Preparation of endotoxin chelator by targeted delivery to ileum and application thereof in treatment of metabolic diseases

Abstract

To provide an endotoxin chelating agent performing targeted delivery to an ileum, and to provide a method for preparing the same and application thereof.SOLUTION: An agent according to the present invention comprises a specific macromolecular copolymer, which has a three-dimensional frame composed of organic carbon-carbon chemical bonds and contains amino groups. An objective of the present invention is to deliver polymeric chelators to a distal part of a small intestine to chelate pathogen-associated molecular patterns produced by intestinal microorganisms, including endotoxins and CpG-DNA. A complex of the polymeric chelating agent and the pathogen molecular pattern released in an ileum is discharged from a digestive tract, thereby reducing inflammation in the body and being used for alleviation of various metabolic diseases.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and relates to an endotoxin chelating agent for target delivery to the ileum, a method for producing the same, and further an application in therapeutic agents for metabolic diseases. A specific polymer chelating agent can be delivered to the terminal part of the small intestine and released, chelate pathogen morphologies generated by intestinal microorganisms, that is, bacterial endotoxins and CpG-DNA, and excrete them outside the body, thereby contributing to the alleviation and treatment of multiple metabolic diseases.

Background Art

[0002] Metabolic abnormalities that appear in the form of obesity, fatty liver disease, type 2 diabetes, etc. pose a major health problem and a heavy economic burden on societies worldwide (The metabolic syndrome: prevalence in worldwide populations, Endocrinol Metab Clin North Am 2004; 33:351-75)(The global burden of metabolic disease: Data from 2000 to 2019. Cell Metab 2023;35:414-428). According to research, metabolic diseases affect many adults. Based on large-scale surveys, the prevalence varies from about 10% to over 30%, specifically depending on the surveyed region and population. In the United States, the metabolic syndrome is a major public health problem (Trends in Metabolic Syndrome Among US Youth, From 1999 to 2018, JAMA 2022;176:043-1045). Contributing factors to the high prevalence include a diet rich in processed foods, high-fat and high-sugar intake, and a relatively sedentary lifestyle. Also, the high obesity rate in the United States greatly promotes the onset of the metabolic syndrome and diabetes.

[0003] Alcoholic liver disease (ALD) includes various liver diseases. According to epidemiological surveys, it is estimated that 60 million people in China are suffering from different degrees of alcoholic liver disease (Epidemiological characteristics of alcohol-related liver disease in China: a systematic review and meta-analysis, BMC 2023;23:1276). Heavy drinking can cause extensive hepatocyte necrosis and liver failure (Alcoholic liver disease: pathogenesis and new therapeutic targets, Gastroenterology 2011;141:1572-85).

[0004] Alcohol consumption is associated with a wide range of diseases and health problems (Global, regional, and national comparative risk assessment of 84 behavioral, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017, Lancet 2018;392:1923-1994). First, excessive alcohol consumption can cause the accumulation of fat in the liver. Heavy drinkers may progress to alcoholic hepatitis (AH), which is characterized by jaundice, abdominal pain, and fatigue. Persistent alcoholic hepatitis can progress to liver inflammation and cirrhosis, which can lead to other complications such as liver failure, cancer, and bleeding from dilated blood vessels in the esophagus. Alcohol consumption can also cause cardiovascular disease (CVD), arrhythmia (irregular heartbeat), myocardial injury, esophagitis, gastritis, peptic ulcer, pancreatitis, and even cancer.

[0005] Blood test indicators for alcoholic liver disease include elevated levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (GGT), total bilirubin (TBil), prothrombin time (PT), mean corpuscular volume (MCV), and carbohydrate-deficient transferrin (CDT).

[0006] In a normal physiological state, a large amount (1 - 2 kilograms) of gut microbiota is mainly restricted to the large intestine, and the upper part of the small intestine is almost sterile. This gating mechanism is partially mediated by α-defensins 5 / 6 secreted by Paneth cells present in the gaps between microvilli (Paneth cell alpha-defensins: peptide mediators of innate immunity in the small intestine. Springer Semin Immunopathol, 2005;27:133 - 46). Loss of Paneth cell function or reduced expression of α-defensins is usually associated with many metabolic abnormalities. Furthermore, intestinal damage is often associated with aging, and intestinal leakage due to loss of tight junctions of intestinal epithelial cells, which is common in patients with metabolic disorders, is also seen (The ageing gastrointestinal tract, Curr Opin Clin Nutr Metab Care 2016;19:12 - 8).

[0007] Previous studies have shown that vitamin D signaling controls the function of Paneth cells to some extent by promoting the expression of α-defensins. Usually, vitamin D deficiency associated with metabolic disorders may reduce the expression of defensins and cause small intestinal bacterial overgrowth (SIGO). This may promote endotoxemia and cause insulin resistance, metabolic disorders, and hepatic steatosis (Vitamin D Signaling through Induction of Paneth Cell Defensins Maintains Gut Microbiota and Improves Metabolic Disorders and Hepatic Steatosis in Animal Models, Front Physiol 2016;7:498).

[0008] Once pathogen-associated molecular patterns (PAMPs) derived from gut microbiota enter the human body, they may promote metabolic disorders through various mechanisms. For example, PAMPs can activate innate immunity through pattern recognition receptors (such as Toll-like receptors) on immune cells (PAMPs and DAMPs: signal 0s that spur autophagy and immunity. Immunol Rev 2012;249:158-75). The interaction between the ligand and the receptor causes a series of signal events, leading to the release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), which may initiate an inflammatory response.

[0009] From a biochemical perspective, endotoxin is lipopolysaccharide (LPS), which is derived from the cell wall of Gram-negative bacteria. LPS released from dead bacteria or the cell wall of living organisms increases circulating endotoxin levels, which is called metabolic endotoxin (Gut-derived low-grade endotoxaemia, atherothrombosis and cardiovascular disease, Nat Rev Cardiol 2023;20:24-37). LPS levels in the peripheral circulation usually increase after a meal. LPS is taken up by chylomicrons, crosses the intestinal barrier and enters the lymphatic system, and then enters the bloodstream. In the blood, endotoxin binds to LPS-binding protein called LBP and several other factors (such as high-density lipoprotein cholesterol) (Chylomicrons promote intestinal absorption of lipopolysaccharides, J Lipid Res 2009;50:90-7).

[0010] In the physiological state of healthy subjects, endotoxin bound to the above-mentioned high-density lipoprotein cholesterol or LBP is transported to the liver, where it is either degraded by specific liver enzymes (such as acyl-oxyacyl hydroxylase and alkaline phosphatase) or excreted into bile via the clearance receptor. Kupffer cells in the liver can detoxify endotoxin by phagocytosis. However, with increasing age and various forms of liver injury, hepatocytes are unable to eliminate circulating endotoxin, and excess endotoxin leaks into the systemic circulation, causing mild endotoxaemia. This further promotes insulin resistance in type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). The pro-inflammatory signal induced by LPS occurs when its lipid moiety, lipid A, binds to TLR4 and binds to the membrane-bound co-receptor CD14. When LPS binds to TLR4, the adaptor protein MyD88 is recruited to the cytoplasmic domain of TLR4, activating transcription factors such as NF-κB and triggering a transcriptional cascade reaction and an inflammatory response.

[0011] Endotoxins in the small intestine may be absorbed by the liver via the portal vein, especially significantly in cases of aging and intestinal damage caused by various reasons.

[0012] Numerous clinical studies have revealed that endotoxemia is strongly associated with various diseases, especially liver diseases. Long-term alcohol consumption, aging, viral infection, vitamin D deficiency, etc. can impair intestinal integrity and cause intestinal leakage. Under such circumstances, bacteria in the large intestine may move into the small intestine, which is called small intestinal bacterial overgrowth (SIBO). As a result, endotoxins and other pathogen-associated molecular patterns accumulate in the distal part of the small intestine.

[0013] Numerous studies have shown that the plasma endotoxin levels in patients with alcoholic liver disease are several times higher than those in healthy subjects (Endotoxemia in patients with alcoholic and non-alcoholic cirrhosis and in subjects with no evidence of chronic liver disease following acute alcohol excess. J Hepatol 1987;4:8-14). The plasma endotoxin levels in normal subjects range from 0.3 to 10.4 picograms / milliliter, while those in patients with alcoholic liver disease range from 8.5 to 206 picograms / milliliter (Plasma endotoxin and serum cytokine levels in patients with alcoholic hepatitis: relation to severity of liver disturbance. Alcohol Clin Exp Res 2000;24:48S-54S). Although endotoxin shows a large variation among individuals, it has been confirmed that the plasma endotoxin levels in patients with alcoholic liver disease are always 5 to 20 times higher than those in normal subjects. Animal experimental models of alcoholic liver injury have confirmed endotoxemia in alcoholic liver disease. Based on male rat models, some reports through excessive alcohol consumption have shown that acute or chronic ethanol administration increases plasma endotoxin levels, and this level is closely related to the progression of liver injury (Exacerbation of alcoholic liver injury by enteral endotoxin in rats. Hepatology 2000;32:1008-17).In another study, in rats fed ethanol, increased endotoxemia and lipid peroxidation in female rats stimulated the activation of NF-κB and the production of chemokines, indicating a worsening of liver injury (Increased severity of alcoholic liver injury in female rats: role of oxidative stress, endotoxin, and chemokines. Am J Physiol Gastrointest Liver Physiol 2001;281:G1348-56). The alleviation of alcohol-induced endotoxemia and liver injury by antibiotics indicates that endotoxin has an important causal relationship in alcoholic liver injury. In fact, it has been shown that alcoholic hepatitis in Wistar rats is improved by sterilizing intestinal microorganisms and reducing endotoxin by orally administering a large amount of polymyxin B (Antibiotics prevent liver injury in rats following long-term exposure to ethanol. Gastroenterology 1995;108:218-24). According to newly presented evidence, LPS and ethanol show a synergistic effect on hepatocytes. LPS alone cannot mimic ethanol-induced steatosis or hepatitis, but co-administration of ethanol and LPS effectively induces liver injury.Feeding ethanol increases the susceptibility of the livers of experimental animals to cell damage induced by LPS (Chronic ethanol feeding increases activation of NADPH oxidase by lipopolysaccharide in rat Kupffer cells: role of increased reactive oxygen in LPS-stimulated ERK1 / 2 activation and TNF-alpha production. J Leukoc Biol 2006;79: 1348-56)(Ethanol feeding enhances inflammatory cytokine expression in lipopolysaccharide-induced hepatitis. J Gastroenterol Hepatol 1997;12:305-13).

