Methods for Treating Intestinal Motility Disorders

Abstract

The present disclosure is directed to compounds and compositions for treating intestinal motility disorders, such as, for example, achalasia, Hirschsprung's disease, intestinal pseudo-obstruction, gastroesophageal reflux disease (GERD), functional dysphagia, functional dyspepsia, irritable bowel syndrome (IBS), gastroparesis, functional constipation, functional diarrhea, and fecal incontinence. This summary is intended as a scanning tool for purposes of searching in a particular technical field and is not intended to limit the invention.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 296,151, filed January 3, 2022, pursuant to 35 U.S.C. 119(e), which is incorporated by reference in its entirety.

[0002] Statement regarding federally funded research This invention was made with Government support under Grant Nos. DP2NS116769 and R01CA240984 awarded by the National Institutes of Health (NIH) and Grant No. R01DK121169 awarded by the National Institute of Diabetes and Digestive and Kidney Diseases. The Government has certain rights in this invention. [Background technology]

[0003] background The enteric nervous system (ENS) is the largest and most complex division of the autonomic nervous system (De Giorgio, 2006). Over 500 million enteric neurons and approximately seven times as many enteric glia form interconnected enteric ganglia embedded in two distinct layers within the intestinal wall: the myenteric plexus, located between the longitudinal and circular muscles, and the submucosal plexus, located between the circular muscle and the mucosa (Grubisic and Gulbransen, 2017; Grundmann et al., 2019; Hamnett et al., 2021; Sasselli et al., 2012).

[0004] The ENS does not depend on input from the central nervous system (CNS) to govern the function of the gastrointestinal (GI) tract (Furness et al., 2014). This autonomy is exemplified by studies in which gut segments removed from the body continue to generate complex motor patterns ex vivo. ENS autonomy is the result of highly diverse neuronal and glial cell types with distinct neurochemical signatures that work together in unison (Brehmer, 2021; Qu et al., 2008; Fung and Vanden Berghe, 2020). Thus, the ENS is equipped to control complex gut functions, including motility, secretion, absorption, blood flow regulation, and support of barrier function. Furthermore, the ENS extrinsically communicates with the CNS, enteroendocrine system, immune system, and gut microbiota to maintain vitality and proper intestinal homeostasis ( Furness et al., 2014 , Long-Smith et al., 2020 , Muller et al., 2014 , Obata and Pachnis, 2016 , Schneider et al., 2019 , Yoo and Mazmanian, 2017 ).

[0005] Although the neurochemical and functional complexity of the ENS is similar to the CNS (Gershon, 1999), progress in the field of ENS research has been much slower. Despite being the largest and most complex section of the peripheral nervous system and playing a central role in the development and progression of enteric neuropathies and gut-brain axis diseases, ENS research has been unduly impacted by multiple long-standing technical challenges. For example, enteric neurons are diluted throughout the GI tract, constituting less than 1% of intestinal tissue (Drokhlyansky et al., 2020). Thus, to access ENS tissue, scientists must rely on samples collected during GI resection surgery rather than more routine GI biopsies. Furthermore, it is difficult to isolate the ENS without significant sampling bias associated with harsh tissue dissociation techniques that damage fragile neurites, and we lack reliable surface markers suitable for FACS purification of enteric neurons and glia.

[0006] The complex developmental process and elaborate cellular structure of the ENS, as well as its remarkable communication with the rest of the body, provide a wide range of possibilities for abnormalities to occur. Enteric neuropathy, also known as disorders of gut-brain interaction (DGBI), including some of the most challenging clinical disorders, result from the loss, degeneration, or dysfunction of ENS cell types (De Giorgio et al., 2016; Niesler et al., 2021). This incomplete understanding of ENS development and function is responsible for the long-term morbidity and mortality of GI diseases, as well as the limited availability of therapeutic interventions. Specifically, there is extraordinary interest in gaining a better understanding of intestinal nitrergic neurons (NO neurons), which release nitric oxide (NO) to relax smooth muscle tissue and promote GI motility. This is due to selective dysfunction and degeneration of NO neurons in different forms of DGBI, including esophageal atachalasia, infantile hypertrophic pyloric stenosis, and gastroparesis (Bodi et al., 2019; Rivera et al., 2011). Thus, there remains a need for compounds and compositions that can modulate NO neuronal activity, and methods of making and using them. Summary of the Invention

[0007] overview In accordance with the object(s) of the disclosure, as embodied and broadly described herein, the disclosure relates in some embodiments to compounds and compositions useful for treating intestinal motility disorders, such as, for example, atlantasia, Hirschsprung's disease, intestinal pseudo-obstruction, gastroesophageal reflux disease (GERD), functional dysphagia, functional dyspepsia, irritable bowel syndrome (IBS), gastroparesis, functional constipation, functional diarrhea, and fecal incontinence.

[0008] Accordingly, provided herein is a method of treating an intestinal motility disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of carprofen, mefenamic acid, phenacetin, valdecoxib, fenoldopam mesylate, fluphenazine hydrochloride, bupivacaine HCl, phenazopyridine HCl, alverine citrate, nitenpyram, 4-aminobutyric acid (GABA), PF-3845, esmolol HCl, cimetidine, conivaptan HCl, (+)-MK-801, maleate, MK-0752, R04929097, rosmarinic acid, theophylline, aripiprazole, flopropion, latrepirdine 2HCl, ADX-47273, MPEP, nefopam HCl, phenazopyridine HCl, epinephrine, (+)-matrine, phenothiazines, naproxen sodium, AMG-517, isoliquiritigenin, nilvadipine, prednisone, and simvastatin, or a pharma- ceutically acceptable salt thereof.

[0009] In some embodiments, the compound is selected from aripiprazole, dexmedetomidine, matrine, and MPEP, or a pharma- ceutically acceptable salt thereof. In some embodiments, the compound is dexmedetomidine. In some embodiments, the intestinal motility disorder is selected from achalasia, Hirschsprung's disease, intestinal pseudo-obstruction, gastroesophageal reflux disease (GERD), functional dysphagia, functional dyspepsia, irritable bowel syndrome (IBS), gastroparesis, functional constipation, functional diarrhea, and fecal incontinence. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject has been diagnosed with a need for treatment of an intestinal motility disorder prior to the administering step. In some embodiments, the method further comprises identifying a subject in need of treatment of an intestinal motility disorder. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is a prophylactically effective amount.

[0010] Also provided is a method of modulating NO neuron activity in a subject having a gut motility disorder, the method comprising administering to the subject an effective amount of carprofen, mefenamic acid, phenacetin, valdecoxib, fenoldopam mesylate, fluphenazine hydrochloride, bupivacaine HCl, phenazopyridine HCl, alverine citrate, nitenpyram, 4-aminobutyric acid (GABA), PF-3845, esmolol HCl, cimetidine, conivaptan HCl, (+)-MK-801 maleate, The method includes administering a compound selected from MK-0752, R04929097, rosmarinic acid, theophylline, aripiprazole, flopropion, latrepirdine 2HCl, ADX-47273, MPEP, nefopam HCl, phenazopyridine HCl, epinephrine, (+)-matrine, phenothiazines, naproxen sodium, AMG-517, isoliquiritigenin, nilvadipine, prednisone, and simvastatin, or a pharma- ceutically acceptable salt thereof.

[0011] In some embodiments, the compound is selected from aripiprazole, dexmedetomidine, matrine, and MPEP, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is dexmedetomidine. In some embodiments, modulating NO neuronal activity induces colon activity.

[0012] Also provided are Carprofen, Mefenamic Acid, Phenacetin, Valdecoxib, Fenoldopam Mesylate, Fluphenazine Hydrochloride, Bupivacaine HCl, Phenazopyridine HCl, Alverine Citrate, Nitenpyram, 4-Aminobutyric Acid (GABA), PF-3845, Esmolol HCl, Cimetidine, Conivaptan HCl, (+)-MK-801 Maleate, MK-0752, R04929097, Rosmarinic Acid, Theophylline, Aripiprazole, Flopropion, Latrepirdine 2HCl, A kit comprising a compound selected from ADX-47273, MPEP, nefopam HCl, phenazopyridine HCl, epinephrine, (+)-matrine, phenothiazine, naproxen sodium, AMG-517, isoliquiritigenin, nilvadipine, prednisone, and simvastatin, and one or more selected from (a) an agent known to treat an intestinal motility disorder, (b) instructions for treating an intestinal motility disorder, and (c) instructions for administering the compound in association with treating an intestinal motility disorder.

[0013] In some embodiments, the agent is selected from a parasympathomimetic agent, a prokinetic agent, an opioid antagonist, an antidiarrheal, and an antibiotic. In some embodiments, the agent is selected from neostigmine, bethanechol, metoclopramide, cisapride, and loperamide. In some embodiments, the compound and agent are co-packaged. In some embodiments, the compound and agent are co-formulated.

[0014] Further objects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description. Only the preferred embodiment has been shown and described herein, which is merely an illustration of the best mode. As will be realized, the present disclosure is capable of other different embodiments, and its several details are capable of modification in various obvious respects, without departing from the present disclosure. The description is therefore to be regarded as illustrative in nature, and not as restrictive. [Brief description of the drawings]

[0015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects and, together with the description, serve to explain the principles of the present disclosure.

[0016] [Figure 1A] Figures 1A-H show representative data illustrating the identification of enteric NO neuron modulators by functional high-throughout screening. Specifically, Figure 1A shows flow cytometry quantification of stage 2 enteric ganglioid cFOS expression in response to epinephrine. Mean and SEM error bars are shown. ****: p-value <0.0001. Figures 1B-C show multi-electrode array (MEA) analysis (Figure 1B) and quantification of changes in neuronal firing (Figure 1C) in response to epinephrine in stage 1 enteric ganglioid NO neurons. Figure 1D shows the identification of candidate neuromodulators that induce cFOS expression in stage 2 enteric ganglioid NO neurons by HTS. Figure 1E shows the identification of candidate neuromodulators that induce NO release in stage 1 2D ENS cultured NO neurons by HTS. Figure 1F shows the predicted targets and FDA approval status of hits identified in the cFOS induction and NO release screen. Figure 1G shows snRNA-seq analysis violin plots of module scoring for all predicted NO neuronal activity driving receptor genes in stage 1 gut ganglioid NO subtypes compared to other neurons. Figure 1H shows snRNA-seq analysis dot plots of average module scores for individual categories of NO neuronal activity driving receptor gene categories. [Figure 1B] Please see the legend to FIG. 1A. [Figure 1C] Please see the legend to FIG. 1A. [Figure 1D] Please see the legend to FIG. 1A. [Figure 1E] Please see the legend to FIG. 1A. [Figure 1F] Please see the legend to FIG. 1A. [Figure 1G] Please see the legend to FIG. 1A. [Figure 1H]Please see the legend to FIG. 1A. [Figure 2A]Figures 2A-N show representative data illustrating that NO neuron-specific functional screens identify regulators of colonic motility. Specifically, Figure 2A shows a schematic of a high-throughput flow cytometry-based screen to identify compounds that induce cFOS expression in hESC-derived stage 2 enteric ganglioid NO neurons. Figure 2B shows the target classes of hits identified in the enteric NO neuron cFOS induction screen (Figure 1D, red dots). Figure 1C shows a schematic of a high-throughput calorimetry-based screen to identify compounds that induce NO release in hESC-derived stage 1 2D ENS cultures. Figure 2D shows the target classes of hits identified in the NO release screen (Figure 1E, red dots). Protein classes in common with (Figure 2B) are indicated with asterisks. Figure 1E shows feature plots showing predicted responsiveness of subclustered stage 1 ganglioid enteric NO neurons to neurotransmitters by module scoring of neurotransmitter receptor gene families. FIG. 2F shows the expression of genes belonging to the target classes shown in FIG. 2B and FIG. 2D in subtypes of hESC-derived stage 1 and primary human intestinal nitrergic neurons compared to all other neurons. FIG. 2G shows the combined protein target analysis for selected screening hits showing the shared protein classes. The color code matches the target classes in FIG. 2B and FIG. 2D. FIG. 2H shows a schematic diagram for testing the effect of selected candidate hits (listed in (FIG. 2G)) on mouse colonic motility ex vivo. FIG. 2I shows a representative spatiotemporal map of mouse colonic contraction along the proximal-distal axis over a 10 min period. FIG. 2J shows the quantification of colonic migrating motor complex (CMMC) intervals (FIG. 3A) at the 75th percentile of CMMC cumulative percentage for selected hit compounds. FIG. 2K shows the experimental design for measuring the effect of selected candidate hits on mouse colonic motility ex vivo. Representative spatiotemporal map of mouse colonic contraction along the proximal-distal axis over a 26 min period. Three representative longitudinal contraction events (LCEs) are shown per condition (arrows).Figure 2L shows a diagram of quantification of CMMC cumulative percentiles and CMMC intervals (time difference between two consecutive contractions) at the 75th percentile of dexmedetomidine. Mean and SEM error bars for five pairs of untreated and drug-treated mouse colons are shown. Figure 2M shows the total number of colonic longitudinal contraction events (LCEs) within each 6-minute treatment condition for five dexmedetomidine-treated mouse colons measured from spatiotemporal maps. *: p-value < 0.05. Figure 2N shows the average of LCE durations calculated for three LCEs within each 6-minute treatment (one at the beginning, one in the middle, and one at the end of each spatiotemporal map; see Figure 2K). Data are shown for five dexmedetomidine-treated mouse colons. SEM error bars are shown. [Figure 2B] Please see the legend to FIG. 2A. [Figure 2C] Please see the legend to FIG. 2A. [Figure 2D] Please see the legend to FIG. 2A. [Figure 2E] Please see the legend to FIG. 2A. [Figure 2F] Please see the legend to FIG. 2A. [Figure 2G] Please see the legend to FIG. 2A. [Figure 2H] Please see the legend to FIG. 2A. [Figure 2I] Please see the legend to FIG. 2A. [Figure 2J] Please see the legend to FIG. 2A. [Figure 2K] Please see the legend to FIG. 2A. [Figure 2L] Please see the legend to FIG. 2A. [Figure 2M] Please see the legend to FIG. 2A. [Figure 2N] Please see the legend to FIG. 2A. [Figure 3A]Figures 3A-C show representative data illustrating testing of selected HTS hits in an ex vivo mouse colonic motility assay. Specifically, Figures 3A-B show plots of cumulative percentiles of CMMC (Figure 3A) and slow waves (Figure 3B). Figure 3C shows quantification of the latency during slow waves at the 75th percentile for selected HTS hits. From left: aripiprazole, dexmedetomidine, latrepirdine, matrine, MPEP, and naproxen. Unidirectional SEM error bars are shown in Figures 3A-B. [Figure 3B] Please see the legend to FIG. 3A. [Figure 3C] Please see the legend to FIG. 3A. [Figure 4A] Figures 4A-F show representative data illustrating the effect of candidate drugs on mouse colonic migratory motor complexes (CMMCs) ex vivo. Specifically, Figures 4A-F show plots of CMMC cumulative percentiles and quantification of CMMC intervals at the 75th percentile for aripiprazole (Figures 4A-B), matrine (Figures 4C-D), and MPEP (Figures 4E-F). Compounds were used at 1 μM, and the mean and unidirectional SEM error bars are shown in Figures 4A, 4C, and 4E. [Figure 4B] Please see the legend to FIG. 4A. [Figure 4C] Please see the legend to FIG. 4A. [Figure 4D] Please see the legend to FIG. 4A. [Figure 4E] Please see the legend to FIG. 4A. [Figure 4F] Please see the legend to FIG. 4A. [Figure 5A] Figures 5A-B show representative data illustrating the effect of candidate drugs on ex vivo mouse colon CMMC and slow wave intervals at the 75th percentile. Specifically, Figures 5A-B show quantification of CMMC (Figure 5A) and slow wave (Figure 5B) intervals (time difference between two successive contractions) at the 75th percentile for dexmedetomidine, aripiprazole, matrine, and MPEP. In each panel, the top row (blue) shows the control colon and the bottom row (red) shows the treated colon within each pair. Compounds were used at 1 μM. [Figure 5B] Please see the legend to FIG. 5A. [Figure 6A] Figures 6A-H show representative data illustrating the effect of candidate drugs on mouse colonic slow waves ex vivo. Specifically, Figures A-H show quantification of slow wave cumulative percentiles and latencies between slow waves at the 75th percentile for aripiprazole (Figures 6A-B), dexmedetomidine (Figures 6C-D), matrine (Figures 6E-F), and MPEP (Figures 6G-H). Compounds were used at 1 μM, and mean and unidirectional SEM error bars are shown in Figures 6A, 6C, 6E, and 6G. [Figure 6B] Please see the legend to FIG. 6A. [Figure 6C] Please see the legend to FIG. 6A. [Figure 6D] Please see the legend to FIG. 6A. [Figure 6E] Please see the legend to FIG. 6A. [Figure 6F] Please see the legend to FIG. 6A. [Figure 6G] Please see the legend to FIG. 6A. [Figure 6H] Please see the legend to FIG. 6A. [Figure 7A]Figures 7A-P show representative data illustrating that PDGFR inhibition promotes enteric NO neuron induction. Specifically, Figure 7A shows a schematic of a high-throughput pharmacological screen to identify compounds that enrich for NO neurons in hESC-derived 2D ENS cultures. Figure 7B shows combined protein target analysis of the HTS top 12 hits, showing shared protein classes between structurally similar hits. Figure 7C shows the effect of PP121 treatment duration on NOS1::GFP induction efficiency. Figure 7D shows immunofluorescence staining of NOS1 and neuronal TUBB3 in stage 1 enteric gangliodes treated with or without PP121 from day 15-20. Figure 7E shows split UMAP of cell types present in stage 1 control (top) and PP121-treated (bottom) enteric gangliodes cultures. Figure 7F shows dot plots of mean module scores of control-only enteric gangliodes subtype transcriptional signatures in PP121-treated gangliodes subtypes. FIG. 7G shows the splut UMPA of neuronal subtypes present in stage 1 control (top) and PP121-treated (bottom) intestinal ganglioid cultures. FIG. 7H shows a dot plot of the mean module score of the control-only neuronal subtype transcriptional signature in PP121-treated ganglioid neuronal subtypes. FIG. 7I shows the distribution of NO neuronal subtypes in control compared to PP121-treated stage 1 intestinal ganglioid cultures. FIG. 7J shows the splut UMAP of the subclustered NO subtypes present in stage 1 control (top) and PP121-treated (bottom) intestinal ganglioid cultures. FIG. 7K shows a dot plot of the mean module score of the control-only NO neuronal subtype transcriptional signature in PP121-treated ganglioid NO neuronal subtypes. FIG. 7L shows feature plots showing the expression of ERBB, PDGFR, and VEGFR in D15 subclustered intestinal crestspheres. FIG. 7M shows a schematic diagram of receptor tyrosine kinase (RTK) natural agonists and selected pharmacological antagonists, including the top hit PP121, which enriches for NO neurons.Figure 7N shows the effect of RTK ligand treatment on stage 1 enteric ganglioside NO neuron induction. Figures 7O and 7P show the effect of knockout of PDGFRA (Figure 7O) and PDGFRB (Figure 7P) in D15 enteric crestspheres on stage 1 enteric ganglioside NO neuron enrichment as measured by flow cytometry. [Figure 7B] Please see the legend to FIG. 7A. [Figure 7C] Please see the legend to FIG. 7A. [Figure 7D] Please see the legend to FIG. 7A. [Figure 7E] Please see the legend to FIG. 7A. [Figure 7F] Please see the legend to FIG. 7A. [Figure 7G] Please see the legend to FIG. 7A. [Figure 7H] Please see the legend to FIG. 7A. [Figure 7I] Please see the legend to FIG. 7A. [Figure 7J] Please see the legend to FIG. 7A. [Figure 7K] Please see the legend to FIG. 7A. [Figure 7L] Please see the legend to FIG. 7A. [Figure 7M] Please see the legend to FIG. 7A. [Figure 7N] Please see the legend to FIG. 7A. [Figure 7O] Please see the legend to FIG. 7A. [Figure 7P] Please see the legend to FIG. 7A. [Figure 8A]Figures 8A-D show representative data illustrating that small molecule high throughput screening identifies compounds that enrich for NO neurons in hESC-derived ENS cultures. Specifically, Figure 8A shows the identification of candidate compounds that enrich for NO neurons in stage 1 2D ENS cultures. Figure 8B shows HTS hits with a greater than 8.0-fold increase in the percentage of NOS1+ cells. Figures 8C and 8D show that PP121 induction of NOS1 expression is dose and time dependent. Representative flow diagram showing NOS1 expression in stage 1 enteric neurons when cultures were treated with different PP121 concentrations at D15-D20 (Figure 8C) or with 2 μM PP121 over different time frames (Figure 8D). [Figure 8B] Please see the legend to FIG. 8A. [Figure 8C] Please see the legend to FIG. 8A. [Figure 8D] Please see the legend to FIG. 8A. [Figure 9-1] Figures 9A-F show representative data illustrating that PP121 treatment enriches for NO neurons without affecting the overall cellular diversity of neurons. Specifically, Figure 9A shows the distribution of cell types in control compared to PP121-treated stage 1 enteric ganglioid cultures. Figure 9B shows the correlation of mean gene expression in matched controls compared to PP121-treated enteric ganglioid cell types. Figure 9C shows the distribution of neuronal subtypes in control compared to PP121-treated stage 1 enteric ganglioid cultures. Figure 9D shows the correlation of mean gene expression in matched controls compared to PP121-treated enteric ganglioid neuronal subtypes. Figure 9E shows the distribution of NO neuronal subtypes in control compared to PP121-treated stage 1 enteric ganglioid cultures. Figure 9F shows the correlation of mean gene expression in matched controls compared to PP121-treated enteric ganglioid NO neuronal subtypes. [Figure 9-2] Please refer to the description of Figure 9-1. [Figure 9-3] Please refer to the description of Figure 9-1. [Figure 10A] Figures 10A-E show representative data illustrating that human surface marker antibody screening identifies NO neuron-specific surface markers. Specifically, Figure 10A shows a schematic of using enriched enteric NO neuron cultures to identify their specific surface markers by flow cytometry-based high-throughput antibody screening. Figure 10B shows surface markers expressed in at least 50% of NOS1+ cells, i.e., showing greater than 50% sensitivity (top), filtered based on their specificity for NOS1+ cells (at least 70% of all stained cells were NOS1+, i.e., greater than 70% specificity, intermediate). Figure 10C shows the surface marker screen top hits with the highest specificity and sensitivity for identifying enteric NO neurons. Figure 10D shows violin plot stacks showing expression (left) and scaled mean expression (right) of surface marker screen hits in control and PP121-treated NO neuron subtypes compared to other neurons. Figure 10E shows violin plot stacks showing expression of surface marker screen hits in adult human NO neuron subtypes compared to other neurons. FIG. 10F shows immunofluorescence analysis of the expression of neuronal markers TUBB3, NOS1, and CD47 in adult human primary colonic tissue. Myenteric plexus ganglions are shown. Arrows indicate colocalization of CD47 and NOS1. FIG. 10G shows surface markers that stained at least 70% of all cells as potential pan-ENS cell markers. FIG. 10H shows CD24 expression in stage 1 enteric ganglioid clusters in PP121-treated and untreated samples. FIG. 10I shows a representative immunofluorescence analysis of CD24 and neuronal marker TUBB3 expression in human primary colonic sections showing myenteric plexus ganglions. [Figure 10B] Please see the legend to FIG. 10A. [Figure 10C] Please see the legend to FIG. 10A. [Figure 10D] Please see the legend to FIG. 10A. [Figure 10E]Please see the legend to FIG. 10A. [Figure 11A] Figures 11A-C show representative data illustrating extensive engraftment of hESC-derived ganglioids in adult mouse colon. Specifically, Figure 11A shows a schematic showing transplantation of hESC-derived stage 1 intestinal ganglioids into mouse proximal colon. Figure 11B shows engraftment of hESC-derived stage 1 intestinal ganglioid cells throughout the entire length of the mouse colon, as indicated by expression of the human cytoplasmic marker SC121 in red. Figure 11C shows immunohistochemical analysis of human cytoplasmic protein SC121, and NO neuronal marker NOS1, in Nos1- / - mouse colon 8 weeks after transplantation. [Figure 11B] Please see the legend to FIG. 11A. [Figure 11C] Please see the legend to FIG. 11A. [Figure 12] Representative data are shown illustrating hESC-derived intestinal gangliosides engrafted in adult mouse colon. Specifically, immunohistochemistry analysis of neuronal TUBB3, human cytoplasmic protein SC121, and NO neuronal marker NOS1 in Nos1- / - mouse colon (not treated with cyclosporine A, not manipulated, top) 8 weeks after transplantation (bottom). Purple arrows point to NOS1+ cells.

