Combination therapy for the treatment of neuropathy

Abstract

The present application relates to a method for treating a human subject suffering from an enteric neuropathy or enteric hypoganglionosis, such as Hirschsprung's disease (HSCR), by administration of an effective dose of a combination comprising a glial cell line-derived neurotrophic factor (GDNF) polypeptide and a short chain fatty acid, such as butyrate.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 202,549, filed Jun. 16, 2021, which is incorporated herein by reference.

[0002] The present disclosure generally relates to the treatment of intestinal neuropathies such as Hirschsprung's disease (HSCR) and intestinal aganglionosis.

Background Art

[0003] The enteric nervous system (ENS) extends along the entire gastrointestinal tract and controls intestinal motility, blood flow, and epithelial activity in response to sensory stimuli (1). Interconnected intestinal ganglia containing neurons and glia develop from neural crest - derived precursors that migrate through the gut during prenatal development. Incomplete colonization of the distal colon by ENS precursors causes Hirschsprung's disease (HSCR), a condition that affects 1 in 5000 newborns (2, 3). In HSCR, the distal colon without neuronal ganglia (i.e., the aganglionic colon) remains in a tonic contraction, does not propagate contractions, and causes functional intestinal obstruction. Symptoms of HSCR include refractory constipation with fecal and air retention, abdominal distension, failure to thrive, occasional vomiting, intestinal inflammation (enteritis), and the risk of bacterial translocation into the bloodstream, which can cause sepsis and early death (2).

[0004] HSCR is clinically subdivided into short-range (S-HSCR) and long-range (L-HSCR) (4). S-HSCR, which occurs in >80% of cases, means that ENS is absent in the rectum and sigmoid colon. L-HSCR means that the longer region of the distal intestine is aganglionate. The etiology of HSCR is still not fully understood, and many genes influence HSCR risk (2). Furthermore, genetically risky variants, in combination with non-genetic factors, can inhibit complete intestinal colonization by ENS precursors (5). This non-Mendelian inheritance occurs because many proteins need to function together for normal ENS development.

[0005] Since 1948, most children with HSCR have been saved by surgical removal of the distal aganglionic duct (16, 17). However, this procedure is far from ideal. Postoperative complications are common and can be long-lasting, affecting survival rates (e.g., enteritis) and / or quality of life (e.g., fecal incontinence or obstructive symptoms) (18-20). One promising approach would be "regenerative medicine" to reconstruct the ENS and reduce the need for surgery. This idea has led many groups to develop HSCR therapies based on cell transplantation (21). However, despite many promising results, several challenges still remain (22). The optimal source of stem cells, ideal amplification and / or differentiation strategies before transplantation, methods of cell delivery, and post-transplant cell function and final outcomes are still not well defined. In addition, non-autonomous cell transplantation may require immunosuppression.

[0006] Gangliocellular dysplasia, also known as enteric gangliocellular dysplasia, is a disorder that causes a reduction in the number of nerve cells in the intestinal wall. Enteric gangliocellular dysplasia can closely resemble HSCR, and patients with both conditions may present with chronic constipation, intestinal obstruction, and colitis (inflammation of the intestines). Patients with gangliocellular dysplasia may also suffer from severe complications, including faecaloma (hardening of the stool in the colon), intestinal bleeding or perforation, and respiratory distress resulting from colonic distension. The exact cause of gangliocellular dysplasia is often unknown. In some cases, it is due to factors present at birth (congenital), but in other cases, it is thought to be an acquired condition. Management of isolated gangliocellular dysplasia generally involves surgery to remove the affected area of ​​the intestine.

[0007] Therefore, there is a clear need for alternative treatments for intestinal neuropathy such as HSCR and enteric ganglion cell dysplasia, and for treatments aimed at inducing neurogenesis in the distal colon, particularly to restore distal colon motility in patients with HSCR and enteric ganglion cell dysplasia.

[0008] This document references several sources, the contents of which are incorporated herein by reference in their entirety. [Overview of the Initiative]

[0009] This disclosure relates to the use of GDNF and short-chain fatty acids for the treatment of one or more pathological features of intestinal neuropathy, such as Hirschsprung's disease.

