Methods and compositions related to hybrid GPCR peptides

Abstract

A composition that can activate both a class B and class C G-protein-coupled receptor (GPCR) simultaneously is described herein. This composition can be used in a variety of methods, and is exemplified by a HYBD-HEXA peptide, which is found in SEQ ID NO: 1.

Description

Attorney Docket No. 10063-095W01METHODS AND COMPOSITIONS RELATED TO HYBRID GPCR PEPTIDESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 693,887, filed September 12, 2024, incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on September 11, 2025, is entitled “10063-095W01_ST26.xml”, and is 22,354 bytes in size.BACKGROUND

[0003] The development of type 2 diabetes occurs slowly without obvious symptoms, and the Centers for Disease Control and Prevention (CDC) estimates that approximately 8.1 million people do not even know that they have diabetes. With 21 million people in the U.S. diagnosed with diabetes, it is likely that every person may know someone who has type 2 diabetes, or at least someone who has one or more of the risk factors for developing type 2 diabetes. The risk factors for developing type 2 diabetes have been attributed to a genetic predisposition or lifestyle behaviors, and these risk factors include: having a parent, brother, or sister with diabetes; high blood pressure or abnormal cholesterol levels; a history of diabetes during pregnancy, a history of heart disease or stroke, or polycystic ovary syndrome; steroid treatment; being overweight, obese, age 45 or older, of a certain ethnicity (African American, Alaska Native, American Indian, Asian American, Hispanic / Latino, Native Hawaiian, or Pacific Islander American ethnicity), or physically inactive. It has been shown that regular physical activity can reduce excess weight and obesity and can temporarily reverse type 2 diabetes, but the prevalence of physical inactivity in the U.S. is ever-increasing and can be attributed, at least in part, to the increasing use of modern technologies, leading to a more sedentary lifestyle. Exercise leads to the contraction of skeletal muscle, which pulls on the bones, prompting the release of the bone- derived hormone osteocalcin into the blood circulation. Then, osteocalcin signals to the pancreas to produce and secrete insulin by enhancing pancreatic beta cell mass and function, with subsequent reduction in blood glucose levels. In type 2 diabetes, pancreatic beta cell mass can be reduced due to a loss in cell identity, wherein functional pancreatic beta cells becomeAttorney Docket No. 10063-095W01 dysfunctional insulin non-producing cells. Circulating levels of osteocalcin in the blood have been shown to be associated with a lower risk of developing type 2 diabetes. Unfortunately, osteocalcin levels decline with age, coinciding with decreased exercise capacity.

[0004] G protein coupled receptors (GPCRs) are the targets of a large fraction of marketed drugs (-34% of drugs approved by the US Food and Drug Administration). The GPCR superfamily can be broadly categorized into the following classes: A, Bl, B2, C and F. To date, diabetes therapeutics have predominantly targeted class A and B GPCRs. Targeting other GPCR classes, such as class C, remains elusive.

[0005] Thus, there exists a need for hybrid GPCR agonists that can provide a combination of activating both a B-Class GPCR and a C-Class GPCR simultaneously.SUMMARY

[0006] Disclosed herein is a composition that can activate both a class B and class C G- protein-coupled receptor (GPCR) simultaneously. This is referred to herein as a “GPCR Class B-C Agonist.”

[0007] Also disclosed is a polypeptide comprising 90% or more identity to SEQ NO: 1. This is also referred to herein as the HYBD-HEXA peptide.

[0008] Further disclosed is a polypeptide comprising a class B 1 GPCR agonist and a class C GPCR agonist, wherein the class Bl GPCR agonist comprises 88% or more identity to SEQ ID NO: 2, and wherein the class C GPCR agonist comprises SEQ ID NO: 3.

[0009] Disclosed herein is a method of activating a class B GPCR and a class C GPCR simultaneously, the method comprising exposing the GPCRs to a composition which comprises both a class B GPCR activator and a class C GPCR activator (GPCR Class B-C Agonist).

[0010] Further disclosed is a method of treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is associated with both a class B GPCR and a class C GPCR, the method comprising administering to the subject a composition comprising a peptide, wherein the peptide can simultaneously activate both a class B GPCR and a class C GPCR (GPCR Class B-C Agonist).

[0011] Also disclosed is a method of restoring pancreatic P cell function, mass, and / or identity, the method comprising exposing a pancreatic cell to a composition, wherein the composition comprises a peptide, wherein the peptide can simultaneously activate both a class B GPCR and a class C GPCR.

[0012] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailedAttorney Docket No. 10063-095W01 description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

[0014] Figure 1 shows rat (SEQ ID NO: 13), mouse (SEQ ID NO: 14), human (SEQ ID NO: 15) and bovine (SEQ ID NO: 16) osteocalcin was aligned using sequence available at UniProt database. The c-terminal bio-active core amino acids are displayed in a box (SEQ ID NO: 17). The core amino acids are 100% homologous in human and bovine but not when compared to the same region with rat and mouse. Rat and mouse are 100% aligned at the bioactive core similar to human and bovine.

[0015] Figure 2 shows amino acid sequences of native bioactive OCN (SEQ ID NO: 4) and the newly generated hybrid HEXA (SEQ ID NO: 1). Note the HEXA peptide amino acid sequence is placed at the C-terminal of the hybrid HEXA. The physical properties of the peptides are shown in the table. Both in native OCN and in hybrid-HEXA, the two ends N and C terminal are separated allowing the C-terminal region to act independently for any interaction.

[0016] Figure 3A-B shows a dose response of HYBD-HEXA in HepG2 (A) and 1.2B4 (B) cells. Various doses of HYBD-HEXA were used to measure cAMP in presence of IBMX. Values are mean±SD, n=2. The equation used to generate the response curve was Sigmoidal, 4Pl / y2, X is concentration.

[0017] Figure 4A-B shows cAMP response in human islet. (A) Forskolin at 5nM (B) response of HYBD-HEXA in islet. Values are mean SD, n=3. *P<0.05

[0018] Figure 5 shows intracellular calcium and cell viability in human islets: Intracellular calcium induced by HYBD-HEXA peptides. Values are mean±SD, n=3. *P<0.05.

[0019] Figure 6 shows glucose stimulated insulin release in human islets (A) Islets were challenged with low and high glucose and insulin released was measured in the medium.

[0020] Figure 7 shows human islets (approximately 1000) were treated with lOOpM of HYBD_HEXA or control with saline for 5 days In CMRL medium. RNA was extracted and used to reverse transcribe to cDNA using random hexamer primer by reverse transcriptase (NEB: E3010S) in a 20pl reaction. The transcribed cDNA was used to PCR amplify PDX1, FOXO1, Insulin and actin using gene specific primers. All amplification were done at 94 °CAttorney Docket No. 10063-095W01(94°C / 30sec,62°C / 30sec,72730sec) x22cyc, 7275min. The PCR Product was resolved in 1% agarose and stained with ethidium bromide and images were captured in BioRad imager. The density of bands was analyzed by Image-J, followed by normalization with actin as loading and internal control.

[0021] Figure 8A-B shows HepG2 cells were seeded in 96 well dish and grown overnight. Following overnight growth cells were starved for FBS and grown in medium containing 1.5mM Glucose for 24h. The cells were then treated with PA 250pM, 0.4ng / ml HEXA and various concentration of HYBD_HEXA for 24h in medium supplemented with 16.7mM glucose and 3% FBS (A). All values are compared with Con (3%FBS) Values (B) are mean SD, n=4-6. *P0.05 (compared to 3% FBS Control). Values were compared with PA (250pMmedium and with PA (250pM). Values are mean SD, n=4-6. All values are significant at P<0.05 (compared to PA (250pM).

