Targeted ileal delivery of endotoxin sequestering agent drugs and methods of making and using the same

Abstract

The present application relates to a targeted ileum delivery endotoxin sequestering agent drug and its preparation method and application, belong to the technical field of pharmaceutical preparation, the drug includes specific high molecular copolymer, the high molecular copolymer includes the three-dimensional framework of organic carbon-carbon chemical bond, and includes amino group. It is aimed at transporting high molecular chelating agent to the distal small intestine and releasing, and chelating pathogen-associated molecular patterns including endotoxin and CpG-DNA produced by intestinal microorganisms. The complex formed by the high molecular chelating agent released in the ileum and the pathogenic molecular pattern can be discharged from the digestive tract, so as to reduce the body inflammation and be used for relieving various metabolic diseases.

Description

Technical Field

[0001] This invention belongs to the technical field of pharmaceutical formulations, and relates to a targeted ileal delivery endotoxin chelating agent drug, its preparation method, and its application in the treatment of metabolic diseases. It can deliver a specific polymer chelating agent to the end of the small intestine for release, so as to chelate and eliminate pathogenic molecular patterns produced by intestinal microorganisms, including bacterial endotoxins and CpG-DNA, and excrete them from the body, for the purpose of alleviating and treating a variety of metabolic diseases. Background Technology

[0002] Metabolic disorders, manifesting as obesity, fatty liver disease, and type 2 diabetes, pose significant health challenges and a heavy economic burden to 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). Studies show that metabolic diseases affect a large segment of the adult population. Based on large-scale surveys, prevalence ranges from approximately 10% to over 30%, depending on the region and population studied. In the United States, metabolic syndrome is a major public health issue (Trends in Metabolic Syndrome Among US Youth, From 1999 to 2018, JAMA 2022; 176:043-1045). This high prevalence is at least partly attributed to factors such as diets rich in processed foods, high-fat, high-sugar intake, and a relatively sedentary lifestyle. Furthermore, the high obesity rate in the United States has greatly contributed to the development of metabolic syndrome and diabetes.

[0003] Alcohol consumption is associated with a wide range of diseases and health problems (Global, regional, and national comparative risk assessment of 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 leads to fat accumulation in the liver. Heavy drinkers may develop alcoholic hepatitis (AH), characterized by jaundice, abdominal pain, and fatigue. Persistent alcoholic hepatitis can progress to liver inflammation and cirrhosis, potentially leading to liver failure, cancer, and other complications such as bleeding from enlarged blood vessels in the esophagus. Alcohol consumption can also cause cardiovascular disease (CVD), arrhythmias (irregular heartbeat), myocardial damage, esophagitis and gastritis, peptic ulcers, pancreatitis, and even cancer.

[0004] Blood tests for alcoholic liver disease include elevated levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), total bilirubin (TBil), prothrombin time (PT), mean corpuscular volume (MCV), and glucose-deficient transferrin (CDT).

[0005] Under normal physiological conditions, a large number (1-2 kg) of gut microbiota are mostly confined to the large intestine, while the upper small intestine is almost sterile. This gating mechanism is partly mediated by α-defensins 5 / 6 secreted by Paneth cells located in the microvilli crypts (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 and decreased α-defensin expression are often associated with many metabolic disorders. In addition, intestinal damage is often associated with aging and leaky gut due to loss of tight junctions in intestinal epithelial cells, which is common in patients with metabolic disorders (The ageing gastrointestinal tract, Curr Opin Clin Nutr Metab Care 2016; 19:12-8).

[0006] Previous studies have shown that vitamin D signaling controls Paneth cell function to some extent by promoting the expression of α-defensins. Vitamin D deficiency, which is usually associated with metabolic disorders, leads to downregulation of defensin expression and small intestinal bacterial overgrowth (SIGO), which may in turn promote endotoxemia, resulting in 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).

[0007] Once pathogen-associated molecular patterns (PAMPs) originating from gut microbiota enter the human body, they can promote metabolic disorders through a variety of mechanisms. For example, PAMPs can activate innate immunity through pattern recognition receptors on immune cells (such as Toll-like receptors) (PAMPs and DAMPs: signal 0s that spur autophagy and immunity. Immunol Rev2012; 249:158-75). The interaction between ligands and receptors triggers a series of signaling events that may lead to the release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), thereby initiating an inflammatory response.

[0008] From a biochemical perspective, endotoxins are lipopolysaccharides (LPS), derived from the cell walls of Gram-negative bacteria. LPS shed from the cell walls of dead bacteria and organisms leads to elevated circulating endotoxin levels, a condition known as metabolic endotoxin (Gut-derived low-grade endotoxaemia, atherothrombosis and cardiovascular disease, Nat Rev Cardiol 2023; 20:24-37). Peripheral circulating LPS levels typically rise after eating. LPS is incorporated into chylomicrons, crosses the intestinal barrier into the lymphatic system, and then enters the bloodstream. In the blood, endotoxins are transported by LPS-binding proteins called LBPs, as well as others such as high-density lipoprotein cholesterol (Chylomicrons promote intestinal absorption of lipopolysaccharides, J Lipid Res 2009; 50:90-7).

[0009] In healthy subjects, endotoxins bound to high-density lipoprotein cholesterol (HDL-C) or LBP are transported to the liver, where they are degraded by specific hepatic enzymes such as acylhydroxylase and alkaline phosphatase, or excreted into the bile via scavenger receptors. Kupffer cells in the liver detoxify endotoxins through phagocytosis. However, with age or various forms of liver injury, hepatocytes become unable to clear circulating endotoxins, leading to their spillover into the systemic circulation and causing mild endotoxemia. This further contributes to insulin resistance in type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). The pro-inflammatory signaling exerted by LPS is generated through the binding of its lipid fraction, lipid A, to TLR4 and its membrane-bound co-receptor CD14. LPS binding to TLR4 leads to the recruitment of the adaptor protein MyD88 to the cytoplasmic domain of TLR4, thereby activating transcription factors such as NF-κB, triggering a transcriptional cascade and inflammatory response.

[0010] Endotoxins in the small intestine can also be absorbed into the liver via the portal vein, especially in cases of aging and intestinal damage caused by various factors.

[0011] Numerous clinical studies have found a strong association between endotoxemia and various diseases, particularly liver disease. Chronic alcohol consumption, aging, viral infections, and vitamin D deficiency can impair intestinal integrity, leading to leaky gut. In these cases, bacteria from the large intestine can migrate into the small intestine, a condition known as small intestinal bacterial overgrowth (SIBO), resulting in the accumulation of toxins and other pathogen-associated molecular patterns in the distal small intestine.

[0012] Numerous studies have shown that plasma endotoxin levels in patients with alcoholic liver disease are several times higher than in healthy subjects (Endotoxemia in patients with alcoholic and non-alcoholic cirrhosis and insubjects with no evidence of chronic liver disease following acute alcohol excess. J Hepatol 1987; 4:8-14). Plasma endotoxin levels in normal subjects range from 0.3 to 10.4 picograms per milliliter, while those in patients with alcoholic liver disease range from 8.5 to 206 picograms per 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 levels vary considerably among individuals, plasma endotoxin levels in patients with alcoholic liver disease are consistently 5-20 times higher than in normal subjects. Animal models of alcoholic liver injury have confirmed endotoxemia in alcoholic liver disease. Based on a male rat model and through binge drinking, one report showed that acute or chronic ethanol administration increases plasma endotoxin levels, and these levels are closely associated with the development of liver injury (Exacerbation of alcoholic liver injury by enteral endotoxin in rats. Hepatology 2000; 32:1008-17). Another study showed that ethanol-fed rats exhibited increased endotoxemia and lipid peroxidation stimulating NF-κB activation and chemokine production, enhancing liver injury (Increased severity of alcoholic liver injury in female rats: role of oxidative stress, endotoxin, and chemokines. Am JPhysiol Gastrointest Liver Physiol 2001; 281:G1348-56). The relief of alcohol-induced endotoxemia and liver injury by antibiotics suggests that endotoxins play a crucial causal role in alcoholic liver injury.In fact, oral administration of large doses of polymyxin B to sterilize the intestinal microbiota and reduce endotoxins can improve alcoholic hepatitis in Wistar rats (Antibiotics prevent liver injury in rats following long-term exposure to ethanol. Gastroenterology 1995; 108:218-24). Emerging evidence suggests that LPS and ethanol have a synergistic effect on hepatocytes. LPS alone cannot mimic ethanol-induced steatosis or hepatitis, but ethanol and LPS together can effectively induce liver injury. Ethanol feeding sensitizes the livers of experimental animals to LPS-induced cell damage (Chronic ethanolfeeding 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)(Ethanolfeeding enhances inflammatory cytokine expression in lipopolysaccharide-induced hepatitis. J Gastroenterol Hepatol 1997; 12:305-13).

[0013] In the circulatory system, lipopolysaccharide-binding protein (LBP) presents bacterial toxins to cellular receptors CD14 and TLR-4. Therefore, prolonged ethanol administration enhances the expression of LBP and CD14. Conversely, LBP, CD14, and TLR-4 knockout mice exhibit resistance to ethanol stimulation, suggesting a causal role of endotoxins in promoting liver injury (Role of lipopolysaccharide-binding protein in early alcohol-induced liver injury in mice. J Immunol 2002. 168; 6:2963-9).

