TWI796875B - 一種醫藥組合物用於製備眼表損傷之藥物的用途 - Google Patents
一種醫藥組合物用於製備眼表損傷之藥物的用途 Download PDFInfo
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Abstract
本發明提供一種醫藥組合物用於製備治療由眼病或眼損傷如乾眼症、化學或物理損傷、感染、神經感覺異常和未指定病因引起的眼表損傷藥物的用途,其中該醫藥組合物包含治療有效劑量的microRNA-328拮抗劑。
Description
本發明提供醫藥組合物用於製備眼表損傷之藥物的用途,涉及生醫領域和醫藥的應用,目的是提供一種在用於製備治療由眼病或眼損傷如乾眼症、化學或物理損傷、感染、神經感覺異常和未指定病因引起的眼表損傷藥物的用途,其中該醫藥組合物包含治療有效劑量的microRNA-328拮抗劑。
眼睛的表面由角膜和結膜組成,眼表損傷會導致疼痛、發紅、視力不佳,並最終導致永久性角膜或結膜損傷。
許多眼部疾病或眼部損傷可能導致眼表損傷的發生,乾眼症是最常見的眼表疾病,它是由淚液和眼表等多因素導致的疾病,會產生不適、視力障礙和淚膜不穩定的症狀。
國際淚膜和眼表協會(TFOS)組織的國際乾眼研討會(DEWS)報告了“TFOS DEWS II定義和分類”(Ocul Surf.2017 Jul;15(3):276-283)並提供定義本發明所提到的乾眼症,環境因素也經常與乾眼症有關,包括暴露於污染物、紫外線(UV)輻射和臭氧,以及長期使用含防腐劑的眼藥水,例如治療青光眼的眼藥水,這些因素會增加氧化壓力和眼表炎症,
從而導致乾眼症的發展。
最近,瞼板腺功能障礙被認為是乾眼症的另一個主要危險因素,乾眼症的典型症狀是乾燥、灼熱和沙質眼刺激,隨著時間的推移會變得更糟,雙眼通常都會受到影響,眼睛乾澀一段時間會導致眼睛表面的微小擦傷、角膜糜爛和點狀角膜病變,在晚期病例中,上皮發生病理變化,即上皮細胞轉變成鱗狀化和杯狀細胞數目減少。
化學傷害包括對眼睛的鹼和酸傷害,對眼表造成廣泛的傷害,如果角膜修復不完整,這種損傷可能會導致永久性視力損傷,從異物、創傷或金屬碎片導致的物理傷害都可能對眼表造成不同程度的損傷,眼部感染可導致角膜糜爛,如果角膜上皮不重新生長,那麼角膜糜爛可能發展成角膜潰瘍,再未經治療的話,角膜潰瘍則會導致嚴重的視力喪失。
神經營養性角膜病變,也稱為神經營養性角膜炎(neurotrophic keratopathy,NK),其特徵在於角膜敏感性降低或缺失,因為角膜神經支配受損,角膜感覺的喪失可能導致上皮性角膜病變、上皮缺損、間質潰瘍,最終導致角膜穿孔,這是一種罕見疾病,估計患病率低於5/10,000。
NK的病因包括但不限於皰疹性角膜炎(帶狀皰疹和單純性)、長期使用含有苯扎氯銨(benzalkonium chloride,BAC)的眼藥、化學和物理燒傷、隱形眼鏡濫用、角膜手術如激光原位角膜磨鑲術(laser in situ keratomileusis,LASIK)等,人類神經生長因子(NGF)已被證明可有效治療NK患者,含有重組人神經生長因子(rhNGF)的Cenegermin(OxervateTM)是2018年8月22日在美國獲批用於治療神經營養性角膜炎的首個NK局部用藥。
杯狀細胞局限於結膜上皮,杯狀細胞的主要功能是產生和分泌粘蛋白,從而滋潤和潤滑眼表,粘蛋白是高度糖基化的糖蛋白,粘蛋白分為兩種不同的類型:跨膜粘蛋白和分泌型粘蛋白,MUC5AC是研究最多的粘蛋白之一,它是一種在結膜中發現在大型凝膠形成時所分泌的粘蛋白,在乾眼症患者中已經證明結膜中杯狀細胞數量減少,此外,隱形眼鏡的使用會改變杯狀細胞的密度。