[0022] Figure 9A-C shows values represent means ± SEM. Baseline values (just before first dosing on day +1) were compared with results at (day +28 (9A) and +63 (9B)). Dosedependent treatment effects (HFD + treatment) versus obese vehicle control group (chow) were analyzed with two-way ANOVA at study end (day +26 and day + 63) followed by Dunnett’s test for pairwise comparisons between each treated and obese control group. *p < 0.05, **p < 0.01, ***p < 0.001, and****p < 0.0001 versus obese vehicle control (9C).

[0023] Figure 10A-D shows EchoMRI-3inl small animal Quantitative Magnetic Resonance (QMR) machine was used to determine percent body fat (10A, 10C) and Lean (10B, 10D) in the whole animal. Vehicle control (chow diet) and Obese (HFD) +Dose-dependent treatment effects versus Obese group were analyzed with one-way ANOVA at (day +28 and +63). Values represent means + SD. *p < 0.05, **p < 0.01, ***p < 0.001, and****p < 0.0001.

[0024] Figure 11A-C shows Hybrid Hexa decreases Liver weight and hepatic fat content. The weight of the liver in the Obese and Dose-dependent treatment effects versus vehicle control group were compared at the end of the study (+day 63, Fig. 11 A) and found that only obese shows a significant increase in liver weight vs. control (11B). To further investigate the effects on liver metabolism and NASH, H&E of the liver from all groups were compared. All different doses of treatment showed complete protection from high fat diet comparable to the Control (Chow) (11C). Obese (HFD) and Dose-dependent treatment effects versus vehicle control (chow) group were analyzed with one-way ANOVA at study end (day + 63). Values represent means + SD. Ns not significant and****p < 0.0001.

[0025] Figure 12A-D shows Hybrid Hexa protects the heart from cardiomegaly. The weight of the heart in the Obese and Dose-dependent treatment effects versus vehicle controlAttorney Docket No. 10063-095W01 group at the end of the study (+day63, Fig. 12A) and found that only obese shows a significant increase in heart weight and size vs. treatment (Fig. 12B & 12C). Moreover, the increase in weight and heart size was accompanied by a significant increase in cardiac myocytes size (heart muscle cells) (Fig 12D).

[0026] Figure 13A-B shows Hybrid Hexa protects the % Beta cell per islet. The average islet areas and the percent of beta cell per islet between groups measured by the endocrine staining synaptophysin (Syp) and insulin (INS) are shown in 13 A. Interestingly while treatment with as low as 3ng / g, resulted in comparable percentage of Beta cell per islet to the lean control (chow) group, higher doses showed effect in the islet area (13B).

[0027] Figure 14A-B shows the effect of Hybrid Hexa on glucose metabolism. Fig. 14A shows that for the GTT, mice n = 8 per group were injected with Ig / kg and blood glucose levels in response were determined at 0, 15, 30, 60, 90 and 120-minute post-injection. Fig. 14B shows that for the ITT, mice were injected with 0.75U / kg insulin and blood glucose levels were determined at 0, 15, 30, 60, 90 and 120-minute post-injection. While all treatment doses showed improvement in both tests, higher doses of 10 and 30ng / g were comparable to the lean group (chow) in the GTT (14A) and in the case of ITT, treatment with 3ng / g were comparable to the lean group (chow (14B)). n = 8 per group. Statistical analysis was performed using ANOVA and Tukey's post hoc test. Dose-dependent treatment effects (HFD + treatment) and obese vehicle versus control group (chow) were analyzed with two-way ANOVA at study end (day + 63) followed by Dunnett’s test.DETAILED DESCRIPTION

[0028] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable sub-combination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.DEFINITIONS

[0029] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:Attorney Docket No. 10063-095W01

[0030] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, nonlimiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0031] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a peptide”, includes, but is not limited to, two or more such compounds, compositions, or peptides, and the like.

[0032] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0033] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.Attorney Docket No. 10063-095W01

[0034] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0035] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0036] The term “isolated” as used herein means having been removed from its natural environment.

[0037] The term “purified,” as used herein relates to the isolation of a molecule or composition in a form that is substantially free of contaminants normally associated with the molecule or composition in a native or natural environment and means having been increased in purity as a result of being separated from other components of the original composition. The term “purified peptide” is used herein to describe a peptide which has been separated from other compositions including, but not limited to nucleic acid molecules, lipids and carbohydrates.

[0038] As used herein, the term “peptide” encompasses a sequence of 2 or more amino acids and typically less than 50 amino acids, wherein the amino acids are naturally occurring or coded or non-naturally occurring or non-coded amino acids. Non-naturally occurring aminoAttorney Docket No. 10063-095W01 acids refer to amino acids that do not naturally occur in vivo but which, nevertheless, can be incorporated into the peptide structures described herein. “Non-coded” as used herein refer to an amino acid that is not an L-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Vai, Trp, Tyr.

[0039] As used herein, the terms “polypeptide” “peptide” and “protein” are terms that are used interchangeably to refer to a polymer of amino acids, without regard to the length of the polymer. Typically, polypeptides and proteins have a polymer length that is greater than that of “peptides.” In some instances, a protein comprises more than one polypeptide chain covalently or noncovalently attached to each other.

[0040] As used herein, the term “peptide complex” or simply “complex,” refers to a group of two or more associated peptide chains. Peptides in a peptide complex can be linked by non- co valent protein-protein interactions. It will be understood that the complex may be a multimeric complex comprising two, three, four, five, six or more peptides. Also, the complex may additionally comprise a non-proteinaceous molecule.

[0041] As used herein, the terms “nucleic acid molecule,” “polynucleotide,” “polynucleic acid,” or “nucleic acid” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

[0042] As used herein, the term “ligand” or “receptor ligand” means a molecule that specifically binds to a GPCR, either intracellularly or extracellularly. A ligand may be, without the purpose of being limitative, a protein, a (poly)peptide, a lipid, a small molecule, a protein scaffold, an antibody, an antibody fragment, a nucleic acid, a carbohydrate. A ligand may be synthetic or naturally occurring. The term “ligand” includes a “native ligand” which is a ligand that is an endogenous, natural ligand for a native GPCR. In most cases, a ligand is a “modulator” that increases or decreases an intracellular response when it is in contact with, for example binds to, a GPCR that is expressed in a cell. Examples of ligands that are modulators include agonists, partial agonists, inverse agonists, and antagonists, of which a more detailed description can be found further in the specification.Attorney Docket No. 10063-095W01

[0043] A “deletion” is defined here as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a parental polypeptide or nucleic acid. Within the context of a protein or a fragment thereof, a deletion can involve deletion of about 2, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A protein or a fragment thereof may contain more than one deletion. Within the context of a GPCR, a deletion may in particular be a loop deletion, or an N- and / or C-terminal deletion.

[0044] An “insertion” or “addition” is that change in an amino acid or nucleotide sequence which has resulted in the addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a parental protein. “Insertion” generally refers to addition to one or more amino acid residues within an amino acid sequence of a polypeptide, while “addition” can be an insertion or refer to amino acid residues added at an N- or C-terminus, or both termini. Within the context of a protein or a fragment thereof, an insertion or addition is usually of about 1, about 3, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A protein or fragment thereof may contain more than one insertion.

