Compositions and methods for treating metabolic diseases

Abstract

The invention provides compositions and methods for inducing satiety or treating a metabolic condition. The compositions include polypeptides, which can be used to provide treatment for obesity and metabolism related disorders.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING METABOLIC DISEASES

[0002] Sequence Listing

[0003] This application contains a Sequence Listing which has been filed electronically in Extensible Markup Language (XML) format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 14, 2025, is named 51547-021 WO2_Sequence_Listing_4_14_25. XML and is 4,462 bytes in size.

[0004] Background of the Invention

[0005] The prevalence of obesity continues to increase worldwide. However, there is still a lack of effective, long-term, non-invasive treatments for obesity and other metabolism related disorders. Current treatments for metabolic syndrome, diabetes, obesity, and obesity-related disorders with existing therapeutics do not produce suitable therapeutic effects and often cause severe side effects such as nausea. Accordingly, new treatments without side effects are needed.

[0006] Summary of the Invention

[0007] In one aspect, the invention features a polypeptide of SEQ ID NO: 1 .

[0008] In another aspect, the invention features a polypeptide of SEQ ID NO: 2.

[0009] In another aspect, the invention features a polypeptide of SEQ ID NO: 3.

[0010] In another aspect, the invention features a pharmaceutical composition (e.g., formulated for local oral administration, e.g., topical-lingual administration) that includes the polypeptide of SEQ ID NO: 1 and a pharmaceutically acceptable excipient.

[0011] In another aspect, the invention features a pharmaceutical composition (e.g., formulated for local oral administration, e.g., topical-lingual administration) that includes the polypeptide of SEQ ID NO: 2 and a pharmaceutically acceptable excipient.

[0012] In another aspect, the invention features a pharmaceutical composition (e.g., formulated for local oral administration, e.g., topical-lingual administration) that includes the polypeptide of SEQ ID NO: 3 and a pharmaceutically acceptable excipient.

[0013] In some embodiments, the composition is configured for administration without substantially changing the concentration of the polypeptide in the blood of a subject. In some embodiments, the composition is configured to administration without the polypeptide substantially appearing in the blood of the subject.

[0014] In some embodiments, the composition is formulated for local administration.

[0015] In some embodiments, the composition is formulated for oral administration.

[0016] In some embodiments, the composition is formulated to topical lingual administration.

[0017] In some embodiments, the composition is formulated as an oral dissolving tablet, a lozenge, a film, a spray, a semisolid, a particulate, or in a lipid-based carrier.

[0018] In some embodiments, the pharmaceutically acceptable excipient includes one or more of propylene glycol, potassium sorbate, l-arginine, edetate disodium, monosodium phosphate, polysorbate 20, gelatin, mannitol, dextran, alginate, polyvinyl alcohol, polyvinylpyrrolidone, acacia, aspartame, sodium methylparaben, sodium propylparaben, phenylalanine, water, or a combination thereof.

[0019] In some embodiments, the pharmaceutically acceptable excipient includes one or more of gelatin, mannitol, dextran, alginate, polyvinyl alcohol, polyvinylpyrrolidone, acacia, aspartame, sodium methylparaben, sodium propylparaben, phenylalanine, water, or a combination thereof.

[0020] In some embodiments, the pharmaceutically acceptable excipient includes one or more of a stabilizer, a preservative, an antioxidant, a buffer, a surfactant, a rheology modifier, and a mucosal permeation enhancer.

[0021] In some embodiments, the preservative includes sodium methyl paraben or sodium propyl paraben.

[0022] In some embodiments, the stabilizer includes mannitol or sucrose.

[0023] In some embodiments, the mucosal permeation enhancer includes gelatin.

[0024] In some embodiments, the pharmaceutically acceptable excipient further includes a flavoring or sweetening agent.

[0025] In some embodiments, the flavoring agent includes mint (e.g., peppermint or spearmint), cinnamon, vanilla, cherry, strawberry, or lemon.

[0026] In some embodiments, the sweetening agent includes sorbitol, sucrose, or aspartame.

[0027] In some embodiments, the composition is formulated for topical administration to the Gl tract, for intranasal administration, or for intrarectal administration.

[0028] In some embodiments, the composition is formulated as a gastrointestinal patch.

[0029] In some embodiments, the composition is formulated as a suppository.

[0030] In another aspect, the invention features a method for inducing satiety or treating a disease or disorder selected from a metabolic syndrome, obesity, an obesity-related disorder, diabetes, fatty liver disease, nonalcoholic steatohepatitis, chronic kidney disease, polycystic ovary syndrome, cardiovascular disease, obstructive sleep apnea, retinopathy, peripheral vascular disease, peripheral artery disease, and neuropathy in a subject in need thereof by administering to the subject a pharmaceutical composition as described herein, e.g., of any of the above embodiments.

[0031] In some embodiments, the composition is administered locally.

[0032] In some embodiments, the composition is administered orally.

[0033] In some embodiments, the composition is administered topically on the tongue.

[0034] In some embodiments, the composition is administered topically to the Gl tract, intranasally, or intrarectally.

[0035] In some embodiments, the composition provides treatment without substantially changing the concentration of the polypeptide in the blood of the subject. In some embodiments, the composition provides treatment without the polypeptide substantially appearing in the blood of the subject.

[0036] In some embodiments, the concentration in blood of the polypeptide of the does not exceed an endogenous PYY level in the subject by more than 100%. Definitions

[0037] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the invention. Terms such as "a," "an," and "the" are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.

[0038] As used herein, the term “about” refers to a value that is within 10% above or below the value being described.

[0039] As used herein, the term “metabolic disorder” refers to a human or animal condition or disease associated with and / or resulting from abnormal function or control of the metabolic system (e.g., obesity, diabetes, fatty liver disease, nonalcoholic steatohepatitis, polycystic ovary syndrome, elevated blood glucose levels, chronic kidney disease, cardiovascular disease, obstructive sleep apnea, retinopathy, neuropathy (e.g., diabetic neuropathy), peripheral artery disease, and / or peripheral vascular disease). The term “disorder” generally refers to disruption to regular bodily structure and function or a pathophysiological response to internal or external factors.

[0040] As used herein, the term “subject,” refers to a human or non-human animal (e.g., a mammal).

[0041] As used herein, the terms “topical-lingual administration,” “topically to the tongue,” or variations thereof, refer to the local administration of an agent (e.g., a polypeptide, e.g., a PYY variant) to the epithelium of the mouth and / or tongue of a subject with substantially no change in the levels of the agent in the blood of the subject (e.g., substantially no systemic exposure).

[0042] As used herein, the phrases “without substantially changing the concentration of the agent in the blood or plasma of the subject” and “without substantially appearing in the blood or plasma of the subject” refer to an increase of the agent level (e.g., PYY variant) in the blood and / or plasma by no more than up to 10% (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01% or less) of the preadministration level of the PYY variant. For example, a PYY variant administered to a subject topically to the tongue (e.g., topically to the lingual epithelium) at any dosage would reach at most a peak level of one tenth that of the endogenous level of PYY and decrease substantially thereafter. In some embodiments, the blood and / or plasma level of the agent does not surpass 20 pM.

[0043] Brief Description of the Drawings

[0044] FIG. 1 A is a table showing the functional potency of PYY(3-36), PYY-1 , PYY-2, and PYY-3 on hNPY2R.

[0045] FIG. 1B is a table showing the binding affinity of PYY(3-36), PYY-1 , PYY-2, and PYY-3 to hNPY2R.

[0046] FIG. 1C is a table showing the selectivity of PYY(3-36), PYY-1 , PYY-2, and PYY-3 on hNPY1 R, hNPY4R, and hNPY5R relative to hNPY2R.

[0047] FIG. 2A is a table showing the turbidity of PYY(3-36), PYY-1 , PYY-2, and PYY-3 as an indicator of solubility at different pH values. FIG. 2B is a table showing the fibrillation profile of PYY(3-36), PYY-1 , PYY-2, and PYY-3 in three different buffer solutions.