[0017] Additional advantages of the present disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the present disclosure. The advantages of the present disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure as claimed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description The present invention may be understood more readily by reference to the following detailed description of the disclosure and the examples included therein.

[0019] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods, unless otherwise specified, or to specific reagents, unless otherwise specified, as such, can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure, exemplary methods and materials are now described.

[0020] Although embodiments of the present invention may be described and claimed in a particular legal class, such as a system legal class, this is for convenience only, and one of ordinary skill in the art will understand that each embodiment of the present invention may be described and claimed in any legal class. Unless expressly stated otherwise, it is not intended that any method or embodiment described herein be interpreted as requiring that its steps be performed in a particular order. Thus, unless specifically set forth by a method claim in the claims or description that the steps should be limited to a particular order, no order is intended to be imposed in any respect. This also applies to any possible implicit criteria of interpretation, including logical matters regarding the organization of steps or workflows, general meanings derived from grammatical organization or punctuation, or the number or type of embodiments described herein.

[0021] Throughout this application, various publications are referenced. The disclosures of these publications are incorporated by reference into this application in order to more fully describe the state of the art to which this specification pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior invention. Further, the dates of publications provided herein may be different from the actual publication dates and may require independent confirmation.

[0022] A.Definition Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification, unless limited in specific instances either individually or as part of a larger group.

[0023] As used herein, the term "a" or "an" means "at least one" or "one or more," unless the context dictates otherwise. The term "and / or" as used in the present specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements, whether related or unrelated to those specifically identified elements, may optionally be present, other than the elements specifically identified, unless there is a clear indication to the contrary. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," can refer in various embodiments to A without B (optionally including elements other than B), in other embodiments to B without A (optionally including elements other than A), and in yet other embodiments to both A and B (optionally including other elements).

[0024] The term "or" as used herein should only be construed as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by the terms of exclusivity, "either," "one of," "only one of," or "exactly one of."

[0025] As used herein, the terms "comprising" (and any form of including, such as "comprise," "comprises," and "comprised"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0026] As used herein, the term "about" means that the numerical values ​​are approximate and that small variations would not significantly affect the practice of the disclosed embodiments. When numerical limitations are used, unless otherwise indicated by context, "about" means that the numerical values ​​can vary by ±10% and remain within the scope of the disclosed embodiments.

[0027] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulas set forth herein are constructed according to the standard rules of chemical valency known to the chemical arts.

[0028] References in this specification and in the concluding claims to parts by weight of a particular element or component in a composition indicate the weight relationship between the element or component and any other elements or ingredients in the composition or article for which the parts by weight are expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0029] Weight percentages (wt %) of components are based on the total weight of the formulation or composition in which the component is included, unless otherwise specified.

[0030] As used herein, the term "optional" or "optionally" means that the event or circumstance described thereafter may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not occur.

[0031] As used herein, the term "diagnosed" means that a subject has undergone a physical examination by a person skilled in the art, e.g., a physician, and has been found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. In some embodiments of the disclosed methods, the subject has been diagnosed with a need for treatment of a disorder associated with NO neuron activity, e.g., gut motility disorder, prior to the administration step. As used herein, phrases such as "identified as in need of treatment for a disorder" refer to selecting a subject based on the need for treatment of the disorder. It is contemplated that the identification, in some embodiments, can be performed by a person different from the person making the diagnosis. In further embodiments, it is also contemplated that the administration can be performed by the person who subsequently performed the administration.

[0032] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, intraocular administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injections such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration may be continuous or intermittent. In various embodiments, the preparation may be administered therapeutically, i.e., administered to treat an existing disease or condition. In further various embodiments, the preparation may be administered prophylactically, i.e., administered for the prevention of a disease or condition.

[0033] As used herein, the term "contacting" refers to bringing together a disclosed compound and a cell, target receptor, or other biological entity in such a way that the compound can affect the activity of the target (e.g., a receptor, a cell, etc.) either directly (i.e., by interacting with the target itself) or indirectly (i.e., by interacting with another molecule, cofactor, factor, or protein on which the activity of the target depends).

[0034] As used herein, "IC 50 " is intended to refer to the concentration of a substance (e.g., a compound or drug) required for 50% inhibition of a biological process or a component of a process, including proteins, subunits, organelles, ribonucleoproteins, etc. In some embodiments, the IC 50 may refer to the concentration of a substance required for 50% inhibition in vivo, as further defined elsewhere herein.

[0035] As used herein, "EC 50" is intended to refer to the concentration of a substance (e.g., a compound or drug) that produces a half-maximal response (i.e., 50% of the maximal response) of a biological process or component of a process, including proteins, subunits, organelles, ribonucleoproteins, etc. In some embodiments, EC 50 may refer to the concentration of a substance required to achieve 50% of the maximal response in vivo, as further defined elsewhere herein.

[0036] Compounds according to the present disclosure may form prodrugs at hydroxyl or amino functionalities using groups such as alkoxy, amino acids, etc. as prodrug-forming moieties. For example, hydroxymethyl positions may form monophosphates, diphosphates, or triphosphates, and these phosphates may also form prodrugs. The preparation of such prodrug derivatives is discussed in various literature sources (examples include: Alexander et al., J. Med. Chem. 1988, 31, 318; Aligas-Martin et al., PCT WO2000 / 041531, p. 30). The nitrogen functional group that is converted in preparing these derivatives is one (or more) of the nitrogen atoms of the compounds of the present disclosure.

[0037] The "derivatives" of the compounds disclosed herein are pharmaceutically acceptable salts, prodrugs, deuterated forms, radiolabeled forms, isomers, solvates, and combinations thereof. In this context, the term "combination" refers to derivatives that fall into at least two of the following groups: pharmaceutically acceptable salts, prodrugs, deuterated forms, radiolabeled forms, isomers, and solvates. Examples of radiolabeled forms include compounds labeled with tritium, phosphorus-32, iodine-129, carbon-11, fluorine-18, and the like.

[0038] The term "leaving group" refers to an atom (or group of atoms) with electron-withdrawing capability that can take up and replace bonding electrons as a stable species. Examples of suitable leaving groups include sulfonate esters, including triflates, mesylates, tosylates, brosylates, and halides.

[0039] As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad embodiment, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, for example, those described below. The permissible substituents can be one or more of the same or different suitable organic compounds. For purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents, and / or any permissible substituent of organic compounds described herein that satisfies the valence of the heteroatom. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. The terms "substituted" or "substituted with" also include the implicit proviso that such substitution is subject to the permissible valence of the substituted atom and substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like. Also, in some embodiments, unless expressly stated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or not substituted).

[0040] In defining various terms, 1 ","A 2 ","A 3 " and "A 4 " are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, including but not limited to those disclosed herein, and where in one instance they are defined to be certain substituents, in another instance they may be defined as several other substituents.

[0041] The terms "halo" and "halogen" as used herein refer to an atom selected from fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), and iodine (iodine, -I).

[0042] The terms "aliphatic" or "aliphatic group," as used herein, refer to a hydrocarbon moiety that may be straight-chained (i.e., unbranched), branched, or cyclic (including fused, bridged, and spiro-fused polycyclic), may be fully saturated, or may contain one or more units of unsaturation, but is not aromatic. Unless otherwise specified, aliphatic groups contain 1-20 carbon atoms. Aliphatic groups include, but are not limited to, linear or branched alkyl, alkenyl, and alkynyl groups, as well as hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0043] As used herein, the term "alkyl" refers to a monovalent, saturated, straight or branched chain hydrocarbon radical having 1 to 6 carbon atoms, unless otherwise specified. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, n-pentyl, tert-pentyl, neopentyl, sec-pentyl, 3-pentyl, sec-isopentyl, hexyl, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, 2,3-dimentivutane, and the like. Alkyl groups can be substituted or unsubstituted. For example, alkyl groups can be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, or thiol, as described herein. A "lower alkyl" group is an alkyl group containing 1 to 6 (e.g., 1 to 4) carbon atoms. The term alkyl group may be C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, etc., up to and including C1-C24 alkyl.

[0044] Throughout this specification, "alkyl" is used generally to refer to both unsubstituted and substituted alkyl groups, however, substituted alkyl groups are also specifically referred to herein by identifying the particular substituent(s) on the alkyl group. For example, the term "halogenated alkyl" or "haloalkyl" specifically refers to an alkyl group substituted with one or more halides, such as fluorine, chlorine, bromine, or iodine. Alternatively, the term "monohaloalkyl" specifically refers to an alkyl group substituted with a single halide, such as fluorine, chlorine, bromine, or iodine. The term "polyhaloalkyl" specifically refers to an alkyl group independently substituted with two or more halides, i.e., each halide substituent need not be the same halide as another halide substituent, and the multiple instances of the halide substituent need not be on the same carbon. The term "alkoxyalkyl" specifically refers to an alkyl group substituted with one or more alkoxy groups, as described below. The term "aminoalkyl" specifically refers to an alkyl group substituted with one or more amino groups. The term "hydroxyalkyl" specifically refers to an alkyl group substituted with one or more hydroxy groups. If "alkyl" is used in one instance and a specific term such as "hydroxyalkyl" is used in another instance, this does not mean that the term "alkyl" does not refer to the specific term such as "hydroxyalkyl."

[0045] This practice is also used for other groups described herein. That is, a term such as "cycloalkyl" refers to both unsubstituted and substituted cycloalkyl moieties, but in addition, substituted moieties can be specifically identified herein, e.g., a particular substituted cycloalkyl can be referred to as, e.g., an "alkylcycloalkyl". Similarly, a substituted alkoxy can be specifically referred to as, e.g., a "halogenated alkoxy", a particular substituted alkenyl can be, e.g., an "alkenylalcohol", etc. Again, the practice of using a general term such as "cycloalkyl" and a specific term such as "alkylcycloalkyl" does not mean that the general term does not also include the specific term.

[0046] The term "alkenyl" as used herein refers to a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon double bond. 1 A 2 )C=C(A 3 A 4 Asymmetric structures such as , are intended to include both the E and Z isomers. This can be inferred in structural formulas herein where an asymmetric alkene is present or is explicitly indicated by the bond symbol C=C. Alkenyl groups can be substituted with one or more groups as described herein, including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfoxo, or thiol.

[0047] The term "alkynyl" as used herein is a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon triple bond. Alkynyl groups can be unsubstituted or substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfoxo, or thiol, as described herein.

[0048] As used herein, the term "heteroalkyl" refers to an alkyl group containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, with nitrogen, phosphorus, and sulfur atoms being optionally oxidized, and nitrogen heteroatoms being optionally quaternized. Heteroalkyl can be substituted as defined above for alkyl groups.

[0049] The term "haloalkyl" includes mono-, poly-, and perhaloalkyl groups, where the halogens are independently selected from fluorine, chlorine, bromine, and iodine.

[0050] "Alkoxy" is an alkyl group attached to another moiety via an oxygen linker (-O(alkyl)). Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy.

[0051] "Haloalkoxy" includes, but is not limited to, -OCHCF 2 Or -OCF 3 and the like. A haloalkyl group is attached to another moiety through an oxygen atom, such as:

[0052] The term "9-10 membered carbocyclyl" refers to a 9- or 10-membered monocyclic, bicyclic (e.g., bridged or spiro bicyclic ring), polycyclic (e.g., tricyclic), or fused hydrocarbon ring system that is saturated or partially unsaturated. The term "9- to 10-membered carbocyclyl" also includes saturated or partially unsaturated hydrocarbon rings that are fused to one or more aromatic or partially saturated hydrocarbon rings (e.g., dihydroindenyl and tetrahydronaphthalenyl). Bridged bicyclic cycloalkyl groups include, but are not limited to, bicyclo[4.3.1]decanyl, and the like. Spiro bicyclic cycloalkyl groups include, for example, spiro[3.6]decanyl, spiro[4.5]decanyl, spiro[4.4]nonyl, and the like. Fused cycloalkyl rings include, for example, decahydronaphthalenyl, dihydroindenyl, decahydroazulenyl, octahydroazulenyl, tetrahydronaphthalenyl, and the like. It will be understood that, when specified, optional substituents on a carbocyclyl (e.g., in the case of an optionally substituted cycloalkyl) may be present at any substitutable position, including, for example, the position at which the carbocyclyl group is attached.

[0053] The term "cycloalkyl" as used herein is a non-aromatic carbon-based ring of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, and the like. The term "heterocycloalkyl" is a type of cycloalkyl group as defined above and is included within the meaning of the term "cycloalkyl", in which at least one of the ring carbon atoms is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkyl and heterocycloalkyl groups can be substituted or unsubstituted. Cycloalkyl and heterocycloalkyl groups can be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, or thiol, as described herein. In various aspects, cycloalkyl and heterocycloalkyl groups can be monocyclic, bicyclic (e.g., bridged, such as bicyclo[4.3.1]decanyl, or spiro, such as spiro[3.6]decanyl, spiro[4.5]decanyl, spiro[4.4]nonyl, etc.), polycyclic (e.g., tricyclic), or fused hydrocarbon ring systems that are saturated or partially unsaturated (e.g., decahydronaphthalenyl, dihydroindenyl, decahydroazulenyl, octahydroazulenyl, tetrahydronaphthalenyl).

[0054] The term "cycloalkenyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bond, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term "heterocycloalkenyl" is a type of cycloalkenyl group defined above and is included within the meaning of the term "cycloalkenyl", in which at least one of the ring carbon atoms is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkenyl and heterocycloalkenyl groups can be substituted or unsubstituted. The cycloalkenyl and heterocycloalkenyl groups can be substituted with one or more groups described herein, including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfoxo, or thiol.

[0055] The term "cycloalkynyl" as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bond. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term "heterocycloalkynyl" is a type of cycloalkenyl group defined above and is included within the meaning of the term "cycloalkynyl", in which at least one of the ring carbon atoms is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkynyl and heterocycloalkynyl groups can be substituted or unsubstituted. Cycloalkynyl and heterocycloalkynyl groups can be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfoxo, or thiol, as described herein.

