Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/48—Nerve growth factor [NGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C53/00—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen
  • C07C53/124—Acids containing four carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• the present disclosure generally relates to the treatment of enteric neuropathies such as Hirschsprung disease (HSCR) and intestinal hypoganglionosis.
• enteric neuropathies such as Hirschsprung disease (HSCR) and intestinal hypoganglionosis.
• the enteric nervous system extends along the entire gastrointestinal tract to control bowel motility, blood flow and epithelial activity in response to sensory stimuli (1).
• Interconnected enteric ganglia containing neurons and glia develop from neural crest-derived progenitors that migrate through the intestine during prenatal development. Incomplete colonization of distal colon by ENS progenitors causes Hirschsprung disease (HSCR), a condition affecting 1 in 5000 newborns (2,3).
• HSCR distal colon without neural ganglia (i.e., aganglionic colon) remains tonically contracted and does not propagate contractions, causing functional intestinal obstruction.
• HSCR symptoms include refractory constipation with retention of stool and air, abdominal distension, growth failure, occasional vomiting, bowel inflammation (enterocolitis) and a risk of bacterial translocation into blood causing sepsis and premature death (2).
• HSCR is clinically subdivided into short-segment (S-HSCR) and long-segment forms (L- HSCR) (4).
• S-HSCR which occurs in >80% of cases, means the ENS is absent from rectum and sigmoid colon.
• L-HSCR means longer regions of distal bowel are aganglionic.
• HSCR etiology remains incompletely understood, but many genes influence HSCR risk (2).
• genetic risk variants may combine with non-genetic factors to prevent full bowel colonization by ENS progenitors (5). This non-Mendelian inheritance occurs because many proteins must work together for normal ENS development.
• hypoganglionosis also known as intestinal hypoganglionosis, is a disorder causing a reduced number of nerves in the intestinal wall.
• Intestinal hypoganglionosis can mimic HSCR; patients with both conditions may present with chronic constipation, intestinal obstruction, and enterocolitis (inflammation of the intestines).
• Patients with hypoganglionosis may also suffer from severe complications including fecaloma (hardening of the feces inside the colon), bleeding or perforation of the intestine, and breathing problems resulting from a distended colon.
• the exact cause of hypoganglionosis is often not known. In some cases, it is due to factors present at birth (congenital), while other times it is believed to be an acquired condition.
• the management of isolated hypoganglionosis generally involves surgery to remove the affected bowel segment.
• the present disclosure relates to the use of a combination of GDNF and a short-chain fatty acid for the treatment of one or more pathological features of enteric neuropathies such as Hirschsprung disease.
• the present disclosure relates to the following items 1 to 50:
• a method for treating a human subject suffering from an enteric neuropathy comprising administrating to the subject an effective amount of (i) a Glial cell line-Derived Neurotrophic Factor (GDNF) polypeptide; and (ii) a short chain fatty acid (SCFA), or a pharmaceutically acceptable salt or ester thereof.
• GDNF Glial cell line-Derived Neurotrophic Factor
• SCFA short chain fatty acid
• GDNF polypeptide comprises an amino acid sequence having at least 70% identity with amino acids 78-211 of SEQ ID NO:1 (FIG. 3A).
• GDNF polypeptide comprises an amino acid sequence having at least 90% identity with amino acids 78-211 of SEQ ID NO:1 (FIG. 3A).
• GDNF polypeptide comprises an amino acid sequence having at least 95% identity with amino acids 78-211 of SEQ ID NO:1 (FIG. 3A).
• the pharmaceutically acceptable carrier comprises a saline solution or a gelling agent.
• SCFA are administered into the rectum and/or the sigmoid colon.
• a combination for use in treating a human subject suffering from an enteric neuropathy comprising (i) a GDNF polypeptide; and (ii) an SCFA, or a pharmaceutically acceptable salt or ester thereof.
• the GDNF polypeptide comprises an amino acid sequence having at least 70% identity with amino acids 78-211 of SEQ ID NO:1
• GDNF polypeptide comprises an amino acid sequence having at least 90% identity with amino acids 78-211 of SEQ ID NO:1 (FIG. 3A).
• GDNF polypeptide comprises an amino acid sequence having at least 95% identity with amino acids 78-211 of SEQ ID NO:1 (FIG. 3A).
• FIG. 1A is a graph showing the survival of homozygous Holstein T9/T 9 mice following administration of vehicle, butyrate (5 mM), GDNF (1 pg/mI) and a combination of butyrate + GDNF.
• FIG. 1B depicts the statistical analysis of survival assay shown in FIG. 1A.
• FIG. 1C depicts the survival rate per specific time points shown in FIG. 1A.
• FIG. 2A is a graph showing the survival of homozygous Holstein T9/T 9 mice following administration of GDNF alone or in combination with various neurotrophic factors.