[0048] FIG. 2C is a table showing the solubility and chemical and physical stability as a function of the percent change in high molecular weight PYY(3-36), PYY-1 , PYY-2, and PYY-3 as measured by size exclusion chromatography.a2mg / mL in 5mM phosphate buffer pH 7;1SEC determining HMW (high molecular weight): >0.5% is a medium risk; Percentages of HMW.

[0049] FIG. 2D is a table showing the loss of main peak of PYY(3-36), PYY-1 , PYY-2, and PYY-3 as measured by reverse-phase (RP) chromatography.a2mg / mL in 5mM phosphate buffer pH 7;2RP determining loss of main peak: >3% is a medium risk.

[0050] FIG. 3A is a table showing the visual inspection results of Placebo, PYY(3-36), PYY-1 , and PYY-3 under 2-8°C and 25°C at 1 , 2, and 3 months after formulation. Placebo samples are duplicated aliquots containing inactive ingredients only. +, Formulation vial contains small quantity of flake-like visible particles; CCS, clear colorless solution.

[0051] FIG. 3B is a table showing the solubility of Placebo, PYY(3-36), PYY-1 , and PYY-3 under 2- 8°C and 25°C at 1 , 2, and 3 months after formulation. Placebo contains inactive ingredients only. Boxes are shaded based on <10 NTU (shade 1 ), 10-15 NTU (shade 2), 15-25 NTU (shade 3), or >25 NTU (shade 4); higher NTUs indicate more turbid.

[0052] FIG. 4 is a table showing the pH measurements of Placebo, PYY(3-36), PYY-1 , and PYY-3 under 2-8°C and 25°C at 1 , 2, and 3 months after formulation. Placebo contains inactive ingredients only.

[0053] FIG. 5A is a table showing the percent change in high molecular weight relative to t=0 of PYY(3-36), PYY-1 , and PYY-3 as measured by size exclusion chromatography under 2-8°C and 25°C at 1 , 2, and 3 months after formulation.1SEC determining HMW % change relative to TO.

[0054] FIG. 5B is a graph showing the percent change in high molecular weight relative to t=0 of PYY(3-36), PYY-1 , and PYY-3 as measured by size exclusion chromatography under 2-8°C at 1 , 2, and 3 months after formulation.

[0055] FIG. 5C is a graph showing the percent change in high molecular weight relative to t=0 of PYY(3-36), PYY-1 , and PYY-3 as measured by size exclusion chromatography under 25°C at 1 , 2, and 3 months after formulation.

[0056] FIG. 6A is a table showing the percent change in loss of main peak relative to t=0 of PYY(3- 36), PYY-1 , and PYY-3 as measured by reverse-phase chromatography under 2-8C° and 25°C at 1 , 2, and 3 months after formulation.1RP determining loss of main peak (%) relative to TO.

[0057] FIG. 6B is a graph showing the percent change in loss of main peak relative to t=0 of PYY(3- 36), PYY-1 , and PYY-3 as measured by reverse-phase chromatography under 2-8C° at 1 , 2, and 3 months after formulation.

[0058] FIG. 6C is a graph showing the percent change in loss of main peak relative to t=0 of PYY(3- 36), PYY-1 , and PYY-3 as measured by reverse-phase chromatography under 25°C at 1 , 2, and 3 months after formulation. FIG. 7A is a table showing the percent increase in degradant relative to t=0 of PYY(3-36), PYY-1 , and PYY-3 as measured by reverse-phase chromatography under 2-8°C and 25°C at 1 , 2, and 3 months after formulation.1RP determining degradant % increase relative to TO

[0059] FIG. 7B is a graph showing the percent increase in degradant relative to t=0 of PYY(3-36), PYY-1 , and PYY-3 as measured by reverse-phase chromatography under 2-8°C at 1 , 2, and 3 months after formulation.

[0060] FIG. 7C is a graph showing the percent increase in degradant relative to t=0 of PYY(3-36), PYY-1 , and PYY-3 as measured by reverse-phase chromatography under 25°C at 1 , 2, and 3 months after formulation.

[0061] FIG. 8A is a table showing the percent change in recovery relative to t=0 of PYY(3-36), PYY- 1 , and PYY-3 as measured by size exclusion chromatography under 2-8°C and 25°C at 1 , 2, and 3 months after formulation.1Recovery (%) = Total peak areas at time point / total peak areas at TO.

[0062] FIG. 8B is a graph showing the percent change in recovery relative to t=0 of PYY(3-36), PYY- 1 , and PYY-3 as measured by size exclusion chromatography under 2-8°C and 25°C at 1 , 2, and 3 months after formulation.

[0063] FIG. 8C is a table showing the percent change in recovery relative to t=0 of PYY(3-36), PYY- 1 , and PYY-3 as measured by reverse-phase chromatography under 2-8°C and 25°C at 1 , 2, and 3 months after formulation.1Recovery (%) = Total peak areas at time point / total peak areas at TO.

[0064] FIG. 8D is a graph showing the percent change in recovery relative to t=0 of PYY(3-36), PYY- 1 , and PYY-3 as measured by reverse-phase chromatography under 2-8°C and 25°C at 1 , 2, and 3 months after formulation.

[0065] FIG. 9A is a graph showing the blood glucose level over 120 minutes following subcutaneous delivery of vehicle, extendin-4 (Ex-4) at 30 nmol / kg, or PYY(3-36) at 30, 100, 300, or 1000 nmol / kg in normal lean mice (*, p < 0.05).

[0066] FIG. 9B is a graph showing the acetaminophen level over 120 minutes following subcutaneous delivery of vehicle, Ex-4 at 30 nmol / kg, or PYY(3-36) at 30, 100, 300, or 1000 nmol / kg in normal lean mice (*, p < 0.05).

[0067] FIG. 10A is a graph showing the blood glucose level over 120 minutes following subcutaneous delivery of vehicle, Ex-4 at 30 nmol / kg, or PYY-1 at 30, 100, 300, or 1000 nmol / kg in normal lean mice (*, p < 0.05).

[0068] FIG. 10B is a graph showing the acetaminophen level over 120 minutes following subcutaneous delivery of vehicle, Ex-4 at 30 nmol / kg, or PYY-1 at 30, 100, 300, or 1000 nmol / kg in normal lean mice (*, p < 0.05).

[0069] FIG. 11 A is a graph showing the blood glucose level over 120 minutes following subcutaneous delivery of vehicle, Ex-4 at 30 nmol / kg, or PYY-2 at 30, 100, 300, or 1000 nmol / kg in normal lean mice (*, p < 0.05).

[0070] FIG. 11B is a graph showing the acetaminophen level over 120 minutes following subcutaneous delivery of vehicle, Ex-4 at 30 nmol / kg, or PYY-2 at 30, 100, 300, or 1000 nmol / kg in normal lean mice (*, p < 0.05). FIG. 12A is a graph showing the blood glucose level over 120 minutes following subcutaneous delivery of vehicle, Ex-4 at 30 nmol / kg, or PYY-3 at 30, 100, 300, or 1000 nmol / kg in normal lean mice (*, p < 0.05).

[0071] FIG. 12B is a graph showing the acetaminophen level over 120 minutes following subcutaneous delivery of vehicle, Ex-4 at 30 nmol / kg, or PYY-3 at 30, 100, 300, or 1000 nmol / kg in normal lean mice (*, p < 0.05).

[0072] FIG. 13A is a graph showing the cumulative food intake level up to 1 hour after subcutaneous delivery of vehicle or PYY(3-36) at 3, 10, 30, 100, 300, or 1000 nmol / kg in overnight-faster male C57BL / 6 mice (*, p < 0.05). Data are expressed as mean ± SEM.

[0073] FIG. 13B is a graph showing the cumulative food intake level up to 2 hours after subcutaneous delivery of vehicle or PYY(3-36) at 3, 10, 30, 100, 300, or 1000 nmol / kg in overnight- faster male C57BL / 6 mice (*, p < 0.05). Data are expressed as mean ± SEM.

[0074] FIG. 14A is a graph showing the cumulative food intake level up to 1 hour after subcutaneous delivery of vehicle or PYY-1 at 3, 10, 30, 100, 300, or 1000 nmol / kg in overnight-faster male C57BL / 6 mice (*, p < 0.05). Data are expressed as mean ± SEM.

