Application of rosaxostat in preparation of medicine for relieving CKD kidney inflammation level

Abstract

The invention relates to application of rosaxostat in preparation of a medicine for relieving the CKD kidney inflammation level. The application finds that 1, the plasma SDF-1 level of a CKD patient taking the rosaxostat is remarkably increased, and the proportion of peripheral blood anti-inflammatory myeloid suppressor cells is remarkably increased; 2, a mouse renal fibrosis model research finds that the proportion of anti-inflammatory myelosuppression cells in the spleen, peripheral blood and kidney of a mouse is increased after the mouse takes the rosaxostat; the expression of HIF-1alpha in the spleen and the kidney is increased, but the expression of SDF-1 in the spleen is increased, and the expression of SDF-1 in the kidney is not obviously changed. It is shown that the Rosaxostat relieves the CKD kidney inflammation level by regulating and controlling the spleen-derived myeloid inhibition cell typing.

Description

Technical Field

[0001] The present invention relates to use of roxadustat in preparing a medicine for alleviating CKD kidney inflammation level. Background Art

[0002] Roxadustat is an oral hypoxia-inducible factor-prolyl hydroxylase inhibitor (HIF-PHI) currently used to treat renal anemia. Its mechanism of action is to inhibit the ubiquitination and degradation of hypoxia-inducible factor, maintain the stability of hypoxia-inducible factor 1α (HIF-1α), promote the expression of erythropoietin (EPO) and its receptor, improve iron absorption, utilization and transport, and comprehensively regulate erythropoiesis.

[0003] Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immune cells. During chronic inflammation, MDSCs are activated and induced to differentiate, primarily into two subpopulations: pro-inflammatory (human phenotype: CD11b+CD14-CD15+, mouse phenotype: CD11b+ly6C-ly6G+, G-MDSCs) and anti-inflammatory (human phenotype: CD11b+CD14+CD15-, mouse phenotype: CD11b+ly6C+ly6G-, M-MDSCs). Activated HIF-1α can activate the downstream factor SDF-1, which, through binding to the CXCR4 receptor, promotes the accumulation and migration of MDSCs, exerting anti-inflammatory and immunosuppressive effects.

[0004] However, no studies at home and abroad have yet clarified whether rosuvastatin-induced HIF-1α activation regulates the inflammatory state of CKD by transforming MDSCs into different subpopulations through the SDF-1 / CXCR4 signaling pathway. Summary of the Invention

[0005] The present invention has discovered a new pharmaceutical use of roxadustat, and the specific technical solution includes:

[0006] Use of roxadustat or a pharmaceutically acceptable salt thereof in the preparation of a medicament, wherein the medicament is used for:

[0007] (a) Upregulates the proportion of anti-inflammatory myeloid-derived suppressor cells (MDSCs) by activating the HIF-1α / SDF-1 signaling pathway;

[0008] Alternatively, (b) treating chronic kidney disease (CKD)-associated renal inflammation and / or fibrosis.

[0009] The increase in the proportion of anti-inflammatory MDSCs is due to the upregulation of HIF-1α and SDF-1 expression in the spleen, and the MDSCs migrate to the kidneys and inhibit local inflammatory responses.

[0010] The drug works through the following mechanisms:

[0011] (i) Activate HIF-1α in the spleen and promote the expression of SDF-1;

[0012] (ii) inducing the migration of spleen-derived anti-inflammatory MDSCs to the kidney;

[0013] (iii) Reduce the level of local pro-inflammatory factors in the kidney and inhibit the progression of fibrosis.

[0014] The drug is an oral preparation, and the effective dosage range of the drug containing roxadustat is 20-150 mg / day.

[0015] A pharmaceutical composition for treating chronic kidney disease (CKD), comprising:

[0016] (a) roxadustat or a pharmaceutically acceptable salt thereof;

[0017] (b) at least one additional therapeutic agent selected from an EPO analog, an iron agent, an anti-fibrotic agent, or an immunomodulatory agent;

[0018] (c) a pharmaceutically acceptable carrier.

[0019] The additional therapeutic agent is an anti-fibrotic agent, and the composition reduces renal inflammation and fibrosis levels by synergistically inhibiting the HIF-1α / SDF-1 pathway and the fibrosis pathway.

