KR102046422B1 - A pharmaceutical composition for preventing or treating sepsis or septic shock comprising N-(Cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]4-pyrimidinyl]-benzamide or a pharmaceutically acceptable salt thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating sepsis or septic shock, comprising N-(cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]4-pyrimidinyl]-benzamide or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutical composition for preventing or treating bacteremia or endotoxemia. It is confirmed that N-(cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]4-pyrimidinyl]-benzamide (CYT387) is used for treating myelofibrosis and some cancers, prevents lipopolysaccharide-induced maturation, and inhibits activation of lipopolysaccharide (LPS)-induced signaling pathway, thereby reducing expression of proinflammatory cytokines. Thus, the pharmaceutical composition of the present invention is expected to be variously used for treating sepsis, bacteremia and endotoxemia infection and for developing pharmaceutical.

Description

N- (Cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide or a pharmaceutically acceptable salt thereof as an active ingredient A pharmaceutical composition for preventing or treating sepsis or septic shock comprising N- (Cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino ] 4-pyrimidinyl] -benzamide or a 黄 acceptable salts as an active ingredient}

The present invention is effective to N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide or a pharmaceutically acceptable salt thereof. It relates to a pharmaceutical composition for the prevention or treatment of sepsis or septic shock, and a pharmaceutical composition for the prevention or treatment of bacteremia or endotoxin.

Sepsis is an inflammatory reaction caused by excessive activation of the body's immune system by acting as a toxin of lipopolysaccharide (LPS), a cell wall component, when infected with pathogenic Gram-negative bacteria. It can be accompanied by shock if it is causing or severe symptoms. In particular, sepsis may include humoral immune dysfunction or cells such as malignant tumor, leukemia, malignant lymphoma, AIDS, collagen disease, renal failure, liver disease, cerebrovascular disorder, diabetes, and other basic diseases such as elderly, premature infants, and the like. It occurs mainly when a host with weak resistance with sexual immunity is treated with adrenal steroid or anti-tumor chemotherapy, cobalt irradiation, radiotherapy or chemotherapy, hemodialysis, organ transplantation, cardiac surgery, etc. do. Sepsis is a major cause of death for patients admitted to hospital intensive care units, and is a very serious disease with a mortality rate of more than 30%. Despite advances in medical technology, sepsis occurs because of infections after surgery worldwide, and when people with weak immunity, such as newborns or the elderly, are infected with sepsis. Typically, neonatal sepsis is known to occur in about 3 out of 1,000 full-term infants, premature infants are known to increase the incidence 3 to 4 times. If you have sepsis, you will be treated with antibiotics. However, if the proper treatment is delayed and the bacteria have multiplied, or if you have been infected by a strain that is resistant to antibiotics, antibiotics alone will not be able to treat you effectively. The pathogens having a gradual increase is an urgent situation to develop a new sepsis treatment.

N- (Cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] -4-pyrimidinyl] -benzaamide has been used to treat myelofibrosis since 2011. 10-2013-0137011) and is a promising molecule that has been shown to destroy metastatic KRAS-mutated non-small cell lung cancers and metastatic pancreatic ductal adenocarcinoma. However, despite the effects on the immune response and various important pathways, there was no study on the treatment of sepsis.

The present inventors have conducted research to satisfy the above requirements, and as a result, N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl Benzamide inhibits lipopolysaccharide-induced maturation and inhibits the activation of lipopolysaccharide-induced signaling pathways. By reducing the expression was confirmed that there is an excellent therapeutic effect on sepsis, bacteremia and endotoxins, the present invention was completed.

Accordingly, the present invention relates to N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide or a pharmaceutically acceptable salt thereof. An object of the present invention is to provide a pharmaceutical composition for preventing or treating sepsis or septic shock, which is included as an active ingredient.

The present invention also provides N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide or a pharmaceutically acceptable salt thereof. An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bacteremia or endotoxemia comprising as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl]- It provides a pharmaceutical composition for the prevention or treatment of sepsis or septic shock comprising benzamide or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the sepsis or septic shock may be caused by Gram-negative bacteria.

In another embodiment of the present invention, the causative bacteria of the Gram-negative bacteria may be one or more causative bacteria selected from the group consisting of Escherichia coli, Salmonella, D. cholera, and Typhoid.

In another embodiment of the invention, the N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide is It may be one that interferes with lipopolysaccharide-induced maturation.

In another embodiment of the invention, the N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide is lipopoly It may be to inhibit the activation of the saccharide-induced signaling pathway (lipopolysaccharide-induced signaling pathway).

The present invention also relates to N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide or a pharmaceutically acceptable salt thereof. It provides a pharmaceutical composition for the prevention or treatment of bacteremia or endotoxins comprising as an active ingredient.

In one embodiment of the present invention, the endotoxin may be lipopolysaccharide-induced endotoxin.

The present inventors have conducted research to satisfy the above requirements, and as a result, N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl Benzamide is not only used to treat myelofibrosis and some cancers, but also interferes with lipopolysaccharide-induced maturation and lipopolysaccharide-induced signaling pathways. It has been confirmed that it inhibits the activation of -induced signaling pathway, and thus, it is expected to reduce the expression of proinflammatory cytokines, which can be used in the treatment of sepsis, bacteremia and endotoxins, and in the development of medicines.

