WO2013188999A1 - 线叶旋覆花内酯a在制备治疗多发性硬化症药物中的应用 - Google Patents

线叶旋覆花内酯a在制备治疗多发性硬化症药物中的应用 Download PDF

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WO2013188999A1
WO2013188999A1 PCT/CN2012/001411 CN2012001411W WO2013188999A1 WO 2013188999 A1 WO2013188999 A1 WO 2013188999A1 CN 2012001411 W CN2012001411 W CN 2012001411W WO 2013188999 A1 WO2013188999 A1 WO 2013188999A1
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extract
lactone
tablet
petroleum ether
inflorescence
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French (fr)
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张卫东
单磊
苏娟
金慧子
李慧梁
沈云亨
徐希科
柳润辉
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Second Military Medical University SMMU
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Priority to EP12879377.5A priority Critical patent/EP2865379B1/en
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Priority to US14/576,164 priority patent/US9278948B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/93—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems condensed with a ring other than six-membered
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • A61K9/2018—Sugars, or sugar alcohols, e.g. lactose, mannitol; Derivatives thereof, e.g. polysorbates
    • 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
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2059—Starch, including chemically or physically modified derivatives; Amylose; Amylopectin; Dextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps

Definitions

  • the invention relates to a traditional Chinese medicine, in particular to the application of the linear inulin a lactone A in the preparation of a medicament for treating multiple sclerosis.
  • MS Multiple sclerosis
  • the disease is a chronic, autoimmune disease that affects normal nerve transmission by destroying the nerve fiber protective layer of myelin in the brain, spinal cord, and optic nerve, leading to physical disability.
  • the disease has become one of the most common and important neurological diseases due to its extremely high recurrence rate and disability rate, chronic disease duration and proneness to young people. Since the peak incidence of the disease is around 30 years old, the serious impact on the productivity and quality of life of the young and middle-aged population also imposes a great burden on the state and the government, restricting social and economic development.
  • Glucocorticoids are mainly administered in the acute phase, and the main treatments for relapsing-remission are: interferon-gamma, glatiramer acetate and the monoclonal antibody natalizumab. Severe patients can also consider autologous stem cell transplantation. Although Western medicine's hormone therapy can alleviate acute symptoms, it has obvious side effects, so it is not recommended for long-term use.
  • the immunomodulators interferon IFN-P, GA and natalizumab are not only expensive, but also long-term use for a single target. Drugs are prone to tolerance.
  • Inula lineariifolia Turcz. (syn. Inula /inariaefo/''a) is a perennial herb of the genus Compositae, a perennial herb, commonly known as a narrow-leaved invertebrate, a leaf-spreading flower, a small flowering Cover flower. It is widely distributed in northeastern, northern, central and eastern China, such as Henan and Hebei. Also distributed in Mongolia, North Korea, Russia (Far East) and Japan. Very common in hillsides, wasteland, roadsides, river banks, etc.
  • the traditional Chinese medicine invertebrate is the head flower of the invertebrate flower or the large flower invertebrate, and the plant whole grass (gold boiled grass) is also used for medicinal purposes.
  • the invertebrate flower is also used as a spinach flower in some parts of China's East China, and as a medicine for lowering, running water, anti-inflammatory, and softening, it was once included in the 1963 edition of the Chinese Pharmacopoeia (Part I), but due to patient service After nausea, vomiting, etc. It has been discontinued before.
  • the present inventors conducted a systematic chemical composition study on the invertebrate flower, and isolated a large number of sesquiterpenoids, and published relevant research results [Li-Yue Nie, Jiang-Jiang Qin, Lan Yan, Yu-Bo Liu,
  • the technical problem to be solved by the present invention is to study the application of the extract of the invertebrate, the invertebrate, in the preparation of a medicament for treating multiple sclerosis.
