Methods and pharmaceutical compositions for treating chronic kidney disease

Abstract

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

Description

Technical Field

[0001] The present invention relates to the fields of disease treatment and drugs, and particularly to the treatment of chronic kidney disease, especially diabetic kidney disease. Background Art

[0002] Chronic Kidney Disease (CKD) is a disease that seriously affects human health, especially the health of the elderly. According to statistics, the prevalence of CKD in American adults has reached as high as 11.3%, while the estimated prevalence of CKD in China is about 10%. Diabetes and hypertension are considered the main inducing factors of CKD.

[0003] Diabetes is one of the common diseases that affect human health. In 2015, there were nearly 420 million diabetic patients globally. It is estimated that the number of diabetic patients will reach 640 million by 2040, and it is expected that 20% - 40% of diabetic patients may develop diabetic kidney disease. Diabetic kidney disease is a major complication of diabetes and is also known as DKD (Diabetic Kidney Disease) or DN (Diabetic Nephropathy) in the literature. Diabetic kidney disease is a pathological change of abnormal structure and function of the kidney caused by chronic microvascular lesions of diabetes. The clinical manifestations are mainly hypertension, proteinuria, edema, etc. The pathological manifestations are damage to glomerular blood vessels, sclerosis to form nodular lesions, which then lead to abnormal renal function and persistent proteinuria, and ultimately result in renal failure to form end-stage renal disease (ESRD), with a relatively high fatality rate.

[0004] DKD is a complex disease jointly participated by environmental and genetic factors. The reported mechanisms of its occurrence and development are very complex, involving multiple pathological and pathogenic changes such as oxidative stress, inflammation, renal function injury, renal interstitial fibrosis, hemodynamic changes, and genetic factors. Among them, oxidative stress plays an important role in the pathogenesis and is considered to be closely related to the occurrence and development of DKD.

[0005] Currently, the main DKD management strategies are to achieve optimal blood glucose and blood pressure control, correct lipid metabolism disorders, reverse insulin resistance, and reduce proteinuria (using angiotensin-converting enzyme inhibitors and angiotensin receptor blockers), thereby delaying renal function decline and reducing cardiovascular risk events. Inhibiting the activation of the renin-angiotensin-aldosterone system (RAAS) by applying angiotensin-converting enzyme inhibitor ACEI and / or angiotensin receptor antagonist ARB is the main means of treating DKD currently. Valsartan is a commonly used ARB in clinical practice. It continuously reduces urinary albumin by improving the selective permeability of glomeruli and keeping the radius of glomerular filtration pores unchanged, and does not cause a decrease in glomerular filtration rate (eGFR), and is a commonly used drug for treating DKD currently. However, the existing treatment methods and drugs still perform poorly in terms of preventing and controlling the progression and effectiveness of diabetic nephropathy. Some studies have shown that ARB does not increase the risk of myocardial infarction and can further reduce the risks of heart failure and stroke, but this study also found that ARB cannot reduce the all-cause mortality of the included patients. At the same time, for diabetic patients with normal blood pressure and no proteinuria, using ACEI or ARB drugs cannot prevent the occurrence of microalbuminuria.

[0006] Therefore, there is an urgent clinical need to develop new drugs that can effectively treat CKD and / or DKD and effectively delay the development of CKD / DKD to ESRD. Summary of the Invention

[0007] Therefore, an object of the present invention is to provide a better or alternative treatment method and drug for treating chronic kidney disease (especially diabetic nephropathy).

[0008] The inventors surprisingly found that the combination of doxazosin, pramipexole and metoprolol has significant efficacy in the treatment of chronic kidney disease, especially diabetic nephropathy.

[0009] Therefore, the first aspect of the present invention relates to a pharmaceutical composition for treating chronic kidney disease, which comprises doxazosin (or its pharmaceutically acceptable salt), pramipexole or its pharmaceutically acceptable salt, and metoprolol (or its pharmaceutically acceptable salt) as active ingredients.

[0010] The second aspect of the present invention relates to the use of the combination of doxazosin (or its pharmaceutically acceptable salt), pramipexole (or its pharmaceutically acceptable salt), and metoprolol (or its pharmaceutically acceptable salt) in the preparation of a pharmaceutical composition for treating chronic kidney disease, especially diabetic nephropathy, in a subject in need thereof.

[0011] The third aspect of the present invention 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 kidney disease, especially diabetic kidney disease, in a subject in need thereof.

[0012] The fourth aspect of the present invention relates to a method for treating chronic kidney disease, especially diabetic kidney disease, the method comprising administering a therapeutically effective amount of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof), and metoprolol (or a pharmaceutically acceptable salt thereof) to a subject in need thereof.

[0013] The inventors used spontaneously occurring chronic diabetic nephropathy rhesus monkeys as an animal model to simulate human DKD patients and unexpectedly found that the combination of doxazosin, pramipexole, and metoprolol can significantly reduce proteinuria and significantly improve glomerular filtration rate in the animal model, thereby controlling or delaying the development of DKD to end-stage renal disease (ESRD), and no drug-related adverse reactions were observed during the administration period, showing good safety. Therefore, the drug combination of the present invention represents a novel therapy for chronic kidney disease, especially diabetic kidney disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following will, with reference to the accompanying drawings, elaborate on the specific embodiments of the present invention, where:

[0015] Figure 1 Shows the comparison of the changes in albuminuria (UACR, urinary albumin excretion rate) of each group of rhesus monkeys after 30 days (D30) of drug administration treatment (**, p < 0.01 compared with the placebo group; *, p < 0.05 compared with the placebo group);

[0016] Figure 2 Shows the comparison of the changes in albuminuria (UACR, urinary albumin excretion rate) of each group of rhesus monkeys after 58 days (D58) of drug administration treatment (**, p < 0.01 compared with the placebo group; *, p < 0.05 compared with the placebo group);

[0017] Figure 3 Shows the changes in eGFR of each group of rhesus monkeys after drug administration (the selected eGFR of each group: 30 - 59 ml / min / 1.73m 2 for analysis, the DOX + PMP + MTP1# experimental group and the DOX + PMP + MTP2# experimental group were combined into one group for analysis; *, p < 0.05 for the DOX + PMP + MTP group compared with the placebo group). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] The present application relates to combinations of various GPCR drugs and corresponding combination treatment methods.

[0019] G protein (guanine nucleotide-binding protein)-coupled receptors (GPCRs) are a large class of transmembrane proteins that regulate intracellular signal transduction and are essential for intracellular homeostasis. Previous studies have shown that the GPCR signaling pathway is associated with diabetes-induced peroxide production (Du, Y., et al., Adrenergic and serotonin receptors affect reti / l superoxide generation in diabetic mice: Relationship to capillary degeneration and permeability. 2015. 29(5): p. 2194.). Although oxidative stress is an important mechanism in the development of DKD, and GPCR drugs that regulate diabetes-induced peroxide production may theoretically have a certain impact on the development of DKD, current research is limited to the pathogenesis level, and the therapeutic effect of GPCR drugs on DKD remains to be studied. In addition, GPCR drugs cover hundreds of different substances, and it is extremely difficult to screen out drugs that are clinically effective for DKD.

