Treatment of gastrointestinal diseases

Abstract

The invention discloses application of thiazolidinedione medicines to treatment of digestive system diseases including gastroparesis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 332,778, filed on April 20, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to therapeutic agents and methods for treating gastrointestinal disorders, such as gastroparesis. Background Art

[0004] Digestive disease is a condition of the digestive tract, which is also called the gastrointestinal (GI) tract. GI disease and disorders are any conditions related to the GI system, which includes the entire GI tract, from mouth to anus, including the esophagus, stomach, and intestines. Diagnoses may include acute, short-term illnesses. GI disease may also include more chronic diagnoses that may require long-term, specialized treatment.

[0005] Conditions can range from mild to severe. Some common problems include heartburn, cancer, irritable bowel syndrome (IBS), and lactose intolerance. Other digestive disorders include gallstones, cholecystitis, cholangitis, rectal disorders (such as anal fissures, hemorrhoids, proctitis, and rectal prolapse), esophageal disorders (such as strictures and achalasia and esophagitis), stomach disorders (such as gastritis, gastric ulcers often caused by Helicobacter pylori infection, and cancer), liver disorders (such as hepatitis B or C, cirrhosis, liver failure, and autoimmune and alcoholic hepatitis), pancreatitis and pancreatic pseudocysts, intestinal disorders (such as polyps and cancer, infections, celiac disease, Crohn's disease, ulcerative colitis, diverticulitis, malabsorption, short bowel syndrome, and intestinal ischemia), gastroesophageal reflux disease (GERD), peptic ulcer disease, and hiatal hernia.

[0006] Among all digestive disorders, gastroparesis is a condition characterized by delayed gastric emptying (DGE) in the absence of mechanical obstruction. Symptoms are chronic with intermittent exacerbations. Idiopathic gastroparesis accounts for the largest number of cases and affects mainly young adult women. Gastroparesis is also frequently associated with diabetes (diabetic gastroparesis), which may occur due to impaired neural control of gastric motility. In addition, acute hyperglycemia may slow gastric emptying and reduce the effectiveness of prokinetic drugs.

[0007] On a molecular level, gastroparesis may be caused by a loss of neural nitric oxide expression, as cells in the GI tract secrete nitric oxide. This important signaling molecule plays a variety of roles in the GI tract and muscles throughout the body. When nitric oxide levels are low, smooth muscle and other organs may not function properly. Other important components of the stomach are the interstitial cells of Cajal (ICC), which act as pacemakers as they transmit signals from motor neurons to create electrical rhythms in smooth muscle cells. Lower nitric oxide levels have also been associated with a loss of ICC cells, which can ultimately lead to a loss of smooth muscle function in the stomach as well as other areas of the GI tract. The pathogenesis of diabetic gastroparesis symptoms includes (1) loss of gastric neurons containing nitric oxide synthase (NOS), which results in defective regulatory reflexes and leads to early satiety and postprandial fullness; (2) impaired electromechanical activity of the myenteric plexus, resulting in delayed gastric emptying, which causes nausea and vomiting; (3) sensory neuropathy of the gastric wall, which may be the cause of epigastric pain; and (4) abnormal pacemaker activity (bradycardia), which may generate noxious signals to the central nervous system (CNS) causing nausea and vomiting.

[0008] Macrophages also play a role in the development and progression of gastroparesis. Specifically, macrophages infiltrate the gastric smooth muscle layer in patients with gastroparesis and release pro-inflammatory molecules that can cause gastric muscle damage and dysfunction. In the case of diabetic gastroparesis, high blood sugar levels activate macrophages, leading to inflammation and damage to the gastric nerves and muscles. There are two different subtypes of macrophages that have different functions in the immune system, namely M1 and M2 macrophages. M1 macrophages are involved in inflammatory responses and are activated in response to pro-inflammatory signals. They are important in fighting infections. M1 macrophages produce pro-inflammatory cytokines and reactive oxygen species that help kill invading pathogens and promote the recruitment of other immune cells to the site of infection. On the other hand, M2 macrophages are involved in tissue repair and immune regulation and are activated in response to anti-inflammatory signals. They play an important role in resolving inflammation and promoting tissue remodeling and healing. M2 macrophages produce anti-inflammatory cytokines and growth factors that help repair damaged tissues and promote angiogenesis. Overall, M1 and M2 macrophages represent two different functional states of macrophages, allowing them to respond to different signals and play different roles in the immune system. The balance between M1 and M2 macrophages is important for maintaining immune homeostasis and promoting appropriate immune responses. Drugs that can inhibit M1 macrophages, promote M2 macrophages, and / or repolarize M1 to M2 macrophages may be able to treat digestive diseases including gastroparesis.

[0009] Because the signs and symptoms of gastroparesis overlap with other GI conditions, gastroparesis is sometimes misdiagnosed as intestinal obstruction, functional dyspepsia, irritable bowel syndrome, or peptic ulcer disease. In patients with signs and symptoms of gastroparesis, the finding of DGE in the absence of obstruction or an alternative diagnosis provides critical support for the diagnosis of gastroparesis, which can be confirmed using gastric emptying scintigraphy, gastric emptying breath testing, or SmartPill TM Motion test system for evaluation.

[0010] Approved drugs for treating gastroparesis can be divided into two different groups. The first group of drugs only temporarily relieves some of the disease symptoms, such as nausea and vomiting, and includes diphenhydramine and ondansetron as well as prochlorperazine. The second group of drugs is used to stimulate the stomach muscles and includes metoclopramide (e.g. ) and erythromycin. However, both metoclopramide and erythromycin carry the risk of serious side effects. Clearly, there is an urgent need to develop safe and effective therapies to treat patients with gastroparesis.

[0011] Thiazolidinediones represent a novel therapeutic strategy that may slow, stop, or reverse the underlying disease processes involved in digestive disorders such as gastroparesis. Summary of the invention

[0012] Disclosed herein is a method for treating a GI disease, comprising administering to a subject in need thereof an effective amount of a thiazolidinedione selected from one or more of pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, riglitazone, nyglitazone, riglitazone, troglitazone, and balaglitazone. In some embodiments, the thiazolidinedione is lobeglitazone. In some embodiments, the gastrointestinal disease is gastroparesis. In some embodiments, gastroparesis is idiopathic gastroparesis, diabetic gastroparesis, postoperative gastroparesis, or drug-induced gastroparesis.

[0013] Lobeglitazone is a thiazolidinedione with the chemical name 5-(4-(2-((6-(4-methoxyphenoxy)pyrimidin-4-yl)(yl)amino)ethoxy)benzyl)thiazolidine-2,4-dione. Lobeglitazone is approved as an antidiabetic drug under the brand name As an agonist of PPARα and PPARγ, it acts as an insulin sensitizer by binding to PPAR receptors in adipocytes and making the cells more sensitive to insulin. Lobeglitazone exhibits much higher potency than pioglitazone in PPARγ activity. The chemical structure of lobeglitazone is as follows:

[0014]

[0015] Gastroparesis is a condition characterized by delayed gastric emptying (DGE) in the absence of mechanical obstruction. Symptoms are chronic with intermittent exacerbations. Idiopathic gastroparesis accounts for the largest number of cases and affects primarily young adult females. Gastroparesis is also frequently associated with diabetes (diabetic gastroparesis), which may occur due to impaired neural control of gastric motility. Macrophages play a role in the development and progression of gastroparesis.

[0016] In some embodiments, the GI disease affects a digestive organ or tissue selected from the group consisting of the oral cavity, pharynx (throat), esophagus, stomach, small intestine, large intestine, rectum, anus, salivary glands, liver, gall bladder, and pancreas.

[0017] In some embodiments, the method comprises treating a cell of the GI system selected from absorptive cells (enterocytes), goblet cells, pancreatic islet cells, enteroendocrine cells, hepatocytes, Paneth cells, fenestrated liver endothelial cells, Kupffer cells, serous cells, gastric chief cells, mucous cells, smooth muscle cells, gastric parietal cells, myoepithelial cells, stem cells, gastric surface mucous cells, pancreatic acinar cells, taste buds, interstitial cell (ICC) types of Cajal, and neuronal cells.

[0018] In some embodiments, the cell is an animal cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is treated in vitro. In some embodiments, the cell is treated ex vivo. In some embodiments, the cell is treated in vivo.

[0019] In some embodiments, the cell is located in or from a subject suffering from or at risk of suffering from a gastrointestinal disease or disorder. One or more selected from the group consisting of abdominal adhesions, acid reflux in adults (gastroesophageal reflux disease or GERD), acid reflux in infants (GERD), lower gastrointestinal tract anatomy problems, appendicitis, Barrett's esophagus, bowel control problems (fecal incontinence), celiac disease, colon polyps, constipation, Crohn's disease, cyclic vomiting syndrome, diarrhea, diverticulosis and diverticulitis, dumping syndrome, food poisoning, gallstones, flatulence, gastritis, gastroparesis, GI bleeding, hemorrhoids, digestive disorders (dyspepsia), inguinal hernia, intestinal pseudo-obstruction, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), lactose intolerance, liver disease, microscopic colitis, enterostomy, pancreatitis, peptic ulcer (gastric ulcer), proctitis, short bowel syndrome, ulcerative colitis, viral gastroenteritis, Zollinger-Ellison syndrome, or conditions related thereto. In some embodiments, the disease or disorder is gastroparesis. In some embodiments, the gastroparesis is idiopathic gastroparesis, diabetic gastroparesis, postoperative gastroparesis, or drug-induced gastroparesis.

[0020] Also disclosed herein is the use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, neglitazone, riglitazone, troglitazone, and balaglitazone to alleviate one or more signs and / or symptoms of gastroparesis. In some embodiments, the thiazolidinedione is lobeglitazone. In some embodiments, the signs and symptoms of gastroparesis are measured by the ANMS GCSI-DD. In some embodiments, the signs and symptoms of gastroparesis are measured by the change in GCSI-DD. In some embodiments, the signs and symptoms of gastroparesis are measured by the change in GCSI-DD score from baseline to 4 weeks of treatment. In some embodiments, the signs and symptoms of gastroparesis are measured by the change in GCSI-DD score from baseline to 8 weeks of treatment. In some embodiments, the signs and symptoms of gastroparesis are measured by the change in GCSI-DD score from baseline to 12 weeks of treatment. In some embodiments, signs and symptoms of gastroparesis are measured by the change in GCSI-DD score from baseline to 1 year of treatment. In some embodiments, signs and symptoms of gastroparesis are measured by the change in GCSI-DD score from baseline to more than 1 year of treatment.

[0021] Also disclosed herein is the use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, neglitazone, riglitazone, troglitazone and balaglitazone to reduce the severity of nausea in gastroparesis subjects. In some embodiments, the thiazolidinedione is roglitazone. In some embodiments, the reduction in nausea severity is measured by the change in nausea severity from baseline to 4 weeks of treatment. In some embodiments, the reduction in nausea severity is measured by the change in nausea severity from baseline to 8 weeks of treatment. In some embodiments, the reduction in nausea severity is measured by the change in nausea severity from baseline to 12 weeks of treatment. In some embodiments, the reduction in nausea severity is measured by the change in nausea severity from baseline to 1 year of treatment.

