Use of GALR2 agonists for the treatment of gastrointestinal and / or endocrine disorders

Abstract

The present invention provides agonists for galanin receptor type 2 (GALR2 agonists) and methods of using such agonists to treat gastrointestinal and / or endocrine disorders. The GALR2 agonists described herein are distinct (functionally and / or structurally) when compared to other GALR2 ligands (e.g., wild-type spexin and / or galanin), and therefore more effective in treating the diseases described herein.

Description

Detailed Description of the Invention

[0001] [Technical Field] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims priority to U.S. Provisional Application No. 63 / 385,363, filed November 29, 2022, the entire text of which is incorporated herein by reference.

[0002] Electronically Submitted Sequence Listing Reference The contents of the Sequence Listing have been submitted electronically with this application (Filename: 3763_021PC01_Seqlisting_ST26.xml; Size: 108,231 bytes; Creation Date: November 27, 2023), the entire contents of which are incorporated herein by reference.

[0003] The present invention provides galanin receptor type 2 (GALR2) specific agonists and their use for the treatment of a wide range of diseases, including gastrointestinal and / or endocrine disorders.

[0004] [Background technology] Diseases and disorders of the gastrointestinal and / or endocrine systems are major health problems worldwide. For example, bowel symptoms are very common in people suffering from central nervous system injuries or diseases (Emmanuel, A., F1000 Research 8 (F1000 Faculty Rev): 1800 (2019)). In fact, in a significant number of spinal cord injury patients, bowel dysfunction is reported as a much greater problem than bladder dysfunction, sexual dysfunction, pain, fatigue, or body image issues. These symptoms are very common in many endocrine disorders (Maser et al. World J Gastroenterol 12 (20): 3174-3179 (2006)). Thus, there remains a need for safe and effective alternative treatments for gastrointestinal and / or endocrine disorders.

[0005] Summary of the Invention Provided herein are methods of treating a gastrointestinal disorder in a subject in need thereof, the methods comprising administering to the subject a galanin receptor type 2 (GALR2) agonist, a nucleic acid encoding the GALR2 agonist, or a vector comprising the nucleic acid, wherein the GALR2 agonist is selected from the group consisting of X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ) or citrulline (Cit); X 3 is threonine (T), alanine (A), or lysine (K); X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); X 5 is asparagine (N) or glutamine (Q); X 6 is alanine (A) or serine (S); X 7 is alanine (A) or methionine (M); X 8 is leucine (L), glutamine (Q) or glycine (G); X 11 is leucine (L), phenylalanine (F), tyrosine (Y) or aspartic acid (D); X 12 is glycine (G) or alanine (A); X 13 is proline (P), arginine (R), or alanine (A); X 14 is glutamine (Q), histidine (H), or valine (V), and wherein the GALR2 agonist specifically activates GALR2.

[0006] In some aspects, the gastrointestinal disorder comprises constipation, neurogenic bowel dysfunction (NBD), or all of these. In some aspects, the constipation comprises opioid-induced constipation (OIC).

[0007] The present invention also provides a method for regulating defecation in a subject in need thereof, the method comprising administering to the subject a galanin receptor type 2 (GALR2) agonist, a nucleic acid sequence encoding the GALR2 agonist, or a vector comprising the nucleic acid, wherein the GALR2 agonist is selected from the group consisting of X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ) or citrulline (Cit); X 3 is threonine (T), alanine (A), or lysine (K); X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); X 5 is asparagine (N) or glutamine (Q); X 6 is alanine (A) or serine (S); X 7 is alanine (A) or methionine (M); X 8 is leucine (L), glutamine (Q) or glycine (G); X 11 is leucine (L), phenylalanine (F), tyrosine (Y) or aspartic acid (D); X 12 is glycine (G) or alanine (A); X13 is proline (P), arginine (R), or alanine (A); X 14 is glutamine (Q), histidine (H), or valine (V), and wherein the GALR2 agonist specifically activates GALR2.

[0008] In some aspects, modulating bowel movement comprises modulating colonic transit time in the subject. In some aspects, after administration of the GALR2 agonist, the colonic transit time in the subject is reduced compared to a reference subject (e.g., the subject before administration and / or a matched subject not receiving the administration). In some aspects, the colonic transit time is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the reference subject.

[0009] Also provided herein is a method of treating an endocrine disorder in a subject in need thereof, the method comprising administering to the subject a galanin receptor type 2 (GALR2) agonist, a nucleic acid sequence encoding the GALR2 agonist, or a vector comprising the nucleic acid sequence, wherein the GALR2 agonist is selected from the group consisting of X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ) or citrulline (Cit); X 3 is threonine (T), alanine (A), or lysine (K); X 4is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); X 5 is asparagine (N) or glutamine (Q); X 6 is alanine (A) or serine (S); X 7 is alanine (A) or methionine (M); X 8 is leucine (L), glutamine (Q) or glycine (G); X 11 is leucine (L), phenylalanine (F), tyrosine (Y) or aspartic acid (D); X 12 is glycine (G) or alanine (A); X 13 is proline (P), arginine (R), or alanine (A); X 14 is glutamine (Q), histidine (H), or valine (V), and wherein the GALR2 agonist specifically activates GALR2.

[0010] In some aspects, the endocrine disorder comprises chronic renal failure, hypercalcemia, or all of these.

[0011] In some aspects of the methods provided herein (e.g., methods provided above), X 1 In some aspects, N at position 2 of SEQ ID NO: 1 is D-tryptophan. 4 A in X is D-alanine, D-glutamic acid, or D-arginine. 4 In some aspects, V is D-valine. 6 In some aspects, A in X is D-alanine. 11 In some aspects, K is D-lysine. 12 In some aspects, A in X is D-alanine. 13 In some aspects, A in X is D-alanine.14 Q is D-glutamine.

[0012] In some aspects of the methods provided herein (e.g., the methods provided above), the GALR2 agonist does not activate (i) galanin receptor type 1 (GALR1), (ii) galanin receptor type 3 (GALR3), or (iii) all of (i) and (ii).

[0013] In some aspects of the methods provided herein (e.g., methods provided above), X 7 is A and X 11 is F. In some respects, X 5 is N and X 7 is A and X 11 is F. In some respects, X 5 is N and X 7 is A and X 11 is F and X 13 is P.

[0014] In some aspects of the methods provided herein (e.g., methods provided above), the amino acid sequence of the GALR2 agonist comprises the sequence set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, or SEQ ID NO:91.

[0015] In some aspects of the methods provided herein (e.g., methods provided above), the amino acid sequence of the GALR2 agonist is attached to polyethylene glycol (PEG), an acetyl (Ac) group, or Fmoc. 1 is N protected with polyethylene glycol (PEG), acetyl (Ac) group or Fmoc.

[0016] In some aspects of the methods provided herein (eg, those provided above), the amino acid sequence of the GALR2 agonist is attached to the C-terminal NH2.

[0017] In some aspects of the methods provided herein (e.g., methods provided above), the GALR2 agonist is administered to the subject intranasally, intrasphincterically, intramuscularly, subcutaneously, intraocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraventricularly, intraspinally, intraventricularly, intracisternally, intracapsularly, topically, orally, or a combination thereof. In some aspects, the GALR2 agonist is administered to the subject subcutaneously, intranasally, or intraperitoneally.

[0018] In some aspects of the methods provided herein (e.g., the methods provided above), the GALR2 agonist is administered to a subject one, two, three, four, five, six, or seven or more times.

[0019] In some aspects of the methods provided herein (e.g., methods provided above), the method further comprises administering an additional therapeutic agent to the subject. In some aspects, the additional therapeutic agent comprises a laxative (e.g., bisacodyl), an opioid receptor antagonist (e.g., naloxone methiodide), douching (e.g., anal or colonic douching), electrical stimulation, or a combination thereof. In some aspects, the additional therapeutic agent and the GALR2 agonist are administered to the subject simultaneously. In some aspects, the additional therapeutic agent and the GALR2 agonist are administered to the subject sequentially. In some aspects, the GALR2 agonist is administered to the subject as a lyophilized powder or a solution.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides a diagram of the general experimental design for evaluating the effects of the GALR2 agonists described herein in normal, unsensitized ICR mice. The upper arrows (seven in total) indicate daily administration of the GALR2 agonist. Control animals received a control vehicle (negative control) or bisacodyl (positive control). Thirty minutes after the last administration, glass beads were inserted into the animals' rectums. Colonic transit time was assessed by measuring the delay in expelling the glass beads.

[0021] Figure 2 shows colonic transit time (seconds) in normal, unsensitized ICR mice administered a PEGylated GALR2 (PEG-GALR2) agent via intraperitoneal or intranasal administration. Treatment groups were as follows: (G1) control vehicle (intraperitoneal), (G2) PEG-GALR2 agent (1 mg / kg; intraperitoneal), (G3) control vehicle (intranasal), (G4) PEG-GALR2 agent (10 pg / mouse; intranasal), and (G5) bisacodyl (100 mg / kg; oral). The overall experimental design was as shown in Figure 1. Statistical analysis was performed using Student's t-test. * indicates a significant difference at the p<0.05 level compared to G3.

[0022] Figure 3 shows the dose-dependent effect of a PEGylated GALR2 (PEG-GALR2) agent on colonic transit time in normal, non-sensitized ICR mice. Treatment groups were as follows: (G1) control vehicle (intraperitoneal), (G2) PEG-GALR2 agent 0.1 mg / kg (intraperitoneal), (G3) PEG-GALR2 agent 0.3 mg / kg (intraperitoneal), (G4) PEG-GALR2 agent 1 mg / kg (intraperitoneal), (G5) PEG-GALR2 agent 1 mg / kg (subcutaneous), and (G6) bisacodyl (100 mg / kg; oral). The overall experimental design was as shown in Figure 1. Statistical analysis was performed using Student's t-test. * indicates a significant difference at the p<0.05 level compared to G1.

[0023] Figure 4 provides a diagram illustrating the overall experimental design for evaluating the effects of the GALR2 agonists described herein in a mouse model of opioid-induced constipation (OIC). The upper arrows (seven in total) indicate daily administration of the GALR2 agonist. The GALR2 agonist administered to the relevant group was PEGylated. Control animals received a control vehicle (negative control) or bisacodyl (positive control). Animals receiving a single dose of naloxone methiodide (e.g., on day 7) were also used as a positive control. Ten minutes after the last administration, each animal received morphine (3 mg / kg; subcutaneously). Thirty minutes after the last administration, glass beads were inserted into the animals' rectums. Colonic transit time was assessed by measuring the delay in expelling the glass beads.

[0024] Figure 5 shows the colonic transit time in mice administered PEGylated GALR2 (PEG-GALR2) agonists via various routes of administration (e.g., intraperitoneal, intranasal, or subcutaneous). Treatment groups were as follows: (G1) normal, non-sensitized mice (no OIC); (G2) control vehicle (intraperitoneal); (G3) PEG-GALR2 agonist (1 mg / kg; intraperitoneal); (G4) control vehicle (intranasal); (G5) PEG-GALR2 agonist (10 pg; intranasal); (G6) bisacodyl (100 mg / kg; oral) (positive control group); and (G7) naloxone methiodide (10 mg / kg; intraperitoneal) (positive control group). The overall experimental design was as shown in Figure 4. Statistical analysis was performed using Student's t-test. "*** / ** / *" = Significant difference at p<0.001 / 0.01 / 0.05 levels when compared to G1. "###" = Significant difference at p<0.001 level when compared to G2. "$$$" = Significant difference at p<0.001 level when compared to G4.

[0025] FIG. 6 shows the dose-dependent effect of a GALR2 agonist on colonic transit time in OIC mice. The treatment groups were as follows: (G1) normal, non-sensitized mice (no OIC), (G2) control vehicle (intraperitoneal), (G3) 0.1 mg / kg PEG-GALR2 agent (intraperitoneal), (G4) 0.3 mg / kg PEG-GALR2 agent (intraperitoneal), (G5) 1 mg / kg PEG-GALR2 agent (intraperitoneal), (G6) control vehicle (intranasal), (G7) 1 pg PEG-GALR2 agent (intranasal), (G8) 3 pg PEG-GALR2 agent (intranasal), (G9) 10 pg PEG-GALR2 agent (intranasal), (G10) bisacodyl (100 mg / kg; oral), and (G11) naloxone methiodide (10 mg / kg; intraperitoneal). The overall experimental design is shown in Figure 4. Statistical analysis was performed using Student's t-test. "*** / **" = Significant difference at p<0.001 / 0.01 levels when compared to G1, respectively. "### / ## / #" = Significant difference at p<0.001 / 0.01 / 0.05 levels when compared to G2, respectively. "$$$ / $$ / $" = Significant difference at p<0.001 / 0.01 / 0.05 levels when compared to G6, respectively.

[0026] Figure 7 shows the effect of administration route (intraperitoneal vs. subcutaneous) on the dose-dependent effect of GALR2 agonists on colonic transit time in mice with OIC. Treatment groups were as follows: (G1) normal, non-sensitized mice (no OIC), (G2) control vehicle (intraperitoneal), (G3) 0.5 mg / kg PEG-GALR2 agonist (intraperitoneal), (G4) 1 mg / kg PEG-GALR2 agonist (intraperitoneal), (G5) control vehicle (subcutaneous), (G6) 0.5 mg / kg PEG-GALR2 agonist (subcutaneous), (G7) 1 mg / kg PEG-GALR2 agonist (subcutaneous), (G8) bisacodyl (100 mg / kg; oral), and (G9) naloxone methiodide (10 mg / kg; intraperitoneal). The overall experimental design was as shown in Figure 4. Statistical analysis was performed using Student's t-test. "*** / **" = significant difference at p<0.001 / 0.01 level when compared to G1. "###" = significant difference at p<0.001 level when compared to G2. "$$$" = significant difference at p<0.001 level when compared to G5.

