CN109893656B

CN109893656B - Application of miR-327 inhibitor and/or FGF10 promoter in preparation of medicines for preventing and/or treating lipodystrophy

Info

Publication number: CN109893656B
Application number: CN201711310808.XA
Authority: CN
Inventors: 杨云龙; 曹义海
Original assignee: Yihui Technology Shenzhen Co ltd
Current assignee: Yihui Technology Shenzhen Co ltd
Priority date: 2017-12-11
Filing date: 2017-12-11
Publication date: 2021-07-20
Anticipated expiration: 2037-12-11
Also published as: CN109893656A

Abstract

The invention provides a new application of a miR-327 inhibitor and/or FGF10 promoter, in particular to an application in preparing a medicament for preventing and/or treating diabetes. The miR-327 inhibitor and the FGF10 accelerant can promote the increase of FGF10 in a stromal cell component of adipose tissue, promote an adipocyte precursor of the stromal cell component to be differentiated into brown adipocytes, improve energy consumption, promote fat metabolism, and further achieve the effect of preventing and/or treating fat metabolism. The invention also provides a medicament for preventing and/or treating lipodystrophy, which comprises the miR-327 inhibitor and a pharmaceutically acceptable carrier thereof.

Description

Application of miR-327 inhibitor and/or FGF10 promoter in preparation of medicines for preventing and/or treating lipodystrophy

Technical Field

The invention relates to the technical field of medicines, in particular to application of a miR-327 inhibitor and/or FGF10 promoter in medicines for preventing and/or treating lipodystrophy.

Background

Fat metabolism is the process of digestion, absorption, synthesis and decomposition of fat in organisms with the help of various related enzymes, and is processed into substances required by organisms, so that the normal physiological function operation is ensured, and the fat metabolism has important significance on life activities. Diseases caused by abnormal fat metabolism are common diseases in modern society. Among them, obesity is a common disorder of fat metabolism characterized by an abnormal increase in the volume and number of fat cells in the body, resulting in an abnormally high percentage of body fat to body weight and excessive deposition of fat in some parts. Statistically, obesity is associated with 15% to 20% of patients who die due to the disease. Obesity is the cause of the onset of cardiovascular disease, particularly coronary heart disease. Obesity also easily causes diseases such as fatty liver, cholecystitis and the like, so the treatment of obesity is particularly important for the health of people.

Currently, there are many methods for treating obesity, including dietary management, drug therapy, surgical treatment, and the like. Among them, the therapeutic effects of drugs are more remarkable than those of dietary control, and are also strongly sought after without the high cost of surgical treatment. Most of the commercially available drugs for treating obesity are based on methods such as emetic and appetite suppression to reduce energy intake in vivo and achieve the purpose of losing weight, but the drugs cause certain harm to health and cannot radically change obesity. Therefore, there is a need for a drug that fundamentally prevents and/or treats lipodystrophy without harming physical health.

Disclosure of Invention

In order to solve the problems, the invention provides application of a miR-327 inhibitor in preparation of a medicament for preventing and/or treating lipodystrophy. The miR-327 inhibitor can obviously inhibit the expression of miR-327 in a stromal vascular fraction in adipose tissue, reduce the expression of miR-327, promote the increase of fibroblast growth factor 10(FGF10) which is a target of miR-327 in non-adipose cells (such as various cells in the stromal vascular fraction), promote the differentiation of adipose cell precursors in the stromal vascular fraction to brown adipose cells (namely promote the browning of the adipose cell precursors), improve energy consumption, promote the metabolism of fat and reduce the volume of the adipose cells, thereby effectively improving and treating fat metabolism, particularly obesity.

In a first aspect, the invention provides application of a miR-327 inhibitor and/or FGF10 promoter in preparation of a medicament for preventing and/or treating lipodystrophy.

The miR-327 inhibitor is used for inhibiting expression of miR-327 in a fat matrix vascular component (SVF), promoting increase of FGF10 serving as a miR-327 target, promoting differentiation of fat cell precursors in the SVF to brown fat cells, improving energy consumption and promoting fat metabolism. Further, the effect of preventing and/or treating the fat metabolism disease can be achieved.

