WO2021239106A1 - 钾atp通道调节剂在制备抗糖尿病肾病药物中的应用 - Google Patents

钾atp通道调节剂在制备抗糖尿病肾病药物中的应用 Download PDF

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WO2021239106A1
WO2021239106A1 PCT/CN2021/096736 CN2021096736W WO2021239106A1 WO 2021239106 A1 WO2021239106 A1 WO 2021239106A1 CN 2021096736 W CN2021096736 W CN 2021096736W WO 2021239106 A1 WO2021239106 A1 WO 2021239106A1
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diabetic nephropathy
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rat
pharmaceutical composition
diabetic
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陈渊
朱江
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杭州起岸生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61K31/54—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 sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/549—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 sulfur as the ring hetero atoms, e.g. sulthiame having two or more nitrogen atoms in the same ring, e.g. hydrochlorothiazide
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the invention relates to the technical field of chemical medicine for potassium ATP (K ATP ) channel modulators, in particular to a new pharmaceutical application of a K ATP channel modulator, that is, the application of the potassium ATP channel modulator in the preparation of anti-diabetic nephropathy drugs.
  • K ATP potassium ATP
  • Diabetic Kidney Disease is one of the most important microvascular complications of diabetes, the main cause of chronic kidney disease, and the most common disease leading to end-stage renal disease (ESRD).
  • ESRD end-stage renal disease
  • most diabetic patients who died of cardiovascular disease also suffered from diabetic nephropathy, and the cause of death was closely related to diabetic nephropathy (Afkarian M, Sachs MC, Kestenbaum B, Hirsch IB, Tuttle KR, Himmelfarb J, de Boer IH.Kidney disease and increased mortality risk in type 2diabetes[J].Journal of the American Society of Nephrology.2013,24(2):302-308.).
  • Type 1 diabetes T1D
  • type 2 diabetes T2D
  • urine albumin Dwyer JP, Parving HH, Hunsicker LG, Ravid M, Remuzzi G, Lewis JB. Renal dysfunction in the presence of normoalbuminuria in type 2 diabetes: results from the DEMAND Study[J]. Cardiorenal Med. 2012, 2(10):1-10.
  • Diabetic nephropathy has been called a "global medical disaster".
  • type 2 diabetes is the leading cause of kidney disease and the fifth fastest-growing cause of death in the world.
  • the pathological changes of diabetic nephropathy are mainly caused by long-term hyperglycemia.
  • the main functional unit of the kidney is the glomerulus, which is composed of approximately 1 million glomeruli.
  • the pathological changes are mainly manifested as the initial compensatory high filtration, which gradually becomes low filtration over time. This is mainly due to the thickening of the glomerular basement membrane and the widening of the mesangium, until the whole can be closed. Filtration of the glomerulus.
  • Stage I proliferative hyperfiltration stage: At this stage, the structure of the glomerulus is normal and there is no pathological change, but the kidneys are enlarged and the glomerular filtration rate (GFR) increases. After insulin treatment, the After blood sugar control, GFR can decrease;
  • Stage II Histological changes occur at this stage. Pathological examination can reveal that the glomerular basement membrane (GBM) is slightly thickened, and the urine albumin excretion rate (UAE) in the kidney is normal ( ⁇ 30mg/24h) (such as At rest), or intermittent microalbuminuria (such as after exercise, stress state), but the disease is still reversible;
  • GBM glomerular basement membrane
  • UAE urine albumin excretion rate
  • Stage III (early diabetic nephropathy stage): Pathological examination at this stage can reveal thickening of glomerular basement membrane and further widening of mesangium, UAE is 30 ⁇ 300mg/24h, showing persistent microalbuminuria and elevated blood pressure;
  • Stage IV (clinical diabetic nephropathy stage): Pathological examination at this stage can reveal severe changes in glomerulopathy (such as glomerulosclerosis, focal tubular atrophy, and interstitial fibrosis), persistent proteinuria, and sustainability. Hypertension, edema, dyslipidemia, and decreased GFR;
  • Stage V renal insufficiency: end-stage renal failure, elevated serum creatinine, high blood pressure, clinical manifestations of uremia; GFR ⁇ 15 or need dialysis.
  • Insulin resistance is associated with the development of hypercompensatory glomerular filtration, which is common in the initial stage of diabetic nephropathy (Mogensen CE. Early glomerular hyperfiltration in insulin-dependent diabetes and late nephropathy [J]. Scandinavian Journal of Clinical and Laboratory Investigation. 1986 , 46(3):201-206.), the interaction of metabolism and hemodynamics plays a key role in the pathophysiological mechanisms leading to the progression of kidney disease (Caramori ML, Fioretto P, Mauer M. Low glomerular filtration rate in normoalbuminuric type 1 diabetic patients: an indicator of more advanced glomerular Lesions[J].Diabetes.2003,52(4):1036-1040.).
  • type 2 diabetes patients account for a large proportion. But in fact, the incidence of diabetic nephropathy in type 2 diabetes is less than that in type 1 diabetes. The incidence in type 2 diabetes is about 20% to 25%, while the incidence of type 1 diabetes is about 20% to 25%. The prevalence is about 10% higher than that of type 2 diabetes; however, because there are many more patients with type 2 diabetes than type 1 diabetes, there are more patients with type 2 diabetes suffering from diabetic nephropathy in the total number.
  • Hyperglycemia is the most important risk factor for diabetic nephropathy. Other risk factors include high blood pressure, smoking, dyslipidemia, proteinuria, glomerular hyperfiltration and dietary factors.
  • nephropathy mainly manifests as thickening of the glomerulus and tubule basement membrane, accompanied by progressive glomerular expansion (diffuse or nodular), resulting in a gradual decrease in the filtration surface of the glomerulus, and at the same time Changes in interstitial morphology and hyalinization of afferent and efferent glomerular arterioles occur.
  • kidney damage is heterogeneous and more complicated than in individuals with type 1 diabetes.
  • ACE inhibitors drugs for the treatment of diabetic nephropathy
  • SGLT-2 inhibitors drugs for the treatment of diabetic nephropathy
  • Captopril is a new drug developed by Bristol-Myers Squibb (BMS) and was approved by the US FDA for the treatment of type 1 diabetes complications in October 1993 Diabetic nephropathy.
  • BMS Bristol-Myers Squibb
  • K ATP-sensitive potassium (K ATP ) channels play an important role in a variety of tissues by coupling cell metabolism and electrical activity.
  • K ATP channels are assembled into different subtypes or subtypes in various combinations of SUR and Kir subunits.
  • the combination of SUR1 and Kir6.x subunits usually forms adipocytes and pancreatic B-cell type K ATP channels, while the combination of SUR2A and Kir6.x, and the combination of SUR2B and Kir6.x usually form heart and smooth muscle type K ATP channels (Babenko AP , Aguilar-Bryan L, Bryan JA view of sur/kir 6.x, KATP channels. Annu Rev Physiol 1998; 60:667-687).
  • Such potassium channels are inhibited by intracellular ATP and activated by intracellular diphosphate nucleotides.
  • This K ATP channel connects the metabolic state of the cell with the plasma membrane potential, and in this way plays a major role in regulating cell activity.
  • K ATP channel Since the K ATP channel can open and close the channel by sensing the ratio of ADP to ATP in the cell, when at rest, K ATP activation causes membrane hyperpolarization, and its inhibitory effect produces membrane depolarization. Therefore, it can be metabolized by the cell.
