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WO2014063602A1 - New drug targeting delivery adjuvant - Google Patents

New drug targeting delivery adjuvant Download PDF

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Publication number
WO2014063602A1
WO2014063602A1 PCT/CN2013/085627 CN2013085627W WO2014063602A1 WO 2014063602 A1 WO2014063602 A1 WO 2014063602A1 CN 2013085627 W CN2013085627 W CN 2013085627W WO 2014063602 A1 WO2014063602 A1 WO 2014063602A1
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Prior art keywords
drug
sugar
new type
targeted transport
drugs
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PCT/CN2013/085627
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French (fr)
Chinese (zh)
Inventor
尹海芳
韩刚
王青松
Original Assignee
Yin Haifang
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Publication of WO2014063602A1 publication Critical patent/WO2014063602A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention belongs to the field of life sciences and pharmaceutical carriers.
  • the targeted transport efficiency of drugs is an important factor in determining the drug efficacy.
  • the methods commonly used to optimize gene therapy drugs are: optimizing the structure of the drug itself; using macromolecular polymers as transport carriers, the most common of which are nanomaterials;
  • the use of tissue targeting peptides is the covalent attachment of peptides to drugs.
  • Duxing Muscular Dystrophy is a fatal neuromuscular disorder inherited from the human X chromosome. The main symptoms are continuous consumption and degeneration of the whole body muscle. Most patients have a life span of only 20 years old. Left and right, seriously endanger people's health.
  • the cause of the disease is the mutation of the DMD gene encoding the dystrophin dystrophin, which causes the splicing change of the corresponding mRNA precursor and the destruction of the reading frame, and cannot be encoded by the biological function of dys t rophin protein.
  • the full-length gene is 2.4 Mbp, which consists of 79 exons. In addition to inheritance from the parents, it also has a high probability of spontaneous mutation.
  • 65% of the mutations are deletion mutations, and these mutations will cause changes in the gene coding reading frame, resulting in unstable, non-functional dys trophin protein. Deletion of the dys trophin protein causes instability of the monthly/1 meat fiber membrane and a large influx of extracellular Ca 2+ , which causes muscle degeneration and atrophy, and is clinically characterized by severe Duchen muscle atrophy.
  • the gene-encoded dys t rophin protein is composed of four structural regions, including functional heavy Description
  • the N-terminus - the actin-binding domain (ac t in- b inding doma in ), the large span of the rod doma in , the cysteine - rich region and the C - terminal - dys t rophin Related protein complex binding regions.
  • studies have shown that the mutation hotspots of genes are mainly concentrated in the circular region, while the circular region has little effect on the function of dys t rophin protein, so some of its deletions have no obvious effect on the function of dys trophin protein.
  • antisense oligonucleotides prevent the transcriptional splicing complex from targeting to externalization by binding to targeted exons and adjacent regions.
  • Antisense oligonucleotide-mediated exon skipping in Duchen muscle atrophy the earliest evidence comes from in vitro experiments with human lymphocytes and mouse myofibroblasts. These results demonstrate antisense oligo The feasibility of a glycoside-mediated exon skipping method.
  • antisense oligonucleotide-mediated exon skipping has become one of the most successful and promising treatments in the field of gene therapy for Duchenne muscular atrophy.
  • two antisense oligonucleotides have entered the clinical phase 11 / 111 trial, and the results are gratifying.
  • a novel drug-targeted transport adjuvant is at least one of five carbon sugars, six carbon sugars, sugar alcohols, and disaccharides.
  • the five-carbon sugar is at least one selected from the group consisting of xylose and arabinose; the six-carbon sugar is at least one selected from the group consisting of glucose, galactose, fructose, and mannose; and the disaccharide is selected from the group consisting of sucrose and lactose. At least one of maltose and trehalose; and the sugar alcohol is at least one selected from the group consisting of sorbitol, mannitol, erythritol, maltitol, lactitol, and xylitol.
  • the invention also provides a method for using the novel drug targeted transportation auxiliary agent, which comprises the following steps:
  • sugar solution is prepared according to the ratio, the monosaccharide concentration is 50 mg/ml, the mixed sugar is mixed according to the ratio, and the total sugar content is l OOmg/ml;
  • the invention relates to a novel drug targeted transportation auxiliary agent used in transporting medicine.
  • a novel drug-targeted transport adjuvant for use in transporting drugs preferably for targeting antisense oligonucleotide drugs PM0, PNA, 2'ome RNA, peptide-PM0, s iRNAs, and macromolecular plasmids .
  • an antisense oligonucleotide drug PM0 (hosphorodi ami date mrphol ino ol igomer s ), PNA, 2'ome RNA, moon-PMO, s iRNAs and large for targeted transportation therapy Duchen muscle atrophy Molecular plasmid.
  • PMO is a novel antisense oligonucleotide that inhibits the natural RNA splicing process and produces various mRNAs that inhibit gene expression, such as inhibition of protooncogene expression.
  • the doublet formed by the binding of PM0 to specific viral mRNA can effectively block viral RNA transcription and inhibit viral replication.
  • This compound has good stability, solubility and cell permeability and is now used in viral infections, cancer, muscular dystrophy and premature aging syndrome. Description
  • V ome RNA 2'- 0-thiolated RNA antisense oligonucleotide
  • PNA is peptide nucleic acid
  • peptide-PM0 is PMO linked to cell shuttle peptide or tissue targeting peptide
  • s iRNA is a class Double-stranded RNA molecules, all of which are common antisense oligonucleotide drug species, are capable of regulating mRNA expression and are commercially available.