[0075] FIG. 14B is a graph showing the cumulative food intake level up to 2 hours after subcutaneous delivery of vehicle or PYY-1 at 3, 10, 30, 100, 300, or 1000 nmol / kg in overnight-faster male C57BL / 6 mice (*, p < 0.05). Data are expressed as mean ± SEM.

[0076] FIG. 15A is a graph showing the cumulative food intake level up to 1 hour after subcutaneous delivery of vehicle or PYY-2 at 3, 10, 30, 100, 300, or 1000 nmol / kg in overnight-faster male C57BL / 6 mice (*, p < 0.05). Data are expressed as mean ± SEM.

[0077] FIG. 15B is a graph showing the cumulative food intake level up to 2 hours after subcutaneous delivery of vehicle or PYY-2 at 3, 10, 30, 100, 300, or 1000 nmol / kg in overnight-faster male C57BL / 6 mice (*, p < 0.05). Data are expressed as mean ± SEM.

[0078] FIG. 16A is a graph showing the cumulative food intake level up to 1 hour after subcutaneous delivery of vehicle or PYY-3 at 3, 10, 30, 100, 300, or 1000 nmol / kg in overnight-faster male C57BL / 6 mice (*, p < 0.05). Data are expressed as mean ± SEM.

[0079] FIG. 16B is a graph showing the cumulative food intake level up to 2 hours after subcutaneous delivery of vehicle or PYY-3 at 3, 10, 30, 100, 300, or 1000 nmol / kg in overnight-faster male C57BL / 6 mice (*, p < 0.05). Data are expressed as mean ± SEM.

[0080] FIG. 17A is a graph showing the cumulative food intake level over 35 days following topical lingual delivery of vehicle, topical lingual delivery of PYY-3 at 30 nmol / kg, or subcutaneous delivery of semaglutide at 1 nmol / kg in mice.

[0081] FIG. 17B is a graph showing the cumulative food intake level over 35 days following topical lingual delivery of vehicle, topical lingual delivery of PYY-3 at 30 nmol / kg, or subcutaneous delivery of semaglutide at 1 nmol / kg in mice. The data is from FIG. 17A with vehicle subtracted.

[0082] FIG. 17C is a graph showing the change in body weight over 35 days following topical lingual delivery of vehicle, topical lingual delivery of PYY-3 at 30 nmol / kg, or subcutaneous delivery of semaglutide at 1 nmol / kg in mice. Detailed Description of the Invention

[0083] In general, the invention features new Peptide YY (PYY) variants and methods of use thereof for inducing satiety and treating a condition affecting metabolism, such as metabolic syndrome, diabetes, obesity, and obesity-related disorders. PYY is a 36 amino acid metabolic hormone that is released in the gastrointestinal tract in response to feeding and reduces appetite. The two N-terminal residues are cleaved by dipeptidyl peptidase IV (DPPIV) to produce PYY(3-36), which is highly selective for the Neuropeptide Y receptor type 2 (Y2R) that has been implicated in regulating food intake.

[0084] The invention is based in part upon the surprising discovery that certain variants of PYY(3-36) have particularly advantageous properties, such as enhanced potency, solubility and stability, binding affinity, and receptor selectivity, e.g., as compared to native PYY(3-36). Such polypeptides can be administered to a subject, e.g., via oral administration (e.g., topical lingual administration) to effectively treat the disease or disorder.

[0085] In some embodiments, by administering the PYY variant via topical lingual administration routes, the composition can provide treatment to the subject without substantially changing the concentration of the polypeptide in the blood of the subject and / or without the polypeptide substantially appearing in the blood of the subject. Furthermore, these routes of administration avoid systemic exposure, which is known to produce unwanted side effects, such as nausea, injection site pain, or malaise.

[0086] Administration of the PYY variant via a non-systemic pathway allows the polypeptide to target specific satiety centers in the brain, activating critical brain regions to create a feeling of fullness, while avoiding neural or non-neural targets that can cause clinical risk (e.g., toxicity) or side effects, such as nausea. For example, systemic administration of a metabolic hormone activates neural receptors in the hypothalamus, nucleus tractus solitarius (NTS), and area postrema, whereas non-systemic routes of administration as described herein activates neural receptors in the hypothalamus and NTS, but substantially avoids the area postrema. By targeting neural receptors, e.g., in the mouth (e.g., tongue), nose, rectum, or Gl tract, the neural receptors and connections can sufficiently activate the satiety centers in the CNS, which control food intake. Accordingly, activation of these satiety centers may provide treatment for a metabolic disorder or a satiety disorder associated with dysregulated pleasure centers in the brain. The compositions and methods are described in more detail below.

[0087] PYY Variants

[0088] The polypeptides described herein include variants of PYY. In particular, the PYY variants include the following PYY(3-36) variants: Table 1. PYY Variants

[0089] The polypeptides methods described herein include administration of PYY variants as described herein. The PYY variant targets (e.g., binds, associates, or interacts with) a Y2 receptor in the oral, nasal cavity, Gl tract, or rectum of a subject.

[0090] In some embodiments, the PYY variant has the amino acid sequence set forth in SEQ ID NO:

[0091] 1 . The PYY variant of SEQ ID NO: 1 may be formulated in a pharmaceutical composition, e.g., a topical lingual formulation.

[0092] In some embodiments, the PYY variant has the amino acid sequence set forth in SEQ ID NO:

[0093] 2. The PYY variant of SEQ ID NO: 2 may be formulated in a pharmaceutical composition, e.g., a topical lingual formulation.

[0094] In some embodiments, the PYY variant has the amino acid sequence set forth in SEQ ID NO:

[0095] 3. The PYY variant of SEQ ID NO: 3 may be formulated in a pharmaceutical composition, e.g., a topical lingual formulation.

[0096] Pharmaceutical Compositions and Routes of Administration

[0097] The PYY variants described herein can be formulated as pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.

[0098] The compositions described herein may be administered to a subject (e.g., a human) in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compositions described herein may be administered, for example, by any route that allows the composition (e.g., the PYY variant) to reach the target receptor without substantially changing the concentration of the polypeptide in the blood of the subject. The composition may be administered by, for example, topical-lingual (e.g., topically to the tongue, e.g., to the lingual epithelium), intranasal, intrarectal, or topical Gl routes.

[0099] In some embodiments, the compositions described herein are formulated for delivery to the oral cavity, e.g., intraoral, oromucosal, topical lingual, gargles, mouthwashes, gingival solutions, oromucosal solutions and oromucosal suspensions, semi-solid oromucosal preparations (including for example gingival gel, gingival paste, oromucosal gel, oromucosal paste), oromucosal drops, oromucosal sprays and sublingual sprays (including oropharyngeal sprays), dry powder sprays, lozenges and pastilles, compressed lozenges, sublingual tablets and buccal tablets, oromucosal capsules, mucoadhesive preparations). See, e.g., Oromucosal Preparations, (Ph Eur monograph 1807). In some embodiments, the PYY variant in the pharmaceutical composition is adapted for binding to the Y2 receptors expressed in the oral cavity (e.g., to the tongue). In some embodiments, the pharmaceutical composition is formulated as a lozenge, a film, a spray, or an orally dissolvable tablet (ODT). In some embodiments, the pharmaceutical composition is formulated as an ODT using a formulation that rapidly dissolves on the tongue of a subject. For example, the formulation may have partially hydrolyzed gelatin at a concentration of from 1% to 6% w / v (e.g., 1%, 2%, 3%, 4%, 5%, or 6%), mannitol, hydrolyzed dextran, alginate, polyvinyl alcohol, polyvinylpyrrolidone, acacia, aspartame, sodium methylparaben, sodium propylparaben, phenylalanine, water, or a combination thereof. In some embodiments, the ODT is formulated using the ZYDIS® formulation, as described in U.S. Patent Nos. 4,305,502, 4,371 ,516, and 5,738,875, herein incorporated in their entirety by reference.

