WO2017186141A1 - 二苯甲酮类化合物在制药中的应用 - Google Patents

二苯甲酮类化合物在制药中的应用 Download PDF

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WO2017186141A1
WO2017186141A1 PCT/CN2017/082173 CN2017082173W WO2017186141A1 WO 2017186141 A1 WO2017186141 A1 WO 2017186141A1 CN 2017082173 W CN2017082173 W CN 2017082173W WO 2017186141 A1 WO2017186141 A1 WO 2017186141A1
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compared
diabetic nephropathy
rats
acute pyelonephritis
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French (fr)
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李青山
冯秀娥
张圆琳
韩玲革
常熔熔
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山西医科大学
山西中医学院
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Priority claimed from CN201610278158.4A external-priority patent/CN105816446B/zh
Priority claimed from CN201710278835.7A external-priority patent/CN107007610B/zh
Application filed by 山西医科大学, 山西中医学院 filed Critical 山西医科大学
Priority to US16/097,348 priority Critical patent/US20190133968A1/en
Priority to JP2019507980A priority patent/JP6698214B2/ja
Publication of WO2017186141A1 publication Critical patent/WO2017186141A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone

Definitions

  • the invention belongs to the field of new application of compounds, and relates to two compounds having the prevention and treatment of type II diabetic nephropathy, acute pyelonephritis and chronic nephritis, in particular compound 4,5,2'-trihydroxy-2,5'-dibromo Preparation and prevention of type 2 diabetic nephropathy, acute pyelonephritis and chronic nephritis by benzophenone and 4,5,2'-tris(4-morpholinyloxy)-2,5'-dichlorobenzophenone Application in medicine.
  • Diabetic nephropathy is one of the major complications of diabetes, which ultimately leads to end-stage renal disease (ESRD).
  • ESRD end-stage renal disease
  • DN Diabetic nephropathy
  • ESRD end-stage renal disease
  • DN is the leading cause of ESRD, accounting for 25 to 42%, and is also the main cause of ESRD in China.
  • the prevalence of DN in type 2 diabetes is about 20 to 25%.
  • DN can be caused by the initial appearance of urinary protein to hypertension, nephrotic syndrome, and ultimately to renal failure and death.
  • DN blood sugar, lower blood lipids, inhibit inflammation, and resist oxidative stress.
  • drugs mainly uses angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist (ACEI/ARB), and even
  • ACEI/ARB angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist
  • the combination of sugar and drug, and the main function of ACEI / ARB is to reduce glomerular osmotic pressure by lowering blood pressure and urine microalbumin, while improving insulin utilization.
  • electrolyte disorders may occur after the application of ACEI/ARB drugs, which may be complicated by hyperkalemia, and may cause side effects such as dry cough and orthostatic hypotension.
  • Acute pyelonephritis is mainly an inflammatory disease of the renal tubules, renal interstitial and renal parenchyma caused by various pathogenic bacteria, and the glomerular part is generally less damaged. If the treatment is not timely, it can be changed from acute to chronic, and finally to renal failure.
  • the pathogenesis of acute pyelonephritis mainly involves bacterial pathogenicity, inflammation and immune response, and the latter two are increasingly valued by researchers.
  • the most suitable clinical antibacterial drugs are used to treat acute pyelonephritis.
  • drug-resistant strains continue to appear, and are prone to recurrence after treatment, which has affected their clinical efficacy.
  • Chronic glomerulonephritis is the abbreviation of chronic glomerulonephritis. It is an immune-mediated inflammatory disease. It is clinically characterized by proteinuria, hematuria, etc. It can have varying degrees of renal dysfunction, has a tendency to deteriorate renal function and will eventually develop chronic. A group of glomerular diseases of renal failure. In recent years, the incidence of chronic nephritis has gradually increased, and tends to be younger, bringing huge economic burden to families and society. Recent studies suggest that urinary protein is not only a marker of glomerular disease, but also chronic and ongoing Risk factors for the development of patients with renal failure. A large number of clinical and experimental studies have found that urine protein itself has nephrotoxicity and promotes renal failure.
  • reducing and eliminating urine protein is one of the important measures for the treatment of chronic nephritis.
  • the treatment of chronic nephritis is usually treated with classic hormones and immunosuppressive agents. Although it has certain curative effect, it is difficult to fundamentally prevent the development of chronic nephritis due to serious side effects and recurrence.
  • the object of the present invention is to provide a benzophenone compound 4,5,2'-trihydroxy-2,5'-dibromobiphenyl for the treatment of deficiency of type II diabetic nephropathy, acute pyelonephritis and chronic nephritis.
  • Another object of the present invention is to provide a novel anti-type II diabetic nephropathy drug and a medicament for treating acute pyelonephritis and chronic nephritis, which is a compound 4,5,2'-trihydroxy-2,5'-di Bromobenzophenone or 4,5,2'-tris(4-morpholinyloxy)-2,5'-dichlorobenzophenone is the active ingredient, and the drug also includes the activity A common pharmaceutical carrier or excipient used in combination with the ingredients.
  • the drug can be prepared into a conventional preparation such as a solid dispersion, a tablet, a pill, a capsule or an injection according to different requirements.
  • the compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone and 4,5,2'-tris(4-morpholinyloxy)-2,5'-di Chlorobenzophenone is a two benzophenone compound well known to those skilled in the art, and its preparation method is also easy to implement.
  • the invention establishes a solid dispersion of the above two compounds by different doses of the SD rat type II diabetic nephropathy model, the acute pyelonephritis model and the chronic nephritis model, respectively, during which blood glucose, blood fat, urine volume, urine, and dirt are administered.
  • Indicators urine and tissue related indicators of bacteria, renal function, blood CD 4 and CD 8 , inflammatory factors and other indicators were tested to confirm the two compounds for type II diabetic nephropathy, acute pyelonephritis and chronic nephritis rats It has strong renal protective effect and shows its important application prospects in the prevention and treatment of diabetic nephropathy, acute pyelonephritis and chronic nephritis.
  • the main innovations of the present invention are as follows: 1) It is found that the two compounds have remarkable pharmacological activity against type II diabetic nephropathy and significant pharmacological activity for treating acute pyelonephritis and chronic nephritis, and have good development and application value. 2) The two compounds have strong effects of lowering blood sugar, lowering blood fat, anti-oxidation and anti-inflammatory, and their active diversity is consistent with the pathogenesis of DN. 3) Both compounds can effectively improve renal function in rats with type II diabetic nephropathy by administration of solid dispersion.
