Medicine for treating chronic nephritis and composition thereof

Abstract

The invention belongs to the field of medical technology, and discloses a medicine for treating chronic nephritis and a composition thereof. (R)-3-cyclopentyl-3-[4-(7H-pyrigo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propionitrile (ruxolitinib) can significantly reduce urine protein, serum BUN, CRE content and spleen index of rats with nephropathy, has a tendency to reduce serum CHO level, has a good therapeutic effect on rat nephropathy induced by doxorubicin, and ruxolitinib or a pharmaceutically acceptable salt thereof has a synergistic effect with quercetin, coumarin 6, catechin, silymarin, asiatic acid, β-nicotinamide mononucleotide, puerarin and hibiscus flower extract, and has a good therapeutic effect on nephropathy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a medicine for treating chronic nephritis and a composition thereof. Background Art

[0002] Chronic nephritis is a group of glomerular diseases with multiple causes, mainly chronic glomerular lesions. However, the cause of most patients is unknown and there is no clear relationship with streptococcal infection. According to statistics, only 15% to 20% of patients develop chronic nephritis. In addition, most patients with chronic nephritis have no history of acute nephritis. Therefore, many scholars currently believe that there is no definite association between chronic glomerulonephritis and acute nephritis. It may be caused by various bacteria, viruses or protozoa infections through immune mechanisms, inflammatory mediators and non-immune mechanisms. In recent years, due to the application of immunofluorescence technology and electron microscopy, modern medicine has conducted systematic and in-depth research on the immune pathogenesis, pathological morphology and functional changes of chronic nephritis, but there is still a lack of specific drugs for treatment.

[0003] Ruxolitinib ((R)-3-cyclopentyl-3-[4-(7H-pyrigo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propionitrile) is an oral JAK1 and JAK2 tyrosine kinase inhibitor indicated for the treatment of splenomegaly or other symptoms associated with primary myelofibrosis (PMF) (also known as chronic idiopathic myelofibrosis), myelofibrosis secondary to polycythemia vera (PPV-MF), or myelofibrosis secondary to primary thrombocytosis (PET-MF) in adult patients with intermediate- or high-risk primary myelofibrosis (PMF) (also known as chronic idiopathic myelofibrosis).

[0004] To date, there have been no reports on the association between ruxolitinib and its pharmaceutically acceptable salts and chronic nephritis. Summary of the invention

[0005] The purpose of the present invention is to provide a medicine for treating chronic nephritis and a composition thereof to solve the problems existing in the above-mentioned prior art. (R)-3-cyclopentyl-3-[4-(7H-pyrido[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propionitrile and a pharmaceutically acceptable salt thereof can effectively reduce the content of urine protein, increase the content of BUN (blood urea nitrogen), CRE (creatinine) and CHO (total cholesterol) in the blood, and have a good therapeutic effect on chronic kidney disease.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention: Use of (R)-3-cyclopentyl-3-[4-(7H-pyrog[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propionitrile or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating chronic nephritis.

[0008] Furthermore, the pharmaceutically acceptable salt includes pharmaceutically acceptable organic salts or inorganic salts; the organic salts include sulfonates, carboxylates, amino acid salts and fatty acid salts; the inorganic salts include hydrochlorides, bromates, iodates, sulfates, hydrogen sulfates, phosphates, hydrogen phosphates, dihydrogen phosphates or nitrates.

[0009] Technical solution 2 of the present invention: A pharmaceutical composition for treating chronic nephritis, comprising any one of the following combinations:

[0010] (1) A combination of (R)-3-cyclopentyl-3-[4-(7H-pyrido[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propionitrile and any one of the natural extracts;

[0011] (2) A combination of a pharmaceutically acceptable salt of (R)-3-cyclopentyl-3-[4-(7H-pyrog[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propionitrile and any natural extract;

[0012] The natural extracts include quercetin, coumarin 6, catechin, silymarin, asiatic acid, β-nicotinamide mononucleotide, puerarin and hibiscus flower extract.

[0013] Furthermore, pharmaceutically acceptable excipients are also included.

[0014] Furthermore, the second technical solution of the present invention also includes the use of the pharmaceutical composition in the preparation of a drug for preventing or treating chronic nephritis. The chronic nephritis is also called chronic kidney disease.

[0015] Furthermore, kidney disease is treated by:

[0016] (1) Reduce the content of urine protein;

[0017] (2) Increase blood urea nitrogen levels;

[0018] (3) Increase creatinine levels in the blood;

[0019] (4) Increase the total cholesterol level in the blood.

[0020] Furthermore, the chronic kidney disease includes kidney disease induced by birth defects, genetic factors, obesity, diabetes, hypertension, immune system diseases or aging.

[0021] Birth defects: Premature birth, low birth weight and often renal dysplasia.

[0022] Genetic factors: causing nephrocalcinosis or cystic lesions, reduced epithelial integrity, abnormal metabolites or glycoproteins.

[0023] Obesity, diabetes: Diabetes is the primary cause of CKD. Hyperglycemia can activate sodium / glucose co-transporter 2 (SGLT2), promote sodium reabsorption in the proximal tubule, and then inhibit tubuloglomerular feedback and activate the RAS system, ultimately leading to dilation of the afferent arterioles, constriction of the efferent arterioles, and increase in GFR.

[0024] Hypertension: causes progressive microvascular damage and loss. Active control of blood pressure can reduce the risk of cardiovascular events and even death.

[0025] Diseases related to the immune system (eg, glomerulonephritis, systemic lupus).

[0026] Aging: Aging not only causes the loss of nephrons, but is also associated with a decrease in podocytes.

