Application of bile acid and derivatives thereof in prevention or treatment of intracranial aneurysm

Abstract

The invention belongs to the field of biomedicine, and provides application of bile acid and derivatives thereof in prevention or treatment of intracranial aneurysm. The invention particularly relates to application of ursodesoxycholic acid and bile acid combined with ursodesoxycholic acid in preparation of drugs for preventing or treating intracranial aneurysm. According to the method, metabonomics analysis is carried out on a human blood sample, and the early warning effect of ursodesoxycholic acid and bile acid combined with ursodesoxycholic acid on intracranial aneurysm rupture is found; the verification of the influence effect of ursodesoxycholic acid and bile acid combined with ursodesoxycholic acid on the rupture probability of intracranial aneurysm in a mouse intracranial aneurysm model proves that the substance has a protective effect on intracranial aneurysm, namely the risk of intracranial aneurysm rupture is reduced; and an experimental basis is provided for the drug treatment of intracranial aneurysm and the reduction of clinical rupture risk.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of ursodeoxycholic acid and its combined bile acid in preventing or treating intracranial aneurysms. Background Art

[0002] Intracranial aneurysm is the primary cause of spontaneous intracranial subarachnoid hemorrhage. This type of disease is not a tumor disease, but an intracranial arterial vascular disease. Because the intracranial arterial wall bulges outward to form a weak point in the cerebral blood circulation, once ruptured, blood will enter the subarachnoid space, forming subarachnoid hemorrhage. The incidence of intracranial aneurysms in the Chinese population is about 7%, and its annual rupture rate is reported to be about 1%. Once ruptured and bleeding occurs, the mortality and disability rate of subarachnoid hemorrhage caused by it is very high, which seriously endangers the health of the general public. At present, due to the popularization of imaging examinations and the improvement of people's health awareness, more and more unruptured aneurysms are detected. Faced with a large number of unruptured intracranial aneurysms, how doctors and patients make scientific diagnosis and treatment decisions is the key to the current clinical diagnosis and treatment of unruptured intracranial aneurysms. Because a considerable number of aneurysms may remain stable for a long time, that is, long-term carrying or living with such unruptured intracranial aneurysms will not have any impact. Once these patients receive treatment, they will bear unnecessary surgical risks, which will also cause a huge waste of medical resources. Therefore, how to reduce the rupture risk of unruptured aneurysms is the focus and difficulty in current clinical work.

[0003] At present, the main methods for treating intracranial aneurysms are surgical clipping and intravascular embolization, but these methods have certain risks and limitations. Therefore, it is particularly important to find effective drug treatments. Finding or developing key drugs that can reduce the incidence of intracranial aneurysm rupture is of great significance to reducing the survival threat of patients with intracranial aneurysms and improving the quality of life with aneurysms. Summary of the invention

[0004] The present invention investigates the effects of ursodeoxycholic acid (UDCA) and its conjugated bile acids on the probability of intracranial aneurysm rupture. Metabolomics analysis of human blood samples is performed to find the early warning effect of UDCA and its conjugated bile acids on intracranial aneurysm rupture. The effects of UDCA and its conjugated bile acids on the probability of intracranial aneurysm rupture are verified in a mouse intracranial aneurysm model, proving the protective effect of this type of substance on intracranial aneurysms, namely reducing the risk of intracranial aneurysm rupture, providing an experimental basis for the drug treatment of intracranial aneurysms and reducing the risk of clinical rupture.

[0005] UDCA is a bile acid derivative with anti-inflammatory, antioxidant and cytoprotective effects, and is widely used in the treatment of cholestatic liver diseases. UDCA and its conjugated bile acids (such as tauroursodeoxycholic acid (TUDCA) and glycoursodeoxycholic acid (GUDCA)) have a close relationship in chemical structure and physiological function. UDCA is a naturally occurring bile acid with relatively low hydrophilicity and moderate cytotoxicity. In the human intestinal cavity, chenodeoxycholic acid (CDCA) is activated by 7-hydroxysteroid dehydrogenase (7-Hydroxysteroid Under the catalysis of 7-HSDH), 7α-hydroxy isomerizes to 7β-hydroxy to form UDCA (UDCA). CDCA and UDCA are used together in the clinical treatment of gallstones, but CDCA is inferior to UDCA in efficacy, and about 18%-30% of patients have obvious side effects, with diarrhea and transient reversible aminotransferase elevation being the most common. Obvious liver damage can be observed in experiments on various mammals with CDCA, which has not been observed in humans. UDCA is currently more commonly used as a treatment for gallstones because it is better than CDCA in dissolving stones and has fewer side effects.

