Self-assembling depots of adjuvant peptide therapies

Abstract

Described herein are compositions, formulations, and methods for treating diabetes or inducing weight loss. In one aspect, the formulation releases the therapeutic agent over 40 days.

Description

[0001] SELF-ASSEMBLING DEPOTS OF ADJUVANT PEPTIDE THERAPIES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 560,432, filed on March 1 , 2024, U.S. Provisional Patent Application No. 63 / 678,973, filed on August 2, 2024, and U.S. Provisional Patent Application No. 63 / 679,013, filed on August 2, 2024, each of which is incorporated by reference herein in its entirety.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] This application was filed with a Sequence Listing XML in ST.26 XML format accordance with 37 C.F.R. § 1.831 and PCT Rule 13ter. The Sequence Listing XML file submitted in the USPTO Patent Center, “092012-0041-W001_sequence_listing_xml_24-FEB-2025.xml,” was created on February 24, 2025, contains 17 sequences, has a file size of 36.0 kilobytes (36,864 bytes), and is incorporated by reference in its entirety into the specification.

[0006] BACKGROUND

[0007] The global incidence of diabetes and metabolic disorders is steadily increasing; type 2 diabetes, characterized by insulin resistance and hyperglycemia, comprises approximately 90%- 95% of all diagnosed diabetes cases. Glucagon-like peptide-1 (GLP-1), a natural incretin hormone containing 30 amino acids, is produced by intestinal L-cells and functions to reduce blood glucose by stimulating insulin production and suppressing glucagon secretion (FIG. 1A; SEQ ID NO: 1). Accordingly, synthetic GLP-1 receptor agonists have emerged as a therapeutic approach to treat type 2 diabetes and obesity. These agents also act to reduce appetite and inhibit upper gastrointestinal secretion, leading to reduction of body weight. However, GLP-1 receptor agonists can be susceptible to enzymatic degradation and hydrolysis, which impact their stability in vivo. In addition, the relatively small molecular weight of GLP-1 (~3 kDa), results in rapid clearance from circulation.

[0008] Using methods of both sequence and prosthetic modification, new classes of GLP-1 receptor agonists have been developed that offer enhanced enzymatic stability and prolonged circulation time. Of recent relevance, semaglutide (Ozempic®) was approved by the FDA in 2021 to treat type 2 diabetes with a once-weekly administration schedule. Currently, semaglutide is among the most effective drugs to reverse symptoms of early to moderate diabetes. Semaglutide was designed by modifying the native GLP-1 sequence to promote chemical stability and also introduce a prosthetic PEG-linked Cis dicarboxylic fatty acid; this motif binds circulating serum albumin to prolong in vivo circulation (FIG. 8; SEQ ID NO: 2). Semaglutide has also been used both off-label and in reformulated products (Wegovy®) to promote weight loss. Its clinical impact is due, in large part, to its once-weekly administration schedule, offering improvement over the daily or even twice-daily dosing schedules of earlier GLP-1 receptor agonist designs. However, in spite of its improved convenience, there remains low therapeutic persistence in its use; only 45% of individuals that receive a prescription stay on-treatment. Thus, a continued need exists to develop more effective GLP-1 receptor agonists, with ongoing efforts aimed at further reducing treatment burden, limiting side-effects, and improving therapeutic adherence.

[0009] Controlled release approaches have been explored to extend the drug release of GLP-1 receptor agonists while also minimizing side-effects. Notably, one of the first approved GLP-1 receptor agonists for twice-daily use (exenatide; FIG. 18; SEQ ID NO: 7) was loaded into poly(D,i_- lactic-co-glycolic acid) microspheres to achieve a therapeutic (Bydureon®) with once-weekly injection through controlled release; reduced HbA1c levels along with a simultaneous reduction in side-effects of nausea and vomiting were reported from this controlled release approach. Recently, injectable polymer-nanoparticle hydrogels were reported to encapsulate semaglutide and liraglutide, achieving sustained release over several weeks with functional validation in a rat model of type 2 diabetes. Accordingly, material-based encapsulation and controlled delivery offers one option to extend the functional therapeutic half-life, reduce injection frequency, and perhaps also limit the side-effects of GLP-1 receptor agonists.

[0010] SUMMARY

[0011] One embodiment described herein is a therapeutic conjugate comprising: a peptide-based therapeutic moiety; a peptide amphiphile moiety; and a linker attaching the peptide-based therapeutic moiety to the peptide amphiphile moiety, wherein the peptide amphiphile moiety comprises: an alkyl moiety of formula: O attached to the N-terminus of a sequence of

[0012] 3-10 amino acid residues; the sequence comprising: a hydrophobic subsequence of 2-6 hydrophobic amino acid residues, wherein the N- terminus of the hydrophobic subsequence is attached to the alkyl moiety; a hydrophilic subsequence of 1-4 hydrophilic amino residues, wherein the hydrophilic subsequence is attached to the linker. In another aspect, the peptide-based therapeutic moiety is a natural or synthetic hormone moiety.

[0013] In another aspect, the natural or synthetic hormone moiety is a GLP-1 , GIP, and / or glucagon receptor agonist moiety.

[0014] In another aspect, the GLP-1 , GIP, and / or glucagon receptor agonist moiety is: In another aspect, the peptide amphiphile moiety is a peptide moiety of formula (I): wherein:

[0015] Y1is the alkyl moiety of formula: is the sequence of amino acid residues; wherein: is the hydrophobic subsequence; and

[0016] 5— AA4— AA5— f

[0017] ’ ’ is the hydrophilic subsequence.

[0018] In another aspect, AA1, AA2, and AA3are each independently a glycine residue, an alanine residue, a valine residue, an isoleucine residue, or a leucine residue.

[0019] In another aspect, AA4and AA5are each independently a glutamic acid residue, an aspartic acid residue, a lysine residue, or an arginine residue.

[0020] In another aspect, the peptide amphiphile moiety is a peptide moiety of formula (l-a):

[0021] In another aspect, R1, R2, and R3are each

[0022] In another aspect, R4and R5are each

[0023] In another aspect, the peptide amphiphile moiety is:

[0024]

[0025] Another embodiment described herein is a hydrogel comprising: a therapeutic conjugate; and optionally, a diluent peptide amphiphile, the diluent peptide amphiphile comprising: an alkyl moiety of formula: O attached to the N-terminus of a sequence of

[0026] 3-10 amino acid residues; the sequence comprising: a hydrophobic subsequence of 2-6 hydrophobic amino acid residues, wherein the N- terminus of the hydrophobic subsequence is attached to the alkyl moiety; a hydrophilic subsequence of 1-4 hydrophilic amino residues, wherein the N-terminus of the hydrophilic subsequence is attached to the C-terminus of the hydrophobic subsequence; and

[0027] O O the C-terminus of the hydrophilic subsequence is

[0028] In another aspect, the therapeutic conjugate of clause 1 is present at about 0.1-10 wt%; and the diluent peptide amphiphile is present at about 0-10 wt%.

[0029] In another aspect, the diluent peptide amphiphile is a diluent peptide amphiphile of formula (II):

[0030] Y1— AA1— AA2— AA3— AA4— AA5(H), wherein:

[0031] Y1is the alkyl moiety of formula: is the hydrophilic subsequence; and

[0032] In another aspect, the diluent peptide amphiphile is a diluent peptide amphiphile of formula (I l-a): wherein:

[0033] R1, R2, and R3are each independently hydrogen, methyl,

[0034] R4and R5are each independently

[0035] Another embodiment described herein is a pharmaceutical composition comprising a therapeutic conjugate or a hydrogel; and a pharmaceutically acceptable excipient.

[0036] Another embodiment described herein is a method for treating or ameliorating the symptoms of diabetes or inducing weight loss, the method comprising administering a therapeutically effective amount of a therapeutic conjugate, a hydrogel, or a pharmaceutical composition to a subject in need thereof.

[0037] Another embodiment described herein is a kit comprising a therapeutic conjugate, a hydrogel, or a pharmaceutical composition; delivery or administration apparata or devices; and optionally, packaging, a label, or instructions for use. DESCRIPTION OF THE DRAWINGS

[0038] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0039] FIG. 1A-B shows an overview of the GLP-1 therapeutics described herein. FIG. 1A shows the glucagon-like peptide-1 (GLP-1) (residues 7-37; SEQ ID NO: 1). FIG. 1 B schematically illustrates the supramolecular nanofiber depots formed from GLP-1 therapeutics.

[0040] FIG. 2 schematically illustrates the formation of supramolecular nanofiber depots from GLP-1 receptor agonists. As shown, a GLP-1 receptor agonist moiety, e.g., a semaglutide moiety, may be attached to a peptide amphiphile (PA) moiety using a polyethylene glycol (PEG) linker (“PA-GLP1 ,” top). As further shown, to facilitate efficient nanofiber assembly and hydrogelation, the PA-GLP1 may be co-formulated with a diluent peptide amphiphile (“dPA,” middle). Mixing of these two components enables formation of nanofibers that can physically entangle to form injectable hydrogels. Upon injection, the hydrogels form a subcutaneous depot to provide long- lasting therapeutic release of PA-GLP1 (bottom).

[0041] FIG. 3 shows cell activity of PA-GLP1 (a semaglutide moiety attached to a PA moiety through a PEG linker) compared to semaglutide itself using a receptor activation assay in a human reporter cell line expressing the human GLP-1 receptor.

[0042] FIG. 4A-D show hydrogelation and analysis of hydrogels. FIG. 4A shows the hydrogelation of co-formulated PA-GLP1 / dPA, with the mass fraction of PA-GLP1 in dPA labeled for each, assessed by vial inversion. FIG. 4B shows rheological frequency sweep (at 0.5% strain) of co-formulated PA-GLP1 / dPA hydrogels mixed at different mass fractions, where closed symbols are G' and open symbols are G". FIG. 4C shows storage modulus (G', solid, left axis) and complex viscosity (q*, open, right axis) for co-formulated PA-GLP1 / dPA hydrogels mixed at different mass fractions following equilibration over time at 0.5% strain and 1 rad / s. FIG. 4D shows shear viscosity flow ramp 15% PA-GLP1 / dPA hydrogel to demonstrate shear-thinning behavior. FIG. 3E shows step-strain cycling between 0.5% and 100% strain and strain recovery for a 15%PA-GLP1 / dPA hydrogel. The total peptide concentration in all hydrogels was 2% (w / v).

[0043] FIG. 5A-C shows analysis of dPA, PA-GLP1 / dPA, and semaglutide. FIG. 5A shows circular dichroism (CD) spectroscopy of dPA, PA-GLP1 , and semaglutide at 0.2% (w / v) concentrations. FIG. 5B shows CD spectroscopy of co-formulated PA-GLP1 / dPA mixtures at 0.2% (w / v) concentrations and different mass fractions. FIG. 5C shows transmission electron microscopy (TEM) of dPA, PA-GLP1 / dPA mixtures, and PA-GLP1 alone at 0.05% (w / v) peptide. FIG. 6 shows the release profile of methoxycoumarin (MCA)-labeled PA-GLP1 from coformulated hydrogels of PA-GLP1 / dPA at 2% (w / v) total peptide content, and exemplary release of semaglutide at 15% in physical mixture with a dPA at 2% (w / v) total peptide content.

[0044] FIG. 7A-D show the in vivo evaluation of PA-GLP1 / dPA hydrogel formulation in a type 2 diabetic rat model. FIG. 7A shows model development using nicotinamide (NA) and streptozotocin (STZ) and the schedule for treatment with daily semaglutide (20 pg / day) or a single injection at day 0 of the PA-GLP-1 / dPA hydrogel (gel). Evaluation schedule is shown for blood glucose and weight measurements and serum collection for concentration analysis (n = 8-9 rats / group). FIG. 7B shows serum concentrations obtained from a semaglutide ELISA throughout the study for each treatment (correlated to a standard curve of semaglutide, PA-GLP1 , or rat GLP- 1 according to the treatment / control group). FIG. 7C shows blood glucose levels expressed relative to their starting point for each treatment throughout the study. FIG. 7D shows rat weights expressed relative to their starting point for each treatment throughout the study.

[0045] FIG. 8 shows the chemical structure of semaglutide (SEQ ID NO: 2).