  • the main therapeutic indicators are uric acid, urine protein, microalbuminuria, growth factor TGF- ⁇ 1, blood glucose, low density.
  • Lipoproteins are superior to the current clinical first-line drug captopril. 4) The two compounds have strong antibacterial and immunomodulatory effects. 5) So far, there is no relevant report on the pharmacological effects of two compounds against type II diabetic nephropathy, acute pyelonephritis and chronic nephritis.
  • Figure 1 shows the hematoxylin-eosin (HE) staining, and the renal pathology of the rats in each group of diabetic nephropathy was observed under electron microscope (HE ⁇ 200).
  • HE hematoxylin-eosin
  • Figure 2 is a Masson staining, electron microscopic observation, renal pathology comparison of rats in each group of diabetic nephropathy (Masson ⁇ 400).
  • Figure 3 shows the bacterial culture of urine in rats with acute pyelonephritis.
  • Figure 4 shows the bacterial culture of kidney extract in rats with acute pyelonephritis.
  • Figure 5 shows the hematoxylin-eosin (HE) staining, and the renal pathology of the rats in each group of acute pyelonephritis (HE ⁇ 200).
  • the active ingredient is compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone, or compound 4, 5,2'-Tris(4-morpholinyloxy)-2,5'-dichlorobenzophenone.
  • the usual pharmaceutical carriers or excipients used in combination with the active ingredient.
  • the medicament can be prepared into a common dosage form such as an injection, a tablet, a pill, a solid dispersion or a capsule according to specific requirements.
  • the compound of the invention 4,5,2'-trihydroxy-2,5'-dibromobenzophenone and 4,5,2'-tris(4-morpholinyloxy)-2,5'-di Chlorine
  • benzophenone in anti-type II diabetic nephropathy and in the treatment of acute pyelonephritis and chronic nephritis will be illustrated by the following specific examples. It should be noted that the following examples are illustrative and are not intended to limit the invention.
  • mice male SD rats weighing 180-220 g, animal source: China Food and Drug Control Research Institute, license number: SCXK-(Beijing) 2014-0013, certificate number: 11400500008465, the animals used in this test and related disposals
  • the requirements for animal welfare are subject to the ethical review of the Animal Welfare Committee of this institution before the experiment is carried out.
  • Feed high sugar and high fat feed formula: 10% lard, 20% sucrose, 2.5% cholesterol, 0.5% sodium cholate, 67% base stock.
  • Feed source Beijing Keao Xieli Feed Co., Ltd., license number: SCXK-(Beijing) 2014-0010, certificate number: 11002900016120.
  • LM49 prepared by Shanxi Medical University Pharmaceutical Chemistry Laboratory, purity ⁇ 99.5%, batch number 20150320
  • 4,5, 2'-Tris(4-morpholinoyloxy)-2,5'-dichlorobenzophenone (hereinafter referred to as A8, prepared by Shanxi Medical University Pharmaceutical Chemistry Laboratory, purity ⁇ 99.5%, batch number 20150112)
  • PVP K30 polyvinylpyrrolidone (PVP K30)
  • USP26 Jiangyin Jiafeng Chemical Co., Ltd.
  • Tween 80 pharmaceutical grade, Sichuan Jinshan Pharmaceutical Co., Ltd.
  • anhydrous ethanol anhydrous ethanol (analytical grade, Beijing Chemical Reagent Company); Cattop Li (Shanghai Xudong Haipu Pharmaceutical Co., Ltd., production batch number 140603); streptozotocin (Sigma, production batch number 1126C038); rat interle
  • Constant temperature water bath (Shanghai Hecheng Instrument Manufacturing Co., Ltd.); BP-121S electronic balance (Shanghai Precision Scientific Instrument Company); TU-1810 UV-visible spectrophotometer (Beijing General Analysis Instrument Co., Ltd.); blood glucose meter (American Johnson & Johnson) ); microplate reader (Rayto, RT-6100); low-speed automatic balance centrifuge (Baiyang centrifuge factory in Anxin County, Hebei province); ultra-low temperature refrigerator (Haier); Konelab Prime 30 automatic biochemical analyzer (United States Thermo).
  • Diabetic nephropathy animal model preparation
  • Healthy adult male Sprague-Dawley rats (180-220 g) were fed with high-sugar and high-fat diet for 4 weeks, fasted overnight, intraperitoneally injected with streptozotocin (STZ) 40 mg/kg, and the normal group was injected with the same volume of sodium citrate buffer. After 72 hours, fasting for 12 hours, the fasting blood glucose of the model rats was measured, and three consecutive fasting blood glucose >16.7mmol/L was considered to be successful in modeling diabetes. Rats after successful modeling continued to feed high-fat diet during the administration period, and the normal group was fed the normal diet.
  • STZ streptozotocin
  • kidneys were washed with physiological saline, the weight of the kidneys was recorded, and the kidney coefficient (several kidney weight/body weight) was calculated.
  • the same site of renal tissue (including glomeruli and tubules) was immersed in 10% neutral formalin for pathomorphological analysis. The other part was used for biochemical indicators testing, and the right kidney was used for mechanism studies and stored in a -80 °C refrigerator.
  • LM49 solid dispersion low dose group (medicine content is 3mg/kg)
  • LM49 solid dispersion medium dose group (medicine content is 9mg/kg)
  • LM49 solid dispersion high dose group (medicine content is 27mg/kg)
  • A8 solid dispersion low dose group (medicine content is 3mg/kg)
  • A8 solid dispersion medium dose group (medicine content 9mg/kg)
  • A8 solid dispersion high dose group (medicine content is 27 mg/kg)
  • captopril control group (containing 15 mg/kg).
  • Route of administration intragastric administration.
  • Dosing volume normal control and model group were given normal saline 1.0ml/100g, positive control, LM49 solid dispersion high, medium and low dose groups, A8 solid dispersion high, medium and low dose groups were given different concentrations of drugs, given The drug volume is 1.0 ml / 100 g.
  • Drug formulation The solid dispersion was dissolved in physiological saline and the positive control was administered by suspension with 0.5% CMC-Na.
  • Dosing time The administration is started at the end of modeling.
  • Frequency of administration 7 days of dosing per week for a total of 6 weeks.
  • TCHO serum cholesterol
  • HDL-C high density lipoprotein
  • LDL-C low density lipoprotein
  • LM49 has a clear anti-diabetic nephropathy effect, which can effectively alleviate renal tissue lesions, and can significantly reduce serum uric acid, blood sugar, cholesterol, triglyceride, low-density lipoprotein levels, and growth and transformation in kidney tissue.