[0014] In the circulatory system, lipopolysaccharide-binding protein (LBP) presents bacterial toxins to the cell receptor CD14 and TLR-4. Therefore, long-term ethanol administration increases the expression of LBP and CD14. Conversely, gene knockout mice of LBP, CD14, and TLR-4 show resistance to ethanol stimulation, indicating that endotoxin plays a causal role in promoting liver injury (Role of lipopolysaccharide-binding protein in early alcohol-induced liver injury in mice. J Immunol 2002;168;6:2963-9).

[0015] Various methods capable of reducing endotoxin in the blood have been shown to be effective in the treatment of severe liver diseases. For this reason, polymyxin B-immobilized fiber columns (PMX) in extracorporeal circulation devices are clinically used to remove endotoxin from the bodies of patients with liver failure and septic shock (Efficacy of polymyxin B-immobilized fiber column direct hemoperfusion for non-endotoxin-associated severe septic shock. Pediatr Int 2016;58:1346-1347). Extracorporeal circulation and plasma exchange are used to reduce hepatotoxins and endotoxins for the treatment of various diseases such as liver failure, multiple organ failure, and sepsis. However, a major obstacle to extracorporeal circulation methods including PMX is the high cost and surgical risk. Importantly, these treatment methods cannot prevent the influx of gut-derived endotoxin into the body. Therefore, the development of oral drugs capable of chelating enteric pathogen-associated molecular patterns containing endotoxin has profound potential application value in the prevention and treatment of various metabolic disorders including liver diseases.

[0016] Bile acid sequestrants with various different structures include carboxyalkylamine, carboxymethylcellulose, and carboxybenzamine. These are biocompatible resins that are not absorbed by the human body and are completely excreted from the digestive tract when bound to bile acids. Such high molecular weight copolymers are clinically used to reduce hypercholesterolemia by chelating bile acids, and in the process, cholesterol is compensatorily converted to bile acids.

Summary of the Invention

Problems to be Solved by the Invention

[0017] The content of the present invention has demonstrated that a specific copolymer can be used as an active pharmaceutical ingredient and can chelate enteric-derived endotoxins and other pathogen-associated molecular patterns (PAMPs). Furthermore, the content of the present invention has further demonstrated a system capable of targeted delivery of the aforementioned chelating agent to the distal part of the small intestine, thereby realizing a highly efficient chelation reaction and treatment potential. The content of the present invention not only improves the chelation efficiency against endotoxins and other PAM in the ileum, but also significantly reduces the dosage, reduces the adsorption of water-soluble nutrients, and improves the safety of treatment. These two elements form the basis of a new treatment method called pathogen adsorption and sequestration agents (PASA) therapy. In this application, a method for targeted delivery of an endotoxin chelating agent to the ileum and related therapeutic uses are proposed.

[0018] The present invention relates to a method for manufacturing a drug and its potential therapeutic applications. Specifically, it aims to effectively chelate endotoxins and other pathogen-associated molecular patterns (PAMPs) and be used in treatment by using a specific copolymer as an active pharmaceutical ingredient and targeted delivery of the drug to the ileum. This innovative method has distinct advantages in the pharmaceutical field, realizing accurate and effective drug delivery for chelating abundant endotoxins in the distal part of the small intestine. The required method has great potential in the treatment of various diseases and represents a novel and valuable contribution to drug manufacturing and therapeutic application technologies.

[0019] On the one hand, the present invention provides a chelating agent composition for a subject having metabolic abnormalities caused by or promoted by metabolic endotoxins and other gut-derived PAMPs. This pharmaceutical composition variously contains biocompatible particles and polymers that function as active pharmaceutical ingredients (APIs), and these are not digested and absorbed in the digestive tract of the subject. The chelating agent composition binds at least endotoxin or lipopolysaccharide (LPS) and other PAMPs (e.g., CpG-DNA derived from gut microbiota) in the form of a chelating agent-PAMP complex, and this complex can include covalent or non-covalent bonds. By forming the chelating agent-PAMP complex, gut PAMPs containing endotoxin are eliminated from the digestive tract together with the chelating agent complex.

[0020] Tablets or capsules for chelating endotoxin among other PAMPs are composed of a core granule containing a specific copolymer as the API, a separating layer, and a pH-sensitive coating. The insoluble and indigestible copolymer in the formulation is target-delivered to the distal part of the small intestine and can chelate specific pathogen-associated molecular patterns such as endotoxin.

[0021] On the other hand, the present invention discloses a method for treating a subject having metabolic abnormalities that are closely related to persistent inflammation and the accompanying insulin resistance and are partially driven by metabolic endotoxins. Metabolic abnormalities are a significant cause and basis for complications and liver diseases (alcoholic hepatitis, metabolic-associated steatohepatitis (MASH), liver fibrosis, cirrhosis, liver failure, and even liver cancer). Furthermore, metabolic abnormalities caused by endotoxins and other PAMPs in the gut may promote complications such as cardiovascular disease (CVD), type 2 diabetes (T2D), and central obesity. Therefore, the principle of PASA therapy and the pH sensitivity and ileum target delivery of endotoxin chelating agents containing specific copolymers can be utilized for the treatment of these metabolic diseases.

Means for Solving the Problems

[0022] The following are the innovative points related to the disclosure of the present invention.

[0023] Most existing treatment methods for metabolic diseases (non-alcoholic steatohepatitis / metabolic associated steatohepatitis, alcoholic hepatitis) and new drug development efforts focus on specific cellular targets, including important enzymes and receptors that control metabolic pathways. Metabolic diseases include many alcoholic and non-alcoholic liver diseases and have diverse genetic and epigenetic factors. Therefore, it is difficult to obtain broad effects with drugs targeting a single target. For example, obeticholic acid (OCA) is a new drug used for the treatment of NASH and is a farnesoid X receptor (FXR) agonist. FXR is a nuclear receptor that plays an important role in bile acid, lipid, and glucose metabolism. By activating FXR, OCA is expected to improve liver function and reduce inflammation and fibrosis in NASH patients. Although OCA has shown some advantages in NASH patients, the degree of improvement may be insufficient to meet the approved regulatory requirements. Elafibranor is a dual peroxisome proliferator-activated receptor (PPAR) α / δ agonist aimed at improving insulin sensitivity, promoting lipid metabolism, and reducing inflammation, but it failed in clinical trials. Due to genetic and environmental factors, different patients may show different responses, making it difficult to obtain consistent results among different patient groups. Hundreds of genes or single nucleotide polymorphisms (SNPs) are associated with the pathogenesis of NASH. For example, genes involved in lipid metabolism include PNPLA3 (patatin-like phospholipase domain-containing protein 3), TM6SF2 (transmembrane 6 superfamily member 2), and MBOAT7 (containing membrane-bound O-acyltransferase domain 7), which are closely associated with an increased risk of NASH. These genes are involved in processes such as triglyceride metabolism and the formation of lipid droplets. Furthermore, genes related to inflammation and immune responses, such as genes encoding cytokines and chemokines, also contribute to the pathogenesis of NASH. In fact, genes involved in the tumor necrosis factor-α (TNF-α) pathway and interleukin-6 (IL-6) signaling may affect inflammation in the liver.In addition, genes involved in oxidative stress and mitochondrial function, such as SOD2 (superoxide dismutase 2), may also be associated with NASH. Mitochondrial dysfunction and related genes may also cause an increase in oxidative stress and contribute to liver damage. These small molecule drugs targeting specific pathways are insufficiently effective in addressing the extensive diversity of the NASH pathogenesis mechanism. The same is true for other metabolic diseases. Therefore, it is necessary to creatively consider and solve the upstream causes and factors of liver diseases. In this regard, this article proposes a new treatment method called PASA (pathogen adsorption and chelating agent) therapy. This method shows promise in the treatment of alcoholic hepatitis and related metabolic abnormalities. By targeting the delivery of a copolymer-based chelating agent to the distal part of the small intestine, this drug can effectively adsorb and chelate microbial-derived pathogens such as endotoxins, block the upstream of inflammation, and remove the intestinal-derived effects, providing a potential means to address the root cause of metabolic diseases.

