Vestiggrid for treatment of chronic intestinal pseudoobstruction (CIPO)

Abstract

In particular, the present invention relates to 1-isopropyl-2-oxo-1, 2-dihydroquinoline-3-carboxylic acid {(1S, 3R, 5R)-8-[(R)-2-hydroxy-3-(methylsulfonyl-methyl-amino) propyl]-8-azabicyclo [3.2. 1] oct-3-yl} amide (vestritagel), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof. Methods for treating chronic ileus pseudosciatica (CIPO), neurological chronic ileus pseudosciatica, or chronic ileus pseudosciatica secondary to a neurodegenerative or demyelinating disease in a patient suffering from such a disease or condition.

Description

Technical Field

[0001] The present invention relates to vistrag (1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methylsulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]octan-3-yl}amide), its pharmaceutically acceptable salts and compositions comprising said compounds for use in the treatment of chronic intestinal pseudo-obstruction (CIPO), in particular neuropathic CIPO or idiopathic CIPO. Thus, the present invention is in the field of methods for treating altered gastrointestinal motility conditions and disorders, such as chronic intestinal pseudo-obstruction (or colonic pseudo-obstruction) and disorders and conditions associated with constipation, such as constipation associated with the use of opioid analgesics, postoperative constipation, and constipation associated with neuropathic disorders and other conditions. Background Art

[0002] Intestinal pseudo-obstruction is a rare, serious disorder characterized by dysmotility and symptoms of intestinal obstruction, but without signs of mechanical obstruction. The disorder is caused by abnormalities in the neuromuscular and / or autonomic innervation of the intestine; it is the most severe form of gastrointestinal dysmotility, leading to debilitating and life-threatening consequences.

[0003] When the condition persists for more than 6 months, chronic intestinal pseudo-obstruction (CIPO) occurs, which is one of the most important causes of chronic intestinal failure in children (15%) and adults (20%). Since affected individuals are usually unable to maintain a normal oral diet and maintain an appropriate body weight, severe clinical symptoms characterized by disabling digestive symptoms occur in patients, leading to a serious deterioration in the patient's quality of life and eventually death.

[0004] A distinction is made between primary and secondary forms of CIPO. Primary CIPO is caused by an intrinsic defect (congenital or acquired) and can be divided, depending on the type of damage (which may be relevant), into: visceral myopathy (related to muscle damage; e.g. MNGIE: mitochondrial neuro-gastrointestinal neuropathy, MMIHS: megacystis-microcolon-hypomotility syndrome, idiopathic), visceral neuropathy (related to autonomic nerve damage; e.g. dysplasia of the myenteric plexus, Hirschsprung's disease, sequelae of necrotizing enterocolitis, idiopathic) or mesenchymal disease (related to damage to Cajal cells in the digestive tract).

[0005] The etiological classification of CIPO is evolving as new genetic entities are discovered, especially through studies of familial forms. Examples of genes involved are the TYMP gene (mutated in MNGIE), the ACTG2 gene (mutated in megacystis-microcolon-hyperistaltic syndrome (MMIHS)), the SGOL1 gene (mutated in chronic atrial and intestinal arrhythmia syndrome (CAID)), and the POLG gene (mutated in Alpers' disease). Nevertheless, the etiology of most cases of CIPO is currently unknown, and these cases are referred to as idiopathic cases. Therefore, they are classified in the primary CIPO group.

[0006] Secondary CIPO is associated with underlying systemic neurological, endocrine, and connective tissue diseases or malignancies. In these diseases, intestinal motility can be affected by factors involving the autonomic nervous system (stroke, encephalitis, orthostatic hypotension), the intestinal nervous system (paraneoplastic syndromes, viral infections, iatrogenic, diabetes, Chagas' disease, Von Recklinghausen's disease), the intestinal muscular layer (myotonic dystrophy, progressive systemic sclerosis), mixed enteric nervous system and smooth muscle layer (scleroderma, dermatomyositis, amyloidosis, Ehlers-Danlos syndrome, jejunal diverticulosis, radiation enteropathy), and ultimately unknown mechanisms (hypothyroidism, hypoparathyroidism, pheochromocytoma, antidepressants, antineoplastic drugs or bronchodilators) [Antonucci A. et al. Chronic intestinal pseudo-obstruction." World journal of gastroenterology 2008;14:2953-61; Zhu CZ et al. Latest developments in chronic intestinal pseudo-obstruction. World J Clin Cases. 2020; 8: 5852-5865; Billiauws L. et al. Small intestinal motility disorders: Chronic intestinal pseudo-obstruction. J Visc Surg. 2022; 159(1S): S22-S27].

[0007] There are no clear epidemiological data; it is estimated that approximately 100 infants are affected by intestinal pseudo-obstruction each year in the United States; the adult incidence is 0.2 (males) and 0.24 (females) per 100,000 patient-years, and the adult prevalence is estimated to be 0.2 to 0.9 per 100,000 population [Di Nardo G. et al. Pharmacological and nutritional therapy of children and adults with chronic intestinal pseudo-obstruction. Expert Review of Gastroenterology&Hepatology. Vol. 17, No. 2023-4].

[0008] Typically, the most commonly reported symptoms are non-colicky abdominal pain and bloating (80%), which is constant and worsens after eating. These symptoms are usually localized to the umbilicus or epigastrium and gradually spread to include the entire abdomen. Other typical symptoms include nausea (75%), vomiting (40%-50%), constipation (40%), and diarrhea (20%-30%).

[0009] About 30% of patients with typical symptoms also have small intestinal bacterial overgrowth (SIBO); this condition causes further mucosal damage, steatorrhea, diarrhea, and intestinal damage with chronic distension of the intestine, leading to malabsorption, vitamin deficiencies, and weight loss. Extraintestinal manifestations may also occur (e.g., bladder and ureters in MMIHS; ophthalmoplegia, ptosis, and peripheral polyneuropathy in MNGI; depression or other psychological disturbances due to long-term illness).

[0010] The classic clinical presentation of CIPO is recurrent episodes of pseudo-obstruction characterized by abdominal pain, distention, and inability to defecate, with or without vomiting, mimicking a mechanical incomplete obstruction. Important diagnostic signs of this pathological condition during acute episodes are the presence of bowel distension and radiographic markers of air-fluid levels in the upright position. In the most severe cases, the bowel is chronically distended and air-fluid levels are detected.

[0011] Between acute episodes, patients are asymptomatic or experience a variety of gastrointestinal symptoms, primarily related to the location and extent of GI involvement.

[0012] Because CIPO symptoms overlap with other serious digestive symptoms and because of the lack of biomarkers, patients often undergo futile and potentially dangerous abdominal surgery before the cause of their recurrent subacute obstructive episodes is known. The average time to a correct final diagnosis is 8 years.

[0013] The diagnosis of CIPO is primarily clinical, based on radiographic documentation of intestinal dilatation with air-fluid levels, after exclusion of organic lesions obstructing the bowel lumen detected by radiology and / or endoscopy. It is important to determine the possible cause of the secondary form. Manometry can be used to differentiate between mechanical and functional obstruction and to identify the underlying pathophysiological mechanisms. Full-thickness biopsies should be obtained from dilated or non-dilated tracts of the digestive tract in all patients suspected of CIPO who undergo surgery for an unexplained obstructive event [Antonucci A. et al. Chronic intestinal pseudo-obstruction." World journal of gastroenterology 2008;14:2953-61].

[0014] The primary goal of CIPO treatment is to enhance gastrointestinal motility, improve nutritional status, and maintain a stable intestinal environment. Of course, for patients with secondary CIPO, the primary disease must be actively treated to eliminate the cause.

[0015] For cases of acute intestinal pseudo-obstruction, surgery should be minimized as much as possible because surgery can inhibit intestinal motility and even induce further intestinal failure, and the re-intervention rate in such cases is high.

[0016] For the treatment of visceral pain in patients with CIPO, especially chronic pain, low-dose tricyclic antidepressants and gabapentin are used, and opioids are not recommended because they inhibit gastrointestinal motility. However, as the disease progresses, about 25% of patients gradually increase their tolerance to analgesics, so it is recommended that a team including pain specialists and psychologists participate in appropriate treatment management [Stanghellini V. et al. Natural History of Chronic Idiopathic Intestinal Pseudo-Obstruction in Adults: A Single Centre Study, Clinical Gastroenterology and Hepatology, 2005; 3: 449-458].

[0017] To combat concomitant SIBO, the most common complication of chronic intestinal distension, with bacterial concentrations often above 10 3 CFU / mL to 10 5CFU / mL, prescribe oral antibiotic treatment, there are antibiotic-based treatment options, such as amoxicillin-clavulanic acid (500mg, three times a day), ciprofloxacin (500mg, twice a day), doxycycline (100mg, twice a day), metronidazole (250mg, three times a day), neomycin (500mg, twice a day), rifaximin (550mg, twice a day) and tetracycline (250mg, four times a day). Usually, antibiotics are prescribed for 7 to 10 days a month, and the antibiotic type is changed every month for 5 to 6 months to avoid the emergence of drug resistance.

[0018] In addition, fecal microbiota transplantation is also considered a new method for treating CIPO. Studies have shown that fecal microbiota transplantation can significantly relieve abdominal distension and abdominal pain in patients, improve patients' tolerance to enteral nutrition, and prevent and treat related SIBO [Gu L. et al. Serial Frozen Fecal Microbiota Transplantation in the Treatment of Chronic Intestinal Pseudo-obstruction: A Preliminary Study. J Neurogastroenterol Motil. 2017; 23: 289–297].

[0019] Intestinal transplantation can save children's lives but is reserved for patients who are unable to proceed or cannot safely continue long-term parenteral nutrition, including those who have liver complications from parenteral nutrition, difficult central line access, or those who have poor quality of life and increasing pain despite aggressive medical management [Camilleri et al. Chronic Intestinal Pseudo-obstruction: Management, updated February 17, 2022].

[0020] Since CIPO is characterized by impaired intestinal propulsive activity, in the absence of any effective and curative medical treatment, drugs that promote gastrointestinal motility, such as amoxicillin-clavulanate [Gomez R. et al. Effect of Amoxicillin / Clavulanate on Gastrointestinal Motility in Children, Journal of Pediatric Gastroenterology and Nutrition 2012; 54: 780-784] and erythromycin, a macrolide antibiotic and motilin receptor activator [Emmanuel AV et al. Erythromycin for the treatment of chronic intestinal pseudo-obstruction: description of six cases with a positive response. Aliment Pharmacol Ther. 2004; 19: 687-94] are commonly used. However, long-term treatment with erythromycin does not seem feasible due to the phenomenon of tachyphylaxis (associated with downregulation of motilin receptors) [Dhir R, Richter JE. Erythromycin in the short-and long-term control of dyspepsia symptoms in patients with gastroparesis. J Clin Gastroenterol 2004;38:237–42].

[0021] There is insufficient evidence to treat CIPO with metoclopramide and domperidone, two antiemetic and prokinetic drugs, and long-term use of the former carries a risk of tardive dyskinesia. Acetylcholinesterase inhibitors (neostigmine, 8 mg / day, intravenous injection; pyridostigmine, 20 mg / day, oral administration) have also been used to treat CIPO in adults.

[0022] Serotonin (5-HT) is a neurochemical associated with the control of intestinal motility; however, the functional role of endogenous 5-HT is not fully understood, as depletion of neuronal or mucosal 5-HT has little or no effect on intestinal motility. On the other hand, the role of endogenous 5-HT produced by enterochromaffin cells in regulating intestinal motility remains unclear.

[0023] 5-HT receptors are widely expressed in the gastrointestinal tract, and 5 of the 7 known families (5-HT 1 , 5-HT 2 , 5-HT3 , 5-HT 4 and 5-HT 7 Receptors) are expressed in the intestine and can affect intestinal function. 3 and 5-HT 4 The receptor subtypes are the most extensively studied and are used to treat diarrhea and constipation, respectively.

[0024] In addition, it has been described that endogenous 5-HT can activate 5-HT in the enteric nervous system (ENS). 3 and 5-HT 4 In this tightly controlled system, disturbances in these regulatory mechanisms are associated with intestinal motility disorders [Waclawiková B. et al. Gut bacteria-derived 5-hydroxyindole is a potent stimulant of intestinal motility via its action on L-type calcium channels. PLoS Biol. 2021; 19(1): e3001070].

[0025] Prucalopride is a highly selective 5-HT 4 Receptor agonist, no cardiotoxicity, has significant neuroprotective effects on human enteric neurons [Bianco F. et al. Prucalopride exerts neuroprotection in human enteric neurons. Am J Physiol Gastrointest Liver Physiol. 2016; 310: G768-75], and has shown promising results in children and adults with acute and chronic intestinal pseudo-obstruction. Prucalopride is safe, effective and well tolerated for children with acute intermittent or chronic intestinal pseudo-obstruction [Mutalib M. et al. Prucalopride in intestinal pseudo-obstruction, paediatric experience and systematic review. Acta Gastroenterol Belg. 2021; 84: 429-434].

[0026] In addition, Emmanuel AV et al. described a phase II, double-blind, placebo-controlled, two-treatment, four-cycle crossover trial in which the clinical safety, tolerability, and efficacy of prucalopride in improving CIPO-related symptoms were studied. Subjects received four cycles of treatment, each of 12 weeks, with 2 mg of prucalopride (2 cycles) or placebo (2 cycles) in each treatment period. There was no washout period. A total of 7 subjects were randomized; 2 were assigned to PLA-PRU-PLA-PRU, 2 were assigned to PRU-PLA-PRU-PLA, 2 were assigned to PLA-PRU-PRU-PLA, and 1 was assigned to the PRU-PLA-PLA-PRU sequence group. Three patients withdrew from the study after the first cycle: one due to serious adverse events that were considered unrelated to the study drug (feeding tube infection, sepsis, bronchopneumonia, and malnutrition), and two due to withdrawal of consent (placebo and prucalopride, respectively). The other 4 patients completed the study. The symptoms analyzed were pain, vomiting, nausea, and abdominal distension. The study was limited in the number of participants; however, the results of this long-term study suggest that prucalopride relieves CIPO symptoms: prucalopride significantly improved pain in three of four patients, nausea in two patients, vomiting in one patient, and abdominal distension in four patients. The number of analgesic intakes was significantly reduced during prucalopride treatment compared with the placebo treatment period. However, prucalopride did not affect stool frequency and consistency, and there was no evidence of a significant prokinetic effect on gastrointestinal transit. The incidence of pseudo-obstruction episodes in patients with CIPO was not analyzed in this study. [Emmanuel AV, et al. Randomised clinical trial: the efficacy of prucalopride in patients with chronic intestinal pseudo-obstruction--a double-blind, placebo-controlled, cross-over, multiple n=1 study. Aliment Pharmacol Ther. 2012;35:48-55;NCT00793247].

[0027] Other 5-HT 4 Receptor agonists such as cisapride and tegaserod are effective but are contraindicated because of associated fatal arrhythmias.

[0028] Due to 5-HT 4 Alternative splicing of receptor-related genes, 5-HT 4There are several variants of the receptor, which explains the different effects of the reference compounds used on the agonist / antagonist activity of the 5-HT4 splice variants. This is also the reason why the various variants of the receptor are highly unpredictable in terms of targeting of therapeutic effects. The importance of variants introduced by splicing on the pharmacology of the receptor can help to understand the contradictory results of 5-HT4 receptor ligands in different model systems [Takaki M. et al. The 5-hydroxytryptamine 4 Receptor Agonist-induced Actions and EntericNeurogenesis in the Gut. J Neurogastroenterol Motil. 2014; 20: 17-30].

[0029] Clearly, to date, there remains an unmet medical need for patients with CIPO. Despite extensive research, medical treatment options remain limited and are often accompanied by severe side effects. This means that to date, there is no satisfactory treatment for CIPO that is effective in improving its main symptoms. As mentioned above, prior art attempts to treat CIPO have only produced unsatisfactory results or have been accompanied by severe side effects. In particular, there are no treatments that show a prokinetic effect. Furthermore, there are no treatments for damage to the neuroenteric system and neurodegeneration of intestinal dysmotility.

[0030] Vistrepol is a highly selective 5-hydroxytryptamine isoform 4 (5-HT 4 ) receptor agonist. The chemical name of Vistreg is 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methylsulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]octan-3-yl}amide, and its chemical structure is shown in Formula I:

[0031]

[0032] Vistragl was previously disclosed in US Patent Application No. 11 / 100,113 filed April 6, 2005; corresponding to EP 1 735 304, and the corresponding crystalline form was disclosed in US Patent Application No. 11 / 398,119 filed April 5, 2006; corresponding to EP 1 874 766.

[0033] In preclinical models and healthy humans, vestregl has been shown to have potent gastrointestinal prokinetic effects in vivo [Smith JAM et al. The in vitro pharmacological profile of TD-5108, a selective. Naunyn-Schmiedeberg's Arch Pharmacol. 2008; 378: 125–137; Beattie DT, et al. The in vivo gastrointestinal activity of TD-5108, a selective 5-HT 4 Receptor agonist with high intrinsic activity. Naunyn-Schmiedeberg's ArchPharmacol. 2008;378:139–147].

[0034] 5-HT 4 Activation of the receptor is associated with inhibition of contractions of isolated guinea pig colonic longitudinal muscles and of isolated human colonic circular muscles, either electrically evoked or spontaneously. 4 Receptor agonism results in increased colonic transit in guinea pigs, esophageal relaxation in rats, and increased motility in the upper and lower gastrointestinal tracts in dogs.

[0035] Vistregr has been evaluated for the treatment of gastrointestinal (GI) tract motility disorders, including chronic idiopathic constipation (CIC) and gastroparesis (GP).

[0036] WO 2015 / 175997 describes a method for treating or ameliorating the effects of a disorder with altered serotonin transporter (SERT) activity (i.e., autism), which damages the enteric nervous system and thereby impairs intestinal motility. The method comprises administering a 5-HT4 agonist to a subject. CIPO is also mentioned in the disease with altered SERT activity, and vistra is also disclosed in the 5-HT4 agonist, but no specific description of an example of vistra being used for CIPO is given.

[0037] Clinical study NCT00391820 showed that patients with less than 3 spontaneous bowel movements (SBM) per week had a statistically and clinically significant increase in spontaneous bowel movement frequency per week after taking 15, 30, and 50 mg per day for 4 consecutive weeks compared with patients receiving placebo [Goldberg M. et al. Clinical trial: the efficacy and tolerability of velusetrag, a selective 5-HT4 agonist with high intrinsic activity, in chronic idiopathic constipation–a 4-week, randomized, double-blind, placebo-controlled, dose–response study. Alimentary Pharmacology & Therapeutics 2010; 31: 1102-1112].

[0038] Ahn A. et al. disclosed the prokinetic effect of Velusetrag in the upper gastrointestinal tract by evaluating the gastric emptying (GE) time in subjects with diabetes and idiopathic gastroparesis. The subjects were divided into 4 groups and received 5 mg, 15 mg, 30 mg of Velusetrag and placebo, respectively. The results of the study showed that the doses of Velusetrag used were effective in accelerating gastric emptying. All doses were well tolerated, but the 30 mg dose reached a significant level [Ahn A. et al. Su 1426 Velusetrag improves gastric emptying time in subjects with diabetic or idiopathic gastroparesis. Gastroenterology 2015; 148: S-507].

[0039] Clinical study NCT02267525 and patent application WO 2019 / 027881 (corresponding to EP 18 756 041.2) teach the use of visconazole or a pharmaceutically acceptable salt thereof for preventing, alleviating, improving or alleviating, and treating the core symptoms (consisting of postprandial fullness, early satiety, abdominal distension, upper abdominal pain, heartburn, nausea and vomiting) of diabetic or idiopathic human patients. The method comprises administering velusetrag at a specific dose of 5 mg / day for a treatment period of one week, two weeks, four weeks, eight weeks or twelve weeks [Abell T. et al. Velusetrag improves gastroparesis both in symptoms and gastric emptying in patients with diabetic or idiopathic gastroparesis in a 12-week global phase 2B study. Abstract for oral presentation at DDW (Digestive Disease Week) Meeting, San Diego (CA). May 18, 2019. Abell T. et al. Efficacy of velusetrag treatment in patients with idiopathic gastroparesis: subgroup analysis of a phase 2b study, UEG Week 2019 Abstract, 26-04-2019].