  • Factors such as factor, urine protein and urinary albumin significantly inhibited the expression of inflammatory factors such as TNF- ⁇ , NF- ⁇ B, IL-6 and IL-1 ⁇ in renal tissues, and decreased renal tubular water-like lesions and glomerular fibrosis levels. .
  • LM49 has a strong anti-diabetic nephropathy effect, and its main therapeutic indicators are uric acid, urine protein, urinary microalbumin, growth factor TGF- ⁇ 1, blood glucose, low-density lipoprotein, etc.
  • the first-line drug captopril shows an important application prospect.
  • A8 has stronger anti-diabetic nephropathy effect, can effectively alleviate renal tissue lesions, and can significantly reduce uric acid, glycosylated hemoglobin, blood sugar, cholesterol, triglyceride, low-density lipoprotein levels and growth and transformation in kidney tissue.
  • Factors such as factor, urine protein and urinary albumin significantly inhibited the expression of inflammatory factors such as TNF- ⁇ , NF- ⁇ B, IL-6 and IL-1 ⁇ in renal tissues, and decreased renal tubular water-like lesions and glomerular fibrosis levels. .
  • A8 has a strong anti-diabetic nephropathy effect, and its main therapeutic indicators are kidney index, uric acid, urine protein, microalbuminuria, growth factor TGF- ⁇ 1, blood glucose, low-density lipoprotein, etc.
  • the first-line clinical drug captopril shows an important application prospect.
  • Animals 200 SPF male SD rats, weighing (200+20) g, purchased from Beijing Huakangkang Biotechnology Co., Ltd., experimental animal license number: SCXK (Beijing) 2014-0008, certificate number: 11003800008312 .
  • Animal feeding and breeding environment clean and constant temperature environment, the average temperature of the breeding environment is 25 ° C, the average humidity is 37%, 15 to 20 times / hour fresh air. Light: 12 hours and 12 hours dark. Free drinking water to eat.
  • Animal feed purchased from Beijing Huakangkang Biotechnology Co., Ltd., license number: SCXK (Beijing) 2014-0008, certificate number: 11003800008312.
  • Drugs and reagents LM49, A8, provided by Shanxi Medical University; Norfloxacin capsule (Shanxi Taiyuan Pharmaceutical Co., Ltd., batch number: 150401), dexamethasone acetate tablets (Zhejiang Xianyi Pharmaceutical Co., Ltd., batch number: 141006) Escherichia coli ATCC25922 standard strain (Shanghai Luwei Technology Co., Ltd., batch number: A1008B); serum creatinine, urinary creatinine ELISA kit (Nanjing Jiancheng Technology Co., Ltd., batch number: 20160421), IL-1 ⁇ (Xinbosheng Biotechnology Co., Ltd.) , batch number: 160429-009a), IL-1 ⁇ (Xinbosheng Biotechnology Co., Ltd., batch number: 160429-007a), IL-6 (Xinbosheng Biotechnology Co., Ltd., batch number: 160429-003a), IL-10
  • the positive model was successfully prepared, and the unqualified ones were removed, and the number of animals was re-surgery.
  • the successfully prepared models were randomly divided into model group, norfloxacin group (66.7 mg/kg), LM49 solid dispersion low dose group (medicine content 3 mg/kg), medium dose group (medicine content 9 mg/kg). High-dose group (containing 27mg/kg), low-dose A8 solid dispersion (containing 3mg/kg), medium-dose group (containing 9mg/kg), high-dose group (containing 27mg/dose) Kg).
  • the sham operation group and the model group were given an equal volume of normal saline by intragastric administration, and the other groups were given the corresponding doses of the drug once a day for continuous administration for 10 days.
  • Passive Heymann nephritis is a classic model for studying chronic kidney disease in humans. Therefore, the present invention uses this model to study the prevention and treatment effects of the two compounds of the present invention on chronic nephritis.
  • the blank group and the model group were given an equal volume of physiological saline, and the other groups were given corresponding doses of the drug, and administered intragastrically every day for 4 weeks from the day of modeling.
  • All groups of rats were collected urine after the end of the administration (observation of bacterial growth), and abdominal aortic blood was collected (blood routine, CD 4 /CD 8 and serum urinary creatinine, serum creatinine; ELISA for serum IL) -1 ⁇ , IL-1 ⁇ , IL-6, IL-10, MCP-1, etc.), bilateral kidneys were cut (a part of the homogenate was used for bacterial culture, and the other part was observed for pathological changes).
  • the two compounds can significantly reduce the kidney index and bladder index of rats with acute pyelonephritis (see Table 2), reduce the degree of bacterial infection in kidney tissue and urine (Table 12, Figure 1 and Figure 2), and significantly reduce kidney tissue.
  • Middle-medullary myelin structure and histological changes such as renal pelvis and renal pelvis and chronic inflammatory cell infiltration or abscess (Fig. 3).
  • the therapeutic effect of the two compounds on acute pyelonephritis is mainly to increase the autoimmune function of the model rats by regulating CD 4 , CD 8 and CD 4 /CD 8 and inhibit the inflammatory factors such as IL-1 ⁇ , IL-1 ⁇ and IL-6. The expression is achieved by reducing the inflammatory response.
  • the above results showed that the high dose group of LM49 and A8 regulated CD 4 /CD 8 significantly better than the positive drug, showing an important application prospect.

