CN113209091B

CN113209091B - Application of fenticonazole nitrate in preparation of antidiabetic drugs

Info

Publication number: CN113209091B
Application number: CN202110309100.2A
Authority: CN
Inventors: 易伟; 马磊; 周志; 侯宁; 唐君源
Original assignee: Guangzhou Medical University
Current assignee: Guangzhou Medical University
Priority date: 2021-03-23
Filing date: 2021-03-23
Publication date: 2022-05-27
Anticipated expiration: 2041-03-23
Also published as: CN113209091A

Abstract

The invention discloses application of fenticonazole nitrate in preparation of an antidiabetic medicament. Fenticonazole nitrate has stronger affinity with PPAR gamma under the action of 10 mu M concentration. Experiments show that compared with rosiglitazone, fenticonazole nitrate has weaker capacity of inducing adipocyte differentiation, and fenticonazole nitrate weakly activates the expression of a PPAR gamma downstream adipogenic gene; in db/db model mice, fenticonazole nitrate does not cause the weight increase of the mice, and has no obvious side effect on all internal organs of the mice; meanwhile, the blood sugar reducing agent has good blood sugar reducing effect under the concentration of 10 mg/kg. The invention discovers for the first time that fenticonazole nitrate has high-efficiency hypoglycemic effect and low toxic and side effect, and can be developed as antidiabetic medicine.

Description

Application of fenticonazole nitrate in preparation of antidiabetic drugs

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of fenticonazole nitrate in preparation of an antidiabetic medicine.

Background

Diabetes mellitus is a common metabolic disease that is rising at an alarming rate in the world's population and is characterized by hyperglycemia and its associated symptoms due to inadequate insulin secretion, inadequate insulin action, or both. The proportion of non-insulin dependent type 2 diabetes mellitus exceeds 90% in all diabetic patients. In type 2 diabetic patients, who require more insulin to maintain physiological balance of glucose metabolism in vivo due to hyperglycemia caused by insulin resistance in the liver and peripheral tissues, blood glucose levels can be lowered by increasing insulin secretion (endogenous production) of pancreatic islets or by direct injection of recombinant peptides. However, both of these processes lead to pancreatic beta cell dysfunction. Therefore, improving insulin resistance is of great significance in the treatment of type 2 diabetes. Thiazolidinedione drugs (such as rosiglitazone and pioglitazone) which take PPAR gamma as a target have the function of strongly improving insulin sensitivity, thereby achieving the function of reducing blood sugar. Prolonged administration of thiazolidinediones can lead to side effects such as obesity, weight gain, edema, bladder cancer, cardiac enlargement, and heart failure. These side effects have severely hampered the clinical utility of thiazolidinediones. Therefore, the development of a novel oral anti-II diabetes drug which replaces thiazolidinediones and has high-efficiency blood sugar reduction and low toxic and side effects and takes PPAR gamma as a target has become urgent needs of the medical field.

The fenticonazole nitrate is an excellent antifungal drug and is mainly applied to the treatment of female gynecological diseases. Fenticonazole nitrate is an imidazole-based spectrum antifungal agent and is used for treating superficial fungal and vaginal candidiasis infections. The drug was approved for marketing in Italy in 1986, and the dosage forms include cream, spray and pessary. The action mechanism is that the hydroxylation of cytochrome p-450 of the fungal cell is inhibited to enable the phospholipid of the cell wall to be in an unstable state, and further the fungal cell is broken and necrotized.

Disclosure of Invention

The invention aims to provide application of fenticonazole nitrate in preparation of antidiabetic drugs.

