WO2019080284A1 - 一种药物作用靶点的组合物和应用 - Google Patents

一种药物作用靶点的组合物和应用

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WO2019080284A1
WO2019080284A1 PCT/CN2017/115764 CN2017115764W WO2019080284A1 WO 2019080284 A1 WO2019080284 A1 WO 2019080284A1 CN 2017115764 W CN2017115764 W CN 2017115764W WO 2019080284 A1 WO2019080284 A1 WO 2019080284A1
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fkbp10
pcolce
drug
composition
genes
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PCT/CN2017/115764
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English (en)
French (fr)
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黄晓璐
梁筱
李青峰
柴邦达
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上海交通大学医学院附属第九人民医院
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Priority to US16/759,362 priority Critical patent/US12006501B2/en
Publication of WO2019080284A1 publication Critical patent/WO2019080284A1/zh

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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y502/00—Cis-trans-isomerases (5.2)
    • C12Y502/01—Cis-trans-Isomerases (5.2.1)
    • C12Y502/01008—Peptidylprolyl isomerase (5.2.1.8), i.e. cyclophilin

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a composition and application of a drug target.
  • Pathological scar is a skin fibrosis disease that exists in the world, and its incidence varies in different races. Previous reports have reported that the incidence of pathological scars in the Caucasian population is 5% to 37%. The incidence of patients after routine surgery is 36 to 64%, and the incidence in deep burn patients can be as high as 91%. Pathological scars are caused by traumatic inflammatory reactions, such as uncontrolled proliferation of fibroblasts (Fb) and excessive production and deposition of extracellular matrix (ECM) such as collagen. Metabolic diseases, clinically manifested as scar tissue above the surface of the wound that does not exceed the wound surface, early congestion is obvious, may be accompanied by pain or itching, and the joint site may cause activity dysfunction.
  • Fb fibroblasts
  • ECM extracellular matrix
  • the pathogenesis of pathological scars is still under investigation.
  • the main pathogenic factors involved include abnormal ECM metabolism, cytokines (such as TGF- ⁇ , platelet-derived growth factor and insulin-like growth factor), hypoxia and free radicals, and genes. Level changes, etc., in which ECM metabolic abnormalities are the most important pathogenic factors.
  • cytokines such as TGF- ⁇ , platelet-derived growth factor and insulin-like growth factor
  • hypoxia and free radicals and genes.
  • levels changes, etc. in which ECM metabolic abnormalities are the most important pathogenic factors.
  • the synthesis and decomposition of ECM in the human body is in a dynamic equilibrium, thus maintaining relatively stable.
  • pathological scars or keloids may form. Therefore, inhibition of excessive deposition of collagen in ECM can be considered as an important target for the prevention and treatment of pathological scars.
  • FKBP10 is an immunophilin belonging to the immunophilin FKBP subfamily, which localizes to the protein on the rough endoplasmic reticulum of fibroblasts and is an enzyme involved in the post-transcriptional modification of type I collagen, which can assist The precursor collagen folds to form mature type I collagen, and is positively correlated with the secretion of type I collagen.
  • the mutation of FKBP10 in human body is closely related to osteogenic imperfect (OI). Due to the lack of FKBP10, the patient lacks the synthesis and secretion of type I collagen in skin fibroblasts and bone tissue, thus showing skin fragility and lack of elasticity. A series of hoof tissue related symptoms such as easy fracture and key flexion difficulty, and FKBP10 expression in human pathological scar is significantly higher than normal skin.
  • PCOLCE is an enzyme that binds to type I precursor collagen and cleaves and modifies it. After C-type precursor collagen was cleaved by PCOLCE, its C-terminal protease activity increased significantly, and N-terminal and C-terminal cleavage were completed, thereby completing collagen III. Polymerization, polymerization to form mature collagen fibers. PCOLCE is closely related to the degradation of extracellular matrix and is significantly higher in hypertrophic scar than normal skin.
  • glucocorticoid treatment can produce skin atrophy, hypopigmentation, telangiectasia, female menstrual disorders, injection site ulceration or calcification and other complications; laser therapy, clinical efficiency is low, easy to induce new scars; Anti-tumor drug treatment has a great impact on normal cell metabolism, and its clinical value is still not clear; tacrolimus, statins, tamoxifen and other drugs have limited clinical efficacy, and the therapeutic target is not yet clear.
  • the technical problem to be solved by the present invention is to provide a composition and application of a drug target, which inhibits the formation of tissue fibrosis by interfering with FKBP10 and PCOLCE, inhibits proliferation, differentiation and anti-aging of skin fibroblasts.
  • the biological behaviors such as death and secretion of a large amount of extracellular matrix are achieved, thereby achieving the effect of treating fibrosis-related diseases.
  • a composition for medicinal action of the present invention comprises a vector and a drug having a FKBP10 and PCOLCE gene and/or a protein encoded thereby as a drug target.
  • the drug having the FKBP10 and PCOLCE genes and/or a protein encoded thereby as a drug target is an inhibitor of the FKBP10 and PCOLCE genes and/or a protein encoded thereby.
  • the drug targeting the FKBP10 and PCOLCE genes and/or the protein encoded thereby as a drug target includes, but is not limited to, a neutralizing antibody, a small molecule drug, which interferes with the expression and function of the FKBP10 and PCOLCE genes and/or their encoded proteins, Small nucleotides, tiny ribonucleic acids, antisense molecules or polypeptides.
