TW201415028A - 調控細胞表面受體以預防或減少發炎之方法 - Google Patents

調控細胞表面受體以預防或減少發炎之方法 Download PDF

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TW201415028A
TW201415028A TW102144529A TW102144529A TW201415028A TW 201415028 A TW201415028 A TW 201415028A TW 102144529 A TW102144529 A TW 102144529A TW 102144529 A TW102144529 A TW 102144529A TW 201415028 A TW201415028 A TW 201415028A
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Abstract

本發明包括一種在口腔細菌菌種之間進行區辨以測定某一菌種是否對口腔有害的方法。此種方法包括將至少一種細菌或一種屬於口腔細菌菌種之細菌的部分與齒齦細胞接觸；並偵測一種指標化合物的存在。基本上沒有指標物質存在表示該細菌菌種並非有害菌種。同時也包含在本發明之範疇中的是測定一種藥劑的抗發炎效果之方法。此種方法包括在有害的細菌或此種細菌之部分存在下將細胞與該藥劑接觸，並偵測一種指標化合物的存在。基本上沒有指標物質存在即表示該藥劑是一種抗發炎的藥劑。

Description

調控細胞表面受體以預防或減少發炎之方法
本申請案主張2006年1月10日申請之美國臨時專利申請案序號60/757,751之利益；其內容以參考資料併入本說明書中。
本發明包括一種在口腔細菌菌種間進行區辨以測定某一菌種是否對口腔有害的方法。此種方法包括將至少一種屬於口腔細菌菌種之細菌或細菌之一部分與齒齦細胞接觸；並偵測一種指標化合物的存在。基本上沒有指標物質存在表示該細菌菌種並非有害菌種。同時也包含在本發明之範疇中的是測定一種藥劑的抗發炎效果之方法。此種方法包括在有害的細菌或此種細菌之部分存在下將細胞與該藥劑接觸，並偵測一種指標化合物的存在。基本上沒有指標物質表示該藥劑是一種抗發炎的藥劑。
已知哺乳動物口腔之軟組織會展現發炎的最早期指標。這種普遍化的低程度發炎可導致齒齦炎或牙周炎。此種發炎普遍被相信至少部分是存在於口腔的細菌所造成。而且，口腔組織發炎可因手術、局部損傷、創傷、壞死、不正確的口腔衛生或各種全身性的起源而引起。
吾人一般相信與這些疾病和病症牽連的細胞成分包括表皮組織、齒齦的纖維母細胞、和循環的白血球，它們均對於宿主對細菌產生之致病因子的反應有貢獻。某些牽連在這些口腔感染的細菌病原體是已知的，雖然有許多仍然保持未知或未經特徵鑑認。雖然在這許多口腔疾病中被某些型細菌感染經常是病原學的事件，但是這些疾病狀態或病況的致病機制卻是由宿主反應所調節。使用抗細菌劑會降低某一口腔中細菌的量並且會使發炎降低。然而，這種方法有缺點，因為此種殺菌是以不經區辨的方式(有利的口腔細菌和有害的口腔細菌都會死亡)來達成的，並且這種方式對於劑量和時間很敏感。
細菌感染口腔組織會刺激宿主的免疫反應並且藉由向上調節能引起顯著組織破壞的發炎調節因子來減短痊癒過程。這些代謝物已經被顯示為齒齦炎、牙周炎、骨髓炎和其他發炎性疾病的主要調節因子。
已經有報告指出一種發炎的機制是經由哺乳動物之某些穿越細胞膜的受體所調節。例如，似關卡受體(“TLRs”)即為糖苷化之穿越細胞膜的蛋白質，且其一旦受到配體引發寡聚作用的活化就會使細胞內的免疫反應開始發生，最後造成細胞介素、介白素、 和其他調節發炎狀態之分子的表現。
在本技藝中需要有用於診斷、治療和預防此種發炎作用的藥劑和技術。
本發明之簡短說明
本發明包括一種在口腔細菌菌種間進行區辨以測定某一菌種是否對口腔有害的方法。此種方法包括將至少一種細菌或一種屬於口腔細菌菌種之細菌的部分與齒齦細胞接觸；並偵測一種指標化合物的存在。基本上沒有指標物質存在表示該細菌菌種並非有害菌種。
同時也包含在本發明之範疇中的是測定一種藥劑的抗發炎效果之方法。此種方法包括在有害的細菌或此種細菌之部分存在下將細胞與該藥劑接觸，並偵測一種指標化合物的存在。基本上沒有指標物質表示該藥劑是一種抗發炎的藥劑。
本發明之詳細說明
本發明提供(1)區辨有害之細菌菌種和有利之口腔細菌的方法和(2)測定特定藥劑之抗發炎效果的方法。本發明同時提供利用三氯沙(triclosan)防止細胞中似關卡受體受到調控的方法。
在本說明書中所使用的「發炎」係指由損傷或破壞組織所引起之局部保護反應，其係用於破壞、稀釋或使有害物質和受損組織隱退。在急性形式當中，其特徵為疼痛、熱、發紅和腫脹。就組織學而言，發炎涉及一串複雜的事件，包括以增加的滲透性和血流使小動脈、微血管和小靜脈擴大；液體包括血漿蛋白滲出，以 及白血球移動到發炎的部位。發炎作用等於原發炎細胞調節因子或例如因為抗原與抗體作用或藉由抗原與致敏化的淋巴球作用所釋放的物質之量提高。延伸來說，抗發炎劑是任何相較於未經以藥劑處理之同樣細胞能在細胞中降低這些組織學作用之質和/或量的用劑。
