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WO2025175250A1 - Analogues de la mélanocortine d'origine non naturelle administrables par voie orale et leurs utilisations pour moduler la perte de poids - Google Patents

Analogues de la mélanocortine d'origine non naturelle administrables par voie orale et leurs utilisations pour moduler la perte de poids

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Publication number
WO2025175250A1
WO2025175250A1 PCT/US2025/016151 US2025016151W WO2025175250A1 WO 2025175250 A1 WO2025175250 A1 WO 2025175250A1 US 2025016151 W US2025016151 W US 2025016151W WO 2025175250 A1 WO2025175250 A1 WO 2025175250A1
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WO
WIPO (PCT)
Prior art keywords
dphe
dpro
naturally occurring
arg
asp
Prior art date
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PCT/US2025/016151
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English (en)
Inventor
Russell Potterfield
Jordan DELEV
Daniel Marks
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Endevica Bio Inc
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Endevica Bio Inc
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Publication of WO2025175250A1 publication Critical patent/WO2025175250A1/fr
Pending legal-status Critical Current
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • weight loss agents have been developed in an attempt to treat such conditions. Although some weight loss agents have been approved for ongoing use, many are not sufficiently effective in achieving desired amounts or types of weight loss. Further, commonly used weight loss agents are administered subcutaneously, requiring self-administered injections on a daily or weekly basis which may cause administration site irritation and discomfort.
  • the present technology comprises methods of reducing body weight and/or fat mass in a subject in need thereof, comprising administering to the subject a non-naturally occurring melanocortin analog.
  • the present technology comprises a method of reducing body weight and/or fat mass in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I): X 1 -R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8 -R 9 -Y 1 -Y 2 -Y 3 -Y 4 (I), wherein: X 1 is absent or is selected from the group consisting of norleucine (Nle), arginine (Arg), and D-arginine (dArg); R 1 is selected from the group consisting of Nle, Arg, dArg, aspartic acid (Asp), alanine (Ala), lysine (Lys), D-lysine (dLys), histidine (His), and D-histidine (dHi
  • the present technology comprises a method of reducing body weight and/or fat mass in an obese subject, comprising orally administering a non- naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I)
  • the present technology comprises a method of treating, preventing, or reducing hyperplasia in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of treating, preventing, or reducing hypothalamic obesity in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of treating, preventing, or reducing a proopiomelanocortin (POMC) deficiency in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • POMC proopiomelanocortin
  • the present technology comprises a method of treating, preventing, or reducing a leptin deficiency in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of treating, preventing, or reducing a syndromic obesity in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of treating, preventing, or reducing non-alcoholic steatohepatitis (NASH) in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • NASH non-alcoholic steatohepatitis
  • the present technology comprises a method of treating, preventing, or reducing hyperinsulinism in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of treating, preventing, or reducing a cardiovascular disease in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of treating, preventing, or reducing osteoarthritis in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of treating, preventing, or reducing a cancer in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of treating, preventing, or reducing erectile dysfunction in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of treating, preventing, or reducing body weight gain after a weight loss in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of maintaining body weight after a weight loss in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of treating, preventing, or reducing fat mass gain after a weight loss in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of treating, preventing, or reducing glucose intolerance and/or diabetes mellitus in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of reducing obesity-related inflammation in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of preserving or improving kidney function in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin 4 receptor agonist to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the present technology comprises a method of reducing body weight and/or fat mass in a subject in need thereof, comprising orally administering about 15 mg/kg to about 100 mg/kg of a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (IF).
  • the present technology comprises a method of treating, preventing, or reducing glucose intolerance and/or diabetes mellitus in a subject in need thereof, comprising orally administering about 15 mg/kg to about 100 mg/kg of a non- naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (IF).
  • a non-naturally occurring melanocortin analog comprising a sequence of Formula (IF).
  • the present technology comprises a method of reducing obesity-related inflammation in a subject in need thereof, comprising orally administering about 15 mg/kg to about 100 mg/kg of a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (IF).
  • a method of preserving or improving kidney function in a subject in need thereof comprising orally administering about 15 mg/kg to about 100 mg/kg of a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (IF).
  • FIG. 1 illustrates 24-hour plasma concentrations of non-naturally occurring melanocortin analogs of the present technology following oral administration of Compound Ac-Nle-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 14; C2) to Cynomolgus Monkeys at 30.0 mg/kg.
  • FIGS. 1 illustrates 24-hour plasma concentrations of non-naturally occurring melanocortin analogs of the present technology following oral administration of Compound Ac-Nle-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 14; C2) to Cynomolgus Monkeys at 30.0 mg/kg.
  • 2A-2F illustrates 24-hour plasma concentrations of non-naturally occurring melanocortin analogs of the present technology following PO administration of Compound E (Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2 (SEQ ID NO: 43; O10)) to Cynomolgus Monkeys at 3 mg/kg, 10.0 mg/kg, 30.0 mg/kg, or 60 mg/kg. [0032] FIGS.
  • FIGS. 4A-4G illustrate plasma and CSF concentrations of non-naturally occurring melanocortin analogs of the present technology.
  • FIGS. 4A-4G illustrate 24-hour plasma and CSF concentrations of non-naturally occurring melanocortin analogs following IP administration of Compound E of FIGS.2A-2I to Rats at 1.0 mg/kg, 3.0 mg/kg, and 10.0 mg/kg.
  • FIGS.4H-4K illustrate 24-hour plasma and cerebrospinal fluid (CSF) concentrations of non-naturally occurring melanocortin analogs of the present technology following IP administration of Compound E (“O10”) at 10 mg/kg relative to setmelanotide.
  • the shaded areas of 4D-4G represent the drug exposure at concentrations greater than the EC50. Area under the depicted lines represent the non-naturally occurring melanocortin analog exposure at concentrations greater than the EC50.
  • FIGS. 5A-5C illustrates 24-hour plasma and CSF concentrations of non- naturally occurring melanocortin analogs of the present technology following PO administration of Compound E of FIGS.2A-2I to Rats at 10.0 mg/kg and 30.0 mg/kg.
  • FIGS.6A and 6B show preliminary weight loss and daily food intake results in animals administered 3 mg/kg of O10 (Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro- NH2; SEQ ID NO: 43). -8- 170456481.42 Docket No.: 146316.8023.WO00 [0036]
  • FIG.7A shows net weight of food intake among individual monkeys.
  • FIG.7B shows net weight of food intake for each group average.
  • FIG.7C shows cumulative food intake among individual monkeys.
  • FIG.7D shows cumulative food intake for each group average.
  • FIGS. 8A-8H show food intake (g) in the same monkeys as FIGS. 7A-7D administered 1 mg/kg or 3 mg/kg of O10 (Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal- dPro-NH2; SEQ ID NO: 43) on day 8 (D8).
  • FIG.8A shows net weight of food intake among individual monkeys at day 8 (D8).
  • FIG.8B shows net weight of food intake for each group average of monkeys administered the same compound at D8.
  • FIG. 8C shows cumulative food intake among individual monkeys of FIG. 8A at D8.
  • FIG. 8D shows cumulative food intake for each group average of monkeys of FIG.8C administered the same compound at D8.
  • FIG. 8E shows net weight of food intake among individual monkeys at day 12 (D12).
  • FIG.8F shows net weight of food intake for each group average of monkeys administered the same compound at D12.
  • FIG. 8G shows cumulative food intake among individual monkeys at D12.
  • FIG.8H shows cumulative food intake for each group average of monkeys administered the same compound at D12.
  • FIGS. 10A-10C show changes in daily caloric intake (FIG.
  • FIGS. 10A 10A), cumulative caloric intake (FIG.10B), percent change in caloric consumption from baseline through day 6 (FIG.10C) for diet-induced obese monkeys orally administered 10 mg/kg of or O10 (Ac- Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2; SEQ ID NO: 43) (QD: once daily). [0040] FIGS.
  • 11A-11P show changes in cumulative caloric intake, percent change in caloric consumption from baseline, food intake of a normal diet, food intake of a high fat diet, food intake in calories, and food preference for diet-induced obese monkeys orally administered O10 (Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2; SEQ ID NO: -9- 170456481.42 Docket No.: 146316.8023.WO00 43), saline, or semaglutide (sema) (PO: oral administration; SC: subcutaneous administration; QD: once daily; BID: twice daily; BIW: twice weekly). [0041] FIG.
  • FIG. 11R shows an average percent change in caloric consumption from baseline in moneys orally administered O10 (Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]- dVal-dPro-NH2; SEQ ID NO: 43) at a starting dose of 10 mg/kg and increased to 20 mg/kg and 30 mg/kg.
  • FIGS.12A-12K show changes in body weight for diet included obese monkeys administered O10 (Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2; SEQ ID NO: 43) orally and/or subcutaneously.
  • FIGS. 12A and 12B Shown are changes in daily body weight and percent change in body weight from baseline (FIGS. 12A and 12B) for the diet-induced obese monkeys of FIGS.11A-11P through day 8.
  • FIGS.12C shows percent change in body weight as a percentage of initial weight for the monkeys of FIGS. 12A and 12B through day 15 compared to those administered semaglutide (sema) alone.
  • FIG. 12D shows body weight change as a percentage of initial weight in diet induced obese monkeys orally administered O10 at increasing dosages or administered semaglutide.
  • FIG. 12E shows body weight change in the monkeys of FIG.12D.
  • FIG.12F shows body weight change as a percentage of initial weight in diet included obese monkeys administered O10 at changing doses and administration frequencies.
  • FIG.12G shows body weight changes as a percentage of initial in monkeys orally administered O10 (PO) with dose changes or semaglutide.
  • FIG. 12H shows body weight change after removal of O10 administration for the monkeys of FIG. 12G.
  • FIG.12I shows percent change in body weight as a percentage of initial weight for diet induced obese monkeys orally administered O10 followed by subcutaneous administration.
  • FIG. 12J shows body weight percent change after removal of O10 treatment in monkeys.
  • FIG.12K shows body weight percent change at an oral O10 dosing regimen in accordance with the embodiments of the present technology.
  • FIG. 12K shows body weight percent change at an oral O10 dosing regimen in accordance with the embodiments of the present technology.
  • 12L shows changes in body weight as a percentage of day 1 in rats administered saline, a melanocortin 4 receptor (MC4R) selective agonist (A07D (Ac-Nle- c[Asp-Pro-dPhe-Arg-Trp-Lys]-dVal-dPro-NH2), O7 (Ac-Nle-c[Glu-Pro-p(F)dPhe-Arg-Trp- Orn]-dVal-dPro-NH2; SEQ ID NO: 106), or O11 (Ac-Nle-c[Glu-Pro-dPhe-Arg-Trp-Orn]-dVal- dPro-NH2; SEQ ID NO: 62)), or an equal dose of a melanocortin 3 receptor (MC3R)/MC4R coagonist ((O10)(Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2;
  • FIG. 12M shows an average percent change in normalized body weight from baseline in moneys orally administered O10 (Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]- dVal-dPro-NH2; SEQ ID NO: 43) at a starting dose of 10 mg/kg and increased to 20 mg/kg and 30 mg/kg.
  • FIGS.13A-13N show changes in body composition for the diet-induced obese monkeys of FIGS.10A-10C.
  • FIGS. 15A-15C show measurements of systolic blood pressure (FIG. 15A), diastolic blood pressure (FIG. 15B), and heart rate (FIG.
  • FIGS.16A-16D show measurements of diastolic blood pressure (DBP), systolic blood pressure (SBP), heart rate, and heart rate corrected QT interval (QTc) in rats administered setmelanotide at 0.5 mg/kg, 1.0 mg/kg, 3.0 mg/kg, or 6.0 mg/kg, relative to control rats administered saline.
  • DBP diastolic blood pressure
  • SBP systolic blood pressure
  • QTc heart rate corrected QT interval
  • FIGS.17A-17H show measurements of normalized heart rate (FIG.17A), QTc (FIG. 17B), systolic blood pressure (FIG. 17C), diastolic blood pressure (FIG.
  • SC subcutaneously
  • N 3 to 5 animals per group.
  • FIG. 18 shows results of O10 (Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal- dPro-NH2; SEQ ID NO: 43) screened for binding against a panel of 87 targets at 10 ⁇ M.
  • X- axis represents the percent inhibition of control at 10 ⁇ M.
  • M1R melanocortin 1 receptor
  • MC4R melanocortin 4 receptor
  • FIG. 19 shows exemplary adipose tissue staining in diet-induced obese monkeys orally administered O10 (Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro- NH2; SEQ ID NO: 43) at 10 mg/kg.
  • FIG. 20 shows plasma concentration of O10 (Ac-Nle-c[Glu-His-p(F)dPhe-Arg- Trp-Orn]-dVal-dPro-NH2; SEQ ID NO: 43) in monkeys orally (PO) administered 3mg/kg, 10 mg/kg, 30 mg/kg, or 60 mg/kg O10.
  • the present technology comprises methods of treating, preventing, or reducing one or more symptoms or conditions associated with metabolic dysfunction in a subject in need thereof using a non-naturally occurring melanocortin analog.
  • the non-naturally occurring melanocortin analog is administered orally.
  • the method comprises suppressing appetite in a subject in need thereof using a non-naturally occurring melanocortin analog in accordance with the present technology.
  • the method comprises promoting fat loss in a subject in need thereof using a non-naturally occurring melanocortin analog in accordance with the present technology.
  • the method comprises reducing body weight and/or fat mass in a subject in need thereof using a non-naturally occurring melanocortin analog, or a pharmaceutical composition thereof, of the present technology.
  • any of the steps or functions thereof may be outsourced to or performed by one or more third parties.
  • the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • the term “about” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by acceptable levels in -13- 170456481.42 Docket No.: 146316.8023.WO00 the art. Typically, such variation may be as much 10% above and below a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length and such variation may be influenced by standard applicable measurement practices.
  • the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.
  • administering include delivery of therapies (e.g., combination therapies, non-naturally occurring melanocortin analogs (also referred to herein as peptides and synthetic peptides), of the present technology to a subject either by local or systemic administration. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer), intratracheal, intranasal, epidermal and transdermal, oral or parenteral.
  • therapies e.g., combination therapies, non-naturally occurring melanocortin analogs (also referred to herein as peptides and synthetic peptides)
  • Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer),
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • active ingredient and “active compound” refer to a biologically active substance, whether naturally or non-naturally occurring, that is the main component of the pharmaceutical composition which elicits the intended effect of an administered therapeutic. This may be any component that drives the pharmacological activity or direct effect in the diagnosis, cure, mitigation, treatment, or prevention of the conditions associated with the present technology, such as but not limited to, reduced appetite and weight loss.
  • composition or a “pharmaceutical composition” refers to a mixture of the active ingredient with other chemical components, such as pharmaceutically acceptable carriers and/or excipients.
  • a “pharmaceutically acceptable carrier” of the first or the pharmaceutical composition refers to a carrier or diluent that does not cause significant irritation to an organism, does not abrogate the biological activity and properties of the administered active ingredient, and/or does not interact in a deleterious manner with the other components of the composition in which it is contained.
  • carrier encompasses any excipient, binder, diluent, filler, salt, buffer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations.
  • carrier encompasses any excipient, binder, diluent, filler, salt, buffer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations.
  • the choice of a -14- 170456481.42 Docket No.: 146316.8023.WO00 carrier for use in a composition will depend upon the intended route of administration for the composition.
  • the preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005, which is incorporated herein by reference in its entirety).
  • physiologically acceptable carriers include antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICSTM (BASF; Florham Park, N.J.).
  • antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglob
  • excipient of the first or the pharmaceutical composition refers to an inert substance added to a composition to further facilitate administration of a compound.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • weight loss agent refers to a therapeutic agent useful for the treatment or prevention of metabolic dysfunction or one or more symptoms associated with metabolic dysfunction in a subject. In some embodiments, such weight loss agents may be effective to treat, reduce, prevent, or otherwise be useful for a subject having a disease or condition that is not metabolic dysfunction, or besides metabolic dysfunction.
  • the present technology is expected to be useful for subjects that may receive, have received, or are receiving one or more doses of a weight loss agent regardless of the underlying disease or condition that the subject has or develops.
  • the terms “treat,” “treatment,” and “treating” may also refer to the reduction or inhibition of the progression and/or duration of a disease (e.g., metabolic dysfunction), the reduction or amelioration of the severity of the disease, and/or the amelioration of one or more symptoms thereof resulting from the administration of one or more therapies. Specifically, these terms may refer to: (1) a stabilization or reduction (e.g.
  • treat include prophylactic and/or therapeutic treatments. If it is administered prior to clinical manifestation of a condition, the treatment is considered prophylactic.
  • Therapeutic treatment includes, e.g., ameliorating or reducing the severity of a disease, or shortening the length or frequency of the disease.
  • prevent means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.
  • the terms “effective amount” or “therapeutically effective amount ,” refer to that amount of the active ingredient being administered which will relieve to some extent one or more of the symptoms of the disease being treated. The result may be a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • An appropriate “effective amount” may differ -16- 170456481.42 Docket No.: 146316.8023.WO00 from one individual to another.
  • An appropriate “effective amount” in any individual case may be determined using techniques, such as a dose escalation study.
  • after administration refers to any duration of time after the non- naturally occurring melanocortin analog or pharmaceutical composition thereof, and/or the weight loss agent has been administered to a subject. “After administration” may also refer to the duration of time after one dose has been completed or after more than one dose has been completed, such as two doses, three doses, four doses, and the like. In some embodiments, “after administration” refers to completion of dosing regimen that includes one or more doses. Likewise, the term “prior to administration” refers to any duration of time before the non-naturally occurring melanocortin analog or pharmaceutical composition thereof, and/or the weight loss agent has been administered to a subject.
  • BMI Body Mass Index
  • m2 body weight
  • Normal BMI ranges are known to a person of ordinary skill in the art and consider factors such as patient sex, age, height, race, and body type. Typically, a normal BMI range is about 18.5 kg/m2 to about 25 kg/m2.
  • control subject refers to any subject used as a basis for comparison to the subject (e.g., test subject).
  • a control subject includes, but is not limited to, any subject who has not been administered the therapeutic or pharmaceutical composition (e.g., the non-naturally occurring melanocortin analog, a therapeutically effective amount of the non-naturally occurring melanocortin analog or a pharmaceutical composition thereof) or administered a placebo.
  • Melanocortin analogs are used interchangeably and refer to melanocortin-receptor ligands, which are macromolecules containing at least one melanocortin pharmacophore. Melanocortin analogs are typically peptides that bind melanocortin receptors under physiological conditions.
  • Melanocortin analogs include naturally occurring non-naturally occurring melanocortin analogs (i.e., “synthetic peptides” or “synthetic analogs”) and truncated and/or modified versions of melanocortin full-length protein or peptides.
  • synthetic peptides or “synthetic analogs”
  • truncated and/or modified versions of melanocortin full-length protein or peptides For example, the full-length pro- opiomelanocortin protein (POMC), prior to proteolytic cleavage of “sub-peptides,” consists of 241 amino acids. Tissue-specific proteolytic cleavage of POMC yields peptides ranging in size from 13 amino acids to 76 amino acids. See Bicknell and Lawry, Encyclopedia of Stress, vol. 3, 257-265, Academic Press (2000).
  • POMC pro- opiomelanocortin protein
  • Synthesized, non-naturally occurring melanocortin analogs having increased melanocortin receptor activity as discussed herein are approximately 7-12 amino acids in size. Melanocortin analogs exhibit binding functionality with melanocortin receptors. The binding to the melanocortin receptor is inhibitory (antagonist).
  • the non-naturally occurring melanocortin analogs include small molecule analogs of melanocortin or portions thereof comprised of organic compounds, inorganic compounds, or combinations of peptide and small molecule—i.e., peptide mimetics, or various combinations thereof.
  • Non-naturally occurring melanocortin analogs may be structurally similar and/or functionally similar to biological melanocortin proteins in their ability to bind melanocortin receptors. Further, the non- naturally occurring melanocortin analogs generally contain the pharmacophore: His-Phe- Arg-Trp (SEQ ID NO: 1) or a modified version thereof, or a structural or functional peptide mimetic thereof.
  • a “pharmacophore” is the minimum set of amino acid residues necessary to achieve a physiological effect; or a small molecule that is (with respect to a receptor) a structural mimic of the amino acid residues required for binding to and activation of a receptor.
  • non-naturally occurring melanocortin pharmacophore analogs may be small peptides or organic molecules designed to mimic the -18- 170456481.42 Docket No.: 146316.8023.WO00 appearance or function (including activation or deactivation of receptor activity) of the melanocortin pharmacophore core sequence peptide.
  • a “melanocortin receptor agonist” or “melanocortin agonist” is a naturally occurring substance or manufactured drug substance or composition that may interact with a melanocortin receptor and initiate a pharmacological response characteristic of the melanocortin receptor.
  • the terms “bind,” “binding,” “complex,” and “complexing,” refer to all types of physical and chemical binding, reactions, complexing, attraction, chelating, and the like.
  • the “peptides” of the present technology may be (a) naturally occurring, (b) produced by chemical synthesis, (c) produced by recombinant DNA technology, (d) produced by biochemical or enzymatic fragmentation of larger molecules, (e) produced by methods resulting from a combination of methods (a) through (d) listed above, or (f) produced by any other means for producing peptides.
  • the term “peptide” as used herein includes any structure comprised of two or more amino acids, including chemical modifications and derivatives of amino acids.
  • amino acids forming all or a part of a peptide may be naturally occurring amino acids, stereoisomers and modifications of such amino acids, non-protein amino acids, post- translationally modified amino acids, enzymatically modified amino acids, constructs, or structures designed to mimic amino acids, and the like, so that the term “peptide” includes pseudopeptides and peptidomimetics, including structures which have a non-peptidic backbone.
  • the term “peptide” also includes dimers or multimers of peptides.
  • a “manufactured” peptide includes a peptide produced by chemical synthesis, recombinant DNA technology, biochemical, or enzymatic fragmentation of larger molecules, combinations of the foregoing or, in general, made by any other method.
  • peptide includes peptides containing a variable number of amino acid residues, optionally with non- amino acid residue groups at the N- and C-termini, such groups including acyl, acetyl, alkenyl, alkyl, N-alkyl, amine, or amide groups, among others.
  • groups including acyl, acetyl, alkenyl, alkyl, N-alkyl, amine, or amide groups, among others.
  • amino acids are molecules containing an amine group, a carboxylic acid group, and a side-chain that is specific to each amino acid.
  • the key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen and have the generic formula H2N—CHR— COOH, wherein R represents a side chain group.
  • R represents a side chain group.
  • the various ⁇ -amino acids differ in the side-chain moiety that is attached to the ⁇ -carbon.
  • the “amino acids” of the present technology include the known naturally occurring protein amino acids, which are referred to by both their common three letter abbreviation and single letter abbreviation. See generally Synthetic Peptides: A User’s Guide, G. A. Grant, editor, W.H.
  • amino acid also includes stereoisomers and modifications of naturally occurring protein amino acids, non- protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like. Modified and unusual amino acids are described generally in Synthetic Peptides: A User’s Guide, supra; Hruby et al., Biochem. J.268:249-262 (1990); and Toniolo, Int. J.
  • amino acid side chain moiety used herein, including as used in the specification and claims, includes any side chain of any amino acid, as the term “amino acid” is defined herein. This thus includes the side chain moiety present in naturally occurring amino acids. It further includes side chain moieties in modified naturally occurring amino acids, such as glycosylated amino acids. It further includes side chain moieties in stereoisomers and modifications of naturally occurring protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs, or structures designed to mimic amino acids, and the like.
  • the side chain moiety of any amino acid disclosed herein is included within the definition.
  • a “derivative” of an amino acid side chain moiety is included within the definition of an amino acid side chain moiety.
  • the “derivative” of an amino acid side chain moiety includes any modification to or variation in any amino acid side chain moieties, including a modification of naturally occurring amino acid side chain moieties.
  • derivatives of amino acid side chain moieties include straight chain or branched, cyclic or noncyclic, substituted or unsubstituted, saturated or unsaturated, alkyl, aryl, or aralkyl moieties.
  • L-Phe or “lPhe” is L-phenylalanine
  • D-Phe or “dPhe” is D- phenylalanine
  • dVal is D-valine
  • dPro is D-proline
  • D-/L-Phe or “d/lPhe” is D-phenylalanine, L-phenylalanine, or combinations thereof
  • Phe is also D-phenylalanine, L-phenylalanine, or combinations thereof, and so on.
  • An alpha ( ⁇ )-amino acid has the generic formula H2N—C ⁇ HR—COOH, where R is a side chain moiety and the amino group is attached to the carbon atom immediately adjacent to the carboxylate group (i.e., the ⁇ -carbon).
  • R is a side chain moiety and the amino group is attached to the carbon atom immediately adjacent to the carboxylate group (i.e., the ⁇ -carbon).
  • Other types of amino acids exist when the amino group is attached to a different carbon atom.
  • beta ( ⁇ )-amino acids the carbon atom to which the amino group is attached is separated from the carboxylate group by one carbon atom, C ⁇ .
  • ⁇ -amino acids When ⁇ -amino acids are incorporated into peptides, two main types of ⁇ - peptides exist: those with the side chain residue, R, on the carbon next to the amine are called ⁇ 3 peptides and those with the side chain residue on the carbon next to the carbonyl group are called ⁇ 2 amino acids. Further, ⁇ -amino acids may adopt L- or D- stereochemistry. Unless otherwise indicated, all ⁇ -amino acid abbreviations represent either isomer, i.e., the L-isomer, the D-isomer, or combinations thereof.
  • Gamma ( ⁇ )-amino acids are amino acids where the carbon atom to which the amino group attaches is separated from the carboxylate moiety by two carbon atoms.
  • acyl includes a group RCO—, where R is an organic group.
  • R is an organic group.
  • An example is the acetyl group CH3CO—, referred to herein as “Ac.”
  • a peptide or aliphatic moiety is “acylated” when an alkyl or substituted alkyl group as defined above is bonded through one or more carbonyl ⁇ —(C ⁇ O)— ⁇ groups.
  • a peptide is most usually acylated at the N-terminus.
  • An “amine” includes compounds that contain an amine group (—NH2).
  • An “amide” includes compounds that have a trivalent nitrogen attached to a carbonyl group (i.e., —CO—NH2), such as for example methylamide, ethylamide, propylamide, and the like.
  • a peptide is most usually amidated at the C-terminus by the addition of an amine (—NH2) moiety to the C-terminal carboxyl group.
  • Amino acids including stereoisomers and modifications of naturally occurring amino acids, protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs, or structures designed to mimic amino acids (peptide mimetics), and the like, including all of the foregoing, are sometimes referred to herein as “residues.”
  • “Substantial degradation” refers to the degradation of the N-terminal extension, the C-terminal extension, both N- and C-terminal degradation or degradation to other regions -22- 170456481.42 Docket No.: 146316.8023.WO00 of the melanocortin peptide by physiological enzymes and other factors, in such a manner or to a degree that side effects appear.
  • a melanocortin analog having a C-terminal extension that resists substantial degradation is one where no more than 50% of the administered peptide causes side effects and/or displays a low half-life. In some aspects, no more than 25% of the administered peptide causes side effects and/or displays a low half-life. More preferably, in some aspects, less than 10% of the administered peptide causes side effects and/or displays a low half-life, as compared to a melanocortin analog that lacks a C-terminal extension.
  • a “composition” refers to a mixture of the active ingredient with other chemical components.
  • Non-naturally Occurring Melanocortin Analogs may comprise a non-naturally occurring melanocortin analog or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.
  • Non-naturally occurring melanocortin analogs of the present technology may be selective for the melanocortin 4 receptor (MC4R) and/or melanocortin 3 receptor (MC3R) over other melanocortin receptors, i.e., the melanocortin 1 receptor (MC1R), the melanocortin 2 receptor (MC2R), and the melanocortin 5 receptor (MC5R).
  • M1R melanocortin 1 receptor
  • M2R melanocortin 2 receptor
  • M5R melanocortin 5 receptor
  • Some of the non-naturally occurring melanocortin analogs may bind the MC4R with greater affinity than the MC3R.
  • some melanocortin analogs may bind the MC3R with the same or generally similar affinity as the MC4R.
  • the non-naturally occurring melanocortin analogs of the present technology may be full agonists for one or more melanocortin receptors.
  • a full agonist may comprise a non-naturally occurring melanocortin analog having a maximum effect (Emax) agonist value of greater than or equal to 85%.
  • Emax maximum effect
  • the non-naturally occurring melanocortin analogs of the present technology may be partial agonists.
  • a partial agonist may comprise a non-naturally occurring melanocortin analog having a maximum effect Emax agonist value of less than 85%.
  • the non-naturally occurring melanocortin analog may be classified as an agonist (e.g., a full agonist or a partial agonist).
  • the non-naturally occurring melanocortin analogs of the present technology may be one or more of (i) a full MC4R agonist (ii) a full MC3R agonist; and (iii) a partial MC3R agonist with no MC3R antagonist activity.
  • the non-naturally occurring melanocortin is a full MC4R agonist.
  • the non-naturally occurring melanocortin agonist is a full MC3R agonist and a full MC3R agonist.
  • the non-naturally occurring melanocortin analog is a full MC4R agonist and partial MC3R agonist with no MC3R antagonist activity.
  • the non- naturally occurring melanocortin analogs of the present technology have an MC3R Emax agonist value of 50-84%.
  • the non-naturally occurring melanocortin analogs of the present technology have a MC3R:MC4R half maximal effective concentration (EC50) selectivity ratio of less than or equal to 49.
  • the non-naturally occurring melanocortin analogs of the present technology comprise non-naturally occurring MC4R agonists or non-naturally occurring MC3R agonists.
  • the non-naturally occurring melanocortin analogs in accordance with the present technology may have structural features that impart specific properties on the analogs, such as, for example, degradation resistance, enhanced epithelial, gastrointestinal, and/or blood brain barrier transport, and binding affinity for the MC4R and/or MC3R.
  • the non-naturally occurring melanocortin analogs comprise one or more of (i) blood brain barrier passage capabilities, (ii) one or more pharmaceutical- like pharmacokinetic measurements (e.g., a pharmacokinetic measurement of a weight loss agent pharmaceutical composition or an oral non-naturally occurring melanocortin analog pharmaceutical), (iii) degradation resistance; (iv) equipotency on MC3R and MC4R activity, or (v) oral potency.
  • the oral potency is greater than a subcutaneous administration potency for an equivalent dose.
  • the non-naturally -24- 170456481.42 Docket No.: 146316.8023.WO00 occurring melanocortin analogs comprise two or more of (i)-(v). In some embodiments, he non-naturally occurring melanocortin analogs comprise each of (i)-(v). Accordingly, in some embodiments, the non-naturally occurring melanocortin analogs have one or more beta hairpin ( ⁇ -hairpin) and/or beta turn ( ⁇ -turn) structures.
  • amino acids that are structurally rigid such as, for example, Pro, dPro, transPro(guan), and cisPro(guan)
  • disulfide bridges e.g., cyclization via disulfide bond
  • beta-turn structures of the non-naturally occurring melanocortin analogs.
  • cyclization and D-amino acids may induce and/or stabilize beta-turns.
  • melanocortin analogs include a D-valine-D-proline (dVal- dPro) chain as their C-terminus, which may provide enhanced transport and resistance to degradation.
  • dVal- dPro D-valine-D-proline
  • the presence of certain structural features may impart the non-naturally occurring melanocortin analogs of the present technology with specific binding properties. For example, inclusion of p(F)dPhe or dPhe at the R 4 position may result in enhanced binding and activation of the melanocortin 4 receptor. Accordingly, melanocortin analogs having p(F)dPhe or dPhe at the R 4 position may be full agonists on MC4R.
  • Non-naturally occurring melanocortin analogs in accordance with the present technology may induce or increase body weight and/or fat mass loss in a subject in need thereof while reducing lean mass loss, maintaining lean mass, or promoting lean mass gain in the subject. Additionally, the non-naturally occurring melanocortin analogs of the present technology may avoid cardiac activation typically seen in conventional melanocortin peptide and small molecule agonists.
  • the non-naturally occurring melanocortin analog comprises a sequence according to Formula (I): -25- 170456481.42 Docket No.: 146316.8023.WO00 X 1 -R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8 -R 9 -Y 1 -Y 2 -Y 3 -Y 4 (I), wherein: X 1 is absent or is selected from the group consisting of norleucine (Nle), arginine (Arg), and D-arginine (dArg); R 1 is selected from the group consisting of Nle, Arg, dArg, aspartic acid (Asp), alanine (Ala), lysine (Lys), D-
  • the non-naturally occurring melanocortin analog comprises a sequence according to Formula (I), wherein: X 1 is absent or is selected from the group consisting of norleucine (Nle), arginine (Arg), and D-arginine (dArg); R 1 is selected from the group consisting of Nle, Arg, dArg, aspartic acid (Asp), alanine (Ala), lysine (Lys), D-lysine (dLys), histidine (His), and D-histidine (dHis); R 2 is selected from the group consisting of Asp, D-cysteine (dCys), Ala, D-alanine (dAla), proline (Pro), glutamic acid (Glu), and phenylalanine (Phe); R 3 is selected from the group consisting of His, dHis, Pro, Phe, and glutamine (Gln); R 4 is selected from the group consisting of D-phenyla
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (I) wherein R 4 is dPhe or p(F)dPhe.
  • R 4 is dPhe or p(F)dPhe in the sequence of Formula (I)
  • R 9 is absent.
  • the sequence of Formula (I) is a sequence of Formula (IA): X 1 -R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8 -Y 1 -Y 2 -Y 3 -Y 4 (IA), wherein: X 1 is absent or is selected from the group consisting of Nle, Arg, and dArg; R 1 is selected from the group consisting of Nle, Arg, dArg, Asp, Ala, Lys, dLys, His, and dHis; R 2 is selected from the group consisting of Asp, Ala, dAla, Pro, and Glu R 3 is selected from the group consisting of His, dHis, Pro, and Gln; R 4 is dPhe or p(F)dPhe; R 5 is selected from the group consisting of Arg, His, cisPro(guan), and transPro(guan); -33- 1704
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (IA): X 1 -R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8 -Y 1 -Y 2 -Y 3 -Y 4 (IA), wherein: X 1 is absent or is selected from the group consisting of Nle, Arg, and dArg; R 1 is selected from the group consisting of Nle, Arg, dArg, Asp, Ala, Lys, dLys, His, and dHis; R 2 is selected from the group consisting of Asp, Ala, dAla, Pro, and Glu R 3 is selected from the group consisting of His, dHis, Pro, and Gln; R 4 is dPhe or p(F)dPhe; R 5 is selected from the group consisting of Arg, His, cisPro(guan), and transPro(guan
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (IA), wherein: X 1 is absent or is selected from the group consisting of Nle, Arg, and dArg; R 1 is selected from the group consisting of Nle, Arg, dArg, Asp, Ala, Lys, dLys, His, and dHis; R 2 is selected from the group consisting of Asp, Ala, dAla, Pro, and Glu R 3 is selected from the group consisting of His, dHis, Pro, and Gln; R 4 is dPhe or p(F)dPhe; R 5 is selected from the group consisting of Arg, His, cisPro(guan), and transPro(guan); -35- 170456481.42 Docket No.: 146316.8023.WO00 R 6 is Trp or dNal(2’); R 7 is selected from the group consisting of Pro, Orn, and
