Controlled-release formulations of GLP-1 receptor agonists utilizing self-assembling peptides (SAPS)

Abstract

The disclosure provides slow-release formulations of Glucagon-like peptide- 1 receptor agonist (GLP-1 RA), such as for example, semaglutide, tirzepatide, and survodutide, for treating obesity, type II diabetes and other metabolic disorders. The formulations are based on self- assembling peptides compositions, such as, for example, RADA16 and IEIK13.

Description

CONTROLLED-RELEASE FORMULATIONS OF GLP-1 RECEPTOR AGONISTS UTILIZING SELF-ASSEMBLING PEPTIDES (SAPS)PRIORITYThis Application claims priority to US Provisional Application Nos. 63 / 558,878, filed February 28, 2024; 63 / 702,382, filed October 2, 2024; and 63 / 702,388, filed October 2, 2024; the entire contents of each of which are incorporated herein by reference.SEQUENCE LISTINGThe instant application contains a Sequence Listing which is being submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on January 21, 2025, is named 3DM-24-01-SMGL_PCT-SequenceListing.xml and is 40,055 bytes in size.TECHNICAL FIELDThe technical field relates to the methods of making and using controlled-release pharmaceutical drug formulations, more particularly, the use of self-assembling peptide hydrogel solutions, as a depot, for slow-release formulations of Glucagon-like peptide- 1 receptor agonist (GLP-1 RA), for example, for treatment of obesity, type II diabetes, and other metabolic disorders.BACKGROUNDSelf-Assembling Peptides— Self-assembling peptides (“SAPs”) are a type of peptide which assemble spontaneously into highly organized nanostructures when placed in aqueous environment and a chemical or physical change in surrounding conditions occur. One other well- known structure is a nanofibrous biopolymer structure formed by natural collagen. A class of SAPs relevant to this invention consists of alternating hydrophilic and hydrophobic amino acidresidues capable of forming beta-sheets. They autonomously assemble into well-ordered nanostructures in neutral water, and can temporarily disassemble when high shearing force is applied to them. SAPs can form a hydrogel (also known as SAP gels) depending on their environment such as pH and / or osmolality; for example, they are capable of forming a hydrogel in an aqueous solution when they are placed in the body at near neutral pH. SAP gels have been previously described as being used for a variety of medical applications, e.g., improved wound healing, inducement of hemostasis; reduction of adhesion in interior tissues, and particularly, in the context of surgery; as temporary tissue- void matrix fillers, facilitating ingrowth of natural tissue into such a void. Particular SAPs are described in US Patent Nos. 5,670,483; 5,955,343; 9,724,448; 10,596,225 and IntT Pat. Appln. Pub. W02014 / 136081; and foreign equivalents thereof. SAP hydrogels may also be used as a depot for drug delivery, providing controlled release in various applications.Purastat® is one such commercially available peptide hydrogel available from 3-D Matrix, Ltd. which contains a 16-amino acid polypeptide with a repeating sequence of arginine, alanine, and aspartic acid, specifically, Ac-RAD ARAD ARAD ARADA-NH2 (SEQ ID NO: 1) (also referred to as “RADA16”) sold at a concentration of 2.5% w / v. Other SAPs include but are not limited to, Ac-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys- Ile-Glu-Ile-Lys-Ile- NH2 (SEQ ID NO:2) (also referred to as IEIK13) and AC-QLELQLELQLEL-NH2 (SEQ ID NOG) (also referred to herein as QLEL12). Other peptide hydrogels exhibiting similar beneficial properties as discussed herein may also be used and are discussed below.GLP-1 receptor agonists— Obesity and type II diabetes arc highly prevalent, chronic, relapsing diseases requiring long-term management. Specifically, the clinical complications of obesity affect almost every organ system, and the impact of obesity on morbidity, mortality and health care costs is substantial. The prevalence of obesity has risen globally for the past several decades, a trend predicted to continue. Worldwide obesity prevalence is 13%, but many countries have a much higher prevalence; for example, prevalence in adults increased from 31% to 42% between 1999-2000 and 2017-2018 in the USA, with an increase from 10% to 40% across mostEuropean countries over the 10-year period up to 2017. (Chide Kel Bergmann et al., DiabetesObe’s. Metab. 2023 Jan; 25(1): 18-35)Older pharmacological options for chronic weight management, such as orlistat, phentermine-topiramate and naltrexone-bupropion, typically show moderate efficacy (~3%-9% mean weight loss over that achieved with lifestyle intervention alone). Liraglutide 3.0 mg once daily administered subcutaneously was the first GLP-1 receptor agonist (GLP-1 RA) to be approved for weight management, after demonstrating weight losses of 4%-6% over those achieved with lifestyle intervention alone in clinical trials of 20-56 weeks’ duration. In these trials, 46%-76% of participants lost >5% and 23%-37% lost >10% of their baseline body weight. Semaglutide is a GLP-1 analogue shown to reduce energy intake, reduce hunger, and increase feelings of satiety and fullness. Id. This effect has been shown to arise via GLP-1 receptor activation in the central nervous system, with further indirect modulation of neuronal activity involved in appetite regulation and food intake and preference. Id. The terms “weight management,” “treatment of obesity,” and “weight reduction” are used interchangeably herein.Table 1 below presents a partial list of GLP-1 RAs, available on the market.Table 1.Most of the available GLP-1 RAs, e.g., tirzepatide (10 mg or 15 mg), Wegovy® (semaglutide 2.4 mg), Ozempic® (semaglutide 0.5, 1, or 2 mg), exenatide (2 mg), require weekly injections by a patient, resulting in lower levels of compliance and higher levels of side effects. Therefore, there exists a need to provide improved formulations of GLP-1 receptor agonists, particularly, those offering controlled, slowed release of the active drug.In addition to disorders and conditions listed in Table 1, these and newly developed, GLP-1 RA’s have been approved or are being developed for other indications. For example, Zepbound® (tirzepatide) was recently approved for treatment of obstructive sleep apnea in obese patients (Malhotra et al., New England J. Med. (2024) 391(13): 1193-1205; Eli Lilly report dated April 17, 2024) which may result from loss of weight. Other indications for administration of exenatide may include sleep enhancement (US Application Publication No. 2012 / 0231022A1) and treatment of pancreatitis caused by type 2 diabetes (US Application Publication No.2011 / 0306549A1). It has also been reported that semaglutide minimizes metabolic dysfunction- associated steaotic liver disease severity in HIV positive patients (V. Salib, Tech Target: Pharma Life Sciences, March 7, 2024) and it has received FDA approval for kidney and heart protection in patients with type 2 diabetes (A. Kaylor, Tech Target: Pharma Life Sciences, January 29, 2025). Further, Ozempic® and Wegovy® may help prevent major cardiovascular events in diabetics with existing cardiovascular disease (A. Kaylor, Tech Target: Pharma Life Sciences, May 14, 2024).Also reported are a lower risk of diagnosis of anxiety and depression with some GLP-1 RAs in diabetic and nondiabetic patients (V. Salib, Tech Target: Pharma Life Sciences, February 8, 2024), treatment of breast cancer, a cancer for which obesity and type 2 diabetes correlate with outcome of treatment (US Application Publication No. 2011 / 0046071 Al

