A method of regulating neuropathy

Abstract

The present invention relates to a method for regulating neuropathy, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

Description

[0001] Related Applications

[0002] This application is a divisional application of the Chinese invention patent application filed on May 20, 2022, with invention name “A method for regulating neuropathy” and application number 202280027840.X.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to the Chinese invention patent application No. 202110560773.5 filed on May 21, 2021, the contents of which are incorporated herein by reference in their entirety.

[0005] This application claims priority to the Chinese invention patent application with application number 202210110224.2 filed on January 28, 2022, the contents of which are incorporated herein by reference in their entirety.

[0006] This application claims that the Chinese invention patent application with application number 202010794864.0 filed on August 10, 2020 is incorporated herein by reference in its entirety.

[0007] This application claims that the international patent application with application number PCT / CN2021 / 106355 filed on July 14, 2021 is incorporated herein by reference in its entirety. Field of the Invention

[0008] The present invention relates to the field of neuropathy, and more particularly to methods for regulating neuropathy, especially methods for treating, preventing and improving diseases, disorders or conditions associated with neuropathy. Background Art

[0009] Neuropathy includes central neuropathy and peripheral neuropathy. Common and severe central neuropathy in clinical practice includes cerebrovascular disease (such as stroke, traumatic brain injury), neurodegenerative diseases, optic neuropathy, etc. Cerebrovascular disease causes more than 1.5 million deaths each year, of which stroke is one of the main clinical types. About 82% of stroke patients suffer from ischemic stroke. Clinical treatment measures mainly include intravenous thrombolysis and intravascular interventional therapy, timely restoration of cerebral blood flow, reconstruction of blood circulation in the ischemic area, and rescue of the ischemic penumbra. Traumatic brain injury is the leading cause of death and disability. In 2019, there were approximately 61,000 TBI-related deaths in the United States. There are approximately 166 deaths related to traumatic brain injury (TBI) every day.

[0010] Currently, intravenous thrombolysis (IVT) such as alteplase for injection (produced by Boehringer Ingelheim Pharma GmbH & Co. KG) is given within 3 hours after the onset of acute ischemic stroke. Alteplase is the only drug approved by the FDA for the treatment of acute ischemic stroke. Due to the strict time window limit of intravenous thrombolysis, only about 3% of patients benefit from this treatment. Currently, 70% of stroke patients still have varying degrees of labor loss, of which more than 40% are severely disabled. At the same time, some studies have shown that intravenous thrombolysis cannot reduce the 3-6 month mortality rate of patients after stroke (EMBERSON J, LEES KR, LYDEN P, et al. Effect of treatment delay, age, and stroke severity on the effects of intravenous thrombolysis with alteplase for acute ischaemic stroke: a meta-analysis of individual patient data from randomised trials [J]. Lancet, 2014, 384(9958): 1929-1935.). In particular, in patients with large vessel occlusion or severe disease, the effect of intravenous thrombolysis is often poor, with a vascular recanalization rate of only 13% to 18% (GUPTA R, JOVIN T G. Endovascular management of acute ischemic stroke: advances in patient and treatment selection [J]. Expert Rev Neurother, 2007, 7(2): 143-153).

[0011] One of the main goals of treating stroke is neuroprotection. For patients who have already suffered a stroke, it is extremely important to prevent and delay the death of nerve cells, which can retain more nerve cells for functional recovery and buy more time for treatment. However, there is currently a lack of safe and effective neuroprotectants, and neuroprotection is a serious unmet medical need in the clinic (Wu, S. et al. Stroke in China: advances and challenges in epidemiology, prevention, and management. Lancet Neurol. 18, 394–405 (2019).; Kim, AS, Cahill, E. & Cheng, NT Global Stroke Belt: Geographic Variation in Stroke Burden Worldwide. Stroke 46, 3564–3570 (2015)).

[0012] Dementia is a syndrome of cognitive, emotional and social function decline. It is the most common neurodegenerative disease and is currently the seventh leading cause of death among all diseases. It is also one of the main causes of disability and dependence among the elderly worldwide. According to statistics from the World Health Organization (WHO), there are currently about 55 million people suffering from dementia in the world, and there are nearly 10 million new cases each year. The global annual burden is estimated to be about 1 trillion US dollars. It is estimated that there will be 135 million dementia patients in the world by 2050.

[0013] There are many types of dementia, mainly including Alzheimer's disease, vascular dementia, Lewy body dementia and frontotemporal dementia. Dementia may also occur after a stroke or in certain infections, repetitive physical trauma to the brain, or nutritional deficiencies. The boundaries between different forms of dementia are blurred, but cerebrovascular damage is often present in various dementias (Gg A, CqA, Lfa B. Prevention of dementia in an ageing world: Evidence and biological rationale. Ageing Research Reviews, 2020, 64: 101045).

[0014] In the past 40 years, a large number of clinical trials for the treatment of dementia have failed. Currently, there is a lack of safe and effective neuroprotective agents in clinical practice, and neuroprotection is a serious unmet medical need in clinical practice (Jca B, Gl B, Ar B, et al. Alzheimer's disease drug development pipeline: 2019. Alzheimer's & Dementia: Translational Research & Clinical Interventions, 2019, 5: 272-293; Panza F, Lozupone M, Logroscino G, et al. A critical appraisal of amyloid-β-targeting therapies for Alzheimer disease. Nature Reviews Neurology, 2019, 15(2): 73-88).

[0015] Retinal ischemia-reperfusion (I / R) injury is a pathophysiological process of cell damage that leads to a variety of ocular diseases such as optic neuropathy, including glaucoma, ischemic optic neuropathy, retinal vascular occlusion, and various retinopathy (Khanh Vu, Dong Feng Chen. "CD4+T-Cell Responses Mediate Progressive Neurodegeneration in Experimental Ischemic Retinopathy." The American Journal of Pathology 190, no. 8 (August 2020): 1723–34). Retinal ischemia-reperfusion (I / R) induces damage to retinal ganglion cells (RGCs) and the inner retina, which is a common cause of visual impairment in middle-aged people, irreversible visual impairment and blindness in the elderly. It is a serious unmet medical need and there is no effective treatment (Soares ROS, Losada DM, Jordani MC, et al. Ischemia / Reperfusion Injury Revisited: An Overview of the Latest Pharmacological Strategies[J]. International Journal of Molecular Sciences, 2019, 20(20): 5034).

[0016] Glaucoma is a group of eye diseases characterized by progressive retinal ganglion cell (RGC) death and typical visual field loss, and has become the leading cause of irreversible blindness worldwide. Currently, clinical treatment options are mainly based on lowering intraocular pressure, which only protects the visual function of some glaucoma patients. Therefore, it is crucial to develop new treatments to provide safe and effective RGC neuroprotection. In short, there is still a lack of safe and effective drugs for these diseases in clinical practice, so there is an urgent need to find new drugs and therapies.

[0017] Retinal vein occlusion (RVO) is a retinal vascular disease that causes severe vision loss. Patients with RVO may develop neovascular glaucoma (NVG) and become completely blind. Currently, intravitreal injection of anti-VEGF drugs is the first-line treatment. However, due to the short half-life of the drug, the drug requires frequent intraocular injection, which increases the potential risk of endophthalmitis and produces problems such as easy rebound. Long-term use can cause partial atrophy of the pigment epithelium and photoreceptor layer. Therefore, there is an urgent need to find new drugs for the treatment of RVO.

[0018] Diabetes is an increasingly prevalent disease that has a significant impact on human health and quality of life. It is estimated that by 2040, the number of people with diabetes between the ages of 20 and 79 will reach 642 million worldwide, accounting for about 8.8% of the global population. Up to 50% of diabetic patients will develop diabetic peripheral neuropathy (DPN). DPN patients require higher care costs, and patients will incur huge physical, psychological and economic burdens.

[0019] Existing treatments and drugs still perform poorly in preventing and controlling the progression and effectiveness of DPN. Epalrestat is a novel aldose reductase inhibitor of the polyol pathway that can improve the symptoms of diabetic peripheral neuropathy and peripheral nerve conduction velocity. However, the use of Epalrestat can cause some side effects, especially long-term use can cause allergies, liver and kidney function and digestive system adverse reactions, resulting in poor treatment effect and failure to control the progression of neuropathy in the long term (Hotta, Nigishi, Akanuma et al. Long-Term Clinical Effects of Epalrestat, an Aldose Reductase Inhibitor, on Diabetic Peripheral Neuropathy. [J]. Diabetes Care, 2006; Sato K, Yama K, Yu M, et al. Epalrestat increases intracellular glutathione levels in Schwann cells through transcription regulation [J]. Redox Biology, 2014; Epalrestat. Reactions Weekly, 2011, 1369 (1): 18-18; Fujiise Y, Koda M, Kato J, et al. Drug-induced hepatic injury caused by an aldose reductase inhibitor, epalrestat[J]. Kanzo, 2011, 52(6): 351-355.). Currently, epalrestat has not been approved by the US FDA. Therefore, there is currently no drug that can control the progression of neuropathy in patients with DPN for a long time, and the clinical prevention and treatment of diabetic peripheral neuropathy urgently needs to find new drugs and therapies (Zakin E, Abrams R, Simpson DM. Diabetic Neuropathy[J]. Seminars in Neurology, 2019, 39(05): 560-569).

[0020] A considerable number of DPN patients suffer from severe neuropathic pain, namely diabetic peripheral neuropathy (DPNP). This places a huge burden on patients' lives, leading to rising unemployment rates and an increase in the proportion of mental health disorders and physical comorbidities. Therapies for diabetic painful peripheral neuropathy aim to relieve symptoms, but current drugs are only effective for one-third of patients, and cannot completely relieve pain, and are accompanied by side effects or poor efficacy. Therefore, it is crucial to develop new potential therapeutic targets to alleviate diabetic painful peripheral neuropathy (Gordon S, Pallai S, Dinesh S, et al. A new look at painful diabetic neuropathy [J]. Diabetes Research and Clinical Practice, 2018, 144: 177-191.).

[0021] Doxazosin is clinically used for symptomatic treatment of benign prostatic hyperplasia and hypertension. It is usually taken orally in adults, with an initial treatment dose of 1 mg once a day, and the dose is adjusted after 1-2 weeks based on clinical response and tolerance; the maintenance treatment dose is 1-8 mg once a day, with a maximum dose of 16 mg / day. Doses exceeding 4 mg are likely to cause postural hypotension.

[0022] Pramipexole is clinically used to treat the signs and symptoms of idiopathic Parkinson's disease in adults, and is also used for the symptomatic treatment of moderate to severe idiopathic restless legs syndrome. Adults are usually given orally three times a day, with an initial dose of 0.375 mg per day, and then the dose is increased every 5-7 days, with a maximum daily dose of 4.5 mg; the maintenance treatment dose is between 0.375 mg and 4.5 mg per day.

[0023] Metoprolol is an antiarrhythmic drug clinically, and its indications include hypertension, angina pectoris, etc. The common dose for treating hypertension in adults is 100-200 mg / time, twice a day. When used to treat acute myocardial infarction, it is recommended to use this drug in the early stage, that is, in the first few hours. The general usage is to first intravenously inject metoprolol 2.5-5 mg / time (within 2 minutes), once every 5 minutes, for a total of 3 times with a total dose of 10-15 mg. After 15 minutes, start taking this drug orally at 25-50 mg / time, once every 6-12 hours, for a total of 24-48 hours, and then take 50-100 mg / time orally, twice a day.

[0024] U.S. Patent No. US10117868 B2 mentions a method for treating retinal light-induced retinal degeneration, abnormal all-trans retinal clearance or ocular diseases related to the production of reactive oxygen species using a combination of doxazosin and metoprolol. There are also literature reports on the effect of a combination of metoprolol, bromocriptine and doxazosin on inhibiting early diabetic retinopathy (DR) in diabetic mice (Kern TS, Du Y, Tang J, et al. Regulation of adrenergic, serotonin and dopamine receptors to inhibit diabetic retinopathy: monotherapies versus combination therapies [J]. Molecular Pharmacology, 2021: MOLPHARM-AR-2021-000278.). SUMMARY OF THE INVENTION

[0025] The present invention provides a method for regulating neuropathy, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof.

[0026] The method for regulating neuropathy involves inhibiting neuroinflammation or repairing nerve damage. In certain embodiments, the method inhibits neuroinflammation to treat, prevent or improve diseases associated with neuroinflammation, including ischemic stroke, hemorrhagic stroke, transient ischemic attack, craniocerebral injury, vascular dementia, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, glaucoma, ischemic optic neuropathy, optic neuritis, optic nerve tumors, traumatic optic neuropathy, retinal artery occlusion, retinal vein occlusion and retinopathy of premature children.

[0027] In certain embodiments, the method of regulating neuropathy also relates to treating, preventing or ameliorating diseases, disorders or conditions associated with neuropathy, wherein the diseases, disorders or conditions include central nervous system diseases, disorders or conditions and peripheral neuropathy, wherein peripheral neuropathy includes diabetic peripheral neuropathy. In certain embodiments, the method of treating, preventing and / or ameliorating peripheral neuropathy relates to improving nerve conduction velocity.

[0028] In certain embodiments, diabetic peripheral neuropathy is diabetic distal symmetrical polyneuropathy or diabetic mononeuropathy or multiple mononeuropathy. In certain embodiments, the symptoms of the subject are symmetrical, and the subject's symptoms include paresthesia of the distal limbs, paresthesia of both lower limbs, stocking-like sensory disturbances, glove-like sensory disturbances, involvement of the median nerve, ulnar nerve, and common peroneal nerve, sensory dysfunction, motor dysfunction, insensitivity to trauma, and neuropathic pain. In certain embodiments, diabetic peripheral neuropathy is diabetic painful peripheral neuropathy. In certain embodiments, the subject's sleep is affected by pain caused by neuropathy and abnormal nerve conduction, and suffers from depression and anxiety caused by pain or neuropathy.

[0029] In certain embodiments, the method for treating, preventing or improving peripheral neuropathy, especially diabetic peripheral neuropathy, involves: improving peripheral nerve conduction velocity, significantly improving sensory nerve conduction velocity of sural nerve. In certain embodiments, the effects of the method on the subject include: improving microcirculation, inhibiting nerve inflammation, inhibiting central nervous inflammation, repairing damaged nerve tissue, long-term relief of symptoms of peripheral neuropathy, long-term control of the progression of neuropathy, and long-term control of the progression of neuropathic pain.

[0030] In certain embodiments, the central nervous system disease, disorder or condition comprises cerebrovascular disease. Cerebrovascular disease comprises stroke and transient ischemic attack, wherein stroke comprises ischemic stroke and hemorrhagic stroke, and hemorrhagic stroke comprises the condition of cerebral hemorrhage and / or subarachnoid hemorrhage.

[0031] In some embodiments, the subject of ischemic stroke includes a subject within 7 hours after the onset of stroke, or a subject within 5 hours. In some embodiments, the subject's National Institutes of Health Stroke Score (NIHSS) is 20 points or within 20 points, or 15 points or within 15 points, or 8 points to 15 points. The subject can also be a subject with moderate to severe stroke with persistent neurological deficits. In some embodiments, the subject is a subject with anterior circulation large vessel occlusion. In some embodiments, the subject has symptoms of occlusion in the middle cerebral artery M1 segment or M2 segment.

[0032] In some embodiments, the subject with ischemic stroke is a subject with ischemic reperfusion injury, wherein reperfusion includes reperfusion by intravascular therapy, reperfusion by administration of thrombolytic agents, and / or reperfusion by angioplasty. In some embodiments, the subject with ischemic stroke is a subject with ischemia without reperfusion and / or a subject with large cerebral infarction.

[0033] In certain embodiments, the method for treating, preventing or ameliorating stroke, particularly ischemic stroke, involves: improving the acute phase cerebral tissue blood perfusion of the subject, inhibiting the reduction of local cerebral blood volume (rCBV) around the infarcted brain tissue, improving the acute phase ischemic state and saving the ischemic penumbra; reducing the brain edema / infarction volume of the subject; inhibiting central nervous system inflammation, inhibiting the overexpression of glial fibrillary acid protein (GFAP, a marker of astrocyte activation); reducing the neurobehavioral score of stroke, improving the neurological deficit of the subject, improving and restoring the finger function and limb strength of the affected limb of the subject, and reducing the disability rate of the subject; preventing, treating and improving the cognitive dysfunction of the subject, including memory, visual space and understanding judgment, preventing and stopping the development of dementia; and / or treating the destructive effects of ischemia on the central nervous system of the subject.

[0034] In certain embodiments, the method for treating, preventing or ameliorating transient ischemic attack involves: improving the acute phase brain tissue blood perfusion of the subject, inhibiting the reduction of local cerebral blood volume (rCBV) in the damaged area of ​​brain tissue, improving the acute phase (75 hours after stroke) ischemic state and saving the ischemic penumbra; inhibiting central nervous system inflammation, inhibiting the overexpression of glial fibrillary acid protein (GFAP); reducing the neurobehavioral score of stroke, improving the neurological deficit of the subject, improving and restoring the finger function and limb strength of the affected limb of the subject, and reducing the disability rate of the subject; preventing, treating and improving the cognitive dysfunction of the subject, including memory, visual space and understanding and judgment, preventing and stopping the development of dementia; and / or treating the destructive effects of ischemia on the central nervous system of the subject.

[0035] In certain embodiments, the central nervous system disease, disorder or condition comprises craniocerebral injury, wherein craniocerebral injury comprises traumatic brain injury. In certain embodiments, the method for treating, preventing or ameliorating traumatic brain injury involves: inhibiting central nervous system inflammation, and / or inhibiting GFAP overexpression.

[0036] In certain embodiments, the central nervous system disease, disorder or condition comprises a neurodegenerative disease, the neurodegenerative disease comprises dementia, wherein dementia comprises vascular dementia, Alzheimer's disease, Lewy body dementia, and frontotemporal dementia.

[0037] In certain embodiments, the method for preventing, treating and improving dementia involves: preventing, treating and improving cognitive dysfunction in the subject, including memory, visual-spatial and comprehension judgment, especially visual-spatial cognitive dysfunction, preventing and stopping the development of dementia; improving the acute phase cerebral tissue blood perfusion of the subject, inhibiting the reduction of local cerebral blood volume (rCBV) around the infarcted brain tissue, improving the ischemic state and saving the ischemic penumbra; reducing the brain edema / infarction volume of the subject; inhibiting central nervous system inflammation, inhibiting GFAP overexpression; reducing neurobehavioral scores and improving neurological deficits, and / or reducing the overall disability rate of the subject.

[0038] In certain embodiments, the central nervous system disease, disorder or condition comprises optic neuropathy, wherein optic neuropathy comprises glaucoma, ischemic optic neuropathy, optic neuritis, optic nerve tumor and traumatic optic neuropathy. In certain embodiments, the method for treating, preventing and / or improving optic neuropathy relates to inhibiting or reducing retinal ganglion cell (RGC) damage or severe axonal damage in a subject.

[0039] In certain embodiments, the glaucoma includes primary glaucoma, secondary glaucoma and / or developmental glaucoma, wherein primary glaucoma includes open-angle glaucoma, closed-angle glaucoma and special types of glaucoma.

[0040] In certain embodiments, the method for preventing, treating and improving glaucoma involves: slowing down the loss of retinal ganglion cells (RGCs), protecting visual function, inhibiting or reducing the severity of secondary neuronal damage; slowing down internal retinal damage, reducing retinal edema, and slowing down the reduction of retinal thickness; reducing optic nerve fiber layer edema, maintaining the thickness of the optic nerve fiber layer and preventing the thinning of the optic nerve fiber layer; inhibiting optic nerve inflammation and GFAP overexpression, inhibiting the expression level of at least one inflammatory factor related to nerve cell damage, the inflammatory factor including TNF-α, IL-1β, and / or IFN-γ; alleviating optic nerve function reduction including improving full-field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators, and / or protecting the overall retinal function.

[0041] In certain embodiments, the ischemic optic neuropathy comprises anterior ischemic optic neuropathy and / or posterior ischemic optic neuropathy.

[0042] In certain embodiments, the method for preventing, treating and improving ischemic optic neuropathy involves: slowing down the loss of retinal ganglion cells (RGC), protecting visual function, inhibiting or reducing the severity of secondary neuronal damage; slowing down internal retinal damage, reducing retinal edema, and slowing down the reduction of retinal thickness; reducing optic nerve fiber layer edema, maintaining the thickness of the optic nerve fiber layer and preventing the thinning of the optic nerve fiber layer; inhibiting optic nerve inflammation and GFAP overexpression, inhibiting the expression level of at least one inflammatory factor related to nerve cell damage, the inflammatory factor including TNF-α, IL-1β, and / or IFN-γ; alleviating optic nerve function reduction includes improving full-field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators, and protecting the overall retinal function.

[0043] In certain embodiments, the method for preventing, treating and ameliorating optic neuritis involves: inhibiting or reducing retinal ganglion cell (RGC) damage or severe axonal damage in a subject; inhibiting overexpression of GFAP, including I / R-induced GFAP; inhibiting the expression level of at least one inflammatory factor associated with nerve cell damage, including TNF-α, IL-1β, and / or IFN-γ.

[0044] In certain embodiments, the method for preventing, treating and ameliorating optic nerve tumors involves: inhibiting or reducing retinal ganglion cell (RGC) damage or severe axonal damage in a subject; inhibiting overexpression of GFAP, including I / R-induced GFAP; and / or inhibiting the expression level of at least one inflammatory factor associated with nerve cell damage, including TNF-α, IL-1β, and IFN-γ.

[0045] In certain embodiments, the method for preventing, treating and ameliorating traumatic optic neuropathy involves: inhibiting or reducing retinal ganglion cell (RGC) damage or severe axonal damage in a subject; inhibiting overexpression of GFAP, including I / R-induced GFAP; and / or inhibiting the expression level of at least one inflammatory factor associated with nerve cell damage, including TNF-α, IL-1β, and IFN-γ.

[0046] In certain embodiments, the central nervous system disease, disorder or state comprises retinopathy. In certain embodiments, the method for treating, preventing and / or ameliorating retinopathy relates to inhibiting or alleviating retinal ganglion cell (RGC) damage or severe axonal damage in a subject. Retinopathy comprises retinal vascular occlusive disease and / or retinopathy of immature children. Retinal vascular occlusive disease comprises retinal artery occlusion and / or retinal vein occlusion, wherein the retinal artery occlusion comprises central artery occlusion, branch artery occlusion, ciliary artery occlusion and / or retinal precapillary arteriole occlusion; the retinal vein occlusion comprises central vein occlusion and / or branch vein occlusion.

[0047] In certain embodiments, the method for preventing, treating and improving retinal vascular occlusion disease and / or premature retinopathy involves: slowing down the loss of retinal ganglion cells (RGCs), protecting visual function, inhibiting or reducing the severity of secondary neuronal damage; slowing down internal retinal damage, reducing retinal edema, and slowing down the reduction of retinal thickness; reducing optic nerve fiber layer edema, maintaining the thickness of the optic nerve fiber layer and preventing the thinning of the optic nerve fiber layer; significantly inhibiting optic nerve inflammation and GFAP overexpression, inhibiting the expression levels of inflammatory factors related to nerve cell damage such as TNF-α, IL-1β, IFN-γ; and / or alleviating optic nerve function reduction, including improving full-field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators, and protecting the overall retinal function.

[0048] In certain embodiments, the subject comprises a mammal; wherein the mammal comprises a primate, particularly a human.

[0049] In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, in combination with a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and further in combination with a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof.

[0050] In certain embodiments, the method involves administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, simultaneously or separately. When administered simultaneously, the three drug components may be formulated into a single pharmaceutical composition for administration to a subject.

[0051] In certain embodiments, the three drug components or the pharmaceutical composition as described above are formulated as oral dosage forms, intravenous dosage forms, subcutaneous injection dosage forms or fundus injection dosage forms, preferably oral dosage forms. The above three drug components or pharmaceutical compositions can be formulated into tablets, capsules, syrups, suspensions; intravenous injections, subcutaneous injections, intramuscular injections, intraperitoneal injections; creams, jelly, powders, patches; inhalation powders, sprays, suspensions or rectal suppositories, preferably tablets. The above three drug components or pharmaceutical compositions can be administered to a subject orally, intravenously, subcutaneously or intramuscularly, preferably in the form of oral administration.

[0052] In certain embodiments, the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, phosphate, pyrophosphate, hydrobromide or nitrate, citrate, fumarate, maleate, malate, ascorbate, succinate, tartrate, benzoate, acetate, methanesulfonate, ethanesulfonate, salicylate, stearate, benzenesulfonate and / or p-toluenesulfonate. In certain embodiments, the pharmaceutically acceptable salt form of doxazosin is methanesulfonate. In certain embodiments, the pharmaceutically acceptable salt form of pramipexole is hydrochloride. In certain embodiments, the pharmaceutically acceptable salt form of metoprolol is tartrate.

[0053] In certain embodiments, the daily dose of doxazosin ranges from 0.4 mg to 16 mg, preferably 0.5 mg to 8 mg, the daily dose of pramipexole ranges from 0.03 mg to 4.5 mg, preferably 0.1 mg to 1 mg, and the daily dose of metoprolol ranges from 2 mg to 200 mg, preferably 10 mg to 100 mg.

[0054] In certain embodiments, the therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, the therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and the therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, are administered to the subject once, twice, three times, or four times a day.

[0055] In certain embodiments, the therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, the therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and the therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, are administered to the subject in the form of a long-acting oral formulation.

[0056] The present invention also provides a method for inhibiting neuroinflammation, which comprises administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0057] The present invention also provides a method for repairing nerve damage, which comprises administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0058] The present invention also provides a method for treating, preventing or improving cerebrovascular diseases, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof. Cerebrovascular diseases include stroke and transient ischemic attack, wherein stroke includes ischemic stroke and hemorrhagic stroke, and hemorrhagic stroke includes a state of cerebral hemorrhage and / or subarachnoid hemorrhage.

[0059] In some embodiments, the subject of the ischemic stroke attack includes a subject within 7 hours after the stroke attack, or a subject within 5 hours. In some embodiments, the baseline National Institutes of Health Stroke Score (NIHSS) of the subject is 20 points or within 20 points, or 15 points or within 15 points, or 8 points to 15 points. The subject can also be a subject with moderate to severe stroke with persistent neurological deficits. In some embodiments, the subject is a subject with anterior circulation large vessel occlusion. In some embodiments, the subject has symptoms of intracranial occlusion in the middle cerebral artery M1 segment or M2 segment.

[0060] In some embodiments, the subject with ischemic stroke is a subject with ischemic reperfusion injury, wherein reperfusion includes reperfusion by intravascular therapy, reperfusion by administration of thrombolytic agents, and / or reperfusion by angioplasty. In some embodiments, the subject with ischemic stroke is a subject with ischemia without reperfusion and / or a subject with large cerebral infarction.

[0061] In certain embodiments, the method for treating, preventing or ameliorating stroke, particularly ischemic stroke, involves: improving the acute phase brain tissue blood perfusion of the subject, inhibiting the reduction of local cerebral blood volume (rCBV) around the brain tissue infarction, improving the ischemic state in the acute phase after stroke (75 hours after stroke) and saving the ischemic penumbra; reducing the brain edema / infarction volume of the subject; inhibiting central nervous system inflammation, inhibiting the overexpression of glial fibrillary acid protein (GFAP); reducing the neurobehavioral score of stroke, improving the neurological deficit of the subject, improving and restoring the finger function and limb strength of the affected limb of the subject, and reducing the disability rate of the subject; preventing, treating and improving the cognitive dysfunction of the subject, including memory, visual space and understanding and judgment, preventing and stopping the development of dementia; and / or treating the destructive effects of ischemia on the central nervous system of the subject.

[0062] In certain embodiments, the method for treating, preventing or ameliorating transient ischemic attack involves: improving the acute phase cerebral tissue blood perfusion of the subject, inhibiting the reduction of local cerebral blood volume (rCBV) in the damaged area of ​​brain tissue, improving the acute phase ischemic state and saving the ischemic penumbra; inhibiting central nervous system inflammation, inhibiting the overexpression of glial fibrillary acid protein (GFAP); reducing the neurobehavioral score of stroke, improving the neurological deficit of the subject, improving and restoring the finger function and limb strength of the affected limb of the subject, and reducing the disability rate of the subject; preventing, treating and improving the cognitive dysfunction of the subject, including memory, visual space and understanding and judgment, preventing and stopping the development of dementia; and / or treating the destructive effects of ischemia on the central nervous system of the subject.

[0063] The present invention also provides a method for treating, preventing or ameliorating craniocerebral injury, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof. Wherein craniocerebral injury includes traumatic brain injury. In certain embodiments, the method for treating, preventing or ameliorating traumatic brain injury involves: inhibiting central nervous system inflammation, and / or inhibiting GFAP overexpression.

[0064] The present invention also provides a method for treating, preventing or ameliorating neurodegenerative diseases, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof. Neurodegenerative diseases include dementia, wherein dementia includes vascular dementia, Alzheimer's disease, Lewy body dementia and / or frontotemporal dementia.

[0065] In certain embodiments, the method for preventing, treating and improving dementia involves: preventing, treating and improving the cognitive dysfunction of the subject, including memory, visual-spatial and comprehension judgment, especially visual-spatial cognitive dysfunction, preventing and stopping the development of dementia; improving the blood perfusion of the subject's brain tissue, inhibiting the reduction of local cerebral blood volume (rCBV) around the infarct of brain tissue, improving the ischemic state and saving the ischemic penumbra; reducing the subject's brain edema / infarction volume; inhibiting central nervous inflammation, inhibiting GFAP overexpression; reducing neurobehavioral scores and improving neurological deficits, and / or reducing the overall disability rate of the subject.

[0066] The present invention also provides a method for treating, preventing or improving glaucoma, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof. The glaucoma includes primary glaucoma, secondary glaucoma and / or developmental glaucoma, wherein primary glaucoma includes open-angle glaucoma, closed-angle glaucoma and / or special types of glaucoma.

