Antisense oligonucleotide targeting CTGF and application of antisense oligonucleotide in preparation of medicine for treating fibrosis diseases

Abstract

The invention provides antisense oligonucleotide targeting CTGF and application of antisense oligonucleotide in preparation of medicine for treating fibrosis diseases, and belongs to the technical field of biological medicine. The fibrotic diseases can be treated by reducing the activity or expression of the CTGF. Experiments prove that the antisense oligonucleotide targeting the CTGF has a good effect on organ fibrosis, has no adverse reaction, can improve bleomycin-induced pulmonary fibrosis, mouse and rabbit ear skin scar hyperplasia and posterior cataract diseases, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to antisense oligonucleotides targeting CTGF and applications thereof in the preparation of drugs for treating fibrotic diseases. Background Art

[0002] Fibrosis is a medical concept that can occur in multiple organs. The main pathological changes are the increase of fibrous connective tissue and the decrease of parenchymal cells in organ tissues. Continuous progression can lead to organ structural destruction and functional impairment, and even failure, which seriously threatens human health and life.

[0003] Connective tissue growth factor (CTGF) is a multifunctional protein of the CCN family that regulates a variety of biological processes, including cell proliferation, differentiation, and adhesion. Currently, CTGF as a potential drug for the treatment of fibrosis needs further development. Summary of the invention

[0004] The purpose of the present invention is to provide an antisense oligonucleotide targeting CTGF and its application in preparing a medicine for treating fibrotic diseases.

[0005] The present invention provides antisense oligonucleotides targeting CTGF, including a first antisense oligonucleotide, a second antisense oligonucleotide, a third antisense oligonucleotide, a fourth antisense oligonucleotide, a fifth antisense oligonucleotide, a sixth antisense oligonucleotide, a seventh antisense oligonucleotide, an eighth antisense oligonucleotide, a ninth antisense oligonucleotide, a tenth antisense oligonucleotide and an eleventh antisense oligonucleotide, whose nucleotide sequences are respectively shown in SEQ ID NO.1 to SEQ ID NO.11.

[0006] Preferably, the antisense oligonucleotide has at least one modification group; the modification group includes one or more of locked nucleic acid, 2'-MOE and thiophosphate.

[0007] Preferably, 6 bases at the 5' end and 3' end of the antisense oligonucleotide are modified with locked nucleic acid and / or 2'-MOE.

[0008] Preferably, each base of the antisense oligonucleotide is modified with phosphorothioate.

[0009] The present invention also provides the use of the antisense oligonucleotide described in the above scheme in the preparation of drugs for treating fibrotic diseases.

[0010] Preferably, the fibrotic disease includes one or more of posterior capsule opacity, pulmonary fibrosis, skin fibrosis, intestinal fibrosis and liver fibrosis.

[0011] The present invention also provides a medicine for treating fibrotic diseases, which comprises an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises the antisense oligonucleotide described in the above scheme.

[0012] Preferably, the pharmaceutically acceptable excipients include one or more of salts, esters and hydrates; the salts include organic salts and / or inorganic salts; the organic salts include one or more of methanesulfonate, formates, acetates, trifluoroacetates, maleates, tartrates, succinates, fumarates, citrates, benzenesulfonates, p-toluenesulfonates, naphthalenesulfonates, lactates and benzoates; the inorganic salts include one or more of hydrochlorides, hydrobromides, sulfates and phosphates.

[0013] Preferably, the drug is in a dosage form suitable for topical, parenteral, oral, pulmonary, intratracheal, intranasal, transdermal or intraduodenal administration.

[0014] Preferably, the drug is a unit dose preparation; the unit dose preparation is formulated in a dosage form of 0.25 to 10 μg / dose.

[0015] The present invention provides antisense oligonucleotides targeting CTGF, including a first antisense oligonucleotide, a second antisense oligonucleotide, a third antisense oligonucleotide, a fourth antisense oligonucleotide, a fifth antisense oligonucleotide, a sixth antisense oligonucleotide, a seventh antisense oligonucleotide, an eighth antisense oligonucleotide, a ninth antisense oligonucleotide, a tenth antisense oligonucleotide and an eleventh antisense oligonucleotide, whose nucleotide sequences are respectively shown in SEQ ID NO.1 to SEQ ID NO.11. The present invention can treat fibrotic diseases by reducing the activity or expression of CTGF. It has been verified that the antisense oligonucleotides targeting CTGF of the present invention have good efficacy on organ fibrosis and have no adverse reactions, can improve bleomycin-induced pulmonary fibrosis, mouse and rabbit ear skin scar hyperplasia and secondary cataract disease, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0017] Figure 1 Schematic diagram of the antisense oligonucleotide targeting CTGF in Example 1;

[0018] Figure 2 This is a diagram showing the appearance changes of mouse skin scars at different stages;

[0019] Figure 3 The results of the determination of hydroxyproline content in mouse skin scars are shown in Figure 2; **p<0.01, ***p<0.001;

[0020] Figure 4 This is the result of HE and Masson staining of mouse skin scar;

