TWI281859B - Medical composition for treating respiratory allergic disease - Google Patents
Medical composition for treating respiratory allergic disease Download PDFInfo
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- TWI281859B TWI281859B TW94119802A TW94119802A TWI281859B TW I281859 B TWI281859 B TW I281859B TW 94119802 A TW94119802 A TW 94119802A TW 94119802 A TW94119802 A TW 94119802A TW I281859 B TWI281859 B TW I281859B
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Description
1281859 九、發明說明: 【發明所屬之技術領域】 本發明係關於一種治療呼吸道過敏疾病的醫藥組成 物’係包含有效量之咖啡酸落^^ ( CAPE)’及一醫樂上 可接受之載劑。 【先前技術】 氣喘為一種呼吸道過敏#病,在現代社會中已經是 一個常見的疾病,且越是工業化的社會其盛行率越高。 造成氣喘的原因相當複雜,玎能的因素包含有遺傳因 素、環境因素(嬰幼兒早期生活環境、病毒、過敏原與 職業性的暴露)等。美國NIH於1997年出版的 Guidelines for the Diagn〇sis and Management of Asthma定義氣喘為一個呼吸道仗性發炎的症狀。病人在 清晨或深夜時會發生呼吸困難、呼吸時發出氣喘聲、咳 嗷等症狀,尤其是早晚溫差變化大的時候。 過敏性氣喘屬於呼吸道發炎的疾病’在氣喘的免疫組 織病理學上發現,氣管組織中有嗜中性白血球、嗜酸性 白血球與去顆粒化之肥大細胞浸潤的現象,下基底膜 (sub-basement-membrane)增厚,支氣管内腔因黏液過 度分泌而閉塞,且支氣管平滑肌增生、肥大(Busse and Lemanske,2001)。許多研究指出氣喘疾病發生與免疫系 統Th1/Th2細胞扮演的角色有關(Holtzman # 5入, 1996 ; Borish and Rosenwasser,1997),體内免疫系統 對抗環境中抗原時,偏向Th2類型免疫反應是氣喘發生的 重要因素(Hansen以a/·,1999),在氣喘患者支氣管 1281859 切片、肺部沖洗液(bronchoalveolar lavage fluids, BALF)或週邊血液中,可發現活化的τ細胞與嗜酸性白 血球(eosinophils)數目明顯增加(Azzawi a/·, 1990 ; Wilsorl以a/·,1992),此外患者BALF中更有大 量的介白質四號(interieukin-4,IL-4)以及IL-5 (Chung and Barnes, 1999 ; Busse and Lemanske, 2001) 〇 目前對於氣喘的治療大部分偏重於抑制氣管的發炎 現象’以及舒缓氣管的收縮程度,而吸入性類固醇 (corticosteroids)的使用為目前對於氣喘症狀控制和 肺功能改善最有效的方法之一,其主要的機制是藉由抑 制轉錄因子(transcription fact〇rs)如 NF-kB 或 STAT factor荨來调控氣。