CN113648318B

CN113648318B - Use of enhancing cell thermogenesis and treating diseases

Info

Publication number: CN113648318B
Application number: CN202010399851.3A
Authority: CN
Inventors: 丁秋蓉; 邱艳; 应浩
Original assignee: Shanghai Institute of Nutrition and Health of CAS
Current assignee: Shanghai Institute of Nutrition and Health of CAS
Priority date: 2020-05-12
Filing date: 2020-05-12
Publication date: 2023-08-25
Anticipated expiration: 2040-05-12
Also published as: CN113648318A

Abstract

The present invention provides a use of an agent for increasing UCP1 expression or activity in a cell, or for increasing thermogenesis in a cell, or for the preparation of an agent for treating or preventing a disease benefiting from thermogenesis of fat, inhibiting weight gain in a subject, or reducing fat content in a subject, said agent being selected from one or more of the following: (1) a compound of formula (I) or a pharmaceutically acceptable salt or solvate or analog thereof, wherein R1-R10 are each independently selected from hydrogen, halogen, hydroxy, and alkyl, (2) an agent that upregulates expression or activity of a calcium channel-associated protein or subunit thereof, (3) a calcium channel activator, (4) an agent that upregulates expression or activity of calcineurin, and (5) an agent that upregulates expression or activity of NFAT.

Description

Use of enhancing cell thermogenesis and treating diseases

Technical Field

The invention relates to application of enhancing cell heat production and treating diseases, in particular to application of an agent in enhancing cell heat production and treating obesity and related metabolic diseases.

Background

Glyburide (gliclazide)Phenylurea, also known as eudragit) is a sulfonylurea hypoglycemic agent that has been approved by the FDA for the treatment of type 2 diabetes. Glibenclamide targets islet beta cells and closes the islet beta cells by binding to the SUR1 subunit of ATP-sensitive potassium ion channel (KATP) on the cell membrane, thereby secreting insulin and reducing blood glucose. It is considered that the exchange factor (Epac 2) of guanosine small-molecule G protein Rap1 is a direct binding target of sulfonylurea hypoglycemic compound, and can promote insulin secretion. Caveolin (cavolin) is thought to play an important role in transmembrane signal transduction by sulfonylurea hypoglycemic agents, cavolin 1 (Cav 1) is a major integral membrane protein of cell surface cave-like invagination, cav1 is expressed in large amounts in adipocytes, and plays an important role in signal transduction. Glibenclamide promotes Ca ²⁺ Through voltage gate control of calcium ion channel, ca in cell is further obtained ²⁺ Increased levels and with higher glibenclamide concentration, ca entered the cells ²⁺ The more this is, which can be inhibited by the calcium channel inhibitor Nitrendipine (nitendipine). Studies report that TRPV2, a non-selective cation channel, has an important role in the non-shivering thermogenesis of brown adipose tissue in mice. In addition, glibenclamide is considered to be also useful in the treatment of cerebral ischemia and stroke, where the target is SUR1-TRPM4 ion channel.

Obesity is a chronic metabolic disease caused by a variety of factors and is characterized by an abnormally high percentage of body fat based on body weight due to increased volume and cell number of fat cells in the body, and by the deposition of fat in some localized areas. Current anti-obesity agents primarily limit energy intake by reducing fat absorption or suppressing appetite, however clinical effects are not yet apparent and have side effects. No medicine for treating obesity by increasing energy utilization exists clinically, and the energy of the body is consumed in the form of heat energy by increasing the heat generating function of brown adipose tissue BAT, so that the medicine can be used as a target point for treating obesity and related metabolic diseases. The heat generating function of brown fat is realized by mostly depending on uncoupling protein UCP1, so UCP1 is a marker protein of brown fat and can be used as a target for treating obesity and related metabolic diseases.

Disclosure of Invention

In a first aspect, the present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof for increasing UCP1 expression or activity in a cell, or for increasing thermogenesis in a cell, or for the manufacture of a medicament for treating or preventing a disease that benefits from thermogenesis in fat, for inhibiting weight gain in a subject, or for reducing fat content in a subject,

R1-R10 are each independently selected from hydrogen, halogen, hydroxy and C1-C3 alkyl, R11 is pyrrole, pyrroline, phenyl substituted with one or more substituents selected from oxygen, C1-C3 alkyl, C1-C3 alkoxy, halogen, hydroxy.

In one or more embodiments, R1-R10 are each independently selected from hydrogen and halogen, R11 is phenyl substituted with one or more substituents selected from halogen, C1-C3 alkoxy. In one or more embodiments, R1-R10 are each independently selected from hydrogen and halogen, R11 is phenyl substituted with one or more substituents selected from chloro, methoxy. In one or more embodiments, R1-R10 are hydrogen and R11 is 2-methoxy-5-chlorophenyl.

In one or more other embodiments, R1-R10 are each independently selected from hydrogen and C1-C3 alkyl, R11 is pyrroline substituted with one or more substituents selected from oxygen, C1-C3 alkyl. In one or more embodiments, R1-R10 are each independently selected from hydrogen and methyl, R11 is pyrroline substituted with one or more substituents selected from oxygen, methyl, ethyl. In one or more embodiments, R1-R3 and R5-R10 are hydrogen, R4 is methyl, and R11 is 3-ethyl-4-methyl-2-oxo-3-pyrroline.

In one or more embodiments, the compound is a compound of formula (II) below

Wherein R1-R10 are each independently selected from hydrogen, halogen, hydroxy and C1-C3 alkyl.

In one or more embodiments, R1-R4 are each independently selected from hydrogen, halogen, hydroxy, and C1-C3 alkyl, and R5-R10 are hydrogen.

In one or more embodiments, R3-R6 are each independently selected from hydrogen, halogen, hydroxy, and C1-C3 alkyl, and R1-R2, R7-R10 are hydrogen.

In one or more embodiments, R1-R4 are each independently selected from F and Cl, and R5-R10 are hydrogen.

In one or more embodiments, R1-R2 are each independently selected from F and Cl, and R3-R10 are hydrogen.

In one or more embodiments, R3-R4 are each independently selected from F and Cl, and R1-R2 and R5-R10 are hydrogen.

In one or more embodiments, R1-R10 are hydrogen.

In one or more embodiments, the compound is a compound of formula (III) below

R1-R10 are each independently selected from hydrogen and C1-C3 alkyl.

In one or more embodiments, R1-R10 are each independently selected from hydrogen and methyl.

In one or more embodiments, R1-R3 and R5-R10 are hydrogen and R4 is methyl.

In one or more embodiments, the compound is glibenclamide, glimepiride, gliquidone, tolbutamide, gliclazide.

In one or more embodiments, the disease that benefits from fat thermogenesis is a disease that benefits from brown fat thermogenesis. In one or more embodiments, the disease that benefits from fat thermogenesis is obesity and/or metabolic disease.

In one or more embodiments, the metabolic disease is a metabolic disorder and/or an exuberance of metabolism.

In one or more embodiments, the metabolic disease is selected from: type 2 diabetes, diabetic ketoacidosis, fatty liver, hyperuricemia, hypertonic syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin a deficiency, scurvy, vitamin D deficiency, osteoporosis, hyperlipidemia, metabolic encephalopathy, hepatic encephalopathy, congenital metabolic disorders.

In one or more embodiments, the subject is a high fat diet subject or a non-high fat diet subject.

In one or more embodiments, the cell is an adipocyte. In one or more embodiments, the adipocytes are brown adipocytes and/or white adipocytes.

The invention also provides the use of an agent that upregulates expression or activity of a calcium channel associated protein or subunit thereof or a calcium channel activator in increasing UCP1 expression or activity in a cell, or in increasing thermogenesis in a cell, or in the manufacture of an agent for treating or preventing a disease benefiting from brown fat thermogenesis, inhibiting weight gain in a subject, or reducing fat content in a subject.