[0021] Figure 5 This is a diagram showing the appearance changes of rabbit ear scars at different stages;

[0022] Figure 6 The results of hydroxyproline content in rabbit ear scars are shown in Figure 2; **p<0.01, ***p<0.001;

[0023] Figure 7 This is the result of HE and Masson staining of rabbit ear scar;

[0024] Figure 8 The figure shows the weight measurement results of keloid in nude mice; ***p<0.001;

[0025] Fig. 9 The results of the determination of hydroxyproline content in keloids of nude mice are shown in Figure 2; ***p<0.001;

[0026] Fig.10 The results of immunohistochemical staining of eyeball tissues of mice in the normal group, postoperative cataract model group and drug-treated group;

[0027] Fig.11 The results of slit lamp observation of the pathological conditions of the mouse eyes in the normal group, postoperative cataract model group and drug-treated group;

[0028] Fig.12 The results of histopathological section staining of eyeball tissues of mice in the normal group, postoperative cataract model group and drug-treated group;

[0029] Fig.13 The figure is the effect of the antisense oligonucleotide targeting CTGF on the activity of mouse embryonic fibroblasts and the expression of CTGF and Col-1 proteins in Example 1; the left figure is the effect of the antisense oligonucleotide targeting CTGF on the activity of mouse embryonic fibroblasts in Example 1, and the right figure is the effect of the antisense oligonucleotide targeting CTGF on the expression of CTGF and Col-1 proteins in Example 1;

[0030] Fig.14 This is a graph showing the effect of the antisense oligonucleotide targeting CTGF on the mRNA levels of CTGF, α-SMA and Col-1 in Example 1;

[0031] Fig.15The figure is the effect of the antisense oligonucleotide (ASO) targeting CTGF on the migration ability of mouse embryonic fibroblasts in Example 1 and the analysis result diagram; the left figure is a cell migration experiment, and the right figure is an analysis diagram of the migration experiment in the left figure;

[0032] Fig.16 This is the result of small animal CT detection of lung fibrosis progression in each group of mice;

[0033] Fig.17 The results of HE, Masson's staining and Sirius red staining to detect the effect of the antisense oligonucleotide (ASO) targeting CTGF in Example 1 on pulmonary fibrosis;

[0034] Fig.18 This is a graph showing the effect of the antisense oligonucleotide (ASO) targeting CTGF on the lung function of mice in Example 1. DETAILED DESCRIPTION

[0035] The present invention provides antisense oligonucleotides targeting CTGF, including a first antisense oligonucleotide, a second antisense oligonucleotide, a third antisense oligonucleotide, a fourth antisense oligonucleotide, a fifth antisense oligonucleotide, a sixth antisense oligonucleotide, a seventh antisense oligonucleotide, an eighth antisense oligonucleotide, a ninth antisense oligonucleotide, a tenth antisense oligonucleotide and an eleventh antisense oligonucleotide, whose nucleotide sequences are respectively shown in SEQ ID NO.1 to SEQ ID NO.11.

[0036] In the specific implementation of the present invention, the nucleotide sequence of the first antisense oligonucleotide is as shown in SEQ ID NO.1, specifically: 5'-TATGTCTTCATGCTGGTGCAG-3'.

[0037] In the specific implementation of the present invention, the nucleotide sequence of the second antisense oligonucleotide is as shown in SEQ ID NO.2, specifically: 5'-TTCTTCTTCATGACCTCGCCG-3'.

[0038] In the specific implementation of the present invention, the nucleotide sequence of the third antisense oligonucleotide is as shown in SEQ ID NO.3, specifically: 5'-TTGATGAACATCATGTTCT-3'.

[0039] In the specific implementation of the present invention, the nucleotide sequence of the fourth antisense oligonucleotide is as shown in SEQ ID NO.4, specifically: 5'-AAAGATGTCATTGTCTCCGGG-3'.

[0040] In the specific implementation of the present invention, the nucleotide sequence of the fifth antisense oligonucleotide is as shown in SEQ ID NO.5, specifically: 5'-TTAATGTCTCTCACTCTCTGG-3'.

[0041] In the specific implementation of the present invention, the nucleotide sequence of the sixth antisense oligonucleotide is as shown in SEQ ID NO.6, specifically: 5'-TAACATTCTTCAAACCAGTGT-3'.

[0042] In the specific implementation of the present invention, the nucleotide sequence of the seventh antisense oligonucleotide is as shown in SEQ ID NO.7, specifically: 5'-TTAAGGAACAACTTGACTCAG-3'.

[0043] In the specific implementation of the present invention, the nucleotide sequence of the eighth antisense oligonucleotide is as shown in SEQ ID NO.8, specifically: 5'-TTCCTGAACAGTGTCATTC-3'.

[0044] In the specific implementation of the present invention, the nucleotide sequence of the ninth antisense oligonucleotide is as shown in SEQ ID NO.9, specifically: 5'-TAAATTAACTTAGATAACTGT-3'.

