Use of loganic acid in preparation of drugs for preventing and treating renal anemia and renal fibrosis and the drugs

Abstract

The application relates to the field of medicines, and provides an application of loganic acid in preparation of medicines for preventing and treating renal anemia and renal fibrosis and the medicines. The application proves that the loganic acid can significantly improve the performance of mouse renal anemia, restore the kidney function, and reduce the area of renal fibrosis by constructing a chronic kidney disease animal model, and the mechanism of action is related to the regulation of Wnt, tryptophan metabolism, arginine and proline metabolism, and cytokine-receptor interaction signal paths. The loganic acid monomer can be used as an effective component for preventing and treating renal anemia and renal fibrosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to the application of loganic acid in the preparation of drugs for the prevention and treatment of renal anemia and renal fibrosis, and to a drug containing loganic acid for the prevention and treatment of renal anemia and renal fibrosis. Background Technology

[0002] The pathogenesis of renal anemia is complex, mainly including insufficient erythropoietin (EPO) production, impaired iron utilization (such as elevated hepcidin levels), lack of hematopoietic raw materials, chronic inflammatory response, oxidative stress, and accumulation of uremic toxins. Recent studies have found that the long-term microinflammatory state in CKD patients not only directly inhibits the proliferation and differentiation of bone marrow erythroid progenitor cells through inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but also upregulates hepcidin expression, leading to functional iron deficiency.

[0003] Current treatments primarily include iron supplementation, folic acid and vitamin B12 (hematopoietic raw materials), and the use of erythropoietin stimulants (ESAs). Improper use of iron supplements can lead to severe gastrointestinal reactions and oxidative stress. Although the use of erythropoietin stimulants (ESAs) reduces patients' transfusion needs, high-dose ESA treatment is associated with an increased risk of cardiovascular events and tumor recurrence. Furthermore, hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHI), such as roxadustat, have been used in recent years to treat CKD-related anemia by stabilizing hypoxia-inducible factor (HIF), activating endogenous EPO production, improving iron metabolism, and promoting erythropoietin production. Meta-analyses in recent years have found that roxadustat use may cause adverse reactions such as thrombosis, hyperkalemia, and changes in blood pressure. In addition, adverse events associated with roxadustat in phase III clinical trials included hypertension, hyperkalemia, and upper respiratory tract infections. Other studies have found that roxadustat has a stronger inhibitory effect on prolyl hydroxylase domain protein 2 (PHD2), and PHD2 plays a key role in regulating the activity of hypoxia-inducible factor 1α (HIF1α). HIF1α can induce the expression of vascular endothelial growth factor (VEGF), promote retinal neovascularization, and may aggravate retinal hemorrhage. Therefore, achieving effective and safe clinical treatment for renal anemia remains a serious challenge.

[0004] Renal fibrosis is a common pathological process in the progression of kidney disease to end-stage renal failure, characterized by the abnormal deposition of extracellular matrix (ECM) in renal tissue. Fibroblast activation and their transformation into myofibroblasts are the core components, while renal inflammation and oxidative stress under continuous injury play key driving roles in this process. Currently, angiotensin-converting enzyme inhibitors / angiotensin II receptor antagonists (ACEIs / ARBs) can only partially delay this process, and there is still a lack of specific treatments that can effectively slow down the progression of renal fibrosis. Summary of the Invention

[0005] To address the problems of significant side effects and incomplete treatment in existing drugs for the prevention and treatment of renal anemia and renal fibrosis, this invention provides an application of loganic acid in the preparation of drugs for the prevention and treatment of renal anemia and renal fibrosis, and the effects of loganic acid in preventing and treating renal anemia and renal fibrosis are significant.

[0006] To solve the above problems, the present invention adopts the following technical solution.

[0007] Application of a strychnine in the preparation of drugs for the prevention and treatment of renal anemia and renal fibrosis.

[0008] In some embodiments, the loganic acid accounts for 1-99% of the total mass of the drug.

[0009] In some implementations, the purity of the loganic acid raw material is greater than 90%.

[0010] In some embodiments, the loganic acid has the following structural formula: .

[0011] In some embodiments, the drug is in the form of granules, tablets, powders, capsules, oral liquids, injections, sprays, aerosols, powder inhalers, emulsions, nasal drops, suppositories, pills, microspheres, or tinctures.