[0020] A method for screening candidate drugs for treating chronic kidney disease (CKD), comprising the following steps:

[0021] (a) Testing the ability of candidate compounds to activate the HIF-1α / SDF-1 pathway in vitro or in animal models;

[0022] (b) detecting whether the candidate compound induces an increase in the proportion of anti-inflammatory MDSCs;

[0023] (c) Select compounds that can simultaneously activate the HIF-1α / SDF-1 pathway and upregulate anti-inflammatory MDSCs as potential drugs for the treatment of CKD.

[0024] The animal model is a renal fibrosis model, and the detection indicators include the SDF-1 expression level in the spleen, the MDSCs infiltration ratio in the kidney and the inflammatory factor level.

[0025] The beneficial effect of the present invention is that it indicates that rosuvastatin alleviates the level of kidney inflammation in CKD by regulating the typing of spleen-derived myeloid suppressor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Comparison of experimental results on the effect of roxadustat on the proportion of MDSCs subpopulations in peripheral blood of CKD patients; flow cytometry showed that the proportion of G-MDSCs subpopulation in peripheral blood of CKD patients was significantly increased. After roxadustat treatment, the proportion of G-MDSCs subpopulation and M-MDSCs subpopulation in peripheral blood was significantly increased (*P<0.05vs CKD).

[0027] Figure 2: Comparison of experimental results showing the effect of roxadustat on plasma SDF-1 levels in CKD patients. Oral roxadustat treatment significantly increased plasma SDF-1 levels in CKD patients (*P<0.05 vs CKD, EPO). Roxadustat treatment significantly increased plasma SDF-1 levels, whereas EPO treatment had no such effect.

[0028] Figure 3a : Comparison of experimental results of the effect of roxadustat on the proportion of G-MDSCs in different tissues of UUO mice; Comparison of the proportion of G-MDSCs subpopulations in the spleen of UUO model mice and UUO model mice treated with roxadustat;

[0029] Figure 3b :Comparison of experimental results of the effect of roxadustat on the proportion of G-MDSCs in different tissues of UUO mice; Comparison of the proportion of G-MDSCs subpopulations in the blood of UUO model mice and UUO model mice treated with roxadustat

[0030] Figure 3c :Comparison of experimental results of the effect of roxadustat on the proportion of G-MDSCs in different tissues of UUO mice; Comparison of the proportion of G-MDSCs subpopulations in the kidneys of UUO model mice and UUO model mice treated with roxadustat

[0031] Figure 4 Comparison of HIF-1α and SDF-1 expression in the spleens of UUO mice. Splenic HIF-1α and SDF-1 levels were significantly elevated in UUO mice after oral administration of rosuvastatin, but remained unchanged after administration of EPO. Addition of a CXCR4 inhibitor significantly decreased SDF-1 expression in the spleen, but did not significantly alter HIF-1α expression (#P>0.05 vs UUO, EPO; *P<0.05 vs UUO, EPO, CXCR4 inhibitor).

[0032] Figure 5Comparison of HIF-1α and SDF-1 expression in the kidneys of UUO mice. HIF-1α expression in the kidneys of UUO mice was higher than that in the control and sham groups, and oral administration of roxadustat had no significant effect on HIF-1α expression in the kidneys. However, SDF-1 expression in the kidneys of UUO mice was significantly decreased, and oral administration of roxadustat did not increase SDF-1 expression in the kidneys (*P>0.05 vs UUO, EPO, CXCR4 inhibitor)

[0033] Figure 6 Comparison of inflammatory factors in the kidneys of mice undergoing UUO. The UUO group showed decreased levels of the anti-inflammatory cytokine IL-10 and increased levels of the pro-inflammatory cytokine TNF-α. There were no significant differences between the EPO and UUO groups. However, the rosuvastatin group showed significantly increased IL-10 levels and significantly decreased TNF-α levels compared with the EPO and UUO groups. This inflammatory response was reversed by the addition of a CXCR4 inhibitor (*P < 0.05 vs. UUO, EPO, and CXCR4 inhibitor). DETAILED DESCRIPTION

[0034] Roxadustat is a HIF-1α agonist currently indicated for renal anemia caused by chronic kidney disease (CKD). It primarily increases endogenous EPO production through HIF-1α, thereby elevating hemoglobin. The HIF-1α / SDF-1 signaling pathway is also a classic one. The SDF-1 / CXCR4 signaling pathway is one of the key molecules regulating myeloid-derived suppressor cells.

[0035] (1) The proportion of M-MDSCs subpopulation in the peripheral blood of CKD patients increased significantly after oral rosuvastatin treatment.