[0024] Intestinal-derived pathogen-associated molecular patterns are a common source of inflammation in various metabolic diseases. Bacterial-derived endotoxins and other pathogen-associated molecular patterns (PAMPs) that have migrated from the large intestine to the small intestine may enter the circulatory system and the liver mainly through two pathways. They may be adsorbed by the lymphatic system or enter the circulatory system via the portal vein respectively. This high level of intestinal-derived metabolic endotoxin becomes a major source of systemic and tissue inflammation. For example, metabolic endotoxins may promote insulin resistance by inactivating the insulin-mediated signaling pathway. This forms the basis of type 2 diabetes and other related metabolic abnormalities.

[0025] Our research has shown that carboxyalkylamine and carboxymethylcellulose, which are bile acid sequestrants, have the ability to effectively bind and chelate endotoxin and bacteria-derived CpG-DNA. Furthermore, we have found that oral intake of these chelating agents can reduce metabolic endotoxin levels in other pathological conditions such as metabolic syndrome, liver fibrosis, and pancreatic cancer. However, the high molecular weight chelating agents in these original formulations are designed to be released in the upper gastrointestinal tract and are inefficient at capturing endotoxin, which is mainly abundant in the distal part of the small intestine. Additionally, previous formulations of these chelating agents disperse in the upper part of the small intestine and may chelate hydrophobic nutrients such as vitamin D and vitamin K, which can cause some adverse effects. To address these problems, the present invention proposes new methods for the manufacture, composition, and formulation of pharmaceutical preparations for new medical applications and indications against new pathogens.

[0026] In the first part of the present invention, a method for manufacturing a pH-sensitive and time-dependent chelating agent formulation for targeted delivery to the distal part of the small intestine is described. This method can improve the limitations of existing drugs, enhance the efficiency of endotoxin removal in the distal part of the small intestine, and minimize unnecessary chelation with hydrophobic nutrients.

[0027] In the second part of the present invention, it is demonstrated for the first time that a chelating agent can function as a treatment for chelator-related metabolic disorders (such as alcoholic hepatitis) through targeted delivery in the ileum. In particular, it has been discovered that drugs such as carboxyalkylamine and carboxymethylcellulose targeted to the ileum can suppress systemic inflammation and hepatitis. This is a significant factor in many diseases. These findings form the basis for expanding the indications for reducing other liver diseases.

[0028] From a molecular mechanism perspective, it has been discovered that endotoxin chelating agents targeting the ileum can regulate many of the following pathological pathways: (1) hepatocellular carcinoma, (2) diabetic complications, (3) p53, (4) Hippo, (5) insulin resistance, (6) cellular senescence, (7) rheumatoid arthritis, (8) drug metabolism, (9) chemokine signaling, (10) differentiation of Th1 and Th2 cells. The overall regulation of this liver pathway lays the foundation for the wide application of PASA therapy as a new treatment method.

[0029] An object of the present invention is to provide a drug, a manufacturing method, and its application for treating metabolic disorders caused by intestinal pathogen-associated molecular patterns. The specific invention content is as follows.

[0030] The drug contains a specific polymer copolymer, which has a three-dimensional framework composed of organic carbon-carbon chemical bonds and contains amino groups.

[0031] The dosage form of the drug is microspheres, tablets, or capsules.

[0032] The microspheres or tablets are composed of a core granule, an isolation layer, and a pH-sensitive coating; the core granule contains a polymer copolymer as an active drug ingredient.

[0033] The polymer copolymer contains any one or any combination of carboxymethyl cellulose, carboxyalkylamine, carboxybenzamine, and carboxymide.

[0034] The pH-sensitive coating contains methyl methacrylate and methacrylic acid.

[0035] The pH-sensitive coating ensures that the drug is released within the pH range of 6.5 to 7.5.

[0036] A method for manufacturing a drug for treating metabolic disorders caused by enteric pathogen-related molecular patterns, comprising the following steps.

[0037] (1) Manufacture of core granules: Weigh carboxymethyl cellulose or carboxyalkylamine, microcrystalline cellulose, cross-linked sodium carboxymethyl cellulose, and hydroxypropyl methylcellulose, mix for 15 minutes to obtain a premix powder. Then, add magnesium stearate, mix for 5 minutes to obtain the overall mixed powder, and tableting is carried out. (2) Coating with an isolation layer: Pour absolute ethanol into a stainless-steel bucket, start the stirrer, add a methacrylic acid-methyl methacrylate copolymer, and continue stirring until it is completely dissolved. After dissolution, add triethyl citrate while stirring, and continue stirring for 1 hour after addition to produce an isolation layer coating solution; Start a high-efficiency coater and preheat the equipment under idling. When the exhaust temperature reaches 30 °C or higher, place the core granules manufactured in step 1 into the high-efficiency coater for coating. Adjust the weight gain range of the isolation layer to 2-3% to obtain isolation-coated granules; (3) pH-sensitive coating: A. Put pure water into a stainless-steel bucket, add polyethylene glycol 6000 to the pure water partially, and continue stirring until it is completely dissolved to obtain an aqueous solution of polyethylene glycol 6000. B. Take another portion of pure water in a stainless-steel bucket, add talc powder to the pure water, and stir for 30 minutes after addition to obtain a suspension of talc powder. C. Add the aqueous solution of polyethylene glycol 6000 to the cooled talc powder suspension, stir for 30 minutes, then add triethyl citrate, and stir for another 30 minutes to obtain suspension 3. D. Weigh an aqueous dispersion of a methacrylic acid-ethyl acrylate copolymer, put it into a stainless-steel bucket, add pure water, and stir for 30 minutes to obtain an aqueous dispersion suspension of the methacrylic acid-ethyl acrylate copolymer. E. Add suspension 3 to the aqueous dispersion suspension of the methacrylic acid-ethyl acrylate copolymer, continuously stir for 60 minutes to obtain an enteric coating solution with a concentration of 20.0% (w / w). F. Perform enteric layer coating. Start the high-efficiency coater, preheat the equipment by idling, place the isolated coating particles in the high-efficiency coater, adjust the atomization and fan pressure to perform coating, adjust the weight gain of the enteric layer to 8.0 - 10.0% to obtain the drug. Application in a therapeutic agent for metabolic diseases of drugs for treating metabolic disorders caused by enteric pathogen-associated molecular patterns.

[0038] The present invention provides a method for manufacturing a drug for treating metabolic disorders caused by enteric pathogen-associated molecular patterns (PAMPs). This method includes administering to a subject a drug with a special formulation aimed at delivering a specific polymeric chelating agent to the distal part of the small intestine, which chelates PAMPs including endotoxin and CpG-DNA by intestinal microorganisms. The complex formed by the binding of the chelating agent and PAMPs can be excreted from the digestive tract to the outside of the body.

[0039] The specific polymeric chelating agent drug is target-delivered from the pH-sensitive coating in the ileum, effectively neutralizes pathogen-associated molecular patterns at the end of the small intestine, and is used for the treatment of corresponding diseases.

[0040] Metabolic diseases include alcoholic hepatitis, metabolic-associated steatohepatitis (MASH), liver fibrosis and cirrhosis, liver dysfunction, liver cancer, cardiovascular disease (CVD), type 2 diabetes (T2D), and central obesity.

Advantages of the Invention

[0041] The beneficial effects of the present invention are as follows:

[0042] The drug of the present invention effectively adsorbs and chelates pathogens derived from microorganisms such as endotoxins, thereby blocking upstream and gut-derived effects of inflammation and providing a potential means to address the root causes of metabolic diseases. The present invention improves on the limitations of existing drugs, enhances the efficiency of endotoxin removal in the distal part of the small intestine, and minimizes unnecessary chelation with hydrophobic nutrients. We have demonstrated for the first time that a chelating agent can function as a treatment for chelation-related metabolic disorders (such as alcoholic hepatitis) through targeted delivery in the ileum. We have also discovered that drugs such as carboxyalkylamine or carboxymethylcellulose targeted to the ileum suppress systemic inflammation and hepatitis.

[0043] The present invention chelates pathogen-associated molecular patterns (including endotoxins and CpG-DNA) generated by gut microorganisms by delivering and releasing a specific polymeric chelating agent to the distal part of the small intestine. The specific polymeric chelating agent released in the ileum forms a complex with the pathogen molecular pattern and is excreted from the gastrointestinal tract, thereby reducing inflammation in the body and contributing to the alleviation of various metabolic diseases.

Brief Description of the Drawings

[0044]

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Mode for Carrying Out the Invention

[0045] The present invention will be described in detail below based on the accompanying drawings and specific examples. Obviously, the described examples are some of the examples of the present invention, not all of them. Based on the examples of the present invention, all other examples obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present invention.

Example

[0046] Manufacture and formulation of pH-sensitive ileum-targeted release formulations.

[0047] In the present invention, selected specific polymer polymers (such as carboxyalkylamine, carboxymethylcellulose, carboxybenzamine, etc.) are used as active pharmaceutical ingredients (APIs) to manufacture formulations or drugs that are pH-sensitive and targeted for delivery to the ileum.

[0048] One of the important methods for pH-sensitive formulations is based on the Eudragit system. Specifically, Eudragit S100 and Eudragit L100 are two commonly used polymers, both belonging to the Eudragit series and being polymethylacrylate copolymers for drug delivery systems. These two types of polymers have different properties and uses in drug delivery. Eudragit S100 is an anionic polymer, composed of methyl methacrylate (MMA) and methacrylic acid (MAA) in a molar ratio of about 1:2, and has a molecular weight of about 135 kDa. Since Eudragit S100 dissolves in an environment where the pH exceeds 7, it is suitable for a colon-targeted drug delivery system, and the pH in the colon usually exceeds 7. Eudragit L100 is also an anionic polymer, but the molar ratio of MAA to MMA is about 1:1, and the molecular weight is also about 135 kDa. Eudragit L100 is usually a copolymer of methacrylic acid and methyl methacrylate. The chemical formula of methacrylic acid is C 4 H 6 O 2 and it is composed of a carboxylic acid group (-COOH) bonded to a carbon chain with a double bond. The chemical formula of methyl methacrylate is C5 H 8 O 2 It is the methyl ester of methacrylic acid. Eudragit L100 dissolves in an environment where the pH ranges from 6 to 7.