[0056] The terms "heterocycle" or "heterocyclyl" as used herein may be used interchangeably and refer to mono- and polycyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Thus, the term includes, but is not limited to, "heterocycloalkyl", "heteroaryl", "bicyclic heterocycle", and "polycyclic heterocycle". Heterocycles can be monocyclic, bicyclic (e.g., spiro or bridged), polycyclic, or saturated or partially saturated fused systems. Heterocycles include pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole (including 1,2,3-oxadiazole, 1,2,5-oxadiazole, and 1,3,4-oxadiazole), thiadiazole (including 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole), triazole (including 1,2, 3-triazole, including 1,3,4-triazole, tetrazole (including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole), pyridazine, pyrazine, triazine (including 1,2,4-triazine and 1,3,5-triazine), tetrazine (including 1,2,4,5-tetrazine), pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, dioxane, and the like. The term heterocyclyl group may also be C2 heterocyclyl, C2-C3 heterocyclyl, C2-C4 heterocyclyl, C2-C5 heterocyclyl, C2-C6 heterocyclyl, C2-C7 heterocyclyl, C2-C8 heterocyclyl, C2-C9 heterocyclyl, C2-C10 heterocyclyl, C2-C11 heterocyclyl, etc., including up to C2-C18 heterocyclyl. For example, C2 heterocyclyl includes groups having two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, dihydrodiazetyl, oxiranyl, thiaranyl, etc.Alternatively, for example, C5 heterocyclyl includes groups having five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, pyridinyl, etc. It is understood that a heterocyclyl group may be attached, if chemically possible, either through a heteroatom within the ring or through one of the carbons comprising the heterocyclyl ring.

[0057] The term "bicyclic heterocycle" or "bicyclic heterocyclyl" as used herein refers to a ring system in which at least one of the ring members is other than carbon. Bicyclic heterocyclyl includes ring systems in which an aromatic ring is fused to another aromatic ring or an aromatic ring is fused to a non-aromatic ring. Bicyclic heterocyclyl includes ring systems in which a benzene ring is fused to a 5- or 6-membered ring containing 1, 2, or 3 ring heteroatoms, or a pyridine ring is fused to a 5- or 6-membered ring containing 1, 2, or 3 ring heteroatoms. Bicyclic heterocyclic groups include, but are not limited to, indolyl, indazolyl, pyrazolo[1,5-a]pyridinyl, benzofuranyl, quinolinyl, quinoxalinyl, 1,3-benzodioxolyl, 2,3-dihydro-1,4-benzodioxinyl, 3,4-dihydro-2H-chromenyl, 1H-pyrazolo[4,3-c]pyridin-3-yl; 1H-pyrrolo[3,2-b]pyridin-3-yl; and 1H-pyrazolo[3,2-b]pyridin-3-yl.

[0058] The term "heterocycloalkyl" as used herein refers to an aliphatic, partially unsaturated or fully saturated 3-14 membered ring system, including monocyclic and bicyclic and tricyclic ring systems of 3-8 atoms. Heterocycloalkyl ring systems contain 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur, where the nitrogen and sulfur heteroatoms can be optionally oxidized and the nitrogen heteroatom can be optionally substituted. Representative heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolidinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0059] The term "9-membered fused heterocyclyl" refers to a 9-membered saturated or partially unsaturated fused monocyclic heterocycle containing at least one oxygen heteroatom and, optionally, 2 to 4 additional heteroatoms independently selected from N, O, and S. The terms "heterocycle", "heterocyclyl", "heterocyclyl ring", "heterocyclic group", "heterocyclic moiety", and "heterocyclic radical" are used interchangeably herein. A heterocyclyl ring may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Examples of fused saturated or partially unsaturated heterocyclic radicals containing at least one oxygen atom include, but are not limited to, dihydrobenzofuranyl, dihydrofuropyridinyl, octahydrobenzofuranyl, and the like. When specified as optionally substituted, the substituents on the heterocyclyl (e.g., in the case of an optionally substituted heterocyclyl) may be present at any substitutable position, including, for example, the position to which the heterocyclyl group is attached.

[0060] The term "aromatic group" as used herein refers to a ring structure having a cyclic cloud of delocalized π-electrons above and below the plane of the molecule, where the π-cloud contains (4n+2) π-electrons. Further discussion of aromaticity is found in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter 13, "Aromaticity," pages 477-497, which is incorporated herein by reference. The term "aromatic group" includes both aryl and heteroaryl groups.

[0061] The term "aryl" as used herein refers to any carbon-based aromatic group-containing group, including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. Aryl groups can be substituted or unsubstituted. Aryl groups include alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, -NH, as described herein. 2 The aryl group may be substituted with one or more groups, including, but not limited to, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfoxo, or thiol. The term "biaryl" is a specific type of aryl group and is included in the definition of "aryl". In addition, an aryl group may be a single ring structure, or may be a fused ring structure, or may contain multiple ring structures linked through one or more bridging groups, such as carbon-carbon bonds. For example, a biaryl may be two aryl groups linked together through a fused ring structure, such as naphthalene, or linked through one or more carbon-carbon bonds, such as biphenyl.

[0062] The term "heteroaryl" as used herein refers to an aromatic group having at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, with N-oxides, sulfur oxides, and dioxides being permissible heteroatom substitutions. Heteroaryl groups can be substituted or unsubstituted. Heteroaryl groups can be substituted with one or more groups, including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, or thiol, as described herein. Heteroaryl groups can be monocyclic, or alternatively, fused ring systems. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridinyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, isoquinolinyl, pyrazolyl, triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, benzoxolyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, imidazopyridinyl, pyrazolopyridinyl, and pyrazolopyrimidinyl. Further non-limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, pyrazolyl, imidazolyl, benzo[d]oxazolyl, benzo[d]thiazolyl, quinolinyl, quinazolinyl, indazolyl, imidazo[1,2-b]pyridazinyl, imidazo[1,2-a]pyrazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl.

[0063] The term "5- or 6-membered heteroaryl" refers to a 5- or 6-membered aromatic radical containing 1 to 4 heteroatoms selected from N, O, and S. Non-limiting examples include thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, and the like. When specified, optional substituents on a heteroaryl group may be present at any substitutable position, including, for example, the position to which the heteroaryl is attached.

[0064] The term "aldehyde" as used herein is represented by the formula -C(O)H. Throughout this specification, "C(O)" is a shorthand notation for a carbonyl group, i.e., C=O.

[0065] As used herein, the term "amine" or "amino" refers to a group of the formula -NA 1 A 2 Represented by A 1 and A 2 may independently be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. 2 It is.

[0066] The term "alkylamino" as used herein is represented by the formula -NH(-alkyl), where alkyl is a as described herein. Representative examples include, but are not limited to, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, (sec-butyl)amino, (tert-butyl)amino, pentylamino, isopentylamino, (tert-pentyl)amino, hexylamino, and the like.

[0067] As used herein, the term "dialkylamino" refers to a group of the formula -N(-alkyl) 2and alkyl is a as described herein. Representative examples include, but are not limited to, a dimethylamino group, a diethylamino group, a dipropylamino group, a diisopropylamino group, a dibutylamino group, a diisobutylamino group, a di(sec-butyl)amino group, a di(tert-butyl)amino group, a dipentylamino group, a diisopentylamino group, a di(tert-pentyl)amino group, a dihexylamino group, an N-ethyl-N-methylamino group, an N-methyl-N-propylamino group, and an N-ethyl-N-propylamino group.

[0068] The term "carboxylic acid" as used herein is represented by the formula -C(O)OH.

[0069] As used herein, the term "ester" refers to an ester of the formula -OC(O)A 1 or -C(O)OA 1 In the formula, A 1 may be an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. As used herein, the term "polyester" refers to a group of compounds of the formula -(A 1 O(O)CA 2 -C(O)O) a -or- (A 1 O(O)CA 2 -OC(O) a In the formula, A 1 and A 2 can be independently an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein, and "a" is an integer from 1 to 500. "Polyester" is a term used to describe groups produced by the reaction between a compound having at least two carboxylic acid groups and a compound having at least two hydroxyl groups.

[0070] As used herein, the term "ether" refers to a compound of formula A 1 Office Automation 2 In the formula, A1 and A 2 may be independently an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. As used herein, the term "polyether" refers to a group having the formula -(A 1 Office Automation 2 O) a In the formula, A 1 and A 2 may be independently an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein, and "a" is an integer from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.

[0071] As described herein, the compounds of the present disclosure may contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at all positions. Combinations of substituents envisioned by the present invention are preferably those that result in the formation of stable or chemically feasible compounds. Also, in certain embodiments, unless expressly indicated to the contrary, individual substituents may be further optionally substituted (i.e., further substituted or unsubstituted).

[0072] In some embodiments, the structure of the compound has the following formula: TIFF2025503568000002.tif13128 and is understood to be equivalent to the following formula: TIFF2025503568000003.tif22128 where n is typically an integer. That is, Rn is a group consisting of five independent substituents, R n(a) , R n(b) , R n(c) , R n(d) , R n(e) In each such case, the five R n Each of R can be hydrogen or a recited substituent. By "independent substituents" it is meant that each R substituent can be independently defined. For example, in one example, R n(a) In the example, when is halogen, R n(b) is not necessarily a halogen.

[0073] In still some further embodiments, the structure of the compound has the following formula: TIFF2025503568000004.tif16128, where R y For example, A 1 , A 2 , and A 3 which are understood to be equivalent to the group of the formula: TIFF2025503568000005.tif169164

[0074] Again, "independent substituents" means that each R substituent can be defined independently. For example, in one example, R y1 A 1 In that example, R y2 is not necessarily A 1 This is not necessarily the case.

[0075] In some further embodiments, the structure of the compound has the following formula: TIFF2025503568000006.tif21128, where, for example, Q includes three substituents independently selected from hydrogen and A, which are understood to be equivalent to the following formula: TIFF2025503568000007.tif21128

[0076] Again, "independent substituents" means that each Q substituent is independently defined as hydrogen or A, which are understood to be equivalent to the group of the formula: TIFF2025503568000008.tif55165

[0077] In some embodiments, the disclosed compounds exist as geometric isomers. "Geometric isomer" refers to an isomer that differs in the orientation of the substituent atoms relative to the cycloalkyl ring, i.e., cis or trans isomers. When the disclosed compounds are named or depicted by structure without showing a specific cis or trans geometric isomeric form, it should be understood that the name or structure encompasses one geometric isomer without including other geometric isomers, mixtures of geometric isomers, or mixtures enriched in one geometric isomer compared to the corresponding geometric isomer. When a specific geometric isomer, i.e., cis or trans, is depicted, the depicted isomer is at least about 60%, 70%, 80%, 90%, 99%, or 99.9% pure by weight compared to the other geometric isomer.

[0078] The compounds described herein may exist in the form of pharmaceutically acceptable salts. For use in medicine, the salts of the compounds described herein refer to non-toxic "pharmaceutically acceptable salts". The pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic / anionic or basic / cationic salts. Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include, for example, salts of inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and sulfuric acid) and salts of organic acids (such as acetic acid, benzenesulfonic acid, benzoic acid, methanesulfonic acid, and p-toluenesulfonic acid). Examples of pharmaceutically acceptable base addition salts include, for example, sodium, potassium, calcium, ammonium, organic amino, or magnesium salts.

[0079] The term "pharmaceutical acceptable carrier" refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound to be formulated. Pharmaceutically acceptable carriers, adjuvants, or vehicles that can be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0080] As used herein, the phrase "pharmacologically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals. In some embodiments, "pharmacologically acceptable" means approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, and more specifically in humans.

[0081] Disease, disorder, and condition are used interchangeably herein.

[0082] As used herein, the terms "treatment", "treat" and "treating" refer to reversing, alleviating, delaying the onset of, reducing, ameliorating, preventing or inhibiting the progression of a disease or disorder or one or more symptoms thereof, as described herein. "Treating" may refer to the administration of a compound, composition or pharmaceutical composition as described herein. "Treating" includes the concept of "alleviating", which refers to reducing the frequency of occurrence or recurrence of any symptoms or other adverse effects associated with intestinal motility disorders, or their severity. The term "treating" also includes the concept of "managing", which refers to reducing the severity of a particular disease or disorder in a patient, or delaying its recurrence, for example, extending the period of remission in a patient who has been affected by the disease. It is understood that treating a disorder or condition does not require, but is not excluded, the complete elimination of the disorder, condition, or symptoms associated therewith. In some embodiments, treatment may be administered after one or more symptoms have developed, i.e., therapeutic treatment. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of the symptoms and / or exposure to a particular organism or other susceptibility factors), i.e., prophylactic treatment. Treatment may also be continued after symptoms have resolved, e.g., to delay their recurrence.

[0083] As used herein, the term "prevent" or "preventing" refers to making impossible, avoiding, forestalling, preventing, stopping, or impeding something from happening, especially by prior action. It is understood that where reduce, inhibit, or prevent are used herein, the use of the other two words is also expressly disclosed unless otherwise indicated. The term "preventing" refers to preventing the occurrence of a disease, disorder, or condition and / or inhibiting, i.e., arresting the occurrence of, a disease, disorder, or condition in a human or animal that may have a predisposition to, but has not yet been diagnosed as having, the disease, disorder, and / or condition.

[0084] The term "therapeutic effect" as used herein is meant to refer to some degree of alleviation of one or more of the symptoms of a disorder (e.g., intestinal motility disorder) or its associated pathology. The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to achieve a desired result (e.g., a dosage of 0.01-100 mg / kg body weight / day that will elicit a biological or medical response in the subject) or to affect an undesirable condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic result or to have an effect on an undesirable condition, but generally not sufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the particular composition used; the age, weight, general health, sex, and diet of the patient; the time of administration; the route of administration; the excretion rate of the particular compound used; the duration of treatment; drugs used in combination with or concurrently with the particular compound used, as well as similar factors well known in the medical arts. For example, it is well within the skill of the art to begin dosing the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. If necessary, the effective daily dose can be divided into multiple doses for administration purposes. Thus, single dose compositions can contain such amounts or submultiples thereof to constitute a daily dose. Dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary and can be administered in one or more dose administrations per day for one or several days. Guidance can be found in the literature for appropriate dosages for a given class of pharmaceuticals. In further various embodiments, the preparation can be administered in a "prophylactically effective amount", i.e., an amount effective for the prevention of a disease or condition.

[0085] As used herein, the term "sample" generally refers to a limited amount of something, which is intended to resemble and represent a larger amount of something. In this disclosure, a sample is a collection, swab, brushing, scraping, biopsy, removed tissue, or surgical resection that is tested for the absence, presence, or grading of intestinal motility disorder. In some embodiments, the sample is taken from a patient or subject believed to have intestinal motility disorder.

[0086] As used herein, the term "salt" refers to an acid or base salt of a compound used in the method of the present disclosure. Illustrative examples of acceptable salts are salts of inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, etc.), salts of organic acids (such as acetic acid, propionic acid, glutamic acid, citric acid, etc.), and salts of quaternary ammonium (such as methyl iodide, ethyl iodide, etc.).

[0087] The terms "subject" and "patient" may be used interchangeably and refer to a mammal in need of treatment, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). Typically, the subject is a human in need of treatment.

[0088] The term "associated with" or "associated with" in the context of a substance or activity or function of a substance associated with a disease (e.g., a protein-associated disease, a symptom associated with intestinal motility disorder, a symptom associated with NO neuron activity) means that the substance or activity or function of the substance causes (in whole or in part) the disease (e.g., intestinal motility disorder) or causes the symptoms of the disease. For example, the symptoms of an intestinal motility disease or condition can be symptoms that are caused (in whole or in part) by regulating NO neuron activity (e.g., induction of colonic motility). As used herein, what is described as being associated with a disease can be a target for treating the disease if it is a causative agent. For example, intestinal motility disorder can be treated with an agent (e.g., a compound described herein) that is effective in regulating NO neuron activity (e.g., effective in inducing colonic motility).

[0089] "Control" or "control experiment" is used according to its plain and ordinary meaning to refer to an experiment in which the experimental subjects or agents are treated as in a parallel experiment, except for the omission of an experimental procedure, agent, or variable. In some cases, a control is used as a standard of comparison in evaluating the effect of an experiment.

[0090] "Contacting" is used according to its plain and ordinary meaning and refers to a process that allows at least two different species (e.g., chemical compounds, including biomolecules, or cells) to come into sufficient proximity to react, interact, or physically touch. However, it should be understood that the resulting reaction product can be generated directly from the reaction between the added reagents, or from an intermediate from one or more of the added reagents that can be generated in the reaction mixture. The term "contacting" can include allowing two species to react, interact, or physically touch, and the two species can be a compound described herein and a protein or enzyme (e.g., PINK1). In some embodiments, contacting includes allowing a compound described herein to interact with a protein or enzyme involved in a signal transduction pathway.

[0091] As used herein, the terms "inhibit," "inhibit," "inhibiting," and the like in relation to protein-inhibitor (e.g., antagonist) interactions refer to adversely affecting (e.g., decreasing) the activity or function of a protein compared to the activity or function of the protein in the absence of the inhibitor. In some embodiments, inhibition refers to a reduction in a disease or a symptom of a disease. In some embodiments, inhibition refers to a reduction in a signaling pathway or the activity of a signaling pathway. Thus, inhibition includes, at least in part, partially or completely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating a signaling or enzymatic activity or the amount of a protein.

[0092] symbol TIFF2025503568000009.tif3128 indicates the point of attachment of a chemical moiety to the remainder of a molecule or chemical formula.

[0093] As used herein, the terms "activation," "activating," "activating," and the like in relation to protein-activator (e.g., agonist) interactions refer to adversely affecting (e.g., increasing) the activity or function of a protein compared to the activity or function of the protein in the absence of the activator. In some embodiments, activation refers to an increase in the activity of a signal transduction pathway or a signal transduction pathway. Thus, activation may include, at least in part, partially or fully increasing a stimulus, increasing or enabling activation, or activating, sensitizing, or upregulating a signal transduction or enzymatic activity or amount of a protein that is decreased in a disease. Activation may include, at least in part, partially or fully increasing a stimulus, increasing or enabling activation, or activating, sensitizing, or upregulating a signal transduction or enzymatic activity or amount of a protein, which may regulate the level of another protein or increase cell survival.

[0094] The term "modulator" refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule. In some embodiments, the modulator is a modulator of NO neuron activity. In some embodiments, the modulator is a compound that is a modulator of NO neuron activity and reduces the severity of one or more symptoms of a disease associated with NO neuron activity. In some embodiments, the modulator is a compound that reduces the severity of one or more symptoms of a gut motility disorder that is not caused by or characterized by NO neuron activity but can benefit from the modulation of NO neuron activity (e.g., induction of colonic motility).

[0095] "Patient" or "subject in need thereof" refers to an organism suffering from or susceptible to a disease or condition that can be treated by administration of a compound or pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the patient is a human.

[0096] "Disease" or "condition" refers to an existing or health state of a patient or subject that can be treated with a compound, pharmaceutical composition, or method provided herein. In some embodiments, the disease is a disease associated with (e.g., characterized by) modulation of NO neuronal activity. In some embodiments, the disease is a gut motility disorder.

[0097] As used herein, the term "signal transduction pathway" refers to a series of interactions between cellular and optionally extracellular components (e.g., proteins, nucleic acids, small molecules, ions, lipids) that transmit a change in one component to one or more other components, which in turn may transmit the change to further components, and optionally are propagated to other signal transduction pathway components.