  • the intervention involves the simultaneous intervention of the FKBP10 and PCOLCE genes and/or the proteins encoded thereby, or the intervention of the FKBP10 gene and/or its encoded protein followed by the PCOLCE gene and/or its encoded protein.
  • the administration of the FKBP10 and PCOLCE genes and/or the protein encoded thereby as a drug target is administered by local injection or smear.
  • Such vectors include, but are not limited to, histidine polypeptides, lysine polypeptides, branched histidine-lysine copolymer polypeptides, cationic polymers, silicon nanoparticles, lipid carriers, or viral vectors.
  • Other pharmaceutically acceptable carriers are acceptable.
  • composition of a drug action target of the present invention is characterized in that the composition is used for the preparation of a medicament for treating a fibrosis-related disease.
  • the present invention inhibits the formation of tissue fibrosis by interfering with FKBP10 and PCOLCE, and inhibits skin fibrillation.
  • Biological effects such as cell proliferation, differentiation, anti-apoptosis, and secretion of a large amount of extracellular matrix, thereby achieving effects of treating fibrosis-related diseases, such as pathological scars;
  • the drug according to the present invention has a clear therapeutic target relative to tacrolimus, a statin, a tamoxifen, and the like, and avoids disadvantages such as unclear therapeutic mechanism, low efficiency, and easy side effects;
  • the drug of the present invention circumvents a series of side effects that the hormone may bring with respect to the glucocorticoid drug
  • the drug according to the present invention circumvents the possibility of damaging normal cells of the human body relative to antitumor drugs and radiation therapy;
  • the present invention circumvents the disadvantages of shallow treatment depth and the possibility of inducing new scar formation
  • the medicament of the invention can treat a large area of pathological scar and avoid the defect of small treatment range;
  • the medicament of the present invention can be treated after pathological scar formation, circumventing the limitation that only early prevention can be avoided;
  • the drug according to the present invention has a significant cost advantage over the current drug, and the synthesis cost is low.
  • Figure 1 is a comparison of the expression of FKBP10 and PCOLCE in human hypertrophic scars and normal skin;
  • Figure 2 shows that knockdown of FKBP10 and/or PCOLCE expression inhibits the expression of fibrotic markers in human hypertrophic scar fibroblasts
  • Figure 3 is a graph showing that small nucleotide interference against human FKBP10 and/or PCOLCE inhibits the activity of human hypertrophic scar fibroblasts;
  • Figure 4 shows that small nucleotide interference against mouse FKBP10 and/or PCOLCE can inhibit the formation of hypertrophic scars in mice; among them, A: human hypertrophic scar and normal skin HE staining; B: mouse model in each intervention mode Lower HE staining; C: Masson staining in mouse model in each intervention mode; D: Statistical analysis of collagen content in Masson staining in mouse model;
  • Figure 5 is a small nucleotide interference against mouse FKBP10 and/or PCOLCE which inhibits the expression of mouse skin fibrosis markers.
  • FKBP10 and PCOLCE genes and/or their encoded proteins there are many ways to intervene in the expression and function of the FKBP10 and PCOLCE genes and/or their encoded proteins, such as neutralizing antibodies, small molecule drugs, small nucleotides, microRNAs, polypeptides, etc., in this example, small nucleosides
  • acid drugs human hypertrophic scar fibroblasts (HHSF) were used as target cells, and it was confirmed that intervention of FKBP10 and PCOLCE expression can inhibit the activity of HHSF and reduce the expression of fibrosis-related markers.
  • HHSF human hypertrophic scar fibroblasts
  • Small nucleotide injections on the local skin confirmed that interference with FKBP10 and PCOLCE inhibited the formation of hypertrophic scars in mice.
  • RNA interference refers to the endogenous or exogenous double-stranded RNA (dsRNA)-mediated degradation of intracellular mRNA, which leads to the silencing of target gene expression and the corresponding loss of functional phenotype. .
  • Antisense strand 5'-AAUGGAGGGAUGAUAAUCUUCCUUC-3';
  • mouse FKBP10 mRNA Its sequence for mouse FKBP10 mRNA is:
  • Antisense strand 5'-AUAGGUAUUGUAGGUGUGGTT-3';
  • Antisense strand 5'-GGAAGGAGGUCUCUCGAAA-3';
  • Antisense strand 5'-GGACCGUUGGUUCACUGAA-3'.
  • the histidine-lysine polymer (HKP) which has been used as a siRNA transfection vector has a lysine skeleton including a branch containing a plurality of recombinant amino acids, lysine or aspartic acid.
  • HKP was dissolved in DEPC water to prepare a DEPC aqueous solution, and then mixed with an aqueous solution of siRNA (purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.) at a mass ratio of 4:1 and a volume ratio of 1:1 to form an average diameter of 150-200 nm. Nanoparticles.
  • the HKP-siRNA aqueous solution is translucent and has no significant accumulation of precipitates and can be stored at 4 ° C for at least three months.
  • siRNA aqueous solution and HKP aqueous solution were prepared, and then mixed with siRNA aqueous solution at a mass ratio of 4:1 and a volume ratio of 1:1 to form nanoparticles with an average diameter of 150-200 nm.
