阅读说明：本技术 D609在制备预防和治疗视网膜损伤性疾病药物中的应用 (Application of D609 in preparing medicine for preventing and treating retina injury diseases ) 是由 欧阳宏 王力 于 2019-11-18 设计创作，主要内容包括：本发明公开了D609在制备预防和治疗视网膜损伤性疾病药物中的应用。D609其中文名为三环葵烷-9-基-二硫代碳酸酯钾盐,是一种抗病毒及抗肿瘤的小分子化合物,属于一种磷脂酰胆碱特异性磷脂酶(PC-PLC)的选择性抑制剂。本发明经过广泛而深入的研究,首次出乎意料地发现,D609通过上调金属硫蛋白表达,能有效抑制RPE细胞氧化损伤,从而可用于预防或治疗氧化应激损伤视网膜色素上皮细胞引起的视网膜损伤性疾病,尤其是可极其有效地用于预防或治疗视网膜黄斑变性疾病,且无明显药物毒性,能够有效控制视网膜损伤性疾病的发生、发展,为开发D609未知的生物活性及将来的临床治疗作用提供新的理论支持。(The invention discloses an application of D609 in preparing a medicament for preventing and treating retina injury diseases. D609 is named as tricyclodecane-9-yl-dithiocarbonate potassium salt in Chinese, is a small molecular compound with antiviral and antitumor effects, and belongs to a selective inhibitor of phosphatidylcholine-specific phospholipase (PC-PLC). Through extensive and intensive research, the invention discovers for the first time unexpectedly that D609 can effectively inhibit RPE cell oxidative damage by up-regulating metallothionein expression, so that the D609 can be used for preventing or treating retinal damage diseases caused by oxidative stress damage of retinal pigment epithelial cells, particularly can be used for preventing or treating retinal macular degeneration diseases extremely effectively, has no obvious drug toxicity, can effectively control the occurrence and development of the retinal damage diseases, and provides new theoretical support for developing unknown bioactivity of D609 and future clinical treatment effect.)

The application of D609 or pharmaceutically acceptable salts or stereoisomers thereof in preparing a medicament for preventing and/or treating retinal injury diseases singly or in combination with an anti-retinal injury protective medicament, wherein D609 is tricyclodecane-9-yl-dithiocarbonate potassium salt.

2. The use according to claim 1, wherein the retinal damage disease is a retinal damage disease caused by damage to retinal pigment epithelial cells.

3. The use according to claim 2, wherein the retinal damage disease is a retinal damage disease caused by oxidative stress damage to retinal pigment epithelial cells.

4. The use according to claim 2 or 3, wherein the retinal pigment epithelial cells are ARPE-19.

5. The use according to claim 2 or 3, wherein the retinal damaging disease comprises one or more of age-related macular degeneration disease, retinitis pigmentosa disease or chorioretinopathy disease.

6. Use according to claim 5, wherein the age-related macular degeneration disease comprises wet age-related macular degeneration disease and/or dry age-related macular degeneration disease.

7. Use according to claim 1, characterized in that the object applied is a human or an animal.

8. The use of claim 1, wherein the anti-retinal damage protective drug is a cartesian compound, a photosensitizer, an integrin, an antioxidant, an interferon, a xanthopterin derivative, a growth hormone, a neurotrophic factor, a regulator of neovascularization, an anti-VEGF antibody, a prostaglandin, an antibiotic, a phytoestrogen, an anti-inflammatory compound, or an anti-angiogenic compound.

9. The use according to any one of claims 6 to 8, wherein the effective dose of D609 is 10 to 100mg/kg, preferably 20 to 80mg/kg, more preferably 25 to 50mg/kg when the subject is an animal.

10. A pharmaceutical composition for preventing and/or treating a retinal damaging disease, which comprises D609 and a pharmaceutically acceptable carrier; the preparation form is selected from tablets, solutions, suspensions, emulsions, powders, granules, capsules, microcapsules, microspheres, injection, liposomes or aerosols; wherein D609 is used as an active ingredient and accounts for 0.0001-50 wt%, preferably 0.001-20 wt%, and more preferably 0.01-10 wt% of the total weight of the preparation.