  • the beneficial effects of the invention are as follows: the targeted transportation effect is good, and the effect of the medicine in the target tissue can be significantly improved; the natural product-sugar is basically harmless to the human body, and the toxic and side effects are small.
  • Figure 1 is the result of immunohistochemical analysis
  • Figure 2 is a quantitative analysis of the number of muscle fibers positive for dys trophin in the anterior humerus L section.
  • Figure 3 is an RT-PCR analysis chart.
  • FIG. 4 is a quantitative analysis of the transport effect of the four different antisense oligonucleotide drugs in Example 2.
  • FIG. 5 is a RT-PCR analysis diagram of the transport effect of the four different antisense oligonucleotide drugs in Example 2.
  • 6 is a quantitative analysis of the transport effects of s iRNA drugs capable of regulating GAPDH housekeeping gene expression in Examples 2, 4, 5, 6, 7, 11, and 12.
  • the above code F1 is physiological saline, which is a commonly used drug transport carrier, and other commonly used drug carriers also have a PBS solution, and the effect is consistent with physiological saline.
  • the ratio of the above Examples 10 and 11 is the mass ratio of glucose: fructose: sucrose.
  • the monosaccharides of Examples 1-9 and 12 are prepared by preparing a monosaccharide solution having a concentration of 50 mg/ml using a mother liquor of a pre-formulated sugar, and directly adding the drug to the monosaccharide solution according to the amount of administration. can.
  • glucose, fructose, and sucrose are required to be 30 mg/ml, 50 mg/ml, and 20 mg/min, respectively, in a ratio of 3:5:2. Ml. According to the ratio of 4:2:1, glucose, fructose and sucrose are required to be 57 mg/ml and 29 mg/mU 14 mg/ml, respectively.
  • P007 is a cell shuttle peptide, specifically disclosed in Human Molecular Genet i cs, 2008, Vol. 17, No. 24 3909-3918.
  • the dystrophin gene contains a nonsense point mutation in exon 23, which leads to the early termination of dystrophin (anti-a few atrophin) protein translation, thus unable to translate the meaningful dystrophin protein. Therefore, this study used mdx mice as test animals. Purchased from the Nanjing Model Animal Institute of China.
  • morpholino (PM0), was purchased from Gene Tools, USA.
  • Figure 1 is a graph showing the results of immunohistochemical analysis, in which a white grid appears as dystrophin-positive muscle fibers; c57 is a wild-type C57BL6 mouse, and it can be seen that after the PM0 transport using the drug of the embodiment of the present invention, small In the anterior tibial muscle tissue of the mouse, the dystrophin-positive muscle fibers increased significantly, further indicating that PM0 can significantly restore the expression level of dystrophin protein under the aid of the transport of this example, which can be visually compared with the physiological saline of F1.
  • the number of dystrophin-positive muscle fibers of Examples 6, 7, 10, 11, and 12 was significantly improved, and other examples achieved at least the same effect.
  • Fig. 2 is a quantitative analysis of the number of dystrophin-positive muscle fibers in the anterior tibia; L-face, and it can be seen that the number of dystrophin-positive muscle fibers in the above embodiment is greatly increased relative to physiological saline, indicating that the drug delivery auxiliary of the present invention has a large effect. Better than saline.
  • Figure 3 is a RT-PCR analysis diagram in which fuU-length indicates the PCR product not skipped; exon23 is the PCR product for exon 23 skipping; exon22 & 23 is the PCR for exon 22 and 23 skipping.
  • the product, C57 was a wild-type C57BL6 mouse and Ve was a negative control. It can be seen that in the case of transportation of the transport adjuvants of Examples 1-12, a significant exon skipping occurred, resulting in an increase in the expression of dys trophin-positive muscle fibers.
  • Example 1 In order to demonstrate the remarkable effect of the drug delivery adjuvant of the present invention in the transport of other antisense oligonucleotide drugs, a comparative test was carried out using Example 1 , F3, and physiological saline.
  • Figure 4 shows the quantitative analysis of dys trophin-positive genomic fibers.
  • the four groups of data show the transport effect of Example 2 with physiological saline on 2, ome RNA, PNA, PMO, P007-PMO, which is directly expressed as dys t rophin positive. As the number of muscle fibers increased, four sets of data were seen, and the effect of Example 2 of the present invention was significantly greater than that of physiological saline.
  • Fig. 5 is a diagram showing the results of RT-PCR of the effects of physiological saline and the above 2, ome RNA, PNA, PMO, and P007-PMO drugs, and it can be seen that antisense oligonucleotide drug-mediated exons The efficiency of skipping is significantly improved.
  • the applicant selected the GAPDH housekeeping gene for testing, and selected a class of s iRNA drugs which can down-regulate the expression level.
  • the sugar solutions used were Examples 2, 4, 5, 6, 7, 11, 12, and the basic experimental operations were the same as above.
  • Figure 6 is a quantitative analysis of GAPDH housekeeping gene expression levels. It can be seen that the illustrated examples have different degrees of down-regulation, especially in Example 2, i.e., F3 is the best, and this test expands the scope of application of the present invention.

