Velsetrag for use in the treatment of chronic intestinal pseudo-obstruction (CIPO)

Abstract

The present invention relates to 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide (velcetrag) or a pharmaceutically acceptable salt thereof for use in the treatment of idiopathic chronic intestinal pseudo-obstruction (CIPO), neuropathic chronic intestinal pseudo-obstruction, or chronic intestinal pseudo-obstruction secondary to a neurodegenerative or demyelinating condition in patients suffering from said disease or condition.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to velusetrag [1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide], pharmaceutically acceptable salts thereof, and compositions comprising said compound(s), for use in the treatment of chronic intestinal pseudo-obstruction (CIPO), in particular neuropathic or idiopathic CIPO. Accordingly, 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 constipation-related disorders and conditions, such as, for example, constipation associated with the use of opiate analgesics, post-surgical constipation, constipation associated with neurological disorders and other conditions. [Background technology]

[0002] prior art Intestinal pseudo-obstruction is a rare and severe condition characterized by disordered peristalsis with symptoms of intestinal obstruction, but without signs of mechanical obstruction. The disorder results from abnormalities in the enteric neuromusculature and / or its autonomic innervation. It is the most severe form of gastrointestinal dysmotility, which can be debilitating and life-threatening.

[0003] If this condition persists for more than six months, chronic intestinal pseudo-obstruction (CIPO) develops, which is one of the most important causes of chronic intestinal failure in both pediatric (15%) and adult (20%) cases. Because affected individuals are often unable to take a normal oral diet and maintain an adequate weight, they develop a severe clinical picture characterized by disabling gastrointestinal symptoms, which severely reduces the patient's quality of life and can lead to death.

[0004] A distinction is made between primary and secondary CIPO. Primary CIPO is due to an intrinsic defect (congenital or acquired) and can be classified by the type of (potentially associated) damage: abdominal myopathy (related to muscle damage; e.g., MNGIE: mitochondrial neurogastrointestinal encephalomyopathies, MMIHS: megacystis-microcolon-intestinal hypoperistalsis syndrome, idiopathic), abdominal neuropathy (related to autonomic nerve damage; e.g., myenteric plexus dysgenesis, Hirschsprung's disease, sequelae of necrotizing enterocolitis, idiopathic), or mesenchymopathy (related to damage to the Cajal cells of the gastrointestinal tract).

[0005] The etiological classification of CIPO has evolved with the discovery of new genetic entities, particularly through the study of familial forms. Examples of implicated genes include the TYMP gene (mutated in MNGIE), the ACTG2 gene (mutated in megacystis-microcolon-hypoperistalsis 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 the majority of CIPO cases is currently unknown, and these cases are referred to as idiopathic. Therefore, these cases are included in the primary CIPO group.

[0006] Secondary CIPO is associated with underlying systemic neurological, endocrine and connective tissue disorders or malignancies. In these diseases, gastrointestinal motility may be affected by the involvement of the autonomic nervous system (stroke, encephalitis, orthostatic hypotension), the nervous system of the intestinal wall (paraneoplastic syndromes, viral infections, iatrogenic diseases, diabetes, Chagas disease, von Recklinghausen disease), the muscular layer of the intestinal wall (myotonic dystrophy, progressive systemic sclerosis), a mixture of the enteric nervous system and smooth muscle layer (scleroderma, dermatomyositis, amyloidosis, Ehlers-Danlos syndrome, jejunal diverticulum, radiation enteritis), and finally, unknown mechanisms (hypothyroidism, hypoparathyroidism, pheochromocytoma, antidepressants, antineoplastic agents, bronchodilators) [Antonucci A. et al., "Chronic intestinal pseudo-obstruction." World journal of gastroenterology 2008; 14: 2953-61 (Non-Patent Document 1); Zhu CZ et al., "Latest developments in chronic intestinal pseudo-obstruction. World J Clin Cases. 2020; 8: 5852-5865 (Non-patent document 2); Billiauws L. et al., Small intestine motility disorders: Chronic intestinal pseudo-obstruction. J Visc Surg. 2022;159(1S):S22-S27 (Non-patent document 3)].

[0007] Although there are no clear epidemiological data, it is estimated that in the United States, approximately 100 infants are affected by intestinal pseudo-obstruction annually, and the incidence (prevalence) in adults is 0.2 (male) and 0.24 (female) per 100,000 patient-years, with an estimated prevalence in adults of 0.2-0.9 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. Volume 17, 2023 - Issue 4 (Non-Patent Document 4)].

[0008] In general, the most commonly reported symptoms are non-colic abdominal pain and distension (80%), which are persistent and worsened by eating. These symptoms are generally localized to the umbilical or epigastric region and gradually spread to involve the entire abdomen. Other typical symptoms are nausea (75%), vomiting (40%-50%), constipation (40%), and diarrhea (20%-30%).

[0009] Approximately 30% of patients with typical symptoms also exhibit small intestinal bacterial overgrowth (SIBO), a condition that contributes to further mucosal damage, steatorrhea, diarrhea, and intestinal damage; chronically dilated loops of intestine contribute to malabsorption, vitamin deficiencies, and weight loss. Extraintestinal manifestations are possible (e.g., bladder and ureter in MMIHS; ophthalmoplegia, ptosis, and peripheral polyneuropathy in MNGI; depression or other psychological disorders with the long-term course of the disease).

[0010] The typical clinical manifestation of CIPO is recurrent pseudo-obstructive episodes, characterized by abdominal pain, abdominal distension, and inability to defecate, with or without vomiting, resembling mechanical sub-occlusion. In acute episodes, key diagnostic markers of this pathological condition are radiological evidence of distended intestinal loops and the presence of an air-fluid level in the upright position. In the most severe cases, the intestinal loops are chronically distended, and a niveau is detectable.

[0011] During an acute episode, patients may be asymptomatic or suffer from a variety of gastrointestinal symptoms that are primarily related to the location and extent of the gastrointestinal (gut) tract involved.

[0012] Because CIPO symptoms overlap with severe forms of other gastrointestinal disorders and there are no biomarkers, patients often undergo unnecessary and potentially dangerous abdominal surgery before the cause of their recurrent subacute obstructive episodes is suspected. The average time to a correct, definitive diagnosis is 8 years.

[0013] The diagnosis of CIPO is primarily made in clinics and is supported by radiographic documentation of a dilated bowel with niveau after excluding organic lesions obstructing the intestinal lumen detected by radiological and / or endoscopic examinations. Identifying possible causes of secondary cases is important. Intestinal manometry may help distinguish between mechanical and functional obstruction and identify the underlying pathophysiological mechanism. In all patients suspected of CIPO undergoing surgery for unexplained obstructive episodes, full-thickness biopsies should be obtained from the dilated or non-dilated tract of the gastrointestinal tract [Antonucci A. et al., "Chronic intestinal pseudo-obstruction," World Journal of Gastroenterology 2008; 14: 2953-61 (Non-Patent Document 5)].

[0014] The primary goals of CIPO therapy are to enhance gastrointestinal motility, improve nutritional status, and maintain a stable intestinal environment. In patients with secondary CIPO, the primary disease must, of course, be aggressively treated to eliminate its cause.

[0015] In acute pseudo-obstruction episodes, the use of surgery should be minimized as it may inhibit intestinal peristalsis and even induce further intestinal failure, and the reintervention rate in such cases is high.

[0016] Low-dose tricyclic antidepressants and gabapentin are used to treat visceral pain, especially chronic pain, in patients with CIPO. However, opioids are not recommended because they inhibit gastrointestinal motility. However, as the disease progresses, approximately 25% of patients gradually develop analgesic tolerance, and the involvement of a team including a pain specialist and psychologist is recommended for appropriate treatment management [Stanghellini V. et al., Natural History of Chronic Idiopathic Intestinal Pseudo-Obstruction in Adults: A Single Center Study, Clinical Gastroenterology and Hepatology, 2005; 3: 449-458 (Non-Patent Document 6)].

[0017] Bacterial concentration is usually 10 3 ~10 5Oral antibiotic therapy is prescribed to control concomitant SIBO, the most common complication of chronic intestinal distension, which exceeds CFU / mL. Antibiotic-based treatment plans include amoxicillin-clavulanate (500 mg tid (three times daily)), ciprofloxacin (500 mg bid (twice daily)), doxycycline (100 mg bid), metronidazole (250 mg tid), neomycin (500 mg bid), rifaximin (550 mg bid), and tetracycline (250 mg qid (four times daily)). Antibiotics are typically prescribed for 7–10 days per month, and the antibiotic type is changed monthly for 5–6 months to avoid resistance.

[0018] Fecal bacterial transplantation has also been proposed as a new approach to the treatment of CIPO. Studies have shown that fecal bacterial transplantation significantly reduces abdominal distension and pain, improves tolerance to enteral nutrition, and prevents and treats associated 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 (Non-Patent Document 7)).

[0019] Intestinal transplantation has the potential to save a child's life, but is only indicated for patients in whom long-term parenteral nutrition (intravenous nutrition) is not feasible or cannot be safely continued, including those who develop liver complications due to parenteral nutrition, those with difficult central line access, or those who experience poor quality of life and worsening pain despite aggressive medical management (Camilleri et al., Chronic Intestinal Pseudo-obstruction: Management, UpToDate, Feb 17, 2022).

[0020] Because CIPO is characterized by impaired intestinal propulsive activity, in the absence of an effective and resolvable treatment for this condition, drugs 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 (Non-Patent Document 9)] 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 (Non-Patent Document 10)] are commonly used to promote gastrointestinal motility. However, long-term treatment with erythromycin does not appear to be feasible due to tachyphylaxis (associated with downregulation of the motilin receptor) [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 (Non-patent Document 11)].

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

[0022] Serotonin (5-HT) is a neurochemical involved in the regulation of intestinal motility. However, the functional role of endogenous 5-HT has not yet been fully elucidated, as depletion of neuronal or mucosal 5-HT has little or no effect on intestinal motility. Meanwhile, 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 five of the seven known families, 5-HT1 receptor, 5-HT2 receptor, 5-HT3 receptor, 5-HT4 receptor, and 5-HT7 receptor, are expressed in the intestine and may affect intestinal function. The 5-HT3 receptor subtype and the 5-HT4 receptor subtype have been most extensively studied in the intestine and are targeted for the treatment of diarrhea and constipation, respectively.

[0024] Furthermore, it has been reported that endogenous 5-HT can act as a modulator of gastrointestinal motility through activation of 5-HT3 and 5-HT4 receptors in the enteric nervous system (ENS). Disturbance of these regulatory mechanisms within this tightly controlled system is associated with intestinal motility disorders [Waclawikova 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 (Non-Patent Document 12)].

[0025] Prucalopride, a non-cardiotoxic, highly selective 5-HT receptor agonist, exerts 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). It has shown promising results in children and adults with acute and chronic intestinal pseudo-obstruction. Prucalopride is safe, effective, and well tolerated in children with acute intermittent or chronic intestinal pseudo-obstruction (Mutalib M. et al., Prucalopride in intestinal pseudo-obstruction, pediatric experience and systematic review. Acta Gastroenterol Belg. 2021;84:429-434).

[0026] Furthermore, Emmanuel AV et al. described a phase II, double-blind, placebo-controlled, two-treatment, four-period crossover trial that investigated the clinical safety, tolerability, and efficacy of prucalopride in improving symptoms associated with CIPO. Subjects received either prucalopride 2 mg (Period 2) or placebo (Period 2) for four 12-week periods. There was no washout period. A total of seven subjects were randomized: two to the PLA-PRU-PLA-PRU sequence, two to the PRU-PLA-PRU-PLA sequence, two to the PLA-PRU-PRU-PLA sequence, and one to the PRU-PLA-PLA-PRU sequence. Three patients discontinued the study after the first period. One of the deaths was due to a serious adverse event considered unrelated to the study drug (feeding line infection, sepsis, bronchopneumonia, and malnutrition), and two were due to consent withdrawal (one each for placebo and prucalopride). The remaining four patients completed the study. Symptoms analyzed were pain, vomiting, nausea, and flatulence. This study suffers from a limited number of participants. However, the results of this long-term study demonstrate that prucalopride alleviated CIPO symptoms. Prucalopride significantly improved pain in three of four patients, nausea in two, vomiting in one, and flatulence in four. The frequency of analgesic intake was significantly reduced during prucalopride treatment compared with the placebo period. However, prucalopride did not affect stool frequency or consistency, and there was no evidence of a major prokinetic (enhancing) effect on gastrointestinal transit.The incidence of pseudo-obstruction episodes was not analyzed in this study of patients with CIPO [Emmanuel AV et al., Randomized 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 (Non-Patent Document 15)].

[0027] Other 5-HT4 receptor agonists, such as cisapride and tegaserod, are effective but are contraindicated due to associated fatal arrhythmias.

[0028] The 5-HT4 receptor has several variants due to alternative splicing of the corresponding gene, which explains the different effects of reference compounds used in terms of agonist / antagonist activity against 5-HT4 splice variants. This makes targeting various variants of the receptor highly unpredictable in terms of therapeutic efficacy. The importance of variation introduced by splicing to receptor pharmacology may help understand the contradictory results observed with 5-HT4 receptor ligands in different model systems [Takaki M. et al., The 5-hydroxytryptamine 4 receptor agonist-induced actions and enteric neurogenesis in the gut. J Neurogastroenterol Motil. 2014; 20:17-30 (Non-Patent Document 16)].

[0029] To date, it is clear that unmet medical needs remain for patients with CIPO. Despite extensive research, pharmacological treatment options remain limited and are often associated with severe side effects. This means that, to date, there have been no satisfactory treatments for CIPO aimed at effectively ameliorating the disease's core symptoms. As outlined above, prior art attempts to treat CIPO have either yielded unsatisfactory results or been associated with severe side effects. In particular, no treatments have demonstrated prokinetic effects. Furthermore, there have been no treatments that target the neurodegeneration underlying neuroenteric system disorders and gut dysmotility conditions.

[0030] Velusetrag is a highly selective 5-hydroxytryptamine subtype 4 (5-HT4) receptor agonist with prokinetic activity. The chemical name of velusetrag is 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide, and its chemical structure is shown in Formula I below:

[0031] [ka]

[0032] Velsetrag was previously disclosed in U.S. patent application Ser. No. 11 / 100,113, filed April 6, 2005 (corresponding to EP 1 735 304), and a related crystalline form was disclosed in U.S. patent application Ser. No. 11 / 398,119, filed April 5, 2006 (corresponding to EP 1 874 766).

[0033] The robust gastrointestinal motility-promoting effects of velusetrag have been confirmed in vivo in both preclinical models and healthy humans (Smith JAM et al., The in vitro pharmacological profile of TD-5108, a selective. Naunyn-Schmiedeberg's Arch Pharmacol. 2008; 378: 125-137 (Non-Patent Document 17); Beattie DT et al., The in vivo gastrointestinal activity of TD-5108, a selective 5-HT4 receptor agonist with high intrinsic activity. Naunyn-Schmiedeberg's Arch Pharmacol. 2008; 378: 139-147 (Non-Patent Document 18)).

[0034] Activation of 5-HT4 receptors is associated with contractions of the isolated longitudinal muscle of the guinea pig colon and inhibition of electrically evoked or spontaneous contractions of the isolated circular muscle of the human colon. In vivo 5-HT4 receptor agonism increases colonic transit in guinea pigs, relaxes the esophagus in rats, and enhances upper and lower gastrointestinal (GI) tract motility in dogs.

[0035] Velsetrag is being evaluated for the treatment of gastrointestinal (GI) motility disorders, including chronic idiopathic constipation (CIC) and gastroparesis (GP).

[0036] WO 2015 / 175997 (Patent Document 5) describes a method for treating or ameliorating the effects of autism, a condition in which serotonin transporter (SERT) activity is altered, which impairs the enteric nervous system and results in decreased gastrointestinal motility. The method comprises administering a 5-HT4 agonist to a subject. CIPO is also mentioned as a disease associated with altered SERT activity, and velusetrag is disclosed as a 5-HT4 agonist, but no specific examples of velusetrag being used for CIPO are described.

[0037] Clinical trial NCT00391820 showed that patients with fewer than three spontaneous bowel movements (SBMs) per week were administered 15 mg, 30 mg, and 50 mg daily for four weeks, which resulted in a statistically and clinically significant increase in weekly SBM frequency compared to 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 (Non-Patent Document 19)].

[0038] Ahn A. et al. disclosed the prokinetic effects of velusetrag on the upper gastrointestinal tract by evaluating gastric emptying (GE) time in both diabetic and idiopathic gastroparesis subjects randomized into four groups receiving velusetrag 5 mg, 15 mg, 30 mg, and placebo. The study results showed that the doses of velusetrag used were effective in promoting gastric emptying. All doses were well tolerated, with a significant level reached at the 30 mg dose [Ahn A. et al., Su 1426 Velusetrag improves gastric emptying time in subjects with diabetic or idiopathic gastroparesis. Gastroenterology 2015; 148: S-507 (Non-Patent Document 20)].

[0039] Clinical trial NCT02267525 and patent application WO 2019 / 027881 (corresponding to EP 18 756 041.2 (Patent Document 7)) teach the use of velusetrag or a pharmaceutically acceptable salt thereof in a method for preventing, reducing, ameliorating, alleviating, or treating the core symptoms of gastroparesis, consisting of postprandial fullness, early satiety, flatulence, upper abdominal pain, epigastric burning, nausea, and vomiting, in diabetic or idiopathic human patients. This method involves administering velusetrag at a specific dose of 5 mg / day for a 1-week, 2-week, 4-week, 8-week, or 12-week administration period (treatment period) [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 (Non-Patent Document 21); Abell T. et al., Efficacy of velusetrag treatment in patients with idiopathic gastroparesis: subgroup analysis of a phase 2b study, Abstract at UEG Week 2019, 26-04-2019 (Non-Patent Document 22)].

[0040] Like other agonists of the 5-HT4 receptor, velusetrag works by inducing the release of neurotransmitters such as acetylcholine from enteric motor neurons and calcitonin gene-related peptide from sensory neurons in the gastrointestinal tract. By selectively activating 5-HT4 receptors in the gastrointestinal tract, velusetrag enhances the peristaltic reflex, stimulates intestinal secretions (enteroendocrine secretion), and inhibits visceral sensation.