本發明之一部份提供一種評估特定細菌菌種或菌株以測定是否此種菌種或菌株在口腔中有害的方法。「有害」一詞特意指其存在會造成受到影響之細胞產生發炎調節因子的致病細菌。某些細菌菌株/菌種已經被報導為有害者，包括以下表I中的細菌：
圖1是一張顯示細胞表面與微生物接觸的流程圖(發明人：Prem Sreenivasan；標題：調控細胞表面受體以預防或減少發炎之方法；快遞編號：EV 360790848 US；申請日期：2006年1月10日)。
圖2顯示似關卡受體(TLR)1、2、3、4、5、6、7和9的信息傳遞過程，以及藉由調節每種TLR所產生的分子(發明人：Prem Sreenivasan；標題：調控細胞表面受體以預防或減少發炎之方法；快遞編號：EV 360790848 US；申請日期：2006年1月10日)。
本方法包括使至少一種屬於待評估口腔細菌菌種或菌株的細菌與至少一種齒齦細胞接觸。或者，該齒齦細胞可與該細菌的一部分、細胞膜、細胞質內容物、該細菌的代謝物、終產物和/或副產物和/或致病因子接觸。
這個步驟可發生在體外，例如藉由維持細菌或細菌之一部分的培養物和齒齦細胞的培養物，並將此種培養物合併在一起。或者，此種接觸可發生在體內，其藉著把裝在適當遞送載體中的細 菌培養物施加到口腔。
在此種接觸以後，可以偵測到一種或多於一種指標物質。此種指標物質包括任何已知或是待發現之由宿主免疫反應之一般過程中產生的任何物質，包括中間體化合物、酵素、蛋白質、RNAs、DNAs、和涉及細胞生產細胞介素和其他原發炎調節因子之其他分子。較佳指標物質是由調控似關卡受體所產生者(例如圖2所示)，包括例如TLRs2、3、4、5、7和9；NFκ-B途徑的分子、細胞介素、介白素(例如1、6、8、12)、腫瘤壞死因子(TNF)、肽水解酶、以及編碼介白素或TNF次單元體和/或介白素、趨化素(例如CCL5、CCL4、CCL3、和CCL10)、和基質金屬蛋白酶的mRNAs。
偵測被挑選之指標物質可藉由本技藝中已知或是待研發的方式來實行，並且可以是一種相對或一種絕對的測量方法。偵測可藉由直接測量被挑選的指標物質的量來達成。或者，其可利用其他的偵測系統如放射和/或螢光標記物、抗體、特定基因的基因表現程度、mRNA分析、微陣列分析、和/或抗氧化劑狀態的變化來進行間接測量。例如，若該指標物質是一種酵素，吾人可將該試樣用一種適當的受質處理並且測量酵素催化反應的終產物以測定該酵素指標物質的存在與否。在本發明之此方面的實行上，基本上被挑選之指標物質的不存在即證明該細菌菌種/菌株並非是有害的-亦即不會以使細胞開始產生任何原發炎調節因子的方式影響該細胞。
本發明也包括評估一種特定藥劑之抗發炎能力或效果，亦即該藥劑在暴露於致病細菌的組織內降低或縮減發炎之能力的方法。該方法包括挑選一種待評估的藥劑。此種藥劑可為蛋白質、肽、有機分子、無機分子或任何者些物質的連接體。
在某些情況下，可能較好是該被挑選之待評估的藥劑並不會在口腔結構中表現出顯著的殺細菌效果。例如，該藥劑可能是從表現出最小抑制濃度(MIC)為0，小於大約5%，小於大約10%，小於大約20%和小於大約30%的用劑之中挑選出來的。
MIC研究一般包括在適當的溶劑中製備活性劑的溶液，並且緊接著連續稀釋溶離的活性劑。將被挑選細菌(這些細菌可能是一群細菌，每種都需要其本身特殊化的培養和處理方法)的標準懸浮液添加到每一種濃度的稀釋活性劑中。在37℃的適當條件下培育細菌+活性劑並且典型的在培育48小時之後監測細菌的生長情形。
MIC研究的對照組包括監測不添加任何溶劑或賦形劑的情況下細菌生長情形的生長對照組。其他對照組則監測用於試驗之培養基的無菌性。用以溶離細菌體上的活性劑之溶劑效果是最後一系列被包含於這些研究的對照組。
在培育之後，將培養盤在微盤分光光度計中讀取610nm波長的吸光值。其結果被解釋成活性劑抑制細菌生長之最低濃度。在高活性劑的濃度下細菌不會生長(以低的吸光值讀數值顯示)，而在低活性劑濃度下則會增殖(以高的吸光值讀數值顯示)。阻止細菌生長之最低活性劑濃度如MIC所定義。對照組應以如下方式進行：培養基之無菌性-沒有細菌生長
監測細菌生長-結果顯示極大量的生長
用於溶離活性劑的溶劑不應抑制細菌(因為它們必須本質上是無害的)。
評估特定藥劑之抗發炎能力或效果的方法包括使細胞與致病的或有害的細菌接觸之步驟。將被接觸的細胞可以是在體外或體內、原核或真核的、並且可得自細胞培養株或臨床試樣。
用於本方法之適當致病或有害的細菌包括任何本技藝中已知或待發現者，其以誘使免疫反應的方式來影響被挑選的細胞。此種細菌可包括例如列於以上表I中的細菌。
評估特定藥劑之抗發炎能力或效果的方法包括偵測一種或多於一種指標化合物的存在與否。指標化合物和偵測方法及系統可為任何以上那些經說明過者。
本發明亦包括含有被發現能藉由以上說明之檢驗展現抗發炎效果之藥劑的口服調配物，以及利用三氯沙(triclosan)與口腔組織細胞接觸來預防口腔組織細胞上似關卡受體被調控的方法。
本發明亦提供藉著以低於MIC的濃度將一種化合物如三氯沙施用到組織以降低或預防口腔組織發炎的方法。