• FIG. 2B depicts the survival rate per specific time points shown in FIG. 2A.
• FIG. 2C depicts the statistical analysis of survival assay shown in FIG. 2A.
• FIG. 3A shows the amino acid sequence of human GDNF isoform 1 (UniProtKB accession No. P39905, SEQ ID NO:1), with the sequence corresponding to the signal peptide underlined (residues 1-19), the sequence corresponding to the propeptide italicized (residues 20- 75) and the sequence corresponding to the mature polypeptide in bold (residues 78-211).
• FIGs. 3B-C show the nucleotide sequence of the cDNA encoding human GDNF isoform 1 (RefSeq accession No.
• NM_000514.4 SEQ ID NO:2
• sequence encoding the signal peptide underlined amino acid sequence encoding the signal peptide underlined
• sequence encoding the propeptide italicized nucleotides 619-786
• sequence encoding the mature polypeptide in bold nucleotides 793-1194.
• the term “about” has its ordinary meaning.
• the term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).
• recombinant GDNF in combination with short chain fatty acid may be used for the treatment of enteric neuropathies (e.g., ENS defects such as HSCR), i.e., for improving one or more of the pathological features of enteric neuropathies and/or survival, in human patients.
• enteric neuropathies e.g., ENS defects such as HSCR
• the present disclosure provides a method for treating a human subject suffering from an enteric neuropathy (e.g., Hirschsprung disease (HSCR) or intestinal hypoganglionosis), the method comprising administrating to the subject an effective amount of (i) Glial cell line-Derived Neurotrophic Factor (GDNF) and (ii) a short chain fatty acid (SCFA), or a pharmaceutically acceptable salt or ester thereof.
• GDNF Glial cell line-Derived Neurotrophic Factor
• SCFA short chain fatty acid
• the present disclosure also provides the use of (i) GDNF and (ii) an SCFA, or a pharmaceutically acceptable salt or ester thereof, for treating a human subject suffering from an enteric neuropathy (e.g., Hirschsprung disease (HSCR) or intestinal hypoganglionosis).
• the present disclosure also provides the use of (i) GDNF and (ii) an SCFA, or a pharmaceutically acceptable salt or ester thereof, for the manufacture of a medicament for treating a human subject suffering from an enteric neuropathy (e.g., Hirschsprung disease (HSCR) or intestinal hypoganglionosis).
• an enteric neuropathy e.g., Hirschsprung disease (HSCR) or intestinal hypoganglionosis
• the present disclosure also provides a combination for use in treating a human subject suffering from an enteric neuropathy (e.g., Hirschsprung disease (HSCR) or intestinal hypoganglionosis), the combination comprising (i) GDNF and (ii) an SCFA, or a pharmaceutically acceptable salt or ester thereof.
• the present disclosure also provides a method for restoring distal colon motility and/or epithelial barrier in a human subject suffering from an enteric neuropathy (e.g., HSCR or intestinal hypoganglionosis), the method comprising administrating an effective amount of (i) Glial cell line- Derived Neurotrophic Factor (GDNF) and (ii) a short chain fatty acid (SCFA), or a pharmaceutically acceptable salt or ester thereof.
• an enteric neuropathy e.g., HSCR or intestinal hypoganglionosis
• GDNF Glial cell line- Derived Neurotrophic Factor
• SCFA short chain fatty acid
• the present disclosure also provides the use of an effective amount of (i) GDNF; and (ii) a short chain fatty acid (SCFA), or a pharmaceutically acceptable salt or ester thereof, for restoring distal colon motility in a human subject suffering from an enteric neuropathy (e.g., HSCR or intestinal hypoganglionosis).
• an enteric neuropathy e.g., HSCR or intestinal hypoganglionosis
• the present disclosure also provides the use of an effective amount of (i) GDNF; and (ii) a short chain fatty acid (SCFA), or a pharmaceutically acceptable salt or ester thereof for the manufacture of a medicament for restoring distal colon motility in a human subject suffering from an enteric neuropathy (e.g., HSCR or intestinal hypoganglionosis).
• the present disclosure also provides a combination for use in restoring distal colon motility in a human subject suffering from an enteric neuropathy (e.g., HSCR or intestinal hypoganglionosis), the combination comprising (i) GDNF and (ii) an SCFA, or a pharmaceutically acceptable salt or ester thereof.
• an enteric neuropathy e.g., HSCR or intestinal hypoganglionosis
• the combination comprising (i) GDNF and (ii) an SCFA, or a pharmaceutically acceptable salt or ester thereof.
