Compositions and methods for controlling food intake, energy expenditure, and body weight for the treatment of obesity and metabolic disease

Abstract

Provided herein are compositions and methods for the treatment of obesity and metabolic disorders using GPR75 inhibitors.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 384,272, filed November 18, 2022, the entire contents of which are incorporated herein by reference as if set forth in full. Incorporation by Reference of Materials Submitted in Electronic Format

[0002] The contents of the electronic sequence listing (UPNK-113-PCT.xml; size: 10,298 bytes; and creation date: November 20, 2023) are incorporated herein by reference in their entirety.

[0003] The present invention relates to the fields of weight loss, weight maintenance, obesity, and metabolic disease treatment. More specifically, the present invention provides peptide sequences and variants thereof capable of inhibiting G protein receptor 75 (GPR75). [Background technology]

[0004] Several publications and patent documents are cited throughout this specification in order to describe the state of the art to which this invention pertains, all of which are incorporated herein by reference.

[0005] Obesity and its cardiometabolic complications, particularly type 2 diabetes and coronary artery disease, account for significant morbidity and mortality worldwide. There is a significant unmet medical need for safe and effective weight loss approaches and strategies for maintaining weight loss.

[0006] Lifestyle interventions, such as diet and physical activity therapy, are the first-line options for managing obesity, but their effectiveness is limited, and weight regain is common. Bariatric surgery is highly effective in reducing weight in severely obese and high-risk patients, but its use is limited by its invasive nature, cost, and risk of perioperative adverse events, including perioperative death. While several medications have proven effective in reducing weight, pharmacotherapy for obesity treatment is limited by the slow weight loss induced by most medications, the development of drug dependency, side effect profiles, contraindications, poor compliance, and social barriers to treatment.

[0007] G protein receptor 75 (GPR75) is a member of the G protein-coupled receptor family. GPRs are cell surface receptors that activate guanine-nucleotide-binding proteins upon ligand binding. GPR75 likely couples to heterotrimeric Gq proteins, and upon activation, stimulates inositol triphosphate production and calcium mobilization. Various experiments have demonstrated a strong association between GPR75 and obesity and metabolic diseases, yet no molecules that antagonize GPR75 are known. Clearly, GPR75 inhibitors are urgently needed for the treatment of obesity and metabolic diseases. Summary of the Invention

[0008] According to the present invention, isolated or purified peptides of SEQ ID NO: 1 or SEQ ID NO: 2, sequences having at least 95% identity to SEQ ID NO: 1 or SEQ ID NO: 2, and functional fragments thereof are provided for effective weight management and treatment of obesity.

[0009] In certain embodiments, the peptide has anti-obesity activity. In certain embodiments, the peptide comprises one or more modified amino acids selected from Gln (alkyne), Ala (alkyne), Gly (alkyne), Lys (N3), and the modified amino acids listed in Table 2 for bioconjugation, including lipidation and / or fluorescent tagging. In certain embodiments, the modified amino acids facilitate bioconjugation of agents selected from one or more of N- and / or C-terminal protection moieties, lipids, recombinant FC-peptides, and cell-penetrating peptides, and enhance one or more of the function, stability, and bioavailability of the peptide. In preferred embodiments, the peptide is delivered in a pharmaceutically acceptable carrier. In certain embodiments, the composition further comprises at least one of HEPES buffer and acetate. In certain embodiments, the composition is formulated to maintain a neutral pH. In certain embodiments, the composition is formulated for intracerebroventricular injection or peripheral administration.

[0010] In another aspect, isolated nucleic acids encoding the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 are also disclosed. In certain embodiments, the nucleic acid is encapsulated in a liposome or extracellular vesicle, or affixed to a nanoparticle or lipid particle. In other aspects, the isolated nucleic acid is present in a vector for robust expression and production in an organism of interest. In certain embodiments, the vector is a plasmid vector, a lentiviral vector, or an AAV vector. In certain embodiments, the vector is encapsulated in a liposome or extracellular vesicle, or affixed to a nanoparticle or lipid nanoparticle. Administration of the peptide, nucleotide, vector, or composition can be via any suitable route, such as systemic, intramuscular, topical, oral, parenteral, transdermal patch, aerosolized, pulmonary, ocular, buccal, and lingual.

[0011] In yet another embodiment, a method of treating obesity in a subject in need thereof is disclosed comprising administering an effective amount of the above-described peptide.

[0012] Also provided is a method for treating a metabolic disease or disorder in a subject in need thereof, comprising administering an effective amount of a peptide of SEQ ID NO: 1 or SEQ ID NO: 2, or a functional variant thereof. The metabolic disease or disorder to be treated includes, but is not limited to, obesity, diabetes, dyslipidemia, insulin resistance, hepatic steatosis, hypercholesterolemia, and non-alcoholic fatty liver disease. In certain embodiments, the disease is diabetes selected from type 1 diabetes or type 2 diabetes. In other embodiments, the method can further comprise administering a second therapeutic agent that treats or inhibits obesity. In certain embodiments, the method further comprises mandating a lifestyle intervention involving diet and / or physical activity in the subject. In a preferred embodiment, the patient loses weight after administration of the peptide.

[0013] In certain embodiments, the patient experiences a decrease in food intake for at least 1, 3, 6, and / or 24 hours after administration of the peptide compared to untreated controls. In another aspect, the method can further include evaluating the patient for alleviation of obesity symptoms. In certain embodiments, the patient is evaluated for alleviation of symptoms of obesity, diabetes, dyslipidemia, insulin resistance, hepatic steatosis, hypercholesterolemia, or non-alcoholic fatty liver disease. In certain embodiments, the peptide, nucleotide, vector, or composition can be administered via any suitable route, such as systemic, intramuscular, topical, oral, parenteral, transdermal patch, aerosolized, pulmonary, ocular, buccal, and lingual. In certain embodiments, administration is via intraventricular injection or peripheral administration.

[0014] In certain embodiments, the peptide and the second therapeutic agent act synergistically to increase weight loss. [Brief explanation of the drawings]

[0015] [Figure 1]Synthesis and purity of peptides. (Figure 1A) SU75-36 (4894.1 g / mol; 17.084 min TR; 100% purity), (Figure 1B) SU75-37 (4462.9 g / mol; 15.381 min TR; 98.3% purity). [Figure 2] Blind in silico docking of SU75-36 / GPR75 receptor using the HPEPDOCK program. SU75-36 (aquamarine; see arrow) surface binding of GPR75 is consistent with surface plasmon resonance (SPR) binding (Figure 3). Docking score: 0.884. [Figure 3] Surface plasmon resonance (SPR) assay tracking the binding of SU75-36 to GPR75. SU75-36 binds to GPR75 with a KD of 7.76 μM. [Figure 4] Surface plasmon resonance (SPR) assay tracking the binding of SU75-37 at GPR75. SU75-37 binds to GPR75 with a KD of 23.8 μM. [Figure 5] Surface plasmon resonance (SPR) assay comparing the binding of SU75-36 to hGLP-1R with a positive Ex-4 control, a negative ODN control, and SU75-37, which does not bind to hGLP-1R. [Figure 6] Surface plasmon resonance (SPR) assay tracking the binding of SU75-36 at hGLP-1R. SU75-36 binds to hGLP-1R with a KD of 182 μM. [Figure 7] Fourth ventricle GPR75 ligands suppress food intake and body weight in high-fat diet (HFD)-fed rats. Effects of fourth ventricle administration of SU75-36 (20, 100, or 200 μg / 2 μL in aCSF) and SU75-37 (20 μg / 2 μL in aCSF) on 24-hour food intake (Figure 7A) and body weight change (Figure 7B) in HFD-fed rats. All data are mean ± SEM. [Figure 8]Lateral ventricle injection of GPR75 ligands suppresses food intake and body weight in standard chow and HFD-fed rats. The effects of lateral ventricle administration of SU75-36 (20 μg or 100 μg / 2 μL in aCSF) or SU75-37 (20 μg / 2 μL in aCSF) on 24-h food intake in standard chow-fed (Figure 8A) and HFD-fed rats (Figure 8B), kaolin intake (standard chow-fed rats: Figure 8C, high-fat diet-fed rats: Figure 8D), and body weight change (standard chow-fed rats: Figure 8E, high-fat diet-fed rats: Figure 8F). All data are mean ± SEM. [Figure 9] Secondary structure analysis of SU75-36 and SU75-37 (40 μM in 0.5% saline) by CD spectroscopy. The helicity fractions were 20.9% for SU75-36 and 21.3% for SU75-37. [Figure 10] Centrally administered 36 suppressed 24-h food intake in rats in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. Effects of intravenous administration of 36 (150 μg / 3 μL) into the lateral ventricle on 24-h food intake (Figure 10A) and body weight (Figure 10B) in male HFD-fed rats. All data are mean ± SEM. [Figure 11] Centrally administered 37 suppresses 24-hour food intake in rats in HEPES buffer. Effects of intravenous administration of 37 (150 μg / 3 μL) into the lateral ventricle on 24-hour food intake (Figure 11A) and body weight (Figure 11B) in male HFD-fed rats. All data are mean ± SEM. [Figure 12] Peripherally administered 36 and 37 suppress 24-hour food intake in mice. Effects of IP 36 (0.5 and 5 mg / kg) on ​​24-hour food intake (Figure 12A) and body weight (Figure 12B) and 37 (0.5 and 5 mg / kg) on ​​24-hour food intake (Figure 12C) and body weight (Figure 12D) in HFD-fed male mice. All data are mean ± SEM. [Figure 13]Peripherally administered IP 37 suppresses 24-hour food intake in mice. Effects of IP 37 (10 mg / kg) treatment on 24-hour food intake (A) and body weight (B) in HFD-fed male mice. All data are means ± SEM. DETAILED DESCRIPTION OF THE INVENTION

[0016] As demonstrated herein, this application provides novel, non-natural peptide ligands that antagonize the highly anticipated orphan receptor GPR75. These GPR75 inhibitors represent a major drug discovery opportunity for the pharmaceutical industry, given the association of GPR75 with obesity and metabolic diseases.