[0040] As an alternative agonist of the 5-HT4 receptor, Vistragliol works by triggering the release of neurotransmitters, such as acetylcholine from enteric motor neurons and calcitonin gene-related peptide from gastrointestinal sensory neurons. 4 Receptors, vistra enhances peristaltic reflexes, stimulates intestinal secretion, and inhibits visceral sensitivity.

[0041] The inventors have demonstrated that vistrag-induced 5-HT4 activation enhances specific protective responses in different neuronal cells analyzed, including human enteric neurons. These findings open new avenues for the treatment of gastrointestinal disorders and homeostasis, especially those related to neurodegeneration that often leads to severe intestinal dysmotility. Summary of the invention

[0042] The present invention relates to vistragliflozin or a pharmaceutically acceptable salt thereof for use in treating idiopathic chronic intestinal pseudo-obstruction (CIPO), neurogenic chronic intestinal pseudo-obstruction, or chronic intestinal pseudo-obstruction secondary to neurodegeneration or secondary to autoimmune disease or secondary to connective tissue disease or secondary to demyelinating disease.

[0043] According to one embodiment, the pharmaceutically acceptable salt is a hydrochloride. In addition, the present invention also relates to the above use, wherein vistrag is in a crystalline form and / or a hydrated form.

[0044] The present invention also relates to the above use, wherein CIPO is idiopathic CIPO, neurogenic CIPO or CIPO secondary to neurodegeneration or secondary to autoimmune disease or secondary to connective tissue disorder or secondary to demyelinating disease, or any CIPO caused by autonomic nervous system disease, such as stroke, encephalitis, basal ganglia calcification, orthostatic hypotension; CIPO caused by intestinal wall nervous system disease, such as paraneoplastic syndrome, viral infection, iatrogenic disease, congenital megacolon, Chagas disease, von Recklinghausen disease; CIPO caused by intestinal wall muscular layer disease, such as myotonic myotrophy or CIPO caused by an unknown mechanism, such as hypothyroidism, hypoparathyroidism, pheochromocytoma, antidepressants, antineoplastic agents, or bronchodilators; or CIPO caused by an immune-mediated connective tissue disorder or disease, such as paraneoplastic diseases (central nervous system tumors, lung microstoma, bronchial carcinoid, leiomyosarcoma, systemic lupus erythematosus).

[0045] The present invention also relates to the above use, wherein the patient is a subject suffering from CIPO. Preferably, the patient is an adult subject or a pediatric subject.

[0046] The present invention also relates to the above use, wherein the patient has a history of chronic CIPO or a history of CIPO secondary to a neurodegenerative disease or a demyelinating disease.

[0047] In addition, the present invention relates to the above-mentioned use, wherein at least one CIPO symptom (e.g., vomiting, abdominal distension, abdominal pain) or exacerbation of the condition (e.g., pseudo-obstruction) is alleviated or improved. In addition, the present invention also relates to the above-mentioned use, wherein at least one CIPO symptom selected from gastrointestinal motility abnormalities, increased dilatation of the proximal colon and / or distal small intestine, changes in intestinal contractility, ulcers, inflammation of the proximal colon and / or distal small intestine, pseudo-obstruction and mortality is alleviated or improved; and / or the number and / or frequency of hospitalizations related to and / or caused by CIPO is reduced.

[0048] In addition, the present invention relates to the above-mentioned use, wherein the dosage of vistrag administered is 0.5 mg to 30 mg, preferably 5 mg to 15 mg, based on the weight of the free base. These dosages refer to the daily dosage for ordinary adult patients. However, the dosage can be changed and / or adjusted according to the severity of the disease, the specific patient condition, the specific administration route selected.

[0049] The present invention further relates to the above-mentioned purposes, wherein the treatment duration can be changed and / or adjusted according to the severity of the disease or the specific patient condition. Preferably, the treatment is applied for at least 1 week to 24 weeks, preferably at least 24 weeks, preferably at least 2 weeks or at least 4 weeks, preferably at least 6 weeks, preferably at least 12 weeks, or at least 14 weeks, preferably at least 16 weeks, preferably at least 18 weeks, 20 weeks, 22 weeks. Preferably 24 weeks or at least 24 weeks, preferably 26 weeks, 28 weeks, 30 weeks or longer. Treatment can also be carried out in a repeated cycle, at least continuing 1 week to 24 weeks, preferably at least 24 weeks, preferably at least 2 weeks or at least 4 weeks, preferably at least 6 weeks, preferably at least 12 weeks, or at least 14 weeks, preferably at least 16 weeks, preferably at least 18 weeks, 20 weeks, 22 weeks. Preferably 24 weeks or at least 24 weeks, preferably 26 weeks, 28 weeks, 30 weeks or longer.

[0050] The administration dose of Vistrag is preferably 15 mg per day, wherein the dosage is taken once a day, preferably in the form of 3 x 5 mg tablets. The preferred administration route is oral administration.

[0051] In another embodiment of the present invention, pharmaceutical compositions for different routes of administration of Vistrag are contemplated for use in human patients. The routes of administration particularly include oral, parenteral, buccal, sublingual, rectal, intraperitoneal or intratracheal routes of administration. For example, parenteral administration may be performed by infusion, injection or implantation. Parenteral administration may also include administration subcutaneously, intramuscularly, intravenously, transdermally or by implantation routes. If Vistrag is used parenterally, it may be in liquid, solid or gel form. Similarly, if Vistrag is administered orally, it may be in the form of a liquid, capsule, tablet, chewable tablet or soluble film.

[0052] In one embodiment, the product comprises an amount of about 0.5 mg to about 30 mg of vistraglobulin labeled for treatment of symptoms of CIPO. In another embodiment, the product comprises about 0.5 mg to about 15 mg, about 0.5 mg to about 5 mg, about 5 mg to about 15 mg, or about 5 mg, or about 15 mg of vistraglobulin labeled for treatment of symptoms of CIPO.

[0053] In one embodiment, the present invention provides a kit comprising instructions for use and dosage on a package insert of a pharmaceutical product containing vistrag of the present invention. In another embodiment, the package insert instructs the patient to administer vistrag during the above treatment period.

[0054] Vistrag can be used in combination with other drugs that are not contraindicated with Vistrag. It is preferred that Vistrag not be taken together with opioids and / or other 5-HT4 receptor agonists (e.g., prucalopride, cisapride, clebopride, cilnipride). BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In the figure below, Normal = wild-type mice, and groups G2, G3, and G4 are PrP-SCA7-92Q transgenic mice.

[0056] Figure 1 : Shown are the dilatation of the distal small intestine (DSI) and proximal colon measured in diameter (mm) in two mouse CIPO models treated with 1 mg / kg and 3 mg / kg vistra. 1A: Rb1cKO mice (CKO) ***P<0.001 vs. normal group. 1B: PrP-SCA7-92Q transgenic mice. *P<0.05, ***P<0.001; one-way ANOVA; vs G2 (vehicle).

[0057] Figure 2 : Shown are the histological analysis scores of DSI and proximal colon in two mouse CIPO models treated with 1 mg / kg and 3 mg / kg vistrag (H&E total score is defined as in Example 3). 2A: Histological analysis scores of DSI and proximal colon in the CIPO model of Rb1cKO mice treated with vistrag *P<0.05, ***P<0.001; one-way ANOVA; vs vehicle. 2B: Histological analysis scores of DSI and proximal colon in the CIPO model of PrP-SCA7-92Q transgenic mice treated with vistrag, *P<0.05, ***P<0.001; T test; vs G2.

[0058] Figure 3: Protein levels of p-mTOR, mTOR, p-Akt, Akt, and p-P70S6 in the distal small intestine. The expression levels of p-mTOR and mTOR were normalized to actin. The expression levels of p-Akt and Akt were normalized to actin. The expression level of p-P70S6 was normalized to actin. The results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, vs vehicle group, n = 10.

[0059] 3A: refers to the fold changes in the expression levels of various proteins in DSI of wild-type (normal) and Rb1cKO (CKO) mice treated with 1 mg / kg and 3 mg / kg of vistragliflozin.

[0060] 3B: refers to the fold changes in the expression levels of various proteins in DSI of wild-type (normal) and PrP-SCA7-92Q transgenic mice (G2, G3, G4) treated with 1 mg / kg and 3 mg / kg of vistragliflozin.

[0061] Figure 4 : Quantitative analysis of MAP2 neurons in the DSI and proximal colon of two CIPO mouse models treated with 1 mg / kg and 3 mg / kg vistrag is shown. 4A: Rb1 cKO mice. 4B: PrP-SCA7-92Q transgenic mice. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, vs vehicle group, n = 10.

[0062] Figure 5 : Immunofluorescence of SOX10 and Hu+ neurons in DSI and colon of wild-type (normal) and Rb1cKO mice treated with vistra (1 mg / kg or 3 mg / kg). Quantitative analysis of ganglion neurons in the distal small intestine (A) and colon (B). Glial cells / Hu+ neuron ratio in the distal small intestine (C) and colon (D). Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, vs vehicle group, n=10.

[0063] Figure 6 : Shown is the 5HT4 receptor mRNA expression in DSI and colon of Rb1cKO mice treated with vistrag (1 mg / kg or 3 mg / kg). The results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, vs vehicle group, n=10.

[0064] Figure 7: Immunofluorescence colocalization of Ataxin-7 nuclear inclusions and Hu+ cytoplasmic markers in the distal small intestine and colon. Quantitative analysis of Hu+ neuron counts in the distal small intestine (A) and colon (B) of PrP-SCA7-92Q transgenic mice treated with Vistrag (1 mg / kg and 3 mg / kg). Percentage of colocalized cells in Hu+ cells in the distal small intestine (C) and colon (D). Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, vs vehicle group, n=10.

[0065] Figure 8 : Immunofluorescence of ataxin-7 and nNOS+ cytoplasmic markers in the distal small intestine and colon of PrP-SCA7-92Q transgenic mice treated with vistrag (1 mg / kg and 3 mg / kg). Quantitative analysis of nNOS+ neuron counts in the distal small intestine (A) and colon (B). Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, vs vehicle group, n=10.

[0066] Fig. 9 : Quantitative analysis of CHAT+ neuron counts in the distal small intestine (A) and colon (B) of Rb1cKO mice treated with vistraglobulin (1 mg / kg and 3 mg / kg).

[0067] Fig.10 : Immunofluorescence colocalization of ataxin-7 nuclear inclusions (red) with CHAT+ cytoplasmic markers in the distal small intestine and colon of PrP-SCA7-92Q transgenic mice treated with vistrag (1 mg / kg and 3 mg / kg). Quantitative analysis of CHAT+ neuron counts in the distal small intestine (A) and colon (B) of PrP-SCA7-92Q transgenic mice treated with vistrag (1 mg / kg and 3 mg / kg). Percentage of colocalized cells in CHAT+ cells in the distal small intestine (C) and colon (D). Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, vs vehicle group, n=10.

[0068] Fig.11 : Immunofluorescence staining of calretinin neurons in PrP-SCA7-92Q transgenic mice treated with vistrag (1 mg / kg and 3 mg / kg). Calretinin neurons in the distal small intestine (A) and calretinin neurons in the colon (B). Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, vs vehicle group, n = 10.

[0069] Fig.12: Shows the cytoplasmic expression of n-NOS and CHAT in the proximal colon of PrP-SCA7-92Q transgenic mice treated with vistrag (1 mg / kg and 3 mg / kg). 12A: Cytoplasmic nNOS / actin. 12B: Cytoplasmic CHAT / actin.

[0070] Fig.13 : Body weight of PrP-SCA7-92Q transgenic mice after 5 weeks of treatment.

[0071] Fig.14 : Immunofluorescence of SOX10 and Hu+ neurons in the small intestine and colon of wild-type (normal) and PrP-SCA7-92Q transgenic mice treated with vistra. Quantitative analysis of ganglion neurons in the distal small intestine (A) and colon (B). Glial cells / Hu+ neuron ratio in the distal small intestine (C) and colon (D). Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, vs vehicle group, n=10.

[0072] Fig.15 : mRNA expression of 5HT4 receptors in the distal small intestine and colon of wild-type (normal) and PrP-SCA7-92Q transgenic mice treated with vistragol. Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, vs vehicle group, n=10.

[0073] Fig.16 :Clinical study phase II scheme. Patients were randomly divided into 4 different groups and received 4 different treatment sequences. The study consisted of a 7-day screening period (day -7 to day -1), followed by 4 treatment periods, each of which lasted 4 weeks, in which subjects received 15 mg of vestragel (VEL) (2 cycles) or placebo (PLA) (2 cycles), with a 2-week washout period between treatment periods, followed by a 2-week follow-up period (about 175 days in total). Scheduled visits (V=visits) for the corresponding days after the start of the study were also shown.

[0074] Fig.17 : Line graph showing the mean WGGSAIS values ​​for subjects treated with vistraglobulin or placebo in the mFAS1 population (30 pairs of observations).

[0075] D7 = day 7; D14 = day 14; D21 = day 21; EoT = end of treatment; PLA = placebo; PRE = before treatment; VEL = vistraglobate; WO-D7 = washout on day 7; WO-D14 = washout on day 14.

[0076] Paired t-test: p value = 0.0310, 95% CI for the mean difference in change between vistraglita and placebo -0.741; -0.038.

[0077] Fig.18 : Line graph showing the mean of each individual symptom score for subjects treated with vistraglobulin or placebo in the mFAS1 population.

[0078] D7 = Day 7; D14 = Day 14; D21 = Day 21; EoT = End of Treatment; mFAS1 = Modified Full Analysis Set 1; PLA = Placebo; PRE = Before Treatment; VEL = Vistregr; WO-D7 = Day 7 Washout; WO-D14 = Day 14 Washout. Only data constituting evaluable pairs for each individual symptom, including Day 7 Washout and Day 14 Washout, were considered in the analysis. Paired t-test: p-value = 0.0164

[0079] Fig.19 : Line graph showing the mean values ​​of bowel habit scores for subjects treated with vistraglobulin or placebo in the mFAS1 population.

[0080] D7 = Day 7; D14 = Day 14; D21 = Day 21; EoT = End of Treatment; PLA = Placebo; PRE = Before Treatment; VEL = Vistregr; WO-D7 = Day 7 Washout; WO-D14 = Day 14 Washout. Only data constituting pairs evaluable for weekly bowel movements or complete voiding, which contained both Day 7 Washout and Day 14 Washout, were considered in the analysis. DETAILED DESCRIPTION

[0081] As shown above, Vistralig is a compound of Formula I and forms a crystalline hydrochloride salt of Formula II:

[0082]

[0083] The present invention generally relates to vistrag for use in the treatment of idiopathic chronic intestinal pseudo-obstruction (CIPO), neurogenic chronic intestinal pseudo-obstruction, or chronic intestinal pseudo-obstruction secondary to neurodegeneration or secondary to autoimmune diseases or secondary to connective tissue disorders or secondary to demyelinating diseases. In addition, the present invention relates to the above-mentioned use, wherein at least one CIPO symptom (e.g., vomiting, abdominal distension, abdominal pain, constipation, etc.) or exacerbation of the condition (e.g., pseudo-obstruction attack) is alleviated or improved.

[0084] By said use, the main symptoms of CIPO, i.e., gastrointestinal motility disorders, increased dilatation of the proximal colon and / or distal small intestine, changes in intestinal contractility, ulcers, inflammation of the proximal colon and / or distal small intestine, and lethality, can be alleviated or improved. In addition, the use of Vistreg according to the present invention allows for a reduction in the need for "artificial food" (i.e., parenteral nutrition at home or in hospital). Vistreg also reduces the number / frequency and duration of CIPO-related hospitalizations (i.e., hospitalizations directly caused by CIPO, hospitalizations directly caused by CIPO, and / or hospitalizations caused by complications of CIPO), hospitalizations caused by CIPO (i.e., hospitalizations directly caused by CIPO), and improves the quality of life.

[0085] To evaluate the preclinical pharmacodynamics of vistrag in neuropathic CIPO, two models were used. One of them is the Rb1 retinoblastoma knockout (Rb1cKO or CKO) mouse, which has a retinoblastoma mutation in the enteric nervous system. This model established by Fu et al. allows highlighting pseudo-obstruction associated with important neuronal dysfunction and altered intestinal contractility [Fu M, et al. Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction. J Clin Invest. 2013; 123: 5152–5164].

[0086] Retinoblastoma Rb1 cKO mice die prematurely due to pseudo-intestinal obstruction, so 50% of the mice die by postnatal day 30. From postnatal day 8 to 30, the distal small intestine (DSI) and proximal colon begin to dilate, and hard black fecal masses appear in the colon.

[0087] As in mice, patients are diagnosed with CIPO when defects in intestinal motility lead to functional rather than mechanical obstruction, resulting in abdominal distension, pain, malnutrition, and, in severe cases, the need for parenteral nutrition or intestinal transplantation for survival. A variety of genetic, infectious, autoimmune, metabolic, and toxic insults are likely causes of CIPO.

[0088] It is well known that intestinal motility is controlled by an interconnected intrinsic network of neurons and glial cells called the enteric nervous system (ENS). Normally, the ENS is formed by neural crest-derived cells that migrate through the fetal intestine, proliferate in large numbers, and then exit the cell cycle to differentiate into many different neuronal subtypes. If certain enteric neurons become defective, it can be life-threatening because neurons in the ENS must be present in the proper proportions for the intestine to function properly. The signals that control the proliferation and cell cycle exit of ENS precursors are not fully understood.

[0089] Based on the above observations, Rb1 retinoblastoma cKO mice were generated, because Rb1 plays a role in cell cycle checkpoints at S and G2 / M phases by preventing cells from entering S phase, thereby participating in cell cycle exit, maintaining development, terminal differentiation and tissue homeostasis, and its mutation leads to tissue-specific defects. Indeed, Rb1 inactivation in the ENS leads to progressive lethal defects in a subset of NO-producing enteric myoneural neurons, which inhibits intestinal contraction. These cells undergo endoreplication, forming large, irregularly shaped nuclei similar to those seen in progeria. In addition, Rb1 is also deleted in enteric glial cells and other types of enteric neurons that do not undergo endoreplication, highlighting the differential dependence of different cell types in the ENS lineage on Rb1.

[0090] In terms of translational evidence for this relevant Rb1 cKO mouse CIPO model, after only two weeks of treatment, vistrapol protected mice from mortality and antagonized glial activation, a marker of intestinal inflammation in cKO mice, in addition to reducing proximal colon distension. The effect of vistrapol in reversing intestinal distension was associated with a specific anti-inflammatory protective effect, which can reasonably be inferred to have disease-modifying effects in addition to its prokinetic effects.

[0091] Another preclinical pharmacodynamic model is the transgenic mouse PrP-SCA7-92Q, also referred to herein as the human ATXN7 transgenic mouse, which develops pseudo-obstruction and signs of visceral neuropathy [Clarke CM et al. Visceral neuropathy and intestinal pseudo-obstruction in a murine model of anuclear inclusion disease. Gastroenterology. 2007; 133(6): 1971–1978]. These animals exhibit many features of the human polyglutamine neurodegenerative disease, spinocerebellar ataxia type 7 (SCA7), as well as nuclear inclusions in a subset of cholinergic enteric ganglion cells that contain transgenic-derived ataxin-7 and pathogenic polyglutamine expansion. Transgenic mice develop marked dilatation of the distal small intestine beginning at 13 weeks of age.

[0092] Ataxin-7 inclusions were evident in the nuclei of a subset of enteric ganglion cells and myoenteric neurons that co-expressed the cytoplasmic marker choline acetyltransferase. The density of calretinin-immunoreactive myoenteric ganglion cells in the proximal colon was significantly reduced in transgenic mice at 13 weeks. Nerve fiber loss in the myoenteric plexus and delayed gastrointestinal transit were also observed. In this transgenic model, oral delivery of vistrag once daily for 5 weeks was shown to inhibit distal small intestinal distension, antagonize neurodegeneration of ChAT, nNOS, and calretinin neurons, and the appearance of ataxin-7 inclusions in enteric neurons.

[0093] Furthermore, visconazole increased the levels of cytoplasmic ChAT and nNOS in the proximal colon, suggesting the ability to alter neuronal plasticity toward normalization and enhance intestinal motility.

[0094] The doses of vistragliflozin for both preclinical mouse models were calculated based on the doses administered to patients during clinical development, i.e., 15 mg and 5 mg / patient [FDA Guidance US FDA, 2005; Nair and Jacob, 2016].