小分子核糖核酸(microRNA,miRNA)是長度約21-23個核苷酸的非編碼單鏈RNA分子,在動物體內成熟的miRNA會與一種或多種信使核醣核酸(message RNA,mRNA)的3'端的非轉譯區(untranslated region,UTR)互補,miRNA與其目標mRNA的黏合(annealing)導致蛋白質轉譯和/或mRNA裂解的抑制,先前曾報導過microRNA-328(miR-328)是近視的危險因素(Chen等人,Invest.Ophthalmol.Vis.Sci.,53:2732-2739,2012)和Anti-miR-328寡核苷酸用在治療近視(Juo等人,US10179913B2,2019)。
本發明提供一種醫藥組合物用於製備治療由眼病或眼損傷如乾眼症、化學或物理損傷、感染、神經感覺異常和未指定病因引起的眼表損傷之藥物的用途,其中該醫藥組合物包含有效劑量的microRNA-328拮抗劑。
本發明之說明書中所述的「受試者」在此指的是動物,尤其是哺乳動物,於一實施例中,所述的「受試者」泛指人類,且不限於特定一個或是多個。
本發明之說明書中使用的術語「治療有效」旨在限定每種藥
劑的劑量,實現降低疾病嚴重程度為目標,同時避免不良副作用,例如通常與替代療法相關的副作用。
於一實施例中,本發明之組合進一步包含醫藥上可接受的鹽類、載劑、佐劑或賦形劑。
另一實施例中,本發明之miR-328拮抗劑是Anti-miR-328寡核苷酸,其中包含一段核苷酸序列,且與miR-328或前驅物互補。
於一實施例中,Anti-miR-328寡核苷酸序列(15-22個核苷酸長度)根據人類成熟miR-328(mature miR-328)的序列(SEQ ID NO:1)設計,Anti-miR-328寡核苷酸序列的長度為15-22個核苷酸長度(SEQ ID NOs:2-9),該等序列見於下表1,本發明提到的Anti-miR-328寡核苷酸序列皆在先前專利US10179913B2已被揭露,該專利全文併入本發明說明書中供參考。
於一實施例中,Anti-miR-328為15-22個核苷酸長度;於另一實施例中,Anti-miR-328為16或17個核苷酸長度,於較佳實施例中,Anti-miR-328寡核苷酸係由SEQ ID NO:3或SEQ ID NO:4組成;於另一段實施例當中,Anti-miR-328寡核苷酸係由SEQ ID NO:3組成。
於一實施例中,本發明之一種醫藥組合物用於製備治療眼表損傷的藥物的用途,其中Anti-miR-328寡核苷酸的濃度為1-500uM。
另一實施例中,本發明之一種醫藥組合物用於製備治療眼表損傷的藥物的用途,其中Anti-miR-328寡核苷酸的濃度為10-160uM。
本發明提供一種醫藥組合物,包含Anti-miR-328寡核苷酸以及醫藥上可接受的載體,其用於治療眼部疾病的方式係透過局部的溶液或軟膏,於一實施例中,本發明之醫藥組合物施用於眼睛;於另一實施例中,該醫藥組合物的投藥方式係以眼藥水的形式。
圖1為兔角膜細胞株miR-328表現量在不同濃度的BAC的長條圖。
圖2為兔角膜細胞株中Anti-miR-328濃度與NGF的表現量關係之長條圖。
圖3為磷酸鹽緩衝鹽水(PBS)或Anti-miR-328治療乾眼症兔後的角膜螢光染色的代表圖。
圖4(A)為正常、PBS和Anti-miR-328組中角膜的代表性H&E染色;4(B)為不同組的上皮層和基質厚度的散點圖。
圖5(A)為不同組組織染色圖;(B)為不同組間角膜細胞凋亡
的散點圖;(C)為不同組間的基質細胞凋亡的散點圖。
圖6為PBS與Anti-miR-328對於兔瞼板腺孔口的染色圖。
圖7為第14天時,不同劑量染色圖。
圖8為Anti-miR-328修復小鼠眼中的角膜磨損的結果染色圖,(A)為第0天染色圖;(B)為第3天染色圖。
以下的實施例並沒有限制的作用或功能,僅代表本發明之多個面向及特色的示例以及文字說明。
實施例1,Anti-miR-328寡核苷酸在活體外的研究。