Wherein the FGF10 promoter can promote the increase of FGF10 in fat Stromal Vascular Fraction (SVF), promote the differentiation of fat cell precursors in SVF to brown fat cells, improve energy consumption and promote fat metabolism. Further, the effect of preventing and/or treating the fat metabolism disease can be achieved.

Optionally, the medicament for preventing and/or treating the lipodystrophy simultaneously contains the miR-327 inhibitor and the FGF10 promoter. Under the condition of the two components existing at the same time, the fat metabolism can be synergistically promoted.

Wherein the lipodystrophy includes one or more of obesity, fatty liver, hyperlipidemia, hyperlipoproteinemia, cardiovascular diseases, ketosis, and hypoproteinemia, but is not limited thereto.

In the present invention, miR-327 is a micro RNA (MicroRNA), a short non-coding RNA, that regulates protein expression post-transcriptionally.

Optionally, the miR-327 inhibitor comprises one or more of a chemical drug, a polypeptide drug, a protein drug and a gene drug that inhibits miR-327.

Further, the chemical drugs for inhibiting miR-327 generally refer to small molecule organic compounds. The protein drug for inhibiting miR-327 comprises natural protein and recombinant protein, including but not limited to antibody. The miR-327 inhibition "gene medicine" comprises but is not limited to nucleic acid fragments, such as DNA fragments and RNA fragments.

In one embodiment of the invention, the miR-327 inhibitor is an LNA inhibitor probe for antagonizing miR-327. The miR-327 inhibitor can target and inhibit miR-327, and the miR-327 inhibitor is complementary to the sequence of miR-327. Wherein, the nucleotide sequence of miR-327 comprises ACUUGAGGGGCAUGAGGAU.

Optionally, the FGF10 promoter comprises one or more of a chemical drug, a polypeptide drug, a protein drug, and a gene drug that promotes the increase of FGF 10.

Further, the chemical agents that promote the increase of FGF10 generally refer to small molecule organic compounds. The protein drugs promoting the increase of FGF10 comprise natural proteins and recombinant proteins, including but not limited to antibodies. The "gene-like drug" promoting the increase of FGF10 includes, but is not limited to, nucleic acid fragments, such as DNA fragments, RNA fragments. Specifically, the FGF10 promoter comprises FGF10 protein.

Optionally, the pharmaceutically acceptable carrier includes at least one of a solvent, a polymer, a liposome, a recombinant viral vector, and a eukaryotic recombinant expression vector, but is not limited thereto.

Wherein the solvent includes, but is not limited to, water, physiological saline, and other non-aqueous solvents. The recombinant viral vector may include one or more of a lentiviral vector, an adenoviral vector, and a retroviral vector, but is not limited thereto.

Wherein the polymer comprises one or more of polylysine, polyethyleneimine (branched and/or chain) and modified substances thereof, polyamidoamine dendrimer (PAMAM) and derivatives thereof, polypropyleneimine dendrimer (PPI) and derivatives thereof, chitosan, polylactic-co-glycolic acid (PLGA), polylactic acid, gelatin, cyclodextrin, sodium alginate, albumin and hemoglobin, but is not limited thereto. Among them, polyethyleneimine and its modified products, PAMAM and its derivatives, PPI and its derivatives, chitosan, etc. may be referred to as cationic polymers.

The liposome can be prepared by self-assembly of cationic lipid, neutral auxiliary lipid, cholesterol, and phospholipid (such as soybean lecithin, yolk lecithin, cephalin, etc.), or can be prepared by inserting distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) into phospholipid layer formed by phospholipid molecules.

In the present invention, the "pharmaceutically acceptable carrier" is used to transport the drug of the present invention to exert its intended effect. In general, delivery is from one organ or portion to another, and the carrier must be compatible with the pharmaceutical composition, not interfere with the biological activity of the drug, and be relatively non-toxic, e.g., the carrier enters the body without causing toxic side effects or having a severe reaction with the drug it carries, which does not adversely affect the patient.