[0095] Furthermore, the effect of vistrag administration on activation of the PI3K / AKT / mTOR signaling pathway was evaluated in these two mouse models.

[0096] mTOR is a component of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which are widely distributed throughout the body and control multiple functions such as gene transcription, metabolism, cell survival, and cell senescence. mTOR promotes neuronal cell regeneration through stem cell renewal and oversees key pathways such as apoptosis, autophagy, and programmed necrosis to promote protection against neurodegenerative conditions through its relationship with phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (Akt) and multiple downstream signaling pathways such as p70 ribosomal S6 kinase (p70S6K) and 40 kDa proline-rich Akt substrate (PRAS40). Overall, mTOR is an important neuroprotective pathway, but it must be carefully targeted to maximize clinical efficacy and eliminate any clinical toxicity. [Maiese K. Driving neural regeneration through themammalian target of rapamycin. Neural Regen Res. 2014; 1; 9: 1413-7.]

[0097] Neurological diseases are often associated with changes or disruptions in homeostatic processes controlled by glial cells (even if not caused by them). In the ENS, these roles are played by a unique population of peripheral glial cells called enteric glial cells. Enteric glial cells regulate gastrointestinal motility through bidirectional communication with enteric neurons and contribute to the formation of neuroinflammation. Glial cell mechanisms can lead to gastrointestinal dysmotility, and data from animal models support this view, indicating that altered glial function disrupts motility, promotes neurodegeneration during acute colitis, and affects immune responses. However, the specific mechanism by which enteric glial cells cause dysmotility remains unclear. [Ahmadzai MM et al., J Clin Invest. 2022 Feb 15; 132(4): e149464]. The effects of vestregr on neurons and glial cells in the Rb1 retinoblastoma knockout (cKO) mouse model were evaluated by immunohistochemistry of distal small intestine and proximal colon samples using antibodies against the glial cell marker SOX10 and the neuronal protein HuCD, showing pseudo-obstruction associated with neuronal dysfunction and altered intestinal contractility, as well as signs of pseudo-obstruction and visceral neuropathy in the transgenic mouse PrP-SCA7-92Q model.

[0098] Experimental results showed that administration of vistra for 14 days could reduce the expression of relevant receptors, especially in the distal small intestine (DSI) area associated with neuronal dysfunction and altered intestinal contractility in the CIPO model.

[0099] RB1 cKO significantly induced DSI and colonic distension in mice. Surprisingly, the study found that in RB1 cKO mice treated with 1mg / kg and 3mg / kg of vistrag, there was a statistically significant trend towards suppression of colonic distension after administration with 3mg / kg. Prp-SCA7-92Q transgenic mice also showed increased DSI and colonic distension compared to normal mice. DSI distension was significantly improved after treatment with 1mg / kg or 3mg / kg of vistrag. Treatment with 1mg / kg or 3mg / kg of vistrag was able to reduce proximal colonic distension.

[0100] The number of glial cells and the ratio of glial cells to neurons were increased in the DSI and colon of RB1 cKO vehicle group mice. Surprisingly, the number of glial cells and the ratio of glial cells to neurons in the DSI and proximal colon were significantly reduced after treatment with 3 mg / kg visconazole. In Prp-SCA7-92Q or human ATXN7 transgenic mice, the ratio of glial cells to neurons in the DSI and colon of the vehicle group was significantly increased. The ratio of glial cells to neurons was significantly reduced after treatment with 1 mg / kg or 3 mg / kg visconazole.

[0101] The number of MAP2-stained neurons in the DSI and colon was reduced in the RB1 cKO vehicle group. After treatment with 1mg / kg and 3mg / kg of vistra, the number of MAP2-stained neurons in the DSI and proximal colon increased. In Prp-SCA7-92Q transgenic mice, the number of MAP2-stained neurons in the DSI and proximal colon was reduced in the vehicle group. After treatment with 1mg / kg and 3mg / kg of vistra, the number of MAP2-stained neurons in the DSI and proximal colon increased.

[0102] The number of CHAT-stained neurons in the DSI and colon was reduced in the RB1 cKO vehicle group. After treatment with 1 mg / kg and 3 mg / kg vistrag, the number of CHAT-stained neurons in the DSI and proximal colon increased.

[0103] In hematoxylin-eosin staining (HE), the DSI and colon of both the RB1 cKO vehicle group and the PrP-SCA7-92Q transgenic vehicle group showed obvious inflammatory infiltration and ulceration. Surprisingly, in both CIPO animal models, the disease scores of the 1mg / kg and 3mg / kg groups of Vistragliflozin were significantly reduced after treatment.

[0104] The 5HT4 receptor mRNA levels in DSI and colon were increased in the RB1 cKO vehicle group, but not significantly different. After treatment with vistrag 3 mg / kg, the 5HT4 receptor mRNA levels in DSI were significantly decreased.

[0105] The p-mTOR, p-AKT, p-P70S6, mTOR, and AKT proteins increased in the RB1 cKO vehicle group, among which the p-Akt, p-P70S6 proteins, and p-AKT / AKT ratio of DSI were significantly increased compared with the normal group. After treatment with 3 mg / kg vistrag, the p-mTOR, p-AKT, p-P70S6, mTOR, and AKT proteins were significantly decreased.

[0106] The p-Akt, Akt, p-P70S6, p-mTOR and mTOR protein levels of DSI in the PrP-SCA7-92Q transgenic vehicle group were increased, where p-Akt, p-P70S6 and Akt were significantly different compared to the normal group. The p-Akt / AKT ratio of DSI in the vehicle group was significantly increased. Treatment with 3 mg / kg visconazole reduced p-Akt, Akt, p-P70S6, p-mTOR and mTOR protein levels, where p-Akt, Akt and p-P70S6 were significantly different. Treatment with 1 mg / kg visconazole significantly reduced the p-mTOR / mTOR ratio.

[0107] The number of neurons stained with calretinin, Hu, nNOS, and CHAT in the DSI and colon of PrP-SCA7-92Q transgenic mice was reduced. After treatment with 1 mg / kg and 3 mg / kg vestragel, the number of neurons stained with calretinin, Hu, nNOS, and CHAT in the DSI and / or colon was significantly increased.

[0108] The unexpected experimental results further showed that both doses of Vistrag (i.e., 1 mg / kg and 3 mg / kg) were effective in improving weight loss, intestinal distension, and mortality, while the higher dose of 3 mg / kg was more effective in terms of inflammation and ulcers, restoring the state of healthy animals. As for the effects of Vistrag on neuronal parameters, such as the increase in nitrergic neurons or the increase in cholinergic neurons, both doses provided similar effects. Therefore, Vistrag is particularly effective in treating neurogenic CIPO, and in addition has a significant anti-inflammatory effect on the intestine.

[0109] Overall, the results of the experiments performed and presented herein indicate that vistragliflozin provides modulatory, neuroprotective and neurotrophic effects on the ENS in both CIPO animal models tested. The improvements in the enteric nervous system and the reduction in inflammatory symptoms following treatment suggest that vistragliflozin has value in the treatment of neuroenteric disorders associated with intestinal dysmotility disorders.

[0110] Based on the demonstrated technical efficacy, the proposed use of Vistrex is for the treatment of idiopathic chronic intestinal pseudo-obstruction, or chronic intestinal pseudo-obstruction secondary to neurodegeneration, or secondary to autoimmune diseases, or secondary to connective tissue disorders, or secondary to demyelinating diseases affecting the intestine. Diseases that may benefit from such use are idiopathic chronic intestinal pseudo-obstruction or chronic intestinal pseudo-obstruction secondary to neurodegeneration or secondary to autoimmune disease or secondary to connective tissue disorders or secondary to demyelinating diseases that affect intestinal motility by one or more of the following: autonomic nervous system diseases (i.e., stroke, encephalitis, basal ganglia calcification, orthostatic hypotension); intestinal wall nervous system diseases (i.e., paraneoplastic syndromes, viral infections, iatrogenic diseases, Hirschsprung's disease, Chagas disease, von Recklinghausen disease); intestinal wall muscular layer diseases (i.e., myotonic dystrophy, progressive systemic sclerosis); mixed intestinal nervous system and smooth muscle layer diseases (scleroderma, dermatomyositis, amyloidosis, Ehlers-Danlos syndrome); and mechanisms unknown (i.e., hypothyroidism, hypoparathyroidism, pheochromocytoma, antidepressants, antineoplastic drugs, bronchodilators).

[0111] Due to the technical effect provided by the use of Vistrag, syndromes caused by or characterized by inflammatory / immune infiltration of neurons located in the submucosal layer and myenteric ganglia of the enteric nervous system caused by cellular infiltration of circulating anti-neuronal antibodies, which leads to intestinal dysmotility, can be significantly improved from the above treatment. The treatment of the present invention is effective for both acute and chronic forms of the above forms of CIPO.

[0112] Pharmaceutical compositions and preparations

[0113] The present invention also relates to a pharmaceutical composition containing vistraglita or a pharmaceutically acceptable salt thereof for treating CIPO.

[0114] The term "pharmaceutically acceptable" refers to a substance that is not biologically or otherwise unacceptable when used in the present invention. For example, the term "pharmaceutically acceptable carrier" refers to a substance that can be incorporated into a composition and administered to a patient without causing an unacceptable biological effect or interacting with other components of the composition in an unacceptable manner. Such pharmaceutically acceptable substances generally meet the required standards of toxicology and manufacturing testing, and include materials identified by the U.S. Food and Drug Administration as suitable inactive ingredients.

[0115] The term "pharmaceutically acceptable salt" refers to a salt prepared from a base or an acid that is acceptable for administration to a patient (such as a mammal) (e.g., a salt with acceptable mammalian safety for a given dosage regimen). Pharmaceutically acceptable salts can be derived from pharmaceutically acceptable inorganic or organic bases, and pharmaceutically acceptable inorganic or organic acids. In addition, when a compound contains a basic moiety (such as an amine, pyridine or imidazole) and an acidic moiety (such as a carboxylic acid or tetrazole), zwitterions can be formed and are included in the term "salt" as used herein. Salts derived from pharmaceutically acceptable inorganic bases include ammonium salts, calcium salts, copper salts, ferrous salts, ferrous salts, lithium salts, magnesium salts, hexavalent manganese salts, divalent manganese salts, potassium salts, sodium salts, and zinc salts, etc. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines, and the like, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methyl reduced glucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. Salts derived from pharmaceutically acceptable inorganic acids include the following salts: boric acid, carbonic acid, hydrohalic acid (hydrobromic acid, hydrochloric acid, hydrofluoric acid or hydroiodic acid), nitric acid, phosphoric acid, aminosulfonic acid and sulfuric acid. Salts derived from pharmaceutically acceptable organic acids include salts of aliphatic hydroxy acids (e.g., citric acid, gluconic acid, glycolic acid, lactic acid, lactobionic acid, malic acid, and tartaric acid), aliphatic monocarboxylic acids (e.g., acetic acid, butyric acid, formic acid, propionic acid, and trifluoroacetic acid), amino acids (e.g., aspartic acid and glutamic acid), aromatic carboxylic acids (e.g., benzoic acid, p-chlorobenzoic acid, diphenylacetic acid, gentisic acid, hippuric acid, and triphenylacetic acid), aromatic hydroxy acids (e.g., o-hydroxybenzoic acid, p-hydroxybenzoic acid, 1-hydroxynaphthalene-2-carboxylic acid, and 3-hydroxynaphthalene-2-carboxylic acid), ascorbic acid, dicarboxylic acids (e.g., fumaric acid, maleic acid, oxalic acid, and succinic acid), glucuronic acid, mandelic acid, mucic acid, nicotinic acid, orotic acid, pamoic acid, acid), pantothenic acid, sulfonic acids (e.g., benzenesulfonic acid, camphorsulfonic acid, edisylic acid, ethanesulfonic acid, isethionic acid, methanesulfonic acid, naphthalenesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2,6-disulfonic acid, and p-toluenesulfonic acid), xinafoic acid, and the like.

[0116] The terms "treat" or "treatment" include preventing, alleviating, ameliorating, or alleviating symptoms associated with CIPO, as assessed by the change in the mean weekly Global Gastrointestinal Symptom Index score from the beginning to the end of each treatment period.

[0117] "Symptoms associated with CIPO" include abdominal pain, bloating, nausea, and vomiting.

[0118] According to the invention, a therapeutic effect is present if at least one therapeutic effect is observed:

[0119] - Change in waist circumference from the beginning to the end of each treatment period,

[0120] - An improvement of 1 point in the mean Global Gastrointestinal Symptom Index score per week from the beginning to the end of each treatment period,

[0121] - Changes in individual symptom scores for abdominal pain, bloating, nausea, and vomiting from the beginning to the end of each treatment period.

[0122] - Change in the number of bowel movements per week from the start to the end of each treatment period (only for subjects with a Bristol Scale type 1 or 2 at the start of the treatment period),

[0123] - the change in the number of complete voidings per week from the beginning to the end of each treatment period,

[0124] - Change in stool type on the Bristol Stool Scale from the beginning to the end of each treatment period,

[0125] - the weekly change in bowel habit satisfaction scores from the beginning to the end of each treatment period, measured on a scale of 0 to 10,

[0126] - change in oro-cecal transit time measured using the lactulose breath test from the start to the end of the first treatment period,

[0127] - The proportion of days with medication changes that relieved the main gastrointestinal symptoms of CIPO during each treatment period, washout period, and follow-up period (proportion of days with dose increases or decreases compared to the start of the treatment period, proportion of days with medication added or removed compared to the start of the treatment period,

[0128] - Change in quality of life (SF-12) from the beginning to the end of each treatment period,

[0129] - Number of CIPO-related hospitalizations during treatment,

[0130] - changes in demand for artificial foods,

[0131] - Change in the number of pseudo-obstruction episodes from the beginning to the end of each treatment period, based on the investigator's judgment,

[0132] - Change from the end of each treatment period to the end of the first and second weeks of the washout period, or during the follow-up period:

[0133] Weekly abdominal pain scores,

[0134] Weekly bloating scores,

[0135] Weekly nausea rating,

[0136] Weekly vomiting scores,

[0137] Average weekly overall gastrointestinal symptom index score,

[0138] Number of bowel movements per week,

[0139] The number of complete voidings per week,

[0140] Stool type on the Bristol Stool Scale,

[0141] Weekly bowel habit satisfaction score, 0 to 10 points.

[0142] The SF-12 Health Survey is a simplified version of its predecessor, the SF-36, which was derived from the International Medical Outcomes Study used to assess patients' quality of life [Gandek B. et al. Cross-Validation of Item Selection and Scoring for the SF-12 Health Survey in Nine Countries: Results from the IQOLA Project, Journal of Clinical Epidemiology, 1998; 51: 1171-1178].

[0143] The Bristol scale or chart is a clinical assessment tool developed in 1997 that is designed to categorize stool into seven groups. [Russo M. et al. Stool Consistency, but Not Frequency, Correlates with Total Gastrointestinal Transit Time in Children. The Journal of Pediatrics, 2013; 162: 1188-1192]. This tool categorizes a patient's excretions based on the shape and type of stool, which correlates well with the time it takes for food to pass through the gastrointestinal tract and be eliminated from the body as waste.

[0144] The term "unit dosage form" refers to physically discrete units suitable for administration to a patient, ie, each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic effect either alone or in combination with one or more other units.

[0145] The crystalline hydrochloride form of Vistrag is usually administered to patients in the form of a pharmaceutical composition. Such pharmaceutical compositions can be administered to patients by any acceptable route of administration, including but not limited to oral, rectal, vaginal, intranasal, inhalation, topical (including transdermal) and parenteral administration modes.

[0146] Therefore, in one aspect of the composition, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a therapeutically effective amount of a crystalline hydrochloride salt of a compound of formula I. Optionally, such pharmaceutical compositions may contain other therapeutic agents and / or compounding agents, if desired.

[0147] The pharmaceutical compositions of the present invention generally contain a therapeutically effective amount of a crystalline salt of the present invention. Typically, such pharmaceutical compositions will contain from about 0.1% by weight to about 95% by weight of an active agent; from about 1% by weight to about 70% by weight, such as from about 5% by weight to about 60% by weight of an active agent.

[0148] Any conventional carrier or excipient can be used in the pharmaceutical composition of the present invention. The selection of a specific carrier or excipient, or a combination of carriers or excipients will depend on the mode of administration for treating a specific patient or a specific type of medical condition or state. In this regard, it is entirely within the scope of a technician in the medical field to prepare a suitable pharmaceutical composition for a specific mode of administration. In addition, the ingredients of such compositions can be purchased from, for example, Sigma, PO Box 14508, St. Louis, MO 63178. As a further illustration, conventional formulation techniques are described in Remington: The Science and Practice of Pharmacy, 20th Edition, Lippincott Williams & White, Baltimore, Maryland (2000); and HC Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippincott Williams & White, Baltimore, Maryland (1999).

[0149] Representative examples of substances that can serve as pharmaceutically acceptable carriers include, but are not limited to, the following: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) celluloses such as microcrystalline cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, Olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffered saline; and (21) other nontoxic compatible substances employed in pharmaceutical compositions.

[0150] The pharmaceutical composition of the present invention is generally prepared by fully and uniformly mixing or blending the compound of the present invention with a pharmaceutically acceptable carrier and one or more optional ingredients. When necessary or desired, the resulting uniformly blended mixture can then be formed into tablets, capsules, pills, etc. or loaded into tablets, capsules, pills, etc. using conventional steps and equipment.

[0151] The pharmaceutical composition of the present invention is preferably packaged in unit dosage form. For example, such unit dosage form can be capsules, tablets, pills, etc.

[0152] In a preferred embodiment, the pharmaceutical composition of the present invention is suitable for oral administration. Suitable pharmaceutical compositions for oral administration can be in the form of capsules, tablets, pills, lozenges, cachets, sachets, stick-packs, dragees, powders, granules; or as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion; or in the form of an elixir or syrup, etc.; each containing a predetermined amount of a compound of the present invention as an active ingredient.

[0153] When intended for oral administration in a solid dosage form (ie, capsules, tablets, pills, etc.), the pharmaceutical compositions of the invention will generally comprise a compound of the invention as an active ingredient and one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate. Optionally or alternatively, such solid dosage forms may also contain: (1) fillers or extenders, such as starch, microcrystalline cellulose, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and / or sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and / or glyceryl monostearate; (8) adsorbents, such as kaolin and / or bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and / or mixtures thereof; (10) coloring agents; (11) buffering agents.

[0154] Release agents, wetting agents, coating agents, sweeteners, flavoring agents and aromatics, preservatives and antioxidants may also be present in the pharmaceutical composition of the present invention. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. Coating agents for tablets, capsules, pills and the like include those used for enteric coatings, such as cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), hydroxypropyl methylcellulose phthalate, methacrylate, cellulose acetate trimellitate (CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose succinate (HPMCAS) and the like.

[0155] If desired, the pharmaceutical composition of the invention can also be formulated to provide slow or controlled release of the active ingredient, for example, using hydroxypropylmethylcellulose in varying proportions; or other polymer matrices, liposomes and / or microspheres.

[0156] In addition, the pharmaceutical composition of the present invention may optionally contain an opacifier and may be formulated to release the active ingredient only in a certain portion of the gastrointestinal tract, or preferably in a certain portion of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in a microencapsulated form together with one or more of the above-mentioned excipients, if necessary.

[0157] Suitable liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Such liquid dosage forms typically include an active ingredient and an inert diluent, such as water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuranyl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. In addition to the active ingredient, the suspension may also contain suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth and mixtures thereof.

[0158] Alternatively, the pharmaceutical compositions of the present invention are formulated to be administered by inhalation. Suitable pharmaceutical compositions administered by inhalation will generally be in the form of an aerosol or powder. Such compositions are generally administered using a known delivery device, such as a metered dose inhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

[0159] When administered by inhalation from a pressurized container, the pharmaceutical compositions of this invention will generally contain the active ingredient and a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

[0160] Alternatively, the pharmaceutical composition may be in the form of capsules or cartridges (eg made from gelatin) containing the compound of the invention and a powder suitable for use in a powder inhaler. Suitable powder bases include, for example, lactose or starch.

[0161] The compounds of the present invention can also be administered transdermally using known transdermal delivery systems and excipients. For example, the compounds of the present invention can be mixed with penetration enhancers (e.g., propylene glycol, polyethylene glycol monolaurate, azacycloalkane-2-one, etc.) and then added to a patch or similar delivery system. If desired, other excipients, including gelling agents, emulsifiers, and buffers, can be used in such transdermal compositions.