[0022] Also disclosed herein is the use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, riglitazone, neglitazone, riglitazone, troglitazone and balaglitazone to reduce the severity of early satiety in gastroparesis subjects. In some embodiments, the thiazolidinedione is roglitazone. In some embodiments, the reduction in the severity of early satiety is measured by the change in the severity of early satiety from baseline to 4 weeks of treatment. In some embodiments, the reduction in the severity of early satiety is measured by the change in the severity of early satiety from baseline to 8 weeks of treatment. In some embodiments, the reduction in the severity of early satiety is measured by the change in the severity of early satiety from baseline to 12 weeks of treatment. In some embodiments, the reduction in the severity of early satiety is measured by the change in the severity of early satiety from baseline to 1 year of treatment. In some embodiments, the reduction in the severity of early satiety is measured by the change in the severity of early satiety from baseline to more than 1 year of treatment.

[0023] Also disclosed herein is the use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, neglitazone, riglitazone, troglitazone and balaglitazone to reduce the severity of postprandial fullness in gastroparesis subjects. In some embodiments, the thiazolidinedione is roglitazone. In some embodiments, the reduction in the severity of postprandial fullness is measured by the change in the severity of postprandial fullness from baseline to 4 weeks of treatment. In some embodiments, the reduction in the severity of postprandial fullness is measured by the change in the severity of postprandial fullness from baseline to 8 weeks of treatment. In some embodiments, the reduction in the severity of postprandial fullness is measured by the change in the severity of postprandial fullness from baseline to 12 weeks of treatment. In some embodiments, the reduction in the severity of postprandial fullness is measured by the change in the severity of postprandial fullness from baseline to 1 year of treatment. In some embodiments, the reduction in severity of postprandial fullness is measured by the change in severity of postprandial fullness from baseline to a treatment period of more than 1 year.

[0024] Also disclosed herein is the use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, riglitazone, neglitazone, riglitazone, troglitazone and balaglitazone to reduce the severity of upper abdominal pain in gastroparesis subjects. In some embodiments, the thiazolidinedione is lobeglitazone. In some embodiments, the reduction in the severity of upper abdominal pain is measured by the change in the severity of upper abdominal pain from baseline to 4 weeks of treatment. In some embodiments, the reduction in the severity of upper abdominal pain is measured by the change in the severity of upper abdominal pain from baseline to 8 weeks of treatment. In some embodiments, the reduction in the severity of upper abdominal pain is measured by the change in the severity of upper abdominal pain from baseline to 12 weeks of treatment. In some embodiments, the reduction in the severity of upper abdominal pain is measured by the change in the severity of upper abdominal pain from baseline to 1 year of treatment. In some embodiments, the reduction in upper abdominal pain severity is measured by the change in upper abdominal pain severity from baseline to a treatment period of more than 1 year.

[0025] Also disclosed herein is the use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, neglitazone, riglitazone, troglitazone, and balaglitazone to reduce the frequency of vomiting attacks in gastroparesis subjects. In some embodiments, the thiazolidinedione is roglitazone. In some embodiments, the reduction in the severity of vomiting attacks is measured by the change in the severity of vomiting attacks from baseline to 4 weeks of treatment. In some embodiments, the reduction in the severity of vomiting attacks is measured by the change in the severity of vomiting attacks from baseline to 8 weeks of treatment. In some embodiments, the reduction in the severity of vomiting attacks is measured by the change in the severity of vomiting attacks from baseline to 12 weeks of treatment. In some embodiments, the reduction in the severity of vomiting attacks is measured by the change in the severity of vomiting attacks from baseline to 1 year of treatment. In some embodiments, the reduction in the severity of vomiting attacks is measured by the change in the severity of vomiting attacks from baseline to more than 1 year of treatment.

[0026] Also disclosed herein is the use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, riglitazone, neglitazone, riglitazone, troglitazone, and balaglitazone to reduce the overall severity of gastroparesis. In some embodiments, the thiazolidinedione is lobeglitazone. In some embodiments, the reduction in the overall severity of gastroparesis is measured by the change in severity of the overall severity of gastroparesis from baseline to 4 weeks of treatment. In some embodiments, the reduction in the overall severity of gastroparesis is measured by the change in severity of the overall severity of gastroparesis from baseline to 8 weeks of treatment. In some embodiments, the reduction in the overall severity of gastroparesis is measured by the change in severity of the overall severity of gastroparesis from baseline to 12 weeks of treatment. In some embodiments, the reduction in the overall severity of gastroparesis is measured by the change in severity of the overall severity of gastroparesis from baseline to 1 year of treatment. In some embodiments, the reduction in the overall severity of gastroparesis is measured by the change in severity of the overall severity of gastroparesis from baseline to a treatment period greater than 1 year.

[0027] Also disclosed herein is the use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, riglitazone, nyglitazone, riglitazone, troglitazone and balaglitazone to slow down, stop or reverse the progression of GI disease. In some embodiments, the thiazolidinedione is roglitazone.

[0028] Also disclosed herein is a method for treating a mammal GI disease, comprising administering to a mammal an effective amount of a thiazolidinedione selected from one or more of pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, riglitazone, nyglitazone, riglitazone, troglitazone, and balaglitazone. In some embodiments, the GI disease is gastroparesis or IBD. In some embodiments, the thiazolidinedione is roglitazone.

[0029] In some embodiments, the gastroparesis is idiopathic gastroparesis, diabetic gastroparesis, postoperative gastroparesis, or drug-induced gastroparesis.

[0030] Also disclosed herein are methods of inhibiting M1 macrophage differentiation in an individual, comprising administering to the individual an effective amount of roglitazone. In some embodiments, due to the inhibition of M1 macrophage differentiation, a GI system disorder in the individual is treated.

[0031] Also disclosed herein is a method of promoting M1 macrophage differentiation in an individual, comprising administering to the individual an effective amount of pioglitazone or rosiglitazone.

[0032] Also disclosed herein is a method for promoting the differentiation of M2 macrophages in an individual, comprising administering an effective amount of rosiglitazone, pioglitazone or rosiglitazone to the individual. In some embodiments, due to promoting the differentiation of M2 macrophages, the GI system disorders of the individual are treated. Also disclosed herein is a method for promoting the repolarization of M1-to-M2 macrophages in an individual, comprising administering an effective amount of rosiglitazone, pioglitazone or rosiglitazone to the individual. In some embodiments, due to promoting the repolarization of M1-to-M2 macrophages, the GI system disorders of the individual are treated. In some embodiments, the thiazolidinedione is rosiglitazone. In some embodiments, the GI system disorder is selected from abdominal adhesions, gastric acid reflux (GERD) in adults, gastric acid reflux (GERD) in infants, lower GI tract anatomy problems, appendicitis, Barrett's esophagus, bowel control problems (fecal incontinence), celiac disease, colon polyps, constipation, Crohn's disease, cyclic vomiting syndrome, diarrhea, diverticulosis and diverticulitis, dumping syndrome, food poisoning, gallstones, flatulence, gastritis, gastroparesis, GI bleeding, hemorrhoids, digestive disorders (dyspepsia), inguinal hernia, intestinal pseudo-obstruction, IBD, IBS, lactose intolerance, liver disease, microscopic colitis, enterostomy, pancreatitis, peptic ulcer (gastric ulcer), proctitis, short bowel syndrome, ulcerative colitis, viral gastroenteritis, Zollinger-Ellison syndrome, or a disease related thereto. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Depicted is the experimental timeline for the study of roglitazone in a mouse inflammatory bowel disease (IBD) model.

[0034] Figure 2 The animal body weights (mpk means mg / kg) of the untreated group, 5% dextran sulfate sodium (DSS) model group (Model), roglitazone group and cyclosporine A group are depicted. *p<0.05, **p<0.01 vs. model control group.

[0035] Figure 3 The changes in animal body weight of the untreated group, 5% DSS model group, roglitazone group, and cyclosporine A group are depicted. *p<0.05, **p<0.01 vs. model control group.

[0036] Figure 4 The disease activity index (DAI) scores of the untreated group, 5% DSS model group, roglitazone group, and cyclosporine A group are depicted. **p<0.01, ***p<0.001 vs. the model control group.

[0037] Figure 5The post-treatment colon weights of the untreated group, 5% DSS model group, roglitazone group, and cyclosporine A group are depicted.

[0038] Figure 6 The colon lengths after treatment of the untreated group, 5% DSS model group, roglitazone group, and cyclosporine A group are depicted. *p<0.05, ***p<0.001 vs. the model control group.

[0039] Figure 7 The colon weight / length ratio after treatment in the untreated group, 5% DSS model group, lobeglitazone group, and cyclosporine A group is depicted. *p<0.05 vs. model control group.

[0040] Figure 8 The TNF-α gene expression in colon after treatment in untreated group, 5% DSS model group, lobeglitazone group and cyclosporine A group is depicted. *p<0.05, ***p<0.001 vs. model control group.

[0041] Fig. 9 The IL-10 gene expression in the colon of the untreated group, 5% DSS model group, lobeglitazone group, and cyclosporine A group after treatment was depicted.

[0042] Fig.10 The CCL2 gene expression in the colon after treatment of the untreated group, 5% DSS model group, roglitazone group, and cyclosporine A group is depicted. *p<0.05, **p<0.01 vs. the model control group.

[0043] Fig.11 The IL-1β gene expression in the colon after treatment in the untreated group, 5% DSS model group, roglitazone group, and cyclosporine A group is depicted. ***p<0.001 vs. model control group.

[0044] Fig.12 Post-treatment inflammation / tissue damage scores (total hematoxylin and eosin (H&E) scores) of the untreated group, 5% DSS model group, roglitazone group, and cyclosporine A group are depicted. **p<0.01, **p<0.001 vs. model control group.

[0045] Fig.13 The goblet cell numbers after treatment in the untreated group, 5% DSS model group, roglitazone group, and cyclosporine A group are depicted (periodic acid-Schiff (PAS) staining). *p<0.05 vs. model control group.

[0046] Fig.14 Expression of the cytokine IL-23 by M1 macrophages after differentiation with rosiglitazone, pioglitazone, or rosiglitazone is depicted.

[0047] Fig.15Expression of the cytokine IL-1b by M1 macrophages after differentiation with rosiglitazone, pioglitazone, or rosiglitazone is depicted.

[0048] Fig.16 Expression of the cytokine TNF-alpha (TNF-alpha) by M1-like macrophages after differentiation with roglitazone, pioglitazone, or rosiglitazone is depicted.

[0049] Fig.17 Expression of the cytokine IL-12p70 by M1-like macrophages after differentiation with rosiglitazone, pioglitazone, or rosiglitazone is depicted.

[0050] Fig.18 Depicted is the expression of the cytokine IL-6 by M1-like macrophages after differentiation with rosiglitazone, pioglitazone, or rosiglitazone.

[0051] Fig.19 Depicted are the expression of M2 macrophage surface markers following treatment with rosiglitazone, pioglitazone, or rosiglitazone (Donor 1).

[0052] Fig. 20 Depicted are the expression of M2 macrophage surface markers following treatment with rosiglitazone, pioglitazone, or rosiglitazone (Donor 2).

[0053] Fig.21 Depicted are the expression of M2 macrophage surface markers following treatment with rosiglitazone, pioglitazone, or rosiglitazone (Donor 3).