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Abstract

本发明公开了两种二苯甲酮类化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮和4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮的医药新用途，具体为在制备抗Ⅱ型糖尿病肾病、治疗急性肾盂肾炎和慢性肾炎药物中的应用，同时本发明也公开了以上述两种化合物为活性成分的抗Ⅱ型糖尿病肾病药物及治疗急性肾盂肾炎和慢性肾炎的药物。这两种化合物均能有效改善Ⅱ型糖尿病肾病模型大鼠、急性肾盂肾炎模型大鼠和慢性肾炎模型大鼠的的肾功能，显示了在抗糖尿病肾病、治疗急性肾盂肾炎和慢性肾炎方面重要的应用前景。

Description

二苯甲酮类化合物在制药中的应用 技术领域
本发明属于化合物新用途领域，涉及两种具有预防和治疗Ⅱ型糖尿病肾病、急性肾盂肾炎和慢性肾炎的化合物，具体为化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮和4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮在制备预防和治疗Ⅱ型糖尿病肾病、急性肾盂肾炎和慢性肾炎药物中的应用。
背景技术
糖尿病肾病(diabetic nephropathy,DN)是糖尿病常见的主要并发症之一，最终导致肾病(end-stage renal disease,ESRD)。目前中国已有1.14亿糖尿病患者，1.4亿糖尿病前期患者，随着全球范围内的糖尿病患者的不断增加，DN的患病率也随之提高。在西方国家DN是导致ESRD的首要病因，占25～42％，也是我国ESRD的主要病因。DN在Ⅱ型糖尿病中的患病率约为20～25％，DN可由最初出现尿蛋白到高血压、肾病综合征，最终导致肾衰和死亡。
现阶段治疗DN主要是控制血糖、降低血脂、抑制炎症、抗氧化应激，目前临床用药主要是采用血管紧张素转换酶抑制剂/血管紧张素受体拮抗剂(ACEI/ARB)，甚至和降糖药联合用药，而ACEI/ARB的主要功能是通过降低血压和尿微量白蛋白，降低肾小球渗透压，同时改善对胰岛素利用。但是，对多数DN患者，特别是DN IV期以后的患者，应用ACEI/ARB类药物以后多会出现电解质紊乱，易合并高血钾症，同时有引起干咳、体位性低血压等副作用。
急性肾盂肾炎主要是由多种致病菌侵袭引起的肾小管、肾间质和肾实质的炎症性疾病，一般肾小球部位较少受损。若治疗不及时，可由急性转慢性，最后发展为肾功能衰竭。急性肾盂肾炎的发病机制主要涉及细菌致病能力、炎症和免疫反应等方面，而后两者越来越受到研究者的重视。目前临床上主要选用合适的抗菌药物治疗急性肾盂肾炎。但是，随着抗菌药的滥用，耐药菌株不断出现，治后容易复发等，使其临床疗效受到了影响。
慢性肾炎是慢性肾小球肾炎的简称，是由免疫介导的炎症性疾病，临床表现为蛋白尿、血尿等，可有不同程度的肾功能减退，具有肾功能恶化倾向和最终将发展为慢性肾衰竭的一组肾小球病。近年来，慢性肾炎的发病率逐渐升高，并趋向于年轻化，给家庭和社会带来了巨大的经济负担。最近研究提示，尿蛋白不仅是肾小球疾病的标志，还是慢性和进行 性肾功能衰竭患者病情发展的危险因子。大量临床和实验研究发现，尿蛋白本身具有肾毒性，促进肾衰竭。所以，减少和消除尿蛋白，是治疗慢性肾炎的重要措施之一。目前治疗慢性肾炎通常采用经典的激素和免疫抑制剂治疗，虽有一定疗效，但由于存在严重的副作用及复发性，难以从根本上阻止慢性肾炎的发展。
因此，针对上述三种肾病的危害性以及目前临床上该类疾病在药物治疗中存在的缺陷，寻找合适的治疗药物已成为当下医药界的热点研究方向。
发明内容
本发明的目的是针对现有治疗Ⅱ型糖尿病肾病、急性肾盂肾炎和慢性肾炎的不足，提供二苯甲酮类化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮和4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮的新的药理用途，具体为在制备抗Ⅱ型糖尿病肾病及治疗急性肾盂肾炎和慢性肾炎药物中的应用。
本发明的另一个目的在于提供一种新的抗Ⅱ型糖尿病肾病的药物及治疗急性肾盂肾炎和慢性肾炎的药物，该药物以化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮或4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮为活性成分，并且该药物中还包括与所述活性成分组合使用的普通药用载体或赋形剂。该药物能够根据不同的要求制成固体分散体、片剂、滴丸剂、胶囊剂或注射剂等常用制剂。
其中，化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮(LM49)的结构式如下：
Figure PCTCN2017082173-appb-000001
化合物4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮(A8)的结构式如下：
Figure PCTCN2017082173-appb-000002
所述化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮和4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮是本领域技术人员所熟知的两种二苯甲酮类化合物，其制备方法也是容易实现的。
本发明通过分别建立SD大鼠Ⅱ型糖尿病肾病模型、急性肾盂肾炎模型和慢性肾炎模 型，灌胃给予不同剂量上述两种化合物的固体分散体，期间对血糖、血脂、尿量、尿液、脏器指数、尿液及组织中细菌、肾功能相关指标、血液中CD4及CD8、炎症因子等各项指标进行检测，证实两种化合物对Ⅱ型糖尿病肾病、急性肾盂肾炎和慢性肾炎大鼠具有很强的肾脏保护作用，显示了其在预防和治疗糖尿病肾病、急性肾盂肾炎和慢性肾炎方面重要的应用前景。
本发明的主要创新点在于：1)发现两种化合物具有显著的抗Ⅱ型糖尿病肾病的药理活性和显著的治疗急性肾盂肾炎和慢性肾炎的药理活性，具有很好的开发应用价值。2)两种化合物具有很强的降血糖、降血脂、抗氧化、抗炎等作用，其活性多样性与DN的发病机制多重契合。3)两种化合物以固体分散体给药均能有效改善Ⅱ型糖尿病肾病大鼠的肾功能，其主要治疗指标尿酸、尿蛋白、尿微量白蛋白、生长转化因子TGF-β1、血糖、低密度脂蛋白等优于目前临床一线药物卡托普利。4)两种化合物具有很强的抗菌、免疫调节等作用。5)到目前为止，对于两种化合物的抗Ⅱ型糖尿病肾病、治疗急性肾盂肾炎和慢性肾炎的药理作用国内外尚无相关报道。
附图说明
图1为苏木精-伊红(HE)染色，电镜下观察，各组糖尿病肾病模型大鼠肾脏病理学比较(HE×200)。