[0027] Figure 8 compares colonic transit time in mice treated with subcutaneous administration of wild-type spexin peptide or a GALR2 agonist described herein. The GALR2 agonist was either PEGylated (PEG-GALR2 agonist) or unPEGylated (GALR2 agonist). Treatment groups were as follows: (G1) normal, non-sensitized mice (no OIC), (G2) control vehicle, (G3) 0.5 mg / kg PEG-GALR2 agonist, (G4) 1 mg / kg PEG-GALR2 agonist, (G5) 0.5 mg / kg GALR2 agonist, (G6) 1 mg / kg GALR2 agonist, (G7) 0.5 mg / kg wild-type spexin, (G8) 1 mg / kg wild-type spexin, (G9) bisacodyl (100 mg / kg; orally administered), (G10) nanoparticles (orally administered), and (G11) nanoparticles (orally administered). Xon methiodide (10 mg / kg; intraperitoneal administration). The overall experimental design is as shown in Figure 4. "*** / ** / *" = Significant difference (t-test) at p<0.001 / 0.01 / 0.05 levels when compared to G1, respectively. "### / ## / #" = Significant difference (t-test) at p<0.001 / 0.01 / 0.05 levels when compared to G2, respectively. "$" = Significant difference (G4, G6, G8, one-way ANOVA, Bonferroni's multiple comparison test) at p<0.05 level when compared to G8.

[0028] Figures 9a and 9b show the effects of administration schedule and route of administration on GALR2 agonist-mediated colonic transit time regulation in OIC mice. Figure 9a is a schematic diagram of the overall experimental design. Non-pegylated GALR2 agonists were administered to OIC mice as follows: (i) daily subcutaneous administration (1 mg / kg per dose) for 7 days ("G3"), (ii) a single subcutaneous administration (1 mg / kg) on ​​day 7 ("G4"), and (iii) a single intranasal administration (10 pg / animal) on day 7 ("G5"). Some animals received a single dose of Fc-conjugated non-pegylated GALR2 (GALR2-Fc) agonist (35 mg / kg) on ​​day 4 ("G6"). Normal, non-sensitized mice (no OIC, "G1") and OIC mice subcutaneously injected with control vehicle ("G2") served as controls. Ten minutes after the last dose, each animal received morphine (3 mg / kg; subcutaneous). Thirty minutes after the last dose, a glass bead was inserted into the animal's rectum. Colonic transit time was assessed by measuring the delay in the animal's expulsion of the glass bead. Figure 9b shows a comparison of colonic transit time between the various treatment groups. "*** / **" = significant difference at the p<0.0001 / 0.01 level when compared to G1. "###" = significant difference at the p<0.001 level when compared to G2.

[0029] Figures 10a, 10b, and 10c show the effects of GALR2 agonists on colonic transit time after multiple inductions of opioid-mediated constipation. Figure 10a shows the overall experimental design. Mice were administered a single dose of GALR2 agonists subcutaneously (1 mg / kg) (G3) or intranasally (10 pg / animal) (G4). Normal, non-sensitized mice (without OIC) (G1) and OIC mice treated with a control vehicle (deionized water) (G2) served as controls. For the first OIC induction, morphine (3 mg / kg) was administered subcutaneously to the animals 4 hours after administration, and a glass bead was inserted into the rectum 30 minutes later. The first colonic transit time of the animals was then assessed for 60 minutes. At 48 hours after administration, a second dose of morphine (3 mg / kg) was administered subcutaneously, and after the second OIC induction, a glass bead was inserted into the rectum again 30 minutes later. The animals were then evaluated for second colonic transit time for 60 minutes. Colonic transit time was assessed by measuring the delay in the animals expelling the glass beads. Figure 10b compares first colonic transit times. Figure 10c compares second colonic transit times. "***" = significant difference (T-test) when compared to G1, p<0.001. "###" = significant difference (T-test) when compared to G2, p<0.001.

[0030] Figure 11 compares the in vitro efficacy of three different GALR2 agonist peptides with specific amino acid substitutions at the fourth amino acid position. Specifically, the GALR2 agonist peptides are as follows: (1) nWTaNAALYLFGPq-NFE (D-alanine substitution; triangles), (2) PEG2-NWTeNAALYLFGPq-NH2 (D-glutamic acid; filled circles), and (3) PEG2-NWTrNAALYLFGPq-NH2 (D-arginine; open circles). The efficacy of the GALR2 agonists was assessed by measuring SRE luciferase activity in the peptide-treated mGqi-hGALR2-SRE Luc-expressing cell line.

[0031] [Mode for Carrying Out the Invention] The present invention generally relates to methods of treating a variety of diseases and disorders (e.g., gastrointestinal, endocrine, and / or metabolic disorders) comprising administering to a subject an agonist of galanin receptor type 2 (a "GALR2 agonist"). As further described herein, the GALR2 agonists of the invention exhibit one or more properties (e.g., structural and / or functional) that distinguish them from other GALR2 ligands (e.g., wild-type spexin). Additional aspects of the invention are presented throughout this application.

[0032] To facilitate understanding of the subject matter disclosed herein, a number of terms and syntax are defined. Additional definitions are set forth throughout the detailed description.

[0033] I. Definition Throughout this specification, the terms "a," "an," "an," "an," "an," "an," "an," "one," "one or more," "at least one," etc. can be used interchangeably herein.

[0034] Also, as used herein, "and / or" should be construed as specifically disclosing each of the two specified features or components with or without the other one. Thus, as used herein, the term "and / or" in a phrase such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" in a phrase such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0035] As used herein, aspects described with the expression "comprising" are understood to also disclose other similar aspects described with the terms "consisting of" and / or "consisting essentially of."

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology (Juo, Pei-Show, 2nd ed., 2002, CRC Press); Dictionary of Cell and Molecular Biology (3rd ed., 1999, Academic Press); and Oxford Dictionary of Biochemistry and Molecular Biology (Revised, 2000, Oxford University Press) provide general dictionaries for many of the terms used in the present invention.

[0037] Units, prefixes, and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers determining the range. Unless otherwise specified, amino acid sequences are written from left to right in an amino-terminal to carboxy-terminal orientation. The headings presented herein do not limit the various aspects of the invention available throughout the specification. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.

[0038] The term "about" is used herein to mean approximately, roughly, on the order of, or in the range of. When the term "about" is used in conjunction with a numerical range, it extends the boundaries above and below the stated numerical values, modifying that range. In general, the term "about" can modify a numerical value above and below the stated value, for example, by as much as 10 percent above or below (to be higher or lower).

[0039] As used herein, the term "galanin receptor type 2 agonist" or "GALR2 agonist" refers to any molecule capable of binding to GALR2 and activating a GALR2-mediated signaling pathway. As described herein, in some aspects, the GALR2 agonists described herein do not activate GALR1 and GALR3 (i.e., are specific for GALR2). Additionally, unless otherwise specified, the GALR2 agonists of the present invention include one or more modifications (e.g., amino acid modifications) described herein. Non-limiting examples of such modifications are provided throughout the present invention.

[0040] The term "galanin receptor type 2" (GALR2) refers to the G-protein-coupled galanin (GAL) receptor encoded by the GALR2 gene. In addition to GALR2, two other subtypes of GAL receptors exist: galanin receptor type 1 (GALR1) and galanin receptor type 3 (GALR3). The human GALR1 gene contains three exons and translates into a protein consisting of 349 amino acids (see Table 1). The interspecies homology between rat and human GALR1 is 93%. GALR1 expression is regulated by cAMP through the transcription factor CREB. Human GALR2 shares 92% sequence identity with rat GALR2, but human GALR2 has a 15-amino acid extension at its C-terminus. The amino acid sequence of human GALR2, consisting of 387 amino acids, is presented in Table 1. The GALR2 gene is more widely expressed than the GALR1 gene, as it is found in many peripheral tissues, including the central nervous system, pituitary gland, gastrointestinal tract, skeletal muscle, heart, kidney, uterus, ovaries, and testes. Finally, human GALR3 consists of 368 amino acids (see Table 1) and shares 36% identity with human GALR1, 58% with human GALR2, and approximately 90% with rat GALR3. [Table 1]

[0041] Natural ligands for GAL receptors (including GALR2) are known and include galanin and spexin. Galanin is an important neuromodulator widely distributed throughout the body (e.g., the brain, gastrointestinal system, and hypothalamic-pituitary axis). Sipkova, J. et al., Physiol Res 66:729-740 (2017), the entire contents of which are incorporated herein by reference. At least in humans, galanin is a 30-amino acid peptide with a non-amidated C-terminus that plays a role in many biological functions, including somatosensory transmission, smooth muscle contraction, hormone secretion, and food intake. The amino acid sequence of human galanin is presented in Table 2. The precursor peptide is 123 amino acids long (SEQ ID NO: 83), and undergoes proteolytic degradation to produce the mature galanin peptide (SEQ ID NO: 84). More specifically, (i) amino acids 1 to 19 correspond to the signal peptide, (ii) amino acids 20 to 30 correspond to the propeptide, (iii) amino acids 33 to 62 correspond to the galanin peptide (SEQ ID NO: 84), and (iv) amino acids 65 to 123 correspond to the galanin message-associated peptide. [Table 2]

[0042] "Spexin" (also known as NPQ, SPX, and neuropeptide Q) is a more recently discovered neuropeptide that shares many similarities with galanin. Like galanin, spexin is distributed in diverse tissues and plays a variety of biological functions (e.g., gastrointestinal motility, energy balance and weight loss, fatty acid absorption, glucose homeostasis, pain sensation, and cardiovascular / renal function). However, unlike galanin, which can activate all GALR subtypes, spexin is specific only for GALR2 and GALR3, but not for GALR1. The mature spexin peptide sequence consists of 14 amino acids formed by cleavage of a dibasic amino acid by a proprotein convertase (see Table 3) and is highly conserved across common vertebrate species and humans. [Table 3]

[0043] As used herein, the term "gastrointestinal disorder" refers to any disease or disorder affecting a subject's upper and / or lower gastrointestinal tract. Non-limiting examples of such disorders include: heartburn, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peptic ulcer, stress ulcer, bleeding peptic ulcer, duodenal ulcer, infectious enteritis, colitis, diverticulitis, hyperacidity, dyspepsia, gastrointestinal paralysis, Zollinger-Ellison syndrome, and gastroesophageal reflux disease ("GERD") (i.e., acid reflux), including, but not limited to, hypersecretory states associated with Helicobacter pylori-associated disease, systemic mastocytosis, or basophilic leukemia, and hyperhistamineemia caused by neurosurgery, head injury, severe physical trauma, or burns. In some aspects, gastrointestinal disorders do not include irritable bowel syndrome (IBS). As is apparent from the present invention, in some aspects, the gastrointestinal disorder may be associated with abnormal neural function. Unless otherwise specified, such gastrointestinal disorders are also referred to herein as "neuropathic bowel dysfunction." In some aspects, gastrointestinal disorders treatable via the present invention are associated with intestinal motility disorders. Without being bound by any theory, in some aspects, the GALR2 agonists described herein can treat such gastrointestinal disorders by promoting (e.g., increasing) intestinal motility.

[0044] As used herein, the term "lower gastrointestinal tract" refers to the ileum, colon, cecum, and / or rectum. As used herein, the term "upper gastrointestinal tract" refers to the esophagus, stomach, duodenum, and / or jejunum.

[0045] A "polypeptide" refers to a chain composed of at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues in a protein may be modified, including, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides. Unless otherwise specified, the terms "polypeptide" and "protein" are used interchangeably herein.

[0046] As used herein, the terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably and are intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0047] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting other nucleic acids. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Yet another type of vector is a viral vector, into which additional DNA segments can be ligated. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell after introduction, and thus are replicated along with the host genome. Some vectors are also capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably as the plasmid is the most frequently used form of vector. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions are also included.

[0048] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell that contains a nucleic acid that is not naturally occurring within it, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms refer not only to the particular cell, but also to the progeny of that cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may not be identical to the parent cell, although these cells are still included within the scope of the term "host cell" as used herein.

[0049] As used herein, the term "linked" or "conjugated" means that two or more molecules are joined together. The linkage can be covalent or non-covalent. The linkage can also be genetic (i.e., recombinant fusion). Such linkage can be achieved using a variety of recognized techniques, including chemical conjugation and recombinant protein production.

[0050] As used herein, the term "administration" refers to the physical introduction of a therapeutic agent or a composition containing a therapeutic agent into a subject, and can be accomplished using a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration for the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration (e.g., injection or infusion). The phrase "parenteral administration," as used herein, generally refers to modes of administration other than enteral and topical administration via injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, epidural, and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the antibodies described herein can be administered parenterally, e.g., via topical, epidermal, or mucosal routes of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or local routes. Administration may be, for example, once, multiple times and / or over one or more periods of time.

[0051] As used herein, the terms "treat" and "treatment" refer to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the progression, onset, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease. Treatment can be performed on subjects suffering from a disease, or on subjects not suffering from a disease (e.g., for prophylaxis).

[0052] As used herein, the term "subject" includes all humans and non-human animals. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and worms.

[0053] As used herein, the term "therapeutically effective amount" refers to an amount of a drug, alone or in combination with other therapeutic agents, that is effective to "treat" a disease or disorder in a subject or to reduce the risk, latency, likelihood, or occurrence of a disease or disorder (e.g., a gastrointestinal and / or endocrine disorder). A "therapeutically effective amount" includes an amount of a drug or therapeutic agent that provides some degree of improvement or benefit to a subject suffering from or at risk of developing a disease or disorder (e.g., a gastrointestinal and / or endocrine disorder). Thus, a "therapeutically effective" amount refers to an amount that reduces the risk, latency, likelihood, or occurrence of a disease, or that alleviates or relieves / mitigates to some extent a disease or disorder, or that reduces / reduces at least one indicator, or that reduces at least one clinical symptom of a disease or disorder.