[0041] The present inventors demonstrated that velusetrag-induced 5-HT4 activation enhances specific defensive responses in different neuronal cells analyzed, including human enteric neurons. These findings open new avenues for the treatment of gastrointestinal disorders and homeostasis, particularly in the context of neurodegeneration, which often underlies severe intestinal motility disorders. [Prior art documents] [Patent documents]

[0042] [Patent Document 1] U.S. Patent Application No. 11 / 100,113 [Patent Document 2] EP 1 735 304 [Patent Document 3] U.S. Patent Application No. 11 / 398,119 [Patent Document 4] EP 1 874 766 [Patent Document 5] WO 2015 / 175997 [Patent Document 6] WO 2019 / 027881 [Patent Document 7] EP 18 756 041.2 [Non-patent literature]

[0043] [Non-Patent Document 1] Antonucci A. et al., Chronic intestinal pseudo-obstruction." World journal of gastroenterology 2008; 14: 2953-61 [Non-patent document 2] Zhu CZ et al. Latest developments in chronic intestinal pseudo-obstruction. World J Clin Cases. 2020; 8: 5852-5865 [Non-patent document 3] Billiauws L. et al., Small intestine motility disorders: Chronic intestinal pseudo-obstruction. J Visc Surg. 2022 ;159(1S):S22-S27 [Non-patent document 4] Di Nardo G. et al., Pharmacological and nutritional therapy of children and adults with chronic intestinal pseudo-obstruction. Expert Review of Gastroenterology & Hepatology. Volume 17, 2023 - Issue 4 [Non-Patent Document 5] Antonucci A. et al., Chronic intestinal pseudo-obstruction." World journal of gastroenterology 2008; 14: 2953-61 [Non-patent document 6] Stanghellini V. et al., Natural History of Chronic Idiopathic Intestinal Pseudo-Obstruction in Adults: A Single Center Study, Clinical Gastroenterology and Hepatology, 2005; 3: 449-458 [Non-Patent Document 7] 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 [Non-patent document 8] Camilleri et al., Chronic Intestinal Pseudo-obstruction: Management, UpToDate, Feb 17, 2022 2022 [Non-Patent Document 9] Gomez R. et al., Effect of Amoxicillin / Clavulate on Gastrointestinal Motility in Children, Journal of Pediatric Gastroenterology and Nutrition 2012; 54: 780-784 [Non-Patent Document 10] 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 [Non-Patent Document 11] 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 [Non-Patent Document 12] Waclawikova 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 [Non-Patent Document 13] Bianco F. et al., Prucalopride exerts neuroprotection in human enteric neurons. Am J Physiol Gastrointest Liver Physiol. 2016; 310: G768-75 [Non-Patent Document 14] Mutalib M. et al. Prucalopride in intestinal pseudo-obstruction, paediatric experience and systematic review. Acta Gastroenterol Belg. 2021; 84: 429-434 [Non-Patent Document 15] Emmanuel AV et al., Randomized 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 [Non-Patent Document 16] Takaki M. et al., The 5-hydroxytryptamine 4 Receptor Agonist-induced Actions and Enteric Neurogenesis in the Gut. J Neurogastroenterol Motil. 2014; 20:17-30 [Non-Patent Document 17] Smith JAM et al., The in vitro pharmacological profile of TD-5108, a selective. Naunyn-Schmiedeberg's Arch Pharmacol. 2008; 378:125-137 [Non-Patent Document 18] Beattie DT et al. The in vivo gastrointestinal activity of TD-5108, a selective 5-HT4 receptor agonist with high intrinsic activity. Naunyn-Schmiedeberg's Arch Pharmacol. 2008; 378: 139-147 [Non-Patent Document 19] 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 [Non-Patent Document 20] Ahn A. et al., Su 1426 Velusetrag improves gastric emptying time in subjects with diabetic or idiopathic gastroparesis. Gastroenterology 2015; 148: S-507 [Non-Patent Document 21] 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. [Non-Patent Document 22] Abell T. et al., Efficacy of velusetrag treatment in patients with idiopathic gastroparesis: subgroup analysis of a phase 2b study, Abstract at UEG Week 2019, 26-04-2019 Summary of the Invention

[0044] Summary of the Invention The present invention relates to velusetrag or a pharmaceutically acceptable salt thereof for use in the treatment of idiopathic chronic intestinal pseudo-obstruction (CIPO), neuropathic chronic intestinal pseudo-obstruction, or chronic intestinal pseudo-obstruction secondary to neurodegeneration, secondary to an autoimmune condition, secondary to a connective tissue disorder, or secondary to a demyelinating condition.

[0045] According to one embodiment, the pharmaceutically acceptable salt is the hydrochloride salt. The present invention also relates to the above-mentioned use, wherein velusetrag is in crystalline form and / or hydrate form.

[0046] The present invention also relates to the above-mentioned use, wherein the CIPO is idiopathic CIPO, neuropathic CIPO, or CIPO secondary to neurodegeneration, secondary to an autoimmune condition, secondary to a connective tissue disorder, or secondary to a demyelinating condition, or any CIPO resulting from the following diseases or mechanisms: Autonomic nervous system disorders such as stroke, encephalitis, calcification of basal ganglia, and orthostatic hypotension Paraneoplastic syndromes, viral infections, iatrogenic disorders, diseases of the intestinal wall nervous system such as Hirschsprung's disease, Chagas' disease, and von Recklinghausen's disease, Diseases of the intestinal wall muscle layer, such as myotonic dystrophy and progressive systemic sclerosis, Mixed enteric nervous system and smooth muscle layer diseases such as scleroderma, dermatomyositis, amyloidosis, and Ehlers-Danlos syndrome, unknown mechanism, such as hypothyroidism, hypoparathyroidism, pheochromocytoma, antidepressants, antineoplastic drugs, or bronchodilators; or Paraneoplastic disease (immune-mediated and connective tissue disorder or disease, such as central nervous system neoplasms, lung microstoma, bronchial carcinoid, leiomyosarcoma, and systemic lupus erythematosus)

[0047] The present invention also relates to the above-mentioned use, wherein the patient is a subject with CIPO. Preferably, the patient is an adult or child subject.

[0048] The present invention also relates to the above-mentioned use, wherein the patient is a patient with a history of chronic CIPO or CIPO secondary to a neurodegenerative disease or demyelinating disease (demyelinating disease).

[0049] The present invention also relates to the above-mentioned use, which alleviates or ameliorates at least one of the symptoms of CIPO (e.g., vomiting, flatulence, abdominal pain) or exacerbations of the disease (e.g., pseudo-obstructive episodes). The present invention also relates to the above-mentioned use, which alleviates or ameliorates at least one of the symptoms of CIPO selected from abnormal gastrointestinal motility, increased dilation of the proximal colon and / or distal small intestine, modified intestinal contractility, ulcer formation, inflammation of the proximal colon and / or distal small intestine, pseudo-obstructive episodes, and mortality, and / or reduces the number and / or frequency of CIPO-related and / or CIPO-caused hospitalizations.

[0050] Additionally, the present invention relates to the above-mentioned use, wherein velusetrag is administered in a dose amount ranging from 0.5 mg to 30 mg, preferably from 5 mg to 15 mg, based on the weight of the free base. These doses represent daily doses for an average adult human patient. However, the dose can be modified and / or adapted depending on the severity of the disease, the condition of the particular patient, and the particular route of administration selected.

[0051] The present invention also relates to the above-mentioned use, wherein the treatment period (administration period or treatment period, treatment duration) can be changed and / or adapted depending on the severity of the disease or the condition of a particular patient. Preferably, the treatment (administration or treatment) is carried out for at least 1 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, or 22 weeks. Preferably, the treatment (administration or treatment) is carried out for 24 weeks or at least 24 weeks, preferably 26 weeks, 28 weeks, 30 weeks, or more. The treatment (administration or treatment) may be carried out in repeated cycles over at least 1 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, or 22 weeks. Preferably, the treatment is carried out for 24 weeks or at least 24 weeks, preferably 26 weeks, 28 weeks, 30 weeks, or more.

[0052] Velusetrag is preferably administered at a dose of 15 mg per day, taken once daily, preferably in the form of 3 x 5 mg tablets. The preferred route of administration is oral.

[0053] Another embodiment of the present invention encompasses pharmaceutical compositions that vary in the route of administration of velusetrag to a human patient. Routes of administration include, inter alia, oral, parenteral, buccal, sublingual, rectal, intraperitoneal, or intratracheal administration. For example, parenteral administration may be by infusion, injection, or implant. Parenteral administration also includes subcutaneous, intramuscular, intravenous, transdermal, or percutaneous administration via implant routes. When velusetrag is administered parenterally, it may be in the form of a liquid, solid, or gel. Similarly, when velusetrag is administered orally, it may be in the form of a liquid, capsule, tablet, chewable tablet, or dissolvable film.

[0054] In one embodiment, the product contains velusetrag in an amount of about 0.5 mg to about 30 mg labeled for the treatment of symptoms of CIPO. In yet another embodiment, the product contains velusetrag in an amount of 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 labeled for the treatment of symptoms of CIPO.

[0055] In one embodiment, the present invention provides a kit comprising a pharmaceutical product containing velusetrag according to the present invention with instructions for use and administration on the package insert. In another embodiment, the package insert instructs the patient to administer velusetrag for the treatment (administration or treatment) period described above.

[0056] Velusetrag may be administered in combination with other drugs that are not contraindicated with the administration of velusetrag. Preferably, velusetrag is not administered with opioids and / or other 5-HT4 receptor agonists (e.g., prucalopride, cisapride, clebopride, cinitapride). [Brief explanation of the drawings]

[0057] BRIEF DESCRIPTION OF THE DRAWINGS In the figures below, normal = wild type mice, and groups G2, G3 and G4 are PrP-SCA7-92Q transgenic mice. [Figure 1] Figure 1: Dilatation measured as diameter (mm) of the distal small intestine (DSI) and proximal colon in two CIPO mouse models treated with velusetrag at 1 mg / kg and 3 mg / kg. 1A: Rb1cKO mice (CKO) ***P<0.001 vs. normal. 1B: PrP-SCA7-92Q transgenic mice. *P<0.05, ***P<0.001; one-way ANOVA; vs. G2 (vehicle). [Figure 2] Figure 2: Histological analysis scores (H&E total score as defined in Example 3) for DSI and proximal colon in two CIPO mouse models treated with velusetrag at 1 mg / kg and 3 mg / kg. 2A: Histological analysis scores for DSI and proximal colon in the Rb1cKO mouse model of CIPO treated with velusetrag. *P<0.05, ***P<0.001; one-way ANOVA; vs. vehicle. 2B: Histological analysis scores for DSI and proximal colon in the PrP-SCA7-92Q transgenic mouse model of CIPO treated with velusetrag. *P<0.05, ***P<0.001; T-test; vs. G2. [Figure 3A] Figure 3: Protein levels of p-mTOR, mTOR, p-Akt, Akt, and p-P70S6 in the distal small intestine. Expression levels of p-mTOR and mTOR were normalized to actin. Expression levels of p-Akt and Akt were normalized to actin. Expression levels of p-P70S6 were normalized to actin. Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs. vehicle group, n=10. 3A: Fold change in various protein expression levels in DSI of wild-type (normal) mice and Rb1cKO (CKO) mice treated with velusetrag at 1 mg / kg and 3 mg / kg is shown. [Figure 3B]Figure 3: Protein levels of p-mTOR, mTOR, p-Akt, Akt, and p-P70S6 in the distal small intestine. Expression levels of p-mTOR and mTOR were normalized to actin. Expression levels of p-Akt and Akt were normalized to actin. Expression levels of p-P70S6 were normalized to actin. Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs. vehicle group, n=10. 3B: Fold changes in expression levels of various proteins in DSI of wild-type (normal) mice and PrP-SCA7-92Q transgenic mice (G2, G3, G4) treated with velusetrag at 1 mg / kg and 3 mg / kg are shown. [Figure 4] Figure 4: Quantitative analysis of MAP2 neurons in the DSI and proximal colon of two CIPO mouse models treated with velusetrag at 1 mg / kg and 3 mg / kg. 4A: Rb1cKO mice. 4B: PrP-SCA7-92Q transgenic mice. Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs. vehicle, n=10. [Figure 5] Figure 5: Immunofluorescence of SOX10 and Hu+ neurons in the DSI and colon in wild-type (normal) and Rb1cKO mice treated with velusetrag (1 mg / kg or 3 mg / kg). Quantitative analysis of ganglionic neurons in the distal small intestine (A) and colon (B). Ratio of glial / Hu+ neurons 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, n=10. [Figure 6] Figure 6: 5HT4 receptor mRNA expression in the DSI and colon of Rb1cKO mice treated with velusetrag (1 mg / kg or 3 mg / kg). Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs. vehicle group, n=10. [Figure 7]Figure 7: Immunofluorescence colocalization of ataxin-7 intranuclear inclusions with Hu+ cytoplasmic markers in the distal small intestine and colon. Quantitative analysis of the number of Hu+ neurons in the distal small intestine (A) and colon (B) of PrP-SCA7-92Q transgenic mice treated with velusetrag (1 mg / kg and 3 mg / kg). Percentage of colocalized cells among 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. [Figure 8] 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 velusetrag (1 mg / kg and 3 mg / kg). Quantitative analysis of the number of nNOS+ neurons 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. [Figure 9] Figure 9: Quantitative analysis of CHAT+ neuronal cell numbers in the distal small intestine (A) and colon (B) of Rb1cKO mice treated with velusetrag (1 mg / kg and 3 mg / kg). [Figure 10] Figure 10: Immunofluorescence colocalization of ataxin-7 intranuclear inclusions (red) with the CHAT+ cytoplasmic marker in the distal small intestine and colon of velusetrag-treated PrP-SCA7-92Q transgenic mice (1 mg / kg and 3 mg / kg). Quantitative analysis of the number of CHAT+ neurons in the distal small intestine (A) and colon (B) of velusetrag-treated PrP-SCA7-92Q transgenic mice (1 mg / kg and 3 mg / kg). Percentage of colocalized 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. [Figure 11]Figure 11: Immunofluorescence staining of calretinin neurons in PrP-SCA7-92Q transgenic mice treated with velusetrag (1 mg / kg and 3 mg / kg). Calretinin neurons 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. [Figure 12] Figure 12: Cytoplasmic expression of n-NOS and CHAT in the proximal colon of PrP-SCA7-92Q transgenic mice treated with velusetrag (1 mg / kg and 3 mg / kg). 12A: Cytoplasmic nNOS / actin. 12B: Cytoplasmic CHAT / actin. [Figure 13] Figure 13: Body weight of PrP-SCA7-92Q transgenic mice 5 weeks after treatment. [Figure 14] Figure 14: Immunofluorescence for SOX10 and Hu+ neurons in the small intestine and colon in wild-type (normal) mice and PrP-SCA7-92Q transgenic mice treated with velusetrag. Quantitative analysis of ganglionic neurons in the distal small intestine (A) and colon (B). Ratio of glia / Hu+ neurons 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. [Figure 15] Figure 15: 5HT4 receptor mRNA expression in the distal small intestine and colon of wild-type (normal) mice and PrP-SCA7-92Q transgenic mice treated with velusetrag. Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs. vehicle group, n=10. [Figure 16]Figure 16: Clinical trial phase II scheme. Patients were randomized into four groups, each with a different dosing sequence. The study consisted of a screening period of up to 7 days (days -7 to -1), followed by four 4-week treatment periods. In each period, subjects received either velusetrag (VEL) 15 mg (period 2) or placebo (PLA) (period 2), with a 2-week washout period between treatment periods and a 2-week follow-up period (total of approximately 175 days). Scheduled visits (V = visit) on the corresponding days from the start of the study are also shown. [Figure 17] Figure 17: Line graph of the mean WGGSAIS for subjects receiving velusetrag or placebo by dose (30 observed pairs) in the mFAS1 population. D7 = Day 7; D14 = Day 14; D21 = Day 21; EoT = End of treatment; PLA = Placebo; PRE = Pre-treatment; VEL = velusetrag; WO-D7 = Washout Day 7; WO-D14 = Washout Day 14. Paired t-test: p-value = 0.0310, 95% CI for the mean difference in change between velusetrag and placebo equals -0.741; -0.038. [Figure 18] Figure 18: Line graph of the mean individual symptom scores for subjects receiving velusetrag or placebo by dose in the mFAS1 population. D7 = Day 7; D14 = Day 14; D21 = Day 21; EoT = End of Treatment; mFAS1 = Modified Full Analysis Set 1; PLA = Placebo; PRE = Pre-dose; VEL = Velusetrag; WO-D7 = Washout Day 7; WO-D14 = Washout Day 14. In analyses including Washout Day 7 and Washout Day 14, only evaluable pairs of data for each individual symptom were considered. Paired t-test: p-value = 0.0164. [Figure 19]Figure 19: Line graph of mean bowel habit scores for subjects receiving velusetrag or placebo by dose in the mFAS1 population. D7 = Day 7; D14 = Day 14; D21 = Day 21; EoT = End of treatment; PLA = Placebo; PRE = Pre-dose; VEL = velusetrag; WO-D7 = Washout Day 7; WO-D14 = Washout Day 14. In analyses including Washout Day 7 and Washout Day 14, only pairs of data that were evaluable for weekly bowel movements or complete bowel movements were considered. DETAILED DESCRIPTION OF THE INVENTION

[0058] Detailed Description of the Invention As outlined above, velusetrag is a compound of formula I, which forms a crystalline hydrochloride salt shown in formula II.

[0059] [ka]

[0060] The present invention relates generally to velusetrag for use in a method for treating idiopathic chronic intestinal pseudo-obstruction (CIPO), neuropathic chronic intestinal pseudo-obstruction, or chronic intestinal pseudo-obstruction secondary to neurodegeneration, autoimmune conditions, connective tissue disorders, or demyelinating conditions, and the invention also relates to the above uses, wherein at least one of the symptoms of CIPO (e.g., vomiting, flatulence, abdominal pain, constipation, etc.) and / or disease exacerbations (e.g., pseudo-obstruction episodes) is reduced or ameliorated.

[0061] Such use can reduce or ameliorate the cardinal symptoms of CIPO, including gastrointestinal motility abnormalities, increased distension of the proximal colon and / or distal small intestine, altered intestinal contractility, ulcer formation, inflammation of the proximal colon and / or distal small intestine, and fatality. Furthermore, the use of velusetrag according to the present invention can reduce the need for "artificial food" (i.e., home- or hospital-based parenteral nutrition). Velusetrag also reduces the number / frequency and duration of CIPO-related hospitalizations (i.e., hospitalizations that are a direct consequence of CIPO, directly result from CIPO, and / or complicated by CIPO) and CIPO-attributed hospitalizations (i.e., hospitalizations directly resulting from CIPO), improving quality of life.

[0062] Two types of models have been used to evaluate the preclinical pharmacodynamics of velusetrag in neuropathic CIPO. One of these is the Rb1 retinoblastoma knockout (Rb1cKO or CKO) mouse, which is mutated in the retinoblastoma of the enteric nervous system. This model, established by Fu et al., allowed the identification of intestinal pseudo-obstruction associated with significant 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].

[0063] Retinoblastoma Rb1 cKO mice died early due to intestinal pseudo-obstruction, with 50% of mice not surviving past postnatal day P30 (postnatal day 30). The distal small intestine (DSI) and proximal colon were dilated, and hard, black, rabbit-like feces emerged from the colon between postnatal days P8 and P30.