[0020] In addition, most current 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, due to the complex pathogenesis of diabetic nephropathy, which involves multiple links, it is difficult for these animal models to accurately reflect the course of DKD in patients and to be used to evaluate the effectiveness of disease treatment methods. Non-human primates are extremely similar to humans in terms of physiology, biochemistry, and systems biology. There have been many research reports that the disease characteristics of spontaneously chronic diabetic nephropathy rhesus monkeys are very similar to those of clinical DKD patients, also showing characteristics such as moderately to severely increased urinary albumin and decreased eGFR. Therefore, spontaneously chronic diabetic nephropathy rhesus monkeys provide an important research tool for CKD / DKD research and new drug development (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).

[0021] Considering the limitations of rodent animal models in studying human diabetic nephropathy, the inventors of the present invention used spontaneously chronic diabetic nephropathy rhesus monkeys as animal models to simulate human patients and conducted a large number of detailed studies and extensive screenings on many GPCR drugs. Unexpectedly, it was found that certain combinations of GPCR drugs have obvious curative effects on DKD. Specifically, it has been found that the combination of doxazosin, pramipexole, and metoprolol can significantly reduce proteinuria (UACR) in a rhesus monkey animal model with stage G3 of CKD-EPI rating and moderate to severe proteinuria, significantly improve the glomerular filtration rate eGFR, and inhibit the development of DKD to end-stage renal disease (ESRD), and its curative effect is comparable to that of valsartan, a commonly used first-line drug in clinical practice. Based on this, the present application discloses a pharmaceutical composition for treating chronic kidney disease, especially DKD, and its medical uses.

[0022] I. Pharmaceutical Composition

[0023] The first aspect of the present invention provides a pharmaceutical composition for treating chronic kidney disease, especially diabetic nephropathy, which comprises 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.

[0024] 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.

[0025] The terms "comprising", "including", "having" are used in an inclusive, open sense, which means that additional elements may be included in addition to those specified. The terms "such as", "for example" used herein are non-restrictive and are for illustrative purposes only. "Including" and "including but not limited to" are used interchangeably.

[0026] Unless the context clearly indicates otherwise, the term "or", as used herein, should be understood to mean "and / or".

[0027] The term "treatment" refers to inhibiting a disease, disorder and / or symptom in a subject, such as hindering its progression; and alleviating a disease, disorder and / or symptom, such as causing the regression of a disease, disorder and / or symptom. Treating a disease or disorder includes improving at least one symptom of a specific disease or disorder, even if the underlying pathophysiology is not affected. In particular, in this application, "treatment" also covers the meaning of reducing the risk of onset of a certain disease by administering drugs, that is, "treatment" includes both prevention before onset and remission, inhibition and cure of the disease after onset.

[0028] The term "pharmaceutical composition" refers to a combination of various substances having a specific medical or biological use, and is generally expected to have a therapeutic or preventive effect on a specific disease after being administered to a subject. A pharmaceutical composition may contain only the specified active ingredient (bioactive substance), or 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 ingredient (and optionally a pharmaceutically acceptable carrier) can be mixed together and provided in the form of a mixture in which the components are indistinguishable. As another implementation of the pharmaceutical composition of the present invention, the specified active ingredients can also be individually packaged in small portions and then combined and accommodated in a larger container to provide the "pharmaceutical composition" of the present invention.

[0029] The terms "active ingredient" and "bioactive substance" refer to molecules and other reagents of biological, physiological or pharmaceutical active substances that act on treating a disease or disorder (such as diabetic nephropathy) in a patient or subject. This term is used relative to terms such as "pharmaceutically acceptable carrier", "excipient", "adjuvant", etc. "Active ingredient" (or "bioactive substance") includes but is not limited to its pharmaceutically acceptable salts and prodrugs. These reagents 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 bioactivated when administered to a patient or subject.

[0030] As used herein, the term "doxazosin" refers to the molecule with the English name Doxazosin (abbreviated as DOX), IUPAC name (RS)-2-[4-(2,3-dihydro-1,4-benzodioxin-2-carbonyl)piperazin-1-yl]-6,7-dimethoxy-4-amine (molecular formula C 23 H 25 N 5 O 5 , molecular weight: 451.475 g / mol) as follows:

[0031]

[0032] . The term "doxazosin" also encompasses its isotopically labeled compounds, or its optical isomers, geometric isomers, tautomers or mixtures of isomers, or its prodrugs (i.e., compounds that yield the above molecules through in vivo reactions).

[0033] Doxazosin is an approved marketed drug by major pharmaceutical regulatory agencies (such as the FDA). It is a selective α1 receptor antagonist that inhibits the binding of norepinephrine (released from sympathetic nerve endings) to α-1 receptors on the cell membrane of vascular smooth muscle and is commonly used to treat essential hypertension.

[0034] The pharmaceutical composition of the present invention may contain doxazosin or its pharmaceutically acceptable salt as an active ingredient. Commercially available doxazosin drugs are mostly in the form of salts, especially its mesylate salt, such as the controlled-release tablets of doxazosin mesylate (Cardura) provided by Pfizer Pharmaceuticals, the tablets of doxazosin mesylate provided by Hangzhou Conba Pharmaceutical Co., Ltd., etc.

[0035] As used herein, the term "pramipexole" refers to the molecule with the English name Pramipexole (abbreviated as PMP), IUPAC name (S)-N6-propyl-4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-diamine (molecular formula C 10 H 17 N 3 S, molecular weight: 211.324 g / mol) as follows:

[0036]

[0037] The term "pramipexole" also encompasses its isotopically labeled compounds, or its optical isomers, geometric isomers, tautomers or mixtures of isomers, or its prodrugs (i.e., compounds that yield the above-mentioned molecules through in vivo reactions).

[0038] Pramipexole is an approved marketed drug by major pharmaceutical regulatory agencies (such as the FDA). It is an antihistamine and, as a dopamine receptor D2 / D3 agonist, is mainly used clinically for the treatment of Parkinson's disease, either alone (without levodopa) or in combination with levodopa. In the literature, pramipexole is sometimes also referred to as "Mirapexin" or "Mirapex", "Mirapexin", "Sifrol", etc.

[0039] The pharmaceutical composition of the present invention may contain pramipexole or a pharmaceutically acceptable salt thereof as an active ingredient. Commercially available drugs of pramipexole are mostly in the form of salts, especially its hydrochloride salt, such as pramipexole hydrochloride tablets (Sifrol) provided by Boehringer Ingelheim Pharmaceuticals, Inc., etc.

[0040] The term "metoprolol" as used herein refers to the molecule with the English name Metoprolol (abbreviated as MTP), IUPAC name (RS)-1-[4-(2-methoxyethyl)phenoxy]-3-[(propan-2-yl)amino]propan-2-ol (molecular formula C 15 H 25 NO 3 , molecular weight 267.37 g / mol):

[0041]

[0042] The term "metoprolol" also encompasses its isotopically labeled compounds, or its optical isomers, geometric isomers, tautomers or mixtures of isomers, or its prodrugs (i.e., compounds that yield the above-mentioned molecules through in vivo reactions).