[0045] A “substitution,” as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity. By conservative substitutions is intended combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, or trp.

[0046] The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Determining the percentage of sequence identity can be done manually, or by making use of computer programs that are available in the art.Attorney Docket No. 10063-095W01

[0047] As used herein, the terms “determining,” “measuring,” “assessing,” “monitoring,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

[0048] The term “biologically active,” with respect to a GPCR, refers to a GPCR having a biochemical function (e.g., a binding function, a signal transduction function, or an ability to change conformation as a result of ligand binding) of a naturally occurring GPCR.

[0049] The terms “therapeutically effective amount,” “therapeutically effective dose” and “effective amount,” as used herein, mean the amount needed to achieve the desired result or results.

[0050] The term “pharmaceutically acceptable,” as used herein, means a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0051] As used herein, the term “treatment” or “treating,” is defined as the application or administration of a therapeutic agent, i.e., a composition of the invention (alone or in combination with another pharmaceutical agent), to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject (e.g., for diagnosis or ex vivo applications), with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a disease or disorder associated with GPCRs, as described elsewhere herein.

[0052] As used herein, the term “prevent” or “prevention” 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.

[0053] As used herein, the term “patient” or “subject” refers to a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. The patient or subject can be human.

[0054] As used herein, “linking group” or “linker” is a molecule or group of molecules that binds two separate entities to one another. Linking groups may provide for optimal spacing of the two entities or may further supply a labile linkage that allows the two entities to be separated from each other. Labile linkages include hydrolyzable groups, photocleavable groups, acid- labile moieties, base-labile moieties and enzyme cleavable groups for example.

[0055] As used herein, the term “prodrug” is defined as any compound that undergoes chemical modification before exhibiting its full pharmacological effects.Attorney Docket No. 10063-095W01

[0056] As used herein, a “dipeptide” is the result of the linkage of an a-amino acid or a- hydroxyl acid to another amino acid, through a peptide bond.

[0057] As used herein the term “chemical cleavage” absent any further designation encompasses a non-enzymatic reaction that results in the breakage of a covalent chemical bond.COMPOSITIONS

[0058] The G-protein-coupled receptors (GPCRs) form the largest class of cell surface receptors and play a major role in cellular perception of the environment 1. GPCRs are sensitive to a diverse range of ligands that include light (photons), ions, amino acids and large proteins. (Chun, et al. Structure and ligand recognition of class C GPCRs. Acta Pharmacol Sin 33, 312— 323 (2012). The GPCRs can be classified into five families based on the sequence phylogeny of a conserved heptahelical transmembrane domain (7TM) (Pin et al. Evolution, structure, and activation mechanism of family 3 / C G-protein-coupled receptors. Pharmacol Ther 2003; 98: 325-54).

[0059] Class B 1 GPCRs are important regulators of many physiological functions such as glucose homeostasis, which is mainly mediated by three peptide hormones, i.e., glucagon-like peptide-1 (GLP-1), glucagon (GCG), and glucose-dependent insulinotropic polypeptide (GIP). They trigger a cascade of signaling events leading to the formation of an active agonist- receptor-G protein complex (Cong et al. Molecular features of the ligand-free GLP-1 R, GCGR and GIPR in complex with Gs proteins. Cell Discov 10, 18 (2024)).

[0060] Class C GPCRs are characterized by two unique features: the large extracellular and ligand-binding N-terminal domain in addition to the generic 7TM, and the formation of homo- and heterodimers where orthosteric and allosteric ligands can occupy the same receptor or a canonical transmode (Clemmensen et al. The GPCR, class C, group 6, subtype A (GPRC6A) receptor: from cloning to physiological function. Br J Pharmacol. 2014 Mar; 171(5): 1129-41). The peptide osteocalcin has been shown to be an endogenous GPRC6A agonist. Osteocalcin is a small protein (49 amino acids) encoded by the BGLAP gene synthesized by osteoblasts, and is present in two forms: Carboxylated (cOC) and undercarboxylated (ucOC) (Diaz-Franco et al Osteocalcin-GPRC6A: An update of its clinical and biological multi-organic interactions. Mol Med Rep. 2019 Jan;19(l): 15-22).

[0061] Disclosed herein is a composition that can activate both a class B and class C G- protein-coupled receptor (GPCR) simultaneously. This composition is referred to herein as a “GPCR Class B-C agonist.” This composition comprises the essential amino acids that are required for the activation of GPCR Class B receptors (such as class Bl receptors GLP1, GIP,Attorney Docket No. 10063-095W01 and glucagon, either alone or in combination), and it also comprises amino acids which can target (and activate) GPCR C class receptors. An example of a C class targeting peptide can be found in osteocalcin.

[0062] The GPCR Class B-C agonist described herein can treat or prevent a variety of diseases and disorders associated with GPCRs, which are described in more detail below. An example of one such composition is referred to herein as “HYBD-HEXA” (SEQ ID NO: 1) and its structure and sequence can be found in Figure 2. Contemplated herein are variants of SEQ ID NO: 1, wherein 1, 2, 3, 4, 5, 6, or more amino acids are substituted, added, or deleted from SEQ ID NO: 1. Put another way, SEQ ID NO: 1 can vary by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and still function as GPCR Class B-C agonist.

[0063] The GPCR Class B-C agonists described herein can be comprised of a peptide, wherein the peptide has multiple, functional parts. The first part is an agonist of a class B GPCR, such as a Bl GPCR. The Bl GPCR(s) which are targeted by the peptide can comprise the glucagon, GLP1, and / or GIP receptors. In a specific example, all three receptors (glucagon, GLP1, and GIP) are simultaneously targeted by the GPCR Class B-C agonist described herein.

[0064] In a particular embodiment, the portion of the GPCR Class B-C agonist which targets the Class B receptor (such as glucagon, GLP1, and / or GIP receptors) is represented by SEQ ID NO: 2 (HCDGTFTSDGSQEFYRWL) and is referred to herein as the “HYBD” portion of the GPCR Class B-C agonist, with the “HYBD” portion representing the Class B targeting portion of the GPCR Class B-C agonist. Also contemplated are variants of SEQ ID NO: 2, wherein 1, 2, 3, 4, 5, 6, or more amino acids are substituted, added, or deleted from SEQ ID NO: 2. Put another way, SEQ ID NO: 2 can vary by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and still function as an agonist of GPCR Class B receptor. The Class B GPCR portion of the GPCR Class B-C agonist can be on either the N-terminal portion or the C-terminal portion of the peptide.

[0065] Furthermore, the GPCR Class B-C agonist comprises a C Class targeting portion of the peptide. In a particular embodiment, the Class C GPCR component comprises GPRC6A, and the portion of the composition which targets GPRC6A comprises a C-terminal derived peptide from osteocalcin. This is represented, for example, by SEQ ID NO: 3 (RFYGPV), which is referred to herein as the “HEXA” portion of the GPCR Class B-C agonist. Also contemplated are variants of SEQ ID NO: 3, wherein 1 or more amino acids are substituted or deleted, and wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or more amino acids can be added to SEQ ID NO: 3. Put another way, SEQ ID NO: 3 can vary by 80%, 81%, 82%, 83%, 84%,Attorney Docket No. 10063-095W0185%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and still function as an agonist of GPCR Class C receptor. The Class C GPCR portion of the GPCR Class B-C agonist can be on either the N-terminal portion or the C-terminal portion of the peptide.

