7-ketolithocholic acid for treatment of intestinal dysbacteriosis and for use as probiotic

Abstract

The present invention relates to the use of 3, 7-ketolithocholic acid (7-KCLA, 3 [alpha]-hydroxy-7-oxo-5 [beta]-cholanic acid) and pharmaceutically acceptable salts and glycine or taurine conjugates thereof in the prevention or treatment of intestinal dysbacteriosis, diseases and conditions associated with poor intestinal health, or in the improvement of the health of the intestinal microbiome.

Description

[0001] The present invention relates to compounds that exhibit prebiotic effects. In particular, the present invention relates to 7-ketolithocholic acid and its salts and glycine and taurine conjugates, which are useful as prebiotics, particularly for improving the health of the intestinal microbiome and for treating intestinal dysbiosis and diseases and conditions associated with poor intestinal health. The present invention also relates to prebiotic compositions comprising 7-ketolithocholic acid and its salts and glycine and taurine conjugates. Background of the Invention

[0003] The gut microbiome consists of communities of microorganisms in the small and large intestines. The microorganisms in the gut, collectively referred to as the microbiota, play a vital role in human and animal health and can indeed influence the development and progression of many diseases and conditions (Vyas, 2012). The number of species in the gut microbiota varies between individuals, but more than 1,000 different species of gut bacteria have been identified, and approximately 160 species are typically present in an individual's gut ( 2014). Examples of species that may be present in the gut microbiome include:

[0004] Beneficial bacteria, such as Bifidobacterium species, Lactobacillus species, Roseburia species, Eubacterium recale, and Faecalibacterium prausnitzii;

[0005] commensal species, such as Bacteroides and Enterococcus; and

[0006] · Pathogenic species, such as Desulfovibrionales, Clostridium histolyticum, Atopobium and Coriobacterium species.

[0007] Wilmanski (2021) believes that a healthy intestinal microbiome can successfully maintain long-term stability, resist invading pathogens, provide key nutrients to the host (including vitamins and fermentation byproducts), and help maintain host metabolic and immune homeostasis. Indicators of a healthy intestinal microbiome include high levels of certain beneficial microbial species, low levels of harmful microbial species, and diversity of microbial species.

[0008] The gut microbiota is essential for host digestion and complements the activity of mucosal mammalian enzymes in the liver and intestine (Rowland, 2018). For example, it plays a role in the breakdown of polysaccharides and polyphenols and the synthesis of vitamins. There is also evidence that the gut microbiota plays a role in a range of gastrointestinal diseases and disorders, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), colon cancer, and antibiotic-associated diarrhea, as well as obesity and diabetes (Greenblum, 2012; Marchesi, 2016).

[0009] Bacterial fermentation of dietary carbohydrates and resistant starch in the large intestine produces short-chain fatty acids, especially lactic acid, acetic acid, propionic acid, and butyric acid.

[0010] Butyrate is produced by Faecalibacterium spp., Roseburia spp., and Eubacterium spp. Butyrate participates in the acetyl CoA-butyl CoA pathway, which can both increase satiety and reduce inflammation. Other beneficial effects of butyrate include the prevention and treatment of diet-induced obesity and insulin resistance (Steliou, 2012; De Vadder, 2014), which is important for the prevention of metabolic syndrome and type II diabetes. Butyrate can also enhance the intestinal barrier function of intestinal epithelial cells and exert anti-inflammatory effects (Ma, 2012; Matter, 2005), in addition, it also has potential anti-cancer activity (Steliou, 2012; 2013).

[0011] Gut dysbiosis refers to changes in the bacterial colonisation of the intestine that are associated with disease manifestations. The composition of the changes may include a decrease in the number of beneficial microorganisms (such as beneficial bacteria), an increase in the number of pathogenic microorganisms (such as pathogenic bacteria), or a decrease in the total number of microorganisms that make up the intestinal microbiota. Studies have shown that gut dysbiosis can lead to one or more conditions selected from the following:

[0012] Obesity, hyperglycemia, metabolic syndrome, prediabetes, and type 2 diabetes ( 2018; Wu, 2020), inflammatory bowel disease (including ulcerative colitis and Crohn's disease), irritable bowel syndrome and other inflammatory conditions, and cardiovascular and central nervous system disorders (including anxiety and depression ( 2018, Flux, 2021), bipolar disorder (Wilmanski, 2021), sleep disorders and cognitive dysfunction (Pferschy-Wenzig, 2022), and schizophrenia (Flux, 2021). Martinez (2021) taught that gut microbiota dysbiosis is believed to contribute to the development of various immune-mediated conditions, including inflammatory bowel disease (IBD), rheumatoid arthritis, type 1 diabetes, multiple sclerosis, celiac disease, and systemic lupus erythematosus (SLE).

[0013] Mahmud (2022) taught that there is a connection between the gut microbiota and the skin microbiota, and that gut dysbiosis may lead to skin diseases including psoriasis, atopic dermatitis, acne vulgaris, rosacea, alopecia areata, and hidradenitis suppurativa.

[0014] There is also evidence that an imbalance in the gut microbiota may contribute to cancers such as colon cancer ( 2018) and liver disease.

[0015] Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (Hill, 2014), and probiotic microorganisms include strains of Bifidobacterium and Lactobacillus as well as Akkermansia muciniphila, Faecalibacterium prausnitzii, and Roseburia.

[0016] Research is also ongoing on prebiotics, which were originally defined as indigestible food ingredients that exert beneficial effects on the host by selectively stimulating the growth and / or activity of one or a limited number of bacterial species already resident in the colon, thereby attempting to improve the health of the host (Gibson, 1995). The original prebiotics were almost exclusively dietary fiber, but more recently it has been recognized that other substances may also have prebiotic effects, and prebiotics have recently been redefined as substances that can be selectively utilized by the host microbiome to confer health benefits (Gibson, 2017).

[0017] Bile acids play a role in glucose and lipid metabolism in humans and other mammals through a complex signaling system. The primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized in the liver from cholesterol. Before secretion into bile, taurine or glycine conjugates of the primary bile acids (primary bile salts) are formed, and these substances are then transported to the gallbladder and then secreted into the stomach as part of the digestive process. Most bile acids are recycled back to the liver in the enterohepatic circulation, but a small portion of bile acids enter the colon and interact with the intestinal microbiota.

[0018] Primary bile acids and bile salts are essential for the absorption of fats and fat-soluble vitamins as part of the digestive process, and they act as metabolic sensing molecules that transmit signals through G protein-coupled receptors (GPCRs) and nuclear receptors (NRs) to regulate lipid and glucose homeostasis. Primary bile acids and bile salts have cleansing properties and have antimicrobial effects that can inhibit microbial proliferation in the intestine. CA and CDCA are converted into secondary bileacids deoxycholic acid (DCA) and lithocholic acid (LCA) by intestinal bacteria, and these secondary bile acids act as second-line signaling molecules.

[0019] Recently, it has been discovered that in addition to playing a role in glucose and lipid metabolism, bile acids also affect the gut microbiome. For example, male patients who had previously undergone colorectal adenoma resection and were subsequently treated with ursodeoxycholic acid (UDCA) for three years were found to show an increased incidence of the beneficial microorganism Faecalibacterium prausnitzii (Pearson, 2019). UDCA has also been shown to reduce the levels of hydrogen- and methanogenic bacteria in the small intestine (Kim, 2020). Therefore, it has been proposed that it is possible to use UDCA as a prebiotic substance that can be selectively utilized by the host microbiome to promote beneficial bacteria in the intestine and bring health benefits.

[0020] The bile acid 7-ketolithocholic acid (7-KLCA) is metabolized in the intestine to produce UDCA, and the inventors surprisingly discovered that 7-KLCA has particularly advantageous prebiotic properties, having a positive effect on the growth of beneficial microorganisms in the intestinal microbiota. Summary of the invention

[0021] In a first aspect of the present invention, there is provided 7-ketolithocholic acid (7-KCLA, 3α-hydroxy-7-oxo-5β-cholanoic acid) having the formula:

[0022]

[0023] Or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, for preventing or treating intestinal flora imbalance and diseases and disorders associated with poor intestinal health, or for improving the health of the intestinal microbiome.

[0024] Surprisingly, the study found that 7-KLCA was able to improve the health of the gut microbiome by selectively promoting the growth of beneficial microorganisms, especially beneficial bacterial species.

[0025] Therefore, there is also provided a method for adjusting the composition of the intestinal microbiota of an individual to contain a greater proportion of beneficial microorganisms, the method comprising administering 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof to the individual.

[0026] definition

[0027] In this specification, unless the context requires otherwise due to expressive language or necessary meaning, the word "comprise" or variations such as "include" or "contain" are used in an inclusive sense, i.e., specifying the presence of the described features but not excluding the presence or addition of other features in various embodiments of the invention.

[0028] In the present specification, prebiotics are compounds that promote the growth of beneficial microorganisms in the intestine and have a prebiotic effect. Beneficial microorganisms will be discussed in more detail below, and beneficial microorganisms include, in particular, beneficial bacteria, including but not limited to Bifidobacteria species, Faecalibacterium prausnitzii, Eubacterium rectum, Roseburia, Lactobacillus, Bacteroides xylanisolvens and Eubacterium eligens.

[0029] The most suitable individuals for administration of prebiotics are humans, but in some cases, prebiotics can be used to treat other animals, especially for agricultural purposes. For example, prebiotics can be used to treat mammals, including pigs and ruminants, such as cattle, sheep and goats; poultry, such as chickens, turkeys and geese; and aquatic animals, such as fish.

