WO2024005157A1 - Adipocyte activator - Google Patents

Abstract

The purpose of the present invention is to provide: a novel adipocyte activator; and a use thereof. The present invention provides an adipocyte activator containing SerpinA1, a SerpinA1 expression promoter, or a SerpinA1 agonist, wherein the adipocyte is selected from among a brown preadipocyte, a brown adipocyte, and a beige adipocyte. An adipose tissue activator according to the present invention has an action that enhances a heat production action in adipose tissue, and can be used as a pharmaceutical composition for preventing and/or treating obesity and/or obesity-related diseases.

Description

fat cell activator

The present invention relates to an adipocyte activator and its uses.

Obesity and its accompanying metabolic syndrome and type 2 diabetes have become major problems in modern society. These are said to be major obstacles to extending healthy life expectancy, as they increase the risk of cardiovascular disease, kidney disease, and malignant tumors.

Mammalian adipose tissue is broadly classified into two types, white adipose tissue and brown adipose tissue, depending on its function and histological characteristics. At present, there are three types of adipocytes: white adipocytes present in white adipose tissue, brown adipocytes present in brown adipose tissue, and beige adipocytes that are scatteredly induced within white adipose tissue. classified as adipocytes. These three types of adipocytes are the same in that they contain neutral fat within their cells, but their localized sites and functions are significantly different.

White fat cells exist in various areas such as the subcutaneous tissue, visceral tissue, and intraperitoneal cavity, and store excess energy in the body as fat, and release it into the blood as free fatty acids when nutrition is needed, such as during times of starvation. On the other hand, when humans are young, brown fat cells exist abundantly between the shoulder blades and play a role in burning fat and producing heat, but as they grow, they decrease, and in adults, brown fat cells are present in abundance between the shoulder blades and play a role in burning fat and producing heat. It is present in the suprafossa, around the spine, and around the heart. Brown fat cells are abundant in newborns and hibernating animals and are thought to be important for maintaining body temperature. In this way, the physiological role of brown fat cells is completely opposite to that of white fat cells, and they consume and dissipate energy as heat when stimulated by sympathetic nerves. Brown adipocytes are characterized by having multilocular lipid droplets and abundant mitochondria, and UCP1 (uncoupling protein 1) present in the mitochondria plays a role in dissipating energy as heat.

Beige adipocytes are UCP1-positive adipocytes that exist scattered within white adipose tissue, especially subcutaneous adipose tissue. Brown adipocytes and beige adipocytes have similar morphological characteristics of being multivesicular and containing many mitochondria, and both have the function of thermogenic adipocytes, but their origins and localization are different. Brown adipocytes are derived from muscle progenitor cells that express Myogenic factor 5 (Myf5), which is common to skeletal muscle, whereas beige adipocytes, like white adipocytes, are derived from Myf5-negative preadipocytes. However, at present, it has not yet been determined whether beige adipocytes are formed from preadipocytes, through transdifferentiation from mature white adipocytes, or both. In this way, beige adipocytes are said to have characteristics similar to white adipocytes and brown adipocytes.

Brown adipose tissue was thought to exist only in youth and degenerate with age, but in recent years, the function of brown adipose tissue and beige adipose tissue has attracted attention even in adult humans, and their activity has been linked to obesity and insulin resistance. It has been shown that there is a negative correlation with Therefore, activation (browning) of fat cells is expected to have preventive and therapeutic effects on many lifestyle-related diseases and obesity, and is attracting attention (Non-Patent Document 1, Non-Patent Document 2). , Non-Patent Document 3, Patent Document 1).

α1-antitrypsin belongs to the Serpin superfamily, and its function as a protease inhibitor has been revealed from the protein structure. In humans, it is encoded by the SERPINA1 gene. The previously known function of α1-antitrypsin as an inhibitor of enzyme activity in the blood is thought to protect surrounding tissues from neutrophil elastase. When there is an insufficient amount of α1-antitrypsin in the blood or a functionally defective SERPNA1 (α1-antitrypsin deficiency), neutrophil elastase degrades elastin excessively, resulting in lung It is thought that this decrease in elasticity may cause respiratory complications of chronic obstructive pulmonary disease (COPD) in adults. Many things remain unknown, such as what kind of physiological activity this kind of protein degradation inhibition actually has and what kind of organs it targets.

WO2022/045074

An object of the present invention is to provide a new adipocyte activator and its uses.

The present inventor established IR/IGF1R-inducible double knockout mice that can induce organ-specific insulin receptor/IGF-1 receptor (IR/IGF1R) deficiency after growing into adults, and rapidly developed adipose tissue. It was found that the disappearance of . In these mice, we confirmed that insulin resistance in adipose tissue is the primary cause of metabolic syndrome, and also demonstrated that brown adipose tissue can recover its function even in such a severe disease state. I found out. Therefore, we extracted 1,469 types of serum proteins from serum proteomics analysis using IR/IGF1R-inducible double knockout mice and conducted intensive research on factors that induce the proliferation and activation of brown fat cells. The present invention was completed based on the discovery that SerpinA1, a physiologically active factor derived from SerpinA1, is a molecule related to the activation of adipocytes (brown adipocytes and/or beige adipocytes). SerpinA1 is also known as α1-antitrypsin. Therefore, in this specification, SerpinA1 and α1-antitrypsin are used to mean the same thing, and mean terms that can be replaced with each other.

The present invention includes the following.
[1] An activator for adipocytes containing SerpinA1 and/or a SerpinA1 expression promoter, wherein the adipocytes are adipocytes selected from brown adipocytes, brown adipocytes, and beige adipocytes. Cell activator.
[2] The adipocyte activator according to [1] above, which has an effect of promoting UCP1 (uncoupling protein 1) expression.
[3] The adipocyte is a brown preadipocyte, and the adipocyte activator according to [1] or [2] above has an effect of promoting proliferation of the brown preadipocyte.
[4] The fat cells are beige fat cells, and the fat cells according to [1] or [2] above have an effect of promoting beigeization of white fat cells (transdifferentiation of white fat cells into beige fat cells). Activator.
[5] The adipocyte activator according to [1] above, wherein the SerpinA1 is SerpinA1 (α1-antitrypsin) purified from mammalian plasma (preferably human plasma).
[6] The SerpinA1 expression promoter is a substance that promotes the expression of the SerpinA1 gene (for example, a substance that promotes transcription initiation of the SerpinA1 gene, a substance that inhibits degradation of SerpinA1 gene mRNA, transcription from the mRNA to SerpinA1 protein) The adipocyte activating agent according to [1] above, which is a SerpinA1 nucleic acid construct.
[7] An activator for adipocytes containing a SerpinA1 agonist, wherein the adipocytes are adipocytes selected from brown adipocytes, brown adipocytes, and beige adipocytes. .
[8] The SerpinA1 agonist binds to EphB2 receptor (Ephrin type-B receptor 2) and has the activity of (1) enhancing expression and/or phosphorylation of EphB2 receptor, or (2) brown fat The adipocyte activator according to [7] above, which is a substance having any of the following activities: promoting the expression of UCP1 (uncoupling protein 1) in cells.
[9] The adipocyte activator according to [7] or [8] above, wherein the SerpinA1 agonist is selected from the group consisting of low molecular weight substances, high molecular weight substances, nucleic acid molecules, proteins, and peptides.
[10] The adipocyte activator according to any one of [1] to [9] above, which has an effect of enhancing thermogenic action in adipose tissue.
[11] A pharmaceutical composition for preventing and/or treating obesity and/or obesity-related diseases, comprising the adipocyte activator according to any one of [1] to [10] above.
[12] The obesity-related diseases include diabetes, non-alcoholic liver disease, hypertension, dyslipidemia, heart disease (myocardial infarction, angina pectoris, etc.), cerebrovascular disease, fatty liver, obesity-related kidney disease, and high uric acid. The pharmaceutical composition according to the above [11], which is a disease selected from the group consisting of bloodemia.