[0066] As described above, the GPCR Class B-C agonist of the present invention, such as the HYBD-HEXA peptide, can be engineered for optimal functionality. Thus, when referring to the function or activity of “modified peptide,” one of ordinary skill in the art would understand that this includes, for example, a peptide that 1) performs the same activity or has the same specificity as the unmodified peptide (in the case of HYBD-HEXA, SEQ ID NO: 1), but that may have a different level of activity; and 2) possesses an additional advantage over the unmodified peptide. Determination of activity may be achieved using assays familiar to those of skill in the art, particularly with respect to the peptide's activity.

[0067] As mentioned above, modified GPCR Class B-C agonists of the present invention may possess deletions and / or substitutions of amino acids; thus, a peptide with a deletion, a peptide with a substitution, and a peptide with a deletion and a substitution are modified peptides. In some embodiments these modified peptides may further include insertions or added amino acids, such as with fusion proteins or proteins with linkers, for example. A “modified deleted peptide” lacks one or more residues of the original peptide (such as HYBD-HEXA), but possesses the specificity and / or activity of the original GPCR Class B-C agonist.

[0068] Substitutional or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the peptide and may be designed to modulate one or more properties of the peptide, particularly to reduce its immunogenicity / antigenicity, reduce any side effects in a subject, or increase its efficacy. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. An antigenic region of a polypeptide may be substituted for a less antigenic region; the less antigenic region may contain residues that are identical to the corresponding residues in the native protein, yet also contain some conservative substitutions and / or nonconservative substitutions.Attorney Docket No. 10063-095W01

[0069] In addition to a deletion or substitution, a modified peptide may possess an insertion of residues, which typically involves the addition of at least one residue in the peptide. This may include the insertion of a targeting peptide or simply a single residue.

[0070] Mutants and variants as described herein, which can function the same as the original or native molecule, are referred to as “functional equivalents” (also referred to herein as a “functional derivative, fragment, or variant thereof’). This term is well understood in the art and is further defined in detail herein.

[0071] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.

[0072] Any portion of the GPCR Class B-C agonist can be replaced or substituted by a non-naturally occurring amino acid. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C- terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).Attorney Docket No. 10063-095W01

[0073] The GPCR Class B-C agonist can also comprise a linker. This linker can, for example, be between the Class B targeting portion of the composition and the Class C targeting portion of the composition. The linker can be any length, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acids in length.

[0074] The GPCR Class B-C agonist described herein can further comprise additional elements, such as other additional modulators of additional receptors, or additional modulators of the same receptors. Also contemplated are that the GPCR Class B-C agonists of the present invention can further comprise other targeting moieties, detection moieties, or other elements which increase its usefulness, targeting ability, or effectiveness.

[0075] Also disclosed herein are nucleic acids which encode the GPCR Class B-C agonists described herein. These nucleic acids can be used in vectors and inserted in cells so that the cells can produce the GPCR Class B-C agonists described herein.METHODS

[0076] Disclosed herein is a method of activating a class B GPCR and a class C GPCR simultaneously, the method comprising exposing the GPCRs to a composition which comprises both a class B GPCR activator and a class C GPCR activator (referred to herein as a GPCR Class B-C agonist). By “activating” is meant that the GPCR carries out a function or activity at a higher rate than it was before activation. Examples of activating these receptors are given below.

[0077] The GPCR Class B-C agonist can elicit insulin production and / or secretion in human pancreatic cells. This increase can be a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% increase, or a 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more increase as compared to a control.

[0078] The GPCR Class B-C agonist can also increase glucose uptake and cause an increase in cAMP and intracellular calcium. This increase can be a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,Attorney Docket No. 10063-095W0140%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% increase, or a 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more increase as compared to a control.

[0079] The GPCR Class B-C agonist can also reduce at least one (NASH) component. This reduction can be a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% reduction, or a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, or 10-fold or more reduction as compared to a control. These NASH components can comprise, but are not limited to, steatosis, ballooning degeneration, inflammation, and fibrosis. NASH and treatment thereof is described in more detail below.

[0080] The GPCR Class B-C agonist can also reduce nuclear expression of FOXO1 in pancreatic P cells. This reduction in nuclear expression can be a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% reduction, or a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more reduction as compared to a control.

[0081] The GPCR Class B-C agonist can also restore cytoplasmic expression of PDX-1 in pancreatic cells. This restoration of cytoplasmic expression can be a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,Attorney Docket No. 10063-095W0187%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% restoration, or a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more restoration as compared to a control.

[0082] The GPCR Class B-C agonist can also restore human pancreatic P cell function, mass, and / or identity. This restoration of human pancreatic cell function, mass, and / or identity can be a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% restoration, or a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more restoration as compared to a control.

[0083] Disclosed is a method of treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is associated with both a class B GPCR and a class C GPCR, the method comprising administering to the subject a composition comprising a peptide, wherein the peptide can simultaneously activate both a class B GPCR and a class C GPCR.

[0084] The GPCR Class B-C agonists disclosed herein can be given in a dose which provides benefit to the subject. One of skill in the art can determine an appropriate dose based on specific disease indications, as well as the specific biometrics of the subject. By way of example, the GPCR Class B-C agonists of the present invention can be given at a dose in the range of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or more or less ng / ml.

[0085] The GPCR Class B-C agonists disclosed herein can be delivered in pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier” is a carrier, such as a solvent, suspending agent or vehicle, for delivering the disclosed compositions to a subject in need thereof. The carrier can be liquid or solid and is selected with the planned manner of administration in mind. Liposomes and nanoparticles are also a pharmaceutical carrier. As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.

[0086] Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueousAttorney Docket No. 10063-095W01 sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question.

[0087] Specifically, the compositions of the present invention can be formulated for oral administration, parenteral administration, inhalation administration, topical administration, mucosal administration, or the like. In some embodiments, the administering is via a route selected from peroral, intravenous, intraperitoneal, inhalation, intranasal, or intraprostatic. The present invention is not limited by the route of administration. Compositions formulated for oral delivery can, for example, include an enteric coat, to ensure that peptides contained therein reach the intestine and beyond. Enteric formulations such as gastro resistant capsules for oral administration, suppositories for rectal or vaginal administration also form part of this disclosure. Compositions formulated for topical delivery can be, for example, suspended in a gel or cream, coated on a microneedle, or infused into a bandage or topical patch, to extend the duration of action of the peptides contained therein. Any inhalable formulation which can provide for an aerosolized form including a subject peptide for delivery to a patient via the intrapulmonary route may be used in conjunction with the present disclosure.

[0088] The compositions disclosed herein can be delivered to a cell via a peptide-based drug delivery system. Examples of this can be found in Berillo et al. Peptide-Based Drug Delivery Systems. Medicina (Kaunas). 2021 Nov 5;57(11): 1209, herein incorporated by reference in its entirety for its teaching concerning delivery methods. The carrier can be a nanocarrier, such as various types of lysosomes, solid lipid nanoparticles, dendrimers, polymeric micelles, virus-based nanoparticles, inorganic and organic / inorganic hybrid nanocarriers. Extracellular vesicles (EVs) derived from cells can also be utilized for effective delivery. Also disclosed are cell penetrating peptides (CPPs) for use with the GPCR Class B-C agonists of the invention.