[0043] Metoprolol is an approved marketed drug by major pharmaceutical regulatory agencies (such as the FDA). It is a selective beta-1 adrenergic receptor blocker and is commonly used for the treatment of hypertension and angina. The pharmaceutical composition of the present invention may contain metoprolol or a pharmaceutically acceptable salt thereof as an active ingredient. Commercially available drugs of metoprolol are mostly in the form of salts, especially tartrate salts, such as metoprolol tartrate tablets (Betaloc) provided by AstraZeneca Pharmaceuticals Co., Ltd., metoprolol tartrate controlled-release tablets (Lijunning) provided by Guangzhou Baiyunshan Tianxin Pharmaceutical Co., Ltd., etc.

[0044] The active ingredients (doxazosin, pramipexole, metoprolol) contained in the composition of the present invention can all 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 salts used herein are salts formed with pharmaceutically acceptable acids or bases. 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. For the avoidance of doubt, the "pharmaceutically acceptable salts" of a certain active ingredient described in the present invention also include pharmaceutically acceptable salts formed from isotopically labeled compounds of the active ingredient, or its optical isomers, geometric isomers, tautomers or mixtures of isomers, or its prodrugs.

[0045] Pharmaceutically acceptable salts can be synthesized from the parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, these salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both; generally, 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, the salts can include, but are not limited to, hydrochloride, tartrate, mesylate, etc. of the compounds of the present invention.

[0046] It should be understood that all references to the various active ingredients or pharmaceutically acceptable salts include solvate addition forms (solvates, such as hydrates, ethanol solvates, acetone solvates, etc.) of the same active ingredient or salt or its various crystal forms (such as amorphous, polymorphs, etc.).

[0047] The contents 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, according to the administration mode of the pharmaceutical composition (oral or injection, etc.), the content or ratio of each drug in the pharmaceutical composition can be changed.

[0048] In some embodiments, the pharmaceutical composition of the present invention generally comprises 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.

[0049] In the pharmaceutical composition of the present invention, the amount of pramipexole or a pharmaceutically acceptable salt thereof is generally 0.5 to 45 parts by weight, for example, it can be 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, 1 to 15 parts by weight, 1 to 10 parts by weight, 2 to 45 parts by weight, 2 to 40 parts by weight, 2 to 35 parts by weight, 2 to 30 parts by weight, 2 to 25 parts by weight, 2 to 20 parts by weight, 2 to 15 parts by weight, 2 to 10 parts by weight, 5 to 45 parts by weight, 5 to 40 parts by weight, 5 to 35 parts by weight, 5 to 30 parts by weight, 5 to 25 parts by weight, 5 to 20 parts by weight, 5 to 15 parts by weight, 5 to 10 parts by weight, 10 to 40 parts by weight, 10 to 30 parts by weight, 10 to 25 parts by weight, etc. In a preferred embodiment of the present invention, the content of pramipexole or a pharmaceutically acceptable salt thereof is 1 to 10 parts by weight.

[0050] In the pharmaceutical composition of the present invention, the usual amount of doxazosin or its pharmaceutically acceptable salt is 5 parts by weight to 160 parts by weight, for example, it can be 5 parts by weight to 150 parts by weight, 5 parts by weight to 130 parts by weight, 5 parts by weight to 120 parts by weight, 5 parts by weight to 100 parts by weight, 5 parts by weight to 80 parts by weight, 5 parts by weight to 60 parts by weight, 5 parts by weight to 50 parts by weight, 5 parts by weight to 40 parts by weight, 10 parts by weight to 150 parts by weight, 10 parts by weight to 130 parts by weight, 10 parts by weight to 120 parts by weight, 10 parts by weight to 100 parts by weight, 10 parts by weight to 80 parts by weight, 10 parts by weight to 60 parts by weight, 10 parts by weight to 50 parts by weight, 10 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 invention, the content of doxazosin or its pharmaceutically acceptable salt is 5 parts by weight to 80 parts by weight.

[0051] In the pharmaceutical composition of the present invention, the amount of metoprolol or its pharmaceutically acceptable salt is generally 50 parts by weight to 2000 parts by weight, for example, it can be 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 800 parts by weight, 50 parts by weight to 600 parts by weight, 50 parts by weight to 500 parts by weight, 50 parts by weight to 400 parts by weight, 100 parts by weight to 1800 parts by weight, 100 parts by weight to 1600 parts by weight, 100 parts by weight to 1500 parts by weight, 100 parts by weight to 1300 parts by weight, 100 parts by weight to 1200 parts by weight, 100 parts by weight to 1000 parts by weight, 100 parts by weight to 800 parts by weight, 100 parts by weight to 600 parts by weight, 100 parts by weight to 500 parts by weight, 100 parts by weight to 400 parts by weight, 150 parts by weight to 2000 parts by weight, 150 parts by weight to 1800 parts by weight, 150 parts by weight to 1600 parts by weight, 150 parts by weight to 1500 parts by weight, 150 parts by weight to 1300 parts by weight, 150 parts by weight to 1200 parts by weight, 150 parts by weight to 1000 parts by weight, 150 parts by weight to 800 parts by weight, 150 parts by weight to 600 parts by weight, 200 parts by weight to 2000 parts by weight, 200 parts by weight to 1800 parts by weight, 200 parts by weight to 1600 parts by weight, 200 parts by weight to 1500 parts by weight, 200 parts by weight to 1300 parts by weight, 200 parts by weight to 1200 parts by weight, 200 parts by weight to 1000 parts by weight, 200 parts by weight to 800 parts by weight, 200 parts by weight to 600 parts by weight, etc. In a preferred embodiment of the present invention, the content of doxazosin or its pharmaceutically acceptable salt is 100 parts by weight to 1000 parts by weight.

[0052] II. Pharmaceutical Dosage Form

[0053] In some embodiments, the pharmaceutical composition of the present invention may be provided in the form of an active pharmaceutical ingredient (e.g., a homogeneous mixture or individual packages of the components). In other embodiments, the pharmaceutical composition of the present invention may 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 may be used as the "pharmaceutically acceptable carrier". In the present application, the phrase "pharmaceutically acceptable" means a compound, composition, polymer and other materials that are compatible with other components of the composition of the present application within the scope of reasonable medical judgment, and are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications. In certain 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 carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) maltodextrin; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, sunflower seed oil, sesame oil, olive oil, corn oil and soybean oil; (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) buffering agents, 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 and compatible substances used in pharmaceutical preparations.

[0054] The pharmaceutical composition of the present invention can be formulated into dosage forms suitable for administration routes such as oral administration, parenteral (including subcutaneous, intramuscular, intracutaneous and intravenous) administration, bronchial administration, intravitreal injection or nasal administration as needed. Among them, preferably, the pharmaceutical composition of the present invention is formulated into a dosage form (preparation) suitable for oral administration.

[0055] If a solid carrier is used, the preparation can be in the form of tablets, placed in hard gel capsules in the form of powder or granules, or in the form of troches or lozenges. The solid carrier can include conventional excipients such as binders, fillers, tablet lubricants, disintegrants, wetting agents, and the like. If desired, the tablets can be film-coated by conventional techniques. If a liquid carrier is used, the preparation can be in the form of syrup, emulsion, soft gel capsules, a sterile carrier for injection, an aqueous or non-aqueous liquid suspension, or can be a dry product to be reconstituted with water or other suitable carrier before use. The liquid preparation can contain conventional additives such as suspending agents, emulsifying agents, wetting agents, non-aqueous carriers (including edible oils), preservatives, and flavoring and / or coloring agents. For parenteral administration, usually the carrier consists at least mostly of sterile water, but saline solutions, glucose solutions, etc. can also be used. Injectable suspensions can also be used, in which case conventional suspending agents can be used. Conventional preservatives, buffering agents, etc. can also be added to parenteral dosage forms.