• the present disclosure also provides a method for inducing enteric neurogenesis in an aganglionic or hypoganglionic segment of the distal colon of a human subject suffering from an enteric neuropathy (e.g., HSCR or intestinal hypoganglionosis), the method comprising administering an effective amount of (i) GDNF; and (ii) a short chain fatty acid (SCFA), or a pharmaceutically acceptable salt or ester thereof.
• an enteric neuropathy e.g., HSCR or intestinal hypoganglionosis
• SCFA short chain fatty acid
• the present disclosure also provides the use of (i) GDNF; and (ii) a short chain fatty acid (SCFA), or a pharmaceutically acceptable salt or ester thereof, for inducing enteric neurogenesis in an aganglionic or hypoganglionic segment of the distal colon of a human subject suffering from an enteric neuropathy (e.g., HSCR or intestinal hypoganglionosis), wherein the composition is for administration into the distal colon of the subject.
• an enteric neuropathy e.g., HSCR or intestinal hypoganglionosis
• the present disclosure also provides the use of (i) GDNF; and (ii) a short chain fatty acid (SCFA), or a pharmaceutically acceptable salt or ester thereof, for the manufacture of a medicament for inducing enteric neurogenesis in an aganglionic or hypoganglionic segment of the distal colon of a human subject suffering from an enteric neuropathy (e.g., HSCR or intestinal hypoganglionosis).
• an enteric neuropathy e.g., HSCR or intestinal hypoganglionosis
• the present disclosure also provides a combination for use in inducing enteric neurogenesis in an aganglionic or hypoganglionic segment of the distal colon of a human subject suffering from an enteric neuropathy (e.g., HSCR or intestinal hypoganglionosis), the combination comprising (i) GDNF and (ii) an SCFA, or a pharmaceutically acceptable salt or ester thereof.
• an enteric neuropathy e.g., HSCR or intestinal hypoganglionosis
• SCFA a pharmaceutically acceptable salt or ester thereof.
• SCFA short chain fatty acid
• acetic acid C2
• propionic acid C3
• butyric acid C4
• valeric acid isovaleric acid
• 2-methylbutanoic acid C5
• Pharmaceutically acceptable salts of SCFAs such as pharmaceutically acceptable acetates, proprionates, butyrates, isobutyrates, valerates, isovalerates and 2-methylbutanoates may also be administered/used in the methods and uses described herein.
• salts are intended to mean those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are derived from addition of an inorganic base or an organic base to the organic acid.
• 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 aluminium salts, iron salts, zinc salts, copper salts, nickel salts and a cobalt salts; inorganic amine salts such as ammonium or substituted ammonium salts, such as trimethylammonium salts; and salts with organic bases (for example, organic amines) such as chloroprocaine salts, dibenzylamine salts, dicyclohexylamine salts, dicyclohexylamines, diethanolamine salts, ethylamine salts (including diethylamine salts and triethylamine salts), ethylenediamine salts, glucosamine salts, guanidine salts, methylamine salts (including dimethylamine salts and trimethylamine salts), morpholine salts, morpholine salts, N,N'-dibenzyl
• Salts of SCFAs may be formed, for example, by reacting the SCFA with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
• the pharmaceutically acceptable salt of the SCFA is a metal salt, such as a sodium, potassium, lithium, ammonium, calcium or magnesium salt. In a further embodiment, the pharmaceutically acceptable salt of the SCFA is a sodium salt.
• Esters of SCFAs may also be administered/used in the methods and uses of the present disclosure.
• SCFA esters are triglycerides of SCFAs that are composed of the SCFA (three molecules) and glycerol.
• Triglycerides of SCFAs are metabolized in vivo to release the three SCFAs molecules and are thus considered prodrugs of SCFAs.
• Examples of triglycerides of SCFAs include triacetin, triproprionin, tributyrin, triisobutyrin, trivalerin and triisovalerin.
• enteric neuropathy refers to a disease associated with abnormalities in the ENS, including abnormal development of the ENS, e.g., abnormal number of neurons (hypoganglionosis, aganglionosis) and/or abnormal differentiation of neurons.
• enteric neuropathies include enteric dysganglionoses such as HSCR and intestinal hypoganglionosis.
• the enteric neuropathy is HSCR.
• the enteric neuropathy is intestinal hypoganglionosis.
• the expression “inducing enteric neurogenesis” as used herein refers to an increase in the production of enteric neurons and/or enteric glial cells relative to prior to treatment with the composition comprising a human GDNF polypeptide.
• the enteric nervous system comprises various types of neurones including enteric primary afferent neurons (EPANs), excitatory circular muscle motorneurons, inhibitory circular muscle motorneurons, longitudinal muscle motorneurons, ascending interneurons, descending interneurons, secretomotor and vasomotor neurons, and intestinofugal neurons, as well as enteric glial cells (EGCs) that provide structural support to neurons and contribute to neuronal 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 administration/use of the combination described herein reduces the infiltration of inflammatory or immune cells (e.g., neutrophils) in the colon (e.g., distal colon).