[0100] In some embodiments, the compositions described herein are formulated for intranasal delivery. The intranasal composition may be formulated, e.g., as a spray, a semisolid, a particular, or in a lipid-based carrier. The formulation may be, e.g., a solution, a suspension, a powder, or a gel. Suitable intranasal formulations are described, e.g., in Marx et al. Drug Discov Dev 299-320, 2015, which is hereby incorporated by reference in its entirety. In some preferred embodiments, the compositions described herein are administered via inhalation, e.g., via nasal inhalation. An inhalable composition described herein may be provided as a liquid dosage form or dry powder dosage form. A dry powder composition may be, e.g., administered by inhalation as is or after reconstitution in a vehicle, e.g., saline (e.g., isotonic saline), phosphate-buffered saline, or water. In some embodiments, the intranasal formulation does not include insulin.

[0101] In some embodiments, the compositions described herein are formulated for topical administration to the Gl tract. The topical composition may be formulated, e.g., as a Gl patch. Suitable Gl patch formulations are described, e.g., in Tao, et al Drug discovery Today 10:909-915, 2005, which is hereby incorporated by reference it its entirety.

[0102] In some embodiments, the compositions described herein are formulated for intrarectal delivery. The intrarectal composition may be formulated, e.g., as a suppository, an enema, an ointment, or a rectal foam. Suitable intrarectal formulations are described, e.g., in Hua Front. Pharmacol. 10: 1196, 2019, which is hereby incorporated by reference in its entirety. Compositions for rectal administration may be in the form of suppositories containing a conventional suppository base, such as cocoa butter.

[0103] Solutions of a composition described herein can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO, and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington’s Pharmaceutical Sciences (2012, 22nd ed.) and in The United States Pharmacopeia: The National Formulary (USP 41 NF 36), published in 2018. The composition described herein may be administered to an animal, e.g., a human, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which is determined by the solubility and chemical nature of the composition, chosen route of administration, and standard pharmaceutical practice. In some embodiments, the compositions include excipients that increase the time the PYY variant is in contact with the mucosa (e.g., oral mucosa, nasal mucosa, Gl mucosa, or rectal mucosa). The excipients may provide viscosity enhancement, encapsulation, and controlled release. In some embodiments, increasing the contact time of the pharmaceutical formulation with the mucosa leads to increased binding of the PYY variant to its receptor. Suitable excipients for viscosity enhancement include rheology modifiers which also may be mucoadhesive such as methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, alginic acid, polyvinylpyrrolidone, and sodium carboxymethylcellulose. Suitable excipients for modified release of the PYY variant in the mucosa include mucoadhesive permeation enhancers such as 23-lauryl ether, aprotinin, azone, benzalkonium chloride, cetylpyridinium chloride, cetyltrimethylammonium bromide, cyclodextrins, dextran sulfate, and lauric acid. Other suitable mucoadhesive polymers used for the compositions described herein include agarose, chitosan, gelatin, hyaluronic acid, gums (e.g. guar, hakea, xanthan, gellan, carrageenan, pectin, and sodium alginate), cellulose derivatives (e.g., CMC, thiolated CMC, sodium CMC, HEC, HPMC, MC, methylhydroxylethylcellulose), poly (acrylic acid)-based polymers (e.g., CP, PC, PAA, polyacrylates, poly(methylvinylether-co-methacrylic acid), poly(2-hydroxyethyl methacryalate), poly(alkylcyanoacryalate), poly(isohexylcyanocrylate), poly(isobutylcyanoacrylate), copolymer of acrylic acid and PEG, poly(N-2-hydroxypropyl methacrylamide), PHPMAm, polyoxyethylene, PVA, PVP, and other thiolated polymers; scleroglucan, PVA, steroidal detergents, non-ionic surfactants, laureth-9, sodium fusidate, included sodium lauryl, sodium laurate (e.g., pH 8.9), palmitoyl carnitine, lauric acid / propylene glycol vehicle, Brij 78, sodium deoxycholate, sodium lauryl sulfate, lecithin and PVP. See, e.g., International Journal of Pharmaceutics, Volume 53, Issue 3, 1 August 1989, Pages 227-235.

[0104] In some embodiments, the pharmaceutical compositions include excipients that increase the residence time of the PYY variant in the saliva (e.g., the amount of time the PYY variant remains in the saliva without significant degradation of the peptide). Without being bound by theory, it is believed that increasing the residence time of PYY variant in the saliva increases the opportunity for the PYY variant to bind its receptor on the tongue. The residence time in the saliva can optionally be adjusted to avoid increasing systemic exposure to the PYY variant through, for example, swallowing.

[0105] A composition containing a PYY variant as described herein can include one or more pharmaceutically acceptable excipients, such as propylene glycol, potassium sorbate, l-arginine, edetate disodium, monosodium phosphate, and polysorbate 20. In some embodiments, propylene glycol is present in a concentration of 100 mg / ml, l-arginine is present in a concentration of 25 mg / ml, potassium sorbate is present in a concentration of 2 mg / ml, edetate disodium is present in a concentration of 1 .2 mg / ml, sodium phosphate monobasic dihydrate is present in a concentration of 7.8 mg / ml, and polysorbate is present in a concentration of 5 mg / ml. Furthermore, the compositions described herein can include co-solvent stabilizers like propylene glycol or other suitable co-solvent stabilizers (e.g., lower molecular weight polyethylene glycols (PEG) such as PEG 200 and 400, glycerin, and ethanol. In some embodiments, compositions described herein include amino acid stabilizers like L-arginine or other suitable amino acid stabilizers (e.g., alanine, aspartic acid, glycine, lysine, proline, or methionine). In some embodiments, compositions described herein can include preservatives like potassium sorbate, or other suitable preservatives (e.g., ascorbic acid, benzyl alcohol, benzoic acid, citric acid, chlorobutanol, m-cresol, glutathione, methionine, methylparaben, propylparaben, sodium sulfite, parahydroxybenzoate esters (methylhydroxybenzoate and propylhydroxybenzoate), boric acid and borate salts, sorbic acid and other sorbate salts besides potassium, and phenolics), compositions described herein can include antioxidants such as edetate disodium or another suitable antioxidant (e.g., sodium formaldehyde sulphoxylate, butylated hydroxyanisole, and butylated hydroxytoluene). In some embodiments, the compositions described herein include buffers (e.g., acetate, carbonate, citrate, citrate-phosphate, glycine, HEPES, histidine, maleate, phosphate, succinate, tartrate, and triethanolamine (Tris)). In some embodiments, the compositions described herein can include surfactants, such as polysorbate 20 or other suitable surfactants (e.g., Poloxamer 188 / 407, polysorbate 40 or 80, or sodium lauryl sulfate).

[0106] In some embodiments, the excipients include flavorings to increase compliance with ingesting the composition. For example, the flavorings can be used to mask bitter or other undesirable flavor properties, or to make the composition compatible with the flavor of food that may be ingested before or after administration of the composition. Compatible flavorings include, for example, apple, banana, bubblegum, cherry, chocolate, grape, lemon, mango, orange, raspberry, strawberry, vanilla, watermelon, mint (e.g., peppermint or spearmint), cinnamon, or a combination of the above flavors. In another aspect, these flavorings are dye-free, sugar-free, hypoallergenic, gluten-free, and casein-free.

[0107] In some embodiments, the pharmaceutical composition may be administered as in a unit dose form or as a dose per mass or weight of the subject.

[0108] The dosage of the PYY variant described herein, can vary depending on many factors, such as the pharmacodynamic properties of PYY variant, the mode of administration, the age, health, and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment, and the type of concurrent treatment, if any, and the clearance rate of the composition in the animal to be treated. The compositions described herein may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In some embodiments, the dosage of the PYY variant is a prophylactically or a therapeutically effective amount. Furthermore, it is understood that all dosages may be continuously given or divided into dosages given per a given time frame. The composition can be administered, for example, every hour, day, week, month, or year. In some embodiments, the composition may be administered continuously. For example, a rectal formulation or Gl patch may be present on the subject for a sustained amount of time (e.g., for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or longer).

[0109] The pharmaceutical compositions described herein (e.g., containing a PYY variant variant) may be provided in a kit that includes the pharmaceutical composition (e.g., in a container) and instructions for use thereof. The kit may contain one or more containers, in which each container contains a different composition of the invention. The instructions enclosed with the kit may be used to instruct a user to perform a method as described herein.