而的發炎反應(Barnes and Adcock, 1998),但此類藥物主要以免疫抑制方式控制病情,未能 根治氣喘’並且經常伴隨著副作用的產生,故坊間有不 少非傳統辅助療法’例如中草藥或保健食品等(Ziment and Tashkin,2000 ; Bielory and Up〇li,1999)。 過去臨床研究發現,目前廣泛應用作治療氣喘病、 肺氣腫及其他慢性啤吸系統疾病的吸入式類固醇 (inhaled steroid )藥物,在病發時雖然可以纾缓症 狀,但卻不可延緩病情惡化。而且據美國一項醫學研究 發現’長期使用更會引致皮膚受損及骨質疏鬆等副作 用。此外,研九發現長期使用吸入式類固醇經常導致口 腔黏膜感染白色念珠菌的症狀。對兒童而言,長期使用 類固醇有可此景^響骨路之發育,或造成「庫辛氏症」, 即出現臉部浮腫、軀幹腫胖的現象。 1281859 方式或藥 前醫學上 因此’尋求一個有效治療氣喘的有效治療 物,同時降低副作用的發生或嚴重程度,為當 極為重要的課題。 $ 【發明内容】 本發明係根據一項超乎預期的發現,即蜂膠口 解氣%模式貫驗小鼠之氣喘症狀,降低其呼^;父緩 值。進一步的研究發現,蜂膠中的有效活性成八1阻力 酸苯乙酯。實驗發現,以咖啡酸苯乙酯餵食氣二^咖啡 鼠確實可以達到緩解氣喘症狀的功效。 而果式小 本發明之目的係關於一種用於治療呼吸道過敏; 的醫樂組合物,包括治療有效量的咖啡酸笨乙酯 ^ = 樂上可接受之載劑。 ^ 、本發明之醫藥組成物係可為乾燥型態(如錠型或粉末型 或液型態(如飲料或糖漿),其可為飲食補充劑或藥劑,亦可為 飲料或食品,例如:茶(如茶飲料或茶包成份)、汽水、果汁(如 水果萃取物或果汁飲料)、牛奶、咖啡、餅乾、麥片、巧克力及 點心棒。本發明之醫藥組成物在任何前述形式下皆可用於治療 呼吸道過敏疾病,包括,但不限於氣喘病。 本發明之另一目的係關於一種用於治療呼吸道過敏 疾病的化合物,其中該化合物包括咖啡酸苯乙酯或其衍 生物。 在本發明中,咖啡酸苯乙酯係由蜂膠中萃取而得, ί 2取ί法係為已知,在此不作贅述。蜂膠自古以來即 ^區^美等地的廣泛地使用於傳統醫療中,由於蜂膠具有 几菌的特殊效果,因此被視為提高抵抗力的極佳營養補 1281859 充品。由此可見食用蜂膠或攝取其活性成分-咖啡酸苯乙 酯並不會產生極大之副作用,具有取代吸入式類固醇而 成為治療氣喘用藥的潛力。 【實施方式】 本發明之目的在於提供一種用於治療呼吸道過敏疾 病的醫藥組合物,包括治療有效量的咖啡酸苯乙酯,及 一醫藥上可接受之載劑。本發明亦關於一種用於治療呼 吸道過敏疾病的化合物,其_該化合物包括咖啡酸苯乙 酷或其衍生物。其中前述呼吸道過敏疾病可為氣喘病。 前述醫藥組合物的形式可為錠型、液型或粉末型。在本 發明中,咖啡酸苯乙酯係由蜂膠中萃取而得。咖啡酸苯 乙酯的每曰/每70公斤體重的有效劑量可為38. 8毫克至 155. 16毫克,較佳為77.58毫克至155. 16毫克,更佳為 38.8毫克至77.58毫克。 當過敏原侵入人體時,過敏原會被存在氣管中的樹 突細胞(dendritic cel Is)帶到附近的淋巴結(lymph node),利用其表面第二型組織抗原分子 (major histocompatibility complex class 11 molecules, MHC class 11)將過敏原呈現給T細胞,使natve T細胞(未 被活化過的T細胞)偏向分化成Th2淋巴球,受活化的 Th2淋巴球則會分泌大量的IL-4、IL-5、IL-10及IL-13 等細胞激素,刺激T細胞與B細胞交互作用,製造許多 IgE抗體。誘發B細胞產生IgE抗體則需要兩個訊息傳 遞,第一為Th2細胞分泌的細胞激素IL-4、IL-13與B細 胞上的受體結合後會藉由 STAT-6 (signal 1281859 transduct ion-act ivi ted transcription-6)傳遞訊號 (Willseh/.,1998)。第二是來自T細胞表面上的CD40 ligand (CD40L)和Β細胞表面的CD40結合後所傳遞的 訊號(Bacharier ei a/·,1998)。
