WO2017036398A1 - Small interfering rna, pharmaceutical composition, and application thereof - Google Patents

Abstract

Provided is a siRNA that inhibits RRM2 gene expression. A sense-strand base sequence of the siRNA is as shown in SEQ ID NO:2, an antisense-strand base sequence of the siRNA is as shown in SEQ ID NO:3, and a phosphate-sugar backbone of the siRNA may further have a modified group; also provided is a pharmaceutical composition comprising the siRNA, which can inhibit tumor cell proliferation and facilitate tumor cell apoptosis, thereby inhibiting growth of tumor tissues.

Description

一种小干扰核酸和药物组合物及其用途Small interfering nucleic acid and pharmaceutical composition and use thereof 技术领域Technical field

本发明涉及生物医学技术领域，具体地，涉及一种siRNA(小干扰核酸)、该siRNA的RNA干扰靶核酸、一种药物组合物以及它们的用途。The present invention relates to the field of biomedical technology, and in particular to an siRNA (small interfering nucleic acid), an RNA interference target nucleic acid of the siRNA, a pharmaceutical composition, and uses thereof.

背景技术Background technique

肿瘤目前主要治疗手段仍然是手术、放疗和化疗。虽然目前的治疗方法较以往提高患者的生存率，但仍存在着盲目性，针对性不强。新的化疗药物的不断问世，耐药性成了一个不可忽视的重要问题。以胰腺癌为例，目前，胰腺癌治疗的首选是手术切除，但仅有15-20％的患者确诊时可行手术治疗，胰腺癌患者5年生存率低于5％。胰腺癌对于放疗和化疗均不甚敏感，多数药物客观有效率低于10％。吉西他滨是目前标准胰腺癌化疗的一线治疗药物，但其对于改善患者生存期和生活质量的疗效有限。在进展期的胰腺癌患者，吉西他滨的客观有效率仅12％左右，生存期仅仅轻度延长。总体上说，目前胰腺癌患者仍缺乏有效的治疗手段。The main treatments for cancer are still surgery, radiotherapy and chemotherapy. Although the current treatment method improves the survival rate of patients compared with the past, it still has blindness and is not targeted. With the advent of new chemotherapy drugs, drug resistance has become an important issue that cannot be ignored. Taking pancreatic cancer as an example, at present, the first choice for the treatment of pancreatic cancer is surgical resection, but only 15-20% of patients are diagnosed with surgery, and the 5-year survival rate of patients with pancreatic cancer is less than 5%. Pancreatic cancer is not very sensitive to radiotherapy and chemotherapy, and most drugs are objectively less than 10% effective. Gemcitabine is currently the first-line treatment for standard pancreatic cancer chemotherapy, but its efficacy in improving patient survival and quality of life is limited. In patients with advanced pancreatic cancer, the objective rate of gemcitabine is only about 12%, and the survival period is only slightly prolonged. In general, patients with pancreatic cancer still lack effective treatment.

近年来研究表明，细胞信号传导中相关因子的表达异常，肿瘤细胞DNA的合成和修复及其他相关基因的表达异常与耐药的产生存在密切关系。其中，核糖核苷酸还原酶是生物体内唯一的催化四种核糖核苷酸还原、生成相应的脱氧核糖核苷酸的酶。该酶是DNA合成和修复的关键酶和限速酶，对细胞的增殖和分化起着调控作用。核糖核苷酸还原酶(Ribonucleotide Reductase，RR)由大亚基M1和小亚基M2组成，两者均为二聚体结构，通常称为RRM1和RRM2。RRM1具有与底物及变构效应物结合的部位，且含有直接供给电子的硫基，控制着底物的特异性及酶活性的开关是肿瘤抑制基因，也是吉西他滨 (Gemcitabine)的分子靶点。RRM2是一种铁硫蛋白，它通过络氨酸残基的苯环形成特异的自由基而参加催化反应，既是负责底物转化的催化区，又携带接触抑制区。RRM2是一个同源二聚体，形成两个相同的二价铁中心，稳定一个络氨酰自由基，这些对催化过程中触发电子运输非常重要。而且，目前已经发现RRM2在肿瘤中具有高表达；虽然有文献(CN200680018408.5)报道了含有RRM2的siRNA药物组合物可以用于治疗肝癌，但是，其siRNA体内活性不高；同时由于给药载体的自身缺陷，只能瘤内局部给药，2个siRNA组成的cocktail用药量还在2.5mg/kg，且给药频率高，为每日一次，连续给药3次。这种肝内动脉施用法，施用频率高，给药量大，难以满足临床需求。Recent studies have shown that the expression of related factors in cell signaling is abnormal, and the synthesis and repair of tumor cell DNA and the abnormal expression of other related genes are closely related to the emergence of drug resistance. Among them, ribonucleotide reductase is the only enzyme in vivo that catalyzes the reduction of four ribonucleotides to produce the corresponding deoxyribonucleotides. The enzyme is a key enzyme and rate-limiting enzyme for DNA synthesis and repair, and plays a regulatory role in cell proliferation and differentiation. Ribonucleotide Reductase (RR) consists of a large subunit M1 and a small subunit M2, both of which are dimeric structures, commonly referred to as RRM1 and RRM2. RRM1 has a site that binds to a substrate and an allosteric effector, and contains a sulfur group directly supplying electrons. The switch that controls the specificity and enzyme activity of the substrate is a tumor suppressor gene and is also gemcitabine. (Gemcitabine) molecular target. RRM2 is an iron-sulfur protein which participates in a catalytic reaction by forming a specific free radical through the benzene ring of a tyrosine residue, which is both a catalytic region responsible for substrate conversion and a contact inhibition region. RRM2 is a homodimer that forms two identical ferrous iron centers that stabilize a lysyl radical, which is important for triggering electron transport during the catalytic process. Moreover, RRM2 has been found to have high expression in tumors; although it is reported in the literature (CN200680018408.5) that siRNA pharmaceutical compositions containing RRM2 can be used to treat liver cancer, their siRNA activity is not high in vivo; The self-defect can only be administered locally in the tumor. The dose of the cocktail composed of 2 siRNAs is still 2.5 mg/kg, and the frequency of administration is high, once a day, and 3 times for continuous administration. This method of intrahepatic artery administration has a high frequency of administration and a large dose, which is difficult to meet clinical needs.

发明内容Summary of the invention

本发明的目的是提供一种针对RRM2基因的高效siRNA序列，并提供包含有该siRNA序列的药物组合物，该药物组合物可以有效抑制肿瘤细胞增殖、促进肿瘤细胞凋亡，从而抑制肿瘤组织生长。本发明与现有技术相比，提供了全新高效的siRNA及其药物组合物，通过***给药，降低了给药剂量，并延长了给药间隔，具备临床研究及商业化开发的现实性。The object of the present invention is to provide a high-efficiency siRNA sequence against the RRM2 gene, and to provide a pharmaceutical composition comprising the siRNA sequence, which can effectively inhibit tumor cell proliferation, promote tumor cell apoptosis, thereby inhibiting tumor tissue growth. . Compared with the prior art, the present invention provides a new and high-efficiency siRNA and a pharmaceutical composition thereof, which can reduce the dosage of administration and prolong the administration interval by systematic administration, and has the reality of clinical research and commercial development.

第一方面，本发明提供了一种siRNA，其中，该siRNA的正义链碱基序列如SEQ ID NO：2所示，且该siRNA的反义链碱基序列如SEQ ID NO：3所示；且所述siRNA的磷酸-糖骨架分别具有或不具有修饰基团。In a first aspect, the present invention provides an siRNA, wherein the sense strand base sequence of the siRNA is set forth in SEQ ID NO: 2, and the antisense strand base sequence of the siRNA is set forth in SEQ ID NO: 3; And the phosphate-sugar backbone of the siRNA has or does not have a modifying group, respectively.

第二方面，本发明还提供了一种RNA干扰靶核酸，其中，该RNA干扰靶核酸如SEQ ID NO：4所示。In a second aspect, the invention also provides an RNA interference target nucleic acid, wherein the RNA interferes with the target nucleic acid as set forth in SEQ ID NO:4.

第三方面，本发明提供了一种药物组合物，该药物组合物含有如上所述的siRNA和药学上可接受的载体，其中，所述药学上可接受的载体含有有机胺、胆固醇和聚乙二醇化磷脂；其中，所述有机胺为如式(1)所示的化合物和/或它 的药学上可接受的盐：In a third aspect, the present invention provides a pharmaceutical composition comprising the siRNA as described above and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier contains an organic amine, cholesterol and polyethylene a glycolated phospholipid; wherein the organic amine is a compound as shown in formula (1) and/or Pharmaceutically acceptable salt:

其中：R₁和R₂各自独立地为C₁₀-C₂₀的直链烷基；n为1-6的整数；R₃、R₄、R₅和R₆各自独立地为氢或Rx；且R₃、R₄、R₅和R₆中至少一个为Rx；Wherein: R ₁ and R ₂ are each independently a C ₁₀ -C ₂₀ linear alkyl group; n is an integer from 1 to 6; and R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or Rx; At least one of R ₃ , R ₄ , R ₅ and R ₆ is Rx;

Rx为

或

Ry为碳链数为C₁₂-C₂₀的直链烷基。Rx is

or

Ry is a linear alkyl group having a carbon number of C ₁₂ - C ₂₀ .

第四方面，本发明提供了如上所述的siRNA和/或如上所述的药物组合物在制备治疗癌症的药物中的用途。In a fourth aspect, the invention provides the use of a siRNA as described above and/or a pharmaceutical composition as described above for the manufacture of a medicament for the treatment of cancer.

第五方面，本发明提供了一种治疗癌症的方法，该方法包括将如上所述的siRNA和/或如上所述的药物组合物给予有需要的患者。In a fifth aspect, the present invention provides a method of treating cancer comprising administering an siRNA as described above and/or a pharmaceutical composition as described above to a patient in need thereof.

第六方面，本发明提供了一种抑制细胞中RRM2基因表达的方法，该方法包括将如上所述的siRNA和/或如上所述的药物组合物导入所述细胞。In a sixth aspect, the invention provides a method of inhibiting expression of a RRM2 gene in a cell, the method comprising introducing an siRNA as described above and/or a pharmaceutical composition as described above into the cell.

通过上述技术方案，本发明提供了一种对癌症的新的有效治疗手段，该手段可以通过***给药，降低给药量，拉长给药间隔。具体地，本发明的siRNA通过静脉给药，在给药量为1mg/kg，给药间隔为每周2次的情形下，在体内能取得高达82％的肿瘤抑制率，显著优于文献中瘤旁给药、3天内连续给药3次，每天每次给药2.5mg/kg的治疗方案；同时，采取文献中的siRNA，经过与本发明相似的修饰手段得到的修饰后siRNA，在相同的给药方式下，68％的肿瘤抑制率也远不及本发明的siRNA取得的85％的抑制效率。Through the above technical solutions, the present invention provides a new and effective treatment method for cancer, which can be administered by system, reducing the dose and lengthening the administration interval. Specifically, the siRNA of the present invention can be administered intravenously at a dose of 1 mg/kg and the administration interval is twice a week, and a tumor inhibition rate of up to 82% can be obtained in vivo, which is significantly superior to the literature. Paraneoplastic administration, continuous administration 3 times in 3 days, 2.5 mg/kg of treatment per administration per day; meanwhile, the siRNA in the literature was modified by the modification method similar to the present invention. Under the mode of administration, 68% of the tumor inhibition rate was far less than the 85% inhibition efficiency achieved by the siRNA of the present invention.