[0012] The present invention also provides a drug for preventing and treating renal anemia and renal fibrosis, wherein the active ingredient of the drug includes loganic acid.

[0013] In some embodiments, loganic acid constitutes 1-99% of the total mass of the drug.

[0014] In some implementations, the drug is an oral or injectable formulation.

[0015] In some implementations, the drug also includes pharmaceutical excipients.

[0016] In some embodiments, the pharmaceutical excipient is one or more of a pharmaceutical carrier, diluent, adjuvant, and excipient.

[0017] In some embodiments, the pharmaceutical carrier includes, but is not limited to, liposomes, liposomes, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, and mesoporous silica nanoparticles.

[0018] In some embodiments, the diluents, adjuvants, and excipients may specifically include one or more of the following: pharmaceutical preservatives, antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coatings, antibacterial agents, isotonic agents, and absorption retardants.

[0019] In some embodiments, the filler is one or more of microcrystalline cellulose, pregelatinized starch, hydroxypropyl cellulose, and polyethylene glycol.

[0020] In some embodiments, the dosage form of the drug is granules, tablets, powders, capsules, oral liquids, injections, sprays, aerosols, powder inhalers, emulsions, nasal drops, suppositories, pills, microspheres, or tinctures.

[0021] The present invention determines the specific pharmaceutical excipients, their dosage, and preparation methods according to different drug dosage forms, without any special limitations.

[0022] The present invention does not have any special requirements for the preparation method of the drug for preventing and treating renal anemia and renal fibrosis; the preparation method of the corresponding dosage form known to those skilled in the art can be used.

[0023] In some implementations, the advantages of the present invention compared to the prior art are: The pharmacological results of this invention show that loganic acid can increase hemoglobin levels, hematocrit, and erythrocyte count in CKD model mice, suggesting that it has an effect on improving renal anemia. Simultaneously, loganic acid can reduce the kidney index (kidney index = kidney weight / body weight × 100%), blood urea nitrogen (BUN), serum creatinine (Scr), and 24-hour urinary protein (24-hour U-TP) levels in CKD model mice, improving renal function impairment and alleviating pathological changes related to renal fibrosis. These results indicate that loganic acid has pharmacological activity in improving renal anemia and inhibiting the progression of renal fibrosis. Attached Figure Description

[0024] Figure 1 The changes in RBC, Hb, and HCT in each group of mice in Example 1; Figure 2 The changes in serum BUN, Scr, and urinary U-TP in each group of mice in Example 2 are shown. Figure 3 The images show the pathological changes in the kidney tissue of mice in each group in Example 3.

[0025] Figure 4 Volcano plots of RNA-Seq results from mouse kidney tissues in each group in Example 4. Figure 5 Bubble chart showing gene enrichment analysis of mouse kidney tissues in each group in Example 4 using Ontology / Kyoto Encyclopedia of Genes and Genomes (GO / KEGG); Figure 6 The image shows Venn diagrams and heatmaps of differentially expressed genes related to the Wnt signaling pathway in the kidney tissues of mice in Example 4. Detailed Implementation

[0026] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments and accompanying drawings, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1: The effect of loganic acid on improving renal anemia in an adenine-induced CKD mouse model (a) Establishment of animal models Fifty healthy male SPF-grade C57BL / 6 mice, aged 6 weeks and weighing 18-22 g, were selected and acclimatized to a standard diet for one week. Ten mice were then randomly selected as the control group. The control group mice were administered an equal volume of 0.5% CMC-Na solution by gavage, while the remaining mice were administered adenine suspension (prepared with 0.5% CMC-Na solution) at a dose of 80 mg / kg body weight by gavage daily for four consecutive weeks. Blood samples were collected from the submandibular region at week 4, and RBC, Hb, and HCT levels were measured using an automated blood analyzer.

[0028] (II) Grouping and Administration The model animals were randomly divided into 4 groups, and an equal number of normal mice were selected as the normal group. Each group received a corresponding intervention for 4 weeks. The specific grouping and intervention measures are as follows: Normal control group: Administered intraperitoneal injection of the same volume of normal saline as the drug intervention; Adenine model group (ADE group): Administered intraperitoneal injection of the same volume of physiological saline as the drug intervention; Low-dose loganic acid group (LA-L group): Loganic acid was dissolved in physiological saline and administered intraperitoneally at a dose of 25 mg / kg / day. High-dose loganic acid group (LA-H group): Loganic acid was dissolved in physiological saline and administered intraperitoneally at a dose of 50 mg / kg / day.