[0045] In the specific implementation of the present invention, the nucleotide sequence of the tenth antisense oligonucleotide is shown in SEQ ID NO.10, specifically: 5'-TTACATTCTACCTATGGTGTT-3'.

[0046] In the specific implementation of the present invention, the nucleotide sequence of the eleventh antisense oligonucleotide is shown in SEQ ID NO.11, specifically: 5'-TTGAACGATCAGACAAGCTTT-3'.

[0047] In a specific implementation of the present invention, the agent for negatively regulating CTGF includes the antisense oligonucleotide targeting CTGF described in the above scheme.

[0048] In the specific implementation of the present invention, the antisense oligonucleotide has at least one modification group; the modification group includes one or more of locked nucleic acid (LNA), 2'-MOE and thiophosphate.

[0049] In the specific implementation of the present invention, the number of bases modified with locked nucleic acid and / or 2'-MOE in the antisense oligonucleotide is 6. Preferably, 6 bases at the 5' end and 3' end of the antisense oligonucleotide are modified with locked nucleic acid and / or 2'-MOE.

[0050] In a specific implementation of the present invention, each base of the antisense oligonucleotide is modified with thiophosphate.

[0051] In one embodiment of the present invention, the antisense oligonucleotide is a first antisense oligonucleotide; 6 bases at the 5′ end and 3′ end of the antisense oligonucleotide are modified with locked nucleic acid and each base in the entire sequence is modified with thiophosphate.

[0052] LNA modification is: a methylene bridge is introduced at the 2' oxygen atom and 4' carbon atom position of the carbon ring to form a ring structure. 2'-MOE (2-O-(2-methoxyethyl)) modification: It is a common oligonucleotide modification, mainly used to improve the stability and target binding affinity of oligonucleotides. This modification is achieved by introducing a 2'-MOE group into the sugar ring portion of the oligonucleotide, and the 2'-MOE group replaces the 2'-OH group in the sugar ring. Phosphorothioate modification: It is mainly used in biochemistry to enhance the stability of nucleic acid drugs and their interaction with organisms. This modification changes the chemical structure of DNA or RNA by replacing one of the non-bridging oxygen atoms in the phosphodiester bond in DNA or RNA with a sulfur atom to form a phosphorothioate diester bond.

[0053] In the present invention, 6 bases at the 5' and 3' ends of the antisense oligonucleotide are modified with locked nucleic acids, and the methylene bridge between the 2' oxygen atom and the 4' carbon atom in the ribose locks the sugar ring into a double-ring molecular pattern, thereby effectively limiting the flexibility of the sugar ring, reducing the flexibility of the ribose structure, and increasing the stability of the local structure of the phosphate backbone. The new combined force of the sequence binding to DNA and RNA is increased, the sequence specificity and thermal stability are enhanced, and the nuclease resistance is enhanced. Phosphorothioate (PS) backbone modification is used to enhance the properties of nucleic acid drugs including antisense oligonucleotides (ASOs). PS modification can improve the metabolic stability of nucleic acid drugs and enhance the interaction of nucleic acid drugs with plasma, cell surface and intracellular proteins, which helps their tissue distribution and cellular uptake.

[0054] The present invention also provides the use of the antisense oligonucleotide described in the above scheme in the preparation of drugs for treating fibrotic diseases.

[0055] In the specific implementation of the present invention, the fibrosis includes organ fibrosis; the fibrotic disease includes one or more of posterior capsule opacity (after-cataract), pulmonary fibrosis, skin fibrosis, intestinal fibrosis and liver fibrosis.

[0056] In the specific implementation of the present invention, the pulmonary fibrosis includes bleomycin-induced pulmonary fibrosis; the secondary cataract includes anterior capsule-induced secondary cataract; the skin fibrosis includes skin scar hyperplasia and / or keloid.

[0057] In the specific implementation process of the present invention, the treatment of skin scar hyperplasia includes any one of the following: 1) reducing the area of ​​skin scar; 2) inhibiting collagen deposition in scar; 3) repairing accessory tissue; 4) reducing the thickness of the dermis; 5) reducing the number of inflammatory cells and / or fibroblasts; 6) restoring fiber order; 7) alleviating scar progression; the accessory tissue includes hair follicles and / or sebaceous glands.

[0058] In the specific implementation of the present invention, the treatment of the anterior capsule-induced after-cataract includes any one of the following: 1) alleviating anterior capsule fibrosis; 2) alleviating the migration of lens epithelial cells; 3) shearing the formation of capsule fibrosis scars.

[0059] In the specific implementation of the present invention, the treatment of the bleomycin-induced pulmonary fibrosis includes any one of the following: 1) inhibiting cell migration under TGF-β1 stimulation; 2) reducing collagen deposition; 3) restoring lung function.

[0060] The present invention also provides a medicine for treating fibrotic diseases, which comprises an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises the antisense oligonucleotide described in the above scheme.

[0061] In the present invention, the antisense oligonucleotide is the only active ingredient of the drug.

[0062] In the specific implementation of the present invention, the dosage form of the drug is a dosage form suitable for topical, parenteral, oral, pulmonary, intratracheal, intranasal, transdermal or intraduodenal administration.