The agent that upregulates expression of the calcium channel associated protein or subunit thereof is an expression vector for the calcium channel associated protein or subunit thereof.

The subunit of the calcium ion channel-associated protein is selected from the group consisting of: alpha self-subunit, alpha-subunit, beta-subunit, gamma-subunit.

Agents that up-regulate the activity of a calcium channel-associated protein or subunit thereof are agonists of the calcium channel-associated protein or subunit thereof.

In one or more embodiments, the calcium channel activator is selected from nifedipine, amlodipine, and lacidipine.

The invention also provides the use of an agent that upregulates calcineurin expression or activity in a cell to increase UCP1 expression or activity in a cell, or to increase thermogenesis in a cell, or in the manufacture of an agent for treating or preventing a disease that benefits from thermogenesis of fat, inhibiting weight gain in a subject, or reducing fat content in a subject.

An agent that upregulates expression of calcineurin is an expression vector for calcineurin.

Agents that up-regulate calcineurin activity are calcineurin agonists.

The invention also provides the use of an agent that upregulates expression or activity of NFAT in increasing UCP1 expression or activity in a cell, or in increasing thermogenesis in a cell, or in the manufacture of an agent that treats or prevents a disease that benefits from thermogenesis of fat, inhibits weight gain in a subject, or reduces fat content in a subject.

In one or more embodiments, the agent that upregulates expression of NFAT is an expression vector of NFAT.

In one or more embodiments, the agent that upregulates NFAT activity is an NFAT agonist.

In one or more embodiments, NFAT is selected from NFAT1, NFAT2, NFAT3, and NFAT4.

In one or more embodiments, NFAT1 is as shown in NCBI gene accession number 18019.

In one or more embodiments, NFAT2 is as shown in NCBI gene accession number 18018.

In one or more embodiments, NFAT3 is as shown in NCBI gene accession number 73181.

In one or more embodiments, NFAT4 is as shown in NCBI gene accession number 18021.

The invention also provides a pharmaceutical composition comprising one or more of the following and other agents selected from the group consisting of agents that treat or prevent a disease that benefits from fat thermogenesis, inhibit weight gain or reduce fat content in a subject, and pharmaceutically acceptable excipients:

(1)

R1-R10 are each independently selected from hydrogen, halogen, hydroxy and C1-C3 alkyl, R11 is pyrrole, pyrroline, phenyl substituted with one or more substituents selected from oxygen, C1-C3 alkyl, C1-C3 alkoxy, halogen, hydroxy,

(2) An agent that upregulates expression or activity of a calcium channel associated protein or subunit thereof,

(3) A calcium ion channel activator which is used for activating the calcium ion channel,

(4) Agents that up-regulate the expression or activity of calcineurin,

(5) An agent that upregulates expression or activity of NFAT.

In one or more embodiments, the compound is as described in the first aspect herein.

In one or more embodiments, the additional agent that treats or prevents a disease that benefits from thermogenesis of fat, inhibits weight gain in the subject, or reduces fat content in the subject is an agent that reduces fat absorption and/or inhibits appetite.

In one or more embodiments, the additional agent that treats or prevents a disease that benefits from thermogenesis of fat, inhibits weight gain in a subject, or reduces fat content in a subject is selected from the group consisting of centrally acting appetite suppressants, appetite regulating hormones, lipase inhibitors, 5-HT2C receptor agonists, ghrelin antagonists, MCHR1 antagonists, SGLT2 inhibitors, agents that reduce fat synthesis, agents that promote fat hydrolysis, enzymes on the lipid metabolism pathway.

In one or more embodiments, the additional agent that treats or prevents a disease that benefits from thermogenesis of fat, inhibits weight gain in a subject, or reduces fat content in a subject is selected from bupropion, naltrexone, zonisamide, atoxetine, meltreprostine, TM-30339, exenatide, liraglutide, albiglutide, cetilistat, pramlintide, alkoxyphenoxythiazoles.

The present invention also provides a method of increasing UCP1 expression or activity in mammalian tissue or cells in vitro, or enhancing thermogenesis in mammalian tissue or cells, comprising one or more steps selected from the group consisting of:

(1) Culturing the tissue or cell in the presence of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,

(2) Up-regulating the expression or activity of a calcium channel associated protein or subunit thereof of said tissue or cell,

(3) Culturing the tissue or cell in the presence of a calcium channel activator,

(4) Up-regulating the expression or activity of calcineurin in the tissue or cell,

(5) Up-regulate the expression or activity of NFAT in the tissue or cell.

In one or more embodiments, the tissue is adipose tissue. In one or more embodiments, the adipocytes are brown adipose tissue and/or white adipose tissue.

In one or more embodiments, step (2) comprises transferring an expression vector of a calcium ion channel-associated protein or subunit thereof into the tissue or cell or culturing the tissue or cell in the presence of an agonist of a calcium ion channel-associated protein or subunit thereof.

In one or more embodiments, step (3) comprises transferring an expression vector for calcineurin into the tissue or cell or culturing the tissue or cell in the presence of an agonist of calcineurin.

In one or more embodiments, step (4) comprises transferring an expression vector of NFAT into the tissue or cell or culturing the tissue or cell in the presence of an agonist of NFAT.

In one or more embodiments, the method is non-therapeutic or non-diagnostic.

The present invention also provides a method for screening for a compound that enhances tissue or cell thermogenesis in a mammal, treats or prevents a disease that benefits from fat thermogenesis, inhibits weight gain in a subject, or reduces fat content in a subject, comprising the steps of:

(1) Incubating a tissue or cell in the presence of the compound,

(2) Detecting UCP1 expression or activity of the tissue or the cell,

wherein upregulation of UCP1 expression or activity indicates that said compound is a compound that enhances thermogenesis in said tissue or cell, treats or prevents a disease that would benefit from thermogenesis of fat, inhibits weight gain in a subject, or reduces fat content in a subject.

In one or more embodiments, the detection is detecting the amount or activity of mRNA or protein of UCP1 in a tissue or cell.

In one or more embodiments, methods for detecting the mRNA or protein content of UCP1 include, but are not limited to, southern, RT-PCR, western; methods for detecting the activity of the mRNA or protein content of UCP1 include, but are not limited to, cold stimulation experiments, hippocampal experiments.

Drawings

Fig. 1: ucp1-GFP mouse construction strategy.

Fig. 2: PCR results of Ucp-GFP mice were constructed.

Fig. 3: ucp1-GFP adipocyte construction strategy.

Fig. 4: glibenclamide can promote UCP1 expression of brown adipocytes in vitro.

Fig. 5: glimepiride can promote UCP1 expression of brown adipocytes in vitro.

Fig. 6: glibenclamide can promote the expression of UCP1 in white adipocytes in vitro.

Fig. 7: glibenclamide can inhibit weight gain of mice under high-fat feeding condition.

Fig. 8: the decrease in body weight of glibenclamide-treated high-fat diet mice was due to the decrease in fat content.

Fig. 9: the triglyceride content in the blood of glibenclamide-treated mice on a high-fat diet was significantly reduced.

Fig. 10: the basal metabolism of glibenclamide-treated mice on high-fat diet was increased.

Fig. 11: on a high fat diet, the three adipose tissue weights were significantly reduced in glibenclamide-treated mice (upper left, lower left) and the three adipose tissue cell sizes were significantly smaller (right panel) compared to the control group.

Fig. 12: on a high-fat diet, the glibenclamide-treated mice showed significantly up-regulated UCP1 protein expression in brown adipose tissue, compared to the control group.

