Albumin-binding polymer triple agonist that activates GLP-1 / GIP / glucagon receptors

Abstract

The pharmaceutical composition comprises a GPCR agonist fusion protein in which a GPCR agonist peptide is covalently coupled to albumin via a linker in a manner resistant to retro-Michael addition. Advantageously, the compositions presented herein avoid separation of the agonist from albumin while retaining the agonist in a steric relationship with albumin that allows for effective binding and activation of the GPCR, as well as enabling gp60-mediated transcytosis and FcRn-mediated albumin recycling. These properties enable ultra-low doses of the GPCR agonist fusion protein to produce therapeutic effects, while substantially reducing or even completely avoiding the adverse effects generally associated with otherwise unbound agonists. Such retro-Michael-resistant compositions are generally achieved by conformational modification of albumin, resulting in sterically selective coupling of the linker to albumin.

Description

[Technical Field]

[0001] This application is a U.S. Provisional Patent Application No. 63 / 523,324 filed on June 26, 2023; a U.S. Provisional Patent Application No. 63 / 619,584 filed on January 10, 2024; a U.S. Provisional Patent Application No. 63 / 624,692 filed on January 24, 2024; a U.S. Provisional Patent Application No. 63 / 625,169 filed on January 25, 2024; and a U.S. Provisional Patent Application No. 63 / 625,169 filed on February 8, 2024. Provisional Patent Application No. 63 / 551,315, U.S. Provisional Patent Application No. 63 / 551,334 filed on February 8, 2024, U.S. Provisional Patent Application No. 63 / 551,370 filed on February 8, 2024, U.S. Provisional Patent Application No. 63 / 561,187 filed on March 4, 2024, U.S. Provisional Patent Application No. 63 / 564,426 filed on March 12, 2024, 2 U.S. Provisional Patent Application No. 63 / 564,941 filed March 13, 2024, U.S. Provisional Patent Application No. 63 / 566,870 filed March 18, 2024, U.S. Provisional Patent Application No. 63 / 567,402 filed March 19, 2024, U.S. Provisional Patent Application No. 63 / 568,988 filed March 22, 2024, and U.S. Provisional Patent Application No. 63 / 568,988 filed April 9, 2024 This invention claims priority to U.S. Provisional Patent Application No. 63 / 631,777, U.S. Provisional Patent Application No. 63 / 634,379 filed on 15 April 2024, U.S. Provisional Patent Application No. 63 / 641,782 filed on 2 May 2024, and U.S. Provisional Patent Application No. 63 / 650,095 filed on 21 May 2024, each of which is incorporated herein by reference.

[0002] Sequence List The contents of the sequence listing XML file designated 1026900063PCT-C (size 981,726 bytes), which was created on June 25, 2024, and submitted electronically through the Patent Center together with this application, are incorporated herein by reference in their entirety.

[0003] The field of the present invention is pharmaceutical compositions and methods relating particularly to agonists, dual agonists, and triple agonists of glucagon-like peptide 1 (GLP-1) receptors, glucose-dependent insulin-secreting polypeptide (GIP) receptors, and / or glucagon (GcG) receptors. [Background technology]

[0004] The background art description contains information that may be useful in understanding the present invention. It is not acknowledged that any information provided herein constitutes prior art, or is related to the claimed invention, or that any publication specifically or implicitly referenced constitutes prior art.

[0005] All publications and patent applications herein are incorporated by reference to the same extent as each individual publication or patent application is specifically and individually indicated as being incorporated herein by reference. If a definition or use of a term in an incorporated reference is inconsistent with or contradicts a definition of that term provided herein, the definition provided herein shall apply, and the definition in the reference shall not apply.

[0006] Obesity, a global health crisis, is worsening at an alarming rate. As of 2020, 41.9% of adults in the United States were classified as obese. Globally, more than one billion people, including 650 million adults, are affected by obesity. This condition is associated with numerous health complications, including hyperglycemia, type 2 diabetes, hypertension, dyslipidemia, obstructive sleep apnea, atherosclerosis, osteoarthritis, urinary incontinence, non-alcoholic steatohepatitis, cardiovascular disease, certain cancers, and premature death. Furthermore, obesity negatively impacts both physical and mental health, leading to a decline in quality of life and cardiopulmonary function, ultimately increasing the risk of cardiovascular disease and overall mortality.

[0007] Similarly, diabetes is becoming an increasingly common disease, with 537 million adults worldwide affected in 2021 and projected to reach 783 million by 2045. Of these patients, approximately 90% have type 2 (or non-insulin-dependent) diabetes. Often associated with obesity, this disease is characterized by elevated blood glucose levels, decreased insulin action, and impaired glucose tolerance. Treatment typically involves diet, weight loss, and exercise regimens, as well as the administration of antidiabetic drugs, including metformin to reduce hepatic glucose excretion, sulfonylurea and metformin to increase insulin secretion, and / or thiazolidinediones to enhance insulin sensitivity.

[0008] While these drug treatment options are often effective, the disease can progress despite treatment, and patients may eventually require daily insulin administration. Therefore, novel therapeutic strategies in the form of insulin-secreting peptides, including glucagon-like peptide 1 (GLP-1), gastric suppressor polypeptide (GIP), and analogs of glucose-regulating peptides such as glucagon and amylin, are being developed for the treatment of type 2 diabetes.

[0009] GLP-1 is a naturally occurring hormone released by L cells in the lower intestine in response to nutrient intake. In addition to its potent insulin-secreting effect, GLP-1 has been shown to suppress glucagon, stimulate pancreatic β-cell proliferation, inhibit gastric emptying, and reduce gastrointestinal (GI) motility. All of these effects contribute to glucose-dependent postprandial maintenance of normal blood glucose levels and regulation of appetite. Due to these multifaceted effects, GLP-1 analogs are attractive candidates for the treatment of type 2 diabetes.

[0010] A major drawback of using native GLP-1 is its extremely short half-life of several minutes, due to cleavage at the N-terminal alanine residue by the ubiquitous proteolytic enzyme dipeptidyl peptidase IV. In contrast to the short lifespan of the GLP-1 peptide, exendin-4 (a 39-amino acid peptide agonist of the glucagon-like peptide 1 (GLP-1) receptor) is less susceptible to degradation by dipeptidyl peptidase IV, and has a circulating half-life of approximately 33 minutes in humans. Exendin-4 induces similar physiological effects to GLP-1 via the GLP-1 receptor and is more potent than native GLP-1. Synthetic exendin-4 (Byetta) has been used as adjunctive therapy with oral antidiabetic agents in the treatment of non-insulin-dependent diabetes mellitus (T2DM). As an approach to further extend the half-life of exendin-4, this agonist has been encapsulated in poly-(DL-lactide-coglycolide) microspheres (Bydureon).

[0011] As there was a need for longer-lasting active formulations, recombinant proteins such as the fusion protein described in CA2434237 (not commercially available) and the fusion protein albiglutide (TANZEUM®, GlaxoSmithkline, discontinued), as described in U.S. Patent No. 7141547, were developed. Albiglutide is a GLP-1 albumin fusion protein in which a tandem repeat of Gly8 GLP-1 is directly fused to the N-terminus of human serum albumin. Here, the tandem repeat is intended to improve affinity for the GLP-1 receptor by creating a long distance between albumin and the distal GLP-1 peptide. The half-life was extended to 6-8 days, making it suitable for once-weekly administration, but the potency of albiglutide was significantly reduced (albiglutide has a GLP-1 receptor affinity of 20 nM, compared to 0.02 nM for exenatide). This is thought to be due to the combination of Gly8 modification and direct covalent bonding with albumin. When administered as monotherapy, the reduction in HbA1c from baseline was -0.7% at week 52 with a weekly dose of 30 mg. With a weekly dose of 50 mg, the change in HbA1c from baseline was -0.9%. Despite the administration of high doses of GLP-1 (30 mg and 50 mg), no reduction in HbA1c from baseline of more than 1% was achieved. Based on these findings, Knudsen et al. (Knudsen LB, Lau J. The Discovery and Development of Liraglutide and Semaglutide. Front Endocrinol (Lausanne). 2019 Apr 12;10:155.doi:10.3389 / fendo.2019.00155.PMID:31031702;PMCID:PMC6474072.) concluded that, assuming that GLP-1 agonists may bind to albumin or the GLP-1 receptor but not both, fusing GLP-1 agonists to albumin does not yield an effective composition.Specifically, the industry opposed irreversible binding to albumin and instead implemented the idea of ​​"constructing reversible binding to albumin as a solution for systemic survival extension of GLP-1 analogs." The main challenge identified in previous studies was that strong binding to albumin negatively impacted the potency of the compound against GLP-1R due to competition between binding to albumin and binding to the receptor. The theory was that only the free fraction in plasma that did not bind to albumin was available to activate GLP-1R. Therefore, the stronger the affinity for albumin, the smaller the free and active circulating fraction of the GLP-1 peptide. This phenomenon had been previously observed with liraglutide analogs, and there was a clear trend that longer fatty acids with improved affinity for albumin were associated with reduced potency against GLP-1R.

[0012] Based on this assumption, a modified approach by the pharmaceutical industry was adopted that utilized the reversible non-covalent binding of GLP-1 analogs to albumin. In this approach, GLP-1 agonists were acylated to facilitate the non-covalent binding of acylated GLP-1 agonists to seven fatty acid binding sites on albumin, thereby extending the half-lives of these compositions. Examples of such approaches include VICTOZA (liraglutide, Novo Nordisk, albumin containing a modified GLP-1 agonist), OZEMPIC (trademark) / WEGOVY (trademark) (semaglutide, Novo Nordisk, albumin containing a modified GLP-1 agonist), MOUNJARO (trademark) / ZEPBOUND (trademark) (chilzepatide, Eli Lilly, albumin containing a modified GLP-1 / GIP dual agonist), Pemvidutide (AltImmune, albumin containing a modified GLP-1 / glucagon dual agonist), efinopegdutide (Merck, albumin containing a modified GLP-1 / glucagon dual agonist), and survodutide (Zealand, Boehringer, albumin containing a modified GLP-1 / glucagon dual agonist). While at least some of these formulations resulted in increased stability and serum half-life, receptor activation still required the dissociation of the modified agonist from the albumin carrier. This reversible approach resulted in 99% binding to albumin, leaving only 1% of the active substance available for binding to the GLP-1 receptor, thus leading to renal clearance of the agonist and the delivery of high doses of GLP-1. Furthermore, numerous adverse effects, including nausea, vomiting, and diarrhea, are common with formulations that have reversible and non-covalent binding of GLP-1 analogs at these high doses. To improve the tolerability of such compositions, it is routinely recommended to dose subjects up to a tolerable therapeutic dose, which may take several weeks.

[0013] Furthermore, in other known compositions, exendin-4 is linked to albumin via a linker, as described in International Publication No. 2007 / 053946, International Publication No. 2009 / 075859 and International Publication No. 2011 / 109787. 34 Covalently bound to albumin, CA2501421 and CA2550050 teach further insulin secretagogues having linkers that can be coupled to albumin. Conceptually attractive, but albumin's Cys 34 The covalent bond in this compound generally undergoes retro-Michael addition, which releases the previously bound exendin-4, leading to rapid renal clearance and potential adverse effects by the unbound GLP-1 agonist, thus leaving the same difficulties observed with the acylated GLP-1 agonists mentioned above.

[0014] Therefore, despite the fact that various compositions and methods of GPCR agonists, particularly GLP-1, GIP, and glucagon receptor agonists, are known in the art, all or almost all of them have several drawbacks. In particular, small agonist peptides such as GLP-1 are rapidly degraded by endopeptidases, while small agonist peptide analogs such as exendin-4 are more stable, but still have rapid renal clearance. On the other hand, GLP-1 albumin direct fusion proteins have a remarkably long serum half-life, but are sterically hindrance and require very high doses, as demonstrated with albiglutide. Furthermore, fatty acid-modified GLP-1 and GLP-1 analogs, which are non-covalently bound to the hydrophobic pocket of other albumins, need to dissociate from albumin to activate their target receptors. Unfortunately, the dissociated free fatty acid-modified GLP-1 and its analogs, once separated from albumin, produce adverse effects and do not penetrate the blood-brain barrier (BBB). Similarly, linker-bound exendin-4 in albumin via Michael addition is linked to Cys 34When coupled, free exendin-4 is encountered again for retro-Michael addition, leading to rapid exendin-4 clearance and adverse effects from the unbound agonist once more. [Overview of the project] [Problems that the invention aims to solve]

[0015] Therefore, there is still a need to provide an improved composition in which the GPCR agonist has high stability in plasma / serum, exhibits agonist activity at low concentrations while rebinding stably to the carrier, and can easily move from the blood compartment to the target tissue, or cross the blood-brain barrier to enter nerve tissue and be delivered both subcutaneously, subdermally, intranasally, and orally. [Means for solving the problem]

[0016] The subject of the present invention relates to various compositions and methods of GPCR agonists, particularly insulin-secreting agonists, that have high stability in plasma / serum, remain stably bound to a carrier and possess agonist activity, allowing the GPCR agonist to easily move from blood compartments to target tissues or even cross the blood-brain barrier to enter nerve tissue. Advantageously, the compositions presented herein are retro-Michael resistant and therefore reduce or completely avoid the adverse effects typically caused by unbound or free agonists.

[0017] In one aspect of the subject matter of the present invention, the inventors envision a reaction intermediate comprising (a) conformationally modified albumin isolated in a solvent, wherein at least one hydrophobic pocket of the albumin is not occupied by a non-covalently bonded lipid, and (b) a Michael acceptor reagent.

[0018] Most typically, the isolated conformation-modified albumin is recombinant human albumin, and in certain embodiments, the isolated conformation-modified albumin is defatted albumin. The solvent is more preferably (but not required) an aqueous solvent with a pH of pH < 7.0. Furthermore, the Michael acceptor comprises a planar acceptor group (e.g., a maleimide group or a bromomaleimide group), and in most embodiments, the Michael acceptor reagent is intended to further comprise a linker covalently coupled to a class BG protein-coupled receptor (GPCR) agonist peptide.

[0019] From a different perspective, the inventors of Cys 34 Albumin is covalently bonded to the chiral carbon atom of the coupling group Cys 34 The aim is to provide a chemically modified albumin in which a coupling group is added and the chiral atom has a preferred stereochemical configuration. For example, a preferred stereochemical configuration may be at least 70:30 or at least 90:10. As described above, the albumin is typically human albumin and may be recombinant or not. It is even more preferable that the albumin is conformationally modified albumin and / or that the albumin is in an aqueous solvent having pH < 7.0. Furthermore, the albumin is unreacted Cys 34 The albumin is contained in an aqueous solvent further containing albumin having the group (free albumin), and the ratio of free albumin to chemically modified albumin is intended to be at least 1:1.2. In further intended embodiments, the coupling group may be covalently coupled to a linker, and the linker may be covalently coupled to a class B GPCR agonist peptide.

[0020] Therefore, the present inventors believe that albumin is Cys 34It should be understood that chemically modified albumin modified with a Michael addition conjugate and resistant to retro-Michael addition reaction is also contemplated. Most preferably, the Michael addition conjugate includes hydrophilic linkers of various lengths, and the hydrophilic linkers may be further covalently bonded to class B GPCR agonist peptides. In still other contemplated embodiments, at least one hydrophobic pocket of albumin is not occupied by non-covalently bound lipids. Thus, the albumin can be at least partially (or completely) defatted albumin. Further, it is also contemplated that the chiral carbon atom of the Michael addition conjugate forms a covalent bond with the sulfur of Cys34 and the chiral carbon atom has a preferred stereochemical configuration (e.g., the preferred stereochemical configuration is preferred in a ratio of at least 70:30).