[0094] ), and liraglutide in doses lower than administered for treatment of diabetes and obesity for treatment of impaired small or large intestine (e.g., Inflammatory Bowel Disease; US Patent No. 9,168,288B2).There are also plentiful reports of GLP-1 RAs acting in combination with other drugs for treatment of various conditions and the same acting in other ways in addition to interaction with GLP-1 receptors.SUMMARYThe present invention is based at least in part on the discovery that certain selfassembling peptide solutions may be advantageous for Glucagon-like peptide- 1 receptor agonist (GLP-1 RA) delivery, specifically, by providing controlled-release formulations, thereby reducing the frequency of injections, thereby increasing patient’s quality of life, compliance and improving the ease of use of GLP-1 RA drugs that are normally delivered by relatively frequent subcutaneous injections. In particular, the properties of various self-assembling peptide solutions, including but not limited to, those of RADA16 and IEIK13, which when mixed with a GLP-1 RA may provide prolonged time-delayed near-linear release of the active drug. The terms “controlled release,” “delayed release,” “slow release,” “prolonged release,” and “extended release” are used interchangeably herein. The term “extended” release refers to a release that occurs over one week or longer, preferably, over two weeks, more preferably over four, five, six, seven, eight, nine, ten, eleven, twelve weeks or more. In the formulation of the invention, SAP (e.g., RADA or IEIK13) and PLG-1 RA (semaglutide, tirzepatide, or survodutide, etc.) are mixed at a predetermined ratio. Generally, the invention features “SAP-rich mixing ratios” meaning that the weight to weight ratio of SAP to GLP-1 RA is greater than 1, 1.1, 1.2, 1.3, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more.Furthermore, in some embodiments, depending on the depot used and the concentration ratio of SAP: drug, near-linear release may occur after a potentially non-linear release following 12 hours, and within the first 12 hours no more than 30% of drug is released.Accordingly, the invention provides a delayed-release formulation, comprising a mixture of Glucagon-like peptide- 1 receptor agonist (GLP-1 RA) and a self-assembling peptide (SAP), wherein upon subcutaneous administration using a needle wherein the mixture comes in contact with biological fluids, the SAP forms a depot releasing no more than 30% GLP-1 RA over a period of 12 hours following administration, and thereupon continues to release GLP-1 RA, followed preferably by a near-linear release over an extended period of time as described above. Characteristics of the undesirable non-linear and desirable near-linear release are described in Example 7 herein. As shown in that example, unlike RADA16 and IEIK13, QLEL12 exhibited acceptable release within the first 12 hours, however, following that, the release was highly nonlinear and unacceptably slow.In some embodiments, GLP-1 RAs include those listed in Table 1 , including but not limited to scmaglutidc, tirzepatide, and survodutidc (sec also Figures 1-3).As a peptide hydrogel solution can withstand shear stress this property may be exploited to facilitate easy injection through a narrow needle. In at least some embodiments, their stimuli- responsive gelation after being injected into the body can be utilized to make a stable hydrogel depot in the subcutaneous area. Accordingly, viscous peptide solutions may be utilized for the delivery of GLP- 1 RA with the controllable therapeutic release over time and with methods disclosed herein.The invention further provides methods of making and methods of using the formulations for the invention.In some embodiments, the invention provides a method of making the delayed-release formulation by mixing to a homogenous solution or homogenous suspension an SAP and a GLP- 1 RA, wherein said solution is sterile and is suitable for subcutaneous administration into a human subject.In some embodiments, the invention provides any of the GLP-1 RAs listed in Table 1 or known in the art, can be used for treating suffering from one or more metabolic or other disorders, such as, for example, obesity and / or type II diabetes as well as other disorders, including but not limited to: a) obstructive sleep apnea in obese patients, b) for sleep enhancement, c) pancreatitis caused by type 2 diabetes metabolic dysfunction-associated steaotic liver disease severity in HIV positive patients; d) for preventing major cardiovascular events in diabetics with existing cardiovascular disease; e) anxiety and f) depression. Examples of specific but non-limiting uses are described in the Background Section herein.In some embodiments, the subject is human, while in other embodiments the subject is a mammal, preferably a primate such a chimpanzee, a monkey, etc.More detailed description of various embodiments is provided below. In the event of any discrepancies between this document and its priority applications, this document supersedes all prior applications.BRIEF DESCRIPTION OF THE FIGURESFig. 1 depicts the chemical structure of semaglutide.Fig. 2 depicts the chemical structure of tirzepatide.Fig. 3 depicts the chemical structure of survodutide.Fig. 4 depicts the graph of injection force vs time / injection volume of 2.5% w / v RADA 16 (PuraStat®) repeated with four 32G 4mm hypodermic needles.Fig. 5 shows the appearance of certain SAPs / semaglutide mixtures in aqueous solution. Panel A shows a mixture of 2.5% w / v RADA16 and 2% w / v semaglutide exhibiting an opaque suspension. Panel B shows a mixture of 1.3% w / v IEIK13 and 4% w / v semaglutide exhibiting homogeneous transparent solution. Panel C shows 2% w / v QLEL12 and 4% w / v semaglutide exhibiting homogeneous transparent solution.Fig. 6 shows a graph of in vitro release of semaglutide (13.3 mg / ml) from RADA16 (1.5% w / v) gel.Fig. 7 shows a graph of in vitro release of semaglutide (13.3 mg / ml) from RADA16 (2.5% w / v) gel.Fig. 8 shows a graph of in vitro release of semaglutide (20 mg / ml) from RADA 16 (25 mg / ml) gel.Fig. 9 shows a graph of in vitro release of semaglutide (23 mg / mL) from RADA 16 (25 mg / ml) gel.Fig. 10 shows a graph of in vitro release of semaglutide (13.3 mg / ml) from IEIK13 (7.5 mg / ml) gel.Fig. 11 shows a graph of in vitro release of semaglutide (20 mg / ml) from IEIK13 (0.75% w / v) gel.Fig. 12 shows a graph of in vitro release of semaglutide (20 mg / ml) from IEIK13 (1.3% w / v) gel.Fig. 13 shows a graph of in vitro release of semaglutide (40 mg / ml) from IEIK13 (1.3% w / v) gel.Fig. 14 shows a graph of in vitro release of semaglutide (40 mg / ml) from QLEL12 (2% w / v) gel.Fig. 15 shows a graph of in vitro release of gentamicin sulfate (1 mg / ml) from RADA 16 (0.9% w / v) gel.DETAILED DESCRIPTIONRelease ProfileThe invention provides a delayed-release formulation, comprising a mixture of a Glucagon- like peptide- 1 receptor agonist (GLP-1 RA, also referred to as “drug”) and a self-assembling peptide (SAP, also referred to as “depot”), e.g., in aqueous solution, wherein upon subcutaneous administration using a needle wherein the mixture comes in contact with biological fluids, thereby the SAP forms a depot releasing no more than 50% GLP-1 RA over a period of 20 