KR100755872B1 - Pharmaceutical composition for preventing and treating inflammation containing midazolam - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating inflammation containing an effective amount of midazolam and a pharmaceutically acceptable carrier, diluent or excipient and having an anti-inflammatory effect through inhibition of inflammation.

The composition according to the present invention inhibits inflammation by inhibiting iNOS or COX-2 gene expression, inhibiting NF-kB activity, inhibiting NF-kB p65 migration into the cell nucleus, inhibiting phosphorylation of MAPK p38 or inhibiting the production of free radicals. Effect.

Description

A pharmaceutical composition for preventing and treating inflammation containing midazolam {A Pharmaceutical Composition for Preventing or Remedying Inflammation}

1 is a blot photograph and graph showing that midazolam inhibits proinflammatory mediator production of macrophages.

2 is a blot picture, graph and immunofluorescence results showing that midazolam inhibits LPS-induced NF-kB activity.

3 is a blot photograph showing that midazolam inhibits LPS-induced p38MAPK activity.

4 is a graph showing that midazolam inhibits the production of free radicals induced by LPS.

The present invention relates to a pharmaceutical composition for preventing, inhibiting and treating inflammatory diseases comprising midazolam as an active ingredient.

Inflammatory diseases are reactions in the human body, including infections, trauma, and immunological reactions. Acute inflammatory symptoms such as fever, redness, swelling, and pain are present. Later in the inflammatory process, white blood cells move to the inflammatory site and cytokines, degradative enzymes, and bioactive lipid intermediates. Intracellular changes occur, such as the generation of transient reactive oxygen species and lymphocytes (sensitized lmphocytes). In chronic inflammatory diseases, cell activity and apoptosis occur due to infiltration of white blood cells. Prostaglandins (PG) and nitric oxide (NO) are known as important mediators of carcinogenesis and inflammation.

Nonsteroidal antiinflammatory drugs (NSAIDs), the most commonly used drugs that control inflammation or pain, are pharmacological by inhibiting cyclooxygenase (COX), which produces PG in the body. It was found to exist as two isomers, -1 and COX-2. COX-1 is an enzyme that is involved in the production of PGs, which are involved in the protection and regulation of gastrointestinal mucosa, platelets, and kidneys in the normal state, whereas COX-2 is very low in normal cells but in inflammation-related cells. It is induced by various stimuli (cytokine, endotoxin, mitogen) and is involved in the production of PGs that are involved in pain or inflammation. Nitric oxide synthase (NOS) is also involved in the regulation of blood vessel tone, neurotransmission, killing and homeostasis of bacteria and cancer cells. High levels of NO also occur in physiological reactions such as circulatory shock, inflammation and carcinogenesis. Neuronal NOS (Neuronal NOS) and endothelial NOS (endothelial NOS) are present in the normal state, while iNOS (NOS type2) is induced and expressed by LPS, cytokine (cytokine).

Sepsis is one of the leading causes of multi organ failure and still has a high mortality rate. Lipopplysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, has been known to be the most important factor causing sepsis. Activated macrophage (macrophage) is involved in the inflammatory response and produces a variety of cytokines (cytokine) secretion into the circulatory system affects the physiological characteristics that occur in sepsis. LPS, the main outer membrane of Gram-negative bacteria, activates various mammalian cells such as monocytes / macrophages and endothelial cells and causes systematic mutations in sepsis. LPS has been known to initiate signaling pathways that induce the activation of NF-kB and MAPK. NF-kB activation refers to the phosphorylation and phosphorylation of serine residues 32 and 36 of IkB followed by ubiquitination, degradation of IkB, and subsequent secretion and migration of NF-kB.

Proinflammatory mediators such as cytokines and nitric oxides (NO) play a central role in various inflammatory diseases. In sepsis, overexpression of NO is considered to be one of the most important factors affecting tissue damage. Inflammatory stimuli, such as mitogen, cytokine or bacterial LPS, cause macrophages to express COX-2 or iNOS. Accumulated data suggest that COX-2 and iNOS are involved in many inflammatory processes and cause various cancers, indicating that COS-2 and iNOS play an important role in inflammation and cancer induction. Therefore, if these inflammatory factors can be tuned, it is thought that the LPS-induced inflammatory response can be improved.

Macrophages are one of the major components of the inflammatory response to tissue damage. Sudden increases in reactive oxygen species (ROS) are caused by inflammatory stimuli such as LPS and pro-inflammatory cytokine (e.g. TNF-α or IL-1β), and are associated with numerous factors such as DNA synthesis, transcription factor activation, gene expression and proliferation. Causes changes in cell function. The production of MAPK pathways and pro-inflammatory mediators mediated by MAPK has been known as redox, which depends at least in part on NF-kB / ROS sensitive mechanisms.