[0054] II. The Methods of the Invention Some aspects of the present invention relate to methods for treating a disease or disorder in a subject in need thereof. Unless otherwise specified, diseases or disorders that may be treated by the present invention do not include any of the following: attention deficit hyperactivity disorder (ADHD), bipolar disorder, body dysmorphic disorder, bulimia nervosa and other eating disorders, cataplexy, dysthymia, generalized anxiety disorder, hypersexuality, irritable bowel syndrome, impulse control disorder (MDD), kleptomania, migraine, major depressive disorder, narcolepsy, obsessive-compulsive disorder, oppositional defiant disorder, panic disorder, post-traumatic stress disorder (PTSD), premenstrual dysphoric disorder (PMDD), social anxiety disorder, chronic pain, intermittent explosive disorder, pathological gambling, personality disorder, pyromania, substance abuse and addiction, trichotillomania, Alzheimer's disease, or obesity disorders. In some aspects, diseases or disorders that may be treated by the present invention do not include attention deficit hyperactivity disorder (ADHD). In some aspects, diseases or disorders that may be treated by the present invention do not include bipolar disorder. In some aspects, diseases or disorders that may be treated via the present invention do not include body dysmorphic disorder. In some aspects, diseases or disorders that may be treated via the present invention do not include binge eating disorder. In some aspects, diseases or disorders that may be treated via the present invention do not include eating disorders (e.g., eating disorder nervosa). In some aspects, diseases or disorders that may be treated via the present invention do not include cataplexy. In some aspects, diseases or disorders that may be treated via the present invention do not include dysthymia. In some aspects, diseases or disorders that may be treated via the present invention do not include generalized anxiety disorder. In some aspects, diseases or disorders that may be treated via the present invention do not include hypersexuality. In some aspects, diseases or disorders that may be treated via the present invention do not include irritable bowel syndrome. In some aspects, diseases or disorders that may be treated via the present invention do not include impulse control disorder (MDD). In some aspects, diseases or disorders that may be treated via the present invention do not include kleptomania. In some aspects, diseases or disorders that may be treated via the present invention do not include migraine. In some aspects, diseases or disorders that may be treated via the present invention do not include major depressive disorder. In some aspects, diseases or disorders that may be treated via the present invention do not include narcolepsy.In some aspects, diseases or disorders that may be treated via the present invention do not include obsessive-compulsive disorder. In some aspects, diseases or disorders that may be treated via the present invention do not include oppositional defiant disorder. In some aspects, diseases or disorders that may be treated via the present invention do not include panic disorder. In some aspects, diseases or disorders that may be treated via the present invention do not include post-traumatic stress disorder (PTSD). In some aspects, diseases or disorders that may be treated via the present invention do not include premenstrual dysphoric disorder (PMDD). In some aspects, diseases or disorders that may be treated via the present invention do not include social anxiety disorder. In some aspects, diseases or disorders that may be treated via the present invention do not include chronic pain. In some aspects, diseases or disorders that may be treated via the present invention do not include intermittent explosive disorder. In some aspects, diseases or disorders that may be treated via the present invention do not include pathological gambling. In some aspects, diseases or disorders that may be treated via the present invention do not include personality disorders. In some aspects, diseases or disorders that may be treated via the present invention do not include pyromania. In some aspects, diseases or disorders that may be treated via the present invention do not include substance abuse and addiction. In some aspects, diseases or disorders that may be treated via the present invention do not include trichotillomania. In some aspects, diseases or disorders that may be treated via the present invention do not include Alzheimer's disease. In some aspects, diseases or disorders that may be treated via the present invention do not include obesity disorders.

[0055] II.A. Gastrointestinal Disorders In some aspects, a disease or disorder that may be treated via the present invention includes a gastrointestinal disorder in a subject in need thereof, and the method of treatment of the present invention includes administering to the subject one of the galanin receptor type 2 (GALR2) agonists described herein. As will be apparent from the present invention, in some aspects, the GALR2 agonist is administered to the subject as a protein. In some aspects, the GALR2 agonist is administered to the subject in the form of a nucleic acid (e.g., encoding one of the GALR2 agonists provided herein). In some aspects, administering the GALR2 agonist to the subject includes administering a vector comprising a nucleic acid encoding one of the GALR2 agonists described herein. Thus, unless otherwise specified, "administering a GALR2 agonist" includes (i) administering the GALR2 agonist itself to the subject (e.g., as a protein), (ii) administering a nucleic acid encoding a GALR2 agonist, (iii) administering a vector comprising a nucleic acid encoding a GALR2 agonist, and (iv) any combination of (i)-(iii).

[0056] In some aspects, gastrointestinal disorders treatable via the present invention are associated with abnormal neural function. For example, in some aspects, the gastrointestinal disorder refers to a gut motility disorder, where the gut motility disorder is associated with (e.g., caused by) abnormal neural function. Without being bound by any theory, in some aspects, the gastrointestinal disorder may result from an inability to regulate one or more portions of the gastrointestinal tract (e.g., the large intestine) due to abnormal neural function. As described herein, such gastrointestinal disorders are referred to herein as "neuropathic bowel dysfunction" or "NBD." Accordingly, in some aspects, the present invention provides methods of treating neuropathic bowel dysfunction in a subject in need thereof, the methods comprising administering to the subject one of the GALR2 agonists described herein.

[0057] As can be seen from at least the foregoing disclosure, the GALR2 agonists of the present invention can be used to treat neuropathic bowel dysfunction associated with all types of neurological dysfunction. For example, in some aspects, the NBD is associated with physical injury (e.g., spinal cord injury). In some aspects, the NBD is associated with a neurological disorder. Non-limiting examples of such neurological disorders include multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), stroke, spina bifida, valvular encephalopathy, Parkinson's disease, diabetes, and combinations thereof. In some aspects, the NBD is associated with both physical injury and a neurological disorder.

[0058] As can be seen from the present invention, in some aspects, gastrointestinal disorders treatable via the present invention are not associated with abnormal neuronal function, e.g., in some aspects, the gastrointestinal disorder manifests as a gut motility disorder, where the gut motility disorder is not associated with (e.g., is not caused by) abnormal neuronal function.

[0059] In some aspects, administering a GALR2 agonist described herein to a subject can promote improvement (e.g., alleviation) of one or more symptoms associated with gastrointestinal disorders. For example, in some aspects, after administering a GALR2 agonist described herein, the subject experiences improvement in one or more symptoms of NBD. Non-limiting examples of such symptoms include constipation, diarrhea, and fecal incontinence. See, e.g., Emmanuel, A., F1000 Research 8 (F1000 Faculty Rev): 1800 (2019), the entire contents of which are incorporated herein by reference.

[0060] In some aspects, the methods provided herein are useful for treating constipation in a subject in need thereof. In some aspects, such methods include administering to the subject one of the GALR2 agonists described herein, where, following administration, the subject's constipation is improved (e.g., reduced). As used herein, the term "constipation" refers to a physical condition that includes at least one of the following: decreased frequency of bowel movements, hardened stool, and difficulty in passing stool. Generally, individuals with constipation often strain during bowel movements or feel a sense of incomplete bowel movement after defecation. In some aspects, constipation refers to a subject experiencing an average of fewer than three rapid-response-free bowel movements (RFBMs) per week, where "response-free bowel movements" refers to the passage and expulsion of stool, i.e., relaxation. Thus, in some aspects, improved constipation includes: (i) more frequent and regular bowel movements (e.g., an average of 3 or more RFMBs per week), (ii) softening of stool, (iii) reduced difficulty in passing stool, or (iv) any combination thereof.

[0061] As can be clearly seen from the present invention, the methods provided herein can be used to treat constipation associated with a wide variety of causes. In some aspects, the constipation includes opioid-induced constipation. As used herein, the term "opioid-induced constipation" refers to any constipation caused by the use of opioid drugs. As used herein, the term "opioid" refers to a compound that binds to an opioid receptor. Unless otherwise specified, the term "opioid drug" as used herein includes all natural and synthetic opioids. In some aspects, opioid drugs include not only drugs that act on opioid receptors present in the central nervous system and / or peripheral nervous system, but also drugs that act on opioid receptors present in the gastrointestinal tract. Non-limiting examples of natural opioids include morphine, codeine, thebaine, and salvinorin A. Non-limiting examples of synthetic opioids include semi-synthetic opium alkaloid derivatives such as heroin (diacetylmorphine), dihydrocodeine, hydromorphone, nicomorphine, and oxycodone. Examples of fully synthetic opioid drugs include, but are not limited to, anilidopiperidines (e.g., pentanyl), phenylpiperidines (e.g., pethidine), diphenylpropylamine derivatives (e.g., loperamide), benzomorphan derivatives (e.g., dezocine), oripavine derivatives (e.g., buprenorphine), and morphinan derivatives (e.g., butorphanol).

[0062] Without being bound by any theory, in some aspects, the GALR2 agonists described herein can treat gastrointestinal disorders (e.g., neuropathic bowel dysfunction) by regulating bowel movements in a subject. As used herein, the term "defecation" refers to the expulsion of stool from the gastrointestinal tract. For example, if a subject is experiencing decreased bowel movements (e.g., constipation), in some aspects, administering a GALR2 agonist described herein can increase the subject's bowel movements. Thus, in some aspects, the present invention relates to a method for regulating bowel movements in a subject experiencing constipation, the method comprising administering to the subject one of the GALR2 agonists described herein, wherein after said administration, the subject experiences an increase in bowel movements. In some aspects, the constipation includes opioid-induced constipation. As can be clearly seen from the present invention, in some aspects, an increase in bowel movements refers to an increase in the number of bowel movements absent from emergency situations. In some aspects, the subject's bowel movements are increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference subject (e.g., a subject not administered a GALR2 agonist and / or a subject prior to administration of a GALR2 agonist). In some aspects, after administration of the GALR2 agonist, the subject experiences, on average, three or more emergency-free bowel movements per week.

[0063] Without being bound by any theory, in some aspects, gastrointestinal disorders (e.g., neurogenic bowel dysfunction, e.g., opioid-induced constipation) can be treated by decreasing a subject's colonic transit time. As used herein, the term "colonic transit time" refers to the time it takes for a substance to pass through the colon. As can be clearly seen from the present invention, in some aspects, a decrease in colonic transit time can increase intestinal motility and bowel movement frequency. In some aspects, after administration of a GALR2 agonist described herein, the subject's colonic transit time is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference subject (e.g., a subject not administered a GALR2 agonist and / or a subject prior to administration). Colonic transit time can be assessed using any suitable method known in the art (e.g., using radiopaque markers, radioisotopes, radiomobility capsules), non-limiting examples of which are described herein (see, e.g., Example 2).

[0064] II.B. Endocrine Disorders Some aspects of the present invention relate to methods of treating an endocrine disorder in a subject in need of such treatment, the method comprising administering to the subject one of the GALR2 agonists described herein. Some aspects of the present invention relate to methods of treating a metabolic disorder in a subject in need of such treatment, the method comprising administering to the subject one of the GALR2 agonists described herein. Some aspects of the present invention relate to methods of treating an endocrine and metabolic disorder in a subject in need of such treatment, the method comprising administering to the subject one of the GALR2 agonists described herein. As used herein, the term "endocrine disorder" refers to a disease or condition of the endocrine system. The "endocrine system" refers to the various glands and organs that make and secrete hormones that regulate various aspects of the body, including, but not limited to, growth and development, metabolism, and reproduction. In some aspects, the endocrine disorder includes (i) endocrine hypofunction / hypocretion (leading to hormone deficiency); (ii) endocrine hyperfunction / hyperocretion (leading to hormone excess); (iii) endocrine tumor (positive or malignant); or (iv) any combination of (i)-(iii).

[0065] Thus, in some aspects, the GALR2 agonists described herein may be useful for treating any endocrine disorder known in the art. Non-limiting examples of endocrine disorders include glucose homeostasis disorders (e.g., diabetes mellitus, hypoglycemia, and glucagonoma), thyroid disorders (e.g., goiter, hyperthyroidism, hypothyroidism, thyroiditis, thyroid cancer, and thyroid hormone insensitivity), calcium homeostasis disorders and metabolic bone disease (e.g., hypercalcemia, parathyroid disorders, osteoporosis, osteitis deformans, rickets, and osteomalacia), posterior pituitary disorders (e.g., diabetes insipidus, syndrome of inappropriate antidiuretic hormone secretion (SIADH)), anterior pituitary disorders (e.g., hypopituitarism and pituitary tumors), sex hormone disorders (e.g., disorders of sex development or intersexual disorder, hypogonadism, pubertal disorders, menstrual dysfunction, or reproductive disorders), kidney disorders (e.g., chronic renal failure), multiple endocrine neoplasms, carcinoid syndrome, and combinations thereof. In some aspects, endocrine disorders that can be treated using the methods provided herein include both chronic renal failure and hypercalcemia. In some aspects, the endocrine disorder is chronic renal failure. In some aspects, the endocrine disorder is hypercalcemia.

[0066] In any of the treatment methods provided herein, a GALR2 agonist described herein can be administered to a subject (e.g., a subject suffering from a gastrointestinal disorder and / or an endocrine disorder) via any suitable route. Non-limiting examples of such routes of administration include intrasphincteric, intramuscular, subcutaneous, intraocular, intravenous, intraperitoneal, intradermal, intraorbital, intracerebral, intracranial, intraventricular, intraspinal, intraventricular, intraspinal, intracisternal, intracapsular, topical, or a combination thereof. In some aspects, a GALR2 agonist described herein is administered subcutaneously to a subject. In some aspects, a GALR2 agonist is administered intranasally to a subject. In some aspects, a GALR2 agonist described herein is administered intraperitoneally to a subject.

[0067] As demonstrated herein, in some aspects, a GALR2 agonist described herein may be administered to a subject multiple times. For example, in some aspects, a GALR2 agonist is administered to a subject at least about two times, at least about three times, at least about four times, at least about five times, at least about six times, at least about seven times, at least about eight times, at least about nine times, or at least about ten times. In some aspects, a GALR2 agonist is administered to a subject daily for seven consecutive days. Also, as demonstrated herein, in some aspects, a GALR2 agonist described herein may be administered to a subject once. In some aspects, a GALR2 agonist described herein is administered to a subject at a dose of about 0.01 mg / kg to about 100 mg / kg. In some aspects, a GALR2 agonist described herein is administered to a subject at a dose of about 0.05 mg / kg to about 0.5 mg / kg. In some aspects, the GALR2 agonist is administered to a subject at a dose of about 0.05 mg / kg. In some aspects, the GALR2 agonist is administered to the subject at a dose of about 0.1 mg / kg. In some aspects, the GALR2 agonist is administered to the subject at a dose of about 0.2 mg / kg. In some aspects, the GALR2 agonist is administered to the subject at a dose of about 0.3 mg / kg. In some aspects, the GALR2 agonist is administered to the subject at a dose of about 0.4 mg / kg. In some aspects, the GALR2 agonist is administered to the subject at a dose of about 0.5 mg / kg. In some aspects, the GALR2 agonist is administered to the subject at a dose of about 0.6 mg / kg. In some aspects, the GALR2 agonist is administered to the subject at a dose of about 0.7 mg / kg. In some aspects, the GALR2 agonist is administered to the subject at a dose of about 0.8 mg / kg. In some aspects, the GALR2 agonist is administered to the subject at a dose of about 0.9 mg / kg. In some aspects, the GALR2 agonist is administered to the subject at a dose of about 1 mg / kg.

