CN113143787A - Local fat-reducing composition and application thereof - Google Patents

Abstract

The invention discloses a local fat-reducing composition, which comprises a solvent, transdermal peptide and small interfering RNA aiming at an AdPLA gene, wherein the solvent is prepared from the following raw materials: water, glycerol, tranexamic acid, glutathione, butanediol, taurine, potassium methoxysalicylate, glyceryl caprylate, caprylyl hydroximic acid, p-hydroxyacetophenone, carnosine, Alcaligenes polysaccharide, dipotassium glycyrrhizinate, and phenoxyethanol; the ratio of vehicle, transdermal peptide and small interfering nucleic acid to the AdPLA gene was 1g vehicle: 0.1-0.5mg transdermal peptide: 10-100ng of a small interfering nucleic acid directed against the AdPLA gene. The invention also provides application of the fat reducing composition in preparing a medicine for treating the thyroid eye disease and a local fat reducing product.

Description

Local fat-reducing composition and application thereof

Technical Field

The invention relates to the technical field of cosmetics, in particular to a local fat-reducing composition and application thereof in the field of treatment of thyroid-related ophthalmopathy and local fat reduction.

Background

Thyroid-associated ophthalmopathy is the most common autoimmune ophthalmopathy of adults closely related to thyroid diseases, and is manifested by eyeball protrusion, eyelid recession and retardation, periorbital swelling, restrictive extraocular muscle lesions, exposed keratitis, and compressive optic neuropathy. Orbital adiposity, infiltration of inflammatory cells in the orbital adipose connective tissue and extraocular muscles, and polysaccharide deposition cause herniation of the eye. About 52% of patients have elevated orbital fat which causes the eyeball to bulge. Orbital fat augmentation is a result of both increased numbers of fat cells and increased volume of fat cells. Both increased adipogenesis and decreased hydrolysis within adipocytes can lead to increased adipocyte volume. Studies have demonstrated accelerated adipogenesis in orbital adipocytes of patients with thyroid-related eye disease.

Inhibition of adipogenesis only inhibits new adipocyte increases and does not treat the existing orbital adipose tissue accumulation. Therefore, only the promotion of orbital fat hydrolysis can treat the eyeball protrusion caused by the orbital fat tissue accumulation. The fat is hydrolyzed to change triglyceride in fat cells into free fatty acid and glycerol and release the free fatty acid and glycerol into circulation, so that the volume of the fat cells is reduced, the content of the orbit is reduced, and the exophthalmos returns to normal. The process of fat hydrolysis is regulated by a number of endocrine factors and hormones, fat specific phospholipase (AdPLA) being among the most important autocrine and paracrine regulatory factors of fat hydrolysis, hydrolyses the sn-2 position of phospholipids to produce arachidonic acid and free fatty acids, arachidonic acid produces prostaglandin E2 by cyclooxygenase (cox), which binds to Gai-coupled prostaglandin receptor 3(EP3) on the surface of adipocytes, inhibiting adenylate cyclase and reducing fat hydrolysis.

RNA interference techniques, also referred to figuratively as gene knock-down or gene silencing (PTGS), are a typical method of post-transcriptional gene regulation, also known as post-transcriptional gene silencing (PTGS). The double-stranded siRNA forms an RNA-induced silencing complex (RISC) with associated enzymes and proteins of cell origin. During RNA interference, the sense strand of the double-stranded siRNA is excluded from the complex, the antisense strand directs RISC to bind to the homologous site of the target mRNA, and the target mRNA is then degraded by ribonuclease III in the complex, thereby turning off the expression of the target gene.

Research shows that AdPLA plays a key role in the fat cell fat hydrolysis of a mouse model, and the AdPLA-deficient mouse has unlimited whole-body fat hydrolysis. However, this method is not suitable for the treatment of thyroid-related eye disease fat accumulation because the technique hydrolyzes systemic fat and is uncontrolled. Inspired by the research, the RNA interference technology is used for silencing AdPLA gene in orbit fat only, and only hydrolyzing the orbit fat without affecting the whole body fat, so that the aim of treating the exophthalmos is achieved, and the whole body side effect is not generated. Therefore, the fat-reducing mask can be popularized to local fat-reducing of other parts of the body, and the effect of beautifying the body is achieved.