NSAIDs, among the drugs that control inflammation and pain, inhibit the production of PG related to gastric mucosal protection and platelet function in addition to PGs involved in inflammatory reactions in the human body, and also have side effects such as gastrointestinal disorders and bleeding.

Therefore, there is a need for the development of new inflammation prevention / treatment agents that have no side effects and can selectively inhibit the activity of inflammation-related genes and / or related substances.

The present inventors have found for the first time the fact that midazolam, a derivative that has a sedative effect of the benzodiazepine family, widely used as an anesthetic and has not been reported as a side effect for the first time, deeply studies the fact that it has an inflammatory and therapeutic effect. As a result, the present invention has been completed.

An object of the present invention is to identify the anti-inflammatory action of midazolam in cultured macrophages.

Still another object of the present invention is to discover a new function of midazolam, which is used as a conventional anesthetic, and to provide a pharmaceutical composition for preventing, inhibiting and treating an inflammatory disease comprising midazolam as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical agent for preventing and treating inflammation containing an effective amount of midazolam and a pharmaceutically acceptable carrier, diluent or excipient and having an anti-inflammatory effect through the inhibition of inflammation. It relates to a composition.

In the present invention, the inhibition of the inflammation-induced ① inhibition of iNOS (inducible nitric oxide synthase) or COX-2 (cyclooxygenase-2) gene ② inhibition of activity of NF-kB (Nuclear factor-kB) ③ NF-kB p65 (Nuclear) Factor-kB p65) inhibition of migration to the cell nucleus ④ MAPK p38 (mitogen-activated protein kinase p38) is characterized in that it is made through the inhibition of phosphorylation or ⑤ the generation of free radicals.

In the pharmaceutical composition according to the present invention, the effective content of midazolam is preferably 0.001 mg / ml to 0.01 mg / ml.

The following experiments were carried out to confirm the effect of the midazolam used in the pharmaceutical composition according to the present invention.

RAW264.7 cells, one of the macrophage cell lines, were used to measure the effects of midazolam on mitogen-activated protein kinase and proinflammatory mediators by western blot analysis. Activation of nuclear factor-kB (NF-kB) and migration of p65 subunits of NF-kB were measured using luciferase assay and immunocytochemistry. The production of free radicals was measured using lucigenin chemiluminescence.

Several experiments confirmed that midazolam inhibits overregulation of COX-2 and iNOS induced by LPS (lipopolysaccharide) in a concentration-dependent manner (about 3-30 μM). The degradation of IkB-α induced by LPS and NF-kB transcriptional activity were also inhibited by midazolam. The migration of NF-kB to the cell nucleus of the p65 subunit was also inhibited by midazolam. Midazolam also inhibited the phosphorylation of p38 MAPK and the production of free radicals induced by LPS.

The pharmaceutical composition according to the present invention has an anti-inflammatory or anti-inflammatory effect because it contains an effective amount of midazolam to inhibit gene expression of inflammatory-induced biofactors. At this time, the pharmaceutical composition according to the present invention is a pharmaceutically acceptable pharmaceutical preparation, such as injections, solutions, syrups, tablets, capsules, medicinal herbs, etc. together with midazolam and pharmaceutically acceptable carriers, diluents or excipients. It can be formulated as a drug for the treatment and prevention of inflammatory diseases.

In addition, the pharmaceutical composition according to the present invention may be administered according to a conventional administration method by the method of intravenous, spinal cavity, oral, abdominal cavity, subcutaneous administration, etc. when clinically administered.

In addition, the clinical dosage of the pharmaceutical composition according to the present invention can be variously adjusted according to the age, symptoms, dosage form or type of drug of the patient. Determining the desired dosage in a particular condition corresponds to known techniques.

Hereinafter, the present invention will be described in more detail with reference to the following examples. The embodiments are only listed by way of example to illustrate the present invention, thereby not changing or reducing the scope or content of the technical spirit of the present invention. In the Examples, only the mechanism and effect of midazolam were examined and tested, but the composition of the midazolam, which was confirmed to have anti-inflammatory action by the Example, at an appropriate concentration, and the resulting composition can be administered in various ways. It would be easy for me.

The cell lines and materials used in the examples and the experimental and analytical methods were as follows.