[0017] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings that are commonly understood by those of ordinary skill in the art. In addition to the definitions contained in this subsection, additional term definitions are scattered throughout the text.

[0018] As used herein, "a," "or," and "an" can mean "at least one," "one or more," etc., unless the context clearly indicates otherwise. The term "or" means "and / or" unless otherwise specified. However, in the case of multiple dependent claims, the use of the term "or" refers back to multiple preceding claims in the alternative only.

[0019] Furthermore, a compound "selected from the group consisting of" refers to one or more of the compounds in the following list, including mixtures (i.e., combinations) of two or more compounds. According to the present invention, an isolated or biologically pure molecule is a compound that has been removed from its natural environment. As such, "isolated" or "biologically pure" does not necessarily reflect the extent to which a compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthesis techniques, or can be produced by such chemical synthetic routes.

[0020] The terms "agent" and "test compound" refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (especially mammalian) cells or tissues. Biological macromolecules include peptides, peptide / DNA complexes, siRNAs, shRNAs, antisense oligonucleotides, and nucleic acid-based molecules encoding proteins described herein.

[0021] Additionally, the term "compound" or "compounds" refers to the compounds discussed herein and is intended to include precursors and derivatives of the compounds, as well as pharmaceutically acceptable salts of the compounds, precursors and derivatives.

[0022] The phrase "consisting essentially of" when referring to a particular nucleotide or amino acid means a sequence having the characteristics of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the functional and novel characteristics of the sequence.

[0023] A "derivative" of a polypeptide, polynucleotide, or fragment thereof refers to a sequence that has been altered by changing the sequence of the construct, for example, by manipulating the nucleic acid encoding the protein or by altering the protein itself. A "derivative" of a gene or nucleotide sequence refers to an isolated nucleic acid molecule that has significant sequence similarity to the gene or nucleotide sequence, or a portion thereof. Furthermore, "derivative" includes such isolated nucleic acids that contain modified nucleotides or mimetics of naturally occurring nucleotides.

[0024] As used herein, the term "functional" means that the nucleic acid or amino acid sequence is functional for a stated assay or purpose.

[0025] For purposes of the present invention, the terms "nucleic acid," "nucleotide sequence," or "nucleic acid molecule" as used herein refer to any DNA or RNA molecule, either single-stranded or double-stranded, and, if single-stranded, to the molecule of complementary sequence, either linear or circular. In discussing nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule may be described herein according to the usual convention of providing the sequence in the 5' to 3' direction. The term "isolated nucleic acid" is sometimes used with respect to the nucleic acids of the present invention. When applied to DNA, this term refers to a DNA molecule separated from sequences with which it is immediately adjacent in the naturally occurring genome of the organism from which it originates. For example, an "isolated nucleic acid" can include a DNA molecule inserted into a vector, such as a plasmid or viral vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. Alternatively, the term can refer to DNA that has been sufficiently separated (e.g., substantially free from) other cellular components with which the DNA would naturally be associated.

[0026] "Isolated" does not mean artificial or synthetic mixture with other compounds or materials, or exclusive of the presence of impurities that do not interfere with basic activity and that may be present, for example, due to incomplete purification. When applied to RNA, the term "isolated nucleic acid" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term can refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it is associated in its natural state (i.e., in cells or tissues). Isolated nucleic acid (either DNA or RNA) can also refer to a molecule produced directly by biological or synthetic means and separated from other components present during its production.

[0027] A "specific binding pair" comprises a specific binding member (sbm) and a binding partner (bp) that have a particular specificity for each other and that, under normal conditions, bind preferentially to one another over other molecules. Examples of specific binding pairs include antigens and antibodies, biotin and streptavidin, ligands and receptors, and complementary nucleotide sequences. Those skilled in the art will recognize many other examples. Furthermore, the term "specific binding pair" also applies when either or both of the specific binding member and binding partner form part of a larger molecule. In embodiments where the specific binding pair comprises nucleic acid sequences, they will be of a length sufficient to hybridize to each other under assay conditions, preferably at least 10 nucleotides in length, more preferably at least 15 or 20 nucleotides in length.

[0028] According to the present invention, an isolated or biologically pure molecule or cell is a compound that has been removed from its natural environment. Therefore, "isolated" or "biologically pure" does not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthesis techniques, or can be produced by such a chemical synthesis route.

[0029] As used herein, the term "delivery" refers to the introduction of a foreign molecule (i.e., a miRNA encoding a polypeptide of interest) into a cell. As used herein, the term "administration" refers to the introduction of a foreign molecule into a cell. This term is intended to be synonymous with the term "delivery."

[0030] peptide The peptides of the present invention inhibit or modulate GPR75 activity. The terms "inhibition" or "suppression" refer to the reduction or cessation of some event (such as protein-ligand binding), or the reduction or cessation of some phenotypic characteristic, or the reduction or cessation of the incidence, degree, or likelihood of that characteristic. "Reduce" or "inhibit" refers to the decrease, reduction, or cessation of an activity, function, and / or amount compared to a baseline. Inhibition or reduction need not be complete. For example, in certain embodiments, "reduce" or "inhibit" refers to the ability to cause an overall decrease of 20% or more. In other embodiments, "reduce" or "suppress" refers to the ability to cause an overall decrease of 50% or more. In yet other embodiments, "reduce" or "suppress" refers to the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or more.

[0031] As used herein, the term "modulation" refers to the ability of a compound to measurably alter an activity relative to an appropriate control. The presence of a compound in an assay may result in an increase or decrease in activity relative to a control in which the compound is absent. Preferably, an increase in activity is at least 25%, more preferably at least 50%, and most preferably at least 100% compared to the activity level in the absence of the compound. Similarly, a decrease in activity is preferably at least 25%, more preferably at least 50%, and most preferably at least 100% compared to the activity level in the absence of the compound. A compound that increases a known activity is an "agonist." An agent that decreases or blocks a known activity is an "antagonist."

[0032] The term "inhibitor" refers to an agent that slows or blocks a particular chemical reaction, signaling pathway, or other process, or reduces the activity of a particular reactant, catalyst, or enzyme.

[0033] "G protein receptor 75" or "GPR75" refers to a member of the G protein-coupled receptor family. GPRs are cell surface receptors that activate guanine-nucleotide binding proteins upon ligand binding. GPR75 is a protein-encoding gene. Its associated pathways include class A / 1 (rhodopsin-like receptor) and 15q13.3 copy number variant syndrome. Gene Ontology (GO) annotations associated with this gene include G protein-coupled receptor activity and CC chemokine receptor activity.

[0034] "G protein receptor 75 inhibitors" or "GPR75 inhibitors" refer to a group of drugs that inhibit the action of GPR75. Peptides of interest herein are GPR75 inhibitors, each of which adopts an α-helical secondary structure (Figure 9). Exemplary GPR75-binding peptides are shown in Table 1. [Table 1]

[0035] In certain embodiments, the residues of a protein or peptide are contiguous, with no non-genetically encoded amino acids or synthetic amino acids interrupting the sequence of amino acid residues. In other embodiments, the sequence may contain one or more non-genetically encoded amino acid moieties or synthetic amino acid moieties. In certain embodiments, the sequence of residues of a peptide may be interrupted by one or more non-genetically encoded amino acids or synthetic amino acid moieties, including, but not limited to, those shown in Table 2. [Table 2]

[0036] In certain embodiments, the peptides disclosed herein can be modified to optimize peptide function, stability, formulation, or solubility using the approaches described in Table 3. [Table 3]

[0037] Such improvements result in analogs with long half-lives that can be administered, for example, once a week.

[0038] In certain embodiments, the present invention includes peptides having at least 80% identity to any of the peptides described herein. The phrases "% sequence identity," "percent identity," or "% identity" refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well known. Some alignment techniques take into account conservative amino acid substitutions. Such conservative substitutions generally maintain the charge and hydrophobicity at the substitution site, preserving the structure (and therefore function) of the polypeptide. Percent amino acid sequence identity can be determined as understood in the art. In certain embodiments, peptides of the invention have a sequence identity of at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity.