[13] A pharmaceutical composition for preventing and/or treating obesity and/or obesity-related diseases, comprising a SerpinA1 expression promoter, wherein the SerpinA1 expression promoter is a substance that promotes SerpinA1 gene expression. (For example, a substance that promotes transcription initiation of the SerpinA1 gene, a substance that inhibits degradation of mRNA of the SerpinA1 gene, a substance that promotes transcription from the mRNA to SerpinA1 protein) or a pharmaceutical composition that is a SerpinA1 nucleic acid construct.
[14] The pharmaceutical composition according to the above [13], wherein the SerpinA1 nucleic acid construct is carried in a nucleic acid vector and/or included in a lipid nanoparticle.
[15] The obesity-related diseases include diabetes, non-alcoholic liver disease, hypertension, dyslipidemia, heart disease (myocardial infarction, angina pectoris, etc.), cerebrovascular disease, fatty liver, obesity-related kidney disease, and hyperuricemia. The pharmaceutical composition according to [13] or [14] above, which is a disease selected from the group consisting of:

[16] A method for screening drugs for preventing or treating obesity or obesity-related diseases, using the ability to promote the action of SerpinA1 as an indicator, and screening candidate substances having this ability to prevent or treat obesity or obesity-related diseases. A method, characterized in that it is identified as a drug for preventing or treating.
[17] The indicator has the ability to promote the expression of SerpinA1 gene (for example, the ability to promote the production of SerpinA1 gene mRNA, the ability to promote the transcription initiation of SerpinA1 gene, the ability to inhibit the degradation of SerpinA1 gene mRNA, the method according to [16] above.
[18] The method according to [16] above, wherein the indicator is the effect of serepin A1 as an agonist on EphB2 receptor.
[19] The SerpinA1 agonist action binds to the EphB2 receptor and (1) has the activity of enhancing the expression and/or phosphorylation of the EphB2 receptor, or (2) inhibits UCP1 (depletion) in brown adipocytes. The method according to [18] above, wherein the method has any of the following activities: promoting the expression of coupling protein 1).
[20] The method according to any one of [16] to [19] above, wherein the candidate substance is selected from the group consisting of a low molecular weight substance, a high molecular weight substance, a nucleic acid molecule, a protein, and a peptide.
[21] The above-mentioned [16] to [20], wherein the drug to be screened is a drug for improving obesity by enhancing lipolysis, improving insulin resistance, or acquiring obesity resistance by turning into beige adipocytes. Any one of the methods described.
[22] The method according to any one of [16] to [21] above, wherein the drug to be screened is a drug for preventing or treating obesity and/or obesity-related diseases.
[23] The obesity-related diseases include diabetes, non-alcoholic liver disease, hypertension, dyslipidemia, heart disease (myocardial infarction, angina pectoris, etc.), cerebrovascular disease, fatty liver, obesity-related kidney disease, and hyperuricemia. The method according to [22] above, wherein the disease is a disease selected from the group consisting of:

[24] A method for treating or preventing obesity or obesity-related diseases, which comprises administering to a subject in need thereof a therapeutically effective amount of a substance selected from the group consisting of SerpinA1, a SerpinA1 agonist, and a SerpinA1 expression promoter. A method comprising administering.
[25] The method according to [24] above, wherein the SerpinA1 is SerpinA1 (α1-antitrypsin) purified from mammalian plasma (preferably human plasma).
[26] The SerpinA1 agonist binds to EphB2 receptor (Ephrin type-B receptor 2) and has the activity of (1) enhancing the expression and/or phosphorylation of EphB2 receptor, or (2) brown fat. The method according to [24] above, wherein the substance has any of the following activities: promoting the expression of UCP1 (uncoupling protein 1) in cells.
[27] The SerpinA1 expression promoter may be a substance that promotes SerpinA1 gene expression (for example, a substance that promotes transcription initiation of the SerpinA1 gene, a substance that inhibits degradation of SerpinA1 gene mRNA, transcription from the mRNA to SerpinA1 protein) The method according to [24] above, which is a SerpinA1 nucleic acid construct.
[28] The obesity-related diseases include diabetes, non-alcoholic liver disease, hypertension, dyslipidemia, heart disease (myocardial infarction, angina pectoris, etc.), cerebrovascular disease, fatty liver, obesity-related kidney disease, and hyperuricemia. The method according to any one of [24] to [27] above, wherein the disease is a disease selected from the group consisting of:

According to the present invention, it is possible to activate brown adipocytes and beige adipocytes and obtain effects such as improving obesity by increasing intracellular fat decomposition or acquiring obesity resistance by turning into beige adipocytes. Furthermore, according to the present invention, it is possible to provide new drugs for obesity-related diseases.

Figure 1 shows the amino acid sequence of SerpinA1. Figure 2 shows the base sequence of SerpinA1. The left panel (A) in FIG. 3 shows the results of cold stimulation after tamoxifen induction, and the right panel (B) in FIG. 3 shows the results of cold stimulation after a long period of time. This is the result of imaging the neck of a mouse using a thermography camera. Control is the result of cold stimulation using a control mouse, and Ai-DKO is a mouse in which IR and IGF1R deficiency has been induced by administration of tamoxifen (IGF1R ^−/− IR ^−/− ). Darker (red) areas indicate higher temperatures. The results of analysis of the expression of SerpinA1 in the liver due to cold stimulation in mice are shown. The left panel shows SerpinA1 mRNA expression. The upper right figure shows the results of confirming SerpinA1 protein expression by Western blot analysis. The lower right figure is a graph of the Western blot analysis results. Inguinal white adipose tissue; iWAT, epididymal white adipose tissue; eWAT, cervical brown adipose tissue; iBAT, liver: Liver. Data represent mean ± SEM, black circles indicate individual subjects (mice) (unless otherwise stated, the same applies in the following figures). *: P<0.05, **: P<0.01, ***: P<0.001, n. s. indicates that there is no significant difference (the same applies to the following drawings unless otherwise specified). The results of analysis of UCP1 expression in fat induced by cold stimulation in mice are shown. The left panel shows UCP1 mRNA expression. The upper right figure shows the results of confirming UCP1 protein expression by Western blot analysis. The lower right figure is a graph of the Western blot analysis results. These are the results of analyzing SerpinA1 expression in a mouse pathological model (high-fat diet loading model). CD is a normal diet group, and HFD is a high-fat diet group. The left panel shows SerpinA1 mRNA expression. The center figure shows the results of Western blot analysis confirming protein expression of SerpinA1 in the liver. The figure on the right is a graph of the Western blot analysis results. iWAT, eWAT, iBAT and Liver are the same as above. These are the results of confirming the proliferation effect of SerpinA1 protein on brown preadipocytes. A shows the results of measuring the ratio of EdU to DAPI. B shows the measurement results of UCP1 mRNA expression and protein expression. C is the measurement result of oxygen consumption rate. These are the results of confirming the proliferation effect of SerpinA1 protein on white adipocytes. A shows the results of measuring the ratio of EdU to DAPI. B shows the measurement results of UCP1 mRNA expression and protein expression. C is the measurement result of oxygen consumption rate. These are the results of comparing wild-type mice and liver-specific SerpinA1 overexpressing Tg mice (SerA1Tg mice). The left figure shows the measurement results of random blood sugar and fasting blood sugar. The upper right figure shows the results of the insulin tolerance test (ITT), and the lower right figure shows the results of the glucose tolerance test (GTT). These are the results of analyzing mRNA expression in brown adipose tissue of wild-type mice and SerA1Tg mice. A shows the results of qPCR, and B shows the results of immunostaining. These are the results of wild-type mice and SerA1Tg mice in response to cold stimulation. The figure on the left shows the rectal temperature, and the figure on the right shows the result of imaging the neck with a thermography camera. A is the result of qPCR analysis of mRNA expression in subcutaneous white tissues of wild-type mice and SerA1Tg mice. B is the result of measuring the amount of subcutaneous fat per body weight. The left panel C shows the results of HE staining the tissue. The right panel C shows the results of measuring the diameter of a lipid droplet from an HE-stained image. These are the results of high-fat diet feeding to wild-type mice and SerA1Tg mice. A is the measurement result of fasting blood sugar, B is the result of glucose tolerance test (GTT), and C is the result of insulin tolerance test (ITT). A shows an outline of the SerpinA1 gene KOed using CRISPR-Cas9 technology. B and C are the results of glucose tolerance test (GTT) and insulin tolerance test (ITT) in wild-type mice and SerA1KO mice, respectively. These are the results of comparing wild-type mice and SerA1KO mice. A is the measurement result of oxygen intake. B and C are the results of measuring rectal temperature after cold stimulation and imaging the neck with a thermography camera, respectively. These are the results of Western blot analysis after inducing differentiation of brown adipocyte precursor cells into brown adipocytes, adding SerpinA1 at various concentrations. The left figure shows the expression of EphB2 receptor, and the right figure shows the expression of tyrosine-phosphorylated EphB2 receptor. These are the results of Western blot analysis using brown adipose tissue taken from wild-type mice and SerpinA1Tg mice. The left figure shows the expression of EphB2 receptor, and the right figure shows the expression of tyrosine-phosphorylated EphB2 receptor. These are the results of measuring oxygen consumption rates using brown preadipocyte cell lines established from wild-type mice and EphB2 receptor KO mice. These are the results of measuring the expression of UCP1 using brown preadipocyte cell lines established from wild-type mice and EphB2 receptor KO mice. These are the results of cold stimulation in wild-type mice in which physiological saline (control) or human SerpinA1 recombinant protein was administered to the left and right cervical brown adipose tissue. The above figure shows rectal temperature and interscapular BAT temperature. The figure below shows the results of an image of the neck taken with a thermography camera. FIG. 2 is a diagram showing the correlation between serum SerpinA1 concentration and HbA1c or BMI in metabolic patients with type 2 diabetes and obesity. This figure shows the results of feeding high-fat diet to SerA1KO mice. The upper figure shows the weight change, and the lower figure shows the weight of subcutaneous fat (iWAT) and visceral fat (eWAT). WT is the result of control wild type mice.