  • the invention provides the application of the wire-flower inulin a lactone A in the preparation of a medicament for treating multiple sclerosis.
  • the line configuration is as follows:
  • the wire inflorescence lactone A of the present invention is prepared by the following method:
  • the extract is equivalent to lml containing linear leaves of 0.8-1.2g, and the extract is diluted with 1 ⁇ 3 times of water, and then extracted with petroleum ether 0.5 ⁇ 2 times V/V for 3 ⁇ 5 times.
  • Petroleum ether site The above petroleum ether fraction was subjected to silica gel column chromatography, and eluted with a gradient of petroleum ether/ethyl acetate system in a volume ratio of 100:0 to 1:1, and detected by thin layer chromatography.
  • the invention provides the animal efficacy test of the prepared invertebrate lactone A, and the results show that the cyclosporine A has a good therapeutic effect on the experimental autoimmune encephalomyelitis (EAE) model in mice.
  • EAE experimental autoimmune encephalomyelitis
  • the EAE model is an ideal animal model for human multiple sclerosis (MS) and is commonly used for mechanisms of immune activation and immunosuppression. Therefore, the linear inulinate A can be used to prepare a medicament for treating multiple sclerosis.
  • the medicament for treating the multiple sclerosis of the present invention is a pharmaceutical composition prepared from the active ingredient of the invertebrate, as an active ingredient, and a conventional pharmaceutical carrier.
  • the pharmaceutical composition may be a tablet, a dispersible tablet, a tablet, an orally disintegrating tablet, a sustained release tablet, a capsule, a soft capsule, a dropping pellet, a granule, an injection, a powder injection or an aerosol.
  • the invention provides a new medicine for treating multiple sclerosis, and has great clinical application value.
  • Fig.1 The change of EAE incidence score in mice after intragastric administration of inflorescence A lactone A was 50 mg/kg) (The three lines from top to bottom are: line inflorescence A group, model group, Blank group).
  • Fig.2 HE staining of brain tissue sections of mice after intragastric administration of the invertebrate A (dose: 50 mg/kg) 0
  • the obtained compound is firstly determined by mass spectrometry, molecular weight 366, molecular formula C 19 H 28 0 7 , and then subjected to nuclear magnetic resonance analysis to obtain carbon spectrum, hydrogen spectrum and two-dimensional spectrum data, for structural analysis, and known compounds in the line of leaves The data for ester A was consistently confirmed).
  • the mouse experimental autoimmune encephalomyelitis (EAE) model is an ideal animal model of human multiple sclerosis (MS) and is commonly used for mechanisms of immune activation and immunosuppression.
  • mice C57BL/6 mice, M0G35-55 300 ug/only, Mycobacterium tuberculosis H37Ra 400 yg/only, emulsified, 100 ⁇ , subcutaneous injection; Pertussis toxin ⁇ 500 ng/only, day 0 and day 2 intraperitoneal injection Shoot 200 ⁇ 1 . Animals were grouped into groups of 6 each.
  • Example 1 45.3 g (manufactured in Example 1) suspended in 0.5% CMC-Na (carboxymethylcellulose sodium) formulated into a 10 mg/ml solution, with 4 concentration gradients, The drug was administered once a day from the 0th day of immunization for 30 days.
  • CMC-Na carboxymethylcellulose sodium
  • Preparation method the wire leaf inulin a lactone A, lactose and corn starch are mixed, and evenly moistened with water, The wetted mixture was sieved and dried, sieved, magnesium stearate was added, and the mixture was tableted, each weighing 300 mg, and the inflorescence A lactone A content was 25 mg.
  • the preparation method comprises the following steps: dissolving the inflorescence A and glucose in an appropriate amount of water for injection, filtering the obtained solution, and filling the infusion bottle (100 ml per bottle) under aseptic conditions, each bottle containing a leaf-spreading flower Lactone A 5 mg.
  • the preparation method comprises the following steps: dissolving the inulin and the mannitol in an appropriate amount of water for injection, filtering the solution, and filling it into a vial (10 ml vial, 2 ml per bottle) under aseptic conditions, and lyophilizing. Each line contains 10 mg of inflorescence A.