[0006] In hepatocytes, by combining UDCA with organic acids (such as taurine or glycine), the conjugated bile acids formed have higher polarity and hydrophilicity, thus having higher solubility in aqueous solution. In the intestine, the conjugated bile acids such as TUDCA and GUDCA as upstream substances will be converted into UDCA to exert pharmacological effects (Ding Jinsong, Wang Anna, Huang Liang, et al. Human ursodeoxycholic acid metabolism and technical challenges of bioequivalence study [J]. Acta Pharmaceutica Sinica, 2020, 55(9): 2070-2079. doi: 10.16438 / j.0513-4870.2020-0447). This chemical combination significantly reduces cytotoxicity, allowing TUDCA and GUDCA to be safely used at higher concentrations. In addition, due to their complete ionization and water solubility, these conjugated bile acids can be better absorbed by the intestine and liver under different pH conditions and are more evenly distributed. Clinical trials have shown that TUDCA and GUDCA have higher absorption efficiency and lower toxicity in the treatment of primary biliary cholangitis (PBC) and other hepatobiliary diseases, and are better than UDCA. On the other hand, it has also been proven that the core part of the therapeutic effect of this type of bile acid comes from UDCA, and the conjugated product can produce a better effect. The prior art has not reported the relationship between UDCA and its conjugated bile acid and intracranial aneurysms.

[0007] Metabolomics is a science that conducts quantitative and qualitative analysis of all metabolites of an organism or cell to reflect the changes in an organism in response to external stimuli or genetic modification. Metabolites are small molecular weight compounds that participate in the metabolism of an organism, maintain normal growth function and development, including peptides, sugars, amino acids, lipids, etc., and are downstream products and final products of the genome. The characteristic of the metabolome is the key to connecting genotype and phenotype, and it is closest to and can best reflect phenotypic characteristics; changes in gene and protein levels ultimately produce effects as changes in metabolic processes, and metabolites can reflect the physiological and pathological states of the body. From the detection aspect, because small changes in gene expression will be amplified in metabolites, they are also easier to detect, and their types are far less than the number of genes and proteins. Metabolomics methods have been widely used in the study of many diseases, and are an important means to discover key factors in the occurrence or prognosis of diseases, and to develop and explore key drugs or compounds.

[0008] At present, there are few reports on metabolomics studies and risk metabolite screening studies related to intracranial aneurysms. Xiong Y et al. conducted a proteomic analysis of patients with intracranial aneurysms (ruptured aneurysms, unruptured aneurysms) and healthy people, intending to find protein markers for intracranial aneurysms and their rupture. In their study, a group of protein combinations were reported to predict aneurysm rupture, but there were no reported results for substances such as bile acids (doi:10.15252 / emmm.202114713.Epub 2022Jan 3.). Su J et al. conducted a metabolite study of unruptured aneurysms. They found that a group of metabolite combinations could diagnose unruptured intracranial aneurysms. Lactate, glutamine, isoarginine and 3-methylglutaric acid were identified as metabolic biomarker panels, showing satisfactory diagnostic results. However, this study did not include ruptured aneurysms and did not discuss the risk of rupture, and did not find a significant effect of bile acids, especially UDCA and its conjugated bile acids (doi:10.1002 / smtd.202201486.). Liu Q et al.'s study on metabolites of intracranial aneurysm instability risk had a relatively large sample size. Although animal models were validated, this study did not find any relevant effects of UDCA and its conjugated bile acids on the rupture of unruptured aneurysms (doi:10.1016 / j.scib.2023.05.001.). Sun HT et al. conducted a gut flora and metabolomics study on ruptured and unruptured aneurysm cases and found that tryptophan and its metabolites could distinguish the rupture status. Similarly, UDCA and its conjugated bile acids were not studied (doi:10.1136 / gutjnl-2024-332245).

[0009] In the existing metabolomics studies of intracranial aneurysms, a large part of the grouping methods or research purposes are aimed at "whether the subject has an intracranial aneurysm" and "proving whether the subject carries an intracranial aneurysm by detecting metabolic markers". The cohort of patients with unruptured intracranial aneurysms and the cohort of healthy people are used for group comparative analysis. The clinical value of this research purpose and the detected target products is limited. At present, the gold standard for the detection of intracranial aneurysms is cerebral vascular imaging examinations, such as head CTA examinations, which are highly accurate, safe, fast, and convenient, and can meet clinical needs. Therefore, the clinical value of the above research results is limited, the application prospects are insufficient, and the most critical and most harmful clinical problem of intracranial aneurysms - rupture - is not solved.

[0010] Research or research results on the rupture problem and how to reduce the rupture rate of intracranial aneurysms will play an important role in the future treatment of such diseases.

[0011] In view of this, the first aspect of the present disclosure provides a use of bile acid and its derivatives in the preparation of a pharmaceutical composition for preventing and / or treating aneurysms.