Disclosure of Invention

The present invention provides a lipid-reducing composition comprising a small interfering nucleic acid molecule directed against the AdPLA gene and the use of the composition for improving the ocular process symptoms of thyroid eye disease or for local lipid reduction.

The local fat-reducing composition provided by the invention comprises a solvent, transdermal peptide (also called transdermal decapeptide-4 in the invention) and small interfering nucleic acid (AdPLA gene siRNA) aiming at an AdPLA gene, wherein the solvent is prepared from the following raw materials: water, glycerol, tranexamic acid, glutathione, butanediol, taurine, potassium methoxysalicylate, glyceryl caprylate, caprylyl hydroximic acid, p-hydroxyacetophenone, carnosine, Alcaligenes polysaccharide, dipotassium glycyrrhizinate, and phenoxyethanol; the ratio of the solvent, transdermal decapeptide-4 and AdPLA gene siRNA is 1g of solvent: 0.1-0.5mg transdermal decapeptide-4: 10-100ng AdPLA gene siRNA. Specifically, 1g of the vehicle comprises the following raw materials:

0.93 g of water, 0.05 g of glycerol, 0.003 g of tranexamic acid, 0.002 g of glutathione, 0.001 g of butanediol, 0.001 g of taurine, 0.001 g of potassium methoxysalicylate, 0.001 g of glyceryl caprylate, 0.002 g of caprylyl hydroxamic acid, 0.001 g of p-hydroxyacetophenone, 0.001 g of carnosine, 0.002 g of alcaligenes polysaccharides, 0.003 g of dipotassium glycyrrhizinate and 0.002 g of phenoxyethanol. .

The invention also provides a preparation method of the local fat-reducing composition, which comprises the following steps of adding raw materials for preparing a solvent into a water phase pot of a homogenizing and emulsifying machine according to the following formula, and stirring at 400 revolutions per minute: 0.93 g of water, 0.05 g of glycerol, 0.003 g of tranexamic acid, 0.002 g of glutathione, 0.001 g of butanediol, 0.001 g of taurine, 0.001 g of potassium methoxysalicylate, 0.001 g of glycerol caprylate, 0.002 g of caprylyl hydroxamic acid, 0.001 g of p-hydroxyacetophenone, 0.001 g of carnosine, 0.002 g of alcaligenes polysaccharides, 0.003 g of dipotassium glycyrrhizinate and 0.002 g of phenoxyethanol, stirring for twenty minutes, and then adding transdermal decapeptide-4 and AdPLA gene siRNA, wherein the proportion of the three is 1g of solvent: 0.1-0.5mg transdermal decapeptide-4: 10-100ng AdPLA gene siRNA, and stirring for ten minutes.

The raw materials used for preparing the solvent in the invention are all purchased from Guangzhou Yuanji Biotechnology GmbH, and the catalog numbers of the products are as follows:

water catalog number: 06260

Glycerol catalog number: 02421

Tranexamic acid catalog number: 04866

Glutathione catalog number: 02586

Butanediol catalog number: 0003

Taurine catalog number: 04876

Catalog number of potassium methoxysalicylate: 03354

Catalog number for glyceryl caprylate: 02478

Catalog number for caprylyl hydroxamic acid: 07218

Catalog number of p-hydroxyacetophenone: 02021

Carnosine catalogue number: 03150

Alcaligenes polysaccharide catalogue number: 01616

Catalog number of dipotassium glycyrrhizinate: 02397

Catalogue number of phenoxyethanol: 01294

The sequence of transdermal decapeptide-4 was synthesized by Guangzhou Yuanji Biotech, Inc., as shown in Chinese granted patent CN 106084006B.

AdPLA Gene siRNA was synthesized by Ribo Biotech, Inc., Guangzhou.