[0064] As in mice, CIPO in patients is diagnosed when bowel motility defects cause functional, but not mechanical, obstruction, leading to abdominal distension, pain, and malnutrition, and in severe cases to reliance on parenteral nutrition or intestinal transplantation for survival. A variety of genetic, infectious, autoimmune, metabolic, and toxic insults are all thought to contribute to the pathogenesis of CIPO.

[0065] Gastrointestinal motility is known to be controlled by an interconnected intrinsic network of neurons and glia called the enteric nervous system (ENS). Generally, the ENS is formed from neural crest-derived cells that migrate through the fetal intestine, proliferate extensively, and then exit the cell cycle and differentiate into many different neuronal subtypes. Because proper intestinal function requires the presence of an appropriate ratio of neurons within the ENS, even a partial loss of enteric neurons can be life-threatening. The signals that control the proliferation and cell cycle exit of ENS precursors are not entirely clear.

[0066] Based on these findings, cKO mice for Rb1 retinoblastoma were generated. This is because Rb1 is involved in cell cycle exit by preventing cells from entering S phase at the S and G2 / M cell cycle checkpoints, thereby maintaining development, terminal differentiation, and tissue homeostasis, and its mutation causes tissue-specific defects. Indeed, Rb1 inactivation in the ENS leads to progressive and fatal loss of a subset of NO-producing myenteric neurons, which inhibit intestinal contractions. These cells undergo endoreduplication and develop large, irregularly shaped nuclei similar to those seen in progeria. Furthermore, Rb1 is also deleted in enteric glia and other types of enteric neurons that do not undergo endoreduplication, highlighting differences in Rb1 dependency in different cell types within the ENS lineage.

[0067] Regarding translational evidence for this relevant Rb1 cKO mouse model of CIPO, velusetrag was able to reduce proximal colon distension after just 2 weeks of treatment, as well as protect mice from lethality and suppress glial activation, an indicator of intestinal inflammation evident in cKO mice. The ability of velusetrag to reverse intestinal distension associated with specific anti-inflammatory protection may suggest disease-modifying effects in addition to its prokinetic effects.

[0068] Another preclinical pharmacodynamic model is the transgenic mouse PrP-SCA7-92Q, also referred to herein as the human ATXN7 transgenic mouse, which exhibits signs of intestinal pseudo-obstruction and visceral neuropathies [Clarke CM et al., Visceral neuropathy and intestinal pseudo-obstruction in a murine model of a nuclear inclusion disease. Gastroenterology. 2007; 133(6): 1971-1978]. These animals exhibit many aspects of the human polyglutamine neurodegenerative disorder spinocerebellar ataxia type 7 (SCA7), including a subset of cholinergic enteric ganglion cells with nuclear inclusions containing transgene-derived ataxin-7 and pathogenic polyglutamine expansions. Transgenic mice exhibit marked distension of the distal small intestine from 13 weeks of age.

[0069] Ataxin-7 inclusions were clearly observed in the nuclei of a subset of enteric ganglion cells and myenteric neurons that coexpressed the cytoplasmic marker choline acetyltransferase. The density of calretinin-immunoreactive myenteric ganglion cells was also significantly reduced in the proximal colon of transgenic mice at 13 weeks. Loss of nerve fibers in the myenteric plexus and delayed gastrointestinal transit were also observed. In this transgenic model, oral administration of velusetrag once daily for 5 weeks inhibited distal small intestinal distension and prevented neurodegeneration of ChAT, nNOS, and calretinin neurons, as well as the appearance of ataxin-7 inclusions in enteric neurons.

[0070] Velsetrag also increases cytosolic ChAT and nNOS levels in the proximal colon, suggesting its ability to modify neuronal plasticity toward normal intestinal motility.

[0071] The dose of velusetrag for both preclinical mouse models selected for treatment was calculated according to the dose administered during clinical development in patients, i.e., 15 mg / patient and 5 mg / patient [FDA Guidelines US FDA, 2005; Nair and Jacob, 2016].

[0072] Furthermore, the effect of velusetrag administration on activation of the PI3K / AKT / mTOR signaling pathway was evaluated in these two mouse models.

[0073] mTOR is a component of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), protein complexes that are ubiquitously distributed throughout the body and regulate multiple functions, including gene transcription, metabolism, cell survival, and cellular senescence. Through its association with phosphoinositide 3-kinase (PI 3-K) and protein kinase B (Akt), as well as multiple downstream signaling pathways, including p70 ribosomal S6 kinase (p70S6K) and proline-rich Akt substrate 40 kDa (PRAS40), mTOR promotes neuronal regeneration by stem cell renewal and oversees key pathways, such as apoptosis, autophagy, and necroptosis, promoting protection against neurodegenerative disorders. Overall, mTOR is an essential neuroprotective pathway that needs to be carefully targeted to maximize clinical efficacy and eliminate any toxic side effects [Maiese K., Driving neural regeneration through the mammalian target of rapamycin. Neural Regen Res. 2014;1;9 :1413-7.]

[0074] Nervous system disorders are often associated with, if not caused by, alterations or disruptions in homeostatic processes controlled by glia. In the ENS, a unique population of peripheral glia called enteric glia fulfills these roles. Enteric glia regulate gastrointestinal motility through bidirectional communication with enteric neurons and are involved in the establishment of neuroinflammation. Glial mechanisms may be involved in disrupting gastrointestinal motility, as supported by data from animal models showing that alterations in glial function disrupt motility, promote neurodegeneration during acute colitis, and affect immune responses. However, the specific mechanisms by which enteric glia may contribute to disrupted motility remain largely unknown [Ahmadzai MM et al., J Clin Invest. 2022 Feb 15; 132(4): e149464]. The effects of velusetrag on neurons and glial cells were assessed by immunohistological analysis of distal small intestine and proximal colon samples using antibodies against the glial cell marker SOX10 and the neuronal protein HuCD in a mouse model of Rb1 retinoblastoma knockout (cKO) mice, which exhibit intestinal pseudo-obstruction associated with neuronal dysfunction and altered intestinal contractility, and in a transgenic mouse model of PrP-SCA7-92Q, which exhibits signs of intestinal pseudo-obstruction and visceral neuropathy.

[0075] Experimental results showed that velusetrag administered for 14 days was able to reduce the expression of relevant receptors, particularly in the distal small intestine (DSI) region in a model of CIPO, which is associated with neuronal dysfunction and altered intestinal contractility.

[0076] RB1 cKO significantly induced distension in the DSI and colon of mice. Surprisingly, treatment of RB1 cKO mice with velusetrag at 1 mg / kg and 3 mg / kg tended to inhibit distension, with a statistically significant difference observed in the colon after administration of 3 mg / kg. Increased distension in the DSI and colon was also observed in Prp-SCA7-92Q transgenic mice compared with normal mice. After treatment with velusetrag at 1 mg / kg or 3 mg / kg, DSI distension was significantly improved. Treatment with velusetrag at 1 mg / kg or 3 mg / kg reduced distension in the proximal colon.

[0077] The number of glia and the glia / neuron ratio were increased in the DSI and colon of RB1 cKO vehicle mice. Surprisingly, after treatment with velusetrag at 3 mg / kg, the number of glia and the glia / neuron ratio were significantly decreased in the DSI and proximal colon. In Prp-SCA7-92Q or human ATXN7 transgenic mice, the glia / neuron ratio was significantly increased in the DSI and colon of the vehicle group. After treatment with velusetrag at 1 mg / kg or 3 mg / kg, the glia / neuron ratio was significantly decreased.

[0078] The number of MAP2-stained neurons was decreased in the DSI and colon of the RB1 cKO vehicle group. After treatment with velusetrag at 1 mg / kg and 3 mg / kg, the number of MAP2-stained neurons increased in the DSI and proximal colon. In Prp-SCA7-92Q transgenic mice, the number of MAP2-stained neurons was decreased in the DSI and proximal colon of the vehicle group. After treatment with velusetrag at 1 mg / kg and 3 mg / kg, the number of MAP2-stained neurons increased in the DSI and proximal colon.

[0079] The number of CHAT-stained neurons was decreased in the DSI and colon of the RB1 cKO vehicle group. After treatment with velusetrag at 1 mg / kg and 3 mg / kg, the number of CHAT-stained neurons increased in the DSI and proximal colon.

[0080] Hematoxylin and eosin staining (HE) revealed clear inflammatory infiltrates and ulcers in the DSI and colon in both the RB1 cKO vehicle and PrP-SCA7-92Q transgenic vehicle groups. Surprisingly, disease scores in the velusetrag 1 mg / kg and 3 mg / kg groups were significantly reduced after treatment in both animal models of CIPO.

[0081] The 5HT4 receptor mRNA levels in DSI and colon in the RB1 cKO vehicle group were increased but not significantly different. After treatment with velusetrag at 3 mg / kg, the 5HT4 receptor mRNA levels in DSI were significantly decreased.

[0082] P-mTOR, p-AKT, p-P70S6, mTOR, and AKT proteins were increased in the RB1 cKO vehicle group, and showed significant increases in p-Akt, p-P70S6 proteins, and the p-AKT / AKT ratio in DSI compared with the normal group. After treatment with velusetrag at 3 mg / kg, p-mTOR, p-AKT, p-P70S6, mTOR, and AKT proteins were all significantly decreased.

[0083] The protein levels of p-Akt, Akt, p-P70S6, p-mTOR, and mTOR in DSI were increased in the PrP-SCA7-92Q transgenic vehicle group, with significant differences in p-Akt, p-P70S6, and Akt compared with the normal group. The p-Akt / AKT ratio in DSI was significantly increased in the vehicle group. Treatment with velusetrag at 3 mg / kg reduced the protein levels of p-Akt, Akt, p-P70S6, p-mTOR, and mTOR, with significant differences in p-Akt, Akt, and p-P70S6. Treatment with velusetrag at 1 mg / kg significantly reduced the p-mTOR / mTOR ratio.

[0084] The numbers of calretinin-, Hu-, nNOS-, and CHAT-stained neurons were decreased in the DSI and colon of PrP-SCA7-92Q transgenic mice. After treatment with velusetrag at 1 mg / kg and 3 mg / kg, the numbers of calretinin-, Hu-, nNOS-, and CHAT-stained neurons were significantly increased in the DSI and / or colon.

[0085] Furthermore, unexpected experimental results indicate that while both doses of velusetrag, i.e., 1 mg / kg and 3 mg / kg, were effective in improving body weight loss, intestinal distension, and lethality, the higher dose of 3 mg / kg was more effective in reducing inflammation and ulcer formation, restoring the animals to the state of healthy animals. Regarding the effects of velusetrag on neuronal parameters, such as the increase in nitrergic neurons or the increase in cholinergic neurons, similar results were obtained at both doses. Thus, velusetrag is particularly effective in treating neuropathic CIPO and, in addition, has a significant anti-inflammatory effect on the intestine.

[0086] Overall, the results of the experiments performed and presented herein demonstrate that velusetrag has modulatory, neuroprotective, and neurotrophic effects on the ENS in both CIPO animal models tested. The improvement in the enteric nervous system and reduction in inflammatory signs following treatment demonstrate the utility of velusetrag for treating neuroenteric disorders correlated with intestinal motility disorders.

[0087] Taking into account the demonstrated technical effect, the proposed use of velusetrag is the treatment of idiopathic chronic intestinal pseudo-obstruction or diseases secondary to neurodegeneration, or secondary to autoimmune conditions, or secondary to connective tissue disorders, or secondary to demyelinating conditions affecting gastrointestinal motility. Conditions that may benefit from this use include idiopathic chronic intestinal pseudo-obstruction or diseases secondary to neurodegeneration, or secondary to autoimmune conditions, or secondary to connective tissue disorders, or secondary to demyelinating conditions affecting gastrointestinal motility, due to one or more of the following: diseases of the autonomic nervous system (i.e., stroke, encephalitis, basal ganglia calcification, orthostatic hypotension); diseases of the enteric nervous system (i.e., paraneoplastic syndromes, viral infections, iatrogenic disorders, Hirschsprung's disease, Chagas' disease, von Recklinghausen's disease); diseases of the intestinal muscularis (i.e., myotonic dystrophy, progressive systemic sclerosis); mixed diseases of the enteric nervous system and smooth muscle (scleroderma, dermatomyositis, amyloidosis, Ehlers-Danlos syndrome); and diseases of unknown mechanism (i.e., hypothyroidism, hypoparathyroidism, pheochromocytoma, antidepressants, antineoplastic agents, bronchodilators).

[0088] Due to the technical effect brought about by the use of velusetrag, syndromes resulting in gastrointestinal motility disorders caused by or characterized by inflammatory / immune infiltration of neurons localized in the submucosal and myenteric ganglia of the enteric nervous system due to cellular infiltration of circulating antineuronal antibodies may be able to greatly benefit from the above-mentioned treatment. The treatment of the present invention is effective for both acute and chronic forms of the above-mentioned forms of CIPO.

[0089] Pharmaceutical Compositions and Formulations The present invention also relates to a pharmaceutical composition comprising velusetrag or a pharmaceutically acceptable salt thereof in the treatment of CIPO.

[0090] The term "pharmaceutically acceptable," as used herein, refers to a material that is not biologically or otherwise unacceptable. For example, the term "pharmaceutically acceptable carrier" refers to a material that can be incorporated into a composition and administered to a patient without causing unacceptable biological effects or interacting in an unacceptable manner with other components of the composition. Such pharmaceutically acceptable materials typically meet the necessary standards of toxicology and manufacturing testing and include materials identified as suitable inactive ingredients by the U.S. Food and Drug Administration.

[0091] The term "pharmaceutically acceptable salt" refers to a salt prepared from a base or acid acceptable for administration to a patient, such as a mammal (e.g., a salt that has acceptable safety for a mammal at a given dosing regimen). Pharmaceutically acceptable salts may be derived from pharmaceutically acceptable inorganic or organic bases or from pharmaceutically acceptable inorganic or organic acids. Furthermore, when a compound contains both a basic moiety, such as an amine, pyridine, or imidazole, and an acidic moiety, such as a carboxylic acid or tetrazole, zwitterions may be formed, and zwitterions are included in the term "salt" as used herein. Salts derived from pharmaceutically acceptable inorganic bases include ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, and zinc salts. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary, and tertiary amines, including substituted amines, cyclic amines, and naturally occurring amines such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. Salts derived from pharmaceutically acceptable inorganic acids include salts of boric acid, carbonic acid, hydrohalic acid (hydrobromic acid, hydrochloric acid, hydrofluoric acid, or hydroiodic acid), nitric acid, phosphoric acid, sulfamic acid, and sulfuric acid.Pharmaceutically acceptable salts derived from organic acids include 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 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, 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.

[0092] The terms "treating" or "treatment" include preventing, alleviating, ameliorating, or giving relief to symptoms associated with CIPO as assessed by the change in the weekly global gastrointestinal symptoms average index score from the beginning to the end of each treatment period.

[0093] The term "symptoms associated with CIPO" includes abdominal pain, bloating, nausea, and vomiting.

[0094] According to the present invention, a therapeutic effect (treatment effect) exists when at least one of the following is observed: - Change in waist circumference from the start to the end of each treatment period, A one-point improvement in the weekly global gastrointestinal symptom mean index score from the beginning to the end of each treatment period; -Changes in individual symptom scores for abdominal pain, bloating, nausea, and vomiting from the beginning to the end of each treatment period; Change in weekly bowel movements from the beginning to the end of each treatment period (only for subjects with a Bristol scale type 1 or 2 at the beginning of the treatment period), Change in the number of complete bowel movements per week from the beginning to the end of each treatment period; -Change in stool type on the Bristol stool scale from the start to the end of each administration period, Change in weekly bowel habit satisfaction scores from the beginning to the end of each treatment period, measured using a scale of 0 to 10; Change in oro-cecal transit time from the beginning to the end of the first treatment period (initial treatment period), as measured using the lactulose breath test; - The percentage of days during each treatment period, washout period, and follow-up period when medications used to relieve the main CIPO gastrointestinal symptoms were changed (the percentage of days when the dose was increased or decreased compared to the beginning of the period, and the percentage of days when medications were added or removed compared to the beginning of the period), Changes in quality of life (SF-12) from the start to the end of each treatment period the number of CIPO-related hospitalizations during the treatment period; · Changes in artificial dietary needs, - Change in the number of pseudo-obstructive episodes from the beginning to the end of each treatment period based on the investigator's judgment (investigation's judgment), Changes in the following from the end of each treatment period to the first and second weeks of washout or the end of the follow-up period: weekly abdominal pain score, weekly flatulence score, weekly nausea scores, weekly vomiting scores, Weekly overall gastrointestinal symptom mean index score, The number of bowel movements per week, Number of complete bowel movements per week, Bristol stool scale stool type, Weekly bowel habit satisfaction score measured using a 0–10 scale.

[0095] The SF-12 Health Survey is a shortened version of its predecessor, the SF-36, which itself evolved from the Medical Outcomes Study, used to assess patient quality of life internationally [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].

[0096] The Bristol Scale or Chart is a clinical assessment tool developed in 1997 and designed to classify 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 classifies a patient's stool based on the shape and type of stool, and this classification correlates well with the time it takes food to pass through the gastrointestinal tract and be excreted as feces.

[0097] The term "unit dosage form" refers to physically discrete units suitable for administration to a patient, i.e., each unit containing a predetermined quantity of active agent calculated to produce a desired therapeutic effect alone, or in combination with one or more additional units.

[0098] The crystalline hydrochloride salt form of velusetrag is typically administered to a patient in the form of a pharmaceutical composition. Such a pharmaceutical composition may be administered to a patient by any acceptable route of administration, including, but not limited to, oral, rectal, vaginal, nasal, inhalation, topical (including transdermal), and parenteral modes of administration.

[0099] Accordingly, in one of its composition aspects, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a therapeutically effective amount of the crystalline hydrochloride salt of a compound of Formula I. Optionally, such a pharmaceutical composition may contain other therapeutic and / or formulating agents, if desired.

[0100] Pharmaceutical compositions of the invention typically contain a therapeutically effective amount of a crystalline salt of the invention. Typically, such pharmaceutical compositions will contain from about 0.1 to about 95% by weight of the active agent, such as from about 1 to about 70% by weight of the active agent, for example, from about 5 to about 60% by weight of the active agent.

[0101] Any conventional carrier or excipient may be used in the pharmaceutical compositions of the present invention. The selection of a particular carrier or excipient, or combination of carriers or excipients, will depend on the mode of administration used to treat a particular patient, or the type of medical condition or disease state. In this regard, the preparation of a pharmaceutical composition suitable for a particular mode of administration is within the skill of one of ordinary skill in the pharmaceutical arts. Furthermore, ingredients for such compositions are commercially available, for example, from Sigma, PO Box 14508, St. Louis, MO 63178. For further illustration, conventional formulation techniques can be found in Remington: The Science and Practice of Pharmacy, 20th Edition, Lippincott Williams & White, Baltimore, Maryland (2000), and H.C. Cansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th Edition, Lippincott Williams & White, Baltimore, Maryland (1999).