[0010] In aspects and embodiments, this disclosure relates to the following clauses 1 to 50: 1. A method for treating a human subject suffering from intestinal neuropathy, comprising administering to the subject an effective amount of (i) glial cell line-derived neurotrophic factor (GDNF) polypeptide and (ii) short-chain fatty acids (SCFAs), or pharmaceutically acceptable salts or esters thereof. 2. The method according to Clause 1 (Figure 3A), wherein the GDNF polypeptide comprises an amino acid sequence having at least 70% identity with amino acids 78-211 of Sequence ID No. 1. 3. The method according to Clause 2 (Figure 3A), wherein the GDNF polypeptide comprises an amino acid sequence having at least 90% identity with amino acids 78-211 of SEQ ID NO: 1. 4. The method according to Clause 3 (Figure 3A), wherein the GDNF polypeptide comprises an amino acid sequence having at least 95% identity with amino acids 78-211 of SEQ ID NO: 1. 5. The method according to clause 4, wherein the GDNF polypeptide contains amino acids 78-211 of SEQ ID NO: 1 (Figure 3A). 6. The method according to any one of clauses 1 to 5, wherein an effective dose of GDNF polypeptide administered to a human subject corresponds to a dose of approximately 5 μg to approximately 20 μg in a mouse pup. 7. The method by which an SCFA is a C3-C5 SCFA as described in any one of clauses 1-6. 8. The method according to any one of clauses 1 to 7, wherein SCFA is butyric acid. 9. The method according to any one of clauses 1 to 8, wherein the GDNF polypeptide and / or SCFA is present in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. 10. The method according to Clause 9, wherein the pharmaceutically acceptable carrier comprises a physiological saline solution or a gelling agent. 11. The method according to any one of clauses 1 to 10, wherein the GDNF polypeptide and / or SCFA is administered rectally via enema. 12. The method according to any one of the claims 1 to 10, wherein GDNF polypeptide and / or SCFA are administered by injection into the distal colon wall. 13. The method according to any one of clauses 1 to 12, wherein GDNF polypeptide and / or SCFA are administered once to a maximum of four times daily. 14. The method according to any one of clauses 1 to 13, wherein GDNF polypeptide and / or SCFA are administered for at least two consecutive days. 15. The method according to any one of the provisions of Clauses 1 to 14, wherein the method is performed prior to the surgical removal of an aganglionic area or an area with few ganglion cells in the subject. 16. The method according to any one of the provisions of 1 to 15, wherein the method is performed after surgical removal of an aganglionic area or an area with few ganglion cells in the subject. 17. The method of Clause 15 or 16, wherein surgical removal of an aganglionic area or a ganglionocyte-deficient area is performed via a pull-through procedure. 18. The method according to any one of the clauses 1 to 17, wherein the intestinal neuropathy is enteric ganglion cell dysplasia. 19. Intestinal neuropathy is Hirschsprung's disease (HSCR) as described in any one of the methods described in clauses 1 through 17. 20. The method described in Clause 19, for subjects suffering from short-range HSCR. 21. The method according to Clause 16 or 17, wherein the HSCR is a collagen VI-related HSCR. 22. The method described in any one of the clauses 1 to 21, wherein the human subject is under 5 years of age. 23. The method of the procedure described in Clause 22, wherein the human subject is less than six months old. 24. The method according to any one of the claims 1 to 23, wherein GDNF polypeptide and / or SCFA are administered rectally and / or to the sigmoid colon. 25. The method according to Clause 29, wherein the pharmaceutical composition is administered to or intended for administration to the rectosigmoid region. 26. A combination for use in the treatment of human subjects suffering from intestinal neuropathy, comprising (i) a GDNF polypeptide and (ii) SCFA, or a pharmaceutically acceptable salt or ester thereof. 27. A combination for use as described in Clause 26 (Figure 3A), wherein the GDNF polypeptide comprises an amino acid sequence having at least 70% identity with amino acids 78-211 of SEQ ID NO: 1. 28. A combination for use as described in Clause 27 (Figure 3A), wherein the GDNF polypeptide comprises an amino acid sequence having at least 90% identity with amino acids 78-211 of SEQ ID NO: 1. 29. A combination for use as described in Clause 28 (Figure 3A), wherein the GDNF polypeptide comprises an amino acid sequence having at least 95% identity with amino acids 78-211 of SEQ ID NO: 1. 30. GDNF polypeptide combination for use as described in Clause 29, comprising amino acids 78-211 of SEQ ID NO: 1 (Figure 3A). 31. A combination of uses described in any one of clauses 26 to 30, wherein the dose of recombinant GDNF polypeptide used corresponds to a dose of approximately 5 μg to approximately 20 μg in a mouse pup. 32. A combination of SCFAs for use as described in any one of the clauses 26-31, where the SCFA is a C3-C5 SCFA. 33. SCFA is butyric acid, in combination for use as described in any one of clauses 26 to 32. 34. A combination for use according to any one of clauses 26 to 33, wherein the GDNF polypeptide and / or SCFA is present in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. 35. A combination for use as described in Clause 34, wherein the pharmaceutically acceptable carrier comprises a physiological saline solution or a gelling agent. 36. GDNF polypeptide and / or SCFA are administered rectally via enema, in combination for use as described in any one of clauses 26-35. 37. GDNF polypeptide and / or SCFA are administered by injection into the distal colon wall, in combination for use as described in any one of clauses 26-35. 38. GDNF polypeptide and / or SCFA in combination for use as described in any one of clauses 26 to 37, administered once to a maximum of four times daily. 39. A combination of GDNF polypeptide and / or SCFA for use as described in any one of clauses 26-38, administered for at least two consecutive days. 40. A combination for use as described in any one of clauses 26 to 39, which is intended for use prior to the surgical removal of an aganglionic or ganglionocyte-deficient area in the subject. 41. The combination according to any one of clauses 26 to 40, for use after surgical removal of an aganglionic region or a hypoganglionosis region in a subject. 42. The method according to clause 40 or 41, wherein the surgical removal of the aganglionic region or the hypoganglionosis region is through a pull-through operation. 43. The combination for use according to any one of clauses 26 to 42, wherein the intestinal neuropathy is intestinal hypoganglionosis. 44. The combination for use according to any one of clauses 26 to 42, wherein the intestinal neuropathy is Hirschsprung's disease (HSCR). 45. The combination for use according to clause 44, wherein the subject has short-segment HSCR. 46. The combination for use according to clause 44 or 45, wherein HSCR is collagen VI-related HSCR. 47. The combination for use according to any one of clauses 26 to 46, wherein the human subject is less than 5 years old. 48. The combination for use according to clause 47, wherein the human subject is less than 6 months old after birth. 49. The combination for use according to any one of clauses 26 to 48, wherein the GDNF polypeptide and / or SCFA is for administration to the rectum and / or sigmoid colon. 50. The combination for use according to clause 49, wherein the GDNF polypeptide and / or SCFA is for administration to the rectal sigmoid region.

[0011] Other objects, advantages, and features of the present invention will become more apparent upon reading the following non-limiting description of specific embodiments, given by way of example only with reference to the accompanying drawings. [[ID=2३]]

Brief Description of the Drawings

[0012] In the accompanying drawings, [Figure 1A]This graph shows the survival of homozygous Holstein Tg / Tg mice after administration of vehicle, butyrate (5 mM), GDNF (1 μg / μl), and a combination of butyrate and GDNF. [Figure 1B] Figure 1A shows the statistical analysis of the survival assay. [Figure 1C] Figure 1A shows the survival rate at a specific point in time. [Figure 2A] This graph shows the survival of homozygous Holstein Tg / Tg mice after administration of GDNF alone or in combination with various neurotrophic factors. [Figure 2B] Figure 2A shows the survival rate at a specific point in time. [Figure 2C] Figure 2A shows the statistical analysis of the survival assay. [Figure 3A] The amino acid sequence of human GDNF isoform 1 (UniProtKB accession number P39905, SEQ ID NO: 1) is shown below. The sequence corresponding to the signal peptide (residues 1-19) is underlined, the sequence corresponding to the propeptide (residues 20-75) is italicized, and the sequence corresponding to the mature polypeptide (residues 78-211) is in bold. [Figure 3B] The nucleotide sequence of the cDNA encoding human GDNF isoform 1 (RefSeq accession number NM_000514.4, SEQ ID NO: 2) is shown, with the sequence encoding the signal peptide (nucleotides 562-618) underlined, the sequence encoding the propeptide (nucleotides 619-786) italicized, and the sequence encoding the mature polypeptide (nucleotides 793-1194) in bold. [Figure 3C] The nucleotide sequence of the cDNA encoding human GDNF isoform 1 (RefSeq accession number NM_000514.4, SEQ ID NO: 2) is shown, with the sequence encoding the signal peptide (nucleotides 562-618) underlined, the sequence encoding the propeptide (nucleotides 619-786) italicized, and the sequence encoding the mature polypeptide (nucleotides 793-1194) in bold.

[0013] Disclosure of the invention In the context of describing the technology (particularly in the context of the following claims), the use of the terms "a," "an," and "the," and similar references, is to be interpreted as encompassing both singular and plural forms, unless otherwise indicated herein or unless it is clearly inconsistent with the context.

[0014] The terms “comprising,” “possessing,” “including,” and “containing” are to be interpreted as open-ended terms (i.e., “including, but not limited to”) unless otherwise noted.

[0015] All methods described herein may be carried out in any preferred order, unless otherwise indicated herein or unless the context clearly contradicts it.

[0016] Any and all embodiments provided herein, or the use of exemplary language ("e.g.", "etc."), are intended merely to better illustrate embodiments of the claimed technology and, unless otherwise claimed, do not constitute a limitation of scope.

