recently, it has been found that an increase in calcium in various types of cells or tissues irreversibly induces toxic reactions. In other words, it is well known that a molecule needs a certain ion to pass through a biological membrane (Suttorp N et al. (1988) Infect. Immun. 56, 2228). In the case of calcium, it is known that some bacterial toxins are involved in the process of entering cells and expressing toxicity. For example, diphtheria and Pseudomonas toxins have been found to increase the concentration of intracellular calcium by these toxins, resulting in cell death. In the case of anthrax toxin, it was found that the removal of calcium from the culture medium during cell culture did not express cytotoxicity caused by lethal factor (Bhatnagar R. et al. (1989) Infect Immun 57, 2107). In addition, blocking the calcium ion channel, which is the calcium channel of the cell wall, with a blocking agent did not show toxicity by lethal toxin. It is known that the external calcium of cells affects the cytotoxic expression caused by lethal toxin, but the exact mechanism is not known how the increase of intracellular calcium is involved in the cytotoxic expression of anthrax toxin.

The present inventors have recently discovered the toxic mechanism of lethal toxin caused by calcium inside the cell, the present invention provides an inhibitor of such toxic mechanism as an toxic antidote of anthrax toxin.

It is therefore an object of the present invention to provide an antidote for anthrax toxin.

More specifically, it is an object of the present invention to provide an antidote for anthrax toxin as an active ingredient an inhibitor of toxic mechanism of anthrax toxin by calcium inside the cell.

1 is a diagram showing the cytoprotective effect of the concentration of dantrolene on the cytotoxicity of anthrax toxin.

Figure 2 shows the cytoprotective effect of the concentration of cyclosporin A on the cytotoxicity of anthrax toxin.

Figure 3 is a diagram showing the cytoprotective effect of the concentration of butacaine on the cytotoxicity of anthrax toxin.

Figure 4 shows the cytoprotective effect of the concentration of 4-bromophenacyl bromide on the cytotoxicity of anthrax toxin.

Figure 5 shows the cytoprotective effect of the concentration of quinacrine on the cytotoxicity of anthrax toxin.

The present invention provides a material for protecting peritoneal macrophages from the cytotoxicity of anthrax toxin:

1. dantrolene, which inhibits calcium mobilization from intracellular calcium stores;

2. Cyclosporin A, a substance that protects the membrane of the mitochondria, the calcium reservoir in the cell, thereby inhibiting the release of calcium from the mitochondria;

3. Present an inhibitor of phospholipase A ₂ which is activated by intracellular calcium increase.

The present invention will be described in detail step by step as follows.

A) Selection of Macrophages

According to the study, lethal toxin was found to be selectively cytotoxic in macrophages, unlike edema toxin (Manna et al. (1993) Proc. Natl. Acad. Sci. 90, 10198). Treatment of these cells with low concentrations (0.1 μg / 10 ⁵ cells each of PA and LF) of lethal toxin (LeTx) has been shown to initiate cell necrosis usually within 1-2 hours (Hanna et al. (1992) Mol. Biol. Cell. 3, 1269). In contrast, other types of cells are known to have no cytotoxicity, even though lethal toxin has been introduced into the cells. Therefore, it is likely that macrophages play an important role in the history of anthrax. For this reason, in the present invention, experiments were performed by selecting macrophages as model cells. Intraperitoneal macrophages include resident cells, elicited cells, and primed cells. In this experiment, thioglycolate-induced macrophages were used. That is, three days prior to using the cells, 3 ml of thioglycolate (2.5%) medium was administered intraperitoneally in 30 g ICR mice, followed by harvesting by washing the abdominal cavity with saline.

B) Selection of Dantrolene

Dantrolene (1-[[5- (p-nitrophenyl) furfurylidene] amino] hydantoin sodium) is known to inhibit or reduce calcium transport in intracellular calcium stores in various cell types (Frandsen (1991) J. Neurochem. 56, 1075). The substance is currently used to treat malignant hyperthermia and to treat muscle paralysis. In the treatment of muscle paralysis, dantrolene releases calcium from the sarcoplasmic reticulum in the muscle cells, thereby releasing the contracted muscles. It is also known to affect calcium secretion and response at nerve endings (Ward et al. (1986) Drug 32, 130).

C) Selection of Cyclosporin A

Cyclosporin A is widely used as an immunosuppressant to prevent hypersensitivity clinically and is also known to have antiphrastic and antimalaria effects (Richter et al. (1990) Biochem. Pharmacol). 40, 799). Recently, it has been reported that this substance inhibits the calcium secretion of mitochondria by oxygen radicals (George et al. (1992) Biochem. Pharmacol. 44, 1995). That is, it is known that the harmful oxygen causes a structural change in the membrane of the mitochondria, resulting in a change in the permeability of the membrane. This change in permeability causes calcium inside the mitochondria to flow into the cytoplasm. In this case, cyclosporin A is believed to play a role in stabilizing the mitochondrial membrane to prevent changes in permeability, but its detailed mechanism is not yet known.