The invention has the advantages of

The local fat-reducing product provided by the invention can efficiently and specifically inhibit the expression of the AdPLA gene and effectively hydrolyze fat. After the product is used for 10 days on experimental animals, the highest fat hydrolysis rate can reach more than 40 percent, and the product can be used for a long time and can achieve the aim of treating both symptoms and root causes.

Detailed Description

The siRNA aiming at the AdPLA gene is designed, the expression of the AdPLA gene can be pertinently reduced or even silenced, fat is hydrolyzed, and the local high-efficiency transdermal penetration composition is matched to play a local fat reduction effect.

The embodiment of the invention discloses a method for designing and obtaining small interfering RNA molecules aiming at an AdPLA gene and a method for preparing a local fat-reducing composition by applying the small interfering RNA molecules. Those skilled in the art can now appreciate from this disclosure that appropriate modifications of the process parameters and adjuvants/starting components can be made, and it is specifically intended that all such similar substitutes and modifications which are obvious to those skilled in the art are deemed to be within the scope of the invention. The invention of small interfering nucleic acid and product composition has been described by preferred embodiments, but the related personnel obviously can not deviate from the content and scope of the invention to the product and method of change or appropriate changes and combinations to realize and apply the technology of the invention. The following examples are merely illustrative, and the present invention is not limited to these examples.

The invention designs and screens active small interfering nucleic acid (siRNA) sequences based on the sequence of an AdPLA Gene (Gene ID: 11145).

AdPLA-siRNA 1: sense strand 5 '-GCGAGCACUUGUGAAUGAdTdT-3' SEQ ID No.1

Antisense strand 5 '-UCAUUCACAAGUGCUCGCdTdT-3' SEQ ID No.2

AdPLA-siRNA 2: sense strand 5 '-GGUCAGGGAUUGUCUUCAdTdT-3' SEQ ID No.3

Antisense chain 5 '-UGAAAGACAUCCCUGACCDTTdT-3' SEQ ID No.4

AdPLA-siRNA 3: sense strand 5 '-CCCACACAAUUCAUAUAUAUAAUdTdT-3' SEQ ID No.5

Antisense strand 5 '-AUUAUGAAUAUUGUGUGGGGdTdT-3' SEQ ID No.6

AdPLA-siRNA 4: sense strand 5 '-GCCGUGACUACAUCUUGTdT-3' SEQ ID No.7

Antisense strand 5 '-ACAAGAUGUAUCCACGGCDTdT-3' SEQ ID No.8

The specific steps of the small interfering nucleic acid chemical synthesis of the nucleotide sequence of the AdPLA gene are as follows:

(1) synthesis of oligoribonucleotides: synthesis of oligoribonucleotides was performed on an automated DNA/RNA synthesizer. The small interfering RNA consists of a section of 19 oligoribonucleotides and 2 deoxythymidylate. Therefore, the starting material is 5 '-0-p-dimethoxy-thymidine connected with a solid phase, and the specific synthesis of each cycle can be completed by four steps, wherein the first step is to elute the protecting group at the 5' position on the thymidine connected with the immobilization under the action of trichloroacetic acid; secondly, coupling 5' -0-p-dimethoxytrityl-thymidine phosphoramidite to the previous thymidine which is deprotected under the action of an active catalyst S-ethyltetrazole to form dithymidine phosphite triester, wherein the coupling time and the coupling frequency are all completed according to the program provided by an instrument manufacturer; the third step is that the coupled dithymidine phosphotriester is oxidized into dithymidine phosphotriester under the action of 0.05M iodine water; the fourth step is acetylation, in which small amount of unreacted active groups (such as hydroxyl and amine groups) on the solid phase are reacted with acetic anhydride to form ester or amide, thereby achieving blocking effect to reduce the generation of overall by-products, and the cycle is repeated until the synthesis of the whole nucleic acid sequence is completed.