[0098] The term "preparation" is intended to include formulations of the active compound with an encapsulating material as a carrier, providing capsules in which the active compound is surrounded by a carrier with or without other carriers, so that the carrier is associated with it. Also included are cachets and lozenges. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0099] As used herein, the term "administering" refers to oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration to a subject, or implantation of a sustained release device, such as a mini-osmotic pump, into a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. By "co-administered" it is meant that the compositions described herein are administered simultaneously with, immediately prior to, or immediately following the administration of one or more additional therapeutic agents (e.g., a therapeutic agent for cardiomyopathy including an angiotensin converting enzyme inhibitor (e.g., enalipril, lisinopril), an angiotensin receptor blocker (e.g., losartan, valsartan), a beta blocker (e.g., lopressor, toprol-XL), digoxin, or a diuretic (e.g., Lasix, or a therapeutic agent for Parkinson's disease including, for example, levodopa, a dopamine agonist (e.g., bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, lisuride), an MAO-B inhibitor (e.g., selegiline or rasagiline), amantadine, an anticholinergic, an anti-inflammatory agent (e.g., clozapine), a cholinergic inhibitor, modafine, or a nonsteroidal anti-inflammatory agent).

[0100] The compounds of the present disclosure can be administered alone or can be co-administered to a patient. Co-administration is meant to include administering the compounds individually or in combination (two or more compounds or agents), simultaneously or sequentially. Thus, the preparations can also be combined with other active substances (e.g., to reduce metabolic degradation) if necessary. The compositions of the present disclosure can be delivered by topical routes, transdermally, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Oral preparations include tablets, pills, powders, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, and the like, suitable for ingestion by the patient. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions. The compositions of the present disclosure can further include components that provide sustained release and / or comfort. Such components include high molecular weight, anionic mucus-mimetic polymers, gelling polysaccharides, and finely divided drug carrier substrates.These components are discussed in more detail in U.S. Patent Nos. 4,911,920, 5,403,841, 5,212,162, and 4,861,760.The entire contents of these patents are incorporated herein by reference in their entirety for all purposes.The disclosed compositions can also be delivered as microspheres for sustained release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres for subcutaneous sustained release (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995), as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995), or as microparticles for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997).In some embodiments, the formulation of the composition of the present disclosure can be delivered by using liposomes that fuse with or are internalized by cell membranes, i.e., by using receptor ligands attached to liposomes that bind to cell surface membrane protein receptors that lead to endocytosis. The use of liposomes can focus the delivery of the composition of the present disclosure to target cells in vivo, especially when the liposome surface carries receptor ligands specific to the target cells or is otherwise preferentially directed to a particular organ. (See, for example, Al-Muhammed, J.Microencapsul.13:293-306,1996; Chonn, Curr.Opin.Biotechnol.6:698-708,1995; Ostro, Am.J.Hosp.Pharm.46,1576-1587,1989). The composition of the present disclosure can also be delivered as nanoparticles.

[0101] The pharmaceutical compositions provided by the present disclosure include compositions in which the active ingredient (e.g., a compound described herein, including an embodiment or example) is contained in a therapeutically effective amount, i.e., an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, among other things, on the condition being treated. When administered in a method for treating a disease, such compositions contain an amount of active ingredient effective to achieve the desired result, e.g., modulating the activity of a target molecule and / or reducing, eliminating, or delaying the progression of disease symptoms. Determination of a therapeutically effective amount of a composition of the present disclosure is well within the capabilities of one of ordinary skill in the art, especially in light of the detailed disclosure herein.

[0102] The dosage and frequency (single or multiple doses) administered to a mammal may vary depending on a variety of factors, such as whether the mammal is suffering from another disease and its route of administration, the size, age, sex, health, weight, body mass index, and diet of the recipient, the nature and extent of symptoms of the disease being treated (e.g., symptoms of intestinal motility disorders), types of concurrent treatments, complications from the disease being treated, or other health-related problems. Other therapeutic regimens or drugs may be used in conjunction with the methods and compounds of Applicant's disclosure. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the capabilities of one of ordinary skill in the art.

[0103] For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays. The target concentration will be the concentration of active compound(s) that can be measured using methods described herein or known in the art to achieve the effects described herein.

[0104] As is well known in the art, the therapeutically effective amount used in humans can also be determined from animal models.For example, human doses can be formulated to achieve a concentration that has been found to be effective in animals.Dosages in humans can be adjusted by monitoring the effectiveness of the compound and adjusting the dosage upwards or downwards, as described above.Based on the above and other methods, it is well within the capabilities of those skilled in the art to adjust dosages to achieve maximum effectiveness in humans.

[0105] Dosage may vary depending on the patient's requirements and the compound used. In light of the present disclosure, the dose administered to a patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The size of the dose will also be determined by the existence, nature, and extent of any adverse side effects. Determining the appropriate dosage for a particular situation is within the capabilities of one of ordinary skill in the art. Generally, treatment is initiated with a smaller dosage, which is less than the optimal dose of the compound. Dosage is then increased by small increments until the optimal effect under the circumstances is reached.

[0106] Dosage amount and interval may be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated, thereby providing a therapeutic regimen appropriate to the severity of the individual's condition.

[0107] Utilizing the teachings provided herein, one can design an effective prophylactic or therapeutic treatment regimen that does not cause substantial toxicity, but is effective in treating the clinical symptoms demonstrated by a particular patient. This plan should involve careful selection of an active compound by considering factors such as the potency of the compound, relative bioavailability, the patient's weight, the presence and severity of adverse side effects, the preferred mode of administration, and the toxicity profile of the selected agent.

[0108] The compounds described herein can be used in combination with each other, in combination with other active agents known to be useful in the treatment of intestinal motility disorders as further described herein, or in combination with adjunct agents that may not be effective alone but may contribute to the effectiveness of the active agent.

[0109] In some embodiments, simultaneous administration includes administering one active agent within about 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of the second active agent. Simultaneous administration includes administering two active agents simultaneously, at about the same time (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order. In some embodiments, simultaneous administration can be achieved by co-formulation, i.e., preparing a single pharmaceutical composition that includes both active agents. In other embodiments, the active agents can be formulated separately. In some embodiments, the active agents and / or adjuncts may be linked or conjugated to each other. In some embodiments, the compounds described herein may be combined with treatments for neurodegeneration, such as surgery. In some embodiments, the compounds described herein may be combined with treatments for cardiomyopathy, such as surgery.

[0110] The term "derivative" when applied to a phosphate-containing, monophosphate, diphosphate, or triphosphate group or moiety refers to a chemical modification of such a group, where the modification may include the addition, removal, or substitution of one or more atoms of the phosphate-containing, monophosphate, diphosphate, or triphosphate group or moiety. In some embodiments, such derivatives are prodrugs of the phosphate-containing, monophosphate, diphosphate, or triphosphate group or moiety that are converted from the derivative to the phosphate-containing, monophosphate, diphosphate, or triphosphate group or moiety after administration to a subject, patient, cell, biological sample, or after contact with a subject, patient, cell, biological sample, or protein (e.g., enzyme). In one embodiment, the triphosphate derivative is a gamma-thiotriphosphate. In one embodiment, the derivative is a phosphoramidate. In some embodiments, derivatives of phosphate-containing, monophosphate, diphosphate, or triphosphate bases or moieties are disclosed in Murakami et al. J. Med Chem., 2011, 54, 5902; Sofia et al., J. Med Chem. 2010, 53, 7202; Lam et al. ACC, 2010, 54, 3187; Chang et al., ACS Med Chem Lett., 2011, 2, 130; Furman et al., Antiviral Res., 2011, 91, 120; Vernachio et al., ACC, 2011, 55, 1843; Zhou et al., AAC, 2011, 44, 76; Reddy et al., BMCL, 2010, 20, 7376; Lam et al., J. Virol., 2011, 85, 12334; Sofia et al. al., J. Med. Chem., 2012, 55, 2481; Hecker et al., J. Med. Chem., 2008, 51, 2328; or Rautio et al., Nature Rev. Drug. Discov., 2008, 7, 255, all of which are incorporated by reference in their entirety for all purposes.

[0111] As used herein, the term "animal" includes, but is not limited to, humans and non-human vertebrate animals, such as wild, domestic, and farm animals.

[0112] As used herein, the term "antagonize" or "antagonizing" means to reduce or completely eliminate an effect.

[0113] As used herein, the term "carrier" refers to a diluent, adjuvant, or excipient with which a compound is administered. Pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Pharmaceutical carriers can also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and colorants can be used.

[0114] As used herein, the terms "comprising" (and any form of including, such as "comprise," "comprises," and "comprised"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0115] As used herein, the term "contacting" refers to bringing two elements together in an in vitro system or in an in vivo system. For example, "contacting" a compound disclosed herein with an individual or patient or cell includes administration of the compound to an individual or patient, such as a human, as well as introducing the compound into a sample that contains, for example, a cell or a purified preparation that contains a compound or pharmaceutical composition disclosed herein.

[0116] As used herein, the terms "individual," "subject," or "patient" are used interchangeably and refer to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, such as humans.

[0117] As used herein, the phrase "in need thereof" means that an animal or mammal has been identified as having a need for a particular method or treatment. In some embodiments, the identification may be by any diagnostic means. The animal or mammal may be in need of any of the methods and treatments described herein. In some embodiments, the animal or mammal is in or will move into an environment where a particular disease, disorder, or condition is prevalent.

[0118] As used herein, the phrase "an integer from X to Y" refers to any integer inclusive of the endpoints. For example, the phrase "an integer from 1 to 5" refers to 1, 2, 3, 4, or 5.

[0119] As used herein, the term "isolated" means that the compounds described herein are separated from other components of either (a) a natural source, such as a plant or cell, or (b) a synthetic organic chemical reaction mixture, for example, by conventional techniques.

[0120] As used herein, the term "mammal" means a rodent (i.e., mouse, rat, or guinea pig), monkey, cat, dog, cow, horse, pig, or human. In some embodiments, the mammal is a human.

[0121] As used herein, the term "prodrug" refers to a derivative of a known direct acting drug, which has enhanced delivery properties and therapeutic value compared to the drug, and is converted to an active drug by enzymatic or chemical processes. The compounds described herein also include derivatives called prodrugs, which can be prepared by modifying functional groups present in the compound such that the modifications are cleaved to the parent compound, either by routine manipulation or in vivo. Examples of prodrugs include the compounds of the present disclosure described herein that contain one or more molecular moieties attached to the hydroxyl, amino, sulfhydryl, or carboxyl groups of the compound, which upon administration to a patient, cleave in vivo to form the free hydroxyl, amino, sulfhydryl, or carboxyl groups, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups in the compounds of the present disclosure. The preparation and use of prodrugs is discussed in T. Higuchi et al., “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the ACS Symposium Series, and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference in their entireties.

[0122] As used herein, the term "purified" means that upon isolation, the isolate contains at least 90%, at least 95%, at least 98%, or at least 99%, by weight of the isolate, of a compound described herein.

[0123] As used herein, the phrase "solubilizing agent" refers to an agent that results in the formation of a micellar solution or a true solution of a drug.

[0124] As used herein, the term "solution / suspension" refers to a liquid composition in which a first portion of an active agent is present in solution and a second portion of the active agent is present in particulate form in suspension in a liquid matrix.

[0125] As used herein, the term "substantially isolated" means a compound that is at least partially or substantially separated from the environment in which it is formed or detected.

[0126] As used herein, the phrase "therapeutically effective amount" refers to an amount of an active compound or agent that induces a biological or pharmaceutical response sought in a tissue, system, animal, individual, or human by a researcher, veterinarian, physician, or other clinician. The therapeutic effect depends on the disorder being treated or the biological effect desired. Thus, the therapeutic effect may be a reduction in the severity of symptoms associated with the disorder and / or inhibition of the progression of the disorder (partially or completely), or an improved treatment, cure, prevention, or elimination of the disorder or side effects. The amount required to induce a therapeutic response may be determined based on the age, health, size, and sex of the subject. The optimal amount may be determined based on monitoring the subject's response to the treatment.

[0127] It is further understood that certain features that are described herein for clarity in the context of separate embodiments can also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable combination of components.

[0128] It should be noted that any embodiment of the present disclosure may, for purposes of claiming subject matter, optionally exclude one or more embodiments.

[0129] In some embodiments, the compound or its salt is substantially isolated. Partial isolation can include, for example, compositions enriched with the compound of the present disclosure. Substantial isolation can include compositions containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compound of the present disclosure, or its salt. Methods for isolating compounds and their salts are routine in the art.

[0130] B. Methods of Treating Intestinal Motility Disorders In some embodiments, the compounds and compositions described herein are useful for treating intestinal motility disorders.Therefore, disclosed herein is a method for treating intestinal motility disorders, which comprises administering to a subject in need thereof a therapeutically effective amount of a compound described herein (e.g., a modulator of COX, dopamine receptor, sodium channel, serotonin receptor, acetylcholine receptor, GABA receptor, FAAH, adrenergic receptor, histamine receptor, vasopressin receptor, NMDAR, beta amyloid, gamma-secretase, IxB / IKK, glutamate receptor, opioid receptor, TRPV, aldose reductase, calcium channel, glucocorticoid receptor, and / or HMG-CoA reductase), or a pharmaceutically acceptable salt thereof, or a composition comprising the disclosed compound or a pharmaceutically acceptable salt thereof. Disorders treatable by the compounds and compositions of the invention include, for example, achalasia, Hirschsprung's disease, intestinal pseudo-obstruction, gastroesophageal reflux disease (GERD), functional dysphagia, functional dyspepsia, irritable bowel syndrome (IBS), gastroparesis, functional constipation, functional diarrhea, and fecal incontinence.

[0131] In some embodiments, the present disclosure relates to any of the above-disclosed methods disclosed herein, wherein the administering step comprises administering a pharmaceutical composition comprising (i) a pharma- ceutical effective amount of any of the disclosed compounds, and (ii) a pharma- ceutical acceptable carrier.

[0132] Accordingly, in various embodiments, a method is disclosed for treating an intestinal motility disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of carprofen, mefenamic acid, phenacetin, valdecoxib, fenoldopam mesylate, fluphenazine hydrochloride, bupivacaine HCl, phenazopyridine HCl, alverine citrate, nitenpyram, 4-aminobutyric acid (GABA), PF-3845, esmolol HCl, cimetidine, conivaptan HCl, (+)- The method includes administering a compound selected from MK-801 maleate, MK-0752, R04929097, rosmarinic acid, theophylline, aripiprazole, flopropion, latrepirdine 2HCl, ADX-47273, MPEP, nefopam HCl, phenazopyridine HCl, epinephrine, (+)-matrine, phenothiazine, naproxen sodium, AMG-517, isoliquiritigenin, nilvadipine, prednisone, and simvastatin, or a pharmaceutically acceptable salt thereof. In a further embodiment, the compound is selected from aripiprazole, dexmedetomidine, matrine, and MPEP, or a pharmaceutically acceptable salt thereof. In yet a further embodiment, the compound is dexmedetomidine.

[0133] In a further embodiment, the compound is FDA approved.

[0134] In further embodiments, administering is accomplished by oral administration, parenteral administration, sublingual administration, transdermal administration, rectal administration, transmucosal administration, topical administration, inhalation administration, buccal administration, intrapleural administration, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, intranasal administration, intrathecal administration, and intraarticular administration, or combinations thereof.

[0135] In various embodiments, the method further comprises administering an effective amount of an agent associated with treating a gut motility disorder.

[0136] Thus, in various embodiments, the method further comprises administering a drug known for treating intestinal motility disorders. Examples of drugs known for treating intestinal motility disorders include, but are not limited to, parasympathomimetics, prokinetics (also called stimulants), opioid antagonists, antidiarrheals, and antibiotics. In further embodiments, the drug is selected from neostigmine, bethanechol, metoclopramide, cisapride, and loperamide.

[0137] In some embodiments, the compound and the agent are administered simultaneously. In some embodiments, the compound and the agent are administered sequentially.

[0138] In some embodiments, the compound and agent are co-packaged. In some embodiments, the compound and agent are co-formulated.

[0139] C. Methods of Modulating NO Neuronal Activity in a Subject In some embodiments, a method of modulating NO neuron activity in a subject is disclosed, the method comprising administering to the subject a therapeutically effective amount of at least one disclosed compound (e.g., a modulator of COX, dopamine receptor, sodium channel, serotonin receptor, acetylcholine receptor, GABA receptor, FAAH, adrenergic receptor, histamine receptor, vasopressin receptor, NMDAR, beta amyloid, gamma-secretase, IxB / IKK, glutamate receptor, opioid receptor, TRPV, aldose reductase, calcium channel, glucocorticoid receptor, and / or HMG-CoA reductase), or a pharma- ceutically acceptable salt thereof.

[0140] Accordingly, in various embodiments, a method is disclosed for modulating NO neuron activity in a subject having a gut motility disorder, the method comprising administering to the subject an effective amount of carprofen, mefenamic acid, phenacetin, valdecoxib, fenoldopam mesylate, fluphenazine hydrochloride, bupivacaine HCl, phenazopyridine HCl, alverine citrate, nitenpyram, 4-aminobutyric acid (GABA), PF-3845, esmolol HCl, cimetidine, conivaptan HCl, (+)-MK- The method includes administering a compound selected from 801 maleate, MK-0752, R04929097, rosmarinic acid, theophylline, aripiprazole, flopropion, latrepirdine 2HCl, ADX-47273, MPEP, nefopam HCl, phenazopyridine HCl, epinephrine, (+)-matrine, phenothiazine, naproxen sodium, AMG-517, isoliquiritigenin, nilvadipine, prednisone, and simvastatin, or a pharmaceutically acceptable salt thereof. In a further embodiment, the compound is selected from aripiprazole, dexmedetomidine, matrine, and MPEP, or a pharmaceutically acceptable salt thereof. In yet a further embodiment, the compound is dexmedetomidine.

[0141] In a further embodiment, modulating NO neuronal activity induces colonic activity.

[0142] As used herein, "modulation" can refer to either inhibition or enhancement of a particular activity. For example, modulation of NO neuron activity can refer to inhibition and / or activation of NO neuron-dependent activity, such as increased colonic motility. In some embodiments, the compounds described herein increase colonic motility by a factor of about 1% to about 50%. NO neuron activity can be measured by any method, including, but not limited to, the methods described herein.

[0143] In a further embodiment, the modulating is inducing colonic motility.

[0144] In a further embodiment, the subject is a mammal. In yet a further embodiment, the subject is a human.

[0145] In a further embodiment, the subject has been diagnosed with a need for treatment of an intestinal motility disorder prior to the administering step. In yet a further embodiment, the method further comprises the step of identifying the subject at risk for developing an intestinal motility disorder prior to the administering step.

[0146] In further embodiments, administering is accomplished by oral administration, parenteral administration, sublingual administration, transdermal administration, rectal administration, transmucosal administration, topical administration, inhalation administration, buccal administration, intrapleural administration, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, intranasal administration, intrathecal administration, and intraarticular administration, or combinations thereof.

[0147] In various embodiments, the method further comprises administering an effective amount of an agent associated with treating a gut motility disorder.

[0148] Thus, in various embodiments, the method further comprises administering a drug known for treating intestinal motility disorders. Examples of drugs known for treating intestinal motility disorders include, but are not limited to, parasympathomimetics, prokinetics, opioid antagonists, antidiarrheals, and antibiotics. In further embodiments, the drug is selected from neostigmine, bethanechol, metoclopramide, cisapride, and loperamide.