[0054] Fig. 22 Depicted is the expression of M1 macrophage-related cytokines in cells from three donors following treatment with rosiglitazone, pioglitazone, or rosiglitazone.

[0055] Fig.23 Depicted is the expression of M2 macrophage-associated cytokines in cells from three donors following treatment with rosiglitazone, pioglitazone, or rosiglitazone.

[0056] Fig.24 Depicted is the expression of macrophage-derived chemokine (MDC) in cells from three donors following treatment with rosiglitazone, pioglitazone, or rosiglitazone.

[0057] Fig.25 Depicted is the expression of Eotaxin-2 in cells from three donors following treatment with rosiglitazone, pioglitazone, or rosiglitazone.

[0058] Fig.26 Depicted is the expression of IL-1RA in cells from three donors following treatment with rosiglitazone, pioglitazone, or rosiglitazone.

[0059] Fig. 27 Depicted are the expression of M2 macrophage surface markers following treatment with rosiglitazone, pioglitazone, or rosiglitazone (Donor 1).

[0060] Fig.28 The expression of M1 macrophage-related cytokines after treatment with rosiglitazone, pioglitazone, or rosiglitazone is depicted.

[0061] Fig.29 The expression of M2 macrophage-related cytokines after treatment with rosiglitazone, pioglitazone, or rosiglitazone is depicted.

[0062] Fig.30 The expression of MDC after treatment with rosiglitazone, pioglitazone, or rosiglitazone is depicted.

[0063] Fig.31 The expression of Eotaxin-2 after treatment with rosiglitazone, pioglitazone, or rosiglitazone is depicted.

[0064] Fig.32 IL-1RA expression after treatment with rosiglitazone, pioglitazone, or rosiglitazone is depicted. DETAILED DESCRIPTION

[0065] Disclosed herein are methods of treating the GI diseases or conditions disclosed herein with thiazolidinediones.

[0066] The term "thiazolidinedione" refers to a class of heterocyclic glitazone compounds comprising a five-membered C3NS ring, including its prodrugs, salts, solvates, hydrates, cocrystals, enantiomers and deuterated forms. As used herein, thiazolidinedione includes but is not limited to one or more of pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, riglitazone, nyglitazone, riglitazone, troglitazone, balaglitazone and other thiazolidinedione molecules. In some embodiments disclosed herein, the method includes the use of any thiazolidinedione. In some embodiments, the thiazolidinedione is roglitazone. In some embodiments, the method explicitly excludes the use of one or more thiazolidinediones disclosed herein. In some embodiments, the thiazolidinedione is not pioglitazone. In some embodiments, the thiazolidinedione is not pioglitazone.

[0067] Roglitazone ( Chong Kun Dang) is a thiazolidinedione with the chemical name 5-(4-(2-((6-(4-methoxyphenoxy)pyrimidin-4-yl)(methyl)amino)ethoxy)benzyl)thiazolidine-2,4-dione. As an agonist of peroxisome proliferator-activated receptor-α (PPARα) and γ (PPARγ), roglitazone acts as an insulin sensitizer by binding to PPAR receptors in adipocytes and making the cells more sensitive to insulin.

[0068] Pioglitazone ( Takeda) is a thiazolidinedione with the chemical name (±)-5-[p-[2-(5-ethyl-2-pyridinyl)ethoxy]benzyl]-2,4-thiazolidinedione monohydrochloride. Pioglitazone is a potent and highly selective PPARγ agonist that increases muscle and adipose tissue sensitivity to insulin and inhibits hepatic gluconeogenesis. Pioglitazone improves glycemic control while reducing circulating insulin levels.

[0069] Rosiglitazone ( GlaxoSmithKline) is a thiazolidinedione with the chemical name (RS)-5-[4-(2-[methyl(pyridin-2-yl)amino]ethoxy)benzyl]thiazolidine-2,4-dione. Like other thiazolidinediones, rosiglitazone's mechanism of action is through activation of intracellular PPAR, especially PPARγ. Rosiglitazone is a selective ligand for PPARγ and has no PPARα binding effect.

[0070] As used herein, the terms "treat," "treating," or "treatment" refer to alleviating, reducing, or eliminating one or more symptoms or features of a disease, and may cure, alleviate, prevent, or slow the progression of a disease.

[0071] The term "effective amount" refers to an amount that can lead to a reduction in digestive system damage (if applicable or specified) and produce a desired effect or result. The term "therapeutically effective amount" refers to the amount of a thiazolidinedione, including but not limited to pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, nyglitazone, riglitazone, troglitazone, balaglitazone and other thiazolidinedione molecules, alone or in combination with other active ingredients, can induce a desired biological or pharmacological response, such as effectively preventing, alleviating or improving the symptoms of a disease or condition; slowing down, stopping or reversing the underlying disease process or progression; partially or completely restoring cell function; or prolonging the survival of a subject receiving treatment. In some embodiments, the thiazolidinedione is not pioglitazone.

[0072] The term "patient" or "subject" includes mammals, including non-human mammals and humans. In one embodiment, the patient or subject is a human.

[0073] The term "significant" or "significantly" was determined by t-test at a significance level of 0.05.

[0074] The present methods are based on the surprising discovery that thiazolidinediones can significantly protect the digestive system from damage, as evidenced by inflammation. Accordingly, disclosed herein is the use of one or more thiazolidinediones for treating GI disorders.

[0075] As used herein, the terms "digestive" and "gastrointestinal" are used interchangeably and refer to the organs, cells, tissues, and diseases associated with the gastrointestinal (GI) tract, including, but not limited to, the mouth, pharynx (throat), esophagus, stomach, small intestine, large intestine, rectum, and anus, as well as the salivary glands, liver, pancreas, and gall bladder.

[0076] Digestive (gastrointestinal) disorders resulting from damage to the digestive system include, but are not limited to, abdominal adhesions, acid reflux in adults (gastroesophageal reflux disease or GERD), acid reflux in infants (GERD), lower gastrointestinal tract anatomy problems, appendicitis, Barrett's esophagus, bowel control problems (fecal incontinence), celiac disease, colon polyps, constipation, Crohn's disease, cyclic vomiting syndrome, diarrhea, diverticulosis and diverticulitis, dumping syndrome, food poisoning, gallstones, flatulence, gastritis, gastroparesis, GI bleeding, hemorrhoids, digestive disorders (dyspepsia), inguinal hernia, intestinal pseudo-obstruction, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), lactose intolerance, liver disease, microscopic colitis, enterostomy surgery, pancreatitis, peptic ulcer (stomach ulcer), proctitis, short bowel syndrome, ulcerative colitis, viral gastroenteritis, Zollinger-Ellison syndrome, or conditions related thereto.

[0077] Intestinal macrophages are key immune cells that maintain intestinal immune homeostasis and play a role in the pathogenesis of GI diseases such as inflammatory bowel disease. Depending on microenvironmental cues, macrophages polarize into two different phenotypes: classically activated (M1) or alternatively activated (M2) macrophages. For example, when the intestinal barrier function is impaired, endocytosis causes macrophages to be functionally biased toward the M2 subtype, producing cytokines, chemokines, and lipid mediators involved in repairing the intestinal mucosal barrier and maintaining homeostasis. Intestinal macrophages can produce a variety of cytokines and other soluble factors, including prostaglandin E2 (PGE2), bone morphogenetic protein 2 (BMP2), and WNT ligands, which stimulate epithelial cell expansion and participate in the enteric nervous system or intestinal mucosal barrier to help maintain tissue homeostasis. Intestinal macrophages promote antigen-specific CD4 + CD25 +Regulatory T (T-reg) cells proliferate to maintain tolerance, primarily by producing IL-10 to suppress unrestrained inflammation against harmless commensal microorganisms.

[0078] Infiltration and activation of macrophages can engulf pathogens and produce various cytokines in specific situations, and cooperate with different immune cells in many aspects of the pathogenesis of GI diseases. Imbalance in macrophage polarization leads to the exacerbation of GI diseases, and the production of specific cytokines and / or chemokines depends on the ratio of pro-inflammatory M1 and anti-inflammatory M2 subsets. Therefore, targeted therapy of macrophages is a new option to regulate the immune microenvironment and reshape intestinal tissue.

[0079] Gastroparesis is a condition characterized by delayed gastric emptying (DGE) in the absence of mechanical obstruction. Symptoms are chronic with intermittent exacerbations. Idiopathic gastroparesis accounts for the largest number of cases and primarily affects young adult women. Gastroparesis is also frequently associated with diabetes (diabetic gastroparesis), which may occur due to impaired neural control of gastric motility. Macrophages play a role in the development and progression of gastroparesis. Specifically, macrophages infiltrate the smooth muscle layer of the stomach in patients with gastroparesis and release pro-inflammatory molecules, leading to gastric muscle damage and dysfunction. There are two different subtypes of macrophages that play different roles in the immune system, namely M1 and M2 macrophages. M1 macrophages participate in inflammatory responses and are activated in response to pro-inflammatory signals. M2 macrophages produce anti-inflammatory cytokines and growth factors that help repair damaged tissues and promote angiogenesis. Overall, M1 and M2 macrophages represent two different functional states of macrophages, allowing them to respond to different signals and play different roles in the immune system. The balance between M1 and M2 macrophages is important for maintaining immune homeostasis and promoting appropriate immune responses. Drugs that can inhibit M1 macrophages, promote M2 macrophages, and / or repolarize M1-M2 macrophages may be able to treat digestive diseases including gastroparesis.

[0080] Therefore, the present disclosure also provides the use of thiazolidinediones in inhibiting or promoting M1 macrophage differentiation and / or promoting M2 macrophage differentiation. In some embodiments, the thiazolidinedione is lobeglitazone.

[0081] Signs and symptoms of gastroparesis are nausea, vomiting, postprandial fullness, early satiety, and epigastric pain. Patients may experience any combination of signs and symptoms that vary in severity. Pain is more prevalent in patients with idiopathic gastroparesis than in patients with diabetic gastroparesis. Glycemic control may be further disturbed in patients with diabetic gastroparesis due to unpredictable gastric emptying and altered absorption of oral glucose-lowering medications.

[0082] Understanding the symptoms associated with gastroparesis is important in treating patients with this condition. In gastroparesis, symptom experience and severity are obtained from the patient. Therefore, patient-reported symptom scales are needed to capture overall gastroparesis severity to evaluate treatments for gastroparesis. Well-defined patient-reported outcome (PRO) instruments that measure clinically important signs and symptoms of gastroparesis would be a useful assessment tool for clinical trials to support label claims for treatments for gastroparesis. The American Society of Neurogastroenterology and Motility Gastroparesis Cardinal Symptom Index-Daily Diary (ANMS GCSI-DD) is a patient-reported outcome instrument that captures daily symptoms associated with gastroparesis. The ANMS GCSI-DD is designed to assess GI symptoms associated with idiopathic and diabetic gastroparesis. The ANMS GCSI-DD is designed to assess symptoms associated with idiopathic and diabetic gastroparesis. The ANMS GCSI-DD includes 5 items, namely, nausea, vomiting, early satiety, postprandial fullness, and epigastric pain. Four of the items (nausea, early satiety, postprandial fullness, and epigastric pain) are rated on a scale of none (0), mild (1), moderate (2), severe (3), and very severe (4) according to the worst severity of the symptom in the past 24 hours. Vomiting is assessed as the number of vomiting episodes in the past 24 hours, with a maximum upper limit of 4 (range 0 to 4). The ANMS GCSI-DD total score is a useful patient-reported outcome for gastroparesis and can be used as an endpoint in clinical trials of gastroparesis.

