Combined medicine for treating nephropathy, medicine kit and application

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a combined medicine for treating nephropathy, a medicine kit and application. The combined medicine is prepared from uremia clearing and empagliflozin. The mass ratio of the ureamethoxam to the empagliflozin is (10-40) g: (5-25) mg. The invention also provides a pharmaceutical kit. The pharmaceutical kit comprises a composition A, a composition B and a composition C, the composition B comprises empagliflozin and pharmaceutically acceptable auxiliary materials. The invention further provides application of the combined medicine or the medicine kit in preparation of medicines for treating nephropathy. The drug combination has a certain synergistic promotion effect on apoptosis resistance, oxidation resistance and membrane charge of kidney-derived cells as well as sugar transport and sugar utilization of liver cells, and has a synergistic treatment effect on an adenine-induced rat chronic renal failure animal model; uric acid, serum creatinine, urea nitrogen, low-density lipoprotein and 24-hour total protein level of a nephrotic patient can be reduced, and kidney injury is improved.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a combined drug for treating kidney disease, a drug kit and an application thereof. Background Art

[0002] Urea Clearing Granules can clear the bowels and reduce turbidity, strengthen the spleen and eliminate dampness, and activate blood circulation and remove blood stasis. It is commonly used for patients with chronic renal failure, azotemia and early uremia, and those with spleen deficiency, dampness and turbidity and spleen deficiency and blood stasis according to TCM syndrome differentiation.

[0003] Empagliflozin is a sodium-glucose co-transporter 2 (SGLT2) inhibitor. It is used in combination with exercise and diet as an adjunct therapy to improve blood sugar control in adult patients with type 2 diabetes. Empagliflozin belongs to the class of sodium-glucose co-transporter-2 (SGLT-2) inhibitors, which can block the reabsorption of glucose in the kidneys and excrete excess glucose out of the body, thereby achieving the effect of lowering blood sugar levels, and this blood sugar-lowering effect is independent of beta cell function and insulin resistance. Empagliflozin not only has a good blood sugar-lowering effect, but also protects against cardiovascular disease, reduces the risk of heart failure, and reduces various mortality rates. Empagliflozin also has the function of protecting the kidneys. It can be used alone or in combination with other hypoglycemic drugs.

[0004] The adenine-induced rat renal injury model is characterized by tubular destruction as the main pathological feature. Different adenine dosages and courses of treatment will lead to different degrees of renal function impairment, which can be used to study some new drugs to restore tubular function. Adenine is one of the main components of nucleic acids. When the body ingests large doses of adenine, the abnormally high concentration of adenine is converted into 2,8-dihydroxyadenine, which is extremely difficult to dissolve in water, under the action of xanthine oxidase. 2,8-dihydroxyadenine is deposited in the glomeruli and renal interstitium, forming foreign body granulomatous inflammation, and blocking the renal tubular lumen, causing corresponding cystic expansion or atrophy of the renal tubular lumen, interstitial edema, lymphocyte infiltration and interstitial microscopic tissue hyperplasia. As the disease progresses, a large number of renal units are lost, leading to azotemia, toxin accumulation, electrolyte and amino acid metabolism disorders, and then renal failure. Adenine-induced rat renal injury model often causes changes in blood urea nitrogen (BUN), serum creatinine (sCr), uric acid (UA), endogenous creatinine clearance (Ccr), urine protein, urine N-acetyl-BD-glucosaminyl glucosidase (NAG), and blood albumin (ALB); electrolyte indicators such as blood potassium and phosphorus levels; blood routine indicators such as red blood cell count (RBC), hemoglobin (HGB), and hematocrit (HCT) and other physiological and biochemical indicators; at the same time, it can change the renal weight index, and pathological histological observations will also show different degrees of lesions in the glomeruli and renal tubules. The characteristics are similar to human renal failure. The animal model of chronic renal failure was established by gavage of adenine in SD rats to provide reliable experimental subjects for the results; and the rat chronic renal failure model induced by adenine gavage has the advantages of easy operation, sensitive indicators, stable course of disease, and high success rate. Therefore, it is often used as a renal injury model for related research.