此外，由有机胺、辅助脂质、聚乙二醇化磷脂形成的药学上可接受的载体与本发明的siRNA形成的特定药物组合物，与其它常规载体与本发明的siRNA形成的药物组合物相比，具有极高的生物活性，其mRNA抑制效率是其它常规载体形成的药物组合物的2-20倍。 Further, a specific pharmaceutical composition formed from a pharmaceutically acceptable carrier formed of an organic amine, a helper lipid, or a pegylated phospholipid, and a siRNA of the present invention, and a pharmaceutical composition formed by the other conventional carrier and the siRNA of the present invention It has a very high biological activity and its mRNA inhibition efficiency is 2-20 times that of other conventional carrier-formed pharmaceutical compositions.

本发明的其他特征和优点将在随后的具体实施方式部分予以详细说明。Other features and advantages of the invention will be described in detail in the detailed description which follows.

附图说明DRAWINGS

附图是用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。在附图中：The drawings are intended to provide a further understanding of the invention, and are intended to be a In the drawing:

图1是显示siRRM2-M和无关siRNA(siNC)对PANC-1细胞集落成集的抑制作用的结果图。Figure 1 is a graph showing the results of inhibition of PANC-1 cell colony assembly by siRRM2-M and irrelevant siRNA (siNC).

图2是显示阿霉素与RBP131载体递送的siRRM2-M联用对胰腺癌肿瘤组织生长的抑制作用的肿瘤体积结果图。Figure 2 is a graph showing tumor volume results of inhibition of pancreatic cancer tumor tissue growth by the combination of doxorubicin and RBP131 vector-delivered siRRM2-M.

图3是显示阿霉素与RBP131载体递送的siRRM2-M联用对胰腺癌肿瘤组织生长的抑制作用的肿瘤重量结果图。Figure 3 is a graph showing tumor weight results of inhibition of pancreatic cancer tumor tissue growth by the combination of doxorubicin and RBP131 vector-delivered siRRM2-M.

图4是显示阿霉素与RBP131载体递送的siRRM2-M联用对胰腺癌肿瘤组织生长的抑制作用的肿瘤块照片结果图。Figure 4 is a photograph showing the results of tumor block photographs showing inhibition of pancreatic cancer tumor tissue growth by the combination of doxorubicin and RBP131 vector-delivered siRRM2-M.

图5是显示RBP131载体递送的siRRM2-M对肝癌肿瘤组织生长的抑制作用的肿瘤重量结果图。Figure 5 is a graph showing tumor weight results showing inhibition of hepatocarcinoma tumor tissue growth by siRRM2-M delivered by RBP131 vector.

具体实施方式detailed description

以下结合附图对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative and not restrictive.

本发明中，RRM2是指cDNA序列如Genebank注册号:NM_001034.3所示的基因，其RNA序列如SEQ ID NO:1所示。In the present invention, RRM2 refers to a cDNA sequence such as the gene shown in Genebank Registry No.: NM_001034.3, and the RNA sequence thereof is shown in SEQ ID NO: 1.

第一方面，本发明提供了一种siRNA，其中，该siRNA的正义链碱基序列如SEQ ID NO：2所示，且该siRNA的反义链碱基序列如SEQ ID NO：3所示； 且所述siRNA的磷酸-糖骨架分别具有或不具有修饰基团。In a first aspect, the present invention provides an siRNA, wherein the sense strand base sequence of the siRNA is set forth in SEQ ID NO: 2, and the antisense strand base sequence of the siRNA is set forth in SEQ ID NO: 3; And the phosphate-sugar backbone of the siRNA has or does not have a modifying group, respectively.

其中，本发明的siRNA含有磷酸-糖骨架和碱基。本发明的siRNA含有修饰基团，所述修饰基团不会导致所述siRNA抑制RRM2基因表达的功能明显削弱或丧失。目前，本领域存在多种可用于修饰siRNA的方式，具体可如文献(Watts,J.K.,G.F.Deleavey,and M.J.Damha,Chemically modified siRNA:tools and applications.Drug Discov Today,2008.13(19-20):p.842-55)所记载。在本发明所述siRNA的一些实施方式中，所述修饰基团为任选取代的糖基及任选的取代的酯基，但不限于此。Among them, the siRNA of the present invention contains a phosphate-sugar skeleton and a base. The siRNA of the present invention contains a modifying group which does not cause a significant weakening or loss of the function of the siRNA to inhibit expression of the RRM2 gene. Currently, there are a variety of ways in the art that can be used to modify siRNA, as in the literature (Watts, JK, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008. 13 (19-20): p .842-55). In some embodiments of the siRNA of the invention, the modifying group is an optionally substituted sugar group and an optionally substituted ester group, but is not limited thereto.

其中，优选地，该siRNA的磷酸-糖骨架分别具有如下修饰基团：正义链第1、6、14、16和18位的糖基为2’-甲氧基核糖基；反义链第2位的糖基为2’-甲氧基核糖基，反义链第3位的糖基为2’-氟代核糖基。Wherein, preferably, the phosphate-sugar backbone of the siRNA has the following modifying groups: the glycosyl group at the 1, 6, 14, 16 and 18 positions of the sense strand is a 2'-methoxyribosyl group; the antisense strand is 2 The glycosyl group at the position is a 2'-methoxyribosyl group, and the glycosyl group at the third position of the antisense strand is a 2'-fluororibosyl group.

其中，2’-甲氧基核糖基指核糖基的2’-OH被甲氧基取代后形成的基团；2’-氟代核糖基指核糖基的2’-OH被氟取代后形成的基团。在核糖基团的2’-羟基位置引入例如甲氧基或氟后，可使血清中的核糖核酸酶不易切割核酸，由此增加核酸的稳定性，使核酸具有更强的抵抗核酸酶水解的性能。Wherein 2'-methoxyribosyl refers to a group formed by substitution of a 2'-OH of a ribose group with a methoxy group; 2'-fluororibose group refers to a group formed by substitution of a 2'-OH of a ribose group with fluorine Group. The introduction of, for example, a methoxy group or a fluorine at the 2'-hydroxyl position of the ribose group makes it difficult for the ribonuclease in the serum to cleave the nucleic acid, thereby increasing the stability of the nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis. performance.

其中，优选地，该siRNA的正义链和/或反义链的第20位和第21位之间的酯基为硫代磷酸酯基。Among them, preferably, the ester group between the 20th and 21st positions of the sense strand and/or the antisense strand of the siRNA is a phosphorothioate group.

其中，硫代磷酸酯基是指磷酸二酯基中的一个氧原子被硫原子取代后形成的基团，具体如式(5)所示：Wherein, the phosphorothioate group refers to a group formed by replacing one oxygen atom in the phosphodiester group with a sulfur atom, as shown in the formula (5):

第二方面，本发明还提供了一种RNA干扰靶核酸，其中，该RNA干扰靶核酸如SEQ ID NO：4所示。其中，所述RNA干扰靶核酸是指RRM2 mRNA 中，与如SEQ ID NO：3所示的反义链中第1至19位的核酸相互杂交的片段。In a second aspect, the invention also provides an RNA interference target nucleic acid, wherein the RNA interferes with the target nucleic acid as set forth in SEQ ID NO:4. Wherein the RNA interference target nucleic acid refers to RRM2 mRNA In the fragment which hybridizes to the nucleic acids of positions 1 to 19 in the antisense strand as shown in SEQ ID NO: 3.

第三方面，本发明提供了一种药物组合物，该药物组合物含有如上所述的siRNA和药学上可接受的载体。In a third aspect, the invention provides a pharmaceutical composition comprising an siRNA as described above and a pharmaceutically acceptable carrier.

所述药学上可接受的载体可以是siRNA给药领域常规使用的载体，例如但不限于磁性纳米粒(magnetic nanoparticles，如Fe₃O₄、Fe₂O₃)、碳纳米管(carbon nanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphate nanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1,2-二油酰基-3-三甲铵丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N,N-二甲氨基乙酯)(poly(2-dimethylaminoethyl methacrylate)，PDMAEMA)以及它们的衍生物等。在本发明的药物组合物中，对siRNA和药学上可接受的载体的含量没有特别要求，一般地，siRNA与药学上可接受的载体的重量比可以为1:(1-500)，优选为1:(1-50)。The pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (such as Fe ₃ O ₄ , Fe ₂ O ₃ ), carbon nanotubes, Mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, polylysine (poly (L-lysine), PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly D or L Type lactic acid/glycolic acid copolymer (poly(D&L-lactic/glycolic acid) copolymer, PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA) and poly(methacrylic acid) -N,N-dimethylaminoethyl ester (poly(2-dimethylaminoethyl methacrylate), PDMAEMA), and derivatives thereof. In the pharmaceutical composition of the present invention, the content of the siRNA and the pharmaceutically acceptable carrier is not particularly required, and generally, the weight ratio of the siRNA to the pharmaceutically acceptable carrier may be 1: (1 to 500), preferably 1: (1-50).

在本发明的药物组合物中，还可以包含药学上可接受的其它辅料，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种。例如，所述药学上可接受的其它辅料可以包括pH值缓冲液、保护剂和渗透压调节剂中的至少一种。所述pH值缓冲液可以为pH值7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH值5.5-8.5的磷酸盐缓冲液，优选为pH值5.5-8.5的磷酸盐缓冲液。所述保护剂可以为肌醇、山梨醇、蔗糖、海藻糖、甘露糖、麦芽糖、乳糖和葡糖糖中的至少一种。以所述药物组合物的总重量为基准，所述保护剂的含量可以为0.01-30重量％。所述渗透压调节剂可以为氯化钠和/或 氯化钾。所述渗透压调节剂的含量使所述药物组合物的渗透压为200-700毫渗摩尔/千克。根据所需渗透压，本领域技术人员可以容易地确定所述渗透压调节剂的含量。The pharmaceutical composition of the present invention may further comprise other excipients which are pharmaceutically acceptable, and the excipients may be one or more of various preparations or compounds conventionally employed in the art. For example, the pharmaceutically acceptable other excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure adjusting agent. The pH buffer may be a trishydroxymethylaminomethane hydrochloride buffer having a pH of 7.5-8.5 and/or a phosphate buffer having a pH of 5.5-8.5, preferably a phosphate having a pH of 5.5-8.5. Buffer. The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The protective agent may be included in an amount of from 0.01 to 30% by weight based on the total weight of the pharmaceutical composition. The osmotic pressure adjusting agent may be sodium chloride and/or Potassium chloride. The osmotic pressure adjusting agent is present in an amount such that the osmotic pressure of the pharmaceutical composition is from 200 to 700 milliosmoles per kilogram. The content of the osmotic pressure adjusting agent can be easily determined by those skilled in the art depending on the desired osmotic pressure.

根据本发明的一些实施方式，所述药物组合物可以为液体制剂，例如注射液；也可以为冻干粉针剂，实施给药时与液体辅料混合，配制成液体制剂。所述液体制剂可以但不限于用于皮下、肌肉或静脉注射给药，也可以但不限于通过喷雾给药到肺脏、或通过喷雾经肺脏给药到其它脏器组织(如肝脏)。优选地，所述药物组合物用于静脉注射给药。According to some embodiments of the present invention, the pharmaceutical composition may be a liquid preparation, such as an injection solution, or may be a lyophilized powder injection, which is mixed with a liquid adjuvant when administered, and formulated into a liquid preparation. The liquid formulation can be, but is not limited to, for subcutaneous, intramuscular or intravenous administration, and can be, but is not limited to, administered to the lungs by spraying, or administered to other organ tissues (such as the liver) via the lungs by spraying. Preferably, the pharmaceutical composition is for intravenous administration.