hours following administration, and thereupon continuing to release GLP- 1 RA, preferably, in a near- linear manner following 12 hours after the injection. In some embodiments, no more than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% of the drug is released from the depot over a period of 20 hours following administration. In some embodiments, no more than 30%, 25%, 20%, 15%, 10% of the drug is released from the depot over a period of 12 hr, 20 hr, 24 hr, 30 hr, 35 hr, or 40 hr (wherein each series of %-release should be understood as explicitly teaching alternative embodiments with respect to each of the aforementioned time points). In preferred embodiments, no more than 10%, 15%, 20%, 25%, or at most 30% of the drug is released over 12 hours following the injection. Additional conditions will be described below.Self-Assembling Peptides for Use With GLP-1 RAThe delayed-release formulation is claimed in the present invention, wherein the SAP is selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:24 (Table 2), but excluding SEQ ID NOG (QLEL12). In general, without undue experimentation, the SAP is selected from the group consisting of or comprising a sequence of amino acid residues conforming to one or more of Formulas I- IV :((XaaneL1-Xaa+)x(XaaneLl-Xaa_)y)n (I) ((Xaaneu-Xaa-)x(Xaaneu-Xaa+)y)u(II) ((Xaa+-Xaaneu)x(Xaa"-Xaaneu)y)n(III)((Xaa--Xaaneu)x(Xaa+-Xaaneu)y)n(IV) where Xaaneurepresents an amino acid residue having a neutral charge at neutral pH; Xaa+represents an amino acid residue having a positive charge at neutral pH; Xaa“represents an amino acid residue having a negative charge at neutral pH; x and y are integers having a value of 1, 2, 3, or 4, independently; and n is an integer having a value of 1-5.In non-limiting exemplary embodiments, the SAP is selected from RADA16 and IEIK13.The rheological properties of self-assembling peptides PURASTAT® (RADA16; (SEQ ID NO:1)), IEIK13 (SEQ ID NO:2), QLEL12 (SEQ ID NO:3) and KLD12 (SEQ ID NO:4) -each having unique physical and biochemical properties, are suited to the present invention and have been disclosed previously. PURASTAT® is comprised of the synthetic peptide Ac- RAD ARAD ARAD ARAD A-NH2 (SEQ ID NO:1), and potentially truncated fragments thereof, and is commercially supplied as a solution at 2.5% wt / vol in water (3-D Matrix, Ltd., Japan). The SAP IEIK13 (SEQ ID NO:2) shows different gelation characteristics than RADA16 when applied as a solution in vitro and in vivo when brought in to contact with biological fluids, such as blood, or in in vzvo-like conditions. Both these SAPs form viscous hydrogels with a nanofibrous matrix in a range of concentrations at about neutral pH. The SAPs disclosed in this application share this and other characteristics despite having different compositions.PURASTAT® is comprised of the amphiphilic self-assembling peptide RADA16 (repeats of arginine, alanine; aspartic acid, and alanine (SEQ ID NO:1)) and is supplied sterile at 2.5% (wt / vol) in water (3-D Matrix, Ltd.). The solution has a pH of 2-3, pKi of 1.79, and pK2 of 12.58. PURASTAT® exhibits different behavior and properties at different pH values. At pH 2.2, PURASTAT® is a viscous solution. Once the gel is touched / broken, the reassembly is slow because of strong repulsive (+) electrostatic interactions. At pH 2.5-4, PURASTAT® forms a semi-rigid, viscous solution. The gel formation is triggered by hydrophobic and charge-charge interactions. Once the gel is broken, the reassembly is fast due to weak electrostatic interactions. Between pH 4 and 7.5, the self-assembling peptides form a rigid hydrogel. Nanofiber formation is due to hydrophobic and attractive charge-charge interactions.Both RADA16 (SEQ ID NO:1) and IEIK13 (SEQ ID NO:2) have shown utility inpromoting rapid hemostasis when applied to oozing biological tissues and, upon forming a hydrogel matrix integral with wounded tissue, promote normal healing over time rather than scar formation or lack of healing. As reported by Katsuyama et al. {Minimally Invasive Therapy & Allied Technologies 29(5): 283-292 (2020)), IEIK13 (called TDM-623 in that reference) forms a stiffer gel (i.e., has a higher storage modulus, G’) compared to RADA 16 (TDM-621 in that reference) when exposed to physiological conditions which correlated with improved hemostasis when the product was applied to liver punch hole injuries in pigs. While reporting statistically significant improvement in hemostasis compared to RADA16, these authors also reported the absence of inflammatory cell infiltration due to the presence of the RADA16 or IEIK13 hydrogel following application of the SAP solutions to the respective wounds in the short-term study. In a more recent study, IEIK13 ((SEQ ID NO:2); (called TDM-623 in that reference) was tested as a hemostatic agent administered endoscopically to oozing wounds created in the walls of the stomach and / or duodenum of pigs with apparent success, although no control group was included (Kubo et al., Endoscopic application of novel, infection-free, advanced hemostatic material: Its usefulness to upper gastrointestinal oozing (2021); doi.org / 10.1002 / deo2.25). As also found in the previously cited study, hemostasis was achieved in both heparinized and non-heparinized animals. It is noteworthy that, given the acidic pH in the stomach, application of IEIK13 still resulted in hemostasis of gastric bleeding.The SAPs used in this invention are wholly synthetic and carry no risk of infection from animal-derived products. In addition, it has been reported in numerous studies that RADA16 and IEIK13 do not themselves promote an immune response or inflammation (e.g., Katsuyama et al.; Kubo et al., supra).The use of D-amino acid containing SAPs, including, RADA16 and IEIK13, as the hydrogel component of the nanocomposite system are included in embodiments of the present invention, particularly where a slower rate of in vivo degradation and / or resorption may be desirable. The appropriate peptide concentrations for RADA16, IEIK13, and all other peptides are listed under “Peptide Concentrations” below.In some embodiments of the present invention, the SAP is KLD12 (SEQ IDNO:4). Tn some particular embodiments, where the SAP is TETK13 (SEQ ID NO:2), peptide concentration is within a range of range of about 0.05% to about 2.0% (wt / vol) in water,Peptide Concentrations— Rheological properties of peptide compositions as described previously (US Patent No. 10,654,893, herein incorporated by reference) may be controlled by selection of peptide concentration, for example as may be specifically preferred for a particular indication or use of compositions, through selection and / or adjustment of peptide concentration. For numerous SAPs, composition stiffness has been shown in vitro to increase substantially linearly with peptide concentration. Any of the peptides recited in Table 2 can be used at concentrations while in non-swollen solution within a range of (all w / v) about 0.05% to about 5%, from about 0.05% to about 4%, from about 0.5% to about 4%, from about 0.5% to about 3.5%, from about 0.5% to about 3%, from about 0.5% to about 2.5%, from about 0.5% to about 2%, from about 0.5% to about 1.5%, or about 1%, about 2%, about 2.5%, about 3%, about 4%, or about 5%.In vitro, the rheological properties