[0005] As chronic kidney disease progresses, renal damage worsens, and the patient's immune system is also affected. If not controlled in time, it may develop into end-stage renal disease. The effect of using empagliflozin or ureachloride alone in the treatment of chronic kidney disease is limited, and it is unclear whether the combined effect has a significant therapeutic effect on adenine-induced chronic kidney disease renal damage. Developing a combination of enhanced and reduced toxicity drugs with enhanced efficacy and reduced side effects to provide data support for clinical application is an urgent problem to be solved in this field. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a combined drug, a drug kit and applications for treating kidney disease.

[0007] In order to achieve the purpose of the present invention, the technical solution adopted is as follows:

[0008] A combined drug for treating kidney disease, comprising ureachloride and empagliflozin.

[0009] Preferably, the mass ratio of Urea Clear and Empagliflozin is 10-25g:5-10mg.

[0010] Preferably, the mass ratio of Urea Clear and Empagliflozin is 12.5g:10g, or 25g:5mg, or 12.5g:5mg, or 25g:10mg; the composition further comprises a pharmaceutically acceptable carrier.

[0011] The second object of the present invention is to provide a drug kit, which comprises composition A: Urea Clear; composition B: Empagliflozin and pharmaceutically acceptable excipients thereof.

[0012] Preferably, the mass ratio of Urea Clear and Empagliflozin is 10-25g:5-10mg.

[0013] The third object of the present invention is to provide the use of the above-mentioned combined drug or the above-mentioned drug kit in the preparation of drugs for treating kidney disease.

[0014] Preferably, the effective human dosage of Urea Clear is 10-25 g / 60 kg, and the effective human dosage of Empagliflozin is 5-10 mg / 60 kg.

[0015] Preferably, the kidney disease is one or more of chronic kidney disease, chronic renal failure, and diabetic nephropathy.

[0016] Preferably, the drug can synergistically promote the absorption and utilization of glucose by insulin-resistant hepatocytes;

[0017] Preferably, the drug can synergistically promote anti-apoptotic activity on podocytes;

[0018] Preferably, the drug can synergistically promote the anti-apoptotic activity of the glomerular mesangial cell line HBZY-1.

[0019] Preferably, the drug can produce a synergistic protective effect on the membrane charge of HBZY-1.

[0020] Preferably, the drug can synergistically improve kidney lesions, and / or the drug can reduce one or more of the levels of uric acid, blood creatinine, urea nitrogen, low-density lipoprotein and 24-hour total protein in patients with kidney disease.

[0021] Preferably, the drug can synergistically improve the tGFR index of the kidney in a nephrotic body.

[0022] The use of Urea Clear in the preparation of products for use in combination with Empagliflozin.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The combined use of Urea Clear and Empagliflozin has a certain synergistic promoting effect on the anti-apoptosis, antioxidant, membrane charge of renal cells and the sugar transport and utilization of hepatocytes.

[0025] (2) The combination of Urea Clear and Empagliflozin has a synergistic therapeutic effect on the adenine-induced rat chronic renal failure animal model. It can reduce the uric acid, serum creatinine, urea nitrogen, low-density lipoprotein and 24-hour total protein levels of patients with kidney disease, increase the renal tGFR index of the renal body, and improve renal pathological changes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 To study the anti-apoptotic activity of each group of drugs on podocytes;

[0027] Figure 2 The anti-apoptotic activity of each group of drugs on rat glomerular mesangial cell line HBZY-1;

[0028] Figure 3 The silver staining results of kidneys of rats in each group;

[0029] Figure 4 The following are the results of HE staining of kidney after medication in each group. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific implementation modes.

[0031] The drug information in the following specific implementation manner is as follows.

[0032]

[0033] Example 1

[0034] Combined drugs: 25g of Urea Clearing Granules and 10mg of Empagliflozin Tablets.

[0035] Example 2

[0036] Combined drugs: Urea Clearing Granules 12.5g, Empagliflozin Tablets 5mg.

[0037] Example 3

[0038] Combined drugs: 25g of Urea Clearing Granules and 5mg of Empagliflozin Tablets.

[0039] Example 4

[0040] Combined drugs: 12.5g of Urea Clearing Granules and 10mg of Empagliflozin Tablets.