[0162] The following formulations illustrate representative pharmaceutical compositions of the present invention:

[0163] Formulation Example A

[0164] Hard gelatin capsules for oral administration are prepared as follows:

[0165]

[0166] Representative steps : The ingredients were mixed thoroughly and then filled into hard gelatin capsules (260 mg composition per capsule).

[0167] Formulation Example B

[0168] Hard gelatin capsules for oral administration are prepared as follows:

[0169]

[0170] Representative steps The ingredients are mixed thoroughly and then passed through a No. 45 mesh U.S. sieve and filled into hard gelatin capsules (200 mg of the composition per capsule).

[0171] Formulation Example C

[0172] Capsules for oral administration are prepared as follows:

[0173]

[0174]

[0175] Representative steps : The ingredients are mixed thoroughly and then packed into gelatin capsules (310 mg composition per capsule).

[0176] Formulation Example D

[0177] Tablets for oral administration are prepared as follows:

[0178]

[0179] Representative steps : The active ingredient, starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. A solution of polyvinyl pyrrolidone is mixed with the resulting powder and the mixture is then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50° C. to 60° C. and then passed through a No. 18 mesh U.S. sieve. Sodium carboxymethyl starch, magnesium stearate and talc (previously passed through a No. 60 mesh U.S. sieve) are then added to the granules. After mixing, the mixture is placed on a tablet press and compressed to obtain tablets weighing 100 mg.

[0180] Formulation Example E

[0181] Tablets for oral administration are prepared as follows:

[0182]

[0183] Representative steps : The ingredients are mixed thoroughly and then compressed to form tablets (440 mg composition per tablet) preparation Example F

[0184] Single scored tablets for oral administration are prepared as follows:

[0185]

[0186] Representative steps : The ingredients are thoroughly blended and compressed to form single scored tablets (215 mg composition per tablet).

[0187] Formulation Example G

[0188] The suspension for oral administration is prepared as follows:

[0189]

[0190] Representative steps : The ingredients are mixed to form a suspension containing 10 mg of active ingredient per 10 mL of suspension.

[0191] Formulation Example H

[0192] The dry powder for administration by inhalation is prepared as follows:

[0193]

[0194] Representative steps : The active ingredient is micronized and then blended with lactose. The blended mixture is then loaded into a gelatin inhalation cartridge. The contents of the cartridge are administered using a powder inhaler.

[0195] Formulation Example I

[0196] A dry powder for administration by inhalation in a metered dose inhaler is prepared as follows:

[0197] Representative steps : A suspension containing 5% by weight of the salt of the invention and 0.1% lecithin is prepared by dispersing 10 g of the active compound as micronized particles with an average size of less than 10 μm in a solution formed by dissolving 0.2 g of lecithin in 200 mL of demineralized water. The suspension is spray dried and the resulting material is micronized into particles with an average diameter of less than 1.5 μm. The particles are loaded into cartridges together with pressurized 1,1,1,2-tetrafluoroethane.

[0198] Formulation Example J

[0199] The injectable formulation is prepared as follows:

[0200]

[0201] Representative steps : Mix the above ingredients and adjust the pH to 4±0.5 using 0.5N HCl or 0.5N NaOH.

[0202] Formulation Example K

[0203] Capsules for oral administration are prepared as follows:

[0204]

[0205] Representative steps : The ingredients were mixed thoroughly and then filled into gelatin capsules (size #1, white, opaque) (264 mg composition per capsule).

[0206] Formulation Example L

[0207] Capsules for oral administration are prepared as follows:

[0208]

[0209] Representative steps : The ingredients were mixed thoroughly and then filled into gelatin capsules (size #1, white, opaque) (148 mg composition per capsule).

[0210] Route of administration

[0211] The present invention also relates to acceptable routes of administering vistra to human patients, including, but not limited to, oral, parenteral, buccal, sublingual, rectal, intraperitoneal or intratracheal routes of administration. For example, parenteral administration can be performed by infusion, injection or implantation. Parenteral administration can also include administration subcutaneously, intramuscularly, intravenously, transdermally or by implantation routes. If vistra is used parenterally, it can be in liquid, solid or gel form. Similarly, if vistra is administered orally, it can be in the form of a liquid, capsule, tablet, chewable tablet or soluble film.

[0212] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention described herein.

[0213] Example

[0214] Animal models

[0215] Rb1 cKO retinoblastoma mice [Fu M, et al. Retinoblastoma protein prevents senteric nervous system defects and intestinal pseudo-obstruction. J Clin Invest. 2013; 123: 5152–5164] constitute a pseudo-obstruction model, especially a model with neuronal dysfunction. In this model, vistrag dissolved in saline was administered intraperitoneally (ip) at one of two doses (1 mg / 10 ml / kg or 3 mg / 10 ml / kg, n=10 animals / group) once a day for 14 days starting from day 15 after birth.

[0216] PrP-SCA7-92Q is described in La Spada AR et al., Polyglutamine-expanded ataxin-7 antagonizes CRX function and induces cone-rod dystrophy in a mouse model of SCA7. Neuron 2001; 31: 913–27. PrP-SCA7-92Q mice represent a model of human pseudo-ileus, specifically visceral neuropathy with intranuclear neuronal inclusions and pseudo-ileus. In this model, vistrapol was orally administered once daily for 5 weeks starting at 8 weeks after birth at one of two doses (1 mg / 10 ml / kg or 3 mg / 10 ml / kg, n=10 animals / group).

[0217] Modified mice were administered vehicle (saline) as a positive control. Unmodified mice were used as negative controls.

[0218] Statistical analysis

[0219] All data in Tables 1 to 17 are mean values ​​± SEM of 10 mice per group. Statistical comparisons were performed by ANOVA (Graph Pad Prism 9.3), where the P value compared with the normal group is represented by the symbol *, and the P value compared with the cKO vehicle is represented by the symbol °. In particular, *P<0.05, **p<0.01, ***p<0.001, ****P<0.0001 vs. normal group; °p<0.05, °°P<0.01, °°°P<0.001, °°°°P<0.0001, vs. cKO+vehicle.

[0220] Example 1 - Effect of Vistralig on Lethality of Animal Model Mice

[0221] C57BL / 6 (Cyagen, China) and Rb1 cKO retinoblastoma mice (Cyagen, China) were used with free access to food and water. Starting from day 15 after birth, vestregr was administered intraperitoneally at one of two doses (3 mg / 10 mL / kg or 1 mg / 10 mL / kg) once a day for 14 days. The survival rate of only 10 mice was monitored within 60 days after birth.

[0222] As reported in Table 1, 40% of the control group (KO animals treated with saline) died within 15 days after the start of treatment. In contrast, only 10% of the knockout mice treated with 1 mg / kg or 3 mg / kg of vistragliflozin died. Thus, vistragliflozin significantly improved mortality.

[0223] Furthermore, at both doses, vistragliflozin was well tolerated as the treated animals did not experience weight loss (Table 1).

[0224] Table 1. Effects of Vistregr on body weight and mortality of Rb1 knockout animals

[0225]

[0226] Similar results were observed in Prp-SCA7-92Q mice.

[0227] To assess whether vistragliflozin is well tolerated in this mouse model, body weights of Prp-SCA7-92Q mice treated with vehicle or 1 mg / kg or 3 mg / kg vistragliflozin were measured throughout treatment. A normal control (NC) group of wild-type mice that did not receive treatment was also examined. There were no significant differences between the groups ( Fig.13 ).

[0228] After 5 weeks of treatment, the body weight of transgenic mice showed no difference compared with the normal control group. Treatment with 1mg / kg and 3mg / kg of vistra was well tolerated, and no weight loss or death was observed during the entire treatment process ( Fig.13 ).

[0229] Example 2 - Effect of Vistregr on Intestinal Distension

[0230] C57BL / 6 and Rb1 cKO retinoblastoma mice (Cyagen, China) were used with free access to food and water. Starting from day 15 after birth, vistra was administered intraperitoneally at one of two doses (3 mg / 10 mL / kg or 1 mg / 10 mL / kg) once a day for 14 days until day 28.

[0231] C57BL / 6 mice (Cyagen, China) and Prp-SCA7-92Q transgenic mice were given free access to water and food, and were orally administered vistragliflozin at two doses (3 mg / 10 mL / kg and 1 mg / 10 mL / kg) once daily starting from 8 weeks after birth for 5 weeks until 13 weeks of age.

[0232] On the day after the last treatment, CO 2 Animals were sacrificed by inhalation. Distal small intestine (DSI) and proximal colon samples were collected and rinsed with PBS. The diameters of DSI and colon were measured using Image J (version 1.53c) according to the scale plate where the tissue was placed when the image was taken. The diameters of the distal small intestine (DSI) and proximal colon were evaluated in five areas and expressed in mm.

[0233] Compared with wild-type normal animals, Rb1 cKO mice showed intestinal distension, with an increase in DSI and proximal colon diameter. In Rb1 cKO mice, administration of vistrag at both tested doses reduced intestinal distension in both the distal small intestine and the proximal colon compared with untreated KO mice. The reduction was dose-dependent, with the effect being more pronounced with the larger dose of vistrag (approximately Figure 1 A and Table 2).

[0234] Table 2. Effects of vistrag on the diameters of the distal small intestine (DSI) and proximal colon in Rb1 cKO mice (expressed in mm).

[0235] Group DSI(mm) Proximal colon (mm) C57BL / 6 (normal), administered with vehicle (sterile saline solution) 1.29±0.01 1.38±0.01 Rb1cKO+Vehicle (sterile saline solution) 1.56±0.02*** 1.73±0.01**** Rb1cKO + Vistregr 1mg / 10mL / kg intraperitoneal injection 1.48±0.02** 1.65±0.01**** Rb1cKO + Vistregr 3mg / 10mL / kg intraperitoneal injection 1.44±0.01** 1.58±0.01***°

[0236] When compared to wild-type normal animals, Prp-SCA7-92Q transgenic mice exhibited intestinal distension, with increased DSI and proximal colon diameter. In Prp-SCA7-92Q transgenic mice, both doses of vistragliflozin significantly reduced distension of the distal small intestine and proximal colon compared to vehicle-treated transgenic mice. In the proximal colon, intestinal distension was restored to values ​​seen in healthy animals after treatment with 3 mg / 10 ml / kg vistragliflozin (Tables 3 and Figure 1 B).

[0237] Table 3. Effects of vestreglo on intestinal distension in Prp-SCA7-92Q transgenic mice

[0238] Group DSI(mm) Proximal colon (mm) C57BL / 6 (normal), administered with vehicle (sterile saline solution) 1.61±0.2 1.72±0.2 Prp-SCA7-92Q+carrier 1.81±0.01* 1.9±0.03 Prp-SCA7-92Q + Vistragl 1 mg / kg oral administration 1.44±0.02°°°° 1.75±0.01 Prp-SCA7-92Q + Vistragl 3 mg / kg oral administration 1.44±0.01°°°° 1.72±0.01

[0239] Vistrepol at both 1 mg / kg and 3 mg / kg doses significantly antagonized the normalization of dilation by normalizing DSI dilation at both doses ( Figure 1 B).

[0240] Example 3 - Effect of Vistralig on Intestinal Morphology (Anti-inflammatory Activity)

[0241] By CO 2 After the mice were killed by inhalation, the small intestine and colon tissues were cut and fixed with 10% neutral buffered formalin for 48 hours. According to standard methods known in the art, paraffin samples were prepared by dehydrating with a series of gradient alcohol solutions. The tissue was cut into 4 μm slices with a rotary slicer and baked at 60 ° C for 2 hours. Through the fully automatic digital pathology section system (KFBIO, KF-PRO-005, China), K-viewer imaging software (KF BiO, V1, China) was used to obtain hematoxylin-eosin (HE) images from each animal at 20x magnification for histological analysis. The ulcers (statistical area of ​​intestinal mucosal damage), inflammation and bleeding of the pathological characteristics were evaluated and scored according to the following table.

[0242] Histological analysis scoring

[0243]

[0244] The anti-inflammatory activity of Vistregr was evaluated by histological analysis of intestinal samples from mice in both models. The presence of inflammation and ulceration was quantified using indices (i.e., ulceration = percentage of intestinal mucosal lesion area, inflammation = necrosis, hemorrhage) with a score of 0 to 3. Inflammation and ulceration scores were derived after section evaluation and analysis.

[0245] All measured parameter values ​​of DSI and proximal colon diameter in Rb1 cKO mice showed increased values ​​compared to wild-type normal animals. Both doses of vistragliflozin reduced inflammation and ulceration in Rb1 cKO mice compared to untreated KO animals (positive control). Treatment with 3 mg / 10 ml / kg vistragliflozin was able to restore the parameters of the distal small intestine and proximal colon to the values ​​of healthy animals (WT, normal mice) (negative control) (Table 4 and Figure 2 A).

[0246] Table 4. Effects of vistraglobulin treatment on intestinal morphology assessment in Rb1 cKO mice

[0247]

[0248] Likewise, the values ​​of all measured parameters of DSI and proximal colon diameter in Prp-SCA7-92Q mice showed increased values ​​compared to wild-type normal animals. In Prp-SCA7-92Q mice, both doses of vestregl determined reduced inflammation and ulceration compared to the positive control, and the 3 mg / 10 ml / kg dose of vestregl significantly improved the values ​​of the distal small intestine and proximal colon compared to the positive control (Tables 5 and Figure 2 B).

[0249] Table 5. Effects of vistraglobulin treatment on intestinal morphology assessment in Prp-SCA7-92Q transgenic mice

[0250]

[0251] Figure 2 A and 2B also show the total scores of the two models.

[0252] These results indicate that CKO and Prp-SCA7-92Q transgenic mice displayed disrupted intestinal structural integrity, and administration of vistragliflozin effectively ameliorated intestinal abnormalities in a dose-dependent manner (P<0.05 and P<0.001 for the two doses, respectively).

[0253] Example 4 - Activity of Vistralig on AKT / mTOR / p70s6k signaling pathway

[0254] The effect of vistrag administration on the activation of the PI3K / AKT / mTOR signaling pathway was evaluated by Western blot analysis of extracts from distal small intestine samples of treated animals using specific antibodies against the proteins: p-Akt, Akt, p-mTOR, m-TOR, p-P70S6, Actin.

[0255] On the day after the last treatment, CO 2 The animals were killed by inhalation.

[0256] According to the manufacturer's instructions, distal small intestinal tissue samples were collected and placed in 10 volumes (w:v) of lysis buffer (Beyotime Biotechnology, China) containing a protease and phosphatase inhibitor cocktail (ThermoScientific, USA). Tissue samples were homogenized on ice for 15 seconds, incubated on ice for 30 minutes, and then centrifuged at 15,000×g for 15 minutes. The supernatant was collected and the protein concentration was determined by the BCA method.

[0257] Proteins were separated on 10% SDS-PAGE gel and transferred to NC membrane. After blocking with 5% BSA in TBST (0.05% Tween-20) for 2 hours at room temperature, the membrane was incubated with the following primary antibodies at 4°C overnight (not less than 15 hours): p-Akt 1:1000 (Cell Signaling Technology, 4060S, USA), Akt 1:1000 (Cell Signaling Technology, 9272, USA), p-mTOR 1:1000 (Cell Signaling Technology, 5536S, USA), mTOR 1:1000 (Cell Signaling Technology, 2972, USA), p-P70S6 1:1000 (Cell Signaling Technology, 9204, USA), nNOS 1:1000 (Abcam, ab76067, USA), ChAT 1:1000 (Millipore, AB144P, USA), actin 1:1000 (Beyotime, AF5003, China) or MAPK 1:1000 (Cell Signaling Technology, 4695, USA). The blots were washed with TBST (3 times, 10 minutes each) and incubated with the following secondary antibodies: goat anti-rabbit IgG H&L ( 800CW) pre-adsorbed at 1:5000 (Abcam, 216773, USA) or donkey anti-goat IgG H&L ( 800CW) 1:5000 (Abcam, 216775, USA) was adsorbed at room temperature for 1 hour. After washing, the membrane was placed in the substrate working solution for 5 minutes and then imaged using the ChemiDoc system (Bio-Rad, 12003154, USA). Among these biomarkers, Akt and mTOR were re-probed after membrane stripping using RestoreTM Western Blot Stripping Buffer (Thermo Fisher Scientific, 21059, USA) according to the manufacturer's protocol. Densitometry analysis of protein bands was performed using QuantityOne software version 4.6.2.

[0258] Compared with wild-type C57Bl / 6 mice, increased expression levels of pAKT, mTOR, p-mTOR, and p70SKal were observed in Rb1 knockout mice treated with vehicle. Treatment with 3mg / 10ml / kg of vestragel inhibited signaling activation, resulting in decreased protein levels of the AKT / mTOR / p70s6k pathway compared with the positive control. The 1mg / kg dose reduced protein expression compared with the positive control, but not significantly (Table 6).

[0259] Table 6. Effects of vistrag on AKT / mTOR signaling pathway in DSI of RB1 cKO mice

[0260]

[0261] Increased expression levels of pAKT, mTOR, p-mTOR, and p70SKal were observed in Prp-SCA7-92Q transgenic mice treated with vehicle compared to wild-type C57Bl / 6 mice. In this model, the highest dose of vistrag resulted in decreased protein levels of the AKT / mTOR / p70s6k pathway compared to the positive control (Prp-SCA7-92Q transgenic mice treated with vehicle). The 1 mg / Kg dose did not appear to have any effect, as values ​​were similar and in some cases even higher compared to the positive control (Table 7).

[0262] Table 7. Effects of Vistrepol on the AKT / mTOR signaling pathway in DSI in Prp-SCA7-92Q transgenic mice

[0263]

[0264] These results are also Figure 3A (Rb1cKO mouse model) and 3B (PrP-SCA7-92Q transgenic mouse model).

[0265] Example 5 - Effect of Vistregr on MAP2 dendrite strength

[0266] Microtubule-associated protein 2 (MAP2) immunostaining mainly visualizes the perinuclear dendritic domains and proximal portions of axons of porcine enteric neurons, thus enabling unequivocal immunocytochemical identification of enteric multi(short) dendritic monoaxial type I neurons. The effects of vestregr treatment on enteric neuronal dendrites were evaluated in whole-mount samples isolated from two CIPO model animals treated as described above.

[0267] On the day after the last treatment, CO 2 Mice were sacrificed by inhalation. Distal small intestine (DSI) and proximal colon samples were collected, washed with PBS, and subsequently subjected to immunohistochemical analysis.

[0268] The tissue was cut along the edge of the mesentery and fixed on one side of a Sylgard plate. After fixation with 4% paraformaldehyde for 30 minutes, the muscle layer was separated from the mucosa and submucosa using a pointed nickel under a dissecting microscope.

[0269] DSI and colon samples were cut into 1 cm segments and stored in 50% glycerol / PBS at -20°C until staining and analysis. Myenteric nerve plexus preparations were washed 3 times with PBST (PBS + 0.5% Triton X 100) and processed as follows:

[0270] The whole tissue was washed three times with PBST (PBS + 0.5% Triton X-100).

[0271] Incubate the tissue with stripping buffer at 37 °C for 40 min.

[0272] The whole tissue was washed three times with PBST (PBS + 0.5% TritonX-100).

[0273] The tissues were incubated with the primary antibody MAP2 at 1:1000 (Abcam, USA) at 4°C overnight.

[0274] The tissue was washed three times with TBST (PBS + 0.5% Tritonx-100 + 0.5% Tween-20).

[0275] The tissues were incubated with the secondary antibody F(ab')2-goat anti-rabbit HRP 1:1000 (Abcam, USA) at room temperature for 1 hour.

[0276] The tissue was washed three times with PBST (PBS + 0.5% Triton X-100).

[0277] The tissues were incubated with TSA670 1:200 (Wi See Biotechnology, China) for 1 h at room temperature in the dark.

[0278] The tissue was washed three times with PBST (PBS + 0.5% Triton X-100).

[0279] The tissues were incubated with DAPI 1:5000 (Invitrogen, USA) for 10 min at room temperature.

[0280] Use a drop of ProLong TM The coverslips were mounted with glass antifade mounting medium (Invitrogen, USA).

[0281] Tissue samples were excited with lasers, and excitation and barrier filters were set according to the specific excitation / emission spectra of each fluorophore. Images of each animal were acquired using a confocal scanning microscope (Operetta CLS high Content Analysis System, PerkinElmer, USA), and cell counting was performed using a water immersion x20 objective. MAP2+ cells were counted.

