CN110833552A - Use of pyrazolopyrimidine derivatives for the treatment of lupus nephritis - Google Patents
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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Abstract
The pyrazolopyrimidine derivative can obviously reduce the levels of anti-ds-DNA antibodies, IL-6, IL-18, TNF- α and IFN-gamma in serum of lupus mice, inhibit the expression of ICAM-1 and IL-18 proteins in renal tissues of the lupus mice, has a certain repairing effect on injury of glomeruli and renal tubules caused by inflammatory reaction, and has a better treatment effect on lupus nephritis.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of pyrazolopyrimidine derivatives in treating lupus nephritis.
Background
Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and multiple system involvement, with diverse clinical manifestations including facial butterfly erythema, photosensitivity, arthritis, and the like. The course of disease is repeated, and can affect multiple systems and multiple organs, wherein the most common kidney is affected, and Lupus Nephritis (LN) occurs in about 50-70% of SLE patients, and kidney biopsy shows that almost all patients have pathological changes of the kidney. It is currently believed that the occurrence and development of Lupus Nephritis (LN) are closely related to the imbalance between renal cell proliferation and apoptosis, which is manifested by an abnormal increase in cell number, while apoptosis is absolutely or relatively deficient, resulting in predominance of cell proliferation.
The pyrazolopyrimidine derivative (formula I) is a small molecule inhibitor targeted on FLT3 kinase, is a novel compound independently developed by the applicant, and the patent of the compound is granted in China, the United states, Japan and other areas at present.
Only the pyrazolopyrimidine derivative is reported to treat acute myelogenous leukemia and psoriasis at present, but the pyrazolopyrimidine derivative has no application in treating lupus nephritis.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides the application of the pyrazolopyrimidine derivative in treating lupus nephritis.
The purpose of the invention can be realized by the following technical scheme:
application of pyrazolopyrimidine derivative shown as formula I or pharmaceutically acceptable salt and hydrate thereof in preparation of medicine for treating lupus nephritis
Preferably, the lupus nephritis is manifested as glomerular injury.
Preferably, the lupus nephritis is manifested as a renal tubular injury.
Preferably, the lupus nephritis is manifested by infiltration of inflammatory cells into renal tissue.
Preferably, the lupus nephritis is manifested by enhanced expression of ICAM-1, IL-18 proteins in kidney tissues.
Preferably, the lupus nephritis is manifested by an increase in serum anti-ds-DNA antibody, IL-6, IL-18, TNF- α, IFN- γ levels.
Preferably, the pharmaceutically acceptable salts include hydrochloride, sulfate, mesylate, phosphate.
The invention also provides application of the pharmaceutical composition in preparing a medicine for treating lupus nephritis, which is characterized in that the pharmaceutical composition contains pyrazolopyrimidine derivative shown as the formula I or pharmaceutically acceptable salt or hydrate thereof as an active ingredient and a pharmaceutically acceptable excipient.
Preferably, the pharmaceutical composition is an injection preparation, an oral preparation, an external preparation, a sustained release preparation, or a controlled release preparation.
Preferably, the oral preparation comprises tablets, granules and capsules.
Preferably, the pharmaceutical composition is a controlled release formulation, a sustained release formulation, an immediate release formulation.
anti-ds-DNA antibodies are closely related to disease activity and lupus nephritis, high titers of anti-ds-DNA antibodies are often found in LN patients and vary with the activity of the disease, the time of appearance, titer and time and severity of renal damage, ds-DNA anti-ds-DNA antibody immune complexes are one of pathogenic pathways of LN, IL-6 promotes cell activation and differentiation and participates in cell-mediated inflammatory reactions such as T, B lymphocytes, IL-6 levels in the serum of LN patients are significantly elevated and positively correlated with disease activity, serum anti-dsDNA antibodies, IL-18 plays a major role in LN inflammatory processes, IL-18 elevation is significantly correlated with lupus disease activity index, IL-18 concentration after mitogen induction of peripheral blood mononuclear cells in LN patients is significantly elevated compared with systemic lupus erythematosus Tumor Necrosis Factor (TNF) is one of the major causes of pathological damage in autoimmune diseases, increased local expression of TNF- α mRNA may cause kidney function and structural changes, Tumor Necrosis Factor (TNF) may further accelerate development of kidney function and kidney sclerosis in mice, IFN- γ -mediated inflammatory processes, IFN-mediated inflammatory processes may promote further development of renal sclerosis and IFN- γ -mediated immune cell infiltration into mouse lung cancer, and IFN- γ -mediated inflammatory processes.
