Use of IAA and / or ILA in the manufacture of a medicament or a skin care product for the treatment of obesity, localised fat accumulation and their complications

Abstract

The application discloses use of IAA and / or ILA in preparation of medicines, foods or skin care products for treating obesity, local fat accumulation and complications thereof. Obesity and complications thereof are effectively treated by a brand-new mechanism of inducing white adipose browning, promoting fat thermogenesis and increasing energy consumption of the body.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and in particular to the use of IAA and / or ILA in the preparation of medicaments or skin care products for the treatment of obesity, localized fat accumulation and their complications. Background Technology

[0002] To maintain homeostasis, the body needs to obtain sufficient nutrients to meet its daily energy requirements. Simultaneously, the body needs to defend against invading pathogens. In mammals, these vital functions are controlled by the metabolic and immune systems, respectively; these two systems are well-known to be closely intertwined and precisely regulated. Imbalances between metabolic and immune responses are a contributing factor to various metabolic and cardiovascular diseases, including obesity and type 2 diabetes. However, the mechanisms underlying the integration of nutrition and immune responses in cases of malnutrition or overnutrition remain a crucial question.

[0003] The mammalian gut comes into extensive contact with pathogens and antigens, and is responsible for the absorption and metabolism of nutrients and drugs. It both senses and metabolizes nutrients and defends against pathogens. Therefore, the gut is a highly immune and nutritionally challenging site. In the context of chronic overnutrition, immune system dysregulation is a key potential mechanism in the pathogenesis of various metabolic diseases. However, the regulatory mechanisms and functions of the interaction between immune and metabolic functions in the gut remain largely unclear.

[0004] The gut microbiota is a key regulator of the host's immune system and metabolism, influencing host immune defense and energy metabolism through complex interactions between gut microbes and their metabolites and host cells. Gut bacteria produce various essential nutrients, such as short-chain fatty acids (SCFAs), branched-chain amino acids (BCAAs), bile acids, vitamins, and tryptophan and indole derivatives, which are crucial for host metabolic homeostasis. However, under abnormal conditions such as antibiotic use, disease, stress, aging, and poor dietary habits, the composition and abundance of the gut microbiota can be altered, leading to damage to the host. In fact, overnutrition has been shown to alter the production of gut metabolites (such as BCAAs, indolepropionic acid, and endocannabinoids), leading to gut microbiota dysbiosis and consequently obesity-related metabolic diseases such as type 2 diabetes. However, the mechanisms of interaction between gut metabolites and host cells in overnutrition-induced metabolic disorders remain largely unknown.

[0005] Circular 2',3'-GMP-AMP (cGAMP) synthase (cGAS) (Gene ID: 214763) is considered an intracellular aberrant double-stranded DNA (dsDNA) sensor that activates the type I interferon (IFN) pathway in response to viral infection. cGAS catalyzes the synthesis of the second messenger cGAMP, which subsequently binds to interferon gene-stimulating factor (STING), stimulating phosphorylation and activation of TANK-bindingkinase 1 (TBK1) and the transcription factor NF-κB, leading to the expression of type I interferon (IFN) and other inflammatory genes essential for immune defense. Therefore, activation of the cGAS / STING signaling pathway triggers an innate immune response involving IFN and pro-inflammatory cytokines to eliminate pathogens. In addition to exogenous pathogen DNA, cGAS can also be activated by endogenous DNA, such as mitochondrial DNA (mtDNA) and nuclear DNA aberrantly localized in the cytoplasm, thereby initiating a widespread inflammatory response leading to metabolic and renal diseases. cGAS is expressed in various metabolic tissues, including adipose tissue and the liver. However, because the gut is not only where bacteria and the host's immune system meet, but also the organ that processes nutrients, little is known about whether and how gut cGAS contributes to obesity and metabolic dysfunction caused by overnutrition.

[0006] This invention, combining in vitro, in vivo, and bioinformatics methods, has revealed the mechanism by which intestinal cGAS regulates energy homeostasis under physiological and pathophysiological conditions. A high-fat diet (HFD) significantly increased cGAS expression in the ileum and colon of mice. Furthermore, intestinal-specific cGAS knockout protected mice from HFD-induced obesity and metabolic dysfunction. Inhibition of cGAS in the gut altered the composition of the microbiota, increasing the production of the intestinal metabolites indole-3-acetic acid (IAA) and indole-3-lactic acid (ILA). IAA and ILA exert their anti-obesity effects by promoting thermogenesis from beige fat. These findings are the first to reveal the crucial role of intestinal cGAS in mediating excess-induced metabolic dysfunction, and further highlight the therapeutic potential of IAA and ILA in treating obesity and related metabolic disorders. Summary of the Invention

[0007] To address the aforementioned technical problems, the first objective of this invention is to provide the use of IAA and / or ILA in the preparation of medicaments or skin care products for treating obesity, localized fat accumulation, and their complications.