[0014] Various methods for reducing endotoxin levels in the blood have proven effective in treating severe liver disease. For this purpose, polymyxin B-immobilized fiber columns (PMX) in extracorporeal circulation devices have been clinically used to remove endotoxins from patients with liver failure and septic shock (Efficacy of polymyxin B-immobilized fiber column direct hemooperfusion for non-endotoxin-associated severe septic shock. Pediatr Int 2016; 58:1346-1347). Extracorporeal circulation or plasma exchange has been used to reduce hepatotoxins and endotoxins to treat various diseases such as liver failure, multiple organ failure, and sepsis. However, high cost and surgical risks are significant obstacles for extracorporeal circulation methods, including PMX. Importantly, these treatments cannot prevent the influx of intestinal-derived endotoxins into the body. Therefore, the development of oral medications capable of chelating molecular patterns associated with intestinal pathogens, including endotoxins, has profound potential value for the prevention and treatment of various metabolic disorders, including liver diseases. Summary of the Invention

[0015] Bile acid chelators with various structures, including cholestyramine, cholesvelam, and colestipol, are biocompatible resins. They are not absorbed by the human body and are completely excreted from the digestive tract after binding with bile acids. These types of high molecular weight copolymers have been used clinically to lower hypercholesterolemia by chelating bile acids, in which cholesterol is converted into bile acids in a compensatory manner.

[0016] This invention demonstrates that specific copolymers can be used as active pharmaceutical ingredients to chelate intestinal-derived endotoxins and other pathogen-associated molecular patterns (PAMPs). Furthermore, this invention further demonstrates a targeted delivery system capable of delivering the aforementioned chelating agent to the distal region of the small intestine to achieve efficient chelation and therapeutic potential. This invention not only improves the chelation efficiency of endotoxins and other PAMPs in the ileum but also significantly reduces the dosage and decreases the adsorption of hydrophobic nutrients, thus improving therapeutic safety. These two factors therefore form the basis of a novel therapeutic approach called PASA (ppathogen adsorption and sequestration agents) therapy. In this current application, we propose a method for preparing a targeted ileal delivery endotoxin chelating agent and its related therapeutic applications.

[0017] This invention relates to a method for preparing a drug and its potential therapeutic applications. Specifically, it involves using a specific copolymer as the active pharmaceutical ingredient and formulating the drug for targeted delivery to the ileum, thereby effectively chelating endotoxins and other pathogen-associated molecular patterns (PAMPs) for therapeutic applications. This innovative method offers significant advantages in the pharmaceutical field, enabling precise and effective drug delivery to chelate abundant endotoxins in the distal small intestine. The claimed method has great potential for treating a variety of conditions and represents a novel and valuable contribution to drug preparation and therapeutic applications.

[0018] On one hand, the present invention provides a chelating agent composition for treating subjects suffering from metabolic disorders caused or promoted by metabolic endotoxins or other PAMPs derived from the gut. The pharmaceutical composition comprises a variety of biocompatible particles and / or polymers as active pharmaceutical ingredients (APIs) that cannot be digested and absorbed by the subject's digestive tract. The chelating agent composition binds to at least endotoxins or lipopolysaccharide (LPS) and other PAMPs (e.g., CpG-DNA derived from gut microbiota) in the form of a chelating agent-PAMP complex, which may include covalent or non-covalent bonds. Due to the formation of the chelating agent-PAMP complex, intestinal PAMPs, including endotoxins, are cleared from the digestive tract along with the chelating agent complex.

[0019] Tablets or capsules used to chelate endotoxins in other PAMPs consist of a core particle containing a specific copolymer as an API, an isolation layer, and a pH-sensitive coating. The insoluble and indigestible copolymer in the formulation can be targeted to the distal region of the small intestine to chelate certain pathogen-associated molecular patterns, such as endotoxins.

[0020] On the other hand, this invention discloses a method for treating subjects with metabolic disorders closely associated with persistent inflammation and resulting insulin resistance, partly driven by metabolic endotoxins. Metabolic disorders are a prominent cause and basis for complications and liver diseases such as alcoholic hepatitis, metabolic-associated steatohepatitis (MASH), liver fibrosis and cirrhosis, liver failure, and even liver cancer. Furthermore, metabolic disorders caused by intestinal endotoxins and other PAMPs can also contribute to complications such as cardiovascular disease (CVD), type 2 diabetes (T2D), and central obesity. Therefore, the principles of PASA therapy and the pH-sensitive, ileal-targeted delivery of endotoxin chelators including specific copolymers can be used to treat these metabolic diseases.

[0021] The following are the innovative points related to the disclosure of this invention:

[0022] Existing treatments for metabolic diseases (such as non-alcoholic steatohepatitis / metabolic steatohepatitis, and alcoholic hepatitis) and efforts to develop new drugs largely target specific cellular targets, including key enzymes and receptors that control metabolic pathways. Metabolic diseases, including various alcoholic liver diseases and non-alcoholic liver diseases, involve multiple genetic and epigenetic factors. Therefore, single-target drugs are unlikely to achieve broad efficacy. For example, obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist, is a novel drug under development for the treatment of NASH. FXR is a nuclear receptor that plays a crucial role in bile acid, lipid, and glucose metabolism. By activating FXR, OCA should improve liver function and reduce inflammation and fibrosis in NASH patients. Although OCA has shown some benefits in NASH patients, the extent of improvement may not be sufficient to meet regulatory approval requirements. Elafibranor, a dual peroxisome proliferator-activated receptor (PPAR) α / δ agonist designed to improve insulin sensitivity, lipid metabolism, and reduce inflammation, failed in clinical trials. Different patients may respond differently due to genetic and environmental factors, making it difficult to achieve consistent results across different patient populations. Hundreds of genes or single nucleotide polymorphisms (SNPs) are involved in the pathogenesis of NASH. For example, genes involved in lipid metabolism, such as PNPLA3 (containing a patatin-like phospholipase domain), TM6SF2 (a member of the transmembrane 6 superfamily), and MBOAT7 (containing a membrane-bound O-acyltransferase domain), are closely associated with an increased risk of NASH. These genes play roles in processes such as triglyceride metabolism and lipid droplet formation. Furthermore, genes associated with inflammation and immune responses, such as those 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 can influence inflammation in the liver. 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 can also lead to increased oxidative stress and contribute to liver damage. Small molecule drugs targeting specific pathways have been less effective in addressing the broad diversity of NASH pathogenesis. The same is true for other metabolic diseases. Therefore, we need to creatively think about and address the upstream sources or causes of liver diseases. In this regard, this article proposes a novel treatment approach called PASA (Pathogen Adsorption and Chelating Agent) therapy. This approach shows promise in treating alcoholic hepatitis and related metabolic disorders. By targeting the release of a copolymer-based chelating agent in the distal small intestine, the drug can effectively adsorb and chelate microbial-derived pathogens, such as endotoxins, thereby blocking the upstream and intestinal sources of inflammation and providing a potential solution to address the root causes of metabolic diseases.

[0023] Gut-derived pathogenic molecular patterns are a common source of inflammation in many metabolic diseases. Endotoxins and other pathogen-associated molecular patterns (PAMPs) originating from bacteria and transferred from the colon to the small intestine can enter the circulatory system and the liver via two main pathways: adsorption by the lymphatic system or via the portal vein. High levels of these gut-derived metabolic endotoxins are a major source of systemic and tissue inflammation. For example, metabolic endotoxins can promote insulin resistance by inactivating insulin-mediated signaling pathways. This is the basis of type 2 diabetes and other related metabolic disorders.

[0024] Our research has found that bile acid sequestrants such as cholestyramine and cholesvelam have the ability to effectively bind and chelate endotoxins and bacterial CpG-DNA. Furthermore, we found that oral administration of these sequestrants can reduce metabolic endotoxin levels in other pathological conditions such as metabolic syndrome, liver fibrosis, and pancreatic cancer. However, the high molecular weight sequestrants in these original formulations are designed for release in the upper gastrointestinal tract, resulting in lower efficiency in capturing endotoxins that are primarily enriched in the distal small intestine. In addition, the previous formulations of these sequestrants, being dispersed in the upper small intestine, may also chelate hydrophobic nutrients such as vitamin D and vitamin K, leading to some adverse effects. To address these issues, this invention proposes a novel method for the preparation, composition, and formulation of pharmaceutical preparations for novel medical applications and indications targeting new pathogens.

[0025] In the first part of this invention, we describe a method for preparing a pH-sensitive and time-dependent formulation of a chelating agent that targets and releases in the distal small intestine. This method overcomes the limitations of existing drugs, improves the chelation efficiency for eliminating endotoxins in the distal small intestine, and minimizes unnecessary chelation of hydrophobic nutrients.

[0026] In the second part of this invention, we demonstrate for the first time that ileal-targeted release of chelating agents can serve as a treatment for related metabolic disorders, such as alcoholic hepatitis. Notably, we have also found that ileal-targeted cholesvelam or cholestyramine drugs can suppress systemic and hepatic inflammation, prominent factors in many diseases. These findings therefore form the basis for expanding indications to alleviate other liver diseases.

[0027] In terms of molecular mechanisms, we found that ileal-targeted endotoxin chelators can modulate many pathological pathways, including (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, and (10) Th1 and Th2 cell differentiation. This global regulation of liver pathways also lays the foundation for the widespread application of PASA therapy as a novel treatment.