[0056] Dosage forms suitable for parenteral injection can 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).

[0057] These pharmaceutical dosage forms can also contain various excipients, e.g., preservatives, wetting agents, emulsifying agents and dispersing agents. The inhibition of the action of microorganisms can be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). Isotonic agents such as sugars, sodium chloride, etc. can also be included. The absorption of injectable pharmaceutical dosage forms can be prolonged by using agents for delayed absorption (e.g., aluminum monostearate and gels).

[0058] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert excipient (or carrier) (e.g., sodium citrate or calcium phosphate dibasic), which may also include: (a) fillers or bulking agents (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid); (b) binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia); (c) humectants (e.g., glycerol); (d) disintegrants (e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain synthetic silicates, and sodium carbonate); (e) solution retarders (e.g., paraffin); (f) absorption promoters (e.g., quaternary ammonium compounds); (g) wetting agents (e.g., cetyl alcohol and glyceryl 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.

[0059] Similar types of solid compositions can also be used as fillers in soft and hard gelatin capsules using, for example, lactose and high molecular weight polyethylene glycols as excipients.

[0060] 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). They can contain opacifying agents and 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 embedding compositions that can be used are polymeric substances and waxes. The active ingredient can also be in microencapsulated form and, if appropriate, can have one or more of the above excipients.

[0061] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, dispersions, syrups, and elixirs. In addition to the active compound, the liquid dosage forms can contain inert diluents commonly used in the art (e.g., water or other solvents), solubilizers, and emulsifying agents (e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide), oils (specifically, cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan or mixtures of these substances.

[0062] In addition to these inert diluents, the pharmaceutical dosage forms can also include, for example, wetting agents, emulsifying and suspending agents, perfuming agents, flavoring agents, and sweetening agents.

[0063] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecanol, polyethylene oxide sorbitol, sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth gum, or mixtures of these substances.

[0064] The pharmaceutical dosage forms of the present invention also include ointments, powders, sprays, and inhalants. The active ingredient is mixed with a physiologically acceptable carrier and any required preservatives, buffers, or propellants under sterile conditions.

[0065] The amount of the active ingredient in the pharmaceutical composition and pharmaceutical dosage form can be appropriately determined by those skilled in the art according to needs. For example, each active ingredient is usually present in the pharmaceutical composition or dosage form in a therapeutically effective amount.

[0066] For example, the pharmaceutical composition of the present invention can be formulated into an oral dosage form (such as an oral dosage form taken 2 to 3 times a day or a long-acting sustained-release oral preparation), an intravenous injection dosage form, or a intramuscular injection dosage form.

[0067] 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 an oral dosage form taken once a day, an oral dosage form taken 2 to 3 times a day, or a long-acting sustained-release oral preparation.

[0068] 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 an oral dosage form taken daily.

[0069] III. Uses of the Pharmaceutical Composition and the Pharmaceutical Dosage Form

[0070] It has been found that the pharmaceutical composition and pharmaceutical dosage form of the present invention can be used to treat chronic kidney disease (CKD), especially diabetic nephropathy (DN or DKD).

[0071] Therefore, a second aspect of the present invention 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 preparation of a pharmaceutical composition for treating chronic kidney disease, especially diabetic nephropathy, in a subject in need thereof.

[0072] A third aspect of the present invention 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 treating chronic kidney disease, especially diabetic nephropathy, in a subject in need thereof.

[0073] The fourth aspect of the present invention relates to a method for treating chronic kidney disease, particularly diabetic nephropathy, the method comprising administering to a subject in need thereof a therapeutically effective amount of doxazosin (or a pharmaceutically acceptable salt thereof), pramipexole (or a pharmaceutically acceptable salt thereof), and metoprolol (or a pharmaceutically acceptable salt thereof).

[0074] The pharmaceutical compositions and dosage forms of the present invention are suitable for the treatment of diabetic nephropathy at all stages and are also suitable for prophylactic treatment before the onset of the disease.

[0075] A "patient" or "subject" treated by the method of the present invention may refer to a human or non-human animal, such as a primate. Preferably, the "patient" or "subject" is a human.

[0076] The treatment method of the present invention involves the combined use of multiple active ingredients, also known as "combination treatment" or "combination therapy". The "combined use of drugs" or "combination treatment" refers to the administration of the multiple active ingredients described in the present invention so that they act together to provide beneficial effects. The beneficial effects of the above combinations include, but are not limited to, the combined effects in terms of pharmacokinetics or pharmacodynamics produced by the combination of the above active ingredients. The combined administration of these active ingredients is usually completed within a specified period of time (usually minutes, hours, days or weeks, depending on the doctor's judgment). "Combined use of drugs" or "combination treatment" is intended to include the administration of these active ingredients in a sequential manner, that is, each of these active ingredients is administered at different times, 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 to the host a single capsule containing a fixed ratio of each active ingredient, or by administering to the host multiple capsules, each capsule containing one of the active ingredients. The sequential administration or substantially simultaneous administration of each active ingredient may be affected by any suitable route, including, but not limited to, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue. The active ingredients may be administered by the same route or 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. Or, for example, all the active ingredients may be administered orally, or all the active ingredients may be administered by intravenous injection. The order of administration of these active ingredients is not strictly limited.

[0077] "Combined therapy" or "combination treatment" also includes co-administering the active ingredients described above further with other bioactive ingredients and non-pharmacological treatments (e.g., surgical treatment or mechanical treatment). When the combined treatment further includes non-pharmacological treatment, the non-pharmacological treatment can be carried out at any appropriate time, as long as the beneficial effects produced by the combined action of the active ingredients and the non-pharmacological treatment can be achieved. For example, in appropriate circumstances, the beneficial effects can still be achieved when the non-pharmacological treatment is temporarily stopped after the administration of the active ingredients, where the stop may be for several days or even several weeks.

[0078] To achieve the desired effect, a therapeutically effective amount of the pharmaceutical composition or dosage form of the present invention or the individual active ingredients alone is usually administered to a patient or subject.

[0079] The phrase "therapeutically effective amount" is a term well recognized in the art. In certain embodiments, the term refers to the amount necessary or sufficient to eliminate, reduce, or maintain the target of a particular treatment regimen. The effective amount can vary depending on factors such as the disease or disorder being treated, the particular targeting construct being administered, the size of the subject, or the severity of the disease or disorder. A person of ordinary skill in the art or a physician can determine the effective amount of a particular compound empirically without undue experimentation. In certain embodiments, the therapeutically effective amount of a therapeutic agent used in vivo may depend on many factors, including: the mode and method of administration; any other materials included in the drug in addition to the agent. In vitro or in vivo assays can optionally be used to assist in determining the optimal dosage range.