[0089] Also provided are liposomal pharmaceutical compositions comprising the GPCR Class B-C agonists disclosed herein. Any convenient nanocarriers and liposomes can be adapted for use in preparing liposomal formulations of the subject peptides, such as those nanocarriers and liposomes described by Arias in“Liposomes in drug delivery: a patent review”. ExpertAttorney Docket No. 10063-095W01Opinion on Therapeutic Patents, 23, 2013, issue 11, p. 1399-1414; and Torchilin in“Multifunctional nanocarriers”, Advanced Drug Delivery Reviews, Volume 58, Issue 14, 1 December 2006, Pages 1532-1555.

[0090] Regarding nanoparticles, these compositions can increase the aqueous solubility of a peptide of interest and can achieve protected, sustained, and targeted delivery of the peptide in therapeutic applications (e.g., as described herein). In some cases, the formulation is a polymer-based nanoparticle formulation. Nanoparticle formulations of interest include albumin nanoparticles, e.g., human serum albumin containing nanoparticle formulations. Particle size, peptide drug release, encapsulation efficiency and peptide drug polymer interactions can be determined and selected using any convenient in vitro methods. Cell culture studies, in vivo pharmacokinetics, e.g., in rats, can be used for biological characterization of a desirable formulation.

[0091] A variety of diseases and disorders can be treated using the compositions and methods disclosed herein. Examples include, but are not limited to, type 2 diabetes mellitus (T2DM), obesity, depression, cancer, migraines, osteoporosis, depression, anxiety, cardiovascular disease, and Alzheimer’s disease. Examples of other diseases and disorders which can be treated with the compositions disclosed herein can be found in Yang et al. (G protein-coupled receptors: structure- and function-based drug discovery. Signal Transduct Target Ther. 2021 Jan 8;6(1):7) and in Hauser et al. (Trends in GPCR drug discovery: new agents, targets and indications. Nat Rev Drug Discov. 2017 Dec;16(12):829-842. doi: 10.1038 / nrd.2017.178), both incorporated by reference in their entirety for their teaching concerning GPCR-associated diseases.

[0092] Also disclosed herein is a method of restoring pancreatic P cell function, mass, and / or identity, the method comprising exposing a pancreatic cell to a GPCR Class B-C agonist, as described herein. This can occur when a pancreatic P cell has lost function, mass, and / or identity. P-cell dysfunction arises from insufficient glucose sensing to stimulate insulin secretion, hence increased glucose levels persist. This process leads to the development of insulin resistance, increased glucose concentrations beyond the physiological state, thereby resulting in the manifestation of hyperglycemia. As a result, P-cells compensate for insulin resistance by hypersecretion of insulin, ultimately leading to P-cell failure. P-cell dysfunction follows insulin resistance during the development and progression of T2D, for example (Dludla et al. Pancreatic P-cell dysfunction in type 2 diabetes: Implications of inflammation and oxidative stress. World J Diabetes. 2023 Mar 15; 14(3): 130-146). ‘Attorney Docket No. 10063-095W01

[0093] This method of restoring pancreatic P cell function, mass, and / or identity can be done by directly exposing a pancreatic cell to a GPCR Class B-C agonist. This can happen in vitro or ex vivo. When this occurs, the dosage can be anywhere in the range of 50pM, lOOpM, 150pM, 200pM, 250pM, 300pM, 350pM, 400pM, 450pM, 500pM, 550pM, 600pM, 650pM, 700pM, 750pM, 800pM, 850pM, 900pM, 950pM, lOOOpM, or any amount above, below, or inbetween these values.

[0094] This method can also be carried out in a subject in vivo). Dosages and methods of providing the GPCR Class B-C agonist to the subject are given above. In one example, the subject can have type II diabetes (T2D). In another example, the subject may have been exposed to a substance which causes loss of pancreatic P cell function, mass, and / or identity. Examples of this include, but are not limited to, exposure to doxorubicin, anti-hypertensive vasodilator diazoxide, beta-blockers, antipsychotics, statins, and / or corticosteroids.EXAMPLESExample 1: HYBRID GPCR PEPTIDE

[0095] A new peptide that combined two classes of G protein coupled receptors (GPCRs): Class Bl and C to effectively treat insulin resistance, decrease fat mass, non-alcoholic fatty liver steatosis and restore pancreatic cell identity and functional mass in pre-diabetic and diabetic patients is described in this example.

[0096] C-terminal derived HEXA peptide from osteocalcin (Figure 1) was found to be effective in inducing insulin secretion, an increase in cAMP and intracellular calcium. It also had significant effect on reducing the expression of FOXO1 and increasing PDX-1 expression which resulted in restoring human P-cell function and identity which is important for an effective diabetes treatment, a study structured a new peptide by adding all the essential amino acids that are required for the activation of all the three receptors namely GLP1, GIP and glucagon receptor (GPCR Class Bl) and incorporating six amino acids at C-terminal with (SEQ ID NO: 3) RFYGPV derived from OCN (GPCR Class C). The complete sequence of the HYBD-HEXA peptide, its physical properties and the ribbon structure is shown in Figure 2. Based on the physical properties the molar absorption coefficient of the HYBD-HEXA was almost four times compared to native OCN. However, the isoelectric pH did not significantly change between the native and the HYBD-HEXA. Based on the ribbon structure it is noteworthy that both the native OCN and the HYBD-HEXA showed that the N and the C terminal were farAttorney Docket No. 10063-095W01 apart from each other, suggesting that the C-Terminal six amino acid is accessible for interacting with receptors.

[0097] To characterize the HYBD HEXA, the study determined the increase in cAMP; a hallmark for insulin release in pancreas by GLP1 like peptides; in two cell lines, HepG2 and 1.2B4. HepG2 cells are derived from human hepatocarcinoma and expression of GLP1 receptor and GPRC6A. 1.2B4 cells are a GPRC6A receptor and GLP1 receptor because of their pancreatic origin. cAMP was measured after treatment with several doses of HYBD_HEXA in two cell lines (Figure 3). Both cell lines showed dose dependent response when cAMP was measured in cell lysate treated in presence of IB MX (Figure 3). In HepG2 cells the EC50 was approximately 0.5nM while in 1.2B4 cells this response could be achieved approximately at lOOpM.

[0098] Moreover, the study measured the elevation of cAMP levels in response to three different doses of HYBD-HEXA on human islets as shown in Figure 4 in order to observe similar effects in human islets. The study treated approximately 100 human islets with 50, 100, 150pM HYBD-HEXA in presence of IB MX and cAMP was measured (Figure 4). The study also used 5nM forskolin as a positive control for cAMP response(Figure 4A).

[0099] Increase in cAMP results in increased free intracellular calcium pool in the cytol of P-cells . The increase in free calcium is necessary to induce insulin release from the granules. To establish this, islets were treated with HYBD-HEXA and the intracellular calcium was measured using FURA-4AM. It was expected that an increase in intracellular calcium pool would be seen upon stimulation with HYBD-HEXA. Indeed, there was significant increase in free calcium in the metabolically active human islet (Figure 5). Metabolically active cells in the islet preparation were also measured by MTT assay.

[0100] Finally, to confirm that HYBD-HEXA is capable of inducing insulin release in response to high glucose the human islets were challenged with low and high glucose and the insulin release in the medium was determined, as shown in figure 6.