(2) Deprotection: putting the synthesized solid phase small interfering nucleic acid into a bottle which can be sealed, adding 1 ml of ethanol/amine (volume ratio is 1: 3), sealing, putting the bottle in an incubator at 55-70 ℃, incubating for 2-30 hours, taking out the solution, rinsing the solid phase with double distilled water again, collecting the eluent, and drying to remove the solvent. Then, 1 ml of tetrahydrofuran solution (IM) of tetrabutylammonium fluoride was added, and the mixture was left at room temperature for 4 to 12 hours, followed by ethanol precipitation to obtain a crude product of small interfering nucleic acid.

(3) And (3) purification and separation: the crude product of small interfering nucleic acid is dissolved in 2 ml of ammonium acetate aqueous solution, then the separation is carried out by reaction C18 high pressure liquid chromatography, the gradient elution method is applied to collect the main product of small interfering nucleic acid (eluent A:0.1M ammonium acetate; eluent B: 20% of 0.1M ammonium acetate and 80% acetonitrile), the solvent in the main product is removed, 5 ml of 80% acetic acid aqueous solution is added, the mixture is kept stand for 15 minutes at room temperature, and then the solution is subjected to anion exchange separation (DEAE-5PW, anion exchange column), thus obtaining the small interfering nucleic acid with the purity of more than 90% (gradient elution, eluent A: 0.025M Tris-HCl,0.025M NaCl, pH 8, 5% acetonitrile; eluent B: 0.025M Tris-HCl, 2OM NaCl, pH 8, 5% acetonitrile).

(4) Desalting: the purified small interfering RNA is dialyzed to remove salts, and then the small interfering RNA solution is filtered, sterilized, dried and crystallized. Then annealing the oligoribonucleotides of the sense strand and the antisense strand to form a stable double-stranded small interfering RNA, by mixing and dissolving the oligoribonucleotides of the sense strand and the antisense strand in 1-2 ml of buffer (10mM Tris, pH 7.5-8.0,50mM NaCl), heating the solution to 95 ℃, slowly cooling the solution to room temperature, and storing the solution in a refrigerator at 4 ℃ for use at any time.

Example 1

Adding the raw materials for preparing the solvent into a water phase pot of a homogenizing and emulsifying machine according to the following formula, and stirring at 400 revolutions per minute: 0.93 g of water, 0.05 g of glycerol, 0.003 g of tranexamic acid, 0.002 g of glutathione, 0.001 g of butanediol, 0.001 g of taurine, 0.001 g of potassium methoxysalicylate, 0.001 g of glycerol caprylate, 0.002 g of caprylyl hydroxamic acid, 0.001 g of p-hydroxyacetophenone, 0.001 g of carnosine, 0.002 g of alcaligenes polysaccharides, 0.003 g of dipotassium glycyrrhizinate and 0.002 g of phenoxyethanol, stirring for 20 minutes, and then adding transdermal decapeptide-4 and AdPLA-siRNA1, wherein the proportion of the three is 1g of solvent: 0.1mg transdermal decapeptide-4: 10ng of AdPLA-siRNA1 and stirring continued for an additional 10 minutes to give the topical fat reduction product of example 1.

Example 2

Essentially the same as example 1, except that: the proportion of the three is 1g of solvent: 0.1mg transdermal decapeptide-4: 25ng of AdPLA-siRNA1, the topical fat reduction product of example 2 was obtained.

Example 3

Essentially the same as example 1, except that: the proportion of the three is 1g of solvent: 0.1mg transdermal decapeptide-4: 50ng of AdPLA-siRNA1, the topical fat reduction product of example 3 was obtained.

Example 4

Essentially the same as example 1, except that: the proportion of the three is 1g of solvent: 0.1mg transdermal decapeptide-4: 75ng AdPLA-siRNA1, resulting in the topical fat reduction product of example 4.

Example 5

Essentially the same as example 1, except that: the proportion of the three is 1g of solvent: 0.1mg transdermal decapeptide-4: 100ng of AdPLA-siRNA1, the topical fat reduction product of example 5 was obtained.

Example 6

Essentially the same as example 1, except that: the proportion of the three is 1g of solvent: 0.5mg transdermal decapeptide-4: 10ng AdPLA-siRNA1, the topical fat reduction product of example 6 was obtained.