图2为Masson染色，电镜下观察，各组糖尿病肾病模型大鼠肾脏病理学比较(Masson×400)。
图3为急性肾盂肾炎模型大鼠尿液细菌培养情况。
图4为急性肾盂肾炎模型大鼠肾脏提取液细菌培养情况。
图5为苏木精-伊红(HE)染色，电镜下观察，各组急性肾盂肾炎模型大鼠肾脏病理学比较(HE×200)。
具体实施方式
化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮和4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮在制备抗Ⅱ型糖尿病肾病药物和治疗急性肾盂肾炎和慢性肾炎药物中的应用。
上述抗Ⅱ型糖尿病肾病的药物和治疗急性肾盂肾炎和慢性肾炎药物，其活性成分为化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮，或者是化合物4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮。该药物中还包括与所述活性成分组合使用的普通药用载体或赋形剂。该药物可根据具体要求制成注射剂、片剂、滴丸剂、固体分散体或胶囊剂等常用剂型。
本发明化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮和4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二 苯甲酮在抗Ⅱ型糖尿病肾病和治疗急性肾盂肾炎和慢性肾炎中的药理作用将通过下述具体实施例进行阐述。需要说明的是，以下实施例都为说明性的，并不对本发明做任何限定。
实施例1
材料：
实验动物：雄性SD大鼠，体重180～220g，动物来源：中国食品药品检定研究院，许可证号：SCXK-(京)2014-0013，合格证号：11400500008465，本试验所用动物及相关处置符合动物福利的要求，实验开展前要经过本机构动物福利委员会的伦理审查。
饲料：高糖高脂饲料配方：10％猪油，20％蔗糖，2.5％胆固醇，0.5％胆酸钠，67％基础料。饲料来源：北京科澳协力饲料有限公司，许可证号：SCXK-(京)2014-0010，合格证号：11002900016120。
药品与试剂：
4,5,2'-三羟基-2,5'-二溴二苯甲酮(以下简称LM49，由山西医科大药物化学实验室制备，纯度≥99.5％，批号为20150320)；4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮(以下简称A8，由山西医科大药物化学实验室制备，纯度≥99.5％，批号为20150112)；聚乙烯吡咯烷酮(PVP K30)(USP26，江阴嘉丰化工有限公司)；吐温80(药用级，四川金山制药有限公司)；无水乙醇(分析纯，北京化学试剂公司)；卡托普利(上海旭东海普药业有限公司，生产批号140603)；链脲佐菌素(Sigma，生产批号1126C038)；大鼠白细胞介素-1β酶联免疫检测试剂盒(南京建成，批号1501261)；大鼠白细胞介素-6酶联免疫检测试剂盒(南京建成，批号1412271)；大鼠微量白蛋白(Alb)酶联免疫检测试剂盒(南京建成，批号1506201)；大鼠可溶性细胞间黏附分子(sICAM-1)检测试剂盒(南京建成，批号1501201)；大鼠白肿瘤坏死因子(TNF-α)酶联免疫检测试剂盒(南京建成，批号1507281)；大鼠白血管内皮细胞黏附分子(sVCAM-1)酶联免疫检测试剂盒(南京建成，批号1508221)；总抗氧化能力(T-AOC)试剂盒(南京建成，批号20151010)；BCA蛋白定量试剂盒(博士德生物，批号10K12B46)；尿素(BUN)试剂盒(上海复星长征医学科学有限公司，批号P1311021)；尿酸(UA)试剂盒(上海复星长征医学科学有限公司，批号PF1405011)；肌酐(CREA)试剂盒(上海复星长征医学科学有限公司，批号D1404063)；临床化学综合质控血清(上海复星长征医学科学有限公司，批号842UN)；丙二醛(MDA)测试盒(南京建成，批号20150126)；大鼠核转录因子(NF-κB)酶联免疫试剂盒(南京建成，批号1507251)；SOD试剂盒(WST-1法，南京建成，批号20150127)；Masson染液(南京建成，批号20150915)。
仪器：
恒温水浴锅(上海和呈仪器制造有限公司)；BP-121S电子天平(上海精密科学仪器公司)；TU-1810紫外可见分光光度计(北京普析通用仪器有限责任公司)；血糖仪(美国强生)；酶标仪(Rayto,RT-6100)；低速自动平衡离心机(河北省安新县白洋离心机厂)；超低温冰箱(Haier)；Konelab Prime 30全自动生化分析仪(美国Thermo)。
固体分散体的制备：
采用溶剂法制备：按LM49、PVPK30、tween80的质量比为1:5:1和A8、PVPK30、tween80的质量比为1:4:1，分别精密称取各药，用适量无水乙醇溶解，不断搅拌混合均匀，置于60℃水浴上蒸发除去无水乙醇，得到粘稠状物，置于-20℃冰箱中冷冻2h，取出置于真空干燥箱中干燥24h，待脆化后粉碎过80目筛，置于干燥器中保存备用。
糖尿病肾病动物模型制备：
健康成年雄性SD大鼠(180～220g)高糖高脂饲料喂食4周，隔夜禁食，腹腔注射链脲佐菌素(STZ)40mg/kg，正常组注射同体积的柠檬酸钠缓冲液，72h后，禁食12h，测造模大鼠空腹血糖，连续3次空腹血糖>16.7mmol/L认为糖尿病造模成功。造模成功后的大鼠，给药期间继续喂食高脂饲料，正常组喂普通饲料。
给药6周后，用代谢笼收集6h尿。
禁食过夜，腹腔麻醉，抽取腹主动脉血检测相关指标。用生理盐水将肾洗净，记录双肾重量，计算肾脏系数(双肾重量/体重)。纵切取左侧肾的同一部位肾组织(包括肾小球和肾小管)浸入10％中性福尔马林中用于病理形态学分析。另一部分用于生化指标检测，右侧肾用于机制研究，储存在-80℃冰箱中。
组别及剂量设计：
造模成功后，除正常组(10只)外，其它动物随机分为8组，每组12只。分为模型组、LM49固体分散体低剂量组(含药量为3mg/kg)、LM49固体分散体中剂量组(含药量为9mg/kg)、LM49固体分散体高剂量组(含药量为27mg/kg)、A8固体分散体低剂量组(含药量为3mg/kg)、A8固体分散体中剂量组(含药量为9mg/kg)、A8固体分散体高剂量组(含药量为27mg/kg)、卡托普利对照组(含药量为15mg/kg)。
给药方法：
给药途径：灌胃。
给药体积：正常对照组和模型组给予生理盐水1.0ml/100g，阳性对照，LM49固体分散体高、中、低剂量组，A8固体分散体高、中、低剂量组分别给予不同浓度的药物，给药体积为1.0ml/100g。
药物配制：固体分散体用生理盐水溶解，阳性对照用0.5％CMC-Na混悬给药。
给药时间：于造模结束开始给药。