[0041] Experimental example

[0042] 1. Preparation of test and reference substances

[0043]

[0044]

[0045] 2 Main instruments

[0046]

[0047] 3 Main reagents

[0048]

[0049] Experiment 1: Experimental methods for the anti-apoptotic activity of empagliflozin and urea-repellent combined with different cell lines

[0050] 1. Test methods and steps:

[0051] (1) Solution preparation

[0052] Preparation of Urea Cleanser: Take 1 mL of Urea Cleanser extract and add 11.6 mL of PBS to completely dissolve it to obtain 0.17 g crude drug / mL mother solution for use. Refrigerate at 4°C for use.

[0053] Preparation of Empa: Weigh about 9 mg of Empa, add 10 mL of PBS, and prepare 2×10 6 The stock solution of nmol / L is kept for later use.

[0054] Preparation of AGEs: 5 g BSA and 9 g D-glucose were dissolved in 100 ml PBS, filtered and sterilized, and then divided into portions. AGEs were incubated in a 37°C incubator for 90 days, and unreacted proteins and glucose were removed by extensive dialysis to obtain AGEs.

[0055] Preparation of vitamin E: Accurately pipette 90.68 μL of vitamin E, add 410 μL of anhydrous ethanol to make a 400 mM mother solution, divide it into portions, and refrigerate at 4°C for later use. When using, take 5 μL of the mother solution and add 1.2 mL of PBS to dilute it to the required concentration.

[0056] AO / EB dye: Acridine orange (AO) and ethidium bromide (EB) were mixed in a 1:1 ratio.

[0057] (2) Cell grouping and dose setting

[0058] The experiment was divided into control group, model group, positive group (vitamin E), urea-clearing group (final concentration 10 -4 g crude drug / mL, empagliflozin group (100nm / L), combined drug group (NDQ 10 -4 gCrude drug / mL+Empa 50nm / L, NDQ 10 -5 g crude drug / mL+Empa50nm / L).

[0059] Experimental procedures

[0060] 1) Cell slides

[0061] After washing and sterilizing the slide, place the slide in a 24-well plate, take HPC cells in the logarithmic growth phase, inoculate the cell suspension, 1.5 ml per well, incubate at 37°C, 5% CO2 until the cells cover about 70%-80% of the slide area, then perform modeling and drug administration.

[0062] 2) Modeling and drug administration

[0063] 150 μL of culture medium was drawn out from each well, 150 μL of PBS was added to the control group, 60 μL of AGEs (final concentration was 25 mg / L) were added to the model group, positive drug group and drug administration group, and then 90 μL of PBS, vitamin E and urea serum (final concentration was 10 mg / L) were added to the model group, positive drug group and drug administration group respectively. -4 g crude drug / mL), empagliflozin (100nm / L), combined drug group 1 (NDQ 10 -4 g crude drug / mL+Empa 50nm / L, combined medication 2 groups (NDQ 10 -5 g crude drug / mL+Empa50nm / L), and two replicate wells were made for each group and incubated in an incubator.

[0064] 3) Observe and take photos

[0065] Take equal volumes of AO (100 μg / ml) and EB (100 μg / ml) and mix them to make AO / EB fluorescent dye solution. Take 5 μL on a glass slide, take out the slides in the 24-well plate, rinse them in PBS working solution, absorb the excess liquid with filter paper, and cover the cells face down on the AO / EB fluorescent dye solution. Observe and take pictures under a fluorescence microscope (blue light filter).

[0066] 1.2 Results and Analysis:

[0067] The ANOVA analysis method in SPSS software was used for data statistical analysis. The data were expressed as mean±SEM. P<0.05 was considered to be statistically significant, and P<0.01 was considered to be an extremely significant difference.

[0068] (1) Study on the anti-apoptotic activity of podocytes by the combination of empagliflozin and urea-drug

[0069] Results Figure 1 Compared with the control group, the apoptosis rate in the model group was significantly increased. Compared with the model group, the apoptosis rate was significantly reduced after treatment with different concentrations of Urea Qing Extract (c = 10 -6 -10 -4 g crude drug / mL). In the combined drug administration, compared with the model group, by reducing the administration concentration of one of the drugs, better anti-apoptotic activity can still be obtained, indicating that the combined drug administration has a certain synergistic effect.

[0070] (2) Study on the anti-apoptotic activity of empagliflozin and urea-drug combination on rat glomerular mesangial cell line HBZY-1

[0071] Results Figure 2 When empagliflozin was used in combination with ureachloride, the cell apoptosis rate was significantly lower than that when either drug was used alone, indicating that the combination of the two drugs has a synergistic effect.