achieved at a particular peptide concentration vary depending on the identity of the peptide. For example, the storage modulus G’ of KLD12 (SEQ ID NO:4) 1.5% in water was found to be about 350 Pa similar to that of 2.5% RADA 16 (SEQ ID NO:1) in water under the same test conditions. The storage modulus G’ of 1% IEIK13 (SEQ ID NO:2) in water (-700 Pa) was found to be similar to that of 2.5% KLD12 (SEQ ID NO:4) in water and higher than that of 2.5% RADA16 (SEQ ID NO: 1) in water (-350 Pa) under the same test conditions (US Patent No. 10,654,893 - see Tables 3 and 3 A therein). Overall, the order of rheological strength among these compositions was IEIK13 (SEQ ID NO:2) > KLD12 (SEQ ID NO:4) > RADA16 (SEQ ID NO:1), so a composition of IEIK13 (SEQ ID NO:2) showed greater rheological strength than did a composition of KLD12 (SEQ ID NO:4), which in turn showed greater rheological strength than did a composition of RADA16 (SEQ ID NO:1) when peptide concentration in water was the same in each case. Given the results obtained by loading GLP-1 PAs in various SAPs, the previous characterization of various SAPs provides a reasonable expectation that the new formulation comprising different SAPs will also show rheological properties similar to or the same as the plain SAPs.The above and other peptides suitable for use in methods of the invention are similar to RADA 16 and include the following peptides listed in Table 2 and modified peptides and peptidomimetics listed further below (all with appropriate A-terminal carboxylation and C- terminal amidation of the peptides) and any of the appropriate concentrations listed under “Peptide Concentrations” above.Table 2. Examples of Self-Assembling PeptidesIn some embodiments, the SAPs may further comprise an amino acid sequence that interacts with the extracellular matrix, wherein the amino acid sequence anchors the SAPs to the extracellular’ matrix (for example, RGD) as for the example described in JP Patent No. JP 6224040, or its US counterpart. Pat. No. US 8,022,178, or other sequence as described therein.In other embodiments, the amino acid residues in the SAPs can be (synthetic or not animal derived) naturally occurring or non-naturally occurring amino acid residues. Naturally occurring amino acids can include amino acid residues encoded by the standard genetic code while non-naturally occurring amino acid include non-standard amino acids (e.g., amino acids having the D-configuration instead of the L-configuration or combinations of D- and L-amino acids), as well as those amino acids that can be formed by modifications of standard amino acids (e.g., pyro lysine or selenocysteine). Suitable non-naturally occurring amino acids include, but are not limited to, D-alloisoleucine(2R,3S)-2-amino-3-methylpentanoic acid, and L-cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid. In some embodiments, the SAPs used in the method of invention comprise only naturally occurring amino-acids, or only unnaturally occurring amino acids (such as D-amino acids, e.g., RADA16 or IEIK13 comprising D-amino acids); or combination of both D- and L- amino acids. RADA16 comprising D-amino acids, as well as other SAPs, could be used in the methods of the invention, to potentially reduce the in vivo degradation of the hydrogel matrix thereby increasing the retention time of the hydrogel at the site of adhesion, which, in turn, could affect the retention of solutes (e.g., drugs) within the gel matrix, ingrowth of tissue into the matrix, and tissue healing.In other embodiments, another class of materials that can self-assemble and mimic the SAPs are peptidomimetics. Peptidomimetics, as used herein, refers to molecules which mimic peptide structure. Peptidomimetics have general features analogous to their parent structures, polypeptides, such as amphiphilicity. Examples of such peptidomimetic materials are described in Moore et al., Chem. Rev. 101(12), 3893-4012 (2001). The peptidomimetic materials, used in the invention, can be classified into four categories: a-peptides, P-peptides, y-peptides, and 5- peptides. Copolymers of these peptides can also be used. Examples of a-peptide peptidomimetics include, but are not limited to, N,N'-linked oligoureas, oligopyrrolinones, oxazolidin-2-ones, azatides and azapeptides. Examples of P-peptides include, but are not limited to, P-peptide foldamers, a-aminoxy acids, sulfur-containing P-peptide analogues, and hydrazino peptides. Examples of y-peptides include, but are not limited to, y-peptide foldamers, oligoureas, oligocarbamates, and phosphodiesters. Examples of 5-peptides include, but are not limited to, alkene-based 5-amino acids and carbopeptoids, such as pyranose-based carbopeptoids and furanose-based carbopeptoids.In certain embodiments, the SAP is AC5®, AC5-V®, AC5-G™ or TK45, also known as AC1, made by and available from Arch Therapeutics, Inc. (see www.archthcrapcutics.com). Each of these self- assembling peptides, and others disclosed herein, are capable of forming a hydrogel when applied to a biological tissue (e.g., in situ) at about neutral pH. Generally, the SAP concentration in water will range from about 1% to about 5% weight / volume although this range is not exclusive. For example, the aqueous concentration at which IEIK13 (SEQ ID N0:2) peptides form a hydrogel matrix when exposed to physiologic conditions is generally between about 0.5 and about 2.5% w / v. However, self-assembly (or not) of this and other peptides in aqueous environments depends on numerous variables including pH, ionic strength, the concentration and composition of the SAP itself, the type of ions present, and numerous other factors. Data characterizing and comparing the properties of RADA16 (SEQ ID NO: 1), IEIK13 (SEQ ID NO:2) and KLDL12 (SEQ ID NO:4) under different conditions can be found in US Patent No. 10,654,893; the entire contents of which are incorporated herein by reference.GLP-1 RA for Use in the Formulations With SAPsIn the formulations of the invention, SAP (e.g., RADA16 or IEIK13) and GLP-1 RA (semaglutide, tirzepatide, or survodutide) in water are mixed at a predetermined ratio. In a majority of the embodiments, SAP: GLP-1 RA weight to weight ratio is greater than 1. Generally, the invention features “SAP-rich mixing ratios” means that the weight to weight ratio of SAP to GLP-1 RA is greater than 1, 1.1, 1.2, 1.3, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or more. The delayed-release formulation may contain GLP-1 RA selected from the GLP-1 RAs listed in Table 1. In some preferred exemplary embodiments, GLP-1 RA is selected from semaglutide, tirzepatide, and survodutide. In some embodiments, the properties of various self-assembling peptide solutions, including but not limited to, those of RADA16 and IEIK13, which when mixed with a GLP- 1 RA may provide prolonged time-delayed release along with near- linear time release of the active drug. The terms “controlled release,” “delayed release,” “slow release,” “prolonged release,” and “extended release” are used interchangeably herein. The term “extended" release refers to a release that occurs over one week or longer, preferably, over two weeks, more preferably over four, five, six, seven, eight, nine, ten, eleven, twelve weeks or more. Depending on the depot used and the ratio of SAP:drug, the