[0012] In one embodiment, the bile acid and its derivatives include free bile acid and / or conjugated bile acid, the free bile acid includes one or more of deoxycholic acid, chenodeoxycholic acid, cholic acid or lithocholic acid, and the conjugated bile acid includes the product formed by the free bile acid conjugated with taurine or glycine.

[0013] In one embodiment, the free bile acid is deoxycholic acid, and the conjugated bile acid is tauroursodeoxycholic acid and / or glycoursodeoxycholic acid.

[0014] In one embodiment, the bile acid and its derivatives are ursodeoxycholic acid and its conjugated bile acids.

[0015] In one embodiment, the bile acid and its derivatives are selected from one or more of ursodeoxycholic acid, its conjugated bile acids tauroursodeoxycholic acid or glycoursodeoxycholic acid.

[0016] In one embodiment, the aneurysm is an intracranial aneurysm.

[0017] In one embodiment, the prevention and / or treatment of aneurysms is prevention of aneurysm rupture and / or reduction of the incidence of aneurysm rupture.

[0018] In one embodiment, the pharmaceutical composition further contains another active ingredient for treating intracranial aneurysms or the use includes the combination of the pharmaceutical composition and other drugs for treating intracranial aneurysms.

[0019] In one embodiment, the pharmaceutical composition further contains a pharmaceutically acceptable carrier or excipient.

[0020] In one embodiment, the pharmaceutical composition is contained in one or more pharmaceutical preparations selected from injections, oral liquids, suspensions, emulsions, extracts, powders, granules, suppositories, aerosols, tablets, capsules, microcapsules, microspheres, nanoparticles, liposomes or capsules.

[0021] In one embodiment, the therapeutically effective amount of the pharmaceutical composition comprises 1-100% bile acid and its derivatives.

[0022] In one embodiment, the therapeutically effective amount of the pharmaceutical composition comprises 1-20% of bile acids and their derivatives.

[0023] In one embodiment, the therapeutically effective amount of the pharmaceutical composition comprises 1-5% bile acid and its derivatives.

[0024] In one embodiment, the therapeutically effective amount of the pharmaceutical composition comprises 1.2% of bile acids and their derivatives.

[0025] Beneficial Effects

[0026] 1. In view of the defects of the above-mentioned prior art, the present invention conducts a large-sample metabolomics analysis, and the grouping method focuses on the analysis of risk factors for intracranial aneurysm rupture. The present invention is based on a metabolomics data analysis of thousands of large sample data, and the results obtained are more accurate and reliable compared with the data analysis of dozens or hundreds of cases.

[0027] 2. Based on the clinical data analysis, this paper further uses animal models to verify the data to prove the reliability and scientific nature of the findings of this study.

[0028] 3. This disclosure clearly analyzes UDCA and its combined bile acids, and conducts animal experiments for in vivo experimental verification. The number of bile acids and their metabolites can reach dozens. If the relevant effects of a certain substance on intracranial aneurysms are not clearly proposed, it will not be able to provide valuable help for the prevention and treatment of intracranial aneurysms, and the clinical application of drugs cannot be realized. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Violin plots of the concentration distribution of bile acid metabolites in the plasma of the unruptured intracranial aneurysm group and the ruptured aneurysm group. Figure 1 A in the figure is the difference in plasma concentration distribution of UDCA between the two groups. Figure 1 Figure B shows the difference in plasma concentration distribution of GUDCA between the two groups.

[0030] Figure 2 Comparison pictures of brain tissues of mice in the normal group, model group and UDCA treatment group.

[0031] Figure 3 The aneurysm rupture rate and the difference were significant between the model group and the UDCA treatment group (p=0.038).

[0032] Figure 4 Representative images of immunofluorescence expression of endothelial cell marker CD31 in brain tissue of mice in the normal group, model group, and UDCA treatment group (DAPI: nucleus-specific staining, scale bar, 50 μm). Independent experiments were repeated at least 5 times.

[0033] Figure 5 Representative images of immunofluorescence expression of CD68, a macrophage marker, in brain tissue of mice in the normal group, model group, and UDCA-treated group (DAPI: specific staining of cell nuclei, scale bar, 50 μm). Independent experiments were repeated at least 5 times. DETAILED DESCRIPTION