[0067] In certain embodiments, the method for preventing, treating and improving glaucoma involves: slowing down the loss of retinal ganglion cells (RGCs), protecting visual function, inhibiting or reducing the severity of secondary neuronal damage; slowing down internal retinal damage, reducing retinal edema, and slowing down the reduction of retinal thickness; reducing optic nerve fiber layer edema, maintaining the thickness of the optic nerve fiber layer and preventing the thinning of the optic nerve fiber layer; inhibiting optic nerve inflammation and GFAP overexpression, inhibiting the expression level of at least one inflammatory factor related to nerve cell damage, the inflammatory factors including TNF-α, IL-1β, and IFN-γ; alleviating optic nerve function reduction including improving full-field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators, and / or protecting the overall retinal function.

[0068] The present invention also provides a method for treating, preventing or improving ischemic optic neuropathy, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof. The ischemic optic neuropathy comprises anterior ischemic optic neuropathy and / or posterior ischemic optic neuropathy.

[0069] In certain embodiments, the method for preventing, treating and improving ischemic optic neuropathy involves: slowing down the loss of retinal ganglion cells (RGCs), protecting visual function, inhibiting or reducing the severity of secondary neuronal damage; slowing down internal retinal damage, reducing retinal edema, and slowing down the reduction of retinal thickness; reducing optic nerve fiber layer edema, maintaining the thickness of the optic nerve fiber layer and preventing the thinning of the optic nerve fiber layer; inhibiting optic nerve inflammation and GFAP overexpression, inhibiting the expression level of at least one inflammatory factor related to nerve cell damage, the inflammatory factors including TNF-α, IL-1β, and IFN-γ; alleviating optic nerve function reduction includes improving full-field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators, and / or protecting the overall retinal function.

[0070] The present invention also provides a method for treating, preventing or improving optic neuritis, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof. In certain embodiments, the method for preventing, treating and improving optic neuritis involves: inhibiting or reducing retinal ganglion cell (RGC) damage or severe axonal damage in a subject; inhibiting overexpression of GFAP, the GFAP including I / R-induced GFAP; and / or inhibiting the expression level of at least one inflammatory factor associated with nerve cell damage, the inflammatory factor including TNF-α, IL-1β, and IFN-γ.

[0071] The present invention also provides a method for treating, preventing or improving optic nerve tumors, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof. In certain embodiments, the method for preventing, treating and improving optic nerve tumors involves: inhibiting or reducing retinal ganglion cell (RGC) damage or severe axonal damage in a subject; inhibiting overexpression of GFAP, the GFAP including I / R-induced GFAP; and / or inhibiting the expression level of at least one inflammatory factor associated with nerve cell damage, the inflammatory factor including TNF-α, IL-1β, and IFN-γ.

[0072] The present invention also provides a method for treating, preventing or improving traumatic optic neuropathy, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof. In certain embodiments, the method for preventing, treating and improving traumatic optic neuropathy involves: inhibiting or reducing retinal ganglion cell (RGC) damage or severe axonal damage in a subject; inhibiting overexpression of GFAP, the GFAP including I / R-induced GFAP; and / or inhibiting the expression level of at least one inflammatory factor associated with nerve cell damage, the inflammatory factor including TNF-α, IL-1β, and IFN-γ.

[0073] The present invention also provides a method for treating, preventing or improving retinopathy, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof. In certain embodiments, the method for treating, preventing and / or improving retinopathy involves inhibiting or alleviating retinal ganglion cell (RGC) damage or severe axonal damage in a subject. Retinopathy includes retinal vascular occlusion disease and / or retinopathy of immature children. Retinal vascular occlusion disease includes retinal artery occlusion and / or retinal vein occlusion, wherein the retinal artery occlusion includes central artery occlusion, branch artery occlusion, ciliary artery occlusion and / or retinal precapillary arteriole occlusion; the retinal vein occlusion includes central vein occlusion and / or branch vein occlusion.

[0074] In certain embodiments, the method for preventing, treating and improving retinal vascular occlusion disease and / or premature retinopathy involves: slowing down the loss of retinal ganglion cells (RGCs), protecting visual function, inhibiting or reducing the severity of secondary neuronal damage; slowing down internal retinal damage, reducing retinal edema, and slowing down the reduction of retinal thickness; reducing optic nerve fiber layer edema, maintaining the thickness of the optic nerve fiber layer and preventing the thinning of the optic nerve fiber layer; significantly inhibiting optic nerve inflammation and GFAP overexpression, inhibiting the expression levels of inflammatory factors related to nerve cell damage such as TNF-α, IL-1β, IFN-γ; alleviating optic nerve function reduction including improving full-field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators, and / or protecting the overall retinal function.

[0075] The present invention also provides a method for inhibiting or reducing retinal ganglion cell (RGC) damage or severe axonal damage in a subject, the method comprising administering to the subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0076] The present invention also provides a method for treating, preventing or improving peripheral neuropathy, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof. In certain embodiments, the method for treating, preventing or improving peripheral neuropathy involves improving nerve conduction velocity.

[0077] The present invention also provides a method for treating, preventing or improving diabetic peripheral neuropathy. In certain embodiments, the diabetic peripheral neuropathy is diabetic distal symmetrical polyneuropathy or diabetic mononeuropathy or multiple mononeuropathy. In certain embodiments, the symptoms exhibited by the subject are symmetrical, and the subject's symptoms include paresthesia of the distal limbs, paresthesia of both lower limbs, stocking-like sensory disturbances, glove-like sensory disturbances, involvement of the median nerve, ulnar nerve, and common peroneal nerve, sensory dysfunction, motor dysfunction, insensitivity to trauma, and neuropathic pain. In certain embodiments, the diabetic peripheral neuropathy is diabetic painful peripheral neuropathy. In certain embodiments, the subject's sleep is affected by pain caused by neuropathy and abnormal nerve conduction, and suffers from depression and anxiety caused by pain or neuropathy.

[0078] In certain embodiments, the method for treating, preventing or improving peripheral neuropathy, especially diabetic peripheral neuropathy, involves: improving peripheral nerve conduction velocity, significantly improving sensory nerve conduction velocity of sural nerve. In certain embodiments, the effects of the method on the subject include: improving microcirculation, inhibiting nerve inflammation, inhibiting central nervous inflammation, repairing damaged nerve tissue, long-term relief of symptoms of peripheral neuropathy, long-term control of the progression of neuropathy, and long-term control of the progression of neuropathic pain.

[0079] The present invention also provides use of the pharmaceutical composition in the preparation of a medicament for regulating neuropathy, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0080] The present invention also provides use of the pharmaceutical composition in preparing a medicament for inhibiting neuroinflammation, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0081] The present invention also provides use of the pharmaceutical composition in preparing a medicament for repairing nerve damage, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0082] The present invention also provides use of the pharmaceutical composition in preparing a drug for treating, preventing or improving cerebrovascular diseases, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0083] The present invention also provides use of the pharmaceutical composition in preparing a drug for treating, preventing or improving craniocerebral injury, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0084] The present invention also provides use of the pharmaceutical composition in preparing a drug for treating, preventing or ameliorating neurodegenerative diseases, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0085] The present invention also provides use of the pharmaceutical composition in preparing a drug for treating, preventing or improving glaucoma, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0086] The present invention also provides use of the pharmaceutical composition in preparing a medicament for treating, preventing or improving ischemic optic neuropathy, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0087] The present invention also provides use of the pharmaceutical composition in preparing a medicament for treating, preventing or improving optic neuritis, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0088] The present invention also provides use of the pharmaceutical composition in preparing a drug for treating, preventing or ameliorating optic nerve tumors, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0089] The present invention also provides use of the pharmaceutical composition in preparing a medicament for treating, preventing or ameliorating traumatic optic neuropathy, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0090] The present invention also provides use of the pharmaceutical composition in preparing a medicament for treating, preventing or improving retinopathy, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0091] The present invention also provides use of the pharmaceutical composition in preparing a medicament for inhibiting or alleviating retinal ganglion cell (RGC) damage or severe axonal damage in a subject, the use comprising administering to the subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0092] The present invention also provides use of the pharmaceutical composition in preparing a medicament for treating, preventing or improving peripheral neuropathy, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0093] The present invention also provides use of the pharmaceutical composition in preparing a medicament for treating, preventing or improving diabetic peripheral neuropathy, the use comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 The occlusion site (distal to the M1 segment of the middle cerebral artery) of the rhesus monkey middle cerebral artery occlusion (MCAO) model is shown. Figure 1 Left: The aneurysm clip occludes the middle cerebral artery at the distal end of the M1 segment and the proximal end of the M2 segment. Figure 1 Right: The middle cerebral artery is occluded with an aneurysm clip.

[0095] Figure 2 Angiography of rhesus monkeys 3-4 hours after MCAO reperfusion is shown. A: MRA images show successful MCAO reperfusion; B: MRA images show no MCAO reperfusion; C: CTA images show successful MCAO reperfusion; D: CTA images show no MCAO reperfusion. The black arrow indicates the location of the middle cerebral artery obstruction, and the white asterisk indicates the MCAO surgical side.

[0096] Figure 3 The method of middle cerebral artery occlusion (MCAO) in rhesus monkeys is shown. A craniotomy was performed in the frontotemporal region, the lateral fissure was opened, and the middle cerebral artery (MCA) was exposed from its origin at the bifurcation of the internal carotid artery, and the orbitofrontal branch and the M1 and M2 segments of the MCA were shown distally. A: Schematic diagram of middle cerebral artery occlusion, with the occlusion site distal to the M1 segment and proximal to the M2 segment of the middle cerebral artery. B: Magnetic resonance angiography (MRA) and computed tomography angiography (CTA) 3-4 hours after reperfusion after 90 minutes of middle cerebral artery occlusion showed blood flow reestablishment in the distal middle cerebral artery. C: Magnetic resonance diffusion-weighted imaging (DWI) high signal showed infarct / edema area 3-4 hours after reperfusion after 90 minutes of middle cerebral artery occlusion (i.e., before drug administration), and the infarct / edema volume was similar in the two groups. NS means "not statistically significant difference".

[0097] Figure 4The experimental process, drug administration and the timetable for testing various indicators of the 90-minute middle cerebral artery occlusion reperfusion model (MCAO) in rhesus monkeys are shown.

[0098] Figure 5 The results show that oral administration of DOX+PMP+MTP starting 5 hours after stroke in rhesus monkeys with 90-minute middle cerebral artery occlusion and continuous treatment for 28 days can significantly reduce the infarct / edema volume in the cerebral reperfusion rhesus monkeys. A: 3-4 hours after 90-minute middle cerebral artery occlusion and reperfusion, the high signal of magnetic resonance diffusion weighted imaging (DWI) showed the infarct area. The average volume of DWI quantitative brain edema / infarction area in the two groups was 8.98±1.14cm 3 and 8.17±0.85cm 3 , there was no statistical difference in edema / infarct volume between the two groups before drug administration. NS means "no statistically significant difference". B: Dynamic changes in brain edema / infarct volume at each time point measured by magnetic resonance DWI and T2FLAIR within 28 days after stroke. DOX+PMP+MTP treatment of MCAO rhesus monkeys showed significant reductions in brain tissue edema / infarct volume on the surgical side (right side) at 75h, 7 days, 14 days and 28 days. (Compared with the placebo group, *p<0.05, **p<0.01) C: Representative MRA images 3-4 hours after reperfusion after 90 minutes of middle cerebral artery occlusion for placebo and DOX+PMP+MTP treatment, and representative T2 FLAIR images at 28 days. T2 FLAIR images are images of different levels of the same animal at 28 days, showing lesions in the temporal lobe, frontal lobe, parietal lobe, and occipital lobe regions. It can be seen that both groups of animals showed reperfusion of the middle cerebral artery after stroke, and 28 days after stroke, the brain edema / infarction area of ​​the rhesus monkeys treated with DOX+PMP+MTP was significantly smaller than that of the rhesus monkeys treated with placebo.

[0099] Figure 6 It is shown that oral administration of DOX+PMP+MTP started 5 hours after stroke in rhesus monkeys with 90-minute middle cerebral artery occlusion and continued for 28 days can significantly reduce the volume of cerebral infarction / edema and significantly improve neurobehavioral function. A: Representative MRA and DWI images 3-4 hours after reperfusion after 90-minute middle cerebral artery occlusion and T2 FLAIR images at different time points within 28 days of treatment. It can be seen that both groups of animals showed middle cerebral artery reperfusion after stroke, and the edema / infarction volume of the two groups of animals before administration was similar. At different times within 28 days of administration, the brain edema / infarction area of ​​rhesus monkeys treated with DOX+PMP+MTP was significantly smaller than that of rhesus monkeys treated with placebo. B: NHPSS observation results within 28 days of administration. DOX+PMP+MTP can significantly reduce the neurobehavioral scores of MCAO rhesus monkeys and significantly improve the neurological deficits of stroke animals (p < 0.01).

[0100] Figure 7 The quantitative analysis of GFAP protein expression in brain tissues around the infarct region 30 days after stroke in the rhesus monkey middle cerebral artery ischemia-reperfusion model was shown. A) Representative images of GFAP protein immunohistochemical staining in brain tissues of the corresponding regions of the temporal lobe and precentral gyrus on the non-surgical side; B) Representative images of GFAP protein immunohistochemical staining in brain tissues of the temporal lobe and precentral gyrus around the infarct region on the surgical side in the placebo group and C) DOX+PMP+MTP group (×400). D) GFAP protein expression in brain tissues of the corresponding regions on the non-surgical side, the surgical side temporal lobe and precentral gyrus around the infarct region in the placebo group and the DOX+PMP+MTP group 30 days after stroke. Data are presented as Mean±SD; **, p<0.01 vs. non-surgical side corresponding region brain tissue; ##, p<0.01 vs. placebo group.

[0101] Figure 8 Shown is a schematic diagram of the preparation of acute retinal ischemia / reperfusion injury (I / R) in rhesus monkeys and a schedule for drug administration and detection of various indicators.

[0102] Fig. 9 Schematic diagram showing the sampling, sectioning and retinal ganglion cell (RGC) counting of rhesus monkey retina.

[0103] Fig.10 Spectral domain-optical coherence tomography (SD-OCT) images of the optic nerve head and retinal layers of a rhesus monkey are shown. (A) Representative SD-OCT images of the optic nerve head of a normal monkey, and concentric SD-OCT scans show the retinal cell layers of a normal eye. (B) Schematic diagram of optical coherence tomography (OCT) measurement of the thickness of the EDTRS region of the retina in the 9th square of a rhesus monkey.

[0104] Fig.11 Representative images of spectral domain-optical coherence tomography (SD-OCT) measuring retinal thickness in the EDTRS region of rhesus monkeys before and after drug administration on day 63 are shown.

[0105] Fig.12 (A) shows the light-adapted 3.0 ERG waveform of rhesus monkeys without ischemic damage, where 1 is the OD right eye and 2 is the OS left eye; (B) shows the flash VEP waveform of rhesus monkeys without ischemic damage.

[0106] Fig.13It was shown that the DOX+MTP+PMP group significantly improved the survival rate of RGCs after retinal I / R injury in rhesus monkeys after 63 days of treatment; (AF) OCT images of the I / R-injured right eye (OD) and left eye (OS non-ischemic control eye) of a representative animal in each of the I / R control group, DOX+MTP+PMP group and DOX+MTP+BRM group on D63 after retinal I / R; (GL) Representative micrographs of Brn3a immunohistochemistry of retinal sections of the same eye, Brn3a: yellow immunolabeled RGCs, magnification (×200). (M) RGC survival rate of rhesus monkeys in each group after 63 days of drug treatment (**, p<0.01 compared with the I / R control group's I / R eyes); ##, p<0.01 compared with the OS non-ischemic contralateral eyes; including I / R control group (N=3), I / R+DOX+MTP+PMP group (N=3), I / R+DOX+MTP+BRM group (N=3), I / R+DOX+MTP group (N=3), I / R+DOX group (N=3), I / R+PMP group (N=1) (Note: BRM is Bromocriptine).

[0107] Fig.14 The expression of GFAP protein in the retina of rhesus monkeys treated with DOX+MTP+PMP group for 63 days (D63) was significantly lower than that in the I / R control group; (AC) Representative micrographs of GFAP immunohistochemistry of retinal sections of I / R-injured right eyes (OD) of one representative animal in the I / R control group and DOX+MTP+PMP group at D63 and the left eye (non-ischemic control eye) of one animal, GFAP: yellow immunolabeled astrocytes, magnification (×200). (D) Expression of GFAP protein in rhesus monkeys with retinal ischemia / reperfusion injury in each group at 63 days (D63) (**, p<0.01 compared with the I / R control group), among which OS non-ischemic control eyes n=7.

[0108] Fig.15 The levels of inflammatory factors (IFN-γ, IL-1β, TNF-α) in each group of rhesus monkeys after drug administration are shown (**, p<0.01 compared with OS non-ischemic control eyes).

[0109] Fig.16 Shown is a schematic diagram of the Wisconsin device and experiment.

[0110] Fig.17It was shown that oral administration of DOX+PMP+MTP about 7 hours after stroke in the rhesus monkey middle cerebral artery occlusion 180 minutes reperfusion model and continuous treatment for 60 days can significantly reduce the infarction / edema volume of the MCAO reperfused rhesus monkey brain. A: 3-4 hours after the middle cerebral artery occlusion 180 minutes reperfusion, magnetic resonance diffusion weighted imaging (DWI) showed abnormal high signals in the right frontal lobe, parietal lobe, and temporal lobe, which is similar to the lesions of middle cerebral artery ischemia in clinical patients. The average volume of DWI quantitative edema / infarction area in the 5 rhesus monkeys in the placebo group and the 4 rhesus monkeys in the DOX+PMP+MTP group was 14.61±2.63cm, respectively. 3 and 13.43±3.14cm 3 , there was no statistical difference in edema / infarct volume between the two groups before administration. NS means "no statistically significant difference". B: Compared with the placebo group, the edema / infarct volume of the brain tissue on the surgical side (right side) in the DOX+PMP+MTP group was significantly reduced at 75h, 7 days, 14 days and 28 days after reperfusion (p<0.05 or p<0.01). C: Representative MRA images of the placebo group and the DOX+PMP+MTP group 3-4 hours after reperfusion after 180 minutes of middle cerebral artery occlusion, and representative T2 FLAIR images at 28 days. The T2 FLAIR images are images of different layers of the same animal at 28 days, showing lesions in the temporal lobe, frontal lobe, parietal lobe, and occipital lobe. It can be seen that both groups of animals showed reperfusion of the middle cerebral artery after stroke, and the brain edema / infarct area of ​​the rhesus monkeys treated with DOX+PMP+MTP was significantly smaller than that of the rhesus monkeys treated with placebo 28 days after stroke. .

[0111] Fig.18 It is shown that oral administration of DOX+PMP+MTP started about 7 hours after stroke in the rhesus monkey middle cerebral artery occlusion 180 minutes reperfusion model, and continued treatment for 60 days can significantly reduce the perfusion defect area and the volume of the non-perfusion area of ​​brain tissue. A: DSC showed that normal animals had no perfusion defects. 75 hours after reperfusion, the rhesus monkeys in the placebo group showed obvious non-perfusion areas, decreased CBF and CBV, and increased MTT and TTP. The perfusion defect area in the DOX+PMP+MTP group was significantly smaller than that in the placebo group. B: Analysis of the volume of the non-perfusion area measured by DSC 75 hours after 180 minutes of middle cerebral artery occlusion and reperfusion. Compared with the placebo group, DOX+PMP+MTP can significantly reduce the volume of the non-perfusion area in MCAO rhesus monkeys (p < 0.05). The unit of the volume of the non-perfusion area in the figure is expressed as pixel value (pixel 2 ), which is converted to volume (cm 3 ) is: rCBV non-perfused area volume (cm 3 ) = pixel value (pixel 2)÷1000×0.2. The volume of the non-perfused area (ischemic core area) measured in the CBV image was 17031±6355 pixels. 2 (After conversion, it is 3.41±1.27cm 3 ), the volume of the non-perfused area was measured to be 4747±3210 pixels in the CBV image on the surgical side (right side) 2 (After conversion, it is 0.95±0.64cm 3 ). C: Analysis of the volume of the non-perfused area measured by DSC 75 hours after reperfusion after 180 minutes of middle cerebral artery occlusion. Compared with the placebo group, DOX+PMP+MTP can significantly reduce the volume of the non-perfused area in MCAO rhesus monkeys (p < 0.05). The unit of the non-perfused area volume in the figure is expressed as cm 3 .

[0112] Fig.19 The results of NHPSS observation within 28 days of administration of DOX+PMP+MTP were shown in the rhesus monkey middle cerebral artery occlusion 180 minutes reperfusion stroke model, which was started by oral administration about 7 hours after treatment for 60 consecutive days. Compared with the placebo group, DOX+PMP+MTP can significantly reduce the neurobehavioral score of MCAO rhesus monkeys and significantly improve the neurological deficit of stroke animals (p < 0.01).

[0113] Fig. 20 Representative FP and FFA images of the RVO rhesus monkey on the day of modeling are shown. FP and FFA images show that the RVO rhesus monkey model was successfully established by laser photocoagulation of the superior branch vein around the optic disc of the left eye. FP showed obvious venous dilation and tortuosity in the retina, and the veins turned white with no blood return. FFA showed delayed filling and fluorescence leakage near the venous occlusion area.

[0114] Fig.21 Representative FP and FFA images of RVO rhesus monkeys in the placebo group and the DOX+MTP+PMP group on the 7th day of administration are shown. A) FP and FFA images of the placebo group showed delayed filling of the superior retinal branch veins, flame-shaped hemorrhages, dilated and tortuous veins, and large-area capillary fluorescence leakage around the occlusion site; B) On the 7th day of treatment in the DOX+MTP+PMP group, delayed filling of the superior retinal branch veins, flame-shaped hemorrhages (slightly milder than the placebo group), dilated and tortuous veins, and capillary fluorescence leakage around the occlusion site were observed, which was significantly milder than the placebo group.

[0115] Fig. 22The changes in retinal thickness around the occlusion site of the superior retinal vein in each group are shown. A) Representative images of changes in retinal thickness around the occlusion site in the placebo group on day 0 and day 14 of administration; B) Representative images of changes in retinal thickness around the occlusion site in the DOX+MTP+PMP group on day 0 and day 14 of administration; C) Changes in retinal edema thickness around the occlusion site in the placebo group and the DOX+MTP+PMP group on day 14 of administration. Data are presented as Mean±SD, *p<0.05vs. placebo group.

[0116] Fig.23 The RNFL thickness changes of the RVO rhesus monkeys in the placebo group and the DOX+MTP+PMP group on day 42 are shown. The data are presented as Mean±SD, *p<0.05 vs. the placebo group.

[0117] Fig.24 Individual data of the effects of 58 days of administration on the sensory conduction velocity of rhesus monkeys with diabetic peripheral neuropathy in each group are shown, including the placebo group (n=4, 7 abnormal nerves), the DOX+PMP+MTP treatment group (n=4, 17 abnormal nerves), the PMP treatment group (n=3, 8 abnormal nerves), the DOX+MTP treatment group (n=3, 9 abnormal nerves), the DOX+PMP treatment group (n=2, 6 abnormal nerves) and the epalrestat group (n=4, 13 abnormal nerves).

[0118] Fig.25 The effects of 58 days of administration to each group on the sensory nerve conduction velocity of rhesus monkeys with diabetic peripheral neuropathy are shown, including the placebo group (n=4, 7 abnormal nerves), the DOX+PMP+MTP treatment group (n=4, 17 abnormal nerves), the PMP treatment group (n=3, 8 abnormal nerves), the DOX+MTP treatment group (n=3, 9 abnormal nerves), the DOX+PMP treatment group (n=2, 6 abnormal nerves) and the epalrestat group (n=4, 13 abnormal nerves).

[0119] Fig.26 Individual data of changes in sensory nerve conduction velocity of the sural nerve in rhesus monkeys with diabetic peripheral neuropathy after 58 days of administration in each group are shown, including the placebo group (n=4, 7 abnormal nerves), the DOX+PMP+MTP treatment group (n=4, 8 abnormal nerves), the PMP treatment group (n=3, 5 abnormal nerves), the DOX+MTP treatment group (n=3, 6 abnormal nerves), the DOX+PMP treatment group (n=2, 2 abnormal nerves) and the epalrestat group (n=4, 7 abnormal nerves).

[0120] Fig. 27The effects of 58 days of administration to each group on the sensory nerve conduction velocity of the sural nerve in rhesus monkeys with diabetic peripheral neuropathy are shown, including the placebo group (n=4, 7 abnormal nerves), the DOX+PMP+MTP treatment group (n=4, 8 abnormal nerves), the PMP treatment group (n=3, 5 abnormal nerves), the DOX+MTP treatment group (n=3, 6 abnormal nerves), the DOX+PMP treatment group (n=2, 2 abnormal nerves) and the epalrestat group (n=4, 7 abnormal nerves). DETAILED DESCRIPTION OF THE INVENTION

[0121] definition

[0122] Certain terms are used in the specification, examples and claims of this application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs.

[0123] As used herein, the term "subject" refers to an animal, including but not limited to a mammal. Mammals include primates or non-primates, for example, monkeys, pigs, sheep, dogs, cats, cows, horses, mice, rats, rabbits or transgenic species thereof. In certain embodiments, the subject is a human.

[0124] As used herein, the term "prevent" includes, but is not limited to, delaying and / or preventing the onset of a disease, disorder or condition and / or its attendant symptoms; preventing a subject from developing a disease, disorder or condition; or reducing the risk of a subject developing a disease, disorder or condition.

[0125] As used herein, the term "treat" includes, but is not limited to, alleviating or eliminating a disease, disorder or condition, or one or more symptoms associated therewith; or alleviating or eradicating the cause of the disease, disorder or condition.

[0126] As used herein, the terms "mitigate," "reduce," or "improve" include, but are not limited to, reducing one or more symptoms of a disease, disorder, or condition (e.g., cerebral edema). These terms may also refer to reducing side effects associated with an active ingredient. Sometimes, the beneficial effects that a subject obtains from a prophylactic or therapeutic agent do not result in a cure of the condition, disease, or disorder.

[0127] As used herein, the term "regulate" refers to the prevention, treatment and / or improvement of a disease, disorder or condition, including but not limited to (a) preventing or delaying the occurrence of a disease, disorder or condition, or reducing the risk of the occurrence of a disease, disorder or condition, (b) preventing the aggravation of a disease, disorder or condition, delaying or slowing the development of a disease, disorder or condition; and (c) reducing the severity of a disease, disorder or condition, causing the regression of a disease, disorder or condition.

[0128] As used herein, the term "administering" refers to the action of delivering, or causing the delivery of, a compound or pharmaceutical component / composition to a subject's body by methods described herein or known in the art. Administering a compound or pharmaceutical component / composition includes prescribing a compound or pharmaceutical component / composition to be delivered into the subject's body. Exemplary forms of administration include oral dosage forms, e.g., tablets, capsules, syrups, suspensions; injectable dosage forms, e.g., intravenous (IV), intramuscular (IM), subcutaneous (SC), or intraperitoneal (IP); transdermal dosage forms, including creams, jellies, powders, or patches; oral dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

[0129] As used herein, the term "therapeutically effective amount" is a term recognized in the art. In certain embodiments, the term refers to an amount necessary or sufficient to eliminate, reduce, or maintain the goal of a particular treatment regimen. The effective amount may vary depending on factors such as the disease or condition being treated, the specific targeting construct being administered, the size of the subject, or the severity of the disease or condition. A person of ordinary skill in the art or a physician can determine the effective amount of a particular compound empirically without excessive experimentation. In certain embodiments, the therapeutically effective amount of a therapeutic agent used in vivo may depend on many factors, including: the manner and method of administration; any other materials contained in the drug in addition to the agent. In vitro or in vivo tests may be used to help determine the optimal dosage range.

[0130] As used herein, the term "about" or "approximately" refers to the acceptable error of a particular value determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" refers to within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" refers to within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0131] When a range of values ​​is listed herein, it is intended to encompass every value and subrange within that range. For example, "1-5 mg" or "about 1 mg to about 5 mg" is intended to encompass 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 1-2 mg, 1-3 mg, 1-4 mg, 1-5 mg, 2-3 mg, 2-4 mg, 2-5 mg, 3-4 mg, 3-5 mg, and 4-5 mg.

[0132] The terms "including," "comprising," and "having" are used in an inclusive, open sense, meaning that additional elements may be included in addition to those specified. The terms "such as," "for example," and "for example" as used herein are non-limiting and are used for illustrative purposes only. "Including" and "including but not limited to" are used interchangeably.

[0133] As used herein, the term "and / or" as used in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0134] As used herein, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements may optionally be present in addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, in one embodiment, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B") may refer to at least one, optionally including more than one, referring to A and the absence of B (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, referring to B and the absence of A (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, referring to A, and at least one, optionally including more than one, referring to B (and optionally including other elements); and so on.

[0135] 1. Methods of regulating neuropathy

[0136] The present invention provides a method for regulating neuropathy, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof.

[0137] Neuropathy refers to damage, disease or dysfunction of one or more nerves, including those of the central and peripheral nervous systems. It usually manifests as burning or shooting pain, numbness, tingling, muscle weakness or atrophy, usually degenerative, and is usually caused by injury, infection, disease, drugs, toxins or vitamin deficiency.

[0138] The regulation of neuropathy described in the present invention includes prevention, treatment and / or improvement thereof. Prevention, treatment and / or improvement of neuropathy include but are not limited to (a) preventing or delaying the occurrence of neuropathy, or reducing the risk of neuropathy, (b) preventing the aggravation of neuropathy, delaying or slowing the development of neuropathy; and (c) reducing the severity of neuropathy and causing the degeneration of neuropathy.

[0139] In certain embodiments, the method of regulating neuropathy described in the present invention involves inhibiting neuroinflammation. Neuroinflammation refers to various inflammatory lesions involving nerves. Neuroinflammation is one of the important pathogenesis of dementia. Neuroinflammation leads to pathological processes such as white matter lesions, hippocampal damage and Aβ deposition, which in turn leads to the occurrence of cognitive impairment-related diseases such as vascular dementia and Alzheimer's disease.

