WO2024045250A1 - Complexe pharmaceutique tétraédrique d'adn pour le traitement de maladies rétiniennes néovasculaires, son procédé de préparation et son utilisation - Google Patents

Complexe pharmaceutique tétraédrique d'adn pour le traitement de maladies rétiniennes néovasculaires, son procédé de préparation et son utilisation Download PDF

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WO2024045250A1
WO2024045250A1 PCT/CN2022/121365 CN2022121365W WO2024045250A1 WO 2024045250 A1 WO2024045250 A1 WO 2024045250A1 CN 2022121365 W CN2022121365 W CN 2022121365W WO 2024045250 A1 WO2024045250 A1 WO 2024045250A1
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dna
group
sirna
bevasiranib
tetrahedral
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罗德伦
徐枭潇
吴大颖
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成都景润泽基因科技有限公司
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the present invention relates to the field of drugs for neovascular retinal diseases, specifically to the field of gene drugs for silencing VEGF.
  • Neovascular retinal disease is a common type of blinding eye disease, including age-related macular degeneration (AMD), diabetic macular edema (DME), etc.
  • AMD age-related macular degeneration
  • DME diabetic macular edema
  • the common feature of these diseases is the generation of new blood vessels, which makes the treatment of the disease very difficult, seriously affects the patient's quality of life, and brings a heavy burden to the patient's family and society.
  • AMD age-related macular degeneration
  • DME diabetic macular edema
  • DME diabetic macular edema
  • VEGF as the most important stimulating factor for neovascularization, plays an important role in retinal neovascularization.
  • new blood vessels are morphologically different from normal blood vessels. Their lumens are irregular and their walls are mostly leaky. This abnormal proliferation of highly permeable or leaky blood vessels often leads to scarring on the retina, which can further detach and affect vision.
  • VEGF inhibitors Based on the pathological role of VEGF in neovascular retinopathy, VEGF inhibitors have become a research and development hotspot. They can block the interaction between VEGF and VEGF receptors on endothelial cells, thereby preventing information transmission mediated by VEGF. , inhibiting the growth of new blood vessels caused by high expression of VEGF, so as to prevent and stop retinal hemorrhage.
  • VEGF inhibitors include Macugen (pegaptanib sodium), Lucentis, Aflibercept, Avastin (bevacizumab) and AdPEDF, etc.
  • VEGF inhibitors Although the emergence of the above-mentioned VEGF inhibitors has brought hope to patients with fundus neovascular diseases, current therapies are not universally effective, and long-term inhibition of VEGF may lead to tissue atrophy and other side effects.
  • monthly injections of anti-VEGF drugs are associated with On-demand injections are more likely to cause geographic atrophy than on-demand injections, and an increase in the number of injections is also significantly related to the progression of retinal pigment epithelial atrophy. Therefore, it is necessary to explore new treatments and drugs to treat abnormal angiogenesis, reduce the burden on patients and doctors as much as possible, prevent the occurrence of retinal atrophy, and maximize the long-term prognosis and quality of life of patients.
  • RNA interference is a method of post-transcriptional gene regulation conserved in many eukaryotes. Endogenous or exogenous dsRNA can be cleaved by specific ribonuclease (Dicer) in the body into small double-stranded fragments with a length of 21 to 23 base pairs. These small double-stranded fragments are called small interfering RNA (siRNA).
  • the double strands of siRNA can be connected to the RNA-induced gene silencing complex (RISC), and after binding to RISC, it targets and cuts specific mRNA into small fragments of 10 to 11 bases, thereby interrupting The translation process of specific mRNA inhibits and silences the expression of the target gene.
  • RISC RNA-induced gene silencing complex
  • siRNA can be recycled, very similar to multiple turnover enzymes.
  • One siRNA molecule can induce the cleavage of about 1,000 mRNA molecules. It can be seen that developing effective siRNA drugs for specific targets can greatly improve drug efficiency. Patients who require long-term inhibition of relevant targets for disease treatment are very promising.