之後IgE抗體會與組織中的肥大細胞或週邊血液中的 嗜鹼性白血球上具高親和力的IgE接受器(FcsRI)結 合。當附著於這些發炎細胞表面的IgE抗體再次接觸過 敏原時,過敏原會與IgE抗體進行交叉鍵結,刺激發炎 細胞活化與去顆粒化作用(degranulation),釋放發炎 物質,如組織胺(histamine)、白三烯素(leukotrienes) 及趨化因子(chemotactic factors)等,導致氣管平滑 肌收縮、黏液增加、肺部呼吸困難。IL-5細胞激素與趨 化因子也會活化嗜酸性白血球,被活化的嗜酸性白血球 不但會釋放發炎物質損害氣管,還會吸引更多發炎性細 胞浸潤至肺部,造成氣管慢性發炎(Barnes 1997 ; Busse and Lemanske, 2001)。 在研究中發現’儀食蜂膠組小氣有較低的呼吸道阻 力值’低劑量蜂膠組小氣血清中’ IgE、IgGi抗體旦 較控制組低,且傲食蜂膠後可增加IFN-γ分、必H = IL-10分泌量,推測蜂膠具有減緩實驗小氣氣—及低 而達到免疫調節的功效。 而、务生’ 口J 只哪又δ又5丨丄七人将ttf -丁次逼發炎模 活體内試驗(//7 F/ra)驗證咖啡酸笨式動物進行 緩解或治療氣喘之活性成分。活體試驗中,:^蜂膠中 予呼吸道發炎模式動物管饒咖啡酸笨乙,、‘五天绐 -曰彳采討吻/啡酸 1281859 笨乙s旨對其免疫調節制之影響。實驗 白(OVA)抗原致敏小鼠產生過敏反應,再白蛋 白方式誘發小鼠呼吸道發炎症狀。小^ P白蛋 析血清中卵白蛋白特異性IgE抗體生成量,分 立成功後,將不同劑量咖啡酸苯乙醋、、果式建 組)或prednisolone氣喘治療藥物(ϋ (,制 銀小氣。動物犧牲後,抽取肺部沖洗液、管 液及脾臟細胞’分析淋巴細胞增生反應、細出 ==質之分泌情形,探討咖啡酸笨乙醋對 叙火杈式動物免疫調節作用之影響。 及k 明奎施㈣僅祕說明本發明’料於限制本說 丄:戶:揭路之内容’任何熟習本技術領域的人士,皆可 根據本s兄明書中揭露之内容,進一步做任何可能 與潤飾以達到最大功效,所有詳列於本說 以全文引人作為參考資I ^者作係 【實施例】 材料與方法 試劑 【血清中卵白蛋白特異性IgE抗體的測定試劑】 1· Mouse IgE ELISA quantitation Kit (Bethyl, E90-115) : g〇at anti-mouse IgE-HRP conjugate 2·塗覆緩衝液(coating buffer) : 0· 012MNa2C〇3, 1281859 0· 028MNaHC〇3,pH 值 9· 6。 3· 10 倍 Tris 緩衝液(Tris buffer)。 4·阻斷緩衝液(Blocking buffer) : 1% (重量/體積) 牛血清(BSA)溶於1倍7^3缓衝液。 5·待測樣品稀釋倍數:稀釋2〇倍。 【細胞激素與發炎相關物質的測定試劑】 l.DuoSet mouse TNF-α (R&D system, DY410)
DuoSet mouse IL-6 (R&D system, DY406)
DuoSet mouse IL-1^ (R&D system, DY401)