The present invention will now be described by way of example embodiments, along with preferred methods and materials that can be used in the practice of the invention, but the invention is not limited to the embodiments described below. It should be noted that, unless otherwise specified herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Also, any materials and methods equivalent or similar to those described herein can also be used in the practice of the invention. In addition, all publications and patents cited herein in connection with the present invention are incorporated herein by reference, e.g., as indicating methods, materials, etc. that can be used in the present invention. It forms part of the book.

In this specification, the description "A to B" indicating a numerical range means a numerical range that includes the end points A and B. The same applies to "A to B". Furthermore, in this specification, "about" is used to mean a tolerance of ±10%.

(SerpinA1, SerpinA1 expression promoter, adipocyte activator containing SerpinA1 agonist)
In one embodiment, the present invention is an adipocyte activator containing SerpinA1. SerpinA1 is a protein that belongs to the Serpin superfamily and is a protease inhibitor. Serpins are classified into AI (9 types), which have multiple members, and 37 types have been identified in humans so far, and each molecule has different structures and expressions, as well as different targets and functions. SerpinA1 is a 52 kDa protein, also called α1-antitrypsin (A1AT). The SerpinA1 gene nucleotide sequence has been registered in GenBank provided by the National Center for Biotechnology Information (NCBI) with the accession number (NM_00112770.1) (if multiple revisions are registered, the latest (understood as referring to revision). The amino acid sequence of SerpinA1 is shown in SEQ ID NO: 1 (natural A1AT of 418 amino acids, transcript variants 1 to 11 have been reported), and its base sequence is shown in SEQ ID NO: 2.

SerpinA1 exists in the blood and is said to protect surrounding tissues from the protein-cleaving enzyme elastase. Neutrophils secrete elastase to degrade connective tissue during inflammation. On the other hand, blood cells migrate to the area surrounding the inflammation, protecting it from foreign substances and repairing degraded tissue. SerpinA1 protects adjacent areas and primarily prevents this neutrophil elastase from spreading throughout the body. Furthermore, SerpinA1 has been reported to have multiple paralogs encoding A1AT in mice, and due to the complexity of this gene structure, detailed analysis using knockout mice has not progressed to date.

As used herein, "activation of adipocytes" means activating one or more of adipocytes such as brown adipocyte precursor cells, brown adipocytes, or beige adipocytes, and is limited thereto. However, it means an action that produces one or more of the following effects. (1) Effect of promoting UCP1 expression in brown adipose progenitor cells, brown adipocytes, or beige adipocytes (UCP1 expression here means UCP1 gene expression and/or UCP1 protein expression) . (2) Effect of promoting proliferation of brown adipose progenitor cells. (3) Effect of promoting transdifferentiation from white adipocytes to beige adipocytes. (4) Effect of promoting fat burning by brown fat cells or beige fat cells and producing heat. (5) Effect of enhancing thermogenic action in brown adipose tissue or white adipose tissue containing beige cells. Therefore, the activator of the present invention activates brown fat cells and beige fat cells, burns intracellular fat, and functions to prevent hypothermia and obesity. In this specification, the terms "activation of brown adipocytes" and "activators of brown adipocytes" are used to include the activation of brown adipocyte precursor cells, which are precursor cells of brown adipocytes; When describing cell activation, the term brown adipocytes is used to include both brown adipocytes and brown preadipocytes, unless the context clearly indicates that only one or the other is meant. be understood. As used herein, the term "adipocyte activator" is used interchangeably with the term "brown adipocyte and/or beige adipocyte activator." In this case as well, brown adipocytes are understood to include brown adipocytes and brown preadipocytes.

SerpinA1 contained in the adipocyte activator of the present invention is not particularly limited as long as it has the amino acid sequence of SerpinA1 and has any one of the effects described in (1) to (5) above, such as , SerpinA1 isolated from a living body, or recombinant SerpinA1 produced using genetic engineering technology. Examples of SerpinA1 isolated from a living body include, but are not limited to, SerpinA1 isolated from pooled mammalian (preferably human) plasma. Methods for isolating SerpinA1 from plasma are known, and SerpinA1 used in the present invention can be produced by appropriately referring to the known methods. For example, it can be carried out with reference to the method described in WO2004/060528 (this description is incorporated herein by reference). Plasma-derived A1AT is commercially available and is also used as a drug for α1-antitrypsin deficiency, and can also be used in the present invention. Recombinant SerpinA1 can be produced using known genetic engineering methods, for example, with reference to the methods described in WO2010/127939 and WO2020/092448 (these descriptions are , incorporated herein by reference).

Here, having the amino acid sequence of SerpinA1 means having substantially the amino acid sequence of SerpinA1, and is not limited to this, but 90% or more, 91% or more of the amino acid sequence of SerpinA1 shown in SEQ ID NO: 1. , 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more, and of the amino acid sequence of SerpinA1 shown in SEQ ID NO: 1. Also includes amino acid sequences with partial substitutions, deletions, and additions (for example, one or more, but not limited to, specifically one, two, three, four, or five amino acids). , and an adipocyte activator containing a mutation in the amino acids surrounding the serine residues of the active center loop involved in suppressing the serine protease enzyme activity of SerpinA1.

In another embodiment of the present invention, the adipocyte activator of the present invention has an effect of promoting the expression of UCP1 (uncoupling protein 1) in brown adipocytes and beige adipocytes. By increasing the expression of UCP1, brown adipocytes and beige adipocytes can be activated. Here, expression of UCP1 is used to include both UCP1 gene expression (that is, mRNA expression) and protein expression.

In another embodiment of the present invention, the adipocyte activator of the present invention has an effect of promoting proliferation of brown preadipocytes. By enhancing proliferation of progenitor cells, brown adipocytes can be activated, and thermogenic action in brown adipose tissue can be enhanced.

In another embodiment of the present invention, the adipocyte activating agent of the present invention has the effect of promoting beigeization of white adipocytes, in other words, the transdifferentiation of white adipocytes into beige adipocytes. Beige adipocytes can be activated by promoting differentiation into beige adipocytes, and thermogenic action in white adipose tissue including beige adipocytes can be enhanced.

In another embodiment of the present invention, the adipocyte activating agent of the present invention has an action of enhancing thermogenic action in adipose tissue. Enhancement of thermogenic action in adipose tissue can be achieved by promoting one or more of the above-mentioned actions, but in addition, UCP1-independent thermogenic mechanisms can be achieved by activating brown fat cells and beige cells. It can also be done by enhancement. Examples of such a thermogenic mechanism include Ca ²⁺ cycling.

In another embodiment of the present invention, the adipocyte activating agent of the present invention is an adipocyte activating agent containing an expression promoter of SerpinA1. In the present invention, a SerpinA1 expression promoter means a substance that promotes the expression of SerpinA1 in cells. For example, but not limited to, a substance that promotes transcription initiation of SerpinA1 in cells, a substance that inhibits degradation of SerpinA1 gene mRNA, a substance that promotes transcription from SerpinA1 mRNA to SerpinA1 protein, or a substance that promotes transcription of SerpinA1 in cells. Examples include substances that promote expression.