[0030] In the present specification, "improving the health of the intestinal microbiome" means adjusting the composition of an individual's intestinal microbiota to include a higher proportion of beneficial microorganisms, especially beneficial bacteria, such as those described above.

[0031] In some cases, improving the health of the gut microbiome can also include adjusting the composition of the individual's gut microbiota to include a smaller proportion of harmful microorganisms, particularly harmful bacteria. Harmful bacteria include Atopobium, Coriobacterium, Clostridium histolyticum, Clostridium perfringens, Desulfovibrionales, Desulfuromonadales, and Dialister pneumosintes, particularly Atopobium, Coriobacterium, Clostridium histolyticum, Clostridium perfringens, Desulfovibrionales, Desulfuromonadales, and Dialister pneumosintes.

[0032] Improving the health of the gut microbiome can also include increasing the overall number of microorganisms in the gut microbiota.

[0033] In this specification, the fasting blood glucose level of a prediabetic patient is 100 to 125 mg / dL, and the fasting blood glucose level of a diabetic patient is 126 mg / dl or higher (American Diabetes Association).

[0034] According to the definition of the International Diabetes Federation, patients with metabolic syndrome typically have a combination of central obesity and any two of the following symptoms: high blood pressure (130 / 85 mmHg or higher), high plasma triglyceride levels (≥150 mg / dL), low plasma HDL cholesterol levels (<40 mg / dL), and elevated fasting plasma glucose (>5.6 mmol / L or 100 mg / dL).

[0035] "Hyperglycemia" is defined as a plasma glucose level of 11.1 mmol / L (200 mg / dL).

[0036] The salts of 7-KLCA are suitably non-toxic and pharmaceutically acceptable. Suitable pharmaceutically acceptable salts are well known to those skilled in the art and are described, for example, by Gupta et al. (2018). Some particularly suitable salts of compounds of formula (I) include base addition salts such as sodium, potassium, calcium, aluminum, zinc, magnesium and other metal salts and choline, diethanolamine, ethanolamine, ethylenediamine and meglumine salts.

[0037] In taurine conjugates of bile acids (including 7-KLCA), the side chain carboxylic acid group is replaced by: C(O)NHCH2CH2S(O)2OH, while in glycine conjugates, the carboxylic acid group is replaced by C(O)NHCH2C(O)OH. In vivo, bile acids are usually present in the form of their glycine or taurine conjugates, and conjugated bile acids are the main components of human bile.

[0038] Thus, one skilled in the art will appreciate that salts or glycine or taurine conjugates of 7-KLCA are expected to have substantially the same activity in vivo as the parent bile acid. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention provides 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, which is used for preventing or treating intestinal flora imbalance and diseases and disorders associated with poor intestinal health, or for improving the health of the intestinal microbiome.

[0041] In one aspect, 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof is provided for use in preventing intestinal flora imbalance.

[0042] In another aspect, 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof is provided for use in treating intestinal flora imbalance.

[0043] In another aspect, 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof is also provided for use in preventing diseases and disorders associated with poor intestinal health.

[0044] In yet another aspect, 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof is provided for use in treating diseases and conditions associated with poor intestinal health.

[0045] In another aspect, 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof is provided for use in improving the health of the intestinal microbiome.

[0046] Also provided is the use of 7-KLCA or a pharmaceutically acceptable salt thereof or a glycine or taurine conjugate thereof in the preparation of a medicament for preventing or treating intestinal flora imbalance and diseases and disorders associated with poor intestinal health, or for improving the health of the intestinal microbiome.

[0047] In one aspect of the present invention, there is provided use of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof in the preparation of a medicament for preventing intestinal flora imbalance.

[0048] In another aspect, the present invention provides use of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof in the preparation of a medicament for treating intestinal flora imbalance.

[0049] In another aspect, there is provided use of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof in the preparation of a medicament for preventing diseases and disorders associated with poor intestinal health.

[0050] Also provided is the use of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof in the preparation of a medicament for treating diseases and disorders associated with poor intestinal health.

[0051] In yet another aspect, the present invention provides use of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof in the preparation of a medicament for improving the health of the intestinal microbiome.

[0052] Also provided is a method for preventing or treating intestinal flora imbalance and diseases and disorders associated with poor intestinal health, or improving the health of the intestinal microbiome, the method comprising administering to an individual an effective amount of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof.

[0053] In one aspect of the present invention, a method for preventing intestinal flora imbalance is provided, the method comprising administering to an individual an effective amount of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof.

[0054] In another aspect, provided is a method for treating intestinal flora imbalance, the method comprising administering to an individual an effective amount of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof.

[0055] In yet another aspect, provided is a method for preventing diseases and disorders associated with poor intestinal health, the method comprising administering to an individual an effective amount of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof.

[0056] In another aspect, provided is a method for treating diseases and conditions associated with poor intestinal health, the method comprising administering to an individual an effective amount of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof.

[0057] In yet another aspect, provided is a method for improving the health of the intestinal microbiome, the method comprising administering to an individual an effective amount of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof.

[0058] As mentioned above, gut dysbiosis is associated with a variety of diseases and conditions, such as obesity, hyperglycemia, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), irritable bowel syndrome and autoimmune disorders (including rheumatoid arthritis, multiple sclerosis, type 1 diabetes, celiac disease, systemic lupus erythematosus), CNS disorders (including anxiety, depression, sleep disorders, cognitive dysfunction, schizophrenia and bipolar disorder), cardiovascular disease and skin diseases (including psoriasis, atopic dermatitis, acne vulgaris, rosacea, alopecia areata and hidradenitis suppurativa).

[0059] In some cases, gut dysbiosis can result from the use of antibiotics to treat bacterial infections. When antibiotics are administered to treat bacterial infections, it can have negative effects on the gut microbiota, including reduced species diversity and altered metabolic activity (often resulting in antibiotic-associated diarrhea), as well as a decrease in the total number of microorganisms present. It can also lead to the proliferation of antibiotic-resistant organisms in the gut microbiota, which may be particularly relevant to prolonged or frequent antibiotic use.

[0060] Since 7-KLCA and its salts and glycine and taurine conjugates promote the proliferation of beneficial microorganisms, such as bacterial species such as Bifidobacterium, Faecalibacterium prausnitzii, Eubacterium rectum, Roseburia, Lactobacillus, Bacteroides xylanolyticus and Eubacterium fussi, especially Bifidobacterium, Faecalibacterium prausnitzii and Roseburia, while inhibiting the proliferation of harmful bacterial species such as Atopobium, Bacillus rubrum, Clostridium histolyticum, Clostridium perfringens, Desulfovibrioles, Desulfovibrioles and Desulfomonasales, especially Atopobium, Bacillus rubrum, Clostridium histolyticum, Clostridium perfringens, Desulfovibrioles and Desulfomonasales, 7-KLCA and its salts, especially its pharmaceutically acceptable salts, and glycine and taurine conjugates are expected to be used to restore the health of the intestinal microbiome after treatment with antibiotics.

[0061] Therefore, 7-KLCA can be administered in combination with or after antibiotic administration to ensure that the gut microbiome is restored to a healthy state after bacterial infection.

[0062] 7-KLCA and its salts and glycine or taurine conjugates can effectively increase the number of beneficial microorganisms in the intestine and reduce the number of harmful microorganisms, and prevent or treat a variety of diseases and conditions associated with poor intestinal health. The inventors have demonstrated that 7-KLCA can selectively enhance the bifidobacterium, Faecalibacterium prausnitzii, Eubacterium rectum, Roseburia, Lactobacillus, Bacteroides xylanolyticus and Eubacterium fusilli in the intestinal microbiome, especially Bifidobacterium, Faecalibacterium prausnitzii and Roseburia (see Examples 1 and 2 below). This is particularly advantageous, as shown in Example 2, and contrary to the findings of Pearson 2019, this was not found after the administration of other bile acids (such as UDCA), at least within the time frame studied by the inventors. The inventors also demonstrated that 7-KLCA reduces the incidence of pathogenic microorganisms in the intestine, such as bacterial species, including Atopobium, Bacillus rubrum, Clostridium histolyticum, Clostridium perfringens, Desulfovibrioles and Desulfomonas order (see Example 2 again).

[0063] Bifidobacteria, such as Bifidobacterium longum subsp. longum, are known to have beneficial effects on the gut microbiome (Wong, 2019) and have been included in various probiotic products.

[0064] It is also known that Faecalibacterium prausnitzii has benefits, especially in reducing metabolic disorders, including obesity, hyperglycemia, insulin resistance, prediabetes, type 2 diabetes, and intestinal inflammation. Studies have found that Faecalibacterium prausnitzii can improve intestinal homeostasis and affect insulin sensitivity by producing SCFA (short-chain fatty acid) butyrate (Maioli, 2021; Wu, 2020). The reduction of SCFA-producing species (such as Faecalibacterium prausnitzii) is also associated with bipolar disorder (Wilmanski, 2021). The gut microbiome is associated with various neurocognitive and mental health disorders through the microbiome-gut-brain axis (Kraeuter, 2020; Flux, 2021; Pferschy-Wenzig, 2022). Therefore, improving the health of the gut microbiome can prevent or improve neurocognitive and mental health-related disorders, including anxiety, depression, sleep disorders, cognitive dysfunction, schizophrenia, and related mental disorders.

[0065] Roseburia is known to improve colonic motility, immune function, and has anti-inflammatory properties. It has been suggested that Roseburia affects various metabolic pathways and that they could be used to prevent and treat irritable bowel syndrome, obesity, prediabetes, type 2 diabetes, and allergies (Tamanai-Shacoori, 2017).