[0063] In the specific implementation of the present invention, the drug is a unit dose preparation based on mice; the unit dose preparation is formulated into a dosage form of 0.25 to 10 μg / dose, preferably a dosage form of 0.5 to 1 μg / dose.

[0064] In the specific implementation of the present invention, the pharmaceutically acceptable excipient includes one or more of salts, esters and hydrates; the salt includes organic salts and / or inorganic salts; the organic salt includes one or more of methanesulfonate, formates, acetates, trifluoroacetates, maleates, tartrates, succinates, fumarates, citrates, benzenesulfonates, p-toluenesulfonates, naphthalenesulfonates, lactates and benzoates; the inorganic salt includes one or more of hydrochlorides, hydrobromides, sulfates and phosphates.

[0065] In the specific implementation of the present invention, the dosage form of the drug includes one or more of oral preparations, injections, implants, external preparations, sprays and inhalants.

[0066] In the specific implementation process of the present invention, the oral preparation includes oral solid preparation or; the oral solid preparation includes tablets, capsules, pills, granules or powders; the oral liquid preparation includes syrup, tincture, wine or dew; the implant includes suppository; the spray includes aerosol; the external preparation includes film.

[0067] The present invention also provides a method for treating fibrotic diseases, characterized in that it comprises the following steps:

[0068] The patient is administered an effective amount of the antisense oligonucleotide described in the above protocol.

[0069] In a specific implementation of the present invention, the antisense oligonucleotide is administered topically, parenterally, orally, pulmonary, intratracheally, intranasally, transdermally or intraduodenally.

[0070] To further illustrate the present invention, the antisense oligonucleotides targeting CTGF provided by the present invention and their use in preparing drugs for treating fibrotic diseases are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0071] Example 1

[0072] In this embodiment, the nucleotide sequence of the antisense oligonucleotide targeting CTGF is as shown in SEQ ID NO.1, and 6 bases at the 5′ end and 3′ end of the antisense oligonucleotide are modified with locked nucleic acid and each base of the entire sequence is modified with thiophosphate, which is synthesized by WuXi AppTec; the nucleotide sequence of the antisense oligonucleotide targeting CTGF and the schematic diagram of the modified structure are shown in Figure 1 shown.

[0073] Example 2

[0074] Effect of antisense oligonucleotides targeting CTGF in Example 1 on mouse hypertrophic scar model

[0075] 1. Mouse Preparation

[0076] C57BL / 6J, (8-10 weeks old) wild-type mice.

[0077] 2. Grouping

[0078] Control group: no treatment;

[0079] Model group: A circular incision was made on the back of the mouse using a biopsy sample with a diameter of 8 mm, and then a 10 mm silicone ring was sutured with 3.0 nylon thread to apply surface tension. The mice were kept for two weeks until scar formation, and then 10 μl of normal saline was injected into the scar.

[0080] The group receiving the antisense oligonucleotide targeting CTGF in Example 1: a circular incision was made on the back of the mouse using a biopsy sample with a diameter of 8 mm, and then a 10 mm silicone ring was sutured with a 3.0 nylon thread to apply surface tension. The mice were kept for two weeks until scars were formed, and then 10 μg of the antisense oligonucleotide targeting CTGF in Example 1 was injected into the scars;

[0081] The drug was administered twice a week, and after four weeks of administration, the animals were sacrificed to collect data. The hydroxyproline content was subsequently detected and pathological sections were stained.

[0082] 3. Hydroxyproline content detection

[0083] Place the mouse skin in an ampoule and dry it in an oven at 120°C for 18 hours. Acid hydrolysis of collagen fibers to release L-hydroxyproline: prepare 6M hydrochloric acid, add 3mL to each ampoule, cover the bottle mouth with plastic film and tighten the lid, and bake in an oven at 120°C for 18 hours (if the hydrochloric acid evaporates completely, open the lid and add 1mL of hydrochloric acid to continue baking). Add 2mL of 6M NaOH and filter into a 10mL centrifuge tube. Adjust the pH to 6.5-8.0, add PBS to adjust the total volume to 10mL, and vortex to mix. Prepare a hydroxyproline standard solution. Dilute the hydroxyproline standard solution by half to prepare 500μL of each solution of 10, 5, 2.5, 1.25, 0.625, and 0μg / mL. Add 350μL to the labeled centrifuge tube. H2O and 50μL of sample. Take 400μL of the standard and add it to a new centrifuge tube. Add 200μL of chloramine T to each tube, vortex, and keep at room temperature for 20 minutes. Add 200μL of perchloric acid, vortex, and keep at room temperature for 5 minutes. Add 200μL of p-dimethylamino-benzaldehyde (P-DMAB), vortex, and place in a 50℃ water bath for 20 minutes. After cooling, take 200μL from each tube and put it into a 96-well plate. Set up 2 to 3 replicate wells for each sample, measure the absorbance at 577nm, and calculate the hydroxyproline content of the tissue to be tested based on the hydroxyproline standard curve.