Examples of the substance that promotes the expression of SerpinA1 in the cells include a nucleic acid construct containing the SerpinA1 gene. By introducing the nucleic acid construct into cells and expressing it within the cells, the expression of SerpinA1 within the cells can be enhanced. The nucleic acid construct is preferably carried in a nucleic acid vector or encapsulated in a lipid nanoparticle. Furthermore, a nucleic acid construct carried by a nucleic acid vector (preferably a viral vector) may be included in the lipid nanoparticle. By administering to a subject in such a state, expression of the SerpinA1 gene can be efficiently promoted within cells.

Examples of viral vectors include adeno-associated virus (AAV) vectors, adenovirus vectors, retrovirus vectors, and lentivirus vectors. AAV vectors include AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh. 64R1, AAVhu. 37, AAVrh. 8, AAVrh. Mention may be made of vectors consisting of 32.33, AAV8, AAV9, AAV-DJ, AAV2/8, AAVrhlO, AAVLK03, AV10, AAV11, AAV12, rh10, and hybrids thereof.

As lipid nanoparticles, any lipid nanoparticles that can encapsulate nucleic acids and can be used for introducing nucleic acids into cells can be used in the present invention without any limitations. Such lipid nanoparticles are known and many reports have been made, and can be used in the present invention.

In addition to the SerpinA1 gene, the nucleic acid construct may have additional sequences, such as an origin of replication, a promoter, and a gene encoding antibiotic resistance. Nucleic acid constructs can be either as naked nucleic acids, loaded onto non-viral vector delivery systems such as non-viral vectors, plasmid vectors, or complexed with delivery vehicles such as liposomes, lipid nanoparticles (LNPs) or poloxamers. or in a viral vector (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus), or even in a vector as described above and complexed with the delivery vehicle as described above. may be administered to a subject at A nucleic acid construct containing the SerpinA1 gene can be produced using known genetic engineering techniques. The production of a nucleic acid construct containing the SerpinA1 gene can be performed, for example, with reference to the methods described in WO2013/106589, WO2016/110565, WO2017/093804, WO2020/082047, and WO2020/092448 ( (These descriptions are incorporated herein by reference).

The expression mode of the SerpinA1 gene within the cell is not particularly limited, and for example, the SerpinA1 gene may be expressed within the cell using an expression vector, or the SerpinA1 gene may be expressed by targeted insertion into a safe harbor locus. Methods for introduction into cells and expression are known and can be carried out using these methods. For example, a method for inserting and expressing the SerpinA1 gene at the human safe harbor locus using a guide RNA is described in WO2017/093804 and WO2020/082047 (these descriptions are incorporated herein by reference). part of the book).

In another embodiment, the present invention is a pharmaceutical composition for treating obesity and/or obesity-related diseases, which comprises a SerpinA1 expression promoter. As the SerpinA1 expression promoter, the SerpinA1 expression promoter described in the adipocyte activator can also be used in the pharmaceutical composition.

In another embodiment of the present invention, the adipocyte activating agent of the present invention is an adipocyte activating agent containing a SerpinA1 agonist. The present inventors have discovered, as one aspect of the present invention, that SerpinA1 binds to EphB2 receptors. This indicates that SerpinA1 regulates EphB2 receptor as a target. Therefore, as used herein, an agonist of SerpinA1 is defined as one that binds to the EphB2 receptor and (1) has the activity of enhancing expression and/or phosphorylation of the EphB2 receptor, or (2) agonist of UCP1 ( A substance having any of the following activities: promoting the expression of uncoupling protein 1).

EphB2 receptor (Ephrin type-B receptor 2) is a single-transmembrane receptor tyrosine kinase encoded by the EPHB2 gene, and is known to bind to a ligand called ephrin B. Eph receptors are classified into two subclasses, EphA and EphB, based on differences in their amino acid sequences and affinity with ligands, and six types of EphB subclasses have been identified in vertebrates (EphB1-6).

In the present invention, an agonist of SerpinA1 means a substance that binds to EphB2 receptor and activates the same intracellular signal transduction system as SerpinA1. For example, it refers to a substance that binds to the EphB2 receptor and has any one or more of the following activities (1) to (3): (1) has the activity of enhancing the expression of the EphB2 receptor; Here, enhancing the expression of EphB2 receptor means enhancing the expression of EphB2 receptor mRNA, suppressing the degradation of EphB2 receptor mRNA, enhancing the expression of EphB2 receptor protein, and suppressing the degradation of EphB2 receptor protein. As a result, it means a case where the agonist has an activity of enhancing EphB2 expression compared to the case where no agonist is used. (2) It has the activity of enhancing the phosphorylation of EphB2 receptor. (3) It has the activity of promoting the expression of UCP1 (uncoupling protein 1) in brown fat cells.

Measurement of the agonist activity of SerpinA1 is not limited to this, but includes, for example, measuring the expression levels of EphB2 receptor mRNA and protein using brown adipocytes expressing EphB2 receptor using known techniques; This can be carried out by measuring the amount of phosphorylation (preferably tyrosine phosphorylation) or by measuring the expression of UCP1 in brown cells.

In the present invention, the substance that is an agonist of SerpinA1 is not particularly limited, and may be any of a low molecular weight substance, a high molecular weight substance, a nucleic acid molecule, a protein, or a peptide.

Another embodiment of the present invention is a pharmaceutical composition containing the adipocyte activating agent of the present invention. The adipocyte activator or pharmaceutical composition of the present invention can be used to prevent or treat obesity or obesity-related diseases. Generally, a state in which there is too much adipose tissue in the body is referred to as "obesity". The term "obesity" refers to a state of excessive body fat, and refers to a case where the body mass index (BMI) is 30 or more, according to the World Health Organization (WHO). Generally, it means that the body weight is higher than the normal value, but even if the body weight is not high, if the body composition has a high proportion of body fat, it is diagnosed as obesity, and it can be diagnosed as obesity in both adults and children. Refers to the disease that occurs. The term "obesity-related disease" encompasses disorders associated with, caused by, or resulting from obesity. Examples of obesity-related diseases include overeating and bulimia, diabetes, hypertension, elevated plasma insulin levels and insulin resistance, dyslipidemia, hyperlipidemia, breast, prostate, endometrial and colon cancers, heart disease, and heart disease. These include vascular disorders, abnormal heart rhythms and arrhythmias, myocardial infarction, congestive heart failure, coronary heart disease, angina, cerebral infarction, cerebral thrombosis and transient ischemic attacks, and osteoarthritis. Other examples include pathological conditions that exhibit decreased metabolic activity or decreased resting energy expenditure as a percentage of total lean mass. Further examples of obesity-related diseases include metabolic syndrome, also known as syndrome Secondary consequences of obesity include sclerosis, hypercholesterolemia, hyperuricemia, and left ventricular hypertrophy. Obesity-related diseases also include obesity-related liver abnormalities such as non-alcoholic fatty liver disease (NAFLD), which is responsible for the increased cirrhosis associated with obesity and metabolic syndrome. NAFLD can exist as simple steatosis or progress to inflammation and steatohepatitis (NASH). "Dyslipidemia" is a major risk factor for coronary heart disease (CHD) and is often associated with obesity. Therefore, when using the adipocyte activator and pharmaceutical composition of the present invention, not only obesity but also the aforementioned obesity-related diseases can be prevented or treated at the same time. It also includes objects that there is a desire to reduce.

Examples of dosage forms for the pharmaceutical composition of the present invention include injections, oral preparations (tablets, granules, powders, capsules), ointments, creams, patches, suppositories, and the like. Among these, injections and oral preparations are preferred.

The pharmaceutical composition of the present invention can be mixed with a pharmaceutically acceptable carrier used to form a pharmaceutical composition. Examples of the carrier include solubilizing agents, excipients, binders, lubricants, disintegrants, coloring agents, flavoring agents, preservatives, and surfactants. When preparing an oral preparation, excipients, binders, lubricants, disintegrants, coloring agents, flavoring agents, etc. are used, and the preparation can be carried out by conventional methods. When preparing an injection, water, a solubilizing agent, etc. are used. The injection is preferably an intravenous injection, a subcutaneous injection, or an intramuscular injection, and examples include freeze-dried preparations and powder fillers.