• the administration/use of the combination described herein restores (partly or completely) the proportions of immune cells in the colon (e.g., distal colon).
• the administration/use of the combination described herein restores epithelial impermeability.
• human GDNF polypeptide refers to the native mature human GDNF protein, or to functional variants or fragments thereof that retain a biological activity of the native mature human GDNF protein, e.g., the ability to bind to a GDNF receptor (particularly the "rearranged during transfection” (RET) proto-oncogene and/or the Neural Cell Adhesion Molecule (NCAM) receptor) and trigger a signal in a cell expressing a GDNF receptor (e.g., RET and/or NCAM).
• RET rearranged during transfection
• NCAM Neural Cell Adhesion Molecule
• the amino acid sequence of native human GDNF protein is depicted in FIG.
• GDNF precursor protein is processed to a mature secreted form that exists as a homodimer.
• Each GDNF monomer contains seven conserved cysteine residues, including Cys-101 , which is used for inter-chain disulfide bridging, and others that are involved in the intramolecular ring formation known as the cysteine-knot configuration.
• the human GDNF polypeptide is a recombinant human GDNF polypeptide.
• the term “recombinant” when made in reference to a protein or a polypeptide refers to a protein or polypeptide molecule that is not isolated from a natural source (e.g., biological sample), e.g., which is expressed from a recombinant nucleic acid construct created by means of molecular biological techniques. Referring to a nucleic acid construct as “recombinant” therefore indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e., by human intervention. Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation (e.g., transduction or transfection).
• Functional variants or fragments of native mature human GDNF protein may include one or more amino acid substitutions, deletions and/or additions relative to the native mature human GDNF protein, and may have a biological activity that is lower, equivalent or higher than that of the native mature human GDNF protein.
• the functional variant or fragment has an activity that is equivalent (e.g., between 90% to 110%) or higher (e.g., more than 110%) to that of the native mature human GDNF protein.
• the variant comprises one or more conservative substitutions.
• Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and or chemical properties is exchanged for another amino acid that has the same chemical or physical properties.
• the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp to 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, lie, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.).
• an amino acid with a polar side chain substituted for another amino acid with a nonpolar side chain e.g., Ala, Gly, Val, lie, Leu, Met, Phe, Pro, Trp, Val, etc.
• a basic amino acid substituted for another basic amino acid e.g., Ala, Gly, Val, lie, Leu, Met, P
• the variants can comprise the amino acid sequence of the native GDNF protein or polypeptide with at least one nonconservative amino acid substitution.
• the non-conservative amino acid substitution(s) enhance(s) the activity of the variant relative to that of the native mature human GDNF protein.
• the human GDNF polypeptide has the ability to bind to the RET receptor.
• the human GDNF polypeptide has the ability to bind to the NCAM receptor.
• the human GDNF polypeptide has the ability to bind to the GDNF family coreceptor alpha (GFRalpha) 1-3.
• the human GDNF polypeptide comprises at least 10, 15 or 20 amino acids (e.g., contiguous amino acids) from the mature human native GDNF protein.
• 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 a!., J Neurosci. 2009 Sep 9; 29(36): 11360-11376).
• the human GDNF polypeptide comprises at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 amino acids (e.g., contiguous amino acids) from the mature human native GDNF protein, including residues 153-167 of SEQ ID NO: 1.
• the human GDNF polypeptide comprises an amino acid sequence that is at least 50%, 60% or 70% identical to the sequence of residues 78-211 depicted in FIG. 3A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 80% identical to the sequence of residues 78-211 depicted in FIG. 3A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 85% identical to the sequence of residues 78-211 depicted in FIG. 3A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of residues 78-211 depicted in FIG.
• the human GDNF polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of residues 78-211 depicted in FIG. 3A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 98% identical to the sequence of residues 78-211 depicted in FIG. 3A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 99% identical to the sequence of residues 78-211 depicted in FIG. 3A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises or consists of the sequence of residues 78-211 depicted in FIG.
• Identity refers to sequence identity between two polypeptides. Identity can be determined by comparing each position in the aligned sequences. Methods of determining percent identity are known in the art, and several tools and programs are available to align amino acid sequences and determine a percentage of identity including EMBOSS Needle, ClustalW, SIM, DIALIGN, etc. As used herein, a given percentage of identity with respect to a specified subject sequence, or a specified portion thereof, may be defined as the percentage of amino acids in the candidate derivative sequence identical with the amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the Smith Waterman algorithm (Smith & Waterman, J. Mol.
• Covalent modifications of the human GDNF polypeptide are included within the scope of this disclosure.
