Use of dehydroepicholesteric acid in the preparation of a medicament for treating diabetes or diabetic nephropathy

Abstract

The application belongs to the field of medicine and relates to application of dehydroepicholesterol in preparation of a medicine for treating diabetes or diabetic nephropathy. The application provides a new use of dehydroepicholesterol: a1. preparation of a medicine for preventing and / or treating diabetes; a2. preparation of a medicine for preventing and / or treating diabetic nephropathy; a3. establishment of an ex vivo model for exciting or activating TGR5 receptors and / or FXR receptors in renal tubular epithelial cells to prevent and / or treat diabetes or diabetic nephropathy. The medicine for preventing and / or treating diabetes or diabetic nephropathy provided by the application contains dehydroepicholesterol as an active ingredient, can activate Takeda G protein-coupled receptor 5 and / or farnesol X receptor, can effectively improve blood glucose, kidney injury and / or liver function of a diabetes and / or diabetic nephropathy model mouse, and has a prospect of being developed into a medicine for treating diabetes and diabetic nephropathy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and in particular relates to the application of dehydroepicholesterol in the preparation of a drug for treating diabetes or diabetic nephropathy. BACKGROUND

[0002] Diabetes mellitus (DM) is a common chronic disease that seriously endangers public health, and long-term existence can lead to multiple complications. Diabetic kidney disease (DKD) is a common complication of diabetes and a common cause of end-stage renal disease (Prevalence of comorbidities in patients with type-2 diabetes mellitus. Prim Care Diabetes. 2020.). In recent years, the incidence of diabetic kidney disease has shown an increasing trend worldwide, which has brought a heavy burden to the quality of life of patients and the medical system. The current treatment of diabetic kidney disease includes control of blood glucose, renin-angiotensin-aldosterone system inhibitors (RAASi), sodium-glucose co-transporter 2 inhibitors (SGLT2i), aldosterone receptor antagonists (MRA), and glucagon-like peptide-1 receptor agonists (GLP-1RA). Each treatment has its advantages and limitations, for example: control of blood glucose can only delay the progression of DKD; RAASi is very effective in protecting the kidneys, but can cause side effects such as hyperkalemia; SGLT2i and GLP-1RA provide additional kidney protection, but can be associated with other types of side effects, such as urogenital infections. Therefore, the development of new drugs for the prevention and treatment of diabetes or diabetic kidney disease is the only way to reduce the incidence of end-stage renal disease and patient mortality.

[0003] Takeda G protein-coupled receptor 5 (TGR5) and farnesol X receptor (FXR) play an important role in glucose metabolism. TGR5 is mainly expressed in the liver, fat, intestine, gallbladder and brain, while FXR is mainly expressed in the liver, intestine and gallbladder. Both TGR5 and FXR have high expression in the kidney. INT-777 is a selective TGR5 agonist, and OCA, GW4064 are selective FXR agonists, and INT-767 is a dual agonist of FXR and TGR5, but due to the non-targeted nature and potential cytotoxicity of the above drugs, they have not been widely used in clinical practice. Activation of intestinal TGR5 can promote GLP-1 secretion and improve insulin resistance, while FXR helps to regulate glucose homeostasis. Therefore, TGR5 and FXR agonists are expected to treat diabetes and improve diabetic complications such as diabetic nephropathy.

[0004] The prior art discloses that bile acid activates FXR and TGR5 receptors to play its biological role, such as Chinese invention patent application publication No. CN112409435A, application date is September 16, 2019, and the invention name is "Bile acid derivative, its composition and application", which discloses a new type of bile acid derivative for treating fatty liver disease, its pharmaceutical composition and application for preparing a medicament for treating and improving diseases and symptoms mediated or caused by FXR or TGR5. The bile acid derivative of the invention inhibits / delays the metabolism of cholic acid by bacterial BSH / 7a dehydroxylase in the intestinal tract, greatly prolongs the effective survival time of bile acid in the intestinal tract, and the bile acid derivative and its pharmaceutical composition can significantly agonize the bile acid membrane receptor TGR5, promote the secretion of glucagon-like peptide 1 by intestinal endocrine cells, improve liver fat accumulation, significantly improve liver function, and have good effect on treating fatty liver disease. The bile acid derivative and its composition are mainly used for treating fatty liver disease, and there is no approved TGR5 and FXR dual agonist drug for treating diabetes or diabetic nephropathy in the clinic at present. SUMMARY