[0084] 4. Staining of pathological sections of mouse skin scar tissue

[0085] The mouse skin scar tissue was fixed with paraformaldehyde, routinely dehydrated, paraffin embedded, sectioned, HE / Masson stained, and sealed with neutral gum, and the HE / Masson staining image data was collected using an upright microscope.

[0086] The experimental results are as follows Figures 2 to 4 As shown by Figure 2It can be seen that scars appeared in the model group at the second week. After 4 weeks of administration, compared with the model group, the group administered with the antisense oligonucleotide targeting CTGF in Example 1 can significantly reduce the scar area of ​​the mouse skin. Figure 3 It can be seen that the antisense oligonucleotide targeting CTGF significantly reduced the hydroxyproline content in the scar tissue of mice, indicating that the antisense oligonucleotide targeting CTGF can inhibit collagen deposition, and p<0.05, that is, there is a statistically significant difference. Figure 4 It can be seen that HE / Masson staining observations showed that the scar tissue in the model group lacked hair follicles, sebaceous glands and other dermal appendages. After four weeks of administration of antisense oligonucleotides targeting CTGF, the scar area was reduced, the skin tissue morphology was close to normal skin tissue, and the hair follicles, sebaceous glands and other appendages began to recover.

[0087] Example 3

[0088] Effect of antisense oligonucleotides targeting CTGF in Example 1 on the rabbit ear hypertrophic scar model

[0089] 1. Rabbit preparation

[0090] New Zealand female rabbit, 2.5kg.

[0091] 2. Grouping

[0092] Control group: no treatment;

[0093] Model group: Six 8mm circular incisions were made on the ventral inner side of each rabbit ear using an 8mm diameter biopsy sampler, and erythromycin was used to prevent wound infection. The rabbits were kept for 4 weeks, and 10μl of normal saline was injected into the scar after scar formation;

[0094] The group receiving the antisense oligonucleotide targeting CTGF in Example 1: Six 8 mm circular incisions were made on the ventral inner side of each rabbit ear using a biopsy sampler with a diameter of 8 mm, and erythromycin was used to prevent wound infection. The rabbits were kept for 4 weeks, and after scar formation, 10 μg of the antisense oligonucleotide targeting CTGF in Example 1 was injected into the scar;

[0095] The drug was administered twice a week, and after four weeks of administration, the animals were sacrificed to collect data. The hydroxyproline content was subsequently detected and pathological sections were stained.

[0096] 3. Hydroxyproline content detection

[0097] Place the rabbit ear scar tissue into an ampoule and dry it in an oven at 120℃ for 18h. Acid hydrolysis of collagen fibers to release L-hydroxyproline: Prepare 6M hydrochloric acid, add 3mL to each ampoule, cover the bottle mouth with plastic film and tighten the lid, and bake in an oven at 120℃ for 18h (if the hydrochloric acid evaporates completely, open the lid and add 1mL of hydrochloric acid to continue baking). Add 2mL of 6M NaOH and filter into a 10mL centrifuge tube. Adjust the pH to 6.5-8.0, add PBS to adjust the total volume to 10mL, and vortex to mix. Prepare a hydroxyproline standard solution. Dilute the hydroxyproline standard solution by half to prepare 500μL of each solution of 10, 5, 2.5, 1.25, 0.625, and 0μg / mL. Add 350μL of H2O and 50μL of sample to the labeled centrifuge tube. Take 400μL of the standard and add it to a new centrifuge tube. Add 200 μL of chloramine T to each tube, vortex, and keep at room temperature for 20 minutes. Add 200 μL of perchloric acid, vortex, and keep at room temperature for 5 minutes. Add 200 μL of p-dimethylamino-benzaldehyde (P-DMAB), vortex, and keep in a 50°C water bath for 20 minutes. After cooling, draw 200 μL from each tube and place it in a 96-well plate. Set up 2 to 3 replicate wells for each sample, measure the absorbance at 577 nm, and calculate the hydroxyproline content of the tissue to be tested based on the hydroxyproline standard curve.

[0098] 4. Staining of rabbit ear scar tissue pathology sections

[0099] The rabbit ear scar tissue was fixed with paraformaldehyde, routinely dehydrated, paraffin-embedded, sectioned, HE / Masson stained, and sealed with neutral gum. The HE / Masson staining image data were collected using an upright microscope.

[0100] The experimental results are as follows Figures 5 to 7 As shown by Figure 5 It can be seen that the scar tissue of the rabbit ears in the model group was obviously raised, the scar tissue was harder, and the color was ruddy. After 8 weeks of administration of the antisense oligonucleotide targeting CTGF in Example 1, the height of the raised scar decreased and the color became lighter. Figure 6 It can be seen that the antisense oligonucleotide targeting CTGF in Example 1 significantly reduced the hydroxyproline content in the scar tissue of mice, indicating that the antisense oligonucleotide targeting CTGF in Example 1 can inhibit collagen deposition, and p<0.05, that is, there is a statistically significant difference. Figure 7It can be seen that HE staining observation shows that the HE staining results show that the thickness of the dermis in the model group increased, and a large number of inflammatory cells and fibroblasts gathered in the scar. After the administration of the antisense oligonucleotide targeting CTGF in Example 1, the thickness of the dermis decreased, and the number of inflammatory cells and fibroblasts decreased. The Masson staining results showed that the collagen fibers in the model group showed a dense and disorderly distribution, and the fiber order was restored after the administration of the antisense oligonucleotide targeting CTGF in Example 1. It shows that the antisense oligonucleotide targeting CTGF in Example 1 can alleviate the collagen deposition in the scar to a certain extent.