[0140] In certain embodiments, the present invention provides a method for inhibiting neuroinflammation, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof. In certain embodiments, the method treats, prevents or improves a disease, disorder or state associated with neuroinflammation. Diseases associated with neuroinflammation include: ischemic stroke, hemorrhagic stroke, transient ischemic attack, craniocerebral injury, vascular dementia, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, glaucoma, ischemic optic neuropathy, optic neuritis, optic nerve tumors, traumatic optic neuropathy, retinal artery occlusion, retinal vein occlusion, and retinopathy of premature children.

[0141] The inventors found that the expression levels of inflammatory factors TNF-α, IL-1β and IFN-γ in the retinal tissue of subjects with retinal I / R attack were at least 5 times, at least 7 times, or even 10 times the normal level (i.e., the expression level of inflammatory factors in the retinal tissue of subjects without retinal I / R attack). Without being bound by theory, cytokines play an important role in the process of neuroinflammation, and proinflammatory cytokines such as IFN-γ, IL-1β, TNF-α and IL-6 are involved in the process of neuroinflammation and can be regarded as biomarkers of neuroinflammation.

[0142] The inventors also found that the expression of GFAP in the damaged area of ​​the retina and brain tissue of subjects with neuroinflammation caused by retinal I / R injury or ischemic stroke was significantly increased compared with the normal expression (i.e., the GFAP expression of the tissue of subjects without neuroinflammation), and the overexpressed GFAP can be measured by conventional immunohistochemical staining. The increased expression of glial fibrillary acid protein (GFAP), a biomarker of astrocytes, is also regarded as one of the signs of neuroinflammation (LiM, Li Z, Yao Y, et al. Astrocyte-derived interleukin-15 exacerbates ischemic brain injury via propagation of cellular immunity. Proc Natl Acad Sci USA, 2017, 114 (3): E396-E405.). GFAP is an astrocyte-specific intermediate filament protein, and its expression is necessary for the normal function of astrocytes, including maintaining the integrity of CNS white matter and blood-brain barrier. It is noteworthy that most astrocytes do not express sufficient GFAP under normal circumstances to be stained by conventional immunohistochemical methods.

[0143] In addition, neuroinflammation leads to pathological processes such as white matter lesions, hippocampal damage and Aβ deposition, which in turn leads to the occurrence of cognitive impairment-related diseases such as vascular dementia and Alzheimer's disease (Chen YH, Lin RR, Tao Q Q. The role of P2X7R in neuroinflammation and implications in Alzheimer's disease[J]. Life Sciences, 2021, 271: 119187.).

[0144] In certain embodiments, the method for regulating neuropathy described in the present invention relates to repairing nerve damage. Nerve injury generally refers to damage to nerve tissue. In certain embodiments, the present invention provides a method for repairing nerve damage, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof.

[0145] In certain embodiments, the methods described herein treat, prevent or ameliorate diseases, disorders or conditions associated with neuropathy. Diseases, disorders or conditions associated with neuropathy include, but are not limited to, diseases, disorders or conditions caused by neuropathy and / or diseases, disorders or conditions with neuropathy as one of the symptoms.

[0146] In certain embodiments, the disease, disorder or state comprises peripheral neuropathy. Peripheral nerve refers to cranial nerves and spinal nerves, autonomic nerves and their ganglia other than the olfactory and optic nerves. Peripheral nerve disease refers to a disease originating from structural or functional damage of the peripheral nervous system. The peripheral nerves are functionally divided into two parts: sensory afferent and motor efferent. In certain embodiments, the method for treating, preventing or improving peripheral neuropathy improves nerve conduction velocity, particularly significantly improves the sensory nerve conduction velocity of the sural nerve, thereby effectively controlling and alleviating the progression of diabetic peripheral neuropathy and neuropathic pain.

[0147] In certain embodiments, the peripheral neuropathy includes ischemic neuropathy, peripheral neuropathy caused by immune-related inflammation (Bourque PR, Chardon JW, Massie R. Autoimmune peripheral neuropathies. Clin Chim Acta. 2015 Sep 20; 449: 37-42.), and peripheral neuropathy caused by trauma (Menorca RM, Fussell TS, Elfar JC. Peripheral Nerve Trauma: Mechanisms of Injury and Recovery. Hand Clin. 2013; 29 (3): 317-330). Ischemic peripheral neuropathy is a common type of polyneuropathy, and its cause is most common in arteriosclerosis and vasculitis (Blaes F. Diagnosis and therapeutic options for peripheral vasculitic neuropathy. Ther Adv Musculoskelet Dis. 2015; 7 (2): 45-55).

[0148] In certain embodiments, the peripheral neuropathy is diabetic peripheral neuropathy. Diabetic peripheral neuropathy (DPN) refers to a chronic microvascular complication of diabetes with a very high incidence and a high disability rate. The disease can cause neurological dysfunction and low nerve sensitivity in the early stage, such as numbness of the limbs, hyperalgesia, foot ulcers, gangrene and other serious lesions. The amputation rate of patients can exceed 80%, of which at least about 20% will develop into neuropathic pain, i.e. diabetic peripheral neuropathy (DPNP, also known as diabetic painful peripheral neuropathy). In certain embodiments, the present invention provides a method for treating, preventing or improving diabetic peripheral neuropathy, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0149] In certain embodiments, the disease, disorder or condition comprises a central nervous system disease, disorder or condition. In certain embodiments, the central nervous system disease, disorder or condition comprises cerebrovascular disease, craniocerebral injury, neurodegenerative disease, optic neuropathy and / or retinopathy.

[0150] 1.1 Cerebrovascular disease

[0151] In certain embodiments, the central nervous system disease, disorder or state is a cerebrovascular disease. In certain embodiments, provided herein is a method for treating, preventing or ameliorating a cerebrovascular disease, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0152] Cerebrovascular disease is a general term for cerebrovascular diseases caused by various reasons. Stroke is the main clinical type of cerebrovascular disease, including ischemic stroke and hemorrhagic stroke, which are a group of cerebrovascular diseases caused by organic brain damage with sudden onset and rapid localized or diffuse brain function impairment as common clinical features. In certain embodiments, the cerebrovascular disease includes stroke and / or transient ischemic attack.

[0153] 1.1.1 Stroke

[0154] In certain embodiments, the cerebrovascular disease is a stroke. Stroke (also known as apoplexy) is a disease caused by impaired blood flow in the central nervous system, with sudden onset and rapid onset of localized or diffuse brain dysfunction as common clinical features. It is a group of cerebrovascular diseases caused by organic brain damage, and its potential causes include embolism, hemorrhage and thrombosis. About 82% of stroke patients suffer from ischemic stroke. The primary treatment measure for ischemic stroke is to restore cerebral blood flow in a timely manner, reconstruct blood circulation in the ischemic area and save the ischemic penumbra. Currently, intravenous thrombolysis (IVT) can save the penumbra within 3 hours after the onset of ischemic stroke. However, due to the lower proportion of vascular reperfusion and the damage caused by reperfusion, as well as the symptoms of bleeding that may accompany reperfusion, 70% of stroke patients still have varying degrees of labor loss.

[0155] Without being bound by theory, the neurons of subjects who have suffered a stroke are not only difficult to survive on their own in the early stage of fighting brain damage, but also rely on the environmental support of various glial cells and immune cells in the later reconstruction and repair of brain tissue. Ischemia-reperfusion (I / R) injury of brain tissue is accompanied by the proliferation and activation of glial cells, neuroinflammation and cytokine cascade reaction, leading to neuronal cell death, which is considered to be an important pathological mechanism of stroke. Astrocytes are important components of the blood-brain barrier. After a stroke, astrocytes increase their uptake of glutamate and lactate and swell, compressing the blood vessels in the ischemic area and aggravating the state of vascular insufficiency. The increased expression of the astrocyte biomarker glial fibrillary acidic protein (GFAP) is regarded as a sign of neuroinflammation, and the expression of multiple proinflammatory cytokines is increased, which aggravates I / R injury. For patients who have already suffered a stroke, it is also extremely important to prevent and delay neuronal cell death, so that more neurons can be retained for functional recovery and more time for treatment.

[0156] In certain embodiments, the stroke includes ischemic stroke and / or hemorrhagic stroke. In certain embodiments, the stroke is an ischemic stroke. Ischemic stroke refers to a type of stroke caused by obstruction of blood flow to the brain. The most common underlying condition for this obstruction is the development of fatty deposits or blood clots lining the blood vessel walls, known as atherosclerosis. These blood clots can cause two types of blockages. One is the formation of blood clots in cerebral blood vessels to block blood vessels. The other refers to blood clots or plaque fragments formed at locations outside the brain entering cerebral blood vessels until the blockage is too small to pass through the blood vessels. The second important cause of blockage is an irregular heartbeat, known as atrial fibrillation. Atrial fibrillation causes blood clots to form in the heart and move to the brain. Other potential causes of ischemic stroke include cerebral hemorrhage, thrombosis, arterial or venous dissection, cardiac arrest, and iatrogenic causes, such as surgical damage to cerebral blood vessels or surgery on the brain or heart. Ischemic stroke accounts for approximately 83% of all stroke cases.

[0157] In some embodiments, the subject with ischemic stroke is a subject within 7 hours after the onset of stroke. It should be noted that the current clinical treatment of stroke has a very narrow dosing window, which has considerable restrictions on the time and conditions of administration, such as requiring that intravenous thrombolytic (IVT) drugs must be given for treatment within 4.5 hours after the onset of acute ischemic stroke symptoms after appropriate imaging examinations have been performed in advance, which has brought great inconvenience to clinical medication. However, the inventors have found that the method for treating, preventing and / or improving ischemic stroke still produces beneficial effects when used for subjects within 7 hours after the onset of stroke. In some embodiments, the subject with ischemic stroke is a subject within 5 hours after the onset of stroke.

[0158] In some embodiments, the National Institute of Health Stroke Scale (NIHSS) of the subject with ischemic stroke is 20 points or less, or 15 points or less, or between 8 and 20 points, or between 15 and 20 points, or between 8 and 15 points, or 8 points. In some embodiments, the subject is a subject with moderate to severe stroke with persistent neurological deficits. The National Institute of Health Stroke Scale (NIHSS) is recommended for use in assessing the extent of cerebral infarction. NIHSS baseline assessment can reflect the severity of stroke at the time of onset, and can also regularly assess the effect after treatment. The range of NIHSS is between 0 and 42. The higher the score, the more severe the stroke (Adams HP Jr, Davis PH, Leira EC, et al. Baseline NIH Stroke Scale score strongly predicts outcome after stroke: A report of the Trial of Org 10172 in Acute Stroke Treatment (TOAST). Neurology. 1999 Jul; 53(1): 126-131.).

[0159] For example: 0-1 points are normal or near normal; 1-4 points are mild stroke / minor stroke; 5-15 points are moderate stroke; 15-20 points are moderate-severe stroke; 20-42 points are severe stroke. For every 1 point increase in NIHSS baseline, the probability of a good prognosis decreases by 17%. Patients with a baseline score greater than 16 are likely to die. Patients with endovascular treatment (EVT) usually have NIHSS scores ≥ 15-20. In addition to mild hemiparesis, there are also neurological abnormalities, which are considered to be moderate to severe stroke (Brott TG, Adams HP Jr, Olinger CP, et al. Measurements of acute cerebral infarction: a clinical examination scale. Stroke 1989; 20: 864–70.).

[0160] In certain embodiments, the subject is a subject with anterior circulation large vessel occlusion. In certain embodiments, the subject is a subject with acute ischemic stroke caused by anterior circulation large vessel occlusion. In certain embodiments, the subject's NIHSS score is between 8 and 20 points (including 8 and 20 points). In certain embodiments, the subject is accompanied by moderate to severe neurological impairment. Anterior circulation large vessel occlusion refers to middle cerebral artery occlusion or internal carotid artery occlusion, mostly middle cerebral artery trunk occlusion, and a few are large cerebral infarctions caused by internal carotid artery siphon segment occlusion. Applying this method to the subject for 75 hours of continuous administration can reduce the subject's brain edema / infarction volume, reduce the volume of the ischemic core area, and significantly improve the ischemic state. After 1 month or 2 months, the subject has a good neurological prognosis (for example, the NIHSS score is reduced to 2 or 3 points), and the neurological deficit of the subject can be improved. Compared with the NIHSS score of a subject who does not implement the present method (eg, a score of about 6 points one month after a stroke), the NIHSS score of the subject who implements the present method can be reduced by about 50%, or even about 70%.

[0161] In certain embodiments, the subject is a subject with anterior circulation large vessel occlusion, particularly a subject with symptoms of occlusion in the M1 or M2 segments of the middle cerebral artery. In certain embodiments, the subject has symptoms of occlusion in the M1 segment of the middle cerebral artery. It should be noted that the middle cerebral artery is a frequent site of ischemic stroke in humans. When the occlusion site is the M1 segment, the peak of edema presents an edema / infarct volume of about 40% of the half brain (1.5 hours of occlusion of the M1 segment of the middle cerebral artery and reperfusion) and / or an edema / infarct volume of about 70% of the half brain volume (3 hours of occlusion of the M1 segment of the middle cerebral artery and reperfusion), with an obvious midline shift. 28 days after the stroke, the infarct area includes cognitive and memory-related brain areas such as the temporal lobe and hippocampus, which can lead to long-term neurological impairment and disability of the affected limbs and / or long-term cognitive decline. The inventors have found that the method for treating, preventing and / or ameliorating ischemic stroke still produces beneficial effects when used for subjects with such severe disabled cerebral edema / infarction, for example, when orally treated about 7 hours after the onset of stroke.

[0162] In certain embodiments, the subject suffering from ischemic stroke is a subject suffering from ischemic reperfusion injury, wherein reperfusion includes reperfusion by intravascular therapy and / or reperfusion by administration of thrombolytic agents. Intravascular therapy includes: intra-arterial thrombolysis, mechanical thrombectomy and emergency angioplasty (Chinese Stroke Society. Chinese Guidelines for Endovascular Treatment of Acute Ischemic Stroke 2018 [J]. Chinese Journal of Stroke, 2018, 013(007): 706-729).

[0163] In certain embodiments, the method of the present invention can be combined with intravenous rt-PA thrombolysis for acute stroke and administered to the subject (eg, within 3 hours after the onset of the stroke). The combined administration can better improve the neurological prognosis of the subject.

[0164] In certain embodiments, if a subject misses the time window for intravenous rt-PA thrombolysis after an acute stroke attack, the method of the present invention can be applied, which can expand the treatment time window, thereby allowing subjects who seek medical treatment beyond the time window for intravenous thrombolysis to obtain the opportunity for intravascular reperfusion therapy.

[0165] In certain embodiments, the subject misses the intravenous rt-PA thrombolysis time window after an acute stroke and adopts an intravascular treatment method (e.g., thrombectomy and reperfusion in subjects with large vessel (carotid artery / middle cerebral artery M1 segment) occlusive ischemic stroke 6-16 hours after the onset of stroke), and the method of the present invention can be applied to the subject in combination with the intravascular treatment method.

[0166] In certain embodiments, the subject is given intravenous rt-PA thrombolysis after acute stroke, and at the same time, an intravascular treatment method is used due to large vessel occlusion or severe condition (for example, thrombectomy and reperfusion are performed on subjects within 6 hours, or within 8 hours, or 6-16 hours after stroke onset). The method of the present invention can also be combined with the above intravenous thrombolysis method and the intravascular treatment method and applied to the subject. The method of the present invention has good safety and does not cause or increase the risk of bleeding, so it can be combined with intravenous thrombolysis and intravascular interventional treatment and applied to the subject.

[0167] In certain embodiments, the subject with ischemic stroke is a subject with ischemia without reperfusion, such as a subject without intravenous rt-PA thrombolysis and / or reperfusion with intravascular therapy. In certain embodiments, the subject with ischemic stroke is a subject with large-area cerebral infarction, also known as malignant middle cerebral artery infarction.

[0168] Large hemispheric infarction (LHI), also known as malignant middle cerebral artery infarction, is an important disease that causes death or disability in humans. Such patients have typical clinical symptoms and often die from transtentorial herniation. In certain embodiments, the method of the present invention treats subjects who have been reperfused 3 hours after occlusion of a large blood vessel of the anterior circulation (e.g., the M1 segment of the middle cerebral artery) confirmed by MRA imaging. In certain embodiments, the method of the present invention treats subjects with edema / infarction volume accounting for about 70% of the volume of the hemibrain at the peak of edema (75 hours after stroke), forming a large area of ​​cerebral infarction. The method of the present invention is applied to the subject for 28 consecutive days, which can significantly reduce the brain edema / infarction volume of the subject, significantly reduce the volume of the ischemic core area and significantly improve the ischemic state at 75 hours, and improve the neurological deficit of the subject. The NIHSS score of the subject who implements the method can be reduced by about 50% or even about 70% compared with the subject who does not implement the method.

[0169] Advantageously, the inventors of the present application have found that the method has beneficial effects in preventing, treating and / or improving stroke, especially ischemic stroke. Without being bound by theory, the drug components administered by the method combine multiple mechanisms such as inhibiting neuroinflammation, resisting oxidative stress, and improving ischemia to protect nerves and prevent the expansion of the infarct core area. In particular, the method for preventing, treating and / or improving cerebral stroke described in the present invention improves the blood perfusion of brain tissue in the acute phase after stroke (i.e., 75 hours after stroke) of the subject, inhibits the reduction of local cerebral blood volume (rCBV) around the infarct of brain tissue, improves the ischemic state in the acute phase (75 hours after stroke) and saves the ischemic penumbra; significantly reduces the brain edema / infarction volume of the subject; inhibits central nervous inflammation and overexpression of glial fibrillary acid protein (GFAP); treats the destructive effects of ischemia on the central nervous system of the subject; reduces the neurobehavioral score of stroke (e.g., NIHSS), improves the neurological deficit of the subject, especially improves and restores the finger function and limb strength of the affected limb of the subject, and reduces the disability rate of the subject; and / or prevents, treats and improves the cognitive dysfunction of the subject, including memory, visual space and understanding and judgment, and prevents and stops the development of dementia.

[0170] In certain embodiments, the method for treating, preventing or improving stroke improves the blood perfusion of brain tissue in the acute phase after stroke (75 hours after stroke) of the subject, inhibits the reduction of local cerebral blood volume (rCBV) around the infarct of brain tissue, improves the ischemic state in the acute phase and saves the penumbra. Local cerebral ischemia is composed of a central necrotic area and a surrounding cerebral ischemic penumbra. Brain cells die in the necrotic area. Due to the presence of collateral circulation, there are still a large number of surviving neurons in the ischemic penumbra. If the blood flow of the ischemic penumbra can be quickly restored in a short period of time, the damage to the brain tissue in this area is reversible, and the nerve cells may survive and restore their metabolic function. Advantageously, the volume of the non-perfused area (i.e., the ischemic core area) of the brain tissue of the subject with stroke in the acute phase (75 hours after stroke) using the method of the present invention is reduced by at least 70% compared to the subject with stroke who does not use the method.

[0171] In certain embodiments, the method for treating, preventing or improving stroke reduces the brain edema / infarction volume of the subject. Brain edema / infarction volume refers to the volume of local brain tissue ischemia and anoxic necrosis due to cerebral blood supply disorder. Ischemic stroke is characterized by significant cell death and obvious inflammatory response in the infarction site. Brain edema volume refers to the excessive accumulation volume of fluid in the intracellular or extracellular space of the brain. Advantageously, the inventors used 3.0T GE MRI to dynamically observe the edema / infarction trajectory of the subject 3-4 hours, 72-75 hours, 7 days, 14 days and 28 days after stroke. MRI imaging sequences include T1WI, T2 FLAIR, DWI, MRA, CTA. Among them, T1WI can display the anatomical structure of the brain, distinguish gray matter and white matter areas, diagnose hemorrhage and ischemia, etc., T2 FLAIR and DWI can quantify the volume of edema / necrosis, and T2 FLAIR can indicate an increase in water content in the tissue, and is used to show lesions such as inflammation, ischemia, and edema. The MRA imaging in the present invention confirms that the T2 FLAIR brain edema / infarction volume of subjects who have undergone stroke and who have been continuously administered the drug for 28 days using the method of the present invention, especially subjects who have been occluded for 3 hours and reperfused by the large blood vessels of the anterior circulation (the M1 segment of the middle cerebral artery) and occluded for 1.5 hours and reperfused and accompanied by moderate or above neurological impairment, is reduced by at least 30% (75 hours after stroke), at least 40% (7 days after stroke), and even close to 50% (28 days after stroke) compared to the T2FLAIR brain edema / infarction volume of subjects who have undergone stroke and who have not adopted the method.

[0172] In certain embodiments, the method for treating, preventing or improving stroke inhibits central nervous inflammation, particularly inhibits overexpression of glial fibrillary acid protein (GFAP). Without being bound by theory, the inventors found that the expression of GFAP in the peri-infarct area of ​​the brain tissue of a subject with a stroke attack was significantly increased compared with that in the contralateral normal brain tissue. Advantageously, the method of the present invention significantly inhibits the increase in GFAP expression in the damaged area of ​​the brain tissue of a subject with neuroinflammation caused by ischemic stroke.

[0173] In certain embodiments, the method for treating, preventing or improving stroke reduces the neurobehavioral score (e.g., NIHSS) of stroke subjects, improves the neurological deficit of the subject, particularly improves and restores the finger function and limb strength of the affected limb of the subject, and reduces the disability rate of the subject. Neurological deficits refer to symptoms that occur after the lesion of the neural structure, and the defect symptoms refer to the weakening or disappearance of normal functions when the neural structure is damaged. Advantageously, the NIHSS score of the subject with stroke using the method of the present invention can be reduced by at least 40% (14 days after stroke), at least 50% (28 days after stroke), or even 60% compared to the NIHSS score of the subject with stroke not using the method. At the same time, the inventor found that the subject who did not use the method had long-term neurological impairment and disability 3 months after the stroke, and the subject's anterior gyrus was involved, resulting in impaired contralateral hand function, such as the loss of finger movement of the thumb and index finger, and could not complete the delicate task of peeling peanuts with both hands. Advantageously, the subjects who adopted the method of the present invention had normal function of the affected hand 3 months after the stroke, the finger movement of the thumb and index finger was not affected, and they could complete the delicate task of peeling peanuts with both hands.

[0174] Cognitive dysfunction and even dementia may occur after a stroke or in certain infections, repetitive physical injuries to the brain, or nutritional deficiencies. In certain embodiments, the method for preventing, treating, and / or improving stroke described in the present invention prevents, treats, and improves cognitive dysfunction in subjects, including memory, visual space, and understanding judgment, especially visual space cognitive dysfunction, and prevents and prevents the development of dementia. Visual spatial dysfunction refers to a functional disorder caused by the patient's inability to accurately judge the position of himself and his objects, which is often manifested as: the patient cannot find a parking space when parking, and gets lost when going home due to misjudging the direction; when laying the tablecloth, the tablecloth cannot be aligned with the table due to the inability to correctly judge the position of the tablecloth and the corner of the table; the pot cannot be accurately placed on the stove and falls to the ground; the patient cannot accurately copy a three-dimensional picture, and in severe cases, even a simple plane picture cannot be drawn; there are difficulties in dressing in life, and the clothes cannot be judged up and down and left and right, and the clothes and pants are worn backwards, etc. Advantageously, in a spatial delayed reaction experiment 3 months after stroke, subjects with stroke who adopted the method of the present invention successfully passed the 1s, 5s, 10s, and 30s delayed task tests, while subjects with stroke who did not adopt the method of the present invention only passed the 1s delayed task test or even failed to complete the 1s delayed task test.

[0175] In certain embodiments, the stroke is a hemorrhagic stroke. Hemorrhagic stroke accounts for about 17% of stroke cases. It is caused by the rupture of fragile blood vessels and bleeding into the surrounding brain. Blood can accumulate and compress the surrounding brain tissue. Two common types of hemorrhagic stroke are intracerebral hemorrhage and subarachnoid hemorrhage. Hemorrhagic stroke is caused by the rupture of weakened blood vessels, and the potential causes of blood vessel rupture include hypertensive bleeding, in which hypertension causes blood vessel rupture, or other potential causes of weakened blood vessels, such as rupture of cerebral vascular malformations, including cerebral aneurysms, arteriovenous malformations (AVMs) or cavernous vascular malformations. Hemorrhagic stroke can also be caused by the hemorrhagic transformation of ischemic stroke, resulting in weakened blood vessels in the infarction, or bleeding of primary or metastatic tumors containing abnormally fragile blood vessels in the central nervous system. Hemorrhagic stroke may also be caused by iatrogenic causes, such as direct surgical damage to cerebral blood vessels. Aneurysms are expansions of weak areas of blood vessels. If left untreated, aneurysms may continue to weaken until it ruptures and bleeds into the brain. An arteriovenous malformation (AVM) is a group of abnormally formed blood vessels. A cavernous malformation is a venous abnormality that can weaken the venous structure leading to bleeding. Any of these blood vessels can rupture and also cause bleeding in the brain. Hemorrhagic strokes can also be caused by physical trauma.

[0176] In certain embodiments, the hemorrhagic stroke includes a state of cerebral hemorrhage and / or a state of subarachnoid hemorrhage. Intracerebral hemorrhage refers to non-traumatic intraparenchymal bleeding. When an intracranial blood vessel ruptures and blood flows into the subarachnoid space, it is called subarachnoid hemorrhage, which is divided into two types: traumatic and spontaneous. The spontaneous type is further divided into two types: primary and secondary. Primary subarachnoid hemorrhage is caused by rupture of vascular lesions at the base of the brain or on the surface of the brain (such as congenital aneurysms, cerebral vascular malformations, microaneurysms caused by hypertensive cerebral arteriosclerosis, etc.), and blood flows into the subarachnoid space, accounting for about 10% of acute strokes: secondary subarachnoid hemorrhage is caused by intracerebral hematoma penetrating brain tissue, and blood flows into the subarachnoid space.

[0177] 1.1.2 Transient ischemic attack

[0178] In certain embodiments, the cerebrovascular disease is a transient ischemic attack. A transient ischemic attack (TIA) is a transient neurological deficit caused by local brain or retinal ischemia, with clinical symptoms generally lasting no more than 1 hour and no more than 24 hours, and is a mild or warning stroke. Subjects undergoing cardiac surgery are particularly susceptible to transient ischemic attacks.

[0179] Advantageously, the inventors of the present application have found that the method has beneficial effects of preventing, treating and / or improving transient ischemic attacks. Particularly advantageously, the method of preventing, treating and / or improving transient ischemic attacks of the present invention improves the blood perfusion of brain tissue in the acute phase after stroke (75 hours after stroke) of the subject, inhibits the reduction of local cerebral blood volume (rCBV) in the damaged area of ​​brain tissue, improves the ischemic state in the acute phase and saves the penumbra; inhibits central nervous inflammation, inhibits the overexpression of glial fibrillary acid protein (GFAP); treats the destructive effect of ischemia on the central nervous system of the subject; reduces the neurobehavioral score of stroke (such as NIHSS), improves the neurological deficit of the subject, especially improves and restores the finger function and limb strength of the affected limb of the subject, and reduces the disability rate of the subject; and / or prevents, treats and improves the cognitive dysfunction of the subject, including memory, visual space and understanding judgment, and prevents and prevents the development of dementia. .

[0180] In certain embodiments, the method for treating, preventing or ameliorating transient ischemic attack improves the blood perfusion of brain tissue in the acute phase after stroke (75 hours after stroke) of the subject, inhibits the reduction of local cerebral blood volume (rCBV) in the damaged area of ​​brain tissue, improves the ischemic state in the acute phase and saves the penumbra. Advantageously, the volume of the non-perfused area (i.e., ischemic core area) of the brain of the subject with transient ischemic attack using the method of the present invention is reduced by at least 70% compared with the subject with transient ischemic attack not using the method.

[0181] In certain embodiments, the method for treating, preventing or improving transient ischemic attack inhibits central nervous inflammation, particularly inhibits overexpression of glial fibrillary acid protein (GFAP). Without being bound by theory, the inventors found that the expression of GFAP in the brain tissue of subjects with transient ischemic attack was significantly increased compared with that in the contralateral normal brain tissue. Advantageously, the method of the present invention significantly inhibits the increase in GFAP expression in the damaged area of ​​brain tissue of subjects with neuroinflammation caused by transient ischemic attack.

[0182] In certain embodiments, the method for treating, preventing or improving transient ischemic attack reduces the neurobehavioral score (e.g., NIHSS) of a subject with stroke, improves the neurological deficit of the subject, particularly improves and restores the finger function and limb strength of the affected limb of the subject, and reduces the disability rate of the subject. Advantageously, the NIHSS score of a subject with transient ischemic attack using the method of the present invention can be reduced by at least 40% (14 days after stroke), at least 50% (28 days after stroke) or even 60% compared to the NIHSS score of a subject with transient ischemic attack not using the method.

[0183] In certain embodiments, the method for preventing, treating and / or improving transient ischemic attack of the present invention prevents, treats and improves cognitive dysfunction of the subject, including cognitive dysfunction in memory, visual space and understanding judgment, especially visual space, and prevents and stops the development of dementia. Advantageously, in the spatial delayed reaction experiment, the subjects with transient ischemic attack using the method of the present invention all successfully passed the delay 1s, 5s, 10s, and 30s task tests, while the subjects with transient ischemic attack not using the method of the present invention only passed the delay 1s task test, and even failed to complete the delay 1s task test.

[0184] 1.2 Brain injury

[0185] In certain embodiments, the central nervous system disease, disorder or state is craniocerebral injury. In certain embodiments, the present invention provides a method for treating, preventing or ameliorating craniocerebral injury, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0186] Craniocerebral injury refers to any trauma to the skull and brain. In certain embodiments, the craniocerebral injury is a traumatic brain injury. Traumatic brain injury (TBI) refers to a non-degenerative, non-reproductive external mechanical force that damages the brain, which may lead to permanent or temporary damage to cognitive, physiological and psychosocial functions, accompanied by a weakening or change in consciousness. Traumatic brain injury is an injury that affects the way the brain works, which may be caused by a collision, blow or shaking of the head, or a penetrating head injury (such as a gunshot). Traumatic brain injury is mainly divided into three types: mild TBI or concussion; moderate TBI; and severe TBI. TBI usually originates from primary injury and then gradually develops into secondary injury. Secondary pathogenesis includes ischemic changes, inflammatory cascades, oxidative stress, etc., which can lead to temporary or permanent neurological deficits. Improving ischemia, inhibiting neuroinflammation, and resisting oxidative stress are the key mechanisms of neuroprotection and repair in TBI (Galgano M, Toshkezi G, Qiu X, et al. Traumatic Brain Injury: Current Treatment Strategies and Future Endeavors[J]. Cell Transplantation, 2017, 26(7): 1118-1130.).

