Use of compound-8 in the preparation of a product for alleviating or treating blue light damage-induced retinal degeneration

Abstract

The application belongs to the technical field of medicines, and particularly relates to application of Compound-8 in preparation of products for relieving or treating blue light damage induced retinal degeneration. It is found for the first time that Compound-8 has a protective effect on blue light damage induced retinal rod cells and cone cells, can increase the length, density and disc thickness of the cilia of the cone cells and the rod cells, can increase the thickness of the outer nuclear layer of the retina, significantly improves the photoreceptor cell damage caused by blue light exposure, can relieve or treat the retinal degeneration caused by blue light damage, and provides a new thought and means for the treatment of blue light damage induced retinal degeneration.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of Compound-8 in the preparation of products that alleviate or treat retinal degeneration induced by blue light damage. Background Technology

[0002] The main source of blue light is sunlight, with a wavelength of 400-500 nm. Currently, most light sources emit potentially harmful blue light (415-455 nm). Studies have shown that the toxic effects of blue light are not limited to the retina; it can also damage structures such as mitochondria, the lens, and the tear film through oxidative stress and inflammatory responses, causing a variety of eye diseases, including age-related macular degeneration and dry eye syndrome.

[0003] The retina, located at the back of the eyeball, is the image-forming screen of the eye and is severely damaged when exposed to light of specific wavelengths or intensities. Because the retina is extremely sensitive to light, the primary damage occurs to photoreceptor cells (including cone and rod cells). However, the molecular mechanisms underlying blue light damage remain unclear. Therefore, in-depth research into the molecular mechanisms by which blue light damage leads to retinal degeneration is crucial for developing new targets and drugs to treat retinal degenerative diseases. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of Compound-8 in the preparation of products that alleviate or treat retinal degeneration induced by blue light damage.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides the use of Compound-8 in the preparation of products that alleviate or treat blue light-induced retinal degeneration.

[0007] The Compound-8 reduces blue light-induced retinal degeneration by maintaining the UFMylation-ciliary axis; specifically, maintaining the UFMylation-ciliary axis involves enhancing UFMylation activity, thereby increasing the stability of the KIF11 protein.

[0008] Compound-8 is a covalent inhibitor of UFM1-specific peptidase 2 (UFSP2), with CAS number 850188-43-1 and chemical structure as follows: Compound-8 can enhance ubiquitination activity and has been shown to enhance the efficacy of anti-PD-1 immunotherapy, inhibiting tumor growth in various tumor models.

[0009] This invention is the first to mechanistically verify that UFL1-mediated UFMylation plays a key role in the pathogenesis of blue light retinal disease (BLD). The invention demonstrates that photoreceptor cilia destruction is a critical event in BLD in a mouse model, and reveals that UFMylation of kinesin family member 11 (KIF11) is regulated under blue light irradiation. Furthermore, it discloses that the unique ligase for UFMylation, UFM1-specific ligase 1 (UFL1), is located in the matrix and is essential for maintaining photoreceptor cilia. Blue light irradiation disrupts the localization of UFL1 in the matrix, leading to cilia defects and subsequent photoreceptor dysfunction. Intravitreal injection of drugs that enhance UFMylation or cilia formation can alleviate the pathological changes caused by blue light irradiation.

[0010] Compound-8 increased UFMylation activity in the retina of BLD mice, thereby increasing the expression of downstream KIF11 protein and alleviating the decrease in KIF11 protein levels caused by blue light exposure, confirming that targeted regulation of UFMylation is a potential strategy for treating this type of disease.

[0011] Furthermore, the product has the following functions:

[0012] (a1) Enhances UFMylation activity in the retina;

[0013] (a2) Enhances the stability of KIF11 protein in the retina and restores the decrease in KIF11 protein levels caused by blue light exposure;

[0014] (a3) Improves the length and density of cilia in cone and rod cells;

[0015] (a4) Improves the thickness of the outer nuclear layer of the retina;

[0016] (a5) Improves the thickness of the membrane discs of rod cells and cone cells.

[0017] This invention demonstrates that Compound-8 has a protective effect on blue light-induced retinal rod and cone cells by injecting Compound-8 into the vitreous cavity of BLD mice. It can improve the ciliary length, density, and disc thickness of cone and rod cells, improve the thickness of the outer nuclear layer of the retina, significantly improve photoreceptor cell damage caused by blue light exposure, and alleviate retinal degeneration caused by blue light damage.