[0083] In some aspects, the disclosure provides a method of exerting a protective effect in a cell, comprising contacting the cell with an effective amount of a thiazolidinedione. As used herein, the term "effective amount" refers to the amount of a thiazolidinedione that will result in a desired effect or outcome, such as an amount that will result in a protective effect.

[0084] In some aspects, the present disclosure provides a method of reducing inflammation, comprising the step of contacting a cell with an effective amount of a thiazolidinedione.

[0085] In some aspects, the present disclosure provides a method of increasing the lifespan of a cell, comprising the step of contacting the cell with an effective amount of a thiazolidinedione.

[0086] In some embodiments, the cell is an animal cell, such as a mammalian cell. In some embodiments, the cell is a human cell or a non-human cell. In some embodiments, the cell is treated in vitro, in vivo, or ex vivo. In some embodiments, the cell is a diseased cell. In some embodiments, the cell is a diseased cell from a patient suffering from a disease or condition disclosed herein.

[0087] Also disclosed herein is a method for treating a mammal suffering from a disease or condition that would benefit from a protective effect on cells, or for preventing or reducing the risk of a mammal developing a disease or condition, the method comprising the step of administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising a thiazolidinedione. In some embodiments, the mammal is a human or non-human mammal. In some embodiments, the mammal is a human. In some embodiments, the disease or condition is caused by damage that reduces digestive system function. In some embodiments, the disease is selected from one or more digestive system diseases or conditions associated therewith. In some embodiments, the disease is gastroparesis. In some embodiments, the disease is idiopathic gastroparesis, diabetic gastroparesis, or media-induced gastroparesis.

[0088] Also disclosed herein are methods of treating a disease or condition that causes damage or inflammation to the digestive system of a mammal, or methods of extending the lifespan of cells in a patient suffering from a disease or condition that causes damage to the digestive system.

[0089] In some embodiments, the method comprises the step of administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising a thiazolidinedione. In some embodiments, the mammal is a human or a non-human mammal. In one embodiment, the disease or condition is selected from, but not limited to, abdominal adhesions, acid reflux in adults (gastroesophageal reflux disease or GERD), acid reflux in infants (GERD), lower gastrointestinal anatomical problems, appendicitis, Barrett's esophagus, bowel control problems (fecal incontinence), celiac disease, colon polyps, constipation, Crohn's disease, cyclic vomiting syndrome, diarrhea, diverticular disease and diverticulitis, dumping syndrome, food poisoning, gallstones, flatulence, gastritis, gastroparesis, gastrointestinal bleeding, hemorrhoids, digestive disorders (dyspepsia), inguinal hernia, intestinal pseudo-obstruction, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), lactose intolerance, liver disease, microscopic colitis, enterostomy, pancreatitis, peptic ulcer (gastric ulcer), proctitis, short bowel syndrome, ulcerative colitis, viral gastroenteritis, Zollinger-Ellison syndrome, or a condition related thereto.

[0090] Also disclosed herein are methods of treating gastroparesis. In some embodiments, gastroparesis includes idiopathic gastroparesis, diabetic gastroparesis, postoperative gastroparesis, and drug-induced gastroparesis. In some embodiments, primary gastroparesis is idiopathic. In some embodiments, secondary gastroparesis is caused by a disease such as diabetes, cancer, or infection, or a side effect of a drug or surgery.

[0091] In another aspect, provided herein are methods of reducing GI tract inflammation to treat digestive disorders such as gastroparesis and IBD.

[0092] In another aspect, provided herein are methods of reducing the risk of nausea, early satiety, postprandial fullness, epigastric pain, vomiting episodes, and overall severity of gastroparesis.

[0093] In another aspect, provided herein are methods of slowing, halting or reversing disease progression to digestive disorders such as gastroparesis and IBD.

[0094] In another aspect, provided herein are methods of slowing, halting or reversing disease progression as indicated by nausea, early satiety, postprandial fullness, epigastric pain, vomiting episodes and overall severity of gastroparesis.

[0095] In another aspect, disclosed herein is a method of treating a mammal suffering from a disease or condition, the symptoms of which can be prevented, alleviated or ameliorated by cytoprotection; or the disease process or progression can be slowed, stopped or reversed by cytoprotection; the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a thiazolidinedione. In some embodiments, the thiazolidinedione is lobeglitazone.

[0096] In a related aspect, disclosed herein are methods of treating gastroparesis. In one embodiment, gastroparesis includes idiopathic gastroparesis, diabetic gastroparesis, postoperative gastroparesis, and drug-induced gastroparesis.

[0097] The disclosure further provides for the use of a thiazolidinedione in the preparation of a medicament for treating a human suffering from any of the diseases or conditions disclosed herein or for use in any of the methods of the disclosure involving administering a thiazolidinedione to a human.

[0098] The pharmaceutical composition of the present disclosure comprises a therapeutically effective amount of a thiazolidinedione and at least one pharmaceutically acceptable excipient. The term "excipient" refers to a pharmaceutically acceptable inactive substance used as a carrier of the pharmaceutically active ingredient thiazolidinedione, including anti-adherents, adhesives, coatings, disintegrants, fillers, diluents, solvents, flavoring agents, expanders, colorants, glidants, dispersants, wetting agents, lubricants, preservatives, adsorbents and sweeteners. The choice of excipient will depend on factors such as the specific mode of administration and the nature of the dosage form. Solutions or suspensions for injection or infusion may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate or phosphate, and an agent for regulating tonicity, such as sodium chloride or glucose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.The parenteral preparation can be enclosed in ampoules, disposable syringes (including autoinjectors) or multiple dose vials made of glass or plastic.

[0099] The pharmaceutical preparation of the present disclosure can be any pharmaceutical dosage form. The pharmaceutical preparation can be, for example, a tablet, a capsule, a nanoparticle material (e.g., a granular particle material or a powder), a freeze-dried material for reconstruction, a liquid solution, a suspension, an emulsion or other liquid form, an injectable suspension, a solution, an emulsion, etc., a suppository, or an external or transdermal preparation or a patch. The pharmaceutical preparation generally contains about 1% to about 99% by weight of a thiazolidinedione and 99% to 1% by weight of a suitable pharmaceutical excipient. In one embodiment, the dosage form is an oral dosage form. In another embodiment, the dosage form is a parenteral dosage form. In another embodiment, the dosage form is an enteral dosage form. In another embodiment, the dosage form is a topical dosage form. In one embodiment, the pharmaceutical dosage form is a unit dose. The term "unit dose" refers to the amount of a thiazolidinedione given to a patient in a single dose.

[0100] In some embodiments, the pharmaceutical compositions disclosed herein are delivered to a subject parenterally, enterally, or topically.

[0101] Examples of parenteral routes suitable for the disclosed pharmaceutical compositions include, but are not limited to, one or more of the following: intraperitoneal, intraamniotic, intraarterial, intraarticular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracoronary, intracoronary, in vivo, intracranial, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intracavitary, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intraocular, intrasinus, intraspinal, intrasynovial, intratendonal, intratesticular, intrathecal, intrathoracic, intraductal, intratumoral, intratympanic, intrauterine, intravascular, intravenous (bolus or infusion), ventricular, intravesical, and / or subcutaneous. In some embodiments, the route of administration is not buccal or sublingual. In some embodiments, the route of administration is not nasal.

[0102] Enteral routes of administration include administration to the GI tract via the mouth (oral), stomach (gastric), and rectum (rectal). Gastric administration typically involves the use of a tube through the nasal passage (NG tube) or a tube in the esophagus that leads directly to the stomach (PEG tube). Rectal administration typically involves rectal suppositories.

[0103] Topical administration includes administration to a body surface, such as the skin or mucous membranes, including pulmonary administration. Transdermal forms include creams, foams, gels, lotions or ointments. Pulmonary forms include liquids and powders, such as liquid sprays.

[0104] Dosages may vary depending on the dosage form employed, sensitivity of the patient, and route of administration. Dosage and administration are adjusted to provide adequate levels of the active agent or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the general health of the subject, the age, weight and sex of the subject, diet, time and frequency of administration, drug combinations, reaction sensitivities, and tolerance / response to treatment.

[0105] In one embodiment, the daily dose of the thiazolidinedione administered to the patient is selected from up to 200 mg, 175 mg, 150 mg, 125 mg, 100 mg, 90 mg, 80 mg, 70 mg, 60 mg, 50 mg, 30 mg, 25 mg, 20 mg, 15 mg, 14 mg, 13 mg, 12 mg, 11 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, 1 mg, 0.9 mg, 0.8 mg, 0.7 mg, 0.6 mg, 0.5 mg, 0.45 mg, 0.4 mg, 0.3 mg, 0.2 mg, 0.1 mg, 0.08 mg, 0.05 mg, 0.03 mg, 0.02 mg or up to 0.01 mg. In another embodiment, the daily dose is at least 0.01 mg, 0.02 mg, 0.05 mg, 0.08 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 5 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, 2,000 mg, 3,000 mg, 4,000 mg or at least 5,000 mg.In another embodiment, the daily dose is 0.01-0.02 mg, 0.02-0.05 mg, 0.05-0.08 mg, 0.08-0.1 mg, 0.1-0.2 mg, 0.2-0.4 mg, 0.4-0.6 mg, 0.6-0.8 mg, 0.8-1 mg, 1-2 mg, 2-4 mg, 1-5 mg, 5-7.5 mg, 7.5-10 mg, 10-15 mg, 10-12.5 mg, 12.5-15 mg, 15-17.7 mg, 17.5-20 mg, 20-25 mg, 20-22.5 mg, 22.5-25 mg, 25-30 mg, 25-27.5 mg, 27.5-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, or 45-50 mg, 50-75 mg, 75-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 5-200 mg, 5-300 mg, 5-400 mg, 5-500 mg, 5-600 mg, 5-700 mg, 5-800 mg, 5-900 mg, 5-1,000 mg, 5-2,000 mg, 5-5,000 mg or more than 5,000 mg, or any range defined by these value pairs.

[0106] In another embodiment, the single dose of thiazolidinedione administered to the patient is selected from the group consisting of: 0.01 mg, 0.02 mg, 0.05 mg, 0.08 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 100 mg, 110 mg, 120 mg, 130 mg, 1 40mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 260 mg, 270mg, 280mg, 290mg, 300mg, 310mg, 320mg, 330mg, 340mg, 350mg, 360mg, 370mg, 380mg, In some embodiments, the thiazolidinedione is lobeglitazone.

[0107] As a non-limiting example, the dosage of a thiazolidinedione (e.g., lobeglitazone) administered orally can be about 0.01 to 50 mg per day, administered in divided doses. The single dose of a thiazolidinedione (e.g., lobeglitazone) administered by subcutaneous injection may be about 0.01-50 mg, preferably about 0.1-10 mg, 0.2-1 mg, 0.3-0.6 mg, or 0.5 mg, or any scope limited by these values. Other embodiments include the scope of about 0.05-5,000 mg, preferably about 0.1-10 mg, 0.2-5 mg, 0.3-1 mg, or 0.5 mg, or any scope limited by these values. For those patients who need to be divided into 10 or more doses of injection every day, subcutaneous infusion may be a better choice.