  • the HKP-siRNA aqueous solution is translucent and has no significant accumulation of precipitates and can be stored at 4 ° C for at least three months.
  • Clinical specimens human hypertrophic scars, keloids or normal skin
  • Dispase II (2 mg/ml, Life Technologies, ThermoFisher)
  • ThermoFisher overnight at 4 ° C
  • the tissue was minced in a sterile environment, soaked with 4 mg/ml collagenase, digested at 37 ° C for 2-4 hours on a shaker, filtered to filter the cell suspension, centrifuged at 1500 rpm for 5 min, the supernatant was removed, and the cell pellet was resuspended in medium and inoculated.
  • DMEM medium Change the fluid every 2 days. Regular passage.
  • mice in the control group, the FKBP10 siRNA alone administration group, and the PCOLCE siRNA alone administration group were injected every 3 days until the 56th day of the model establishment.
  • the FKBP10 siRNA+PCOLCE siRNA was administered at the same time.
  • the final concentration of FKBP10 siRNA was 200 ⁇ g/ml, and the final concentration of PCOLCE siRNA was 50 ⁇ g/ml.
  • the two were mixed with HKP to make HKP-siRNA solution and injected once every 3 days. , until the model is established on the 56th day.
  • FKBP10 siRNA+(24h) PCOLCE siRNA administration group FKBP10 siRNA (200 ⁇ g/ml) was injected on the first day, and PCOLCE siRNA (50 ⁇ g/ml) was injected 24h later. The two drugs were alternately injected every 3 days until The model was built on the 56th day.
  • the cells are exchanged and the HKP-siRNA aqueous solution is added (method refers to Section 1.2).
  • FKBP10 siRNA+PCOLCE siRNA was used in the cells of the intervention group.
  • the final concentration of FKBP10 siRNA in the culture medium was 50 nM, and the final concentration of PCOLCE siRNA was 25 nM.
  • the two were mixed with HKP to make HKP-siRNA solution, and then mixed in the culture medium for 24 hours.
  • FKBP10 siRNA+(24h) cells of PCOLCE siRNA intervention group were cultured with FKBP10 siRNA at a final concentration of 50 nM. After 24 h, the cells were cultured with PCOLCE siRNA at a final concentration of 50 nM, and cultured for 24 h.
  • RNA of tissues or cells was extracted with Trizol (Invitrogen, Grand Island, NY, USA), and RNA concentration and purity were measured by ultraviolet spectrophotometry (ND-1000, Thermo, Rockford, IL, USA).
  • the extracted total RNA was subjected to reverse transcription reaction using an RT-PCR kit (TaKaRa, Shiga, Japan) and an ABI HT7900 PCR instrument (Applied Biosystems, Foster City, CA, USA) to synthesize cDNA.
  • MIX SYBR Premix Ex Taq
  • Upstream primer 0.4 ⁇ l
  • reaction tubes were ligated, mixed by brief centrifugation, and placed in a PCR reactor (Applied Biosystems). Reaction conditions: After 10 s hot start at 95 ° C, the reaction was carried out for 40 cycles: 95 ° C for 15 s, 60 ° C for 30 m, and 72 ° C for 30 m. After the reaction, the results were analyzed by the ⁇ CT Relative Quantitation method using analytical software. Three parallel sub-wells were made for each sample and GADPH primers were used as internal controls.
  • Clinical specimens and animal tissues were routinely fixed, dehydrated, and paraffin-embedded, and 6 ⁇ m sections were prepared. After dewaxing, the sections were blocked with 1% goat serum and incubated overnight at 4 °C. After washing the sections with BSA, they were incubated with a horseradish peroxidase or a fluorescently labeled secondary antibody and incubated at 37 ° C for 2 hours. Develop color and take a photo.
  • the CCK-8 kit was purchased from Dojindo (Tokyo, Japan) and the experimental procedure was carried out in strict accordance with the kit instructions.
  • Human hypertrophic scar fibroblasts are isolated and cultured.
  • the RNAi technique was used to knock down FKBP10 and/or PCOLCE in human hypertrophic scar fibroblasts, and real-time quantitative PCR (q-PCR) was used for fibrosis markers COL1A, COL3A, FN, ⁇ -SMA and TGF- ⁇ 1.
  • the mRNA level is detected.
  • q-PCR results showed that knockdown of FKBP10 and/or PCOLCE significantly reduced fibrosis markers COL1A, COL3A, FN, ⁇ -SMA and TGF- ⁇ 1 Expression (p ⁇ 0.001).
  • the cells treated by these two methods had lower proliferation activity than cells interfering with FKBP10 or PCOLCE alone, ie interfering with FKBP10 and PCOLCE targets.
  • the effect of the point is better than the effect of one of the two targets alone ( Figure 3).
  • a classical mouse hypertrophic scar model was established to mimic the pathological changes in human hypertrophic scars (Fig. 4A-C).
  • HE and Masson staining were performed on the scars of the mice after the samples were taken.
  • the results showed that after FKBP10 and/or PCOLCE intervention, the characteristics of skin hypertrophic scars (characteristics: thickening of the epidermis and dermis, reduction of skin accessory organs, collagen enlargement and disorder, etc.) were significantly inhibited (p ⁇ 0.001).