[0102] Representative examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to: (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, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, and the like. (10) oils such as sesame oil, olive oil, corn oil, and soybean oil, (11) glycols such as propylene glycol, (12) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol, (13) esters such as ethyl oleate and ethyl laurate, (14) buffers such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer, and (21) other non-toxic compatible substances used in pharmaceutical compositions.

[0103] Pharmaceutical compositions of the present invention are typically prepared by thoroughly and intimately mixing or blending a compound of the present invention with a pharmaceutically acceptable carrier and one or more optional ingredients. If necessary or desired, the resulting homogeneously blended mixture can then be shaped or filled into tablets, capsules, pills, etc. using conventional procedures and equipment.

[0104] The pharmaceutical compositions of the present invention are preferably packaged in unit dosage form, such as a capsule, tablet, pill, or the like.

[0105] In a preferred embodiment, the pharmaceutical composition of the present invention is suitable for oral administration. Pharmaceutical compositions suitable for oral administration may be in the form of capsules, tablets, pills, lozenges, cachets, sachets, stick packs, dragees, powders, granules, aqueous or non-aqueous liquid solutions or suspensions, oil-in-water or water-in-oil liquid emulsions, elixirs, syrups, etc., each of which contains a predetermined amount of the compound of the present invention as an active ingredient.

[0106] When intended for oral administration in a solid dosage form (i.e., capsules, tablets, pills, etc.), pharmaceutical compositions of the invention will typically 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 contain the following ingredients: (1) fillers or extenders such as starch, microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants such as glycerol; (4) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and / or sodium carbonate; and (5) solution retarders such as paraffin. agents), (6) absorption enhancers such as quaternary ammonium compounds, (7) wetting agents such as cetyl alcohol and / or glycerol monostearate, (8) absorbents such as kaolin and / or bentonite clay, (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and / or mixtures thereof, (10) coloring agents, and (11) buffering agents.

[0107] Release agents, wetting agents, coating agents, sweeteners, flavoring and perfuming agents, preservatives, and antioxidants may also be present in the pharmaceutical compositions of the present invention. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), dibutylhydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid. Examples of coating agents for tablets, capsules, pills, etc. include coating agents used for enteric coating, such as cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), hydroxypropylmethylcellulose phthalate, methacrylic acid esters, cellulose acetate trimellitate (CAT), carboxymethylethylcellulose (CMEC), and hydroxypropylmethylcellulose acetate succinate (HPMCAS).

[0108] If desired, the pharmaceutical compositions of the present invention may also be formulated for sustained or controlled release of the active ingredient using, for example, hydroxypropyl methylcellulose in various proportions, or other polymer matrices, liposomes and / or microspheres.

[0109] Furthermore, pharmaceutical compositions of the present invention may optionally contain opacifying agents and may be formulated so as to release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally in a delayed manner. Examples of encapsulating compositions that can be used include polymeric substances or waxes. The active ingredient may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0110] Liquid dosage forms suitable for oral administration include, for example, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. Such liquid dosage forms typically contain the active ingredient and an inert diluent, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, 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, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. Suspensions may contain, in addition to the active ingredient, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, and mixtures thereof.

[0111] Alternatively, the pharmaceutical composition of the present invention is formulated for administration by inhalation. Pharmaceutical compositions suitable for administration by inhalation will typically be in the form of an aerosol or powder. Such compositions are generally administered using well-known delivery devices, such as metered dose inhalers, dry powder inhalers, nebulizers or similar delivery devices.

[0112] When administered by inhalation using a pressurized container, pharmaceutical compositions of the invention will typically contain the active ingredient and a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

[0113] Furthermore, the pharmaceutical composition may be in the form of a capsule or cartridge (made, for example, from gelatin) containing a compound of the invention and a powder suitable for use in a powder inhaler. Suitable powder bases include, by way of example, lactose or starch.

[0114] 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 (permeation enhancers) such as propylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-one, etc., and incorporated into a patch or similar delivery system.Additional excipients, including gelling agents, emulsifiers, and buffers, may be used in such transdermal compositions if desired.

[0115] The following formulations illustrate representative pharmaceutical compositions of this invention.

[0116] Prescription Example A Hard gelatin capsules for oral administration are prepared as follows.

[0117] [Table 1]

[0118] Typical Procedure : The ingredients are thoroughly blended and then loaded into hard gelatin capsules (260 mg of composition per capsule).

[0119] Prescription Example B Hard gelatin capsules for oral administration are prepared as follows.

[0120] [Table 2]

[0121] Typical Procedure The ingredients are thoroughly blended, then passed through a No. 45 mesh US sieve and loaded into a hard gelatin capsule (200 mg of composition per capsule).

[0122] Prescription Example C Capsules for oral administration are prepared as follows.

[0123] [Table 3]

[0124] Typical Procedure : The ingredients are thoroughly blended and then loaded into a gelatin capsule (310 mg of composition per capsule).

[0125] Prescription Example D Tablets for oral administration are prepared as follows.

[0126] [Table 4]

[0127] Typical Procedure The active ingredient, starch, and cellulose are passed through a No. 45 mesh US sieve and thoroughly mixed. The polyvinylpyrrolidone solution is mixed with the resulting powder, and the mixture is then passed through a No. 14 mesh US sieve. The granules thus produced are dried at 50-60°C and passed through a No. 18 mesh US sieve. Sodium carboxymethyl starch, magnesium stearate, and talc (previously passed through a No. 60 mesh US sieve) are then added to the granules. After mixing, the mixture is compressed on a tablet machine to yield tablets weighing 100 mg.

[0128] Prescription Example E Tablets for oral administration are prepared as follows.

[0129] [Table 5]

[0130] Typical Procedure The ingredients are thoroughly blended and then compressed to form tablets (440 mg of composition per tablet).

[0131] Prescription Example F Single-scored tablets for oral administration are prepared as follows.

[0132] [Table 6]

[0133] Typical Procedure : The ingredients are thoroughly blended and compressed to form single-scored tablets (215 mg of composition per tablet).

[0134] Prescription Example G A suspension for oral administration is prepared as follows.

[0135] [Table 7]

[0136] Typical Procedure : The ingredients are mixed to form a suspension containing 10 mg of active ingredient per 10 mL of suspension.

[0137] Prescription Example H A dry powder formulation for administration by inhalation is prepared as follows.

[0138] [Table 8]

[0139] Typical Procedure The active ingredient is micronized and then blended with lactose. The blend is then loaded into a gelatin inhalation cartridge. The contents of the cartridge are administered using a powder inhaler.

[0140] Prescription Example I A dry powder formulation for administration by inhalation with a metered dose inhaler is prepared as follows.

[0141] Typical Procedure A suspension containing 5% by weight of the salt of the present invention and 0.1% by weight of 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 containing 0.2 g of lecithin dissolved in 200 mL of demineralized water. The suspension is spray-dried and the resulting material is micronized to particles with an average diameter of less than 1.5 μm. The particles are loaded into cartridges with pressurized 1,1,1,2-tetrafluoroethane.

[0142] Prescription Example J The injectable formulation is prepared as follows.

[0143] [Table 9]

[0144] Typical Procedure : Blend the above ingredients and adjust the pH to 4±0.5 with 0.5N HCl or 0.5N NaOH.

[0145] Prescription Example K Capsules for oral administration are prepared as follows.

[0146] [Table 10]

[0147] Typical Procedure : The ingredients are thoroughly blended and then loaded into a gelatin capsule (size #1, white, opaque) (264 mg of composition per capsule).

[0148] Prescription Example L Capsules for oral administration are prepared as follows.

[0149] [Table 11]

[0150] Typical Procedure : The ingredients are thoroughly blended and then loaded into a gelatin capsule (size #1, white, opaque) (148 mg of composition per capsule).

[0151] Administration route The present invention also relates to acceptable routes of administration of velusetrag to human patients, including, but not limited to, oral, parenteral, buccal, sublingual, rectal, intraperitoneal, or intratracheal administration. For example, parenteral administration may be by infusion, injection, or implant. Parenteral administration also includes subcutaneous, intramuscular, intravenous, transdermal, or transdermal administration via implant routes. When velusetrag is administered parenterally, it may be in the form of a liquid, solid, or gel. Similarly, when velusetrag is administered orally, it may be in the form of a liquid, capsule, tablet, chewable tablet, or dissolvable film.

[0152] The following examples are intended to be illustrative without limiting the scope of the invention described herein. [Example]

[0153] Example Animal models Rb1 cKO retinoblastoma mice [Fu M et al., Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction. J Clin Invest. 2013;123: 5152-5164] constitute a model of intestinal pseudo-obstruction, particularly associated with neurological dysfunction. In this model, velusetrag dissolved in saline was administered intraperitoneally (ip) once daily for 14 days, starting on postnatal day 15, at one of two doses (1 mg / 10 ml / kg or 3 mg / 10 ml / kg, n = 10 animals per group).

[0154] PrP-SCA7-92Q was used as described by 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 ileus pseudo-obstruction, specifically abdominal neuropathy accompanied by intranuclear neuronal inclusions and ileus pseudo-obstruction. In this model, velusetrag was administered orally once daily for 5 weeks, starting at 8 weeks of age, at one of two doses (1 mg / 10 ml / kg or 3 mg / 10 ml / kg, n = 10 animals per group).

[0155] As a positive control, modified mice were administered with a vehicle (physiological saline), and unmodified mice were used as a negative control.

[0156] statistical analysis All data in Tables 1-17 are the mean ± SEM of 10 mice per group. Statistical comparisons were performed by ANOVA (Graph Pad Prism 9.3), where P values ​​relative to normal are indicated by the symbol . * The P values ​​relative to the cKO vehicle (vs. the cKO vehicle) are expressed as °. * P<0.05, ** P<0.01, *** p<0.001, **** P<0.0001; vs. cKO+vehicle, °p<0.05, °°P<0.01, °°°P<0.001, °°°°P<0.0001.

[0157] Example 1 - Effect of velusetrag on lethality in mice in an animal model C57BL / 6 (Cyagen, China) and Rb1 cKO retinoblastoma mice (Cyagen, China) with free access to water and food were used. Velsetrag was administered i.p. once daily for 14 days, starting on postnatal day 15, at one of two doses (3 mg / 10 mL / kg or 1 mg / 10 mL / kg). Ten mice were monitored for survival only up to postnatal day 60.

[0158] As shown in Table 1, 40% of the control group (KO animals treated with saline) died within 15 days of treatment initiation. In contrast, only 10% of the knockout mice treated with velusetrag at 1 mg / kg or 3 mg / kg died. Thus, velusetrag has a significant benefit on mortality.

[0159] Furthermore, at both doses, velusetrag was well tolerated, as treated animals did not experience weight loss (Table 1).

[0160] [Table 12]

[0161] Similar results were observed in Prp-SCA7-92Q mice.

[0162] To assess whether velusetrag was well tolerated in this mouse model, the body weights of Prp-SCA7-92Q mice treated with either vehicle or 1 mg / kg or 3 mg / kg velusetrag were measured during the treatment period. A normal control (NC) group of untreated wild-type mice was also examined. No significant differences were observed between groups (Figure 13).

[0163] After 5 weeks of treatment, the body weight of the transgenic mice showed no difference compared to the normal control group. Treatment with velusetrag at 1 mg / kg and 3 mg / kg was well tolerated, with no reduction in body weight or mortality observed during the treatment period (Figure 13).

[0164] Example 2 - Effect of Velusetrag on Intestinal Distension C57BL / 6 and Rb1 cKO retinoblastoma mice (Cyagen, China) with free access to water and food were used. Velsetrag was administered intravenously once daily at two doses (3 mg / 10 mL / kg or 1 mg / 10 mL / kg) for 14 days, starting from postnatal day 15 to day 28.

[0165] C57BL / 6 mice (Cyagen, China) and Prp-SCA7-92Q transgenic mice with free access to water and food were orally administered velusetrag at two doses (3 mg / 10 mL / kg and 1 mg / 10 mL / kg) once daily for 5 weeks starting at 8 weeks of age until 13 weeks of age.

[0166] The day after the last treatment, the animals were sacrificed by CO2 inhalation. Distal small intestine (DSI) and proximal colon samples were collected and washed with PBS. The diameters of the DSI and colon were measured using Image J (version 1.53c) based on the scale plate on which the tissue was placed at the time of image capture. The diameters of the distal small intestine (DSI) and proximal colon in five regions were evaluated and expressed in mm.

[0167] Rb1 cKO mice exhibit intestinal distension, with increased diameters of both the DSI and proximal colon compared to wild-type normal animals. Following administration of velusetrag at both doses tested, Rb1 cKO mice exhibited reduced intestinal distension in both the distal small intestine and proximal colon compared to untreated KO mice. This reduction was dose-dependent, with greater effects observed at higher doses of velusetrag (Figure 1A and Table 2).

[0168] [Table 13]

[0169] Prp-SCA7-92Q transgenic mice also exhibit intestinal distension, with increased diameters of both the DSI and proximal colon compared with wild-type normal animals. In Prp-SCA7-92Q transgenic mice, velusetrag was shown to significantly reduce distal small intestinal distension at both doses tested, compared with vehicle-treated transgenic mice. In the proximal colon, after treatment with 3 mg / 10 ml / kg velusetrag, intestinal distension returned to the values ​​of healthy animals (Table 3 and Figure 1B).

[0170] [Table 14]

[0171] In both the 1 mg / kg and 3 mg / kg dose groups, velusetrag was able to significantly reduce dilation by normalizing DSI dilation at both doses (Figure 1B).

[0172] Example 3 - Effect of velusetrag on intestinal morphometry (anti-inflammatory activity) After mice were sacrificed by CO2 inhalation, small intestinal and colonic tissues were dissected and fixed in 10% neutral buffered formalin for 48 hours. Paraffin samples were prepared by dehydrating through a series of gradient alcohol solutions according to standard methods known in the art. Tissues were cut into 4 μm sections using a rotary microtome and baked at 60°C for 2 hours. After hematoxylin-eosin staining, images were obtained from each animal at 20x magnification for histological analysis using a fully automatic digital pathology slide system (KF BIO, KF-PRO-005, China) with K-viewer Imaging software (KF BiO, V1, China). Pathological features such as ulcers (the statistical area of ​​intestinal mucosal damage), inflammation, and hemorrhage were evaluated and scored according to the following table.

[0173] [Table 15]

[0174] The anti-inflammatory activity of velusetrag was evaluated by histological analysis of intestinal samples isolated from mice in both models. The presence or absence of inflammation and ulcers was quantified using an index associated with a score of 0 to 3 (i.e., ulcer = area (%) with intestinal mucosal damage; inflammation = necrosis, bleeding). Scoring of inflammation and ulcers was performed after the sections were evaluated and analyzed.

[0175] Rb1 cKO mice showed increased values ​​for all measured parameters, including both DSI and proximal colon diameter, compared with wild-type normal animals. In Rb1 cKO mice, both doses of velusetrag demonstrated reduced inflammation and ulcer formation compared with untreated KO animals (positive control). Treatment with 3 mg / 10 ml / kg velusetrag restored parameters to those of healthy animals (WT, normal mice) (negative control) in both the distal small intestine and proximal colon (Table 4 and Figure 2A).

[0176] [Table 16]

[0177] Similarly, Prp-SCA7-92Q mice showed increased values ​​for all parameters measured, including both DSI and proximal colon diameter, compared with wild-type normal animals. In Prp-SCA7-92Q mice, both doses of velusetrag reduced both inflammation and ulcer formation compared with the positive control, with the 3 mg / 10 ml / kg dose of velusetrag significantly improving values ​​compared with the positive control in both the distal small intestine and proximal colon (Table 5 and Figure 2B).

[0178] [Table 17]

[0179] The overall scores for both models are also shown in Figures 2A and 2B.

[0180] These results indicate that CKO and Prp-SCA7-92Q transgenic mice exhibit impaired intestinal structural integrity, whereas administration of velusetrag effectively ameliorated the intestinal abnormalities in a dose-dependent manner (P<0.05 and P<0.001 for the two doses, respectively).

[0181] Example 4 - Velusetrag activity on the AKT / mTOR / p70s6k signaling pathway The effect of velusetrag administration on activation of the PI3K / AKT / mTOR signaling pathway was assessed by Western blot on extracts of distal small intestine samples from treated animals using specific antibodies against the proteins p-Akt, Akt, p-mTOR, m-TOR, p-P70S6, and actin.

[0182] The day after the last treatment, the animals were sacrificed by CO2 inhalation.

[0183] Distal small intestinal samples were collected and placed in 10 volumes (w:v) of lysis buffer (Beyotime Biotechnology, China) containing a protease and phosphatase inhibitor cocktail (Thermo Scientific, USA) according to the manufacturer's instructions. 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.

[0184] Proteins were fractionated on a 10% SDS-PAGE gel and transferred to an NC membrane. After blocking with 5% BSA in TBST (0.05% Tween-20) for 2 hours at room temperature (RT), the membranes were incubated overnight (≥15 hours) at 4°C with the following primary antibodies: 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), and 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 (three times, 10 min each) and incubated with the following secondary antibodies at room temperature for 1 h: Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed 1:5000 (Abcam, 216773, USA) or Donkey anti-Goat IgG H&L (IRDye® 800CW) 1:5000 (Abcam, 216775, USA). After washing, the membrane was placed in substrate working solution for 5 min and then imaged using a ChemiDoc System (Bio-Rad, 12003154, USA).Among these biomarkers, Akt and mTOR were reprobed after membrane stripping using Restore™ 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. Increased levels of pAKT, mTOR, p-mTOR, and p70SKal expression were observed in vehicle-treated Rb1 knockout mice compared with wild-type C57Bl / 6 mice. Treatment with velusetrag at a dose of 3 mg / 10 ml / kg inhibited signal activation and reduced protein levels of the AKT / mTOR / p70SKal pathway compared with the positive control. A dose of 1 mg / kg reduced protein expression compared with the positive control, but not significantly (Table 6).

[0185] [Table 18]

[0186] Increased levels of pAKT, mTOR, p-mTOR, and p70SKal expression are observed in vehicle-treated Prp-SCA7-92Q transgenic mice compared to wild-type C57B1 / 6 mice. In this model, the highest dose of velusetrag results in decreased protein levels of the AKT / mTOR / p70SKal pathway compared to the positive control (vehicle-treated Prp-SCA7-92Q transgenic mice). The 1 mg / kg dose appears to have no effect, with similar, or in some cases even higher, values ​​observed compared to the positive control (Table 7).

[0187] [Table 19]

[0188] These results are also shown in Figure 3A (Rb1cKO mouse model) and Figure 3B (Prp-SCA7-92Q transgenic mouse model).