[0110] The methods described herein include administration of a PYY variant locally to the mouth (e.g., tongue, salivary glands, lingual and / or sublingual epithelium, or mucosa), rectum, nasal cavity, or Gl tract of a subject, wherein the local administration does not produce substantial change to the level of the PYY variant in the blood and / or plasma of the subject. In general, the PYY variant level in the blood and / or plasma of the subject does not increase more than up to 10% (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) of the pre-administration level of the polypeptide. For example, a PYY variant administered to a subject topically to the tongue (e.g., topically to the lingual epithelium), at any dosage would reach at most a peak level of one tenth that of the endogenous level of PYY and decrease substantially thereafter. In some embodiments, the blood and / or plasma level of the PYY variant does not substantially surpass pre-prandial levels of from 15 pmol / l to 25 pmol / l as reported in Batterham et al, Cell Metabolism, 4:223-233, 2006, herein incorporated by reference, in its entirety, after topical-lingual administration of PYY(3-36).

[0111] Indications

[0112] The methods described herein include the administration of a PYY variant for the induction of satiety and / or treatment of metabolic syndrome, diabetes, obesity, or any obesity-related disorder.

[0113] In some embodiments, the subject in need of treatment has been diagnosed with or it is at risk of a metabolic syndrome, obesity, an obesity-related disorder, diabetes, fatty liver disease, nonalcoholic steatohepatitis, chronic kidney disease, polycystic ovary syndrome, cardiovascular disease, obstructive sleep apnea, retinopathy, peripheral vascular disease, peripheral artery disease, and neuropathy (e.g., diabetic neuropathy). In some embodiments, the methods are used for maintenance of weight or prevention of weight gain. The methods may be used for weight loss or prevention of weight gain.

[0114] Examples

[0115] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0116] Example 1. Evaluation of the functional potency, binding affinity, and selectivity of PYY(3-36), PYY-1, PYY-2, and PYY-3 in vitro

[0117] Objective

[0118] The objective of this study was to determine the in vitro functional potency, binding affinity, and selectivity of the PYY variants.

[0119] Materials and Methods

[0120] Preparation of hNPYIR, hNPY2R, hNPY4R, and hNPY5R

[0121] PYY(3-36) (SEQ ID NO: 4), PYY-1 (SEQ ID NO: 1 ), PYY-2 (SEQ ID NO: 2), and PYY-3 (SEQ ID NO: 3) were synthesized at Lilly Research Laboratories (Indianapolis, IN, USA) and were characterized by LC / MS, NMR, and LC / UV analysis (99.5% purity). Peptide contents were estimated at 80% powder mass. The peptides were prepared as 2 mM stock solution in 100% DMSO and kept frozen at -20°C until just prior to testing in the assays.

[0122] For hNPY1 R, transient overexpression was performed using CHO cells. Stably transfected cell lines were prepared for hNPY2R by subcloning receptor cDNA into pcDNA3.1 expression plasmid and transfecting into human embryonic kidney (HEK) 293 cells followed by selection with Geneticin. hNPY4R was transiently overexpressed in HEK293 cells, and hNPY5R was transiently overexpressed in CHO cells.

[0123] For the preparation of hNPY1 R, hNPY2R, hNPY4R, and hNPY5R crude cell membranes, two different methods (described below) were utilized:

[0124] Method 1 - For hNPY2R membrane, frozen cell pellets were lysed on ice in 10 mL hypotonic homogenization buffer containing 50 mM Tris HCI, pH 7.5, and Roche Complete™ Protease Inhibitors with EDTA (#1 169749001 ) per gram of wet cell paste. The cell suspension was disrupted using a glass Potter-Elvehjem homogenizer fitted with a Teflon® pestle for 25 strokes. The homogenate was centrifuged at 4 °C at 1 ,100 x g for 10 minutes. The supernatant was collected and stored on ice while the pellets were resuspended in homogenization buffer and re-homogenized as described above. The homogenate was centrifuged at 1 100 x g for 10 minutes. The second supernatant was combined with the first supernatant and centrifuged at 35,000 x g for 1 hour at 4 °C. The resulting membrane pellet was resuspended in homogenization buffer containing protease inhibitors at approximately 1 to 3 mg / mL, quickly frozen in liquid nitrogen, and stored as aliquots in a -80°C freezer until use. Protein concentration was determined using a BCA protein assay kit (Pierce, #23225) with BSA as a standard.

[0125] Method 2 - For hNPY1 R, hNPY4R and hNPY5R membranes, frozen cell pellets were lysed on ice in 5 mL hypotonic homogenization Buffer containing 25 mM Tris HCI, pH 7.5, 1 mM MgCl2, 25 units / mL DNase I (Invitrogen, #18047-019) and Roche COMPLETE™ Protease Inhibitors without EDTA (#1 1836170001 ) per gram of wet cell paste. The cell suspension was disrupted using a glass Potter-Elvehjem homogenizer fitted with a Teflon® pestle for 25 strokes. The homogenate is centrifuged at 4 °C at 1 ,800 x g for 15 minutes in a 50 mL conical tube. The supernatant was collected and stored on ice while the pellets were resuspended in homogenization buffer and re-homogenized as described above, except DNase I was not used in the homogenization buffer. The homogenate was centrifuged at 1 ,800 x g for 15 minutes. The second supernatant is combined with the first supernatant and centrifuged at 25,000 x g for 30 minutes at 4 °C. The resulting membrane pellet was resuspended in homogenization buffer containing protease inhibitors at approximately 2 mg / mL, aliquoted, and stored in a -80°C freezer until use. Protein concentration was determined using a BCA protein assay kit (Pierce, #23225) with BSA as a standard.

[0126] Functional potency

[0127] The in vitro functional activity of the PYY variants was assessed by measuring inhibition of forskolin-induced intracellular cAMP production in HEK 293 cells overexpressing recombinant hNPY2R. Human PYY(3-36) and PYY variants were synthesized, characterized, and stored as described above. Receptor cloning - A cell line stably expressing hNPY2R was prepared by subcloning receptor cDNA into pcDNA3.1 expression plasmid and transfecting it into HEK 293 cells followed by selection with Geneticin. Aliquots of cells (1 -2 x 106cells / mL) were made and kept frozen in the vapor phase of a liquid nitrogen tank. These frozen aliquots were used at the time of the assay. Cells maintained greater than 95% viability over several months. hNPY2R cAMP assay - Inhibition of forskolin-induced cAMP production by PYY(3-36) and PYY variants was measured using HEK 293 cells overexpressing recombinant hNPY2R. Frozen aliquots of cells were thawed in a 37°C water bath. Cells were transferred to a 50 mL tube with 10 mL of culture medium (DMEM cell culture medium with 4.5g / L D-Glucose (Gibco Cat #31053) and 10% FBS). The cell suspension was centrifuged for 5 minutes at 1500 rpm in a Beckman tabletop centrifuge. The supernatant was removed, and the cell pellet was resuspended in 10 mL of assay buffer, consisting of DMEM with 4.5g / L D-Glucose (Gibco Cat #31053), 1 % GlutaMax (Gibco Cat #35050), 20 mM HEPES (Gibco Cat #15630-080) and 0.1 % Casein from Bovine Milk (Sigma Cat #C4765), followed by passage through a 40 pm strainer. An accurate count of cell number and cell viability was determined using a Vi-Cell Analyzer from Beckman-Coulter (Vi-Cell XR 2.03), and cells were further diluted into assay buffer to a concentration of 0.2M cells / mL. The cell suspension was added to all wells (10 pL / well) of white 384-well assay plates (Corning, Cat #3570) using a Combi-Tip Dispenser (Thermo Scientific). Plates were centrifuged for 1 minute at 1500 rpm. A 20 point 2-fold dilution of the test peptides in 100% DMSO was added to the cells using acoustic dispensing technology (Labcyte Echo), and assay plates were incubated for 45 minutes at 37°C (final DMSO concentration^ %). Following the incubation, 10 pL / well of assay buffer was supplemented with 0.5 pM forskolin (Tocris Cat #1603) and 250 pM IBMX for 30 minutes at 37°C. The intracellular cAMP was quantified using a Perkin Elmer cAMP Gs dynamic kit (Perkin Elmer Cat #62AM4PEJ) following the manufacturer’s instructions. Briefly, cAMP levels within the cell were detected using the HTRF kit reagents by adding cAMP-d2 conjugate in cell lysis buffer (10 pL / well) followed by adding the antibody anti-cAMP-Eu3+-Cryptate, also in cell lysis buffer (10 pL / well). The resulting competitive assay was incubated for at least 60 minutes at room temperature, then read on a PerkinElmer ENVISION™ instrument with excitation at 320 nm and emission at 665 nm and 620 nm. A standard curve of known cAMP concentrations (0.5 pM to 1 pM) was prepared in assay buffer according to the manufacturer’s instructions.