• the native glycosylation pattern of the human GDNF polypeptide may be modified (Beck et a!., Curr. Pharm. Biotechnol. 9: 482-501 , 2008; Walsh, Drug Discov. Today 15: 773-780, 2010), and linking the human GDNF polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos.
• PEG polyethylene glycol
• polypropylene glycol polypropylene glycol
• polyoxyalkylenes polyoxyalkylenes
• the human GDNF polypeptide may comprise one or more modifications that confer additional biological properties to the polypeptide such as protease resistance, plasma protein binding, increased plasma half-life, tissue or intracellular penetration, etc.
• modifications include, for example, covalent attachment of molecules/moiety to the polypeptide such as fatty acids (e.g., C 6 -Ci 8 ), attachment of proteins such as albumin (see, e.g., U.S. Patent No. 7,268,113); sugars/polysaccharides (glycosylation), biotinylation or PEGylation (see, e.g., U.S. Patent Nos. 7,256,258 and 6,528,485).
• the human GDNF polypeptide may also be conjugated to moieties to induce its multimerization or oligomerization (e.g., tetramerization), for example by fusing the human GDNF polypeptide to an oligomerization domain or to a molecule that may be oligomerized (e.g., biotin that may bind to 4 binding sites on streptavidin).
• moieties e.g., tetramerization
• the human GDNF polypeptide may also be conjugated to moieties that will target the GDNF polypeptide to the distal colon or to specific cells of the distal colon (e.g., Schwann cells and/or precursor thereof, enteric glial cells, pericytes), for example using an antibody, antibody fragment or ligand that binds to a marker present on cells from the distal colon.
• moieties that will target the GDNF polypeptide to the distal colon or to specific cells of the distal colon (e.g., Schwann cells and/or precursor thereof, enteric glial cells, pericytes), for example using an antibody, antibody fragment or ligand that binds to a marker present on cells from the distal colon.
• the human GDNF polypeptide can also be conjugated to one or more therapeutic or active agents (e.g., to a drug, or to another polypeptide to form a fusion polypeptide). Any method known in the art for conjugating the human GDNF polypeptide to another moiety (e.g., active agent) may be employed, including those methods described by Hunter et al. (1962) Nature, 144:945; David etal. (1974) Biochemistry, 13: 1014; Pain etal. (1981) J. Immunol. Meth., 40:219; Nygren, J. Histochem. and Cytochem., 30:407 (1982), and Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.
• the human GDNF polypeptide may be conjugated to another moiety either directly or through a linker.
• the GDNF polypeptide and/or SCFA is formulated in a pharmaceutical composition.
• the GDNF polypeptide and SCFA may be formulated in the same pharmaceutical composition or in distinct pharmaceutical compositions.
• the GDNF polypeptide and the SCFA are formulated in distinct pharmaceutical compositions.
• the GDNF polypeptide and the SCFA are formulated in the same pharmaceutical composition.
• Such pharmaceutical compositions typically comprise one or more pharmaceutically acceptable excipients.
• excipient has its normal meaning in the art and is any ingredient that is not an active ingredient (drug) itself. Excipients include for example buffers, binders, lubricants, diluents, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, stabilizing agent, release-delaying agents and other components. "Pharmaceutically acceptable excipient” as used herein refers to any excipient that does not interfere with effectiveness of the biological activity of the active ingredients and that is not toxic to the subject, i.e., is a type of excipient and/or is for use in an amount which is not toxic to the subject.
• Excipients are well known in the art, and the present composition is not limited in these respects.
• the carrier/excipient can be suitable, for example, for intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal, rectal or pulmonary (e.g., aerosol) administration.
• compositions are prepared using standard methods known in the art by mixing the active ingredient having the desired degree of purity with one or more optional pharmaceutically acceptable carriers, excipients and/or stabilizers (see Remington: The Science and Practice of Pharmacy, by Loyd V Allen, Jr, 2012, 22 nd edition, Pharmaceutical Press; Handbook of Pharmaceutical Excipients, by Rowe etal., 2012, 7 th edition, Pharmaceutical Press).
• the GDNF polypeptide and/or SCFA (or pharmaceutically acceptable salt or ester thereof) is formulated for oral administration.
• Formulations suitable for oral administration may include (a) liquid solutions, such as an effective amount of active agent(s)/composition(s) suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
• Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
• Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient(s), carriers known in the art.
• a flavor e.g., sucrose
• an inert base such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient(s), carriers known in the art.
• the GDNF polypeptide and/or SCFA is formulated for parenteral administration (e.g., injection).
• parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
• Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
• 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 as a gel.
• excipients e.g., lactose
• aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate
• glycocholate and deoxycholate may be oily solutions for administration in the form of nasal drops, or as a gel.