DuoSet mouse IFN-γ (R&D system, DY485)
DuoSet mouse IL-2 (R&D system, DY402)
DuoSet mouse IL-4 (R&D system, DY404)
DuoSet mouse IL-10 (R&D system, DY417)
OptEIA mouse IL-5 Set (PharMingen, 555236)
OptEIA mouse MCP-l Set (PharMingen, 555260) 2. 10 倍鱗酸緩衝液(phosphate buffer sal ine ; PBS), pH值至7· 4。 3. 阻斷緩衝液:1% (重量/體積)牛血清溶於1倍磷 酸缓衝液。 4.待測樣品稀釋倍數: 1281859 IL-6細胞激素:待測樣品稀釋20倍。 IFN-γ細胞激素:待測樣品稀釋10倍。 IL-2細胞激素:待測樣品稀釋20倍。 MCP-1細胞激素:待測樣品稀釋5倍。 其餘五種細胞激素:待測樣品不需稀釋。 【其他實驗試劑】 試驗物質咖啡酸苯乙酯的配製 咖嗜酸苯乙酯粉狀物 (Phenethyl Caffeiate, Cayman, 70750, USA)為無菌真空包裝,以無菌針筒取】 毫升 DMSO (Dimethyl Sulphoxide,Sigma,D2650,USA) 注入無菌真空包裝内,使咖啡酸苯乙酯粉末完全溶於 DMS0,將咖啡酸苯乙酯溶液分裝至1.5毫升棕色離心管, 並保存於-20°C冰箱中備用。使用前以CM-10稀釋至所需 適當濃度。 CM-10的配製 1. RPMI-1640 (HyClone, SH30027.01, USA) 2· L-麩醯胺酸(L-glutamine) (HyClone, SH30034.01, USA) 3. HEPES (HyClone, SH30237.01, USA) 4·乙基硫醇(y5-mercap1:oethanol)l(Sigma,63689,USA) 5· PS 二合一抗生素(HyClone, SV30010,USA) 12 1281859 6.胎牛血清 FBS (Fetal bovine serum, HyClone, CH30160.02, USA) 利用500毫升RPMI-1640 (含有2.05 mM L-麵酸胺酸; HyClone,SH30027.02,USA)作為培養基,加入50毫升 經去補體活化作用處理(將解凍之胎牛血清在56°C水浴 中,經過30分鐘處理)之胎牛血清、5毫升PS二合一抗 生素(penicillin-streptomycin solution)、5 毫升 L-麩醯胺酸、0· 5毫升5 X 10_5M乙基硫醇(少ME, fmercaptoethanol)還原劑上述成份加入至RPMI-1640 培養基中,即可得CM-10 (含有10% FBS之complete medium) 〇 卵白蛋白/氫氧化鋁的配製 分別配製20或50 pg/mL卵白蛋白(Albumin, chicken egg,Sigma,A-5503)配合 2 mg 氫氧化銘(aluminium hydroxide,alum, Pierce,77161)佐劑,每隻小鼠腹 腔注射量為0.2毫升。 HBSS溶液的配製 將一包含 9· 5 公克 Hank’ s balanced salt mixture 之黃色粉末(HyClone,SH30016.01,USA)溶於1公升 去離子水,以0.2微米(μιη)濾膜進行過濾滅菌,再以 滅菌之 7.5% NaHCOs (Merck, 1.06329,Germany)調整 pH值於7. 2至7· 4即可。 紅血球溶解緩衝液的配製 13 1281859 本實驗使用的紅血球溶解緩衝液為ACK溶裂缓衝液
