Methods and pharmaceutical compositions for treating chronic nephropathy

Abstract

The present application relates to a method and pharmaceutical composition for treating chronic nephropathy. The method and pharmaceutical composition of the present application relate to the use of a combination of doxazosin or a pharmaceutically acceptable salt thereof, pramipexole or a pharmaceutically acceptable salt thereof, and metoprolol or a pharmaceutically acceptable salt thereof, which can significantly improve chronic nephropathy, significantly reduce urinary albumin excretion rate (UACR), significantly improve glomerular filtration rate (eGFR), and effectively delay the progression of renal function decline to end-stage renal failure.

Description

[Technical Field]

[0001] This application relates to the field of disease treatment and medicine, and specifically to the treatment of chronic nephropathy, especially diabetic nephropathy. [Background technology]

[0002] Chronic kidney disease (CKD) is a disease that severely affects human health, especially the health of the elderly. According to statistics, the prevalence of CKD among American adults is estimated to be 11.3%, and the prevalence of CKD in China is estimated to be approximately 10%. Diabetes and hypertension are considered to be the main triggers of CKD.

[0003] Diabetes mellitus (DKD) is a common disease affecting human health. In 2015, there were nearly 420 million people with diabetes worldwide, and the number is expected to reach 640 million by 2040. It is estimated that 20% to 40% of diabetic patients will develop diabetic nephropathy. Diabetic nephropathy, also known as diabetic kidney disease (DKD) or diabetic nephropathy (DN), is a major complication of diabetes. Diabetic nephropathy is a condition characterized by abnormalities in renal structure and function due to chronic diabetic microvascular disease. Clinical manifestations include hypertension, proteinuria, and edema. Pathological manifestations include glomerular vascular damage and sclerosis, which leads to nodular lesions, further renal dysfunction and persistent urinary proteinuria. Ultimately, renal failure leads to end-stage renal disease (ESRD), which has a high mortality rate.

[0004] DKD is a complex disease in which environmental and genetic factors interact, and its reported mechanisms of onset and progression are extremely complex, involving a variety of pathological changes, including oxidative stress, inflammation, renal dysfunction, renal interstitial fibrosis, changes in hemodynamics, and genetic factors. Of these, oxidative stress is thought to play an important role in the pathogenesis and is closely related to the onset and progression of DKD.

[0005] Currently, DKD management strategies primarily focus on achieving optimal blood glucose and blood pressure control, correcting blood lipid metabolism abnormalities, reversing insulin resistance, and reducing proteinuria (using angiotensin-converting enzyme inhibitors and angiotensin receptor blockers), thereby delaying renal function decline and reducing cardiovascular risk events. Inhibition of renin-angiotensin-aldosterone system (RAAS) activation by using angiotensin-converting enzyme inhibitors (ACEIs) and / or angiotensin receptor blockers (ARBs) is currently the primary approach to treating DKD. Valsartan, a commonly used ARB, improves glomerular selective permeability, thereby maintaining a constant radius of the glomerular filtration pore and sustainably reducing urinary albumin without causing a decline in glomerular filtration rate (eGFR). However, current treatments and medications are still insufficient in terms of efficacy and prevention of diabetic nephropathy progression. Studies have shown that ARBs do not increase the risk of myocardial infarction and can even reduce the risks of heart failure and stroke, but the study also found that ARBs do not reduce all-cause mortality in patients. Furthermore, administering ACEI or ARB medications to diabetic patients with normal blood pressure and no proteinuria did not prevent the development of trace proteinuria.

[0006] Therefore, in clinical practice, there is an urgent need to develop novel drugs that can effectively treat CKD and / or DKD and effectively delay the progression of CKD / DKD to ESRD. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, one object of the present application is to provide better or alternative therapeutic methods and medicaments for treating chronic nephropathy (particularly diabetic nephropathy). [Means for solving the problem]

[0008] The present inventors have surprisingly found that the combination of doxazosin, pramipexole and metoprolol has significant effects in the treatment of chronic nephropathy, particularly diabetic nephropathy.

[0009] Therefore, a first aspect of the present application relates to a pharmaceutical composition for treating chronic nephropathy, comprising as active ingredients doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole or a pharmaceutically acceptable salt thereof, and metoprolol (or a pharmaceutically acceptable salt thereof).

[0010] A second aspect of the present application relates to the use of a combination of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof) and metoprolol (or a pharmaceutically acceptable salt thereof) in the manufacture of a pharmaceutical composition for treating chronic nephropathy, in particular diabetic nephropathy, in a subject in need thereof.

[0011] A third aspect of the present application relates to the use of a combination of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof) and metoprolol (or a pharmaceutically acceptable salt thereof) in the treatment of chronic nephropathy, particularly diabetic nephropathy, in a subject in need thereof.

[0012] A fourth aspect of the present application relates to a method for treating chronic nephropathy, particularly diabetic nephropathy, said method comprising administering to a subject in need thereof therapeutically effective amounts of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof), and metoprolol (or a pharmaceutically acceptable salt thereof). [Effects of the Invention]

[0013] The present inventors used rhesus monkeys, which suffer from spontaneous chronic diabetic nephropathy, as an animal model to simulate human DKD patients. Surprisingly, they found that the combination of doxazosin, pramipexole, and metoprolol significantly reduced proteinuria and significantly improved glomerular filtration rate in the animal model, thereby preventing or delaying the progression of DKD to end-stage renal disease (ESRD), and exhibited favorable safety with no administration-related side effects. Therefore, the pharmaceutical combination of the present application represents a novel method for treating chronic nephropathy, particularly diabetic nephropathy.

[0014] Hereinafter, specific embodiments of the present invention will be described in detail with reference to the drawings. [Brief explanation of the drawings]

[0015] [Figure 1] The comparison of changes in albuminuria (UACR, urinary albumin excretion rate) on day 30 (D30) of treatment in rhesus monkeys of each group is shown (**: p<0.01 compared with the placebo group; *: p<0.05 compared with the placebo group). [Figure 2] The comparison of changes in albuminuria (UACR, urinary albumin excretion rate) on day 58 (D58) of treatment in rhesus monkeys from each group is shown (**: p<0.01 compared with the placebo group; *: p<0.05 compared with the placebo group). [Figure 3] The changes in eGFR after administration of rhesus monkeys in each group are shown (eGFR included in each group: 30-59 ml / min / 1.73 m2 was analyzed, and the DOX+PMP+MTP1# experimental group and the DOX+PMP+MTP2# experimental group were combined into one group for analysis; *: p<0.05 when comparing the DOX+PMP+MTP group with the placebo group). DETAILED DESCRIPTION OF THE INVENTION

[0016] The present application relates to combinations of GPCR drugs and corresponding combination treatment methods.

[0017] G protein (guanine nucleotide-binding protein)-coupled receptors (GPCRs) are a type of transmembrane protein that regulates intracellular signaling and is essential for maintaining intracellular steady state. Literature studies have shown that GPCR signaling pathways are involved in diabetes-induced superoxide generation (Du, Y., et al., Adrenergic and serotonin receptors affect retinoid superoxide generation in diabetic mice: Relationship to capillary degeneration and permeability. 2015.29(5):p.2194). Oxidative stress is an important mechanism in the development and progression of DKD. GPCR drugs that regulate diabetes-induced superoxide generation could theoretically have a significant impact on DKD progression. However, current research is limited to the pathogenic mechanism, and further investigation into the therapeutic effects of GPCR drugs on DKD is needed. Furthermore, the wide variety of GPCR drugs available makes it difficult to select clinically effective drugs for DKD.