[0282] The results are shown in Table 8.

[0283] Table 8. Effects of Vistrepol on MAP2 dendrite strength in Rb1 cKO mice

[0284]

[0285] The number of MAP2-positive neurons in the DSI and proximal colon was significantly reduced in Rb1 cKO mice compared with the wild-type group (DSIP < 0.05; colon P < 0.01). The number of MAP2-positive neurons in the DSI and proximal colon of Rb1 cKO mice treated with visconazole (at a dose of 1 mg / kg or 3 mg / kg) was significantly increased compared with cKO animals treated with the vehicle (DSI: 1 mg / kg group, P < 0.05; 3 mg / kg group, P < 0.01; colon: 1 mg / kg group, P < 0.05). Then, in the DSI, visconazole administration restored the reduction of MAP2-positive neurons to a level comparable to that of wild-type animals. This effect was slightly lower in the proximal colon (Table 8).

[0286] The same immunostaining experiments were performed on whole-mount tissue samples of intestine isolated from Prp-SCA7-92Q transgenic mice. The results showed that the transgenic animals had reduced dendrites compared to wild-type (control) animals (Table 9).

[0287] Table 9. Effect of Vistregr on the Dendritic Strength of Prp-SCA7-92Q MAP2 Transgenic Mice

[0288]

[0289] The rearrangement of the enteric neuronal network in PrP-SCA7-92Q mice treated with vehicle alone was also observed by immunostaining the intestinal myenteric plexus with MAP2, a cytoskeletal regulator within neuronal dendrites that is involved in the regulation of neurite outgrowth, synaptic plasticity, and protein folding / transport and is a strong somatodendritic marker ( Figure 4B). The number of MAP2+ neurons in the DSI was significantly reduced in transgenic mice treated with vehicle compared with wild-type controls (P<0.001), only in the DSI but not in the proximal colon, suggesting a location-specific reduction in somatodendritic reduction. Vistraligat at doses of 1 and 3 mg / kg had little effect on MAP2 staining ( Figure 4 B).

[0290] Figure 4 A and 4B show the effects of vistrag on DSI and the number of MAP2+ neurons in the proximal colon of mouse model animals. 4A: Rb1cKO mice (*P<0.05, **P<0.01; T test; vs vehicle CKO). 4B: PrP-SCA7-92Q transgenic mice (***P<0.001; one-way ANOVA; vs G2).

[0291] Example 6 - Detection of the effect of vestragel on neurons and glial cells by whole-body immunostaining

[0292] The effects of vestregr on neurons and glial cells in Rb1 retinoblastoma knockout mice were evaluated by immunofluorescence analysis of distal small intestine and proximal colon samples using antibodies against the glial cell marker SOX10 and the neuronal protein HuCD.

[0293] On the day after the last treatment, CO 2Animals were sacrificed by inhalation. Distal ileum and colon segments were placed in phosphate buffer and the mucosa and submucosa were manually removed with fine forceps. The muscularis propria and the encased myenteric nerve plexus were fixed in ice-cold acetone for 10 minutes and then immersed in 1X PBS, followed by blocking of the tissue in 1.5% BSA blocking buffer and 1% Triton X-100 in PBS for 2 hours at room temperature. The samples were incubated with primary antibody against glial cell protein SOX10 (Invitrogen, USA) (1:250) at +4°C overnight; then incubated with F(ab')-goat anti-rabbit HRP secondary antibody (Abcam, USA) (1:1000) at room temperature for 1 hour; washed three times with PBST (PBS + 0.5% Triton X-100), incubated with fluorescent dye TSA520 (Wi See Biotechnology, China) (1:200) at room temperature for 1 hour, avoiding direct light, and then washed three times with PBST (PBS + 0.5% Triton X-100). The neuronal marker HuCD was detected by the following method: incubating with primary antibody HuCD (Abcam, USA) (1:500) at room temperature for 1 hour, washing 3 times with TBST (PBS + 0.5% Triton X-100 + 0.5% Tween 20), incubating with secondary antibody F(ab')-goat anti-rabbit HRP (1:1000) at room temperature for 1 hour, washing 3 times with PBST (PBS + 0.5% Triton X-100); incubating with fluorescent dye TSA570 (Wi See Biotechnology, China) (1:200) at room temperature to avoid direct light for 1 hour; washing 3 times with PBST (PBS + 0.5% Triton X-100); incubating with DAPI (1:5000) at room temperature for 10 minutes. Images were taken with a microscope (Olympus, BX53). Tissue samples were excited with excitation lamps (UW, BWA and GW) and excitation / emission spectra (340-390nm / 420IF; 460-495nm / 510-550nm; 530-550nm / 575IF). SOX10+ and HuCD+ antibodies were conjugated with fluorescent dyes TSA520 and TSA570, with specific excitation / emission spectra of 488 / 519nm and 555 / 570nm. SOX10+ (green) and HuCD+ (red) cells were counted and evaluated per mm 2 The number of SOX10+ and HUCD+ neurons.

[0294] Immunofluorescence analysis of distal ileum and colon segments showed that vistrag reduced glial cells (SOX10+ cells) and the glial cell / neuron ratio. Thus, it reduced inflammation and gastrointestinal dysfunction. Examination of glial and neuronal markers showed that glial cell marker signals (SOX10) and glial cell / neuron ratios (SOX10 / HuCD) were reduced in the distal small intestine and proximal colon at both doses of vistrag compared with untreated knockout animals, but this reduction was more pronounced when the higher dose of vistrag was administered in both intestinal regions (Table 10).

[0295] Table 10. Effects of vestregr on glial cells and glial cell / neuron ratio

[0296]

[0297] Figure 5 Shown are the effects of vistraglobulin treatment on DSI and proximal colonic myenteric ganglia in Rb1 cKO mice (*P<0.05, **P<0.01, ***P<0.001; one-way ANOVA; vs vehicle CKO).

[0298] Neurodegeneration was also investigated by counting the number of neurons and glial cells in whole-mount preparations of the intestine from the myenteric plexus of transgenic treated and untreated mice using pan-neuronal (HuCD) or glial (SOX10) markers ( Fig.14 ). Immunofluorescence staining showed a significant increase in glial cells in the proximal colon of transgenic mice treated with vehicle compared with NC mice, along with a decrease in neuronal cells in the small and large intestines (P<0.001 and P<0.01, respectively). Correspondingly, the ratio of glial cells / Hu+ neurons was increased in both intestinal regions (P<0.001). Both doses of vistra treatment appeared to counteract neuronal degeneration, as shown by the ratio of HuCD:glial cells in the small and large intestines.

[0299] Example 7 - Effect of Vistralig on the serotonin receptor 5-HT4 in enteric neurons of RB1 cKO mice

[0300] The effects of vistrag administered at two tested doses were analyzed by assessing changes in 5-HT4 receptor mRNA levels in enteric neurons of the distal small intestine and proximal colon of wild-type and RB1 knockout mice.

[0301] TRIzol reagent (Invitrogen, USA) was used to measure the concentration of 100 μg / mL of 40 μg / mL of CO from the day after the last treatment. 2Total RNA was extracted from the distal small intestine and proximal colon of mice sacrificed by inhalation. 1 ml of TRIzol was added to the sample (approximately 50 mg of tissue). TM Reagent, using tissue lyser (60 Hz, 1 min, cold), then incubate for 5 min to allow complete dissociation of nucleoprotein complexes. The resulting lysate was centrifuged at 12,000 × g for 5 min at 4°C, and the clear supernatant was transferred to a new tube.

[0302] Chloroform (0.2 mL) was added, mixed gently and incubated at room temperature for 5 minutes.

[0303] After centrifugation at 12,000 x g for 15 min at 4°C, the mixture was separated into a red phenol-chloroform lower phase, an interphase, and a colorless upper aqueous phase (containing RNA), and the RNA was transferred to a new tube.

[0304] Genomic DNA contamination was eliminated using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Japan) according to the manufacturer’s instructions, and mRNA was reverse transcribed into cDNA for real-time RT-PCR (qPCR).

[0305] The obtained cDNA was used as template and 5-HT 4 The specific primers for the receptor coding region were placed in a real-time PCR instrument (Bio-Rad, 1855484, USA) and quantitative PCR reaction (qPCR assay, Applied Biosystems, USA) was performed according to the following reaction settings.

[0306]

[0307] The results showed that the 5-HT4 receptor mRNA level was slightly increased in the Rb1 cKO group in both DSI and colon. After treatment with 3 mg / kg vistrag, the 5-HT4 receptor mRNA level was significantly decreased in DSI. In the proximal colon, no differences between the different groups were observed (Table 11).

[0308] Table 11. Vistrepol for 5-HT 4 Role of receptor subtypes

[0309]

[0310] Treatment with vistragliflozin for 14 days reduced the expression of relevant 5-HT4 receptors, particularly in the DSI region of Rb1 cKO animals.

[0311] Figure 6The results of the effect of vistragliflozin on serotonin receptor mRNA in enteric neurons of Rb1 cKO mice are shown (*P<0.05; T test; vs vehicle CKO).

[0312] To investigate whether desensitization of 5HT4 receptors was evident after 5 weeks of vistragol treatment, the mRNA expression levels of 5HT4 serotonin receptors in DSI and colon tissues of treated and untreated groups were also investigated. Notably, vehicle-treated transgenic mice showed significantly different levels of 5HT4R compared to controls. Administration of vistragol in transgenic mice did not alter this difference ( Fig.15 ).

[0313] Example 8 - Effects of Vistralig on Total Neurons and Ataxin-7 Inclusions in the Prp-SCA7-92Q Mouse Model

[0314] Immunofluorescence analysis of DSI and proximal colon was performed using primary antibodies against HUC / D and Ataxin-7 proteins to detect the presence of neurons in the myenteric plexus and ataxin inclusions typical of the model. Samples were obtained from the day after the last treatment with Vistragel as described in Example 5 by CO 2 Animals killed by inhalation.

[0315] After incubation in blocking buffer, tissue samples were incubated with HuC / D primary antibody (Abcam, USA) (1:500) at room temperature for one hour, followed by incubation with secondary antibody F(ab')2 goat anti-rabbit HRP (Abcam, USA) (1:1000) at room temperature for one hour. Tissues were washed three times with PBST (PBS + 0.5% Triton X-100) and then incubated with TSA520 (1:200) at room temperature for one hour. Tissues were washed three times with PBST (PBS + 0.5% Triton X-100). Primary antibody Ataxin-7 (ThermoFisher, USA) (1:2000) was used for incubation overnight at 4°C, followed by incubation with secondary antibody F(ab')2-goat anti-rabbit HRP (1:1000) at room temperature for one hour to detect Ataxin inclusion bodies. The tissue was incubated with TSA570 (Wi See Biotechnology, China) (1:200) at room temperature for 1 hour and washed three times with PBST (PBS + 0.5% Triton X-100). The tissue was incubated with DAPI (1:5000) at room temperature for 10 minutes. TMGlass Antifade Mountant (Invitrogen, USA) was dropped over the sample and the image was captured with a microscope (Olympus, BX53). Tissue samples were excited with an excitation lamp and barrier filters (UW, BWA and GW), and the excitation / emission spectra were set (340-390nm / 420IF; 460-495nm / 510-550nm; 530-550nm / 575IF). HUC / D and Ataxin-7 antibodies were conjugated with fluorescent dyes TSA520 and TSA570, whose specific excitation and emission spectra were 488 / 519nm and 555 / 570nm, respectively. HuCD (green) and cells co-expressing with ataxin-7 (yellow) were counted.

[0316] The following primary antibodies were used: Hu C / D 1:500 (Abcam, Ab184267, USA); Ataxin-7 1:2000 (ThermoFisher, PA1-749, USA), nNOS 1:500 (Abcam, ab76067, USA), ChAT 1:1000 (Abcam, Ab181023, USA), Calreticulin 1:500 (Merk, MAB1568, USA) and SOX10 1:250 (Invitrogen, MA5-32398, USA).

[0317] Secondary antibodies included F(ab')2-goat anti-rabbit HRP 1:1000 (Abcam, Ab6013, USA), donkey anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 1:500 (Invitrogen, A-21202, USA), donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 594 1:500 (Invitrogen, A-21207, USA). Fluorescent staining used TSA520 1:200 (Wi See Biotechnology, D11013, China), TSA570 1:200 (Wi See Biotechnology, D11011, China).

[0318] The data showed that vistragliflozin was able to counteract neurodegeneration at both doses tested and in DSI and proximal colon. Vistragliflozin also reduced mutant ataxin-7 levels in pan-neurons (Table 12).

[0319] Table 12. Effects of vestreglo on total neurons and ataxin-7 inclusions

[0320]

[0321] To investigate whether this Tg strain accumulates Ataxin-7-positive aged neuronal inclusions, immunofluorescence was performed on whole-mount preparations of the DSI and proximal colon of 13-week-old PrP-SCA7-92Q male mice ( Figure 7 AD). Double staining using ATX-7 and the neuronal marker Hu C / D revealed that approximately 40% of enteric neurons in DSI and approximately 30% of enteric neurons in the proximal colon colocalized with Ataxin-7-positive nuclear inclusions. Surprisingly, Vistregrin significantly reduced these percentages.

[0322] Example 9 - Effects of Vistralig on Nitrergic Neurons in the Prp-SCA7-92Q Mouse Model

[0323] Enteric neurons express nitric oxide synthase nNOS, which catalyzes the production of NO from oxygen and arginine. NO acts as a neurotransmitter responsible for regulating gastrointestinal (GI) motility. Overproduction of NO also impairs normal GI motor activity under certain inflammatory conditions. NOS1+ neurons are in close contact with smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs); it has been observed that loss or damage of these types of neurons leads to impaired motility in the developing GI tract.

[0324] Immunohistochemical analysis of DSI and proximal colon samples using specific nNOS and Ataxin-7 primary antibodies detected the presence of nitrergic neurons and Ataxin inclusions in the myenteric plexus of Prp-SCA7-92Q transgenic animals treated with Vistralig.

[0325] Samples were prepared from C57BL / 6 mice (control) and Prp-SCA7-92Q transgenic mice (Cyagen, China) with free access to water and food. Starting from 8 weeks after birth, vistra was orally administered at two doses (3 mg / 10 mL / kg or 1 mg / 10 mL / kg) once a day for 5 weeks until 13 weeks of age. The mice were treated with CO as described in Example 5 on the second day of the last vistra treatment. 2 Animals were sacrificed by inhalation and treated with specific antibodies.

[0326] After fixation with 4% paraformaldehyde for 30 minutes, the muscularis containing the myenteric plexus was separated from the mucosa and submucosa using a pointed nickel. The samples were washed three times with PBST (PBS + 0.5% Triton X-100) and incubated with blocking buffer (1.5% BSA and 1% Triton X-100 in PBS) for 2 hours at room temperature, and then incubated with nNOS primary antibody (Abcam, USA) (1:500) at room temperature for 1 hour. The tissue samples were washed three times with TBST (PBS + 0.5% Triton X-100 + 0.5% Tween-20) and incubated with F(ab')2-goat anti-rabbit HRP secondary antibody (Abcam, USA) (1:1000) at room temperature for 1 hour. The samples were washed three times with PBST (PBS + 0.5% Triton X-100) and incubated with TSA520 (WI See Biotechnology, China) (1:200) at room temperature for one hour. Ataxin inclusion bodies were detected by incubating with Ataxin-7 primary antibody (Thermo Fisher, USA) (1:2000) at 4°C overnight. The samples were washed three times with TBST (PBS 0.5% Triton X-1000.5% Tween-20). The samples were incubated with F(ab')2-Goat anti-Rabbit HRP secondary antibody (Abcam, USA) (1:1000) at room temperature for 1 hour and washed three times with PBST (PBS + 0.5% Triton X-100). The samples were incubated with TSA570 (Wi See Biotechnology, China) (1:200) at room temperature for one hour, washed three times with PBST (PBS + 0.5% Triton X-100), and incubated with DAPI (1:5000) at room temperature for 10 minutes. ProLong TM Glass Antifade Mountant (Invitrogen, US) was dropped to cover the tissue, and the image was taken with a microscope (Olympus, BX53). The tissue samples were excited with an excitation lamp and barrier filters (UW, BWA and GW), and the excitation / emission spectrum was set (340-390nm / 420IF; 460-495nm / 510-550nm; 530-550nm / 575IF). nNOS and ataxin-7 antibodies were combined with fluorescent dyes TSA520 and TSA570, and the specific excitation and emission spectra were 488 / 519nm and 555 / 570nm, respectively, and nNOS (green) and cells co-expressing ataxin-7 (yellow) were counted. The data are shown in Table 13.

[0327] The following primary antibodies were used: nNOS 1:500 (Abcam, ab76067, USA), ChAT 1:1000 (Abcam, Ab181023, USA), (calretinin 1:500 (Merk, MAB1568, USA) and SOX10 1:250 (Invitrogen, MA5-32398, USA).

[0328] Secondary antibodies included F(ab')2-goat anti-rabbit HRP 1:1000 (Abcam, Ab6013, USA), donkey anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 1:500 (Invitrogen, A-21202, USA), donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 594 1:500 (Invitrogen, A-21207, USA). Fluorescent staining was performed using TSA520 1:200 (Wi See Biotechnology, D11013, China), TSA570 1:200 (Wi See Biotechnology, D11011, China)

[0329] Table 13. Effects of vestregr on nitrergic neurons

[0330]

[0331] Figure 8 The results showed that the number of NOS+ neurons in both regions was significantly reduced in the transgenic model. In addition, both doses of vestregr increased the number of nitrergic neurons in both regions compared with untreated transgenic mice. The effect was slightly higher in the proximal colon and the effect did not appear to be dose-dependent. *P<0.05, ***P<0.001; one-way ANOVA; vs G2.

[0332] Then, treatment with Vistregrin restored normal levels of nitrergic neurons.

[0333] Example 10 - Effects of Vistregr on Cholinergic Neurons in Mouse Model

[0334] Choline acetyltransferase (CHAT) is the enzyme responsible for the biosynthesis of the neurotransmitter acetylcholine. Most acetylcholine is synthesized locally at nerve endings, and CHAT catalyzes the transfer of the acetyl group from acetyl-CoA to choline in a single-step process. CHAT is expressed by cholinergic neurons in the central nervous system (CNS) and peripheral nervous system (PNS) and is involved in learning, memory, movement, and vision.

[0335] Immunostaining with specific CHAT antibodies is often used for morphological studies of cholinergic cell populations. Mice were treated and samples were prepared as described in Example 6.

[0336] After incubation in blocking buffer, tissue samples were incubated with CHAT primary antibody (see above, Abcam, USA) (1:1000) at room temperature for one hour. Samples were washed three times with TBST (PBS + 0.5% Triton X-100 + 0.5% Tween-20) and incubated with secondary antibody F(ab')2-goat anti-rabbit HRP (Abcam, USA) (1:1000) at room temperature for one hour. Tissues were washed three times with PBST (PBS + 0.5% Triton X-100) and incubated with TSA520 (Wi See Biotechnology, China) (1:200) at room temperature for one hour. The method for detecting ataxin inclusion bodies was performed as described above by using Ataxin-7 primary antibody (Thermo Fisher, USA) (1:2000) at 4°C overnight. The samples were washed three times with TBST (PBS + 0.5% Triton X-100 + 0.5% Tween-20), then incubated with F(ab')2-Goat anti-Rabbit HRP secondary antibody (Abcam, USA) (1:1000) at room temperature for 1 hour, and washed three times with PBST (PBS + 0.5% Triton X-100). After incubation with TSA570 (Wi See Biotechnology, China) (1:200) at room temperature for 1 hour, washed three times with PBST (PBS + 0.5% Triton X-100), and incubated with DAPI (1:5000) at room temperature for 10 minutes. TM Glass Antifade Mountant (Invitrogen, USA) was dropped to cover the tissue. Images were taken with a microscope (Olympus, BX53). Tissue samples were excited with an excitation lamp and barrier filters (UW, BWA and GW), and the excitation / emission spectrum was set (340-390nm / 420IF; 460-495nm / 510-550nm; 530-550nm / 575IF). CHAT and Ataxin-7 antibodies were combined with fluorescent dyes TSA520 and TSA570, whose specificity and emission spectra are 488 / 519nm and 555 / 570nm. CHAT (green) and cells co-expressing ataxin-7 (yellow) were counted. The counts detected in Rb1cKO mice are shown in Table 14.