[0066] Bifidobacteria produce the SCFA acetate, which in turn serves as a “fuel” for butyrate-producing species such as Faecalibacterium prausnitzii and Roseburia. The gut microbiome of prediabetic patients is altered, with reduced levels of butyrate-producing bacteria, and this altered microbiome plays a role in the progression of the disease to type 2 diabetes. In addition, as mentioned above, butyrate can also enhance the intestinal barrier function of intestinal epithelial cells and exert anti-inflammatory effects (Ma, 2012; Matter, 2005), and it also has potential anti-cancer activity (Steliou, 2012; 2013).

[0067] There also appears to be a connection between the gut microbiota and the skin microbiota (Mahmud, 2022), such that disruptions to gut integrity and imbalances in the microbial community can have a significant impact on the overall homeostasis of the skin, meaning that improving the health of the gut microbiome may be beneficial in treating or preventing skin diseases. For example, Faecalibacterium prausnitzii and Ruminococcus species have been found to prevent psoriasis by competitively inhibiting pathogens and producing SCFAs. Lactobacillus species reduce skin inflammation and reduce the size of acne lesions by modulating the immune system, while Lactobacillus and Bifidobacterium species can be used to reduce the severity of atopic dermatitis (eczema). Reducing the number of pathogenic species such as Helicobacter pylori can reduce rosacea, while activation of the immune system associated with a healthy gut microbiome can reduce or prevent alopecia areata and hidradenitis suppurativa (Mahmud, 2022).

[0068] Reduced species diversity in the gut microbiota has also been associated with IBD, obesity, and type 2 diabetes (Wilmanski, 2021; Flux, 2021).

[0069] Due to its effect on the intestinal microbiota, 7-KLCA, its glycine and taurine conjugates and salts thereof, especially its pharmaceutically acceptable salts, can be used to prevent and treat a variety of diseases and conditions associated with poor intestinal health, including obesity, hyperglycemia, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), irritable bowel syndrome and autoimmune disorders (including rheumatoid arthritis, multiple sclerosis, type I diabetes, celiac disease, systemic lupus erythematosus), CNS disorders (including anxiety, depression, sleep disorders, cognitive dysfunction, schizophrenia and bipolar disorder), cardiovascular disease and skin diseases (including psoriasis, atopic dermatitis, acne vulgaris, rosacea, alopecia areata and hidradenitis suppurativa).

[0070] In particular, the diseases and conditions include obesity, hyperglycemia, metabolic syndrome, type 2 diabetes, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), irritable bowel syndrome, and autoimmune disorders (including rheumatoid arthritis, multiple sclerosis, type I diabetes, and celiac disease). More particularly, the diseases and conditions include obesity, hyperglycemia, metabolic syndrome, and type 2 diabetes.

[0071] WO 2017 / 019524 and WO 2015 / 183794 both teach that several bile acids have TGR5 agonist activity, so they can be used to treat diseases and conditions such as obesity, diabetes and metabolic syndrome. There are many references in the prior art about the TGR5 agonist activity of bile acids (such as taurolithocholic acid, lithocholic acid, deoxycholic acid, chenodeoxycholic acid and cholic acid) (e.g., Guo, 2016). WO 2017 / 019524 also teaches that some bile acids have FXR agonist activity, and FXR can participate in glucose and insulin metabolism. Bile acids (such as chenodeoxycholic acid) and bile acid derivatives (such as obeticholic acid) are known to have FXR agonist activity (Guo, 2018). However, there is no evidence in the literature that 7-KLCA has significant activity as a TGR5 agonist or as a FXR agonist, and therefore, it cannot be expected from the teachings of documents such as WO 2017 / 019524 and WO 2015 / 183794 that 7-KLCA can be used to treat TGR5 and FXR-mediated disorders such as obesity, diabetes and metabolic syndrome.

[0072] Due to its ability to promote the growth of bifidobacteria and butyrate-producing bacterial species, 7-KLCA and its salts, especially its pharmaceutically acceptable salts, and its glycine and taurine conjugates are also used to enhance the intestinal barrier function of intestinal epithelial cells and as an anti-inflammatory active agent.

[0073] Another aspect of the present invention provides a method for adjusting the composition of an individual's intestinal microbiota to contain a higher proportion of beneficial microorganisms, the method comprising administering 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof to the individual.

[0074] Beneficial microorganisms may include bacteria, for example bacteria selected from the group consisting of Bifidobacterium, Faecalibacterium prausnitzii, Eubacterium rectale, Roseburia, Lactobacillus, Bacteroides xylanisolvens and Eubacterium fusilli, in particular Bifidobacterium, Faecalibacterium prausnitzii and Roseburia.

[0075] In addition, the method may also include adjusting the intestinal microbial composition of the individual to include a lower proportion of pathogenic microorganisms. The pathogenic microorganisms may include bacteria, such as bacteria selected from the following: Atopobium, Bacillus rubrum, Clostridium histolyticum, Clostridium perfringens, Desulfovibrioles, Desulfomonasales and Dialisterella pneumophila, especially Atopobium, Bacillus rubrum, Clostridium histolyticum, Clostridium perfringens, Desulfovibrioles and Desulfomonasales.

[0076] The method may also comprise increasing the number of microorganisms, particularly bacteria, in the intestinal microbiota.

[0077] In some cases, individuals may require modification of their gut microbiota following antibiotic treatment because, as mentioned above, antibiotic treatment may negatively affect the gut microbiota.

[0078] The method may thus comprise administering 7-KLCA concurrently with or subsequent to antibiotic treatment.

[0079] Antibiotics and 7-KLCA or its salt, especially pharmaceutically acceptable salt, or its glycine or taurine conjugate can be administered together and can also be provided in the form of a single composition. Alternatively, they can be provided as separate compositions suitable for separate or sequential administration.

[0080] Suitably, the antibiotic is selected from a macrolide (e.g. clarithromycin or erythromycin), a penicillin (e.g. nafcillin, ampicillin or amoxicillin), a cephalosporin (e.g. cefazolin), a carbepenem (e.g. imipenem or aztreonam), another β-lactam antibiotic, a β-lactam inhibitor (e.g. sulbactam), an oxaline (e.g. linezolid), an aminoglycoside (e.g. gentamicin), chloramphenicol, Sulfonamides (e.g., sulfamethoxazole), glycopeptides (e.g., vancomycin), quinolones (e.g., ciprofloxacin), tetracyclines (e.g., minocycline), fusidic acid, trimethoprim, metronidazole, clindamycin, mupirocin, rifamycins (e.g., rifampicin), streptogramins (e.g., quinupristin and dalfopristin), lipocinoids (e.g., daptomycin), polyenes (e.g., amphotericin B), azoles (e.g., fluconazole), and echinocandins (e.g., caspofungin acetate).

[0081] 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof is intended for use as a prebiotic and is therefore suitably administered as part of a composition, suitably as part of an orally administrable composition.

[0082] Therefore, another aspect of the present invention provides a prebiotic composition comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof as the sole active agent, and a suitable carrier.

[0083] In some embodiments, the composition does not contain water-soluble starch conversion products resulting from partial hydrolysis of starch.Examples of water-soluble starch conversion products include maltodextrin, dextrin, liquid glucose, corn syrup solids, liquid glucose, and soluble starch.

[0084] In some embodiments, the composition is free of maltodextrin.

[0085] In some embodiments, the composition does not contain water-soluble non-starch polysaccharides, such as guar gum, pectin, gum arabic, psyllium, oat gum, soy fiber, oat bran, corn bran, cellulose, or wheat bran.

[0086] In some embodiments, the composition does not contain guar gum, pectin, or gum arabic.

[0087] In some embodiments, the composition contains neither water-soluble starch conversion products obtained by partial hydrolysis of starch nor water-soluble non-starch polysaccharides.

[0088] In some embodiments, the composition contains neither maltodextrin nor any of guar gum, pectin, and gum arabic.

[0089] Suitably, the composition is suitable for oral administration. It can be formulated into separate units, such as capsules, sachets or tablets, each of which contains a predetermined amount of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof; or formulated into a powder or granules.

[0090] For tablets and capsules, suitable carriers include vehicles, such as common excipients, such as adhesives, such as syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinyl pyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sucrose and starch; fillers and carriers, such as corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, calcium hydrogen phosphate, sodium chloride and alginic acid; and lubricants, such as magnesium stearate, sodium stearate and other stearic acid metal salts, stearyl glyceride, stearic acid, silicone oil, talcum wax, oil and colloidal silica. In some compositions, the excipient is not a soluble starch conversion product or a soluble non-starch polysaccharide as described above. In particular, in some embodiments, the excipient may not include maltodextrin. In other embodiments, the excipient does not include one or more of guar gum, pectin and gum arabic. Flavoring agents such as peppermint, wintergreen oil, cherry flavoring agents, etc. can also be used. It may be desirable to add a coloring agent to render the dosage form easily identifiable. The tablets may also be coated by methods well known in the art.

[0091] Tablets can be made by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared by compressing the active agent in a free-flowing form, such as a powder or granules, in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersant. Molded tablets can be prepared by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine. Tablets can be optionally coated or scored, and can be formulated to provide slow release or controlled release of the active agent.

[0092] Other formulations suitable for oral administration include lozenges containing 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, in a flavored base, typically sucrose and acacia or tragacanth; lozenges containing 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, in an inert base, such as gelatin and glycerin, or sucrose and acacia.