near-linear release may occur afterpotentially non-linear release following 12 hours, and within the first 12 hours no more than 30% of drug is released.Accordingly, the invention provides a delayed-release formulation, comprising a mixture of a Glucagon-like peptide- 1 receptor agonist (GLP-1 RA) and a self-assembling peptide (SAP), wherein upon subcutaneous administration to a subject using a needle wherein the mixture comes in contact with biological fluids in vivo, the SAP forms a hydrogel depot releasing no more than 30% GLP-1 RA to the subject over a period of 12 hours following administration, and thereupon continues to release GLP-1 RA, preferably, followed by a near-linear manner over an extended period of time as described above. Characteristics of the undesirable non-liner and desirable near- linear release are described in Example 7. As shown in that Example, unlike RADA16 and IEIK13, QLEL12 exhibited acceptable release within the first 12 hours, however, following that, the release was highly non-linear and unacceptably slow.In some embodiments, GLP-1 RAs include GLP1 RAs listed in Table 1, including but not limited to semaglutide, tirzepatide, and survodutide (see also Figures 1-3).Since a peptide hydrogel solution can withstand shear stress, this property may be exploited to facilitate easy injection through a narrow needle. In at least some embodiments, their stimuli-responsive gelation after being injected into the body can be utilized to make a stable hydrogel depot in the subcutaneous area. Accordingly, viscous peptide solutions may be utilized for the delivery of GLP-1 RA with the controllable therapeutic release over time and with methods disclosed herein.Medical IndicationsIn some embodiments, the invention provides any of the GLP-1 RAs listed in Table 1 or known in the art, can be used for treating suffering from one or more metabolic or other disorders, such as, for example, obesity and / or type II diabetes as well as other disorders, including but not limited to: a) obstructive sleep apnea in obese patients, b) for sleep enhancement (insomnia), c) pancreatitis caused by type 2 diabetes metabolic dysfunction- associated steaotic liver disease severity in HIV positive patients; d) for preventing major cardiovascular events in diabetics with existing cardiovascular disease; e) anxiety and f)depression. Examples of specific but non-limiting uses are described in the Background Section herein.The invention is further explained by non-limiting illustrative examples shown below.Example 1: Injectability of RADA16 and IEIK13 through Hypodermic Needles.Poor injectability to the subcutaneous area can be an obstacle to the use of biomaterials for GLP1-RA delivery. Thus, the injectability of RADA16 and IEIK13 has been studied.Materials and Methods — A DHR1 rheometer (TA Instruments) was used to test the injectability of RADA16 and IEIK13 through hypodermic needles. Sample syringes were placed on the syringe holder and a 40-mm upper plate was loaded to the syringe to push the syringe plunger before tests started. The compression speed was 1.2 mm / sec. Compression tests were performed for 10 seconds, and the force was recorded. The syringes’ inner diameter was 12.4 mm. The injection volume per second was calculated to be approximately 0.1 ml / sec. Maximum force (Fmax) was recorded as the highest force measured. Injection force tests were performed with various needles and the results are shown in Figure 4 and Table 3. It was reported that the average maximum force that can be generated for a typical syringe injection with the thumb pushing on the plunger, while the ipsilateral index and middle fingers are used to stabilize the syringe flanks, is 79.8 N (males: 95.4 N, females: 64.1 N). See ( Astin AD. Finger Force Capability: Measurement and Prediction Using Anthropometric and Myoelectric Measures, Faculty of the Virginia Polytechnic Institute and State University, Blacksburg, Va, USA, 1999: vtechworks.lib.vt.edu / handle / 10919 / 30923). Thus, the required forces for RADA16 and IEIK13 injection through hypodermic needles were under the reported average maximum force.Table 3. Injection Force Test Result Summary. RADA16 2.5% and IEIK13 1.3% with various needles. The injection rate was 1.2 mE / min. (N =4, Ave ± SD)Example 2: Appearance of GLP1-RA and Self-Assembling Peptide MixturesMixing characteristics of GLP-1 RAs were further explored and were found to vary among various peptide hydrogel solutions, including but not limited to. IEIK13, QLEL12, and RADA 16. For example, GLP-1 RAs can be heterogeneously or homogeneously mixed with SAP peptide solutions. Semaglutide was one GLP-1 RA tested in these experiments with 3 different SAPs. Semaglutide showed different mixing phases with RADA 16, IEIK13, and QLEL12. Figure 5 and Table 4 show the appearance of semaglutide mixtures with RADA16, IEIK13, and QLEL12. Semaglutide showed a homogeneous and transparent mixing phase with IEIK13 and QLEL12 at all the tested mixing ratios. However, semaglutide showed a heterogeneous and opaque mixing phase with RADA 16.As shown in the results, GLP-1 RAs can be stably mixed with SAP solutions depending on their mixing ratio. On the other hand, GLP-1 RA can be unstably phased-separated when it is mixed with SAP solutions depending on mixing ratio. For example, while semaglutide was stably suspended in RADA 16 at RADA16-rich mixing ratios; at the same time semaglutide was unstably phase- separated when mixed with RADA16 at semaglutide-rich mixing ratios. (Table 5). Generally, as used herein, “RADA16-rich mixing ratios” means that the weight to weightratio of RADA 16 to semaglutide is greater than 1 ; and conversely, “semaglutide-rich mixing ratios” means that the weight to weight ratio of RADA16 to semaglutide is less than 1.Table 5. Mixing appearance at semaglutide-rich for Self-assembling peptide-rich phaseExample 3: Slow-Release Formulations of GLP1-RA and Self-Assembling Peptide GelsThe disclosed formulations are advantageous in prolonging the interval of injections of GLP-1 RA peptide compared to previously possible methods using weekly injection techniques. It is expected GLP-1 RA when formulated with RADA16 or IEIK13 would stay in the body for up to several months without any functional loss. GLP-1 RA mixed with RADA16 or IEIK13 slowly released GLP-1 RA from the depot located in a subcutaneous area for several weeks. GLP-1 RA can be slowly released via IEIK13 depot diffusion from the peptide hydrogel matrix. GLP-1 RA would be slowly released via hydrogel matrix degradation. GLP-1 RA can be slowly released via both diffusion and matrix degradation together.To carry out drug release tests, a semaglutide / peptide mixture of 100 pL was placed in a round bottom glass tube (4 mm in i.d. and 7 cm in height) and PBS buffer (pH 7.2) of 400 pL was added. The test samples were kept at 37°C. At each time point, all volume of PBS was collected and fresh PBS of 400 pL was replaced. The collected samples were kept at -20°C until tested with HPLC.An Agilent HPLC 1100 (Agilent Technologies) was used for this study. Column temperature was kept at 50 °C. Solvent A was water with 0.1% TFA and Solvent B was 100%Acetonitrile with 0.1% TFA. The gradient of solvent B was controlled from 45% to 70% in 10 min. A Supelco