[0008] The obesity-related comorbidity mentioned refers to metabolic syndrome caused by obesity.

[0009] The treatment includes: promoting beige transformation of white adipose tissue, promoting fat breakdown, reducing fat accumulation, promoting fat thermogenesis, increasing energy expenditure, reducing weight, improving glucose metabolism, lowering blood sugar, and improving fatty liver.

[0010] IAA and / or ILA work by activating the PKA-UCP1 signaling pathway.

[0011] For the purposes described, the IAA and / or ILA are administered orally, intravenously, or subcutaneously.

[0012] As described above, the drug or skin care product includes an effective dose of IAA and / or ILA.

[0013] As described above, the drug also includes pharmaceutically acceptable excipients.

[0014] In the aforementioned applications, the excipients are selected from one or more of fillers, binders, disintegrants, solubilizers, and solvents.

[0015] A second objective of this invention is to provide the use of an agent that inhibits or knocks out the cGAS gene in the preparation of a medicament or skin care product for treating obesity, localized fat accumulation and its complications.

[0016] A third objective of this invention is to provide the use of an agent that reduces intestinal type I interferon in the preparation of medicaments or skin care products for treating obesity, localized fat accumulation and its complications.

[0017] A fourth objective of this invention is to provide the use of an agent that inhibits or knocks out the cGAS gene in the preparation of agents that alter the abundance of gut microbiota.

[0018] A fifth objective of this invention is to provide the use of an agent that increases intestinal tryptophan metabolites in the preparation of medicaments or skin care products for treating obesity, localized fat accumulation and its complications.

[0019] The sixth object of the present invention is to provide the use of an agent that increases intestinal lactobacilli in the preparation of medicaments or skin care products for treating obesity, localized fat accumulation and their complications.

[0020] This invention identifies a specific mechanism by which intestinal cGAS deficiency rebuilds the gut microbiota by reducing type I interferon, leading to an increase in lactobacilli and their metabolites indole-3-acetic acid (IAA) and indole-3-lactic acid (ILA), which directly activates PKA signaling and thermogenesis in adipocytes, ultimately improving obesity and related metabolic syndrome caused by overnutrition.

[0021] This invention found that HFD greatly induces the expression of intestinal cGAS, and in HFD-fed mice, knockout of intestinal cGAS remodeled the gut microbiota and improved metabolic disorders, suggesting that cGAS may be an important regulator between diet and gut microbiota.

[0022] This invention was discovered in cGAS IKO In mice, an increased abundance of *Lactobacillus murineis* was observed at the species level, which is consistent with cGAS. IKO The lean phenotype in mice corresponds to this. On the other hand, we found that ABX treatment or cohabitation with mice could reverse cGAS. IKO Beneficial metabolic effects in mice. Therefore, these findings suggest that gut microbiota, particularly lactobacilli, contribute to the development of beneficial metabolic effects in HFD-fed cGAS. IKO In mice, it plays a key role in mediating the beneficial effects of metabolic improvement induced by intestinal cGAS deficiency.

[0023] This invention discovers that tryptophan metabolites mediated by Lactobacillus murineis can directly target adipocytes to promote thermogenesis and combat obesity; this discovery may be one of the potential regulatory mechanisms by which tryptophan metabolites improve obesity and metabolic disorders.

[0024] This application utilizes a novel mechanism of action—inducing beige transformation of white adipose tissue, promoting fat thermogenesis, and increasing the body's energy expenditure—to effectively treat obesity and its complications.

[0025] This invention does not affect normal eating and digestion, does not affect the central nervous system, avoids serious side effects, and does not depend on significant changes in lifestyle and diet; it has virtually no side effects and can be used to prepare drugs or skin care products for treating obesity and its complications. Therefore, using the IAA and / or ILA described in this invention to prepare weight loss-related drugs has great development potential. Attached Figure Description

[0026] Figure 1 The process of demonstrating the activation of the gut cGAS signaling pathway in obese individuals and mice;

[0027] Figure 2 Gut-specific knockout of cGAS protects mice against HFD-induced obesity and metabolic dysfunction;

[0028] Figure 3 Intestinal-specific cGAS knockout promotes thermogenesis and energy expenditure in mice;

[0029] Figure 4 : Knockout of cGAS in the gut alters the gut microbiota in mice;

[0030] Figure 5Intestinal cGAS knockout increases indole-3-acetic acid levels in mice;

[0031] Figure 6 Indole-3-acetic acid acts directly on adipocytes, promoting thermogenesis and combating obesity.