  • simultaneous interference with FKBP10, PCOLCE two targets and interference with FKBP10 and interference with PCOLCE target after 24h the effect of the two methods was significantly better than the effect of FKBP10 or PCOLCE alone (p ⁇ 0.05).
  • FKBP10 and/or PCOLCE The expression of FKBP10 and/or PCOLCE in the mouse model was interfered with by small nucleotides, and the resulting skin samples were subjected to q-PCR detection.
  • simultaneous interference with FKBP10, PCOLCE two targets and interference with FKBP10 and interference with PCOLCE target after 24h the expression of fibrotic markers in the skin treated by these two methods is lower than that of the fibers in the skin of FKBP10 or PCOLCE alone.
  • the expression of the marker i.e., the effect of interfering with the FKBP10 and PCOLCE dual targets, was superior to the effect of one of the two targets alone (Fig. 5).

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Abstract

本发明公开了一种药物作用靶点的组合物和应用，组合物包括载体和以FKBP10及PCOLCE基因和/或其编码的蛋白作为药物作用靶点的药物。本发明系通过干预FKBP10及PCOLCE以抑制组织纤维化的形成，抑制皮肤成纤维细胞度增殖、分化、抗凋亡、分泌大量细胞外基质等生物学行为，从而达到治疗纤维化相关疾病的效果。

Description

一种药物作用靶点的组合物和应用 技术领域
本发明属于生物技术领域，特别涉及一种药物作用靶点的组合物和应用。
背景技术
病理性瘢痕是存在于全世界范围内的一种皮肤纤维化疾病，在不同种族中其发病率有所差异，以往的报道提到白种人群中的病理性瘢痕发病率在5％～37％，常规手术后病人中的发病率为36～64％，而在深度烧伤病人中的发病率可高达91％。病理性瘢痕是由创伤炎性反应等引起，以成纤维细胞(fibroblast,Fb)增殖失控和胶原等大量细胞外基质(extra cellular matrix,ECM)过度产生和沉积为特征的人类真皮区特有的纤维代谢性疾病，在临床上表现为不超过伤口范围的高出皮肤表面的瘢痕组织，早期充血明显，可伴有疼痛或瘙痒，位于关节部位则可能会引起活动功能障碍。病理性瘢痕产生的不雅外观和不适症状让患者身心都承受着巨大的压力，预防瘢痕不仅是医学问题，也是一个社会问题，其防治对临床工作者来说更是一个挑战。目前病理性瘢痕的治疗和预防方法有多种，主要集中在上皮覆盖创面后，在瘢痕形成前和尚未成熟的阶段尽量去除各种造成瘢痕增生的因素，防止瘢痕对机体造成各种畸形和功能障碍，包括皮质类固醇激素、他克莫司、他汀类药物、他莫昔芬、激光、放射治疗、压力预防、硅胶制品预防等，但防治效果十分有限。因此，寻找新的治疗方法仍具有较高的学术与商业价值。
病理性瘢痕的发病机制目前尚在研究中，参与的致病因素主要包括ECM代谢异常、细胞因子(如TGF-β、血小板源性生长因子和胰岛素样生长因子)、低氧和自由基、基因水平变化等，其中以ECM代谢异常为最主要致病因素。正常情况下，人体内ECM的合成和分解处于动态平衡，从而维持着相对稳定。在创伤愈合的过程中，如果ECM合成明显增多则会形成病理性瘢痕或瘢痕疙瘩。因此，抑制ECM中胶原的过度沉积可被认为是防治病理性瘢痕的重要目标。
FKBP10是一种免疫亲和素，属于免疫亲和素FKBP亚家族，其定位于成纤维细胞中粗面内质网上的蛋白，是一种参与I型胶原的转录后修饰的酶，它能辅助前体胶原折叠形成成熟I型胶原，且与I型胶原的分泌量成正相关。在人体内FKBP10的突变与成骨不全症(osteogenesis imperfect,OI)关系密切，患者因FKBP10缺乏，导致皮肤成纤维细胞及骨组织内I型胶原合成及分泌不足，从而表现出皮肤脆弱缺乏弹性、易骨折、关键屈曲困难等一系列结蹄组织相关症状，而FKBP10在人病理性瘢痕中的表达量显著高于正常皮肤。
PCOLCE是一种可与I型前体胶原结合，并对其进行剪切和修饰的酶。I型前体胶原在经PCOLCE剪切后，其C端蛋白酶活性显著增加，并完成N端与C端的剪切，从而完成胶原三 聚反应，聚合形成成熟的胶原纤维。PCOLCE与细胞外基质的降解关系密切，并在增生性瘢痕中的表达显著高于正常皮肤。