FIG. 1A is a diagram showing hierarchical clustering when lipopolysaccharide (LPS) is treated in dendritic cells (DCs) as in Examples 1-4. FIG.
FIG. 1B is a diagram showing significantly enriched standard paths identified through Ingenuity Pathway Analysis (IPA). FIG.
1C is a diagram showing Gene Ontology (GO) clusters that are significantly associated with lipopolysaccharide (LPS) treatment in mouse DCs as in Examples 1-4. The rod type corresponds to the number of genes in each GO term.
FIG. 1D shows Hematological System Development and Function, Immune Cell Trafficking, and Inflammatory Response Networks treated with LPS to DCs using Ingenuity Pathway Analysis (IPA). When confirmed as the top-level network involved.
1E is a diagram representing upstream regulators predicted after LPS treatment on DCs. All listed kinases were predicted to be more active by IPA.
FIG. 1F shows an infrared heat map showing gene expression of Tbk1 (TANK-binding kinase1) downstream targets.
Figure 1g is a diagram showing the expression of Il6 and Irf7 as a result of real time polymerase chain reaction (Real Time-Polymerase Chain Reaction (RT-PCR) analysis as in Example 1-6 after LPS treatment, compared with the control DCs.
2A is a diagram showing the chemical structure of N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide (CYT387) .
FIG. 2B shows the results of analyzing DCs overnight with CYT387, DMSO and H ₂ O ₂ and analyzing the cytotoxicity of CYT387 on DCs using a luminescent cell viability kit as in Example 1-7. It is also.
FIG. 2C shows pretreatment of DCs at the indicated concentrations of CYT387 for 30 minutes prior to LPS treatment (50 ng · mL ⁻¹ ) and incubated overnight, followed by supernatant collection and levels of TNF, IL-6 and IL-12p70 It is a figure which shows the result measured by ELISA like 1-8.
Figure 3a shows pretreatment of DCs at the indicated concentrations of CYT387 for 30 minutes before LPS treatment (50 ngmL- ¹ ) and incubation overnight, then the supernatant was collected and the amount of IFN-β as ELISA as in Example 1-8. The figure which shows the result measured.
FIG. 3B shows pretreatment of DCs with CYT387 (0.5 μM) for 30 minutes at the indicated time point before LPS stimulation (50 ng · mL ⁻¹ ), followed by p-p38, p38, p-ERK, ERK, p in cell lysate. Figures showing the results of protein expression of -JNK, JNK, p-Akt, Akt, IκBα and α-tubulin were detected by western blotting as in Example 1-9.
FIG. 3C shows pretreatment of DCs with CYT387 (0.5 μM) for 30 minutes prior to LPS treatment (50 ng · mL ⁻¹ ) and incubated overnight, supernatant was collected and level of HMGB1 was determined by ELISA as in Examples 1-8 It is a figure which shows the result of a measurement.
Figure 4a is a view of the abdominal cavity CYT387 (50 mg · kg ^-1) for injection to 1 hour before, mouse LPS (20mg · kg ^-1) showing the injection and observed for survival for 48 hours or more, as a result the abdominal cavity.
Figure 4b after the injection the CYT387 (50 mg · kg ^-1) in mice before injection intraperitoneally 1 hours of LPS (10mg · kg ^-1) and left standing for 2 hours the abdominal cavity, TNF, IL-6 and IL-12p70 Serum cytokine level is shown in the results measured by ELISA as in Example 1-8.
Figure 4c after the scan within the CYT387 (50 mg · kg ^-1) in mice before injection intraperitoneally 1 hours of LPS (10mg · kg ^-1) intraperitoneally and allowed to stand overnight, mice were euthanized flowing through the lungs and fixed in formalin The result which showed the result. They were stained with hematoxylin and eosin (H & E) and representative images of three independent experiments were shown.
Figure 4d is a diagram showing the results of measuring the cytokine level of TNF, IL-1β and IL-6 in the lysate of the lung from the same mouse by ELISA as in Example 1-8.
Figure 4e is a diagram showing the results measured by ELISA as in Examples 1-8 to verify the serum levels of AST, ALT and BUN liver and kidney damage.
Figure 4f is a diagram showing the results of measuring the HMGB1 level in serum as in Example 1-8.
FIG. 5A shows the survival rate of 72 hours or more after mice were intraperitoneally injected with CYT387 (50 mg · kg ⁻¹ ) 1 hour before the intraperitoneal injection of E. coli K1 (1 Х 10 ⁷ CFU · mice ⁻¹ ). to be.
FIG. 5B shows that mice were intraperitoneally injected with CYT387 (50 mg · kg ⁻¹ ) 1 hour before intraperitoneal injection of Escherichia coli K1 (1 Х 10 ⁶ CFU · mice ⁻¹ ) and left for 2 hours, followed by TNF, IL- 6 and IL-12p70 serum cytokine level is shown in the results measured by ELISA as in Example 1-8.
5C shows mice were intraperitoneally injected with CYT387 (50 mg · kg ⁻¹ ) and cultured overnight 1 hour prior to intraperitoneal injection of E. coli K1 (1 Х 10 ⁶ CFU · mice ⁻¹ ). After perfusion and fixation with formalin, the lung sections were stained with hematoxylin and eosin (hematoxylin & eosin, H & E). Shown are representative images of three independent experiments.
Figure 5d is a diagram showing the results of measuring the cytokine level of TNF-α, IL-1β and IL-6 in the lysate of the lung from the same mouse by ELISA as in Example 1-8.
Figure 5e shows the results of measuring the level of serum AST, ALT and BUN by ELISA as in Example 1-8 to identify liver and kidney damage in E. coli K1-induced sepsis model as in Example 1-11 It is also.
FIG. 5F shows the colony counts of bacteria isolated from lung, liver, kidney and spleen measured using LB agar plates between different treatments.
Figure 5g is a diagram showing the results of measuring the serum toxin level using a LAL assay kit (LAL assay kit).
Figure 5h is a diagram showing the result of measuring the serum HMGB1 level by ELISA as in Example 1-8.