[0039] Polypeptide sequence identity may be measured over the length of the entire defined polypeptide sequence, e.g., as defined by a particular SEQ ID NO:, or over a shorter length, such as the length of a fragment taken from a larger, defined polypeptide sequence, e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues. It is understood that such lengths are exemplary, and that any fragment length supported by the sequences set forth herein can be used to describe the length over which percent identity may be measured.

[0040] Preferably, the peptides of the above sequences and their functional equivalents are administered to regulate obesity. As used herein, the term "functional equivalent" is intended to include amino acid sequence variants with amino acid substitutions in part or all of the protein, or amino acid additions or deletions in part of the protein. The amino acid substitutions are preferably conservative substitutions. Examples of conservative substitutions of naturally occurring amino acids include: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn), and sulfur-containing amino acids (Cys, Met). The amino acid deletions are preferably located in regions that are not directly involved in the activity of the peptide.

[0041] As used herein, the term "variant" refers to a nucleic acid sequence or polypeptide that comprises a sequence that differs from a wild-type nucleic acid or polypeptide sequence at one or more nucleic acid or amino acid positions (by deletion, insertion, and / or substitution of a nucleic acid or amino acid, an L or D stereoisomer of an amino acid, or a non-naturally occurring amino acid).

[0042] As used herein, the term "linker" refers to the connection between two protein-coding sequences or their protein products. A linker comprises a contiguous nucleic acid or amino acid sequence and retains at least one cleavage site that allows the genes or their products to be separated by cleavage of the linker. Preferably, the linker comprises a cleavage site at its 5'-terminus and a cleavage site at its 3'-terminus, or a cleavage site at its N-terminus and a cleavage site at its C-terminus.

[0043] The peptide can be fused to biotin, polylysine, lysozyme, green fluorescent protein (and its derivatives), SUMO, or other desired proteinaceous tag. Production of the desired peptide sequence can be performed using existing techniques in E. coli, SF9, Pichia, etc. For example, the protein fusion tag can be removed or left intact. In certain embodiments, the peptide of interest can be fused via a linker.

[0044] The peptide can be fused to one or more cell penetrating peptides (CPPs) useful for facilitating delivery of the peptide to target cells. CPPs are known to those skilled in the art and include, but are not limited to, penetratin (RQIKIWFQNRRMKWKK) (SEQ ID NO: 3), VP22 peptide (DAATATRGRSAASRPTERPRAPARSASRPRRVD) (SEQ ID NO: 4), MAP (KLALKLALKALKAALKLA-amide) (SEQ ID NO: 5), transpectin (GWTLNSAGYLLGKINLKALAALAKKIL-amide) (SEQ ID NO: 6), R7 (RRRRRRR) (SEQ ID NO: 7), MPG (GALFLGWLGAAGSTMGAPKKRKV) (SEQ ID NO: 8), Pep-1 (KETWWETWWTEWSQPKKKRKV) (SEQ ID NO: 9), and tat (YGRKKRRQRRR) (SEQ ID NO: 10).

[0045] The peptides can be expressed as fusions with larger proteins, facilitating large-scale expression, easy purification, and ensuring product quality. The expression system can also be utilized to generate large sequence libraries, enabling directed evolution for targeted properties. Peptides can be produced sustainably using existing environmentally friendly fermentation techniques.

[0046] As noted above, the present invention also encompasses polynucleotides encoding the peptides described herein or fusion proteins containing the peptides. Those skilled in the art understand the degeneracy of the genetic code and understand that various polynucleotides can encode the same polypeptide. In some embodiments, the polynucleotide (i.e., the polynucleotide encoding the fusion polypeptide) can be codon-optimized for expression in a particular cell, including, but not limited to, a plant cell, a bacterial cell, or an algal cell. Any polynucleotide sequence encoding the desired form of the polypeptide described herein can be used. The polynucleotide sequences encoding the polypeptides of the present invention represent sequences that do not occur in nature. Computer programs for generating degenerate coding sequences are available and can be used for this purpose.

[0047] In the present context, the term "codon optimization" refers to changing the codons of a nucleotide sequence to favor expression in a particular species, without changing the amino acid sequence it encodes. Codon optimization is used to increase the abundance of the peptide or protein that a nucleotide sequence encodes, as "rare" codons are removed and replaced with abundant codons.

[0048] With respect to the fusion polypeptides disclosed herein, structural similarity is typically at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity. In vitro peptide synthesis

[0049] Peptides can be chemically synthesized in solution or on solid phase. This process involves the directed and selective formation of an amide bond between an N-protected amino acid and an amino acid bearing a free amino group and a protected carboxylic acid. In solid phase synthesis, the carboxyl protecting group is attached to a polymer support. After bond formation, the amino protecting group of the dipeptide is removed, and the next N-protected amino acid is attached.

[0050] Solid-phase peptide synthesis (SSPS) is the most frequently used method for peptide synthesis due to its efficiency, simplicity, speed, and ease of parallelization. SPPS involves sequential addition of amino and side-chain protected amino acid residues to an amino acid or peptide bound to an insoluble polymeric support. N-α-protection is achieved using the acid-labile Boc group (Boc SPPS) or the base-labile Fmoc group (Fmoc SPPS). After removal of this protecting group, the next protected amino acid is added using a coupling reagent or a preactivated protected amino acid derivative. The C-terminal amino acid is anchored to the resin via a linker, the nature of which determines the conditions required to release the peptide from the support after chain elongation. Side-chain protecting groups are often selected so that they are cleaved simultaneously with the peptide's release from the resin.

[0051] While peptides of 50 amino acids can be routinely prepared, the synthesis of proteins of more than 100 amino acids is commonly reported. Longer proteins can be made by native chemical ligation of fully deprotected peptides in solution. This method allows the synthesis of natural peptides that are difficult to express in bacteria, the incorporation of unnatural amino acids and D-amino acids, and the generation of circular, branched, labeled, and post-translationally modified peptides.

[0052] Solution-phase peptide synthesis, which typically utilizes Boc or Z-amino protection, has largely replaced existing processes for large-scale industrial peptide synthesis, with SPPS replacing existing processes. Desired sequences can be developed by commercial organizations, such as Sigma-Aldrich and Avivasysbio, which offer this service for a fee.

[0053] Vectors and production Transgenic cell expression of the polynucleotides and polypeptides also forms an aspect of the present invention. Transgenic cells can be obtained by introducing a recombinant nucleic acid molecule encoding a protein of the present disclosure. As used herein, the term "recombinant nucleic acid" refers to a polynucleotide that has been manipulated by human intervention. A recombinant nucleic acid molecule can contain two or more nucleotide sequences linked in such a way that the product is not found in a cell in nature. In particular, two or more nucleotide sequences can be operably linked, e.g., to encode a fusion polypeptide. Recombinant nucleic acid molecules can also be based on naturally occurring polynucleotides but engineered to be different from them. For example, a polynucleotide can contain one or more nucleotide changes that bias the first codon normally present in the polynucleotide toward chloroplast codon usage. Alternatively, a sequence of interest, such as a restriction endonuclease recognition site, splice site, promoter, or DNA replication origin, can be introduced into the polynucleotide.

[0054] Any suitable technique for introducing recombinant nucleic acid molecules into cells can be used. Techniques for nuclear and chloroplast transformation are known and include, but are not limited to, electroporation, biological transformation (also called microprojectile / particle bombardment), agitation in the presence of glass beads, and Agrobacterium-based transformation.

[0055] As used herein, the term "construct" refers to a recombinant polynucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded and may represent a sense or antisense strand. A recombinant polynucleotide is a polynucleotide formed by laboratory methods that includes polynucleotide sequences derived from at least two different natural sources, and may be synthetic. Thus, a construct may include, for example, a new modification of an endogenous gene introduced by genome editing technology. A construct also includes, for example, a recombinant polynucleotide produced using recombinant DNA methods.

[0056] A "vector" is a vector capable of transferring a gene sequence into a target cell. Generally, the terms "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct capable of inducing expression of a gene of interest and transferring a gene sequence into a target cell. Thus, the term includes cloning vectors, expression vectors, and integrating vectors.

[0057] The constructs and vectors provided herein can be prepared by methods available to those skilled in the art.In particular, each of the claimed constructs or expression cassettes is a recombinant molecule and does not exist in nature.In general, the nomenclature used herein and the experimental procedures utilized in the present invention include molecular biological, biochemical and recombinant DNA techniques that are well known and commonly employed in the art.Standard techniques available to those skilled in the art can be used for cloning, DNA and RNA isolation, amplification and purification.Such techniques are fully described in the literature.

[0058] The constructs and expression cassettes provided herein can include a promoter operably linked to any one of the polynucleotides described herein, but do not necessarily have a promoter, and can be used for homologous recombination into a cell. The constructs can include a promoter, which can be a heterologous promoter or an endogenous promoter associated with the polypeptide.