(lysis buf fer )。秤得 8· 29 公克、37. 2 公克 Na2EDTA 及1公克KHCO3加入去離子水溶解後,定量至一公升,倒 入有蓋血清瓶中,經過高溫高壓滅菌(autoclave,
Sturdy,SA-400A,Taiwan)處理後,方可使用。 有絲分裂劑配製
伴刀豆球蛋白(]〇11八((]〇1^8113¥81]111八,84111&,0 0412) 籲與植物血球凝集素 PHA(phytohaemagglutinin; lectin, Sigma,L-9132)是一種萃取自豆科植物的有絲分裂劑, 主要可刺激T細胞的活化增生作用。植物血球凝集素LPS (脂多醣 lipopolysaccharides,Sigma, L-2654)純化自 E· coli的細胞壁成份,能促使b細胞增生,並激活巨嗤 細胞。以上有絲分裂劑均為無菌真空包裝,以無菌針筒 取一毫升CM-10培養液注入無菌真空包裝内,分裝保存 於-20 C冰箱中備用。使用前以CM-1 〇稀釋為所需適當濃 度後,再以〇·2μιη濾膜進行過濾,保存於-20°C冰箱中。 φ 離子黴素約鹽Ionomycin calcium salt與孟寧素 monensin (Sigma,Μ-5273,USA)的配製則為利用無菌 針筒取1毫升DMS0注入含有1毫克的PMA包裝内,混合 均勻後分裝保存於-80°C冰箱中備用。使用前以CM-10豨 釋至所需適當濃度後,再以〇· 2 μπι濾膜進行過濾,保存 於-20°C冰箱中。 染色緩衝液(Staining Buffer)的配製 秤得〇· 5公克NaN3與25毫升去補體活化作用處理 14 1281859 之 FBS ’ 加入 1 倍 DPBS 溶液(Dulbecco,s PBS,了人
Msf2+ > r〇2+^ 不含 ^ a肖隹子)至總體積500毫升,待溶液溶解均句 调整pH值至7· 4〜7.6,保存於4°C冰箱。 1 固定緩衝液(Fixat ion Buffer)的配製 祥知4公克三聚曱酸(paraformaldehyde)加入1 倍DPBS溶液100毫升,置於56°C水浴1〜3小時,直到 二聚甲酸粉末溶解,待溶液冷卻後調整pH值至7.4〜 7· 6 ’避光保存於4°c冰箱。 ^透化、、爰衝液(Permeabilization Buffer)的配製 科得10公克BSA、0· 5公克NaNs與0· 5公克saponin, 加入1倍DPBS溶液500毫升,調整pH值至7. 4〜7· 6, 保存於4°C冰箱。 實施例一:建立呼吸道發炎模式動物 小鼠八週齡時,利用卵白蛋白物質作為過敏原,與氫 氧化紹之佐劑混合均勻後,以腹腔注射方式 (intraperitoneal injection, IP)致敏小鼠三次(每 次致敏間隔為二週),第一次致敏每隻小鼠腹腔注射20 Mg卵白蛋白,第二次腹腔注射致敏為50 pg卵白蛋白 並進行一次呼吸道吸入2%卵白蛋白致敏(即 inhalation, IH),模擬氣喘反應的發生,第三次致敏每 隻小鼠再次腹腔注射50 pg卵白蛋白。小鼠致敏之前與 第三次致敏後一週均眼窩採血,利用ELISA方法測得小 15 1281859 鼠血清中卵白蛋白特異性I gE抗體生成量,以確定模式 建立成功。 實施例二:吸入性致敏(Inhalation) 為了模擬氣喘疾病的發生,讓小鼠由呼吸道吸入抗原 而產生呼吸道發炎反應。將2公克卵白蛋白粉末溶於1 倍磷酸緩衝液溶液100毫升中,待其自然溶解後,稍微 搖晃使其混合均勻。隨機將小鼠放置於12 X12 X14立方公 分的有蓋透明塑膠方形筒中,以超音波喷霧化儀器 (DeVilbiss Pulmo-Aide, 5650D,USA)將 2% 印白蛋白 溶液喷霧化,使小鼠在密閉容器中自然地將過敏原經呼 吸道吸入體内。每次吸入性致敏為4〜6隻小鼠,以8 毫升2%卵白蛋白溶液進行喷霧20分鐘。 實施例三:動物分組