  • the electrical activity of the plasma membrane is linked to study K ATP channels.
  • K ATP channels can protect nerve cells from apoptosis after stroke.
  • K ATP channels can also regulate male reproductive behavior. Human memory is also related to K ATP channels in the brain. Related and so on. However, there is no literature report on the correlation between potassium ATP channels and diabetic nephropathy.
  • Diazoxide (Diazoxide) is also known as antihypertensive, chemical name: 7-chloro-3-methyl-2hydro-1,2,4-benzothiadiazine 1,1-dioxide, CAS number: 364-98-7, the molecular formula is: C 8 H 7 ClN 2 O 2 S, the structural formula is:
  • Diazoxide is a K ATP channel agonist, which is known to be used in the treatment of the following diseases: 1) hypertensive emergencies; 2) hyperinsulinemic hypoglycemia; 3) idiopathic hypoglycemia in young children.
  • diseases 1) hypertensive emergencies; 2) hyperinsulinemic hypoglycemia; 3) idiopathic hypoglycemia in young children.
  • relevant literature reports on the application of diazoxide for expansion indications are as follows:
  • the Chinese invention patent with publication number CN 101043879A discloses that diazoxide can be used to treat obesity and mental illness.
  • US invention patent of US 5,629,045 discloses that diazoxide can be used for topical ophthalmic administration.
  • the Chinese invention patent with publication number CN107106500A discloses that diazoxide can be used to treat Prader-Willi syndrome or Smith-Magilli syndrome.
  • K ATP channel openers such as diazoxide, crocaline, pinacidil, Nicotil, Apcarin, etc.
  • K ATP channel openers can be used for the treatment of diabetic nephropathy; in particular, whether diazoxide at a specific dosage of administration is effective in preventing or treating diabetic nephropathy at an early stage is not known to those skilled in the art.
  • the technical problem to be solved by the present invention is to provide a new pharmaceutical application of diazoxide and K ATP channel agonist, which can be used to treat diabetic nephropathy, especially for the early stage of diabetic nephropathy.
  • the invention provides an application of a potassium ATP channel regulator in the preparation of anti-diabetic nephropathy drugs.
  • diabetic nephropathy is diabetic nephropathy complicated by type 1 diabetes and/or type 2 diabetes.
  • staged evolution of the diabetic nephropathy is in stage I, stage II or stage III.
  • the potassium ATP channel modulator includes a potassium ATP channel opener or a potassium ATP channel inhibitor.
  • the potassium ATP channel modulator is selected from one of diazoxide, crocaline, pinacidil, nicotidil, apracarin, quinetazolone, minoxidil, and nicodipine. kind.
  • the potassium ATP channel opener is diazoxide at a dosage of 0.5-5 mg/kg.
  • the present invention also provides a pharmaceutical composition for the treatment of diabetic nephropathy, which contains the aforementioned potassium ATP channel modulator as an active ingredient.
  • the pharmaceutical composition contains pharmaceutically acceptable excipients.
  • the pharmaceutical composition is used to prevent or treat early diabetic nephropathy, specifically referring to stage I, stage II, or stage III in the evolution of stages of diabetic nephropathy.
  • the dosage form of the pharmaceutical composition is selected from one of tablets, capsules, granules, injections, patches, and gels.
  • the aforementioned pharmaceutically acceptable auxiliary materials are one or more of fillers, disintegrants, binders, diluents, lubricants, regulators, solubilizers, cosolvents, and emulsifiers.
  • the "pharmaceutical composition” of the present invention refers to a preparation of one or more compounds of the present invention or their salts and a carrier generally accepted in the art for delivering biologically active compounds to organisms (such as humans).
  • the purpose of the pharmaceutical composition is to facilitate the administration and delivery of the organism.
  • pharmaceutically acceptable carrier refers to a substance that is co-administered with the active ingredient and facilitates the administration of the active ingredient, including but not limited to acceptable use for humans or animals (such as livestock) approved by the China Food and Drug Administration Any glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, disintegrant, suspending agent, stabilizer, isotonic Agent, solvent or emulsifier. Examples include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and various starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • the pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, Inhalants, gels, microspheres and aerosols, etc.
  • the pharmaceutical composition of the present invention can be manufactured by methods well known in the art, such as conventional mixing method, dissolution method, granulation method, sugar-coated pill method, grinding method, emulsification method, freeze-drying method and the like.
  • the administration route of the compound of the present invention or its pharmaceutically acceptable salt or its pharmaceutical composition includes but not limited to oral, rectal, transmucosal, enteral administration, or topical, transdermal, inhalation, parenteral , Sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.
  • the preferred route of administration is oral administration.
  • the pharmaceutical composition can be formulated by mixing the active compound with a pharmaceutically acceptable carrier well known in the art.
  • a pharmaceutically acceptable carrier well known in the art.
  • These carriers enable the compound of the present invention to be formulated into tablets, pills, lozenges, sugar coatings, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.
  • tablets can be obtained by combining the active ingredient with one or more solid carriers, granulating the resulting mixture if necessary, and adding a small amount of excipients if necessary Processed into mixtures or granules to form tablets or tablet cores.
  • the tablet core can be combined with coating materials that are optionally suitable for enteric dissolution, and processed into a coating preparation form that is more conducive to absorption by organisms (such as humans).
  • the present invention has the following beneficial effects:
  • the present invention uses streptozotocin (STZ) to model diabetic rats. After modeling, the rats are given a high-fat diet to form diabetic nephropathy. Urine microalbumin (mALB) is used as an indicator to test whether the rats enter diabetic nephropathy. Rat body weight and blood glucose level are indicators to observe whether the model is successful. The results of the experiment found that the administration of diazoxide to diabetic rats can delay the progression of kidney damage in diabetic rats. Furthermore, use the same diabetic rat model to administer K ATP channel openers such as crocaline, pinacidil, nicotidil, apracarin, quinetazolone, minoxidil, nicotinia, etc. Judging from the results of urine microalbumin detection, there are varying degrees of reducing urine microalbumin in diabetic nephropathy rats.
  • K ATP channel openers such as crocaline, pinacidil, nicotidil, apracarin, quinet
  • Figure 1 shows the effect of each experimental group in Example 2 on blood glucose.
  • Figure 2 shows the effect of each experimental group in Example 2 on body weight.
  • Figure 3 shows the effect of each experimental group in Example 2 on mALB.
  • group A is the blank control group
  • group B is the negative control group
  • group C is the dosing group with 0.5 mg/kg diazoxide
  • group D is the dosing group with 5 mg/kg diazoxide .
  • Figure 4 shows the effect of the experimental group of different K ATP channel openers in Example 3 on mALB.
  • Figure 5 shows the effects of each experimental group in Example 2 on rat kidney glomeruli.
  • Negative control 1) Left: 6-month-old rat, kidney HE ⁇ 400, glomerulus in good condition, obvious glomerular volume enlargement, renal tubule damage; Right: 6-month-old rat, kidney HE ⁇ 400, the glomerulus obviously increases in volume;
  • the sources of the experimental instruments, experimental actuals, and experimental samples involved are as follows:
  • Step 1 Measure 80 mL of deionized water and place it in a plastic beaker (due to the large amount of heat generated during the NaOH dissolution process, the glass beaker is not used to avoid the glass beaker from bursting);
  • Step 2 Weigh 20g of NaOH and slowly add it into the beaker, stirring while adding;
  • Step 3 After the NaOH is completely dissolved, dilute the volume to 100 mL with deionized water;
  • Step 4 Transfer the prepared solution to a plastic container and store at room temperature.