[0187] Without being bound by theory, the neurons of subjects with traumatic brain injury are not only difficult to survive on their own in the early stages of fighting brain damage, but also rely on the environmental support of various glial cells and immune cells in the later reconstruction and repair of brain tissue. Ischemia-reperfusion (I / R) injury of brain tissue is accompanied by the proliferation and activation of glial cells, neuroinflammation and cytokine cascade reactions, leading to neuronal cell death. The increased expression of the astrocyte biomarker glial fibrillary acidic protein (GFAP) is regarded as a sign of neuroinflammation, while the expression of multiple proinflammatory cytokines is increased, which aggravates I / R injury. For patients who have already suffered traumatic brain injury, it is also extremely important to prevent and delay neuronal cell death, so that more neurons can be retained for functional recovery and more time for treatment.

[0188] Without being bound by theory, the inventors of the present application have found that the drug components administered by the method combine multiple mechanisms such as inhibiting neuroinflammation, resisting oxidative stress, and improving ischemia to protect nerves and prevent the expansion of the infarct core area. Advantageously, the inventors of the present application have found that the method has a beneficial effect of preventing, treating and / or improving traumatic brain injury. Particularly advantageously, the method for preventing, treating and / or improving traumatic brain injury described in the present invention inhibits central nervous inflammation, especially inhibiting GFAP overexpression.

[0189] 1.3 Neurodegenerative diseases

[0190] In certain embodiments, the central nervous system disease, disorder or condition is a neurodegenerative disease. In certain embodiments, the present invention provides a method for treating, preventing or ameliorating a neurodegenerative disease, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0191] Neurodegenerative diseases are a group of diseases with unknown causes that cause chronic and progressive damage to nerves and other tissues. Many degenerative diseases may be caused by a series of complex molecular biological disorders in the process of evolution, development, maturation and aging of nerves and other tissues, thus showing changes in structure and function. The degeneration process may involve the entire nerve cell (cell body, nucleus, axon tip) and may also affect other components such as myelin sheath.

[0192] In certain embodiments, the neurodegenerative disease comprises dementia, wherein the dementia comprises vascular dementia, Alzheimer's disease, Lewy body dementia, and / or frontotemporal dementia. Cognitive dysfunction and even dementia may occur after a stroke or in certain infections, repeated physical trauma to the brain, or nutritional deficiencies. The boundaries between different forms of dementia are blurred, but cerebrovascular damage is often present in various dementias. In certain embodiments, the dementia comprises vascular dementia.

[0193] In certain embodiments, the dementia includes Alzheimer's disease. Alzheimer's disease (AD) is a degenerative disease of the central nervous system that occurs in the elderly and pre-elderly, characterized by progressive cognitive dysfunction and behavioral impairment, clinically manifested as memory impairment, aphasia, apraxia, agnosia, visual-spatial ability impairment, abstract thinking and calculation impairment, personality and behavioral changes, etc. AD is the most common type of dementia in the elderly, accounting for about 50% to 70% of senile dementia.

[0194] In certain embodiments, the dementia includes Lewy body dementia. Dementia with Lewy bodies (DLB) is a neurodegenerative disease, which is mainly manifested clinically as fluctuating cognitive impairment, Parkinson's syndrome, and psychiatric symptoms with visual hallucinations as prominent manifestations. The pathological characteristic changes are visible Lewy bodies in the cerebral cortex.

[0195] In certain embodiments, the dementia includes frontotemporal dementia. Frontotemporaldementia refers to a dementia syndrome in which middle-aged and elderly patients slowly develop personality changes, speech disorders, and abnormal behavior. Neuroimaging shows atrophy of the frontotemporal lobes. This disease is a common cause of neurodegenerative dementia, accounting for about 1 / 4 of all dementia patients.

[0196] Without being bound by theory, cytokines play an important role in the neuroinflammatory process, and proinflammatory cytokines such as IFN-γ, IL-1β, TNF-α, and IL-6 are involved in the neuroinflammatory process. In addition, IFN-γ and TNF-α increase NO levels in combination, which participates in the death of dopaminergic neurons and promotes the development of Alzheimer's disease. Increased expression of the astrocyte biomarker glial fibrillary acid protein (GFAP) is considered a hallmark of neuroinflammation. Neuroinflammation leads to pathological processes such as white matter lesions, hippocampal damage, and Aβ deposition, which in turn lead to the occurrence of cognitive impairment-related diseases such as vascular dementia and Alzheimer's disease. Therefore, neuroinflammation plays a key role in the pathogenesis of various dementias and neurodegenerative changes.

[0197] Advantageously, the inventors of the present application have found that the method has beneficial effects in preventing, treating and / or improving dementia. Without being bound by theory, the drug components administered by the method combine multiple mechanisms such as inhibiting neuroinflammation, resisting oxidative stress, and improving ischemia, breaking the vicious cycle between nerve damage and neuroinflammation to protect nerves.

[0198] Particularly advantageously, the method for preventing, treating and / or improving dementia of the present invention prevents, treats and improves cognitive dysfunction of the subject, including cognitive dysfunction in memory, visual space and understanding judgment, especially visual space, and prevents and stops the development of dementia. Advantageously, in the spatial delayed reaction experiment, the subjects with impaired cognitive function who adopted the method of the present invention all successfully passed the delayed 1s, 5s, 10s, and 30s task tests, while the subjects with impaired cognitive function who did not adopt the method of the present invention only passed the delayed 1s task test, or even failed to complete the delayed 1s task test.

[0199] More advantageously, the method for preventing, treating and / or improving dementia described in the present invention also improves the blood perfusion of the subject's brain tissue, inhibits the reduction of local cerebral blood volume (rCBV) around the brain tissue infarction, improves ischemia and saves the penumbra; significantly reduces the subject's brain edema / infarction volume; inhibits central nervous system inflammation, especially inhibits GFAP overexpression; significantly reduces neurobehavioral scores (such as NIHSS) and improves neurological deficits, and / or reduces the subject's overall disability rate.

[0200] In certain embodiments, the method for treating, preventing or improving dementia improves the blood perfusion of the subject's brain tissue, inhibits the reduction of the local cerebral blood volume (rCBV) around the brain tissue infarction, improves ischemia and saves the penumbra. Advantageously, the volume of the non-perfused area (ischemic core area) of the brain of the subject with impaired cognitive function using the method of the present invention is reduced by at least 70% compared to the subject with impaired cognitive function who does not use the method.

[0201] In certain embodiments, the method for treating, preventing or improving dementia reduces the brain edema / infarction volume of the subject. 3.0T GE MRI was used to dynamically observe the edema / infarction trajectory of the subject 3-4 hours, 72-75 hours, 7 days, 14 days and 28 days after the stroke. Advantageously, the inventor confirmed by MRA imaging that the subject who was continuously administered for 60 days by the method of the present invention, especially the subject with 3 hours of occlusion and reperfusion of the large blood vessels of the anterior circulation (M1 segment of the middle cerebral artery) and moderate or above neurological impairment, had a T2 FLAIR brain edema / infarction volume compared to the T2 FLAIR brain edema / infarction volume of the subject with impaired cognitive function who did not adopt the method. It was reduced by at least 30% (72 hours after stroke), at least 40% (7 days after stroke), and even close to 50% (28 days after stroke).

[0202] In certain embodiments, the method for treating, preventing or improving dementia inhibits central nervous inflammation, particularly inhibiting overexpression of glial fibrillary acid protein (GFAP). Without being bound by theory, the inventors found that the expression of GFAP in the peri-infarct area of ​​brain tissue of subjects with impaired cognitive function due to stroke was significantly increased compared with that in the contralateral normal brain tissue. Advantageously, the method of the present invention significantly inhibits the increase in GFAP expression in the damaged area of ​​brain tissue of subjects with impaired cognitive function due to neuroinflammation. .

[0203] In certain embodiments, the method for treating, preventing or improving dementia significantly reduces neurobehavioral scores (e.g., NIHSS) and improves neurological deficits, particularly improves and restores the finger function and limb strength of the affected limbs of the subject, and reduces the overall disability rate of the subject. Advantageously, the subject who adopts the method of the present invention to continuously administer for 60 days, especially the subject who can be confirmed by MRA imaging that the anterior circulation large blood vessels (middle cerebral artery M1 segment) are occluded for 3 hours and reperfused with moderate or above neurological impairment, and the NIHSS score can be reduced by at least 30%, at least 40%, at least 50%, at least 60%, and even close to 70% compared with the NIHSS score of the subject with impaired cognitive function who does not adopt the method. The inventor found that the subject with impaired cognitive function who did not adopt the method had long-term neurological impairment and disability 3 months after the onset of stroke, and the subject's anterior gyrus was affected, resulting in impaired contralateral hand function, such as loss of finger movement of the thumb and index finger, and inability to complete fine tasks. Advantageously, the dementia subject using the method of the present invention has normal function of the affected hand 3 months after the onset of stroke, the finger movement of the thumb and index finger is not affected, and can complete fine tasks of both hands.

[0204] 1.4 Optic neuropathy

[0205] In certain embodiments, the central nervous system disease, disorder or condition is optic neuropathy, including glaucoma, ischemic optic neuropathy, optic neuritis, optic nerve tumors and / or traumatic optic neuropathy. Optic neuropathy refers to damage or disease of the optic nerve that affects the survival and function of retinal neurons, affecting the function of transmitting visual signals from the retina of the eye to the brain.

[0206] In certain embodiments, the method for preventing, treating and / or ameliorating optic neuropathy involves inhibiting or alleviating retinal ganglion cell (RGC) damage or severe axonal damage in a subject.

[0207] In certain embodiments, the present invention provides a method for inhibiting or reducing retinal ganglion cell (RGC) damage or severe axonal damage in a subject, the method comprising administering to the subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof. In certain embodiments, the method treats, prevents, or improves a disease, disorder, or state associated with retinal ganglion cell (RGC) damage or severe axonal damage. Diseases associated with retinal ganglion cell (RGC) damage or severe axonal damage include: glaucoma, ischemic optic neuropathy, optic neuritis, optic nerve tumors, traumatic optic neuropathy, retinal artery occlusion, retinal vein occlusion, and retinopathy of premature children.

[0208] 1.4.1 Glaucoma

[0209] In certain embodiments, the optic neuropathy is glaucoma. The present invention provides a method for treating, preventing or improving glaucoma, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0210] Glaucoma is a group of eye diseases characterized by progressive retinal ganglion cell (RGC) death and typical visual field loss, and has become the leading cause of irreversible blindness worldwide. The main risk factors associated with this disease include ischemia, gliosis, inflammation, excitotoxicity, and oxidative stress. Currently, lowering intraocular pressure is only partially effective in protecting visual function in patients with glaucoma. Therefore, it is crucial to develop new treatments to provide safe and effective RGC neuroprotection. Subjects were diagnosed with glaucoma, including primary open-angle glaucoma, angle-closure glaucoma, secondary glaucoma, normal tension glaucoma, or congenital glaucoma. Glaucoma can be normal intraocular pressure, or high intraocular pressure.

[0211] In certain embodiments, the glaucoma includes primary glaucoma, secondary glaucoma and / or developmental glaucoma. Secondary glaucoma refers to a type of glaucoma caused by other eye diseases or systemic diseases. Developmental glaucoma is a type of glaucoma caused by maldevelopment or abnormal development of the eye's internal chamber angle during the embryonic and developmental periods, and occurs before and after birth, in infancy, and in childhood.

[0212] Primary glaucoma is the main type of glaucoma that occurs in people aged 18 and above. It is usually bilateral, but the onset of the disease in the two eyes may be sequential and the severity is often different. Primary glaucoma is divided into two categories: angle-closure glaucoma and open-angle glaucoma. The etiology of this type of glaucoma has been gradually understood through long-term research, but has not yet been elucidated. In order to distinguish it from secondary glaucoma, it is customarily called primary glaucoma.

[0213] In certain embodiments, the primary glaucoma includes open-angle glaucoma, closed-angle glaucoma and / or special types of glaucoma. Primary open-angle glaucoma refers to a group of syndromes with optic nerve damage and an open anterior chamber angle, and intraocular pressure may be elevated or sometimes within a normal range. Primary angle-closure glaucoma refers to a type of glaucoma in which the anterior chamber angle is blocked by the mechanical system of the surrounding iris tissue due to a pre-existing iris configuration, resulting in obstruction of aqueous humor outflow and increased intraocular pressure.

[0214] The term "special types of glaucoma" refers to a unique type of glaucoma that is still primary and different from angle-closure and open-angle glaucoma. It includes high-fold iris glaucoma, malignant glaucoma, normal-tension glaucoma, pigmentary glaucoma, and exfoliation glaucoma.

[0215] Advantageously, the inventors of the present application have found that the method has a beneficial effect of preventing, treating and / or improving glaucoma. Particularly advantageously, the method for preventing, treating and / or improving glaucoma of the present invention slows down the loss of retinal ganglion cells (RGC), protects visual function, inhibits or reduces the severity of secondary neuronal damage; slows down internal retinal damage, reduces retinal edema, and slows down the reduction of retinal thickness; reduces optic nerve fiber layer edema, maintains optic nerve fiber layer thickness and prevents optic nerve fiber layer thinning; significantly inhibits optic nerve inflammation and GFAP overexpression, inhibits the expression level of inflammatory factors related to nerve cell damage such as IFN-γ, IL-1β, TNF-α; reduces optic nerve function reduction including improving full field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators, and / or protects the overall function of the retina.

[0216] In certain embodiments, the method for treating, preventing or improving glaucoma slows down the loss of retinal ganglion cells (RGC), protects visual function, and inhibits or reduces the severity of secondary neuronal damage. Advantageously, the average loss rate of the number of retinal ganglion cells in glaucoma subjects using the method of the present invention is reduced by at least 80% compared to the placebo group (i.e., subjects not using the method of the present invention), while the single-component control group (i.e., subjects who only use one drug component), the two-component control group (i.e., subjects who only use two components of DOX+MTP) and the three-component control group (i.e., subjects who use three-component drugs DOX+MTP+BRM different from the present invention) only reduce the average loss rate of the number of retinal ganglion cells by 20%, or even only by 15%.

[0217] In certain embodiments, the method for treating, preventing or improving glaucoma significantly inhibits optic nerve inflammation and GFAP overexpression. Astrocytes are the main glial cells in the normal retina, and play a vital role in maintaining the blood-retinal barrier and RGC health. The increased expression of the astrocyte biomarker GFAP is considered a sign of neuroinflammation and a sign of retinal degeneration. Without being bound by theory, the inventors found that the expression of GFAP in the retinal tissue of glaucoma subjects increased to about 5 times the normal expression (i.e., the expression of GFAP in the retinal tissue of subjects who do not suffer from glaucoma), which is considered to be an overexpression. Advantageously, the GFAP expression in the retinal tissue of glaucoma subjects using the method of the present invention is reduced to less than 50% of the overexpression, less than 40%, or even close to 30%.

[0218] In certain embodiments, the method for treating, preventing or ameliorating glaucoma inhibits the increased expression level of at least one inflammatory factor, wherein the inflammatory factor includes IFN-γ, IL-1β, and / or TNF-α. Without being bound by theory, the inventors found that the expression level of inflammatory factors in the retinal tissue of subjects with glaucoma was significantly increased compared with the normal level (i.e., the expression level of inflammatory factors in the retinal tissue of subjects without glaucoma). Advantageously, the expression level of inflammatory factors in the retinal tissue of subjects with glaucoma using the method of the present invention is reduced to near normal levels.

[0219] In certain embodiments, the method for treating, preventing or improving glaucoma slows down the damage to the inner retina, reduces retinal edema, and slows down the reduction in retinal thickness. Advantageously, the reduction in the average thickness of the retinal EDTRS area of ​​the glaucoma subject using the method of the present invention is reduced by at least 70% or even at least 80% compared to the placebo group (i.e., the glaucoma subject not using the method of the present invention), while the single-component control group (i.e., the glaucoma subject who only uses one drug component), the two-component control group (i.e., the glaucoma subject who only uses two components of DOX+MTP) and the three-component control group (i.e., the glaucoma subject who uses the three-component drug DOX+MTP+BRM different from the present invention) only reduces the reduction in the average thickness of the retinal EDTRS area by about 30%, or 20%, or 10%, or 5%, or even does not reduce but increases its reduction. More advantageously, the reduction in the average thickness of the retinal EDTRS area in subjects without glaucoma who adopt the method of the present invention is reduced by at least 30%, at least 40%, at least 50%, at least 60%, or even close to 70% compared to the control group (i.e., subjects without glaucoma who did not adopt the method of the present invention).

[0220] In certain embodiments, the method for treating, preventing or ameliorating glaucoma reduces optic nerve fiber layer edema, maintains optic nerve fiber layer thickness and prevents optic nerve fiber layer thinning. The inventors observed the ocular phenotype of spontaneously glaucoma rhesus monkeys, which exhibited important ophthalmic features of primary open-angle glaucoma in humans, including thinning of the retinal peripapillary nerve fiber layer (RNFL). (Louis R Pasquale et al. Development of Primary Open Angle Glaucoma-Like Features in a Rhesus Macaque Colony From Southern China. [J]. Transl Vis Sci Technol. 2021 Aug 2; 10(9): 20) Advantageously, the reduction in the average thickness of the optic nerve fiber layer (RNFL) of glaucoma subjects using the method of the present invention was reduced by at least 80% compared with the placebo group (i.e., glaucoma subjects who did not use the method of the present invention), while the single-component control group (i.e., glaucoma subjects who only administered one drug component), the two-component control group (i.e., glaucoma subjects who only administered two components, DOX+MTP), and the three-component control group (i.e., glaucoma subjects who administered a three-component drug, DOX+MTP+BRM, different from the present invention) only reduced the reduction in the average thickness of the optic nerve fiber layer (RNFL) by about 30%, or even did not reduce it but increased it.

[0221] In certain embodiments, the method for treating, preventing or improving glaucoma reduces the reduction of optic nerve function, including improving full-field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators, and protecting the overall function of the retina. Advantageously, the amplitude of the a wave and b wave of the full-field electroretinogram (FERG) light-adapted 3.0ERG reaction of the glaucoma subject using the method of the present invention is increased by about 30%, or 50%, or 100%, or 150%, or even close to 200% compared with the control group (i.e., the glaucoma subject not using the method of the present invention). More advantageously, the N2-P2 amplitude of the flash visual evoked potential (FVEP) of the glaucoma subject using the method of the present invention is increased by about 100%, or 200%, or 300%, or 400%, or even close to 500% compared with the control group (i.e., the glaucoma subject not using the method of the present invention).

[0222] 1.4.2 Ischemic optic neuropathy

[0223] In certain embodiments, the optic neuropathy is ischemic optic neuropathy. The present invention provides a method for treating, preventing or ameliorating ischemic optic neuropathy, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0224] Ischemic optic neuropathy is caused by acute circulation disorders in the nutrient vessels of the optic nerve. Clinically, it can be divided into anterior ischemic optic neuropathy and posterior ischemic optic neuropathy. The clinical manifestations are a sudden, isolated, usually painless visual impairment accompanied by optic disc swelling. Anterior ischemic optic neuropathy is a disease in which the optic disc is acutely ischemic and edematous due to insufficient blood supply to the optic disc caused by circulatory disorders in the posterior ciliary arteries. Posterior ischemic optic neuropathy is a disease in which the optic nerve vessels between the lamina cribrosa and the optic chiasm undergo acute circulation disorders, resulting in damage to the optic nerve function due to ischemia.

[0225] Advantageously, the inventors of the present application have found that the method has a beneficial effect of preventing, treating and / or improving ischemic optic neuropathy. Particularly advantageously, the method for preventing, treating and / or improving ischemic optic neuropathy described in the present invention slows down the loss of retinal ganglion cells (RGC), protects visual function, and the severity of secondary neuronal damage associated with optic neuropathy; slows down internal retinal damage, reduces retinal edema, and slows down the reduction of retinal thickness; reduces optic nerve fiber layer edema, maintains optic nerve fiber layer thickness and prevents optic nerve fiber layer thinning; significantly inhibits optic nerve inflammation and GFAP overexpression, inhibits the expression level of inflammatory factors related to nerve cell damage such as IFN-γ, IL-1β, and / or TNF-α; reduces optic nerve function reduction including improving full field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators, and / or protects the overall function of the retina.

[0226] In certain embodiments, the method for treating, preventing or improving ischemic optic neuropathy slows down the loss of retinal ganglion cells (RGC), protects visual function, and inhibits or reduces the severity of secondary neuronal damage associated with optic neuropathy. Advantageously, the average loss rate of the number of retinal ganglion cells in subjects with ischemic optic neuropathy using the method of the present invention is reduced by at least 80% compared to the placebo group (i.e., subjects with ischemic optic neuropathy who do not use the method of the present invention), while the single-component control group (i.e., subjects with ischemic optic neuropathy who only use one drug component), the two-component control group (i.e., subjects with ischemic optic neuropathy who only use two components of DOX+MTP) and the three-component control group (i.e., subjects with ischemic optic neuropathy who use three components of drug DOX+MTP+BRM different from the present invention) only reduce the average loss rate of the number of retinal ganglion cells by 20%, or even by only 15%.

[0227] In certain embodiments, the method for treating, preventing or ameliorating ischemic optic neuropathy significantly inhibits optic nerve inflammation and GFAP overexpression. Without being bound by theory, the inventors found that the expression of GFAP in the retinal tissue of subjects with ischemic optic neuropathy increased to about 5 times the normal expression (i.e., the expression of GFAP in the retinal tissue of subjects without ischemic optic neuropathy), which is considered to be an overexpression. Advantageously, the expression of GFAP in the retinal tissue of subjects with ischemic optic neuropathy using the method of the present invention is reduced to less than 50% of the overexpression, less than 40%, or even close to 30%.

[0228] In certain embodiments, the method for treating, preventing or improving ischemic optic neuropathy inhibits the increased expression level of at least one inflammatory factor, wherein the inflammatory factor includes IFN-γ, IL-1β, and / or TNF-α. Without being bound by theory, the inventors found that the expression level of inflammatory factors in the retinal tissue of subjects with ischemic optic neuropathy was significantly increased compared with the normal level (i.e., the expression level of inflammatory factors in the retinal tissue of subjects without ischemic optic neuropathy). Advantageously, the expression level of inflammatory factors in the retinal tissue of subjects with ischemic optic neuropathy using the method of the present invention is reduced to near normal levels.

[0229] In certain embodiments, the method for treating, preventing or improving ischemic optic neuropathy slows down the damage to the inner retina, reduces retinal edema, and slows down the reduction in retinal thickness. Advantageously, the reduction in the average thickness of the retinal EDTRS area of ​​the subjects with ischemic optic neuropathy using the method of the present invention is reduced by at least 70% or even at least 80% compared to the placebo group (i.e., the subjects with ischemic optic neuropathy who did not use the method of the present invention), while the single-component control group (i.e., the subjects with ischemic optic neuropathy who only used one drug component), the two-component control group (i.e., the subjects with ischemic optic neuropathy who only used two components of DOX+MTP) and the three-component control group (i.e., the subjects with ischemic optic neuropathy who used three components of DOX+MTP+BRM different from the present invention) only reduced the reduction in the average thickness of the retinal EDTRS area by about 30%, or 20%, or 10%, or 5%, or even did not reduce but increased its reduction. More advantageously, the reduction in the average thickness of the retinal EDTRS area of ​​subjects without ischemic optic neuropathy who adopt the method of the present invention is reduced by at least 30%, at least 40%, at least 50%, at least 60%, or even close to 70% compared to the control group (i.e., subjects without ischemic optic neuropathy who did not adopt the method of the present invention).

[0230] In certain embodiments, the method for treating, preventing or improving ischemic optic neuropathy reduces optic nerve fiber layer edema, maintains optic nerve fiber layer thickness and prevents optic nerve fiber layer thinning. Advantageously, the reduction in the average thickness of the optic nerve fiber layer (RNFL) of the subjects with ischemic optic neuropathy using the method of the present invention is reduced by at least 80% compared to the placebo group (i.e., the subjects with ischemic optic neuropathy who did not use the method of the present invention), while the single-component control group (i.e., the subjects with ischemic optic neuropathy who only used one drug component), the two-component control group (i.e., the subjects with ischemic optic neuropathy who only used two components of DOX+MTP) and the three-component control group (i.e., the subjects with ischemic optic neuropathy who used three-component drugs DOX+MTP+BRM different from the present invention) only reduced the average thickness of the optic nerve fiber layer (RNFL) by about 30%, or even did not reduce but increased its reduction.

[0231] In certain embodiments, the method for treating, preventing or improving ischemic optic neuropathy reduces optic nerve function reduction, including improving full-field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators, and protecting the overall function of the retina. Advantageously, the amplitude of the a wave and b wave of the full-field electroretinogram (FERG) light-adapted 3.0ERG response of the subject with ischemic optic neuropathy using the method of the present invention is increased by about 30%, or 50%, or 100%, or 150%, or even close to 200% compared with the control group (i.e., the subject with ischemic optic neuropathy not using the method of the present invention). More advantageously, the N2-P2 amplitude of the flash visual evoked potential (FVEP) of the subject with ischemic optic neuropathy using the method of the present invention is increased by about 100%, or 200%, or 300%, or 400%, or even close to 500% compared with the control group (i.e., the subject with ischemic optic neuropathy not using the method of the present invention).

[0232] 1.4.3 Optic neuritis, optic nerve tumors, and traumatic optic neuropathy

[0233] In certain embodiments, the optic neuropathy is optic neuritis. Optic neuritis refers to a variety of inflammatory lesions involving the optic nerve. In certain embodiments, the optic neuropathy is an optic nerve tumor. Optic nerve tumors are tumors that occur in the optic nerve, and the more common ones are optic nerve gliomas and optic nerve meningiomas. In certain embodiments, the optic neuropathy is traumatic optic neuropathy. Traumatic optic neuropathy refers to impact damage to the optic nerve caused by external force, which can lead to partial or total loss of vision.

[0234] Advantageously, the inventors of the present application have found that the method has a beneficial effect of preventing, treating and / or improving optic neuritis, optic nerve tumors and / or traumatic optic neuropathy. Particularly advantageously, the method of preventing, treating and / or improving optic neuritis, optic nerve tumors and / or traumatic optic neuropathy of the present invention inhibits or reduces retinal ganglion cell (RGC) damage or severe axon damage in the subject; significantly inhibits overexpression of glial fibrillary acid protein (GFAP, a marker of neuroinflammation), wherein GFAP can be induced by I / R; and inhibits the expression level of inflammatory factors related to nerve cell damage, such as IFN-γ, IL-1β, and / or TNF-α.

[0235] In certain embodiments, the method for treating, preventing or ameliorating optic neuritis, optic nerve tumors and / or traumatic optic neuropathy inhibits or reduces retinal ganglion cell (RGC) damage or severe axonal damage in the subject. Advantageously, the mean rate of loss of retinal ganglion cells in subjects with optic neuritis, optic nerve tumors and / or traumatic optic neuropathy who adopt the method of the present invention is reduced by at least 80% compared to the placebo group (i.e., subjects with optic neuritis, optic nerve tumors and / or traumatic optic neuropathy who do not adopt the method of the present invention), while the one-component control group (i.e., subjects with optic neuritis, optic nerve tumors and / or traumatic optic neuropathy who are administered only one drug component), the two-component control group (i.e., subjects with optic neuritis, optic nerve tumors and / or traumatic optic neuropathy who are administered only the two components DOX+MTP) and the three-component control group (i.e., subjects with optic neuritis, optic nerve tumors and / or traumatic optic neuropathy who are administered a three-component drug DOX+MTP+BRM different from the present invention) only reduced the mean rate of loss of retinal ganglion cells by 20%, or even by only 15%.

[0236] In certain embodiments, the method for treating, preventing or ameliorating optic neuritis, optic nerve tumors and / or traumatic optic neuropathy inhibits overexpression of glial fibrillary acidic protein (GFAP), wherein GFAP can be I / R induced. Without being bound by theory, the inventors found that the expression of GFAP in the retinal tissue of subjects with optic neuritis, optic nerve tumors and / or traumatic optic neuropathy increased to about 5 times the normal expression (i.e., the expression of GFAP in the retinal tissue of subjects who do not suffer from optic neuritis, optic nerve tumors and / or traumatic optic neuropathy) is considered to be an overexpression. Advantageously, the expression of GFAP in the retinal tissue of subjects with optic neuritis, optic nerve tumors and / or traumatic optic neuropathy using the method of the present invention is reduced to less than 50% of the overexpression, less than 40%, or even close to 30%.

[0237] In some embodiments, the method for treating, preventing or improving optic neuritis, optic nerve tumors and / or traumatic optic neuropathy suppresses the expression level of at least one inflammatory factor to increase. Inflammatory factor (inflammatory factor) mainly refers to various cytokines involved in inflammatory response. In some embodiments, the inflammatory factor includes IFN-γ, IL-1β, and / or TNF-α. Without being bound by theory, the inventors found that the expression level of inflammatory factors in the retinal tissue of the subject of optic neuritis, optic nerve tumors and / or traumatic optic neuropathy was significantly increased compared with the normal level (i.e., the expression level of inflammatory factors in the retinal tissue of the subject of optic neuritis, optic nerve tumors and / or traumatic optic neuropathy). Advantageously, the expression level of inflammatory factors in the retinal tissue of the subject of optic neuritis, optic nerve tumors and / or traumatic optic neuropathy using the method of the present invention is reduced to near normal levels.