The active pharmaceutical ingredients, namely the miR-327 inhibitor and/or the FGF10 accelerant, can be dispersed or absorbed in the carrier to form a dispersion system, and can also be coated/encapsulated by the liposome, the polymer and the like to form a spherical structure (such as a nano capsule or a micro capsule). For example, when the miR-327 inhibitor is a nucleic acid fragment inhibiting miR-327, the drug for preventing and/or treating lipodystrophy comprises, but is not limited to, a cationic polymer, a polypeptide, a protein drug and the like which are wrapped, combined or blended with the nucleic acid fragment.

The miR-327 inhibitor encapsulated in the spherical structure can be subjected to sustained release, controlled release or targeted release, so that the drug can exert the optimal efficacy, and the stability of the drug can be improved. For example, albumin, gelatin, chitosan, polylactic acid may generally form microspheres that can disperse or encapsulate a pharmaceutically active ingredient.

Optionally, in the medicament for preventing and/or treating lipodystrophy, the content of the miR-327 inhibitor is 5-20 mg/kg. Further preferably 5-10 mg/kg.

Optionally, in the medicament for preventing and/or treating lipodystrophy, the FGF10 promoter is contained in an amount of 1 to 100 mg/kg.

Further, the content ratio of the miR-327 inhibitor to the FGF10 promoter is (0.2-20): 1.

in the medicament for preventing and/or treating the lipodystrophy, the miR-327 inhibitor can be selected as a single active ingredient, the FGF10 promoter can be selected as a single active ingredient, and the medicament can also contain the miR-327 inhibitor and the FGF10 promoter as active ingredients. This is also what is meant by "miR-327 inhibitor and/or FGF10 promoter" in the context of the present invention.

On the basis, the medicament for preventing and/or treating the lipodystrophy further comprises other pharmaceutically acceptable active ingredients. The miR-327 inhibitor and/or FGF10 promoter can act together with other pharmaceutically acceptable active ingredients to achieve the purpose of preventing and/or treating diabetes.

Wherein the other pharmaceutically acceptable active ingredients include one or more of orlistat, mazindol and rimonabant, but are not limited thereto.

The Orlistat (Orlistat) is a gastrointestinal lipase inhibitor, and can directly block the absorption of fat in food by a human body, so that once the absorbed heat energy and fat are less than the consumption, the fat in the body is naturally reduced, thereby achieving the purpose of losing weight. The Mazindol (Mazindol) is an appetite suppressant and acts on non-organic simple obesity. The rimonabant (rimonabant) is 5- (4-chlorphenyl) -1- (2, 4-dichlorophenyl) -4-methyl-N- (1-piperidyl) -1H-pyrazole-3-formamide, is a cannabinoid receptor 1 antagonist, and can effectively reduce the weight of obese patients. Specifically, orlistat, mazindol, rimonabant or other drugs having the function of preventing and/or treating obesity can be selected according to actual needs.

Further, in the medicament for preventing and/or treating lipodystrophy, the mass ratio of the miR-327 inhibitor and/or FGF10 promoter to the other pharmaceutically-acceptable active ingredients is (1-10): 1.

optionally, the medicament for preventing and/or treating lipodystrophy further comprises one or more of a diluent and an excipient.

The primary function of the diluent is to fill the weight or volume of the tablet to facilitate tableting. Optionally, the diluent comprises one or more of starches, sugars, celluloses, and inorganic salts. The excipient is additive except main active ingredients in the medicine, and comprises binder, filler, disintegrating agent, lubricant, wine, vinegar, medicinal juice, etc. in the tablet, matrix part in semi-solid preparation ointment and cream, antiseptic, antioxidant, corrective, aromatic, cosolvent, emulsifier, solubilizer, osmotic pressure regulator, colorant, etc. in the liquid preparation.

Alternatively, the form of the agent for preventing and/or treating lipodystrophy includes a tablet, a capsule, a powder, a granule, a pill, a syrup, a solution, or a suspension. The form of the agent for preventing and/or treating lipodystrophy depends on the actual use.

Alternatively, the agent for preventing and/or treating lipodystrophy is administered orally or by injection.

Preferably, the agent for preventing and/or treating lipodystrophy is administered by injection. Further, in this case, the pharmaceutical form for preventing and/or treating lipodystrophy is preferably a solution, for example, dissolved in water or physiological saline. Further, the injection is administered by intraperitoneal injection, subcutaneous injection, intramuscular injection or intravenous injection.