Fig. 13: the glibenclamide treated group showed significantly up-regulated UCP1 protein expression in brown adipose tissue in mice compared to the control group (solvent treatment), similar to the positive control CL-316243 treated group (left panel). Hematoxylin-eosin (HE) staining also showed that the cell size of glibenclamide treated mice was significantly smaller, multiple lipid droplets appeared within a single cell, and UCP1 staining was increased (right panel).

Fig. 14: compared to the control group (solvent treatment), glibenclamide treated mice were better resistant to cold (4 ℃) (right panel), and mice had significantly increased oxidative phosphorylation-related proteins (left panel).

Fig. 15: glibenclamide up-regulates lipid metabolism related genes in adipocytes, and promotes lipofuscation of adipocytes.

Fig. 16: glibenclamide significantly upregulates pATF2 in brown adipocytes, while downregulating pSTAT3 and pERK, compared to solvent (DMSO). It is suggested that glibenclamide may regulate UCP1 expression by activating ATF2 and inhibiting STAT3 and ERK signaling pathways.

Fig. 17: sgrnas were designed for exon 2 of kir6.1, exon 1 of kir6.2, exon 2 of Sur1 and exon 4 of Sur2 (upper left, lower left) and were assayed for activity in brown adipocytes, respectively. The results showed that all 4 sgrnas had higher cleavage activity (upper right, lower right).

Fig. 18: knocking out Kir and Sur, ucp1 in brown adipocytes, respectively, showed no difference in mRNA and protein expression, and glibenclamide treatment in Kir and Sur knocked out cells showed no difference in Ucp1 expression at mRNA and protein levels. Suggesting that the upregulation of UCP1 by glibenclamide may not depend on ATP-sensitive potassium channels (K) _ATP )。

Fig. 19: only glibenclamide significantly promoted the expression of brown adipocytes Ucp1 at mRNA and protein levels.

Fig. 20: only glibenclamide can promote the expression of white adipocyte UCP1 protein.

Fig. 21: sgrnas were designed for exon 2 of Epac2 and exon 2 of Cav1 (left one, right two), and the activity of each sgRNA in brown adipocytes was examined, respectively. The results showed that all 2 sgrnas had cleavage activity (left two, right one).

Fig. 22: knockout of Epac2, ucp1 in brown adipocytes showed no difference in mRNA and protein levels, and treatment with glibenclamide in Epac2 knockout cells showed no difference in Ucp1 expression at mRNA and protein levels. There was no difference in the expression of UCP1 protein by knocking out Cav1 in brown adipocytes, and there was no difference in the expression of UCP1 protein by treatment with glibenclamide in Cav1 knocked out cells. The above results indicate that glibenclamide upregulation of UCP1 is independent of Epac2 and Cav1.

Fig. 23: inhibition of Ca ²⁺ Entering into the inhibitor can inhibit the upward regulation of UCP1 by glibenclamide.

Fig. 24: glibenclamide upregulates UCP1 independent of Trpv2.

Fig. 25: in brown adipocytes, both nitrendipine and cyclosporin a can significantly inhibit the upregulation of Ucp1 at mRNA and protein levels by glibenclamide (left one, left two), and VIVIT also significantly inhibits the upregulation of Ucp1 at mRNA levels by glibenclamide (right one). Description in brown adipocytes, ca inhibition ²⁺ The Calcineurin-NFAT signal pathway can inhibit the upregulation of UCP1 by glibenclamide.

Fig. 26: both nitrendipine and cyclosporin a can significantly inhibit the upregulation of UCP1 protein by glibenclamide or rosiglitazone in white adipocytes.

Fig. 27: mechanism of glyburide to regulate UCP1 expression.

Detailed Description

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.

The inventors have found that the compound represented by the formula (I) or a pharmaceutically acceptable salt or solvate thereof can be obtained by reacting Ca with ²⁺ The Calcineurin-NFAT signal pathway up-regulates UCP1, thereby promoting thermogenesis of adipocytes, achieving the effect of treating or preventing obesity and metabolic diseases.

Herein, UCP1 is up-regulated by at least 20%, at least 50%, at least 100%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 45-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 85-fold, at least 9-fold, at least 95-fold, at least 10-fold, at least 15-fold, at least 20-fold.

The tissues and cells described herein may be any tissue and cells of a mammal, preferably adipose tissue and cells. Adipose tissue and cells include brown adipose tissue and cells and white adipose tissue and cells. The cells may be isolated cells of an in vitro mature cell line or animal or in vivo cells. Exemplary adipocytes are white or brown adipocytes obtained from mice. Methods for obtaining white or brown adipocytes from living bodies are well known in the art. Brown and white adipocytes have thermogenic effects and can cope with obesity and metabolic diseases. The compounds or agents described herein are therefore useful for treating or preventing a disease that benefits from thermogenesis of fat, inhibiting weight gain in a subject, or reducing fat content in a subject. Herein, diseases benefiting from fat thermogenesis include diseases benefiting from brown or white fat thermogenesis, including obesity and/or metabolic diseases. Metabolic disorders are metabolic disorders and/or profuse metabolism, including type 2 diabetes, diabetic ketoacidosis, fatty liver, hyperuricemia, hypertonic syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin a deficiency, scurvy, vitamin D deficiency, osteoporosis, hyperlipidemia, metabolic encephalopathy, hepatic encephalopathy, congenital metabolic disorders.

Accordingly, the present invention provides use of a compound represented by the following formula (I) or a pharmaceutically acceptable salt or solvate thereof for increasing UCP1 expression or activity in a cell, or for increasing thermogenesis in a cell, or for preparing an agent for treating or preventing a disease benefiting from thermogenesis of fat, inhibiting weight gain in a subject, or reducing fat content in a subject,

wherein R1-R10 are each independently selected from hydrogen, halogen, hydroxy and C1-C3 alkyl, R11 is pyrrole, pyrroline, phenyl substituted with one or more substituents selected from oxygen, C1-C3 alkyl, C1-C3 alkoxy, halogen, hydroxy.

In one or more embodiments, the compound is a compound of formula (II) below

In one or more embodiments, R1-R4 are each independently selected from hydrogen, halogen, hydroxy, and C1-C3 alkyl, R5-R10 are hydrogen; alternatively, R3-R6 are each independently selected from hydrogen, halogen, hydroxy and C1-C3 alkyl, R1-R2, R7-R10 are hydrogen; alternatively, R1-R4 are each independently selected from F and Cl, and R5-R10 are hydrogen; alternatively, R1-R2 are each independently selected from F and Cl, and R3-R10 are hydrogen; alternatively, R3-R4 are each independently selected from F and Cl, and R1-R2 and R5-R10 are hydrogen; alternatively, R1-R10 are hydrogen. In the case where R1 to R10 are hydrogen, the compound represented by the formula (II) is glibenclamide.

In one or more embodiments, the compound is a compound of formula (III) below

Wherein R1-R10 are each independently selected from hydrogen and C1-C3 alkyl.

In one or more embodiments, R1-R10 are each independently selected from hydrogen and methyl. In the case where R1 to R3 and R5 to R10 are hydrogen and R4 is methyl, the compound represented by the formula (III) is glimepiride.

Halogen as described herein includes, but is not limited to F, I, br. The compounds of formula (I) may have chemical modifications common in the art that do not affect the activity of the compounds to increase UCP1 protein expression. The invention also includes analogs or derivatives of glibenclamide, including but not limited to: glimepiride, gliquidone, tolbutamide, gliclazide.