  • STZ damages pancreatic ⁇ -cells rapidly and excessively through chemical toxicity (Saini K, Thompson C, Winterford CM, Walker NI, Cameron DP. Streptozotocin at low doses induces apoptosis and at high doses causes causes necrosis inline, INS ⁇ 1 cell J].International Union of Biochemistry and Molecular Biology Life.1996,39(6):1229-1236.), the pathophysiological characteristics of rats modeled by STZ are similar to that of humans, and blood sampling can be repeated to monitor changes in indicators. Enough kidney tissue can be obtained for subsequent tissue section analysis. Therefore, STZ was chosen to model the diabetic rat model. In addition, due to the greater toxicity of STZ, multiple injections at low doses can better establish a model. After modeling, a high-fat diet was taken every day until the end of the experiment. High-fat diet can cause diabetic nephropathy in STZ rats.
  • Urine microalbumin is an indicator of early kidney damage diagnosis. This experiment uses mALB as an indicator to test whether the rat enters diabetic nephropathy. The weight and blood glucose level of the rat are only used to observe whether the model is successful.
  • mALB is one of the most important monitoring indicators for nephropathy. Clinically, mALB>20 ⁇ g/min is nephropathy, and there is kidney damage. Because the mALB measurement results of humans and mice are different, and different kits have certain errors, this experiment uses the difference between the dosing group (group C) and the negative control group (group B) to judge the test results of this experiment is it effective. In this experiment, the normal value of mALB only uses the data of group A (blank control group) as the reference value.
  • the changes of the glomerulus mainly include the expansion of the mesangial matrix, any degree of hyalinization of small arteries, thickening of the basement membrane and interstitial fibrosis.
  • Rat urine collection Urine was collected through a metabolic cage. Urine was collected 24 hours later, the total amount of urine was recorded, and the 24-hour urine output was calculated. Take 1ml of urine, centrifuge and take the supernatant, and store it at -80°C for testing.
  • Rat blood collection 3mL of rat blood was collected by trimming the tail. The blood was collected in a medical blood test tube containing a coagulant. After standing at room temperature for 2 hours or overnight at 4°C, centrifuged at 1000 rcf for 20 minutes, and the supernatant was taken at -80 Store at °C for testing. Or, collect 3 mL of rat blood by trimming the tail, collect the blood in a test tube of lithium heparin, centrifuge at 1000 rcf for 20 minutes, take the supernatant, and store it at -80°C for testing.
  • Collect 24h urine samples of rats for testing The collected samples are centrifuged at 1000 rcf in a centrifuge at 4°C for 20 minutes within 15 minutes, and the supernatant is taken to test the mALB value, or the mALB value to be measured is stored at -20°C.
  • the standard is diluted to a corresponding gradient. Take a bottle of standard product, add 1mL of standard product diluent (the concentration is 1000 ⁇ g/mL at this time) and mix gently. Let it stand at room temperature for 10 minutes, and gently shake it every 2 minutes. After mixing, dilute it to 100 ⁇ g/mL, take 4 EP tubes (add 600 ⁇ L of standard diluent to each EP tube), and dilute the standard with a concentration of 100 ⁇ g/mL three times to 33.33 ⁇ g/mL, 11.11 ⁇ g/mL, 3.70 ⁇ g/mL, 1.23 ⁇ g/mL, standard dilution as a blank hole (0 ⁇ g/mL).
  • detection solution B Before each use, detection solution B needs to be centrifuged at 5000 rcf for 10 seconds to deposit the liquid on the tube wall or bottle cap to the bottom of the tube. Dilute the test diluent B with 1:100 before use, and mix it thoroughly with a shaker. Before use, calculate the required amount for dilution by 100 ⁇ L/well (add 0.1-0.2mL is required).
  • Concentrated washing liquid Dilute the concentrated washing liquid by 30 times (the amount of dilution is calculated based on the amount of your own sample).
  • this example aims to investigate whether high, medium, and low doses of diazoxide have a therapeutic effect on diabetic nephropathy.
  • group B was the rats that had only been injected with STZ, as a negative control
  • group C was gavage with 0.5 mg/kg of diazoxide per day (low-dose administration group)
  • D Groups were given diazoxide at a dose of 5 mg/kg per day (medium-dose administration group)
  • group E was given diazoxide at a dose of 50 mg/kg per day (high-dose administration group). Due to the large body surface area of rats, the doses of C, D, and E groups after conversion are equivalent to human 0.095mg/Kg, 0.95mg/Kg, 9.5mg/Kg (conversion based on literature: Anroop B Nair, Shery Jacob. A simple practice guide for dose conversion between animals and human.Journal of Basic and Clinical Pharmacy.2016,7(2):27-31.), binding to albumin is 95%, similar to humans. Rats in all groups were fed high-fat diet.
  • the rats in the high-dose E group died before the model of diabetic nephropathy, 13 of them died in the first four months, and the other 5 died in the fifth and sixth months. It is speculated that the death may be related to the blood glucose of the diabetic rats. Excessive elevation is related; therefore, it is not included in the statistics of Table 1, Figure 1, Figure 2, Figure 3, and Figure 5.
  • the average blood glucose of the blank control group ( ⁇ ) is between 4.0 and 4.9 mmol/L, while the STZ control group ( ⁇ ) and the 5 mg/kg dosing group ( ⁇ )
  • the average blood glucose is basically maintained between 18-19mmol/L, which is higher than the 16.7mmol/L specified in the literature (see step 2 of the aforementioned modeling method); moreover, the rats in the STZ control group and the 5mg/kg dosing group are both Already have the symptoms of diabetes, such as irritability, unresponsiveness, dry hair color, and yellowing, indicating that the model of diabetes has been successfully modeled.
  • the weight of the blank control group has been consuming high-fat feed for a long time without any drug effects (including STZ), so the weight has been steadily increasing and has remained the highest; the STZ control group and the 5 mg/kg dosing group are due to STZ damage (the average blood glucose value of the group) It is stable above 16.7mmol/L), so the body weight is maintained at a low level (far lower than the blank control group).
  • STZ damage the average blood glucose value of the group
  • P>0.05 There is no statistically significant difference in the previous body weight between the STZ control group and the 5 mg/kg dosing group (P>0.05) ).
  • the STZ control group and the 5mg/kg dosing group had extremely significant differences in their body weights (p ⁇ 0.01).
  • group C and group D are significantly different (p ⁇ 0.05), indicating that both low-dose and medium-dose diazoxide still have antimicroalbuminuria and diabetes delay effects at this time.
  • group C Compared with group B, the mALB value of group C and group D decreased to a certain extent, among which group D showed a significant difference (p ⁇ 0.05), and group C did not show a significant difference (p>0.05).
  • 2B group (negative control group) has obvious glomerular enlargement and renal tubule damage
  • group C taking diazoxide at a dose of 5 mg/kg as an example, part of renal tubule degeneration in group C (see the right figure of group C in Fig. 5), group B and group C both had renal tubular lesions of varying degrees .