[0018] Currently, most animal models of diabetic nephropathy are rodents, such as drug-induced mouse models, spontaneous db / db mice, ob / ob mice, Agout mutant mice, New Zealand obese mice, and transgenic mouse models. However, because the pathogenesis of diabetic nephropathy is complex and involves many processes, the renal dysfunction in these animal models cannot accurately reflect the course of DKD in patients, making it difficult to evaluate the effectiveness of disease treatments. Non-human primates are extremely similar to humans physiologically, biochemically, and phylogenetically. Numerous published studies have shown that the disease characteristics of rhesus monkeys with spontaneous chronic diabetic nephropathy are very similar to those of clinical DKD patients, with similar features such as moderate to severe increases in urinary albumin and decreased eGFR. Therefore, rhesus monkeys with spontaneous chronic diabetic nephropathy provide an important research tool for CKD / DKD research and the development of new drugs (Najafian, B., et al., Glomerulopathy in spontaneously obese rhesus monkeys with type 2 diabetes: a stereological study. Diabetes Metab Res Rev, 2011.27(4):p.341-7. Liang Y, Yang Z, et al. Diabetic Kidney Disease (DKD) in Nonhuman Primates (NHP's) is Comparable to Humans for Glomerular Filtration Rate (GFR), Histology, and High Risk Factors.ADA2017).

[0019] Considering the limitations of rodent animal models in the study of human diabetic nephropathy, the present inventors used rhesus monkeys with spontaneous chronic diabetic nephropathy as an animal model to mimic human patients. Using these models, they conducted extensive and in-depth research and extensive screening of numerous GPCR-based drugs. Surprisingly, they found that a combination of certain GPCR-based drugs has a significant therapeutic effect on DKD. Specifically, they found that the combination of doxazosin, pramipexole, and metoprolol significantly reduced proteinuria (UACR), significantly improved glomerular filtration rate (eGFR), and inhibited the progression of DKD to end-stage renal disease (ESRD) in a rhesus monkey model with G3 CKD-EPI and moderately severe proteinuria. The therapeutic effects were comparable to those of the commonly used drug valsartan. Based on this, the present application discloses a pharmaceutical composition for treating chronic nephropathy, particularly DKD, and its medical use.

[0020] 1. Pharmaceutical composition A first aspect of the present application provides a pharmaceutical composition for treating chronic nephropathy, particularly diabetic nephropathy, comprising doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof), and metoprolol (or a pharmaceutically acceptable salt thereof) as active ingredients.

[0021] 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 one of ordinary skill in the art.

[0022] The terms "comprise," "contain," and "have" are used in an inclusive and open sense, meaning that additional elements may be included in addition to those specified. As used herein, the terms "such as," "for example," and "for example" are non-limiting and are used for descriptive purposes only. "Including" and "including but not limited to" may be used interchangeably.

[0023] Unless the context clearly indicates otherwise, the term "or," as used herein, should be understood to refer to "and / or."

[0024] The term "treatment" means inhibiting a disease, disease, and / or symptom in a subject, e.g., inhibiting its progression, alleviating the disease, disease, and / or symptom, e.g., causing the regression of the disease, disease, and / or symptom. Treating a disease or disease includes ameliorating at least one symptom of a particular disease or disease, even if the underlying pathophysiology is unaffected. In particular, in this application, "treatment" also includes reducing the risk of developing a disease by administering a pharmaceutical agent; i.e., "treatment" includes not only prevention before the onset of the disease, but also alleviation, suppression, and cure of the disease after the onset of the disease.

[0025] The term "pharmaceutical composition" refers to a combination of multiple substances with specific medicinal or biological uses, generally expected to have a therapeutic or preventive effect against a specific disease after administration to a subject. A pharmaceutical composition may contain only a specific active ingredient (biologically active substance), or may be provided together with a conventional pharmaceutically acceptable carrier for various uses. The term "composition" in this application should be interpreted broadly. In one embodiment of the pharmaceutical composition of this application, for example, the specific active ingredients (and any pharmaceutically acceptable carriers) can be mixed together to provide a mixture in which the components are indistinguishable. In another embodiment of the pharmaceutical composition of this application, each specific active ingredient can be individually packaged in small packages, and these small, independent packages can then be placed together in a larger container to provide the "pharmaceutical composition" of this application.

[0026] The terms "active ingredient" and "biologically active agent" refer to biologically, physiologically, or pharmaceutically active molecules and other agents that act in a patient or subject to treat a disease or condition (e.g., diabetic nephropathy). This term is used in conjunction with terms such as "pharmaceutically acceptable carrier," "excipient," and "auxiliary agent." An "active ingredient" (or "biologically active agent") includes, but is not limited to, pharmaceutically acceptable salts and prodrugs thereof. These agents may be acids, bases, or salts; they may be neutral molecules, polar molecules, or molecular complexes capable of hydrogen bonding; they may also be prodrugs in the form of ethers, esters, amides, etc., which are biologically activated when administered to a patient or subject.

[0027] The term "doxazosin" as used herein refers to the compound whose English name is Doxazosin (abbreviated as DOX) and whose IUPAC name is (RS)-2-[4-(2,3-dihydro-1,4-benzodioxane-2-carbonyl)piperazin-1-yl]-6,7-dimethoxy-4-amine (molecular formula C 23 H 25 The molecule below is N5O5 and has a molecular weight of 451.475 g / mol.

[0028] [ka]

[0029] The term "doxazosin" also includes isotopically labeled compounds thereof, or optical isomers, geometric isomers, tautomers or isomeric mixtures thereof, or precursor drugs thereof (ie, compounds that react in vivo to yield said molecule).

[0030] Doxazosin is a marketed drug approved by major drug regulatory agencies (e.g., the FDA). It is a selective alpha-1 receptor antagonist that inhibits the binding of norepinephrine (released from peripheral sympathetic nerve endings) to alpha-1 receptors on the membrane of vascular smooth muscle cells and is commonly used to treat primary hypertension.

[0031] The pharmaceutical composition of the present application may contain doxazosin or a pharmaceutically acceptable salt thereof as an active ingredient. Most commercially available doxazosin drugs are in the form of salts, particularly its methanesulfonate salt, such as Doxazosin Methanesulfonate Controlled-Release Tablets (Cardura) provided by Pfizer Inc. and Doxazosin Methanesulfonate Tablets provided by Hangzhou Kongenpei Pharmaceutical Co., Ltd., China.

[0032] The term "pramipexole" as used herein refers to the compound whose English name is Pramipexole (abbreviated as PMP) and whose IUPAC name is (S)-N6-propyl-4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-diamine (molecular formula: C 10 H 17 The molecule below is N3S, which has a molecular weight of 211.324 g / mol.

[0033] [ka]

[0034] The term "pramipexole" also includes isotopically labeled compounds thereof, or optical isomers, geometric isomers, tautomers or isomeric mixtures thereof, or precursor drugs thereof (i.e., compounds that react in vivo to yield the molecule).

[0035] Pramipexole is a commercially available drug approved by major drug regulatory agencies (e.g., the FDA). It is an antihistamine and dopamine receptor D2 / D3 agonist primarily used clinically to treat Parkinson's disease, either alone (without levodopa) or in combination with levodopa. In the literature, pramipexole is also referred to as "Mirapex," "Mirapexin," or "Sifrol."

[0036] The pharmaceutical composition of the present application may contain pramipexole or a pharmaceutically acceptable salt thereof as an active ingredient. Most commercially available pramipexole is in the form of a salt, particularly its hydrochloride, such as Pramipexole Hydrochloride Tablets (Sifrol) provided by Boehringer Ingelheim.

[0037] The term "metoprolol" as used herein refers to the compound whose English name is metoprolol (abbreviated as MTP) and whose IUPAC name is RS-1-[4-(2-methoxyethyl)phenoxy]-3-[(propan-2-yl)amino]propan-2-ol (molecular formula C 15 H 25 The molecule below is NO3 and has a molecular weight of 267.37 g / mol.

[0038] [ka]

[0039] The term "metoprolol" also includes isotopically labeled compounds thereof, or optical isomers, geometric isomers, tautomers or isomeric mixtures thereof, or precursor drugs thereof (i.e., compounds that react in vivo to yield the molecule).