[0021] As a result, the inventors also 34 A method of modifying albumin with, comprising the step of providing or preparing conformationally modified albumin in a solvent, wherein at least one hydrophobic pocket of the conformationally modified albumin is not occupied by non-covalently bound lipids and the pH of the solvent is pH < 7.0 is contemplated. Next, in a further step, the sulfur atom of 34 Cys is reacted with a coupling group to thereby produce modified albumin.

[0022] In some embodiments, the conformationally modified albumin is modified by at least partial degreasing, and / or the solvent pH is 4.0–6.0. ​​Typically, though not necessarily, the reaction step is carried out in equimolar ratios between the conformationally modified albumin and the coupling group. As understood, the modified albumin is retro-Michael resistant to the covalently bonded coupling group. Furthermore, with respect to the reaction step, it is intended that the step be at least sterically preferred (e.g., one chiral configuration occurs at least 60%) or even sterically selective. Most typically, the coupling group comprises a planar Michael acceptor group (e.g., a maleimide group or a bromomaleimide group), and the coupling group is further covalently coupled to a class B GPCR agonist peptide.

[0023] Therefore, from a different perspective, the inventors also believe that Cys 34 Cys reacts with Michael's addition reagent. 34 The present invention aims to reduce the retro-Michael addition reaction in modified albumin. Such a method typically involves the steps of preparing conformationally modified albumin in a solvent such that at least one hydrophobic pocket of the conformationally modified albumin is not occupied by non-covalently bonded lipids, wherein the pH of the solvent is pH < 7.0; and Cys 34 The process includes the step of reacting the sulfur atom with the coupling group of a Michael addition reagent to produce modified albumin. Most typically, but not always, the conformationally modified albumin is modified by at least partial degreasing. The pH of the solvent is more preferably 4.0 to 6.0.

[0024] Furthermore, the reaction step is intended to be carried out in equimolar ratios between the conformation-modified albumin and the coupling group. In the intended method, the retro-Michael addition reaction is typically reduced by at least 50%. In yet another embodiment, the Michael addition reagent is intended to include linkers of various lengths covalently coupled to a class B GPCR agonist peptide, and / or the Michael addition reagent is intended to have the structure of any one of SEQ ID NOs: 1 to 471.

[0025] In another aspect of the subject matter of the present invention, the inventors also provide (a) a step of providing or producing conformation-modified albumin and (b) a step of adding a GPCR agonist peptide to conformation-modified albumin via a Michael addition reaction. 34 The present invention proposes a method for producing a class BG protein-coupled receptor (GPCR) agonist fusion protein, comprising the steps of covalent coupling and the coupling step performed at pH < 7.0.

[0026] In at least some embodiments, the conformation-modified albumin is modified by at least partial delipidation, and / or the coupling step is carried out using albumin in molar excess relative to the GPCR agonist peptide. While not essential, the pH is preferably 4.0–6.0. ​​Most typically, the GPCR agonist peptide contains a linker having a planar Michael acceptor group (e.g., a maleimide group or a bromomaleimide group). In such cases, it will be understood that the Michael addition reaction may even produce a sterically preferred or sterically selective chiral carbon atom in the Michael acceptor group. Furthermore, the GPCR agonist peptide is intended to have a structure according to any one of SEQ ID NOs: 1–471.

[0027] Therefore, the inventors also provide a method for producing a retro-Michael addition-resistant class B GPCR fusion protein, comprising the steps of (a) providing or producing conformationally modified albumin, and (b) converting a GPCR agonist peptide to a conformationally modified albumin via a Michael addition reaction. 34 The method includes a step of co-coupling, and the coupling step is intended to be performed at pH < 7.0.

[0028] Preferably, though not essential, the GPCR agonist peptide has the structure of one of SEQ ID NOs: 1 to 471, and / or the conformationally modified albumin is modified by at least partial delipidation. If desired, the coupling step is carried out using albumin in a molar excess relative to the GPCR agonist peptide, and / or the pH is intended to be 4.0 to 6.0. As mentioned above, the GPCR agonist peptide may contain a linker having a planar Michael acceptor group such as a maleimide group or a bromomaleimide group. Thus, the Michael addition reaction produces even sterically preferred or sterically selective chiral carbon atoms in the Michael acceptor group.

[0029] In yet another aspect of the subject matter of the present invention, the inventors also intend to provide a resin-immobilized cysteine-reactive class B GPCR agonist peptide comprising a GPCR agonist peptide containing a Lys amino acid, wherein (a) a linker is covalently bonded via an amide bond formed by the epsilon amino group of the Lys amino acid, and (b) a synthetic resin having a spacer is covalently bonded via an amide bond formed by the distal amino group in the spacer.

[0030] Among other options, particularly intended linkers of various lengths and amino acid compositions include MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA, and (AEEEA)2-OA-(bromo)MPA. Furthermore, the synthetic resin is generally intended to contain a polystyrene resin (e.g., Ramage resin) having a tricyclic spacer with a distal amino group, a resin substitution level of about 0.4-0.6 mmol / gram, and / or the synthetic resin is crosslinked with about 1% divinylbenzene. For example, the intended GPCR agonist peptide may have a structure corresponding to one of SEQ ID NOs: 1 to 471.

[0031] Therefore, from a different perspective, the inventors also intend to provide a peptide solid synthesis intermediate comprising a Ramage resin in which a first amino acid of the peptide is covalently coupled to the C-terminus of the peptide via an amide bond, the first amino acid further covalently coupled to a linker via an amide bond in the side chain of the first amino acid, and the linker comprising a coupling group. Most typically, the first amino acid is lysine, and / or the linker is MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA, and (AEEEA)2-OA-(bromo)MPA. It is more generally preferred that the coupling group includes a planar Michael acceptor group (e.g., a maleimide group or a bromomaleimide group).

[0032] Accordingly, the present inventors also intend a method for synthesizing a peptide containing a linker having a coupling group, comprising the steps of (a) providing a synthetic resin having a spacer containing a distal amino group; (b) covalently coupling a first amino acid to the resin using the distal amino group, wherein the first amino acid has a protected reactive group in its side chain; (c) sequentially attaching a plurality of Fmoc-protected amino acids to the first amino acid using solid-phase synthesis to obtain a protected peptide; (d) selectively deprotecting the protected reactive group in the side chain and reacting the reactive group with a linker, wherein the linker contains a second protected reactive group; (e) selectively deprotecting the second protected reactive group in the linker and reacting the second reactive group with a coupling group; and (f) deprotecting the protected peptide and cleaving the deprotected peptide from the resin, thereby forming a peptide containing a linker having a coupling group.

[0033] Most typically, the synthetic resin in such a method is Ramage resin, the first amino acid is lysine, and / or the linker is MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA, and (AEEEA)2-OA-(bromo)MPA. Similarly, it is preferred that the reactive group in the linker typically contains a planar Michael acceptor group such as a maleimide group or a bromomaleimide group, and / or the protecting peptide contains a class B GPCR agonist peptide.

[0034] Accordingly, the present inventors also intend to provide a class B GPCR agonist peptide having a reactive linker, wherein the GPCR agonist peptide comprises a Lys amino acid to which the linker is covalently bonded via an amide bond formed by the epsilon amino group of the Lys amino acid, the linker further comprises a thiol reactive group, and the GPCR agonist peptide has the structure of any one of SEQ ID NOs: 1 to 471.

[0035] As described above, the thiol-reactive group is intended to include a planar Michael acceptor group such as a maleimide group or a bromomaleimide group, and / or the linker is intended to be selected from the group consisting of MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA, and AEEEA)2-OA-(bromo)MPA.

[0036] In yet another aspect of the subject matter of the present invention, the inventors also intend to provide a class B GPCR agonist peptide having a reactive linker, comprising (a) a GPCR agonist peptide comprising a Lys amino acid, and (b) a linker covalently bonded to the GPCR agonist peptide via an amide bond formed by the epsilon-amino group of the Lys amino acid and the carboxyl group of the linker, wherein the linker further comprises a maleimide group, and the GPCR agonist peptide has the structure of any one of SEQ ID NOs: 1 to 471. The linker in such agonist peptides is preferably MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA, or (AEEEA)2-OA-(bromo)MPA.

[0037] Therefore, taking the above into consideration, the inventors have proposed (a) Cys 34 We also envision a class B GPCR agonist fusion protein comprising (b) a conformation-modified albumin containing an amino acid, a GPCR agonist peptide containing a Lys amino acid, and (c) a linker that covalently couples the conformation-modified albumin to the GPCR agonist peptide, wherein the linker is covalently bonded to the GPCR agonist peptide via an amide bond formed by the epsilon amino group of the Lys amino acid, and the linker has a chiral carbon atom of the coupling group in the linker and Cys 34 It is covalently bonded to albumin via a covalent bond with the sulfur atom in the amino acid. While not essential, preferably, the conformationally modified albumin is at least partially defatted albumin, and / or the chiral carbon atoms have a sterically preferred configuration. Most typically, the linker is retro-Michael resistant.

[0038] In particularly preferred embodiments, the fusion protein is retro-Michael-resistant arbenatide (AB-013-AEEA-succinimide (SEQ ID NO: 2)-albumin), retro-Michael-resistant arbenatidex (AB-014-AEEA-succinimide (SEQ ID NO: 185)-albumin), retro-Michael-resistant albudiplate (AB-029-AEEA-succinimide (SEQ ID NO: 7)-albumin), or retro-Michael-resistant arbutide (AB-044-AEEA-succinimide (SEQ ID NO: 223)-albumin).

[0039] In yet another aspect of the subject matter of the present invention, the inventors also provide a pharmaceutical composition comprising a class BGPCR agonist fusion protein in combination with an aqueous carrier, wherein the GPCR fusion protein is (a) Cys 34 (b) a conformation-modified albumin containing an amino acid, (c) a GPCR agonist peptide containing a Lys amino acid, and a linker that covalently couples the conformation-modified albumin to the GPCR agonist peptide, wherein the linker covalently bonds to the GPCR agonist peptide via an amide bond formed by the epsilon amino group of the Lys amino acid, and the linker connects the chiral carbon atom of the coupling group in the linker with Cys 34 The aim is to create a pharmaceutical composition in which an amino acid is covalently bonded to albumin via a covalent bond with a sulfur atom in the amino acid. Most typically, the aqueous support has a pH < 7.0 (e.g., between 4.0 and 6.0).

[0040] In such compositions, it is generally preferred that the GPCR agonist peptide and the linker have a structure according to one of SEQ ID NOs: 1 to 471. From another point of view, the linker is intended to be MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA, and (AEEEA)2-OA-(bromo)MPA. If desired, the composition contains unreacted Cys 34 It is further intended that the composition may further contain an albumin having an amino acid. Furthermore, it is intended that the composition may be formulated for injection or intranasal administration. Furthermore, in at least some embodiments, the composition may contain a GPCR agonist peptide at a concentration of 1.5 mg / mL or less, and / or the composition may further contain metformin and / or thiazolidinedione.

[0041] In particularly preferred compositions, the GPCR agonist fusion protein is retro-Michael-resistant arbenatide (AB-013-AEEA-succinimide (SEQ ID NO: 2)-albumin), retro-Michael-resistant arbenatidex (AB-014-AEEA-succinimide (SEQ ID NO: 185)-albumin), retro-Michael-resistant albudiplate (AB-029-AEEA-succinimide (SEQ ID NO: 7)-albumin), or retro-Michael-resistant arbutide (AB-044-AEEA-succinimide (SEQ ID NO: 223)-albumin).

[0042] From a different perspective, the inventors envision a pharmaceutical composition comprising a class B GPCR agonist fusion protein combined with an aqueous carrier, wherein the GPCR fusion protein is a Michael adduct of conformationally modified albumin and a linker further covalently bonded to the GPCR agonist peptide, and the fusion protein is retro-Michael addition resistant. Most typically, the composition is formulated for injection or intranasal administration, the composition contains the GPCR agonist peptide at a concentration of 1.5 mg / mL or less, and / or the formulation has a pH between 4.0 and 6.0.

[0043] If desired, the composition contains unreacted Cys 34 It is intended to further contain albumin having amino acids. Furthermore, it is preferable that the Michael adduct is sterically preferred or sterically selective, and / or that the GPCR agonist peptide and linker have a structure according to one of SEQ ID NOs: 1 to 471.

[0044] Therefore, the inventors also provide a pharmaceutical composition comprising a class B GPCR agonist fusion protein combined with an aqueous carrier, wherein the GPCR fusion protein is a Michael adduct of albumin and a linker further covalently bonded to the GPCR agonist peptide, and the composition contains unreacted Cys 34 The aim is to create a pharmaceutical composition that further contains albumin having amino acids, and in which the aqueous carrier has a pH of < 7.0. For example, unreacted Cys 34 The ratio of albumin containing amino acids to albumin bound to linkers and GPCR agonist peptides is at least 0.3:1.0. Furthermore, unreacted Cys 34 Recombinant human albumin is an albumin in which an amino acid-containing albumin is linked to a linker and a GPCR agonist peptide. In at least some embodiments, unreacted Cys 34 The albumin containing amino acids is a conformationally modified albumin, and / or the GPCR agonist peptide and linker have a structure according to one of SEQ ID NOs: 1 to 471.

[0045] From a different perspective, the inventors also envision a pharmaceutical composition comprising a pharmaceutically acceptable liquid carrier in combination with a class BG protein-coupled receptor (GPCR) agonist fusion protein in which a GPCR agonist peptide is covalently coupled to albumin via a linker. The envisioned pharmaceutically acceptable liquid carrier is safe, capable of containing sufficient concentrations of the agonist, and enabling the delivery of the GPCR agonist fusion protein in the form of subcutaneous, intradermal, intranasal, intrathecal, and oral delivery, thereby enabling sufficient systemic, mucosal, or nerve absorption. In such a composition, the pharmaceutically acceptable liquid carrier is substantially free of unbound GPCR agonist peptides. Among other suitable GPCR agonist fusion proteins, particularly preferred GPCR agonist fusion proteins include retro-Michael-resistant albenatide (AB-013-AEEA-succinimide (SEQ ID NO: 2)-albumin), retro-Michael-resistant albenatidex (AB-014-AEEA-succinimide (SEQ ID NO: 185)-albumin), retro-Michael-resistant albudiplate (AB-029-AEEA-succinimide (SEQ ID NO: 7)-albumin), or retro-Michael-resistant albutide (AB-044-AEEA-succinimide (SEQ ID NO: 223)-albumin). Most typically, the amount of unbound GPCR agonist peptide is 1% or less, or 0.1% or less, of the amount of GPCR agonist peptide covalently coupled to albumin via the linker.

[0046] In further aspects of the subject matter of the present invention, the inventors envision a method for producing a pharmaceutical composition comprising the steps of (a) providing a pharmaceutically acceptable carrier having a pH < 7.0, and (b) including a retro-Michael-resistant G protein-coupled receptor (GPCR) agonist fusion protein covalently bound to a GPCR agonist peptide in the pharmaceutically acceptable carrier, wherein the GPCR agonist peptide is present in the composition at a concentration of 1.5 mg / mL or less. Among other suitable routes of administration, it is generally preferred that the composition be formulated for injection or intranasal administration. A pharmaceutically acceptable liquid carrier is envisioned that is safe, capable of containing sufficient concentrations of agonist, and enabling delivery of the GPCR agonist fusion protein in the form of subcutaneous, intradermal, intranasal, intrathecal and oral delivery, thereby enabling sufficient systemic, mucosal, or nerve absorption.