[0068] In some aspects, the methods of treatment described herein (e.g., methods of treating gastrointestinal disorders and / or endocrine disorders) include administering a GALR2 agonist and an additional therapeutic agent to a subject in need of treatment. In some aspects, the GALR2 agonist and the additional therapeutic agent may be administered to a subject simultaneously. For example, in some aspects, the GALR2 agonist and the additional therapeutic agent may be administered to a subject as a single composition, e.g., a pharmaceutical composition containing both the GALR2 agonist and the additional therapeutic agent. In some aspects, the GALR2 agonist and the additional therapeutic agent are administered to a subject simultaneously, but as separate compositions. For example, a first pharmaceutical composition containing the GALR2 agonist and a second pharmaceutical composition containing the additional therapeutic agent may be administered to a subject simultaneously. In some aspects, the GALR2 agonist and the additional therapeutic agent are administered sequentially to a subject. For example, in some aspects, the GALR2 agonist is administered to a subject before the additional therapeutic agent. In some aspects, the GALR2 agonist is administered to the subject after administration of the additional therapeutic agent.

[0069] As will be apparent from the present invention, the additional therapeutic agent can include any treatment known in the art that is suitable for treating the conditions described herein (e.g., gastrointestinal disorders and / or endocrine disorders). For example, if a subject is suffering from a gastrointestinal disorder (e.g., constipation), in some aspects, additional therapeutic agents that can be administered to the subject along with the GALR2 agonist include a laxative (e.g., bisacodyl), an opioid receptor antagonist (e.g., naloxone methiodide), douching (e.g., anal or colonic irrigation), electrical stimulation, or a combination thereof.

[0070] III. GALR2 Agonists As described elsewhere herein, GALR2 agonists useful in the present invention exhibit one or more properties (e.g., structural and / or functional) that distinguish them from other GALR2 ligands. Non-limiting examples of such differences are set forth below. Additional disclosure of useful GALR2 agonists is provided, for example, in U.S. Pat. No. 11,248,023, the entire disclosure of which is incorporated herein by reference.

[0071] III.A. Amino Acid Modifications In some aspects, a GALR2 agonist of the invention comprises one or more amino acid variations (e.g., substitutions, deletions, additions, and / or insertions / deletions) when compared to a reference GALR2 ligand (e.g., wild-type spexin and / or galanin). For example, in some aspects, a GALR2 agonist useful in the invention (e.g., which may be used to treat gastrointestinal disorders, endocrine disorders, or all of these) comprises the amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit); (2) X 3 is threonine (T), alanine (A), or lysine (K); (3) X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); (4) X 5 is asparagine (N) or glutamine (Q); (5) X 6 is alanine (A) or serine (S); (6) X 7 is alanine (A) or methionine (M); (7) X 8 is leucine (L), glutamine (Q), or glycine (G); (8) X 11is leucine (L), phenylalanine (F), tyrosine (Y), or aspartic acid (D); (9) X 12 is glycine (G) or alanine (A); (10)X 13 is proline (P), arginine (R), or alanine (A); (11) X 14 is glutamine (Q), histidine (H), or valine (V); (12) is any combination of (1) to (11).

[0072] Thus, in some aspects, provided herein is a method of treating a gastrointestinal disorder in a subject in need thereof, the method comprising administering to said subject an amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit); (2) X 3 is threonine (T), alanine (A), or lysine (K); (3) X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); (4) X 5 is asparagine (N) or glutamine (Q); (5) X 6 is alanine (A) or serine (S); (6) X 7 is alanine (A) or methionine (M); (7) X 8 is leucine (L), glutamine (Q), or glycine (G); (8) X 11 is leucine (L), phenylalanine (F), tyrosine (Y), or aspartic acid (D); (9) X 12 is glycine (G) or alanine (A); (10)X 13is proline (P), arginine (R), or alanine (A); (11) X 14 is glutamine (Q), histidine (H), or valine (V); (12) is any combination of (1) to (11). In some aspects, provided herein is a method for regulating bowel movement in a subject in need thereof, the method comprising administering to the subject an amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit); (2) X 3 is threonine (T), alanine (A), or lysine (K); (3) X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); (4) X 5 is asparagine (N) or glutamine (Q); (5) X 6 is alanine (A) or serine (S); (6) X 7 is alanine (A) or methionine (M); (7) X 8 is leucine (L), glutamine (Q), or glycine (G); (8) X 11 is leucine (L), phenylalanine (F), tyrosine (Y), or aspartic acid (D); (9) X 12 is glycine (G) or alanine (A); (10)X 13 is proline (P), arginine (R), or alanine (A); (11) X 14is glutamine (Q), histidine (H), or valine (V); (12) any combination of (1) through (11). In some aspects, provided herein are methods of treating an endocrine disorder in a subject in need thereof, the method comprising administering to the subject an amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit); (2) X 3 is threonine (T), alanine (A), or lysine (K); (3) X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); (4) X 5 is asparagine (N) or glutamine (Q); (5) X 6 is alanine (A) or serine (S); (6) X 7 is alanine (A) or methionine (M); (7) X 8 is leucine (L), glutamine (Q), or glycine (G); (8) X 11 is leucine (L), phenylalanine (F), tyrosine (Y), or aspartic acid (D); (9) X 12 is glycine (G) or alanine (A); (10)X 13 is proline (P), arginine (R), or alanine (A); (11) X 14 is glutamine (Q), histidine (H), or valine (V); (12) is any combination of (1) to (11).

[0073] In some aspects, the GALR2 agonist has the amino acid sequence X 1 WX 3 X4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit); (2) X 3 is threonine (T), alanine (A), or lysine (K); (3) X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); (4) X 5 is asparagine (N) or glutamine (Q); (5) X 6 is alanine (A) or serine (S); (6) X 7 is alanine (A) or methionine (M); (7) X 8 is leucine (L), glutamine (Q), or glycine (G); (8) X 11 is leucine (L), phenylalanine (F), tyrosine (Y), or aspartic acid (D); (9) X 12 is glycine (G) or alanine (A); (10)X 13 is proline (P), arginine (R), or alanine (A); (11) X 14 is glutamine (Q), histidine (H), or valine (V); (12) any combination of (1) to (11). In some aspects, the GALR2 agonist comprises the amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit); (2) X 3is threonine (T), alanine (A), or lysine (K); (3) X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); (4) X 5 is asparagine (N) or glutamine (Q); (5) X 6 is alanine (A) or serine (S); (6) X 7 is alanine (A) or methionine (M); (7) X 8 is leucine (L), glutamine (Q), or glycine (G); (8) X 11 is leucine (L), phenylalanine (F), tyrosine (Y), or aspartic acid (D); (9) X 12 is glycine (G) or alanine (A); (10)X 13 is proline (P), arginine (R), or alanine (A); (11) X 14 is glutamine (Q), histidine (H), or valine (V); (12) any combination of (1) through (11). In some aspects, the GALR2 agonist consists essentially of the amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit); (2) X 3 is threonine (T), alanine (A), or lysine (K); (3) X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); (4) X 5 is asparagine (N) or glutamine (Q); (5) X 6 is alanine (A) or serine (S); (6) X 7 is alanine (A) or methionine (M); (7) X 8is leucine (L), glutamine (Q), or glycine (G); (8) X 11 is leucine (L), phenylalanine (F), tyrosine (Y), or aspartic acid (D); (9) X 12 is glycine (G) or alanine (A); (10)X 13 is proline (P), arginine (R), or alanine (A); (11) X 14 is glutamine (Q), histidine (H), or valine (V); (12) is any combination of (1) to (11).

[0074] In some respects, X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit). 3 is threonine (T), alanine (A), or lysine (K). 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V). 5 is asparagine (N) or glutamine (Q). 6 is alanine (A) or serine (S). 7 is alanine (A) or methionine (M). 8 is leucine (L), glutamine (Q), or glycine (G). 11 is leucine (L), phenylalanine (F), tyrosine (Y), or aspartic acid (D). 12 is glycine (G) or alanine (A). 13 is proline (P), arginine (R), or alanine (A). 14 is glutamine (Q), histidine (H) or valine (V).

[0075] Unless otherwise specified, the amino acid at each position may be in the D- or L-form. For example, in some aspects, X 1 is L-asparagine or D-asparagine. 1 is L-asparagine. 1 is D-asparagine. 1 is L-glycine or D-glycine. 1 is L-glycine. 1 is D-glycine. In some aspects, W at position 2 of SEQ ID NO:1 is L-tryptophan or D-tryptophan. In some aspects, W at position 2 of SEQ ID NO:1 is L-tryptophan. In some aspects, W at position 2 of SEQ ID NO:1 is D-tryptophan. In some aspects, X 3 is L-threonine or D-threonine. 3 is L-threonine. 3 is D-threonine. In some aspects, X 3 is L-alanine or D-alanine. 3 is L-alanine. 3 is D-alanine. In some aspects, X 3 is L-lysine or D-lysine. 3 is L-lysine. 3 is D-lysine. 4 is L-proline or D-proline. 4 is L-proline. 4 is D-proline. 4 is L-leucine or D-leucine. 4 is L-leucine. 4 is D-leucine. 4is L-glutamic acid or D-glutamic acid. 4 is L-glutamic acid. 4 is D-glutamic acid. In some aspects, X 4 is L-arginine or D-arginine. 4 is L-arginine. 4 is D-arginine. 4 is L-valine or D-valine. 4 is L-valine. 4 is D-valine. 5 is L-asparagine or D-asparagine. 5 is L-asparagine. 5 is D-asparagine. 5 is L-glutamic acid or D-glutamic acid. 5 is L-glutamic acid. 5 is D-glutamic acid. In some aspects, X 6 is L-alanine or D-alanine. 6 is L-alanine. 6 is D-alanine. In some aspects, X 6 is L-serine or D-serine. 6 is L-serine. 6 is D-serine. 7 is L-alanine or D-alanine. 7 is L-alanine. 7 is D-alanine. In some aspects, X 7 is L-methionine or D-methionine. 7 is L-methionine.7 is D-methionine. 8 is L-leucine or D-leucine. 8 is L-leucine. 8 is D-leucine. 8 is L-glutamic acid or D-glutamic acid. 8 is L-glutamic acid. 8 is D-glutamic acid. In some aspects, X 8 is L-glycine or D-glycine. 8 is L-glycine. 8 is D-glycine. In some aspects, Y at position 9 of SEQ ID NO:1 is L-tyrosine or D-tyrosine. In some aspects, Y at position 9 of SEQ ID NO:1 is L-tyrosine. In some aspects, Y at position 9 of SEQ ID NO:1 is D-tyrosine. In some aspects, L at position 10 of SEQ ID NO:1 is L-leucine or D-leucine. In some aspects, L at position 10 of SEQ ID NO:1 is L-leucine. In some aspects, L at position 10 of SEQ ID NO:1 is D-leucine. In some aspects, X 11 is L-leucine or D-leucine. 11 is L-leucine. 11 is D-leucine. 11 is L-phenylalanine or D-phenylalanine. 11 is L-phenylalanine. In some aspects, X 11 is D-phenylalanine. In some aspects, X 11 is L-tyrosine or D-tyrosine. 11 is L-tyrosine. 11 is D-tyrosine. 11is L-aspartic acid or D-aspartic acid. 11 is L-aspartic acid. 11 is D-aspartic acid. In some aspects, X 12 is L-glycine or D-glycine. 12 is L-glycine. 12 is D-glycine. In some aspects, X 12 is L-alanine or D-alanine. 12 is L-alanine. 12 is D-alanine. In some aspects, X 13 is L-proline or D-proline. 13 is L-proline. 13 is D-proline. 13 is L-arginine or D-arginine. 13 is L-arginine. 13 is D-arginine. 13 is L-alanine or D-alanine. 13 is L-alanine. 13 is D-alanine. In some aspects, X 14 is L-glutamine or D-glutamine. 14 is L-glutamine. In some aspects, X 14 is D-glutamine. In some aspects, X 14 is L-histidine or D-histidine. 14 is L-histidine. 14 is D-histidine. 14 is L-valine or D-valine. 14 is L-valine.14 is D-valine.

[0076] Thus, in some aspects, GALR2 agonists useful in the methods provided herein comprise, consist of, or consist essentially of an amino acid sequence comprising one or more D-amino acids. Without being bound by any theory, in some aspects, modifying a GALR2 agonist described herein to include one or more D-amino acids may, for example, improve the durability of the GALR2 agonist when administered to a subject. For example, the inclusion of D-amino acids may prevent the polypeptide from being degraded by proteases and peptidases in the subject's blood. Thus, in some aspects, a GALR2 agonist described herein comprises, consists of, or consists essentially of an amino acid sequence comprising one or more D-amino acids, and the GALR2 agonist is more resistant to protease and / or peptidase degradation compared to a reference GALR2 ligand. Unless otherwise specified, the reference GALR2 ligand can include: (i) wild-type galanin, (ii) wild-type spexin, (iii) the GALR2 agonist that does not have the one or more D-amino acids, or (iv) any combination of (i) through (iii).