[0032] Figure 7 Intestinal cGAS ablation promotes lactobacillus / indole-3-acetic acid (IAA)-mediated lipid thermogenesis by reducing intestinal type I interferon levels. Detailed Implementation

[0033] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Example 1

[0034] cGAS is expressed in all organs of mice, but it is expressed at a higher level in the intestine. Figure 1 A), especially in the jejunum and ileum, considering the large contact area between the intestine and nutrients and microorganisms, it is necessary to clarify whether excessive nutrition will affect the expression of cGAS in the mouse intestine. 60% high-fat induction for 8 weeks did not affect the expression of cGAS in the duodenum and jejunum of mice. Figure 1 B and 1C), but significantly increased cGAS expression in the ileum and colon ( Figure 1 D and Figure 1 E), and also significantly elevated levels of several pro-inflammatory cytokines (mRNA levels), including IL-1β, IL-6, and tumor necrosis factor-α (TNF-α). Figure 1 F and Figure 1 G), high-fat diets also caused damage to villi in the ileum and colon and loss of crypts in mice. Figure 1 H and Figure 1 I). Consistent with the above results, immunohistochemical staining and immunoblotting showed that the expression level of cGAS in the distal ileum of obese individuals was significantly higher than that in the non-obese control group. Figure 1 In summary, these results suggest a potential link between increased intestinal cGAS expression and impaired intestinal function in obese individuals and mice. Example 2

[0035] To investigate the tissue-specific role of intestinal cGAS, we generated an intestinal-specific cGAS knockout (cGAS) gene by crossing cGASfloxed (Loxp) mice with Villin-cre mice. IKO (mice). cGAS expression in cGAS IKO Different intestinal regions of mice, including the jejunum, ileum, and colon ( Figure 2 It is greatly inhibited in A), but not in other tissues ( Figure 2 B). Under normal feeding conditions, cGAS compared to Loxp control mice. IKO There were no significant differences in food intake, body weight, glucose tolerance, and insulin sensitivity among the mice. Figure 2 CF). High-fat (HFD) fed cGAS IKO There were no significant differences between the mice and the Loxp control group in terms of food intake, fecal output, fecal energy density, physical activity, and body length. Figure 2 G-2K). However, intestinal cGAS deficiency significantly reduced the body weight of HFD-fed mice ( Figure 2 L). Lack of cGAS in the mouse intestine also significantly reduced the volume of white fat (L). Figure 2 M) and weight ( Figure 2 N), body fat ( Figure 2 O) and fat cell size ( Figure 2 P). Compared with HFD-fed Loxp control mice, HFD-fed cGAS IKO Mice showed milder hepatic lipid deposition ( Figure 2 P) and glucose (P) Figure 2 Q) and insulin ( Figure 2 Improved tolerance (R). Intestinal-specific knockout of cGAS also significantly reduced HFD-induced plasma triglycerides (tg). Figure 2 S) and non-esterified fatty acids (NEFA) Figure 2 T), but had little effect on serum cholesterol, total bile acids (TBA), high-density lipoprotein and low-density lipoprotein levels in mice. Figure 2 In summary, these results identify the gut cGAS as a potential mediator of HFD-induced obesity and metabolic dysfunction. Example 3

[0036] To determine the mechanism by which intestinal cGAS knockout inhibits weight gain in mice, and whether intestinal cGAS deficiency affects energy expenditure, cGAS was investigated under thermoneutral conditions (30°C). IKO The energy expenditure of mice was similar to that of control mice, but under room temperature (22°C) or low temperature (6°C) conditions, cGAS IKO Mice have a higher oxygen consumption rate. Figure 3A and 3B), while energy consumption increases ( Figure 3 C). Furthermore, compared to the Loxp control group mice, cGAS IKO The respiratory exchange ratio (RER) of mice was significantly reduced. Figure 3 D), which indicates that cGAS IKO Mice consumed more fat as a fuel source to promote energy expenditure. Consistent with these results, compared with Loxp control mice, cGAS in sWAT and BAT was higher. IKO Mice all showed higher mitochondrial oxygen consumption rates (OCRs). Figure 3 E). Mouse intestinal cGAS knockout also significantly increased the mRNA levels of Ucp1 and Ppargc1-a in sWAT and BAT. Figure 3 F and 3G) and the protein levels of Ucp1 and PGC1a ( Figure 3 (H and 3I). In summary, these data suggest that gut-specific cGAS deficiency may alleviate obesity by promoting thermogenesis and increasing energy expenditure. Example 4