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Description

线叶旋覆花内酯 A在制备治疗多发性硬化症药物中的应用 技术领域
本发明涉及中药，具体涉及线叶旋覆花内酯 A在制备治疗多发性硬化症药物 中的应用。 背景技术
多发性硬化症（multiple sclerosis, MS)于 1868年由法国医生 Charcot首次 提出，至今仍是整个医学界未解的难题之一。该病是一种慢性、自身免疫性疾病， 通过破坏脑、脊髓以及视神经的神经纤维保护层髓磷脂，影响正常的神经传递而 导致身体残疾。由于该病具有极髙的复发率和致残率、呈慢性病程和倾向于年轻 人罹患， 已成为常见的、 重要的神经系统疾病之一。 由于该病的发病高峰在 30 岁左右， 因此， 严重危害青壮年人口的生产力和生活质量， 也给国家和政府造成 极大负担， 制约社会和经济发展。
目前临床上对该病无特效治疗方法， 缺乏令人满意的治疗药物。 急性期主 要给予糖皮质激素， 复发-缓解期主要治疗药物包括：干扰素 -γ， 醋酸格拉替雷和 单克隆抗体那他珠单抗。重度患者还可以考虑自体干细胞移植。西医的激素疗法 虽然能够缓解急性期症状， 但毒副作用明显， 故不推荐长期使用； 免疫调节剂干 扰素 IFN- P、 GA和那他珠单抗等不仅费用昂贵， 且长期使用针对单一靶点药物 容易出现耐受。且上述药物均需经静脉、 或注射给药， 不能有效改善 MS反复复 发和髓鞘脱失的根本问题。 因此， 积极研制能够有效控制 MS的复发、 改善髓鞘 脱失的口服药物具有十分重要的意义。传统中医药具有多靶点、系统调节的作用 特点， 针对 MS的复杂性、 难治性疾病有一定优势。
线叶旋覆花 [Inula lineariifolia Turcz. (syn. Inula /inariaefo/''a)]是菊科旋覆花属 植物， 多年生草本， 俗名窄叶旋覆花、 条叶旋覆花、 小朵旋覆花。广产于中国东 北部、北部、中部和东部，如河南、河北等省。也分布于蒙古、朝鲜、俄罗斯（远 东地区）和日本。 极常见生于山坡、 荒地、 路旁、 河岸等。 中药旋覆花即为旋覆 花或大花旋覆花等的头状花序， 而植物全草 (金沸草)亦供药用。 线叶旋覆花在中 国华东等部分地区也作旋覆花使用， 并且作为下气、 行水、 消炎、 软坚之药，曾 被收载于 1963年版中国药典（一部）， 但由于病人服后有恶心、 呕吐等反应，目 前已停止使用。
本发明人对线叶旋覆花进行了系统的化学成分研究，分离得到大量倍半萜类 化合物， 相关研究结果发表了学术论文 [Li-Yue Nie, Jiang-Jiang Qin, Lan Yan, Yu-Bo Liu,
Yue-Xing Pan, Hui-Zi Jin and Wei-Dong Zhang. Sesquiterpenoids from Inula lineariifolia inhibit nitric Oxide production. Journal of Natural Products, 2010,73:1117-1120】。本发明人公开了线 叶旋覆花内酯 A在制备抗炎药物中的应用， （抗炎化合物线叶旋覆花内酯 A及其 制备方法和应用 申请号： 201010200697.9)。 现进一步研究发现线叶旋覆花 内酯 A具有治疗多发性硬化症的功效， 可以开发新的用途。 发明内容
本发明所要解决的技术问题在于研究设计线叶旋覆花的提取物线叶旋覆花 内酯 A在制备治疗多发性硬化症药物中的应用。
本发明提供了线叶旋覆花内酯 A在制备治疗多发性硬化症的药物中的应用。 线 构式如下：
Figure imgf000004_0001
本发明所述线叶旋覆花内酯 A是通过下列方法制备得到的：
将线叶旋覆花干燥全草粉碎， 以 8~20倍重量的 80~95%乙醇回流提取 1~3 次， 每次 2~3小时， 合并提取液， 提取液减压浓缩成流浸膏， 流浸膏相当于 lml 含线叶旋覆花 0.8-1.2g， 流浸膏加 1~3倍重量的水稀释后， 以石油醚 0.5~2倍量 V/V萃取 3~5次， 得到石油醚部位。 将上述石油醚部位应用硅胶柱层析， 以体积 比为 100: 0~1： 1的石油醚 /乙酸乙酯系统梯度洗脱， 薄层层析检测， 收集含线 叶旋覆花内酯 A的流分， 再经 C18反相柱层析， 以重量比为 50: 100-70： 100的 甲醇 /水进行洗脱， 薄层层析检测， 得线叶旋覆花内酯 A纯品。