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Abstract

Disclosed is a drug targeting delivery adjuvant, which is at least one selected from the group of pentoses selected from xylose and arabinose; hexoses selected from glucose, galactose, fructose and mannose; sugar alcohols selected from sorbitol, mannitol, erythritol, maltitol, lactitol and xylitol; disaccharide selected from sucrose, lactose, maltose and trehalose. Also disclosed is an application of the drug targeting delivery adjuvant in the delivery of drugs, in particular for antisense oligonucleotide drugs PMO, PNA, 2'ome RNA, peptide-PMO, siRNAs and macromolecule plasmids. The adjuvant has an effect of significantly improving the effect of a drug in a target tissue, and small toxic and side effects.

Description

说 明 书  Description
一种新型药物靶向运输辅助剂 技术领域  Novel drug targeted transportation auxiliary agent
本发明属于生命科学及药物载体领域。  The invention belongs to the field of life sciences and pharmaceutical carriers.
背景技术 Background technique
药物的靶向运输效率是决定药物药效的重要因素，现在常用来优化基因治疗 药物的方法有： 对药物本身结构进行优化； 使用大分子聚合物作为运输载体， 其中最常见的就是纳米材料； 组织靶向肽的使用， 即将肽段与药物共价结合。  The targeted transport efficiency of drugs is an important factor in determining the drug efficacy. The methods commonly used to optimize gene therapy drugs are: optimizing the structure of the drug itself; using macromolecular polymers as transport carriers, the most common of which are nanomaterials; The use of tissue targeting peptides is the covalent attachment of peptides to drugs.
其中药物载体的应用最为广泛， 但是现有的药物运输载体存在的不足之处 为：  Among them, drug carriers are the most widely used, but the shortcomings of existing drug delivery vehicles are:
1、 靶向运输效率低；  1. Low efficiency of targeted transportation;
2、 对人体存在潜在的危害。  2. Potential harm to the human body.
以杜兴肌肉萎缩症为例，杜兴肌肉萎缩症是一种与人类 X染色体连锁遗传的 致死性神经肌肉失调症， 主要症状表现为全身肌肉持续消耗和退化， 大多数病 人的寿命仅 20岁左右， 严重地危害人们的健康。  Taking Duxing Muscular Atrophy as an example, Duxing Muscular Dystrophy is a fatal neuromuscular disorder inherited from the human X chromosome. The main symptoms are continuous consumption and degeneration of the whole body muscle. Most patients have a life span of only 20 years old. Left and right, seriously endanger people's health.
疾病发生的原因是编码抗肌萎缩蛋白 dys trophin的 DMD基因发生突变， 造 成相应 mRNA 前体的拼接改变和读码框的破坏， 不能编码有生物学功能的 dys t rophin蛋白所致。 基因全长为 2. 4Mbp , 由 79个外显子组成， 除从亲本 遗传外， 其本身也存在着很高的自发突变几率。 在 基因的突变中， 65%的 突变是缺失突变， 这些突变都会造成 ?基因编码阅读框的改变， 从而产生不 稳定、 无功能活性的 dys trophin蛋白。 dys trophin蛋白的缺失会造成月 /1肉纤维 膜的不稳定和细胞外 Ca2+的大量涌入， 从而引起肌肉退化和萎缩消耗， 临床上表 现为严重的杜兴肌肉萎缩症。 The cause of the disease is the mutation of the DMD gene encoding the dystrophin dystrophin, which causes the splicing change of the corresponding mRNA precursor and the destruction of the reading frame, and cannot be encoded by the biological function of dys t rophin protein. The full-length gene is 2.4 Mbp, which consists of 79 exons. In addition to inheritance from the parents, it also has a high probability of spontaneous mutation. In the mutation of the gene, 65% of the mutations are deletion mutations, and these mutations will cause changes in the gene coding reading frame, resulting in unstable, non-functional dys trophin protein. Deletion of the dys trophin protein causes instability of the monthly/1 meat fiber membrane and a large influx of extracellular Ca 2+ , which causes muscle degeneration and atrophy, and is clinically characterized by severe Duchen muscle atrophy.
基因编码的 dys t rophin蛋白是由四个结构区域组成的，其中包括功能重 说 明 书 The gene-encoded dys t rophin protein is composed of four structural regions, including functional heavy Description
要的 N末端-即肌动蛋白结合区 （ac t in- b inding doma in )、 跨度很大的环状区 域（rod doma in ), 半胱氨酸富集区和 C末端-即 dys t rophin相关蛋白复合物结 合区。 研究表明 基因的突变热点主要集中在环状区域， 而环状区域对 dys t rophin蛋白的功能影响不大，所以其部分缺失对 dys trophin蛋白的功能影 响不明显。 这些都为通过强制性地调控 ?基因 mRNA前体拼接过程来对突变的 ?基因进行修复提供了理论基础。 The N-terminus - the actin-binding domain (ac t in- b inding doma in ), the large span of the rod doma in , the cysteine - rich region and the C - terminal - dys t rophin Related protein complex binding regions. Studies have shown that the mutation hotspots of genes are mainly concentrated in the circular region, while the circular region has little effect on the function of dys t rophin protein, so some of its deletions have no obvious effect on the function of dys trophin protein. These provide a theoretical basis for the repair of mutant genes by forcibly regulating the gene mRNA precursor splicing process.