• the GDNF polypeptide and/or SCFA is formulated for enteric delivery, i.e., delivery into the intestines. This may be achieved by methods well known in the art.
• the GDNF polypeptide and/or SCFA may be coated or encapsulated with an enteric agent or material. Enteric agents for instance allow release at certain pHs or in the presence of degradative enzymes or bacteria that are characteristically present in specific locations of the Gl tract (e.g., small intestine, large intestine, or specific regions thereof) where release is desired.
• the enteric material is pH-sensitive and is affected by changes in pH encountered within the gastrointestinal tract (pH-sensitive release).
• the enteric material typically remains insoluble at gastric pH, then allows for release of the active ingredient in the higher pH environment of the downstream gastrointestinal tract (e.g., often the duodenum, or sometimes the colon).
• the enteric material comprises enzymatically degradable polymers that are degraded by bacterial enzymes (e.g., carbohydrate processing enzymes such as glycosidases, polysaccharide lyases and carbohydrate esterases) present in the lower gastrointestinal tract, particularly in the colon.
• Such enteric materials include, for example, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, and other methacrylic resins that are commercially available under the trade-name Acryl-EZE® (Colorcon, USA), Eudragit® (Rohm Pharma; Westerstadt, Germany), including Eudragit® L30D-55 and L100-55 (soluble at pH 5.5 and above), Eudragit® L
• pH-dependent systems e.g., using pH-dependent polymers
• receptor-mediated systems e.g., using pH-dependent polymers
• magnetically-driven systems e.g., magnetically-driven systems
• delayed or time-dependent systems e.g., microbially triggered drug delivery systems
• microbially triggered drug delivery systems e.g., comprising sugar-based polymers that may be degraded by enzymes produced by the colon microflora such as glucoronidase, xylosidase, arabinosidase, galactosidase), pressure controlled colonic delivery capsule (drug release induced by the higher pressures encountered in the colon), osmotic controlled drug delivery, as well as any combinations of these approaches (e.g., colon targeted delivery system (CODESTM) using a combined approach of pH dependent and microbially triggered drug delivery).
• CODESTM colon targeted delivery system
• the GDNF polypeptide and/or SCFA (or pharmaceutically acceptable salt or ester thereof) is formulated in a capsule made of an enteric material (enteric capsule).
• the GDNF polypeptide and/or SCFA (or pharmaceutically acceptable salt or ester thereof) is formulated for administration into the distal colon of the subject.
• distal colon refers to the last three segments of the colon, namely the descending colon, the sigmoid colon and the rectum.
• the pharmaceutical composition is administered or is for administration into the rectum and/or the sigmoid colon.
• the pharmaceutical composition is administered or is for administration into the rectosigmoid region, which comprises the last part of the sigmoid colon and the beginning of the rectum.
• GDNF polypeptide and/or SCFA may be administered directly into the distal colon, or may be administered at a site away from the distal colon but using suitable means to provide delivery of the GDNF polypeptide and/or SCFA (or pharmaceutically acceptable salt or ester thereof) into the distal colon.
• the formulation(s) may comprise a coating that is specifically degraded under the conditions (e.g., pH, enzymatic environment, bacterial environment, etc.) of the distal colon, and thus the formulation(s) may be administered in another region of the gastro-intestinal system but the GDNF polypeptide and/or SCFA (or pharmaceutically acceptable salt or ester thereof) will only be released once the formulation reaches the colon, and more specifically the distal colon.
• Approaches for colon specific drug delivery are well known in the art (see, e.g., Philip et a!., Oman Med J. 2010 Apr; 25(2): 79-87; Lee et al., Pharmaceutics.
• pH-dependent systems e.g., using pH-dependent polymers
• receptor-mediated systems e.g., using pH-dependent polymers
• magnetically-driven systems e.g., magnetically-driven systems
• delayed or time-dependent systems e.g., microbially triggered drug delivery systems
• microbially triggered drug delivery systems e.g., comprising sugar- based polymers that may be degraded by enzymes produced by the colon microflora such as glucoronidase, xylosidase, arabinosidase, galactosidase
• pressure controlled colonic delivery capsule drug release induced by the higher pressures encountered in the colon
• osmotic controlled drug delivery as well as any combinations of these approaches (e.g., colon targeted delivery system (CODESTM) using a combined approach of pH dependent and microbially triggered drug delivery).
• CODESTM colon targeted delivery system
• Formulations for rectal/distal colon administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non-irritating carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax, or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the appropriate body cavity and release the GDNF polypeptide and/or SCFA (or pharmaceutically acceptable salt or ester thereof). More recently, liquid suppositories have been developed.
• Liquid suppositories typically contain thermosensitive and/or mucoadhesive polymers such as poloxamers, Carbopol ® (crosslinked polyacrylic acid polymers), sodium alginate, polycarbophil, hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose, and methylcellulose.