[0034] I. Definitions

[0035] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the relevant terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0036] In the description of this document, reference is made to “some embodiments”, “some implementation schemes” or “some implementation plans”, which describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0037] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0038] The term "intracranial aneurysm" mostly refers to an abnormal bulge on the wall of an intracranial artery, which is the leading cause of subarachnoid hemorrhage. Intracranial aneurysms are prone to occur at the bifurcation of the arterial circle at the base of the brain and its main branches. About 85% of aneurysms are located in the internal carotid artery system in the anterior half of the Willis artery circle, that is, the intracranial segment of the internal carotid artery, the anterior cerebral artery, the anterior communicating artery, the middle cerebral artery, and the posterior communicating artery. If the arterial wall is asymmetrically saccular, it is called a saccular aneurysm. Small saccular aneurysms with narrow necks are also called berry-shaped aneurysms. The vast majority of intracranial aneurysms are saccular or berry-shaped, or lobed. Other shapes include gourd-shaped, spherical, and sausage-shaped. The aneurysm wall is generally smooth like a capsule, and most of them are composed of abnormally weak vascular walls. They are often located at the bifurcation of larger arteries. The part where the aneurysm connects to the parent artery is relatively narrow, which is called the neck (pedicle) or base of the aneurysm. The width of the neck is very inconsistent. The most prominent part on the far side opposite to the neck is the base (top) of the aneurysm, and the part between the neck and the base of the aneurysm is called the body (sac). The bursa is a small protrusion on the aneurysm sac, which is often the place where the aneurysm ruptures or the remains after the rupture. The size of intracranial aneurysms varies greatly, usually between 0.3 and 2 cm. The rupture of an aneurysm is related to its size. It is generally believed that ruptured aneurysms are larger and unruptured aneurysms are smaller. Aneurysms with a diameter exceeding 0.5 cm have a gradually increasing chance of bleeding. When the diameter exceeds 3.0 cm, the symptoms of increased intracranial pressure replace the bleeding symptoms.

[0039] The diagnosis of intracranial aneurysm includes determining whether it has ruptured and caused subarachnoid hemorrhage (SAH) and / or determining the cause and location of the lesion.

[0040] Among them, determine whether there is subarachnoid hemorrhage: in the acute stage of hemorrhage, CT has a very high positive rate for confirming SAH, which is safe, rapid and reliable. Increased lumbar puncture pressure accompanied by bloody cerebrospinal fluid is often direct evidence for diagnosing subarachnoid hemorrhage after aneurysm rupture. However, when the intracranial pressure is very high, lumbar puncture should be performed with caution. Determine the cause and location of the lesion: Cerebral angiography is the "gold standard" for diagnosing intracranial aneurysms. It can clearly determine the location, shape, size, number of aneurysms, whether there is vascular spasm, and determine the final surgical plan. If the first angiography is negative, the angiography should be repeated after 3 to 4 weeks. CTA can replace cerebral angiography to a certain extent and provide more information for aneurysm treatment decisions.

[0041] The term "bile acid" (BA) is the main organic component of bile and is a general term for several steroid acids with similar structures. In human bile, conjugated bile acids are the main form of existence. Under the pH environment of bile, they almost all exist in the form of sodium or potassium salts, which are called bile salts, or bile salts for short. Bile acids are divided into free bile acids and conjugated bile acids according to their chemical structure. Free bile acids include cholic acid, deoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid and lithocholic acid. Conjugated bile acids are the products of the above-mentioned free bile acids combined with glycine, taurine, sulfuric acid and glucuronic acid. Most of them are combined with glycine and taurine, especially glycine. Only a small amount of bile acids (mainly chenodeoxycholic acid and lithocholic acid) are combined with sulfuric acid and glucuronic acid. There are 16 products according to the arrangement and combination, but the most common ones are 4, including glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid.

[0042] The metabolic process of bile acid generally includes: A: Production of primary bile acid: Hepatocytes use cholesterol as raw material and synthesize primary bile acid through multiple steps, among which the key enzyme is cholesterol 7α-hydroxylase. Converting cholesterol into bile acid is the main way for the liver to remove cholesterol. Early cholesterol-lowering treatment is to use oral bile acid binders to promote hepatocytes to synthesize more bile acid to consume cholesterol. B: Production of secondary bile acid: After bile is discharged into the intestinal cavity, the bound primary bile acid is hydrolyzed by bacteria in the ileum and upper colon into free primary bile acid, which then undergoes 7-position dehydroxylation to form secondary bile acid - bile acid is converted into deoxycholic acid, and chenodeoxycholic acid is converted into lithocholic acid. C: Enterohepatic circulation: About 95% of the various bile acids discharged into the intestine are reabsorbed. The reabsorption in the ileum is active reabsorption, and the rest of the intestine is passive reabsorption. The reabsorbed bile acid enters the liver through the portal vein, where it is reabsorbed by the liver cells, converted into conjugated bile acid and discharged into the intestine again, thus forming the enterohepatic circulation. The physiological significance of the enterohepatic circulation is to make up for the inadequate ability of liver cells to synthesize bile acid. The daily synthesis of bile acid is only 0.4 to 0.6 grams, which is far from meeting the needs of the small intestine for fat emulsification. With the help of the enterohepatic circulation, it is equivalent to 12 to 32 grams of bile acid entering the intestine every day. D: Excretion: The bile acid that is not reabsorbed forms bile acid derivatives under the action of intestinal bacteria and is excreted in the feces. With the help of the enterohepatic circulation, the liver only needs to synthesize this part of the bile acid that is not reabsorbed. The water solubility of bile acid combined with sulfuric acid and glucuronic acid is significantly enhanced, and it can be filtered and discharged by the kidneys. Therefore, the content of such compounds in the urine of patients with bile duct obstruction will increase compensatorily.