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The present inventors have conducted research to satisfy the above requirements, and as a result, N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl Benzamide (CYT387) interferes with lipopolysaccharide-induced maturation and inhibits the activation of lipopolysaccharide-induced signaling pathway By reducing the expression of proinflammatory cytokines was confirmed the excellent therapeutic effect on sepsis, bacteremia and endotoxins was completed the present invention.

In one embodiment of the present invention, RNA is isolated and carried out from immature Dendritic Cells (Day 6) and LPS-induced mature DCs (Day 7) as in Examples 1-4. As in Example 1-5, microarray examination was performed to observe the transcript change induced by LPS, and upstream regulator analysis using IPA revealed that Tbk1 was LPS in DCs. It was confirmed that it plays an important role in transcriptional changes induced by (see Example 2).

In another embodiment of the present invention, the cytokine release was examined by ELISA as in Example 1-8, and CYT387 did not change levels of TNF, IL-6, IL-12p70 in the untreated DCs population, but LPS. It was confirmed that dose-dependent inhibition of the production of these cytokines in activated DCs, and that CYT387 can inhibit LPS-induced DC maturation (see Example 3).

In another embodiment of the present invention, the treatment of mature DCs with LPS in the presence or absence of CYT387 tested whether the effect of CYT387 is associated with NF-κB and MAPK signaling pathways, and significantly inhibited phosphorylation of Akt. As a result of ELISA as in Example 1-8, it was confirmed that CYT387 reduces the secretion of HMGB1 in LPS activated DCs (see Example 4).

In another embodiment of the present invention, ELISA was performed as in Example 1-8 to demonstrate the effect of CYT387 on serum cytokine levels in the LPS-induced endotoxemia mouse model as in Example 1-10. The results showed that TNFγ reduced IL-6 and IL-12p70 levels, and LPS-induced lung pre-inflammatory IL-1β, IL-6 and TNF levels, and influenced the infiltration of inflammatory cells into the lung. For a more extensive evaluation, H & E staining confirmed that CYT387 reduced AST, ALT, BUN, and HMGB1 levels (see Example 5).

In another embodiment of the present invention, CYT387 was performed as in Example 1-8 to analyze serum levels of various pro-inflammatory cytokines in the E. coli K1-induced sepsis model, as in Example 1-11. It was confirmed that this reduced levels of TNF, IL-6 and IL12-p70, and that CYT387 treatment actually reduced the number of bacteria in major internal organs, including inhibiting bacteremia in both lung and liver. It was confirmed that K1-induced sepsis could reduce serum endotoxin levels originally increased (see Example 6).

Accordingly, the present invention relates to N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide (CYT387) or a pharmaceutical thereof It relates to a pharmaceutical composition for the prevention or treatment of sepsis or septic shock, including an acceptable salt as an active ingredient.

The term "N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide" used in the present invention is a Tbk1 inhibitor, Also called CYT387, Momelotinib, INN, or GS-0387. It has been used to treat myelofibrosis since 2011 and has an acceptable safety profile. Functionally, CYT387 acts as an ATP competitor by inhibiting Janus kinase 1 (JAK1) and JAK2, disrupting the JAK-STAT signaling pathway and inhibiting the expression of pro-tumorigenic cytokines. Decrease. It can also suppress abnormal IKK-ε and Tbk1. However, a recent report in 2016 warned of the possibility of treatment failure with CYT387 in a phase 3 trial aimed at alleviating myelofibrosis symptoms. Nevertheless, it is still a promising molecule that has been found to destroy metastatic KRAS-mutated non-small cell lung cancers and metastatic pancreatic ductal adenocarcinoma.

The N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide is used for lipopolysaccharide-induced maturation of cells. interfering with lipopolysaccharide-induced maturation, specifically, the cells are dendritic cells, and interfering with lipopolysaccharide-induced maturation is tumor necrosis factor, interleukin- 6 (Interleukin-6), or interleukin-12p70 (Interleukin-12p70) was confirmed to inhibit the level.

In addition, the N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide is lipopolysaccharide-induced signaling. It is characterized by inhibiting the activation of (lipopolysaccharide-induced signaling pathway), specifically lipopolysaccharide-induced signaling pathway is NF-κB signaling pathway, Akt involved in the NF-κB signaling pathway Or it is characterized by inhibiting the activity of HMGB1 (High-Mobility Group Box 1) to inhibit the activation of NF-κB signaling pathway, but is not limited thereto.

Tbk1 stands for TANK-binding kinase 1, and is a serine / threonine kinase, which plays an essential role in regulating the inflammatory response by TLR to foreign components. Toll-interleukin receptor (TIR) -domain-adapter-induced interferon-β (TRIF) -dependent signaling pathway, shared exclusively by TLR3 and TLR4 receptors Activation of inducing interferon-β (TRIF) -dependent signaling pathways is associated with tumor necrosis factor (TNF) receptor-associated factor 3 (TNF) receptor-associated factor 3 (TRAF3). TRAF3) associated NF-κB activator (TANK)]. This leads to the activation of abnormal Tbk1 and IκB kinase ε (IKKε) that phosphorylates interferon regulatory factor 3 (IRF3) and IRF7. This phosphorylation allows for subsequent homodimerization and nuclear translocation leading to transcriptional activation of pro-inflammatory and antiviral genes including type I and type III interferon (IFN). In addition, IKKε / Tbk1 directly directs Akt by phosphorylation of hydrophobic motifs that depend on phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling pathways. It has been shown to activate. This promotes KRAS-driven tumorigenesis by regulating the self-secreting C-C motif chemokine ligand 5 (CCL5) and interleukin (IL) -6.