Technical Field

The invention belongs to the technical field of biological medicines. More particularly, the invention relates to the application of tricyclodecane-9-yl-dithiocarbonate potassium salt (D609) in preparing a medicament for preventing and treating retinal injury diseases, and develops a new method and thought for preventing and clinically treating ophthalmic related diseases.

Background

The retinal pigment epithelial cells (RPE) are a layer of pigmented cells that lie immediately beneath the retina, and are an aligned monolayer of hexagonal cells located between the retina of the optic nerve and the Brunch's membrane. The maintenance of the normal physiological function of the RPE plays an important role in the normal reception and conduction of light-sensitive signals, which is mainly reflected in: 1) regulating the transport of nutrients from choroidal capillaries to photoreceptor cells; 2) the light is absorbed, the quality of a visual system is improved, and the stimulation of photooxidation is relieved; 3) secreting a number of regulatory factors and signaling molecules to interact with endothelial cells or immune cells; 4) maintaining a fluid environment between the retinal layers, etc. Maintaining the stability of retinal pigment epithelial cells is of great significance to the visual system. Any pathological change in RPE that results in its dysfunction or loss causes secondary damage to the photoreceptor cells. Additional studies have shown that RPE is susceptible to oxidative damage, which plays an important role in the pathogenesis and progression of age-related macular degeneration.

Age-related macular degeneration (AMD), a condition that can lead to blurred vision or central vision impairment, is also known as age-related macular degeneration. In the central area of the fundus of the patient, retinal tissues in the macular area are degenerated and irreversibly diseased, and retinal pigment epithelium area is atrophied. Although the disease does not cause complete loss of vision, the macula lutea is a special structure with a diameter of about 1.5 mm located in the central part of the posterior pole of the retina, and is mainly responsible for fine vision, color vision and the like, as the most acute part of vision, once the macula lutea is damaged, the central vision is seriously affected, and the loss of the central vision may cause obstacles to the daily activities of the patient such as face recognition, driving, reading and the like. The incidence of AMD is mostly age-related and mostly occurs in people over 45 years old, but the precise pathogenesis is not clear in the global scope. AMD is classified as wet AMD (presence of choroidal neovascularization, retinal pigment epithelium detachment, discoid fibrosis, etc.) and dry AMD (presence of soft drusen, geographic atrophy, etc.), wet AMD (wamd) has more severe visual impairment than dry AMD, including resulting in faster vision loss and higher blindness rates. Dry AMD accounts for about 85% to 90% of the total cases of AMD, and dry AMD can be converted to wet AMD.

In developed countries, AMD has become a leading cause of vision loss in the elderly population, and large epidemiological surveys have shown that the prevalence of AMD in western countries is 1.7% to 15.6% in people over 43 years of age. The global AMD sick population reaches 1.7 hundred million, the AMD sick population in China continuously increases along with the aging, and the AMD prevalence rate in the population over 50 years can reach more than 15.5 percent in partial areas. According to the 'prevalence rate of age-related macular degeneration among the elderly population in cities in China' issued by Jiangning ophthalmology, the total number of AMD patients over 50 years old in China is 5082 ten thousand in recent years, and the prevalence rate and the blindness causing rate increase with the age. With the development of socioeconomic level and medical industry, reversible blinding eye diseases such as cataract and keratopathy have been effectively controlled, but AMD, as an irreversible eye disease, has an increasing incidence rate year by year and has become a public health problem. At present, the ophthalmic medicine for treating retinal diseases is mainly a VEGF inhibitor, the traditional AMD treatment methods (laser therapy and photodynamic therapy) have limited effect, are difficult to meet the treatment requirement and are gradually eliminated, and the foreign AMD market takes Noohua (Roche) and regenerative (Bayer) as the leading points, so that the research and development of new AMD treatment methods are increasingly carried out, and the improvement of the curative effect and the compliance are the trend of future development of new AMD medicines.