Example

[0049] For the scheme to produce target carboxyalkylamine or carboxymethylcellulose released at pH 6.5 and pH 7.5 in the ileum using the Eudragit system, refer to Table 1 and Table 2.

[0050] Composition of core particles

[0051]

Table 1

[0052] Composition of pH-sensitive ileum target release coating layer

[0053]

Table 2

[0054] The present invention manufactures carboxyalkylamine or carboxymethylcellulose preparations that are target released at a pH value of about 6.5 - 7.5 in the ileum using the Eudragit system. Specifically, it is as follows: 1. Materials and equipment: (A) Carboxyalkylamine or carboxymethylcellulose powder as the active drug ingredient. (B) Eudragit polymers suitable for pH-dependent release (for example, Eudragit L100 for pH 6.5 and Eudragit S100 for the pH 7.5 version). (C) Organic solvents (for example, ethanol, isopropanol). (D) High-shear mixer. (E) Spray dryer. (F) pH buffer solution for testing. 2. Formulation Ingredients: (A) Carboxyalkylamine or carboxymethylcellulose, an active ingredient intended for release in the ileum. (B) Eudragit polymer: Select a Eudragit polymer that is insoluble under acidic conditions and begins to dissolve in a state close to pH 6.5 to ensure targeted release in the ileum. (C) Additives: Optional additives such as plasticizers, stabilizers, or flow aids can be added to improve the performance of the formulation. 3. Manufacturing Procedure: (A) Dissolve the Eudragit polymer (Eudragit L100 or Eudragit S100) in a suitable organic solvent (e.g., ethanol, isopropanol, acetone) to form a polymer solution. (B) Add the carboxyalkylamine powder to this solution and mix well using a high-shear mixer to ensure that the carboxyalkylamine is uniformly dispersed within the polymer matrix. (C) Adjust the concentration and viscosity of the mixture as needed to achieve the desired coating performance. (D) Use a spray dryer to coat the particles of carboxyalkylamine or carboxymethylcellulose with the Eudragit polymer, forming coated particles with controlled release characteristics through the spray drying process. (E) Optionally, the coated tablets can be further processed through sieving or polishing to obtain a uniform particle size distribution. (F) Test the carboxyalkylamine formulation coated in an acidic to basic pH buffer solution to confirm targeted release at pH 6.5. 4. Quality Control: (A) Particle Size Analysis: Use techniques such as laser diffraction or microscopy to identify the particle size distribution of the coated carboxyalkylamine particles. (B) Encapsulation Efficiency: Evaluate the amount of carboxyalkylamine encapsulated within the Eudragit coating using appropriate analytical methods. (C) In vitro release study: Conduct a release study using a pH buffer solution under simulated gastrointestinal conditions. Monitor the release of carboxyalkylamine over time to ensure targeted release at pH 6.5. (D) Stability test: Store the formulation under different conditions (such as temperature and humidity), and monitor the changes in particle size, encapsulation efficiency, and release characteristics over time. 5. Conclusion: By following this method, it is possible to manufacture target carboxyalkylamine formulations at pH 6.5 and pH 7.5 using the Eudragit system and achieve release in the ileum. This formulation may enhance the therapeutic effect by targeting the release of carboxyalkylamine and other polymers to specific action sites in the gastrointestinal tract.

Example

[0055] Another scheme for manufacturing mini-tablets of carboxyalkylamine and carboxymethylcellulose with pH sensitivity and ileum-targeted release using the Eudragit system is shown. Refer to Table 3 and Table 4.

[0056] In the formulation example of SQ6.5, the carboxyalkylamine is designed to disintegrate at pH 6.5.

[0057]

Table 3

[0058] In the formulation example of SQ7.5, the carboxyalkylamine or carboxymethylcellulose is designed to disintegrate at pH 7.5.

[0059]

Table 4

[0060] (1) Ingredients: The core granules contain a bile acid chelating agent as the active pharmaceutical ingredient and, as other ingredients, lactose, microcrystalline cellulose, pregelatinized starch, starch, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, and magnesium stearate. The pH-sensitive enteric layer contains Eudragit S100 (methacrylic acid-ethyl acrylate) copolymer, hydroxypropyl methylcellulose acetate succinate, carboxylated agarose, carboxymethyl yellow lemon calcium chloride hydrogel, anionic copolymer of methacrylic acid and methyl methacrylate, cellulose phthalate, hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethyl cellulose, cellulose acetate, and maleic acid and phthalic acid derivative copolymer. The coating contains Eudragit L100 (or Eudragit S100) (5% - 7%), triethyl citrate (0.5% - 0.7%), talc (4% - 5%), ethanol (83% - 87%), and pure water (4% - 6%). (2) Process: Mix a predetermined amount of carboxyalkylamine hydrochloride or carboxymethyl cellulose, microcrystalline cellulose, and sodium carboxymethyl cellulose. Rotate the container at a speed of 15 revolutions per minute for 15 minutes. After mixing uniformly, slowly add 5% hydroxypropyl methylcellulose and perform wet granulation. Then, dry in a forced-air drying oven at 60°C, and then add magnesium stearate and mix further. Compress the granules using a 2-millimeter punching head to produce mini-tablets for animal experiments. Next, enteric coat the core granules with Eudragit L100 or Eudragit S100 and other ingredients. (3) Measurement of disintegration time and pH value: The mini-tablets are named SQ6.5, contain carboxyalkylamine as the active drug ingredient, disintegrate at pH 6.5, and SQ7.5 disintegrates at pH 7.5. As shown in Table 5, these two types of tablets can withstand up to 4 hours in 0.1N hydrochloric acid. In contrast, the SQ6.5 mini-tablets disintegrate in a solution at pH 6.5 (50 mM Tris at pH 6.5), while the SQ7.5 mini-tablets are stable at pH 6.5 and completely disintegrate within 30 minutes at pH 7.5. We also evaluated the hardness of tablets containing barium sulfate (Table 6) used for animal experiment follow-up. The prototype mini-tablets of SQ6.5 and SQ7.5 are shown in Figure 4A, where SQ6.5 shows carboxyalkylamine that disintegrates when the pH is 6.5, and SQ7.5 shows carboxyalkylamine released when the pH is 7.5.

[0061] Disintegration conditions of ileum-targeted tablets

[0062]

Table 5

[0063] Hardness of pH-sensitive and ileum-targeted microspheres.

[0064]

Table 6

Example

[0065] Hydroxypropylmethylcellulose phthalate (T-HPMCP, T-type) is often used in pH-sensitive formulations for drug delivery. T-HPMCP is a polymer that exhibits pH-dependent dissolution characteristics. It is insoluble in an acidic environment but dissolves at higher pH values. This property is very suitable for preventing drug degradation in the acidic environment of the stomach and ensuring target release in the high-pH region of the intestine. (1) T-component: For pH-sensitive copolymer drugs targeting the ileum, designing a T-HPMCP formulation can release the drug at the appropriate site, improve the bioavailability of the drug, and reduce side effects. Hydroxypropyl methylcellulose phthalate (HPMCP) is used as an enteric polymer to control release in the ileum. Other additives may include fillers (e.g., microcrystalline cellulose), adhesives (e.g., polyvinylpyrrolidone), lubricants (e.g., magnesium stearate), and disintegrants (e.g., cross-linked sodium carboxymethyl cellulose). (2) Method: Weigh and mix the active pharmaceutical ingredient (such as carboxyalkylamine) and various additives. Granulation is performed if necessary. Compress the mixture to form core granules. Then, apply a T-HPMCP coating to the particles using a coating device or fluidized bed coating technology. Adjust the coating parameters to achieve targeted release of carboxyalkylamine in the ileum. Optimize the coating thickness and composition to ensure that the formulation is completely retained in the stomach and small intestine and the drug is released in the ileum.

Example

[0066] A scheme for manufacturing a pH 6.5 targeted formulation that is released in the ileum using a poly(lactic-co-glycolic acid) (PLGA) system. (1) Introduction: This method describes the steps for creating a formulation aimed at releasing a copolymer as an active pharmaceutical ingredient in the ileum. The pH of the ileum is approximately 6.5. Poly(lactic-co-glycolic acid) (PLGA) is used as a pH-sensitive polymer that can decompose in response to specific pH changes and release the encapsulated substance. (2) Materials and Equipment: Poly(lactic-co-glycolic acid) copolymer with appropriate molecular weight and degradation characteristics; copolymer as the active pharmaceutical ingredient (API) (such as carboxyalkylamine or carboxymethyl cellulose); organic solvents (e.g., dichloromethane or acetone); ultrasonic device; rotary evaporator; freeze dryer; pH buffer solution for testing. (3) Formulation Ingredients: PLGA is used as a pH-dependent release pill coating polymer. The selection of PLGA should be based on its degradation rate at pH 6.5 - 7.5 and the ability to release the active drug ingredient. Different molecular weights and copolymer ratios can be considered to achieve the desired release curve. Carboxyalkylamine or carboxymethylcellulose is used as the active drug ingredient delivered to the ileum and effectively removes PAMPs generated by intestinal microorganisms. Optional additives such as stabilizers, surfactants, or excipients can also be added to the core of the pill to improve the stability and release characteristics of the formulation. (4) Manufacturing Procedure: (A) Dissolve PLGA in a suitable organic solvent (e.g., acetone or ethyl acetate) to form a polymer solution. The concentration of PLGA needs to be optimized to ensure appropriate encapsulation and release characteristics. (B) Add carboxyalkylamine or carboxymethylcellulose to the solution and mix thoroughly using an ultrasonic device or other suitable mixing equipment. This step ensures that the active drug ingredient is uniformly dispersed within the polymer matrix. (C) Use emulsion technology to create an emulsion of the polymer-active drug ingredient solution in the aqueous phase. This can be achieved by adding the polymer solution drop by drop to a stirred aqueous solution containing a surfactant. (D) After stabilizing the emulsion, remove the organic solvent using a rotary evaporator. This step forms PLGA nanoparticles encapsulating the active drug ingredient.