As used herein, the term “pharmaceutically acceptable salts” refers to formulations of compounds that do not cause serious irritation to the organism to which the compound is administered and do not impair the biological activity and properties of the compound. The pharmaceutical salts include acids that form non-toxic acid addition salts containing pharmaceutically acceptable anions, for example inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, and the like, tartaric acid, formic acid, citric acid Sulfur acids such as organic carbonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc., such as acetic acid, trichloroacetic acid, trichloroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc. Acid addition salts formed by phonic acid or the like. For example, pharmaceutically acceptable carboxylic acid salts include metal salts or alkaline earth metal salts formed by lithium, sodium, potassium, calcium, magnesium, amino acid salts such as lysine, arginine, guanidine, dicyclohexylamine, N Organic salts such as -methyl-D-glucamine, tris (hydroxymethyl) methylamine, diethanolamine, choline and triethylamine and the like.

As used herein, the term "septicemia" refers to a condition in which a serious inflammatory response occurs in the whole body by infection with a microorganism. Fever symptoms that rise above 38 degrees or hypothermia below 36 degrees, respiratory rate increases above 24 times per minute (empty breathing), heart rate above 90 beats per minute (tachycardia), and increases or significant decreases in white blood cell counts on blood tests This is called systemic inflammatory response syndrome (SIRS). This systemic inflammatory response syndrome is called sepsis when it is caused by infection of microorganisms. In infectious lesions of the body, pathogens enter the bloodstream continuously or intermittently and settle in various organ tissues, creating lesions and showing severe systemic symptoms. The causative agent of a purulent disease may be introduced into the blood, may be infected through food ingestion, and even if the blood is not directly infected with bacteria, the symptoms may be caused by an inflammatory substance generated from an infectious agent at a site. Alcohol poisoning, malnutrition, liver disease, etc. are less likely to occur in people or newborns with reduced immunity, but are not limited thereto.

The causative agents of the purulent disease include, but are not limited to, Gram-positive bacteria or Gram-negative bacteria, and the positive bacteria include Streptococcus, Staphylococcus and Bacillus subtilis, and Gram-negative bacteria include Escherichia coli, Salmonella, and D. cholera. Preferably, E. coli but not limited thereto.

The present invention also provides N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide or a pharmaceutically acceptable salt thereof. It relates to a pharmaceutical composition for the prevention or treatment of bacteremia or endotoxin, comprising as an active ingredient.

The endotoxin is characterized in that the lipopolysaccharide-induced endotoxin but is not limited thereto.

As used herein, the term "bacteremia" refers to a state in which bacteria enter the blood and circulate all over the body. Even if bacteria originally enter the human body, when they enter the blood vessels, they are soon removed by white blood cells, so there are no bacteria in the blood. However, if there is a lot of bacteria in one or several parts of the body, and it is a lot of bacteria, it travels through blood vessels. This condition is called bacteremia. The most common pathology of bacteremia is the genitourinary system (25%), 20% respiratory system, 10% abscess, and surgery. 5% red wounds, 5% biliary tract, 10% other sites, 25% no cause. Bacteremia occurs when bacteria multiply at rates beyond the repellency of the reticuloendothelial system. The most common route of propagation of blood fungi into the blood through lymphatic vessels is from blood vessels, and bacteremia that spreads directly into the blood due to infective endocarditis, infection of arteriovenous fistula, fungal aneurysms, purulent phlebitis, and various catheters in the blood vessels. However, the present invention is not limited thereto.

The term "endotoxinemia" used in the present invention refers to a condition caused by inflow of endotoxin, a cell wall component of gram-negative bacteria, into blood, and there are infectious (septic) and non-infectious (non-septic) endotoxins. This is not restrictive. Non-infectious endotoxinemia, also called endogenous endotoxinemia, is considered to be derived from bacteria in the digestive tract, and the mechanism of development is called endotoxin production, which is caused by phagocytic disorder, hepatocellular dysfunction, and endotoxin production. This symptom causes disseminated intravascular coagulation, endotoxin shock, multi organ failure (MOF), and the like, but not limited thereto.

As used herein, the term "prevention" refers to any action that inhibits or delays the onset of sepsis infection by preemptively administering the pharmaceutical composition according to the present invention prior to sepsis infection.

As used herein, the term "treatment" refers to any action in which symptoms for sepsis-infected diseases are improved or beneficially altered by administration of the pharmaceutical composition according to the present invention after sepsis infection.

Thus, it may further comprise suitable carriers, excipients, and diluents commonly used to prepare pharmaceutical compositions. It may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions according to conventional methods.

Carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil and the like. In formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.

As used herein, the term "administration" means providing a subject with any of the compositions of the present invention in any suitable manner. The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the type, severity, and activity of the patient's disease. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts.

The pharmaceutical composition according to the present invention may be administered simultaneously, separately, or sequentially with the drug to be used in combination to enhance the therapeutic effect, and may be administered singly or in multiple doses. . Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art. Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in combination.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example by oral administration, intranasal administration, coronary administration, arterial injection, intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. The daily dosage is preferably administered once or several times a day, but is not limited thereto.

The pharmaceutical composition of the present invention is determined according to the type of drug which is the active ingredient, along with various related factors such as the disease to be treated, the route of administration, the age, sex, weight of the patient, and the severity of the disease.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Experiment Preparation and Methods

       1-1. Animals (Animals)

Six week old female C57BL / 6 mice (H-2Kb and I-Ab) and six week old female BALB / c mice were purchased from Orient Bio (Daejeon, Korea). The procedure used in the study was approved and managed by the Institutional Animal Care and Use Committee (IACUC) (IACUC No .: KU17044-2).