[0337] Table 14. Effects of Vistregr on CHAT neurons of Rb1 cKO mice.

[0338]

[0339] The results showed that the number of CHAT+ neurons in the DSI and colon of KO animals was significantly reduced compared with the normal group (DSIP < 0.01; colon P < 0.05). After treatment with 1mg / kg and 3mg / kg visconazole, the number of CHAT neurons in the DSI and proximal colon increased. Especially after 1mg / kg visconazole treatment, the difference in DSI was significant. These results were also shown in Fig. 9 (*P<0.05, **P<0.01; T test; vs G2).

[0340] In Prp-SCA7-92Q transgenic mice, the presence of cholinergic neurons in the myenteric plexus was assessed by immunofluorescence analysis using CHAT and Ataxin-7 primary antibodies on DSI and proximal colon samples isolated from transgenic animals.

[0341] The results are shown in Table 15.

[0342] Table 15. Effects of Vistralig on Cholinergic Neurons and Ataxin-7 Inclusions in Prp-SCA7-92Q Transgenic Mice

[0343]

[0344] The results showed that the number of ChAT+ neurons in the Prp-SCA7-92Q CIPO model was significantly reduced compared with normal wild-type mice. In addition, both doses of vestregrin induced an increase in cholinergic neurons in both the distal small intestine and the proximal colon compared with vehicle-treated transgenic mice. This effect appeared to be dose-independent.

[0345] In addition, the percentage of cholinergic neurons containing Ataxin 7 inclusions was significantly increased in the transgenic animals. Both tested doses of Vistragliol significantly reduced the percentage of cholinergic neurons containing Ataxin 7 inclusions compared to transgenic mice treated with the vehicle. Fig.10 As shown (*P<0.05, ***P<0.001; one-way ANOVA; vs G2).

[0346] Thus, at both doses, vistragliflozin antagonized neurodegeneration of cholinergic neurons in DSI and proximal colon and reduced nuclear inclusions of mutant ataxin-7 in DSI and colon.

[0347] Example 11 - Effects of Vistralig on Calretinin Neurons in the Prp-SCA7-92Q Mouse Model

[0348] Calreticulin is a calcium-binding protein that is abundantly expressed in neurons and plays an important role as a regulator of neuronal excitability.

[0349] Calretinin neurons were detected and their number was determined in the myenteric plexus by immunofluorescence analysis of DSI and proximal colon samples isolated from transgenic animals. Mice were administered vestregr as described above and CO 2 Mice were killed by inhalation. Whole-body tissue samples were prepared as described in Example 5 and washed three times with PBST (PBS + 0.5% Triton X-100).

[0350] After incubation in 10% donkey serum blocking solution (PBS containing 10% donkey serum and 1% Triton X-100) for 2 hours at room temperature, tissue samples were incubated with primary antibody against calreticulin (see above, Merck, USA) diluted 1:500 overnight at 4°C.

[0351] After washing three times with TBST (PBS + 0.5% Tritonx-100 + 0.5% Tween-20), the samples were incubated with donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody Alexa Fluor 488 1:500 (Invitrogen, USA) at room temperature for 1 hour.

[0352] After washing the tissue samples three times with PBST (PBS+0.5% Tritonx-100), they were incubated with DAPI 1:5000 (Invitrogen, USA) at room temperature for 10 minutes.

[0353] Use a drop of ProLong TM Coverslips were mounted with glass antifade mounting medium.

[0354] Tissue specimens were excited by a lamp with excitation and barrier filters (UW, BWA and GW) with excitation / emission spectra of 340-390nm / 420IF; 460-495nm / 510-550nm; 530-550nm / 575IF. Calreticulin antibody was conjugated with AlexaFluor 488 dye with specificity / emission spectra of 499 / 520nm and 590 / 618nm.

[0355] Images of each animal were acquired at 20× magnification through a microscope (Olympus, Bx53, Japan) using CellSens standard imaging software (Ver. 3.2, Olympus, Japan) for cell counting.

[0356] The results are shown in Table 16.

[0357] Table 16 - Effects of Vistralig on Calretinin Neurons in Transgenic Prp-SCA7-92Q Mice

[0358]

[0359] The number of calretinin neurons in the colon of vehicle-treated transgenic mice was significantly reduced compared to the normal group (i.e., vehicle-treated C57BL / 6 mice) (P<0.001). Vistregr treatment induced an increase in the number of calretinin neurons in DSI, but no significant difference was detected. In the colon, 1 mg / kg and 3 mg / kg of Vistregr induced a significant increase in the number of calretinin neurons (P<0.05 and P<0.01, respectively).

[0360] Fig.11 Also shown are changes in DSI and calretinin neurons in the proximal colon of PrP-SCA7-92Q transgenic mice treated with vistrag (1 mg / kg and 3 mg / kg) (**P<0.01, ***P<0.001; one-way ANOVA; vs G2 vehicle).

[0361] Calretinin-expressing neurons (excitatory motor cells) were found to be reduced in the proximal colon of vehicle-treated transgenic mice, but not in DSI, whereas administration of vistra protected this neuronal cell population at both doses ( Fig.11 ).

[0362] These results indicate that both doses of vistragliflozin antagonize neurodegeneration of calretinin neurons in the proximal colon.

[0363] Example 12 - Effect of Vistralig on n-NOS and CHAT protein levels in neuromuscular tissue in the Prp-SCA7-92Q mouse model

[0364] Western blotting was used to detect and analyze the protein levels of n-NOS and CHAT in the cytoplasm of the proximal colon of transgenic mice.

[0365] According to the manufacturer's instructions, the proximal colon samples were placed in 10 volumes (w:v) of RIPA lysis buffer (Beyotime Biotechnology, China) containing a mixture of protease and phosphatase inhibitors (Thermo Scientific, USA). The tissue was homogenized on ice for 15 seconds, incubated on ice for 30 minutes, and then centrifuged at 15,000×g for 15 minutes. The supernatant was collected and analyzed for protein concentration using the BCA method. An appropriate amount of protein loading buffer was added to the supernatant to perform protein denaturation (95°C, 10 minutes). The proteins were separated on a 10% SDS-PAGE gel and transferred to a NC membrane. After the membrane was blocked with 5% BSA in TBST (0.05% Tween-20) for 2 hours at room temperature, the membrane was incubated with primary antibodies nNOS 1:1000 (Abcam, USA), CHAT 1:1000 (Millipore, USA), Actin 1:1000 (Beyotime, China) or MAPK 1:1000 (Cell Signaling Technology, 4695, USA) at 4°C overnight (no less than 15 hours). The blot was washed with TBST (3 times, 10 minutes each time) and then incubated with pre-absorbed secondary antibodies goat anti-rabbit IgG H&L (1:5000) 800CW) (Abcam, USA) or donkey anti-goat IgG H&L ( 800CW) 1:5000 (Abcam, USA) and incubated at room temperature for 1 hour.

[0366] After washing, the membrane was placed in substrate working solution for 5 minutes and then imaged using the ChemiDoc system (Bio-Rad, 12003154, USA). Densitometry analysis of protein bands was performed using QuantityOne software version 4.6.2.

[0367] The data are shown in Table 17.

[0368] Table 17. Effects of Vistralig on cytoplasmic nNOS and ChAT protein levels

[0369]

[0370] In the Prp-SCA7-92Q model, the levels of nNOS and ChAT proteins were significantly decreased. Both doses of vistrapol reversed this effect, as increased expression of nNOS and ChAT proteins was observed in the proximal colon compared with untreated transgenic mice. This effect appeared to be dose-independent.

[0371] These results are Fig.12As shown. *P<0.05, ***P<0.001; T test; vs G2

[0372] Overall results in mouse models

[0373] The efficacy of the 5-HT4 receptor agonist vestregl was evaluated in two CIPO mouse models: RB1 cKO mice and PrP-SCA7-92Q transgenic mice. Histopathological analysis of tissues by hematoxylin and eosin staining (HE) showed that DSI and colonic mucosa showed inflammation, macrophage infiltration, and ulceration in both models, and vestregl was able to ameliorate intestinal damage in a dose-dependent manner.

[0374] By using HuCD antibodies, immunohistochemical staining showed a significant reduction in enteric neurons in transgenic mice. In the distal small intestine and proximal colon of transgenic mice, this pan-neuronal marker involved in intestinal neurogenesis, neuronal survival and plasticity was reduced, indicating a loss of function of Hu proteins that affect neuronal signaling. [Li et al. Scientific Reports | 6: 38216 | DOI: 10.1038 / srep38216 and 25]. In addition, pan-neuronal antibodies showed a significant increase in Ataxin-7 inclusions in DSI and colon, and Vistregr treatment opposed this increase. This specifically refers to the only subpopulation of ganglion cells that express ChAT but not nNOS that contains Ataxin-7 nuclear inclusions.

[0375] Although inhibitory motor neurons expressing nNOS were not affected by ataxin-7 inclusions, these neurons were lost in the DSI and colon of transgenic mice compared to control mice, but these neurons were protected by administration. Neuronal NOS (nNOS), constitutively expressed in peripheral neurons, is involved in synaptic plasticity, central regulation of blood pressure, smooth muscle relaxation, and regulation of vasodilation via peripheral nitrergic nerves. The ability to form nitrergic neurons is a key step in the development of "normal" intestinal circuits, and many intestinal diseases may benefit from the transplantation and implantation of nNOSt cells. [McCann, CJ, et al., Nat Commun, 2017.8: p.15937.]. Nitric oxide (NO) is the major non-adrenergic non-cholinergic inhibitory signal in the peripheral nervous system, including the gastrointestinal (GI) tract and the enteric nervous system (ENS), relaxing gastrointestinal smooth muscle to regulate physiological peristalsis, so the loss of neuronal NO synthase (nNOS) interferes with normal gastrointestinal motility. NO enhances colonic transit in rats by mediating descending relaxation, thereby promoting propulsion of colonic contents. In the present invention, nNOS+ neurons in the ENS of transgenic mice in the vehicle-treated group were reduced, but not after vistragol treatment, indicating that prokinetic drugs are beneficial to NO function in the ENS.

[0376] In the large intestine of mice, the principal neurons expressing calretinin, a calcium-binding protein, were investigated, which is a marker for myenteric motor neurons, interneurons, and most putative primary afferent neurons in the muscularis mucosa and lamina propria. Also in this case, there was a decrease in calretinin-immunoreactive neurons (putative intrinsic primary afferent neurons) in myenteric ganglia in the colon of PrP-SCA7-92Q mice, and vestragel was able to protect such neurons.

[0377] Mammalian microtubule-associated protein 2 (MAP2) is primarily expressed in neurons, but is also expressed in oligodendrocytes, and is present in both early and late stages of neuronal development. Three isoforms are known, MAP2A, B, and C. MAP2C is located in the cell bodies, dendrites, and axons of juvenile neurons, while MAP2A and B are primarily located in the dendrites of mature neurons. The MAP2 / Tau family of proteins was originally discovered and characterized for its ability to bind and stabilize microtubules. In the present invention, the MAP2 staining intensity was reduced in KO and transgenic mice, indicating that the number of mature neurons or dendrites in transgenic mice was smaller. After treatment with visconazole, the MAP2 intensity in the ENS increased, indicating that visconazole can improve the microtubule function of dendrites.

[0378] Enteric glial cells are also considered because they interact with other gastrointestinal cell types, such as epithelial cells and cells of the immune system, to maintain homeostasis (Boesmans et al. Frontiers in cell and Development Biology Vol. 9 | Article 775102).

[0379] In vitro studies have shown that undifferentiated progenitor cells expressing Sox10 in the embryonic intestine give rise to enteric neurons (Sox10-) and glial cells (Sox10+), but the neurogenic potential of these cells in vivo and their temporal regulation during intestinal organogenesis are currently unclear.

[0380] It was found that most of the submucosal HuCD+ neurons and a small number of intestinal muscle neurons co-expressed the glial cell markers Sox10 and S100B (Parathan P. et al. (2020). The Enteric Nervous System Undergoes Significant Chemical and Synaptic Maturation during Adolescence in Mice. Dev. Biol. 458, 75–87.10.1016 / j.ydbio.2019.10.011). These may be newly differentiated neurons that are gradually shutting down the expression of neural cell markers.

[0381] In the present invention, compared with control mice, the number of glial cells in the proximal colon increased. Considering the proportion of glial cells in pan-neurons, both doses of vistra treatment inhibited the increase in the number of glial cells in DSI and colon of KO or transgenic mice. Kulkarni et al. (Proc Natl Acad Sci USA, 2017.114 (18):: E3709-E3718 pages) show that under physiological conditions, adult ENS is maintained by a dynamic balance between neuronal apoptosis and neurogenesis of neuronal Sox10- / Nestin+ precursors from immature glial cells. However, the neurogenesis potential of Sox10 / +Nestin- cells is only activated when injured. In the KO mice and transgenic mice in the present invention, it was observed that Sox10+ glial cells increased. However, vistra treatment resulted in a decrease in Sox10+ glial cells. In transgenic mice, it may be necessary to increase glial cells to compensate for the reduction of some other types of neuronal cells. Because visnatol promotes the production of neuronal cells, the need for glial cells decreases when intestinal damage or dysfunction is reduced.

[0382] Since vistrag is a 5-HT4 receptor (5-HT4R) agonist, the expression of 5-HT4R was studied because this type of receptor 5-HT4 can regulate gastrointestinal motility and reduce visceral sensitivity. In addition, 5-HT4R is related to the development and functional maintenance of enteric nerves. In the present invention, changes in 5HT4R mRNA levels were observed in cKO and transgenic mice, with an increase in 5HT4R mRNA in intestinal tissue and a decrease in 5HT4R mRNA in colon tissue. Obviously, vistrag treatment does not change the mRNA level of 5HT4 in transgenic mice.

[0383] In the present invention, biomarkers related to cell survival, proliferation and metabolism (signaling mTOR / AKT) were also evaluated. Mammalian target of rapamycin (mTOR) is a serine / threonine protein kinase belonging to the phosphatidylinositol 3-kinase (PI3K)-related kinase (PIKK) family, which interacts with other subunits to form two different complexes, mTORC1 and mTORC2. mTORC1 coordinates cell growth and metabolism in response to environmental inputs (including growth factors, amino acids, energy and pressure). mTORC2 controls cell survival and migration mainly by phosphorylating glucocorticoid-regulated kinase (SGK), protein kinase B (Akt) and protein kinase C (PKC) kinase families. mTOR dysregulation is found in many human diseases, including cancer, cardiovascular disease, neurodegenerative diseases and epilepsy. In the present invention, activation (i.e. phosphorylation) of signaling mTOR was observed in DSI of transgenic and cKO mice, and the application of vestragel antagonized this activation. The serine-threonine kinase of AKT, which has three different protein isoforms (AKT1, AKT2 and AKT3), also acts on cell survival, proliferation and metabolism. Also in this case, phosphorylation of AKT was found in DSI of transgenic and cKO mice, and treatment with Vistregrin reduced this phosphorylation. Finally, p70S6 kinase is activated by growth factors and plays a central role in cell growth and proliferation by mediating the phosphorylation of 40S ribosomal protein S6, thereby achieving efficient translation of 5-terminal oligopyrimidine tract mRNA (5-TOP). mTOR promotes translation initiation by phosphorylating two targets, namely ribosomal p70S6 kinase (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). In the present invention, increased p70S6 activity was observed again in transgenic and cKO mice. Vistregrin inhibited this effect. In general, the 3 mg / kg group of vistragliflozin was able to reduce the expression of p-mTOR, p-Akt, and p-P70S6 in the distal small intestine after 5 weeks of treatment.

[0384] Vistregrin significantly improved the biomarkers of CIPO and ameliorates its pathological conditions, and therefore has great potential for the treatment of CIPO.

[0385] Example 13 - Multicenter, double-blind, placebo-controlled, two-treatment, four-period crossover, multiple (n=1) trial to evaluate the efficacy and safety of vistragliflozin in patients with CIPO

[0386] Materials and methods

[0387] A phase II, multicenter, double-blind, placebo-controlled, two-treatment four-period crossover, multiple (n=1) trial was conducted to evaluate the efficacy and safety of 15 mg of vistragliflozin once daily compared with placebo in patients with idiopathic chronic intestinal pseudo-obstruction (CIPO) or CIPO secondary to primary neurodegenerative or demyelinating disease.

[0388] This study evaluated the safety, tolerability, and efficacy of 15 mg of vistragliflozin once daily compared with placebo in improving the severity of CIPO-related symptoms in patients with idiopathic CIPO and CIPO secondary to neurodegenerative disease.

[0389] CIPO is a chronic rare disease with fluctuating symptoms, different underlying pathology, and potentially different responses to active treatment. Therefore, standard parallel-group placebo-controlled studies are unlikely to detect a clear benefit due to the wide variability in clinical presentation.

[0390] The n=1 crossover, placebo-controlled study design can evaluate the treatment effect of each subject, so that each subject can serve as its own control, and even in a single subject, a positive effect can be demonstrated to avoid underestimation of the efficacy. (Emmanuel et al. Randomised clinical trial: the efficacy of prucalopride in patients with chronic intestinal pseudo-obstruction--a double-blind, placebo-controlled, cross-over, multiple n=1 study. Aliment Pharmacol Ther. 2012 January; 35(1): 48-55).

[0391] Placebo was used as a comparator because this allowed the actual efficacy of vistragliflozin to be assessed.

[0392] The study consisted of a 7-day screening period (Day -7 to Day -1), followed by 4 treatment periods of 4 weeks each, in which subjects received either vestreglobate (VEL) 15 mg (2 cycles) or placebo (PLA) (2 cycles), with a 2-week washout period between treatment periods, followed by a 2-week follow-up period (approximately 175 days in total), as Fig.16 Assessments were performed at the beginning of the screening period (Visit 1), at the beginning and end of each treatment period (Visits 2 to 9), and at the end of the follow-up period (Visit 10).

[0393] After a 7-day screening period (from Day -7 to Day -1), eligible subjects were randomly assigned to one of the following four sequences in a 1:1:1:1 ratio at the randomization visit (Visit 2), such as Fig.16 As shown:

[0394] A.VEL-PLA-VEL-PLA

[0395] B.PLA-VEL-PLA-VEL

[0396] C.VEL-PLA-PLA-VEL

[0397] D.PLA-VEL-VEL-PLA

[0398] Among them, VEL = vistra, once a day, 15 mg, for 4 weeks.

[0399] PLA = matching placebo once daily for 4 weeks.

[0400] The severity of gastrointestinal symptoms (abdominal pain, bloating, nausea, and vomiting) and bowel habits (weekly recall) were recorded using an electronic diary according to the assessment schedule on day -1 and weekly after randomization, during the treatment and washout periods until the end of the follow-up period.

[0401] During the study period, the following parameters were collected during the visits:

[0402] Serum nutritional markers (albumin, prealbumin, vitamin B12, and folate) were collected before treatment, every 2 weeks during the washout period, and at the end of follow-up after period 4 or at the early termination visit (ETV) or early switch visit (ESV);

[0403] The number of pseudo-obstruction episodes and hospitalizations were assessed at each visit;

[0404] ·Assess the need for parenteral and enteral nutritional supplements at each visit;

[0405] A lactulose breath test (L-BT) was performed at the screening visit (Visit 1) and at the end of the first 4-week treatment period (Visit 3) to assess oro-cecal transit time;

[0406] Table 18 reports the evaluation schedule for the measured parameters.

[0407]

[0408]

[0409]

[0410] Subjects were also required to record permitted medications taken for the treatment of CIPO gastrointestinal symptoms prior to the start of the first treatment period (Day -1), as well as any changes in treatment dose (increase / decrease) or amount of concomitant medication throughout the treatment and washout periods. In addition, subjects recorded the amount and timing of study product intake daily in an electronic diary during the treatment period.

[0411] Study Group

[0412] A total of 17 patients with a history of chronic idiopathic intestinal pseudo-obstruction or CIPO secondary to neurodegenerative or demyelinating disease were randomly assigned to one of the four groups described above.

[0413] Randomization occurred at Visit 2 (V2 - Day 1) after all screening procedures had been performed and study eligibility had been assessed.