By "therapeutically effective amount" herein is meant an amount of SerpinA1 or its agonist that results in the activation of brown adipocytes and/or beige adipocytes, which provides the desired benefit/risk ratio. It means an amount that is effective to produce a therapeutic effect, such as weight loss. A therapeutically effective amount can vary depending on the route of administration used, as is known to those skilled in the art. Furthermore, the therapeutically effective amount can be appropriately determined depending on the subject to be treated, the severity of the symptoms, the route of administration, the frequency of administration, the judgment of the prescribing physician, and other relevant factors. Generally, a therapeutically effective amount will be about 0.01 to about 1000 mg/kg body weight, preferably about 0.05 to about 500 mg/kg body weight, more preferably about 0.1 to about 100 mg/kg body weight.

The adipocyte activator or pharmaceutical composition of the present invention is an agent other than SerpinA1 or an agonist thereof or a SerpinA1 expression promoter (hereinafter collectively referred to as SerpinA1 drug in this paragraph) for treating obesity and/or obesity-related diseases. Can be administered in combination with drugs. Here, "administering in combination" means (i) administering the SerpinA1 drug and the non-SerpinA1 drug in the same pharmaceutical composition, or (ii) administering the SerpinA1 drug and the non-SerpinA1 drug as separate formulations. (iii) The SerpinA1 drug and the drug other than SerpinA1 may be prepared as separate preparations and administered at separate timings. The usage, dosage, administration timing, administration interval, etc. of the SerpinA1 drug and other drugs to be administered can be determined as appropriate based on the common technical knowledge known to those skilled in the art, depending on the above-mentioned factors.

The adipocyte activator or pharmaceutical composition of the present invention induces differentiation of beige adipocytes in white adipose tissue, and/or activates beige adipocytes and brown adipocytes, and consumes whole body energy, especially through thermogenesis. By promoting metabolism by promoting , obesity can be prevented and/or suppressed (treated). Therefore, in addition to treating and/or preventing obesity, it may also be effective in treating and/or preventing diseases caused by obesity. It can also prevent the body from getting cold by activating brown fat cells and/or beige fat cells and increasing cold-induced heat production.

In another aspect of the invention, the adipocyte activating agents or pharmaceutical compositions of the invention can be used in combination with one or more of the following therapeutic agents in treating obesity or obesity-related disorders: . Such drugs include, but are not limited to, phenylpropanolamine, phentermine, diethylpropion, mazindol, fenfluramine, dexfenfluramine, phentiramine, beta3 adrenoceptor agonist drugs; sibutramine, gastrointestinal lipase inhibitors (eg, orlistat), and leptin. Other drugs used in treating obesity or obesity-related diseases include neuropeptide Y, enterostatin, cholecytokinin, bombesin, amylin, histamine H3 receptors, dopamine, D2 receptor modulators, melanocytes. The stimulating hormones include galanin and gamma-aminobutyric acid (GABA).

As used herein, "treatment" refers to the use of the pharmaceutical composition of the present invention (a pharmaceutical composition containing an adipocyte activator and a pharmaceutical composition containing a SerpinA1 expression promoter) for reducing or maintaining the body weight of an obese subject. means administration. An example of one treatment result is a decrease in the obese subject's weight compared to the subject's weight prior to administration. Another example of a therapeutic outcome may be the prevention of regaining weight that has already been lost as a result of diet, exercise or drug therapy. Another example of a therapeutic outcome may be a reduction in the occurrence and/or severity of obesity-related diseases.

As used herein, "prevention" refers to the administration of the pharmaceutical composition of the present invention to reduce or maintain body weight in a subject at risk of obesity. An example of one preventive outcome is a reduction in the weight of a subject at risk of obesity compared to the subject's weight before administration. Another example of a preventive outcome may be the prevention of regaining weight that has already been lost as a result of diet, exercise or drug therapy. Another example of a preventive outcome may be prevention of the development of obesity when therapeutic administration occurs prior to the onset of obesity development in a subject at risk of obesity. Another example of a preventive outcome may be a reduction in the occurrence and/or severity of obesity-related disorders when therapeutic administration occurs prior to the onset of obesity development in a subject at risk of obesity. Furthermore, if treatment is initiated in a subject who is already obese, such treatment may prevent the onset, progression, or severity of obesity-related diseases.

The subject to whom the pharmaceutical composition of the present invention is administered is any subject for whom diagnosis, prognosis, or treatment is desired, particularly mammals. Mammalian subjects include, but are not limited to, humans, non-human primates, domestic animals, farm animals, companion animals, rodents, etc., which are recipients of certain treatments; Preferably it is a human.

The pharmaceutical composition of the present invention can be used to treat obesity caused by a relatively lower numerical or mass ratio of beige adipocytes or brown adipocytes to white adipocytes than in a healthy state, as well as beige adipocytes or brown adipocytes. Due to low expression of the cell-specific gene UCP1, the numerical or mass ratio of beige or brown adipocytes to white adipocytes is relatively lower than in a healthy state. It can be effective against obesity.

Obesity caused by a relatively lower numerical or mass ratio of beige adipocytes or brown adipocytes to white adipocytes than in a healthy state can be investigated using methods known in the art. For example, obesity can be investigated by quantifying the activity of brown fat cells using a test called FDG-PET that quantifies glucose uptake after applying a cold stimulus. Cold stimulation can be performed under the usual conditions used in the art. The activity of brown adipocytes is quantified by determining that, among the areas that are consistent with adipose tissue in CT values, the areas in which glucose uptake above a threshold value is observed in FDG-PET are determined to be brown adipocytes, and the remaining areas are determined to be white adipocytes. be able to. Therefore, if the above quantitative result in an obese patient is lower than the above quantitative result for one healthy person or the average value of the above quantitative results for multiple healthy individuals, the It can be determined that the patient has obesity caused by a numerical ratio or a mass ratio that is relatively lower than a healthy state. Furthermore, the above-mentioned quantification can be used to confirm that the prevention or treatment of obesity according to the present invention has been achieved by promoting the differentiation of preadipocytes into beige adipocytes.

In one embodiment of the present invention, the present invention is also a method for confirming or evaluating the state of obesity or an obesity-related disease using SerpinA1 in blood or serum as an indicator. Obesity and obesity-related diseases are as described above.

(Method of screening drugs for obesity or obesity-related diseases)
According to the present invention, a method for screening drugs for treating obesity or obesity-related diseases is provided. In this method, the ability to promote the action of SerpinA1 is used as an indicator of the ability of a candidate drug, and candidate drugs having this ability are identified as agents for treating obesity-related diseases. Here, promoting the action of SerpinA1 includes, but is not limited to, the ability to promote SerpinA1 gene expression, binding activity to EphB2 receptor, which is a receptor for SerpinA1, or agonist activity of SerpinA1. can. Examples of the ability to promote SerpinA1 gene expression include the ability to promote SerpinA1 gene transcription initiation, the ability to inhibit the degradation of SerpinA1 gene mRNA, or the ability to promote transcription from SerpinA1 mRNA to SerpinA1 protein. I can do things. The agonist activity of SerpinA1 binds to the EphB2 receptor and can include any of the activities described above.

The screening method of the present invention can be appropriately implemented by those skilled in the art by referring to known techniques in the technical field, depending on the index used for drug selection. For example, by preparing hepatocytes expressing SerpinA1 or cells transformed by gene transfer so that the SerpinA1 gene is expressed, an expression test is performed in the presence and absence of a candidate substance, and the amount of SerpinA1 mRNA is determined. Alternatively, the ability of the candidate substance to promote (enhance) SerpinA1 gene expression can be measured by comparing the protein amounts. The amount of SerpinA1 mRNA can be measured by PCR using primers specific to the SerpinA1 gene according to a conventional method. The amount of SerpinA1 protein can be measured according to a conventional method using an assay system using an antibody that specifically reacts with SerpinA1. Furthermore, a solid-phase binding elastase binding assay (see, for example, WO2012/159700) can also be used to measure the activity of SerpinA1.

In addition, when screening candidate substances using the ability to promote (enhance) the expression of the SerpinA1 gene as an indicator, for example, a DNA fragment containing the transcription regulatory region of the SerpinA1 gene and a DNA fragment containing a reporter gene are operable. By using nucleic acid molecules linked in this manner, candidate substances that promote transcription of the SerpinA1 gene can be screened. The nucleotide sequence of the transcriptional regulatory region of the SerpinA1 gene is available from the NCBI Gene Bank. Furthermore, the reporter gene may be one for which a method for detecting the expression product of the gene is known, such as a photoprotein gene such as a luciferase gene, an enzyme gene such as a β-galactosidase gene, or a chloramphenicol acetyltransferase gene. Genes, fluorescent protein genes such as the green fluorescent protein (GFP) gene, etc. can be used. In detecting and evaluating the ability of a candidate substance to promote SerpinA1 gene expression, an in vitro transcription activity evaluation system into which the nucleic acid molecule has been introduced or cells transformed with the nucleic acid molecule (mammalian cells, insect cells, yeast, Escherichia coli, etc.) can be used. For related genetic manipulation, detection of reporter gene expression, and general techniques for screening candidate substances, techniques well known to those skilled in the art can be used.