[0238] 1.5 Retinopathy

[0239] In certain embodiments, the central nervous system disease, disorder or condition comprises retinopathy. Retinopathy is a disease occurring in the retina, which is the initial site of vision formation and the site of lesions of various blinding eye diseases. The present invention provides a method for treating, preventing or improving retinopathy, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0240] In certain embodiments, the method for preventing, treating and / or improving retinopathy involves inhibiting or reducing retinal ganglion cell (RGC) damage or severe axonal damage in a subject. In certain embodiments, the method treats, prevents or improves a disease, disorder or state associated with retinal ganglion cell (RGC) damage or severe axonal damage. Diseases associated with retinal ganglion cell (RGC) damage or severe axonal damage include: glaucoma, ischemic optic neuropathy, optic neuritis, optic nerve tumors, traumatic optic neuropathy, retinal artery occlusion, retinal vein occlusion, and immature retinopathy.

[0241] 1.5.1 Retinal vascular occlusive disease

[0242] In certain embodiments, the retinopathy includes retinal vascular occlusion disease. The present invention provides a method for treating, preventing or improving retinal vascular occlusion disease, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0243] Retinal vascular occlusion disease (retinal vascular occlusion) is divided into retinal artery or vein obstruction, which can cause retinal ischemia or hemorrhage, and is a common fundus vascular disease. Retinal artery or vein obstruction can cause retinal ischemia, edema or hemorrhage. The main symptoms are decreased central vision or partial visual field loss. The cause is complex and is closely related to retinal inflammation, retinal hypoperfusion, hypertension, arteriosclerosis, high blood viscosity and abnormal hemodynamics.

[0244] Without being bound by theory, retinal ischemia / reperfusion injury is accompanied by glial activation, neuroinflammation and cytokine cascades leading to neurodegeneration and retinal cell death, which are considered to be the main early factors of neuronal death. Astrocytes are the main glial cells in the normal retina and play a vital role in maintaining the blood-retinal barrier and RGC health. Increased expression of astrocyte biomarker glial fibrillary acid protein (GFAP) is regarded as a sign of neuroinflammation and a marker of retinal degeneration. At the same time, the expression of multiple proinflammatory cytokines such as IFN-γ, IL-1β, and / or TNF-α increases, which aggravates I / R injury. Without being bound by theory, targeted inhibition of GFAP proliferation and RGC apoptosis caused by I / R injury, inhibition of the increase of multiple proinflammatory cytokines, and improvement of retinal degeneration after I / R play a very important role in disease treatment.

[0245] In certain embodiments, the retinal vascular occlusion disease includes retinal vein occlusion. Retinal vein occlusion (RVO) is caused by the interruption of venous return of normal retinal tissue. It is a common retinal vascular disease second only to diabetic retinopathy. The vision of the affected eye is easily impaired or even blinded due to complications. According to the location of the vein occlusion, it is divided into central retinal vein occlusion, hemi-central vein occlusion, and branch vein occlusion. The blocked part of central retinal vein occlusion is mostly located in the trunk of the central retinal vein in the lamina cribrosa area or the posterior lamina cribrosa area of ​​the optic disc, and is characterized by high distension and extensive bleeding of the retinal vein. Branch retinal vein occlusion (branch retinal vein occlusion) is more common in patients with arteriosclerosis. The blockage is common in the temporal branch, especially the superior temporal branch. The artery at the blocked site is mostly located in front of the vein, and it occurs at the intersection of the artery and vein from the first branch to the third branch of the vein.

[0246] Retinal vein occlusion is a retinal vascular disease that causes severe vision loss. Its causes include compression of the central retinal vein at or behind the optic disc lamina cribrosa caused by atherosclerosis of the central retinal artery, venous blood stasis, and venous endothelial damage leading to thrombosis. According to the location of the occlusion, it can be divided into central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO). Patients often show a large amount of fundus hemorrhage, venous tortuosity, cotton wool spots, macular edema, and optic disc edema. At the same time, patients with central retinal vein occlusion may develop neovascular glaucoma (NVG) and lead to complete blindness.

[0247] In certain embodiments, the retinal vascular occlusion disease includes retinal artery occlusion. Retinal artery occlusion is an acute fundus disease that severely impairs vision. It can be divided into central retinal artery occlusion, branch retinal artery occlusion, retinal ciliary artery and retinal precapillary arteriolar occlusion according to the source and level of the involved blood vessels. Central retinal artery occlusion refers to an acute ophthalmic disease in which the central retinal artery is partially or completely blocked, causing symptoms such as sudden blindness or sudden vision loss in patients. Branch retinal artery occlusion refers to a disease in which blockage occurs in the branch retinal artery. It is common on the temporal side, especially in the superior temporal branch artery occlusion. The prognosis of vision and fundus changes depend on the location and degree of arterial occlusion. Cilioretinal artery occlusion is a rare critical and severe disease in ophthalmology. If not treated in time, it will seriously threaten vision or even blindness. The ciliary artery is composed of the short posterior ciliary artery, the long posterior ciliary artery and the anterior ciliary artery, which nourishes the entire eyeball except the choroid, the outer layer of the retina and part of the optic nerve. Precapillary arteriolar occlusion (Retinal Precapillary Arteriolar Occlusion) manifests as small gray-white beads or cotton-like spots, which occur in systemic diseases such as diabetes, hypertension, arteriosclerosis, etc.

[0248] Advantageously, the inventors of the present application have found that the method has a beneficial effect of preventing, treating and / or improving retinal vascular occlusion diseases, especially retinal artery occlusion diseases and retinal vein occlusion diseases. Particularly advantageously, the method for preventing, treating and / or improving retinal vascular occlusion diseases of the present invention slows down the loss of retinal ganglion cells (RGCs), protects visual function, inhibits or reduces the severity of secondary neuronal damage; slows down internal retinal damage, reduces retinal edema, and slows down the reduction of retinal thickness; reduces optic nerve fiber layer edema, maintains the thickness of the optic nerve fiber layer and prevents the thinning of the optic nerve fiber layer; significantly inhibits optic nerve inflammation and GFAP overexpression, inhibits the expression level of inflammatory factors related to nerve cell damage such as IFN-γ, IL-1β, and / or TNF-α; reduces optic nerve function reduction, including improving full-field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators, and protects the overall function of the retina.

[0249] In certain embodiments, the method for treating, preventing or ameliorating retinal vascular occlusion disease slows down the loss of retinal ganglion cells (RGC), protects visual function, and inhibits or reduces the severity of secondary neuronal damage. Advantageously, the average loss rate of the number of retinal ganglion cells in subjects with retinal vascular occlusion disease using the method of the present invention is reduced by at least 80% compared to the placebo group (i.e., subjects with retinal vascular occlusion disease who do not use the method of the present invention), while the single-component control group (i.e., subjects with retinal vascular occlusion disease who only use one drug component), the two-component control group (i.e., subjects with retinal vascular occlusion disease who only use two components DOX+MTP) and the three-component control group (i.e., subjects with retinal vascular occlusion disease who use three-component drugs DOX+MTP+BRM different from the present invention) only reduce the average loss rate of the number of retinal ganglion cells by 20%, or even by only 15%.

[0250] In certain embodiments, the method for treating, preventing or ameliorating retinal vascular occlusion disease significantly inhibits optic nerve inflammation and GFAP overexpression. Without being bound by theory, the inventors found that the expression of GFAP in the retinal tissue of subjects with retinal vascular occlusion disease increased to about 5 times the normal expression (i.e., the expression of GFAP in the retinal tissue of subjects without retinal vascular occlusion disease), which is considered to be an overexpression. Advantageously, the expression of GFAP in the retinal tissue of subjects with retinal vascular occlusion disease using the method of the present invention is reduced to less than 50%, less than 40%, or even close to 30% of the overexpression.

[0251] In certain embodiments, the method for treating, preventing or ameliorating retinal vascular occlusion disease inhibits the increase in the expression level of at least one inflammatory factor, wherein the inflammatory factor includes IFN-γ, IL-1β, and / or TNF-α. Without being bound by theory, the inventors found that the expression level of inflammatory factors in the retinal tissue of subjects with retinal vascular occlusion disease was significantly increased compared with the normal level (i.e., the expression level of inflammatory factors in the retinal tissue of subjects without retinal vascular occlusion disease). Advantageously, the expression level of inflammatory factors in the retinal tissue of subjects with retinal vascular occlusion disease using the method of the present invention is reduced to a level close to the normal level.

[0252] In certain embodiments, the method for treating, preventing or improving retinal vascular occlusive disease slows down the damage to the inner retina, reduces retinal edema, and slows down the decrease in retinal thickness. Advantageously, the decrease in the average thickness of the retinal EDTRS area of ​​the subjects with retinal vascular occlusive disease using the method of the present invention is reduced by at least 70% or even at least 80% compared to the placebo group (i.e., the subjects with retinal vascular occlusive disease who do not use the method of the present invention), while the single-component control group (i.e., the subjects with retinal vascular occlusive disease who only use one drug component), the two-component control group (i.e., the subjects with retinal vascular occlusive disease who only use two components of DOX+MTP) and the three-component control group (i.e., the subjects with retinal vascular occlusive disease who use three components of DOX+MTP+BRM different from the present invention) only reduce the decrease in the average thickness of the retinal EDTRS area by about 30%, or 20%, or 10%, or 5%, or even do not reduce but increase the decrease. More advantageously, the reduction in the average thickness of the retinal EDTRS area of ​​subjects without retinal vascular occlusive disease who adopt the method of the present invention is reduced by at least 30%, at least 40%, at least 50%, at least 60%, or even close to 70% compared to the control group (i.e., subjects without retinal vascular occlusive disease who do not adopt the method of the present invention).

[0253] In certain embodiments, the method for treating, preventing or improving retinal vascular occlusive disease reduces optic nerve fiber layer edema, maintains optic nerve fiber layer thickness and prevents optic nerve fiber layer thinning. Advantageously, the average reduction in optic nerve fiber layer (RNFL) thickness of subjects with retinal vascular occlusive disease using the method of the present invention is reduced by at least 80% compared to the placebo group (i.e., subjects with retinal vascular occlusive disease who do not use the method of the present invention), while the single-component control group (i.e., subjects with retinal vascular occlusive disease who only use one drug component), the two-component control group (i.e., subjects with retinal vascular occlusive disease who only use two components of DOX+MTP) and the three-component control group (i.e., subjects with retinal vascular occlusive disease who use three components of DOX+MTP+BRM different from the present invention) only reduce the average reduction in optic nerve fiber layer (RNFL) thickness by about 30%, or even do not reduce but increase the reduction.

[0254] In certain embodiments, the method for treating, preventing or improving retinal vascular occlusion disease reduces optic nerve function by improving full-field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators to protect the overall function of the retina. Advantageously, the amplitudes of the a-wave and b-wave of the light-adapted 3.0 ERG response of the full-field electroretinogram (FERG) of the subject with retinal vascular occlusion disease using the method of the present invention are increased by about 30%, or 50%, or 100%, or 150%, or even close to 200% compared with the control group (i.e., the subject with retinal vascular occlusion disease not using the method of the present invention). More advantageously, the N2-P2 amplitude of the flash visual evoked potential (FVEP) of the subject with retinal vascular occlusion disease using the method of the present invention is increased by about 100%, or 200%, or 300%, or 400%, or even close to 500% compared with the control group (i.e., the subject with retinal vascular occlusion disease not using the method of the present invention).

[0255] 1.5.2 Immature retinopathy

[0256] In certain embodiments, the retinopathy includes retinopathy of prematurity. Children with retinopathy of prematurity (ROP) are mostly under 32 weeks of gestational age and weigh less than 1500g at birth. They are mostly premature infants with a history of inhaling high-concentration oxygen or low-weight infants with developmental delay. ROP is an important cause of blindness in infants and one of the important eye diseases that causes white pupil sign. Premature birth, low birth weight and inhalation of high-concentration oxygen are known pathogenic factors, which are caused by vasoconstriction and vascular proliferation of the incompletely vascularized retina in response to oxygen.

[0257] Advantageously, the inventors of the present application have found that the method has a beneficial effect of preventing, treating and / or improving retinopathy of immature children. Particularly advantageously, the method for preventing, treating and / or improving retinopathy of immature children described in the present invention slows down the loss of retinal ganglion cells (RGCs), protects visual function, inhibits or reduces the severity of secondary neuronal damage; slows down internal retinal damage, reduces retinal edema, and slows down the reduction of retinal thickness; reduces optic nerve fiber layer edema, maintains the thickness of the optic nerve fiber layer and prevents the thinning of the optic nerve fiber layer; significantly inhibits optic nerve inflammation and GFAP overexpression, inhibits the expression level of inflammatory factors related to nerve cell damage such as IFN-γ, IL-1β, and / or TNF-α; reduces optic nerve function reduction, including improving full-field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators, and protects the overall function of the retina.

[0258] In certain embodiments, the method for treating, preventing or improving retinopathy of premature children slows down the loss of retinal ganglion cells (RGC), protects visual function, and inhibits or reduces the severity of secondary neuronal damage. Advantageously, the average loss rate of the number of retinal ganglion cells in subjects with retinopathy of premature children using the method of the present invention is reduced by at least 80% compared to the placebo group (i.e., subjects with retinopathy of premature children who do not use the method of the present invention), while the single-component control group (i.e., subjects with retinopathy of premature children who only use one drug component), the two-component control group (i.e., subjects with retinopathy of premature children who only use two components of DOX+MTP) and the three-component control group (i.e., subjects with retinopathy of premature children who use three components of drugs DOX+MTP+BRM different from the present invention) only reduce the average loss rate of the number of retinal ganglion cells by 20%, or even only by 15%.

[0259] In certain embodiments, the method for treating, preventing or improving retinopathy of premature infants significantly inhibits optic nerve inflammation and GFAP overexpression. Without being bound by theory, the inventors found that the expression of GFAP in the retinal tissue of subjects with retinopathy of premature infants increased to about 5 times the normal expression (i.e., the expression of GFAP in the retinal tissue of subjects who do not suffer from retinopathy of premature infants), which is considered to be an overexpression. Advantageously, the expression of GFAP in the retinal tissue of subjects with retinopathy of premature infants using the method of the present invention is reduced to less than 50% of the overexpression, less than 40%, or even close to 30%.

[0260] In certain embodiments, the method for treating, preventing or improving retinopathy of immature children suppresses the increased expression level of at least one inflammatory factor, wherein the inflammatory factor includes IFN-γ, IL-1β, and / or TNF-α. Without being bound by theory, the inventors found that the expression level of inflammatory factors in the retinal tissue of subjects with retinopathy of immature children was significantly increased compared with the normal level (i.e., the expression level of inflammatory factors in the retinal tissue of subjects who do not suffer from retinopathy of immature children). Advantageously, the expression level of inflammatory factors in the retinal tissue of subjects with retinopathy of immature children using the method of the present invention is reduced to near normal levels.

[0261] In certain embodiments, the method for treating, preventing or improving retinopathy of premature children slows down the damage to the inner retina, reduces retinal edema, and slows down the reduction of retinal thickness. Advantageously, the reduction in the average thickness of the retinal EDTRS area of ​​the subjects with retinopathy of premature children using the method of the present invention is reduced by at least 70% or even at least 80% compared to the placebo group (i.e., the subjects with retinopathy of premature children who do not use the method of the present invention), while the single-component control group (i.e., the subjects with retinopathy of premature children who only use one drug component), the two-component control group (i.e., the subjects with retinopathy of premature children who only use two components of DOX+MTP) and the three-component control group (i.e., the subjects with retinopathy of premature children who use three components of DOX+MTP+BRM different from the present invention) only reduce the reduction in the average thickness of the retinal EDTRS area by about 30%, or 20%, or 10%, or 5%, or even do not reduce but increase the reduction. More advantageously, the reduction in the average thickness of the retinal EDTRS area of ​​subjects without retinopathy of premature infants who adopt the method of the present invention is reduced by at least 30%, at least 40%, at least 50%, at least 60%, and even close to 70% compared to the control group (i.e., subjects without retinopathy of premature infants who did not adopt the method of the present invention).

[0262] In certain embodiments, the method for treating, preventing or improving retinopathy in premature children reduces optic nerve fiber layer edema, maintains optic nerve fiber layer thickness and prevents optic nerve fiber layer thinning. Advantageously, the average reduction in the thickness of the optic nerve fiber layer (RNFL) of subjects with retinopathy in premature children using the method of the present invention is reduced by at least 80% compared to the placebo group (i.e., subjects with retinopathy in premature children who do not use the method of the present invention), while the single-component control group (i.e., subjects with retinopathy in premature children who only use one drug component), the two-component control group (i.e., subjects with retinopathy in premature children who only use two components of DOX+MTP) and the three-component control group (i.e., subjects with retinopathy in premature children who use three components of DOX+MTP+BRM different from the present invention) only reduce the average reduction in the thickness of the optic nerve fiber layer (RNFL) by about 30%, or even do not reduce but increase the reduction.

[0263] In certain embodiments, the method for treating, preventing or improving retinopathy of premature infants reduces optic nerve function by improving full-field electroretinogram (FERG) and flash visual evoked potential (FVEP) functional indicators and protecting the overall function of the retina. Advantageously, the amplitude of the a-wave and b-wave of the full-field electroretinogram (FERG) light-adapted 3.0ERG response of the subjects with retinopathy of premature infants using the method of the present invention is increased by about 30%, or 50%, or 100%, or 150%, or even close to 200% compared with the control group (i.e., the subjects with retinopathy of premature infants who did not use the method of the present invention). More advantageously, the N2-P2 amplitude of the flash visual evoked potential (FVEP) of the subjects with retinopathy of premature infants using the method of the present invention is increased by about 100%, or 200%, or 300%, or 400%, or even close to 500% compared with the control group (i.e., the subjects with retinopathy of premature infants who did not use the method of the present invention).

[0264] 1.6 Diabetic peripheral neuropathy

[0265] In certain embodiments, the peripheral neuropathy is diabetic peripheral neuropathy (DPN). In certain embodiments, a method for treating, preventing or improving diabetic peripheral neuropathy is provided herein, the method comprising administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof or an acceptable form thereof.

[0266] In certain embodiments, diabetic peripheral neuropathy is diabetic distal symmetrical polyneuropathy (DSPN) or diabetic mononeuropathy or multiple mononeuropathy (Zakin E, Abrams R, Simpson DM. Diabetic Neuropathy. Semin Neurol. 2019 Oct; 39(5): 560-569.). Patients with diabetic mononeuropathy or multiple mononeuropathy are often affected by the median nerve, ulnar nerve, and common peroneal nerve, often with insidious onset, and also with acute onset, mainly manifested as sensory and motor dysfunction in the innervated area.

[0267] Diabetic distal symmetric polyneuropathy (DSPN) is the most common type of DPN, present in approximately 50% of diabetic patients. The Toronto Diabetic Neuropathy Consensus Group defines DSPN as "a symmetrical, length-dependent sensorimotor polyneuropathy attributable to metabolic and microvascular alterations resulting from chronic hyperglycemic exposure and cardiovascular risk covariates". DSPN patients include two main clinical manifestations, insensitivity to trauma leading to foot ulcers and extremely painful neuropathic pain. In certain embodiments, DSPN patients present with symmetrical symptoms, and the patient's symptoms include distal limb paresthesias, bilateral lower limb paresthesias, stocking-like dysesthesias, glove-like dysesthesias, involvement of the median nerve, ulnar nerve, and common peroneal nerve, sensory dysfunction, motor dysfunction, insensitivity to trauma, and neuropathic pain.

[0268] Electrophysiological examination (EMG) is widely used to evaluate various types of neurological dysfunction in patients with diabetic peripheral neuropathy. EMG can make an objective and quantitative diagnosis of nerve function. Neuroelectrophysiological studies have shown that the sensory nerve conduction velocity of diabetic patients for 5-10 years is slowed down, that is, these diabetic patients have diabetic peripheral neuropathy. The degree and rate of abnormal sensory nerve conduction velocity (SCV) in patients with diabetic peripheral neuropathy are usually more serious than the abnormal motor nerve conduction velocity (MCV), and the patient's nerve function abnormalities are more serious in the lower limbs than in the upper limbs (Neurological Complications Group of the Diabetes Branch of the Chinese Medical Association. Expert Consensus on the Diagnosis and Treatment of Diabetic Neuropathy (2021 Edition). Chinese Journal of Diabetes, 2021, 13(06): 540-557).

[0269] Advantageously, the inventors of the present application have found that the present method has beneficial effects in preventing, treating and / or improving peripheral neuropathy, especially diabetic peripheral neuropathy. In certain embodiments, the method improves peripheral nerve conduction velocity, significantly improves sensory nerve conduction velocity of the sural nerve, improves microcirculation, inhibits nerve inflammation, inhibits central nervous inflammation, repairs damaged nerve tissue, relieves symptoms of peripheral neuropathy for a long time, controls the progression of neuropathy for a long time, and controls the progression of neuropathic pain for a long time.

[0270] Particularly advantageously, the method for preventing, treating and / or improving diabetic peripheral neuropathy of the present invention improves peripheral nerve conduction velocity, especially significantly improves the sensory nerve conduction velocity of the sural nerve. Compared with before administration, the sensory nerve conduction velocity of the subjects of DSPN using the method of the present invention increased by at least 4.3m / s, even 4.6m / s on average. In contrast, the sensory nerve conduction velocity of the placebo group (i.e., the DSPN subjects who did not use the method of the present invention), the single-component control group (i.e., the subjects who only used one drug component), and the two-component control group (i.e., the subjects who only used two components of DOX+MTP or the subjects who only used two components of DOX+PMP) did not show significant changes before and after administration, and even the conduction velocity after administration was reduced compared with that before administration. Advantageously, in the abnormal sural sensory nerves of the subjects of DSPN, after treatment with the method of the present invention, up to 75% of the nerve numbers were effectively improved, and the effective improvement rate was higher than that of the positive drug control group.

[0271] Advantageously, the method for preventing, treating and / or improving diabetic peripheral neuropathy of the present invention can improve microcirculation, inhibit the reduction of local cerebral blood volume (rCBV) around cerebral tissue infarction, improve the blood perfusion of brain tissue of the subject, improve cerebral edema and brain tissue lesions, and reduce brain tissue necrosis. Without being bound by theory, the present method improves the microcirculation of the patient by inhibiting the reduction of brain volume in patients with diabetic peripheral neuropathy, alleviates the ischemia and hypoxia of neural tissue and the damage of neural structure and function caused by microcirculatory disorders. The DSC-PWI sequence results of MRI scans can be used to judge the situation of cerebral microcirculation. Among them, cerebral blood flow (CBF) refers to the blood flow that flows through a certain amount of brain tissue vascular structure per unit time, and the smaller its value, the lower the brain tissue blood flow; cerebral blood volume (CBV) refers to the blood volume present in a certain amount of brain tissue vascular structure, and the smaller its value, the less brain tissue blood volume; mean transit time (MTT) refers to the average time for the contrast agent to pass through the brain tissue, and peak time (TTP) refers to the time when the contrast agent reaches the peak value in the brain area. When the relative cerebral blood flow (rCBF) is significantly reduced, the relative cerebral blood volume (rCBV) is significantly reduced, the relative mean transit time (rMTT) is prolonged, and the relative time to peak (rTTP) is prolonged, it indicates that the patient has microcirculatory disorders and insufficient blood perfusion. The recovery or increase of relative cerebral blood volume (rCBV) indicates that the patient's microcirculation has improved. DSC-PWI results can provide clinical patients with sensitive and accurate brain microcirculation information (Li Yongli, Yan Fengshan, Dou Shewei, et al. Clinical comparative application of arterial proton spin labeling and dynamic magnetic sensitivity contrast enhancement perfusion imaging in the evaluation of brain microcirculation in patients with cerebral ischemia. Journal of Zhengzhou University: Medical Edition, 2013(4):4; Jing Yanping, Luo Bin, Gao Zhengrong, et al. The application value of DSC-PWI in ischemic cerebrovascular disease. Chinese Journal of Physicians, 2020, 22(3):6). Advantageously, the volume of the non-perfused area (ischemic core area) of the brain of a subject with microcirculatory disorder using the method of the present invention is reduced by at least 70% compared to a subject with microcirculatory disorder not using the method.

[0272] Advantageously, the method for preventing, treating and / or improving diabetic peripheral neuropathy of the present invention can also inhibit neuroinflammation, especially inhibit central nervous inflammation, which is manifested as inhibiting the overexpression of GFAP protein in brain tissue. The inventors found that the expression of GFAP in the tissue surrounding cerebral infarction of subjects with cerebral microcirculation disorders increased to about 3-4 times the normal expression (i.e., the expression of GFAP in the brain tissue of subjects without cerebral microcirculation disorders), which is considered to be an overexpression. Without being bound by theory, the present method inhibits neuroinflammation, inhibits the increase in oxidative stress levels caused by abnormal glucose metabolism, repairs inflammation and damage to the vascular endothelium and blood-brain barrier, and alleviates diabetic neuropathy by inhibiting the overexpression of GFAP in brain tissue. Advantageously, the expression of GFAP in the brain tissue of subjects with cerebral microcirculation disorders using the method of the present invention is reduced to less than 50% of the overexpression, or even less than 40%.

[0273] Advantageously, the method for preventing, treating and / or improving diabetic peripheral neuropathy of the present invention can also repair damaged nerve tissue. Particularly advantageously, the method of the present invention can reduce optic nerve fiber layer edema, maintain optic nerve fiber layer thickness and prevent optic nerve fiber layer thinning. Without being bound by theory, the present method maintains the thickness of the optic nerve fiber layer, slows down edema, degeneration and necrosis of nerve fibers, maintains the structure and function of nerve cells, protects damaged neurons, and repairs damaged nerve tissue. The average thickness reduction of the optic nerve fiber layer (RNFL) of the subjects using the method of the present invention can be reduced by at least 80% compared with the placebo group (i.e., the subjects who did not use the method of the present invention), while the single-component control group (i.e., glaucoma subjects who only used one drug component), the two-component control group (i.e., glaucoma subjects who only used two components of DOX+MTP) and the three-component control group (i.e., glaucoma subjects who used three-component drugs DOX+MTP+BRM different from the present invention) can only reduce the average thickness reduction of the optic nerve fiber layer (RNFL) by about 30%, or even increase the reduction without reducing it.

[0274] Advantageously, the method for preventing, treating and / or improving diabetic peripheral neuropathy of the present invention can relieve the symptoms of peripheral neuropathy for a long time, control the progression of neuropathy for a long time, and show good safety during administration. Without being bound by theory, the method for preventing, treating and / or improving diabetic peripheral neuropathy of the present invention can relieve and control the progression of peripheral neuropathy for a long time by improving cerebral microcirculation, inhibiting central nervous inflammation, and repairing damaged nerve tissue. Advantageously, the method of the present invention will not cause elevated liver enzyme levels and gastrointestinal-related toxic and side effects, and can have the effect of long-term relief and control of neuropathy progression.

[0275] In certain embodiments, diabetic peripheral neuropathy is diabetic peripheral neuropathic pain (DPNP). About 20% of DSPN patients experience neuropathic pain (painful-DSPN), i.e., DPNP, and are referred to as painful DSPN patients. The most common neuropathic symptoms in this type of patient are burning sensation, "electric shock-type" pain, and severe pain. In certain embodiments, the patient's sleep is affected by pain, and they suffer from depression and anxiety caused by pain. More than half of the patients consider their pain level to be "severe," and patients often report that their symptoms are more severe at night. It is predicted that up to three-quarters of patients' sleep will be disturbed, and patients with diabetic painful peripheral neuropathy are also more likely to experience symptoms of depression and anxiety.

[0276] Advantageously, the method for preventing, treating and / or improving diabetic peripheral neuropathy described in the present invention can control the progression of neuropathic pain for a long time and relieve the symptoms of neuropathic pain. Without being bound by theory, the method for preventing, treating and / or improving diabetic peripheral neuropathy described in the present invention protects central nervous system and peripheral neuropathy simultaneously by improving cerebral microcirculation, inhibiting central nervous system inflammation, and repairing damaged nerve tissue, and can treat DPNP based on the above mechanism to relieve the neuropathic pain of the subject. Without being bound by theory, this method can inhibit the overexpression of GFAP protein, inhibit neuroinflammation, repair damaged nerve cells, and relieve the patient's neuropathic pain and other symptoms. Without being bound by theory, the protection of the structure and function of peripheral nerves and central nervous system (CNS) suggests the relief of pain symptoms in DPNP patients.

[0277] 2. Pharmaceutical Composition

[0278] In certain embodiments, the present invention relates to a pharmaceutical composition for regulating neuropathy, comprising a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof.

[0279] The term "pharmaceutical composition" refers to a combination of multiple substances with specific medical or biological uses, which are usually expected to have a therapeutic or preventive effect on a specific disease after being administered to a subject. A pharmaceutical composition may contain only a specified active ingredient (biologically active substance), or it may be provided together with a conventional pharmaceutically acceptable carrier for various purposes. The term "composition" in this application should be interpreted in a broad sense. As one implementation of the pharmaceutical composition of the present invention, for example, the specified active ingredients (and optional pharmaceutically acceptable carriers) can be mixed together to provide a mixture in which the components are indistinguishable. As another implementation of the pharmaceutical composition of the present invention, each specified active ingredient can also be separately packaged into small portions, and these small independent packages can be combined and accommodated together in a larger container to provide the "pharmaceutical composition" of the present invention.

[0280] In certain embodiments, a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof are formulated into a single pharmaceutical composition.

[0281] The terms "active ingredient" and "biologically active substance" refer to molecules and other agents of biologically, physiologically or pharmaceutically active substances that act in a subject to treat a disease or condition (e.g., diabetic retinopathy). The terms are used relative to terms such as "pharmaceutically acceptable carrier", "excipient", "adjuvant", etc. "Active ingredient" (or "biologically active substance") includes, but is not limited to, pharmaceutically acceptable salts and prodrugs thereof. These agents can be acids, bases or salts; they can be neutral molecules, polar molecules or molecular complexes capable of hydrogen bonding; they can be prodrugs in the form of ethers, esters, amides, etc., which are biologically activated when administered to a subject.