[0077] For example, in some aspects, the GALR2 agonist has the amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (i) X 1 is a D-amino acid, (ii) W at position 2 of SEQ ID NO: 1 is D-tryptophan, and (iii) X 4 is a D-amino acid, and (iv) X 6 is a D-amino acid, and (v) X 11 is a D-amino acid, and (vi) X 12is a D-amino acid, and (vii) X 13 is a D-amino acid, and (viii) X 14 is a D-amino acid or any combination of (ix)(i) through (viii). More specifically, in some aspects, the GALR2 agonist comprises the amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (i) X 1 is D-asparagine, (ii) W at position 2 of SEQ ID NO: 1 is D-tryptophan, and (iii) X 4 is D-alanine or D-valine, and (iv) X 6 is D-alanine, (v) X 11 is D-lysine, and (vi) X 12 is D-alanine, and (vii) X 13 is D-alanine, and (viii) X 14 is D-glutamine, or any combination of (ix)(i) through (viii). In some aspects, the GALR2 agonist comprises the amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1 is D-asparagine, (2)X 4 is D-alanine, (3)X 14 is D-glutamine, or (4) any combination thereof. A non-limiting example of such a GALR2 agonist is provided as SEQ ID NO: 87. In some aspects, the GALR2 agonist has the amino acid sequence X 1 WX3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 4 is D-glutamic acid, (2)X 14 is D-glutamine or (3)X 4 is D-glutamic acid, and X 14 is D-glutamine. A non-limiting example of such a GALR2 agonist is provided as SEQ ID NO: 90. In some aspects, the GALR2 agonist has the amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 4 is D-arginine, (2)X 14 is D-glutamine or (3)X 4 is D-arginine, and X 14 is D-glutamine. A non-limiting example of such a GALR2 agonist is provided as SEQ ID NO:91.

[0078] In some aspects, a GALR2 agonist useful in the present invention has the amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit); (2) X 3 is threonine (T), alanine (A), or lysine (K); (3) X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); (4) X 5 is asparagine (N) or glutamine (Q); (5) X 6 is alanine (A) or serine (S); (6) X 7 is alanine (A); (7)X 8 is leucine (L), glutamine (Q), or glycine (G); (8) X 11 is phenylalanine (F); (9)X 12 is glycine (G) or alanine (A); (10)X 13 is proline (P), arginine (R), or alanine (A); (11) X 14 is glutamine (Q), histidine (H), or valine (V); (12) any combination of (1) through (11). For example, in some aspects, a GALR2 agonist useful in the present invention comprises, consists of, or consists essentially of the amino acid sequence NWTPQAALYLFGAQ (SEQ ID NO: 48).

[0079] In some aspects, a GALR2 agonist useful in the present invention has the amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit); (2) X 3 is threonine (T), alanine (A), or lysine (K); (3) X 4is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); (4) X 5 is asparagine (N); (5)X 6 is alanine (A) or serine (S); (6) X 7 is alanine (A); (7)X 8 is leucine (L), glutamine (Q), or glycine (G); (8) X 11 is phenylalanine (F); (9)X 12 is glycine (G) or alanine (A); (10)X 13 is proline (P), arginine (R), or alanine (A); (11) X 14 is glutamine (Q), histidine (H), or valine (V); (12) is a combination of (1) through (11). In some aspects, such a GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence NWTPNAALYLFGAQ (SEQ ID NO: 50).

[0080] In some aspects, a GALR2 agonist useful in the present invention has the amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit); (2) X 3 is threonine (T), alanine (A), or lysine (K); (3) X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); (4) X 5 is asparagine (N); (5)X 6 is alanine (A) or serine (S); (6) X 7is alanine (A); (7)X 8 is leucine (L), glutamine (Q), or glycine (G); (8) X 11 is phenylalanine (F); (9)X 12 is glycine (G) or alanine (A); (10)X 13 is proline (P); (11)X 14 is glutamine (Q), histidine (H), or valine (V); (12) any combination of (1) through (11). In some aspects, such a GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence NWTPNAALYLFGPQ (SEQ ID NO: 51).

[0081] In some aspects, a GALR2 agonist that may be used with the present invention comprises, consists of, or consists essentially of the amino acid sequence set forth in one of the following: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, or SEQ ID NO:91. See Table 4 below. [Table 4] JPEG2025538651000005.jpg236169JPEG2025538651000006.jpg231169

[0082] In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:9. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:10. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:11. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:12. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:13. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:14. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:16. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:17. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 18. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 19. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 20. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 21. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 22. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 23.In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 24. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 25. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 26. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 27. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 31. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 32. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 33. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 37. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 39. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 40. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 41. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 42. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 44. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 45.In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 47. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 48. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 50. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 51. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 52. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 53. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 54. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 55. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 56. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 57. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 58. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 59. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 87. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 88.In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 89. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 90. In some aspects, the GALR2 agonist comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 91.

[0083] As explained and demonstrated herein, in some aspects, one or more of the amino acid variations described above cause the GALR2 agonists described herein to exhibit one or more properties not present in other GALR2 ligands (e.g., wild-type spexin and / or galanin).

[0084] For example, in some aspects, a GALR2 agonist described herein can induce activation of GALR2 and GALR3, but not GALR1. In some aspects, a GALR2 agonist described herein exhibits greater potency (or agonism) (e.g., EC ) on GALR2 when compared to a reference substance (e.g., wild-type spexin and / or galanin). 50 For example, in some aspects, the GALR2 agonists described herein exhibit about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 6-fold or more, about 7-fold or more, about 8-fold or more, about 9-fold or more, about 10-fold or more, about 15-fold or more, about 20-fold or more, about 25-fold or more, about 30-fold or more, about 35-fold or more, about 40-fold or more, about 45-fold or more, about 50-fold or more, about 75-fold or more, or about 100-fold or more potency against GALR2 when compared to a substance (e.g., wild-type spexin and / or galanin). In some aspects, the GALR2 agonists described herein exhibit an efficacy (EC 50 ) can induce activation of GALR2.

[0085] In some aspects, the GALR2 agonists described herein are specific for GALR2, unlike other GALR2 ligands (e.g., wild-type spexin and / or galanin). As used herein, an agonist is "specific for GALR2" if it can preferentially induce activation of GALR2 over other GALR subtypes (GALR1 and GALR3). As is apparent from the present invention, in some aspects, an agonist specific for GALR2 can have a certain degree of agonistic effect on GALR3. However, such an agonist exhibits a much weaker agonistic effect on GALR3 compared to its agonistic effect on GALR2. Thus, in some aspects, the GALR2 agonists described herein exhibit an agonistic effect on GALR2 that is about 1-fold or greater, about 2-fold or greater, about 3-fold or greater, about 4-fold or greater, about 5-fold or greater, about 6-fold or greater, about 7-fold or greater, about 8-fold or greater, about 9-fold or greater, about 10-fold or greater, about 15-fold or greater, about 20-fold or greater, about 25-fold or greater, about 30-fold or greater, about 35-fold or greater, about 40-fold or greater, about 45-fold or greater, about 50-fold or greater, about 75-fold or greater, or about 100-fold or greater when compared to their agonistic effect on GALR3. In some aspects, agonists specific for GALR2 (e.g., GALR2 agonists described herein) do not induce activation of GALR3. Thus, in some aspects, the GALR2 agonists described herein do not induce activation of all of GALR1 and GALR3. Unless otherwise specified, GALR2 agonists do not induce activation of GALR subtypes (GALR1 and / or GALR3). In this case, the efficacy of GALR2 agonists against GALR subtypes (EC 50 ) is greater than about −5 nM.

[0086] III.B. Other Variations As is apparent from at least the above disclosure, in some aspects, GALR2 agonists useful in the present invention include one or more modifications at the N-terminus, C-terminus, or both the N-terminus and C-terminus. In some aspects, such modifications can help increase the stability of the GALR2 agonist. In some aspects, such modifications at the N-terminus and / or C-terminus do not affect the activity of the GALR2 agonist. Instead, such modifications at the N-terminus and / or C-terminus facilitate the synthesis / production of the GALR2 agonist. For example, in some aspects, the N-terminus and / or C-terminus can improve the solubility of the GALR2 agonist. In some aspects, modifications at the N-terminus and / or C-terminus prevent degradation in the blood, for example, when administered to a subject. Thus, in some aspects, modifications at the N-terminus and / or C-terminus can be useful for increasing the half-life of the GALR2 agonist. In some aspects, the GALR2 agonists described herein are modified (or engineered) so that the GALR2 agonist is conjugated to an N-terminal protecting group. Any suitable N-terminal protecting group known in the art may be used. In some aspects, the GALR2 agonist described herein is conjugated at the N-terminus with one of the following: a 9-fluorenylmethoxycarbonyl group (Fmoc group), pyroglutamic acid (pQ), citrulline (Cit), an acetyl (Ac) group, polyethylene glycol (PEG), or a combination thereof. In some aspects, the N-terminus of the GALR2 agonist described herein may be methylated.

[0087] Thus, in some aspects, the GALR2 agonists described herein comprise the amino acid sequence X 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit); (2) X 3 is threonine (T), alanine (A), or lysine (K); (3) X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); (4) X 5 is asparagine (N) or glutamine (Q); (5) X 6 is alanine (A) or serine (S); (6) X 7 is alanine (A) or methionine (M); (7) X 8 is leucine (L), glutamine (Q), or glycine (G); (8) X 11 is leucine (L), phenylalanine (F), tyrosine (Y), or aspartic acid (D); (9) X 12 is glycine (G) or alanine (A); (10)X 13 is proline (P), arginine (R), or alanine (A); (11) X 14 is glutamine (Q), histidine (H), or valine (V); (12) any combination of (1) through (11), wherein the N-terminus of the GALR2 agonist is conjugated to any one of the following: an Fmoc group, pQ, Cit, Ac group, PEG, or a combination thereof. In some aspects, the N-terminus of the GALR2 agonist is conjugated to Fmoc. In some aspects, the N-terminus of the GALR2 agonist is conjugated to pQ. In some aspects, the N-terminus of the GALR2 agonist is conjugated to an Ac group. In some aspects, the N-terminus of the GALR2 agonist is conjugated to PEG. Non-limiting examples of such GALR2 agonists are listed in Table 4 above. See, for example, SEQ ID NOs: 10, 11, 42, 44, 45, 53, 55-59, and 87-89.

[0088] In some aspects, the C-terminus of a GALR2 agonist is modified (or engineered) such that the GALR2 agonist is conjugated to a C-terminal protecting group. Any suitable C-terminal protecting group known in the art may be used. Non-limiting examples of such C-terminal protecting groups include an amine group (-NEE), a streptococcus tag, a His tag, or a combination thereof. In some aspects, the C-terminus of a GALR2 agonist is conjugated to a C-terminal protecting group during synthesis, which is then removed after synthesis.

[0089] In some aspects, the GALR2 agonists described herein are additionally modified, e.g., such that when administered to a subject, the GALR2 agonist exhibits increased survival or half-life. Thus, in some aspects, the GALR2 agonists described herein are conjugated to a half-life extending moiety. For example, in some aspects, the GALR2 agonists described herein are conjugated to a half-life extending moiety. 1 WX 3 X 4 X 5 X 6 X 7 X 8 YLX 11 X 12 X 13 X 14 (SEQ ID NO: 1), wherein: (1) X 1 is asparagine (N), glycine (G), pyroglutamic acid (pQ), or citrulline (Cit); (2) X 3 is threonine (T), alanine (A), or lysine (K); (3) X 4 is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); (4) X 5 is asparagine (N) or glutamine (Q); (5) X 6 is alanine (A) or serine (S); (6) X 7 is alanine (A) or methionine (M); (7) X 8 is leucine (L), glutamine (Q), or glycine (G); (8) X 11is leucine (L), phenylalanine (F), tyrosine (Y), or aspartic acid (D); (9) X 12 is glycine (G) or alanine (A); (10)X 13 is proline (P), arginine (R), or alanine (A); (11) X 14 is glutamine (Q), histidine (H), or valine (V); (12) any combination of (1) to (11), wherein the GALR2 agonist is conjugated to a half-life extending moiety.

[0090] Any suitable half-life extending moiety known in the art may be used in the present invention. Non-limiting examples of such half-life extending moieties include Fc, albumin, albumin-binding polypeptide, Pro / Ala / Ser (PAS), C-terminal peptide of the P subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), long atypical hydrophilic sequences of amino acids (XTEN), hydroxyethyl starch (HES), albumin-binding small molecules, or combinations thereof. In some aspects, the half-life extending moiety is Fc.

[0091] In some aspects, GALR2 agonists useful in the present invention can include one or more additional moieties that, for example, enable the molecule to specifically target various tissues when administered to a subject. For example, in some aspects, GALR2 agonists described herein can include a peptide that enables the agent to cross the blood-brain barrier (also referred to herein as the "BBB shuttle"). Examples of such BBB shuttles are known in the art. Non-limiting examples are listed in Table 5 below. See, e.g., Oiler-Salvia et al., Chem Soc Rev 45:4690 (2016). [Table 5] The nomenclature for cyclic peptides (&) was adapted to the three-letter amino acid code described in Spengler et al., J Pept Res 65:550-555 (2005); [Dap] means diaminopropionic acid.

[0092] IV. Nucleic Acids, Vectors and Cells Some aspects of the present invention relate to one or more nucleic acid molecules (hereinafter also referred to as "nucleic acids" or derivatives thereof) encoding a GALR2 agonist described herein. The nucleic acid may be present in whole cells, a cell lysate, or in a partially purified or substantially pure form. In some aspects, the nucleic acid is a DNA sequence and / or an RNA sequence (e.g., mRNA). In some aspects, the nucleic acid comprises modified nucleotide analogs. A nucleic acid is "isolated" or "substantially pure" when purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to DNA isolated from nature) or proteins using alkali / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other standard techniques well known in the art (see F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York). In some aspects, the nucleic acid molecule may or may not contain intron sequences. In some aspects, the nucleic acid is a cDNA molecule. The nucleic acids described herein can be obtained using standard molecular biology techniques known in the art.

[0093] In some aspects, the present invention relates to a vector comprising an isolated nucleic acid molecule encoding a GALR2 agent described herein. Suitable vectors for the present invention include, but are not limited to, expression vectors, viral vectors, and plasmid vectors. In some aspects, the vector is a viral vector.