[0027] The present invention discloses the following technical effects:

[0028] The ruxolitinib in the present invention can significantly reduce urine protein, serum BUN, CRE content and spleen index of rats with nephrotic syndrome, has a tendency to reduce serum CHO levels, and has a good therapeutic effect on doxorubicin-induced rat nephropathy. At the same time, ruxolitinib or a pharmaceutically acceptable salt thereof has a synergistic effect with quercetin, coumarin 6, catechin, silymarin, asiatic acid, β-nicotinamide mononucleotide, puerarin and hibiscus flower extract, and has a good therapeutic effect on nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The effect of the ruxolitinib composition in Example 3 of the present invention on the expression of p-JNK / JNK in LPS-induced RAW264.7 macrophages;

[0031] Figure 2 The effect of the Ruxolitinib composition in Example 3 of the present invention on the expression of p-NF-κB / NF-κB in LPS-induced RAW264.7 macrophages;

[0032] Figure 3 This is the effect of the ruxolitinib composition in Example 3 of the present invention on the expression of pro-inflammatory factors in LPS-induced RAW264.7 macrophages. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The quercetin, coumarin 6, catechin, silymarin, asiatic acid, β-nicotinamide mononucleotide, puerarin, and hibiscus flower extract used in the following examples were all purchased commercially.

[0039] Experimental Example 1

[0040] (1) Experimental animals:

[0041] Animal quality: SD rats, male, SPF grade, 40, body weight 200-250 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., certificate number: (Beijing) 2012-0001.

[0042] Animal breeding environment: air conditioning, room temperature 21 ~ 25 ℃; humidity 45 ± 20%, ventilation 10 times / h fresh air, artificial lighting 12h, free access to food and water.

[0043] Pre-experimental adaptation period: After the animals were introduced and inspected as required, they were housed in separate cages, with 2 animals in each cage, and were observed for 7 days to observe their general condition. Their body weight was measured at the end of the adaptation period.

[0044] (2) Model establishment:

[0045] Forty rats were randomly divided into 4 groups, with 10 rats in each group. Group 1 was the blank control group, group 2 was the model control group, group 3 was the low-dose ruxolitinib group, and group 4 was the high-dose ruxolitinib group.

[0046] Doxorubicin (purity 98%, product number A74950-1G, CAS number 25316-40-9, Inotech); sodium chloride injection (Shandong Hualu Pharmaceutical Co., Ltd., national medicine standard number H20023428).

[0047] Preparation of doxorubicin sodium chloride injection: dissolve doxorubicin in sodium chloride injection to prepare a concentration of 4 mg / mL doxorubicin sodium chloride injection; dissolve doxorubicin in sodium chloride injection to prepare a concentration of 3.5 mg / mL doxorubicin sodium chloride injection.

[0048] Group 1: Each rat was injected with sodium chloride injection (Shandong Hualu Pharmaceutical Co., Ltd., National Medicine Standard No. H20023428) once through the tail vein, and then injected with sodium chloride injection once through the tail vein after 7 days. The injection volume each time was 1 mL / kg rat. The rats were raised under the animal breeding environment described in step (1) for 7 days.

[0049] Groups 2 to 4: Each rat was injected once with 4 mg / mL doxorubicin sodium chloride injection via the tail vein, with an injection volume of 1 mL / kg rat; 7 days later, each rat was injected once with 3.5 mg / mL doxorubicin sodium chloride injection via the tail vein, with an injection volume of 1 mL / kg rat. The rats were cultured for 7 days under the animal breeding environment described in step (1), and the urine protein content was measured after mixing the urine collected for 24 hours. Compared with the control group, the urine protein content showed a significant difference, indicating that the chronic kidney disease model was successfully established.

[0050] (3) Administration:

[0051] Ruxolitinib: purity 97%, Aladdin, cas number 941678-49-5, product number R171967-100mg.

[0052] Preparation of the low-dose ruxolitinib solution: Dissolve ruxolitinib in sodium chloride injection to prepare a solution with a concentration of 2.25 mg / mL.

[0053] Preparation of the high-dose ruxolitinib solution: Dissolve ruxolitinib in sodium chloride injection to prepare a solution with a concentration of 4.5 mg / mL.

[0054] The four groups of rats treated in step (2) were subjected to the following operations:

[0055] Group 1 and Group 2: intragastric administration of sodium chloride injection, the intragastric volume was 2 mL / kg rat, once a day for 28 consecutive days;

[0056] Group 3: Oral administration of low-dose ruxolitinib solution, with a dose of 2 mL / kg rat, once a day for 28 consecutive days;

[0057] Group 4: The high-dose group of ruxolitinib was gavaged orally at a volume of 2 mL / kg rat, once a day for 28 consecutive days.

[0058] (4) Growth of rats: The average body weight of rats was counted every 7 days after administration in step (3). The statistical results are shown in Table 1.

[0059] Table 1 Body weight changes of rats

[0060]

[0061] Note: “#” indicates p<0.05 compared with the blank control group, “##” indicates p<0.01 compared with the blank control group; “*” indicates p<0.05 compared with the model control group, and “**” indicates p<0.01 compared with the model control group.

[0062] It can be seen from Table 1 that compared with the blank control group, the body weight of the model rats established with doxorubicin was significantly reduced (p < 0.05 or 0.01), and no significant effect was observed on the body weight of the rats after administration of ruxolitinib (p > 0.05).

[0063] (5) Urine protein content: After the last administration, the urine of rats in Groups 1 to 4 was collected for 24 hours under fasting conditions but without deprivation of water. The urine was mixed and the urine protein content was determined. The results are shown in Table 2.

[0064] Table 2

[0065] Group Urine protein (mg / d) Blank control 55.45±17.66 Model control group <![CDATA[258.82±50.53 ## ]]> Ruxolitinib low-dose group <![CDATA[163.52±20.15 * ]]> Ruxolitinib high-dose group <![CDATA[112.73±16.78 ** ]]>

[0066] Note: “#” indicates p<0.05 compared with the blank control group, “##” indicates p<0.01 compared with the blank control group; “*” indicates p<0.05 compared with the model control group, “**” indicates p<0.01 compared with the model control group; the same applies to the following tables.