[0059] As used herein, the terms "heterologous promoter," "promoter," "promoter region," or "promoter sequence" generally refer to the transcriptional regulatory region of a gene, which may be found 5' or 3' to a polynucleotide described herein, within the coding region of the polynucleotide, or within an intron of the polynucleotide. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3') coding sequence. A typical 5' promoter sequence is surrounded at its 3' end by a transcription start site and extends upstream (5') to include the minimum number of bases or elements required to initiate transcription detectable at levels above background. Within the promoter sequence is a transcription start site (easily defined by mapping with nuclease S1) and a protein binding domain (consensus sequence) responsible for binding RNA polymerase.

[0060] In some embodiments, the disclosed polynucleotide is operably linked to a promoter.As used herein, a polynucleotide is "operably linked" or "operably linked" when it is placed in a functional relationship with a second polynucleotide sequence.For example, a promoter is operably linked to a polynucleotide when the promoter is linked to the polynucleotide so as to affect the transcription of the polynucleotide.In various embodiments, a polynucleotide can be operably linked to at least one, at least two, at least three, at least four, at least five, or at least ten promoters.

[0061] Heterologous promoters useful in practicing the present invention include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters. Heterologous promoters can be plant, animal, bacterial, fungal, or synthetic promoters.

[0062] Treatment and administration As used herein, the terms "alleviate" or "inhibit" refer to the administration of a compound prior to or during the onset of clinical symptoms of a disease or condition, so as to reduce the physical manifestations of abnormalities associated with the disease or condition.

[0063] As used herein, a "condition in need of treatment" refers to a determination by a caregiver (e.g., in the case of a human, a physician, nurse, nurse practitioner, or individual; in the case of an animal, including a non-human mammal, a veterinarian) that a subject is in need of or will benefit from treatment. This determination is within the expertise of the caregiver, but is made based on a variety of factors, including knowledge that the subject is ill or will become ill as a result of a condition treatable by the disclosed compounds.

[0064] In the methods described herein, acts may be performed in any order unless a temporal or operational order is expressly recited. Furthermore, certain acts may be performed simultaneously unless express claim language clearly recites them performed separately. For example, a claimed act of performing X and a claimed act of performing Y may be performed simultaneously in a single operation, and the resulting process falls within the literal scope of the claimed process.

[0065] As used herein, a "subject" includes, but is not limited to, animals, plants, bacteria, viruses, parasites, and other organisms or entities. A subject may be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent), fish, bird, reptile, or amphibian. A subject may be an invertebrate, more specifically an arthropod (e.g., an insect or crustacean). The term does not denote a particular age or sex; thus, adults, newborns, and fetuses of either sex are included. A patient refers to a subject suffering from a disease or disorder. The term "patient" includes human and veterinary subjects.

[0066] In some embodiments, treatment results in a measurable therapeutic effect following administration of the agent or a composition comprising the agent, including one or more of: reduction / reduction in obesity, reduction / reduction in the severity of obesity (e.g., reduction or inhibition of growth or obesity), reduction / reduction in symptoms and obesity-related effects, delaying the onset of symptoms and obesity-related effects, reducing the severity of obesity-related symptoms, reducing the severity of acute episodes, reducing the number of symptoms and obesity-related effects, shortening or reducing the latency of symptoms and obesity-related effects, ameliorating symptoms and obesity-related effects, reducing secondary symptoms, reducing secondary infections, preventing relapse of obesity, reducing the number or frequency of recurrent episodes, increasing the time between symptom episodes, increasing the time to sustained progression, promoting remission, inducing remission, enhancing remission, hastening recovery, increasing the effectiveness of alternative therapeutic agents or reducing resistance, and / or increasing the survival time of the affected host animal. A prophylactic effect can consist of completely or partially avoiding / inhibiting or delaying (e.g., completely or partially avoiding / inhibiting or delaying) the onset / progression of obesity or prolonging the survival of an affected host animal following administration of a treatment protocol. Treating obesity includes treating a subject already diagnosed with obesity, regardless of clinical stage or condition, delaying the onset or progression or worsening or progression of symptoms or signs of obesity, and / or preventing and / or reducing the severity of obesity.

[0067] In another aspect, provided herein is a method for treating a patient, comprising administering a peptide of interest.In any of the embodiments described herein, the subject may have obesity or excess weight, elevated BMI, elevated body fat mass, percentage, or volume, and / or excessive food intake.In any of the embodiments described herein, the subject may be obese.In any of the embodiments described herein, the subject may have excess weight.In any of the embodiments described herein, the subject may have elevated BMI.In any of the embodiments described herein, the subject may have elevated body fat mass, percentage, or volume.In any of the embodiments described herein, the subject may have excessive food intake.

[0068] Symptoms of obesity include, but are not limited to, excess body fat accumulation (especially around the waist), shortness of breath, increased sweating, snoring, inability to tolerate sudden exercise, feeling extra tired every day, back and joint pain, and skin problems (due to fluid accumulation in skin folds).

[0069] Provided is a method for treating a subject with obesity, comprising administering a GPR75 inhibitor to the subject.Also disclosed is a method for treating an overweight subject, comprising administering a GPR75 inhibitor to the subject.The present disclosure also provides a method for treating a subject with elevated BMI, comprising administering a GPR75 inhibitor to the subject.Also described is a method for treating a subject with elevated body fat mass, body fat percentage, or elevated body fat mass, comprising administering a GPR75 inhibitor to the subject.Finally, a method for treating a subject with excessive food intake, comprising administering a GPR75 inhibitor, is also disclosed.

[0070] The present disclosure also provides a method of treating a subject to prevent weight gain or maintain weight loss, the method comprising administering to the subject a GPR75 inhibitor.

[0071] In certain embodiments, methods are provided for treating a metabolic disease or disorder in a subject, comprising administering to the subject an effective amount of a GPR75 inhibitor.

[0072] As used herein, the term "metabolic disease" or "metabolic abnormality" refers to a series of abnormal conditions, also known as metabolic syndrome, that increase the risk of cerebrovascular disease and diabetes, such as increased body fat, elevated blood pressure, elevated blood glucose levels, and abnormal blood lipids. Metabolic diseases are not single diseases but are comprehensive diseases caused by genetic predisposition and environmental factors. In the present invention, metabolic diseases may be selected from the group consisting of obesity, diabetes, dyslipidemia, insulin resistance, hepatic steatosis, hypercholesterolemia, and non-alcoholic fatty liver disease, and are more preferably obesity or diabetes, but are not limited thereto.

[0073] As used herein, the term "diabetes" refers to a metabolic disease characterized by hyperglycemia (high blood glucose levels) caused by insufficient or improper insulin secretion, resulting in various symptoms and signs due to hyperglycemia and glucose excretion in the urine. Diabetes is classified into type 1 diabetes, which is caused primarily by destruction of pancreatic beta cells, resulting in insulin secretion failure, and type 2 diabetes, which is caused by insufficient insulin secretion in the body or insulin resistance, which prevents cells from responding to insulin. In the present invention, diabetes includes both type 1 diabetes and type 2 diabetes. In certain embodiments, the method further comprises administering a second therapeutic agent for treating or suppressing obesity. Non-limiting examples of therapeutic agents that treat or inhibit obesity and / or BMI increase include, but are not limited to, GLP-1R agonists, melanocortin 4 receptor (MC4R) agonists, sibutramine, orlistat, phentermine, lorcaserin, naltrexone, liraglutide, diethylpropion, bupropion, metformin, pramlintide, topiramate, and zonisamide, or combinations thereof.

[0074] The administration of the therapeutic agent for treating or suppressing obesity and / or GPR75 inhibitor can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months.The repeated administration can be the same dose or different doses.The administration can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more.For example, according to a specific administration regimen, the subject can be treated for a long period of time, for example, six months, one year, or more.

[0075] Administration of the therapeutic agent for treating or inhibiting obesity and / or GPR75 inhibitor can be by any suitable route, including, but not limited to, parenteral, intravenous, oral, lingual, buccal, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., single dose). Pharmaceutical compositions can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. Formulations will vary depending on the route of administration selected. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

[0076] The compounds may be combined with one or more pharmaceutically acceptable carriers and / or excipients that are believed to be safe and effective and can be administered to an individual without causing unwanted biological side effects or undesirable interactions. A carrier refers to all ingredients present in a pharmaceutical formulation other than the active ingredient. See, for example, Remington's Pharmaceutical Sciences, latest edition, by EW Martin Mack Pub. Co., Easton, PA. This publication discloses typical carriers that can be used in conjunction with the preparation of formulations of the compounds described herein and conventional methods for preparing pharmaceutical compositions, and is incorporated herein by reference. The most common carriers are standard carriers for administering compositions to humans. In one aspect, these include solutions such as sterile water, saline, and buffers at physiological pH. In a preferred embodiment, the composition contains a HEPES buffer. Other compounds are administered according to standard procedures used by those skilled in the art.