[0047] In a particularly preferred method, the GPCR agonist fusion protein is retro-Michael-resistant arbenatide (AB-013-AEEA-succinimide (SEQ ID NO: 2)-albumin), retro-Michael-resistant arbenatidex (AB-014-AEEA-succinimide (SEQ ID NO: 185)-albumin), retro-Michael-resistant albudiplate (AB-029-AEEA-succinimide (SEQ ID NO: 7)-albumin), or retro-Michael-resistant arbutide (AB-044-AEEA-succinimide (SEQ ID NO: 223)-albumin).

[0048] Various objects, features, aspects and advantages of the subject matter of the present invention will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings in which similar reference numerals represent similar components. [Brief explanation of the drawing]

[0049] [Figure 1] A schematic diagram of the structure of retroMichael-resistant arbenatide is shown, highlighting the peptide sequence surrounding Cys34. [Figure 2] A schematic diagram of the structure of retro-Michael-resistant arbenatide is shown. [Figure 3]The schematic overall structure of retroMichael-resistant arbenatide, in which the agonist peptide portion is bound to the GLP-1 receptor, is shown. [Figure 4] This is an exemplary collection of GLP-1 agonist peptides suitable for use in this specification. [Figure 5] This is an exemplary collection of GLP-1 / GIP biagonal agonist peptides suitable for use in this specification. [Figure 6] This is an exemplary set of GLP-1 / GcG biagonal agonist peptides suitable for use in this specification. [Figure 7] This is an exemplary collection of GLP-1 / GIP / GcG triple agonist peptides suitable for use in this specification. [Figure 8] This is an exemplary collection of other GLP-1 / GIP / GcG triple agonist peptides suitable for use in this specification. [Figure 9] This is an exemplary collection of other GLP-1 / GIP / GcG triple agonist peptides suitable for use in this specification. [Figure 10] This is an exemplary collection of yet another GLP-1 / GIP / GcG triple agonist peptide suitable for use in this specification. [Figure 11] This is an exemplary collection of additional GLP-1 / GIP / GcG triple agonist peptides suitable for use in this specification. [Figure 12] This is an exemplary collection of similarly intended agonist peptides that have exemplary linker structures. [Figure 13] This is one exemplary general sequence of a triple agonist peptide suitable for use in this specification. [Figure 14] Another exemplary general sequence of a triple agonist peptide suitable for use in this specification. [Figure 15] Exemplary results regarding the binding energies of the tested dual agonist peptides to the GIP receptor and the GLP-1 receptor are shown. [Figure 16]For comparison, the calculated self-energy of a dual agonist using tilzepatide is shown. [Figure 17] Exemplary results for tilzepatide, GLP-1, and GIP receptors are shown. [Figure 18] Exemplary results for AB-804-1 and the GIP receptor are shown. [Figure 19] Exemplary results for AB-804-1 and the GLP-1 receptor are shown. [Figure 20] Exemplary results regarding binding interactions and interaction pair numbers for AB-804-2 and the GIP receptor are shown. [Figure 21] Exemplary results regarding binding interactions and the number of interaction pairs for AB-804-2 and GLP-1 receptors are shown. [Figure 22] Exemplary results regarding binding interactions and interaction pair numbers for AB-804-3 and the GIP receptor are shown. [Figure 23] Exemplary results regarding binding interactions and interaction pair numbers for AB-804-3 and the GLP-1 receptor are shown. [Figure 24] This paper presents comparative results regarding the pair interaction energies of the innate GLP-1 complex, GIP complex, and GcG complex. [Figure 25] This specification presents an exemplary selection of linkers suitable for use in this specification. [Figure 26] This shows the approximate overall structure of human albumin. [Figure 27] A schematic side view of the Cys34 hydrophobic pocket in conformationally modified human albumin is shown. [Figure 28A] Figure 27 shows a schematic diagram of the MPA coupling group occupying the hydrophobic pocket of unconformation-modified human albumin, with arrows indicating the indiscriminate attack direction of the sulfur atom on the planar maleimide group of the MPA. [Figure 28B-C]Figure 28B: A schematic diagram of the MPA coupling group occupying the hydrophobic pocket of conformationally modified human albumin in Figure 27, with arrows indicating the sterically preferred or sterically selective attack direction of the sulfur atom against the planar maleimide group of MPA. Figure 28C: A schematic superposition of conformationally unmodified human albumin (dark gray) and modified human albumin (light gray), with arrows indicating the position of the sulfur atom at Cys34. [Figure 29] This graph shows exemplary pharmacokinetic data for AB-013 without conformational modification. [Figure 30] This graph shows exemplary results from serum in the experiment shown in Figure 29, as observed on a PAGE gel. [Figure 31] The following shows an exemplary structure of a peptide agonist intermediate bound to Ramage or Rink resin. [Figure 32] This shows a typical agonist peptide intermediate. [Figure 33] The study demonstrated the efficacy of arbenatide in inducing weight loss in a diet-induced obesity (DIO) mouse model, showing it to be equivalent to that of semaglutide. [Figure 34] This shows food and water consumption after arbenatide compared to semaglutide. [Figure 35] This study demonstrates the efficacy of arbenatide in glucose regulation in DIO mice, showing that a covalently bound GLP-1 agonist reacts with the GLP-1 receptor and possesses biological efficacy contrary to current formulation instructions. [Figure 36] Contrary to current formulation instructions, this study demonstrates the efficacy of arbenatide in reducing non-alcoholic steatohepatitis (NASH), using histological evidence of semaglutide as a control and comparator, demonstrating that covalently bound GLP-1 agonists react with GLP-1 receptors and possess biological efficacy. [Figure 37A-B]This study demonstrates improvement in liver function and cholesterol levels in a DIO mouse model after arbenatide administration, showing that covalently bound GLP-1 agonists react with GLP-1 receptors and possess biological efficacy contrary to current formulation instructions. [Figure 37C-D] This study demonstrates improvement in liver function and cholesterol levels in a DIO mouse model after arbenatide administration, showing that covalently bound GLP-1 agonists react with GLP-1 receptors and possess biological efficacy contrary to current formulation instructions. [Figure 38] This figure shows the improvement of anti-inflammatory effects by cytokines after arbenatide administration. [Modes for carrying out the invention]

[0050] The inventors have now discovered that it is possible to prepare various class B GPCR agonist peptide fusion proteins that exhibit high stability in plasma / serum, possess agonist activity at low concentrations while stably bound to a carrier, readily migrate from blood compartments to target tissues, and can even cross the blood-brain barrier. Such fusion proteins are prepared by using a linker that irreversibly and covalently links albumin to a class B GPCR agonist peptide (such as an incretin or insulin secretagogue or its analogue). In this regard, it should be understood that the term “fusion protein” in combination with the compounds presented herein includes proteins in which a first peptide portion (e.g., agonist peptide) is covalently bound to a second peptide portion (e.g., albumin) via a linker portion, and the linker may be a third peptide portion or a non-peptide portion (e.g., AEEA-MPA). Therefore, the term “fusion protein” in combination with the compounds presented herein may also be called a “bioconjugate” or “conjugate.”

[0051] For example, in some preferred embodiments, one end of the linker is covalently bonded to the C-terminal reactive group of the agonist peptide (e.g., via an amide bond), and the other end of the linker is bonded to the carbon atom of the coupling group in the linker, Cys 34 Cys 34 It covalently bonds to albumin. In another preferred embodiment, one end of the linker covalently bonds (e.g., via an amide bond) to a reactive group in the side chain of an intermediate amino acid in the agonist peptide, and the other end of the linker has a carbon atom of the coupling group in the linker that is Cys 34 Regarding the hydrophobic pocket where it is located, Cys has a retro-Michael-resistant thioether bond with a stereospecific configuration that makes the bond retro-Michael-resistant via the retro-Michael-resistant bond. 34 It covalently binds to albumin. Furthermore, a suitable linker provides rotational degrees of freedom and steric distance between the agonist peptide and albumin, allowing the agonist peptide to bind to the target receptor while remaining covalently bound to albumin, thereby activating the target receptor.

[0052] Advantageously, the intended retro-Michael-resistant albumin-binding insulin secretagogue rapidly transcytoses into the pancreatic and brain tissue microenvironments, leaving a plasma compartment via the gp60 / caveoli-1 / caveoli pathway along with long-acting agonists via FcRn rescue. Thus, the intended compounds and compositions can utilize cell-mediated transport, thereby enabling ultra-low doses and ultra-low plasma concentrations, resulting in therapeutic effects with a high therapeutic index and a substantially low GI adverse event profile.

[0053] As should be understood, particularly intended peptide agonists suitable for use in combination with the teachings presented herein include GPCR agonists (and especially class B) and all their derivatives, analogs, and fragments. Therefore, among other intended peptide agonists, GLP-1, GIP, glucagon (GcG), amyrin, NPY2, neuropeptide Y and PYY, as well as their derivatives, analogs, and fragments, are particularly preferred. Furthermore, it should be understood that the peptide agonists intended herein may have binding and activation specificity for one receptor (single agonist), two receptors (dual agonist), or three receptors (triple agonist). In this regard, it should be particularly understood that, contrary to prior knowledge, it is possible to produce compounds in which a ligand covalently and irreversibly binds to albumin, and the same ligand can bind to and activate two or even three different G protein-coupled receptors (GLP-1, GIP, and / or GcG receptors). As a result, it should be recognized that the biological activity of fusion proteins containing such peptide agonists can be modified to suit specific applications (e.g., insulin secretagogues, appetite suppressants, etc.).

[0054] From another perspective, as will be shown in more detail below, highly stable, highly receptor-binding, and peptide-loss-free GPCR (and especially BGPCR) agonist fusion proteins can be prepared that activate GLP-1, GIP, GcG, NPY2, Y2, or PYY as a single agonist, or GLP-1 / GIP, GLP-1 / GcG, GLP-1 / NPY2, GLP-1 / Y2, or GLP-1 / PYY as a dual agonist, or GLP-1 / GIP / GcG as a triple agonist. Furthermore, since such agonist peptides are irreversibly covalently bound to conformationally modified albumin, gp60-mediated transcytosis and FcRn albumin recycling enable these fusion proteins to cross the blood-brain barrier and rapidly move from the blood compartment to the target tissue compartment (e.g., nerve tissue, liver tissue, adipose tissue, and / or pancreatic tissue) while achieving a high half-life.

[0055] In at least some embodiments, the peptide agonist is preferably an exendin-4 derivative containing modified amino acids to avoid peptide cleavage by exendin-4 or dipeptidyl peptidase-4, and containing a C-terminal tryptophan cage ("fish-hook") structure for further enhanced stability. Furthermore, depending on the specific amino acid substitutions (compared to exendin-4), the intended peptide agonist exhibits an affinity for the GLP-1 receptor equivalent to or higher than that of native GLP-1, resulting in higher potency. In addition, one or more amino acid substitutions (compared to exendin-4) would also enable multi-agonist activity (e.g., triple-agonist activity for the GLP-1 / GIP / glucagon receptor).

[0056] In one exemplary fusion protein, retro-Michael-resistant albenatide (acting as a GLP-1 agonist) contains exendin-4 (SEQ ID NO: 3) covalently coupled to a modified lysine at its C-terminus, the modified lysine containing a linker with a coupling group. In this example, the modified lysine forms a peptide bond with the C-terminal serine of exendin-4 and further contains an AEEA-MPA group covalently bonded to the epsilon-amino group of the lysine via an amide bond. Cys in albumin in a sterically preferred or sterically selective manner as described in more detail below 34 Retro-Michael-resistant albenatide (AB-013-AEEA-succinimide-albumin) reacts with the thiol group of the MPA coupling group. Naturally, various alternative linker moieties may be used, and it should be understood that suitable alternative linkers having a coupling group include MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA and (AEEEA)2-OA-(bromo)MPA (where MPA represents maleimidopropionic acid and OA represents 8-aminooctanoate). If desired, modified lysine can also be coupled to the C-terminal serine via a single AEEA group. In such cases, the modified lysine may contain a coupling group again, such as MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA, or (AEEEA)2-OA-(bromo)MPA.

[0057] Retro-Michael-resistant albenatide (AB-013-AEEA-succinimide (SEQ ID NO: 2)-albumin) is a Cys 34Figure 1 illustrates the surrounding peptide sequence, with the # symbol representing a non-racemic chiral atom, and Figure 2 illustrates the overall structure of retro-Michael-resistant arbenatide as shown in Figure 1. Figure 3 shows the overall structure of retro-Michael-resistant arbenatide with the agonist peptide bound to the GLP-1 receptor (note that GIP receptors and GcG receptors are structurally very similar to the GLP-1 receptor, and therefore Figure 3 is also representative of these agonist-receptor complexes). As is readily apparent from Figure 3, the linker provides sufficient flexibility and steric distance between the covalently bound agonist peptide and the target receptor, allowing, for example, effective binding and activation of the receptor while the agonist remains covalently bound to albumin.

[0058] In another exemplary fusion protein, following the same approach, retro-Michael-resistant albenatidex (AB-014-AEEA-succinimide (SEQ ID NO: 185)-albumin) (acting as a GLP-1 agonist) contains a chimeric GLP-1 / exendin-4 moiety covalently coupled to a modified lysine at its C-terminus, the modified lysine containing a linker with a coupling group. In this example, the modified lysine forms a peptide bond with the C-terminal serine of the chimeric peptide, and the modified lysine further contains an AEEA-MPA group covalently bonded via an amide bond to the epsilon amino group of the lysine, as shown in SEQ ID NO: 185. The MPA coupling group and Cys in albumin are described in more detail below. 34Reacting the thiol group with retro-Michael-resistant albenatidex is formed by sterically preferential or sterically selective reaction (AB-014-AEEA-succinimide-albumin). As mentioned above, various alternative linker moieties may be used, and suitable alternative linkers having coupling groups include MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA and (AEEEA)2-OA-(bromo)MPA (where MPA represents maleimidopropionic acid and OA represents 8-aminooctanoate). If desired, modified lysine may also be coupled to a C-terminal serine via a single AEEA group. In such a case, the modified lysine may again contain coupling groups such as MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA, or (AEEEA)2-OA-(bromo)MPA.

[0059] In yet another exemplary fusion protein, following the same approach, the retro-Michael-resistant albumipeptide (AB-029-AEEA-succinimide (SEQ ID NO: 7)-albumin) (acting as a GIP / GLP-1 dual agonist) comprises a modified exendin-4 moiety covalently coupled to a modified lysine at its C-terminus, the modified lysine containing a linker with a coupling group. In this example, the modified lysine forms a peptide bond with the C-terminal serine of the modified exendin-4, and the modified lysine further contains an AEEA-MPA group covalently bonded via an amide bond to the epsilon-amino group of the lysine, as shown in SEQ ID NO: 7. Again, the retro-Michael-resistant albumipeptide (AB-029-AEEA-succinimide-albumin) is formed by sterically preferential or sterically selective reaction of the MPA coupling group with the thiol group of Cys34 in the albumin, as described in more detail below. As already mentioned above, various alternative linker moieties may be used, and suitable alternative linkers having coupling groups include MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA and (AEEEA)2-OA-(bromo)MPA (where MPA represents maleimidopropionic acid and OA represents 8-aminooctanoate). If desired, modified lysine may also be coupled to the C-terminal serine via a single AEEA group. In such cases, the modified lysine may contain a coupling group again, such as MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA, or (AEEEA)2-OA-(bromo)MPA.

