TWI832531B - Rip1抑制劑或mlkl抑制劑用於治療或預防遺傳性視網膜失養症的用途 - Google Patents
Rip1抑制劑或mlkl抑制劑用於治療或預防遺傳性視網膜失養症的用途 Download PDFInfo
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Abstract
本發明提出一種RIP1抑制劑或MLKL抑制劑用於製備治療或預防遺傳性視網膜失養症之醫藥的用途。RIP1抑制劑例如為RIPA-56，而MLKL抑制劑例如為GW806742X。
Description
本發明攸關RIP1抑制劑或MLKL抑制劑的醫藥用途，且特別關於RIP1抑制劑或MLKL抑制劑用於治療或預防遺傳性視網膜失養症的用途。
遺傳性視網膜失養症（hereditary retinal dystrophy，HRD）屬於一群基因異質性與表型異質性的遺傳性視網膜疾病，例如：視網膜色素變性（retinitis pigmentosa，RP）、萊伯氏先天性黑朦症（Leber’s congenital amaurosis，LCA）、尤塞氏症候群（Usher's syndrome）、脈絡膜缺失症（choroideremia）、視網膜裂損症（retinoschisis）、及斯特格病變（Stargardt disease）。雖然醫療技術持續精進，但HRD仍為眼科醫師無法克服的重大障礙。研究發現至今超過280個基因與HRD有關，但目前仍未提出有效的治療方案。無法透徹瞭解HRD的難處在於如此不同且多樣的初級基因缺陷為何會同樣地導致HRD所呈現的表型。
隨著DNA技術的進步便開啟基因治療的發展。然而，對基因表現的認知有限與對基因物質的選擇造成基因治療至今尚未成功。目前著手進行多種疾病的基因治療，眼睛為具特殊免疫特性的小型封閉器官，故其為基因治療的主要標的之一。無創成影技術可比較治療前、後的結果。於確認HRD有關的突變基因後，臨床發展上證實基因治療為有效的。基因治療可產生患者的體細胞以產生特殊的治療蛋白質，進而調節遺傳性疾病。
礙於現今醫學科技對基因治療的認知仍嫌不足，故尋找分子藥物來治療HRD確實有迫切必要性。
本發明之目的在於提出一種RIP1抑制劑或MLKL抑制劑用於製備治療或預防遺傳性視網膜失養症之醫藥的用途。
示範地，所述RIP1抑制劑為RIPA-56。
示範地，所述MLKL抑制劑為GW806742X。
示範地，所述遺傳性視網膜失養症為Pomgnt1突變造成的遺傳性視網膜失養症。
示範地，所述Pomgnt1突變造成的遺傳性視網膜失養症為Pomgnt1
L120R/L120R突變造成的遺傳性視網膜失養症。
示範地，所述遺傳性視網膜失養症包括：視網膜色素變性、萊伯氏先天性黑朦症、尤塞氏症候群、脈絡膜缺失症、視網膜裂損症、或斯特格病變。
示範地，所述醫藥用以降低beclin 1、P62、或LC3B的表現量。
示範地，所述醫藥用以提升視網膜色素上皮細胞的跨膜上皮電阻值。
示範地，所述醫藥用以搭配基因治療。
示範地，所述基因治療包括：投予可表現蛋白質POMGNT1的核酸。
為讓本發明上述及/或其他目的、功效、特徵更明顯易懂，下文特舉較佳實施方式，作詳細說明於下：
本發明乃基於發現RIP1抑制劑或MLKL抑制劑對
POMGNT1基因剔除之人類視網膜色素上皮細胞（human retinal pigment epithelial cell，hRPE）的跨膜上皮電阻值（transepithelial electrical resistance，TEER）與野生型hRPE相當來得知的。
基於此，本發明之實施方式揭露一種RIP1抑制劑或MLKL抑制劑用於製備治療或預防遺傳性視網膜失養症之醫藥的用途。
所用的術語「RIP1抑制劑」意指其能有效抑制RIP1的活性（磷酸化）或能有效降低RIP1與RIP3的相互作用，實例可為但不限於RIPA-56。RIPA-56亦可以下化學式表示：
。
所用的術語「MLKL抑制劑」意指此物質能有效抑制MLKL的活性（磷酸化）或能有效降低MLKL與RIP1或RIP3的相互作用，實例可為但不限於GW806742X。GW806742X亦可以下化學式表示：