[0093] Alternatively, the composition may be a food or beverage product comprising 7-KLCA or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a glycine or taurine conjugate thereof, and a suitable flavoring agent. Foods include, for example, yogurt or bread, while beverages may be milk-based or water-based, and preferably contain flavoring agents and / or taste masking agents to mask the bitter taste of 7-KLCA.

[0094] As mentioned above, one of the uses of 7-KLCA and its salts, especially its pharmaceutically acceptable salts, and glycine and taurine conjugates is to restore the intestinal microbiome to a healthy state during or after the administration of antibiotics. Therefore, the composition may also include an antibiotic, such as one of the antibiotics listed above.

[0095] Also provided is a prebiotic composition comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, and a suitable carrier for preventing or treating intestinal flora imbalance, diseases and disorders associated with poor intestinal health, or for improving the health of the intestinal microbiome.

[0096] In one aspect, a prebiotic composition is provided, comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, for preventing intestinal flora imbalance.

[0097] In another aspect, a prebiotic composition is provided, comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, for use in treating intestinal dysbiosis.

[0098] In another aspect, a prebiotic composition is provided comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, for use in preventing diseases and disorders associated with poor intestinal health.

[0099] In yet another aspect, a prebiotic composition is provided comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, for use in treating diseases and conditions associated with poor intestinal health.

[0100] In another aspect, a prebiotic composition is provided comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, for improving the health of the intestinal microbiome.

[0101] The present invention also provides a use of a prebiotic composition comprising 7-KLCA or a pharmaceutically acceptable salt thereof or a glycine or taurine conjugate thereof and a suitable carrier in the preparation of a medicament for preventing or treating intestinal flora imbalance, diseases and disorders associated with poor intestinal health, or for improving the health of the intestinal microbiome.

[0102] In one aspect of the present invention, there is provided a use of a prebiotic composition comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof in the preparation of a medicament for preventing intestinal flora imbalance.

[0103] In another aspect, the present invention provides a use of a prebiotic composition comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof in the preparation of a medicament for treating intestinal dysbacteriosis.

[0104] In another aspect, there is provided a use of a prebiotic composition comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, in the preparation of a medicament for preventing diseases and disorders associated with poor intestinal health.

[0105] Also provided is the use of a prebiotic composition comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, in the preparation of a medicament for treating diseases and disorders associated with poor intestinal health.

[0106] In yet another aspect, the present invention provides the use of a prebiotic composition comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, in the preparation of a medicament for improving the health of the intestinal microbiome.

[0107] Also provided are methods for preventing or treating intestinal dysbiosis, diseases and conditions associated with poor intestinal health, or for improving the health of the intestinal microbiome, the methods comprising administering to an individual a prebiotic composition comprising 7-KLCA or a salt thereof or a glycine or taurine conjugate and a suitable carrier.

[0108] In one aspect of the invention, there is provided a method for preventing intestinal flora imbalance, the method comprising administering to an individual an effective amount of a prebiotic composition comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof.

[0109] In another aspect, a method of treating intestinal dysbiosis is provided, the method comprising administering to an individual an effective amount of a prebiotic composition comprising 7-KLCA or a salt thereof, particularly a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof.

[0110] In yet another aspect, a method of preventing diseases and conditions associated with poor intestinal health is provided, the method comprising administering to an individual an effective amount of a prebiotic composition comprising 7-KLCA or a salt thereof, particularly a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof.

[0111] In another aspect, a method of treating diseases and conditions associated with poor intestinal health is provided, the method comprising administering to an individual an effective amount of a prebiotic composition comprising 7-KLCA or a salt thereof, particularly a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof.

[0112] In yet another aspect, a method of improving the health of the intestinal microbiome is provided, the method comprising administering to an individual an effective amount of a prebiotic composition comprising 7-KLCA or a salt thereof, particularly a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof.

[0113] Also provided is a method of adjusting the composition of an individual's intestinal microbiota to contain a higher proportion of beneficial microorganisms (e.g., beneficial bacteria), the method comprising administering to the individual a prebiotic composition comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof and a suitable carrier.

[0114] As mentioned above, beneficial bacteria include Bifidobacterium, Faecalibacterium prausnitzii, Eubacterium rectale, Roseburia, Lactobacillus, Bacteroides xylanolyticus, and Eubacterium fusilli.

[0115] The method may also comprise modifying the composition of the individual's intestinal microbiota to contain a lower proportion of pathogenic microorganisms, particularly pathogenic bacteria.

[0116] As mentioned above, pathogenic bacteria include Atopobium, Rhodococcus, Clostridium histolyticum, Clostridium perfringens, Desulfovibrioles, Desulfomonasales, and Dialisterella pneumophila.

[0117] The method may also comprise increasing the number of microorganisms, particularly bacteria, in the intestinal microbiota.

[0118] Regarding the medical use of 7-KLCA or its salt, especially a pharmaceutically acceptable salt, or its glycine or taurine conjugate, other preferred aspects are as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1Graph showing the effect of 7-KLCA and maltodextrin (as carbon source) on various bacterial species in batch culture fermentation slurries from fecal samples 0, 5, 10 and 24 hours after inoculation of the slurry. In the graph, EUB represents total bacteria; BIF represents Bifidobacterium; LAB represents Lactobacillus-Enterococcus; BAC represents Bacteroides-Prevotella; EREC represents Eubacterium rectum-Clostridium coccoides; RREC represents Roseburia and Eubacterium rectum; ATO represents Atopobium-Russobacterium; PROP represents Clostridial cluster IX; FPRAU represents Faecalibacterium prausnitzii; DSV represents Desulfovibrioles and Desulfuromonadales; and CHIS represents Clostridium histolyticum-perfringens.

[0121] Figure 2 is a graph showing the effect of maltodextrin (as a carbon source) alone on various bacterial species in batch culture fermentation slurries from fecal samples 0, 5, 10 and 24 hours after inoculation of the slurry. In this figure, EUB represents total bacteria; BIF represents Bifidobacterium; LAB represents Lactobacillus-Enterococcus; BAC represents Bacteroides-Prevotella; EREC represents Eubacterium rectum-Clostridium cocci; RREC represents Roseburia and Eubacterium rectum; ATO represents Atopobium-Russobacterium; PROP represents Clostridium group IX; FPRAU represents Faecalibacterium prausnitzii; DSV represents Desulfovibrioles and Desulfomonasales; CHIS represents Clostridium histolyticum--Clostridium perfringens.

[0122] Figure 3 is a series of graphs showing the production of the SCFAs acetate, propionate, butyrate and lactate in batch culture fermentation slurry samples from fecal samples and uninoculated control samples (NEG) using 7-KLCA and maltodextrin together (7-KLCA) or maltodextrin alone (MAL) 0 (T0), 5 (T5), 10 (T10) and 24 (T24) hours after inoculation of the slurry.

[0123] Figure 4It is a series of figures showing the flow-FISH analysis results of embodiment 2, and these results are from 3-stage continuous culture fermentation system after 512 hours (8 full volume turnovers, 14 days) reaching the baseline sample of the first stable state (SS1) and UDCA and 7KLCA after 512 hours (8 full volume turnovers, 14 days).These figures show total bacterial community (EUB-I-II-III), Bifidobacterium (Bifidobacterium spp.) (Bif164), Lactobacillus (Lactobacillus spp.) (Lab158) and Bacteroides-Prevotella (Bac303).These figures show the bacterial count in units of log10 (CFU / mL).Error bar (error bar) is SEM.

[0124] Figure 5 It is a series of figures showing the flow-FISH analysis results of embodiment 2, and these results are from the baseline sample of 3-stage continuous culture fermentation system reaching the first stable state (SS1) after 512 hours (8 full volume turnovers, 14 days) and UDCA and 7KLCA through the sample after 512 hours (8 full volume turnovers, 14 days).These figures show rectal eubacterium (Erec482), Roseburia (Rrec584), Atopobium (Atopobium spp.) (Ato291) and Clostridium group IX (Prop853).These figures show the bacterial count in units of log10 (CFU / mL).Error bar is SEM.

[0125] Figure 6 It is a series of figures showing the flow-FISH analysis results of Example 2, and these results are from the baseline sample of the 3-stage continuous culture fermentation system reaching the first stable state (SS1) after 512 hours (8 full volume turnovers, 14 days) and UDCA and 7KLCA after 512 hours (8 full volume turnovers, 14 days). These figures show Faecalibacterium prausnitzii (Fprau655), Desulfovibrioles (DSV687), Clostridium histolyticum (Chis150) and Streptococcus (Streptococcusspp.) (Strp). These figures show the bacterial count in units of log10 (CFU / mL). Error bars are SEM.

[0126] Figure 7 A-7E is a series of graphs showing the concentrations of the bacterial metabolites lactate (7A) and the single-chain fatty acids acetate (7B), butyrate (7C), propionate (7D) and valerate (7E) detected by GC-MS after the first stable state (SS1) and after UDCA and 7KLCA treatment. Error bars are standard error of the mean (SEM).

[0127] FIG8 is two graphs of samples collected from V3 (distal colon) before (upper trace) and after (lower trace) 7-KLCA treatment. 1 Comparison of H NMR spectra, where peaks associated with bacterial metabolites are marked (see Example 2). Fig. 8A The area from 2.5ppm to 0ppm is shown. Figure 8B The region from 4.0 ppm to 2.5 ppm is shown.

[0128] Fig. 9 is a schematic diagram of the study design of Example 3, showing the diets of the test and control group mice.

