TW202239962A - 一種抑制b型肝炎病毒基因表達的rna抑制劑及其應用 - Google Patents
一種抑制b型肝炎病毒基因表達的rna抑制劑及其應用 Download PDFInfo
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Abstract
本發明屬於生物化學領域,具體涉及一種抑制B型肝炎病毒基因表達的RNA抑制劑及其應用,所述的RNA抑制劑由正義鏈和反義鏈通過鹼基配對形成,正義鏈和反義鏈彼此之間至少85%鹼基互補,且部分或全部核苷酸糖基2’位的-OH被氟或甲氧基取代,末端至少有連續3個核苷酸之間的磷酸酯被硫代。本發明的RNA抑制劑的結構中還含有5’MVIP和3'MVIP,使所述RNA抑制劑具有特異性的肝靶向。本發明提供的RNA抑制劑具有當前臨床一線B型肝炎治療藥物所不具有的功效,可以持續高效抑制HBV表面抗原HBsAg的合成,並有表面抗體HBsAb產生。本發明的RNA抑制劑對HBV最常見的A、B、C和D型都具有顯著的抑制作用。本發明的RNA抑制劑與核苷類似物或干擾素聯合使用,可以功能性治癒B型肝炎。
Description
本發明屬於生物化學領域,具體涉及一種抑制B型肝炎病毒基因表達的RNA抑制劑及其應用,所述的RNA抑制劑由正義鏈和反義鏈通過鹼基配對形成,正義鏈和反義鏈彼此之間至少85%鹼基互補,且部分或全部核苷酸糖基2’位的-OH被氟或甲氧基取代,末端至少有連續3個核苷酸之間的磷酸酯被硫代。本發明的RNA抑制劑的結構中還含有使所述RNA抑制劑具有肝靶向特異性的結構5’MVIP和3'MVIP,其中5’MVIP偶聯在所述RNA抑制劑正義鏈和/或反義鏈5’末端,3’MVIP偶聯在所述RNA抑制劑反義鏈和/或正義鏈3’末端,5’MVIP和3’MVIP都包含有肝靶向特異性配體X、支鏈L、接頭B和連接鏈D,5’MVIP還包含與所述RNA抑制劑正義鏈或反義鏈5’末端連接的轉接點R
1,3’MVIP還包含與所述RNA抑制劑正義鏈或反義鏈3’末端連接的轉接點R
2,所述肝靶向特異性配體X、支鏈L或接頭B在5’MVIP與3’MVIP各自的內部或5’MVIP與3’MVIP之間可以相同,也可以不同。本發明提供的RNA抑制劑具有當前臨床一線B型肝炎治療藥物所不具有的功效,可以直接破壞HBV mRNA作為翻譯範本的功能,阻止HBV表面抗原HBsAg的合成。另外,本發明的RNA抑制劑對HBV最常見的A、B、C和D型都有顯著的抑制作用;可以與核苷類似物及干擾素聯合使用;可以持續高效地使HBV小鼠的HBsAg的表達水準減少,並產生表面抗體HBsAb,可以功能性治癒B型肝炎。
RNAi
RNAi(RNA干擾)於1998年由安德魯·法厄(Andrew Z. Fire)等在秀麗隱桿線蟲中進行反義RNA抑制實驗時發現,並將這一過程稱為RNAi。這一發現被《Science》雜誌評為2001年的十大科學進展之一,並名列2002年十大科學進展之首。自此以後,以RNAi為作用機理的RNA抑制劑作為潛在的基因治療藥物得到人們廣泛的關注,2006年,安德魯·法厄與克雷格·梅洛(Craig C. Mello)由於在RNAi機制研究中的貢獻獲得了諾貝爾生理或醫學獎。RNAi在許多生物(包括動物、植物和真菌)中都可由雙鏈RNA(dsRNA)觸發,在RNAi過程中,一種稱為“Dicer”的核酸內切酶將長鏈dsRNA切割或“切丁”成21~25個核苷酸長的小片段。這些小片段,被稱為小干擾RNA(RNA抑制劑),其中的反義鏈(Guide strand)被載入到Argonaute蛋白(AGO2)上。AGO2載入發生在RISC-loading複合物中,這是一個三元複合物,由Argonaute蛋白、Dicer和dsRNA結合蛋白(簡稱為TRBP)組成。在裝載過程中,正義鏈(Passenger strand)鏈被AGO2裂解並排出。然後,AGO2使用反義鏈與包含完全互補序列的mRNA結合,然後催化這些mRNA的切割,致使mRNA分裂喪失翻譯範本的作用,進而阻止相關蛋白質的合成。切割後,被切割的mRNA被釋放,載入著反義鏈的RISC-loading複合物被迴圈用於另一輪的切割。
B型肝炎是由B型肝炎病毒持續感染超過6個月以上,肝臟發生不同程度炎症壞死(或)纖維化的疾病。據世界衛生組織估計全世界已大約有20億人感染,其中每年約4百萬人急性感染,約3.5-4億B型肝炎感染者,非洲區和西太平洋占68%。全球每年約有1百萬人死於B型肝炎感染相關疾病,其中肝硬化占30%,原發性肝細胞癌占45%。我國肝硬化和原發性肝細胞癌患者中,由B型肝炎病毒所致者分別為77%和84%。目前臨床一線用藥包括核苷類(NUC)和干擾素類藥物,最主要藥物仍是核苷類藥物如拉米夫定、恩替卡韋、阿德福韋、替比夫定等。丙酚替諾福韋是最新上市的一款新的NUC類藥物但因有可能造成腎功能損害,所以一定程度上限制了它的使用。核苷類藥物具有生物利用度高、口服較安全的優勢。然而,核苷類藥物雖然能有效地控制病情,但長期使用均可出現耐藥性,另外停藥後出現HBV DNA、ALT及肝組織學的不同程度的反彈;且在長期使用核苷類藥物後出現的較為明顯的副作用,例如腎臟損傷、嬰兒致畸等。病毒耐藥株的出現是長期使用核苷類藥物必須要面對的另一個副作用,耐藥株的出現大大降低治癒率,甚至無效。因為核苷類藥物對病毒複製是可逆的,所以對大部分患者若欲達到最大療效,療程必須在一年以上,如此其耐藥性隨之出現,就達不到預期之效果。核苷類(NUC)藥物需要天天服用,患者用藥依從性差。
B肝表面抗原(HBsAg)是B肝病毒(HBV)的外殼蛋白,是作為第一可檢測病毒的標誌物。HBsAg陽性是判斷HBV感染的金標準。對於B肝患者,如在肝硬化前獲HBsAg清除,則其肝硬化和肝細胞癌發生率將降低60倍。美國肝病研究協會AASLD、亞太肝臟研究協會APASL和歐洲肝臟研究協會EASL的指南中均將HBsAg血清清除作為治療終點判定標準之一。另外,高水準抗原誘導免疫耐受,抗原HBsAg水準的減少可以恢復HBV感染的免疫學控制。目前,臨床一線用藥包括核苷類(NUC)和干擾素類藥物都不具有降低抗原HBsAg水準效果,更談不上清除HBsAg。
B型肝炎的治療目前仍然是個全球性的健康挑戰。因此,本領域急需開發出新的治療機理的抗HBV藥物,通過高效持久降低抗原HBsAg水準,使B型肝炎患者重新產生HBsAb抗體,最終可以被功能性治癒。
本發明涉及一種抑制B型肝炎病毒基因表達的RNA抑制劑及其應用,所述的RNA抑制劑由正義鏈和反義鏈通過鹼基配對形成,正義鏈和反義鏈彼此之間至少85%鹼基互補,且部分或全部核苷酸糖基2’位的-OH被氟或甲氧基取代,末端至少有連續3個核苷酸之間的磷酸酯被硫代,以增強其在體內的穩定性。本發明所述的RNA抑制劑的結構中還含有的5’MVIP和3'MVIP,使所述RNA抑制劑具有肝靶向特異性的結構,其中5’MVIP偶聯在所述RNA抑制劑正義鏈和/或反義鏈5’末端,3’MVIP偶聯在所述RNA抑制劑反義鏈和/或正義鏈3’末端,5’MVIP和3’MVIP都包含有肝靶向特異性配體X、支鏈L、接頭B和連接鏈D,5’MVIP還包含與所述RNA抑制劑正義鏈或反義鏈5’末端連接的轉接點R
1,3’MVIP還包含與所述RNA抑制劑正義鏈或反義鏈3’末端連接的轉接點R
2,所述肝靶向特異性配體X、支鏈L或接頭B在5’MVIP與3’MVIP各自的內部或5’MVIP與3’MVIP之間可以相同,也可以不同。本發明提供的RNA抑制劑具有當前臨床一線B型肝炎治療藥物所不具有的功效,可以直接破壞HBV mRNA作為翻譯範本的功能,阻止HBV表面抗原HBsAg的形成。另外,本發明的RNA抑制劑對HBV最常見的A、B、C和D型都有顯著的抑制作用,可以與核苷類似物及干擾素聯合使用;可以持續高效使HBV小鼠的HBsAg的表達水準減少,並產生表面抗體HBsAb,可以功能性治癒B型肝炎。與已有公開的同類技術相比較,本發明所述的RNA抑制劑最主要的特徵是在體內可以產生表面抗體HBsAb,激發體內重新對HBV產生免疫力,實現功能性治癒B型肝炎。
一方面,本發明提供了一種抑制B型肝炎病毒基因表達的RNA抑制劑或其藥學上可接受的鹽,其中,
所述RNA抑制劑由鏈長為15-30的正義鏈和反義鏈通過鹼基配對形成,其中鏈長優選為19-23。
在上述技術方案中,優選地,所述正義鏈和反義鏈之間至少有85%的鹼基互補;
所述正義鏈或反義鏈的部分或全部核苷酸糖基2’位的-OH可以被取代,其中,所述取代基團為氟或甲氧基;
且所述正義鏈或反義鏈的末端至少有3個相鄰核苷酸之間的磷酸酯鍵可以硫代。
更優選地,所述正義鏈為如下所示的SEQ ID NO. 1或與SEQ ID NO. 1相差一個、兩個或三個核苷酸的序列,所述反義鏈為如下所示的SEQ ID NO. 2或與SEQ ID NO. 2相差一個、兩個或三個核苷酸的序列:
正義鏈:5' ggguuuuucucguugacaa 3' SEQ ID NO.: 1
反義鏈:5' uugucaacgagaaaaacccuu 3' SEQ ID NO.: 2
其中,g=鳥苷酸,a=腺苷酸,u=尿苷酸,c=胞苷酸。
或者,更優選地,所述正義鏈為如下所示的SEQ ID NO. 3或與SEQ ID NO. 3相差一個、兩個或三個核苷酸的序列,所述反義鏈為如下所示的SEQ ID NO. 4或與SEQ ID NO. 4相差一個、兩個或三個核苷酸的序列:
正義鏈:5'ggguuuuucuuguugacaa 3' SEQ ID NO.: 3
反義鏈:5' uugucaacaagaaaaacccuu 3' SEQ ID NO.: 4
其中,g=鳥苷酸,a=腺苷酸,u=尿苷酸,c=胞苷酸。
為了增強上述RNA抑制劑在體內的穩定性,在不影響其活性甚至增強其活性的情況下,可以對上述RNA抑制劑的正義鏈和反義鏈進行修飾,其中的核苷酸可以有修飾基團,可以整條鏈或者部分修飾。
在一個優選的技術方案中,所述RNA抑制劑修飾後的正義鏈為如下所示的SEQ ID NO. 2’或與SEQ ID NO. 2’相差一個、兩個或三個核苷酸的序列,其修飾後的反義鏈為如下所示的SEQ ID NO. 59或與SEQ ID NO. 59相差一個、兩個或三個核苷酸的序列:
正義鏈: 5' Gs fGs G U fU U fU fU fC U C G U U G A Cs As A 3' SEQ ID NO.:2’
反義鏈: 5' Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U 3' SEQ ID NO.:59
其中,G=2'-O-甲基鳥苷酸,A=2'-O-甲基腺苷酸,U=2'-O-甲基尿苷酸,C=2'-O-甲基胞苷酸;Gs=2'-O-甲基-3’-硫代鳥苷酸,As=2'-O-甲基-3'-硫代腺苷酸,Us=2'-O-甲基-3'-硫代尿苷酸,Cs=2'-O-甲基-3'-硫代胞苷酸;fG=2'-氟鳥苷酸,fA=2'-氟腺苷酸,fU=2'-氟尿苷酸,fC=2'-氟胞苷酸;fGs=2'-氟-3'-硫代鳥苷酸,fAs=2'-氟-3'-硫代腺苷酸,fUs=2'-氟-3'-硫代尿苷酸,fCs=2'-氟-3'-硫代胞苷酸。
在另一個優選的技術方案中,所述RNA抑制劑修飾後的正義鏈為如下所示的SEQ ID NO. 142或與SEQ ID NO. 142相差一個、兩個或三個核苷酸的序列,其修飾後的反義鏈為如下所示的SEQ ID NO. 143或與SEQ ID NO. 143相差一個、兩個或三個核苷酸的序列:
正義鏈:5' Gs Gs G U fU U fU fU fC U U G U U G A Cs As A 3' SEQ ID NO.: 142
反義鏈:5' Us Us G U C A fA C A A G fA A fA A A C C Cs Us U 3' SEQ ID NO.: 143
其中,G=2'-O-甲基鳥苷酸,A=2'-O-甲基腺苷酸,U=2'-O-甲基尿苷酸,C=2'-O-甲基胞苷酸;Gs=2'-O-甲基-3’-硫代鳥苷酸,As=2'-O-甲基-3'-硫代腺苷酸,Us=2'-O-甲基-3'-硫代尿苷酸,Cs=2'-O-甲基-3'-硫代胞苷酸;fG=2'-氟鳥苷酸,fA=2'-氟腺苷酸,fU=2'-氟尿苷酸,fC=2'-氟胞苷酸;fGs=2'-氟-3'-硫代鳥苷酸,fAs=2'-氟-3'-硫代腺苷酸,fUs=2'-氟-3'-硫代尿苷酸,fCs=2'-氟-3'-硫代胞苷酸。
在上述技術方案中,優選地,所述RNA抑制劑或其藥學上可接受的鹽還含有5’MVIP和3’MVIP的組合,其中,
所述5’MVIP和3’MVIP為帶有肝靶向特異性配體X的配體結構,其還包含支鏈L、接頭B和連接鏈D;
所述5’MVIP偶聯在所述正義鏈和/或反義鏈5’末端,其還包含與所述正義鏈或反義鏈5’末端連接的轉接點R
1;
所述3’MVIP偶聯在所述反義鏈和/或正義鏈3’末端,其包含與所述正義鏈或反義鏈3’末端連接的轉接點R
2;
其中,
n和m分別為0-4的整數,優選為1-3的整數,且n+m=2-6的整數,優選n+m=2、3或4;
所述轉接點R
1和R
2結構中帶有-NH-、硫原子或氧原子,一般結構中至少有一個-NH-、硫原子或氧原子,R
1和R
2通過結構中-NH-、硫原子或氧原子分別與5'MVIP和3'MVIP的連接鏈D以及正義鏈和/或反義鏈5’末端和3'末端相連,從而引入肝靶向特異性配體X,所述轉接點R
1和R
2可以是直鏈;帶有支鏈的直鏈或者各種環狀結構,環狀結構如飽和或不飽和的脂肪族碳環基,或者含有硫、氧或氮原子的五元或六元雜環基或芳香烴基等等;
R
1優選為-NH(CH
2)
xCH
2O-,其中x為3-12的整數,優選為4-6的整數;
R
2優選為-NH(CH
2)
x1CH(OH)(CH
2)
x2CH
2O-,其中x1為1-4的整數,x2為0-4的整數;
所述肝靶向特異性配體X,選自半乳糖、半乳糖胺、N-乙醯半乳糖胺及其衍生物,優選選自N-乙醯半乳糖胺及其衍生物,並且所述肝靶向特異性配體X在5’MVIP與3’MVIP各自的內部或5’MVIP與3’MVIP之間可以相同,也可以不同;
所述支鏈L是含有-NH-、C=O、O、S、醯胺基、磷醯基、硫代磷醯基、C4-C10脂肪族碳環基、苯基或者這些基團的組合的C4-C18直鏈,所述直鏈可以帶有乙基醇或羧酸類的側鏈,所述支鏈L優選為含醯胺基或六元脂肪族碳環基的C7-C18直鏈,並且所述支鏈L在5’MVIP與3’MVIP各自的內部或5’MVIP與3’MVIP之間可以相同,也可以不同;
其中,A
1和A
2各自獨立地是C、O、S、-NH-、羰基、醯胺基、磷醯基或硫代磷醯基,r為0-4的整數,並且所述接頭B在5’MVIP與3’MVIP之間可以相同,也可以不同;
所述連接鏈D是含有-NH-、C=O、O、S、醯胺基、磷醯基、硫代磷醯基、芳香烴基、C4-C10脂肪族碳環基、含1-3個氮的五元或六元雜環基或者這些基團的組合的C3-C18直鏈,所述C3-C18直鏈也可以帶有甲基醇、甲基叔丁基、甲基苯酚基、C5-C6脂肪環基的側鏈,所述連接鏈D優選為含兩個C=O、六元脂肪族碳環基或苯基的C3-C10直鏈。
在一些實施方案中,所述的n是指同時放在所述RNA抑制劑的正義鏈和反義鏈5’末端5'MVIP中n之和,所述的m是指同時放在所述RNA抑制劑的正義鏈和反義鏈3’末端3'MVIP中m之和。
所述肝靶向特異性配體X選自用于增強肝細胞對RNA抑制劑的攝取的結構,可以是脂質、類固醇、維生素、糖、蛋白質、肽、多胺及肽類比結構。在本發明提供的RNA抑制劑中,引入所述RNA抑制劑正義鏈或反義鏈末端的肝靶向特異性配體配體X可以相同,也可以不同,例如在特性上,有些可以是增強肝靶向性,有些可以是所述RNA抑制劑在體內藥代動力學的調節結構,有些可以是具有體內溶解活性的結構。在一些實施方案中,所述肝靶向特異性配體X選自以下結構中的一種或多種單糖及其衍生物。