Example

[0067] Improved Version of pH-Sensitive Targeted Formulation

[0068] Composition Examples of Carboxyalkylamine (SQ1-T) or Carboxymethylcellulose (SQ1-C) That Disintegrate at pH 6.8

[0069]

Table 7

[0070] (1) Compression of core granules: Weigh carboxyalkylamine or carboxymethyl cellulose (active pharmaceutical ingredient), microcrystalline cellulose, cross-linked sodium carboxymethyl cellulose, and hydroxypropyl methylcellulose based on the specified amounts in Table 7, and mix for 15 minutes to obtain a premix powder. Then, add magnesium stearate and mix for 5 minutes to obtain the overall mixed powder. Test the content of the intermediate product. Based on the content of the intermediate product, calculate the weight of the tablets to be compressed. Control the weight variation of the tablets within ±5.0%. Compress using a circular deep concave punching head and mold with a diameter of 2 millimeters. Control the hardness to 5 - 15 newtons per square millimeter. (2) Coating with an isolation layer: Start the high-efficiency coater and preheat it in a no-load state. When the exhaust temperature reaches 30°C or higher, put the manufactured core granules into the high-efficiency coater, randomly sample and measure the weight. Adjust the atomization pressure and the fan pressure within the range of 0.1 - 0.6 megapascals. During the coating process, control the inlet temperature at 30 ± 10°C, the exhaust temperature at 30 ± 10°C, the pump speed at 1 - 25 revolutions per minute, and the pan speed at 2 - 8 revolutions per minute. Confirm that the weight gain range of the isolation layer is within 2 - 3%. (3) Preparation before coating: (A) Put pure water into a stainless-steel bucket, add polyethylene glycol 6000 little by little to the pure water, and stir until it is completely dissolved to obtain an aqueous solution of polyethylene glycol 6000 (Solution 1). (B) Prepare another portion of pure water in a stainless-steel bucket, add talcum powder little by little to the pure water, then continue stirring for 30 minutes, and use a high-speed homogenizer to homogenize the stirred suspension for 3 minutes (homogenizer speed: 8000 revolutions per minute) to obtain a talcum powder suspension (Suspension 2). (C) Add Solution 1 to the cooled Suspension 2, stir for 30 minutes, then add triethyl citrate and stir for another 30 minutes to obtain Suspension 3. (D) Weigh an aqueous dispersion of a methacrylic acid-ethyl acrylate copolymer, put it into a stainless-steel bucket, add the remaining pure water and stir for 30 minutes to obtain an aqueous dispersion suspension of the methacrylic acid-ethyl acrylate copolymer. (E) Slowly add suspension 3 to suspension 4 and stir for 60 minutes to obtain an enteric coating solution of about 20.0% (w / w). (F) Coating of the enteric layer: Open the high-efficiency coater and preheat it in the unloaded state. When the exhaust temperature reaches 30 °C or higher, put the coated tablets (isolation layer) into the high-efficiency coater and preheat for 5 minutes. After preheating, randomly sample and measure the weight. Adjust the atomization pressure and the fan pressure in the range of 0.1 - 0.6 megapascals. During the coating process, control the inlet temperature at 30 ± 10 °C, the exhaust temperature at 30 ± 10 °C, the pump speed at 10 - 25 revolutions per minute, and the pan speed at 6 - 18 revolutions per minute. The weight increase of the enteric layer should be in the range of 8.0 - 10.0%.

[0071] The present invention describes methods, compositions, and formulations of drugs. The drug formulations contain an endotoxin chelating agent as the active drug ingredient, and these formulations are designed to target release in the distal part of the small intestine. The purpose of these formulations is to chelate and excrete endotoxin and other pathogen-associated molecular patterns. Furthermore, these formulations inhibit the entry of endotoxin into the circulation and are used for the medical prevention and treatment of metabolic disorders including liver diseases.

Examples

[0072] Use an animal model to study the in vivo disintegration situation of carboxyalkylamine and carboxymethylcellulose in the ileum target release.

[0073] As shown in Figure 1, mini - tablets were manufactured as prototypes of three types of pH - sensitive formulations. The blue tablets are barium sulfate which is the target at pH 6.8 and is used to track the dissolution time and location in the body. The white tablets are for SQ1 - C (carboxyalkylamine), and the red tablets are for SQ1 - T (carboxymethylcellulose). Both are designed to disintegrate at pH 6.5. The diameter of the mini - tablets used in the rat experiment is 2 millimeters, the length is 5 millimeters, the hardness is in the range of 5 - 15 newtons per square millimeter, the weight of each tablet is about 8 - 10 milligrams, and the active pharmaceutical ingredient contains 5 - 7 milligrams.

[0074] We measured the disintegration pH values of the mini - tablets. First, it was proved that the three types of tablets could withstand in 0.2N hydrochloric acid for more than 60 minutes. Next, the tablets were transferred to buffer solutions with pH values gradually increasing from 2.5 to 7.5. These buffer solutions contain 120 mM sodium chloride and 50 mM tris (tris - hydroxymethylaminomethane). The dissolution time was recorded, and it was observed that the tablets disintegrated in the buffer solution of pH 6.5. The tablets disintegrated within 30 minutes in the buffer solution of pH 6.5, but the actual in - vivo dissolution time was identified by in - vivo tracking using barium sulfate tablets.

[0075] To identify the disintegration situation in the body, adult SD rats were fasted overnight before orally gavaging 4 - 5 mini - tablets containing barium sulfate at pH 6.8. Then, standard feed and drinking water were added to the cage. X - ray - based CT scans were performed on the anesthetized rats at the designated time points. As shown in Figure 2, the tablets at pH 6.8 began to dissolve within 4 - 6 hours and most of them disintegrated within 8 hours. No tablets were found in the digestive tract 24 hours after administration. Furthermore, to confirm the disintegration situation in the distal part of the small intestine, rats were euthanized and dissected at different time points to identify the location of the tablets. SQ1 - T is a carboxyalkylamine for ileal - targeted release, and SQ1 - C is a carboxymethylcellulose for ileal - targeted release.

Example

[0076] In vitro chelation experiments of endotoxin and CpG-DNA.

[0077] Endotoxin or lipopolysaccharide in solution is measured by the amoeba reagent (LAL) detection method based on the activity of endotoxin. On the other hand, purified endotoxin has a specific adsorption peak at 258 nanometers, which is utilized for our in vitro binding detection to perform adsorption and chelation analysis. As shown in Figure 3A, the in vitro adsorption / neutralization ability of SQ1-CT and the active drug ingredient powder suspension was confirmed. Specifically, LPS (B4, Sigma-Aldrich) is dissolved in pure water, and the specific adsorption peak at 258 nanometers is scanned and recorded. SQ1-CT and the active drug ingredient powder are dissolved in a buffer solution at pH 6.5 (120 mM sodium chloride, 50 mM Tris, pH 6.5). The high molecular weight resin is washed twice with the same buffer solution and then collected by centrifugation. Subsequently, a certain amount of LPS is mixed with a certain volume of resin or the control buffer solution. After incubating the mixture for 1 hour, the system is centrifuged, and the supernatant is collected for spectrophotometric analysis. The results show that the adsorption / binding ability of the SQ-1 formulation to LPS at pH 6.8 is very similar to that of the active drug ingredient copolymer. In particular, according to this detection, 1 gram of SQ1 or carboxyalkylamine can adsorb or chelate approximately 0.1 gram of endotoxin in vitro. In the same way, bacterial genomic DNA is isolated and purified according to the standard process, and the chelating ability is measured as shown in Figure 3B.

Example

[0078] In vitro disintegration experiments of two types of pH-sensitive carboxyalkylamine formulations.

[0079] According to the embodiments of the present invention, two types of carboxyalkylamines are produced using a Eudragit system, designated as SQ6.5 and SQ7.5 respectively, and are designed to disintegrate at pH 6.5 and pH 7.5. The mini-tablets contain 4.8 - 5.6 milligrams of the active pharmaceutical ingredient in a total weight of 10 milligrams (Figure 4). Figure 4A shows the prototypes of two types of pH-sensitive ileal-release pills, where SQ6.5 represents the carboxyalkylamine that disintegrates at pH 6.5, and SQ7.5 represents the carboxyalkylamine that is released at pH 7.5. Figure 4B shows the in vitro disintegration time of two types of pH-sensitive ileal microspheres and the situation of their pH values. SQ6.5 indicates the carboxyalkylamine that disintegrates at pH 6.5, and SQ7.5 indicates the carboxyalkylamine that is released at pH 7.5.