• 確定呼吸道發炎模式動物建立成功後,依據ELISA 方法測得小鼠血清中卵白蛋白特異性IgE抗體生成量, 將小鼠分為六組’包含控制組(Ctrl,每日饒食紅花軒 油)、咖啡酸苯乙酯組(CAPE,每公斤體重小鼠每曰管餘 5、10或20毫克)、藥物治療組(Pred,每曰管饒〇· 1毫 克prednisol〇ne藥物)與未處理(non-treat)組,每 組小鼠為6〜8隻。控制組、咖σ朴酸苯乙醋組與藥物治 療組均為呼吸道發炎模式動物,而未處理組則無接受管 餵或卵白蛋白致敏之處理。本研究中,藥物治療組為正 16 1281859 向控制組,可與咖啡酸苯乙酯組相互比較,另外以未處 理組作為背景對照組,代表健康小鼠之免疫狀態,可與 其他五組有誘發呼吸道發炎模式小鼠作比較。 實施例四:試驗物質 咖啡酸苯乙酯粉狀物(Phenethyl Caffeiate,Cayman, 70750,USA)為無菌真空包裝,以無菌針筒取0.1毫升 DMSO (Dimethyl Sulphoxide, Sigma, D2650, USA)注 入無菌真空包裝内,使咖啡酸苯乙酯粉末完全溶於 DMS0。咖啡酸苯乙酯管灌餵食劑量乃參考2〇〇4年Park 等人之研究(Park 5人,2004),首先將咖啡酸苯乙 酯溶液配合紅花籽油,配製成所需之劑量(每公斤體重 小鼠每天管餵5、10或20毫克),利用3_way st〇pc〇ck (Nipro,03F25,Japan)將咖啡酸苯乙酯溶液與紅花籽 油混合均勻,再以無菌1毫升針筒套上不銹鋼餵食針管, 連續五天每日管餵50毫升體積之紅花籽油(控制組)或 不同劑量之咖啡酸苯乙酯物質。 潑尼松龍(Prednisolone) (Pred,Sigma p〇152 USA)為無菌真空包裝,先以無菌針筒取i毫升輯酒精 (Showa,2841Y,Japan)注入無菌真空包裝内。使25〇 毫克潑尼松龍潑尼松龍粉末完全溶於無水酒精後,再以i 倍PBS溶液配製為溶解完全的溶液。管餵劑量是參考2〇〇2 年Jones等人之人體試驗(J〇nes打200〇,由為 試者所服用的劑量換算至小鼠使用的劑量,每公斤體^ 17 1281859 小鼠每曰管餵5毫克潑尼松龍,連續五天每日管餵200 毫升體積之潑尼松龍水溶液。 統計方法 實驗結果以平均值士標準差(mean ± SD)表示, 利用 SAS (Statistical Analysis System)統計軟體進 行分析··包含Student,s i-test或單因子變異數 (one-way analysis of variance, one-way ANOVA)分 析,當結果有差異時,接著以鄧肯氏多變域測定法 (Duncan s multiple range method)進行試後比較, 當P <0· 05時,表示實驗值之間有顯著差異。*則表示 與控制組有顯著差異。 結果 1·银食咖啡酸苯乙酯對呼吸道發炎模式小鼠生長體重之影 響為觀察小鼠生長狀況有無異常,在實驗期間每兩週秤 量小鼠體重,此外並記錄小鼠餵食咖啡酸苯乙酯前後其 體重變化情形,以瞭解餵食咖啡酸苯乙酯是否影響小氣 生長狀況。如第一圖所示,銀食咖啡酸苯乙g旨(每公斤 小鼠每天5、1〇或20mg)五天後,各組小鼠體重變化與 银食咖啡酸苯乙酯前無顯著差異,顯示银食此劑量範圍 内之咖啡酸苯乙酯不影響小鼠生長情形。 2·假食咖啡酸苯乙酯對呼吸道發炎模式小鼠肺部沖洗液中 免疫相關分析之影響 2· 1肺部沖洗液中各類免疫細胞百分比與細胞數目 18 液,經離心後取得肺部沖 ’計數單核球及嗜酸性白 之百分比與細胞數目,觀 由第二>