[0414] Randomization was stratified according to CIPO diagnosis (idiopathic or secondary to neurodegenerative or demyelinating disease) and 5-HT4 receptor agonist response status (responder / naive or nonresponder) as follows:

[0415] Non-responders to 5-HT4 receptor agonists

[0416] ·5-HT4 receptor agonist responders / first-time users with idiopathic CIPO

[0417] ·5-HT4 receptor agonist responders / naive users or CIPO secondary to neurodegenerative or demyelinating diseases.

[0418] Non-responders were defined as subjects who, based on the investigator's judgment, had a history of lack of benefit from 5-HT4 receptor agonists.

[0419] Main inclusion criteria:

[0420] 1. Male or female aged 18 to 80 years old.

[0421] 2. Subjects with a history of chronic idiopathic intestinal pseudo-obstruction or CIPO secondary to neurodegenerative or demyelinating disease.

[0422] 3. Subjects whose estimated oral caloric intake is at least 30% of the recommended daily caloric intake for age and sex (according to Table 19, stage 0, 1 or 2 of the "Artificial Food Requirements" scale).

[0423] Table 19. Artificial food requirement table

[0424]

[0425] 4. On Day -1, the gastrointestinal symptom questionnaire was collected for subjects with at least 2 of the 4 CIPO gastrointestinal symptoms (i.e., abdominal pain, bloating, nausea, and vomiting), where the scores of both symptoms were ≥3 (on a scale of 0 to 4).

[0426] 5. The subject agrees and is legally competent to provide informed consent to all procedures contained in the protocol.

[0427] 6. All male participants with female partners of reproductive potential who are sexually active must use condoms during sexual intercourse until the 90th day after the end of the entire study.

[0428] 7. All female participants must:

[0429] a. Infertility, i.e.: i) postmenopausal (no natural menstruation for at least 2 years), or ii) surgical sterilization (bilateral fallopian tube occlusion or hysterectomy), or iii) bilateral oophorectomy

[0430] or

[0431] b. Of childbearing potential, but with negative pregnancy test results at screening and randomization, and agree to use highly effective contraceptive methods (i.e., failure rate less than 1% per year) until the end of the study.

[0432] Highly effective contraceptive methods were the main exclusion criteria defined according to the recommendations of the European Union Clinical Trials Promotion Group:

[0433] 1. Subjects with primary CIPO or CIPO secondary to other known endocrine / metabolic, autoimmune diseases, and neurological diseases other than neurodegenerative or demyelinating diseases.

[0434] 2. Subjects with diseases characterized by mechanical intestinal obstruction.

[0435] 3. Placement of a nasogastric, gastrostomy, or jejunostomy feeding tube at the time of randomization or planned throughout the study, or artificial food requirement scale stage 3 ("total non-oral nutrition", see the "Artificial Food Requirement" (AFN) scale for CIPO subjects in Appendix 3 of study protocol 21.3).

[0436] 4. The presence of untreated clinically relevant thyroid dysfunction that is considered clinically significant by the investigator or known thyroid dysfunction that is not well controlled with treatment (e.g., subjects with abnormal thyroid stimulating hormone [TSH] and, if available, abnormal triiodothyronine [T3] and thyroxine [T4] levels).

[0437] 5. Subjects with a history of diabetes at the time of screening.

[0438] 6. Clinically significant electrocardiographic abnormalities during screening and randomization (e.g., ST-segment elevation or depression suggestive of ischemia, partial or complete left bundle branch block [LBBB]).

[0439] 7. During screening, the electrocardiogram shows QTcF>450 milliseconds for males and>470 milliseconds for females, or there is a family history of sudden cardiac death.

[0440] 8. Subjects who require a low-galactose diet.

[0441] 9. Allergy or documented intolerance to lactulose, lactose, or any excipients of the lactulose formulation used in L-BT.

[0442] 10. History of allergy to visconazole or any visconazole excipients or placebo excipients.

[0443] 11. Use of scopolamine or erythromycin within 2 weeks prior to screening and / or planned throughout the study.

[0444] 12. Planned use of 5-HT4 receptor agonists (e.g., prucalopride, cisapride, clebopride, and cinipride) within 5 days before randomization and / or throughout the study.

[0445] 13. Use of opioids within 8 weeks from the screening date and / or plan to use opioids throughout the study period.

[0446] 14. Planned use of strong cytochrome P450 isoenzyme 3A4 (CYP3A4) inhibitors (e.g., clarithromycin, indinavir, itraconazole, ketoconazole, nefazodone, nelfinavir, ritonavir, saquinavir, telithromycin, grapefruit juice) or strong CYP3A4 inducers (e.g., rifampicin, phenytoin, carbamazepine, phenobarbital, St. John's wort) within 2 weeks prior to screening and / or throughout the study.

[0447] 15. Planned use of potent P-glycoprotein (P-gp) transporter inhibitors (e.g., captopril, carvedilol, diltiazem) within 2 weeks prior to screening and / or throughout the study.

[0448] 16. Planned use of potent breast cancer resistance protein (BCRP) transporter inhibitors (e.g., curcumin, cyclosporine A, eltrombopag) within 2 weeks prior to screening and / or throughout the study.

[0449] 17. Current swab positive or suspected (under investigation) COVID19 infection.

[0450] 18. Have had cancer (except non-melanoma skin cancer) and / or required any anti-cancer treatment (including radiotherapy) in the past 5 years.

[0451] 19. Severe renal insufficiency (i.e. estimated glomerular filtration rate <30 ml / min).

[0452] 20. Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) level > 2.5 times the upper limit of normal (ULN); bilirubin (unless considered to be caused by Gilbert's syndrome) or alkaline phosphatase (ALP) > 1.5 times ULN.

[0453] 21. Severe hepatic impairment, defined as Child-Pugh C.

[0454] 22. Any history of heart disease:

[0455] a. Torsades de pointes, ventricular tachycardia, ventricular fibrillation.

[0456] b. Myocardial infarction, unstable angina, acute coronary syndrome, coronary artery or cerebral revascularization surgery, or stroke in the past 18 months.

[0457] c. Angina grade 2-4 in the 12 months prior to screening.

[0458] d. Suffering from congestive heart failure NYHA class III-IV within 18 months prior to screening.

[0459] 23. History of alcohol or drug abuse or dependence in the past year (judged by the researchers).

[0460] 24. Any current significant health condition (e.g., cardiovascular, respiratory, renal, hepatic, neurological, psychiatric, hematologic, oncologic, immune, muscular, and joint, etc.) that the researcher believes may:

[0461] a. endanger the safety of the subjects participating in the trial; or

[0462] b. Makes it more likely that the subject will not be able to complete the study; or

[0463] c. Makes it likely that the subject will be unable to comply with study procedures (e.g., anticipated need for unauthorized treatment, severe disability, terminal illness).

[0464] 25. Pregnant or breastfeeding women.

[0465] 26. Use of any experimental drugs within 12 weeks before screening.

[0466] Ingredients used

[0467] Participants in the study took either 15 mg (3 x 5 mg capsules) of Vistragliol or matching placebo (3 capsules) once daily for 4 weeks over 4 treatment periods. Participants were asked to take three capsules orally with water on an empty stomach at approximately the same time each morning.

[0468] Vistra

[0469] Product active ingredient: Vistralig (ScinoPharm Taiwan, Ltd.) 5 mg. Excipients: lactose monohydrate, microcrystalline cellulose, hypromellose and magnesium stearate.

[0470] Placebo

[0471] Active Product Ingredient: Not present.

[0472] Excipients: lactose monohydrate, microcrystalline cellulose, hypromellose and magnesium stearate.

[0473] Prohibited drugs

[0474] The following medications were not permitted within 2 weeks prior to screening and / or were not planned to be used during the entire study period:

[0475] Scopolamine

[0476] Erythromycin

[0477] Strong CYP3A4 inhibitors or strong CYP3A4 inducers (according to Table 20)

[0478] Potent P-gp transporter inhibitors (according to Table 20)

[0479] Potent BCRP transporter inhibitors (as per Table 20)

[0480] Table 20. Prohibited drugs

[0481]

[0482] Additionally, use:

[0483] · No opioid use was permitted within 8 weeks from screening and / or was not planned during the entire study period.

[0484] The use of 5-HT4 receptor agonists (e.g., prucalopride, cisapride, clebopride, cilnipride) was not permitted from 5 days before randomization until randomization and throughout the study.

[0485] Poorly absorbed orally administered opioids (i.e., loperamide) may be used if medically indicated (for treatment of potential adverse effects).

[0486] If 5-HT was used during the study 4 If the subject did not receive any of the following: 1) antagonists (e.g., prucalopride, cisapride, clebopride, cilnipride) or scopolamine, or erythromycin and opioids for analgesia, the subject was considered a treatment failure and was withdrawn from the study.

[0487] Additional prescription and over-the-counter medications may be used, provided that they are not known to be strong inducers or inhibitors of CYP3A4, P-gp, and BCRP activity.

[0488] Any addition or change in the concomitant medication regimen that affects CYP3A4, P-gp, and BCRP activity met the inclusion or exclusion criteria and was recorded in the source files and eCRF.

[0489] Medications taken during the period 30 days before the screening visit until the end of the follow-up period were recorded. If subjects had previously taken 5-HT4 receptor agonists (e.g., prucalopride, cisapride, clebopride, cilnipride), the last treatment period for each subject (even if it occurred more than 30 days before the screening visit) and the efficacy of such treatment (responder / non-responder) were recorded in the eCRF.

[0490] If clinically possible, subjects were encouraged not to change their current concomitant medication regimen or to start new concomitant medications.

[0491] Permitted treatments

[0492] The use of medications to relieve the main symptoms of CIPO was allowed, and the use of the following concomitant medications was recorded daily in an electronic diary:

[0493] Treatment of nausea, vomiting and / or non-serotonergic prokinetic drugs (e.g. metoclopramide, domperidone, medicinal ginger, pyridostigmine, prochlorperazine, promethazine, ondansetron, and aprepitant).

[0494] Medications to treat constipation (e.g. polyethylene glycol, bisacodyl, linaclotide, laxative enemas).

[0495] Diarrhea treatment (eg, tannates, loperamide).

[0496] Abdominal pain treatment (eg, acetaminophen, NSAIDs, trimebutine, mebeverine, gabapentin, duloxetine, amitriptyline)

[0497] Others (e.g. octreotide, somatostatin, pancreatic enzymes, probiotics, rifaximin, metronidazole, fluconazole).

[0498] · Record in an electronic diary daily the medications taken to treat gastrointestinal symptoms of CIPO before the start of the first treatment period (day -1) and the doses of all concomitant treatments or changes in the quantity of concomitant medications throughout the treatment and washout periods until the end of follow-up.

[0499] Efficacy Endpoints

[0500] Primary Efficacy End Point

[0501] The primary efficacy endpoint was to assess the change in the weekly Global Gastrointestinal Symptom Index Score (WGGSAIS) from the beginning to the end of each treatment period. The score was calculated by averaging the scores of 4 symptoms assessed weekly: abdominal pain, bloating, nausea, and vomiting, with a score range of 0 to 4 (0 - none; 1 - mild (does not interfere with daily activities); 2 - moderate (deviates from daily activities but does not prompt a change in daily activities); 3 - severe (significantly interferes with daily activities, enough to prompt a change in daily activities; 4 - extremely severe (interferes with daily activities)) (Barbara G et al., 2004, Gastroenterology Mar;126(3):693-702).

[0502] Secondary Efficacy End Points

[0503] Secondary efficacy endpoints were assessed based on the following changes from the beginning to the end of each treatment cycle:

[0504] 1. Changes in waist circumference.

[0505] 2. The proportion of subjects with a 1-point improvement in WGGSAIS.

[0506] 3. Changes in individual symptom scores such as abdominal pain, bloating, nausea, and vomiting.

[0507] 4. Change in the number of bowel movements per week (limited to subjects with a Bristol stool scale type 1 or 2 at the start of the treatment period).

[0508] 5. Change in the number of complete voids per week (limited to subjects with Bristol Stool Scale type 1 or 2 at the start of the treatment period).

[0509] 6. Changes in stool type on the Bristol Stool Scale.

[0510] 7. Changes in satisfaction with bowel habits every week, 0 to 10 points.

[0511] 8. Change from baseline in oro-cecal transit time measured using the lactulose breath test (at the end of the first treatment period only).

[0512] 9. Changes in nutritional indicators (serum albumin, prealbumin, vitamin B12, and folate levels) from the beginning of each treatment period to the end of each 2-week washout period (or follow-up period).

[0513] 10. The proportion of days during each treatment and washout period (or follow-up period) on which medication changes were allowed to relieve major CIPO gastrointestinal symptoms:

[0514] a. Proportion of days on which the dose was increased compared to the start of the period.

[0515] b. The proportion of days on which the dose was reduced compared to the beginning of the period.

[0516] c. Proportion of days on which medication was added compared to the start of the period.

[0517] d. The proportion of days on which the drug is removed compared to the start of the cycle.

[0518] 11. Changes in quality of life (12-item Short Form Health Survey - SF-12).

[0519] 12. Number of CIPO-related hospitalizations during treatment.

[0520] 13. Changes in the stages of the “Artificial Food Requirement” scale.

[0521] 14. Number of pseudo-obstruction episodes based on the investigator's judgment.

[0522] 15. Changes from the end of each treatment period to the end of the first and second weeks of the washout period (or follow-up period):

[0523] a. Weekly abdominal pain scores.

[0524] b. Weekly abdominal distension scores.

[0525] c. Weekly nausea scores.

[0526] d. Weekly vomiting scores.

[0527] e. Average weekly overall gastrointestinal symptom index score.

[0528] f. Number of bowel movements per week (limited to subjects with Bristol stool scale type 1 or 2 at the start of treatment).

[0529] g. Number of complete voidings per week (only for subjects with Bristol Stool Scale type 1 or 2 at the start of treatment).

[0530] h. Stool types in the Bristol Stool Scale.

[0531] i. Weekly bowel habit satisfaction score, 0 to 10 points.

[0532] Safety Assessment

[0533] Safety was assessed at each visit, and the following parameters were measured and evaluated for changes from baseline:

[0534] Vital signs (including blood pressure, pulse, temperature).

[0535] The physical examination also includes height and weight.

[0536] · Conventional laboratory parameters (for Blood analysisHematocrit, hemoglobin, red blood cell count, white blood cell count (differential count), and platelet count for biochemical analysis; blood glucose, total cholesterol, triglycerides, serum creatinine, urea or blood urea nitrogen [BUN], sodium, potassium, chloride, AST, ALT, gamma-GT, alkaline phosphatase, total and fractionated bilirubin (direct and indirect), erythrocyte sedimentation rate, PT (or INR), aPTT (biochemical) for biochemical analysis; Urinalysis specific gravity, pH, protein, glucose, ketones, hemoglobin, nitrite, bilirubin, urobilinogen, and microscopy).

[0537] Three ECGs

[0538] Adverse events (AEs)

[0539] · Withdrawal from the study due to AEs.

[0540] Statistical methods

[0541] Determination of sample size

[0542] The sample size was determined based on the primary analysis (t-test) of the primary endpoint, i.e., the difference in WGGSAIS in the evaluable pairs. A pair was considered evaluable when there were subjects available for evaluation within a period (i.e., consecutive visstregol and placebo treatment periods, or vice versa).

[0543] It should be noted that each subject should be assessed twice, and each assessment pair should be assessed once. Therefore, each subject can contribute 0, 1, or 2 pairs, and only if these data constitute assessment pairs and meet the requirements of the primary endpoint analysis, they will be included in this analysis.

[0544] Definition of the main study population

[0545] Safety set (SS): All subjects who received treatment. SS was analyzed based on the actual treatment received.

[0546] Modified Full Analysis Set 1 (mFAS1): All 5HT4 receptor agonist-responsive / naive subjects were randomized and treated, with primary endpoint data collected at least once during vistrag treatment and at least once during placebo treatment in the same cycle.

[0547] Modified full analysis set 2 (mFAS2): All subjects were randomized and treated, with primary endpoint data collected at least once during vistrag treatment and at least once during placebo treatment in the same cycle.

[0548] Per-protocol set (PPS): All mFAS1 subjects who met the study protocol requirements in terms of treatment adherence and primary efficacy data collection without major deviations.

[0549] Primary Efficacy Endpoint Analysis

[0550] The WGGSAIS was calculated by averaging the scores of 4 symptoms (abdominal pain, bloating, nausea, and vomiting) assessed weekly, with lower scores indicating better health status. If at least 2 symptoms were assessed, the mean score was calculated, otherwise it was considered missing.

[0551] Considering the assessment pairs (i.e., vistragliflozin and placebo could be assessed within the cycle), the WGGSAIS was summarized based on the data collected from pre-treatment to the end of each treatment period (4 weeks) and the changes occurring during the washout period, i.e., the WGGSAIS representing the post-treatment and subsequent pre-treatment periods.

[0552] The WGGSAIS difference between visstreg and placebo treatments was calculated in each assessment pair as the change in visstreg minus the change in score that occurred during placebo treatment (for both visstreg and placebo, the change was between the end of treatment and the pre-treatment value). Paired t-tests were used to assess the difference in scores between visstreg and placebo treatments: results are presented as mean differences, relative 95% CIs, and p-values.

[0553] Supportive analyses were performed by mixed models, in which the outcome was the difference between the change in WGGSAIS from pre-treatment to end of treatment for visstreg and placebo within the same period, with period considered as a fixed effect and subject as a random effect. Results of the fixed-effect type III test and estimates of the difference in change from pre-treatment to end of treatment for visstreg and placebo with 95% CIs are presented.

[0554] These analyses were performed on mFAS1 as the primary analysis without any missing data imputation.

[0555] Sensitivity analysis: The same analysis was applied to mFAS2.

[0556] Analyses of Key Secondary Efficacy Endpoints

[0557] · The proportion of subjects with a 1-point improvement in WGGSAIS

[0558] A change of ≤-1 point from the WGGSAIS value before treatment was calculated at the end of treatment and was considered "success", while a change of >-1 was considered "failure". The proportion of subjects with a 1-point improvement in WGGSAIS (assessed weekly by electronic diary) was summarized by treatment sequence, treatment period, and time point.

[0559] Considering the assessment pairs, the proportion of pairs of observations with a 1-point improvement in the WGGSAIS was summarized by treatment and time point. The odds ratio of achieving a 1-point improvement at the end of treatment time point with vistra versus placebo is provided, with corresponding 95% CIs derived using a logistic regression model. The proportion of pairs of observations with a "success" / "failure" between treatments was compared using Fisher's test.

[0560] Changes in individual symptom scores

[0561] Each symptom (abdominal pain, abdominal distension, nausea, vomiting) was analyzed as described for the primary efficacy endpoint analysis.

[0562] Effects on bowel habits in subjects with constipation (i.e. Bristol stool types 1 and 2 before treatment)

[0563] The number of bowel movements and the number of complete voidings were reported weekly in an electronic diary, while stool consistency was assessed according to the Bristol scale. Data collected from before treatment to the end of each treatment period and their changes were pooled according to treatment modality.

[0564] Stool consistency

[0565] Mean stool consistency according to the Bristol scale was recorded in an electronic diary. The Bristol scale categories are as follows: constipation: types 1 and 2; normal: types 3 and 4; diarrhea: types 5, 6, and 7. The distribution of subjects by category from before treatment to the end of treatment is presented. Considering the assessment pairs, the number of subjects by category before treatment for each cycle and the number of subjects who remained or changed category at the end of the treatment period for each cycle are presented. In addition, Fisher's exact test was performed to compare the distribution by category at the end of treatment, and p-values ​​are provided.

[0566] Oro-cecal transit time

[0567] Orocecal transit time was calculated based on the lactulose breath test (L-BT) performed only during the first treatment period. The change in oral transit time from pre-treatment to post-treatment was calculated. A t-test was performed to assess the difference between vistragliflozin and placebo.

[0568] Pseudo-obstructive attack

[0569] Considering the assessment pairs, the number of pseudo-obstruction episodes that occurred during each treatment period and the number of pseudo-obstruction episodes that occurred during the washout / follow-up period were treated as categorical variables, categorized as 0, 1, 2, and >2, and summarized according to treatment modality. In addition, Fisher's exact test was performed to compare the distribution of the number of pseudo-obstruction episodes that occurred during each treatment period, considering the categories and p values ​​provided.