Claims (10)

  1. 一種測定藥劑之抗發炎效果的方法，其包括：將該藥劑在有害細菌或該細菌之一部分的存在下與細胞接觸；並且偵測一種指標化合物的存在，其中基本上沒有該指標物質存即表示該藥劑為抗發炎藥劑。
  2. 根據申請專利範圍第1項的方法，其中該細胞為真核細胞。
  3. 根據申請專利範圍第1項的方法，其中該細胞為原核細胞。
  4. 根據申請專利範圍第1項的方法，其中該指標物質係選自藉由調控似關卡(toll-like)受體產生之分子所組成的群組。
  5. 根據申請專利範圍第1項的方法，該指標物質係選自藉由調控似關卡受體2(TLR 2)、TLR 3、TLR 4、TLR 5、TLR 7及TLR 9產生的分子所組成的群組。
  6. 根據申請專利範圍第1項的方法，其中該指標物質係選自NF-κB途徑之分子所組成的群組。
  7. 根據申請專利範圍第1項的方法，其中該指標物質係選自細胞介素與介白素所組成的群組。
  8. 根據申請專利範圍第1項的方法，其中該指標物質係選自IL-1、IL-6、IL-8、IL-12和TNF-α所組成的群組。
  9. 根據申請專利範圍第1項的方法，其中該指標物質係直接偵測的。
  10. 根據申請專利範圍第1項的方法，其中該藥劑係選自顯示MIC小於大約20%之藥劑。
TW102144529A 2006-01-10 2007-01-09 調控細胞表面受體以預防或減少發炎之方法 TW201415028A (zh)

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