。
所用的術語「遺傳性視網膜失養症」意指基因異質性與表型異質性的遺傳性視網膜疾病。依基因層面而言，實例可為但不限於Pomgnt1突變造成的遺傳性視網膜失養症，如：Pomgnt1
L120R/L120R突變造成的遺傳性視網膜失養症；依病徵層面，實例可為但不限於視網膜色素變性、萊伯氏先天性黑朦症、尤塞氏症候群、脈絡膜缺失症、視網膜裂損症、或斯特格病變。
所用的術語「治療」意指藉由投予醫藥來減輕、改善、或緩解遺傳性視網膜失養症的症狀；所用的術語「預防」意指藉由投予醫藥來抑制或延緩遺傳性視網膜失養症的症狀。具體而言，可投予醫藥至有治療或預防需求的個體來實現治療或預防目的。更具體而言，可投予醫藥至有治療或預防需求的個體以透過抑制RIP1或MLKL來實現治療或預防目的。再具體而言，可投予醫藥至有治療或預防需求的個體以透過降低beclin 1、P62、或LC3B的表現量或改善視網膜色素上皮細胞的跨膜上皮電阻值來達到治療或預防目的。個體的實例可為但不限於哺乳類動物，哺乳類動物的實例可為但不限於靈長類動物、貓、狗、大鼠、小鼠、兔、牛、馬、山羊、綿羊、或豬，而靈長類動物的實例可為但不限於黑猩猩、人類、大猩猩、倭黑猩猩、紅毛猩猩、或猴。
所用的術語「投予」意指藉由適當方式將醫藥引入至個體內，實例可為但不限於經口、舌下、直腸、鼻腔、陰道、腹腔、經皮、表皮、關節內、眼球內、或眼球表面投予。依不同的投予途徑，醫藥可選擇呈現不同劑型，例如為但不限於錠劑、膠囊、顆粒、溶液、乳劑、塞劑、貼布、眼藥水、埋植片、或粉劑。
此外，投予醫藥時可搭配基因治療。基因治療可針對造成遺傳性視網膜失養症的基因缺失進行。例如，針對Pomgnt1
L120R/L120R突變造成的遺傳性視網膜失養症，可投予可表現蛋白質POMGNT1的核酸；至於其他基因突變造成的遺傳性視網膜失養症，可據此理解，無贅述之必要。
茲以下列實施例例示說明本發明：
＜實施例1：
Pomgnt1
L120R/L120R 老鼠具異常的視網膜電圖反應＞
POMGnT1同型合子L120R突變的患者於暗視視網膜電圖（electroretinogram，ERG）測試呈現降低或無法測得的b波，然其並無智力障礙、肌肉無力或萎縮。後肢伸展測試顯示未觀察到
Pomgnt1
L120R/L120R 老鼠具肌肉無力或萎縮。為於體內評估視網膜功能，於暗適應的暗視條件下以不同光刺激強度紀錄ERG。暗視ERG呈現視桿光受體（a波）、來自桿對桿的雙極細胞訊號（b波）、視網膜色素上皮（c波）。於光強度範圍0.0001至10cds/m
2紀錄取得的暗視ERG強度顯示視桿細胞與RPE的功能障礙。如圖1A至1D所示，可見於不同年紀族群（6、9、12個月）之
Pomgnt1
L120R/L120R 的a、b、c波振幅逐步降低。a、b、c波振幅降低的現象說明視桿光受體、桿狀雙極細胞突觸後訊號、與RPE於
Pomgnt1
L120R/L120R 老鼠的視網膜受到極大影響。
＜實施例2：投予AAV8-h
POMGNT1-GFP與RIPA 56至
Pomgnt1
L120R/L120R 老鼠改善電生理特性＞
對3個月大
Pomgnt1
L120R/L120R 老鼠與相同年紀的野生型老鼠注射AAV8-GFP（每μL1x10
9個載體基因組，視網膜下注射）、AAV8-h
POMGNT1-GFP（每μL1x10
9個載體基因組，視網膜下注射）、RIPA 56（老鼠每公斤10mg，每週2次，腹腔注射）、及AAV8-h
POMGNT1-GFP結合RIPA 56，並於注射後評估3、6個月。注射3個月後可觀察到相對AAV8-GFP，AAV8-h
POMGNT1-GFP、RIPA 56、以及AAV8-h
POMGNT1-GFP結合RIPA 56的暗視ERG檢測a、b、c波振幅獲致大幅改善（如圖2A、2B）。此外，AAV8-h
POMGNT1-GFP結合RIPA 56的a、b波振幅接近於相同年紀野生型老鼠的數值。注射6個月後可觀察到相對AAV8-GFP控制組，ERG檢測亦獲得改善（如圖2A、2C）。上述結果顯示注射6個月後僅注射AAV8-h
POMGNT1-GFP可有效地改善
Pomgnt1
