CN115068455B

CN115068455B - Application of BMS-303141 in preparation of medicine for treating chronic kidney disease

Info

Publication number: CN115068455B
Application number: CN202210833590.0A
Authority: CN
Inventors: 孙剑; 詹子顺; 吴学钦; 何金荣
Original assignee: Third Xiangya Hospital of Central South University
Current assignee: Third Xiangya Hospital of Central South University
Priority date: 2022-07-14
Filing date: 2022-07-14
Publication date: 2023-11-07
Anticipated expiration: 2042-07-14
Also published as: CN115068455A

Abstract

The invention discloses an application of BMS-303141 in preparing a medicament for treating chronic kidney disease. The invention screens out the micromolecular compound BMS-303141 for the first time, is an ACL inhibitor for treating obesity type 2 diabetes, can inhibit ectopic lipid deposition, can inhibit inflammatory influence and CKD disease fibrosis progress caused by lipid deposition on animal level, has strong relieving effect, and has no harm to mice. Under the current situation that the number of CKD patients caused by obesity and type 2 diabetes is increased, a new way for relieving disease progress and improving prognosis is found, and the application prospect is wide.

Description

Application of BMS-303141 in preparation of medicine for treating chronic kidney disease

Technical Field

The invention belongs to the technical field of medical application, and particularly relates to application of BMS-303141 in preparation of a medicament for treating chronic kidney disease.

Background

Metabolic syndrome characterized by obesity and type 2 diabetes is becoming more and more common worldwide. The number of obese people in the united states between 20-74 years has doubled more than once, from 15% to 35% in the last three years, and diabetics currently account for 9.3% of their total population (2910 tens of thousands). An increase in obesity and diabetes is associated with an increase in chronic kidney disease (chronic kidney disease, CKD). Obesity and diabetes have become the main causes of CKD, and similar conditions are occurring in china. Obesity-related kidney disease (ORG) is mainly characterized by ectopic lipid accumulation of the kidneys (ectopic lipid accumulation, ELA), glomerular hypertrophy and focal segmental glomerulosclerosis. It is now believed that ELA is a key factor in renal injury caused by obesity and diabetes, as ELA causes mitochondrial dysfunction and increases oxidative and endoplasmic reticulum stress, thereby activating pro-inflammatory and pro-fibrotic signaling pathways, ultimately leading to renal injury.

For the treatment of obesity-related nephropathy, the current approaches are mainly weight reduction, insulin resistance correction and kidney local hemodynamic disorder improvement. For commonly used insulin sensitizers such as thiazolidinediones, the adverse effects are mainly sodium retention, weight gain and the risk of congestive heart failure caused by the patients are obviously increased. ACEI (angiotensin converting enzyme inhibitor) drugs can control hypertension, correct renal local hemodynamic abnormalities and reduce proteinuria, and are often used as the first-choice drugs for ORG patients. However, for patients who have progressed to CKD, the ACEI class of drugs increases the nephrotoxicity of creatinine, urea nitrogen, is apparent.

BMS-303141, its structural formula is:it is named: 3, 5-dichloro-2-hydroxy-N- (4-methoxy [1,1' -biphenyl)]-3-yl) benzenesulfonamide, CAS number: 943962-47-8, which is a potent ATP-citrate lyase (ACL) inhibitor of cell permeability. ATP citrate lyase (ATP citrate synthase, ACL) is a transferase that catalyzes the conversion of citric acid and coa to acetyl-coa, and acts as a key regulator between efficient aerobic glycolysis and lipid re-synthesis in many types of tumor cells. In HepG2 cells, BMS-303141 (ic50=8μΜ) can inhibit cholesterol and fatty acid synthesis. In the high-fat fed mouse obesity model, BMS-303141 can reduce plasma cholesterol, triglyceride and glucose and inhibit weight gain by chronic oral administration (10-100 mg/kg/day), and is a potential obesity therapeutic drug. However, no report has been made so far on BMS-303141 as a drug for alleviating chronic kidney disease.

Therefore, further research on the application of 3, 5-dichloro-2-hydroxy-N- (4-methoxy [1,1' -biphenyl ] -3-yl) benzenesulfonamide and development of the application of the benzenesulfonamide in chronic kidney disease are technical problems to be solved in the field.

Disclosure of Invention

The invention provides application of a small molecular compound BMS-303141 in preparing medicines for treating chronic kidney disease in order to overcome the technical problems.