[0282] The term "its acceptable form" includes, but is not limited to, pharmaceutically acceptable hydrates, solvates, isotope-labeled compounds, optical isomers, geometric isomers, tautomers, isomer mixtures, and / or prodrugs of a certain active ingredient (e.g., doxazosin molecule). Solvates include hydrates, ethanol solvates, acetone solvates, etc.

[0283] The "acceptable form" of an active ingredient described in the present invention also includes a pharmaceutically acceptable salt formed from an isotope-labeled compound of the active ingredient, or its optical isomers, geometric isomers, tautomers or isomer mixtures, or a prodrug thereof.

[0284] It should be understood that all references to various active ingredients or pharmaceutically acceptable salts include various crystalline forms (eg, amorphous forms, polymorphs, etc.) of the same active ingredient or salt.

[0285] The term "doxazosin" as used herein refers to the English name Doxazosin (DOX for short), the IUPAC name (RS)-2-[4-(2,3-dihydro-1,4-benzodioxene-2-carbonyl)piperazin-1-yl]-6,7-dimethoxy-4-amine (molecular formula C 23 H 25 N5O5, molecular weight: 451.475 g / mol) as follows:

[0286]

[0287] The present invention also includes isotope-labeled compounds of doxazosin, or optical isomers, geometric isomers, tautomers or isomer mixtures thereof, or prodrugs thereof (ie, compounds obtained by in vivo reactions).

[0288] Doxazosin is a selective α1 receptor antagonist that inhibits the binding of norepinephrine (released from sympathetic nerve endings) to α1 receptors on the vascular smooth muscle cell membrane and is commonly used to treat essential hypertension.

[0289] The pharmaceutical composition of the present invention may contain doxazosin or a pharmaceutically acceptable salt thereof as an active ingredient. In certain embodiments, the salt form of doxazosin is a mesylate.

[0290] The term "pramipexole" as used herein refers to the English name Pramipexole (PMP for short), the IUPAC name of which is (S)-N6-propyl-4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-diamine (molecular formula C 10 H 17 N3S, molecular weight: 211.324 g / mol) is as follows:

[0291]

[0292] The present invention also includes isotope-labeled compounds of pramipexole, or optical isomers, geometric isomers, tautomers or isomer mixtures thereof, or prodrugs thereof (ie, compounds obtained by in vivo reactions).

[0293] Pramipexole is an antihistamine that acts as a dopamine receptor D2 / D3 agonist and is clinically used to treat Parkinson's disease, either alone (without levodopa) or in combination with levodopa. Pramipexole is sometimes also known as "Mirapa" or "Mirapex", "Mirapexin", "Sifrol", etc.

[0294] The pharmaceutical compositions of the present invention may include pramipexole or a pharmaceutically acceptable salt thereof as an active ingredient. In certain embodiments, the salt form of pramipexole is a hydrochloride. In certain embodiments, the present invention includes a dihydrochloride salt of pramipexole. In certain embodiments, the present invention includes a hydrate form of pramipexole. In certain embodiments, the present invention includes a monohydrate of pramipexole.

[0295] The term "metoprolol" as used herein refers to the English name Metoprolol (MTP for short), the IUPAC name (RS)-1-[4-(2-methoxyethyl)phenoxy]-3-[(propan-2-yl)amino]propan-2-ol (molecular formula C 15 H 25 NO3, molecular weight 267.37g / mol) as follows:

[0296]

[0297] The present invention also includes isotope-labeled compounds of metoprolol, or optical isomers, geometric isomers, tautomers or isomer mixtures thereof, or prodrugs thereof (i.e. compounds obtained by in vivo reactions).

[0298] Metoprolol is a selective adrenergic β1 receptor blocker, commonly used to treat hypertension and angina pectoris. The pharmaceutical composition of the present invention may contain metoprolol or a pharmaceutically acceptable salt thereof as an active ingredient. In certain embodiments, the salt form of metoprolol is tartrate.

[0299] The active ingredients (doxazosin, pramipexole, metoprolol) contained in the composition of the present invention can be replaced by their pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" of a compound refers to a salt that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. The pharmaceutically acceptable salt used herein is a salt formed with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include, but are not limited to, inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, pyrophosphoric acid, hydrobromic acid or nitric acid, and organic acids such as citric acid, fumaric acid, maleic acid, malic acid, ascorbic acid, succinic acid, tartaric acid, benzoic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, salicylic acid, stearic acid, benzenesulfonic acid or p-toluenesulfonic acid. Pharmaceutically acceptable bases include hydroxides of alkali metals (such as sodium or potassium) and alkaline earth metals (such as calcium or magnesium), and organic bases such as alkylamines, arylamines or heterocyclic amines.

[0300] Pharmaceutically acceptable salts can be synthesized from parent compounds containing alkaline or acidic moieties by conventional chemical methods. Typically, these salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent or in a mixture of the two; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. A list of salts can be found in Remington's Pharmaceutical Sciences 18th edition (Mack Publishing Company, 1990). For example, salts may include, but are not limited to, hydrochlorides, tartrates, mesylates, etc. of the compounds of the invention.

[0301] The content of doxazosin (or its pharmaceutically acceptable salt), pramipexole (or its pharmaceutically acceptable salt) and metoprolol (or its pharmaceutically acceptable salt) in the pharmaceutical composition of the present invention can be adjusted according to actual needs. For example, the content or ratio of each drug in the pharmaceutical composition can be changed according to the administration method of the pharmaceutical composition (oral or injection, etc.).

[0302] In the pharmaceutical composition of the present application, if the weight of doxazosin or a pharmaceutically acceptable salt thereof is taken as a reference (=1), the weight ratio of metoprolol or a pharmaceutically acceptable salt thereof to doxazosin or a pharmaceutically acceptable salt thereof is generally in the range of 0.3:1 to 400:1, for example, it may be greater than or equal to 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 10:1, 12.5:1 or 15:1, preferably greater than or equal to 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 10:1, 12.5:1 or 15:1. 1, 4:1, 5:1, 10:1, 12.5:1 or 15:1, and may be less than or equal to 350:1, 300:1, 250:1, 200:1, 150:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1 or 20:1, preferably less than or equal to 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1 or 20:1; similarly, based on the weight of doxazosin or its pharmaceutically acceptable salt as a reference (=1), then pramipexole or its The weight ratio of the pharmaceutically acceptable salt to doxazosin or its pharmaceutically acceptable salt is generally in the range of 0.003:1 to 10:1, for example, it may be greater than or equal to 0.004:1, 0.005:1, 0.006:1, 0.007:1, 0.008:1, 0.009:1, 0.01:1, 0.015:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.1:1 or 0.2:1, preferably greater than or equal to 0.005:1, 0.006:1, 0.007:1, 0.008:1, 0.009:1, 0.01:1, 0.015:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.1:1 or 0.2:1. 1, 0.007:1, 0.008:1, 0.009:1, 0.01:1, 0.015:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1 or 0.1:1, and can be less than or equal to 9:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.6:1, 0.5:1, 0.4:1 or 0.3:1, and is preferably less than or equal to 1:1, 0.9:1, 0.6:1, 0.5:1 or 0.4:1.

[0303] 3. Drug dosage form

[0304] In some embodiments, the pharmaceutical composition of the present invention can be provided as a bulk drug in the form of (e.g., a uniform mixture or an independent package of each component). In other embodiments, the pharmaceutical composition of the present invention can be formulated into various pharmaceutical dosage forms (preparations) by adding a pharmaceutically acceptable carrier as needed. For this purpose, various liquid or solid fillers, diluents, excipients, solvents or encapsulating materials can be used as "pharmaceutically acceptable carriers". In the present application, the phrase "pharmaceutically acceptable" refers to compounds, compositions, polymers and other materials that are compatible with other components of the present application composition and suitable for contact with human and animal tissues without excessive toxicity, stimulation, allergic reactions or other problems or complications within the scope of reasonable medical judgment. In some preferred embodiments, the pharmaceutically acceptable carrier is pyrogen-free. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) maltodextrin; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soy oil. oils; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0305] The pharmaceutical composition of the present invention can be formulated into dosage forms suitable for oral administration, parenteral administration (including subcutaneous, intramuscular, cortical and intravenous), bronchial administration, nasal administration, etc. as needed. Among them, preferably, the pharmaceutical composition of the present invention is formulated into a dosage form (preparation) suitable for oral administration.

[0306] If a solid carrier is used, the preparation can be in tablets, placed in a hard gel capsule in the form of a powder or granules, or in the form of a lozenge or a lozenge. The solid carrier may include conventional excipients, such as adhesives, fillers, tableting lubricants, disintegrants, wetting agents, etc. If necessary, the tablet may be coated with a conventional technique film. If a liquid carrier is used, the preparation may be in the form of a syrup, an emulsion, a soft gel capsule, a sterile carrier for injection, an aqueous or non-aqueous liquid suspension, or may be a dry product reconstituted with water or other appropriate carriers before use. The liquid preparation may include conventional additives, such as suspending agents, emulsifiers, wetting agents, non-aqueous carriers (including edible oils), preservatives, and flavoring agents and / or coloring agents. For parenteral administration, the carrier usually includes at least sterile water for the most part, but saline solutions, glucose solutions, etc. may also be used. Injectable suspensions may also be used, in which case conventional suspending agents may be used. Conventional preservatives, buffering agents, etc. may also be added to parenteral dosage forms.

[0307] Dosage forms suitable for parenteral injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (e.g., olive oil) and injectable organic esters (e.g., ethyl oleate).

[0308] These pharmaceutical dosage forms may also include various excipients, such as preservatives, wetting agents, emulsifiers and dispersants. The inhibition of the effects of microorganisms may be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). Isotonic agents may also be included, such as sugar, sodium chloride, etc. The absorption of injectable pharmaceutical dosage forms may be extended by using delayed absorption agents (e.g., aluminum monostearate and gel).

[0309] The present invention also provides pharmaceutical compositions for administration to humans and animals in unit dosage forms, for example, tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral or suspensions, and oil-water emulsions containing a suitable amount of the compound or its pharmaceutically acceptable salt. Pharmaceutical therapeutically active compounds and their salts are formulated and administered in unit dosage forms or multiple dosage forms. Unit dosage forms as used in the present invention refer to physically separate units suitable for human and animal subjects, and are packaged independently, as known in the art. Each unit dose contains a predetermined amount of therapeutically active compounds sufficient to produce the desired therapeutic effect, associated with a desired pharmaceutical carrier, vehicle, or diluent. Examples of unit dosage forms include ampoules and syringes, and independently packaged tablets or capsules. Unit dosage forms can be administered in parts or multiple administrations thereof. Multiple dosage forms are multiple identical unit dosage forms packaged in a single container, to be administered in separate unit dosage forms. Examples of multiple dosage forms include vials, tablets or capsules, or pint or gallon bottles. Therefore, multiple dosage forms are multiple unit doses that are not separated in packaging.

[0310] In some embodiments, pharmaceutical composition is a long-acting preparation. In some embodiments, long-acting preparation can be a sustained release product. The suitable example of sustained release product includes a semipermeable matrix of a solid hydrophobic polymer comprising a compound provided by the invention, wherein the matrix is ​​in the form of a tangible object, such as a film, or a microcapsule. The example of sustained release matrix includes an iontophoresis patch, polyester, hydrogel (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinyl ethanol)), polylactide, a copolymer of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer, such as LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprorelin acetate) and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules to exceed 100 days, and some hydrogels release proteins in a shorter period of time. When encapsulated compounds are kept in the body for a long time, as a result of exposure to a humid environment at 37°C, they may denature or aggregate, resulting in loss of biological activity and possible changes in their structure. Depending on the mechanism of action involved, a rational strategy can be designed for stability. For example, if it is found that the aggregation mechanism is the formation of intermolecular SS bonds through sulfo-disulfide interchange, stability can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling water content, utilizing appropriate additives, and developing special polymer matrix compositions. In some embodiments, the pharmaceutical composition is a long-acting oral formulation.

[0311] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert excipient (or carrier) (e.g., sodium citrate or dicalcium phosphate), which may also include: (a) fillers or admixtures (e.g., starch, lactose, sucrose, glucose, mannitol and silicic acid); (b) binders (e.g., carboxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic); (c) humectants (e.g., glycerol); (d) disintegrants (e.g., agar); The invention can be prepared by mixing (a) agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain synthetic silicates, sodium carbonate); (e) solution retarder (e.g., paraffin); (f) absorption promoter (e.g., quaternary ammonium compounds); (g) wetting agents (e.g., cetyl alcohol and glycerol monostearate); (h) adsorbents (e.g., kaolin and bentonite) and (i) lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate) or mixtures thereof.

[0312] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or lactose as well as high molecular weight polyethylene glycols.

[0313] Solid dosage forms (e.g., tablets, dragees, capsules, pills and granules) can be prepared using coatings and shells (e.g., enteric coatings and others known in the art). They can contain opacifiers, and they can also be compositions that release the active compound or various active compounds in a delayed manner in a certain part of the intestine. Examples of useful embedding compositions are polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0314] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, dispersions, syrups and elixirs. In addition to the active compound, the liquid dosage form may include inert diluents (e.g., water or other solvents), solubilizers and emulsifiers (e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide ammonium), oils (specifically, cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan or mixtures of these substances, etc., commonly used in the art.

[0315] Besides these inert diluents, pharmaceutical dosage forms may also include, for example, wetting agents, emulsifying and suspending agents, perfuming agents, flavoring agents, and perfuming agents.

[0316] In addition to the active compounds, suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

[0317] The pharmaceutical dosage forms of the present invention also include ointments, powders, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any required preservatives, buffers or propellants.

[0318] The amount of active ingredients in the pharmaceutical composition and dosage form can be appropriately determined by those skilled in the art as needed. For example, each active ingredient is generally present in the pharmaceutical composition or dosage form in a therapeutically effective amount.

[0319] For example, the pharmaceutical composition of the present invention can be formulated into an oral dosage form (preferably 1, 2, 3, or 4 times a day), an intravenous dosage form, a subcutaneous injection dosage form, or an intramuscular injection dosage form. In certain embodiments, the pharmaceutical composition of the present invention is formulated into a long-acting oral preparation.

[0320] In a preferred embodiment of the present invention, the pharmaceutical composition of the present invention is an oral dosage form. In a further preferred embodiment of the present invention, the pharmaceutical composition of the present invention is an oral dosage form for 1, 2, 3 or 4 times a day. In certain embodiments, the pharmaceutical composition of the present invention is a long-acting sustained-release oral preparation.

[0321] In a preferred embodiment of the present invention, the pharmaceutical composition of the present invention is a dosage form for oral administration. In a further preferred embodiment of the present invention, the pharmaceutical composition of the present invention is a daily oral dosage form.

[0322] 4. Dosage

[0323] The treatment methods of the present invention involve the combined use of multiple active ingredients, also known as "combination therapy" or "combination therapy". The "combination therapy" or "combination therapy" refers to the administration of multiple active ingredients described in the present invention so that they work together to provide a beneficial effect. The beneficial effects of the above combination include, but are not limited to, the pharmacodynamic or pharmacodynamic synergy produced by the combination of the above active ingredients. The combined administration of these active ingredients is usually completed within a prescribed period of time (usually minutes, hours, days or weeks, depending on the judgment of the doctor). "Combination therapy" or "combination therapy" is intended to include the administration of these active ingredients in a sequential manner, that is, each of the active ingredients is administered at a different time, and also includes the administration of these active ingredients in a substantially simultaneous manner, or the administration of at least two of these active ingredients. Substantially simultaneous administration can be achieved, for example, by administering a single capsule to the host, wherein the capsule contains a fixed ratio of each active ingredient, or by administering multiple capsules to the host, each capsule containing one of the active ingredients. The sequential or substantially simultaneous administration of each active ingredient may be affected by any suitable route, including, but not limited to, oral, intravenous, intramuscular, intraocular, and direct absorption via mucosal tissue. The active ingredients may be administered by the same route or by different routes. For example, the first active ingredient in the selected combination may be administered by intravenous injection, while the other active ingredients in the combination may be administered orally. Alternatively, for example, all active ingredients may be administered orally, or all active ingredients may be administered by intravenous injection. The order of administration of the active ingredients is not strictly limited.

[0324] "Combination therapy" or "combination therapy" also includes the combination of the active ingredients described above with other biologically active ingredients and non-drug treatments (e.g., surgical treatment or mechanical treatment). When the combination therapy further includes non-drug treatment, the non-drug treatment can be performed at any appropriate time as long as the beneficial effects of the combination of the active ingredients and the non-drug treatment can be achieved. For example, in appropriate circumstances, the beneficial effects can still be achieved after the non-drug treatment is temporarily stopped from the administration of the active ingredients, and the stopping may be for several days or even weeks.

[0325] In order to achieve the desired effect, it is usually necessary to administer a therapeutically effective amount of the pharmaceutical composition or dosage form of the present invention or each active ingredient alone to a subject.

[0326] In certain embodiments, the methods described herein comprise administering to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof in combination with a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and further in combination with a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof.

[0327] In certain embodiments, the methods described herein administer to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, simultaneously.

[0328] In certain embodiments, the methods described herein administer to a subject a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, respectively.

[0329] In some embodiments of the present invention, for an adult with a normal body weight of about 60 kg, when the pharmaceutical dosage form or pharmaceutical composition of the present invention is taken orally, the daily dose of doxazosin can range from 0.4 mg to 16 mg, for example, 0.4 mg to 15 mg, 0.4 mg to 13 mg, 0.4 mg to 12 mg, 0.4 mg to 10 mg, 0.4 mg to 8 mg, 0.4 mg to 6 mg, 0.4 mg to 5 mg, 0.4 mg to 4 mg, 0.5 mg to 16 mg, 0.5 mg to 15 mg, 0.5 mg to 13 mg, 0.5 mg to 12 mg, 0.5 mg to 10 mg, 0.5 mg to 8 mg, 0.5 mg to 6 mg, 0.5 mg to 5 mg , 0.5mg to 4mg, 1mg to 15mg, 1mg to 13mg, 1mg to 12mg, 1mg to 10mg, 1mg to 8mg, 1mg to 6mg, 1mg to 5mg, 1mg to 4mg, 1.5mg to 16mg, 1.5mg to 15mg, 1.5mg to 13mg, 1.5mg to 12mg, 1.5mg to 10mg, 1.5mg to 8mg, 1.5mg to 6mg, 1.5mg to 5mg, 1.5mg to 4mg, 2mg to 16mg, 2mg to 15mg, 2mg to 13mg, 2mg to 12mg, 2mg to 10mg, 2mg to 8mg, 2mg to 6mg, 2mg to 5mg, 2mg to 4mg, etc. In a preferred embodiment of the present invention, the daily dose of doxazosin can be about 2mg. In another preferred embodiment of the present invention, the daily dose of doxazosin is about 8mg.

[0330] In some embodiments of the present invention, for a normal adult with a body weight of about 60 kg, when the pharmaceutical dosage form or pharmaceutical composition of the present invention is taken orally, the daily dose of pramipexole can range from 0.03 mg to 4.5 mg, for example, 0.03 mg to 4.2 mg, 0.03 mg to 4 mg, 0.03 mg to 3.5 mg, 0.03 mg to 3 mg, 0.03 mg to 2.5 mg, 0.03 mg to 2 mg, 0.03 mg to 1.5 mg, 0.03 mg to 1 mg, 0.05 mg to 4.5 mg, 0.05 mg to 4.2 mg, 0.05 mg to 4 mg, 0.05 mg to 3.5 mg, 0.05 mg to 3 mg, 0.05 mg to 2.5 mg, 0.05 mg to 2 mg, 0.05 mg to 1.5 mg, 0.05 mg to 1 mg, 0.1 mg to 4.5 mg, 0.1 mg to 4 .2mg, 0.1mg to 4mg, 0.1mg to 3.5mg, 0.1mg to 3mg, 0.1mg to 2.5mg, 0.1mg to 2mg, 0.1mg to 1.5mg, 0.1mg to 1mg, 0.2mg to 4.5mg, 0.2mg to 4.2mg, 0.2mg to 4mg, 0.2mg to 3.5mg, 0.2mg to 3mg, 0.2mg to 2.5mg, 0.2mg to 2mg, 0.2mg to 1.5mg, 0.2mg to 1.0mg, 0.5mg to 4.5mg, 0.5mg to 4.2mg, 0.5mg to 4mg, 0.5mg to 3.5mg, 0.5mg to 3mg, 0.5mg to 2.5mg, 0.5mg to 2mg, 0.5mg to 1.5mg, 0.5mg to 1mg, 1mg to 4mg, 1mg to 3mg, 1mg to 2.5mg, etc. In a preferred embodiment of the present invention, the daily dose of pramipexole is about 0.0625mg. In another preferred embodiment of the present invention, the daily dose of pramipexole is about 0.125mg.

[0331] The daily dose of metoprolol may range from 2 mg to 200 mg, such as 2.5 mg to 200 mg, for example 5 mg to 200 mg, preferably 5 mg to 100 mg. In some embodiments of the present invention, for a normal adult with a body weight of about 60 kg, when the pharmaceutical dosage form or pharmaceutical composition of the present invention is taken orally, the daily dose of metoprolol can range from 2 mg to 180 mg, 2 mg to 160 mg, 2 mg to 150 mg, 2 mg to 130 mg, 2 mg to 120 mg, 2 mg to 100 mg, 2 mg to 80 mg, 2 mg to 60 mg, 2 mg to 50 mg, 2 mg to 40 mg, 5 mg to 180 mg, 5 mg to 160 mg, 5 mg to 150 mg, 5 mg to 130 mg, 5 mg to 120 mg, 5 mg to 100 mg, 5 mg to 80 mg, 5 mg to 60 mg, 5 mg to 50 mg, 5 mg to 40 mg, 10 mg to 180 mg, 10 mg to 160 mg , 10mg to 150mg, 10mg to 130mg, 10mg to 120mg, 10mg to 100mg, 10mg to 80mg, 10mg to 60mg, 10mg to 50mg, 10mg to 40mg, 15mg to 200mg, 15mg to 180mg, 15mg to 160mg, 15mg to 150mg, 15mg to 130mg, 15mg to 120mg, 15mg to 100mg, 15mg to 80mg, 15mg to 60mg, 20mg to 200mg, 20mg to 180mg, 20mg to 160mg, 20mg to 150mg, 20mg to 130mg, 20mg to 120mg, 20mg to 100mg, 20mg to 80mg, 20mg to 600mg by weight. In a preferred embodiment of the present invention, the daily dose of metoprolol ranges from 10 mg to 100 mg. In another preferred embodiment of the present invention, the daily dose of metoprolol is about 20 mg. In another preferred embodiment of the present invention, the daily dose of metoprolol is about 100 mg.

[0332] The above daily dose can be administered continuously and periodically, for example, once every 2 hours, every 6 hours, every 8 hours, every 12 hours, or about every 24 hours. Preferably, the daily dose can be administered to the subject once, twice, three times, or four times a day, or administered using a sustained-release tablet. The oral daily doses of the above three active ingredients vary greatly, which is determined by the in vivo pharmacokinetic properties of each active ingredient.

[0333] In certain embodiments, a therapeutically effective amount of doxazosin, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, a therapeutically effective amount of pramipexole, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, and a therapeutically effective amount of metoprolol, a pharmaceutically acceptable salt thereof, or an acceptable form thereof, is administered to a subject once, twice, three times, or four times a day.

[0334] Those skilled in the art will appreciate that when the pharmaceutical composition of the present invention is formulated into other dosage forms suitable for intravenous infusion or intramuscular injection, the dosage range of each active ingredient may be different from the oral dosage range given above. Those skilled in the art or doctors can make reasonable determinations based on in vivo and in vitro experiments and taking into account the different pharmacokinetic characteristics of various routes of administration.

[0335] 5. Subjects

[0336] In a preferred embodiment of the present invention, the pharmaceutical composition of the present invention is used in mammals, primate subjects, especially human subjects.

[0337] Over the past decades, more than 1,000 cell experiments and rodent experiments such as ischemia-reperfusion mouse and rat models have demonstrated the effectiveness of neuroprotection, but the results have been poor or failed in clinical trials. The main reason for this is the difference in the anatomical structure between the brains of small animals and humans (Cook DJ, Teves L, Tymianski M. Treatment of stroke with a PSD-95 inhibitor in the gyrencephalic primate brain. Nature. 2012 Feb 29; 483(7388): 213-7.).

[0338] The present invention uses a 90-minute reperfusion model of middle cerebral artery occlusion (MCAO) in rhesus monkeys to evaluate the neuropathy regulation and protection of the composition, especially the therapeutic effect on stroke. The MCAO rhesus monkey model is a primate model with the following advantages:

[0339] (1) Rhesus monkeys have distinct cerebral sulci and gyri, and their brain volume and structure are similar to the human cortical, subcortical, and white matter anatomy, and they can be assessed for cognitive, motor, and sensory deficits through standardized tests.

[0340] (2) Rhesus monkeys can survive for a long time and undergo multiple dynamic MRI examinations. The trajectory of brain tissue degeneration and necrosis, such as ischemia, edema, and necrosis, can be dynamically observed at 3-4 hours, 72-75 hours, 7 days, 14 days, and 28 days after reperfusion. MRI imaging sequences include T1WI, T2 FLAIR, DWI, MRA, and DSC. Among them, T1WI can display the anatomical structure of the brain, distinguish gray matter and white matter areas, and diagnose hemorrhage and ischemia. T2 FLAIR and DWI can quantify the volume of edema / necrosis. T2 FLAIR can indicate an increase in tissue water content and is used to show inflammation, ischemia, edema and other lesions. After cerebral infarction occurs, an inflammatory reaction occurs immediately, and the bound water content increases. Therefore, an abnormally high signal of T2 FLAIR indicates the presence of cerebral infarction and inflammation. DSC can show the blood perfusion of brain tissue and the sensitive cerebral ischemic area after acute ischemic stroke, and can quantitatively evaluate the volume of the ischemic core area of ​​brain tissue. MRI can quantify ischemic penumbra extent and edema / infarct volume, and the results can be highly consistently translated to humans.

[0341] (3) The middle cerebral artery, a large vessel of the anterior circulation, is a common site of ischemic stroke in humans. The present invention adopts a rhesus monkey 90-minute MCAO reperfusion model, and the occlusion site is selected as the M1 segment. The model animal presents an edema / infarction volume of about 40% of the half brain at the peak of edema, and there is an obvious midline shift. The use of this severe brain edema / infarction model can be transformed for more clinical patients to evaluate the regulatory effect of neuropathy. At the same time, an MRA scan is performed 3-4 hours after reperfusion, which can accurately show whether the blood vessels are reperfused and whether collateral circulation is formed. At the same time, the DWI image shows middle cerebral artery ischemic infarction lesions and quantitative edema volume similar to those of clinical patients. This is used as the model success standard to reduce the model variability.

[0342] (4) In this study, the Non-Human Primate Stroke Scale (NHPSS) was used to quantify the neurological deficits in rhesus monkeys. NHPSS is a scoring combination similar to the National Institutes of Health Stroke Scale (NIHSS).

[0343] The embodiment of the present invention also uses the 180-minute reperfusion model of the middle cerebral artery occlusion (MCAO) of rhesus monkeys to evaluate the neuropathy regulating effect of the composition. The 180-minute MCAO reperfusion model can be used as a cognitive dysfunction model to evaluate the neuroprotective effect of the composition and prevent and improve cognitive function. In addition to having the advantages of the 90-minute MCAO reperfusion model as described above, the 180-minute MCAO reperfusion model can form a larger range of edema / infarction at the baseline (i.e., before group administration) than the 90-minute MCAO reperfusion model. The model animal presents an edema / infarction volume of about 70% of the hemisphere at the peak of edema, which can lead to a long-term significant decline in cognitive function and can be used to evaluate the efficacy of neuroprotective agents in preventing and improving cognitive function. In the embodiment of the 180-minute MCAO reperfusion model of the present invention as described below, the spatial delayed response experiment (Spatial Delayed Response testing) is also used to evaluate the cognitive function of monkeys, including learning, memory ability, visual space ability and understanding judgment ability.

[0344] The rhesus monkey panretinal ischemia / reperfusion injury model provides important insights into the mechanisms and treatment strategies of human I / R injury, especially in terms of neurodegenerative damage to the retinal neurovascular unit. The embodiments of the present invention use the rhesus monkey panretinal ischemia / reperfusion injury model. Currently, the commonly used model is the acute intraocular pressure (IOP) reperfusion model in mice and rats, while the advantages of the primate model are: (1) the existence of the rhesus monkey macula, which is an important site of damage; (2) retinal vascular pattern: the retinal vascular morphology of rhesus monkeys is the same as that of humans; (3) rhesus monkey eyes are larger, and the anterior chamber and vitreous volume are closer to human eyes, which provides more opportunities for applying new intervention methods to humans (Pasquale, LR et al. The Prevalence of Primary Open-Angle Glaucoma-Like Features in a Rhesus Macaque Colony from Southern China. Invest. Ophthalmol. Vis. Sci. 61, 1436–1436 (2020); Morgan, J E Optic nerve head structure in glaucoma: Astrocytes as mediators of axonal damage. Eye 14, 437–444 (2000); Tang, Y. et al. Therapeutic Targeting of Retinal Immune Microenvironment With CSF-1Receptor AntibodyPromotes Visual Function Recovery After Ischemic OpticNeuropathy. Front. Immunol. 11, 585918 (2020).).

[0345] Therefore, the present invention uses a rhesus monkey panretinal ischemia / reperfusion injury model to study the apoptosis of retinal neurons and the trajectory of retinal degeneration after I / R injury, and quantitatively and dynamically evaluates the effects on retinal structure on the 7th day (D7), the 21st day (D21), the 35th day (D35) and the 63rd day (D63) after retinal I / R injury in rhesus monkeys by fundus photography (FP) and optical coherence tomography (OCT), including changes in retinal thickness and optic nerve fiber layer (RNFL), and dynamically evaluates optic nerve conduction function and overall retinal function by flash visual evoked potential (FVEP) and full-field electroretinogram (FERG), and simultaneously evaluates whether the composition can inhibit RGC cell apoptosis, retinal gliosis and inflammation to prevent retinal function loss after I / R.