所述單糖選自以下結構中的一種或多種:甘露糖、半乳糖、D-阿拉伯糖、葡萄糖、果糖、木糖、葡糖胺、核糖。甘露糖選自以下結構中的一種或多種:D-吡喃甘露糖、L-吡喃甘露糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖。半乳糖選自以下結構中的一種或多種:L-半乳糖、D-半乳糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖。葡萄糖選自以下結構中的一種或多種:D-葡萄糖、L-葡萄糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖。果糖選自以下結構中的一種或多種:α-D-呋喃果糖、α-D-吡喃果糖。木糖選自以下結構中的一種或多種:D-呋喃木糖、L-呋喃木糖。核糖選自以下結構中的一種或多種:核糖、D-核糖、L-核糖。單糖衍生物選自甘露糖衍生物、半乳糖衍生物、葡萄糖衍生物、核糖衍生物以及其他衍生物。半乳糖衍生物選自α-D-半乳糖胺、N-乙醯基半乳糖胺、4-硫代-β-D-吡喃半乳糖。葡萄糖衍生物可以選自2-氨基-3-O-[(R)-1-羧乙基]-2-去氧-β-D-吡喃葡萄糖、2-去氧-2-甲基氨基-L-吡喃葡萄糖、2-去氧-2-磺氨基-D-吡喃葡萄糖、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙醯基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯。核糖衍生物選自D-4-硫代核糖、L-4-硫代核糖的一種或多種。
其中,W1為氫或羥基保護基,可以相同也可以不同;W為-OH、-NHCOOH或-NHCO(CH
2)
qCH
3,其中q為0-4的整數;W2為-NH-、O、S或C。
其中,W選自-OH、-NHCOOH或-NHCO(CH
2)
qCH
3中的一種或兩種,其中q為0-4的整數。
在一些實施方案中,所述肝靶向特異性配體X在同一個5'MVIP或3'MVIP結構中可以相同,也可以不同。
在一些實施方案中,5'MVIP與3'MVIP彼此之間的X可以相同,也可以不同。
所述支鏈L是含有-NH-、C=O、O、S、醯胺基、磷醯基、硫代磷醯基、C4-C10脂肪族碳環基、苯基或者這些基團的組合的C4-C18的直鏈,所述直鏈可以帶有乙基醇或羧酸類的側鏈,所述支鏈L優選為含醯胺基或六元脂肪族碳環基的C7-C18直鏈,其長短或結構會影響到本發明所述的RNA抑制劑的活性。
在一些實施方案中,所述支鏈L在同一個5'MVIP或3'MVIP結構中可以相同,也可以不同。
在一些實施方案中,5'MVIP與3'MVIP彼此之間的支鏈L可以相同,也可以不同。
其中,r1是1-12的正整數,r2為0-20的整數,Z為H或烷基或醯胺基,如C1-C5烷基、C1-C5的醯胺基,如甲醯胺等。
所述接頭B的結構與能引入的特異性配體X的數量有關,接頭B中含-NH-、C、O、S、醯胺基、磷醯基、硫代磷醯基,當n或m為1時,其為一條直鏈,當n或m為2、3或4時,其分叉的次數分別為2、3或4。所述接頭B可以選自以下結構式:
其中,A
1和A
2各自獨立地是C、O、S、-NH-、羰基、醯胺基、磷醯基或硫代磷醯基,r為0-4的整數。
其中,r為0-4的整數。
所述連接鏈D是含有-NH-、C=O、O、S、醯胺基、磷醯基、硫代磷醯基、芳香烴基、C4-C10脂肪族碳環基、含1-3個氮的五元或六元雜環基或者這些基團的組合的C3-C18直鏈,所述C3-C18直鏈也可以帶有甲基醇、甲基叔丁基、甲基苯酚基、C5-C6脂肪環基的側鏈,所述連接鏈D優選為含兩個C=O、六元脂肪族碳環基或苯基的C3-C10直鏈。
其中,每個n為1-20的正整數,且每個n為相同或不同的整數;s為2-13的整數;Z1和Z2為相同或者不同的取代基團,如C3-C10烷基。
在一些最優選實施方案中,所述連接鏈D為含兩個C=O的C3-C10直鏈。
表1:5’MVIP的
結構
序號 | 代碼 | 結構式 |
1 | 5'YICdd-01 | |
2 | 5'YICd-01 | |
3 | 5'YICc-01 | |
4 | 5'YICa-01 | |
5 | 5'YICa-02 | |
6 | 5'YICa-03 | |
7 | 5'YICa-04 | |
8 | 5'YICa-05 | |
9 | 5'ERCa-01 | |
10 | 5'ERCa-02 | |
11 | 5'ERCa-03 | |
12 | 5'ERCa-04 | |
13 | 5'ERCa-05 | |
14 | 5'ERCdd-01 | |
15 | 5'ERCd-01 | |
16 | 5'ERCc-01 | |
17 | 5'SANCdd-01 | |
18 | 5'SANCd-01 | |
19 | 5'SANCc-01 | |
20 | 5'SANCa-01 | |
21 | 5'SANCa-02 | |
22 | 5'SANCa-03 |
在一些實施方案中,5’MVIP也可以不存在,這時候m可以為2-4的整數。
表2:3’MVIP的
結構
序號 | 代碼 | 結構 |
1 | 3'SANCdd-01 | |
2 | 3'SANCd-01 | |
3 | 3'SANCc-01 | |
4 | 3'SANCa-01 | |
5 | 3'SANCa-02 | |
6 | 3'SANCa-03 | |
7 | 3'ERCdd-01 | |
8 | 3'ERCd-01 | |
9 | 3'ERCc-01 | |
10 | 3'ERCa-01 | |
11 | 3'ERCa-02 | |
12 | 3'ERCa-03 | |
13 | 3'ERCa-04 | |
14 | 3'ERCa-05 | |
15 | 3'YICa-01 | |
16 | 3'YICa-02 | |
17 | 3'YICa-03 | |
18 | 3'YICa-04 | |
19 | 3'YICa-05 | |
20 | 3'YICdd-01 | |
21 | 3'YICd-01 | |
22 | 3'YICc-01 |
在本發明提供的RNA抑制劑中,所述5’MVIP還包含與所述正義鏈或反義鏈5’末端連接或偶聯的轉接點R
1,所述轉接點R
1結構中帶有-NH-、硫原子或氧原子,一般結構中至少有一個-NH-、硫原子或氧原子。R
1通過其結構中-NH-、硫原子或氧原子與5'MVIP的連接鏈D及正義鏈或反義鏈5’末端相連,從而引入肝靶向特異性配體X。所述轉接點R
1可以是直鏈;帶有醯胺基、羧基、烷基支鏈的直鏈或者各種環狀結構,環狀結構如飽和或不飽和的脂肪族碳環基,或者如含有硫、氧或氮原子的五元或六元雜環基或芳香烴基等等。
在一些實施方案中,R
1為-B
1(CH
2)
xCH
2B
2-,其中x為3-10的整數,優選為4-6的整數,基團B
1和B
2可以分別為-NH-、硫原子或氧原子。
在一些實施方案中,R
1為-B
1(CH
2)
xCH(B
3CH
3)B
2-,其中x為3-10的整數,B
1和B
2可以分別為-NH-、硫原子或氧原子,基團B
3為含氮、硫、氧或羧基或甲基類烷基的官能團。
在一些優選的實施方案中,R
1為-NH(CH
2)
xCH
2O-,其中x為3-10的整數,優選為4-6的整數,可以通過以下兩種亞磷醯胺單體引入:
i. 其中的一個氧原子或硫原子用於R
1亞磷醯胺單體的合成,通過固相合成的方法接入RNA抑制劑單鏈的5’端。結構中-NH-、硫原子或氧原子用於與5'MVIP中的連接鏈D連接,從而在RNA抑制劑的5’端引入肝靶向特異性配體X。引入到RNA抑制劑5’端的單體示例性結構如下:
通式中n為1-4時,上述的單體中接頭B部分分別支化1至4次,以獲得對應的單體化合物,借助上述的單體化合物,肝靶向特異性配體X通過固相合成被引入到正義鏈或反義鏈5’末端。
在本發明提供的RNA抑制劑中,所述3’MVIP還包含與所述正義鏈或反義鏈3’末端連接或偶聯的轉接點R
2,所述轉接點R
2結構中帶有-NH-、硫原子或氧原子,一般結構中至少有一個-NH-、硫原子或氧原子。R
2通過結構中-NH-、硫原子或氧原子與3'MVIP的連接鏈D及正義鏈或反義鏈3’末端相連,從而引入肝靶向特異性配體X。所述轉接點R
2可以是直鏈;帶有醯胺基、羧基或烷基的支鏈的直鏈或各種環狀結構,環狀結構如飽和或不飽和的脂肪族碳環基,或者如含有硫、氧或氮原子的五元或六元雜環基或芳香烴基等等。
本發明所述的R
2是通過丁二酸酐與R
2結構中-NH-、硫原子或氧原子成酯或醯胺的同時,又與空白Solid Support中-NH-進行偶聯,形成3'MVIP solid spport,再通過亞磷醯胺固相合成法,將3’MVIP引入到正義鏈或反義鏈的3’末端。
當通式中m為1-4時,上述的單體中接頭B部分分別支化1至4次,以獲得對應的Solid Support。
當通式中m為1-4時,上述的單體中接頭B部分分別支化1至4次,以獲得對應的Solid Support。
當通式中m為1-4時,上述的單體中接頭B部分分別支化1至4次,以獲得對應的Solid Support。
表3:5’MVIP中
與R
1的組合
序號 | 代碼 | R 1 | 5'MVIP代碼 |
1 | 5'YICd-01 | -NH(CH 2) 6O- | 5'MVIP01 |
2 | 5'YICc-01 | -NH(CH 2) 6O- | 5'MVIP02 |
3 | 5'YICa-01 | -O(CH 2) 6O- | 5'MVIP03 |
4 | 5'YICa-02 | -O(CH 2) 6O- | 5'MVIP04 |
5 | 5'YICa-03 | -S(CH 2) 6O- | 5'MVIP05 |
6 | 5'YICa-04 | -NH(CH 2) 6S- | 5'MVIP06 |
7 | 5'YICa-05 | -NH(CH 2) 8O- | 5'MVIP07 |
8 | 5'YICr-06 | -NH(CH 2) 8O- | 5'MVIP08 |
9 | 5'ERCd-01 | -NH(CH 2) 6O- | 5'MVIP09 |
10 | 5'ERCc-01 | -NH(CH 2) 6O- | 5'MVIP10 |
11 | 5'ERCa-01 | -NH(CH 2) 5CH(CH 2CH 3)O- | 5'MVIP11 |
12 | 5'ERCa-02 | -O(CH 2) 6O- | 5'MVIP12 |
13 | 5'ERCa-03 | -S(CH 2) 6O- | 5'MVIP13 |
14 | 5'ERCa-04 | -O(CH 2) 6O- | 5'MVIP14 |
15 | 5'ERCa-05 | -O(CH 2) 6O- | 5'MVIP15 |
16 | 5'ERCr-06 | -S(CH 2) 4CH(CH 3)O- | 5'MVIP16 |
17 | 5'SANCd-01 | -NH(CH 2) 6O- | 5'MVIP17 |
18 | 5'SANCc-01 | -NH(CH 2) 6O- | 5'MVIP18 |
在一些實施方案中,3’MVIP可以不存在,這時候n可以是2-4。
表4:3’MVIP的
與R
2組合
序號 | 代碼 | R 2 | 3'MVIP代碼 |
1 | 3'YICd-01 | 3'MVIP01 | |
2 | 3'YICc-01 | 3'MVIP02 | |
3 | 3'YICa-01 | 3'MVIP03 | |
4 | 3'YICa-02 | 3'MVIP04 | |
5 | 3'YICa-03 | 3'MVIP05 | |
6 | 3'YICa-04 | 3'MVIP06 | |
7 | 3'YICa-05 | 3'MVIP07 | |
8 | 3'YICr-06 | 3'MVIP08 | |
9 | 3'ERCd-01 | 3'MVIP09 | |
10 | 3'ERCc-01 | 3'MVIP10 | |
11 | 3'ERCa-01 | 3'MVIP11 | |
12 | 3'ERCa-02 | 3'MVIP12 | |
13 | 3'ERCa-03 | 3'MVIP13 | |
14 | 3'ERCa-04 | 3'MVIP14 | |
15 | 3'ERCa-05 | 3'MVIP15 | |
16 | 3'ERCr-06 | 3'MVIP16 | |
17 | 3'SANCd-01 | 3'MVIP17 | |
18 | 3'SANCc-01 | 3'MVIP18 | |
19 | 3'SANCa-01 | 3'MVIP19 | |
20 | 3'ERCd-01 | 3'MVIP20 | |
21 | 3'ERCd-01 | 3'MVIP21 | |
22 | 3'ERCd-01 | 3'MVIP22 | |
23 | 3'ERCd-01 | 3'MVIP23 | |
24 | 3'ERCd-01 | 3'MVIP24 | |
25 | 3'ERCd-01 | 3'MVIP25 | |
26 | 3'ERCd-01 | 3'MVIP26 | |
27 | 3'ERCd-01 | 3'MVIP27 |
本發明提供的RNA抑制劑結構中的正義鏈和反義鏈鏈長為15-30,優選19-23,彼此之間至少85%鹼基互補。為了增強正義鏈和反義鏈在體內的穩定性,RNA抑制劑的正義鏈和反義鏈可以在沒有影響活性甚至增強活性的情況下進行修飾,其中的核苷酸可以有修飾基團,可以整條鏈或者部分修飾,優選全部修飾。所述修飾為本領域的研究者易於理解的技術,可以在糖基部分,選至如下任一種或多種:去氧核糖核苷酸、核苷酸模擬物、脫鹼基核苷酸、2’-修飾核苷酸、3’至3’連接(倒置)核苷酸、含非天然鹼基的核苷酸、橋接核苷酸、肽核酸(PNA)、解鎖的核鹼基類似物、鎖定核苷酸、3’-O-甲氧基(2’核苷間連接)核苷酸、2’-F-阿拉伯糖核苷酸、5’-Me/2’-氟帶核苷酸、嗎啉代核苷酸、乙烯基膦酸酯去氧核糖核苷酸、含乙烯基膦酸酯的核苷酸和含環丙基膦酸酯的核苷酸。其中,2’-修飾核苷酸包括但不限於:2’-O-甲基核苷酸、2’-去氧-2’-氟核苷酸、2’-去氧核苷酸、2’-甲氧基乙基核苷酸、2’-氨基核苷酸和2’-烷基核苷酸。在本發明提供的RNA抑制劑中,所述RNA抑制劑的正義鏈和反義鏈均不需要均勻修飾,可在其單個核苷酸中摻入一種以上的修飾。所述的修飾也可在鹼基部分發生,經修飾的核鹼基包括合成和天然的核鹼基,諸如5-取代嘧啶,6-氮雜嘧啶和N-2/N-6和O-6取代嘌呤,5-甲基胞嘧啶,5-羥甲基胞嘧啶,黃嘌呤,次黃嘌呤,2-氨基腺嘌呤,腺嘌呤和鳥嘌呤的6-烷基,腺嘌呤和鳥嘌呤的2-烷基和其他烷基衍生物,2-硫代尿嘧啶,2-硫代胸腺嘧啶,2-硫代胞嘧啶,5-鹵代尿嘧啶,胞嘧啶,5-丙炔基尿嘧啶,5-丙炔基胞嘧啶,6-偶氮尿嘧啶,6-偶氮胞嘧啶,6-偶氮胸腺嘧啶,5-尿嘧啶,4-硫尿嘧啶,8-鹵素、8-氨基、8-巰基、8-硫代烷基、8-羥基和其他8-取代的腺嘌呤和鳥嘌呤,5-鹵、5-三氟甲基和其他5-取代的尿嘧啶和胞嘧啶,7-甲基鳥嘌呤和7-甲基腺嘌呤,8-氮鳥嘌呤和8-氮雜腺嘌呤,7-脫氮鳥嘌呤,7-脫氮腺嘌呤,3-脫氮鳥嘌呤和3-脫氮腺嘌呤。
本發明所述的RNA抑制劑的正義鏈和反義鏈的部分或全部是2’-O-甲基核苷酸和/或2’-去氧-2’-氟核苷酸,且至少連續兩個硫代磷酸酯鍵存在其正義鏈5’端和反義鏈3’端的核苷酸之間,優選末端3個連續的核苷酸之間磷酸酯鍵被硫代。
在本發明提供的RNA抑制劑中,當其一條單鏈上有3’MVIP時,與這條單鏈相互補的另外一條單鏈相應的有5’MVIP或3’MVIP或者沒有;當所述RNA抑制劑的一條單鏈上有5’MVIP時,與其相互補的另外一條單鏈相應的有3’MVIP或5’MVIP或者沒有。5’MVIP和3’MVIP也可以同時連接在正義鏈或反義鏈的對應末端,即正義鏈的5’末端有5’MVIP時,其3'末端也可以有3'MVIP;反義鏈的5’末端有5’MVIP時,其3'末端也可以有3'MVIP。或者,5’MVIP同時放在正義鏈和反義鏈的5’末端。或者,3’MVIP同時放在正義鏈和反義鏈的3’末端。