[0029] Roxadustat group (ROX group): Roxadustat was administered intraperitoneally at a dose of 25 mg / kg / day; Operating instructions: Administer the medication using a 1mL syringe. The medication solution must be prepared fresh for immediate use. The injection procedure and feeding environment should be consistent between the control group and the medication group; the only difference is the intervention variable.

[0030] (III) Detection of peripheral blood RBC, Hb and HCT After drug intervention, mice were fasted for 12 hours, weighed, and their organ indices were analyzed. Blood was collected from the orbital rimi, and peripheral blood RBC, Hb, and HCT levels were measured using a blood analyzer. Results were statistically analyzed using GraphPad Prism v10.0 and subjected to one-way ANOVA. All data are expressed as (±SD). * indicates p<0.05, ** indicates p<0.01, p<0.05 was considered statistically significant, and ns indicated no significant difference. The test results are as follows... Figure 1 As shown.

[0031] Depend on Figure 1 The results showed that, compared with the normal group, the RBC, Hb, and HCT levels of mice in the adenine model group were significantly decreased, indicating that adenine-induced renal anemia was present in the mice. Compared with the adenine model group, intervention with different doses of loganic acid and roxadustat significantly restored RBC, Hb, and HCT levels in mice, suggesting that loganic acid can effectively alleviate adenine-induced renal anemia. Furthermore, the efficacy of loganic acid at doses of 25 mg / kg / d and 50 mg / kg / d in improving renal anemia was superior to that of roxadustat at dose 25 mg / kg / d, verifying the significant anti-renal anemia therapeutic effect of loganic acid.

[0032] Example 2: The effect of loganic acid on renal fibrosis in an adenine-induced CKD mouse model (a) Establishment of animal models Fifty healthy male SPF-grade C57BL / 6 mice, aged 6 weeks and weighing 18-22 g, were selected and acclimatized to a standard diet for one week. Ten mice were then randomly selected as the control group. The control group mice were administered an equal volume of 0.5% CMC-Na solution by gavage, while the remaining mice were administered adenine suspension (prepared with 0.5% CMC-Na solution) at a dose of 80 mg / kg body weight by gavage daily for four consecutive weeks. At the end of the fourth week, 24-hour metabolic monitoring was performed, and serum and urine samples were collected. Serum BUN, Scr, and 24-hour urinary protein levels were measured using an automated biochemical analyzer.

[0033] (II) Grouping and Administration The model animals were randomly divided into 4 groups, and an equal number of normal mice were selected as the normal group. Each group received a corresponding intervention for 4 weeks. The specific grouping and intervention measures are as follows: Normal control group: Administered intraperitoneal injection of the same volume of normal saline as the drug intervention; Adenine model group (ADE group): Administered intraperitoneal injection of the same volume of physiological saline as the drug intervention; Low-dose loganic acid group (LA-L group): Loganic acid was dissolved in physiological saline and administered intraperitoneally at a dose of 25 mg / kg / day. High-dose loganic acid group (LA-H group): Loganic acid was dissolved in physiological saline and administered intraperitoneally at a dose of 50 mg / kg / day.

[0034] Losartan group: Losartan was administered by gavage at a dose of 20 mg / kg / day.

[0035] The operating procedures are the same as those in “Example 1-(II) Grouping and Administration”.

[0036] (III) Detection of serum BUN, Scr and 24-hour urinary protein Twenty-four hours before the end of drug intervention, 24-hour urine samples were collected from mice using metabolic cages. After the experiment, mice were fasted for 12 hours and weighed. Serum BUN, Scr, and 24-hour urinary protein levels were detected using an automated biochemical analyzer. The statistical methods were the same as in "Example 1-(III) Detection of Peripheral Blood RBC, Hb, and HCT". The results are as follows: Figure 2 As shown.