[0129] Fig.10 It is a graph showing the weight change (expressed in g) of mice in the normal diet (CTRL 1), normal diet + 7-KLCA (CTRL 2), HGD diet (CTRL 3), and HGD diet + 7-KLCA (treatment) groups from day 0 to day 60 (experimental endpoint) in Example 3. The data are expressed as the mean ± SD of n = 5 mice per group.

[0130] Fig.11 It is a graph showing blood glucose levels of mice in the normal diet (CTRL 1), normal diet + 7-KLCA (CTRL 2), HGD diet (CTRL 3), and HGD diet + 7-KLCA (treatment) groups of Example 3 on day 60. The values ​​expressed in mg / dl represent n=5 for each group.

[0131] Fig.12 It is a graph showing the HDL levels of mice in the normal diet (CTRL 1), normal diet + 7-KLCA (CTRL 2), HGD diet (CTRL 3), and HGD diet + 7-KLCA (treatment) groups of Example 3 on day 60. The values ​​expressed in mg / dl represent n=5 for each group.

[0132] Fig.13 It is a graph showing the LDL levels of mice on day 60 in the normal diet (CTRL 1), normal diet + 7-KLCA (CTRL 2), HGD diet (CTRL 3), and HGD diet + 7-KLCA (treatment) groups of Example 3. The values ​​expressed in mg / dl represent n=5 for each group.

[0133] Fig.14is a series of graphs showing total cholesterol levels of mice on day 60 in the normal diet (CTRL 1), normal diet + 7-KLCA (CTRL 2), HGD diet (CTRL 3), and HGD diet + 7-KLCA (treatment) groups in Example 3. Values ​​expressed in mg / dl represent n=5 for each group.

[0134] Fig.15 is a Krona plot showing both the abundance and hierarchy of DNA extracted from samples before 7-KLCA treatment. The Krona plot features a color gradient that represents the average e-value of BLAST hits within each taxon.

[0135] Fig.16 is a Krona plot showing both the abundance and hierarchy of DNA extracted from proximal colon (V1) samples after 7-KLCA treatment.

[0136] Fig.17 is a Krona plot showing both the abundance and levels of DNA extracted from transverse colon (V2) samples after 7-KLCA treatment.

[0137] Fig.18 is a Krona plot showing both the abundance and hierarchy of DNA extracted from distal colon (V3) samples after 7-KLCA treatment. Example

[0138] Example 1 - Batch culture fermentation

[0139] Fresh stool samples were collected 1 hour before inoculation from healthy adult male and female donors who had not taken antibiotics and had no history of gastrointestinal disease within 3 months before collection. Stool was diluted in phosphate buffered saline (PBS, pH 7.0) at a ratio of 1:10 and then mixed in a stomacher for 2 minutes to make a stool slurry.

[0140] The method used was as described by Olano-Martin et al. (2000). 9 mL of autoclaved nutrient medium was added to a 10 mL working volume fermentation vessel. The nutrient medium contained (g / L -1 ): peptone water, 2; yeast extract, 2; NaCl, 0.1; K2HPO4, 0.04; KH2PO4, 0.04; MgSO4.7H2O, 0.01; CaCl2.6H2O, 0.01; NaHCO3, 2; hemin (dissolved in a few drops of 1 mol 1 -1 NaOH), 0.05; cysteine ​​HCl, 0.5; bile salts, 0.5; 80,2 and 10 μ L of vitamin K, supplemented to 1 L with ddH2O.By gaseous N2 of culture medium, purge overnight to produce anaerobic conditions.By circulating water bath, container is maintained at 37 ℃, and pH value is maintained between 6.7 and 6.9 to simulate the condition in distal colon using the pH controller connected with 0.25M HCl and NaOH solution.Before inoculating feces sample, immediately 0.1g maltodextrin (1%, w / v) and 5mg 7-KLCA (0.5mg / mL, w / v) are added in container, then 1mL (v / v) feces slurry is added.After inoculating slurry, immediately (0 hour) and take out sample from fermentation tank after 5 hours, 10 hours and 24 hours, to carry out bacterial count and metabolite analysis.

[0141] Bacterial counting by flow cytometry-fluorescence in situ hybridization (Flow-FISH)

[0142] Samples (750 μL) were taken from the in vitro fermentation vessel at 0, 5, 10 and 24 hours, immediately placed on ice, then centrifuged at 13,000 × g for 5 minutes, and the supernatant was discarded. The pelleted bacteria were fixed at 4°C in a 1:3 (v / v) ratio of PBS and 4% (w / v) filtered paraformaldehyde (PFA, pH 7.2) for 4 hours. The samples were washed twice with filtered PBS and resuspended in 300 μL PBS and ethanol (1:1, v / v) and then stored at -20°C for up to 3 months.

[0143] Hybridization was performed using the method described by Rigottier-Gois et al. (2003) using genus- and group-specific 16S rRNA targeting oligonucleotide probes. The primers used were Non-Eub, Eub338, Bif164, Lab158, Bac303, Erec482, Rrec584, Ato291, Prop853, Fprau655, DSV687, and Strc493, as shown in Table 1 below. Flow cytometry (Accurate flow cytometry) was used. TM C6, BD Biosciences, USA) and Accuri TM CFlow software screened samples.

[0144] Table 1 - Fluorescence in situ hybridization oligonucleotide probe sequences (50 ng / μL)

[0145]

[0146]

[0147] R stands for G / A

[0148] Y stands for T / C

[0149] After thawing, vortex the sample for 10 seconds, add 75 μL of sample to 500 μL PBS in a 1.5 mL tube, vortex again, and centrifuge at 13,000 × g for 3 minutes. Remove the supernatant and discard. Add 100 μL Tris-EDTA buffer containing lysozyme (1 mg / mL) to the tube, mix with a pipette, and then incubate in the dark for 10 minutes. Vortex the sample and centrifuge at 13,000 × g for 3 minutes, and remove the supernatant. Add 500 μL PBS to the tube, resuspend the pellet with a pipette, then vortex and centrifuge at 13,000 × g for 3 minutes. Remove the supernatant, resuspend the pellet in 150 μL hybridization buffer (0.9 M NaCl, 0.2 M Tris-HCl (pH 8.0), 0.01% sodium dodecyl sulfate, 30% formamide), vortex and centrifuge at 13,000 × g for 3 minutes. The supernatant was removed again and the pellet was resuspended in 1 mL of hybridization buffer. 4 μL of oligonucleotide probe solution (50 ng / μL) was added to a 1.5 mL centrifuge tube together with 50 μL of sample, vortexed and incubated at 36°C overnight.

[0150] After hybridization is complete, add 125 μL of hybridization buffer to each tube, vortex, centrifuge at 13,000 × g for 3 minutes, and carefully remove the supernatant. Add 175 μL of wash buffer (0.064M NaCl, 0.02M Tris / HCl (pH8.0), 0.5M EDTA (pH8.0), 0.01% sodium dodecyl sulfate) to each tube, keep at 40°C, and resuspend the precipitate. The tubes are then vortexed and incubated at 38°C in the dark for 20 minutes to remove nonspecifically bound primers. The samples are then centrifuged at 13,000 × g for 3 minutes, the supernatant is removed, 300 μL of PBS is added, and then vortexed. The samples are refrigerated in the dark at 4°C before flow cytometric analysis.

[0151] Flow-FISH Overview

[0152] These batch cultures focused on the effect of 7-KLCA on bacterial populations. Samples were taken at 0, 5, 10, and 24 hours for flow-FISH analysis, which showed changes in bacterial composition at different 7-KLCA concentrations. Samples from three donors were used in these fermentations, using vessels containing either 7-KLCA and maltodextrin (as a carbon source), or maltodextrin alone (as a carbon source).

[0153] The results are shown in Table 2 and Figure 1 as well as Figure 2 The analysis showed that in the container containing 7-KLCA, the total bacteria ( Figure 1 and Figure 2EUB in the culture medium, total amount in Table 1) and Bifidobacterium spp. (BIF) increased statistically significantly. The number of Bifidobacterium increased from Log10 6.48±0.14 CFU / mL to Log10 7.45±0.16 CFU / mL at T5, and increased to Log10 7.55±0.21 CFU / mL at T10.

[0154] Meanwhile, the number of bifidobacteria did not increase significantly in the container with maltodextrin alone, only the number of total bacteria and lactobacilli (LAC) increased significantly at 5 h.

[0155] There was also no increase in pathogenic bacterial species, including Atopobium, Rhabditis erythrorhizium, Clostridium histolyticum, Clostridium perfringens, Desulfovibrioles, and Desulfomonalales, in the containers containing 7-KLCA and the control containers.