[0005] 1. PROBLEMS TO BE SOLVED

[0006] In view of the technical problems of non-targeting and potential cytotoxicity of the TGR5 and FXR agonists for preventing or treating diabetes or diabetic nephropathy in the prior art, the application provides the application of dehydrolithocholic acid (DHLCA) as a TGR5 and FXR dual agonist for treating diabetes and / or diabetic nephropathy. The application also provides a medicament for preventing and / or treating diabetes or diabetic nephropathy, wherein the active ingredient of the medicament comprises dehydrolithocholic acid.

[0007] 2. TECHNICAL SCHEME

[0008] To achieve the above-mentioned purpose, the technical scheme provided is as follows:

[0009] The application of dehydrolithocholic acid is as follows a1, a2 or a3,

[0010] a1. preparing a medicament for preventing and / or treating diabetes;

[0011] a2. preparing a medicament for preventing and / or treating diabetic nephropathy;

[0012] a3. establishing an ex vivo model for agonizing or activating TGR5 receptors and / or FXR receptors in renal tubular epithelial cells to prevent and / or treat diabetes or diabetic nephropathy.

[0013] Further, the active ingredient of the medicament comprises dehydrolithocholic acid.

[0014] Further, the dehydroepicholesterol can effectively improve blood glucose, kidney injury and / or liver function of the diabetic and / or diabetic nephropathy model mice.

[0015] The application further provides a medicine for preventing and / or treating diabetes or diabetic nephropathy, wherein an active ingredient of the medicine comprises dehydroepicholesterol.

[0016] Further, the medicine further comprises a pharmaceutically acceptable excipient.

[0017] Preferably, the excipient is selected from one or any combination of a binder, a filler, a plasticizer, a glidant, a disintegrant and a lubricant.

[0018] Further, the medicine is prepared into a pharmaceutically acceptable dosage form by using the pharmaceutically acceptable excipient.

[0019] Further, the dosage form comprises a tablet, a capsule, a granule, an oral solution, an oral suspension, an oral emulsion, an oral liquid, an oral syrup and an injection.

[0020] 3. Advantages

[0021] Compared with the prior art, the technical scheme provided by the application has the following advantages:

[0022] (1) The application of the dehydroepicholesterol can effectively improve blood glucose, kidney injury and liver function of the diabetic and / or diabetic nephropathy model mice by activating TGR5 and / or FXR in renal tubular epithelial cells, and has the prospect of developing into a medicine for treating diabetes and diabetic nephropathy.

[0023] (2) The medicine for preventing and / or treating diabetes or diabetic nephropathy provided by the application, wherein an active ingredient of the medicine comprises dehydroepicholesterol, can improve blood glucose control, reduce kidney injury, reduce proteinuria, improve lipid metabolism, has anti-inflammatory effect and slows down the progression of kidney disease. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Flow chart for intervention of diabetic or diabetic nephropathy mice by dehydroepicholesterol;

[0025] Figure 2 Urinary albumin / creatinine ratio (A) and fasting blood glucose (B) of mice in each group added with different intervention amounts of dehydroepicholesterol;

[0026] Figure 3 Urinary albumin / creatinine ratio (A), urea nitrogen (B), creatinine clearance (C), kidney weight / body weight (D), glutamic-pyruvic transaminase (E), glutamic-oxalacetic transaminase (F), total bile acid (G) and fasting blood glucose (H) of mice in each group;

[0027] Figure 4 The tubulointerstitial injury degree of each group of mice: (A) H&E, PAS, MASSON staining pathological picture;