[0072] Experiment 2: Animal Experiment

[0073] 2.1 Animal grouping

[0074] Male SD rats weighing 280-380 g were selected and randomly divided into groups according to their weight, with 12 rats in each group. The correspondence between the groups and the animal numbers is shown in Table 1.

[0075] Table 1 Group and animal number table

[0076]

[0077] 2.2 Dosage design

[0078] The proposed daily dose of Urea Clearing Granules is 25g / person, which is converted into a rat clinical equivalent dose of 0.45g / rat / day according to the human-rat body surface area conversion coefficient of 0.018, that is, 2.25g / kg. The normal clinical dose of Empagliflozin Tablets is 10mg / person, which is converted into a rat clinical equivalent dose of 0.18mg / rat / day according to the human-rat body surface area conversion coefficient of 0.018, that is, 0.9mg / kg. The specific dosage is shown in Table 2.

[0079] Table 2 Dosage design and administration schedule

[0080]

[0081]

[0082] Note: The dose conversion formula is: logS = 0.8762 + 0.698logW (S: body surface area, unit is cm 2 ; W: body weight, in g), human weight is calculated as 60 kg, and body weight is calculated as kg / animal.

[0083] 2.3 Model preparation

[0084] The rats in the modeling group were gavaged with adenine suspension [150 mg / (kg·d)] once a day according to their body weight for 2 consecutive weeks. In the 3rd and 7th weeks, the dosage of the modeling drug was adjusted according to the animal status combined with the results of urine biochemical indicators, ultrasound and tGFR, and the modeling was changed to gavage 6 times a week.

[0085] 2.4 Animal Dosing

[0086] Starting from the third week, 0.5 ml of the corresponding therapeutic drug was gavaged according to body weight, and different concentrations of the same volume were administered. During the entire experiment, the control group was given the same volume of ultrapure water. The dose of the Uridine Clearance group was 2.25 g of preparation / kg; the dose of the Empagliflozin group was 0.9 mg / kg; the dose of Uridine Clearance combined with Empagliflozin 1-3 groups was Uridine Clearance: Empagliflozin in different ratios (Uroid Clearance combined with Empagliflozin group 1 0.5:1; Uridine Clearance combined with Empagliflozin group 2 1:0.5; Uridine Clearance combined with Empagliflozin group 3 0.5:0.5.

[0087] 2.5 Index detection

[0088] The animals were observed daily and weighed once a week. The 24-hour urine volume was measured once every 2 weeks; the total protein (TP), creatinine (Cr), and urea nitrogen (BUN) in the urine were detected. Four animals were randomly selected from each group for ultrasound angiography of the left kidney 2 weeks after modeling and every 4 weeks during the administration period, and half of the animals were tested for glomerular filtration rate. After the end of the administration, 24-hour urine was collected to detect the total protein (TP) in the urine; animal serum was collected to detect serum creatinine (sCr), blood urea nitrogen (BUN), and blood biochemistry; the left kidney was taken, weighed, and measured in size, cut along the longitudinal section of the kidney at the renal hilum, photographed, half of which was preserved in anhydrous ethanol, and the other half was placed in 4% paraformaldehyde for future use; the right kidney was stored at -80℃. The kidneys preserved in anhydrous ethanol were taken, and urate crystals were observed by Gomori hexamine silver staining, and urate crystal scores were performed; the kidneys preserved in 4% paraformaldehyde were taken, sliced, and HE staining and Masson staining were performed to observe renal pathological changes.

[0089] 2.6 Results and Conclusions

[0090] The ANOVA analysis method in SPSS software was used for data statistical analysis. The data were expressed as mean±SEM. P<0.05 was considered to be statistically significant, and P<0.01 was considered to be an extremely significant difference.

[0091] (1) Silver staining results of kidneys of rats in each group of animal experiments

[0092] Results Figure 3 Compared with the model group, the empagliflozin group, the ureachlorpone group, the ureachlorpone combined with empagliflozin group 1, the ureachlorpone combined with empagliflozin group 2, and the ureachlorpone combined with empagliflozin group 3 all showed significant improvements.