The inventors found that not all existing ACL inhibitors can be used for treating and alleviating chronic kidney disease, for example, 3 existing ACL inhibitors, such as shown in table 1 in example 1, BMS-303141, SB-204990 and ETC-1002 are all ACL inhibitors, but the inventors found that BMS-303141 has excellent therapeutic effect for alleviating chronic kidney disease through affinity screening. Specifically: based on an obesity type 2 diabetes animal model, the inventor obtains that the small molecular compound BMS-303141 has obvious inhibition effect on the local ectopic lipid deposition of the kidney under the oral effective dose, and has alleviation effect on the progress of the fibrosis of the kidney CKD.

The invention provides application of BMS-303141 in preparing a medicament for treating or relieving chronic kidney disease.

In the present invention, the administration mode of the BMS-303141 is not particularly limited, and administration modes well known in the art, such as intraperitoneal injection, oral administration, etc., may be employed.

In the invention, BMS-303141 can be mixed with sterile water for injection and CMC-Na (sodium carboxymethylcellulose) to form suspension for administration, and Tween-80 or PEG with a certain proportion can also be added.

Wherein, in the suspension, the mass percentage of CMC-Na may be 0.5-1.5%, for example 1%.

Wherein the mass percentage of the water in the suspension may be 50-99%, for example 99%.

Wherein, in the suspension, the weight percentage of Tween80 is 0.1-10%, for example, 01 or 5%.

Wherein the PEG may be present in the suspension in a mass percentage of 30-50%, for example 40%.

In the present invention, the BMS303141 can be administered at a dosage of 35-50mg/kg/d.

In the present invention, the chronic kidney disease may be diabetic nephropathy.

Wherein the diabetic nephropathy is a progressive decrease in proteinuria and Glomerular Filtration Rate (GFR) due to prolonged diabetes. Diabetic nephropathy is one of the most important complications for diabetic patients. The early stage often has no obvious symptoms, the middle and late stages often take hypertension edema and a large amount of proteinuria as main, and the later stage develops to the end stage renal disease to cause water electrolyte disturbance and acid-base balance disturbance. Because of complex metabolic disorders, once the kidney disease is developed to the end stage, the kidney disease is more troublesome than the treatment of other kidney diseases, so the timely prevention and treatment are significant for delaying diabetic nephropathy.

The invention also discloses a using method of the BMS-303141, and the BMS-303141 is used before or as early as possible before proteinuria occurs.

The invention also provides a composition comprising BMS-303141, comprising BMS-303141, CMC-Na and water.

Wherein, in the BMS-303141-containing composition, the CMC-Na may be contained in an amount of 0.5 to 1.5% by mass, for example, 1%.

Wherein, in the composition containing BMS-303141, the water may be 50-99% by mass, for example 52 or 99%.

Wherein, the composition containing BMS-303141 can further comprise a surfactant, such as Tween80 and/or PEG.

In the BMS-303141-containing composition, the Tween80 may be present in an amount of 0.1-10%, such as 0.5% or 5%.

The weight percentage of the PEG in the BMS-303141-containing composition may be 30-50%, for example 40%.

In the present invention, the BMS-303141, which may also be referred to as 3, 5-dichloro-2-hydroxy-N- (4-methoxy [1,1' -biphenyl)]-3-yl) benzenesulfonamide of the formula

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

the invention screens out the micromolecular compound BMS-303141 for the first time, is an ACL inhibitor for treating obesity type 2 diabetes, can inhibit ectopic lipid deposition, can inhibit inflammatory influence and CKD disease fibrosis progress caused by lipid deposition on animal level, has strong relieving effect, and has no harm to mice. Under the current situation that the number of CKD patients caused by obesity and type 2 diabetes is increased, a new way for relieving disease progress and improving prognosis is found, and the application prospect is wide.

Drawings

FIG. 1 is a graph showing comparison of body weight of type 2 diabetic mice and mice in a control group of the present invention observed for 8 weeks.

FIG. 2 is a graph showing comparison of blood glucose levels observed in type 2 diabetic mice and mice in the control group for 8 weeks in example 2 of the present invention.

FIG. 3 is a graph showing the comparison of the urinary creatinine ratio (ACR) of type 2 diabetic mice and control mice of example 2 of the present invention observed for 8 weeks.

FIG. 4A is a graph showing comparison of serum total cholesterol observed in type 2 diabetic mice and mice in the control group for 8 weeks in example 2 of the present invention.

FIG. 4B is a comparison of serum triglycerides observed for 8 weeks in type 2 diabetic mice and control mice in example 2 of the present invention.

Fig. 5 is a comparative x500 glomerular transmission electron microscope (labeled with a fat droplet) of type 2 diabetic mice and mice of the control group of the present invention 30 days after administration.