       1-2. Bacteria

E. coli K1 strain RS218 (O18: K1: H7) used in the E. coli K1-induced sepsis mouse model as in Example 1-11 was provided by Dr. Jang Won-won, Kangwon National University (Kangwon-do, Korea).

       1-3. Reagents and antibodies

Recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF), FITC anti-CD11c antibody (clone: N418), PE anti-CD86 antibody (clone: GL-1), PE anti -H-2Kd / H-2Dd antibody (clone: 34-1-2S), and PE anti-IA / IE antibody (clone: M5 / 114.15.2) were purchased from BioLegend (San Diego, CA, USA). To maintain dendritic cells (DCs) as in Examples 1-4, RPMI 1640 medium, fetal bovine serum (FBS) and penicillin-streptomycin solution purchased from Biowest (Nouaille, France) were used. E. coli O111: B4 LPS was used for in vitro experiments from Invivogen (San Diego, CA, USA) and E. coli O127: B8 LPS was used for in vivo experiments using Sigma-Aldrich (St. Louis, MO, USA). CYT387 was purchased from Selleckchem (Houston, TX, USA). MTT Cell viability kit was obtained from Promega (Madison, WI, USA) as in Example 1-7. In western blotting as in Examples 1-9, anti-p-p38 antibody, anti-p38 antibody, anti-p-ERK antibody, anti-ERK antibody, anti-pNNK antibody, anti-JNK antibody, Anti-p-Akt antibodies, anti-Akt antibodies and anti-IκBα antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Anti-α-tubulin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Protease Inhibitor Cocktails Luria-Bertani (LB) broth powder and LB agar powder were both purchased from Biobasic (Amherst, NY, USA) and LAL Endotoxin Detection Kit from Lonza (Basel, Switzerland).

       1-4. Production of Murine Bone Marrow DCs

Tibia and femur bone marrow were used to isolate DCs after euthanasia of C57BL / 6 mice according to IACUC guidelines. Erythrocytes were removed from bone marrow using erythrocyte lysis buffer, leaving only progenitor cells. The progenitor cells were placed in a cell culture plate containing RPMI 1640 (containing 10% FBS, 1% penicillin / streptomycin, 10 ng mL-1 GM-CSF) and incubated for 6 days at 37 ° C. and 5% CO _2. . After 6 days, progenitor cells differentiated into immature DCs. To produce mature DCs immature DCs were treated with 50 ng · mL ⁻¹ LPS and incubated overnight at 37 ° C. under 5% CO ₂ .

       1-5. Microarray assay

RNA purity and integrity were evaluated using an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA). RNA labeling and hybridization were performed using the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology, V 6.5, 2010). Briefly, 100 ng of total RNA was linearly amplified from each sample and labeled with Cy3-dCTP. Labeled cRNA was purified using RNAeasy Mini Kit (Qiagen, Hilden, Germany). The concentration and specific activities of labeled cRNA (pmol Cy3 · g cRNA- ¹ ) were measured using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). Next, 600 ng of each labeled cRNA sample was fragmented by addition of 5 × 10 × blocker and 1 μL of 25 × cleavage buffer before heating at 60 ° C. for 30 minutes. Finally, 25 μL 2 × GE hybridization buffer was added to dilute labeled cRNA. A total of 40 μL of hybridization solution was dispensed onto the gasket slide and inserted into Agilent SurePrint G3 mouse GE 8X60K Microarrays (Agilent Technologies, Santa Clara, Calif., USA).

The slides were incubated at Agilent Hybridization Oven (Agilent Technologies, Santa Clara, Calif., USA) for 17 hours at 65 ° C and according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology, V 6.5, 2010). Wash at room temperature (22 ° C.). Hybridized arrays were immediately scanned with an Agilent Microarray Scanner D (Agilent Technologies, Santa Clara, Calif., USA). Raw data was extracted using Agilent Feature Extraction Software v11.0.1.1 (Agilent Technologies, Santa Clara, Calif., USA), and then automatically summarized using the Agilent feature extraction protocol to generate a raw data text file. . This provided expression data for each gene included in the array for further analysis. Array probes flagged with flag A were removed. Each selected gProcessedSignal value was algebraically converted and normalized using the quantile method. Statistical significance in the expression data was determined by fold change and LPE test, and the null hypothesis was not different between the two groups. Hierarchical cluster analysis was performed using complete connections and Euclidean distances as a measure of similarity. Gene-Enrichment and Functional Annotation analyzes of important probe lists were performed using DAVID (http://david.abcc.ncifcrf.gov/home.jsp). All data analysis and visualization of differentially generated genes was performed using R 3.0.2 (http://www.r-project.org).

       1-6. Real time-polymerase chain reaction (RT-PCR)

Extract total RNA using TRIzol reagent (Thermo Fisher Scientific, San Jose, Calif., USA), digest with DNase I (Biobasic, Amherst, NY, USA), and then use the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster) City, CA, USA). CDNA amplification was performed using the LightCycler 480 II (Roche, Basel, Switzerland) and the LightCycler 480 SYBR Green I Master mix (Roche, Basel, Switzerland) according to the manufacturer's recommendations.

       1-7. Cell viability assay

As in Example 1-4, immature dendritic cells (DCs) were treated with various concentrations of CYT387 (0.1, 0.25, 0.5, 1.1, 2.5, 5.0 μM) and incubated overnight. H ₂ O ₂ was used as a positive control. After incubation, a cell viability assay kit solution was added to the cells. They were then incubated at room temperature for 12 minutes and luminescence was measured using a Veritas Luminometer (Turner Biosystems, Inc. Sunnyvale, Calif., USA).