在一些實施方案中,優選下表5中不同5'MVIP和3'MVIP組合接入RNA抑制劑的正義鏈和或反義鏈的不同位置,考察對HBV的HBsAg水準的影響。
表5:5'MVIP與3'MVIP的組合
序號 | 5’MVIP代碼 | 5’MVIP結構 | 3’MVIP代碼 | 3’MVIP結構 |
1 | 5’MVIP01 | 3’MVIP17 | ||
2 | 5’MVIP09 | 3’MVIP09 | ||
3 | 5’MVIP17 | 3’MVIP01 | ||
4 | 5’MVIP01 | 3’MVIP01 | ||
5 | 5’MVIP01 | 3’MVIP09 | ||
6 | 5’MVIP09 | 3’MVIP01 |
在一些實施方案中,本發明所述的RNA抑制劑或其藥學上可接受的鹽優選以羧酸鹽、鈉鹽、三乙胺鹽或其它可藥用鹽的形式製備或合成。
在一些實施方案中,所述RNA抑制劑或其藥學上可接受的鹽更優選為其鈉鹽或三乙胺鹽。
在一些實施方案中,所述RNA抑制劑的正義鏈選自下表6:
表6:RNA抑制劑的正義鏈
SEQ ID NO. | 單鏈代碼 | 正義鏈序列5'→3'(19mer) |
1 | Ky-S | ggguuuuucucguugacaa |
2’ | Ky-S0 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
3’ | Ky-S1 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A- 3'MVIP17 |
4’ | Ky-S2 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP01 |
5 | Ky-S3 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP09 |
6 | Ky-S4 | 5'MVIP17-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
7 | Ky-S5 | 5'MVIP01-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
8 | Ky-S6 | 5'MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
9 | Ky-S7 | 5'MVIP01-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP01 |
10 | Ky-S8 | 5'MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP09 |
11 | Ky-S9 | 5'MVIP17-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP17 |
12 | Ky-S10 | 5'MVIP01-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP17 |
13 | Ky-S11 | 5'MVIP17-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP01 |
14 | Ky-S12 | 5'MVIP01-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP09 |
15 | Ky-S13 | 5'MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP01 |
16 | Ky-S14 | 5'MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP17 |
17 | Ky-S15 | 5'MVIP17-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP09 |
18 | Ky-S16 | 5'MVIP12-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
19 | Ky-S17 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP19 |
20 | Ky-S18 | 5'MVIP16-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP16 |
21 | Ky-S19 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP17 |
22 | Ky-S20 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP18 |
23 | Ky-S21 | 5' MVIP03-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
24 | Ky-S22 | 5' MVIP08-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
25 | Ky-S23 | 5' MVIP16-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
26 | Ky-S24 | 5'MVIP13-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP06 |
27 | Ky-S25 | 5'MVIP04-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP06 |
28 | Ky-S26 | 5'MVIP11-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
29 | Ky-S27 | 5'MVIP11-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3'MVIP14 |
30 | Ky-S28 | 5'MVIP15-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
31 | Ky-S29 | 5'MVIP02-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
32 | Ky-S30 | 5'MVIP05-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
33 | Ky-S31 | 5'MVIP06-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
34 | Ky-S32 | 5'MVIP07-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
35 | Ky-S33 | 5'MVIP10-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
36 | Ky-S34 | 5'MVIP14-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
37 | Ky-S35 | 5'MVIP18-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
38 | Ky-S36 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP02 |
39 | Ky-S37 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP03 |
40 | Ky-S38 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP04 |
41 | Ky-S39 | 5' MVIP04-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP04 |
42 | Ky-S40 | 5' MVIP03-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP19 |
43 | Ky-S41 | 5' MVIP18-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP18 |
44 | Ky-S42 | 5' MVIP08-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP18 |
45 | Ky-S43 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP05 |
46 | Ky-S44 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP07 |
47 | Ky-S45 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP10 |
48 | Ky-S46 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP11 |
49 | Ky-S47 | 5' MVIP11-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP11 |
50 | Ky-S48 | 5' MVIP15-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP15 |
51 | Ky-S49 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP06 |
52 | Ky-S50 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP08 |
53 | Ky-S51 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP12 |
54 | Ky-S52 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP13 |
55 | Ky-S53 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP14 |
56 | Ky-S54 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP15 |
57 | Ky-S55 | Gs fGs G U fU U fU fU fC U C G U U G A Cs As A -3' MVIP16 |
在一些實施方案中,本發明所述的RNA抑制劑的正義鏈與表6中的各序列相差一個、兩個或三個核苷酸。
在一些實施方案中,所述RNA抑制劑的反義鏈選自下表7:
表7:RNA抑制劑的反義鏈
SEQ ID NO. | 單鏈代碼 | 反義鏈序列5'→3'(21mer) |
2 | Ky-AS | uugucaacgagaaaaacccuu |
59 | Ky-AS0 | 5' Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U 3' |
60 | Ky-AS1 | 5' MVIP01-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
61 | Ky-AS2 | 5' MVIP09-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
62 | Ky-AS3 | 5' MVIP17-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
63 | Ky-AS4 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP01 |
64 | Ky-AS5 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP09 |
65 | Ky-AS6 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP17 |
66 | Ky-AS7 | 5' MVIP01-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP01 |
67 | Ky-AS8 | 5' MVIP09-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP09 |
68 | Ky-AS9 | 5' MVIP17-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP17 |
69 | Ky-AS10 | 5' MVIP01-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP17 |
70 | Ky-AS11 | 5' MVIP17-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP01 |
71 | Ky-AS12 | 5' MVIP01-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP09 |
72 | Ky-AS13 | 5' MVIP09-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP01 |
73 | Ky-AS14 | 5' MVIP09-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP17 |
74 | Ky-AS15 | 5' MVIP17-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP09 |
75 | Ky-AS16 | 5' MVIP12-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
76 | Ky-AS17 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U-3' MVIP19 |
77 | Ky-AS18 | 5'MVIP16- Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3'MVIP16 |
78 | Ky-AS19 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U-3' MVIP17 |
79 | Ky-AS20 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U-3' MVIP18 |
80 | Ky-AS21 | 5' MVIP03-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