[0108] The fine particle dosage of thiazolidinedione (e.g., lobeglitazone) administered by pulmonary administration (e.g., inhalation using a pressurized metered dose inhaler (pMDI), dry powder inhaler (DPI), soft mist inhaler, nebulizer or other device) can be in the range of about 0.1-50 mg, preferably about 0.2-10 mg, 0.3-1 mg or 0.5 mg, or any range defined by these values. Other embodiments include a range of about 0.05-5,000 mg, preferably about 0.1-1,000 mg, 0.2-100 mg, 0.3-1 mg, 0.4-0.5 mg or 0.5 mg, or any range defined by these values. The nominal dose (ND) of a thiazolidinedione drug (e.g., lobeglitazone) administered by pulmonary administration, i.e., the amount of drug measured in the container (also referred to as a metered dose), can be, for example, in the range of 0.1-15 mg, 0.1-10 mg, 0.1-1 mg, 0.2-0.3 mg, 0.3-0.4 mg, 0.4-0.5 mg, 0.5-0.6 mg, 0.6-0.7 mg, 0.7-0.8 mg, 0.8-0.9 mg or 0.9-1 mg, or any range defined by these value pairs. Other embodiments include a range of about 0.05-5,000 mg, preferably about 0.1-1,000 mg, 0.2-10 mg, 0.3-1 mg, 0.4-0.5 mg or 0.5 mg, or any range defined by these value pairs.

[0109] Long-acting pharmaceutical compositions can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more than 10 times daily (preferably ≤ 10 times daily), every other day, every 3 to 4 days, weekly or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

[0110] In embodiments of any of the methods and compositions described above, the thiazolidinedione or salt, solvate, hydrate and cocrystal thereof is a racemic mixture of R and S enantiomers, or is enriched in the R enantiomer (i.e., the ratio of R to S enantiomer administered is from 1.1:1 to 1,000:1, 10:1 to 10,000:1, or 100:1 to 100,000:1, or all thiazolidinedione enantiomers in the composition are at least 98%). R enantiomer, 99% enantiomer, 99.5% enantiomer, 99.9% enantiomer, or do not have any observable amount of the S enantiomer), or is enriched in the S enantiomer (i.e., the ratio of S to R enantiomer is from 1.1:1 to 1,000:1, 10:1 to 10,000:1, or 100:1 to 100,000:1, or at least 98% S enantiomer, 99% enantiomer, 99.5% enantiomer, 99.9% enantiomer of all thiazolidinedione enantiomers in the composition, or do not have any detectable amount of the R enantiomer).

[0111] The present disclosure also provides an in vitro or ex vivo method of reducing cell damage, the method comprising the step of contacting the cell with an effective amount of a thiazolidinedione.

[0112] Suitably, the cell suffers from, is at risk of, or is at risk of developing a disease or condition selected from any one or more of the following: abdominal adhesions, acid reflux in adults (gastroesophageal reflux disease or GERD), acid reflux in infants (GERD), lower gastrointestinal anatomical problems, appendicitis, Barrett's esophagus, bowel control problems (fecal incontinence), celiac disease, colon polyps, constipation, Crohn's disease, cyclic vomiting syndrome, diarrhea, diverticular disease and diverticulitis, dumping syndrome, food poisoning, gallstones, flatulence, gastritis, gastroparesis, gastrointestinal bleeding, hemorrhoids, digestive disorders (dyspepsia), inguinal hernia, intestinal pseudo-obstruction, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), lactose intolerance, liver disease, microscopic colitis, colostomy surgery, pancreatitis, peptic ulcer (gastric ulcer), proctitis, short bowel syndrome, ulcerative colitis, viral gastroenteritis, Zollinger-Ellison syndrome, or a disease related thereto.

[0113] Suitably, in the methods, compositions and / or second medical uses of the compositions disclosed herein, the thiazolidinedione may be administered or formulated for administration at a dose of 0.01 mg or more. Suitably, the thiazolidinedione is administered at a dose of between 0.1-5000 mg / day.

[0114] Suitably, in the methods, compositions and / or second medical uses of the compositions disclosed herein, the thiazolidinedione may be administered or formulated for administration in any suitable manner, such as parenterally, enterally or topically.

[0115] Suitably, in the methods, compositions and / or second medical uses of the compositions disclosed herein, the thiazolidinedione may be administered or formulated for administration by oral, sublingual, buccal, pulmonary, intravenous, intramuscular or subcutaneous administration.

[0116] Another embodiment of the present disclosure includes the use of thiazolidinediones to reduce cell damage or increase cell survival.

[0117] Another embodiment of the present disclosure includes the use of a thiazolidinedione to treat gastroparesis or IBD.

[0118] Another embodiment of the present disclosure includes the use of a thiazolidinedione to reduce the core signs and symptoms of gastroparesis as measured by the ANMS GCSI-DD.

[0119] Another embodiment of the present disclosure includes the use of a thiazolidinedione to reduce the severity of nausea.

[0120] Another embodiment of the present disclosure includes the use of a thiazolidinedione to reduce the severity of premature satiety.

[0121] Another embodiment of the present disclosure includes the use of a thiazolidinedione to reduce the severity of postprandial fullness.

[0122] Another embodiment of the present disclosure includes the use of a thiazolidinedione to reduce the severity of upper abdominal pain.

[0123] Another embodiment of the present disclosure includes the use of a thiazolidinedione to reduce the severity of emetic episodes.

[0124] Another embodiment of the present disclosure includes the use of a thiazolidinedione to reduce the overall severity of gastroparesis.

[0125] Example

[0126] The scope of the present invention is not limited to the specific embodiments described herein. In fact, it will be apparent to those skilled in the art from the above description and accompanying drawings that various modifications of the present invention, except as described herein, are apparent. Such modifications are intended to fall within the scope of the appended claims.

[0127] It should also be understood that all values ​​are approximate and are used for descriptive purposes only.All references cited and discussed in this specification are incorporated herein by reference in their entirety to the same extent as if each reference was incorporated by reference individually.

[0128] Example 1: Efficacy of lobeglitazone in treating GI inflammation

[0129] Methodology

[0130] The aim of this study was to test the efficacy of the compound lobeglitazone in a DSS-induced IBD model in female Balb / c mice following oral administration. This study was performed at an AAALAC-accredited institution and all animal research procedures were approved by the Institutional Animal Care and Use Committee (IACUC).

[0131] This study was conducted according to the procedures outlined below.

[0132] Preparation of carriers and test articles

[0133] A total of 5 g of carboxymethylcellulose (CMC) powder was weighed out and transferred to a clean bottle, followed by the addition of 1 L of MilliQ water. A stirring bar was then added and the solution was stirred for 6 hours until clear. The 0.5% CMC was stored at 4°C for further use.

[0134] Weigh an accurate amount of lobeglitazone sulfate (lot number: EW34258-13-P1; purity: 97.6%) into a clean test tube, add 0.5% CMC and vortex thoroughly, then sonicate for 2-3 minutes to obtain a uniform suspension. The solution concentrations are 0.361 mg / mL, 0.120 mg / mL, and 0.036 mg / mL. The dosing solution is freshly prepared every day until the end of the study. The compound is administered orally (PO) at 10 mL / kg per day for 10 days.

[0135] Weigh 125.82 mg of cyclosporin A (CSA, purity 98%) into a clean test tube, add 2.055 mL of castor oil, then add 39.046 mL of 95% CMC (containing 0.1% Tween 80), vortex thoroughly, and ultrasonicate for 10 minutes to obtain a clear solution. The solution concentration is 3 mg / mL. The compound is taken orally, 10 mL / kg per day, for 10 consecutive days.

[0136] Preparation of 5% DSS: Weigh 5 g of DSS powder into a clean bottle, then add 1 L of Milli Q water and stir until the solution is clear. Then transfer the 5% DSS to the animal's water bottle. Prepare 5% DSS freshly every day until the end of the study.

[0137] Animal Husbandry

[0138] A total of 50 (7-week-old) female Balb / c were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were acclimatized for 1 week before the experiment. All in vivo experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC). All euthanasia was performed by carbon dioxide inhalation, and every effort was made to minimize animal suffering.

[0139] Group and dosage design

[0140] At the beginning of the study, randomization was performed based on body weight. All animals were randomly assigned to 1 of 6 groups (Table 1). Figure 1 shown.

[0141] Table 1. Study groups and dosage design

[0142]

[0143]

[0144] Animal health monitoring

[0145] During the study, the general condition (appearance and activity) of all animals was carefully monitored daily by veterinarians.

[0146] Disease Activity Index (DAI) score

[0147] DAI was assessed using the criteria listed in Table 2. The DAI score was calculated as the sum of the weight loss score, stool consistency score, and occult-gross bleeding score. Weight loss was calculated by comparison with the body weight on day 1. Fecal samples were collected from each animal and assessed for stool consistency and occult-gross bleeding according to Table 2. Fecal consistency was assessed by the appearance of the stool. Occult-gross bleeding was assessed using fecal occult blood test strips. Occult bleeding was scored as 1 if light purple or dark purple appeared within 20 seconds and more than 15 seconds. Occult bleeding was scored as 2 if light purple or dark purple appeared within 15 seconds. Occult-gross bleeding was scored as 3 if blood was visible in the stool.

[0148] Table 2. DAI scores.

[0149]

[0150] Sample collection

[0151] Animals were sacrificed with CO2 at the end of the study. Colon length and colon weight (excluding the cecum) were recorded, and macroscopic changes were scored according to Table 2. The colon was washed with saline and all feces were removed before weighing. The middle part of the colon was collected in a cassette and fixed in 10% neutral buffered formalin (NBF) for H&E and PAS staining. For the left colon, the mucosa was removed from the colon surface and then quickly frozen in liquid nitrogen and stored at -80°C for QT-PCR analysis of TNF-α, IL-10, CCL2 and IL-1β. QT-PCR primers were purchased from Sangon Biotech Co., Ltd.

[0152] Tissue processing before RNA extraction was as follows.

[0153] (a) Removal of mucosa from the colon surface. Cut colon tissue into small pieces on ice.

[0154] (b) Weigh no more than 30 mg of tissue and transfer the tissue to a tube of Lysis Matrix D. Immediately add a total of 700 μL of Trizol to the tissue and Lysis Matrix D.

[0155] (c) Place the Lysis Matrix D tube containing the tissue, lysis matrix, and 700 μL Trizol into the FastPrep TM -24 sample preparation system.

[0156] (d) Set to speed 6 and grind for 2 minutes

[0157] (e) Add 140 μL of chloroform to the tube and shake the tube vigorously by vortexing for 15 seconds. Incubate at room temperature for 2 minutes.

[0158] (f) Centrifugation at 12000 rpm for 10 minutes at 4°C.

[0159] (g) Transfer the supernatant to a 96-well 2.2 mL deep-well plate.

[0160] The RNA sample preparation procedure was as follows.

[0161] (a) Add 600 μL of Binding Buffer to columns 1 and 7.

[0162] (b) Add 600 μL DNase Stop Buffer to columns 2 and 8.

[0163] (c) Add 600 μL of Wash Buffer to columns 3, 4, 9, and 10.

[0164] (d) Add 100 μL of elution buffer to columns 5 and 11.