[0094] As used herein, the term "expression vector" refers to a nucleic acid construct that contains elements necessary for the transcription and translation of an inserted coding sequence, and in the case of an RNA viral vector, contains elements necessary for replication and translation when introduced into an appropriate host cell. Expression vectors can include plasmids, phagemids, viruses, and their derivatives.

[0095] As used herein, the term "viral vector" includes, but is not limited to, nucleic acid sequences from viruses such as retroviruses (e.g., Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus); lentiviruses; adenoviruses; adeno-associated viruses; SV40-type viruses; polyoma viruses; Epstein-Barr virus; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA viruses such as retroviruses. Some viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with a gene of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral integration into host cell DNA.

[0096] In some aspects, the vector is derived from an adeno-associated virus. In some aspects, the vector is derived from a lentivirus. Examples of lentiviral vectors are published in WO9931251, WO9712622, WO9817815, WO9817816, and WO9818934, the entire contents of each of which are incorporated herein by reference.

[0097] Other vectors include plasmid vectors (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989). In recent years, plasmid vectors have proven particularly advantageous for transferring genes to cells in vivo because they cannot replicate or integrate within the host genome. However, such plasmids equipped with a promoter compatible with the host cell are capable of expressing peptides from genes operatively encoded within the plasmid. Commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids include pcDNA3.1 (Catalog No. V79020), pcDNA3.1 / hygro (Catalog No. V87020), pcDNA4 / myc-His (Catalog No. V86320), and pBudCE4.1 (Catalog No. V53220), all available from Invitrogen (Carlsbad, Calif.). Plasmids can also be custom designed to remove and / or add specific DNA fragments using standard molecular biology techniques.

[0098] Also included herein are cells containing nucleic acid molecules encoding the GALR2 agonists described herein. In some aspects, the cells contain vectors containing the nucleic acid molecules. Host cells containing such nucleotide sequences are within the scope of the present specification. Non-limiting examples of host cells that can be used include immortal hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, human amniotic fluid-derived cells (CapT cells), COS cells, or combinations thereof.

[0099] V. Pharmaceutical Compositions Some aspects of the present invention relate to compositions comprising a therapeutic agent described herein (e.g., a GALR2 agonist, a nucleic acid encoding the GALR2 agonist, a vector comprising the nucleic acid, and / or cells comprising the vector) having a desired purity within a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). For example, in some aspects, provided herein are compositions comprising (i) a GALR2 agonist and (ii) a pharmaceutically acceptable carrier, excipient, or stabilizer. In some aspects, the present invention provides compositions comprising a nucleic acid molecule encoding a GALR2 agonist and (ii) a pharmaceutically acceptable carrier, excipient, or stabilizer. In some aspects, the present invention provides compositions comprising a vector comprising a nucleic acid molecule encoding a GALR2 agonist and (ii) a pharmaceutically acceptable carrier, excipient, or stabilizer. In some aspects, the invention provides compositions comprising a cell transformed to contain a vector comprising a nucleic acid molecule encoding a GALR2 agonist and (ii) a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0100] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0101] In some aspects, pharmaceutical compositions useful in the present invention comprise one or more of the therapeutic agents described herein (e.g., a GALR2 agent described herein, a nucleic acid molecule encoding the GALR2 agent, a vector comprising the nucleic acid, and / or cells transformed to contain the vector), and optionally, an additional prophylactic or therapeutic agent, in a pharmaceutically acceptable carrier. In some aspects, pharmaceutical compositions comprise a therapeutic agent described herein (e.g., a GALR2 agent described herein, a nucleic acid molecule encoding the GALR2 agent, a vector comprising the nucleic acid, and / or cells transformed to contain the vector), and optionally, one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In some aspects, a therapeutic agent described herein is the only active ingredient contained in the pharmaceutical composition.

[0102] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed vegetable oil, cottonseed oil, corn oil, sesame oil, and peanut oil. Antibacterial agents may be added to parenteral formulations packaged in multi-dose containers at bacteriostatic or fungistatic concentrations. Such formulations also include phenols, cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic formulations include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers include the miscible vehicles ethyl alcohol, polyethylene glycol, and propylene glycol, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0103] Pharmaceutical compositions may be formulated for any route of administration to a subject. Specific examples of administration routes include intranasal, oral, parenteral, intrathecal, intraventricular, pulmonary, subcutaneous, or intraventricular administration. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables may be prepared in existing forms, such as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquid prior to injection, or emulsions. Injectables, solutions, and emulsions may also contain one or more excipients. Suitable excipients include, for example, water, saline, dextrose, glycerol, or ethanol. If desired, the administered pharmaceutical composition may also contain minor amounts of nontoxic auxiliary substances, such as wetting agents, emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0104] Formulations for parenteral administration of the therapeutic agents described herein (e.g., GALR2 agents, nucleic acids encoding GALR2 agents, vectors containing the nucleic acids, and / or cells containing the vectors) include sterile solutions ready for injection, sterile dry soluble products (including injectable tablets) such as lyophilized powders ready to be mixed with a solvent immediately before use, sterile suspensions ready for injection, sterile dry insoluble products ready to be mixed with a carrier immediately before use, and sterile emulsions. The solutions can be aqueous or insoluble.

[0105] For intravenous injection, suitable carriers include physiological saline or phosphate buffered saline (PBS), including solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0106] Topical mixtures containing therapeutic agents are prepared as described for local and systemic administration. The resulting mixture may be in the form of a solution, suspension, emulsion, or similar, and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, wash, spray, suppository, bandage, skin patch, or other form suitable for topical administration.

[0107] Pharmaceutical compositions can be formulated as aerosols for topical application, such as inhalation (see, e.g., U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923). Such dosage forms for administration to the respiratory tract can be in the form of a nebulizer aerosol or solution, or in the form of a fine powder for inhalation, used alone or with an inert carrier such as lactose. In this case, the particles of the dosage form can, in some aspects, have diameters of less than about 50 microns, e.g., less than about 10 microns.

[0108] Pharmaceutical compositions can be formulated for local or topical application, such as topical application to the skin and mucous membranes (e.g., the eyes) in the form of gels, creams, lotions, or for topical administration, such as ocular, intracisternal, or intraspinal application. Topical administration is contemplated for transdermal delivery, administration to the eyes or mucous membranes, or inhalation therapy. Antibodies can also be administered nasally alone or together with other pharmaceutically acceptable excipients.

[0109] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those skilled in the art and can be used to administer the therapeutic agents described herein. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

[0110] In some aspects, the pharmaceutical compositions described herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures, and may also be reconstituted and formulated in solid or gel form. Lyophilized powders are prepared by dissolving a therapeutic agent described herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some aspects, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable formulations. The solvent may also contain a buffer, such as citric acid, sodium phosphate, potassium phosphate, or other buffers known to those of skill in the art. In some aspects, the buffer has a near-neutral pH. The solution can then be sterile filtered and lyophilized under standard conditions known to those skilled in the art to provide the desired dosage form. In some aspects, the resulting solution can be aliquoted into vials for lyophilization. Each vial may contain a single dose or multiple doses of a therapeutic agent described herein (e.g., a GALR2 agonist, a nucleic acid encoding a GALR2 agonist, a vector containing the nucleic acid, and / or cells containing the vector). The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0111] This lyophilized powder is reconstituted with water for injection to provide a dosage form suitable for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount will vary with the compound selected, and such amount can be determined empirically.

[0112] In some aspects, a pharmaceutical composition containing one of the therapeutic agents described herein can be formulated to target a particular tissue, receptor, or other site in the body of the subject being treated. For non-limiting examples of targeting methods, see, e.g., U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874.

[0113] Compositions to be used for in vivo administration can be sterilized, hi some aspects, such sterilization can be accomplished, for example, by filtration through sterile filtration membranes.

[0114] VI. Kit Also provided herein are kits comprising one or more of the therapeutic agents described herein (e.g., a GALR2 agonist, a nucleic acid encoding a GALR2 agonist, a vector comprising the nucleic acid, and / or cells comprising the vector). As will be apparent from the present invention, in some aspects, kits comprising a GALR2 agonist described herein may be useful for treating a variety of diseases or disorders (e.g., gastrointestinal disorders and / or endocrine disorders). In some aspects, provided herein are pharmaceutical packs or kits comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions described herein, and optionally, instructions for use. In some aspects, the kits comprise a pharmaceutical composition described herein and any prophylactic or therapeutic agent described herein.

[0115] The following examples are offered for purposes of illustration and not limitation.

[0116] [Example] Example 1: Colonic transit time in non-sensitized mice treated with GALR2 agonists To begin evaluating the therapeutic effects of GALR2 agonists, pegylated GALR2 (PEG-GALR2) agonists were administered daily for 7 days to unsensitized mice (without OIC) as described in Figure 1. PEG-GALR2 was administered intraperitoneally (1 mg / kg) or intranasally (10 pg / animal). Control animals received either control vehicle (PBS) (negative control) or bisacodyl (positive control). The various treatment groups are listed in Table 6 below. [Table 6]

[0117] As shown in Figure 2, animals receiving the PEG-GALR2 agent intraperitoneally (i.e., G2) had significantly reduced colonic transit times compared to control animals, whereas animals receiving the PEG-GALR2 agent intranasally (i.e., G4) generally had similar colonic transit times to control animals.

[0118] To further evaluate the effect on colonic transit time after intraperitoneal administration, various doses (0.1 mg / kg, 0.3 mg / kg, or 1 mg / kg) of the PEG-GALR2 agonist were administered intraperitoneally to unsensitized mice again as shown in Figure 1 (i.e., daily for 7 consecutive days). For comparison, the PEG-GALR2 agonist was administered subcutaneously (1 mg / kg) to some animals. The various treatment groups are listed in Table 7 below. [Table 7]

[0119] As shown in Figure 3, after intraperitoneal administration, the effect of the GALR2 agonist on colonic transit time was dose-dependent, with a statistically significant decrease observed in animals administered a 1 mg / kg dose of the GALR2 agonist (i.e., G4). Animals subcutaneously injected with the same dose (1 mg / kg) of the GALR2 agonist showed a trend toward decreased colonic transit time compared to negative control animals (G5 vs. G1).

[0120] The above results demonstrate the ability of the GALR2 agonists described herein to decrease colonic transit time.

[0121] Example 2: Effect of GALR2 agonists on colonic transit time in opioid-induced constipation (OIC) mice An OIC mouse model was used to evaluate whether the GALR2 agonists described herein may be useful for treating opioid-induced constipation. As shown in Figure 4, PEG-GALR2 agonists were administered daily to unsensitized mice for seven consecutive days. To further evaluate whether the route of administration had an effect, PEG-GALR2 agonists were administered intraperitoneally, intranasally, or subcutaneously to mice. After the final administration, the animals were subcutaneously administered morphine (3 mg / kg) to induce constipation ("OIC" mice). Subsequently, glass beads were inserted into the rectum, and colonic transit time was assessed by measuring the delay time until the animals expelled the glass beads. The following animals were used as control groups: (i) normal mice (untreated, no morphine); (ii) OIC mice treated with a control vehicle (negative control group); (iii) OIC mice treated with bisacodyl (positive control group); and (iv) OIC mice treated with naloxone methiodide (an opioid receptor antagonist) (positive control group). A detailed description of the various treatment groups is provided in Table 8 below. [Table 8]

[0122] As shown in Figure 5, intraperitoneal and intranasal administration of GALR2 agonists significantly decreased colonic transit time in OIC mice compared to control animals. The decrease in colonic transit time in animals treated with intraperitoneal GALR2 agonists was similar to that observed in positive control animals (i.e., animals treated with naloxone methiodide). No significant differences were observed between animals receiving intraperitoneal or subcutaneous GALR2 agonists.

[0123] The above results demonstrate that the GALR2 agonists described herein can also reduce colonic transit time in animals experiencing opioid-induced constipation. In contrast to what was observed in non-sensitized animals (see Example 1), administration of a GALR2 agonist in the presence of opioid-induced constipation confirmed little effect of the route of administration.

[0124] Example 3: Dose-dependent effects of GALR2 agonists after intraperitoneal or intranasal administration in OIC mice To further investigate the effects of the GALR2 agonists described herein on opioid-induced constipation, we again used the OIC mouse model. Briefly, as shown in Figure 4, various doses of PEG-GALR2 agonists were administered to mice for 7 consecutive days. The PEG-GALR agonists were administered intraperitoneally (0.1 mg / kg, 0.3 mg / kg, or 1 mg / kg) or intranasally (1 pg, 3 pg, or 10 pg). After the final administration, the animals were subcutaneously administered morphine (3 mg / kg) to induce constipation ("OIC" mice). Subsequently, glass beads were inserted into the rectum, and colonic transit time was assessed by measuring the delay time until the animals expelled the glass beads. The following animals were used as control groups: (i) normal mice (untreated, no morphine); (ii) OIC mice treated with a control vehicle (negative control group); (iii) OIC mice treated with bisacodyl (positive control group); and (iv) OIC mice treated with naloxone methiodide (an opioid receptor antagonist) (positive control group). A detailed description of the various treatment groups is provided in Table 9 below. [Table 9]

[0125] Consistent with previously presented data, all animals treated with the GALR2 agonists described herein had decreased colonic transit times compared to control animals, as shown in Figure 6. Regardless of the route of administration, the greatest decrease in colonic transit time was observed in animals receiving the highest dose of GALR2 agonist, i.e., 1 mg / kg when administered intraperitoneally and 10 pg when administered intranasally.

[0126] The above results demonstrate that the therapeutic effects observed with the GALR2 agonists described herein are dose-dependent, further demonstrating that the route of administration (at least between intraperitoneal and intranasal) can have only a negligible effect on colonic transit time.