[0156] Table 2

[0157]

[0158] Bacteria covered (see Table 1)

[0159] BIF – Bifidobacterium

[0160] LAB – Lactobacillus-Enterococcus

[0161] BAC – Bacteroides – Prevotella

[0162] EREC – Eubacterium rectale – Clostridium cocci

[0163] RREC-Rostermaria and Eubacterium rectale

[0164] ATO – Atopobium – Atopobium rubrum

[0165] PROP–Clostridium Group IX

[0166] FPRAU – Faecalibacterium prausnitzii;

[0167] DSV – Desulfovibrioles and Desulfomonasales

[0168] CHIS – Clostridium histolyticum – Clostridium perfringens

[0169] Gas chromatography-mass spectrometry (GC-MS) for short-chain fatty acids (SCFA) and lactate analysis

[0170] 1.5 mL samples were taken from containers containing 7-KLCA + maltodextrin and maltodextrin alone at 0, 5, 10, and 24 h, centrifuged at 13,000 × g for 10 minutes, and the supernatant was stored at -20 ° C until metabolite analysis was required. After the samples were taken out of -20 ° C storage and thawed, they were vortexed and centrifuged again at 13,000 × g for 10 minutes. Then a 1 mL sample supernatant aliquot was transferred to a flat-bottomed glass tube containing 50 μL of internal standard solution (0.1 M 2-ethylbutyric acid). 500 μL of concentrated hydrochloric acid (HCl) and 3 mL of ether were added to the samples in each glass tube, then vortexed for 1 minute and centrifuged at 2000 × g for 10 minutes. Then 400 μL of the combined ether extract was added to 50 μL of N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) in a GC screw cap vial. External samples of 1 M acetate, butyrate, propionate, and lactate were also prepared using the Richardson SCFA derivatization method. The samples were left at room temperature for 72 h to allow complete derivatization of the metabolites. An Agilent / HP6890 gas chromatograph (Hewlett Packard, UK) equipped with an HP-5MS 30 m×0.25 mm column (coated with a 0.25 μm coating (cross-linked (5%-phenyl)-methyl polysiloxane, Hewlett Packard, UK)) was used for metabolite analysis. Quantification of the samples was obtained by calibration curves of lactate, acetate, propionate, and butyrate at concentrations between 6.25 and 100 mM from external standards.

[0171] Preliminary results from one donor showed that different 7-KLCA concentrations promoted the production of beneficial SCFAs that contribute to health benefits ( Figure 3 ) have different metabolic characteristics.

[0172] Example 2-3 Stage Intestinal Model

[0173] The 3-stage intestinal model is a continuous system designed to reproduce the nutrition, timing and physiological and chemical characteristics of the microbial flora in the large intestine. The 3-stage intestinal model used in the present embodiment is developed by the University of Reading, and has been verified by the chemical and microbiological measurements of the intestinal contents of the victims of sudden death, and it is an excellent model for operation, as an indicator of how the test substrate works in vivo. These experiments take longer than the experiment described in Example 1, because multiple turnovers (trunover) are required before the inoculation of the test compound to reach a stable state, which is important for understanding whether any changes detected are caused by adding the test substrate. The bacterial population in the intestinal model can be visualized using flow-FISH cytometry and 16S rRNA sequencing analysis, which can reveal changes in bacterial population and metabolism when combined with mass spectrometry.

[0174] The aim of the 3-stage gut model experiment was to determine the effects of 7KLCA on the gut microbiome of healthy volunteers through in vitro gut fermentation and downstream analysis of bacterial populations and produced metabolites using flow-FISH cytometry, 16S rRNA sequencing, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy.

[0175] 3-stage intestinal model

[0176] In this example, the test substance was 7-KLCA, and UDCA was used as a control.

[0177] A three-stage continuous culture system was established to simulate the defining sections of the large intestine, proximal colon, transverse colon and distal colon. The containers were set up in order and the nutrition, timing and physiochemical characteristics of the microbial flora in the proximal (V1, 80 ml, pH = 5.5), transverse colon (V2, 100 ml, pH = 6.2) and distal colon (V3, 120 ml, pH = 6.8) were simulated. The system was maintained under anaerobic conditions by purging the container with nitrogen (15 ml / min) and the temperature was maintained at 37°C by a circulating water bath.

[0178] A fecal slurry at a concentration of 6% was inoculated in each of three containers; the samples were donated by three healthy adult donors. The fecal donors had not taken antibiotics within 6 months before the experiment and were not taking prebiotic or probiotic supplements regularly. The donors collected fecal samples in anaerobic jars (AnaeroJarTM 2.5L, Basingstoke, UK, Oxoid Ltd.) and stored under anaerobic conditions using anaerobic bags (AnaeroGen, Oxoid) and used within 1 hour of production. Fecal slurries were prepared in a 1:5 (w:v) ratio of feces to PBS (anaerobic phosphate-buffered saline; 0.1 mol / L; pH 7.4) and homogenized for 2 minutes (240 paddle beats / minute) using a stomacher (Stomacher 400, Seward, West Sussex, UK).

[0179] Samples were collected from V1, 2, and 3 immediately after inoculation of the fermentation system (T-1) and 24 hours after inoculation (T-0). The T0 sample provided ideal starting conditions for continuous culture fermentation, as the bacterial population increased and settled to the species composition found under the conditions and pH conditions in each container. After the T0 sample was collected, the three-stage continuous culture system began the first stable state 1 (SS1), and the nutrient medium began to turn over at a retention time suitable for healthy adults, taking into account the operating volume (300 mL) and retention time (flow rate 6 mL / hour) of the intestinal model system.

[0180] Using standard intestinal model culture medium, the first stable state (SS1) was reached after 512 hours (8 full volume turnovers, equivalent to 14 days). Once equilibrium was reached, bile acids (400 mg / day) were injected into V1 every day, and the intestinal model entered the second stable state (SS2) stage. Again, using standard intestinal model culture medium, bile acids were injected into V1 at the same flow rate at the same time every day during SS2 (512 hours (8 full volume turnovers, 14 days). Samples were collected two days and one day before SS1 sample collection (SS1-2 and SS1-1) to ensure that the intestinal model system had reached equilibrium (using GC-MS). After the second stable state (SS2), samples were also collected from each container of the system.

[0181] Bacterial counting by flow cytometry-fluorescence in situ hybridization (Flow-FISH)

[0182] This was performed by the same method and using the same 16S rRNA-targeting oligonucleotide probe. Gas chromatography Mass spectrometry (GC-MS) for short-chain fatty acids (SCFA) and lactate analysis

[0183] 1.5 mL samples were taken from each container at 0, 5, 10, and 24 h, centrifuged at 13,000 × g for 10 minutes, and the supernatant was stored at -20 ° C until needed for metabolite analysis. Samples were prepared for GC-MS using the SCFA derivatization method described by Richardson et al. (1989). After the samples were taken out of -20 ° C storage and thawed, they were vortexed and centrifuged again at 13,000 × g for 10 minutes. A 1 mL sample supernatant aliquot was then transferred to a flat-bottomed glass tube containing 50 μL of internal standard solution (0.1 M 2-ethylbutyric acid). 500 μL of concentrated hydrochloric acid (HCl) and 3 mL of ether were added to the samples in each glass tube, then vortexed for 1 minute and centrifuged at 2000 × g for 10 minutes. Then 400 μL of the combined ether extract was added to 50 μL of N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) in a GC screw cap vial. External samples of 1 M acetic acid, butyric acid, propionic acid and lactic acid were also prepared using the SCFA derivatization method (Richardson et al., 1989). The samples were placed at room temperature for 72 hours to allow complete derivatization of the metabolites. An Agilent / HP6890 gas chromatograph (Hewlett Packard, UK) equipped with an HP-5MS 30m×0.25mm column (the column surface was coated with a 0.25 μm coating (cross-linked (5%-phenyl)-methylpolysiloxane, Hewlett Packard, UK)) was used for metabolite analysis. The quantification of the samples was obtained by calibration curves of lactic acid, acetic acid, propionic acid, butyric acid with concentrations between 6.25 and 100 mM from external standards.

[0184] DNA extraction for 16S rRNA sequencing

[0185] At each sampling point, 1.5 mL of sample was taken from the batch culture fermentation and centrifuged at 13,000 x g for 10 minutes. The pellet was then stored at -20°C until required for DNA extraction. Provided ProDNA kit for extraction. The pellet samples were thawed on ice, then 800μL of solution CD1 was added to the pellet and homogenized. Each mixture was then pipetted into a PowerBead Pro tube and briefly vortexed to mix. The samples were then loaded into the PowerBead Pro tubes and homogenized using the TissueLyser II set to 30Hz for 2 minutes, then placed on ice for 3 minutes, and then placed in the TissueLyser II for another 2 minutes. The tubes were then centrifuged at 15,000xg for 1 minute, and the supernatant was transferred to a clean 2ml microcentrifuge tube. 200μL of solution CD2 was added to the tube, vortexed for 5 seconds, and then centrifuged at 15,000xg for 1 minute. Up to 700μL of the supernatant in 2ml (Up to 700μL of the supernatant was 2ml) was transferred to a clean microcentrifuge tube, 600μl of solution CD3 was added and vortexed for 5 seconds. All lysate was then loaded onto an MB spin column and centrifuged at 15,000xg for 1 minute to allow the DNA to selectively bind to the silica gel membrane. The MB spin column was then placed in a clean 2ml collection tube and 500μl of Solution EA was added and then centrifuged at 15,000xg for 1 minute. The flow-through was then discarded and the MB spin column was placed back into the same 2ml collection tube. 500μl of Solution C5 was then added to the MB spin column and centrifuged at 15,000xg for 1 minute. The flow-through was then discarded and the MB spin column was placed in a new 2ml collection tube. The tube was then centrifuged at 16,000xg for 2 minutes. The column was then placed in a new 1.5ml elution tube and 50μL of Solution C6 was pipetted onto the center of the white filter membrane. The column was then eluted in a NanoDrop TM The quality of the DNA was analyzed on the PCR agarose gel, and the samples were then frozen at -80 °C and sent to Microsynth AG, Switzerland, for 16S rRNA sequencing.