[0060] Furthermore, in another exemplary fusion protein, also following the same approach, retro-Michael-resistant albutide (AB-044-AEEA-succinimide (SEQ ID NO: 223)-albumin) (acting as a GIP / GLP-1 / GcG triple agonist) contains a modified exendin-4 moiety covalently coupled to a modified lysine at its C-terminus, and the modified lysine contains a linker with a coupling group. In this example, the modified lysine forms a peptide bond with the C-terminal serine of the modified exendin-4, and the modified lysine further contains an AEEA-MPA group covalently bonded via an amide bond to the epsilon-amino group of the lysine, as shown in SEQ ID NO: 223. Again, the MPA coupling group and Cys in albumin, as will be described in more detail below. 34Reacting the thiol group with retro-Michael resistance in a sterically preferential or sterically selective manner forms retro-Michael resistance albumin (AB-044-AEEA-succinimide (SEQ ID NO: 223)-albumin). Furthermore, the closely related retro-Michael resistance fusion protein, AB-045-AEEA-succinimide (SEQ ID NO: 69)-albumin, is particularly targeted. To reiterate, various alternative linker moieties may be used, and suitable alternative linkers having coupling groups include MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA and (AEEEA)2-OA-(bromo)MPA (where MPA represents maleimidopropionic acid and OA represents 8-aminooctanoate). If desired, modified lysine may also be coupled to the C-terminal serine via a single AEEA group. In such cases, modified lysine may again contain coupling groups such as MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA, and (AEEEA)2-OA-(bromo)MPA.

[0061] Therefore, based on the above examples, it should be understood that a number of retro-Michael-resistant fusion proteins with albumin can be prepared, avoiding the drawbacks of fusion proteins that can be deconjugated by agonist peptides. In fact, while a number of class B GPCR agonist peptides are particularly intended herein, a wide variety of alternative drug conjugates are also considered appropriate, including various peptide hormones, cytokines, chemokines, and other biologically active peptides, as well as larger proteins. In each of these fusion proteins, the same or similar linkers can be used as described in more detail below, and the fusion proteins, once prepared, are intended to possess retro-Michael-resistant properties.

[0062] Exemplary agonist peptides, each of which may be further bound to a linker having a coupling group, include, in particular, GLP-1 agonists, double agonists, and triple agonists, as shown in Figures 4 to 11 and listed below. Here, unless otherwise indicated, K* represents modified lysine having an AEEA-MPA, AEEA-AEEA-MPA, AEEA-OA-MPA, or AEEA-OA-AEEA-MPA linker, with or without an AEEA group preceding the lysine. However, it should be understood that various alternative linkers having coupling groups may be used, including MPA, (bromo)MPA, AEEA, AEEA-(bromo)MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-(bromo)MPA, AEEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA, and (AEEEA)2-OA-(bromo)MPA. Figure 12 shows other exemplary agonist and linker groups. Figures 13 and 14 show typical sequences for triple agonists, where X 20 or X 21In the case of lysine, these can be modified lysine having an MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-MPA, AEEA-OA-(bromo)MPA, (AEEEA)2-OA-MPA, and (AEEEA)2-OA-(bromo)MPA linker, and such modified lysine may or may not have a preceding AEEA group.

[0063] The intended agonist peptides can be tested for their binding and activation parameters using in vitro cell-based experiments, SPR experiments, cell-based experiments, or in vivo tests, which are well known in the art. However, it should be understood that the stability and binding of the agonist peptides to their respective receptors can also be investigated in silico by measuring complex binding energies and peptide self-energies. For example, the inventors have found that when covalently bound in a retro-Michael-resistant manner, recombinant albumin's Cys 34 A hydrophobic tryptophan cage with a hydrophilic linker can be utilized for covalent linkage to the free thiol, and a preferred sequence (AB-804-3, SEQ ID NO: 467, possibly without a linker / coupling group) with high binding energy in terms of both complex binding energy and peptide self-energy, even if conformational modification is not required, has been discovered. Studies have shown that all peptide domains of AB-804-3, including S1, S2, S3, S4, the tryptophan cage, and the N and C-terminuses, interact with the extracellular and transmembrane domains of their respective receptors (GLP-1 and GIP), with exemplary data showing AB-804-3 having the highest binding energy.

[0064] In particular, Figure 15 shows the binding energies of the tested dual agonist peptides to the GIP and GLP-1 receptors, and Figure 16 shows the calculated self-energy of the dual agonist using tylzepatide as a control. Binding interactions and interaction pair numbers are shown in Figure 17 for tylzepatide and the GLP-1 and GIP receptors, in Figure 18 for AB-804-1 and the GIP receptor, and in Figure 19 for AB-804-1 and the GLP-1 receptor. Figure 20 shows the binding interactions and interaction pair numbers for AB-804-2 and the GIP receptor, and Figure 21 shows the binding interactions and interaction pair numbers for AB-804-2 and the GLP-1 receptor. Similarly, Figure 22 shows the binding interactions and interaction pair numbers for AB-804-3 and the GIP receptor, and Figure 23 shows the binding interactions and interaction pair numbers for AB-804-3 and the GLP-1 receptor. Figure 24 provides a comparative table of pair interaction energies for native GLP-1, GIP, and GcG complexes.

[0065] Peptide agonists are used in albumin cys 34 With regard to linkers intended to be used for coupling to thiol groups, preferred linkers are generally intended to provide sufficient flexibility and steric distance so that the bound agonist peptide can bind to the GPCR (typically in a two-step process) so that the GPCR is activated. Therefore, in at least some embodiments, the linker is made of albumin's Cys 34The reactive group to be bonded to the thiol group (e.g., a maleimide group) and the group to which the linker is bonded to the agonist peptide (e.g., an amino group) have a linear distance equivalent to at least 6 carbon-carbon bonds, or at least 8 carbon-carbon bonds, or at least 10 carbon-carbon bonds, or at least 12 carbon-carbon bonds, or at least 14 carbon-carbon bonds, or at least 18 carbon-carbon bonds, or at least 24 carbon-carbon bonds. Also, as can be easily understood, the linker may further contain one or more non-carbon atoms to form amide bonds, ether bonds, etc. Therefore, in at least some embodiments, the preferred linker is a hydrophilic linker. Figure 25 shows an exemplary selection of linkers suitable for use herein, where AEEA represents (amide(ethoxy)ethoxyacetate acyl), MPA represents maleimidopropionic acid, OA represents 8-aminooctanoate, and Lys represents lysine.

[0066] In yet another embodiment, the linker is made of albumin Cys 34 The reactive group bonded to the thiol group is intended to be a thiol-reactive group such as a maleimide group, a bromo-maleimide group, or a succinimide group. However, the linker is Cys 34 The reactive group bonded to the thiol group is particularly preferably a group that reacts with cysteine ​​in the Michael addition reaction. The Michael reaction, or Michael 1,4 addition, is a reaction between a Michael donor, such as an enolate, thiolate, or other nucleophile, and a Michael acceptor, which is usually an α,β-unsaturated carbonyl such as MPA, to produce a Michael adduct by creating a carbon-carbon or carbon-sulfur bond at the β-carbon of the acceptor. In this regard, it should be particularly understood that in the Michael reaction between a thiol group and a planar maleimide group, if there is no stereochemically biasing factor, the reaction product will contain a newly formed chiral carbon atom (with a covalently bonded sulfur atom) resulting in a racemic product. From a different perspective, the Michael addition reaction product without a stereochemically biasing factor is an equal mixture of R- and S-configured chiral carbon atoms.

[0067] Furthermore, it should be understood that, unless other factors interfere with such a reaction, Michael addition reactions are reversible (like any other chemical reaction). In fact, such retro-Michael addition reactions are well known in connection with antibody-drug conjugates, in which the drug is readily uncoupled in retro-Michael addition reactions where the drug is bound to an exposed thiol group.

[0068] Considering the above, the inventors have unexpectedly developed a peptide agonist having a linker with a maleimide or bromomaleimide coupling group that sterically selects and retro-Michael-resistant links albumin so that the peptide agonist remains covalently bonded to albumin without decoupling. 34 We discovered that it can be coupled to Cys 34 However, since the gap walls are occupied by multiple amino acid side chains, it is located in a hydrophobic gap with a depth of 9.5-10 Å, 34 We recognized that the spatial organization surrounding the thiol group can substantially affect the stereospecificity of the newly created chiral center. Surprisingly, we found that the spatial organization of the hydrophobic gaps could be modulated by changing the level of sodium octanoate (or other fatty acids or hydrophobic ligands) present in the hydrophobic fatty acid binding pockets of albumin during the bioconjugation process. In particular, we observed that when Michael addition was performed when all or most of the hydrophobic fatty acid binding pockets were occupied by octanoate or other hydrophobic molecules, the reaction product was substantially a racemic mixture (i.e., a 50 / 50 mixture of R- and S-configured chiral carbons in the conjugate), but the stereospecificity of the reaction increased as the level of octanoate in the binding pockets decreased. Furthermore, this unexpected finding was further emphasized when the pH during the conjugation reaction was below pH 7.0. Figure 26 shows the domains D1A-D3B, Cys 34 This shows human albumin having fatty acid binding pockets highlighted by numbers 1-7.

[0069] While we do not wish to be bound by any theory or hypothesis, the inventors believe that fatty acids (or other hydrophobic ligands) that bind to albumin are related to Cys 34 The thiol group of is preferentially or exclusively available for Michael addition reactions in only one plane of the maleimide ring, and therefore, the intention is to change the 3D conformation to preferentially or exclusively produce only one stereoisomer, resulting in a sterically selective chiral product. Furthermore, especially at lower pH (i.e., pH < 7.0), "Cys 34 The resulting microenvironment within the "hydrophobic gap" becomes inhibiting to the mechanistic steps necessary to support the retro-Michael reaction. Therefore, unexpectedly, the stereospecificity and corresponding microenvironment in the conformationally modified albumin guard of the bioconjugated adduct are inhibiting the retro-Michael binding of Cys 34 Protects against sulfur-carbon (MPA) cleavage.

[0070] Figure 27 shows that the thiol points upward near the bottom of the gap, and Asp 38 Cys with side chains pointing to the center 34 A side view of the hydrophobic gap is shown. In this unmodified albumin conformation, the hydrophobic pocket of albumin is occupied by octanoate, and the thiol group can participate in the reaction on both sides of the planar maleimide group. Figure 28A shows Cys 34 Asp 38 Figure 27 shows a schematic detail of the carboxylate moiety and thiol group forming hydrogen bonds with water molecules. As schematically shown by the two arrows in Figure 28A, Michael Donner (Cys 34 The sulfur atom of the acceptor approaches the Michael acceptor group (planar maleimide ring) indiscriminately with respect to the plane of the acceptor. As a result, the reaction product generates a chiral carbon center, and since there is no distinction with respect to the nucleophilic attack surface, the reaction product is a racemic product (i.e., substantially equal proportions of R- and S- configurations). Removal of one or more hydrophobic ligands from the binding pocket in albumin, particularly at pH < 7.0 (Asp 38(resulting in a protonated carboxylate group), the inventors have found that albumin is conformationally modified, and Cys 34 We discovered that the hydrophobic gap not only makes retro-Michael uncoupling stereochemically unfavorable, but also alters the reaction to result in sterically preferred or even sterically selective reaction products, as illustrated by a single arrow in Figure 28B. The conformational changes of albumin were analyzed using the Swiss pdb viewer (v3.7) (FEBS Journal 272(2005)353-362) to analyze the Cys residues in each structure. 34 and His 39 Figure 28C illustrates the structural superposition of unconformation-modified (dark gray) and conformation-modified (light gray) albumin prepared by aligning the skeletal atoms of the albumin.

[0071] Unexpectedly, despite relatively small conformational changes within the hydrophobic pocket, Cys in unmodified albumin 34 The previously "open" environment surrounding Cys has transformed into a stereochemically constrained environment. Furthermore, as can be seen from Figure 28C, 34 The sulfur atom is Tyr 84 They are oriented away from each other. Such changes create a chiral environment in which nucleophilic attack of the sulfur atom is preferred, or even restricted, to a single plane of the planar Michael acceptor, resulting in stereoisomers with retro-Michael resistance due to the steric constraints of the hydrophobic pocket.

[0072] In contrast, retro-Michael reactions in other compositions (e.g., various antibody-drug conjugates) have so far been avoided only by hydrolysis of the succinimide group formed after Michael addition. However, such hydrolysis occurs over a period of 24–60 hours at pH > 9 and temperatures in the range of 37–50°C. Clearly, under such conditions, both the protein and the pharmacologist, particularly in the case of peptide-based compounds, are deamidated at the amino acid amide side chain, resulting in the loss of function of the GPCR agonist compounds presented herein.

[0073] Therefore, the inventors should recognize that they were able to produce an albumin carrier that covalently holds the GPCR agonist so that the GPCR agonist is not substantially released upon administration of the compound, resulting in a highly effective compound without the accompanying release of the agonist, thereby eliminating the adverse effects associated with separately encountered free or deconjugated agonists. Furthermore, it should be understood that, due to the linker binding properties to albumin and the agonist, as will be discussed in more detail below, the agonist is also sterically free to engage with the GPCR so that the agonist can bind with high affinity and the agonist can activate the GPCR in a two-step manner.

[0074] Regarding the removal of octanoic acid or other hydrophobic compounds, including fatty acids, from albumin, all methods capable of reducing octanoate or other hydrophobic compounds without denaturing albumin are considered suitable for use herein, with particularly preferred methods including charcoal filtration, dialysis, and ion-exchange chromatography (see, e.g., Biochim Biophys Acta 1970 Nov 17;221(2):376-8;Nature Communications Materials;(2020)1:45). As is readily apparent, the albumin is preferably human serum albumin, which may be isolated and purified from human serum, or recombinantly produced using a bacterial expression system, or more typically a yeast expression system. Thus, the albumin intended herein may or may not be N or O glycosylated. In this regard, it should be understood that commercially available highly purified natural or recombinant albumins are typically stabilized with exogenously added octanoate or other short-chain or medium-chain fatty acids.

[0075] Furthermore, it should be understood that conformation-modified albumin can be formed by completely or partially degreasing albumin such that at least one, at least two, at least three, at least four, at least five, at least five, at least six, or all of the hydrophobic fatty acid binding pockets of albumin do not contain hydrophobic ligands. The appropriate degree of degreasing is at least partially determined by the desired degree of sterically selective reaction product. Thus, most typically, albumin is degreasing completely (all hydrophobic fatty acid binding pockets of albumin do not contain hydrophobic ligands) or almost completely (at least four hydrophobic fatty acid binding pockets of albumin do not contain hydrophobic ligands). Once degreased, the conformation-modified albumin thus prepared is then reacted with a linker containing a coupling group (typically containing a maleimide group or a bromo-maleimide group) preferably in equimolar ratios and at a pH of preferably pH < 7.0 (e.g., pH 6.0–6.9, or pH 5.5–6.0, or pH 5.0–5.5, or pH 4.5–4.5) according to standard Michael addition reaction conditions. However, an excess of albumin during the coupling reaction, e.g., 1.1 times, 1.2 times, 1.3 times, 1.4 times, or 1.5 times, or even greater molar excess, is intended to be appropriate. 34 Exemplary reaction conditions for coupling are described, for example, in Endocrinology (2005) 146 3052-3058, Bioorg. Med. Chem. Lett. (2003) 13 3571-3575, and Bioconjug. Chem. (2005) 16 1000-1008, each incorporated herein by reference.

[0076] Thus, the present inventors have demonstrated how to easily prepare conformation-modified albumin in solution, and how to perform conformational modifications, particularly Cys 34It should be noted that we have discovered that modifications in the steric environment of the hydrophobic gaps where are located can be used in a variety of biochemical reactions. Most advantageously, such modified environments can yield sterically preferred or even sterically selective reactions, and their enantiomers may yield preferred reaction products or even pure reaction products due to their chirality. For example, if a conformationally modified albumin has at least one (or at least two, at least three, at least four, at least five, or at least six) hydrophobic pockets not occupied by a hydrophobic ligand such as octanoate, sterically preferred or sterically selective Michael addition can be performed using a (typically planar) Michael acceptor reagent. Among other Michael acceptor reagents, if the Michael donor is Cys 34 When the thiol group is present, maleimide or bromomaleimide-containing reagents are particularly intended. As a result, as discussed above, especially when the pH is pH < 7.0, retro-Michael-resistant products (e.g., those containing linkers and class B GPCR agonist peptides as discussed herein) can be easily obtained from the reaction intermediate.