[0101] Example 4

[0102] Effect of antisense oligonucleotides targeting CTGF in Example 1 on nude mouse keloid xenograft model

[0103] 1. Preparation of nude mouse keloid xenograft animal model

[0104] Fresh keloid samples were cut into 5×5×5 mm fragments (weight ranged from 0.07 to 0.1 g) and transplanted subcutaneously into athymic nude BALB / c mice.

[0105] 2. Grouping

[0106] 14 days after the keloid was implanted, 10 μg of the antisense oligonucleotide targeting CTGF in Example 1 was injected into the scar for treatment, and the treatment was performed six times within two weeks, and mice injected with normal saline were used as controls. After the treatment, the mice were anesthetized and killed by cervical dislocation. The keloid was removed by dissection, the graft weight was determined, and histological analysis was performed.

[0107] 3. Hydroxyproline content detection

[0108] Place the rabbit ear scar tissue into an ampoule and dry it in an oven at 120℃ for 18h. Acid hydrolysis of collagen fibers to release L-hydroxyproline: Prepare 6M hydrochloric acid, add 3mL to each ampoule, cover the bottle mouth with plastic film and tighten the lid, and bake in an oven at 120℃ for 18h (if the hydrochloric acid evaporates completely, open the lid and add 1mL of hydrochloric acid to continue baking). Add 2mL of 6M NaOH and filter into a 10mL centrifuge tube. Adjust the pH to 6.5-8.0, add PBS to adjust the total volume to 10mL, and vortex to mix. Prepare a hydroxyproline standard solution. Dilute the hydroxyproline standard solution by half to prepare 500μL of each solution of 10, 5, 2.5, 1.25, 0.625, and 0μg / mL. Add 350μL of H2O and 50μL of sample to the labeled centrifuge tube. Take 400μL of the standard and add it to a new centrifuge tube. Add 200 μL of chloramine T to each tube, vortex, and keep at room temperature for 20 minutes. Add 200 μL of perchloric acid, vortex, and keep at room temperature for 5 minutes. Add 200 μL of p-dimethylamino-benzaldehyde (P-DMAB), vortex, and keep in a 50°C water bath for 20 minutes. After cooling, draw 200 μL from each tube and place it in a 96-well plate. Set up 2 to 3 replicate wells for each sample, measure the absorbance at 577 nm, and calculate the hydroxyproline content of the tissue to be tested based on the hydroxyproline standard curve.

[0109] The experimental results are as follows Figure 8 and Fig. 9 As shown by Figure 8 It can be seen that compared with the control group, the weight of the tissue treated with the antisense oligonucleotide targeting CTGF in Example 1 was reduced. The hydroxyproline content was also significantly lower than that of the control group. Fig. 9 It can be seen that the antisense oligonucleotide targeting CTGF in Example 1 significantly reduced the hydroxyproline content in the scar tissue of mice, indicating that the antisense oligonucleotide targeting CTGF in Example 1 can inhibit collagen deposition, and p<0.05, that is, there is a statistically significant difference.

[0110] In summary, the antisense oligonucleotide targeting CTGF in Example 1 has good efficacy on hypertrophic scars and keloids without adverse reactions, can inhibit collagen deposition in scar tissue, and alleviate scar progression, and has good application prospects in treating, alleviating or improving scars.

[0111] Example 5

[0112] Effects of the antisense oligonucleotide targeting CTGF in Example 1 on after-cataract in mice induced by acupuncture of the anterior capsule.

[0113] 1. Preparation of animal model of secondary cataract

[0114] In C57BL / 6JNifdc wild-type mice (6-8 weeks old), after anesthesia, an insulin needle (29G, 0.33 mm) was inserted perpendicularly into the cornea to a depth of about 1 / 3 of the needle tip, causing a 300 μm deep injury in the center of the anterior capsule. The modeling period was 2 weeks.

[0115] 2. Grouping

[0116] Control group: no treatment;

[0117] Model group: After the model was established by acupuncture of the anterior capsule in the above manner, the same volume of normal saline as that of the drug-treated group was injected into the anterior chamber;

[0118] Drug administration group: After modeling, equal volumes of the antisense oligonucleotide drug solution targeting CTGF in Example 1 were injected into the intracameral chamber at dosages of 0.25 μg / eye, 0.5 μg / eye, 1 μg / eye, and 10 μg / eye, respectively.