[0018] Specifically, improving the cilia length and density of cone and rod cells refers to restoring the reduced cilia length and density of cone and rod cells caused by blue light damage to normal levels. Improving the thickness of the outer nuclear layer of the retina refers to restoring the reduced thickness of the outer nuclear layer of the retina caused by blue light damage to normal levels. Improving the membrane disc thickness of rod and cone cells refers to restoring the reduced membrane disc thickness of rod and cone cells caused by blue light damage to normal levels.

[0019] In some embodiments, the product is a drug.

[0020] In some embodiments, the drug is a formulation prepared with Compound-8 as the active ingredient and pharmaceutically acceptable excipients.

[0021] In some embodiments, the concentration of Compound-8 in the drug is 8 to 9 mol / L, preferably 8.6 mol / L.

[0022] In some embodiments, the formulation is an oral formulation, an injectable formulation, or a transdermal formulation. The injectable formulation is a formulation for subretinal or intravitreal injection.

[0023] In some embodiments, the injectable formulation is a formulation administered via intravitreal injection.

[0024] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0025] This invention is the first to discover that Compound-8 has the effect of protecting retinal photoreceptor cells. Experiments have shown that Compound-8 has a protective effect on blue light-induced retinal rod and cone cells, can improve the ciliary length, density and disc thickness of cone and rod cells, can improve the thickness of the outer nuclear layer of the retina, significantly improve photoreceptor cell damage caused by blue light exposure, and can alleviate retinal degeneration caused by blue light damage. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the construction of the BLD mouse model in an embodiment of the present invention;

[0027] Figure 2 The results of the analysis of endogenous KIF11 UFMylation in the retinas of mice exposed to yellow or blue light in this embodiment of the invention are relative UFMylation levels. In this example, A represents the results of immunoblotting analysis of UFM1, KIF11 and β-actin in the retinas of experimental and control mice, and B represents the relative modification level of KIF11 UFMylation in the retinas of experimental and control mice. ***p<0.001, ****p<0.0001;

[0028] Figure 3 This is a schematic diagram of Compound-8 intravitreal injection in an embodiment of the present invention;

[0029] Figure 4 The present invention provides the results of immunoblotting analysis and relative UFMylation levels of UFM1, KIF11, UFSP2, and β-actin in the retinas of BLD mice in this embodiment of the invention. Specifically, A represents the results of immunoblotting analysis of UFM1, KIF11, UFSP2, and β-actin in the retinas of BLD mice injected with DMSO or Compound-8, and B represents the relative modification level of UFMylation in the retinas of BLD mice injected with DMSO or Compound-8. *** indicates that Compound-8 significantly and persistently increased the overall UFMylation level in the mouse retinas.

[0030] Figure 5 This is a schematic diagram of the KIF11 UFMylation immunoblotting results in HEK293T cells transfected with HA-UFM1 and GFP-KIF11 and treated with DMSO or Compound-8 in an embodiment of the present invention.

[0031] Figure 6 The figures are ERG recordings and B wave amplitudes of the retina of BLD mice after injection of DMSO or Compound-8 in an embodiment of the present invention. A is a schematic diagram of ERG recording, B is a schematic diagram of the a wave result of the retina of BLD mice after injection of DMSO or Compound-8, and C is a schematic diagram of the b wave result of the retina of BLD mice after injection of DMSO or Compound-8. ****p < 0.0001.

[0032] Figure 7 These are retinal histological micrographs and quantitative analyses of BLD mice after injection of DMSO or Compound-8, as assessed by H&E staining, in embodiments of the present invention. A represents retinal histological micrographs assessed by H&E staining; B represents the quantitative analysis of the outer nuclear layer thickness of the mouse retina after injection of DMSO or Compound-8 under 600 lux of blue light; C represents the quantitative analysis of the outer nuclear layer thickness of the mouse retina after injection of DMSO or Compound-8 under 2000 lux of blue light; and D represents the quantitative analysis of the density of nuclear cells in the outer nuclear layer of the mouse retina after injection of DMSO or Compound-8 under 600 or 2000 lux of blue light, respectively. ****p < 0.0001;

[0033] Figure 8The images shown are immunofluorescence images of mice injected with DMSO or Compound-8, and quantitative analysis of ciliary axonal length and ciliary density in the retinas of BLD mice. In this embodiment of the invention, A is an immunofluorescence image, B is a quantitative analysis of ciliary axonal length in the retinas of BLD mice, and C is a quantitative analysis of ciliary density in the retinas of BLD mice. p < 0.0001.

