WO2018171201A1 - Application of dibutyryladenosine cyclophosphate salt in preparing fat-burning and weight-loss drug for external use - Google Patents

Abstract

Provided is an application of a dibutyryladenosine cyclophosphate salt in preparing a fat-burning and weight-loss drug for external use. The dibutyryladenosine cyclophosphate salt can directly enter an adipocyte and activate PKA protein, thereby activating hormone-sensitive lipase (HSL) to catalyze the breakdown of fat into free fatty acid and glycerol. Also, the destructive effect of phosphodiesterase in the body is prevented, and compared to cAMP, the action time is longer, and the speed is faster.

Description

Application of dibutyryl cyclic adenosine monophosphate in preparation of topical cellulite diet Technical field

The invention relates to the field of medicine, in particular to the use of dibutyryl cyclic adenosine monophosphate in the preparation of a topical cellulite diet drug.

Background technique

According to the distribution of systemic adipose tissue, obesity can be divided into two categories: generalized obesity and local obesity (upper body obesity and lower body obesity, abdominal obesity). Systemic obesity has now become a global and serious social public health problem. The statistics released by the World Health Organization show that at least 1 billion adults are overweight and 300 million are obese in the world. Obesity has seriously threatened global development. While local obesity generally does not cause serious health problems, it is a more common social problem. It refers to the obesity that occurs in a certain part of the body where fat accumulates, and the abdomen (general belly / beer belly), arm (Butterfly arm), thigh (elephant leg) and buttocks are the most common. At present, with the continuous improvement of people's living standards, rich diet and over-nutrition, almost everyone has more or less local obesity problems, which seriously affects the overall image and appearance of the individual, causing great trouble to many people!

The current local cellulite cosmetic methods mainly include liposuction, injection of a lipolysis needle, and the like. However, liposuction may cause skin depression, sagging, and even serious side effects such as subcutaneous congestion and bleeding. The fat-soluble component of the lipolysis needle is complex and unclear, and is prone to swelling and pain. Even the serious problem of necrosis has been banned by many countries in the world including China. The vast majority of the market's slimming, slimming and body-building beauty products are ineffective.

In summary, there are currently no safe and effective local weight loss products and technologies on the market. It is undoubtedly a huge market potential to develop safe, convenient and efficient local cellulite beauty products!

The molecular formula of dibutyryl cyclic adenosine salt is C ₁₈ H ₂₃ N ₅ XO ₈ P, and its structural formula is represented by the following formula (1):

Dibutyryl cyclic adenosine can be used for adjuvant treatment of angina pectoris and acute myocardial infarction. It can also be used for myocarditis, cardiogenic shock, subperitoneal hemorrhage and psoriasis after surgery, and can assist other anticancer drugs in the treatment of leukemia. However, there has been no report on the fat-reducing effect of dibutyryl cyclic adenosine monophosphate.

Summary of the invention

In order to solve the above problems, the present invention provides the following technical solutions:

The application of dibutyryl cyclic adenosine monophosphate in the preparation of topical cellulite diet drugs.

A topical cellulite diet drug composition characterized by comprising the above dibutyryl cyclic adenosine salt.

A topical cellulite diet drug composition characterized by comprising the above dibutyryl cyclophosphine Adenosine salt, and one or more of forskolin (or extract of Coleuss chinensis), L-carnitine, and theophylline.

The dibutyryl cyclic adenosine salt is dibutyryl cyclic adenosine monophosphate, dibutyryl cyclic adenosine monophosphate or other form of salt.

Dibutyryl adenosine can directly enter fat cells to activate PKA protein and activate hormone-sensitive esterase (HSL) to catalyze the breakdown of fat into free fatty acids and glycerol. At the same time, it can resist the destruction of phosphodiesterase in the body, and the action time and speed are longer and faster than cAMP.

DRAWINGS

Figure 1 shows the effect of dibutyryl cyclic adenosine monophosphate on fat accumulation.

Figure 2 shows the effect of forskolin on fat accumulation.

Figure 3 shows the effect of theophylline on fat accumulation.

Figure 4 shows the effect of a drug combination on fat accumulation.