[0080] First, in vitro experiments were conducted to measure the disintegration pH values of the mini-tablets in buffer solution while shaking at 37°C. The results showed that the tablets could withstand up to 4 hours in 0.1N hydrochloric acid and were stable. Then, the tablets were transferred to buffer solutions of pH 6.5 and 7.5. This buffer solution contains 120 mM of sodium chloride and 50 mM of tris (tris(hydroxymethyl)aminomethane). Record the dissolution time. As shown in the figure, barium sulfate equivalent to the SQ6.5 tablets dissolved within 2 hours in the pH 6.5 buffer solution, and the SQ7.5 tablets and barium sulfate were stable at pH 6.5 but completely dissolved within 4 hours in the pH 7.5 buffer solution (Figure 5). Figure 5 records the in vitro disintegration situation of the formulation of the microsphere agent. This microsphere was placed in a buffer solution and shaken at 37°C for 6 hours. The buffer solutions were respectively: (a) 0.1 mol / liter hydrochloric acid, (b) a pH 6.5 buffer solution containing 50 mmol / liter of tris (Tris) and 120 mmol / liter of sodium chloride, and (c) a pH 7.5 buffer solution containing 50 mmol / liter of tris (Tris) and 120 mmol / liter of sodium chloride.

[0081] In vivo disintegration experiment of target-release polymer in the ileum. Adult SD rats were fasted overnight. Five tablets each of SQ6.5 and SQ7.5 containing barium sulfate were orally gavaged. Then, standard feed and drinking water were added to the cages. X-ray-based CT scans were performed on the anesthetized rats at the designated time points. As shown in Figure 6, the pH-sensitive tablets began to disintegrate at 8 to 10 hours and dissolved approximately in the distal part of the small intestine. This view was also confirmed by giving the rats mini-tablets and euthanizing and dissecting them at different time points.

Example

[0082] Experimental design for young rats that underwent three additional binge drinking treatments after 3 weeks of alcohol induction.

[0083] The experimental design is to evaluate acute alcoholic hepatitis in young rats and the effect of SQ1. According to reports, after administering ethanol (5 grams / kilogram) intragastrically to Sprague Dawley rats acutely, the endotoxin level in portal vein blood rapidly rises from 0 within 60 minutes and reaches 10 picograms / milliliter at 90 minutes. Here, this method was modified to identify the endotoxin level and treatment effect in rats with 3-week alcoholic hepatitis. As shown in Figure 7, 20 7-week-old SPF-grade SD rats were used. Among them, 5 were assigned to the normal control group, and the remaining 15 were given Lieber-DeCarli standard alcohol liquid (36% of the total calories were from alcohol) for 3 weeks to establish the model. Then, biochemical metabolic indicators were measured. The blood transaminase (ALT) level increased from 25 ± 10 units / milliliter in the normal control to 60 ± 8 units / milliliter, indicating liver damage caused by alcohol. At this point, the model rats were further divided into 3 groups, each containing 5 rats. The model group (ASH) continued to be given alcohol liquid diet for another 3 weeks, while one group was treated with SQ1 (SQ1-CV, 7 mini-tablets per day, the dose of the active drug ingredient was 25 milligrams per day, orally administered), and the third group was treated with the active drug ingredient powder (CV of carboxyalkylamine, 25 milligrams per day, orally administered). Finally, the rats in the three groups induced by alcohol were given an excessive drinking treatment at a dose of 5 grams of alcohol / kilogram (10 milliliters per kilogram of body weight). The rats were euthanized 24 hours after the last excessive drinking, and tissues of blood, liver, and pancreas were collected for analysis.

Example

[0084] Carboxyalkylamine with ileal target release can reduce endotoxemia caused by acute alcoholic hepatitis.

[0085] As shown in Figure 8, we examined the blood endotoxin levels in young rats that had received 6 weeks of alcohol induction followed by three additional episodes of binge drinking. As shown in the figure, the endotoxin levels increased from 0.3 EU / ml in control rats to 2.0 EU / ml in rats with alcoholic liver disease, a six-fold increase. In particular, administration of the SQ1-CV mini-tablets significantly reduced the blood endotoxin levels by approximately 75%, although the active drug ingredient powder (CV) also partially reduced the blood endotoxin levels. After treatment with the carboxyalkylamine with ileal target (SQ1-C) and the corresponding carboxyalkylamine suspension (CV) at pH 6.5, the plasma endotoxin levels were suppressed. The experimental design is shown in Figure 7. Plasma endotoxin levels were measured using the amoeba reagent (amoeba lysate, LAL) detection method.

Example

[0086] Carboxyalkylamines with ileal target release reduce systemic inflammation caused by acute alcoholic hepatitis.

[0087] When bacterial endotoxins enter the bloodstream, they induce the expression of a series of inflammatory factors (such as TNF-α and IL-1) through the activation of TLR-4 / CD14 cell membrane receptors and signal pathways. The characteristics of alcoholic liver disease (ALD) are steatosis and elevated pro-inflammatory cytokines including IL-1β. IL-1β, type I IL-1 receptor (IL-1R1), and IL-1 receptor antagonist (IL-1Ra) are important regulators of the IL-1 signal complex and play important roles in inflammation. IL-1 is significantly associated with the main clinical symptoms of acute alcoholic hepatitis (fever, neutrophilia, and cachexia), and interfering with the IL-1 pathway may be a promising future treatment strategy. The important roles of type I IL-1 cytokines and several inflammasomes have also been confirmed in a mouse model of non-alcoholic fatty liver disease. Carboxyalkylamines targeted to the ileum can effectively reduce blood endotoxin levels. As shown in Figure 9, by measuring the serum level of interleukin-1β, the alcohol-induced systemic inflammation and the inhibitory effects of orally administered ileum-targeted SQ1-C and carboxyalkylamine suspension (CV) were identified. The experimental design is shown in Figure 7. This cytokine is detected using enzyme-linked immunosorbent assay (ELISA). By measuring the LPS / TLR4 / CD14 inflammatory signal pathway in young rats that had three episodes of binge drinking after 6 weeks of alcohol induction, it was confirmed that orally administered ileum-targeted carboxyalkylamine could reduce the IL-1β level in the blood, thus confirming the concept of the present invention.

Example

[0088] Carboxyalkylamines released from the ileum target reduce the interleukin-6 level caused by acute alcoholic hepatitis.

[0089] Interleukin-6 (IL-6) is an important inflammatory cytokine that plays a central role in the development and progression of liver diseases. Compared with the control group, the serum IL-6 concentration in patients with alcoholic or non-alcoholic cirrhosis and toxic hepatitis is significantly increased. IL-6 causes alcoholic liver disease (ALD) by activating signal transducer and activator of transcription 3 (STAT3), and then induces the expression of various hepatoprotective genes in hepatocytes. Furthermore, clinical studies have shown that serum IL-6 levels function as prognostic factors for alcoholic liver disease. On the other hand, endotoxin can rapidly induce the expression of IL-6 in the body. Studies have shown that hepatocytes can express IL-6 by receiving endotoxin stimulation. Therefore, liver injury reduces the ability to remove IL-6 and promotes the pathogenesis mechanism of inflammation. As shown in Figure 10, SQ1-C (carboxyalkylamine that is target-released to the ileum at pH 6.5) and its corresponding active pharmaceutical ingredient (API), CV, can suppress alcohol-induced systemic inflammation, and this inhibitory effect is measured by serum IL-6 levels. The experimental design is shown in Figure 7. This cytokine is detected using enzyme-linked immunosorbent assay (ELISA). By measuring the LPS / TLR4 / CD14 inflammatory signaling pathway, it was found that orally administered ileal-targeted carboxyalkylamine can partially reduce the level of IL-6 inflammatory factors in the blood, confirming the concept of our disclosed content.

Example

[0090] Evaluation of fatty liver in young rats with alcoholic hepatitis.

[0091] To quantitatively evaluate clinical samples and animal experiment data, the International Liver Disease Association classifies the degree of fatty liver into mild, moderate, and severe. The general evaluation indicators are the percentage of fat content in liver tissue or the grade score. Specifically, it is as follows: F1 is mild fatty liver, with the fat content in liver tissue less than 30%. Fat deposition at this level is generally considered to have a very small impact on liver function. F2 is moderate fatty liver, with the fat content in liver tissue ranging from 30% to 60%, which is considered moderate fatty liver. Moderate fat deposition can cause inflammatory reactions and damage, and may affect liver function to a certain extent. F3 is severe fatty liver. When the fat content in liver tissue exceeds 60%, it is considered severe fatty liver. Severe fatty liver usually involves liver inflammation, fibrosis, and cell damage, and has a significant impact on liver function. The evaluation of fatty liver is a qualitative and quantitative process. Usually, medical imaging techniques such as ultrasound, computed tomography (CT), or magnetic resonance imaging (MRI) are used to determine the degree of fat content in liver tissue. The liver tissue of the experiment shown in Figure 7 was stained with hematoxylin and eosin, and the selected sections based on the scoring criteria are shown in Figure 11A.

Example

[0092] The ileum-targeted formulation of carboxyalkylamine can improve alcoholic steatosis.

[0093] As shown in Figure 11B, fatty liver was observed in young rats that had three episodes of excessive alcohol consumption in addition to Lieber-DeCarli alcohol diet. However, there were significant individual differences within the group. As shown in the figure, among the alcoholic liver disease model group, one subject showed F3, two showed F2, and two showed F1. After oral administration of SQ1-CV, two subjects showed F2, and three more showed F1. Importantly, it was confirmed that in the subjects who took SQ1, fatty liver was partially improved and the expression of systemic inflammation and hepatitis factors decreased. Thus, our hypothesis that SQ1-CV adsorbs endotoxin, reduces endotoxemia and systemic inflammation, and contributes to the improvement of fatty liver was further verified.

