WO2016197442A1 - 乌苯美司在制备用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂中的应用 - Google Patents

乌苯美司在制备用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂中的应用 Download PDF

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WO2016197442A1
WO2016197442A1 PCT/CN2015/086002 CN2015086002W WO2016197442A1 WO 2016197442 A1 WO2016197442 A1 WO 2016197442A1 CN 2015086002 W CN2015086002 W CN 2015086002W WO 2016197442 A1 WO2016197442 A1 WO 2016197442A1
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inflammation
pharmaceutical preparation
bestatin
lps
active ingredient
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French (fr)
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钱峰
戈梅
何慧琼
罗敏玉
夏兴
饶敏
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Shanghai Laiyi Center For Biopharmaceutical R&D
Shanghai Jiao Tong University
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Shanghai Laiyi Center For Biopharmaceutical R&D
Shanghai Jiao Tong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate

Definitions

  • 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.
  • 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)
  • LPS Lipopolysaccharides
  • TLRs Toll-like receptors
  • 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- ⁇ , IL-6, IL- ⁇ ).
  • LPS can be regarded as the main pathogenic factor of bacteria and plays an important role in
  • 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).
  • 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.
  • 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.
  • 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:
  • 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 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.
  • 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
  • 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 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.
  • 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.
  • 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 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
  • 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, and the total protein content in the lung lavage lotion was detected, and the activity of myeloperoxidase (MPO) in the lung tissue was detected.
  • MPO myeloperoxidase
  • 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.
  • the cells were lysed and collected, denatured, SDS electrophoresis 80V 2 ⁇ , transfected 80V 1 ⁇ , 5% milk blocked half ⁇ , 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.
  • TBST buffer Tris-Buffered Saline and Tween 20
  • 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.
  • mice 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 50 ⁇ 1;
  • LPS inflammation group tracheal injection 5mg / kg LPS 50 ⁇ 1;
  • LPS + umbryiz treatment group two times before the tracheal injection of LPS (5 mg / kg, about 50 ⁇ 1) 12 hours and 10 minutes, two intraperitoneal injections of umbrel (10 mg / kg each time, about 200 ⁇ l) ;
  • 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.
  • 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 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.
  • 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.
  • 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, ⁇ -actin as a control
  • Figure 3B shows the quantitative analysis of iNOS protein expression, the optical density ratio of iNOS and ⁇ -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 ** ⁇ ⁇ 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. 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 ** ⁇ ⁇ 0.01, *** ⁇ ⁇ 0.005.
  • 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
  • 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.
  • 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.
  • the pharmaceutical preparation comprises a tablet, a capsule, an oral solution, a granule or a dosage form. Injection (such as lyophilized powder injection).

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Abstract

本发明提供了一种乌苯美司（Bestatin）的新应用，即其作为活性成分在制备用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂中的应用。本发明还提供了一种用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂，含有治疗有效量的活性成分和药学上可接受的载体或赋形剂，所述活性成分为乌苯美司、其旋光异构体或其药物学上可接受的盐。