[0189] Example 5 - Effect of velusetrag on MAP2 dendritic strength Microtubule-associated protein 2 (MAP2) immunostaining primarily visualizes the perikarya-dendritic domain and the proximal portion of axons in enteric neurons of the porcine intestine, allowing unambiguous immunocytochemical identification of enteric multi(short)dendritic uniaxonal type I neurons. The effects of velcetrag treatment on enteric neuronal dendrites were evaluated in whole-mount specimens isolated from animals of the two CIPO models treated as described above.

[0190] The day after the last treatment, mice were sacrificed by CO2 inhalation. Samples of the distal small intestine (DSI) and proximal colon were collected, washed with PBS, and then processed for immunohistochemistry.

[0191] The tissue was cut along the mesenteric edge and fixed on one side of a Sylgard plate. After fixation in 4% paraformaldehyde for 30 minutes, the muscle layer was separated from the mucosa and submucosa using fine-tipped forceps under a dissecting microscope.

[0192] DSI and colon samples were cut into 1 cm segments and stored in 50% glycerol / PBS at -20°C until staining and analysis. Myenteric plexus preparations were washed three times with PBST (PBS + 0.5% Triton X 100) and processed as follows:

[0193] Whole-mount tissues were washed three times with PBST (PBS + 0.5% Triton X-100).

[0194] The tissue was incubated with stripping buffer at 37°C for 40 minutes.

[0195] Whole-mount tissues were washed three times with PBST (PBS + 0.5% Triton X-100).

[0196] The tissues were incubated with primary antibody MAP2 1:1000 (Abcam, USA) overnight at 4°C.

[0197] The tissue was washed three times with TBST (PBS + 0.5% Triton x-100 + 0.5% Tween-20).

[0198] 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.

[0199] The tissue was washed three times with PBST (PBS + 0.5% Triton X-100).

[0200] The tissue was incubated with TSA670 1:200 (Wi See Biotechnology, China) at room temperature for 1 hour away from light.

[0201] The tissue was washed three times with PBST (PBS + 0.5% Triton X-100).

[0202] The tissues were incubated with DAPI 1:5000 (Invitrogen, USA) for 10 minutes at room temperature.

[0203] A drop of ProLong™ Glass Antifade Mountant (Invitrogen, USA) was applied to the cover slip.

[0204] Tissue specimens were excited by lasers equipped with excitation and barrier filters set for individual fluorochromes according to their specific excitation / emission spectra. Images were acquired from each animal using a confocal scanning microscope (Operetta CLS high Content Analysis System, PerkinElmer, USA) equipped with a 20x water-immersion objective for cell counting. MAP2+ cells were counted.

[0205] The results are shown in Table 8.

[0206] [Table 20]

[0207] In Rb1 cKO mice, the number of MAP2-positive neurons in the DSI and proximal colon was significantly reduced compared with the wild-type group (DSI P<0.05; colon P<0.01). The number of MAP2-positive neurons in the DSI and proximal colon of Rb1 cKO mice treated with velusetrag at either 1 mg / kg or 3 mg / kg was significantly increased compared with vehicle-treated cKO animals (DSI: 1 mg / kg group, P<0.05; 3 mg / kg group, P<0.01; colon: 1 mg / kg group, P<0.05). Next, in the DSI, velusetrag administration restored the reduction in MAP2-positive neurons to levels comparable to those in wild-type animals. This effect was slightly less pronounced in the proximal colon (Table 8).

[0208] Similar immunostaining experiments were performed on whole-mount intestinal preparations isolated from Prp-SCA7-92Q transgenic mice, and the results showed a reduction in dendrites in the transgenic animals compared with wild-type (control) animals (Table 9).

[0209] [Table 21]

[0210] Reorganization of the enteric neuronal network in PrP-SCA7-92Q mice treated with vehicle alone was also visualized by immunostaining of the myenteric plexus with MAP2. MAP2, a robust somatodendritic marker, is a cytoskeletal regulator within neuronal dendrites involved in regulating neurite outgrowth, synaptic plasticity, and protein folding / trafficking (Fig. 4B). The number of MAP2+ neurons in DSI in vehicle-treated transgenic mice was significantly reduced (P<0.001) compared with wild-type controls, but this was only in the DSI, not the proximal colon, suggesting a site-specific somatodendritic depletion. At both the 1 mg and 3 mg / kg doses, velusetrag had a minimal effect on MAP2 staining (Fig. 4B).

[0211] Figures 4A and 4B show the effect of velusetrag on the number of MAP2+ neurons in the DSI and 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).

[0212] Example 6 - Effects of velusetrag on neurons and glial cells by whole-mount immunostaining The effects of velusetrag on neurons and glial cells in Rb1 retinoblastoma knockout mice were assessed by immunofluorescence analysis of distal small intestine and proximal colon samples using antibodies against the glial cell marker SOX10 and the neuronal protein HuCD.

[0213] The day after the final treatment, animals were sacrificed by CO2 inhalation. Segments of distal ileum and colon were placed in phosphate-buffered saline, and the mucosa and submucosa were manually removed using fine forceps. The muscularis propria and enclosed myenteric plexus were fixed in ice-cold acetone for 10 minutes. After immersion in 1X PBS, the tissues were blocked for 2 hours at room temperature in PBS containing 1.5% BSA blocking buffer and 1% Triton X-100. The samples were incubated overnight at +4°C with a primary antibody against the glial protein SOX10 (Invitrogen, USA) (1:250), then incubated with a 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 the fluorescent dye TSA520 (Wi See Biotechnology, China) (1:200) at room temperature for 1 hour away from direct light, and then washed three times with PBST (PBS + 0.5% Triton X-100). Detection of the neuronal marker HuCD was performed with the primary antibody HuCD (Abcam, USA) (1:500) at room temperature for 1 h, washed three times with TBST (PBS + 0.5% Triton X-100 + 0.5% Tween 20), incubated with the secondary antibody F(ab')-goat anti-rabbit HRP (1:1000) at room temperature for 1 h, washed three times with PBST (PBS + 0.5% Triton X-100), incubated with the fluorescent dye TSA570 (Wi See Biotechnology, China) (1:200) at room temperature for 1 h, washed three times with PBST (PBS + 0.5% Triton X-100), and incubated with DAPI (1:5000) for 10 min at room temperature. Images were captured using an Olympus BX53 microscope. Tissue samples were excited with excitation lamps (UW, BWA, and GW) and excitation / emission spectra (340–390 nm / 420 IF; 460–495 nm / 510–550 nm; 530–550 nm / 575 IF).SOX10+ and HuCD+ antibodies were combined with the fluorescent dyes TSA520 and TSA570, which have specific excitation / emission spectra of 488 / 519 nm and 555 / 570 nm. SOX10+ (green) and HuCD+ (red) cells were counted and analyzed by 1 mm. 2 The numbers of SOX10+ and HUCD+ neurons per 1000 μm were assessed.

[0214] Immunofluorescence analysis of distal ileum and colon segments showed that velusetrag reduced glial cells (SOX10+ cells) and the glial / neuronal ratio. Thus, velusetrag attenuates inflammation and gastrointestinal dysfunction. Detection of both glial and neuronal markers showed that both doses of velusetrag reduced the glial marker signal (SOX10) and the glial / neuronal ratio (SOX10 / HuCD) in both the distal small intestine and proximal colon compared to untreated knockout animals, with this reduction being more pronounced at higher doses of velusetrag in both intestinal regions (Table 10).

[0215] [Table 22]

[0216] Figure 5 shows the effect of treatment with velusetrag on DSI and myenteric ganglion in the proximal colon of Rb1 cKO mice ( * P<0.05, ** P<0.01, *** P<0.001; one-way ANOVA; vs. vehicle CKO).

[0217] Neuronal degeneration was also investigated by counting the number of neurons and glia bearing pan-neuronal (HuCD) or glia (SOX10) markers in intestinal whole-mount preparations of the myenteric plexus from transgenic-treated and untreated mice (Figure 14). Immunofluorescence staining showed a significant increase in glia in the proximal colon, accompanied by a decrease in neurons in both the small and large intestines of vehicle-treated transgenic mice compared with NC mice (P<0.001 and P<0.01, respectively). Accordingly, the glia / Hu+ neuron ratio was elevated in both regions of the intestine (P<0.001). Treatment with velusetrag, at both doses, appeared to attenuate neuronal degeneration in both the small and large intestines, as indicated by the HuCD:glia ratio.

[0218] Example 7 - Effect of velusetrag on serotonin receptor 5-HT4 in enteric neurons of RB1 cKO mice The effects of velusetrag administration at the two doses tested were analyzed by assessing changes in 5-HT4 receptor mRNA levels in enteric neurons in the distal small intestine and proximal colon of wild-type and RB1 knockout mice.

[0219] Total RNA was extracted from distal small intestine and proximal colon samples from mice sacrificed by CO2 inhalation the day after the final treatment using TRIzol Reagent (Invitrogen, USA). 1 ml of TRIzol™ Reagent was added to the sample (approximately 50 mg of tissue), homogenized using a Tissue Lyser (60 Hz, 1 minute, pre-cooled), and incubated for 5 minutes to completely dissociate nucleoprotein complexes. The resulting lysate was centrifuged at 12,000 × g for 5 minutes at 4°C, and the clear supernatant was then transferred to a new tube.

[0220] Chloroform (0.2 mL) was added, mixed gently and incubated at room temperature for 5 minutes.

[0221] After centrifugation at 12,000 × g for 15 min at 4°C, the mixture separated into a red lower phenol-chloroform phase, an interphase, and a colorless upper aqueous phase containing RNA, which was transferred to a new tube.

[0222] Genomic DNA contamination was eliminated by using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Japan), and mRNA was reverse transcribed into cDNA and used for real-time RT-PCR (qPCR) according to the manufacturer's instructions.

[0223] The obtained cDNA was used as a template for quantitative PCR reactions (qPCR assay, Applied Biosystems, USA) using primers specific for the 5-HT4 receptor coding region in a real-time PCR instrument (Bio-Rad, 1855484, USA) according to the following reaction settings:

[0224] [Table 23]

[0225] The results show that 5-HT4 receptor mRNA levels were slightly increased in the DSI and colon in the Rb1 cKO group. After treatment with velusetrag at 3 mg / kg, 5-HT4 receptor mRNA levels in the DSI were significantly decreased. No changes were observed in the proximal colon between the different groups (Table 11).

[0226] [Table 24]

[0227] Treatment with velusetrag administered for 14 days reduces the expression of the related 5-HT4 receptor, particularly in the DSI region of Rb1 cKO animals.

[0228] The results of the effect of velusetrag on serotonin receptor mRNA in enteric neurons of Rb1 cKO mice are shown in Figure 6 ( * P<0.05; T-test; vs. vehicle CKO).

[0229] To determine whether 5HT4 receptor desensitization was evident 5 weeks after velusetrag treatment, we also analyzed the mRNA expression levels of 5HT4 serotonergic receptors in DSI and colon tissues of treated and untreated transgenic mice. Notably, vehicle-treated transgenic mice had significantly different 5HT4R levels compared with the control group. These differences were not altered by velusetrag administration in transgenic mice (Figure 15).

[0230] Example 8 - Effect of velusetrag on total neurons and ataxin-7 inclusions in the Prp-SCA7-92Q mouse model

[0231] The presence of neurons in the myenteric plexus and model-typical ataxin inclusions was detected by immunofluorescence analysis of DSI and proximal colon using primary antibodies against HUC / D and ataxin-7 proteins. Samples were prepared from animals sacrificed by CO2 inhalation the day after the last treatment with velusetrag, as described in Example 5.

[0232] After incubation in blocking buffer, tissue samples were incubated with HuC / D primary antibody (Abcam, USA) (1:500) at room temperature for 1 hour, followed by secondary antibody F(ab')2-Goat anti-Rabbit HRP (Abcam, USA) (1:1000) at room temperature for 1 hour. Tissue samples were washed three times with PBST (PBS + 0.5% Triton X-100) and incubated with TSA520 (1:200) at room temperature for 1 hour. Tissues were washed three times with PBST (PBS + 0.5% Triton X-100). Ataxin inclusions were detected by overnight incubation at 4°C with primary antibody Ataxin-7 (Thermo Fisher, USA) (1:2000) and secondary antibody F(ab')2-Goat anti-Rabbit HRP (1:1000) at room temperature for 1 hour. Tissues were 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). Tissues were then incubated with DAPI (1:5000) at room temperature for 10 minutes. Samples were covered with a drop of ProLong™ Glass Antifade Mountant (Invitrogen, USA), and images were taken using a microscope (Olympus, BX53). The excitation / emission spectrum was set to (340-390 nm / 420 IF; 460-495 nm / 510-550 nm; 530-550 nm / 575 IF), and the tissue samples were excited using excitation lamps and barrier filters (UW, BWA, and GW). The HUC / D and ataxin-7 antibodies were combined with the fluorescent dyes TSA520 and TSA570, which have specific excitation and emission spectra of 488 / 519 nm and 555 / 570 nm, respectively. Cells that co-expressed HuCD (green) and ataxin-7 (yellow) were counted.

[0233] The following primary antibodies were used: Hu C / D 1:500 (Abcam, Ab184267, USA); ataxin-7 1:2000 (Thermo Fisher, PA1-749, USA), nNOS 1:500 (Abcam, ab76067, USA), ChAT 1:1000 (Abcam, Ab181023, USA), calretinin 1:500 (Merck, MAB1568, USA), and SOX10 1:250 (Invitrogen, MA5-32398, USA).

[0234] 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), and Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 1:500 (Invitrogen, A-21207, USA). For fluorescent staining, TSA520 1:200 (Wi See Biotechnology, D11013, China) and TSA570 1:200 (Wi See Biotechnology, D11011, China) were used.

[0235] The data show that velusetrag can attenuate neurodegeneration in both DSI and the proximal colon at both doses tested. Velusetrag also reduces mutant ataxin-7 inclusions in pan-neuronal cells (Table 12).

[0236] [Table 25]

[0237] To examine whether this transgenic mouse line accumulates ataxin-7-positive aging neuronal inclusions, we performed immunofluorescence on whole-mount preparations of myenteric plexus from the DSI and proximal colon of 13-week-old PrP-SCA7-92Q male mice (Fig. 7A–D). Double staining with ATX-7 and the neuronal marker Hu C / D revealed that approximately 40% of enteric neurons in the DSI and 30% of enteric neurons in the proximal colon colocalized with ataxin-7-positive nuclear inclusions. Surprisingly, these percentages were significantly reduced by velsetrag.

[0238] Example 9 - Effect of velusetrag on nitrergic neurons in the Prp-SCA7-92Q mouse model Enteric neurons express nitric oxide synthase (nNOS), an enzyme that catalyzes the production of NO from oxygen and arginine. NO acts as a neurotransmitter and plays a role in regulating gastrointestinal (GI) motility. Excessive production of NO in some inflammatory conditions also impairs normal GI motor activity. NOS1+ neurons form close contacts with smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs). Loss or damage of these types of neurons has been observed to contribute to the development of GI motility disorders.

[0239] The presence of nitrergic neurons and ataxin inclusions was detected in the myenteric plexus of Prp-SCA7-92Q transgenic animals treated with velusetrag by immunohistochemical analysis of DSI and proximal colon samples using specific nNOS and ataxin-7 primary antibodies.

[0240] Samples were prepared from C57BL / 6 mice (control) and Prp-SCA7-92Q transgenic mice (Cyagen, China) with free access to water and food. Velusetrag was administered orally at two doses (3 mg / 10 mL / kg or 1 mg / 10 mL / kg) once daily for 5 weeks, starting at 8 weeks of age and continuing until 13 weeks of age. The day after the last treatment with velusetrag, the animals were sacrificed by CO2 inhalation and treated with specific antibodies, as described in Example 5.

[0241] After fixation with 4% paraformaldehyde for 30 minutes, the muscle layer containing the myenteric plexus was separated from the mucosa and submucosa using fine-tipped forceps. Samples were washed three times with PBST (PBS + 0.5% Triton X-100) and incubated with blocking buffer (PBS containing 1.5% BSA and 1% Triton X-100) for 2 hours at room temperature, followed by incubation with nNOS primary antibody (se, Abcam, USA) (1:500) for 1 hour at room temperature. 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) for 1 hour at room temperature. 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 1 hour. Ataxin inclusions were detected by overnight incubation at 4°C with the primary antibody Ataxin-7 (Thermo Fisher, USA) (1:2000). Samples were washed three times with TBST (PBS + 0.5% Triton X-100 + 0.5% Tween-20). 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). After incubation with TSA570 (Wi See Biotechnology, China) (1:200) at room temperature for 1 hour and washing three times with PBST (PBS + 0.5% Triton X-100), the tissue was incubated with DAPI (1:5000) at room temperature for 10 minutes. The tissue was covered with a drop of ProLong™ Glass Antifade Mountant (Invitrogen, USA), and images were taken with a microscope (Olympus, BX53). The excitation / emission spectrum was set to (340-390 nm / 420 IF; 460-495 nm / 510-550 nm; 530-550 nm / 575 IF), and the tissue samples were excited using excitation lamps and barrier filters (UW, BWA, and GW).The nNOS and ataxin-7 antibodies were combined with the fluorescent dyes TSA520 and TSA570, which have specific excitation and emission spectra of 488 / 519 nm and 555 / 570 nm. Cells that co-expressed nNOS (green) and ataxin-7 (yellow) were counted. The counts are shown in Table 13.

[0242] 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).

[0243] 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), and Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 1:500 (Invitrogen, A-21207, USA). For fluorescent staining, TSA520 1:200 (Wi See Biotechnology, D11013, China) and TSA570 1:200 (Wi See Biotechnology, D11011, China) were used.

[0244] [Table 26]

[0245] Figure 8 shows that the number of NOS+ neurons is significantly reduced in both regions of the transgenic model. Furthermore, both doses of velusetrag increase the number of nitrergic neurons in both regions compared to untreated transgenic mice. This effect is slightly greater in the proximal colon, and the effect does not appear to be dose-dependent. * P<0.05, *** P<0.001; one-way ANOVA; vs. G2.

[0246] Subsequent treatment with velusetrag restored nitregic neuronal levels to normal.

[0247] Example 10 - Effect of velusetrag on cholinergic neurons in a mouse model Choline acetyltransferase (CHAT) is an enzyme involved in the biosynthesis of the neurotransmitter acetylcholine. Most acetylcholine is synthesized locally at nerve terminals, where CHAT catalyzes the transfer of an acetyl group from acetyl-coenzyme A 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.

[0248] 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.