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (I) or (IA) wherein R 4 is dPhe or p(F)dPhe, then R 8 may also be absent. Additionally, the sequence of Formula (I) or (I) may be cyclized through a lactam bond between R 2 and R 7 .
  • the sequence of Formula (I) or (IA) is a sequence of Formula (IB): X 1 -R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -Y 1 -Y 2 -Y 3 -Y 4 (IB), wherein: X 1 is absent or Nle; R 1 is selected from the group consisting of Nle, Arg, dArg, Ala, Lys, dLys, His, and dHis; R 2 is Asp or Glu; R 3 is selected from the group consisting of His, dHis, Pro, and Gln; -39- 170456481.42 Docket No.: 146316.8023.WO00 R 4 is dPhe or p(F)dPhe; R 5 is selected from the group consisting of Arg, His, cisPro(guan), and transPro(guan); R 6 is Trp or dNal(2’); R 7
  • R 3 is His
  • R 5 is Arg
  • R 6 is Trp
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (IC): X 1 -R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -Y 1 -Y 2 -Y 3 -Y 4 (IC), wherein: X 1 is absent or Nle; R 1 is selected from the group consisting of Nle, Arg, dArg, Ala, Lys, dLys, His, and dHis; R 2 is Asp or Glu; R 3 is His; R 4 is p(F)dPhe; R 5 is Arg; R 6 is Trp; R 7 is Orn or Lys; Y 1 is selected from the group consisting of dVal, dPro, and dTle; Y 2 is selected from the group consisting of dVal, dPro, and dTle; Y 3 is absent, dVal, or dPro; Y 4 is absent or dPro
  • the non-naturally occurring melanocortin analog comprises a sequence of any one of Formulae (I)-(IB) wherein R 4 is dPhe.
  • the sequence of any one of Formulae (I)-(IB) is a sequence of Formula (ID): X 1 -R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8 -Y 1 -Y 2 -Y 3 (ID), wherein: X 1 is absent or norleucine (Nle); R 1 is Nle or Asp; R 2 is selected from the group consisting of Asp, Glu, and Pro; R 3 is selected from the group consisting of His, dHis, Pro, and Gln; R 4 is dPhe; R 5 is selected from the group consisting of Arg, His, cisPro(guan), and transPro(guan); R 6 is Trp; R 7 is selected from the group consisting of Orn, Lys, and Pro; R 8 is absent or Lys; Y 1 is dVal or dPro; Y 2 is dVal or dPro; Y 3 is absent or
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (ID): -47- 170456481.42 Docket No.: 146316.8023.WO00 X 1 -R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8 -Y 1 -Y 2 -Y 3 (ID), wherein: X 1 is absent or norleucine (Nle); R 1 is Nle or Asp; R 2 is selected from the group consisting of Asp, Glu, and Pro; R 3 is selected from the group consisting of His, dHis, Pro, and Gln; R 4 is dPhe; R 5 is selected from the group consisting of Arg, His, cisPro(guan), and transPro(guan); R 6 is Trp; R 7 is selected from the group consisting of Orn, Lys, and Pro; R 8 is absent or Lys; Y 1 is dVal or
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (ID), wherein: -48- 170456481.42 Docket No.: 146316.8023.WO00 X 1 is absent or norleucine (Nle); R 1 is Nle or Asp; R 2 is selected from the group consisting of Asp, Glu, and Pro; R 3 is selected from the group consisting of His, dHis, Pro, and Gln; R 4 is dPhe; R 5 is selected from the group consisting of Arg, His, cisPro(guan), and transPro(guan); R 6 is Trp; R 7 is selected from the group consisting of Orn, Lys, and Pro; R 8 is absent or Lys; Y 1 is dVal or dPro; Y 2 is dVal or dPro; Y 3 is absent or dPro; and the non-naturally occurring melanocortin analog is cyclized through a moiety selected from
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (I) or (IA) wherein R 4 is dPhe or p(F)dPhe, then R 8 is present. In further embodiments, R 4 is dPhe.
  • the sequence of Formula (I) or (IA) is a sequence of Formula (IE): X 1 -R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -Y 1 -Y 2 (IE), wherein: X 1 is absent or selected from the group consisting of Nle, Arg, and dArg; R 1 is Asp; R 2 is dAla or Ala; R 3 is His; R 4 is dPhe; -50- 170456481.42 Docket No.: 146316.8023.WO00 R 5 is Arg; R 6 is Trp; R 7 is Lys; Y 1 is dVal; Y 2 is dPro; and the non-naturally occurring melanocortin analog is cyclized through a lactam bridge between Asp at R 1 and Lys at R 7 .
  • IE Formula
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (IE): X 1 -R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -Y 1 -Y 2 (IE), wherein: X 1 is absent or selected from the group consisting of Nle, Arg, and dArg; R 1 is Asp; R 2 is dAla or Ala; R 3 is His; R 4 is dPhe; R 5 is Arg; R 6 is Trp; R 7 is Lys; Y 1 is dVal; Y 2 is dPro; and the non-naturally occurring melanocortin analog is cyclized through a lactam bridge between Asp at R 1 and Lys at R 7 .
  • IE Formula
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (I) that is cyclized between Asp or Glu at R 2 and Orn at -51- 170456481.42 Docket No.: 146316.8023.WO00 R7.
  • R 3 is His and R 4 is p(F)dPhe or p(Cl)dPhe.
  • the sequence of Formula (I) is a sequence of Formula (IF): R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -Y 1 -Y 2 (IF), wherein: R 1 is Nle; R 2 is Asp or Glu; R 3 is His; R 4 is selected from dPhe, p(F)dPhe, and p(Cl)dPhe; R 5 is Arg or His; R 6 is Trp; R 7 is Orn; Y 1 is dVal; Y 2 is dPro; and the non-naturally occurring melanocortin analog is cyclized through a lactam bridge between Asp or Glu at R 2 and Orn at R 7 .
  • IF Formula
  • the non-naturally occurring melanocortin analog compirses a sequence of Formula (IF): R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -Y 1 -Y 2 (IF), wherein: R 1 is Nle; R 2 is Asp or Glu; R 3 is His; R 4 is selected from dPhe, p(F)dPhe, and p(Cl)dPhe; R 5 is Arg or His; R 6 is Trp; -52- 170456481.42 Docket No.: 146316.8023.WO00 R 7 is Orn; Y 1 is dVal; Y 2 is dPro; and the non-naturally occurring melanocortin analog is cyclized through a lactam bridge between Asp or Glu at R 2 and Orn at R 7 .
  • IF Formula
  • the non-naturally occurring melanocortin analog of any one of Formulae (I)-(IF) has one or more beta hairpin ( ⁇ -hairpin) and/or beta turn ( ⁇ -turn) structures.
  • the presence of Pro, dPro, transPro(guan), and/or cisPro(guan) provides the ⁇ -hairpin and/or ⁇ -turn structures of the non-naturally occurring melanocortin analog.
  • the disulfide bond of the sequence according to Formula (I), if present, provides the ⁇ -hairpin and/or ⁇ -turn structures of the non-naturally occurring melanocortin analog.
  • non-naturally occurring melanocortin analogs comprising a sequence of any one of Formulae (I)-(IF), have an N- terminus and a C-terminus.
  • the melanocortin analogs of the present technology are written beginning with the N-terminus at the left-most amino acid residue and ending with the C- terminus at the right most residue.
  • the N-terminus of a non-naturally melanocortin analog comprising a sequence of any one of Formulae (I)-(IF) may be at X 1 or R 1 .
  • the C-terminus of a non-naturally occurring melanocortin analog comprising a sequence of any one of Formulae (I)-(IF) may be at any of R 7 , R 8 , R 9 , Y 1 , Y 2 , Y 3 , and Y 4 .
  • the N-terminus of the non-naturally occurring melanocortin analog, if present, is modified by an acyl group.
  • the acyl group is acetyl ).
  • In some of the non-naturally occurring melanocortin analog, if present, is not modified.
  • Y 1 Y 2 Y 3 Y 4 or a fragment thereof represents a C-terminus of the non-naturally occurring melanocortin analog.
  • Y 1 and Y 2 are present and Y 3 -Y 4 are absent.
  • Y 3 -Y 4 are absent, and Y 1 is D-valine and Y 2 is D-proline.
  • Y 3 -Y 4 are absent, and Y 1 is D-proline and Y 2 is D-valine.
  • Y 3 -Y 4 are absent, and Y 1 is D-tert-leucine and Y 2 is D-proline.
  • Y 1 -Y 3 are present and Y 4 is absent.
  • Y 4 is absent and Y 1 is D-proline, Y 2 is D-valine, and Y 3 is D-proline.
  • Y 4 is absent, and Y 1 is D-tert-leucine, Y 2 is D-tert-leucine, and Y 3 is D-valine.
  • Y 1 -Y 4 are present.
  • Y 1 -Y 4 are present and Y 1 is D-valine, Y 2 is D-valine, Y 3 is D-valine, and Y 4 is D-proline.
  • the C-terminus of the non-naturally occurring melanocortin analog is modified by an amide
  • a non-naturally with a C- terminus modified by an amide may be represented by a terminal -NH2.
  • the C-terminus of the non-naturally occurring melanocortin analog is not modified.
  • a non-naturally occurring melanocortin analog with an unmodified C-terminus may be represented by -OH.
  • Non-naturally occurring melanocortin analogs comprising a sequence of any one of Formulae (I)-(IF) are cyclized.
  • the non-naturally occurring melanocortin analog may be cyclized through a moiety selected from the group consisting of: a disulfide bond between R 2 and R 7 when R 2 is dCys, R 4 is p(Cl)dPhe, and R 7 is Cys; a lactam bridge between R 1 or R 2 and any one of R 7 -R 9 when R 1 or R 2 is Asp, R 4 is dPhe, or p(F)dPhe, and any one of R 7 -R 9 is Lys; and a lactam bridge between R 2 and R 7 when R 2 is Asp or Glu and R 7 is Orn.
  • R 3 is His.
  • R 3 is His and the non-naturally occurring melanocortin analog is cyclized through a lactam bridge between the Asp at R 2 and the Lys at R 7 .
  • R 4 is p(F)dPhe or dPhe.
  • X 1 , Y 3 , and Y 4 are all absent.
  • R 1 is selected from Nle, Ala, Arg, dArg, Lys, dLys, His, and dHis and R 4 is p(F)dPhe.
  • Y 1 is dVal and Y 2 is dPro.
  • sequence of any one of Formulae (I)-(IF) is selected from the group consisting of: Ac-Ala-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 3); Ac-dArg-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 4); Ac-Nle-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 14); Ac-Arg-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 15); Ac-Lys-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dVal
  • Y 1 is dTle
  • Y 2 is dPro.
  • the sequence of any one of Formulae (I)-(IF) is selected from the group consisting of: Ac-Ala-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dTle-dPro-NH2 (SEQ ID NO: 7); Ac-dArg-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dTle-dPro-NH2 (SEQ ID NO: 8); Ac-Nle-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dTle-dPro-NH2 (SEQ ID NO: 24); Ac-Arg-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dTle-dPro-NH2 (SEQ ID NO: 27);
  • R 1 is Nle and R 4 is dPhe.
  • Y 1 is dVal or dPro
  • Y 2 is dPro or dVal.
  • the sequence of any one of Formulae (I)-(IF) is: Ac-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-dPro-dVal-NH2 (SEQ ID NO: 33); or Ac-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 42), wherein c represents cyclization through R 2 and R 7 via a lactam bond.
  • any one of Formulae (I)-(IF) is selected from the group consisting of: Ac-Nle-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dTle-dTle-dVal-NH2 (SEQ ID NO: 6); Ac-Nle-Nle-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 20); Ac-Nle-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dVal-dVal-dPro-NH2 (SEQ ID NO: 22); Ac-Nle-c[Asp-His-p(F)dPhe-Arg-Trp-Lys]-dVal-dVal-dVal-dPro-NH2 (SEQ ID NO: 22); Ac-Nle-c[Asp
  • R 6 when R 3 is His, R 4 is p(F)dPhe or dPhe, and the non- naturally occurring melanocortin analog is cyclized through a lactam bond between Asp at R 2 and Lys at R 7 , then R 6 is Trp. Alternatively, in some embodiments, R 6 is an amino acid other than Trp. In further embodiments, R 6 is dNal(2’).
  • the non-naturally occurring melanocortin analog is cyclized through a disulfide bond between dCys at R 2 and Cys at R 7 .
  • R 4 is p(Cl)dPhe.
  • the sequence of any one of -57- 170456481.42 Docket No.: 146316.8023.WO00 Formulae (I)-(IF) is Ac-Nle-c[dCys-His-p(Cl)dPhe-Arg-Trp-Cys]-dVal-dPro-NH2 (SEQ ID NO: 2), wherein c represents cyclization through R 2 and R 7 via a disulfide bond.
  • the non-naturally occurring melanocortin analog is cyclized through Asp at a residue position other than R 2 and/or Lys at a residue position other than R 7 .
  • the non-naturally occurring melanocortin analog may be cyclized through Asp at R 1 and Lys at R 7 or through Asp at R 2 and Lys at R 8 .
  • the non-naturally occurring melanocortin analog is cyclized through a lactam bond between Asp at R 2 and Lys at R 8 .
  • the sequence of any one of Formulae (I)-(IF) is: Ac-Nle-c[Asp-His-dPhe-Arg-Trp-Pro-Lys]- dVal-dPro-NH2 (SEQ ID NO: 35), wherein c represents cyclization through R 2 and R 8 via a lactam bond.
  • the non-naturally occurring melanocortin analog is cyclized between a lactam bond between Asp at R 1 and Lys at R 7 .
  • R 2 is Ala or dAla and R 4 is dPhe.
  • the sequence of any one of Formulae (I)-(IF) is selected from the group consisting of: Ac-Nle-c[Asp-dAla-His-dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 38); Ac-Arg-c[Asp-dAla-His-dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 39); Ac-dArg-c[Asp-dAla-His-dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 40); and Ac-Nle-c[Asp-Ala-His-dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 41), wherein c represents cyclization through R 1 and R 7 via a lactam bond.
  • R 2 is Pro
  • R 4 is Phe
  • the sequence of any one of Formulae (I)-(IF) is: Ac-Nle-c[Asp-Pro-His-dPhe-Arg-Trp-Lys]- dVal-dPro-NH2 (SEQ ID NO: 13), wherein c represents cyclization through R 1 and R 7 via a lactam bond.
  • R 3 is an amino acid other than His. In some embodiments, R 3 is selected from dHis, Gln and Pro.
  • R 2 is Asp
  • R 4 is dPhe or p(F)dPhe
  • R 5 is Arg
  • R 6 is Trp
  • R 7 is Lys. -58- 170456481.42 Docket No.: 146316.8023.WO00
  • the sequence of any one of Formulae (I)-(IF) is selected from the group consisting of: Ac-Nle-c[Asp-Gln-p(F)dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 9); Ac-Nle-Nle-c[Asp-Pro-dPhe-Arg-Trp-Lys]-dPro-dVal-dPro-NH2 (SEQ ID NO: 10); Ac-Nle-c[Asp-dHis-p(F)dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 32); Ac-Nle-c[A
  • R 3 is an amino acid other than His and R 9 is absent. Alternatively, in some embodiments, R 9 is present.
  • the sequence of any one of Formulae (I)-(IF) is: Ac-Nle-c[Asp-Phe-Phe-Pro- His-dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 (SEQ ID NO: 34), wherein c represents cyclization through R 1 and R 9 via a lactam bond.
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (I).
  • the non-naturally occurring melanocortin analog comprises any one of the sequences of SEQ ID NOs: 3-10, 13-33, and 35-47. [0148] In some embodiments, the non-naturally occurring melanocortin analog comprises a sequence of Formula (IA). In some embodiments, the non-naturally occurring melanocortin analog comprises any one of the sequences of SEQ ID NOs: 3-10, 14-33, 36, 37, and 42-47. [0149] In some embodiments, the non-naturally occurring melanocortin analog comprises a sequence of Formula (IB).
  • the non-naturally occurring melanocortin analog comprises any one of the sequences of SEQ ID NOs: 3-8, 14-31, 43, and 44. [0150] In some embodiments, the non-naturally occurring melanocortin analog comprises a sequence of Formula (IC). In some embodiments, the non-naturally occurring -59- 170456481.42 Docket No.: 146316.8023.WO00 melanocortin analog comprises any one of the sequences of SEQ ID NOs: 10, 13, 33, 35- 37, 42, and 45-47. [0151] In some embodiments, the non-naturally occurring melanocortin analog comprises a sequence of Formula (ID).
  • ID sequence of Formula
  • the non-naturally occurring melanocortin analog comprises any one of the sequences of SEQ ID NOs: 38-41. [0152] In some embodiments, the non-naturally occurring melanocortin analog comprises a sequence of Formula (IF). In some embodiments, the non-naturally occurring melanocortin analog comprises any one of the sequences of SEQ ID NOs: 11, 12, and 43- 45. In some embodiments, the non-naturally occurring melanocortin analog comprises any one of the sequences of SEQ ID NOs: 43-45.
  • Non-naturally occurring melanocortin analogs of any one of Formulae (I)-(IF) may be an agonist of the melanocortin 4 receptor and an agonist of the melanocortin 3 receptor.
  • non-naturally occurring melanocortin analogs of any one of Formulae (I)-(IF) are full agonists of the melanocortin 4 receptor and full agonists of the melanocortin 3 receptor.
  • non-naturally occurring melanocortin analogs of any one of Formulae (I)-(IF) are full agonists of the melanocortin 4 receptor and partial agonists of the melanocortin 3 receptor.
  • Non-Naturally Occurring Melanocortin Analog Synthesis The non-naturally occurring melanocortin analogs of the present technology may be readily synthesized by any known conventional procedure for the formation of a peptide linkage between amino acids. Such conventional procedures include, for example, any solution phase procedure permitting a condensation between the free alpha amino group of an amino acid or residue thereof having the carboxyl group or other reactive groups protected and the free primary carboxyl group of another amino acid or residue thereof having the amino group or other reactive groups protected.
  • the peptides of the present technology may be synthesized by solid-phase synthesis and purified according to methods known in the art.
  • the process for synthesizing the peptides may be carried out by a procedure whereby each amino acid in the desired sequence is added one at a time in succession to another amino acid or residue thereof or by a procedure whereby peptide fragments with the desired amino acid sequence are first synthesized conventionally and then condensed to provide the desired peptide. The resulting peptide is then cyclized to yield a cyclic peptide.
  • Solid phase peptide synthesis methods are well known and practiced in the art. In such methods, the synthesis of peptides may be carried out by sequentially incorporating the desired amino acid residues one at a time into the growing peptide chain according to the general principles of solid phase methods. These methods are disclosed in numerous references, including Merrifield, Angew Chem.24:799-810 (1985) and Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross E. and Meienhofer J., Eds. Academic Press 1-284 (1980), the disclosure of which is incorporated herein by reference in its entirety.
  • Alpha amino groups may be protected by a suitable protecting group, including a urethane-type protecting group, such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p- bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz); aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl, and allyloxycarbonyl.
  • a suitable protecting group including a urethane-type protecting group, such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as
  • Fmoc is useful for alpha amino protection.
  • Guanidino groups may be protected by a suitable protecting group, such as nitro, p-toluenesulfonyl (Tosyl), Z, pentamethylchromanesulfonyl (Pmc), -61- 170456481.42 Docket No.: 146316.8023.WO00 adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and Boc.
  • Pmc is a useful protecting group for Arg.
  • Solid phase synthesis is commenced from the C-terminal end of the peptide by coupling a protected alpha amino acid to a suitable resin.
  • Such starting material is prepared by attaching an alpha amino-protected amino acid by an ester linkage to a p- benzyloxybenzyl alcohol (Wang) resin or a 2-chlorotrityl chloride resin, by an amide bond between an Fmoc-Linker, such as p-[(R,S)- ⁇ -[1-(9H-fluor-en-9-yl)-methoxyformamido]-2,4- dimethyloxybenzyl]-phenoxyacetic acid (Rink linker) to a benzhydrylamine (BHA) resin, or by other means well known in the art.
  • Fmoc-Linker-BHA resin supports are commercially available and generally used when feasible.
  • the resins are carried through repetitive cycles as necessary to add amino acids sequentially.
  • the alpha amino Fmoc protecting groups are removed under basic conditions. Piperidine, piperazine, diethylamine, or morpholine (20- 40% v/v) in N,N-dimethylformamide (DMF) may be used for this purpose.
  • DMF N,N-dimethylformamide
  • the subsequent protected amino acids are coupled stepwise in the desired order to obtain an intermediate, protected peptide-resin.
  • the activating reagents used for coupling of the amino acids in the solid phase synthesis of the peptides are well known in the art.
  • the orthogonally protected side chain protecting groups may be removed using methods well known in the art for further derivatization of the peptide.
  • Reactive groups in a peptide may be selectively modified, either during solid phase synthesis or after removal from the resin.
  • peptides may be modified to obtain N-terminus modifications, such as acetylation, while on resin, or may be removed from the resin by use of a cleaving reagent and then modified. Methods for N-terminus modification, such as acetylation, and for C-terminus modification, such as amidation, are known in the art.
  • the peptide may be cyclized prior to cleavage from the peptide resin.
  • the desired side chains are deprotected, -62- 170456481.42 Docket No.: 146316.8023.WO00 and the peptide suspended in a suitable solvent and a cyclic coupling agent added.
  • suitable solvents include, for example DMF, dichloromethane (DCM) or 1-methyl-2-pyrrolidone (NMP).
  • Suitable cyclic coupling reagents include, for example, 2-(1H-benzotriazol-1-yl)- 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate (HBTU), benzotriazole-1-yl-oxy- tris(dimethylamino)phosphoniumhexafluorophosphate (BOP), benzotriazole-1-yl-oxy- tris(pyrrolidino)phosphoniumhexafluorophosphate (PyBOP), 2-(7-aza-1H-benzotriazol-1- yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TATU), 2-(2-oxo-1 (2H)-pyridyl)-1,1,3,3- tetramethyluronium tetrafluo
  • Coupling is convention initiated by use of a suitable base, such as N,N-diispropylethylamine (DIPEA), sym-collidine, or N-methylmorpholine (NMM).
  • DIPEA N,N-diispropylethylamine
  • NMM N-methylmorpholine
  • the peptide may be purified by any number of methods, such as reverse phase high performance liquid chromatography (RP-HPLC), using a suitable column, such as a C18 column. Other methods of separation or purification, such as methods based on the size or charge of the peptide, may also be employed.
  • the peptide may be characterized by any number of methods, such as high performance liquid chromatograph (HPLC), amino acid analysis, mass spectrometry, and the like.
  • Salt Forms of Non-Naturally Occurring Melanocortin Analogs may be in the form of any salt.
  • pharmaceutically acceptable salts refers to salts prepared from non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like.
  • Exemplary salts are the ammonium, calcium, lithium, magnesium, potassium, and sodium salts.
  • Salts derived from organic non- toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine, -63- 170456481.42 Docket No.: 146316.8023.WO00 diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine
  • acid addition salts may be prepared from non-toxic acids, including inorganic and organic acids.
  • acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, carboxylic, citric, ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, malonic, mucic, nitric, pamoic, pantothenic, phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonic acid, trifluoroacetic acid, and the like.
  • Acid addition salts of the peptides of the present technology are prepared in a suitable solvent from the peptide and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, citric, tartaric, maleic, succinic or methanesulfonic acid.
  • an acid such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, citric, tartaric, maleic, succinic or methanesulfonic acid.
  • suitable salts may include alkali metal salts, such as sodium or potassium salts, or alkaline earth metal salts, such as calcium or magnesium salts.
  • Conjugates [0167] The present technology further includes conjugates comprising a non-naturally occurring melanocortin analog.
  • the non-naturally melanocortin analog is conjugated to a pharmaceutical agent.
  • suitable pharmaceutical agents include peptides, monoclonal antibodies, and small molecules.
  • the non-naturally occurring melanocortin analog is conjugated to semaglutide.
  • the non-naturally occurring melanocortin analog is conjugated to setmelanotide.
  • the non-naturally occurring melanocortin analog is conjugated to a small molecule MCR agonist.
  • Conjugates of the present technology further comprise a linker connecting the non-naturally occurring melanocortin analog to the pharmaceutical agent. In some embodiments, the linker is rigid.
  • the linker is flexible. In some -64- 170456481.42 Docket No.: 146316.8023.WO00 embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises a glycine-serine (Gly/Ser) linker, a proline-threonine-glycine linker, an alanine linker, a lysine linker, a threonine linker, a valine-glycine-serine-threonine linker, an elastin-like peptide linker, a hexahistidine linker, a polyethylene glycol linker, a fatty acid linker, or a hydrocarbon linker.
  • Gly/Ser glycine-serine
  • the linker is a peptide linker.
  • the peptide linkers of the present technology may vary from 2 to 31 amino acids of any primary sequence in length and do not impose any constraints on the conformation or interactions of the linked partners.
  • the linkers vary from about 2-30, 2-29, 2-28, 2-27, 2-26, 2-25, 2-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2- 14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-31, 3-30, 3-29, 3-28, 3-27, 3-26, 3-25, 3-24, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-31, 4-30, 4-29, 4-28, 4-27,
  • the peptide linkers may be designed as appropriate for an intended use.
  • the peptide linkers may comprise one or more of a Gly-rich linker (e.g., a flexible linker connecting various domains in a single protein without interfering with the function of each domain; a linker forming stable covalently linked dimers; a linker to connect two independent domains that create a ligand-binding site or recognition sequence), a Serine linker (e.g., a coiled structure linker); a coiled structure linker comprising a Gln, Arg, Glu, Ser, and/or Pro amino acids; a rigid space linker comprising one or more of a Pro, Arg, Phe, Thr, Glu, and/or Gln residues; a linker comprising a flexible Gly-rich regions that may may generate loops connecting domains; or a linker comprising a Thr, Ser, Gly, and/or Ala residue.
  • the linker comprises an amino acid sequence about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of Linker A, Linker B, Linker C, Linker D, Linker E, Linker F, Linker G, Linker H, Linker I, Linker J, Linker K, Linker L, Linker M, Linker N, Linker O, Linker P, Linker Q, Linker R, or Linker S in Table 0.
  • the linker comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of Linker A, Linker B, Linker C, Linker D, Linker E, Linker F, Linker G, Linker H, Linker I, Linker J, Linker K, Linker L, Linker M, Linker N, Linker O, Linker P, Linker Q, Linker R, or Linker S in Table 0.
  • the linker comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of Linker A, Linker B, Linker C, Linker D, Linker E, Linker F, Linker G, Linker H, Linker I, Linker J, Linker K, Linker L, Linker M, Linker N, Linker O, Linker P, Linker Q, Linker R, or Linker S in Table 0.
  • the non-naturally occurring melanocortin analogs of the present technology are present in a pharmaceutical composition. -67- 170456481.42 Docket No.: 146316.8023.WO00 [0174] In some embodiments, the non-naturally occurring melanocortin analog is present in the pharmaceutical composition in a concentration of 0.1 mg/mL to 500 mg/mL, relative to a total volume of the composition.
  • the non-naturally occurring melanocortin analog is present in the pharmaceutical composition in a concentration of 0.1 mg/mL to 500 mg/mL, 0.5 mg/mL to 250 mg/mL, 1 mg/mL to 100 mg/mL, 2.5 mg/mL to 50 mg/mL, or 5 mg/mL to 25 mg/mL, relative to a total volume of the composition.
  • the non-naturally occurring melanocortin analog is present in the pharmaceutical composition in a concentration of about 50 mg/mL, relative to a total volume of the pharmaceutical composition.
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (IF), and the non-naturally occurring melanocortin analog is present in the pharmaceutical composition in a concentration of 5 mg/mL to 100 mg/mL, relative to a total volume of the pharmaceutical composition.
  • the non-naturally occurring melanocortin analog comprising a sequence of Formula (IF) is present in the pharmaceutical composition in a concentration of 5 mg/mL to 100 mg/mL, 10 mg/mL to 75 mg/mL, 15 mg/mL to 50 mg/mL, 20 mg/mL to 40 mg/mL, or 25 mg/mL to 30 mg/mL, relative to a total volume of the pharmaceutical composition.
  • the non- naturally occurring melanocortin analog comprising a sequence of Formula (IF) is present in the pharmaceutical composition in a concentration of about 50 mg/mL, relative to a total volume of the pharmaceutical composition.
  • the composition comprises the non-naturally occurring melanocortin analog of Formula (I) at a concentration at about 0.001 nmol, 0.005 nmol, 0.01 nmol, 0.02 nmol, 0.05 nmol, 0.1 nmol, 0.25 nmol, 0.5 nmol, 1 nmol, 2.5 nmol, 5 nmol, 10 nmol, 20 nmol, 25 nmol, 50 nmol, 100 nmol, 250 nmol, 500 nmol, or 1000 nmol, or more, depending on the specific peptide selected, the desired response, the route of administration, the formulation and other factors known to those of skill in the art.
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (IF).
  • the non-naturally occurring melanocortin analogs may be formulated with one or more pharmaceutically acceptable carriers and/or excipients.
  • the carriers and/or -68- 170456481.42 Docket No.: 146316.8023.WO00 excipients of the present technology facilitate delivery of the non-naturally occurring melanocortin analog to a subject.
  • Other pharmaceutically acceptable carriers and/or excipients may be included in the pharmaceutical composition to enhance dispersion, solubility, and/or stability of the non-naturally occurring melanocortin analog, and/or to reduce adverse injection site reactions.
  • the pharmaceutical composition comprises 0.1 to 99.9999 wt.%, 1 to 99.999 wt.%, 5 to 99.99 wt.%, 10 to 99.9 wt.%, 15 to 99 wt.%, 20 to 90 wt.%, 30 to 85 wt.%, 40 to 80 wt.%, 50 to 75 wt.%, or 60 to 70 wt.% of the pharmaceutically acceptable carrier and/or excipient relative to a total weight of the pharmaceutical composition.
  • the pharmaceutically acceptable carrier and/or excipient of the pharmaceutical composition is selected from the group consisting of water, a buffer, an inorganic salt, a fatty acid, a vegetable oil, a synthetic fatty ester, a surfactant, and a polymer.
  • the pharmaceutically acceptable carrier and/or excipient of the pharmaceutical composition is water.
  • the pharmaceutically acceptable carrier and/or excipient of the pharmaceutical composition is a buffer.
  • the pharmaceutical composition contains about 0.1 to about 99.9999 wt.%, about 1 to about 99.999 wt.%, about 5 to about 99.99 wt.%, about 10 to about 99.9 wt.%, about 15 to about 99 wt.%, about 20 to about 90 wt.%, about 30 to about 85 wt.%, about 40 to about 80 wt.%, about 50 to about 75 wt.%, or about 60 to about 70 wt.% of one or more pharmaceutically acceptable carriers and/or excipients relative to a total weight of the pharmaceutical composition.
  • the pharmaceutical composition contains at least 0.1 to at least 99.9999 wt.%, at least 1 to at least 99.999 wt.%, at least 5 to at least 99.99 wt.%, at least 10 to at least 99.9 wt.%, at least 15 to at least 99 wt.%, at least 20 to at least 90 wt.%, at least 30 to at least 85 wt.%, at least 40 to at least 80 wt.%, at least 50 to at least 75 wt.%, or at least 60 to at least 70 wt.% of one or more pharmaceutically acceptable carriers and/or excipients relative to a total weight of the pharmaceutical composition.
  • the pharmaceutical composition contains at least about 0.1 to at least about 99.9999 wt.%, at least about 1 to at least about 99.999 wt.%, at least about 5 to at least about 99.99 wt.%, at least about 10 to at least about 99.9 wt.%, at least about 15 to at least about 99 wt.%, at least about 20 to at least about 90 wt.%, at least about 30 to at least about 85 wt.%, at least about 40 to at least about 80 wt.%, at least about 50 to at least about 75 wt.%, or at least about 60 to at least about 70 wt.% of one or more pharmaceutically acceptable carriers and/or excipients relative to a total weight of the pharmaceutical composition.
  • any pharmaceutically acceptable carriers and/or excipients known in the art may be included in the pharmaceutical composition.
  • pharmaceutically acceptable carriers and/or excipients include buffers, binders, excipients, stabilizers, lubricants, oils, adjuvants, preservatives, lipids, and antioxidants.
  • the pharmaceutical composition may comprise any combination of the one or more pharmaceutically acceptable carriers and/or excipients previously described in relation to the first and pharmaceutical compositions.
  • the one or more pharmaceutically acceptable carriers and/or excipients comprise water.
  • the carriers and/or excipients of the composition may generally include one or more of the following components: (i) one or more antioxidants, (ii) one or more preservatives, (iii) one or more buffers, (iv) one or more tonicity adjustors, (v) one or more surfactants, (vi) flavor, (vii) propellants, and/or (viii) a vehicle or solvent.
  • all components are compatible with the non-naturally occurring melanocortin analog (i.e., do not react or cause the non-naturally occurring melanocortin analog to react) and are homogeneously dispersed or dissolved uniformly in the composition.
  • the one or more pharmaceutically acceptable carriers and/or excipients are isotonic. In some embodiments, the carrier and/or excipient is isotonic to nasal fluids.
  • the pharmaceutical composition further comprises a pharmaceutical salt. Any pharmaceutical salt known in the art may be included in the pharmaceutical composition. For examples, to achieve a desirable tonicity, the pharmaceutical composition may include a salt selected from the group consisting of sodium -70- 170456481.42 Docket No.: 146316.8023.WO00 chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate, and calcium chloride.
  • the salt is present in the pharmaceutical composition in a concentration of about 0.1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 25 mg/mL, or about 5 mg/mL to about 10 mg/mL, relative to a total volume of the composition. [0189] In some embodiments, the salt is present in the pharmaceutical composition in a concentration of at least 0.1 mg/mL to at least 50 mg/mL, at least 1 mg/mL to at least 25 mg/mL, or at least 5 mg/mL to at least 10 mg/mL, relative to a total volume of the composition.
  • the salt is present in the pharmaceutical composition in a concentration of at least about 0.1 mg/mL to at least about 50 mg/mL, at least about 1 mg/mL to at least about 25 mg/mL, or at least about 5 mg/mL to at least about 10 mg/mL, relative to a total volume of the composition.
  • Pharmaceutically acceptable carriers and/or excipients that may be included in the pharmaceutical composition generally include a pH buffered aqueous solution comprising one or more of the following components: (a) sodium acetate, (b) Tris, and (c) water.
  • the water may act as a diluent and include, without limitation, water for injection (WFI), sterile water, bacteriostatic water for injection (BWFI), distilled water, bidistilled water, deionized water, deionized distilled water, and reverse osmosis water.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • distilled water bidistilled water
  • deionized water deionized distilled water
  • reverse osmosis water the water present in the pH buffered aqueous solution is water for injection.
  • the pharmaceutical composition includes water in an amount of about 1 wt% to about 90 wt%, about 10 wt% to about 75 wt%, or about 25 wt% to about 50 wt%, relative to a total weight of the composition.
  • the pharmaceutical composition includes water in an amount of at least 1 wt% to at least 90 wt%, at least 10 wt% to at least 75 wt%, or at least 25 wt% to at least 50 wt%, relative to a total weight of the composition.
  • the pharmaceutical composition includes water in an amount of at least about 1 wt% to at least about 90 wt%, at least about 10 wt% to at least about 75 wt%, or at least about 25 wt% to at least about 50 wt%, relative to a total weight of the composition.
  • sodium acetate is present in the pharmaceutical composition in a concentration of 0.5 mg/mL to 50 mg/mL, relative to a total volume of the composition.
  • sodium acetate may be present in the pharmaceutical composition in a concentration of 0.5 mg/mL to 50 mg/mL, 1 mg/mL to 40 mg/mL, 2 mg/mL to 30 mg/mL, 4 mg/mL to 20 mg/mL, 5 mg/mL to 15 mg/mL, 6 mg/mL to 12 mg/mL, or 8 mg/mL to 10 mg/mL, relative to a total volume of the composition.
  • sodium acetate is present in the pharmaceutical composition in a concentration of about 6 mg/mL to about 8 mg/mL, relative to a total volume of the composition.
  • sodium acetate may be present in the pharmaceutical composition in a concentration of 6 mg/mL, 6.5 mg/mL, 7 mg/mL, 7.1 mg/mL, 7.5 mg/mL, or 8 mg/mL, relative to a total volume of the composition.
  • sodium acetate is present in the pharmaceutical composition in a molar concentration of 5 mM to 700 mM, relative to a total volume of the composition.
  • sodium acetate may be present in the pharmaceutical composition in a molar concentration of 5 mM to 700 mM, 10 mM to 600 mM, 20 mM to 500 mM, 30 mM to 400 mM, 40 mM to 300 mM, 50 mM to 200 mM, 60 mM to 100 mM, or 70 mM to 80 mM, relative to a total volume of the composition.
  • sodium acetate is present in the pharmaceutical composition in a molar concentration of about 80 mM to about 100 mM, relative to a total volume of the composition.
  • sodium acetate may be present in the pharmaceutical composition in a molar concentration of 80 mM, 85 mM, 87 mM, 90 mM, 95 mM, or 100 mM, relative to a total volume of the composition.
  • Tris refers to tris(hydroxymethyl)aminomethane, which is also known as Tris buffer, Tris base, TRIS, tromethamine, tromethamine buffer, Trizma®, Trisamine, Trometamol, Tromethane, Trisaminol, or THAM.
  • Tris is present in the pharmaceutical composition in a concentration of 0.5 mg/mL to 50 mg/mL, relative to a total volume of the composition.
  • Tris may be present in the pharmaceutical composition in a concentration of 0.5 mg/mL to 50 mg/mL, 1 mg/mL to 40 mg/mL, 2 mg/mL to 30 mg/mL, 4 mg/mL to 20 mg/mL, 5 mg/mL to 15 mg/mL, 6 mg/mL to 12 mg/mL, or 8 mg/mL to 10 mg/mL, relative to a total volume of the composition.
  • Tris is present in the pharmaceutical composition in a concentration of about 6 mg/mL to about 8 mg/mL, relative to a total volume of the composition.
  • Tris may be present in the pharmaceutical composition in a concentration of 6 mg/mL, 6.5 mg/mL, 7 mg/mL, 7.3 mg/mL, 7.6 mg/mL, or 8 mg/mL, relative to a total volume of the composition.
  • Tris is present in the pharmaceutical composition in a molar concentration of 2 mM to 500 mM, relative to a total volume of the composition.
  • Tris may be present in the pharmaceutical composition in a molar concentration of 2 mM to 500 mM, 5 mM to 400 mM, 10 mM to 300 mM, 20 mM to 200 mM, 30 mM to 150 mM, 40 mM to 100 mM, 50 mM to 80 mM, or 60 mM to 70 mM, relative to a total volume of the composition.
  • Tris is present in the pharmaceutical composition in a molar concentration of about 50 mM to about 70 mM, relative to a total volume of the composition.
  • Tris may be present in the pharmaceutical composition in a molar concentration of 50 mM, 55 mM, 60 mM, 65 mM, or 70 mM, relative to a total volume of the composition.
  • the pH buffered aqueous solution provides the pharmaceutical composition with a pH equivalent or close to the physiological pH levels. This may reduce adverse injection site reactions and also provide the non-naturally occurring melanocortin analog with enhanced stability and resistance to aggregation and degradation.
  • a weight ratio of sodium acetate to Tris is about 1:4 to about 4:1, about 2:7 to about 7:2, about 1:3 to about 3:1, about 2:5 to about 5:2, about 1:2 to about 2:1, about 2:3 to about 3:2, or about 1:1. In some embodiments, the weight ratio of sodium acetate to Tris is about 1:1.
  • a weight ratio of sodium acetate to Tris is at least 1:4 to at least 4:1, at least 2:7 to at least 7:2, at least 1:3 to at least 3:1, at least 2:5 to at least 5:2, at least 1:2 to at least 2:1, at least 2:3 to at least 3:2, or at least 1:1. In some embodiments, the weight ratio of sodium acetate to Tris is at least 1:1.
  • a weight ratio of sodium acetate to Tris is at least about 1:4 to at least about 4:1, at least about 2:7 to at least about 7:2, at least about 1:3 to at least about 3:1, at least about 2:5 to at least about 5:2, at least about 1:2 to at least about 2:1, at least about 2:3 to at least about 3:2, or at least about 1:1. In some embodiments, the weight ratio of sodium acetate to Tris is at least about 1:1.