现有的预防和治疗病理性瘢痕方法的局限性：手术疗法，复发率高且无法适用于大面积瘢痕的患者；加压治疗及硅胶制品治疗，对大面积烧伤患者效果较好，但仅用于早期预防，对已形成的瘢痕治疗效果有限；放射治疗，对患者全身和局部有一定辐射伤害；冷冻治疗，仅适用于面积较小的瘢痕，且治疗可引起皮肤色素加深、皮肤轻度萎缩等并发症；糖皮质激素治疗，可产生皮肤萎缩、色素脱失、毛细血管扩张、女性月经失调、注射部位溃烂或钙化等并发症；激光疗法，临床有效率较低，易诱发新的瘢痕；抗肿瘤药物治疗，对正常细胞代谢影响较大，临床价值尚不确切；他克莫司、他汀类药物、他莫昔芬等药物，临床疗效有限，治疗靶点尚不确切。
发明内容
本发明所要解决的技术问题是提供一种药物作用靶点的组合物和应用，该组合物系通过干预FKBP10及PCOLCE以抑制组织纤维化的形成，抑制皮肤成纤维细胞度增殖、分化、抗凋亡、分泌大量细胞外基质等生物学行为，从而达到治疗纤维化相关疾病的效果。
本发明的一种药物作用靶点的组合物，包括载体和以FKBP10及PCOLCE基因和/或其编码的蛋白作为药物作用靶点的药物。
所述以FKBP10及PCOLCE基因和/或其编码的蛋白作为药物作用靶点的药物为FKBP10及PCOLCE基因和/或其编码的蛋白的抑制剂。
所述以FKBP10及PCOLCE基因和/或其编码的蛋白作为药物作用靶点的药物包括但不限于干预FKBP10及PCOLCE基因和/或其编码蛋白的表达与功能的中和性抗体、小分子药物、小核苷酸、微小核糖核酸、反义分子或多肽。
所述干预方式包括同时干预FKBP10及PCOLCE基因和/或其编码的蛋白，或先干预FKBP10基因和/或其编码的蛋白之后再干预PCOLCE基因和/或其编码的蛋白。
所述以FKBP10及PCOLCE基因和/或其编码的蛋白作为药物作用靶点的药物的给药方式为局部注射或涂抹给药。
所述载体包括但不限于组氨酸多肽、赖氨酸多肽、分支状的组氨酸-赖氨酸共聚多肽、阳离子聚合物、硅纳米颗粒、脂质载体或病毒载体。其他药学上可接受的载体皆可。
本发明的一种药物作用靶点的组合物的应用，其特征在于：所述组合物用于制备治疗纤维化相关疾病的药物。
有益效果
(1)本发明系通过干预FKBP10及PCOLCE以抑制组织纤维化的形成，抑制皮肤成纤维细 胞度增殖、分化、抗凋亡、分泌大量细胞外基质等生物学行为，从而达到治疗纤维化相关疾病的效果，例如病理性瘢痕；
(2)本发明涉及的药物相对于他克莫司、他汀类药物、他莫昔芬等药物而言，治疗靶点明确，规避了治疗机制不明、效率低、易引起副作用等缺点；
(3)相对于糖皮质激素类药物，本发明涉及的药物规避了激素可能带来的一系列副作用；
(4)相对于抗肿瘤类药物和放射疗法，本发明涉及的药物规避了损伤人体正常细胞的可能；
(5)相对于激光治疗，本发明规避了治疗深度浅的缺点以及诱发新瘢痕形成的可能性；
(6)相对于手术治疗和冷冻疗法，本发明涉及的药物可治疗大面积的病理性瘢痕，规避了治疗范围小这一缺陷；
(7)相对于加压疗法和硅胶疗法，本发明涉及的药物可在病理性瘢痕形成后再进行治疗，规避了仅能进行早期预防这一局限性；
(8)本发明涉及的药物相对于目前现行药物具有成本上的显著优势，合成成本较低。
附图说明
图1为人增生性瘢痕与正常皮肤中FKBP10及PCOLCE表达差异的比较；
图2为敲减FKBP10和/或PCOLCE表达可抑制人增生性瘢痕成纤维细胞中纤维化标记物的表达；
图3为针对人FKBP10和/或PCOLCE的小核苷酸干扰可抑制人增生性瘢痕成纤维细胞的活性；
图4为针对小鼠FKBP10和/或PCOLCE的小核苷酸干扰可抑制小鼠增生性瘢痕的形成；其中，A：人增生性瘢痕和正常皮肤HE染色；B：小鼠模型在各干预方式下HE染色；C：小鼠模型在各干预方式下Masson染色；D：小鼠模型Masson染色中胶原含量统计图；
图5为针对小鼠FKBP10和/或PCOLCE的小核苷酸干扰可抑制小鼠皮肤纤维化标记物的表达。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
干预FKBP10及PCOLCE基因和/或其编码的蛋白的表达与功能的方式较多，如中和性抗体、小分子药物、小核苷酸，微小核糖核酸，多肽等，本实施例以小核苷酸药物为例，以人增生性瘢痕成纤维细胞(HHSF)为靶细胞，证实对FKBP10及PCOLCE的表达进行干预能抑制HHSF的活性，并减少纤维化相关标记物的表达。另外，通过建立小鼠增生性瘢痕模型， 对局部皮肤进行小核苷酸注射，证实对FKBP10及PCOLCE进行干扰能抑制小鼠增生性瘢痕的形成。
实施例1
1.实验材料
1.1小核苷酸序列
小核苷酸(Small interfering RNA,siRNA)：是一种小RNA分子(～21-25核苷酸)，由Dicer(RNAaseⅢ家族中对双链RNA具有特异性的酶)加工而成。SiRNA是siRISC的主要成员，激发与之互补的目标mRNA的沉默。RNA干涉(RNAinterference，RNAi)是指内源性或外源性双链RNA(dsRNA)介导的细胞内mRNA发生特异性降解，从而导致靶基因的表达沉默，产生相应的功能表型缺失的现象。
它针对人FKBP10mRNA的序列为：
正义链：5’-GAAGGAAGAUUAUCAUCCCUCCAUU-3’；
反义链：5’-AAUGGAGGGAUGAUAAUCUUCCUUC-3’；
它针对小鼠FKBP10mRNA的序列为：
正义链：5’-CCACACCUACAAUACCUAUTT-3’；
反义链：5’-AUAGGUAUUGUAGGUGUGGTT-3’；
它针对人PCOLCE mRNA的序列为：
正义链：5’-CCUUCCUCCAGAGAGCUUU-3’；