As used herein, the term "alkyl" alone or in combination with other terms refers to a saturated aliphatic alkyl group, including straight or branched chain alkyl groups of 1 to 20 carbon atoms. Preferably, alkyl refers to medium alkyl groups containing 1 to 10 carbon atoms such as methyl, ethyl, propyl, 2-isopropyl, n-butyl, isobutyl, t-butyl, pentyl and the like. More preferably, it means a lower alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, 2-isopropyl, n-butyl, isobutyl, tert-butyl and the like. The alkyl group may be substituted or unsubstituted. When substituted, the number of substituents is 1 or more, preferably 1 to 3, more preferably 1 or 2, and the substituents are independently selected from the group consisting of halogen, hydroxy, lower alkoxy, aryl.

As used herein, the term "aryl", alone or in combination with other terms, refers to an aromatic ring group containing 6 to 14 carbon atoms (e.g., six-membered monocyclic, ten-membered bicyclic, or fourteen-membered tricyclic ring systems), and exemplary aryl groups include phenyl, naphthyl, biphenyl, indenyl, and anthracenyl. As used herein, "phenylene" refers to a phenyl group having two substituents. Aryl is optionally substituted with one or more substituents independently selected from halogen, alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amino, mono-or di-alkylamino, carboxy or N-sulfonamide. In particular, phenyl or phenylene may be optionally substituted with 1 to 4 groups independently selected from C1-C3 alkyl, halogen and hydroxy.

As used herein, the term "alkoxy" refers to-O- (unsubstituted alkyl) and-O- (unsubstituted cycloalkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexyloxy, and the like.

As used herein, the term "substituted" means that a compound has a substituent comprising at least one carbon atom, nitrogen atom, oxygen atom or sulfur atom bearing one or more hydrogen atoms. If a substituent is described as being "substituted," it is meant that a non-hydrogen substituent occupies a hydrogen position on a carbon, nitrogen, oxygen, or sulfur. For example, a substituted alkyl substituent refers to a position in which at least one non-hydrogen substituent occupies a hydrogen on the alkyl group. By way of further illustration, monofluoroalkyl refers to an alkyl substituted with one fluorine and difluoroalkyl refers to an alkyl substituted with two fluorine.

The compounds disclosed herein, or pharmaceutically acceptable salts thereof, may include one or more asymmetric centers and thus may exist in enantiomers, diastereomers, and other stereoisomeric forms that may be defined, and may be classified as (R) -or (S) -or (D) -or (L) -for amino acids, depending on the stereochemistry. The present invention is intended to include all such possible isomers, as well as racemic and optically pure forms. Optically active (+) and (-), (R) -and (S) -or (D) -and (L) -isomers may be prepared by chiral synthons or chiral reagents, or by separation using conventional techniques such as high performance liquid phases using chiral columns. When a compound of the present invention contains an olefinic double bond or other geometric asymmetric center, it is intended that the compound include both E and Z geometric isomers unless otherwise specified. Likewise, all tautomers are also included.

The "pharmaceutically acceptable salts" as used herein include acid salts and basic salts.

By "pharmaceutically acceptable acid salt" is meant a salt that retains the biological activity and properties of the free base without undesirable biological activity or other changes. Such salts may be formed from inorganic acids such as, but not limited to, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like. Such salts may also be formed from organic acids such as, but not limited to, acetic acid, dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphorsulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclic benzoic acid, dodecylsulfonic acid, 1, 2-ethanedisulfonic acid, ethanesulfonic acid, isethionic acid, formic acid, fumaric acid (fiimaric acid), galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutarate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5-disulfonic acid, 2-naphthalenesulfonic acid, 1-naphthol-2-carboxylic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, glutamic acid, salicylic acid, 4-aminosalicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, pyruvic acid, and the like.

By "pharmaceutically acceptable basic salt" is meant a salt that retains the biological activity and properties of the free acid, without undesirable biological activity or other changes. These salts are prepared by adding an inorganic or organic base to the free acid. Salts obtained with inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts. Salts obtained by organic bases include, but are not limited to, primary, secondary, and tertiary ammonium salts, substituted amines including naturally substituted amines, cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dantol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benzamine, N' -dibenzylethylenediamine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, bradykinin, purine, piperazine, piperidine, N-ethylpiperidine, polyamide resins, and the like. Preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

Crystallization typically yields solvated products of the disclosed compounds. As used herein, the term "solvate" refers to a polymer comprising one or more compound molecules of the present disclosure and one or more solvent molecules. The solvent may be water and the solvate may be a hydrate. Alternatively, the solvent may also be an organic solvent. Thus, the compounds of the present disclosure may exist as hydrates, including mono-, di-, hemi-, sesqui-, tri-, tetra-and similar structures, as well as the corresponding solvated products. The compounds disclosed herein may be true solvates, while in other cases, the compounds disclosed herein may also be those that retain only a portion of the water, or a mixture of water and some solvent.

As described above, the inventors have discovered that compounds of formula (I) can up-regulate UCP1 via the Ca2+ -Calcineurin-NFAT signaling pathway, thereby promoting adipocyte thermogenesis. Thus, the present invention also provides for the use of an agent that upregulates expression or activity of a calcium channel associated protein or subunit thereof, or a calcium channel activator, an agent that upregulates expression or activity of calcineurin, and/or an agent that upregulates expression or activity of NFAT, in increasing UCP1 expression or activity of a cell, or in increasing thermogenesis of a cell, or in the manufacture of an agent that treats or prevents a disease that benefits from thermogenesis of fat, inhibits weight gain in a subject, or reduces fat content in a subject.

Typically, the agent that upregulates expression of a polypeptide or protein is an expression vector for said polypeptide or protein. For example, expression vectors suitable for expressing a calcium ion channel-associated protein or subunit thereof, calcineurin, and/or NFAT in a host cell (e.g., an adipocyte) may be constructed using techniques conventional in the art and transferred into the host cell by conventional methods such that the expression vector expresses the molecule in the host cell, thereby effecting upregulation of its expression. The expression vectors contain other components necessary for expression of these genes in the host cell and those of skill in the art will recognize these components. In certain embodiments, expression of genes upstream of these molecules may be regulated, thereby increasing expression of such molecules. For example, in certain embodiments, a viral vector (e.g., a lentiviral vector) that expresses a calcium ion channel associated protein or subunit thereof, calcineurin, and/or NFAT may be administered to a subject cell, thereby increasing the level of expression of the gene in the subject cell. Based on the disclosed gene or amino acid sequence on NCBI, one skilled in the art can obtain expression vectors comprising the coding sequences for calcium channel-related proteins or subunits thereof, calcineurin and/or NFAT. The UCP1 expression of the cells which overexpress the protein is up-regulated, and the heat production of the cells is increased. The coding sequences for calcium channel-related proteins or subunits thereof, calcineurin and/or NFAT are known to the person skilled in the art. Illustratively, NFAT comprises NFAT1, NFAT2, NFAT3, and NFAT4, wherein NFAT1 is as shown in NCBI gene accession number 18019; NFAT2 is shown in NCBI gene accession number 18018; NFAT3 is shown in NCBI gene accession number 73181; NFAT4 is shown as NCBI gene accession number 18021.

Typically, an agent that upregulates the activity of a polypeptide or protein is an agonist of the polypeptide or protein. Agonists of the calcium channel-associated proteins or subunits thereof, calcineurin, and/or NFAT to which the present invention relates are well known in the art. In addition, calcium channel activators may also be used in the methods of the invention to achieve upregulation of UCP1 expression and increased cellular thermogenesis. Calcium ion channel activators include, but are not limited to: nifedipine, amlodipine, and lacidipine.