C18 column (4.6 mm X 250 mm, 5pm, 300 A) was used for this test. Figures 6-14 show the data of the semaglutide release from the various peptide hydrogel matrices. See also Tables 6A and 6B.For example, to test GLP-1 RA release from the peptide solution, semaglutide was mixed with RADA16, IEIK13, and QLEL12 solutions at various mixing ratios. The mixing conditions are listed in Table 6A, along with the percent release of semaglutide at 1 day, 2 weeks, and 12 weeks (Table 6B).Table 6A. Mixing conditions of peptide hydrogel with semaglutideTable 6B: semaglutide release data from self-assembling peptides (A = 3)*: N = 2;#and$: N= 1.As observed from Tables 6A and 6B and Figures 6-14, the peptide concentration in GLP-1 RA / peptide mixtures may impact drug release kinetics. GLP1-RA may be released faster when mixed with a peptide hydrogel matrix at a lower concentration. For example, semaglutide (13.3 mg / ml) was released faster from RADA 16 1 .5% (68% in 12 weeks) than RADA 16 2.5% (29% in 12 weeks). On the other hand, GLP1-RA may be released faster when mixed with a peptide hydrogel matrix at a higher concentration. For example, semaglutide (20 mg / ml) was released faster from IEIK13 1.3% (80% in 12 weeks) than IEIK13 0.75% (66% in 12 weeks)Notably, GLP-1 RA concentration in GLP-1 RA / SAP mixtures may impact drug release kinetics. GLP1-RA at a higher concentration may be released faster when mixed with a peptide hydrogel matrix. For example, semaglutide (23 mg / ml) (62% in 12 weeks) was released faster than semaglutide (20 mg / ml) (49% in 12 weeks) and semaglutide (13.3 mg / ml) (29% in 12 weeks), when mixed with RADA16 2.5% (Figures 6-14 and Tables 6A and 6B). As another example, semaglutide (20 mg / ml) (66% release in 12 weeks) was released faster than semaglutide (13.3 mg / ml) (52% in 12 weeks), when mixed with IEIK13 0.75% (Figures 6-14 and Tables 6A and 6B).On the other hand, peptide characteristics may impact drug release kinetics. GLP-1 RA mixed with RADA16 may be released slower than with IEIK13 and QLEL12. For example, semaglutide (23 mg / ml) in RADA16 2.5% (33% in 2 weeks) was released slower than semaglutide (20 mg / ml) in IEIK13 1.3% (68% in 2 weeks) and semaglutide (40 mg / ml) in QLEL12 2% (70% in 2 weeks) (Figures 6-14 and Tables 6A and 6B).Example 4: Negative Control Formulations of Self-Assembling Peptide GelsAs shown in Example 3, GLP-1 RAs may be slowly released from self-assembling peptide hydrogel matrix. This seems to be related to specific interactions between GLP- 1 RAs and self-assembling peptides. This slow release has not been observed with conventional drugs. For example, as a control, gentamicin sulfate was mixed with RADA 16 hydrogel matrix and therelease of gentamicin sulfate was tested. 1 .8 ml of 1 % RADA16 was mixed with 0.2 ml of 1 % gentamicin sulfate (Sigma- Aldrich) in water. The final concentrations of RADA 16 and gentamicin sulfate were 0.9% and 0.1%, respectively. The mixture of 400 pl was placed in 24 well plates (2 cm2) to make 2 mm height of the mixture and 2 ml of PBS buffer solution was gently added. Four samples were tested in this study. The samples were kept at 37°C. A 1 ml aliquot was taken at each time point and 1 ml PBS buffer solution was gently replaced in the plate. HPLC analysis of the samples was obtained using Agilent Model 1100 Series LC coupled with an lon-trap-electrospray ionization (ESI) mass spectrometer (Agilent 1100 with Ion-Trap XCT and a Hewlett-Packard computer using the ChemStation / MSD Trap control program) was used for data acquisition and analysis.Separation was performed at 30°C on a reversed-phase column, ZORBAX 300 Extended-C18 (4.6 mm X 250 mm, 5pm, 300 A) purchased from Agilent Technologies. The flow rate was 0.5 ml / min. Ionization was performed in positive mode and the ions m / z values were 450.5, 464.5 and 478.5 for gentamicin. The mobile phase was a mixture of pcntafluoropropionic acid (PFPA, Acros Organics) (20 mM in HPLC grade ultrapure water) and methanol (60 / 40 v / v). 15 pl of each sample was injected onto the HPLC column. The release profile of 0.1% (1 mg / ml) gentamicin sulfate from 0.9% RADA 16 is shown in Figure 15. It was found that 80% of gentamicin sulfate was released in 4 hours.Example 5: “Burst Effect”Based on the experiments described above, it was found that peptide characteristics may impact the so-called “burst effect” of semaglutide release. The “burst effect” has been an obstacle to the use of biomaterials for drug delivery systems. See, e.g., Brazel CS, Huang X. The Cost of Optimal Drug Delivery: Reducing and Preventing the Burst Effect in Matrix Systems. Carrier-Based Drug Delivery. Vol 879: American Chemical Society; 2004:267-282.As seen from Figures 6-9, GLP-1 RA can be released from certain hydrogel depots without a burst effect that is shown in usual drug delivery depots. GLP-1 RA can be delivered to patients with near linear release kinetics.Specifically, GLP-1 RA mixed with RADA 16 showed no burst effect unlike IEIK13 and QLEL12 (Figures 10-14). For example, only 1.9% of semaglutide (20 mg / ml) in RADA16 2.5% was released in 1 day, while 30.2 % of semaglutide (20 mg / ml) in IEIK13 1.3% was released in1 day and 33% of semaglutide (40 mg / ml) in QLEL12 2% was released in 1 day (Table 6B), which is not practically useful. GLP-1 RA mixed with RADA 16 did not show burst effect, while GLP-1 RA mixed with IEIK13 and QLEL12 did. Nevertheless, even though the burst effect is present in IEIK13, the following favorable release has long-term near-linear release kinetics. Accordingly, in the order of preference, RADA16-containing GLP-1 RA formulations, and in particular such formulations with RADA16-rich mixing ratios is preferred, followed by IEIK13 formulations. As results show QLEL12 does have both disadvantages, it has both a “burst effect” and fast drug release, making it impractical for use as a depot.Example 6: “Theoretical Rationale”Without wishing to be bound by theory, the inventors hypothesized that GLP-1 RAs may have specific secondary interactions with specific self-assembling peptides. Specific secondary interactions of GLP-1 RAs with self-assembling peptides may determine GLP-1 RA long-term near linear-release rate and whether there is a burst effect,For example, charge-charge interactions between GLP-1 RA and self-assembling peptide may impact their GLP-1 RA release. The net charges of semaglutide, tirzepatide, and survodutide are -3, -3, and -4, respectively. The net charges of RADA16 and IEIK13 at pH 2~3 are + 4 and +3, respectively. Therefore, GLP-1 RAs may have strong charge-charge interactions with RADA 16 and IEIK13 when they are mixed in solution at pH 2~3. Once pH becomes neutral in the body, the charge-charge interaction may become weaker because the net charges of RADA16 and IEIK13 become slightly positive. This weak charge-charge interaction may have an important role in slow GLP-1 RA release from RADA16 and IEIK13 hydrogel matrix. On the other hand, the net charge of QLEL12 at neutral pH is -3. Therefore, GLP-1 RA may not have charge-charge interactions with QLEL12 when they are mixed at pH 7-8 or in the body. The absence of charge-charge interactions between GLP-1 RA and QLEL12 could be attributed to relatively rapid GLP-1 RA release from QLEL12 hydrogel