[0017] No language in this specification should be construed as indicating that any non-claimed element is essential to the implementation of the claimed embodiment of the technology.

[0018] In this specification, the term “approximately” has its usual meaning. The term “approximately” is used to indicate that a value includes inherent variability due to errors in the device or method used to determine the value, or that it includes values ​​close to the enumerated values, for example, values ​​within 10% of the enumerated values ​​(or range of values).

[0019] References to ranges of values ​​in this specification are intended simply as a way of referring individually to each individual value within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually referred to herein. All subsets of values ​​within a range are also incorporated herein as if they were individually enumerated herein.

[0020] If the features or aspects of the disclosure are described in terms of the Markush group or list of substitutes, a person skilled in the art will recognize that the disclosure is also described in terms of any individual component or subgroup of components of the Markush group or list of substitutes.

[0021] Unless otherwise specifically defined, all technical and scientific terms used herein should be interpreted as having the same meaning as commonly understood by those skilled in the art (for example, in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0022] Unless otherwise indicated, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology, A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors), DNA Cloning, A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revisions to date), Ed Harlow and David Lane (editors), Antibodies, A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JEColigan et al. This is described and explained throughout the literature in sources such as al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all revisions to date).

[0023] In the studies described herein using a mouse model of intestinal neuropathy (e.g., Hirschsprung's disease (HSCR)), the inventors demonstrate that administration of recombinant GDNF in combination with short-chain fatty acid (SCFA) butyrate in the distal colon significantly improves survival. Butyrate alone showed no effect on survival, but significantly enhanced the effect of recombinant GDNF. These results provide evidence that administration of recombinant GDNF in combination with short-chain fatty acids can be used for the treatment of intestinal neuropathy (e.g., ENS deficiency such as HSCR), i.e., for the improvement of one or more pathological features of intestinal neuropathy and / or survival in human patients.

[0024] Accordingly, in a first aspect, the Disclosure provides a method for treating a human subject suffering from intestinal neuropathy (e.g., Hirschsprung's disease (HSCR) or enteric ganglion cell atrophy), comprising administering to the subject an effective amount of (i) glial cell line-derived neurotrophic factor (GDNF) and (ii) short-chain fatty acids (SCFA), or pharmaceutically acceptable salts or esters thereof. The Disclosure also provides the use of (i) GDNF and (ii) SCFA, or pharmaceutically acceptable salts or esters thereof, for treating a human subject suffering from intestinal neuropathy (e.g., Hirschsprung's disease (HSCR) or enteric ganglion cell atrophy). The Disclosure also provides the use of (i) GDNF and (ii) SCFA, or pharmaceutically acceptable salts or esters thereof, for the manufacture of a drug for treating a human subject suffering from intestinal neuropathy (e.g., Hirschsprung's disease (HSCR) or enteric ganglion cell atrophy). The disclosure also provides combinations for use in the treatment of human subjects suffering from intestinal neuropathy (e.g., Hirschsprung's disease (HSCR) or enteric ganglion cell dysplasia), comprising (i) GDNF and (ii) SCFA, or pharmaceutically acceptable salts or esters thereof.

[0025] The disclosure also provides a method for restoring distal colon motility and / or epithelial barrier in human subjects suffering from intestinal neuropathy (e.g., HSCR or enteric ganglion cell dysplasia), comprising administering an effective amount of (i) glial cell line-derived neurotrophic factor (GDNF) and (ii) short-chain fatty acids (SCFA), or pharmaceutically acceptable salts or esters thereof. The disclosure also provides the use of an effective amount of (i) GDNF and (ii) short-chain fatty acids (SCFA), or pharmaceutically acceptable salts or esters thereof, for restoring distal colon motility in human subjects suffering from intestinal neuropathy (e.g., HSCR or enteric ganglion cell dysplasia). The disclosure also provides the use of effective amounts of (i) GDNF and (ii) short-chain fatty acids (SCFAs), or pharmaceutically acceptable salts or esters thereof, for the manufacture of agents for restoring distal colon motility in human subjects suffering from intestinal neuropathy (e.g., HSCR or enteric ganglion cell dysplasia). The disclosure also provides combinations for use in restoring distal colon motility in human subjects suffering from intestinal neuropathy (e.g., HSCR or enteric ganglion cell dysplasia), comprising (i) GDNF and (ii) SCFAs, or pharmaceutically acceptable salts or esters thereof.

[0026] The Disclosure also provides a method for inducing intestinal neurogenesis in an aganglionic or ganglionocyte-deficient area of ​​the distal colon of a human subject suffering from intestinal neuropathy (e.g., HSCR or enteric ganglion cell deficiency), comprising administering an effective amount of (i) GDNF and (ii) short-chain fatty acids (SCFAs), or pharmaceutically acceptable salts or esters thereof. The Disclosure also provides a use of (i) GDNF and (ii) short-chain fatty acids (SCFAs), or pharmaceutically acceptable salts or esters thereof, for inducing intestinal neurogenesis in an aganglionic or ganglionocyte-deficient area of ​​the distal colon of a human subject suffering from intestinal neuropathy (e.g., HSCR or enteric ganglion cell deficiency), wherein the composition is for administration to the distal colon of the subject. The disclosure also provides the use of (i) GDNF and (ii) short-chain fatty acids (SCFAs), or pharmaceutically acceptable salts or esters thereof, for the manufacture of agents for inducing intestinal neurogenesis in aganglionic or ganglionocyte-deficient areas of the distal colon in human subjects suffering from intestinal neuropathy (e.g., HSCR or enteric ganglionocyte-deficient areas). The disclosure also provides combinations for use in inducing intestinal neurogenesis in aganglionic or ganglionocyte-deficient areas of the distal colon in human subjects suffering from intestinal neuropathy (e.g., HSCR or enteric ganglionocyte-deficient areas), comprising (i) GDNF and (ii) SCFAs, or pharmaceutically acceptable salts or esters thereof.