Description

乌苯美司在制备用于预防和治疗炎症疾病和与炎症相关 的感染疾病的药物制剂中的应用 技术领域
本发明涉及乌苯美司作为活性成分在制备用于预防和治疗炎症疾病和与炎症相 关的感染疾病的药物制剂中的应用。
背景技术
炎症是一种十分常见而又重要的基本病理过程， 任何能够弓 I起组织损伤的因素 ， 如感染、 非感染性组织损伤 （如创伤、 手术等） 均可导致炎症的发生， 且这 些炎症具有很多相似的特征 (Barton GM. A calculated response: control of inflammation by the innate immune system. J. Clin. Invest., 2008, 118: 413-420 以细菌感染导致的炎症为例， 细菌分泌的内毒素 （细菌脂多糖 lipopolysaccharides ，简称 LPS) 是细菌致病的主要因素， 可引起机体的一系列炎症反应。 LPS诱导的 炎症信号与 Toll样受体 （TLRs) 家族等有关。 当机体受到细菌感染吋， LPS作用 于细胞膜上 TLRs受体， 通过胞内信号传递， 级联基因表达发生变化， LPS可刺 激体内多种细胞因子合成和释放炎性因子 （如 NO， TNF-α, IL-6, IL-Ιβ等） ， 导致全身性炎症反应发生， 引起中毒性休克、 全身炎症反应综合征 （SIRS) 和 多器官功能障碍综合征 （MODS) 等炎症疾病 （王晓东， 内毒素中和蛋白及其在 脓毒症防治中的作用 [J]， 国外医学生理、 病理科学与临床分册， 2001,21(2): 14 4-146； 丽颖、 王兴鹏， 阻断内毒素信号传导通路治疗脓毒症或脓毒性休克的研 究进展 [J]， 中华急诊医学杂志， 2003， 12(2)： 135-137； 王颖、 郭在晨， 感染性 休克的治疗 [J]， 中华实用儿科临床杂志， 2007， 22(6)： 403-405； 张雪梅、 熊焕 章， LPS诱导的炎症反应信号传导通路研究进展， 中国兽医杂志， 2010， 46(7)： 45-47) 。 因此， LPS可视为细菌的主要致病因子， 在细菌感染的发病机理中起 着十分重要的作用。 同样， 在组织损伤吋， 机体会分泌大量细胞因子以修复损 伤， 但当反应过度吋， 也会引发 SIRS、 MODS等炎症疾病。
乌苯美司 （Ubenimex, 又名 Bestatin) 是常见的抗肿瘤辅助药物， 可增强免疫 功能， 用于抗癌化疗、 放疗的辅助治疗等 （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, D.T., Voelkel. N.F. & Fitzpatrick, F.A. (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. Cancer
Figure imgf000004_0001
[0005] 以前的研究表明， 乌苯美司具有良好的体内免疫增强作用， 如能增强 T细胞和 杀伤性 NK细胞活性和功能等， 但对在如细菌弓 I起的急性炎症中的作用尚未见报 道。
技术问题
[0006] 本发明的发明人在对乌苯美司 （Bestatin) 的功能做进一步研究中发现， 其对 于 LPS引起的急性炎症具有明显的抑制作用。 因此， 本发明的目的在于提供一种 乌苯美司 （Bestatin) 作为活性成分在制备用于预防和治疗炎症疾病和与炎症相 关的感染疾病的药物制剂中的新应用。
问题的解决方案
技术解决方案
[0007] 为实现上述目的， 本发明采用以下技术方案： [0008] 本发明的第一个方面是提供一种乌苯美司 （Bestatin) 作为活性成分在制备用 于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂中的应用， 所述乌 苯美司 （Bestatin) 的化学结构式为：
Figure imgf000005_0001
[0010] 其中， 所述炎症疾病和与炎症相关的感染疾病包括细菌感染、 无并发症的膀胱 炎、 支气管炎、 外伤、 术后炎症反应、 意外伤害、 心肌梗塞、 结核病或肉状瘤 病、 脓毒症、 转移性肿瘤、 活动期类风湿性关节炎、 血清阴性的脊椎关节炎、 免疫性血管炎、 风湿性多肌病、 局限性回肠炎、 深部静脉血栓形成的炎症等。
[0011] 发明人通过构建细菌内毒素 LPS诱导的小鼠急性肺损伤模型， 腹腔给予乌苯美 司 （Bestatin) 药物， 检测药物对 LPS引起的急性肺损伤的作用， 以及检测 LPS诱 导的炎性因子 NO表达和释放， 检测后发现， LPS诱导的炎性因子 NO的释放量急 剧减少。
[0012] 还通过髓过氧化物酶 （Myeloperoxidase, ΜΡθ) 活性检测、 肺盥洗液中总蛋白 含量检测， 检验嗜中性粒细胞的浸润情况， 以评价 LPS诱导的急性肺损伤严重程 度。 检测结果表明， 乌苯美司 （Bestatin) 对 LPS诱导的小鼠肺部嗜中性粒细胞 浸润有抑制作用。