  • Bevasiranib is the world's first siRNA drug to enter clinical trials. It is a 21-mer siRNA targeting the VEGF target developed by Opko Health for the treatment of AMD. However, the development path of Bevasiranib was not smooth. Although it showed biological activity in Phase I and II clinical trials, its III clinical trial was eventually terminated due to poor effect in reducing vision loss. So far, the first siRNA drug Treatment attempts failed due to barriers to administration. In view of this, it is an urgent and realistic need to develop an siRNA delivery platform to achieve effective delivery of siRNA drugs in the body.
  • TDNs DNA tetrahedral
  • Patent CN109646450B discloses the use of TDNs in preparing drugs for treating corneal damage
  • patent CN112007044B discloses TDNs-miR155 complexes and their use in preparing drugs for preventing or treating wet macular degeneration
  • patent CN112843085B discloses TDNs- The miR22 complex and its use in the preparation of drugs for treating optic nerve damage. So far, TDNs carrying siRNA have not been seen for the treatment of ophthalmic diseases.
  • the object of the present invention is to provide a DNA tetrahedral drug complex for the treatment of neovascular retinal diseases, which is a complex formed by TDNs carrying Bevasiranib, and further provides the above complex for the treatment of neovascular retinal diseases.
  • a DNA tetrahedral drug complex for the treatment of neovascular retinal diseases which is a complex formed by TDNs carrying Bevasiranib, and further provides the above complex for the treatment of neovascular retinal diseases.
  • a DNA tetrahedral drug complex for treating neovascular retinal diseases includes:
  • siRNA for silencing the VEGF gene comprising a sense strand composed of the nucleotides shown in SEQ ID NO.5 and an antisense strand composed of the nucleotides shown in SEQ ID NO.6;
  • a DNA tetrahedron is formed by complementary base pairing of four single-stranded DNAs; the nucleotide sequences of the four single-stranded DNAs are selected one-to-one from SEQ ID NO. 1 to SEQ The sequence shown in ID NO.4;
  • the siRNA that silences the VEGF gene is connected to at least one single strand of the DNA tetrahedron.
  • the sense strand of the siRNA that silences the VEGF gene is connected to at least one single strand of the DNA tetrahedron through a chemical bond.
  • the DNA tetrahedral drug complex of the present invention wherein the sense strand of the siRNA that silences the VEGF gene is chemically bonded to the single strand of the DNA tetrahedron through the connecting sequence -TTTTT. connect.
  • the DNA tetrahedral drug complex of the present invention wherein the four single-stranded DNAs forming the DNA tetrahedron are placed in an equal molar ratio at a temperature sufficient to denature them to denature them, and then the temperature is lowered to anneal them. Then, a DNA tetrahedral structure is formed through complementary inter-strand base pairing; and then at least one of the four single-stranded DNAs is connected to the siRNA that silences the VEGF gene.
  • a pharmaceutical composition containing the DNA tetrahedral drug complex for treating neovascular retinal diseases of the present invention is provided.
  • Neovascular retinal diseases include age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinal vein occlusion, or treatment failure caused by new blood vessel growth.
  • the DNA tetrahedrons used in the present invention are formed by complementary base pairing of four single-stranded DNAs; the sequences of the four single-stranded DNAs correspond to the sequences of SEQ ID NO. 1 to 4 in sequence, and
  • the target product TDNs can be assembled through denaturation and annealing processes. Specifically, the four single-stranded DNAs of TDNs were maintained at a temperature sufficient to denature them for 10 minutes, and then the temperature was lowered to 2-8°C for more than 20 minutes.
  • the DNA tetrahedron is prepared by denaturing the four DNA single strands at 90-98°C for 10-15 minutes and annealing at 2-8°C for 20-30 minutes.
  • the DNA tetrahedron is prepared by denaturing the four DNA single strands at 95°C for 10 minutes and annealing at 4°C for 20 minutes.
  • the complex of the present invention is a complex composed of DNA tetrahedron and Bevasiranib (or "siRNA that silences the VEGF gene", both of which are interchangeable in the present invention) according to a molar ratio of 1: (1-4), wherein Bevasiranib It is an siRNA duplex, which has a sense strand as shown in SEQ ID NO:5 and an antisense strand as shown in SEQ ID NO:6.
  • the Bevasiranib is connected to four DNA single strands in the DNA tetrahedral structure through chemical bonds.
  • the connecting sequence is a nucleotide sequence, preferably a deoxyribose nucleus.