Alternatively, the agent for preventing and/or treating lipodystrophy may be administered locally or systemically.

Optionally, the amount of the agent for preventing and/or treating lipodystrophy is 5-20mg/kg body weight per day. Specifically, the amount of the agent for preventing and/or treating lipodystrophy to be used depends on various factors including, but not limited to, the desired biological activity and tolerance to the agent of the subject to be administered.

The miR-327 inhibitor and the FGF10 promoter can promote the increase of FGF10 in a fat matrix vascular component, promote fat cell precursors in the matrix vascular component to be differentiated to brown fat cells, improve energy consumption, promote fat metabolism and further achieve the effect of preventing and/or treating fat metabolism. The invention provides a new application of a miR-327 inhibitor and/or FGF10 promoter. The preparation method opens up a new way for treating the fat metabolism disease, especially the obesity and the obesity related diseases, has a more obvious prevention or treatment effect, and can effectively reduce the risk of the people suffering from the fat metabolism disease, especially the obesity and the obesity related diseases.

In a second aspect, the invention provides a medicament for preventing and/or treating lipodystrophy, wherein the medicament for preventing and/or treating lipodystrophy comprises one or more of a miR-327 inhibitor and a FGF10 promoter, and a pharmaceutically acceptable carrier.

The action of the miR-327 inhibitor, the FGF10 promoter and the pharmaceutically-acceptable carrier are all as described in the first aspect of the invention, and are not described in detail herein.

Optionally, in the medicament for preventing and/or treating lipodystrophy, the content of the miR-327 inhibitor is 5-20 mg/kg.

Optionally, the medicament for preventing and/or treating lipodystrophy further comprises other pharmaceutically acceptable active ingredients, wherein the other pharmaceutically acceptable active ingredients comprise one or more of orlistat, mazindol and rimonabant.

Optionally, the mass ratio of the miR-327 inhibitor to the other pharmaceutically-acceptable active ingredients is (1-10): 1.

optionally, the mass ratio of the FGF10 promoter to the other pharmaceutically acceptable active ingredient is (1-10): 1.

the medicament for preventing and/or treating lipodystrophy provided by the second aspect of the invention comprises a miR-327 inhibitor and a pharmaceutically acceptable carrier thereof. The miR-327 inhibitor in the medicine can effectively inhibit the expression of miR-327 in a fat matrix vascular component, promote the increase of FGF10, promote the differentiation of fat cell precursors to brown fat cells, improve energy consumption, promote fat metabolism and further improve fat metabolism.

Advantages of embodiments of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of embodiments of the invention.

Drawings

FIG. 1 shows the results of histological examination of control group 2 and control group 3 in the absence or in the presence of drug stimulation;

FIG. 2 is a graph quantifying the mean diameter of adipocytes in control group 2 and control group 3 that were not subjected to cold stimuli;

FIG. 3 is the results of the adipose tissue weight test of control group 2 and control group 3 given cold stimulation or not;

FIG. 4 shows the results of histological examination of the test group and the control group 1, which were not or not given cold stimuli;

FIG. 5 shows the results of metabolic tests of the experimental group and the control group 1, which were not or not given cold stimuli.

Detailed Description

While the following is a description of the preferred embodiments of the present invention, it should be noted that those skilled in the art can make various modifications and improvements without departing from the principle of the embodiments of the present invention, and such modifications and improvements are considered to be within the scope of the embodiments of the present invention.

Example 1 mice were dosed with drugs to investigate the effect of the drugs on adipose tissue weight and morphology

Experimental groups: an LNA inhibitor probe which is customized by Exiqon and antagonizes miR-327 is selected as a miR-327 inhibitor (the miR-327 inhibitor can target and inhibit miR-327, and the sequence of the miR-327 inhibitor is complementary to the nucleotide sequence of miR-327, wherein the nucleotide sequence of miR-327 comprises ACUUGAGGGGCAUGAGGAU). 12 female C57BL/6 mice, 8 weeks old, were placed under thermoneutral conditions (30 ℃), and the miR-327 inhibitor was injected into the mice 1 time per week at a dose of 8mg/kg, and then 6 of the mice were subjected to cold (4 ℃) stimulation, and 6 of the mice were left at 30 ℃ (i.e., no cold stimulation was given) (note that cold stimulation could also be replaced by drug stimulation, such as β 3-adrenoceptor agonist stimulation).