[0186] NMR sample preparation

[0187] The samples were thawed and vortexed for 10 seconds to resuspend the pellets, and then they were allowed to settle again in the tube. 400 μL of the sample (avoiding the pellet) was then transferred to a new 1.5 mL centrifuge tube containing 200 μL of 0.2 M sodium phosphate buffer solution (pH 7.4), which was made of 100% deuterium oxide (D2O) and also contained 0.01% sodium 3-(trimethylsilyl)[2,2,3,3,-2H4]propionate (TSP) and 3 mM sodium azide (NaN3). D2O is required for field locking of the NMR spectrometer, and TSP is an internal reference standard for calibrating the acquired spectral curves. The mixture was vortexed and centrifuged for 10 seconds, and then 550 μL of the mixture was added to an NMR tube with an outer diameter of 5 mm. Following the established metabolic profiling protocol, a standard one-dimensional NMR pulse sequence pre-saturated with water was used, and samples were ordered in a random order and one-dimensional spectral data were taken.

[0188] result

[0189] Bacterial enumeration by flow-FISH cytometry

[0190] like Figure 4 As shown, use Statistics One-way ANOVA analysis showed statistically significant differences in total bacteria (EUB-I-II-III) between the 7-KLCA-treated intestinal model (Log10 9.35 ± 0.16 CFU / mL) and the UDCA-treated model (Log10 8.74 ± 0.50 CFU / mL) in the proximal colon region (V1). Although there was no significant increase in total bacteria compared to baseline and after bile acid treatment, the number of bacteria was greater after 7-KLCA treatment in all three regions of the model.

[0191] In addition, one-way ANOVA showed that there were statistically significant differences (P<0.05) in the number of Bacteroides-Prevotella (BAC303) flora between the two treatments in the transverse colon (V2), Log10 8.47±0.24 CFU / mL (7-KLCA) and Log10 6.94±0.33 CFU / mL (UDCA).

[0192] Paired sample t-test analysis showed that the Bacteroides-Prevotella population in the distal colon region (V3) was statistically significantly different (P<0.05), Log10 7.69±0.15 CFU / mL (7-KLCA) and 6.51±0.60 CFU / mL (UDCA). Paired sample t-test analysis showed that there was a greater statistical difference between the 7-KLCA-treated intestinal model and the UDCA-treated model in the transverse colon region (P<0.001).

[0193] There was no statistically significant increase in the number of Bifidobacterium spp. (Bif164) at baseline or after 7-KLCA or UDCA treatment, but after 7-KLCA treatment, the number of bacteria increased in all three stages of the intestinal model (V1, 2, and 3), with the greatest difference in the transverse colon region, where the number increased from Log10 7.54±1.06CFU / mL to Log10 8.16±0.13CFU / mL.

[0194] Figure 5 Although not statistically significant, there was an increase in Eubacterium rectale and Roseburia species in V1, V2, and V3 between baseline and after 7-KLCA treatment.

[0195] like Figure 6 As shown, there was a statistically significant increase in the number of butyrate-producing Faecalibacterium prausnitzii in V2 (Log10 7.47±0.91 CFU / mL to Log10 8.81±0.09 CFU / mL) and V3 (Log10 6.95±0.97 CFU / mL to Log10 8.39±0.22 CFU / mL) between baseline and after 7-KLCA treatment. UDCA treatment did not result in an increase in Faecalibacterium prausnitzii.

[0196] Figure 6 It also showed that there was no statistically significant increase in the number of pathogenic bacteria, Clostridium histolyticum, Desulfovibrio, or Streptococcus, with either UDCA or 7-KLCA.

[0197] These results suggest that while both 7-KLCA and UDCA can increase the number of beneficial bacteria in the intestine, 7-KLCA is more effective than UDCA in increasing the total bacterial population (EUB-I-II-III) in the proximal colon region. 7-KLCA also provided superior results to UDCA in increasing the population of Bacteroides-Prevotella (BAC303) in the transverse and distal colon regions. 7-KLCA treatment also resulted in increases in Bifidobacterium (Bif164), Eubacterium rectum, and Roseburia, although these results were not significant. Such increases were not seen after UDCA treatment.

[0198] The results of flow-FISH cytometry showed that both 7-KLCA and UDCA were beneficial, but 7-KLCA was superior to UDCA in increasing some beneficial bacteria.

[0199] Bacterial metabolite quantification by GC-MS

[0200] like Figure 7As shown in A, lactate did not show statistically significant changes in V1, V2, and V3, however an increase in this metabolite was measured after 7KLCA treatment.

[0201] Figure 7 B, 7C, 7D, and 7E show no statistically significant changes in acetate, butyrate, propionate, or valerate concentrations in V1, V2, and V3, respectively, whereas an increase in propionate concentration was measured after 7KLCA treatment.

[0202] Determination of bacterial composition by 16s rRNA sequencing

[0203] The abundance and levels of DNA extracted from healthy in vitro intestinal samples were measured using 16S rRNA sequencing before and after 7-KLCA treatment.

[0204] from Figures 15 to 18 From the Krona plot, it can be seen that before 7-KLCA treatment, the percentage of the probiotic species Faecalibacterium prausnitzii was 4%, Eubacterium rectale was also detected at 4%, Bacteroides xylanisolvens at 1%, and Roseburia species totaled 9%. Dioblastus pneumophila showed pathogenic potential in various parts of the body. Before 7-KLCA treatment, this bacterium accounted for 7% of the total bacterial composition. After 7-KLCA treatment, this percentage dropped to 3% in both the transverse colon and the distal colon (V2 and V3).

[0205] After 7-KLCA treatment, the abundance of Faecalibacterium prausnitzii increased to 17% and that of Eubacterium rectale rose to 18% in the proximal colon region.

[0206] Similarly, in the transverse colon region, Faecalibacterium prausnitzii increased dramatically, reaching 18% of the total bacterial composition. Bacteroides xylanisolvens was measured at 2% before 7-KLCA treatment and increased to 9% after bile acid intervention. Bacteroides xylanisolvens has been described as a new generation probiotic (NGP) for its immunomodulatory and anticancer properties (Ulsemer et al., 2016). Eubacterium fusilli was recorded at 2% before 7-KLCA treatment, however, this species, which has anti-inflammatory activity by promoting IL-10 production in epithelial cells, rose to 5% in V2 and remained at 2% in V1 and V3.

[0207] After 7-KLCA treatment, Faecalibacterium prausnitzii increased from 0.6% to 10% in V3 (distal colon). Bifidobacterium was 5%, which, although lower than baseline, indicates that the species can be successfully grown using 7-KLCA treatment. Sequencing also showed that Clostridium species decreased from 5.6% of the total bacterial species detected to 4.5% after 7-KLCA treatment.

[0208] pass 1 H NMR metabolite profiling

[0209] like Fig. 8A and 8B As shown, before and after 7-KLCA treatment 1 H NMR spectra analysis showed the presence of bacterial metabolites isoleucine and leucine after treatment, two amino acids important for immunity, protein metabolism, fatty acid metabolism and glucose transport. Alanine was also detected after 7-KLCA treatment, a metabolite important for protein synthesis and a source of energy for muscle and central nervous system. The peak of butyrate after 7-KLCA treatment was greater than the peak of these metabolites before bile acid treatment. Butyrate is an important SCFA that helps maintain the barrier function of the intestinal wall, contributes to digestive health, helps control inflammation and prevents disease.

[0210] 1 H NMR spectra analysis also revealed the presence of several other bacterial metabolites, including propionic acid and acetic acid, both of which showed higher peaks after 7-KLCA treatment. Other metabolites detected included: lysine, pyruvic acid, valeric acid, and isovaleric acid, all of which play important roles in the body, including protein synthesis, energy metabolism, and immune regulation.

[0211] Example 3 - Study on the efficacy of lowering blood sugar in mice

[0212] Experimental plan

[0213] The study was conducted in the laboratories of the Department of Medicine and Surgery at the University of Naples Federico II, Italy.

[0214] Study Design:

[0215] CTRL 1 mice were fed a normal diet for 60 days (5 mice)

[0216] CTRL 2 mice were fed a normal diet and given 10 mg / kg of 7KLCA for 60 days (5 mice)

[0217] CTRL 3 mice were fed a normal diet for 30 days, followed by a high-sugar diet for 30 days (5 mice)

[0218] Mice were treated with a normal diet for 30 days, followed by a high-sugar diet for 30 days, and 7KLCA was used throughout the study (5 mice)

[0219] Standard diet (10.6% fat J / J, 29.0% protein J / J and 60.4% carbohydrates J / J).

[0220] High glucose diet (10.5% fat J / J, 16.4% protein J / J and 73.1% carbohydrates J / J).

[0221] All experimental groups had free access to tap water. Experimental animals were housed in resin glass cages with 5 animals / sex / group / cage as a group.

[0222] Experiments were performed in 8-12 week old male CD-1 mice in accordance with the Italian DL No. 116 of January 27, 1992 for the safe use and care of laboratory animals and the relevant European Community Council guidelines (86 / 609 / ECC and 2010 / 63 / UE), including the 3R concept (Kilkenny et al., 2010; McGrath et al., 2015). Animals were housed with free access to food and water and maintained on a 12 h light-dark cycle. The experimental study groups were randomized and blinded.

[0223] The control group animals were treated in the same manner as the treated groups, using the corresponding vehicle and the same administration volume. The tested item formulation or vehicle was administered by oral gavage. Body weight was recorded before assignment to the experimental group, on the first day of administration, and once a week during the treatment period.

