Active ingredients, pharmaceutical compositions and uses for treating age-related macular degeneration

Abstract

The present invention discloses an active ingredient, a pharmaceutical composition and an application for treating age-related macular degeneration, wherein the active ingredient is a small non-coding RNA antagonist, or a derivative of a chemically modified small non-coding RNA antagonist; the small non-coding RNA is a small non-coding RNA derived from tRNA-Glu-CTC; and the small non-coding RNA is a tRF-Glu-CTC having a sequence as shown in SEQ ID No: 1, or is cut from a tRNA-Glu-CTC having a sequence as shown in SEQ ID No: 2, and comprises a 5' end, a length of 23-31nt, and a small non-coding RNA having the same or substantially the same function as the tRF-Glu-CTC. The active ingredient and / or pharmaceutical composition provided by the present invention can act on a small non-coding RNA other than VEGF, and has an efficient and safe therapeutic effect on age-related macular degeneration.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an active ingredient, a pharmaceutical composition and an application thereof for treating age-related macular degeneration. Background Art

[0002] Age-related macular degeneration (AMD) is a progressive blinding eye disease that occurs in the elderly and has threatened the vision of about 200 million patients worldwide. The main characteristics of this disease are degenerative lesions of photoreceptor cells and retinal pigment epithelium in the macula. Patients may experience blurred central vision and distorted vision. In severe cases, vision suddenly decreases, subretinal hemorrhage and detachment, which seriously affect the quality of life. Clinically, this disease can be divided into atrophic (dry) and neovascular (wet) lesions. Among them, neovascular AMD is the primary cause of blindness in patients. Its pathogenesis involves the destruction of the blood-ocular barrier. The abnormal proliferation of choroidal blood vessels causes damage to the neural retina in the macula, which seriously affects the patient's vision. At present, the treatment of AMD in clinical practice mainly targets the abnormal neovascularization of wet AMD. The treatment measures are relatively limited, including intravitreal injection of anti-vascular endothelial growth factor (VEGF), laser, photodynamic therapy and surgical treatment. Among them, laser and surgery are mainly symptomatic treatments with uncertain efficacy. Anti-VEGF drugs are currently the most mainstream treatment methods.

[0003] The principle of anti-VEGF drug treatment is to directly inhibit VEGF, a molecule that plays a key role in abnormal choroidal neovascularization, to promote vascular occlusion and reduce leakage. However, in clinical applications, it was found that some patients have drug resistance or incomplete response, and about 50% of patients still have exudation after 2 years of treatment; secondly, anti-VEGF drugs require repeated intravitreal injections. Frequent treatment leads to increased economic burden and decreased compliance of patients, which in turn affects the treatment effect. In addition, studies have also found that the inhibition of VEGF, a single factor, is not conducive to the recovery of pathological vascular endothelial function. At present, there is a lack of drugs that can both inhibit abnormal neovascularization and protect normal vascular function.

[0004] Therefore, there is an urgent need in this field to develop new, efficient and safe drugs for treating neovascular AMD that act outside the VEGF mechanism. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide an active ingredient and a pharmaceutical composition for treating age-related macular degeneration, which has a target other than VEGF and is highly effective and safe.

[0006] In order to achieve the above object, the present invention provides an active ingredient for treating age-related macular degeneration, wherein the active ingredient is a small non-coding RNA antagonist, or,

[0007] A chemically modified derivative of a small non-coding RNA antagonist; wherein,

[0008] The small non-coding RNA is a small non-coding RNA derived from tRNA-Glu-CTC; and,

[0009] The small non-coding RNA is tRF-Glu-CTC as shown in SEQ ID No: 1, or

[0010] It is cut from tRNA-Glu-CTC with a sequence as shown in SEQ ID No: 2, and contains a 5' end, a small non-coding RNA with a length of 23-31 nt, and a function that is the same or substantially the same as that of the tRF-Glu-CTC.

[0011] Preferably, the sequence of the small non-coding RNA antagonist is shown as SEQ ID No: 3.

[0012] The present invention also provides an active ingredient for treating age-related macular degeneration, wherein the active ingredient is an expression vector comprising the small non-coding RNA antagonist as described above.

[0013] Preferably, the expression vector is capable of expressing an antagonist nucleic acid molecule targeting the tRF-Glu-CTC and a tRF derived from the same precursor tRNA and having similar sequence and function.

[0014] The above-mentioned active ingredient is a small non-coding RNA antagonist, or a chemically modified derivative of the small non-coding RNA antagonist, or an expression vector comprising the small non-coding RNA antagonist as described above, which can be used in the preparation of a drug for treating age-related macular degeneration.

[0015] The present invention also provides a pharmaceutical composition for treating age-related macular degeneration, which comprises the active ingredients described above.

[0016] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or other drugs for treating age-related macular degeneration.

[0017] Preferably, the pharmaceutical composition may exist in the form of salts, hydrates or solvates.

[0018] The present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating age-related macular degeneration, wherein the drug is in a dosage acceptable in pharmacotherapy.