In another aspect, the invention provides a pharmaceutical composition comprising a compound or agent described herein and a pharmaceutically acceptable adjuvant and optionally other agents that treat or prevent a disease that benefits from thermogenesis of fat, inhibit weight gain or reduce fat content in a subject. Herein, "pharmaceutically acceptable excipients" refers to carriers, diluents and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, including, but not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes/colorants, odorants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers. In some embodiments, pharmaceutically acceptable excipients may include one or more inactive ingredients, including but not limited to: stabilizers, preservatives, additives, adjuvants, sprays, compressed air or other suitable gases, or other suitable inactive ingredients for use with the pharmaceutically effective compounds. More specifically, suitable pharmaceutically acceptable excipients may be those commonly used in the art for administration of small molecule drugs or nucleic acids.

Typically, the pharmaceutical compositions comprise a therapeutically effective amount of a compound or agent described herein. A therapeutically effective amount refers to a dose that achieves treatment, prevention, alleviation and/or alleviation of a disease or a disorder in a subject. The therapeutically effective amount may be determined by factors such as the age, sex, severity of the condition, other physical condition of the patient, and the like. The therapeutically effective amount may be administered as a single dose or may be administered in multiple doses depending on the effective treatment regimen. Herein, a subject or patient refers generally to a mammal, particularly a human.

The compounds or agents of the invention may be administered alone or as pharmaceutical compositions. The compounds or agents of the invention may be administered in a manner suitable for the treatment (or prevention) of a disease. The number and frequency of administration will be determined by various factors, such as the condition of the patient, and the type and severity of the patient's disease. Administration of the composition may be carried out in any convenient manner, including by injection, infusion, implantation or implantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous injection or intraperitoneally.

The compounds or agents described herein may be used in combination with other agents for treating or preventing a disease that benefits from thermogenesis of fat, inhibiting weight gain in a subject, or reducing fat content in a subject. Other agents that treat or prevent diseases that benefit from heat production of fat, inhibit weight gain in a subject, or reduce fat content in a subject include agents that reduce fat absorption and/or agents that inhibit appetite, e.g., centrally-located appetite suppressants, appetite regulating hormones, lipase inhibitors, 5-HT2C receptor agonists, ghrelin antagonists, MCHR1 antagonists, SGLT2 inhibitors, agents that reduce fat synthesis, agents that promote fat hydrolysis, enzymes on the lipid metabolic pathway. Herein, other agents for treating or preventing a disease that benefits from thermogenesis of fat, inhibiting weight gain in a subject, or reducing fat content in a subject may be selected from bupropion, naltrexone, zonisamide, atosibiritine, meltretine, TM-30339, exenatide, liraglutide, albiglutide, cetilistat, pramlintide, alkoxyphenoxythiazoles. The dosage of other agents to be administered can be determined by one skilled in the art.

The present invention also includes a method of increasing UCP1 expression or activity in a mammalian tissue or cell, or enhancing thermogenesis in a mammalian tissue or cell, comprising one or more steps selected from the group consisting of: (1) culturing the tissue or cell in the presence of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, (2) up-regulating the expression or activity of a calcium ion channel-associated protein or subunit thereof of the tissue or cell or culturing the tissue or cell in the presence of a calcium ion channel activator, (3) up-regulating the expression or activity of calcineurin of the tissue or cell, (4) up-regulating the expression or activity of NFAT of the tissue or cell. The compounds or reagents that accomplish these steps are described above.

The invention also relates to methods of treating or preventing a disease that benefits from fat thermogenesis, inhibiting weight gain or reducing fat content in a subject. The method comprises administering to a subject in need thereof a therapeutically effective amount of a compound, agent or pharmaceutical composition described herein. The invention also relates to a kit for treating or preventing a disease benefiting from fat-producing fever, inhibiting weight gain in a subject, or reducing fat content in a subject, the kit comprising a compound, agent, or pharmaceutical composition described herein. The invention also relates to the use of a compound, agent or pharmaceutical composition as described herein for the manufacture of a kit for treating or preventing a disease benefiting from fat thermogenesis, inhibiting weight gain or reducing fat content in a subject. In particular, an "effective amount" refers to an amount that is therapeutically functional to a human or animal and acceptable to the animal and human. For example, in a combination of tablets, the content of the compound of formula (1) may be 10mg, 20mg, 50mg, 100mg, 200mg or more.

The mode of administration of the drug of the present invention may include, but is not limited to, subcutaneous injection, transdermal injection, implantation, topical administration, intramuscular injection, sustained release administration, oral administration, and the like. Those skilled in the art are aware of other agents required to administer a drug to a subject in different modes of administration, dosages, sites of administration, etc. Such as dressings, solvents (e.g., water), and the like.

The present invention is described in further detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as being limited to the following examples, but rather should be construed to include any and all variations that become apparent from the teachings provided herein. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.

The present invention also provides a method of screening for a compound that enhances heat production by a tissue or cell (e.g., adipose tissue or cell) of a mammal, comprising the steps of: (1) Incubating a tissue or cell in the presence of said compound, (2) detecting UCP1 expression or activity of the tissue or cell, wherein upregulation of UCP1 expression or activity indicates that said compound is a compound that enhances heat production by said tissue or cell. The assay is to detect the amount or activity of mRNA or protein of UCP1 in a tissue or cell. Methods for detecting the mRNA or protein content of UCP1 include, but are not limited to, southern, RT-PCR, western. The method for detecting the activity of the content of mRNA or protein of UCP1 includes: cold stimulation experiments, hippocampal experiments, and the like. The person skilled in the art knows the reagents necessary to carry out the above-described method.

Examples

Example 1, mouse and cell construction

Construction of Ucp1-GFP mice

The experimental steps are as follows: the knock-in mice with the Ucp1 gene inserted into the P2A-GFP were obtained by CRISPR-Cas9 gene editing. The design strategy is as follows: GFP gene was inserted into the C-terminal of Ucp gene, ucp gene was constructed, P2A-EGFP-inserted transgenic mice were constructed (FIG. 1), and PCR identification was performed on the obtained transgenic mice (FIG. 2). A single-stranded guide RNA (sgRNA) site is designed aiming at the No. 6 exon of Ucp, and the sgRNA sequence is shown as SEQ ID NO. 1. PCR amplified 5' homology arms using the mouse genome as template and primers Ucp1-5 arm-F (SEQ ID NO: 2), ucp1-5 arm-R (SEQ ID NO: 3); PCR amplified 3' homology arms using primers Ucp1-3 arm-F (SEQ ID NO: 4), ucp1-3 arm-R (SEQ ID NO: 5); the fragment 2A-GFP fragment was amplified using the primer 2A-GFP-F (SEQ ID NO: 6), 2A-GFP-R (SEQ ID NO: 7). The P2A-GFP knock-in vector (template plasmid) was obtained by the method of cleavage ligation. And obtaining the offspring mice through fertilized egg male prokaryotic injection and transplantation. Ucp1-GFP positive mice were obtained by identification (Ucp 1-genotype-5 arm-F (SEQ ID NO: 8), ucp1-genotype-5 arm-R (SEQ ID NO: 9), ucp1-genotype-3 arm-F (SEQ ID NO: 10), ucp1-genotype-3 arm-R (SEQ ID NO: 11)).

Experimental results: the PCR results for the insert showed (FIG. 2) that the genome of the transgenic mice we constructed successfully amplified the band, whereas the wild-type mice did not, indicating successful P2A-EGFP insertion and successful construction of the transgenic mice.