Tricyclic decanol-9-yl-dithiocarbonate potassium salt (tricyclic decanol-9-yl-xanthogenate, D609) is a small molecule compound with antiviral and antitumor effects, and belongs to a selective inhibitor of phosphatidylcholine-specific phospholipase (PC-PLC). Chinese patent publication No. CN 200410023498.X discloses that D609 can be used to induce vascular endothelial cells to differentiate towards neurons, and D609 has the effect of treating neurodegenerative diseases. D609 reports that D609 has the effect of resisting amyloid β peptide-induced oxidative stress, but the influence of D609 on retinal pigment epithelial cells under oxidative stress state is not reported at home and abroad as to whether D609 has the possibility of reducing the oxidative stress damage of retinal pigment epithelial cells to delay the occurrence and development of AMD and is yet to be researched.

Disclosure of Invention

The invention aims to provide a new application of D609 in preparing a medicament for preventing and treating retinal injury diseases, and develop a new method and thought for prevention and clinical treatment of ophthalmic related diseases.

The above purpose of the invention is realized by the following technical scheme:

the invention relates to an application of D609 or pharmaceutically acceptable salt or stereoisomer thereof in preparing a medicament for preventing and treating retina injury diseases by using the D609 or the pharmaceutically acceptable salt or the stereoisomer thereof singly or in combination with an anti-retina injury protective medicament.

The inventor of the invention has conducted extensive and intensive studies and unexpectedly found for the first time that D609 can effectively inhibit oxidative damage of RPE cells by up-regulating Metallothionein (Metallothionein) expression, has a protective effect on retinal pigment epithelial cells in an oxidative stress state, and has a good inhibitory effect on oxidative damage of the retinal pigment epithelial cells, so that D609 can be used for preventing or treating retinal damage diseases caused by oxidative stress damage of the retinal pigment epithelial cells, especially can be used for preventing or treating macular degeneration diseases of retina very effectively, has no obvious drug toxicity, can effectively control occurrence and development of the retinal damage diseases, and provides a new theoretical support for developing unknown bioactivity of D609 and future clinical therapeutic effect.

In another preferred embodiment, the retinal damage disease is a retinal damage disease caused by damage to retinal pigment epithelial cells.

In another preferred example, the retinal damage disease is a retinal damage disease caused by oxidative stress damage to retinal pigment epithelial cells.

In another preferred embodiment, the retinal pigment epithelial cell is ARPE-19.

In another preferred example, the retinal damage disease includes one or more of age-related macular degeneration disease, retinitis pigmentosa disease, or chorioretinopathy disease.

In another preferred example, the age-related macular degeneration disease includes a wet age-related macular degeneration disease and/or a dry age-related macular degeneration disease.

In another preferred example, the object applied is a human or an animal. By "animal" in the present invention is meant any animal that benefits from the improvement or reduction of the loss or other deterioration of the animal's age-related visual system, including humans, birds (avians), bovine (bovine), canine (Canine), equine (equine), feline (feline), caprine (hicrine), wolfine (lupine), murine (murine), ovine (ovine), and porcine (porcine), and preferably domestic animals. Animals domesticated therein include animals such as dogs, cats, birds, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, recreational horses, cows, goats, sheep, donkeys, pigs, and more exotic species fed by humans.

In another preferred embodiment, the anti-retinal damage protective drug is a steroid, a light sensitizer, an integrin, an antioxidant, an interferon, a xanthine derivative, a growth hormone, a neurotrophic factor, a regulator of neovascularization, an anti-VEGF antibody, a prostaglandin, an antibiotic, a phytoestrogen, an anti-inflammatory compound, or an antiangiogenic compound.

In another preferred embodiment, thalidomide, verteporfin, a vasostabilizing steroid, rhuFab, interferon-2 α, or pentoxifylline, or a pharmaceutically acceptable salt or stereoisomer thereof.

In another preferred embodiment, when the subject is an animal (e.g., mouse), the effective dose of D609 is 10-100 mg/kg, preferably 20-80 mg/kg, and more preferably 25-50 mg/kg.