  • this example aims to investigate whether other K ATP agonists except diazoxide have a therapeutic effect on diabetic nephropathy.

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Abstract

一种钾ATP通道调节剂的制药新用途，即钾ATP通道调节剂(如二氮嗪、克罗卡林、吡那地尔、尼克地尔、阿普卡林等)在制备抗糖尿病肾病药物中的应用；还提供了一种以钾ATP通道调节剂作为活性成分的药物组合物可用于预防或治疗糖尿病肾病的用途。通过链脲佐菌素(STZ)造模糖尿病大鼠，建模后给予大鼠高脂饮食之形成糖尿病肾病，以尿微量白蛋白(mALB)为指标检验大鼠是否进入糖尿病肾病，以大鼠体重及血糖水平为指标观察模型是否成功。实验结果发现，对糖尿病大鼠给药二氮嗪等钾ATP通道开放剂后，能延缓糖尿病大鼠出现肾损伤的进程。

Description

钾ATP通道调节剂在制备抗糖尿病肾病药物中的应用 技术领域
本发明涉及钾ATP(K ATP)通道调节剂的化药技术领域，具体涉及一种K ATP通道调节剂的制药新用途，即钾ATP通道调节剂在制备抗糖尿病肾病药物中的应用。
背景技术
糖尿病肾病(Diabetic Kidney Disease,DKD)是糖尿病最主要的微血管并发症之一，是慢性肾病的主要原因，也是导致终末期肾病(End stage renal disease，ESRD)的最常见病症。事实上，大部分因心血管疾病死亡的糖尿病患者同时也患有糖尿病肾病，且死亡原因于糖尿病肾病有很大的关联(Afkarian M,Sachs MC,Kestenbaum B,Hirsch IB,Tuttle KR,Himmelfarb J,de Boer IH.Kidney disease and increased mortality risk in type 2diabetes[J].Journal of the American Society of Nephrology.2013,24(2):302-308.)。无论是1型糖尿病(T1D)患者还是2型糖尿病(T2D)患者，都会出现明显的肾功能损害和一定的尿白蛋白(Dwyer JP,Parving HH,Hunsicker LG,Ravid M,Remuzzi G,Lewis JB.Renal dysfunction in the presence of normoalbuminuria in type 2 diabetes:results from the DEMAND Study[J].Cardiorenal Med.2012,2(10):1-10.)。随着糖尿病患者的数量越来越多，糖尿病肾病患者的数量也在逐步增加，糖尿病肾病已被称为“全球性医疗灾难”。在美国，2型糖尿病是导致肾脏疾病的首要原因，同时也是全球增长速度第五快的死亡病因。
糖尿病肾病的病理改变主要由长期的高血糖引起。肾的主要功能单位是肾小球，由大约有100万肾小球组成。病理改变主要表现为初期的代偿性高滤过，随着时间的推移逐渐变成低滤过，这主要是由于肾小球基底膜的增厚及系膜的增宽，直至最终可以关闭整个肾小球的滤过。
根据糖尿病肾病的病理生理特点和演变过程，目前学界采用“Mogensen分期”的分期标准，将糖尿病肾病分为5期(赵进喜,王世东,李靖,黄为钧.糖尿病肾脏病分期辨证规范与疗效评定方案及其研究[J].世界中医药.2017,12(1):1-4.)：
I期(增生高滤期)：此阶段肾小球的结构正常未发生病理性的改变，只是肾脏增大、肾小球的滤过率(Glomerular Filtration Rate，GFR)增加，经胰岛素治疗、高血糖控制后，GFR可下降；
Ⅱ期(临床前期)：此阶段发生组织学变化，病理检查可发现肾小球基底膜(GBM)轻度增厚，肾脏内尿白蛋白排出率(UAE)正常(<30mg/24h)(如休息时)，或呈间歇性微量 白蛋白尿(如运动后，应激状态)，但病变仍属可逆；
Ⅲ期(早期糖尿病肾病期)：此阶段病理检查可发现肾小球基底膜增厚及系膜进一步增宽，UAE为30～300mg/24h，呈持续性微量白蛋白尿，血压升高；
IV期(临床糖尿病肾病期)：此阶段病理检查可发现肾小球病变更重(如肾小球硬化、灶性肾小管萎缩及间质纤维化)，呈持续性蛋白尿，可持续性伴有高血压、水肿、血脂异常，GFR下降；
V期(肾功能不全期)：终末期肾衰竭，血清肌酐升高，高血压，临床表现尿毒症；GFR<15或需要透析。
胰岛素抵抗与肾小球滤过代偿性过高的发展相关，常见糖尿病肾病初始阶段(Mogensen CE.Early glomerular hyperfiltration in insulin-dependent diabetics and late nephropathy[J].Scandinavian Journal of Clinical and Laboratory Investigation.1986,46(3):201-206.)，代谢和血液动力学的相互作用对导致肾脏疾病进展的病理生理机制起关键作用(Caramori ML,Fioretto P,Mauer M.Low glomerular filtration rate in normoalbuminuric type 1 diabetic patients:an indicator of more advanced glomerular lesions[J].Diabetes.2003,52(4):1036-1040.)。在大多数情况下，蛋白尿和肾小球滤过率降低通常同时发生，这也就意味着在产生尿白蛋白时GFR会减少，此时代偿期已过。有研究表明，少数患者可能在不增加UAE的情况下患有糖尿病肾病(Mogensen CE.Glomerular filtration rate and renal plasma flow in short-term and long-term juvenile diabetes mellitus[J].Scandinavian Journal of Clinical&Laboratory Investigation.1971,28(1):91-100.)，也就是说当尿白蛋白值在正常范围内时，一些患者的GFR已经在降低了。大约10％的T2D患者的GFR较低而没有微量白蛋白尿，在T1D患者和微量白蛋白尿的肾病患者中也有观察到这种现象(Perkins BA,Krolewski AS.Early nephropathy in type 1diabetes:the importance of early renal function decline[J].Current Opinion in Nephrology and Hypertension.2009,18(3):233-240.)。因此，检测尿微量白蛋白是早期症状，而肾小球滤过率的筛查可以帮助发现较严重的糖尿病肾病。