[0028] Figure 5 The tubulointerstitial injury degree of each group of mice: (B) IFTA score chart;

[0029] Figure 6 The tubulointerstitial injury degree of each group of mice: (C) KIM-1 or NGAL expression of tubular injury marker;

[0030] Figure 7 The TGR5 and FXR expression of each group of mice: (A) mRNA expression chart;

[0031] Figure 8 The TGR5 and FXR expression of each group of mice: (B) immunohistochemical chart;

[0032] Figure 9 The TGR5 and FXR expression of each group of mice: (C&D) protein expression chart. DETAILED DESCRIPTION

[0033] In order to further understand the content of the present application, the present application will be described in detail in conjunction with examples.

[0034] EXAMPLE

[0035] Experimental materials: C57BL / 6J mice were purchased from Jiangsu Jicui Yekang Biotechnology Co., Ltd.

[0036] Dehydroepiandrosterone, also known as 3-oxo-5β-cholanic acid, has a molecular formula of C 24 H 38 O3, purchased from MCE company (full name MedChemExpress).

[0037] Experimental method:

[0038] S1, 31 four-week-old male C57BL / 6J mice were raised under standard environmental conditions, i.e. temperature 24℃, humidity 40-70% and 12-hour light-dark cycle, with sufficient air exchange.

[0039] S2, the mice were randomly divided into two groups: the control group was 10% fat diet group (LFD, n=5); DKD group (diabetic nephropathy group) was 60% high-fat diet (HFD, n=26) group.

[0040] S3, 14 weeks old, 26 HFD mice in DKD group were intraperitoneally injected with 40 mg / kg streptozotocin (STZ) for 5 consecutive days, and 5 LFD mice in the control group were intraperitoneally injected with sodium citrate buffer for 5 consecutive days.

[0041] S4, 18 weeks old, all mice were fasted for 12 h before detecting the fasting blood glucose (FBG), and the diabetic model was confirmed by the fasting blood glucose level greater than 11.1 mmol / L. The control group of mice was gavaged with CMC-Na for 2 weeks after overnight fasting, and the DKD group was divided into 4 groups according to the fasting blood glucose as the main indicator:

[0042] DKD model group n = 8, mice were gavaged with CMC-Na for 2 weeks after overnight fasting;

[0043] DHLCA experiment 1 group n = 5, mice were gavaged with DHLCA 5 mg / kg for 2 weeks after overnight fasting;

[0044] DHLCA experiment 2 group n = 8, mice were gavaged with DHLCA 10 mg / kg for 2 weeks after overnight fasting;

[0045] DHLCA experiment 3 group n = 5, mice were gavaged with DHLCA 20 mg / kg for 2 weeks after overnight fasting.

[0046] S5, 20 weeks, physiological metabolism cage to collect 24-hour urine of mice, collect plasma (cardiac blood, whole blood at room temperature for 30 min, centrifuged at 3000 r / min for 15 min to get mouse serum sample), feces, bilateral kidneys, etc. for serological and renal histopathological detection, and record the kidney weight and body weight.

[0047] a, urine creatinine was determined by Hitachi automatic analyzer 3500;

[0048] b, urine albumin was determined by MedicalSystem;

[0049] c, urea nitrogen (BUN), serum creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bile acid (TBA) were detected by Hitachi automatic analyzer.

[0050] d, renal histopathological detection

[0051] Half of the unilateral kidney was fixed in formalin solution, and after 24 h of fixation at 4℃, the renal tissue of each group was paraffin-embedded, and the section thickness was 4 μm. Then H&E staining, PAS staining, MASSON staining were performed, and the pathological changes of kidney structure and renal tubulointerstitial injury were observed and histologically analyzed by optical microscope; the tubulointerstitial injury was semi-quantitatively scored by IFTA scoring system.

[0052] The severity of tubulointerstitial injury was assessed according to the interstitial fibrosis and tubular atrophy (IFTA) scoring system. IFTA score was defined as the percentage of total area of involved interstitium and tubules, with 0 representing no IFTA, 1 representing IFTA < 25%, 2 representing 25% < IFTA < 50%, and 3 representing IFTA > 50%.