兔角膜(SIRC)細胞株培養在DMEM的培養基中,並補充10%胎牛血清(FBS)和100U/mL青黴素,在37℃溫度下和5% CO2中培養,細胞暴露於苯扎氯銨(benzalkonium chloride,BAC)10分鐘,然後用新鮮培養基替換原先的培養基,為了測量miR-328表現量,接著將細胞再培養24小時,然後收集這些細胞進行RNA萃取,為了測量NGF的表現,在細胞暴露於BAC10分鐘後,接著細胞使用Anti-miR-328處理持續12小時。
實施例2,利用老鼠模型進行活體研究,評估Anti-miR-328寡核苷酸序列治療乾眼症的效果。
乾眼症的老鼠模型
苯扎氯銨(BAC)是一種常用的眼藥水防腐劑,然而BAC對於眼睛來說是有毒性,包含結膜發炎和纖維化、淚膜不穩定、眼角膜細胞毒性以及前房發炎,也因為毒性作用,高濃度的BAC被廣泛使用於動物模型中誘導乾眼症的發生。
首先是C57BL6小鼠被隨機分配,一個為給予Anti-miR-328組;另一個則是PBS鹽水組,值得注意的是,PBS作為一種Anti-miR-328的眼藥水的溶劑,向C57BL6小鼠每天在雙眼滴入5μL濃度0.2% BAC,持續7天以誘發乾眼症,到了第8天,每隻眼睛開始接受Anti-miR-328(10μM)或是PBS鹽水,並持續2周,而這2周期間仍要持續滴入BAC到雙眼。
這2周實驗期間的治療順序是:首先滴入PBS或是Anti-miR-328,然後在大約10分鐘後滴入BAC,這個策略是模擬患者接受乾眼症治療時無法去除乾眼的原因。
如果有兩組動物在第7天具有可進行比較的角膜染色分數,那麼會使用student-t檢定評估治療效果,否則會使用配對t檢定(paired t-test)用以評估治療效果。
結果評估
藉由連續2週的PBS/Anti-miR-328治療,以評估Anti-miR-328的治療效果,每天進行臨床觀察,每週進行螢光染色,對於角膜螢光素染色,在螢光素紙條(Madhu Instruments Pvt.Ltd.,Okhla Industrial Area,India)上滴入製備1%螢光素鈉,隨後將5μL溶液滴入結膜囊中,在帶有鈷藍濾光片的裂隙燈SL-15(Kowa公司,東京,日本)下對眼睛進行檢查和分級。
根據FDA批准的乾眼症藥物Lifitegrast 3期臨床試驗的角膜螢光素染色分級標準,稍作修改如下,角膜表面被等分為9個區域,每個區域的評分範圍為0到4分(最高分為36分),增量為0.5分,分數越低表示情況越好,評分為:“0”無染色,“1”很少或罕見的點狀病變,“2”離散且可計數的病變,“3”病變數量太多無法計數;和“4”融合病灶。
結果
本次實驗總共有19之公鼠,其中9隻被分類到安慰劑組(PBS)以及10隻小鼠是Anti-miRNA-328組,因此就結果來說,總共有18隻眼睛是滴入安慰劑以及20隻眼睛是滴入Anti-miR-328。
PBS組以及Anti-miR-328組都能夠顯著改善乾眼症在螢光染色的表現,然而Anti-miR-328的治療有達成更優秀的表現,如下表二所示,從第7天和第21天的觀察結果顯示,PBS組中修改後染色分數為31.15和17.94(p=0.0003,配對t檢定,表2)和Anti-miRNA-328組的分數為31.70和8.2(p=1.67 x 10-9,配對t檢定,表2),證明給予Anti-miR-328比給予PBS有更大療效。
實施例3,利用兔子模型進行活體內研究,評估Anti-miR-328寡核苷酸序列對乾眼症的治療效果。
乾眼症的兔子模型
材料和方法
在研究中使用眼睛有色素的雷克斯兔(Rex rabbit),並且使用