Example

[0094] Method for evaluating liver inflammation in an experimental animal model of alcoholic hepatitis.

[0095] As shown in Figure 12, based on the rat liver tissue sections obtained in our experiment and the results of hematoxylin-eosin staining, the liver inflammation index was set as follows: grade 0, less than 2 lesions in 10 fields at a magnification of 200 times; grade 1, exactly 2 lesions in 10 fields at a magnification of 200 times; grade 2, 2 to 4 lesions in 10 fields at a magnification of 200 times. Importantly, the ranking of the liver inflammation index among each group was consistent with the levels of endotoxin and systemic inflammatory factor (IL-1), indicating that endotoxemia caused by intestinal bacterial toxins plays an important role in the development of liver diseases.

Example

[0096] Carboxyalkylamine with ileal target release can suppress the mRNA expression of inflammatory factors in liver tissue in alcoholic liver disease.

[0097] The mRNA expression of two kinds of inflammatory factors in liver tissue was measured. As shown in Figure 13, the exacerbation of liver inflammation was confirmed by measuring the messenger RNA (mRNA) level of interleukin-1β (Figure A) in rats, and improvement was obtained by administering the ileal target carboxyalkylamine preparation SQ1-C at a dose of 91 milligrams / kilogram per day. Also, by measuring the mRNA level of interleukin-6 (Figure B) in rats, the exacerbation of liver inflammation was confirmed, and it was similarly improved by SQ1-C at a dose of 91 milligrams / kilogram per day.

[0098] Six weeks of alcohol feeding and three episodes of binge drinking doubled the expression of cytokine interleukin IL-1β and tripled the expression of IL-6 in liver tissue. Administration of carboxyalkylamine with ileal targeting reduced the expression of these two inflammatory factors in liver tissue. Importantly, the expression of cytokine IL-1 is directly regulated by endotoxin / TLR4 / CD14. Here, it was shown that its expression in the liver of alcoholic liver disease completely coincides with the trend of endotoxin concentration in the blood, demonstrating its causal pathological relationship again. Furthermore, it was confirmed that SQ1 treatment reduced the expression of liver inflammatory genes. This indicates that chelating intestinal endotoxin can reduce the expression of liver inflammatory genes.

Example

[0099] Experimental design of treatment using acute alcoholic hepatitis rats and endotoxin chelators SQ1-C and SQ1-T with ileal targeting.

[0100] As shown in Figure 14, 40 middle-aged rats (10 - 12 months old) were used in this experiment. Among them, 30 rats were subjected to alcohol induction for 10 days with Lieber-DeCarli diet (5% alcohol, accounting for 36% of total calories). Then, the animals were given a single episode of binge drinking at a dose of 5 grams per kilogram of body weight and 10 milliliters. The experiment was terminated within 24 hours. During the experimental period, carboxyalkylamine and carboxymethylcellulose were orally administered using mini-tablets SQ1-C and SQ1-T with ileal targeting at pH 6.5, with a dose of 91 milligrams / kilogram, and n = 10 for each group.

Example

[0101] Endotoxin chelators SQ1-C and SQ1-T with ileal targeting suppress the metabolic endotoxin levels in acute alcoholic hepatitis rats.

[0102] Long-term alcohol consumption impairs the intestinal barrier function and increases its permeability. As a result, bacterial endotoxin (lipopolysaccharide, LPS) leaks from the intestine into the bloodstream. Once in the blood, LPS becomes metabolic endotoxin. LPS binds to Toll-like receptor 4 (TLR4) on immune cells such as Kupffer cells in the liver. This activates the immune system and causes the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Evidence from a large number of clinical studies and animal experiments shows that intestinal damage associated with aging (such as intestinal leakage) further enhances the transfer of endotoxin to the liver and systemic circulation. The release of these cytokines causes liver inflammation and oxidative stress. If this process continues, it causes hepatocyte damage, steatosis (accumulation of fat), inflammation (alcoholic steatohepatitis), fibrosis (scar formation), and ultimately progresses to cirrhosis. As shown in Figure 15, based on the method mentioned in Figure 14, the circulating levels of endotoxin in middle-aged rats with acute alcoholic hepatitis were measured regardless of the presence or absence of drug treatment. Middle-aged rats were subjected to alcohol induction (5% alcohol in the liquid diet, 36% of calories from alcohol) for 10 days, followed by a single binge drinking treatment (5 grams per kilogram of body weight). This significantly increased the plasma endotoxin level, that is, the metabolic endotoxin increased. Notably, it was confirmed that the copolymers SQ1-C and SQ1-T, designed at pH 6.5 and targeted for release in the ileum, could statistically suppress the plasma level of endotoxin, as measured by the amoebocyte lysate (LAL) assay.

Example

[0103] The ileum-targeted endotoxin chelators SQ1-C and SQ1-T at pH 6.5 can suppress systemic inflammation in acute alcoholic hepatitis.

[0104] There is sufficient literature documenting that endotoxin induces direct systemic inflammation in various metabolic disorders, including alcoholic hepatitis. This manifests as an increase in interleukin-1β and tumor necrosis factor-α. In alcoholic liver disease, IL-1β is an important mediator of inflammation. It increases in response to alcohol-induced liver injury. IL-1β is induced by bacterial endotoxins that enter the bloodstream and activates the immune system. IL-1β is significantly associated with fever, neutrophilia (an increase in the number of neutrophils in the blood), and cachexia (weight loss syndrome), which are the main clinical symptoms of acute alcoholic hepatitis. Tumor necrosis factor-α is also an important inflammatory cytokine involved in the inflammatory response of alcoholic diseases. Alcohol consumption increases the production of tumor necrosis factor-α. High levels of tumor necrosis factor-α are associated with the progression of alcoholic liver disease from steatosis (accumulation of fat in the liver) to more severe forms (steatohepatitis: accumulation of fat accompanied by inflammation and damage to the liver), fibrosis (scarring of the liver), and cirrhosis (end-stage scarring of the liver). As shown in Figure 16, the response of middle-aged rats to acute alcoholic hepatitis to the method mentioned in Figure 14 is a rapid increase in the circulating levels of interleukin-1β and tumor necrosis factor-α. In contrast, the pH-sensitive and ileum-targeted carboxyalkylamine (SQ1-C) and carboxymethylcellulose (SQ1-T) were found to be able to suppress systemic inflammation in acute alcoholic hepatitis. First, this result is consistent with the reduction of plasma endotoxin levels by the intestinal chelating action of the two copolymers, as shown through delivery by a pH-sensitive and time-dependent formulation targeted at ileal release. Second, this result showing the reduction of systemic inflammation provides the basis for many treatment possibilities. The cytokine levels in rat blood were measured using enzyme-linked immunosorbent assay. As shown in Figure 14, it was confirmed that this pH-sensitive and ileum-targeted endotoxin chelator can suppress the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1β in acute alcoholic hepatitis by intestinal chelating action.

Example

[0105] The orally administered pH 6.5 ileum-targeted endotoxin chelators SQ1-C and SQ1-T can reduce the increase in plasma transaminase levels in rats with acute alcoholic hepatitis.

[0106] As shown in Figure 17, the pH-sensitive and ileum-targeted copolymers SQ1-C (with carboxyalkylamine as the active drug component) and SQ1-T (with carboxymethylcellulose as the active drug component) can suppress the increase in transaminase levels, reduce liver function, and suppress liver damage in middle-aged rats with acute alcoholic hepatitis. As shown in the figure, in middle-aged rats, a single episode of excessive alcohol treatment after alcohol induction statistically caused an increase in serum liver transaminase levels (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)). It is noted that SQ1-C and SQ1-T can improve liver damage, which is manifested as the suppression of transaminase levels.

Example

[0107] The ileum-targeted endotoxin chelators SQ1-C and SQ1-T can improve the coagulation dysfunction in acute alcoholic hepatitis.

[0108] As shown in the experimental conditions mentioned in Figures 18 and 14, the coagulation system of middle-aged rats with acute alcoholic hepatitis is damaged, and the prothrombin time (PT) and activated partial thromboplastin time (APTT) are increased. Importantly, oral administration of carboxyalkylamine (SQ1-C) and the corresponding carboxymethylcellulose (SQ1-T) released at pH 6.5 in the ileum can restore the coagulation homeostasis in rats with acute alcoholic hepatitis. In the rat model shown in Figure 14, the activated partial thromboplastin time (APTT) prolonged by alcoholic hepatitis was detected by an automated chemical analyzer and was found to be suppressed by orally administered SQ1-C and SQ1-T.

Example

[0109] The orally administered ileum-targeted endotoxin chelators SQ1-C and SQ1-T can improve pancreatic injury in rats with acute alcoholic hepatitis.

[0110] As shown in Figure 19, after 10 days of alcohol induction, a single episode of excessive drinking caused pancreatic injury, and the levels of plasma pancreatic markers α-amylase (AMY) and alkaline phosphatase (ALK) increased. Notably, by orally administering SQ1-C and SQ1-T, which use a high molecular weight copolymer as the active drug ingredient in the ileum-targeted formulation at pH 6.5, pancreatic injury was completely suppressed and the pathological markers decreased. Furthermore, the improvement of pancreatic function by chelation therapy is closely related to the reduction of metabolic endotoxin and systemic inflammation, indicating a potential causal relationship that provides a basis for a new treatment method.