[0128] Data analysis for hNPY2R cAMP assay - The fluorescence emission ratio (665 nM / 620 nm) was calculated and converted to nM cAMP per well using the cAMP standard curve plotted as fluorescence emission ratio versus cAMP concentration. The amount of cAMP generated (nM) in each well was converted to a percent of maximal response, using the positive (saturation concentration of PYY(3-36)) and negative (DMSO only) control wells included on each plate.

[0129] Percent specific inhibition (y-axis) was plotted against the concentration of competitor (x-axis) and analyzed using a four-parameter (curve top, curve bottom, ICso, Hill slope) nonlinear regression routine as defined below: top — bottom yJ= bottom -I - / v \ Hrirllm Sl —ope

[0130] 1 + The relative IC50 value represents the concentration causing 50% inhibition of forskolin- induced cAMP production.

[0131] Reported values for IC50 are calculated as the geometric mean as shown below: Geometric Mean = lQ(Arithmetic Mean of Logl0 lC50 Values)

[0132] Standard error of the mean (SEM) is calculated using the delta method as shown below: . SD of log transformed data ,

[0133] SEM = Geometric Mean x - Sq -u -are root of n x ln(10), where SD is the standard deviation, n is the number of independent runs, and ln(10) is the natural logarithm of 10.

[0134] Binding Affinity and Selectivity

[0135] General binding assay - The equilibrium dissociation constants (Kd) for the various receptor / radioligand interactions were determined from saturation binding analysis and homologous competitive binding analysis using the same reagents and buffers as described below for compound testing. The Kd values determined for the receptor preparations used in this study were as follows: hNPY2R, 0.0047 nM; hNPY1 R, 0.121 nM; hNPY4R, 0.0553 nM; and hNPY5R, 0.18 nM. hNPY1 R binding protocol - The receptor binding affinity (Ki) of PYY(3-36) and PYY variants for hNPY1 R was determined from a competitive radioligand binding assay with human recombinant [125I]-PYYI-36 (#NEX341 , 2,200 Ci / mmol) obtained from Perkin Elmer (Waltham, MA). The assay was performed with a SPA method using polyvinyltoluene (PVT) wheat germ agglutinin-coupled SPA beads (#RPNQ0001 , Perkin Elmer). Assay buffer containing 25 mM HEPES, pH 7.5, 1 mM MgCl2, 2.5 mM CaCl2, and 0.2% w / v BSA (Roche Diagnostics) was used for preparation of reagents. PYY(3-36) and PYY variants were thawed and 3-fold serially diluted in 100% DMSO (10 point concentration response curves) using a Tecan Evo liquid handler. Next, 5 pL serially diluted peptide or DMSO was transferred into a CORNING® 3604 clear bottom assay plate containing 45 pL assay buffer or unlabeled PYY1-36 control (nonspecific binding, at 10 nM final concentration). Then, 50 pL [125l]-PYYi- 36 (0.05 nM final concentration) and 50 pL hNPY1 R membranes (1 .0 pg / well) were added. The final addition was 50 pL of WGA SPA beads (50 pg / well). Final DMSO concentration was 2.5%. Plates were sealed and mixed on a plate shaker (setting 6) for 1 minute and read with a PerkinElmer Trilux MicroBeta® scintillation counter after 10 hours of incubation / bead settling time at room temperature. Final assay concentration ranges for peptides tested in response curves were: PYY variants (10 pM to 0.5 nM) and PYY1-36 (10 nM to 0.5 pM). hNPY2R binding protocol - The receptor binding affinity (Ki) of PYY(3-36) and PYY variants for hNPY2R was determined from a competitive radioligand binding assay as described above for hNPY1 R. 0.2% w / v Bacitracin (RPI, #B32000) was used instead of BSA. A 20-fold step-down dilution of peptide into assay buffer was made to reduce the level of DMSO and peptide concentration prior to addition into the assay plate. Final DMSO concentration was 0.125%. Final assay concentration ranges for peptides tested in response curves were: PYY variants (10 nM pM to 5 pM) and PYY(3-36) (10 nM to 0.5 pM). hNPY4R binding protocol - The receptor binding affinity (Ki) of PP(1 -36) and PYY variants for hNPY4R was determined from a competitive radioligand binding assay as described above for hNPY1 R. 0.2% w / v Bacitracin (RPI, #32000) was used instead of BSA. Final assay concentration ranges for peptides tested in response curves were: PYY variants (10 pM to 0.5 nM) and pancreatic polypeptide (10 nM to 0.5 pM). hNPY5R binding protocol - The receptor binding affinity (Ki) of PYY(3-36) and PYY variants for hNPY5R was determined from a competitive radioligand binding assay with human recombinant [125I]-PYYI-36 (2,200 Ci / mmol) obtained from Vitrax (Placentia, CA). The assay was performed with a SPA method using polyvinyltoluene (PVT) wheat germ agglutinin-coupled SPA beads (#RPNQ0001 , Perkin Elmer). Assay buffer containing 25 mM HEPES, pH 7.5, 1 mM magnesium acetate, 2.5 mM CaCl2, and 0.1% Fatty-acid free BSA (BioWorld) was used for preparation of reagents. PYY(3-36) and PYY variants were thawed and 3-fold serially diluted in 100% DMSO (11 point concentration response curves) using a Tecan Evo liquid handler. A 200-fold step-down dilution of peptide into assay buffer was made to reduce the level of DMSO and peptide concentration prior to addition into the assay plate. Next, 50 pL serially diluted peptide or DMSO was transferred into a CORNING® 3604 clear bottom assay plate containing assay buffer or unlabeled PYY1-36 control (nonspecific binding, at 0.2 pM final concentration). Then, 50 pL [125I]-PYYI-36 (0.1 nM final concentration) and 50 pL hNPY5R membranes (7.0 pg / well) were added. The final addition was 50 pL of WGA SPA beads (300 pg / well). Final DMSO concentration was 0.5%. Plates were sealed and mixed on a plate shaker (setting 6) for 1 minute and read with a PerkinElmer Trilux MicroBeta® scintillation counter after 3 hours of incubation / bead settling time at room temperature. Final assay concentration ranges for peptides tested in response curves were: PYY variants (3 pM to 30 pM) and PYY(3-36) (1 pM to 10 pM).

[0136] Results

[0137] PYY-1 , PYY-2, and PYY-3 possessed similar or better functional potency (FIG. 1 A) and similar binding affinity to hNPY2R (FIG. 1B) when compared to PYY(3-36). PYY-1 , PYY-2, and PYY-3 exhibited reduced binding affinity for hNPY1 R, hNPY4R, and hNPY5R when compared to PYY(3-36) (FIG. 1C).

[0138] Representative concentration response curves by PYY(3-36) or PYY-1 , PYY-2, PYY-3 in a competitive radioligand binding assay with human recombinant [125I]-PYYI-36 in membranes expressing human NPY receptors (hNPY2R, hNPY1 R, hNPY4R, and hNPY5R). Data shown as Geo mean, SEM (n). A summary of binding affinities are shown in Table X below:

[0139] Table 2: Human NPY2 receptor binding affinity and selectivity of PYY analogs to other NPY receptors.

[0140] 1Note potency for hNPY2R is pM, while for hNPY1 R, hNPY4R, and hNPY5R is nM.

[0141] 2A value higher than one indicates more potent than PYY (3-36)

[0142] Conclusion

[0143] PYY-1 , PYY-2, and PYY-3 were more efficient agonists of hNPY2R than PYY(3-36).