D) Selection of Phospholipase A ₂ Inhibitors

As already known, one of the cytotoxic mechanisms caused by the increase of intracellular calcium is the activation of phospholipidase phospholipase or protease protease. These enzymes promote cell necrosis by damaging cell membranes (Arsenious (1985) TIPS BEST Supple 15, 18). In particular, phospholipase A _2, which acts to release one of the cell membrane components, arachidonic acid from the cell membrane, is easily activated by calcium and destroys the cell membrane. In the present invention, butacaine, 4-bromophenacyl bromide, and quinacrine, which are inhibitors of activation of this enzyme, were selected.

E) Cytotoxicity and Cytoprotective Evaluation Test

To assess the cytotoxicity of anthrax lethal toxin, lactate dehydrogenase (LDH) leakage was used. In general, the degree of cell death can be determined by measuring the degree of damage to the plasma membrane. LDH is a stable enzyme present in the cytoplasm inside the cell and leaks out of the cell depending on the degree of damage to the cell membrane. In other words, as the number of necrotic cells increases, or as cell membranes with damaged cell membranes increase, the amount of LDH leaking out of the cells increases. At this time, by collecting and quantifying only the LDH leaked out of the cell can determine the toxicity of the cell (cf. Murphy et al., (1993) Neurosci 55, 597). Using this method, the degree of cytotoxicity was evaluated by measuring and comparing the change of cell viability by time, concentration and mortality by lethal toxin. In addition, as a screening method for a substance that inhibits cytotoxicity caused by lethal toxin, the LDH method was used as a cytoprotective material to increase cell viability.

The present invention will be described in more detail with reference to the following Examples.

Example 1. Isolation and Culture of Macrophages

To obtain macrophages satisfying the experimental conditions, 6-7 week old male ICR mice weighing about 30 g were selected. To get induced macrophages intraperitoneally, 3 ml of 2.5% thioglycolate medium was administered intraperitoneally of the mice 3 days before harvesting the cells. After 3 days, the abdominal cavity was washed with 8 ml of saline to obtain cells. The resulting cells were passed through four layers of gauze and harvested into 50 ml conical centrifuge tubes. After centrifugation (400 xg) of the harvested cell solution for 5 minutes, the supernatant was removed and the precipitate was taken and the cell culture medium (10% fetal bovine serum and penicillin (100 U / ml) and streptomycin (100 μg /) in RPMI 1640 medium. ml) were added simultaneously and dissolved in the one prepared on the day of the experiment). After titration, the cell number was adjusted to 10 ⁵ / ml, and the cells were divided into microplates and incubated with the cell culture solution for 3 hours in an incubator with 95% oxygen and 5% carbon dioxide at 37 ° C. After 3 hours, the microplates were washed three times with RPMI 1640 medium to obtain only cells fixed on the bottom of the microplates. After the cells were washed, freshly prepared cell culture solution was added thereto, followed by culturing in an incubator in which 95% carbon dioxide and 5% oxygen were injected. After 4 hours, 100 μl of the cell culture solution was taken and used as a sample for cytotoxicity evaluation or cytoprotective evaluation.

Example 2. Cytotoxicity Assessment of Lethal Toxin

To evaluate the cytotoxicity, the cell survival rate was measured after incubating the experimental and control groups treated with lethal factor for a certain time. For cell viability measurement, the LDH assay kit (product number: 1 664 793) manufactured by Boeringer Manheim of Germany was used. After the lethal toxin treatment was performed with 0.1 μg / ml (10 ⁵ cells) of lethal factor and protective antigen, respectively, the cell viability was decreased over 1 hour. This markedly decreased. Therefore, cell survival rate was compared 4 hours after lethal toxin treatment for evaluation of cell protection ability of candidates.

Example 3. Evaluation of Cytoprotective Capacity

Cell protective material dissolved in dimethyl sulfoxide (DMSO) was added to the cell culture as a protective agent, and then suspended immediately by the addition of lethal toxin, followed by incubation in the cell incubator for 4 hours. The amount of DMSO was used not to exceed 0.2% of the total. In the control group, as much DMSO and lethal toxin as used in the protective group were added. In other words, the cell viability was measured by analyzing and analyzing the cell viability after incubating into an experimental group to which the lethal toxin and the protective agent were added, the control group containing only the lethal toxin and DMSO, and the control group to which the lethal toxin and DMSO were not added. Was evaluated. Each group was repeated three or more times, three times each. Evaluation results for each cell protective agent are shown in FIGS. 1 to 5.