[0149] In some embodiments, the compound and the agent are administered simultaneously. In some embodiments, the compound and the agent are administered sequentially.

[0150] In some embodiments, the compound and agent are co-packaged. In some embodiments, the compound and agent are co-formulated.

[0151] D. Pharmaceutical Compositions Also provided herein is a pharmaceutical composition comprising the compounds disclosed herein, or pharma- ceutically acceptable salts thereof, and a pharma- ceutically acceptable carrier. Thus, in various embodiments, a pharmaceutical composition is disclosed comprising a therapeutically effective amount of at least one disclosed compound (e.g., a modulator of COX, dopamine receptor, sodium channel, serotonin receptor, acetylcholine receptor, GABA receptor, FAAH, adrenergic receptor, histamine receptor, vasopressin receptor, NMDAR, beta amyloid, gamma-secretase, IxB / IKK, glutamate receptor, opioid receptor, TRPV, aldose reductase, calcium channel, glucocorticoid receptor, and / or HMG-CoA reductase) and a pharma- ceutically acceptable carrier. In further embodiments, a pharmaceutical composition can be provided comprising a therapeutically effective amount of at least one disclosed compound. In yet further embodiments, a pharmaceutical composition can be provided comprising a prophylactically effective amount of at least one disclosed compound. In yet further embodiments, the present disclosure relates to a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a disclosed compound, wherein the compound is present in an effective amount.In yet further embodiments, the pharmaceutical composition is useful for modulating NO neuron activity (e.g., inducing colonic motility).In still further embodiments, the pharmaceutical composition is useful for treating intestinal motility disorders.

[0152] Thus, in various embodiments, carprofen, mefenamic acid, phenacetin, valdecoxib, fenoldopam mesylate, fluphenazine hydrochloride, bupivacaine HCl, phenazopyridine HCl, alverine citrate, nitenpyram, 4-aminobutyric acid (GABA), PF-3845, esmolol HCl, cimetidine, conivaptan HCl, (+)-MK-801 maleate, MK-0752, R04929097, rosmarinic acid, theophylline, aripidis, Provided herein are pharmaceutical compositions comprising a therapeutically effective amount of a compound selected from prazole, flopropion, latrepirdine 2HCl, ADX-47273, MPEP, nefopam HCl, phenazopyridine HCl, epinephrine, (+)-matrine, phenothiazine, naproxen sodium, AMG-517, isoliquiritigenin, nilvadipine, prednisone, and simvastatin, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0153] Pharmaceutically acceptable salts of compounds are conventional acid or base addition salts, which retain the biological effectiveness and properties of the compounds and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Exemplary acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, iodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, and fumaric acid. Exemplary base addition salts include those derived from, for example, ammonium, potassium, sodium, and quaternary ammonium hydroxides, for example, tetramethylammonium hydroxide. Chemical modification of pharmaceutical compounds into salts is a known technique to obtain improved physical and chemical stability, hygroscopicity, flowability, and solubility of the compounds. See, e.g., pages 196 and 1456-1457 of H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995).

[0154] The pharmaceutical composition comprises the compound in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carrier refers to sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and sterile powder for reconstitution into sterile injectable solution or dispersion immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. The compound can be formulated with pharmaceutically acceptable carriers or diluents, and any other known adjuvants and excipients according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

[0155] In a further embodiment, the pharmaceutical composition is administered to a mammal. In a still further embodiment, the mammal is a human. In an even further embodiment, the human is a patient.

[0156] In a further embodiment, the pharmaceutical composition is administered after identification of a mammal in need of treatment for a disorder associated with NO neuronal activity, hi yet a further embodiment, the mammal has been diagnosed with a need for treatment for a disorder associated with NO neuronal activity prior to the administering step.

[0157] In a further embodiment, the pharmaceutical composition is administered after identification of a mammal in need of treatment for an intestinal motility disorder, hi yet a further embodiment, the mammal has been diagnosed with a need for treatment for an intestinal motility disorder prior to the administering step.

[0158] In various embodiments, the disclosed pharmaceutical compositions comprise a disclosed compound (including its pharma- ceutically acceptable salt(s)) as an active ingredient, a pharma- ceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, as well as the nature and severity of the condition for which the active ingredient is to be administered. The pharmaceutical compositions are conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

[0159] The selection of carrier can be determined in part by the specific method used to administer the composition.Therefore, there are a wide variety of suitable formulations of the pharmaceutical composition of the present invention.The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, rectal and vaginal administration are merely exemplary and are in no way limiting.

[0160] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in a diluent, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient as a solid or granules; (c) powders; (d) suspensions in a suitable liquid; and (e) suitable emulsions. Liquid formulations can contain diluents such as water, cyclodextrin, dimethyl sulfoxide, and alcohols, including ethanol, benzyl alcohol, propylene glycol, glycerin, and polyethylene glycols, with or without the addition of a pharma-ceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type, containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, coloring agents, diluents, buffers, disintegrants, humectants, preservatives, flavorings, and pharmacologically compatible carriers.Lozenge forms can include candies containing the active ingredient in a flavor, usually sucrose and acacia or tragacanth, and a gel containing the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing the active ingredient plus a carrier as known in the art.

[0161] The compounds of the present disclosure can be made into aerosol preparations, alone or in combination with other suitable components, to be administered via inhalation.These aerosol preparations can be placed in pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen.They can also be formulated as pharmaceuticals for non-pressurized preparations, such as nebulizers or atomizers.

[0162] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. The compounds may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, alcohols, e.g., ethanol, isopropanol, or hexadecyl alcohol, glycols, e.g., propylene glycol or polyethylene glycols, e.g., poly(ethylene glycol) 400, glycerol ketals, e.g., 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, with or without the addition of a pharma- ceutically acceptable surfactant, such as a soap or detergent, suspending agents, e.g., pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents, as well as other pharmaceutical auxiliary agents.

[0163] The oil that can be used in parenteral formulations includes petroleum, animal, vegetable, or synthetic oils.Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral.The fatty acids suitable for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include: (a) cationic detergents, such as, for example, dimethyldialkylammonium halides, and alkylpyridinium halides; (b) anionic detergents, such as, for example, alkyl, aryl, and olefin sulfonates, alkyl olefin, ether, and monoglyceride sulfates, and sulfosuccinates; (c) nonionic detergents, such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers; (d) amphoteric detergents, such as, for example, alkyl β-aminopropionates, and 2-alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0164] Parenteral formulations typically contain about 0.5% to about 25% by weight of the active ingredient in solution. Suitable preservatives and buffers can be used in such formulations. To minimize or eliminate irritation at the injection site, such compositions may contain one or more non-ionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations ranges from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with hydrophobic bases formed by condensation of propylene glycol and propylene oxide.

[0165] Pharmaceutically acceptable excipients are also well known to those skilled in the art. The choice of excipient will be determined in part by the specific compound and the specific method used to administer the composition. Therefore, there are a wide variety of suitable formulations of the pharmaceutical composition of the present disclosure. The following methods and excipients are merely exemplary and in no way limiting. Pharmaceutically acceptable excipients preferably do not interfere with the action of the active ingredient and do not cause adverse side effects. Suitable carriers and excipients include solvents such as water, alcohol, and propylene glycol, solid absorbents and diluents, surface active agents, suspending agents, tableting binders, lubricants, flavoring agents, and coloring agents.

[0166] The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid vehicle for injection, e.g., water, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art. Pharmaceutics and Pharmacy Practice, JBLippincott Co., Philadelphia, PA; Banker and Chalmers, Eds., 238-250 (1982); and ASHP Handbook on Injectable Drugs, Toissel, 4 th ed., 622-630 (1986).

[0167] Formulations suitable for topical administration include lozenges containing the active ingredient in a flavored agent, usually sucrose and acacia or tragacanth; pastilles containing the active ingredient in an inert base, for example, gelatin and glycerin, or sucrose and acacia; and mouthwashes containing the active ingredient in a suitable liquid carrier; and creams, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.

[0168] Moreover, formulations suitable for rectal administration may be presented as suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or sprays containing, in addition to the active ingredient, such carriers as are known in the art as appropriate.

[0169] Those skilled in the art will appreciate that suitable methods of exogenously administering the compounds of the present disclosure to animals are available, and that more than one route may be used to administer a particular compound, with a particular route providing a more immediate and effective response than another route.

[0170] For these uses, the method includes administering to an animal, particularly a mammal, more particularly a human, a therapeutically effective amount of a compound effective for treating (e.g., prophylactically or therapeutically) intestinal motility disorder. The method also includes administering to an animal, particularly a human, a therapeutically effective amount of a compound for treating a patient predisposed to suffering from intestinal motility disorder. In the context of the present invention, the dose administered to an animal, particularly a human, should be sufficient to affect a therapeutic response in the animal in a reasonable time frame. Those skilled in the art will recognize that the dosage depends on various factors, including the condition of the animal, the weight of the animal, and the severity and stage of the disorder.

[0171] The total amount of the compound of the present disclosure administered in a typical treatment is preferably about 1 mg / kg body weight to about 100 mg / kg body weight in mice, and about 10 mg / kg body weight to about 50 mg / kg body weight, and about 20 mg / kg body weight to about 40 mg / kg body weight in humans, per daily dose. In some embodiments, the subject is a human and the dose is about 10 mg / kg body weight to about 90 mg / kg body weight. In some embodiments, the subject is a human and the dose is about 10 mg / kg body weight to about 80 mg / kg body weight. In some embodiments, the subject is a human and the dose is about 10 mg / kg body weight to about 70 mg / kg body weight. In some embodiments, the subject is a human and the dose is about 10 mg / kg body weight to about 60 mg / kg body weight. In some embodiments, the subject is a human and the dose is about 1 mg / kg human body weight to about 300 mg / kg human body weight. This total amount is typically, but not necessarily, administered as a series of smaller doses from about once per day to about three times per day for a period of about 24 months, and twice per day for a period of about 12 months.

[0172] The size of the dose will also be determined by the route, timing, and frequency of administration, as well as the existence, nature, and extent of any adverse side effects that may accompany the administration of the compound, and the desired physiological effect.Those of skill in the art will appreciate that various conditions or disease states, particularly chronic conditions or disease states, may require prolonged treatment involving multiple administrations.

[0173] In certain embodiments, the compositions described herein are formulated for administration to a patient in need of such compositions. The compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intraperitoneally, or intravenously. Sterile injectable forms of the compositions described herein may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.

[0174] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, excretion rate, drug combination, and the judgment of the treating physician, as well as the severity of the particular disease being treated. The amount of a compound described herein in the composition will also depend on the particular compound in the composition.

[0175] The compounds described herein can be administered alone or can be administered simultaneously with additional therapeutic agents.Thus, the preparation can also be combined with other active substances (e.g., to reduce metabolic degradation) as needed.The additional therapeutic agents include, but are not limited to, other active substances known to be useful for treating intestinal motility disorders, as further described herein.

[0176] In some embodiments, the compounds described herein can be delivered in vesicles, in particular liposomes (see Langer, Science, 1990, 249, 1527-1533; Treat et al., Lopez-Berestein, ibid., pp. 317-327 in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); see generally ibid.).

[0177] Suitable compositions include, but are not limited to, oral non-absorbable compositions, including, but not limited to, saline, water, cyclodextrin solutions, and buffer solutions of pH 3-9.

[0178] The compounds described herein, or pharma- ceutically acceptable salts thereof, can be formulated with a number of excipients, including purified water, propylene glycol, PEG 400, glycerin, DMA, ethanol, benzyl alcohol, citric acid / sodium citrate (pH 3), citric acid / sodium citrate (pH 5), tris(hydroxymethyl)aminomethane HCl (pH 7.0), 0.9% saline, 1.2% saline, acetate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate, acid bitartrate, bromide, camsylate, carbonate, chloride, citrate, decanoate, edetate, esylate, fumarate, gluceptate, gluconate. In some embodiments, the excipient is selected from propylene glycol, purified water, and glycerin.

[0179] In some embodiments, the formulation can be lyophilized to a solid and reconstituted, for example with water, prior to use.

[0180] When administered to mammals (eg, animals for veterinary use or humans for clinical use), the compounds may be administered in isolated form.

[0181] When administered to humans, the compound can be sterile.When the compound of formula I is administered intravenously, water is a suitable carrier.For injection solutions, physiological saline and aqueous dextrose and glycerol solutions are also used as liquid carriers.Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc.If desired, the composition can also contain small amounts of wetting or emulsifying agents, or pH buffering agents.

[0182] The compositions described herein can take the form of a solution, suspension, emulsion, tablet, pill, pellet, capsule, capsule containing liquid, powder, sustained release formulation, suppository, aerosol, spray, or any other form suitable for use. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. R. Gennaro (Editor) Mack Publishing Co.

[0183] In some embodiments, the compound is formulated according to the usual procedures as a pharmaceutical composition suitable for administration to humans. Typically, the compound is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent. Compositions for intravenous administration may optionally include a local anesthetic, such as lidocaine, to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in a unit dosage form, e.g., as a dry lyophilized powder or a water-free concentrate in a sealed container, such as an ampoule or sachet indicating the quantity of active agent. When the compound is administered by infusion, it may be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When the compound is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration.

[0184] Pharmaceutical compositions can be in unit dosage form.In such form, the composition can be divided into unit doses containing appropriate amounts of active ingredients.The unit dosage form can be a packaged preparation, the package containing discrete amounts of the preparation, for example, packeted tablets, capsules, and powders in vials or ampoules.The unit dosage form can also be a capsule, cachet, or tablet itself, or the appropriate number of any of these packaged forms.

[0185] In some embodiments, the compositions of the present disclosure are in liquid form and the active agent is present in a solution, suspension, emulsion, or solution / suspension. In some embodiments, the liquid composition is in the form of a gel. In other embodiments, the liquid composition is aqueous. In other embodiments, the composition is in the form of an ointment.

[0186] In some embodiments, the composition is in the form of a solid article. For example, in some embodiments, the ocular composition is a solid article that can be inserted into a suitable position in the eye, such as between the eyelid and the eyelid, or into the conjunctival sac, and releases an active agent, for example, as described in U.S. Pat. Nos. 3,863,633, 3,867,519, 3,868,445, 3,960,150, 3,963,025, 4,186,184, 4,303,637, 5,443,505, and 5,869,079. Such an article usually releases the active agent to the cornea, either through the tear fluid that bathes the surface of the cornea, with which the solid article is generally in intimate contact, or directly through the cornea itself. A solid article suitable for implantation into the eye in such a manner is generally composed mainly of a polymer, and can be bioerodible or non-bioerodible. Bioerodible polymers that can be used in the preparation of ocular implants carrying one or more of the compounds described herein in accordance with the present disclosure include, but are not limited to, aliphatic polyesters such as poly(glycolide), poly(lactide), poly(epsilon caprolactone), poly-(hydroxybutyrate), and poly(hydroxyvalerate) polymers and copolymers, polyamino acids, polyorthoesters, polyanhydrides, aliphatic polycarbonates, and polyether lactones. Suitable non-bioerodible polymers include silicone elastomers.

[0187] The compositions described herein can contain preservative.Suitable preservatives include, but are not limited to, mercury-containing substances such as phenylmercurate (e.g., phenylmercurate acetate, borate and nitrate) and thimerosal; stabilized chlorine dioxide; quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride; imidazolidinyl urea; parabens such as methylparaben, ethylparaben, propylparaben and butylparaben and their salts; phenoxyethanol; chlorophenoxyethanol; phenoxypropanol; chlorobutanol; chlororesol; phenylethyl alcohol; disodium EDTA; and sorbic acid and its salts.

[0188] It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be used in the disclosed methods of use.

[0189] E.Kit In some embodiments, kits are disclosed that include a disclosed compound (e.g., a modulator of COX, a dopamine receptor, a sodium channel, a serotonin receptor, an acetylcholine receptor, a GABA receptor, FAAH, an adrenergic receptor, a histamine receptor, a vasopressin receptor, NMDAR, beta amyloid, gamma-secretase, IxB / IKK, a glutamate receptor, an opioid receptor, TRPV, aldose reductase, a calcium channel, a glucocorticoid receptor, and / or HMG-CoA reductase), or a pharma- ceutically acceptable salt thereof, and one or more selected from (a) an agent known to treat an intestinal motility disorder, (b) instructions for treating an intestinal motility disorder, and (c) instructions for administering the compound in conjunction with treating an intestinal motility disorder.

[0190] Thus, in various embodiments, carprofen, mefenamic acid, phenacetin, valdecoxib, fenoldopam mesylate, fluphenazine hydrochloride, bupivacaine HCl, phenazopyridine HCl, alverine citrate, nitenpyram, 4-aminobutyric acid (GABA), PF-3845, esmolol HCl, cimetidine, conivaptan HCl, (+)-MK-801 maleate, MK-0752, R04929097, rosmarinic acid, theophylline, aripiprazole, flopropione, latrepirdine 2HCl, ADX-4727 3, MPEP, Nefopam HCl, phenazopyridine HCl, epinephrine, (+)-matrine, phenothiazine, naproxen sodium, AMG-517, isoliquiritigenin, nilvadipine, prednisone, and simvastatin, or a pharma- ceutically acceptable salt thereof, and one or more selected from (a) an agent known to treat an intestinal motility disorder, (b) instructions for treating an intestinal motility disorder, and (c) instructions for administering the compound in conjunction with treating an intestinal motility disorder.

[0191] In a further embodiment, the kit comprises a drug known for treating intestinal motility disorders. Examples of drugs known for treating intestinal motility disorders include, but are not limited to, parasympathomimetics, prokinetics, opioid antagonists, antidiarrheals, and antibiotics. In yet a further embodiment, the drug known for treating intestinal motility disorders is selected from neostigmine, bethanechol, metoclopramide, cisapride, and loperamide.

[0192] In a further embodiment, the compound and at least one agent are co-formulated. In a further embodiment, the compound and at least one agent are co-packaged.

[0193] The kits can also include compounds and / or products that are co-packaged, co-formulated, and / or co-delivered with other ingredients. For example, a pharmaceutical manufacturer, pharmaceutical reseller, physician, compounding store, or pharmacist can provide a kit that includes the disclosed compounds and / or products and another ingredient for delivery to a patient.

[0194] It is understood that the disclosed kits can be prepared from the disclosed compounds, products, and pharmaceutical compositions. It is also understood that the disclosed kits can be used in conjunction with the disclosed methods of use.

[0195] The foregoing description illustrates and describes the present disclosure. Moreover, the present disclosure shows and describes only preferred embodiments, but as stated above, it is to be understood that the present disclosure can be used in various other combinations, modifications, and environments, and that changes or modifications are possible within the scope of the concept of the disclosure expressed herein, consistent with the teachings above and / or the skill or knowledge of the relevant art. The embodiments described herein above are further intended to describe the best mode known to the applicant and to enable others skilled in the art to utilize the present disclosure in such or other embodiments, and with various modifications as required by their particular application or use. Thus, the description is not intended to limit the present disclosure to the form disclosed herein. Also, it is intended that the appended claims be construed to include alternative embodiments.