糖尿病肾病患者中，2型糖尿病病人占很大的比率。但实际上，糖尿病肾病在2型糖尿病中的患病几率会比在1型糖尿病中的患病几率小，在2型糖尿病中的病率大约在20％到25％左右，而1型糖尿病的患病率会比2型糖尿病高大约10％；但是由于2型糖尿病患者比1型糖尿病患者多得多，在总数上2型糖尿病病人患糖尿病肾病就显得多多了。高血糖是诱发糖尿病肾病的最主要危险因素，其他风险因素还包括高血压，吸烟，血脂异常，蛋白尿，肾小球高滤过和饮食因素等等。
糖尿病肾病前期的治疗主要是延缓疾病的发展或进展。1型糖尿病患者中，肾病主要表 现为肾小球和肾小管基底膜增厚，伴有渐进性肾小球膜扩张(弥漫性或结节性)，导致肾小球滤过面逐渐减少，同时发生间质形态改变和传入和传出肾小球小动脉的透明化。在患有2型糖尿病的患者中，肾损伤是异质的并且比患有1型糖尿病的个体更复杂。实现最佳代谢控制，使用阻断肾素—血管紧张素—醛固酮系统的药物治疗高血压(<130/80mmHg)和血脂异常(LDL胆固醇<100mg/dl)是有效的策略，使其能够预防微量白蛋白尿的发展，延缓肾病患者更晚期的进展。
目前，治疗糖尿病肾病的药物有两类：ACE抑制剂和SGLT-2抑制剂。ACE抑制剂中的代表药为卡托普利(Captopril)，这是一款由百时美-施贵宝(BMS)公司研发的新药，于1993年10月被美国FDA批准用于治疗1型糖尿病并发的糖尿病肾病。ACE抑制剂治疗糖尿病肾病的机理目前学术界尚未明确，多数学者认为它干预的肾素血管紧张素系统，降低了肾小管的出球小动脉而降低肾小管压力，实现治疗糖尿病肾病；临床表现为Captopril可以延迟、减慢白蛋白尿的进展(万晓琳.卡托普利治疗糖尿病性肾病[J].医药导报,1995(03):126)。SGLT-2抑制剂中的代表药为卡格列净(canagliflozin)，这是一款由强森集团旗下的杨森公司研发的新药，于2013年3月被美国FDA批准用于治疗2型糖尿病，于2019年10月被美国FDA批准用于治疗2型糖尿病并发的糖尿病肾病，这也是目前唯一一款获批在2型糖尿病(T2D)患者中既可以治疗糖尿病肾病，又能够降低因心力衰竭住院风险的降糖疗法。
ATP敏感性钾(K ATP)通道通过将细胞代谢与电活动偶联而在多种组织中起重要的作用。K ATP通道以SUR和Kir亚单位的各种组合装配而成得不同的亚型或亚类存在。SUR1与Kir6.x亚单位得组合通常形成脂细胞和胰腺B-细胞类型K ATP通道，而SUR2A与Kir6.x组合、SUR2B与Kir6.x组合通常形成心脏型和平滑肌类型K ATP通道(Babenko AP，Aguilar-Bryan L，Bryan J.A view of sur/kir6.x,KATP channels.Annu Rev Physiol 1998；60:667-687)。这类钾通道受细胞内ATP抑制，且由细胞内二磷酸核苷酸激活。这种K ATP通道使细胞的代谢状态与血浆膜电势相联系，并以该方式在调节细胞活性中起主要的作用。
由于K ATP通道能够通过感知细胞内的ADP和ATP比值来开放和闭合通道，静止时，K ATP激活引起膜超极化，而其抑制作用则产生膜去极化，因此可以通过将细胞代谢与质膜的电活动相联系来研究K ATP通道。近年来，人们研究了K ATP通道在葡萄糖稳态和缺血保护的作用，发现了磺酰脲类药物可以降低血糖。此外，人们还发现了K ATP通道的一些其他作用，例如，通过K ATP通道可以保护中风后神经细胞的凋亡，K ATP通道还可以调节男性生殖行为，人类记忆也与大脑中的K ATP通道有关等等。但是，尚未有文献报道钾ATP通道与糖尿病肾病的相关性。
二氮嗪(Diazoxide)别名降压嗪，化学名为：7-氯-3-甲基-2氢-1,2,4-苯并噻二嗪1,1-二 氧化物，CAS号为：364-98-7，分子式为：C 8H 7ClN 2O 2S，结构式为：
Figure PCTCN2021096736-appb-000001
二氮嗪系K ATP通道激动剂，已知可用于如下病症的治疗：1)高血压急症；2)高胰岛素性低血糖病症；3)幼儿特发性低血糖病症。此外，关于二氮嗪的扩增适应症应用，相关文献报道如下：
公开号为CN 101043879A的中国发明专利，公开了二氮嗪可用于治疗肥胖和精神病。
US 5629045的美国发明专利，公开了二氮嗪可用于局部眼科施用。
公开号为CN 107106500A的中国发明专利，公开了二氮嗪可用于治疗普拉德-威利综合征或史密斯-马吉利综合征。
经申请人检索发现，目前尚未有文献报道K ATP通道激动剂与糖尿病肾病的治疗相关性，也未见文献报道K ATP通道开放剂(如二氮嗪、克罗卡林、吡那地尔、尼克地尔、阿普卡林等)可用于治疗糖尿病肾病；尤其是选择特定给药剂量的二氮嗪对预防或治疗早期阶段的糖尿病肾病是否有效，本领域技术人员无法知悉。
发明内容
本发明要解决的技术问题在于，提供了一种二氮嗪以及K ATP通道激动剂的制药新用途，可用于治疗糖尿病肾病，特别针对糖尿病肾病的早期阶段。
为此，本发明采取了以下技术方案：
本发明提供了一种钾ATP通道调节剂在制备抗糖尿病肾病药物中的应用。
优选的，糖尿病肾病为1型糖尿病和/或2型糖尿病并发的糖尿病肾病。
更优选的，该糖尿病肾病的分期演变过程处于I期、Ⅱ期或Ⅲ期。
优选的，钾ATP通道调节剂包括钾ATP通道开放剂或钾ATP通道抑制剂。
更优选的，钾ATP通道调节剂选自二氮嗪、克罗卡林、吡那地尔、尼克地尔、阿普卡林、喹乙唑酮、米诺地尔、尼古地平中的一种。
优选的，钾ATP通道开放剂为给药剂量为0.5～5mg/kg的二氮嗪。
本发明还提供了一种治疗糖尿病肾病的药物组合物，该药物组合物中包含有如前所述的钾ATP通道调节剂作为活性成分。
优选的，该药物组合物中包含有药学上可接受的辅料。
优选的，该药物组合物用于预防或治疗早期的糖尿病肾病，具体指糖尿病肾病分期演变过程中的I期、Ⅱ期或Ⅲ期。
优选的，该药物组合物的剂型选自片剂、胶囊剂、颗粒剂、注射剂、贴剂、凝胶剂中的一种。
优选的，前述药学上可接受的辅料为填充剂、崩解剂、粘合剂、稀释剂、润滑剂、调节剂、增溶剂、助溶剂、乳化剂中的一种或几种。
相关术语定义：
本发明所述的“药物组合物”，指一种或多种本发明的化合物或其盐与在本领域中通常接受的用于将生物活性化合物输送至有机体(例如人)的载体的制剂。药物组合物的目的是有利于对有机体给药输送。
术语“药学上可接受的载体”，指与活性成份共同给药的、且有利于活性成份给药的物质，包括但不限于药监局许可的可接受的用于人或动物(例如家畜)的任何助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味增强剂、表面活性剂、润湿剂、分散剂、崩解剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。例如，包括但不限于碳酸钙、磷酸钙、各种糖和各类淀粉、纤维素衍生物、明胶、植物油和聚乙二醇。