Figure 5 is a graph showing the effects of drug combinations on body weight and abdominal subcutaneous fat in obese mice (A: body weight, *P < 0.05, **P < 0.01; B: subcutaneous fat, *P < 0.05).

Figure 6 is a graph showing the effects of a pharmaceutical composition on heart, liver, kidney, spleen, lung and testis of obese mice.

detailed description

Example: Preparation of dibutyrylcyclophosphazate

In a dry reaction flask, 3.2 g (0.01 mol) of cyclic adenosine triethylamine salt, 100 ml of chloroform, 4 g of pyridine (0.05 ml) and 15.82 g (0.10 mol) of n-butyric anhydride were added and stirred at 60 ° C for 48 h. After completion of the reaction, after recovering the solvent, 100 ml of water was added, and the mixture was extracted with isohexanone several times to discard. The aqueous layer was concentrated under reduced pressure to 15 ml, extracted with chloroform (30 ml×3), and the organic layer was combined and recovered. After the solvent, 30 ml of water was added to the residue, and the mixture was stirred and dissolved, then purified by Amberlite IR-120 resin, concentrated and cooled, and the solid was precipitated and dried to obtain 4.32 g (88%) of dibufenyl adenosine salt, mp 240 ° C (dec) ).

Experimental preparation: 3T3-L1 preadipocytes were induced to differentiate into adipocytes. The high glucose DMEM rat fibroblast cell line 3T3-L1 cells were seeded at a density of 5 × 10 ⁴ / ml in 12-well plates in culture, containing 10% fetal bovine serum (FBS, GIBCO, Life technology) of Culture in medium (GIBCO, Life technology). The medium was changed to 1 μg/ml insulin, 0.25 μmol/L dexamethasone and 0.5 mmol/L 3-isobutyl-1-methylxanthine when the cells were grown to 70% confluency. (IBMX) induced differentiation in new DMEM medium (containing 10% FBS); after 2 days, the medium was changed to 1 μg/ml insulin in new DMEM medium (containing 10% FBS); after 2 days, the medium was replaced with Normal DMEM medium (containing 10% FBS) was observed until fat cells were formed.

Experimental method 1: The accumulation of fat was detected by oil red O staining and a microplate reader. Oil red O purchased from sigma was used, and a solution of 10 mg/ml was prepared with isopropyl alcohol, and stored in the dark after filtration. 3T3-L1 fat cells were washed twice with phosphate buffered saline (PBS), fixed with 10% formaldehyde for more than 30 minutes; diluted with oil red: deionized water = 3:2, filtered through filter paper, left at room temperature for 10 min; cell stained for about 10 min. Then rinse with 75% alcohol/60% isopropanol to remove excess dye; microscope observation and photographing. After photographing, it was dissolved in 100% isopropanol, and the solution was harvested, and the absorbance was measured at a wavelength of 510 nm by a microplate reader. The treatment group is a group in which each component is used singly or in combination, and the control group is a group to which no component is added.

Experimental Method 2: Establishment and Grouping of Obese Animal Models: Preparation of Animal Models and Grouping: 4-week-old C57/BJ6L male mice were fed normal diet for 7 days in the test environment, weighed, and mice with the same body weight were selected to feed with high-fat diet for 8 weeks, and the body weight was 20% higher than that of mice fed with normal diet. The mice were obese mice and were included in the experimental group: obese control group and drug combination treatment group. The mice were fed in cages and fed freely; the natural day and night lighting was performed at room temperature of 18 ° C to 25 ° C; the drug combination treatment group was applied to the abdomen of the mice by smearing on a daily basis, and the hair was removed before application. Change food every 2-3 days, change water every other day; relative humidity is about 50%; the experiment lasts for 30 days. Animals in each group were fed a high fat diet. Body weight was measured after the treatment was completed, and then the mice were sacrificed to take their abdominal skin, heart, liver, kidney, spleen, lungs, and testes. The abdominal skin was fixed and embedded, and subjected to H&E staining to photograph and calculate the thickness of subcutaneous fat. Other tissues were fixed and embedded for H&E staining and photographed for tissue morphology. Note: Ordinary feed: moisture: 8.4%, crude protein 25.8%, crude fat 6.53%, coarse ash 6.2%, crude fiber 4.12%, nitrogen-free extract 46.8%, calcium 1.26%, phosphorus 0.89%, lysine 1.36 %. High-fat diet: moisture: 8.6%, crude protein 18.8%, crude fat: 18.83%, coarse ash 5.2%, crude fiber 3.98%, nitrogen-free extract 45.2%, calcium 1.24%, phosphorus 0.83%, lysine 1.38 %.