[0249] After incubation in blocking buffer, tissue samples were incubated with CHAT primary antibody (see above; Abcam, USA) (1:1000) at room temperature for 1 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 1 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 1 hour. Ataxin inclusions were detected with primary antibody Ataxin-7 (Thermo Fisher, USA) (1:2000) overnight at 4°C as described. After washing the samples three times with TBST (PBS + 0.5% Triton X-100 + 0.5% Tween-20), they 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). After incubating with TSA570 (Wi See Biotechnology, China) (1:200) at room temperature for 1 hour, the samples were washed three times with PBST (PBS + 0.5% Triton X-100) and incubated with DAPI (1:5000) at room temperature for 10 minutes. The tissues were covered with a drop of ProLong™ Glass Antifade Mountant (Invitrogen, USA). Images were taken using an Olympus microscope (BX53). The tissue samples were excited using excitation lamps and barrier filters (UW, BWA, and GW) with excitation / emission spectra set to (340-390 nm / 420 IF; 460-495 nm / 510-550 nm; 530-550 nm / 575 IF). CHAT and ataxin-7 antibodies were combined with the fluorescent dyes TSA520 and TSA570, which have specific excitation and emission spectra of 488 / 519 nm and 555 / 570 nm, respectively. Cells coexpressing CHAT (green) and ataxin-7 (yellow) were counted. The counts detected in Rb1 cKO mice are shown in Table 14.

[0250] [Table 27]

[0251] The results show that the number of CHAT+ neurons in the DSI and colon of KO animals is significantly reduced compared to the normal group (DSI P<0.01; colon P<0.05). The number of CHAT neurons in the DSI and proximal colon increases after treatment with 1 mg / kg and 3 mg / kg velusetrag. In particular, the difference is significant in the DSI after treatment with 1 mg / kg velusetrag. These results are also shown in Figure 9 ( * P<0.05, ** P < 0.01; T test; vs. G2).

[0252] In Prp-SCA7-92Q transgenic mice, the presence of cholinergic neurons in the myenteric plexus was assessed by immunofluorescence analysis using primary antibodies CHAT and ataxin-7 on DSI and proximal colon samples isolated from transgenic animals.

[0253] The results are shown in Table 15.

[0254] [Table 28]

[0255] Results show that the number of ChAT+ neurons in the Prp-SCA7-92Q model of CIPO is significantly reduced compared to normal wild-type mice. Furthermore, both doses of velusetrag induce an increase in cholinergic neurons in both the distal small intestine and proximal colon compared to vehicle-treated transgenic mice. This effect does not appear to be dose-dependent.

[0256] Furthermore, the percentage of cholinergic neurons with ataxin 7 inclusions is significantly increased in transgenic animals. Velsetrag, at both doses tested, significantly reduces the percentage of cholinergic neurons with ataxin 7 inclusions compared to vehicle-treated transgenic mice. The results are shown in Figure 10 ( * P<0.05, *** P<0.001; one-way ANOVA; vs. G2).

[0257] Thus, velusetrag attenuates cholinergic neuronal degeneration in both DSI and proximal colon at both doses tested, and reduces mutant ataxin-7 nuclear inclusions in DSI and colon.

[0258] Example 11 - Effect of velusetrag on calretinin neurons in the Prp-SCA7-92Q mouse model Calretinin is a calcium-binding protein abundantly expressed in neurons, and plays an important role as a modulator of neuronal excitability.

[0259] Calretinin-expressing neurons in the myenteric plexus were detected and quantified by immunofluorescence analysis of DSI and proximal colon samples isolated from transgenic animals. Mice were administered velusetrag as described above and sacrificed by CO2 inhalation. Whole-mount tissue samples were prepared as described in Example 5 and washed three times with PBST (PBS + 0.5% Triton X-100).

[0260] After incubation in 10% donkey serum blocking (PBS containing 10% donkey serum and 1% Triton X-100) for 2 h at room temperature, tissue samples were incubated overnight at 4°C with a 1:500 dilution of calretinin primary antibody (see above; Merck, USA).

[0261] After washing three times with TBST (PBS + 0.5% Triton x-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) for 1 h at room temperature.

[0262] After washing three times with PBST (PBS + 0.5% Triton x-100), tissue samples were incubated with DAPI 1:5000 (Invitrogen, USA) for 10 min at room temperature.

[0263] A drop of ProLong™ Glass Antifade Mountant was applied to the cover slip.

[0264] The tissue specimens were excited by a lamp using excitation and barrier filters (UW, BWA, and GW) with excitation / emission spectra set to 340-390 nm / 420 IF; 460-495 nm / 510-550 nm; and 530-550 nm / 575 IF. The calretinin antibody was combined with Alexa Fluor 488 dye, which has specific excitation / emission spectra of 499 / 520 nm and 590 / 618 nm.

[0265] Images from each animal were acquired at 20x magnification under a microscope (Olympus, BX53, Japan) using CellSens Standard Imaging software (Ver. 3.2, Olympus, Japan), and cells were counted.

[0266] The figures are shown in Table 16.

[0267] [Table 29]

[0268] 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). Treatment with velusetrag induced an increase in the number of calretinin neurons in DSI, but no significant difference was detected. In the colon, velusetrag at 1 mg / kg and 3 mg / kg induced a significant increase in the number of calretinin neurons (P<0.05 and P<0.01, respectively).

[0269] The variations in calretinin neurons in the DSI and proximal colon of PrP-SCA7-92Q transgenic mice treated with velusetrag (1 mg / kg and 3 mg / kg) are also shown in Figure 11 ( ** P<0.01, *** P<0.001; one-way ANOVA; vs. G2 vehicle).

[0270] Calretinin-expressing neurons (excitable motor cells) were reduced in the proximal colon of vehicle-treated, but not DSI-treated, transgenic mice, whereas administration of velusetrag at both doses protected this neuronal population (Figure 11).

[0271] These results indicate that velusetrag at both doses prevents neurodegeneration of calretinin neurons in the proximal colon.

[0272] Example 12 - Effect of velusetrag on n-NOS and CHAT protein levels in neuromuscular tissue of the Prp-SCA7-92Q mouse model The protein levels of n-NOS and CHAT in the cytoplasm of the proximal colon of transgenic mice were detected and analyzed by Western blotting.

[0273] Proximal colon samples were placed in 10 volumes (w:v) of RIPA lysis buffer (Beyotime Biotechnology, China) containing a protease and phosphatase inhibitor cocktail (Thermo Scientific, USA) according to the manufacturer's instructions. 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 subjected to protein concentration analysis by the BCA method. An appropriate amount of protein loading buffer was added to the supernatant for protein denaturation (95°C, 10 minutes). Proteins were fractionated on a 10% SDS-PAGE gel and transferred to a NC membrane. After blocking with 5% BSA in TBST (0.05% Tween-20) for 2 hours at room temperature, the membrane was incubated overnight (over 15 hours) at 4°C 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). The blot was washed with TBST (three times, 10 minutes each) and then incubated with secondary antibodies: Goat anti-Rabbit IgG H&L (IRDye® 800CW) serum-adsorbed 1:5000 (Abcam, USA) or Donkey anti-Goat IgG H&L (IRDye® 800CW) 1:5000 (Abcam, USA) for 1 hour at room temperature.

[0274] After washing, the membrane was placed in substrate working solution for 5 min before being imaged using a ChemiDoc System (Bio-Rad, 12003154, USA). Densitometric analysis of protein bands was performed using QuantityOne software version 4.6.2.

[0275] The data are shown in Table 17.

[0276] [Table 30]

[0277] nNOS and ChAT protein levels are significantly reduced in the Prp-SCA7-92Q model. Compared to untreated transgenic mice, increased expression of nNOS and ChAT proteins was observed in the proximal colon, and velusetrag reversed this effect at both doses. This effect did not appear to be dose-dependent.

[0278] These results are shown graphically in FIG. * P<0.05, *** P<0.001; T-test; vs. G2.

[0279] Overall results obtained in mouse models The efficacy of velusetrag, a 5-HT4 receptor agonist, was evaluated in two CIPO mouse models: RB1 cKO mice and PrP-SCA7-92Q transgenic mice. Histopathological analysis of tissues by HE staining revealed DSI and colonic mucosal inflammation, macrophage infiltration, and ulceration in both models, and velusetrag was able to ameliorate intestinal injury in a dose-dependent manner.

[0280] Immunohistochemical staining, as demonstrated by the use of HuCD antibodies, revealed a significant reduction in enteric neurons in transgenic mice. This pan-neuronal marker, involved in intestinal neurogenesis, neuronal survival, and plasticity, was reduced in the distal small intestine and proximal colon of transgenic mice, suggesting a loss of function of Hu proteins affecting neuronal signaling [Li et al., Scientific Reports | 6:38216 | DOI: 10.1038 / srep38216 and 25]. Furthermore, the pan-neuronal antibody demonstrated a significant increase in ataxin-7 inclusions in DSI and the colon, which was suppressed by velsetrag treatment. This was specifically associated with a unique subpopulation of ganglion cells with ataxin-7 intranuclear inclusions that express ChAT but not nNOS.

[0281] Although nNOS-expressing inhibitory motor neurons were not affected by ataxin-7 inclusions, they were lost in the DSI and colon of transgenic mice compared with control mice, and treatment with velusetrag protected against this loss. Neuronal NOS (nNOS), constitutively expressed in peripheral neurons, is involved in synaptic plasticity, central regulation of blood pressure, smooth muscle relaxation, and peripheral nitrergic nerve-mediated vasodilation. The ability to form nitrergic neurons is a critical step in the development of "normal" intestinal circuitry, and many intestinal disorders may benefit from the transplantation and engraftment of nNOSt cells [McCann, CJ et al., Nat Commun, 2017. 8: p. 15937.] Nitric oxide (NO) acts as a major nonadrenergic, noncholinergic inhibitory signal in the peripheral nervous system, including the gastrointestinal (GI) tract and the enteric nervous system (ENS), relaxing GI smooth muscle and regulating physiological peristalsis. Therefore, loss of neuronal NO synthase (nNOS) may disrupt normal GI motility. NO mediates descending relaxation, facilitating colonic transit and promoting the propulsion of colonic contents in rats. In the present study, we found that nNOS+ neurons were reduced in the ENS of vehicle-treated transgenic mice, but not after velsetrag treatment, suggesting that prokinetic drugs may be beneficial to NO function in the ENS.

[0282] In the mouse large intestine, we investigated the major population of neurons expressing the calcium-binding protein calretinin as a marker for the majority of myenteric motor neurons, interneurons, and putative primary afferent neurons in the muscularis mucosae and lamina propria. In this case, calretinin-immunoreactive neurons in the myenteric ganglion (putative intrinsic primary afferent neurons) were reduced in the colon of PrP-SCA7-92Q mice, but velusetrag was able to protect them.

[0283] Mammalian microtubule-associated protein 2 (MAP2), which is primarily expressed in neurons but also in oligodendrocytes, is present in both early and late stages of neuronal development. Three isoforms, MAP2A, B, and C, are known. MAP2C is localized in the soma, dendrites, and axons of immature neurons, whereas MAP2A and B are primarily localized in the dendrites of mature neurons. MAP2 / Tau family proteins were originally discovered and characterized for their ability to bind and stabilize microtubules. In the present study, the intensity of MAP2 staining was reduced in knockout and transgenic mice, indicating that transgenic mice had fewer mature neurons or dendrites. MAP2 intensity in the ENS increased after treatment with velusetrag, suggesting that velusetrag may improve dendritic microtubule function.

[0284] Enteric glial cells were taken into consideration because they interact with other gastrointestinal (gastrointestinal) cell types, such as the epithelium and immune system, to maintain homeostasis (Boesmans et al., Frontiers in cell and Development Biology Volume 9 | Article 775102).

[0285] In vitro studies suggest that Sox10-expressing undifferentiated progenitor cells in the fetal intestine generate both enteric neurons (Sox10-) and glia (Sox10+), but the neurogenic potential and temporal regulation of these cells in vivo during intestinal organogenesis are currently unknown.

[0286] A large proportion of submucosal HuCD+ neurons and a small subpopulation of myenteric neurons were found to co-express the glial 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 are likely newly differentiated neurons in the process of turning off glial marker expression.

[0287] In the present study, we observed an increase in the number of glia in the proximal colon compared to control mice. Considering the ratio of glia to all neurons, the increase in DSI and colonic DSI in both KO and transgenic mice was inhibited by treatment with any dose of velusetrag. Kulkarni et al. (Proc Natl Acad Sci USA, 2017. 114(18):: pp. E3709-E3718) showed that under physiological conditions, the adult ENS is maintained by a dynamic balance between neuronal apoptosis and neurogenesis from Sox10- / Nestin+ (neuronal) precursors, but not mature glial cells. However, the neurogenic potential of Sox10 / +Nestin- cells is activated only upon injury. In the present study, we observed an increase in Sox10+ glial cells using KO and transgenic mice. However, treatment with velusetrag resulted in a decrease in Sox10+ glial cells. It is thought that in transgenic mice, increased glial cells may be needed to compensate for the loss of several other types of neurons. Because velusetrag can promote neuronal production, there is less need for glial cells when intestinal damage or dysfunction is reduced.

[0288] Because velusetrag is a 5-HT4 receptor (5-HT4R) agonist, 5-HT4R expression was investigated because this type of receptor, 5-HT4, can regulate gastrointestinal movement and reduce visceral sensitivity (visceral hypersensitivity). Furthermore, 5-HT4R is involved in the development and maintenance of enteric nerve function. In this study, we observed changes in 5HT4R mRNA levels in cKO and transgenic mice, with an increase in intestinal tissue and a decrease in colonic tissue. Clearly, treatment with velusetrag did not alter 5HT4 mRNA levels in transgenic mice.

[0289] In this study, biomarkers related to cell survival, proliferation, and metabolism (mTOR / AKT signaling) 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. It interacts with other subunits to form two distinct complexes, mTORC1 and mTORC2. mTORC1 regulates cell growth and metabolism in response to environmental inputs, including growth factors, amino acids, energy, and stress. mTORC2 primarily controls cell survival and migration through phosphorylation of the glucocorticoid-regulated kinase (SGK), protein kinase B (Akt), and protein kinase C (PKC) kinase families. Dysregulation of mTOR has been observed in many human diseases, including cancer, cardiovascular disease, neurodegenerative diseases, and epilepsy. In this study, activation of mTOR signaling, as expressed by phosphorylation, was observed in DSI in transgenic and cKO mice, and this was suppressed by administration of velusetrag. AKT, a serine-threonine kinase with three distinct protein isoforms (AKT1, AKT2, and AKT3), also plays a role in cell survival, proliferation, and metabolism. In this case, AKT phosphorylation was observed in DSI in transgenic and cKO mice, but was reduced by treatment with velusetrag. Finally, p70S6 kinase is activated by growth factors and plays a central role in cell growth and proliferation by mediating the phosphorylation of the 40S ribosomal protein S6, thereby enabling efficient translation of 5-terminal oligopyrimidine tract mRNA (5-TOP). mTOR promotes translation initiation by phosphorylating two targets: ribosomal p70S6 kinase (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). Furthermore, increased p70S6 activation was observed in transgenic and cKO mice. This effect was inhibited by velusetrag.Overall, the 3 mg / kg velusetrag group was able to reduce the expression of p-mTOR, p-Akt, and p-P70S6 in the distal small intestine after 5 weeks of treatment.

[0290] Velsetrag significantly improves biomarkers and ameliorates the pathological state of CIPO, and therefore holds high therapeutic potential for the treatment of CIPO.

[0291] Example 13 - A multicenter, double-blind, placebo-controlled, two-treatment, four-period crossover, multiple (n=1) trial to evaluate the efficacy and safety of velusetrag in patients with CIPO Materials and Methods A Phase II, multicenter, double-blind, placebo-controlled, two-arm, four-period crossover multiple (n=1) study was conducted to evaluate the efficacy and safety of velusetrag 15 mg once daily compared with placebo in subjects with idiopathic chronic intestinal pseudo-obstruction (CIPO) or CIPO secondary to a primary neurodegenerative or demyelinating condition.

[0292] This study evaluated the safety, tolerability, and efficacy of velusetrag 15 mg once daily compared with placebo in improving the severity of symptoms associated with CIPO in subjects with idiopathic CIPO and CIPO secondary to neurodegenerative conditions.

[0293] CIPO is a chronic, rare disease with variable symptoms and different underlying pathologies that may respond differently to aggressive treatment. Therefore, standard parallel-group, placebo-controlled trials have not been able to detect clear benefits due to the wide variability in clinical presentation.

[0294] The n=1 crossover, placebo-controlled study design allows evaluation of treatment effects in all subjects, allowing each subject to serve as their own control, demonstrating a positive effect even in a single subject, and avoiding underestimation of treatment efficacy (Emmanuel et al., Randomized 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 Jan;35(1):48-55).

[0295] Placebo was used as a comparator because it allows estimation of the actual therapeutic effect of velusetrag.

[0296] This study consisted of a maximum 7-day screening period (Day -7 to Day -1) followed by four 4-week treatment periods. Subjects received either velusetrag (VEL) 15 mg (Period 2) or placebo (PLA) (Period 2), with a 2-week washout period between treatment periods and a 2-week follow-up period (total of approximately 175 days), as shown in Figure 16. Evaluations were conducted at the beginning of the screening period (Visit 1), the beginning and end of each treatment period (Visits 2 to 9), and the end of the follow-up period (Visit 10).

[0297] After a screening period of up to 7 days (Day -7 to Day -1), at the randomization visit (Visit 2), eligible subjects were randomly assigned in a 1:1:1:1 ratio to one of the following four sequences, as shown in Figure 16:

[0298] A. VEL-PLA-VEL-PLA B. PLA-VEL-PLA-VEL C. VEL-PLA-PLA-VEL D. PLA-VEL-VEL-PLA VEL = velusetrag 15 mg once daily for 4 weeks PLA = matching placebo once daily for 4 weeks

[0299] Gastrointestinal symptom severity (abdominal pain, flatulence, nausea, and vomiting) and bowel habits (weekly recall) were recorded according to the Schedule of Assessments using an e-diary on Day -1 and weekly after randomization, during both the treatment and washout periods, as appropriate, until the end of the follow-up period.

[0300] During the study, the following parameters were collected during visits: Serum nutritional marker (albumin, prealbumin, vitamin B12, and folate) levels were collected before treatment, during each 2-week washout period, and at the end of follow-up or early termination visit (ETV) or early switch visit (ESV) after the fourth period. The number of pseudo-obstructive episodes and hospitalizations was assessed at each visit. The need for parenteral and enteral nutritional supplements was assessed at each visit. A lactulose breath test (L-BT) was performed to assess oro-cecal transit time at the screening visit (Visit 1) and at the end of the first 4-week treatment period (Visit 3).

[0301] Table 18 shows the Schedule of Assessments of the measured parameters.

[0302] [Table 31]

[0303] [Table 32]

[0304] [Table 33]

[0305] [Table 34]

[0306] Subjects were also required to record any permitted medications for CIPO gastrointestinal symptoms taken immediately prior to the start of the first dosing period (Day -1), as well as any changes (increases / decreases) in dose or number of concomitant medications during the entire dosing and washout periods. Additionally, the amount and time of study medication intake were recorded by subjects in their e-diary every day during the dosing period.