本发明将制备的线叶旋覆花内酯 A进行动物药效试验， 结果表明线叶旋覆花 内酯 A对小鼠实验性自身免疫性脑脊髓炎 （EAE)模型有较好的治疗作用， 而 EAE 模型是人类多发性硬化（MS)的理想动物模型， 常用于免疫激活和免疫抑制方面 的机制研究。 因此， 线叶旋覆花内酯 A可用于制备治疗多发性硬化症的药物。 本发明所述线叶旋覆花内酯 A在制备治疗多发性硬化症的药物为由线叶旋覆 花内酯 A作为活性成分与常规药用载体制成的药物组合物。 所述药物组合物可以 是片剂、 分散片、 含片、 口崩片、 缓释片、 胶囊剂、 软胶囊剂、 滴丸、 颗粒剂、 注射剂、粉针剂或气雾剂等。本发明为治疗多发性硬化症提供了新的药物， 有较 大的临床应用价值。 附图说明
图 1灌胃给予线叶旋覆花内酯 A后小鼠 EAE发病评分变化剣量 50 mg/kg) (三条线从上到下依次为： 线叶旋覆花内酯 A组、 模型组、 空白组）。
图 2灌胃给予线叶旋覆花内酯 A后小鼠脑组织切片 HE染色（剂量： 50 mg/kg)0 具体实施方式
实施例 1 线叶旋覆花内酯 A的制备
将线叶旋覆花干燥全草 50Kg粉碎， 以 90%乙醇 750L回流提取 2次， 每次 2小时， 合并提取液， 提取液减压浓缩成流浸膏， 流浸膏相当于 lml含线叶旋覆 花 1.0g， 流浸膏加水 50L稀释后， 以石油醚每次 50L萃取 5次， 得到石油醚部 分。 将石油醚部分应用硅胶柱层析， 以体积比为 100: 0-1： 1的石油醚 /乙酸乙 酯系统梯度洗脱， 薄层层析检测， 收集含线叶旋覆花内酯 A的流分， 再经 C18 反相柱层析， 以重量比为 50: 100-70： 100的甲醇 进行洗脱， 薄层层析检测， 得线叶旋覆花内酯 Α纯品 45.3g。
(得到的化合物先采用质谱测定分子量 366， 分子式 C19H2807， 再进行核磁 共振分析得到碳谱、氢谱以及二维谱数据， 进行结构解析， 与已知化合物线叶旋 覆花内酯 A的数据一致得到确证）。
实施例 2线叶旋覆花内酯 A动物药效试验
小鼠实验性自身免疫性脑脊髓炎 （EAE)模型是人类多发性硬化（MS) 的理 想动物模型， 常用于免疫激活和免疫抑制方面的机制研究。
2. 1 EAE造模方法
C57BL/6小鼠， M0G35-55 300 u g/只， 结核分支杆菌 H37Ra 400 y g/只， 乳化， 100 μ ΐ, 皮下注射； 百日咳毒素 ΡΤ 500 ng/只， 第 0天和第 2天腹腔注 射 200 μ 1。 动物分组， 每组 6只。
2. 2给药方式
线叶旋覆花内酯 A 45.3g (实施例 1制得） 悬于 0. 5%的 CMC- Na (羧甲基纤维 素钠） 配制成 10mg/ml的溶液中， 设 4个浓度梯度， 从免疫第 0天开始每天给 药 1次， 连续给药 30天。
( 1 ) 25 rag/kg每天
(2) 50 mg/kg每天
(3 ) 100 mg/kg每天
(4) 200 mg/kg每天
2. 3实验结果
50 mg/kg · d"1 ( 50 mg/kg每天 1次）及更高剂量的线叶旋覆花内酯 A灌胃 给药， 可明显延迟 EAE发病时间， 并且降低发病严重程度（见图 1 )。模型组 EAE 小鼠脑组织炎症细胞浸润， 血管、 组织排列松散， 边缘不清晰； 50 mg/kg - d"1 及更高剂量的线叶旋覆花内酯 A灌胃给药，线叶旋覆花内酯 A处理后，炎症细胞 减少， 血管、 组织基本恢复正常状态（见图 2)。
上述实验重复进行 3次。 效果同上所述。
2. 4毒性
小鼠 200 mg/kg « (Γ灌胃给药， 未观察到明显毒性反应。
2. 5 结论
线叶旋覆花内酯 Α灌胃给药 50-200 mg/kg · d"1能较好的阻止 EAE的发病过 程。提示线叶旋覆花内酯 A对多发性硬化症有较好的治疗作用。说明线叶旋覆花 内酯 A可用于制备治疗多发性硬化症的药物。 实施例 3 片剂制备
线叶旋覆花内酯 A 25g
乳糖 210g
玉米淀粉 60g
硬脂酸镁 5g
制备方法： 将线叶旋覆花内酯 A、 乳糖和玉米淀粉混合， 用水均匀湿润， 把湿润后的混合物过筛并干燥， 再过筛， 加入硬脂酸镁， 然后将混合物压片，每 片重 300mg， 线叶旋覆花内酯 A含量为 25mg。
实施例 4: 注射液制备
线叶旋覆花内酯 A 5g
葡萄糖 50g
制备方法：将线叶旋覆花内酯 A和葡萄糖溶解于适量的注射用水中，过滤所 得溶液， 在无菌条件下装入输液瓶（每瓶 100ml) 中， 每瓶含线叶旋覆花内酯 A 5mg。
实施例 5: 注射用冻干粉针剂制备
线叶旋覆花内酯 A 10g
甘露醇 30g
制备方法：将线叶旋覆花内酯 A和甘露醇溶解于适量的注射用水中，过滤所 得溶液， 在无菌条件下装入西林瓶（10ml西林瓶， 每瓶 2ml)中， 冻干， 每支含 线叶旋覆花内酯 A 10mg。