在本发明的药物组合物的一个优选实施方式中，所述药物组合物可以为脂质体制剂的形式。在一个更优选的实施方式中，所述脂质体制剂中使用的药学上可接受的载体包含含胺的转染化合物(下文也可将其称为有机胺)、辅助脂质和/或聚乙二醇化磷脂。其中，所述有机胺、辅助脂质和聚乙二醇化磷脂可分别选自于CN201180060664.1(通过引用将其整体并入本文)中所描述的含胺的转染化合物或其药学上可接受的盐或衍生物、辅助脂质和聚乙二醇化磷脂中的一种或多种。In a preferred embodiment of the pharmaceutical composition of the invention, the pharmaceutical composition may be in the form of a liposomal formulation. In a more preferred embodiment, the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid and/or a poly Ethylene glycolated phospholipids. Wherein the organic amine, helper lipid, and pegylated phospholipid are each selected from the group consisting of amine-containing transfection compounds described in CN201180060664.1 (hermby incorporated by reference herein in its entirety) One or more of a salt or a derivative, a helper lipid, and a pegylated phospholipid.

根据本发明的药物组合物一种优选的实施方式，其中，所述药学上可接受的载体含有有机胺、胆固醇和聚乙二醇化磷脂；其中，所述有机胺为如式(1)所示的化合物和/或它的药学上可接受的盐：A preferred embodiment of the pharmaceutical composition according to the present invention, wherein the pharmaceutically acceptable carrier contains an organic amine, cholesterol and a pegylated phospholipid; wherein the organic amine is as shown in formula (1) a compound and/or a pharmaceutically acceptable salt thereof:

其中：R₁和R₂各自独立地为C₁₀-C₂₀的直链烷基；n为1-6的整数；R₃、R₄、R₅和R₆各自独立地为氢或Rx；且R₃、R₄、R₅和R₆中至少一个为Rx；Wherein: R ₁ and R ₂ are each independently a C ₁₀ -C ₂₀ linear alkyl group; n is an integer from 1 to 6; and R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or Rx; At least one of R ₃ , R ₄ , R ₅ and R ₆ is Rx;

Rx为

或

Ry为碳链数为C₁₂-C₂₀的直链烷基。 Rx is

or

Ry is a linear alkyl group having a carbon number of C ₁₂ - C ₂₀ .

特别优选地，所述有机胺为如式(2)所示的有机胺和/或如式(3)所示的有机胺：Particularly preferably, the organic amine is an organic amine as shown in formula (2) and/or an organic amine as shown in formula (3):

所述聚乙二醇化磷脂为1,2-二棕榈酰基-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。The pegylated phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

其中，所述药物组合物中，所述有机胺、胆固醇和所述聚乙二醇化磷脂三者之间的摩尔比可以为(19.7-80)：(19.7-80)：(0.3-50)。Wherein, in the pharmaceutical composition, the molar ratio between the organic amine, cholesterol and the pegylated phospholipid may be (19.7-80): (19.7-80): (0.3-50).

其中，作为本发明特别优选的一种实施方式，所述药物组合物中，所述有机胺、胆固醇和所述聚乙二醇化磷脂三者之间的摩尔比为(50-70)：(20-40)：(3-20)。 Wherein, as a particularly preferred embodiment of the present invention, the molar ratio between the organic amine, cholesterol and the PEGylated phospholipid is (50-70) in the pharmaceutical composition: (20) -40): (3-20).

由本发明的siRNA与上述载体形成的脂质体颗粒具有约30nm至约200nm的平均直径，通常为约40nm至约135nm，更通常地，该脂质体颗粒的平均直径是约50nm至约120nm、约50nm至约100nm、约60nm至约90nm或约70nm至约90nm，例如，该脂质体颗粒的平均直径是约30、40、50、60、70、75、80、85、90、100、110、120、130、140、150或160nm。The liposome particles formed from the siRNA of the present invention and the above carrier have an average diameter of from about 30 nm to about 200 nm, typically from about 40 nm to about 135 nm, and more typically, the liposome particles have an average diameter of from about 50 nm to about 120 nm, From about 50 nm to about 100 nm, from about 60 nm to about 90 nm, or from about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.

在脂质体制剂形式的药物组合物中，本发明的siRNA与全部脂质(例如有机胺、辅助脂质和/或聚乙二醇化脂质)的重量比(重量/重量比)在从约1:1至约1:50、从约1:1至约1:30、从约1:3至约1:20、从约1:4至约1:18、从约1:5至约1:17、从约1:5至约1:15、从约1:5至约1:12、从约1:6至约1:12或从约1:6至约1:10的范围内，例如，本发明的siRNA与全部脂质的重量比为约1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12、1:13、1:14、1:15、1:16、1:17或1:18。In a pharmaceutical composition in the form of a liposome formulation, the weight ratio (weight/weight ratio) of the siRNA of the invention to all lipids (eg, organic amines, helper lipids, and/or PEGylated lipids) is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1 :17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12 or from about 1:6 to about 1:10, For example, the weight ratio of the siRNA of the present invention to the total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13 , 1:14, 1:15, 1:16, 1:17 or 1:18.

根据本发明特别优选的一种实施方式，其中，所述药物组合物还包括至少一种额外的抗癌化疗剂，该化疗剂与核酸以附加或协同的方式抑制癌细胞。核酸与化疗剂可预先制备为组合制剂，或可以独立地制备并选择适当的时间与剂量施用，以达到组合的效果。优选的，所述抗癌化疗剂为阿霉素。在该优选实施方式中，本发明的siRNA和阿霉素能够发挥优异的增效效果。其中，阿霉素可以独立存放。其中，siRNA可以与阿霉素混合后同时给药，siRNA也可以与阿霉素分别先后给药。A particularly preferred embodiment of the invention, wherein the pharmaceutical composition further comprises at least one additional anti-cancer chemotherapeutic agent that inhibits cancer cells in an additive or synergistic manner with the nucleic acid. The nucleic acid and the chemotherapeutic agent can be prepared in advance as a combined preparation, or can be prepared independently and selected for appropriate time and dosage to achieve the combined effect. Preferably, the anti-cancer chemotherapeutic agent is doxorubicin. In this preferred embodiment, the siRNA of the present invention and doxorubicin are capable of exerting an excellent synergistic effect. Among them, doxorubicin can be stored separately. Among them, siRNA can be administered simultaneously with doxorubicin, and siRNA can also be administered sequentially with doxorubicin.

第四方面，本发明提供了如上所述的siRNA和/或如上所述的药物组合物在制备治疗癌症的药物中的用途。In a fourth aspect, the invention provides the use of a siRNA as described above and/or a pharmaceutical composition as described above for the manufacture of a medicament for the treatment of cancer.

其中，所述癌症包括但不限于白血病、淋巴瘤、多发性骨髓瘤、脑瘤、乳腺癌、肾上腺瘤、甲状腺癌、胰腺癌、垂体癌、***、卵巢癌、食管癌、胃癌、结肠癌、直肠癌、肝癌例、胆囊癌、肺癌、睾丸癌、***癌、头颈癌(包 括口腔癌等)、皮肤癌、肾癌。优选地，如上所述的siRNA和/或如上所述的药物组合物特别适合用于肝癌和/或胰腺癌的治疗。The cancer includes, but is not limited to, leukemia, lymphoma, multiple myeloma, brain tumor, breast cancer, adrenal adenoma, thyroid cancer, pancreatic cancer, pituitary cancer, cervical cancer, ovarian cancer, esophageal cancer, gastric cancer, colon cancer. , rectal cancer, liver cancer, gallbladder cancer, lung cancer, testicular cancer, prostate cancer, head and neck cancer Including oral cancer, etc.), skin cancer, kidney cancer. Preferably, the siRNA as described above and/or the pharmaceutical composition as described above is particularly suitable for the treatment of liver cancer and/or pancreatic cancer.

第五方面，本发明提供了一种治疗癌症的方法，该方法包括将如上所述的siRNA和/或如上所述的药物组合物给予有需要的患者。In a fifth aspect, the present invention provides a method of treating cancer comprising administering an siRNA as described above and/or a pharmaceutical composition as described above to a patient in need thereof.

适于本发明方法的给药途径包括局部给药和全身给药。可通过本领域已知的任何合适途径向受试者给药，所述途径包括但不仅限于：口服或胃肠外途径，包括静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、肺部给药、鼻部给药、直肠给药和局部给药(包括口腔含化给药和舌下给药)。Routes of administration suitable for the methods of the invention include topical administration and systemic administration. Administration can be administered to a subject by any suitable route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal. Drug, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including buccal administration and sublingual administration).

适于本发明方法的给药剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是受试者的年龄、体重和性别来确定。可基于由细胞培养分析和动物研究得到的数据得出人用剂量的范围。Dosages suitable for the methods of the invention may be those conventional in the art, which may be determined based on various parameters, particularly the age, weight and sex of the subject. The range of human doses can be derived based on data obtained from cell culture assays and animal studies.

第六方面，本发明提供了一种抑制细胞中RRM2基因表达的方法，该方法包括将如上所述的siRNA和/或如上所述的药物组合物导入所述细胞。In a sixth aspect, the invention provides a method of inhibiting expression of a RRM2 gene in a cell, the method comprising introducing an siRNA as described above and/or a pharmaceutical composition as described above into the cell.

其中，所述细胞包括但不限于人肝癌细胞系HepG2、人***细胞系Hela、人胰腺癌细胞系PANC-1、人乳腺癌细胞系MDA-MB-231。Wherein, the cells include, but are not limited to, human hepatoma cell line HepG2, human cervical cancer cell line Hela, human pancreatic cancer cell line PANC-1, and human breast cancer cell line MDA-MB-231.

以下，结合实施例进一步说明本发明。除非特别说明，本发明所用到的试剂、培养基等实验材料均为市售商品。Hereinafter, the present invention will be further described with reference to the examples. Unless otherwise specified, the experimental materials such as reagents and culture media used in the present invention are commercially available products.

制备例1Preparation Example 1

siRNA的序列如表1所示，该siRNA的一条单链具有由SEQ ID NO:2表示的序列，与RRM2的mRNA序列中相应的RNA干扰靶核酸(如SEQ ID NO:4所示)相同，另一条单链具有由SEQ ID NO:3表示的序列，与RRM2的mRNA序列中相应的RNA干扰靶核酸互补。 The sequence of the siRNA is shown in Table 1. One single strand of the siRNA has the sequence represented by SEQ ID NO: 2, which is identical to the corresponding RNA interference target nucleic acid (shown as SEQ ID NO: 4) in the mRNA sequence of RRM2. The other single strand has the sequence represented by SEQ ID NO: 3, which is complementary to the corresponding RNA interference target nucleic acid in the mRNA sequence of RRM2.

siRNA的寡聚核苷酸单链按照本领域公知的方法进行化学合成。合成时，在寡聚核苷酸单链的3′末端加上两个脱氧胸腺嘧啶核苷酸dTdT。互补的寡聚核苷酸单链退火形成双链，双链的两端分别具有dTdT的3′突出端。合成的寡聚核苷酸的序列见表1。Oligonucleotide single strands of siRNA are chemically synthesized according to methods well known in the art. For the synthesis, two deoxythymidine dTdTs are added to the 3' end of the single strand of the oligonucleotide. The complementary oligonucleotides are single-stranded to form a double strand, and the two ends of the double strand have a 3' overhang of dTdT, respectively. The sequences of the synthesized oligonucleotides are shown in Table 1.

表1Table 1

实施例1Example 1

本实施例用来检测siRNA在体外对RRM2mRNA表达水平的抑制效率。This example was used to examine the inhibitory efficiency of siRNA against RRM2 mRNA expression levels in vitro.