[0028] The purpose of this invention is to provide a drug, its preparation method, and its application for treating metabolic disorders caused by intestinal pathogen-associated molecular patterns. The specific contents of the invention are as follows:

[0029] The drug comprises a specific polymer copolymer containing a three-dimensional framework of organic carbon-carbon chemical bonds and containing amino groups.

[0030] The drug is available in the form of pellets, tablets, or capsules.

[0031] The microcapsules or tablets consist of a core particle, an isolation layer, and a pH-sensitive coating; the core particle contains a polymer copolymer as the active pharmaceutical ingredient.

[0032] The polymer copolymers include one or any combination of cholesvelam, cholestyramine, colestipol, and cholesamide.

[0033] The pH-sensitive coating comprises methyl methacrylate and methacrylic acid.

[0034] The pH-sensitive coating ensures that the drug is released within a pH range of 6.5 to 7.5.

[0035] A method for preparing a drug for treating metabolic disorders caused by intestinal pathogen-associated molecular patterns includes the following steps:

[0036] (1) Preparation of core particles:

[0037] Weigh out cholesvelam or cholestyramine, microcrystalline cellulose, croscarmellose sodium and hydroxypropyl methylcellulose and mix for 15 minutes to obtain a premixed powder. Then, add magnesium stearate and mix for 5 minutes to obtain a total mixed powder, and compress into tablets.

[0038] (2) Coating with an isolation layer:

[0039] Pour anhydrous ethanol into a stainless steel container, turn on the stirrer and add methacrylic acid-methyl methacrylate copolymer. Continue stirring until completely dissolved. Then, while stirring, continue to add triethyl citrate. After the addition is complete, continue stirring for 1 hour to prepare the isolation layer coating solution.

[0040] Turn on the high-efficiency coating machine, preheat the machine without air, and when the exhaust temperature reaches above 30°C, take the core particles prepared in step 1) and place them in the high-efficiency coating machine for coating, so that the weight gain of the isolation layer is between 2 and 3%, and obtain the isolation coated sheet.

[0041] (3) pH-sensitive coating:

[0042] A puts purified water into a stainless steel bucket, adds polyethylene glycol 6000 to part of the purified water and stirs continuously until dissolved to obtain an aqueous solution of polyethylene glycol 6000 for later use;

[0043] B takes another portion of purified water from a stainless steel bucket, adds talcum powder to the purified water, and stirs for 30 minutes to obtain a talcum powder suspension.

[0044] C. Add the aqueous solution of polyethylene glycol 6000 to the cooled talc suspension and stir for 30 minutes. Then add triethyl citrate and stir for another 30 minutes to obtain suspension 3 for later use.

[0045] D. Weigh out the aqueous dispersion of methacrylic acid-ethyl acrylate copolymer and put it into a stainless steel bucket. Add purified water and stir for 30 minutes to obtain a suspension of the aqueous dispersion of methacrylic acid-ethyl acrylate copolymer.

[0046] E. Add suspension 3 to the aqueous dispersion suspension of methacrylate-ethyl acrylate copolymer and stir continuously for 60 min to obtain an enteric-coated solution with a concentration of 20.0% (w / w) d.

[0047] F. Coat the enteric layer, turn on the high-efficiency coating machine, preheat the machine empty, add the isolation coating tablet into the high-efficiency coating machine, adjust the atomization and fan-shaped pressure, coat, so that the weight gain of the enteric layer is 8.0-10.0%, and obtain the drug.

[0048] Application of a drug for treating metabolic disorders caused by intestinal pathogen-associated molecular patterns in the treatment of metabolic diseases.

[0049] The present invention also provides a method for preparing a drug for treating metabolic disorders caused by intestinal pathogen-associated molecular patterns (PAMPs), the method comprising administering to a subject a specially formulated drug designed to deliver a specific polymer chelating agent to a distal region of the small intestine to chelate PAMPs of intestinal microorganisms, including endotoxins and CpG-DNA, wherein the complex of the chelating agent and PAMPs bound is excreted from the digestive tract.

[0050] The specific polymer chelating agent is targeted for release from the pH-sensitive coating in the ileum to effectively neutralize pathogenic molecular patterns at the end of the small intestine, for the treatment of the corresponding disease.

[0051] Metabolic diseases include alcoholic hepatitis, metabolic-associated steatohepatitis (MASH), liver fibrosis and cirrhosis, liver failure, liver cancer, cardiovascular disease (CVD), type 2 diabetes (T2D), and central obesity.

[0052] The beneficial effects of this invention are:

[0053] This invention provides a drug that effectively adsorbs and chelates microbial-derived pathogens, such as endotoxins, thereby blocking upstream and intestinal sources of inflammation and offering a potential solution to address the root causes of metabolic diseases. This invention overcomes the limitations of existing drugs, improves the chelation efficiency for eliminating endotoxins in the distal small intestine, and minimizes unnecessary chelation of hydrophobic nutrients. We demonstrate for the first time that ileal-targeted release of chelating agents can serve as a treatment for related metabolic disorders, such as alcoholic hepatitis. We also found that ileal-targeted cholesvelam or cholestyramine drugs can suppress systemic and hepatic inflammation.

[0054] This invention delivers and releases a specific polymer chelating agent to the distal small intestine, thereby chelating pathogen-associated molecular patterns produced by intestinal microorganisms, including endotoxins and CpG-DNA. The complex formed by the specific polymer chelating agent released in the ileum and the pathogen molecular patterns can be excreted from the digestive tract, thereby reducing inflammation and alleviating various metabolic diseases. Attached Figure Description

[0055] Figure 1 A diagram of an enteric-coated polyamine drug SQ1 prepared in an embodiment of the present invention.

[0056] Figure 2 Computed tomography-based X-ray imaging was used to determine the disintegration time and location map of endotoxin chelators targeted for ileal release.

[0057] Figure 3 A graph illustrating the ability of ileal-released cholestyramine formulations to chelate lipopolysaccharide (LPS) and CpG-DNA fragments in vitro.

[0058] Figure 4 Schematic diagram of two pH-sensitive ileal-released pill prototypes and their in vitro disintegration.

[0059] Figure 5 A chart recording the in vitro disintegration of the micro-pellet formulation.

[0060] Figure 6 The in vivo disintegration of barium sulfate-containing pH-released microspheres is illustrated by X-ray imaging of Sprague-Dawley rats at specific time points.

[0061] Figure 7 The therapeutic effects of cholesvelam released at pH 6.5 and the active pharmaceutical ingredient (API) powder suspension on acute alcoholic hepatitis in young rats are illustrated.

[0062] Figure 8 The effect of ileal release of colesvelam in reducing alcohol-induced endotoxemia is illustrated in the diagram.

[0063] Figure 9 The diagram shows the effect of ileal release of colesvelam in reducing alcohol-induced systemic inflammation.

[0064] Figure 10 This is a diagram showing the effect of ileal release of colesvelam in reducing alcohol-induced systemic inflammation.

[0065] Figure 11 This is an image showing the effect of ileal release of colesvelam in reducing alcohol-induced fatty liver.

[0066] Figure 12 This is a diagram showing the effect of ileal release of colesvelam in reducing alcohol-induced inflammation.

[0067] Figure 13 This is a diagram showing the effect of ileal release of colesvelam in reducing alcohol-induced liver tissue inflammation.

[0068] Figure 14 This is a graph showing the therapeutic effects of cholesvelam and cholestyramine, both pH-sensitive and targeting the ileum, on acute alcoholic hepatitis in middle-aged rats.

[0069] Figure 15 This is a diagram showing the effect of ileal release of colesvelam in reducing acute alcohol-induced endotoxemia.

[0070] Figure 16 This is a diagram showing the effect of ileal release of colesvelam in reducing systemic inflammation induced by acute alcoholic hepatitis.

[0071] Figure 17 This is a diagram showing the effect of ileal release of colesvelam in reducing the elevation of transaminase levels induced by acute alcoholic hepatitis.

[0072] Figure 18 This is a diagram showing the effect of ileal release of colesvelam in reducing coagulation disorders induced by acute alcoholic hepatitis.

[0073] Figure 19 This is a diagram showing the effect of ileal release of colesvelam in reducing pancreatic damage induced by acute alcoholic hepatitis.

[0074] Figure 20 The image shows the effect of ileal release of colesvelam in reducing hyperlipidemia induced by acute alcoholic hepatitis.

[0075] Figure 21 This is a graph showing the analysis of liver gene expression in middle-aged rats under acute alcoholic hepatitis conditions compared to the control group.

[0076] Figure 22 This is a Kyoto Gene and Genome Encyclopedia (KEGG) enrichment analysis of liver gene expression in middle-aged rats under acute alcoholic hepatitis conditions compared to rats treated with endotoxin chelators targeting the ileum.

[0077] Figure 23 This is a diagram showing the effect of ileal release of colesvelam in reducing the expression of liver inflammation genes induced by acute alcoholic hepatitis.

[0078] Figure 24 This is a diagram showing the formation and pathological progression of liver fibrosis. Detailed Implementation

[0079] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0080] Example 1

[0081] Preparation and formulation of pH-sensitive targeted ileal release formulations.

[0082] In this invention, specific polymers selected, such as cholestyramine, cholesvelam, and colestipol, are used as active pharmaceutical ingredients (APIs) to prepare pH-sensitive formulations or drugs that target the ileum.