[0144] Example 2. Evaluation of solubility and stability of PYY(3-36), PYY-1, PYY-2, and PYY-3 Objective

[0145] The objective of this study was to determine the pH-dependent solubility, fibril formation, and chemical and physical stability of the PYY variants.

[0146] Materials and Methods

[0147] Micro-Turbidity

[0148] Micro-turbidity assay was performed to evaluate pH-dependent solubility of PYY(3-36) and PYY variants in low and high pH buffer systems. This assay monitored the opaqueness of a solution due to the presence of suspend solid and is measured in terms of nephelometric turbidity unit (NTU).

[0149] PYY(3-36) and PYY variants were dissolved in H2O or suitable buffers (e.g., 40 mM Tris, pH 9) as stock solution with a staring concentration that is 10 times higher than the final concentration in plate. The dilution of the PYY peptide stock solution to final concentration was conducted in a clear flat-bottom 96-well plate with assay buffer (20 mM citrate-phosphate) ranging from pH 2 to pH 8 and 1X PBS. The total volume in each well was 100 pL. After the plate was sealed, the plate was shaken at room temperature for 15 minutes and incubated at 37 °C for 30 minutes. The sample plate was then loaded onto multimode plate reader (Synergy Neo2, BioTek) and the turbidity was measured as the endpoint OD at 350 nm and 500 nm at room temperature. The readouts of absorbance at 350 nm were converted to turbidity in NTU using a standard curve that is derived from turbidity standard solutions. Sample solution was considered turbid if the reading was higher than the threshold cutoff.

[0150] Fibrillation ThT Assay

[0151] Non-seeding ThT assay was performed to detect fibrillation formation propensity of PYY(3-36) and PYY variants.

[0152] PYY(3-36) and PYY variants were dissolved in suitable buffers (e.g., 5 mM phosphate at pH 6 (P6), pH 7 (P7), and 1XPBS) to which Thioflavin T (ThT) was added to the final concentration of 2 mg / mL at time 0. Samples were loaded in triplicate in a clear flat-bottom 384-well plate, sealed and stressed at 37 °C for 36 hours. Fluorescence measurements (ex = 450 nm; em = 480 nm) were recorded every 15 min via multimode plate reader (Synergy Neo2, BioTek) to plot the kinetics of fibril formation.

[0153] Stability

[0154] Accelerated stability study was performed to evaluate the chemical and physical stability risk for the PYY variants. The stability of PYY(3-36) and PYY variants was assessed at 2 mg / mL in each of the following formulation conditions: 5 mM phosphate, pH 6 (P6); 5 mM phosphate, pH 7 (P7); 5 mM phosphate, pH 8 (P8). The solution was filtered using Millivex 0.22pM filleter (PVDF membrane). The final stability samples were held for 3 days at 5 °C and 40 °C. Sample stability was measured via RP-HPLC and SEC-HPLC at time 0 and 3 days after incubation. RP-HPLC assay was applied to analyze the loss of main peak and SEC-HPLC assay was applied to determine the growth of high molecular weight (HMW) aggregate.

[0155] RP-HPLC assay conditions: Analytical column Agilent Zorbax Bonus RP, 1 .8pm, 3.0 x 100mm, 828668-301 ; Mobile phase A: 0.03% TFA in H2O; Mobile phase B: 0.03% TFA in 70:30 CAN, H2O; Column Temp: 60 °C; Wavelength: 214 nm; Flow rate: 0.5 mL / min.

[0156] Table 3: RP-HPLC gradient:

[0157] SEC-HPLC assay conditions: Analytical column X Bridge BEH125A SEC 3.5pm, P / N: 186007637; Mobile phase: PBS with 20% CAN; Column Temp: ambient; Wavelength: 214 nm; Gradient: isocratic for 40 min. Results

[0158] Micro-Turbidity

[0159] PYY-1 , PYY-2, and PYY-3 showed reduced turbidity and wider soluble pH windows than PYY(3-36) (FIG. 2A).

[0160] Fibrillation ThT assay

[0161] PYY(3-36) showed fibril formation in P7 and PBS whereas PYY-1 , PYY-2, and PYY-3 showed no fibril formation in P6, P7, or PBS (FIG. 2B).

[0162] Stability

[0163] PYY-2 demonstrated mid-high risk while PYY-1 and PYY-3 demonstrated low-risk (FIGS. 2C and 2D).

[0164] Conclusion

[0165] PYY-1 , PYY-2, and PYY-3 showed improved solubility profiles, wider soluble pH windows, and enhanced chemical and physical stability when compared to PYY(3-36), suggesting that the PYY variants are suitable peptides for drug formulation.

[0166] Example 3. Evaluation of long-term stability of PYY(3-36), PYY-1 , and PYY-3 formulations

[0167] Objective

[0168] The objective of this study was to determine the long-term stability of the PYY variants.

[0169] Materials and Methods

[0170] Long-term stability study was performed to evaluate the chemical and physical stability risk for PYY variants for 3 months after formulation. The stability of PYY(3-36), PYY-1 , PYY-3, and placebo samples was assessed at 2.5 mg / mL in a buffer containing inactive ingredients: propylene glycol (100 mg / mL), polysorbate 20 (5 mg / mL), disodium edetate dihydrate (1 .2 mg / mL), sodium phosphate monobasic dihydrate (7.8 mg / mL), potassium sorbate (2 mg / mL) and H2O for injection. Stability samples had a pH of 7.0 ± 0.1 . The solution was filtered using Millivex 0.22pM filleter (PVDF membrane) and filled in glass vials. The final stability samples were held for 3 months at 2-8 °C and 25 °C. Samples were collected at time 0, 1 -month, 2-month, and 3-months for stability assessment including visual inspection, micro-turbidity, pH measurement, and chromatography-based analysis by RP-HPLC and SEC-HPLC.

[0171] Visual Inspection

[0172] Visual inspection was performed on light inspection chamber for turbidity, particulates, gel formation, phase separation, and color change.

[0173] Micro-Turbidity

[0174] Micro-turbidity assay was performed as described above. pH Measurement pH measurement was conducted with a benchtop pH meter. Stability

[0175] Chromatography-based stability analyses were conducted by RP-HPLC and SEC-HPLC.

[0176] Stability samples were diluted to 1 mg / mL for HPLC analysis.

[0177] Table 4: RP-HPLC assay conditions:

[0178] Table 5: RP-HPLC gradient:

[0179] Table 6: SEC-HPLC assay conditions:

[0180] Results

[0181] Visual inspection

[0182] PYY-1 and PYY-3 showed reduced precipitation and cloudiness under 25°C at 1 , 2, and 3 months after formulation when compared to PYY(3-36) (FIG. 3A).

[0183] Micro-Turbidity

[0184] PYY-1 and PYY-3 showed reduced turbidity under 2-8°C and 25°C at 1 , 2, and 3 months after formulation when compared to PYY(3-36) (FIG. 3B). pH measurement

[0185] No significant pH changes were observed across all samples under 2-8°C and 25°C conditions for 3 months (FIG. 4).

[0186] SEC and RP

[0187] PYY-1 and PYY-3 showed reduced levels of aggregation, main peak loss, and degradation under 25°C over 3 months when compared to PYY(3-36) (FIGS. 5A-8D).

[0188] Conclusion

[0189] PYY-1 and PYY-3 were more stable under 25°C at 3 months after formulation, suggesting the improved feasibility of drug formulation of PYY-1 and PYY-3 compared to PYY(3-36).

[0190] Example 4. Evaluation of the potency, efficacy, and time of action of PYY(3-36), PYY-1, PYY-2, and PYY-3 in vivo

[0191] Objective

[0192] The objective of this study was to examine the potency, efficacy, and time of action of the PYY variants in mice.

