WO2016197443A1

WO2016197443A1 - New use of 3-amino-2-hydroxy-4-phenyl-valyl-isoleucine

Info

Publication number: WO2016197443A1
Application number: PCT/CN2015/086003
Authority: WO
Inventors: 戈梅; 钱峰; 饶敏; 何慧琼; 王涛; 罗敏玉
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: CN104984318A; CN104984318B

Abstract

Disclosed is a new use of 3-amino-2-hydroxy-4-phenyl-valyl-isoleucine, i.e. the use of same as an active ingredient in preparing a pharmaceutical preparation for preventing and treating inflammatory diseases and infectious diseases associated with inflammation, and a pharmaceutical preparation for preventing and treating inflammatory diseases and infectious diseases associated with inflammation. The pharmaceutical preparation containing a therapeutically effective amount of the active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient is 3-amino-2-hydroxy-4-phenyl-valyl-isoleucine, optical isomers thereof or a pharmaceutically acceptable salt thereof.

Description

New application of 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine

[0001] The present invention belongs to the field of medicine, and in particular, relates to 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine as an active ingredient in preparation for prevention and treatment of inflammatory diseases and inflammation New applications in pharmaceutical preparations related to infectious diseases.

Background technique

[0002] 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 cause inflammation, and These inflammations have many similar features (Barton GM. A calculated response: control of inflammation by the innate immune system. J. Clin. Invest., 2008, 118: 413-420, for example, inflammation caused by bacterial infection, bacterial secretion Endotoxin (lipopolysaccharides, abbreviated as LPS) is a major cause of bacterial pathogenesis and can cause a series of inflammatory reactions in the body. LPS-induced inflammatory signals are associated with Toll-like receptors (TLRs), etc. When the body is infected with bacteria吋, LPS acts on TLRs receptors on the cell membrane, and 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., causes systemic inflammatory reactions, causing toxic shock, systemic inflammatory response syndrome (SIRS) and Inflammatory diseases such as multiple organ dysfunction syndrome (MODS) (Wang Xiaodong, Endotoxin Neutralizing Protein and Its Role in Prevention and Treatment of Sepsis[J], Foreign Medical Physiology, Pathology 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; Ying, 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, which plays an important role in the pathogenesis of bacterial infection. Similarly, in tissue damage, the body will secrete a large number of cells. Factor to repair damage, but when the reaction is excessive, it can also cause inflammatory diseases such as SIRS and MODS.

[0003] 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] 3-Amino-2-hydroxy-4-phenyl-prolyl-isoleucine is a novel derivative similar in structure to Bestatin, and the applicant of the present invention is in the application number 201110076554.6 For the first time, the Chinese invention patent discloses 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, which has the chemical structural formula:

[0006]

[0007] The application of the compound 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine as an aminopeptidase inhibitor is further disclosed in Chinese Patent Application No. 201110076555.0, the activity thereof It is superior to Be statin and can be used as a tumor treatment or adjuvant therapy. It has better in vivo immune enhancement, such as enhancing the activity and function of T cells and killer NK cells, but The role of acute inflammation has not been reported.

technical problem

The inventors of the present invention have further studied the function of the compound 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, and found that the compound has obvious effects on acute inflammation caused by LPS. Inhibition. Accordingly, it is an object of the present invention to provide a 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine as an active ingredient in the preparation of an inflammatory inhibitor or a medicament for the prevention and treatment of an inflammatory disease. New applications in formulations. Problem solution

Technical solution

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

A first aspect of the present invention provides 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine as an active ingredient in the preparation of a medicament for the prevention and treatment of inflammatory diseases and inflammation For use in a pharmaceutical preparation for infecting a disease, the chemical structural formula of the 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine is:

[0012] 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.

[0013] The inventors administered a bacterial endotoxin LPS-induced acute lung injury model in mice, intraperitoneal administration of 3-amino- 2-hydroxy-4-phenyl-prolyl-isoleucine drug, detecting the effect of drugs on acute lung injury caused by LPS, and testing for 3-amino-2-hydroxy-4-phenyl-prolyl- The effect of isoleucine on the expression of cytokines, inflammatory molecules and the like. In the same manner, the anti-inflammatory effect of 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine was also evaluated, and the inventors selected the mouse mononuclear-macrophage cell line Raw264.7 for testing. The expression and release of inflammatory factor NO induced by LPS were detected, and it was found that the release of inflammatory factor NO induced by LPS was greatly reduced.

