Application of H008 in treating age-related macular degeneration (AMD)

Abstract

The invention provides application of H008 in preparation of medicines for preventing and treating age-related macular degeneration, and belongs to the technical field of chemical medicines. It is reported for the first time that the small molecule medicine H008 can improve apoptosis of H2O2-induced ARPE-19 cells and improve the retinal function and form of AMD model mice, and a guiding effect is provided for treatment of age-related macular degeneration.

Description

Technical Field

[0001] This invention belongs to the field of chemical drug technology, specifically relating to the application of H008 in the treatment of age-related macular degeneration (AMD). Background Technology

[0002] Age-related macular degeneration (AMD) is a degenerative disease of the retinal tissue in the macular region. The retina is a crucial structure for vision formation and is also highly susceptible to light damage. The pathogenesis of AMD is related to the cumulative effects of long-term exposure to low-intensity light. The location of retinal damage is similar to the pathological changes in AMD and other diseases, with the outer retina, especially the outer segment of the photoreceptors, being affected earliest. In all stages of age-related macular degeneration, retinal pigment epithelial (RPE) cells are one of the central components of the disease process.

[0003] The active ingredient of Kemprazan hydrochloride tablets is 1-[5-(2-fluorophenyl)-1-((3-(3-methoxypropoxy)phenyl)sulfonyl)-1H-pyrrolo-3-yl]-N-methylmethylamine hydrochloride (hereinafter referred to as H008). Chinese patent CN201580069871.1 discloses the synthesis method of this compound, and its molecular formula is C008. 22 H 25 FN2O4S·HCl.

[0004] For AMD, a disease that severely impairs vision, there is currently no ideal treatment. Therefore, research on the prevention and treatment of AMD is of great significance for preventing and treating blindness. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a medicament for the prevention or treatment of age-related macular degeneration (AMD), comprising: a compound represented by Formula I or a pharmaceutically acceptable salt thereof.

[0006]

[0007] In one embodiment, the pharmaceutically acceptable salt of the compound of Formula I is one of hydrochloride, fumarate, succinate, and methanesulfonate, preferably hydrochloride. The structure is shown in Formula II.

[0008]

[0009] This invention reports that H008 can improve hydrogen peroxide-induced apoptosis in human retinal pigment epithelial cells (ARPE-19).

[0010] This invention reports that a single intravitreal injection of H008 can improve retinal function and morphology in a mouse model of AMD.

[0011] This invention provides the application of H008 in the treatment of age-related macular degeneration (AMD). This invention is the first to report that the small molecule drug H008 can improve hydrogen peroxide-induced apoptosis in ARPE-19 cells and improve retinal function and morphology in AMD mice, providing guidance for the treatment of AMD. Detailed Implementation

[0012] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0013] Example 1

[0014] To verify the protective effect of H008 on human retinal pigment epithelial cells (ARPE-19), this study investigated the effect of H008 on hydrogen peroxide-induced apoptosis in ARPE-19 cells. ARPE-19 cells were cultured in DMEM complete medium at 37°C in a 5% CO2 cell culture incubator, digested with trypsin, and passaged. Cells with vigorous proliferation and good condition were used for the experimental study. The experimental groups were divided into five groups: ① control group; ② H2O2 group; ③ Group A (H2O2 + 25 μmol / L H008); ④ Group B (H2O2 + 50 μmol / L H008); ⑤ Group C (H2O2 + 100 μmol / L H008). Each treatment group was first cultured with the corresponding concentration of H008 for 24 h. After 24 h of H008 treatment, H2O2 was simultaneously added to both the treatment groups and the H2O2 group, and the cells were cultured for another 24 h. Then, the apoptosis rate was measured.

[0015] Apoptosis rate was detected by flow cytometry: Logarithmic growth phase cells were seeded into 6-well culture plates and cultured at 37°C with 5% CO2. After adhesion, cells were treated with 0, 25, 50, or 100 μmol / L H008 for 24 h, respectively, followed by H2O2 treatment and incubation for 24 h. Culture was terminated at the same time for all groups. Cells were digested with trypsin and collected into 1.5 mL Eppendorf tubes. Cells were washed with PBS, the supernatant was discarded, and cells were resuspended in Binding Buffer. Annexin V-FITC and PI were added and mixed. Flow cytometry analysis was performed, and data were analyzed. SPSS 17.0 statistical software was used for data processing. All data are expressed as mean ± SD. One-way ANOVA was used to compare differences between groups, and P < 0.05 was considered statistically significant. The results showed that H2O2 significantly increased the apoptosis rate of ARPE-19 cells, and the difference was statistically significant compared with the control group (P<0.05). Incubation with different concentrations of H008 significantly reduced the apoptosis rate of ARPE-19 cells, and the difference was statistically significant compared with the H2O2 group (P<0.05), and the effect was dose-dependent (see Table 1).

[0016] Table 1. Effect of H008 on H2O2-induced apoptosis rate of ARPE-19 cells (mean ± SD, n = 8)

[0017]

[0018] Compared with the control group, *P<0.05; compared with the H2O2 group, # P < 0.05.