[0043] In this article, the term "cohort study" refers to an observational research method that divides a specific population into different subgroups according to whether they are exposed to a suspected factor or the degree of exposure, tracks and observes the occurrence of outcomes (such as diseases) in members of two or more groups, and compares the differences in the incidence of outcomes between the groups, so as to determine whether there is a causal relationship between these factors and the outcome and the degree of association.

[0044] In this article, "case-control study" is also called retrospective study, which compares the percentage difference of patients with a certain disease and controls without a certain disease exposed to a possible risk factor, and analyzes whether these factors are related to the disease. It is one of the most basic and important research types in epidemiological analysis methods.

[0045] The term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium representative of carriers that can deliver an effective amount of the active substance (UDCA and its conjugated bile acid) disclosed herein, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or subject, including water, oil, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity enhancers, transdermal enhancers, etc. Their preparations are well known to those skilled in the field of cosmetics or topical medicines.

[0046] The term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a non-toxic drug or medicament that can achieve the desired effect. In embodiments of the present disclosure, when treating a patient according to the present disclosure, the amount of a given drug depends on many factors, such as a specific dosing regimen, the type of disease or condition and its severity, the uniqueness of the subject or host to be treated (e.g., body weight), but, according to specific surrounding conditions, including, for example, a specific drug that has been adopted, a route of administration, the condition to be treated, and the subject or host to be treated, the dosage can be routinely determined by methods known in the art. Typically, for dosages used in adult treatment, the dosage is typically in the range of 0.02-5000 mg / day, such as about 1-1500 mg / day. The desired dose can be conveniently expressed as one dose, or simultaneously administered (or in a short period of time) or in divided doses at appropriate intervals, such as two, three, four, or more divided doses per day. It will be appreciated by those skilled in the art that, although the above-mentioned dosage range is given, the specific effective amount can be appropriately adjusted according to the patient's condition and in conjunction with the physician's diagnosis.

[0047] As used herein, the term "subject" refers to any animal that is about to or has been administered the UDCA and its conjugated bile acid or pharmaceutical composition according to the embodiments of the present disclosure, preferably a mammal, and most preferably a human. As used herein, the term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., with humans being the most preferred.

[0048] II. Embodiment

[0049] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below. The described embodiments should not be regarded as limiting the present invention. All other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0050] Before further describing the embodiments of the present invention in detail, the nouns and terms involved in the embodiments of the present invention are described. The nouns and terms involved in the embodiments of the present invention are subject to the following interpretations.

[0051] The raw materials and equipment used in the specific embodiments of the present disclosure are all known products, which are obtained by purchasing commercially available products.

[0052] Example 1: Metabolome analysis of clinical samples

[0053] Study subjects

[0054] Patients diagnosed with intracranial aneurysms were diagnosed based on head vascular imaging (including CTA, MRA, and DSA). Blood samples were collected from patients for analysis. The clinical information, imaging information, medication information, and laboratory tests of all enrolled patients were recorded in detail.

[0055] 1.1: Inclusion criteria:

[0056] 1. Patients diagnosed with intracranial aneurysm; 2. Patients with intracranial aneurysm who have not received treatment; 3. Patients with complete medical history information; 4. Patients and their families were informed and agreed to participate in this study.

[0057] Exclusion criteria:

[0058] 1. Dynamic aneurysms with cerebral vascular malformations or cerebral arteriovenous fistulas; 2. Traumatic or false aneurysms.

[0059] 1.2: Research Methods

[0060] This study is a case-control study, not a cohort study. It does not involve follow-up observations of intracranial aneurysms, or ethical issues caused by the failure to treat high-risk aneurysms. It also does not involve the treatment of aneurysm patients. Enrollment in this study will not affect the normal diagnosis and treatment plan and process of patients, and will not have any intervention measures on the treatment of patients.

[0061] This study divided the cases into groups based on the characteristics of the aneurysm cases collected at the time of enrollment, i.e., unruptured aneurysms and ruptured aneurysms, and compared the differences in metabolic markers between the groups.