Example

[0111] The orally administered ileum-targeted endotoxin chelators SQ1-C and SQ1-T can reduce hyperglycemia in alcoholic hepatitis.

[0112] As shown in Figure 20, drinking and excessive drinking significantly increased the blood glucose level in middle-aged rats, causing a hyperglycemic state. An increase in blood glucose level, i.e., hyperglycemia, is a common feature of alcoholic hepatitis. Notably, by orally administering carboxyalkylamine (SQ1-C) and carboxymethylcellulose (SQ1-T) released from the ileum at pH 6.5, hyperglycemia in the rat model shown in Figure 14 can be reduced. The levels of triglyceride and glucose in plasma are measured using an automated chemical analyzer.

Example

[0113] Gene ontology (GO) enrichment and KEGG enrichment analysis of acute alcoholic hepatitis rats and PASA treatment.

[0114] According to the experimental design shown in Fig. 14, RNA was extracted from liver tissues and RNA-seq analysis was performed. Based on the sparse data obtained from RNA-seq, gene ontology (GO) enrichment analysis was further carried out to provide insights into the biological processes regarding the differences in regulation between the model group (SD rats with acute alcoholic hepatitis) and the control group (Fig. 21). Also, the results of the KEGG enrichment analysis of "Model_vs_control_all_1" provide insights into the differences between the animal model (model) with excessive alcohol intake and the control group. The results indicate that alcohol consumption activates the following pathways: xenobiotic catabolic process, sterol and steroid biosynthetic process, and the process by which cells respond to glucocorticoid stimulation and ketones.

[0115] Fig. 21A shows the gene ontology (GO) analysis of gene expression in the liver of middle-aged rats in the state of acute alcoholic hepatitis. Gene ontology (GO) enrichment analysis helps to deepen the understanding of the biological processes differentially regulated between Sprague-Dawley rats with alcoholic hepatitis (model group) and the control group. Fig. 21B shows the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of gene expression in the liver of middle-aged rats in the state of acute alcoholic hepatitis. The results of the KEGG enrichment analysis between the alcohol model group and the control group contribute to a deeper understanding of the differences existing between the excessive alcohol-consuming rats and the control group rats in the experiment shown in Fig. 14.

Example

[0116] KEGG enrichment analysis of liver gene expression in the treatment of acute alcoholic hepatitis rats and ileum-targeted endotoxin chelators SQ1-C and SQ1-T.

[0117] KEGG enrichment analysis provides information on the biological pathways involved in chelation therapy with SQ1-C or SQ1-T in middle-aged rats with alcoholic hepatitis. The experimental conditions are shown in Figure 14. This analysis shows that excessive alcohol consumption affects multiple pathways related to diseases and biological processes (Figure 22). Chelation of endotoxin by SQ1-C and SQ1-T in the ileal target acts on these pathways, counteracts the adverse effects of alcohol, and may have the potential effect of preventing or treating various diseases. Treatments with SQ1-C and SQ1-T seem to affect pathways related to hepatocellular carcinoma, diabetic complications, p53 and Hippo, insulin resistance, cellular senescence, rheumatoid arthritis and drug metabolism, chemokine signaling, and differentiation of Th1 and Th2 cells.

[0118] Figure 22A shows the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of liver gene expression compared with treatment with the ileal target endotoxin chelator SQ1-C in middle-aged rats in the state of acute alcoholic hepatitis. Figure 22B shows another KEGG enrichment analysis of liver gene expression compared with treatment with the ileal target endotoxin chelator SQ1-T in middle-aged rats in the state of acute alcoholic hepatitis.

Example

[0119] The orally administered ileal target endotoxin chelators SQ1-C and SQ1-T can suppress the expression of TNF-α receptor and thrombospondin 1 (TSP-1) in the liver of rats with acute alcoholic hepatitis.

[0120] As shown in Figure 14, middle-aged rats were alcohol-induced for 10 days and then underwent a single binge drinking treatment. Subsequently, gene expression in liver tissue was analyzed by RNA-seq. As shown in Figure 23, alcohol treatment significantly increased the expression of TNF-α and thrombospondin 1 in the liver, consistent with systemic inflammation caused by endotoxin levels and white blood cells in the body. Here, it is shown that liver tissue has the ability to endogenously express the TNF-α receptor, indicating that the liver is a source of inflammation. TSP-1 promotes the fibrinolytic system, affects thrombus dissolution, and may increase systemic inflammation and the severity of the disease state in acute-chronic liver failure. LPS is known to induce the production of TSP-1. Notably, the two orally administered ileum-targeted polymeric copolymers SQ1-C and SQ1-T can suppress the expression of TNF-α (Figure A) and thrombospondin 1 (Figure B) in the liver.

Example

[0121] Orally administered ileum-targeted intestinal endotoxin chelators can reduce liver fibrosis in rats with alcoholic hepatitis.

[0122] The experiments and treatments are described in Figure 14. As shown in Figure 24, rats with acute alcoholic hepatitis showed liver fibrosis and pathological progression, with increased mRNA levels of lysyl oxidase 2 (Lox2, Figure A) and transforming growth factor β-2 (Tgfb2, Figure B) in their livers. Strong evidence from clinical relevance and genetic experiments indicates that the endotoxin signal plays an important role in liver fibrosis and the activation of hepatic stellate cells through the Lox2 and TGF-beta signals.

Claims

1. A pharmaceutical for treating metabolic disorders caused by enteric pathogen-associated molecular patterns, characterized in that the pharmaceutical comprises a specific polymer copolymer, which has a three-dimensional framework composed of organic carbon-carbon chemical bonds and contains an amino group.

2. A pharmaceutical for treating metabolic disorders caused by enteric pathogen-associated molecular patterns as described in claim 1, characterized in that the pharmaceutical is in the form of microballs, tablets or capsules.

3. The pharmaceutical for treating metabolic disorders caused by enteric pathogen-associated molecular patterns according to claim 2, characterized in that the microballs or tablets are composed of a core granule, an isolating layer and a pH-sensitive coating, and the core granule contains a polymeric copolymer as an active drug component.

4. The pharmaceutical for treating metabolic disorders caused by enteric pathogen-associated molecular patterns as described in claim 3 is characterized in that the polymer copolymer contains any one or any combination of carboxymethylcellulose, carboxyalkylamine, carboxybenzamine and carboximide.

5. The pharmaceutical for treating metabolic disorders caused by enteric pathogen-associated molecular patterns as described in claim 3, characterized in that the pH-sensitive coating comprises methyl acrylate and methacrylic acid.

6. A pharmaceutical for treating metabolic disorders caused by enteric pathogen-associated molecular patterns as described in claim 3, characterized in that the pH-sensitive coating ensures that the pharmaceutical is released within a pH value range of 6.5 to 7.

5.

7. The pharmaceutical for treating metabolic disorders caused by enteric pathogen-associated molecular patterns as described in claim 3 is characterized in that the isolation layer comprises a methacrylic acid-ethyl acrylate copolymer or a methacrylic acid-methyl acrylate copolymer.

8. A method for producing a medicine for treating metabolic disorders caused by enteric pathogen-associated molecular patterns, the method comprising the steps of: (1) Preparation of core granules: Carboxymethylcellulose or carboxyalkylamine, microcrystalline cellulose, cross-linked sodium carboxymethylcellulose and hydroxypropylmethylcellulose are weighed and mixed to obtain premixed powder, then magnesium stearate is added and mixed to obtain the whole mixed powder, which is then compressed into tablets. (2) Coating with an isolation layer: Pour absolute ethanol into a stainless steel bucket, start the stirrer, add methacrylic acid-methyl acrylate copolymer and continue stirring until it is completely dissolved, then add triethyl citrate while stirring. After adding, stir for another hour to produce the isolation layer coating solution. Start the high-efficiency coater and preheat the equipment by idle running. When the exhaust temperature reaches 30°C or above, place the core granules produced in step 1 into the high-efficiency coater and coat them, and adjust the increase range of the isolation layer to 2-3% to obtain isolation coated granules. (3) pH-sensitive coating: A. Put pure water into a stainless steel bucket, add polyethylene glycol 6000 partially to the pure water, and continue stirring until dissolved to obtain an aqueous solution of polyethylene glycol 6000. B. Take another portion of the pure water in a stainless steel bucket, add the talcum powder to the pure water, and stir for 30 minutes after adding to obtain a suspension of the talcum powder. C. Add an aqueous solution of polyethylene glycol 6000 to the cooled talcum suspension and stir for 30 minutes, then add triethyl citrate and stir for 30 minutes to obtain Suspension 3. D. The aqueous dispersion of methacrylic acid-ethyl acrylate copolymer is weighed and placed in a stainless steel bucket, and purified water is added and stirred for 30 minutes to obtain an aqueous dispersion suspension of methacrylic acid-ethyl acrylate copolymer. E. Add suspension 3 to the aqueous dispersion suspension of methacrylic acid-ethyl acrylate copolymer and stir continuously for 60 minutes to obtain an enteric coating solution with a concentration of 20.0% (w / w). F. Enteric layer coating: Start the high-efficiency coater, preheat the equipment by idle running, place the isolation coating granules into the high-efficiency coater, adjust the atomization and fan pressure to coat, and make the enteric layer gain 8.0-10.0% to obtain the said medicine.

9. Application of drugs to treat metabolic disorders caused by enteric pathogen-associated molecular patterns in metabolic disease therapeutics.

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