[0067] It can be seen from Table 2 that compared with the blank control group, the protein level of the model rats established with doxorubicin was significantly increased (p < 0.01), but the administration of ruxolitinib can significantly reduce the urine protein content of doxorubicin nephropathy rats (p < 0.05), and the effect of the high-dose ruxolitinib group is better than that of the low-dose ruxolitinib group, indicating that ruxolitinib can treat rat kidney disease.

[0068] (6) Blood biochemical test: After the last administration, the serum of rats in Group 1 to Group 4 was collected to measure the levels of BUN, CRE and CHO in the serum. The results are shown in Table 3.

[0069] Table 3 Rat serum biochemical test results

[0070]

[0071] Note: “#” indicates p<0.05 compared with the blank control group, “##” indicates p<0.01 compared with the blank control group; “*” indicates p<0.05 compared with the model control group, “**” indicates p<0.01 compared with the model control group; the same applies to the following tables.

[0072] As can be seen from Table 3, compared with the blank control group, the levels of BUN, CRE and CHO in the model rats established with doxorubicin were significantly increased (p < 0.05 or 0.01), but the administration of ruxolitinib can significantly reduce the levels of BUN, CRE and CHO in doxorubicin nephropathy rats (p < 0.05 or 0.01), and the effect of the high-dose ruxolitinib group is better than that of the low-dose ruxolitinib group. The levels of BUN, CRE and CHO in the high-dose ruxolitinib rats are almost equivalent to those in the blank control group, indicating that ruxolitinib can effectively treat rat nephropathy.

[0073] (7) Organ index: After the last administration, the kidneys, spleens, and thymus of the rats in Groups 1 to 4 were collected, and the proportion of the organs in the rat body weight was calculated. The results are shown in Table 4.

[0074] Table 4 Rat organ index

[0075]

[0076] Note: “#” indicates p<0.05 compared with the blank control group, “##” indicates p<0.01 compared with the blank control group; “*” indicates p<0.05 compared with the model control group, “**” indicates p<0.01 compared with the model control group; the same applies to the following tables.

[0077] It can be seen from Table 4 that compared with the blank control group, the spleen index of the model rats established with doxorubicin was significantly increased, while the spleen index of the rats with nephrotic syndrome was significantly reduced after administration of ruxolitinib (p < 0.05 or 0.01); there was no significant effect on the renal index and thymus index of the rats with nephrotic syndrome after administration of ruxolitinib.

[0078] It can be seen from Tables 2 to 4 that the high-dose and low-dose groups of ruxolitinib can significantly reduce urine protein, serum BUN, CRE content and spleen index of rats with nephrotic syndrome after administration, and have a trend of reducing serum CHO levels. Compared with the low-dose ruxolitinib group, the effect of the high-dose ruxolitinib group is more obvious; it can be seen that ruxolitinib has a good therapeutic effect on doxorubicin-induced rat nephropathy.

[0079] Experimental Example 2

[0080] Effects of compounds on rat renal tubular epithelial NRK-52E cell injury:

[0081] (1) Rat renal tubular epithelial NRK-52E cells were purchased from the Cell Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences. The density of NRK-52E cells in the logarithmic growth phase was adjusted to 0.5×10 5 100 μL per well was inoculated into a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 4 h to adhere to the plate.

[0082] (2) Grouping and drug pre-protection: The NRK-52E cells prepared in step (1) were grouped into a blank control group, a model control group, a positive control group and a compound group.

[0083] A. Preparation of drug solution:

[0084] Dimethyl sulfoxide solution: dilute dimethyl sulfoxide in culture medium to prepare a dimethyl sulfoxide solution with a final concentration of 10 umol / L.

[0085] Dexamethasone solution: Dexamethasone was dissolved in dimethyl sulfoxide to prepare a dexamethasone stock solution with a concentration of 10 mmol / L. When used, it was diluted in culture medium to prepare a dexamethasone solution with a final concentration of 10 umol / L.

[0086] Preparation of quercetin solution: dissolve quercetin in dimethyl sulfoxide to prepare a stock solution with a concentration of 10 mmol / L. When used, dilute it in culture medium to prepare quercetin solutions with final concentrations of 10umol / L, 20umol / L, 50umol / L, and 100umol / L.

[0087] The preparations of coumarin 6 solution, catechin solution, silymarin solution, asiatic acid solution, β-nicotinamide mononucleotide solution, puerarin solution and hibiscus flower extract solution are the same as the preparation of quercetin solution.

[0088] B. Pre-protection by adding medicine

[0089] Blank control group: 100 μL of dimethyl sulfoxide solution with a final concentration of 10 umol / L was added to the cells of the blank control group and pre-incubated in an incubator at 37° C. and 5% CO 2 for 1 hour.

[0090] Model control group: 100 μL of dimethyl sulfoxide solution with a final concentration of 10 umol / L was added to the cells of the model control group, and cultured in an incubator at 37°C and 5% CO2 for 1 hour.

[0091] Positive control group: 100 μL of 0.1 um / μL dexamethasone solution was added to the cells of the model control group.

[0092] Quercetin group: 100 μL of quercetin solution with a final concentration of 10 umol / L, 20 umol / L, 50 umol / L, and 100 umol / L was added to the cells in the quercetin group, respectively, and cultured in an incubator at 37°C and 5% CO2 for 1 h.

[0093] The treatments of the coumarin 6 group, catechin group, silymarin group, asiatic acid group, β-nicotinamide mononucleotide group, puerarin group, and hibiscus flower extract group were the same as the quercetin group.