[0077] Therefore, from a different perspective, albumin is Cys 34 By reacting the sulfur atom with the coupling group, Cys 34Albumin can be modified in such a way that it should be understood that the modified albumin is a conformationally modified albumin in which at least one hydrophobic pocket of the conformationally modified albumin is not occupied by a non-covalently bonded lipid. As previously mentioned, such reactions are preferably carried out at a pH < 7.0, more typically 4.0 to 6.0. Advantageously, such reactions result in retro-Michael-resistant reaction products in which the coupling group is a planar Michael acceptor such as a maleimide or bromomaleimide group. Thus, these reactions can be used to reduce, or even completely avoid, retro-Michael reactions in albumin with Michael conjugates, particularly when such conjugates have therapeutic uses, such as agonist peptides having linkers and reactive groups, as enumerated in SEQ ID NOs: 1 to 471.

[0078] In yet another unexpected discovery, the inventors found that even if the octanoate level is higher during conjugation, unreacted Cys in the standard purified pharmaceutical formulation nevertheless... 34 We discovered that retro-Michael resistance fusion proteins can be generated via sterically selective translocation to unreacted albumin containing a group (which may or may not be conformationally modified). Alternatively, if the purification step is ultrafiltration or diafiltration, unreacted Cys 34 By adjusting the conjugation ratio of a peptide intermediate having an MPA as a linker and a reactive coupling moiety to albumin, a formulation containing an excess amount of unreacted albumin (which may or may not be conformationally modified) can be obtained. This unexpected discovery was based on the following observations.

[0079] Using an albumin-exendin-4 MPA conjugate construct very similar to AB-013, where the albumin was not conformationally modified and the construct was in an aqueous solvent at pH 7.4, it was observed that such a construct underwent significant and extensive retro-Michael deconjugation within the first 10 minutes after intravenous injection into mice, as determined by sandwich ELISA. Based on this observation, it was concluded that the conjugate peptide is cleaved from albumin in the retro-Michael reaction, and the only therapeutic method to prevent such a reaction is hydrolysis of the succinimide ring in the coupling group by stabilizing the thiol bond by increasing the pH at high temperature for an extended period (i.e., pH > 9).

[0080] To evaluate the pharmacokinetic profiles of both the unconformation-modified AB-013 conjugate and any free unconjugated peptide having linker and MPA groups that would be released into plasma, the inventors conducted studies over 96 hours after subcutaneous or intravenous injection. For this purpose, rats were subjected to intravenous injection of the unconformation-modified AB-013 conjugate at a dose of 100 nmol / kg and subcutaneous injection at a dose of 500 nmol / kg. The unconformation-modified AB-013 conjugate and the expected retro-Michael-free peptide were quantified separately by ELISA tests, but within the same timeframes: 2 minutes, 30 minutes, 1, 2, 4, 8, 24, 48, 72, and 96 hours for both administration routes.

[0081] From this study, the inventors observed that at the first intravenous (IV) time point, the concentrations of the unconformation-modified AB-013 intermediate and the free unconjugated AB-013 intermediate were 2528.8 nmol / L and 24.9 nmol / L, respectively (resulting in a ratio of 0.98%). Furthermore, from the pharmacokinetic profiles, the total area under the curve (AUC) for unconformation-modified AB-013 in intravenous time was 37418 (nmol / L*hours), while the AUC for the free unconjugated material was 53.69 (nmol / L*hours), resulting in a ratio of 0.14%. Surprisingly, unconformation-modified AB-013 and the unconjugated "free" peptide accounted for 99.86% and 0.14% of the drug's presence in plasma intravenously, respectively. Similarly, in the case of subcutaneous (SC) administration, the pharmacokinetic profile showed that the total area under the curve (AUC) of unconformed AB-013 was 18944 (nmol / L*hour), while the AUC of free unconjugated peptide was 143 (nmol / L*hour), representing a ratio of 0.7%.

[0082] Surprisingly, in contrast to the above observation of retro-Michael decoupling in the range of 20-40% during the first 10 minutes after intravenous injection, albumin cys 34 Such a retro-Michael reaction resulting in cleavage between the thiol and the reactive moiety MPA was not observed in this particular experiment. Furthermore, the inventors did not observe any pharmacokinetic parallelism between unconformationally modified AB-013 and the unconjugated or newly formed retro-Michael product. Such parallelism suggested that the retro-Michael reaction plays a major role in the degradation of unconformationally modified AB-013. Figure 29 shows exemplary results from the above experiment.

[0083] In a further experimental setup utilizing an unconformationally modified albumin-exendin-4 MPA-driven covalent construct very similar to the conformationally modified AB-013, it was reported that retro-Michael deconjugation occurred when such unconformationally modified constructs were placed in a pH > 9 solution and heated at 37°C for 20 hours, and that the deconjugation could be monitored by mass spectral analysis. Interestingly, the retro-Michael process occurred in only 50% of the MPA-driven covalent constructs used in that study, suggesting that some hydrolysis of the succinimide ring had occurred.

[0084] To further investigate the apparent contradiction, supplemental electrophoresis experiments were performed on plasma samples collected from rats that underwent subcutaneous and intravenous injection as described above, and the exemplary results are shown in Figure 30. As is readily apparent from the gel images, when unconformationally modified AB-013 was tested by electrophoresis, only one band was observed in the region where the albumin-exendin-4 MPA covalent construct is observed. Quite unexpectedly, all time points obtained during the experiment, including the earliest ones, had two bands in the region associated with the albumin-exendin-4 MPA covalent construct. The first band observed by electrophoresis was associated with unconformationally modified AB-013, and the second band immediately above it was associated with the exendin-4 MPA covalent construct, but covalently bound to rat albumin, while unconformationally modified AB-013 was covalently bound to human albumin.

[0085] Therefore, the inventors have surprisingly found that the retro-Michael deconjugated exendin-4 MPA construct converts human albumin to rat albumin Cys 34We discovered that it directly transfers in vivo. Interestingly, although the intensities of the two bands observed on the electrophoretic gel were similar (approximately 50-50), no detectable levels of free (decoupled) exendin-4 MPA construct were present. This initial observation was further supported when the dynorphin-A-MPA peptide was intravenously injected into rats. Importantly, this dynorphin-A-MPA peptide was not conjugated to human albumin or any other type of albumin. Therefore, it was injected with the intention of in vivo bioconjugation to rat albumin. In vivo bioconjugation did indeed occur in rat albumin monitored via the electrophoretic gel (data not shown). Interestingly, at each time point tested, only one band was observed on the electrophoretic gel when the dynorphin-A-MPA peptide was intravenously injected. In a similar manner, the dynorphin-A-MPA peptide was also conjugated in vitro to human unconformation-modified albumin, and the cystic compounds of human albumin were also conjugated. 34 A covalent bond was formed between them. The dynorphin A-human albumin covalent construct was intravenously injected into rats. Once again, the inventors then used human albumin's Cys 34 We observed direct in vivo transfer from covalently bound MPA peptide to rat albumin.

[0086] These results further support our findings that Michael addition occurring in the sterically constrained hydrophobic pocket of albumin leads to the formation of a chiral center, and that conformational modification of albumin leads to a conformationalized hydrophobic pocket that favors or even completely forms retro-Michael-resistant stereoisomers. Furthermore, the retro-Michael reaction may be even more unfavorable at lower pH levels, particularly below 7.0. From another perspective, the retro-Michael reaction requires a proper conformational structure within the hydrophobic pocket, along with proper stereochemical orientation. Indeed, even when experiments are designed to force the retro-Michael reaction under harsh conditions (pH > 9, high temperature (e.g., 50°C) and long duration (about 20 hours)), the retro-Michael reaction is observed in only about 50% of the theoretically possible retro-Michael reaction yield, strongly suggesting the importance of the chirality generated during the conjugation process and the limiting nature of the sterically constrained hydrophobic pocket.

[0087] Therefore, considering the above, Cys 34 Albumin is modified so that the sulfur atom of the coupling group is covalently bonded to the chiral carbon atom of the coupling group, and the chiral atom has a preferred stereochemical configuration (instead of the racemic carbon atom in which the thiol group is not sterically constrained as described above) by Cys 34 It should be recognized that chemical modification is possible with coupling groups. In fact, preferred stereochemistry can be preferred in ratios of at least 60:40, at least 70:30, at least 80:20, at least 85:15, at least 90:10, or at least 95:5, especially as the degree of degreasing increases (i.e., at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or all of the hydrophobic bonding pockets are not occupied by non-covalently bonded lipids).

[0088] Therefore, the present inventors have found that albumin is a Michael-added conjugate of Cys34 It should be noted that we have discovered that by modifying albumin with cystic linkage, we can produce chemically modified albumin in which the Michael addition conjugate is resistant to retro-Michael addition reactions. In fact, we have found that the coupling group (a hydrophilic linker and optionally a peptide coupled to the linker) can be modified in a sterically preferred manner (i.e., at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of all chiral carbon atoms in the CS bond in the coupling group have a retro-Michael resistant configuration) or sterically selective (98-100% of all chiral carbon atoms in the CS bond in the coupling group have a retro-Michael resistant configuration) to modify albumin with Cystic linkage. 34 It should be understood that it is now possible to prepare compositions that can covalently bond to a coupling group, thereby making the molecular entity bonded to the coupling group resistant to RetroMichael. While this chemically modified albumin is used for the delivery of insulin affinity agents, it is intended that this form of albumin, together with the linker described herein, may also function as a delivery system for cytotoxic agents, chemotherapeutic agents in the form of targeted therapies, or other drug conjugates, including antibody-drug conjugates (ADCs) in the field of cancer.

[0089] From a different perspective, in an isotonic buffer (pH 7.0) solution containing a retro-Michael-resistant conjugate of albumin and agonist peptide of interest, less than 40%, 30%, 20%, 15%, 10%, 8%, 6%, 4%, or 2% of all bound agonist peptides dissociate from albumin via the retro-Michael reaction over a 24-hour period at room temperature. Therefore, most linkers with covalently bound GPCR agonist peptides dissociate from albumin via the retro-Michael-resistant reaction. 34 It is important to understand that it binds to amino acids. Therefore, GPCR agonist peptides are sterically preferred or sterically selective in their configuration to bind to albumin's Cys 34Pharmaceutical compositions covalently bonded to amino acids substantially contain unbound GLP-1 agonist peptides (for example, 10% or less, or 7% or less, or 5% or less, or 3% or less, or 2% or less of the total agonist peptides in the composition are unbound).

[0090] As already mentioned above, such compositions have coupling groups and linkers (whether or not there is a bound peptide or other molecular entity) of conformationally modified albumin. 34 By reacting with, Michael addition is performed in the presence of free albumin in a molar excess relative to the coupling group, and / or by reacting with Cys 34 It can be prepared by adding free albumin to the Michael adduct produced by reacting with a coupling group and a linker (with or without other molecular entities such as a bound peptide or linker-bound GPCR agonist peptide). As can be easily understood, Cys 34 The ratio of free albumin to albumin with the bound ligand can vary, with suitable ratios typically being 1:5, 1:4, 1:3, 1:2, 1:1.5, 1:1.2, 1:1, 1.2:1, 1.5:1, 2:1, 3:1, 4:1, or 5:1. In each case where additional albumin is used, such albumin may and / or may not be conformationally modified. Furthermore, it should be recognized that once the coupling reaction between the linker (typically including the agonist peptide) and albumin is complete, the GPCR agonist fusion protein can be purified using standard methods well known in the art. Additionally, if desired, octanoate or a hydrophobic agent can be added to the fusion protein to reoccupy the hydrophobic binding site, thereby improving the stability of the fusion protein.

[0091] Returning to the intended linker, it should be noted that the linker coupled to the conformation-modified albumin is typically already covalently bonded to the GPCR agonist peptide, preferably via the epsilon amino group of lysine located at the C-terminus of the agonist peptide.

[0092] While at least conceptually simple, the synthesis of GPCR agonists containing reactive linkers is no trivial matter, especially when automated Merrifield-type synthesis is desired. Synthesis becomes even more complex when the agonist is exendin-4 or an exendin-4 derivative containing a tryptophan cage at the C-terminus, as this exhibits substantial steric hindrance to any chemistry proximal to the C-terminus. Furthermore, differential protection and deprotection are required, particularly when the agonist peptide is bound to the linker via a second reactive group, which can also be difficult to achieve in close proximity to the synthetic resin. From a different perspective, while linear GLP-1 analogs have been prepared on a solid phase, as described in U.S. Patent No. 6,514,500, no protocol has been established for performing orthogonal site-specific chemical reactions on the first amino acid immobilized on a solid support, and there are no known synthetic protocols for performing orthogonal site-specific chemical reactions on the first amino acid immobilized on a solid support when the peptide structure is highly organized at the C-terminus.

[0093] Considering the above difficulties, the inventors unexpectedly discovered that a successful protocol for carrying out orthogonal site-specific chemical reactions on a first amino acid immobilized on a solid support requires a favorable distance between the solid support (here, polystyrene beads) and the immobilization site of the first amino acid. In one particularly preferred embodiment, a tricyclic amide resin (Ramage resin) provided the necessary and preferred distance for successfully carrying out orthogonal protection / deprotection. Advantageously, the inventors also found that such tricyclic amide resins are suitable for large-scale and cGMP-scale production.

[0094] In the following examples, small-scale peptide synthesis of the AB-013 intermediate was performed on a modified tricyclic amide resin (Ramage resin) using an automated solid-phase procedure in a Symphony® peptide synthesizer with manual intervention to carry out orthogonal chemistry incorporating a linker such as AEEA (amide(ethoxy)ethoxy)acetate acyl and a reactive moiety such as maleimidopropionate acyl. As can be easily understood, the same synthetic strategy can be carried out for other GPCR agonist peptides presented herein.

[0095] As is readily apparent, the appropriate selection of the resin substitution level is a function of the desired synthetic efficiency, the length of the peptide intermediate, and the complexity of the structural conformation combined with the position of such complexity along the peptide chain. However, preferred agonist peptides have a length of 30–45 amino acids with significant structural conformational complexity (here, a Trp cage) at the C-terminus of the peptide. Furthermore, in most embodiments (though not all intended embodiments), orthogonal chemistry is performed on the first amino acid immobilized in the resin. Therefore, a suitable distance between the polystyrene and the immobilization site of the first amino acid is preferred, as described above. Advantageously, the modified Ramage resin structure and substitution level address synthetic efficiency, the length of the peptide intermediate, the complexity / position of the structural conformation, and the orthogonal chemistry on the first amino acid immobilized in the resin. Preferably, though not necessarily, the substitution level of the Ramage resin is about 0.4–0.6 mmol / gram.

[0096] Regarding scale-up, it should be noted that the batch size of the peptide intermediate depends on the scale of the synthesis and the substitution of the Ramage resin. This provides the necessary information regarding the amount of Ramage resin for a particular batch. For example, a 100 mmol synthesis scale using a modified Ramage resin with a substitution level of 0.5 mmol / g requires approximately 200 g of starting Ramage resin and has a theoretical yield of approximately 8.5% for a 40-mer peptide. Therefore, a 100 mmol batch size is a suitable large-scale (e.g., cGMP) batch size for process validation, while a batch size of 100 µl / g of modified Ramage resin is appropriate for study scales.