BAC誘導乾眼症之前,將兔子隨機分配到PBS治療組或Anti-miR-328治療組,乾眼症是透過每天兩次(9:00和17:00)滴入20μL濃度為0.15% BAC(Sigma-Aldrich,St.Louis,MO,USA)誘導產生,持續1週後,在第8天,兔子開始接受PBS(安慰劑)或Anti-miR-328治療,每天兩次,持續2週,而BAC仍在這兩週內保持每天兩次滴入眼中,於2週期間的治療順序是先眼部滴入PBS或Anti-miR-328,然後再間隔10分鐘滴入BAC。
結果評估
如前段所述,持續2週的Anti-miR-328/PBS處理後,評估其治療效果,同時使用稱為“眼部總分”的第二個分級來評估對兔眼的治療效果。
“眼部總分”由角膜和隱形眼鏡研究單位(CCLRU)分級量表和已發布的指南修改而來(參見Takamura E.等,日本過敏性結膜疾病指南2017.Allergol Int.2017;66:220-229;and Su G.,Wei Z.,Wang L.,et al.Evaluation of Toluidine Blue-Mediated Photodynamic Therapy for Experimental Bacterial Keratitis in Rabbits.Transl Vis Sci Technol.2020;9:13),並且量表基於以下4個領域:角膜緣充血、球結膜充血、跗結膜充血和角膜炎,前3個域有4個嚴重級別(從0-3),角膜炎有5個嚴重級別(從0-4),兔眼也用於組織學檢查,包括角膜和瞼板腺。
組織學分析
第21天的時候,將兔子進行人道處理,接著將右眼摘除並進入至浸入戴維森固定液(Davidson's fixative)(20ml 37%福爾馬林、100ml冰醋酸、350ml 95%酒精和530ml水),組織在進行修剪和加工前,先將組織
固定48小時,然後用清水沖洗並轉移至10%的中性溶液-福馬林。
此外,眼睛的附屬結構移除後立即固定在甲醛溶液24小時,隨後,收集的樣本(角膜、結膜和瞼板腺)用梯度序列的乙醇脫水並包埋在石蠟中,切片機切片後,包埋的組織塊用蘇木精和伊紅(H&E)染色進行組織學檢查,在自動數位載玻片掃描儀(Pannoramic mini II,3dhitech Ltd.,Budapest,Hunger)下觀察所有標本的組織學圖像,並使用CaseViewer軟體(https://www.3dhistech.com/caseviewer)進行可視化和測量。
TUNEL分析是用來呈現評估角膜上皮和基質中細胞凋亡的工具,根據製造商的說明(Roche,Indianapolis,IN),使用原位細胞死亡檢測試劑盒POD(編號11684 817910)進行TUNEL測定,在40倍大小的顯微鏡下,三個隨機選擇的視野中計數凋亡的細胞。
評估上瞼瞼板腺孔口角化過度,係以計算1200μm組織學載玻片上每個孔口角化過度造成阻塞的百分比,一張載玻片中所有孔口阻塞的平均值,代表其對這一特定眼睛的治療效果。
結膜印跡細胞學
在第0、7、14和21天收集結膜印跡細胞學的標本,在滴入0.5%愛爾卡因(Alcaine)並擦去眼睛中過量的液體後,用半圓形的硝酸纖維素濾紙(Toyo Roshi Kaisha,Ltd.,日本)直徑5.5mm置於上球結膜上,將濾紙通過輕微的壓力保持在原位1分鐘,然後從眼睛上剝離並立即用10%中性緩衝液-福馬林固定,接著按照製造商的說明(PAS-2-IFU,ScyTek Laboratories,Inc.,Logan,U.S.A.)用PAS試劑盒對紙進行染色,利用PAS試劑對組織染色後,在400倍顯微鏡下計數杯狀細胞的數量,染色後定量杯
狀細胞的密度。
實施例4,利用物理損傷導致角膜擦破以及使用Anti-miR-328寡核苷酸的療效評估。
角膜擦破的小鼠模型
材料和方法
C57BL/6小鼠先處於麻醉狀態,並在雙眼滴入愛爾卡因,用以減少角膜物理損傷期間的任何後續不適,為了製造角膜擦破,使用已經清潔過的眼部毛刺(Algerbrush),用手指分別握住眼瞼,一次睜開一隻眼睛,然後將Algerbrush牢牢地接觸到角膜,並在眼表上前後和側向移動Algerbrush以引起角膜擦破,手術後進行角膜螢光染色,之後每天進行角膜螢光染色,每一隻老鼠左眼滴PBS,右眼滴Anti-miR-328,採取每天兩次(上午9點和下午5點)。