本发明所述的药物组合物，可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、溶液剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶等等。
本发明所述的药物组合物，可以采用本领域熟知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。
本发明所述的化合物或其药学上可接受的盐或其药物组合物的给药途径，包括但不限于口服、直肠、透黏膜、经肠给药，或者局部、经皮、吸入、肠胃外、舌下、阴道内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。优选的给药途径是口服给药。
对于口服给药，可以通过将活性化合物与本领域熟知的药学上可接受的载体混合，来配制该药物组合物。这些载体能使本发明的化合物被配制成片剂、丸剂、锭剂、糖衣剂、胶囊剂、液体、凝胶剂、浆剂、悬浮剂等，以用于对患者的口服给药。例如，用于口服给药的药物组合物，可采用如下方式获得片剂：将活性成分与一种或多种固体载体合并，如果需要将所得混合物制粒，并且如果需要加入少量的赋形剂加工成混合物或颗粒，以形成片剂或片芯。片芯可与任选适合肠溶的包衣材料结合，加工成更有利于有机体(例如人)吸收的包衣制剂形式。
综上，与现有技术相比，本发明的有益效果在于：
本发明通过链脲佐菌素(STZ)造模糖尿病大鼠，建模后给予大鼠高脂饮食之形成糖尿病肾病，以尿微量白蛋白(mALB)为指标检验大鼠是否进入糖尿病肾病，以大鼠体重及血糖水平为指标观察模型是否成功。实验结果发现，对糖尿病大鼠给药二氮嗪后，能延缓糖尿病大鼠出现肾损伤的进程。进一步，用同样的糖尿病大鼠模型给药克罗卡林、吡那地尔、尼克地尔、阿普卡林、喹乙唑酮、米诺地尔、尼古地平等K ATP通道开放剂，从尿微量白蛋白的检测结果来看，有不同程度的降低糖尿病肾病大鼠尿微量白蛋白的效果。
附图说明
图1为实施例2的各实验组对血糖的影响。
图2为实施例2的各实验组对体重的影响。
图3为实施例2的各实验组对mALB的影响。
图3中，A组为空白对照组，B组为阴性对照组，C组为给药0.5mg/kg二氮嗪的加药组，D组为给药5mg/kg二氮嗪的加药组。
图4为实施例3的不同K ATP通道开放剂实验组对mALB的影响。
图5为实施例2的各实验组对大鼠肾脏肾小球的影响。
图5中，A)空白对照组：1)左：六个月大鼠，肾HE×100，肾小球明显体积增大，肾小球内细胞增生；2)右：实验六个月大鼠，肾HE×400，肾小管状态良好；
B)阴性对照：1)左：实验六个月大鼠，肾HE×400,肾小球状态良好，肾小球明显体积增大，肾小管损伤；右：六个月大鼠，肾HE×400，肾小球明显体积增大；
C)中等剂量加药组：1)左：六个月大鼠，肾HE×400，部分肾小球明显体积增大，细胞数增多，部分肾小球形态良好；右：六个月大鼠，肾HE×400，肾小球体积增大，肾小管空泡变性。
具体实施方式
以下是本发明的具体实施例，对本发明的技术方案做进一步的描述，但是本发明的保护范围并不限于这些实施例。凡是不背离本发明构思的改变或等同替代均包括在本发明的保护范围之内。
本发明中，所涉及的实验仪器、实验实际、实验样品，来源如下：
1、动物材料
分两大组在不同时间进行实验，共205只大鼠，其中75只用于研究二氮嗪，另外130分 别用于其他K ATP通道激动剂，如克罗卡林、吡那地尔、尼克地尔、阿普卡林、喹乙唑酮、米诺地尔、尼古地平的研究。
选用8周SD雄性大鼠205只(由浙江省医学科学院动物实验中心提供)，体重为200g～250g。饲养条件：室温保持在25℃左右，湿度60％左右，昼夜各12h，24h循环透气通风，除特殊实验(如禁食测血糖等)外，所有动物自由进食，自由饮水。
2、实验主要药品及试剂
表1 药品及试剂
Figure PCTCN2021096736-appb-000002
3、实验主要仪器及设备
表1 仪器及设备
Figure PCTCN2021096736-appb-000003
Figure PCTCN2021096736-appb-000004
4、实验主要试剂的配制
1)链脲佐菌素(STZ)
用天平称取500mg的STZ，加入50mL浓度为0.1mol/L橼酸钠缓冲液(pH＝4.5)，配制成浓度为10mg/ml的溶液，装入50mL离心管(外包锡箔纸)，并在10min内使用完毕，以防止STZ失效。
2)0.5％CMC-Na溶液
称取500mg CMC-Na，加入100mL超纯水，配制溶液终浓度为0.5％。将溶液放入超声机内充分震荡，超声机温度设为50℃以上以便于溶解。
3)氢氧化钠溶液的配制
步骤1：量取80mL去离子水置于塑料烧杯中(由于NaOH溶解过程中大量放热，因此不使用玻璃烧杯，避免玻璃烧杯炸裂)；
步骤2：称取20g NaOH缓慢加入到烧杯中，边加边搅拌；
步骤3：待NaOH完全溶解后，用去离子水定容至100mL；
步骤4：将配好的溶液转移至塑料容器中，室温保存即可。
4)二氮嗪
称取1g二氮嗪，加入6mL NaOH溶液，再加入450mL超纯水，放入超声机内充分震荡至透明无沉淀。加入适量5uM的HCl溶液，调节pH至7.5，再定容到500mL，配制成浓度为5mg/kg的溶剂。
5)葡萄糖溶液
称取25g葡萄糖，加入45mL超纯水，将溶剂放入超声机内充分震荡，超声机温度设为50℃以便于溶解。充分溶解后，定容至50ml。注意及时使用，若隔夜使用须放入冰箱，于4℃保存。
实施例1糖尿病肾病模型的建立与药物检测方法
1、糖尿病模型的建立
STZ通过化学毒性急剧和过度地损害胰岛β细胞(Saini K,Thompson C,Winterford CM,Walker NI,Cameron DP.Streptozotocin at low doses induces apoptosis and at high doses causes necrosis in a murine pancreatic β cell line,INS1[J].International Union of Biochemistry and Molecular Biology Life.1996,39(6):1229-1236.)，通过STZ造模的大鼠，其病理生理学特征与人类较为接近，可以重复采血监测指标变化，并能获得足够的肾组织进行后续组织切片分析。因此，选用STZ造模糖尿病大鼠模型。此外，由于STZ毒性较大，通过低剂量多次注射能更好的建立模型。建模后每天高脂饮食，直到实验结束。高脂饮食可以使STZ大鼠形成糖尿病肾病。
具体建模方式见实施例2。
2、糖尿病肾病模型的建立及测量指标的确立
尿微量白蛋白(mALB)是早期肾脏损伤诊断指标，本实验以mALB为指标检验大鼠是否进入糖尿病肾病，大鼠体重及血糖水平仅用于观察模型是否成功。