FIG. 6 is a comparison of kidney cortical oil red o sections of type 2 diabetic mice and control mice of example 3 of the present invention after 30 days of administration.

FIG. 7 is a comparative graph of HE stained sections of type 2 diabetic mice and control mice of example 3 of the present invention, observed for renal injury 30 days after administration.

FIG. 8 is a comparative chart showing the observation of PAS staining sections of kidney damage after 30 days of administration to type 2 diabetic mice and control mice in example 3 of the present invention

FIG. 9 is a comparison of Masson's stained sections of type 2 diabetic mice and control mice of example 3 of the present invention, observed for kidney damage 30 days after administration.

FIG. 10 is a graph showing comparison of mRNA of the target gene Acl of the inhibitor in renal cortex observed in type 2 diabetic mice and mice of a control group of the present invention 30 days after administration.

FIG. 11 is a schematic diagram showing the simulated docking effect of mouse ACL protein with BMS-303141 in example 1 of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1

ACL inhibitor affinity identification:

affinity calculations between the chemical components shown in table 1 and the mouse ACL protein receptor were performed using AutoDock software with the LGA (Lamarckian Genetic Algorithm) algorithm, and the results were plotted by Pymol software. Schematic of the simulated docking of the mouse ACL protein with BMS-303141 is shown in fig. 11.

Among them, molecular docking calculations can be referred to as affinity mimetic binding between small molecule compounds and large molecule proteins: forsli, stefano et al, "Computational protein-ligand docking and virtual drug screening with the AutoDock suite," Nature protocols vol.11,5 (2016): 905-19.Doi:10.1038/nprot.2016.051; alternatively, zhang, xue-Yan et al, "Biological, clinical and epidemiological features of COVID-19,SARS and MERS and AutoDock simulation of ACE2," Infectious diseases of poverty vol.9, 199.20Jul.2020, doi:10.1186/s40249-020-00691-6.

TABLE 1

As can be seen from table 1: BMS-303141, SB-204990 and ETC-1002 all act as ACL inhibitors, but have different protein affinities, and BMS-303141 has the most excellent protein affinity.

Example 2

(1) Small molecule compound BMS-303141 was dissolved and formulated:

the suspension is prepared by adopting 1% CMC-Na and 99% sterile water for injection (1% of CMC-Na in the suspension refers to the mass percentage of BMS-303141 in the suspension; the concentration of BMS-303141 in the suspension can be adjusted according to the weight of a mouse and the dosage of administration, and the single gastric lavage volume of the mouse is generally controlled to be 500-600 mu L of the suspension), and Tween80 can be added appropriately to be fixed to 0.5%.

(2) Animal disease model construction:

the db/m mice and db/db mice (model mice for type II diabetes, purchased from Jiangsu Jiuzhikang biotechnology Co., ltd.) were fed on a common diet to 12 weeks of age.

The experimental group included: a 12 week old db/m control mice group, a 12 week old db/db control mice group, and a 12 week old db/db drug treated mice group (30 days (50 mg/kg/d, i.g.) treated with BMS 303141) (3 groups, n=8 for each group, 40 total). Wherein: the db/m control group and the db/db control group were not administered with the drug, and only 1% CMC-Na+99% sterile water for injection was administered.

(3) These mice were measured monthly for body weight, blood glucose, plasma triglycerides, serum free fatty acids and serum cholesterol (serum acquisition procedure is the same as in example 3) from 8 weeks to 16 weeks of age, urine microalbumin/creatinine ratio (uACR).

Example 3

1. The mice in example 2 were subjected to blood sampling and measurement from 8 weeks to 16 weeks of age, and the measurement was performed once a month, which was 8 weeks, 12 weeks, and 16 weeks; serum was collected (orbit blood was collected) as follows:

(1) Disinfecting periocular coat with iodophor;

(2) Penetrating the posterior edge of the orbit with a 0.5 x 100mm sized capillary tube to a depth of about 2-3 mm;

(3) Pulling out the hemostix after taking 200-300 mu L of blood, pressing the cleaning cotton ball on the eyes for a moment, and disinfecting the eyes by using iodophor;

(4) Standing the obtained blood at room temperature for 2 hours, and then putting the blood into a centrifugal machine to centrifuge at 3500rpm for 20 minutes;

(5) The supernatant was taken in a clean centrifuge tube and stored at-80 ℃.

2. The mice in example 2, after 30 days of treatment (from 12 weeks of age to 16 weeks of age), were kidney harvested; the renal cortex was treated as follows:

(1) Extracting RNA by using Trizol reagent; a pair of real-time fluorescent quantitative PCR primers is designed based on the standard quality grain sequence. A standard curve is drawn by Q-PCR of a standard plasmid, and the result shows that a good linear relation exists between the CT value and the standard substance. All experiments were performed in 3 replicates.