  • a weight ratio of the non-naturally occurring melanocortin analog to sodium acetate is about 1:1 to about 20:1, about 3:2 to about 15:1, about 2:1 to about 12:1, about 3:1 to about 10:1, about 4:1 to about 9:1, about 5:1 to about 8:1, or about 6:1 to about 7:1. In some embodiments, the weight ratio of the non-naturally occurring melanocortin analog to sodium acetate is about 7:1.
  • a weight ratio of the non-naturally occurring melanocortin analog to sodium acetate is at least 1:1 to at least 20:1, at least 3:2 to at least 15:1, at least 2:1 to at least 12:1, at least 3:1 to at least 10:1, at least 4:1 to at least 9:1, at least 5:1 to at least 8:1, or at least 6:1 to at least 7:1.
  • the weight ratio of the non-naturally occurring melanocortin analog to sodium acetate is at least 7:1.
  • a weight ratio of the non-naturally occurring melanocortin analog to sodium acetate is at least about 1:1 to at least about 20:1, at least about 3:2 to at least about 15:1, at least about 2:1 to at least about 12:1, at least about 3:1 to at least about 10:1, at least about 4:1 to at least about 9:1, at least about 5:1 to at least about 8:1, or at least about 6:1 to at least about 7:1.
  • the weight ratio of the non-naturally occurring melanocortin analog to sodium acetate is at least about 7:1.
  • a weight ratio of the non-naturally occurring melanocortin analog to Tris is about 1:1 to about 20:1, about 3:2 to about 15:1, about 2:1 to about 12:1, about 3:1 to about 10:1, about 4:1 to about 9:1, about 5:1 to about 8:1, or about 6:1 to about 7:1. In some embodiments, the weight ratio of the non-naturally occurring melanocortin analog to Tris is about 7:1.
  • a weight ratio of the non-naturally occurring melanocortin analog to Tris is at least 1:1 to at least 20:1, at least 3:2 to at least 15:1, at least 2:1 to at least 12:1, at least 3:1 to at least 10:1, at least 4:1 to at least 9:1, at least 5:1 to at least 8:1, or at least 6:1 to at least 7:1. In some embodiments, the weight ratio of the non-naturally occurring melanocortin analog to Tris is at least 7:1.
  • a weight ratio of the non-naturally occurring melanocortin analog to Tris is at least about 1:1 to at least about 20:1, at least about 3:2 to at least about 15:1, at least about 2:1 to at least about 12:1, at least about 3:1 to at least about 10:1, at least about 4:1 to at least about 9:1, at least about 5:1 to at least about 8:1, or at least about 6:1 to at least about 7:1.
  • the weight ratio of the non- naturally occurring melanocortin analog to Tris is at least about 7:1.
  • the pharmaceutical composition may include other buffering agents.
  • additional buffering agents include saline, phosphate, phosphoric acid, citrate, succinate, gluconate, histidine, acetic acid, ascorbate, tartartic acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, bicarbonate, carbonic acid, succinic acid, sodium benzoate, benzoic acid, gluconate, edetate, malate, imidazole, and mixtures thereof.
  • the pharmaceutical composition comprises acetic acid as an additional buffering agent.
  • the pharmaceutical composition may further comprise one or more chelating agents.
  • Suitable chelating agents include, but are not limited to edetate disodium dihydrate, calcium disodium edetate, sodium edetate, calcium versetamide sodium, calteridol, and diethylenetriaminepentaacetic acid.
  • the pharmaceutical composition further comprises edetate disodium dihydrate. -75- 170456481.42 Docket No.: 146316.8023.WO00 [0216]
  • the pharmaceutical composition may further comprise a preservative agent.
  • Exemplary preservative agents include, but are not limited to, ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, phenol, m-cresol, benzyl alcohol, alpha-tocopherol, citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, benzalkonium chloride, phenoxyethanol, and methyl paraben.
  • ascorbic acid cysteine hydrochloride
  • sodium bisulfate sodium metabisulfite
  • sodium sulfite sodium sulfite
  • ascorbyl palmitate butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl
  • the preservative agent when the pharmaceutical composition comprises a preservative agent, is phenol, benzyl alcohol, or a combination thereof.
  • the concentration of the preservative agent may range from 0.001 mg/mL to 50 mg/mL, 0.01 mg/mL to 25 mg/mL, 0.1 mg/mL to 10 mg/mL, or 1 mg/mL to 5 mg/mL, relative to a total volume of the composition.
  • the pharmaceutical composition may further comprise an emulsifier.
  • Non- limiting examples of emulsifiers that may be included in the pharmaceutical composition include sodium carboxymethylcellulose, cetyl alcohol, glycerol monostearate, methylcellulose, and stearic acid. In some embodiments, when the pharmaceutical composition comprises an emulsifier, the emulsifier is sodium carboxymethylcellulose.
  • the pharmaceutical composition may further comprise a lipid. Lipids may enhance solubility and/or improve permeability of the non-naturally occurring melanocortin analog. In some embodiments, the lipid is a phospholipid.
  • Non-limiting examples of phospholipids that may be included in the pharmaceutical composition include egg phosphatidylcholine, hydrogenated soybean phoshphaditylcholine, glycerophosphocholine, lecithin, and N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-glycero-3- phosphoethanolamine sodium salt.
  • the pharmaceutical composition when the pharmaceutical composition comprises a lipid, the lipid is N-(carbonyl-methoxypolyethylene glycol 2000)- 1,2-distearoyl-glycero-3-phosphoethanolamine sodium salt
  • the pharmaceutical composition may further comprise a bulking agent. Inclusion of a bulking agent may increase the stability of the pharmaceutical composition.
  • Non-limiting examples of bulking agents that may be included in the pharmaceutical -76- 170456481.42 Docket No.: 146316.8023.WO00 composition include sucrose, lactose, trehalose, mannitol, sorbitol, glucose, raffinose, glycine, histidine, and polyvinyl pyrrolidone.
  • the pharmaceutical composition comprises a bulking agent
  • the bulking agent is mannitol.
  • the pharmaceutical composition is in the form of an aqueous solution or a suspension.
  • the pharmaceutical composition is in the form of an emulsion.
  • the pharmaceutical composition is in the form of an aqueous solution.
  • the pharmaceutical composition is in the form of an aqueous solution which is clear, colorless, and/or free of visible foreign matter.
  • the pharmaceutical composition has a pH ranging from about 6.5 to about 8.5. In some embodiments, the pharmaceutical composition has a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. [0223] In some embodiments, the pharmaceutical composition has a pH ranging from at least 6.5 to at least 8.5.
  • the pharmaceutical composition has a pH of at least 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.
  • the pharmaceutical composition has a pH ranging from at least about 6.5 to at least about 8.5.
  • the pharmaceutical composition has a pH of at least about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.
  • the pharmaceutical composition is basic and has a pH of about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the pharmaceutical composition has a pH ranging from about 7.3 to about 7.4. In some embodiments, the pharmaceutical composition has a pH of 7.3 or 7.4. [0226] In some embodiments, the pharmaceutical composition is basic and has a pH of at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the pharmaceutical composition has a pH ranging from at least 7.3 to at least 7.4.
  • the pharmaceutical composition has a pH of 7.3 or 7.4. -77- 170456481.42 Docket No.: 146316.8023.WO00 [0227]
  • the pharmaceutical composition is basic and has a pH of at least about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.
  • the pharmaceutical composition has a pH ranging from at least about 7.3 to at least about 7.4.
  • the pharmaceutical composition has a pH of 7.3 or 7.4.
  • the pharmaceutical composition has an osmolality ranging from 250 mOsm/kg to 350 mOsm/kg.
  • the pharmaceutical composition may have an osmolality ranging from 250 mOsm/kg to 360 mOsm/kg, 260 mOsm/kg to 340 mOsm/kg, 270 mOsm/kg to 330 mOsm/kg, 280 mOsm/kg to 320 mOsm/kg, 290 mOsm/kg to 310 mOsm/kg, or about 300 mOsm/kg.
  • the pharmaceutical composition has an osmolarity of about 250 mOsm/kg, about 260 mOsm/kg, about 270 mOsm/kg, about 280 mOsm/kg, about 290 mOsm/kg, about 300 mOsm/kg, about 310 mOsm/kg, about 320 mOsm/kg, about 330 mOsm/kg, about 340 mOsm/kg, about 350 mOsm/kg, or about 360 mOsm/kg.
  • the pharmaceutical composition has an osmolality ranging from about 275 mOsm/kg to about 330 mOsm/kg.
  • the pharmaceutical composition has an osmolality of about 279 mOsm/kg, about 314 mOsm/kg, or about 329 mOsm/kg. [0229] In some embodiments, the pharmaceutical composition has an osmolality ranging from 250 mOsm/kg to 350 mOsm/kg.
  • the pharmaceutical composition may have an osmolality ranging from 250 mOsm/kg to 360 mOsm/kg, 260 mOsm/kg to 340 mOsm/kg, 270 mOsm/kg to 330 mOsm/kg, 280 mOsm/kg to 320 mOsm/kg, 290 mOsm/kg to 310 mOsm/kg, or at least 300 mOsm/kg.
  • the pharmaceutical composition has an osmolarity of at least 250 mOsm/kg, at least 260 mOsm/kg, at least 270 mOsm/kg, at least 280 mOsm/kg, at least 290 mOsm/kg, at least 300 mOsm/kg, at least 310 mOsm/kg, at least 320 mOsm/kg, at least 330 mOsm/kg, at least 340 mOsm/kg, at least 350 mOsm/kg, or at least 360 mOsm/kg.
  • the pharmaceutical composition has an osmolality ranging from at least 275 mOsm/kg to at least 330 mOsm/kg. In some embodiments, the pharmaceutical composition has an osmolality of at least 279 mOsm/kg, at least 314 mOsm/kg, or at least 329 mOsm/kg. -78- 170456481.42 Docket No.: 146316.8023.WO00 [0230] In some embodiments, the pharmaceutical composition has an osmolality ranging from 250 mOsm/kg to 350 mOsm/kg.
  • the pharmaceutical composition may have an osmolality ranging from 250 mOsm/kg to 360 mOsm/kg, 260 mOsm/kg to 340 mOsm/kg, 270 mOsm/kg to 330 mOsm/kg, 280 mOsm/kg to 320 mOsm/kg, 290 mOsm/kg to 310 mOsm/kg, or at least about 300 mOsm/kg.
  • the pharmaceutical composition has an osmolarity of at least about 250 mOsm/kg, at least about 260 mOsm/kg, at least about 270 mOsm/kg, at least about 280 mOsm/kg, at least about 290 mOsm/kg, at least about 300 mOsm/kg, at least about 310 mOsm/kg, at least about 320 mOsm/kg, at least about 330 mOsm/kg, at least about 340 mOsm/kg, at least about 350 mOsm/kg, or at least about 360 mOsm/kg.
  • the pharmaceutical composition has an osmolality ranging from at least about 275 mOsm/kg to at least about 330 mOsm/kg. In some embodiments, the pharmaceutical composition has an osmolality of at least about 279 mOsm/kg, at least about 314 mOsm/kg, or at least about 329 mOsm/kg. [0231] In some embodiments, the pharmaceutical composition has a viscosity ranging from about 0.5 cP to about 5 cP.
  • the pharmaceutical composition may have a viscosity ranging from about 0.5 cP to about 5 cP, about 0.75 cP to about 4.5 cP, about 1.0 cP to about 4 cP, about 1.2 cP to about 3.5 cP, about 1.3 cP to about 3 cP, about 1.4 cP to about 2.5 cP, about 1.5 cP to about 2 cP, or about 1.6 cP to about 1.8 cP.
  • the pharmaceutical composition has a viscosity of about 0.5 cP, 0.6 cP, 0.7 cP, 0.8 cP, 0.9 cP, 1.0 cP, 1.1 cP, 1.2 cP, 1.3 cP, 1.4 cP, 1.5 cP, 1.6 cP, 1.7 cP, 1.8 cP, 1.9 cP, or 2.0 cP.
  • the pharmaceutical composition has a viscosity of about 1.4 cP or about 1.6 cP.
  • the pharmaceutical composition has a viscosity ranging from at least 0.5 cP to at least 5 cP.
  • the pharmaceutical composition may have a viscosity ranging from at least 0.5 cP to at least 5 cP, at least 0.75 cP to at least 4.5 cP, at least 1.0 cP to at least 4 cP, at least 1.2 cP to at least 3.5 cP, at least 1.3 cP to at least 3 cP, at least 1.4 cP to at least 2.5 cP, at least 1.5 cP to at least 2 cP, or at least 1.6 cP to at least 1.8 cP.
  • the pharmaceutical composition has a viscosity of at least 0.5 cP, 0.6 cP, 0.7 cP, 0.8 cP, 0.9 cP, 1.0 cP, 1.1 cP, 1.2 cP, 1.3 cP, 1.4 cP, 1.5 cP, -79- 170456481.42 Docket No.: 146316.8023.WO00 1.6 cP, 1.7 cP, 1.8 cP, 1.9 cP, or 2.0 cP. In some embodiments, the pharmaceutical composition has a viscosity of at least 1.4 cP or at least 1.6 cP.
  • the pharmaceutical composition has a viscosity ranging from at least about 0.5 cP to at least about 5 cP.
  • the pharmaceutical composition may have a viscosity ranging from at least about 0.5 cP to at least about 5 cP, at least about 0.75 cP to at least about 4.5 cP, at least about 1.0 cP to at least about 4 cP, at least about 1.2 cP to at least about 3.5 cP, at least about 1.3 cP to at least about 3 cP, at least about 1.4 cP to at least about 2.5 cP, at least about 1.5 cP to at least about 2 cP, or at least about 1.6 cP to at least about 1.8 cP.
  • the pharmaceutical composition has a viscosity of at least about 0.5 cP, 0.6 cP, 0.7 cP, 0.8 cP, 0.9 cP, 1.0 cP, 1.1 cP, 1.2 cP, 1.3 cP, 1.4 cP, 1.5 cP, 1.6 cP, 1.7 cP, 1.8 cP, 1.9 cP, or 2.0 cP. In some embodiments, the pharmaceutical composition has a viscosity of at least about 1.4 cP or at least about 1.6 cP.
  • the pharmaceutical composition disclosed is formulated for parenteral administration, such as, for example, in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions.
  • parenteral includes subcutaneous, intravenous, intraperitoneal, intramuscular, and intralesional, or infusion techniques.
  • the active ingredient(s) e.g., the non- naturally occurring melanocortin analog
  • the aforementioned carrier and/or excipient may be dissolved or suspended in the aforementioned carrier and/or excipient.
  • Additional aqueous or non-aqueous carriers that may facilitate dissolution of the active ingredient include, but are not limited to, ethanol, benzyl alcohol, DMSO, polyethylene glycol, propylene glycol, corn oil, cottonseed oil, peanut oil, sesame oil, and/or various buffers.
  • the pharmaceutical composition is formulated for parenteral administration (e.g., subcutaneous administration) and comprises a non-naturally occurring melanocortin analog in a concentration of about 0.001 nmol, 0.005 nmol, 0.01 nmol, 0.02 nmol, 0.05 nmol, 0.1 nmol, 0.25 nmol, 0.5 nmol, 1 nmol, 2.5 nmol, 5 nmol, 10 nmol, 20 nmol, 25 nmol, 50 nmol, 100 nmol, 250 nmol, 500 nmol, or 1000 nmol, or even -80- 170456481.42 Docket No.: 146316.8023.WO00 more, depending on the specific peptide selected, the desired therapeutic response, the route of administration, the formulation and other factors known to those of skill in the art.
  • the pharmaceutical composition is formulated for parenteral administration (e.g., subcutaneous administration) and comprises sodium acetate in a concentration of about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM
  • the pharmaceutical composition is formulated for parenteral administration (e.g., subcutaneous administration) and comprises Tris in a concentration of about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, or 120 mM.
  • the pharmaceutical composition comprises one or more antioxidants.
  • the pharmaceutical composition may comprise ascorbic acid, cysteine, sodium metabisulfite, propyl gallate, butylated hydroxytoluene, and/or butylated hydroxyanisole.
  • the pharmaceutical composition comprises a surfactant, such as a sorbitan ester.
  • the pharmaceutical composition comprises a flavoring or scent, such as an aromatic oil.
  • the non-naturally occurring melanocortin analog is solubilized or suspended in a solvent or vehicle.
  • the solvent or vehicle may be purified water, ethyl alcohol, and/or propylene glycol.
  • the pharmaceutical composition comprises between 0.03 wt% and 1 wt% melanocortin analog solubilized or suspended in a solvent or vehicle.
  • the pharmaceutical composition may comprise the non-naturally occurring melanocortin analog in an amount of about 0.03 wt%, -81- 170456481.42 Docket No.: 146316.8023.WO00 about 0.05 wt%, about 0.1 wt%, about 0.15 wt%, about 0.2 wt%, about 0.25 wt%, about 0.3 wt%, about 0.35 wt%, about 0.4 wt%, about 0.45 wt%, about 0.5 wt%, about 0.55 wt%, about 0.6 wt%, about 0.65 wt%, about 0.7 wt%, about 0.75 wt%, about 0.8 wt%, about 0.85 wt%, about 0.9 wt%, about 0.95 wt%, or about 1
  • the pharmaceutical composition may comprise the non- naturally occurring melanocortin analog in an amount of at least 0.03 wt%, at least 0.05 wt%, at least 0.1 wt%, at least 0.15 wt%, at least 0.2 wt%, at least 0.25 wt%, at least 0.3 wt%, at least 0.35 wt%, at least 0.4 wt%, at least 0.45 wt%, at least 0.5 wt%, at least 0.55 wt%, at least 0.6 wt%, at least 0.65 wt%, at least 0.7 wt%, at least 0.75 wt%, at least 0.8 wt%, at least 0.85 wt%, at least 0.9 wt%, at least 0.95 wt%, or at least 1 wt%.
  • the pharmaceutical composition may comprise the non- naturally occurring melanocortin analog in an amount of at least about 0.03 wt%, at least about 0.05 wt%, at least about 0.1 wt%, at least about 0.15 wt%, at least about 0.2 wt%, at least about 0.25 wt%, at least about 0.3 wt%, at least about 0.35 wt%, at least about 0.4 wt%, at least about 0.45 wt%, at least about 0.5 wt%, at least about 0.55 wt%, at least about 0.6 wt%, at least about 0.65 wt%, at least about 0.7 wt%, at least about 0.75 wt%, at least about 0.8 wt%, at least about 0.85 wt%, at least about 0.9 wt%, at least about 0.95 wt%, or at least about 1 wt%.
  • the non-naturally occurring melanocortin analogs of the present technology may be formulated for administration using any means known in the art, including orally, rectally, vaginally, ocularly, intranasally, topically, parenterally, or by injection. If administered by injection, the peptide injection may be intravenous (IV), subcutaneous (SC), intramuscular (IM), intraperitoneal (IP), intracerebroventricular (ICV), or other means known in the art.
  • IV intravenous
  • SC subcutaneous
  • IM intramuscular
  • IP intraperitoneal
  • ICV intracerebroventricular
  • the non-naturally occurring melanocortin analog of the combination therapy may be formulated by any means known in the art, including but not limited to formulation as tablets, capsules, caplets, suspensions, powders, lyophilized preparations, suppositories, pessaries, ocular drops, skin patches, orally soluble formulations, enteric formulations, solutions sprays, aerosols and the like, and may be mixed and formulated with buffers, binders, excipients, stabilizers, lubricants, oils, adjuvants, anti-oxidants and other agents known in the art.
  • Administration includes topical delivery.
  • Administration includes delivery across the blood brain barrier.
  • Administration includes delivery through mucous membranes, buccal administration, ophthalmic administration, oral administration, dermal administration, inhalation administration, nasal administration, urethral administration, vaginal administration, rectal administration, and the like.
  • the pharmaceutical composition formulated for intranasal administration comprises a non-naturally occurring melanocortin analog at a concentration at about 0.001 nmol, 0.005 nmol, 0.01 nmol, 0.02 nmol, 0.05 nmol, 0.1 nmol, 0.25 nmol, 0.5 nmol, 1 nmol, 2.5 nmol, 5 nmol, 10 nmol, 20 nmol, 25 nmol, 50 nmol, 100 nmol, 250 nmol, 500 nmol, or 1000 nmol, or more, depending on the specific peptide selected, the desired therapeutic response, the route of administration, the formulation and other factors known to those of skill in the art.
  • the pharmaceutical composition is formulated for oral administration.
  • the pharmaceutical composition may be in the form of a tablet, capsule, lozenge, pill, sachet, or any other orally deliverable form know in the art.
  • the composition may be formulated to be delivered by nose drop, spray device, or topical solution.
  • the pharmaceutical composition may be formulated as an aerosol, atomizer, inhalation, insufflation, metered-dose inhaler, or nebulizer.
  • the pharmaceutical composition includes a propellant, such as hydrofluoroalkane.
  • the pharmaceutical composition may be configured to be administered using a spray device or nasal inhaler.
  • the spray device or nasal inhaler may be configured to deliver 1ug to 100ug per spray.
  • the spray device or nasal inhaler may be configured to deliver 1ug to 100ug, 5ug to 90ug, 10ug to 80ug, 15ug to 70ug, 20ug to 60ug, 25ug, to 50ug, or 30ug to 40ug per spray.
  • the non-naturally occurring melanocortin analog or a pharmaceutical composition thereof is administered hourly (such as every hour, every 2 hours, every 4 hours, every 8 hours, etc.), once a day, -83- 170456481.42 Docket No.: 146316.8023.WO00 or twice a day.
  • the non-naturally occurring melanocortin analog is administered every morning, every evening, or every afternoon.
  • the non-naturally occurring melanocortin analog is administered before a meal, after a meal, or with a meal.
  • the non-naturally occurring melanocortin analog or pharmaceutical composition thereof may be administered as a dosing regimen comprising once, twice, or three times daily administration on a (i) weekly; (ii) every other week; (iii) one week of therapy followed by two, three or four weeks off; (iv) two weeks of therapy followed by one, two, three or four weeks off; (v) three weeks of therapy followed by one, two, three, four or five week off; (vi) four weeks of therapy followed by one, two, three, four or five week off; (vii) five weeks of therapy followed by one, two, three, four or five week off; or (viii) monthly schedule.
  • a dosing regimen comprising once, twice, or three times daily administration on a (i) weekly; (ii) every other week; (iii) one week of therapy followed by two, three or four weeks off; (iv) two weeks of therapy followed by one, two, three or four weeks off; (v) three weeks of therapy followed by one, two, three, four
  • the (i)-(viii) schedules may be repeated 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times or more.
  • the non-naturally occurring melanocortin analog or pharmaceutical composition thereof is administered at various dosages during the dosing regimen (e.g., a first dose with an effective amount of 10 mg/kg and a second dose with an effective amount of 5 mg/kg).
  • the non-naturally occurring melanocortin analog or pharmaceutical composition thereof is administered once every other day, once every 2 or 3 days, once every third day, once per week, once every other week, once every third week, once every month, once every six weeks, once every other month, once every three months, once every six months, or once per year.
  • Administration of the non-naturally occurring melanocortin analog or pharmaceutical composition thereof at any of the dosing frequencies of the present technology may be repeated for a total of about 2 dosages, about 3 dosages, about 4 dosages, about 5 dosages, about 10 dosages, about 15 dosages, about 20 dosages, about 30 dosages, about 40 dosages, about 50 dosages or more.
  • Administration of the non-naturally occurring melanocortin analog or pharmaceutical composition thereof at any of the dosing frequencies of the present technology may be repeated for a total of at least 2 dosages, at least 3 dosages, at least 4 -84- 170456481.42 Docket No.: 146316.8023.WO00 dosages, at least 5 dosages, at least 10 dosages, at least 15 dosages, at least 20 dosages, at least 30 dosages, at least 40 dosages, at least 50 dosages or more.
  • Administration of the non-naturally occurring melanocortin analog or pharmaceutical composition thereof at any of the dosing frequencies of the present technology may be repeated for a total of at least about 2 dosages, at least about 3 dosages, at least about 4 dosages, at least about 5 dosages, at least about 10 dosages, at least about 15 dosages, at least about 20 dosages, at least about 30 dosages, at least about 40 dosages, at least about 50 dosages or more.
  • the frequency of dosages of the non-naturally occurring melanocortin analog or pharmaceutical composition thereof is the same during a treatment regimen. In other embodiments, the frequency of dosages of the non-naturally occurring melanocortin analog or pharmaceutical composition thereof is different during a treatment regimen.
  • the non-naturally occurring melanocortin analog is administered at least once daily in an amount ranging from 0.001 mg/kg to 25 mg/kg, 0.01 mg/kg to 20 mg/kg, 0.05 mg/kg to 15 mg/kg, 0.075 mg/kg to 10 mg/kg, 0.1 mg/kg to 8 mg/kg, 0.2 mg/kg to 6 mg/kg, 0.3 mg/kg to 4 mg/kg, 0.4 mg/kg to 2 mg/kg, or 0.5 mg/kg to 1 mg/kg per body weight of the subject.
  • the non-naturally occurring melanocortin analog is administered at least once daily in an amount ranging from about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 7.5 mg/kg, or about 2.5 mg/kg to about 5 mg/kg per body weight of the subject.
  • the methods disclosed herein may be performed on the subject for about 1 day, 1 week, 1 month, 3 months, 6 months, 1 year, or 5 years.
  • the combination therapy (i.e., pharmaceutical combination) is administered to the subject for about 1 day, about 2 days, about 5 days, about 6 days, about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years.
  • the methods disclosed herein may be performed on the subject for at least 1 day, 1 week, 1 month, 3 months, 6 months, 1 year, or 5 years.
  • the combination therapy (i.e., pharmaceutical combination) is administered to the subject for at least 1 day, at least 2 days, at least 5 days, at least 6 days, at least 1 week, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.
  • the methods disclosed herein may be performed on the subject for at least about 1 day, 1 week, 1 month, 3 months, 6 months, 1 year, or 5 years.
  • the combination therapy i.e., pharmaceutical combination
  • a method disclosed herein is performed on the subject for 1 day, 7 days, 14 days, 21 days, 28 days, 35 days, 40 days, 45 days, 50 days, 60 days, 75 days, 90 days, 100 days, 110 days, or 120 days.
  • at least one active ingredient of the combination therapy is administered to a subject in a continuous doing schedule.
  • a “continuous dosing schedule” is an administration or dosing regimen without dose interruptions, e.g., without days off treatment. Repetition of 21-day or 28-day treatment cycles without dose interruptions is an exemplary continuous dosing schedule.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 15 mg/kg to about 100 mg/kg per body weight of the subject once daily. In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 30 mg/kg or 60 mg/kg per body weight of the subject once daily. -86- 170456481.42 Docket No.: 146316.8023.WO00 [0264] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 0.1 mg to about 100 mg once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 10 mg or about 50 mg once daily. [0265] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 5 mg/kg to about 1000 mg/kg per body weight of the subject once daily. In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 10 mg/kg to about 550 mg/kg per body weight of the subject once daily. In some embodiments, non-naturally occurring melanocortin analog is administered at a dose of about 15 mg/kg to about 100 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 20 mg/kg to about 75 mg/kg per body weight of the subject once daily. In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 30 mg/kg or 60 mg/kg per body weight of the subject once daily. [0266] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least 5 mg/kg to at least 1000 mg/kg per body weight of the subject once daily. In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least 10 mg/kg to at least 550 mg/kg per body weight of the subject once daily.
  • non-naturally occurring melanocortin analog is administered at a dose of at least 15 mg/kg to at least 100 mg/kg per body weight of the subject once daily. In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least 20 mg/kg to at least 75 mg/kg per body weight of the subject once daily. In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least 30 mg/kg or 60 mg/kg per body weight of the subject once daily. [0267] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least about 5 mg/kg to at least about 1000 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of at least about 10 mg/kg to at least about -87- 170456481.42 Docket No.: 146316.8023.WO00 550 mg/kg per body weight of the subject once daily. In some embodiments, non-naturally occurring melanocortin analog is administered at a dose of at least about 15 mg/kg to at least about 100 mg/kg per body weight of the subject once daily. In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least about 20 mg/kg to at least about 75 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of at least about 30 mg/kg or 60 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 50 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 250 mg/kg, about 300 mg/kg, about 350 mg/kg, about 400 mg/kg, about 450 mg/kg, or about 500 mg/kg, about 550 mg/kg, about 600 mg/kg, about
  • the non-naturally occurring melanocortin analog is administered at a dose of at least 5 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, at least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 50 mg/kg, at least 65 mg/kg, at least 70 mg/kg, at least 75 mg/kg, at least 80 mg/kg, at least 85 mg/kg, at least 90 mg/kg, at least 95 mg/kg, at least 100 mg/kg, at least 125 mg/kg, at least 150 mg/kg, at least 175 mg/kg, at least 200 mg/kg, at least 250 mg/kg, at least 300 mg/kg, at least 350 mg/kg, at least 400 mg/kg, at least 450 mg/kg, or at least 500 mg/kg, at least 550 mg/kg, at least 600 mg/kg, at least 650 mg/kg, at least 700 mg/kg, at least
  • the non-naturally occurring melanocortin analog is administered at a dose of at least about 5 mg/kg, at least about 10 mg/kg, at least about 15 mg/kg, at least about 20 mg/kg, at least about 25 mg/kg, at least about 30 mg/kg, at least about 35 mg/kg, at least about 40 mg/kg, at least about 50 mg/kg, at least about 65 mg/kg, -88- 170456481.42 Docket No.: 146316.8023.WO00 at least about 70 mg/kg, at least about 75 mg/kg, at least about 80 mg/kg, at least about 85 mg/kg, at least about 90 mg/kg, at least about 95 mg/kg, at least about 100 mg/kg, at least about 125 mg/kg, at least about 150 mg/kg, at least about 175 mg/kg, at least about 200 mg/kg, at least about 250 mg/kg, at least about 300 mg/kg, at least about 350 mg/kg, at least about 400 mg/kg
  • the non-naturally occurring melanocortin analog is administered at a dose of about 5 mg/kg per body weight of the subject once daily. [0272] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 10 mg/kg per body weight of the subject once daily. [0273] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 15 mg/kg per body weight of the subject once daily. [0274] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 20 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 25 mg/kg per body weight of the subject once daily. [0276] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 30 mg/kg per body weight of the subject once daily. [0277] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 35 mg/kg per body weight of the subject once daily. [0278] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 40 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 50 mg/kg per body weight of the subject once daily. -89- 170456481.42 Docket No.: 146316.8023.WO00 [0280] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least 5 mg/kg per body weight of the subject once daily. [0281] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least 10 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of at least 15 mg/kg per body weight of the subject once daily. [0283] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least 20 mg/kg per body weight of the subject once daily. [0284] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least 25 mg/kg per body weight of the subject once daily. [0285] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least 30 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of at least 35 mg/kg per body weight of the subject once daily. [0287] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least 40 mg/kg per body weight of the subject once daily. [0288] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least 50 mg/kg per body weight of the subject once daily. [0289] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least about 5 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of at least about 10 mg/kg per body weight of the subject once daily. [0291] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least about 15 mg/kg per body weight of the subject once daily. [0292] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of at least about 20 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of at least about 25 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of at least about 30 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of at least about 35 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of at least about 40 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of at least about 50 mg/kg per body weight of the subject once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 50 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 250 mg/kg, about 300 mg/kg, about 350 mg/kg, about 400 mg/kg, about 450 mg/kg, about 500 mg/kg, about 550 mg/kg, about 600 mg/kg, about 650 mg/kg, about 700 mg/kg, about 750 mg/kg, about 800 mg/kg, about 850 mg/kg, about 900 mg/kg, about 950 mg/kg, or
  • the non-naturally occurring melanocortin analog is administered at a dose of about 5 mg/kg per body weight of the subject twice daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 10 mg/kg per body weight of the subject twice daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 15 mg/kg per body weight of the subject twice daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 20 mg/kg per body weight of the subject twice daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 25 mg/kg per body weight of the subject twice daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 30 mg/kg per body weight of the subject twice daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 35 mg/kg per body weight of the subject twice daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 40 mg/kg per body weight of the subject twice daily. [0307] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 50 mg/kg per body weight of the subject twice daily. [0308] In some embodiments, the non-naturally occurring melanocortin analog is administered at one or more doses. In some embodiments, the non-naturally occurring melanocortin analog is administered at two or more doses. [0309] In some embodiments, the non-naturally occurring melanocortin analog is administered at a first dose once daily or twice daily.
  • the non- naturally occurring melanocortin analog is administered at a second dose once daily or twice daily.
  • the non-naturally occurring melanocortin analog is administered at a dose that is a projected human equivalent dose (HED) based on a nonhuman primate dose.
  • HED human equivalent dose
  • the HED based on a 10 mg/kg nonhuman primate dose ranges from about 300 mg to about 550 mg.
  • the non-naturally occurring melanocortin analog is administered at a first dose once daily for about 5 days to about 10 days.
  • the non-naturally occurring melanocortin analog is administered at a second dose once daily for about 5 days to about 10 days, after administration of the first dose. In some embodiments, the non-naturally occurring melanocortin analog is administered at a third dose twice daily for about 7 days to about 21 days, after administration of the second dose. In some embodiments, the second dose is greater than the first dose. In some -92- 170456481.42 Docket No.: 146316.8023.WO00 embodiments, the third dose comprises a cumulative dose that is greater than the second dose.
  • the non-naturally occurring melanocortin analogs or a pharmaceutical composition thereof is administered at a second dose that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% greater than the first dose.
  • the non-naturally occurring melanocortin analogs or a pharmaceutical composition thereof is administered at a third dose that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% greater than the first dose and/or the second dose.
  • the dosage of the second dose is greater than the dosage of the first dose.
  • the first dose is administered about once a day and the second dose is administered about twice a day.
  • the dosage of the third dose is greater than the dosage of the first dose and/or the second dose. In some embodiments, the first dose and/or the second dose is administered about once a day, and the third dose is administered about twice a day.
  • the non-naturally occurring melanocortin analog is administered using two or more different administration routes. The two or more different administration routes may comprise an oral administration and a subcutaneous administration. The oral administration may occur before, during, or after the subcutaneous administration.
  • the first dose and the second dose comprise different routes of administration. In some embodiments, the first dose comprises an oral dose and the second dose comprises a subcutaneous dose.
  • the first dose and/or second dose and the third dose comprise different routes of administration.
  • the first dose and/or the second dose comprises an oral dose and the third dose comprises a subcutaneous dose.
  • the second dose is administered at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks after the first dose.
  • the third dose is administered at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks after the first dose and/or the second dose.
  • the non-naturally occurring melanocortin analog or a pharmaceutical composition thereof e.g., the pharmaceutical composition
  • the first dosage amount, the second dosage amount, and the third dosage amount may each be greater than, less than, equal to, or substantially equivalent to another dosage amount.
  • the first dosage amount is less than a second dosage amount which is greater than a third dosage amount also greater than the first dosage amount.
  • the dosage amounts refer to doses administered more than once over a period of time, such as a first dosage amount administered daily, BID, TID, or weekly, a second dosage amount administered daily, BID, TID, or weekly, and a third dosage amount administered daily, BID, TID, or weekly.
  • the first dosage amount, the second dosage amount, or the third dosage amount may be administered every other day, every second day, every third day, twice per week, or twice per month.
  • the first dosage amount of 10 mg/kg may be administered once a day for 7 days, followed by the second dosage amount of 20 mg/kg once a day for 7 days, followed by the third dosage amount of 15 mg/kg twice a day for at least about 7 days.
  • the third dosage amount is administered for at least about 7 days, at least about 14 days, or at least about 21 days.
  • the non-naturally occurring melanocortin analog or a pharmaceutical composition thereof e.g., the pharmaceutical composition
  • the first dosage -94- 170456481.42 Docket No.: 146316.8023.WO00 amount and the second dosage amount may be administered by two different routes of administration.
  • first dosage amount and the second dosage amount administered via different routes of administration may be the same or different dosage amounts.
  • the first dosage amount is administered orally and the second dosage amount is administered subcutaneously.
  • the dosage frequency and/or duration of the dosage amounts may be the same or may be different.
  • the frequency of each dosage amount may be more than once per day, once per day, once every other day, once per week, or once every other week, or once per month.
  • the duration of each dosage amount may be one day, two days, three days, more than three days, one week, more than one week, two weeks, more than two weeks, one month, or more than one month.
  • a dosing regimen may comprise a first dosage amount administered once a day or twice a day for about 5 weeks, and a second dosage amount administered subcutaneously once a day or twice a day for about 1 week.
  • the second dosage amount is administered the day after the duration of the first dosage amount is complete, or two days, three days, more than three days, one week, more than one week, two weeks, more than two weeks, one month, or more than one month after the first dosage amount is complete.
  • the second dosage amount is administered at least about 1 week after administration of the first dosage is complete.