81 | Ky-AS22 | 5' MVIP08-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
82 | Ky-AS23 | 5'MVIP16-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
83 | Ky-AS24 | 5'MVIP13-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U-3'MVIP06 |
84 | Ky-AS25 | 5'MVIP04-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U-3'MVIP06 |
85 | Ky-AS26 | 5' MVIP11-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
86 | Ky-AS27 | 5' MVIP11-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U-3' MVIP14 |
87 | Ky-AS28 | 5' MVIP15-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
88 | Ky-AS29 | 5' MVIP02-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
89 | Ky-AS30 | 5' MVIP05-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
90 | Ky-AS31 | 5' MVIP06-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
91 | Ky-AS32 | 5' MVIP07-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
92 | Ky-AS33 | 5' MVIP10- Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
93 | Ky-AS34 | 5' MVIP14- Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
94 | Ky-AS35 | 5' MVIP18- Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U |
95 | Ky-AS36 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP02 |
96 | Ky-AS37 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U-3' MVIP03 |
97 | Ky-AS38 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP04 |
98 | Ky-AS39 | 5' MVIP04-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U-3' MVIP04 |
99 | Ky-AS40 | 5' MVIP03-Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP19 |
100 | Ky-AS41 | 5' MVIP18- Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U-3' MVIP18 |
101 | Ky-AS42 | 5' MVIP08- Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U-3' MVIP18 |
102 | Ky-AS43 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP05 |
103 | Ky-AS44 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP07 |
104 | Ky-AS45 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP10 |
105 | Ky-AS46 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP11 |
106 | Ky-AS47 | 5' MVIP11- Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U-3' MVIP11 |
107 | Ky-AS48 | 5' MVIP15- Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U-3' MVIP15 |
108 | Ky-AS49 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP06 |
109 | Ky-AS50 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP08 |
110 | Ky-AS51 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP12 |
111 | Ky-AS52 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP13 |
112 | Ky-AS53 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP14 |
113 | Ky-AS54 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP15 |
114 | Ky-AS55 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP16 |
在一些實施方案中,本發明所述的RNA抑制劑的反義鏈與表7中的各序列相差一個、兩個或三個核苷酸。
在一些體內外效果考察實施方案中,所述RNA抑制劑的正義鏈或反義鏈選自下表8:
表8:RNA抑制劑的正義鏈或反義鏈
SEQ ID NO. | 單鏈代碼 | 單鏈序列5'→3' |
115 | Ky-S56 | Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A |
116 | Ky-S57 | Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A - 3' MVIP17 |
117 | Ky-S58 | Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A -3'MVIP01 |
118 | Ky-S59 | Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A -3' MVIP09 |
119 | Ky-S60 | 5' MVIP17-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A |
120 | Ky-S61 | 5' MVIP01-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A |
121 | Ky-S62 | 5' MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A |
122 | Ky-S63 | 5' MVIP01-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A -3' MVIP01 |
123 | Ky-S64 | 5' MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A -3'MVIP09 |
124 | Ky-S65 | 5' MVIP17-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A -3' MVIP17 |
125 | Ky-S66 | 5' MVIP01-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A -3' MVIP17 |
126 | Ky-S67 | 5' MVIP17-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A -3' MVIP01 |
127 | Ky-S68 | 5' MVIP01-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A -3' MVIP09 |
128 | Ky-S69 | 5' MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A -3' MVIP01 |
129 | Ky-S70 | 5' MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A -3'MVIP17 |
130 | Ky-S71 | 5' MVIP17-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A -3' MVIP09 |
131 | Ky-S72 | 5' MVIP09-G fG G U fU U fU fU fC U C G U U G A Cs As A |
132 | Ky-AS56 | Us Us G U C A fA C G A G fA A fA fA A C C C U U -3' MVIP09 |
133 | Ky-AS57 | Us Us G U C A fA C G A G fA A fA fA A C C Cs Gs C -3' MVIP09 |
134 | Ky-S73 | 5' MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A C A A A As Us A |
135 | Ky-S74 | 5' MVIP01-Gs fGs G U fU U fU fU fC U C G U U G A C A As As A |
136 | Ky-AS58 | Us Us U U G U fC A A C G fA G fA fA A A A C C Cs Us U -3' MVIP09 |
137 | Ky-S75 | 5' MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A C A A A A Us Cs C |
138 | Ky-S76 | 5' MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A C A A A A U C Cs Us A |
139 | Ky-AS59 | Us As G G A U fU U U G U fC A fA fC G A G A A A A A C C Cs Us U-3' MVIP09 |
3 | Ky-S77 | g g g u u u u u c u u g u u g a c a a |
4 | Ky-AS60 | u u g u c a a c a a g a a a a a c c c u u |
142 | Ky-S78 | Gs Gs G U fU U fU fU fC U U G U U G A Cs As A |
143 | Ky-AS61 | Us Us G U C A fA C A A G fA A fA A A C C Cs Us U |
144 | Ky-S79 | 5'MVIP09-Gs Gs G U fU U fU fU fC U U G U U G A Cs As A |
145 | Ky-AS62 | Us Us G U C A fA C A A G fA A fA A A C C Cs Us U -3' MVIP09 |
146 | Ky-S80 | 5'MVIP09-CsUsUACGCUGAGfUACUUCGAsAsA |
147 | Ky-AS63 | UsCsGAAGfUACUfCAGCGfUAAGsUsU-3'MVIP09 |
在一些實施方案中,本發明所述的RNA抑制劑的正義鏈或反義鏈與表8中的各序列相差一個、兩個或三個核苷酸。
在一些實施方案中,使用細胞株HepG2.2.15評估將5'MVIP和3'MVIP放在正義鏈(SEQ ID NO: 2’)和/或反義鏈(SEQ ID NO: 59)對應末端,評估所得RNA抑制劑對HBV的HBsAg水準降低效果影響。RNA抑制劑代碼、所含的單鏈及SEQ ID NO.如下表9:
表9
RNA抑制劑代碼 | 單鏈代碼 | SEQ ID NO. |
Ky-00 | Ky-S0 | 2’ |
Ky-AS0 | 59 | |
Ky-01 | Ky-S1 | 3’ |
Ky-AS0 | 59 | |
Ky-02 | Ky-S1 | 3’ |
Ky-AS1 | 60 | |
Ky-03 | Ky-S1 | 3’ |
Ky-AS2 | 61 | |
Ky-04 | Ky-S1 | 3’ |
Ky-AS3 | 62 | |
Ky-05 | Ky-S0 | 2’ |
Ky-AS3 | 62 | |
Ky-06 | Ky-S2 | 4’ |
Ky-AS3 | 62 | |
Ky-07 | Ky-S3 | 5 |
Ky-AS3 | 63 | |
Ky-08 | Ky-S3 | 5 |
Ky-AS1 | 60 | |
Ky-09 | Ky-S3 | 5 |
Ky-AS2 | 61 | |
Ky-10 | Ky-S2 | 4’ |
Ky-AS2 | 61 | |
Ky-11 | Ky-S0 | 2’ |
Ky-AS2 | 61 | |
Ky-12 | Ky-S3 | 5 |
Ky-AS0 | 59 | |
Ky-13 | Ky-S2 | 4’ |
Ky-AS1 | 60 | |
Ky-14 | Ky-S4 | 6 |
Ky-AS0 | 59 | |
Ky-15 | Ky-S4 | 6 |
Ky-AS4 | 63 | |
Ky-16 | Ky-S4 | 6 |
Ky-AS5 | 64 | |
Ky-17 | Ky-S4 | 6 |
Ky-AS6 | 65 | |
Ky-18 | Ky-S0 | 2’ |
Ky-AS6 | 65 | |
Ky-19 | Ky-S5 | 7 |
Ky-AS6 | 65 | |
Ky-20 | Ky-S6 | 8 |
Ky-AS6 | 65 | |
Ky-21 | Ky-S6 | 8 |
Ky-AS4 | 63 | |
Ky-22 | Ky-S6 | 8 |
Ky-AS5 | 64 | |
Ky-23 | Ky-S5 | 7 |
Ky-AS5 | 64 | |
Ky-24 | Ky-S0 | 2’ |
Ky-AS5 | 64 | |
Ky-25 | Ky-S6 | 8 |
Ky-AS0 | 59 | |
Ky-26 | Ky-S5 | 7 |
Ky-AS4 | 63 | |
Ky-27 | Ky-S7 | 9 |
Ky-AS0 | 59 | |
Ky-28 | Ky-S0 | 2’ |
Ky-AS7 | 66 | |