[0046] FIG. 9 shows the chemical structure of PA-GLP1 .

[0047] FIG. 10 shows the chemical structure of an example dPA.

[0048] FIG. 11 shows the chemical structure of MCA-PA-GLP1 (MCA-labeled PA-GLP1).

[0049] FIG. 12 shows the chemical structure of Dan-PA-GLP1 (Dansyl (Dan)-labeled PA-GLP1).

[0050] FIG. 13 shows the hydrogelation test of PA-GLP1 alone without dPA by gross inspection using vial inversion. Peptides were prepared in 20 mM HEPES, 150 mM NaCI, 15 mM CaCL.

[0051] FIG. 14A-E show the fluorescence emission spectra of 5% Dan-PA-GLP1 / dPA (0.1 mg / mL Dan-PA-GLP1) (FIG. 14A), 10% mass fraction of Dan-PA-GLP1 with dPA (0.2 mg / mL Dan-PA- GLP1) (FIG. 14B), 15% mass fraction of Dan-PA-GLP1 with dPA (0.3 mg / mL Dan-PA-GLP1) (FIG. 14C), 20% mass fraction of Dan-PA-GLP1 with dPA (0.4 mg / mL Dan-PA-GLP1) (FIG. 14D), and 40% mass fraction of Dan-PA-GLP1 with dPA (0.1 mg / mL Dan-PA-GLP1) (FIG. 14E).

[0052] FIG. 15 shows the in vitro release profile of physical mixture of 5%, 10%, 20%, and 40% MCA-Semaglutide as a mass fraction combined with dPA. Total peptide concentrations: 2% w / v.

[0053] FIG. 16 shows the hydrogelation test by gross inspection using vial inversion of MCA- Semaglutide mixed with dPA at various noted mass fractions. Peptide concentrations: 2% w / v.

[0054] FIG. 17 shows an evaluation of injectability for 15%PA-GLP1 / dPA hydrogels prepared at 2% (w / v) and extruded into 20 mM HEPES, 150 mM NaCI, pH 7.4.

[0055] FIG. 18 shows the chemical structure of exenatide (SEQ ID NO: 7).

[0056] FIG. 19A shows the chemical structure of tirzepatide (SEQ ID NO: 8). FIG. 19B shows the chemical structure of a tirzepatide moiety attached to a PA moiety through a PEG linker (“PA- Tir”). FIG. 19C shows the hydrogelation test of PA-Tir / dPA prepared at noted mass fractions by gross inspection using vial inversion. Peptide concentrations: 2% w / v. FIG. 19D shows CD spectroscopy of co-formulated PA-Tir / dPA prepared at noted mass fractions. Peptide concentrations: 0.2% w / v.

[0057] FIG. 20A shows the chemical structure of retatrutide (SEQ ID NO: 10). FIG. 20B shows the chemical structure of a retatrutide moiety attached to an attached to a PA moiety through a PEG linker (“PA-Reta”). FIG. 20C shows the hydrogelation test of PA-Reta / dPA by gross inspection using vial inversion. Peptide concentrations: 2 wt%. FIG. 20D shows CD spectroscopy of co-assembled PA-Reta / dPA formulations. Peptide concentrations: 0.2 wt%.

[0058] FIG. 21A shows an example structure of an alternate version of PA-GLP1 prepared with an ester linker between the PA module and the GLP-1 receptor agonist module, termed PA-ester- GLP1. FIG. 21 B shows gelation tests of PA-ester-GLP1 mixed with dPA at different stated mass fractions, with gels prepared at a total peptide concentration of 2% w / v. FIG. 21C shows release testing of MCA-labeled PA-ester-GLP1 mixed with dPA at different stated mass fractions, with gels prepared at a total peptide concentration of 2% w / v (left) or 3% w / v (right).

[0059] FIG. 22A shows an example structure of an alternate version of PA-GLP1 prepared with an alternate p-sheet hydrogen bonding domain (V4E3) and three PEGs linkers, termed PA(V4E3)- (PEGS)3-GLP1. FIG. 22B shows hydrogelation tests of single-component hydrogels prepared from only PA bearing a GLP-1 receptor agonist with no diluent PA. This sample is PA( 4E2)- (PEGS)3-GLP1 prepared at concentrations of 2%, 5%, and 10% w / v.

[0060] FIG. 23A shows an example structure of an alternate version of PA-GLP1 prepared with an alternate [3-sheet hydrogen bonding domain (V4E2) and three PEGs linkers, termed PA(V4E2)- (PEGS)3-GLP1. FIG. 23B shows hydrogelation tests of single-component hydrogels prepared from only PA bearing a GLP-1 receptor agonist with no diluent PA. This sample is PA(V4E3)- (PEG5)3-GLP1 prepared at concentrations of 2%, 5%, and 10% w / v.

[0061] DETAILED DESCRIPTION

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0063] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0064] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of’ the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open- ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim.

[0065] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0066] As used herein, the term “or” can be conjunctive or disjunctive.

[0067] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0068] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0069] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.” All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0070] As used herein, the terms “room temperature,” “RT,” or “ambient temperature” refer to the typical temperature in an indoor laboratory setting. In one aspect, the laboratory setting is climate controlled to maintain the temperature at a substantially uniform temperature or with a specific range of temperatures. In one aspect, “room temperature” refers a temperature of about 15-30 °C, including all integers and endpoints within the specified range. In another aspect, “room temperature” refers a temperature of about 15-30 °C; about 20-30 °C; about 22-30 °C; about 25-30 °C; about 27-30 °C; about 15-22 °C; about 15-25 °C; about 15-27 °C; about 20-22 °C; about 20-25 °C; about 20-27 °C; about 22-25 °C; about 22-27 °C; about 25-27 °C; about 15 °C ± 10%; about 20 °C ± 10%; about 22 °C ± 10%; about 25 °C ± 10%; about 27 °C ± 10%; ~20 °C, ~22 °C, ~25 °C, or ~27 °C, at standard atmospheric pressure.

[0071] As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.

[0072] As used herein, the terms “control,” or “reference” are interchangeable. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.

[0073] As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein.

[0074] As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

[0075] As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.

[0076] As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), nonhuman primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.

[0077] As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0078] As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0079] As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.

[0080] Definitions of specific functional groups and chemical terms are described in more detail herein. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5thed, John Wiley & Sons, Inc., New York, 2001 ; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rded, Cambridge University Press, Cambridge, 1987.

[0081] The term “alkyl,” as used herein, means a straight or branched, saturated hydrocarbon chain. The term “lower alkyl” or “Ci-ealkyl” means a straight or branched chain hydrocarbon containing from 1 to 6 carbon atoms. The term “Ci.4alkyl” means a straight or branched chain hydrocarbon containing from 1 to 4 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, / so-propyl, n-butyl, sec-butyl, / so-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n- heptyl, n-octyl, n-nonyl, and n-decyl.

[0082] The term “halogen” or “halo,” as used herein, means Cl, Br, I, or F.

[0083] The term “haloalkyl,” as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by a halogen.

[0084] The term “haloalkoxy,” as used herein, means at least one haloalkyl group, as defined herein, is appended to the parent molecular moiety through an oxygen atom.

[0085] The term “substituted” refers to a group that may be further substituted with one or more non-hydrogen substituent groups. Substituent groups include, but are not limited to, halogen, =0 (oxo), =S (thioxo), cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0086] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0087] The term “peptide,” as used herein, means a compound comprising two or more amino acids. As used herein, the term “peptide” encompasses peptides terminated with a carboxyl group and peptides terminated with a free amide group (-C(0)NH2).

[0088] The term “amino acid,” as used herein, means an organic compound that contains both amino and carboxyl functional group. As used herein, the term “amino acid” may refer to a naturally occurring amino acid residue (i.e. , a proteogenic amino acid residue) or a non-naturally occurring amino acid residue (i.e., a non-proteogenic amino acid residue). A “naturally occurring amino acid” is an amino acid that is encoded by the genetic code, as well as those amino acids that are encoded by the genetic code that are modified after synthesis, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. An amino acid analog is a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e. , an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, ornithine, methionine sulfoxide, methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but will retain the same basic chemical structure as a naturally occurring amino acid.

[0089] A “non-naturally occurring amino acid” is a compound that has the same basic chemical structure as a naturally occurring amino acid but is synthetically or enzymatically prepared. “Non- naturally occurring amino acid” includes, but is not limited to, amino acids that occur by modification (e.g., posttranslational modifications) of a naturally encoded amino acid (including but not limited to, the 20 common amino acids) but are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. Non-limiting examples of non-naturally occurring amino acids that can be inserted into a polypeptide sequence or substituted for a wildtype residue in polypeptide sequence include D-amino acids, p-amino acids, homoamino acids, cyclic amino acids, or amino acids with derivatized side chains.

[0090] The term “amino acid residue,” as used herein, means the part of an amino acid that remains after two or more amino acids combine to form a peptide. As used herein, the term “amino acid residue” may refer to a naturally occurring amino acid residue (i.e., a proteogenic amino acid residue) ora non-naturally occurring amino acid residue (i.e., a non-proteogenic amino acid residue).

[0091] The term “N-terminus,” as used herein, means the amino terminus of an amino acid, peptide, or protein.

[0092] The term “C-terminus,” as used herein, means the carboxyl terminus of an amino acid, peptide, or protein.

[0093] The term “peptide-based therapeutic moiety,” as used herein means a peptide-based moiety that is capable of treating a disorder or disease.

[0094] The term “agonist moiety” as used herein refers to a moiety that binds to a receptor or enzyme and activates the receptor to produce a biological response. As used herein, the term “agonist moiety” encompasses full agonist moieties and partial agonist moieties.

[0095] “GLP-1 ,” as used herein, means glucagon-like peptide-1.

[0096] “GIP,” as used herein, means glucose-dependent insulinotropic polypeptide. Diabetes and obesity have emerged as major global health concerns. Glucagon-like peptide-1 (GLP-1), a natural incretin hormone, stimulates insulin production and suppresses glucagon secretion to stabilize and reduce blood glucose levels and control appetite. The therapeutic use of GLP-1 receptor agonists (e.g., semaglutide) has transformed the standard of care in recent years for treating type 2 diabetes and reversing obesity. The native GLP-1 sequence has a very short half-life, and therapeutic advances have come from molecular engineering to alter the pharmacokinetic profile of synthetic GLP-1 receptor agonists to enable once-weekly administration, reduce the frequency of injection, and improve adherence. Efforts to further extend this profile would offer additional convenience or enable entirely new treatment modalities. Here, an injectable GLP-1 receptor agonist depot is engineered through integration of a prosthetic self-assembling peptide motif to enable supramolecular nanofiber formation and hydrogelation. This supramolecular GLP-1 receptor agonistic (PA-GLP1) offers sustained release in vitro for multiple weeks, supporting long-lasting therapeutic release. Moreover, in a rat model of type 2 diabetes, a single injection of the supramolecular PA-GLP1 formulation achieved sustained serum concentrations for at least 40 days, with an overall reduction in blood glucose levels and reduced weight gain, comparing favorably to daily injections of semaglutide. The general and modular approach is also extensible to other next-generation peptide therapies. Accordingly, the formation of supramolecular nanofiber depots offers a more convenient and long- lasting therapeutic option to manage diabetes and treat metabolic disorders.

[0097] This work sought to create a supramolecular GLP-1 receptor agonist designed to selfassemble into nanofibers and form hydrogels to enable depot formation and controlled release (FIG. 2). This work was inspired by peptide amphiphile (PA) supramolecular materials that consist of a short peptide sequence linked to an aliphatic tail to drive nanofibrillar self-assembly in water, with physical entanglement of the nanofibers leading to hydrogel formation. The amphiphilicity of the PA design also enables incorporation of large bioactive peptide sequences for presentation on the surface of assembled nanofibers. As peptides, GLP-1 receptor agonists were reasoned feasible to incorporate into PA supramolecular self-assemblies through synthetic modification. Accordingly, the peptide sequence of semaglutide was conjugated to a PA module in place of its usual prosthetic albumin-binding motif, yielding a GLP-1 receptor agonist PA (PA-GLP1). This approach offers a route to achieve peptide-only depot formation in order to extend drug release without the need for encapsulation of the agent in a secondary material. The bulky nature of the receptor agonist necessitated the inclusion of a diluent PA (dPA) molecule to reduce frustration in molecular packing and promote more effective nanofiber formation and hydrogelation. The design facilitated tunable, controlled release of PA-GLP1 into a bulk phase over a period of weeks. When explored in a rat model of type 2 diabetes, a single administration of the PA-GLP1 formulation was able to support steady and long-lasting serum concentrations for at least 40 days and resulted in therapeutic efficacy in controlled blood glucose levels and reduced weight gain, comparing favorably to daily administration of semaglutide. The platform nature of this approach was further validated by including a dual GLP-1 and gastric inhibitory polypeptide (GIP) agonist (tirzepatide) for presentation on the PA platform, as well as a triple GLP-1 , GIP, and glucagon agonist (retatrutide) for presentation.