In the present invention, agonists of serepin A1 can also be screened as candidate substances using an evaluation system using the EphB2 receptor. For example, using brown adipocytes that express the EphB2 receptor, the expression levels of EphB2 receptor mRNA and protein can be measured using known techniques or the genetic engineering technique described above for SerpinA1, and the phosphorylation of the EphB2 receptor ( Preferably, this can be carried out by measuring the amount of tyrosine phosphorylation) or by measuring the expression of UCP1 in brown cells.

In the screening method of the present invention, various types of substances can be used as drug candidates, such as low-molecular substances, high-molecular substances, nucleic acid molecules, proteins, and peptides (including cyclic peptides).

(Method for treating or preventing obesity or obesity-related diseases)
In another aspect, the present invention provides a method for treating or preventing obesity or obesity-related diseases, wherein a subject in need thereof is given a therapeutically effective amount of SerpinA1, a SerpinA1 agonist, or a SerpinA1 expression promoter. The method includes administering a substance selected from the group consisting of: Here, obesity-related diseases include the diseases described regarding the adipocyte activating agent of the present invention and the pharmaceutical composition containing the activating agent, which also apply to the treatment or prevention method of the present invention. Similarly, regarding SerpinA1, SerpinA1 agonists, or SerpinA1 expression promoters, the contents described regarding the adipocyte activating agent of the present invention and the pharmaceutical composition containing the activating agent also apply to the treatment or prevention method of the present invention. Applicable.

Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to the following Examples.

(Example 1) Preparation of IR/IGF1R-inducible double knockout mice In which organ-specific insulin receptor/IGF-1 receptor (IR/IGF1R) deficiency can be induced with tamoxifen, IR/IGF1R- Inducible double knockout mice were generated (see Sakaguchi et al., Cell Metabolism 25, 448-462, 2017, which is incorporated herein by reference).
By crossing mice with floxed insulin and/or IGF1 receptor alleles (foxedIR/IGF1R) and mice carrying a tamoxifen-inducible Cre ER ^T2 transgene under the control of the adiponectin promoter (AdipoqCreER ^T2 ). established a mouse model in which IR and/or IGF1R are specifically deleted in adipose tissue when induced. Mice carrying the floxed allele but not the Cre transgene served as controls. Control mice were also given tamoxifen, which under the conditions used showed no adverse effects.
When 8-week-old mice carrying foxedIR/IGF1R and AdipoqCreER ^T2 transgenes (Ai-DKO mice) were treated with tamoxifen, rapid loss of adipose tissue was observed. In addition, glucose intolerance and insulin resistance were induced, and at the same time, brown fat weight was decreased, and cold tolerance was significantly decreased by cold stimulation. In these mice, insulin resistance in adipose tissue was shown to be the primary cause of metabolic syndrome.
However, when these Ai-DKO mice were observed for a long period of time, it was found that the brown adipose tissue regenerated and the cold tolerance improved to an almost normal state. Figure 3A shows the results of cold stimulation (2 hours of exposure to an 8°C environment) 9 days after tamoxifen induction. Thereafter, after a long period of time (90 days), cold stimulation was applied again, and the results are shown in FIG. 3B. It was suggested that brown adipocytes disappear due to tamoxifen induction, but that brown adipocytes regenerate after a long period of time. Recalling the existence of physiologically active factors involved in the regeneration of brown adipose tissue, the factors were identified as follows.

(Example 2) Identification of brown fat cell regeneration factor Serum was collected from control mice and Ai-DKO mice during the acute phase (adipose tissue loss phase) and brown adipose tissue regeneration phase, analyzed by mass spectrometry, and proteomics. From the analysis, we searched for physiologically active substances whose expression significantly increases in serum during brown adipose tissue regeneration. 1469 types of serum proteins were extracted. We narrowed down the factors that induce mouse brown fat cell proliferation and activation, and identified SerpinA1 (Serpin Family A Member 1) (α1-antitrypsin) as a physiologically active factor derived from the liver. As a result of measuring the mRNA expression of SerpinA1 in various organs of mice, it was found that SerpinA1 is a liver-derived factor that is selectively expressed in the liver, and mRNA expression in other organs is minimal.

(Example 3) Expression of SerpinA1 in a cold environment The influence of cold stimulation on the expression of SerpinA1 was analyzed.
After exposing 12-week-old C57BL/6 mice to environments of 23°C and 4°C for 48 hours, SerpinA1 expression in the liver and UCP1 (Uncoupled protein 1) expression in fat were analyzed. The results of SerpinA1 expression in the liver are shown in FIG. It was found that mRNA expression and protein expression of SerpinA1 in the liver were significantly increased by cold stimulation. Furthermore, the results of UCP1 expression in fat are shown in FIG. It was found that cold stimulation significantly increased UCP1 mRNA and protein expression in subcutaneous fat and brown adipose tissue. These results revealed that there was a positive correlation between SerpinA1 expression in the liver and UCP1 expression in brown adipose tissue.

(Example 4) Expression of SerpinA1 in a pathological model For analysis in a pathological model, SerpinA1 expression in each tissue was analyzed using C57BL/6 mice after being fed a high-fat diet for 4 months from the age of 5 weeks. did. The results of analyzing SerpinA1 mRNA expression by qPCR are shown in FIG. There were no significant differences in subcutaneous fat, visceral fat, and brown fat between the normal diet group and the high-fat diet group, but in the liver, SerpinA1 expression was significantly decreased in the high-fat diet group. I understand. Furthermore, the results of Western blot analysis confirmed that protein expression was also significantly reduced in the high-fat diet loaded group.

(Example 4) Effect of SerpinA1 on brown adipocytes Brown adipose progenitor cells were loaded with SerpinA1 recombinant protein (purchased from Bio Vision (product number 7294-1000)) at various concentrations (50, 100 μg/mL) for 18 hours. , and staining with EdU for 2 hours to measure the ratio of EdU to DAPI. The results are shown in Figure 7A. SerpinA1 was confirmed to have an effect of inducing proliferation of brown preadipocytes.
Next, SerpinA1 recombinant protein was loaded for 12 hours (200 μg/mL) or 24 hours (300 μg/mL) into mature brown adipocytes in which brown preadipocytes were induced to differentiate, and UCP1 mRNA and protein expression was confirmed. However, there was a significant increase. The results are shown in Figure 7B. In addition, mature brown adipocytes were loaded with SerpinA1 for 16 hours (300 μg/mL), and in the respiratory chain complex (electron transfer assay), an inhibitor (oligo) of the electron transport chain and oxidative phosphorylation coupling (coupling assay) was used. Oxygen consumption rate (OCR) was measured while adding mycin, FCCR, rotenone, and antimycin A), and the effect on mitochondrial activity was investigated. It turned out to be effective. The results are shown in Figure 7C.

(Example 5) Effect of SerpinA1 on white adipocytes White preadipocytes were loaded with SerpinA1 recombinant protein at various concentrations (100, 200 μg/mL) for 18 hours, and stained with EdU for 2 hours to determine the ratio of EdU to DAPI. It was measured. The results are shown in Figure 8A. SerpinA1 was confirmed to have an effect of inducing proliferation of white preadipocytes.
Next, SerpinA1 recombinant protein was loaded for 16 hours (200,500 μg/mL) or 36 hours (500 μg/mL) into mature white adipocytes that had been induced to differentiate from white preadipocytes, and UCP1 mRNA and protein expression was inhibited. When I checked, it was found to have increased significantly. The results are shown in Figure 8B. In addition, when mature brown adipocytes were loaded with SerpinA1 (500 μg/mL) for 36 hours and the effect on mitochondrial activity was investigated using oxygen consumption rate (OCR) as an index, it was found that SerpinA1 increases mitochondrial activity in mature white adipocytes. It turned out to be effective. The results are shown in Figure 8C.
From these results, SerpinA1 showed an effect on white adipocytes as well as brown adipocytes.