[0040] Metoprolol is a selective adrenergic β1-receptor blocker commonly used to treat hypertension and angina pectoris, a commercially available drug approved by major drug regulatory authorities (e.g., the FDA). The pharmaceutical composition of the present application may contain metoprolol or a pharmaceutically acceptable salt thereof as an active ingredient. Most commercially available metoprolol formulations are in the form of salts, particularly the tartrate salt, such as metoprolol tartrate tablets (Betaloc) from AstraZeneca and metoprolol tartrate controlled-release tablets (Lijuning) from Guangzhou Baiyunshan Tianxin Pharmaceutical Co., Ltd., China.

[0041] Any of the active ingredients (doxazosin, pramipexole, metoprolol) contained in the compositions of the present application can be replaced with a pharmaceutically acceptable salt thereof. The term "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. As used herein, a pharmaceutically acceptable salt is a salt formed with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable salts 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 (e.g., sodium or potassium) and alkaline earth metals (e.g., calcium or magnesium), and organic bases such as alkylamines, arylamines, or heterocyclic amines. For the avoidance of doubt, references to "pharmaceutically acceptable salts" of an active ingredient described herein also include pharmaceutically acceptable salts formed from isotopically labeled compounds of the active ingredient, or optical isomers, geometric isomers, tautomers, or isomeric mixtures thereof, or precursor drugs thereof.

[0042] Pharmaceutically acceptable salts can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Typically, these salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or in a mixture of the two. Non-aqueous media such as ethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are usually preferred. A list of salts can be found in Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing Company, 1990). For example, salts can include, but are not limited to, hydrochloride, tartrate, methanesulfonate, and the like, of the compounds described herein.

[0043] It should be understood that all references to various active ingredients or pharmaceutically acceptable salts include solvent addition forms (solvates such as hydrates, ethanol solvates, acetone solvates, etc.) of the same active ingredient or salt or its various crystalline forms (e.g., amorphous, polycrystalline, etc.).

[0044] In the pharmaceutical composition of the present application, the contents of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof), and metoprolol (or a pharmaceutically acceptable salt thereof) can be adjusted according to actual needs. For example, the contents and proportions of each drug in the pharmaceutical composition can vary depending on the method of administration of the pharmaceutical composition (oral administration, injection, etc.).

[0045] In some embodiments, the pharmaceutical composition of the present application typically contains 0.5 to 45 parts by weight (preferably 1 to 10 parts by weight) of pramipexole or a pharmaceutically acceptable salt thereof, 5 to 160 parts by weight (preferably 5 to 80 parts by weight) of doxazosin or a pharmaceutically acceptable salt thereof, and 50 to 2000 parts by weight (preferably 100 to 1000 parts by weight) of metoprolol or a pharmaceutically acceptable salt thereof.

[0046] In the pharmaceutical composition of the present application, the amount of pramipexole or a pharmaceutically acceptable salt thereof is usually 0.5 to 45 parts by weight, for example, 0.5 to 42 parts by weight, 0.5 to 40 parts by weight, 0.5 to 35 parts by weight, 0.5 to 30 parts by weight, 0.5 to 25 parts by weight, 0.5 to 20 parts by weight, 0.5 to 15 parts by weight, 0.5 to 10 parts by weight, 1 to 45 parts by weight, 1 to 42 parts by weight, 1 to 40 parts by weight, 1 to 35 parts by weight, 1 to 30 parts by weight, 1 to 25 parts by weight, 1 to 20 parts by weight, The amount may be 1 part by weight to 15 parts by weight, 1 part by weight to 10 parts by weight, 2 parts by weight to 45 parts by weight, 2 parts by weight to 40 parts by weight, 2 parts by weight to 35 parts by weight, 2 parts by weight to 30 parts by weight, 2 parts by weight to 25 parts by weight, 2 parts by weight to 20 parts by weight, 2 parts by weight to 15 parts by weight, 2 parts by weight to 10 parts by weight, 5 parts by weight to 45 parts by weight, 5 parts by weight to 40 parts by weight, 5 parts by weight to 35 parts by weight, 5 parts by weight to 30 parts by weight, 5 parts by weight to 25 parts by weight, 5 parts by weight to 20 parts by weight, 5 parts by weight to 15 parts by weight, 5 parts by weight to 10 parts by weight, 10 parts by weight to 40 parts by weight, 10 parts by weight to 30 parts by weight, 10 parts by weight to 25 parts by weight, etc. In a preferred embodiment of the present application, the content of pramipexole or a pharmaceutically acceptable salt thereof is 1 part by weight to 10 parts by weight.

[0047] In the pharmaceutical composition of the present application, the amount of doxazosin or a pharmaceutically acceptable salt thereof is usually 5 to 160 parts by weight, for example, 5 to 150 parts by weight, 5 to 130 parts by weight, 5 to 120 parts by weight, 5 to 100 parts by weight, 5 to 80 parts by weight, 5 to 60 parts by weight, 5 to 50 parts by weight, 5 to 40 parts by weight, 10 to 150 parts by weight, 10 to 130 parts by weight, 10 to 120 parts by weight, 10 to 100 parts by weight, 10 to 80 parts by weight, 10 to 60 parts by weight, 10 to 50 parts by weight, 10 to 50 parts by weight, 10 to 60 parts by weight, parts by weight to 40 parts by weight, 15 parts by weight to 160 parts by weight, 15 parts by weight to 150 parts by weight, 15 parts by weight to 130 parts by weight, 15 parts by weight to 120 parts by weight, 15 parts by weight to 100 parts by weight, 15 parts by weight to 80 parts by weight, 15 parts by weight to 60 parts by weight, 15 parts by weight to 50 parts by weight, 15 parts by weight to 40 parts by weight, 20 parts by weight to 160 parts by weight, 20 parts by weight to 150 parts by weight, 20 parts by weight to 130 parts by weight, 20 parts by weight to 120 parts by weight, 20 parts by weight to 100 parts by weight, 20 parts by weight to 80 parts by weight, 20 parts by weight to 60 parts by weight, 20 parts by weight to 50 parts by weight, 20 parts by weight to 40 parts by weight, etc. In a preferred embodiment of the present application, the content of doxazosin or a pharmaceutically acceptable salt thereof is 5 parts by weight to 80 parts by weight.

[0048] In the pharmaceutical composition of the present application, the amount of metoprolol or a pharmaceutically acceptable salt thereof is usually 50 parts by weight to 2000 parts by weight, for example, 50 parts by weight to 1800 parts by weight, 50 parts by weight to 1600 parts by weight, 50 parts by weight to 1500 parts by weight, 50 parts by weight to 1300 parts by weight, 50 parts by weight to 1200 parts by weight, 50 parts by weight to 1000 parts by weight, 50 parts by weight to 80 parts by weight, 0 parts by weight, 50 parts by weight ~ 600 parts by weight, 50 parts by weight ~ 500 parts by weight, 50 parts by weight ~ 400 parts by weight, 100 parts by weight ~ 1800 parts by weight, 100 parts by weight ~ 1600 parts by weight, 100 parts by weight Parts by weight ~ 1500 parts by weight, 100 parts by weight ~ 1300 parts by weight, 100 parts by weight ~ 1200 parts by weight, 100 parts by weight ~ 1000 parts by weight, 100 parts by weight ~ 800 parts by weight, 100 parts by weight ~ 600 parts by weight, 100 parts by weight ~ 500 parts by weight, 100 parts by weight ~ 400 parts by weight, 150 parts by weight ~ 2000 parts by weight, 150 parts by weight ~ 1800 parts by weight, 150 parts by weight ~ 1600 parts by weight Parts by weight, 150 parts by weight - 1500 parts by weight, 150 parts by weight - 1300 parts by weight, 150 parts by weight - 1200 parts by weight, 150 parts by weight - 1000 parts by weight, 150 parts by weight - 800 parts by weight, 1 The amount may be 50 to 600 parts by weight, 200 to 2000 parts by weight, 200 to 1800 parts by weight, 200 to 1600 parts by weight, 200 to 1500 parts by weight, 200 to 1300 parts by weight, 200 to 1200 parts by weight, 200 to 1000 parts by weight, 200 to 800 parts by weight, 200 to 600 parts by weight, etc. In a preferred embodiment of the present application, the content of doxazosin or a pharmaceutically acceptable salt thereof is 100 to 1000 parts by weight.