[0098] Therapeutic Conjugates

[0099] In one aspect, the present disclosure provides therapeutic conjugates. As used herein, the term “therapeutic conjugate” refers to a compound comprising a therapeutic moiety linked to another molecular moiety, e.g., a peptide amphiphile (PA) moiety. The therapeutic conjugates described herein comprise: a peptide-based therapeutic moiety; a peptide amphiphile moiety; and a linker attaching the peptide-based therapeutic moiety to the peptide amphiphile moiety, wherein the peptide amphiphile moiety comprises:

[0100] C6-24a Iky I an alkyl moiety of formula: O attached to the N-terminus of a sequence of

[0101] 3-10 amino acid residues; the sequence comprising: a hydrophobic subsequence of 2-6 hydrophobic amino acid residues, wherein the N- terminus of the hydrophobic subsequence is attached to the alkyl moiety; a hydrophilic subsequence of 1-4 hydrophilic amino residues, wherein the hydrophilic subsequence is attached to the linker.

[0102] In various instances, the peptide-based therapeutic moiety is a natural or synthetic hormone moiety.

[0103] In various instances, the natural or synthetic hormone moiety is a GLP-1 , GIP, and / or glucagon receptor agonist moiety.

[0104] In various instances, the GLP-1 , GIP, and / or glucagon receptor agonist moiety is:

[0105] In various instances, the linker comprises wherein n is 1-10.

[0106] In various instances, the peptide amphiphile moiety is a peptide moiety of formula (I): wherein:

[0107] Y1is the alkyl moiety of formula: is the hydrophilic subsequence.

[0108] In various instances, AA1, AA2, and AA3are each independently a glycine residue, an alanine residue, a valine residue, an isoleucine residue, or a leucine residue.

[0109] In various instances, AA4and AA5are each independently a glutamic acid residue, an aspartic acid residue, a lysine residue, or an arginine residue.

[0110] In various instances, the peptide amphiphile moiety is a peptide moiety of formula (l-a): wherein:

[0111] R1, R2, and R3are each independently hydrogen, methyl,

[0112] R4and R5are each independently

[0113] In some instances, R1, R2, and R3are each

[0114] In some instances, R4and R5are each

[0115] In some instances, the peptide amphiphile moiety is:

[0116]

[0117] Diluent Peptide Amphiphiles (dPAs)

[0118] The present disclosure provides further diluent peptide amphiphiles (dPAs). As used herein, the term “diluent peptide amphiphile” refers to a non-bioactive peptide amphiphile (PA) that acts to co-assemble alongside the PA moiety of the active therapeutic conjugate to facilitate molecular spacing within an assembled structure. Exemplary diluent peptide amphiphiles (dPAs) may be used in conjunction with a therapeutic conjugate described above. The diluent peptide amphiphiles (dPAs) disclosed herein comprise:

[0119] C 6-2481 ky I an alkyl moiety of formula: O attached to the N-terminus of a sequence of

[0120] 3-10 amino acid residues; the sequence comprising: a hydrophobic subsequence of 2-6 hydrophobic amino acid residues, wherein the N- terminus of the hydrophobic subsequence is attached to the alkyl moiety; a hydrophilic subsequence of 1-4 hydrophilic amino residues, wherein the N- terminus of the hydrophilic subsequence is attached to the C-terminus of the hydrophobic subsequence; and

[0121] 0 O the C-terminus of the hydrophilic subsequence is

[0122] In some instances, the diluent peptide amphiphile is a diluent peptide amphiphile of formula (II):

[0123] Y1— AA1— AA2— AA3— AA4— AA5(||)iwherein:

[0124] Y1is the alkyl moiety of formula: is the hydrophilic subsequence; and the C-terminus

[0125] In some instances, the diluent peptide amphiphile is a diluent peptide amphiphile of formula (ll-a): wherein:

[0126] R1, R2, and R3are each independently hydrogen, methyl,

[0127] R4and R5are each independently

[0128] The therapeutic conjugates and peptide amphiphiles may exist as stereoisomers wherein asymmetric or chiral centers are present. The terms “R” and “S” used herein are configurations as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure AppL Chem., 45: 13-30 (1976). The assignment “R” or “S” depends on the configuration of substituents around the chiral carbon atom. The disclosure contemplates various stereoisomers and mixtures thereof and these are specifically included within the scope of this invention. Stereoisomers include enantiomers and diastereomers, and mixtures of enantiomers or diastereomers. Individual stereoisomers of the compounds may be prepared synthetically from commercially available starting materials, which contain asymmetric or chiral centers or by preparation of racemic mixtures followed by methods of resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and optional liberation of the optically pure product from the auxiliary as described in Furniss, Hannaford, Smith, and Tatchell, “Vogel’s Textbook of Practical Organic Chemistry”, 5th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns or (3) fractional recrystallization methods.

[0129] It should be understood that the therapeutic conjugates and peptide amphiphiles may possess tautomeric forms, as well as geometric isomers, and that these also constitute an aspect of the invention.

[0130] In the therapeutic conjugates and peptide amphiphiles, any “hydrogen” or “H,” whether explicitly recited or implicit in the structure, encompasses hydrogen isotopes1H (protium) and2H (deuterium).

[0131] The present disclosure also includes isotopically-labeled therapeutic conjugates and peptide amphiphiles (e.g., deuterium labeled), where an atom in the isotopically-labeled therapeutic conjugates and peptide amphiphiles is specified as a particular isotope of the atom. Examples of isotopes suitable for inclusion in the compounds described herein are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to,2H,3H,13C,14C,15N,180,170,31P,32P,35S,18F, and36CI, respectively. The isotope may be a stable isotope or a radioactive isotope, or a combination thereof.

[0132] Isotopically-enriched forms of therapeutic conjugates and peptide amphiphiles may generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using an appropriate isotopically-enriched reagent in place of a non-isotopically-enriched reagent. The extent of isotopic enrichment can be characterized as a percent incorporation of a particular isotope at an isotopically-labeled atom (e.g., % deuterium incorporation at a deuterium label).

[0133] Pharmaceutical Salts

[0134] The disclosed therapeutic conjugates and peptide amphiphiles may exist as pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to salts or zwitterions of the compounds which are water or oil-soluble or dispersible, suitable for treatment of disorders without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio and effective for their intended use. The salts may be prepared during the final isolation and purification of the therapeutic conjugates and peptide amphiphiles or separately by reacting an amino group of the therapeutic conjugates and peptide amphiphiles with a suitable acid. For example, a therapeutic conjugate or peptide amphiphile may be dissolved in a suitable solvent, such as but not limited to methanol and water and treated with at least one equivalent of an acid, like hydrochloric acid. The resulting salt may precipitate out and be isolated by filtration and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide a salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, thrichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric and the like. The amino groups of the compounds may also be quaternized with alkyl chlorides, bromides, and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like.

[0135] Basic addition salts may be prepared during the final isolation and purification of the disclosed therapeutic conjugates and peptide amphiphiles by reaction of a carboxyl group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts can be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, / V, / V-dimethylaniline, / V-methylpiperidine, A / -methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, A / , / V-dibenzylphenethylamine, 1 -ephenamine and A / ,A / '-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.

[0136] General Synthesis

[0137] The therapeutic conjugates and peptide amphiphiles disclosed herein may be prepared according to peptide synthesis methods known in the art. In some instances, the therapeutic conjugates and peptide amphiphiles may be prepared using solid-phase synthesis. Optimum reaction conditions and reaction times for each individual step can vary depending on the particular reactants employed and substituents present in the reactants used. Specific procedures are provided in the Examples section. Reactions can be worked up in the conventional manner, e.g., by eliminating the solvent from the residue and further purified according to methodologies generally known in the art such as, but not limited to, crystallization, distillation, extraction, trituration, and chromatography. Unless otherwise described, the starting materials and reagents are either commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature.

[0138] Starting materials, if not commercially available, can be prepared by procedures selected from standard organic chemical techniques, techniques that are analogous to the synthesis of known, structurally similar therapeutic conjugates and peptide amphiphiles, or techniques that are analogous to the procedures described in the Examples below.

[0139] Routine experimentations, including appropriate manipulation of the reaction conditions, reagents and sequence of the synthetic route, protection of any chemical functionality that cannot be compatible with the reaction conditions, and deprotection at a suitable point in the reaction sequence of the method are included in the scope of the invention. Suitable protecting groups and the methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which can be found in PGM Wuts and TW Greene, in Greene’s book titled Protective Groups in Organic Synthesis (4thed.), John Wiley & Sons, NY (2006), which is incorporated herein by reference in its entirety. Synthesis of the compounds of the invention can be accomplished by methods analogous to those described in the synthetic schemes described hereinabove and in specific examples.

[0140] When an optically active form of a disclosed peptide amphiphile is required, it can be obtained by carrying out one of the procedures described herein using an optically active starting material (prepared, for example, by asymmetric induction of a suitable reaction step), or by resolution of a mixture of the stereoisomers of the peptide amphiphile or intermediates using a standard procedure (such as chromatographic separation, recrystallization, or enzymatic resolution).

[0141] Similarly, when a pure geometric isomer of a peptide amphiphile is required, it can be obtained by carrying out one of the above procedures using a pure geometric isomer as a starting material, or by resolution of a mixture of the geometric isomers of the peptide amphiphile or intermediates using a standard procedure such as chromatographic separation.

[0142] It can be appreciated that the synthetic schemes and specific examples as described are illustrative and are not to be read as limiting the scope of the invention as it is defined in the appended claims. All alternatives, modifications, and equivalents of the synthetic methods and specific examples are included within the scope of the claims.

[0143] Hydrogels

[0144] In another aspect, the present disclosure provides hydrogels comprising a therapeutic conjugate. Optionally, the hydrogels may further comprise a diluent peptide amphiphile. In various instances, the hydrogels may further comprise a buffer solution. The buffer solution may be a HEPES (A / -2-hydroxyethylpiperazine-A / '-2-ethanesulfonic acid) buffer solution.

[0145] In various instances, the therapeutic conjugate may be present in the hydrogel at about 0.1-10 mass percent (e.g., wt%). In various instances, the therapeutic conjugate may be present in the hydrogel at about 0.5-9.5 wt%; about 1.0-9.0 wt%; about 1.5-8.5 wt%; about 2.0-8.0 wt%; about 2.5-7.5 wt%; about 3.0-7.0 wt%; about 3.5-6.5 wt%; about 4.0-6.0 wt%; about 4.5-6.5 wt%; or about 5.0-6.0 wt%. In various instances, the therapeutic conjugate may be present in the hydrogel at no greater than about 10 wt%; no greater than about 9.0 wt%; no greater than about 8.0 wt%; no greater than about 7.0 wt%; no greater than about 6.0 wt%; no greater than about 5.0 wt%; no greater than about 4.0 wt%; no greater than about 3.0 wt%; no greater than about 2.0 wt%; no greater than about 1.0 wt%; or no greater than about 0.5 wt%. In various instances, the therapeutic conjugate may be present in the hydrogel at no less than about 0.1 wt%; no less than about 0.5 wt%; no less than about 1.0 wt%; no less than about 2.0 wt%; no less than about 3.0 wt%; no less than about 4.0 wt%; no less than about 5.0 wt%; no less than about 6.0 wt%; no less than about 7.0 wt%; no less than about 8.0 wt%; or no less than about 9.0 wt%.