[0019] Example 2

[0020] To verify the effect of H008 on AMD in rats, a retinal photodamage model was established by continuously irradiating SD rats with a white cold light source for 24 hours. H008 or a solvent control (sterile phosphate-buffered saline (PBS) less than 1% DMSO) was injected intravitreally once. One week after intravitreal injection of 320 μg of H008 in each eye, retinal function was examined using electroretinography (ERG). Electrophysiological instruments were used to record ERG signal responses, and all ERG procedures were performed in a dark room under dim red light. After 2 hours of dark adaptation, both eyes were instilled with compound tropicamide eye drops to dilate the pupils once, followed by anesthesia 5 minutes later, and then compound tropicamide was instilled again. Once the rats' breathing was stable and they were under anesthesia, both eyes were topically anesthetized with tetracaine. The rats were fixed on the examination table, and a self-made ring-shaped platinum wire electrode was placed in the measuring eye, ensuring maximum contact with the cornea, and lubricated with methylcellulose. The reference electrode was a needle placed under the tongue, and the grounding electrode was placed at the tail. The skin was wiped with alcohol to ensure maximum contact between the electrodes and the skin. During the procedure, red light was turned off, and a single white flash stimulus was applied. Each eye was measured three times, with a 20-second interval between each stimulus. The scotopic mixed response was recorded, and the b-wave amplitude was measured and statistically analyzed. Results showed that the b-wave amplitude of the scotopic mixed response in normal rats was (286.453±88.340) μV, while in the model control group, the b-wave amplitude decreased to (91.549±41.376) μV, which was 31.96% of the amplitude before light exposure. This difference was significant compared to the normal group (P<0.05). The b-wave amplitude of the scotopic mixed response in the H008 group was 49.3% of the amplitude before light exposure, which was significantly different from both the normal group and the model control group (P<0.05). (See Table 2). This indicates that H008 can improve retinal function in AMD model mice.

[0021] Table 2. Changes in b-wave amplitude in ERG scotopic mixed response (mean ± SD, n = 8)

[0022]

[0023] Compared with the control group, *P<0.05; compared with the model control group, # P < 0.05.

[0024] Morphological observation: Eyeballs were immediately removed after cervical dislocation and fixed in a mixed fixative solution. After 3 hours, they were transferred to 4% paraformaldehyde for overnight fixation. The eyeballs were dissected along the marked suture line and the optic nerve line, and the temporal side was harvested. The lens was removed, and the superior side of the eyeballs was dissected along the marked suture line and the optic nerve line again. The eyes were dehydrated with graded alcohols, cleared with xylene, embedded in paraffin, and sectioned to a thickness of 5 μm. The sections were routinely dewaxed, hydrated with double-distilled water, and stained with hematoxylin and eosin (HE). The sections were mounted with neutral resin. Photographs were acquired using a digital imaging system.

[0025] The electron microscopy examination procedure is as follows: the animal is deeply anesthetized, euthanized by cervical dislocation, and the eyeball is immediately removed and fixed overnight in 3% glutaraldehyde. Under a dissecting microscope, the anterior segment of the eye is removed, the eye cup is preserved, the optic disc is removed and fixed, and the eyeball is dehydrated stepwise with acetone and alcohol. The eyeball is embedded in epoxy resin, ultrathin sections are prepared, and the sections are stained with uranium acetate and lead citrate. The ultrastructure is observed under a transmission electron microscope, and satisfactory areas are selected for imaging.

[0026] The results showed: ① Normal control group (light microscopy): The retina was morphologically normal, with clear structural layers. The photoreceptors were arranged neatly and regularly in the inner and outer segments, with clear layers, distinct structure, and uniform staining. The outer nuclear layer was regular in shape, approximately 12-13 nuclei thick. The RPE layer was neatly arranged and normal in shape. (Electron microscopy): The outer segment membrane discs were densely arranged in a lamellar pattern. The inner segment mitochondria were abundant, regularly arranged, with continuous cristae, clear structure, and oval or elongated spindle shape. The outer nuclear layer nuclei were regular in shape, and the chromatin was evenly distributed. ② Model control group (light microscopy): The inner and outer segments of the retina were disordered, with unclear boundaries. The outer segment structure was blurred, the stacked structure was dissociated, and it showed vacuolar degeneration with no normal structure observed. The inner segment mitochondria were swollen, and the inner cristae were widened. The outer nuclear layer was edematous, degenerated, and thinned, approximately 5-6 nuclei thick, with large intercellular spaces, chromatin aggregation, nuclear membrane shrinkage, and many condensed nuclei were visible. Electron microscopy: The outer segment membrane of the photoreceptor is swollen and blurred, with disordered arrangement, large intermembranous spaces, and separation, disintegration, and vacuolar degeneration. The inner segment mitochondria are swollen, with cristae broken, and some show vacuolar degeneration. The outer nuclear layer shows extensive nuclear membrane shrinkage and invagination, chromatin aggregation and uneven concentration, and numerous apoptotic cells. ③ H008 group (light microscopy): The retinal structure is less damaged, the boundary between the inner and outer segments is clear, the structure is distinct, and the outer nuclear layer is neatly arranged, approximately 10-11 nuclei thick. Electron microscopy: The retinal damage is mild, showing only slight disordered arrangement of the outer segment membrane discs and slight swelling of the inner segment mitochondria. No obvious changes in apoptotic cells are seen in the outer nuclear layer. The RPE layer of the retina in all groups is relatively neatly arranged and morphologically regular, with no obvious pathological changes. No obvious pathological changes, inflammation, or necrotic cells are seen in the inner nuclear layer and ganglion cell layer.

[0027] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A medicament for preventing or treating age-related macular degeneration (AMD) comprising: a compound represented by Formula I or a pharmaceutically acceptable salt thereof, 2. The medicament according to claim 1, characterized in that, the pharmaceutically acceptable salt of the compound of Formula I is one of hydrochloride, fumarate, succinate, and methanesulfonate, and is preferably hydrochloride.

3. The medicament according to claim 1, characterized in that, Apoptosis of human retinal pigment epithelial cells (ARPE-19) induced by hydrogen peroxide can be improved.

4. The medicament according to claim 1, characterized in that, Retinal function and morphology in an AMD model mouse model can be improved after a single intravitreal injection.

5. The medicament according to claim 1, characterized in that, It can be used for preventing or treating age-related macular degeneration.

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