When the application object is a human, the dosage can be correspondingly converted into the dosage of the human body according to the relevant administration standard in the pharmaceutical field. As another preferred example, when the subject is a human, the effective dose of D609 is about 1000-2500 mg, and in the case of a single dose, the dose can be administered several times a day.

In another preferred embodiment, when the subject is human or animal, the effective concentration of D609 is 1-50 μ g/mL, preferably 5-30 μ g/mL, more preferably 10-20 μ g/mL.

In another preferred embodiment, the pharmaceutical preparation is in the form of tablet, solution, suspension, emulsion, powder, granule, capsule, microcapsule, microsphere, injection, liposome or aerosol.

In another preferred embodiment, the drug is a sustained or controlled release formulation or not.

The invention also relates to a pharmaceutical composition for preventing and/or treating the retinal injury diseases, which is characterized by comprising D609 and a pharmaceutically acceptable carrier.

In another preferred embodiment, in the pharmaceutical composition of the present invention, D609 is used as the only active ingredient and accounts for 0.0001 to 50 wt% (preferably 0.001 to 20 wt%, more preferably 0.01 to 10 wt%) of the total weight of the preparation, and the rest is pharmaceutically acceptable carriers and other additives.

In another preferred embodiment, in the pharmaceutical composition of the present invention, D609 is used as one of the active ingredients to constitute the pharmaceutical composition together with other active ingredients, wherein D609 is used as one of the active ingredients to constitute 0.0001 to 50 wt% (preferably 0.001 to 20 wt%, more preferably 0.01 to 10 wt%) of the total weight of the preparation.

In another preferred embodiment, the site of application of D609 may be applied directly to the retina or its surrounding tissue. When used for the prevention or treatment of diseases associated with retinal cell damage, intraocular administration (e.g., intravitreal administration) is preferred. Thus, the pharmaceutical composition may be in the form of an ophthalmic formulation, such as an intraocular injection. Any pharmaceutical carrier suitable for intraocular injection is useful.

Compared with the prior art, the invention has the following beneficial effects:

the invention unexpectedly discovers for the first time that D609 can effectively inhibit oxidative damage caused by iodate on RPE cells, remarkably improves the survival rate of PRE cells, can remarkably reduce the release level of lactate dehydrogenase under oxidative stress, has stronger protective effect on the damage of the RPE cells after D609 is given, and can restore the lactate dehydrogenase under the oxidative stress to a normal level, thereby effectively reducing the damage degree of the RPE cells, and has no obvious toxic effect on normal ARPE-19 cells, which shows that D609 can effectively treat the damage of retinal pigment epithelium. In addition, experiments in animal bodies show that compared with a control group, an experimental group which injects D609 into the tail vein of a macular degeneration model mouse at the same time can effectively prevent the damage of the retinal pigment epithelium layer by the D609, has higher drug effect and no obvious drug toxicity, and the D609 can become a strong and effective drug for clinically treating AMD in the future.

Drawings

FIG. 1 shows the screening process of D609.

FIG. 2 shows the cell number and morphology change of ARPE-19 in each experimental group after D609 (10. mu.g/mL) is administered for 12h and 24 h.

FIG. 3 is a graph showing the cell survival (%) of ARPE-19 in each experimental group measured by CCK8 at 18 hours and 24 hours after D609 (10. mu.g/mL) administration.

FIG. 4 is a graph showing the lactate dehydrogenase release levels in each experimental group 24h after D609 (10. mu.g/mL) administration.

FIG. 5 shows the immunofluorescence staining pattern of DAPI and HMBG1 for ARPE-19 cells of each experimental group.

FIG. 6 is a graph showing the expression level of Metallothionein (MT) protein in ARPE-19 cells damaged by D609 (10. mu.g/mL) in response to SI oxidative stress.

FIG. 7 is a graph showing immunofluorescence staining of F-actin and ZO-1 on eyeball retinal pigment epithelial layer tissues of mice in each experimental group.