[0165] (e) Add 190 μL DEPC water and 10 μL Reagents

[0166] (f) Transfer 300 μL of the colorless upper phase containing RNA to columns 1 and 7.

[0167] (g) Place the test plate into the nucleic acid purification instrument

[0168] (h) Set up the instrument as follows according to Table 3.

[0169] Table 3.

[0170]

[0171]

[0172] The eluate was transferred to a clean nuclease-free microcentrifuge tube and the RNA samples were stored at -80 °C.

[0173] To use Takara Prime Script TM For reverse transcription systems, mix and briefly centrifuge each component before use. Mix the following components in the volumes listed in Table 4.

[0174] Table 4.

[0175] Reaction components Volume (μL) 5x gDNA Wipe Buffer 2 gDNA Eraser 1 Total RNA 1 μg x Nuclease-free water x Final volume 10

[0176] (a) Store on ice until adding the reverse transcription mix. Centrifuge for 10 seconds in a microcentrifuge. Heat in a 42°C heat block for 2 minutes and immediately cool in 4°C ice water for at least 5 minutes.

[0177] (b) Prepare the reverse transcription reaction mixture, 10 μL per cDNA reaction. Mix on ice in the order listed. Volumes of components for step b) (Table 5).

[0178] Table 5.

[0179] Reaction components Volume (μL) 5x PrimeScript Buffer 4 PrimeScript RT Enzyme Mix 1 RT Primer Mix 1 Nuclease-free water 4 Final volume 10

[0180] (c) Combine 20 μL of the reverse transcription mixture and centrifuge in a microcentrifuge for 10 seconds.

[0181] (d) Anneal in a heating block at 37°C for 15 min, 85°C for 5 s, and store in S100 at 4°C. TM on a thermal cycler.

[0182] (e) Aliquot the cDNA samples and store at -20°C.

[0183] QT-PCR Procedure

[0184] (a) Prepare qPCR reaction mixture in one tube according to the assay number and replicates.

[0185] (b) Prepare primers and master mix according to the manufacturer's instructions and dispense 9 μL of the mixture into each well of a 384 qPCR plate.

[0186] (c) Transfer 1 μL of cDNA to each well of a 384 qPCR plate containing 9 μL of the mixture. Briefly centrifuge the plate.

[0187] (d) Sealing the plate

[0188] (e) Mix thoroughly and swirl briefly

[0189] (f) Run PCR reaction

[0190] (g) Place the reaction plate into the instrument.

[0191] (h) Set up Applied Biosystems ViiA according to Table 6 TM 7 loop conditions.

[0192] Table 6.

[0193]

[0194] Statistics of real-time quantitative QT-PCR

[0195] Data are presented as mean ± SD. mRNA expression levels were analyzed using the 2-ΔΔCT method. Results were normalized to RNA input using glyceraldehyde-3-phosphate dehydrogenase (GAPDH or other internal controls). Statistical analysis was performed using one-way ANOVA, followed by post hoc Dunnett's test if significant. When N was too small or the data did not follow a Gaussian distribution, nonparametric tests such as the Mann-Whitney and Kruskal-Wallis tests were used. Differences were considered significant when p < 0.05.

[0196] Histological analysis

[0197] H&E staining: Colon sections were cut into 4 μm thickness, dried in an oven for 1 hour, and stained with H&E solution according to our standard protocol. Briefly, sections were stained with hematoxylin solution for 90 seconds. Then they were stained in eosin solution for 30 seconds. They were then dehydrated and coverslipped for subsequent image analysis. H&E staining images were scanned by GT450. The severity of inflammatory cell infiltration and tissue damage was semi-quantitatively evaluated according to the pathological scoring criteria in Table 7.

[0198] Table 7.

[0199] score Inflammatory cell infiltration Tissue damage 0 Occasional or no infiltration No mucosal damage 1 Infiltration into the lamina propria Localized crypt lesions 2 Infiltration into the submucosal layer Mucosal erosion or ulcer 3 Transmural infiltration Extensive damage affecting the submucosa

[0200] PAS staining: Colon sections were cut into 4 μm thickness, dried in an oven for 1 hour, and stained with a PAS kit (Gefan #M035) using our standard protocol. Briefly, sections were stained with periodic acid working solution for 8 minutes. Then washed with distilled water for 5 minutes, three times. Stained in Schiff solution for 10 minutes. Switched to distilled water for 5 minutes, three times. Then dehydrated and coverslipped for subsequent image analysis.

[0201] The entire length of the colon was measured with a ruler and then divided into three sections. 100 μm in the middle of each section was taken to count goblet cells. In each 100 μm of colon tissue, the number of goblet cells was counted, and the average number of goblet cells in the three sections was taken for the next analysis.

[0202] Data analysis

[0203] The data in the animals are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparisons. When N was too small or the data did not follow a Gaussian distribution, the nonparametric test of Whitney et al. was used. Differences were considered significant when p < 0.05.

[0204] result

[0205] Weight and weight change

[0206] like Figure 2 As shown in Figure 2, there was no significant difference in body weight among the groups. Figure 3 As shown, body weight changes were significantly reduced after treatment with 3.61 mg / kg roglitazone on day 2 and 30 mg / kg CSA on days 2, 3, 4, 5, and 6 compared to control (untreated) mice. No significant changes in body weight were observed after treatment with 1.20 mg / kg and 0.36 mg / kg roglitazone compared to control mice.

[0207] DAI Rating

[0208] like Figure 4 As shown, DSS treatment significantly increased the DAI of mice on days 7, 8, 9, and 10. Compared with the control group, 30 mg / kg CSA treatment significantly reduced the DAI of mice on days 8, 9, and 10. Roglitazone treatment did not significantly reduce the DAI scores of animals.

[0209] Colon length and colon weight

[0210] like Figure 5 As shown in Figure 2, DSS treatment did not significantly change colon weights. Treatment with 30 mg / kg CSA and roglitazone did not significantly change colon weights compared to the control group. Figure 6 As shown in Figure 2, DSS treatment significantly shortened colon length. Treatment with 30 mg / kg CSA significantly increased colon length compared to the control group. Treatment with roglitazone did not significantly increase colon length. Figure 7 As shown, DSS treatment significantly increased the colon length / weight ratio. Treatment with 30 mg / kg CSA significantly reduced the colon length / weight ratio compared to the control group. Treatment with roglitazone did not significantly reduce the colon length / weight ratio.

[0211] Gene expression levels in the colon

[0212] like Figure 8 As shown, the TNF-α gene expression level in the colon was significantly increased after DSS treatment compared to the untreated group. Treatment with roglitazone (0.36 and 1.20 mg / kg) significantly reduced the TNF-α gene expression level. Treatment with 30 mg / kg CSA reduced TNF-α expression. Treatment with roglitazone (3.61 mg / kg) did not show a significant change in TNF-α gene expression levels.

[0213] like Fig. 9 As shown, the IL-10 gene expression level was decreased after DSS treatment compared with the untreated group, while no significant changes were observed after roglitazone and 30 mg / kg CSA treatment.

[0214] like Fig.10 As shown, the CCL2 gene expression level in the colon was significantly increased after DSS treatment compared to the untreated group. The CCL2 gene expression level was significantly decreased after treatment with 3.61 mg / kg roglitazone and 30 mg / kg CSA. Treatment with roglitazone (0.36 and 1.20 mg / kg) resulted in a decrease in the CCL2 gene expression level.

[0215] like Fig.11 As shown in the figure, the IL-1β gene expression level in the colon was significantly increased after DSS treatment compared with the untreated group. The IL-1β gene expression level was significantly decreased after treatment with the compound roglitazone (0.36 and 1.20 mg / kg) and 30 mg / kg CSA, while the 3.61 mg / kg roglitazone treatment did not change the IL-1β gene expression level.

[0216] Colon H&E staining and scoring

[0217] The untreated group had intact colon mucosal structure and abundant goblet cells. The 5% DSS-induced model group showed severe colonic inflammatory cell infiltration, gland separation, crypt loss, mucosal structural destruction, and a significant increase in the total H&E score. The total H&E score was significantly reduced after treatment with 30 mg / kg CSA. Roglitazone (0.36, 1.20, and 3.61 mg / kg) treatment resulted in a decrease in the total H&E score ( Fig.12 ).

[0218] The number of goblet cells in the DSS-treated group was significantly reduced compared to the untreated group. After treatment with 30 mg / kg CSA, goblet cells increased significantly. Treatment with the compound roglitazone (0.36 and 1.20 mg / kg) tended to increase the number of goblet cells, while treatment with 3.61 mg / kg roglitazone did not change the number of goblet cells ( Fig.13 ).

[0219] in conclusion

[0220] Rosiglitazone was evaluated at 0.36 and 1.20 and 3.61 mg / kg in a mouse IBD model (DSS model). The results showed that treatment with rosiglitazone significantly reduced GI inflammation, as evidenced by gene expression data. Rosiglitazone did not significantly reduce DAI scores and colon weight / length ratio.

[0221] Example 2: M1 macrophage differentiation assay

[0222] Methodology

[0223] The aim of this study was to evaluate the effects of rosiglitazone and other thiazolidinediones on M1 macrophage differentiation. The test articles and vehicles used in this study were rosiglitazone sulfate, pioglitazone hydrochloride, and rosiglitazone.

[0224] Media and solution reconstitution: (1) Prepare complete medium: add 50 mL HI FBS (10%) and 5 mL (1%) 100X Pen / Strep to a 500 mL RPMI-1640 bottle; (2) Reconstitute LPS with sterile water to a stock concentration of 1 mg / ml; (3) Dissolve human IL-4 in sterile water by pipette to reconstitute at 0.1 mg / ml; (4) Dissolve human M-CSF in sterile water by pipette to a concentration of 0.1 mg / ml; (5) Dissolve human GM-CSF in sterile water by pipette to reconstitute at a concentration of 0.1 mg / ml; (6) Reconstitute the test article (M107, pioglitazone, rosiglitazone) with DMSO to achieve a stock concentration of 10 mM.

[0225] The timetable for the M1 macrophage differentiation assay is shown in Table 8 below.

[0226] Table 8.

[0227]

[0228] The drug concentrations tested in the M1 differentiation assay are shown in Table 9.

[0229] Table 9.

[0230] drug Test concentration (μM) Roglitazone sulfate 0,0.025,0.05,0.1,0.2,0.4,0.8,1.6,3.2,6.4 Pioglitazone 0,2.5,5,10,20,40 Rosiglitazone 0,0.4,0.8,1.6,3.2,6.4

[0231] Experimental Procedure

[0232] (1) Thawing frozen monocytes.

[0233] (2) Count the cells and adjust the cell density to 0.5 x 10 with fresh complete differentiation medium (RPMI1640 + 10% FBS + 1% PS plus GM-CSF or M-CSF, concentration of 50 ng / ml). 6 (3) 100 μL of the separated monocytes were inoculated into each well of a 96-well plate.

[0234] (4) Add the test articles as plate map using D300e (final concentration in μM).

[0235] (5) Monocytes were differentiated into macrophages in the presence of GM-CSF to M1 or M-CSF to M2 for 5 days.

[0236] (6) On day 5, cytokines were added at 2 times the final concentration.

[0237] (6a) Prepare a medium containing GM-CSF for M1 and a medium containing M-CSF for M2.