将人类肝癌细胞株HepG2用含10％胎牛血清、2mM L-谷胺酰胺、100U/mL青霉素、100g/mL链霉素的DMEM完全培养基接种于24孔板，细胞密度为4×10⁵/孔，每孔0.5mL，37℃培养过夜。The human hepatoma cell line HepG2 was inoculated into a 24-well plate with DMEM complete medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, and the cell density was 4×10 ^{5 .} /well, 0.5 mL per well, incubated overnight at 37 °C.

转染的具体操作步骤如下：将100ng制备例1中的siRNA稀释于50μL DEME无血清培养基中，同时将1μL Lipofectamine^TM 2000(Invitrogen公司)稀释于50μL DEME无血清培养基中，将上述两种溶液在室温下孵育5分钟后混合。混合溶液于室温静置20分钟后，把100μL的上述混合溶液加到接种有PANC-1细胞的24孔板中。siRNA的最终浓度约为10nM。细胞于37℃培养4小时，再加入1mL含10％胎牛血清、2mM的L-谷胺酰胺、100U/mL青霉素、 100g/mL链霉素的DMEM完全培养基，然后在37℃再培养24小时。Transfected specific steps are as follows: 100ng diluted in Preparation Example 1 siRNA in serum-free medium 50μL DEME while 1μL Lipofectamine ^TM 2000 (Invitrogen) was diluted in serum-free medium 50μL DEME, the above two The solution was incubated after 5 minutes at room temperature. After the mixed solution was allowed to stand at room temperature for 20 minutes, 100 μL of the above mixed solution was added to a 24-well plate inoculated with PANC-1 cells. The final concentration of siRNA was approximately 10 nM. The cells were cultured at 37 ° C for 4 hours, and then 1 mL of DMEM complete medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 g/mL streptomycin was added, and then cultured at 37 ° C. hour.

通过荧光定量实时PCR(Quantitative Real-Time PCR)分别检测转染了siNC、siPC1、siPC2、siRRM2的HepG2细胞中RRM2 mRNA的表达量。具体步骤如下：培养转染的细胞24小时后，用RNeasy mini Kit(Qiagen公司，货号74106)提取细胞中的总RNA。用紫外分光光度计测定提取的RNA样品的OD280和OD260的吸光度，并利用公式：RNA浓度(μg/μL)＝0.04×OD260×稀释倍数，计算RNA样品的浓度。然后用PrimeScript^TM II 1st Strand cDNA Synthesis Kit(Takara公司，货号6210A)合成cDNA，每个样品使用2μg总RNA。在合成cDNA后，按照

 Premix Ex Taq^TM II(Takara公司，货号DRR081A)试剂盒的说明书，进行荧光定量实时PCR反应。其中，用于扩增RRM2和作为定量PCR反应的内参的β-actin的PCR引物如表2所示。The expression of RRM2 mRNA in HepG2 cells transfected with siNC, siPC1, siPC2, and siRRM2 was detected by Quantitative Real-Time PCR. The specific procedure was as follows: After culturing the transfected cells for 24 hours, total RNA in the cells was extracted with RNeasy mini Kit (Qiagen, Cat. No. 74106). The absorbance of OD280 and OD260 of the extracted RNA sample was measured by an ultraviolet spectrophotometer, and the concentration of the RNA sample was calculated using the formula: RNA concentration (μg/μL) = 0.04 × OD260 × dilution factor. Then ^{PrimeScript TM II 1st Strand cDNA Synthesis Kit} (Takara Company, Catalog No. 6210A) synthesis of the cDNA, each sample using 2μg of total RNA. After synthesizing cDNA, follow

The instructions of the Premix Ex Taq ^TM II (Takara, Cat. DRR081A) kit were used for fluorescence quantitative real-time PCR reactions. Among them, PCR primers for amplifying RRM2 and β-actin as an internal reference for quantitative PCR reaction are shown in Table 2.

表2Table 2

siRNA对RRM2 mRNA表达水平的抑制率按如下等式计算：抑制率＝[1-(实验孔RRM2 mRNA的表达量/实验孔β-Actin mRNA的表达量)/(阴性对照孔RRM2 mRNA的表达量/阴性对照孔β-Actin mRNA的表达量)]×100％。结果如表3所示。The inhibition rate of siRNA on RRM2 mRNA expression level was calculated as follows: inhibition rate = [1-(experimental RRM2 mRNA expression level / experimental hole β-Actin mRNA expression level) / (negative control well RRM2 mRNA expression level) / Negative control well β-Actin mRNA expression level)] × 100%. The results are shown in Table 3.

表3table 3

siRNAsiRNA	mRNA抑制率(％)mRNA inhibition rate (%)
siNC siNC	00
siPC1siPC1	65.765.7
siPC2siPC2	82.482.4
siRRM2siRRM2	91.391.3

从表1可以看出，本发明siRNA的活性显著高于文献(CN200680018408.5) 已公开的序列，可以更加高效地抑制靶基因RRM2的表达。As can be seen from Table 1, the activity of the siRNA of the present invention is significantly higher than that of the literature (CN200680018408.5) The disclosed sequence can inhibit the expression of the target gene RRM2 more efficiently.

制备例2Preparation Example 2

合成表4中所列的寡聚核苷酸。表4中的寡聚核苷酸包含修饰的核苷酸残基，互补的寡聚核苷酸链退火形成修饰的siRNA，分别记为siRRM2-M、siPC1-M和siPC2-M。其中，(OMe)代表它左边的核苷酸残基中戊糖基团为2’-甲氧基核糖基，(F)代表它左边的核苷酸残基中戊糖基团为2’-氟代核糖基；(S)代表它两边的脱氧核糖核苷酸dTdT之间的酯基为硫代磷酸酯基。这些siRNA未加修饰前的核苷酸序列分别对应于制备例1中的siRRM2、siPC1和siPC2。The oligonucleotides listed in Table 4 were synthesized. The oligonucleotides in Table 4 contain modified nucleotide residues, and the complementary oligonucleotide strands anneal to form modified siRNAs, designated siRRM2-M, siPC1-M, and siPC2-M, respectively. Wherein (OMe) represents a pentose group in the nucleotide residue to the left of which is a 2'-methoxyribosyl group, and (F) represents a pentose group in the nucleotide residue to the left of which is 2'- The fluororibose group; (S) represents an ester group between the deoxyribonucleotides dTdT on both sides thereof is a phosphorothioate group. The nucleotide sequences before the modification of these siRNAs corresponded to siRRM2, siPC1 and siPC2 in Preparation Example 1, respectively.

表4Table 4

实施例2Example 2

本实施例用来检测化学修饰对siRNA血清稳定性的影响。This example was used to examine the effect of chemical modification on siRNA serum stability.

对制备例2中得到的siRRM2-M、siPC1-M和siPC2-M，以及制备例1中得到的siRRM2、siPC1和siPC2测定其在血清环境中的稳定性。具体步骤如下。The stability of the siRRM2-M, siPC1-M and siPC2-M obtained in Preparation Example 2, and siRRM2, siPC1 and siPC2 obtained in Preparation Example 1 were measured in a serum environment. Specific steps are as follows.

将10μL上述修饰和未修饰的siRNA(20μM)分别与90μL 50％人血浆(Human plasma，HP，PBS稀释)混合后，在37℃下体外孵育0、2、4、8、24、48和72小时后得到处理样品。处理样品取样10μL，随即进行液氮速冻，冻存于-80℃备用。配制20重量％的非变性聚丙烯酰胺凝胶，将10μL 5倍稀释的上述样品(稀释液1×PBS，pH 7.4)与4μl上样缓冲液(20mM EDTA，36重量％甘油，0.06重量％溴酚蓝)混合，然后上样，在80mA恒流条件下电泳60分钟 左右。电泳结束后，用1×Sybr Gold染料(Invitrogen公司，货号11494)染色15分钟后成相，通过定量读取电泳主条带(最长片段)的灰度值，计算siRNA降解率。具体的，将0小时的样品作为对照，以处理样品的电泳主条带的灰度值与对照样品的电泳主条带的灰度值的比值计算降解率：72小时降解率＝[1-(72小时的电泳主条带灰度/0小时的电泳主条带灰度)]×100％。结果如表5所示。10 μL of the above modified and unmodified siRNA (20 μM) were mixed with 90 μL of 50% human plasma (Human plasma, HP, PBS diluted), respectively, and incubated at 37 ° C for 0, 2, 4, 8, 24, 48 and 72 in vitro. The treated sample was obtained after an hour. The sample was sampled and sampled at 10 μL, and immediately frozen in liquid nitrogen, and stored at -80 ° C for use. Prepare 20% by weight of non-denaturing polyacrylamide gel, 10 μL of 5-fold diluted sample (diluent 1×PBS, pH 7.4) and 4 μl loading buffer (20 mM EDTA, 36% by weight glycerol, 0.06 wt% bromine) Phenol blue) mixed, then loaded and electrophoresed for 60 minutes under constant flow conditions of 80 mA about. After completion of electrophoresis, the cells were stained with 1×Sybr Gold dye (Invitrogen, Cat. No. 11494) for 15 minutes, and the siRNA degradation rate was calculated by quantitatively reading the gray value of the electrophoresis main band (longest fragment). Specifically, the 0 hour sample was used as a control to calculate the degradation rate by the ratio of the gray value of the electrophoresis main strip of the treated sample to the gray value of the electrophoretic main strip of the control sample: 72 hours degradation rate = [1-( 72 hours of electrophoresis main strip gray scale / 0 hour electrophoresis main strip gray scale)] × 100%. The results are shown in Table 5.

表5table 5

由表5可以看出，在人血清环境中，修饰的siRNA的稳定性相比未修饰的siRNA明显增强。As can be seen from Table 5, the stability of the modified siRNA was significantly enhanced in the human serum environment compared to the unmodified siRNA.

实施例3Example 3

本实施例用来检测化学修饰前后siRNA在体外对RRM2 mRNA表达水平的抑制效率。This example was used to detect the inhibition efficiency of siRNA on the expression level of RRM2 mRNA in vitro before and after chemical modification.

按照与实施例1相同的方法，对制备例2中得到的siRRM2-M、siPC1-M和siPC2-M，以及制备例1中得到的siRRM2、siPC1和siPC2分别测定其对RRM2mRNA表达水平的抑制效率。转染和荧光定量实时PCR的具体步骤如同实施例1中所述。转染时，对于上述各siRNA采用梯度剂量进行转染，使其终浓度分别为0.5nM、1nM和10nM。作为阴性对照，采用与实施例1中相同的siRNA(siNC)。siRNA对RRM2 mRNA表达水平的抑制率按如下等式计算：抑制率＝[1-(实验孔RRM2 mRNA的表达量/实验孔β-Actin mRNA的表达量)/(阴性对照孔RRM2 mRNA的表达量/阴性对照孔β-Actin mRNA的表达量)]×100％。结果 如表6所示。The inhibitory efficiency of the expression levels of RRM2 mRNA was determined by siRRM2-M, siPC1-M and siPC2-M obtained in Preparation Example 2, and siRRM2, siPC1 and siPC2 obtained in Preparation Example 1, respectively, in the same manner as in Example 1. . The specific steps of transfection and fluorescence quantitative real-time PCR are as described in Example 1. At the time of transfection, the above siRNAs were transfected with a gradient dose to a final concentration of 0.5 nM, 1 nM and 10 nM, respectively. As a negative control, the same siRNA (siNC) as in Example 1 was used. The inhibition rate of siRNA on RRM2 mRNA expression level was calculated as follows: inhibition rate = [1-(experimental RRM2 mRNA expression level / experimental hole β-Actin mRNA expression level) / (negative control well RRM2 mRNA expression level) / Negative control well β-Actin mRNA expression level)] × 100%. Result As shown in Table 6.