1281859 小鼠犧牲後抽出肺部沖洗 出細胞,進行細胞固定及染色 士球細胞,所佔肺部沖洗液中 察小鼠肺部支氣管發炎情形。 原致敏為呼吸道發炎::二且:鼠 P 1、^二 式故其肺部沖洗液中主要為淋 血球二二^早核球’沒有性白血球或嗜中性白 常多二=Ϊ現象1制組小鼠肺部沖洗液中則有非 /1、。铲二二韭2球與單核球聚集,淋巴球細胞反而減 :·,.n西,本乙酯組小鼠,隨咖啡酸苯乙酯劑量提 ^ ^ H 〇 ^ ^ 艮b芜克咖啡酸笨乙酯組即達到顯 對u老^ κ占二毛+見到飯食咖啡酸笨乙酯(2〇呵/岐BW/day) 抑制效果與藥物組相當;由第二圖 球細胞數量較控制組提升;而由第二圖(c) ::: 亦低於控制組。藥物治療組之嗜酸性白 球數目及早核球數量亦顯著較控制組低。綜上所述, 於1吸這發炎錢肺部沖㈣之倾㈣分析 儀食咖啡酸苯以旨可顯著降低嗜酸性白血球與單桉^ 潤至肺部支氣管,由此情_測咖啡酸苯u旨可能是= 接影響嗜酸性白血球之活性,或者咖啡酸苯乙目旨誘 酸性白血球走向細胞計,以至於產生如此之結9 19 1281859 激氣喘小鼠腹腔抽出細胞,培養24小時後收集細胞培養上清 液。腹腔中以巨噬細胞為主,當巨噬細胞受到活化時,會分泌 IL-6與TNF-α等促發炎細胞激素(Marin eia/·,1997)。 結果顯不’如第三圖所示,傲食咖啡酸苯乙g旨組(1 〇 或20 mg/kg BW/day)小鼠之腹腔細胞,以LPS刺激培養 上清液中IL-6分泌量顯著低於控制組。第四圖結果中, 餵食20 mg咖啡酸苯乙酯/kg BW/day組小鼠其細胞培養 0 上清液中TNF-α分泌量有較控制組降低之趨勢 (ρ=0· 0501 )。藥物組之腹腔細胞培養上清液中促發炎細 胞激素分泌量,皆與控制組無顯著差異。 由上述實驗結果可推測咖啡酸苯乙酯可能具有抑制 巨噬細胞活化之作用,具有抗發炎之功效。 4·餵食咖啡酸苯乙酯對呼吸道發炎模式小鼠脾臟細胞免疫 相關分析之影響 4· 1以不同有絲分裂劑刺激脾臟細胞培養之增生反應 Φ 實驗利用不同有絲分裂劑刺激脾臟細胞,共同培養 48小時後,以3Η-胸腺嘲咬攝入測試(沱―thymidine incorporation assay)方法測定細胞增生反應。 將脾臟細胞以5 pg/mL伴刀豆球蛋白或1〇 μδ/ιηί 植物血球凝集素刺激培養之增生反應,代表τ細胞族群 的增生反應,以10 Mg/mL脂多醣刺激培養之反應代表Β 細胞族群的增生反應。結果如弟五圖所示,第五圖(Β) 為以植物血球凝集素(ΡΗΑ)刺激培養下,饒食咖啡酸苯 20 1281859 乙酯組(5、10或20 mg/kg BW/day)與藥物治療組小鼠 之脾臟增生反應皆顯著高於控制組。第五圖(A)及(C) 分別為以伴刀豆球蛋白(Con A)或脂多酿(LPS)刺激 培養下,儀食10 mg咖°非酸苯乙酯/kg BW/day組小鼠之 脾臟增生反應亦顯著高於控制組,顯示咖啡酸苯乙酯具 有活化T與B淋巴細胞增生之功能。 4. 2利用免疫螢光染色法測定脾臟細胞内細胞激素分泌情 • 形 實驗利用淋巴球活化劑植物血球凝集素與離子黴素 刺激呼吸道發炎模式小鼠脾臟細胞,共同培養6小時後, 先進行細胞表面抗原CD3染色,再將細胞内IFN-γ細胞激 素染色,最後以流式細胞計數儀分析結果,即可了解脾 臟細胞中會分泌IFN-γ細胞激素的細胞百分比,以及CD3 Τ 細胞中會分泌IFN-γ細胞激素的細胞百分比。 