Ky-29 | Ky-S8 | 10 |
Ky-AS0 | 59 | |
Ky-30 | Ky-S0 | 2’ |
Ky-AS8 | 67 | |
Ky-31 | Ky-S9 | 11 |
Ky-AS0 | 59 | |
Ky-32 | Ky-S0 | 2’ |
Ky-AS9 | 68 | |
Ky-33 | Ky-S10 | 12 |
Ky-AS0 | 59 | |
Ky-34 | Ky-S0 | 2’ |
Ky-AS10 | 69 | |
Ky-35 | Ky-S11 | 13 |
Ky-AS0 | 69 | |
Ky-36 | Ky-S0 | 2’ |
Ky-AS11 | 70 | |
Ky-37 | Ky-S12 | 14 |
Ky-AS0 | 59 | |
Ky-38 | Ky-S0 | 2’ |
Ky-AS12 | 71 | |
Ky-39 | Ky-S13 | 15 |
Ky-AS0 | 59 | |
Ky-40 | Ky-S0 | 2’ |
Ky-AS13 | 72 | |
Ky-41 | Ky-S14 | 16 |
Ky-AS0 | 59 | |
Ky-42 | Ky-S0 | 2’ |
Ky-AS14 | 73 | |
Ky-43 | Ky-S15 | 17 |
Ky-AS0 | 59 | |
Ky-44 | Ky-S0 | 2’ |
Ky-AS15 | 74 | |
Ky-45 | Ky-S5 | 7 |
Ky-AS1 | 60 | |
Ky-46 | Ky-S6 | 8 |
Ky-AS2 | 61 | |
Ky-47 | Ky-S4 | 6 |
Ky-AS3 | 62 | |
Ky-48 | Ky-S5 | 7 |
Ky-AS3 | 62 | |
Ky-49 | Ky-S5 | 7 |
Ky-AS2 | 61 | |
Ky-50 | Ky-S6 | 8 |
Ky-AS3 | 62 | |
Ky-51 | Ky-S6 | 8 |
Ky-AS1 | 60 | |
Ky-52 | Ky-S4 | 6 |
Ky-AS1 | 60 | |
Ky-53 | Ky-S4 | 6 |
Ky-AS2 | 61 | |
Ky-54 | Ky-S2 | 4’ |
Ky-AS4 | 63 | |
Ky-55 | Ky-S3 | 5 |
Ky-AS5 | 64 | |
Ky-56 | Ky-S1 | 3’ |
Ky-AS6 | 65 | |
Ky-57 | Ky-S2 | 4’ |
Ky-AS6 | 65 | |
Ky-58 | Ky-S2 | 4’ |
Ky-AS5 | 64 | |
Ky-59 | Ky-S3 | 5 |
Ky-AS6 | 65 | |
Ky-60 | Ky-AS5 | 5 |
Ky-AS4 | 63 | |
Ky-61 | Ky-S1 | 3’ |
Ky-AS5 | 64 | |
Ky-62 | Ky-S1 | 3’ |
Ky-AS4 | 63 |
在一些實施方案中,優選出組合5’MVIP01/3'MVIP01、5’MVIP01/3'MVIP17、5’MVIP09/3'MVIP09放在正義鏈的5’末端和反義鏈的3’末端。
在一些實施方案中,優選出5’MVIP01/3'MVIP09、5’MVIP09/3'MVIP01放在正義鏈的5’末端和3’末端。
另一方面,本發明還提供了一種上述RNA抑制劑或其藥學上可接受的鹽在製備用於治療肝源性疾病的藥物中的應用,其中,所述肝源性疾病包括但不限於肝炎、肝腫瘤、肝硬化、黃疸、二型糖尿病、脂肪肝、血液系統的凝血性疾病、血液白蛋白及球蛋白相關的疾病、高血脂、動脈粥樣硬化、原發性高血壓。
再一方面,本發明提供了一種藥物組合物,該藥物組合物包括上述RNA抑制劑或其藥學上可接受的鹽和藥學上可接受的輔料,其劑型為口服劑、靜脈注射劑或者皮下或肌內注射劑,優選為皮下注射劑。
又一方面,本發明提供了一種藥物組合物,該藥物組合物包含上述RNA抑制劑或其藥學上可接受的鹽和其他治療B型肝炎的藥物。其他治療B型肝炎藥物,包括但不限於已在臨床上使用的核苷類似物或干擾素類,也包括在研當中的一些候選B肝治療藥物,如免疫調節劑在一個實施方案中,在轉基因小鼠模型上比對了本發明所述的RNA抑制劑與當前慢性B肝治療所用的一線用藥替諾福韋對HBV的HBsAg的抑制效果。試驗結果證實了,抗B肝藥物核苷類似物對HBV HBsAg沒有抑制效果,聯合使用時,也不影響本發明所述的RNA抑制劑對HBsAg的抑制效果。
在一些實施方案中,本發明所述的RNA抑制劑與當前慢性B肝治療所用的一線用藥恩替卡韋或干擾素進行聯合使用,考察對HBV的抑制效果及是否有相互干擾作用。試驗在廣泛使用的HepG2.2.15細胞株中評估了本發明所述的RNA抑制劑與不同濃度的恩替卡韋或干擾素聯合用給藥對HBV的抑制效果。
在一些實施方案中,使用陰性對照siRNA,正義鏈為SEQ ID NO.: 146,反義鏈為SEQ ID NO.: 147,考察本發明的RNA抑制劑對HBV 4種常見的亞型A、B、C和D型的抑制效果。
在一些實施方案中,使用細胞株HepG2.2.15考察了5'MVIP和/或3'MVIP結構中X、L、B、D、R
1和R
2不同對所述RNA抑制劑正義鏈(SEQ ID NO: 2’)、反義鏈(SEQ ID NO: 59)對應末端,評估所得RNA抑制劑對HBV的HBsAg水準降低效果影響,當X、L、B、D、R
1和R
2之一不同時,相應的5'MVIP和/或3'MVIP中其它部分與5’MVIP09/3’MVIP09的相同。
在一些實施方案中,使用細胞株HepG2.2.15考察了不同肝靶向特異性配體X對所述的RNA抑制劑降低HBV的HBsAg水準效果的影響:
在一些實施方案中,使用細胞株HepG2.2.15考察了不同支鏈L對所述的RNA抑制劑作用效果的影響:
表11
L代碼 | RNA抑制劑代碼 | L結構 |
L1 | Ky-22 | |
L2 | Ky-22-L2 | |
L3 | Ky-22-L3 | |
L4 | Ky-22-L4 | |
L5 | Ky-22-L5 | |
L6 | Ky-22-L6 | |
L7 | Ky-22-L7 | |
L8 | Ky-22-L8 | |
L9 | Ky-22-L9 | |
L10 | Ky-22-L10 | |
L11 | Ky-22-L11* | S的5' MVIP 中L: AS的3'MVIP中L : |
L12 | Ky-22-L12* | S的5' MVIP 中L: AS的3'MVIP中L : |
L13 | Ky-22-L13* | S的5'MVIP中L : AS的3'MVIP中L : |
L14 | Ky-22-L14 |
備註:標有*的RNA抑制劑,表示在同一個5'MVIP或3'MVIP結構中或5'MVIP與3'MVIP彼此L結構不同。
在一些實施方案中,使用細胞株HepG2.2.15考察了接頭B對所述的RNA抑制劑降低HBV的HBsAg水準效果的影響:
表12
B代碼 | RNA抑制劑代碼 | B結構 |
B1 | Ky-22 | |
B2 | Ky-22-B2 | |
B3 | Ky-22-B3 | |
B4 | Ky-22-B4 | |
B5 | Ky-22-B5 | |
B6 | Ky-22-B6 | |
B7 | Ky-22-B7 | |
B8 | Ky-19* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B9 | Ky-19-B2* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B10 | Ky-19-B3* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B11 | Ky-19-B4* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B12 | Ky-19-B5* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B13 | Ky-19-B6* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B14 | Ky-19-B7* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B15 | Ky-19-B8* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B16 | Ky-19-B9* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B17 | Ky-19-B10* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B18 | Ky-19-B11* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B19 | Ky-19-B12* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B20 | Ky-26 | |
B21 | Ky-26-B2 | |
B22 | Ky-26-B3 | |
B23 | Ky-26-B4 | |
B24 | Ky-26-B5 | |
B25 | Ky-26-B6 | |
B26 | Ky-26-B7 | |
B27 | Ky-37* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B28 | Ky-37-B2* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B29 | Ky-37-B3* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B30 | Ky-37-B4* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B31 | Ky-37-B5* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B32 | Ky-37-B6* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B33 | Ky-39* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B34 | Ky-39-B2* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B35 | Ky-39-B3* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B36 | Ky-39-B4* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B37 | Ky-39-B5* | S的5'MVIP中B: |
AS的3'MVIP中B: | ||
B38 | Ky-39-B6* | S的5'MVIP中B: |
AS的3'MVIP中B: |
備註:標有*的RNA抑制劑,表示5'MVIP與3'MVIP彼此之間的接頭B結構不同。
在一些實施方案中,使用細胞株HepG2.2.15考察了連接鏈D對所述的RNA抑制劑降低HBV的HBsAg水準效果的影響:
表13
D代碼 | RNA抑制劑代碼 | D結構 |
D1 | Ky-22* | S的5'MVIP中D: |
AS的3'MVIP中D: | ||
D2 | Ky-22-D2* | S的5'MVIP中D: |
AS的3'MVIP中D: | ||
D3 | Ky-22-D3* | S的5'MVIP中D: |
AS的3'MVIP中D: | ||
D4 | Ky-22-D4* | S的5'MVIP中D: |
AS的3'MVIP中D: | ||
D5 | Ky-22-D5* | S的5'MVIP中D: |
AS的3'MVIP中D: |
備註:標有*的RNA抑制劑,表示5'MVIP與3'MVIP彼此之間的連接鏈D結構不同。
在一些實施方案中,使用細胞株HepG2.2.15考察了不同轉接點R
1對所述的RNA抑制劑降低HBV的HBsAg水準效果的影響:
表14
R 1代碼 | RNA抑制劑代碼 | R 1結構 |
R1-1 | Ky-22 | -NH(CH 2) 6O- |
R1-2 | Ky-22-R1-1 | -O(CH 2) 6O- |
R1-3 | Ky-22-R1-2 | -S(CH 2) 6O- |
R1-4 | Ky-22-R1-3 | -NH(CH 2) 8O- |
R1-5 | Ky-22-R1-4 | -NH(CH 2) 5CH(CH 2CH 3)O- |
R1-6 | Ky-22-R1-5 | -S(CH 2) 4CH(CH 3)O- |
在一些實施方案中,使用細胞株HepG2.2.15考察了不同轉接點R
2對所述的RNA抑制劑降低HBV的HBsAg水準效果的影響:
表15
R 2代碼 | RNA抑制劑代碼 | R 2結構 |
R2-1 | Ky-22 | |
R2-2 | Ky-22-R2-1 | |
R2-3 | Ky-22-R2-2 | |
R2-4 | Ky-22-R2-3 | |
R2-5 | Ky-22-R2-4 | |
R2-6 | Ky-22-R2-5 | |
R2-7 | Ky-22-R2-6 | |
R2-8 | Ky-22-R2-7 | |
R2-9 | Ky-22-R2-8 | |
R2-10 | Ky-22-R2-9 | |
R2-11 | Ky-22-R2-10 | |
R2-12 | Ky-22-R2-11 |
在一些實施方案中,對本發明所述的RNA抑制劑Ky-22的序列進行了進一步優化調整,包括序列mer數、容許的核苷酸可相差個數、氟代及末端硫代個數的考察,這些調整對所述的RNA抑制劑在降低HBsAg水準效果及效果持續性的影響,序列見表16。
表16:Ky-22的序列調整表
RNA抑制劑代碼 | 序列5'→3' |
Ky-22 | 5'MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP09 | |
Ky-2201 | 5'MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us A |
Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP09 | |
Ky-2202 | 5'MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A C A As Us U |
Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP09 | |
Ky-2203 | 5'MVIP09-Gs fGs G U fU U fU fU fC U U G U U G A Cs As A |
Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP09 | |
Ky-2204 | 5'MVIP09-Gs fGs G U fU U fU fU fC U U G U U G A C A As Gs C |
Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP09 | |
Ky-2205 | 5'MVIP09-Gs fGs G U fU U fU fU fC U U G U U G A C A As Us A |
Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U -3' MVIP09 | |
Ky-2206 | 5'MVIP09-Gs fGs G U fU U fU fU fC U C G U U G A Cs As A |
Us Us G U C A fA C G A G fA A fA fA A C C Cs Gs C -3' MVIP09 | |
Ky-2207 | 5'MVIP09-G fG G U fU U fU fU fC U C G U U G A Cs As A |
Us Us G U C A fA C G A G fA A fA fA A C C C U U -3' MVIP09 | |
Ky-2208 | 5'MVIP09-Gs Gs G U fU U fU fU fC U U G U U G A Cs As A |
Us Us G U C A fA C A A G fA A fA A A C C Cs Us U -3' MVIP09 |
實施方案結果顯示,與Ky-22相比較,正義鏈長度為21-mer的Ky-2201在降低HBsAg水準及效果持續性上並沒有顯著提高,甚至稍微降低了一些,所以本發明提供的RNA抑制劑正義鏈長最優選19-mer;與Ky-22相比較,正義鏈和反義鏈中各有一個核苷酸變化的Ky-2203,其降低HBsAg水準及效果持續性上並沒有明顯影響;在Ky-2203設計的基礎上,正義鏈長度為21-mer的Ky-2204與Ky-2203的作用效果無顯著差異;在Ky-2203基礎上調整氟代個數,氟代個數相對少的Ky-2208的作用效果稍稍優於Ky-2203;改變Ky-2204正義鏈3'末端的兩個懸垂核苷酸得到的RNA抑制劑Ky-2205和改變Ky-22反義鏈3'末端的兩個核苷酸得到的RNA抑制劑Ky-2206以及改變Ky-2201正義鏈3'末端的兩個懸垂核苷酸得到的RNA抑制劑Ky-2202與變換前作用效果無顯著差異,說明本發明的RNA抑制劑容許正義鏈或反義鏈各出現1至3個的核苷酸的相差。與Ky-22相比較,正義鏈的5'末端和反義鏈3'末端的連續三個核苷酸之間的磷酸酯鍵的硫代被取消所得的Ky-2207在降低HBsAg水準效果及效果持續時間上影響顯著。
本發明優選正義鏈的鏈長為19-mer、反義鏈的鏈長為21-mer的序列,允許其中有1至3個核苷酸的差異。
在一些實施方案中,將所述的RNA抑制劑Ky-2208與核苷類似物替諾福韋(TDF)在轉基因小鼠模型中進行了抗B肝病毒藥效比對及聯合用藥的研究,實施結果顯示替諾福韋(TDF)不具有減少HBsAg的功效,Ky-2208可以高效地降低HBsAg的水準,最高可以降低99.98%,與替諾福韋(TDF)合用,本發明的RNA抑制劑的效果不受影響。
在一些實施方案中,考察了本發明提供的RNA抑制劑中的Ky-08、Ky-10、Ky-19、Ky-13、Ky-21、Ky-22、Ky-23、Ky-26、Ky-27、Ky-29、Ky-37及Ky-39對HBV轉基因小鼠模型的HBsAg的抑制效果。實施結果顯示,其中RNA抑制劑Ky-19、Ky-26、Ky-37和Ky-39在HBV轉基因小鼠體內可以至少連續4周使HBsAg的表達水準減少93.0%-99.5%以上。
在一些實施方案中,本發明所提供的RNA抑制劑Ky-2208在AAV-HBV小鼠體內可以持續140天左右使HBsAg水準減少98.2-99.6%,並使其體內產生表面抗體HBsAb,顯示功能性治癒B型肝炎的可能。
以下實施例說明了本發明公開的一些實施方案,但並不局限於這些。此外,在提供具體實施方案時,本發明人預期了那些具體實施方案的應用。例如具有具體同類或類似化學結構的RNA抑制劑,用於不同的肝源性疾病的治療。
說明:
DMSO的中文名稱為二甲基亞碸;
DMF的中文名稱為N,N-二甲基甲醯胺;
HOBt的中文名稱為1-羥基苯並三氮唑;
HBTU的中文名稱為O-苯並三氮唑-四甲基脲六氟磷酸酯;
DIPEA(DIEA)的中文名稱為N,N-二異丙基乙胺;
DCM的中文名稱為二氯甲烷;
DMAP的中文名稱為4-二甲氨基吡啶;
DMT-CL的中文名稱為4,4'-二甲氧基三苯基氯甲烷;
MEOH的中文名稱為甲醇;
TBTU的中文名稱為O-苯並三氮唑-N,N,N',N'-四甲基脲四氟硼酸;
實施例一、
RNA
抑制劑
Ky-19
、
Ky-22
、
Ky-2208
、
Ky-26
、
Ky-37
和
Ky-39
的合成
本發明所述的RNA抑制劑是通過固相亞磷醯胺法得到各自的正義鏈和反義鏈,正義鏈與對應的反義鏈互補退火得到終產品。固相亞磷醯胺法基本步驟包括:1)脫保護:脫掉起始單體Solid Support羥基保護基(DMTr);2)偶聯:加上第一個亞磷醯胺單體,通過3’至5’方向發生偶聯反應;3)氧化:將所得的核苷亞磷酸酯氧化成更穩定的核苷磷酸酯(即三價磷氧化成五價磷);4)封閉:將沒有反應的前一步核苷酸單體5’-OH加冒封死,使其不再進一步參與反應;重複上述步驟,直至最後一個亞磷醯胺單體的接入。然後用甲胺水溶液和氨水裂解Solid Support與起始單體之間酯鍵,並將所得寡聚核苷酸上的各個鹼基與磷酸上的保護基氰乙基(P)、苯甲醯基(mA、fA)、乙醯基(mC)脫掉。經HPLC分離純化後,過濾除菌,凍幹得到相應的正義鏈或反義鏈。
退火 精確測定正義鏈和反義鏈複溶溶液的濃度,按等摩爾濃度進行混合後,加入該體積1/20體積的1 M PBS溶液再次混勻,將混合體系加熱至95°C,持續5min,然後自然降溫3h至40℃或者室溫時,進行HPLC檢測,若單鏈殘餘<5%即可認為反應完成。
m為1-4時,通式中接頭B部分分別支化1至4次,以獲得對應的3’MVIP的Solid Support。
當m為1時,所得的Solid Support作為RNA抑制劑Ky-26的反義鏈和Ky-39正義鏈固相合成的起始單體;當m為2時,所得的Solid Support作為RNA抑制劑Ky-37的正義鏈和Ky-22和Ky-2208的反義鏈的固相合成起始單體;當m為3時,所得的Solid Support作為RNA抑制劑Ky-19的反義鏈的固相合成起始單體。
n為1-4時,通式中接頭B部分分別支化1至4次,以獲得對應的5’MVIP亞磷醯胺單體。
當n為1時,所得的5’MVIP亞磷醯胺單體作為RNA抑制劑Ky-19、Ky-26和Ky-37的正義鏈固相合成的最後一個單體;當n等於2時,所得的5’MVIP亞磷醯胺單體作為Ky-39、Ky-22和Ky-2208的正義鏈固相合成的最後一個單體。
本發明所述的這些RNA抑制劑的正義鏈和反義鏈在進行亞磷醯胺固相合成之前,需要先化學合成相應的3’MVIP的Solid Support和5’MVIP亞磷醯胺單體。化學合成過程敘述如下:
1. 3’MVIP的Solid Support的合成
合成過程描述:
稱取2-氨基-1,3-丙二醇(5.0g,54.9mmol)加入DMSO 50mL,氫氧化鈉溶液(1g/mL)5mL,降溫到0℃,滴加丙烯酸叔丁酯(20mL,137.8mol)2小時加完,室溫反應48h,加石油醚(100mL),飽和食鹽水洗2次,有機層乾燥。過層析柱(洗脫液:乙酸乙酯:石油醚=25%-75%),上柱加0.05%的三乙胺,得無色油狀物6.2g。
稱取ERC-01-c1(6.2g,17.9mmol),加二氯甲烷50mL,碳酸鈉溶液(25%)23mL,室溫滴加氯甲酸苄酯(8.2mL,57.4mmol),2小時滴加完,室溫反應過夜,飽和食鹽水洗滌3次,無水硫酸鈉乾燥,蒸幹溶劑,過層析柱(乙酸乙酯:石油醚=5%-30%)得油狀物4.0g。
取ERC-01-c2(4.0g,8.3mmol)加甲酸12mL,室溫反應過夜,減壓蒸幹溶劑,得產品2.8g。
將化合物ERC-01-c3(1.11g,3.0mmol)和dlSANC-c4(3.6g,8.04mmol)加到DMF(60 mL)中,然後加入HOBt(2.24g)和HBTU(3.36g),然後緩慢加入DIEA(4.16mL)。反應液室溫下攪拌反應3小時。然後加入水,水層用二氯甲烷萃取(2x10mL)。合併有機層,然後依次用飽和碳酸氫鈉(80 mL)、水(2x60 mL)、飽和食鹽水(60mL)洗。用無水硫酸鈉乾燥,減壓蒸幹,用矽膠柱層析純化(洗脫液:3-15 % MeOH in DCM)。得淡黃色固3.24g。
ERCd-01-c1(3.24g,2.6mmol)用甲醇(60mL)溶解,加入10%鈀碳(0.3g),乙酸(2.0mL)。然後常壓下加氫,反應過夜。反應液用矽藻土過濾,濾液減壓蒸幹,得油狀物ERCd-01-c2 2.9g,其高分辨質譜圖見圖1。
向反應瓶內依次加入SANCd-01-c0(0.824g,1.5mmol)和ERCd-01-c2(1.09g,1.0mmol),再加入10mL的DCM,攪拌溶解,再依次加入TBTU(0.963g)和DIPEA(0.517g),反應過夜,加水,用DCM萃取,有機相再用飽和食鹽水洗滌,乾燥、過濾、濃縮,最後過矽膠柱進行純化,得產品1.3g。
1.1.7. 3’MVIP09-c2的合成
向反應瓶內依次加入3’MVIP09-c1(1.62g,1µmol)和10mL的DCM,室溫攪拌溶解,再依次加入DMAP(0.366g)和丁二酸酐(0.2g,3µmol),室溫攪拌反應,TLC分析,反應合格濃縮掉DCM,加水,用DCM萃取,有機相再用飽和食鹽水洗滌,有機相經無水硫酸鈉乾燥、過濾、濃縮,最後過矽膠柱進行純化,得到產品為1.55g。
向反應瓶內依次加入3’MVIP09-c2(0.86g,0.5µmol)和10mL DMF,溶解,再依次加入HBTU(0.19g)、DIPEA(0.194g)和大孔氨甲基樹脂(2.0g),搖床24h,過濾,樹脂用10%甲醇/DCM洗滌,再用25%醋酸/吡啶進行封端,取代度150µmol/g。
合成步驟參照實施例1中1.1.1. ERC-01-c1的合成。
合成步驟參照實施例1中1.1.2. ERC-01-c2的合成。
合成步驟參照實施例1中1.1.3. ERC-01-c3的合成。
合成步驟參照實施例1中 1.1.4. ERCd-01-c1的合成。
合成步驟參照實施例1中 1.1.5. ERCd-01-c2的合成。
合成步驟參照實施例1中 1.1.6. 3’MVIP09-c1的合成,合成所得3’MVIP17-c1的高分辨質譜圖見圖2。
合成步驟參照實施例1中 1.1.7. 3’MVIP09-c2的合成。
合成步驟參照實施例1中1.1.8 3’MVIP09的Solid Support合成。
合成過程描述:
合成步驟參照實施例1中1.1.6. 3’MVIP09-c1的合成。
合成步驟參照實施例1中 1.1.7. 3’MVIP09-c2的合成。
合成步驟參照實施例1中 1.1.8. 3’MVIP09的Solid Support合成。
2. 5’MVIP亞磷醯胺單體的合成
稱量ERCd-01-c2 (2.18g,2.0mmol)溶於DMF(50mL),加戊二酸單苄酯(0.53g,2.4mmol)、DIPEA(0.78g)與TBTU(0.84g),室溫攪拌過夜,加水淬滅(50mL),DCM(30mL*3)萃取,10%檸檬酸(50mL*3)、飽和碳酸氫鈉50mL和吡啶100mL洗滌,無水硫酸鈉乾燥,過濾,旋蒸,過柱純化得產品5’MVIP09-ERCd-PFP-c1 (2.15g)。
稱量5’MVIP09-ERCd-PFP-c1 (2.15g,1.66mmol)和10%鈀碳(0.21g),加甲醇(50mL),室溫攪拌加氫過夜,反應結束後矽藻土過濾鈀碳,旋蒸得5’MVIP09-ERCd-PFP-c2粗品(1.9g),其高解析度質譜圖如圖3所示。
稱量5’MVIP09-ERCd-PFP-c2 粗品(1.9g,1.58mmol)溶於DCM(60mL),加DIPEA(1.33g),冷卻,加三氟乙酸五氟苯酚酯(2.21g,7.9mmol),室溫攪拌反應2h後旋蒸,再溶於DCM(60mL),飽和碳酸氫鈉(30mL*3)、10%檸檬酸(30mL*1)、飽和食鹽水(50mL*1)洗滌,無水硫酸鈉乾燥,過濾,旋蒸得5’MVIP09-ERCd-PFP粗品(2.35g),抽幹後無純化直接用於下一步反應。
5’MVIP09-ERCd-PFP粗品(2.35g,1.58mmol)溶於DCM(60mL),加DIPEA(0.82g,6.32mmol)、6-氨基-1-己醇(0.37g,3.16mmol),室溫攪拌反應過夜。加10%檸檬酸(30mL),DCM(30mL*3)萃取,飽和食鹽水(50mL)洗滌,無水硫酸鈉乾燥,過濾、旋蒸,過柱純化得產品5’MVIP09單體-c1 (1.73g)。
稱量5’MVIP09亞磷醯胺單體-c1(1.3g、1.0mmol)溶於乙腈(30mL),加入二異丙胺三氮唑(0.22g),冰浴下滴加雙-(二異丙基氨基)(2-氰基乙氧基)膦(0.36g,1.2mmol),室溫反應4h,HPLC中控,反應合格後,濃縮過柱純化得到產品5’MVIP09單體(1.2g)。
5'MVIP01的亞磷醯胺單體稱量YICd-01-c2 (1.12g,2.0mmol),剩餘操作參照2.1.1.~ 2.1.5.。
實施例二:體外試驗
實施例 1 :使用細胞株HepG2.2.15評估5'MVIP和3'MVIP與正義鏈和反義鏈不同末端偶聯所得的RNA抑制劑降低HBV的HBsAg水準效果。
試驗過程描述:按實施例一所述的方法製備相應的RNA抑制劑Ky-00~Ky-26,配製含10%胎牛血清的DMEM培養基。用培養液配製含0.05、0.5、5nM RNA抑制劑樣品的培養基。以10
5細胞密度接種HepG2.2.15細胞,10%胎牛血清DMEM培養基,37℃,5%CO
2,培養24h後,加不同濃度的上述RNA抑制劑樣品干預,孵育72h後,取上清,用HBsAg檢測試劑盒(上海科華,ELISA法),與未干預HepG2.2.15細胞上清對比,標定樣品干預組的HBsAg相對百分率。
所得試驗資料見圖4。如圖4所示,其中Ky-19、Ky-22和Ky-26對HBsAg的抑制效果優於其它化合物。
實施例2
:使用細胞株HepG2.2.15評估5'MVIP和3'MVIP同時放在RNA抑制劑的正義鏈或反義鏈的兩個末端上或5'MVIP或3'MVIP同時放在反義鏈和正義鏈的同一個末端,如3’末端或5’末端,考察所得的RNA抑制劑降低HBV的HBsAg水準效果。
試驗過程描述:按實施例一所述的方法製備相應的RNA抑制劑Ky-27~Ky-44,配製含10%胎牛血清的DMEM培養基。用培養液配製含0.05、0.5、5nM RNA抑制劑樣品的培養基。以10
5細胞密度接種HepG2.2.15細胞,10%胎牛血清DMEM培養基,37℃,5%CO
2,培養24h後,加不同濃度的上述RNA抑制劑樣品干預,孵育72h後,取上清,用HBsAg檢測試劑盒(上海科華,ELISA法),與未干預HepG2.2.15細胞上清對比,標定樣品干預組的HBsAg相對百分率。所得試驗資料見圖5。
如圖5所示,其中Ky-37和Ky-39對HBsAg的抑制效果優於其它化合物。
實施例
3
使用細胞株
HepG2.2.15
評估不同肝靶向特異性配體
X
對
RNA
抑制劑降低
HBV
的
HBsAg
水準效果的影響
考察不同肝靶向特異性配體X對RNA抑制劑降低HBV的HBsAg水準效果的影響,所得的RNA抑制劑中Ky-22、Ky-22-X2~Ky-22-X6除X結構改變外,L、B、D及R
1/R
2與組合5’MVIP09 /3’MVIP09中的一致。
試驗所涉及的RNA抑制劑中的正義鏈為SEQ ID NO:2’,反義鏈為SEQ ID NO:59,正義鏈5'末端偶聯5'MVIP,反義鏈3'末端偶聯3'MVIP。
試驗過程描述:按實施例一所述的方法製備相應的RNA抑制劑,配製含10%胎牛血清的DMEM培養基。用培養液配製含10nM RNA抑制劑樣品的培養基。以10
5細胞密度接種HepG2.2.15細胞,10%胎牛血清DMEM培養基,37℃,5%CO