表6Table 6

由表6可知，siRRM2-M相对于siPC1-M和siPC2-M具有更强的抑制效率，采用1nM和10nM剂量时，siRRM2-M对于RRM2 mRNA表达量具有与siRRM2类似的抑制效果。在0.5nM剂量时，siRRM2-M相比对应的siRRM2具有更为优异的抑制效果(46％vs.0％)。这或许是由于修饰增强了siRNA的稳定性，并由此增长了siRNA在细胞内的存留时间，从而提高了siRNA的活性抑制。As can be seen from Table 6, siRRM2-M has a stronger inhibitory efficiency against siPC1-M and siPC2-M, and siRRM2-M has a similar inhibitory effect on siRRM2 as the expression of RRM2 mRNA at doses of 1 nM and 10 nM. At a dose of 0.5 nM, siRRM2-M had a more superior inhibitory effect (46% vs. 0%) than the corresponding siRRM2. This may be due to the fact that the modification enhances the stability of the siRNA and thereby increases the retention time of the siRNA in the cell, thereby increasing the inhibition of the activity of the siRNA.

实施例4Example 4

本实施例用来检测修饰后的siRNA对于肝癌肿瘤细胞生长的抑制作用，具体地使用MTT检测siRRM2-M对HepG2细胞的生长抑制作用。This example was used to detect the inhibitory effect of the modified siRNA on the growth of liver cancer tumor cells, and specifically, the growth inhibitory effect of siRRM2-M on HepG2 cells was examined using MTT.

取HepG2细胞5×10³/mL,100μL/孔接种于96孔微量培养板内，培养24小时后，转染制备例2中得到的siRRM2-M、siPC1-M和siPC2-M，每种siRNA的终浓度依次各自为50nM、100nM和200nM。另设无细胞调零孔。肿瘤细胞在37℃、5％CO₂条件下培养48小时后，加入MTT(Sigma公司，货号M5655)20μL/孔(用生理盐水配制，5mg/mL)；在培养箱孵育4小时后，弃上清液，加入二甲基亚砜(DMSO)150μL/孔，将细胞培养板在微孔振荡器上震荡5分钟后， 用酶标仪测定570nm处的吸光值。试验中每个浓度重复6孔，每次实验至少重复3次。按照下列公式计算被测物对癌细胞生长的抑制率：肿瘤抑制率＝(对照组OD值-给药组OD值)/对照组OD值×100％。细胞活性用上述MTT法评价，siRRM2-M的IC₅₀为65.0nmol/L，siPC1-M的IC₅₀为143.7nmol/L，siPC2-M的IC₅₀为129.2nmol/L。可见，本发明提供的siRRM2-M相对于siPC1-M和siPC2-M具有显著抑制肝癌细胞生长的效果。平行地，在胰腺癌细胞系PANC-1上的实验结果类似(结果未显示)。HepG2 cells were taken at 5×10 ³ /mL, 100 μL/well were seeded in 96-well microplates, and after 24 hours of culture, siRRM2-M, siPC1-M and siPC2-M obtained in Preparation Example 2 were transfected, each siRNA. The final concentrations were in turn 50 nM, 100 nM and 200 nM, respectively. There is also a cell-free zeroing hole. Tumor cells were cultured for 48 hours at 37 ° C, 5% CO ₂ , and then added to MTT (Sigma, Cat. No. M5655) 20 μL / well (prepared with physiological saline, 5 mg / mL); after incubating in the incubator for 4 hours, discard The supernatant was added with 150 μL/well of dimethyl sulfoxide (DMSO), and the cell culture plate was shaken on a microplate shaker for 5 minutes, and the absorbance at 570 nm was measured with a microplate reader. 6 wells were repeated for each concentration in the experiment, and each experiment was repeated at least 3 times. The inhibition rate of the test substance on the growth of cancer cells was calculated according to the following formula: tumor inhibition rate = (control group OD value - administration group OD value) / control group OD value × 100%. Cell viability was evaluated by the above MTT method, the IC ₅₀ of siRRM2-M was 65.0 nmol/L, the IC ₅₀ of siPC1-M was 143.7 nmol/L, and the IC ₅₀ of siPC2-M was 129.2 nmol/L. It can be seen that the siRRM2-M provided by the present invention has an effect of significantly inhibiting the growth of liver cancer cells relative to siPC1-M and siPC2-M. In parallel, the experimental results on the pancreatic cancer cell line PANC-1 were similar (results not shown).

实施例5Example 5

本实施例用来检测修饰后siRNA对胰腺癌肿瘤细胞周期的阻滞作用，具体地使用流式细胞术检测siRRM2-M对PANC-1细胞周期的阻滞作用。This example was used to detect the blocking effect of the modified siRNA on the cell cycle of pancreatic cancer cells, and specifically, the effect of siRRM2-M on the cell cycle arrest of PANC-1 was detected by flow cytometry.

转染前24小时，在6孔板中以每孔2×10⁵个细胞的密度铺板，培养基为含10％FBS、1％青霉素/链霉素的完全培养基DMEM，培养于37℃的含5％CO₂的细胞培养箱中。使用Lipofectamine 2000转染siRNA，使siNC终浓度为50nM，siRRM2-M终浓度为25nM和50nM；4小时后，补加2mL含10％FBS的DMEM培养基。转染后72小时，收集细胞以1000rpm/min，离心5min，弃去上清；用预冷的PBS洗涤一次，1000rpm/min，离心5min，弃去上清；再用500μL预冷PBS吹打均匀，每个样品再加3mL 70％预冷乙醇固定细胞，轻轻吹打几次，-20℃冻存3小时。2000rpm/min，离心5min，留少量的液体弹匀后，加入5mL含1％FBS的PBS，洗涤细胞一次，2000rpm/min，离心5min。每个样品加入200μL PBS吹打均匀，加入6μL 20mg/mL的RNase，37℃消化30min。在悬液里加入20μL 500μg/mL的荧光染料PI(碘化丙啶，Propidium Iodide，购自Sigma，货号P4170)，避光孵育30min。2000rpm/min，5min离心，去上清，每样品加600mL PBS吹打均匀。转移至流式管中，使用流式细胞仪(购自BD公司，型 号FACSCalibur^TM/Calibur)进行DNA含量检测与细胞周期分析，得到DNA含量的分布直方图，计算S期细胞所占比例，结果如表7所示。24 hours before transfection, plated at a density of 2 × 10 ⁵ cells per well in a 6-well plate. The medium was DMEM containing 10% FBS, 1% penicillin/streptomycin, and cultured at 37 °C. In a cell culture incubator containing 5% CO ₂ . siRNA was transfected with Lipofectamine 2000 to give a final concentration of siNC of 50 nM, a final concentration of siRRM2-M of 25 nM and 50 nM; after 4 hours, 2 mL of DMEM medium containing 10% FBS was added. 72 hours after transfection, the cells were collected at 1000 rpm/min, centrifuged for 5 min, and the supernatant was discarded; washed once with pre-cooled PBS, centrifuged at 1000 rpm/min for 5 min, and the supernatant was discarded; then uniformly sprayed with 500 μL of pre-cooled PBS. Each sample was further fixed with 3 mL of 70% pre-chilled ethanol, gently pipetted several times, and frozen at -20 ° C for 3 hours. After centrifugation for 5 min at 2000 rpm/min, a small amount of liquid was shaken, and 5 mL of PBS containing 1% FBS was added, and the cells were washed once, 2000 rpm/min, and centrifuged for 5 min. Each sample was pipetted with 200 μL of PBS, and 6 μL of 20 mg/mL RNase was added and digested at 37 ° C for 30 min. 20 μL of 500 μg/mL fluorescent dye PI (propidium iodide, purchased from Sigma, Cat. No. P4170) was added to the suspension, and incubated for 30 min in the dark. Centrifuge at 2000 rpm/min, 5 min, remove the supernatant, and mix 600 mL PBS per sample. Transfer to a flow tube, use a flow cytometer (purchased from BD, model FACSCalibur ^TM /Calibur) for DNA content detection and cell cycle analysis, obtain a histogram of the distribution of DNA content, calculate the proportion of cells in the S phase, and the results As shown in Table 7.

表7Table 7

siRNAsiRNA	S期细胞比例(％)S phase cell ratio (%)
无处理组No processing group	24.7424.74
siNC(50nM)siNC (50nM)	27.9927.99
siRRM2-M(25nM)siRRM2-M (25nM)	57.4257.42
siRRM2-M(50nM)siRRM2-M (50nM)	63.4663.46

结果显示，与siNC处理的细胞相比，用siRRM2-M在25nM、50nM转染浓度下处理细胞后，可显著将细胞捕获在S期，抑制细胞增殖。平行地，在肝癌细胞系HepG2上的实验结果类似(结果未显示)。The results showed that compared with siNC-treated cells, cells treated with siRRM2-M at 25 nM, 50 nM transfection concentration significantly arrested the cells in the S phase and inhibited cell proliferation. In parallel, the experimental results on the liver cancer cell line HepG2 were similar (results not shown).

实施例6Example 6

本实施例用来检测修饰后siRNA对胰腺癌肿瘤细胞集落成集的抑制作用。This example was used to detect the inhibitory effect of modified siRNA on colony assembly of pancreatic cancer tumor cells.

取指数生长期细胞，按传代培养法收集细胞。用10mL的10％FBS、1％青霉素/链霉素的DMEM重悬细胞，进行细胞计数。用6孔板铺板，每孔细胞数分别为300个。铺板后24小时，用Lipofectamine 2000转染siRRM2-M与无关siRNA对照(siNC)，转染浓度为50nM，每个siRNA做3个复孔。转染后72h，用新鲜培养液(10％FBS、1％青霉素/链霉素的DMEM)换液。换液后继续培养10天，每三天换一次新鲜培养液。培养10天，采用结晶紫染色法染色：先除去培养基，用PBS洗1-2次，加入0.1％的结晶紫染液(用20％乙醇配结晶紫染液)，染色10分钟，着色足够时弃去培养液，用PBS洗2-3次，待稍干后进行克隆计数、拍照。细胞数≥30时，为一个集落。The exponential growth phase cells were taken and the cells were collected by subculture. The cells were resuspended in 10 mL of 10% FBS, 1% penicillin/streptomycin in DMEM, and cell counts were performed. The plate was plated with a 6-well plate, and the number of cells per well was 300. 24 hours after plating, siRRM2-M was transfected with Lipofectamine 2000 and an unrelated siRNA control (siNC) at a transfection concentration of 50 nM, and 3 replicate wells per siRNA. 72 h after transfection, the medium was replaced with fresh medium (10% FBS, 1% penicillin/streptomycin in DMEM). The culture was continued for 10 days after changing the solution, and the fresh medium was changed every three days. Culture for 10 days, staining by crystal violet staining: first remove the medium, wash 1-2 times with PBS, add 0.1% crystal violet dye solution (with 20% ethanol with crystal violet dye solution), stain for 10 minutes, coloring enough When the culture solution was discarded, it was washed 2-3 times with PBS, and after being slightly dried, the clone was counted and photographed. When the number of cells is ≥30, it is a colony.

图1显示siRRM2-M对PANC-1细胞集落成集的抑制作用。结果显示，相比于无关siRNA(siNC)处理的细胞，用siRRM2-M处理的细胞的集落生成数显著减少，证明抑制RRM2的表达后可以抑制PANC-1细胞的生长。平行地，在肝 癌细胞系HepG2上的实验结果类似(结果未显示)。Figure 1 shows the inhibitory effect of siRRM2-M on PANC-1 cell colony assembly. The results showed that the number of colony formation of cells treated with siRRM2-M was significantly reduced compared to cells treated with irrelevant siRNA (siNC), demonstrating that inhibition of RRM2 expression inhibits the growth of PANC-1 cells. Parallel, in the liver The experimental results on the cancer cell line HepG2 were similar (results not shown).