最为典型的应用反义寡核苷酸调控 基因 mRNA前体拼接的过程是， 反义寡 核苷酸通过与靶向外显子及其邻近区域的结合， 阻止转录拼接复合体对靶向外 显子的识别， 从而将其从成熟的转录本切除掉， 完成跳读过程， 产生读码框正 确的转录本， 最终生成短的有生物活性的 dys t rophin 蛋白。 反义寡核苷酸介导 的外显子跳读方法在杜兴肌肉萎缩症的应用， 最早的证据来自于人类淋巴细胞 和 小鼠肌纤维细胞的体外试验， 这些实验结果证明了反义寡核苷酸介导的外 显子跳读方法的可行性。 目前反义寡核苷酸介导的外显子跳读已经成为杜兴肌 肉萎缩症基因治疗领域中最为成功、 最有应用前景的治疗方法之一。 并且已有 两种反义寡核苷酸进入临床 11 / 111期试验， 试验结果喜人。  The most typical application of antisense oligonucleotides to regulate gene mRNA precursor splicing is that antisense oligonucleotides prevent the transcriptional splicing complex from targeting to externalization by binding to targeted exons and adjacent regions. The recognition of the child, thereby excising it from the mature transcript, completes the skipping process, produces the correct transcript of the reading frame, and finally produces a short biologically active dys t rophin protein. Antisense oligonucleotide-mediated exon skipping in Duchen muscle atrophy, the earliest evidence comes from in vitro experiments with human lymphocytes and mouse myofibroblasts. These results demonstrate antisense oligo The feasibility of a glycoside-mediated exon skipping method. At present, antisense oligonucleotide-mediated exon skipping has become one of the most successful and promising treatments in the field of gene therapy for Duchenne muscular atrophy. And two antisense oligonucleotides have entered the clinical phase 11 / 111 trial, and the results are gratifying.
然而， 临床测试和临床前研究结果都表明 , 系统运输效率低是目前包括反 义寡核苷酸在内的各种基因治疗药物亟需解决的难题， 药物运输效率的高低是 决定其治疗效果的关键因素之一 , 也是目前基因治疗的瓶颈。 因此发展新的运 输系统， 提高药物运输效率是当务之急。  However, clinical trials and preclinical studies have shown that low systemic transport efficiency is an urgent problem to be solved for various gene therapy drugs including antisense oligonucleotides. The efficiency of drug transport is determined by its therapeutic effect. One of the key factors is also the bottleneck of current gene therapy. Therefore, it is imperative to develop new transportation systems and improve the efficiency of drug transportation.
发明内容 Summary of the invention
本发明的目的是提供一种靶向运输效率高、 制备简单、 副作用小的新型药 物靶向运输辅助剂。  SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel drug targeted transport adjuvant which is targeted for high transport efficiency, simple preparation, and low side effects.
为实现上述的发明目的， 本发明的技术方案如下： 说 明 书 In order to achieve the above object, the technical solution of the present invention is as follows: Instruction manual
一种新型药物靶向运输辅助剂， 为五碳糖、 六碳糖、 糖醇、 二糖中的至少 一类。  A novel drug-targeted transport adjuvant is at least one of five carbon sugars, six carbon sugars, sugar alcohols, and disaccharides.
所述五碳糖选自木糖、 阿拉伯糖中的至少一种； 所述六碳糖选自葡萄糖、 半乳糖、 果糖、 甘露糖中的至少一种； 所述二糖选自蔗糖、 乳糖、 麦芽糖、 海 藻糖中的至少一种； 所述糖醇选自山梨糖醇、 甘露醇、 赤藓糖醇、 麦芽糖醇、 乳糖醇、 木糖醇中的至少一种。  The five-carbon sugar is at least one selected from the group consisting of xylose and arabinose; the six-carbon sugar is at least one selected from the group consisting of glucose, galactose, fructose, and mannose; and the disaccharide is selected from the group consisting of sucrose and lactose. At least one of maltose and trehalose; and the sugar alcohol is at least one selected from the group consisting of sorbitol, mannitol, erythritol, maltitol, lactitol, and xylitol.
优选地， 使用如下重量分数组合： 葡萄糖、 果糖、 蔗糖 =3: 5： 2。  Preferably, the following weight fraction combination is used: glucose, fructose, sucrose = 3: 5: 2.
优选地， 使用如下重量分数组合： 葡萄糖、 果糖、 蔗糖 =4: 2： 1。  Preferably, the following weight fraction combination is used: glucose, fructose, sucrose = 4: 2: 1.
本发明还提供所述的一种新型药物靶向运输辅助剂的使用方法， 包括如下 步骤：  The invention also provides a method for using the novel drug targeted transportation auxiliary agent, which comprises the following steps:
1 )制备糖溶液： 按照比例配制糖溶液， 单糖浓度为 50mg/ml， 混合糖按照 比例混合， 总糖含量为 l OOmg/ml ;  1) Preparation of sugar solution: The sugar solution is prepared according to the ratio, the monosaccharide concentration is 50 mg/ml, the mixed sugar is mixed according to the ratio, and the total sugar content is l OOmg/ml;
2 )按照步骤 1中所述浓度的糖溶液运输药物， 按照要求剂量给药。  2) The drug is transported according to the concentration of the sugar solution described in step 1, and administered as required.