[0146] In various instances, the diluent peptide amphiphile may be present in the hydrogel at about 0-10 wt%. In some instances, the diluent peptide amphiphile may be present in the hydrogel at about 0.1-10 wt%; about 0.5-9.5 wt%; about 1.0-9.0 wt%; about 1 .5-8.5 wt%; about 2.0-8.0 wt%; about 2.5-7.5 wt%; about 3.0-7.0 wt%; about 3.5-6.5 wt%; about 4.0-6.0 wt%; about 4.5-6.5 wt%; or about 5-6 wt%. In various instances, the diluent peptide amphiphile may be present in the hydrogel at no greater than about 10 wt%; no greater than about 9.0 wt%; no greater than about 8.0 wt%; no greater than about 7.0 wt%; no greater than about 6.0 wt%; no greater than about 5.0 wt%; no greater than about 4.0 wt%; no greater than about 3.0 wt%; no greater than about 2.0 wt%; no greater than about 1 .0 wt%; no greater than about 0.5 wt%; or no greater than about 0.1 wt%. In various instances, the diluent peptide amphiphile may be present in the hydrogel at no less than about 0.1 wt%; no less than about 0.5 wt%; no less than about 1.0 wt%; no less than about 2.0 wt%; no less than about 3.0 wt%; no less than about 4.0 wt%; no less than about 5.0 wt%; no less than about 6.0 wt%; no less than about 7.0 wt%; no less than about 8.0 wt%; or no less than about 9.0 wt%.

[0147] In various instances, the buffer solution may be present in the hydrogel at about 80-99.9 wt%. In various instances, the buffer solution may be present in the hydrogel at about 81-99 wt%; 82-98 wt%; 83-97 wt%; 84-96 wt%; 85-95 wt%; 86-94 wt%; 87-93 wt%; 88-92 wt%; or 89-91 wt%. In various instances, the buffer solution may be present in the hydrogel at no greater than about 99.9 wt%; no greater than about 99 wt%; no greater than about 98 wt%; no greater than about 97 wt%; no greater than about 96 wt%; no greater than about 95 wt%; no greater than about 94 wt%; no greater than about 93 wt%; no greater than about 92 wt%; no greater than about 91 wt%; no greater than about 90 wt%; no greater than about 89 wt%; no greater than about 88 wt%; no greater than about 87 wt%; no greater than about 86 wt%; no greater than about 85 wt%; no greater than about 84 wt%; no greater than about 83 wt%; no greater than about 82 wt%; or no greater than about 81 wt%. In various instances, the buffer solution may be present in the hydrogel at no less than about 80 wt%; no less than about 81 wt%; no less than about 82 wt%; no less than about 83 wt%; no less than about 84 wt%; no less than about 85 wt%; no less than about 86 wt%; no less than about 87 wt%; no less than about 88 wt%; no less than about 89 wt%; no less than 90 wt%; no less than 91 wt%; no less than 92 wt%; no less than 93 wt%; no less than 94 wt%; no less than 95 wt%; no less than 96 wt%; no less than 97 wt%; no less than 98 wt%; or no less than 99 wt%.

[0148] Pharmaceutical Compositions

[0149] Pharmaceutical compositions described herein may comprise a therapeutic conjugate or a hydrogel comprising a therapeutic conjugate.

[0150] The therapeutic conjugate or hydrogel comprising the therapeutic conjugate may be incorporated into pharmaceutical compositions suitable for administration to a subject (such as a patient, which may be a human or non-human). The pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactically effective amount” of the active agent (therapeutic conjugate). A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A “therapeutically effective amount” is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactical ly effective amount will be less than the therapeutically effective amount.

[0151] The pharmaceutical compositions may include pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0152] Thus, the hydrogels and their physiologically acceptable salts and solvates may be formulated for administration by, for example, solid dosing, eyedrop, in a topical oil-based formulation, injection, inhalation (either through the mouth or the nose), implants, or oral, buccal, parenteral, or rectal administration. Techniques and formulations may generally be found in ‘ Remington’s Pharmaceutical Sciences," (Meade Publishing Co., Easton, Pa.). Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage.

[0153] The route by which the hydrogel-encapsulated glucagon / glucagon analogue is administered, and the form of the composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).

[0154] Carriers for systemic administration typically include at least one of diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, and others. All carriers are optional in the compositions. Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol. The amount of diluent(s) in a systemic or topical composition is typically about 50 to about 90%.

[0155] Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma. The amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10%.

[0156] Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder(s) in a systemic composition is typically about 5 to about 50%.

[0157] Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of disintegrant(s) in a systemic or topical composition is typically about 0.1 to about 10%. Suitable colorants include a colorant such as an FD&C dye. When used, the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1%. Suitable flavors include menthol, peppermint, and fruit flavors. The amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0%.

[0158] Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s) in a systemic or topical composition is typically about 0.001 to about 1 %. Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically about 0.1 to about 5%. Suitable preservatives include benzalkonium chloride, methyl paraben and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5%. Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5%.

[0159] Suitable solvents include water, isotonic saline, ethyl oleate, glycerine, hydroxylated castor oils, alcohols such as ethanol, and phosphate buffer solutions. The amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100%. Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of Philadelphia, PA) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%. Suitable surfactants include lecithin, Polysorbate 80, and sodium lauryl sulfate, and the TWEENs from Atlas Powder Company of Wilmington, Delaware. Suitable surfactants include those disclosed in the C. T.F.A. Cosmetic Ingredient Handbook, 1992, pp.587-592; Remington’s Pharmaceutical Sciences, 15th Ed. 1975, pp. 335-337; and McCutcheon’s Volume 1 , Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. The amount of surfactant(s) in the systemic or topical composition is typically about 0.1 % to about 5%.

[0160] Although the amounts of components in the systemic compositions may vary depending on the type of systemic composition prepared, in general, systemic compositions include 0.01% to 50% of actives and 50% to 99.99% of one or more carriers. Compositions for parenteral administration typically include 0.1 % to 10% of actives and 90% to 99.9% of a carrier including a diluent and a solvent.

[0161] Compositions for oral administration can have various dosage forms. For example, solid forms include tablets, capsules, granules, and bulk powders. These oral dosage forms include a safe and effective amount, usually at least about 5%, and more particularly from about 25% to about 50% of actives. The oral dosage compositions include about 50% to about 95% of carriers, and more particularly, from about 50% to about 75%.

[0162] Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically include an active component, and a carrier comprising ingredients selected from diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, glidants, and combinations thereof. Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose. Specific binders include starch, gelatin, and sucrose. Specific disintegrants include alginic acid and croscarmellose. Specific lubricants include magnesium stearate, stearic acid, and talc. Specific colorants are the FD&C dyes, which can be added for appearance. Chewable tablets preferably contain sweeteners such as aspartame and saccharin, or flavors such as menthol, peppermint, fruit flavors, or a combination thereof.

[0163] Capsules (including implants, time release and sustained release formulations) typically include an active and a carrier including one or more diluents disclosed above in a capsule comprising gelatin. Granules typically comprise an active, and preferably glidants such as silicon dioxide to improve flow characteristics. Implants can be of the biodegradable or the non- biodegradable type.

[0164] The selection of ingredients in the carrier for oral compositions depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this disclosure. Solid compositions may be coated by conventional methods, typically with pH or timedependent coatings, such that the hydrogel-encapsulated glucagon / glucagon analogue is released in the gastrointestinal tract in the vicinity of the desired application, or at various points and times to extend the desired action. The coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, EUDRAGIT coatings (available from Rohm & Haas G. M.B.H, of Darmstadt, Germany), waxes and shellac.

[0165] Compositions for oral administration can have liquid forms. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non- effervescent granules, suspensions reconstituted from non-effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like. Liquid orally administered compositions typically include the hydrogel-encapsulated glucagon / glucagon analogue and a carrier, namely, a carrier selected from diluents, colorants, flavors, sweeteners, preservatives, solvents, suspending agents, and surfactants. Peroral liquid compositions preferably include one or more ingredients selected from colorants, flavors, and sweeteners.

[0166] Other compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms. Such compositions typically include one or more of soluble filler substances such as diluents including sucrose, sorbitol, and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose. Such compositions may further include lubricants, colorants, flavors, sweeteners, antioxidants, and glidants.

[0167] The disclosed compositions can be topically administered. Topical compositions that can be applied locally to the skin may be in any form including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-on and rinse-out hair conditioners, milks, cleansers, moisturizers, sprays, skin patches, and the like. Topical compositions include: a disclosed hydrogel and a carrier. The carrier of the topical composition preferably aids penetration of the hydrogels into the skin. The carrier may further include one or more optional components.

[0168] The amount of the carrier employed in conjunction with the hydrogel-encapsulated glucagon / glucagon analogue is sufficient to provide a practical quantity of composition for administration per unit dose of the medicament. Techniques and compositions for making dosage forms useful in the methods described herein are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nded., (1976). A carrier may include a single ingredient or a combination of two or more ingredients. In the topical compositions, the carrier includes a topical carrier. Suitable topical carriers include one or more ingredients selected from phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil, combinations thereof, and the like. More particularly, carriers for skin applications include propylene glycol, dimethyl isosorbide, and water, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols.

[0169] The carrier of a topical composition may further include one or more ingredients selected from emollients, propellants, solvents, humectants, thickeners, powders, fragrances, pigments, and preservatives, all of which are optional.

[0170] Suitable emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane-1 , 2-diol, butane-1 ,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate, and combinations thereof. Specific emollients for skin include stearyl alcohol and polydimethylsiloxane. The amount of emollient(s) in a skin-based topical composition is typically about 5% to about 95%.

[0171] Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof. The amount of propellant(s) in a topical composition is typically about 0% to about 95%.

[0172] Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinations thereof. Specific solvents include ethyl alcohol and homotopic alcohols. The amount of solvent(s) in a topical composition is typically about 0% to about 95%.

[0173] Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof. Specific humectants include glycerin. The amount of humectant(s) in a topical composition is typically 0% to 95%. The amount of thickener(s) in a topical composition is typically about 0% to about 95%. Suitable powders include beta-cyclodextrins, hydroxypropyl cyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium polyacrylate, tetra alkyl ammonium smectites, trialkyl aryl ammonium smectites, chemically-modified magnesium aluminum silicate, organically- modified Montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate, and combinations thereof. The amount of powder(s) in a topical composition is typically 0% to 95%. The amount of fragrance in a topical composition is typically about 0% to about 0.5%, particularly, about 0.001% to about 0.1 %. Suitable pH adjusting additives include HCI or NaOH in amounts sufficient to adjust the pH of a topical pharmaceutical composition.

[0174] Methods of Treatment

[0175] The disclosed compositions may be used to treat various disorders. Example disorders that may be treated using the therapeutic conjugates and hydrogels disclosed herein include diabetes (e.g., type 2 diabetes), hypertension, hypoglycemia, hyperlipidemia (dyslipidemia), atherosclerosis, coronary artery disease, cardiovascular disorders, abnormal blood clotting, obesity, diabetic complications, diabetic retinopathy, liver disease, hepatobiliary disease, fatty liver, alcoholic steatohepatitis, chronic kidney disease, insulin resistance and impaired glucose tolerance.

[0176] The disclosed compositions may be used to treat or ameliorate the symptoms of diabetes and / or induce weight loss in a subject. The method may comprise administering a pharmaceutical composition comprising a therapeutically effective amount of a therapeutic conjugate or a hydrogel comprising a therapeutic conjugate, as described herein, to a subject in need thereof. In various instances, the subject in need thereof may have an insulin disorder, such as diabetes. In various instances, the subject in need thereof may have a metabolic disorder, such as obesity. In various instances, the subject in need thereof may be at risk of experiencing a cardiovascular disease, stroke, blindness, infection, a non-healing wound, amputation, neuropathy.