如第六圖所示,實驗結果發現,相較於控制組之下, φ 傲食20 mg咖啡酸苯乙酯/kg BW/day組之小鼠脾臟細胞 (第六圖(B))與CD3 T細胞(第六圖(A))中,會分泌 IFN-γ細胞激素的細胞百分比顯著提高,non-treat組小 鼠脾臟細胞中,會分泌IFN-γ細胞激素的細胞百分比亦顯 著高於控制組。至於藥物組IFN-γ的細胞百分比,皆與控 制組無顯著差異。 4. 3以伴刀豆球蛋白刺激脾臟細胞培養上清液中細胞激素 分泌量 21 ^81859 嗜酸性白血球之活性,亦或 亡,而降低嗜酸性白血球浸潤5 V嗜酸性白血球細周 巨t細胞之活化。本發明揭:(㈣ ’提供了治療氣喘病新方:所未 療物質的種種副作用。 又免習知治 23 1281859 【圖式之簡單說明】 第一圖係顯示咖啡酸苯乙酯對小鼠生長體重的影響。 第二圖(A)係顯示餵食咖啡酸苯乙酯對呼吸道發炎小 鼠肺部沖洗液中對嗜酸性白血球細胞聚集之影響。 第二圖(B)係顯示银食咖啡酸苯乙醋對呼吸道發炎小 鼠肺部沖洗液中淋巴球細胞聚集之影響。 第二圖(C )係顯示餵食咖啡酸苯乙酯對呼吸道發炎小 鼠肺部沖洗液中單核球細胞聚集之影響。 Φ 第三圖係顯示餵食咖啡酸苯乙酯對呼吸道發炎小鼠 腹腔細胞以脂多醣刺激培養上清液中IL-6分泌量之影 響。 第四圖係顯示餵食咖啡酸苯乙酯對呼吸道發炎小鼠 腹腔細胞以脂多醣刺激培養上清液中TNF-α分泌量之影 響。 第五圖(A)係顯示餵食咖啡酸苯乙酯對呼吸道發炎小 鼠脾臟細胞以伴刀豆球蛋白(Con A)有絲分裂劑刺激增 生反應之影響,。 ® 第五圖(B)係顯示餵食咖啡酸苯乙酯對呼吸道發炎小 鼠脾臟細胞以植物血球凝集素(PHA)有絲分裂劑刺激增 生反應之影響。 第五圖(C)係顯示餵食咖啡酸苯乙酯對呼吸道發炎小 鼠脾臟細胞以脂多醣(LPS)有絲分裂劑刺激增生反應之 影響。 第六圖(A)係顯示餵食咖啡酸苯乙酯對呼吸道發炎小 鼠CD3 T細胞内IFN-r表現之影響。 24 1281859 第六圖(B)係顯示餵食咖啡酸苯乙酯對呼吸道發炎小 鼠脾臟細胞内IFN-r表現之影響。 第七圖係顯示餵食咖啡酸苯乙酯對呼吸道發炎小鼠 脾臟細胞以半刀豆球蛋白刺激培養上清液中IFN-r分泌 量之影響。 第八圖係顯示餵食咖啡酸苯乙酯對呼吸道發炎小鼠 脾臟細胞以半刀豆球蛋白刺激培養上清液中IL-5分泌量 之影響。
25
Claims (1)
1281859 十、申請專利範圍: m(更)正本 Mill丨 1公告本丨 1. 一種用於治療呼吸道過敏疾病的醫藥組合物,包括治療 有效量的咖啡酸苯乙酯,及一醫藥上可接受之載劑。 2. 如申請專利範圍第1項所述之醫藥組合物,其中前述呼 吸道過敏疾病為氣喘病。 3. 如申請專利範圍第1項所述之醫藥組合物,其中前述醫 藥組合物的形式可為錠型、液型或粉末型。
4. 如申請專利範圍第1項所述之醫藥組合物,其中前述咖 啡酸苯乙酯的每日/每70公斤體重的有效劑量為38.8毫 克至155.16毫克。 5. 如申請專利範圍第1項所述之醫藥組合物,其中前述咖 啡酸苯乙酯的每日/每70公斤體重的有效劑量為77.58毫 克至155.16毫克。 6. 如申請專利範圍第1項所述之醫藥組合物,其中前述咖 啡酸苯乙酯的每日/每70公斤體重的有效劑量為38.8毫 克至77.58毫克。 7. 如申請專利範圍第1項所述之醫藥組合物,其中前述咖 啡酸苯乙酯係由蜂膠中萃取而得。 26
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