[0093] (2) HE staining to observe the effects of empagliflozin and urea-repellent on the kidneys of rats in each group of animal experiments

[0094] Results Figure 4Compared with the model group, the pathological changes in the empagliflozin group, the ureachlorpone group, the ureachlorpone combined with empagliflozin group 1, the ureachlorpone combined with empagliflozin group 2, and the ureachlorpone combined with empagliflozin group 3 were alleviated to a certain extent.

[0095] (3) Detection of renal physiological and biochemical indicators in animal experiments

[0096] The main detection indicators were uric acid (UA), serum creatinine (sCr), urea nitrogen (BUN) and 24h total protein (24hTP).

[0097] The results are shown in Table 3. Compared with the model group, empagliflozin alone can only reduce the content of uric acid, while urea clearing alone can reduce the levels of uric acid (UA), serum creatinine (sCr), and urea nitrogen (BUN).

[0098] Compared with the model group, the Urea Clear combined with Empagliflozin 1 group could significantly reduce uric acid (UA), serum creatinine (sCr), urea nitrogen (BUN) and 24h total protein (24h TP), and all four indicators were statistically significant.

[0099] Compared with the model group, the two groups of Niaoduqing combined with Empagliflozin could significantly reduce uric acid (UA), serum creatinine (sCr) and 24h total protein (24h TP, with statistical significance;

[0100] Compared with the model group, the three groups of Urea Clear combined with Empagliflozin could significantly reduce uric acid (UA), creatinine (SCr), urea nitrogen (BUN) and 24h total protein (24h TP) at the same time, which were all statistically significant.

[0101] Table 3 Effects of different treatment groups on renal physiological and biochemical parameters of adenine-induced CKD rats

[0102]

[0103] Note: vs normal control group: #p<0.05, ##p<0.01; vs model group: *p<0.05, **p<0.01.

[0104] Conclusion: Urea clearing and the combination of Urea clearing and empagliflozin have certain promoting effects on the anti-apoptosis, antioxidant effect, membrane charge of renal-derived cells and sugar transport and sugar utilization of hepatocytes; Urea clearing and the combination of Urea clearing and empagliflozin have certain therapeutic and alleviating effects on the adenine-induced chronic renal failure animal model in rats.

[0105] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A combined drug for treating kidney disease, characterized in that: The combined drug includes ureachlorhydrin and empagliflozin.

2. The combined drug according to claim 1, characterized in that: The mass ratio of Urea Clear and Empagliflozin is 10-40g:5-25mg.

3. The combined drug according to claim 1, characterized in that: The mass ratio of Urea Clear and Empagliflozin is 12.5g:10g, or 25g:5mg, or 12.5g:5mg, or 25g:10mg; the composition also includes a pharmaceutically acceptable carrier.

4. A drug kit, characterized in that: The invention comprises composition A: Urea Detoxification; composition B: Empagliflozin and pharmaceutically acceptable excipients thereof.

5. The drug kit according to claim 4, characterized in that The mass ratio of Urea Clear and Empagliflozin is 10-40g:5-25mg.

6. Use of the combined drug according to any one of claims 1 to 3 or the drug kit according to any one of claims 4 to 5 in the preparation of a drug for treating kidney disease.

7. The use according to claim 6, characterized in that: The effective human dosage of Urea Clear is 10-40 g / 60 kg, and the effective human dosage of Empagliflozin is 5-25 mg / 60 kg.

8. The use according to claim 6, characterized in that: The kidney disease is one or more of chronic kidney disease, chronic renal failure, and diabetic nephropathy.

9. The use according to claim 6, characterized in that: The drug can synergistically promote the absorption and utilization of glucose by insulin-resistant hepatocytes; and / or, the drug can synergistically promote anti-apoptotic activity on podocytes; and / or, the drug can synergistically promote the anti-apoptotic activity of the glomerular mesangial cell line HBZY-1; and / or, the drug can produce a synergistic protective effect on the HBZY-1 membrane charge; And / or, the drug can synergistically improve kidney lesions, and / or, the drug can reduce one or more of the levels of uric acid, blood creatinine, urea nitrogen, low-density lipoprotein and 24-hour total protein in patients with kidney disease; And / or, the drug can synergistically improve the tGFR index of the kidney of a nephrotic body.

10. The use of Urea Clear in the preparation of products for use in combination with Empagliflozin.

Images