[0177] In various instances, the therapeutically effective amount comprises 0.2-10 mg. In various instances, the therapeutically effective amount comprises 0.5-9.5 mg; 1-9 mg; 1.5-8.5 mg; 2-8 mg; 2.5-7.5 mg; 3-7 mg; 3.5-6.5 mg; 4-6 mg; or 4.5-5.5 mg. In various instances, the therapeutically amount comprises no greater than 10 mg; no greater than 9 mg; no greater than 8 mg; no greater than 7 mg; no greater than 6 mg; no greater than 5 mg; no greater than 4 mg; no greater than 3 mg; no greater than 2 mg; or no greater than 1 mg. In various instances, the therapeutically amount comprises no less than 0.2 mg; no less than 0.5 mg; no less than 1 mg; no less than 2 mg; no less than 3 mg; no less than 4 mg; no less than 5 mg; no less than 6 mg; no less than 7 mg; no less than 8 mg; or no less than 9 mg. In various instances, following administration of the pharmaceutical composition, the subject has blood glucose levels of about 60-200 mg / dL. In various instances, following administration of the pharmaceutical composition, the subject has blood glucose levels of about 70-190 mg / dL; 80-180 mg / dL; 90-170 mg / dL; 100-160 mg / dL; 110-150 mg / dL; or 120-140 mg / dL. In various instances, following administration of the pharmaceutical composition, the subject has blood glucose levels of no greater than 200 mg / dL; no greater than 190 mg / dL; no greater than 180 mg / dL; no greater than 170 mg / dL; no greater than 160 mg / dL; no greater than 150 mg / dL; no greater than 140 mg / dL; no greater than 130 mg / dL; no greater than 120 mg / dL; no greater than 110 mg / dL; no greater than 100 mg / dL; no greater than 90 mg / dL; no greater than 80 mg / dL; or no greater than 70 mg / dL. In various instances, following administration of the pharmaceutical composition, the subject has blood glucose levels of no less than 60 mg / dL; no less than 70 mg / dL; no less than 80 mg / dL; no less than 90 mg / dL; no less than 100 mg / dL; no less than 110 mg / dL; no less than 120 mg / dL; no less than 130 mg / dL; no less than 140 mg / dL; no less than 150 mg / dL; no less than 160 mg / dL; no less than 170 mg / dL; no less than 180 mg / dL; or no less than 190 mg / mL

[0178] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof. Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0179] Clause 1 . A therapeutic conjugate comprising: a peptide-based therapeutic moiety; a peptide amphiphile moiety; and a linker attaching the peptide-based therapeutic moiety to the peptide amphiphile moiety, wherein the peptide amphiphile moiety comprises: an alkyl moiety of formula: O attached to the N-terminus of a sequence of 3-10 amino acid residues; the sequence comprising: a hydrophobic subsequence of 2-6 hydrophobic amino acid residues, wherein the N-terminus of the hydrophobic subsequence is attached to the alkyl moiety; a hydrophilic subsequence of 1-4 hydrophilic amino residues, wherein the hydrophilic subsequence is attached to the linker.

[0180] Clause 2. The therapeutic conjugate of clause 1 , wherein the peptide-based therapeutic moiety is a natural or synthetic hormone moiety.

[0181] Clause 3. The therapeutic conjugate of clause 2, wherein the natural or synthetic hormone moiety is a GLP-1 , GIP, and / or glucagon receptor agonist moiety.

[0182] Clause 4. The therapeutic conjugate of clause 3, wherein the GLP-1 , GIP, and / or glucagon receptor agonist moiety is:

[0183] Clause 5. The therapeutic conjugate of any one of clauses 1—4, the linker comprising wherein n is 1-10.

[0184] Clause 6. The therapeutic conjugate of any one of clauses 1-5, wherein the peptide amphiphile moiety is a peptide moiety of formula (I): wherein:

[0185] Y1is the alkyl moiety of formula: is the hydrophilic subsequence.

[0186] Clause 7. The therapeutic conjugate of clause 6, wherein AA1, AA2, and AA3are each independently a glycine residue, an alanine residue, a valine residue, an isoleucine residue, or a leucine residue.

[0187] Clause 8. The therapeutic conjugate of clause 6 or 7, wherein AA4and AA5are each independently a glutamic acid residue, an aspartic acid residue, a lysine residue, or an arginine residue.

[0188] Clause 9. The therapeutic conjugate of any one of clauses 1-8, wherein the peptide amphiphile moiety is a peptide moiety of formula (l-a): wherein:

[0189] Clause 10. The therapeutic conjugate of clause 9, wherein R1, R2, and R3are each

[0190] Clause 11. The therapeutic conjugate of clause 9 or 10, wherein R4and R5are each

[0191] Clause 12. The therapeutic conjugate of any one of clauses 1-11 , wherein the peptide amphiphile moiety is:

[0192] Clause 13. A hydrogel comprising: the therapeutic conjugate of any one of clauses 1-12; and optionally, a diluent peptide amphiphile, the diluent peptide amphiphile comprising: an alkyl moiety of formula: O attached to the N-terminus of a sequence of 3-10 amino acid residues; the sequence comprising: a hydrophobic subsequence of 2-6 hydrophobic amino acid residues, wherein the N-terminus of the hydrophobic subsequence is attached to the alkyl moiety; a hydrophilic subsequence of 1-4 hydrophilic amino residues, wherein the N-terminus of the hydrophilic subsequence is attached to the C-terminus of the hydrophobic subsequence; and the C-terminus of the hydrophilic subsequence is

[0193] Clause 14. The hydrogel of clause 13, wherein: the therapeutic conjugate of clause 1 is present at about 0.1-10 wt%; and the diluent peptide amphiphile is present at about 0-10 wt%.

[0194] Clause 15. The hydrogel of clause 13 or 14, wherein the diluent peptide amphiphile is a diluent peptide amphiphile of formula (II):

[0195] Y1— AA1— AA2— AA3— AA4— AA5(H), wherein:

[0196] Y1is the alkyl moiety of formula: is the hydrophilic subsequence; and the C-terminus

[0197] Clause 16. The hydrogel of any one of clauses 13-15, wherein the diluent peptide amphiphile is a diluent peptide amphiphile of formula (I l-a):

[0198] Clause 17. A pharmaceutical composition comprising: the therapeutic conjugate of any one of clauses 1-12 or the hydrogel of any one of clauses 13-16; and a pharmaceutically acceptable excipient.

[0199] Clause 18. A method for treating or ameliorating the symptoms of diabetes or inducing weight loss, the method comprising administering a therapeutically effective amount of the therapeutic conjugate of any one of clauses 1-12, the hydrogel of any one of clauses 13- 16, or the pharmaceutical composition of clause 17 to a subject in need thereof.

[0200] Clause 19. A kit comprising the therapeutic conjugate of any one of clauses 1-12, the hydrogel of any one of clauses 13-16, or the pharmaceutical composition of clause 17; delivery or administration apparata or devices; and optionally, packaging, a label, or instructions for use.

[0201] Clause 20. Use of the therapeutic conjugate of any one of clauses 1-12, the hydrogel of any one of clauses 13-16, or the pharmaceutical composition of clause 17 for the preparation of a medicament for treating diabetes or inducing weight loss.

[0202] EXAMPLES

[0203] Example 1

[0204] Peptide Synthesis and Purification Peptides were synthesized with a Liberty Blue microwave peptide synthesizer using standard solid-phase Fmoc peptide synthesis procedures. Fmoc groups were deprotected in 20% (v / v) piperidine in A / ,A / -dimethylformamide (DMF) for 5 min and repeated 2 times. Diisopropylcarbodiimide (DIC) was used as the coupling reagent and reacted with Fmoc- protected amino acids in the presence of oxyma with a molar ratio of 1 :1 :1 (amino acid:DIC:oxyma). For PA-GLP1 (FIG. 9), the main GLP-1 receptor agonist segment was synthesized by the automated peptide synthesizer. The PA motif was subsequently conjugated by dendritic growth from the s-amine of a lysine residue using removal of a selectively labile Mtt protecting group following by standard manual coupling methods; an Fmoc-PEGs-COOH was added for the initial spacer, followed by the PA component. After completion of synthesis, the product was cleaved using a mixture of trifluoroacetic acid (TFA), triisopropanolsilane (TIS), and H2O (95:2.5:2.5, v / v / v) for 3 h at room temperature. The cleavage solution was collected, and the resin washed with dichloromethane (DCM). The TFA / DCM solution was concentrated under vacuum and the residual peptide solution was precipitated in cold diethyl ether, followed by centrifugation and washing with cold diethyl ether and drying under vacuum overnight. The crude peptide was dissolved in hexafluoro-2-propanol (HFIP) at a concentration of 100-150 mg / mL and purified using a preparative reversed phase bio-Cis cartridge (25 g) column with a linear gradient of water to acetonitrile, both containing 0.1 % NH4OH, at a flow rate of 40 mL / min on a Biotage® Isolera. Elution was monitored at 220 nm and 260 nm. The collected fractions were verified as pure by electrospray ionization mass spectrometry (ESI-MS, Advion) and high performance-liquid chromatography (HPLC) using a C Gemini (Phenomenex) column. Fractions with verified purity were combined and evaporated to remove most of the acetonitrile, and then lyophilized to yield a white powder. A diluent PA (dPA) of sequence (C16V3E2, FIG. 10) was synthesized by standard microwave-assisted methods. To synthesize a fluorescent methoxycoumarin (MCA)-PA-GLPI , MCA-lysine (Sigma) was inserted in place of the tryptophan residue at position 25 (FIG. 11). The Dan-PA-GLP1 was synthesized by coupling the Dansyl group on an additional lysine residue that was between PA and PEG groups (FIG. 12).

[0205] GLP-1 Receptor Activation Assay

[0206] A HEK293 / GLP1 R stable cell line (Eurofins DiscoverX, Fremont, CA) was cultured in selective media with 10% Fetal Bovine Serum, 100 pg / mL hygromycin B, 200 pg / mL G418, 1 pg / mL puromycin added to Dulbecco’s modified eagle’s medium with L-glutamine and 4.5 g / L glucose without sodium pyruvate. The day before an assay, cells were plated on 384-black / pclear tissue culture-treated plates at a density of 15,000 cells / well in 50 pL culture media with 2 pg / mL poly-D-lysine and left to incubate overnight at 37 °C and 5% CO2. The following day, media was removed from the wells and the cells were washed with 50 pL of Hank’s Buffered Saline Solution (HBSS) supplemented with 50 mM phosphate buffer. Then, 20 pL of dye-loading buffer containing FluoForte reagent (Enzo Life Sciences, Farmingdale, NY) was added to each well, and cells were incubated at 37 °C and 5% CO2 for 45 min. Agonist plates were prepared in HBSS with 50 mM phosphate buffer. Triton X-100 was used as a positive control in addition to a plate blank and n = 3 sample concentrations per column. The fluorescence (Ex. 485 nm, Em. 525 nm, Filter 515 nm) over time in each well was monitored on a FlexStation® 3 (Molecular Devices, San Jose, CA), quantifying calcium mobilization arising from receptor stimulation. Fluorescence was continuously read using flex mode, with liquid handling features introducing 20 pL of buffer at 19 seconds and 20 pL of agonist at 40 seconds and a full read time of 2 minutes per column. All curves have a minimum of three experimental replicates collected on different days, where each replicate is the average of at least 3 technical replicates per concentration. Raw data from the assay was adjusted according to the max fluorescence in each well, correcting for well-specific baseline, subtracting the plate blank wells containing only cells and loading dye solution, and then normalizing to the Triton X-100 average max value. Using GraphPad Prism, a non-linear fit of the resulting 4-parameter logistic curves was found using the “log(agonist) vs. response - Find ECanything” model. This EC50 represents the concentration of agonist needed to reach halfmaximum receptor stimulation. EC50 error is expressed as plus or minus one-half of the symmetrical 95% confidence interval range. The EC50 of PA-GLP1 was compared to that for a commercially sourced semaglutide standard (Adipogen). The actual peptide content of PA-GLP1 was measured using a tryptophan standard curve (E = 5307 M-1cm-1) and calculated as 78%; this value was used to obtain accurate known concentrations for experimental samples.

[0207] Preparation of PA-GLP1 / dPA Hydrogels

[0208] The individual components of PA-GLP1 and dPA were fully dissolved in a mixture of 1 :1 (v / v) water and acetonitrile to achieve a “molecularly mixed” initial state. A series of mixed PA- GLP1 / dPA formulations with various mass ratio of PA-GLP1 were prepared and followed by lyophilization and rehydration in HEPES buffer (20 mM HEPES, 150 mM NaCI, 15 mM CaCh) to form self-supporting hydrogels.