[0049] In the pharmaceutical composition of the present application, the weight ratio of metoprolol or a pharmaceutically acceptable salt thereof to doxazosin or a pharmaceutically acceptable salt thereof, based on the weight of doxazosin or a pharmaceutically acceptable salt thereof (=1), is generally within the range of 0.3:1 to 400:1, and may be, for example, 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 or more, and preferably 1:1 , 1.5:1, 2:1, 3:1, 4:1, 5:1, 10:1, 12.5:1 or 15:1 or more, and may be 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 or less, preferably 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1 or 20:1 or less, and similarly, based on the weight of doxazosin or a pharmaceutically acceptable salt thereof (=1) In this regard, the weight ratio of pramipexole or a pharmaceutically acceptable salt thereof to doxazosin or a pharmaceutically acceptable salt thereof is generally within the range of 0.003:1 to 10:1, and may be, for example, 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 or more, and preferably The ratio may be 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 or 0.1:1 or more, and may be 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 or less, and is preferably 1:1, 0.9:1, 0.6:1, 0.5:1 or 0.4:1 or less.

[0050] (ii) Pharmaceutical dosage form In some embodiments, the pharmaceutical compositions of the present application may be provided in the form of bulk drugs (e.g., a homogeneous mixture or individual components packaged separately). In other embodiments, the pharmaceutical compositions of the present application may be formulated into various pharmaceutical dosage forms (formulations) by adding pharmaceutically acceptable carriers, if necessary. For this purpose, various liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials may be used as "pharmaceutically acceptable carriers." As used herein, the phrase "pharmaceutically acceptable" refers to compounds, compositions, polymers, and other materials that, within the scope of reasonable medical judgment, are compatible with the other ingredients of the compositions of the present application and are suitable for contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problems or complications. In some preferred embodiments, the pharmaceutically acceptable carrier is pyrogen-free. Some examples of materials useful 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, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) maltodextrin; (6) gelatin; (7) talc; (8) adjuncts such as cocoa butter and suppository wax; and (9) peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, and cocoa. (10) diols such as propylene glycol; (11) polyhydric alcohols such as glycerin, 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; and (21) other non-toxic, compatible substances used in pharmaceutical formulations.

[0051] The pharmaceutical composition of the present application may be formulated, as needed, into a dosage form suitable for administration routes such as oral administration, parenteral (including subcutaneous, intramuscular, cortical, and intravenous) administration, bronchial administration, intravitreal injection, or nasal administration. Here, the pharmaceutical composition of the present application is preferably formulated into a dosage form (preparation) suitable for oral administration.

[0052] When a solid carrier is used, the formulation may be tableted, placed in a hard gel capsule in powder or granular form, or in the form of a sugar-coated tablet or tablet. Solid carriers can contain conventional excipients such as binders, fillers, tableting lubricants, disintegrants, and wetting agents. The tablets may be film-coated, if desired, by conventional techniques. When a liquid carrier is used, the formulation may be in the form of a syrup, emulsion, softgel capsule, sterile injectable carrier, aqueous or non-aqueous liquid suspension, or a dry product to be reconstituted with water or another suitable carrier before use. Liquid formulations may contain conventional additives such as suspending agents, emulsifiers, wetting agents, non-aqueous carriers (including edible oils), preservatives, and flavorings and / or colorants. For parenteral administration, carriers typically comprise at least sterile water, although saline solutions, glucose solutions, and the like can also be used. Injectable suspensions can also be used, in which case conventional suspensions can be used. Conventional preservatives, buffers, and the like can also be added to parenteral dosage forms.

[0053] Dosage forms suitable for parenteral injection 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, and solvents include water, ethanol, polyols (propylene glycol, polyethylene glycol, propanetriol, etc.), suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate).

[0054] These pharmaceutical dosage forms may also contain various excipients such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action can be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). Isotonicity adjusting agents such as sugars, sodium chloride, etc. may also be included. Absorption delaying agents (e.g., aluminum monostearate and gels) can be used to prolong absorption of the injectable pharmaceutical dosage form.

[0055] Oral solid dosage forms 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) and contains at least one of: (a) fillers or fillers (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid); (b) binders (e.g., carboxymethylcellulose, alginate esters, gels, polyvinylpyrrolidone, sucrose, and gum arabic); (c) humectants (e.g., propanetriol); and (d) disintegrants (e.g., agar-agar, calcium carbonate, jasmine, etc.). It may further comprise: (a) potato or tapioca starch, alginic acid, certain synthetic silicates, sodium carbonate; (e) solution blocking agents (e.g., paraffin); (f) absorption promoters (e.g., quaternary ammonium compounds); (g) wetting agents (e.g., cetanol and propanetriol 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.

[0056] Solid compositions of a similar type may also be employed as fillers in soft and hard gel capsules using, for example, lactose, high molecular weight polyethylene glycols and the like as excipients.

[0057] Solid dosage forms (e.g., tablets, dragees, capsules, pills, and granules) can be prepared with coatings and shells (e.g., enteric coatings and others known in the art). These may contain light-blocking agents and may release the active compound or compositions of various active compounds in a delayed manner in a certain part of the intestinal tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0058] Oral liquid dosage forms include pharmaceutically acceptable emulsions, solutions, dispersions, syrups, and elixirs. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art (e.g., water or other solvents), solubilizing agents and emulsifiers (e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzoyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformylammonium), oils (specifically, cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil), propanetriol, tetrahydrofuran alcohol, polyethylene glycol, and fatty acid esters of sorbitan, or mixtures thereof.

[0059] In addition to these inert diluents, pharmaceutical dosage forms may contain, for example, wetting agents, emulsifying and suspending agents, flavoring agents, taste-improving agents, and flavor-adding agents.

[0060] In addition to the active compound, suspensions may contain suspending agents such as ethoxylated isooctadecanol, polyethylene oxide sorbitol, sorbitan esters, microcrystalline fibers, aluminum metahydroxide, bentonite, agar-agar or tragacanth, or mixtures thereof.

[0061] Pharmaceutical dosage forms of the present application also include ointments, powders, sprays, and inhalants. The active ingredient is admixed under sterile conditions with a physiologically acceptable carrier and any desired preservatives, buffers, or propellants.

[0062] The amounts of active ingredients in pharmaceutical compositions and dosage forms 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.

[0063] For example, the pharmaceutical composition of the present application can be formulated into an oral dosage form (e.g., an oral dosage form administered twice or three times daily or a long-term sustained-release oral dosage form), an intravenous injection dosage form, or an intramuscular injection dosage form.

[0064] In a preferred embodiment of the present application, the pharmaceutical composition of the present application is in an oral dosage form. In a more preferred embodiment of the present application, the pharmaceutical composition of the present application is in a once-daily oral dosage form, a two- to three-times-daily oral dosage form, or a long-term sustained-release oral dosage form.

[0065] In a preferred embodiment of the present application, the pharmaceutical composition of the present application is in an oral dosage form. In a more preferred embodiment of the present application, the pharmaceutical composition of the present application is in a daily oral dosage form.

[0066] 3. Use of pharmaceutical compositions and pharmaceutical dosage forms It is known in the art that the pharmaceutical compositions and pharmaceutical dosage forms of the present application can be used to treat chronic nephropathy (CKD), particularly diabetic nephropathy (DN or DKD).

[0067] Accordingly, a second aspect of the present application relates to the use of a combination of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof), and metoprolol (or a pharmaceutically acceptable salt thereof) in the manufacture of a pharmaceutical composition, said pharmaceutical composition being used to treat chronic nephropathy, in particular diabetic nephropathy, in a subject in need thereof.

[0068] A third aspect of the present application relates to the use of a combination of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof) and metoprolol (or a pharmaceutically acceptable salt thereof) in the treatment of chronic nephropathy, particularly diabetic nephropathy, in a subject in need thereof.