[0013] 本发明的第二个方面是提供一种用于预防和治疗炎症疾病和与炎症相关的感染 疾病的药物制剂， 含有治疗有效量的活性成分和药学上可接受的载体或赋形剂 ， 所述活性成分为乌苯美司 （Bestatin) 、 其旋光异构体或其药物学上可接受的
[0014] 优选地， 所述药物制剂的剂型包括片剂、 胶囊剂、 口服液、 颗粒剂或注射剂 （ 如冻干粉针剂） 。 [0015] 药物学上可接受的盐可以是药理学上和其药物学上都接受的。 药理学上和其药 物学上可接受的盐可以是碱金属盐或碱土金属盐， 优选钠盐、 钾盐、 镁盐或钙
[0016] 所述药物制剂涉及经肠的 （例如口服、 舌下含服或直肠给药） 、 胃肠外的或局 部的 （如透皮药物制剂） 剂型。 不与活性成分反应的有机或无机物可以作为载 体， 如水、 油、 苯甲醇、 聚乙二醇、 甘油三乙酸酯或其它脂肪酸甘油酯、 明胶 、 卵磷脂、 环糊精、 乳二糖或淀粉等糖类、 硬脂酸镁、 滑石或纤维素。 口服用 药优选片剂、 糖衣丸剂、 胶囊、 粉、 糖浆、 浓缩剂或滴剂， 直肠给药优选栓剂 ， 胃肠外给药优选水溶液或油溶液， 或冻干剂。 也可以使用混悬剂、 乳剂或植 入剂， 局部用药可以用贴剂或乳膏剂。 胃肠外使用的药物制剂包括活性化合物 的无菌含水或无水注射液， 在此优选与受体血液等渗的溶液。
[0017] 这些药物制剂可以含有稳定剂、 控制药学活性化合物释放的添加剂、 抗氧剂、 缓冲剂、 抑菌剂和用于制备等渗溶液的佐剂。 含水和无水的无菌混悬剂可以含 有混悬添加剂和增稠剂。 药物制剂可以分装在单剂量或多剂量容器如密封剂瓶 中， 也可以作为经冻干的制品保存， 如需要， 使用吋可用无菌液体如水或盐溶 液配制。 还可以以同样的方式使用无菌粉剂、 颗粒剂或片剂。
[0018] 所述药物制剂可用于人和动物中预防和治疗炎症和炎症相关疾病。
发明的有益效果
对附图的简要说明
附图说明
[0019] 图 1为构建的 LPS诱导的小鼠急性肺损伤模型中， 髓过氧化物酶 （MPO) 活性 检测结果图表。
[0020] 图 2A和图 2B为构建的 LPS诱导的小鼠急性肺损伤模型中， 肺盥洗液中总蛋白含 量检测结果图表； 其中， 图 2A为 LPS给药 4小吋后肺盥洗液的总蛋白浓度测定， 图 2B为 LPS给药 12小吋后肺盥洗液的总蛋白浓度测定。
[0021] 图 3A为 LPS诱导的 Raw264.7细胞中， iNOS蛋白表达的两次免疫印迹结果图表
[0022] 图 3B为 LPS诱导的 Raw264.7细胞中， iNOS蛋白表达的量化分析图表； 本发明的实施方式
[0024] 下面通过具体实施例对本发明进行详细和具体的介绍， 以使更好的理解本发明
， 但是下述实施例并不限制本发明范围。
[0025] 以下实施例中所使用的检测方法的具体操作如下：
[0026] 1、 构建 LPS诱导的急性炎症模型：
[0027] 将 C57/BL小鼠分组， 每组至少 5只， 称重， 分别用生理盐水 （阴性对照组） 和 5 mg/kg的 LPS (炎症组） 进行气管给药， 在 LPS注射前 12小吋， 10分钟后以及 12 小吋后给予乌苯美司 （Bestatin) 腹腔注射 （阴性对照组除外） ， 0、 4、 24小吋 后处死， 心脏取血， 用 lml的 PBS反复冲洗肺部， 收集盥洗液后取肺组织， 检测 肺盥洗液中总蛋白的含量， 进行肺组织髓过氧化物酶 （MPO) 活性的检测。
[0028] 2、 荧光定量 PCR (QPCR) ：
[0029] Trizol裂解细胞， 抽提 RNA， 逆转录 PCR获得 cDNA， 以此为模板进行荧光定量 PCR, 检验药物作用前后诱导的炎症相关细胞因子和炎性分子等的 DNA转录水 平变化。
[0030] 3、 免疫印迹 （Western Blot) ：
[0031] 裂解并收集细胞， 变性处理， SDS电泳 80V 2小吋， 转膜 80V 1小吋， 5%牛奶封 闭半小吋， 4°C孵育一抗过夜， TBST缓冲液 (Tris-Buffered Saline and Tween 20 ) 洗膜 3次， 常温孵育二抗 1小吋， TBST洗膜 3次， Odyssey远红外观测仪上观察 药物作用前后诱导的一氧化氮合成酶 iNOS蛋白表达水平变化等。
[0032] 4、 Griess法检测 NO释放量：
[0033] NO在水溶液中极易形成 NO 2-， 在酸性条件下， NO 2 -与重氮盐磺胺发生重氮反 应， 生成重氮化合物， 进一步与萘基乙烯基二胺发生偶合反应， 其产物浓度与 N 0 2-浓度具有线性关系， 在 540-560nm处有最大吸收峰。 利用此原理可检测细胞 上清中 NO的释放量。
[0034] 棚列 1、 (Bestatin) 隱 LPSi秀 至删市咅 耆 瞧田隱闰：
[0035] 将 C57/BL小鼠 30只， 随机分成 3组， 每组 10只， 称重， 分别进行如下操作： [0036] 阴性对照组： 气管注射 0.9%的生理盐水 50μ1;
[0037] LPS炎症组： 气管注射 5mg/kg的 LPS 50μ1;
