WO2016197443A1 - Nouvelle utilisation de 3-amino-2-hydroxy-4-phényl-valyl-isoleucine - Google Patents

Nouvelle utilisation de 3-amino-2-hydroxy-4-phényl-valyl-isoleucine Download PDF

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Publication number
WO2016197443A1
WO2016197443A1 PCT/CN2015/086003 CN2015086003W WO2016197443A1 WO 2016197443 A1 WO2016197443 A1 WO 2016197443A1 CN 2015086003 W CN2015086003 W CN 2015086003W WO 2016197443 A1 WO2016197443 A1 WO 2016197443A1
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Prior art keywords
hydroxy
isoleucine
phenyl
amino
inflammation
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PCT/CN2015/086003
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English (en)
Chinese (zh)
Inventor
戈梅
钱峰
饶敏
何慧琼
王涛
罗敏玉
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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
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Publication of WO2016197443A1 publication Critical patent/WO2016197443A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides

Definitions

  • 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.
  • 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.
  • TLRs Toll-like receptors
  • 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- ⁇ , IL-6, IL- ⁇ , 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
  • Ubenimex also known as Bestatin
  • Bestatin is a common anti-tumor adjuvant that enhances immunity. Function, for anticancer chemotherapy, adjuvant therapy for radiotherapy, etc.
  • 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).
  • 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
  • the Chinese invention patent discloses 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, which has the chemical structural formula:
  • 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
  • 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
  • the chemical structural formula of the 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine is:
  • 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.
  • 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 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.
  • 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.
  • the pharmaceutical preparation comprises a tablet, a capsule, an oral solution, a granule or an injection (such as a lyophilized powder injection).
  • 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.
  • 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.
  • MPO myeloperoxidase
  • FIG. 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.
  • LYR 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine
  • FIG. 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
  • FIG. 6 is a graph showing the detection results of the NO release amount in the culture supernatant by the Griess method.
  • 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.
  • the inventors numbered 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine as LYRM03.
  • the specific operation of the detection method used in the following embodiments is as follows:
  • 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.
  • saline negative control group
  • LPS inflammation group
  • 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- ⁇ , IL-12, etc.).
  • 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.
  • 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.
  • WM 3- 3 ⁇ 4-2-hydroxy 3 ⁇ 4-4-benzene 3 ⁇ 4 -decanoyl-iso-t-acid reduces LPS induction to the market ⁇ ⁇ ⁇ ⁇ ⁇ :
  • LPS inflammation group tracheal injection of 5 mg / kg LPS 50 ⁇ 1;
  • LPS+LYRM03 treatment group LYRM03 (10 mg/kg each time, about 200 ⁇ l) was intraperitoneally injected 12 hours before and 10 minutes after intratracheal injection of LPS (5 mg/kg, about 50 ⁇ l);
  • MPO myeloperoxidase
  • FIG. 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.
  • Example 3 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.
  • 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.
  • Example 3 3- 3 ⁇ 4-2-hydroxy 3 ⁇ 4-4-benzene 3 ⁇ 4-quinoyl-iso-acid (LYRM03) anti-inflammatory effect 3 ⁇ 4 :
  • 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).
  • 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, ⁇ -actin as a control
  • Figure 5B shows the quantitative analysis of iNOS protein expression, the optical density ratio of iNOS and ⁇ -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 ** ⁇ ⁇ 0.01.
  • 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.
  • the release of NO has a reduction of more than 50%.
  • 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.
  • 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.
  • the pharmaceutical preparation comprises a tablet, a capsule, an oral solution, a granule or an injection (e.g., a lyophilized powder injection).

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une nouvelle utilisation de 3-amino-2-hydroxy-4-phényl-valyl-isoleucine, à savoir l'utilisation de celle-ci en tant que substance active dans la préparation d'une préparation pharmaceutique pour la prévention et le traitement de maladies inflammatoires et de maladies infectieuses liées à l'inflammation, et une préparation pharmaceutique pour la prévention et le traitement de maladies inflammatoires et de maladies infectieuses associées à une inflammation. La préparation pharmaceutique contenant une quantité thérapeutiquement efficace de la substance active et un véhicule ou excipient pharmaceutiquement acceptable, la substance active étant la 3-amino-2-hydroxy-4-phényl-valyl-isoleucine, ses isomères optiques ou un sel pharmaceutiquement acceptable de celle-ci.
PCT/CN2015/086003 2015-06-12 2015-08-04 Nouvelle utilisation de 3-amino-2-hydroxy-4-phényl-valyl-isoleucine WO2016197443A1 (fr)

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CN201510323395.3A CN104984318B (zh) 2015-06-12 2015-06-12 3‑氨基‑2‑羟基‑4‑苯基‑缬氨酰‑异亮氨酸的新应用
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102477067A (zh) * 2010-11-19 2012-05-30 上海来益生物药物研究开发中心有限责任公司 3-氨基-2-羟基-4-苯基-缬氨酰-异亮氨酸及其制备方法和应用
CN102631664A (zh) * 2011-01-28 2012-08-15 上海来益生物药物研究开发中心有限责任公司 3-氨基-2-羟基-4-苯基-缬氨酰-异亮氨酸的应用

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US4370318A (en) * 1980-07-07 1983-01-25 Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai Bestatin-related compounds as immunopotentiator
DD282710A5 (de) * 1989-04-27 1990-09-19 Adw Ddr Verfahren zur herstellung von bestatin
JPH05117146A (ja) * 1991-04-19 1993-05-14 Microbial Chem Res Found 抗真菌剤
JPH05310668A (ja) * 1992-05-07 1993-11-22 Meiji Milk Prod Co Ltd 新規ロイシン誘導体並びにそれを含有する抗アレルギー剤及び抗炎症剤

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CN102477067A (zh) * 2010-11-19 2012-05-30 上海来益生物药物研究开发中心有限责任公司 3-氨基-2-羟基-4-苯基-缬氨酰-异亮氨酸及其制备方法和应用
CN102631664A (zh) * 2011-01-28 2012-08-15 上海来益生物药物研究开发中心有限责任公司 3-氨基-2-羟基-4-苯基-缬氨酰-异亮氨酸的应用

Non-Patent Citations (2)

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Title
WANG, TING ET AL.: "A method for Detecting the Binding Activitics of Aminopeptidase N Inhibitors to Tumor Cell by Fluorescein", PROGRESS IN MODERN BIOMEDICINE, vol. 12, no. 4, 28 February 2014 (2014-02-28), pages 634 - 638 and page 636 *
WANG, TING ET AL.: "A method for Detecting the Binding Activities of Aminopeptidase N Inhibitors to Tumor Cell by Fluorescein", PROGRESS IN MODERN BIOMEDICINE, vol. 12, no. 4, 28 February 2014 (2014-02-28), pages 634 - 638 *

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