WO2016197442A1

WO2016197442A1 - Use of bestatin in the preparation of pharmaceutical preparation for the prevention and treatment of inflammatory diseases and infectious diseases associated with inflammation

Info

Publication number: WO2016197442A1
Application number: PCT/CN2015/086002
Authority: WO
Inventors: 钱峰; 戈梅; 何慧琼; 罗敏玉; 夏兴; 饶敏
Original assignee: 上海来益生物药物研究开发中心有限责任公司; 上海交通大学
Priority date: 2015-06-12
Filing date: 2015-08-04
Publication date: 2016-12-15
Also published as: CN104887655A

Abstract

Provided in the present invention is a new use of Bestatin in the preparation of a pharmaceutical preparation for the prevention and treatment of inflammatory diseases and infectious diseases associated with inflammation as an active ingredient. Also provided in the present invention are a pharmaceutical preparation for the prevention and treatment of inflammatory diseases and infectious diseases associated with inflammation, comprising a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient is Bestatin, an optical isomer thereof or a pharmaceutically acceptable salt thereof.

Description

Application of umbrel in the preparation of a pharmaceutical preparation for preventing and treating inflammatory diseases and infectious diseases associated with inflammation

The present invention relates to the use of umbrel as an active ingredient in the preparation of a pharmaceutical preparation for the prevention and treatment of inflammatory diseases and infectious diseases associated with inflammation.

Background technique

Inflammation is a very common and important basic pathological process. Any factor that can cause tissue damage, such as infection, non-infectious tissue damage (such as trauma, surgery, etc.) can lead to inflammation, and these inflammations have A lot of similar features (Barton GM. A calculated response: control of inflammation by the innate immune system. J. Clin. Invest., 2008, 118: 413-420) In the case of inflammation caused by bacterial infection, endotoxin secreted by bacteria ( Lipopolysaccharides (LPS) is a major cause of bacterial pathogenesis and can cause a series of inflammatory reactions in the body. LPS-induced inflammatory signals are related to Toll-like receptors (TLRs) family. When the body is infected with bacteria, LPS Acting on the TLRs receptors on the cell membrane, the expression of cascaded genes changes through intracellular signaling. LPS can stimulate the synthesis and release of inflammatory factors (such as NO, TNF-α, IL-6, IL-Ιβ). Etc.), causing systemic inflammatory reactions, causing toxic shock, systemic inflammatory response syndrome (SIRS) and multiple devices Inflammatory diseases such as dysfunction syndrome (MODS) (Wang Xiaodong, Endotoxin Neutralizing Protein and Its Role in Prevention and Treatment of Sepsis[J], Foreign Medical Physiology, Pathology Science and Clinical Section, 2001, 21(2): 14 4-146; Li Ying, Wang Xingpeng, Research progress in blocking endotoxin signaling pathway in the treatment of sepsis or septic shock [J], Chinese Journal of Emergency Medicine, 2003, 12(2): 135-137; Guo Zaichen, Treatment of septic shock [J], Chinese Journal of Practical Pediatrics, 2007, 22(6): 403-405; Zhang Xuemei, Xiong Huanzhang, Progress in LPS-induced inflammatory response signaling pathway, Chinese Journal of Veterinary Medicine, 2010, 46 (7): 45-47) Therefore, LPS can be regarded as the main pathogenic factor of bacteria and plays an important role in the pathogenesis of bacterial infection. Similarly, in the case of tissue damage, the body secretes a large number of cytokines. Repairing the damage, but when the reaction is excessive, it can also cause inflammatory diseases such as SIRS and MODS.

Ubenimex (also known as Bestatin) is a common anti-tumor adjuvant that enhances immunity. Function, for anticancer chemotherapy, adjuvant therapy for radiotherapy, etc. (Umezawa, H., Aoyagi, T., Suda, H.,

Hamada, M. & Takeuchi, T. (1976). Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes, (29). Pp. 97-99; Muskardin, DT, Voelkel. NF & Fitzpatrick, FA (1994). Modulation of pulmonary leukotriene formation and perfusion pressure by bestatin, an inhibitor of leukotriene A4 hydrolase. (48).

Pp.131-137; Hirayama, Y; Sakamaki, S; Takayanagi, N; Tsuji, Y; Sagawa, T; Chiba, H; Matsunaga, T; Niitsu, Y. (2003). "Chemotherapy with ubenimex corresponding to patient age And organ disorder for 18 cases of acute myelogeneous leukemia in elderly atients- effects, complications and long-term survival". Gan to kagakuryoho.