[0014] The neutrophil infiltration was also observed and tested by myeloperoxidase (ΜΡθ) activity assay, total protein content detection in lung lavage lotion, and HE stained tissue sections to evaluate LP S-induced The severity of acute lung injury. The results showed that ₃ -amino-2-hydroxy-4-phenyl-prolyl-isoleucine 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 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, an optical isomer thereof or a pharmaceutically acceptable salt thereof.

[0016] Preferably, the pharmaceutical preparation comprises a tablet, a capsule, an oral solution, a granule or an injection (such as a lyophilized powder injection).

[0017] Pharmaceutically acceptable salts can be 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.

[0019] These pharmaceutical preparations may contain stabilizers, additives to control the release of pharmaceutically active compounds, antioxidants, buffers, bacteriostats, and adjuvants for the preparation of isotonic solutions. Aqueous and anhydrous sterile suspensions may contain There are suspension additives and thickeners. The pharmaceutical preparations may be dispensed in single or multi-dose containers, such as sealant bottles, or as lyophilized preparations, if desired, using sterile liquids such as water or saline solutions. 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 inflammation and inflammation-related diseases 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 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 pulmonary 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 3 shows the presence or absence of 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine (LYR) after 4 hours of LPS induction.

In the case of M03), a graph of the expression levels of cytokines and inflammatory factors in mouse lung tissue.

4 is a graph showing the results of immunoblotting of lNOS-induced expression of iNOS and COX-2 protein in LPS-induced lung tissue by 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine (LYRM03).

5A is a graph showing the results of two immunoblotting results of iNOS protein expression in LPS-induced Raw264.7 cells.

Figure 5B is a quantitative analysis chart of iNOS protein expression in LPS-induced Raw264.7 cells;

6 is a graph showing the detection results of the NO release amount in the culture supernatant by the Griess method.

Embodiments of the invention

The present invention will be described in detail and by the following detailed description of the preferred embodiments of the invention.

The present invention provides a pharmaceutical preparation for preparing an infectious disease for preventing and treating inflammatory diseases and inflammation, as an active ingredient of 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine. Application in . In the present example, the inventors numbered 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine as LYRM03. [0030] The specific operation of the detection method used in the following embodiments is as follows:

[0031] 1. Constructing an acute inflammation model induced by LPS:

[0032] 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, the total protein content in the lung lavage fluid was detected, the activity of myeloperoxidase (MPO) in the lung tissue was detected, and the tissue sections were prepared, and the cytokines and inflammation in the lung tissue were detected by real-time PCR. The expression of factors (mainly TNF-ot, IL-6, IL-Ιβ, IL-12, etc.).

[0033] 2. Fluorescence quantitative PCR (QPCR):

[0034] 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.

[0035] 3. Western Blot:

[0036] Lysis and collection of cells, denaturing treatment, SDS electrophoresis 80V 2 吋, transfection 80V 1 吋, 5% milk closed 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.

[0037] 4, Griess method to detect the amount of NO released:

[0038] 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 has a maximum absorption peak at 540-560 nm. Using this principle, the amount of NO released from the cell supernatant can be detected.

[0039] WM 3- 3⁄4-2-hydroxy 3⁄4-4-benzene 3⁄4 -decanoyl-iso-t-acid (LYRM03) reduces LPS induction to the market 咅瞧闰闰闰:

[0040] 30 C57/BL mice were randomly divided into 3 groups, 10 rats in each group, weighed, and the following operations were performed respectively: [0041] Negative control group: tracheal injection 0.9% physiological saline 50 μl;

[0042] LPS inflammation group: tracheal injection of 5 mg / kg LPS 50μ1; [0043] LPS+LYRM03 treatment group: LYRM03 (10 mg/kg each time, about 200 μl) was intraperitoneally injected 12 hours before and 10 minutes after intratracheal injection of LPS (5 mg/kg, about 50 μl);

[0044] 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, Figure 2, and Figure 3, respectively.

[0045] Neutrophil infiltration was examined by the above three tests to evaluate the severity of LPS-induced acute lung injury.

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

[0047] From the test results of FIG. 1, after LPS (5 mg/kg) tracheal administration for 4 hours and 24 hours, the lung MPO activity was 3 times higher than that of the saline control group (Saline). The increase in left and right, and the intervention of LYRM0 3 (I0mg/kg), the MPO activity increased by no more than 2 times (where ** Ρ <0.01, *** Ρ

<0.005,

.

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 pulmonary 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.

[0049] The results of measuring the total protein content in the lung lavage lotion of FIGS. 2A and 2B show that LYRM03 (10 mg/kg)

Increased LPS-induced increase in total lung protein concentration by approximately 50% (Fig. 2A: 4 吋) and close to 100% (Fig. 2)

2B: 24 hours) inhibition.