(1) Cell lines and materials used

In this example, a rat macrophage line RAW264.7 was used. RAW264.7 cell line is an immortalized macrophage cell line isolated from BALB / c mice transfected with Abelson Ieukemia virus (Nramp 1 ^{D169 / D169} ). RAW264.7 and human embryonic kidney 293 (HEK293) cell lines were divided from the American Type Culture Collection (Rockvile, MD). These cell lines were maintained and cultured in a 37 ° C. water incubator environment with 5% carbon dioxide and 95% air. Dulbecco's Modified Eagle's Medium (Gibco, Grand Island, NY) supplemented with 10% Fetal Bovine Serum, penicillin 100U / ml, and streptomycin 100µg / ml were used.

In preliminary experiments, dilution of midazolam with 0.9% NaCl had no effect on the solubility of midazolam. In addition, treatment of cells with 0.9% NaCl showed no effect on the expression of LPS-inducible COX-2 (data not shown). For this reason, midazolam (Roche) was used by dissolving in 0.9% NaCl aqueous solution, and 0.9% NaCl aqueous solution was selected as a solvent control.

Bacterial endotoxin LPS (300 ng / ml) was used to induce macrophage activation. Used for LPS of other E. coli serotype O26: B6, human TNF-α, β-actin, reduced form of NADPH (Sigma), Horseradish peroxidase-labeled anti-rabbit and anti-mouse antibody (Amersham, UK), Western blot Antibodies such as anti-iNOS, IkB-α (Santa Cruz), anti-COX-2 (BD Biosciences), anti-phospho-p38 and anti-extracellular signal-regulated kinase (Cell Signaling) were used.

(2) Experimental method and analysis method

① Western blot analysis

For Western blot analysis, RAW264.7 cells were treated with 20 mM Tris-Cl, pH 7.5 100 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM Na ₃ VO ₃ , 1 mM β-glycerophosphate, 4 mM Na pyrophosphate, 5 mM NaF, 1% Triton X- 100 μl of lysis buffer containing protease inhibitor cocktail was harvested. Lysate was centrifuged at 12,000 rpm for 20 minutes to separate the supernatant. 40 μg of proteins (9% iNOS, 10% COX-2, 12.5% IkB-α, phospho-p38 and phosphoextracellular signal-regulatedkinase) were isolated using SDS-PAGE and electrophoresed on polyvinylidene difluoride membrane. After blocking for 2 hours at room temperature with 5% skim milk, the blot was incubated overnight at 4 ° C at a ratio of 1: 1000 with specific primary antibodies, and detected with horseradish peroxidase-conjugated secondary antibody. ECL kit (Pierce) was used for the blot.

② Transfection and Reporter assay

To observe the effect of midazolam on the activation of NF-kB, a reporter assay was performed using HEK293 cells infected with NF-kB. Toll-like receptors of LPS were not expressed in HEK293 cells, and since TNF-α is produced by LPS in macrophages, TNF-α was used to stimulate NF-kB activation instead of LPS. For infection, 5 × 10 ⁴ cells were infected with 1 μg NF-kB luciferase or Renilla luciferase using Lipofectamine 2000 (Invitrogen) in 6 well plates. For cotransfection experiments, the same molar amount of each plasmid was infected at 1 µg / well. After 1 day of infection, cells were cultured for 8 hours in a midazolam-treated and non-moldazol-treated environment with TNF-α (15ng / ml) treatment to allow luciferase production to be measured. Cells were harvested and subjected to dual luciferase assay according to the manufacturer's manual. Luciferase activity was measured using Luminoskan Ascent (Thermo Labsystems).

③ Immunofluorescent staining

The effect of midazolam on the migration of NF-kB p65 induced by LPS was measured by immunofluorescent staining of RAW264.7 cells. Since NF-kB migration occurs within 30 minutes after LPS treatment, RAW264.7 cells were treated with LPS (300 ng / ml) for 30 minutes in the presence or absence of midazolam. For immunofluorescence staining, RAW265.7 cells were grown on glass coverslip. After exposure to midazolam or LPS, cells were fixed with 1% glutaldehyde, cell permeability was enhanced with 0.25% triton X-100, and blocked with 2% bovin serum albumin for 1 hour. Coverslip was incubated overnight at 4 ° C using rabbit anti-p65 (1: 100) primary antibody in 1% bovin serum albumin. The cells were washed and incubated for 1 hour in fluorescein isothiocyanate-labeled secondary antibody. The cells were washed again and stained for 10 minutes to see the nuclei with 5 μg / ml propidium iodide and washed. Fluorescence of the coverslip was observed with a Zeiss confocal microscope (Oberkochen).