[0101] Next, the effect of the peptide HYBD-HEXA on human pancreatic J3-cells functional identity was tested by measuring the expression of PDX1, FOXO1, and insulin using RT-PCR. The results showed a significant increase in PDX1 and insulin and decreased FOXO1 expression in islets treated with lOOpM of HYBD-HEXA compared to untreated islets. (Fig. 7). The loss of FOXO1 and increase in PDX1 expression signifies transcription of insulin mRNA because of its promoter activation. Indeed RT-PCR results showed increased insulin mRNA in islets treated with HYBD-HEXA.Attorney Docket No. 10063-095W01

[0102] Chronic non-alcoholic fatty liver disease (NAFLD) leads to liver failure. Based on previous work on the effect of Osteocalcin on NAFLD in an animal model that showed administration of osteocalcin improves insulin sensitivity, decreases liver inflammation, and improve liver function, the study sought to investigate the ability of HEXA or HYBD_HEXA to rescue Palmitic acid (PA) induced cell death in HepG2 cells. Results from such an experiment shown that both HEXA and HYBD_HEXA rescued the cells from PA induced toxicity indicating that these peptides could be an alternative treatment for NAFLD patients. HYBD_HEXA is a new class of peptide that can elicit insulin response in human beta cells and is beneficial in increasing insulin sensitivity, weight loss and improving pathologically and clinical manifestation of NAFLD.Example 2: Metabolic effects of Hybrid Peptide in a high fat diet (HFD)-induced obesity mouse model

[0103] Design: 7-week-old Female C57BL / 6NHsd were divided into two initial groups lean and high fat diet-induced obese (DIO) mice. Based on group assignment Mice were fed for 18 weeks either standard chow (Teklad Global Diets Rodent 2014 ad libitum) to serve as age matched controls while DIO mice had received a high fat diet (TD97366 ad libitum) to induce the DIO phenotype), All Mice were kept on a 12-h / 12-h light-dark cycle in a controlled room (temperature 20 to 26 C, relative humidity range of 30 to 70%) with free access to food and water. At approximately 25 weeks of age DIO mice were randomized into three treatment groups (n = 8 / group) while on high fat diet (HFD) and treated for 63 days with different subcutaneous (s.c.) doses of (3, 10, or 30 ng / g) of the Hybrid peptide administered twice daily (bid) or with vehicle.

[0104] Results: Hybrid peptide has profound effects in mouse model of diet-induced obesity (DIO) on all aspects of metabolic syndrome and obesity.Attorney Docket No. 10063-095W01

[0105] Effect of Hybrid peptide on body weight: Compared to weight at baseline, regular chow fed animals-maintained body weight, while the obese control group (vehicle treated) exhibited a body weight increase of +8.1% and 14% by days 23 and 63 respectively (Figure 1A &B). Dramatic reductions in body weight were observed on day 23 and 63 with increasing doses of Hybrid peptide; (3ng / g) weight reduction of -8.2%, -20.4%; (lOng / g) were -0.8%, -23.9%; and at (30 ng / g) -12.3%, and -20.4%, respectively. All Hybrid Hexa doses of 3 ng / g or greater showed statistically significant reductions in body weight (g) compared with vehicle-treated obese controls (Figure 9C).

[0106] Effect of Hybrid Hexa on body composition: Furthermore, the percentage body fat and lean body mass in the animals using an EchoMRI-3inl for small animal Quantitative Magnetic Resonance (QMR) machine. Interestingly, not only was there a decrease in % body fat but more notably increase in % lean body mass as early as day 23 post treatment with more significant improvement at day 63 post treatment as seen in Figure 10.

[0107] Effect of Hybrid Hexa on Liver weight and hepatic fat content: The weight of the liver in the chow control mice, the vehicle treated Obese (HFD; sham controls), and the various peptide treatment groups were compared at the end of the study and at animal sacrifice (+day 63). Only obese vehicle treated mice demonstrated a significant increase in liver weight vs. chow control (Fig. 1 IB), while all hybrid peptide treated groups-maintained liver weight similar to the chow controls. To further investigate the effects of the experimental treatments on liver histological structure and NASH, H&E of biopsies of the liver were obtained from all animals at the end of the study. All animals treated with hybrid peptide demonstrated complete protection from high fat diet induced (HFD; vehicle treated controls) changes in the liver such as fat deposition, hepatocyte injury, inflammation and their liver histology were comparable to the Chow fed Controls (Fig 11C). A similar improvement in Plasma triglycerides, phospholipids, cholesterol, non-esterified fatty acids (NEFA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels is expected.

[0108] Hybrid Hexa protects from cardiomegaly. Cardiac enlargement and dysfunction are now recognized manifestations of obesity; "obesity cardiomyopathy," is a newly recognized form of heart failure which develops independent of hypertension, coronary heart disease, and other heart diseases. We compared the weight of the heart in the Obese and Dose-dependent treatment groups to the vehicle treated obese controls and chow fed animals group at the end of the study (+day63). Only obese vehicle treated animals (HFD) had a significant increase in heart weight and size vs. hybrid peptide treated groups (Fig. 12A, B & C); a sign of heart enlargement, known as cardiomegaly, can be a serious health concern. This enlargement can progress toAttorney Docket No. 10063-095W01 obesity cardiomyopathy; a condition associated with increased risk of sudden cardiac death. Moreover, the increase in weight and heart size in obese vehicle treated animals was accompanied by a significant increase in cardiac myocytes size as shown in Figure 12D. These changes are often associated with cardiac hypertrophy, where the heart muscle enlarges, and can lead to impaired heart function. Interestingly, treatment with as low as 3ng / g, resulted in comparable heart weight, size, and cardiac myocytes size to the lean control (chow) group.

[0109] Hybrid Hexa protects HFD-induced islet size overexpansion and Loss of % Beta cell per islet. Islet areas were significantly increased in obese mice on HFD in comparison to lean mice (chow). However, this increase in Islet size measured by the endocrine staining synaptophysin (Syp, red) was accompanied by decrease in % Beta cell per islet identified by insulin staining (INS, green) as shown in Figure 13. Interestingly treatment with as low as 3ng / g, resulted in comparable % Beta cell per islet to the lean control (chow) group, while the higher doses showed effect in the islet area as shown in Figure 13.