L120R/L120R 老鼠的電生理表現。
＜實施例3：細胞程序性壞死為
Pomgnt1
L120R/L120R 老鼠視網膜變性的主因之一＞
S-arrestin（Sag）為一種G蛋白偶聯受體（G-protein coupled receptor），其於視紫紅質（rhodopsin）去敏感化過程扮演重要角色，以於視桿光受體中抑制光激活的光傳導。先前已發現Sag透過網格蛋白（clathrin）依賴型胞吞作用形成的視紫紅質-arrestin穩定複合體參與光誘導的光受體細胞凋亡。此外，先前亦已確認Sag為自體抗原，會於虹彩炎（uveitis）患者中啟動免疫反應。
Enolase 1（Eno 1）為一種糖解胞質酶，並隨處表現於不同組織。Sag與Eno 1的作用已證實可降低Eno 1的催化活性約25％。光受體為體內最耗能的細胞之一，其於暗適應條件下消耗10
8ATP/秒/細胞。有氧糖酵解代謝效率的交替可對光受體產生極大衝擊。基於本發明人的研究，期望測試以下假設：L120R突變強化並穩定Sag-Eno 1複合體，進而於視網膜內降低糖解活性並啟動發炎反應。後續的能量缺乏與發炎會活化細胞程序性壞死。
Eno 1與Sag蛋白質的表現於
Pomgnt1
L120R/L120R 視網膜均向上調控（如圖3A、3B）。自噬反應相關蛋白質的表現，如：beclin 1、P62、與LC3B，於
Pomgnt1
L120R/L120R 視網膜均提升，此意謂自噬反應流量受抑制。RIP3與MLKL於野生型視網膜均維持於低表現量，但於
Pomgnt1
L120R/L120R 視網膜卻顯著提升。RIP3為細胞程序性壞死的關鍵調控因子，其表現量呈現與細胞程序性壞死一致。MLKL為於細胞程序性壞死過程中RIP3的主要下游影響。免疫組織化學與免疫點墨表明細胞程序性壞死調控的訊號於
Pomgnt1
L120R/L120R 模型為視網膜神經元死亡的重要驅動子。
基於上述發現，提出
Pomgnt1
L120R/L120R 模型神經元死亡的機制（如圖4）。根據此機制，L120R突變蛋白質可強化並穩定Sag-Eno 1複合體，以於視網膜中驅動發炎反應並降低糖解活性。後續的ATP減少與持續的發炎反應進一步活化RIP3與MLKL，並抑制自噬反應流量，從而導致視網膜神經元細胞的細胞程序性壞死而死亡。
＜實施例4：治療策略的發展＞
1、於
Pomgnt1基因剔除人類RPE細胞轉移AAV調控的
Pomgnt1基因
市售購買編碼人類Pomgnt1編碼序列的質體（GeneCopeia, Inc. Rockville，Maryland，USA），並包裝至血清第8型的AAV載體。質體含有EF1a啟動子與融合至Pomgnt1下游的增強綠色螢光蛋白（enhanced green fluorescent protein，eGFP）（AAV8-Pomgnt1-eGFP）。含有包含eGFP之相同調控序列但無Pomgnt1的空白載體（AAV8-eGFP）作為控制組。
AAV轉導後，於
POMGNT1基因剔除人類RPE細胞中的eGFP免疫螢光染色呈現高品質的eGFP表現（如圖5）。RPE為極化的單層上皮，其介於光受體與脈絡膜間。RPE為透過維持許多重要功能使神經視網膜健康的關鍵，這些功能包含：光吸收、跨膜上皮輸送、吞噬、與分泌生長因子。健康RPE的特徵在於：透過介於上皮細胞間之特殊緊密連接（tight junction）形成的跨膜上皮電阻值。使用具穿孔培養的Millicell ERS Voltmeter（MERS00002，EMD Millipore）於野生型單層RPE與於
POMGNT1基因剔除細胞的單層RPE檢測跨膜上皮電阻值，進而評估單層細胞的完整性。如圖6所示，相對野生型，TEER程度於
POMGNT1基因剔除細胞顯著降低。此外，AAV8-Pomgnt1-eGFP轉導後，於
POMGNT1基因剔除細胞可觀察到TEER程度大幅改善。
高基氏體（Golgi complex，GC）由一或多個扁平內腔的堆疊，其主要分布於核周圍。於神經退化過程中，GC會破碎成許多的小型分離組成。GC碎片化為許多神經退化性疾病的早發事件。共軛焦顯微鏡照片顯示GM130染色的小點於