[0346] The embodiments of the present invention also use a spontaneous diabetic DPN rhesus monkey model to evaluate the effectiveness and safety tolerability of the composition on peripheral neuropathy. The advantages of this primate model are that rhesus monkeys are extremely similar to humans in physiology, biochemistry and systems biology, and it has been found that the disease characteristics of spontaneous DPN rhesus monkeys are very similar to those of clinical human DPN patients. In 1989, Cornblath et al. (Cornblath DR, Hillman MA, Stiffler JS, et al. Peripheral neuropathy in diabetic monkeys [J]. Diabetes, 1989, 38 (11): 1365-1370.; Cornblath DR, Dellon AL, Mackinnon SE. Spontaneous diabetes mellitus in a rhesus monkey: Neurophysiological studies [J]. Muscle & Nerve, 1989, 12 (3): 233-235.) reported a spontaneous diabetic DPN rhesus monkey model, which showed a significant decrease in motor and sensory nerve conduction velocity compared with non-diabetic patients, and the pathogenesis and clinical features were similar to those of human patients. In addition, the DPN animal models commonly used in current research are mostly rodents, such as ischemia-reperfusion, nerve resection, simple STZ (streptozotocin) induction, and transgenic techniques to establish a variety of rat and mouse models. However, due to the complex pathogenesis of DPN and the many links involved, the damage to nerve function in these animal models is difficult to accurately reflect the course of DPN in patients. Therefore, the spontaneous diabetic DPN rhesus monkey model can provide an important research tool for DPN research and new drug development, and provide more accurate conclusions than other animal models.

[0347] It will be appreciated by those skilled in the art that the various aspects of the invention described herein can be combined in various ways that are apparent to those skilled in the art without departing from the subject matter and ideas of the present application. Such combinations are also included within the scope of the present application. For example, the dosage range of certain components of the present invention includes any combination of any lower limit and any upper limit mentioned in the specification, and also includes any range formed by combining the specific content of the component in each specific embodiment as an upper limit or a lower limit; all of these ranges are covered within the scope of the present invention. In addition, each feature of the present invention listed in the specification can be combined with any other feature of the invention, and such combinations are also within the scope of the disclosure of the present invention. The specific embodiments of the present application are described in detail below in conjunction with the accompanying drawings. Example

[0348] 1. Example 1

[0349] The inventors used the 90-minute middle cerebral artery ischemia-reperfusion model of rhesus monkeys to evaluate the effects of doxazosin (DOX), pramipexole (PMP) and metoprolol (MTP) combined with central nervous system diseases, especially the prevention, treatment and / or improvement of ischemic stroke, including the effects on reducing edema / infarction volume and improving neurological deficits after stroke. Evaluation indicators include:

[0350] (1) 3.0T GE MRI was used to dynamically observe the edema / infarction trajectory 3-4 hours, 72-75 hours, 7 days, 14 days and 28 days after reperfusion; MRI imaging sequences included T1WI, T2 FLAIR, DWI, MRA and CTA. Among them, T1WI can display the anatomical structure of the brain, distinguish gray matter and white matter areas, diagnose hemorrhage and ischemia, etc. T2FLAIR and DWI can quantify the volume of edema / necrosis, and T2 FLAIR can indicate an increase in water content in the tissue, which is used to show lesions such as inflammation, ischemia and edema.

[0351] (2) MRA and CTA scans performed 3-4 hours after stroke can accurately show whether the blood vessels are reperfused and whether collateral circulation is formed. At the same time, DWI images show middle cerebral artery ischemic infarction lesions and quantitative edema volume similar to those of clinical patients. This is used as the success standard of the model to reduce the variability of the model. The design of administering the combination 5 hours after stroke is more clinically transferable than preventive administration.

[0352] 1.1 Experimental Materials

[0353] Test sample 1:

[0354] Name or abbreviation (English name): Doxazosin mesylate (DOX)

[0355] Purity: 99.6% HPLC

[0356] Manufacturer: Zhejiang Xinsaike Pharmaceutical Co., Ltd.

[0357] Test sample 2:

[0358] Name or abbreviation (English name): Pramipexole 2HCL Monohydrate (PMP)

[0359] Purity: 99.55% HPLC

[0360] Production unit: Shanghai Ziqi Biotechnology Co., Ltd.

[0361] Test sample 3

[0362] Name or abbreviation (English name): Metoprolol tartratesalt (MTP)

[0363] Purity: 100% HPLC

[0364] Manufacturer: Sun Pharmaceutical Industries Ltd.

[0365] 1.2 Specific experimental plan

[0366] 1.2.1 Experimental animals

[0367] Twelve healthy rhesus monkeys, Macaca mulatta (Rhesus Macaque), 5-7 years old, normal grade, weight: 6-9 kg, provided by the Prime Primate Research Center were selected.

[0368] The breeding environment level during the experimental period: ordinary level. Temperature: 18-26℃. Relative humidity: 40% to 70%. Ventilation: No less than 8 air changes per hour, using 100% fresh air (no air circulation). Lighting time: automatic lighting, alternating light and dark every 12 hours. Animal cages: 850*900*2365mm double-layer stainless steel cages. Stocking density: 1 / cage. All operations have been reviewed and approved by the IACUC Committee of the Primate Research Center of Prime.

[0369] 1.2.2 Establishment of 90-minute reperfusion model of middle cerebral artery occlusion (MCAO) in rhesus monkeys

[0370] The MCAO model of rhesus monkeys was established by craniotomy. Figure 1 and Figure 2 ). The monkeys were fasted but not watered for at least 8 hours before surgery. After anesthesia with propofol 10 mg / kg + 2% isoflurane, the head was shaved and the skin of the surgical area was disinfected. The animals were placed supine on a thermostatic operating table. The skin was incised from the right top of the head to the ear to expose the skull. A hole was made in the skull with an electric drill, and a 4×5 cm bone flap was removed with bone forceps to expose the dura mater. After cutting the dura mater, the middle cerebral artery was located along the lateral cerebral groove under a surgical microscope, and the M1 distal end and M2 branches of the middle cerebral artery were determined. The middle cerebral artery was clamped with an aneurysm clip for 90 minutes, and then the aneurysm clip was released to restore blood supply for reperfusion. The dura mater, muscles, and skin were sutured and disinfected.

[0371] 3h-4h after MCAO surgery (reperfusion) in rhesus monkeys, brain magnetic resonance angiography (MRA) and CT angiography (CTA) were performed. The angiography images were used to determine whether the middle cerebral artery of the rhesus monkeys was reperfused. Figure 2As shown in A and C, both middle cerebral arteries are clearly visible, indicating successful reperfusion (see Figure 3 ).like Figure 2 As shown in B and D, the middle cerebral artery on the operated side is not visible compared with the contralateral side, indicating that reperfusion has not occurred. At the same time, DWI images are shown 3-4 hours after reperfusion. Figure 3 C shows abnormal high signals in the right frontal lobe, parietal lobe and temporal lobe, which is similar to the lesions of middle cerebral artery ischemia in clinical patients. The 12 animals selected in this example were all rhesus monkeys with MCAO occlusion for 90 minutes and reperfusion.

[0372] 1.2.3 Grouping and Dosing

[0373] Twelve rhesus monkeys with MCAO model formed by 90 minutes of occlusion and reperfusion were divided into groups based on DWI quantitative edema volume 3-4 hours after reperfusion. DWI is the only imaging method that can reflect the diffusion characteristics of water molecules. Its high signal reflects the infarct area with limited diffusion movement of water molecules. Changes can occur 2 hours after ischemia, and early brain edema that cannot be found in T2 images can be found. Therefore, DWI quantitative edema / infarction area volume is used as the basis for grouping. The 12 MCAO rhesus monkeys were divided into a model control group of 6 and a DOX+PMP+MTP group of 6. The average volume of DWI quantitative edema / infarction area in the two groups was 8.98±1.14cm, respectively. 3 and 8.17±0.85cm 3 There was no statistical difference between the two groups. Figure 3 .

[0374] Animals in the drug administration group were orally administered approximately 5 hours after stroke (i.e., the time of aneurysm clipping was the time of stroke onset), 2-4 times a day at intervals, for 28 consecutive days. The following is the dosage and method of each administration:

[0375] Model control group : Give empty capsules.

[0376] DOX+PMP+MTP treatment group :DOX: 0.266mg / kg to 0.532mg / kg; PMP: 0.002 to 0.004mg / kg; MTP: 1.667mg / kg

[0377] 1.2.4 Test process

[0378] Magnetic resonance imaging was performed at 3-4h, 72-75h, 7 days, 14 days, and 28 days after MCAO reperfusion, and brain edema / infarction volume was measured and neurobehavioral score (NHPSS) was performed. Figure 4 .

[0379] 1.3 Observation indicators and monitoring methods

[0380] 1.3.1 Main efficacy indicators

[0381] 1.3.1.1 3.0T GE 750w MRI to scan brain tissue and quantify brain edema / infarction volume

[0382] After rhesus monkeys were anesthetized with 5-10 mg / kg propofol and 2% isoflurane, head scans were performed on a 3.0T GE 750w magnetic resonance imaging instrument using a customized head coil for non-human primates. The scanning sequences included: T1WI, T2FLAIR, DWI, and 3D-TOF. The scanning range covered the entire brain tissue, including the cerebral cortex, cerebellum, and temporal lobe. The scanning parameters of T2FLAIR are: TE 120ms, TR 8000ms, matrix 288×288, slice thickness 1.5mm, FOV 14cm, resolution 273μm×273μm×1500μm; the scanning parameters of DWI are: TE 85ms, TR 7000ms, b=1000s, matrix 140×140, slice thickness 2.0mm, FOV 14cm, resolution 1094μm×1094μm×2000μm. These two sequences are used to measure the infarct volume.

[0383] GE MRI's Advantage Workstation 4.7 workstation software was used for image processing. In each scan layer image, the boundary of the high signal area of ​​the brain tissue on the surgical side was manually outlined as the region of interest (ROI). The software automatically calculated the area of ​​the ROI, which was the area of ​​cerebral infarction at that layer. The method for calculating the volume of cerebral infarction was as follows: infarction volume V = t × (A1 + A2 + A3·····An). t is the scanning layer thickness, and A is the area of ​​cerebral infarction at each layer.

[0384] 1.3.1.2 NHPSS neurobehavioral score

[0385] The Non-Human Primate Stroke Scale (NHPSS) was used to quantitatively evaluate the neurological deficits of the animals, with 41 points indicating severe bilateral neurological damage and 0 points indicating normal. The testing time points were: 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 7 days, 14 days, and 28 days after MCAO.

[0386] The single-blind method was used to evaluate the neurobehavioral characteristics of monkeys after cerebral ischemia surgery, that is, the experimental designer marked the animals by group, and the experimenter who scored the neurobehavioral characteristics did not know the grouping of the animals. After the scoring, the scorer submitted the scoring results to the designer, who unblinded and obtained the scores of each animal in each experimental group.

[0387] The National Institute of Health Stroke Scale (NIHSS) is recommended for assessing the severity of cerebral infarction. The NIHSS baseline assessment can reflect the severity of stroke at the onset and can also regularly assess the effect of treatment. The NIHSS ranges from 0 to 42. The higher the score, the more severe the stroke.

[0388] The Non-Human Primate Stroke Scale (NHPSS) is a scoring combination similar to the National Institutes of Health Stroke Scale (NIHSS). Existing literature shows that the scoring indicators of NHPSS and NIHSS are basically consistent (Brott TG, Adams HP Jr, Olinger CP, et al. Measurements of acute cerebral infarction: clinical examination scale. Stroke 1989; 20: 864–70.; ASE K. Clinical interpretation and use of stroke scales. [J]. The Lancet Neurology, 2006, 5 (7): 603-612.).

[0389] NHPSS is used to dynamically evaluate the improvement of neurological deficits at 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 7 days, 14 days and 28 days after stroke. The NHPSS score is composed of a comprehensive score of consciousness, defensive reaction, grasping reflex, limb movement, gait, rotational movement, bradykinesia, balance, reaction to the outside world and facial muscle strength. The total score is 41 points, with 0 points for normal behavior and 41 points for severe bilateral nerve damage. The specific scoring items of NHPSS are shown in Table 1 (Roitberg B, Khan N, Tuccar E, et al. Chronic ischemic stroke model in cynomolgus monkeys: Behavioral, neuroimaging and anatomical study [J]. Neurological Research, 2003, 25 (1): 68-78.).

[0390] Table 1 Neurobehavioral score table (NHPSS) of cerebral ischemia monkeys

[0391]

[0392]

[0393] The following are the scoring indicators of the National Institutes of Health Stroke Scale (NIHSS).

[0394] 1. Level of consciousness (LOC)

[0395] 1a: LOC-reaction

[0396] score Test Results 0 Awake and responsive 1 Sleepy, can be awakened by slight stimulation, can answer questions and follow instructions 2 Lethargy or unresponsiveness: Nonstereotypic responses require repeated, intense, or painful stimulation 3 Coma: only reflex activity or spontaneous response or complete lack of response, limp, and no reflexes

[0397] 1b: LOC-Ask a Question

[0398]

[0399] 1c: LOC-Instructions

[0400] score Test Results 0 Both are correct 1 A correct 2 Both are incorrect

[0401] 2. Gaze

[0402]

[0403]

[0404] 3. Field of view

[0405] score Test Results 0 No visual field loss 1 Clearly asymmetric blindness (including quadrantanopia) or partial hemianopia or impending death 2 Complete hemianopia 3 Bilateral hemianopia (including cortical blindness) or total blindness of any cause

[0406] 4. Facial paralysis

[0407] score Test Results 0 normal 1 Mild (flattening of the nasolabial folds when smiling, asymmetry) 2 Partial (complete or almost complete paralysis of the lower face) 3 Complete (paralysis on one or both sides of the face, lack of movement in the upper and lower face)

[0408] 5. Upper limb exercise

[0409]

[0410] 6. Lower limb exercise

[0411]

[0412]

[0413] 7. Limb ataxia

[0414] score Test Results 0 No ataxia: movements are smooth and accurate 1 Ataxia in one limb: stiff or inaccurate movements 2 Ataxia in 2 or more limbs: stiff or inaccurate movements on one side UN Amputation or joint fusion (recorded)

[0415] 8. Feeling

[0416]

[0417] 9. Language

[0418]

[0419] 10. Dysarthria

[0420]

[0421]

[0422] 11. Ignore

[0423]

[0424] 1.3.1.3 Quantification of GFAP expression in the peri-infarct area by immunohistochemistry

[0425] 30 days after stroke in the 90-minute middle cerebral artery ischemia-reperfusion model of rhesus monkeys, one monkey in the placebo group and one monkey in the DOX+PMP+MTP group were euthanized under pentobarbital anesthesia. After dissection, the whole brain tissue was removed and fixed in 4% paraformaldehyde for more than 72 hours. Brain tissue was obtained from the infarcted area of ​​the cerebral hemisphere on the surgical side, and brain tissue was obtained from the corresponding area of ​​the cerebral hemisphere on the non-surgical side. Fourteen coronal brain sections (4 mm thick) were prepared using MSM-2500 brain matrix, and the peri-infarct areas in the temporal lobe and precentral gyrus were dehydrated, wax-impregnated, embedded, and sectioned (3 slices, 5 μm / slice) in sequence for GFAP immunohistochemical staining. After routine dewaxing to water, the paraffin sections were subjected to EDTA hot water bath antigen retrieval for 20 minutes and cooled to room temperature; after PBS washing, 3% H2O2 was incubated at room temperature for 20 minutes; after PBS washing, normal goat serum was added dropwise and blocked at 37°C for 20 minutes. Then, the primary antibody (GFAP, 1:250) was added and incubated at 4°C overnight. After washing with PBS, the reaction enhancement solution was added and incubated at 37°C for 20 min; after washing with PBS, the secondary antibody was added and incubated at 37°C for 20 min; after washing with PBS, the DAB colorimetric agent was added and the color was developed at room temperature, controlled under the microscope, and the color was terminated with distilled water. Hematoxylin counterstaining was performed for 2 min, alcohol dehydration was performed, and xylene was transparent; neutral gum sealing was performed. A negative control was set for each experiment, and the primary antibody was replaced with PBS solution. The other steps were exactly the same. Three immunohistochemical slides were taken under an optical microscope for GFAP protein quantification. Two peri-infarction areas were selected for each slice, and images were collected under a 200x field of view. Image Pro plus 6.0 morphological analysis software was used to quantify the GFAP protein expression in six peri-infarction areas.

[0426] 1.3.2 Nursing of rhesus monkeys with stroke

[0427] MCAO rhesus monkeys were immediately transferred to the neuro-ICU ward for care after surgery, and heart rate, blood oxygen saturation, blood pressure, electrocardiogram, urine volume and body temperature were monitored regularly. Biochemical and hematological examinations were performed 3h after MCAO (before MRI examination) and 16h, 27h, 48h, 72h, 96h, 7d (7 days), 14d (14 days), and 28d (28 days) after surgery. No drugs to reduce intracranial pressure or cerebral edema were used in the experiment.

[0428] None of the 12 MCAO rhesus monkeys selected for this experiment died during the 28-day trial period, and all 12 animals survived.

[0429] 1.3.3 Data processing

[0430] Statistical analysis software was used, and quantitative data were expressed as mean ± standard deviation. One-way analysis of variance was used for cerebral infarction volume and neurological deficit symptom scores. P < 0.05 was defined as a significant difference.

[0431] 1.4 Experimental Results and Discussion

[0432] 1.4.1 Effects on brain edema / infarct volume and brain tissue at various time points after stroke

[0433] The changes of brain edema / infarct volume at different time points after stroke in each group of subjects are shown in Figure 5 .

[0434] In the model control group (N = 6), the average DWI volume of brain edema / infarction on the surgical side (right side) 3-4 hours after reperfusion was 8.98 ± 1.14 cm 3 The average brain edema / infarct volume reached its peak within 75 hours after stroke, and was 21.57±4.31cm 3 The edema volume accounted for about 40% of the half brain volume, and there was an obvious midline shift. MRI imaging examination showed lesions in the frontal lobe, parietal lobe, and temporal lobe on the surgical side (right side). The average volume of T2 FLAIR cerebral edema / infarction on the 7th day after stroke (D7) was 20.60±6.45cm 3 At the same time, necrotic liquefaction foci appeared in some brain tissues in the frontal lobe area. From the 14th day (D14) to the 28th day (D28) after stroke, the brain tissue edema / infarction volume gradually decreased to 10.34±3.91cm 3 and 7.93±3.39cm 3 At the same time, the boundaries between the liquefaction necrosis area and the infarction area and the healthy side (left side) brain tissue are clearer.

[0435] DOX+PMP+MTP treatment group (N=6): 6 MCAO rhesus monkeys were orally administered with DOX starting about 5 hours after stroke for 28 consecutive days. The edema / infarction volume of the brain tissue on the surgical side (right side) was significantly reduced at 75h, 7 days, 14 days and 28 days after stroke (p<0.05). The average volume of T2 FLAIR cerebral edema / infarction at the peak of edema at 75h and 7 days (D7) after stroke was 13.86±3.63cm 3 and 11.62±3.63cm 3 The mean volume of T2 FLAIR edema infarction on the 14th day (D14) and the 28th day (D28) after stroke was 5.83±2.24cm 3and 4.34±1.20cm 3 .

[0436] The above data show that the combination containing three drugs can significantly reduce brain edema / infarct volume in the 90-minute MCAO reperfusion rhesus monkey model when administered 5 hours after stroke for 28 consecutive days.

[0437] 1.4.2 Effects on NHPSS neurobehavioral scores at different time points after stroke

[0438] The changes of NHPSS neurobehavioral scores at different time points after stroke in each group of subjects are shown in Figure 6 .

[0439] Model control group (N = 6): NHPSS scores at 24 hours, 72 hours and 7 days (D7) after stroke were 15.4 ± 2.9, 12.0 ± 1.6 and 7.4 ± 2.6, mainly manifested by weak defense reaction, decreased strength of upper and lower limbs on the contralateral side (affected side) compared with the healthy side, weakness of the affected arm and inability to lift over the head, neurological deficits in the affected fingers and inability to grasp food, slow movements, and significantly reduced balance and rotational motor abilities. NHPSS scores gradually decreased on the 14th day (D14) and 28th day (D28) after stroke, which were 3.6 ± 1.8 and 1.8 ± 0.4, respectively. The fine function of the affected side (left) fingers and arm strength of the MCAO rhesus monkeys on the 28th day (D28) were still slightly worse than those on the healthy side (right side).

[0440] DOX+PMP+MTP group (N=6): NHPSS scores at 24 hours, 72 hours and 7 days (D7) after stroke were 10.3±3.1, 5.3±2.4 and 2.7±1.9, which were significantly lower than those in the model control group at the same time points (p<0.05 or p<0.01). NHPSS scores at 14 days (D14) and 28 days (D28) after stroke were 1.2±0.4 and 0.7±0.5, respectively.

[0441] As can be seen from the above, the DOX+PMP+MTP treatment group, which was administered 5 hours after stroke, significantly reduced the neurobehavioral scores of MCAO rhesus monkeys and significantly improved the neurological deficits of stroke animals. The recovery of finger function and limb strength of the affected limb was significantly faster than that of the model control group, and the imaging results were highly correlated with the NHPSS scores.

[0442] 1.4.3 Effect on GFAP protein expression

[0443] Results Figure 7In the placebo group, the expression of GFAP protein in the brain tissue around the infarct area increased significantly 30 days after stroke, which was 3-4 times higher than that in the corresponding area of ​​the non-operated cerebral hemisphere (p<0.01). The expression of GFAP protein was 9.79±1.93. At the same time, the morphology of reactive astrocytes was changed, with hypertrophy, swelling and increased protrusions of the cell body.

[0444] The GFAP expression level in the brain tissue around the infarction area in the DOX+PMP+MTP group was significantly lower than that in the placebo group 30 days after stroke (p<0.01), and the GFAP expression level was 4.21±0.75. Treatment with the DOX+PMP+MTP combination for 30 days significantly inhibited the increase in GFAP protein expression in the brain tissue around the infarction area of ​​rhesus monkeys with stroke, and suppressed neuroinflammation.

[0445] 1.5 Safety and tolerance

[0446] The safe dosage range and toxic and side effects of DOX, PMP, and MTP are known. The commonly used tablet dose of DOX is 4-8 mg / time, twice a day. The recommended individual dose of PMP is 0.375 mg to 4.5 mg per day. When used to treat idiopathic Parkinson's disease, the starting dose is 0.375 mg per day, and then the dose is increased every 5-7 days, with a maximum dose of 1.5 mg. The incidence of drowsiness increases when the daily dose is higher than 1.5 mg. The recommended dose of MTP is 100-200 mg / day, which can reach 300 mg / day or 400 mg / day if necessary. The known adverse reaction is hypotension.

[0447] The human dosage calculated based on the dosage of rhesus monkeys in the experiment was lower than the maximum value of the above-mentioned recommended dosage, so theoretically the three-drug combination of DOX+PMP+MTP of the present invention has very good safety.

[0448] During the above experiment, magnetic resonance imaging of all 6 animals in the DOX+PMP+MTP group showed no adverse events related to medication, such as intracranial hemorrhage, during the entire medication period, which further confirmed that the DOX+PMP+MTP triple drug combination of the present invention has good safety.

[0449] 2. Example 2

[0450] The inventors used a retinal ischemia-reperfusion (I / R) rhesus monkey model to evaluate the effects of a pharmaceutical combination of doxazosin + pramipexole + metoprolol, a combination of doxazosin + metoprolol + bromocriptine, a combination of doxazosin + metoprolol, doxazosin alone, and pramipexole alone on the prevention, treatment and / or improvement of optic neuropathy and retinopathy, particularly ischemic optic neuropathy.

[0451] Main efficacy indicators: 1) Fundus photography (FP) and optical coherence tomography (OCT) were used to quantitatively and dynamically evaluate the effects of retinal structure on rhesus monkeys at D7, D21, D35 and D63 after retinal I / R injury, including changes in retinal thickness and nerve fiber layer (RNFL) thickness; 2) Flash visual evoked potential (FVEP) and full-field electroretinogram (FERG) were used to dynamically evaluate the optic nerve conduction function and overall retinal function; 3) Immunohistochemistry was used to quantify the number of retinal ganglion cells (RGC) and the expression of glial fibrillary acidic protein (GFAP) at D63 after retinal I / R, and real-time fluorescence quantitative PCR was used to detect the expression of inflammatory factors in ischemic retinopathy.

[0452] 2.1 Experimental Materials

[0453] Test sample 1:

[0454] Name or abbreviation (English name): Doxazosin mesylate (DOX)

[0455] Purity: 99.6% HPLC

[0456] Manufacturer: Zhejiang Xinsaike Pharmaceutical Co., Ltd.

[0457] Test sample 2:

[0458] Name or abbreviation (English name): Pramipexole 2HCL Monohydrate (PMP)

[0459] Purity: 99.55% HPLC

[0460] Production unit: Shanghai Ziqi Biotechnology Co., Ltd.

[0461] Test sample 3

[0462] Name or abbreviation (English name): Metoprolol tartratesalt (MTP)

[0463] Purity: 100% HPLC

[0464] Manufacturer: Sun Pharmaceutical Industries Ltd.

[0465] Test sample 4

[0466] Name or abbreviation (English name): Bromocriptine Mesilate (BRM)

[0467] Specification: 2.5mg / tablet

[0468] Manufacturer: Hungary Gedeon Pharmaceuticals. The starting clinical dose of bromocriptine mesylate is 1.25 mg, 2-3 times a day. The conventional dose of bromocriptine mesylate tablets is 4-16 tablets a day (10-40 mg of bromocriptine mesylate).

[0469] 2.2 Specific experimental plan

[0470] 2.2.1 Experimental animals

[0471] Twenty-three healthy rhesus monkeys Macaca mulatta (Rhesus Macaque) aged 4-6 years old, of ordinary grade, were selected and provided by the Prime Primate Research Center. The breeding environment level during the experimental period: ordinary grade. Temperature: 18-26°C. Relative humidity: 40% to 70%. Ventilation: The number of air changes per hour is not less than 8 times, using 100% fresh air (no air circulation). Lighting time: automatic lighting, alternating light and dark every 12 hours. Animal cages: 850*900*2365mm double-layer stainless steel cages. Stocking density: 1 / cage. All procedures involving animals are in accordance with the ARVO statement on the use of animals in ophthalmic and visual research, and all operations have been reviewed and approved by the IACUC Committee of the Prime Primate Research Center.

[0472] 2.2.2 Retinal ischemia / reperfusion injury (I / R) model in rhesus monkeys

[0473] Acute global retinal ischemia / reperfusion injury (I / R) in rhesus monkeys Figure 8 . The right eye was anesthetized with 8 mg / kg of compound ketamine, and retinal ischemia was induced unilaterally, while the contralateral eye served as a non-ischemic control. After one drop of compound tropicamide eye drops (Santen Pharmaceutical) was dripped into the right eye, the pupil was dilated in a dark room. When the pupil of the right eye was larger than 6 mm, the anterior chamber was injected with water and pressurized to create a model. 0.9% sodium chloride injection (Sichuan Kelun Pharmaceutical Co., Ltd.) was injected into a 250 ml infusion bag (Sichuan Kangning Medical Equipment Co., Ltd.), and a 30G needle (BD BD) Connect the end of the infusion tube, insert a needle into the right anterior chamber of the eye and inject 0.9% sterile saline. As the saline bag rises to 1.9 meters, the intraocular pressure (IOP) of the rhesus monkeys rises acutely to 140 mmHg, and the duration of ischemia is 90 minutes. Observation of corneal edema is considered a sign of successful induction of retinal ischemia. After the ischemia is completed, slowly lower the saline reservoir, remove the needle from the eye, and restore IOP to form retinal reperfusion.

[0474] 2.2.3 Grouping and Dosing

[0475] 23 acute retinal I / R rhesus monkeys were divided into 6 groups, namely I / R control group (n=6), I / R+DOX+MTP+PMP group (n=6), I / R+DOX+MTP group (n=3), I / R+DOX+MTP+BRM group (n=3), I / R+DOX group (n=3) and I / R+PMP group (n=2). Animals in each administration group were orally administered 4-5 hours after retinal I / R injury (pulling the needle out of the eye was the starting point of I / R injury) for 63 consecutive days. At the end of the experiment, some animals were euthanized to complete pathology.

[0476] The following are the doses and frequency of each administration:

[0477] I / R control group: given fruits.

[0478] I / R+DOX+PMP+MTP group: DOX: 0.266 to 0.532 mg / kg; PMP: 0.002 to 0.004 mg / kg; MTP: 1.667 mg / kg; administration 2-3 times a day.

[0479] I / R+DOX+MTP group: DOX: 0.266 to 0.532 mg / kg; MTP: 1.667 to 3.33 mg / kg; administration 2-3 times a day.

[0480] I / R+DOX group: 0.266 to 0.532 mg / kg, 2-3 times a day.

[0481] I / R+PMP group: 0.002 to 0.004 mg / kg, 2-3 times a day.

[0482] I / R+DOX+MTP+BRM group: Ratio: DOX: 0.266 to 0.532 mg / kg; MTP: 1.667 mg / kg; BRM: 0.03 mg / kg, 2-3 times a day.

[0483] Table 2 Test drugs and their dosages

[0484]

[0485] Note: The monkey dose and human dose can be converted using the body surface area method or the in vivo drug concentration exposure method.

[0486] 2.3 Observation indicators and monitoring methods

[0487] 2.3.1FP / OCT examination of retinal thickness / optic nerve fiber layer thickness

[0488] Both eyes were scanned with a Kowa VX-20 fundus camera and Heidelberg SPECTRALIS plus OCT before modeling and on the 7th day (D7), 21st day (D21), 35th day (D35) and 63rd day (D63) after retinal I / R injury. The right eye was the I / R-injured eye, and the left eye was the non-ischemic control eye.