[0062] 1.2.1: Study cohort and sample collection

[0063] The patients enrolled in this study were divided into a screening cohort and a validation cohort, and each cohort was divided into a bleeding group and a non-bleeding group according to whether bleeding occurred. The groups were matched based on factors such as patient age, gender, medical history, medication, location and nature of aneurysm lesions to improve the effectiveness of screening results. A total of 637 patients were enrolled in this study cohort, including 480 patients with unruptured aneurysms and 157 patients with ruptured aneurysms. All blood samples were collected, plasma was immediately separated by centrifugation, and stored at -80°C until analysis.

[0064] 1.2.2: Sample preparation

[0065] (1) Preparation of standard solution:

[0066] Ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, tauroursodeoxycholic acid, cholic acid, deoxycholic acid, and glycodeoxycholic acid were fully dissolved in methanol, mixed, and prepared into a 1 mg / ml solution, which was the concentrated stock solution, and prepared into a series of concentrations for preparing a standard curve.

[0067] (2) Preparation of internal standard solution:

[0068] Ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, tauroursodeoxycholic acid, cholic acid, deoxycholic acid, and glycodeoxycholic acid are fully dissolved in methanol, mixed, and prepared into a 1 mg / ml solution, i.e., a concentrated stock solution, which is diluted to a certain concentration and reacted with carbon 13-labeled 3-nitrophenylhydrazine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to obtain the obtained solution.

[0069] (3) Preparation of derivatization reagents:

[0070] 3-Nitrophenylhydrazine was prepared into a 200 mmol solution with 75% methanol aqueous solution, mixed evenly, sealed and stored at 4°C for later use.

[0071] (4) Preparation of reaction catalyst for derivatization:

[0072] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide was prepared into a 120 mmol solution with pyridine, mixed well, sealed and stored at 4°C for later use.

[0073] (5) Sample testing

[0074] Serum samples were subjected to metabolomics analysis using the Q300 metabolite analysis kit (Human Metabolomics Institute, Inc., Shenzhen, Guangdong, China). Samples were thawed in an ice bath to reduce sample degradation. 25 ml of supernatant sample was added to each 96-well plate. The plates were then transferred to the Biomek 4000 workstation (Biomek 4000, Beckman Coulter, Inc., Brea, California, USA). Ice-cold methanol and ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, cholic acid, deoxycholic acid, and glycodeoxycholic acid internal standards were automatically added to each sample and vortexed vigorously for 5 minutes. The plate was centrifuged at 4000g for 30 minutes (Allegra X-15R, Beckman Coulter, Inc., Indianapolis, IN, USA). The plate was then returned to the workstation. 30 ml of supernatant was transferred to a clean 96-well plate and 20 ml of freshly prepared derivatization reagent was added to each well. After the plate was sealed, derivatization was performed at 30°C for 60 minutes. After derivatization, 350 ml of ice-cold 50% methanol solution was added to dilute the sample. The plate was then stored at -20°C for 20 minutes and subsequently centrifuged at 4000 g for 30 minutes at 4°C. 135 ml of supernatant was transferred to a new 96-well plate containing 15 ml of internal standard per well. Serial dilutions of the derivatized standard were added to the left well. Finally, the plate was sealed for UPLC-MS analysis.

[0075] 1.2.3: UPLC-MS analysis

[0076] Ultra-high performance liquid chromatography (UPLC) analysis was performed using a Waters Acquity UPLC system (Waters Corp., Milford, MA, USA) using an Acquity UPLC BEH C18 column (2.1 mm×100 mm, 1.7 μm). Mobile phase A was 0.1% formic acid in water, B was 0.1% formic acid in acetonitrile, and the gradient elution program was 0-2 minutes 5% B, 2-7 minutes 5%-95% B, 7-8 minutes 95% B, 8-10 minutes 5% B (re-equilibration). The flow rate was set at 0.3 mL / min, the column temperature was 40°C, and the injection volume was 5 μL. Mass spectrometry (MS) detection was performed using a Waters Xevo TQ-S triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA), with electrospray ionization (ESI) operated in negative ion mode, with a capillary voltage of −2.5 kV, a cone voltage of 30 V, an ion source temperature of 150 ° C, a backflush gas (N2) flow rate of 600 L / h, and a temperature of 350 ° C. The multiple reaction monitoring (MRM) mode was used to select characteristic ion pairs of target metabolites and internal standards for detection.

[0077] Data acquisition and processing were performed using MassLynx software (Waters Corp., Milford, MA, USA), and data processing and analysis were performed using TargetLynx version 4.1 application manager (Waters Corp., Milford, MA, USA). Quantitative analysis was performed by comparing the peak area of ​​the target compound in the sample with that of the internal standard, and metabolite identification was performed based on mass-to-charge ratio (m / z) and fragment information.