2,培養24h後,加藥干預,孵育72h後,取上清,用HBsAg檢測試劑盒(上海科華,ELISA法),與未干預HepG2.2.15細胞上清對比,標定樣品干預組的HBsAg相對百分率。
所得試驗資料見圖6。結果表明,當X分別為半乳糖、半乳糖胺、N-乙醯半乳糖胺及其衍生物時,所得RNA抑制劑優選N-乙醯半乳糖胺及其衍生物作為配體。
實施例
4
使用細胞株
HepG2.2.15
評估不同支鏈
L
對
RNA
抑制劑降低
HBV
的
HBsAg
水準效果的影響
考察不同支鏈L對RNA抑制劑降低HBV HBsAg水準效果的影響,所得RNA抑制劑中Ky-22、Ky-22-L2~Ky-22-L14除L結構改變外,X、B、D及R
1/R
2與組合5’MVIP09 /3’MVIP09中的一致。
試驗所涉及的RNA抑制劑中的正義鏈為SEQ ID NO:2’,反義鏈為SEQ ID NO:59,正義鏈5'末端偶聯5'MVIP,反義鏈3'末端偶聯3'MVIP。
試驗過程描述:按實施例一所述的方法製備相應的RNA抑制劑,配製含10%胎牛血清的DMEM培養基。用培養液配製含10nM RNA抑制劑樣品的培養基。以10
5細胞密度接種HepG2.2.15細胞,10%胎牛血清DMEM培養基,37℃,5%CO
2,培養24h後,加藥干預,孵育72h後,取上清,用HBsAg檢測試劑盒(上海科華,ELISA法),與未干預HepG2.2.15細胞上清對比,標定樣品干預組的HBsAg相對百分率。
所得試驗資料見圖6。結果表明,L的長短對RNA抑制劑的作用效果影響較大,L鏈不能太短也不能太長;當含有-NH-、C=O、O、S、醯胺基、磷醯基、硫代磷醯基、脂肪族碳環基如環己烷或者這些基團的組合時,或者在同一個5'MVIP、3'MVIP結構中或5'MVIP與3'MVIP彼此L結構不同、在鏈長為C7-C18這個範圍,所得RNA抑制劑對HBV的HBsAg水準降低效果相差不大。
實施例
5
使用細胞株
HepG2.2.15
評估接頭
B
對
RNA
抑制劑降低
HBV
的
HBsAg
水準效果的影響
考察不同接頭B對RNA抑制劑降低HBV的HBsAg水準效果的影響,所得RNA抑制劑Ky-22、Ky-22-B2~ Ky-22-B7、Ky-19、Ky-19-B2~ Ky-19-B12、Ky-26、Ky-26-B2~ Ky-26-B7、Ky-37、Ky-37-B2~ Ky-37-B6、Ky-39、Ky-39-B2~ Ky-39-B6,除B結構改變外,X、L、D及R
1/R
2與組合5’MVIP09/3’MVIP09中的一致。
試驗所涉及的RNA抑制劑中的正義鏈為SEQ ID NO:2’,反義鏈為SEQ ID NO:59,正義鏈5'末端偶聯5'MVIP,反義鏈3'末端偶聯3'MVIP。
試驗過程描述:按實施例一所述的方法製備相應的RNA抑制劑,配製含10%胎牛血清的DMEM培養基。用培養液配製含10nM RNA抑制劑樣品的培養基。以10
5細胞密度接種HepG2.2.15細胞,10%胎牛血清DMEM培養基,37℃,5%CO
2,培養24h後,加藥干預,孵育72h後,取上清,用HBsAg檢測試劑盒(上海科華,ELISA法),與未干預HepG2.2.15細胞上清對比,標定樣品干預組的HBsAg相對百分率。
所得試驗資料見圖7。結果表明,除接頭B結構改變外,而X、L、D及R
1/R
2與組合5’MVIP09/3’MVIP09中的一致時,接頭B中通式中的A
1和A
2各自獨立地是C、O、S、-NH-、羰基、醯胺基、磷醯基或硫代磷醯基,r為0-4的整數,並且接頭B在5’MVIP與3’MVIP之間相同或不同時,對HBsAg水準降低效果相差不大。
實施例
6
使用細胞株
HepG2.2.15
評估連接鏈
D
對
RNA
抑制劑降低
HBV
的
HBsAg
水準效果的影響
考察不同連接鏈D對RNA抑制劑降低HBV的HBsAg水準效果的影響,所得RNA抑制劑Ky-22、Ky-22-D2~ Ky-22-D5除D結構改變外,X、L、B及R
1/R
2與最優選MVIP組合5’MVIP09/3’MVIP09中的一致。
試驗所涉及的RNA抑制劑中的正義鏈為SEQ ID NO:2’,反義鏈為SEQ ID NO:59,正義鏈5'末端偶聯5'MVIP,反義鏈3'末端偶聯3'MVIP。
試驗過程描述:按實施例一所述的方法製備相應的RNA抑制劑,配製含10%胎牛血清的DMEM培養基。用培養液配製含10nM RNA抑制劑樣品的培養基。以10
5細胞密度接種HepG2.2.15細胞,10%胎牛血清DMEM培養基,37℃,5%CO
2,培養24h後,加藥干預,孵育72h後,取上清,用HBsAg檢測試劑盒(上海科華,ELISA法),與未干預HepG2.2.15細胞上清對比,標定樣品干預組的HBsAg相對百分率。
所得試驗資料見圖6。結果表明,在MVIP結構及RNA抑制劑相同的情況下,不同的連接鏈D會對RNA抑制劑抑制HBsAg的效果有影響,其中D1、D2、D4的效果接近且優於D3。
實施例
7
:使用細胞株
HepG2.2.15
評估不同
R
1
對
RNA
抑制劑降低
HBV
的
HBsAg
水準效果的影響
考察不同轉接點R
1對RNA抑制劑降低HBV的HBsAg水準效果的影響,所得RNA抑制劑Ky-22、Ky-22-R1-1~Ky-22-R1-5除R
1結構改變外,X、L、B、D及R
2與最優選MVIP組合5’MVIP09/ 3’MVIP09中的一致。
試驗所涉及的RNA抑制劑中的正義鏈為SEQ ID NO:2’,反義鏈為SEQ ID NO:59,正義鏈5'末端偶聯5'MVIP,反義鏈3'末端偶聯3'MVIP。
試驗過程描述:按實施例一所述的方法製備相應的RNA抑制劑,配製含10%胎牛血清的DMEM培養基。用培養液配製含10nM RNA抑制劑樣品的培養基。以10
5細胞密度接種HepG2.2.15細胞,10%胎牛血清DMEM培養基,37℃,5%CO
2,培養24h後,加藥干預,孵育72h後,取上清,用HBsAg檢測試劑盒(上海科華,ELISA法),與未干預HepG2.2.15細胞上清對比,標定樣品干預組的HBsAg相對百分率。
所得試驗資料見圖8。結果表明,不同的轉接點R
1會對RNA抑制劑抑制HBsAg的效果有影響,其中R1-1作為轉接點的降低HBsAg水準效果最佳。
實施例
8
:使用細胞株
HepG2.2.15
評估不同
R
2
對
RNA
抑制劑降低
HBV
的
HBsAg
水準效果的影響
考察不同轉接點R
2對RNA抑制劑降低HBV的HBsAg水準效果的影響,所得RNA抑制劑Ky-22、Ky-22-R2-1~Ky-22-R2-11除R
2結構改變外,X、L、B、D及R
1與最優選MVIP組合5’MVIP09/3’MVIP09中的一致。按實施例一所述的方法製備得到相應的RNA抑制劑。
試驗所涉及的RNA抑制劑中的正義鏈為SEQ ID NO:2’,反義鏈為SEQ ID NO:59,正義鏈5'末端偶聯5'MVIP,反義鏈3'末端偶聯3'MVIP。
試驗過程描述:按實施例一所述的方法製備相應的RNA抑制劑,配製含10%胎牛血清的DMEM培養基。用培養液配製含10nM RNA抑制劑樣品的培養基。以10
5細胞密度接種HepG2.2.15細胞,10%胎牛血清DMEM培養基,37℃,5%CO
2,培養24h後,加藥干預,孵育72h後,取上清,用HBsAg檢測試劑盒(上海科華,ELISA法),與未干預HepG2.2.15細胞上清對比,標定樣品干預組的HBsAg相對百分率。
所得試驗資料見圖8。結果表明,不同的轉接點R
2會對RNA抑制劑降低HBsAg水準的效果有影響,其中R2-1作為轉接點時的降低HBsAg水準效果最佳。
實施例
9 Ky-22
與當前慢性
B
肝治療所用的一線用藥恩替卡韋或干擾素聯合使用,考察對
HBV
的抑制效果是否有相互干擾作用
試驗在廣泛使用的HepG2.2.15細胞株中評估了本發明所述的RNA與不同濃度的恩替卡韋(ETV)或干擾素(IFN-a)聯合用給藥對HBV的抑制效果。
試驗所涉及的RNA抑制劑Ky-22中的正義鏈為SEQ ID NO:2’,反義鏈為SEQ ID NO:59,正義鏈5'末端偶聯5'MVIP,反義鏈3'末端偶聯3'MVIP。
試驗過程描述:配製含10%胎牛血清的DMEM培養基。用培養液配製含10nM RNA抑制劑Ky-22樣品的培養基。以10
5細胞密度接種HepG2.2.15細胞,10%胎牛血清DMEM培養基,37℃,5%CO
2,培養24h後,加藥干預,孵育72h後,取上清檢測HBsAg、HBeAg、HBV DNA,與未干預HepG2.2.15細胞上清對比,標定樣品干預組的HBsAg、HBeAg、HBV DNA相對百分率。
加藥濃度為:
ETV:10μM、1μM、0.1μM;
IFN-a:1000IU/mL 、100IU/mL、10IU/mL;
Ky-22:0.125μg/mL;
ETV+Ky-22:10μM+0.125μg/mL、1μM+0.125μg/mL、0.1μM+0.125μg/mL;
IFN-a +Ky-22:1000IU/mL+0.125μg/mL、100IU/mL+0.125μg/mL、10IU/mL+0.125μg/mL
RNA抑制劑Ky-22對HepG2.2.15細胞中HBsAg和HBeAg水準的影響分別如圖9和圖10所示。結果表明,單獨使用恩替卡韋或干擾素對HBsAg和HBeAg無顯著抑制效果,恩替卡韋或干擾素聯合使用Ky-22則對HBsAg和HBeAg表現出顯著抑制效果,且抑制程度與恩替卡韋或干擾素的濃度沒有相關性。恩替卡韋或干擾素不會影響本發明的RNA抑制劑的對HBsAg和HBeAg的效果;RNA抑制劑Ky-22與恩替卡韋或干擾素聯合使用,也不影響恩替卡韋或干擾素對HBV DNA的抑制效果,甚至會強化干擾素對HBV DNA的作用效果,資料結果見圖11。本發明的RNA抑制劑可以和恩替卡韋和干擾素聯合使用。
實施例
10 Ky-22
對
4
種不同基因型
(A
、
B
、
C
、
D)HBV
細胞株的抑制效果研究
試驗過程描述:
細胞株構建:選擇在HepG2細胞的基礎上,用sleeping beauty的轉座子系統進行HBV基因的整合;細胞培養條件:DMEM+10%FBS,37°C、5% CO
2。4種不同基因型(A、B、C、D)的HBV1.3倍體基因通過Gibson Assembly® Master Mix連接到PT2/HB載體上,同時連接紅色螢光蛋白和嘌呤黴素抗性基因,作為細胞株篩選時的標記;構建好的質粒分別與pCMV(CAT)T7-SB100用轉染試劑X-tremeGENE HP DNA Transfection Reagent共轉染到HepG2細胞中,轉染方法為:按說明書配製好一塊10cm培養皿的細胞轉染所需的轉染體系,靜止20min,將匯合度達到70%的HepG2細胞消化成細胞懸液,加入配製好的轉染體系,混合均勻後放入培養箱培養。轉染後48h用2μg/mL嘌呤黴素抗性進行篩選,不表達嘌呤黴素抗性的細胞即不整合HBV的細胞死亡,擴增整合HBV的細胞,並通過流式細胞儀分選出紅色螢光強度高的細胞,即HBV整合拷貝數高的細胞,獲得4種不同基因型的HBV穩定整合細胞株。
將對數生長期的A、B、C、D 4種基因型的HBV穩定整合細胞消化成細胞懸液,加入48孔板中(300μl/孔),每孔約300000個細胞,待細胞匯合度達到70%(約鋪板24h後),加入以下濃度的Ky-22或者陰性對照siRNA(正義鏈SEQ ID NO.: 146,反義鏈為SEQ ID NO.: 147):
4.1μg/mL 、2.2μg/mL、1.1μg/mL 、0.6μg/mL 、0.3μg/mL、0.15μg/mL、0.0725μg/mL、0.03625μg/mL、0.018125μg/mL、 0.0090625μg/mL 、0.00453125μg/mL 、0.00226563μg/mL、0.00113281μg/mL 、0.000566406μg/mL、0.000283203μg/mL
或不加藥,分別於24h,48h,72h將上清收集後保存至-20℃,並更換新鮮不加藥的培養基。檢測細胞上清中HBsAg的含量。
試驗資料見圖12。結果有顯示與陰性對照siRNA處理組(Control)相比,Ky-22對於A、B、C和D基因型的HBV均具有顯著的抑制效果,EC50 (ng/mL)分別為22.72、25.45、29.06和23.35。
實施例三
體內藥效研究
實施例
1
:
RNA
抑制劑對
HBV
轉基因小鼠模型的
HBsAg
的降低效果研究
按實施例一所述的方法製備得到相應的RNA抑制劑Ky-08、Ky-10、Ky-13、Ky-19、Ky-21、Ky-22、Ky-23、Ky-26、Ky-27、Ky-29、Ky-37及Ky-39,選取65只、雄性、體重25~35g、周齡為8-10w的HBV轉基因小鼠,飼養於符合SPF級標準動物房,濕度16~26 ℃,溫度40~70%,迴圈光照(明暗各12小時),自由攝食飲水。
動物分組前檢測HBV HBsAg,按HBV HBsAg表達量進行隨機分組,儘量保持各組HBV HBsAg的平均水準一致。將小鼠分為13組,每組5只,對照組(生理鹽水)、給藥組1-給藥組12。給藥劑量均為3mg/kg,單次給藥,給藥當天設置為d0,各組小鼠d0按0.04mL/10g皮下注射給予相應受試品液。動物觀察時間4-6周不等,采血時間點d0、d7、d14、d21、d28、d35和d42。各組各采血時間通過小鼠眼眶靜脈叢採集全血,3000×g離心5min,取上清,檢測HBV HBsAg表達量。
將各給藥組動物的HBsAg水準歸一至給藥前和對照組,試驗資料如圖13所示。
研究結果顯示,本發明所述的RNA抑制劑在前三周均呈現出對HBV HBsAg水準顯著的減少效果,其中最佳的降低率可達99.8%。因與5'MVIP和或3'MVIP偶聯的位置不同,各RNA抑制劑對HBsAg的降低效果的持續時間不一致,其中Ky-19、Ky-22、Ky-26、Ky-29、Ky-37及Ky-39在d28仍維持在HBV HBsAg水準減少93%以上的效果,且Ky-22效果的持續性最好,在d35還維持在HBV HBsAg水準減少91%以上的效果。
實施例
2
:考察
Ky-22
序列調整對
HBV
轉基因小鼠
HBsAg
抑制效果影響
按實施例一所述的方法製備得到相應的RNA抑制劑Ky-22、Ky-2201~Ky-2208,選取50只、雄性、體重25~35g、周齡為8-13w的HBV轉基因小鼠,飼養於符合SPF級標準動物房,濕度16~26 ℃,溫度40~70%,迴圈光照(明暗各12小時),自由攝食飲水。
動物分組前檢測HBV HBsAg,按HBV HBsAg表達量進行隨機分組,儘量保持各組HBV HBsAg 的平均水準一致。將小鼠分為10組,每組5只,對照組(生理鹽水)、給藥組(9組)。給藥劑量均為3mg/kg,單次給藥,給藥當天設為d0,各組小鼠d0按0.04mL/10g皮下注射給予相應受試品液。動物觀察時間6周,采血時間點d0、d7、d14、d21、d28、d35和d42。各組各采血時間通過小鼠眼眶靜脈叢採集全血,3000×g離心5min,取上清,檢測HBV HBsAg表達量。
分組 | 小鼠數量/組 | 給藥時間 | 給藥劑量 | 采血時間點 | 給藥方式 |
對照組 | 5 | d0 | - | d0、d7、d14、d21、d35、d42 | 皮下注射,單次 |
Ky-22 | 5 | 3mg/kg | |||
Ky-2201 | 5 | ||||
Ky-2202 | 5 | ||||
Ky-2203 | 5 | ||||
Ky-2204 | 5 | ||||
Ky-2205 | 5 | ||||
Ky-2206 | 5 | ||||
Ky-2207 | 5 | ||||
Ky-2208 | 5 |
將各給藥組動物的HBsAg水準歸一至給藥前和對照組。