[0034] Figure 9 These are immunofluorescence images after intravitreal injection of DMSO or Compound-8 in this embodiment of the invention, and quantitative analysis of the thickness of rod and cone cell membrane discs in the retina of BLD mice. In this example, A is an immunofluorescence image, B is a quantitative analysis of the thickness of rod cell membrane discs in the retina of BLD mice, and C is a quantitative analysis of the thickness of cone cell membrane discs in the retina of BLD mice. ****p < 0.0001. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1: Construction of a mouse model and verification of the UFMylation process of KIF11 under blue light irradiation

[0039] 1. Constructing a BLD mouse model:

[0040] The BLD mouse model was established using C57BL / 6J mice. All experiments used mice with a balanced male-to-female ratio. Eight-week-old mice were used for the light exposure experiment. Mice were divided into experimental and control groups. The experimental group was further divided into a weak blue light group (BL-600, 600 lux blue light exposure) and a strong blue light group (BL-2000, 2000 lux blue light exposure). The control group was divided into a weak yellow light group (YL-600, 600 lux yellow light exposure) and a strong yellow light group (YL-2000, 2000 lux yellow light exposure). The construction process is as follows: Figure 1 As shown. After one day of dark acclimatization, pupil dilation was performed 30 minutes before light exposure using 1% cyclopentanol hydrochloride (Mita Pharmaceutical, Osaka, Japan). The light source was either a blue LED (456 nm, Cree, Durham, North Carolina, USA) or a yellow LED (583 nm, Toshiba, Tokyo, Japan), fixed to the cage wall (10 cm from the top of the cage) and 30 cm away from the mice. The ambient temperature was maintained at 25 ± 1.5 °C during the light exposure. The experimental group mice were exposed to blue light (600 or 2000 lux) for 2 hours daily for 3 days. The control group mice were exposed to yellow light (600 or 2000 lux) for 2 hours daily for 3 days. The 600 and 2000 lux values ​​were chosen because previous studies have shown that these intensities induce photoreceptor cell degeneration; 600 lux simulates indoor lighting conditions, and 2000 lux simulates strong light conditions. After light exposure, the mice underwent a 3-day routine light-dark cycle, followed by a 1-day dark adaptation period.

[0041] 2. Verification of the UFMylation process of KIF11 under blue light irradiation:

[0042] To investigate the molecular mechanism of the decrease in KIF11 protein levels under blue light conditions, further analysis of KIF11 UFMylation was conducted. Western blot analysis showed... Figure 2 As shown in Figures A and B, the UFMylation level of KIF11 in the retina of mice in the blue light irradiation group was significantly lower than that in the same yellow light irradiation group. This indicates that blue light conditions disrupt the UFMylation process of KIF11 and reduce its stability.

[0043] Example 2 verifies the involvement of UFL1-mediated UFMylation in BLD-related retinal functional defects.

[0044] Given that UFL1 depletion leads to retinal dysfunction reproducing BLD-related pathological features, this study investigated whether enhancing UFMylation could improve these retinal abnormalities. To verify this hypothesis, BLD mice were intravitreally injected with the known UFSP2 inhibitor Compound-8 to enhance UFMylation activity and increase the UFMylation level of downstream substrates. The intravitreal injection method of Compound-8 was as follows: Figure 3As shown, specifically: Mice were anesthetized with 2% isoflurane and 0.5% hydroxybuspirone (Sandton Pharmaceuticals), and the medication was applied to the corneal surface. Iris dilation was achieved using 0.5% tropicamide (Sandton), followed by intravitreal injection of 0.5 μL of Compound-8 solution (8.6 mol / L). The Compound-8 solution was prepared by dissolving Compound-8 (PC-20117; ProbeChem, Shanghai Pudong) in DMSO (HY-Y0320C, MCE) and diluting it with 0.9% physiological saline at a ratio of 1:10. Mice were injected intravitreal with 0.5 μL of Compound-8 solution or an equivalent volume of DMSO every two days. After injection, 0.5% prapaccaine and tobradex (Gennan, Arkansas, Switzerland) were applied to the eyes to reduce pain and lower the risk of inflammation. Immunoblot analysis was performed on the retinas of BLD mice injected with DMSO or Compound-8 to detect UFM1, KIF11, UFSP2, and β-actin. Figure 4 As shown in A, Compound-8 significantly and persistently increased the overall UFMylation level in the mouse retina. Figure 4 As shown in Figure B, Compound-8 significantly increased the expression level of KIF11 in the retina of BLD mice. Analysis of KIF11 UFMylation in HEK293T cells transfected with HA-UFM1 and GFP-KIF11 and treated with DMSO or Compound-8 yielded the following results: Figure 5 As shown, Compound-8 significantly promoted UFMylation of KIF11. These results confirm that UFMylation enhances the stability of the KIF11 protein and that enhancing UFMylation can reverse the decrease in KIF11 protein levels caused by blue light exposure.