[0238] (6b) Add the corresponding activators to reach 2 times the final working concentration: (i) IFNγ stock solution 0.2mg / ml: add 2.5μL stock solution to 5ml complete differentiation medium, the concentration is 100ng / ml; (ii) LPS stock solution 1.0mg / ml: mix 2μL stock solution into 100μl medium and dilute to 20μg / ml; then add 10μl 20μg / ml LPS to 5ml complete differentiation medium, the concentration is 40ng / ml; (iii) IL-4 stock solution 0.1mg / ml: dilute 2μL stock solution to 5ml complete differentiation medium, the concentration is 40ng / ml. (6c) Add 100μL differentiation medium containing cytokines to each well, the total volume of each well is 200μL.

[0239] (6d) Add the test compound again using D300e. Make sure the final volume is set to 200 μL. (7) Incubate the cytokines and test compound for 48 hours.

[0240] (8) On day 7, the culture plates were spun at 300 x g for 5 min and the supernatants were collected for quantification of cytokines using MSD.

[0241] (9) Quantification of M1-related macrophage cytokines TNF-α, IL-1b, IL-6, IL-12, and IL-23.

[0242] result

[0243] The results of cytokine expression in M1 macrophages were as follows: Fig.14 The results of the M1-like macrophage cytokine expression assay are shown in Figures 14 to 18 as shown in .

[0244] in conclusion

[0245] This study evaluated the effects of rosiglitazone (M107), pioglitazone (Pigl), and rosiglitazone (Rosig) on ​​M1 and M2 macrophage differentiation by cytokine assays. Among the three tested samples, pioglitazone and rosiglitazone promoted M1 macrophage cytokines in a dose-dependent manner, while rosiglitazone did not show a promoting effect on M1 macrophage cytokines.

[0246] Example 3: M2 macrophage differentiation assay

[0247] Methodology

[0248] The aim of this study was to evaluate the effects of rosiglitazone and other thiazolidinediones on M2 macrophage differentiation. The test articles and vehicles used in this study were rosiglitazone sulfate, pioglitazone hydrochloride, and rosiglitazone.

[0249] Media and solution reconstitution: (1) Prepare complete medium: add 50 mL HI FBS (10%) and 5 mL (1%) 100X Pen / Strep to a 500 mL RPMI-1640 bottle; (2) Reconstitute LPS with sterile water to a stock concentration of 1 mg / ml; (3) Dissolve human IL-4 in sterile water by pipette to reconstitute at 0.1 mg / ml; (4) Dissolve human M-CSF in sterile water by pipette to reconstitute at a concentration of 0.1 mg / ml; (5) Dissolve human GM-CSF in sterile water by pipette to a concentration of 0.1 mg / ml; (6) Reconstitute the test article (M107, pioglitazone, rosiglitazone) with DMSO to achieve a stock concentration of 10 mM.

[0250] The timetable for the M2 macrophage differentiation assay is shown in Table 10 below.

[0251] Table 10.

[0252]

[0253] The drug concentrations tested in the M2 differentiation assay are shown in Table 11.

[0254] Table 11.

[0255] drug Test concentration (μM) Lobeglitazone sulfate 0.025,0.05,0.1,0.2,0.4,0.8,1.6 Pioglitazone 2.5,5,7.5,10 Rosiglitazone 0.4,0.8,1.6,3.2

[0256] Experimental procedures (monocyte isolation, processing and seeding)

[0257] (1) Use Monocyte Isolation Kit Isolate monocytes from PBMCs

[0258] (2) Count the cells and adjust the cell density to 0.5 x 10 6 To each 5 ml cell mixture, add 5 μL of M-CSF stock solution (final concentration is 100 μg / ml). (3) Inoculate 100 μL of the separated monocytes into each well of a 96-well plate.

[0259] (4) Add the test articles as plate map using D300e (final concentration in μM).

[0260] (5) Monocytes were differentiated into macrophages in the presence of M-CSF to M4 for 4 days.

[0261] (6) On day 4, add additional culture medium containing M-CSF.

[0262] (7) Add test compounds using D300e (final volume = 100 μL).

[0263] (8) On day 6, add IL-4 at 5 times the final concentration (IL-4 stock solution 0.1 mg / ml: dilute 10 μl of stock solution into 10 ml of culture medium to a concentration of 100 ng / ml. Add 50 μl of 100 ng / ml IL-4 to each well containing 200 μL of culture medium to reach 20 ng / ml).

[0264] (9) On day 8, the culture plate was spun at 300 x g for 5 min and the supernatant was collected for cytokine quantification using MSD.

[0265] (10) Table 12 below lists the M2-related macrophage cytokines of interest.

[0266] Table 12.

[0267]

[0268] (11) Collect cells for flow cytometry analysis of viability / CD11c / CD163 / CD206.

[0269] result

[0270] M2 repolarization was characterized by flow cytometry and surface markers CD163, CD11c, and CD206. No staining and all stainings were performed with surface marker antibodies to confirm optimization of the gating strategy.

[0271] Detailed cell phenotypes such as Fig.19 , Fig. 20 and Fig.21 shown.

[0272] The expression of cytokines and chemokines was quantified using MSD. Fig. 22 and Fig.23 The expression of overall M1 and M2-related cytokines is shown separately. The detailed M2-related cytokines MDC, Eotaxin-2, and IL-1RA are shown in Fig.24 , Fig.25 and Fig.26 What is presented.

[0273] in conclusion

[0274] This study evaluated the effects of rosiglitazone, pioglitazone, and rosiglitazone on M2 macrophage differentiation using flow cytometry and cytokine assays. As shown in flow cytometric studies, M2 macrophage surface marker characterization showed that M107 promoted M2 repolarization at lower concentrations (~0.025 μM) in donors 1 and 2 compared to pioglitazone hydrochloride (~10 μM) and rosiglitazone (~0.4 μM). In cytokine analysis, M107 also showed significant promotion of MDC expression (in donor 3) and Eotaxin-2 expression (in donor 1).

[0275] Example 4: M1-to-M2 macrophage repolarization assay

[0276] Methodology

[0277] The aim of this study was to evaluate the effects of rosiglitazone and other thiazolidinediones on M1-to-M2 macrophage repolarization. The test articles and vehicles used in this study were rosiglitazone sulfate, pioglitazone hydrochloride, and rosiglitazone.

[0278] Culture medium and solution reconstitution: (1) Prepare complete culture medium: add 50 mL HI FBS (10%) and 5 mL (1%) 100X Pen / Strep to 500 mL bottle of RPMI-1640; (2) Reconstitute LPS with sterile water to a stock concentration of 1 mg / ml; (3) Reconstitute hIL-4 with sterile water to a stock concentration of 100 μg / ml; (4) Dissolve human M-CSF in sterile water with a pipette to reconstitute at a concentration of 0.1 mg / ml; (5) Dissolve human GM-CSF in sterile water with a pipette to reconstitute at a concentration of 0.1 mg / ml; (6) Dissolve the test samples (M107, pioglitazone, rosiglitazone) with DMSO until a stock concentration of 10 mM is reached.

[0279] The timetable for the M1 to M2 macrophage repolarization assay is shown in Table 13 below.

[0280] Table 13.

[0281]

[0282] The drug concentrations tested in the M1 differentiation assay are shown in Table 14.

[0283] Table 14.

[0284] drug Test concentration (μM) Roglitazone sulfate 0.025,0.05,0.1,0.2,0.4,0.8,1.6,3.2 Pioglitazone 2.5,5,10 Rosiglitazone 0.2,0.4,0.8,1.6,3.2,6.4

[0285] Experimental procedures (monocyte isolation, processing and seeding)

[0286] (1) Use The Monocyte Isolation Kit isolates monocytes from PBMCs.

[0287] (2) Count the cells and adjust the cell density to 0.5 x 10 6 cells / ml (add 10 μl of M-CSF stock solution (100 ug / ml) to 10 ml of cell mixture to a final concentration of 50 ng / ml).

[0288] (3) 100 μL of the separated monocytes were seeded into each well of a 96-well plate.

[0289] (4) On day 4, add 100 ul of additional culture medium containing M-CSF

[0290] (5) On day 6, add stimuli at 5 times the final concentration (add 200 μL of culture medium to each well) to make the final concentration reach 50 ng / ml.

[0291] (6) On day 8, the culture medium was removed and supplemented with 200 μL of cytokine-free culture medium, followed by the addition of test compounds (final concentration in μM) and incubation for 4 days.

[0292] (7) On day 12, IL-4 was added and incubated for 2 days (50 μL of 100 ng / ml IL-4 was added to each well containing 200 μL of medium to reach 20 ng / ml).

[0293] (8) On day 14, the culture plate was spun at 300 x g for 5 min and the supernatant was collected for cytokine quantitative analysis.

[0294] (9) The M1 and M2 related macrophage cytokines of interest are shown in Table 15 below.

[0295] Table 15.

[0296]

[0297] Cells were then collected for flow analysis of viability / CD11c / CD163 / CD206.

[0298] result

[0299] M2 repolarization was characterized by flow cytometry using cell surface markers CD163, CD11c, and CD206. Controls included no staining and staining with antibodies to all surface markers to confirm optimization of the gating strategy. Detailed cell phenotypes are shown in Fig. 27 shown.

[0300] Cytokine and chemokine expression was quantified using MSD. Fig.28 and Fig.29The overall M1 and M2-related cytokine expressions are shown separately. The detailed M2-related cytokines MDC, Eotaxin-2, and IL-1RA are shown in Figure 30-32 shown.

[0301] in conclusion

[0302] This study evaluated the effects of rosiglitazone, pioglitazone, and rosiglitazone on M1-to-M2 repolarization using flow cytometry and cytokine assays. M2 macrophage surface marker characterization showed that M107 promoted M1-to-M2 repolarization at concentrations as low as ∼0.05 μM, as shown in flow cytometric studies. In cytokine analysis, M107 also showed significant promotion of MDC expression at 0.1 μM, compared with 5 μM and 6.4 μM for pioglitazone and rosiglitazone, respectively.

[0303] Unless otherwise stated, all the numbers used in the specification and claims that represent the number of ingredients, properties (such as molecular weight, reaction conditions, etc.) should be understood to be modified by the term "about" in all cases. The "about" in the term "about" and "approximately" means within the range of 10% to 15%, preferably within the range of 5% to 10%. Therefore, unless otherwise stated, the numerical parameters listed in the specification and the attached claims are approximate values ​​and may vary depending on the desired properties to be obtained by the present invention. At least, it is not intended to limit the application of the principle of equivalents to the scope of the claims, and each numerical parameter should at least be interpreted according to the number of reported significant figures and the application of ordinary rounding techniques. Although the numerical ranges and parameters that illustrate the wide range of the present invention are approximate values, the numerical values ​​listed in the specific examples are reported as accurately as possible. However, any numerical value inherently contains certain errors, which are inevitably caused by the standard deviation in its respective test measurements.

[0304] The terms "one", "the" and similar designations used in the context of describing the present invention (especially in the context of the following claims) should be interpreted as covering the singular and the plural, unless otherwise specified herein or the context is clearly contradictory. The description of the range of values ​​herein is intended only to be used as a shorthand method for each individual value in the range of individual references. Unless otherwise specified herein, each individual value is incorporated into the specification as if it were individually described herein. All methods described herein can be performed in any suitable order, unless otherwise specified herein or the context is clearly contradictory. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the claimed invention. Any language in the specification should not be interpreted as indicating that any unclaimed element is essential to the practice of the present invention.