[0069] A fourth aspect of the present application relates to a method for treating chronic nephropathy, particularly diabetic nephropathy, said method comprising administering to a subject in need thereof therapeutically effective amounts of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof), and metoprolol (or a pharmaceutically acceptable salt thereof).

[0070] The pharmaceutical compositions and dosage forms of the present application are suitable for the treatment of diabetic nephropathy at various stages, and also for preventive treatment before the onset of the disease.

[0071] A "patient" or "subject" treated by the methods of the present application may be a human or non-human animal, such as a primate. Preferably, the "patient" or "subject" is human.

[0072] The therapeutic method of the present application relates to the co-administration of multiple active ingredients, also referred to as "combination therapy." The term "co-administration" or "combination therapy" refers to the administration of multiple active ingredients described herein to achieve a beneficial effect through their synergistic action. The beneficial effect of the combination includes, but is not limited to, the synergistic action of the pharmacological activity or efficacy of the combined active ingredients. The combined administration of these active ingredients is typically completed within a predetermined period (usually minutes, hours, days, or weeks, as determined by the physician). "Co-administration" or "combination therapy" is intended to include sequential administration of these active ingredients, i.e., administration of each active ingredient at different times, substantially simultaneous administration of these active ingredients, or administration of at least two of these active ingredients. Substantially simultaneous administration can be achieved, for example, by administering to the host a single capsule containing each active ingredient in a fixed ratio, or by administering to the host multiple capsules, each containing a single active ingredient. The sequential or substantially simultaneous administration of each active ingredient can be effected by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The active ingredients may be administered by the same route or different routes. For example, a first active ingredient in a selected combination may be administered by intravenous injection, and 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.

[0073] "Co-administration" or "combined treatment" also includes administering the aforementioned active ingredients in combination with other biologically active ingredients and non-pharmaceutical therapies (e.g., surgical or mechanical treatments). When the combined treatment further includes a non-pharmaceutical therapy, the non-pharmaceutical therapy can be administered at any appropriate time, as long as a beneficial effect is achieved through the synergistic action of the combination of the active ingredients and the non-pharmaceutical therapy. For example, in appropriate cases, such beneficial effect can be achieved even after temporarily suspending the non-pharmaceutical therapy from the administration of the active ingredients, for several days or even weeks.

[0074] To achieve the desired effect, it is usually necessary to administer to a patient or subject a therapeutically effective amount of the pharmaceutical composition or dosage form of the present application, or of each active ingredient taken alone.

[0075] The phrase "therapeutically effective amount" is a term well known in the art. In some embodiments, the term refers to an amount necessary or sufficient to eliminate, reduce, or maintain a target of a particular therapeutic procedure. The effective amount can vary depending on factors such as the disease or condition being treated, the specific targeting construct being administered, the age of the subject, or the severity of the disease or condition. A skilled artisan or physician can empirically determine the effective amount of a particular compound without undue experimentation. In some embodiments, the therapeutically effective amount of a therapeutic agent used in vivo also depends on many factors, including the mode and method of administration and any other materials contained in the pharmaceutical formulation in addition to the drug. In vitro or in vivo testing can be used to determine optimal dosage ranges.

[0076] In some embodiments of the present application, for an adult with a normal body weight of about 60 kilograms, when the pharmaceutical dosage form or pharmaceutical composition of the present application is orally administered, the daily dose range of doxazosin or a pharmaceutically acceptable salt thereof may be 0.5 mg to 16 mg, for example, 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, 0.5 mg to 4 mg, 1 mg to 15 mg, 1 mg to 13 mg, 1 mg to 1 ... 2 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 6 mg, 1 mg to 5 mg, 1 mg to 4 mg, 1.5 mg to 16 mg, 1.5 mg to 15 mg, 1.5 mg to 13 mg, 1.5 mg to 12 mg, 1.5 mg to 10 mg, 1.5 mg to 8 mg, 1.5 mg to 6 mg, 1.5 mg to 5 mg, 1.5 mg to 4 mg, 2 mg to 16 mg, 2 mg to 15 mg, 2 mg to 13 mg, 2 mg to 12 mg, 2 mg to 10 mg, 2 mg to 8 mg, 2 mg to 6 mg, 2 mg to 5 mg, 2 mg to 4 mg, etc. In a preferred embodiment of the present application, the daily dosage range of doxazosin or a pharmaceutically acceptable salt thereof may be about 2 mg. In another preferred embodiment of the present application, the daily dosage of doxazosin is about 4 mg.

[0077] In some embodiments of the present application, for an adult with a normal body weight of about 60 kilograms, when the pharmaceutical dosage form or pharmaceutical composition of the present application is orally administered, the daily dose range of pramipexole or a pharmaceutically acceptable salt thereof may be 0.05 mg to 4.5 mg, for example, 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 4.2 mg, 0.1 mg to 4 mg, 0.1 mg to 3.5 mg, 0.1 mg to 3 mg, 0.1 mg The amount may be 0.5 mg to 2.5 mg, 0.1 mg to 2 mg, 0.1 mg to 1.5 mg, 0.1 mg to 1 mg, 0.2 mg to 4.5 mg, 0.2 mg to 4.2 mg, 0.2 mg to 4 mg, 0.2 mg to 3.5 mg, 0.2 mg to 3 mg, 0.2 mg to 2.5 mg, 0.2 mg to 2 mg, 0.2 mg to 1.5 mg, 0.2 mg to 1.0 mg, 0.5 mg to 4.5 mg, 0.5 mg to 4.2 mg, 0.5 mg to 4 mg, 0.5 mg to 3.5 mg, 0.5 mg to 3 mg, 0.5 mg to 2.5 mg, 0.5 mg to 2 mg, 0.5 mg to 1.5 mg, 0.5 mg to 1 mg, 1 mg to 4 mg, 1 mg to 3 mg, or 1 mg to 2.5 mg. In a preferred embodiment of the present application, the daily dose range of pramipexole or a pharmaceutically acceptable salt thereof is about 0.1 mg to 1 mg. In a preferred embodiment of the present application, the daily dose of pramipexole is about 0.0625 mg. In another preferred embodiment of the present application, the daily dose of pramipexole is about 0.125 mg.

[0078] In some embodiments of the present application, for an adult with a normal body weight of about 60 kilograms, when the pharmaceutical dosage form or pharmaceutical composition of the present application is orally administered, the daily dose range of metoprolol or a pharmaceutically acceptable salt thereof may be 5 mg to 200 mg, for example, 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, 10 mg to 150 mg, 10 mg to 130 mg, 10 mg In a preferred embodiment of the present application, the daily dose range of metoprolol or a pharmaceutically acceptable salt thereof may be 10 mg to 100 mg. In a preferred embodiment of the present application, the daily dose of metoprolol is about 20 mg. In another preferred embodiment of the present application, the daily dose of metoprolol is about 40 mg.

[0079] The daily dose can be administered continuously at regular intervals, for example, every 2 hours, every 6 hours, every 8 hours, every 12 hours, and about every 24 hours. Preferably, the daily dose can be administered to patients two to three times a day, or can be administered using sustained-release tablets. The oral dosages of the above three active ingredients vary significantly, depending on the metabolic kinetics of each active ingredient.

[0080] As can be understood by those skilled in the art, when the pharmaceutical composition of the present application is formulated to be compatible with other dosage forms such as intravenous infusion or intramuscular injection, the dosage range of each active ingredient may differ from the oral dosage range described above, and a person skilled in the art or a physician can reasonably determine this range by combining in vivo and in vitro experiments and taking into account the different metabolic kinetic characteristics of drugs for various administration routes.

[0081] In a preferred embodiment of the present application, the pharmaceutical compositions of the present application are used in primate subjects, particularly human subjects.