[0083] A key approach 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 family, and are polymethacrylate copolymers used in drug delivery systems. These two polymers have different properties and applications in drug delivery. Eudragit S100 is an anionic polymer composed of methyl methacrylate (MMA) and methacrylic acid (MAA) in a molar ratio of approximately 1:2, with a molecular weight of approximately 135 kDa. Eudragit S100 dissolves in environments with a pH greater than 7, making it suitable for colon-targeted drug delivery systems, as the pH in the colon is typically higher than 7. Eudragit L100 is also an anionic polymer, but with a MAA to MMA molar ratio of approximately 1:1, and its molecular weight is also approximately 135 kDa. Eudragit L100 is typically a copolymer of methacrylic acid and methyl methacrylate. Methacrylic acid has the chemical formula C4H6O2. It consists of carboxylic acid groups (-COOH) attached to a carbon chain with a double bond. Methyl methacrylate has the chemical formula C5H8O2. It is the methyl ester of methacrylic acid. Utec L100 dissolves in environments with a pH between 6 and 7.

[0084] Example 2

[0085] The formulations for preparing targeted cholesvelam or cholestyramine at pH 6.5 and pH 7.5 for ileal release using the Utec system are shown in Tables 1 and 2.

[0086] Table 1 Core Particle Composition

[0087] Element gram API, polymer 70.0 microcrystalline cellulose 25 Sodium carboxymethyl cellulose 4.5 magnesium stearate 0.5 common 100

[0088] Table 2 pH-sensitive ileal release

[0089]

[0090] This protocol uses the Eutectic system to prepare cholesvelam or cholestyramine formulations that are targeted for release in the ileum at a pH of approximately 6.5–7.5, as detailed below:

[0091] 1. Materials and Equipment:

[0092] (A) Cholesvelam or cholestyramine powder as the active pharmaceutical ingredient.

[0093] (B) Eutectic polymers suitable for pH-dependent release (e.g., Eutectic L100 for pH 6.5 and Eutectic S100 for pH 7.5).

[0094] (C) Organic solvents (e.g., ethanol, isopropanol).

[0095] (D) High-shear mixer.

[0096] (E) Spray dryer.

[0097] (F) pH buffer solution used for testing.

[0098] 2. Formula composition:

[0099] (A) Cholesvelam or cholestyramine, which is an active ingredient designed to be released in the ileum.

[0100] (B) Eutrich polymer: Select an utrich polymer that is insoluble under acidic conditions and begins to dissolve at a pH close to 6.5, which will ensure targeted release in the ileum.

[0101] (C) Additives: Optional additives, such as plasticizers, stabilizers or flow aids, may be added to improve the performance of the formulation.

[0102] 3. Preparation steps:

[0103] (A) Dissolve Eudragit polymer (Eudragit L00 or Eudragit S100) in a suitable organic solvent, such as ethanol, isopropanol, or acetone, to form a polymer solution.

[0104] (B) Add colesvelam powder to the solution and mix thoroughly using a high-shear mixer to ensure that colesvelam is uniformly dispersed in the polymer solution.

[0105] (C) Adjust the concentration and viscosity of the mixture as needed to achieve the desired coating performance.

[0106] (D) Using a spray dryer to coat colesvelam or colesylamine particles with Utec polymer, the spray drying process will result in the formation of coated particles with controlled-release properties.

[0107] (E) Optionally, the coated tablets may be further processed by sieving or grinding to obtain a uniform particle size distribution.

[0108] (F) Coated cholesvelam formulations were tested in pH buffer solutions ranging from acidic to alkaline to confirm targeted release at pH 6.5.

[0109] 4. Quality Control:

[0110] (A) Particle size analysis: The particle size distribution of coated colesvelan particles was determined using techniques such as laser diffraction or microscopy.

[0111] (B) Encapsulation efficiency: The amount of colesvelan encapsulated within the Urtig coating was evaluated using appropriate analytical methods.

[0112] (C) In vitro release studies: Release studies were conducted in pH buffer solutions simulating gastrointestinal conditions. The release of colesvelam over time was monitored, and targeted release at pH 6.5 was ensured.

[0113] (D) Stability testing: Store the formulation under different conditions (e.g., temperature, humidity) and monitor changes in particle size, encapsulation efficiency and release characteristics over time.

[0114] 5. Conclusion: By following this protocol, targeted cholesvelam formulations at pH 6.5 and pH 7.5 can be prepared using the Eutectic system for release in the ileum. This formulation has the potential to improve therapeutic efficacy by targeting cholesvelam or other polymers to specific sites of action in the gastrointestinal tract.

[0115] Example 3

[0116] Another approach to preparing pH-sensitive, ileal-targeted cholesvelam and cholestyramine microtablets using the Utex system is shown in Tables 3 and 4.

[0117] Table 3 shows the formulation examples for SQ6.5, designed to cause cholesvelam to disintegrate at pH 6.5.

[0118]

[0119] Table 4 shows the formulation examples for SQ7.5, designed to disintegrate cholesvelam or cholestyramine at pH 7.5.

[0120]

[0121] (1) Ingredients:

[0122] The core particles contain bile acid chelators as the active pharmaceutical ingredient, as well as other ingredients including lactose, microcrystalline cellulose, pregelatinized starch, starch, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, and magnesium stearate.

[0123] The pH-sensitive enteric coating contains Utec 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 acetate, hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethyl cellulose, cellulose acetate, and copolymers of maleic acid and phthalic acid derivatives.

[0124] The coating contains Eutech L100 (or Eutech S100) (5%–7%), triethyl citrate (0.5%–0.7%), talc (4%–5%), ethanol (83%–87%), and purified water (4%–6%).

[0125] (2) Process: Mix the specified amounts of cholesvelam hydrochloride or cholestyramine, microcrystalline cellulose, and sodium carboxymethyl cellulose. Rotate the container at 15 rpm for 15 minutes. After uniform mixing, slowly add 5% hydroxypropyl methylcellulose and wet granulate, then dry in a forced-air drying oven at 60°C, followed by further mixing with magnesium stearate. To prepare microtablets for animal experiments, granules are pressed using a 2 mm punch. The core granules are then enteric-coated with Eutectic L100 or Eutectic S100 and other ingredients.

[0126] (3) Measurement of disintegration time and pH: The microtablets, designated SQ6.5 containing cholesvelam as the active pharmaceutical ingredient, disintegrated at pH 6.5, and SQ7.5, disintegrated at pH 7.5. As shown in Table 5, both tablets were tolerated for up to 4 hours in 0.1N hydrochloric acid. Conversely, the SQ6.5 microtablet disintegrated in a solution at pH 6.5 (50 mM Tris at pH 6.5), while the SQ7.5 microtablet was stable at pH 6.5 but completely disintegrated in less than 30 minutes at pH 7.5. We also evaluated the tablet stiffness, including barium sulfate-containing tablets used for animal testing (Table 6). Prototype microtablets of SQ6.5 and SQ7.5 are shown below. Figure 4 As shown in A, SQ6.5 represents colesvelan that disintegrates at pH 6.5, and SQ7.5 represents colesvelan that is released at pH 7.5.

[0127] Table 5 Disintegration conditions of targeted ileal tablets

[0128]

[0129] Table 6. Hardness of pH-sensitive microcapsules targeting the ileum

[0130] Name, API Hardness, Newton SQ6.5 Disintegration of Kolai Velen 25.04-32.23 SQ7.5 Disintegration of Kolai Velen 27.12-33.09 SQ6.5 disintegrating barium sulfate 100.9-112.3 SQ6.5 disintegrating barium sulfate 99.8-110.6

[0131] Example 4

[0132] Hydroxypropyl methylcellulose phthalate (T-HPMCP, T-type) is commonly used in pH-sensitive formulations for drug delivery. T-HPMCP is a polymer that exhibits pH-dependent solubility. It is insoluble in acidic environments but becomes soluble at higher pH values. This property makes it ideal for protecting drugs from degradation in the acidic environment of the stomach and ensuring targeted release in the higher pH regions of the intestine.

[0133] (1) T-component: For pH-sensitive copolymer drugs targeting the ileum, T-HPMCP formulations can be designed to release the drug at the appropriate site, improving drug bioavailability and reducing side effects. Hydroxypropyl methylcellulose phthalate (HPMCP) is used as the enteric polymer to control release in the ileum. Other excipients may include fillers (e.g., microcrystalline cellulose), binders (e.g., polyvinylpyrrolidone), lubricants (e.g., magnesium stearate), and disintegrants (e.g., croscarmellose sodium).

[0134] (2) Method: Weigh and mix the copolymer and various excipients as the active pharmaceutical ingredient (e.g., cholesvelam). Granulation is performed if necessary. The mixture is pressed into core particles. Then, a T-HPMCP coating is applied to the particles using techniques such as pan coating or fluidized bed coating. Coating parameters are adjusted to achieve targeted release of cholesvelam in the ileum. The coating thickness and composition can be optimized to ensure the formulation remains intact in the stomach and small intestine and releases the drug in the ileum.

[0135] Example 5

[0136] A protocol for preparing a targeted formulation at pH 6.5 for ileal release using a polylactic-co-glycolic acid copolymer (PLGA) system.

[0137] (1) Introduction: This protocol describes the steps for creating a formulation designed to release a copolymer as an active pharmaceutical ingredient in the ileum, where the pH is approximately 6.5. Polylactic-co-glycolic acid copolymer (PLGA) is used as the pH-sensitive polymer because it is capable of degrading and releasing the encapsulated substance in response to specific pH changes.