④ Free radical measurement

To determine the effect of midazolam on the free radicals produced by LPS (O ^2- ), Raw264.7 cells were exposed to LPS for 18 hours in and without midazolam and Lucigenin-enhanced chemiluminescence assay. Was implemented. Lucigenin-enhanced chemiluminescene assays were performed to analyze the extent of free radical production, as reported previously (17–19). Lucigenin (bis-N-methylacridinium nitrate) specifically emits light in the presence of free radicals. Briefly, RAW264.7 (1 × 10 ⁵ cells) was added to Krebs-HEPES buffer (100 mM NaCl, 4.7 mM KCl, 1.97 mM CaCl ₂ , 1.2 mM MgSO ₄ , 1.03 mM K ₂ HPO ₄ , 25 mM NaHCO ₃ , 20 mM Na-HEPES, pH 7.4) was transferred to a scintillation vial containing lucigenin to a final concentration of 5 μM. Chemiluminescence was recorded for 2 minutes after the addition of 100μM NADPH. The chemiluminescence was used to determine whether free radicals were generated by subtracting the amount of light emitted from the blank. The figures are 1x10 ⁵ It is expressed as the relative amount of light per cell.

⑤ Statistical analysis

The results are expressed as mean ± standard deviation. Student t test was used to verify statistical significance between the control and drug treatment groups. It was determined that P value <0.05 was significant.

Example 1 Experiment Inhibitory Effect of COX-2 and iNOS Induced by LPS

The effect of midazolam on the expression of pro-inflammatory mediators in macrophages was investigated.

Macrophages were pretreated with midazolam (3-30 μM) and exposed to 300 ng / ml LPS for 18 hours. Expression levels of iNOS and COX-2 were measured by Western blot analysis (FIG. 1). In the figure, (A) midazolam is a Western blot photograph showing the expression of iNOS and COX-2 proteins induced by LPS in RAW264.7 cells (in which β-Actin is used as a control), and (B) is densitometry data. Mean ± SEM is shown for each of the three experiments. * And # mean significant data with p <0.05.

As shown in FIG. 1, LPS induced expression of iNOS and COX-2, and iNOS and COX-2 were not detected in cells not stimulated by LPS. However, pretreatment with midazolam inhibited the expression of COX-2 and iNOS even in LPS-stimulated cells in a concentration-dependent manner (about 3 to 30 μM). In particular, high concentrations (30 μM) of midazolam completely inhibited iNOS and COX-2 expression induced by LPS in RAW264.7 cells.

In summary, midazolam inhibited the expression of iNOS and COX-2, known as pro-inflammatory mediators, in the range of 3-30 μM. The plasma concentration of the benzodiazepine family used in clinical practice is 0.1 ~ 50μM. This example suggests that midazolam administered clinically can exhibit anti-inflammatory activity through inhibition of pro-inflammatory mediators in macrophages. Peripheral benzodiazepine binding sites are present in mouse macrophages. Therefore, this example shows that midazolam has an anti-inflammatory effect in macrophages through peripheral benzodiazepine binding sites.

Example 2 NF-kB activity inhibitory effect experiment

(1) In order to confirm whether midazolam directly inhibits the degradation of IkB, the macrophage RAW264.7 was exposed to LPS for 30 minutes, and then the change in the amount of IkB-α protein was measured (A of FIG. 2). FIG. 2A is a Western blot photograph showing the effect of midazolam on the degradation of IkB-α in RAW264.7 cells, wherein β-Actin was used as a control.

As can be seen in A of FIG. 2, LPS caused degradation of IkB-α. However, pretreatment with midazolam significantly inhibited the degradation of IkB-α. Therefore, it can be inferred that midazolam can inhibit NF-kB activity by blocking the degradation of IkB-α induced by LPS.

(2) NF-kB is well known to bind to cis- elements of promoter genes of COX-2 and iNOS and regulate their transcription. In order to confirm this, the effect of midazolam on NF-kB luciferase activity was confirmed (B of FIG. 2). In Figure 2 B, luciferase activity is expressed as fold change%. Enzyme values were expressed as mean ± standard deviation for 4 separate experiments.

As shown in B of FIG. 2, midazolam completely inhibited the increase of luciferase activity induced by TNF-α. According to this example, midazolam is believed to inhibit NF-kB activity by inhibiting LPS-induced IkB degradation and / or inhibiting its ability to bind promoter elements.

(3) NF-kB p50 / p65 heterodimers must be transferred to the nucleus for NF-kB mediated transcription after exposure to LPS. To observe the effect of midazolam on NF-kB migration, we examined the effect of midazolam on p65 nuclear migration in LPS-stimulated RAW264.7 macrophages.