[0307] Study population A total of 17 patients with a history of chronic idiopathic intestinal pseudo-obstruction or CIPO secondary to neurodegenerative or demyelinating disease were randomized and assigned to one of the four groups described above.

[0308] Randomization took place at Visit 2 (V2 - Day 1) after all screening procedures were performed to assess study eligibility.

[0309] Randomization was stratified by CIPO diagnosis (idiopathic or secondary to a neurodegenerative or demyelinating disease) and 5-HT4 receptor agonist responder status (responder / naive or non-responder) as follows:

[0310] 5-HT4 receptor agonist non-responders 5-HT4 receptor agonist responder / naive and idiopathic CIPO 5-HT4 receptor agonist responder / naive, and CIPO secondary to neurodegenerative or demyelinating diseases

[0311] Non-responders were defined as any subject who, based on the investigator's judgment, had previously failed to benefit from a 5-HT4 receptor agonist.

[0312] Main selection criteria: 1. Male or female, aged 18-80. 2. Subjects with a history of chronic idiopathic intestinal pseudo-obstruction or CIPO secondary to neurodegenerative or demyelinating disease. 3. Subjects whose estimated oral caloric intake is at least 30% of the recommended daily caloric intake for their age and sex (stages 0, 1, or 2 on the "artificial food need" scale according to Table 19).

[0313] [Table 35]

[0314] 4. Subjects with at least two of the four CIPO gastrointestinal symptoms (i.e., abdominal pain, bloating, nausea, and vomiting), each with a score ≥ 3 (on a scale of 0 to 4) collected on the gastrointestinal symptom questionnaire on Day -1. 5. Subjects are willing and legally able to provide free informed consent for all procedures included in the protocol. 6. All sexually active male participants who were partners of women of childbearing potential were required to use condoms during sexual intercourse until 90 days after the end of the entire study. 7. All female participants had to meet the following criteria: a. Individuals of no childbearing potential, i.e., (i) postmenopausal (no natural menstruation for at least two years) or (ii) surgically sterilized (tubal ligation or hysterectomy) or (iii) removal (ablation) of both ovaries or b. Individuals of childbearing potential who have a negative pregnancy test result at the time of screening and randomization and who agree to use highly effective contraceptive methods (i.e., failure rate <1% per year) throughout the entire study. Highly effective contraceptive methods were defined according to the recommendations of the EU Clinical Trial Facilitation Group.

[0315] Main exclusion criteria 1. Subjects with primary CIPO or CIPO secondary to other known endocrine / metabolic, autoimmune diseases, and CIPO secondary to neurological conditions other than neurodegenerative or demyelinating diseases. 2. Subjects with a condition characterized by mechanical ileus. 3. A nasogastric, gastrostomy, or jejunostomy feeding tube is in place at the time of randomization or planned for the duration of the study, or the artificial food needs scale is stage 3 ("total parenteral nutrition"; see the artificial food needs (AFN) scale for CIPO subjects in 21.3 Appendix 3 of the protocol). 4. Presence of untreated clinically relevant thyroid dysfunction or known thyroid dysfunction not adequately controlled by treatment, deemed clinically significant by the investigator (e.g., subjects with abnormal thyroid-stimulating hormone [TSH], and, if available, triiodothyronine [T3] and thyroxine [T4] levels). 5. Subjects with a history of diabetes at the time of screening. 6. Clinically significant ECG abnormalities (e.g., ST-segment elevation or depression suggestive of ischemia, partial or complete left bundle branch block [LBBB]) at the time of screening and randomization. 7. QTcF > 450 msec in men or QTcF > 470 msec in women on screening ECG or family history of sudden cardiac death. 8. Subjects requiring a low galactose diet. 9. Hypersensitivity or documented intolerance to lactulose, lactose, or any of the excipients in the lactulose formulations used in L-BT. 10. History of sensitivity to velusetrag or to any of the excipients in velusetrag or placebo. 11. Has used scopolamine or erythromycin within 2 weeks prior to screening and / or plans to use them during the study period. 12. Have used a 5-HT4 receptor agonist (e.g., prucalopride, cisapride, clebopride, and cinitapride) within 5 days prior to randomization and / or plan to use it during the study. 13. Have used opioids within 8 weeks of screening and / or plan to use them during the study period. 14. Have received and / or will receive during the study period a strong cytochrome P450-isoenzyme 3A4 (CYP3A4) inhibitor (e.g., clarithromycin, indinavir, itraconazole, ketoconazole, nefazodone, nelfinavir, ritonavir, saquinavir, telithromycin, grapefruit juice) or a strong CYP3A4 inducer (e.g., rifampicin, phenytoin, carbamazepine, phenobarbital, St. John's wort) within 2 weeks prior to screening. 15. Has received and / or will receive during the study period a strong P-glycoprotein (P-gp) transporter inhibitor (e.g., captopril, carvedilol, diltiazem) within 2 weeks prior to screening. 16. Has received and / or will receive during the study period a potent breast cancer resistance protein (BCRP) transporter inhibitor (e.g., curcumin, cyclosporine A, eltrombopag) within 2 weeks prior to screening. 17. Currently have a positive swab for COVID-19 infection or are suspected of having the infection (currently undergoing testing). 18. Cancer (excluding non-melanoma skin cancer (skin cancer other than malignant melanoma)) and / or required anti-cancer treatment (including radiation therapy) within the past 5 years. 19. Severe (critical) renal impairment (i.e., estimated glomerular filtration rate <30 ml / min). 20. Aspartate aminotransferase (AST) or alanine transaminase (ALT) levels >2.5 times the upper limit of normal (ULN), bilirubin (except when considered due to Gilbert's syndrome) or alkaline phosphatase (ALP) levels >1.5 times the ULN. 21. Severe liver damage defined as Child-Pugh C. 22. History of any of the following cardiac disorders: a. Torsades de pointes, ventricular tachycardia, ventricular fibrillation. b. History of myocardial infarction, unstable angina, acute coronary syndrome, coronary or cerebral revascularization, or stroke (attack) within the past 18 months. c. Class 2-4 angina in the past 12 months prior to screening. d. NYHA class III-IV congestive heart failure in the past 18 months prior to screening. 23. History of alcohol or drug abuse or dependence within the past year (as determined by the investigator). 24. Current significant health conditions (e.g., cardiovascular, respiratory, renal, hepatic, neurological, psychiatric, hematological, oncological, immune, muscular and joint, etc.) that, in the investigator's judgment, may include: a. Compromises the subject's safe participation in the study; or b. The subject is unlikely to complete the study, or c. The subject is unlikely to comply with study procedures (e.g., strong anticipated need for unauthorized treatment, significant disability, terminal illness). 25. Pregnant or breastfeeding women. 26. Use of any experimental medication within 12 weeks prior to screening.

[0316] Configuration used Subjects enrolled in the study received 15 mg (3 x 5 mg capsules) of velusetrag or a matching placebo (3 capsules) once daily for 4 weeks in each of the four treatment periods. Subjects were instructed to take 3 capsules orally with water once daily on an empty stomach at approximately the same time each morning.

[0317] Belsetrag Active ingredient: velcetrag (ScinoPharm Taiwan, Ltd.) 5mg Excipients: lactose monohydrate, microcrystalline cellulose, hypromellose and magnesium stearate

[0318] placebo Active ingredients: None Excipients: lactose monohydrate, microcrystalline cellulose, hypromellose and magnesium stearate

[0319] Prohibited drugs Use of the following medications was not permitted within 2 weeks prior to screening and / or was not scheduled during the study period:

[0320] Scopolamine ·erythromycin Strong CYP3A4 inhibitors or strong CYP3A4 inducers (see Table 20) Potent P-gp transporter inhibitors (according to Table 20) Potent BCRP transporter inhibitors (see Table 20)

[0321] [Table 36]

[0322] Additionally, the use of: Opioids were not permitted within 8 weeks of screening and / or were not scheduled during the study period. 5-HT4 receptor agonists (e.g., prucalopride, cisapride, clebopride, cinitapride) were not permitted from 5 days prior to randomization through the duration of the study. · If medically indicated (treatment of underlying AEs), poorly orally absorbed opioids (i.e., loperamide) may be used.

[0323] If 5-HT4 receptor agonists (e.g., prucalopride, cisapride, clebopride, cinitapride) or scopolamine, or erythromycin and opioids for analgesic use were taken during the study, the subject was considered a treatment failure and was excluded from the study.

[0324] Additional prescription and over-the-counter medications were permitted, provided that such drugs were not known to be strong inducers or inhibitors of CYP3A4, P-gp, and BCRP activity.

[0325] Addition of concomitant medications or changes in regimen that affect CYP3A4, P-gp, and BCRP activity were in accordance with the inclusion or exclusion criteria and were documented in the source documents and eCRF.

[0326] Medications taken from 30 days before the screening visit through the end of the follow-up period were recorded. If subjects had previously taken a 5-HT4 receptor agonist (e.g., prucalopride, cisapride, clebopride, cinitapride), the last period of administration (even if it occurred more than 30 days before the screening visit) and the effectiveness of such administration (responder / non-responder) for each subject were recorded on the eCRF.

[0327] If clinically permitted, subjects were encouraged not to change their current concomitant medication regimen or start any new concomitant medications.

[0328] Permitted Treatments Medications used to alleviate the main symptoms of CIPO were permitted, and the use of the following concomitant medications was recorded daily in the e-diary:

[0329] Treatment of nausea and vomiting and / or non-serotonergic gastroprokinetic drugs (e.g., metoclopramide, domperidone, pharmaceutical ginger preparations, pyridostigmine, prochlorperazine, promethazine, ondansetron, and aprepitant) Treatment of constipation (e.g., macrogol, bisacodyl, linaclotide, laxatives, enemas) Treatment of diarrhea (e.g., tannates, loperamide) Treatment of abdominal pain (e.g., paracetamol, NSAIDs, trimebutine, mebeverine, gabapentin, duloxetine, amitriptyline) Other (e.g., octreotide, somatostatin, pancreatic enzymes, probiotics, rifaximin, metronidazole, fluconazole) All permitted medications for CIPO gastrointestinal symptoms taken immediately before the start of the first treatment period (day -1), as well as any changes in concomitant dose or number of medications during the entire treatment and washout periods, were recorded daily in the e-diary until the end of follow-up.

[0330] Efficacy endpoints Primary efficacy endpoint The primary efficacy outcome was the change in the weekly Global Gastrointestinal Symptoms Index Score (WGGSAIS) from the beginning to the end of each treatment period. Scores were obtained by averaging the scores for each of four symptoms (abdominal pain, bloating, nausea, and vomiting) rated weekly on a scale of 0 to 4: 0—absent; 1—mild (not interfering with usual activities); 2—moderate (deviating from usual activities but not enough to force a change); 3—severe (significantly interfering with usual activities and forcing a change); and 4—extremely severe (interfering with daily activities) (Barbara G et al., 2004, Gastroenterology Mar;126(3):693-702).

[0331] Secondary efficacy endpoints Secondary efficacy endpoints were assessed as the following changes from the start to the end of each treatment period:

[0332] 1. Changes in waist circumference 2. Percentage of subjects who achieved a 1-point improvement in WGGSAIS 3. Changes in individual symptom scores for abdominal pain, flatulence, nausea, and vomiting 4. Change in the number of weekly bowel movements (only for subjects with Bristol Stool Scale type 1 or 2 at the start of the treatment period) 5. Change in number of complete bowel movements per week (only for subjects with Bristol Stool Scale type 1 or 2 at the start of the treatment period) 6. Change in stool type on the Bristol Stool Scale Change in weekly bowel habit satisfaction score measured using a 7.0 to 10 scale 8. Change from baseline in oro-cecal transit time measured using the lactulose breath test (end of first treatment period only) 9. Changes in nutritional markers (serum albumin, prealbumin, vitamin B12, and folate levels) from the start of each treatment period to the end of each 2-week washout period (or follow-up period) 10. Percentage of days during each treatment period and washout period (or follow-up period) when permitted medications used to relieve major CIPO gastrointestinal symptoms were changed: a. Percentage of days when the dose was increased compared to the beginning of the period b. The percentage of days when the dose was reduced compared to the beginning of the period c. The percentage of days on which medication was added compared to the beginning of the period d. Percentage of days the drug was eliminated compared to the beginning of the period 11. Changes in quality of life (Short 12-item Health Survey - SF-12) 12. Number of hospitalizations related to CIPO during the treatment period 13. Change in stage on the "need for artificial food" scale 14. Number of pseudo-obstructive episodes based on investigator judgment 15. Changes in the following from the end of each treatment period to the end of the first and second washout weeks (or follow-up period): a. Weekly abdominal pain score b. Weekly flatulence score c. Weekly nausea score d. Weekly vomiting score e. Weekly global gastrointestinal symptom mean index score f. Number of weekly bowel movements (only for subjects with a Bristol Stool Scale type 1 or 2 at the start of the treatment period) g. Number of complete bowel movements per week (only for subjects with a Bristol Stool Scale type 1 or 2 at the start of the treatment period) h. Bristol stool scale stool type i. Weekly bowel habit satisfaction score measured using a scale of 0 to 10

[0333] Safety evaluation Safety was assessed at each visit to measure and evaluate changes from baseline in the following parameters: Vital signs (including blood pressure, pulse, and temperature) Physical examination, including height and weight Routine laboratory parameters ( Hematology Hematocrit, hemoglobin, red blood cell count, white blood cell count including differential count, and platelet count; Biochemistry blood glucose, total cholesterol, triglycerides (neutral fats), 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; Urine tests specific gravity, pH, protein, glucose, ketones, hemoglobin, nitrites, bilirubin, urobilinogen and microscopy) Triplicate ECG Adverse Events (AEs) Withdrawal from the study due to AEs

[0334] statistical methods Determining sample size Sample size was based on the primary analysis (t-test) of the primary endpoint, i.e., the difference in WGGSAIS between evaluable pairs. Pairs were considered evaluable if there were assessments available for subjects in one cycle, i.e., consecutive velusetrag and placebo periods of administration (or vice versa).

[0335] Of note, each subject was required to be assessed twice, once for each evaluable pair. Thus, each subject could contribute to 0, 1, or 2 pairs, and only data comprising evaluable pairs for the primary endpoint were considered in this analysis.

[0336] Definition of the main study population Safety analysis set (SS): All subjects who received treatment. Analysis of SS was performed according to the actual treatment received. Modified Full Analysis Set 1 (mFAS1): All randomized, treated subjects who are responders / naive to 5HT4 receptor agonists and for whom data on the primary endpoint were obtained at least once during the velusetrag treatment period and at least once during the placebo treatment period of the same cycle. Modified Full Analysis Set 2 (mFAS2): All randomized and treated subjects for whom data on the primary endpoint were obtained at least once during the velusetrag treatment period and at least once during the placebo treatment period within the same cycle. Per Protocol Set (PPS): All subjects in mFAS1 who met protocol requirements with regard to dosing compliance and collection of primary efficacy data and who had no major deviations.

[0337] Analysis of primary efficacy endpoints The WGGSAIS was obtained by averaging the scores for each of the four symptoms (abdominal pain, bloating, nausea, and vomiting) assessed each week, with lower scores representing better health. If at least two symptoms were assessed, the mean score was calculated; otherwise, the mean score was considered missing.

[0338] Considering the evaluable pairs (i.e., assessments available for both velusetrag and placebo within a cycle), the WGGSAIS was summarized by dose, taking into account data collected from pre-dose to the end of each period (4 weeks) and changes that occurred during the washout period, to present the WGGSAIS for the post-dose period followed by the pre-dose period.

[0339] The difference in WGGSAIS between velusetrag and placebo was calculated within each evaluable pair as the change in velusetrag minus the change in scoring that occurred during placebo treatment (for both velusetrag and placebo, the change is between the end-of-treatment value and the pre-treatment value). Paired t-tests were used to assess the difference in scoring between velusetrag and placebo treatment. Results are provided as mean differences with relative 95% CI and p-values.

[0340] Supportive analyses were performed using mixed models, and the outcome was the difference in WGGSAIS between velusetrag and placebo in the change from pre-dose to end-of-dose in the same cycle, with cycle considered as a fixed effect and subject as a random effect. Results of the fixed-effect Type III study and estimates of the difference between velusetrag and placebo in the change from pre-dose to end-of-dose are provided with 95% CIs.

[0341] These analyses were presented for mFAS1 without imputing missing data as the primary analysis.

[0342] Sensitivity analysis: The same analysis was provided for mFAS2.

[0343] Analysis of Key Secondary Efficacy Endpoints Percentage of subjects with a 1-point improvement in WGGSAIS A calculated change in WGGSAIS score ≤ -1 point between end of treatment and pretreatment was considered a "success," whereas a change > -1 was considered a "failure." The proportion of subjects with a 1-point improvement in WGGSAIS was summarized by treatment sequence, period, and time point (weekly assessment by e-diary).

[0344] Considering evaluable pairs, the proportion of paired observations with a 1-point improvement on the WGGSAIS is summarized by treatment and time point. The odds ratio of achieving a 1-point improvement with velusetrag compared with placebo is provided with the corresponding 95% CI using the logistic model at the end of treatment. Fisher's test was applied to compare "success" / "failure" paired observations between treatments.

[0345] Changes in individual symptom scores Each symptom (abdominal pain, flatulence, nausea, and vomiting) was analyzed as described in the analysis of the primary efficacy endpoint.

[0346] Effect on bowel habits in subjects with constipation (i.e., Bristol stool types 1 and 2 at baseline) The number of bowel movements and complete bowel movements, along with an assessment of stool consistency according to the Bristol Scale, were reported weekly in an e-diary. These numbers were summarized by treatment, taking into account data collected from pre-treatment through the end of each treatment period and any changes therein.

[0347] Stool consistency Average stool consistency was recorded in an e-diary according to the Bristol Scale. The Bristol Scale was classified as follows: constipation: types 1 and 2, normal: types 3 and 4, and diarrhea: types 5, 6, and 7. The distribution of subjects by category from pre-treatment to end-of-treatment is provided. Considering evaluable pairs, the number of subjects by category at pre-treatment for each cycle is provided, along with the number of subjects whose category remained unchanged or changed at the end of the treatment period for each cycle. Additionally, a Fisher Exact test was performed to compare the distribution by category at end-of-treatment, and p-values ​​were provided.

[0348] ·Oral-caecal transit time Oral-cecal transit time was calculated based on the lactulose breath test (L-BT), which was performed only during the first treatment period. Changes in oroceccal transit time were calculated between post-treatment and pre-treatment. A t-test was performed to assess the difference between velsetrag and placebo.