  • the non-naturally occurring melanocortin analog or a pharmaceutical composition thereof is administered at a dose of (i) 10 mg/kg once a day for 7 days, followed by (ii) 20 mg/kg once a day for 7 days, followed by (iii) 15 mg/kg twice a day for at least about 7 days.
  • (iii) is administered for at least about 7 days, at least about 14 days, or at least about 21 days.
  • the non-naturally occurring melanocortin analog or a pharmaceutical composition thereof comprises a (i) first dose administered orally once a day or twice a day for about 5 weeks, and (ii) second dose administered subcutaneously once a day or twice a day for about 1 week.
  • the second dose is administered at least about 1 week after administration of the first dose is complete.
  • the non-naturally occurring melanocortin analog is administered (i) at a first dose of about 5 mg/kg to about 20 mg/kg administered once a day for about 5 days to about 10 days; (ii) at a second dose of about 10 mg/kg to about 40 mg/kg administered once a day after administration of the first dose for about 5 days to about 10 days; (iii) at a third dose of about 5 mg/kg to about 30 mg/kg administered twice a day after administration of the second dose for at least about 5 days to about 10 days. In some embodiments, the third dose is administered twice a day for at least about 7 days or more.
  • the non-naturally occurring melanocortin analog is administered (i) at a first dose of about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, or about 20 mg/kg administered once a day for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days; (ii) at a second dose of about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, or about 40 mg/kg administered once a day after administration of the first dose about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days; (iii) at a third dose of about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, or about 30 mg/kg administered twice a day after administration of the second dose for at least about 5 days, about, 6 days, about 7 days, about 8 days, about
  • the third dose is administered twice a day for at least about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days or more.
  • the non-naturally occurring melanocortin analog is administered (i) at a first dose of about 5 mg/kg administered once a day for about 7 days; (ii) at a second dose of about 15 mg/kg administered once a day after administration of the first dose for about 7 days; (iii) at a third dose of about 10 mg/kg administered twice a day after administration of the second dose for at least about 7 days.
  • the third dose is administered twice a day for at least about 14 days or more.
  • the non-naturally occurring melanocortin analog is administered (i) at a first dose of about 10 mg/kg administered once a day for about 7 days; (ii) at a second dose of about 20 mg/kg administered once a day after administration of the first dose for about 7 days; (iii) at a third dose of about 15 mg/kg administered twice a day -96- 170456481.42 Docket No.: 146316.8023.WO00 after administration of the second dose for at least about 7 days.
  • the third dose is administered twice a day for at least about 14 days or more.
  • the non-naturally occurring melanocortin analog is administered (i) at a first dose of about 15 mg/kg administered once a day for about 7 days; (ii) at a second dose of about 25 mg/kg administered once a day after administration of the first dose for about 7 days; (iii) at a third dose of about 20 mg/kg administered twice a day after administration of the second dose for at least about 7 days. In some embodiments, the third dose is administered twice a day for at least about 14 days or more. [0329] In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 0.1 mg to about 100 mg once daily.
  • the non-naturally occurring melanocortin analog is administered at a dose of about 0.5 mg to about 75 mg once daily. In some embodiments, the non-naturally occurring melanocortin analog is administered at a dose of about 5 mg to about 60 mg once daily. In some embodiments, wherein the non-naturally occurring melanocortin analog is administered at a dose of about 10 mg or about 50 mg once daily. [0330] In some embodiments, the non-naturally occurring melanocortin analog is administered as a single dosage unit. In some embodiments, the non-naturally occurring melanocortin analog dose is administered as multiple dosage units.
  • the multiple dosage units individually comprise about 2.5 mg/mL to about 100 mg/mL of the non-naturally occurring melanocortin analog. In some embodiments, the multiple dosage units individually comprise about 2.5 mg/mL to about 10 mg/mL of the non-naturally occurring melanocortin analog. In some embodiments, the multiple dosage units individually comprise about 6 mg/mL of the non-naturally occurring melanocortin analog. In some embodiments, the non-naturally occurring melanocortin analog is administered as about 5 dosage units to about 10 dosage units. In some embodiments, the non-naturally occurring melanocortin analog is administered as 5 dosage units. In some embodiments, the non-natural occurring melanocortin analog is administered as 10 dosage units.
  • the non-naturally occurring melanocortin analog is present in the pharmaceutical composition in a concentration of 5 mg/mL to 100 mg/mL, relative to a total volume of the pharmaceutical composition.
  • the non- -97- 170456481.42 Docket No.: 146316.8023.WO00 naturally occurring melanocortin analog is present in the pharmaceutical composition in a concentration of about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, or about 60 mg/mL, relative to a total volume of the pharmaceutical composition.
  • the non-naturally occurring melanocortin analog is present in the pharmaceutical composition in a concentration of about 50 mg/mL, relative to a total volume of the pharmaceutical composition. [0332] In some embodiments, the non-naturally occurring melanocortin analog is present in the pharmaceutical composition at a dose of about 5 mg to about 1000 mg in a dose volume of about 1 mL to 20 mL, and wherein the administration is performed once, twice, 3 times, 4 times, 5 times, or 10 times per day.
  • the non-naturally occurring melanocortin analog is present in the pharmaceutical composition at a dose of about 50 mg to about 100 mg in a dose volume of about 2 mL to 10 mL, and wherein the administration is performed once, twice, 3 times, 4 times, 5 times, or 10 times per day. In some embodiments, the non-naturally occurring melanocortin analog is present in the pharmaceutical composition at a dose of about 75 mg in a dose volume of about 5 mL, and wherein the administration is performed once, twice, 3 times, 4 times, 5 times, or 10 times per day. In some embodiments, the administration is performed once per day.
  • the present technology also provides methods of treating, preventing, reducing, or otherwise ameliorating one or more symptoms or conditions associated with metabolic dysfunction comprising administering a non-naturally occurring melanocortin analog to a subject in need thereof.
  • the methods of the present technology may promote fat loss, prevent muscle loss, decrease body weight, accelerate weight loss, decrease fat mass to lean mass ratio, improve body composition and/or BMI, reduce waist circumference, or any combination thereof.
  • the present technology comprises methods of reducing body weight and/or fat mass in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog of the present technology to the subject.
  • the present technology comprises methods of reducing lean mass loss, maintaining lean mass, or promoting lean mass gain in the subject.
  • the lean mass is lean muscle mass.
  • the present technology comprises methods of treating, preventing, reducing, or otherwise ameliorating glucose intolerance and/or diabetes mellitus in the subject.
  • the present technology comprises a method of reducing body weight and/or fat mass in an obese subject (e.g., an obese subject without diabetes mellitus or an obese subject having diabetes mellitus), comprising administering a non- naturally occurring melanocortin analog to the subject.
  • the methods of the present technology (i) reduce a lipid droplets size (e.g., an average lipid droplet size); (ii) increase an adipose tissue mitochondrial number or adipose tissue mitochondrial concentration; (iii) increase an adipose tissue vascularization level; (iv) increase, maintain, or prevent reduction of a beige or brown adipocyte level; (v) increase a metabolic rate; (vi) increase a calorie burn level; and/or (vii) increase, maintain, or prevent reduction of an insulin sensitivity level, relative to a control.
  • a lipid droplets size e.g., an average lipid droplet size
  • the methods of the present technology comprise reducing a lipid droplet, or an average lipid droplet, size by about 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or 95%, relative to a control.
  • the methods of the present technology comprise reducing a lipid droplet, or an average lipid droplet, size by at least 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or 95%, relative to a control.
  • the methods of the present technology comprise reducing a lipid droplet, or an average lipid droplet, size by at least about 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or 95%, relative to a control.
  • the methods of the present technology comprise one or more of (i) increasing an adipose tissue mitochondrial number; (ii) increasing an adipose tissue mitochondrial concentration; (iii) increasing an adipose tissue vascularization level; -99- 170456481.42 Docket No.: 146316.8023.WO00 increasing a beige adipocyte level; (iv) increasing a brown adipocyte level; (v) increasing a metabolic rate; (vi) increasing a calorie burn level; or (vii) increasing an insulin sensitivity level, by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more, relative to a control.
  • the methods of the present technology comprise one or more of (i) increasing an adipose tissue mitochondrial number; (ii) increasing an adipose tissue mitochondrial concentration; (iii) increasing an adipose tissue vascularization level; increasing a beige adipocyte level; (iv) increasing a brown adipocyte level; (v) increasing a metabolic rate; (vi) increasing a calorie burn level; or (vii) increasing an insulin sensitivity level, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more, relative to a control.
  • the methods of the present technology comprise one or more of (i) increasing an adipose tissue mitochondrial number; (ii) increasing an adipose tissue mitochondrial concentration; (iii) increasing an adipose tissue vascularization level; increasing a beige adipocyte level; (iv) increasing a brown adipocyte level; (v) increasing a metabolic rate; (vi) increasing a calorie burn level; or (vii) increasing an insulin sensitivity level, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more, relative to a control.
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating hyperplasia in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating hypothalamic obesity (e.g., congenital hypothalamic damage and/or or damage from tumors or trauma) in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • hypothalamic obesity e.g., congenital hypothalamic damage and/or or damage from tumors or trauma
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating a proopiomelanocortin (POMC) deficiency in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • POMC proopiomelanocortin
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating a leptin deficiency in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating a syndromic obesity in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • syndromic obesity include Prader–Willi syndrome, Wilms tumor, Aniridia, Genitourinary, Range of Developmental Delays (WAGR) syndrome, Bardet- Biedl syndrome, Fragile X syndrome, Cohen syndrome, Proprotein Convertase Subtilisin/Kexin Type 1 (PCSK1) deficiency, Alstrom syndrome, MC4 haploinsufficiency, 17p11.2 deletion syndrome, and 2q37 deletion syndrome.
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating non-alcoholic steatohepatitis (NASH) (i.e., metabolic dysfunction-associated steatohepatitis (MASH)), in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • NASH non-alcoholic steatohepatitis
  • MASH metabolic dysfunction-associated steatohepatitis
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating hyperinsulinism in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating Type 2 Diabetes in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating a cardiovascular disease (e.g., stroke and/or heart attack) in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • a cardiovascular disease e.g., stroke and/or heart attack
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating osteoarthritis in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • a method of preventing, reducing, or otherwise ameliorating a cancer e.g., decreasing a risk of cancer
  • administering a non-naturally occurring melanocortin analog to the subject comprising administering a non-naturally occurring melanocortin analog to the subject.
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating erectile dysfunction in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • the method further comprises reducing obesity-related inflammation in the subject.
  • method further comprises preserving or improving kidney function in the subject.
  • the present technology also provides methods of reducing obesity-related inflammation in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin analog to the subject.
  • the present technology further provides methods of preserving or improving kidney function in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin analog to the subject.
  • the method comprises orally administering a non-naturally occurring melanocortin analog comprising a sequence of Formula (I) to the subject. Any of the sequences of Formula (I) listed above may be used in the methods of the present technology.
  • the method comprises orally administering a non- naturally occurring melanocortin analog comprising a sequence of Formula (IA) to the subject. Any of the sequences of Formula (IA) listed above may be used in the methods of the present technology.
  • the present technology also provides methods of reducing body weight and/or fat mass in a subject in need thereof, comprising orally administering about 15 mg/kg to about 100 mg/kg of a non-naturally occurring melanocortin analog to the subject.
  • the non-naturally occurring melanocortin analog comprises a sequence of -102- 170456481.42 Docket No.: 146316.8023.WO00 Formula (I).
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (IA).
  • the present technology also provides methods of treating, preventing, or reducing glucose intolerance and/or diabetes mellitus in a subject in need thereof, comprising orally administering about 15 mg/kg to about 100 mg/kg of a non-naturally occurring melanocortin analog to the subject.
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (I).
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (IA).
  • the present technology also provides methods of reducing obesity-related inflammation in a subject in need thereof, comprising orally administering about 15 mg/kg to about 100 mg/kg of a non-naturally occurring melanocortin analog to the subject.
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (I). In some embodiments, the non-naturally occurring melanocortin analog comprises a sequence of Formula (IF).
  • the present technology also provides methods of preserving or improving kidney function in a subject in need thereof, comprising orally administering about 15 mg/kg to about 100 mg/kg of a non-naturally occurring melanocortin analog to the subject.
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (I). In some embodiments, the non-naturally occurring melanocortin analog comprises a sequence of Formula (IF).
  • the present technology also provides methods of reducing body weight and/or fat mass in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin analog having a sequence selected from the group consisting of SEQ ID NOs: 11, 12, and 43-45 to the subject.
  • the method further comprises reducing lean mass loss, maintaining lean mass, or promoting lean mass gain in the subject.
  • the lean mass is lean muscle mass.
  • the method further comprises treating, preventing, or reducing glucose intolerance and/or diabetes mellitus in the subject.
  • the present technology also provides methods of treating, preventing, or reducing glucose intolerance and/or diabetes mellitus in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin analog having a sequence selected from the group consisting of SEQ ID NOs: 11, 12, and 43-45 to the subject.
  • the method further comprises reducing lean mass loss, maintaining lean mass, or promoting lean mass gain in the subject.
  • the lean mass is lean muscle mass.
  • the method further comprises reducing body weight and/or fat mass in the subject.
  • the method further comprises reducing obesity-related inflammation in the subject. In some embodiments, the method further comprises preserving or improving kidney function in the subject. [0372] The present technology also provides methods of reducing obesity-related inflammation in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin analog to the subject. [0373] The present technology also provides methods of preserving or improving kidney function in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin analog to the subject.
  • the subject’s body weight is more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, or more than 40% reduced from baseline prior to administration of the non-naturally occurring melanocortin analog.
  • the subject’s fat mass is more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, or more than 40% reduced from baseline prior to administration of the non-naturally occurring melanocortin analog.
  • the subject exhibits about a 1%, about a 2%, about a 3%, about a 4%, about a 5%, about a 6%, about a 7%, about an 8%, about a 9%, or about a 10% reduction in body weight about 10 days, about 20 days, or about 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology.
  • the subject exhibits at least a 1%, at least a 2%, at least a 3%, at least a 4%, at least a 5%, at least a 6%, at least a 7%, at least an 8%, at least a 9%, or at least a 10% reduction in body weight at least 10 days, at least 20 days, or at least 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology.
  • the subject exhibits at least about a 1%, at least about a 2%, at least about a 3%, at least about a 4%, at least about a 5%, at least about a 6%, at least about a 7%, at least about an 8%, at least about a 9%, or at least about a 10% reduction in body weight at least about 10 days, at least about 20 days, or at least about 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology.
  • the subject exhibits about a 1%, about a 2%, about a 3%, about a 4%, about a 5%, about a 6%, about a 7%, about an 8%, about a 9%, about a 10%, about a 12.5%, about a 15%, about a 17.5%, about a 20%, about a 25%, or about a 30% reduction in fat mass about 10 days, about 20 days, or about 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology.
  • the subject exhibits at least a 1%, at least a 2%, at least a 3%, at least a 4%, at least a 5%, at least a 6%, at least a 7%, at least an 8%, at least a 9%, at least a 10%, at least a 12.5%, at least a 15%, at least a 17.5%, at least a 20%, at least a 25%, or at least a 30% reduction in fat mass at least 10 days, at least 20 days, or at least 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology.
  • the subject exhibits at least about a 1%, at least about a 2%, at least about a 3%, at least about a 4%, at least about a 5%, at least about a 6%, at least about a 7%, at least about an 8%, at least about a 9%, at least about a 10%, at least about a 12.5%, at least about a 15%, at least about a 17.5%, at least about a 20%, at least about a 25%, or at least about a 30% reduction in fat mass at least about 10 days, at least about 20 days, or at least about 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology.
  • the subject exhibits no change, about a 1%, about a 2%, about a 3%, about a 4%, about a 5%, about a 6%, about a 7%, about an 8%, about a 9%, or about a 10% reduction in lean mass loss about 10 days, about 20 days, or about 30 days after administration of a first dose of a non- naturally occurring melanocortin analog of the present technology.
  • the subject exhibits no change, at least a 1%, at least a 2%, at least a 3%, at least a 4%, at least a 5%, at least a 6%, at least a 7%, at least an 8%, at least a 9%, or at least a 10% reduction in lean mass loss at least 10 days, at least 20 days, or at least 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology.
  • the subject exhibits no change, at least about a 1%, at least about a 2%, at least about a 3%, at least about a 4%, at least about a 5%, at least about a 6%, at least about a 7%, at least about an 8%, at least about a 9%, or at least about a 10% reduction in lean mass loss at least about 10 days, at least about 20 days, or at least about 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology.
  • the subject exhibits no change, about a 1%, about a 2%, about a 3%, about a 4%, about a 5%, about a 6%, about a 7%, about an 8%, about a 9%, or about a 10% increase in lean mass about 10 days, about 20 days, or about 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology.
  • the subject exhibits no change, at least a 1%, at least a 2%, at least a 3%, at least a 4%, at least a 5%, at least a 6%, at least a 7%, at least an 8%, at least a 9%, or at least a 10% increase in lean mass at least 10 days, at least 20 days, or at least 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology.
  • the subject exhibits no change, at least about a 1%, at least about a 2%, at least about a 3%, at least about a 4%, at least about a 5%, at least about a 6%, at least about a 7%, at least about an 8%, at least about a 9%, or at least about a 10% increase in lean mass at least about 10 days, at least about 20 days, or at least about 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology.
  • the subject exhibits about a 5%, about a 10%, about a 12.5%, about a 15%, about a 17.5%, about a 20%, about a 25%, about a 30%, about a 35%, about a 40%, about a 45%, about a 50%, about a 60%, or about a 70% reduction in food intake per day about 10 days, about 20 days, or about 30 days after administration of a first dose of the combination therapy.
  • the reduction in food intake occurs about 0.5 hours, about 1 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or about 6 weeks after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology.
  • Food intake may reflect cumulative food intake, net food intake, or both. Cumulative food intake may represent total food intake. Net food intake may represent the difference between total food intake and energy expenditure.
  • Cumulative and net food intake may represent a food intake measurement from start to end of dosing, from start to an intermediate time point of dosing (i.e., a time point after the start of dosing but prior to the end of dosing), from an intermediate time point to end of dosing, or between two or more intermediate time points.
  • -107- 170456481.42 Docket No.: 146316.8023.WO00 The methods of the present technology alter a subject’s preference for food having high-fat content or low-fat content or is otherwise calorically dense or less calorically dense.
  • the methods of the present technology increase a subject’s preference for one or more of a lower-fat food or a less calorically dense food relative to the food preference of the subject at baseline or of a control. In some embodiments, the methods of the present technology decrease the subject’s preference for one or more of a higher-fat food or a more calorically dense food relative to the food preference of the subject at baseline or of a control. [0391] In some embodiments of the methods of the present technology, the blood glucose level of the subject is decreased following administration of the combination therapy. In some embodiments, the blood glucose level of the subject is decreased following administration of a first dose of the combination therapy.
  • the subject exhibits at least about a 1%, about a 2%, about a 3%, about a 4%, about a 5%, about a 6%, about a 7%, about an 8%, about a 9%, about a 10%, about a 15%, about a 20%, about a 25%, or about a 30% reduction in a blood glucose level about 1 day, 2 days, 3 days, 4 days, 8 days, 10 days, 12 days, 14 days, 20 days, or about 30 days after administration of a first dose of a non-naturally occurring melanocortin analog, relative to a control.
  • the subject exhibits at least at least a 1%, at least a 2%, at least a 3%, at least a 4%, at least a 5%, at least a 6%, at least a 7%, at least an 8%, at least a 9%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, or at least a 30% reduction in a blood glucose level at least 1 day, 2 days, 3 days, 4 days, 8 days, 10 days, 12 days, 14 days, 20 days, or at least 30 days after administration of a first dose of a non-naturally occurring melanocortin analog, relative to a control.
  • the subject exhibits at least about at least about a 1%, at least about a 2%, at least about a 3%, at least about a 4%, at least about a 5%, at least about a 6%, at least about a 7%, at least about an 8%, at least about a 9%, at least about a 10%, at least about a 15%, at least about a 20%, at least about a 25%, or at least about a 30% reduction in a blood glucose level at least about 1 day, 2 days, 3 days, 4 days, 8 days, -108- 170456481.42 Docket No.: 146316.8023.WO00 10 days, 12 days, 14 days, 20 days, or at least about 30 days after administration of a first dose of a non-naturally occurring melanocortin analog, relative to a control.
  • the subject of the methods of the present technology may experience no change in or a reduction in one or more of an alkaline phosphatase level, a total protein level, an albumin level, a globulin level, a gamma-glutamyl transferase (GGT), a glucose level, a urea nitrogen level, a creatine level, a urea acid level, or a C-reactive protein level in a biological sample from the subject.
  • biological samples include a circulatory fluid sample (e.g., blood, serum, lymphatic fluid), a urine sample, or a tissue sample.
  • the subject exhibits at least about a 1%, about a 2%, about a 3%, about a 4%, about a 5%, about a 6%, about a 7%, about an 8%, about a 9%, about a 10%, about a 15%, about a 20%, about a 25%, or about a 30% reduction in an alkaline phosphatase level, a total protein level, an albumin level, a globulin level, a GGT, a glucose level, a urea nitrogen level, a creatine level, a urea acid level, or a C-reactive protein level in the biological sample about 1 day, 2 days, 3 days, 4 days, 8 days, 10 days, 12 days, 14 days, 20 days, or about 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology, relative to a control.
  • the subject exhibits at least at least a 1%, at least a 2%, at least a 3%, at least a 4%, at least a 5%, at least a 6%, at least a 7%, at least an 8%, at least a 9%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, or at least a 30% reduction in an alkaline phosphatase level, a total protein level, an albumin level, a globulin level, a GGT, a glucose level, a urea nitrogen level, a creatine level, a urea acid level, or a C-reactive protein level in the biological sample at least 1 day, 2 days, 3 days, 4 days, 8 days, 10 days, 12 days, 14 days, 20 days, or at least 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology, relative to a control.
  • the subject exhibits at least about at least about a 1%, at least about a 2%, at least about a 3%, at least about a 4%, at least about a 5%, at least about a 6%, at least about a 7%, at least about an 8%, at least about a 9%, at least about a 10%, at least about a 15%, at least about a 20%, at least about a 25%, or at least about a -109- 170456481.42 Docket No.: 146316.8023.WO00 30% reduction in an alkaline phosphatase level, a total protein level, an albumin level, a globulin level, a GGT, a glucose level, a urea nitrogen level, a creatine level, a urea acid level, or a C-reactive protein level in the biological sample at least about 1 day, 2 days, 3 days, 4 days, 8 days, 10 days, 12 days, 14 days, 20 days, or
  • the subject of the methods of the present technology experiences no change or a reduction in one or more of a total white blood cell count level, a neutrophil level, a lymphocyte level, a monocyte level, an eosinophil level, a basophil level, a red cell distribution width level, a platelet count level, a mean platelet volume in a biological sample from the subject.
  • the subject exhibits at least about a 1%, about a 2%, about a 3%, about a 4%, about a 5%, about a 6%, about a 7%, about an 8%, about a 9%, about a 10%, about a 15%, about a 20%, about a 25%, or about a 30% reduction in a total white blood cell count level, a neutrophil level, a lymphocyte level, a monocyte level, an eosinophil level, a basophil level, a red cell distribution width level, a platelet count level, a mean platelet volume in a biological sample from the subject.
  • the reduction occurs about 1 day, 2 days, 3 days, 4 days, 8 days, 10 days, 12 days, 14 days, 20 days, or about 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology, relative to a control.
  • the subject exhibits at least at least a 1%, at least a 2%, at least a 3%, at least a 4%, at least a 5%, at least a 6%, at least a 7%, at least an 8%, at least a 9%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, or at least a 30% reduction in a total white blood cell count level, a neutrophil level, a lymphocyte level, a monocyte level, an eosinophil level, a basophil level, a red cell distribution width level, a platelet count level, a mean platelet volume in a biological sample from the subject.
  • the reduction occurs at least 1 day, 2 days, 3 days, 4 days, 8 days, 10 days, 12 days, 14 days, 20 days, or at least 30 days after administration of a first dose of a non- naturally occurring melanocortin analog of the present technology, relative to a control.
  • the subject exhibits at least about at least about a 1%, at least about a 2%, at least about a 3%, at least about a 4%, at least about a 5%, at least about a 6%, at least about a 7%, at least about an 8%, at least about a 9%, at least about a 10%, at least about a 15%, at least about a 20%, at least about a 25%, or at least about a 30% reduction in a total white blood cell count level, a neutrophil level, a lymphocyte level, a monocyte level, an eosinophil level, a basophil level, a red cell distribution width level, a platelet count level, a mean platelet volume in a biological sample from the subject.
  • the reduction occurs at least about 1 day, 2 days, 3 days, 4 days, 8 days, 10 days, 12 days, 14 days, 20 days, or at least about 30 days after administration of a first dose of a non-naturally occurring melanocortin analog of the present technology, relative to a control.
  • the subject of the methods of the present technology experiences no change or an increase in one or more of a red blood cell count, a hemoglobin level, a hematocrit level, a mean corpuscular volume level, a mean corpuscular hemoglobin level, or a mean corpuscular hemoglobin concentration level in a biological sample from the subject, relative to a control.
  • the red blood cell count, a hemoglobin level, the hematocrit level, the mean corpuscular volume level, the mean corpuscular hemoglobin level, or the mean corpuscular hemoglobin concentration level is increased in the biological sample by at least about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 300%, after administration of a first dose of a non- naturally occurring melanocortin analog, relative to a control.
  • the subject of the methods of the present technology may experience no change in or an increase in an albumin to globulin ratio in a biological sample from the subject, relative to a control.
  • the albumin to globulin ratio is increased in the biological sample by at least about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 300%, after administration of a first dose of a non-naturally occurring melanocortin analog, relative to a control.
  • the subject administered a non-naturally occurring melanocortin analog of the present technology exhibits one or more of: -111- 170456481.42 Docket No.: 146316.8023.WO00 (a) a reduction in fat mass by at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% relative to baseline or a control; (b) a reduction in epididymal fat mass at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% relative to baseline or a control; (c) a reduction in perirenal fat mass at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
  • any one of (a)-(nn) is maintained for at about 2 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 1 year, or 5 years, after administration of the non-naturally occurring melanocortin analog.
  • any one of (a)-(nn) is maintained for at least 2 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 1 year, or 5 years, after administration of the non-naturally occurring melanocortin analog.
  • any one of (a)-(nn) is maintained for at least about 2 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 1 year, or 5 years, after administration of the non-naturally occurring melanocortin analog.
  • any one of (a)-(nn) occurs during or after administration of a first, a second, and/or a third dose of the non-naturally occurring melanocortin analog.
  • any one of (a)-(nn) is greater during or after administration the second dose relative to during or after administration of the first dose.
  • any one of (a)-(nn) is greater during or after administration the third dose relative to during or after administration of the first dose or the second dose.
  • the muscle mass is cardiac muscle mass, skeletal muscle mass, or both.
  • the cardiac muscle mass is determined by measuring change in heart weight.
  • the skeletal muscle mass is determined by measuring change in gastrocnemius tissue weight.
  • the muscle mass change is cardiac muscle mass change, skeletal muscle mass change, or -116- 170456481.42 Docket No.: 146316.8023.WO00 both.
  • cardiac muscle is determined by heart weight.
  • skeletal muscle mass is determined by gastrocnemius tissue weight.
  • the muscle mass changes may be assessed by using magnetic resonance imaging (MRI) and/or or nuclear magnetic resonance (NMR).
  • Weight Loss Agents [0411] Administration of the non-naturally occurring melanocortin analogs of the present technology may comprise rebound-resistant weight loss (i.e., prevention of weight gain after treatment completion) and/or increased muscle retention, relative to a control (e.g., administration of a conventional weight loss agent alone).
  • the rebound resistant weight loss may comprise a prevention or a reduction in fat mass gain and/or BMI increase, relative to the control.
  • the non-naturally occurring melanocortin analog is administered as a combination therapy with a weight loss agent, where the combination therapy achieves the same weight loss as administration of the weight loss agent alone, but at a reduced dose or dosing frequency relative to the weight loss agent.
  • the rebound-resistant weight loss is maintained for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks after administration of the non-naturally occurring melanocortin analog is complete and/or discontinued.
  • the rebound-resistant weight loss is maintained for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks after administration of the non-naturally occurring melanocortin analog is complete and/or discontinued. [0414] In some embodiments, the rebound-resistant weight loss is maintained for at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks after administration of the non-naturally occurring melanocortin analog is complete and/or discontinued.
  • the subject’s weight increases no more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than about 1%.
  • -117- 170456481.42 Docket No.: 146316.8023.WO00 [0416]
  • the present technology comprises a method of suppressing appetite in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject prior to, during, and/or after administration of the weight loss agent.
  • the weight loss agent is a GLP-1 receptor agonist and the non-naturally occurring melanocortin analog is a melanocortin agonist and administration of the combination therapy produces an additive effect on suppressing the subject’s appetite.
  • the weight loss agent is a dual GLP-1/GIP receptor agonist and the non-naturally occurring melanocortin analog is a melanocortin agonist and administration of the combination therapy produces a synergistic effect on suppressing the subject’s appetite.
  • the method reduces food intake of the subject.
  • Administration of the non-naturally occurring melanocortin analog prior to, during, and/or after administration of the weight loss agent may produce an additive or synergistic effect on reducing the subject’s food intake.
  • the method reduces fat body mass and/or body weight of the subject.
  • the reduction in food intake is a reduction in caloric intake.
  • the subject’s weight increases no more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than about 1% at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks after administration of the non-naturally occurring melanocortin analog is complete and/or discontinued.
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating body weight gain a during or after use of a weight loss in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • the present technology comprises a method of maintaining body weight after use of a weight loss in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • a weight loss in a subject in need thereof comprising administering a non-naturally occurring melanocortin analog to the subject.
  • -118- 170456481.42 Docket No.: 146316.8023.WO00 [0421]
  • the present technology comprises a method of preventing, reducing, or otherwise ameliorating a fat mass gain after a weight loss in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject.
  • the methods of the present technology may decrease the risk and/or incidence of adverse cardiovascular events associated with the use of a weight loss agent including, but not limited to, cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, angina, sinus tachycardia, sinus bradycardia, coronary artery bypass grafting, percutaneous coronary intervention, heart failure, carotid endarterectomy, peripheral vascular disease, or any combination thereof.
  • Cardiac Effects [0423] The methods of the present technology may prevent or decrease the risk and/or incidence of adverse cardiac effects associated with the use of conventional melanocortin analogs, melanocortin peptides, and/or melanocortin receptor agonists.
  • the subject of the present technology experiences no or substantially no adverse cardiac effects and/or no change in cardiac outcomes during or after administration of a non-naturally occurring melanocortin analog of the present technology, relative to a control (e.g., a subject administered conventional melanocortin analogs, melanocortin peptides, and/or melanocortin receptor agonists).
  • a control e.g., a subject administered conventional melanocortin analogs, melanocortin peptides, and/or melanocortin receptor agonists.
  • the adverse cardiac effect and/or change in cardiac outcomes comprises one or more of a change in blood pressure (e.g., systolic blood pressure, diastolic blood pressure, mean arterial blood pressure), heart rate, QT interval, QRS interval, RR interval, or QTc, relative to a control.
  • the adverse cardiac effect and/or change in cardiac outcomes is a transient change in blood pressure (e.g., systolic blood pressure, diastolic blood pressure, mean arterial blood pressure), heart rate, QT interval, QRS interval, RR interval, or QTc, relative to a control.
  • the transient change in blood pressure comprises an increase in blood pressure in the evening, at night, during a sleep cycle, or during a dark cycle after administration of the conventional melanocortin analogs, melanocortin peptides, and/or melanocortin receptor agonists.
  • the present technology comprises a method of inducing a tanning effect in a subject during or after administration of a non-naturally occurring melanocortin analog.
  • the tanning effect comprises skin darkening without hyperpigmentation (e.g., an uneven distribution of melanin) compared to subjects who have not received the non-naturally occurring melanocortin analog or compared to the same subject at baseline (e.g., prior to receiving a dose of the non-naturally occurring melanocortin analog).