[0209] Circular Dichroism Spectroscopy

[0210] Circular Dichroism (CD) spectroscopy was performed on a Jasco 710 CD spectrometer. Samples were prepared in 20 mM HEPES, pH 7.4, at a concentration of 0.2% w / v. The CD spectra were collected from 250 nm to 190 nm at room temperature (RT) using a 0.1 mm cuvette, a bandwidth at 0.1 nm, scan rate at 50 nm / min and a response time of 2 s. Each spectrum was averaged from three scans. A background of 20 mM HEPES buffer was subtracted from all samples. Data in mDeg was converted to mean residue ellipticity (MRE) via the formula 0= (1000 x mDeg) / (c n / ) where (c) is the concentration of the peptide solution expressed in mM, (n) is the number of amino acids in the peptide sequence, and ( / ) is the path length of the cell used in the unit of millimeter.

[0211] Transmission Electron Microscopy (TEM).

[0212] Samples were prepared in 20 mM HEPES, pH 7.4 at a concentration of 0.05% w / v. 10 pL of peptide solution was pipetted onto a lacey carbon grid (Ted Pella 01824). After 2 min, excess solution was carefully removed by wicking with filter paper. 10 pLof 2 wt% uranyl acetate aqueous solution was pipetted onto the grid for negative staining. After 2 min, excess staining solution was removed by wicking with filter paper and grids were dried overnight before imaging on a Thermo Scientific™ Tales F200i (S)TEM 20-200 kV field emission (scanning) TEM.

[0213] Rheological Characterization

[0214] The rheological properties of PA-GLP1 / dPA hydrogels were evaluated with a TA Instruments Discovery HR-2 rheometer fitted with a Peltier stage using a parallel plate geometry with a diameter of 25 mm. PA-GLP1 hydrogels at 2% (w / v) were prepared as described above prior to measurements. A strain amplitude sweep was performed to determine the linear viscoelastic range for each PA-GLP1 hydrogel formulation, and then a frequency sweep was performed at constant strain of 0.5%. Subsequently, a time-sweep (0.5% strain, 1 rad / s angular frequency) was performed to measure and compare the storage modulus (G'), loss modulus (G"), and complex viscosity (q*) of PA-GLP1 hydrogels over time. A step-strain cycling study alternating between 0.5% strain for 150 s and 100% strain for 30 s at angular frequency of 1 rad / s was performed for 15% PA-GLP1 hydrogel. The shear-thinning property of 15% PA-GLP1 hydrogel was evaluated under steady shear flow over a shear rate range of 0.0001 to 80 s-1.

[0215] In vitro PA-GLP1 Release

[0216] To evaluate PA-GLP1 release, PA-GLP1 / dPA hydrogels were prepared at a concentration of 2% (w / v). These samples used a variant of PA-GLP1 modified with a fluorescent lysine-linked 7-methoxycoumarin-4-acetic acid (MCA) group inserted in place of tryptophan at position 25 of the semaglutide-derived segment. Hydrogels of PA-GLP1 / dPA mixed at various mass ratios at a total volume of 50 pL were incubated within 12-well plates in 2 mL of 20 mM HEPES, pH 7.4, 150 mM NaCI). At each time point, a 200 pL aliquot was taken for fluorescence analysis (Ex: 324 nm, Em: 380 nm) to determine released PA-GLP1 concentration through aid of a standard curve. With each sampling, half the volume of the buffer was taken and replaced with an equal volume of fresh HEPES buffer to create a bulk dilution condition. The release profile of physically mixed MCA-labeled Semaglutide in dPA hydrogels were evaluated by these same methods to assess the impact of supramolecular incorporation of PA-GLP1 on release.

[0217] In vivo Evaluation

[0218] Male Sprague Dawley rats (Charles River) were used for experiments. Animal studies were performed in accordance with guidelines for the care and use of laboratory animals and protocols were approved by the University of Notre Dame Institutional Animal Care and Use Committee (IACUC). The type 2 diabetes rat models were established by following an established protocol. Masiello et al., Diabetes 47(2): 224-229 (1998). Briefly, male Sprague Dawley rats (280- 380 g) were weighed and fasted for 8 h before injection of nicotinamide (NA) and streptozotocin (STZ). Each rat was sequentially injected intraperitoneally (i.p.) with 200 mg / kg NA and then 65 mg / kg STZ 15 min after NA administration. After STZ injection, rats were provided with water containing 10% sucrose for 24 h. Blood glucose levels were monitored for a week and rats having three consecutive fasting blood glucose levels of 130-200 mg / dL were selected for the following in vivo study. Selected rats were randomized into three groups and treated with single subcutaneous (s.c.) injection of HEPES buffer; a single s.c. injection of PA-GLP1 / dPA hydrogel (PA-GLP1 : 2 mg); and daily s.c. injection of semaglutide (20 pg). For each of the treatment groups (n = 8-9), blood was collected from the tail vein three times each week and blood glucose levels were measured by a handheld blood glucose monitor over the course of the study. Once blood glucose was measured, the rat weights were measured, and blood samples were collected from the tail vein to measure serum semaglutide concentrations using a semaglutide ELISA kit. The standard curve for this ELISA kit was chosen to reflect its different sensitivity for semaglutide, PA- GLP1 in the two treated groups, as well as the presence of native rat GLP-1 in the control.

[0219] Design of PA-GLP1

[0220] The chemical structure of authentic semaglutide consists of a 31 amino acid peptide segment resembling the structure of native GLP-1 , with substitutions of an a-aminobutyric acid (Aib) residue at position 2 and an arginine at position 28; lysine at position 20 is furthermore modified from its s-amine with two consecutive PEG2 spacers and finally a Cis diacid (FIG. 8; SEQ ID NO: 2). Inspired by the structure of this remarkably successful therapeutic, the design for PA- GLP1 conserved the 31 amino acid active segment of semaglutide while modifying the lysine at position 20 with a self-assembling peptide amphiphile (PA) motif linked to the active signaling component via a PEGs linker (FIG. 9). The PA-GLP1 sequence was synthesized according to standard solid-phase methodology, first preparing the active semaglutide-derived segment terminated with a Boc-protected histidine and then synthesizing the PA module linked via a PEG spacer to a selectively deprotected e-amine of a lysine (Mtt) inserted at position 20.

[0221] The PA motif consisted of the V3E2peptide sequence terminated with a Ci6 alkyl chain (FIG. 2, FIG. 9). The presence of three valine (V) residues adjacent to the alkyl segment is known to promote stable [3-sheet-like hydrogen bonding to drive one-dimensional assembly of PA molecules into high aspect-ratio nanofibers. Meanwhile, the two glutamic acid (E) residues were intended to promote directional organization of the PA module in water, and furthermore enable control over self-assembly and hydrogelation of PA designs by controlling the charge state of the carboxylate side chains through pH or introduction of divalent cations (e.g., Ca2+). The C16-V3E2 PA sequence used for the PA module here has been used to encapsulate and functionally deliver an anti-hypertensive therapeutic within hydrogels of supramolecular PA nanofibers, exhibiting good hemocompatibility and cytocompatibility. To aid in release study quantification, an alternate variant placed a fluorescent MCA group at position 25 in place of tryptophan (FIG. 11).

[0222] Considering the possibility of steric hindrance from its bulky GLP-1 receptor agonist motif that may limit the ability of PA-GLP1 to self-assemble into nanofibers on its own, a diluent PA (dPA) was also synthesized consisting of the C16-V3E2 PA sequence (FIG. 2, FIG. 10). In conserving the PA motif between dPA and PA-GLP1 , the desire was to take advantage of the typical thermodynamic preference for self-interactions in self-assembly motifs to promote coassembly by limiting the possibility of kinetically trapped or segregated nanostructures.

[0223] PA-GLP1 Receptor Signaling

[0224] To ensure this new sequence maintained its function, the ability of PA-GLP1 to activate the GLP-1 receptor was compared to authentic semaglutide in a reporter cell-based assay measuring intracellular calcium flux in response to receptor signaling (FIG. 3). On the basis of the half-maximal effective concentration (EC50), PA-GLP1 (52.4 ± 17.9 nM) showed a reduction in potency compared to authentic semaglutide (8.8 ± 7.2 nM). Given a higher extent of modifications to append the PA component in the PA-GLP1 design, some reduction in receptor binding may be expected due to steric effects. Moreover, as the PA component is designed explicitly to associate and self-assemble, it is possible that some association or aggregation of PA-GLP1 in the assay media limits the effective functional concentration of the active ligand available for receptor binding. Accordingly, PA-GLP1 retains the ability to activate the GLP-1 receptor, though does exhibit some reduction in its ultimate and / or effective potency.

[0225] PA-GLP1 Self-Assembly

[0226] It was hypothesized that the self-assembly of PA-GLP1 could enable ultra-long lasting therapeutic function through the formation of injectable hydrogel depots. As such, PA-GLP1 was first assessed on its own by gross inspection using vial inversion for its ability to form a self- supporting hydrogel from prepared solutions of various concentrations ranging from 1-6 mg / mL in a buffer consisting of 20 mM HEPES, 150 mM NaCI, and 15 mM CaCh (FIG. 13). PA-GLP1 formed cloudy suspensions at each tested concentration, suggestive of the formation of aggregates, yet was unable to form hydrogels that would be indicative of physically entangled nanostructures. To facilitate gelation, the dPA sequence was next mixed with PA-GLP1. The intention of mixing these two PA molecules was to de-frustrate the molecular-scale packing of PA- GLP1 arising from its bulky GLP-1 receptor agonist domain and thereby promote increased [3- sheet cohesion. Solutions of the two peptides were prepared at 4% w / v in DI water and pH was adjusted to neutral. After mixing the two peptide solutions in DI water at their desired component mass ratios, the mixtures were further diluted 1:1 with a 2* HEPES buffer (40 mM HEPES, 300 mM NaCI, 30 mM CaCh) to yield mixed samples of the two PAs at 2% (w / v) in 1* HEPES buffer. Samples prepared with 5%, 10%, 15%, 20%, and 40% of PA-GLP1 by weight, with a balance of dPA, formed self-supporting hydrogels (FIG. 4A). Hydrogels tended to flow slowly upon vial inversion as the fraction of PA-GLP1 was increased. Accordingly, PA-GLP1 both alone and at high proportion in mixtures with dPA appears to generally frustrate self-assembly and inhibit gelation.

[0227] To further quantify the properties of PA-GLP1 / dPA hydrogels that were initially evident in inspection by vial inversion, oscillatory rheology was next performed. The PA-GLP1 / dPA hydrogel mixtures were freshly prepared with 5%, 10%, 15%, 20%, and 40% of PA-GLP1 by weight in dPA at a final concentration of 2% (w / v) in 1 * HEPES buffer, as described above, and placed onto the rheometer stage. A frequency sweep of all hydrogels performed within the linear viscoelastic region demonstrated hydrogelation (FIG. 4B), with the storage modulus (G') in excess of the loss modulus (G") for the full range of frequencies evaluated. Limited frequency-dependent behavior and lack of an observable G'-G" crossover supports viscoelastic supramolecular hydrogels with very slow dynamics of physical crosslinking. When compared on the basis of their high-frequency G' value (FIG. 4C), dPA alone formed very stiff hydrogels (29,682 Pa); stiffness decreased substantially upon incorporation of even 5% PA-GLP1 in the mixture (766 Pa) and continued to decrease as the fraction of PA-GLP1 was increased up to 40% (55 Pa). The complex viscosity (q*) offers a measure of the total resistance to flow as a function of angular frequency. This value demonstrated similar trends, as dPA alone had a q* of 30,596 Pa s, decreasing to 782 Pa s for the 5% PA-GLP1 mixture and 57 Pa s for the 40% mixture. Accordingly, inclusion of even a small fraction of PA-GLP1 has a dramatic impact on the rheological properties of the resulting mixtures compared to dPA alone; this finding indirectly supports an interaction between the two PA molecules and seemingly suggests PA-GLP1 frustrates the otherwise efficient assembly and hydrogelation of dPA.