[0489] Methods: Rhesus monkeys were anesthetized by intramuscular injection of 8 mg / kg of compound ketamine. 1-2 drops of compound tropicamide eye drops were added to each eye to dilate the pupil. After dilation, if the pupil is larger than 6 mm under light, the fundus can be examined. The right and left eyes were opened in turn with an eyelid speculum, and the fundus was photographed using a fundus camera (Kowa VX-20, Japan) based on the optic disc. After that, an OCT examination was performed. The thickness of the EDTRS area of ​​the retinal 9-square map and the thickness of the optic nerve fiber layer (RNFL) in each quadrant were measured using OCT. The analysis method is shown in Fig.10 The mean RNFL thickness was calculated by averaging the thickness values ​​measured over 360 degrees. The thickness of each region was calculated by averaging the temporal (T; 315 degrees to 45 degrees), superior temporal (TS; 45 degrees to 90 degrees), inferior temporal (TI; 90 degrees to 135 degrees), nasal (N; 135 degrees to 225 degrees), superior nasal (NS; 225 degrees to 270 degrees), and inferior nasal (NI; 270 degrees to 315 degrees) measurements according to the instrument vendor's instructions.

[0490] 2.3.2 FERG and FVEP dynamic assessment of optic nerve conduction function and overall retinal function

[0491] Acute retinal ischemia can cause damage to the neural retina, including photoreceptors, whose function can be evaluated by FERG and FVEP. Flash visual evoked potential (FVEP) and full-field electroretinogram (FERG) were performed dynamically using an ocular electrophysiology instrument (Roland Consult Stasche&Finger GmbH, Ganzfeld Q450 SC) on the 7th day (D7), 21st day (D21), and 63rd day (D63) after I / R injury. All test parameters were set according to the latest standards of the International Society of Electrophysiology (ISCEV).

[0492] Methods: The anesthesia method was the same as that of FP / OCT examination. The electrode sites were disinfected with 75% alcohol. The eyelids were opened with an eyelid opener. The recording electrode was placed 2-3 cm from the occipital protuberance, the reference electrode was placed in the middle of the forehead, and the ground electrode was placed at the base of the tail. The non-test eye was covered, and FVEP was recorded in the order of the right eye first and the left eye. After completing the recording of FVEP, the pupil was fully dilated with compound tropicamide eye drops. The ground electrode was connected to the middle of the forehead, and the reference electrode was placed at the outer canthus of both eyes. After using Benoxite eye surface anesthesia, two drops of sodium hydroxymethylcellulose eye drops were dripped on the corneal contact electrode, which was placed in both eyes and ensured to be in full contact with the cornea. The light-adapted 3.0 ERG and light-adapted 30HZ oscillation potential were recorded in sequence. The recording electrode for detecting ERG was a ring corneal electrode, and the reference electrode and ground electrode were needle electrodes (rhesus monkey). The VEP electrode was a needle electrode, and the self-impedance of each electrode was less than 5KΩ.

[0493] 2.3.3 Immunohistochemistry and retinal ganglion cell counting

[0494] On the 63rd day (D63) after retinal I / R injury, 16 rhesus monkeys were euthanized and divided into I / R control group (n=3, 6 eyes), I / R+DOX+MTP+PMP group (n=3, 6 eyes), I / R+DOX+MTP group (n=3, 6 eyes), I / R+DOX+MTP+BRM group (n=3, 6 eyes), I / R+DOX group (n=3, 6 eyes) and I / R+PMP group (n=1, 2 eyes).

[0495] Methods: The eyeball was placed in FAA fixative and removed after 72 hours. Fig.10 The retina was cut within the range shown (red box), and a rectangular (approximately: 0.8cm×1.6cm) eyeball wall tissue containing the optic disc and macular area was obtained for immunohistochemical staining. The sampled retina was embedded and sliced, and continuous slices were made at the fovea of ​​the macula. The fovea was approximately 0.5mm=500μm, and approximately 100 slices (5μm / slice) could be made. The slices were divided into 10 rounds (10 slices / round), and one continuous slice was taken every 50μm (i.e., each round) for Brn-3a staining and GFAP protein staining. 8-10 immunohistochemical slides were selected for ganglion cell counting and GFAP protein quantification. The number of temporal and nasal ganglion cells within 1.5mm from the fovea was counted for each slide. The operation process is shown in the figure. Fig. 9 The number of RGCs in each image was calculated by subtracting the number of RGCs in the retina of the ischemic eye (OD) from the number of RGCs in the retina of the contralateral non-ischemic eye (OS) of the same rhesus monkey and dividing it by the number of RGCs in the contralateral eye (OS), which was the percentage of RGC loss. All quantitative procedures were performed by two researchers in a double-blind manner.

[0496] 2.3.4 Real-time fluorescence quantitative PCR detection of inflammatory cytokine expression in ischemic retinopathy

[0497] Methods: RNA was extracted from retinal tissue using a total RNA extraction kit (Foregene) and its absorbance was measured. Reverse transcription was performed using 2ug of total RNA as a template. The primers in Table 3 and EvaGreen Express 2×qPCR were used.

[0498] MasterMix-No Dye fluorescent dye (abm) was used for relative quantitative PCR detection, and β-Actin was used as an internal reference. Reaction conditions: 95℃ pre-denaturation for 10 min; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles; melting curve (60℃ to 95℃, 0.3℃ every 15 s). Primer design was performed using Primer Premier 5.0 software, and the primers were synthesized by Shanghai Shenggong Biotechnology Technology Service Co., Ltd.

[0499] Table 3 Primer sequences

[0500]

[0501] 2.3.5 Statistical analysis

[0502] All results are expressed as mean ± SEM. Statistical analysis was performed by two-tailed T test using Graphpad Prism (version 8.0). p < 0.05 was considered statistically significant.

[0503] 2.4 Experimental Results and Discussion

[0504] 2.4.1 Effects on retinal ganglion cells

[0505] Histopathological analysis of Brn3a immunohistochemistry was performed on day 63 (D63) after retinal I / R injury to evaluate the number of retinal ganglion cells (RGCs). The results of each group are shown in Fig.13 Compared with OS eyes without ischemic injury, the I / R eyes in the I / R control group (N=3) had an average loss of 75.7±7.5% of RGC cells on day 63 (D63). The I / R+DOX+MTP+PMP group (N=3) showed a significant improvement in the survival rate of retinal ganglion cells (RGCs) compared with the I / R control group after 63 days of treatment (p<0.01), with an average loss of 13.4±5.5% of RGC cells.

[0506] The average RGC cell losses in I / R eyes in the I / R+DOX+MTP+BRM group, I / R+DOX+MTP group, I / R+DOX group and I / R+PMP group were 73.3±6.0%, 60.0±14.6%, 63.9±14.1% and 60.0%, respectively, which were not statistically different from the I / R control group.

[0507] 2.4.2 Effect on GFAP protein expression

[0508] Results Fig.14 The retinal GFAP protein expression of non-ischemic OS control eyes (N=7 eyes) was quantified to 2.04±0.13, and GFAP staining was only observed in RGCs and nerve fiber layers; compared with non-ischemic OS control eyes, the GFAP protein expression in the retina of I / R eyes (N=3) in the I / R control group was significantly increased to 10.66±1.41 (p<0.01), and the signal reached the inner plexiform layer. Compared with the I / R eyes in the I / R control group, the I / R+DOX+MTP+PMP group (N=3) significantly inhibited the overexpression of GFAP protein in the I / R eyes after 63 days of treatment (p<0.01), and the GFAP was quantified to 3.29±0.36.

[0509] As can be seen above, the DOX+MTP+PMP group significantly improved the survival rate of ganglion cells (RGC) after retinal I / R injury after 63 days of treatment, and significantly inhibited the increase in GFAP protein expression in retinal tissue induced by I / R and inhibited neuroinflammation. At the same time, the experimental data showed that the DOX+MTP+BRM group, the DOX+MTP group, the DOX group alone, and the PMP group alone had weak protection for retinal ganglion cells.

[0510] 2.4.3 Changes in retinal thickness

[0511] The results of quantitative analysis of the average thickness of the retinal EDTRS area in 9 images after I / R injury using OCT are shown in Table 4. Fig.10 and Fig.11 Compared with the pre-modeling, the average thickness of the retinal EDTRS area in the I / R control group (N=6) was significantly reduced on the 7th day (D7), 21st day (D21), 35th day (D35) and 63rd day (D63) (p<0.05), and the average thickness was reduced by 78±24 μm on the 63rd day (D63), indicating that all layers of the retina were severely degenerated and the structure was severely damaged. Compared with the I / R control group, the I / R+DOX+MTP+PMP group (N=6) significantly prevented the reduction of the retinal EDTRS area thickness in the I / R eyes (p<0.01), and the average thickness of the retinal EDTRS area on the 7th day (D7), 21st day (D21), 35th day (D35) and 63rd day (D63) was reduced by 7±6 μm, 19±13 μm, 21±15 μm and 21±19 μm, respectively.

[0512] In addition, compared with before modeling, the average thickness of the retinal EDTRS area in the I / R+DOX+MTP+BRM group, I / R+DOX+MTP group, I / R+DOX group and I / R+PMP group was significantly reduced at each detection time point, and the degree of reduction at each time point was not statistically different from that of the I / R control group.

[0513] 2.4.4 Changes in nerve fiber layer (RNFL) thickness

[0514] The OCT quantitative results are shown in Tables 5 and Fig.10 .

[0515] Compared with the pre-modeling, the I / R eyes in the I / R control group showed edema in the retinal NFL and RGCs layers on the 7th day (D7), and then the retinal RNFL thickness gradually became thinner on the 21st day (D21), 35th day (D35) and 63rd day (D63). By the 63rd day (D63), the global average thickness of the optic nerve fiber layer (RNFL) decreased by 32±17 microns. Compared with the I / R eyes in the I / R control group, the I / R+DOX+MTP+PMP group significantly prevented the significant decrease of the retinal RNFL on the 63rd day (D63) (p<0.05), and by the 63rd day (D63), it decreased by 5±4 microns on average.

[0516] In addition, compared with the I / R control group, the average retinal RNFLGlobal thickness of the I / R+DOX+MTP+BRM group, I / R+DOX+MTP group, I / R+DOX group and I / R+PMP group was significantly reduced on the 7th day (D7), 21st day (D21), 35th day (D35) and 63rd day (D63), and the degree of reduction at each time point was not statistically different from that of the I / R control group.

[0517] 2.4.5 Full-field electroretinogram (FERG) test results

[0518] I / R induced neuronal damage and visual impairment in different retinal diseases. The a-wave amplitude and peak time of the light-adapted 3.0 ERG response reflected the function of photoreceptors, and the b-wave amplitude and peak time reflected the function of cones. The results are shown in Table 6. Compared with the uninjured eyes OS, the a-wave and b-wave amplitudes of the light-adapted 3.0 ERG response of the I / R control group (N=6) were reduced at D7 and D21 after I / R injury. The retinal function recovered but was still significantly reduced on the 63rd day (D63); no significant changes were observed in the uninjured eyes during the experimental period.

[0519] Compared with the I / R control group, the amplitudes of the a-wave and b-wave of the light-adapted 3.0ERG reaction in the DOX+MTP+PMP group (N=6) were significantly increased on the 7th day (D7), 21st day (D21) and light-adapted 3.0ERG reaction after I / R injury (p<0.05), and the combination significantly inhibited the reduction of retinal function after I / R. Compared with the I / R control group, the amplitudes of the a-wave and b-wave of the light-adapted 3.0ERG reaction in the I / R+DOX+MTP+BRM group (N=3) at each time point after injury were significantly reduced, and no significant difference was observed. The retinal function recovered but still decreased on the 63rd day (D63).

[0520] 2.4.6 Results of photopic visual evoked potential (FVEP) test

[0521] FVEP reflects the conduction function of the entire visual pathway from the ganglion cells of the retina to the occipital visual cortex of the brain. The results are shown in Table 7.

[0522] Compared with the uninjured eye OS, the P2 amplitude of the I / R eyes (N=4) in the I / R control group was reduced on the 21st day (D21) and the 63rd day (D63). Compared with the I / R control group, the P2 amplitude (N2-P2) of the I / R eyes (N=4) in the I / R+DOX+MTP+PMP group on the 21st day (D21) and the 63rd day (D63) after administration was significantly increased (p<0.05), and the combination significantly improved retinal function.

[0523] 2.4.7 Effects on inflammatory factors after retinal I / R injury in rhesus monkeys

[0524] Results Fig.15 Compared with the non-ischemic control eyes (N=6), the results of the I / R eyes (N=3) in the I / R control group on day 63 (D63) showed that retinal I / R caused a significant upregulation of TNF-α, IL-1β and IFN-γ levels (p<0.01 or p<0.05); compared with the I / R eyes in the I / R control group, the I / R eyes in the DOX+MTP+PMP group (N=3) significantly reduced the increased expression levels of TNF-α, IL-1β and IFN-γ caused by retinal I / R (p<0.05), and there was no statistical difference in the expression levels compared with the non-ischemic control eyes.

[0525] The above shows that the DOX+MTP+PMP combination protected the loss of RGC cells and overexpression of GFAP after panretinal I / R injury in rhesus monkeys, significantly reduced the increased expression levels of TNF-α, IL-1β and IFN-γ caused by retinal I / R, maintained retinal thickness and reduced neuronal damage, alleviated the decline in optic nerve function caused by I / R including FERG and FVEP destruction, and preserved the overall function of the retina.

[0526]

[0527]

[0528]

[0529]

[0530] 3. Example 3

[0531] The inventors used a rhesus monkey middle cerebral artery M1 segment occlusion for 180 minutes and reperfusion to cause cognitive dysfunction model to evaluate the effect of combined use of doxazosin (DOX), pramipexole (PMP) and metoprolol (MTP) on the prevention, treatment and / or improvement of neurodegenerative diseases. Rhesus monkey middle cerebral artery M1 segment occlusion for 180 minutes and reperfusion can form a larger range of infarction, and at the peak of edema, the edema / infarction volume accounts for about 70% of the half brain, which can lead to long-term significant cognitive decline, and can be used to evaluate the efficacy of neuroprotective agents in preventing and improving cognitive function. Evaluation indicators include:

[0532] 1) 3.0T GE MRI was used to dynamically observe the edema / infarction trajectory 3-4 hours, 72-75 hours, 7 days, 14 days and 28 days after reperfusion; MRI imaging sequences included T1WI, T2 FLAIR, DWI, MRA, and DSC. Among them, T1WI can display the anatomical structure of the brain, distinguish gray matter and white matter areas, diagnose hemorrhage and ischemia, etc. T2FLAIR and DWI can quantify the volume of edema / necrosis, and T2 FLAIR can indicate increased water content in the tissue, which is used to show inflammation, ischemia, edema and other lesions. DSC can show the blood perfusion of brain tissue after acute ischemic stroke and the sensitive reaction brain ischemic area, and can quantitatively evaluate the volume of the ischemic core area of ​​brain tissue.

[0533] 2) MRA scans were performed 3-4 hours after stroke (ischemia for 180 minutes followed by reperfusion) to accurately show whether the blood vessels were reperfused and whether collateral circulation was formed. DWI showed ischemic infarction lesions of the anterior circulation middle cerebral artery similar to those in clinical patients 3-4 hours after stroke. This was used as the model success criterion to reduce model variability.

[0534] 3) The design of administering the combination within 7 hours of stroke is more clinically translatable than preventive administration.

[0535] 4) NHPSS was used to dynamically evaluate the improvement of neurological deficits at 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 7 days, 14 days, 21 days and 28 days after stroke.

[0536] 5) Perform a spatial delayed response test 3 months after stroke to evaluate the improvement effect on cognitive dysfunction.

[0537] 3.1 Experimental Materials

[0538] The experimental materials are the same as described in Example 1.1.

[0539] 3.2 Specific experimental plan

[0540] 3.2.1 Experimental animals

[0541] Nine healthy rhesus monkeys, Macaca mulatta (Rhesus Macaque), aged 5-7 years, normal grade, weight: 6-9 kg, provided by the Prime Primate Research Center. The breeding environment level during the experimental period: normal grade. Temperature: 18-26℃. Relative humidity: 40% to 70%. Ventilation: No less than 8 air changes per hour, using 100% fresh air (no air circulation). Lighting time: automatic lighting, alternating light and dark every 12 hours. Animal cages: 850*900*2365mm double-layer stainless steel cages. Stocking density: 1 / cage. All operations have been reviewed and approved by the IACUC Committee of the Prime Primate Research Center.

[0542] 3.2.2 Establishment of 180-minute middle cerebral artery occlusion and reperfusion model (MCAO) in rhesus monkeys

[0543] The method for making the MCAO model is the same as described in Example 1.2.2, except that in Example 3, the time for clamping the M1 segment of the middle cerebral artery is 180 minutes of reperfusion.

[0544] 3.2.3 Grouping and Dosing

[0545] Nine rhesus monkeys were selected as model animals with 180-minute MCAO and reperfusion. The edema volume was quantified by DWI 3-4 hours after reperfusion and divided into groups. The nine rhesus monkeys were scanned with 3.0T GEMRI 3h-4h after 180-minute MCAO and reperfusion. The use of brain magnetic resonance angiography (MRA) and CT angiography (CTA) showed that both middle cerebral arteries were clearly visible, which was judged as successful reperfusion. Fig.17 Nine MCAO rhesus monkeys were divided into a model control group (5) and a DOX+PMP+MTP group (4). The average volume of edema / infarction area in the two groups was 14.61±2.63cm 3 and 13.43±3.14cm 3 There was no statistical difference between the two groups.

[0546] Animals in the drug administration group were orally administered approximately 7 hours after stroke (i.e., the time of aneurysm clipping was the time of stroke onset), 2-4 times a day at intervals, for 60 consecutive days. The following is the dosage and method of each administration:

[0547] Model control group (Placebo group) : Give empty capsules.

[0548] DOX+PMP+MTP treatment group :DOX: 0.266mg / kg to 0.532mg / kg; PMP: 0.002 to 0.004mg / kg; MTP: 1.667mg / kg to 3.334mg / kg.

[0549] 3.2.4 Test process

[0550] Magnetic resonance imaging was performed 3-4h, 72-75h, 7 days, 14 days, and 28 days after MCAO-180-minute reperfusion surgery to measure brain edema / infarct volume and perform neurobehavioral score (NHPSS). To investigate long-term neurological deficits, cognitive function was assessed using the spatial delayed response test 3 months after stroke.

[0551] 3.3 Observation indicators and monitoring methods

[0552] 3.3.1 Main efficacy indicators

[0553] 3.3.1.1 Scanning brain tissue and quantification of brain edema / infarct volume

[0554] Same as described in Example 1.3.1.1.

[0555] 3.3.1.2 NHPSS neurobehavioral score

[0556] Same as described in Example 1.3.1.2.

[0557] 3.3.1.3 Quantitative evaluation of brain microcirculatory perfusion and sensitive response to cerebral ischemic areas

[0558] Dynamic susceptibility contrast-enhanced magnetic resonance imaging (MRI-DSC) is an examination method that uses rapid scanning sequence imaging to obtain a series of dynamic images when the contrast agent passes through the examined tissue for the first time after intravenous bolus injection of paramagnetic contrast agent. It is a MRI functional imaging technology that reflects the distribution of tissue microvessels and blood perfusion, and provides important imaging basis for selecting treatment plans and judging the efficacy of patients with ischemic cerebrovascular disease.

[0559] MRI-DSC imaging is post-processed by the workstation to analyze and calculate cerebral hemodynamic parameters, which can produce four main indicators reflecting local tissue blood supply: 1) Regional cerebral blood volume (rCBV) refers to the blood volume existing in a certain amount of brain tissue vascular structure. 2) Regional cerebral blood flow (rCBF) refers to the blood flow that flows through a certain amount of brain tissue vascular structure per unit time. The smaller the value, the lower the brain tissue blood flow. 3) Regional mean transit time (rMTT) refers to the time it takes for contrast agents to pass through capillaries when blood flows through vascular structures (the time it takes for blood to flow from arteries to veins). A large value indicates poor microcirculation. 4) Regional time to peak (rTTP) refers to the time it takes for contrast agents to reach the peak value of ROI in the brain. The larger the value, the later the time it takes for the contrast agent peak to reach the brain tissue. 75 hours after stroke, the volume of the ischemic core area was quantified in the MRI-DSC imaging to evaluate the efficacy of improving ischemia.

[0560] The area with significantly decreased CBV and CBF and significantly prolonged MTT and TTP was selected as the non-perfused area (ischemic core area) (see Fig.18, CBV, CBF, MTT, TTP dark areas). GE MRI Advantage Workstation 4.7 software was used to measure the non-perfusion area of ​​each layer of CBV. The ischemic area of ​​each layer was added and multiplied by the layer thickness to obtain the cerebral ischemic volume. (Wintermark M, Sesay M, Barbier E, et al. Comparative overview of brain perfusion imaging techniques. [J]. Journal of Neuroradiology, 2005, 32(9): 294-314.; Shih LC, Saver JL, Alger JR, et al. Perfusion-Weighted MagneticResonance Imaging Thresholds Identifying Core, Irreversibly Infarcted Tissue[J].Stroke,2003,34(6):1425-1430.;Oppenheim C,Peeters A,Cosnard G,et al.WhichMR-derived perfusion parameters are the best predictors of infarct growth inhyperacute stroke between relative and Comparative study? quantitativemeasurements.[J].Radiology,2002,223(2):361-370.; Jing Yanping, Luo Bin, Gao Zhengrong, et al. Application value of DSC-PWI in ischemic cerebrovascular disease[J]. Chinese Journal of Physician,2020,22(3):6.).

[0561] 3.3.1.4 Cognitive function testing

[0562] The spatial delayed response test (Spatial Delayed Response testing) was used to evaluate the cognitive functions of monkeys, including learning, memory, visual-spatial ability, and comprehension and judgment abilities (Bo, Zhang, Fei, et al. Chronicphencyclidine treatment impairs spatial working memory in rhesus monkeys. [J]. Psychopharmacology, 2019.; Buccafusco J J. Working Memory Delayed Response Tasks in Monkeys--Methods of Behavior Analysis in Neuroscience [J]. CRC Press, 2009.).

[0563] Using a modified Wisconsin apparatus (such as Fig.16 The spatial delayed reaction experiment was conducted in two containers (as shown). Under the gaze of the monkey, food was randomly placed in one of the two containers, the lid was closed, and then the baffle was lowered to block the monkey's sight. After a certain delay, the animal was allowed to choose the container with food. The animal must remember which container the food was placed in in order to make the correct choice and get the food. The delay time was 1s, 5s, 10s, and 30s, respectively. Three months after the stroke, a total of 6 animals in the model control group (N=3) and the DOX+PMP+MTP group (N=3) were tested 30 times a day. If the animal's accuracy rate in the test for two consecutive days reached more than 80%, the delay time was extended successively. The efficacy indicators are the accuracy rate of completing the test at different delay times and the longest time interval that can be achieved in memory as cognitive function indicators.

[0564] In the previous study, the inventor selected 10 healthy rhesus monkeys aged 5-7 to complete the spatial delayed reaction experiment, and the longest delay time of all animals was ≥30s. When the interval time is more than 30s, the animals' lack of concentration affects the accuracy of the test, so in this embodiment, the inventor selected a test with a maximum delay of 30 seconds.

[0565] 3.3.2 Rhesus monkey care

[0566] Same as described in Example 1.3.2.

[0567] 3.3.3 Data Analysis

[0568] Same as described in Example 1.3.3.

[0569] 3.4 Experimental Results and Discussion

[0570] 3.4.1 Effect on brain edema / infarct volume

[0571] Results Fig.17 .

[0572] Model control group (N = 5): DWI of the operated side (right side) 3-4 hours after reperfusion showed that the average volume of b...

Claims

1. A therapeutically effective amount of doxazosin or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of pramipexole or a pharmaceutically acceptable salt thereof, and Use of a therapeutically effective amount of metoprolol or a pharmaceutically acceptable salt thereof in preparing a medicament for treating ischemic stroke in a subject.

2. The method according to claim 1, wherein the subject is a subject within 7 hours after the onset of stroke.

3. The method of claim 1 or 2, wherein the subject has a National Institutes of Health Stroke Scale (NIHSS) score of 20 or less.

4. The method according to claim 1 or 2, wherein the subject is a subject with moderate to severe stroke accompanied by persistent neurological deficit.

5. The method according to claim 3, wherein the subject is a subject with moderate to severe stroke accompanied by persistent neurological deficit.

6. The use according to claim 1 or 2, wherein the subject is a subject with anterior circulation large vessel occlusion.

7. The use according to claim 3, wherein the subject is a subject with anterior circulation large vessel occlusion.

8. The use according to claim 4, wherein the subject is a subject with anterior circulation large vessel occlusion.

9. The use according to claim 5, wherein the subject is a subject with anterior circulation large vessel occlusion.

10. The use according to claim 6, wherein the subject has symptoms of occlusion in the M1 segment of the middle cerebral artery.

11. The use according to claim 7, wherein the subject has symptoms of occlusion in the M1 segment of the middle cerebral artery.

12. The use of claim 8, wherein the subject has symptoms of occlusion in the M1 segment of the middle cerebral artery.

13. The use of claim 9, wherein the subject has symptoms of occlusion in the M1 segment of the middle cerebral artery.

14. The use of claim 6, wherein the subject has symptoms of occlusion in the middle cerebral artery M2 segment.

15. The use of claim 7, wherein the subject has symptoms of occlusion in the middle cerebral artery M2 segment.

16. The use of claim 8, wherein the subject has symptoms of occlusion in the middle cerebral artery M2 segment.

17. The use of claim 9, wherein the subject has symptoms of occlusion in the middle cerebral artery M2 segment.

18. The use according to claim 1 or 2, wherein the subject is a subject suffering from ischemia-reperfusion injury.

19. The use according to claim 3, wherein the subject is a subject suffering from ischemia-reperfusion injury.

20. The use according to claim 4, wherein the subject is a subject suffering from ischemia-reperfusion injury.

21. The use according to claim 5, wherein the subject is a subject suffering from ischemia-reperfusion injury.

22. The use according to claim 6, wherein the subject is a subject suffering from ischemia-reperfusion injury.

23. The use according to claim 7, wherein the subject is a subject suffering from ischemia-reperfusion injury.

24. The use according to claim 8, wherein the subject is a subject suffering from ischemia-reperfusion injury.

25. The use according to claim 9, wherein the subject is a subject suffering from ischemia-reperfusion injury.

26. The use according to claim 10, wherein the subject is a subject suffering from ischemia-reperfusion injury.

27. The use according to claim 11, wherein the subject is a subject suffering from ischemia-reperfusion injury.

28. The use according to claim 12, wherein the subject is a subject suffering from ischemia-reperfusion injury.

29. The use according to claim 13, wherein the subject is a subject suffering from ischemia-reperfusion injury.

30. The use according to claim 14, wherein the subject is a subject suffering from ischemia-reperfusion injury.

31. The use according to claim 15, wherein the subject is a subject suffering from ischemia-reperfusion injury.

32. The use according to claim 16, wherein the subject is a subject suffering from ischemia-reperfusion injury.

33. The use according to claim 17, wherein the subject is a subject suffering from ischemia-reperfusion injury.

34. The use according to claim 1 or 2, wherein the subject is a subject suffering from ischemia without reperfusion.

35. The use according to claim 3, wherein the subject is a subject suffering from ischemia without reperfusion.

36. The use according to claim 4, wherein the subject is a subject suffering from ischemia without reperfusion.

37. The use according to claim 5, wherein the subject is a subject suffering from ischemia without reperfusion.

38. The use according to claim 6, wherein the subject is a subject suffering from ischemia without reperfusion.

39. The use according to claim 7, wherein the subject is a subject suffering from ischemia without reperfusion.

40. The use according to claim 8, wherein the subject is a subject suffering from ischemia without reperfusion.

41. The use according to claim 9, wherein the subject is a subject suffering from ischemia without reperfusion.

42. The use according to claim 10, wherein the subject is a subject suffering from ischemia without reperfusion.

43. The use according to claim 11, wherein the subject is a subject suffering from ischemia without reperfusion.

44. The use according to claim 12, wherein the subject is a subject suffering from ischemia without reperfusion.

45. The use according to claim 13, wherein the subject is a subject suffering from ischemia without reperfusion.

46. ​​The use according to claim 14, wherein the subject is a subject suffering from ischemia without reperfusion.

47. The use according to claim 15, wherein the subject is a subject suffering from ischemia without reperfusion.

48. The use according to claim 16, wherein the subject is a subject suffering from ischemia without reperfusion.

49. The use according to claim 17, wherein the subject is a subject suffering from ischemia without reperfusion.

50. The use according to claim 1 or 2, wherein the subject is a subject with large-area cerebral infarction.

51. The use of claim 3, wherein the subject is a subject with large-area cerebral infarction.

52. The use of claim 4, wherein the subject is a subject with large-area cerebral infarction.

53. The use of claim 5, wherein the subject is a subject with large-area cerebral infarction.

54. The use according to claim 6, wherein the subject is a subject with large-area cerebral infarction.

55. The use according to claim 7, wherein the subject is a subject with large-area cerebral infarction.

56. The use of claim 8, wherein the subject is a subject with large-area cerebral infarction.

57. The use of claim 9, wherein the subject is a subject with large-area cerebral infarction.

58. The use according to claim 10, wherein the subject is a subject with large-area cerebral infarction.

59. The use according to claim 11, wherein the subject is a subject with large-area cerebral infarction.

60. The use according to claim 12, wherein the subject is a subject with large-area cerebral infarction.

61. The use according to claim 13, wherein the subject is a subject with large-area cerebral infarction.

62. The use according to claim 14, wherein the subject is a subject with large-area cerebral infarction.

63. The use of claim 15, wherein the subject is a subject with large-area cerebral infarction.

64. The use according to claim 16, wherein the subject is a subject with large-area cerebral infarction. The use according to claim 17 , wherein the subject is a subject with large-area cerebral infarction.

66. A therapeutically effective amount of doxazosin or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of pramipexole or a pharmaceutically acceptable salt thereof, and Use of a therapeutically effective amount of metoprolol or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating ischemic optic neuropathy in a subject.

67. The use according to claim 66, wherein the ischemic optic neuropathy is anterior ischemic optic neuropathy.

68. The use of claim 66, wherein the ischemic optic neuropathy is posterior ischemic optic neuropathy.

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