[0127] Example 4: Dose-dependent effects of GALR2 agonists after subcutaneous administration to OIC mice To further investigate the effects of the GALR2 agonists described herein on opioid-induced constipation, we again used the OIC mouse model. Briefly, mice were subcutaneously administered with PEG-GALR2 agonists (0.5 mg or 1 mg) for seven consecutive days. For comparison, some animals were intraperitoneally administered with PEG-GALR2 agonists. After the final administration, the animals were subcutaneously administered with morphine (3 mg / kg) to induce constipation ("OIC" mice). Subsequently, glass beads were inserted into the rectum and colonic transit time was assessed by measuring the latency time until the animals expelled the glass beads. The following animals were used as controls: (i) normal mice (untreated, no morphine); (ii) OIC mice treated with a control vehicle (negative control group); (iii) OIC mice treated with bisacodyl (positive control group); and (iv) OIC mice treated with naloxone methiodide (an opioid receptor antagonist) (positive control group). A detailed description of the various treatment groups is provided in Table 10 below. [Table 10]

[0128] As shown in Figure 7, all animals administered with the GALR2 agonist exhibited reduced colonic transit time compared to animals treated with PBS. As previously observed (Example 3), among animals administered intraperitoneally with the GALR2 agonist, the greatest therapeutic effect was observed in mice administered a higher dose (i.e., 1 mg / kg), confirming the dose-dependent effect previously described. A similar dose-dependent effect was also observed in mice subcutaneously injected with the GALR2 agonist.

[0129] These results further demonstrate the dose-dependent therapeutic effect of the GALR2 agonists described herein on colonic transit time. As in Example 3, these results suggest that the influence of the specific administration route may be insignificant (at least between intraperitoneal and subcutaneous administration).

[0130] Example 5: Comparison of PEG-GALR2, GALR2, and wild-type spexin To compare the therapeutic effects of the GALR2 agonists provided herein with wild-type spexin, the OID mouse model was again used. Briefly, as shown in Table 11 below, each animal received one of the following treatments subcutaneously daily for 7 consecutive days: (i) PEG-GALR2 (0.5 or 1 mg / kg); (ii) non-PEGylated GALR2 ("GALR2") (0.5 or 1 mg / kg); and (iii) wild-type spexin (0.5 or 1 mg / kg). OIC was then induced in each animal, and colonic transit time was measured as described in the previous example (see Example 2). A detailed description of the various treatment groups is provided in Table 11 below. [Table 11]

[0131] As shown in Figure 8, colonic transit time decreased in a dose-dependent manner in all treated animals (i.e., G3 to G8). When compared with various treatment regimens, the therapeutic effect on colonic transit time in animals treated with GALR2 agonists was much greater than that in animals treated with wild-type spexin. No significant difference was observed between PEGylated and non-PEGylated GALR2.

[0132] The above results confirm the superior therapeutic effects associated with the GALR2 agonists described herein. When compared to wild-type spexin (the natural ligand for GALR2), the GALR2 agonists of the present invention are significantly more effective in reducing colonic transit time in patients experiencing opioid-induced constipation.

[0133] Example 6: Effect of administration schedule of GALR2 agonist on therapeutic effect To assess whether administration frequency could be reduced without affecting therapeutic efficacy, the GALR2 agonists described herein were conjugated to Fc (GALR2-Fc). Mice were then administered GALR2-Fc or non-Fc-conjugated GALR2, as detailed in Table 12 below and shown in Figure 9a. After the final administration, OIC was induced in each animal, and colonic transit time was measured as described in previous examples (see, e.g., Example 2). [Table 12]

[0134] As shown in Figure 9b, a single subcutaneous administration of a GALR2 agonist described herein (i.e., G4) resulted in a similar decrease in colonic transit time compared to animals that received a total of 7 days of subcutaneous administration of the GALR2 agonist daily (i.e., G3). A similar decrease was also observed in animals that received a single intranasal administration of the GALR2 agonist (i.e., G5). A single subcutaneous administration of Fc-conjugated GALR2 resulted in a significant decrease in colonic transit time compared to control animals (i.e., G2).

[0135] The above results suggest that a therapeutic effect (eg, a reduction in colonic transit time) can be achieved with a single administration of the GALR2 agonists described herein.

[0136] Example 7: Effect of GALR2 agonist administration on repeated OIC induction To evaluate the long-term therapeutic effect of GALR2 administration, an OIC mouse model was used. As shown in Figure 10a, mice were administered a single dose of a GALR2 agonist (subcutaneously or intranasally). The following animals were used as controls: (i) normal mice (untreated, no morphine); and (ii) OIC mice treated with a control vehicle (negative control group). OIC was then induced, and colonic transit time was measured as described in previous examples (see, e.g., Example 2). After a certain period of time (approximately 44 hours), OIC was again induced in each animal, and the animals' second colonic transit time was measured.

[0137] Consistent with the results of previous Examples (see, e.g., Example 5), a single administration of a GALR2 agonist reduced colonic transit time compared to control OIC mice (see Figure 10b). This reduction was observed regardless of whether the GALR2 agonist was administered subcutaneously or intranasally. A similar reduction in colonic transit time was observed after a second OIC induction (see Figure 10c).

[0138] Collectively, the above results demonstrate that a single administration of a GALR2 agonist can have a significant, long-lasting effect on colonic transit time, further confirming that the GALR2 agonists described herein have excellent therapeutic potential for treating diseases associated with constipation.

[0139] Example 8: Effect of substitutions at the fourth amino acid position of GALR2 agonists and changes in solubility To further investigate the properties of the GALR2 agonists provided herein, solubility analyses were performed. Specifically, three GALR2 agonist peptides differing in the fourth amino acid position were tested: (1) nWTaNAALYLFGPq (i.e., "D-alanine"; SEQ ID NO: 87), (2) NWTeNAALYLFGPq (i.e., "D-glutamic acid"; SEQ ID NO: 88), and (3) NWTrNAALYLFGPq (i.e., "D-arginine"; SEQ ID NO: 89). In each amino acid sequence shown, lowercase letters indicate D-amino acids. The D-glutamic acid and D-arginine peptides were additionally PEGylated at the N-terminus. In all three tested GALR2 agonist peptides, an NH group was attached to the C-terminus to block C-terminal degradation of the synthesized peptides, but this group is unnecessary for the functional activity of the peptides. The specific structures of the three GALR2 agonist peptides tested were as follows: (1) nWTaNAALYLFGPq-NH2, (2) PEG2-NWTeNAALYLFGPq-NH2, and (3) PEG2-NWTrNAALYLFGPq-NH2.

[0140] The solubility of each GALR2 agonist peptide was evaluated using two different solvents: solvent 1: pH 5.5, L-histidine (10 mM) and solvent 2: pH 6.0, phosphate buffer. The three different GALR2 agonist peptides were evaluated at three different concentrations: 1.0 mg / 100.0 pL, 1.0 mg / 1.0 mL, and 0.1 mg / 1.0 mL.

[0141] The peptides required for each concentration were added to each solvent and mixed by vigorously shaking for 30 seconds every 5 minutes at 20±5°C. After about 30 minutes, the dissolution state of the peptide protein powder in the solvent was confirmed. [Table 13] An "O" in the middle right column (Buffer 1) indicates complete dissolution of the peptide upon visual inspection. An "X" in the far right column (Buffer 2) indicates poor peptide solubility upon visual inspection.

[0142] As observed in Table 13 above, both the D-glutamic acid peptide (i.e., containing a D-glutamic acid substitution at the fourth amino acid position) and the D-arginine peptide (i.e., containing a D-arginine substitution at the fourth amino acid position) exhibited improved solubility compared to the D-alanine peptide (i.e., containing a D-alanine substitution at the fourth amino acid position). Specifically, improved solubility was observed for the D-glutamic acid peptide when dissolved in solvent 2 at a concentration of 0.1 mg / 1.0 mL. Improved solubility was observed for the D-arginine peptide when dissolved in solvent 1 at a concentration of 0.1 mg / 1.0 mL. Improved solubility was observed for the D-arginine peptide in both solvent 1 and solvent 2 when dissolved at a concentration of 0.1 mg / 1.0 mL.

[0143] Overall, the above results indicate that certain amino acid substitutions (eg, D-glutamic acid or D-arginine) at the fourth amino acid substitution may be useful in improving the solubility of GALR2 agonists.

[0144] Example 9: Effect of substitution at the fourth amino acid position of GALR2 agonists and in vitro efficacy In addition to solubility (as described above in Example 8), the in vitro efficacy of the three GALR2 agonist peptides described in Example 7 was evaluated as follows: Specifically, a cell line expressing mGqi-hGALR2-SRELuc was treated with various peptides, and SRE luciferase activity was measured (by measuring intracellular activity mediated through the GALR2 agonist peptides).

[0145] E of three GALR2 agonist peptides max The logEC values ​​were found to be similar. 50 The (mean ± SEM) values ​​are presented in Table 14. Also, as shown in Figure 11, the D-glutamic acid peptides had similar or even lower EC 50It was confirmed that the D-arginine peptide had a higher EC than the D-alanine peptide. 50 The value increased by approximately 3.5 times. [Table 14]

[0146] The above results further confirm that a fourth amino acid substitution (eg, D-arginine) improves the properties (eg, efficacy) of GALR2 agonist peptides.

[0147] It should be understood that the Detailed Description, but not the Abstract, is intended to be used to interpret the claims. The Abstract does not present every exemplary embodiment of the invention contemplated by the inventors, but may present one or more, and is therefore not intended to limit the scope of the present disclosure and the appended claims in any manner.

[0148] The present invention has been described above with the aid of functional components that illustrate the implementation of specific functions and relationships thereof. The boundaries of such functional components have been arbitrarily defined for the convenience of description. Other boundaries may be defined as long as the specified functions and relationships are appropriate.

[0149] The foregoing description of specific embodiments sufficiently illustrates the general characteristics of the present invention, so that others, applying knowledge of the art, can readily modify and / or adapt such specific embodiments to various fields of application without undue experimentation without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments based on the disclosure and guidance presented herein. It is to be understood that the phrases and terms used herein are for purposes of description and not of limitation. Accordingly, the phrases and terms used herein should be interpreted by those skilled in the art in light of the disclosure and guidance herein.