[0036] Peripheral blood was collected from 9 patients with CKD who were treated in the First Affiliated Hospital of Wannan Medical College (Yijishan Hospital). The oral dosage of rosuvastatin was 100 mg, TIW. The subcutaneous EPO dosage was 6000 U, once every two weeks. Patients who underwent health examinations at the physical examination center of the First Affiliated Hospital of Wannan Medical College (Yijishan Hospital) were selected as healthy controls. The proportion of G-MDSCs subpopulation in the peripheral blood of CKD patients was significantly higher than that of healthy controls (76.8±1.6% vs 0.8±0.3%). After oral rosuvastatin treatment, the proportion of G-MDSCs subpopulation in the peripheral blood was significantly decreased (4.3±0.6% vs 76.8±1.6%), and the proportion of M-MDSCs subpopulation was significantly increased (23.67±3.51% vs 0.18±0.04%) ( Figure 1 Rosuvastatin regulates the HIF pathway (such as promoting HIF-1α stability), affects the differentiation or migration of MDSCs, and promotes the transformation of the immunosuppressive phenotype from pro-inflammatory G-MDSCs to anti-inflammatory M-MDSCs, thereby improving the inflammatory state of CKD.

[0037] (2) Changes in plasma SDF-1 concentration in patients after oral rosuvastatin treatment.

[0038] Fifty patients with chronic kidney disease and renal anemia were selected. Among them, 19 patients took rosuvastatin orally (dose of 100 mg, TIW), 22 patients received subcutaneous injection of EPO (6000 U, once every two weeks), and 9 patients did not receive rosuvastatin or EPO treatment. Eight healthy controls without chronic kidney disease were also selected, and after collecting venous plasma, the SDF-1 concentration in the plasma was examined by ELISA. The results showed that the plasma SDF-1 level of CKD patients taking rosuvastatin (89.77±44.97pg / ml) was significantly higher than that of patients with subcutaneous injection of EPO (46.85±24.06pg / ml) and patients who did not receive treatment (59.53±17.07pg / ml), and was also significantly higher than that of healthy controls (51.77±15.04pg / ml). Figure 2) Further comparison of plasma SDF-1 levels before and after treatment in 6 patients who took rosuvastatin orally and 11 patients who took subcutaneous EPO showed no statistically significant difference in plasma SDF-1 concentration before and after treatment in patients who took subcutaneous EPO (73.44±48.48pg / mL vs 68.26±49.22pg / mL), while plasma SDF-1 levels were significantly increased after taking rosuvastatin orally (61.26±53.31pg / mL vs 108.89±49.28pg / mL) ( Figure 2) This indicates that rosuvastatin can significantly activate the SDF-1 signaling pathway in CKD patients.

[0039] (3) A unilateral ureteral occlusion (UUO)-induced mouse renal fibrosis model was established. A blank control group (Control) and a sham operation group (Sham) were set up. The mice were given roxadustat by gavage, EPO by subcutaneous injection, or roxadustat by gavage + intraperitoneal injection of CXCR4 inhibitor AMD3100, respectively. Drug administration began one week after UUO model establishment. The roxadustat group was given roxadustat by gavage at a dose of 12.5 mg / kg, the roxadustat + CXCR4 antagonist AMD3100 group was given roxadustat by gavage at a dose of 12.5 mg / kg, and the CXCR4 receptor antagonist AMD3100 was intraperitoneally injected at a dose of 5 mg / kg. The EPO group was given EPO by subcutaneous injection at a dose of 1000 IU / kg. The drug was administered three times a week for a total of 2 weeks before the mice were euthanized. The proportions of different subpopulations of MDSCs in the spleen, blood and kidneys of the mice were detected. The results showed that the proportions of G-MDSCs subpopulations in the spleen, blood and kidneys of the UUO group were increased, with the proportions in the spleen, blood and kidneys being 50.13±1.36%, 30.23±1.37% and 43.27±2.40%, respectively. There was no significant change in the old G-MDSCs subpopulations in the spleen, blood and kidneys of the EPO group, which were 48.40±2.29%, 31.90±1.38% and 40.70±0.60%, respectively. In the roxadustat group, the proportions of G-MDSCs in the spleen, blood, and kidney were significantly reduced, reaching 4.73±0.31%, 1.50±1.32%, and 4.10±0.36%, respectively, and the proportions of the M-MDSCs subpopulation were significantly increased, reaching 21.63±1.05%, 72.77±6.28%, and 47.67±2.52%, respectively (Figure 3). After the use of AMD3100, the above effects of roxadustat were reversed, indicating that roxadustat regulates the transformation of spleen-derived MDSCs into anti-inflammatory subpopulations through the SDF-1 / CXCR4 pathway.