[0005] Previous studies have shown that umbrel has a good in vivo immune enhancement, such as enhancing the activity and function of T cells and killer NK cells, but has not yet played a role in acute inflammation such as bacterial arches. See the report.

technical problem

The inventors of the present invention found in the further study of the function of Bestatin that it has a significant inhibitory effect on acute inflammation caused by LPS. Accordingly, it is an object of the present invention to provide a novel use of Bestatin as an active ingredient in the preparation of a pharmaceutical preparation for preventing and treating inflammatory diseases and infectious diseases associated with inflammation.

Problem solution

Technical solution

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: [0008] A first aspect of the present invention provides a use of Bestatin as an active ingredient in the preparation of a pharmaceutical preparation for preventing and treating inflammatory diseases and infectious diseases associated with inflammation, The chemical structural formula of Bestatin is:

[0010] wherein the inflammatory disease and infectious diseases associated with inflammation include bacterial infection, uncomplicated cystitis, bronchitis, trauma, postoperative inflammatory reaction, accidental injury, myocardial infarction, tuberculosis or sarcoidosis, Sepsis, metastatic tumors, active rheumatoid arthritis, seronegative spinal arthritis, immune vasculitis, rheumatic polymyopathy, Crohn's disease, inflammation of deep vein thrombosis, etc.

[0011] The inventors established a mouse model of acute lung injury induced by bacterial endotoxin LPS, intraperitoneal administration of Bestatin, the effect of drugs on acute lung injury caused by LPS, and detection of LPS-induced inflammation. Factor NO expression and release, after detection, found that LPS-induced release of inflammatory factor NO decreased dramatically.

[0012] The infiltration of neutrophils was also examined by detection of myeloperoxidase (ΜΡθ) activity, total protein content in the lung lavage lotion, to evaluate the severity of acute lung injury induced by LPS. The results showed that Bestatin inhibited LPS-induced neutrophil infiltration in mice.

A second aspect of the present invention provides a pharmaceutical preparation for preventing and treating an inflammatory disease and an infectious disease associated with inflammation, comprising a therapeutically effective amount of the active ingredient and a pharmaceutically acceptable carrier or excipient The active ingredient is Bestatin, an optical isomer thereof or a pharmaceutically acceptable

[0014] Preferably, the pharmaceutical preparation comprises a tablet, a capsule, an oral solution, a granule or an injection (such as a lyophilized powder injection). [0015] Pharmaceutically acceptable salts can be both pharmacologically and pharmaceutically acceptable. The pharmacologically and pharmaceutically acceptable salt thereof may be an alkali metal or alkaline earth metal salt, preferably a sodium salt, a potassium salt, a magnesium salt or a calcium salt.

The pharmaceutical preparations involve enteral (e.g., oral, sublingual or rectal administration), parenteral or topical (e.g., transdermal pharmaceutical formulations) dosage forms. An organic or inorganic substance which does not react with the active ingredient may be used as a carrier such as water, oil, benzyl alcohol, polyethylene glycol, triacetin or other fatty acid glycerides, gelatin, lecithin, cyclodextrin, lactose or A sugar such as starch, magnesium stearate, talc or cellulose. The oral administration is preferably a tablet, a dragee, a capsule, a powder, a syrup, a concentrate or a drop, a suppository for rectal administration, an aqueous solution or an oil solution for parenteral administration, or a lyophilizate. Suspensions, emulsions or implants may also be employed, and patches or creams may be used for topical administration. Pharmaceutical preparations for parenteral administration comprise sterile aqueous or anhydrous injections of the active compound, preferably a solution which is isotonic with the blood of the recipient.

[0017] These pharmaceutical preparations may contain a stabilizer, an additive that controls the release of the pharmaceutically active compound, an antioxidant, a buffer, a bacteriostatic agent, and an adjuvant for preparing an isotonic solution. Aqueous and anhydrous sterile suspensions may contain suspending additives and thickening agents. The pharmaceutical preparation may be dispensed in a single or multiple dose container such as a sealant bottle, or may be stored as a lyophilized product, if desired, in a sterile liquid such as water or a salt solution. Sterile powders, granules or tablets can also be used in the same manner.

The pharmaceutical preparation can be used for the prevention and treatment of diseases related to inflammation and inflammation in humans and animals.

Advantageous effects of the invention

Brief description of the drawing

DRAWINGS

1 is a graph showing the results of detection of myeloperoxidase (MPO) activity in a model of acute lung injury induced by LPS in mice.

2A and FIG. 2B are graphs showing the results of detection of total protein content in lung lavage fluid in a model of LPS-induced acute lung injury in mice; wherein, FIG. 2A is a total of lung lavage fluid after 4 hours of administration of LPS. Protein concentration determination, Figure 2B is the determination of the total protein concentration of the lung lavage fluid after 12 hours of LPS administration.