[0094] (3) Cell injury model establishment

[0095] 100 μL of doxorubicin with a final concentration of 0.5 ug / mL was added to the cells of the model control group, positive control group, and compound group for treatment;

[0096] (4) After incubation for 24 h, the culture medium was removed and the cells were washed with PBS and then the activity of NRK-52E cells was detected by CCK8.

[0097] (5) The cell recovery effect and cell activity of NRK-52E cells treated in step (4) were calculated. The results are shown in Tables 5 to 12.

[0098] Table 5 Effect of quercetin on doxorubicin-induced rat renal tubular epithelial NRK-52E cell injury model

[0099] Group concentration Cell Restoration Cell viability Blank control group - 0.36±0.03 0.36±0.03 Model control group - <![CDATA[0.18±0.05 ### ]]> - Positive control group 10um <![CDATA[0.24±0.03 *** ]]> 0.41±0.03 Low dose group 10um 0.19±0.02 0.36±0.08 Low-medium dose group 20um 0.21±0.05 0.43±0.07 Medium and high dose group 50um <![CDATA[0.25±0.03 ** ]]> 0.45±0.07 High dose group 100um <![CDATA[0.31±0.05 *** ]]> 0.44±0.04

[0100] Note: “#” indicates p<0.05 compared with the blank control group, “##” indicates p<0.01 compared with the blank control group; “###” indicates p<0.001 compared with the blank control group; “*” indicates p<0.05 compared with the model control group, “**” indicates p<0.01 compared with the model control group; “***” indicates p<0.001 compared with the model control group; the same applies to the following tables.

[0101] As can be seen from Table 5, compared with the blank control group, the activity of NRK-52E cells after doxorubicin induction was significantly reduced (p < 0.001); compared with the model control group, the positive drug dexamethasone can significantly increase the activity of NRK-52E cells after doxorubicin induction (p < 0.001); compared with the model control group, the medium-high and high concentrations of quercetin can significantly reduce doxorubicin-induced rat tubular epithelial NRK-52E cell damage (p < 0.01, 0.001); at low, medium-low, medium-high and high concentrations, quercetin did not show obvious cytotoxicity.

[0102] Table 6 Effect of coumarin 6 on doxorubicin-induced rat renal tubular epithelial NRK-52E cell injury model

[0103] Group concentration Cell Restoration Cell viability Blank control group - 0.25±0.03 0.27±0.05 Model control group - <![CDATA[0.12±0.00 ### ]]> - Positive control group 10um <![CDATA[0.16±0.01 *** ]]> 0.32±0.02 Low dose group 10um <![CDATA[0.17±0.02 ** ]]> 0.32±0.05 Low-medium dose group 20um <![CDATA[0.19±0.01 *** ]]> 0.24±0.07 Medium and high dose group 50um <![CDATA[0.21±0.02 *** ]]> 0.31±0.03 High dose group 100um <![CDATA[0.29±0.03 *** ]]> 0.37±0.03

[0104] Note: “#” indicates p<0.05 compared with the blank control group, “##” indicates p<0.01 compared with the blank control group; “###” indicates p<0.001 compared with the blank control group; “*” indicates p<0.05 compared with the model control group, “**” indicates p<0.01 compared with the model control group; “***” indicates p<0.001 compared with the model control group; the same applies to the following tables.

[0105] As can be seen from Table 6, compared with the blank control group, the activity of NRK-52E cells induced by doxorubicin was significantly reduced (p < 0.001); compared with the model control group, the positive drug dexamethasone can significantly increase the activity of NRK-52E cells induced by doxorubicin (p < 0.001); compared with the model control group, low, medium, medium-high and high concentrations of coumarin 6 can significantly reduce doxorubicin-induced rat tubular epithelial NRK-52E cell damage (p < 0.01, 0.001), and the effect increases with the increase of concentration; at low, medium-low, medium-high and high concentrations, coumarin 6 did not show obvious cytotoxicity.

[0106] Table 7 Effects of catechins on doxorubicin-induced rat renal tubular epithelial NRK-52E cell injury model

[0107] Group concentration Cell Restoration Cell viability Blank control group - 0.26±0.01 0.17±0.00 Model control group - <![CDATA[0.14±0.02 ### ]]> - Positive control group 10um <![CDATA[0.20±0.00 *** ]]> 0.21±0.02 Low dose group 10um <![CDATA[0.17±0.01 ** ]]> 0.19±0.04 Low-medium dose group 20um <![CDATA[0.21±0.02 *** ]]> 0.22±0.03 Medium and high dose group 50um <![CDATA[0.25±0.03 *** ]]> 0.22±0.04 High dose group 100um <![CDATA[0.29±0.01 *** ]]> 0.19±0.03

[0108] Note: “#” indicates p<0.05 compared with the blank control group, “##” indicates p<0.01 compared with the blank control group; “###” indicates p<0.001 compared with the blank control group; “*” indicates p<0.05 compared with the model control group, “**” indicates p<0.01 compared with the model control group; “***” indicates p<0.001 compared with the model control group; the same applies to the following tables.

[0109] As can be seen from Table 7, compared with the blank control group, the activity of NRK-52E cells after doxorubicin induction was significantly reduced (p < 0.001); compared with the model control group, the positive drug dexamethasone can significantly increase the activity of NRK-52E cells after doxorubicin induction (p < 0.001); compared with the model control group, low, medium-low, medium-high and high concentrations of catechins can significantly reduce doxorubicin-induced rat renal tubular epithelial NRK-52E cell damage (p < 0.01, 0.001); at low, medium-low, medium-high and high concentrations, catechins did not show obvious cytotoxicity.