[0196] All publications and patent applications cited in this specification are incorporated by reference herein for any and all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between the present disclosure and any publication or patent application incorporated by reference herein, the present disclosure controls. EXAMPLES

[0197] F. Working Example The enteric nervous system (ENS) plays a central role in intestinal physiology and mediating crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS remains elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here, we performed an unbiased screen to identify drug candidates that modulate the activity of NO neurons. Their potential to promote motility was then demonstrated in mouse colon tissue ex vivo. To define the developmental programs involved in NO neuron specification, a high-throughput strategy was created and it was discovered that PDGFR inhibition promotes the induction of NO neurons in intestinal ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice resulted in extensive tissue engraftment, providing a xenograft model for the study of the human ENS in vivo, and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathy.

[0198] Representative examples of the disclosed compounds are illustrated in the following non-limiting methods, schemes, and examples.

[0199] 1. Method a. Cultivation and maintenance of undifferentiated human stem cells Human embryonic stem cell (hESC) line H9 (WAe009-A, and derivative hSYN::ChrR2-EYFP, a reporter expressing NOS1::GFP) and induced pluripotent stem cell (hiPSC) line WTC-11 (UCSFi001-A) were seeded on Geltrex™ coated plates and maintained in chemically defined medium (E8) as previously described (Barber et al., 2019). Maintenance cultures were tested for mycoplasma every 30 days.

[0200] b. Enteric neural crest (ENC) induction When monolayer cultures of hPSCs reached approximately 70% confluency, a previously established 12-day enteric neural crest (ENC) induction protocol was performed by aspirating the maintenance medium (E8) and replacing it with neural crest induction medium A [BMP4 (1 ng ml ) in Essential 6 Medium]. <2049> -1<!--2049--> ), SB431542 (10 μM), and CHIR99021 (600 nM) in Essential 6 medium] (Barber et al., 2019, Fattahi et al., 2016) (D0). Cultures were then fed with neural crest induction medium B [SB431542 (10 μM) and CHIR99021 (1.5 μM) in Essential 6 medium] on ENC induction days D2 and D4, and medium C [medium B containing retinoic acid (1 μM)] on D6, D8, and D10. ENC crest spheres were then formed during D12–D15 to facilitate selection for ENC lineages and against contaminating them in our cultures. At that time, ENC induction crest medium C was removed on D12, and ENC monolayers were detached using Accutase (30 min, 37 °C, 5% CO2). After centrifugation of the samples at 290 × g for 1 min, the ENC cells were resuspended in NC-C medium [FGF2 (10 ng ml -1 ), CHIR 99021 (3 μM), N2 supplement (10 μl ml -1 ), B27 supplement (20 μl ml -1 ), Glutagro (10 μl ml -1 ), and MEM NEAA (10 μl ml -1 )] and transferred to ultra-low attachment plates to form free-floating 3D intestinal crestspheres. At D14, when free-floating intestinal crestspheres could be observed, they were gently collected in the center of each well using a swirling motion. Then, the old medium was carefully aspirated from the periphery of each well without removing the crestspheres. After adding fresh NC-C medium, the cultures were incubated for 24 h (37 °C and 5% CO2) before the enteric neuron induction phase.

[0201] c. Induction of enteric neurons from the enteric neural crest On day 15, intestinal crestspheres were collected in the center of the well using a swirling motion and the NC-C medium was removed using a P1000 micropipette in a slow circular motion, avoiding free-floating crestspheres. At this step, the protocol varied depending on the final culture layout desired (2D ENS cultures vs. 3D intestinal gangliosides). For 2D ENS cultures, after washing the intestinal crestspheres with PBS, Accutase (Stemcell Technologies, 07920) was added and the plate was incubated at 37 °C for 30 min to dissociate the crestspheres. The remaining spheroids were then cultured in ENC medium [GDNF (10 ng ml ) in neurobasal medium]. -1 ), ascorbic acid (100 μM), N2 supplement (10 μl ml -1 ), B27 supplement (20 μl ml -1 ), Glutagro (10 μl ml -1 ), and MEM NEAA (10 μl ml -1 The cells were disrupted by pipetting 200 µL of ethanol (pH 7.5). The cells were spun down (2 min, 290 × g, 20–25 °C) and the supernatant was removed. The pellet was resuspended in ENC medium and the cells were plated on poly-L-ornithine (PO) / laminin / fibronectin (FN) plates at 1 cm <2091> 2<!--2091--> For 3D intestinal gangliospheres, Accutase treatment was avoided and intestinal crestspheres were seeded with the same volume of ENC medium [GDNF (10 ng ml) in neurobasal medium]. -1 ), ascorbic acid (100 μM), N2 supplement (10 μl ml -1 ), B27 supplement (20 μl ml -1 ), Glutagro (10 μl ml -1 ), and MEM NEAA (10 μl ml -1 Continue feeding with ENC medium every other day until D30-D40, after which the feeding frequency can be reduced to 1-2 times per week, but with a larger amount of medium.

[0202] d. Immunofluorescence For immunofluorescence (IF) staining, cells were first fixed in 4% PFA in PBS (30 min, room temperature (RT) and then blocked and permeabilized with permeabilization buffer (PB) (Foxp3 / transcription factor staining buffer set, 00-5523) for another 30 min at RT. After the fixation and permeabilization step, cells were incubated in primary antibody solution overnight at 4° C. and then washed three times with PB before incubation with fluorophore-conjugated secondary antibodies at room temperature. Before imaging, stained cells were incubated with DAPI fluorescent nuclear stain and washed three more times. A list of antibodies and working dilutions is not shown.

[0203] e. Preparation of intestinal ganglioid frozen sections hPSC-derived gangliosides were collected at stage 1 (days 37-50) and stage 2 (days 70-90), rinsed twice in PBS, and fixed in 4% PFA (SCBT sc-281692) for 3 h on ice, followed by replacement of 90% of the supernatant with PBS for storage at 4° C. for up to 6 months. Gangliosides were treated with 5% sucrose (RPI Research Products 524060) in PBS for 10 min at room temperature, followed by 10% sucrose in PBS for 2 h at room temperature, and 20% sucrose overnight at 4° C. Sucrose-treated ganglioids were placed in cryomolds (Tissue-Tek® Cryomold® medium, VWR25608-924), all 20% sucrose was removed, and incubated in 2:1 20% sucrose:OCT (Tissue Plus OCT Compound Fisher HealthCare 5484) for 2 hours at room temperature before flash freezing in ethanol / dry ice. 1220 μM sections were collected on a cryostat (Leica 3050S) attached to a Superfrost® Plus Micro Slide, Premium (VWR 48311-703) and dried on a 42°C slide dryer for up to 2 hours before being stored at -80°C for up to 1 year.

[0204] f. Preparation of Paraffin-Embedded Human Colon Sections Human sigmoid colon tissue was received from the International Institute for the Advancement of Medicine (IIAM), which provides non-transplant organs from organizations that procure organs for biomedical research purposes. Colon tissue was obtained under sterile conditions, flushed with isotonic solution, immersed in organ transplantation solution, and shipped to the laboratory on ice within 24 hours after death. Full-thickness tissue pieces (approximately 2 cm2) were fixed overnight (<24 hours) in 10% neutral buffered formalin (Cancer Diagnostics, FX1003). Samples were transferred to 70% ethanol prior to paraffin embedding (Leica ASP6025, tissue processor). Transverse tissue sections approximately 5 μM thick were cut onto coated glass slides (Superfrost® Plus Micro Slide; VWR, 48311-703) and air-dried overnight. The following slide preparation steps were all performed at room temperature. Slides with paraffin sections were washed three times with clean xylene substitute (Sigma A5597) and then washed once each with 100% ethanol, 95% ethanol, and 70% ethanol. Slides were then passed under house deionized water for 5 minutes before being placed in 1x PBS for storage at 4°C for up to 4 weeks. Prior to staining, paraffin sections underwent antigen retrieval in either citrate buffer (Vector Laboratories Antigen Unmasking Solution H-3300) or TE buffer (Thermo 17890, pH 9.0 with 1M NaOH). Slides were incubated in the buffer at 95°C for 10 minutes using a Pelco BioWave Pro+ set at 400 watts.

[0205] g. Intestinal ganglioid staining of frozen sections and paraffin-embedded human colon sections All steps were performed at room temperature unless otherwise specified. Ganglioid cryosections and paraffin-embedded human normal colon sections were prepared as above, then washed three times in PBS and blocked for 1–2 h in serum (10% donkey or 10% goat) with 0.5% (v / v) Triton X-100 (VWR0694). Slides were then incubated with primary antibodies (10% donkey or 10% goat) diluted in serum with 0.1% Triton X-100 for 12–20 h at 4 °C. Slides were washed six times for 20 min each in PBS with 0.1% Tween-20 (Sigma P1379) and incubated with Alexa Fluor-conjugated secondary antibodies for 1 h. The diluted secondary antibody solution was removed and replaced with 1.0 μg / mL DAPI in water for 10 min. Slides were washed six times for 20 min each in PBS containing 0.1% Tween-20 and coverslips were mounted with Fluoromount-G (Southern Biotech 0100-01). A list of antibodies and working dilutions is not shown. Images were acquired on a Leica SP8 inverted confocal or an Echo Revolve. Leica's LAS X tile feature or the Grid / Pairwise Stitch plugin for FIJI (PMID19346324) were used to stitch images.

[0206] h. Two-photon fluorescence imaging Imaging experiments were performed on a custom-built upright two-photon microscope operated with μManager software (San Francisco, CA). The excitation source was a two-photon Coherent Chameleon Vision II laser (Coherent, Santa Clara, CA) operating at 760 nm. Images were collected using an Olympus LWD 1.05 NA water immersion lens (Olympus, Tokyo Japan). To image DAPI, an emission filter collecting light from 380 nm to 420 nm (Chroma, Bellow Falls VT) was used, whereas the fluorescent emission of Alexa 568 was collected using a 565 nm to 635 nm filter (Chroma, Bellow Falls VT).

[0207] i. Macro fluorescence imaging Images were taken with a Nikon AZ100M "macro" laser scanning confocal configured with a long working distance low magnification lens. The microscope is equipped with standard 405 nm, 488 nm, 561 nm, and 640 nm laser lines and has a PMT detector with a detection range of 400-700 nm. An optical zoom factor of 2.1x was used to reduce signal falloff at the image edges, and a digital zoom factor of 1.873x was used to increase lateral resolution.

[0208] j.Flow cytometry For preparation of samples for flow cytometry analysis, cells were first dissociated into a single cell suspension by Accutase treatment (Stemcell Technologies, 07920, 30-60 min, 37 °C, 5% CO2) and then fixed and permeabilized using Fixation / Permeabilization Buffer (Foxp3 / Transcription Factor Staining Buffer Set, 00-5523). Cells were stained with primary and secondary antibodies as described above for immunofluorescence. Flow cytometry was performed using a BD LSRFortessa cell analyzer and data were analyzed using Flowjo™ (FlowJo™ Software version 8.7). A list of antibodies and working dilutions is not shown.

[0209] k. Human synapsin::CHANNELRHODOPSIN2-EYFP gut ganglioid blue light activation Intestinal ganglioids were either exposed to blue light (100% laser intensity, 3 x 1 min exposures with 30 sec intervals, EVOS FL) or left in ambient light. Intestinal ganglioids were then incubated at 37°C for 45 min before being dissociated, fixed and permeabilized for flow cytometry (see above). Cells were stained using antibodies against cFos (abcam, ab190289) and TUBB3 (Biolegend, 801202).

[0210] l. Bulk RNA-seq data analysis Total RNA was extracted using PureLink™ RNA Mini Kit. First strand cDNA was then synthesized with Lexogen's Quantseq Forward Library Preparation Kit. Illumina-compatible RNA sequencing libraries were prepared with Quantseq and pooled and sequenced on an Illumina Hiseq 4000 platform at the UCSF Center for Advanced Technology. UMIs were extracted from fastq files using umi_tools and short and low quality reads were removed using cutadapt. Reads were aligned to the human GENCODE v.34 reference genome using STAR aligner and duplicated reads were collapsed using umi_tools. Gene level counts were measured using HTSeq and compared using DESeq2.

[0211] m. Single-cell and single-nucleus RNA sequencing sample preparation and data collection All tubes and pipette tips used for cell harvest were pretreated with 1% BSA in 1× PBS. Cells were dissociated in Accutase (Stem Cell) at 37°C with end-to-end rotation in 10 min increments until a single cell suspension was obtained. Cells were washed with cell staining buffer (Biolegend) and stained with TotalSeq HTO antibody for 30 min on ice. Cells were washed twice in cell staining buffer and filtered through a 40 μm pipette tip strainer (BelArt). Cells were counted using Trypan Blue dye and a hemocytometer and pooled for sequencing. scRNA-seq libraries were prepared with Chromium Next GEM Single Cell 3′ Kit v3.1 (10× Genomics) with custom amplification of TotalSeq HTO sequences (Biolegend). Libraries were sequenced on an Illumina NovaSeq sequencer at the Center for Advanced Technologies (UCSF). Cell feature matrices were extracted using kallisto / bustools and demultiplexed using seurat.

[0212] Quality control and cell filtration Datasets were analyzed with Seurat v4 in R v4.0.3 (Hao et al., 2021). The "PercentageFeatureSet" function was used to calculate the number of reads mapping to mitochondrial and ribosomal gene transcripts per cell. Cells were identified as low quality and then removed independently for each dataset based on the number of unique features captured per cell, the number of UMIs captured per cell, and the percentage of reads mapping to mitochondrial transcripts per cell. Dataset-specific quality control metric cutoffs are not shown.

[0213] o. Dimensionality reduction, clustering, and annotation When applicable, biological replicate samples were first merged using the base R "merge" function. Count matrices were log-normalized with a scaling factor of 10,000 and 2,000 variable features were identified using the "vst" method. Count matrices of biological replicate samples were unified using the Seurat unification function with default parameters. Cell cycle was predicted using the "CellCycleScoring" function with Seurat's S and G2M features provided in "cc.genes". The variable feature set was scaled and centered, and the following variables were regressed: nFeatures, nCounts, mitochondrial gene percentage, ribosomal gene percentage, S score, and G2M score. Principal component analysis (PCA) was performed using default settings, and Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction was performed using PCA reduction. Shared nearest neighbor (SNN) graphs were calculated using default settings, and cell clustering was performed using the default Leuven algorithm. Quality control metrics were visualized for each cluster to identify and remove clusters of low-quality cells (lower than average nFeatures or nCounts, and higher than average mitochondrial and ribosomal gene percentages). The above pipeline was run again on the dataset for subclustering analysis of enteric neural crest, enteric neurons, nitrergic neurons, and enteric glia after removing any low-quality cell clusters. The number of principal components used for UMAP reduction and SNN calculation was determined by the standard deviation of the principal components and varied for each dataset. The number of principal components used for SNN and UMAP calculation, and the resolution used for clustering each dataset are not shown. Cluster markers were found using Wilcoxon rank sum test, and clusters were annotated based on the expression of known cell type marker genes. Following cell type annotation, gene dropout values ​​were imputed using adaptively thresholded low-rank approximation (ALRA) (Linderman et al., 2018).The rank-k approximation was automatically selected for each dataset, and all other parameters were set as default values. Imputed gene expression is shown in all plots and was used in all downstream analyses unless otherwise specified.

[0214] p. Analysis of published datasets Quality control. Low-quality cells were identified and removed using criteria used by the original authors of each dataset. Dataset-specific quality control metric cutoffs are not shown.

[0215] Dimensionality Reduction and Clustering. The dataset was analyzed with Seurat using the methods and parameters described by the original authors.

[0216] Morarach et al.: For all datasets, count matrices were normalized and regressed on mitochondrial gene percentages to return 3000 variable features using the "SCTransform" function. Highly expressed specific and immediate early genes (Xist, Gm13305, Tsix, Eif253y, Ddx3y, Uty, Fos, Jun, Junb, Egr1) were removed from the variable feature list before performing PCA. Dataset-specific parameters used for the "RunUMAP", "FindNeighbors", and "FindClusters" functions are not shown. Cell annotations determined by the authors were used for cell type and neuronal subtype.

[0217] Drokhlyansky et al.: For all datasets, count matrices were log-normalized with a scaling factor of 10,000 and 2,000 variable features were identified using the "vst" method. Batch correction by "Unique_ID" was performed using mutual nearest neighbor correction (MNN) with the "RunFastMNN" Seurat Wrappers function. Dataset-specific parameters used for the "RunUMAP", "FindNeighbors", and "FindClusters" functions are not shown. Cell annotations determined by the authors were used for cell types and neuronal subtypes. For consistency of comparisons, gene dropout values ​​were imputed using ALRA for all published datasets using an automatically determined rank-k approximation and all other default values. Imputed gene expression is shown in all plots and was used in all downstream analyses unless otherwise specified.

[0218] Glia subclustering analysis. Glia were subclustered using a method similar to the original analysis pipeline described by each of the authors above.

[0219] Morarach et al.: Because the E18 dataset contained a single transcriptionally homogenous glial cluster, glial and progenitor populations were subclustered together to provide the comparative cell populations required for downstream analysis. Subset datasets were then normalized, regressed on mitochondrial gene percentages, and returned 3000 variable features using the "SCTransform" function. Highly expressed specific and immediate early genes (Xist, Gm13305, Tsix, Eif253y, Ddx3y, Uty, Fos, Jun, Junb, Egr1) were removed from the variable feature list before running PCA. Dataset-specific parameters used for the "RunUMAP", "FindNeighbors", and "FindClusters" functions are not shown.

[0220] Drokhlyansky et al.: The glial subset dataset was log-normalized with a scaling factor of 10,000 and 2,000 variable features were identified using the "vst" method. Batch correction by "Unique_ID" was performed using mutual nearest neighbor correction (MNN) with the "RunFastMNN" Seurat Wrappers function. Dataset-specific parameters used for the "RunUMAP", "FindNeighbors", and "FindClusters" functions are not shown.

[0221] q. Characterization of gene expression Gene lists were compiled for genes belonging to 10 different functional groups (transcription factors, neurotransmitter synthesis, neuropeptides, neurotransmitter receptors, neuropeptide receptors, cytokines, cytokine receptors, secreted signaling ligands, ligand receptors, and surface markers). For each dataset, gene lists were filtered to remove low abundance genes (detected in less than 25% of cells in each cluster). Genes from these lists were determined to be exclusively expressed by a cluster if more than 25% of cells in only a single cluster expressed the gene.

[0222] r. Cell type transcriptional signature scoring To find transcriptionally similar cell populations between the two datasets, first, the differentially expressed (DE) genes of the reference dataset were calculated from the unimputed gene counts using the Wilcoxon rank sum test with the "FindAllMarkers" function, returning only genes with positive fold changes. The DE gene list was first filtered to remove genes not present in the query dataset. Then, for each cell cluster of the reference dataset, a transcription signature gene list is created from the top 100 DE genes sorted by increasing adjusted p-values. The query dataset is then scored for the transcription signature gene list of each reference dataset's cell cluster based on the imputed gene counts of the query dataset using the "AddModuleScore" function.

[0223] s. Spearman's rank correlation coefficient Transcriptional correlations of cell clusters in the two datasets were calculated from the unimputed gene counts, and the unification function in Seurat was used to first find 3,000 anchor features based on the first 30 dimensions of the canonical correlation analysis, and then the two datasets were unified using the same number of dimensions. The expression of these 3000 anchor features was then scaled and centered on the unified data object, and the scaled average expression of each anchor feature was calculated for cell clusters of interest in each dataset using the "AverageExpression" function. A Spearman correlation matrix comparing all cell clusters to all cell clusters was generated based on the scaled average expression of the 3000 anchor features.