Claims

权利 要求
1、线叶旋覆花内酯 A在制备治疗多发性硬化症药物中的应用，其特征在于， 所述线叶旋覆花内酯 A的结构式如下-
Figure imgf000008_0001
2、 根据权利要求 1所述的应用， 其特征在于， 所述线叶旋覆花内酯 A通过 下列方法制备得到：
将线叶旋覆花干燥全草粉碎， 以 8~20倍重量的 80~95%乙醇回流提取 1~3 次， 每次 2~3小时， 合并提取液， 提取液减压浓缩成流浸膏， 流浸膏相当于 lml 含线叶旋覆花 0.8-1.2g， 流浸膏加 1~3倍重量的水稀释后， 以石油醚 0.5~2倍量 V/V萃取 3~5次， 得到石油醚部位； 将石油醚部位应用硅胶柱层析， 以体积比为 100： 0~1： 1的石油醚 /乙酸乙酯系统梯度洗脱， 薄层层析检测， 收集含线叶旋 覆花内酯 A的流分， 再经 C18反相柱层析， 以重量比为 50: 100-70： 100的甲醇 / 水进行洗脱， 薄层层析检测， 得线叶旋覆花内酯 A纯品。
3、根据权利要求 1所述的应用，其特征在于，所述药物为由线叶旋覆花内酯 A作为唯一活性成分与药用载体制成的药物组合物。
4、根据权利要求 3所述的应用， 其特征在于， 所述药物组合物为片剂、分散 片、 含片、 口崩片、 缓释片、 胶囊剂、 软胶囊剂、 滴丸、 颗粒剂、 注射剂、 粉针 剂或气雾剂。
PCT/CN2012/001411 2012-06-21 2012-10-22 线叶旋覆花内酯a在制备治疗多发性硬化症药物中的应用 Ceased WO2013188999A1 (zh)

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