[0110] Table 8 Effect of silymarin on doxorubicin-induced rat renal tubular epithelial NRK-52E cell injury model

[0111]

[0112]

[0113] Note: “#” indicates p<0.05 compared with the blank control group, “##” indicates p<0.01 compared with the blank control group; “###” indicates p<0.001 compared with the blank control group; “*” indicates p<0.05 compared with the model control group, “**” indicates p<0.01 compared with the model control group; “***” indicates p<0.001 compared with the model control group; the same applies to the following tables.

[0114] As can be seen from Table 8, compared with the blank control group, the activity of NRK-52E cells after doxorubicin induction was significantly reduced (p < 0.001); compared with the model control group, the positive drug dexamethasone can significantly increase the activity of NRK-52E cells after doxorubicin induction (p < 0.001); compared with the model control group, the medium-high and high concentrations of silymarin can significantly reduce the doxorubicin-induced rat tubular epithelial NRK-52E cell damage (p < 0.01, 0.001); at low, medium-low, medium-high and high concentrations, silymarin did not show obvious cytotoxicity.

[0115] Table 9 Effects of asiatic acid on doxorubicin-induced rat renal tubular epithelial NRK-52E cell injury model

[0116] Group concentration Cell Restoration Cell viability Normal control group - 0.42±0.04 0.42±0.03 Model control group - <![CDATA[0.21±0.00 ### ]]> - Positive control group 10um <![CDATA[0.26±0.02 *** ]]> 0.44±0.02 Low dose group 10um <![CDATA[0.28±0.02 *** ]]> 0.49±0.03 Low-medium dose group 20um <![CDATA[0.31±0.02 *** ]]> 0.51±0.01 Medium and high dose group 50um <![CDATA[0.34±0.02 *** ]]> 0.49±0.03 High dose group 100um 0.11±0.00 <![CDATA[0.01±0.00 ### ]]>

[0117] Note: “#” indicates p<0.05 compared with the blank control group, “##” indicates p<0.01 compared with the blank control group; “###” indicates p<0.001 compared with the blank control group; “*” indicates p<0.05 compared with the model control group, “**” indicates p<0.01 compared with the model control group; “***” indicates p<0.001 compared with the model control group; the same applies to the following tables.

[0118] As can be seen from Table 9, compared with the blank control group, the activity of NRK-52E cells after doxorubicin induction was significantly reduced (p < 0.001); compared with the model control group, the positive drug dexamethasone can significantly increase the activity of NRK-52E cells after doxorubicin induction (p < 0.001); compared with the model control group, the medium, high and high concentrations of Centella asiatica acid can significantly reduce the doxorubicin-induced damage to rat renal tubular epithelial NRK-52E cells (p < 0.01, 0.001); at high concentrations, Centella asiatica acid showed certain cytotoxicity (p < 0.001).

[0119] Table 10 Effects of β-nicotinamide mononucleotide on doxorubicin-induced rat renal tubular epithelial NRK-52E cell injury model

[0120] Group concentration Cell Restoration Cell viability Blank control group - 0.22±0.02 0.22±0.01 Model control group - <![CDATA[0.10±0.05 ### ]]> - Positive control group 10um <![CDATA[0.16±0.02 *** ]]> 0.20±0.03 Low dose group 10um 0.13±0.00 0.26±0.04 Low-medium dose group 20um 0.15±0.00 0.24±0.03 Medium and high dose group 50um <![CDATA[0.17±0.01 ** ]]> 0.27±0.03 High dose group 100um <![CDATA[0.22±0.02 *** ]]> 0.20±0.00

[0121] Note: “#” indicates p<0.05 compared with the blank control group, “##” indicates p<0.01 compared with the blank control group; “###” indicates p<0.001 compared with the blank control group; “*” indicates p<0.05 compared with the model control group, “**” indicates p<0.01 compared with the model control group; “***” indicates p<0.001 compared with the model control group; the same applies to the following tables.

[0122] As can be seen from Table 10, compared with the blank control group, the activity of NRK-52E cells after doxorubicin induction was significantly reduced (p < 0.001); compared with the model control group, the positive drug dexamethasone can significantly increase the activity of NRK-52E cells after doxorubicin induction (p < 0.001); compared with the model control group, the medium-high and high concentrations of β-nicotinamide mononucleotide can significantly reduce doxorubicin-induced rat renal tubular epithelial NRK-52E cell damage (p < 0.01, 0.001); at low, medium-low, medium-high and high concentrations, β-nicotinamide mononucleotide did not show obvious cytotoxicity.

[0123] Table 11 Effect of puerarin on doxorubicin-induced rat renal tubular epithelial NRK-52E cell injury model

[0124] Group concentration Cell Restoration Cell viability Blank control group - 0.26±0.03 0.26±0.03 Model control group - <![CDATA[0.14±0.02 ### ]]> - Positive control group 10um <![CDATA[0.18±0.03 *** ]]> 0.31±0.02 Low dose group 10um 0.14±0.02 0.27±0.04 Low-medium dose group 20um 0.15±0.04 0.25±0.02 Medium and high dose group 50um 0.17±0.02 0.25±0.01 High dose group 100um <![CDATA[0.18±0.02 *** ]]> 0.27±0.04

[0125] Note: “#” indicates p<0.05 compared with the blank control group, “##” indicates p<0.01 compared with the blank control group; “###” indicates p<0.001 compared with the blank control group; “*” indicates p<0.05 compared with the model control group, “**” indicates p<0.01 compared with the model control group; “***” indicates p<0.001 compared with the model control group; the same applies to the following tables.

[0126] As can be seen from Table 11, compared with the blank control group, the activity of NRK-52E cells after doxorubicin induction was significantly reduced (p < 0.001); compared with the model control group, the positive drug dexamethasone can significantly increase the activity of NRK-52E cells after doxorubicin induction (p < 0.001); compared with the model control group, high concentrations of puerarin can significantly reduce doxorubicin-induced rat renal tubular epithelial NRK-52E cell damage (p < 0.01, 0.001); at low, medium-low, medium-high, and high concentrations, puerarin did not show obvious cytotoxicity.