[0097] The N-terminus of an amino acid is relatively easily removed to enable peptide bond formation and is therefore protected by a group called a “temporary” protecting group. Two common N-terminal protecting groups are tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc), each having distinct characteristics that determine their use. Boc requires a moderately strong acid, such as trifluoroacetic acid (TFA), to be removed from the newly added amino acid, while Fmoc is a base-unstable protecting group that is removed with a mild base, such as piperidine. Due to the mild deprotection conditions, Fmoc chemistry is more commonly used in commercial settings for higher quality and higher yield, but Boc is preferred for complex peptide synthesis or when a base-sensitive non-natural peptide or analog is required. Thus, the protecting groups intended herein fall into two categories: base-unstable and acid-unstable. Base-unstable protecting groups are typically used to block an α-amino group during a coupling reaction and are then removed in a deblocking step to allow the introduction of the next amino acid in the sequence. For example, 9-fluorenylmethoxycarbonyl (Fmoc) can be used as a base-unstable α-amino protecting group. In another example, acid-unstable protecting groups (e.g., triphenylmethyl (Trt) / t-butyl (tBu) / t-butyl ester (OtBu) / t-butyloxycarbonyl (Boc) / 2,2,5,7,8, pentamethylchroman-6-sulfonyl (Pmc) / methyltrityl (Mt)) can be used to protect the side-chain reactive functional groups of amino acids during synthesis and must be resistant to deblocking mixtures (e.g., 20% piperidine, v / v in dimethylformamide (DMF)). Once peptide synthesis is complete, these protecting groups are removed by a strong acid (e.g., an 85% aqueous trifluoroacetic acid solution (TFA) containing a scavenger).

[0098] One particularly preferred method for preparing the peptide agonists presented herein involves solid-phase peptide synthesis in which the amino acid N-terminus is protected by an acid or base-sensitive group, while the peptide chain being extended can be easily removed without disrupting the chain or racemizing any of the chiral centers contained therein. Examples of N-protecting groups and carboxyl protecting groups are disclosed by reference in Greene, "Protecting Groups in Organic Synthesis," (John Wiley & Sons, New York, pp. 152-186 (1981)), which is incorporated herein by reference.Examples of N-protecting groups include, but are not limited to, lower alkanoyl groups such as formyl, acetyl("Ac"), propionyl, pivaloyl, and t-butylacetyl; other acyl groups include 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, o-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, etc.; benzenesulfonyl, p-toluenesulfonyl, o-nitrophenylsulfonyl, 2,2,5,7, Sulfonyl groups such as 8-pentamethylchroman-6-sulfonyl (pmc); t-amyloxycarbonyl, benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 1,2,4-dimethoxybenzyloxycarbonyl, 4-ethoxybenzyloxycarbonyl, 2- Nitro-4,5-dimethoxybenzyloxycarbonyl, 1,3,4,5-trimethoxybenzyloxycarbonyl, 1,1-(p-biphenylyl)-1-methylethoxycarbonyl, a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, carbamate-forming groups such as 2,2,2, -trichloroethoxycarbonyl Examples include carbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, isobromooxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, etc.; arylalkyl groups, such as benzyl, biphenylisopropyloxycarbonyl, triphenylmethyl, benzyloxymethyl, 9-fluorenylmethyloxycarbonyl (Fmoc), etc.; and silyl groups, such as trimethylsilyl.Preferred aN-protecting groups include o-nitrophenylsulfenyl, 9-fluorenylmethyloxycarbonyl, t-butyloxycarbonyl (boc), isobutyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, t-amyloxycarbonyl, and 2-cyano-t-butyloxycarbonyl, with 9-fluorenylmethyloxycarbonyl (Fmoc) being more preferred. On the other hand, preferred side-chain N-protecting groups include 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, and 4-methoxybenzene-sulfonyl, due to the presence of amino side-chain groups such as lysine and arginine. It includes honyl, Cbz, Boe, and adamantyloxycarbonyl; in the case of tyrosine, benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl, and acetyl (Ac); serine is t-butyl, benzyl, and tetrahydropyranyl; in the case of trityl, benzyl, Cbz, p-toluenesulfonyl, and histidine, it is 2,4-dinitrophenyl; tryptophan is formyl or Boc; aspartic acid and glutamic acid are benzyl and t-butyl; and cysteine ​​is triphenylmethyl (trityl).

[0099] A carboxyl protecting group conventionally refers to a carboxylic acid-protected ester or amide group. Such carboxyl protecting groups are well known to those skilled in the art and are widely used for protecting carboxyl groups in the penicillin and cephalosporin fields, as described, for example, in U.S. Patent No. 3,840,556 and U.S. Patent No. 3,719,667, both of which are incorporated herein by reference. Typical carboxy protecting groups are not particularly limited, but include C1-C8 lower alkyl groups, arylalkyl groups, e.g., phenethyl or benzyl and their substituted derivatives, e.g., alkoxybenzyl or nitrobenzyl groups; aryl alkenyls such as phenylethenyl; aryls and their substituted derivatives, e.g., 5-indanyl; dialkylaminoalkyls such as dimethylaminoethyl; alkoxymethyl, butyryloxymethyl, valeryloxymethyl, isobutyryloxymethyl, isovaleryloxymethyl, 1-(propionyloxy)-1-ethyl, 1-(pivaloyloxy)-1-ethyl, 1-methyl1-(propionyloxy)-1-ethyl, pivaloyloxymethyl, propionyloxymethyl and other alkanoyloxyalkyl groups; cyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxymethyl, cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxymethyl Cycloalkanoyloxyalkyl groups such as tyl; aroyloxyalkyl groups such as benzoyloxymethyl and benzoyloxyethyl; arylalkylcarbonyloxyalkyl groups, e.g., benzylcarbonyloxymethyl, 2-benzylcarbonyloxyethyl; alkoxycarbonylalkyl groups or cycloalkyloxycarbonylalkyl groups, e.g., methoxycarbonylmethyl, cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl; alkoxycarbonyloxyalkyl groups or cycloalkyloxycarbonyloxyalkyl groups, e.g., methoxycarbonyloxymethyl, t-butyloxycarbonyloxymethyl, 1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl; aryloxycarbonyloxyalkyl groups such as 2-(phenoxycarbonyloxy)ethyl and 2-(5-indanyloxycarbonyloxy)ethyl;Alkoxyalkylcarbonyloxyalkyls such as 2-(1-methoxy-2-methylpropane-2-yloxy)ethyl; arylalkyloxycarbonyloxyalkyls such as 2-(benzyloxycarbonyloxy)ethyl; arylalkenyloxycarbonyloxyalkyls such as 2-(3-phenylpropene-2-yloxycarbonyloxy)ethyl; alkoxycarbonylaminoalkyls such as t-butyloxycarbonylaminomethyl; alkylaminocarbonylaminoalkyls such as methylaminocarbonylaminomethyl; acetylaminomethyl and other alkoxycarbonylaminoalkyls. Examples include canoylaminoalkyl groups; heterocyclic carbonyloxyalkyl groups such as 4-methylpiperazinylcarbonyloxymethyl; dialkylaminocarbonylalkyl groups such as dimethylaminocarbonylmethyl and diethylaminocarbonylmethyl; (5-(lower alkyl)-2-oxo-1,3-dioxolenn-4-yl)alkyl groups such as (5-t-butyl-2-oxo-1,3-dioxolenn-4-yl)methyl; and (5-phenyl-2-oxo-1,3-dioxolenn-4-yl)alkyl groups such as (5-phenyl-2-oxo-1,3-dioxolenn-4-yl)methyl. Typical amide carboxy protecting groups include, but are not limited to, aminocarbonyl groups and lower alkylaminocarbonyl groups. Among the carboxyl protecting groups mentioned above, lower alkyl, cycloalkyl, or arylalkyl esters, such as methyl esters, ethyl esters, propyl esters, isopropyl esters, butyl esters, sec-butyl esters, isobutyl esters, amyl esters, isoamyl esters, octyl esters, cyclohexyl esters, phenylethyl esters, etc., or alkanoyloxyalkyl, cycloalkanoyloxyalkyl, aroyloxyalkyl, or arylalkylcarbonyloxyalkyl esters are preferred. The preferred amide carboxyl protecting group is the lower alkylaminocarbonyl group.

[0100] The C-terminal amino acid is N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), or O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium-hexafluorophosphate (HBTU), 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), benzotriazole-1-yloxytris(dimethylamino)phosphonium-hexafluorophosphate (BOP), or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), or oxymethylcyanohydroxyiminoacetate, or HATU (1-[bis(dimethylamino)methylene]-1H The resin is bonded using -l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, or TBTU 2-(1H-benzotriazolyl)-1,1,3,3-tetramethylaminium tetrafluoroborate, or TNTU 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethyluronium tetrafluoroborate, or PyOxim 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidinophosphonium hexafluorophosphate, or PyClock 6-chloro-benzotriazolyloxy-tris-pyrrolidinophosphonium hexafluorophosphate diethylamine (DEA).

[0101] The coupling is carried out at a temperature of 10°C to 50°C for approximately 1 to 24 hours in a solvent such as dichloromethane, DMF, and NMP, and / or a solvent mixture suitable for amino acid coupling and / or acylation.

[0102] During the activation / coupling step, the deprotected α-amino group is acylated by the next activated amino acid in the sequence. As is easily understood, the reagents used to achieve acylation are selected to create optimal reaction conditions and to facilitate the removal of excess reagent at the end of the coupling reaction. Activation of each acid coupling is carried out using TBTU / HBTU and DIPEA. DIPEA is used as an additive to complete the reaction. TBTU / HBTU and DIPEA are dissolved in DMF:NMP (1:1) and added to the appropriate Fmoc-protecting amino acid to generate an acylation process for the free Nα-amino functional group attached to the fully protected peptide that remains extended while attached to the modified Ramage resin.

[0103] During the deprotection step, the base instability protecting group (Fmoc) is cleaved from the α-amino functional group of the N-terminal amino acid on the elongating peptide chain by treating the resin twice with a 20% solution of piperidine in DMF (v / v). Typically, the resin is stirred for 20 minutes, then the 20% piperidine / DMF solution is filtered, and the treatment is repeated for a second period of 20 minutes with a fresh 20% piperidine / DMF solution. This treatment is typically sufficient to allow complete cleavage of the Fmoc protecting group without affecting the stability of the acid instability protecting group present on the elongating peptide chain.

[0104] In one example, the synthesis of the AB-013 intermediate was carried out using a tricyclic amide resin consisting of a Ramage "connector" structurally defined as (9H-fluoren-9-yl)methyl-N-(6-{[(1-{[(4-methylphenyl)(phenyl)methyl]carbamoyl}pentyl)carbamoyl]methoxy}tricyclo[9.4.0.03,8]pentadeca-1(11),3(8),4,6,12,14hexaen-2-yl)carbamate bonded to a polystyrene support crosslinked with 1% divinylbenzene (DVB). Next, the Ramage "connector" was modified to 2-[2-({2-aminotricyclo[9.4.0.03,8]pentadeca-1(11),3(8),4,6,12,14hexaen-6-yl}oxy)acetamide]-N[(4methylphenyl)(phenyl)methyl]hexaneamide (modified Ramage resin), enabling the immobilization of the first amino acid onto a solid support under standard coupling conditions.

[0105] The peptide synthesis methodology utilized a conventional Fmoc-protected amino acid approach using appropriately orthogonally protected amino acids. Coupling was achieved by using O-benzotriazole-1-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA) as an activator mixture in N,N-dimethylformamide (DMF) solution. The Fmoc protecting group was removed using 20% ​​piperidine / DMF (v / v). A Boc-protected amino acid was used at the N-terminus of the peptide on a solid support to generate a free Nα-terminus after AB-013 was cleaved from the resin. All amino acids used during synthesis were L-amino acids, and a sigma-coated glass reaction vessel was used during synthesis. As described above, the synthetic processes for producing various peptide analogs and derivatives vary greatly depending on the properties of the various elements contained in the peptide analog or derivative, namely the peptide analog sequence, coupling groups, and reactive entities. In most situations, chemically reactive coupling groups (such as MPA or bromo-MPA) are coupled in the final stage of synthesis. As should be understood, the chemically reactive coupling group should be positioned at a site that allows the peptide to bind to conformationally modified albumin while maintaining its biological activity.

[0106] Peptide elongation on a solid support: Solid-phase peptide synthesis was performed on a 100 μmol scale using modified Ramage resin (substitution level 0.45 mmol / g). The following protective amino acids were sequentially added to the resin: Fmoc-Lys40(Aloc)-OH; Fmoc-Ser(tBu)-OH; Fmoc-Pro-OH; Fmoc-Pro-OH; Fmoc-Ala-OH.(H2O); Fmoc-Gly-OH; Fmoc-Ser(tBu)-OH; Fmoc-Ser(tBu)-OH; Fmoc-Pro-OH; Fmoc-Gly-OH; Fmoc-Gly-OH; Fmoc-Asn(Trt)-OH; Fmoc-Lys(Boc)-OH; Fmoc-Leu-OH; Fmoc-Trp(Boc)-OH; Fmoc-Glu(OtBu)-OH(H2O); Fmoc-Ile-OH; Fmoc-Phe-OH; Fmoc-Leu-OH; Fmoc-Arg(P mc)-OH;Fmoc-Val-OH;Fmoc-Ala-OH.(H2O);Fmoc-Glu(OtBu)-OH(H2O);Fmoc-Glu(OtBu)-OH(H2O );Fmoc-Glu(OtBu)-OH(H2O);Fmoc-Met-OH;Fmoc-Gln(Trt)-OH;Fmoc-Lys(Boc)-OH;Fmoc-Ser(tB u)-OH;Fmoc-Leu-OH;Fmoc-Asp(OtBu)-OH;Fmoc-Ser(tBu)-OH;Fmoc-Thr(tBu)-OH;Fmoc-Phe-OH ;Fmoc-Thr(tBu)-OH;Fmoc-Gly-OH;Fmoc-Glu(OtBu)-OH(H2O);Fmoc-Gly-OH;Boc-His1(Boc)-OH.

[0107] A molar excess of Fmoc-amino acid / HBTU / DIEA was used for each amino acid coupling (3-5 equivalents), dissolved in N,N-dimethylformamide (DMF), and activated in order with O-benzotriazole-1-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was performed with a 20% piperidine / DMF solution for 20 minutes. After each coupling and / or Fmoc removal, the modified Ramage resin was subjected to a washing cycle of 3×DMF, followed by 3×isopropanol. This was repeated three more times, with a final wash of 1×DMF to swell the resin and prepare it for the next chemical step. It should be noted that the first amino acid immobilized on the solid support may also be Fmoc-Lys(Mtt)-OH or Fmoc-Lys(ivDDE)-OH as a substitute for Fmoc-Lys(Alloc)-OH, or any Nα-Fmoc-amino acid having a well-protected orthogonal functional group that can result in the execution of orthogonal chemistry incorporating a linker such as AEEA and a moiety reactive to maleimide propion acyl (MPA).

[0108] Orthogonal chemistry, specifically the removal of orthogonal protecting groups from a first amino acid immobilized on a solid support, followed by coupling of the linker AEEA with the reactive moiety MPA, was selectively performed on the first amino acid immobilized on a modified Ramage resin. The orthogonal protecting group Alloc on Lys40 was removed over 1 hour with 0.2 equivalents of tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) and 2 equivalents of tributyltin hydride (Bu3SnH) in dry DCM. The resin was subjected to a washing cycle of 3×DMF followed by 3×isopropanol. This was repeated three more times, with a final wash of 1×DMF to swell the resin and prepare it for the next chemical step. Next, the free amino functional groups on the side chains of Lys40 directly immobilized on the modified Ramage resin were acylated with Fmoc-AEEA-COOH under standard HBTU / DIEA coupling conditions, followed by a standard washing cycle. Next, the Fmoc protecting the AEEA hydrophilic linker was removed using 20% ​​piperidine / DMF for 20 minutes, followed by a standard washing cycle. Subsequently, the free terminal amino portion of AEEA was acylated under standard HBTU / DIEA coupling conditions to obtain the AB-013 intermediate.