制备例3Preparation Example 3

本制备例用来制备siRNA药物组合物RBP131/siRNA及RBP130/siRNA。This preparation was used to prepare siRNA pharmaceutical compositions RBP131/siRNA and RBP130/siRNA.

将三种干粉脂质化合物(即，有机胺(如式(2)或式(3)所示的物质，其制备方法参见CN201180060664.1中的化合物87或72)、胆固醇、聚乙二醇化脂质(1,2-二棕榈酰基-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)-2000]))以59:29:12的摩尔比悬浮于乙醇中并混合，三种脂质化合物的总质量浓度约为8.85mg/ml。将待测siRNA(制备例1中的siNC、siRRM2和制备例2中的siRRM2-M、siPC1-M、siPC2-M)溶解于200mM醋酸钠(pH 5.2)溶液中，使siRNA的浓度为0.2mg/ml。以1:3的体积比，快速混合所得到的脂质乙醇溶液和siRNA醋酸钠水溶液。在表8中描述了混合后得到的脂质体制剂的具体组成。Three dry powder lipid compounds (ie, organic amines (such as those shown in formula (2) or formula (3), the preparation method thereof, see compound 87 or 72 in CN201180060664.1), cholesterol, PEGylated lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000]))) suspended in ethanol at a molar ratio of 59:29:12 And mixed, the total mass concentration of the three lipid compounds was about 8.85 mg/ml. The siRNA to be tested (siNC, siRRM2 in Preparation Example 1 and siRRM2-M, siPC1-M, siPC2-M in Preparation Example 2) was dissolved in a 200 mM sodium acetate (pH 5.2) solution to have a siRNA concentration of 0.2 mg. /ml. The obtained lipid ethanol solution and siRNA aqueous sodium acetate solution were quickly mixed at a volume ratio of 1:3. The specific composition of the liposome preparation obtained after mixing is described in Table 8.

表8脂质体制剂的组成Table 8 Composition of liposome preparations

将混合后得到的脂质体制剂(即，有机胺、胆固醇、聚乙二醇化脂质与siRNA的组合物)在约50℃孵育10分钟。孵育后，使用

切相流***，中空纤维柱100KDa超滤，超滤交换溶液为pH 7.4的PBS。超滤的同时可以将制剂浓缩或稀释到期望的siRNA浓度。超滤后的制剂在0.22μm滤器上过滤除菌。将由式(2)所示的有机胺、胆固醇、1,2-二棕榈酰基-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)-2000]组成的脂质混合物称为RBP131，由式(3)所示的有机胺、 胆固醇、1,2-二棕榈酰基-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)-2000]组成的脂质混合物称为RBP130。所得RBP131/siRNA或RBP130/siRNA脂质体制剂在使用前储存在4℃，并检测相关理化性质，RBP131/siRNA和RBP130/siRNA脂质体制剂的理化参数相似，检测结果见表9。The liposome preparation obtained after mixing (i.e., a combination of an organic amine, cholesterol, PEGylated lipid and siRNA) was incubated at about 50 ° C for 10 minutes. After incubation, use

The phase-cut flow system, the hollow fiber column 100KDa ultrafiltration, the ultrafiltration exchange solution was pH 7.4 PBS. The preparation can be concentrated or diluted to the desired siRNA concentration while ultrafiltration. The ultrafiltered preparation was sterilized by filtration on a 0.22 μm filter. A lipid composed of an organic amine represented by the formula (2), cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy (polyethylene glycol)-2000] The mixture is called RBP131, an organic amine represented by formula (3), cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy (polyethylene glycol)-2000 The composition of the lipid mixture is called RBP130. The obtained RBP131/siRNA or RBP130/siRNA liposome preparations were stored at 4 ° C before use, and the relevant physical and chemical properties were examined. The physical and chemical parameters of the RBP131/siRNA and RBP130/siRNA liposome preparations were similar, and the test results are shown in Table 9.

表9RBP131/siRNA和RBP130/siRNA脂质体制剂的理化参数Table 9 Physicochemical parameters of RBP131/siRNA and RBP130/siRNA liposome preparations

检测指征Detection indication	参数范围Parameter range
pHpH	7.2-7.67.2-7.6
包封率(％)Encapsulation rate (%)	>90％>90%
siRNA浓度(mg/ml)siRNA concentration (mg/ml)	0.10-0.150.10-0.15
粒径(nm)Particle size (nm)	60-10060-100
PDIPDI	<0.20<0.20
渗透压(mOsm/kg)Osmotic pressure (mOsm/kg)	300-400300-400

实施例7Example 7

本实施例用来检测修饰后siRNA与广谱抗癌药物阿霉素联合使用，对抑制胰腺癌肿瘤组织生长的协同作用。This example was used to detect the synergistic effect of the modified siRNA in combination with the broad-spectrum anticancer drug doxorubicin on inhibiting the growth of pancreatic cancer tumor tissue.

(1)siRNA药物组合物的制备。(1) Preparation of siRNA pharmaceutical composition.

按照制备例3所述的方法，制备药物组合物RBP131/siNC和RBP131/siRRM2-M。The pharmaceutical compositions RBP131/siNC and RBP131/siRRM2-M were prepared according to the method described in Preparation 3.

(2)裸鼠皮下胰腺癌模型的制备。(2) Preparation of a subcutaneous pancreatic cancer model in nude mice.

用BLAB/c裸鼠(购于维通利华)，皮下接种胰腺癌细胞：取培养好的PANC-1细胞，用无菌PBS稀释成5×10⁶细胞/100μL，接种于BALB/c小鼠右腋皮下，细胞接种量为100μL/只。测量瘤体大小：皮下接种约2～3天后，观察皮下实体瘤形成情况，待实体瘤长到平均50mm³后备用(检测肿瘤体积的计算方法V＝0.5×a×b×b，其中a为长度，b为宽度)。Pancreatic cancer cells were subcutaneously inoculated with BLAB/c nude mice (purchased from Vitalivo): the cultured PANC-1 cells were diluted with sterile PBS to 5×10 ⁶ cells/100 μL, and inoculated into BALB/c. Under the skin of the right axilla, the amount of cells inoculated was 100 μL/head. Measurement of tumor size: After subcutaneous inoculation for about 2 to 3 days, observe the formation of subcutaneous solid tumors, and wait until the solid tumor grows to an average of 50 mm ³ (the method for calculating the tumor volume is V = 0.5 × a × b × b, where a is Length, b is the width).

(3)siRNA药物组合物与阿霉素联合给药的方法及检测。(3) Method and detection of siRNA pharmaceutical composition in combination with doxorubicin.

动物被随机分到5组，每组8只小鼠。这5组动物做如下处理：(1)1×PBS组；(2)阿霉素(ADM，购于克拉玛尔klamar公司，货号25316-40-9)单用组，剂 量为1mg/kg；(3)阿霉素与无关siRNA(siNC)联用组，其中阿霉素剂量为1mg/kg，siNC剂量为5μg/只；(4)阿霉素与siRRM2-M联用组，其中阿霉素剂量为1mg/kg，siRRM2-M剂量为2μg/只；(5)阿霉素与siRRM2-M联用组，其中阿霉素剂量为1mg/kg，siRRM2-M剂量为5μg/只。阿霉素采用腹腔注射给药，一周给三次药物；siRNA均是RBP131包裹的制剂(即RBP131/siNC和RBP131/siRRM2-M)，采用瘤旁注射给药，注射体积为30μL，一周给药两次。给药过程中一直用数显游标卡尺测量肿瘤大小；给药第25天取材，取材时对小鼠进行称重、麻醉和拍照，之后处死小鼠，分离肝脏、脾脏与肿瘤组织，对肝脏、脾脏、肿瘤组织进行称重。Animals were randomly assigned to 5 groups of 8 mice each. The five groups of animals were treated as follows: (1) 1×PBS group; (2) doxorubicin (ADM, purchased from Klamal klamar, article number 25316-40-9) single use group, agent The dose was 1 mg/kg; (3) the combination of doxorubicin and unrelated siRNA (siNC), wherein the dose of doxorubicin was 1 mg/kg, the dose of siNC was 5 μg/head; (4) the combination of doxorubicin and siRRM2-M In the group, the dose of doxorubicin was 1 mg/kg, and the dose of siRRM2-M was 2 μg/head; (5) the combination of doxorubicin and siRRM2-M, wherein the dose of doxorubicin was 1 mg/kg, and the dose of siRRM2-M It is 5 μg/only. Doxorubicin was administered by intraperitoneal injection, and the drug was administered three times a week; the siRNAs were all RBP131-encapsulated preparations (ie, RBP131/siNC and RBP131/siRRM2-M), administered by intratumoral injection, with an injection volume of 30 μL, two doses per week. Times. The tumor size was measured with a digital vernier caliper during the administration; the mice were weighed on the 25th day of administration, the mice were weighed, anesthetized and photographed, and then the mice were sacrificed to isolate the liver, spleen and tumor tissue, and the liver and spleen. Tumor tissue was weighed.

图2、图3和图4显示阿霉素与RBP131载体递送的siRRM2-M联用对肿瘤生长的抑制。结果显示给药期间组(4)和组(5)，即阿霉素联用的RBP131/siRRM2-M(2μg或5μg)组比其他组肿瘤生长速度缓慢。阿霉素与RBP131/siRRM2-M(5μg)联用组，与阿霉素与RBP131/siNC(5μg)联用组相比，瘤重亦有显著下降。给药期间没有发现行为、精神和粪便等异常。肝体比、脾体比正常，体重无明显变化。Figures 2, 3 and 4 show the inhibition of tumor growth by the combination of doxorubicin and RPR131 vector delivered siRRM2-M. The results showed that the group (4) and the group (5) during the administration period, that is, the RBP131/siRRM2-M (2 μg or 5 μg) group in which doxorubicin was used was slower in growth rate than the other groups. The combination of doxorubicin and RBP131/siRRM2-M (5 μg) showed a significant decrease in tumor weight compared with the combination of doxorubicin and RBP131/siNC (5 μg). No abnormalities such as behavior, spirit, and feces were found during the administration. The ratio of liver to body and spleen were normal, and there was no significant change in body weight.

实施例8Example 8

本实施例用来检测修饰后siRNA对肝癌肿瘤组织生长的抑制作用。This example was used to detect the inhibitory effect of modified siRNA on the growth of liver cancer tumor tissue.

(1)siRNA药物组合物的制备。按照制备例3所述的方法，制备药物组合物RBP131/siNC、RBP131/siRRM2和RBP131/siRRM2-M。(1) Preparation of siRNA pharmaceutical composition. The pharmaceutical compositions RBP131/siNC, RBP131/siRRM2 and RBP131/siRRM2-M were prepared according to the method described in Preparation 3.

(2)裸鼠肝原位移植瘤模型的制备。(2) Preparation of a nude mouse liver orthotopic transplantation tumor model.