[0077] These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0078] In some embodiments, a therapeutic agent for treating or inhibiting obesity and / or a GPR75 inhibitor (any of the peptide ligands disclosed herein) is administered intrathecally (i.e., into the subarachnoid space of the spinal cord or into the spinal canal, or into an anatomical or potential space within the sheaths, including, by way of non-limiting example, the arachnoid membrane, of the brain or spinal cord, so that the therapeutic agent can reach the cerebrospinal fluid of the subject). In some embodiments, intrathecal administration results in the therapeutic agent acting in, but not limited to, the cerebral cortex, cerebellum, striatum, cervical, lumbar, or thoracic vertebrae. A therapeutic agent administered intrathecally may ultimately affect targets throughout the central nervous system. In some embodiments, intrathecal administration is into (the retrocerebellomedullary cisterna magna) or through the lumbar region or area. In some embodiments, intrathecal administration into the lumbar region or area delivers the therapeutic agent to the distal spinal canal.

[0079] Exemplary methods of intrathecal administration are described, for example, in Lazorthes et al., Advances in Drug Delivery Systems and Applications in Neurosurgery, 143-192. In some embodiments, intrathecal administration is via injection, bolus injection, catheter, or pump. In some embodiments, intrathecal administration is via lumbar puncture. In some embodiments, the pump is an osmotic pump. In some embodiments, the pump is implanted in the subarachnoid space of the spinal canal, under the skin in the abdomen, or behind the chest wall. In some embodiments, intrathecal administration is via an intrathecal delivery system for the therapeutic agent, including a reservoir containing a volume of the therapeutic agent and a pump configured to deliver at least a portion of the therapeutic agent contained in the reservoir. In some embodiments, intrathecal administration is via intermittent or continuous access to an implanted intrathecal drug delivery device (IDDD). In some embodiments, the therapeutic agent is an inhibitory nucleic acid molecule. In some embodiments, the amount of nucleic acid molecule or peptide molecule administered intrathecally ranges from about 10 μg to about 2 mg, about 50 μg to about 1500 μg, or about 100 μg to about 1000 μg. In some embodiments, the therapeutic agent is disposed in a pharmaceutical composition. In some embodiments, the pharmaceutical composition does not contain a preservative.

[0080] Throughout this document, values ​​expressed in range format should be interpreted flexibly to include not only the numerical values ​​expressly stated as the limits of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly stated. For example, the range "about 0.1% to about 5%" or "about 0.1% to about 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3%, 4%) and subranges within the stated range (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). Parenteral formulations

[0081] The peptides described herein can be formulated for parenteral administration, including intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intravitreal, intratumoral, intramuscular, subcutaneous, intralingual, subconjunctival, intravesical, intrapericardial, and intraumbilical administration by injection and infusion.

[0082] Parenteral preparations can be prepared as aqueous compositions by using techniques known in the art.Typically, these compositions can be prepared as injectable preparations, for example, solution or suspension; the solid form that is suitable for being used to prepare solution or suspension by adding reconstitution medium before injection; emulsion, such as water-in-oil (w / o) emulsion, oil-in-water (o / w) emulsion and their microemulsions, liposome or emulsosome.

[0083] For intravenous administration, the composition is prepared in a sterile isotonic aqueous buffer solution. In certain embodiments, the composition is prepared in a solution containing HEPES buffer. If necessary, the composition can also contain a solubilizing agent. The components of the composition are supplied in unit dosage form, for example, as a dry lyophilized powder or a concentrated solution, separately or mixed together in a sealed container such as an ampoule or sachet indicating the amount of active agent. When the composition is administered by infusion, it can be administered using an infusion bottle containing sterile pharmaceutical water or saline. When the composition is administered by injection, an ampoule of sterile water or saline can be provided so that the components can be mixed before injection.

[0084] The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), oil, for example, vegetable oil (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and / or by using surfactants. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride.

[0085] Solutions and dispersions of the active compounds as free acids or bases or pharmacologically acceptable salts thereof can be prepared in water or other solvent or dispersion medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersing agents, emulsifying agents, pH adjusters, viscosity adjusters, and combinations thereof.

[0086] Suitable surfactants include anionic, cationic, amphoteric, or nonionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate, and sulfate ions. Examples of anionic surfactants include long-chain alkylsulfonates, such as sodium, potassium, and ammonium, and alkylarylsulfonates, such as sodium dodecylbenzenesulfonate; dialkylsodium sulfosuccinates, such as sodium dodecylbenzenesulfonate; dialkylsodium sulfosuccinates, such as sodium bis(2-ethylthio)sulfosuccinate; and alkylsulfates, such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyldimethylbenzylammonium chloride, polyoxyethylene, and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl 4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine.

[0087] The formulation may contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. The formulation may also contain an antioxidant to prevent the decomposition of the active agent.

[0088] The formulation is typically buffered to a pH of 3-8 upon reconstitution for parenteral administration. Suitable buffers include, but are not limited to, phosphate buffer, acetate buffer, and citrate buffer. In certain embodiments, the formulation comprises a HEPES buffer.

[0089] Water-soluble polymers are commonly used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, polyethylene glycol, etc.

[0090] Sterile injectable solution can be prepared by mixing the required amount of active compound in suitable solvent or dispersion medium, and optionally with one or more of the above-mentioned additives, and then filter sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains basic dispersion medium and other necessary ingredients from the above-mentioned.For the sterile powder to prepare sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, and obtains powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized by filtering.Powder can be prepared in a way that makes particles porous, which can increase the solubility of particles.The method of producing porous particles is well known in the art.

[0091] The parenteral formulations described herein can be formulated for controlled release, including immediate release, delayed release, sustained release, pulsatile release, and combinations thereof.

[0092] Nanoparticles and microparticles For parenteral administration, one or more compounds, and optionally one or more additional active agents, can be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release of the compounds and / or one or more additional active agents. In forms in which the formulation includes two or more peptides, the peptides can be formulated for the same type of controlled release (e.g., delayed, extended, immediate, or pulsatile), or the peptides can be independently formulated for different types of release (e.g., immediate and delayed, immediate and extended, delayed and extended, delayed and pulsatile, etc.).

[0093] For example, the compound and / or one or more additional active agents can be incorporated into polymeric microparticles that provide controlled release of the peptide. Release of the peptide is controlled by diffusion of the protein from the microparticle and / or degradation of the polymeric microparticle by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives.

[0094] Polymers that dissolve slowly and form gels in aqueous environments, such as hydroxypropylmethylcellulose and polyethylene oxide, are also suitable materials for protein-containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyric acid (PHB) and copolymers thereof, poly-4-hydroxybutyric acid (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.

[0095] Alternatively, proteins can be incorporated into microparticles prepared from materials that are insoluble in aqueous solutions or slowly soluble in aqueous solutions but decompose in the digestive tract by means including enzymatic degradation, the surfactant action of bile acids, and / or mechanical erosion. As used herein, the term "slowly soluble in water" refers to a material that does not dissolve in water within 30 minutes. Preferred examples include fats, fatty substances, waxes, wax-like substances, and mixtures thereof. Suitable fats and fatty substances include fatty alcohols (such as lauryl, myristylstearyl, cetyl, or cetostearyl alcohol), fatty acids and their derivatives, including, but not limited to, fatty acid esters, fatty acid glycerides (mono-, di-, and triglycerides), and hydrogenated fats and oils. Specific examples include, but are not limited to, hydrogenated vegetable oil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oils available under the trade name Sterotex®, stearic acid, cocoa butter, stearyl alcohol, and the like. Suitable waxes and wax-like substances include natural or synthetic waxes, hydrocarbons, and conventional waxes. Specific examples of waxes include beeswax, glycowax, castor oil wax, carnauba wax, paraffin, candelilla wax, etc. As used herein, wax-like substances are defined as any substance that is normally solid at room temperature and has a melting point of about 30-300°C.

[0096] In some cases, it may be desirable to change the rate at which water penetrates the microparticles. For this purpose, a rate-controlling agent (wicking agent) can be added along with the oils and waxes mentioned above. Examples of rate-controlling materials include certain starch derivatives (e.g., waxy maltodextrin and drum-dried cornstarch), cellulose derivatives (e.g., hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, and carboxymethylcellulose), alginic acid, lactose, and talc. Furthermore, a pharmaceutically acceptable surfactant (e.g., lecithin) may be added to promote the disintegration of such microparticles.

[0097] Water-insoluble proteins, such as zein, can also be used as materials for forming protein-containing microparticles. Furthermore, water-soluble proteins, polysaccharides, and combinations thereof can be formulated into microparticles with peptides and then crosslinked to form an insoluble network. For example, cyclodextrins can be complexed with individual drug molecules and then crosslinked. Nano- and micro-particle manufacturing method

[0098] Encapsulation or incorporation of a drug into a carrier material to produce drug-containing microparticles can be achieved by known pharmaceutical formulation techniques. When formulated with oils, waxes, or wax-like substances, the carrier material is generally heated above its melting point, and the drug is added to form a mixture containing drug particles suspended in the carrier material, drug dissolved in the carrier material, or a mixture thereof. Microparticles can be formulated by several methods, including, but not limited to, solidification, extrusion, spray cooling, and aqueous dispersion. In a preferred process, wax is heated above its melting temperature, the drug is added, and the molten wax-drug mixture is allowed to solidify under constant agitation while cooling. Alternatively, the molten wax-drug mixture can be extruded and spheronized to form pellets or beads. These processes are known in the art.