mALB是肾病最重要的监测指标之一，临床上mALB＞20μg/min即为肾病，存在肾损伤。由于人类与鼠类的mALB的测量结果不同，而且不同试剂盒检测有一定的误差，因此本实验以加药组(C组)与阴性对照组(B组)的区别，来判断本实验检测结果是否有效。本实验mALB正常值仅以A组(空白对照组)数据作为参考值。
肾小球的变化主要有系膜基质的扩张，任何程度的小动脉透明样化，基底膜增厚和间质纤维化等。
3、血液及尿液样本的采集以及组织标本留取
实验开始时采集所有大鼠的尿液及血液用于检测所需指标，初期指标作为横向参考大鼠 的病情进展。实验开始后所有大鼠每隔一个月用Elisa试剂盒抽样检测一次mALB等数据。
大鼠尿液收集：尿液通过代谢笼收集，24h后收集尿液，记录尿液总量，计算24h尿量。取1ml尿液，离心后取上清液，于-80℃保存待测。
大鼠血液收集：通过剪尾收集大鼠血液3mL，血液收集在含促凝剂的医用血液试管中，室温静置2h或者4℃过夜后，1000rcf离心20分钟，取上清液，于-80℃保存待测。或，通过剪尾收集大鼠血液3mL，血液收集在肝素锂的试管中，1000rcf离心20分钟，取上清液，于-80℃保存待测。
4、检测方法
成模时采集所有大鼠血液与尿液，Elisa试剂盒抽样检测mALB等生化指标。实验开始后每隔一个月检测一次所有以上指标，直至大部分实验鼠糖尿病肾病。
收集大鼠的24h尿液样本用于检测，采集到的样本于15min内、4℃离心机1000rcf离心20min，取上清液检测mALB值，或-20℃保存待测mALB值。
实验开始前需要做如下准备：
(1)将所有的试剂(包括标本)置于室内，均衡至室温(18-25℃)方可使用。
(2)标准品稀释成相应梯度。取一瓶标准品，加入标准品稀释液1mL(此时浓度为1000μg/mL)后轻轻混匀，室温静置10分钟，每隔2分钟轻轻摇晃混匀。混匀后将其稀释至100μg/mL，取4个EP管(每个EP管中加入600μL的标准品稀释液)，将浓度为100μg/mL的标准品依次三倍稀释成33.33μg/mL，11.11μg/mL，3.70μg/mL，1.23μg/mL，标准品稀释液作为空白孔(0μg/mL)。
(3)检测溶液A工作液：检测溶液A母液中加入150μL试剂稀释液，室温静置10分钟，每隔2分钟轻轻摇晃混匀。临用前，用检测稀释液A以1:100稀释，震荡机充分混匀。使用前按50μL/孔计算出所需用量进行稀释(需多配制0.1-0.2mL)。
(4)检测溶液B工作液：检测溶液B在每次使用前需5000rcf离心10s，以使管壁或瓶盖的液体沉积到管底。临用前用检测稀释液B以1:100稀释，震荡机充分混匀。使用前按100μL/孔计算出所需用量进行稀释(需多配制0.1-0.2mL)。
(5)浓洗涤液：将浓洗涤液进行30倍稀释(稀释量根据自己样本量计算)。
所有用品准备好后，进行实验操作，具体操作步骤如下：
(1)设置标准孔、待测样品孔、空白孔后开始加样。设标准孔5孔，依次加入50μL不同浓度的标准品(也可以设置10孔，每个标准品设置两孔，最后取平均值)。空白孔加50μL标准品稀释液，待测样品孔加待测样品50μL(做好样本标记，以免后期搞混)。随后立即每孔加检测溶液A工作液50μL，轻轻振动，混匀，注意不要有气泡，酶标板加上覆膜(切勿 触碰到酶标板底部，以免吸光不准确)，37℃温育1小时。
(2)洗涤液洗涤。温育完成后，弃去孔内液体(可用移液枪吸出，也可在吸水纸上轻拍甩出孔内液体)，每孔用350μL的洗涤液洗涤，浸泡1-2分钟，在吸水纸上轻拍酶标板来移除孔内所有液体。重复以上操作三次(最后一次洗涤应用移液枪吸出或倒出剩余的全部洗涤缓冲液)。
(3)每孔加检测溶液B工作液100μL，酶标板加上覆膜(切勿触碰到酶标板底部，以免吸光不准确)，37℃温育30分钟。
(4)移液枪吸出或吸水纸上轻拍甩干，重复洗板5次(操作步骤同步骤2)。
(5)每孔加底物溶液90μL，酶标板加上覆膜，37℃避光显色(反应时间控制在10-20分钟，不要超过30分钟(当后三个浓度的标准孔有明显的梯度蓝色，前3孔梯度不明显时，即可终止)。
(6)每孔加终止溶液50μL，终止反应(此时蓝色立转黄色)。
(7)在确保酶标板底无水滴及孔内无气泡后，立即用酶标仪在450nm波长测量各孔的光密度(OD值)。
(8)根据各标准品及样本OD值扣除空白孔OD值后作图(如设置复孔，则应取其平均值计算)，绘制标准曲线后将样品的OD值代入方程式，计算出样品浓度。
实施例2研究二氮嗪对糖尿病肾病的影响
【实验目的】
在实施例1构建的糖尿病肾病大鼠模型及检测方法的基础上，本实施例旨在考察高、中、低剂量的二氮嗪是否对糖尿病肾病有治疗效果。
【实验方法】
SD大鼠适应性饲养7天后，随机选出10只作为空白对照组(A组)，即不注射STZ。其余的65只大鼠随机分为4组，其中14只为阴性对照组，其余每组各17只，高脂饲料喂养2周诱导出胰岛素抵抗后，开始建立糖尿病模型，具体造模方式如下：
(1)禁食至少12h后，按30mg/kg的剂量注射STZ，注射后自由饮食、饮水；
(2)测注射STZ 48h后空腹血糖，血糖值高于16.7mmol/L即可；
(3)血糖值未达标的大鼠，禁食12h后，继续按30mg/kg的剂量注射STZ，直至血糖符合要求；
(4)所有符合要求大鼠一周后检测血糖是否仍达标，未达标的实验鼠重复以上操作。此时，造模成功的老鼠，尿量明显增多，出现糖尿病的“三多”症状。
将所有达标的糖尿病大鼠随机分组4组：B组为仅注射过STZ的大鼠，作为阴性对照；C组每天灌胃0.5mg/kg剂量的二氮嗪(低剂量给药组)；D组每天灌胃5mg/kg剂量的二氮嗪(中剂量给药组)；E组每天灌胃50mg/kg剂量的二氮嗪(高剂量给药组)。由于大鼠体表面积较大，C，D和E组的剂量换算后相当于人类的0.095mg/Kg、0.95mg/Kg、9.5mg/Kg(换算依据文献：Anroop B Nair,Shery Jacob.A simple practice guide for dose conversion between animals and human.Journal of Basic and Clinical Pharmacy.2016,7(2):27-31.)，与白蛋白结合力为95％，和人类类似。所有组大鼠均喂养高脂饲料。
【实验结果】
实验所得数据均用Micorosoft Office Excle软件，进行记录处理，计算P值，以P<0.05判断是否存在显著差异。
实验结果显示，在注射STZ半个月(2周)后，各实验组大鼠尿液明显增多；空白对照组(A组，下同)大鼠24h平均尿液量为6.50±0.52mL，STZ对照组(B组，下同)大鼠和加药组(包含C组、D组和E组)大鼠24h平均尿液量为27.23±3.82mL，与空白对照组相比，存在极显著性差异(p＜0.01)。
此外，高剂量E组的大鼠在糖尿病肾病成模前均死亡，其中13只在前四个月死亡，另5只在第五、第六个月死亡，推测死亡可能与糖尿病大鼠的血糖升高过高有关；故未纳入表1、图1、图2、图3、图5的统计。