  • the tanning effect is not clinically significant, occurs in response to UV or sun exposure, and/or is not substantially ectopic.
  • the tanning effect may occur in combination with weight loss.
  • the weight loss may comprise a retention in muscle mass (e.g., lean muscle mass) and a loss of fat mass.
  • the tanning effect is observed about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 days after administration of a non- naturally occurring melanocortin analog.
  • the tanning effect occurs at a dose of greater than 100 mg or at least about 200 mg. [0425]
  • the tanning effect is reduced after completion and/or discontinuation of the non-naturally occurring melanocortin analog dosing.
  • the tanning effect is reduced at least about 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 week, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 10 weeks, 15 weeks, or 20 weeks after completion and/or discontinuation of the non-naturally occurring melanocortin analog dosing.
  • the reduced tanning effect comprises reversal of about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 95%, 99%, or 100% of the total area of skin darkening.
  • the tanning effect does not comprise ectopic skin hyperpigmentation and/or tanning effects (e.g., of the palms or gums).
  • the reduced tanning effect comprises reversal of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 95%, 99%, or 100% of the total area of skin darkening.
  • the tanning effect does not comprise ectopic skin hyperpigmentation and/or tanning effects (e.g., of the palms or gums).
  • the reduced tanning effect comprises reversal of at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 95%, 99%, or 100% of -120- 170456481.42 Docket No.: 146316.8023.WO00 the total area of skin darkening.
  • the tanning effect does not comprise ectopic skin hyperpigmentation and/or tanning effects (e.g., of the palms or gums).
  • the non-naturally occurring melanocortin analog may be useful in treating, preventing, or otherwise ameliorating one or more hypopigmentation diseases.
  • hypopigmentation disease examples include albinism (e.g., oculocutaneous albinism), vitiligo, piebaldism, nevus depigmentosus, hypomelanosis of Ito, phenylketonuria, tuberous sclerosis, dyschromatosis symmetrica hereditaria, Waardenburg syndrome, Hermansky- Pudlak syndrome, Chediak-Higashi syndrome, Griscelli syndrome, and Tietz syndrome, and acquired hypopigmentary diseases such as leukoderma Sutton, Vogt-Koyanagi Harada syndrome, leukoderma senile, leukoderma after sea bathing, and leukoderma syphiliticum.
  • albinism e.g., oculocutaneous albinism
  • vitiligo e.g., piebaldism
  • nevus depigmentosus e.g., hypomelanosis of Ito
  • phenylketonuria phenylket
  • the methods of the present technology may prevent or decrease the risk and/or incidence of hyperpigmentation associated with the use of conventional melanocortin analogs, melanocortin peptides, and/or melanocortin receptor agonists.
  • the subject of the present technology experiences no or substantially no hyperpigmentation during or after administration of a non-naturally occurring melanocortin analog of the present technology, relative to a control (e.g., a subject administered conventional melanocortin analogs, melanocortin peptides, and/or melanocortin receptor agonists).
  • the hyperpigmentation comprises one or more of hyperpigmentation of the skin, nose, cheeks, or site of administration of the non-naturally occurring melanocortin analog.
  • the subject has a body mass index (BMI) of 18.5 kg/m 2 to 35 kg/m 2 . In some embodiments, the subject has a BMI of less than 20 kg/m 2 . In some embodiments, the subject has a BMI of less than 18.5 kg/m 2. [0432] In some embodiments, the subject has a BMI of greater than 24.9 kg/m 2 . In some embodiments, the subject has a BMI of 25 kg/m 2 to 29.9 kg/m 2 .
  • the subject has a BMI of greater than or equal to 30 kg/m 2 . -121- 170456481.42 Docket No.: 146316.8023.WO00 [0433]
  • the subject is an overweight subject. Overweight subjects include those having a body weight about 3% or more, 5% or more, 10% or more, 20% or more, or 30% or less, than the upper end of "normal" BMI (e.g., 24.9 kg/m2).
  • the subject is an obese subject. Obese subjects include those having a BMI of greater than or equal to 30 kg/m2.
  • the subject has a metabolic dysfunction.
  • the metabolic dysfunction is selected from the group consisting of obesity, diabetes mellitus, metabolic syndrome, insulin resistance, non-alcoholic fatty liver disease, polycystic ovarian syndrome, metabolic acidosis, hypothyroidism, hyperlipidemia, Cushing Syndrome, and metabolic myopathies.
  • the subject has another condition which is not metabolic dysfunction, but administration of the weight loss agent provides therapeutic benefit to the non-metabolic dysfunction condition.
  • the subject with a metabolic dysfunction has previously received treatment with the weight loss agent.
  • a subject who has previously received treatment with the weight loss agent has completed at least one full treatment cycle with the weight loss agent.
  • the subject with a metabolic dysfunction is currently receiving treatment with the weight loss agent.
  • a subject who is currently receiving treatment with the weight loss has received at least one dose of the weight loss agent.
  • the subject with a metabolic dysfunction has not received any treatment with the weight loss agent.
  • a subject who has not received any treatment with the weight loss agent has not received even a single dose of the weight loss agent.
  • a subject who has been treated with a weight loss agent has received a weight loss agent in an amount sufficient to induce at least minimum weight loss.
  • a subject how is currently undergoing treatment with a weight loss agent is receiving a weight loss agent in an amount sufficient to induce at least minimum weight loss.
  • the subject has previously received treatment with the weight loss agent and has not lost body weight and/or fat mass.
  • the subject has previously received treatment with the weight loss agent and -122- 170456481.42 Docket No.: 146316.8023.WO00 has lost body weight and/or fat mass.
  • the subject may still be overweight or obese.
  • the subject may have stopped treatment with the weight loss agent because they were unable to continue losing body weight and/or fat mass with the weight loss agent.
  • the subject’s body weight may be more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, or more than 40% reduced from baseline prior to administration of the non-naturally occurring melanocortin analog.
  • the subject in which the subject has previously received treatment with the weight loss agent, the subject’s fat mass may be more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, or more than 40% reduced from baseline prior to administration of the non-naturally occurring melanocortin analog.
  • the subject is currently receiving treatment with the weight loss agent and has not lost any body weight.
  • the subject is currently receiving treatment with the weight loss agent and has lost body weight and/or fat mass.
  • the subject is currently receiving treatment with the weight loss agent, has lost body weight and/or fat mass, and has failed to lose sufficient body weight and/or fat mass following administration of the weight loss agent.
  • the subject who is currently receiving treatment with the weight loss agent may still be overweight or obese.
  • the subject’s body weight may be more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, or more than 40% reduced from baseline prior to administration of the non-naturally occurring melanocortin analog.
  • the subject in embodiments in which the subject is currently receiving treatment with the weight loss agent, the subject’s fat mass may be more than 5%, more than 10%, more than -123- 170456481.42 Docket No.: 146316.8023.WO00 15%, more than 20%, more than 25%, more than 30%, more than 35%, or more than 40% reduced from baseline prior to administration of the non-naturally occurring melanocortin analog.
  • the subject has not received any treatment with the weight loss agent and has failed to lose sufficient or any weight using other weight loss methods.
  • the subject has not received any treatment with the weight loss agent and has not attempted to lose weight using any other methods.
  • the subject may begin treatment with non-naturally occurring melanocortin analog and the weight loss agent at the same time.
  • a subject who begins administration of the weight loss agent and the non-naturally occurring melanocortin analog at the same time, and continues with concurrent administration of the two therapies may experience greater body weight and/or fat mass loss compared to a subject who is administered only one of the therapies, e.g., only the weight loss agent or only the non-naturally occurring melanocortin analog.
  • the subject’s body weight may be more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, or more than 55% reduced from baseline prior to administration of the non-naturally occurring melanocortin analog.
  • the subject begins treatment with the non-naturally occurring melanocortin analog and the weight loss agent at the same time
  • the subject’s fat mass may be more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, or more than 55% reduced from baseline prior to administration of the non-naturally occurring melanocortin analog.
  • the method prevents or reduces muscle mass loss (e.g., involuntary muscle mass loss) in the subject.
  • the subject’s muscle mass is not more than 5%, not more than 10%, not more than 15%, not more than 20%, not -124- 170456481.42 Docket No.: 146316.8023.WO00 more than 25%, or not more than 30% reduced from baseline prior to administration of the non-naturally occurring melanocortin analog and/or the weight loss agent.
  • the weight loss agent is administered to the subject for treating or preventing one of one or more metabolic dysfunctions.
  • Nonlimiting examples of metabolic dysfunctions include obesity, diabetes mellitus, metabolic syndrome, insulin resistance, non-alcoholic fatty liver disease, polycystic ovarian syndrome, metabolic acidosis, hypothyroidism, hyperlipidemia, Cushing Syndrome, and metabolic myopathies.
  • the subject of the present technology may be a mammal, including but not limited to a human, a non-human primate such as a chimpanzee, a domestic livestock or a farm animal such as a cow, a bison, sheep, a pig, a goat, a horse, a chicken, and a rooster, a domestic pet animal such as a dog, a cat, a rat, a mouse, and a rabbit, and a laboratory subject such as a rodent, including a rat, a mouse, and a guinea pig.
  • the subject is a human.
  • the subject is an animal such as a rat or a dog.
  • the non-naturally occurring melanocortin analogs of the present technology exhibit pharmacokinetic (pK) and/or pharmacodynamic (pD) parameters.
  • pK and/or pD may be expressed or otherwise determined relative to a control, which, in some instances, may be a non-naturally occurring melanocortin analog lacking one or more features of the non-naturally occurring melanocortin analogs of the present technology.
  • the pK and/or pD of the non-naturally occurring melanocortin analogs may be assessed using concentration and/or temporal measurements (e.g., Tfinal, Cmax, T 1 ⁇ 2 (h)), AUC, or Tmax.
  • concentration and/or temporal measurements e.g., Tfinal, Cmax, T 1 ⁇ 2 (h)
  • AUC e.g., AUC, or Tmax.
  • the non-naturally occurring melanocortin analogs have reduced clearance and/or metabolism, increased uptake, absorption, and/or stability, relative to a control. Clearance [0452] “Clearance” may refer to the elimination, absorption, and/or metabolism of the non-naturally occurring melanocortin analogs in the subject’s plasma.
  • Clearance may be assessed as volume of plasma cleared of the non-naturally occurring melanocortin analogs -125- 170456481.42 Docket No.: 146316.8023.WO00 over time (e.g., mL/min, L/hr, or L/day) and/or may be normalized to body weight of the subject (e.g., mL/min/kg). Reduced clearance may also be represented by an increase in half-life or volume of distribution (Vd).
  • measuring clearance comprises measuring a terminal elimination rate constant ( ⁇ z) or an inter-compartmental clearance (Q).
  • the reduction in clearance comprises a measurement about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 day, 6 days, or 1 week after administration of the non-naturally occurring melanocortin analogs.
  • the reduced clearance comprises an intestinal fluid clearance, a gastric fluid clearance, a liver clearance, or a liver microsome clearance.
  • the reduction in clearance comprises a measurement during administration of the non-naturally occurring melanocortin analogs.
  • the reduction in clearance comprises a measurement at the completion of administration of the non-naturally occurring melanocortin analogs.
  • Concentration may comprise a measurement reflecting one or more of the absolute amounts of the non-naturally occurring melanocortin analogs, the absorption of the non-naturally occurring melanocortin analogs, the metabolism of non-naturally occurring melanocortin analogs, or the elimination of non-naturally occurring melanocortin analogs.
  • the aqueous concentration may comprise an intestinal fluid concentration or a gastric fluid concentration.
  • the increased tissue, plasma, and/or serum concentration may be an increase in concentration of the non-naturally occurring melanocortin analogs at a given time point relative to a control administered at the same dose and measured at the same time point.
  • the concentration may be measured at an intermediate time point or a final time point and may be measured as a mean residence time (MRT), an average concentration (Cavg), a -126- 170456481.42 Docket No.: 146316.8023.WO00 trough concentration (Ctrough), or a concentration at the end of administration (e.g., infusion) time (CT).
  • MRT mean residence time
  • Cavg average concentration
  • Ctrough a concentration at the end of administration (e.g., infusion) time
  • CT concentration at the end of administration (e.g., infusion) time
  • CT concentration at the end of administration (e.g., infusion) time
  • CT concentration at the end of administration (e.g., infusion) time
  • CT concentration at
  • Cmax may suggest increased absorption, reduced metabolism, or slower elimination of the non-naturally occurring melanocortin analogs, relative to a control.
  • Cmax comprises a dose normalized Cmax (DNCmax).
  • the increased concentration is reflected by an increase in minimum plasma concentration (Cmin).
  • Cmin may suggest increased absorption, reduced metabolism, or slower elimination of the non-naturally occurring melanocortin analogs, relative to a control.
  • Cmin comprises a dose normalized Cmin (DNCmin).
  • Tmax time to reach Cmax
  • a reduced Tmax may suggest increased absorption, reduced metabolism, or slower elimination of the non-naturally occurring melanocortin analogs, relative to a control.
  • the increased concentration is reflected by a final measurable concentration (Tfinal).
  • Tfinal may suggest increased absorption, reduced metabolism, or slower elimination of the non-naturally occurring melanocortin analogs, relative to a control.
  • the increased concentration is reflected by an increase in area under the curve (AUC).
  • AUC area under the curve
  • An increase in AUC may signify increased exposure to the non-naturally occurring melanocortin analogs and/or may suggest increased absorption, reduced metabolism, or slower elimination of the non-naturally occurring melanocortin analogs, relative to a control.
  • the AUC measurement may comprise an Area Under the Curve for Concentration of Drug in Non-Compartmental Analysis (DNAUC).
  • DNAUC Area Under the Curve for Concentration of Drug in Non-Compartmental Analysis
  • the increased concentration is reflected by a reduction in a partition coefficient or an increase in a partition coefficient, relative to a control.
  • the reduced partition coefficient may reflect an increase in aqueous solubility (e.g., an intestinal -127- 170456481.42 Docket No.: 146316.8023.WO00 fluid or a gastric fluid), relative to the control.
  • the increased partition coefficient may reflect an increase in membrane permeability, relative to the control.
  • the increase in tissue, plasma, and/or serum concentration comprises a measurement about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 day, 6 days, or 1 week after administration of the non- naturally occurring melanocortin analogs.
  • the increase in tissue, plasma, and/or serum concentration comprises a measurement at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 day, 6 days, or 1 week after administration of the non- naturally occurring melanocortin analogs.
  • the increase in tissue, plasma, and/or serum concentration comprises a measurement at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 day, 6 days, or 1 week after administration of the non-naturally occurring melanocortin analogs.
  • the increase in tissue, plasma, and/or serum concentration comprises a measurement during administration of the non-naturally occurring melanocortin analogs.
  • the increase in tissue, plasma, and/or serum concentration comprises a measurement at the completion of administration of the non- naturally occurring melanocortin analogs.
  • the concentration of the non-naturally occurring melanocortin analog in the plasma or a tissue of the subject is at least about 5 ng/mL to at least about 2000 ng/mL about 24 hours after administration of the non-naturally occurring melanocortin analog.
  • the non-naturally occurring melanocortin analog is administered to the subject until the concentration of the non-naturally occurring -128- 170456481.42 Docket No.: 146316.8023.WO00 melanocortin analog in the plasma or a tissue of the subject is at least about 5 ng/mL to at least about 2000 ng/mL after administration of the non-naturally occurring melanocortin analog. In some embodiments, the concentration is at least about 5 ng/mL after administration of the non-naturally occurring melanocortin analog. In some embodiments, the concentration is at least about 10 ng/mL after administration of the non-naturally occurring melanocortin analog.
  • the concentration is at least about 50 ng/mL after administration of the non-naturally occurring melanocortin analog. In some embodiments, the concentration is at least about 100 ng/mL after administration of the non- naturally occurring melanocortin analog. In some embodiments, the concentration is at least about 200 ng/mL after administration of the non-naturally occurring melanocortin analog. In some embodiments, the concentration is at least about 300 ng/mL after administration of the non-naturally occurring melanocortin analog. In some embodiments, the concentration is at least about 400 ng/mL after administration of the non-naturally occurring melanocortin analog.
  • the concentration is at least about 500 ng/mL after administration of the non-naturally occurring melanocortin analog. In some embodiments, the wherein concentration is at least about 750 ng/mL after administration of the non- naturally occurring melanocortin analog. In some embodiments, the concentration is at least about 1000 ng/mL after administration of the non-naturally occurring melanocortin analog. In some embodiments, the is at least about 1500 ng/mL after administration of the non- naturally occurring melanocortin analog. In some embodiments, the concentration is at least about 2000 ng/mL after administration of the non-naturally occurring melanocortin analog. In some embodiments, the concentration is a Cmax.
  • the non-naturally occurring melanocortin analogs of the present technology comprise an increased distribution relative to a control.
  • the increased distribution may be an increase in distribution of the non-naturally occurring melanocortin analogs at a given time point relative to a control administered at the same dose and measured at the same time point.
  • the distribution may be measured at an intermediate time point or a final time point.
  • the measurement of distribution comprises measuring a volume of distribution at the terminal phase (Vd or Vdß), a central volume of distribution (V), a peripheral volume of distribution (V2), an apparent volume of distribution (Vz), or a measurement of distribution comprises measuring a volume of distribution at steady state (Vss).
  • Vd or Vdß a volume of distribution at the terminal phase
  • V central volume of distribution
  • V2 a peripheral volume of distribution
  • Vz an apparent volume of distribution
  • a measurement of distribution comprises measuring a volume of distribution at steady state (Vss).
  • Vd, Vdß, Vz, and/or Vss may suggest large distribution beyond the tissue, plasma, and/or serum compartment, relative to the control.
  • the increase in distribution comprises a measurement about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 day, 6 days, or 1 week after administration of the non-naturally occurring melanocortin analogs.
  • the increase in distribution comprises a measurement at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 day, 6 days, or 1 week after administration of the non-naturally occurring melanocortin analogs.
  • the increase in distribution comprises a measurement at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 day, 6 days, or 1 week after administration of the non-naturally occurring melanocortin analogs.
  • the increase in distribution comprises a measurement during administration of the non-naturally occurring melanocortin analogs.
  • the increase in distribution comprises a measurement at the completion of administration of the non-naturally occurring melanocortin analogs.
  • the non-naturally occurring melanocortin analog of the present technology exhibits one or more of the following: (a) an increase in half-life relative to a control; (b) a reduction in clearance relative to a control; (c) an increase in tissue concentration relative to a control; -130- 170456481.42 Docket No.: 146316.8023.WO00 (d) an increase in plasma concentration relative to a control; (e) an increase in serum concentration relative to a control; (f) an increase in distribution relative to a control; (g) an increase in an AUC measurement relative to a control; (h) an increase in a DNAUC measurement relative to a control; (i) an increase in Tfinal relative to a control; (j) an increase in Cmax relative to a control; (k) an increase in Cmin relative to a control; (l) an increase in DNCmin relative to a control; (m) an increase in MRT relative
  • the non-naturally occurring melanocortin analog of the present technology exhibits one or more of the following, relative to a control: (a) an increase in half-life by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000% relative to a control; (b) a reduction in clearance by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to a control; -131- 170456481.42 Docket No.: 146316.8023.WO00 (c) an increase in tissue concentration by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%
  • Example 1 Peptide Synthesis-Generic [0480]
  • the non-naturally occurring melanocortin analogs of the present technology were synthesized by conventional procedures (e.g., solution-phase procedure, solid-phase synthesis) for the formation of a peptide linkage between amino acids.
  • the solution-phase procedure involved a condensation between the free alpha amino group of an amino acid or derivative thereof having the carboxyl group or other reactive groups protected and the free primary carboxyl group of another amino acid or derivative thereof having the amino group or other reactive groups protected.
  • the solid-phase synthesis utilized a variety of resins and reagents and may involve additional purification steps.
  • the process for synthesizing the non-naturally occurring melanocortin analogs was generally performed by a procedure as follows. Each amino acid in the desired sequence of the non-naturally occurring melanocortin analogs was added one at a time in succession to another amino acid or derivative thereof or by a procedure whereby peptide fragments with the desired amino acid sequence were first synthesized conventionally and then condensed to provide the desired peptide. In most cases, the resulting peptide was then cyclized to yield a cyclic peptide.
  • Solid-phase peptide synthesis was carried out by sequentially incorporating the desired amino acid residues one at a time into the growing peptide chain coupled to a solid- phase support according to the general principles of solid phase methods (see Merrifield, Angew Chem.24:799-810 (1985) and Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross E. and Meienhofer J., Eds. Academic Press 1-284 (1980)).
  • An exemplary solid-phase synthesis of non-naturally occurring melanocortin analogs is provided below.
  • the C-terminal amino acid residue of the non-naturally occurring melanocortin analog was coupled to a solid-phase support, e.g., a solid-phase resin.
  • a solid-phase support e.g., a solid-phase resin.
  • Coupling of the C-terminal amino acid residue and the solid-phase support may be carried out according to any method know in the art.
  • the alpha- -134- 170456481.42 Docket No.: 146316.8023.WO00 amine of the C-terminal amino acid residue may or may not be protected with an amine protecting group, as described below.
  • the carboxyl group of the amino acid residue may or may not be activated prior to coupling to the solid-phase support in order to increase its electrophilicity.
  • an amino acid residue may be coupled to a p-benzyloxybenzyl alcohol resin (Wang) or a 2-chlorotrityl chloride resin via an ester linkage.
  • an amino acid residue may be coupled to a benzhydrylamine (BHA) resin through an Fmoc-linker such as, for example, p- [(R,S)- ⁇ -[1-(9H-fluor-en-9-yl)-methoxyformamido]-2,4-dimethyloxybenzyl]-phenoxyacetic acid (Rink linker) via an amide linkage.
  • BHA benzhydrylamine
  • Fmoc-linker such as, for example, p- [(R,S)- ⁇ -[1-(9H-fluor-en-9-yl)-methoxyformamido]-2,4-dimethyloxybenzyl]-phenoxyacetic acid (Rink linker) via an amide linkage.
  • the peptide was cleaved from the solid-phase support and purified by methods known in the art, such as, for example, reverse phase high performance liquid chromatography (RP-HPLC) using a suitable column, such as a C18 column. Additionally, or alternatively, other methods of separation or purification were employed, including, but not limited to, methods based on the size or charge of the peptide.
  • RP-HPLC reverse phase high performance liquid chromatography
  • HPLC high-performance liquid chromatograph
  • amino acid analysis amino acid analysis
  • mass spectrometry mass spectrometry
  • Example 2 Peptide Synthesis-Protecting Groups
  • reactive side chain groups of the various amino acid residues were protected with suitable protecting groups, which prevented undesirable chemical reaction from occurring at that site until the protecting group was removed.
  • protection of the alpha amino group of an amino acid residue or fragment was performed while that entity reacting with the carboxyl group, followed by the selective removal of the alpha amino protecting group to allow a subsequent reaction to take place at that site.
  • Specific protecting groups for solid phase synthesis methods and solution -135- 170456481.42 Docket No.: 146316.8023.WO00 phase synthesis methods are known to those having ordinary skill in the art.
  • Alpha amino groups were protected by a suitable protecting group, including a urethane-type protecting group, such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p- chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p- biphenyl-isopropoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and p- methoxybenzyloxycarbonyl (Moz); aliphatic urethane-type protecting groups, such as t- butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl, and allyloxycarbonyl.
  • a urethane-type protecting group such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p- chlorobenzyloxycarbony
  • Fmoc was also used for alpha amino protection. Guanidino groups, if present, were protected by a suitable protecting group, such as nitro, p-toluenesulfonyl (Tos), Z, pentamethylchromanesulfonyl (Pmc), adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and Boc. Pmc was used as a protecting group for Arg.
  • a suitable protecting group such as nitro, p-toluenesulfonyl (Tos), Z, pentamethylchromanesulfonyl (Pmc), adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and Boc.
  • Pmc was used as a protecting group for Arg.
  • Alpha aminoprotecting groups may be removed under basic conditions, such as, for example, using a solution of piperidine, piperazine, diethylamine, or morpholine (20- 40% v/v) in N,N-dimethylformamide (DMF).
  • DMF N,N-dimethylformamide
  • alpha amino protecting groups were used, protecting groups were removed after synthesis of the peptide and before or after cleavage of the solid-phase support.
  • Example 3 Peptide Synthesis-Additional Modifications
  • the peptides were further modified to obtain N-terminus modifications, such as acetylation, while on resin, or were removed from the resin by use of a cleaving reagent and then modified.
  • C-terminus modification (e.g., amidation), was performed if needed.
  • C-terminus modification e.g., amidation
  • the cyclized peptide structures were obtained prior to cleavage from the peptide resin.
  • the desired side chains were deprotected, and the peptide suspended in a suitable solvent and a cyclic coupling agent added.
  • suitable solvents for example DMF, dichloromethane (DCM) or 1- methyl-2-pyrrolidone (NMP), were used for the cyclization.
  • Suitable cyclic coupling reagents e.g., 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(1H- benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), benzotriazole- 1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP), benzotriazole-1-yl- -136- 170456481.42 Docket No.: 146316.8023.WO00 oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate (PyBOP), 2-(7-aza-1H-benzotriazol- 1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TATU), 2-(2-oxo-1 (2H)-pyr
  • exemplary non-naturally occurring melanocortin analogs at the melanocortin receptors were measured via cAMP accumulation assay, according to the following procedure. Experimental design and execution were conducted by Epics Therapeutics S.A. EuroscreenFast (Bruxelles, Belgium).
  • Compound Handing Compounds were delivered as powder (1 mg) or 10 mM solutions (100 ⁇ l) in 100% DMSO. Powders were solubilized in 100% DMSO at a concentration of 10 mM (master solution) in a solvent volume defined. Serial dilutions were performed from master solution in 100% DMSO to obtain intermediate concentrations 200-, 300- or 400-fold higher than the concentrations to be tested, depending on the assay. Each sample was diluted 100- fold in the assay buffer and dispensed in a test plate. Amounts, solvents, and dilutions were estimated based on standard small-molecule drugs. Cell lines used for functional assays are shown in Table 1. Table 1.
  • Cyclic AMP Homogenous Time-Resolved Fluorescence (HTRF) assay for Gs coupled receptor: [0494] CHO-K1 cells expressing recombinant human receptor grown prior to the test in media without antibiotic were detached by gentle flushing with PBS-EDTA (5 mM EDTA), recovered by centrifugation and resuspended in assay buffer (KRH: 5 mM KCl, 1.25 mM MgSO4, 124 mM NaCl, 25 mM HEPES, 13.3 mM Glucose, 1.25 mM KH2PO4, 1.45 mM CaCl2, 0.5 g/l BSA, supplemented with 1mM IBMX or 25 ⁇ M Rolipram).
  • Group A melanocortin analogs also comprise Ala or dArg at the N-terminus and dVal-dPro or dTle-dPro at the C-terminus.
  • Group A non-naturally occurring melanocortin analogs are provided in Table 2. Table 2.
  • Group A non-naturally occurring melanocortin analogs Melanocortin SEQ ID Formula analo NO p y g g , all of which are cyclic peptides comprising the motif c[Asp-His-p(F)dPhe-Arg-Trp-Lys] (SEQ ID NO: 161).
  • Group B melanocortin analogs also comprise Arg, Lys, dLys, His, or dHis at the N-terminus and dVal-dPro or dTle-dPro at the C-terminus.
  • Group B non-naturally occurring melanocortin analogs are provided in Table 3. -139- 170456481.42 Docket No.: 146316.8023.WO00 Table 3.
  • Group B non-naturally occurring melanocortin analogs Melanocortin SEQ ID Formula analog NO 2 2 2 2 l of which are cyclic peptides comprising the motif c[Asp-His-p(F)dPhe-Arg-Trp-Lys] (SEQ ID NO: 161).
  • Group C melanocortin analogs also comprise Nle at the N-terminus.
  • Group C non-naturally occurring melanocortin analogs are provided in Table 4. Table 4.
  • Group C non-naturally occurring melanocortin analogs Melanocortin SEQ ID n l NO Formula 2 2 2 2 o- -140- 170456481.42 Docket No.: 146316.8023.WO00 [0505]
  • Group D included non-naturally occurring melanocortin analogs D1 to D4, all of which are cyclic peptides comprising a derivative of the motif c[Asp-His-p(F)dPhe-Arg-Trp- Lys] (SEQ ID NO: 161).
  • Derivatives of the motif of SEQ ID NO: 161 may include substitution of His with another amino acid, for example, Gln or dHis, substitution of Arg with another amino acid, for example, His, or substitution of Trp with another amino acid, for example, dNal(2’).
  • Group D non-naturally occurring melanocortin analogs are provided in Table 5. Table 5.
  • Group D non-naturally occurring melanocortin analogs Melanocortin SEQ ID Formula analog NO H2 l of which are cyclic peptides comprising the motif c[Asp-His-dPhe-Arg-Trp-Lys] (SEQ ID NO: 164) or a derivative thereof.
  • Derivatives of the motif of SEQ ID NO: 164 included in Group E melanocortin analogs include insertions of a Pro between Asp and His or between Trp and Lys.
  • Group E non-naturally occurring melanocortin analogs are provided in Table 6. Table 6.
  • Group E non-naturally occurring melanocortin analogs Melanocortin SEQ ID l NO Formula [050 ]
  • Group ncuded non-naura y occurrng meanocor n anaogs o , al of which are cyclic peptides comprising a derivative of the motif c[Asp-His-dPhe-Arg-Trp-Lys] (SEQ ID NO: 164).
  • Derivatives of the motif of SEQ ID NO: 164 may include substitution of His with another amino acid, for example, Gln, Pro or dHis, or substitution of Arg with another -141- 170456481.42 Docket No.: 146316.8023.WO00 amino acid, for example, cisPro(guan) or transPro(guan).
  • Group F non-naturally occurring melanocortin analogs are provided in Table 7. Table 7.
  • Group F non-naturally occurring melanocortin analogs Melanocortin SEQ ID Formula analog NO 2 2 s a cyclic peptide comprising a derivative of the motif c[Asp-His-dPhe-Arg-Trp-Lys] (SEQ ID NO: 164).
  • the derivative of the motif of SEQ ID NO: 164 included in the Group G melanocortin analog includes at least one insertion between Asp and His.
  • the Group G non-naturally occurring melanocortin analog is provided in Table 8. Table 8.
  • Group G non-naturally occurring melanocortin analog Melanocortin SEQ ID Formula analo NO , l of which are cyclic peptides comprising the motif dAla-His-dPhe-Arg-Trp (SEQ ID NO: 163), or a derivative thereof. Derivatives of the motif of SEQ ID NO: 163 may include substitution of dAla with a similar amino acid, for example, Ala.
  • Group H non-naturally occurring melanocortin analogs are provided in Table 9. Table 9.
  • Group H non-naturally occurring melanocortin analogs Melanocortin SEQ ID Formula 2 2 -142- 170456481.42 Docket No.: 146316.8023.WO00 H3 40 Ac-dArg-c[Asp-dAla-His-dPhe-Arg-Trp-Lys]-dVal-dPro-NH2 of which are cyclic peptides comprising the motif His-p(Cl)dPhe-Arg-Trp (SEQ ID NO: 165). Group I melanocortin analogs also comprise Nle at the N-terminus. Group I non-naturally occurring melanocortin analogs are provided in Table 10. Table 10.
  • Group I non-naturally occurring melanocortin analogs Melanocortin SEQ ID analog NO Formula 2 2 2 nd O10, all of which are cyclic peptides comprising the motif His-p(F)dPhe-Arg-Trp-Orn (SEQ ID NO: 162), or a derivative thereof. Derivatives of SEQ ID NO: 162 may include substitution of Arg with a similar amino acid, for example, His.
  • Group O non-naturally occurring melanocortin analogs are provided in Table 11. Table 11.
  • Group O non-naturally occurring melanocortin analogs Melanocortin SEQ ID Formula l NO 2 2 go s ac y o ea oco a aogs o ea oco ecepo a ea oco n 5 receptor [0512] Administration of some non-naturally occurring melanocortin analogs activated melanocortin 1 receptor (MC1R) and melanocortin 5 receptor (MC5R) activity, as measured by cAMP levels (Table 12). -143- 170456481.42 Docket No.: 146316.8023.WO00 Table 12.
  • O10 was compared to sequences with different lactam cyclization types, different residues at the R 4 and R 3 positions, and different C-terminal residues.
  • the agonist activity of O10 and comparative sequences is provided in Table 15, below. -149- 170456481.42 Docket No.: 146316.8023.WO00 Table 15.
  • results from this assay are provided in Table 22.
  • Table 22 Dose-response results of melanocortin analogs and control against QRFP Synthetic Max EC50 (nM)* Hill Top (%) -153- 170456481.42 Docket No.: 146316.8023.WO00 Antagonist activity on QRFP [0524] The antagonist activity of some of the non-naturally occurring melanocortin analogs was measured on QRFP by Aequorin assay. Results from this assay are provided in Table 23. Table 23.
  • results from this assay are provided in Table 27.
  • Table 27 Dose-response results of melanocortin analogs and control against NPFF2S Synthetic Max IC50 (nM)* Hill Top (%) Ag -155- 170456481.42 Docket No.: 146316.8023.WO00 [0529] The agonist activity of some of the non-naturally occurring melanocortin analogs was measured on NPY1 by cAMP assay. Results from this assay are provided in Table 28. Table 28.
  • results from this assay are provided in Table 30.
  • Table 30 Dose-response results of melanocortin analogs and control against NPFF1 Synthetic Max EC50 (nM)* Hill Top (%)
  • the antagonist activity of some of the non-naturally occurring melanocortin analogs was measured on NPFF1 by cAMP assay. Results from this assay are provided in Table 31. -156- 170456481.42 Docket No.: 146316.8023.WO00 Table 31.
  • Remaining Test Article dry powder or solid
  • Remaining test article will be stored at room temperature desiccated, and protected from light until shipment or discard.
  • Animals will be fasted overnight through 4 hours post dosing.
  • Vehicle and Formulation Preparation [0538] Appropriate amount of test article will be accurately weighed and mixed with appropriate volume of vehicle to get a clear solution or suspension.
  • Formulation samples will be removed from each of the formulation solutions, transferred into 1.5 mL of polypropylene microcentrifuge tubes and run dose validation by LC/UV or LC-MS/MS.
  • Cynomolgus Monkey Specifications Species Cynomolgus Monkeys K in -160- 170456481.42 Docket No.: 146316.8023.WO00 Method of Identification Unique skin tattoo on chest Number of Animals for 4 males e or al ff g e
  • Environmental Conditions Environment controls will be set to maintain a temperature range of 20-26°C, a relative humidity range of 40 to 70%, and a 12-hour light/12-hour dark cycle. The light/dark cycle may be interrupted for study-related activities.
  • Housing Animals will be group-housed (up to four animals/sex/cage) in polysulfone cages with certified aspen shaving bedding or corncob bedding during acclimation and study period.
  • While animals may be individually housed after surgery or when there is special requirement in protocol, as well as, for behavioral or health reasons or due to cage mate death.
  • Diet and Feeding Animals were offered certified rodent breeding and growth diet ad libitum every day, unless fasted for study procedures. Each lot of the diet is analyzed for nutrients, chemical contaminant and microorganisms, the results are reviewed and evaluated by veterinarians before provided to animals.
  • Drinking Water Autoclaved RO (reverses osmosis) water will be available to all animals, ad libum.
  • Feed and Water Analyses Autoclaved RO water will be provided ad libitum via water bottle. Water samples are periodically analyzed by a certified laboratory for specified microorganisms and environment contaminants. The diet is routinely analyzed by the manufacturer for specified microorganisms, nutritional components and environmental contaminants. [0547] Environmental Enrichment: Enrichment toys will be provided. [0548] Dose Administration: The dose volume will be determined by the animals' body weight collected on the morning of dosing day. Animal Specifications: Rats [0549] Rat specifications are outlined in Table 36. Table 36.