以上述角膜上面螢光染色的大小作為結果評估。
結果
BAC對於miR-328的影響
兔角膜細胞株(SIRC)用不同濃度的BAC處理,miR-328表現量隨著BAC的濃度增加(圖1),數據,平均值±SEM,來自3個獨立實驗,Anti-miR-328可以增加BAC處理後的兔角膜細胞SIRC的NGF表現量(圖2)。
總共40隻眼接受PBS治療和42隻眼睛接受Anti-miR-328的治療,Anti-miR-328顯示其具有減少兔眼角膜染色的治療作用,如圖3所示,本發明中使用PBS作為陰性對照,從第0天到第21天,BAC誘導乾眼症,從第
8天到第21天開始使用PBS(上圖)或Anti-miR-328(下圖)眼藥水,綠色螢光是角膜染色,這表明由於乾眼症造成的角膜損傷,在第21天,Anti-miR-328處理的兔子沒有角膜染色,而PBS組仍有角膜染色,經過治療2週後,使用Anti-miR-328眼藥水顯著降低了改良染色評分(p=0.038,配對t檢定,表3),但PBS對角膜染色沒有影響(p=0.699,配對t檢定,表3)。
當以眼部總分分析數據時,觀察到類似的結果,該分數的平均值在PBS組中顯著變差的現象(p=7.4 x 10-8,配對t檢定,表3),而Anti-miR-328組有邊際改善(p=0.053,配對t檢定,表3)。
*PBS組顯著更糟
角膜厚度
使用Anti-miR-328或PBS(安慰劑)眼藥水治療的乾眼症兔,在角膜上皮的平均厚度有顯著差異,PBS處理的眼睛中,其角膜上皮變薄
(36.4±1.2μm vs 25.6±1.7μm,p=9.4x10-5),而正常兔子為45.4±1.2μm(圖4(A)),正常、PBS處理和Anti-miR-328處理的眼睛之間的基質沒有統計學差異(p=0.34)(578.8±25.0μm vs 539.5±31.8μm,圖4(B))。
圖5(A)為顯示三種不同組的組織染色圖,正常兔眼的基質厚度為521.2±20.4μm,在TUNEL檢測顯示PBS組比Anti-miR-328組在角膜上皮(53±3個細胞對39±3個細胞,P=0.002)(圖5(B))和基質(84±7個細胞對65±5個細胞),P=0.029)(圖5(C))凋亡細胞的情況,與PBS處理的眼睛相比,Anti-miR-328處理的眼睛在角膜上皮和基質層中的凋亡細胞更少,而正常眼睛幾乎沒有凋亡細胞。
瞼板腺組織學
從組織學顯示Anti-miR-328組相比PBS組具有更低的平均阻塞百分比(56.4%±7.59% vs 78.5%±8.17%,p=0.059),如圖6所示,儘管差異並未達到0.05的顯著水準,在PBS處理的兔眼中顯示瞼板腺口中的角化過度(箭頭所指的位置),但在Anti-miR-328治療的兔眼中沒有出現角化過度的現象。
結膜杯狀細胞
Anti-miR-328組的結膜杯狀細胞密度顯著高於PBS組(26 vs 19cell/mm2,p=0.005),這一發現與先前關於乾眼症中杯狀細胞密度低的報告一致。
劑量依賴效應
為了找到對乾眼症達到最大治療效果的劑量,本發明在患有乾眼症的小鼠中測試以下Anti-miR-328劑量,如圖7所示,分別是:10、30、60、90、120和160μM,且也持續給予濃度為0.2% BAC,數據顯示Anti-miR-328對減少角膜染色具有劑量依賴性,並且用劑量160μM的Anti-miR-328的眼藥水治療的眼睛在第14天幾乎沒有螢光染色。
通過BAC誘導小鼠眼睛患有乾眼症,並同時用Anti-miR-328(間隔10分鐘)每天兩次治療,持續14天,於第14天拍攝,數據顯示160μM的劑量治療效果最好,因此,160μM的Anti-miR-328可能是治療小鼠乾眼症的最佳劑量。