[0078] 1.3: Statistical analysis methods

[0079] Sample distribution was determined by the Kolmogorov-Smirnov normality test. Normally distributed continuous variables were expressed as mean ± standard deviation (SD). Continuous variables that did not pass the normality test were expressed as median with interquartile range (IQR). For statistical comparison, normally distributed variables were compared using one-way ANOVA. Non-normally distributed data were analyzed using the Kruskal-Wallis test and were analyzed using SPSS26.0 (IBM SPSS, Chicago, IL, USA).

[0080] 1.4: Results and Analysis

[0081] 1) A panel of bile acid biomarkers that can be used to distinguish unruptured from ruptured intracranial aneurysms

[0082] The differences in bile acid abundance between the intracranial aneurysm group and patients with aneurysmal subarachnoid hemorrhage were compared to screen for alternative differential bile acid metabolic markers. Metabolites with fold change (FC) satisfying FC>1.2 or FC<0.83 were screened, and the U test P<0.05 was used as the screening threshold. Metabolites that met both conditions were considered candidate differential metabolites. The results showed that there were two bile acid metabolites with significantly decreased abundance after intracranial aneurysm rupture, namely UDCA and GUDCA (as shown in Table 1 and Table 2). Figure 1 shown).

[0083] Table 1: A panel of bile acid biomarkers that distinguish unruptured from ruptured intracranial aneurysms

[0084] Bile acid biomarkers FC(aSAH / IA) p-value Differential metabolites UDCA 0.71 0.037 conform to GUDCA 0.69 0.00013 conform to TUDCA 0.83 0.00021 exclude CDCA 0.84 0.0054 exclude TCDCA 1.07 0.0003 exclude GCDCA 1.08 0.25 exclude CA (cholic acid) 0.96 <0.0001 exclude DA (deoxycholic acid) 0.93 <0.0001 exclude GDA (Glycodeoxycholic acid) 1.02 <0.0001 exclude

[0085] Example 2: Verification of UDCA in reducing the rupture rate of intracranial aneurysms in mice

[0086] 2.1: Experimental Purpose

[0087] The aim of the study was to investigate and verify whether UDCA has a protective effect on aneurysms and can reduce the aneurysm rupture rate in the model.

[0088] 2.2: Experimental animals

[0089] C57BL / 6 mice, 10 weeks old, 25 g, SPF grade.

[0090] 2.3: Experimental Grouping

[0091] (1) Normal group (n=10): Sham surgery related to intracranial aneurysm modeling (incision and muscle separation on the back, neck, and scalp, and stereotactic injection of normal saline).

[0092] (2) Intracranial aneurysm group (model group, n=19): Intracranial aneurysm was induced by intragastric administration of 0.2 mL 6% sodium bicarbonate solution (once a day).

[0093] (3) Intracranial aneurysm + UDCA group (treatment group, n = 19): Intracranial aneurysm was induced, and 100 mg / kg UDCA (dissolved in 0.2 mL 6% sodium bicarbonate solution) was administered orally (once a day).

[0094] 2.4: Observation indicators

[0095] (1) Basal blood pressure: measured at 0, 1, 2, and 3 weeks after modeling (blood pressure is a key factor in inducing aneurysm models. By comparing the blood pressure between the model group and the UDCA treatment group, it was demonstrated whether the protective effect of UDCA depends on its antihypertensive effect).

[0096] (2) Neurological symptom scoring: The neurological symptoms of mice were observed and evaluated daily. A score of 1 or above indicated aneurysm rupture. Bromophenol blue gelatin solution was immediately perfused to observe brain hemorrhage and aneurysm formation. Asymptomatic mice were perfused 21 days after aneurysm induction.

[0097] Table 2: Scoring criteria for neurological symptoms in mice

[0098] score Neurological symptom scoring criteria 0 points Functional 1 point Decreased activity or weight loss of more than 2 grams (about 10% of body weight) within 24 hours 2 points Lift the animal by the tail with the trunk and forelimbs flexed 3 points While resting, walk in a normal circle to one side 4 points Leaning to one side when resting 5 points No spontaneous activity 6 points Spontaneous sudden death

[0099] (3) Tumor formation rate and rupture rate (draw a bar graph).

[0100] (4) Aneurysm size: Take a photo and use a ruler as a reference for measurement.

[0101] (5) Aneurysm grading. The grading criteria are shown in Table 2.

[0102] Table 3: Aneurysm grading criteria

[0103] level Grading Standards Level 1 Normal artery Level 2 Arterial dilatation without aneurysm formation Level 3 Unruptured aneurysm Level 4 Ruptured aneurysm

[0104] (6) The severity of subarachnoid hemorrhage (SAH) is graded from 0 to 3. The grading criteria are shown in Table 3 below.