[0109] Cleavage of the AB-013 intermediate from the resin and removal of all orthogonal protecting groups: The AB-013 intermediate was cleaved from the resin using 85% trifluoroacetic acid (TFA) / 5% triisopropylsilane (TIS) / 5% thioanisole and 5% phenol, followed by precipitation using ice-cold (0-4°C) ethyl ether (Et2O). The crude peptide was recovered in a polypropylene sintered funnel, dried, redissolved in a 40% mixture of acetonitrile (CH3CN) in water (0.1% TFA), and freeze-dried to produce the corresponding crude material used in the purification process.

[0110] Purification: The AB-013 intermediate was purified by preparative reverse-phase HPLC using a preparative two-component HPLC system. Purification was performed using a 10μ phenylhexyl, 50mm x 250mm column (10μ) equilibrated with a water / TFA mixture (0.1% TFA in H2O (solvent A) and acetonitrile / TFA (0.1% TFA in CH3CN (solvent B))). Elution was achieved at 50 mL / min by running a 28–38% B gradient over 180 minutes. Fractions containing the peptide were detected by UV absorbance at 214 nm and 254 nm. The fractions were collected in 25 mL aliquots. The molecular weight of the peptide was determined by quadrupole electrospray mass spectrometry. Fractions containing the desired product were identified by liquid chromatography-mass spectrometry (LC-MS). Subsequently, the selected fractions were eluted by analytical HPLC (20–60% B gradient over 20 minutes; Phenomenex Luna The fractions with a purity exceeding 90% for pooling were identified by analysis using a 5μF phenylhexyl column (10mm × 250mm column, 0.5mL / min). The pooled fractions were homogenized, freeze-dried using liquid nitrogen, and then freeze-dried for 2-3 days to obtain a white amorphous powder.

[0111] Of course, the above examples and considerations mainly relate to the synthesis of linear peptides in which the reactive coupling group is covalently bonded to the epsilon amino group of the C-terminal lysine amino acid. However, it should also be recognized that the reactive coupling group may also be covalently bonded to the epsilon amino group of a lysine amino acid at a position other than the terminal position (e.g., between the 2nd and 10th amino acids, or between the 11th and 20th amino acids, or between the 21st and 30th amino acids, or between the 31st and 39th amino acids). Figure 31 shows exemplary structures of peptide agonist intermediates and protected intermediates of AB-013 bound to Ramage resin or Rink resin. Most typically, when the linker and the reactive coupling group are at the C-terminus of the agonist peptide, Ramage resin is typically preferred. On the other hand, when the linker and the reactive coupling group are coupled to the agonist at an intermediate position (and typically, beyond the tryptophan cage if present), Rink (amide) resin can be used. As can be readily appreciated, all resin-bound intermediates may be fully protected or at least partially deprotected. Thus, an exemplary intended agonist peptide intermediate may have the sequence of SEQ ID NO: 322 shown generally and schematically in Figure 32. Thus, when bound to the resin, the intermediate may also have the sequences of SEQ ID NO: 322 and SEQ ID NO: 323 shown below, having the variable amino acids shown in Figure 32 and listed in the sequence listing. X1X2X3GTFTSDX4X5X6X7LX8X9X 10 X 11 X 12 X<0000o82>X 14 FX 15 X 16 WLX 17 X 18 GX 19 PSSGAPPPSX 20 -Ramage resin SEQ ID NO: 322 X1X2X3GTFTSDX4X5X6X7LX8X9X 10 X 11 X 12 X 13 X14 FX 15 X 16 WLX 17 X 18 GX 19 PSSGAPPPS-Rink resin SEQ ID NO: 323

[0112] Furthermore, it should be recognized that the agonist peptide can be synthesized in a single linear Merrifield synthesis process or in a synthesis process in which two or more partial sequences are condensed into the final sequence product. For example, the first peptide partial sequence of SEQ ID NO: 324 can be coupled to Ramage resin or Rink resin, while the second and third partial sequences of protected SEQ ID NO: 325 and SEQ ID NO: 326 are prepared on their respective chlorotrityl resins using variable amino acids as shown in FIG. 32 as follows. NH2X 19 PSSGAPPPSX 20 -Ramage resin SEQ ID NO: 324 Fmoc-X 11 X 12 X 13 X 14 FX 15 X 16 WLX<{ 17 X 18 G-chlorotrityl resin SEQ ID NO: 32^ Boc-X1X2X3GTFTSDX4X5X6X7LX8X9X 10 -chlorotrityl resin SEQ ID NO: 326

[0113] After cleavage of the second and third partial sequences from the resin, resulting in SEQ ID NO: 325 with a protected N-terminus and SEQ ID NO: 326 with an unprotected C-terminus, respectively, Fmoc-X 11 X 12 X 13 X 14 FX 15 X 16 WLX 17 X 18 G-COOH SEQ ID NO: 325 Boc-X1X2X3GTFTSDX4X5X6X7LX^X9X 10-COOH Sequence ID 326 The second subsequence, SEQ ID NO: 325, is condensed with the first subsequence, SEQ ID NO: 324, to produce the resin-bound SEQ ID NO: 328 shown below. Of course, other first peptide subsequences are also considered appropriate, and it should be understood that the exemplary subsequence is SEQ ID NO: 327 bound to the Rink resin. FmocX 11 X 12 X 13 X 14 FX 15 X 16 WLX 17 X 18 G-X19PSSGAPPPSX20-Ramage Resin, Sequence Number 328 While the reaction product remains on the resin, the N-terminal protecting group is removed, resulting in a deprotected SEQ ID NO: 328 having a free NH2 group at the N-terminus. NH2-X 11 X 12 X 13 X 14 FX 15 X 16 WLX 17 X 18 GX 19 PSSGAPPPSX 20 - Ramage resin, SEQ ID NO: 328 This is obtained, to which a third deprotected subsequence, SEQ ID NO: 326, having a deprotected C-terminus, is bound, resulting in the final resin-bound SEQ ID NO: 329 having variable amino acids, as shown in Figure 32. Boc-X1X2X3GTFTSDX4X5X6X7LX8X9X 10 -X 11 X 12 X 13 X 14 FX 15 X 16 WLX 17 X 18 GX 19 PSSGAPPPSX 20 - Ramage resin, Sequence ID 329

[0114] From the above, it can be understood that a peptide solid synthesis intermediate can be easily prepared, which contains a Ramage resin in which the first amino acid of the peptide is covalently coupled to the C-terminus of the peptide via an amide bond, and the first amino acid is further covalently coupled to a linker via an amide bond in the side chain of the first amino acid, and the linker contains a reactive coupling group.

[0115] As already mentioned above, agonists having linkers and reactive coupling groups can also be prepared, where the linkers and reactive coupling groups are located in intermediate positions in the agonist peptide. For example, the synthesis of intermediates with reactive coupling groups usually follows known methodologies (see, e.g., Stewart et al. in "Solid Phase Peptide Synthesis", WH Freeman Co. (San Francisco), 1963 and Meienhofer, Hormonal Proteins and Peptides, 1973, 2 46. For classical solution synthesis, see, e.g., Schroder et al. in "The Peptides", volume 1, Academic Press (New York)). Such methodologies typically involve the sequential addition of one or more amino acids or appropriately protected amino acids to the elongating peptide chain. Usually, the amino or carboxyl group of the first amino acid is protected by an appropriate protecting group. Next, the protected or derivatized amino acids are made available in solution by attaching them to an inert solid support, or by adding the next amino acid in a sequence having a properly protected complementary (amino or carboxyl) group under conditions suitable for forming an amide bond. Then, the protecting group is removed from this newly added amino acid residue, and the next amino acid (properly protected) is added, and so on. After all the desired amino acids have been linked in the appropriate sequence, any remaining protecting groups (and any solid support) are removed sequentially or simultaneously to obtain the final polypeptide. Simple modifications to this general procedure make it possible to add two or more amino 5 acids to the growth chain at once, for example, by coupling a protected tripeptide with a properly protected dipeptide (under conditions that do not racemize the chiral center) to form a pentapeptide after deprotection.

[0116] Using a solid support 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamide MBHA resin (linkamide MBHA resin), the Fmoc group is cleaved with a secondary amine, preferably piperidine in DMF (20% v:v in DMF), before coupling with the α-C-terminal amino acid. Methods for coupling to the deprotected 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamide MBHA resin include O-benzotriazole-1-yl N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), diisopropylethylamine (DIEA), and optionally 1-hydroxybenzotriazole (HOBT) in DMF. Coupling of successive protected amino acids can be carried out by conventional methods well known in the art, using automated and / or semi-synthetic and / or manual peptide synthesizers. Removal of the Fmoc protecting group from the αN-terminus of the growing peptide is conventionally achieved, for example, by treatment with a secondary amine, preferably piperidine, in DMF (20% v:v). Each protecting amino acid is then introduced in a 1-5 molar excess, and coupling is preferably carried out in DMF and / or NMP. Upon completion of peptide elongation on a solid support and the addition of orthogonal functional groups such as linkers and reactive moieties to the fully protected peptide, it is removed from the resin and deprotected in either a continuous or single operation. Removal and deprotection of the polypeptide can be achieved in a single operation, as conventionally, by treating the resin-bound polypeptide with cleavage reagents including thioanisole, triisopropylsilane, phenol, and trifluoroacetic acid. If the aC-terminus of the polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide can be removed, for example, by transesterification with methanol, followed by aminolysis or direct amide exchange. The protected peptide may be purified at this point, or it may proceed directly to the next step. The removal of the side chain protecting group is achieved using the cleavage mixture described above.

[0117] Completely deprotected peptides can be purified by a series of chromatographic steps using one or all of the following types: ion exchange with a weakly basic resin (acetate form); hydrophobic adsorption chromatography with non-derivative polystyrene-divinylbenzene (e.g., Amberlite XAD®); silica gel adsorption chromatography; ion exchange chromatography with carboxymethylcellulose; partition chromatography in countercurrent distribution, e.g., Sephadex G-25®, LH-20®; or high-performance liquid chromatography (HPLC), particularly reversed-phase HPLC with octyl- or octadecylsilyl silica bonded-phase column packing. Anyone skilled in the art will readily be able to determine which chromatographic steps or sequences are preferred to obtain an acceptable purification of the peptide analog.

[0118] For example, small-scale peptide synthesis of the AB-092 intermediate can be performed as follows.

[0119] Peptide elongation on a solid support: Solid-phase peptide synthesis was performed on a 100 μmol scale using modified Rink MBHA resin (substitution level 0.5 mmol / g). The following protective amino acids were sequentially added to the resin: Fmoc-Ser39(tBu)-OH; Fmoc-Pro-OH; Fmoc-Pro-OH; Fmoc-Ala-OH.(H2O); Fmoc-Gly-OH; Fmoc-Ser(tBu)-OH; Fmoc-Ser(tBu)-OH; Fmoc-Pro-OH; Fmoc-Gly-OH; Fmoc-Gly-OH; Fmoc-Glu(OtBu)-OH(H2O); Fmoc-Leu-OH; Fmoc-Leu-OH; Fmoc-Tyr(OtBu)-OH; Fmoc-Glu(OtBu)-OH(H2O); Fmoc-Ile-OH; Fmoc-Phe-OH; Fmoc-Ala-OH.(H2O); Fmoc-Aib- OH;Fmoc-Gln(Trt)-OH;Fmoc-Ala-OH(H2O);Fmoc-Lys(Alloc)-OH;Fmoc-Lys(Boc)-OH;Fmoc- Asp(OtBu)-OH;Fmoc-Leu-OH;Fmoc-αMetyl-Leu-OH;Fmoc-Ile-OH;Fmoc-Ser(tBu)-OH;Fmoc-T yr(OtBu)-OH;Fmoc-Asp(OtBu)-OH;Fmoc-Ser(tBu)-OH;Fmoc-Thr(tBu)-OH;Fmoc-Phe-OH;Fm oc-Thr(tBu)-OH;Fmoc-Gly-OH;Fmoc-Gln(Trt)-OH(H2O);Fmoc-Aib-OH;Boc-Tyr1(OtBu)-OH. Molar excess Fmoc-amino acids / HBTU / DIEA were used for each amino acid coupling (3-5 equivalents). These were dissolved in N,N-dimethylformamide (DMF) and activated in order with O-benzotriazole-1-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Fmoc protecting groups were removed using a 20% piperidine / DMF solution for 20 minutes. After each coupling and / or Fmoc removal, the modified Ramage resin was subjected to a washing cycle of 3×DMF, followed by 3×isopropanol.This process was repeated three more times, followed by a final wash with 1×DMF to swell the resin and prepare it for the next chemical step. Alternatively, the first amino acid immobilized on the solid support could be Fmoc-Lys(Mtt)-OH or Fmoc-Lys(ivDDE)-OH instead of Fmoc-Lys(Alloc)-OH, or any Nα-Fmoc-amino acid having a appropriately protected orthogonal functional group, thereby enabling orthogonal chemical reactions to incorporate a linker (not limited to AEEA) and a reactive moiety (not limited to maleimidopropionate acyl (MPA)).

[0120] Following the removal of orthogonal protecting groups on the first amino acid immobilized on a solid support, the linker AEEA and the reactive moiety MPA were coupled twice. A selective orthogonal chemical reaction was performed on the first amino acid immobilized on the Rink amide MBHA resin. The orthogonal protecting group Alloc on Lys17 was removed over 1 hour with 0.2 equivalents of tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) and 2 equivalents of tributyltin hydride (Bu3SnH) in dry DCM. The resin was subjected to a washing cycle of 3×DMF followed by 3×isopropanol. This was repeated three more times, with a final wash of 1×DMF to swell the resin and prepare it for the next chemical step. Lys17 was directly immobilized on the Rink amide MBHA resin. 17 The free amino functional group on the side chain was acylated twice with Fmoc-AEEA-COOH under standard HBTU / DIEA coupling conditions. A standard washing cycle was then followed. Next, Fmoc protecting the AEEA hydrophilic linker was removed with 20% piperidine / DMF for 20 minutes, followed by a standard washing cycle. Then, the free terminal amino moiety of the second AEEA was acylated under standard HBTU / DIEA coupling conditions.

[0121] Cleavage of the AB-092 intermediate from the resin and removal of all orthogonal protecting groups: The AB-092 intermediate was cleaved from the resin using 85% trifluoroacetic acid (TFA) / 5% triisopropylsilane (TIS) / 5% thioanisole and 5% phenol, followed by precipitation using ice-cold (0-4°C) ether (Et2O). The crude peptide was recovered in a polypropylene sintered funnel, dried, redissolved in a 40% mixture of acetonitrile (CH3CN) in water (0.1% TFA), and freeze-dried to produce the corresponding crude material used in the purification process.

[0122] Purification protocol for small-scale synthesis: The AB-092 intermediate was purified by preparative reverse-phase HPLC using a preparative two-component HPLC system. Purification was performed using a 10μ phenylhexyl, 50mm x 250mm column (10μ) equilibrated with a water / TFA mixture (0.1% TFA in H2O (solvent A) and acetonitrile / TFA (0.1% TFA in CH3CN (solvent B))). Elution was achieved at 50 mL / min by running a 28–38% B gradient over 180 minutes. Fractions containing the peptide were detected by UV absorbance at 214 nm and 254 nm. The fractions were collected in 25 mL aliquots. The molecular weight of the peptide was determined by quadrupole electrospray mass spectrometry. Fractions containing the desired product were identified by liquid chromatography-mass spectrometry (LC-MS). Subsequently, the selected fractions were eluted by analytical HPLC (20–60% B gradient over 20 minutes; Phenomenex Luna The fractions with a purity exceeding 90% for pooling were identified by analysis using a 5 μF phenylhexyl column (10 mm × 250 mm column, 0.5 mL / min). The pooled fractions were homogenized, freeze-dried using liquid nitrogen, and then freeze-dried for at least 2-3 days to obtain a white amorphous powder.