[0027] As used herein, the term “short-chain fatty acids” (SCFAs) refers to fatty acids with 2 to 5 carbon atoms, including acetic acid (C2), propionic acid (C3), butyric acid and isobutyric acid (C4), as well as valeric acid, isovaleric acid and 2-methylbutanoic acid (C5). pharmaceutically acceptable salts of SCFAs, such as pharmaceutically acceptable acetates, propionates, butyrates, isobutyrates, valiates, isovalerates, and 2-methylbutanoates, may also be administered / used in the methods and uses described herein. As used herein, the term “pharmaceutically acceptable salt” is intended to mean a salt that retains the biological efficacy and properties of the free acid and is not biologically or otherwise undesirable. These salts are derived from the addition of inorganic or organic bases to organic acids. Such salts include alkali metal salts such as sodium, lithium, and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, and cobalt salts; inorganic amine salts such as ammonium or substituted ammonium salts such as trimethylammonium salt; and chloroprocaine salt, dibenzylamine salt, dicyclohexylamine salt, dicyclohexylamine, diethanolamine salt, ethylamine salt (including diethylamine salt and triethylamine salt), ethylenediamine salt, glucosamine salt, guanidine salt, methyl This includes salts having organic bases such as amine salts (including dimethylamine salt and trimethylamine salt), morpholine salt, morpholine salt, N,N'-dibenzylendylamine salt, N-benzylphenylethylamine salt, N-methylglucamine salt, phenylglycine alkyl ester salt, piperazine salt, piperidine salt, procaine salt, t-butylamine salt, tetramethylammonium salt, t-octylamine salt, tris-(2-hydroxyethyl)amine salt, tris(hydroxymethyl)aminomethane salt, and amino acid salts such as L-ornithine salt, L-arginine salt, and L-lysine salt. Such salts can be formed very easily by standard techniques to those skilled in the art.In fact, chemically modifying pharmaceutical compounds (i.e., drugs) into salts is a well-known technique among medicinal chemists (for example, H. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6). th See Ed. 1995 at pp. 196 and 1456-1457). Salts of SCFA can be formed, for example, by reacting SCFA with an equal amount of acid or base in a medium that precipitates the salt or in an aqueous medium, followed by freeze-drying.

[0028] In one embodiment, the pharmaceutically acceptable salt of SCFA is a metal salt such as sodium, potassium, lithium, ammonium, calcium, or magnesium salt. In a further embodiment, the pharmaceutically acceptable salt of SCFA is a sodium salt.

[0029] Esters of SCFA may also be administered / used in the methods and uses of this disclosure. Examples of SCFA esters are triglycerides of SCFA, which consist of SCFA (three molecules) and glycerol. Triglycerides of SCFA are metabolized in vivo to release three SCFA molecules and are therefore considered prodrugs of SCFA. Examples of triglycerides of SCFA include triacetin, trippropionine, triplutyrine, triisobutyline, trivalerine, and triisovalerine.

[0030] As used herein, the term “intestinal neuropathy” refers to disorders associated with abnormalities in the ENS, including abnormal development of the ENS, such as abnormalities in the number of neurons (ganglionic dysplasia, anangillosis), and / or abnormalities in neuronal differentiation. Examples of intestinal neuropathy include intestinal ganglion cell abnormalities such as HSCR and enteric ganglion dysplasia. In one embodiment, intestinal neuropathy is HSCR. In another embodiment, intestinal neuropathy is enteric ganglion dysplasia.

[0031] As used herein, the expression "induces intestinal neurogenesis" refers to an increase in the production of intestinal neurons and / or intestinal glial cells compared to before treatment with a composition containing human GDNF polypeptide. The intestinal nervous system includes various types of neurons, including enteric primary afferent neurons (EPANs), excitatory circular myomotor neurons, inhibitory circular myomotor neurons, longitudinal myomotor neurons, ascending interneurons, descending interneurons, secretory and vasomotor neurons, and intestinofugal neurons, as well as intestinal glial cells (EGCs) that provide structural support to neurons and contribute to their maintenance, survival, and function (Costa et al., Gut 2000; (Suppl IV) 47:iv15-iv19, De Giorgio et al., American Journal of Physiology - Gastrointestinal and Liver Physiology, Vol.303, No.8:G887-G893, 2012). The production of one or more of these cell types may be induced by administration / use of the compositions described herein.

[0032] In another embodiment, administration / use of the compositions described herein reduces the infiltration of inflammatory cells or immune cells (e.g., neutrophils) in the colon (e.g., distal colon). In another embodiment, administration / use of the compositions described herein restores (partially or completely) the proportion of immune cells in the colon (e.g., distal colon). In another embodiment, administration / use of the combinations described herein restores epithelial impermeability.

[0033] As used herein, the term “human GDNF polypeptide” refers to the naturally mature human GDNF protein, or a functional variant or fragment thereof that retains the biological activity of the naturally mature human GDNF protein, e.g., the ability to bind to the GDNF receptor (in particular, the “rearranged during transfection” (RET) proto-oncogene and / or neuron cell adhesion molecule (NCAM) receptor) and to induce signaling in cells expressing the GDNF receptor (e.g., RET and / or NCAM). The amino acid sequence (isoform 1, canonical sequence) of the naturally mature human GDNF protein is shown in Figure 3A (SEQ ID NO: 1), with the sequence corresponding to the mature protein (residues 78-211) highlighted in bold. The GDNF precursor protein is processed into the mature secretory form, which exists as a homodimer. Each GDNF monomer contains seven conserved cysteine ​​residues, including Cys-101, which is used for interchain disulfide crosslinking, and others involved in intermolecular ring formation known as the cysteine-knot configuration.

[0034] In one embodiment, the human GDNF polypeptide is a recombinant human GDNF polypeptide. When referring to proteins or polypeptides, the term “recombinant” refers to a protein or polypeptide molecule that is not isolated from a natural source (e.g., a biological sample) but is expressed from a recombinant nucleic acid construct, for example, produced by means of molecular biological techniques. Thus, referring to a nucleic acid construct as “recombinant” indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e., by human intervention. Recombinant nucleic acid constructs can be introduced into host cells, for example, by transformation (e.g., transduction or transfection).

[0035] Functional variants or fragments of naturally mature human GDNF protein may contain one or more amino acid substitutions, deletions, and / or additions compared to naturally mature human GDNF protein and may have lower, equivalent, or higher biological activity than naturally mature human GDNF protein. In one embodiment, the functional variant or fragment has equivalent (e.g., 90% to 110%) or higher (e.g., greater than 110%) activity than naturally mature human GDNF protein. In one embodiment, the variant includes one or more conservative substitutions. Conservative amino acid substitutions include amino acid substitutions known in the art in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same chemical or physical properties. For example, a conserved amino acid substitution could be an acidic amino acid substituted for another acidic amino acid (e.g., Asp-Glu or vice versa), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (e.g., Lys, Arg), or an amino acid with a polar side chain substituted for another amino acid with a polar side chain (e.g., Asn, Cys, Gln, Ser, Thr, Tyr, etc.). In another embodiment, the variant may include an amino acid sequence of a native GDNF protein or polypeptide having at least one non-conserved amino acid substitution. Preferably, the non-conserved amino acid substitution enhances the activity of the variant compared to that of a native mature human GDNF protein. In one embodiment, the human GDNF polypeptide has the ability to bind to the RET receptor. In another embodiment, the human GDNF polypeptide has the ability to bind to the NCAM receptor. In one embodiment, human GDNF polypeptides have the ability to bind to GDNF family coreceptors alpha (GFR alpha) 1-3.

[0036] In one embodiment, the human GDNF polypeptide comprises at least 10, 15, or 20 amino acids (e.g., sequential amino acids) from a mature human native GDNF protein. In one embodiment, the human GDNF polypeptide comprises the sequence ETTYDKILKNLSRNR(gliafin) which corresponds to residues 153-167 of SEQ ID NO: 1 and is the putative binding domain of human GDNF to the NCAM receptor (see Nielsen et al., J Neurosci. 2009 Sep 9;29(36):11360-11376). In other embodiments, the human GDNF polypeptide comprises at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids (e.g., sequential amino acids) from a mature human native GDNF protein, including residues 153-167 of SEQ ID NO: 1.