The injection is performed for 2 weeks, and after 2 weeks, the mice are subjected to metabolic capacity test, specifically to maximum oxygen uptake capacity (VO)₂) Then using excess CO₂Mice were sacrificed and their subcutaneous fat was dissected carefully, the weight was measured on a precision balance and statistically analyzed to obtain mouse adipose tissue weighing results. Meanwhile, the histological detection of the adipose tissues of the mouse specifically comprises the following steps: adipose tissue was fixed using 4% PFA, sliced, digested with cathepsin K (20mM), and permeabilized with 100% methanol. The samples were blocked overnight with 3% milk in 0.1% Triton X-100PBS at 4 deg.C with a primary antibody, such as anti-CD 31(1:100, Cat. AF3628, R)&D) And anti-Perilipin (1:200, catalog No. 20R-PP004, Fitzgerald Industries), anti-UCP 1(1:200, Abcam), etc. After washing with PBS, the samplesBlocked with 3% milk and incubated with a fluorescently labeled secondary antibody (1: 300, Thermo Fisher Scientific Inc.) for 2 hours at room temperature. After washing, the samples were mounted, observed and measured on a Confocal microscope (LSM510Confocal, Zeiss).

Control group 1: non-functional miR inhibitor drugs (random sequences) that do not inhibit miR-327 were injected subcutaneously every week at a dose of 8mg/kg into 8-week-old 12C 57BL/6 mice, 6 of which were then given cold (4 ℃) stimulation, and another 6 were left at 30 ℃ (i.e., no cold stimulation was given). 1 injection per week for a total of 2 weeks; VO was performed on mice 2 weeks later₂Testing, and then reusing excess CO₂The mice were sacrificed and their adipose tissues were weighed and examined histologically as mentioned above.

Control group 2: adenovirus vectors (Vector Biosystems) carrying non-functional miR at 1X 10⁹PFU/dose was injected subcutaneously into white fat of 24C 57BL/6 mice 8 weeks old, 6 mice were given cold (4 ℃) challenge one week later, and another 6 mice were left at 30 ℃; 6 of the mice were given drug stimulation (β 3-adrenoceptor agonists (e.g., CL316243)), and the remaining 6 mice were injected with physiological saline for comparison. After 2 weeks excess CO was used₂The mice were sacrificed and their adipose tissues were weighed and examined histologically as mentioned above.

Control group 3: adenovirus overexpressing miR-327 (Ad-EF1 a-mmu-miR-327-eGFP; Vector Biosystems) at 1X 10⁹PFU/dose was injected into white fat of 12C 57BL/6 mice 8 weeks old, 6 of which were given cold (4 ℃) challenge one week later and 6 were left at 30 ℃; 6 of the mice were given drug (e.g., CL316243) stimulation, and the remaining 6 mice were injected with saline for comparison. After 2 weeks excess CO was used₂The mice were sacrificed and their adipose tissues were weighed and examined histologically as mentioned above.

The histological results of the control group 2 and the control group 3 are shown in fig. 1 and 2, and fig. 1 is the histological result of the control group 2 and the control group 3 when the drug is not stimulated. Fig. 2 is a graph showing the quantification of the mean diameter of adipocytes in the absence and presence of cold stimuli given to control group 2 and control group 3. Wherein Ad-miRNC represents a control group 2, and Ad-miR-327 represents a control group 3. In fig. 1, H & E represents morphological staining of adipose tissue using hematoxylin and eosin. UCP1 staining showed brownish locations, PERI showed adipocyte morphology, DAPI counterstained the nuclei.

As can be seen from FIGS. 1 and 2, in the control group 3, after injecting the miR-327-expressing adenovirus into mice and expressing miR-327 in its adipose tissue (which can be abbreviated as Ad-miR-327), the size of the adipocytes was unchanged compared to the control group 2 without the stimulation of cold, drugs, etc. to promote browning. In the presence of cold, drugs and other stimuli promoting browning, the cell diameter of miR-327-expressing adipose tissues is not reduced (i.e., slightly increased), UCP1 staining shows that the browning degree is reduced, and arrows indicate sites with obvious browning.