FIG. 8 shows the effect of different concentrations of D609(25mg/kg, 50mg/kg) on mice in each experimental group; wherein, as can be seen from the A diagram of FIG. 8, the optimal concentration of D609 is 25mg/kg, and the morphology of the cells is not good at 25mg/kg until 50 mg/kg; while in the B diagram of FIG. 8, the SI of a given concentration is matched with D609 of a different concentration, and the best effect of 25mg/kg can be seen; fig. 8, panel C, shows that both SI and D609 had no significant effect on organs such as the liver and kidney.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

D609 (tricyclodecane-9-yl-dithiocarbonate potassium salt)

D609 is a selective phosphatidylcholine-specific phospholipase C (phosphatidyl choline-specific phospholipase C) inhibitor. D609 has the following chemical formula:

an isomer, racemate, pharmaceutically acceptable salt, hydrate or precursor of D609 is also included in the present invention as long as they have the same or close function of preventing cell damage as D609. The "pharmaceutically acceptable salt" refers to a salt formed by reacting D609 or an analogue thereof with an inorganic acid, an organic acid, an alkali metal, an alkaline earth metal or the like. These salts include (but are not limited to): (1) salts with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts with organic acids such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine. By "precursor" is meant a precursor of the compound which, when administered by an appropriate method, undergoes a metabolic or chemical reaction in the mammal to convert the compound to a compound of formula (I) or an analog thereof.

The D609 or the isomer, racemate, pharmaceutically acceptable salt, hydrate or precursor thereof can be obtained by chemical synthesis.

Use for preventing retinal damage

The invention provides a use of D609 for preparing a preparation for preventing or treating diseases related to the retinal damage of a mammal.

To demonstrate the above-mentioned use of D609, the present inventors applied D609, and observed that D609 could protect retinal pigment epithelial cells from toxic substance invasion and oxidative damage through its interventional study in a model of sodium iodate-damaged retinal pigment epithelial cells; in a cell experiment, D609 is proved to protect cells from the generation of cell mitochondrial damage and cell apoptosis and necrosis caused by sodium iodate; and revealed that D609 antagonizes the occurrence of cell death by oxidative stress by up-regulating the expression of an antioxidant protein family, metallothionein.

Examples of anti-retinal damage protective drugs include, but are not limited to, common therapeutic agents for treating or preventing macular degeneration, such as steroids, photosensitizers, integrins, antioxidants, interferons, xanthine derivatives, growth hormones, neurotrophic factors, neovascularization modulators, anti-VEGF antibodies, prostaglandins, antibiotics, phytoestrogens, anti-inflammatory compounds, or antiangiogenic compounds specific examples of anti-retinal damage protective drugs include, but are not limited to, thalidomide, verteporfin, a vasostabilizing steroid, rhuFab, interferon-2 α, or pentoxifylline, or a pharmaceutically acceptable salt or stereoisomer thereof.

As used herein, unless otherwise indicated, the term "treating a retinal damaging disorder" refers to administering a compound of the invention or other active agent after the onset of symptoms of the retinal damaging disorder, while "preventing" refers to administering the compound or other active agent prior to the onset of symptoms, particularly to a patient at risk of a retinal damaging disorder. As used herein, unless otherwise indicated, the term "controlling a retinal damaging disorder" includes preventing the recurrence of a retinal damaging disorder in a patient who has suffered from a retinal damaging disorder, and/or prolonging the time a patient who has suffered from a retinal damaging disorder remains in remission.

The present invention includes methods of treating, preventing and managing retinal damage diseases and related syndromes in patients with various stages and specific types of disease, including but not limited to wet age-related macular degeneration disease, dry age-related macular degeneration disease, age-related maculopathy (ARM), Choroidal Neovascularization (CNVM), retinal Pigment Epithelial Detachment (PED), and Retinal Pigment Epithelial (RPE) atrophy.