[0305] The grouping of the alternative elements or embodiments of the present invention disclosed herein should not be construed as limiting. Each group member may be cited and claimed individually, or with other members or other elements in the group found herein in any combination and claimed. It is foreseeable that, for reasons of convenience and / or patentability, one or more group members may be included in the group, or deleted therefrom. When any such inclusion or deletion occurs, the specification is deemed to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

[0306] Certain embodiments of the present invention are described herein, including the best mode of implementing the present invention known to the inventor. Of course, variations of these described embodiments will be apparent to those of ordinary skill in the art after reading the above description. The inventors hope that skilled technicians will be able to adopt such variations as appropriate, and the inventors hope to implement the present invention in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter described in the appended claims as permitted by applicable law. In addition, unless otherwise stated herein or the context is clearly contradictory, the present invention encompasses any combination of the above elements in all possible variations thereof.

[0307] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in a claim, whether as filed or added by amendment, the transition term "consisting of" excludes any element, step, or ingredient not specified in the claim. The transition term "consisting essentially of" limits the scope of a claim to the specified materials or steps and to those materials or steps that do not materially affect the basic and novel characteristics. Embodiments of the claimed invention are inherently or expressly described and enabled herein.

[0308] In addition, throughout the specification, patents and printed publications have been cited several times. Each of the above references and printed publications is incorporated herein by reference individually.

[0309] Finally, it should be understood that the embodiments of the present invention disclosed herein are only intended to illustrate the principles of the present invention. Other modifications that may be employed are within the scope of the present invention. Therefore, as an example, but not for limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Therefore, the present invention is not limited to the precise configuration shown and described.

Claims

1. A method for protecting the digestive system from damage, comprising contacting cells of the gastrointestinal (GI) system with an effective amount of a thiazolidinedione selected from one or more of pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, riglitazone, nyglitazone, riglitazone, troglitazone and balaglitazone.

2. The method of claim 1, wherein the thiazolidinedione is roglitazone.

3. The method of claim 1, wherein the cells are from the oral cavity, pharynx (larynx), esophagus, stomach, small intestine, large intestine, rectum, anus, salivary gland, liver, gall bladder or pancreas.

4. The method of claim 1, wherein the cell of the GI system is an absorptive cell (enterocyte), goblet cell, islet cell, enteroendocrine cell, hepatocyte, Paneth cell, fenestrated liver endothelial cell, Kupffer cell, serous cell, chief cell, mucous cell, smooth muscle cell, parietal cell, myoepithelial cell, stem cell, gastric surface mucous cell, pancreatic acinar cell, taste bud, interstitial cell of Cajal (ICC), or neuronal cell.

5. The method according to any one of claims 1 to 4, wherein the cell is a mammalian cell. The method of claim 5 , wherein the cells are human cells.

7. The method according to any one of claims 1 to 6, wherein the cells are treated in vitro.

8. The method according to any one of claims 1-6, wherein the cells are treated ex vivo.

9. The method of any one of claims 1-6, wherein the cells are treated in vivo.

10. The method of any one of claims 1-9, wherein the cell is in a subject having or at risk of having the disease or disorder of the GI system.

11. The method of claim 10, wherein the GI system disease or disorder is selected from the group consisting of abdominal adhesions, acid reflux in adults (gastroesophageal reflux disease or GERD), acid reflux in infants (GERD), lower GI tract anatomy problems, appendicitis, Barrett's esophagus, bowel control problems (fecal incontinence), celiac disease, colon polyps, constipation, Crohn's disease, cyclic vomiting syndrome, diarrhea, diverticular disease and diverticulitis, dumping syndrome, food poisoning, gallstones, flatulence, gastritis, gastroparesis, GI bleeding, hemorrhoids, digestive disorders (dyspepsia), inguinal hernia, intestinal pseudo-obstruction, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), lactose intolerance, liver disease, microscopic colitis, enterostomy, pancreatitis, peptic ulcer (gastric ulcer), proctitis, short bowel syndrome, ulcerative colitis, viral gastroenteritis, and Zollinger-Ellison syndrome, or a disorder related thereto.

12. The method of claim 11, wherein the GI disease or disorder is gastroparesis.

13. The method of claim 12, wherein the gastroparesis is idiopathic gastroparesis, diabetic gastroparesis, postoperative gastroparesis, or drug-induced gastroparesis.

14. A method of treating a GI system disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising one or more thiazolidinediones selected from the group consisting of pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, rivoglitazone, nyglitazone, rivoglitazone, troglitazone, and beglitazone.

15. The method of claim 14, wherein the thiazolidinedione is lobeglitazone.

16. The method of any one of claims 14 or 15, wherein the GI system disease or disorder is selected from abdominal adhesions, gastric acid reflux (GERD) in adults, gastric acid reflux (GERD) in infants, lower GI tract anatomy problems, appendicitis, Barrett's esophagus, bowel control problems (fecal incontinence), celiac disease, colon polyps, constipation, Crohn's disease, cyclic vomiting syndrome, diarrhea, diverticular disease and diverticulitis, dumping syndrome, food poisoning, gallstones, flatulence, gastritis, gastroparesis, GI bleeding, hemorrhoids, digestive disorders (dyspepsia), inguinal hernia, intestinal pseudo-obstruction, IBD, IBS, lactose intolerance, liver disease, microscopic colitis, enterostomy, pancreatitis, peptic ulcer (gastric ulcer), proctitis, short bowel syndrome, ulcerative colitis, viral gastroenteritis and Zollinger-Ellison syndrome, or a disorder related thereto.

17. The method of claim 16, wherein the GI disease or disorder is gastroparesis.

18. The method of claim 17, wherein the gastroparesis is idiopathic gastroparesis, diabetic gastroparesis, postoperative gastroparesis, postoperative gastroparesis, or drug-induced gastroparesis.

19. The method of any one of claims 14-18, wherein the subject is a mammal.

20. The method of claim 19, wherein the mammal is a non-human mammal.

21. The method of claim 19, wherein the mammal is a human.

22. The method of any one of claims 14-21, wherein the thiazolidinedione is administered at a dose of 0.01 mg or more.

23. The method of any one of claims 14-21, wherein the thiazolidinedione is administered at a dose of 0.01-5000 mg / day.

24. The method of any one of claims 14-21, wherein the thiazolidinedione is administered parenterally, enterally, topically, orally, sublingually, buccally, intrapulmonary, intranasally, intravenously, intramuscularly, or subcutaneously.

25. Use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, rivoglitazone, nyglitazone, rivoglitazone, troglitazone and balaglitazone for relieving the signs and symptoms of gastroparesis.

26. The use according to claim 25, wherein the severity of the signs and symptoms of gastroparesis is measured by the American College of Neurogastroenterology and Motility Medicine Gastroparesis Cardinal Symptom Index-Daily Diary (GCSI-DD).

27. The use according to claim 25, wherein the severity of the signs and symptoms of gastroparesis is measured by the change in GCSI-DD score from baseline to 4 weeks of treatment, 8 weeks of treatment, 12 weeks of treatment or 1 year of treatment.

28. Use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, nyglitazone, riglitazone, troglitazone and balaglitazone to reduce the severity of nausea in a subject with gastroparesis.

29. Use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, nyglitazone, riglitazone, troglitazone and balaglitazone to reduce the severity of early satiety in a subject with gastroparesis.

30. Use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, nyglitazone, riglitazone, troglitazone and balaglitazone to reduce the severity of postprandial fullness in a subject with gastroparesis.

31. Use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, nyglitazone, riglitazone, troglitazone and balaglitazone to reduce the severity of upper abdominal pain in a subject with gastroparesis.

32. Use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, nyglitazone, riglitazone, troglitazone and balaglitazone to reduce the severity or frequency of vomiting in a subject with gastroparesis.

33. Use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, nyglitazone, riglitazone, troglitazone and balaglitazone to reduce the overall severity of gastroparesis.

34. Use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, rivoglitazone, nyglitazone, rivoglitazone, troglitazone and balaglitazone to reduce gastrointestinal inflammation as indicated by one or more reductions in TNF-α, IL-10, CCL2 and IL-1β gene expression in cells of a patient experiencing GI inflammation.

35. Use of one or more thiazolidinediones selected from pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, nyglitazone, riglitazone, troglitazone and balaglitazone to slow, stop or reverse the progression of gastroparesis.

36. The use according to any one of claims 25 to 35, wherein the thiazolidinedione is roglitazone.

37. A method for treating a GI disease in a mammal, comprising administering to the mammal an effective amount of a thiazolidinedione selected from one or more of pioglitazone, rosiglitazone, roglitazone, ciglitazone, darglitazone, deuterium-stabilized R-pioglitazone, englitazone, roglitazone, nyglitazone, riglitazone, troglitazone and balaglitazone.

38. The method of claim 37, wherein the GI disease is gastroparesis.

39. The method of claim 38, wherein the gastroparesis is idiopathic gastroparesis, diabetic gastroparesis, postoperative gastroparesis, or drug-induced gastroparesis.

40. The method of any one of claims 37-39, wherein the thiazolidinedione is lobeglitazone.

41. A method of inhibiting M1 macrophage differentiation in an individual, comprising administering to the individual an effective amount of lobeglitazone.

42. The method of claim 41, wherein the subject is treated for a disorder of the GI system due to inhibition of M1 macrophage differentiation.

43. A method of promoting M1 macrophage differentiation in an individual, comprising administering to the individual an effective amount of pioglitazone or rosiglitazone.

44. A method of promoting M2 macrophage differentiation in an individual, comprising administering to the individual an effective amount of rosiglitazone, pioglitazone, or rosiglitazone.

45. The method of claim 44, wherein the subject is treated for a disorder of the GI system due to the promotion of M2 macrophage differentiation.

46. ​​A method of promoting M1-to-M2 macrophage repolarization in an individual, comprising administering to the individual an effective amount of rosiglitazone, pioglitazone, or rosiglitazone.

47. The method of claim 46, wherein the subject's GI system disorder is treated due to promotion of M1-to-M2 macrophage repolarization.

48. The method of claim 44 or 46, wherein the thiazolidinedione is lobeglitazone.

49. The method of claim 45 or 47, wherein the GI system disorder is selected from abdominal adhesions, gastric acid reflux (GERD) in adults, gastric acid reflux (GERD) in infants, lower GI tract anatomy problems, appendicitis, Barrett's esophagus, bowel control problems (fecal incontinence), celiac disease, colon polyps, constipation, Crohn's disease, cyclic vomiting syndrome, diarrhea, diverticular disease and diverticulitis, dumping syndrome, food poisoning, gallstones, flatulence, gastritis, gastroparesis, GI bleeding, hemorrhoids, digestive disorders (dyspepsia), inguinal hernia, intestinal pseudo-obstruction, IBD, IBS, lactose intolerance, liver disease, microscopic colitis, enterostomy, pancreatitis, peptic ulcer (gastric ulcer), proctitis, short bowel syndrome, ulcerative colitis, viral gastroenteritis and Zollinger-Ellison syndrome, or a disorder related thereto.

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