[0045] Example 3: Evaluation of retinal structure and function in BLD mice after injection of Compound-8

[0046] Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital before ERG analysis. The corneas of mice were desensitized with 0.5% oxybuprocaine hydrochloride, and the pupils were dilated with 0.5% tropicamide-phenylephrine solution. A feedback-controlled temperature control system (TC-100, Eaton) was used to maintain body temperature at 37.0 ± 0.5 °C. Reference electrodes from the RetiMINER-C visual electrophysiology system (IRC Technologies, Bangkok, Thailand) were placed subcutaneously below both ears to ensure a stable reference baseline. Gold wire electrodes were inserted into the tail as grounding electrodes, and recording electrodes were precisely placed on the corneal surface of both eyes. Responsive stimulation to white flashes (3 cd·s / m²) was recorded. 2The dark-adapted electroretinogram (ERG) response. The a-wave amplitude is measured from baseline to the a-wave trough, and the b-wave amplitude is measured from the a-wave trough to the b-wave peak. ERG recordings are as follows: Figure 6 As shown in A, B, and C, blue light irradiation caused a decrease in the amplitude of waves a and b, which was significantly improved after Compound-8 injection. Correspondingly, the histopathological analysis of the H&E-stained sections is as follows: Figure 7 As shown in Figure A, the thinning of the outer nuclear layer of the retina caused by blue light irradiation was largely restored after injection of Compound-8. Quantitative analysis results of the outer nuclear layer thickness and outer nuclear cell density in BLD mice after injection of DMSO or Compound-8 are shown below. Figure 7 As shown in B, C, and D, under blue light irradiation of 600 or 2000 lux, BLD mice injected with Compound-8 showed significant improvements in the thickness of the outer nuclear layer of the retina and the density of nuclear cells in the outer nuclear layer compared to the control group. Immunofluorescence images showing the effect of Compound-8 on ciliary integrity are shown below. Figure 8 As shown in Figure A, the shortening and damage of retinal cilia caused by blue light exposure were alleviated in BLD mice after injection of Compound-8, with both the reduction in cilia length and strength in the mouse retina being mitigated. Quantitative analysis results of ciliary axonal length and ciliary density in the mouse retina are shown below. Figure 8 As shown in B and C, the reduction in ciliary length and strength in cone cells was alleviated under blue light conditions. Immunofluorescence microscopy and quantitative analysis of rod and cone cell membrane disc thickness in the retina of BLD mice after intravitreal injection of DMSO or Compound-8 are shown in Figures [Figure Number]. Figure 9 As shown in A, B, and C, Compound-8 significantly ameliorated the membrane disc damage to rod and cone cells induced by blue light exposure. Overall, these results indicate that UFL1-mediated UFMylation plays a crucial role in the pathogenesis of blue light-induced retinopathy, and targeted regulation of UFMylation may be a potential strategy for treating this type of disease.

[0047] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of Compound-8 in the preparation of drugs to alleviate blue light-induced retinal degeneration, characterized in that, The Compound-8 structure is as follows: 。 2. The use of Compound-8 in the preparation of drugs for treating blue light-induced retinal degeneration, characterized in that the Compound-8 structure is as follows: 。 3. The application as described in claim 1 or claim 2, characterized in that, The Compound-8 reduces blue light-induced retinal degeneration by maintaining the UFMylation-ciliary axis.

4. The application as described in claim 3, characterized in that, Maintaining the UFMylation-cilia axis specifically involves enhancing UFMylation activity, thereby increasing the stability of the KIF11 protein.

5. The application as described in claim 1 or claim 2, characterized in that, The drug has the following functions: (a1) Enhances UFMylation activity in the retina; (a2) Enhances the stability of KIF11 protein in the retina; (a3) Improves the length and density of cilia in cone and rod cells; (a4) Improves the thickness of the outer nuclear layer of the retina; (a5) Improves the thickness of the membrane discs of rod cells and cone cells.

6. The application as described in claim 1 or claim 2, characterized in that, The drug is a formulation prepared with Compound-8 as the active ingredient and pharmaceutically acceptable excipients.

7. The application as described in claim 6, characterized in that, The preparation is an oral preparation, an injectable preparation, or a transdermal preparation.

8. The application as described in claim 7, characterized in that, The injectable formulation is a formulation that can be injected subretinally or intravitreally.

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