皮下移植瘤的制备与处理：人肝癌细胞HepG2经传代培养，收集对数生长期的细胞制成单细胞悬液，分别接种于4只裸鼠背部皮下，每只裸鼠注射0.2mL，含细胞数2×10⁶个，继续常规饲养，观察裸鼠背部皮下成瘤情况。待肿瘤体积达 1000mm³时，采用5％水合氯醛腹腔注射(0.7mL/100g)，对裸鼠进行麻醉。无菌条件下取出肿瘤，去除坏死组织，用眼科剪将肿瘤组织剪切成1mm×1mm×1mm大小的瘤块，置于无血清DMEM培养基中备用。Preparation and treatment of subcutaneous xenografts: Human hepatoma cells HepG2 were subcultured, and cells in logarithmic growth phase were collected into single cell suspensions, which were inoculated subcutaneously into the back of 4 nude mice. Each nude mouse was injected with 0.2 mL, containing cells. The number was 2 × 10 ⁶ , and the conventional feeding was continued to observe the subcutaneous tumor formation in the back of the nude mice. When the tumor volume reached 1000 mm ³ , the nude mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (0.7 mL/100 g). The tumor was removed under aseptic conditions, necrotic tissue was removed, and the tumor tissue was cut into 1 mm × 1 mm × 1 mm tumor tissue with ophthalmic scissors, and placed in serum-free DMEM medium for use.

裸鼠肝原位移植瘤模型制备：50只裸鼠，5％水合氯醛(0.7mL/100g)腹腔注射麻醉，腹部正中横切口暴露肝脏，用10mL注射器针头在肝大叶做1个深1～2mm的隧道，将肿瘤组织植入隧道内，明胶海绵压迫止血后全层关腹。手术后，B超监测肿瘤生长情况，21天左右形成可见肿瘤，肿瘤体积平均约为50mm³。Preparation of orthotopic liver transplantation model of nude mice: 50 nude mice, 5% chloral hydrate (0.7mL/100g) were anesthetized by intraperitoneal injection, the liver was exposed to the midline of the abdomen, and the liver was made with a 10mL syringe needle. In the tunnel of ~2mm, the tumor tissue was implanted into the tunnel, and the gelatin sponge was pressed to stop bleeding and the whole layer was closed. After the operation, B-ultrasound monitors the tumor growth, and visible tumors are formed in about 21 days. The tumor volume averages about 50 mm ³ .

(3)siRNA药物组合物的处理方法及检测。(3) Processing method and detection of siRNA pharmaceutical composition.

将裸鼠随机分成5组，每组10只，这5组动物做如下处理：(1)PBS对照组；(2)RBP131/siNC无关对照组；(3)RBP131/siRRM2样品组；(4)RBP131/siRRM2-M样品组；(5)5-Fu阳性对照组。5-Fu(氟尿嘧啶)是常用的化疗抗肿瘤化合物之一，购自天津金耀氨基酸有限公司。上述5组动物分别通过尾静脉注射进行给药。siRNA给药量为1mg/kg，给药体积为10mL/kg，每周给药2次，共计5次；5-Fu给药量为5mg/kg，给药体积为10mL/kg，每两天给药1次，共计8次。给药后对肿瘤大小进行B超检测，以确定肿瘤生长情况。首次给药后第20天取材，取材时对小鼠进行称重、麻醉和拍照，之后处死小鼠，分离肝脏、脾脏与肿瘤组织，对肝脏、脾脏、肿瘤组织进行称重。Nude mice were randomly divided into 5 groups of 10 animals. The 5 groups of animals were treated as follows: (1) PBS control group; (2) RBP131/siNC-independent control group; (3) RBP131/siRRM2 sample group; (4) RBP131/siRRM2-M sample group; (5) 5-Fu positive control group. 5-Fu (fluorouracil) is one of the commonly used anti-tumor compounds of chemotherapy, purchased from Tianjin Jinyao Amino Acid Co., Ltd. The above five groups of animals were each administered by tail vein injection. The siRNA was administered in an amount of 1 mg/kg, the administration volume was 10 mL/kg, and the administration was performed twice a week for 5 times; the 5-Fu dose was 5 mg/kg, and the administration volume was 10 mL/kg every two days. The drug was administered once for a total of 8 times. B-ultrasound was performed on tumor size after administration to determine tumor growth. On the 20th day after the first administration, the mice were weighed, anesthetized and photographed, and then the mice were sacrificed, and the liver, spleen and tumor tissues were separated, and the liver, spleen and tumor tissues were weighed.

图5显示了RBP131载体递送的siRRM2-M siRNA对肝脏肿瘤生长的抑制情况。结果表明，与PBS对照组相比，通过尾静脉注射***给予siRRM2-M的药物组合物可以有效抑制肿瘤组织生长，瘤重减小了82％，接近化疗剂5-Fu的水平；而未修饰的siRRM2瘤重只减少了45％。Figure 5 shows inhibition of liver tumor growth by siRRM2-M siRNA delivered by RBP131 vector. The results showed that the pharmaceutical composition administered siRRM2-M by the tail vein injection system can effectively inhibit tumor tissue growth compared with the PBS control group, and the tumor weight is reduced by 82%, which is close to the level of the chemotherapeutic agent 5-Fu; The siRRM2 tumor weight was only reduced by 45%.

实施例9 Example 9

本实施例用于对比不同的siRNA对肝癌肿瘤组织生长的抑制效率。This example was used to compare the inhibition efficiency of different siRNAs on the growth of liver cancer tumor tissues.

按照制备例3所述的方法，制备siRNA药物组合物RBP130/siRRM2-M、RBP130/siRRM2-M2、RBP130/siPC1-M、RBP130/siPC2-M和RBP130/si501/842；参见实施例8所述的方法，制备裸鼠肝原位移植瘤模型。siRNA pharmaceutical compositions RBP130/siRRM2-M, RBP130/siRRM2-M2, RBP130/siPC1-M, RBP130/siPC2-M and RBP130/si501/842 were prepared according to the method described in Preparation Example 3; The method of preparing a nude mouse liver orthotopic transplantation tumor model.

其中，siRRM2-M2是siRRM2的另一种修饰形式，与siRRM2-M相比，其反义链的修饰方式不同；si501/842采用US8946176B2中公开的方法进行修饰，具体的寡聚核苷酸序列见表10所述。Among them, siRRM2-M2 is another modified form of siRRM2, and its antisense strand is modified differently than siRRM2-M; si501/842 is modified by the method disclosed in US8946176B2, specific oligonucleotide sequence See Table 10.

表10Table 10

表10中的寡聚核苷酸包含修饰的核苷酸残基，互补的寡聚核苷酸链退火形成修饰的siRNA。其中，(OMe)代表它左边的核苷酸残基中戊糖基团为2’-甲氧基核糖基，(F)代表它左边的核苷酸残基中戊糖基团为2’-氟代核糖基；S代表它两边的脱氧核糖核苷酸dTdT之间的酯基为硫代磷酸酯基；p代表第一位核苷酸残基中5'-端连接磷酸基团(序列中不含p的，表示不含5’-磷酸基团)。The oligonucleotides in Table 10 contain modified nucleotide residues that are annealed to form modified siRNA. Wherein (OMe) represents a pentose group in the nucleotide residue to the left of which is a 2'-methoxyribosyl group, and (F) represents a pentose group in the nucleotide residue to the left of which is 2'- Fluororibosyl; S represents the ester group between the deoxyribonucleotides dTdT on both sides thereof is a phosphorothioate group; p represents the 5'-terminal linked phosphate group in the first nucleotide residue (in the sequence) Without p, it means no 5'-phosphate group.

将裸鼠随机分成6组，每组6只，这6组动物做如下处理：(1)PBS对照组；(2)RBP130/siRRM2-M样品组；(3)RBP130/siRRM2-M2样品组；(4)RBP130/siPC1-M样品组；(5)RBP130/siPC2-M样品组；(6)RBP130/si501/842样品组。上述6组动物分别通过尾静脉注射进行给药。siRNA给药量为1mg/kg，给药体积为10mL/kg，每周给药2次，共计5次。给药后对肿瘤大小进行B超检测，以确定肿瘤生长情况。首次给药后第20天取材，取材时对小鼠进行称重、麻醉和拍照，之后处死小鼠，分离肝脏、脾脏与肿瘤组织，对肝脏、脾脏、肿瘤组织进行称重，瘤重抑制率见表11所述。 Nude mice were randomly divided into 6 groups of 6 animals. The 6 groups of animals were treated as follows: (1) PBS control group; (2) RBP130/siRRM2-M sample group; (3) RBP130/siRRM2-M2 sample group; (4) RBP130/siPC1-M sample set; (5) RBP130/siPC2-M sample set; (6) RBP130/si501/842 sample set. The above 6 groups of animals were each administered by tail vein injection. The amount of siRNA administered was 1 mg/kg, and the administration volume was 10 mL/kg, which was administered twice a week for a total of 5 times. B-ultrasound was performed on tumor size after administration to determine tumor growth. On the 20th day after the first administration, the mice were weighed, anesthetized and photographed. The mice were sacrificed, and the liver, spleen and tumor tissues were isolated. The liver, spleen and tumor tissues were weighed, and the tumor weight inhibition rate was determined. See Table 11.

表11Table 11

分组Grouping	瘤重抑制率Tumor weight inhibition rate
1×PBS1×PBS	0％0%
RBP130/siRRM2-MRBP130/siRRM2-M	85％85%
RBP130/siRRM2-M2RBP130/siRRM2-M2	46％46%
RBP130/siPC1-MRBP130/siPC1-M	53％53%
RBP130/siPC2-MRBP130/siPC2-M	68％68%
RBP130/si501/842RBP130/si501/842	83％83%

与PBS对照组相比，通过尾静脉注射***给予RBP130/siRRM2-M的药物组合物可以有效抑制肿瘤组织生长，瘤重减小了85％，与RBP130/si501/842的药物组合物对肿瘤组织的生长抑制相当；而给予了RBP130/siPC1-M的药物组合物的处理组中，瘤重减小了53％，给予了RBP130/siPC2-M的药物组合物的处理组中，瘤重减小了68％，给予了RBP130/siRRM2-M2的药物组合物的处理组中，瘤重只减小了46％。通过以上结果可以得知，通过利用本发明的修饰方式，使得含有较少修饰位点的siRRM2-M能达到与含有较多修饰位点的si501/842相当的体内活性，而同时，虽然siRRM2-M2修饰位点也少于si501/842，但体内活性较差。Compared with the PBS control group, the pharmaceutical composition administered RBP130/siRRM2-M by the tail vein injection system can effectively inhibit tumor tissue growth, the tumor weight is reduced by 85%, and the drug composition with RBP130/si501/842 is on the tumor tissue. The growth inhibition was comparable; in the treatment group administered with the RBP130/siPC1-M pharmaceutical composition, the tumor weight was reduced by 53%, and the tumor weight was decreased in the treatment group administered with the RBP130/siPC2-M pharmaceutical composition. In the treatment group of 68% of the pharmaceutical composition administered with RBP130/siRRM2-M2, the tumor weight was only reduced by 46%. From the above results, it was found that by using the modification method of the present invention, siRRM2-M containing fewer modification sites can achieve in vivo activity comparable to si501/842 containing more modification sites, while at the same time, although siRRM2- The M2 modification site was also less than si501/842, but the activity in vivo was poor.

实施例10Example 10

本实验实施例用来检测不同厂商生产的商业化siRNA体内给药载体，包括Invivo fectamine 2.0(购自Life technology)、invivo jetPEI(购自PolyPlus-transfection(PT))、Entranster(购自Engreen Biosystem)以及本发明所述的RBP131，包载携带siRRM2-M后，在裸鼠原位肝癌模型(构建方法见实施例8)中对肿瘤大小及RRM2 mRNA表达量的抑制效率。This experimental example was used to detect commercial siRNA in vivo delivery vectors produced by different manufacturers, including Invivo fectamine 2.0 (purchased from Life technology), invivo jetPEI (purchased from PolyPlus-transfection (PT)), and Entranster (purchased from Engreen Biosystem). And the RBP131 of the present invention, after carrying the siRRM2-M, inhibits the tumor size and the RRM2 mRNA expression level in the nude mouse orthotopic liver cancer model (see Example 8 for the construction method).