[0224] Preparation of test formulations

[0225] The 7-KLCA dose used in this study was 10 mg / kg / day. The vehicle used was DMSO in saline (NaCl 0.9%), 1 / 3 w / w. 7-KLCA was dissolved in DMSO (1 part w / w) and then resuspended in saline (3 parts w / w). The solution (200 μl / mouse) was freshly prepared every day and kept under magnetic stirring during daily administration.

[0226] Test system

[0227] Species / strain Healthy CD-1 mice source Charles River, Italy gender male Age at start of treatment About 8-12 weeks old Body weight of animals when assigned to experimental groups 26g±2g

[0228] Living and feeding conditions

[0229] Air-conditioned room is completely isolated

[0230] temperature 22±3℃ Relative humidity 55±10％ Artificial light 12 hours light 12 hours dark Ventilation 10 times / hour

[0231] weight

[0232] Body weights were recorded before assignment to experimental groups, on the first day of administration, and weekly thereafter.

[0233] Clinical observation

[0234] All animals were observed for clinical symptoms throughout the treatment period (60 days). General clinical observations were performed at least once a day at the same time. The health status of the animals was recorded.

[0235] result

[0236] After an overnight fast, the animals' blood was collected in serum separator tubes. Fig.10 and 13 Shown are the changes in body weight of the mice in the study. Fig.10 It can be seen that in the group of mice fed a normal diet for 60 days, there was no significant difference in weight gain between the 5 mice in the control group (CTRL 1) and the 5 mice treated with 10 mg / kg 7KLCA (CTRL 2). Fig.13 As shown, in the groups of mice fed a normal diet for 30 days and then switched to an HGD diet for 30 days, there was no significant difference in weight gain between the five mice in the CTRL 3 group and the five mice in the treatment group during the initial 30 days that the mice were fed a standard diet. However, when switched to an HGD diet, the mice treated with 7-KLCA gained a similar amount of weight as the mice on a normal diet (CTRL 1 and CTRL 2), but the untreated mice (CTRL 3) gained significantly more weight.

[0237] like Fig.11 As shown, in mice fed a normal diet for 60 days, there was no significant difference in blood glucose levels between the untreated group CTRL 1 (normal diet) and the treated control group CTRL 2 (normal diet + 7KLCA), and Fig.12 , 13 and 14 showed that there were no significant differences in the levels of HDL cholesterol, LDL cholesterol, and total cholesterol between the two groups.

[0238] Fig.11 It also showed that in the group of mice fed a normal diet for 30 days and then switched to a HGD diet for 30 days, the blood glucose level in the treated group (HGD+7KLCA) after 60 days was significantly lower than that in the CTRL 3 group (HGD). Fig.12 As shown in the figure, the HDL cholesterol level of the treated group (HGD+7KLCA) mice was slightly increased compared with the CTRL 3 group (HGD), but it was not significant. Compared with the untreated CTRL 3 group (HGD) mice, the LDL cholesterol ( Fig.13 ) and total cholesterol ( Fig.14 ) levels were significantly reduced.

[0239] At the selected dose of 10 mg / kg, no toxicity was observed in mice treated with 7-KLCA.

[0240] in conclusion

[0241] The results showed that mice treated with 7-KLCA gained similar amounts of weight, whether they were fed a normal diet (CTRL 2) or an HGD diet (treatment). The weight gain was also similar to that of untreated mice fed a normal diet. In contrast, untreated mice fed an HGD diet (CTRL 3) gained significantly more weight than mice fed a normal diet (CTRL 1). In addition, in mice fed a normal diet for 30 days and then an HGD diet for 30 days, 7-KLCA treatment resulted in a statistically significant decrease in blood sugar levels and an upward trend in insulin levels. The HGD+7KLCA group of mice also had lower LDL and total cholesterol levels compared to the CTRL 3 group of mice.

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[0289] All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

1. 7-ketolithocholic acid (7-KCLA, 3α-hydroxy-7-oxo-5β-cholanoic acid) having the following formula: Or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof, for preventing or treating intestinal flora imbalance and diseases and disorders associated with poor intestinal health, or for improving the health of the intestinal microbiome. 2.7-KLCA or its salt, especially a pharmaceutically acceptable salt, or its glycine or taurine conjugate, for use in preventing intestinal flora imbalance. 3.7-KLCA or its salt, especially a pharmaceutically acceptable salt, or its glycine or taurine conjugate, for use in treating intestinal flora imbalance. 4.7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof for use in preventing diseases and disorders associated with poor intestinal health. 5.7-KLCA or its salts, especially pharmaceutically acceptable salts, or its glycine or taurine conjugates for use in treating diseases and conditions associated with poor intestinal health. 6.7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof for use in improving the health of the intestinal microbiome.

7. Use of 7-KLCA or a pharmaceutically acceptable salt thereof or a glycine or taurine conjugate thereof in the preparation of a medicament for preventing or treating intestinal flora imbalance and diseases and conditions associated with poor intestinal health, or for improving the health of the intestinal microbiome.

8. A method for preventing or treating intestinal flora imbalance and diseases and conditions associated with poor intestinal health, or improving the health of the intestinal microbiome, the method comprising administering to an individual an effective amount of 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof.

9. 7-KLCA for use, use or method according to any one of claims 1 to 3, 7 or 8, wherein the intestinal flora imbalance is associated with one or more diseases or conditions selected from the group consisting of obesity, hyperglycemia, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), irritable bowel syndrome and autoimmune disorders (including rheumatoid arthritis, multiple sclerosis, type 1 diabetes, celiac disease, systemic lupus erythematosus), CNS disorders (including anxiety, depression, sleep disorders, cognitive dysfunction, schizophrenia and bipolar disorder), cardiovascular disease and skin diseases (including psoriasis, atopic dermatitis, acne vulgaris, rosacea, alopecia areata and hidradenitis suppurativa).

10. 7-KLCA for use, use or method according to claim 9, wherein the intestinal flora imbalance is caused by the use of antibiotics to treat bacterial infection.

11. 7-KLCA for use, use or method according to claim 10, wherein the 7-KLCA is administered in combination with or after antibiotic administration.

12. The 7-KLCA for use, use or method according to any one of claims 1, 4, 5, 7 or 8, wherein the diseases and conditions associated with poor gut health are selected from the group consisting of obesity, hyperglycemia, insulin resistance, metabolic syndrome, prediabetes, type 2 diabetes, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), irritable bowel syndrome and autoimmune disorders (including rheumatoid arthritis, multiple sclerosis, type 1 diabetes, celiac disease, systemic lupus erythematosus), CNS disorders (including anxiety, depression, sleep disorders, cognitive dysfunction, schizophrenia and bipolar disorder), cardiovascular disease and skin diseases (including psoriasis, atopic dermatitis, acne vulgaris, rosacea, alopecia areata and hidradenitis suppurativa).

13. 7-KLCA for use, use or method according to claim 12, wherein the diseases and conditions associated with poor gut health are selected from obesity, hyperglycemia, metabolic syndrome, type 2 diabetes, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), irritable bowel syndrome and autoimmune disorders (including rheumatoid arthritis, multiple sclerosis, type 1 diabetes and celiac disease).

14. A method for adjusting the composition of an individual's intestinal microbiota to contain a higher proportion of beneficial microorganisms, the method comprising administering 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof to the individual.

15. The method of claim 14, wherein the beneficial microorganisms comprise bacteria.

16. The method according to claim 15, wherein the bacteria is selected from the group consisting of Bifidobacterium, Faecalibacterium prausnitzii, Eubacterium rectale, Roseburia, Lactobacillus, Bacteroides xylanisolvens and Eubacterium fusilli.

17. The method according to claim 16, wherein the bacteria is selected from the genera Bifidobacterium, Faecalibacterium prausnitzii and Roseburia.

18. The method according to any one of claims 14 to 17, further comprising adjusting the composition of the intestinal microbiota of the individual to contain a lower proportion of pathogenic microorganisms.

19. The method of claim 18, wherein the pathogenic microorganism comprises bacteria.

20. The method of claim 20, wherein the bacteria is selected from the group consisting of Atopobium, Bacillus rubrum, Clostridium histolyticum, Clostridium perfringens, Desulfovibrioles, Desulfomonasales, and Dialisteria pneumophila.

21. The method of claim 20, wherein the bacteria is selected from the group consisting of Atopobium, Bacillus rubrum, Clostridium histolyticum, Clostridium perfringens, Desulfovibrioles, and Desulfomonasales.

22. The method according to any one of claims 14 to 22, further comprising increasing the number of microorganisms, in particular bacteria, in the intestinal microbiota.

23. The method according to any one of claims 14 to 23, wherein the 7-KLCA is administered simultaneously with or after antibiotic treatment.

24. A prebiotic composition comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt, or a glycine or taurine conjugate thereof as the sole active agent and a suitable carrier.

25. A prebiotic composition according to claim 24 wherein the composition does not contain maltodextrin.

26. A prebiotic composition according to claim 24 or 25 suitable for oral administration.

27. A prebiotic composition according to claim 26, which is a food or beverage product comprising 7-KLCA or a salt thereof, especially a pharmaceutically acceptable salt or a glycine or taurine conjugate thereof, and a suitable flavoring agent.

28. A prebiotic composition according to any one of claims 24 to 27 further comprising an antibiotic.

29. A prebiotic composition according to any one of claims 24 to 28 for use in preventing or treating intestinal dysbiosis, diseases and conditions associated with poor intestinal health, or for improving the health of the intestinal microbiome.

30. A method of modifying the composition of an individual's intestinal microbiota to contain a higher proportion of beneficial microorganisms, the method comprising administering to the individual a prebiotic composition according to any one of claims 24 to 28.

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