[0119] Two weeks later, the mouse eyes were observed using a slit lamp, and the mice were killed by cervical dislocation and the eyeballs were removed for fixation, dehydration, embedding, slicing, staining and other treatments to detect the effects of the antisense oligonucleotide targeting CTGF in Example 1 on CTGF silencing and improvement of fibrosis.

[0120] 3. Slit lamp observation

[0121] The model was established by acupuncture of the anterior capsule. Two weeks later, a slit lamp was used to observe the anterior capsule damage in the control group, model group, and drug administration group (the dosages were 0.25 μg / eye, 0.5 μg / eye, and 1 μg / eye of the antisense oligonucleotide drug solution targeting CTGF in Example 1, respectively).

[0122] 4. Staining of eyeball tissue pathology sections

[0123] The mouse eyeballs were fixed with paraformaldehyde, routinely dehydrated, paraffin-embedded, sectioned, H&E stained, and sealed with neutral gum, and H&E staining image data were collected using an upright microscope.

[0124] 5. Immunohistochemical staining

[0125] The mouse eyeballs were fixed with paraformaldehyde, routinely dehydrated, paraffin-embedded, sliced, hydrated, and sodium citrate antigen repaired, then blocked with endogenous peroxidase blocker for 10 minutes, washed 3 times with PBS, and blocked with nonspecific binding blocker for 10 minutes, and then added with CTGF primary antibody at 4°C overnight. After being taken out and warmed up for half an hour, the primary antibody was recovered, and the eyeballs were washed 3 times with PBST, and biotin-labeled secondary antibody was added for 10 minutes. The eyeballs were washed 3 times with PBS, and streptavidin-biotin-peroxidase was added for 10 minutes. The eyeballs were washed 3 times with PBS, and freshly prepared DBA was added for staining. The eyeballs were counterstained with hematoxylin for 5 seconds and then washed with water to reverse blue. The eyeballs were dehydrated with 95% ethanol and 100% ethanol, and transparentized with xylene. After the eyeballs were sealed with neutral resin, the immunohistochemical staining image data were collected using an upright microscope.

[0126] The experimental results are as follows Figures 10 to 12 As shown by Fig.10 It can be seen that after the model was established by acupuncture of the anterior capsule, CTGF immunohistochemical staining showed that the lens, anterior capsule, and scar hyperplasia in the model group showed a significantly higher yellow color than that in the normal group, and after the administration group was injected with 10 μg / eye of the antisense oligonucleotide targeting CTGF in Example 1 into the anterior chamber after modeling, the yellow aggregation was significantly reduced, indicating that the antisense oligonucleotide targeting CTGF in Example 1 has a significant silencing effect on the increased CTGF content in the lens caused by secondary cataract. Fig.11 It can be seen that slit lamp observation showed that the anterior capsule of the normal group of mice was not damaged, while the model group had obvious fibrous scars. The area of ​​fibrous scars in the drug-treated groups (the dosage of the antisense oligonucleotide drug solution targeting CTGF in Example 1 was 0.25 μg / eye, 0.5 μg / eye, and 1 μg / eye, respectively) was significantly reduced with the increase of the dosage, indicating that the antisense oligonucleotide targeting CTGF in Example 1 has a significant effect on reducing the fibrosis of the anterior capsule of mice. Fig.12 It can be seen that H&E staining observation showed that the cornea, capsule and lens structures of the control group mice were intact, and obvious puncture wounds and cell proliferation appeared in the capsule of the model group. After the administration of the antisense oligonucleotide targeting CTGF in Example 1, the puncture wound was significantly reduced, and the amount of epithelial cell proliferation in the capsule and around the lens was greatly reduced, indicating that the antisense oligonucleotide targeting CTGF in Example 1 can reduce the migration of lens epithelial cells and the formation of capsule fibrosis scars to a certain extent.

[0127] In summary, the antisense oligonucleotide targeting CTGF in Example 1 has a good effect on secondary cataracts, has no adverse reactions, can silence the increased expression of CTGF in the anterior capsule and lens of mice with secondary cataracts, and reduce the acupuncture wound and the migration of lens epithelial cells and the formation of fibrotic scars on the capsule. It has a good application prospect in the treatment, relief or improvement of secondary cataracts.

[0128] The above are only specific implementation modes of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0129] Example 6

[0130] Effects of antisense oligonucleotides targeting CTGF in Example 1 on bleomycin-induced pulmonary fibrosis in vivo and in vitro models

[0131] 1. Verification of cell silencing efficacy

[0132] This example first verified the silencing effect and cytotoxicity of ASO in NIH-3T3 cells. PCR and WB results showed that LNA-CTGF ASO inhibited CTGF expression in a gradient manner and showed no obvious cytotoxicity in the range of 10 to 200 nM ( Fig.13 ).