Application of preventing and treating age-related macular degeneration diseases

In a particularly preferred embodiment of the present invention, D609 is particularly effective in preventing damage to retinal pigment epithelial cells (RPE), and thus can be used for prevention and treatment of age-related macular degeneration (AMD).

Retinal pigment epithelial cells play an important role in human eye imaging, and their functions include: 1) transport of nutritional and metabolic waste products in the retina and capillaries; 2) adjusting the components and volume of the electrolyte around the retina; 3) phagocytosis of shed tissue fragments; 4) participate in the metabolic cycle of vitamin A and its analogues. Although the pathogenesis of AMD involves different clinical symptoms, a decline in RPE cells is often observed in the early stages of pathogenesis. The primary AMD pathogenesis includes abnormal RPE morphology and pigmentation and lipofuscin accumulation within the RPE cells.

AMD is mainly characterized in that the phagocytic and digestive capacity of retinal pigment epithelial cells to the outer segment disc membrane of the eye cells is reduced, and as a result, the residual bodies of the disc membrane which are not completely digested are retained in the primary pulp of basal cells and are discharged out of the cells and are deposited on the Bruch membrane to form drusen. This change is more pronounced due to the structural and functional particularities of the macula. Drusen are also found in the elderly with normal vision, but as a result of the secondary pathological changes, macular degeneration occurs.

Oxidative damage is closely related to age-related macular disease (AMD), such as solar radiation, air pollution, smoking, and low intake of antioxidant nutrients. Sodium iodate can cause oxidative damage to retinal pigment epithelial cells.

There is no effective treatment and fundamental preventive measure in the art to date. In recent years, most researchers have advocated the early laser photocoagulation of new blood vessels to exudative patients to avoid the disease from deteriorating. Argon laser can effectively seal the subretinal new blood vessels, so that the argon laser is applied more at present. But has certain damage to the nerve epithelial layer, and laser photocoagulation is only used for sealing existing new blood vessels and cannot block the formation of the new blood vessels, so that the laser photocoagulation is symptomatic treatment. Meanwhile, the laser can generate new blood vessels in the choroid by itself in a slight excess.

The invention surprisingly finds for the first time that D609 is particularly effective in preventing damage to retinal pigment epithelial cells and is thus useful in the prevention and treatment of age-related macular degeneration.

Composition comprising a metal oxide and a metal oxide

The invention provides a pharmaceutical composition comprising: (a) an effective amount of D609, or a pharmaceutically acceptable salt or stereoisomer thereof; and (b) a pharmaceutically acceptable carrier or excipient.

In the present invention, the term "comprising" means that various ingredients can be used together in the mixture or composition of the present invention. Thus, the terms "consisting essentially of … …" and "consisting of … …" are included in the term "comprising".

In the present invention, a "pharmaceutically acceptable" component is a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. A "pharmaceutically acceptable carrier" is a pharmaceutically or comestibly acceptable solvent, suspending agent or excipient for delivering 609, or a physiologically acceptable salt thereof, of the invention to an animal or human. The carrier may be a liquid or a solid.

The dosage form of the pharmaceutical composition of the present invention may be various, and any dosage form may be used as long as it can allow the active ingredient to efficiently reach the body of a mammal. Such as may be selected from: tablets, solutions, suspensions, emulsions, powders, granules, capsules, microcapsules, microspheres, injections, liposomes or aerosols. Wherein 609 may be present in a suitable solid or liquid carrier or diluent.

When used for the prevention or treatment of diseases associated with retinal cell damage, intraocular administration (e.g., intravitreal administration) is preferred. Thus, the pharmaceutical composition may be in the form of an ophthalmic formulation, such as an intraocular injection. Any pharmaceutical carrier suitable for intraocular injection is useful.

D609 of the present invention and compositions thereof may also be stored in a sterile device suitable for injection or instillation. Generally, in the pharmaceutical composition of the present invention, D609 is 0.0001 to 50 wt% (preferably 0.001 to 20 wt%, more preferably 0.01 to 10 wt%) of the total weight of the preparation as an active ingredient, and the rest is pharmaceutically acceptable carriers and other additives.