[0037] In one embodiment, the human GDNF polypeptide contains an amino acid sequence that is at least 50%, 60%, or 70% identical to residues 78-211 of the sequence (SEQ ID NO: 1) shown in Figure 3A. In another embodiment, the human GDNF polypeptide contains an amino acid sequence that is at least 80% identical to residues 78-211 of the sequence (SEQ ID NO: 1) shown in Figure 3A. In another embodiment, the human GDNF polypeptide contains an amino acid sequence that is at least 85% identical to residues 78-211 of the sequence (SEQ ID NO: 1) shown in Figure 3A. In another embodiment, the human GDNF polypeptide contains an amino acid sequence that is at least 90% identical to residues 78-211 of the sequence (SEQ ID NO: 1) shown in Figure 3A. In another embodiment, the human GDNF polypeptide contains an amino acid sequence that is at least 95% identical to residues 78-211 of the sequence (SEQ ID NO: 1) shown in Figure 3A. In another embodiment, the human GDNF polypeptide contains an amino acid sequence that is at least 98% identical to residues 78-211 of the sequence (SEQ ID NO: 1) shown in Figure 3A. In another embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 99% identical to residues 78-211 of the sequence shown in Figure 3A (SEQ ID NO: 1). In another embodiment, the human GDNF polypeptide comprises or consists of residues 78-211 of the sequence shown in Figure 3A (SEQ ID NO: 1). "Identity" refers to sequence identity between two polypeptides. Identity can be determined by comparing each position in an aligned sequence. Methods for determining identity percentages are known in the art, and several tools and programs are available for aligning amino acid sequences and determining identity percentages, including EMBOSS Needle, ClustalW, SIM, and DIALIGN.As used herein, a given percentage of identity with respect to a particular target sequence or a particular portion thereof may be defined as the percentage of amino acids in a candidate inducer sequence that are identical to the amino acids in the target sequence (or a particular portion thereof), after the sequences have been aligned and gaps introduced, if necessary, to achieve the maximum percentage sequence identity generated by the Smith-Waterman algorithm (Smith & Waterman, J. Mol. Biol. 147:195-7 (1981)) using the BLOSUM substitution matrix (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9 (1992)) as a similarity measure. The "identity value %" is determined by dividing the number of matching identical amino acids by the sequence length for which the identity percentage is reported.

[0038] Covalent modification of human GDNF polypeptides is included within the scope of this disclosure. For example, the natural glycosylation pattern of human GDNF polypeptides can be modified (Beck et al., Curr. Pharm. Biotechnol. 9:482-501, 2008; Walsh, Drug Discov. Today 15:773-780, 2010), and human GDNF polypeptides can be linked to a variety of non-proteinoid polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner described in U.S. Patents No. 4,640,835, No. 4,496,689, No. 4,301,144, No. 4,670,417, No. 4,791,192, or No. 4,179,337. Human GDNF polypeptides may contain one or more modifications that confer additional biological properties to the polypeptide, such as protease resistance, plasma protein binding, increased plasma half-life, and permeability into tissues or cells. Such modifications include, for example, fatty acids (e.g., C6-C). 18Examples include covalent bonding of molecules / parts to polypeptides such as ), binding of proteins such as albumin (see, for example, U.S. Patent No. 7,268,113), sugar / polysaccharide (glycosylation), biotinylation, or PEGylation (see, for example, U.S. Patent Nos. 7,256,258 and 6,528,485).

[0039] Human GDNF polypeptides can also be conjugated to a portion for inducing their multimerization or oligomerization (e.g., tetramerization) by fusing the human GDNF polypeptide to an oligomerization domain or a molecule that can be oligomerized (e.g., biotin that can bind to four binding sites on streptavidin). Human GDNF polypeptides can also be conjugated to a portion that will target the distal colon or specific cells of the distal colon (e.g., Schwann cells and / or their precursors, intestinal glial cells, pericytes) by using, for example, antibodies, antibody fragments, or ligands that bind to markers present on cells from the distal colon.

[0040] Human GDNF polypeptides can also be conjugated to one or more therapeutic or activating agents (e.g., drugs, or other polypeptides for forming a fusion polypeptide). Any method known in the art for conjugating human GDNF polypeptides to another part (e.g., an activating agent) can be used, including those methods described by Hunter et al. (1962) Nature, 144:945, David et al. (1974) Biochemistry, 13:1014, Pain et al. (1981) J. Immunol. Meth., 40:219, Nygren, J. Histochem. and Cytochem., 30:407 (1982), and Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego. Human GDNF polypeptides can be conjugated to another part directly or via a linker.

[0041] In one embodiment, the GDNF polypeptide and / or SCFA (or its pharmaceutically acceptable salt or ester) are formulated into a pharmaceutical composition. The GDNF polypeptide and SCFA (or its pharmaceutically acceptable salt or ester) may be formulated into the same pharmaceutical composition or different pharmaceutical compositions. In one embodiment, the GDNF polypeptide and SCFA (or its pharmaceutically acceptable salt or ester) are formulated into different pharmaceutical compositions. In one embodiment, the GDNF polypeptide and SCFA (or its pharmaceutically acceptable salt or ester) are formulated into the same pharmaceutical composition. Such a pharmaceutical composition typically comprises one or more pharmaceutically acceptable excipients.

[0042] As used herein, the term “excipient” has the common meaning in the art and refers to any component other than the active ingredient (drug) itself. Excipients include, for example, buffers, binders, lubricants, diluents, fillers, thickeners, disintegrants, plasticizers, coatings, barrier formulations, stabilizers, release retarders, and other components. As used herein, “pharmaceutically acceptable excipient” means an excipient that does not interfere with the efficacy of the biological activity of the active ingredient and is non-toxic to the subject; that is, a type of excipient and / or any excipient to be used in an amount that is non-toxic to the subject. Excipients are well known in the art and this composition is not limited in these respects. Carriers / excipients may be suitable for administration, for example, intravenously, parenterally, subcutaneously, intramuscularly, intracranially, intraorbitally, ophthalmologically, intraventricularly, intrasacrally, intraspinally, intrathecally, epidurally, intracystically, intraperitoneally, intranasally, rectally, or pulmonaryly (e.g., aerosol). The therapeutic composition is prepared by mixing an active ingredient of the desired purity with one or more pharmaceutically acceptable carriers, excipients, and / or stabilizers using standard methods known in the art (Remington: The Science and Practice of Pharmacy, by Loyd V Allen, Jr, 2012, 22 ndedition,Pharmaceutical Press;Handbook of Pharmaceutical Excipients,by Rowe et al.,2012,7 th edition,Pharmaceutical Press).