Figure 3A is a graph showing the results of two immunoblotting results of iNOS protein expression in LPS-induced Raw264.7 cells.

3B is a quantitative analysis chart of iNOS protein expression in LPS-induced Raw264.7 cells; Embodiments of the invention

[0024] The present invention will be described in detail and by way of specific examples in order to provide a better understanding of the invention.

However, the following examples do not limit the scope of the invention.

[0025] The specific operation of the detection method used in the following embodiments is as follows:

[0026] 1. Constructing an acute inflammation model induced by LPS:

[0027] C57/BL mice were grouped into groups of at least 5, weighed, and administered intratracheally with saline (negative control group) and 5 mg/kg LPS (inflammation group), respectively, before LPS injection. Xiaoyan, 10 minutes later, and 12 hours later, intraperitoneal injection of Bestatin (except for the negative control group), 0, 4, 24 hours later, the heart was taken, and the lungs were repeatedly washed with 1 ml of PBS. The lung tissue was collected after collecting the lavage solution, and the total protein content in the lung lavage lotion was detected, and the activity of myeloperoxidase (MPO) in the lung tissue was detected.

[0028] 2. Fluorescence quantitative PCR (QPCR):

[0029] Trizol lysed cells, extracted RNA, and reverse-transcribed PCR to obtain cDNA, which was used as a template for real-time PCR to examine DNA transcription levels of inflammation-related cytokines and inflammatory molecules induced before and after drug action.

[0030] 3. Western Blot:

[0031] The cells were lysed and collected, denatured, SDS electrophoresis 80V 2 吋, transfected 80V 1 吋, 5% milk blocked half 吋, 4°C incubation primary overnight, TBST buffer (Tris-Buffered Saline and Tween 20) Wash the membrane 3 times, incubate the secondary antibody for 1 hour at room temperature, wash the membrane 3 times with TBST, observe the change of the expression level of nitric oxide synthase iNOS protein induced by the drug before and after the action on the Odyssey far-infrared observer.

[0032] 4, Griess method to detect the amount of NO released:

[0033] NO easily forms NO ₂ - in an aqueous solution, and under acidic conditions, NO ₂ - reacts with a diazonium salt sulfonamide to form a diazo compound, and further undergoes a coupling reaction with naphthyl vinyl diamine. The product concentration has a linear relationship with the N 0 ₂ -concentration and a maximum absorption peak at 540-560 nm. Using this principle, the amount of NO released from the cell supernatant can be detected.

[0034] shed column 1, (Bestatin) hidden LPSi show to delete the market 耆瞧闰闰闰:

[0035] Thirty C57/BL mice were randomly divided into 3 groups, 10 in each group, and weighed, and the following operations were performed respectively: [0036] negative control group: tracheal injection of 0.9% physiological saline 50μ1;

[0037] LPS inflammation group: tracheal injection 5mg / kg LPS 50μ1;

[0038] LPS + umbryiz treatment group: two times before the tracheal injection of LPS (5 mg / kg, about 50 μ1) 12 hours and 10 minutes, two intraperitoneal injections of umbrel (10 mg / kg each time, about 200 μl) ;

[0039] 0, 4, 24 small sputum was sacrificed, blood was taken from the heart, and the lung tissue of the mice was taken, and the activity of myeloperoxidase (ΜΡΟ) in lung tissue and the total protein content in the lung lavage were detected. The test results are shown in Figure 1, Figure 2, and Figure 2, respectively.

[0040] The infiltration of neutrophils was examined by the above three tests to evaluate the severity of acute lung injury induced by LPS.

Figure 1 is a graph showing the results of myeloperoxidase (MPO) activity assay in a model of acute lung injury induced by LPS in mice.

[0042] From the test results of FIG. 1, after LPS (5 mg/kg) tracheal administration for 4 hours and 24 hours, the MPO activity in the lungs of the mice was 3 times compared with the saline control group (Saline). The increase in left and right, and the intervention of Bestatin (lOmg/kg), the MPO activity increased by no more than 2 times (where ** Ρ <0.01 *** _ρ< 0.οο5, η>=5) .

2A and FIG. 2B are graphs showing the results of detection of total protein content in lung lavage fluid in a model of LPS-induced acute lung injury in mice; wherein, FIG. 2A is the total amount of lung lavage fluid after 4 hours of administration of LPS. Protein concentration determination, Figure 2B is the determination of the total protein concentration of the lung lavage fluid after 12 hours of LPS administration.