[0099] Depending on the carrier material, it may be desirable to prepare drug-containing microparticles using a solvent evaporation method, in which the drug and carrier material are co-dissolved in a mutual solvent, after which the microparticles can be produced by several techniques, including, but not limited to, forming an emulsion in water or other suitable medium, spray drying, or evaporating the solvent from a bulk solution and milling the resulting material.

[0100] In some embodiments, the particulate drug is uniformly dispersed in a water-insoluble or slowly water-soluble substance. To minimize the size of the drug particles in the composition, the drug powder itself may be milled to produce fine particles before formulation. For this purpose, a jet milling process known in the pharmaceutical arts may be used. In other embodiments, the particulate drug is uniformly dispersed in a wax or wax-like substance by heating the wax or wax-like substance above its melting point and adding the drug particles while stirring the mixture. In this case, a pharmaceutically acceptable surfactant may be added to the mixture to promote dispersion of the drug particles.

[0101] Particles can also be coated with one or more modified-release coatings. Solid esters of fatty acids hydrolyzed by lipases can be spray-coated onto microparticles or drug particles. Zein is an example of a naturally occurring, water-insoluble protein. It can be coated onto drug-loaded microparticles or drug particles using spray coating or wet granulation techniques. In addition to naturally water-insoluble substances, digestive enzyme substrates also include those that form water-insoluble networks through crosslinking. Many methods for crosslinking proteins have been reported, initiated by both chemical and physical means. One of the most common methods for achieving crosslinking is the use of chemical crosslinkers. Examples of chemical crosslinkers include aldehydes (glutaraldehyde, formaldehyde), epoxy compounds, carbodiimides, and genipin. In addition to these crosslinkers, oxidized and native sugars have been used to crosslink gelatin. Crosslinking can also be achieved by enzymatic methods. For example, transglutaminase has been approved as a GRAS substance for crosslinking in seafood. Finally, crosslinking can be initiated by physical means such as heat treatment, UV irradiation, or gamma irradiation.

[0102] To prepare a coating layer of crosslinked protein surrounding drug-containing microparticles or drug particles, a water-soluble protein can be spray-coated onto the microparticles and then crosslinked by one of the methods described above. Alternatively, drug-containing microparticles can be microencapsulated in a protein by coacervation-phase separation (e.g., by adding salt) and then crosslinked. Suitable proteins for this purpose include gelatin, albumin, casein, and gluten.

[0103] Polysaccharides can also be cross-linked to form water-insoluble networks. In many polysaccharides, the main polymer chains can be cross-linked by reaction with calcium salts or polyvalent cations. Pectin, alginate, dextran, amylose, and guar gum cross-link in the presence of polyvalent cations. Complexes can also be formed between polysaccharides with opposite charges; for example, pectin and chitosan can complex through electrostatic interactions.

[0104] Injectable / implantable preparations The compounds described herein can be incorporated into injectable / implantable solid or semi-solid implants, such as polymeric implants. In some embodiments, the compounds are incorporated into polymers that are liquid or pasty at room temperature but increase in viscosity and form semi-solids or solids upon contact with aqueous media, such as physiological fluids. Exemplary polymers include, but are not limited to, hydroxyalkanoic acid polyesters, derived from the copolymerization of at least one unsaturated hydroxy fatty acid copolymerized with a hydroxyalkanoic acid. The polymer can be melted, mixed with an active agent, and cast or injection molded into a device. Such melt processing requires a polymer with a melting point below the temperature at which the polymer decomposes or reacts with the feed material. Devices can also be prepared by solvent casting, in which a polymer is dissolved in a solvent, the drug is dissolved or dispersed in the polymer solution, and the solvent is then evaporated. Solvent processing requires that the polymer be soluble in an organic solvent. Another method is compression molding of a mixed powder of polymer and drug, or polymer particles loaded with an active agent.

[0105] Alternatively, the compounds can be incorporated into a polymer matrix and molded, compressed, or extruded into a solid device at room temperature. For example, the compounds can be incorporated into biodegradable polymers such as polyanhydrides, polyhydroalkanoates (PHAs), PLA, PGA, PLGA, polycaprolactones, polyesters, polyamides, polyorthoesters, polyphosphazenes, proteins, and polysaccharides such as collagen, hyaluronic acid, albumin, gelatin, and combinations thereof, and compressed into a solid device such as a disk or extruded into a device such as a rod.

[0106] The release of one or more compounds from the implant can be varied by the choice of polymer, the molecular weight of the polymer, and / or modifying the polymer to facilitate degradation (such as by forming pores and / or incorporating hydrolyzable linkages). Methods for altering the properties of biodegradable polymers to alter the release profile of compounds from the implant are well known in the art.

[0107] Enteral / Oral / Lingual Formulations Oral / lingual formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, sodium saccharin, starch, magnesium stearate, cellulose, magnesium carbonate, etc. Such compositions will contain a therapeutically effective amount of the compound and / or antibiotic with an appropriate amount of carrier to provide an appropriate form for the patient based on the mode of administration being used.

[0108] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, lozenges, etc. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can be prepared using techniques well known in the art as hard or soft capsule shells that can enclose liquid, solid, or semi-solid fills.

[0109] The formulation can be prepared using a pharmaceutically acceptable carrier.The term "carrier" as generally used herein includes but is not limited to diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof.

[0110] The carrier also includes all components of the coating composition, including plasticizers, pigments, colorants, stabilizers, lubricants, and the like.

[0111] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, methacrylic resins commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, polysaccharides.

[0112] Additionally, the coating agent may include conventional carriers such as plasticizers, pigments, colorants, lubricants, stabilizers, pore formers, surfactants, and the like.

[0113] "Diluents," also known as "fillers," are typically required to increase the bulk of a solid dosage form to provide a practical size for tablet compression or bead or granule formation. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starch, pregelatinized starch, silicon dioxide, titanium oxide, magnesium aluminum silicate, powdered sugar, and the like.

[0114] "Binders" are used to impart cohesion to solid dosage forms, preventing tablets, beads, or granules from crumbling after formulation. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose, and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia and tragacanth, celluloses such as sodium alginate, hydroxypropylmethylcellulose, hydroxypropylcellulose, and ethylcellulose, and synthetic polymers such as veegum, acrylic and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylic acid copolymers, aminoalkyl methacrylic acid copolymers, polyacrylic acid / polymethacrylic acid, and polyvinylpyrrolidone.

[0115] "Lubricants" are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, mineral oil, and the like.

[0116] "Disintegrants" are used to facilitate the breakup or "break-up" of a dosage form after administration and typically include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clay, cellulose, arginine, gums, or cross-linked polymers such as cross-linked PVP (Polyplasdone® XL from GAF Chemical Corp).

[0117] "Stabilizers" are used to inhibit or slow down decomposition reactions of pharmaceuticals, such as oxidation. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT), ascorbic acid, its salts and esters, vitamin E, tocopherol and its salts, sulfites such as sodium metabisulfite, cysteine ​​and its derivatives, citric acid, propyl gallate, and butylated hydroxyanisole (BHA).

[0118] Oral dosage forms such as capsules, tablets, solutions, and suspensions can be formulated for controlled release. For example, one or more compounds and optionally one or more additional active agents can be formulated into nanoparticles, microparticles, and combinations thereof, and then encapsulated in soft or hard gelatin or non-gelatin capsules, or dispersed in a dispersion medium to form oral suspensions or syrups. Particles can be formed with a drug and a release-controlling polymer or matrix. Alternatively, drug particles can be coated with one or more release-controlling coatings before being incorporated into a finished dosage form.

[0119] In another embodiment, one or more compounds and optionally one or more additional active agents are dispersed in a matrix material that gels or emulsifies upon contact with an aqueous medium, such as physiological fluid. In the gel form, the matrix swells to encase the active agent, slowly releasing it over time by diffusion or degradation of the matrix material. Such matrices can be formulated as tablets or as fillers in hard or soft capsules.

[0120] In yet another embodiment, one or more compounds, and optionally one or more additional active agents, are formulated into a commercial oral dosage form, such as a tablet or capsule, and the solid dosage form is coated with one or more controlled-release coatings, such as delayed-release or sustained-release coatings. The coatings may also include the compounds and / or additional active agents.

[0121] The following materials and methods are provided to facilitate the practice of the present invention.