[0228] Gross inspection of PA-GLP1 / dPA mixtures and further rheological characterization supported hydrogelation in the mixtures that was not achieved for PA-GLP1 alone. However, molecular scale mixing and co-assembly was inconclusive from these studies. To probe molecular-scale interactions in these mixtures, a Dansyl (Dan) conjugated PA-GLP1 was synthesized (FIG. 12). The fluorescence intensity was measured and compared for mixtures of Dan-labeled PA-GLP1 and dPA at 5%, 10%, 15%, 20%, and 40% mass ratio, as well as the Dan- labeled PA-GLP1 alone at the same total concentrations as it was found in each of the mixtures (FIG. 14). The mixed samples exhibited higher fluorescence emission intensity compared to the Dan-labeled PA-GLP1 at all concentrations. As Dan molecules self-quench when in close proximity, this result suggests increased spacing of the Dan fluorophore upon mixture with dPA. While not a direct indication of a homogenous co-assembly, this result nevertheless points to molecular-scale dilution of PA-GLP1 within dPA, evident by a reduction of self-interactions of the Dan dye and supports a lack of complete self-sorting of the peptides in these mixtures (FIG. 14). These studies therefore corroborate evidence from rheology in support of an interaction between PA-GLP1 and dPA when the two PA molecules are mixed.

[0229] With a goal of eventual therapeutic application, PA-GLP1 / dPA hydrogels should be shearthinning to facilitate syringe-based injection and demonstrate recovery of their properties following cessation of high strain associated with such a process. The 2% (w / v) hydrogel prepared from a mixture of 15% PA-GLP1 / dPA was thus studied as an exemplar of these properties. Under conditions of continuously increasing shear rates, the viscosity of the hydrogel was dramatically reduced by over three orders of magnitude from “zero-shear” viscosity of -3490 Pa-s to <1 Pa s at 8.8 s’1(FIG. 4D). To assess the ability of the network to recover from high strain, a step-strain experiment was also performed by cycling between 0.5% and 100% strain at a constant frequency of 1 rad / s (FIG. 4E). The hydrogel structure was disrupted at high strain (G" > G'), and gel character was restored (G' > G") immediately after cessation of high strain, an effect repeated over multiple cycles. A gradual reduction in the low-strain G' was observed with each cycle, a common observation for hydrogels prepared from physically entangled one-dimensional peptide assemblies that likely arises from a reduction in nanofiber length and the corresponding extent of physical crosslinking possible from these shorter fibers.

[0230] Semaglutide and other GLP-1 receptor agonists have an a-helical secondary structure, while PA molecules typically adopt a p-sheet-like structure. As such, the impact of conjugating a PA motif to a GLP-1 receptor agonist on the secondary structure was of interest to study. Authentic semaglutide, dPA, PA-GLP1 , and PA-GLP1 / dPA mixtures were thus evaluated by circular dichroism spectroscopy (CD). From its CD spectrum (FIG. 5A), semaglutide showed an expected a-helical secondary structure, with two negative peaks at 207 nm and 223 nm. Meanwhile, dPA had a typical p-sheet secondary structure, with a pronounced negative peak at 220 nm and a corresponding positive peak at 203 nm. PA-GLP1 alone also showed a signal indicative of p- sheet secondary structure, though it had a wider negative peak at 220 nm. It has been reported that self-assembling peptide motifs can alter the secondary structures of conjugated proteins due to both strong hydrogen bonding and hydrophobic ordering of these motifs. As such, the p-sheet secondary structure arising from intermolecular associations in the PA motif may alter the ability of the conjugated GLP-1 receptor agonist to form its preferred a-helix. An altered secondary structure could also contribute to the reduced potency of receptor activation for PA-GLP1 compared to authentic semaglutide. Samples of PA-GLP1 / dPA mixtures all showed more typical P-sheet secondary structures than PA-GLP1 alone, with negative peaks at 220 nm (FIG. 5B). This is to be expected due to dPA being the majority species, especially when considered in terms of its molar ratio. For PA-GLP1 / dPA mixtures, the negative peaks at 220 nm widened as the fraction of PA-GLP1 was increased, while the positive peaks at 203 nm were reduced. These features suggest that p-sheets become less ordered and / or less abundant as the amount of PA- GLP1 is increased, possibly as a result of more frustrated molecular packing due to steric crowding of the large GLP-1 receptor agonist. Less abundant and / or ordered p-sheets could also contribute to the reduced rheological properties observed for PA-GLP1 / dPA mixtures as the fraction of PA-GLP1 was increased.

[0231] The nanostructures of different PA-GLP1 / dPA mixtures were next characterized by transmission electron microscopy (TEM). Both dPA alone, PA-GLP1 alone, and all PA-GLP1 / dPA mixtures formed filamentous nanofibers (FIG. 5C). As PA-GLP1 composition increased, the nanofibers became wider with a flatter appearance, while the apparent nanofiber length and density of fibers observable on the grid decreased. This result is presumably due to more frustrated packing of the bulky PA-GLP1 , which has an increasing impact on the resulting nanostructure as its composition is increased. The reduction in nanofiber length observed for PA- GLP1 / dPA mixtures as the PA-GLP1 component increased in relative proportion to dPA should strongly contribute to the reduction in rheological properties of the resultant hydrogels, as longer and denser nanofibers would be expected to form a higher extent of physical crosslinks and entanglements supporting gelation and leading to a higher storage modulus than would be possible from networks of shorter and less entangled nanofibers.

[0232] PA-GLP1 Release from Hydrogels

[0233] Strategies for the sustained release of GLP-1 receptor agonists may offer more convenient dosing regimens and reduce the extent of side-effects associated with therapy, as demonstrated by clinical use of polymeric controlled release formulations. To probe PA-GLP1 / dPA hydrogels for their ability to function as depot materials for long-term drug release, both semaglutide and PA- GLP1 were synthesized and modified with MCA. This small fluorescent molecule enabled the release profile of the active component of the hydrogel formulations to be monitored. The release profile of PA-GLP1 from hydrogel formulations of this molecule mixed with dPA was first assessed at varying PA-GLP1 content, expressed in terms of the weight fraction of PA-GLP1 (FIG. 6). The cumulative release of PA-GLP1 from these formulations was enhanced as PA-GLP1 content was increased. Approximately 70% of PA-GLP1 was released rapidly from 40% PA-GLP1 / dPA hydrogels after only 6 d, with continual gradual release ultimately resulting in 83% of the agent released from the hydrogels by 40 d, adhering to first-order release kinetics. As the mass fraction of PA-GLP1 was reduced to 20% by weight in mixture with dPA, first-order kinetics were maintained with a lower rate and extent of both initial and overall release, ultimately leading to about 69% of PA-GLP1 released over 40 d. Further reduction of the PA-GLP1 content to 15% resulted in minimal initial burst release and the appearance of zero-order release over the majority of the 40 d period assessed, culminating in about 40% of drug released by 40 d. Even greater reduction to 10% and 5% PA-GLP1 resulted in very slow, effectively zero-order, drug release that was comparable for both materials, with approximately 20% and 17% of drug released over the duration of the study for the 10% and 5% PA-GLP1 hydrogels, respectively. Thus, as the proportion of PA-GLP1 was reduced in the formulations, its release was slowed; moreover an initial phase of burst release was reduced or essentially eliminated at lower PA-GLP1 content, indicating more stable inclusion in the material when PA-GLP1 was present at a lower relative content to dPA in the 2% w / v hydrogels.

[0234] To verify that supramolecular peptide incorporation contributed to the controlled release of PA-GLP1 , an MCA-labeled semaglutide variant was combined with dPA hydrogels by following the same mixing approach and the same mass fraction as was used to prepare PA-GLP1 / dPA materials. These physical mixtures were then assessed for release (FIG. 15), with an exemplary release profile for the 15% physical mixture shown alongside PA-GLP1 / dPA release data (FIG. 6). Whereas PA-GLP1 / dPA formulations offered sustained release for the duration of a 40 d study, release from physical mixtures at all semaglutide levels showed very rapid release, reaching nearcomplete release in a matter of days. Interestingly, the presence of semaglutide impacted the gelation capacity of dPA hydrogels (FIG. 16). While formulations with up to 20% semaglutide were able to form self-supporting hydrogels, the formulations with 40% semaglutide formed viscous flowing suspension. As the remaining dPA in this 40% formulation is still present at 1.2% (w / v), well above its critical gelation concentration, this result indicates some interaction between semaglutide and dPA that disrupts its otherwise efficient self-assembly and hydrogelation. It is thus likely that the dicarboxylic fatty acid side chain of semaglutide, included to facilitate hydrophobic associations with drug-binding pockets on circulating serum albumin, is able to interact with dPA assemblies and disrupt its ability to self-assemble into high aspect-ratio nanofibers and hydrogels. However, given the very rapid release of free semaglutide in this physical mixture relative to the slow, controlled release of PA-GLP1 , such interactions appear much less stable, likely due to a lack of the cohesive p-sheet interactions arising from the supramolecular peptide module of PA-GLP1.

[0235] Functional Efficacy of PA-GLP1 Hydrogels. Long-lasting release of PA-GLP1 from hydrogels in vitro may support multi-month therapeutic delivery and, in so doing, reduce the dosing frequency and promote improved adherence. To further evaluate therapeutic use of PA- GLP1 / dPA hydrogels, a model of insulin-resistant diabetes was created in Sprague Dawley rats (FIG. 7A) by administering a combination of nicotinamide (NA) and streptozotocin (STZ). STZ is a well-known chemical agent with cytotoxic effects on pancreatic [3-cells, while NA offers partial protection to these same cells to modulate the toxic action of STZ. The sequential administration of NA and STZ thus leads to impaired |3-cell function and compromised insulin signaling, mimicking a Type 2 diabetic phenotype. Rats were monitored daily for a week and were included in the study upon three consecutive fasting blood glucose readings in the range of 130-200 mg / dL.

[0236] In order to evaluate function, three different treatments were applied (n = 8-9 / group): 1) daily injection of 20 pg semaglutide, 2) a single injection of the 15% PA-GLP1 / dPA hydrogel of 2% (w / v) at a total dose of 2 mg PA-GLP1 in the formulation, and 3) a single injection of HEPES buffer as an untreated control. While semaglutide can be dosed once-weekly in humans, daily dosing is required in rats due to a much shorter circulation half-life, on the order of 8 h; 20 pg per day in rats was predicted to replicate the current once-weekly clinical treatment regimen in humans. With an in vitro release profile of ~1 % / day, the 15% PA-GLP1 / dPA hydrogel formulation was selected in order to recreate a scenario of the agent dosed clinically every 3 months. Other formulations released either too fast (e.g., 40% PA-GLP1) or too slow (e.g., 5% PA-GLP1) to achieve this desired dosing schedule, at least in vitro. The evaluation protocol (FIG. 7A) consisted of fasting blood glucose readings three times per week using a handheld glucose meter and blood collection via a venous draw to obtain serum. From serum samples, a commercial semaglutide ELISA kit was used to measure serum concentrations with the aid of standard curves for semaglutide, PA-GLP1 , or rat GLP-1. Individual standard curves were necessary to account for the reduced sensitivity of the kits for PA-GLP1 , perhaps due to the PA module, alteration in the secondary structure of the appended GLP-1 receptor agonist module, or molecular assembly reducing the effective concentration available for antibody recognition. Meanwhile, a standard curve of recombinant rat GLP-1 was used in the untreated control to account for any background in the assay arising from the presence of native GLP-1 in rats.