The inventor utilizes a computer to virtually screen PPAR gamma ligand from a drug library (Shanghai ceramic Biotechnology Co., Ltd.) in high flux so as to obtain a drug with potential hypoglycemic effect, and then obtains an antibacterial drug Fenticonazole Nitrate (FN) which is imidazole derivative through TR-FRET experiment preliminary screening, wherein the Fenticonazole Nitrate has strong affinity with PPAR gamma under the action of 10 mu M concentration. This suggests that fenticonazole nitrate may have excellent hypoglycemic activity. Experiments show that compared with rosiglitazone (Rosi), Fenticonazole Nitrate (FN) has weaker capacity of inducing adipocyte differentiation, fenticonazole nitrate weakly activates the expression of a PPAR gamma downstream adipogenic gene, fenticonazole nitrate does not cause the increase of the weight of a mouse in a db/db model mouse, and fenticonazole nitrate also has good hypoglycemic effect at the concentration of 10 mg/kg. Fenticonazole nitrate also did not cause significant changes in the weight of the mouse major internal organs and also reduced the weight of white adipose tissue in mice at a dose of 50 mg/kg.

The first purpose of the invention is to provide the application of fenticonazole nitrate in preparing anti-diabetic drugs.

Preferably, the antidiabetic agent is an anti-type II diabetes drug targeting PPAR γ.

The invention also provides an antidiabetic medicament which contains an effective amount of fenticonazole nitrate as an active ingredient and a pharmaceutically acceptable carrier.

The invention discovers for the first time that fenticonazole nitrate has high-efficiency hypoglycemic effect and low toxic and side effects, and can be developed as antidiabetic drugs.

Drawings

FIG. 1 shows the PPAR γ activating ability of fenticonazole nitrate;

FIG. 2 is the result of TR-FRET competitive binding experiment for detecting the binding capacity of fenticonazole nitrate to PPAR gamma;

FIG. 3 is a result of oil red O staining detection of fat differentiation-causing ability of fenticonazole nitrate;

FIG. 4 shows the expression of adipogenic genes measured by real-time fluorescent quantitative PCR;

FIG. 5 is a graph of the effect of fenticonazole nitrate on reducing blood glucose in db/db model mice;

FIG. 6 is a graph of the effect of fenticonazole nitrate on the major internal organs and adipose tissues of mice in db/db model mice;

FIG. 7 is a graph showing the effect of fenticonazole nitrate administration on CDK 5-mediated phosphorylation of PPAR γ -Ser-273 and insulin-sensitive gene expression in mouse liver and adipose tissue in db/db model mice.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Fenticonazole nitrate is an antifungal drug, and the chemical structural formula of fenticonazole nitrate is shown as formula I:

the following examples are provided to illustrate a novel use of fenticonazole nitrate provided by the present invention.

Example 1

First, the PPAR gamma activation ability of fenticonazole nitrate (luciferase activity assay method)

Cos-7 cells were purchased from ATCC, cultured in 10% FBS antibiotic-free DMEM, 37 ℃, 5% CO₂An incubator. Plasmid co-transfection (50ng full length hPPAR γ, 100ng PPAR γ, 5ng renilla luciferase plasmid) was performed according to the instructions of lipofectamine2000(Invitrogen) when cells were seeded into 24-well plates in logarithmic growth phase and cells were fused to about 70%. After 24h, the transfected cells are respectively intervened by 1 mu M fenticonazole nitrate, wherein 1 mu M rosiglitazone is used as a positive control, and a solvent DMSO is used as a negative control. Luciferase activity was assayed 24h after the intervention as described in the Reporter luciferase assay kits (Promega) protocol, with 3 independent test wells per group. The experimental results are shown in table 1 and fig. 1, and show that fenticonazole nitrate only weakly activates PPAR γ, unlike rosiglitazone which fully activates PPAR γ function.

TABLE 1 Ppar gamma activating ability of fenticonazole nitrate

	1μM	10μM
			DMSO
	0％	0％
			Rosiglitazone (Rosi)	100％	100％
Fenticonazole Nitrate (FN)	28％	35％
			PPAR gamma agonist VSP-51	34％	42％

Second, TR-FRET experiment is used for detecting the binding capacity of fenticonazole nitrate and PPAR gamma

1. Fenticonazole nitrate was diluted to 1mM with DMSO. DMSO is used as a negative control, and rosiglitazone is used as a positive control.

2. The diluted fenticonazole nitrate was diluted again to 2 μ M with TR-FRET buffer.

3. Fluormone TM Pan-PPAR Green solution (20nM) was prepared using TR-FRET buffer as solvent.

4. 20nM Tb anti-GST antibody and 4. mu.M PPAR γ -LBD protein were prepared in TR-FRET buffer.