Example 7

Essentially the same as example 1, except that: the proportion of the three is 1g of solvent: 0.5mg transdermal decapeptide-4: 25ng of AdPLA-siRNA1, the topical fat reduction product of example 7 was obtained.

Example 8

Essentially the same as example 1, except that: the proportion of the three is 1g of solvent: 0.5mg transdermal decapeptide-4: 50ng of AdPLA-siRNA1, the topical fat reduction product of example 8 was obtained.

Example 9

Essentially the same as example 1, except that: the proportion of the three is 1g of solvent: 0.5mg transdermal decapeptide-4: 75ng AdPLA-siRNA1, to give the topical fat reduction product of example 9.

Example 10

Essentially the same as example 1, except that: the proportion of the three is 1g of solvent: 0.5mg transdermal decapeptide-4: 100ng of AdPLA-siRNA1, the topical fat reduction product of example 10 was obtained.

Example 11

Essentially the same as example 10, except that: AdPLA-siRNA1 was replaced by AdPLA-siRNA2, in a ratio of 1g vehicle: 0.5mg transdermal decapeptide-4: 100ng of AdPLA-siRNA2, the topical fat reduction product of example 11 was obtained.

Example 12

Essentially the same as example 10, except that AdPLA-siRNA1 was replaced with AdPLA-siRNA3, the topical fat reduction product of example 12 was obtained.

Example 13

Essentially the same as example 10, except that AdPLA-siRNA1 was replaced with AdPLA-siRNA4, the topical fat reduction product of example 13 was obtained.

Example 14 thyroid eye disease nude mouse experiment

Orbital fat was obtained from patients undergoing orbital decompression surgery. The tissues were washed for volume measurement and implanted under the ventral skin of 70 nude mice. Topical fat-reducing compositions prepared according to examples 1-13 were numbered 1-13, respectively, and vehicle-only control group 14. Nude mice were randomly divided into different groups of 5 mice each. Groups of products were applied to the inoculation area once a day. After 10 days of application of the above formulation, nude mice were sacrificed and subcutaneous fat was removed. RNA was extracted from a portion of the tissues and expression of ADPLA mRNA was measured by QRT-PCR, and the remaining tissue sections were oil red stained: preparing oil red O diluent: 6mL of the oil red saturated solution was mixed with 4mL of distilled water, and the mixture was filtered. ② the prepared frozen sections are fully washed, and oil red O diluent is dyed for 10min in dark. ③ 60 percent ethanol is differentiated under a mirror and washed with water. And fourthly, re-staining the nuclei with hematoxylin, washing with water, and sealing with 70% glycerol. The photographs were taken under microscopic observation for 2h, followed by washing with PBS, extraction of oil red O by addition of 100-.

TABLE 1 Effect of inhibition of AdPLA mRNA in each group

TABLE 2 oil Red OD values and fat degradation rates for each group

According to the results shown in tables 1 and 2, the inhibition rate of AdPLA mRNA in the case of only the solvent is 0, the orbital fat of the transplanted mouse is obviously reduced 10 days after the product provided by the invention is used, and the combination of 10 g of the solvent, 0.5mg of transdermal peptide and 100ng of AdPLA gene siRNA No.1 has the best degradation effect on fat, and the degradation rate is as high as 46.84%.

Example 15 subcutaneous fat assay in mice

The examples shown above give topical reduced fat compositions 1-13, with a vehicle only control group of 14. 70 adult female C57BL/6 mice were housed in an environmentally controlled animal care laboratory using a standard mouse diet. The hair was carefully trimmed with scissors in a 1cm x 1cm area of the abdomen (without using any mice with evidence of skin damage). Mice were selected and randomized into different groups of 5 mice each. Groups of products were applied once daily to the abdomen in areas where hair was removed. After 10 days of application of the above formula, mice were sacrificed and subcutaneous fat was removed. The sections were stained with oil red as described above.