matrix.Furthermore, the hydrophobic interactions between GLP-1 RA and self-assembling peptide may also have an important role on their mixing phase and GLP-1 RA release. IEIK13 and QLEL12 have stronger hydrophobic side groups of isoleucine (i.e., I in IEIK13) and leucine (L in QLEL12), while RADA16 has a weaker hydrophobic side group of alanine (A in RADA 16). Therefore, IEIK13 and QLEL12 may have stronger hydrophobic interaction withGLP-1 RAs that have a hydrophobic fat group in their side chain compared to RADA 16. The strong hydrophobic interaction may result in the homogeneous mixing of GLP- 1 RA with IEIK13 and QLEL12 (as stated below such a homogenous mixture presents as a heterogeneous / suspension in the case of RADA16). Accordingly, GLP-1 RA molecules are homogeneously solvated in RADA16 hydrogel matrix. Therefore, the release of GLP-1 RA from IEIK13 and QLEL12 hydrogel matrix seems to be controlled by the diffusion of GLP-1 RA. This may result in the usual burst effect of GLP-1 RA from IEIK13 and QLEL12 hydrogel matrix. On the other hand, the weak hydrophobic interaction between GLP-1 RA and RADA 16 may have an influence on their poor mixing with a heterogeneous phase. In the case of GLP-1 RA (i.e., semaglutide-rich phase), RADA 16 cannot be a main matrix to hold GLP-1 RA, resulting in phase separation.Therefore, to make an effective drug delivery system, RADA 16 w / w amount should be more than GLP-1 RA (i.e., RADA16-rich mixture). In the case of RADA16-rich-mixing ratios, RADA 16 hydrogel mixture was found to be a heterogeneous / suspension solution with nearly perfect release profile from the hydrogel. On the other hand, GLP-1 RA exists as heterogeneous / precipitated particles in RADA 16 hydrogel matrix with GLP-1 RA-rich mixing ratios. In such a case, the suspension may be useful as well because the release of GLP-1 RA from RADA16 hydrogel matrix should be controlled by the slow dissolution of GLP-1 RA particles rather than the diffusion of GLP-1 RA. This specific interaction between GLP-1 RA and RADA 16 may result in slower release and no burst effect of GLP-1 RA from RADA 16 hydrogel matrix. -Example 7: Linear Regression Analysis of Release ProfilesRegular formulation of semaglutide is injected into patients and can stay in the body only for 1 week. Thus, semaglutide (alone) needs to be injected into patients every week. The purpose of using SAP hydrogels is to provide a sustained release of semaglutide even after 1 week. Accordingly, the semaglutide injection interval can be extended probably up to 2 weeks, 1 month, 2 months, or 3 months. Thus, it is important to determine the remaining semaglutide after 1 week and its linear release between 2 weeks and 12 weeks (3 months). Table 5 shows the remaining semaglutide after 1 week. Table 7 shows linear regression analysis of semaglutide release data from self- assembling peptides between 2 weeks and 12 weeks.After one week, the remaining scmaglutidc % from RADA 16, IEIK13, and QLEL12 hydrogels were 80-97.8%, 42-63%, and 40%, respectively (calculated from Table 7 below). This indicates that a significant amount of semaglutide can stay in the body with these peptide hydrogels even after one week.Table 7: Linear regression analysis of semaglutide release data from self-assembling peptides (A = 3)*: N = 2;#and$: N = 1.&: Intercept was set at 0.In liner regression analysis, the R2coefficient of determination is a statistical measure of how well the regression predictions approximate the real data points. (See: Schneider A, Hommel G, Blettner M. Linear regression analysis: part 14 of a series on evaluation of scientific publications. Dtsch Arztebl Int. 2010 Nov;107(44):776-82. doi: 10.3238 / arztebl.2010.0776. Epub 2010 Nov 5. See also: Chicco D, Warrens MJ, Jurman G. The coefficient of determination R-squarcd is more informative than SMAPE, MAE, MAPE, MSE and RMSE in regressionanalysis evaluation. Peer, J Comput Sei. 2021 Jul 5;7:e623. doi: 10.7717 / peerj-cs.623.) If R2is close to 1, it indicates that the regression predictions well fit the data. In contrast, if R2is close to 0, it indicates that the regression predictions barely fit the data. For example, an R2value of 0.80 or above might be considered good, indicating that a large proportion of the variance in the dependent variable is explained by the independent variables in the regression model.RADA16 data show the R2values are around and above 0.9 (i.e., 0.8806 - 0.9778) in their regression models between 0 and 12 weeks (all data). However, IEIK13 and QLEL12 data show their R2values are below 0.8 (i.e., 0.6107 -0.7886 and 0.7303, respectively) in their regression models between 0 and 12 weeks (all data; Table 7).Hence, semaglutide release from RADA16 hydrogel could be considered to have good linearity between 0 and 12 weeks, while semaglutide release from IEIK13 and QLEL13 hydrogels could be considered to have poor linearity. This may be because RADA16 hydrogel does not show a burst effect of semaglutide release but IEIK13 and QLEL12 do.The R2values of semaglutide release from RADA16 hydrogel between 2 and 12 weeks were around or above 0.8 (i.e., 0.7666-0.9217). The R2values of semaglutide release from IEIK13 hydrogel between 2 and 12 weeks were above 0.9 (i.e., 0.9340-0.9886). However, The R2value of semaglutide release from QLEL12 hydrogel between 2 and 12 weeks was only 0.3839.Thus, RADA16 and IEIK13 hydrogels could be considered to indicate linear semaglutide release between 2 weeks and 12 weeks (3 months). However, QLEL12 hydrogel could not be considered to indicate linear semaglutide release between 2 weeks and 12 weeks (3 months).It should be noted that the average semaglutide release rates from RADA 16 hydrogels between 2 and 12 weeks (0.27-0.37 % / day) were higher than those from IEIK13 and QLEL12 hydrogels between 2 and 12 weeks (0.14-0.26 % / day and 0.09 % / day, respectively), even though they (0.27-0.37 % / day) decreased compared to those from RADA16 hydrogels between 0 and 12 weeks (0.41-1.1 % / day).In summary, RADA16 shows: 1) more than 80% semaglutide remains in hydrogel depot after one week (80-97.8%), 2), good linear semaglutide release between 0 and 12 weeks (R2= 0.8806 - 0.9778), 3), good near-linear semaglutide release between 2 and 12 weeks (R2= 0.7666-0.9217), 4) still significant rate of semaglutide release between 2 and 12 weeks (0.27-0.37 % / day) compared to those between 0 and 12 weeks (0.41-1.1 % / day).IEIK13 shows: 1 ) around half of semaglutide remains after one week (42-63%) in hydrogel depot, 2) poor linear semaglutide release between 0 and 12 weeks (R2= 0.6107 -0.7886), 3) good near-linear semaglutide release between 2 and 12 weeks (R2= 0.9340-0.9886), 4) still significant release rate of semaglutide release (0.14-0.26 % / day) compared to those between 0 and 12 weeks (0.75-1.2 % / day), but slower release than RADA16 between 2 and 12 weeks (0.27-0.37 % / day).QLEL12 shows: 1) 40% of semaglutide remains in hydrogel depot after one week, 2) however, it exhibits poor linear release between 0 and 12 weeks (R2= 0.7303), 3) and, additional, poor, non-linear, release between 2 and 12 weeks (R2= 0.3839), and 4) very slow release rate of semaglutide (0.09 % / day) compared to between 2 and 12 weeks (1.3 % / day), as compared to RADA16 and IEIK13 formulation..