The results of fig. 1 and 2 demonstrate that miR-327 introduced into mouse adipose tissue by using a viral vector can significantly inhibit the browning of white adipocytes and hinder the fat burning effect.

Fig. 3 is the results of the adipose tissue weight test in the absence and presence of cold stimulation given to control group 2 and control group 3. As can be seen from FIG. 3, the introduction of miR-327 into mouse adipose tissues using a viral vector suppresses the decrease in adipose tissue weight due to cold stimulation, and thus inhibits the effect of fat burning.

The results of histological detection and metabolic test of the experimental group and the control group 1 are shown in fig. 4 and fig. 5, wherein Inh-miR-NC represents the control group 1, and Inh-miR-327 represents the experimental group.

As can be seen from fig. 4, after the mice are injected with the miR-327 inhibitor, the size of the fat cells is significantly reduced compared with the control group 1 if the mice are not stimulated by cold, drugs and the like to promote browning, and the staining of UCP1 shows that the browning degree is significantly increased. In the presence of cold, drugs and other stimuli promoting browning, mice injected with the miR-327 inhibitor further decrease in adipocyte size, and staining by UCP1 shows that the degree of browning is further increased, with arrows indicating sites of marked browning.

As shown in fig. 5, after the miR-327 inhibitor is injected into the mice, the metabolic index thereof is significantly improved compared to the control group 1 without the stimulation of promoting browning, such as cold, drugs, and the like. If the mice are stimulated by cold, medicaments and the like to promote browning, the metabolic indexes of the mice injected with the miR-327 inhibitor are further improved.

Therefore, the results of fig. 4-5 show that the miR-327 inhibitor can significantly promote the browning of white fat of mice, promote fat metabolism and reduce fat accumulation.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

Use of a miR-327 inhibitor or a combination of a miR-327 inhibitor and a FGF10 agonist in the manufacture of a medicament for treating a disorder of fat metabolism, wherein the disorder of fat metabolism includes one or more of obesity, fatty liver, hyperlipidemia, hyperlipoproteinemia, ketosis, and proteinemia.
2. The use of claim 1, wherein the miR-327 inhibitor and the FGF10 promoter both promote an increase in FGF10 in the stromal vascular fraction of adipose tissue, promote differentiation of adipocyte precursors to brown adipocytes in the stromal vascular fraction, increase energy expenditure, promote fat metabolism; wherein the miR-327 inhibitor promotes an increase in FGF10 as a target of miR-327 by inhibiting expression of miR-327 in the stromal vascular fraction.
3. The use of claim 1, wherein said miR-327 inhibitor comprises one or more of a chemical, polypeptide, protein, and gene drug that inhibits miR-327;

the FGF10 promoter includes one or more of a chemical drug, a polypeptide drug, a protein drug, and a gene drug that promotes the increase of FGF 10.
4. The use of claim 1, wherein the medicament for the treatment of lipodystrophy further comprises a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier comprises at least one of a solvent, a polymer, a liposome, a recombinant viral vector and a eukaryotic recombinant expression vector.
5. The use of claim 1, wherein the medicament for the treatment of lipodystrophy comprises a miR-327 inhibitor and a FGF10 promoter.
6. The use of claim 1, wherein the medicament for the treatment of lipodystrophy further comprises other pharmaceutically acceptable active ingredients, said other pharmaceutically acceptable active ingredients comprising one or more of orlistat, mazindol and rimonabant.
7. The medicine for treating lipodystrophy is characterized by comprising a miR-327 inhibitor, an FGF10 promoter and a pharmaceutically acceptable carrier.
8. The medicament for treating lipodystrophy of claim 7 wherein said miR-327 inhibitor is present in an amount of 20-50 mg/kg.
9. The agent for treating lipodystrophy of claim 7 wherein the form of said agent for treating lipodystrophy comprises tablets, capsules, powders, granules, pills, syrups, solutions or suspensions;

the medicament for treating lipodystrophy is administered by oral administration or injection.