[0138] (2) Materials and equipment: Polylactic acid-glycolic acid copolymer with appropriate molecular weight and degradation properties; copolymers as active pharmaceutical ingredients (APIs), such as cholesvelam and cholestyramine; organic solvents, such as dichloromethane or acetone; ultrasonic instrument; rotary evaporator; freeze dryer; pH buffer solution for testing.

[0139] (3) Formulation Composition: PLGA is used as the encapsulating polymer for pH-dependent release pills. The selection of PLGA should be based on its degradation rate at pH 6.5–7.5 and its ability to release the active pharmaceutical ingredient. Different molecular weights and copolymer ratios can be considered to achieve the desired release profile. Cholesvelam or cholestyramine is used as the active pharmaceutical ingredient delivered in the ileum to effectively eliminate PAMPs generated by intestinal microorganisms. Optional additives, such as stabilizers, surfactants, or excipients, can also be added to the pill core to enhance the stability and release characteristics of the formulation.

[0140] (4) Preparation steps:

[0141] (A) Dissolve PLGA in a suitable organic solvent (such as acetone or ethyl acetate) to form a polymer solution. The concentration of PLGA should be optimized to ensure proper encapsulation and release properties.

[0142] (B) Add cholesvelam or cholestyramine to the solution and mix thoroughly using an ultrasonic device or other suitable mixing equipment. This step ensures that the active pharmaceutical ingredient is uniformly dispersed in the polymer matrix.

[0143] (C) Using emulsification techniques, an emulsion of the polymer-active pharmaceutical ingredient solution is created in an aqueous phase. This can be achieved by adding the polymer solution dropwise to a stirred aqueous solution containing a surfactant.

[0144] (D) The emulsion is stabilized, and then the organic solvent is removed using a rotary evaporator. This step results in the formation of PLGA nanoparticles encapsulating the active pharmaceutical ingredient.

[0145] Example 6

[0146] Improved versions of pH-sensitive targeted formulations (Table 7).

[0147] Table 7. Composition examples of cholestyramine (SQ1-T) or cholesvelam (SQ1-C) that disintegrate at pH 6.8.

[0148]

[0149] (1) Compressing core particles:

[0150] Weigh out cholesvelam or cholestyramine (active pharmaceutical ingredient), microcrystalline cellulose, croscarmellose sodium, and hydroxypropyl methylcellulose according to the quantities specified in Table 7, and mix for 15 minutes to obtain a premixed powder. Then, add magnesium stearate and mix for 5 minutes to obtain a total mixed powder. Test the content of intermediate products. Calculate the weight of tablets to be compressed based on the intermediate product content. Control the tablet weight variation within ±5.0%. Compress using a 2 mm diameter circular deep-dip punch and die. Control the hardness within 5-15 N / mm².

[0151] (2) Coating with an isolation layer:

[0152] Turn on the high-efficiency coating machine and preheat it under no-load conditions. When the exhaust temperature reaches above 30℃, place the prepared core particles into the high-efficiency coating machine and randomly sample and weigh them. Adjust the atomization and fan pressure to the range of 0.1-0.6 MPa. During the coating process, control the inlet air temperature at 30±10℃, the exhaust temperature at 30±10℃, the peristaltic pump speed at 1-25 rpm, and the pot speed at 2-18 rpm. Ensure that the weight gain of the isolation layer is between 2-3%.

[0153] (3) Preparation of the coating:

[0154] (A) Put purified water into a stainless steel bucket, slowly add polyethylene glycol 6000 to part of the purified water and stir continuously until dissolved to obtain an aqueous solution of polyethylene glycol 6000 (solution 1) for later use.

[0155] (B) Take another portion of purified water in a stainless steel bucket, slowly add talc powder to the purified water, and continue stirring for 30 minutes. Use a high-speed homogenizer to homogenize the stirred suspension for 3 minutes (homogenizer speed: 8000 rpm) to obtain talc powder suspension (suspension 2).

[0156] (C) Add solution 1 to the cooled suspension 2 and stir for 30 minutes. Then add triethyl citrate and stir for another 30 minutes to obtain suspension 3 for later use.

[0157] (D) Weigh the aqueous dispersion of methacrylic acid-ethyl acrylate copolymer and put it into a stainless steel bucket. Add the remaining purified water and stir for 30 minutes to obtain a suspension of the aqueous dispersion of methacrylic acid-ethyl acrylate copolymer.

[0158] (E) Slowly add suspension 3 to suspension 4 and stir continuously for 60 minutes to obtain an enteric-coated solution with a concentration of approximately 20.0% (w / w).

[0159] (F) Apply the enteric coating. Turn on the high-efficiency coating machine and preheat it under no-load conditions. When the exhaust temperature reaches above 30°C, add the coated tablets (isolation layer) to the high-efficiency coating machine and preheat for 5 minutes. After preheating, randomly sample and weigh them. Adjust the atomization and fan pressure to the range of 0.1-0.6 MPa. During the coating process, control the inlet air temperature at 30±10°C, the exhaust temperature at 30±10°C, the peristaltic pump speed at 10-25 rpm, and the reactor speed at 6-18 rpm. The weight gain of the enteric coating should be between 8.0-10.0%.

[0160] This invention describes methods, compositions, and formulations of pharmaceuticals. The pharmaceutical formulations comprise endotoxin chelators as active pharmaceutical ingredients, and these formulations are designed for targeted release in the distal region of the small intestine. The purpose of these formulations is to chelate and excrete endotoxins and other pathogen-associated molecular patterns. Furthermore, these formulations inhibit endotoxin entry into circulation and are used for the medical prevention and treatment of metabolic disorders, including liver disease.

[0161] Example 7

[0162] The in vivo disintegration of colesvelam and cholestyramine via ileal-targeted release was studied using animal models.

[0163] like Figure 1 As shown, prototypes of three pH-sensitive formulations were prepared as microtablets. The blue tablet was for targeted barium sulfate at pH 6.8, used to track dissolution time and location in vivo. The white tablet was for SQ1-C (cholesvelam), and the red tablet was for SQ1-T (cholestyramine), both designed to disintegrate at pH 6.5. The microtablets used in rat experiments were 2 mm in diameter, 5 mm in length, with a hardness between 5 and 15 N / mm², each containing approximately 8-10 mg of active pharmaceutical ingredient and 5-7 mg of active pharmaceutical ingredient.

[0164] We measured the disintegration pH of the microtablets. First, we demonstrated that the three types of tablets could withstand 0.2N hydrochloric acid for more than 60 minutes. Subsequently, the tablets were transferred to buffer solutions with gradually increasing pH values ​​from 2.5 to 7.5, containing 120 mM sodium chloride and 50 mM tris(hydroxymethyl)aminomethane. Dissolution times were recorded, and tablet disintegration was observed in the buffer solution at pH 6.5. Although the tablets disintegrated within 30 minutes in the buffer solution at pH 6.5, the actual in vivo dissolution time was determined by in vivo tracking using barium sulfate tablets.

[0165] To determine in vivo disintegration, adult SD rats were fasted overnight before being orally administered 4-5 microtablets containing barium sulfate at pH 6.8. Standard feed and drinking water were then provided to the cages. Anesthetized rats underwent X-ray-based CT scans at specified time points. Figure 2As shown, tablets at pH 6.8 began to dissolve within 4–6 hours and largely disintegrated within 8 hours. No tablets were found in the gastrointestinal tract 24 hours after administration. To further confirm disintegration in the distal small intestine, rats were sacrificed at different time points and dissected to determine tablet location. SQ1-T is cholestyramine targeting ileal release, and SQ1-C is cholesvelam targeting ileal release.

[0166] Example 8

[0167] In vitro chelation experiment of endotoxin and CpG-DNA.

[0168] Endotoxins or lipopolysaccharides in solution were measured using a horseshoe crab (LAL) assay, based on the activity of the endotoxin. Alternatively, purified endotoxins exhibit a specific adsorption peak at 258 nm, which was used in our in vitro binding assay for adsorption and chelation analysis. Figure 3 As shown in Figure A, we determined the in vitro adsorption / neutralization capacity of SQ1-CT and the active pharmaceutical ingredient powder suspension. Specifically, LPS (B4, Sigma-Aldrich) was dissolved in pure water, and specific adsorption peaks were scanned and recorded at 258 nm. SQ1-CT and the active pharmaceutical ingredient powder were dissolved in a buffer solution (120 mM sodium chloride, 50 mM tris(hydroxymethyl)aminomethane, pH 6.5) at pH 6.5. The high molecular weight resin was washed twice with the same buffer solution, then centrifuged and collected. Next, a certain amount of LPS was mixed with a certain volume of resin or control buffer. After incubating the mixture for 1 hour, the system was centrifuged, and the supernatant was collected for spectrophotometric analysis. The results showed that the adsorption / binding capacity of the SQ-1 formulation for LPS at pH 6.8 was very similar to that of the active pharmaceutical ingredient copolymer. In particular, as this assay showed, 1 gram of SQ1 or cholesvelam could adsorb or chelate approximately 0.1 grams of endotoxin in vitro. In a similar manner, bacterial genomic DNA was isolated and purified according to standard procedures, and as... Figure 3 B shows the measurement of chelating ability.

[0169] Example 9

[0170] In vitro disintegration assays of two pH-sensitive cholesvelam formulations.