[0082] As will be understood by those skilled in the art, the various aspects of the present application described herein can be combined in various ways known to those skilled in the art without departing from the subject matter and spirit of the present application. Such combinations are also within the scope of the present application. For example, the range of the amount of some components used in the present application includes any combination of any lower limit and any upper limit set forth in the specification, and the specific content of the component in each specific example includes any range formed by combining the upper or lower limits, and all of these ranges are within the scope of the present application. Furthermore, each feature of the present application listed in the specification can be combined with any other feature of the invention, and such combinations are also within the scope of the present application. [Example]

[0083] The present inventors used rhesus monkeys with spontaneous chronic diabetic nephropathy as experimental animals to investigate the efficacy, safety, and tolerability of the combined administration of three commercially available GPCR signaling pathway drugs, doxazosin (DOX), pramipexole (PMP), and metoprolol (MTP), for diabetic nephropathy. Valsartan, a drug commonly used to treat the common form of DKD, was used as a positive control in the experiment.

[0084] 1. Experimental materials Specifically, the following three commercially available drugs were used as test samples in the experiment. [Table 1]

[0085] Test sample 1: Name or abbreviation (English name): Doxazosin mesylate (DOX) Purity: 98% HPLC Manufacturer: Shanghai Ziqi Biotechnology Co., Ltd., China

[0086] Test sample 2: Name or abbreviation (English name): Pramipexole 2HCL Monohydrate (PMP) Purity: 99.55% HPLC Manufacturer: Shanghai Ziqi Biotechnology Co., Ltd., China

[0087] Test sample 3: Name or abbreviation (English name): Metoprolol tartratesalt (MTP) Purity: 98% HPLC Manufacturer: Aladdin (Shanghai) Reagent Co., Ltd.

[0088] Positive control drug: Valsartan Name or abbreviation (English name): Valsartan Manufacturer: Shanghai Bide Pharmatech Co., Ltd., China [[ID=4U]]

[0089] 2. Specific experimental scheme Experimental animals: Animal species: Rhesus Macaque, Macaca mulatta Level: Normal level. Passed the quarantine before the test. The quarantine contents include physical examination, two tuberculosis tests, parasite, Salmonella, Shigella, and B virus tests. Animal labeling: Stainless steel numbered plates engraved with Arabic numerals were attached to the neck rings, and tattoos were made on the chest. Supplier: Ya'an Prime Biotech Co., Ltd., China Production license number: SCXK(Sichuan) 2019-027 The test system complies with AAALAC requirements and GLP standards.

[0090] Breeding environment: Environmental level: Normal level. Temperature: 18~26℃. Relative humidity: 40~70%. Ventilation: At least 8 changes per hour, with 100% fresh air used (no air circulation). Lighting schedule: Automatic lighting, alternating between light and dark every 12 hours, lights off at 7:30 PM and lights on at 7:30 AM the next day. Animal cage: Double-layered stainless steel cage, 850*900*2365 mm. Stocking density: 1 animal per cage.

[0091] The study included 16 rhesus monkeys with spontaneous chronic diabetic nephropathy (DKD), with the following inclusion criteria: 1) 16 males / females, aged 14-24 years (equivalent to an adult age of 40-75 years); 14 males, weighing 7-13 kg; 2 females, weighing 6-8 kg. 2) Diabetes history of 2 years or more: fasting plasma glucose (FPG): over 4.8mmol / L, compared with 4.1±0.3mmol / L in age-matched controls. 3) CKD-EPI grades G3a-G3b are defined as having a glomerular filtration rate (eGFR) of 30-59 ml / min / 1.73 m 2 vs. age-matched controls, 92±11 ml / min / 1.73 m 2 or moderately elevated proteinuria (A2-A3) with a urinary albumin / creatinine ratio (UACR) of 15 mg / g to 350 mg / g (4-6 hour urine collection) vs. 3 ± 2 mg / g in age-matched controls. 4) Normal blood pressure, grade I or II hypertension (consistent with clinical patient criteria).

[0092] The exclusion criteria were as follows: 1) Severe liver dysfunction 2) Other diseases that may affect the evaluation of drug efficacy

[0093] Table 1 below shows the baseline period - the underlying etiology and symptoms of rhesus monkeys suffering from spontaneous chronic diabetic nephropathy in each group.

[0094] [Table 2]

[0095] Grouping and Dosing Schedule: This study consisted of a DOX+PMP+MTP group (n=8), a Valsartan group (n=4), and a placebo group (n=4). The DOX+PMP+MTP group was further divided into experimental group 1 and experimental group 2. The specific administration schedule for each group is as follows, and the administration route was oral. The baseline period was one month, followed by 58 consecutive days of oral administration.

[0096] 1# Experimental group: 4 animals D0~D14:DOX+PMP+MTP:0.133+0.002+0.833mg / kg, twice a day D15~D28:DOX+PMP+MTP:0.266+0.002+1.667mg / kg, twice a day D29-D58: DOX+PMP+MTP: 0.266+0.004+0.833mg / kg, DOX and MTP twice daily, PMP once daily

[0097] 2# Experimental group: 4 animals D0~D14:DOX+PMP+MTP:0.133+0.002+0.833mg / kg, twice a day D15~D28:DOX+PMP+MTP:0.133+0.002+1.667mg / kg, twice a day D29~D58:DOX+PMP+MTP:0.266+0.002+1.667mg / kg, twice a day

[0098] Valsartan group: 4 animals, dose 2.67 mg / kg (equivalent to a clinical dose of 40-80 mg), administered daily from D0 to D58, once daily from weeks 1 to 2, and twice daily from weeks 3 to 8.

[0099] Placebo group: Four animals were provided with drinking water and fruit and observed for 58 consecutive days.

[0100] To further evaluate the effect of each active ingredient in the pharmaceutical composition on the efficacy, another group of rhesus monkeys suffering from spontaneous chronic diabetic nephropathy was selected for the study, in which the effects of the DOX+MTP composition and placebo administered separately were compared in parallel.

[0101] The specific administration schedule for each group was as follows, and the administration route was oral administration: The baseline period was 1 month, followed by oral administration for 60 consecutive days.

[0102] DOX+MTP experimental group: 4 animals D0~D30:DOX+MTP:0.266mg / kg+1.667mg / kg, twice a day D30~D58:DOX+MTP:0.532mg / kg+3.334mg / kg, twice a day

[0103] Placebo group: Three animals were provided with drinking water and fruit and observed for 60 consecutive days.

[0104] Table 1A below shows the underlying etiology and symptoms during the baseline period for each experimental animal in the follow-up study.

[0105] [Table 3]

[0106] 3. Main observation indicators and monitoring methods 1. Main efficacy indicators Glomerular filtration rate (eGFR): Creatinine (Cr-P), blood urea nitrogen (BUN), and cystatin (CysC) were measured to calculate eGFR. Tests were performed once during the baseline period before administration and at designated times after administration (e.g., D14, D28, D45, D58, etc.). The eGFR calculation formula, based on reference (Levey AS, Stevens LA, Schmid CH et al. A new equation to eatimate glomerular filtration rate. Ann Intern Med 2009;150:604-612), is as follows: eGFR male =135×min(Cr / 0.9,1) -0.207 ×max(Cr / 0.9,1) -0.601 ×min(CysC / 0.8,1) -0.375 ×max(CysC / 0.8,1) -0.711 ×0.995 Age×3 eGFR female =135×min(Cr / 0.7,1) -0.284 ×max(Cr / 0.7,1) -0.601 ×min(CysC / 0.8,1) -0.375 ×max(CysC / 0.8,1) -0.711 ×0.995 Age×3 ×0.969

[0107] Urinary albumin excretion rate (UACR): Urine was collected at 4 and 6 hours, and urinary microalbumin (Malb) and creatinine (Cr-U) were tested to calculate the UACR value. Tests were conducted once before administration and at predetermined times after administration (e.g., once on day 30 and once on day 58). UACR = Malb / Cr-U.

[0108] Blood pressure: After anesthesia, blood pressure tests including systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP) and heart rate (HR) were performed once before administration and once on day 58 (end of administration).