如图1所示，观察第3～9周的血糖数据，空白对照组(■)的平均血糖在4.0～4.9mmol/L之间，而STZ对照组(▲)和5mg/kg加药组(●)的平均血糖基本保持在18～19mmol/L之间，高于文献规定的16.7mmol/L(见前述建模方式步骤2)；而且，STZ对照组和5mg/kg加药组大鼠均已具备糖尿病“三多”症状，以及出现易怒、反应迟钝、发色干枯发黄等症状，表明糖尿病模型的建模成功。
如图2所示，由大鼠体重趋势图可得：空白对照组(■)＞STZ对照组(▲)＝5mg/kg加药组(●)。空白对照组体重由于长期食用高脂饲料且没有任药物作用(包括STZ)，所以体重呈现出稳定上升且一直保持最高；STZ对照组和5mg/kg加药组因STZ的损伤(组平均血糖值稳定在16.7mmol/L以上)，因此体重维持较低水平(远低于空白对照组)，STZ对照组和5mg/kg加药组之间的前期体重无统计学上的显著差异(P>0.05)。但是，STZ对照组和5mg/kg加药组各自与空白对照组相比，其体重均存在极显著差异(p<0.01)。
如表1和图3所示，分析mALB数据可知：
①实验第24周，随机抽样检测大鼠的mALB值，发现B组的尿微量白蛋白(mALB)为61.9±14.7μg/mL，约第20周时的mALB值6倍量；C组的mALB值为18.2±4.7μg/mL， D组的mALB值为19.8±4.3μg/mL，均与第20周时的mALB值相当；表明第24周时B组大鼠已出现肾损伤，而C组、D组大鼠尚未出现糖尿病肾病的症状。
与B组比较，加药组C组、D组均有显著性差异(p<0.05)，表明二氮嗪能延缓糖尿病大鼠出现肾损伤的进程，对早期阶段的糖尿病肾病患者起到预防或治疗作用。
②实验第26周，同样随机抽样检测大鼠的mALB值，发现与A组比较，B组的mALB值呈极显著差异(p＜0.05)，但C和D组未呈显著差异(p＞0.05)。
与B组比较，C组和D组均呈显著性差异(p<0.05)，表明此时低剂量和中剂量的二氮嗪仍都均具有抗微蛋白尿和延缓糖尿病作用。
③实验第32周，同样观察大鼠的mALB值，B组的mALB值很高，表明B组大鼠已彻底进入糖尿病肾病。
与B组比较，C组和D组的mALB值均有一定程度的降低，其中D组呈显著性差异(p<0.05)，C组未呈显著性差异(p>0.05)。
④实验结束前，对所有剩余大鼠采样检测结果如表1所示，与B组比较，中剂量D组仍有显著性差异(p＜0.05)，低剂量C组在32周及35周虽有一定程度的降低作用，接近但没有达到显著性差异(p＜0.05)。另外，B组呈现代偿性肾小球滤过率增高现象而加药组(C组、D组)可以降低肾小球滤过率的增高。
⑤实验表明，给药中剂量0.5mg/kg的二氮嗪能够延迟糖尿病肾病的进展，对糖尿病大鼠的肾脏具有持续保护作用。
表1 各实验组的在不同时间下的尿微量白蛋白检测数据(mean±SE)
Figure PCTCN2021096736-appb-000005
【注】
a：与A组相比， #p＜0.05， ##p＜0.01；与B组相比，*p＜0.05，**p＜0.01；
b：“NA”表示未测。
此外，临床上肾病患者早期会出现肾小球体积增大，细胞数增多，随病情进展会出现系膜区增宽，系膜细胞和系膜基质会增多，肾小管上皮细胞会出现细胞水肿，也就是常说的颗粒变性或空泡变性，肾小管间质纤维化等症状。本实验拟通过HE染色观察到了大鼠肾切片的肾小球体积变化，各实验组的肾染色切片结果见图5。
如图5所示，所有模型鼠的肾小球有明显体积增大和细胞增多，表明糖尿病肾病模型的建模成功。
此外，从图5中可知：
①A组(空白对照组)肾小球体积轻微增大，但其近曲小管及远曲小管状态良好；
②B组(阴性对照组)肾小球明显体积增大，肾小管损伤；
③C组以加药中剂量5mg/kg的二氮嗪为例，C组部分肾小管空泡变性(见图5之C组的右侧图)，B组和C组均出现不同程度肾小管病变。
实施例3研究其余K ATP激动剂对糖尿病肾病的影响
【实验目的】
在实施例1构建的糖尿病肾病大鼠模型及检测方法的基础上，本实施例旨在考察除二氮嗪外的其余K ATP激动剂是否对糖尿病肾病有治疗效果。
【实验方法】
130只SD大鼠适应性饲养7天后，随机选出10只作为空白对照组，即不注射STZ。其余的120只大鼠随机分组8组(包括：阴性对照组、1mg/Kg克罗卡林组、1mg/Kg吡那地尔组、0.5mg/Kg尼克地尔组、1mg/Kg阿普卡林组、1mg/Kg喹乙唑酮组、0.5mg/Kg米诺地尔组、5mg/Kg尼古地平组)，每组15只，高脂饲料喂养2周诱导出胰岛素抵抗后，开始建立糖尿病模型，具体造模方式和检测方法同实施例2。
【实验结果】
实验所得数据均用Micorosoft Office Excle软件，进行记录处理，计算P值，以P<0.05判断是否存在显著差异。实验第30周的mALB值结果见表2和图4。
表2 各K ATP激动剂的尿微量白蛋白检测数据(mean±SE)
Figure PCTCN2021096736-appb-000006
【注】与阴性对照组比较，*p＜0.05，**p＜0.01。
从表2和图4中可知，与阴性对照组比较，K ATP激动剂克罗卡林、吡那地尔、尼克地尔、阿普卡林、尼古地平均有显著降低糖尿病肾病尿微量白蛋白量的的效果(p＜0.05)。

Claims (10)

  1. 钾ATP通道调节剂在制备抗糖尿病肾病药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述的糖尿病肾病为1型糖尿病和/或2型糖尿病并发的糖尿病肾病，优选处于I期、Ⅱ期或Ⅲ期的糖尿病肾病。
  3. 根据权利要求1或2中所述的应用，其特征在于，所述的钾ATP通道调节剂包括钾ATP通道开放剂或钾ATP通道抑制剂。
  4. 根据权利要求3所述的应用，其特征在于，所述的钾ATP通道调节剂选自二氮嗪、克罗卡林、吡那地尔、尼克地尔、阿普卡林、喹乙唑酮、米诺地尔、尼古地平中的一种。
  5. 根据权利要求3所述的应用，其特征在于，所述的钾ATP通道开放剂为给药剂量为0.5～5mg/kg的二氮嗪。
  6. 一种预防或治疗糖尿病肾病的药物组合物，包括如权1～5中任一项所述的钾ATP通道调节剂作为活性成分。
  7. 根据权利要求6所述的药物组合物，其特征在于，还包括药学上可接受的辅料。
  8. 根据权利要求6所述的药物组合物，其特征在于，所述的药物组合物用于预防或治疗早期的糖尿病肾病，包括糖尿病肾病分期演变过程中的I期、Ⅱ期或Ⅲ期。
  9. 根据权利要求6所述的药物组合物，其特征在于，所述的药物组合物剂型选自片剂、胶囊剂、颗粒剂、注射剂、贴剂、凝胶剂中的一种。
  10. 根据权利要求6所述的药物组合物，其特征在于，所述的药学上可接受的辅料为填充剂、崩解剂、粘合剂、稀释剂、润滑剂、调节剂、增溶剂、助溶剂、乳化剂中的一种或几种。
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