[0193] Materials and Methods

[0194] Gastric emptying

[0195] Normal lean C57BL / 6 mice were fasted for 17-18 hours prior to subcutaneous delivery of vehicle, exendin-4 (Ex-4) at 30 nmol / kg, or PYY variants at 30, 100, 300, or 1000 nmol / kg. After 15 minutes, the animals received an oral bolus of 100 mg / kg acetaminophen in 2 g / kg glucose. At 0, 15, 30, 60, and 120 minutes following the acetaminophen / glucose dose, EDTA-treated plasma was collected from the animals for mass spec analysis of acetaminophen levels and blood glucose level was accessed via glucometers. Data were used to generate area under the curve (AUC) calculations from the glucose concentrations measured between 0 and 120 minutes. Statistical analysis was performed using one-way ANOVA in GraphPad Prism v9.0.

[0196] Food intake

[0197] Overnight-fasted C57BL / 6 mice (n=7-9) were single housed in a normal light / dark cycle prior to subcutaneous (SC) delivery of vehicle or PYY variants at 3, 10, 30, 100, 300, or 1000 nmol / kg. After 15 minutes, the animals were given access to pre-weighed food pellets and the level of food intake was measured at 1 hour and 2 hours following food access. Data were used to generate area under the curve (AUC) calculations from food intake measured between 0-1 hour and 0-2 hours, as well as percent reductions versus vehicle group. Statistical analysis was performed using one-way ANOVE in GraphPad Prism v9.0.

[0198] Results

[0199] Gastric emptying

[0200] Compared to PYY(3-36), PYY-1 , PYY-2, and PYY-3 showed reduced gastric emptying (FIGS. 9A-12B).

[0201] Food intake

[0202] Compared to PYY(3-36), PYY-1 , PYY-2, and PYY-3 showed improved lowering of food intake (FIGS. 13A-16B). Results are summarized in Table 7.

[0203] Table 7: Changes in food intake:

[0204] *P < 0.05 versus vehicle

[0205] Conclusion

[0206] The in vivo potency, efficacy, and time of action of PYY-1 , PYY-2, and PYY-3 were similar or superior to PYY(3-36) in normal lean mice after subcutaneous delivery of PYY.

[0207] Example 5. Evaluation of the chronic effects of PYY-3 in vivo

[0208] Objective

[0209] The objective of this study was to examine the chronic effects of PYY-3 in mice.

[0210] Materials and Methods

[0211] 40 young male C57BL / 6 (JAX 000664) mice were acclimated for 7 days to light a cycle starting at 01 :00am and ending at 1 :00pm. These 8-10 week old male C57BL / 6 mice were housed in groups of four and divided into three treatment groups - VEH, P30, and Serna. The VEH and P30 groups were pre-treated with topical lingual delivery of vehicle daily for 3 days before the day of treatment. The Serna group was pre-treated with subcutaneous delivery of 1 nmol / kg of semaglutide once 3 days before the day of treatment. On the day of treatment, VEH group received topical lingual delivery of vehicle, P30 group received topical lingual delivery of PYY-3 at 30 nmol / kg, and Serna group received subcutaneous injection of semaglutide at 1 nmol / kg. PYY-3 was delivered directly to the tongue with special attention given to avoid delivery of the peptide to the roof of the mouth. For the following 28 days, VEH group received topical lingual delivery of vehicle daily, P30 group received topical lingual delivery of PYY-3 at 30 nmol / kg and subcutaneous delivery of vehicle every 3 days, and Serna group received subcutaneous injection of semaglutide at 1 nmol / kg every 3 days. For all groups, the treatment was delivered 15 minutes before the start of the dark cycle.

[0212] All animals received standard rodent chow diet on days 1 -14 post-treatment and high fat diet on days 15-28. The level of food intake and body weight were measured daily for 35 days. Food consumption was measured every 24h throughout the duration of the study while body weights were also recorded every 24 h for the first 28 days of the study. To reduce the handling of the animals during the final 14 days of the study, daily body weight measurements were switched to every 3rd day concurrent with SC dosing.

[0213] Results

[0214] Compared to VEH, both P30 and Serna reduced the cumulative food intake by mice. Compared to semaglutide, PYY-3 was more effective at reducing the cumulative food intake by mice (FIGS. 17A and 17B). Compared to VEH, both P30 and Serna reduced the weight gain in mice. Compared to PYY-3, semaglutide was more effective at reducing weight gain in mice (FIG. 17C).

[0215] Conclusion Similar to GLP-1 agonists such as semaglutide, PYY-3 was effective at reducing the food intake by mice. PYY-3 was also effective at reducing weight gain.

[0216] Other Embodiments

[0217] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.

Claims

CLAIMS1 . A polypeptide of SEQ ID NO: 1 .

2. A polypeptide of SEQ ID NO: 2.

3. A polypeptide of SEQ ID NO: 3.

4. A pharmaceutical composition comprising the polypeptide of any one of claims 1 -3 and a pharmaceutically acceptable excipient.

5. The pharmaceutical composition of claim 4, wherein the composition is configured for administration without substantially changing the concentration of the polypeptide in the blood of a subject.

6. The pharmaceutical composition of claim 4 or 5, wherein the composition is formulated for local administration.

7. The pharmaceutical composition of any one of claims 4-6, wherein the composition is formulated for oral administration.

8. The pharmaceutical composition of claim 7, wherein the composition is formulated to topical lingual administration.

9. The composition of any one of claims 4-8, wherein the composition is formulated as an oral dissolving tablet, a lozenge, a film, a spray, a semisolid, a particulate, or in a lipid-based carrier.

10. The composition of any one of claims 4-9, wherein the pharmaceutically acceptable excipient comprises one or more of propylene glycol, potassium sorbate, l-arginine, edetate disodium, monosodium phosphate, polysorbate 20, gelatin, mannitol, dextran, alginate, polyvinyl alcohol, polyvinylpyrrolidone, acacia, aspartame, sodium methylparaben, sodium propylparaben, phenylalanine, water, or a combination thereof.11 . The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable excipient comprises one or more of gelatin, mannitol, dextran, alginate, polyvinyl alcohol, polyvinylpyrrolidone, acacia, aspartame, sodium methylparaben, sodium propylparaben, phenylalanine, water, or a combination thereof.

12. The composition of any one of claims 4-11 , wherein the pharmaceutically acceptable comprises one or more of a stabilizer, a preservative, an antioxidant, a buffer, a surfactant, a rheology modifier, and a mucosal permeation enhancer.

13. The pharmaceutical composition of claim 12, wherein the preservative comprises sodium methyl paraben or sodium propyl paraben.

14. The pharmaceutical composition of claim 12, wherein the stabilizer comprises mannitol or sucrose.

15. The pharmaceutical composition of claim 12, wherein the mucosal permeation enhancer comprises gelatin.

16. The pharmaceutical composition of any one of claims 4-15, wherein the pharmaceutically acceptable excipient further comprises a flavoring or sweetening agent.

17. The pharmaceutical composition of claim 16, wherein the sweetening agent comprises sorbitol, sucrose, or aspartame.

18. The pharmaceutical composition of claim 17, wherein the flavoring agent comprises mint, cinnamon, vanilla, cherry, strawberry, or lemon.

19. The composition of any one of claims 4-6, wherein the composition is formulated for topical administration to the Gl tract, for intranasal administration, or for intrarectal administration.

20. The composition of claim 19, wherein the composition is formulated as a gastrointestinal patch.21 . The composition of claim 19, wherein the composition is formulated as a suppository.

22. A method for inducing satiety or treating a disease or disorder selected from a metabolic syndrome, obesity, an obesity-related disorder, diabetes, fatty liver disease, nonalcoholic steatohepatitis, chronic kidney disease, polycystic ovary syndrome, cardiovascular disease, obstructive sleep apnea, retinopathy, peripheral vascular disease, peripheral artery disease, and neuropathy in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any one of claims 4-21 .

23. The method of claim 22, wherein the composition is administered locally.

24. The method of claim 22 or 23, wherein the composition is administered orally.

25. The method of claim 24, wherein the composition is administered topically to the tongue.

26. The method of claim 22 or 23, wherein the composition is administered topically to the Gl tract, intranasally, or intrarectally.

27. The method of any one of claims 21 -25, wherein the composition provides treatment without substantially changing the concentration of the polypeptide in the blood of the subject.

28. The method of any one of claims 21 -27, wherein the concentration in blood of the polypeptide of the does not exceed an endogenous PYY level in the subject by more than 100%.

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