       1-8. ELISA

Levels of various proinflammatory cytokines, including TNF, IL-1β, IL-6 and IL-12p70, were assessed by sandwich ELISA (eBioscience, San Diego, Calif., USA). Levels of HMGB1 were assessed by sandwich ELISA (Cusabio Biotech, Wuhan, China). Optical density at 450 nm was measured using a Sunrise Spectrophotometer (TECAN, Mδnnedorf, Switzerland).

       1-9. Western Blotting

CYT387 / LPS-treated DCs and LPS-treated DCs were simultaneously harvested and washed with PBS. The cells were then treated with RIFA Buffer [0.5% NP-40, 0.01% protease inhibitor cocktail, 1 mM EDTA, 0.5 M NaF, 120 mM NaCl, 50 mM Tris-HCl and 0.5 mM PMSF for 30 minutes at 4 ° C. pH 8.0]. The lysates were then centrifuged at 13,000 rpm for 15 minutes, aliquots of protein lysates were quantified and separated by SDS-PAGE using a 10% polyacrylamide gel. Proteins were transferred to PVDF membranes, blocked with 5% skim milk for 1 hour, and washed three times with Tris buffered saline containing 0.1% Tween-20 (TBS-T) for 10 minutes each. Antibodies specific for p-p38, p-38, p-ERK, ERK, p-JNK, JNK, p-Akt, Akt, IκBα or α-tubulin were incubated with membranes overnight at 4 ° C. The membrane was washed with TBS-T and incubated with HRP-labeled anti-rabbit or anti-mouse secondary antibody for 1 hour at room temperature. ECL solution was added to the membrane and then washed with TBS-T. Bands were detected using LAS4000 (Fuji Film, Tokyo, Japan).

       1-10. LPS-induced endotoxemia mouse model

In the endotoxin model, mice (6 week-old female BALB / c) were injected with CYT387 (50 mg · kg ⁻¹ ), LPS (20 mg · kg ⁻¹ ), or both. To assess serum levels of inflammatory cytokines, blood was collected 2 hours after injection. Serum was then separated, diluted with PBS, and inflammatory cytokines were determined using ELISA as in Examples 1-8. At the indicated time points, mice were sacrificed and blood and lung tissue harvested. The lungs were divided into two halves, half stained with hematoxylin and eosin (H & E), the other half homogenized with stainless beads, and then centrifuged at 3,000 rpm for 5 minutes to increase proinflammatory cytokine levels. The supernatant used for measurement was obtained. Serum was also separated from serum and diluted with PBS. Liver and kidney injury indexes were measured using medical laboratory equipment including Total Laboratory Automation System (Hitachi, Tokyo, Japan) and TBA-200FR NEO (Toshiba, Tokyo, Japan).

       1-11. E. coli K1-induced sepsis mouse model E. coli  K1-induced sepsis mouse model)

Experiments similar to those performed in the LPS-induced endotoxemia mouse model as in Examples 1-10 were also performed in the E. coli K1-induced sepsis mouse model. Blood, lung, liver, kidney and spleen samples were removed from mice sacrificed after overnight injection of E. coli K1. Serum toxin levels were measured using the same LAL assay kit (Lonza, Basel, Switzerland) as before. To assess bacterial load, major organs including lung, liver, kidney and spleen were homogenized with stainless beads and the homogenate diluted with PBS. The homogenate was then incubated overnight at 37 ° C. on an LB agar plate. The colonies were then counted to estimate the relative bacterial populations of major organs.

       1-12. Statistical analysis

All experiments were repeated at least three times with consistent results. Unless otherwise specified, data are expressed as mean ± SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) by Dunnett's test using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Survival rates were compared using the Mantel-Cox log lank test and were used to identify differences between survival curves. The threshold of statistical significance was set below P <0.05 and was considered significant.

Example 2 Tbk1 downstream signaling, a major pathway affected by lipopolysaccharide (LPS) treatment on dendritic cells (DCs)

For microarray examination as in Example 1-5 to understand transcript change induced by LPS, RNA was subjected to immature DCs (6 days) and LPS-induced maturation as in Examples 1-4. Isolation from DC (day 7) (FIGS. 1A-1F). Since LPS treatment induced a relatively large change in gene expression in DCs, cutoff and 0.05 significance thresholds for the 4-fold expression change were used to identify more important mediators (FIG. 1A). Using the Ingenuity Pathway Analysis (IPA), investigating the impact on canonical pathways showed that the "dendritic cell maturation" pathway was significantly altered as expected (FIG. 1B). To further investigate the effect of LPS exposure on DCs, Gene-Enrichment analysis and Functional Annotation analysis were performed on the most important and differentially expressed genes. This analysis revealed several Gene Ontology (GO) categories related to LPS exposure, the top eight clusters involved in glycoproteins, hydrolase, immune response, cell cycle, lipoproteins, and cytokines (FIG. 1c). Finally, network analysis using IPA suggested that there were some and cytokine and transcription regulators known to be involved in the LPS response (FIG. 1D), which produced a truly representative LPS-dependent transcript in DCs. To identify key signal molecules involved, upstream regulator analysis using IPA was also performed. Serine / threonine kinase Tbk1 (serine / threonine kinase Tbk1) was identified as the most important kinase among the signal enzymes (FIG. 1E). Examining this data, the expression of genes downstream of Tbk1 was upregulated in DCs after LPS treatment (FIG. 1F). In order to verify this prediction, transcript data were confirmed by confirming expression changes of two downstream genes (Il6 and Irf7) through real-time polymerase chain reaction as in Example 1-6 (FIG. 1G). In summary, these data show that Tbk1 plays an important role in the transcriptional changes induced by LPS in DCs.