  • the nucleotide sequence is more preferably -TTTTT- (that is, five consecutive thymidine deoxynucleotide sequences).
  • the method for preparing the above-mentioned complex involves placing the four single-stranded DNAs of the DNA tetrahedron at a temperature sufficient to denature them for more than 10 minutes, and then lowering the temperature to 2 to 8° C. for more than 20 minutes; Bevasiranib is linked to one of the four single-stranded DNAs.
  • the DNA tetrahedron is prepared by denaturing the four DNA single strands at 90-98°C for 10-15 minutes and annealing at 2-8°C for 20-30 minutes, one of which is connected to Bevasiranib.
  • the DNA tetrahedron is prepared by denaturing the four DNA single strands at 95°C for 10 min and annealing at 4°C for 20 min, one of which is connected to Bevasiranib.
  • the present invention provides the use of the above-mentioned complex in the preparation of drugs for neovascular retinal diseases, including but not limited to age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinal vein occlusion, and treatment failure caused by new blood vessel growth. .
  • Neovascular retinal disease refers to a blinding vitreoretinal disease caused by the growth of new blood vessels accompanied by pathological changes such as hemorrhage, exudation, and proliferation. Neovascularization is a common pathological change in many important eye diseases.
  • Age-related macular degeneration is a pathological aging change in the structure of the macular area. It can be divided into 2 types: dry (non-exudative) or wet (exudative or neovascular). Wet AMD is characterized by choroidal neovascularization. In the middle and late stages of disease development, when pathological changes continue to worsen, Bruch's membrane ruptures. Choroidal capillaries pass through the ruptured Bruch's membrane and enter under the RPE or under the retinal neuroepithelium, forming choroidal neovascularization. (CNV), which is also the most direct factor affecting vision.
  • CNV choroidal neovascularization
  • Diabetic retinopathy is a relatively serious microvascular complication. Its pathological characteristics are mainly neovascularization and destruction of the retinal blood-retinal barrier (BRB). Diabetic retinopathy (DR) is the most common microvascular complication of diabetes. One of the symptoms is the leakage and obstruction of retinal microvessels caused by chronic progressive diabetes, which leads to a series of fundus lesions, such as microaneurysms, hard exudates, cotton wool spots, new blood vessels, vitreous proliferation, macular edema and even retinal detachment. DR is judged based on whether there are abnormal new blood vessels emanating from the retina and can be divided into proliferative diabetic retinopathy and non-proliferative diabetic retinopathy.
  • Retinal vein occlusion is a common fundus vascular disease with a long course and long-term retinal ischemia, which can induce neovascularization after ischemia.
  • the present invention also introduces treatment methods for related diseases.
  • the above-mentioned complex can be administered to patients in need through a variety of different administration routes, including but not limited to intravenous administration, intravitreal injection, and other ocular administration.
  • the local delivery method is based on a certain dosage form to achieve the purpose of treating eye diseases.
  • the DNA tetrahedron-Bevasiranib complex provided by the present invention carries siRNA through the DNA tetrahedron, which can significantly improve the in vitro cell entry efficiency of Bevasiranib. It not only achieves excellent VEGF protein silencing effect in vitro, but also achieves excellent VEGF protein silencing effect in vivo.
  • the complex exhibited an inhibitory effect on neovascularization that was comparable to or even better than that of the positive control aflibercept, and significantly improved fundus leakage, showing a very prominent retinal repair effect.
  • Figure 1 shows the polyacrylamide gel electrophoresis images of TDN and TDN-Beva
  • Lane 1 is the TDN sample
  • lanes 2-6 are TDN-Beva samples.