[0080] In some embodiments of the present invention, for an adult with a normal body weight of about 60 kilograms, when taking the pharmaceutical dosage form or pharmaceutical composition of the present invention orally, the daily dose range of doxazosin or its pharmaceutically acceptable salt can be from 0.5 mg to 16 mg, for example, it can be from 0.5 mg to 15 mg, from 0.5 mg to 13 mg, from 0.5 mg to 12 mg, from 0.5 mg to 10 mg, from 0.5 mg to 8 mg, from 0.5 mg to 6 mg, from 0.5 mg to 5 mg, from 0.5 mg to 4 mg, from 1 mg to 15 mg, from 1 mg to 13 mg, from 1 mg to 12 mg, from 1 mg to 10 mg, from 1 mg to 8 mg, from 1 mg to 6 mg, from 1 mg to 5 mg, from 1 mg to 4 mg, from 1.5 mg to 16 mg, from 1.5 mg to 15 mg, from 1.5 mg to 13 mg, from 1.5 mg to 12 mg, from 1.5 mg to 10 mg, from 1.5 mg to 8 mg, from 1.5 mg to 6 mg, from 1.5 mg to 5 mg, from 1.5 mg to 4 mg, from 2 mg to 16 mg, from 2 mg to 15 mg, from 2 mg to 13 mg, from 2 mg to 12 mg, from 2 mg to 10 mg, from 2 mg to 8 mg, from 2 mg to 6 mg, from 2 mg to 5 mg, from 2 mg to 4 mg, etc. In a preferred embodiment of the present invention, the daily dose range of doxazosin or its pharmaceutically acceptable salt of doxazosin can be about 2 mg. In another preferred embodiment of the present invention, the daily dose of doxazosin is about 4 mg..

[0081] In some embodiments of the present invention, for an adult with a normal body weight of about 60 kilograms, when the pharmaceutical dosage form or pharmaceutical composition of the present invention is taken orally, the daily dose range of pramipexole or a pharmaceutically acceptable salt thereof can be from 0.05 mg to 4.5 mg, for example, it can be from 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 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, 1 mg to 2.5 mg, etc. In a preferred embodiment of the present invention, 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 invention, the daily dose of pramipexole is about 0.0625 mg. In another preferred embodiment of the present invention, the daily dose of pramipexole is about 0.125 mg.

[0082] 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 range of metoprolol or a pharmaceutically acceptable salt thereof can be 5 mg to 200 mg, for example, it can be 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 to 120 mg, 10 mg to 100 mg, 10 mg to 80 mg, 10 mg to 60 mg, 10 mg to 50 mg, 10 mg to 40 mg, 15 mg to 200 mg, 15 mg to 180 mg, 15 mg to 160 mg, 15 mg to 150 mg, 15 mg to 130 mg, 15 mg to 120 mg, 15 mg to 100 mg, 15 mg to 80 mg, 15 mg to 60 mg, 20 mg to 200 mg, 20 mg to 180 mg, 20 mg to 160 mg, 20 mg to 150 mg, 20 mg to 130 mg, 20 mg to 120 mg, 20 mg to 100 mg, 20 mg to 80 mg, 20 mg to 60 mg. In a preferred embodiment of the present invention, the daily dose range of metoprolol or a pharmaceutically acceptable salt thereof is 10 mg to 100 mg. In a 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 40 mg.

[0083] The above-mentioned daily dose can be administered continuously 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 patient in the form of 2 to 3 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 pharmacokinetics of each active ingredient in the body.

[0084] Those skilled in the art can understand that when the pharmaceutical composition of the present invention is formulated into other dosage forms suitable for intravenous drip or intramuscular injection, etc., the dose ranges of the respective active ingredients may be different from the oral dose ranges given above. Those skilled in the art or doctors can reasonably determine it by combining in vivo and in vitro experiments and considering the different pharmacokinetic characteristics of various administration routes.

[0085] In a preferred embodiment of the present invention, the pharmaceutical composition of the present invention is used for primate subjects, especially human subjects.

[0086] Those skilled in the art can understand that various aspects of the present invention described herein can be combined separately in various ways that are clear to those skilled in the art without departing from the subject matter and ideas of this application. Such combinations are also within the scope of this application. For example, the dosage ranges of certain components involved in the present invention include any combination of any lower limit and any upper limit mentioned in the specification, and also include any range formed by combining the specific content of this component in each specific embodiment as the upper limit or lower limit; all 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.

[0087] Example

[0088] The inventors used spontaneously chronic diabetic nephropathy rhesus monkeys as experimental animals to verify the effectiveness and drug safety tolerance of the combined use of three marketed GPCR signaling pathway drugs, doxazosin (DOX), pramipexole (PMP), and metoprolol (MTP), for diabetic nephropathy through experiments. In the experiment, valsartan, a commonly used first-line drug for DKD currently, was used as a positive control.

[0089] I. Experimental Materials

[0090] Specifically, the following three marketed drugs were used as test articles in the experiment:

[0091]

[0092] Note: The dosage of monkeys can be converted with the dosage of humans using the body surface area method or in vivo drug concentration exposure.

[0093] Test article 1:

[0094] Name or abbreviation (English name): Doxazosin mesylate (DOX)

[0095] Purity: 98% HPLC

[0096] Manufacturer: Shanghai Ziqi Biotechnology Co., Ltd.

[0097] Test article 2:

[0098] Name or abbreviation (English name): Pramipexole 2HCL Monohydrate (PMP)

[0099] Purity: 99.55% HPLC

[0100] Manufacturer: Shanghai Ziqi Biotechnology Co., Ltd.

[0101] Test article 3

[0102] Name or Abbreviation (English name): Metoprolol tartrate salt, MTP

[0103] Purity: 98% HPLC

[0104] Manufacturer: Aladdin (Shanghai) Reagent Co., Ltd.

[0105] Positive control drug: Valsartan

[0106] Name or Abbreviation (English name): Valsartan

[0107] Manufacturer: Shanghai Bide Pharmaceutical Technology Co., Ltd.

[0108] II. Specific Experimental Protocols

[0109] Experimental animals:

[0110] Animal species: Rhesus Macaque, Macaca mulatta.

[0111] Grade: Ordinary grade. Quarantined and passed before the experiment, including physical examination, 2 tuberculosis tests, parasites, Salmonella, Shigella and B virus examinations.

[0112] Animal identification: Wear a stainless steel number plate engraved with Arabic numerals on the neck ring and have a chest tattoo

[0113] Supplier: Ya'an Prime Biotech Co., Ltd.

[0114] Production license number: SCXK(Sichuan)2019 - 027

[0115] The experimental system follows the requirements of AAALAC and GLP standards

[0116] Feeding environment:

[0117] Environmental grade: 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, turning off the lights at 7:30 PM and turning on the lights at 7:30 AM the next day. Animal cages: Double - layer stainless steel cages of 850*900*2365mm. Feeding density: 1 animal per cage.