[0044] The results of measuring the total protein content in the pulmonary lavage lotion of FIG. 2A and FIG. 2B show that the estimatum (Bestatin)

(I0mg/kg) The LPS-induced increase in total lung protein concentration was approximately 50% (Fig. 2A: 4 吋) and nearly 90% (Fig. 2B: 24 吋).

[0045] These results indicate that Bestatin has a significant inhibitory effect on LPS-induced neutrophil infiltration in mice.

[0046] Example 2. Anti-inflammatory effect of Bestatin 3⁄4 _:

[0047] This example uses the Raw264.7 mouse macrophage cell line as an example to compare the effects of Bestatin and its two derivative compounds on the expression of inflammatory factor NO induced by LPS, including 24 hours of LPS stimulation. The expression of iNOS protein in cells after sputum (Fig. 3A and Fig. 3B) and the amount of NO released from the cell culture supernatant (Fig. 4).

[0048] The structural formula of Bestatin according to this embodiment is as follows:

[0050] As shown in FIG. 3A and FIG. 3B, LPS-induced detection of protein expression levels of iNOS in Raw264.7 cells.

Raw264.7 cells were seeded in a 12-well plate at a density of 3.5× ^{10 5} /well, cultured overnight with 1640 medium containing 10% fetal bovine serum, and Bestatin was pre-incubated at a concentration of 0.2 mg/ml. For 30 minutes, the control group was an equal volume of medium, and 24 hours of induction was induced by stimulation with Olg/ml of LPS. The supernatant was collected and lysed for immunoblot analysis. Figure 3A shows the results of two immunoblots of iNOS protein expression, β-actin as a control; Figure 3B shows the quantitative analysis of iNOS protein expression, the optical density ratio of iNOS and β-actin was calculated by Image J software, and three independent experiments were performed. The results were calculated for the mean standard error, and the results of the Bestatin and the LPS group were compared for significant difference analysis, where ** Ρ < 0.01.

[0051] As shown in FIG. 4, the Griess method was used to detect the amount of NO released from the culture supernatant. The cell culture and stimulation induction were as described in Fig. 3A and Fig. 3B, and the culture supernatant was taken, and the amount of NO released therein was detected by a kit. The mean standard error was calculated from the results of three independent experiments. The results of Bestatin and LPS were compared, and ** Ρ <0.01, *** Ρ <0.005.

[0052] From the results of FIG. 3Α, FIG. 3Β, and FIG. 4, it was found that Bestatin has a protein expression level in LPS-induced Raw264.7 cells and a release amount of LPS-induced inflammatory factor NO. More than 50% reduction

Accordingly, the present invention provides a novel use of Bestatin, which is useful as an active ingredient in the preparation of a pharmaceutical preparation for preventing and treating inflammatory diseases and infectious diseases associated with inflammation. Accordingly, the present invention also provides a pharmaceutical preparation for preventing and treating an inflammatory disease and an infectious disease associated with inflammation, comprising a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable carrier or excipient, said activity The ingredient is umbrel, its optical isomer or a pharmaceutically acceptable salt thereof. Further preferably, preferably, the pharmaceutical preparation comprises a tablet, a capsule, an oral solution, a granule or a dosage form. Injection (such as lyophilized powder injection).

The specific embodiments of the present invention have been described in detail above, but by way of example only, the invention is not limited to the specific embodiments described above. Any equivalent modifications and substitutions of the present invention are also within the scope of the invention. Accordingly, equivalent changes and modifications may be made without departing from the spirit and scope of the invention.

Claims

Claim

[Claim 1] The use of umbrel as an active ingredient in the preparation of a pharmaceutical preparation for preventing and treating an inflammatory disease and an infectious disease associated with inflammation, wherein the chemical structural formula of ursin is:

[Claim 2] The use according to claim 1, wherein the inflammatory disease and infectious diseases associated with inflammation include bacterial infection, uncomplicated cystitis, bronchitis, trauma, postoperative inflammatory reaction, Accidental injury, myocardial infarction, tuberculosis or sarcoidosis, sepsis, metastatic tumor, active rheumatoid arthritis, seronegative spinal arthritis, immune vasculitis, rheumatic polymyopathy, localized gyrus Inflammation of enteritis and deep vein thrombosis

[Claim 3] A pharmaceutical preparation for preventing and treating an inflammatory disease and an infectious disease associated with inflammation, comprising a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable carrier or excipient, The active ingredient is umbrel, its optical isomer or a pharmaceutically acceptable salt thereof.

[Claim 4] The pharmaceutical preparation according to claim 3, wherein the pharmaceutical preparation comprises a tablet, a capsule, an oral solution, a granule or an injection.