Example 2, 3- 3⁄4-2-hydroxy 3⁄4-4-benzene 3⁄4-quinoyl-iso-t-acid (LYRM03) Inhibition of LPS-induced fine sputum factor, inflammatory molecular table i large:

Referring to the method of Example 1, after inducing 4 hours of LPS (5 mg/kg), the right lobe lung tissue of the mouse was taken, and in the presence or absence of LYRM03, the cells in the lung tissue of the mouse were detected by real-time PCR. A graph of the expression levels of factors and inflammatory factors, the results are shown in Figure 3.

The method of administration was the same as that described in Example 1. 4 hours and 24 hours after LPS (5mg/kg) induction, the largest lung lobe on the left side of the mouse was taken, lysed after homogenization, total protein concentration was determined, and iNOS and COX-2 were detected by western blot. Protein expression, actin was loaded and the results are shown in Figure 4.

[0053] The results of the detection of the expression levels of cytokines and inflammatory factors from FIG. 3 showed that 3-6 hours after LPS stimulation, Cytokines and inflammatory factors (such as TNF-a, IL-6, IL-Ιβ, cyclooxygenase CO X-2, etc.) in the lungs and blood of mice have a significant increase in expression, which is detected by real-time PCR. The application of LYRM03 can effectively inhibit the expression of these inflammatory factors in mouse lung tissue. For example, LYRM03 (I0mg/kg) was injected 12 hours before LPS stimulation and 10 minutes later. The expression of TNF-α in the lung tissue of mice was 3 1.3% in the LPS group. The expression of IL-Ιβ was in the LPS group. 36.8<3⁄4, the expression level of cyclooxygenase COX-2 was 59% in the LPS group, and the expression levels of other cytokines were also significantly reduced.

[0054] The immunoblot assay of Figure 4 showed that the expression levels of NO synthase iNOS and COX-2 protein in mouse lung tissue decreased under the influence of LYRM03.

[0055] The above results indicate that LYRM03 can alleviate the acute inflammatory injury response of bacterial endotoxin LPS, including neutrophil infiltration and expression of inflammatory molecules, indicating that LYRM03 has an inflammatory therapeutic effect.

[0056] Example 3, 3- 3⁄4-2-hydroxy 3⁄4-4-benzene 3⁄4-quinoyl-iso-acid (LYRM03) anti-inflammatory effect 3⁄4 _:

[0057] This example uses the Raw264.7 mouse macrophage cell line as an example to compare the effect of LYRM03 on the expression of inflammatory factor NO induced by LPS, including the expression of iNOS protein in cells after 24 hours of LPS stimulation (Fig. 5A and Figure 5B) and the amount of NO released in the cell culture supernatant (Figure 6).

[0058] As shown in Figures 5A and 5B, 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 LYRM03 was incubated for 30 minutes at a concentration of 0.2 mg/ml. An equal volume of medium was induced by stimulation with Olg/ml of LPS for 24 hours. The supernatant was collected and lysed for immunoblot analysis. Figure 5A shows the results of two immunoblots of iNOS protein expression, β-actin as a control; Figure 5B shows the quantitative analysis of iNOS protein expression, the optical density ratio of iNOS and β-a _C ti _n using Im _a g _e J software Calculate, take the results of three independent experiments to calculate the standard error of the mean, compare the LYRM03 results with the L PS group, and make a significant difference analysis, where ** Ρ <0.01.

[0059] As shown in FIG. 6, 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. 5A and Fig. 5B, 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 LYRM03 results were compared with the LPS group for significant difference analysis, <0.05, * <0.01.

[0060] As seen from the results of the examination of FIG. 5A, FIG. 5B, and FIG. 6, 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine ( LYRM03) Protein expression levels in LPS-induced Raw264.7 cells and LPS-induced inflammatory factors

The release of NO has a reduction of more than 50%.

Accordingly, the present invention provides a novel use of 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, which is prepared as an active ingredient for the prevention and treatment of inflammatory diseases and Use in pharmaceutical preparations for inflammation-related infectious diseases. 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 component is 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, an optical isomer thereof or a pharmaceutically acceptable salt thereof. Further preferably, preferably, the pharmaceutical preparation comprises a tablet, a capsule, an oral solution, a granule or an injection (e.g., a 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] 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine as an active ingredient in the preparation of a pharmaceutical preparation for preventing and treating inflammatory diseases and infectious diseases associated with inflammation Application, the chemical structural formula of the 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine 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 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, an optical isomer thereof 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.