  • FIG. 2 shows the capillary electrophoresis patterns of TDN and TDN-Beva
  • Figure 3 shows the transmission electron microscope images of TDN and TDN-Beva
  • Figure 4 shows the cell entry efficiency of Bevasiranib, Bevasiranib-LipoRNAiMAX complex, TDN, and TDN-Bevasiranib complex in different cells (HEK-293 cells, HUVEC cells and HREC cells);
  • Figure 5 shows the VEGF gene silencing effect in different cells after each administration group, including the blank group (control), Bevasiranib group, Bevasiranib+Lipo group, and different concentrations of TDN groups (5nmol/L, 10nmol/L, 15nmol/ L), different concentrations of TDN-Beva groups (5nmol/L, 10nmol/L, 15nmol/L);
  • Figure 6 shows the statistics of the inhibition level of VEGF protein expression in different cells (HEK-293 cells, HUVEC cells and HREC cells) after each administration group, including the blank group (control), Bevasiranib group, Bevasiranib+Lipo group, and TDN group , TDN-BEVA group;
  • Figure 7 shows the neovascularization inhibition level of each administration group after being applied to the chick chorioallantoic membrane model, including the blank group (control), Aflibercept (positive control) group, Bevasiranib group, Bevasiranib+Invivofectamine (transfection reagent), TDN group, TDN-Bevasiranib group;
  • Figure 8 shows the expression levels of VEGF protein in the retina of mice in each group after each administration group was administered to the mouse OIR model, including the blank group (control), Bevasiranib group, Bevasiranib+Invivofectamine group, TDN group, and TDN-Bevasiranib group. ;
  • Figure 9 shows the statistical results of the new blood vessel area in the mouse retina of each drug group after each administration group was administered to the mouse OIR model, including the blank group (control), Aflibercept (positive control) group, Bevasiranib+Invivofectamine group, TDN group, TDN -Bevasiranib group;
  • Figure 10 shows the retinal neovascularization and recovery of avascular areas of mice in each administration group in the OIR model
  • FIG 11 is a schematic diagram of TDN-Bevasiranib connection.
  • HEK-293 was purchased from Shanghai Jingze Biotechnology Co., Ltd.;
  • HREC cells were purchased from angiopromie company
  • HUVEC cells were purchased from Aucells Biotechnology;
  • LipoRNAiMAX (Lipo) was purchased from Thermo Fisher;
  • Invivofectamine reagent was purchased from Thermo Fisher;
  • Aflibercept injection (Aflibercept): purchased from Bayer Healthcare Co., Ltd., the specification is 40mg/ml/bottle;
  • C57/BL mice were purchased from Spefford (Beijing) Biotechnology Co., Ltd.
  • the 5' end of S1 is optionally connected to a Cy5 fluorescent labeling group for tracking TDNs.
  • Example 1 On the basis of Example 1, the S1 sequence was replaced with S1-Bevasiranib, in which S1 was connected to the sense chain of Bevasiranib through a chemical bond through the connecting sequence - TTTTT-, and S1-Bevasiranib, S2, S3, and S4 were added in an equal molar ratio ( Add 1 ⁇ l of 100 ⁇ M stock solution to each single strand into a 200 ⁇ l EP tube containing 95 ⁇ l of TM buffer (10 mM Tris-HCl, 50 mM MgCl 2 , pH 8.0). Heat the reaction solution to 95°C for 10 min, and then quickly The temperature was lowered to 4°C and maintained for 20 minutes to synthesize TDN-Bevasiranib (TDN-Beva). The connection method is shown in the schematic diagram ( Figure 11).
  • the sense strand of Bevasiranib (SEQ ID NO.5): ACCUCACCAAGGCCCAGCAC
  • Antisense strand of Bevasiranib (SEQ ID NO.6): GUGCUGGCCUUGGUGAGGU-dTdT
  • TDN-Beva Use capillary electrophoresis and PAGE electrophoresis to detect DNA single strands and synthesized TDN-Beva; use transmission electron microscopy to detect the shapes of TDNs and TDN-Beva; use dynamic light scattering to detect the zeta potential and particle size of TDNs and TDN-Beva.
  • the electrophoresis results indicate that the molecular weight of the TDNs-Beva band is consistent with the complex situation of TDNs and Beva, indicating that Bevasiranib has been successfully connected to TDNs.
  • the tetrahedral structure particles of TDNs and TDNs-Beva can be observed in the transmission electron microscope image.
  • the particle sizes of TDNs particles and TDNs-Beva particles are approximately 10nm-15nm, with the zeta potential of the former being -6.41mV and the zeta potential of the latter being -18.9mV.