[0040] (4) Rosuvastatin can increase the levels of HIF-1α and SDF-1 in the spleen of UUO mice.

[0041] The levels of HIF-1α and SDF-1 in the spleen of UUO mice were significantly increased after oral administration of rosuvastatin, but no significant changes were observed after administration of EPO. The expression of SDF-1 in the spleen was significantly reduced after the addition of CXCR4 antagonist, but the expression of HIF-1α did not change significantly, indicating that rosuvastatin can significantly increase the level of SDF-1 in the spleen of UUO mice, and this effect can be blocked by CXCR4 receptor antagonist ( Figure 4 ).

[0042] (5) Roxadustat had no significant effect on the levels of HIF-1α and SDF-1 in the kidneys of UUO mice.

[0043] The expression of HIF-1α in the kidneys of UUO mice was higher than that in the control and sham groups. Roxadustat gavage had no significant effect on the expression of HIF-1α in the kidneys. However, the expression of SDF-1 in the kidneys of UUO mice was significantly reduced. Roxadustat gavage did not increase the expression level of SDF-1 in the kidneys. Figure 5 ).

[0044] (6) Rosuvastatin upregulates IL-10 expression and downregulates TNF-α expression in the kidneys of UUO mice.

[0045] The anti-inflammatory factor IL-10 in the kidneys of mice in the UUO group was reduced, while the pro-inflammatory factor TNF-α was increased. There was no significant difference between the EPO group and the UUO group. The IL-10 level in the rosuvastatin group was significantly higher than that in the EPO and UUO groups, while the TNF-α level was significantly lower than that in the EPO and UUO groups. This effect of reducing inflammation was reversed after the addition of a CXCR4 antagonist ( Figure 6 ). This indicates that rosuvastatin can reduce the level of inflammation in the kidneys of UUO mice.

Claims

1. A use of roxadustat or a pharmaceutically acceptable salt thereof in the preparation of a medicament, characterized in that: The drug is used to: (a) Upregulates the proportion of anti-inflammatory myeloid-derived suppressor cells (MDSCs) by activating the HIF-1α / SDF-1 signaling pathway; (b) Treatment of renal inflammation and / or fibrosis associated with chronic kidney disease (CKD).

2. The use according to claim 1, characterized in that The increase in the proportion of anti-inflammatory MDSCs is due to the upregulation of HIF-1α and SDF-1 expression in the spleen, and the MDSCs migrate to the kidneys and inhibit local inflammatory responses.

3. The use according to claim 1 or 2, characterized in that The drug works through the following mechanisms: (i) Activate HIF-1α in the spleen and promote the expression of SDF-1; (ii) inducing the migration of spleen-derived anti-inflammatory MDSCs to the kidney; (iii) Reduce the level of local pro-inflammatory factors in the kidney and inhibit the progression of fibrosis.

4. The use according to any one of claims 1 to 3, characterized in that The drug is an oral preparation, and the effective dosage range of the drug containing roxadustat is 20-150 mg / day.

5. A pharmaceutical composition for treating chronic kidney disease (CKD), characterized in that Include: (a) roxadustat or a pharmaceutically acceptable salt thereof; (b) at least one additional therapeutic agent selected from an EPO analog, an iron agent, an anti-fibrotic agent, or an immunomodulatory agent; (c) a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that The additional therapeutic agent is an anti-fibrotic agent, and the composition reduces renal inflammation and fibrosis levels by synergistically inhibiting the HIF-1α / SDF-1 pathway and the fibrosis pathway.

7. A method for screening candidate drugs for treating chronic kidney disease (CKD), characterized in that: The following steps are involved: (a) Testing the ability of candidate compounds to activate the HIF-1α / SDF-1 pathway in vitro or in animal models; (b) detecting whether the candidate compound induces an increase in the proportion of anti-inflammatory MDSCs; (c) Select compounds that can simultaneously activate the HIF-1α / SDF-1 pathway and upregulate anti-inflammatory MDSCs as potential drugs for the treatment of CKD.

8. The method according to claim 7, characterized in that The animal model is a renal fibrosis model, and the detection indicators include the SDF-1 expression level in the spleen, the MDSCs infiltration ratio in the kidney and the inflammatory factor level.

Images