  • Rat Specifications Species Rat in o e e is -162- 170456481.42 Docket No.: 146316.8023.WO00 Observations and Examinations [0550] Clinical Observations: All animals will be observed at dosing and each scheduled collection. All abnormalities will be recorded. [0551] Body Weight: All animals will be weighed on the dosing day prior to dosing to determine the dose volume to be administered. Sample Collection and Processing [0552] Blood Sample Collection and Process: At least 0.1 mL blood will be collected at each time point. All blood samples will be collected via jugular vein.
  • All blood samples will be transferred into low binding EP tube with anticoagulant (0.5 M Potassium (K2) EDTA will be pre-added as a ratio of 50:1 for blood: anticoagulant), 0.05% Triton X-100 (e.g., 100uL Blood+2uL 2.5% Triton X-100) will be used for desorption the blood samples will be placed on wet ice.
  • Anticoagulant 0.5 M Potassium (K2) EDTA will be pre-added as a ratio of 50:1 for blood: anticoagulant
  • Triton X-100 e.g., 100uL Blood+2uL 2.5% Triton X-100
  • Plasma samples will be transferred into their respective pre-labeled low binding EP tube and immediately frozen over dry ice. The plasma samples will be stored lower than -60°C until bioanalysis.
  • LC-MS/MS method development [0555] A LC-MS/MS method for the quantitative determination of test compound in biological matrix will be developed. [0556] N in 1 cassette LC-MS/MS method may be developed for samples coming from different studies as long as these studies belong to the same sponsor. [0557] Cassette administration assay could be performed if the mass difference ( ⁇ Mass) among different analytes is ⁇ 4 Da. In this case, interference evaluation is not necessary. [0558] If ⁇ Mass among different analytes is less than 4 Da, there is a potential risk that interference would occur during LC-MS/MS analysis.
  • sample number within a batch is > 12, one standard curve and two sets of QCs with low, middle and high concentrations will be applied for bioanalysis. Meanwhile, QCs number should be more than 5% of study sample number.
  • Samples, coming from one client with the same type of matrix in different studies, are allowed to be quantified in one analysis run by using the developed N in 1 cassette LC-MS/MS method.
  • the calibration curve will be truncated.
  • the truncated calibration curve should consist of at least 75% of the initial STDs.
  • Carryover The mean calculated carryover peak area in the double blanks or blanks immediately after the highest standard injection should be less than that of LLOQ. -164- 170456481.42 Docket No.: 146316.8023.WO00 If the carryover could’t meet the criteria, the impact of the carryover on unknown samples should be re-evaluated according to the below procedure: [0570] Carryover should be re-evaluated based on absolute carryover.
  • Absolute carryover is calculated by carryover contribution multiplies carryover impact, where the carryover contribution is calculated by the area ratio of the double blank or blank with the highest carryover (Area max of carryover blank) to the ULOQ with the minimum calculated value (Area min of ULOQ), and the carryover impact is calculated by the area ratio of one injection (Area of one injection) to the following injection (Area of the following injection).
  • the absolute carryover should be below the acceptable accuracy of the studies (e.g., 20% or 25%).
  • Carryover contribution Areamax of carryover blank / Areamin of ULOQ
  • Carryover impact Area of one injection / Area of the following injection
  • Absolute carryover Carryover contribution * Carryover impact Data Analysis
  • Plasma concentration versus time data for Compounds A-C in Rats and Cynomolgus Monkeys will be plotted in graph and analyzed by non-compartmental approaches.
  • Related PK parameters will be calculated according to dosing route, e.g., Cl, Vdss and C0 for intravenous administration, Cmax, Tmax or %F for extravascular administration, and T1 ⁇ 2, AUC(0-t), AUC(0-inf), MRT(0-t), MRT(0-inf) for all routes.
  • PK parameters were calculated according to dosing route, e.g., Cl, Vdss and C0 for intravenous administration, Cmax, Tmax or %F for extravascular administration, and T1 ⁇ 2, AUC(0-t), AUC(0-inf), MRT(0-t), MRT(0-inf) for all routes.
  • Plasma Pharmacokinetic Results of Compound E (O10) in Cynomolgus Monkeys after 24 hours AUC0- Dose Cmax AUC 0-inf CE of administration of Compound E (Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2; O10), and in Rats are shown in Table 39 and Table 40. Graphs of plasma concentration and CSF concentration in Rats are shown in FIGS 3A-3C (SC administration), FIGS.4A-4G (IP administration), and FIGS.5A-5C (PO administration). [0575] Plasma and CSF concentrations over 24 hours after administration of Compound E (O10 or setmelanotide were also assessed (FIGS. 4H-4K).
  • Table 44 Pharmacokinetic Data Compound 30 mg/kg -168- 170456481.42 Docket No.: 146316.8023.WO00
  • the antagonist activity of exemplary non-naturally occurring melanocortin analogs at specific ion channels was measured using the Qube electrophysiological platform.
  • the non-naturally occurring melanocortin analogs and specific ion channel targets are provided in Table 45.
  • Table 45. Ion channel targets and melanocortin analogs Melanocortin Ion channel targets MW + Saly Condition Analog
  • the non-naturally occurring melanocortin analogs identified above were tested for antagonist activity at various ion channels at concentrations ranging from 0.1 mM to 30 mM.
  • hNav1.5 Sodium Channel Assay – Qube APC Onset and steady state block of peak Nav1.5 current is measured using a pulse pattern, repeated every 5 sec, consisting of a hyperpolarizing pulse to -120mV for a 200ms duration, depolarization to -15mV amplitude for a 40ms duration, followed by step to 40mV for 200ms and finally a 100ms ramp (1.2 V/s) to a holding potential of -80mV. Peak current is measured during the step to -15mV.
  • hKv4.3/hKChIP2 Potassium Channel Assay – Qube APC After whole cell configuration is achieved, the cells are held at -80mV. Onset and steady state block of hKv4.3 current is measured using a pulse pattern from -80mV to 40mV amplitude for a 110ms duration, and finally a 100ms ramp (1.2 V/s) to -80mV. This paradigm is delivered once every 5s to monitor the current amplitude. [0583] The parameters measured were the maximum outward current evoked on stepping to 40mV from holding potential of -80mV. All data were filtered for seal quality, seal drop, and current amplitude.
  • Control data is the mean Kv4.3/KChIP2 current amplitude collected 10 seconds at the end of the vehicle control period;
  • Test compound data is the mean Kv4.3/KChIP2 current amplitude collected 10 seconds at the end of test concentration application for each concentration.
  • hCav1.2 (L-type) CiPA Calcium Channel Assay – Qube APC Onset and steady state block of peak hCav1.2 current is measured using a pulse pattern, repeated every 15 sec.
  • Cells were held at -80mV for a 50ms before stepping to -90mV for 100ms to measure leak current and then stepped back to -80mV for 50ms, depolarization to 0mV amplitude for a 40ms duration, followed by step to 40mV for 200ms and finally a 100ms ramp (1.2 V/s) to a holding potential of -80 mV. Peak current is measured during the step to 0mV. Each concentration is applied for 5 minutes. [0585] The calcium current amplitude is calculated by measuring the difference between the peak inward current on stepping to 0mV or the peak inward current at the ramp (i.e. peak of the current) and the leak current.
  • Onset and steady state block of Late Nav1.5 current is measured using a pulse pattern, repeated every 5 sec, consisting of a hyperpolarizing pulse to -120mV for a 200ms duration, depolarization to -15mV amplitude for a 40ms duration, followed by step to 40mV for 200ms and finally a 100ms ramp (1.2 V/s) to a holding potential of -80mV.
  • Late current is measured as charge current elicited during the ramp with 50nM ATXII.
  • the parameters measured were the ramp current charge (AUC) evoked on ramping back to -80mV from 40mV test pulse in the presence of 50nM ATXII. All data were filtered for seal quality, seal drop, and current. The peak and ramp current amplitude was calculated before and after compound addition and the amount of current was assessed by dividing the Test compound current by the Control current.
  • Control data is the mean hNav1.5 late current collected 15 seconds at the end of 50nM ATXII application (50nM ATXII control); Test compound data is the mean ramp hNav1.5 current collected 15 seconds at the end of test concentration application for each concentration.
  • hERG Potassium Channel Assay - Qube APC After whole cell configuration is achieved, the cells are held at -80mV. Cells are held at this voltage for 50ms to measure the leak current, which is subtracted from the tail current on-line. The cells are depolarized to +40mV for 500ms and then to -80 mV over a 100ms ramp to elicit the hERG tail current. This paradigm is delivered once every 8s to monitor the current amplitude. All compounds were tested in the presence of 0.1% Pluronic F-68 Non-Ionic Surfactant and at approximately room temperature. [0589] The parameters measured were the maximum tail current evoked ramping back to -80mV from the test pulse of 40mV.
  • Control data is the mean hERG current amplitude collected for three pulses (24 seconds) at the end of the vehicle control;
  • Test compound data is the mean hERG current amplitude collected for three pulses (24 seconds) at the end of test concentration application for each concentration.
  • KCNQ1/minK currents are evoked by a 1000ms pulse from -80mV to 60mV followed by a ramp from 60mV to -80mV over 115ms with the outward peak currents measured upon depolarization of the cell membrane. This paradigm is delivered once every 15s to monitor the current amplitude.
  • the parameters measured were the maximum outward current evoked on stepping to +60mV from a holding potential of -80mV. All data were filtered for seal quality, seal drop, and current amplitude. The peak current amplitude was calculated before and after compound addition and the amount of block was assessed by dividing the Test compound current amplitude by the Control current amplitude.
  • Control data is the mean hKCNQ1/hminK current amplitude collected 30 seconds at the end of vehicle control period;
  • Test compound data is the mean hKCNQ1/hminK current amplitude collected 30 seconds at the end of test concentration application for each concentration.
  • hKir2.1 Potassium Channel Assay – Qube APC [0592] After whole cell configuration is achieved, the cells are held at -30mV. Kir2.1 currents are evoked by a single 500ms pulse to -120mV before returning to the holding potential of -30mV. This paradigm is delivered once every 20s to monitor the current amplitude.
  • the parameters measured were the maximum inward current elicited on stepping to -120mV for 500ms from a holding potential of -30 mV. All data were filtered for seal quality, seal drop, and current amplitude. The peak current amplitude was calculated before and after compound addition. Residual non-Kir2.1 current was eliminated via normalization to residual current after application of 100uM Barium Chloride. The amount of Test compound effect was then assessed by dividing the Test compound current amplitude by the Control current amplitude. Control data is the mean Kir2.1 current amplitude collected 40 seconds at the end of the vehicle control; Test compound data is the mean Kir2.1 current amplitude collected 30 seconds at the end of test concentration application for each concentration.
  • ⁇ M solubility
  • chromatographic purity was defined as the peak area of the principal peak relative to the total integrated peak area in the HPLC chromatogram of the calibration standard.
  • Partition Coefficient [0600] The total amount of compound was determined as the peak area of the principal peak in a calibration standard (100 ⁇ M) containing organic solvent (methanol/water, 60/40, v/v). The amount of compound in buffer was determined as the combined, volume-corrected, and weighted areas of the corresponding peaks in the aqueous phases of three organic- aqueous samples of different composition. An automated weighting system was used to ensure the preferred use of raw data from those samples with well quantifiable peak signals. The amount of compound in organic was calculated by subtraction. Subsequently, Log D was calculated as the Log10 of the amount of compound in the organic phase divided by the amount of compound in the aqueous phase.
  • Fluorescein was used as the cell monolayer integrity marker. Fluorescein permeability assessment (in the A-B direction at pH 7.4 on both sides) was performed after the permeability assay for the test compound. The cell monolayer that had a fluorescein permeability of less than 1.5 x 10 -6 cm/s for Caco-2 and MDR1-MDCKII cells and 2.5 x 10 -6 cm/s for MDCKII cells was considered intact, and the permeability result of the test compound from intact cell monolayer is reported. [0611] Results of the in vitro absorption assessments described above are provided in Table 53.
  • the half-life (T1/2) was estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming the first-order kinetics.
  • O10 may be efficiently manufactured using standard solid-phase peptide synthesis (SPPS).
  • SPPS solid-phase peptide synthesis
  • B07 a melanocortin antagonist, has high structural homology to O10.
  • B07 has been made in GMP batches up to 1 kg and it is amenable to efficient SPPS with high overall yields. Non-canonical residues and cyclic structure may restricts fermentation-based approaches, and peptide purification is performed using standard RP-HPLC techniques.
  • O10 may be amenable to liquid-phase peptide synthesis (LPPS) for late-stage clinical and commercial manufacturing, offering potential for significant reductions in manufacturing cost. It is anticipated that typical scale-up cost reductions (e.g., use of bulk residues, process optimization, captive manufacturing cost setups) will emerge.
  • LPPS liquid-phase peptide synthesis
  • Exemplary melanocortin analogs O7 and O10 and comparator melanocortin analog TCMCB07 were tested at concentrations ranging from 0.1 mM to 100 mM for inhibition of the seven CYPs.
  • the CYP inhibition assays were performed using human liver microsomes (HLM) and human recombinant CYP isozymes in 96-well plate format. Direct inhibition (e.g., zero-min incubation) and time-dependent (e.g., 30 min preincubation) inhibition assays were performed.
  • Example 10 Body Weight, Complete Blood Count, Clinical Chemistry, and Food Intake in an Animal Model Objective [0619] The objective of this study was to evaluate the pharmacodynamic (PD) characteristics and effects on food intake, body composition, and clinical chemistry following administration of exemplary non-naturally occurring melanocortin analogs of the present technology.
  • Non-naturally occurring melanocortin analogs were administered to Cynomolgus monkeys via two subcutaneous (s.c.) injections at different doses.
  • Primate specifications are outlined in Table 58.
  • Body weight, clinical observations, and food intake observations were recorded daily. Measurement of initial body composition using Dual-Energy X-Ray Absorptiometry (DEXA) was recorded at least once during this time. Standard blood markers (e.g., glucose, insulin, HDL-c, LDL-c, triglycerides, total cholesterol, alanine aminotransferase, aspartate aminotransferase) were measured once. [0626] Treatment Phase (Days 8-49): Body weight was recorded twice a week, clinical observation and food intake were recorded once a day. Standard hematology and clinical chemistry readouts as above were conducted before dosing and at the conclusion of the study on day 49. DEXA measurements of body composition were conducted biweekly.
  • DEXA Dual-Energy X-Ray Absorptiometry
  • Food intake measurement was observed at about 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 24 hours, 48 hours, and 72 hours after administration of saline or non- naturally occurring melanocortin analogs at 1 mg/kg and 3 mg/kg (Day 8 and Day 11) dosing.
  • Day 8 and Day 11 On Days 4-7, monkeys were acclimatized to a normal diet of two meals per day.
  • the lower dose of the non-naturally occurring melanocortin analogs were administered, and observations continued through Day 10.
  • the higher dose of the non-naturally occurring melanocortin analogs was administered, and observations continued through Day 13, the conclusion of the study.
  • Blood collection was conducted for the following: clinical chemistry (Serum), hematology (whole blood). Whole blood was collected at each time point with syringe via the cephalic or saphenous veins into labeled polypropylene tubes. -185- 170456481.42 Docket No.: 146316.8023.WO00 Table 60. Blood Sampling Animal Blood Minimum Volume Time Point Test Type Gr S m l T f Bl d S m les [0629] Clinical chemistry and hematology assessments were conducted, assessing the parameters outlined in Tables 61 and 62.
  • Table 61 Clinical Chemistry Parameter Abbreviation Unit -186- 170456481.42 Docket No.: 146316.8023.WO00 C-reactive Protein CRP mg/L
  • Clinical Chemistry including triglycerides, creatine kinase, lactate dehydrogenase, alanine amino transferase, aspartate amino transferase, bilirubin, alkaline phosphatase, total protein, albumin, globulin, glucose, urea nitrogen, creatine, urea acid, and C-reactive protein was assessed in monkey serum on days 1, 8 (2h), and 11 (2h). Results of each measurement are outlined in Tables 71-76.
  • Diet-induced obese monkeys administered O10 showed a 7.5% weight loss in 5 weeks, relative to baseline. -198- 170456481.42 00 O W . 320 8 . 61364 1: . o N . . . t A 72 A 01 6 . 1 9 . 3 03 ekco D ya M 1 D P 7 . y . 6 a M 4 D P 2 4 . 9 . 0 . 0 3 8 5 1 4 9 4 - 0 2 1 0 M 4 4 2 7 M 2 7 9 1 2 - A . 9 . 4 A 5 . 2 6 . 2 7 .
  • Table 107 Clinical Chemistry Day 1 (Part A) Clinical Glu Alanine Aspartate Alkaline Gamma- Total Total Trigly Chemis cos Aminotran Aminotransf Phospha glutamyl Biliru Prote cerid ol 71 5 41 9 .6 80 3 Table 108: Clinical Chemistry Day 1 (Part B) Clinical (Total) Alb High density Low density Lactate Cre Urea Creatin e -215- 170456481.42 Docket No.: 146316.8023.WO00 um mmol Day 1 mmol/L g/L mmol/L mmol/L U/L ol/L /L U/L M 75 95 3 62 5 33 6 56 4 Table 109: Clinical Chemistry Day 15 (Part A) Clinical Meas Glu Alanine Aspartate Alkaline Gamma- Total Total Trigl Chemi urem cos Aminotran Aminotrans Phos ha lutam l Biliru Prot ceri o 5 5 7 3
  • the tanning effect comprised skin darkening without hyperpigmentation (e.g., an uneven distribution of melanin) compared to baseline. Notably, the tanning effect was only seen in typically sun- exposed skin, with no observation of ectopic tanning (e.g., palms, gums, etc.). Monkeys -224- 170456481.42 Docket No.: 146316.8023.WO00 were never exposed to sunlight. Pigmentation resolved back to baseline levels rapidly after treatment was discontinued. [0643] The slope of the O10 weight loss curve (FIG.16I) suggests that a lower dose of O10 may be employed for the same efficacy. This suggests presence of a therapeutic window between weight loss and pigmentation (i.e., tanning effect).
  • rats were subcutaneously administered 0.5 mg/kg of A07D, O7, O10, or O11 for days 1-7 and 1 mg/kg for days 8-17.
  • O11 and O7 did not appear to influence food consumption as a percent of food consumption in saline-treated rats, relative to O10 (FIG.11Q).
  • O10 treated rats gained only about 0.64% of their body weight compared to saline, which gained about 8.98% body weight (**) (FIG.12L).
  • Example 13 Cardiac Effects [0645] Changes in cardiac effects were assessed in rats subcutaneously administered O10 at either 0.5 mg/kg, 1 mg/kg, or 3 mg/kg. Control rats were administered saline. Animal specifications are outlined in Table 119.
  • BP blood pressure
  • ECG blood pressure
  • SBP stolic blood pressure
  • DBP diastolic blood pressure
  • HR heart rate
  • QRS, ST, QT, P wave, T wave, and HR were assessed with ECG measurements.
  • Recording duration occurred from -15 min (pre-dose) to 180 min (after dosing).
  • Blood Collection [0653] After recording, animals were euthanized with CO2 immediately and blood was collected for in vitro analysis, including plasma corticosterone by ELISA Kit. Data Processing and Analysis [0654] All values were expressed as mean ⁇ SEM.
  • Table 121 Blood Pressure Analysis (Part 1) Animal SBP(mmHg) i -227- 170456481.42 Docket No.: 146316.8023.WO00 SEM 1 0 2 1 3 1 2 2 2 G5: Mean 105 104 104 103 107 106 106 110 109 Table 122: Blood Pressure Analysis (Part 2) Animal DBP(mmHg) Group Measureme 0mi 5mi 15mi 30mi 60mi 90mi 120mi 150mi 180mi Table 123: Blood Pressure Analysis (Part 3) Animal HR(BPM) mi -228- 170456481.42 Docket No.: 146316.8023.WO00 G6: O10 SEM 20 32 33 34 42 38 18 19 21 1 k Animal Heart Rate (BPM) Group Measureme 0mi 5mi 15mi 30mi 60mi 90mi 120mi 150mi 180mi Animal PR Interval (s) Group Measurem 15mi 30mi 60mi 90mi 120mi 150mi
  • O10 was assessed for interaction with RFamide receptors, which are hypothesized to cause cardiac activation in MC4R-targeting small molecules and peptides (Table 129).
  • Table 129 O10 Assessment for RFamide Receptor Binding Antagonist mode, IC50 (nM) Agonist mode, EC50 (nM) F 0 [0658]
  • O10 did not agonize or antagonize RFamide receptors up to 1000 nM (top concentration tested), 1000x-100,000x above their respective EC50s on MC4R.
  • O10 was next screened for binding against a panel of 87 receptors and channels at 10 ⁇ M. Results showing an inhibition or stimulation higher than 50% were considered to represent significant effects of the test compounds. O10 only showed >50% inhibition of control on melanocortin receptors (FIG. 18). O10 was shown to have minimal inhibition at 10 ⁇ M for cytochrome P450 (CYP) enzymes (Table 130 and Table 131). O10 showed no meaningful impact on CYP induction or inhibition at physiologically relevant concentrations, demonstrating minimal drug-drug interactions.
  • CYP cytochrome P450
  • Example 15 Assessing Adipose Tissue Effects of O10 [0660]
  • fat tissue was harvested from diet-induced obese cynomolgus monkeys orally administered 10 mg/kg O10 or saline control.
  • O10-treated monkeys showed (1) smaller lipid droplets, (2) darker, dense staining likely attributed to higher mitochondrial concentrations, and (3) increased vascularization, relative to saline controls (FIG 20; H&E staining, 200x), consistent with beige/brown adipocytes. This suggests that O10 administration may stimulate metabolic remodeling through adipose tissue browning, which in turn may increase metabolic rate and calorie burn and may improve insulin sensitivity.
  • Example 16 Assessing absorption, distribution, metabolism, and excretion of non- naturally occurring melanocortin analogs
  • Non-naturally occurring melanocortin analogs of the present technology, O10 and C2 were assessed for solution properties, in vitro absorption, and in vitro metabolism for absorption, distribution, metabolism, and excretion (ADME) properties.
  • ADME absorption, distribution, metabolism, and excretion
  • Aqueous solubility was determined by comparing the peak area of the principal peak in a calibration standard (200 ⁇ M) containing organic solvent (methanol/water, 60/40, v/v) with the peak area of the corresponding peak in a buffer sample. Additionally, chromatographic purity (%) was defined as the peak area of the principal peak relative to the total integrated peak area in the HPLC chromatogram of the calibration standard. A chromatogram of the calibration standard of each test compound, along with a UV/VIS spectrum with labeled absorbance maxima, was generated.
  • the amount of compound in buffer was determined as the combined, volume-corrected, and weighted areas of the corresponding peaks in the aqueous phases of three organic- aqueous samples of different composition. An automated weighting system was used to ensure the preferred use of raw data from those samples with well quantifiable peak signals. -236- 170456481.42 Docket No.: 146316.8023.WO00
  • the amount of compound in organic was calculated by subtraction. Subsequently, LogD was calculated as the Log10 of the amount of compound in the organic phase divided by the amount of compound in the aqueous phase.
  • Half-Life Determination At the end of the incubation at each of the time points, an equal volume of an organic mixture (acetonitrile/methanol, 50/50, v/v) was added to the incubation mixture. Samples were analyzed by HPLC-MS/MS and corresponding peak areas were recorded for each analyte. The ratio of precursor compound remaining after each time point relative to the amount present at time 0, expressed as percent, is reported as chemical stability. The half-life (T1/2) was estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) versus time, assuming first-order kinetics.
  • ADME-Tox In Vitro Absorption Permeability [0667]
  • CR,end is the concentration of the test compound in the receiver chamber at the end time point.
  • ⁇ t is the incubation time.
  • A is the surface area of the cell monolayer.
  • CD,mid is the calculated mid-point concentration of the test compound in the donor side, which is the mean value of the donor concentration at time 0 minute and the donor concentration at the end time point.
  • CD,end is the concentration of the test compound in the donor sample at the end time point.
  • CR,end is the concentration of the test compound in the receiver sample at the end time point.
  • CD0 is the concentration of the test compound in the donor sample at time zero. Concentrations of the test compound are expressed as peak areas of the test compound.
  • Fluorescein Assessment for Permeability Assays [0669] Fluorescein was used as the cell monolayer integrity marker. Fluorescein permeability assessment (in the A-B direction at pH 7.4 on both sides) was performed after the permeability assay for the test compound. The cell monolayer that had a fluorescein permeability of less than 1.5 x 10 -6 cm/s for Caco-2 and MDR1-MDCKII cells and 2.5 x 10 -6 cm/s for MDCKII cells was considered intact, and the permeability result of the test compound from intact cell monolayer is reported.
  • ADME-Tox In Vitro Metabolism Intrinsic Clearance (microsomes, S9, cryopreserved hepatocytes, recombinant CYP, recombinant UGT) [0670] Metabolic stability, expressed as percent of the parent compound remaining, was calculated by comparing the peak area of the compound at the time point relative to that at time-0. The half-life (T1/2) was estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming first-order kinetics.
  • E - E - E - E - E - s t c - - P : - - 0 1 M 0 . 1 M 0 . 1 0 . 1 0 . 1 0 . E 1 0 . y s no n E 1 t i l i e o T C i t 0 . E 2 0 . y E 2 t i l 0 . E 2 0 .
  • O10 is (1) about 408x (x, times) more potent on MC3R, (2) about 120x more potent on MC4R, and (3) has superior PK parameters as shown in at least rats, where the CSF half-life of O10 is about 2x longer, the plasma half- life is about 3x longer, the CSF AUC is about 1.5x higher, and the plasma AUC is 4.5x higher (also see Table 133 and Table 134) compared to setmelanotide.
  • Table 133 cAMP Signaling Assay – Agonist Mode; EC50 (nM) Setmelanotide O10 Table 134: 10mg/kg SC (rat) PK Parameters Setmelanotide O10 PK Mean CSF Mean Mean CSF Mean Parameters Plasma Plasma Cmax 43.2 1164 21.3 1366 (ng/mL) Tmax (h) 1.00 1.00 6.00 2.00 T1/2 (h) 2.79 0.938 5.62 2.78 Tlast (h) 4.00 8.00 10.0 24.0 AUC0-last 83.1 2396 121 10731 (ng.h/mL) AUC0-inf 154 2407 226 10781 (ng.h/mL) [0672] Second, as shown by at least the drug exposure over the EC50 analyses, O10 is present at efficacious doses in CSF for periods of time longer than setmelanotide, O10 remains present in the CSF at concentrations higher than the EC50.
  • O10 -248- 170456481.42 Docket No.: 146316.8023.WO00 will be more efficacious for general obesity (e.g., obesity not caused by genetic factors related to the melanocortin system) due to higher affinity for MC3R and MC4R and PK values in plasma and CSF (as shown in the rat) compared to setmelanotide.
  • O10 reduces food intake in a single dose via 1 and 3 mg/kg SC injection in DIO cynomolgus monkeys, whereas setmelanotide did not affect food intake in 39-week toxicity study via 3 mg/kg/day SC injection in standard BMI cynomolgus monkeys. It is predicted that the 10 and 30 mg/kg oral administration doses of O10 will be efficacious considering the PK data and superior potency of O10 on MC3R and MC4R activation (Table 135).
  • Table 135 PL parameters O1030 mg/kg, oral administration in monkeys PK Mean SD CV (%) Paramet [0674]
  • the oral PK results suggest that O10 will also be efficacious at 10 and 30 mg/kg PO administration.
  • the plasma PKs of a 1 mg/kg SC injection of O10 to an oral 10 mg/kg and 30 mg/kg administration of O10 were above the EC50. Food intake may thus be reduced following oral administration of O10. -249- 170456481.42 Docket No.: 146316.8023.WO00 [0675]
  • (1) O10 is present in efficacious levels in CSF by oral administration.
  • a non-naturally occurring melanocortin analog comprising a sequence according to Formula (IF), R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -Y 1 -Y 2 (IF), wherein: R 1 is Nle; R 2 is Asp or Glu; R 3 is His; R 4 is selected from dPhe, p(F)dPhe, and p(Cl)dPhe; R 5 is Arg or His; R 6 is Trp; R 7 is Orn; Y 1 is dVal; Y 2 is dPro; and the non-naturally occurring melanocortin analog is cyclized through a lactam bridge between Asp or Glu at R 2 and Orn at R 7 .
  • Formula (IF) Formula (IF), R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -Y 1 -Y 2 (IF), wherein: R 1 is Nle; R 2 is Asp or
  • the non-naturally occurring melanocortin analog of embodiment 1, comprising a sequence selected from the group consisting of: Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2 (SEQ ID NO: 43); Ac-Nle-c[Asp-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2 (SEQ ID NO: 44); Ac-Nle-c[Glu-His-dPhe-His-Trp-Orn]-dVal-dPro-NH2 (SEQ ID NO: 45); Ac-Nle-c[Asp-His-p(Cl)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2 (SEQ ID NO: 11); and Ac-Nle-c[Glu-
  • the non-naturally occurring melanocortin analog of embodiment 5, comprising a sequence of: Ac-Nle-c[Glu-His-dPhe-His-Trp-Orn]-dVal-dPro-NH2 (SEQ ID NO: 45), wherein c represents cyclization through R2 and R7 via a lactam bond.
  • R4 is p(F)dPhe or p(Cl)dPhe.
  • the non-naturally occurring melanocortin analog of embodiment 9, comprising a sequence selected from the group consisting of: Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2 (SEQ ID NO: 43); Ac-Nle-c[Asp-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2 (SEQ ID NO: 44); Ac-Nle-c[Asp-His-p(Cl)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2 (SEQ ID NO: 11); and Ac-Nle-c[Glu-His-p(Cl)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2 (SEQ ID NO: 12), wherein c represents cyclization through R2 and R7 via a lactam bond.
  • a non-naturally occurring melanocortin analog for reducing body weight and/or fat mass in an obese subject, comprising orally administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • 20. A method of treating, preventing, or reducing hyperplasia in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • Non-naturally occurring melanocortin analog for treating, preventing, or reducing hyperplasia in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a method of treating, preventing, or reducing hypothalamic obesity in a subject in need thereof comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • [0699] 23 A method of treating, preventing, or reducing hypothalamic obesity in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a non-naturally occurring melanocortin analog for treating, preventing, or reducing hypothalamic obesity in a subject in need thereof, comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • the hypothalamic obesity comprises congenital hypothalamic damage, damage from tumors, or damage from trauma.
  • a method of treating, preventing, or reducing a syndromic obesity in a subject in need thereof comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a method of treating, preventing, or reducing non-alcoholic steatohepatitis (NASH) in a subject in need thereof comprising administering a non- naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • NASH non-alcoholic steatohepatitis
  • a method of treating, preventing, or reducing hyperinsulinism in a subject in need thereof comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a method of treating, preventing, or reducing a cardiovascular disease in a subject in need thereof comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a method of treating, preventing, or reducing osteoarthritis in a subject in need thereof comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a method of treating, preventing, or reducing a cancer in a subject in need thereof comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I). -256- 170456481.42 Docket No.: 146316.8023.WO00 [0709] 44.
  • a method of treating, preventing, or reducing erectile dysfunction in a subject in need thereof comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a method of treating, preventing, or reducing body weight gain after a weight loss in a subject in need thereof comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a method of maintaining body weight after a weight loss in a subject in need thereof comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a method of treating, preventing, or reducing fat mass gain after a weight loss in a subject in need thereof comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a method of treating, preventing, or reducing fat mass gain after a weight loss in a subject in need thereof comprising administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a non-naturally occurring melanocortin analog for treating, preventing, or reducing glucose intolerance and/or diabetes mellitus in a subject in need thereof comprising orally administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a non-naturally occurring melanocortin analog for treating, preventing, or reducing glucose intolerance and/or diabetes mellitus in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a
  • a method of reducing obesity-related inflammation in a subject in need thereof comprising orally administering a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • a method of preserving or improving kidney function in a subject in need thereof comprising orally administering a non-naturally occurring melanocortin 4 receptor agonist to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (I).
  • third dose of about 15 mg/kg administered twice a day after administration of the second dose for at least about 7 days.
  • any one of embodiments 1-70 wherein the non-naturally occurring melanocortin analog is administered (i) at a first dose of about 15 mg/kg administered once a day for about 7 days; (ii) at a second dose of about 25 mg/kg administered once a day after administration of the first dose for about 7 days; (iii) at a third dose of about 20 mg/kg administered twice a day after administration of the second dose for at least about 7 days. [0730] 75.
  • the non-naturally occurring melanocortin analog comprises a sequence of Formula (I), wherein: X 1 is absent or is selected from the group consisting of norleucine (Nle), arginine (Arg), and D-arginine (dArg); R 1 is selected from the group consisting of Nle, Arg, dArg, aspartic acid (Asp), alanine (Ala), lysine (Lys), D-lysine (dLys), histidine (His), and D-histidine (dHis); R 2 is selected from the group consisting of Asp, D-cysteine (dCys), Ala, D-alanine (dAla), proline (Pro), glutamic acid (Glu), and phenylalanine (Phe); R 3 is selected from the group consisting of His, dHis, Pro, Phe, and glutamine (Gln); -259- 17045648
  • the non-naturally occurring melanocortin analog comprises a sequence according to Formula (IE): X 1 -R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -Y 1 -Y 2 (IE), wherein: X 1 is absent or selected from the group consisting of Nle, Arg, and dArg; R 1 is Asp; R 2 is dAla or Ala; R 3 is His; R 4 is dPhe; R 5 is Arg; R 6 is Trp; R 7 is Lys; Y 1 is dVal; Y 2 is dPro; and -269- 170456481.42 Docket No.: 146316.8023.WO00 the non-naturally occurring melanocortin analog is cyclized through a lactam bridge between Asp at R 1 and Lys at R 7 .
  • IE Formula
  • the non-naturally occurring melanocortin analog comprises a sequence according to Formula (IF): R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -Y 1 -Y 2 (IF), wherein: R 1 is Nle; R 2 is Asp or Glu; R 3 is His; R 4 is selected from dPhe, p(F)dPhe, and p(Cl)dPhe; R 5 is Arg or His; R 6 is Trp; R 7 is Orn; Y 1 is dVal; Y 2 is dPro; and the non-naturally occurring melanocortin analog is cyclized through a lactam bridge between Asp or Glu at R 2 and Orn at R 7 .
  • Formula (IF) R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -Y 1 -Y 2 (IF)
  • any one of embodiments 13-105 wherein the non-naturally occurring melanocortin analog comprises a sequence of any of one SEQ ID NOs: 11, 12, and 43-45.
  • a method of reducing body weight and/or fat mass in a subject in need thereof comprising orally administering about 15 mg/kg to about 100 mg/kg of a non- naturally occurring melanocortin analog to the subject, -275- 170456481.42 Docket No.: 146316.8023.WO00 the non-naturally occurring melanocortin analog comprising a sequence of Formula (IF).
  • IF Formula
  • a non-naturally occurring melanocortin analog for reducing body weight and/or fat mass in a subject in need thereof, comprising orally administering about 15 mg/kg to about 100 mg/kg of a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (IF).
  • IF a sequence of Formula
  • a non-naturally occurring melanocortin analog for treating, preventing, or reducing glucose intolerance and/or diabetes mellitus in a subject in need thereof, comprising orally administering about 15 mg/kg to about 100 mg/kg of a non- naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (IF).
  • IF insulin receptor
  • a method of reducing obesity-related inflammation in a subject in need thereof comprising orally administering about 15 mg/kg to about 100 mg/kg of a non- naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (IF).
  • a method of preserving or improving kidney function in a subject in need thereof comprising orally administering about 15 mg/kg to about 100 mg/kg of a non- naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (IF).
  • a non-naturally occurring melanocortin analog for preserving or improving kidney function in a subject in need thereof, comprising orally administering about 15 mg/kg to about 100 mg/kg of a non-naturally occurring melanocortin analog to the subject, the non-naturally occurring melanocortin analog comprising a sequence of Formula (IF).
  • a non-naturally occurring melanocortin analog comprising a sequence of: Ac-Nle-c[Glu-His-p(F)dPhe-Arg-Trp-Orn]-dVal-dPro-NH2 (SEQ ID NO: 43), wherein c represents cyclization through R2 and R7 via a lactam bond.
  • [0778] 123 A method of reducing body weight and/or fat mass in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin analog comprising a sequence of SEQ ID NO: 43 to the subject.
  • [0780] 125 The method or the use of embodiment 123 or 124, wherein the method or the use further comprises reducing lean mass loss, maintaining lean mass, or promoting lean mass gain in the subject.
  • a non-naturally occurring melanocortin analog for treating, preventing, or reducing glucose intolerance and/or diabetes mellitus in a subject in need thereof, comprising orally administering a non-naturally occurring melanocortin analog comprising a sequence of SEQ ID NO: 43 to the subject.
  • the method or the use of embodiment 128 or 129, wherein the method or the use further comprises reducing lean mass loss, maintaining lean mass, or promoting lean mass gain in the subject.
  • 131 The method or the use of embodiment 130, wherein the lean mass is lean muscle mass.
  • 132 The method or the use of embodiment 130, wherein the lean mass is lean muscle mass.
  • a lipid droplets size e.g., an average lipid droplet size
  • any one of embodiments 13-144 wherein the concentration of the non-naturally occurring melanocortin analog in the plasma or a tissue of the subject is at least about 5 ng/mL to at least about 2000 ng/mL about 24 hours after administration of the non-naturally occurring melanocortin analog.
  • the non-naturally occurring melanocortin analog is administered to the subject until the concentration of the non-naturally occurring melanocortin analog in the plasma or a tissue of the subject is at least about 5 ng/mL to at least about 2000 ng/mL after administration of the non-naturally occurring melanocortin analog.