  • HREC cells human embryonic kidney cells 293 (HEK-293), human retinal endothelial cells (HREC cells) and human umbilical vein endothelial cells (HUVEC) as experimental cells
  • mice blank group, Bevasiranib (Cy5 labeled) group, Bevasiranib (Cy5 labeled) + LipoRNAiMAX (transfection reagent) group, TDN (Cy5 labeled) group, TDN-Bevasiranib (Cy5 labeled) group.
  • the method of using the transfection reagent is as follows: change the cell medium and add 1.7 mL of culture medium with 3% serum. Then add 147 ⁇ l of serum-free medium and 3 ⁇ l of Bevasiranib and mix well. Add 141 ⁇ l of serum-free medium and 9 ⁇ l of transfection reagent and mix evenly. Mix the two tubes of liquid, incubate for 5 minutes, and add dropwise to the cells.
  • HREC cells human embryonic kidney cells 293 (HEK-293), human retinal endothelial cells (HREC cells) and human umbilical vein endothelial cells (HUVEC) as experimental cells
  • the trend of gene silencing in each cell is roughly the same.
  • the BEVA group treated without adding transfection reagent did not show obvious differential effects, while the BEVA+Lipo group was different from the blank control.
  • the group showed a certain gene silencing effect.
  • the TDN-BEVA group showed obvious gene silencing effects as the concentration increased, and was equivalent to or even better than the BEVA+Lipo group at the same concentration.
  • HREC cells human embryonic kidney cells 293 (HEK-293), human retinal endothelial cells (HREC cells) and human umbilical vein endothelial cells (HUVEC) as experimental cells
  • the protein quantification results are basically consistent with the aforementioned gene silencing effects.
  • the TDN-Beva group showed a comparable or even lower expression of VEGF than the Bevasiranib+Lipo group.
  • Experimental groups blank group, Aflibercept (positive control) group (1nmol/L), Bevasiranib group (1nmol/L), Bevasiranib (1nmol/L)+Invivofectamine (transfection reagent), TDN group (1nmol/L), TDN- Bevasiranib group (1nmol/L)
  • Wipe the eggs with 1:1000 Xinjie Er Killer Clean the surface of the purchased SPF eggs. After drying, use an egg camera to check whether the eggs are intact. Mark the name of the experiment with a pencil. Then put the eggs in the incubator to hatch. The conditions are set to 37.0 ⁇ 0.5°C, relative humidity 60%, the instrument is set to transfer eggs every two hours, and continue to incubate for 4-5 days.
  • the chicken embryos used in the experiment were randomly divided into 6 groups, 5 in each group.
  • Use a marker pen to mark the air chamber under the egg illuminator and draw the window opening position.
  • use a syringe needle to gently drill a small hole in the eggshell, and then use ophthalmic tweezers to slowly tear off the eggshell. Open a window about 1cm in diameter (use a grinding wheel, and then use ophthalmic tweezers to gently peel off the egg membrane to expose the chorioallantoic membrane of the chicken embryo. Be careful not to damage the blood vessels.
  • the calculation method is as follows :
  • Angiogenesis inhibition rate (MVD value of blank group - MVD value of experimental group)/MVD value of blank group * 100%
  • the Aflibercept positive control group showed significant neovascularization inhibitory effect, while the Bevasiranib+Invivofectamine group showed better neovascularization inhibitory activity than the Bevasiranib group, indicating the role of bevasiranib in transfection reagents. It can effectively enter cells and produce a certain inhibitory effect on new blood vessels by silencing the expression of VEGF.
  • TDN alone exhibited neovascular inhibitory activity almost equivalent to that of the positive control group, and its specific mechanism of action remains to be further explored.
  • the TDN-Beva group showed extremely excellent neovascularization inhibitory activity, which was not only significantly better than the positive control group, but also better than the Bevasiranib+Invivofectamine group and the TDN group.
  • OIR vasculoproliferative retinopathy
  • C57/BL mice and mothers on the 7th day after birth were placed in a breeding box with an oxygen volume fraction of 75% ⁇ 3% for 5 consecutive days, and the breeding temperature was maintained at (25 ⁇ 2)°C. Lighting was provided for 12 hours every day, and an automatic oxygen analyzer was used to monitor the oxygen content in the box.
  • P12 the mice were put back into normal air and raised. At this time, the mouse retina was in a relatively hypoxic state.
  • P17 On the 17th day after birth (P17), a large number of new blood vessels were formed in the retina.