[0122] Peptide synthesis and purification Solid-phase peptide synthesis was performed on ProTide Rink amide resin (CEM Corporation cat # R002) using a microwave-assisted CEM Liberty Blue peptide synthesizer (Matthews, NC). Fmoc-protected amino acids were coupled to the resin using 0.25 M Oxyma Pure (CEM Corporation cat # S001) and 0.125 M N,N'-diisopropylcarbodiimide (Sigma-Aldrich cat # D125407) as the activator and activated base, respectively. Fmoc was removed during coupling with 20% piperidine (Sigma-Aldrich cat # 8.22299.0500). Global deprotection and cleavage of the peptide from the solid support resin was achieved using a CEM Razor instrument by incubation for 40 min at 40 °C in a mixture of 95% TFA (Sigma-Aldrich cat # 8.08260.2501), 2.5% TIPS (Sigma-Aldrich cat # 233781), and 2.5% water. The peptide was purified on an Agilent 1200 Series high-performance liquid chromatography (HPLC) instrument (10-75% HPLC-grade acetonitrile (VWR cat # BDH83639.400)) using an Agilent Zorbax C18 column (5 μm, 9.4 x 250 mm) at a flow rate of 2 mL / min for 20 min, monitoring at 280 nm. Binding analysis of peptides in GPR75

[0123] SU75-36 binding of human GPR75 (372-540 aa region; available at antibodies-online.com, cat # ABIN5709609) was measured using His-tagged GPR75 bound to an NTA-coated gold sensor (Nicoya cat # SEN-AU-100-10-NTA, range = 4.08-204 μM) in HBSS (in-house) at a flow rate of 20 μL / min using a Nicoya OpenSPR instrument. Data were fitted with a global one-to-one model using Nicoya OpenSPR software. Binding analysis of peptides in hGLP-1R

[0124] Binding of SU75-36, SU75-37, Ex-4, and ODN to human GLP-1R (region 21-139aa; available at rndsystems.com, cat # 10956-GL) was measured using His-tagged hGLP-1R coupled to an NTA-coated gold sensor (Nicoya cat # SEN-AU-100-10-NTA) in HBSS (in-house) at a flow rate of 20 μL / min on a Nicoya Open SPR instrument. Data were fitted with a global one-to-one model using Nicoya OpenSPR software. Ex-4 was used as a positive control, and ODN was used as a negative control.

[0125] Circular dichroism (CD) spectroscopy Peptides were prepared at 40 μM in 0.5% saline (pH 7.4) for folding state analysis using a Chirascan VX (Applied Photophysics, Leatherhead, Surrey, UK) spectropolarimeter. Samples were analyzed as duplicate sets of four replicates using a 1 cm quartz cell, a 200–260 nm measurement range, a 100 nm / min scan speed, a 1 nm bandwidth, a 4 s response time, and a 1 nm data pitch. Averaged data output was calculated as ΔE(M -1 cm -1 ) was converted to molar ellipticity to obtain the corresponding percent helicity value.

[0126] animal Adult male Sprague-Dawley rats (Charles River) were housed individually in a temperature- and humidity-controlled satellite vivarium under a 12-hour light:12-hour dark cycle and provided with ad libitum access to water and a standard diet (5001, LabDiet) or a 60% high-fat diet (HFD; D12492, Research Diets). Kaolin pellets (K50001, Research Diets) were also available ad libitum as needed. Rats were exposed to kaolin for at least 5 days before measuring kaolin intake in the pica test. For all feeding tests, except those performed on the bioDAQ, rats were housed in hanging wire cages to allow accurate measurement of food spillage. Experiments were conducted in accordance with the National Institutes for Health Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the University of Pennsylvania Institutional Animal Care and Use Committee.

[0127] surgery For cannula implantation, rats were anesthetized with an intraperitoneal injection of a mixture (KAX) containing ketamine (90 mg / kg, Butler Animal Health Supply), xylazine (2.7 mg / kg, Anased), and acepromazine (0.64 mg / kg, Butler Animal Health Supply) and placed in a stereotaxic apparatus. Each rat received a 26-gauge guide cannula (Plastics One) stereotaxically implanted into the fourth ventricle (guide cannula coordinates: midline, 2.5 mm anterior to the occipital suture, 5.2 mm ventral to the skull; internal cannula coordinates: 7.2 mm ventral to the skull) or lateral ventricle (guide cannula coordinates: 1.5 mm lateral to the midline, 0.9 mm posterior to the sternum, 1.8 mm ventral to the skull; internal cannula coordinates: 3.8 mm ventral to the skull). For all cannulae, a dummy cannula (not protruding beyond the guide) was inserted into the guide cannula and left in place until fluid infusion was administered. In all surgeries, rats received postoperative temperature support and received analgesics (2 mg / kg meloxicam) immediately after surgery and for 2 days postoperatively. Studies on food and kaolin intake

[0128] In all studies measuring food intake after drug administration, central injections were made in a volume of 2 μL using a Hamilton syringe terminating in a syringe tip extending 2.0 mm outward from the guide cannula. On acute treatment days, rats were food-deprived for 2 h before the dark cycle, and injections were given immediately before the onset of the dark cycle. Food intake and kaolin intake were measured 1, 3, 6, and 24 h after the end of injection, and food crumbs were weighed and accounted for between time points. Body weights were measured during injection and 24 h after injection. Injection treatments were administered in a balanced within-subject design and separated by at least 72 h.

[0129] Pharmaceuticals All drugs used in these studies (SUODN36 (SU75-36) and SUODN37 (SU75-37)) were synthesized by the Doyle laboratory at Syracuse University. In both cases, the drugs were dissolved in artificial cerebrospinal fluid (aCSF, Harvard Apparatus). The sequences of SU75-36 and SU75-37 are shown in Table 1.

[0130] The following examples are provided to illustrate certain embodiments of the present invention, but are not intended to limit the invention in any way. [Example]

[0131] Example 1: Peptide ligands that bind to GPR75

[0132] Here, we describe novel non-natural peptide ligands for the highly sought-after orphan GPR75, which has become a major drug discovery target for the pharmaceutical industry due to its association with obesity and metabolic diseases.

[0133] The non-naturally occurring peptides were prepared as described above. Table 4 lists two novel non-natural peptide sequences that antagonize GPR75. [Table 4]

[0134] The synthesis and purity of the peptides were confirmed using high-performance liquid chromatography (HPLC). Figure 1A shows the synthesis of SU75-36 with 100% purity, and Figure 1B shows the synthesis of SU75-37 with 98.3% purity.

[0135] Next, we modeled the binding of SU75-36 to GPR75 using HPEPDOCK. Notably, blind in silico docking of SU75-36 / GPR75 receptor using HPEPDOCK yielded a docking score of 0.884. The successful administration of SU75-37 as shown herein indicates that SU75-37 binds similarly to the GPR75 receptor.

[0136] The binding of SU75-36 to GPR75 was further analyzed using a surface plasmon resonance (SPR) assay to track the binding of SU75-36 to GPR75 over time. The results showed that SU75-36 exhibited a K of 7.76 μM. D The assay was further analyzed using Nicoya OpenSPR software, which showed optimal fit parameters using a one-to-one model. The data obtained with this software are shown in Tables 5 and 6. D is 7.73 x 10 -6 M (7.76 μM). [Table 5]

[0137] [Table 6]

[0138] The SPR assay was repeated to measure the binding of SU75-37 to GPR75 over time. This assay revealed that SU75-37 had a K of 23.8 μM. D SU75-36 was shown to bind to GPR75 (Figure 4). Similar to SU75-36, this assay was further analyzed using Nicoya OpenSPR software. Data obtained with this software are shown in Table 7. D is 2.38 x 10 -5 M (23.8 μM). [Table 7]

[0139] To confirm the specificity of peptide ligand binding, the SPR assay was repeated with both SU75-36 and SU75-37, and binding at the hGLP-1R was determined using Ex-4 and ODN as positive and negative controls, respectively. SU75-37 did not bind to the hGLP-1R receptor at all (Figure 5). SU75-36 showed binding ability to the hGLP-1R receptor, but K D 182 μM, indicating poor binding to this receptor (Figure 7).

[0140] To analyze the folding states of SU75-36 and SU75-37, circular dichroism (CD) spectroscopy was used. Each peptide exhibited a typical α-helical spectrum, with a helicity percentage of over 20% (Figure 9).

[0141] Example 2: Administration of peptide ligands to rats Here, we describe the administration of novel non-natural peptide ligands as GPR75 inhibitors for the treatment of obesity.

[0142] Diet-induced obese (DIO) rats (n = 26) were intracerebroventricularly injected into the fourth ventricle with 20 μg of SU75-36, 100 μg of SU75-36, 200 μg of SU75-36, or 20 μg of SU75-37 and observed for 24 hours. One group of rats received vehicle without a GPR75 inhibitor as a negative control. All rats had free access to water and a 60% high-fat diet (HFD). Food intake was monitored for each rat at 1, 3, 6, and 24 hours after GPR75 inhibitor administration (Figure 7A). Rats in each group treated with the GPR75 inhibitor showed a significant decrease in food intake compared to the control group. Rats treated with 200 μg of SU75-36 showed a greater decrease than any other group.

[0143] The rats were weighed at the start of the experiment and 24 hours after administration of the GPR75 inhibitor. The weight change for each rat over the 24-hour period was then calculated. Rats in the control group showed a slight increase in weight after 24 hours. Rats administered any dose of SU75-36 showed a significant decrease in weight after 24 hours (Figure 7B).