[0224] t.SWNE projection The count matrices of the reference and query datasets are first filtered to include only genes detected in both datasets. A similarly weighted non-negative embedding (SWNE) is then generated for the reference dataset using the SWNE v0.6 package. First, non-negative matrix factorization (NFM) generates constituent factors from 3000 variable features calculated from the unimputed gene counts of the reference dataset. A two-dimensional constituent factor embedding is calculated using Sammon mapping to embed cells and identified key genes in two dimensions for the constituent factors. Finally, an SNN network is calculated from the reference dataset and used to smooth the cell positions. The query dataset is then mapped to the 2D constituent factor space of the reference dataset by first projecting the query dataset onto the NFM factors of the reference dataset. The resulting cell embeddings of the query dataset are then smoothed by projection onto the SNN network of the reference dataset.

[0225] u. Intestinal muscular and submucosal scoring Neurons or glia were grouped separately by tissue layer origin using patient metadata published by the authors. Pan-neuronal and pan-glial myenteric and submucosal gene signatures were created by performing Wilcoxon rank-sum tests to identify DE genes between myenteric and submucosal cell groups. Neuronal and glial datasets were scored with the cell type-specific tissue layer signatures by first ordering the gene list by increasing the adjusted p-value and removing genes not detected in the dataset being scored. Cells were then scored for the 100 most significantly enriched genes in each tissue layer using the "AddModuleScore" function.

[0226] v. Neurochemical identification of neurons Neurochemical identification of neurons was performed independently for each neurotransmitter to accommodate multiple neurochemical identities. For each neurotransmitter, a core set of genes consisting of the rate-limiting synthetic enzyme(s), metabolic enzymes, and transport proteins was selected. First, cells were scored for each neurotransmission-related gene set using the "AddModuleScore" function. Then, cells were annotated as "x-ergic" if the cell's expression of the rate-limiting enzyme was greater than 0 and the cell's module score of the corresponding gene set was greater than 0. If both criteria were not met, the cell was annotated as "other". Multiple neurochemical identities were determined by concatenating the individually determined single neurochemical identities of each cell. The overall prevalence of each neurochemical identity per dataset was calculated by summing the total number of cells annotated for each single identity and calculating the percentage of each "x-ergic" identity from this sum.

[0227] W. Neurotransmitter Response Scoring Separate gene lists were created containing all receptors activated by each neurotransmitter. Cells were scored for expression of each neurotransmitter receptor family gene set using the "AddModuleScore" function.

[0228] x. Glial Gene Set Enrichment Analysis (GSEA) Hierarchical Clustering For each subclustered glial dataset, the DE genes for each glial subtype were calculated using the "FindAllMarkers" function. GSEA on the MSigDB gene ontology sets was performed on the upregulated DE genes (positive log2 fold change only) for each glial subtype that were sorted by decreasing log2 fold change using fgsea v1.16. Normalized enrichment scores (NES) were calculated for gene sets that contained at least 15 genes in the DE gene list, with scoreType set to "positive". GSEA results for each glial subtype were filtered to include only biological process gene sets, but not filtered based on significance to not limit the results to pathways enriched with the highest fold change genes. The NES of the filtered GSEA results for all glial subtypes were then integrated, and pathways that were not detected in a glial subtype were assigned an NES of 0. Hierarchical clustering was then performed based on NES to cluster both gene ontology pathways and glial subtypes. After the glial classes were determined by clustering, pathways enriched in each class were identified by filtering for pathways with an NES greater than 1.1 in all subtypes of a given class.

[0229] y.PP121 vs. control gene expression correlation To compare gene expression between control and PP121-treated cell types, neuronal subtypes, and NO neuronal subtypes, subset data sets were first created for each cell type and subtype annotation. Then, for each subset, the unimputed average expression of all genes was calculated for control and PP121-treated cells using the "AverageExpression" function and natural log transformed for plotting. R was used to compare control and PP121 natural log expression values. 2 Values ​​were calculated from linear modeling using the "y~x" formula.

[0230] Expression screening of z.CFO Stage 2 intestinal gangliosides were dissociated using Accutase and single cell suspensions (in ENC medium) were distributed into wells of a V-bottom 96-well plate. Using a pin tool, compounds from the Neuronal Signaling Compound Library (Selleckchem, USA) were added at 1 pM and cells were incubated for 75 min at 37 °C. Cells were then washed with PBS and immediately fixed for flow cytometry.

[0231] aa.NO release assay For high-throughput measurements of nitric oxide (NO) release, stage 1 2D ENS cultures (96-well plates) were used. After washing the cells with Tyrode's solution [NaCl (129 mM), KCl (5 mM), CaCl2 (2 mM), MgCl2 (1 mM), glucose (30 mM), and HEPES (25 mM), pH 7.4], 70 pl / well of Tyrode's solution is added to each well. Neuronal signaling compounds (Selleckchem, USA) were added at 1 pM using a pin tool. After 45 min of incubation at 37 °C, the supernatants are used to determine NO release using a NO assay kit (Invitrogen, EMSNO). Briefly, the kit uses the enzyme nitrate reductase, which converts nitrate to nitrite, which is later detected as a colored azo dye that absorbs light at 540 nm. NO release for each compound is presented as A540 nm relative to vehicle (DMSO).

[0232] bb. High-throughput screening to identify compounds that enrich in NO neurons Day 15 H9 hESC-derived intestinal crestspheres were dissociated into single cells (Accutase, Stemcell Technologies, 07920, 30 min, 37°C), resuspended in ENC medium and transferred to 384-well plates. Plates were incubated for 2 h to allow cells to attach. Using a pin tool, drugs from a library of 1694 inhibitors (SelleckChem, USA) were added to the wells at a final concentration of 1 pM, plates were incubated with drugs until D20, and medium was changed to drug-free ENC. At day 40, cells were fixed, stained for NOS1 and imaged using an InCellAnalyzer 2000 (GE Healthcare, USA). Hits showed significantly higher NOS1 expression compared to vehicle (DMSO)-treated wells. + Selection was based on the doubling percentage of cells.

[0233] cc. Surface marker screening For human surface marker screening, PP121-treated NOS1::GFP intestinal gangliosides from four independent differentiations were pooled, dissociated into single cells (Accutase, Stemcell Technologies, 07920, 30-60 min, 37 °C, 5% CO2) and fixed (Foxp3 / transcription factor staining buffer set, 00-5523, 30 min, 4 °C). Cells were permeabilized and blocked (same staining kit) before incubation with anti-GFP antibody (abcam, ab13970, 4 °C). After washing three times, cells were stained with Alexa Fluor 488-conjugated secondary antibodies (40 min, RT). The secondary antibody solution was removed (washed three times) and cells were incubated in blocking buffer containing PBS and 2% FBS (30 min, on ice). Cells were split in a ratio of 240:16, corresponding to the number of library antibodies produced in mouse and rat, and received anti-mouse and anti-rat Alexa Fluor 647-conjugated secondary antibodies, respectively. They were then distributed into V-bottom 96-well plates and treated with library antibodies for 30 min on ice (BD Biosciences, 560747). After two washes, surface marker and GFP signals were quantified by high-throughput flow cytometry (BD LSRFortessa). + GFP + Highest percentage of cells) and highest specificity (CD + GFP - Based on the lowest percentage of cells, NO neuron-specific surface markers were identified.

[0234] dd. Drug-target interaction prediction Canonical SMILES of the hits were obtained from PubChem ( De Giorgio et al., 2016 ; Niesler et al., 2021 ), and lists of their known and predicted targets were generated by combining data from the following databases: BindingDB ( https: / / www.bindingdb.org / ), Carlsbad ( http: / / carlsbad.health.unm.edu / ), and DINIES. (https: / / www.genome.jp / tools / dinies / ), PubChem BioAssay (https: / / pubchem.ncbi.nlm.nih.gov / , filtered for active interactions), SEA (http: / / sea.bkslab.org / , filtered for MaxTC > 0.4), SuperDRUG2 (http: / / cheminfo.charite.de / superdrug2 / ), and SwissTargetPrediction (http: / / www.swisstargetprediction.ch / ).

[0235] ee. In vivo cell transplantation Specific pathogen-free (SPF) homozygous neuronal nitric oxide synthase knockout mice (B6.129S4-Nos1 tm1Plh / J, nNos1 - / - ) were bred and maintained for use as recipients in individually ventilated cages (IVC). Animals used in these studies were maintained and experiments carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and approved by the University College London Biological Services Ethical Review Process. Animal husbandry at UCL Biological Services complied with the UK Home Office Designation Certificate Nos. - / - Because the mice were immunocompetent, cyclosporine A (250 μg / ml in drinking water) was administered orally 2 days before transplantation to reduce the possibility of rejection of the donor human cells. - / -Mice were randomly selected within litter groups and stage 1 intestinal ganglioids were implanted into P23–P27 mice via laparotomy under isoflurane anesthesia. Briefly, the distal colon was exposed and then 0.5–1 M cell-laden intestinal ganglioids were implanted onto the serosal surface of the distal colon by mouth pipetting using a pulled glass micropipette. Each implanted tissue typically received three ganglioids that were manipulated onto the surface of the distal colon using a 30 G needle bevel to ensure proper positioning. Implanted Nos1 - / - Mice were maintained for up to 8 weeks post-transplantation with continued free access to cyclosporine A (250 jig / ml)-treated drinking water to ensure prolonged immunosuppression before being sacrificed and the colon removed for analysis. As cyclosporine A can affect several signaling pathways and induce gene expression changes, it will be important to validate the immunofluorescence results using appropriate controls, such as tissues from cyclosporine A-treated non-transplanted animals, in follow-up studies. In addition, other immunodeficient backgrounds (e.g., NSG) will be important to further validate these engraftment results.

[0236] ff. Tissue preparation and fixation After resection, the entire colon was pinned onto a Sylgard (Dow, MI, USA)-lined Petri dish and opened along the mesenteric border, after which the mucosa was removed by sharp dissection and the tissue was fixed in 4% PFA in PBS (45 min to 1 h, 22 °C) for further processing.

[0237] gg.Tissue staining Colonic longitudinal myenteric plexus (LMMP) tissue was fixed (1 h on ice) with 4% PFA (Thermo Scientific, J19943-K2), blocked and permeabilized with a buffer containing 1% BSA and 1% Triton X-100 (in PBS, 45 min, RT). Tissue was then incubated with primary antibody solution (in the same buffer, overnight, 4°C) and washed three times before treatment with fluorophore-conjugated secondary antibodies (1 h, RT). Samples were stained with DAPI and washed using vectashield (Vector Laboratories, H-1400) before mounting. Antibody list not shown.

[0238] hh. Multielectrode array (MEA) analysis Data Acquisition: Neuronal activity was recorded using an Axion Maestro Edge on Cytoview MEA 24-well plates with 1-hour recording sessions for each condition. Neuromodulators or vehicle were added by removing the plate from the Maestro Edge, replacing half of the pre-warmed medium with 2x concentrated neuromodulator or vehicle, immediately placing the plate back into the Axion, and resuming recording. Optogenetic stimulation was performed with an Axion Lumos attachment, stimulating all wells of the plate 30 times with 488 nm light at 50% intensity, 1 second on, 4 seconds off.

[0239] Data processing: Raw data were first sorted in a modified version of SpikeInterface (https: / / github.com / SpikeInterface) using MountainSort to identify high-quality units by manually scoring based on amplitude, waveform shape, firing frequency, and interspike interval contamination. For pharmacology experiments, neurons were matched between vehicle and neuromodulator recordings by examining all units detected on a particular electrode after spike scoring and identifying units with identical waveforms. The firing frequencies of these "paired" units from all wells that received treatment were compared across control and neuromodulator conditions. Positive responders were units with firing frequency changes of more than +0.1 Hz, negative responders had firing frequency changes of less than -0.1 Hz, and neutral responders had firing frequency changes between -0.1 and +0.1 Hz. For optogenetic experiments, individual units were again extracted in SpikeInterface and manually scored. Recordings were divided into "on" times when the LED was active and "off" times when the LED was inactive. All units were pooled together and the firing frequency of each unit was compared during the ON and OFF windows.

[0240] ii. Ex vivo colonic motility assay Preparation of solutions: Krebs buffer [NaCl (117 mM), KCl (4.7 mM), NaH 2 PO 4 (1.2 mM), MgCl 2 (1.5 mM), CaCl 2 .2H 2 O (2.5 mM), NaHCO 3 (25 mM), glucose (11 mM, pH 7.4) were placed in a 37 °C water bath and kept at 95% O before the start of the experiment. 2and 5% CO2 (carbon dioxide) gas mixture for at least 30 minutes. "Drug" treatment solutions were freshly prepared by adding drug compounds to Krebs buffer before data acquisition began. A solution containing NOS1 inhibitor was prepared by adding N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) to the drug solution making "Drug+L-NAME".

[0241] Tissue dissociation: For each experimental replicate, a pair of 8-week-old wild-type C57BL6 mice (male) were placed in a sealed chamber and euthanized using CO2 asphyxiation followed by cervical dislocation. The lower GI tract (cecum and colon) was removed and immediately transferred to charred Krebs buffer at 37°C with fecal material still inside. Adipose tissue and mesentery were removed prior to placing the colon in the organ bath reservoir of a gastrointestinal motility monitor (GIMM) apparatus. The GIMM had two reservoirs allowing for simultaneous acquisition of control and drug-treated colons.

[0242] Experimental setup and procedure: The GIMM was designed based on a previously reported model (Swaminathan et al., 2016). The organ reservoir of the GIMM has two chambers for simultaneous recording of two specimens. It is connected to the working solution kept at 37 °C via a four-channel peristaltic pump (WPI, PERIPRO-4LS). The lower GI tract was harvested and transferred to an organ bath with flowing Krebs buffer. The cecum was pinned at the proximal tip and the distal end of the colon was pinned through the serosa / mesentery. Five 10 min (for the first experiment) or sequential 6 min (for sequential drug treatments in the presence and absence of L-NAME) videos were recorded using IC Capture software (Imaging Source) with a high-resolution monochromatic firewire industrial camera (Imaging Source®, DMK41AF02) connected to a 2 / 3 inch 16 mm f / 1.4 C-mount fixed focus lens (Fujinon HF16SA1). Tissue in the control chamber was exposed only to Krebs solution, while the order of solutions in the experimental chambers was Krebs, drug compound, Krebs (6 min each), L-NAME (2 min), L-NAME in the presence of drug compound (6 min), and Krebs (6 min). Chambers were cleaned after each acquisition.

[0243] Data and statistical analysis: Spatiotemporal maps (STM) of each acquisition were generated using VolumetryG9a (Spear et al., 2018). Slow wave (SW) and colonic migrating motor complex (CMMC) data were generated from the STM. Statistical analysis was performed using PRISM.

[0244] 2. hPSC-derived ENS model identifies regulators of NO neurons that promote colonic motility Given the important role of enteric NO neurons in GI motility and their selective vulnerability in a wide range of congenital and acquired enteric neuropathy (Bodi et al., 2019; Rivera et al., 2011), there is great interest in establishing strategies to modulate their function. Factors that modulate NO neuron activity and increase NO release would facilitate the identification of potential drug targets for the treatment of enteric neuropathy. Therefore, we utilized our scalable ENS culture platform to screen for compounds that induce NO neuron activity.

[0245] A screening strategy for NO neuronal activity was developed based on induction of cFOS expression as a readout. To evaluate cFOS as an accurate readout of neurochemically induced activity, cFOS flow cytometry analysis and MEA neuronal firing measurements were performed in parallel in cultures treated with epinephrine, which is known to stimulate enteric neurons. Epinephrine induced neuronal cFOS expression and led to an increase in the electrical firing of ganglioid neurons. This provides a scalable readout of activity suitable for high-throughput screens. (Figure 1A-C).

[0246] First, we performed a cFos induction screen in which we exposed NOS1::GFP gut gangliosides to a library of 582 neuromodulators (Selleck neuronal signaling library™) and measured co-expression of cFOS and GFP to quantify NO neuronal activity (Figures 2A and 1D). + We identified 20 compounds that increased the proportion of NO neurons in cells with a z-score of >1.5. To identify the mechanisms involved in cFOS expression in NO neurons, we compiled and categorized the list of target proteins, finding multiple shared protein classes. Notably, these proteins converged on serotonin receptors, sodium channels, acetylcholine receptors, glutamate receptors, adrenergic receptors, histamine and opioid receptors, and dopamine receptors (Figure 2B).

[0247] In an independent functional screen, we established a high-throughput readout to assess NO neuronal activity. We utilized a commercially available kit that allows for NO detection in culture medium. Upon release into the medium, NO is spontaneously oxidized to nitrate. The kit uses nitrate reductase to convert nitrate to nitrite, which is later detected as a colored azo dye. 2D ENS cultures were incubated with a neuromodulator library and NO release was measured using calorimetry (Figure 2C). We identified 17 compounds that significantly enhanced NO concentrations in the supernatant, with z-scores >2.0 (Figure 1E). Neuromodulators that induced NO release in ENS cultures were diverse but predicted common target protein classes, including serotonin receptors, sodium channels, acetylcholine receptors, glutamate receptors, adrenergic receptors, and opioid receptors (Figure 2D).

[0248] Interestingly, there was a high degree of similarity between predicted targets from cFOS induction and the NO release screen (Figure 2B, Figure 2D, and Figure 1F). These targets included receptors for neurotransmitters such as serotonin and dopamine. A module scoring neurotransmitter receptor gene families in hPSC-derived stage 1 enteric NO neuron snRNA-seq data confirmed that NO neurons extensively express receptors for NO, serotonin, GABA, glutamate, acetylcholine, or dopamine (Figure 2E). Interestingly, compared to other neuronal subtypes, the stage 1 NO neuron cluster was enriched for all predicted hit targets (Figure 1G). Notably, the NO 3 cluster scored highly for expression of the majority of NO neuron regulator target classes (Figure 1H). Profiling the expression of individual genes of each target protein category in ganglioids and primary human ENS revealed striking subtype-specific expression patterns among NO neurons. For example, GABA receptor genes were expressed primarily by the NO2, NO3, and pNO4 subtypes, whereas acetylcholine receptor gene expression was less specific to a particular subtype (Figure 2F).