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  • Urology & Nephrology (AREA)
  • Addiction (AREA)
  • Toxicology (AREA)
  • Rheumatology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Psychiatry (AREA)
  • Zoology (AREA)
  • Emergency Medicine (AREA)
  • Endocrinology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pain & Pain Management (AREA)
  • Child & Adolescent Psychology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des méthodes de traitement, de prévention ou de réduction d'une ou de plusieurs affections ou d'un ou de plusieurs symptômes associés à un dysfonctionnement métabolique, tels que l'obésité, chez un sujet qui en a besoin à l'aide d'un analogue de la mélanocortine d'origine non naturelle. Dans certains modes de réalisation, l'analogue de la mélanocortine d'origine non naturelle est administré par voie orale. Dans certains modes de réalisation, la méthode comprend la suppression de l'appétit chez un sujet qui en a besoin à l'aide d'un analogue de la mélanocortine d'origine non naturelle selon la présente technologie.
PCT/US2025/016151 2024-02-16 2025-02-14 Analogues de la mélanocortine d'origine non naturelle administrables par voie orale et leurs utilisations pour moduler la perte de poids Pending WO2025175250A1 (fr)

Applications Claiming Priority (30)

Application Number Priority Date Filing Date Title
US202463554944P 2024-02-16 2024-02-16
US63/554,944 2024-02-16
US202463557409P 2024-02-23 2024-02-23
US63/557,409 2024-02-23
US202463572896P 2024-04-01 2024-04-01
US63/572,896 2024-04-01
US202463573433P 2024-04-02 2024-04-02
US63/573,433 2024-04-02
US202463632496P 2024-04-10 2024-04-10
US63/632,496 2024-04-10
US202463637285P 2024-04-22 2024-04-22
US63/637,285 2024-04-22
US202463640864P 2024-04-30 2024-04-30
US63/640,864 2024-04-30
US202463647545P 2024-05-14 2024-05-14
US63/647,545 2024-05-14
US202463650368P 2024-05-21 2024-05-21
US63/650,368 2024-05-21
US202463654876P 2024-05-31 2024-05-31
US63/654,876 2024-05-31
US202463656579P 2024-06-05 2024-06-05
US63/656,579 2024-06-05
US202463663689P 2024-06-24 2024-06-24
US63/663,689 2024-06-24
US202463675219P 2024-07-24 2024-07-24
US63/675,219 2024-07-24
US202463681055P 2024-08-08 2024-08-08
US63/681,055 2024-08-08
US202463692605P 2024-09-09 2024-09-09
US63/692,605 2024-09-09