[0118] A total of 16 Rhesus Macaques with spontaneous chronic diabetic kidney disease (DKD) were enrolled in the experiment. The inclusion criteria are as follows:

[0119] 1) 16 males / females, aged 14 to 24 years (equivalent to 40 to 75-year-old adults); 14 males, weighing 7 to 13 kg; 2 females: 6 to 8 kg

[0120] 2) Diabetes duration of more than 2 years: Fasting plasma glucose (FPG): greater than 4.8 mmol / L, vs age-matched controls 4.1 ± 0.3 mmol / L;

[0121] 3) CKD-EPI rating G3a to G3b with estimated glomerular filtration rate eGFR: 30 - 59 ml / min / 1.73m 2 vs age-matched controls 92 ± 11 ml / min / 1.73m 2 ; or moderate to severe increase in proteinuria (A2 to A3) with urinary albumin / creatinine ratio (UACR): 15 mg / g to 350 mg / g (4 to 6-hour urine collection), vs age-matched controls 3 ± 2 mg / g.

[0122] 4) Normal blood pressure, stage 1 or stage 2 hypertension (consistent with clinical patient criteria).

[0123] Exclusion criteria are as follows:

[0124] 1) Severe liver function abnormalities

[0125] 2) Any other diseases that may affect the evaluation of drug efficacy

[0126] Table 1 below shows the underlying causes and characteristics of the spontaneous chronic diabetic nephropathy rhesus monkeys in each group during the baseline period.

[0127]

[0128] Grouping and Administration Regimen:

[0129] This trial set up a DOX + PMP + MTP group (n = 8), a Valsartan group (n = 4), and a placebo group (n = 4). Among them, the DOX + PMP + MTP group was further divided into an experimental group 1# and an experimental group 2#. The specific dosing regimens for each group are described as follows, and the administration route is oral administration. The baseline period is 1 month, followed by continuous oral administration for 58 days.

[0130] Experimental group 1#: Number of animals 4

[0131] D0 to D14: DOX + PMP + MTP: 0.133 + 0.002 + 0.833 mg / kg, twice a day;

[0132] D15 to D28: DOX + PMP + MTP: 0.266 + 0.002 + 1.667 mg / kg, twice daily;

[0133] D29 to D58: DOX + PMP + MTP: 0.266 + 0.004 + 0.833 mg / kg, DOX and MTP drugs twice daily, PMP drug once daily

[0134] Group 2# experimental group: Number of animals: 4

[0135] D0 to D14: DOX + PMP + MTP: 0.133 + 0.002 + 0.833 mg / kg, twice daily

[0136] D15 to D28,: DOX + PMP + MTP: 0.133 + 0.002 + 1.667 mg / kg, twice daily

[0137] D29 to D58: DOX + PMP + MTP: 0.266 + 0.002 + 1.667 mg / kg, twice daily

[0138] Valsartan group: Number of animals: 4, dose: 2.67 mg / kg (equivalent to clinical equivalent dose of 40 to 80 mg), administered daily from D0 to D58; once daily in the first to second week of the administration period, twice daily in the third to eighth week.

[0139] Placebo group: Number of animals: 4, given drinking water or fruits, continuously observed for 58 days.

[0140] III. Main Observation Indicators and Monitoring Methods

[0141] 1. Main pharmacodynamic indicators

[0142] Glomerular filtration rate eGFR: Detect creatinine (Cr-P), blood urea nitrogen (BUN), cystatin (CysC), and calculate the eGFR value. Detect once at the baseline period before drug administration, and at D14, D28, D45, and D58 after drug administration. Referring to the reference (Levey AS, Stevens LA, Schmid CH et al. A new equation to estimate glomerular filtration rate. Ann Intern Med 2009; 150: 604 - 612), the eGFR calculation formula is as follows:

[0143] eGFR male = 135 × min(Cr / 0.9, 1) -0.207 × max(Cr / 0.9, 1) -0.601 × min(CysC / 0.8, 1)-0.375 ×

[0144] max(CysC / 0.8, 1) -0.711 ×0.995 Age×3

[0145] eGFR female = 135 × min(Cr / 0.7, 1) -0.284 × max(Cr / 0.7, 1) -0.601 × min(CysC / 0.8, 1) -0.375 ×

[0146] max(CysC / 0.8, 1) -0.711 ×0.995 Age×3 ×0.969

[0147] Urinary albumin excretion rate UACR: Urine samples were collected over 4 h / 6 h. Microalbuminuria (Malb) and urinary creatinine (Cr-U) were measured, and the UACR value was calculated. Samples were collected once before dosing, once on day 30 after dosing, and once on D58. UACR = Malb / Cr-U.

[0148] Blood pressure: Blood pressure, including systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), and heart rate (HR), was measured after anesthesia. Samples were collected once before dosing and once on day 58 after dosing (at the end of dosing).

[0149] 2. Secondary pharmacodynamic and safety indicators

[0150] Serum potassium: Samples were collected once before dosing and once on D14, D28, D45, and D58 after dosing;

[0151] Glucose and lipid metabolism and liver function: FPG, FRA, LDL-c, HDL-c, TG, TC, ALT, AST, etc. Samples were collected once before dosing and once on D28 and D58 after dosing;

[0152] Hematological indices: Samples were collected once before dosing and once on D28 and D58 after dosing;

[0153] Body weight: Samples were collected once before dosing and once every 2 weeks during the dosing period;

[0154] Food intake, behavior, etc. were observed for 24 hours every day after dosing.

[0155] 3. Sample collection and preservation

[0156] Method for collecting blood samples: Animals were fasted overnight before blood collection without anesthesia. After animal domestication, the backboard was gently squeezed for fixation, and blood was collected from the forearm vein. After collection, the blood collection site was gently pressed with a sterilized dry cotton ball to stop bleeding.

[0157] Urine sample collection method: The animals were fasted overnight before the urine collection operation. On the day of the operation, they were transferred to a metabolic cage, and all urine excreted in 4 to 6 hours was collected into a urine collection bag.

[0158] Sample processing method: See Table 3-1

[0159] Table 3-1 Sample collection and processing information

[0160]

[0161] 4. Detection methods and equipment

[0162] Detection methods: See Table 3-2 and Table 3-3

[0163] Blood routine detection instrument: Siemens ADVIA 2120i Hematolagy Systems

[0164] Blood biochemical and urine index detection instrument: Roche cobas6000 analyzer series C501 module detection, NT-proBNP was detected using an ELISA kit.

[0165] Table 3-2 Biochemical detection items

[0166]

[0167] Table 3-3 Hematological detection items

[0168]

[0169] 5. Blood pressure detection

[0170] Detection method: The animals were anesthetized by intramuscular injection of 15 mg / kg ketamine hydrochloride. After anesthesia, they were placed in a supine position, and a cuff of appropriate size was tied on according to the standard. The blood oxygen probe was clipped on the finger or toe (except the left hand) of the animal, with the red photosensitive surface on the side of the finger pulp. The blood pressure of the animal was continuously detected 3 times in automatic mode, with an interval of 1 min each time. If the differences in SBP, DBP, and MBP of the three blood pressure measurements were not significant (the difference between the highest value and the lowest value was less than 15 mmHg), the measurement was ended; otherwise, the measurement continued.

[0171] Detection indicators: The detection indicators include systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), and heart rate (HR)

[0172] Detection instrument: GE B40i electrophysiological monitor

[0173] 6. Clinical observation

[0174] Observation frequency: Observed once a day.

[0175] Observation method: Observation through the cage partition

[0176] Observation content: Injection site, skin, hair, eyes, ears, nose, oral cavity, chest, abdomen, urogenital part, limbs and other parts, as well as respiration, movement, urination, defecation and behavioral changes, etc.