本发明所述的一种新型药物靶向运输辅助剂在运输药物中的应用。  The invention relates to a novel drug targeted transportation auxiliary agent used in transporting medicine.
一种新型药物靶向运输辅助剂在运输药物中的应用， 优选地， 用于靶向运 输反义寡核苷酸药物 PM0、 PNA、 2'ome RNA、 肽- PM0、 s iRNAs以及大分子质粒。  A novel drug-targeted transport adjuvant for use in transporting drugs, preferably for targeting antisense oligonucleotide drugs PM0, PNA, 2'ome RNA, peptide-PM0, s iRNAs, and macromolecular plasmids .
进一步地， 用于靶向运输治疗杜兴肌肉萎缩症的反义寡核苷酸药物 PM0 ( hosphorodi ami date mrphol ino ol igomer s )、 PNA、 2'ome RNA、月太- PMO 、 s iRNAs 以及大分子质粒。  Further, an antisense oligonucleotide drug PM0 (hosphorodi ami date mrphol ino ol igomer s ), PNA, 2'ome RNA, moon-PMO, s iRNAs and large for targeted transportation therapy Duchen muscle atrophy Molecular plasmid.
PMO为一种新型反义寡核苷酸， 抑制天然 RNA剪接过程， 产生各种 mRNA, 可抑制基因表达， 如抑制原癌基因的表达。 PM0与特异病毒 mRNA结合形成的双 联物可有效阻断病毒 RNA转录， 抑制病毒复制。 此化合物又具有很好的稳定性、 溶解度和细胞渗透性， 现已用于病毒感染、 癌症、 肌营养不良症和早老综合征 说 明 书 PMO is a novel antisense oligonucleotide that inhibits the natural RNA splicing process and produces various mRNAs that inhibit gene expression, such as inhibition of protooncogene expression. The doublet formed by the binding of PM0 to specific viral mRNA can effectively block viral RNA transcription and inhibit viral replication. This compound has good stability, solubility and cell permeability and is now used in viral infections, cancer, muscular dystrophy and premature aging syndrome. Description
治疗药物的研究中。  In the study of therapeutic drugs.
V ome RNA的全称为： 2'- 0-曱基化 RNA反义寡核苷酸， PNA为肽核酸， 肽 -PM0为连接了细胞穿梭肽或组织靶向肽的 PMO , s iRNA为一类双链 RNA分子， 上 述物质均为常见的反义寡核苷酸药物种类， 能够调控 mRNA的表达， 均可由市场 购得。  The full name of V ome RNA: 2'- 0-thiolated RNA antisense oligonucleotide, PNA is peptide nucleic acid, peptide-PM0 is PMO linked to cell shuttle peptide or tissue targeting peptide, s iRNA is a class Double-stranded RNA molecules, all of which are common antisense oligonucleotide drug species, are capable of regulating mRNA expression and are commercially available.
本发明的有益效果是： 靶向运输效果好， 能够显著提高药物在目标组织中的 作用效果； 取自天然产物一糖类， 对人体基本无伤害， 毒副作用小。  The beneficial effects of the invention are as follows: the targeted transportation effect is good, and the effect of the medicine in the target tissue can be significantly improved; the natural product-sugar is basically harmless to the human body, and the toxic and side effects are small.
附图说明  DRAWINGS
图 1为免疫组化分析结果图  Figure 1 is the result of immunohistochemical analysis
图 2为对前胫骨 L 切面 dys trophin阳性的肌纤维数的定量分析图 图 3 为 RT-PCR分析图  Figure 2 is a quantitative analysis of the number of muscle fibers positive for dys trophin in the anterior humerus L section. Figure 3 is an RT-PCR analysis chart.
图 4为实施例 2对四种不同反义寡核苷酸药物的运输效果定量分析图 图 5为实施例 2对四种不同反义寡核苷酸药物的运输效果的 RT-PCR分析图 图 6为实施例 2、 4、 5、 6、 7、 11、 12对能够调控 GAPDH持家基因表达的 s iRNA药物的运输效果定量分析图  4 is a quantitative analysis of the transport effect of the four different antisense oligonucleotide drugs in Example 2. FIG. 5 is a RT-PCR analysis diagram of the transport effect of the four different antisense oligonucleotide drugs in Example 2. 6 is a quantitative analysis of the transport effects of s iRNA drugs capable of regulating GAPDH housekeeping gene expression in Examples 2, 4, 5, 6, 7, 11, and 12.
具体实施方式  detailed description
以下对本发明的原理和特征进行描述， 所举实例只用于解释本发明， 并非 用于限定本发明的范围。  The principles and features of the present invention are described below, and the examples are intended to be illustrative only and not to limit the scope of the invention.
说明  Description
下表为实施例 1-12选用的糖  The following table shows the sugars selected in Examples 1-12.
Figure imgf000005_0001
说 明 书
Figure imgf000005_0001
Instruction manual
上述代码 F1为生理盐水， 为常用的药物运输载体， 其他常用的药物载体还 有 PBS溶液等， 效果与生理盐水一致。  The above code F1 is physiological saline, which is a commonly used drug transport carrier, and other commonly used drug carriers also have a PBS solution, and the effect is consistent with physiological saline.