(2) Preparing kidney lysate for protein analysis; a small amount of tissue blocks are placed at the spherical part in a 1-2 ml homogenizer, and the tissue blocks are sheared as much as possible by clean scissors. mu.L of detergent lysate (containing PMSF) was added to the homogenizer for homogenization. And then placed on ice. Grinding again after 10 minutes, and placing on ice, and repeating grinding for several times to grind the tissue as much as possible. After 30min of lysis, the lysate was transferred to a 1.5ml centrifuge tube by a pipette, and centrifuged at 14000rpm for 15min at 4℃and the supernatant was dispensed into 0.5ml centrifuge tubes and stored at-20 ℃.

(3) 4% formaldehyde (PBS, pH 7.4) was fixed, paraffin embedded for histological and electron microscopic analysis;

(4) OCT compound is embedded into frozen sections and stained with oil red O;

(5) Lipid extraction.

Example 4

The preparation method of this example comprises administering the small molecule compound BMS-303141 by intraperitoneal injection (administration concentration of 35mg/kg/d, preparation method: 3% DMSO+40% PEG300+5% Tween80+52% ddH) ₂ O), or mixed feed (the specific preparation method is as follows: mixing the medicinal powder with mouse feed at a dose of 8Each squirrel cage is placed with 100g mixed feed per day at a drug concentration of 12% at 0mg/kg/d. ) Similar effects to those of example 2 were obtained in the same manner as in example 2.

Effect example 1

(1) Weight comparison of mice of each group from 8 weeks to 16 weeks of age in example 2

TABLE 2

In table 2, each value is expressed as an average of three tests. Vehicle refers to 1% CMC-Na+99% sterile water for injection. Comparison tests were performed using two-factor anova, representing db+bms303141 at 16 weeks with p less than 0.05 compared to db+vecle.

As can be seen from table 2 and fig. 1, the experimental group mice showed a significant trend of change in body weight compared with the control group mice after 30 days of administration of BMS-303141 by gastric lavage, and the compound was expected to have a greater alleviation effect on the body weight of obese mice and reduce systemic fat deposition, although there was a greater difference between the body weight of the experimental group mice and the control group mice compared with the control group mice.

(2) Blood glucose comparison from 8 weeks to 16 weeks of age for each group of mice in example 2

TABLE 3 Table 3

In table 3, each value is expressed as an average of three tests. Vehicle refers to 1% CMC-Na+99% sterile water for injection.

As can be seen from table 3 and fig. 2, the blood glucose of the mice in the experimental group was not significantly changed from that of the mice in the control group after 30 days of administration of BMS-303141 by stomach irrigation, and thus it was inferred that the compound had no effect of alleviating the blood glucose of the obese mice.

(3) Urinary creatinine ratio (ACR) comparison of mice of each group from 8 weeks of age to 16 weeks of age in example 2

TABLE 4 Table 4

In table 4, each value is expressed as an average of three tests. Vehicle refers to 1% CMC-Na+99% sterile water for injection. Comparison tests were performed using two-factor anova, representing db+bms303141 at 16 weeks with p less than 0.01 compared to db+vehicle.

As can be seen from table 4 and fig. 3, after 30 days of administration of BMS-303141, the mice in the experimental group had a clear trend of change in urinary microalbumin/creatinine ratio (ACR) compared with the mice in the control group, and the compound was shown to have a greater alleviation effect on urinary protein symptoms in obese mice than the mice in the control group, and to alleviate proteinuria caused by glomerular hypertrophy due to deposition of glomerular ectopic lipids.

(4) Comparison of blood lipid (Total cholesterol and Triglycerides) from 8 weeks of age to 16 weeks of age in mice of each group of example 2

TABLE 5

Group of	Serum total cholesterol (mmol/l)	Serum triglyceride (mmol/l)
			db/m+vehicle	2.16	0.9
db/db+vehicle	4.6	2.61
			db/db+BMS-303141	2.7**	1.2**

In table 5, each value is expressed as an average of three tests. Vehicle refers to 1% CMC-Na+99% sterile water for injection. Comparison tests were performed using two-factor anova, representing db+bms303141 at 16 weeks with p less than 0.01 compared to db+vehicle.

As can be seen from table 5, fig. 4A and fig. 4B, the experimental mice showed a significant trend of change in serum total cholesterol and triglyceride levels compared with the control mice 30 days after administration of BMS-303141 by intragastric administration. It is inferred that the compound has a great alleviation effect on hyperlipidemia symptoms in obese mice, can alleviate systemic lipid deposition, and helps to prevent a series of complications caused by hyperlipidemia, such as coronary atherosclerotic plaque formation.