Example 3 CYT387 Interferes with Lipopolysaccharide (LPS) -Induced Maturation of Bone Marrow-Derived Dendritic Cells (DCs)

Transcriptome analysis confirmed that Tbk1 may be a key molecule involved in LPS-induced DCs maturation (FIGS. 1A-1G), so it was decided to test if Tbk inhibitors could affect DCs maturation. Among the Tbk inhibitors, CYT387 (FIG. 2A) was selected due to its relatively high potential and selectivity. First, MTT analysis was performed to measure the cytotoxicity of CYT387 in DCs using H ₂ O ₂ as a positive control group. CYT387 concentration ranges of up to 5 μM were tested and no toxicity was observed in DCs up to 1 μM (FIG. 2B). Therefore, the maximum concentration of CYT387 was set to 1 μM in the experiment.

Next, the release of some important cytokines was examined by ELISA as in Example 1-8. These DC-released cytokines play a pivotal role in both the adaptive and innate immune responses. In particular, IL-12 released by DCs induces differentiation of naive T cells with IFN-γ-producing Th1 phenotype. In contrast, pro-inflammatory cytokines such as TNF and IL-6 induce an innate immune response. In this study, we found that CYT387 did not alter levels of TNF, IL-6, IL-12p70 in the untreated DC population but dose-dependently inhibited the production of these cytokines in LPS-activated DCs (FIG. 2C). ). The results also confirmed that CYT387 could inhibit LPS-induced DC maturation, which reduced the inflammatory response of mature DCs.

Example 4 CYT387 Regulates Activation of LPS-Induced Signaling Molecules in Dendritic Cells (DCs)

Next, we wanted to investigate further the mechanism of Tbk1 inhibition by CYT387. Both Tbk1 and IKK-i have been shown to activate IRF3, the primary transcription factor that regulates type I IFN. Tbk1 is particularly important for IRF3 activation and IFNβ production in response to LPS due to TLR4 ligand stimulation. The data revealed that CYT387 treatment on LPS inhibited the production of IFNβ, confirming previous results that CYT387 is a potential inhibitor of Tbk1 (FIG. 3A).

LPS has also been shown to strongly induce DCs maturation and production of proinflammatory cytokines by TLR4-Myd88 activation, which depends on NF-κB and MAPK signaling pathways. To test whether the effect of CYT387 on LPS-induced DCs is related to NF-κB and MAPK signaling pathways, mature DCs were treated with LPS in the presence or absence of CYT387. Phosphorylation of several MAPKs was examined by Western blot analysis as in Examples 1-9, including ERK, JNK and p38. Unexpectedly, this showed that CYT387 did not inhibit the phosphorylation of ERK, p38 or JNK in LPS-activated DCs (FIG. 3B). In contrast, CYT387 significantly inhibited phosphorylation of Akt and slightly affected the degradation of IκBα in LPS-activated DCs in a time-dependent manner (FIG. 3B).

Under inflammatory conditions, high-mobility group box 1 (HMGB1) can be passively released from damaged cells or actively secreted from activated immune cells. Extracellular HMGB1 acts as a prototypical hazard signal that activates TLR2 and TLR4 as well as receptor RAGE, inducing NF-kB signaling, which induces downstream NF-kB signaling. This in turn regulates the secretion of proinflammatory cytokines and chemokines. Previously, pharmacological or genetic blockade of JAK / STAT signals has been shown to cancel HMGB1 secretion. Thus, the possibility of CYT387 blocking LPS-induced secretion of HMGB1 in mouse DCs was considered. In order to solve this problem, ELISA was performed as in Example 1-8, and it was confirmed that CYT387 reduced HMGB1 secretion in LPS activated DCs (FIG. 3C). These results indicated that CYT387 inhibits proinflammatory gene expression by interfering with Akt and NF-κB signaling cascades in LPS-activated DCs independent of MAPK signaling pathway. In addition, CYT387 was found to reduce the level of extracellular HMGB1, which may induce or increase inflammatory mediator production.

Example 5 CYT387 Having a Therapeutic Effect in a Lipopolysaccharide (LPS) -Induced Lethal Endotoxemia Mouse Model

Tbk1 inhibition has been shown to effectively inhibit polyinosinic-polycytidylic acid-induced immune activation in vitro and in vivo, suggesting a TLR4 signaling pathway. Additional experimental evidence has shown that inhibiting Tbk1 with Compound II can serve as an effective treatment for TREX1-related autoimmune diseases and other interferonopathies. This study provides further support for Tbk1 inhibition as a therapeutic approach for inflammatory response related diseases. Considering in vitro data demonstrating that CYT387 suppresses the inflammatory response by reducing cytokine and dendritic cell surface molecule expression, CYT387 can neutralize the inflammatory response and limit the pathology in vivo when sepsis is present. Considered the possibility. To resolve this, first, the effect of CYT387 on the overall mortality in the LPS-induced endotoxemia mouse model was examined as in Example 1-10. This confirmed that CYT387 treatment improved survival (FIG. 4A). Next, ELISA was performed as in Example 1-8 to demonstrate the effect of CYT387 on serum cytokine levels in the LPS-induced endotoxemia mouse model as in Example 1-10. Consistent with in vitro observations, CYT387 treatment inhibited the increase in TNFγ IL-6 and IL-12p70 levels found in the serum of LPS-induced endotoxemia mice as in Example 1-10 (FIG. 4B). Treatment also reduced LPS-induced lung internal pro-inflammatory IL-1β, IL-6 and TNF levels (FIG. 4D).