POMGNT1基因剔除細胞中廣泛分布。AAV8-Pomgnt1-eGFP的轉導可於POMGNT1缺乏的細胞中大幅地降低GC碎片化（如圖7A、7B）。
2、RIP1抑制劑（RIPA-56）與MLKL抑制劑（GW806742X）對
Pomgnt1基因剔除人類RPE細胞的影響
依先前的結果可知，細胞程序性壞死為
Pomgnt1
L120R/L120R 老鼠視網膜壞死的主因。投予RIPA-56或GW806742X至
POMGNT1基因剔除的人類RPE細胞，其中RIPA-56為有潛力且可選擇的RIP1抑制劑，GW806742X為MLKL抑制劑。如圖8所示，於投予RIPA-56或GW806742X後，
POMGNT1基因剔除細胞的TEER程度大幅改善。
圖3A、3B的結果呈現自噬反應相關蛋白質的表現，如：beclin 1、P62、與LC3B，於
Pomgnt1
L120R/L120R 視網膜均提升，藉此可抑制自噬流量。先前的結果提出細胞程序性壞死調控的訊號於
Pomgnt1
L120R/L120R 老鼠模型為視網膜神經元死亡的重要驅動子。
POMGNT1基因剔除人類RPE細胞的免疫點墨分析亦呈現beclin 1與P62的表現量增加、LC3BII/LC3BI比值的增加、與細胞程序性壞死標誌的活化，如：磷酸化-RIP3與磷酸化-MLKL（如圖9A、9B）。投予RIPA-56可於
POMGNT1基因剔除人類RPE細胞中大幅降低beclin 1、P62、與LC3B的表現量與抑制磷酸化-RIP3與磷酸化-MLKL，從而改善TEER程度。
惟以上所述者，僅為本發明之較佳實施例，但不能以此限定本發明實施之範圍；故，凡依本發明申請專利範圍及發明說明書內容所作之簡單的等效改變與修飾，皆仍屬本發明專利涵蓋之範圍內。
無
圖1A為一暗視ERG，呈現不同年紀族群之
Pomgnt1
L120R/L120R 老鼠的a、b波；
圖1B為一曲線圖，比較不同年紀族群之
Pomgnt1
L120R/L120R 老鼠的a、b波振福；
圖1C為一暗視ERG，呈現不同年紀族群之
Pomgnt1
L120R/L120R 老鼠的c波；
圖1D為一曲線圖，比較不同年紀族群之
Pomgnt1
L120R/L120R 老鼠的c波振福；
圖2A為一暗視ERG，呈現
Pomgnt1
L120R/L120R 老鼠經不同處理後3、6個月的a、b波；
圖2B為一曲線圖，比較
Pomgnt1
L120R/L120R 老鼠經不同處理後3個月的a、b波振福；
圖2C為一曲線圖，比較
Pomgnt1
L120R/L120R 老鼠經不同處理後6個月的a、b波振福；
圖3A為一免疫點墨照片圖，呈現
Pomgnt1
L120R/L120R 老鼠的視網膜蛋白質表現；
圖3B為一長條圖，比較
Pomgnt1
L120R/L120R 老鼠之視網膜的蛋白質相對表現量；
圖4為一示意圖，說明
Pomgnt1
L120R/L120R 老鼠神經元死亡的機制；
圖5為一螢光顯微鏡照片圖，呈現AAV轉導至細胞後的結果；
圖6為一長條圖，呈現AAV轉導至細胞後的TEER結果；
圖7A為一共軛焦顯微鏡照片，呈現AAV轉導至細胞後高基氏體的狀態；
圖7B為一長條圖，呈現AAV轉導至細胞後高基氏體的破碎程度；
圖8為一長條圖，呈現RIPA-56或GW806742X投予至細胞後的TEER結果；
圖9A為一免疫點墨照片圖，呈現RIPA-56或GW806742X投予至細胞後的蛋白質表現；
圖9B為一長條圖，比較RIPA-56或GW806742X投予至細胞後的蛋白質相對表現量。
Claims (3)
- 一種RIP1抑制劑用於製備治療遺傳性視網膜失養症之醫藥的用途，該遺傳性視網膜失養症為視網膜色素變性，該RIP1抑制劑為RIPA-56。
- 如請求項1所述之用途，其中該遺傳性視網膜失養症為Pomgnt1突變造成的遺傳性視網膜失養症。
- 如請求項2所述之用途，其中該Pomgnt1突變造成的遺傳性視網膜失養症為Pomgnt1L120R/L120R突變造成的遺傳性視網膜失養症。
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