[0110] Hybrid Hexa improves glucose metabolism. GTT (Glucose Tolerance Test) and a week later ITT (Insulin Tolerance Test) were performed; both in vivo metabolic tests used to assess glucose homeostasis and insulin sensitivity. GTT measures the efficacy of glucose uptake and the body's ability to clear glucose from the blood after a glucose load, while ITT assesses how effectively the body responds to insulin. For both tests mice fasted for 4 hours in the early morning. For the GTT, mice were injected with Ig / kg and blood glucose levels in response were determined at 0, 15, 30, 60, 90 and 120-minute post-injection (Fig.l4A). For the ITT, mice were injected with 0.75U / kg insulin and blood glucose levels were determined at 0, 15, 30, 60, 90 and 120-minute post-injection (Fig.l4B). While all treatment doses showed improvement in both tests, higher doses of 10 and 30ng / g were comparable to the lean group (chow) in the GTT and in the case of ITT, treatment with 3ng / g were comparable to the lean group (chow). Results reflect how well the pancreas responds to the glucose challenge by secreting insulin and how efficiently the liver and other tissues remove glucose from the bloodstream, an indication of insulin sensitivity.Attorney Docket No. 10063-095W01REFERENCESOh DY, Olefsky JM. G protein-coupled receptors as targets for anti-diabetic therapeutics. Nat Rev Drug Discov. 2016; 15:161-172.Alejandra Tomas, Ben Jones, Colin Leech, New Insights into Beta-Cell GLP-1 Receptor and cAMP Signaling. Journal of Molecular Biology, Volume 432, Issue 5, 2020, Pages 1347- 1366,Bossart M, Wagner M, Elvert R, Evers A, Hiibschle T, Kloeckener T, Lorenz K, Moessinger C, Eriksson O, Velikyan I, Pierrou S, Johansson L, Dietert G, Dietz-Baum Y, Kissner T, Nowotny I, Einig C, Jan C, Rharbaoui F, Gassenhuber J, Prochnow HP, Agueusop I, Porksen N, Smith WB, Nitsche A, Konkar A. Effects on weight loss and glycemic control with SAR441255, a potent unimolecular peptide GLP-l / GIP / GCG receptor triagonist. Cell Metab. 2022 Jan 4;34(l):59-74.el0.Kanazawa S, Tsunoda T, Murakami M, et al. Forskolin-Stimulateddenylylcyclase Activity: A Marker to Assess Islet CellViability Following Cold Storage in Different Solutions and to Predict Islet Cell Function following Transplantation. Cell Transplantation. 1999;8(4):383- 388.M. Prentki, F.M. Matschinsky, S.R. Madiraju. Metabolic signaling in fuel- induced insulin secretion. Cell Metabolism, 18 (2013), pp. 162-185.Min Pi, Karan Kapoor, Ruisong Ye, Satoru Kenneth Nishimoto, Jeremy C. Smith, Jerome Baudry, Leigh Darryl Quarles, Evidence for Osteocalcin Binding and Activation of GPRC6A in P-Cells, Endocrinology, Volume 157, Issue 5, 1 May 2016, Pages 1866-1880,Paul V. Sabatini, Thilo Speckmann, Francis C. Lynn. Friend and foe: P-cell Ca2+ signaling and the development of diabetes. Molecular Metabolism. 21, 2019, 1-12.Sabek OM, Nishimoto SK, Fraga D, Tejpal N, Ricordi C, Gaber AO. Osteocalcin Effect on Human P-Cells Mass and Function. Endocrinology. 2015 Sep;156(9):3137-46.Anisha A. Gupte, * Omaima M. Sabek, * Daniel Fraga, Laurie J. Minze, Satoru K. Nishimoto, Joey Z. Liu, Solmaz Afshar, Lillian Gaber, Christopher J. Lyon, A. Osama Gaber, and Willa A. Hsueh Osteocalcin Protects Against Nonalcoholic Steatohepatitis in a Mouse Model of Metabolic Syndrome. Endocrinology. 2014 Dec; 155(12): 4697-4705.Attorney Docket No. 10063-095W01SEQUENCESSEQ ID NO 1 (HYBD-HEXA): HCDGTFSDGSQEFYRWLRFYGPVSEQ ID NO: 2 (HYBD): HCDGTFSDGSQEFYRWESEQ ID NO: 3 (HEXA): RFYGPVSEQ ID NO: 4 (NATIVE OCN, BIOACTIVE DOMAIN):DCDELADHIGFOEAYRRFYGPVSEQ ID NO: 5 (TABLE 1 PRIMER): GAAGTCTACCAAAGCTCACGCGSEQ ID NO: 6 (TABLE 1 PRIMER): GGAACTCCTTCTCCAGCTCTAGSEQ ID NO: 7 (TABLE 1 PRIMER): AAGAGCGTGCCCTACTTCAASEQ ID NO: 8 (TABLE 1 PRIMER): GTTGTTGTCCATGGATGCAGSEQ ID NO: 9 (TABLE 1 PRIMER): CCCTGCAGAAGCGTGGCATTSEQ ID NO: 10 (TABLE 1 PRIMER): CCATCTCTCTCGGTGCAGGASEQ ID NO: 11 (TABLE 1 PRIMER): AGCACAGAGCCTCGCCTTSEQ ID NO: 12 (TABLE 1 PRIMER): CATCATCCATGGTGAGCTGGSEQ ID NO: 13 (OSTCN RAT, FIGURE 1):MRTLSLLTLLALTAFCLSDLAGAKPSDSESDKAFMSKQEGSKVVNRLRRYLNNGLGAPAPYPDPLEPHREVCELNPNCDELADHIGFQDAYKRIYGTTVSEQ ID NO: 14 (OSTCN MOUSE, FIGURE 1):MRTIFLLTLLTLAALCLSDLTDAKPSGPESDKAFMSKQEGNKVVNRLRRYLGASVPSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITISEQ ID NO: 15 (OSTCN HUMAN, FIGURE 1):MRALTLLALLALAALCIAGQAGAKPSGAESSKGAAFVSKQEGSEVVKRPRRYLYQWLGAPVPYPDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPVSEQ ID NO: 16 (OSTCN BOVINE, FIGURE 1):MRTPMLLALLALATLCLAGRADAKPGDAESGKGAAFVSKQEGSEVVKRLRRYLDHWLGAPAPYPDPLEPKREVCELNPDCDELADHIGFQEAYRRFYGPVSEQ ID NO: 17 (CLEAVED BIOACTIVE PEPTIDE, FIGURE 1):YLYQWLGAPVPYPDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPV

Claims

Attorney Docket No. 10063-095W01CLAIMSWhat is claimed is:

1. A composition that can activate both a class B and class C G-protein-coupled receptor (GPCR) simultaneously.

2. The composition of claim 1, wherein the composition comprises a peptide.

3. The composition of claim 1 or 2, wherein the class B GPCR is a Bl GPCR.

4. The composition of claim 3, wherein the composition targets more than one class Bl GPCR.

5. The composition of claim 3 or 4, wherein the Bl GPCR(s) comprises glucagon, GLP1, and / or GIP receptors.

6. The composition of claim 5, wherein all three of glucagon, GLP1, and GIP receptors are simultaneously targeted by the composition.

7. The composition of claim 6, wherein a portion of the composition which targets glucagon, GLP1, and GIP receptors is 88% or more identical to SEQ ID NO: 2.

8. The composition of any one of claims 1-7, wherein the class C GPCR comprises GPRC6A.

9. The composition of claim 8, wherein a portion of the composition which targets GPRC6A comprises a C-terminal derived peptide from osteocalcin.

10. The composition of claim 9, wherein the C-terminal derived peptide from osteocalcin comprises SEQ ID NO: 3.

11. The composition of any one of claims 1-10, wherein the composition elicits insulin production and / or secretion in human pancreatic cells.

12. The composition of any one of claims 1-11, wherein the composition increases glucose uptake.

13. The composition of any one of claims 1-12, wherein the composition causes an increase in cAMP and intracellular calcium.

14. The composition of any one of claims 1-13, wherein the composition reduces at least one nonalcoholic steatohepatitis (NASH) component.

15. The composition of claim 14, wherein said NASH components comprise steatosis, ballooning degeneration, inflammation, and fibrosis.Attorney Docket No. 10063-095W0116. The composition of any one of claims 1-15, wherein the composition reduces nuclear expression of F0X01 and restores cytoplasmic expression of PDX-1 in pancreatic P cells.

17. The composition of any one of claims 1-16, wherein the composition restores human pancreatic cell function, mass, and / or identity.

18. The composition of any one of claims 1-17, wherein the composition comprises at least one synthetic (non-naturally occurring) peptide.

19. The composition of any one of claims 1-18, wherein the composition comprises a pharmaceutically acceptable carrier.

20. The composition of any one of claims 1-19, wherein the composition comprises a nanocarrier.

21. The composition of any one of claims 1-20, wherein the composition comprises a targeting peptide and / or cell penetrating peptide.