• thermosensitive and/or mucoadhesive polymers such as poloxamers, Carbopol ® (crosslinked polyacrylic acid polymers), sodium alginate, polycarbophil, hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose, and methylcellulose.
• Formulations for rectal/distal colon administration may also be presented as an enema, a liquid-drug solution, suspension or emulsion that is injected into the rectum and the distal colon.
• the liquid in which the GDNF polypeptide and/or SCFA (or pharmaceutically acceptable salt or ester thereof) is diluted may be water or a saline solution or suspension, for example.
• Formulations for rectal/distal colon administration may also be in the form of a rectal foam or gel. Rectal gels are semi-solid formulations that contain a solvent trapped within a polymer network to create a viscous consistency.
• Viscosity of the gel can be modified by the addition of co-solvents (e.g., glycerin and propylene glycol) and electrolytes.
• Foams comprise a hydrophilic liquid continuous phase containing a foaming agent and a gaseous dispersion phase distributed throughout. Following rectal administration, they transition from a foam state to a liquid or semi-solid state on the mucosal surface.
• Foaming agents are typically amphiphilic substances that are important for foam generation and stabilization.
• the molecules contain hydrophilic components that are soluble in the aqueous phase and hydrophobic components that form micelles to minimize contact with the aqueous phase.
• Administration into the rectum/distal colon may be performed using currently available endoscopes or specialized catheters designed for rectal administration or injection into the distal colon wall of medications and liquids, which may be placed safely and remain comfortably in the rectum for repeated use.
• the GDNF polypeptide and/or SCFA may be administered according to any suitable dosage regimen, for example fourtimes- a-day, twice-a-day, once-a-day, twice-a-week, once-a-week, etc.
• the treatment may be performed for any suitable period of time to achieve the desired effect, for example for 1 week, 2 weeks, 3 weeks or more.
• the effective dose of GDNF polypeptide administered or for administration to the human subject corresponds to a dose of about 5 pg to about 20 pg in a mouse pup, which is the range shown to be effective in the studies described herein.
• a 10 pi enema comprising a recombinant GDNF solution was administered to mouse pups. 10 pi is estimated to correspond to the volume necessary to fill the distal colon and rectum of the pups.
• administration of 5 pg GDNF in mice is achieved by administering 10 pi of a 0.5 pg/pl GDNF solution
• administration of 20 pg GDNF in mice is achieved by administering 10 pi of a 2.0 pg/pl GDNF solution.
• the volume required to fill the distal colon and rectum of a human baby may be estimated using the formula: 10ml x weight of the baby (in kg). Accordingly, a dose of 5 pg GDNF in mice corresponds to about 5 mg per kg in a human baby, and a dose of 20 pg GDNF in mice corresponds to about 20 mg per kg in a human baby.
• the effective dose of GDNF polypeptide administered or for administration to the human subject is about 5 mg to about 20 mg per kg, preferably about 10 mg to about 15 mg per kg.
• the GDNF polypeptide is administered or is for administration through a 0.5 mg/ml to 2 mg/ml composition (solution or gel).
• the combination of therapeutic agents may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form.
• Co- administration in the context of the present disclosure refers to the use of more than one therapy in the course of a coordinated treatment to achieve an improved clinical outcome.
• the combination of GDNF polypeptide and SCFA or pharmaceutically acceptable salt or ester thereof described herein may be used in combination with other therapies or drugs, for example analgesics or anti-inflammatory agents.
• the above-mentioned treatment with a combination comprising a GDNF polypeptide and SCFA or pharmaceutically acceptable salt or ester thereof may be performed in combination with surgery (e.g., pull-through surgery of the Swenson, Soave or Duhamel type).
• surgery e.g., pull-through surgery of the Swenson, Soave or Duhamel type.
• clinicians often recommend a trial of daily enema treatments priorto surgery.
• Addition of the combination of GDNF + SCFA disclosed herein to the enema might increase the likelihood that children with HSCR responded well to preoperative enema therapy.
• the above-mentioned treatment with a combination comprising a GDNF polypeptide and SCFA or pharmaceutically acceptable salt or ester thereof is performed prior to pull-through surgery.
• Saline enemas are also commonly used in children with HSCR after pull-through surgery.
• Post-surgical problems in HSCR patients are believed to be due at least in part to hypoganglionosis in retained distal bowel, the so-called “transition zone”.
• addition of combination comprising a GDNF polypeptide and SCFA or pharmaceutically acceptable salt or ester thereof to the enema may be useful to correct the hypoganglionosis in retained distal bowel and/or the neuronal subtype imbalance in the transition zone after surgery.