[0144] In a separate experiment, standard diet-maintained (n = 10) and diet-induced obese (DIO) rats (n = 12) were intracerebroventricularly injected with 20 μg of SU75-36, 200 μg of SU75-36, or 20 μg of SU75-37 into the lateral ventricle and observed for 24 hours. One group of rats received vehicle without a GPR75 inhibitor as a negative control. All rats had ad libitum access to water, kaolin pellets to assess palatability, and food (either a standard chow diet (standard diet) or a 60% high-fat diet (HFD)). At 1, 3, 6, and 24 hours after GPR75 inhibitor administration, each rat's standard chow intake (Figure 8A) or HFD intake (Figure 8B), as well as kaolin intake in standard diet-fed rats (Figure 8C) and HFD-fed rats (Figure 8D), were monitored. Rats in each group treated with GPR75 inhibitors showed a significant decrease in food intake compared to controls. Rats treated with 200 μg of SU75-36 showed an even greater decrease compared to all other groups. Furthermore, rats showed no ingestion of kaolin, an established model of fatigue and nausea. This indicates that antagonizing GPR75 with these novel antagonists suppresses energy balance without causing nausea / fatigue.

[0145] The rats were weighed at the start of the experiment and 24 hours after administration of the GPR75 inhibitor. The weight change for each rat over 24 hours was then calculated. Control rats fed an HFD diet showed a slight increase in weight after 24 hours. Rats treated with either SU75-36 or SU75-37 at any dose showed a significant decrease in weight after 24 hours when fed a standard diet or an HFD diet (Figures 8E and 8F).

[0146] Example 3: Administration of peptide ligands in HEPES buffer to rats Next, we analyzed the appetite suppressant effects of the novel non-naturally occurring peptide ligands in different solutions, including HEPES buffer and DMSO.

[0147] Diet-induced obese (DIO) rats (n = 4) were intracerebroventricularly injected with vehicle or 150 μg / 3 μL of SU75-36 in HEPES buffer and observed for 24 hours. One group of rats received a negative control, a vehicle solution of 50% DMSO in HEPES. All rats had free access to water and a 60% HFD. Food intake was monitored at 1, 3, 6, and 24 hours after administration (Figure 10A). Rats in each SU75-36-treated group showed a significant decrease in food intake compared to the control group. Rats in the SU75-36-treated HEPES buffer showed a greater decrease than any other group.

[0148] The rats were weighed at the start of the experiment and 24 hours after administration. The weight change for each rat over 24 hours was then calculated. Rats in the control group showed a slight increase in weight after 24 hours. Rats administered SU75-36 showed a significant decrease in weight after 24 hours. Rats administered SU75-36 together with HEPES buffer showed a further decrease compared to all other groups (Figure 10B).

[0149] In another experiment, diet-induced obese (DIO) rats (n = 4) were intracerebroventricularly injected with SU75-37 at 133 μg / 2 μL in HEPES buffer and observed for 24 hours. One group of rats received vehicle without a GPR75 inhibitor as a negative control. All rats had free access to water and a 60% high-fat diet (HFD). SU75-37 was optimized using acetate precipitation rather than TFA precipitation to maintain a neutral pH > 4.5 in HEPES buffer. Food intake was monitored for each rat at 1, 3, 6, and 24 hours after administration (Figure 11A). Rats administered SU75-37 in HEPES buffer showed significantly reduced food intake compared to controls.

[0150] The rats were weighed at the start of the experiment and 24 hours after administration. The weight change for each rat over 24 hours was then calculated. Rats in the control group showed a slight increase in weight after 24 hours. Rats administered SU75-37 in HEPES buffer showed a significant decrease in weight after 24 hours (Figure 11B).

[0151] Example 4: Peripheral administration of peptide ligand to rats Next, we analyzed the appetite suppressant effects of peripherally administered novel non-naturally occurring peptide ligands.

[0152] DIO mice were intraperipherally injected with 0.5 mg / kg or 5.0 mg / kg of SU75-36 or SU75-37 together with vehicle and observed for 24 hours. One group received vehicle as a negative control. All mice had free access to water and a 60% HFD. Food intake for each mouse was monitored 3 and 24 hours after administration (Figures 12A and 12C). Mice administered 5 mg / kg of SU75-36 or SU75-37 showed a significant decrease in food intake compared to controls. The mice were weighed at the start of the experiment and 24 hours after administration. The weight change for each mouse over 24 hours was then calculated. Mice in the control group showed either an increase or a slight decrease in weight over 24 hours. Mice administered SU75-36 showed a significant decrease in weight over 24 hours (Figures 12B and 12D).

[0153] This experiment was repeated with a higher dose of SU75-37 (10 mg / kg). At this higher dose, mice showed a significant decrease in food intake compared to controls (Figure 13A). Mice also showed a significant decrease in body weight after 24 hours (Figure 13B).

[0154] Example 5: Administration of peptide ligands to human patients The above information can be clinically applied to patients for therapeutic intervention. A preferred embodiment of the present invention involves clinically applying the information described herein to patients. This may occur after a patient presents to a clinic and exhibits symptoms of obesity or metabolic disorders. A non-limiting example of an effective dose range for the therapeutic compounds described herein is about 0.1 to 5,000 mg / kg body weight / day. One of ordinary skill in the art would be able to examine the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0155] The therapeutic peptides described herein have been shown to be well tolerated, and symptoms were assessed using a clinical scoring system. Treatment protocols can also optionally include the administration of an effective amount of one or more therapeutic agents to treat or inhibit obesity. Such agents include, but are not limited to, bupropion-naltrexone, liraglutide (Saxenda), orlistat (Xenical, Alli), Wegovy, and phentermine-topiramate. Treatment protocols can also optionally include lifestyle modifications or surgery to help manage weight gain.

[0156] While certain features of the invention have been described herein, many modifications, substitutions, changes, and equivalents will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

1. An isolated or purified peptide having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a functional sequence variant thereof having at least 95% identity thereto.

2. The peptide of claim 1 , wherein the peptide has anti-obesity activity.

3. 3. The peptide of claim 1 or claim 2, comprising one or more modified amino acids selected from Gln (alkyne), Ala (alkyne), Gly (alkyne), Lys (N3) and the modified amino acids listed in Table 2 for bioconjugation, including lipidation and / or fluorescent tagging.

4. 4. The peptide of claim 3, wherein the modified amino acids facilitate bioconjugation of one or more agents selected from N- and / or C-terminal protection moieties, lipids, recombinant FC-peptides, cell-penetrating peptides, and enhance one or more of the function, stability, and bioavailability of the peptide.

5. A composition comprising the peptide of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. 6. The composition of claim 5, further comprising at least one of a HEPES buffer and an acetate salt.

7. The composition of claim 5 , wherein the composition is formulated to maintain a neutral pH.

8. The composition of any one of claims 5 to 7, wherein the composition is formulated for intraventricular injection or peripheral administration.

9. A nucleic acid sequence encoding the peptide of claim 1 or claim 2.

10. 10. The nucleic acid of claim 9, encapsulated in a liposome or extracellular vesicle, or attached to a nanoparticle or lipid nanoparticle.

11. A vector comprising the nucleic acid of claim 9.

12. The vector of claim 11, which is a plasmid vector, a lentiviral vector, or an AAV vector.

13. 13. The vector of claim 11 or 12, encapsulated in a liposome or extracellular vesicle, or attached to a nanoparticle or lipid nanoparticle.

14. A method for treating obesity in a subject in need thereof, comprising administering an effective amount of a peptide according to any one of claims 1 to 13.

15. A method for treating a metabolic disease or disorder in a subject in need thereof, comprising administering an effective amount of a peptide according to any one of claims 1 to 13.

16. 16. The method of claim 15, wherein the metabolic disease or disorder is selected from obesity, diabetes, dyslipidemia, insulin resistance, hepatic steatosis, hypercholesterolemia, and non-alcoholic fatty liver disease.

17. 17. The method of claim 16, wherein the diabetes is selected from type 1 diabetes or type 2 diabetes.

18. The method of any one of claims 14 to 17, further comprising administering a second therapeutic agent that treats or inhibits obesity.

19. 19. The method of any one of claims 14 to 18, further comprising administering a lifestyle intervention regarding diet and / or physical activity to the subject.

20. 20. The method of any one of claims 14 to 19, wherein the patient has reduced food intake 1, 3, 6, and / or 24 hours after administration of the peptide compared to untreated controls.

21. 21. The method of any one of claims 14 to 20, wherein the patient loses weight after administration of the peptide.

22. 22. The method of any one of claims 14-21, further comprising evaluating the patient for reduction in symptoms of obesity, diabetes, dyslipidemia, insulin resistance, hepatic steatosis, hypercholesterolemia, or non-alcoholic fatty liver disease.

23. 23. The method of any one of claims 14 to 22, wherein the administration is via a route selected from systemic, intramuscular, topical, oral, parenteral, transdermal patch, aerosolized, pulmonary, ocular, buccal, and lingual.

24. 24. The method of claim 23, wherein the administration is intraventricular injection or peripheral administration.

25. 20. The method of claim 18, wherein the peptide and the second therapeutic agent act synergistically to increase weight loss.

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