[0053] Data processing:

[0054] Normally distributed continuous variables were expressed as mean ± standard deviation (SD), and non-normally distributed continuous variables were expressed as median (interquartile range). Normality was assessed using the Kolmogorov-Smirnov test. Comparisons between two independent groups used t-test or Mann-Whitney U test, while differences between multiple groups used one-way analysis of variance (ANOVA) or Kruskal-Wallis test, with post-hoc pairwise comparisons. * P < 0.05 compared with the control group, # P < 0.05 compared with the DKD group, ** P < 0.01 compared with the control group, ## P < 0.01 compared with the DKD group, *** P < 0.001 compared with the control group.

[0055] Experimental results:

[0056] 1. Selection of optimal dose of dehydroepicholesterol intervention

[0057] Figure 1 The flow chart of dehydroepicholesterol intervention in mice. The results showed that the DKD group receiving 10 mg / kg DHLCA showed the best kidney protection and improvement of glucose metabolism compared with the 5 mg / kg and 20 mg / kg DHLCA groups, reflected in the improvement of indicators such as urinary albumin / creatinine ratio (UACR), fasting blood glucose (FBG), etc. Figure 2 A, Figure 2 B). Therefore, 10 mg / kg DHLCA was finally selected as the optimal intervention dose for the DKD+DHLCA group.

[0058] 2. Comparison of urine and blood biochemistry of mice in each group (DHLCA dose in DKD+DHLCA group is 10 mg / kg)

[0059] Compared with the DKD group, the UACR, kidney weight / body weight ratio, total bile acid (TBA), and FBG levels of the DKD+DHLCA group were significantly reduced (P < 0.05, Figure 3 A, Figure 3 D, Figure 3 G, Figure 3H). But DHLCA intervention did not affect creatinine clearance rate (Ccr) and urea nitrogen (BUN) levels (P>0.05, Figure 3 C, Figure 3 B). In terms of liver indicators, ALT and AST levels in the DKD group were significantly higher than those in the control group (P<0.05, Figure 3 E, Figure 3 F). The ALT level in the DKD+DHLCA group was significantly lower than that in the DKD group (P<0.05, Figure 4 E), indicating that DHLCA can improve liver function.

[0060] 3, Comparison of renal histopathology in each group of mice (DHLCA dosage 10 mg / kg in DKD+DHLCA group)

[0061] H&E, PAS and Masson staining results show (as Figure 5 ), compared with the DKD group, the renal tubular epithelial cell morphology in the DKD+DHLCA group is improved, the renal tubular epithelial cells change from the original flat, exfoliated state to a more regular, cuboidal or columnar morphology, the lumen expansion and notch are reduced; the morphology and distribution of the brush border are restored to normal, the vacuolization and cast formation are reduced; the proliferation and deposition of collagen fibers (presented in blue or green in Masson staining) are reduced, etc., indicating that the morphology and function of renal tubular epithelial cells are recovering, and the degree of renal interstitial fibrosis is reduced, so DHLCA can alleviate the renal tubular injury in DKD mice.

[0062] IFTA score in the DKD+DHLCA group is lower than that in the DKD group (P<0.05, Figure 6 ), IFTA score is an important indicator for evaluating the severity of renal tubulointerstitial injury, which reflects the degree of renal tubulointerstitial fibrosis and renal tubular atrophy. A lower IFTA score indicates a lighter degree of renal tubulointerstitial fibrosis and renal tubular atrophy, i.e., a lighter degree of renal tubulointerstitial injury.

[0063] The expression of KIM-1 and NGAL is also significantly reduced in the DKD+DHLCA group (as Figure 8 shown).

[0064] KIM-1 (Kidney Injury Molecule-1) is a transmembrane glycoprotein expressed on renal proximal tubular epithelial cells, belonging to the immunoglobulin gene superfamily. Under normal circumstances, the expression of KIM-1 in the kidney is very low, but after kidney damage, its expression in damaged proximal tubular epithelial cells will significantly increase. KIM-1 can quickly, sensitively and specifically reflect the injury and recovery process of various kidney diseases, so it can be used as a reliable biological marker for detecting early kidney injury.