  • Intravitreal injection is performed when P12 is released from the oxygen box.
  • the groups are as follows:
  • Bevasiranib group Intravitreal injection of 1 ⁇ L of 75 ⁇ mol/L Bevasiranib in both eyes
  • Bevasiranib+Invivofectamine group Intravitreal injection of 1 ⁇ L siRNA-Invivofectamine complex into both eyes, in which the concentration of Bevasiranib is 75 ⁇ mol/L
  • TDN group intravitreal injection of 1 ⁇ L 75 ⁇ mol/L TDNs in both eyes
  • TDN-Bevasiranib group intravitreal injection of 1 ⁇ L 75 ⁇ mol/L TDN-Bevasiranib complex in both eyes
  • mice from each group were taken. After anesthesia and sacrifice, the eyeballs of both eyes of a total of 6 mice were taken out and put into 250 ⁇ L of cell lysis solution. They were crushed by ultrasonic and ultracentrifuged at low temperature for 30 min. The supernatant was collected and stored in a low-temperature refrigerator. ELISA kit was used to detect the expression of VEGF in retinal protein extract.
  • the Bevasiranib-Invivofectamine group showed a significant inhibitory effect on VEGF expression compared with the blank group, while the TDN-Bevasiranib group showed a better inhibitory effect on VEGF protein expression than the Bevasiranib-Invivofectamine group, indicating that the TDN vector was used to carry Bevasiranib can effectively promote its entry into cells and silence VEGF gene expression, thereby reducing its protein expression.
  • Intravitreal injection is performed when P12 is released from the oxygen box.
  • the groups are as follows:
  • Aflibercept (positive control) group Intravitreal injection of 1 ⁇ L 40mg/mL aflibercept in both eyes
  • Bevasiranib+Invivofectamine group Intravitreal injection of 1 ⁇ L siRNA-Invivofectamine complex into both eyes, in which the concentration of Bevasiranib is 75 ⁇ mol/L
  • TDN group intravitreal injection of 1 ⁇ L 75 ⁇ mol/L TDNs in both eyes
  • TDN-Bevasiranib group intravitreal injection of 1 ⁇ L 75 ⁇ mol/L TDN-Bevasiranib complex in both eyes
  • mice from each group were taken. After anesthesia and sacrifice, a total of 6 eyeballs from both eyes were taken out and fixed in 10% formaldehyde at room temperature for half an hour. The cornea, iris and lens were removed under a microscope, and the retina was carefully and completely peeled off. A radial incision was made from the ora serrata of the retina to the equator of the four quadrants. The retina was laid flat on a glass slide, sealed with water-soluble mounting agent, and covered with a coverslip. Fluorescence microscopy was used to detect the tiled retina, and Image-Pro Plus (Media Cybernetics, USA) software was used to measure the area of retinal neovascularization.
  • Image-Pro Plus Media Cybernetics, USA
  • the blank group pictures show that the retinas of mice that underwent OIR modeling developed obvious lesions at P17, and their retinas formed an avascular area (in a small circle) and a neovascular area in the posterior pole. (Between small circle and large circle).
  • the Aflibercept (positive control) group After administration in the Aflibercept (positive control) group, it can inhibit the formation of new blood vessels to a certain extent and promote the normalization of blood vessels in the avascular area.
  • the TDN group showed an inhibitory effect on neovascularization that was close to that of the Aflibercept (positive control) group. It is worth noting that the TDN-bevasiranib group showed a significantly better inhibitory effect on neovascularization than the positive control group, and could significantly promote the normalization of the avascular zone, showing an excellent repair effect on the diseased retina.

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  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Cardiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Complexe pharmaceutique tétraédrique d'ADN pour le traitement des maladies néovasculaires de la rétine, qui est un complexe constitué par des TDN porteurs de Bevasiranib. Le complexe présente un excellent effet inhibiteur sur la néovascularisation, améliore significativement les fuites du fond d'œil et présente un effet réparateur rétinien très important.
PCT/CN2022/121365 2022-09-01 2022-09-26 Complexe pharmaceutique tétraédrique d'adn pour le traitement de maladies rétiniennes néovasculaires, son procédé de préparation et son utilisation WO2024045250A1 (fr)

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