给药频次：每周给药7天，共给药6周。
统计学方法：
运用IBM SPSS Statistics 20软件进行数据处理，数据以
Figure PCTCN2017082173-appb-000003
表示，各组间比较采用单因素方差分析(ANOVA)，根据方差齐性检验结果，如方差齐采用LSD检验，如方差不齐采用Dunnett's T3检验，以P<0.05为差异有统计学意义。
结果与分析：
1、LM49、A8对糖尿病肾病大鼠肾功能的影响
表1 LM49、A8对糖尿病肾病大鼠肾功能的影响(
Figure PCTCN2017082173-appb-000004
n＝6)
Figure PCTCN2017082173-appb-000005
注：与正常组比较，##P<0.01,##P<0.05；与模型组比较，□□P<0.01，□P<0.05。
表2 LM49、A8对糖尿病肾病大鼠尿酸及生长转化因子的影响(
Figure PCTCN2017082173-appb-000006
n＝6)
Figure PCTCN2017082173-appb-000007
注：与正常组比较，##P<0.01,##P<0.05；与模型组比较，□□P<0.01，□P<0.05；与低剂量组比较，▲▲P<0.01，▲P<0.05；与中剂量组比较，○○P<0.01，○P<0.05；与卡普利组比较，●●P<0.01，●P<0.05。
表3 LM49、A8对糖尿病肾病大鼠尿液中尿蛋白及微量白蛋白的影响(
Figure PCTCN2017082173-appb-000008
n＝6)
Figure PCTCN2017082173-appb-000009
注：与正常组比较，##P<0.01,##P<0.05；与模型组比较，□□P<0.01，□P<0.05；与低剂量组比较，▲▲P<0.01，▲P<0.05；与中剂量组比较，○○P<0.01，○P<0.05；与卡普利组比较，●●P<0.01，●P<0.05。
2、LM49、A8对糖尿病肾病大鼠血清中血糖水平的影响
表4 LM49、A8对糖尿病肾病大鼠血清中血糖的影响(
Figure PCTCN2017082173-appb-000010
n＝6)
Figure PCTCN2017082173-appb-000011
注：与正常组比较，##P<0.01,##P<0.05；与模型组比较，□□P<0.01，□P<0.05；与低剂量组比较，▲▲P<0.01，▲P<0.05；与中剂量组比较，○○P<0.01，○P<0.05；与卡普利组比较，●●P<0.01，●P<0.05。
3、LM49、A8对糖尿病肾病大鼠血清中胆固醇(TCHO)、甘油三脂(TG)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)水平的影响
表5 LM49、A8对糖尿病肾病大鼠血清中清中胆固醇及甘油三酯水平的影响(
Figure PCTCN2017082173-appb-000012
n＝6)
Figure PCTCN2017082173-appb-000013
注：与正常组比较，##P<0.01,##P<0.05；与模型组比较，□□P<0.01，□P<0.05；与低剂量组比较，▲▲P<0.01，▲P<0.05；与中剂量组比较，○○P<0.01，○P<0.05；与卡普利组比较，●●P<0.01，●P<0.05。
表6 LM49、A8对糖尿病肾病大鼠血清中HDL-C,LDL-C水平的影响(
Figure PCTCN2017082173-appb-000014
n＝6)
Figure PCTCN2017082173-appb-000015
注：与正常组比较，##P<0.01,##P<0.05；与模型组比较，□□P<0.01，□P<0.05；与低剂量组比较，▲▲P<0.01，▲P<0.05；与中剂量组比较，○○P<0.01，○P<0.05；与卡普利组比较，●●P<0.01，●P<0.05
4、LM49、A8对糖尿病肾病大鼠肾组织中炎症因子的影响
表7 LM49、A8对糖尿病肾病大鼠肾组织中炎症因子TNF-α,NF-κB及粘附分子ICAM-1的影响(
Figure PCTCN2017082173-appb-000016
n＝6)
Figure PCTCN2017082173-appb-000017
注：与正常组比较，##P<0.01,##P<0.05；与模型组比较，□□P<0.01，□P<0.05；与低剂量组比较，▲▲P<0.01，▲P<0.05；与中剂量组比较，○○P<0.01，○P<0.05；与卡普利组比较，●●P<0.01，●P<0.05。
表8 LM49、A8对糖尿病肾病大鼠肾组织中炎症因子IL-1β,IL-6及粘附分子VCAM-1的影响(
Figure PCTCN2017082173-appb-000018
n＝6)
Figure PCTCN2017082173-appb-000019
注：与正常组比较，##P<0.01,##P<0.05；与模型组比较，□□P<0.01，□P<0.05；与低剂量组比较，▲▲P<0.01，▲P<0.05；与中剂量组比较，○○P<0.01，○P<0.05；与卡普利组比较，●●P<0.01，●P<0.05。
5、LM49、A8对糖尿病肾病大鼠肾组织中氧化因子的影响
表9 LM49、A8对糖尿病肾病大鼠肾组织中氧化因子的影响(
Figure PCTCN2017082173-appb-000020
n＝6)
Figure PCTCN2017082173-appb-000021
上述研究表明LM49有明确的抗大鼠糖尿病肾病作用，可有效减轻肾组织病变，能显著降低大鼠血清中尿酸、血糖、胆固醇、甘油三脂、低密度脂蛋白水平，及肾组织中生长转化因子、尿蛋白和尿微量白蛋白水平，显著抑制肾组织中TNF-α、NF-κB、IL-6、IL-1β等炎症因子的表达，降低肾小管水样病变、肾小球纤维化水平。同时上述结果也表明，LM49有很强的抗大鼠糖尿病肾病作用，其主要治疗指标尿酸、尿蛋白、尿微量白蛋白、生长转化因子TGF-β1、血糖、低密度脂蛋白等优于目前临床一线药物卡托普利，显示了重要的应用前景。
A8有更强的抗大鼠糖尿病肾病作用，可有效减轻肾组织病变，能显著降低大鼠血清中尿酸、糖化血红蛋白、血糖、胆固醇、甘油三脂、低密度脂蛋白水平及肾组织中生长转化因子、尿蛋白和尿微量白蛋白水平，显著抑制肾组织中TNF-α、NF-κB、IL-6、IL-1β等炎症因子的表达，降低肾小管水样病变、肾小球纤维化水平。同时上述结果表明，A8有很强的抗大鼠糖尿病肾病作用，其主要治疗指标肾脏指数、尿酸、尿蛋白、尿微量白蛋白、生长转化因子TGF-β1、血糖、低密度脂蛋白等明显优于目前临床一线药物卡托普利，显示了重要的应用前景。
结论：
通过上述实验可以说明化合物LM49和A8具有明确的抗Ⅱ型糖尿病肾病的功能。尤其是高剂量组效果更显著，化合物A8比LM49具有更高的抗Ⅱ型糖尿病肾病活性，显示了很好的应用开发前景。