[0037] Depend on Figure 2 The results showed that, compared with the normal group, the adenine model group mice had significantly elevated serum BUN, Scr, and 24-hour urinary protein levels, indicating that the adenine model group mice exhibited kidney damage. Compared with the adenine model group, intervention with different doses of loganic acid and losartan significantly reduced serum BUN, Scr, and 24-hour urinary protein levels in mice, suggesting that loganic acid intervention significantly alleviated adenine-induced kidney damage. Furthermore, the intervention effect of high-dose loganic acid (50 mg / kg / d) on mice with kidney damage was similar to that of losartan (20 mg / kg / d), indicating that the two drugs had comparable efficacy in improving renal function, confirming that loganic acid has a significant nephroprotective effect.

[0038] Example 3: Effects of loganic acid on the pathological characteristics of renal tissue in animal models of renal fibrosis (a) Establishment of animal models Mice were treated according to the modeling, grouping, and drug intervention methods described in Example 2.

[0039] (II) Experimental methods and results After the drug intervention ended, the mice were fasted for 12 hours, and the kidney tissue was separated, weighed, and sectioned and pathologically stained for analysis (the extraction and sectioning of mouse kidney tissue can be carried out using existing techniques, so they are not described in detail).

[0040] HE (hematoxylin-eosin) staining: After dewaxing paraffin sections to water, the cell nuclei are stained blue with basic hematoxylin, differentiated by hydrochloric acid alcohol, and then the cytoplasm and extracellular matrix are stained red to different degrees with acidic eosin. After dehydration and clearing, the sections are mounted.

[0041] Masson trichrome staining: The sections are dewaxed to water, and the nuclei are first stained blackish-blue with Weigert iron hematoxylin (or a similar staining solution). Then, the cytoplasm, myofibrils and erythrocytes are stained with Ponceau S-acid fuchsin. After differentiation with phosphomolybdic acid, the collagen fibers are finally stained bright blue with aniline blue.

[0042] PAS (periodic acid-Schiff) staining: After dewaxing the sections to water, periodic acid is used to oxidize the vicinal diol structure in the sugar molecule to generate an aldehyde group. The aldehyde group reacts with Schiff's reagent (colorless fuchsin) to form a purple-red insoluble product. Finally, hematoxylin is used to counterstain the cell nucleus.

[0043] Staining results as follows Figure 3 As shown.

[0044] HE staining results showed that in the adenine model group, the renal tissue of mice exhibited renal tubular dilation, vacuolar degeneration of epithelial cells, inflammatory cell infiltration, and interstitial fibrosis. Compared with the adenine model group, low-dose loganic acid (LA-L) intervention could partially alleviate renal tubular dilation, vacuolar degeneration, and inflammatory infiltration, and improve interstitial fibrosis; high-dose loganic acid (LA-H) had a more significant effect, with the renal tissue structure becoming more intact, and the degree of inflammatory infiltration and fibrosis further reduced, showing a dose-dependent improvement trend; losartan intervention also significantly reduced renal tissue pathological damage, and renal tubular structural abnormalities and interstitial lesions were improved.

[0045] Masson staining showed that the adenine model group mice had significantly increased collagen deposition in the renal interstitium and peritubular region (blue-stained area). Compared with the adenine model group, collagen deposition was reduced in the LA-L group, but focal fibrosis was still visible; collagen fiber deposition was further reduced in the LA-H group, the blue-stained area in the renal interstitium and peritubular region was significantly reduced, and fibrotic lesions were significantly improved; the losartan group also reduced renal interstitial collagen deposition, and the overall renal tissue morphology was significantly restored compared with the model group.

[0046] PAS staining showed that in the adenine model group, the renal tubular structure was disordered, the brush border was damaged, and the deposition of PAS-positive substances in the renal tubules and interstitial regions was increased. Compared with the adenine model group, the LA-L group could partially reduce the deposition of PAS-positive substances and improve renal tubular damage, but abnormal deposition still existed locally; in the LA-H group, the renal tubules were more regularly arranged, the brush border damage was reduced, and the deposition of PAS-positive substances was further reduced, with a more significant improvement effect; the losartan group could also improve the pathological damage of renal tissue and reduce brush border damage and abnormal deposition.

[0047] In summary, loganic acid intervention significantly improved renal tissue damage, glycogen deposition, and renal fibrosis in model mice. Moreover, compared with losartan intervention, loganic acid's intervention effects in improving renal tubular damage, reducing collagen deposition, and inhibiting renal interstitial fibrosis were similar to those of losartan intervention, suggesting that loganic acid can effectively inhibit the progression of renal fibrosis.