反义链：5’-GGAAGGAGGUCUCUCGAAA-3’；
它针对小鼠PCOLCE mRNA的序列为：
正义链：5’-CCUGGCAACCAAGUGACUU-3’；
反义链：5’-GGACCGUUGGUUCACUGAA-3’。
1.2小核苷酸/组氨酸-赖氨酸聚合物(HKP)纳米颗粒/甲基纤维素溶液配制方法
已用作siRNA转染载体的组氨酸-赖氨酸聚合物(HKP)具有赖氨酸骨架，该赖氨酸骨架包括含有多重组氨酸、赖氨酸或天冬氨酸的支链。将HKP溶于DEPC水制成DEPC水溶液，然后以质量比为4:1、体积比为1:1与siRNA(购自上海吉玛制药技术有限公司)水溶液混合，形成平均直径为150-200nm的纳米颗粒。HKP-siRNA水溶液为半透明状，没有明显的沉淀物聚集，可以在4℃下储存至少三个月。
1.3小核苷酸针剂配制方法
体内实验小核苷酸针剂的配制：用DEPC-5％葡萄糖溶液溶解经过特殊修饰的siRNA(购 自锐博生物科技有限公司)和HKP，分别制成siRNA水溶液和HKP水溶液，然后以质量比为4:1、体积比为1:1与siRNA水溶液混合，形成平均直径为150-200nm的纳米颗粒。HKP-siRNA水溶液为半透明状，没有明显的沉淀物聚集，可以在4℃下储存至少三个月。
2.实验方法
2.1人成纤维细胞的提取和培养
取临床标本(人增生性瘢痕、瘢痕疙瘩或正常皮肤)用Dispase II(2mg/ml,Life Technologies,ThermoFisher)浸泡，4℃过夜，并剥离、去除表皮。无菌环境下切碎组织，用4mg/ml胶原酶浸泡，摇床37℃消化2-4小时，滤网过滤细胞悬液，1500rpm离心5min，去除上清，并用培养基重悬细胞沉淀，接种至DMEM培养基。每2天换液一次。常规传代。
2.2小鼠增生性瘢痕模型的建立
将12周龄的C57/BL6小鼠麻醉，背部备皮，做一1cm纵行切口，将注满博来霉素溶液(2.8mg/ml)的微型渗透胶囊(Alzet Model 1004；Durect Corp.,Cupertino,CA,U.S.A.)埋置在皮下。缝合切口。56天后安乐死小鼠，并取材。
2.3小核苷酸针剂注射
小鼠瘢痕建立开始的当天，即开始小核苷酸针剂注射，注射方法为皮内注射。对照组、FKBP10siRNA单独给药组、PCOLCE siRNA单独给药组的小鼠，每3天注射一次，至模型建立第56天取材为止。FKBP10siRNA+PCOLCE siRNA同时给药组，配制注射针剂中FKBP10siRNA终浓度为200μg/ml，PCOLCE siRNA终浓度为50μg/ml，两者分别与HKP制成HKP-siRNA溶液后混合注射，每3天注射一次，至模型建立第56天取材为止。FKBP10siRNA+(24h)PCOLCE siRNA给药组，于第一天注射FKBP10siRNA(200μg/ml)，24h后进行PCOLCE siRNA(50μg/ml)注射，两种药分别按每3天注射一次的规律交替进行，至模型建立第56天取材为止。
2.4体外培养细胞的siRNA转染
待细胞密度至约70％时，对细胞进行换液，并加入HKP-siRNA水溶液(方法参照1.2部分)。对照组、FKBP10siRNA单独给药组、PCOLCE siRNA单独给药组的细胞，培养液中siRNA终浓度为50nM，培养24h后进行检测。FKBP10siRNA+PCOLCE siRNA同时干预组的细胞，培养液中FKBP10siRNA终浓度为50nM，PCOLCE siRNA终浓度为25nM，两者分别与HKP制成HKP-siRNA溶液后于培养液中混合，培养24h后进行检测。FKBP10siRNA+(24h)PCOLCE siRNA干预组的细胞，先配制FKBP10siRNA终浓度为50nM的培养液培养细胞，24h后换成PCOLCE siRNA终浓度为50nM的培养液培养细胞，继续培养24h后进行检测。
2.5实时定量PCR
用Trizol(Invitrogen,Grand Island,NY,USA)提取组织或细胞的总RNA，通过紫外分光光度法对RNA浓度和纯度进行测量(ND-1000,Thermo,Rockford,IL,USA)。用RT-PCR试剂盒(TaKaRa,Shiga,Japan)及ABI HT7900PCR仪(AppliedBiosystems,Foster City,CA,USA)对提取的总RNA进行逆转录反应，合成cDNA。以上述cDNA为模板进行实时定量PCR，20μl反应体系：
MIX(SYBR Premix Ex Taq)：10μl；
上游引物：0.4μl；
下游引物：0.4μl；
ROX II：0.4μl；
cDNA：2μl；
去离子水：6.8μl。
各反应管配齐，短暂离心混匀后置于PCR反应仪中运行(Applied Biosystems)。反应条件：95℃10s热启动后进入40个循环的反应：95℃15s，60℃30m，72℃30m。反应结束后用分析软件以ΔΔCT Relative Quantitation法分析结果。每个样本做三个平行副孔，并以GADPH引物作为内参。
2.6免疫组化/荧光染色
临床标本、动物组织取材后常规固定、脱水、石蜡包埋，并制备6μm切片。脱蜡后切片以1％羊血清进行封闭，并一抗4℃孵育过夜。以BSA清洗切片后，用带有辣根过氧化物酶或荧光标记的二抗进行孵育，37℃孵育2小时。显色，并进行拍照。
2.7CCK-8细胞活性检测
对于体外培养的细胞，采用CCK-8法对细胞的增殖活性进行检测。CCK-8试剂盒购自Dojindo(Tokyo,Japan)实验步骤按照试剂盒说明书严格进行。