[0019] The small non-coding RNA antagonist provided by the present invention is a synthetic nucleic acid preparation, which has a simple structure, an easy preparation method and a variety of administration forms; at the same time, the small non-coding RNA antagonist provided by the present invention makes up for the limitations of current anti-VEGF drugs, has the effect of significantly inhibiting abnormal choroidal neovascularization, and can restore the normal function of the vascular endothelium, improve the hypoxia level in the neovascularization area, and prevent the further progression of fundus lesions, providing a candidate target with great clinical transformation potential for the development of new drugs for neovascular AMD, and laying a corresponding theoretical foundation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0021] Figure 1 It is a structural diagram of tRF-Glu-CTC and its precursor tRNA-Glu-CTC provided by the present invention;

[0022] Figure 2A is an image of choroidal neovascularization taken under a fluorescence microscope in Example 1 of the present invention;

[0023] Figure 2B This is a statistical result diagram in Example 1 of the present invention;

[0024] Figure 3A is an image of choroidal neovascularization taken under a fluorescence microscope in Example 2 of the present invention;

[0025] Figure 3B This is a statistical result diagram in Example 2 of the present invention;

[0026] Figure 4A-4D is a graph showing the migration and tube formation characteristics of vascular endothelial cells in Example 3 of the present invention;

[0027] Figure 5A and Figure 5B This is a result diagram of the neovascularization area of ​​the mouse fundus in Example 4 of the present invention. DETAILED DESCRIPTION

[0028] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0029] The present invention proposes a target for treating age-related macular degeneration and an active ingredient for treating age-related macular degeneration, wherein the active ingredient is selected from a) or b), specifically: a) is a small non-coding RNA antagonist or a chemically modified derivative thereof having the same or similar function, wherein the targeting molecule is a small non-coding RNA derived from tRNA-Glu-CTC. The small non-coding RNA is tRF-Glu-CTC (the core sequence is shown in SEQ ID No: 1, specifically: 5'-TCCCTGGTGGTCTAGTGGTTAGGATTCGGCG-3', see Figure 1 ); or a small non-coding RNA cleaved from tRNA-Glu-CTC (core sequence as shown in SEQ ID No: 2, specifically: 5'-TCCCTGGTGGTCTAGTGGTTAGGATTCGGCGCTCTCACCGCCGCGGCC CGGGTTCGATTCCCGGTCAGGGAA-3') and comprising a 5' end sequence, a length of 23-31 nt, and a function identical or substantially identical to tRF-Glu-CTC. The small non-coding RNA antagonist is a small molecule nucleic acid complementary to the base pairing of tRF-Glu-CTC, and the core sequence is shown in SEQ ID No: 3, specifically 5'-CGCCGAATCCTAACCACTAGACCACCAGGGA-3'. b) is an expression vector containing the tRF-Glu-CTC antagonist sequence described in a), which can express an antagonist nucleic acid molecule targeting the tRF-Glu-CTC and a tRF derived from the same precursor tRNA and having a similar sequence and function.

[0030] The present invention also provides a pharmaceutical composition, which includes a first active ingredient, a second active ingredient and a pharmaceutically acceptable carrier. Specifically, the first active ingredient here is the active ingredient as described above, the second active ingredient is other anti-VEGF drugs acceptable in therapy, and the carrier includes a pharmaceutically acceptable salt, hydrate or solvate, and other pharmaceutically acceptable carriers.

[0031] The present invention also proposes the use of the small non-coding RNA antagonist described above or its chemically modified derivatives with the same or similar functions, and / or pharmaceutical compositions in inhibiting choroidal neovascularization or preventing and / or treating age-related macular degeneration. The application method specifically uses the pharmaceutical composition described above to a subject in need, or administers the active ingredient described above. The pharmaceutical preparation used in this application can be any acceptable dosage form in pharmacotherapy. Preferably, it is an injectable preparation. The various dosage forms of the drug of the present invention can be prepared according to methods well known in the pharmaceutical field. The dosage used can be any acceptable dosage in pharmacotherapy.

[0032] The following is a detailed description through specific examples.

[0033] Example 1: Effects of tRF-Glu-CTC and its antagonists on choroidal neovascularization

[0034] Wild-type C57BL / 6 male 6-week-old mice were subjected to laser-induced retinal neovascularization model. The specific method is to anesthetize the mice, drip mydriatic drugs, place them in front of the slit lamp for mice, and use a laser with an intensity of 110mW and a duration of 100ms to irradiate the fundus of the mice to destroy the blood-eye barrier and induce neovascularization. The tRF-Glu-CTC antagonist was injected into the vitreous cavity of the mice by intravitreal injection, and the corresponding negative control (no treatment) or antagonist control (the injected drug is a random nucleic acid sequence to verify nonsense) mice were set as controls. Among them, the concentration of tRF-Glu-CTC antagonist and antagonist control was 250μM. The administration method is to inject 1μL of the drug solution into the vitreous cavity.