[0171] As described in the Example section of this invention, two types of colesvelam, designated SQ6.5 and SQ7.5, were prepared using the Eutectic system for disintegration at pH 6.5 and pH 7.5, respectively. The microtablets contain 4.8-5.6 mg of the active pharmaceutical ingredient in a total weight of 10 mg. Figure 4 ). Figure 4A shows two pH-sensitive ileal-release pill prototypes, SQ6.5 representing colesvelam that disintegrates at pH 6.5 and SQ7.5 representing colesvelam that releases at pH 7.5. Figure 4 B shows the in vitro disintegration time and pH value of two pH-sensitive ileal microspheres. SQ6.5 represents colesvelam that disintegrates at pH 6.5, and SQ7.5 represents colesvelam that is released at pH 7.5.

[0172] First, we measured the disintegration pH of the microtablets in buffer solution using in vitro experiments at 37°C with shaking. The results showed that the tablets were tolerable and stable in 0.1N hydrochloric acid for up to 4 hours. Subsequently, the tablets were transferred to buffer solutions with pH values ​​of 6.5 and 7.5, containing 120 mM sodium chloride and 50 mM tris(hydroxymethyl)aminomethane. Dissolution times were recorded. As shown in the figure, SQ6.5 tablets and their equivalent barium chloride dissolved in buffer solution at pH 6.5 within 2 hours, while SQ7.5 tablets and barium chloride were stable at pH 6.5 but completely dissolved in buffer solution at pH 7.5 within 4 hours. Figure 5 ). Figure 5 The in vitro disintegration record of the microparticle formulation is shown. The microparticles were placed in buffer solutions and shaken at 37°C for 6 hours. The buffer solutions were: (a) 0.1 mol / L hydrochloric acid, (b) a buffer solution with pH 6.5 containing 50 mmol / L tris(hydroxymethyl)aminomethane (Tris) and 120 mmol / L sodium chloride, and (c) a buffer solution with pH 7.5 containing 50 mmol / L tris(hydroxymethyl)aminomethane (Tris) and 120 mmol / L sodium chloride.

[0173] In vivo disintegration assay of a polymer released at a specific point in the ileum. Adult SD rats were fasted overnight. Five tablets of SQ6.5 and SQ7.5 containing barium sulfate were administered by gavage. Standard feed and drinking water were then added to the cages. Anesthetized rats underwent X-ray-based CT scans at specified time points. Figure 6 As shown, pH-sensitive tablets begin to dissolve after 8–10 hours, likely in the distal small intestine. We also confirmed this finding by feeding rats the microtablets and then sacrificing and dissecting them at different time points.

[0174] Example 10

[0175] Experimental design for young mice that underwent three weeks of alcohol induction followed by three cycles of binge eating.

[0176] Experimental design, acute alcoholic hepatitis in young rats, and evaluation of the efficacy of SQ1. It has been reported that in Sprague Dawley rats, endotoxin levels in portal venous blood rapidly increase from 0 to 10 picograms / mL within 60 minutes after acute intragastric administration of ethanol (5 g / kg). Here, we modified the method to determine endotoxin levels and treatment efficacy in rats with alcoholic hepatitis after three weeks. Figure 7 As shown, twenty 7-week-old SPF-grade SD rats were used. Five rats were assigned to the normal control group, while the remaining fifteen rats were fed Lieber-DeCarli standard alcoholic liquid (containing 36% total calories from alcohol) for three weeks to establish the model. Subsequently, biochemical metabolic parameters were measured. Serum transaminase (ALT) levels increased from 25 ± 10 units / mL in the normal control group to 60 ± 8 units / mL, indicating that alcohol induced liver damage. At this point, the model rats were further divided into three groups of five rats each. While the model group (ASH) continued to be fed the alcoholic liquid diet for another three weeks, one group was treated with SQ1 (SQ1-CV, 7 microtablets daily, active pharmaceutical ingredient dose of 25 mg daily, administered by gavage); the third group was treated with active pharmaceutical ingredient powder (colevelam CV, 25 mg daily, administered by gavage). Finally, the three groups of alcohol-induced rats were subjected to binge eating treatment at a dose of 5 g alcohol / kg (10 mL per kg body weight). The rats were euthanized 24 hours after their last binge eating episode, and blood, liver, and pancreatic tissue were collected for analysis.

[0177] Example 11

[0178] Targeted ileal release of colesvelam can reduce toxemia caused by acute alcoholic hepatitis.

[0179] like Figure 8 As shown, we determined as follows Figure 7 The blood endotoxin levels in young rats that underwent six weeks of alcohol induction followed by three binge-eating episodes were analyzed. As shown in the figure, endotoxin levels increased sixfold from 0.3 EU / mL in control rats to 2.0 EU / mL in rats with alcoholic liver disease. Notably, administration of SQ1-CV microtablets significantly reduced blood endotoxin levels by approximately 75%, while the active pharmaceutical ingredient powder (CV) also partially reduced blood endotoxin levels. Plasma endotoxin levels were inhibited after treatment with cholesvelam (SQ1-C) at pH 6.5 and targeting the ileum, along with the corresponding cholesvelam suspension (CV). The experimental design was as follows. Figure 7 Plasma endotoxin levels were determined using the Limulus amebocyte lysate (LAL) assay.

[0180] Example 12

[0181] Targeted ileal release of colesvelam can reduce systemic inflammation caused by acute alcoholic hepatitis.

[0182] Once bacterial endotoxins enter the bloodstream, they can induce the expression of a series of inflammatory factors, such as TNF-α and IL-1, by activating TLR-4 / CD14 cell membrane receptors and signaling pathways. Alcoholic liver disease (ALD) is characterized by steatosis and the upregulation of 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 signaling complex and play a role in inflammation. Since IL-1 is significantly associated with key clinical symptoms of acute alcoholic hepatitis (such as fever, neutropenia, and cachexia), interfering with the IL-1 pathway may be an attractive therapeutic strategy for the future. The important roles of type I IL-1 cytokines and certain inflammasomes have also been demonstrated in mouse models of non-alcoholic fatty liver disease. Cholesvelam, targeting the ileum, can effectively reduce blood endotoxin levels. Figure 9 As shown, the study determined the inhibitory effects of alcohol-induced systemic inflammation and oral administration of SQ1-C and cholesvelam suspension (CV), which target the ileum, by detecting serum levels of interleukin-1β. The experimental design was as follows. Figure 7 The cytokine was detected by enzyme-linked immunosorbent assay (ELISA). The concept of the invention was confirmed by measuring the LPS / TLR4 / CD14 inflammatory signaling pathway in young mice that had undergone six weeks of alcohol induction followed by three cycles of binge eating. Oral administration of cholesvelam, targeting the ileum, reduced blood IL-1β levels.

[0183] Example 13

[0184] Targeted ileal release of colesvelam can reduce interleukin-6 levels induced by acute alcoholic hepatitis.

[0185] Interleukin-6 (IL-6) is a key inflammatory cytokine that plays a central role in the development and progression of liver diseases. Serum IL-6 concentrations are significantly increased in patients with alcoholic or non-alcoholic cirrhosis and toxic hepatitis compared to controls. IL-6 can induce alcoholic liver disease (ALD) by activating signal transduction and transcription activator 3 (STAT3), subsequently inducing the expression of various hepatoprotective genes in hepatocytes. Furthermore, clinical studies have shown that serum IL-6 levels can serve as a prognostic factor for alcoholic liver disease. On the other hand, endotoxins can rapidly induce IL-6 expression in vivo. Studies have found that hepatocytes can express IL-6 under endotoxin stimulation. Therefore, liver injury also reduces the ability to clear IL-6, promoting inflammatory pathogenesis. Figure 10As shown, SQ1-C (i.e., cholesvelam with a pH of 6.5 and targeting the ileum) and its corresponding active pharmaceutical ingredient (API) named CV can inhibit alcohol-induced systemic inflammation. This inhibitory effect is measured by serum levels of interleukin-6. The experimental design is as follows: Figure 7 The cytokine was detected by enzyme-linked immunosorbent assay (ELISA). Oral administration of colesvelam, targeting the ileum, partially reduced blood levels of the IL-6 inflammatory cytokine by measuring the LPS / TLR4 / CD14 inflammatory signaling pathway, confirming the concept of our disclosure.

[0186] Example 14

[0187] To evaluate fatty liver in young mice with alcoholic hepatitis.

[0188] To quantitatively assess clinical samples and animal experimental data, the International Association for the Study of the Liver (IASL) classifies fatty liver disease into mild, moderate, and severe degrees. Common assessment indicators are the percentage of fat content in liver tissue or a grading score. Specifically: F1 is mild fatty liver, with less than 30% fat in the liver tissue. This level of fat deposition is generally considered to have minimal impact on liver function. F2 is moderate fatty liver, with a fat content between 30% and 60% in the liver tissue. Moderate fat deposition may cause inflammatory responses and damage, affecting liver function to some extent. F3 is severe fatty liver, when the fat content in the liver tissue exceeds 60%. Severe fatty liver is usually accompanied by liver inflammation, fibrosis, and cell damage, significantly impacting liver function. The assessment of fatty liver is a qualitative and quantitative process, typically using medical imaging techniques such as ultrasound, computed tomography (CT), or magnetic resonance imaging (MRI) to determine the degree of fat content in liver tissue. Figure 7 The liver tissue used in the experiment was stained with hematoxylin and eosin, and... Figure 11 Image A shows the slices selected based on the scoring criteria.

[0189] Example 15

[0190] Colesvelan's targeted ileal formulation can improve alcoholic steatosis.