[0037] To determine whether altered gut microbiota helps improve cGAS IKO Metabolic phenotype of mice treated with broad-spectrum antibiotics (ABX) for cGAS IKO Mice and Loxp control mice. ABX treatment completely reversed the effects in cGAS. IKO The increased beneficial effects in mice included reduced body weight and fat mass, as well as improved glucose tolerance and insulin sensitivity. Figure 4 A-4D). Similar results were observed in cohabitation experiments ( Figure 4 E-4H). To further evaluate the effect of symbiotic gut bacteria on improving cGAS. IKO The contribution of mouse metabolic phenotype was determined using data from cGAS. IKO Fecal microbiota colonization of mice or Loxp control mice in antibiotic-treated (germ-free) mice Figure 4 I). Compared with mice receiving fecal microbiota from Loxp control mice (FMT-Loxp), mice receiving cGAS IKO Mouse (FMT-cGAS) IKO Fever microbiota-induced weight loss in HFD-fed mice ( Figure 4 J), fat weight reduction ( Figure 4 K), improved glucose tolerance ( Figure 4 L), increased insulin sensitivity ( Figure 4 M), UCP1 protein levels are elevated in adipose tissue ( Figure 4 In summary, these findings demonstrate the role of the gut microbiota in HFD-fed cGAS. IKOThe beneficial effects of cGAS deficiency-induced metabolic improvement in mice. To analyze the gut microbiota affected by cGAS deficiency, we used 16S rRNA gene sequencing to investigate the effects of cGAS deficiency. IKO The microbial communities of mice and the Loxp control group were analyzed. (In cGAS) IKO A significant difference in β-diversity of the gut microbiota was detected between mice and the Loxp control group. Figure 4 O), but α-diversity did not differ significantly ( Figure 4 P), indicating that the lack of cGAS altered the abundance of the gut microbiota but not its diversity. Linear discriminant analysis (LDA) effect size (LEfSe) analysis showed that the characteristic gut microbiota of the Loxp group was mainly composed of *Parabacteroides goldsteinii* from the phylum Bacteroidetes. On the other hand, cGAS... IKO The main species in the differential flora belong to the phylum Firmicutes, including *Lactobacillus murinus*, *Candidatus Arthromitus*, and *Eubacterium plexicaudatum*. Figure 4 Q and 4R). Through metagenomic analysis, in cGAS IKO At the species level in mice, the increased abundance of *Lactobacillus murinus* was further confirmed. However, *Lactobacillus murinus* in mice showed high homology with *Lactobacillus salivarius* and *Lactobacillus ruminans* in humans. Compared with normal controls, obese individuals showed a significant reduction in both *Lactobacillus salivarius* and *Lactobacillus ruminans*. Figure 4 These results suggest that increased lactobacilli may mediate cGAS (S and 4T). IKO It plays a key role in the beneficial metabolic effects in mice. Example 5

[0038] To elucidate the mechanism by which intestinal cGAS deficiency promotes thermogenesis in adipocytes, we investigated cGAS... IKO Non-targeted metabolomics analysis was performed on the feces of mice and control mice. To maximize cGAS... IKO The differences between LOXP mice and normalized untargeted metabolomics data were analyzed by pairwise orthogonal projection-potential structure discriminant analysis (OPLS-DA). Figure 5 A). Volcano plot analysis of non-target metabolomics data with folding changes > 2 and p-value < 0.05 identified 108 differentially expressed metabolites, of which 82 were upregulated and 26 were downregulated. Figure 5B). Hierarchical cluster analysis of the differentially metabolites obtained from the above analysis showed that six metabolites (L-Dopa, 5α-dihydrotestosterone, trigonelline, salsolinol, indole-3-acetic acid, and indole-3-lactic acid) were significantly different in cGAS. IKO Significant differences were found in the feces of mice compared to the control group. Figure 5 C). Tryptophan metabolism-related pathways have been primarily found in lactic acid bacteria (Gao et al., 2020), consistent with our findings that cGAS, compared to Loxp mice, […]. IKO The number of *Lactobacillus murinus* in mice increased significantly. Figure 5 D). Further correlation analysis between metabolites and the gut microbiota revealed that the tryptophan metabolites indole-3-acetic acid (IAA) and indole-3-lactic acid (ILA) showed a stronger correlation with Lactobacillus. Figure 5 E). Notably, compared to Loxp mice, cGAS IKO The level of IAA in the serum of mice was significantly higher than that in Loxp mice. Figure 5 F) suggests that IAA may be an important gut microbial metabolite involved in the metabolic improvements mediated by cGAS deficiency. Consistent with data from mice, human feces ( Figure 5 G) and serum ( Figure 5 H) IAA levels were negatively correlated with BMI. In summary, these findings suggest that an increase in IAA may be related to cGAS. IKO An important reason for obesity and improved metabolic phenotype in mice. Example 6