[0127] Table 12 Effect of hibiscus flower extract on doxorubicin-induced rat renal tubular epithelial NRK-52E cell injury model

[0128] Group concentration Cell Restoration Cell viability Blank control group - 0.35±0.05 0.35±0.05 Model control group - <![CDATA[0.18±0.01 ### ]]> - Positive control group 10um <![CDATA[0.22±0.03 *** ]]> 0.41±0.04 Low dose group 10um 0.18±0.02 0.39±0.06 Low-medium dose group 20um <![CDATA[0.44±0.07 *** ]]> 0.64±0.08 Medium and high dose group 50um <![CDATA[0.55±0.02 *** ]]> 0.62±0.10 High dose group 100um <![CDATA[0.56±0.05 *** ]]> 0.64±0.04

[0129] Note: “#” indicates p<0.05 compared with the blank control group, “##” indicates p<0.01 compared with the blank control group; “###” indicates p<0.001 compared with the blank control group; “*” indicates p<0.05 compared with the model control group, “**” indicates p<0.01 compared with the model control group; “***” indicates p<0.001 compared with the model control group; the same applies to the following tables.

[0130] As can be seen from Table 12, compared with the blank control group, the activity of NRK-52E cells after doxorubicin induction was significantly reduced (p < 0.001); compared with the model control group, the positive drug dexamethasone can significantly increase the activity of NRK-52E cells after doxorubicin induction (p < 0.001); compared with the model control group, the medium-low, medium-high and high concentrations of hibiscus flower extract can significantly reduce doxorubicin-induced rat tubular epithelial NRK-52E cell damage (p < 0.01, 0.001); at low, medium-low, medium-high and high concentrations, hibiscus flower extract did not show obvious cytotoxicity.

[0131] As can be seen from Tables 5 to 12, quercetin, coumarin 6, catechin, silymarin, asiatic acid, β-nicotinamide mononucleotide, puerarin, and hibiscus flower extract can significantly alleviate doxorubicin-induced rat renal tubular epithelial NRK-52E cell injury, and the cell recovery effect of the compound is enhanced with the increase of concentration.

[0132] Experimental Example 3

[0133] Effects of Ruxolitinib Combinations on Mouse Peritoneal Macrophage Cell Lines:

[0134] (1) Mouse peritoneal macrophage cell line (RAW264.7 macrophages)

[0135] (2) Adjust the density of RAW264.7 macrophages in the logarithmic growth phase to 1×10 6 / mL, 100 μL (100,000) were inoculated in a 12-well plate at each well, and cultured in an incubator at 37°C, 5% CO2 for 2 hours to adhere to the wall; and the cells were divided into a blank control group, a model control group, a ruxolitinib combination control group, and a ruxolitinib combination treatment group.

[0136] (3) Administration: using the RAW264.7 macrophages prepared in step (2);

[0137] A. Preparation of drug solution:

[0138] Dimethyl sulfoxide solution: dilute dimethyl sulfoxide in culture medium to prepare a dimethyl sulfoxide solution with a final concentration of 10 umol / L.

[0139] Dexamethasone solution: Dexamethasone was dissolved in dimethyl sulfoxide to prepare a dexamethasone stock solution with a concentration of 10 mmol / L. When used, it was diluted in culture medium to prepare a dexamethasone solution with a final concentration of 10 umol / L.

[0140] Preparation of ruxolitinib composition solution: ruxolitinib and hibiscus flower extract are dissolved in dimethyl sulfoxide to prepare a mixed stock solution with a concentration of 10 mmol / L for ruxolitinib and hibiscus flower. When used, dilute it in culture medium to prepare a ruxolitinib composition solution with a final concentration of 10 umol / L for ruxolitinib and hibiscus flower.

[0141] B. Pre-protection by adding medicine

[0142] Blank control group: 100 μL of dimethyl sulfoxide solution with a final concentration of 10 umol / L was added to the cells of the blank control group and pre-incubated in an incubator at 37° C. and 5% CO 2 for 1 hour.

[0143] Model control group: 100 μL of dimethyl sulfoxide solution with a final concentration of 10 umol / L was added to the cells of the model control group, and cultured in an incubator at 37°C and 5% CO2 for 1 hour.

[0144] Ruxolitinib composition control group: 100 μL of ruxolitinib composition solution with a final concentration of 10 umol / L was added to the cells and incubated in an incubator at 37° C. and 5% CO 2 for 1 hour.

[0145] Ruxolitinib combination treatment group: 100 μL of ruxolitinib combination solution with a final concentration of 10 umol / L was added to the cells and incubated in an incubator at 37° C. and 5% CO 2 for 1 h.

[0146] C. Lipopolysaccharide

[0147] Blank control group: add 100 μL culture medium and incubate for 30 min;

[0148] Model control group: add 100 μL of lipopolysaccharide solution with a final concentration of 10 ng / mL and incubate for 30 min;

[0149] Ruxolitinib combination control group: add 100 μL of culture medium and incubate for 30 min;

[0150] Ruxolitinib combination treatment group: add 100 μL of lipopolysaccharide solution with a final concentration of 10 ng / mL and incubate for 30 min.

[0151] (4) The cells treated in step (3) were washed twice with PBS, and the cell proteins were collected. The expression of p-JNK and p-NF-κB in RAW264.7 macrophages was detected by western blot. RNA samples were collected and pro-inflammatory factors were detected. The results are shown in Table 1. Figures 1 to 3 .