实施例2
1.实验材料、仪器及试剂
动物：SPF级雄性SD大鼠200只，体重(200+20)g，购自北京华阜康生物科技股份有限公司，实验动物许可证编号：SCXK(京)2014-0008，合格证号：11003800008312。
动物饲养及饲养环境：清洁恒温环境，饲养环境平均温度25℃，平均湿度37％，15～20次/小时全新风。光照：12小时明12小时暗。自由饮水进食。
动物饲料：购自北京华阜康生物科技股份有限公司，许可证编号：SCXK(京)2014-0008，合格证号：11003800008312。
药品与试剂：LM49、A8，由山西医科大学提供；诺氟沙星胶囊(山西太原药业有限公司，批号：150401)，醋酸地塞米松片(浙江仙琚制药股份有限公司，批号：141006)；大肠杆菌ATCC25922标准菌株(上海鲁微科技有限公司，批号：A1008B)；血肌酐、尿肌酐ELISA试剂盒(南京建成科技有限公司，批号：20160421)，IL-1α(欣博盛生物科技有限公司，批号：160429-009a)，IL-1β(欣博盛生物科技有限公司，批号：160429-007a)，IL-6(欣博盛生物科技有限公司，批号：160429-003a)，IL-10(欣博盛生物科技有限公司，批号：160429-004a)，MCP-1(欣博盛生物科技有限公司，批号：160429-113a)，CXCL-2(博士德生物工程公司，批号：3771190428)；血常规试剂包(美国DREW科技有限公司，批号：6572)；anti-Rat CD4(eBioscience公司，批号：46-0040)，anti-Rat CD8-PE(eBioscience公司，批号：12-0084)。
主要仪器：ynergy H1微孔板检测仪(美国伯腾仪器有限公司)；LRH-150B生化培养箱(广东韶关泰宏医疗器械有限公司)；Thermo 1300系列Π级A2型生物安全柜(美国赛默飞世尔科技)；JK-CC30A菌落计数器(上海精科仪器公司)；HEMAVET950FS全自动血液分析仪(美国DREW公司)；CYTOMICS FC500流式细胞仪(美国Beckman Coulter公司)。
2.固体分散体制备
采用溶剂法制备。按LM49、PVPK30、tween80的质量比为1:5:1，A8、PVPK30、tween80的质量比为1:4:1，分别精密称取各药，用适量无水乙醇溶解，不断搅拌混合均匀，置于60℃水浴上蒸发除去无水乙醇，得到粘稠状物，置于-20℃冰箱中冷冻2h，取出置于真空干燥箱中干燥24h，待脆化后粉碎过80目筛，置于干燥器中保存备用。
3.模型的制备及给药
1)急性肾盂肾炎模型大鼠的制备及分组给药
SD大鼠90只，雄性，体重180～220g。选取10只作为假手术组(只结扎，不注射大肠杆菌)。剩余大鼠按照(中国实验动物学杂志,1998,8(1):31-34.)中方法，注射浓度108/ml的大肠杆菌ATCC25922菌液以制备急性肾盂肾炎模型。
3d天后收集尿液，做细菌培养，观察细菌生长情况，以出现阳性为模型制备成功，不合格者剔除，同时再次手术补足动物数。将制备成功的模型随机分为模型组、诺氟沙星组(66.7mg/kg)，LM49固体分散体低剂量组(含药量3mg/kg)、中剂量组(含药量9mg/kg)、高剂量组(含药量27mg/kg)，A8固体分散体低剂量组(含药量3mg/kg)、中剂量组(含药量9mg/kg)、高剂量组(含药量27mg/kg)。
假手术组和模型组灌胃给予等体积生理盐水，其余组给予相应剂量药物，每天给药一次，连续灌胃给药10天。
2)慢性肾炎模型大鼠的制备及分组给药
被动型Heymann肾炎是研究人类慢性肾病的经典模型。因此本发明用该模型研究本发明中两种化合物对慢性肾炎的防治作用。
雄性SD大鼠90只，体重180～220g。选取10只作为空白组，剩余大鼠按照(Journal of Proteomics,2012,75(3):3866-3876)方法制备大鼠Heymann肾炎模型。随机分为模型组、阳性对照组(地塞米松，0.1mg/kg)，LM49固体分散体低剂量组(含药量3mg/kg)、中剂量组(含药量9mg/kg)、高剂量组(含药量27mg/kg)，A8固体分散体低剂量组(含药量3mg/kg)、中剂量组(含药量9mg/kg)、高剂量组(含药量27mg/kg)。
空白组和模型组给予等体积生理盐水，其余组给予相应剂量药物，于造模当日起每天灌胃给药，共4周。
4.样品收集及相关指标检测
1)急性肾盂肾炎模型大鼠
所有组大鼠均于给药结束后收集尿液(观察细菌生长状况)，抽取腹主动脉血(检测血常规、CD4/CD8及血清中尿肌酐、血肌酐；ELISA法检测血清中IL-1α、IL-1β、IL-6、IL-10、MCP-1等的含量)，切取双侧肾脏(一部分匀浆用于细菌培养，另一部分观察病理形态学变化)。
表10肾脏倾注培养细菌生长情况评分标准
Figure PCTCN2017082173-appb-000022
2)慢性肾炎模型大鼠
所有组大鼠均于造模开始第2和第4周采血并收集24小时尿液，分别检测尿白蛋白和血清中血肌酐(Scr)、尿素氮(BUN)、IL-1α、IL-1β、IL-6、IL-10等的含量。
5.统计学方法
数据采用均数±标准差
Figure PCTCN2017082173-appb-000023
表示，用SPSS 19.0统计软件处理，多组比较采用One-way ANOVA单因素方差分析，然后采用LSD-Test进行两两比较。显著性差异水平以P＜0.05和P＜0.01为标准。
6.结果与分析
1)两种化合物对急性肾盂肾炎治疗作用的结果
表11各剂量组对急性肾盂肾炎大鼠脏器指数的影响(
Figure PCTCN2017082173-appb-000024
n＝10)
Figure PCTCN2017082173-appb-000025
注：与假手术组比较：**P<0.01，*P<0.05；与模型组比较：△△P<0.01，△P<0.05；与阳性药组比较：##P<0.01，#P<0.05。
表12各剂量组对急性肾盂肾炎大鼠尿液及肾脏细菌培养情况的影响(
Figure PCTCN2017082173-appb-000026
n＝10)
Figure PCTCN2017082173-appb-000027
注：与假手术组比较：**P<0.01，*P<0.05；与模型组比较：△△P<0.01，△P<0.05；与阳性药组比较：##P<0.01，#P<0.05。
表13各剂量组对急性肾盂肾炎大鼠血常规指标的影响(
Figure PCTCN2017082173-appb-000028
n＝10)
Figure PCTCN2017082173-appb-000029
注：与假手术组比较：**P<0.01，*P<0.05；与模型组比较：△△P<0.01，△P<0.05；与阳性药组比较：##P<0.01，#P<0.05。
表13(续)各剂量组对急性肾盂肾炎大鼠血常规指标的影响(