3.实验结果
3.1人增生性瘢痕与正常皮肤中FKBP10及PCOLCE表达差异的比较。
对临床样本(人增生性瘢痕、正常皮肤)进行免疫组化检测。免疫组化结果显示，人增生性瘢痕中的FKBP10及PCOLCE的表达量均显著高于正常皮肤(p<0.001)(图1)。
3.2敲减FKBP10和/或PCOLCE表达可抑制人增生性瘢痕成纤维细胞中纤维化标记物的表达
分离并培养人增生性瘢痕成纤维细胞。采用RNAi技术对人增生性瘢痕成纤维细胞中的FKBP10和/或PCOLCE进行敲减，并采用实时定量PCR(q-PCR)对纤维化标记物COL1A、COL3A、FN、α-SMA和TGF-β1的mRNA水平进行检测。q-PCR结果显示，对FKBP10及/或PCOLCE进行敲减均可显著降低纤维化标记物COL1A、COL3A、FN、α-SMA和TGF-β1 的表达(p<0.001)。其中，同时干扰FKBP10、PCOLCE两个靶点以及先干扰FKBP10并于24h后干扰PCOLCE靶点，这两种方法处理的细胞中纤维化标记物的表达，低于单独干扰FKBP10或PCOLCE组细胞中纤维化标记物的表达，即干预FKBP10和PCOLCE双靶点的效果优于单独干预这两个靶点其中之一的效果(图2)。
3.3针对人FKBP10和/或PCOLCE的小核苷酸干扰可抑制人增生性瘢痕成纤维细胞的活性
分离并培养人增生性瘢痕成纤维细胞，用小核苷酸对其中FKBP10和/或PCOLCE的表达进行干扰，采用CCK-8法对人增生性瘢痕成纤维细胞的增殖活性进行检测。结果显示，敲减FKBP10和/或PCOLCE后的细胞，其增殖活性在干预后的5d和7d均显著低于对照组。同时干扰FKBP10、PCOLCE两个靶点以及先干扰FKBP10并于24h后干扰PCOLCE靶点，这两种方法处理的细胞，其增殖活性低于单独干扰FKBP10或PCOLCE组细胞，即干预FKBP10和PCOLCE双靶点的效果优于单独干预这两个靶点其中之一的效果(图3)。
3.4针对小鼠FKBP10和/或PCOLCE的小核苷酸干扰可抑制小鼠增生性瘢痕的形成
建立经典的小鼠增生性瘢痕模型，来模拟人增生性瘢痕的病理学改变(图4A-C)。对取材后的小鼠瘢痕进行了HE和Masson染色。结果显示，干预FKBP10和/或PCOLCE后，皮肤增生性瘢痕的特征(特征包括：表皮及真皮变厚、皮肤附属器官减少、胶原增多及排列紊乱，等)被显著抑制(p<0.001)。其中，同时干扰FKBP10、PCOLCE两个靶点以及先干扰FKBP10并于24h后干扰PCOLCE靶点，这两种方法所得效果显著优于单独干预FKBP10或PCOLCE的效果(p<0.05)。
3.5针对小鼠FKBP10和/或PCOLCE的小核苷酸干扰可抑制小鼠皮肤纤维化标记物的表达
用小核苷酸对小鼠模型中FKBP10和/或PCOLCE的表达进行干扰，将所得皮肤样本进行q-PCR检测。结果显示，对FKBP10和/或PCOLCE进行干扰，可显著降低胶原I、胶原III、纤连蛋白、α-SMA和TGF-β1的表达(p<0.001)。其中，同时干扰FKBP10、PCOLCE两个靶点以及先干扰FKBP10并于24h后干扰PCOLCE靶点，这两种方法处理的皮肤中纤维化标记物的表达，低于单独干扰FKBP10或PCOLCE组皮肤中纤维化标记物的表达，即干预FKBP10和PCOLCE双靶点的效果优于单独干预这两个靶点其中之一的效果(图5)。

Claims (7)

  1. 一种药物作用靶点的组合物，其特征在于：包括载体和以FKBP10及PCOLCE基因和/或其编码的蛋白作为药物作用靶点的药物。
  2. 根据权利要求1所述的一种药物作用靶点的组合物，其特征在于：所述以FKBP10及PCOLCE基因和/或其编码的蛋白作为药物作用靶点的药物为FKBP10及PCOLCE基因和/或其编码的蛋白的抑制剂。
  3. 根据权利要求1所述的一种药物作用靶点的组合物，其特征在于：所述以FKBP10及PCOLCE基因和/或其编码的蛋白作为药物作用靶点的药物为干预FKBP10及PCOLCE基因和/或其编码蛋白的表达与功能的中和性抗体、小分子药物、小核苷酸、微小核糖核酸、反义分子或多肽。
  4. 根据权利要求3所述的一种药物作用靶点的组合物，其特征在于：所述干预方式包括同时干预FKBP10及PCOLCE基因和/或其编码的蛋白，或先干预FKBP10基因和/或其编码的蛋白之后再干预PCOLCE基因和/或其编码的蛋白。
  5. 根据权利要求1所述的一种药物作用靶点的组合物，其特征在于：所述以FKBP10及PCOLCE基因和/或其编码的蛋白作为药物作用靶点的药物的给药方式为局部注射或涂抹给药。
  6. 根据权利要求1所述的一种药物作用靶点的组合物，其特征在于：所述载体为组氨酸多肽、赖氨酸多肽、分支状的组氨酸-赖氨酸共聚多肽、阳离子聚合物、硅纳米颗粒、脂质载体或病毒载体。
  7. 一种如权利要求1所述的药物作用靶点的组合物的应用，其特征在于：所述组合物用于制备治疗纤维化相关疾病的药物。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023175277A1 (fr) 2022-03-17 2023-09-21 Nvh Medicinal Ligands spécifiques de la glycoprotéine pcpe-1 et leurs utilisations