[0152] from Figures 1-2 It can be seen that RAW264.7 macrophages were stimulated with LPS, and proteins were extracted to detect the expression of p-JNK and p-NF-κB in the inflammatory signaling pathway. It was found that after LPS stimulation, the expression of p-JNK and p-NF-κB in RAW264.7 macrophages increased significantly (p < 0.001); pre-incubation with the ruxolitinib composition can inhibit LPS-induced RAW264.7 cell inflammation at the protein level, and significantly reduced the expression of p-JNK and p-NF-κB (p < 0.01 or 0.001).

[0153] from Figure 3It can be seen that after stimulating RAW264.7 macrophages with LPS, RNA was extracted to detect changes in the expression levels of inflammatory factors in cells. It was found that after LPS stimulation, proinflammatory factors in RAW264.7 macrophages, such as IL1β and IL6, increased significantly (p < 0.001); after pre-incubation with the ruxolitinib combination, proinflammatory factors in RAW264.7 macrophages decreased significantly (p < 0.01 or 0.001).

[0154] from Figures 1 to 3 It can be seen that pre-incubation of the ruxolitinib combination can significantly reduce LPS-induced RAW264.7 cell inflammation, and the anti-inflammatory effect may be exerted by reducing the protein expression of p-JNK and p-NF-κB, and inhibiting the expression of pro-inflammatory factor RNA levels.

[0155] Experimental Example 4

[0156] (1) Experimental animals:

[0157] Animal quality: SD rats, male, SPF grade, 40, body weight 200-250 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., certificate number: (Beijing) 2012-0001.

[0158] Animal breeding environment: air conditioning, room temperature 21 ~ 25 ℃; humidity 45 ± 20%, ventilation 10 times / hour fresh air, artificial lighting 12 hours, free access to food and water.

[0159] Pre-experimental adaptation period: After the animals were introduced and inspected as required, they were housed in separate cages, with 2 animals in each cage, and were observed for 7 days to observe their general condition. Their body weight was measured at the end of the adaptation period.

[0160] (2) Model establishment:

[0161] Forty rats were randomly divided into 4 groups, 10 rats in each group. Group 1 was the blank control group, group 2 was the model control group, group 3 was the ruxolitinib phosphate group; group 4 was the ruxolitinib and its phosphate group;

[0162] Preparation of doxorubicin sodium chloride injection: dissolve doxorubicin in sodium chloride injection to prepare a concentration of 4 mg / mL doxorubicin sodium chloride injection; dissolve doxorubicin in sodium chloride injection to prepare a concentration of 3.5 mg / mL doxorubicin sodium chloride injection.

[0163] Group 1: Each rat was injected with sodium chloride injection once through the tail vein, and then injected with sodium chloride injection again through the tail vein 7 days later. The injection volume each time was 1 mL / kg rat. The rats were raised under the animal breeding environment described in step (1) for 7 days.

[0164] Groups 2 to 4: Each rat was injected once with 4 mg / mL doxorubicin sodium chloride injection via the tail vein, with an injection volume of 1 mL / kg rat; 7 days later, each rat was injected once with 3.5 mg / mL doxorubicin sodium chloride injection via the tail vein, with an injection volume of 1 mL / kg rat, and the rats were cultured for 7 days under the animal breeding environment described in step (1).

[0165] (3) Administration:

[0166] Preparation of ruxolitinib phosphate solution: dissolve ruxolitinib phosphate with sodium chloride injection to prepare a ruxolitinib phosphate solution with a concentration of 4.5 mg / mL.

[0167] Preparation of the mixed solution of ruxolitinib and its phosphate: Mix ruxolitinib and ruxolitinib phosphate in a mass ratio of 1:1, dissolve them in sodium chloride injection, and prepare a mixed solution with a concentration of 4.5 mg / mL.

[0168] The four groups of rats treated in step (2) were subjected to the following operations:

[0169] Group 1 and Group 2: intragastric administration of sodium chloride injection, the intragastric volume was 2 mL / kg rat, once a day for 28 consecutive days;

[0170] Group 3: intragastric administration of ruxolitinib phosphate solution at a volume of 2 mL / kg rat, once a day for 28 consecutive days;

[0171] Group 4: intragastric administration of a mixture of ruxolitinib and ruxolitinib phosphate at a dose of 2 mL / kg rat, once a day for 28 consecutive days;

[0172] (4) Urine protein content: After the last administration, urine of rats in Group 1 to Group 4 was collected and the urine protein content was measured. The results are shown in Table 13.

[0173] Table 13

[0174]

[0175]

[0176] As can be seen from Table 13, compared with the blank control group, the urine protein content of the rats in the model control group was significantly increased (p < 0.001); compared with the model control group, the urine protein content of the rats in the low-concentration ruxolitinib group was significantly decreased (p < 0.01); compared with the model control group, the urine protein content of the rats in the high-concentration ruxolitinib group was significantly decreased (p < 0.001); and compared with the low concentration, the high concentration of ruxolitinib had a more significant effect on reducing the urine protein in rats (p < 0.01).

[0177] (5) Blood biochemical test: After the last administration, the serum of rats in Group 1 to Group 4 was collected and the levels of BUN, CRE and CHO in the serum were measured. The results are shown in Table 14.

[0178] Table 14 Rat serum biochemical test results

[0179]

[0180] As can be seen from Table 14, compared with the blank control group, the blood BUN, CRE, and CHO levels of the rats in the model control group were significantly increased (p < 0.001); compared with the model control group, the blood BUN and CRE levels of the rats in the low-concentration ruxolitinib group were significantly decreased (p < 0.001); compared with the model control group, the BUN, CRE, and CHO levels of the rats in the high-concentration ruxolitinib group were significantly decreased (p < 0.001); and compared with the low concentration, the high concentration of ruxolitinib had a more significant effect on reducing the BUN, CRE, and CHO levels of rats (p < 0.01).

[0181] Experimental Example 5

[0182] (1) Experimental animals:

[0183] Animal quality: SD rats, male, SPF grade, 280, body weight 200-250 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., certificate number: (Beijing) 2012-0001.