Figure PCTCN2017082173-appb-000030
n＝10)
Figure PCTCN2017082173-appb-000031
注：与假手术组比较：**P<0.01，*P<0.05；与模型组比较：△△P<0.01，△P<0.05；与阳性药组比较：##P<0.01，#P<0.05。
表13(续)各剂量组对急性肾盂肾炎大鼠血常规指标的影响(
Figure PCTCN2017082173-appb-000032
n＝10)
Figure PCTCN2017082173-appb-000033
注：与假手术组比较：**P<0.01，*P<0.05；与模型组比较：△△P<0.01，△P<0.05；与阳性药组比较：##P<0.01，#P<0.05。
表14各剂量组对急性肾盂肾炎大鼠CD4、CD8及其比值的影响(
Figure PCTCN2017082173-appb-000034
n＝10)
Figure PCTCN2017082173-appb-000035
注：与假手术组比较：**P<0.01，*P<0.05；与模型组比较：△△P<0.01，△P<0.05；与阳性药组比较：##P<0.01，#P<0.05。
表15各剂量组对急性肾盂肾炎大鼠血清中Ucr、Scr、IL-1α、IL-1β含量的影响(
Figure PCTCN2017082173-appb-000036
n＝10)
Figure PCTCN2017082173-appb-000037
注：与假手术组比较：**P<0.01，*P<0.05；与模型组比较：△△P<0.01，△P<0.05；与阳性药组比较：##P<0.01，#P<0.05。
表15(续)各剂量组对急性肾盂肾炎大鼠血清中IL-6、IL-10、MCP-1、CXCL-2含量的影响(
Figure PCTCN2017082173-appb-000038
n＝10)
Figure PCTCN2017082173-appb-000039
注：与假手术组比较：**P<0.01，*P<0.05；与模型组比较：△△P<0.01，△P<0.05；与阳性药组比较：##P<0.01，#P<0.05。
结果表明：两种化合物能显著降低急性肾盂肾炎模型大鼠肾脏指数和膀胱指数(见表2)，减轻肾脏组织及尿液细菌感染程度(表12，图1和图2)，明显减轻肾脏组织中皮髓结构及肾盂、肾盏等组织结构病变及慢性炎细胞浸润或脓肿(图3)。而且，两种化合物对急性肾盂肾炎的治疗效果主要是通过调节CD4、CD8及CD4/CD8提高模型大鼠自身免疫功能，抑制IL-1α、IL-1β及IL-6等炎症因子的表达，降低炎症反应来实现的。同时上述结果表明，化合物LM49及A8高剂量组调节CD4/CD8明显优于阳性药，显示了重要的应用前景。
2)两种化合物对慢性肾炎治疗作用的结果
表16各剂量组对慢性肾炎大鼠尿液中24h尿蛋白定量的影响(
Figure PCTCN2017082173-appb-000040
n＝10)
Figure PCTCN2017082173-appb-000041
注：与对照组比较：**P<0.01，*P<0.05；与模型组比较：△△P<0.01，△P<0.05；与阳性药组比较：##P<0.01，#P<0.05。
表17各剂量组对慢性肾炎大鼠血清中血肌酐(Scr)、尿素氮(BUN)的影响(
Figure PCTCN2017082173-appb-000042
n＝10)
Figure PCTCN2017082173-appb-000043
注：与对照组比较：**P<0.01，*P<0.05；与模型组比较：△△P<0.01，△P<0.05；与阳性药组比较：##P<0.01，#P<0.05。
表18各剂量组对慢性肾炎大鼠血清中IL-1α、IL-1β含量的影响(
Figure PCTCN2017082173-appb-000044
n＝10)
Figure PCTCN2017082173-appb-000045
注：与对照组比较：**P<0.01，*P<0.05；与模型组比较：△△P<0.01，△P<0.05；与阳性药组比较：##P<0.01，#P<0.05。
表18(续)各剂量组对慢性肾炎大鼠血清中IL-6、IL-10含量的影响(
Figure PCTCN2017082173-appb-000046
n＝10)
Figure PCTCN2017082173-appb-000047
注：与对照组比较：**P<0.01，*P<0.05；与模型组比较：△△P<0.01，△P<0.05；与阳性药组比较：##P<0.01，#P<0.05。
结果表明：两种化合物均能显著降低尿蛋白量，显著改善肾功能，高剂量组明显优于阳性药组(表16)。而且，两种化合物对慢性肾炎的治疗效果是通过抑制IL-1α、IL-1β及IL-6等炎症因子的表达，降低炎症反应来实现的。
结论：
两种化合物对急性肾盂肾炎模型大鼠和慢性肾炎模型大鼠均具有明确的治疗作用，尤其是高剂量组效果更显著。这也显示了该化合物具有很好的应用开发前景。

Claims (8)

  1. 化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮和4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮在制备抗Ⅱ型糖尿病肾病药物中的应用。
  2. 抗Ⅱ型糖尿病肾病的药物，所述药物的活性成分为化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮或4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮。
  3. 根据权利要求2所述的抗Ⅱ型糖尿病肾病的药物，其特征是该药物中还包括与所述活性成分组合使用的普通药用载体或赋形剂。
  4. 根据权利要求2或3所述的抗Ⅱ型糖尿病肾病的药物，其特征是所述药物为注射剂、片剂、滴丸剂、固体分散体或胶囊剂。
  5. 化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮和4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮在制备治疗急性肾盂肾炎和慢性肾炎药物中的应用。
  6. 治疗急性肾盂肾炎和慢性肾炎的药物，所述药物的活性成分为化合物4,5,2'-三羟基-2,5'-二溴二苯甲酮或4,5,2'-三(4-吗啉甲酰氧基)-2,5'-二氯二苯甲酮。
  7. 根据权利要求6所述的治疗急性肾盂肾炎和慢性肾炎的药物，其特征是该药物中还包括与所述活性成分组合使用的普通药用载体或赋形剂。
  8. 根据权利要求6或7所述的治疗急性肾盂肾炎和慢性肾炎的药物，其特征是所述药物为固体分散体、片剂、滴丸剂、胶囊剂或注射剂。
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