[0249] We then selected a subset of hits representing different target classes, prioritizing FDA-approved compounds for follow-up analysis (Figure 2K and Figure 1F). For selected compounds, we performed a more comprehensive and unified target analysis by combining reported experimental data (Combined DB) and computational methods (SEA, Carlsbad, Dinies, Swisstarget, Superdrug, Pubchem Bioassays, Figure 2G). The effects of these compounds were tested on colonic motility in organ bath assays (Figure 2H). In these assays, excised segments of mouse colon were maintained in physiological buffer to study motility patterns using video recordings. In a first experiment, the effects of all selected drug candidates were tested ex vivo on mouse colonic motility (Figure 2I). In each experiment, untreated control and drug-treated colon samples were tested in parallel during five consecutive 10-min acquisitions. Instead of analyzing the fecal output, which is generally variable, for each acquisition, a sophisticated contraction analysis was performed by generating spatiotemporal maps from the video data based on the change in colon diameter over time, which were used to calculate the velocities of colonic migrating motor complexes (CMMCs) and slow waves (SWs). CMMCs are rhythmic propulsive contractions initiated by the ENS, whereas SWs are mediated through the pacemaking activity of interstitial cells of Cajal (Barajas-Lopez and Huizinga, 1989; Burns et al., 1996; Fida et al., 1997; Lyster et al., 1995; Smith et al., 1987). To examine the dynamics of CMMC and SW events during each acquisition, cumulative percentage graphs were generated (Figure 3A-B) and intervals were calculated at the 75th percentile (Figure 2J and Figure 3C). Compounds that showed promising effects on lowering CMMC intervals compared to the untreated condition were selected for follow-up evaluation (i.e., aripiprazole, dexmedetomidine, matrine, MPEP) (Figure 2J and Figure 3A-C). To assess whether the compounds mediated their effects on CMMC by modulating NO release, sequential drug treatments were performed in the presence and absence of the NOS1 inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME).Each experiment consisted of four 6-min acquisitions of five independent sample pairs in control and drug-treated groups (Figure 2K). Of the tested drugs, the adrenergic receptor agonist dexmedetomidine decreased CMMC intervals in four of five colonic samples, an effect that was absent when colons were simultaneously treated with dexmedetomidine + L-NAME (Figure 2L, Figure 4A-F, and Figure 5A-B). SW was not affected by drug treatment (Figure 6A-H). In addition to CMMCs, we quantified the effect of compounds on colonic motility by annotating and measuring anterograde contractile events detected in the spatiotemporal maps. These events were termed "longitudinal contractile events" (LCEs) and are highlighted by representative arrows in Figure 2K. In dexmedetomidine-treated colons, a decrease in the total number of LCEs (Figure 2M) and a trend toward an increase in their mean duration (Figure 2N) were observed in all five replicates. These effects were reversed after drug removal and blocked by L-NAME cotreatment (Figure 2M-N). These results provide a blueprint for leveraging in vitro human ENS models to uncover mechanisms regulating GI motility that can identify therapies targeting specific ENS populations.

[0250] 3. High-throughput small molecule screen reveals PDGFR inhibition as a driver of NO neuronal induction To evaluate the potential of hPSC-derived cultures to model human ENS development, we decided to define the mechanism of NO neuron specification in vitro. We explored pathways regulating NO neuron differentiation and performed a high-throughput small molecule screen. Identifying different pathways and chemical regulators that promote NO neuron induction can provide insights into NO neuron development and provide strategies to derive ENS cultures enriched for NO neurons.

[0251] To identify compounds that induce NO neuron differentiation, we treated enteroclastospheres with 1694 compounds from the Selleck inhibitor library™ to induce NOS1 +We identified 12 hit compounds that increased the ratio of NO neurons by at least 8-fold (Figure 7A and Figure 8A-B). To clarify the mechanism by which these hit compounds enhanced NO neuronal induction, we performed target prediction analysis by combining reported experimental data (JointDB) and computational methods (SEA, Carlsbad, Dinies, Swisstarget, Superdrug, Pubchem Bioassays) (Figure 7B). After clustering the predicted protein targets, common patterns emerged for a subset of compounds. For example, PP121, ibrutinib, afatinib, and AMG-458 were all predicted to interact with EGFR, ERBB, MAP, and TEC family kinases, among others (Figure 7B). For follow-up analysis, we selected the compounds with the highest %NOS1 in this subset of compounds. + We chose to focus on PP121, a hit with a fold increase. PP121 showed a dose-dependent effect on NO neuron induction efficiency as measured by flow cytometry (Figure 8C). To find the most effective treatment period for PP121-induced NO neuron induction, differentiating cultures were treated for 5 days at various time points. Measurement of GFP signal in stage 1 NOS1::GFP intestinal gangliosides showed the highest induction efficiency for cells treated on days 15-20 (Figures 7C-D and 8D).

[0252] To make the enrichment protocol reliable, it was important to ensure that PP121 treatment did not alter cell type identity. To compare PP121-treated and untreated stage 1 intestinal ganglioids at single-cell resolution, snRNA-seq was performed and both datasets were combined. This analysis revealed that all cell types were represented in both conditions (Figure 7E-F and Figure 9A). Importantly, comparison of the average expression of all genes for matched PP121-treated and untreated cell types showed highly similar transcriptomes (R2 correlation >0.91), indicating that PP121 treatment did not alter the transcriptional identity of cell types (Figure 9B). Interestingly, subclustering of the combined control and PP121-treated neurons revealed nine neuronal subtypes, EN AI (Figure 7G). The PP121-treated dataset subtypes showed high transcriptional similarity to EN1-8 in the control-only dataset (Figure 7H). EN cluster I, consisting mainly of PP121-treated cells and few control cells, showed moderate transcriptional similarity with control-only EN cluster 4, suggesting that this neuronal subtype was present but rare in control cultures, clustering these neurons with the most similar subtype, EN 4 (Figures 7H and 9C). Along with EN I, which was approximately 25% nitrergic, PP121 treatment also enriched the cultures for neuronal subtypes EN D and H (approximately 50% and 25% nitrergic, respectively), whereas EN A and G were less represented (Figures 7I and 9C). Again, despite changes in subtype abundance, control and PP121-treated neurons of the same subtype showed similar transcriptomes (R2 correlation > 0.88) (Figure 9D). Further subclustering of combined control and PP121-treated nitrergic neurons revealed enrichment for nitrergic B (most similar to control-only nitrergic 2) and a rare population, nitrergic C (most similar to control-only nitrergic 3) (Figure 7K and Figure S9E).Comparison of the transcriptomes again showed very similar gene expression of control and PP121-treated nitrergic neurons of the same subtype (R2 correlation > 0.7), with the greatest variance between nitrergic C neurons, likely due to the small number of neurons in this cluster (Figure 9F). Taken together, this data suggests that early ganglioside treatment with PP121 induces changes in the abundance of neuronal subtypes normally found in untreated cultures, without affecting the gene expression patterns of the resulting subtypes.

[0253] The ability to purify enteric NO neurons is of great interest, especially for applications such as cell therapy. Access to the NOS1::GFP reporter line and the ability to direct differentiation towards NO neurons using PP121 allowed us to search for FACS-compatible surface markers on these cells. A panel of 242 antibodies was screened for human cell surface molecules (BD lyoplate) and GFP and surface antigen expression signals were measured by flow cytometry (Figure 10A). 27 antibodies were identified that stained at least 50% of NOS1 neurons (GFP + %CD in the population + GFP + , Fig. 10B, top). To identify the most specific candidates among these hits, we selected hits with CD > 70%. + GFP + Against CD +We identified antibodies with staining ratios (Figure 10B, bottom). CD47, CD49e, CD59, CD90, and CD181 fulfilled both criteria (Figure 10C). Expression and enrichment of CD47, CD49e, CD59, and CD90 was further confirmed in stage 1 ganglioid and NO neuron clusters in human primary snRNA-seq datasets (Figure 10D-E). As an example, localization of CD47 was further confirmed in NO neurons in human primary colonic myogangliomas using immunohistochemistry (Figure 10F). In addition to identifying antibodies that specifically enrich for NO neurons, we found 12 antibodies that stained >70% of ganglioside cells and could serve as pan-enteric neuron surface markers (CD24, CD45RA, CD57, CD63, CD71, CD121b, CD147, CD164, CD184, CD193, CD243, CD275) (Figure S10G). Enriched expression of CD24 was also confirmed in enteric neurons (snRNA-seq data) and primary human colonic muscle ganglions (Figure S10H-I).

[0254] To determine the mechanism by which PP121 induced NO neuronal enrichment in gangliosides, a combination of pharmacological and genetic approaches was used. PP121 is a multitargeted receptor tyrosine kinase (RTK) inhibitor with known inhibitory activity against PDGFR, VEGFR, and EGFR (Apsel et al., 2008). CrestSphere snRNA-seq analysis confirmed expression of PDGFRA, PDGFRB, ERBB2, and ERBB3, whereas VEGFR mRNA was undetectable (Figure 7L). The efficiency of induction of NO neurons in response to PDGF (PDGFR agonist), sunitinib (PDGFR and VEGFR antagonist), NRG1 (ERBB agonist), and sapitinib (ERBB antagonist) was assessed (Figure 7M). NRG1 and sapitinib had no significant effect on NO neuron induction, whereas treatment with PDGFR and sunitinib resulted in lower and higher NO neuron ratios, respectively (Figure 7N). To genetically confirm the role of PDGFR signaling in NO neuron induction, we used CRISPR-Cas9 to knock out PDGFRA and PDGFRB in intestinal crestspheres and analyzed the percentage of NO neurons in stage 1 gangliosides. In both PDGFRA and PDGFRB knockout cultures, NO neurons were enriched, further confirming the mechanism of action of PP121 (Figure 7O-P).

[0255] 4. hESC-derived NOS1 neurons express NOS1 - / - Engraftment in mouse colon Developing an experimental system to study the human ENS in vivo will open up a wide range of basic science and clinical opportunities. For example, human ENS xenografts would enable the study of human neuronal circuits in vivo, as well as the investigation of ENS-CNS and ENS-immune system-microbiome communication. They also provide a platform for disease modeling and drug development. In addition, the limited regenerative capacity of the ENS highlights the importance of developing cell therapy approaches to replace lost populations of neurons. Currently, there are no clinical interventions to replace damaged or lost neurons caused by inherited and acquired ENS pathologies such as Hirschsprung's disease and diabetes. It has previously been shown that hPSC-derived ENC precursors can successfully engraft in vivo (Fattahi et al., 2016). McCann et al. also demonstrated that Nos1 - / - We have shown that transplanted ex vivo cultured mouse neurospheres can rescue GI motility defects in mice (McCann et al., 2017). However, these neurospheres are a heterogeneous population containing only a small percentage of NO neurons. Furthermore, obtaining sufficient numbers of neurospheres from human primary tissue poses a significant limitation for eventual regenerative applications. Compared to ENC precursors, transplantation of mature neurons provides a postmitotic source of cells with a low clinical risk of tumor formation. Obtaining highly enriched NO neuron cultures facilitated the transplantability of intestinal gangliosides evaluated. PP121-treated intestinal gangliosides were compared with Nos1. - / - (B6.129S4-Nos1 tm1Plh / J ) into the wall of the distal colon of mice. Eight weeks after surgery, animals were sacrificed and colonic longitudinal myenteric plexus (LMMP) preparations were evaluated by fluorescence microscopy (Figure 11A). Transplanted cells were distinguished by expression of the human cytoplasmic marker SC121. Notably, there was a significant number of SC121 cells uniformly distributed along the length of the colon. +Cells were observed (FIG. 11B). Engrafted cells were detected both within and outside of myenteric ganglia, and many expressed NOS1, confirming NO fate commitment (FIGS. 11C and 12). In addition to clinically significant cell therapy applications, the developed human intestinal ganglioid xenografts provide previously unattainable opportunities for understanding the development, physiology, and pathophysiology of the human ENS in vivo.

[0256] 5. Discussion An exceptional advantage of hPSC-derived cultures is their scalability. This is especially important when the desired cell type is rare and has very limited regenerative and proliferation capacity, such as neural tissue. The ENS culture platform used herein has repeatedly proven reliable in providing a scalable source of ENS cell types compatible with applications that would be very challenging to implement, such as high-throughput screens. In particular, we used 2D ENS cultures to screen thousands of inhibitors to identify compounds that direct differentiation towards clinically valuable NO neurons. Examination of the mechanism of action of the top hits revealed pathways that are important in NO neuron fate determination specification. Using a combination of pharmacological and genetic approaches, we discovered that one such pathway, PDGFR signaling, contributes to inducing NO neurons, highlighting the remarkable potential of hPSC-based platforms to uncover developmental mechanisms.

[0257] Herein, a functional screening platform is used to reveal candidate drugs that specifically modulate the activity of NO neurons. Interestingly, the hit compounds generally target adrenergic, cholinergic, and serotonergic receptors, as well as sodium channels. Notably, these targets are over-represented in NO neurons, highlighting the specificity of these compounds and their potential for further therapeutic development for GI indications. By testing a subset of these neuromodulators, it was further demonstrated that these candidate drugs are capable of affecting colonic motility patterns in an ex vivo organ bath assay. This is the first example of identifying a candidate drug for modulating GI motility by targeting a specific enteric neuron subtype. These findings demonstrate the reliability, robustness, and scalability of the hPSC-derived ENS model.

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[0322] It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the disclosure. Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the disclosure being indicated by the following claims.

Claims

1. A pharmaceutical composition for use in the treatment of bowel motility disorders in a subject, comprising an effective amount of at least one modulator of nitric oxide (NO) neuronal activity or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to claim 1, wherein at least one modulator of NO neuronal activity is selected from a modulator of COX, a modulator of dopamine receptors, a modulator of sodium channels, a modulator of serotonin receptors, a modulator of acetylcholine receptors, a modulator of GABA receptors, a modulator of FAAH, a modulator of adrenaline receptors, a modulator of histamine receptors, a modulator of vasopressin receptors, a modulator of NMDAR, a modulator of beta-amyloid, a modulator of gamma-secretase, a modulator of IxB / IKK, a modulator of glutamate receptors, a modulator of opioid receptors, a modulator of TRPV, a modulator of aldose reductase, a modulator of calcium channels, a modulator of glucocorticoid receptors, a modulator of HMG-CoA reductase, and pharmaceutically acceptable salts thereof.

3. The at least one modulator of NO neuronal activity is carprofen, mefenamic acid, phenacetin, valdecoxib, phenoldopam mesylate, fluphenazine hydrochloride, bupivacaine HCl, phenazopyridine HCl, alberin cyanide, nitenpyram, 4-aminobutyric acid (GABA), PF-3845, esmolol HCl, cimetidine, conivaptan HCl, (+)-MK-801 maleate, MK-0752, R0492 The pharmaceutical composition according to claim 1, selected from 9097, rosmarinic acid, theophylline, aripiprazole, flopropion, latrepyridine 2HCl, ADX-47273, MPEP, nehopam HCl, phenazopyridine HCl, epinephrine, (+)-matrine, phenothiazine, naproxen sodium, AMG-517, isoliquitigenin, nilvadipine, prednisone, simvastatin, and pharmaceutically acceptable salts thereof.

4. The pharmaceutical composition according to claim 1, wherein at least one modulator of NO neuronal activity is selected from aripiprazole, dexmedetomidine, matrine, MPEP, and pharmaceutically acceptable salts thereof.

5. The pharmaceutical composition according to claim 1, wherein at least one modulator of NO neuronal activity is dexmedetomidine or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition according to claim 1, wherein the bowel motility disorder is selected from atonostomy, Hirschsprung's disease, pseudo-obstruction, gastroesophageal reflux disease (GERD), functional dysphagia, functional indigestion, irritable bowel syndrome (IBS), gastroparesis, functional constipation, functional diarrhea, and fecal incontinence.

7. The pharmaceutical composition according to claim 1, wherein the subject is a human.

8. The pharmaceutical composition according to claim 1, wherein, prior to administration of the pharmaceutical composition, the subject has been diagnosed as needing treatment for the intestinal motility disorder.

9. The pharmaceutical composition according to claim 1, wherein the effective amount is a therapeutically effective amount.

10. The pharmaceutical composition according to claim 1, wherein the effective amount is a preventive effective amount.

11. A pharmaceutical composition for regulating NO neuron activity in a subject having bowel motility disorders, comprising an effective amount of at least one modulator of NO neuron activity or a pharmaceutically acceptable salt thereof.

12. The pharmaceutical composition according to claim 11, wherein at least one modulator of NO neuronal activity is selected from a modulator of COX, a modulator of dopamine receptors, a modulator of sodium channels, a modulator of serotonin receptors, a modulator of acetylcholine receptors, a modulator of GABA receptors, a modulator of FAAH, a modulator of adrenaline receptors, a modulator of histamine receptors, a modulator of vasopressin receptors, a modulator of NMDAR, a modulator of beta-amyloid, a modulator of gamma-secretase, a modulator of IxB / IKK, a modulator of glutamate receptors, a modulator of opioid receptors, a modulator of TRPV, a modulator of aldose reductase, a modulator of calcium channels, a modulator of glucocorticoid receptors, a modulator of HMG-CoA reductase, and pharmaceutically acceptable salts thereof.

13. The at least one modulator of NO neuronal activity is aripiprazole, dexmedetomidine, matrine, MPEP, carprofen, mefenamic acid, phenacetin, valdecoxib, phenoldopam mesylate, fluphenazine hydrochloride, bupivacaine HCl, phenazopyridine HCl, alberin cyanide, nitenpyram, 4-aminobutyric acid (GABA), PF-3845, esmolol HCl, cimetidine, conivaptan HCl, (+)-MK-80 The pharmaceutical composition according to claim 11, selected from 1-maleate, MK-0752, R04929097, rosmarinic acid, theophylline, phlopropion, latrepyridine 2HCl, ADX-47273, nehopam HCl, phenazopyridine HCl, epinephrine, (+)-matrine, phenothiazine, naproxen sodium, AMG-517, isoliquitigenin, nilvadipine, prednisone, simvastatin, and pharmaceutically acceptable salts thereof.

14. The pharmaceutical composition according to claim 11, wherein at least one modulator of NO neuronal activity is dexmedetomidine or a pharmaceutically acceptable salt thereof.

15. The pharmaceutical composition according to claim 11, wherein regulating NO neuron activity induces colonic motility.

16. At least one regulator of NO neuronal activity, (a) an agent for treating a bowel motility disorder, (b) a prescription for treating the bowel motility disorder, and (c) one or a combination selected from the above for administering at least one modulator of NO neuronal activity in connection with treating the bowel motility disorder. A kit that includes this.

17. The kit according to claim 16, wherein at least one modulator of NO neuronal activity is selected from a modulator of COX, a modulator of dopamine receptors, a modulator of sodium channels, a modulator of serotonin receptors, a modulator of acetylcholine receptors, a modulator of GABA receptors, a modulator of FAAH, a modulator of adrenaline receptors, a modulator of histamine receptors, a modulator of vasopressin receptors, a modulator of NMDAR, a modulator of beta-amyloid, a modulator of gamma-secretase, a modulator of IxB / IKK, a modulator of glutamate receptors, a modulator of opioid receptors, a modulator of TRPV, a modulator of aldose reductase, a modulator of calcium channels, a modulator of glucocorticoid receptors, a modulator of HMG-CoA reductase, and pharmaceutically acceptable salts thereof.

18. The at least one modulator of NO neuronal activity is aripiprazole, dexmedetomidine, matrine, MPEP, carprofen, mefenamic acid, phenacetin, valdecoxib, phenoldopam mesylate, fluphenazine hydrochloride, bupivacaine HCl, phenazopyridine HCl, alberin cyanide, nitenpyram, 4-aminobutyric acid (GABA), PF-3845, esmolol HCl, cimetidine, conivaptan HCl, (+)-MK- A kit according to claim 16, selected from 801 maleate, MK-0752, R04929097, rosmarinic acid, theophylline, phlopropion, latrepyridine 2HCl, ADX-47273, nehopam HCl, phenazopyridine HCl, epinephrine, (+)-matrine, phenothiazine, naproxen sodium, AMG-517, isoliquitigenin, nilvadipine, prednisone, simvastatin, and pharmaceutically acceptable salts thereof.

19. The kit according to claim 16, wherein the drug is selected from parasympathetic stimulants, exercise promoters, opioid antagonists, antidiarrheal agents, and antibiotics.

20. The kit according to claim 16, wherein at least one regulator of NO neuron activity and the drug are co-formulated.

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