22. A polypeptide comprising 90% or more identity to SEQ NO: 1.

23. A polypeptide comprising SEQ ID NO: 1.

24. A polypeptide comprising a class B 1 GPCR agonist and a class C GPCR agonist, wherein the class Bl GPCR agonist comprises 88% or more identity to SEQ ID NO: 2, and wherein the class C GPCR agonist comprises SEQ ID NO: 3.

25. The polypeptide of claim 24, wherein the polypeptide further comprises a linker between the class Bl GPCR agonist comprising 88% or more identity to SEQ ID NO: 2 and the class C GPCR agonist comprising SEQ ID NO: 3.

26. The polypeptide of claim 24 or 25, wherein the polypeptide further comprises an additional modulator of an additional receptor.

27. A nucleic acid encoding the polypeptide of any one of claims 24-26.

28. A vector comprising the nucleic acid of claim 27.

29. A method of activating a class B GPCR and a class C GPCR simultaneously, the method comprising exposing the GPCRs to a composition which comprises both a class B GPCR activator and a class C GPCR activator.

30. The method of claim 29, wherein the composition comprises a peptide.

31. The method of claim 29 or 30, wherein the class B GPCR is a Bl GPCR.Attorney Docket No. 10063-095W0132. The method of claim 31 , wherein the composition targets more than one class B 1 GPCR.

33. The method of claim 31 or 32, wherein the Bl GPCR(s) comprises glucagon, GLP1, and / or GIP receptors.

34. The method of claim 33, wherein all three of glucagon, GLP1, and GIP receptors are simultaneously targeted by the composition.

35. The method of claim 34, wherein a portion of the composition which targets glucagon, GLP1, and GIP receptors is 88% or more identical to SEQ ID NO: 2.

36. The method of any one of claims 29-35, wherein the class C GPCR comprises GPRC6A.

37. The method of claim 36, wherein a portion of the composition which targets GPRC6A comprises a C-terminal derived peptide from osteocalcin.

38. The method of claim 37, wherein the C-terminal derived peptide from osteocalcin comprises SEQ ID NO: 3.

39. The method of any one of claims 29-38, wherein the composition elicits insulin production and / or secretion in human pancreatic P cells.

40. The method of any one of claims 29-39, wherein the composition increases glucose uptake.

41. The method of any one of claims 29-40, wherein the composition causes an increase in cAMP and intracellular calcium.

42. The method of any one of claims 29-41, wherein the composition reduces at least one nonalcoholic steatohepatitis (NASH) component.

43. The method of claim 42, wherein said NASH components comprise steatosis, ballooning degeneration, inflammation, and fibrosis.

44. The method of any one of claims 29-43, wherein the composition reduces expression of FOXO1 and increases PDX-1 expression in pancreatic cells.

45. The method of any one of claims 29-44, wherein the composition restores human pancreatic P cell function, mass, and / or identity.

46. The method of any one of claims 29-45, wherein the composition comprises at least one synthetic (non-naturally occurring) peptide.Attorney Docket No. 10063-095W0147. The method of any one of claims 29-46, wherein the composition comprises a pharmaceutically acceptable carrier.

48. The method of any one of claims 29-47, wherein the composition comprises a nanocarrier.

49. The method of any one of claims 29-48, wherein the composition comprises a targeting peptide and / or cell penetrating peptide.

50. A method of treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is associated with both a class B GPCR and a class C GPCR, the method comprising administering to the subject a composition comprising a peptide, wherein the peptide can simultaneously activate both a class B GPCR and a class C GPCR.

51. The method of claim 50, wherein the disease or disorder comprises type 2 diabetes mellitus (T2DM), obesity, depression, cancer, cardiovascular disease, or Alzheimer’s disease.

52. The method of claim 50 or 51, wherein the class B GPCR is a Bl GPCR.

53. The method of claim 52, wherein the composition targets more than one class Bl GPCR.

54. The method of claim 52 or 53, wherein the Bl GPCR(s) comprises glucagon, GLP1, and / or GIP receptors.

55. The method of claim 54, wherein all three of glucagon, GLP1, and GIP receptors are simultaneously targeted by the composition.

56. The method of claim 55, wherein a portion of the composition which targets glucagon, GLP1, and GIP receptors is 88% or more identical to SEQ ID NO: 2.

57. The method of any one of claims 50-56, wherein the class C GPCR comprises GPRC6A.

58. The method of claim 57, wherein a portion of the composition which targets GPRC6A comprises a C-terminal derived peptide from osteocalcin.

59. The method of claim 58, wherein the C-terminal derived peptide from osteocalcin comprises SEQ ID NO: 3.

60. The method of any one of claims 50-59, wherein the composition comprises at least one synthetic (non-naturally occurring) peptide.Attorney Docket No. 10063-095W0161. The method of any one of claims 50-60, wherein the composition comprises a pharmaceutically acceptable carrier.

62. The method of any one of claims 50-61, wherein the composition comprises a nanocarrier.

63. The method of any one of claims 50-62, wherein the composition comprises a targeting peptide and / or cell penetrating peptide.

64. The method of any one of claims 50-62, wherein the composition is targeted to the brain.

65. A method of restoring pancreatic P cell function, mass, and / or identity, the method comprising exposing a pancreatic cell to a composition, wherein the composition comprises a peptide, wherein the peptide can simultaneously activate both a class B GPCR and a class C GPCR.

66. The method of claim 65, wherein the pancreatic P cell has lost function, mass, and / or identity.

67. The method of claim 65 or 66, wherein the cell is in vitro or ex vivo.

68. The method of claim 65 or 66, wherein the cell is in a subject in vivo).

69. The method of claim 68, wherein the subject has type II diabetes.

70. The method of claim any one of claims 65-69, wherein the subject has been exposed to a substance which causes loss of pancreatic P cell function, mass, and / or identity.

71. The method of claim 70, wherein the substance comprises doxorubicin, antihypertensive vasodilator diazoxide, and / or corticosteroids.

72. The method of any one of claims 65-71, wherein the class B GPCR is a Bl GPCR.

73. The method of claim 72, wherein the composition targets more than one class Bl GPCR.

74. The method of claim 72 or 73, wherein the Bl GPCR(s) comprises glucagon, GLP1, and / or GIP receptors.

75. The method of claim 74, wherein all three of glucagon, GLP1, and GIP receptors are simultaneously targeted by the composition.

76. The method of claim 75, wherein a portion of the composition which targets glucagon, GLP1, and GIP receptors is 88% or more identical to SEQ ID NO: 2.Attorney Docket No. 10063-095W0177. The method of any one of claims 65-76, wherein the class C GPCR comprises GPRC6A.

78. The method of claim 77, wherein a portion of the composition which targets GPRC6A comprises a C-terminal derived peptide from osteocalcin.

79. The method of claim 78, wherein the C-terminal derived peptide from osteocalcin comprises SEQ ID NO: 3.

80. The method of any one of claims 65-79, wherein the composition comprises at least one synthetic (non-naturally occurring) peptide.

81. The method of any one of claims 65-80, wherein the composition comprises a pharmaceutically acceptable carrier.

82. The method of any one of claims 65-81, wherein the composition comprises a nanocarrier.

83. The method of any one of claims 65-82, wherein the composition comprises a targeting peptide and / or cell penetrating peptide.

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