Construction of Ucp1-GFP adipocytes

The experimental steps are as follows: taking brown adipocytes as an example, brown lipid vascular matrix fraction SVF (Stromal Vascular Fraction) was isolated from postnatal 2-day male mice, and the resulting tissue was digested with the separate solutions (123 mM sodium chloride, 5mM potassium chloride, 1.3mM calcium chloride, 5mM glucose, 50mM HEPES,4%BSA,1.5mg/mL collagenase A,2mg/mL neutral protease II). The digested tissue cells were filtered through a 70 μm filter plug, the resulting cells were cultured in primary culture medium (high sugar DMEM containing 20% fbs), and retrovirus expressing T antigen was transfected, and subjected to G418 screening to obtain monoclonal cells, which were then stored in liquid nitrogen as a cell expansion culture medium. In the same manner, we obtained Ucp1-GFP white adipocyte strain (FIG. 3).

Example 2 in vitro validation

1) Glibenclamide and glimepiride can promote UCP1 expression of brown adipocytes in vitro

The experimental steps are as follows: glibenclamide (10. Mu.M in DMSO) or glimepiride (10. Mu.M in DMSO) was added to the constructed Ucp-GFP brown adipocytes during in vitro differentiation, with solvent DMSO as a control. Glibenclamide was added 7 days after cell differentiation, and cells were collected 1 day after the addition. Total RNA of the cells was extracted by Trizol (ThermoFisher), and was inverted into cDNA, and the expression of the marker gene Ucp and the gene involved in brown lipid differentiation was detected by qPCR. Meanwhile, the total protein of the cells is extracted by RIPA lysate (Millipore), and the UCP1 protein expression is detected by western blot. The primers for the qPCR described above were as follows: (5'-3')

Ucp1-F:GCATTCAGAGGCAAATCAGC(SEQ ID NO:12)；

Ucp1-R:GCCACACCTCCAGTCATTAAG(SEQ ID NO:13)；

Cidea-F:GAATAGCCAGAGTCACCTTCG(SEQ ID NO:14)；

Cidea-R:AGCAGATTCCTTAACACGGC(SEQ ID NO:15)；

Prdm16-F:CCGACTTTGGATGGGAGATG(SEQ ID NO:16)；

Prdm16-R:CACGGATGTACTTGAGCCAG(SEQ ID NO:17)；

Pgc1a-F:TCACGTTCAAGGTCACCCTA(SEQ ID NO:18)；

Pgc1a-R:TCTCTCTCTGTTTGGCCCTT(SEQ ID NO:19)；

Ppara-F:CATTTCCCTGTTTGTGGCTG(SEQ ID NO:20)；

Ppara-R:ATCTGGATGGTTGCTCTGC(SEQ ID NO:21)。

The experimental results are shown in fig. 4 and 5. Fig. 4 shows that glibenclamide treatment can significantly up-regulate the expression of marker gene Ucp at mRNA and protein levels during brown adipocyte differentiation in vitro, while other genes associated with brown lipid differentiation (Cidea, pgc1 a, ppra, prdm 16) are also significantly up-regulated. FIG. 5 shows that glimepiride treatment can significantly up-regulate marker genes during in vitro differentiation of brown adipocytesUcp1Expression at the mRNA and protein levels.

2) Glibenclamide can promote UCP1 expression of white adipocytes in vitro

The experimental steps are as follows: glibenclamide (10. Mu.M in DMSO) was added to the constructed Ucp-GFP white adipocytes during in vitro differentiation, with solvent DMSO as a control. Glibenclamide was added 6 days after cell differentiation, and cells were collected 4 days after the addition. RNA was extracted and inverted to cDNA, and expression of marker gene Ucp1 and brown lipid differentiation-related genes was detected by qPCR. Simultaneously extracting protein, and detecting the expression of UCP1 protein by western blot.

The experimental results are shown in FIG. 6. Figure 6 shows that glibenclamide treatment can significantly up-regulate the expression of marker gene Ucp at mRNA and protein level during white adipocyte differentiation in vitro, while other genes associated with brown lipid differentiation (Cidea, pgc1 a) are also significantly up-regulated.

Example 3 in vivo verification

1) Glibenclamide can inhibit weight gain of mice under high-fat feeding condition

The experimental steps are as follows: under high fat (Research Diets,60kcal% fat) diet, 2mg/kg of glibenclamide per day was administered by gavage to 8 week old C57BL/6J male mice (Schlemk), and the control group was given an equal volume of solvent (4.5 wt% DMSO,1wt% Tween, water) per day. Food intake and body weight were counted weekly. Each group had 12 mice.

The experimental results are shown in FIG. 7. Figure 7 shows that glibenclamide-treated mice significantly lose weight under high fat diet compared to control. The mice lost weight significantly after 1 week of dosing, and the weight gain was more and more variable over time (left panel). Glibenclamide-treated mice lost 9% of their body weight after 11 weeks of dosing compared to control mice (left and middle panels). While there was no difference in feeding between the two groups of mice (right panel).

2) The reduction in body weight of glibenclamide-treated high-fat diet mice was due to the reduction in fat content

The experimental steps are as follows: on a high-fat diet, 2mg/kg of glibenclamide per day was administered to mice by gavage, and after 8 weeks, the body fat and muscle content of the mice were examined by nuclear magnetic resonance apparatus NMR (Echo MRI). Each group had 12 mice.

The experimental results are shown in FIG. 8. Figure 8 shows that glibenclamide treated mice had 29.52% less body fat content under high fat diet compared to control group, without significant difference in muscle content. Indicating that the decrease in body weight of the administered mice was due to the decrease in fat content.

3) Triglyceride content in blood of glibenclamide-treated mice on high-fat diet is significantly reduced

The experimental steps are as follows: on a high fat diet, 2mg/kg of glibenclamide per day was administered to mice by gavage, and after 11 weeks, the mice were starved for 4 hours, blood was taken through tail veins of the mice, blood samples were placed on ice, and centrifuged at 3000rpm for 15 minutes at 4 ℃, and supernatants were taken. Triglyceride and cholesterol levels in the blood of mice were measured using the triglyceride and cholesterol measurement kit (Shanghai Shen Suoyou Fu Co.). Each group had 12 mice.

The experimental results are shown in FIG. 9. Figure 9 shows that the triglyceride content in the blood of glibenclamide-treated mice was reduced by 35.92% compared to the control group under high-fat diet conditions (left panel), but the cholesterol content in the blood was not significantly changed (right panel).

4) Elevated basal metabolism in glibenclamide-treated mice on high-fat diet

The experimental steps are as follows: on a high-fat diet, 2mg/kg of glibenclamide per day was administered to mice by gavage, and after 11 weeks, the oxygen consumption, carbon dioxide excretion and exercise amount of the mice were measured by a metabolic cage (Columbus Instruments). 8 mice per group.

The experimental results are shown in FIG. 10. Fig. 10 shows that, under high fat diet conditions, both oxygen consumption and carbon dioxide excretion from glibenclamide-treated mice were significantly increased (upper left, lower left) and both day and night oxygen consumption and carbon dioxide excretion were significantly increased (upper right ). While mice had no difference in activity (lower right).

5) Reduced lipid droplet accumulation in adipose tissue of glibenclamide-treated mice on high-fat diet and increased expression of brown fat UCP1

The experimental steps are as follows: on a high fat diet, 2mg/kg of glibenclamide per day was administered to mice by gavage, and after 11 weeks, brown adipose tissue, subcutaneous white adipose tissue, and epididymal white adipose tissue of the mice were taken and weighed, respectively. Fresh three adipose tissues were simultaneously fixed with 4% pfa (paraformaldehyde, national pharmaceutical chemicals limited) overnight and stained with hematoxylin-eosin. And extracting the protein of brown adipose tissue of the mice, and detecting the expression of UCP1 protein by western blot.

The experimental results are shown in fig. 11 and 12. Figure 11 shows that three adipose tissue weights were significantly reduced (upper left, lower left) in glibenclamide-treated mice and three adipose tissue cell sizes were significantly smaller (right panel) in high-fat diet compared to control group. Fig. 12 shows that glibenclamide treated mice significantly up-regulate UCP1 protein expression in brown adipose tissue under high fat diet compared to control group.