The pyrazolopyrimidine derivative is originally an FLT3 inhibitor, and researches show that the pyrazolopyrimidine derivative can obviously reduce the levels of anti-ds-DNA antibodies, IL-6, IL-18, TNF- α and IFN-gamma in the serum of a lupus mouse, inhibit the expression of ICAM-1 and IL-18 proteins in the renal tissue of the lupus mouse, has a certain repairing effect on the injury of glomeruli and renal tubules caused by inflammatory reaction, and has a better treatment effect on lupus nephritis.
Compared with the prior art, the method has the following advantages:
the pyrazolopyrimidine derivative provided by the invention is a novel medicine for treating lupus nephritis, not only expands the selectivity of the existing medicine for treating lupus nephritis, but also provides more medicine choices for effective treatment of lupus nephritis, and further expands the application range of the pyrazolopyrimidine derivative as an FLT3 inhibitor.
Drawings
FIG. 1 is a histomorphological observation of kidney tissue of mice in a blank control group stained by HE.
FIG. 2 is a histomorphological observation of kidney tissue of model group mice stained by HE.
FIG. 3 is a histomorphological observation of positive drug group mouse kidney tissue by HE staining.
FIG. 4 is histomorpheopathological observation of HE staining of renal tissue in a high dose group of mice with pyrazolopyrimidine derivative.
FIG. 5 is histomorpheopathological observation of renal tissues of pyrazolopyrimidine derivative low dose group mice by HE staining.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 Effect on apoptosis of mouse lupus nephritis
1. Experimental animals: selecting a SPF-level systemic lupus erythematosus model BXSB mouse, wherein the mouse is male, the body mass (28 +/-2) g and the age of the mouse is 18 weeks; clean grade C57BL/6 mice, male, body mass (20. + -.2) g; purchased from laboratory animal technology, Inc. of Wei Tong Li Hua, Beijing.
2. Experimental materials: pyrazolopyrimidine derivative was synthesized by the inventors. Pyrazolopyrimidine derivative was synthesized by the inventors. Mixing castor oil with ethanol 1: 1, passing through a sterile filter membrane of 0.22 mu m to obtain an ELE solution, weighing a certain mass of pyrazolopyrimidine derivative powder, dissolving the powder by using the ELE solution, adding a certain volume of sterilized water after the powder is completely dissolved, and uniformly mixing for later use. The volume ratio of the ELE solution to the sterile water is 1: 3.
3. animal grouping and administration: the BXSB mice are randomly divided into a model group, a pyrazolopyrimidine derivative high-dose group (hereinafter referred to as a high-dose group), a pyrazolopyrimidine derivative low-dose group (hereinafter referred to as a low-dose group) and a positive drug group, and C57BL/6 mice are taken as a blank control group, and 10 mice are taken in each group. The volume ratio of the ELE solution to the sterile water in the model group and the blank control group is 1: 3, the high-dose group is intragastrically administered with 16mg/kg/d (1.6mg/mL liquid medicine), the low-dose group is intragastrically administered with 8mg/kg/d (0.8mg/mL liquid medicine) of pyrazolopyrimidine derivative, and the positive drug group is administered with 5mg/kg/d of prednisone. The administration is performed by gavage 1 time daily (10mL/kg), and the administration is performed continuously for 4 weeks. After 4 weeks, the materials are taken, and the corresponding detection indexes are collected.
4. Detecting the index
(1) And (3) detecting kidney pathology: taking a mouse kidney, removing a coating, longitudinally cutting the kidney into two halves, fixing the kidney in 4% paraformaldehyde for 12-24 hours, soaking 20% and 30% sucrose for precipitation, embedding a tissue normal-temperature embedding agent, quenching the tissue by using liquid nitrogen, then slicing the tissue by using a freezing microtome, wherein the thickness of the slice is 6 mu m, baking the slice at the temperature of below 40 ℃, performing HE dyeing, dehydrating, transparentizing and sealing the slice, and observing pathological changes of the kidney under a 400X high-power visual field optical microscope.
(2) ELISA detection, which is to detect the contents of mouse serum ds-DNA antibody, IL-6, IL-18, TNF- α and IFN-gamma by ELISA method.
(3) Detection of glomerular ICAM-1 expression: conventional paraffin section; blocking endogenous peroxidase activity with 3% hydrogen peroxide methanol; performing antigen heat restoration by a microwave oven method; after the slices are treated by the antigen repairing liquid, sequentially dripping goat anti-mouse (ICAM-1) IgG and biotin-labeled rabbit anti-goat IgG; horseradish enzyme is labeled with streptavidin and instant DAB developing solution; the results of the staining intensity/staining area of optionally 5 glomeruli in each specimen were measured by HPIAS-1000 high-resolution color pathology image-text analysis system and averaged.