(5) Comparative glomerular transmission electron microscopy of 16 week old mice of each group of example 2

Under tissue transmission electron microscope scanning (see fig. 5), oral administration of BMS-303141 can alleviate cortical glomerular podocyte podophy fusion and mesangial cell proliferation.

(6) Comparison of 16-week-old sections of kidney cortical oil Red o from mice of each group in example 2

As can be seen from fig. 6, under the fatty oil red O staining: oral administration of BMS-303141 significantly reduced cortical glomerular and tubular lipid deposition.

(7) Comparison of 16-week-old kidney injury HE staining sections of mice of each group in example 2

It was observed under tissue HE staining (see fig. 7) that oral BMS-303141 significantly reduced tissue inflammatory cell aggregation and glomerular hypertrophy, and basement membrane rupture.

(8) Comparison of 16-week-old kidney injury PAS staining sections of mice of each group in example 2

As can be seen from fig. 8, oral BMS-303141 can alleviate CKD tissue fibrosis, basement membrane thickening, cortical glomerulosclerosis, and mesangial matrix proliferation.

(9) Comparison of 16-week-old kidney injury Masson stained sections from mice of each group in example 2

As can be seen from fig. 9, oral BMS-303141 can alleviate CKD tissue fibrosis, basement membrane thickening, cortical glomerulosclerosis, and mesangial matrix proliferation.

(10) mRNA comparison of Acl, the target gene of the inhibitor, in renal cortex

TABLE 6

Group of	mRNA relative expression fold relationship of gene Acl
		db/m+vehicle	1
db/db+vehicle	5.7
		db/db+BMS-303141	3.5

In table 6, each value is expressed as an average of three tests. Vehicle refers to 1% CMC-Na+99% sterile water for injection.

As is clear from Table 6 and FIG. 10, the relative expression level of the target gene Acl was significantly changed in the experimental mice as compared with the control mice after 30 days of administration of BMS-303141 by gastric lavage. The inhibitor does not directly inhibit the expression of the gene, but inhibits the downstream protein after transcription and translation, and is supposed to act similar to negative feedback regulation, so that the expression level of the gene is changed.

The above results demonstrate that the present invention demonstrates that the small molecule compound BMS-303141 inhibits ectopic lipid deposition by inhibiting abnormally high expression of ACL at the renal tissue level.

By administering a small molecular compound BMS-303141 at an oral dose of 50mg/kg/day in a mouse model of obese type 2 diabetes (db/db mouse), blood cholesterol, blood triglycerides and systemic body weight elevation can be substantially reduced, proteinuria can be improved, renal function can be alleviated, and there is no harm to the mouse.

Further, it is known from the mouse kidney pathological tablet that: (1) The oral administration of BMS-303141 under the staining of fatty oil red O can remarkably reduce the lipid deposition of cortical glomeruli and tubular; (2) Under tissue HE staining, the oral administration of BMS-303141 can obviously relieve tissue inflammatory cell aggregation and glomerular hypertrophy, and the basement membrane fracture phenomenon; (3) Under tissue Masson staining and PAS staining, oral administration of BMS-303141 can relieve fibrosis of CKD tissue, thickening of basement membrane, cortical glomerulosclerosis and mesangial matrix proliferation.

Further, it is known that oral administration of BMS-303141 can alleviate the fusion of cortical glomerular podocyte podophy and proliferation of mesangial cells under tissue transmission electron microscopy.

The present invention is not particularly limited in the mode of administration of the drug, and modes of administration well known in the art may be employed.

In the foregoing, the protection scope of the present invention is not limited to the preferred embodiments of the present invention, and any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention disclosed in the present invention fall within the protection scope of the present invention.

Claims

1. Use of BMS-303141 in the manufacture of a medicament for the treatment of chronic kidney disease;

the chronic kidney disease is diabetic nephropathy.

2. The use according to claim 1, wherein the BMS303141 is administered at a dose of 35-50mg/kg/d.

3. The use according to any one of claims 1-2, wherein the medicament is administered in the form of a suspension further comprising one or more of CMC-Na, water and "Tween-80 or PEG".

4. The use according to claim 3, wherein the CMC-Na content in the suspension is 0.5-1.5% by mass;

and/or, in the suspension, the mass percentage of the water is 50-99%;

and/or, in the suspension, the weight percentage of the Tween80 is 0.1-10%; or, in the suspension, the mass percentage of the PEG is 30-50%.