5. Mu.l of the solution prepared in step 2, 10. mu.l of the solution prepared in step 3 and 10. mu.l of the solution prepared in step 4 were mixed in a 384-well plate and shaken for 6 hours.

6. Read on a microplate reader.

The results of the experiment are shown in table 2 and fig. 2, and show that fenticonazole nitrate has strong binding property with PPAR γ at a concentration of 10 μ M.

TABLE 2 binding Capacity of fenticonazole nitrate to PPAR γ

	1μM	10μM
			DSMO	0.835258	0.815266
Rosiglitazone (Rosi)	0.290064	0.284495
			Fenticonazole Nitrate (FN)	0.678865	0.35403

Third, oil red staining detection of fat differentiation capability caused by fenticonazole nitrate

3T3-L1 preadipocytes were purchased from ATCC and cultured in 10% FBS DMEM containing penicillin-streptomycin double antibody at 37 ℃ in 5% CO₂An incubator. The cells were plated on a culture plate, and the induction solution (10% FBS DMEM containing 0.5mmol/L IBMX (3-isobutyl-1-methylxanthine), 1. mu. mol/L DEX (dexamethasone), 850nmol/L insulin) was added 2 days after the cells had grown to the confluence point. After 72h, the medium was changed to 10% FBS high-glucose DMEM containing 850nmol/L insulin every 2 days. 1 mu M rosiglitazone is used as a positive control, DMSO is used as a negative control, and the sample group is 1 mu M fenticonazole nitrate. Oil red O staining was performed on day 8 from the initiation of induction, and photographed by a microscope (OLYMPUS), and the adipocyte differentiation rate was calculated. The results of the experiment are shown in fig. 3, and show that Fenticonazole Nitrate (FN) has a weak ability to induce differentiation of adipocytes (the differentiation rate of adipocytes is only 9.04%) compared to rosiglitazone (Rosi) (the differentiation rate of adipocytes is 70.82%).

Fourthly, real-time fluorescence quantitative PCR determination of expression of adipogenic gene

Inducing and differentiating 3T3-L1 preadipocytes according to the scheme, extracting total RNA of cells, performing Real time PCR according to the scheme of a kit (takara), applying a delta-Ct method, taking beta-actin as an internal reference, and calculating the relative expression of mRNA of PPAR gamma and downstream adipogenic genes (PPAR gamma, CD36, ap2, FASN, LPL and C/EBPa). The results of the experiment are shown in fig. 4, and show that Fenticonazole Nitrate (FN) weakly activates the expression of the PPAR γ downstream adipogenic gene, compared to rosiglitazone (Rosi).

Fifthly, detecting the hypoglycemic effect of fenticonazole nitrate in db/db model mice

Six weeks of db/db model mice were purchased and randomly assigned. Intraperitoneal injection solvent group (Vehicle), Fenticonazole Nitrate (FN) administration group (10mg/kg, 20mg/kg, 50mg/kg), PPAR gamma agonist VSP-51 control group (10 mg/kg); the positive control group was orally administered rosiglitazone (Rosi) (5 mg/kg). The drug is administered once a day, and fasting blood sugar, fasting body weight and glucose tolerance level of mice of each experimental group are detected after four weeks. The results are shown in fig. 5, and show that Fenticonazole Nitrate (FN) treated mice did not show significant weight gain compared to Vehicle control (Vehicle) treated mice (fig. 5A). In terms of reducing blood glucose, Fenticonazole Nitrate (FN) significantly reduced fasting blood glucose levels in mice even at low doses (10mg/kg) and showed dose-dependence (fig. 5B). Mice fasted overnight for 16h were first tested for fasting blood glucose levels as baseline (0min) and then injected intraperitoneally with glucose (iPGTT method) at a dose of 2g glucose/kg body weight. Blood glucose levels were measured 30, 90 and 120min after glucose injection, respectively. The results show that Fenticonazole Nitrate (FN) already showed a better clearance of blood glucose at low doses (10mg/kg), was able to improve glucose tolerance, and showed dose dependence (fig. 5C).