根据生产商提供的标准操作流程，将本发明的siRRM2-M包裹进上述商业化载体，制备相应的载体/siRNA组合物，分别标记为IVF2.0/RRM2、invivo  jetPEI/RRM2、Entranster/RRM2。同时，按照制备例3的方法，制备RBP131/RRM2和RBP130/RRM2组合物。The siRRM2-M of the present invention was packaged into the above commercial carrier according to standard operating procedures provided by the manufacturer to prepare corresponding vector/siRNA compositions, which were labeled as IVF2.0/RRM2, invivo, respectively. jetPEI/RRM2, Entranster/RRM2. Meanwhile, the RBP131/RRM2 and RBP130/RRM2 compositions were prepared in accordance with the method of Preparation 3.

将36只荷瘤裸鼠(肿瘤体积平均约为50mm³)，随机分成6组(每组6只，均为雄性)，分别为PBS对照组、IVF2.0/RRM2、invivo jetPEI/RRM2、Entranster/RRM2、RBP131/RRM2和RBP130/RRM2样品组。所有动物根据体重计算药量，尾静脉给药，每周2次，共给药6次。IVF2.0/RRM2、invivo jetPEI/RRM2、Entranster/RRM2三组的给药剂量为2.5mg/kg(siRNA)，RBP131/RRM2和RBP130/RRM2组的给药剂量为0.5mg/kg(siRNA)，所有6组的给药体积为10mL/kg。末次给药后第3天将动物处死，处死前对动物进行称重、麻醉和拍照，对动物进行大体解剖，观察体内脏器是否有病变，对肉眼观察有病变的组织用10％***保存进一步进行病理观察，收集肝脏、脾脏、肿瘤组织并称重，部分肝组织和肿瘤组织用RNA later(Sigma Aldrich公司)保存；用组织匀浆仪匀浆肿瘤组织，再用Trizol根据总RNA提取的标准操作步骤提取得到总RNA。Thirty-six tumor-bearing nude mice (average tumor volume of approximately 50 mm ³ ) were randomly divided into 6 groups (6 in each group, all male), respectively, PBS control group, IVF2.0/RRM2, invivo jetPEI/RRM2, Entranster /RRM2, RBP131/RRM2 and RBP130/RRM2 sample sets. All animals were dosed according to body weight, administered intravenously in the tail vein twice a week for a total of 6 doses. The IVF2.0/RRM2, invivo jetPEI/RRM2, and Entranster/RRM2 groups were administered at a dose of 2.5 mg/kg (siRNA), and the RBP131/RRM2 and RBP130/RRM2 groups were administered at a dose of 0.5 mg/kg (siRNA). The dosing volume of all 6 groups was 10 mL/kg. The animals were sacrificed on the third day after the last administration, and the animals were weighed, anesthetized and photographed before sacrifice. The animals were grossly dissected to observe whether the organs in the body had lesions, and 10% of the tissues were observed by the naked eye. The forest was further preserved for pathological observation. The liver, spleen and tumor tissues were collected and weighed. Some liver tissues and tumor tissues were preserved with RNA later (Sigma Aldrich); the tumor tissues were homogenized with a tissue homogenizer, and then total RNA was extracted with Trizol. Total RNA was extracted from standard extraction procedures.

采用实时荧光定量PCR检测肝组织中RRM2mRNA的表达水平。检测方法同实施例1所述，检测引物同表2所述。不同的siRNA药物组合物对肿瘤的抑制效率用瘤重变化表示，方法同实施例8。Real-time quantitative PCR was used to detect the expression level of RRM2 mRNA in liver tissue. The detection method was the same as that described in Example 1, and the detection primers were as described in Table 2. The inhibition efficiency of tumors by different siRNA pharmaceutical compositions is expressed by the change in tumor weight in the same manner as in Example 8.

表12显示了所测各组供试品对原位肝癌动物肿瘤组织中RRM2 mRNA表达抑制活性，以及瘤重抑制率的检测结果。Table 12 shows the results of the detection of RRM2 mRNA expression in the tumor tissues of orthotopic liver cancer animals and the detection rate of tumor weight inhibition.

表12Table 12

结果显示，RBP131/RRM2和RBP130/RRM2对基因表达及肿瘤大小的抑制效果相似。与由其它商业化载体形成的药物组合物中抑制mRNA表达效果最好的IVF2.0/RRM2组相比，RBP131/RRM2和RBP130/RRM2组对肝癌模型小鼠中RRM2mRNA的抑制率也都提高了35％以上；同时，RBP131/RRM2和RBP130/RRM2组的抑制活性是invivo jetPEI/RRM2组的2倍、是Entranster/RRM2组的近20倍。瘤重抑制率结果与mRNA抑制率相似。进而，考虑到RBP131/RRM2和RBP130/RRM2组的给药剂量仅为其它三组的1/5，相较于由其它商业化载体形成的药物组合物而言，RBP131/RRM2和RBP130/RRM2显示出极高的生物活性。The results showed that RBP131/RRM2 and RBP130/RRM2 had similar inhibitory effects on gene expression and tumor size. The RBP2/RRM2 and RBP130/RRM2 groups also increased the inhibition rate of RRM2 mRNA in liver cancer model mice compared with the IVF2.0/RRM2 group which inhibited mRNA expression in the pharmaceutical composition formed by other commercial carriers. At the same time, the inhibitory activity of the RBP131/RRM2 and RBP130/RRM2 groups was twice that of the invivo jetPEI/RRM2 group and nearly 20 times that of the Entranster/RRM2 group. The tumor weight inhibition rate results were similar to the mRNA inhibition rate. Furthermore, considering that the doses of the RBP131/RRM2 and RBP130/RRM2 groups are only 1/5 of the other three groups, RBP131/RRM2 and RBP130/RRM2 are displayed compared to the pharmaceutical compositions formed by other commercial carriers. Very high biological activity.

以上结合附图详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the specific details of the embodiments described above, and various modifications may be made to the technical solutions of the present invention within the scope of the technical idea of the present invention. These simple variations are within the scope of the invention.

另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。It should be further noted that the specific technical features described in the above specific embodiments may be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention has various possibilities. The combination method will not be described separately.

此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。 In addition, any combination of various embodiments of the invention may be made as long as it does not deviate from the idea of the invention, and it should be regarded as the disclosure of the invention.

Claims (10)

1. 一种siRNA，其中，该siRNA的正义链碱基序列如SEQ ID NO：2所示，且该siRNA的反义链碱基序列如SEQ ID NO：3所示；且所述siRNA的磷酸-糖骨架分别具有或不具有修饰基团。An siRNA, wherein the sense strand base sequence of the siRNA is as shown in SEQ ID NO: 2, and the antisense strand base sequence of the siRNA is set forth in SEQ ID NO: 3; and the phosphoric acid-sugar of the siRNA The backbone has or does not have a modifying group, respectively.
2. 根据权利要求1所述的siRNA，其中，该siRNA的磷酸-糖骨架分别具有如下修饰基团：The siRNA according to claim 1, wherein the phosphate-sugar backbone of the siRNA has the following modifying groups:

   正义链第1、6、14、16和18位的糖基为2’-甲氧基核糖基；反义链第2位的糖基为2’-甲氧基核糖基，反义链第3位的糖基为2’-氟代核糖基。The glycosyl group at positions 1, 6, 14, 16 and 18 of the sense strand is 2'-methoxyribosyl; the glycosyl group at position 2 of the antisense strand is 2'-methoxyribosyl, and the antisense strand is 3 The glycosyl group at the position is a 2'-fluororibosyl group.
3. 根据权利要求2所述的siRNA，其中，该siRNA的正义链和/或反义链的第20位和第21位之间的酯基为硫代磷酸酯基。The siRNA according to claim 2, wherein the ester group between the 20th and 21st positions of the sense strand and/or the antisense strand of the siRNA is a phosphorothioate group.
4. 一种RNA干扰靶核酸，其特征在于，该RNA干扰靶核酸如SEQ ID NO：4所示。An RNA interference target nucleic acid, characterized in that the RNA interference target nucleic acid is as shown in SEQ ID NO:4.
5. 一种药物组合物，其特征在于，该药物组合物含有权利要求1-3中任意一项所述的siRNA和药学上可接受的载体；所述siRNA与所述药学上可接受的载体的重量比为1:(1-500)；A pharmaceutical composition comprising the siRNA of any one of claims 1 to 3 and a pharmaceutically acceptable carrier; the weight of the siRNA and the pharmaceutically acceptable carrier The ratio is 1: (1-500);

   其中，优选地，所述药学上可接受的载体含有有机胺、胆固醇和聚乙二醇化磷脂；其中，所述有机胺为如式(1)所示的化合物和/或它的药学上可接受的盐：Wherein, preferably, the pharmaceutically acceptable carrier contains an organic amine, cholesterol and a pegylated phospholipid; wherein the organic amine is a compound represented by formula (1) and/or pharmaceutically acceptable thereof Salt:

   

   

   其中：R₁和R₂各自独立地为C₁₀-C₂₀的直链烷基；n为1-6的整数；R₃、R₄、R₅和R₆各自独立地为氢或Rx；且R₃、R₄、R₅和R₆中至少一个为Rx；Wherein: R ₁ and R ₂ are each independently a C ₁₀ -C ₂₀ linear alkyl group; n is an integer from 1 to 6; and R ₃ , R ₄ , R ₅ and R ₆ are each independently hydrogen or Rx; At least one of R ₃ , R ₄ , R ₅ and R ₆ is Rx;

   Rx为

   

   Ry为碳链数为C₁₂-C₂₀的直链烷基。Rx is

   

   Ry is a linear alkyl group having a carbon number of C ₁₂ - C ₂₀ .
6. 根据权利要求5所述的药物组合物，其中，所述有机胺为如式(2)所示的有机胺和/或如式(3)所示的有机胺：The pharmaceutical composition according to claim 5, wherein the organic amine is an organic amine represented by the formula (2) and/or an organic amine represented by the formula (3):

   

   

   

   

   所述聚乙二醇化磷脂为1,2-二棕榈酰基-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。The pegylated phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.
7. 根据权利要求5或6所述的药物组合物，其中，所述药物组合物中，所述有机胺、胆固醇和所述聚乙二醇化磷脂三者之间的摩尔比为(19.7-80)：(19.7-80)：(0.3-50)，The pharmaceutical composition according to claim 5 or 6, wherein the molar ratio between the organic amine, cholesterol and the PEGylated phospholipid is (19.7-80) in the pharmaceutical composition: (19.7-80): (0.3-50),

   优选地，所述药物组合物中，所述有机胺、胆固醇和所述聚乙二醇化磷脂三者之间的摩尔比为(50-70)：(20-40)：(3-20)。Preferably, in the pharmaceutical composition, the molar ratio between the organic amine, cholesterol and the pegylated phospholipid is (50-70): (20-40): (3-20).
8. 根据权利要求5所述的药物组合物，其中，所述药物组合物还包括至少一种额外的抗癌化疗剂，优选地，所述额外的抗癌化疗剂为阿霉素。The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition further comprises at least one additional anti-cancer chemotherapeutic agent, preferably the additional anti-cancer chemotherapeutic agent is doxorubicin.
9. 权利要求1-3中任意一项所述的siRNA和/或权利要求5-8中任意一项所述的药物组合物在制备治疗癌症的药物中的用途。Use of the siRNA according to any one of claims 1 to 3 and/or the pharmaceutical composition according to any one of claims 5 to 8 for the preparation of a medicament for treating cancer.
10. 根据权利要求9所述的用途，其中，所述癌症为肝癌和/或胰腺癌。 The use according to claim 9, wherein the cancer is liver cancer and/or pancreatic cancer.

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