6) Alternarin promotes mouse subcutaneous white fat UCP1 expression by in situ injection administration

The experimental steps are as follows: glibenclamide (1 mg/kg) was administered by injection in situ at the subcutaneous inguinal fat of 7-8 week old male mice. Each mouse was injected once in the bilateral groin, 50. Mu.L each, with solvent and 1mg/kg CL-316243 (Tocres) as negative and positive controls, respectively. Mice were kept under conventional (rat breeder feed, streikang) diet. After 4 days, cold tolerance experiments were performed and subcutaneous white adipose tissue was taken and fresh adipose tissue was simultaneously stained with hematoxylin-eosin (HE) and UCP 1. Meanwhile, tissue protein is extracted, and UCP1 and oxidative phosphorylation related protein expression are detected.

The experimental results are shown in fig. 13 and 14. Fig. 13 shows that the glibenclamide treated group significantly up-regulated the expression of UCP1 protein in brown adipose tissue of mice compared to the negative control group, similar to the positive control CL-316243 treated group (left panel). Hematoxylin-eosin (HE) staining also showed that the cell size of glibenclamide treated mice was significantly smaller, multiple lipid droplets appeared within a single cell, and UCP1 staining was increased (right panel). FIG. 14 shows that the glibenclamide treated mice are better tolerant to cold (4 ℃) than the negative control group (right panel), and that the oxidative phosphorylation related proteins of the mice are significantly increased (left panel).

Example 4 mechanism of action of glibenclamide

1) Glibenclamide up-regulates lipid metabolism related genes in adipocytes, and promotes browning of adipocytes

The experimental steps are as follows: to know the mechanism of action of glibenclamide in upregulating UCP1 in brown and white adipocytes, we first performed RNAseq sequencing analysis on glibenclamide or DMSO-treated brown and white adipocytes. Wherein brown adipocytes were treated with 10 μm glibenclamide or DMSO 7 days after cell differentiation, and harvested after 1 day of addition; white adipocytes were treated with 10. Mu.M glibenclamide or DMSO 6 days after cell differentiation, and harvested 4 days after addition. Then total RNA of the cells is extracted, and then the extracted RNA is subjected to RNAseq sequencing analysis (Shanghai Meiji pharmaceutical technologies Co., ltd.). GO analysis was performed on genes enriched in glibenclamide-treated brown adipocytes or white adipocytes.

The experimental results are shown in FIG. 15. Figure 15 shows that in brown adipocytes, the glibenclamide up-regulated genes are significantly enriched in lipid metabolism-related pathways, non-shivering thermogenesis, steroid metabolism, adipocyte differentiation and redox reactions (upper left) compared to control (DMSO). In white adipocytes, the glibenclamide up-regulated genes are significantly enriched in lipid metabolism-related pathways, fatty acid catabolism, brown adipocyte differentiation, insulin response, and redox reaction (upper right) compared to controls (DMSO). There are 605 genes up-regulated by glibenclamide in brown adipocytes, 504 genes up-regulated by glibenclamide in white adipocytes (lower left), and 100 genes co-up-regulated in both cells, and these co-regulated genes are partially enriched in lipid metabolism regulation pathways (lower right). These results suggest that glibenclamide upregulates lipid metabolism-related genes in adipocytes, promoting adipocyte browning.

2) Glibenclamide up-regulates pATF2 while down-regulating pSTAT3 and pERK

The experimental steps are as follows: to know which signal pathways, specifically, were regulated by glibenclamide in brown adipocytes, we next analyzed proteins of glibenclamide or DMSO-treated brown adipocytes by western blot. Wherein brown adipocytes were treated with 10. Mu.M glibenclamide or DMSO 7 days after cell differentiation, and cells were collected 1 day after the addition. Extracting total protein of the cells, and detecting the expression of the corresponding signal protein.

The experimental results are shown in fig. 16. FIG. 16 shows that glibenclamide significantly upregulates pATF2 in brown adipocytes, while downregulating pSTAT3 and pERK, compared to solvent (DMSO). It is suggested that glibenclamide may regulate UCP1 expression by activating ATF2 and inhibiting STAT3 and ERK signaling pathways.

3) Glibenclamide upregulation of UCP1 independent of ATP-sensitive potassium channels (K) _ATP )

The experimental steps are as follows: to detect whether the target of glibenclamide up-regulation of UCP1 is ATP-sensitive potassium channel (K) _ATP ) We first put K in brown adipocytes _ATP The Kir and SUR subunits of the channel were knocked out. We designed and screened for high activity sgRNAs for Kir6.1, kir6.2, sur1 and Sur2 genes:

Kir6.1：CGAAGAGCAGCCAGCTGCAG(SEQ ID NO:22)；

Kir6.2：GAAGATGCAGCCCAGCATGA(SEQ ID NO:23)；

Sur1：CAGGACAAAGAGCAGCATGA(SEQ ID NO:24)；

Sur2：CAAACGTCAGAATCCATCTC(SEQ ID NO:25)。

Kir6.1-sgRNA and Kir6.2-sgRNA were then introduced simultaneously into brown adipocytes via lentiviral vectors, and Sur1-sgRNA and Sur2-sgRNA were introduced simultaneously into brown adipocyte precursors, and Kir (both Kir6.1 and Kir6.2) and Sur (both Sur1 and Sur 2) were knocked out in brown adipocyte precursors, respectively, using CRISPR-Cas9 technology. The knocked-out cells were then differentiated according to standard differentiation protocols, and Ucp1 expression was examined 8 days after cell differentiation. Cells knocked out of Kir or Sur were also tested for Ucp1 expression under stimulation of glibenclamide (added 7 days after cell differentiation, 1 day of treatment).

The experimental results are shown in fig. 17 and 18. FIG. 17 shows that sgRNAs (upper left first, upper left second, lower left first, lower left second) were designed against exon 2 of Kir6.1, exon 1 of Kir6.2, exon 2 of Sur1 and exon 4 of Sur2, and the activity of each sgRNA in brown adipocytes was examined, respectively. The results showed that all 4 sgrnas had higher cleavage activity (upper right, lower right). FIG. 18 shows that knocking out Kir and Sur, ucp1 in brown adipocytes, respectively, did not differ in mRNA and protein expression, and that there was no difference in mRNA and protein levels of Ucp1 in Kir and Sur knocked out cells treated with glibenclamide. Suggesting that the upregulation of UCP1 by glibenclamide may not depend on ATP-sensitive potassium channels (K) _ATP )。

4) The up-regulation of UCP1 by glibenclamide is independent of Epac2 and Cav1

The experimental steps are as follows: to further verify whether glibenclamide upregulates UCP1 through K _ATP Or Epac2, we selected the following 4 compounds for validation:

compounds of formula (I)	Known targets	Structural information
			Glibenclamide	K _ATP ，Epac2	With sulfonylurea core clusters
Gliclazide	K _ATP ，not Epac2	Having larger sulfonylurea core groups
			Repaglinide	K _ATP ，not Epac2	Sulfonylurea-free core groups
Nateglinide	K _ATP ，not Epac2	Sulfonylurea-free core groups
			Toluene sulfobutyl urea	K _ATP ，Epac2	Having sulfonylurea core groups