[0184] Animal breeding environment: air conditioning, room temperature 21 ~ 25 ℃; humidity 45 ± 20%, ventilation 10 times / hour fresh air, artificial lighting 12 hours, free access to food and water.

[0185] Pre-experimental adaptation period: After the animals were introduced and inspected as required, they were housed in separate cages, with 2 animals in each cage, and were observed for 7 days to observe their general condition. Their body weight was measured at the end of the adaptation period.

[0186] (2) Model establishment:

[0187] 280 rats were randomly divided into 28 groups, 10 rats in each group. The groups are shown in Table 15.

[0188] Preparation of doxorubicin sodium chloride injection: dissolve doxorubicin in sodium chloride injection to prepare a concentration of 4 mg / mL doxorubicin sodium chloride injection; dissolve doxorubicin in sodium chloride injection to prepare a concentration of 3.5 mg / mL doxorubicin sodium chloride injection.

[0189] Group 1: Each rat was injected with sodium chloride injection once through the tail vein, and then injected with sodium chloride injection again through the tail vein 7 days later. The injection volume each time was 1 mL / kg rat. The rats were raised under the animal breeding environment described in step (1) for 7 days.

[0190] Groups 2 to 28: Each rat was injected once with 4 mg / mL doxorubicin sodium chloride injection via the tail vein, with an injection volume of 1 mL / kg rat; 7 days later, each rat was injected once with 3.5 mg / mL doxorubicin sodium chloride injection via the tail vein, with an injection volume of 1 mL / kg rat, and the rats were cultured for 7 days under the animal breeding environment described in step (1).

[0191] (3) Administration:

[0192] The mass ratio of ruxolitinib to quercetin salt, coumarin 6, catechin, silymarin, asiatic acid, β-nicotinamide mononucleotide, puerarin or hibiscus flower extract was 1:1, and they were used to prepare the following mixed solutions.

[0193] The mass ratio of ruxolitinib phosphate, quercetin, coumarin 6, catechin, silymarin, asiatic acid, β-nicotinamide mononucleotide, puerarin or hibiscus flower extract was 1:1 and was used to prepare the following mixed solutions.

[0194] The preparation and administration methods of all drug solutions are the same as those in Example 4, and the drug solution concentrations are shown in Table 15.

[0195] Table 15

[0196]

[0197]

[0198] (4) Urine protein content: After the last administration, urine of rats in Group 1 to Group 28 was collected and the urine protein content was measured. The results are shown in Table 16.

[0199] Table 16

[0200]

[0201]

[0202] As can be seen from Table 16, compared with the blank control group, the urine protein level of the rats in the model control group was significantly increased (p < 0.001); compared with the model control group, the urine protein level of the rats in the ruxolitinib group, ruxolitinib phosphate group, quercetin group, coumarin 6 group, catechin group, silymarin group, asiatic acid group, β-nicotinamide mononucleotide group, puerarin group or hibiscus flower extract group was significantly decreased (p < 0.01, 0.001); compared with the model control group, ruxolitinib and quercetin group, coumarin 6 group, catechin group, silymarin group, asiatic acid group, β-nicotinamide mononucleotide group, puerarin group or hibiscus flower extract group had a significant effect on the urine protein level. The urine protein level of the rats in the nucleotide group, puerarin group or hibiscus flower extract group was significantly reduced (p < 0.001), and the reduction level was significantly higher than that in groups 3-12 (p < 0.01, 0.001); compared with the model control group, the urine protein level of the rats in the ruxolitinib phosphate and quercetin group, coumarin 6 group, catechin group, silymarin group, asiatic acid group, β-nicotinamide mononucleotide group, puerarin group or hibiscus flower extract group was significantly reduced (p < 0.001), and the reduction level was significantly higher than that in groups 3-12 (p < 0.01, 0.001);

[0203] (5) Blood biochemical test: After the last administration, the sera of rats in Group 1 to Group 28 were collected to measure the levels of BUN, CRE and CHO in the sera. The results are shown in Table 17.

[0204] Table 17 Rat serum biochemical test results

[0205]

[0206]

[0207] As can be seen from Table 17, compared with the blank control group, the blood BUN, CRE, and CHO levels of the rats in the model control group were significantly increased (p < 0.001); compared with the model control group, the blood BUN and CRE levels of the rats in the ruxolitinib group, ruxolitinib phosphate group, quercetin group, coumarin 6 group, catechin group, silymarin group, asiatic acid group, β-nicotinamide mononucleotide group, puerarin group or hibiscus flower extract group were significantly decreased (p < 0.05, 0.01); compared with the model control group, ruxolitinib and quercetin group, coumarin 6 group, catechin group, silymarin group, asiatic acid group, β-nicotinamide mononucleotide group, The blood BUN, CRE, and CHO levels of the composite rats in the puerarin group or the hibiscus flower extract group were significantly reduced (p<0.05, 0.001), and the reduction level was significantly higher than that in the 3rd-12th groups (p<0.01, 0.001); compared with the model control group, the blood BUN, CRE, and CHO levels of the composite rats in the ruxolitinib phosphate and quercetin group, coumarin 6 group, catechin group, silymarin group, asiatic acid group, β-nicotinamide mononucleotide group, puerarin group or hibiscus flower extract group were significantly reduced (p<0.001), and the reduction level was significantly higher than that in the 3rd-12th groups (p<0.01, 0.001);

[0208] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of (R)-3-cyclopentyl-3-[4-(7H-pyridine-4-yl)-1H-pyrazol-1-yl]propionitrile or its phosphate and quercetin in the preparation of drugs for preventing or treating chronic nephritis; The mass ratio of the (R)-3-cyclopentyl-3-[4-(7H-pyridine[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propionitrile or its phosphate to quercetin is 1:1.

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