[0038] LPS+乌苯美司治疗组： 在气管注射 LPS (5mg/kg， 约 50μ1) 前 12小吋和 10分钟 后， 两次腹腔注射乌苯美司 （每次 10mg/kg， 约 200μ1) ；
[0039] 0、 4、 24小吋后处死， 心脏取血， 同吋取小鼠肺组织， 分别进行肺组织髓过氧 化物酶 （ΜΡΟ) 活性的检测和肺盥洗液中总蛋白的含量检测， 检测结果分别如 图 1、 图 2Α、 图 2Β所示。
[0040] 通过上述三种检测来检验嗜中性粒细胞的浸润情况， 以评价 LPS诱导的急性肺 损伤严重程度。
[0041] 图 1为构建的 LPS诱导的小鼠急性肺损伤模型中， 髓过氧化物酶 （MPO) 活性 检测结果图表。
[0042] 从图 1的检测结果显示， LPS (5mg/kg) 气管给药 4小吋和 24小吋后， 与生理盐 水对照组 （Saline) 相比， 小鼠肺部 MPO活性均有 3倍左右的增加， 而在乌苯美 司 （Bestatin) (lOmg/kg) 干预下， MPO活性增加不超过 2倍 （其中， ** Ρ<0.01 *** ρ<0.οο5， η>=5) 。
[0043] 图 2Α和图 2Β为构建的 LPS诱导的小鼠急性肺损伤模型中， 肺盥洗液中总蛋白含 量检测结果图表； 其中， 图 2A为 LPS给药 4小吋后肺盥洗液的总蛋白浓度测定， 图 2B为 LPS给药 12小吋后肺盥洗液的总蛋白浓度测定。
[0044] 从图 2A和图 2B的肺盥洗液中总蛋白的含量测定结果显示， 乌苯美司 （Bestatin
) (I0mg/kg) 对 LPS诱导的肺部总蛋白浓度增加有约 50% (图 2A: 4小吋） 和接 近 90% (图 2B: 24小吋) 的抑制。
[0045] 上述这些检测结果说明， 乌苯美司 （Bestatin) 对 LPS诱导的小鼠肺部嗜中性粒 细胞浸润有明显的抑制作用。
[0046] 实施例 2、 乌苯美司 (Bestatin) 的抗炎效 ¾:
[0047] 本实施例以 Raw264.7小鼠巨噬细胞系为例， 比较乌苯美司 （Bestatin) 及其两 个衍生化合物对 LPS诱导的炎性因子 NO表达的影响， 包括 LPS刺激 24小吋后细胞 中 iNOS蛋白的表达 （图 3A和图 3B) 和细胞培养上清中 NO的释放量 （图 4) 。
[0048] 本实施例涉及的乌苯美司 （Bestatin) 的结构式如下：
Figure imgf000009_0001
[0050] 如图 3A和图 3B所示为 LPS诱导的 Raw264.7细胞中 iNOS的蛋白表达水平检测。
将 Raw264.7细胞以 3.5X10 5/孔的密度接种于 12孔板中， 用含 10%胎牛血清的 1640 培养液培养过夜， 乌苯美司 （Bestatin) 以 0.2mg/ml的浓度提前孵育 30分钟， 对 照组为等体积培养液 （medium) ， 用 O.l g/ml的 LPS刺激诱导 24小吋。 收集上清 ， 裂解细胞进行免疫印迹分析。 其中， 图 3A为 iNOS蛋白表达的两次免疫印迹结 果， β-actin为对照； 图 3B为 iNOS蛋白表达的量化分析， iNOS和 β-actin的光密度 比值用 Image J软件测算， 取三次独立实验的结果计算平均值标准误差， 比较乌 苯美司 （Bestatin) 结果与 LPS组， 进行显著性差异分析， 其中 ** Ρ<0.01。
[0051] 如图 4所示为 Griess法检测培养上清中 NO释放量。 细胞培养和刺激诱导如图 3A 和图 3B所述， 取培养上清， 试剂盒检测其中 NO的释放量。 取三次独立实验的结 果计算平均值标准误差， 比较乌苯美司 （Bestatin) 结果与 LPS组， 进行显著性 差异分析， 其中 ** Ρ<0.01， *** Ρ<0.005。
[0052] 由图 3Α、 图 3Β、 图 4检测结果看出， 乌苯美司 （Bestatin) 对 LPS诱导的 Raw264 .7细胞中的蛋白表达水平和 LPS诱导的炎性因子 NO的释放量均有超过 50%的减少
[0053] 因此， 本发明提供了一种乌苯美司 （Bestatin) 的新应用， 即其作为活性成分 在制备用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂中的应用 。 由此， 本发明还提供了一种用于预防和治疗炎症疾病和与炎症相关的感染疾 病的药物制剂， 含有治疗有效量的活性成分和药学上可接受的载体或赋形剂， 所述活性成分为乌苯美司、 其旋光异构体或其药物学上可接受的盐。 进一步优 选地， 优选地， 所述药物制剂的剂型包括片剂、 胶囊剂、 口服液、 颗粒剂或注 射剂 （如冻干粉针剂） 。
以上对本发明的具体实施例进行了详细描述， 但其只是作为范例， 本发明并不 限制于以上描述的具体实施例。 对于本领域技术人员而言， 任何对本发明进行 的等同修改和替代也都在本发明的范畴之中。 因此， 在不脱离本发明的精神和 范围下所作的均等变换和修改， 都应涵盖在本发明的范围内。