[0065] NGAL (Neutrophil Gelatinase-Associated Lipocalin), NGAL is a small molecule lipocalin protein, which is secreted by neutrophils, and is closely related to inflammatory injury, immune response, tumor and kidney damage. In kidney injury, the level of NGAL in blood and urine will increase significantly, and it can appear in the early stage of kidney injury. The detection of NGAL has important significance in the early diagnosis, treatment and prognosis of various kidney diseases.

[0066] 4. Comparison of TGR5 and FXR expression in each group of mice (DHLCA dose 10 mg / kg in DKD+DHLCA group)

[0067] The results of kidney tissue immunohistochemistry and immunofluorescence showed that in the DKD+DHLCA group, the staining of TGR5 protein in renal tubular epithelial cells was deeper and more extensive, and the positive staining intensity of FXR in renal tubular epithelial cells was increased, indicating that after DHLCA intervention, the expression of TGR5 and FXR in renal tubular epithelial cells was significantly increased (as shown in Figure 7 The mRNA expression levels of TGR5 and FXR in the DKD+DHLCA group were higher than those in the DKD group (P<0.05, Figure 9 ). The protein expression levels of TGR5 and FXR in the DKD+DHLCA group were also higher than those in the DKD group (P<0.05, ​ ).

[0068] TGR5 is a G protein-coupled receptor, which is widely expressed in the human and animal body, including the kidney. It plays an important role in regulating glucose metabolism, inflammation and oxidative stress. In the state of diabetes, the expression of kidney TGR5 protein is down-regulated, which may be related to kidney injury. The present application activates kidney tubular TGR5 by dehydroepiandrosterone, increases the mRNA and protein expression levels of kidney tubular TGR5, and plays a protective role in the occurrence and development of diabetic nephropathy.

[0069] FXR is another nuclear hormone receptor, which is mainly expressed in the liver, intestine and kidney. It regulates diabetic nephropathy by regulating various signaling pathways, including blood glucose, blood lipids, inflammation and fibrosis, effectively controlling the occurrence and development of diabetic nephropathy. Dehydroepiandrosterone is a secondary bile acid, and the structure and properties of different bile acids may lead to different binding strengths and activation efficiencies of receptors, thereby affecting their biological effects. For example, intestinal flora can regulate the composition of bile acids, change the properties of bile acids and their effects on bile acid receptors, and the composition and activity of intestinal flora may affect the metabolism and function of bile acids, and further affect the activation mechanism of FXR and TGR5. The present application activates kidney tubular FXR by dehydroepiandrosterone, increases the mRNA and protein expression levels of kidney tubular FXR, and plays a protective role in the occurrence and development of diabetic nephropathy.

[0070] The present application provides methods for treating, preventing or ameliorating a condition, disease or disorder treatable by activation or agonism of TGR5 and FXR, including diabetes and diabetic nephropathy, comprising administering (e.g., a therapeutically effective amount of) dehydroepicholesterol to the patient.

[0071] The above described embodiments only express the preferred embodiments of the present application, which are described in more detail and in more specifically, but can not be understood as the limitation of the patent scope of the present application. It should be noted that for those skilled in the art, several modifications, improvements and substitutions can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. The application of dehydrolithocholic acid, characterized in that: For example, a1 or a2. a1. To prepare drugs for the prevention and / or treatment of type 2 diabetes; a2. To prepare drugs for the prevention and / or treatment of diabetic nephropathy.

2. The application of dehydrolithocholic acid according to claim 1, characterized in that: The active ingredient of the drug includes dehydrolithocholic acid.

3. The application of dehydrolithocholic acid according to claim 1, characterized in that: The dehydrolithocholic acid can effectively improve blood glucose, kidney damage and / or liver function in mice with type II diabetes and / or diabetic nephropathy.

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