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111840311B (zh) * 2020-07-31 2021-11-12 上海交通大学医学院附属第九人民医院 siRNA分子组合物在制备针对HDAC5抑制瘢痕形成的药物中的用途
CN112538479A (zh) * 2020-12-22 2021-03-23 纳肽得有限公司 治疗增生性瘢痕、疤痕的小干扰核苷酸双链及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102031260A (zh) * 2010-08-24 2011-04-27 苏州圣诺生物医药技术有限公司 促进皮肤伤口无疤痕愈合的siRNA及应用
CN103642800A (zh) * 2013-11-19 2014-03-19 上海交通大学医学院附属第九人民医院 siRNA分子组合物及其在治疗增生性瘢痕中的用途
CN106755395A (zh) * 2016-12-16 2017-05-31 山东省医药生物技术研究中心 Xi型成骨不全致病基因fkbp10的突变位点及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008118915A (ja) * 2006-11-10 2008-05-29 Kazuto Nishio 胃癌高発現遺伝子特定による胃癌診断および創薬への利用
US8735567B2 (en) * 2007-11-06 2014-05-27 Patrick Y. Lu Multi-targeted RNAi therapeutics for scarless wound healing of skin
CN103223175B (zh) * 2013-05-23 2015-07-22 中国人民解放军第三军医大学第三附属医院 抗瘢痕和组织纤维化寡聚双链核苷酸药物及其应用
KR20170068452A (ko) * 2014-09-08 2017-06-19 미라젠 세러퓨틱스 인코포레이티드 Mir-29 모방체 및 이의 용도
SI3218478T1 (sl) * 2014-11-12 2020-08-31 Lek Pharmaceuticals D.D. Predvidevanje produktivnosti zgodaj v razvoju celične linije

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102031260A (zh) * 2010-08-24 2011-04-27 苏州圣诺生物医药技术有限公司 促进皮肤伤口无疤痕愈合的siRNA及应用
CN103642800A (zh) * 2013-11-19 2014-03-19 上海交通大学医学院附属第九人民医院 siRNA分子组合物及其在治疗增生性瘢痕中的用途
CN106755395A (zh) * 2016-12-16 2017-05-31 山东省医药生物技术研究中心 Xi型成骨不全致病基因fkbp10的突变位点及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
VICTOR W. WONG: "Transcriptional Profiling of Rapamycin-Treated Fibroblasts From Hypertrophic and Keloid Scars", ANNALS OF PLASTIC SURGERY, vol. 72, no. 6, 30 June 2014 (2014-06-30) - 31 May 2016 (2016-05-31), pages 711 - 719, XP055593862, ISSN: 0148-7043, DOI: 10.1097/SAP.0b013e31826956f6 *
XIAO LIANG ET AL.: "Inhibition of FKBP10 Attenuates Hypertrophic Scarring th- rough Suppressing Fibroblast Activity and Extracellular Matrix Deposition", JOURNAL OF INVESTIGATIVE DERMATOLOGY, vol. 137, no. 11, 27 September 2017 (2017-09-27) - November 2017 (2017-11-01), pages 2326 - 2335, XP055593856, ISSN: 0022-202X, DOI: 10.1016/j.jid.2017.06.029 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023175277A1 (fr) 2022-03-17 2023-09-21 Nvh Medicinal Ligands spécifiques de la glycoprotéine pcpe-1 et leurs utilisations
FR3133609A1 (fr) * 2022-03-17 2023-09-22 Nvh Medicinal Ligands spécifiques de la glycoprotéine PCPE-1 et leurs utilisations

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