[0043] In one embodiment, the GDNF polypeptide and / or SCFA (or a pharmaceutically acceptable salt or ester thereof) is formulated for oral administration. Suitable formulations for oral administration include (a) liquid solutions such as the activator / composition suspended in an effective amount of water, saline, or a diluent such as PEG400; (b) capsules, sachets, or tablets, each containing a predetermined amount of the active ingredient as a liquid, solid, granule, or gelatin; (c) suspensions in a suitable liquid; and (d) suitable emulsions. Tablet forms may contain one or more of the following: lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and pharmaceutically acceptable carriers. The lozenge form may include aromatic tablets containing an active ingredient in a flavor (e.g., sucrose), as well as the active ingredient in addition to the active ingredient, in an inert base such as gelatin and glycerin, or sucrose and acacia emulsion, gel, which contains a carrier known in the art.

[0044] In one embodiment, the GDNF polypeptide and / or SCFA (or its pharmaceutically acceptable salts or esters) are formulated for parenteral administration (e.g., injection). Formulations for parenteral administration may contain excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, plant-derived oils, or hydrogenated naphthalene. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compound. Other potentially useful parenteral delivery systems include ethylene vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients (e.g., lactose), or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops or gels.

[0045] In one embodiment, the GDNF polypeptide and / or SCFA (or its pharmaceutically acceptable salt or ester) is formulated for intestinal delivery, i.e., delivery to the intestinal tract. This can be achieved by methods well known in the art. For example, the GDNF polypeptide and / or SCFA (or its pharmaceutically acceptable salt or ester) can be coated or encapsulated with an enteric coating or enteric material. The enteric coating allows for release, for example, at a specific pH or in the presence of digestive enzymes or bacteria characteristically present at a specific location in the GI tubule where release is desired (e.g., the small intestine, large intestine, or a specific region thereof). In one embodiment, the enteric material is pH-sensitive and affected by pH changes encountered in the gastrointestinal tract (pH-sensitive release). The enteric material typically remains insoluble at the pH of the stomach and then allows for the release of the active ingredient in the higher pH environment of the downstream gastrointestinal tract (e.g., often the duodenum, or sometimes the colon). In another embodiment, the enteric-coated material comprises an enzymatically degradable polymer that is broken down by bacterial enzymes present in the lower gastrointestinal tract, particularly the colon (e.g., carbohydrate-processing enzymes such as glycosidases, polysaccharide lyases, and carbohydrate esterases).Examples of such enteric-coated materials include cellulose polymers such as hydroxypropylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose phthalate, methylcellulose, ethylcellulose, cellulose acetate, cellulose acetate phthalate, cellulose trimellitic acetate, and sodium carboxymethylcellulose; preferably acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, meth Methyl acrylate and / or ethyl methacrylate, as well as Eudragit® L30D-55 and L100-55 (soluble at pH 5.5 or higher), Eudragit® L-IOO (soluble at pH 6.0 or higher), Eudragit® S (soluble at pH 7.0 or higher, as a result of higher esterification), and Eudragit® NE, RL, and RS (water-insoluble polymers with varying degrees of permeability and expandability), trade names Acryl-EZE® (Colorcon, USA), Eudragit® (Rohm Acrylic acid polymers and copolymers formed from other methacrylic resins commercially available in Pharma (Westerstadt, Germany); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinyl acetate phthalate, vinyl acetate crotonic acid copolymer, and ethylene vinyl acetate copolymer; enzymatically digestible polymers such as azopolymers, pectin, chitosan, amylose, and guar gum; and zein and shellac. Combinations of different enteric coating materials may also be used.Approaches for colon-specific drug delivery are well known in the art (see, for example, Philip et al., Oman Med J. 2010 Apr;25(2):79-87; Lee et al., Pharmaceutics. 2020 Jan;12(1):68), and include pH-dependent systems (e.g., using pH-dependent polymers), receptor-mediated systems, magnetically driven systems, delayed or time-dependent systems, microbially induced drug delivery systems (e.g., including polymers based on sugars that can be broken down by enzymes produced by the colonic microbiota, such as glucoronidase, xylosidase, arabinosidase, and galactosidase), pressure-controlled colon delivery capsules (drug release induced by higher pressures encountered in the colon), osmotically controlled drug delivery, and any combination of these approaches (e.g., colon-targeted delivery systems (CODES™) using approaches that combine pH-dependent and microbially induced drug delivery).

[0046] In one embodiment, the GDNF polypeptide and / or SCFA (or a pharmaceutically acceptable salt or ester thereof) is formulated into a capsule made of enteric material (enteric-coated capsule).

[0047] In one embodiment, the GDNF polypeptide and / or SCFA (or a pharmaceutically acceptable salt or ester thereof) is formulated for administration to the distal colon of the subject.

[0048] As used herein, the term “distal colon” ​​refers to the last three segments of the colon, namely the descending colon, the sigmoid colon, and the rectum. In one embodiment, the pharmaceutical composition is administered to or for administration to the rectum and / or sigmoid colon. In one embodiment, the pharmaceutical composition is administered to or for administration to the rectosigmoid region, which includes the last part of the sigmoid colon and the beginning of the rectum. Those skilled in the art will understand that GDNF polypeptide and / or SCFA (or its pharmaceutically acceptable salts or esters) may be administered directly to the distal colon, or to a site distant from the distal colon, using preferred means that provide delivery of GDNF polypeptide and / or SCFA (or its pharmaceutically acceptable salts or esters) to the distal colon. For example, the formulation may contain a coating that is specifically degraded under distal colon conditions (e.g., pH, enzymatic environment, bacterial environment, etc.), and therefore the formulation may be administered to other areas of the gastrointestinal system, but the GDNF polypeptide and / or SCFA (or its pharmaceutically acceptable salts or esters) will only be released when the formulation reaches the colon, more specifically the distal colon. Approaches for colon-specific drug delivery are well known in the art (see, for example, Philip et al., Oman Med J. 2010 Apr;25(2):79-87; Lee et al., Pharmaceutics. 2020 Jan;12(1):68), and include pH-dependent systems (e.g., using pH-dependent polymers), receptor-mediated systems, magnetically driven systems, delayed or time-dependent systems, microbially induced drug delivery systems (e.g., including polymers based on sugars that can be broken down by enzymes produced by the colonic microbiota, such as glucoronidase, xylosidase, arabinosidase, and galactosidase), pressure-controlled colon delivery capsules (drug release induced by higher pressures encountered in the colon), osmotically controlled drug delivery, and any combination of these approaches (e.g., colon-targeted delivery systems (CODES™) using approaches that combine pH-dependent and microbially induced drug delivery).