上述实施例 10和 11的比例为葡萄糖： 果糖： 蔗糖的质量比。  The ratio of the above Examples 10 and 11 is the mass ratio of glucose: fructose: sucrose.
所述实施例的新型药物靶向运输辅助剂的使用方法为：  The method of using the novel drug-targeted transport adjuvant of the embodiment is:
如实施例 1-9及 12的单糖的制备方法为： 使用预先配制好的糖的母液配制 得到浓度为 50mg/ml的单糖溶液， 按照给药量将药物直接加入该单糖溶液中即 可。  The monosaccharides of Examples 1-9 and 12 are prepared by preparing a monosaccharide solution having a concentration of 50 mg/ml using a mother liquor of a pre-formulated sugar, and directly adding the drug to the monosaccharide solution according to the amount of administration. can.
对于实施例 10、 11的混合糖溶液，要达到 100mg/ml的总糖浓度，则按照 3: 5： 2的比例， 需要葡萄糖、 果糖、 蔗糖分别为 30mg/ml、 50mg/ml、 20mg/ml。 按照 4: 2： 1的比例， 则需要葡萄糖、 果糖、 蔗糖分别为 57 mg/ml、 29 mg/mU 14 mg/ml。 For the mixed sugar solutions of Examples 10 and 11, to achieve a total sugar concentration of 100 m g / ml, glucose, fructose, and sucrose are required to be 30 mg/ml, 50 mg/ml, and 20 mg/min, respectively, in a ratio of 3:5:2. Ml. According to the ratio of 4:2:1, glucose, fructose and sucrose are required to be 57 mg/ml and 29 mg/mU 14 mg/ml, respectively.
使用的几大类药物， 具体序列为：  The major classes of drugs used, the specific sequence is:
PM0 5' ggccaaacc tcggct tacctgaaa 13  PM0 5' ggccaaacc tcggct tacctgaaa 13
PNA 5' ggccaaacc tcggct tacct 3'  PNA 5' ggccaaacc tcggct tacct 3'
2' OMe RNA 5' GGCCAAACCUCGGCUUACCU3'  2' OMe RNA 5' GGCCAAACCUCGGCUUACCU3'
P007为一段细胞穿梭肽，具体披露在 Human Molecular Genet i cs , 2008, Vol. 17, No. 24 3909-3918中。  P007 is a cell shuttle peptide, specifically disclosed in Human Molecular Genet i cs, 2008, Vol. 17, No. 24 3909-3918.
试验一  Test one
针对所述实施例的药物靶向运输辅助剂在运输药物中的应用， 尤其是运输 治疗杜兴肌肉萎缩症的反义寡核苷酸药物 PM0中的作用 , 进行了下述实验：  For the application of the drug-targeted transport adjuvant of the above embodiment in transporting drugs, particularly the role of the antisense oligonucleotide drug PM0 for the treatment of Duchen muscle atrophy, the following experiments were carried out:
1材料和方法：  1 Materials and methods:
1 ) Mdx小鼠：  1) Mdx mice:
目前杜兴肌肉萎缩症基因治疗研究中最为常用的动物模型， 在其 说 明 书 Currently the most commonly used animal model for Duchen Muscular Dystrophy gene therapy research, in its Description
dystrophin基因外显子 23上含有一个无义点突变， 导致 dystrophin (抗几萎 缩蛋白）蛋白翻译提前终止， 从而不能翻译有意义的 dystrophin蛋白， 因此本 研究以 mdx小鼠为测试动物， 该小鼠购自中国南京模式动物研究所。 The dystrophin gene contains a nonsense point mutation in exon 23, which leads to the early termination of dystrophin (anti-a few atrophin) protein translation, thus unable to translate the meaningful dystrophin protein. Therefore, this study used mdx mice as test animals. Purchased from the Nanjing Model Animal Institute of China.
2) 测试药物：  2) Test drugs:
以临床试验中的一种反义寡核苷酸药物- morpholino ( PM0 ) 为测试药物， 该药物购自美国 Gene Tools公司。  One of the antisense oligonucleotide drugs in clinical trials, morpholino (PM0), was purchased from Gene Tools, USA.
3 )试验方法：  3) Test method:
对不同的糖类化合物在小鼠的前胫骨肌运输 PM0的效率进行试验。  The efficiency of PM0 transport of different saccharide compounds in the tibialis anterior muscle of mice was tested.
将 2ug PM0分别与实施例 1-12的一定浓度的糖溶液混合， 局部注射到 mdx 小鼠的前胫骨肌中， 单次注射， 2周后， 收取注射过的前胫骨肌组织， 通过免疫 组化一细胞核染色 （scale bar=200um), RT- PCR检测 PM0介导的外显子跳读效 率和恢复 dystrophin蛋白的表达水平， 通过与对照溶液-生理盐水的比较， 确 定不同糖溶液对 PM0的运输效率的提高作用。  2ug PM0 was mixed with a certain concentration of sugar solution of Example 1-12, and injected locally into the anterior tibialis anterior muscle of mdx mice. After a single injection, 2 weeks later, the injected anterior tibial muscle tissue was collected and passed through the immunization group. A nuclear staining (scale bar=200um), RT-PCR detection of PM0-mediated exon skipping efficiency and recovery of dystrophin protein expression level, by comparison with the control solution - physiological saline, determine the different sugar solutions for PM0 The effect of improving transportation efficiency.