由物理損傷引起的角膜擦破和Anti-miR-328寡核苷酸的功效評估
為了證明Anti-miR-328對角膜修復的影響,小鼠角膜經由Algerbrush造成損傷,之後於第0天透過角膜染色觀察在整個角膜上的螢光,顯示角膜完全擦破(圖8(A)),從發生角膜擦破的同一天開始,每天滴兩次眼藥水,左眼滴PBS,右眼滴Anti-miR-328(160μM),在第3天時候,發現使用Anti-miR-328治療的眼睛比給予PBS治療的眼睛恢復更快更好(圖8(B))。
本發明以及製造和使用它的方法和過程,現在以完整、清晰、簡潔和準確的術語進行描述,以使所屬領域中的任何技術人員能夠製作和使用本發明之內容,應當被理解的是前面描述了本發明的優選實施例,並且可以在不脫離申請專利範圍的前題下,闡述本發明的範圍和對其進行修改,為了特別指出並清楚地要求保護被視為發明的主題,接著以申請專利範圍總結本發明之說明書。
<110> 晴航生物醫學股份有限公司
<120> 一種醫藥組合物用於製備眼表損傷之藥物的用途
<130> 3717
<150> US63134599
<151> 2021-01-07
<160> 9
<170> Patentln version 3.5
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<212> RNA
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<212> RNA
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<212> RNA
<213> Homo sapiens
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<211> 18
<212> RNA
<213> Homo sapiens
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<212> RNA
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<212> RNA
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<213> Homo sapiens
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Claims (4)
- 一種醫藥組合物用於製備治療眼表損傷的藥物的用途,其中該醫藥組合物包含治療有效劑量之microRNA-328的拮抗劑,該microRNA-328的拮抗劑為一Anti-miR-328寡核苷酸,且該Anti-miR-328寡核苷酸是由SEQ ID NO:3或SEQ ID NO:4所組成,其中該眼表包含角膜、結膜、淚腺、以及眼皮內的瞼板腺和睫毛根部組織。
- 如請求項1所述之用途,其中該眼表損傷係由乾眼症、化學或物理損傷、瞼板腺功能障礙、感染、神經營養性角膜病變、神經感覺異常或未指定病因所引起。
- 如請求項1所述之用途,其中該Anti-miR-328寡核苷酸的濃度為1-500μM。
- 如請求項1所述之用途,其中該Anti-miR-328寡核苷酸的濃度為10-160μM。
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期刊 王婷等, "microRNA146a在乾眼中的表達及作用", 《眼科新進展》 第38卷第10期 2018年 930-934。 |
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