[0191] like Figure 11As shown in Figure B, fatty liver developed in young Lieber-DeCarli mice subjected to alcohol feeding followed by three binge-eating episodes. However, significant individual differences existed within the groups. As shown in the figure, in the alcoholic liver disease model group, one subject was classified as F3, two as F2, and two as F1. After oral administration of SQ1-CV, two subjects were classified as F2, and three as F1. Importantly, the partial improvement in fatty liver in subjects taking SQ1 was consistent with a decrease in systemic inflammation and the expression of hepatic inflammatory factors, further validating our working hypothesis that SQ1-CV can absorb endotoxins, reduce endotoxemia and systemic inflammation, thereby contributing to the improvement of fatty liver.

[0192] Example 16

[0193] A method for assessing liver inflammation in experimental animal models of alcoholic hepatitis.

[0194] like Figure 12 As shown, based on rat liver sections and hematoxylin-eosin staining results obtained in our experiment, we established the following liver inflammation index: Grade 0, fewer than 2 lesions in 10 fields of view at 200x magnification; Grade 1, exactly 2 lesions in 10 fields of view at 200x magnification; Grade 2, 2 to 4 lesions in 10 fields of view at 200x magnification. Importantly, the ranking of liver inflammation index among the groups was consistent with the levels of endotoxin and systemic inflammatory factor (IL-1), further illustrating the causal relationship in the pathogenesis, namely the key role of endotoxemia caused by intestinal bacterial toxins in the development of liver disease.

[0195] Example 17

[0196] Targeted ileal release of colesvelam can inhibit the mRNA expression of inflammatory factors in liver tissue during alcoholic liver disease.

[0197] The mRNA expression of two inflammatory factors in liver tissue was measured. Figure 13 As shown, the exacerbation of liver inflammation, determined by detecting the messenger ribonucleic acid (mRNA) levels of interleukin-1β (Figure A) in rats, could be alleviated by daily administration of the ileum-targeting cholesvelam preparation SQ1-C at a dose of 91 mg / kg. The exacerbation of liver inflammation, determined by detecting the messenger ribonucleic acid (mRNA) levels of interleukin-6 (Figure B) in rats, could also be alleviated by daily administration of the ileum-targeting cholesvelam preparation SQ1-C at a dose of 91 mg / kg.

[0198] Six weeks of alcohol-feeding combined with three episodes of binge eating resulted in a two-fold increase in the expression of interleukin-1β (IL-1β) and a three-fold increase in the expression of IL-6 in liver tissue. Administration of cholesvelam, targeting the ileum, reduced the expression of these two inflammatory factors in liver tissue. Importantly, the expression of the cytokine IL-1 is directly regulated by endotoxin / TLR4 / CD14. Here, we show that its expression in the liver of patients with alcoholic liver disease follows a trend perfectly consistent with blood endotoxin concentrations, further demonstrating its causal pathological relationship. Furthermore, we demonstrate that SQ1 treatment can reduce the expression of hepatic inflammatory genes. This suggests that chelating intestinal endotoxins can be used to alleviate the expression of hepatic inflammatory genes.

[0199] Example 18

[0200] Experimental design for rats with acute alcoholic hepatitis and their treatment with ileal-targeting endotoxin chelators SQ1-C and SQ1-T.

[0201] like Figure 14 As shown, this experiment used forty middle-aged rats (10-12 months old). Thirty of them were induced with alcohol for ten days on a Lieber-DeCarli diet (containing 5% alcohol, accounting for 36% of total calories). Then, the animals were given a binge-feeding dose of 5 g / kg and 10 ml / kg body weight. The experiment was terminated within 24 hours. During the experiment, microtablets SQ1-C and SQ1-T, containing cholesvelam and cholestyramine respectively, were administered orally at a dose of 91 mg / kg, with n=10 in each group.

[0202] Example 19

[0203] Metabolic endotoxin levels in rats with acute alcoholic hepatitis were inhibited using the ileum-targeting endotoxin chelators SQ1-C and SQ1-T.

[0204] Chronic alcohol consumption damages the intestinal barrier, increasing its permeability. This allows bacterial endotoxins (lipopolysaccharide, LPS) to leak from the gut into the bloodstream. Once in the bloodstream, LPS becomes a metabolic endotoxin. It binds to Toll-like receptor 4 (TLR4) on immune cells such as Kupffer cells in the liver. This activates the immune system and triggers the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Numerous clinical studies and animal experiments have shown that age-related intestinal damage, such as leaky gut, further enhances the transport of endotoxins to the liver and systemic circulation. The release of these cytokines leads to liver inflammation and oxidative stress. If this process continues, it can lead to hepatocellular damage, steatosis (fat accumulation), inflammation (steatohepatitis), fibrosis (scarring), and ultimately cirrhosis. Figure 15 As shown, for Figure 14 Using the method described above, we measured circulating metabolic endotoxin levels in middle-aged rats with acute alcoholic hepatitis, with and without drug treatment. The rats were induced with alcohol (a liquid diet containing 5% alcohol, with 36% of their calories derived from alcohol) for ten days, followed by a binge-feeding treatment (5 g / kg body weight). This resulted in a significant increase in plasma endotoxin levels, i.e., metabolic endotoxins. Notably, the pH 6.5-designed copolymers SQ1-C and SQ1-T, targeting the ileum, statistically inhibited plasma endotoxin levels, as measured by the Limulus Amebocyte Lysate (LAL) assay.

[0205] Example 20

[0206] The endotoxin chelators SQ1-C and SQ1-T, with a pH of 6.5 and targeting the ileum, can inhibit systemic inflammation in acute alcoholic hepatitis.

[0207] Well-documented in the literature, endotoxins can directly induce systemic inflammation in various metabolic disorders, including alcoholic hepatitis, manifested as elevated levels of interleukin-1β and tumor necrosis factor-α (TNF-α). In alcoholic liver disease, IL-1β is a crucial mediator of inflammation. It is upregulated in the response to alcohol-induced liver damage. IL-1β can be induced by bacterial endotoxins that enter the bloodstream and activate the immune system. IL-1β is significantly associated with key clinical symptoms of acute alcoholic hepatitis, such as fever, neutropenia (an increase in the number of neutrophils in the blood), and cachexia (wasting syndrome). TNF-α is also a key inflammatory cytokine involved in the inflammatory response of alcoholic diseases. Alcohol consumption leads to increased TNF-α production. High levels of TNF-α are associated with the progression of alcoholic liver disease from steatosis (fat accumulation in the liver) to more severe forms such as steatohepatitis (inflammation and damage in the liver accompanied by fat accumulation), fibrosis (scarring of the liver), and cirrhosis (late-stage scarring of the liver). Figure 16 As shown, for Figure 14 The method described above showed that in middle-aged rats with acute alcoholic hepatitis, the circulating levels of interleukin-1β and tumor necrosis factor-α increased rapidly. Conversely, pH-sensitive, ileum-targeting cholesvelam (SQ1-C) and cholestyramine (SQ1-T) both suppressed systemic inflammation in acute alcoholic hepatitis. First, this result is consistent with the reduction of plasma endotoxin levels through intestinal chelation of the two copolymers, delivered via targeted delivery using pH-sensitive and time-dependent formulations designed for ileal release. Second, the results showing a reduction in systemic inflammation provide a basis for numerous therapeutic potentials. Cytokine levels in rat blood were measured using enzyme-linked immunosorbent assay (ELISA). Figure 14The endotoxin chelator, which is pH sensitive and targets the ileum, can inhibit the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1β in acute alcoholic hepatitis through intestinal chelation.

[0208] Example 21

[0209] Oral administration of the endotoxin chelators SQ1-C and SQ1-T, which are at pH 6.5 and target the ileum, can reduce elevated plasma transaminase levels in rats with acute alcoholic hepatitis.

[0210] like Figure 17 As shown, pH-sensitive copolymers SQ1-C (with cholesvelam as the active pharmaceutical ingredient) and SQ1-T (with cholestyramine as the active pharmaceutical ingredient), which target the ileum, can inhibit the increase of transaminase levels in middle-aged rats with acute alcoholic hepatitis, reduce liver function, and inhibit liver damage. As shown in the figure, in middle-aged rats, alcohol induction combined with a single binge-eating treatment statistically led to an increase in serum liver transaminase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels. Notably, SQ1-C and SQ1-T can improve liver damage, manifested by inhibiting transaminase levels.

[0211] Example 22

[0212] Endotoxin chelators SQ1-C and SQ1-T targeting the ileum can improve coagulation dysfunction in acute alcoholic hepatitis.

[0213] like Figure 18 and Figure 14 The experimental conditions mentioned above indicate that the coagulation system of middle-aged rats with acute alcoholic hepatitis is impaired, manifested by increased prothrombin time (PT) and activated partial thromboplastin time (APTT). Importantly, oral administration of cholesvelam (SQ1-C) and its corresponding cholestyramine (SQ1-T) at pH 6.5 and targeting ileal release can restore coagulation homeostasis in rats with acute alcoholic hepatitis. Figure 14 In the rat model described above, the prolongation of activated partial thromboplastin time (APTT) caused by alcoholic hepatitis was detected by an automated chemical analyzer, and it was found that it could be inhibited by oral administration of SQ1-C and SQ1-T.

[0214] Example 23

[0215] Oral administration of the ileum-targeting endotoxin chelators SQ1-C and SQ1-T can improve pancreatic damage in rats with acute alcoholic hepatitis.