(Example 6) Examination of SerpinA1 overexpression transgenic mice This is a vector in which a liver-specific albumin promoter is integrated after adding the 3xFlag gene to human SerpinA1 using p3X Flag-CMV-14 Expression Vector (Sigma). A liver-specific SerpinA1 overexpressing Tg mouse (SerA1Tg mouse) was created using a transgene created by inserting it into pLive vector (Minus Bio).
As a result of measuring blood sugar levels in Tg mice, no significant difference was observed in casual blood sugar, but a significant decrease in fasting blood sugar was observed in Tg mice compared to wild type WT. In addition, in the insulin tolerance test (ITT), there was almost no difference between the two, but in the glucose tolerance test (GTT), the blood sugar levels at 30, 60, and 120 minutes after intraperitoneal glucose injection were higher than those in WT. It was significantly decreased in Tg mice. A significant decrease in AUC (area under the curve) was also observed in Tg mice, indicating that SerA1Tg mice had higher glucose tolerance compared to WT. The results are shown in FIG.

Next, the SerpinA1Tg mouse was dissected and the tissue was analyzed. There was no significant difference in liver tissue amount per body weight between WT and Tg mice. However, regarding the subcutaneous fat of SerpinA1Tg mice, the amount of tissue per body weight was significantly reduced compared to WT. Furthermore, when subcutaneous adipose tissue was stained with HE and observed, it was confirmed that fat droplets were smaller in Tg mice than in WT mice. Furthermore, regarding intraperitoneal fat, although no significant difference in weight was observed, HE staining showed a tendency for the fat droplets in Tg mice to be smaller compared to WT mice. Regarding brown fat, there was no significant difference in weight, but HE staining showed that fat droplets were denser in the brown fat of Tg mice than in WT mice. This suggests that this change in adipose tissue is related to the improvement of glucose tolerance in SerpinA1Tg mice.

Furthermore, mRNA expression in the brown adipose tissue of SerA1Tg mice was analyzed by qPCR. The results are shown in FIG. Although there was no significant change in the expression of genes such as Adiponectin and Leptin, the expression of UCP1 was significantly increased in SerA1Tg mice (FIG. 10A). This was also confirmed by immunostaining of brown adipose tissue. The protein expression of UCP1, which glows red after merging with green Perilipin, was increased in the Tg mouse on the right compared to the WT on the left (FIG. 10B).

In addition, when subjected to cold stimulation at 4°C, the rectal temperature of SerA1Tg mice remained higher than that of WT after 30 minutes, and observation using a thermography camera also showed that the temperature in the BAT of the neck of SerA1Tg mice at 180 minutes was high. Production was on the rise. The results are shown in FIG. This indicates that in SerA1Tg mice, UCP1 expression increases in brown adipose tissue and thermogenesis also increases.

When mRNA expression in the subcutaneous white adipose tissue of SerA1Tg mice was analyzed by qPCR, the expression of UCP1 was significantly increased (FIG. 12A), and the increase in UCP1 protein expression was also confirmed by immunostaining. Furthermore, the subcutaneous fat weight per body weight was significantly lower in SerA1Tg mice than in WT (FIG. 12B). Furthermore, when the tissue is stained with HE. It was confirmed that the lipid droplets had become smaller (FIG. 12C). The right diagram in FIG. 12C shows the results of measuring the diameter of lipid droplets, and it can be seen that the graph shifts to the left (fatty size becomes smaller) in SerA1Tg mice.
This suggests that subcutaneous white adipose tissue becomes beige due to SerpinA1, and this is thought to affect the phenotype of SerA1Tg mice.

WT mice and SerA1Tg mice were fed a high-fat diet for 3 months from the age of 5 weeks, and glucose tolerance and insulin resistance were confirmed. The results are shown in FIG. A is the fasting blood sugar level, B is the result of the glucose tolerance test (GTT), and C is the result of the insulin tolerance test (ITT). In SerA1Tg mice, while an improvement in fasting blood sugar was observed, similar to when ingesting normal food, no significant differences were observed in random blood sugar or body weight changes, but blood sugar improvement was observed not only in GTT but also in ITT. , not only showed high glucose tolerance but also improved insulin resistance.

(Example 6) Study of SerpinA1 knockout mouse The mouse SerpinA1 gene has five paralogs. As shown in Figure 14A, we created a quintiple knockout mouse (SerA1 KO mouse) in which all five paralogs encoding the mouse SerpinA1 gene were knocked out using CRISPR-Cas9 technology, and Western blot analysis revealed that SerpinA1 in the liver It was confirmed that there was no protein expression.
When GTT and ITT analyzes were performed using these mice, a significant increase in blood sugar was observed in KO mice compared to WT in GTT, and the AUC was also significantly higher, indicating that SerA1 KO mice had decreased glucose tolerance (Figure 14B ). In addition, in ITT, blood glucose levels at 15, 30, and 60 minutes after intraperitoneal insulin injection were significantly higher in KO mice than in WT, and a significant increase in AUC was observed in KO mice. Resistance was observed (Fig. 14C).

When SerA1 KO mice were placed in a metabolic cage and monitored under free feeding, a decrease in oxygen uptake was observed in the dark period (active period) compared to WT (Figure 15A). Furthermore, when cold stimulation was performed at 4°C, the rectal temperature of SerA1KO mice tended to decrease compared to WT, and a significant difference was observed in rectal temperature after 90 minutes (Figure 15B), and thermography also confirmed a decrease in body temperature. It was completed (Fig. 15C). The results of KO mice also support the thermogenic effect of SerpinA1 in brown adipose tissue in vivo.

(Example 7) Identification of SerpinA1 receptor SerpinA1 with a FLAG tag (3xFLAG) was introduced into brown preadipocytes using an adenovirus vector. As a control, an adenovirus vector carrying only FLAG (3xFLAG) was introduced into cells. Brown preadipocytes overexpressing FLAG-tagged SerpinA1 were prepared, treated with DTSSP, a chemical cross-linker, and FLAG-specific immunoprecipitation was performed, followed by proteomic analysis by mass spectrometry. As a result, EphB2 receptor was identified as a candidate molecule for SerpinA1 complex formation. Western blot analysis of the immunoprecipitated sample confirmed that SerpinA1 and EphB2 receptor were co-precipitated. From this, the EphB2 receptor was identified as a molecule that forms a complex with SerpinA1 and functions as a receptor.

(Example 8) Effect of SerpinA1 on EphB2 receptor The EphB2 receptor, which is a single-transmembrane receptor tyrosine kinase, transmits a signal into cells by binding to a ligand, ephrin. In the EphB2 receptor, tyrosine located near the transmembrane region is phosphorylated upon binding of a ligand. Therefore, the influence of SeprinA1 on the expression and phosphorylation of EphB2 was confirmed in vitro and in vivo as follows.
In an in vitro experiment, isolated brown preadipocytes were induced to differentiate into brown adipocytes, then SerpinA1 (300 μg/mL) was loaded and Western blot analysis was performed. As a result, the expression of EphB2 receptor was significantly higher in the SerpinA1-loaded group, and tyrosine phosphorylation of EphB2 receptor was also significantly enhanced in the SerpinA1-loaded group. The results are shown in FIG.

In in vivo experiments, Western blot analysis was performed using brown adipose tissue taken from WT and SerpinA1Tg mice. EphB2 receptor expression was significantly higher in the SerpinA1Tg mouse group, and tyrosine phosphorylation of the EphB2 receptor was also significantly enhanced in the SerpinA1Tg mouse group. The results are shown in FIG. From this, it was found that SerpinA1 enhances the expression and phosphorylation of EphB2 receptor in brown fat.

(Example 9) Study on EphB2 receptor KO mice In order to verify whether the effect of SerpinA1 to increase UCP1 expression on mature brown adipocytes is mediated by the EphB2 receptor, the EphB2 receptor was knocked out using CRISPR-Cas9 technology ( EphB2 receptor KO) brown adipose progenitor cell line was established. The expression of EphB2 receptor protein in the established cells was measured by Western blot analysis, and it was confirmed that the EphB2 receptor gene was knocked out. Mitochondrial activity was analyzed using the EphB2 receptor KO brown adipose progenitor cell line using OCR as an indicator. As shown in FIG. 18, it was found that the increase in oxygen consumption rate after FCCP administration due to SerpinA1 loading was attenuated by knockout of EphB2 receptor, and the maximum respiratory capacity and preliminary respiratory capacity were also attenuated. Furthermore, the results of analyzing UCP1 mRNA expression by qPCR are shown in FIG. 19. It was found that the increase in UCP1 expression caused by SerpinA1 loading was attenuated by knockout of the EphB2 receptor. These results suggested that the effect of SerpinA1 to increase UCP1 expression on mature brown adipocytes is mediated by EphB2.