[0177] 7. Body weight measurement

[0178] Weighing time: Before feeding on the same day

[0179] Measurement method: The animals are fasted for 14 to 16 hours before weighing. In the awake state, after the animals enter the transfer cage, they are weighed with a large animal scale.

[0180] Measuring instrument: METTLER TOLEDO electronic platform scale

[0181] 8. Data processing

[0182] The experimental results are presented in the form of individual data. Each measurement data is expressed as "Mean±SD" (mean ± standard deviation). The paired T-test analysis of variance method is used to perform statistical analysis on the indicators at each time point before and after drug administration. P < 0.05 indicates a significant difference and has statistical significance.

[0183] IV. Experimental Results and Discussions

[0184] 1. Effects on urinary albumin-creatinine ratio (UACR)

[0185] Protein in urine can increase the osmotic pressure of urine, affect the concentrating function of the kidneys, and damage the functions of glomeruli and renal tubules. Proteinuria is an independent risk factor for glomerular fibrosis. Therefore, controlling UACR in clinical treatment to reduce the risk of renal fibrosis and delay the progression of end-stage renal failure is a very important treatment strategy.

[0186] The effects of drug administration in each group on the urinary albumin excretion rate (UACR) are shown in Figure 1 、 Figure 2 and Table 4-1. In each group, DKD rhesus monkeys with moderate to severe increase in proteinuria, manifested as baseline urinary albumin / creatinine ratio (UACR): 15 mg / g to 350 mg / g (urine collected for 4 to 6 hours), were selected to analyze the changes in UACR at baseline, D30 of drug administration, and D58 of drug administration. The results are as follows:

[0187] Placebo group (n = 4): Compared with the baseline, the UACR (mg / g) at the end of the trial period D58 increased by an average of 27.16 ± 23.57%; the UACR levels of 4 animals fluctuated stably within a certain range and did not show rapid progression.

[0188] Valsartan group (n = 4, 3 / 4 with moderate to severe proteinuria increase were included in the statistics): Compared with the baseline, the average UACR decreased by about 30% at D30 of drug administration, and remained significantly decreased to an average UACR decrease of 40.71 ± 8.27% at D58, showing a highly significant decrease compared with the placebo group (p < 0.01).

[0189] DOX + PMP + MTP1# experimental group (n = 4, 4 / 4 with moderate to severe proteinuria increase were included in the statistics): Compared with the baseline, the average UACR decreased by 50.57 ± 23.86% at D30 after drug administration, showing a highly significant decrease compared with the placebo group (p < 0.01), and remained significantly decreased to an average UACR decrease of 68.75 ± 12.73% at D58, showing a highly significant decrease compared with the placebo group (p < 0.01).

[0190] DOX + PMP + MTP2# experimental group (n = 4, 3 / 4 with moderate to severe proteinuria increase were included in the statistics): Compared with the baseline, the average UACR decreased by 41.56 ± 17.57% at D30 after drug administration, showing a highly significant decrease compared with the placebo group (p < 0.01), and remained significantly decreased to an average UACR decrease of 42.37 ± 3.50% at D58, showing a highly significant decrease compared with the placebo group (p < 0.01).

[0191] In summary, administration of DOX + PMP + MTP for 30 days can significantly reduce proteinuria, and the efficacy can be maintained until 58 days after drug administration, showing a time-dependent effect.

[0192]

[0193] 2. Effect on glomerular filtration rate eGFR

[0194] The effects of each group on eGFR are shown in Figure 3 , Table 4-2 and Table 4-3. The selected eGFR for each group was 30 - 59 ml / min / 1.73m 2 , and the changes in glomerular filtration rate at baseline and D58 after drug administration were analyzed.

[0195] Placebo group (n = 4): Compared with the baseline, the glomerular filtration rate eGFR of 4 animals fluctuated stably within a certain range without showing rapid progression.

[0196] Valsartan group (n = 4, 3 / 4 animals with eGFR: 30 - 59 ml / min / 1.73m 2 were included in the statistics): Compared with the baseline, the average glomerular filtration rate eGFR increased by 10 ± 7 ml / min / 1.73m 2 at D58 after drug administration, showing a significant increase compared with the change value of the placebo group (p < 0.05), significantly improving glomerular deterioration, and showing a time-dependent effect.

[0197] DOX + PMP + MTP1 # Experimental group and 2 # Experimental group (enrolled eGFR: 30 - 59 ml / min / 1.73m 2 , counted according to n = 5, the DOX + PMP + MTP1 # Experimental group and the DOX + PMP + MTP2 # Experimental group were combined into one group for analysis): Compared with the baseline, the mean glomerular filtration rate eGFR increased by 10 ± 4 ml / min / 1.73m on D58 after drug administration 2 , showing a highly significant increase compared with the change value of the placebo group (p < 0.01), and the efficacy showed timeliness.

[0198] In summary, the administration of D58 in the DOX + PMP + MTP experimental group can significantly improve the glomerular filtration rate eGFR.

[0199] Table 4 - 2 Effects of DOX + PMP + MTP administered on D58 on eGFR in spontaneously chronic diabetic nephropathy rhesus monkeys

[0200]

[0201] Note: 1. "a" compared with the baseline.

[0202] Table 4-3 DOX+PMP+MT Change value of eGFR in rhesus monkeys with spontaneously chronic diabetic nephropathy after P administered on D58

[0203]

[0204] Note: Animals with eGFR between 30 and 59 were included in the statistics for the DOX + PMP + MTP group. 1. "a" compared with the baseline. 2. Change value = test time point - baseline value

[0205] 3. Effects on CysC, Cr - P and BUN

[0206] The effects of drug administration in each group on CysC, Cr - P and BUN are shown in Tables 4 - 4 to 4 - 6. CysC is an endogenous marker reflecting changes in glomerular filtration rate. Cr - P has been used as the main evaluation index for renal function for more than 40 years. In this experiment, the glomerular filtration rate eGFR was calculated through CysC and Cr - P. Although BUN was first used as an evaluation index for renal function, it does not meet the requirements of an endogenous GFR marker and was only used as an auxiliary index in this experiment.

[0207] Table 4 - 4 Effects of drug administration for 58 days in each group on CysC in spontaneously chronic diabetic nephropathy rhesus monkeys

[0208]

[0209] Note: 1. "a" compared with the baseline.

[0210] Effect of Administration for 58 Days in Each Group on CR-P of Spontaneous Chronic Diabetic Nephropathy Rhesus Monkeys

[0211]

[0212] Table 4-6 Effect of Administration for 58 Days in Each Group on BUN of Spontaneous Chronic Diabetic Nephropathy Rhesus Monkeys

[0213]

[0214] 4. Effect on Blood Pressure

[0215] The effects of drug administration in each group on blood pressure are shown in Table 4-7 and Table 4-8

[0216] Placebo group (n = 4, 2 / 4 hypertension): Compared with the baseline, the blood pressure of 4 animals fluctuated stably within a certain range

[0217] Valsartan group (n = 4, 2 / 4 hypertension): Compared with the baseline, on D58 after administration, the mean SBP decreased by 13 ± 6 mmHg, and the decrease was significantly lower than that of the placebo group (P < 0.05); the mean DBP decreased by 8 ± 5 mmHg, and the change value was extremely significantly lower than that of the placebo group (p < 0.01).