[0109] 2. Secondary efficacy and safety indicators Blood potassium: Tested once before administration and at predetermined times after administration (e.g., once each on D14, D28, D45, and D58). Glycolipid metabolism and liver function: FPG, FRA, LDL-c, HDL-c, TG, TC, ALT, AST, etc. were examined once before administration and at specified times after administration (e.g., once on D28 and once on D58). Hematological indices: Tested once before administration and at specified times after administration (e.g., once on D28 and once on D58). Body weight: Once before administration, and once every two weeks during the administration period. After administration, changes in feeding and behavior were observed 24 hours a day.

[0110] 3. Sample collection and storage Blood sample collection method: Before the blood collection procedure, the animals were fasted overnight without anesthesia. After the animals were acclimatized, they were restrained by gently pressing the back plate, and blood was collected from the forearm vein. After blood collection, the blood collection site was lightly pressed with a sterile dry cotton ball to stop bleeding.

[0111] Urine sample collection method: Animals were fasted overnight before the procedure. On the day of the procedure, they were transferred to metabolic cages, and all urine excreted over 4 to 6 hours was collected in urine collection bags.

[0112] Sample processing: See Table 3-1

[0113] [Table 4]

[0114] 4. Inspection methods and equipment Testing method: See Table 3-2 and Table 3-3 Blood analyzer: Siemens ADVIA 2120i Hematolagy Systems Blood chemistry and urine indicators were tested using the Roche cobas 6000 analyzer series C501 module, and NT-proBNP was tested using an ELISA kit.

[0115] [Table 5]

[0116] [Table 6]

[0117] 5. Blood pressure test Testing method: The animals were anesthetized with an intramuscular injection of 15 mg / kg ketamine hydrochloride, and then placed in a supine position. An appropriate cuff was then fastened according to the standard. A blood oxygen concentration probe was placed on the animal's finger or toe (except the left hand), with the red photosensitive surface placed on the ventral side of the finger. Using the automatic mode, the animal's blood pressure was measured three times consecutively at 1-minute intervals. If the differences between the three blood pressure readings (SBP, DBP, and MBP) were not significant (the difference between the maximum and minimum values ​​was less than 15 mmHg), the measurement was terminated; otherwise, the measurement continued.

[0118] Test indices: Test indices include systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), and heart rate (HR). Testing equipment: GE B40i electrophysiology signal monitor

[0119] 6 Clinical Observations Number of observations: Once a day Observation method: Observation through a cage Observations include: injection site, skin, fur, eyes, ears, nose, mouth, chest, abdomen, genital tract, limbs, etc., as well as breathing, movement, urinary system, defecation, and behavioral changes.

[0120] 7. Weight measurement Weighing time: before feeding on the day Measurement method: The animals were fasted for 14 to 16 hours before weighing. After placing the animals in a conscious state in a transport cage, they were weighed using a large animal scale. Measuring instrument: METTLER TOLEDO electronic platform scale

[0121] 8. Data Processing The experimental results are presented in the form of individual data. Each measurement data is expressed as "Mean ± SD" (mean ± standard deviation). Statistical analysis was performed on the indicators at each time point before and after administration using the paired t-test analysis of variance. P<0.05 indicates a significant difference, and statistical significance is observed.

[0122] 4. Experimental results and discussion 1. Effect on proteinuria (UACR) Urinary protein increases urinary osmolality, adversely affecting the renal concentrating function and damaging glomerular and tubular function. Proteinuria is an independent risk factor for glomerular fibrosis. Therefore, in clinical treatment, controlling UACR can reduce the risk of renal fibrosis and delay progression to end-stage renal failure, making it a very important therapeutic strategy.

[0123] The effects of administration of each group on the urinary albumin excretion rate (UACR) are shown in Figures 1 and 2 and Table 4-1. For each group, DKD rhesus monkeys showing a moderate increase in proteinuria, with a baseline urinary albumin / creatinine ratio (UACR) of 15 mg / g to 350 mg / g (4-6 hours of urine collection), were selected, and changes in UACR between baseline and D30 and D58 were analyzed. The results are shown below.

[0124] Placebo group (n = 4): Compared with baseline, UACR (mg / g) on ​​D58 after the end of the study period increased by an average of 27.16 ± 23.57%, and the UACR levels of four animals fluctuated stably within a certain range and did not show rapid progression.

[0125] Valsartan group (n=4, 3 / 4 animals had moderately severe increases in proteinuria and were included in the statistics): Compared to baseline, UACR on day 30 of administration decreased by an average of approximately 30%, and a significant decrease was maintained until day 58, with an average decrease of 40.71±8.27%, showing a highly significant decrease compared to the placebo group (p<0.01).

[0126] DOX+PMP+MTP1# experimental group (n=4, 4 / 4 animals had moderately severe increases in proteinuria and were included in the statistics): Compared to baseline, UACR on D30 after administration decreased by an average of 50.57±23.86%, showing a significantly decreased value compared to the placebo group (p<0.01). UACR remained significantly decreased until D58, decreasing by an average of 68.75±12.73%, showing a significantly decreased value compared to the placebo group (p<0.01).

[0127] DOX+PMP+MTP2# experimental group (n=4, 3 / 4 animals had moderately severe increases in proteinuria and were included in the statistics): Compared to baseline, UACR on day 30 after administration decreased by an average of 41.56±17.57%, showing a significantly decreased value compared to the placebo group (p<0.01). UACR remained significantly decreased until day 58, decreasing by an average of 42.37±3.50%, showing a significantly decreased value compared to the placebo group (p<0.01).

[0128] In summary, administration of DOX+PMP+MTP for 30 days significantly reduced proteinuria, and the therapeutic effect was maintained up to 58 days after administration, indicating that the therapeutic effect was time-dependent.

[0129] The UACR experimental data for the additional experiment conducted alone (DOX + MTP group vs. placebo group) are summarized in Table 4-1A. Placebo group (n = 3): Compared to baseline, UACR (mg / g) at the end of the test period, D58, increased by an average of 44.26 ± 52.39.

[0130] DOX+MTP group (n=4): Compared to baseline, UACR on D30 after administration increased by a mean of 27.10±75.86 (mg / g), with no statistical difference compared to the placebo group. Compared to baseline, UACR on D58 after administration increased by a mean of 11.44±38.86 (mg / g), with no statistical difference compared to the placebo group.

[0131] Experimental data showed that administration of DOX+MTP for 58 days did not significantly reduce proteinuria in rhesus monkeys with diabetic nephropathy.

[0132] [Table 7]

[0133] [Table 8]

[0134] 2. Effect on glomerular filtration rate (eGFR) The effect on eGFR in each group is shown in Figure 3, Table 4-2, and Table 4-3. eGFR included in each group: 30-59 ml / min / 1.73 m 2 The changes in glomerular filtration rate between baseline and D58 after administration were analyzed.

[0135] Placebo group (n=4): Compared with baseline, the glomerular filtration rate (eGFR) of the four animals fluctuated stably within a certain range and did not show rapid progression.

[0136] In the Valsartan group (n = 4, 3 / 4 animals had an eGFR of 30–59 ml / min / 1.73 m 2 (not included in the statistics): Compared with baseline, the glomerular filtration rate (eGFR) at D58 after administration was an average of 10 ± 7 ml / min / 1.73 m 2 The increase was significant compared to the change in the placebo group (p<0.05), and glomerular deterioration was significantly improved, and the therapeutic effect was time-dependent.

[0137] DOX + PMP + MTP 1# experimental group and 2# experimental group (inclusion eGFR: 30-59 ml / min / 1.73 m 2 , n=5, and the DOX+PMP+MTP1# experimental group and the DOX+PMP+MTP2# experimental group were combined and analyzed as one group): Compared with baseline, the glomerular filtration rate (eGFR) on day 58 of administration was an average of 10±4 ml / min / 1.73 m 2 The increase was significant compared to the change in the placebo group (p<0.01), and the therapeutic effect was time-dependent.