[0237] Serum concentrations (FIG. 7B) were assessed to quantify the bioavailable therapy following dosing by the different protocols described. With daily dosing of 20 pg semaglutide, a gradual increase in serum concentration was observed with continued dosing; approximately 290 ng / mL was measured after 1 d and this level generally increased to reach a value of approximately 560 ng / mL by the end of study at 40 d. These values were of the same order, though slightly higher, than those in a prior report that also evaluated daily dosing with 20 pg semaglutide in this same rat model. Comparatively, the profile for treatment with the PA-GLP1 / dPA hydrogel showed a maximum concentration of approximately 391 .5 ng / mL after 1 d, likely attributable to the same initial burst release following injection, similar to that observed during in vitro release studies. However, following this initial burst phase, a continuous and steady-state serum concentration persisted for the remainder of the study, leading to an average value of approximately 61 ng / mL from 8 d to the 40 d endpoint. Though lower than the concentration arising from daily semaglutide administration, it was still within the range of serum levels predicted to be therapeutically relevant in this rat model. Future work is needed to establish exact dosing levels of PA-GLP1 / dPA depots so as to precisely tune its steady-state serum concentration. The baseline level for rat GLP-1 in the untreated control was effectively stable over the 40 d study, with average value of approximately 7 ng / mL. Accordingly, this control data supports a conclusion that the majority of detectable serum GLP-1 levels in the two treated groups arises from administered compounds present in excess of baseline levels of native GLP-1.

[0238] Serum quantification showed stable concentrations for at least 40 d following a single injection of the 15% PA-GLP1 / dPA hydrogel depot formulation, remaining at a steady level until the endpoint of the study when protocol constraints limited further monitoring. However, a 40 d timeline of stable serum availability was consistent with results shown recently for a polymeric hydrogel used for controlled release of semaglutide over a 42 d study; pharmacokinetic modeling in this work suggested such a profile may translate to stable levels for at least 120 d in humans. Accordingly, the approach using a supramolecular peptide-based PA-GLP1 / dPA injectable depot could have similar relevance for long-lasting therapeutic use in humans on the basis of the results here.

[0239] The ability of 15% PA-GLP1 / dPA hydrogels to exert a therapeutic effect was also monitored through regular blood glucose measurements and monitoring of rat weight. Blood glucose levels tracked relative to their starting value for each rat were used to assess the progression of an insulin-resistant disease phenotype (FIG. 7C). Though some day-to-day fluctuation was observed by this measure, the untreated control generally showed an average increase of approximately 8% over the period of 20-40 d of the study. Meanwhile, daily treatment with semaglutide resulted in an average blood glucose level that was reduced by approximately 14% over this same period. A single injection of the PA-GLP1 / dPA hydrogel formulation compared favorably, showing a reduction in blood glucose of approximately 11% over the period of 20-40 d after treatment. Accordingly, treatment with both daily semaglutide and a single dose of the PA- GLP1 / dPA hydrogel accounted for an average reduction in blood glucose of approximately 19- 22% relative to an untreated diabetic control. Though the progression in hyperglycemia for the present study was more pronounced in the untreated control, results for daily semaglutide treatment were comparable to those observed by others for this same model and dosing scheme. A single dose of the PA-GLP1 / dPA hydrogel formulation also compared favorably in its function to certain of the controlled release formulations reported in this prior work.

[0240] As another phenotypic indicator of therapeutic function, rat weights were also monitored throughout the study (FIG. 7D). The untreated control rats had an increase in body weight of approximately 50% over the course of the 40 d study. Meanwhile, daily dosing of semaglutide resulted in a body weight gain of only 20% over this same period, including an initial 9% weight loss at day 3 during the initiation phase of treatment. Following initial weight loss, rats treated with daily semaglutide began to gain weight at a more gradual rate than controls. Treatment with a single dose of the PA-GLP-1 / dPA hydrogel resulted in an intermediate level of weight gain, increasing by approximately 34% over the duration of study. This treatment also included a small reduction in weight, in this case about 1%, at day 3 after the single treatment was administered. Contextualizing these data is challenging given that weight gain in excess of that caused by typical growth is a hallmark of this specific rat model, though rats of this age will continue to grow even when fully healthy. Moreover, GLP-1 receptor agonists function in part by controlling food intake, yet nausea and inappetence are also known side-effects of treatment with GLP-1 receptor agonists that are also manifest in animal models. This known effect may contribute in part to the reduced weight gain with daily semaglutide treatment, especially given the initial reduction in rat weight upon initiation of treatment. A single treatment with PA-GLP1 / dPA hydrogels, meanwhile, showed less initial weight loss and more steady weight gain at a rate still below that of the untreated diabetic control. Indeed, a reduction in weight gain of 20% for treatment with PA- GLP1 / dPA hydrogels aligns well with that reported for controlled release of semaglutide from polymeric materials applied in this same animal model. Moreover, clinical evidence supports a reduction in side-effects of nausea and vomiting with use of controlled release formulations, and such an effect may underlie the results seen for the PA-GLP1 / dPA hydrogels evaluated here due to their steady serum profile over the duration of the study.

[0241] Extension to Emerging Peptide Therapeutics

[0242] The modular design of PA materials allows the approach used for PA-GLP1 to be applied to other peptide therapeutics. One such peptide of specific relevance in the context of diabetes, metabolic disease, and obesity is the clinically approved once-weekly dual agonist therapy, tirzepatide (Mounjaro®), that functions by signaling receptors for both GLP-1 and gastric inhibitory polypeptide (GIP). Like GLP-1 , GIP is also a natural incretin protein that supports insulin secretion and regulates gastric function. Tirzepatide has been shown to improve blood glucose control as a treatment for type 2 diabetes, while promoting weight loss when used once weekly to treat obesity. Indeed, tirzepatide has shown improvement relative to semaglutide in clinical comparisons. Similar to semaglutide, tirzepatide contains a pendant C20 fatty diacid to facilitate albumin binding and prolonged circulation. As such, this site was explored for modification with the same PA module as that used for PA-GLP1 to create PA-Tir (FIG. 19BA). Like for PA-GLP1 , mixing PA-Tir with dPA up to 20% by weight enabled formulation of self-supporting hydrogels at 2% (w / v) total peptide (FIG. 19C). While PA-Tir alone had an a-helical secondary structure, its mixture with dPA resulted in a formation of p-sheets (FIG. 19D). As further demonstration of the modularity of this approach, the integration of a next-generation receptor agonist peptide, retatrutide (LY3437943), was also explored. In addition to activating receptors for GLP-1 and GIP, retatrutide is also designed to activate the glucagon receptor for triple agonist function, with data from clinical trials pointing to efficacy in treating diabetes and obesity. Similar to semaglutide and tirzepatide, retatrutide is modified with a pendant C20 fatty diacid group to extend circulation. This same site was modified with the PA module to prepare PA-Reta, which also formed hydrogels upon mixture with dPA (FIG. 20). As such, this modular and generalizable approach can incorporate emerging therapeutic peptides for their long-lasting delivery, as exemplified here using three different receptor agonist peptide therapeutics that treat diabetes, obesity, and metabolic disorder.

[0243] The emergence of peptide therapeutics to treat diabetes, obesity, and metabolic disorders has transformed the standard of care in recent years. Yet, poor adherence to therapy, arising from side-effects or failure to conform to a once-weekly dosing schedule, remains an issue limiting the impact of these approaches for many. Controlled release strategies offer a route to both extend the time between injections and have shown promising clinical precedent to reduce the side-effects of these therapies. State of the art GLP-1 receptor agonists are presently modified with prosthetic groups to extend their circulation half-life through albumin binding. The present study sought to explore alternative modification at these same sites with a peptide amphiphile (PA) module to endow the therapeutic with the capacity for supramolecular assembly into nanofibrillar hydrogels. In so-doing, this approach allows the therapeutic to “self-depot” upon injection of hydrogel formulations prepared from the peptide itself, contrasting with other approaches to achieve controlled release of GLP-1 receptor agonists through encapsulation in polymers. In order to facilitate efficient self-assembly and hydrogelation, a diluent PA was added to the PA-GLP1 to reduce the steric barrier to self-assembly. The resultant materials formed hydrogels incorporating a tunable amount of PA-GLP1 , with the release rate of the PA-GLP1 component directly related to its relative amount in the hydrogels. When these formulations were evaluated in a rat model of type 2 diabetes, stable serum levels persisted for at least 40 d after a single injection, whereas semaglutide requires daily injection for stable serum presence. In terms of phenotypic indicators of function, a single injection of PA-GLP1 demonstrated promise in reducing blood glucose levels and reducing the rate of weight gain compared to an untreated diabetic rat. The modularity of this approach was furthermore amenable to integration of nextgeneration agonist therapeutics.

Claims

CLAIMSWhat is claimed:

1. A therapeutic conjugate comprising: a peptide-based therapeutic moiety; a peptide amphiphile moiety; and a linker attaching the peptide-based therapeutic moiety to the peptide amphiphile moiety, wherein the peptide amphiphile moiety comprises:an alkyl moiety of formula: O attached to the N-terminus of a sequence of 3-10 amino acid residues; the sequence comprising: a hydrophobic subsequence of 2-6 hydrophobic amino acid residues, wherein the N-terminus of the hydrophobic subsequence is attached to the alkyl moiety; a hydrophilic subsequence of 1-4 hydrophilic amino residues, wherein the hydrophilic subsequence is attached to the linker.

2. The therapeutic conjugate of claim 1 , wherein the peptide-based therapeutic moiety is a natural or synthetic hormone moiety.

3. The therapeutic conjugate of claim 2, wherein the natural or synthetic hormone moiety is a GLP-1 , GIP, and / or glucagon receptor agonist moiety.

4. The therapeutic conjugate of claim 3, wherein the GLP-1 , GIP, and / or glucagon receptor agonist moiety is:

5. The therapeutic conjugate of claim 1 , the linker comprisingwherein n is 1-10.

6. The therapeutic conjugate of claim 1, wherein the peptide amphiphile moiety is a peptide moiety of formula (I):wherein:Y1is the alkyl moiety of formula:is the hydrophilic subsequence.

7. The therapeutic conjugate of claim 6, wherein AA1, AA2, and AA3are each independently a glycine residue, an alanine residue, a valine residue, an isoleucine residue, or a leucine residue.

8. The therapeutic conjugate of claim 6, wherein AA4and AA5are each independently a glutamic acid residue, an aspartic acid residue, a lysine residue, or an arginine residue.

9. The therapeutic conjugate of claim 1, wherein the peptide amphiphile moiety is a peptide moiety of formula (l-a):The therapeutic conjugate of claim 9, wherein R1, R2, and R3are each11. The therapeutic conjugate of claim 9, wherein R4and R5are each12. The therapeutic conjugate of claim 1 , wherein the peptide amphiphile moiety is:

13. A hydrogel comprising: the therapeutic conjugate of claim 1 ; and optionally, a diluent peptide amphiphile, the diluent peptide amphiphile comprising:an alkyl moiety of formula: O attached to the N-terminus of a sequence of 3-10 amino acid residues; the sequence comprising: a hydrophobic subsequence of 2-6 hydrophobic amino acid residues, wherein the N-terminus of the hydrophobic subsequence is attached to the alkyl moiety; a hydrophilic subsequence of 1-4 hydrophilic amino residues, wherein the N-terminus of the hydrophilic subsequence is attached to the C-terminus of the hydrophobic subsequence; and the C-terminus of the hydrophilic subsequence is14. The hydrogel of claim 13, wherein: the therapeutic conjugate of claim 1 is present at about 0.1-10 wt%; and the diluent peptide amphiphile is present at about 0-10 wt%.

15. The hydrogel of claim 13, wherein the diluent peptide amphiphile is a diluent peptide amphiphile of formula (II):Y1— AA1— AA2— AA3— AA4— AA5(||), wherein:Y1is the alkyl moiety of formula:is the hydrophilic subsequence; and the C-terminus16. The hydrogel of claim 13, wherein the diluent peptide amphiphile is a diluent peptide amphiphile of formula (ll-a):

17. A pharmaceutical composition comprising: the therapeutic conjugate of claim 1 or the hydrogel of claim 13; and a pharmaceutically acceptable excipient.

18. A method for treating or ameliorating the symptoms of diabetes or inducing weight loss, the method comprising administering a therapeutically effective amount of the therapeutic conjugate of claim 1 , the hydrogel of claim 13, or the pharmaceutical composition of claim 17 to a subject in need thereof.

19. A kit comprising the therapeutic conjugate of claim 1, the hydrogel of claim 13, or the pharmaceutical composition of claim 17; delivery or administration apparata or devices; and optionally, packaging, a label, or instructions for use.

20. Use of the therapeutic conjugate of claim 1, the hydrogel of claim 13, or the pharmaceutical composition of claim 17 for the preparation of a medicament for treating diabetes or inducing weight loss.

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