RAW264.7 cells were first treated with LPS (300 ng / ml) for 30 minutes in the presence or absence of midazolam (30 μM). The treated cells were fixed and stained with nuclei with NF-kB p65 antibody (green) and propidium iodide (PI) and observed by immunofluorescence microscopy (FIG. 2C). According to FIG. 2C, the treatment of RAW cells by LPS obviously induces migration of the NF-kB p65 subunit to the nucleus, and midazolam blocks this migration.

Example 3 Inhibition of Phosphorylation of p38 MAPK

Experiments were conducted to determine whether midazolam inhibits the activity of MAPK (mitogen-activated protein kinase) induced by LPS.

To this end, the degree of phosphorylation of p38 MAPK was observed in macrophages treated with midazolam (FIG. 3). FIG. 3 is a Western blot photograph showing the effect of midazolam on the activation of MAPK and ERK in RAW264.7 cells using β-Actin as a control.

Phosphorylation of p38 MAPK was induced 30 minutes after LPS treatment and then returned to baseline within 50 minutes. Treatment of midazolam completely inhibited p38 activity induced by LPS (FIG. 3A). Midazolam, on the other hand, did not inhibit the activation of extracellular signal-regulated kinase (ERK) induced by LPS (FIG. 3B).

The p38MAPK pathway is known to be involved in cytokine production in cells of the monocyte-macrophage lineage. In this example, midazolam inhibited p38 activation induced by LPS. MAPKs such as c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38 are known to play an important role in the expression of COX-2 and iNOS mediated by LPS. In particular, inhibition of p38MAPK is known to prevent the production of pro-inflammatory cytokine through inhibition of promoter activity of iNOS and COX-2. Thus, inhibition of p38 by midazolam reduced the expression of iNOS and COX-2 and ultimately showed anti-inflammatory effect.

Example 4 Experiment to Inhibit the Production of Reactive Oxygen Species

In order to study whether midazolam inhibits the production of free radicals, the production of free radicals by NADPH was measured according to the lucigenin-enhanced chemiluminescence method (FIG. 4).

LPS caused the production of free radicals by NADPH in RAW 264.7 cells. In the midazolam treatment, the production of free radicals by basic NADPH was not inhibited, but the production of free radicals induced by LPS was significantly suppressed.

The results of Examples 3 and 4 show that p38 MAPK activation and production of free radicals induced by LPS in macrophages are inhibited and blocked by midazolam. Therefore, it can be inferred that inhibition of p38 MAPK is due to inhibition of free radical production induced by midazolam.

Possible mechanisms that explain the anti-inflammatory action of midazolam include inhibition of free radicals induced by LPS. According to the examples, the ROS production in the ground state was not significantly inhibited by midazolam, but the active oxygen increased by LPS was greatly inhibited by the pretreatment of midazolam, which was induced by LPS in macrophages. Suggests specific inhibition of signaling pathways. Overproduction of ROS by activation of macrophages plays an important role in the development of inflammation and tissue damage. Thus, inhibition of ROS production by midazolam would be effective for organ dysfunction due to oxidative stress.

According to the present invention, a new pharmaceutical composition for preventing and treating inflammation containing midazolam, which can selectively inhibit the activity of inflammation-related genes and / or related substances, can be selected.

According to the present invention, it is possible to obtain an effect of widening the choice of drugs for preventing / treating inflammation for inflammatory patients.

Claims (7)

A pharmaceutical composition for preventing and treating inflammation containing an effective amount of midazolam and a pharmaceutically acceptable carrier, diluent or excipient and having an anti-inflammatory effect through inhibition of inflammation. The method of claim 1, Inhibition of the inflammation is a pharmaceutical composition for preventing and treating inflammation, characterized in that made through the inhibition of the expression of iNOS (inducible nitric oxide synthase) or COX-2 (cyclooxygenase-2) gene. The method of claim 1, Inhibition of the inflammation induced pharmaceutical composition for inflammation prevention and treatment, characterized in that made through the inhibition of the activity of NF-kB (Nuclear factor-kB). The method of claim 1, Inhibition of the inflammation induced by the NF-kB p65 (Nuclear factor-kB p65) of the pharmaceutical composition for preventing and treating inflammation, characterized in that made through the inhibition of migration to the cell nucleus. The method of claim 1, Inhibition of the inflammation is a pharmaceutical composition for preventing and treating inflammation, characterized in that through the inhibition of phosphorylation of MAPK p38 (mitogen-activated protein kinase p38). The method of claim 1, Inhibition of the inflammation induced pharmaceutical composition for inflammation prevention and treatment, characterized in that made through the inhibition of the production of free radicals. The method according to any one of claims 1 to 6, The effective amount of the midazolam is 0.001mg / ㎖ ~ 0.01mg / ㎖ pharmaceutical composition for the prevention and treatment of inflammation.

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