图 1为免疫组化分析结果图， 其中白色网格状显示为 dystrophin阳性的肌 纤维； c57为野生型的 C57BL6小鼠， 可以看到， 使用本发明所述实施例的药物 进行 PM0运输后， 小鼠的前胫骨肌组织中 , dystrophin阳性的肌纤维大量增加， 进一步表明 PM0在本实施例的运输辅助下， 能够显著恢复 dystrophin蛋白的表 达水平， 与 F1 的生理盐水相比， 可以直观地看到， 实施例 6、 7、 10、 11、 12 的 dystrophin阳性肌纤维数量有了明显的提高， 其他实施例也达到了至少等同 的效果。  Figure 1 is a graph showing the results of immunohistochemical analysis, in which a white grid appears as dystrophin-positive muscle fibers; c57 is a wild-type C57BL6 mouse, and it can be seen that after the PM0 transport using the drug of the embodiment of the present invention, small In the anterior tibial muscle tissue of the mouse, the dystrophin-positive muscle fibers increased significantly, further indicating that PM0 can significantly restore the expression level of dystrophin protein under the aid of the transport of this example, which can be visually compared with the physiological saline of F1. The number of dystrophin-positive muscle fibers of Examples 6, 7, 10, 11, and 12 was significantly improved, and other examples achieved at least the same effect.
图 2为前胫骨月; L 切面 dystrophin阳性的肌纤维数的定量分析图， 可以看 到上述实施例的 dystrophin阳性的肌纤维数相对生理盐水有大幅度增加， 表明 本发明的药物运输辅助剂的效果大大优于生理盐水。 说 明 书 Fig. 2 is a quantitative analysis of the number of dystrophin-positive muscle fibers in the anterior tibia; L-face, and it can be seen that the number of dystrophin-positive muscle fibers in the above embodiment is greatly increased relative to physiological saline, indicating that the drug delivery auxiliary of the present invention has a large effect. Better than saline. Instruction manual
图 3为 RT- PCR分析图， 图中 fuU- length表示未跳读的 PCR产物； exon23 表示为外显子 23跳读的 PCR产物； exon22 & 23为外显子 22和 23都跳读的 PCR 产物， C57为野生型的 C57BL6小鼠， Ve为阴性对照。 可以看到在实施例 1-12的 运输辅助剂进行运输的情况下， 发生了很明显的外显子跳读， 从而导致 dys t rophin阳性的肌纤维表达量增加。  Figure 3 is a RT-PCR analysis diagram in which fuU-length indicates the PCR product not skipped; exon23 is the PCR product for exon 23 skipping; exon22 & 23 is the PCR for exon 22 and 23 skipping. The product, C57, was a wild-type C57BL6 mouse and Ve was a negative control. It can be seen that in the case of transportation of the transport adjuvants of Examples 1-12, a significant exon skipping occurred, resulting in an increase in the expression of dys trophin-positive muscle fibers.
试验二  Test 2
为了证明本发明的药物运输辅助剂在运输其他反义寡核苷酸药物中的显著 作用 , 使用实施例 1即 F3与生理盐水进行了对比试验。  In order to demonstrate the remarkable effect of the drug delivery adjuvant of the present invention in the transport of other antisense oligonucleotide drugs, a comparative test was carried out using Example 1 , F3, and physiological saline.
图 4为 dys trophin阳性的月几纤维的定量分析， 四组数据分别表示实施例 2 与生理盐水对 2，ome RNA、 PNA、 PMO , P007-PMO 的运输效果， 直接表现为 dys t rophin 阳性的肌纤维的数量增加， 可以看到四组数据， 使用本发明的实施 例 2后效果均比生理盐水有大幅增加。  Figure 4 shows the quantitative analysis of dys trophin-positive genomic fibers. The four groups of data show the transport effect of Example 2 with physiological saline on 2, ome RNA, PNA, PMO, P007-PMO, which is directly expressed as dys t rophin positive. As the number of muscle fibers increased, four sets of data were seen, and the effect of Example 2 of the present invention was significantly greater than that of physiological saline.
图 5为生理盐水与实施例 2对上述 2，ome RNA、 PNA、 PMO , P007-PMO四种药 物运输效果的 RT- PCR结果图， 可以看出反义寡核苷酸药物介导外显子跳读效率 明显提高。  Fig. 5 is a diagram showing the results of RT-PCR of the effects of physiological saline and the above 2, ome RNA, PNA, PMO, and P007-PMO drugs, and it can be seen that antisense oligonucleotide drug-mediated exons The efficiency of skipping is significantly improved.
试验三  Trial three
为了证明本发明的运输辅助剂对治疗其他疾病的其他类型药物的运输效 果， 申请人选用 GAPDH持家基因进行了试验， 选用一类可以下调其表达水平的 s iRNA药物进行试验。 使用的糖溶液为实施例 2、 4、 5、 6、 7、 11、 12， 基本实 验操作同上。 图 6为 GAPDH持家基因表达水平定量分析图， 可以看到， 图示的 实施例均有不同程度的下调， 尤以实施例 2即 F3效果最佳， 这一试验扩大了本 发明的应用范围。  In order to demonstrate the transport effect of the transport adjuvant of the present invention on other types of drugs for treating other diseases, the applicant selected the GAPDH housekeeping gene for testing, and selected a class of s iRNA drugs which can down-regulate the expression level. The sugar solutions used were Examples 2, 4, 5, 6, 7, 11, 12, and the basic experimental operations were the same as above. Figure 6 is a quantitative analysis of GAPDH housekeeping gene expression levels. It can be seen that the illustrated examples have different degrees of down-regulation, especially in Example 2, i.e., F3 is the best, and this test expands the scope of application of the present invention.