(4) Detection of IL-18 protein expression level in renal tissue: dewaxing and dehydrating a paraffin section with the thickness of 5 mu m; after conventional treatment, dropwise adding a goat anti-mouse IL-18 polyclonal antibody (1:40) at 4 ℃ overnight; dripping biotin-labeled donkey anti-sheep secondary antibody and SP reagent in sequence, and then carrying out DAB color development and periwinkle counterstaining; PBS was used as a negative control instead of primary antibody for each staining; IL-18 positive cells are light yellow to tan in the cytoplasm; negative control has no positive reaction; tissue immunostaining was scored semi-quantitatively with reference to the Budwit-Novotny method.
5. The statistical method comprises the following steps: processing data by SPSS17.0, expressing measurement data by mean + -standard deviation, comparing measurement data among multiple groups by t test, and comparing count data by χ test2And (6) checking. P<0.05 difference is statistically significant。
6. Results of the experiment
(1) Influence on histopathological changes of kidney of lupus nephritis mouse
As shown in fig. 1 to 5, compared with the blank control group, the kidney of the model group had a part of glomerular volume increase, a narrow lumen, perivascular inflammatory cell infiltration, and involvement of renal interstitium and glomerulus; the glomerulus and interstitial tissue of the low-dose group have slight lymphocyte infiltration, and the structures of the glomerulus and renal tubule are basically recovered to be normal after the high-dose group and the positive medicine are taken.
(2) Effect on mouse serum anti-ds-DNA antibodies, IL-6, IL-18, TNF- α, IFN-. gamma
TABLE 1 Effect on anti-ds-DNA antibodies, IL-6, IL-18, TNF- α, IFN-. gamma.
P <0.05 compared to model group.
As shown in Table 1, compared with the model group, the high-dose group and the low-dose group can obviously reduce the levels (P is less than 0.05) of anti-ds-DNA antibodies, IL-6, IL-18 and TNF- α in the serum of the lupus nephritis mouse, the high-dose group can obviously reduce the content (P is less than 0.05) of IFN-gamma in the serum, and the low-dose group only shows a reduction trend.
(3) Effect on ICAM-1, IL-18 protein expression in mouse Kidney tissue
TABLE 2 Effect on ICAM-1, IL-18 protein expression
P <0.05 compared to model group.
As shown in Table 2, compared with the model group, the high-dose group and the low-dose group can remarkably reduce the expression of ICAM-1 and IL-18 proteins (P is less than 0.05) in the kidney tissues of mice with lupus nephritis;
conclusion
The pyrazolopyrimidine derivative has a good treatment effect on lupus nephritis, can remarkably reduce the levels of anti-ds-DNA antibodies, IL-6, IL-18, TNF- α and IFN-gamma in the serum of a lupus mouse, inhibits the expression of ICAM-1 and IL-18 proteins in the kidney tissue of the lupus mouse, and has a certain repair effect on the injury of glomeruli and renal tubules caused by inflammatory reaction.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (11)
1. Application of pyrazolopyrimidine derivative shown as formula I or pharmaceutically acceptable salt and hydrate thereof in preparation of medicine for treating lupus nephritis
2. The use of claim 1, wherein the lupus nephritis is manifested as glomerular injury.
3. The use of claim 1, wherein the lupus nephritis is manifested as renal tubular injury.
4. The use of claim 1, wherein the lupus nephritis is manifested by inflammatory cell infiltration of kidney tissue.
5. The use according to claim 1, wherein the lupus nephritis is manifested by increased expression of ICAM-1, IL-18 proteins in kidney tissue.
6. The use according to claim 1, wherein the lupus nephritis is manifested as an increase in serum anti-ds-DNA antibodies, IL-6, IL-18, TNF- α, IFN- γ levels.
7. Use according to claim 1, wherein the pharmaceutically acceptable salts comprise hydrochloride, sulfate, mesylate, phosphate.
8. The use of a pharmaceutical composition in the preparation of a medicament for treating lupus nephritis, wherein the pharmaceutical composition comprises the pyrazolopyrimidine derivative represented by formula i in claim 1, or a pharmaceutically acceptable salt or hydrate thereof, as an active ingredient, and a pharmaceutically acceptable excipient.
9. The use according to claim 8, wherein the pharmaceutical composition is an injection preparation, an oral preparation, an external preparation, a sustained-release preparation, or a controlled-release preparation.
10. The use according to claim 9, wherein the oral formulation comprises tablets, granules, capsules.
11. The use according to claim 8, wherein the pharmaceutical composition is a controlled release formulation, a sustained release formulation, an immediate release formulation.
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