[0049] Formulations for rectal / distal colon administration may be presented as suppositories, which may be prepared by mixing the composition with one or more suitable non-irritating carriers, for example, cocoa butter, polyethylene glycol, suppository wax, or salicylate, the non-irritating carriers being solid at room temperature but liquid at body temperature, and thus melting in a suitable body cavity to release GDNF polypeptide and / or SCFA (or its pharmaceutically acceptable salts or esters). More recently, liquid suppositories have been developed. Liquid suppositories typically contain thermosensitive and / or mucosal-adhering polymers such as poloxamer, Carbopol® (cross-linked polyacrylic acid polymer), sodium alginate, polycarbophil, hydroxypropyl methylcellulose (HPMC), hydroxyethylcellulose, and methylcellulose.

[0050] Formulations for rectal / distal colon administration may also be presented as enemas, liquid drug solutions, suspensions, or emulsions to be injected into the rectum and distal colon. The liquid in which the GDNF polypeptide and / or SCFA (or its pharmaceutically acceptable salts or esters) are diluted may be, for example, water, or saline solution or suspension.

[0051] Formulations for rectal / distal colon administration may also be in the form of rectal foam or gel. Rectal gels are semi-solid formulations containing a solvent trapped within a polymer network to create viscous consistency. The viscosity of the gel can be altered by the addition of cosolvents (e.g., glycerin and propylene glycol) and electrolytes. Foams consist of a hydrophilic liquid continuous phase and a gaseous dispersed phase containing a foaming agent, distributed throughout. Following rectal administration, they transition from a foamy state to a liquid or semi-solid state on the mucosal surface. The foaming agent is typically an amphiphilic substance that is important for foam formation and stabilization. The molecule contains a hydrophilic component that is soluble in the aqueous phase and a hydrophobic component that forms micelles to minimize contact with the aqueous phase.

[0052] Rectal / distal colon administration may be performed using currently available endoscopes or specialized catheters designed for rectal administration or injection into the distal colon wall of pharmaceuticals or liquids, which can be safely positioned and comfortably held within the rectum for repeated use.

[0053] GDNF polypeptide and / or SCFA (or its pharmaceutically acceptable salts or esters) may be administered according to any preferred dosing regimen, for example, four times a day, twice a day, once a day, twice a week, once a week, etc. Treatment may be carried out for any preferred period, for example, one week, two weeks, three weeks or more, to achieve the desired effect.

[0054] In one embodiment, an effective dose of GDNF polypeptide administered to or for administration to human subjects corresponds to a dose of approximately 5 μg to approximately 20 μg in a mouse pup, which is the range shown to be effective in the studies described herein. A 10 μl enema containing recombinant GDNF solution was administered to a mouse pup. 10 μl is estimated to be the volume required to fill the distal colon and rectum of the pup. Therefore, an administration of 5 μg of GDNF in a mouse pup is achieved by administering 10 μl of a 0.5 μg / μl GDNF solution, and an administration of 20 μg of GDNF in a mouse pup is achieved by administering 10 μl of a 2.0 μg / μl GDNF solution. The volume required to fill the distal colon and rectum of a human infant can be estimated using the following formula: 10 ml × infant weight (kg). Therefore, a dose of 5 μg of GDNF in a mouse is equivalent to approximately 5 mg per kg in a human infant, and a dose of 20 μg of GDNF in a mouse is equivalent to approximately 20 mg per kg in a human infant. Thus, in one embodiment, the effective dose of GDNF polypeptide administered to or for administration to a human subject is approximately 5 mg to approximately 20 mg per kg, preferably approximately 10 mg to approximately 15 mg per kg. In one embodiment, the GDNF polypeptide is administered or for administration through a composition (solution or gel) at a concentration of 0.5 mg / ml to 2 mg / ml.

[0055] In one embodiment, the combination of therapeutic agents (GDNF polypeptide and SCFA or a pharmaceutically acceptable salt or ester thereof) may be administered in any conventional dosage form or may be administered concurrently (e.g., sequentially, simultaneously at different times). Concurrent administration in the context of this disclosure refers to the use of two or more therapies in a course of treatment adjusted to achieve improved clinical outcomes. The combination of GDNF polypeptide and SCFA or a pharmaceutically acceptable salt or ester thereof described herein may be used in combination with other therapeutic agents or drugs, such as analgesics or anti-inflammatory agents.

[0056] In one embodiment, the above-described treatment with a combination of GDNF polypeptide and SCFA or a pharmaceutically acceptable salt or ester thereof may be performed in combination with surgery (e.g., Swenson, Soave, or Duhamel type pull-through surgery). Especially in neonates with HSCR, clinicians often recommend trying a trial of daily enema therapy before surgery. Adding the GDNF+SCFA combination disclosed herein to the enema may increase the likelihood that children with HSCR will respond well to preoperative enema therapy. Therefore, in one embodiment, the above-described treatment with a combination of GDNF polypeptide and SCFA or a pharmaceutically acceptable salt or ester thereof is performed before pull-through surgery. Saline enemas are also commonly used in children with HSCR after pull-through surgery. Postoperative problems in HSCR patients are thought to be at least partially due to a lack of ganglion cells remaining in the distal intestine, the so-called "transit section". Therefore, adding a combination of GDNF polypeptide and SCFA or a pharmaceutically acceptable salt or ester thereof to an enema may be useful in correcting ganglion cell deficiency and / or neuronal subtype imbalance in the retained distal bowel after surgery. Accordingly, in another embodiment, the above treatment with a combination of GDNF polypeptide and SCFA or a pharmaceutically acceptable salt or ester thereof is performed after pull-through surgery.

[0057] In one embodiment, the above-described treatment with a combination of GDNF polypeptide and SCFA or a pharmaceutically acceptable salt or ester thereof is carried out in combination with ENS stem cell-based therapies being investigated for the treatment of HSCR. GDNF may be a useful adjunct to these therapies to promote engraftment.

[0058] HSCR is clinically subdivided into short-range (S-HSCR) and long-range (L-HSCR). S-HSCR, which occurs in >80% of cases, means the absence of ENS in the rectum and sigmoid colon. L-HSCR means that the longer region of the distal intestine is aganglionoid. The methods / uses described herein are for the treatment of human patients with S-HSCR or L-HSCR. In one embodiment, the methods / uses described herein are for the treatment of human patients with S-HSCR. In one embodiment, the methods / uses described herein are for the treatment of human patients with L-HSCR.

[0059] HSCR patients can be adult patients or pediatric patients. In one embodiment, the HSCR patient is a pediatric patient, preferably a patient under 5, 4, 3, or 2 years of age, more preferably a patient under 1 year of age or under 6 months of age. In another embodiment, the patient is male.

[0060] HSCR is associated with mutations in RET, EDNRB, SOX10, PHOX2B, and ZFHX1B, as well as Down syndrome or trisomy 21 (collagen VI-related HSCR). There is also a significant sex difference, with a male-to-female ratio of approximately 5:1. In one embodiment, HSCR is collagen VI-related HSCR. In another embodiment, HSCR is EDNRB mutation-related HSCR. In one embodiment, HSCR is HSCR that is predominantly male. In one embodiment, HSCR is RET mutation-related HSCR, for example, HSCR associated with mutations that reduce RET expression and / or activity in cells from the distal colon. The mutation may be in RET or a protein involved in RET signaling. In one embodiment, the mutation is in the RET protein. Mutations in the RET gene on chromosome 10q11.2 have been shown to account for 50% of familial cases and 15-20% of sporadic cases of HSCR, with the majority (approximately 75%) associated with L-HSCR.