[0570] The effect of stopping vistra during a 2-week washout period (or follow-up period) at the end of each treatment period

[0571] definition

[0572] When assessing the impact of interrupting treatment, the following previously described variables were taken into account:

[0573] Weekly abdominal pain score

[0574] Weekly bloating score

[0575] Weekly Nausea Rating

[0576] Weekly vomiting score

[0577] Average weekly overall gastrointestinal symptom index score

[0578] ·Number of bowel movements per week (only for subjects with Bristol stool scale type 1 or 2 before treatment)

[0579] The number of complete voids per week (only for subjects with a Bristol Stool Scale type 1 or 2 at the start of treatment).

[0580] Stool Types on the Bristol Stool Scale

[0581] Weekly bowel habits satisfaction score, 0 to 10 points

[0582] The same descriptive analyses described above were repeated, focusing on changes from the end of each treatment period to the end of each week of the washout or follow-up period, rather than changes between post-treatment and pre-treatment.

[0583] Safety statistical analysis

[0584] Adverse events (AEs)

[0585] Adverse events that occurred on or after the first dose of the investigational product were considered treatment-emergent adverse events (TEAEs). Verbatim terms for AEs were coded using the most recent version of the Medical Dictionary for Regulatory Activities (MedDRA) codes. In addition, events were classified as TEAE-induced if the last treatment administered before the onset of the AE was TEAE and as placebo-induced if the last treatment administered before the onset of the adverse event was placebo.

[0586] The absolute and relative frequencies of subjects with treatment-related TEAEs, serious TEAEs, treatment-related serious TEAEs, TEAEs leading to temporary interruption of treatment, TEAEs leading to treatment discontinuation, serious TEAEs leading to temporary interruption of treatment, serious TEAEs leading to treatment discontinuation, and fatal TEAEs are provided.

[0587] Laboratory parameters

[0588] Laboratory values ​​(hematology, biochemistry, and urinalysis) at each visit, including changes in continuous parameters from baseline, were summarized descriptively. Frequency tables for categorical parameters were prepared. Clinical laboratory values ​​outside the normal range were presented. For selected laboratory data, significant abnormalities were summarized using change tables.

[0589] Electrocardiogram

[0590] ECG recordings were performed in triplicate and the average of the three readings was used to determine ECG parameters (e.g., HR, PR, QRS, QT, QTcF). ECGs were reviewed at the clinical center, and the final interpretation of all ECGs was completed by a central reviewer and sent to the study site for evaluation and archiving. A list of subjects whose 12-lead ECG results were considered "abnormal and not clinically significant" or "abnormal and clinically significant" was provided.

[0591] result

[0592] Study Group

[0593] Seventeen subjects were randomized. Sixteen subjects completed the study (mFAS2 population), and one subject discontinued the study due to withdrawal of informed consent after the first month of treatment.

[0594] Of the 16 randomized subjects who completed the study, one randomized subject had not previously responded to a 5-HT4 receptor agonist, while the other 15 subjects were either 5-HT4 receptor agonist-responsive or naive (mFAS1 population). 4 In the responder status, 10 subjects (66.67%) had 4 Of the subjects with a history of benefit from agonists, 5 subjects (33.33%) had never received treatment (non-users).

[0595] All subjects were diagnosed with idiopathic CIPO. The mean number of pseudo-obstruction episodes and CIPO-related hospitalizations in the 6 months before consent were 1.8 ± 2.34 (range: 0; 6) and 0.3 ± 0.62 (range: 0; 2), respectively.

[0596] Adherence in the mFAS1 population was consistently above 80% across all treatment sequences and treatment periods. Similar results were observed in mFAS2 and PPS.

[0597] The demographic characteristics of the mFAS1 group (main group) are shown in Table 21.

[0598] The mean age was 56.7 ± 10.65 years (range: 40; 74 years), and the majority of subjects were female (12, 80.00%) and not Hispanic or Latino (11, 73.33%).

[0599] Table 21. Population characteristics; modified full analysis set 1

[0600]

[0601] Efficacy Endpoints

[0602] Primary Efficacy End Point

[0603] A summary of the main efficacy results is shown in the following table.

[0604] At the end of treatment, mean WGGSAIS in both the visstregol and placebo groups remained lower than before treatment, indicating improvement in symptoms. The magnitude of the decrease was consistently greater in the visstregol group.

[0605] Fig.17 Shown is a line graph of the WGGSAIS mean by treatment (30 observation pairs); mFAS1 population.

[0606] Table 22 reports the summary of WGGSAIS by treatment in the mFAS1 population (30 observation pairs).

[0607] Table 22. Summary of WGGSAIS by treatment modality (30 observation pairs); mFAS1 population

[0608]

[0609] For mFAS2 and PPS, missing interpolation methods can also be considered for boundary cases.

[0610] In mFAS2, the mean change from pre-treatment to end-of-treatment was -0.529 ± 0.7956 (range: -2.67; 0.75) for visstreg and -0.187 ± 0.7137 (range: -2.25; 1.75) for placebo, resulting in a mean difference between treatments of -0.342 ± 0.9343 (range: -2.92; 1.50). Thus, the decrease was greater with visstreg (paired t-test: p value = 0.0469, 95% CI for the mean difference in change between visstreg and placebo equals -0.679; -0.005; mixed model: p value = 0.0563, 95% CI for the mean difference in change between visstreg and placebo equals -0.69; 0.01).

[0611] Secondary End Points

[0612] The proportion of subjects with at least 1 point improvement in WGGSAIS

[0613] Subjects with available baseline / reference missing data values ​​greater than or equal to 1 were identified as evaluable for achieving at least a 1-point improvement.

[0614] Based on the WGGAIS value before treatment, “success” at each time point indicated an improvement of at least 1 point on the WGGAIS, whereas “failure” indicated no improvement of at least 1 point.

[0615] A total of 20 evaluable observation pairs were identified in the 15 subjects in mFAS1 for assessing success / failure in order to achieve a 1-point improvement in the WGGAIS.

[0616] Table 23 reports the proportion of pairs that improved by 1 point on the WGGAIS with treatment at each time point among the 20 evaluable observation pairs in the mFAS1 population.

[0617] At the end of treatment, the proportion of "success" in the vistrag group was 30.00% (6 pairs) and in the placebo group was 5.00% (1 pair). A greater probability of "success" was observed in the vistrag group (Fisher's exact test: p value = 0.0915; odds ratio estimated by logistic regression model = 8.14, 95% CI 0.88; 75.48).

[0618] These data suggest that, although a placebo effect was detected, Vistregr is more effective than placebo in improving the symptoms analyzed.

[0619] Table 23. Proportion of pairs of observations with a 1-point improvement in WGGAIS score by treatment; mFAS1 (20 evaluable pairs of observations)

[0620]

[0621] Changes in individual symptom scores

[0622] Monitor each individual for symptoms such as bloating, abdominal pain, nausea, and vomiting during treatment.

[0623] Fig.18 Shown are the means of each individual symptom score by treatment sequence and treatment period from pre-treatment to the end of each treatment period, also taking into account washout / follow-up assessments for a total of 23 observation pairs in the mFAS1 population.

[0624] Of the four symptoms monitored, vomiting showed a statistically significant improvement.

[0625] In subjects taking vistrag, the mean vomiting symptom score decreased from 1.3±1.58 (range: 0; 4) before treatment to 0.5±0.95 (range: 0; 3) at the end of treatment.

[0626] In subjects who received placebo, mean vomiting symptom scores remained stable: 1.1 ± 1.42 (range: 0; 4) before treatment and 1.1 ± 1.25 (range: 0; 4) at the end of treatment.

[0627] The mean difference in change between pre-treatment and end-of-treatment for visstreg and placebo was -0.8 ± 1.44 (range: -4; 1), indicating a greater decrease with visstreg (paired t-test: p value = 0.0164, 95% CI equal to -1.4; -0.2; mixed model: p value = 0.0177, 95% CI equal to -1.4; -0.2).

[0628] Similar results were observed when the same analysis was performed for each individual symptom score in the mFAS2 population (24 observation pairs). In particular, vomiting decreased slightly from pre-treatment to end-of-treatment during treatment with vistrag, whereas scores remained stable during treatment with placebo, such that the differences between treatments were statistically significant.

[0629] Effects on bowel habits in patients with constipation (ie, number of bowel movements per week and number of complete voids per week)

[0630] Among the 15 subjects in the mFAS1 population, 4 subjects with constipation were identified as having at least one Bristol stool scale equal to 1 or 2 before treatment. Six pairs were counted among these 4 subjects, considering the number of bowel movements per week and the number of complete voidings per week, to analyze the effect of treatment on bowel habits.

[0631] In subjects treated with vistrag, the mean number of bowel movements per week increased from 3.2 ± 1.72 (range: 1; 6) before treatment to 4.8 ± 2.14 (range: 2; 8) at the end of treatment, with a mean change of 1.7 ± 2.34 (range: -1; 5). In the same group, the mean number of complete voids per week increased from 1.5 ± 1.05 (range: 0; 3) before treatment to 2.2 ± 1.17 (range: 1; 4) at the end of treatment, with a mean change of 0.7 ± 1.21 (range: -1; 2).

[0632] In subjects treated with placebo, the mean number of bowel movements per week increased from 2.3 ± 1.03 (range: 1; 4) before treatment to 3.2 ± 1.17 (range: 3; 5) at the end of treatment, resulting in a mean change of 0.8 ± 0.98 (range: 0; 2). In the same treatment group, the mean number of complete voids per week was 1.0 ± 1.26 (range: 0; 3) before treatment and 1.5 ± 1.05 (range: 0; 3) at the end of treatment, resulting in a mean change of 0.5 ± 0.84 (range: 0; 2).

[0633] For both treatments, the number of bowel movements per week and the number of complete voids per week increased, with slightly greater increases for vistraglobulin.

[0634] Fig.19 A line graph showing the mean number of bowel movements per week and the mean number of complete voiding per week in the mFAS1 population after treatment.

[0635] Stool consistency

[0636] Subjects in the mFAS1 population were stratified according to stool consistency classified by the Bristol Stool Scale:

[0637] "Constipation" (ie, Bristol Scale type 1 or 2)

[0638] “Normal” (i.e. Bristol Scale type 3 or 4)

[0639] "Diarrhea" (ie, Bristol scale type 5, 6, or 7)

[0640] Twenty-three evaluable pairs were observed in the mFAS1 population.

[0641] Before treatment, stool consistency distributions for the 23 observation pairs were similar for vistraglita and placebo.

[0642] The frequency of normal bowel movements increased to 26.09% at the end of treatment in subjects taking Vistrag, while the frequency of constipation decreased to 0% at the end of treatment. Small fluctuations were observed in subjects taking placebo.

[0643] Among subjects treated with vistraglita, all 4 pairs of subjects with constipation before treatment and 1 of 16 pairs of subjects with diarrhea had normal bowel movements at the end of treatment.

[0644] Among the subjects who received placebo, at the end of treatment, 2 of the 17 pairs of subjects with diarrhea before treatment had normal stools, 2 developed constipation, and 13 had persistent diarrhea. Among the 3 pairs of subjects with normal stools before treatment, 1 developed diarrhea at the end of treatment.

[0645] In summary, the proportion of subjects with normal bowel movements increased and the proportion with constipation decreased for vistraglita, whereas small fluctuations were observed for placebo.

[0646] Table 24 reports the change in stool consistency at the end of treatment and before treatment by treatment regimen in the mFAS1 population (23 evaluable observation pairs).

[0647] Table 24. Stool consistency: change between end of treatment and pre-treatment by treatment modality (23 evaluable observation pairs); mFAS1

[0648]

[0649] The same analysis was performed for the mFAS2 population (considering 24 pairs). A similar frequency distribution of pairs classified as "constipation", "normal" and "diarrhea" was observed before treatment.

[0650] Among subjects administered Vistragl, 0, 6 (25.00%), and 18 (75.00%) pairs were classified as "constipation," "normal," and "diarrhea," respectively.

[0651] Among the subjects administered placebo, 6 (25.00%), 4 (16.67%), and 14 (58.33%) were classified as "constipation", "normal", and "diarrhea", respectively.

[0652] For this group, the difference in stool consistency before and at the end of treatment was statistically significant (p value = 0.0339).

[0653] Pseudo-obstruction

[0654] Considering 15 subjects in mFAS1 (30 observation pairs), the number of pseudo-obstruction episodes during the treatment period and the washout period was evaluated.

[0655] Two pairs (6.67%) in the visstreg group experienced one pseudo-obstruction, seven pairs (23.33%) in the placebo group experienced one pseudo-obstruction, and one pair (3.33%) in the visstreg group experienced two pseudo-obstructions. During the washout period, only one pair (3.33%) experienced one pseudo-obstruction after receiving placebo.

[0656] Table 25 reports the distribution of pairs according to the number of pseudo-obstructions by treatment modality, where “pseudo-obstruction episodes during treatment” refer to data collected at the “end of treatment” visit, and “pseudo-obstruction episodes during washout” refer to data collected at the “start of treatment” visit.

[0657] Overall, the incidence of pseudoocclusive episodes was lower during treatment with visstreg than with placebo: four episodes in the visstreg group and eight in the placebo group.

[0658] Table 25. Summary of pseudo-obstruction episodes by treatment modality (30 observation pairs); mFAS1

[0659]

[0660] Oro-cecal transit time

[0661] Oro-cecal transit time was calculated based on the lactulose breath test (L-BT). The L-BT test was performed only during the first treatment period of the study.

[0662] In the mFAS1 population, after administration of Vistra Among treated subjects, mean orocecal transit time was 169.3 ± 60.44 minutes (range: 90; 240 minutes) at visit 1 (screening) and 162.9 ± 67.32 minutes (range: 90; 240 minutes) at visit 3 (end of treatment 1), resulting in Decreased by -6.4 ±118.70 minutes (range: -150; 150 minutes).

[0663] In the same population, among subjects taking placebo, mean oro-cecal transit time was 167.5 ± 61.79 minutes (range: 90; 240 minutes) at visit 1 and 173.6 ± 52.58 minutes (range: 105; 240 minutes) at visit 3. Increased by 10.0 ±55.50 minutes (range: -60; 105 minutes).

[0664] The difference in change between treatments was -16.4 (95% CI: -133.0; 100.1)

[0665] Table 26 reports the summary of oro-cecal transit time by treatment for the mFAS1 population and the change between post-treatment (i.e., end of the first treatment period at Visit 3 (End of Treatment-1)) and pre-treatment (i.e., screening value at Visit 1).

[0666] The same analysis was repeated excluding subjects who had taken antibiotics in the two weeks before LB-T as a sensitivity analysis. Five subjects were excluded as well as one subject who underwent LB-T but for whom the exit-cecal transit time could not be calculated. Also in this case, a decrease in the oro-cecal transit time (minutes) between visit 3 and visit 1 was observed, with a larger mean change (-63.0 ± 83.79 minutes for visstregol and 18.8 ± 68.60 minutes for placebo, with a mean change between visstregol and placebo of -81.8). These data suggest that visstregol has prokinetic activity.

[0667] Table 26. Summary of Orocecal Transit Time by Treatment Modality; mFAS1

[0668]

[0669] Effects of treatment interruption

[0670] The changes after the end of treatment for the identified pairs are described, taking into account the electronic diary data collected during the washout period or the follow-up period. Considering the WGGSAIS for each symptom, a slight increase in the mean was observed for visstreg, indicating a worsening of the condition, while the mean for placebo remained stable. Similarly, for the WGGSAIS total score, the mean change at the end of the washout period was 0.54 ± 0.736 for visstreg and -0.06 ± 0.815 for placebo. For the placebo group, the changes observed in stool consistency were: 2 constipated pairs with an improvement in stool consistency, which subsequently became normal stools; 4 normal pairs with a worsening of stool consistency, which became constipation (2) or diarrhea (2). For the visstreg group, the changes observed were: 1 pair with diarrhea with an improvement in condition, which subsequently became normal stools; 3 normal pairs with a worsening of condition, which became constipation (2) or diarrhea (1). In subjects with constipation, a slight decrease in both the number of bowel movements per week and the number of complete voids per week was observed from the end of treatment to the end of washout for both the visstregol and placebo groups, with a slightly greater decrease in the visstregol group.

[0671] Safety Assessment

[0672] The safety set included 17 subjects; 7 (41.18%) experienced a total of 29 TEAEs classified as visstreptomycin-emergent (the last treatment received before the TEAE onset date was visstreptomycin), while 10 subjects (58.82%) experienced a total of 38 TEAEs classified as placebo-emergent (the last treatment received before the TEAE onset date was placebo). No deaths, serious TEAEs, or TEAEs leading to treatment discontinuation or interruption were reported.

[0673] In addition, no treatment-related TEAEs and no cardiovascular adverse reactions were observed.

[0674] The study found that vistra was safe and well tolerated in all study regimens.

Claims

1. 1-Isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methylsulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]octan-3-yl}amide (vistrag) or a pharmaceutically acceptable salt thereof, for use in the treatment of idiopathic chronic intestinal pseudo-obstruction (CIPO), neurogenic chronic intestinal pseudo-obstruction, or chronic intestinal pseudo-obstruction secondary to neurodegeneration or secondary to autoimmune disease or secondary to connective tissue disease or secondary to demyelinating disease.

2. The Vistregr for use according to claim 1, wherein: The pharmaceutically acceptable salt is hydrochloride.

3. Vistralig for use according to claim 1 or 2, wherein: Vistragl is in crystalline form and / or in hydrated form.

4. Vistralig for use according to one of claims 1 to 3, wherein The idiopathic CIPO, neurological CIPO, or CIPO secondary to neurodegeneration or secondary to autoimmune disease or secondary to connective tissue disorder or secondary to demyelinating disease is CIPO caused by autonomic nervous system disease, such as stroke, encephalitis, basal ganglia calcification, orthostatic hypotension; CIPO caused by intestinal wall nervous system disease, such as paraneoplastic syndrome, viral infection, iatrogenic disease, Hirschsprung's disease, Chagas disease, von Recklinghausen disease; CIPO caused by intestinal wall muscular layer disease, such as myotonic dystrophy, progressive systemic or CIPO caused by a mixed enteric nervous system and smooth muscle layer disease, such as scleroderma, dermatomyositis, amyloidosis, Ehlers-Danlos syndrome; or CIPO caused by an unknown mechanism, such as hypothyroidism, hypoparathyroidism, pheochromocytoma, antidepressants, antineoplastic drugs, or bronchodilators; or CIPO caused by an immune-mediated connective tissue disorder or disease, such as paraneoplastic diseases (central nervous system tumors, pulmonary foramina, bronchial carcinoids, leiomyosarcoma, systemic lupus erythematosus).

5. Vistralig for use according to one of claims 1 to 4, wherein The use is directed to adult patients or pediatric patients.

6. Vistregr for use according to claim 5, wherein: The patient is one with a history of chronic CIPO or CIPO secondary to a neurodegenerative or demyelinating disease.

7. Vistralig for use according to one of claims 1 to 6, wherein At least one CIPO symptom selected from gastrointestinal dysmotility, increased distension of the proximal colon and / or distal small intestine, altered intestinal contractility, ulcers, inflammation of the proximal colon and / or distal small intestine, pseudo-obstruction episodes, vomiting, abdominal distension, abdominal pain, mental health, quality of life and mortality is alleviated / improved; and / or wherein the number and / or frequency of CIPO-related and / or CIPO-induced hospitalizations is reduced.

8. Vistralig for use according to one of claims 1 to 6, wherein The administration dose of vistrag is 0.5 mg / day to 30 mg / day, preferably 5 mg / day to 15 mg / day, more preferably 15 mg / day, based on the weight of the free base.

9. Vistralig for use according to one of claims 1 to 8, wherein The administration period of vistragliflozin is 2 weeks to 12 weeks, preferably 2 weeks to 6 weeks.

10. Vistralig for use according to one of claims 1 to 9, wherein The vistrag is administered orally, parenterally, buccally, sublingually, rectally, intraperitoneally or intratracheally.

11. Vistregr for use according to claim 10, wherein: The transdermal administration is subcutaneous, intramuscular, intravenous, transdermal or by implantation.

12. Vistralig for use according to claim 10, wherein: The vistrag is administered orally in the form of a liquid, capsule, tablet, chewable tablet, soluble film, pill, lozenge, cachet, dragee, powder, granules, solution, suspension, oil-in-water or water-in-oil liquid emulsion, elixir or syrup.

13. Vistralig for use according to claim 10, wherein: The vistragliflozin is administered parenterally in the form of a liquid, solid or gel.

14. Vistralig for use according to one of claims 1 to 13, wherein The vistragliflozin is administered orally with or without food, preferably once daily.

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