以上所述仅为本发明的较佳实施例， 并不用于限制本发明， 凡在本发明的 说 明 书 The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and is in the present invention. Description
精神和原则之内， 所做的任何修改和等同替换， 均应包含在本发明的保护范围 之内。 Any modifications and equivalent substitutions made within the spirit and principles are intended to be included within the scope of the invention.

Claims

权 利 要 求 书 claims
1、 一种新型药物靶向运输辅助剂， 其特征在于： 为五碳糖、 六碳糖、 糖醇、 二糖中的至少一类。 1. A new type of drug targeted transport auxiliary agent, characterized in that: it is at least one type of five-carbon sugar, six-carbon sugar, sugar alcohol, and disaccharide.
2、 根据权利要求 1 所述的一种新型药物靶向运输辅助剂， 其特征在于： 所述 五碳糖选自木糖、 阿拉伯糖中的至少一种； 所述六碳糖选自葡萄糖、 半乳糖、 果糖、 甘露糖中的至少一种； 所述二糖选自蔗糖、 乳糖、 麦芽糖、 海藻糖中的 至少一种； 所述糖醇选自山梨糖醇、 甘露醇、 赤藓糖醇、 麦芽糖醇、 乳糖醇、 木糖醇中的至少一种。 2. A new type of drug targeted transport auxiliary according to claim 1, characterized in that: the five-carbon sugar is selected from at least one of xylose and arabinose; the six-carbon sugar is selected from glucose, At least one of galactose, fructose, and mannose; The disaccharide is selected from at least one of sucrose, lactose, maltose, and trehalose; The sugar alcohol is selected from sorbitol, mannitol, and erythritol , at least one of maltitol, lactitol, and xylitol.
3、 根据权利要求 2所述的一种新型药物靶向运输辅助剂， 其特征在于： 优选 地， 使用如下重量分数组合： 葡萄糖、 果糖、 蔗糖 =3: 5： 2。 3. A new type of drug targeted transport auxiliary according to claim 2, characterized in that: Preferably, the following weight fraction combination is used: glucose, fructose, sucrose =3:5:2.
4、 根据权利要求 2所述的一种新型药物靶向运输辅助剂， 其特征在于： 优选 地， 使用如下重量分数组合： 葡萄糖、 果糖、 蔗糖 =4: 2： 1。 4. A new type of drug targeted transport auxiliary according to claim 2, characterized in that: Preferably, the following weight fraction combination is used: glucose, fructose, sucrose =4: 2: 1.
5、根据权利要求 1-4任一项所述的一种新型药物靶向运输辅助剂的使用方法， 其特征在于： 包括如下步骤： 5. The method of using a new type of drug targeted transport auxiliary according to any one of claims 1 to 4, characterized in that: it includes the following steps:
1 )制备糖溶液： 按照比例配制糖溶液， 单糖浓度为 50mg/ml , 混合糖按照比例 混合， 总糖含量为 l OOmg/ml ; 1) Prepare the sugar solution: Prepare the sugar solution according to the proportion, the monosaccharide concentration is 50 mg/ml, and mix the mixed sugar according to the proportion, the total sugar content is 100 mg/ml;
2 )按照步骤 1中所述浓度的糖溶液运输药物， 按照要求剂量给药。 2) Transport the drug in a sugar solution of the concentration described in step 1 and administer the drug in the required dosage.
6、 根据权利要求 1-4任一项所述的一种新型药物靶向运输辅助剂在运输药物 中的应用。 6. Application of a new type of drug targeted transport auxiliary agent in transporting drugs according to any one of claims 1-4.
7、 根据权利要求 1-4任一项所述的一种新型药物靶向运输辅助剂在运输药物 中的应用， 其特征在于： 优选地， 用于靶向运输反义寡核苷酸药物 PM0、 PNA、 2'ome RNA、 肽- PM0、 s iRNAs以及大分子质粒。 7. Application of a new type of drug targeted transport auxiliary in transporting drugs according to any one of claims 1 to 4, characterized in that: Preferably, it is used for targeted transport of antisense oligonucleotide drugs PMO , PNA, 2'ome RNA, peptide-PM0, siRNAs and macromolecular plasmids.
权 利 要 求 书 Rights demand letter
8、 根据权利要求 1-4任一项所述的一种新型药物靶向运输辅助剂在运输药物 中的应用， 其特征在于： 优选地， 用于靶向运输治疗杜兴月几肉萎缩症和其他月几 肉相关疾病的反义寡核苷酸药物 PM0、 PNA、 2，ome RNA、 肽- PM0、 s i RNAs以及 大分子质粒。 8. Application of a new type of drug targeted transport auxiliary in transporting drugs according to any one of claims 1 to 4, characterized in that: Preferably, it is used for targeted transport in the treatment of Duchenne atrophy. Antisense oligonucleotide drugs PM0, PNA, 2,ome RNA, peptide-PM0, si RNAs and macromolecular plasmids for several meat-related diseases.
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