(Example 10) SerpinA1 administration to brown adipose tissue Physiological saline or human SerpinA1 recombinant protein (1 mg) was administered to the brown adipose tissue (BAT) between the left and right shoulder blades of living mice, and the brown adipose tissue (BAT) was browned by cold stimulation. The thermogenic capacity of adipose tissue was evaluated. The results are shown in FIG. After 120 minutes after cold stimulation at 4°C, the rectal temperature of SerpinA1-administered mice remained higher than that of saline-administered mice. Furthermore, observation using a thermography camera also showed that heat production in the cervical BAT of SerpinA1-administered mice increased at 240 minutes. This indicates that SerpinA1 promotes thermogenesis in brown adipose tissue.

(Example 11) Analysis of Serum SerpinA1 Concentration in Patients with Type 2 Diabetes and Obesity After obtaining approval from the Ethics Committee of Kumamoto University, serum SerpinA1 was analyzed in metabolic syndrome patients with type 2 diabetes and obesity who were visiting the outpatient clinic. The concentration was verified. The results are shown in FIG. SerpinA1 values in serum were low in patients with poor diabetes (high HbA1c values), and an inverse correlation between HbA1c values and SerpinA1 was confirmed. Furthermore, the body mass index (BMI) and serum SerpinA1 concentration of diabetic patients were analyzed. As a result, it was confirmed that BMI and SerpinA1 concentration showed an inverse correlation. These results confirmed that SerpinA1 is also useful as a marker for diagnosing chronic metabolic diseases and understanding pathological conditions.

(Example 12) Analysis by loading SerA1KO mice with a high-fat diet SerpinA1 knockout (SerA1KO) mice were fed a high-fat diet for 3 months from the age of 5 weeks, and analyzed as a pathological model of obesity and diabetes. The results are shown in FIG. 22. A significant weight increase was observed in SerA1KO mice compared to WT. Furthermore, in SerA1KO mice fed a high-fat diet, an increase in the weight of white fat such as subcutaneous fat (iWAT) and visceral fat (eWAT) was also observed.

This application is based on Japanese Patent Application No. 2022-105460 (filing date: June 30, 2022) filed in Japan, and the contents thereof are all included in this specification.

The above detailed description is merely illustrative of the purpose and subject matter of the invention and is not intended to limit the scope of the appended claims. Various modifications and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings provided herein without departing from the scope of the appended claims.

The present invention provides a new adipocyte activator. The adipocyte activating agent of the present invention is useful as a pharmaceutical composition for preventing and/or treating obesity-related diseases.

Claims (23)

1. An activator for adipocytes containing SerpinA1 and/or a SerpinA1 expression promoter, wherein the adipocytes are adipocytes selected from brown adipocytes, brown adipocytes, and beige adipocytes. agent.
2. The adipocyte activator according to claim 1, which has an effect of promoting UCP1 (uncoupling protein 1) expression.
3. The adipocyte activator according to claim 1, wherein the adipocytes are brown preadipocytes, and the adipocyte activator has an effect of promoting proliferation of brown preadipocytes.
4. The adipocyte activator according to claim 1, wherein the adipocytes are beige adipocytes and have an effect of promoting beigeization of white adipocytes (transdifferentiation of white adipocytes into beige adipocytes).
5. The adipocyte activator according to claim 1, wherein the SerpinA1 is SerpinA1 (α1-antitrypsin) purified from mammalian plasma.
6. The SerpinA1 expression promoter is a substance selected from the group consisting of a substance that promotes transcription initiation of the SerpinA1 gene, a substance that inhibits degradation of mRNA of the SerpinA1 gene, and a substance that promotes transcription from the mRNA to SerpinA1 protein, or The adipocyte activator according to claim 1, which is a SerpinA1 nucleic acid construct.
7. An activator for adipocytes containing a SerpinA1 agonist, wherein the adipocytes are adipocytes selected from brown preadipocytes, brown adipocytes, and beige adipocytes.
8. The SerpinA1 agonist binds to EphB2 receptor (Ephrin type-B receptor 2) and has the activity of (1) enhancing expression and/or phosphorylation of EphB2 receptor, or (2) UCP1 in brown adipocytes. 8. The adipocyte activator according to claim 7, which is a substance having an activity of promoting the expression of (uncoupling protein 1).
9. The adipocyte activator according to claim 8, wherein the SerpinA1 agonist is selected from the group consisting of low molecular weight substances, high molecular weight substances, nucleic acid molecules, proteins, and peptides.
10. The adipocyte activator according to any one of claims 1 to 9, which has the effect of enhancing thermogenic action in adipose tissue.
11. A pharmaceutical composition for preventing and/or treating obesity and/or obesity-related diseases, comprising the adipocyte activator according to any one of claims 1 to 9.
12. The obesity-related disease is a disease selected from the group consisting of diabetes, non-alcoholic liver disease, hypertension, dyslipidemia, heart disease, cerebrovascular disorder, fatty liver, obesity-related kidney disease, and hyperuricemia. Item 12. Pharmaceutical composition according to item 11.
13. A pharmaceutical composition for preventing and/or treating obesity and/or obesity-related diseases, comprising a SerpinA1 expression promoter, wherein the SerpinA1 expression promoter is a substance that promotes transcription initiation of the SerpinA1 gene, SerpinA1. A pharmaceutical composition that is a substance selected from the group consisting of a substance that inhibits degradation of gene mRNA, a substance that promotes transcription of the mRNA to SerpinA1 protein, or a SerpinA1 nucleic acid construct.
14. The pharmaceutical composition according to claim 13, wherein the SerpinA1 nucleic acid construct is carried in a nucleic acid vector and/or included in a lipid nanoparticle.
15. The obesity-related disease is a disease selected from the group consisting of diabetes, non-alcoholic liver disease, hypertension, dyslipidemia, heart disease, cerebrovascular disorder, fatty liver, obesity-related kidney disease, and hyperuricemia. The pharmaceutical composition according to item 13 or 14.
16. A method of screening for drugs for preventing or treating obesity or obesity-related diseases, using the ability to promote the action of SerpinA1 as an indicator, and screening candidate substances having the ability to prevent or treat obesity or obesity-related diseases. A method, characterized in that it is identified as a drug for treatment.
17. The index is the ability to promote the expression of the SerpinA1 gene, and the ability to promote the expression of the SerpinA1 gene includes the ability to promote the production of SerpinA1 gene mRNA, the ability to promote transcription initiation of the SerpinA1 gene, and the ability to promote the SerpinA1 gene expression. 17. The method according to claim 16, wherein the ability is selected from the group consisting of the ability to inhibit the degradation of mRNA of SerpinA1, and the ability to promote transcription of the mRNA to SerpinA1 protein.
18. 17. The method according to claim 16, wherein the indicator is the action of SerpinA1 as an agonist on the EphB2 receptor.
19. The agonist action of SerpinA1 binds to the EphB2 receptor and (1) has the activity of enhancing the expression and/or phosphorylation of the EphB2 receptor, or (2) has the activity of enhancing UCP1 (uncoupling protein 1) in brown adipocytes. 19. The method according to claim 18, wherein the method has any of the following activities.
20. The method according to any one of claims 16 to 19, wherein the candidate substance is selected from the group consisting of a low molecular weight substance, a high molecular weight substance, a nucleic acid molecule, a protein, and a peptide.
21. According to any one of claims 16 to 19, the drug to be screened is a drug for improving obesity by enhancing lipolysis, improving insulin resistance, or acquiring obesity resistance by turning into beige adipocytes. the method of.
22. 22. The method according to claim 21, wherein the drug to be screened is a drug for preventing or treating obesity or obesity-related diseases.
23. The obesity-related disease is a disease selected from the group consisting of diabetes, non-alcoholic liver disease, hypertension, dyslipidemia, heart disease, cerebrovascular disorder, fatty liver, obesity-related kidney disease, and hyperuricemia. The method according to item 22.

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