To assess more broadly the effect of CYT387 on the infiltration of inflammatory cells into the lung, H & E staining was performed. This data showed that infiltration of polymorphonuclear (PMN) cells into the lung by LPS infusion was reduced by CYT387 treatment (FIG. 4C). Next, the levels of AST, ALT and BUN secreted from the bloodstream were measured to demonstrate the effect of CYT387 treatment on liver and kidney damage due to LPS. All levels of these markers were alleviated by CYT387 treatment and were similar to the levels detected in control mice (FIG. 4E). According to in vitro data, CYT387 reduced the secretion of HMGB1 levels found in the serum of LPS-induced endotoxemia mouse model as in Example 1-10 (FIG. 4F). In addition, these findings support the hypothesis that Tbk1 inhibition by CYT387 directly attenuates the inflammatory condition that promotes severe sepsis.

Example 6 CYT387 with a Pure Therapeutic Effect in an E. Coli K1-Induced Sepsis Mouse Model

Since sepsis is often caused by bacterial infections, especially Gram-negative bacteria; Next, the clinical efficacy of CYT387 was studied using a Gram-negative Escherichia coli K1 (E. coli K1) sepsis mouse model as in Example 1-11, but not the LPS model. Initially, the effect of the treatment of CYT387 on the survival rate was examined to confirm that CYT387 had a positive effect (FIG. 5A). As mentioned previously, ELISA was performed as in Examples 1-8 to analyze serum levels of various pro-inflammatory cytokines in the E. coli K1-induced sepsis model as in Examples 1-11. This again showed that CYT387 reduced the expression of TNF, IL-6 and IL12-p70 in the sepsis model (FIG. 5B). H & E staining of lung samples confirmed that CYT387 alleviated polymorphonuclear (PMN) infiltration into the lungs of mice with sepsis induced by E. coli K1 (FIG. 5C). CYT387 treatment suggested that lung injury was reduced by reducing TNF, IL-1β and IL-6 levels in lung lysates from sepsis mice induced by E. coli K1 (FIG. 5D). Finally, the results of investigating the levels of AST, ALT and BUN damage markers in E. coli K1 inducible mice as in Example 1-11 confirmed that CYT387 treatment reduced the markers respectively (FIG. 5E).

During sepsis, bacteremia can occur when microbial agents enter the bloodstream at one or more infected sites. This can occur regardless of location, forming localization of other tissues and secondary sites of infection. Several studies have found a correlation between bacteremia and mortality in sepsis patients. Therefore, quantitative bacterial population counts were performed to determine if CYT387 could contribute to reducing lethal signs during sepsis using the E. coli K1-induced sepsis mouse model. The data indicated that CYT387 treatment actually reduced the number of bacteria in the major organs, including inhibiting bacteremia in both lung and liver (FIG. 5F).

In addition, the morbidity of sepsis is not always due to an excessive inflammatory response, and there is substantial evidence indicating that most sepsis patients cannot become immunosuppressed and eradicate infectious pathogens. The potential mechanism of this immunosuppression is that antigen presenting cells (APCs) cannot activate T cells, but the most important is the generation of resistance to endotoxins, which leads to secondary endotoxin testing. Induce white blood cells that cannot respond. Endotoxins are molecules that are closely related to the cytoplasm or cell wall of microorganisms that induce inflammation through interaction with specific high-affinity receptors found in white blood cells. Clinically in Gram-negative bacterial sepsis, endotoxins are often secondary to infection when the causative organism is eliminated by the immune system or antibiotics, and large amounts of endotoxins are released into the bloodstream. Therefore, using the E. coli K1 inducible mouse model as in Example 1-11, it was investigated whether CYT387 treatment could enhance the immune response to endotoxin and affect the serum of sepsis mice. LAL analysis demonstrated that CYT387 treatment could reduce serum endotoxin levels originally increased by E. coli K1 induced sepsis (FIG. 5G). Consistent with the in vitro results, CYT387 treatment excluded the secretion of HMGB1 found in the serum of E. coli K1 inducible mouse model as in Example 1-11 (FIG. 5H). Taken together, the results obtained from the E. coli K1 inducible mouse model as in Example 1-11 indicate that Tbk1 is a major factor in the pathologic aspects and immunosuppression found in sepsis, and that CYT387 may serve as a potential therapeutic intervention. Suggest.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

N- (Cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide or a pharmaceutically acceptable salt thereof as an active ingredient Pharmaceutical composition for the prevention or treatment of sepsis or septic shock.
The method of claim 1,
The sepsis or septic shock is characterized in that caused by Gram-negative bacteria, pharmaceutical composition for the prevention or treatment of sepsis or septic shock.
The method of claim 1,
The N- (cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide is a lipopolysaccharide-induced signaling pathway ( A pharmaceutical composition for the prevention or treatment of sepsis or septic shock, characterized by inhibiting the activation of lipopolysaccharide-induced signaling pathway.
N- (Cyanomethyl) -4- [2-[[4- (4-morpholinyl) phenyl] amino] 4-pyrimidinyl] -benzaamide or a pharmaceutically acceptable salt thereof as an active ingredient Pharmaceutical composition for the prevention or treatment of bacteremia or endotoxin.
The method of claim 4, wherein
The endotoxin is characterized in that lipopolysaccharide-induced endotoxin, pharmaceutical composition for the prevention or treatment of bacteremia or endotoxin.

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