[0039] To investigate the cellular mechanism of IAA-induced thermogenesis in adipocytes, we treated primary white adipocytes with IAA (500 nM) for 24 hours. IAA treatment significantly stimulated the mRNA production of Ucp1 in primary white and brown adipocytes. Figure 6 A) and protein ( Figure 6 Expression of B and 6C), and phosphorylation of protein kinase A (PKA) substrate and camp-responsive element-binding protein (CREB). Figure 6 B and 6C). Furthermore, treatment of cells with the PKA inhibitor H89 significantly blocked IAA-induced phosphorylation of PKA substrates and CREB, as well as UCP1 expression. Figure 6 B and 6C), indicating that IAA may promote adipocyte thermogenesis by activating the PKA signaling pathway. To determine whether IAA-stimulated adipocyte thermogenesis could alleviate obesity in vivo, we administered IAA or a control vector (1 mg / kg, twice weekly for 6 weeks) to HFD-fed obese mice via gavage. Oral administration of IAA (1 mg / kg, twice weekly for 6 weeks) significantly reduced the body weight of HFD mice (B and 6C). Figure 6 D) Fat size and weight ( Figure 6 E and 6F) and adipocyte size ( Figure 6 G), which is related to improving liver osteoproliferation ( Figure 6 G) Glucose intolerance ( Figure 6 H) and insulin resistance (H) Figure 6 I) is related. Importantly, IAA processing also significantly increases sWAT ( Figure 6 J) and BAT ( Figure 6 UCP1 expression in K). Intravenous injection of IAA (100 µg / kg, twice a week for 6 weeks) significantly reduced body weight (K). Figure 6 L), improved HFD-induced metabolic disorders ( Figure 6 M-6Q), which is associated with increased UCP1 protein expression in adipose tissue. Figure 6 (R and 6S). In summary, these results reveal IAA as a promising target for anti-obesity drugs, alleviating obesity by promoting thermogenesis. Example 7

[0040] To explore the mechanism by which gut cGAS deficiency enhances *Lactobacillus murineis* and IAA, we investigated the potential involvement of the cGAS-sting signaling pathway in regulating *Lactobacillus murineis* abundance. We found that, compared to the control group, cGAS... IKO The cGAS-STING signaling pathway was significantly inhibited in the ileum and colon of mice. Figure 7 Consistent with this result, deficiency of intestinal cGAS in mice significantly reduced the mRNA levels of type I interferon-a (IFN-α) and -b (IFN-β) in the ileum. Figure 7 C), but not in the colon ( Figure 7 D). However, intestinal cGAS deficiency has little effect on serum IFN levels ( Figure 7 E), but significantly reduced mouse ileum ( Figure 7 F and 7G) and feces ( Figure 7 The levels of IFN-α and IFN-β in H) were determined. To determine whether IFN in the gut contributes to increasing cGAS IKO In comparing Lactobacillus murineis / IAA levels in mice, we first compared Loxp and cGAS. IKO Effects of mouse cecal extract on the growth of *Lactobacillus murineis*. We found that compared with Loxp mice, cGAS... IKO Mouse cecal extract has a greater effect on the growth rate and maximum growth concentration of Lactobacillus murineis. Figure 7 I). Furthermore, type I IFNs, especially IFN-α, directly inhibit the growth of lactobacilli in a dose-dependent manner. Figure 7 J), while reducing the generation of IAA (J). Figure 7 In summary, these results suggest that the improvement in IAA production and metabolic phenotype induced by intestinal cGAS deficiency is most likely mediated by intestinal type I IFN deficiency. Rectal administration of IFN-α significantly reversed the improved cGAS. IKO Mouse metabolic phenotypes, including weight loss ( Figure 7 L), fat content ( Figure 7 M), improved glucose tolerance ( Figure 7 N) and improved insulin sensitivity ( Figure 7 O), and elevated UCP1 protein levels in adipose tissue ( Figure 7 These results strongly suggest that the cGAS-STING-IFN signaling pathway in the gut negatively regulates fat thermogenesis by inhibiting the production of IAA in *Lactobacillus murineis* and mediating diet-induced obesity and its associated metabolic syndrome.

Claims

1. The use of a preparation for inhibiting the cGAS gene in the preparation of a drug for treating obesity; wherein the preparation for inhibiting the cGAS gene is: a cre / LoxP system reagent for knocking out the cGAS gene.

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