Sixthly, detecting the influence of fenticonazole nitrate on main internal organs and adipose tissues of mice after administration in db/db model mice

On the basis of completing evaluation of the blood glucose reducing effect of Fenticonazole Nitrate (FN), mice of each experimental group are dissected, and main internal organs of heart, liver, spleen, lung and kidney and white adipose tissues are completely taken out and weighed. By statistical analysis, the results in fig. 6 show that Fenticonazole Nitrate (FN) does not cause significant changes in the weight of major internal organs even under high dose conditions (50mg/kg), while reducing the weight of white adipose tissue.

Seventhly, detecting the influence of fenticonazole nitrate on CDK5 mediated PPAR gamma-Ser-273 phosphorylation and insulin sensitive gene expression in liver and fat tissues of mice after administration in db/db model mice

The expression of PPAR gamma-S273 phosphorylated protein in liver tissues of mice of each treatment group is detected. As shown in fig. 7A, Fenticonazole Nitrate (FN) was able to effectively block CDK 5-mediated expression of PAR γ -Ser-273 phosphorylation, as was the effect of rosiglitazone. Total RNA of liver tissue and white adipose tissue was extracted and subjected to real-time fluorescence quantitative PCR, as shown in FIGS. 7B-C, it was revealed that Fenticonazole Nitrate (FN) only slightly induces the expression of key lipid-forming genes such as PPAR γ, AP2, CD36, LPL, C/EBP α and FASN in liver and white adipose, and increases the expression levels of insulin sensitivity-related gene Glut4 and adiponectin.

The above experimental results show that:

(1) fenticonazole nitrate has weak activation capability on PPAR gamma, and the anti-diabetic medicine rosiglitazone is taken as a positive control (defined as 100 percent), and the fenticonazole nitrate activates the PPAR gamma values of only 28 percent and 35 percent at the concentrations of 1 mu M and 10 mu M, which indicates that the fenticonazole nitrate is a partial agonist of the PPAR gamma and can reduce potential side effects compared with complete agonists such as rosiglitazone and the like.

(2) Fenticonazole nitrate has strong binding force with PPAR gamma, and the anti-diabetic medicine rosiglitazone is taken as a positive control (the TR-FRET 520/495 value is 0.284495), and the TR-FRET 520/495 value of fenticonazole nitrate which is combined with PPAR gamma under the concentration of 10 mu M is 0.35403, which indicates that fenticonazole nitrate can well bind with target protein, thereby exerting the hypoglycemic activity of fenticonazole nitrate.

(3) Fenticonazole nitrate has weak adipogenic cell differentiation ability. When the antidiabetic rosiglitazone is used as a positive control (the differentiation rate of fat cells is 70.82%), the differentiation rate of the fat cells of fenticonazole nitrate is far lower than that of the rosiglitazone under the concentration of 10 mu M and is only 9.04%.

(4) Fenticonazole nitrate weakly activates the expression of the downstream adipogenic gene of PPAR gamma.

(5) The fenticonazole nitrate can effectively reduce fasting blood glucose of mice and improve the glucose tolerance in diabetes mellitus model mice. And does not cause weight gain. The fenticonazole nitrate is shown to have good hypoglycemic effect, and the side effect is small.

(6) The weight of main internal organs of a diabetes model mouse treated by fenticonazole nitrate by taking the antidiabetic medicine rosiglitazone as a positive control is not obviously changed, and the weight of white adipose tissues can be reduced under the dosage of 50mg/kg, which indicates that the fenticonazole nitrate does not have obvious side effect after being administrated.

(7) Fenticonazole nitrate exerts a hypoglycemic effect by selectively up-regulating the expression of insulin sensitivity-related genes Glut4 and adiponectin and inhibiting PPAR γ -Ser-273 phosphorylation mediated by CDK 5.

In conclusion, Fenticonazole Nitrate (FN) has a new application, namely the hypoglycemic effect.

Claims

1. The application of fenticonazole nitrate in preparing an anti-diabetic medicament is an anti-II type diabetes medicament which takes PPAR gamma as a target.