TABLE 3 oil Red OD values and fat degradation rates for the respective groups

According to the results shown in Table 3, the subcutaneous fat of the mice is obviously reduced 10 days after the product provided by the invention is used, and the combination of 10 g of solvent, 0.5mg of transdermal peptide and 100ng of AdPLA gene siRNA No.1 has the best degradation effect on the subcutaneous fat, and the degradation rate is as high as 45.71%.

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Claims (9)

1. A topical lipid-lowering composition comprising a vehicle, a transdermal peptide and a small interfering nucleic acid directed against the AdPLA gene.

2. The topical lipid-reducing composition according to claim 1, characterized in that the small interfering nucleic acid against the AdPLA gene is selected from the group consisting of:

AdPLA-siRNA 1: sense strand 5 '-GCGAGCACUUGUGAAUGAdTdT-3' SEQ ID No.1

Antisense strand 5 '-UCAUUCACAAGUGCUCGCdTdT-3' SEQ ID No.2

Or AdPLA-siRNA 2: sense strand 5 '-GGUCAGGGAUUGUCUUCAdTdT-3' SEQ ID No.3

Antisense chain 5 '-UGAAAGACAUCCCUGACCDTTdT-3' SEQ ID No.4

Or AdPLA-siRNA 3: sense strand 5 '-CCCACACAAUUCAUAUAUAUAAUdTdT-3' SEQ ID No.5

Antisense strand 5 '-AUUAUGAAUAUUGUGUGGGGdTdT-3' SEQ ID No.6

Or AdPLA-siRNA 4: sense strand 5 '-GCCGUGACUACAUCUUGTdT-3' SEQ ID No.7

The antisense strand 5 '-ACAAGAUGUAUCCACGGTD-3' SEQ ID No. 8.

3. The topical lipid-reducing composition according to any one of claims 1 or 2, wherein the small interfering nucleic acid directed against the AdPLA gene reduces the expression level of the AdPLA gene mRNA by 60% or more.

4. The topical lipid-reducing composition according to claim 1, wherein 1 gram of the vehicle contains 0.1-0.5mg of the transdermal peptide and 10-100ng of the small interfering nucleic acid against the AdPLA gene.

5. A topical fat-reducing composition according to claim 1, characterized in that 1 gram of vehicle contains 0.93 gram of water, 0.05 gram of glycerol, 0.003 gram of tranexamic acid, 0.002 gram of glutathione, 0.001 gram of butylene glycol, 0.001 gram of taurine, 0.001 gram of potassium methoxysalicylate, 0.001 gram of glyceryl caprylate, 0.002 gram of octanoyl hydroxamic acid, 0.001 gram of p-hydroxyacetophenone, 0.001 gram of carnosine, 0.002 gram of alcaligenes polysaccharides, 0.003 gram of dipotassium glycyrrhizinate and 0.002 gram of phenoxyethanol.

6. The topical fat-reducing composition according to claim 1, wherein the composition has a subcutaneous fat degradation rate of 21% or more after 10 days of use.

7. A process for the preparation of a topical lipid-lowering composition, characterized in that 0.93 g of water, 0.05 g of glycerol, 0.003 g of tranexamic acid, 0.002 g of glutathione, 0.001 g of butanediol, 0.001 g of taurine, 0.001 g of potassium methoxysalicylate, 0.001 g of glyceryl caprylate, 0.002 g of octanoyl hydroxamic acid, 0.001 g of p-hydroxyacetophenone, 0.001 g of carnosine, 0.002 g of alcaligenes polysaccharides, 0.003 g of dipotassium glycyrrhizinate and 0.002 g of phenoxyethanol are added to an aqueous phase pot of a homogenizer and stirred at 400 rpm, and after stirring for twenty minutes, the transdermal peptide and AdPLA gene siRNA are added in a ratio of 1g of solvent: 0.1-0.5mg of transdermal peptide: 10-100ng AdPLA gene siRNA, and stirring for ten minutes.

8. Use of a topical fat-reducing composition as claimed in any one of claims 1 to 6 in the manufacture of a medicament for the treatment of thyroid-related eye disease.

9. Use of a topical fat-reducing composition as claimed in any one of claims 1 to 6 in the manufacture of a topical fat-reducing product.