We first treated with these 4 compounds (added 7 days after cell differentiation at an action concentration of 10. Mu.M) during brown adipocyte differentiation for 1 day, and examined Ucp1 expression. Simultaneously, these 4 compounds (added after cell differentiation for 6 days and at an action concentration of 10 μm) were also added during the differentiation of white adipocytes for 4 days, and the expression of Ucp1 was examined. And simultaneously knocking out the Epac2 in brown fat cells, designing sgRNA aiming at an Epac2 gene, screening the sgRNA with high activity, then introducing the Epac2-sgRNA into brown fat precursor cells through a lentiviral vector, and knocking out the Epac2 in the brown fat precursor cells by using a CRISPR-Cas9 technology. The knocked-out cells were then differentiated according to standard differentiation protocols, and Ucp1 expression was examined 8 days after cell differentiation. Cells knocked out of Epac2 were also tested for Ucp1 expression under stimulation with glibenclamide (added 7 days after cell differentiation, 1 day of treatment). Furthermore, to examine whether glibenclamide upregulates UCP1 through Cav1, we knocked out Cav1 in brown adipocytes. We designed and screened for high activity sgrnas for Cav1 gene:

Epac2：

Cav1：

cav1-sgRNA was then introduced into brown fat precursor cells via lentiviral vectors, and Cavl was knocked out in the brown fat precursor cells using CRISPR-Cas9 technology. The knocked-out cells were then differentiated according to standard differentiation protocols, and Ucp1 expression was examined 8 days after cell differentiation. Cells knocked out of Cav1 were also examined for Ucp1 expression under stimulation of glibenclamide (added 7 days after cell differentiation, 1 day of treatment).

The experimental results are shown in fig. 19, 20, 21 and 22. Fig. 19 shows that only glibenclamide can significantly promote the expression of brown adipocytes Ucp1 at mRNA and protein levels. FIG. 20 shows that only glibenclamide can promote the expression of UCP1 protein in white adipocytes. FIG. 21 shows that sgRNAs were designed for exon 2 of Epac2 and exon 2 of Cav1 (left one, right two) and were tested for their activity in brown adipocytes, respectively. The results showed that all 2 sgrnas had cleavage activity (left two, right one). Fig. 22 shows that knockout of Epac2, ucp1 in brown adipocytes showed no difference in mRNA and protein levels, and that treatment with glibenclamide in Epac2 knockout cells showed no difference in Ucp1 expression at mRNA and protein levels. There was no difference in the expression of UCP1 protein by knocking out Cav1 in brown adipocytes, and there was no difference in the expression of UCP1 protein by treatment with glibenclamide in Cav1 knocked out cells. The above results indicate that glibenclamide upregulation of UCP1 is independent of Epac2 and Cav1.

5) Inhibition of Ca ²⁺ Entering into the preparation of the anti-up-regulating UCP1 of glibenclamide

The experimental steps are as follows: to examine whether glibenclamide up-regulates UCP1 from intracellular Ca ²⁺ Level regulation we first validated with the calcium channel inhibitor Nitrendipine (nitendipine) and the ATP-sensitive potassium channel activator Diazoxide (Diazoxide) in vitro differentiated brown adipocytes, respectively. The expression of Ucp was examined after 1 day by treatment with ganciclovir (30 μm) or diazoxide (10 μm) under glibenclamide stimulation 7 days after brown adipocyte differentiation.

The experimental results are shown in FIG. 23. Fig. 23 shows that only the calcium channel inhibitor nitrendipine can significantly inhibit glibenclamide-induced upregulation of UCP1 in brown adipocytes. Suggesting inhibition of Ca ²⁺ Entering into the inhibitor can inhibit the upward regulation of UCP1 by glibenclamide.

6) Glibenclamide up-regulation of UCP1 independent of Trpv2

The experimental steps are as follows: to examine whether the target of glibenclamide up-regulation of UCP1 is TRPV2 (a non-selective cation channel), we first knocked out TRPV2 in brown adipocytes. We designed and screened for high activity sgRNA against Trpv2 gene, then introduced Trpv2-sgRNA (5'-CCCCATGGAGTCTCCCTTCC-3', SEQ ID NO: 28) into brown adipocytes by lentiviral vector, and knocked out Trpv2 in brown adipocytes using CRISPR-Cas9 technology. The knocked-out cells were then differentiated according to standard differentiation protocols, and Ucp1 expression was examined 8 days after cell differentiation. While detecting Ucp1 expression by Trpv2 knocked-out cells stimulated with glibenclamide (10. Mu.M glibenclamide added 7 days after cell differentiation, 1 day of treatment).

The experimental results are shown in FIG. 24. FIG. 24 shows that there is no difference in expression of the UCP1 protein in brown adipocytes, and that there is no difference in expression of the UCP1 protein in cells from which Trpv2 was knocked out by treatment with glibenclamide (left one). The sgrnas were designed for exon 3 of Trpv2 (left two) and tested for activity in brown fat. The results showed that Trpv2-sgRNA had cleavage activity (right one). These results indicate that glibenclamide up-regulates UCP1 independent of Trpv2.

7) Glibenclamide up-regulates UCP1 through Ca ²⁺ -Calcineurin-NFAT signaling pathway

The experimental steps are as follows: to detect whether glibenclamide is likely to pass Ca ²⁺ The Calcineurin-NFAT signaling pathway regulates UCP1 expression. We have chosen and validated the inhibitors of Calcineurin (Calcineurin), cyclosporin a (CsA, cycloporine a) and NFAT, VIVIT (MCE), respectively. VIVIT is a cell permeable peptide inhibitor of NFAT that selectively inhibits calcineurin-mediated NFAT dephosphorylation. We first detected Ucp1 expression 7 days after brown adipocyte differentiation by treatment with cyclosporin a or VIVIT (10 μm) under glibenclamide stimulation. Meanwhile, after white adipocytes were differentiated for 6 days, cyclosporin A (10. Mu.M) was added for treatment under the stimulation of glibenclamide or rosiglitazone, and after 4 days, the expression of Ucp1 was examined.

The experimental results are shown in fig. 25 and 26. Fig. 26 shows that both nitrendipine and cyclosporin a significantly inhibited the upregulation of Ucp1 at mRNA and protein levels by glibenclamide (left one, left two) and that VIVIT also significantly inhibited the upregulation of Ucp1 at mRNA levels by glibenclamide (right one) in brown adipocytes. Description in brown adipocytes, ca inhibition ²⁺ The Calcineurin-NFAT signal pathway can inhibit the upregulation of UCP1 by glibenclamide. Fig. 27 shows that both nitrendipine and cyclosporin a can significantly inhibit the upregulation of UCP1 protein by glibenclamide or rosiglitazone in white adipocytes.

Taken together, our studies indicate that Ca inhibition by different inhibitors in brown fat and white fat cells ²⁺ The Calcineurin-NFAT signaling pathway prevents the upregulation of UCP1 by glibenclamide, suggesting that glibenclamide may pass through Ca ²⁺ The Calcineurin-NFAT signaling pathway regulates UCP1 expression (FIG. 27).

Sequence listing

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Claims

1. Use of an agent comprising glibenclamide in the manufacture of a medicament for the treatment or prevention of obesity that benefits from browning of adipocytes.

2. The use of claim 1, wherein the agent further comprises one or more selected from the group consisting of:

(1) Expression vectors or agonists for calcium channel-associated proteins or subunits thereof,

(2) A calcium ion channel activator which is used for activating the calcium ion channel,

(3) Expression vectors or agonists for calcineurin,

(4) Expression vectors or agonists of NFAT.

3. Non-therapeutic and non-diagnostic use of an agent comprising glibenclamide in promoting browning of adipocytes.

4. The use of claim 3, wherein the agent further comprises one or more selected from the group consisting of:

(3) Expression vectors or agonists for calcineurin,

(4) Expression vectors or agonists of NFAT.

5. The use according to claim 2 or 4, wherein the calcium channel activator is selected from nifedipine, amlodipine, and lacidipine.