Experimental Example 1: The effect of dibutyryl cyclic adenosine monophosphate on lipogenesis. In order to determine the effect of dibutyryl cyclic adenosine monophosphate on promoting lipolysis in fat cells, 3T3-L1 adipocytes differentiated in experimental preparation were used. The cell culture medium was changed to 10 mg/ml dibutyryl adenosine monophosphate in a new DMEM medium (containing 10% FBS), and then the accumulation of fat in the fat cells was measured according to Test Method 1. The result is shown in Figure 1.

The results in Figure 1 show that the fat content of the fat cells after dibutyryl adenosine monophosphate treatment is significantly lower than that of the control group, and the effect of decomposing fat is better than that of the known comparative control caffeine which has a decomposed fat effect.

Experimental Example 2: The effect of forskolin on fat decomposition. To determine the effect of forskolin on promoting lipolysis in adipocytes, 3T3-L1 adipocytes differentiated in experimental preparation were used. The cell culture medium was changed to a new DMEM medium (containing 10% FBS) of 8.2 μg/liter forskolin, and then the accumulation of fat in the fat cells was measured according to Experimental Method 1. The result is shown in Figure 2.

The results in Figure 2 show that the amount of neutral fat in the fat cells after forskolin treatment was somewhat reduced compared to the control group.

Experimental Example 3: The effect of theophylline on promoting lipolysis. To determine the effect of theophylline on promoting lipolysis in adipocytes, 3T3-L1 adipocytes differentiated in experimental preparation were used. The cell culture medium was changed to a new DMEM medium (containing 10% FBS) of 20 mg/L of theophylline, and then the accumulation of fat in the adipocytes was measured according to Experimental Method 1. The result is shown in Figure 3.

The results in Fig. 3 show that the amount of fat in the fat cells after theophylline treatment was somewhat reduced as compared with the control group.

Experimental Example 4: Synergistic action of dibutyryl cyclic adenosine monophosphate, forskolin, L-carnitine and theophylline. In order to confirm whether the dibutyryl cyclic adenosine salt, forskolin, L-carnitine and theophylline have a synergistic effect in decomposing fat, the 3T3-L1 adipocytes induced by the experimental preparation were treated with the above-described respective component compositions. The concentration of each component was the same as that of the individual treatment. The result is shown in Figure 4.

The results in Fig. 4 show that the amount of fat in the fat cells treated with the composition was significantly lower than that in the control group, and the effect of decomposing fat was significantly better than that of the positive comparative control caffeine which was known to have a fat-decomposing effect.

Experimental Example 5: Efficacy of lipolysis of obese mice by the pharmaceutical composition described in Example 4. In order to examine the effect of the pharmaceutical composition on promoting abdominal fat decomposition in obese mice, obese mice developed in Experimental Method 2 were used, and the body weight and abdominal subcutaneous fat thickness were calculated according to the method of Experimental Method 2, and obese mice were observed. , liver, kidney, spleen, lung and testicular tissue morphology. The results are shown in Figures 5 and 6.

The results in Figure 5 show that the pharmaceutical composition is capable of significantly reducing the body weight of the mouse and the thickness of the abdominal subcutaneous fat.

The results in Figure 6 show that the pharmaceutical composition has no significant effect on the tissue morphology of heart, liver, kidney, spleen, lung and testis in obese mice.

Claims (4)

The application of dibutyryl cyclic adenosine monophosphate in the preparation of topical cellulite diet drugs. A topical cellulite diet drug composition comprising the dibutyryl cyclophosphazate salt of claim 1. A topical cellulite diet composition comprising the dibutyryl adenosine salt according to claim 1, and one or more of forskolin, L-carnitine and theophylline. The dibutyryl cyclic adenosine salt according to any one of claims 1 to 3 is dibufenyl adenosine monophosphate or dibutyryl cyclic adenosine monophosphate.

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