The survival rate of the cells was measured as the degree of extracellular leakage of LDH in the cells, where 100% LHD leakage measured the amount of LDH as it flowed out of the cell after completely damaging the membrane with a detergent (1% Triton X-100). to be. In general, about 10% LDH flows from the standard group during cell culture (Frandson et al. (1991) J. Neurochem. 56, 1975).

Figure 1 shows the cytoprotective effect by dantrolene, a substance that inhibits the movement of intracellular calcium against the cytotoxicity of anthrax lethal toxin by concentration. It can be seen that there is a clear protective effect in the experimental group with a concentration of dantrolene of 10μM or more.

Figure 2 shows the cytoprotective effect of each concentration by cyclosporin A, a protective agent of the mitochondrial membrane, showing an increase in cell viability at low concentrations. It shows a clear cytoprotective effect at the concentration of 0.5 μM.

Figure 3 shows the cytoprotective effect of the concentration by the phosphatase A ₂ inhibitor phospholipase A _{2 but the} cell protective effect in a concentration range of 10-100 μM and anthrax under concentrations of 100 μM or less It can be seen that the toxin-induced toxicity is reduced to 50%.

Figure 4 shows the cytoprotective effect of each concentration by 4-bromophenacyl bromide, a phospholipidase A ₂ inhibitor, phospholipidase of the cell membrane, showing a clear protective effect at a concentration of 10 μM, caused by lethal toxin It can be seen that the cytotoxicity is reduced by about 60%.

Figure 5 shows the cytoprotective effect of each concentration by the phospholipase A ₂ inhibitor quinacrine, a phospholipidase of the cell membrane, showing a clear protective effect at a concentration of 20 μM, showing almost no necrosis of cells by lethal toxin 100% protection.

In addition, the results of the complex treatment of these materials in different combinations are shown in Table 1 below.

Antidote complex treatment result process LDH leak (%) Standard group ¹ 10 LeTx ² 40 Quinacrine 13 Quinacrine + Dantrolene 15 Quinacrine + Cyclosporin A 15

¹ Standard group with no lethal factor.

² LeTx is a control group treated with lethal factor only.

Other groups treated with lethal factor and quinacrine alone or with calcium transfer inhibitors.

As shown in the above results, the combination of the protective agent shows the same degree of cytoprotective effect as quinacrine alone treatment.

As an antidote for peritoneal macrophage protection from anthrax toxin, dantrolene is 0.1-500 μM, cyclosporin A is 0.01-50 μM, butacacaine is 0.1-100 μM, 4-bromophenacyl bromide is 0.5-500 μM, and Quinacrine is preferably used at a concentration of 0.5-500 μM. Limiting concentrations above suggests concentrations from minimally effective to concentrations where the drug itself is not toxic.

Toxicity of antidote agents of anthrax toxin according to the present invention

Dantrolene: Currently in clinical use up to 10 mg / kg (Ward et al., Drugs 32: 130-168, 1986)

LD ₅₀ of cyclosporin A: 107 mg / kg

Butacaine LD ₅₀ : 12.4 mg / kg

4-bromophenacyl bromide: Experimental results showed no cytotoxicity even when treated to 500 μM (Sevanian et al., 1983 Arch Biochem Biophys 223: 441-452; Donal et al., 1990 J. Newrochem. 55: 1716- 1726)

Quinacrine: Experimental results showed no cytotoxicity after treatment up to 250 μM (Sevanian et al., 1983 Arch Biochem Biophys 223: 441-452; Lazarewicz et al., 1990 J. Neurochem 55; 1875-1881)

The present invention is based on the toxic mechanism of anthrax lethal toxin caused by calcium inside the cell, the intracellular calcium migration inhibitor, mitochondrial membrane protective agent, or phospholipid degrading enzyme that can suppress such toxic mechanism an inhibitor of the flow port lipase a ₂ (phospholipase a ₂₎ providing an antidote to protect the cells from, anthrax toxin as an active ingredient. The antidote of the present invention can effectively protect the macrophages of the abdominal cavity, which are the main targets of anthrax toxin, and thus are useful as an antidote for anthrax toxin.

Claims (3)

Cytotoxic antidote to anthrax lethal toxin containing a phospholipase A ₂ inhibitor as an active ingredient. The antidote of claim 1, wherein the phospholipase A ₂ inhibitor is butacaine, quinacrine or 4-bromophenacyl bromide. The antidote according to claim 1 or 2, wherein the phospholipase A ₂ inhibitor is quinacrine and additionally contains cyclosporin A or dantrolene.

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