[0048] Example 4: RNA-Seq analysis of the effects of loganic acid on related pathways in kidney tissue of model animals (a) Establishment of animal models The normal control group (Normal group), adenine model group (ADE group), and loganic acid group (ADE_LA group) were constructed according to the modeling, grouping, and drug intervention methods in Example 2; the dosage of loganic acid group (ADE_LA group) was 50 mg / kg / d.

[0049] (II) Experimental Methods and Results After a 12-hour fast following drug intervention, kidney tissue was isolated for RNA-Seq analysis. Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions. RNA purity and quantification were determined using a NanoDrop 2000 spectrophotometer (ThermoScientific, USA), and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Transcriptome libraries were constructed using the VAHTS Universal V5 RNA-seq Library Prep kit according to the manufacturer's instructions. Transcriptome sequencing and analysis were performed by Shanghai Ouyi Biotechnology Co., Ltd. (Shanghai, China).

[0050] Test results as follows Figures 4-6 As shown: Volcano plot of RNA-Seq analysis results from mouse kidney tissue ( Figure 4 Compared to the normal group (NORMAL), the adenine model group (ADE group) showed a large number of significantly differentially expressed genes, indicating that adenine causes gene expression disorder. After loganic acid intervention, the number of differentially expressed genes was significantly reduced compared to the adenine model group, and the fold change of most genes regressed to normal levels, suggesting that loganic acid can effectively reverse adenine-induced transcriptional dysregulation. GO / KEGG enrichment ( Figure 5 Further analysis suggests that the effects of loganic acid in preventing and treating renal anemia and renal fibrosis may be related to the regulation of signaling pathways such as Wnt, tryptophan metabolism, arginine and proline metabolism, and cytokine-receptor interactions; Venn diagram ( Figure 6Part A) and heat map ( Figure 6 Part B results suggest that loganic acid intervention may regulate... Apcdd1、 Axin2, Bambi, Ccn4, Ctnnd2, Frzb, Fzd2, Lef1, Mmp7, Nfatc4, Tcf7, Wnt11, Wnt2 and wnt4 Fourteen genes related to the Wnt signaling pathway were identified, playing a role in improving renal anemia and renal fibrosis. Studies have shown that persistent abnormal activation of the Wnt signaling pathway in the progression of CKD directly leads to renal interstitial fibrosis by mediating renal tubular epithelial-interstitial transdifferentiation (EMT), driving fibroblast activation, and promoting abnormal extracellular matrix (ECM) deposition. The RNA-Seq results suggest that the regulatory role of loganic acid in Wnt signaling transduction may be an important intervention pathway for alleviating renal anemia and renal fibrosis and delaying the progression of CKD.

[0051] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. The application of loganic acid in the preparation of drugs for the prevention and treatment of renal anemia and renal fibrosis.

2. The application of loganic acid according to claim 1 in the preparation of drugs for the prevention and treatment of renal anemia and renal fibrosis, characterized in that: Loganilic acid comprises 1-99% of the total mass of the drug.

3. The application of loganic acid according to claim 1 in the preparation of drugs for the prevention and treatment of renal anemia and renal fibrosis, characterized in that: The drug is in the form of granules, tablets, powders, capsules, oral liquids, injections, sprays, aerosols, powder inhalers, emulsions, nasal drops, suppositories, pills, microspheres, or tinctures.

4. A drug for preventing and treating renal anemia and renal fibrosis, characterized in that: The active ingredient in the drug includes loganic acid.

5. A drug for preventing and treating renal anemia and renal fibrosis according to claim 4, characterized in that: Loganilic acid accounts for 1-99% of the total mass of the drug.

6. The drug for preventing and treating renal anemia and renal fibrosis according to claim 4, characterized in that: The drug is an oral or injectable formulation.

7. A drug for preventing and treating renal anemia and renal fibrosis according to claim 4, characterized in that: The drug also includes pharmaceutical excipients.

8. A drug for preventing and treating renal anemia and renal fibrosis according to claim 4, characterized in that: The pharmaceutical excipient is one or more of the following: pharmaceutical carrier, diluent, adjuvant, and excipient.

Images