[0349] Pseudo-obstructive episodes Considering the assessable pairs, the number of pseudo-obstructive episodes occurring within each treatment period and the number of pseudo-obstructive episodes occurring within washout / follow-up were considered categorical variables categorized as 0, 1, 2, and >2 and summarized by treatment. Furthermore, Fisher's exact test comparing the distribution of the number of pseudo-obstructive episodes occurring within each treatment period was performed considering the categories, and p-values ​​were provided.

[0350] -Effects of discontinuing velusetrag administration during the 2-week washout period (or follow-up period) at the end of each treatment period

[0351] definition The effect of discontinuing treatment was assessed taking into account the following set of variables: Weekly abdominal pain score Weekly flatulence score Weekly nausea score Weekly vomiting score Weekly overall gastrointestinal symptom mean index score Number of weekly bowel movements (only for subjects with Bristol Stool Scale type 1 or 2 at baseline) Number of complete bowel movements per week (only for subjects with a Bristol Stool Scale type 1 or 2 at the start of the treatment period) Bristol stool scale stool type Weekly bowel habit satisfaction score measured using a scale of 0 to 10

[0352] The same descriptive analyses as above were repeated, focusing on changes from the end of each dosing period to the end of each week of the washout or follow-up period, rather than changes between post-dose and pre-dose.

[0353] Statistical analysis of safety Adverse events (AEs) Adverse events beginning with or after the first dose of the investigational product were considered treatment-emergent AEs (TEAEs). Reporter verbatim terms for AEs were coded using the most recent version of the Medical Dictionary for Regulatory Activities (MedDRA) coding. Furthermore, events were classified as velusetrag-emergent if the last dose taken before the AE began was velusetrag, and as placebo-emergent if the last dose taken before the AE began was placebo.

[0354] Absolute and relative frequencies of subjects with administration-related TEAEs, serious TEAEs, administration-related serious TEAEs, TEAEs leading to temporary interruption of administration, TEAEs leading to discontinuation of administration, serious TEAEs leading to temporary interruption of administration, serious TEAEs leading to discontinuation of administration, and fatal TEAEs were provided.

[0355] Laboratory parameters Laboratory values ​​(hematology, chemistry, and urinalysis), including changes from baseline in continuous parameters, are summarized descriptively at each scheduled visit. Frequency tables are generated for categorical parameters. Laboratory values ​​outside the normal range are listed. For selected laboratory data, significant abnormalities are summarized using shift tables.

[0356] electro-cardiogram Triplicate interpretable ECG recordings were performed, 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 final interpretation of all ECGs was completed by a central reviewer and sent to the site for evaluation and filing. A list of subjects whose 12-lead ECG results were deemed "abnormal and not clinically significant" or "abnormal and clinically significant" was provided.

[0357] result Study population Seventeen subjects were randomized: 16 subjects completed the study (mFAS2 population), and one subject discontinued the study after the first month of treatment due to withdrawal of informed consent.

[0358] Of the 16 randomized subjects who completed the study, one randomized subject had previously failed to respond to 5-HT4 receptor agonists, while the remaining 15 were 5-HT4 receptor agonist responders or naive (mFAS1 population). Regarding 5-HT4 responder status, 10 subjects (66.67%) had previously benefited from 5-HT4 receptor agonists, and 5 subjects (33.33%) were treatment-naive.

[0359] All subjects were diagnosed with idiopathic CIPO. During the 6 months prior to consent, the mean number of pseudo-obstructive episodes was 1.8 ± 2.34 (range: 0; 6), and the mean number of CIPO-related hospitalizations was 0.3 ± 0.62 (range: 0; 2).

[0360] Compliance in the mFAS1 population was consistently above 80% across all dosing sequences and durations, with similar results observed in the mFAS2 and PPS.

[0361] The demographic characteristics of the mFAS1 population (primary population) are shown in Table 21.

[0362] The mean age was 56.7 ± 10.65 years (range: 40 years; 74 years), and most subjects were female (12, 80.00%) and not Hispanic or Latino (11, 73.33%).

[0363] [Table 37]

[0364] Efficacy endpoints Primary efficacy endpoint A summary of the primary efficacy results is shown in the table below.

[0365] Mean WGGSAIS scores were consistently lower at the end of treatment than at the beginning for both velusetrag and placebo, indicating symptomatic improvement. Greater reductions were consistently observed with velusetrag.

[0366] FIG. 17 shows a line graph of the mean WGGSAIS by dose (30 pairs observed); mFAS1 population.

[0367] Table 22 shows a summary of WGGSAIS by administration in the mFAS1 population (30 pairs observed).

[0368] [Table 38]

[0369] For mFAS2 and PPS, approaches involving imputation of missing values ​​may also consider borderline situations.

[0370] On the mFAS2, the mean change from pre-dose to end-of-dose was -0.529 ± 0.7956 (range: -2.67; 0.75) for velusetrag 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 reduction was greater with velusetrag (paired t-test: p=0.0469, 95% CI for the mean difference in change between velusetrag and placebo equals -0.679; -0.005; mixed model: p=0.0563, 95% CI for the mean difference in change between velusetrag and placebo equals -0.69; 0.01).

[0371] Secondary endpoints Percentage of subjects with at least a 1-point improvement in WGGSAIS Subjects with an available baseline / reference missing data value of 1 or greater were identified as evaluable as achieving at least a 1-point improvement.

[0372] Based on pre-dose WGGAIS values, at each time point, "success" indicated at least a 1-point improvement in the WGGAIS and "failure" indicated no improvement of at least a 1-point.

[0373] Among the 15 subjects on mFAS1, a total of 20 evaluable pairs of observations were identified for assessment of “success” / “failure” achieving a 1-point improvement on the WGGAIS.

[0374] Table 23 shows the proportion of pairs with a 1-point improvement in WGGAIS per dose at each time point for the 20 evaluable observation pairs in the mFAS1 population.

[0375] At the end of treatment, the proportion of "successful" pairs was 30.00% (6 pairs) for velusetrag and 5.00% (1 pair) for placebo. A higher probability of "success" was observed with velusetrag (Fisher's exact test: p-value = 0.0915; odds ratio estimate from the logistic model = 8.14, 95% CI 0.88; 75.48).

[0376] These data suggest that velusetrag is more effective than placebo in improving the symptoms analyzed, although a placebo effect was detected.

[0377] [Table 39]

[0378] Changes in individual symptom scores Individual symptoms of flatulence, abdominal pain, nausea and vomiting were monitored during dosing.

[0379] Figure 18 shows the mean individual symptom scores by dosing sequence and period from pre-dose to the end of each dosing period for a total of 23 observation pairs in the mFAS1 cohort, taking into account washout / follow-up assessments.

[0380] Of the four symptoms monitored, vomiting showed a statistically significant improvement.

[0381] In subjects receiving velusetrag, the mean emesis 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.

[0382] In subjects receiving placebo, the mean emetic symptom score remained stable at 1.1 ± 1.42 (range: 0; 4) before treatment and 1.1 ± 1.25 (range: 0; 4) at the end of treatment.

[0383] The mean difference between velusetrag and placebo in the change between pre-dose and end-of-dose was -0.8 ± 1.44 (range: -4; 1), indicating a greater decrease with velusetrag (paired t-test: p-value = 0.0164, 95% CI equals -1.4; -0.2; mixed model: p-value = 0.0177, 95% CI equals -1.4; -0.2).

[0384] Similar results were observed when performing the same analyses on the mFAS2 population (24 observation pairs) for individual symptom scores. In particular, for vomiting, a small decrease from pre- to end-of-treatment was observed during velusetrag treatment, whereas scores remained stable during placebo treatment, resulting in a statistically significant difference between treatments.

[0385] Effect on bowel habits (i.e., weekly bowel movements and weekly complete bowel movements) in subjects with constipation Of the 15 subjects in the mFAS1 population, 4 subjects were identified who had constipation with at least one pre-treatment Bristol Stool Scale value of type 1 or 2. Six pairs from these 4 subjects were counted to analyze the effect of treatment on bowel habits, taking into account the number of weekly bowel movements and the number of weekly complete bowel movements.

[0386] In subjects treated with velusetrag, the mean number of weekly bowel movements increased from 3.2 ± 1.72 (range: 1; 6) at baseline to 4.8 ± 2.14 (range: 2; 8) at end of treatment, a mean change of 1.7 ± 2.34 (range: -1; 5). In the same treatment group, the mean number of weekly complete bowel movements increased from 1.5 ± 1.05 (range: 0; 3) at baseline to 2.2 ± 1.17 (range: 1; 4) at end of treatment, a mean change of 0.7 ± 1.21 (range: -1; 2).

[0387] In subjects receiving placebo, the mean number of weekly bowel movements increased from 2.3 ± 1.03 (range: 1; 4) at baseline to 3.2 ± 1.17 (range: 3; 5) at end of treatment, a mean change of 0.8 ± 0.98 (range: 0; 2). In the same treatment group, the mean number of complete weekly bowel movements was 1.0 ± 1.26 (range: 0; 3) at baseline and 1.5 ± 1.05 (range: 0; 3) at end of treatment, a mean change of 0.5 ± 0.84 (range: 0; 2).

[0388] Thus, both treatments increased the number of weekly bowel movements and the number of weekly complete bowel movements, with slightly greater increases for velusetrag.

[0389] FIG. 19 shows line graphs of mean weekly bowel movements and weekly complete bowel movements by dose in the mFAS1 population.

[0390] Stool consistency Subjects in the mFAS1 population were also stratified for stool consistency and classified according to the Bristol Stool Scale as follows: "Constipation" (i.e., Bristol scale type 1 or 2) "Normal" (i.e., Bristol scale type 3 or 4) "Diarrhea" (i.e., Bristol scale types 5, 6, or 7)

[0391] In the mFAS1 population, 23 evaluable pairs were observed.

[0392] The distribution of stool consistency among the 23 observation pairs was similar between velusetrag and placebo at baseline.

[0393] In subjects receiving velusetrag, the frequency of normal stools increased to 26.09% at the end of treatment, while the frequency of constipation decreased to 0% at the end of treatment. Minimal fluctuations were observed in subjects receiving placebo.

[0394] Among subjects receiving velusetrag, all four pairs with constipation at baseline and one of 16 pairs with diarrhea had normal stools at the end of treatment.

[0395] Among the subjects receiving placebo, of 17 pairs with diarrhea at baseline, 2 pairs had normal stools, 2 pairs had constipation, and 13 pairs continued to have diarrhea at the end of treatment. Of the 3 pairs with normal stools at baseline, 1 pair had diarrhea at the end of treatment.

[0396] In conclusion, velsetrag increased the frequency of subjects with normalized stool and reduced the frequency of constipation, while minimal changes were observed with placebo.

[0397] Table 24 shows the shift (change) in stool consistency between end of treatment and pre-treatment for each treatment in the mFAS1 population (23 evaluable observation pairs).

[0398] [Table 40]

[0399] The same analysis was performed on the mFAS2 population, considering 24 pairs. Similar frequency distributions of pairs classified as "constipated," "normal," and "diarrhea" were observed at pre-treatment.

[0400] In subjects receiving velusetrag, 0 pairs, 6 pairs (25.00%), and 18 pairs (75.00%) were classified as "constipated," "normal," and "diarrhea," respectively.

[0401] In subjects receiving placebo, 6 pairs (25.00%), 4 pairs (16.67%), and 14 pairs (58.33%) were classified as "constipated," "normal," and "diarrhea," respectively.

[0402] For this population, a statistically significant difference in stool consistency emerged between pre-treatment and end-of-treatment (p-value=0.0339).

[0403] pseudo-occlusion The assessment of the number of pseudo-occlusive episodes during both the administration and washout periods was carried out considering 15 subjects (30 observation pairs) in mFAS1.

[0404] Pseudo-occlusion occurred once in two pairs (6.67%) in the velusetrag group and once in seven pairs (23.33%) in the placebo group, while it occurred twice in one pair (3.33%) in the velusetrag group. During washout, only one pair (3.33%) experienced one pseudo-occlusion after placebo administration.

[0405] Table 25 shows the distribution of pairs by number of pseudo-occlusions per treatment, where "pseudo-occlusion episodes during treatment" refers to data collected at the "end of treatment" visit and "pseudo-occlusion episodes during washout" refers to data collected at the "start of treatment" visit.

[0406] In conclusion, there were four pseudo-obstructive episodes in the velusetrag group and eight in the placebo group, meaning fewer pseudo-obstructive episodes were reported during velusetrag treatment compared with placebo.

[0407] [Table 41]

[0408] Orocecal transit time Oral-cecal transit time was calculated based on the lactulose breath test (L-BT), which was only performed during the first dosing period of the study.

[0409] In the mFAS1 population, in subjects receiving velusetrag, the mean orocecal transit time was 169.3 ± 60.44 minutes (range: 90 minutes; 240 minutes) at Visit 1 (screening) and 162.9 ± 67.32 minutes (range: 90 minutes; 240 minutes) at Visit 3 (end of treatment -1), resulting in a decrease of -6.4 ± 118.70 minutes (range: -150 minutes; 150 minutes).

[0410] In the same population, in subjects receiving placebo, mean orocecal transit time was 167.5 ± 61.79 minutes (range: 90 minutes; 240 minutes) at visit 1 and 173.6 ± 52.58 minutes (range: 105 minutes; 240 minutes) at visit 3, an increase of 10.0 ± 55.50 minutes (range: −60 minutes; 105 minutes).

[0411] The difference in change between treatments was -16.4 (95% CI: -133.0; 100.1).

[0412] Table 26 shows a summary of oro-cecal transit times by dose, along with the change between post-dose (i.e., end of the first dosing period at Visit 3 (End of Dosing-1)) and pre-dose (i.e., screening value at Visit 1) for the mFAS1 population.

[0413] As a sensitivity analysis, the same analysis was repeated excluding subjects who had taken antibiotics within 2 weeks prior to LB-T. Five subjects and one subject who underwent LB-T but for whom oro-cecal transit time could not be calculated were excluded. Again, a decrease in oro-cecal transit time (minutes) was observed between Visit 3 and Visit 1, with a larger mean change (-63.0 ± 83.79 minutes for velusetrag vs. 18.8 ± 68.60 minutes for placebo; mean change between velusetrag and placebo: -81.8). These data suggest a prokinetic activity of velusetrag.

[0414] [Table 42]

[0415] Effect of discontinuation Considering e-diary data collected during washout or follow-up, changes from the end of treatment were described for identified pairs. Considering the WGGAIS for each symptom, a small mean increase was observed for velusetrag, indicating a worsening of symptoms, while the mean remained stable for placebo. Similarly, for the WGGSAIS total score, the mean change at the end of washout was 0.54 ± 0.736 for velusetrag and -0.06 ± 0.815 for placebo. For placebo, changes in stool consistency were observed: two constipated pairs improved and subsequently returned to normal stools, while four normal pairs worsened to constipation (two pairs) or diarrhea (two pairs). For velusetrag, one diarrheal pair improved and subsequently returned to normal stools, while three normal pairs worsened to constipation (two pairs) or diarrhea (one pair). In subjects with constipation, small decreases from end of treatment to end of washout in both weekly bowel movements and weekly complete bowel movements were observed with velusetrag and placebo, with slightly greater decreases seen with velusetrag.

[0416] Safety evaluation The safety analysis set included 17 subjects, of whom 7 (41.18%) had a total of 29 TEAEs classified as velusetrag-emergent (the last dose taken before the TEAE onset date was velusetrag) and 10 subjects (58.82%) had a total of 38 TEAEs classified as placebo-emergent (the last dose taken before the TEAE onset date was placebo). No deaths, serious TEAEs, or TEAEs leading to treatment discontinuation or interruption were reported.

[0417] Additionally, no administration-related TEAEs were observed, and no cardiovascular adverse reactions were observed.

[0418] Velusetrag was found to be safe and well tolerated in all regimens tested.

Claims

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

2. velusetrag for use according to claim 1, wherein the pharmaceutically acceptable salt is the hydrochloride salt.

3. 3. Velusetrag for use according to claim 1 or 2, wherein the Velusetrag is in a crystalline form and / or a hydrate form.

4. Idiopathic CIPO, neuropathic CIPO, or CIPO secondary to neurodegeneration, secondary to an autoimmune condition, secondary to a connective tissue disorder, or secondary to a demyelinating condition, It may be due to disorders of the autonomic nervous system such as stroke, encephalitis, calcification of basal ganglia, or orthostatic hypotension, It may be due to paraneoplastic syndromes, viral infections, iatrogenic disorders, or diseases of the intestinal wall nervous system such as Hirschsprung's disease, Chagas' disease, or von Recklinghausen's disease, It may be caused by diseases of the intestinal wall muscle layer, such as myotonic dystrophy or progressive systemic sclerosis, Caused by a mixed enteric nervous system and smooth muscle layer disease, such as scleroderma, dermatomyositis, amyloidosis, or Ehlers-Danlos syndrome; or due to unknown mechanisms such as hypothyroidism, hypoparathyroidism, pheochromocytoma, antidepressants, antineoplastic agents, or bronchodilators; or Paraneoplastic diseases (central nervous system tumors, lung microstoma, bronchial carcinoid, leiomyosarcoma, systemic lupus erythematosus) caused by immune-mediated and connective tissue disorders or diseases Velsetrag for use according to any one of claims 1 to 3.

5. Velsetrag for use according to any one of claims 1 to 4, wherein the use is intended for adult or pediatric patients.

6. 6. The method of claim 5, wherein the patient has a history of chronic CIPO or CIPO secondary to a neurodegenerative or demyelinating disease.

7. alleviated and / or ameliorated at least one symptom of CIPO selected from abnormal gastrointestinal motility, increased distension of the proximal colon and / or distal small intestine, modified intestinal contractility, ulcer formation, inflammation of the proximal colon and / or distal small intestine, pseudo-obstructive episodes, vomiting, bloating, abdominal pain, mental health, quality of life, and mortality; and / or Velsetrag for use according to any one of claims 1 to 6 to reduce the number and / or frequency of CIPO-related and / or CIPO-caused hospitalisations.

8. 7. The method of claim 1, wherein velusetrag is administered at a dose of 0.5 to 30 mg / day, preferably 5 to 15 mg / day, more preferably 15 mg / day, based on the weight of the free base.

9. 9. The method of claim 1, wherein the velusetrag is administered for a period of 2 to 12 weeks, preferably 2 to 6 weeks.

10. 10. Velusetrag for use according to any one of claims 1 to 9, wherein the velusetrag is administered orally, parenterally, bucally, sublingually, rectally, intraperitoneally or intratracheally.

11. 11. The method of claim 10, wherein the transdermal administration is by subcutaneous administration, intramuscular administration, intravenous administration, transdermal absorption administration or implantation.

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

13. 11. The method of claim 10, wherein velusetrag is administered parenterally in the form of a liquid, solid or gel.

14. 14. Velusetrag for use according to any one of claims 1 to 13, wherein the Velusetrag is taken orally with or without food, preferably in a single daily dose.

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