Claims

CLAIMS1. A delayed-release formulation, comprising a mixture of a Glucagon-like peptide- 1 receptor agonist (GLP-1 RA) and a self-assembling peptide (SAP), wherein upon subcutaneous administration using a needle wherein the mixture comes in contact with biological fluids, the SAP forms a depot releasing no more than 30% GLP-1 RA over a period of 12 hrs following administration, and thereupon continues to release GLP-1 RA, which preferably occurs in a near-linear manner, for at least a pre-determined time period.

2. The delayed-release formulation of claim 1, wherein SAP is selected from the group consisting of the peptide sequence as set out in SEQ ID NO:1 through SEQ ID NO:24 (Table 2), but excluding SEQ ID NO:3 (QLEL12).

3. The delayed-release formulation of claim 1, wherein SAP is selected from the group consisting of a sequence of amino acid residues conforming to one or more of Formulas I-IV:((Xaaneu-Xaa+)x(Xaaneu-Xaa-)y)u(I) ((Xaaneu-Xaa")x(Xaaneu-Xaa+)y)n (II) ((Xaa+-Xaaneu)x(Xaa--Xaaneu)y)n(III) ((Xaa"-Xaaneu)x(Xaa+-Xaaneu)y)n(IV) where Xaalieurepresents an amino acid residue having a neutral charge; Xaa+represents an amino acid residue having a positive charge; Xaa“represents an amino acid residue having a negative charge; x and y are integers having a value of 1, 2, 3, or 4, independently; and n is an integer having a value of 1-5.

4. The delayed release formulation of claim 1, wherein the SAP is selected from the group consisting of RADA16 (SEQ ID NO:1) and IEIK13 (SEQ ID NO:2).

5. The delayed-release formulation as in any one of the preceding claims, wherein the GLP- 1 RA is selected from the GLP-1 RAs listed in Table 1.

6. The delayed-release formulation as in any one of the preceding claims, wherein the GLP- 1 RA is selected from the group consisting of semaglutide, tirzepatide, and survodutide.

7. The delayed-release formulation of any one of the preceding claims, wherein the SAP to GLP-1 RA weight to weight ratio of RADA16 (SEQ ID NO:1) to the GLP-1 RA in the mixture is greater than 18. The delayed-release formulation of claim 7, wherein the GLP-1 RA is selected from semaglutide, tirzepatide, and survodutide.

9. The delayed-release formulation of claim 7, wherein the SAP is RADA16 (SEQ ID NO:1) or IEIK13 (SEQ ID NO:2) and wherein GLP-1 RA is selected from the group consisting of semaglutide, tirzepatide, and survodutide.

10. The delayed-release formulation of claim 9, wherein the GLP-1 RA is semaglutide.I L A method of making the delayed-release formulation of any one of the preceding claims, the method comprising mixing to a homogenous solution or homogeneous suspension a SAP and a GLP-1 RA, wherein said solution is sterile and is suitable for subcutaneous administration into a human subject.

12. A method of treating a subject, said subject suffering from one or more metabolic disorders, said method comprising administering subcutaneously to the subject a delayed release composition of any one of claims 1-10.

13. The method of claim 12, wherein the subject is human.

14. The method of claims 13, wherein the metabolic disorder is obesity.

15. The method of claims 13, wherein the metabolic disorder is type II diabetes.

16. The method of any one of claims 12-15, wherein the multiple administrations of the delayed release formulation are performed at least 2 weeks apart.

17. The method of claim 16, wherein the metabolic disorder of the subject upon the course of treatment with the delayed-release composition is improved and / or the amount and severity of the side effect is reduced as compared to a control group treated with a regular solution of the same GLP-1 RA.

18. A delayed-release formulation, comprising a mixture of semaglutide and RADA 16 (SEQ ID NO: 1) at a predetermined ratio.

19. A delayed-release formulation, comprising a mixture of semaglutide and IEIK13 (SEQ ID NO: 2) at a predetermined ratio.

20. A method of treating a human subject diagnosed with obesity and / or type II diabetes, said method comprising administering to the subject the delayed-release formulation of claim 18 or 19, thereby treating the subject’s obesity and / or type II diabetes..

21. A method of treating a human subject diagnosed with a disorder or condition selected from the group consisting of: a) obstructive sleep apnea in obese patients, b) insomnia, c) pancreatitis caused by type 2 diabetes metabolic dysfunction-associated steaotic liver disease severity in HIV positive patients; d) for preventing major cardiovascular events in diabetics with existing cardiovascular disease; e) anxiety and f) depression;the method comprising administering to the subject the delayed-release formulation of claim 18 or 19, thereby treating the subject’s said disorder or condition.