[0061] In another embodiment, the HSCR is not a RET mutation-associated HSCR. [Examples]

[0062] The present invention is illustrated in more detail by the following non-limiting examples.

[0063] Example 1: Materials and Method Mouse. Homozygous Holstein. Tg / Tg Mouse (Hol Tg / TgModels of Trisomy21 [Collagen VI]-related HSCR were treated as controls with either a 10 μl GDNF enema (1 μg / μl) or 10 μl GDNF combined with 5 mM aminobutyrate (butyrate) or 1 μg / μl of another growth factor, or a vehicle (1X PBS). The enema was administered once daily from postnatal day (P) 4 to P8, and mice were monitored daily until P60 or megacolon-related death. Survival graphs and statistical analyses were generated using GraphPad Prism software. One-way ANOVA followed by a post-hoc Tukey multiple comparison test was performed, with a significance level of P<0.05. The number of biological copies (n) for each cohort is shown in the figures where relevant.

[0064] Example 2: Hol Tg / Tg Evaluation of the effects of GDNF combined with neurotrophic factors on mouse survival. In a mouse model of Trisomy21[CollagenVI]-associated HSCR, we investigated whether molecules known to have neurotrophic effects could improve the effect of GDNF on survival (39). In a previous study using the same mouse model, the combination of GDNF with three molecules known to have neurotrophic effects (vitamin C, serotonin, and endothelin 3) did not improve mouse survival compared to GDNF alone (see Soret et al., Gastroenterology. 2020 Nov;159(5):1824-1838.e17.doi:10.1053 / j.gastro.2020.07.018.Epub 2020 Jul 17, Supplementary Figure 1G).

[0065] The molecules tested in this study were as follows: bone morphogenetic protein 4 (BMP4), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), midbrain stellate cell-derived neurotrophic factor (MANF), brain dopamine neurotrophic factor (CDNF), epidermal growth factor (EGF), neuturin (NRTN), fibroblast growth factor 2 (FGF2), butyrate, and retinoic acid (RA). These molecules have been reported to exhibit neurotrophic activity under various conditions (Table 1). [Table 1A] [Table 1B]

[0066] The results shown in Figures 1A-1C indicate that butyrate alone does not affect mouse survival (similar to the vehicle), but it significantly improves the effect of GDNF, and the GDNF + butyrate combination shows a synergistic effect on survival. As demonstrated by the results shown in Figures 2A-2C, none of the other neurotrophic factors tested significantly improved the effect of GDNF on survival in this mouse model. In fact, combining GDNF with some neurotrophic factors can even decrease survival compared to administering GDNF alone (Figure 2C).

[0067] The results presented herein indicate that butyrate, rather than other molecules known to exhibit neurotrophic activity, acts synergistically with GDNF, and Hol Tg / Tg This study demonstrates that the combination of GDNF and butyrate improves mouse survival and therefore may be useful in treating human intestinal neuropathy, such as Hirschsprung's disease.

[0068] While the present invention has been described herein by the specific embodiments described above, it may be modified without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word “including” is used as an open-ended term substantially equivalent to the phrase “including, but not limited to.” The singular forms “a,” “an,” and “the” include the corresponding plural subject matter unless the context clearly indicates otherwise.

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Claims

1. A combination for use in the treatment of human subjects suffering from intestinal neuropathy, comprising (i) a GDNF polypeptide having an amino acid sequence having at least 90% identity with amino acids 78-211 of SEQ ID NO: 1, and (ii) butyric acid or a pharmaceutically acceptable salt or ester thereof.

2. The combination for use according to claim 1, wherein the GDNF polypeptide comprises an amino acid sequence having at least 95% identity with amino acids 78-211 of SEQ ID NO:

1.

3. The combination for use according to claim 2, wherein the GDNF polypeptide comprises amino acids 78 to 211 of SEQ ID NO:

1.

4. A combination for use according to any one of claims 1 to 3, comprising a pharmaceutically acceptable salt of butyric acid.

5. The combination for use according to any one of claims 1 to 3, wherein the GDNF polypeptide and / or the butyric acid or a pharmaceutically acceptable salt or ester thereof is present in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

6. The combination for use according to claim 5, wherein the pharmaceutically acceptable carrier comprises a physiological saline solution or a gelling agent.

7. The combination for use according to any one of claims 1 to 3, wherein the GDNF polypeptide and / or the butyric acid or a pharmaceutically acceptable salt or ester thereof is intended for rectal administration via enema.

8. The combination for use according to any one of claims 1 to 3, wherein the GDNF polypeptide and / or the butyric acid or a pharmaceutically acceptable salt or ester thereof is intended for administration by injection into the distal colon wall.

9. A combination for use according to any one of claims 1 to 3, wherein the GDNF polypeptide and / or the butyric acid or a pharmaceutically acceptable salt or ester thereof is for administration once to a maximum of four times per day.

10. The combination for use according to any one of claims 1 to 3, wherein the GDNF polypeptide and / or the butyric acid or a pharmaceutically acceptable salt or ester thereof is for administration over at least two consecutive days.

11. The combination for use according to any one of claims 1 to 3, wherein the combination is intended for use prior to surgical removal of an aganglionic area or a ganglion cell-deficient area in the subject.

12. The combination for use according to any one of claims 1 to 3, wherein the combination is intended for use after surgical removal of an aganglionic area or a ganglion cell-deficient area in the subject.

13. The combination for use according to claim 11, wherein the surgical removal of the aganglionic area or ganglionocyte-deficient area is performed through a pull-through procedure.

14. The combination for use according to claim 12, wherein the surgical removal of the aganglionic area or area with few ganglion cells is performed through a pull-through procedure.

15. The combination for use according to any one of claims 1 to 3, wherein the intestinal neuropathy is enteric ganglion cell dysplasia.

16. The combination for use according to any one of claims 1 to 3, wherein the intestinal neuropathy is Hirschsprung's disease (HSCR).

17. The combination for use according to claim 16, wherein the subject is suffering from short-range HSCR.

18. The combination for use according to claim 16, wherein the HSCR is a collagen VI-related HSCR.

19. The combination for use according to any one of claims 1 to 3, wherein the human subject is under 5 years of age.

20. The combination for use according to claim 19, wherein the human subject is less than six months old.

21. The combination for use according to any one of claims 1 to 3, wherein the GDNF polypeptide and / or the butyric acid or a pharmaceutically acceptable salt or ester thereof is for administration to the rectum and / or sigmoid colon.

22. The combination for use according to claim 21, wherein the GDNF polypeptide and / or the butyric acid are for administration to the rectal sigmoid region.

23. A combination for use according to any one of claims 1 to 3, comprising sodium butyrate.

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