試驗資料見圖14,實施結果顯示,與Ky-22相比較,正義鏈長度為21-mer的Ky-2201在降低HBsAg水準及效果持續性上並沒有顯著提高,甚至稍微降低了一些,所以本發明提供的RNA抑制劑正義鏈長最優選19-mer;與Ky-22相比較,正義鏈和反義鏈中各有一個核苷酸變化的Ky-2203的,其降低HBsAg水準及效果持續性上並沒有明顯影響;在Ky-2203設計的基礎上,正義鏈長度為21-mer的Ky-2204與Ky-2203的作用效果無顯著差異;在Ky-2203基礎上調整氟代個數,氟代個數相對少的Ky-2208的作用效果稍稍優於Ky-2203;變換正義鏈或反義鏈3'末端的兩個懸垂核苷酸得到的RNA抑制劑Ky-2205和Ky-2206與變換前作用效果無顯著差異;說明本發明的RNA抑制劑容許正義鏈和反義鏈各出現1至3個的核苷酸的相差。與Ky-22相比較,正義鏈的5'末端和反義鏈3'末端的連續三個核苷酸之間的磷酸酯鍵的硫代被取消所得的Ky-2207在降低HBsAg水準效果及效果持續時間上影響顯著。
本發明優選正義鏈的鏈長為19-mer、反義鏈的鏈長為21-mer的序列,允許其中有1至3個核苷酸的差異。
實施例
3
:
AAV-HBV
小鼠模型中,考察
Ky-2208
的劑效回應、單劑量重複給藥對
HBsAg
的降低效果及是否能產生表面抗體
HBsAb
試驗過程描述:取36只適齡小鼠,屏障設施內飼養約7天,日常觀察,無明顯異常後進行實驗。先將HBV病毒4℃依次解凍,用胰島素注射器在小鼠尾靜脈注射rAAV8-1.3HBV(五加和公司,ayw,病毒批號:A2020051801),每只小鼠注射1×10
11v.g.。在造模後於第4周對動物進行采血、離心和收集血清並檢測HBsAg指標。造模後第6周采血檢測血清中HBsAg,根據HBsAg的檢測結果,挑選30只小鼠隨機分5組,儘量保持各組HBsAg的平均水準一致。分組後的第2周開始給藥,給藥當天采血檢測HBsAg,設為d0天。各組給藥資訊和采血點如下表:
分組 | 小鼠數量/組 | 給藥時間 | 給藥劑量 | 采血時間點 | 給藥方式 |
對照組 | 6 | d0 | - | d0、d7、d14、d21、d28、d35、d42、d49、d63、d70、d77、d91、d98、d105、d112、d126、d133、d140 | 皮下注射,單次 |
給藥組1 | 6 | 1mg/kg | d0、d7、d14、d21、d35、d42、d49、d63、d77 | 皮下注射,單次 | |
給藥組2 | 6 | 3 mg/kg | 皮下注射,單次 | ||
給藥組3 | 6 | 9 mg/kg | d0、d7、d14、d21、d35、d42、d49、d63、d77、d91、d98、d126、d133、d140 | 皮下注射,單次 | |
給藥組4 | 6 | 3mg/kg | d0、d7、d14、d21、d35、d49、d63、d70、d98、d105、d112 | 皮下注射,每週一次,連續三次 |
將各給藥組動物的HBsAg水準歸一至給藥前和對照組,所得試驗資料HBsAg和HBsAb分別見圖15和圖16。
試驗結果顯示,在整個140天的考察期內,Ky-2208的9mg/kg組可以使AAV-HBV小鼠模型體內HBsAg水準降低範圍在93.1%-99.6%,重複給藥組考察至112天,抑制效果仍保持在95%以上;至98天,單劑量已在HBV模型鼠體內測出表面抗體HBsAb,使小鼠體內產生新的抗HBV免疫力。
實施例
4
:
Ky-2208
與當前慢性
B
肝治療所用的一線用藥替諾福韋(
TDF
)比對研究和聯合使用,在
HBV
轉基因小鼠模型中考察對
HBV
的
HBsAg
抑制效果及是否有干擾作用
試驗過程描述:HBV-Tg雄性小鼠,48只,體重25~35g,周齡8-13w,飼養於符合SPF級標準動物房,濕度16~26 ℃,溫度40~70%,迴圈光照(明暗各12小時),自由攝食飲水。化合物配製的溶劑為生理鹽水,工作液濃度為0.75mg/mL。動物分組前檢測HBV HBsAg,按HBV HBsAg表達量將48只雄性小鼠隨機分為6組,每組8只,儘量保持各組HBV HBsAg 的平均水準一致。實驗共設6個組,分為1個對照組(0.9%生理鹽水)和5個給藥組。d0天單次給藥,各組小鼠d0按0.04mL/10g皮下注射給予相應受試品液。分別在給藥前d0,給藥後d7、d14、d21、d28通過小鼠眼眶靜脈叢採集全血,3000×g離心5min,取上清,d0、d7、d14、d21、d28送樣檢測HBV HBsAg。
具體給藥方案見下表:
序號 | 試驗藥物 | 給藥劑量 | 小鼠數量/組 | 給藥途徑 | 給藥頻率 | 溶媒 |
1 | 生理鹽水 | / | 8 | sc | 單次 | 生理鹽水 |
2 | TDF | 15mpk | 8 | po | daily | |
3 | Ky-2208 | 3mpk | 8 | sc | 單次 | |
4 | Ky-2208 | 9mpk | 8 | sc | 單次 | |
5 | Ky-2208 +TDF | 3 mpk +15 mpk | 8 | sc/po | Ky-2208單次/TDF daily | |
6 | Ky-2208 +TDF | 9 mpk + 15mpk | 8 | sc/po | Ky-2208單次/TDF daily |
備註:sc為皮下注射,po為灌胃。
所得試驗資料見圖17。試驗結果證實了核苷類似物抗B肝藥物TDF對HBV HBsAg沒有抑制效果,聯合使用時,也不影響本發明所述的RNA抑制劑對HBsAg的抑制效果。單用Ky-2208或Ky-2208與TDF聯合使用,最高分別可以使HBsAg的水準降低99.95%和99.98%。
無
為了使本發明的目的、技術方案和有益效果更加清楚,本發明提供如下附圖:
圖1為實施例一1.1.5中合成的ERCd-01-c2的高分辨質譜圖;
圖2為實施例一1.2.6中合成的3’MVIP17-c1的高分辨質譜圖;
圖3為實施例一2.1.2中合成的5’MVIP09-ERCd-PFP-c2的高分辨質譜圖;
圖4為實施例二實施例1中的Ky-00~ Ky-26對細胞株HepG2.2.15中HBsAg水準抑制效果圖;
圖5為實施例二實施例2中的Ky-27~ Ky-44對細胞株HepG2.2.15中HBsAg水準抑制效果圖;
圖6為實施例二實施例3、4和6中不同X/L/D對RNA抑制劑降低HBV的HBsAg水準效果的影響;
圖7為實施例二實施例5中接頭B對RNA抑制劑降低HBV的HBsAg水準效果的影響圖;
圖8為實施例二實施例7和8中不同轉接點R
1/R
2對RNA抑制劑降低HBV的HBsAg水準效果的影響圖;
圖9為實施例二實施例9中Ky-22與恩替卡韋或干擾素聯用對HepG2.2.15細胞中HBsAg的抑制效果圖;
圖10為實施例二實施例9中Ky-22與恩替卡韋或干擾素聯用對HepG2.2.15細胞中HBeAg的抑制效果圖;
圖11為實施例二實施例9中Ky-22與恩替卡韋或干擾素聯用對HepG2.2.15細胞中HBV DNA抑制效果圖;
圖12為實施例二實施例10中Ky-22對4種不同基因型(A、B、C、D)HBV細胞株的抑制效果圖;
圖13為實施例三實施例1中的RNA抑制劑對HBV轉基因小鼠模型的HBsAg抑制效果圖;
圖14為實施例三實施例2中的Ky-22序列調整對HBV轉基因小鼠HBsAg的抑制效果圖;
圖15為實施例三實施例3中的Ky-2208在AAV-HBV小鼠模型上的劑效考察的結果圖;
圖16為實施例三實施例3中的Ky-2208在AAV-HBV小鼠模型上產生的HBsAb柱狀圖;
圖17為實施例三實施例4中的Ky-2208與TDF在HBV-Tg小鼠上比對及聯合用藥研究的結果圖。
Claims (12)
- 一種抑制B型肝炎病毒基因表達的RNA抑制劑或其藥學上可接受的鹽,其中, 所述RNA抑制劑由鏈長為15-30的正義鏈和反義鏈通過鹼基配對形成,其中鏈長優選為19-23。
- 根據請求項1所述的RNA抑制劑或其藥學上可接受的鹽,其中, 所述正義鏈和反義鏈之間至少有85%的鹼基互補; 所述正義鏈或反義鏈的部分或全部核苷酸糖基2’位的-OH可以被取代,其中,所述取代基團為氟或甲氧基; 且所述正義鏈或反義鏈的末端至少有3個相鄰核苷酸之間的磷酸酯鍵可以硫代。
- 根據請求項2所述的RNA抑制劑或其藥學上可接受的鹽,其中,所述正義鏈為SEQ ID NO. 1或與其相差一個、兩個或三個核苷酸的序列;所述反義鏈為SEQ ID NO. 2或與其相差一個、兩個或三個核苷酸的序列: 正義鏈:5' ggguuuuucucguugacaa 3' SEQ ID NO.: 1 反義鏈:5' uugucaacgagaaaaacccuu 3' SEQ ID NO.: 2 其中,g=鳥苷酸,a=腺苷酸,u=尿苷酸,c=胞苷酸。
- 根據請求項3所述的RNA抑制劑或其藥學上可接受的鹽,其中,所述正義鏈為SEQ ID NO. 2’或與其相差一個、兩個或三個核苷酸的序列;所述反義鏈為SEQ ID NO. 59或與其相差一個、兩個或三個核苷酸的序列: 正義鏈:5' Gs fGs G U fU U fU fU fC U C G U U G A Cs As A 3' SEQ ID NO.:2’ 反義鏈:5' Us Us G U C A fA C G A G fA A fA fA A C C Cs Us U 3' SEQ ID NO.:59 其中,G=2'-O-甲基鳥苷酸,A=2'-O-甲基腺苷酸,U=2'-O-甲基尿苷酸,C=2'-O-甲基胞苷酸;Gs=2'-O-甲基-3’-硫代鳥苷酸,As=2'-O-甲基-3'-硫代腺苷酸,Us=2'-O-甲基-3'-硫代尿苷酸,Cs=2'-O-甲基-3'-硫代胞苷酸;fG=2'-氟鳥苷酸,fA=2'-氟腺苷酸,fU=2'-氟尿苷酸,fC=2'-氟胞苷酸;fGs=2'-氟-3'-硫代鳥苷酸,fAs=2'-氟-3'-硫代腺苷酸,fUs=2'-氟-3'-硫代尿苷酸,fCs=2'-氟-3'-硫代胞苷酸。
- 根據請求項2或3所述的RNA抑制劑或其藥學上可接受的鹽,其中,所述正義鏈為SEQ ID NO. 3或與其相差一個、兩個或三個核苷酸的序列;所述反義鏈為SEQ ID NO. 4或與其相差一個、兩個或三個核苷酸的序列: 正義鏈:5' ggguuuuucuuguugacaa 3' SEQ ID NO.: 3 反義鏈:5' uugucaacaagaaaaacccuu 3' SEQ ID NO.: 4 其中,g=鳥苷酸,a=腺苷酸,u=尿苷酸,c=胞苷酸。
- 根據請求項5所述的RNA抑制劑或其藥學上可接受的鹽,其中,所述正義鏈為SEQ ID NO. 142或與其相差一個、兩個或三個核苷酸的序列;所述反義鏈為SEQ ID NO. 143或與其相差一個、兩個或三個核苷酸的序列: 正義鏈:5' Gs Gs G U fU U fU fU fC U U G U U G A Cs As A 3' SEQ ID NO.: 142 反義鏈:5' Us Us G U C A fA C A A G fA A fA A A C C Cs Us U 3' SEQ ID NO.: 143 其中,G=2'-O-甲基鳥苷酸,A=2'-O-甲基腺苷酸,U=2'-O-甲基尿苷酸,C=2'-O-甲基胞苷酸;Gs=2'-O-甲基-3’-硫代鳥苷酸,As=2'-O-甲基-3'-硫代腺苷酸,Us=2'-O-甲基-3'-硫代尿苷酸,Cs=2'-O-甲基-3'-硫代胞苷酸;fG=2'-氟鳥苷酸,fA=2'-氟腺苷酸,fU=2'-氟尿苷酸,fC=2'-氟胞苷酸;fGs=2'-氟-3'-硫代鳥苷酸,fAs=2'-氟-3'-硫代腺苷酸,fUs=2'-氟-3'-硫代尿苷酸,fCs=2'-氟-3'-硫代胞苷酸。
- 根據請求項1-6中任一項所述的RNA抑制劑或其藥學上可接受的鹽,其中,所述RNA抑制劑還含有5’MVIP和3’MVIP的組合,其中, 所述5’MVIP和3’MVIP為帶有肝靶向特異性配體X的配體結構,其還包含支鏈L、接頭B和連接鏈D; 所述5’MVIP偶聯在所述正義鏈和/或反義鏈5’末端,其還包含與所述正義鏈或反義鏈5’末端連接的轉接點R 1; 所述3’MVIP偶聯在所述反義鏈和/或正義鏈3’末端,其包含與所述正義鏈或反義鏈3’末端連接的轉接點R 2; 所述5’MVIP的結構如通式I所示,所述3’MVIP結構如通式II所示, I II 其中, n和m分別為0-4的整數,優選為1-3的整數,且n+m=2-6的整數,優選n+m=2、3或4,更優選為4; 所述轉接點R 1和R 2結構中帶有-NH-、硫原子或氧原子,一般結構中至少有一個-NH-、硫原子或氧原子,R 1和R 2通過結構中-NH-、硫原子或氧原子分別與5'MVIP和3'MVIP的連接鏈D以及正義鏈和/或反義鏈5’末端和3'末端相連,所述轉接點R 1和R 2可以是直鏈;帶有醯胺基、羧基或烷基類支鏈的直鏈或者各種環狀結構,環狀結構如飽和或不飽和的脂肪族碳環基,或者含有硫、氧或氮原子的五元或六元雜環基或芳香烴基; R 1優選為-NH(CH 2) xCH 2O-,其中x為3-12的整數,優選為4-6的整數; R 2優選為-NH(CH 2) x1CH(OH)(CH 2) x2CH 2O-,其中x1為1-4的整數,x2為0-4的整數; 所述肝靶向特異性配體X選自半乳糖、半乳糖胺、N-乙醯半乳糖胺及其衍生物,優先選自N-乙醯半乳糖胺及其衍生物,並且所述肝靶向特異性配體X在5’MVIP與3’MVIP各自的內部或5’MVIP與3’MVIP之間可以相同,也可以不同; 所述支鏈L是含有-NH-、C=O、O、S、醯胺基、磷醯基、硫代磷醯基、C4-C10脂肪族碳環基、苯基或者這些基團的組合的C4-C18直鏈,所述C4-C18直鏈可以帶有乙基醇或羧酸類的側鏈,所述支鏈L優選為含醯胺基或六元脂肪族碳環基的C7-C18直鏈,並且所述支鏈L在5’MVIP與3’MVIP各自的內部或5’MVIP與3’MVIP之間可以相同,也可以不同; 所述接頭B選自以下結構: 其中,A 1和A 2各自獨立地是C、O、S、-NH-、羰基、醯胺基、磷醯基或硫代磷醯基,r為0-4的整數,並且所述接頭B在5’MVIP與3’MVIP之間可以相同,也可以不同; 所述連接鏈D是含有-NH-、C=O、O、S、醯胺基、磷醯基、硫代磷醯基、芳香烴基、C4-C10脂肪族碳環基、含1-3個氮的五元或六元雜環基或者這些基團的組合的C3-C18直鏈,所述C3-C18直鏈可以帶有甲基醇、甲基叔丁基、甲基苯酚基、C5-C6脂肪環基的側鏈,所述連接鏈D優選為含兩個C=O、六元脂肪族碳環基或苯基的C3-C10直鏈,最優選含兩個C=O的C3-C10直鏈。
- 根據請求項8所述的RNA抑制劑或其藥學上可接受的鹽,其中,所述正義鏈5’MVIP和反義鏈3’MVIP的組合為5’MVIP01/3’MVIP01、5’MVIP01/3’MVIP17或5’MVIP09/3’MVIP09,或者所述正義鏈5’MVIP和正義鏈3’MVIP的組合為5’MVIP01/3'MVIP09或5’MVIP09/3'MVIP01。
- 如請求項1-9中任一項所述的RNA抑制劑或其藥學上可接受的鹽在製備用於治療肝源性疾病的藥物中的應用,其中,所述肝源性疾病包括但不限於肝炎、肝腫瘤、肝硬化、黃疸、二型糖尿病、脂肪肝、血液系統的凝血性疾病、血液白蛋白及球蛋白相關的疾病、高血脂、動脈粥樣硬化、原發性高血壓。
- 一種藥物組合物,該藥物組合物包括請求項1-9中任一項所述的RNA抑制劑或其藥學上可接受的鹽和藥學上可接受的輔料,其劑型為口服劑、靜脈注射劑或者皮下或肌內注射劑,優選為皮下注射劑。
- 一種藥物組合物,該藥物組合物包含請求項1-9中任一項所述的RNA抑制劑或其藥學上可接受的鹽和治療慢性B型肝炎的藥物核苷類似物或干擾素。
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