[0105] Table 4: Subarachnoid hemorrhage grading criteria

[0106] level Grading Standards Level 0 No bleeding Level 2 Local bleeding, thin SAH Level 3 Multiple or extensive hemorrhage, diffuse thick SAH Level 4 Heavy bleeding

[0107] (7) Immunofluorescence: The brain vessels of mice in the normal group, model group, and treatment group were sampled, with 4 mice in each group. After fixation with 4% paraformaldehyde, the samples were routinely paraffin-embedded, then 4 μm paraffin sections were routinely dewaxed to water. The dewaxed brain tissue sections were immersed in 0.1 M PBS, 5% FBS, and 0.3% Triton X solution for 1 h at room temperature. The sections were incubated in CD31 and CD68 antibodies at 4°C overnight. Subsequently, the samples were incubated in Alexa Fluor 594-double antibody for 2 h at room temperature. Finally, they were stained with DAPI for 10 min. The sections were observed under a microscope (Olympus, Japan).

[0108] 2.5: Results and Analysis

[0109] Intracranial aneurysm formation rate and rupture rate in mice

[0110] Depend on Figure 2 It can be seen that compared with the normal group, the model group had more aneurysms and a wide area of ​​ruptured bleeding; compared with the model group, the UDCA treatment group had fewer aneurysms and intact vascular structure. Figure 3It can be seen that compared with the model group, the aneurysm rupture rate in the UDCA treatment group (68.75% VS 29.41%) was significantly reduced. The results showed that UDCA treatment significantly reduced the rupture rate of intracranial aneurysms in mice (p = 0.038).

[0111] Depend on Figure 4 It can be seen that compared with the normal group, the expression of endothelial cell marker CD31 was weakened in the aneurysm wall of the model group, the endothelial layer was intermittently defective, the morphology was incomplete, and the aneurysm cavity area was enlarged. Compared with the model group, the endothelial layer of the vascular wall in the UDCA treatment group basically maintained its complete morphology, the expression of endothelial cell marker CD31 was enhanced compared with the model group, and the aneurysm cavity was smaller than that of the model group. The results show that UDCA can significantly improve endothelial cell damage in the vascular wall and maintain the integrity of the vascular endothelial layer.

[0112] Depend on Figure 5 It can be seen that the expression of macrophage marker CD68 on the aneurysm wall of the model group was significantly increased compared with the normal group, and infiltrated into the entire layer of the blood vessel. After UDCA treatment, the expression of macrophage marker CD68 on the aneurysm wall was significantly reduced and only scattered around the aneurysm wall. The results show that UDCA can inhibit the production and infiltration of macrophages on the aneurysm wall. The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and illustration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is obvious that many changes and variations can be made according to the above teachings. The purpose of selecting and describing exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can implement and utilize various exemplary embodiments of the present invention and various selections and changes. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. Use of bile acid and its derivatives in the preparation of a pharmaceutical composition for preventing and / or treating aneurysms.

2. The use according to claim 1, wherein: The bile acid and its derivatives include free bile acid and / or conjugated bile acid, the free bile acid includes one or more of deoxycholic acid, chenodeoxycholic acid, cholic acid or lithocholic acid, and the conjugated bile acid includes the product formed by the free bile acid conjugated with taurine or glycine.

3. The use according to claim 2, wherein: The free bile acid is deoxycholic acid, and the conjugated bile acid is tauroursodeoxycholic acid and / or glycoursodeoxycholic acid.

4. The use according to claim 2, wherein: The bile acid and its derivatives are ursodeoxycholic acid and its conjugated bile acid.

5. The use according to claim 4, wherein: The bile acid and its derivatives are selected from one or more of ursodeoxycholic acid, its conjugated bile acid tauroursodeoxycholic acid or glycoursodeoxycholic acid.

6. The use according to claim 1, wherein: The aneurysm is an intracranial aneurysm.

7. The use according to claim 1, wherein: The prevention and / or treatment of aneurysms is to prevent aneurysm rupture and / or reduce the incidence of aneurysm rupture.

8. The use according to claim 1, wherein: The pharmaceutical composition also contains another active ingredient for treating intracranial aneurysms, or the application includes the combination of the pharmaceutical composition and other drugs for treating intracranial aneurysms.

9. The use according to claim 8, wherein: The pharmaceutical composition further contains a pharmaceutically acceptable carrier or excipient; Preferably, the pharmaceutical composition is contained in one or more pharmaceutical preparations selected from injection, oral liquid, suspension, emulsion, extract, powder, granule, suppository, aerosol, tablet, capsule, microcapsule, microsphere, nanoparticle, liposome or capsule.

10. The use according to claim 9, wherein: The therapeutically effective amount of the pharmaceutical composition comprises 1-100% bile acid and its derivatives; Preferably, it contains 1-20% bile acid and its derivatives; Preferably, it contains 1-5% bile acid and its derivatives; Preferably, it contains 1.2% of bile acids and their derivatives.

Images