• the above-mentioned treatment with a combination comprising a GDNF polypeptide and SCFA or pharmaceutically acceptable salt or ester thereof is performed after pull-through surgery.
• the above-mentioned treatment with a combination comprising a GDNF polypeptide and SCFA or pharmaceutically acceptable salt or ester thereof is performed in combination with ENS stem cell-based therapies, which are being considered for the treatment of HSCR.
• GDNF may be a useful adjunct to these therapies to promote engraftment.
• HSCR is clinically subdivided into short-segment (S-HSCR) and long-segment forms (L- HSCR).
• S-HSCR which occurs in >80% of cases, means the ENS is absent from rectum and sigmoid colon.
• L-HSCR means longer regions of distal bowel are aganglionic.
• the method/use described herein is for the treatment of a human patient suffering from S-HSCR or L-HSCR. In an embodiment, the method/use described herein is for the treatment of a human patient suffering from S-HSCR. In an embodiment, the method/use described herein is for the treatment of a human patient suffering from L-HSCR.
• the HSCR patient may be an adult patient or pediatric patient.
• the HSCR patient is a pediatric patient, preferably a patient that is less than 5, 4, 3 or 2 year-old, more preferably a patient that is less than 1 year-old or less than 6 month-old.
• the patient is a male.
• HSCR has been associated with mutations in RET, EDNRB, SOX10, PHOX2B, and ZFHX1B, as well as with Down syndrome or Trisomy 21 (Collagen Vl-associated HSCR). There is also a significant sex difference with male to female ratio as high as 5 to 1.
• the HSCR is Collagen Vl-associated HSCR.
• the HSCR is EDNRB mutation-associated HSCR.
• the HSCR is male-biased HSCR.
• the HSCR is a RET mutation-associated HSCR, e.g., HSCR associated with a mutation that reduces RET expression and/or activity in cells from the distal colon.
• the mutation may be a mutation in RET or in a protein involved in RET signaling.
• the mutation is a mutation in the RET protein. Mutations in the RET gene on chromosome 10q11.2 have been shown to account for 50% of familial and 15-20% of sporadic cases of HSCR, most of which (-75%) were associated with L-HSCR.
• the HSCR is not a RET mutation-associated HSCR.
• mice Homozygous Holstein Ts/Ts mice (Hol Tg Tg ; a model for Trisomy 21 [Collagen VI]- associated HSCR) were treated with either 10 pi enemas of GDNF (1 pg/mI), or 10 mI of GDNF combined with 5 mM of amino-butyrate acid (Butyrate) or other growth factors at 1 mg/ml, or vehicle (1X PBS) as control. Enemas were administered once a day from postnatal day (P) 4 until P8, and mice were monitored daily until P60 or megacolon-associated death. Survival graphs and statistical analyses were generated with GraphPad Prism software. One-way ANOVA followed by post-hoc Tukey multiple comparison test was carried out, and significance level was set at P ⁇ 0.05. Number of biological replicates (n) of each cohort is indicated where relevant in Figures.
• Example 2 Assessment of the effect of GDNF in combination with neurotrophic factors on the survival of Hol Tg/Tg mice
• BMP4 Bone morphogenetic protein 4
• NGF Nerve growth factor
• BDNF Brain-derived neurotrophic factor
• MEF Mesencephalic Astrocyte Derived Neurotrophic Factor
• CDNF Cerebral Dopamine Neurotrophic Factor
• EGF Epidermal Growth Factor
• NRTN Neurturin
• FGF2 Fibroblast growth factor 2
• RA butyrate and Retinoic Acid
• FIGs. 1A-C show that whereas butyrate alone has no effect on mouse survival (similar to vehicle), it significantly improves the effect of GDNF, indicating that the GDNF + butyrate combination exhibits a synergistic effect on survival.
• FIGs. 2A-C none of the other neurotrophic factors tested were able to significantly improve the effect of GDNF on survival in this mouse model. Actually, combining GDNF with some of the neurotrophic factors even reduce survival relative to administration of GDNF alone (FIG. 2C).
• Gdnf is mitogenic, neurotrophic, and chemoattractive to enteric neural crest cells in the embryonic colon. Dev Dyn 240, 1402-1411 (2011).
• GDNF is a chemoattractant for enteric neural cells. Dev Biol 229, 503- 516 (2001).
• Glial cell line-derived neurotrophic factor alters axon schwann cell units and promotes myelination in unmyelinated nerve fibers. J Neurosci 23, 561-567 (2003).
• NCAM neural cell adhesion molecule NCAM is an alternative signaling receptor for GDNF family ligands. Ce// 113, 867-879 (2003).
• NCAM neural cell adhesion molecule
• Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential, and factor responsiveness. Neuron 35, 657-669 (2002).
• RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons. Development 128, 3963-3974 (2001).

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