[0218] DOX+PMP+MTP 1# experimental group (n = 4, 2 / 4 hypertension): Compared with the baseline, on D58 of administration, the mean SBP decreased by 10 ± 3 mmHg, and the decrease was significantly lower than that of the placebo group (P < 0.05); DBP did not show a decreasing activity. There is no risk of hypotension in animals with normal blood pressure

[0219] DOX+PMP+MTP 2# experimental group (n = 4, 2 / 4 hypertension): Compared with the baseline, on D58 of administration, the mean SBP decreased by 5 ± 5 mmHg, and the decrease had no statistical significance compared with the placebo group. DBP did not show a decreasing activity. The antihypertensive activity is weak

[0220] In conclusion, the DOX+PMP+MTP composition has a certain antihypertensive activity of 5 to 10 mmHg

[0221] Table 4-7 Classification of Hypertension in Rhesus Monkeys

[0222]

[0223] Note: When SBP and DBP belong to different levels, the higher level shall prevail

[0224] Table 4-8 Effect of Administration for 58 Days in Each Group on Blood Pressure of Spontaneous Chronic Diabetic Nephropathy Rhesus Monkeys

[0225]

[0226] Note: "a" compared with the placebo group.

[0227] 5. Effect on K+

[0228] The effects of drug administration in each dosing group on K+ are shown in Table 4-9. Compared with the baseline period, the serum K+ levels of animals in each test group on day D58 of drug administration did not show significant changes. In summary, there is no risk of causing hyperkalemia in the DOX+PMP+MTP treatment group on day D58 of drug administration.

[0229] Table 4-9 Effects of drug administration for 58 days in each group on K+ ions in rhesus monkeys with spontaneous chronic diabetic nephropathy

[0230]

[0231] 6. Safety tolerance study

[0232] As marketed drugs, the safe dose ranges and toxic and side reactions of DOX, PMP, and MTP are known. The common tablet dose of DOX is 4 to 8 mg per time, twice a day. The recommended individual dose of PMP should be between 0.375 mg and 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 to 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 50 - 200 mg / day, and can reach 300 mg / day or 400 mg / day if necessary. The known adverse reaction is hypotension.

[0233] In this experiment, the calculated equivalent human doses for clinical patients of the combined administration of DOX, PMP, and MTP in treating rhesus monkeys with chronic diabetic nephropathy are far lower than the maximum values of the above recommended doses. Therefore, theoretically, the combined use of DOX+PMP+MTP in the present invention has very good safety.

[0234] In addition, no drug-related adverse events were observed during drug administration, and there were no significant changes in liver function, kidney function, body weight, and biochemical indexes (including FPG, LDL, HDL, TC, TG, ALT, AST, TP, ALB, etc.), further confirming that the combined use of DOX+PMP+MTP in the present invention indeed has good biosafety indexes.

[0235] V. Conclusions

[0236] The above experimental results show that: oral administration of three GPCR agonists (DOX + PMP + MTP) for 58 days in the treatment of spontaneous diabetic nephropathy in rhesus monkeys can significantly reduce moderate to severe proteinuria, significantly improve the deterioration of glomerular filtration rate, and inhibit the occurrence of end-stage renal failure; at the same time, it has good safety and tolerance, and no hyperkalemia or fluid retention was observed during the treatment period.

[0237] VI. Example 1 of the Pharmaceutical Composition

[0238] For a 10-kg monkey, 1.5 mg of doxazosin mesylate (DOX) raw material powder, 0.02 mg of pramipexole dihydrochloride (PMP) raw material powder, and 8 mg of metoprolol tartrate salt (MTP) raw material powder can be taken and evenly mixed to obtain Example 1 of the pharmaceutical composition. This pharmaceutical composition can be directly mixed into food for feeding animals, or can be made into oral tablets after being formulated with appropriate excipients or additives.

[0239] Although the specific implementation manners have been specifically described above, those skilled in the art can understand that, according to the disclosure and guidance of the above description, those skilled in the art of the present invention can also make appropriate changes and modifications to the above implementation manners. Therefore, the present invention is not limited to the specific implementation manners disclosed and described above, and some modifications and changes to the present invention also fall within the protection scope of the claims of the present invention.

Claims

1. A pharmaceutical composition for treating diabetic nephropathy, wherein the active ingredients thereof consist of doxazosin or a pharmaceutically acceptable salt thereof, pramipexole or a pharmaceutically acceptable salt thereof, and metoprolol or a pharmaceutically acceptable salt thereof, wherein the amount of doxazosin or a pharmaceutically acceptable salt thereof is 20 parts by weight to 80 parts by weight, the amount of pramipexole or a pharmaceutically acceptable salt thereof is 0.5 parts by weight to 1 part by weight, and the amount of metoprolol or a pharmaceutically acceptable salt thereof is 100 parts by weight to 500 parts by weight.

2. The pharmaceutical composition according to claim 1, which is in an oral dosage form or for intravenous injection.

3. The pharmaceutical composition according to any one of claims 1 to 2, wherein the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, phosphate, pyrophosphate, hydrobromide, nitrate, citrate, fumarate, maleate, malate, ascorbate, succinate, tartrate, benzoate, acetate, mesylate, esylate, salicylate, stearate, benzenesulfonate or p-toluenesulfonate.

4. 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 preparation of a pharmaceutical composition for treating diabetic nephropathy in a subject in need thereof, wherein the amount of doxazosin or a pharmaceutically acceptable salt thereof is 20 parts by weight to 80 parts by weight, the amount of pramipexole or a pharmaceutically acceptable salt thereof is 0.5 parts by weight to 1 part by weight, and the amount of metoprolol or a pharmaceutically acceptable salt thereof is 100 parts by weight to 500 parts by weight.

5. The use according to claim 4, wherein the pharmaceutical composition is in an oral dosage form or an intravenous injection dosage form.

6. The use according to any one of claims 4 to 5, wherein the pharmaceutical composition is applicable to primate subjects.

7. The use according to any one of claims 4 to 5, wherein the pharmaceutical composition is applicable to human subjects.

8. The use according to any one of claims 4 to 5, wherein the pharmaceutical composition is applicable to human subjects, the daily dose range of doxazosin or a pharmaceutically acceptable salt thereof is 2 mg to 8 mg, the daily dose range of pramipexole or a pharmaceutically acceptable salt thereof is 0.05 mg to 0.1 mg, and the daily dose range of metoprolol or a pharmaceutically acceptable salt thereof is 10 mg to 50 mg, and the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, phosphate, pyrophosphate, hydrobromide, nitrate, citrate, fumarate, maleate, malate, ascorbate, succinate, tartrate, benzoate, acetate, mesylate, esylate, salicylate, stearate, benzenesulfonate or p-toluenesulfonate.

9. The use according to any one of claims 4 to 5, wherein the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, phosphate, pyrophosphate, hydrobromide, nitrate, citrate, fumarate, maleate, malate, ascorbate, succinate, tartrate, benzoate, acetate, mesylate, esylate, salicylate, stearate, benzenesulfonate or p-toluenesulfonate.

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