[0150] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0151] All publications, patents, patent applications, internet sites, and deposit numbers / database sequences (including all polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or deposit number / database sequence was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0152] [Figure 1] FIG. 1 provides the general experimental design for evaluating the effects of the GALR2 agonists described herein in normal, non-sensitized ICR mice. The upper arrows (7 in total) indicate daily administration of the GALR2 agonist. Control animals received a control vehicle (negative control) or bisacodyl (positive control). Thirty minutes after the last administration, glass beads were inserted into the animals' rectums. Colonic transit time was assessed by measuring the delay in expelling the glass beads. [Figure 2] This figure shows colonic transit time (seconds) in normal, non-sensitized ICR mice administered a PEGylated GALR2 (PEG-GALR2) agent via intraperitoneal or intranasal administration. Treatment groups were as follows: (G1) control vehicle (intraperitoneal), (G2) PEG-GALR2 agent (1 mg / kg; intraperitoneal), (G3) control vehicle (intranasal), (G4) PEG-GALR2 agent (10 pg / mouse; intranasal), and (G5) bisacodyl (100 mg / kg; oral). The overall experimental design was as shown in Figure 1. Statistical analysis was performed using Student's t-test. * indicates a significant difference at the p<0.05 level when compared to G3. [Figure 3]This figure shows the dose-dependent effect of a PEGylated GALR2 (PEG-GALR2) agent on colonic transit time in normal, unsensitized ICR mice. Treatment groups were as follows: (G1) control vehicle (intraperitoneal), (G2) PEG-GALR2 agent 0.1 mg / kg (intraperitoneal), (G3) PEG-GALR2 agent 0.3 mg / kg (intraperitoneal), (G4) PEG-GALR2 agent 1 mg / kg (intraperitoneal), (G5) PEG-GALR2 agent 1 mg / kg (subcutaneous), and (G6) bisacodyl (100 mg / kg; oral). The overall experimental design was as shown in Figure 1. Statistical analysis was performed using Student's t-test. * indicates a significant difference at the p<0.05 level compared to G1. [Figure 4] Figure 1 provides the general experimental design for evaluating the effects of the GALR2 agonists described herein in a mouse model of opioid-induced constipation (OIC). The upper arrows (seven in total) indicate daily administration of the GALR2 agonist. The GALR2 agonist administered to the group was PEGylated. Control animals received a control vehicle (negative control) or bisacodyl (positive control). Animals receiving a single dose of naloxone methiodide (e.g., on day 7) were also used as a positive control. Ten minutes after the last administration, each animal received morphine (3 mg / kg; subcutaneously). Thirty minutes after the last administration, a glass bead was inserted into the animal's rectum. Colonic transit time was assessed by measuring the delay time it took for the animal to expel the glass bead. [Figure 5]This figure shows colonic transit time in mice administered PEGylated GALR2 (PEG-GALR2) agonists via various routes of administration (e.g., intraperitoneal, intranasal, or subcutaneous). Treatment groups were as follows: (G1) normal, non-sensitized mice (no OIC); (G2) control vehicle (intraperitoneal); (G3) PEG-GALR2 agonist (1 mg / kg; intraperitoneal); (G4) control vehicle (intranasal); (G5) PEG-GALR2 agonist (10 pg; intranasal); (G6) bisacodyl (100 mg / kg; oral) (positive control group); and (G7) naloxone methiodide (10 mg / kg; intraperitoneal) (positive control group). The overall experimental design was as shown in Figure 4. Statistical analysis was performed using Student's t-test. "*** / ** / *" = Significant difference at p<0.001 / 0.01 / 0.05 levels when compared to G1. "###" = Significant difference at p<0.001 level when compared to G2. "$$$" = Significant difference at p<0.001 level when compared to G4. [Figure 6]FIG. 1 shows the dose-dependent effect of a GALR2 agonist on colonic transit time in OIC mice. The treatment groups were as follows: (G1) normal, non-sensitized mice (no OIC), (G2) control vehicle (intraperitoneal), (G3) 0.1 mg / kg PEG-GALR2 agent (intraperitoneal), (G4) 0.3 mg / kg PEG-GALR2 agent (intraperitoneal), (G5) 1 mg / kg PEG-GALR2 agent (intraperitoneal), (G6) control vehicle (intranasal), (G7) 1 pg PEG-GALR2 agent (intranasal), (G8) 3 pg PEG-GALR2 agent (intranasal), (G9) 10 pg PEG-GALR2 agent (intranasal), (G10) bisacodyl (100 mg / kg; oral), and (G11) naloxone methiodide (10 mg / kg; intraperitoneal). The overall experimental design is shown in Figure 4. Statistical analysis was performed using Student's t-test. "*** / **" = Significant difference at p<0.001 / 0.01 levels when compared to G1, respectively. "### / ## / #" = Significant difference at p<0.001 / 0.01 / 0.05 levels when compared to G2, respectively. "$$$ / $$ / $" = Significant difference at p<0.001 / 0.01 / 0.05 levels when compared to G6, respectively. [Figure 7]This figure shows the effect of administration route (intraperitoneal vs. subcutaneous) on the dose-dependent effect of GALR2 agonists on colonic transit time in mice with OIC. Treatment groups were as follows: (G1) normal, non-sensitized mice (no OIC), (G2) control vehicle (intraperitoneal), (G3) 0.5 mg / kg PEG-GALR2 agonist (intraperitoneal), (G4) 1 mg / kg PEG-GALR2 agonist (intraperitoneal), (G5) control vehicle (subcutaneous), (G6) 0.5 mg / kg PEG-GALR2 agonist (subcutaneous), (G7) 1 mg / kg PEG-GALR2 agonist (subcutaneous), (G8) bisacodyl (100 mg / kg; oral), and (G9) naloxone methiodide (10 mg / kg; intraperitoneal). The overall experimental design is as shown in Figure 4. Statistical analysis was performed using Student's t-test. "*** / **" = significant difference at p<0.001 / 0.01 level when compared to G1. "###" = significant difference at p<0.001 level when compared to G2. "$$$" = significant difference at p<0.001 level when compared to G5. [Figure 8]FIG. 1 compares colonic transit time in mice treated with subcutaneous administration of wild-type spexin peptide or a GALR2 agonist described herein. The GALR2 agonist was either PEGylated (PEG-GALR2 agonist) or non-PEGylated (GALR2 agonist). Treatment groups were as follows: (G1) normal, non-sensitized mice (no OIC), (G2) control vehicle, (G3) 0.5 mg / kg PEG-GALR2 agonist, (G4) 1 mg / kg PEG-GALR2 agonist, (G5) 0.5 mg / kg GALR2 agonist, (G6) 1 mg / kg GALR2 agonist, (G7) 0.5 mg / kg wild-type spexin, (G8) 1 mg / kg wild-type spexin, (G9) bisacodyl (100 mg / kg; orally administered), (G10) nanoparticles (orally administered), and (G11) nanoparticles (orally administered). Xon methiodide (10 mg / kg; intraperitoneal administration). The overall experimental design is as shown in Figure 4. "*** / ** / *" = Significant difference (t-test) at p<0.001 / 0.01 / 0.05 levels when compared to G1, respectively. "### / ## / #" = Significant difference (t-test) at p<0.001 / 0.01 / 0.05 levels when compared to G2, respectively. "$" = Significant difference (G4, G6, G8, one-way ANOVA, Bonferroni's multiple comparison test) at p<0.05 level when compared to G8. [Figure 9a]Figure 9 shows the effects of administration schedule and route of administration on GALR2 agonist-mediated colonic transit time regulation in OIC mice. Figure 9a is a schematic diagram of the overall experimental design. Non-PEGylated GALR2 agonists were administered to OIC mice as follows: (i) daily subcutaneous administration (1 mg / kg per dose) for 7 days ("G3"), (ii) a single subcutaneous administration (1 mg / kg) on ​​day 7 ("G4"), and (iii) a single intranasal administration (10 pg / animal) on day 7 ("G5"). Some animals received a single dose of Fc-conjugated non-PEGylated GALR2 (GALR2-Fc) agonist (35 mg / kg) on ​​day 4 ("G6"). Normal, non-sensitized mice (no OIC, "G1") and OIC mice subcutaneously injected with control vehicle ("G2") served as controls. Ten minutes after the last administration, each animal received morphine (3 mg / kg; subcutaneous). Thirty minutes after the last administration, a glass bead was inserted into the animal's rectum. Colonic transit time was assessed by measuring the delay in expelling the glass bead. [Figure 9b] Figure 9b shows the effect of administration schedule and route of administration on GALR2 agonist-mediated colonic transit time regulation in OIC mice. Figure 9b compares colonic transit time between various treatment groups. "*** / **" = significant difference at p<0.0001 / 0.01 level when compared to G1. "###" = significant difference at p<0.001 level when compared to G2. [Figure 10a]This figure shows the effect of GALR2 agonists on colonic transit time after multiple inductions of opioid-mediated constipation. Figure 10a shows the overall experimental design. Mice were administered a single dose of GALR2 agonists subcutaneously (1 mg / kg) (G3) or intranasally (10 pg / mouse) (G4). Normal, non-sensitized mice (without OIC) (G1) and OIC mice treated with a control vehicle (deionized water) (G2) served as controls. For the first OIC induction, morphine (3 mg / kg) was administered subcutaneously to the animals 4 hours after administration, and a glass bead was inserted into the rectum 30 minutes later. The first colonic transit time of the animals was then assessed for 60 minutes. At 48 hours after administration, morphine (3 mg / kg) was administered subcutaneously for a second time, and after the second OIC induction, a glass bead was inserted into the rectum again 30 minutes later. The second colonic transit time of the animals was then assessed for 60 minutes. Colonic transit time was assessed by measuring the delay time it took the animals to expel the glass beads. [Figure 10b] Figure 10b shows the effect of GALR2 agonists on colonic transit time after multiple inductions of opioid-mediated constipation. Figure 10b compares the first colonic transit time. "***" = significant difference (T-test) when compared to G1, p<0.001. "###" = significant difference (T-test) when compared to G2, p<0.001. [Figure 10c] Figure 10 shows the effect of GALR2 agonists on colonic transit time after multiple inductions of opioid-mediated constipation. Figure 10c compares secondary colonic transit times. "***" = significant difference (T-test) when compared to G1, p<0.001. "###" = significant difference (T-test) when compared to G2, p<0.001. [Figure 11]This figure compares the in vitro efficacy of three different GALR2 agonist peptides with specific amino acid substitutions at the fourth amino acid position. Specifically, the GALR2 agonist peptides are: (1) nWTaNAALYLFGPq-NFE (D-alanine substitution; triangles), (2) PEG2-NWTeNAALYLFGPq-NH2 (D-glutamic acid; filled circles), and (3) PEG2-NWTrNAALYLFGPq-NH2 (D-arginine; empty circles). The efficacy of the GALR2 agonists was assessed by measuring SRE luciferase activity in peptide-treated mGqi-hGALR2-SRE Luc-expressing cell lines.

Claims

1. 1. A method of treating a gastrointestinal disorder in a subject in need thereof, comprising administering to the subject a galanin receptor type 2 (GALR2) agonist, a nucleic acid encoding the GALR2 agonist, or a vector comprising the nucleic acid, wherein the GALR2 agonist is selected from the group consisting of X １ WX ３ X ４ X ５ X ６ X ７ X ８ YLX １１ X １２ X １３ X １４ (SEQ ID NO: 1), wherein: X １ is asparagine (N), glycine (G), pyroglutamic acid (pQ) or citrulline (Cit); X ３ is threonine (T), alanine (A) or lysine (K); X ４ is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); X ５ is asparagine (N) or glutamine (Q); X ６ is alanine (A) or serine (S); X ７ is alanine (A) or methionine (M); X ８ is leucine (L), glutamine (Q) or glycine (G); X １１ is leucine (L), phenylalanine (F), tyrosine (Y) or aspartic acid (D); X １２ is glycine (G) or alanine (A); X １３ is proline (P), arginine (R) or alanine (A); X １４ is glutamine (Q), histidine (H) or valine (V), Here, the GALR2 agonist specifically activates GALR2.

2. 10. The method of claim 1, wherein the gastrointestinal disorder comprises constipation, neurogenic bowel dysfunction (NBD), or all of these.

3. 3. The method of claim 2, wherein the constipation comprises opioid-induced constipation (OIC).

4. 1. A method for regulating bowel movements in a subject in need thereof, comprising administering to the subject a GALR2 (galanin receptor type 2) agonist, a nucleic acid sequence encoding the GALR2 agonist, or a vector comprising the nucleic acid, wherein the GALR2 agonist is selected from the group consisting of X １ WX ３ X ４ X ５ X ６ X ７ X ８ YLX １１ X １２ X １３ X １４ (SEQ ID NO: 1), wherein: X １ is asparagine (N), glycine (G), pyroglutamic acid (pQ) or citrulline (Cit); X ３ is threonine (T), alanine (A) or lysine (K); X ４ is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); X ５ is asparagine (N) or glutamine (Q); X ６ is alanine (A) or serine (S); X ７ is alanine (A) or methionine (M); X ８ is leucine (L), glutamine (Q) or glycine (G); X １１ is leucine (L), phenylalanine (F), tyrosine (Y) or aspartic acid (D); X １２ is glycine (G) or alanine (A); X １３ is proline (P), arginine (R) or alanine (A); X １４ is glutamine (Q), histidine (H) or valine (V), Here, the GALR2 agonist specifically activates GALR2.

5. 5. The method of claim 4, wherein said bowel control comprises controlling colonic transit time in said subject.

6. The method of claim 5, wherein after administration of the GALR2 agonist, the colonic transit time of the subject is reduced compared to the colonic transit time of a reference subject (e.g., the subject before administration and / or a corresponding subject not receiving administration).

7. 7. The method of claim 6, wherein the colonic transit time is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% when compared to the reference subject.

8. 1. A method of treating an endocrine disorder in a subject in need thereof, comprising administering to the subject a galanin receptor type 2 (GALR2) agonist, a nucleic acid sequence encoding the GALR2 agonist, or a vector comprising the nucleic acid sequence, wherein the GALR2 agonist is selected from the group consisting of X １ WX ３ X ４ X ５ X ６ X ７ X ８ YLX １１ X １２ X １３ X １４ (SEQ ID NO: 1), wherein: X １ is asparagine (N), glycine (G), pyroglutamic acid (pQ) or citrulline (Cit); X ３ is threonine (T), alanine (A) or lysine (K); X ４ is proline (P), leucine (L), glutamic acid (E), arginine (R), alanine (A), or valine (V); X ５ is asparagine (N) or glutamine (Q); X ６ is alanine (A) or serine (S); X ７ is alanine (A) or methionine (M); X ８ is leucine (L), glutamine (Q) or glycine (G); X １１ is leucine (L), phenylalanine (F), tyrosine (Y) or aspartic acid (D); X １２ is glycine (G) or alanine (A); X １３ is proline (P), arginine (R) or alanine (A); X １４ is glutamine (Q), histidine (H) or valine (V), Here, the GALR2 agonist specifically activates GALR2.

9. 5. The method of claim 4, wherein the endocrine disorder comprises chronic renal failure, hypercalcemia, or all of these.

10. X １ 10. The method of claim 1, wherein N is D-asparagine.

11. 11. The method of any one of claims 1 to 10, wherein W at position 2 of SEQ ID NO: 1 is D-tryptophan.

12. X ４ The method according to any one of claims 1 to 11, wherein A is D-alanine, D-glutamic acid, or D-arginine.

13. X ４ 12. The method of any one of claims 1 to 11, wherein V is D-valine.

14. X ６ 14. The method according to any one of claims 1 to 13, wherein A is D-alanine.

15. X １１ 15. The method of any one of claims 1 to 14, wherein K is D-lysine.

16. X １２ 15. The method according to any one of claims 1 to 14, wherein A is D-alanine.

17. X １３ 16. The method of any one of claims 1 to 15, wherein A is D-alanine.

18. X １４ 17. The method of any one of claims 1 to 16, wherein Q is D-glutamine.

19. 19. The method of any one of claims 1 to 18, wherein the GALR2 agonist does not activate (i) galanin receptor type 1 (GALR1), (ii) galanin receptor type 3 (GALR3), or (iii) all of (i) and (ii).

20. X ７ is A and X １１ The method of any one of claims 1 to 19, wherein is F.

21. X ５ is N and X ７ is A and X １１ The method of any one of claims 1 to 19, wherein is F.

22. X ５ is N and X ７ is A and X １１ is F and X １３ The method of any one of claims 1 to 19, wherein is P.

23. 20. The method of any one of claims 1 to 19, wherein the amino acid sequence of the GALR2 agonist comprises the sequence set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, or SEQ ID NO:

91.

24. 24. The method of any one of claims 1 to 23, wherein the amino acid sequence of the GALR2 agonist is attached to a polyethylene glycol (PEG), an acetyl (Ac) group, or Fmoc.

25. X １ 25. The method of claim 24, wherein N is protected with polyethylene glycol (PEG), an acetyl (Ac) group, or Fmoc.

26. The amino acid sequence of the GALR2 agonist is ２ 25. The method of any one of claims 1 to 24, wherein the adhesive is attached to a

27. 27. The method of any one of claims 1 to 26, wherein the GALR2 agonist is administered to the subject intranasally, intrasphincterically, intramuscularly, subcutaneously, intraocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraventricularly, intraspinally, intraventricularly, intraspinal, intracisternally, intracapsularly, topically, orally, or a combination thereof.

28. 28. The method of claim 27, wherein the GALR2 agonist is administered subcutaneously, intranasally, or intraperitoneally to the subject.

29. 29. The method of any one of claims 1 to 28, wherein the GALR2 agonist is administered to the subject one, two, three, four, five, six, or seven or more times.

30. 30. The method of any one of claims 1 to 29, further comprising administering to the subject an additional therapeutic agent.

31. 31. The method of claim 30, wherein the additional therapeutic agent comprises a laxative (e.g., bisacodyl), an opioid receptor antagonist (e.g., naloxone methiodide), a douche (e.g., anal or colonic douche), electrical stimulation, or a combination thereof.

32. 31. The method of claim 30, wherein the additional therapeutic agent and the GALR2 agonist are administered to the subject simultaneously.

33. 32. The method of claim 31, wherein the additional therapeutic agent and the GALR2 agonist are administered sequentially to the subject.

34. 33. The method of any one of claims 1 to 32, wherein the GALR2 agonist is administered to the subject as a lyophilized powder or a solution.

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