[0133] 2. In vitro anti-fibrosis effect

[0134] Then, in this example, TGF-β1 was used to induce the cell state of NIH-3T3 cells under the pathological conditions of IPF. After LNA-CTGFASO was transfected into the cells in a gradient manner for 24 hours, the expression of fibrosis factors such as CTGF, α-SMA, and Col-1 was also inhibited in a gradient manner ( Fig.14 ). In order to verify whether ASO has an inhibitory effect on 3T3 cell migration induced by TGF-β1, a cell scratch test was set up in this example. The results showed that compared with cells treated with TGF-β1, the scratch healing speed of cells treated with Nin and ASO was slower, proving that ASO can effectively inhibit cell migration under TGF-β1 stimulation ( Fig.15 ).

[0135] 3. Bleomycin-induced mouse pulmonary fibrosis model:

[0136] Mice were randomly divided into the following groups: CTL, BLM, ASO (n=8). Seven days after modeling, mice in the ASO and ZMO-E5-ASO groups were treated with 2.5 mg / kg LNA-CTGF ASO via tail vein injection once every two days, and the BLM group was injected with normal saline via tail vein injection as a positive control. CT scans, lung function tests, and samples were taken after 14 days of modeling.

[0137] 4. CT results

[0138] On the 13th day of modeling, this embodiment also took lung CT scans of mice to observe the development of IPF. After data reconstruction, the CT perspective image showed that the BLM group mice had severe pulmonary fibrosis and lung parenchyma loss, and after drug administration, their pulmonary fibrosis was significantly improved. After data processing, a 3D image of the lungs was constructed according to the three parts of complete ventilation volume (red), complete non-ventilation volume (yellow), and incomplete ventilation volume (blue). After data analysis of the images and group lung volumes, it was found that the complete non-ventilation volume of the lungs of the ASO group mice was reduced compared with that of the BLM group mice ( Fig.16 ).

[0139] 5. Staining of lung tissue pathology sections

[0140] Then, this example evaluated the pulmonary fibrosis and collagen deposition in mice by H&E, Masson and Sirius red staining. The BLM group had severe loss of lung parenchyma, increased alveolar diameter, and thicker alveolar walls than the normal group. At the same time, inflammatory cell infiltration, including macrophages, was also observed in the lung tissue. Compared with the BLM group, the necrotic area in the ASO group was reduced. In addition, Masson and Sirius red staining showed that the inflammatory cell infiltration of the lung tissue in the ASO group was reduced, and the bronchiolar structure returned to normal. In addition, compared with the BLM group, the collagen deposition shown in the blue area of ​​the ASO group after treatment was significantly reduced compared with the BLM group ( Fig.17 ).

[0141] 6. Pulmonary function test

[0142] Before killing the mice, the lung function of the mice in each group was measured. Compared with the BLM group, the lung function of the mice in the ASO group was restored ( Fig.18 ).

[0143] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antisense oligonucleotide targeting CTGF, characterized in that: It includes a first antisense oligonucleotide, a second antisense oligonucleotide, a third antisense oligonucleotide, a fourth antisense oligonucleotide, a fifth antisense oligonucleotide, a sixth antisense oligonucleotide, a seventh antisense oligonucleotide, an eighth antisense oligonucleotide, a ninth antisense oligonucleotide, a tenth antisense oligonucleotide and an eleventh antisense oligonucleotide, whose nucleotide sequences are respectively shown as SEQ ID NO.1 to SEQ ID NO.

11.

2. The antisense oligonucleotide according to claim 1, characterized in that The antisense oligonucleotide has at least one modification group; the modification group includes one or more of locked nucleic acid, 2'-MOE and thiophosphate.

3. The antisense oligonucleotide according to claim 2, characterized in that Six bases at the 5' end and 3' end of the antisense oligonucleotide are modified with locked nucleic acid and / or 2'-MOE.

4. The antisense oligonucleotide according to claim 2 or 3, characterized in that Each base of the antisense oligonucleotide is modified with phosphorothioate.

5. Use of the antisense oligonucleotide according to any one of claims 1 to 4 in the preparation of a drug for treating fibrotic diseases.

6. The use according to claim 5, characterized in that: The fibrotic disease includes one or more of posterior capsule opacity, pulmonary fibrosis, skin fibrosis, intestinal fibrosis and liver fibrosis.

7. A drug for treating fibrotic diseases, characterized in that: The drug comprises an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises the antisense oligonucleotide according to any one of claims 1 to 4.

8. The drug according to claim 7, characterized in that The pharmaceutically acceptable excipients include one or more of salts, esters and hydrates; the salts include organic salts and / or inorganic salts; the organic salts include one or more of methanesulfonates, formates, acetates, trifluoroacetates, maleates, tartrates, succinates, fumarates, citrates, benzenesulfonates, p-toluenesulfonates, naphthalenesulfonates, lactates and benzoates; the inorganic salts include one or more of hydrochlorides, hydrobromides, sulfates and phosphates.

9. The drug according to claim 7, characterized in that The drug is in a dosage form suitable for topical, parenteral, oral, pulmonary, intratracheal, intranasal, transdermal or intraduodenal administration.

10. The drug according to claim 7, characterized in that The drug is a unit dose preparation; the unit dose preparation is formulated into a dosage form of 0.25 to 10 μg / dose.

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