[0138] In summary, in the DOX+PMP+MTP experimental group, glomerular filtration rate (eGFR) was significantly improved on Day 58 of administration.

[0139] [Table 9]

[0140] [Table 10]

[0141] The eGFR experimental data for the additional experiment (DOX+MTP group vs. placebo group) conducted alone is summarized in Table 4-2A. The data in Table 4-2A show that no significant changes in eGFR were observed after 30 and 58 days of DOX+MTP administration compared to the placebo group.

[0142] [Table 11]

[0143] 3. Effects on CysC, Cr-P, and BUN The effects of administration of each group on CysC, Cr-P, and BUN are shown in Tables 4-4 to 4-6. CysC is an endogenous marker that reflects changes in glomerular filtration rate, and Cr-P has been used as a primary evaluation index of renal function for over 40 years. In this study, glomerular filtration rate (eGFR) was calculated from CysC and Cr-P. Although BUN is primarily used as an evaluation index of renal function, it does not meet the requirements as an endogenous GFR marker, and so was used as a secondary indicator in this study.

[0144] [Table 12]

[0145] [Table 13]

[0146] [Table 14]

[0147] 4. Effects on blood pressure The effects on blood pressure of each dose group are shown in Tables 4-7 and 4-8.

[0148] Placebo group (n=4, 2 / 4 hypertensive): Compared with baseline, the blood pressure of 4 animals fluctuated stably within a certain range.

[0149] Valsartan group (n=4, 2 / 4 hypertension): Compared to baseline, on D58 after administration, SBP decreased by an average of 13±6 mmHg, which was significantly lower than that of the placebo group (P<0.05), and DBP decreased by an average of 8±5 mmHg, which was highly significantly lower than that of the placebo group (P<0.01).

[0150] DOX+PMP+MTP1# experimental group (n=4, 2 / 4 hypertensive): Compared to baseline, on day 58 of administration, SBP decreased by an average of 10±3 mmHg, which was significantly lower than that of the placebo group (P<0.05). DBP did not show any hypotensive activity. There was no risk of hypotension in normotensive animals.

[0151] DOX+PMP+MTP2# experimental group (n=4, 2 / 4 hypertensive): Compared to baseline, SBP decreased by an average of 5±5 mmHg on day 58 of administration, which was not statistically significant compared with the placebo group. DBP did not show any hypotensive activity. The blood pressure-lowering activity was low.

[0152] In summary, the DOX+PMP+MTP composition reduced hypertension activity by 5-10 mmHg.

[0153] [Table 15]

[0154] [Table 16]

[0155] 5. Impact on K+ The effects of administration of each administration group on K+ are shown in Table 4-9. Compared with the baseline period, no significant changes were observed in the serum K+ levels of animals in each test group on administration D58. In summary, no risk of hyperkalemia was observed in the DOX+PMP+MTP treatment group due to administration on D58.

[0156] [Table 17]

[0157] 6 Safety and tolerability studies The safe dosage ranges and toxic side effects of commercially available drugs, DOX, PMP, and MTP, are known. DOX tablets are typically administered at 4–8 mg twice daily. The recommended individual dose of PMP is 0.375–4.5 mg daily. For the treatment of idiopathic Parkinson's disease, the starting dose is 0.375 mg daily, followed by a maximum dose of 1.5 mg every 5–7 days. Drowsiness has been associated with increased incidence of drowsiness when daily doses exceed 1.5 mg. The recommended dose of MTP is 50–200 mg daily, increased to 300 mg or 400 mg daily if necessary. Hypotension is a known side effect.

[0158] In this study, the doses of DOX, PMP, and MTP administered in combination to treat rhesus monkeys with chronic diabetic nephropathy were estimated to be equivalent to the doses administered to clinical patients, all of which were much lower than the maximum recommended doses mentioned above. Therefore, theoretically, the triple-drug combination of DOX, PMP, and MTP used in this study is very safe.

[0159] Furthermore, no adverse events related to administration were observed during the administration period, and no significant changes were observed in liver function, renal function, body weight, or biochemical indicators (including FPG, LDL, HDL, TC, TG, ALT, AST, TP, ALB, etc.), demonstrating that the present triple-drug combination of DOX, PMP, and MTP indeed has good biological safety indicators.

[0160] V. Conclusion The above experimental results demonstrated that the treatment of rhesus monkeys with spontaneous diabetic nephropathy with oral administration of three types of GPCR agonists (DOX+PMP+MTP) for 58 days significantly reduced moderate to severe proteinuria, significantly improved the deterioration of glomerular filtration rate, and suppressed the occurrence of end-stage renal failure. Furthermore, the treatment had a good safety and tolerability profile, and no hypertension or fluid retention was observed during the treatment period.

[0161] 6. Pharmaceutical Composition Example 1 A pharmaceutical composition was obtained by uniformly mixing 1.5 mg of doxazosin mesylate (DOX) bulk powder, 0.02 mg of pramipexole dihydrochloride (PMP) bulk powder, and 8 mg of metoprolol tartrate (MTP) bulk powder for a 10 kg monkey. The pharmaceutical composition of the present application may be fed to animals by directly mixing it with their food, or may be formulated into oral tablets by mixing with appropriate excipients and additives.

[0162] Although the above has specifically described specific embodiments, it will be understood that a person skilled in the art of the present application can appropriately change and modify the above embodiments based on the disclosure and teachings of the above specification. Therefore, the present application is not limited to the specific embodiments disclosed and described above, and any modifications and changes to the present application will also fall within the patentable scope of the claims of the present application.

Claims

1. A pharmaceutical composition for treating diabetic nephropathy, comprising: The active ingredients include doxazosin or a pharmaceutically acceptable salt thereof, pramipexole or a pharmaceutically acceptable salt thereof, and metoprolol or a pharmaceutically acceptable salt thereof; the doxazosin or pharmaceutically acceptable salt thereof has a daily dosage range of 2 to 4 mg; The pramipexole or pharmaceutically acceptable salt thereof has a daily dosage range of 0.0625 to 0.125 mg; The metoprolol or pharmaceutically acceptable salt thereof has a daily dosage range of 10 to 50 mg. Pharmaceutical compositions.

2. comprising 0.5 to 45 parts by weight of pramipexole or a pharmaceutically acceptable salt thereof, 5 to 160 parts by weight of doxazosin or a pharmaceutically acceptable salt thereof, and 50 to 2000 parts by weight of metoprolol or a pharmaceutically acceptable salt thereof; The pharmaceutical composition of claim 1.

3. A composition comprising 1 to 10 parts by weight of pramipexole or a pharmaceutically acceptable salt thereof, 5 to 80 parts by weight of doxazosin or a pharmaceutically acceptable salt thereof, and 100 to 1000 parts by weight of metoprolol or a pharmaceutically acceptable salt thereof. The pharmaceutical composition of claim 2.

4. an oral dosage form or an intravenous dosage form; The pharmaceutical composition according to any one of claims 1 to 3.

5. 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 or p-toluenesulfonate; The pharmaceutical composition according to any one of claims 1 to 3.

6. 1. Use of a combination of doxazosin or a pharmaceutically acceptable salt thereof, pramipexole or a pharmaceutically acceptable salt thereof, and metoprolol or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition, comprising: The pharmaceutical composition is used to treat diabetic nephropathy in a subject in need thereof; the doxazosin or pharmaceutically acceptable salt thereof has a daily dosage range of 2 to 4 mg; The pramipexole or pharmaceutically acceptable salt thereof has a daily dosage range of 0.0625 to 0.125 mg; The metoprolol or pharmaceutically acceptable salt thereof has a daily dosage range of 10 to 50 mg. use.

7. The pharmaceutical composition is in an oral dosage form or an intravenous dosage form.

7. The use according to claim 6.

8. The pharmaceutical composition is suitable for use in primate subjects.

7. The use according to claim 6.

9. The pharmaceutical composition is suitable for human subjects.

9. The use according to claim 8.

10. 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 or p-toluenesulfonate; Use according to any one of claims 6 to 9.

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