[0216] like Figure 19As shown, 10 days of alcohol-induced pancreatic damage followed by a single binge-eating episode resulted in elevated plasma levels of pancreatic markers α-amylase (AMY) and alkaline phosphatase (ALK). Notably, oral administration of SQ1-C and SQ1-T, two substances with high molecular weight copolymers in a pH 6.5-targeted ileal formulation as active pharmaceutical ingredients, completely inhibited pancreatic damage and reduced pathological markers. Furthermore, the improvement in pancreatic function through chelation therapy was closely associated with reduced metabolic endotoxins and systemic inflammation, providing a potential causal relationship for the basis of novel therapies.

[0217] Example 24

[0218] Oral administration of the ileum-targeting endotoxin sequestrants SQ1-C and SQ1-T can reduce hyperglycemia in alcoholic hepatitis.

[0219] like Figure 20 As shown, alcohol consumption combined with overeating significantly increases blood glucose levels in middle-aged rats, i.e., hyperglycemia. Elevated blood glucose levels or hyperglycemia are common characteristics of alcoholic hepatitis. Notably, oral administration of cholesvelam (SQ1-C) and cholestyramine (SQ1-T) at pH 6.5 and targeting the ileum can alleviate [the symptoms]. Figure 14 The hyperglycemia in the rat model described herein was assessed by measuring plasma triglyceride and glucose levels using an automated chemical analyzer.

[0220] Example 25

[0221] Gene Ontology (GO) and KEGG enrichment analyses in rats with acute alcoholic hepatitis and those treated with PASA.

[0222] like Figure 14 As shown in the experimental design, RNA was extracted from liver tissue for RNA-seq analysis. Rare data from the RNA-seq were further subjected to gene ontology (GO) enrichment analysis, which can provide insights into the biological processes regulating the differentially expressed differences between the model group (SD rats in acute alcoholic hepatitis) and the control group. Figure 21 The KEGG enrichment analysis of “Model_vs_control_all_1” provides insights into the differences between animals that binge consume alcohol (model) and the control group in an animal model. The results indicate that alcohol consumption mobilizes the following pathways: exogenous substance catabolic processes, sterol and steroid biosynthesis, cellular responses to glucocorticoid stimulation, and ketones.

[0223] in, Figure 21Figure A shows a gene ontology (GO) analysis of liver gene expression in middle-aged rats under acute alcoholic hepatitis compared to the control group. GO enrichment analysis helps to gain insights into the biological processes differentially regulated between the model group (Sprague-Dowley rats with alcoholic hepatitis) and the control group. Figure 21 B shows the Kyoto Genome and Genome Encyclopedia (KEGG) analysis of liver gene expression in middle-aged rats under acute alcoholic hepatitis conditions compared to the control group. The KEGG enrichment analysis results for the alcohol model group and the control group contribute to a deeper understanding of... Figure 14 The experiment revealed differences between rats that consumed excessive alcohol and those in the control group.

[0224] Example 26

[0225] KEGG enrichment analysis of liver gene expression in rats with acute alcoholic hepatitis and treated with ileal endotoxin chelators SQ1-C and SQ1-T.

[0226] KEGG enrichment analysis provided information on the biological pathways involved in SQ1-C or SQ1-T chelation therapy in middle-aged rats with alcoholic hepatitis. Experimental conditions were as follows: Figure 14 The analysis indicates that excessive alcohol consumption can affect multiple pathways associated with disease and biological processes. Figure 22 The chelation of endotoxins by SQ1-C and SQ1-T targeting the ileum may act on these pathways to counteract the negative effects of alcohol and potentially prevent or treat various diseases. Treatment with SQ1-C and SQ1-T appears to have effects on pathways related to hepatocellular carcinoma, diabetic complications, p53 and Hippo, insulin resistance, cellular senescence, rheumatoid arthritis, as well as drug metabolism, chemokine signaling, and Th1 and Th2 cell differentiation.

[0227] in, Figure 22 A shows the Kyoto Gene and Genome Encyclopedia (KEGG) enrichment analysis of liver gene expression in middle-aged rats under acute alcoholic hepatitis conditions compared with those treated with the ileum-targeted endotoxin chelator SQ1-C. Figure 22 B shows another Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of liver gene expression in middle-aged rats under acute alcoholic hepatitis conditions compared to those treated with the ileum-targeting endotoxin chelator SQ1-T.

[0228] Example 27

[0229] Oral administration of the endotoxin sequestrant SQ1-C, which targets the ileum, can inhibit the expression of hepatic TNF-α receptor and platelet-reactive protein 1 (TSP-1) in rats with acute alcoholic hepatitis.

[0230] like Figure 14 As described above, middle-aged rats were induced with alcohol for ten days and then subjected to a binge-eating treatment. Gene expression in liver tissue was analyzed using RNA-seq. Figure 23 As shown, alcohol treatment significantly increased the expression of TNF-α and thromboretin 1 in the liver, consistent with endotoxin levels and systemic inflammation induced by leukocytes. Here we demonstrate that liver tissue has an intrinsic capacity to express TNF-α receptors, indicating that the liver is a source of inflammation. TSP-1 can promote the fibrinolytic system, affect clot dissolution, and enhance systemic inflammation and disease severity in acute-to-chronic liver failure. LPS is known to induce TSP-1 production. Notably, oral administration of two ileal-targeting polymers, SQ1-C and SQ1-T, inhibited the expression of TNF-α (Figure A) and thromboretin 1 (Figure B) in the liver.

[0231] Example 28

[0232] Oral administration of an intestinal endotoxin chelator targeting the ileum can reduce liver fibrosis in rats with alcoholic hepatitis.

[0233] Experiments and treatments in Figure 14 As described in the text. Figure 24 As shown, rats with acute alcoholic hepatitis exhibited liver fibrosis and pathological progression, with increased mRNA expression levels of lysine oxidase 2 (Lox 2, Figure A) and transforming growth factor β-2 (Tgfb2, Figure B) in their livers. Strong evidence from clinical association and genetic experiments suggests that endotoxin signaling plays a crucial role in liver fibrosis and hepatic stellate cell activation through Lox2 and TGF-beta signaling.

Claims

1. An ileal-targeted release chelating agent for use in the preparation of a drug for treating acute alcoholic hepatitis, said chelating agent comprising core particles, an isolation layer, and a pH-sensitive coating, said core particles comprising 5.6 mg of cholesvelam or cholestyramine, 1 mg of microcrystalline cellulose UF711, 0.6 mg of microcrystalline cellulose 200, 3.4 mg of microcrystalline cellulose KG1000, 0.9 mg of hydroxypropyl methylcellulose, 0.2 mg of croscarmellose sodium, 2.5 mg of lactose, and 0.1 mg of magnesium stearate; The isolation layer comprises 0.61 mg of methacrylate-methyl methacrylate copolymer, 0.11 mg of triethyl citrate, and 8.22 mg of ethanol; the pH-sensitive coating comprises 5.84 mg of methacrylate-ethyl acrylate copolymer aqueous dispersion, 0.28 mg of triethyl citrate, 0.27 mg of talc, 0.01 mg of polyethylene glycol 6000, and 5.14 mg of water, wherein the molar ratio of methacrylic acid to methyl methacrylate in the methacrylate-methyl methacrylate copolymer is 1:

1.

2. The preparation method of the ileal-targeted release chelating agent as described in claim 1 is as follows: (1) Preparation of core particles: Weigh out cholesvelam or cholestyramine, microcrystalline cellulose, croscarmellose sodium, lactose, and hydroxypropyl methylcellulose and mix them to obtain a premixed powder. Then add magnesium stearate and mix to obtain a total mixed powder, and compress into tablets. (2) Coating with an isolation layer: Pour anhydrous ethanol into a stainless steel bucket, turn on the stirrer and add methacrylic acid-methyl methacrylate copolymer and stir continuously until completely dissolved. Then, continue to add triethyl citrate while stirring. After the addition is complete, continue stirring for 1 hour to prepare the isolation layer coating solution. Turn on the high-efficiency coating machine, preheat the machine without air, and when the exhaust temperature reaches above 30°C, take the core particles prepared in step 1) and place them in the high-efficiency coating machine for coating. The weight gain of the isolation layer is between 2-3%, and the isolation coated sheet is obtained. (3) pH-sensitive coating: A puts purified water into a stainless steel bucket, adds polyethylene glycol 6000 to part of the purified water and stirs continuously until dissolved to obtain an aqueous solution of polyethylene glycol 6000 for later use; B takes another portion of purified water from a stainless steel bucket, adds talc powder to the purified water, and stirs for 30 minutes to obtain a talc powder suspension. C. Add the aqueous solution of polyethylene glycol 6000 to the cooled talc suspension and stir for 30 minutes. Then add triethyl citrate and stir for another 30 minutes to obtain suspension 3 for later use. D. Weigh out the aqueous dispersion of methacrylic acid-ethyl acrylate copolymer and put it into a stainless steel bucket. Add purified water and stir for 30 minutes to obtain a suspension of the aqueous dispersion of methacrylic acid-ethyl acrylate copolymer. E. Add suspension 3 to the aqueous dispersion suspension of methacrylate-ethyl acrylate copolymer and stir continuously for 60 min to obtain an enteric coating solution with a concentration of 20.0% w / w. F. Enzyme enteric coating layer, turn on high-efficiency coating machine, preheat empty machine, add isolation coating sheet into high-efficiency coating machine, adjust atomization and fan-shaped pressure, coating, so that the weight gain of enteric coating layer is 8.0-10.0%, to obtain the chelating agent.

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