Claims

权利要求书
[权利要求 1] 乌苯美司作为活性成分在制备用于预防和治疗炎症疾病和与炎症相关 的感染疾病的药物制剂中的应用， 所述乌苯美司的化学结构式为：
Figure imgf000011_0001
[权利要求 2] 根据权利要求 1所述的应用， 其特征在于， 所述炎症疾病和与炎症相 关的感染疾病包括细菌感染、 无并发症的膀胱炎、 支气管炎、 外伤、 术后炎症反应、 意外伤害、 心肌梗塞、 结核病或肉状瘤病、 脓毒症、 转移性肿瘤、 活动期类风湿性关节炎、 血清阴性的脊椎关节炎、 免疫 性血管炎、 风湿性多肌病、 局限性回肠炎、 深部静脉血栓形成的炎症
[权利要求 3] —种用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂， 其特征在于， 含有治疗有效量的活性成分和药学上可接受的载体或赋 形剂， 所述活性成分为乌苯美司、 其旋光异构体或其药物学上可接受 的盐。
[权利要求 4] 根据权利要求 3所述的药物制剂， 其特征在于， 所述药物制剂的剂型 包括片剂、 胶囊剂、 口服液、 颗粒剂或注射剂。
PCT/CN2015/086002 2015-06-12 2015-08-04 乌苯美司在制备用于预防和治疗炎症疾病和与炎症相关的感染疾病的药物制剂中的应用 Ceased WO2016197442A1 (zh)

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