[0035] 7 days after mouse modeling, the eyeballs were taken to make mouse RPE-choroid-sclera flat sheets, and the blood vessels were marked with isolectin B4 (IB4) to evaluate the area of ​​choroidal neovascularization. The specific operation process is as follows: after the mice were killed by cervical dislocation, the mouse eyeballs were enucleated and fixed in 4% paraformaldehyde solution for 40 minutes. After that, the anterior segment structure and retina of the eyeball were removed in turn, and the posterior optic cup was made into RPE-choroid-sclera flat sheets. After incubation at 37 degrees Celsius for 1 hour with 0.5% Trion-X100 and 5% BSA in PBS solution, IB4 was used for staining overnight. The images of choroidal neovascularization were taken under a fluorescence microscope and the blood vessel area was counted, as shown in Figure 2. Figure 2A shown.

[0036] The statistical results are as follows Figure 2B As shown, tRF-Glu-CTC antagonist can effectively inhibit choroidal neovascularization, which is reduced by about 50% compared with the control group.

[0037] Example 2: Effect of tRF-Glu-CTC antagonist combined with anti-VEGF drug on choroidal neovascularization

[0038] Wild-type C57BL / 6 male 6-week-old mice were subjected to laser-induced retinal neovascularization model. The specific method is to anesthetize the mice and drip mydriatic drugs, place them in front of the mouse slit lamp, and use a laser with an intensity of 110mW and a duration of 100ms to irradiate the mouse fundus to destroy the blood-eye barrier and induce neovascularization. The tRF-Glu-CTC antagonist (or antagonist control) was combined with the anti-VEGF drug bevacizumab (bevacizumab) and injected into the vitreous cavity of the mice by intravitreal injection, and the untreated mice were set as negative controls. Among them, the concentration of bevacizumab was 30μg / μL. The concentration of tRF-Glu-CTC antagonist and antagonist control was 250μM. The combined preparation was a PBS solution containing 30μg / μL of bevacizumab and 250μM of tRF-Glu-CTC antagonist (or antagonist control). The administration method was intravitreal injection of 1μL.

[0039] Seven days after the mouse model was established, the eyeballs were removed to prepare mouse RPE-choroid-sclera flat mounts, and the blood vessels were marked with isolectin B4 (IB4) staining to evaluate the area of ​​choroidal neovascularization. Figure 3A and 3B As shown, tRF-Glu-CTC antagonist has similar anti-angiogenic effect as bevacizumab at the experimental concentration.

[0040] Example 3: tRF-Glu-CTC antagonist effectively inhibits endothelial cell migration and tube formation

[0041] Human umbilical vein endothelial cells were used to simulate the characteristics of choroidal endothelial cells. The tRF-Glu-CTCinhibitor was combined with a cell transfection reagent and the cells were transfected to inhibit the tRF-Glu-CTC level. The Transwell assay and angiogenesis assay were used to evaluate the migration and tube-forming properties of endothelial cells, reflecting the angiogenesis ability of the endothelium. Untreated cells were set as negative controls, and cells transfected with antagonist controls (random nucleic acid sequences to verify nonsense) were set as antagonist controls. The results are shown in Figure 2. Figure 4A-4D As shown, compared with the control group, tRF-Glu-CTC antagonist significantly inhibited the migration ability (p<0.05) and tube formation ability (p<0.01) of vascular endothelial cells.

[0042] Example 4: tRF-Glu-CTC antagonist reduces fundus hypoxia levels

[0043] Wild-type C57BL / 6 male 6-week-old mice were modeled with laser-induced retinal neovascularization. Hypoxyprobe was used to evaluate the hypoxia level in the fundus neovascular area after intravitreal injection of tRF-Glu-CTC antagonist or bevacizumab. On the fourth day after modeling, mice were intraperitoneally injected with pimonidazole hydrochloride solution (60 mg / kg). One hour later, the mice were killed by cervical dislocation, and the eyeballs were taken to make RPE-choroid-sclera flat sheets. Monoclonal antibodies targeting pimonidazole, fluorescent groups, and IB4 were incubated overnight, and the fundus fluorescence area was photographed and counted the next day. Since pimonidazole can accumulate in hypoxic cells by covalently binding to macromolecules or by being reduced to metabolites, it can be used as a hypoxia marker to evaluate the hypoxia level in the neovascular area. Among them, mice without treatment were set as negative controls. Bevacizumab was used at a concentration of 30 μg / μL. The concentration of tRF-Glu-CTC antagonist used was 250 μM. The combined preparation was a PBS solution containing 30 μg / μL bevacizumab and 250 μM tRF-Glu-CTC antagonist. The administration method was intravitreal injection of 1 μL. Figure 5A As shown in Figure 5B, the results showed that compared with bevacizumab, tRF-Glu-CTC antagonist can significantly reduce the level of fundus hypoxia and improve the perivascular microenvironment while inhibiting pathological angiogenesis.

[0044] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0045] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0046] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. Use of a small non-coding RNA antagonist in the preparation of a drug for treating neovascular AMD; the sequence of the small non-coding RNA antagonist is shown in SEQ ID No: 3.

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