Publications (1)

Publication Number Publication Date
WO2025175250A1 true WO2025175250A1 (fr) 2025-08-21

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Family Applications (4)

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PCT/US2025/016127 Pending WO2025175235A1 (fr) 2024-02-16 2025-02-14 Analogues de mélanocortine d'origine non naturelle à administration orale
PCT/US2025/016113 Pending WO2025175224A1 (fr) 2024-02-16 2025-02-14 Analogues de la mélanocortine d'origine non naturelle administrables par voie orale et méthodes associés pour moduler le gain de poids
PCT/US2025/016115 Pending WO2025175226A1 (fr) 2024-02-16 2025-02-14 Analogues de mélanocortine d'origine non naturelle pouvant être administrés par voie orale et leurs utilisations pour traiter des troubles liés à l'usage de substances
PCT/US2025/016151 Pending WO2025175250A1 (fr) 2024-02-16 2025-02-14 Analogues de la mélanocortine d'origine non naturelle administrables par voie orale et leurs utilisations pour moduler la perte de poids

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PCT/US2025/016127 Pending WO2025175235A1 (fr) 2024-02-16 2025-02-14 Analogues de mélanocortine d'origine non naturelle à administration orale
PCT/US2025/016113 Pending WO2025175224A1 (fr) 2024-02-16 2025-02-14 Analogues de la mélanocortine d'origine non naturelle administrables par voie orale et méthodes associés pour moduler le gain de poids
PCT/US2025/016115 Pending WO2025175226A1 (fr) 2024-02-16 2025-02-14 Analogues de mélanocortine d'origine non naturelle pouvant être administrés par voie orale et leurs utilisations pour traiter des troubles liés à l'usage de substances

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WO (4) WO2025175235A1 (fr)

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US20150045293A1 (en) * 2012-03-13 2015-02-12 Tensive Controls Inc. Melanocortin analogs having enhanced activity and transport
US20150297672A1 (en) * 2014-04-22 2015-10-22 Txp Pharma Gmbh Alpha- and gamma-MSH analogues
WO2024015880A2 (fr) * 2022-07-12 2024-01-18 Rhythm Pharmaceuticals, Inc. Méthodes de traitement de l'obésité avec un agoniste mc4r
WO2024030977A2 (fr) * 2022-08-03 2024-02-08 Sightstream Biotherapeutics, Inc. Nouveaux analogues de la mélanocortine

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US7084111B2 (en) * 2003-06-23 2006-08-01 University Of Florida Research Foundation, Inc. Melanocortin receptor templates, peptides, and use thereof
US8058240B2 (en) * 2006-04-18 2011-11-15 University Of Florida Research Foundation, Inc. Biological active ligands of melanocortin receptors
EP2440572B1 (fr) * 2009-06-08 2017-04-05 Palatin Technologies, Inc. Peptides spécifiques des récepteurs aux mélanocortines à pont lactame
US20210221867A1 (en) * 2018-05-15 2021-07-22 Novo Nordisk A/S Compounds Capable of Binding to Melanocortin 4 Receptor
WO2024177907A2 (fr) * 2023-02-21 2024-08-29 Endevica Bio, Inc. Méthodes de traitement du cancer et/ou d'états pathologiques liés à la chimiothérapie à l'aide d'analogues de mélanocortine d'origine non naturelle

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150045293A1 (en) * 2012-03-13 2015-02-12 Tensive Controls Inc. Melanocortin analogs having enhanced activity and transport
US20150297672A1 (en) * 2014-04-22 2015-10-22 Txp Pharma Gmbh Alpha- and gamma-MSH analogues
WO2024015880A2 (fr) * 2022-07-12 2024-01-18 Rhythm Pharmaceuticals, Inc. Méthodes de traitement de l'obésité avec un agoniste mc4r
WO2024030977A2 (fr) * 2022-08-03 2024-02-08 Sightstream Biotherapeutics, Inc. Nouveaux analogues de la mélanocortine

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WO2025175235A1 (fr) 2025-08-21
WO2025175226A1 (fr) 2025-08-21

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