[0123] Therefore, in consideration of the above, it should be understood that the intended resin-immobilized cysteine-reactive class BGPCR agonist peptide includes a GPCR agonist peptide containing a Lys amino acid, in which (a) the linker is covalently bonded via an amide bond formed by the epsilon amino group of the Lys amino acid, and (b) the synthetic resin having a spacer is covalently bonded via an amide bond formed by the distal amino group in the spacer. The same considerations provided above apply to appropriate GPCR agonist peptides, linkers, and resins.

[0124] Regarding suitable resin beads, it is generally known that using smaller beads generally results in faster reaction rates due to a larger surface area-to-volume ratio. However, in practice, beads that are too small can lead to longer filtration times. Therefore, suitable resin beads are those with a diameter in the range of 75 to 150 microns, which offer a good balance between reaction rate and reliability, or 35 to 75 microns, respectively. Typically, the resin has a substitution level that varies between 0.1 to 2.0 mmol / g, more preferably between 0.4 to 0.6 mmol / g.

[0125] It should be further understood that peptides have either a carboxylic acid group (-COOH) or an amide group (-CONH2) at their C-terminus. Therefore, common resins for preparing peptide amides include MBHA, BHA, Rink amides, Knorr, DCHD, PAL, Seiber, and Ramage (tricyclic amide) resins. Alternatively, when preparing smaller peptides with less steric demands, suitable resins include Merrifield, hydroxymethyl polystyrene, Wang, and 2-chlorotrityl resins. Among other factors, the appropriate resin can be selected based on the following considerations: Aminomethyl (AM) resins have long been used in solid-phase peptide synthesis as core resins that can bond various linkers via stable amide bonds. However, 4-methylbenzhydrylamine (MBHA) resins were initially developed to form peptide amides using a Boc-N protection / TFA deprotection strategy. These resins form very stable amide or amine bonds to either carboxylic acid or electrophilic alkyl group materials. Generally, strong acid conditions are required to cleave the substrate from these resins. Solid-phase supports for amide product formation, including Rink and Ramage resins, were initially developed for peptide amide synthesis using the Fmoc strategy. These resins are preferred due to their higher acid instability. Cleavage can be carried out under mild conditions such as 1% TFA. Sever resins are useful for preparing amides and amines, as well as fully protected peptide amide fragments. The products can be cleaved under mild conditions using 1% TFA in DCM. This resin exhibits less steric hindrance than Rink resins and therefore allows for higher loading in sterically demanding applications. MBHA (methylbenzhydrylamine) is a structurally similar amide-forming resin to Rink, but the conditions for cleaving peptide products from MBHA are much more stringent than those for Rink, as MBHA requires treatment with hydrofluoric acid or trifluoromethanesulfonic acid (TFMSA). Trityl and 2-chlorotrityl resins are widely used in peptide chemistry.These resins are highly acid-unstable and can be cleaved with acetic acid, and the protected peptides can be cleaved with 1:4v / v hexafluoroisopropyl alcohol / dichloromethane, leaving all side-chain protecting groups intact, even the trityl group on the sulfhydryl function of homocysteine. Trityl and 2-chlorotrityl resins are particularly useful when the substrate after cleavage requires protecting groups that are not acid-unstable, and are especially useful when they yield fully protected short peptides and / or peptide fragments / components that can undergo hybrid manufacturing processes to obtain the desired larger peptide structure.

[0126] Therefore, taking the above into consideration, preferred resins for solid-phase synthesis of peptide intermediates include MBHA, BHA, Rink amide, Knorr, DCHD, PAL, Seiber, and Ramage (tricyclic amide) resins, particularly Ramage (tricyclic amide) resins. Furthermore, preferred resins are crosslinked by about 1% with a mesh size of 200-400.

[0127] To further understand, the peptide intermediates presented herein can be produced using one of three synthetic methods: solid-phase peptide synthesis (SPPS), liquid-phase peptide synthesis (LPPS), or a hybrid approach. LPPS can produce large quantities of high-quality short peptides and / or peptide fragments / components of larger peptide structures. Alternatively, SPPS can provide longer peptides, but depending on the structure, the yield may be lower due to the challenge of removing impurities closely related to the desired product. The hybrid approach can combine SPPS and LPPS methodologies to produce peptides. For example, to construct a 40-amino acid peptide intermediate, SPPS is used to generate small peptides of 10 to 15 amino acid segments, and then segment condensation is performed in LPPS to construct the complete peptide intermediate sequence as illustrated above.

[0128] Therefore, considering the above, class B GPCR agonist fusion proteins provide or generate conformation-modified albumin as described above, and GPCR agonist peptides, via Michael addition reactions, typically at pH < 7.0, to convert conformation-modified albumin to Cys 34 It should be understood that this can be easily prepared by covalent coupling. The same considerations as above apply to GPCR agonist peptides, linkers, reactive coupling groups, and albumin.

[0129] For example, particularly suitable agonist peptides are provided in Table 1 below, with amino acids and modification letters as provided in the sequence listing of this application. Furthermore, it should be understood that (1) sequences in the table without linker modification may also include linker modifications as described herein, particularly when the linker modification is covalently bonded to the epsilon-amino group of lysine, and suitable linker modifications include MPA, (bromo)MPA, AEEA, AEEA-MPA, AEEA-(bromo)MPA, (AEEA)2-MPA, (AEEA)2-(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA)2-OA-MPA, and (AEEEA)2-OA-(bromo)MPA. (2) Any specific linker modification shown in the following table, indicated by a specific modification letter (e.g., AEEA-MPA), may be replaced with any other linker modification intended herein (e.g., AEEA-AEEA-MPA). (3) The C-terminal amino acid may preferably be covalently bonded to the solid phase (e.g., modified Ramage resin) via a spacer group. (4) Any sequence in the following table may be linked via the linkers described herein. 34 (5) Any sequence in the following table having linker modifications may also be intended as an agonist peptide sequence without linker modifications.

[0130] Table 1

[0131] Table 2

[0132] Table 3

[0133] Table 4

[0134] Table 5

[0135] Table 6

[0136] Table 7

[0137] Table 8

[0138] Table 9

[0139] Table 10

[0140] In a further aspect of the subject matter of the present invention, it should be understood that the compositions and compounds presented herein have significant biological activity in vivo when administered to a subject. For example, when retro-Michael-resistant arbenatide (AB-013-AEEA-succinimide (SEQ ID NO: 2)-albumin) was administered as a single dose of 100 nmol / kg (subcutaneous injection) to mice of a diet-induced obesity model, retro-Michael-resistant arbenatide had significant physiological activity with respect to weight loss, comparable to semaglutide over 16 and 26 days, as shown in Figure 33, which is also reflected in the cumulative reduction in food and water consumption shown in Figure 34. Furthermore, in this model, retro-Michael-resistant arbenatide was also observed to have a significant and sustained effect on blood glucose levels, as exemplified in Figure 35.

[0141] Furthermore, in addition to the metabolic regulation described above, retro-Michael-resistant arbenatide also had a significant effect on hepatic lipid metabolism, as can be easily seen from Figure 36. Here, a single dose of retro-Michael-resistant arbenatide resulted in a substantial reduction in liver weight comparable to that achieved with semaglutide. This weight reduction was attributed to the amount of lipid vesicles in the parenchyma, as seen in the micrograph in Figure 36. Notably, thus retro-Michael-resistant arbenatide was effective in weight management and blood glucose reduction, and was even capable of reversing fatty liver. Consistent with these observations, there were also data shown in Figures 37A and 37B demonstrating a significant reduction in serum AST (37A) and ALT (37B). Similarly, administration of retro-Michael-resistant arbenatide further resulted in significantly reduced total serum cholesterol and LDL cholesterol, as shown in the data in Figures 37C and 37D, respectively.

[0142] Furthermore, the inventors also observed that retro-Michael-resistant arbenatide could reduce the levels of pro-inflammatory cytokines IL-1α and IL-6, as shown in Figure 38. In this regard, it should be particularly noted that since cytokines were measured at the end of the study, the reduction and more compact data range are noteworthy and suggest a stronger effect closer to the administration time.

[0143] Further embodiments, examples, and methods of use of the compounds presented herein are described in the applicant's international application dated June 25, 2024, titled "Albumin Bound Macromolecule Tri-Agonist Activating GLP-1 / GIP / Glucagon Receptors And Methods Therefor," which is incorporated herein by reference.

[0144] It will be understood that the present invention extends to any nucleic acid or peptide or its variants, derivatives, or analogs substantially comprising an amino acid or nucleic acid sequence of any sequence (including its variants or fragments) referred to herein. The terms “substantially amino acid / nucleotide / peptide sequence,” “variant,” and “fragment” may refer to sequences having at least 40% sequence identity with any one of the amino acid / nucleotide / peptide sequences referred to herein, such as 40% sequence identity with the sequences identified as SEQ ID NOs: 1 to 471.

[0145] Amino acid / polynucleotide / polypeptide sequences having more than 65%, more preferably more than 70%, even more preferably more than 75%, and even more preferably more than 80% sequence identity with any of the sequences mentioned herein are also conceivable. Preferably, the amino acid / polynucleotide / polypeptide sequences have at least 85% identity, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity with any of the sequences mentioned herein.

[0146] Those skilled in the art will understand how to calculate the percentage of identity between two amino acid / polynucleotide / polypeptide sequences. To calculate the percentage of identity between two amino acid / polynucleotide / polypeptide sequences, one must first prepare an alignment of the two sequences and then calculate the sequence identity value. The percentage of identity between two sequences can take different values ​​depending on: (i) the method used to align the sequences, e.g., ClustalW, BLAST, FASTA, Smith-Waterman (performed with different programs), or structural alignment from 3D comparison; (ii) the parameters used by the alignment method, e.g., local vs. global alignment, the pair score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, e.g., functional form and constant. After alignment, there are many different methods for calculating the percentage of identity between the two sequences. For example, the number of identical residues can be divided by the following: (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of non-gap positions; or (v) the number of equivalent positions excluding overhangs. Furthermore, it will be understood that the identity percentage also depends strongly on length. Thus, the shorter the pair of sequences, the higher the sequence identity expected to occur by chance. Thus, it will be understood that the accurate alignment of protein or DNA sequences is a complex process. The general multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred method for generating multiple alignments of protein or DNA according to the present invention. Suitable parameters for ClustalW are as follows: For DNA alignment: gap open penalty = 15.0, gap extension penalty = 6.66, matrix = identity.For protein alignment: gap open penalty = 10.0, gap extension penalty = 0.2, matrix = Gonnet. For DNA and protein alignment: ENDGAP = -1, GAPDIST = 4. Those skilled in the art will recognize that it may be necessary to vary these and other parameters for optimal sequence alignment.

[0147] Preferably, the percentage of identity between two amino acid / polynucleotide / polypeptide sequences can then be calculated from an alignment such as (N / T)*100, where N is the number of positions in which the sequences share identical residues, and T is the total number of positions to be compared, including gaps and including or excluding overhangs. Preferably, overhangs are included in the calculation. Thus, the most preferred method for calculating the percentage of identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program with an appropriate set of parameters, for example, as described above; and (ii) inserting the values ​​of N and T into the following formula: - Sequence identity = (N / T)*100. Alternative methods for identifying similar sequences are known to those skilled in the art. For example, substantially similar nucleotide sequences are encoded by DNA sequences or sequences that hybridize to their complements under stringent conditions. Stringent conditions mean that nucleotides hybridize to DNA or RNA bound to the filter in 3× sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by at least one wash in 0.2× SSC / 0.1% SDS at approximately 20-65°C.

[0148] Alternatively, substantially similar polypeptides may differ from, for example, the amino acid sequences shown in Sequence IDs 1-471 by at least one, but fewer than 5, 10, 20, 50, or 100 amino acids. Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be altered or modified without substantially affecting the sequence of the protein it encodes to provide functional variants thereof. Suitable nucleotide variants have a sequence altered by the substitution of a different codon encoding the same amino acid in the sequence, thus resulting in a silent change. Other suitable variants have a homologous nucleotide sequence but include all or part of a sequence altered by substituting a different codon encoding an amino acid with a side chain having similar biophysical properties to the amino acid it substitutes for, in order to result in a conservation change. Examples of small nonpolar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, it is understood which amino acids can be replaced with those having similar biophysical properties, and those skilled in the art will know the nucleotide sequences encoding these amino acids.

[0149] It should be noted that any language relating to computers should be read to include any appropriate combination of computing devices, including servers, interfaces, systems, databases, agents, peers, engines, modules, controllers, or other types of computing devices operating individually or collectively. It should be understood that a computing device comprises a processor configured to execute software instructions stored in tangible, non-temporary, computer-readable storage media (e.g., hard drives, solid-state drives, RAM, flash memory, ROM, etc.). The software instructions preferably configure the computing device to provide roles, responsibilities, or other functions with respect to the disclosed device as described below. In particularly preferred embodiments, various servers, systems, databases, or interfaces exchange data using standardized protocols or algorithms based, optionally on HTTP, HTTPS, AES, public-private key exchange, web service APIs, known financial transaction protocols, or other electronic information exchange methods. Data exchange preferably takes place over a packet-switched network, the Internet, a LAN, a WAN, a VPN, or other types of packet-switched networks.

[0150] In some embodiments, numbers representing properties such as quantities and concentrations of components, reaction conditions, etc., used to describe and claim specific embodiments of the present invention should be understood to be modified in some cases by the term “about”. As used herein, the terms “about” and “approximately” mean, when referring to a specified measurable value (e.g., parameter, quantity, duration, etc.), to include the specified value and variations from the specified value, e.g., variations of + / - 10%, + / - 5%, + / - 1%, or + / - 0.1%, insofar as such variations are appropriate for performing in the disclosed embodiments. Thus, the values ​​themselves that the modifiers “about” or “approximately” refer to are also specifically disclosed. The enumeration of value ranges herein is intended merely as a convenient way to refer individually to each distinct value that falls within that range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually enumerated herein.

[0151] Where used herein, the term “administer” a pharmaceutical composition or drug means both direct and indirect administration of the pharmaceutical composition or drug, where direct administration is typically performed by a healthcare professional (e.g., a physician, nurse, etc.), and indirect administration includes the step of providing or making available the pharmaceutical composition or drug to a healthcare professional for direct administration (e.g., via injection, infusion, oral delivery, local delivery, etc.). It should be further noted that the terms “prognose” or “predict” a condition, susceptibility to the onset of a disease, or response to an intended treatment mean that the act of predicting or forecasting a condition, susceptibility and / or response (but not treatment or diagnosis thereof), including the rate of progression, improvement and / or duration of the condition in the subject.

[0152] All methods described herein may be performed in any suitable order unless otherwise indicated herein or unless it is clearly inconsistent with the context. The use of any examples or exemplary language (e.g., "etc.") provided in reference to specific embodiments herein is intended solely to better illustrate the invention and not to limit the claimed scope of the invention. No language herein should be construed as indicating an unclaimed element essential to the practice of the invention.

[0153] As used herein and in the subsequent claims, “a,” “an,” and “the” include the plural unless the context clearly indicates otherwise. Also, as used herein, “in” includes “in” and “on” unless the context explicitly indicates otherwise. Furthermore, as used herein, unless the context indicates otherwise, the term “coupled to” is intended to include both direct coupling (two coupled elements touching each other) and indirect coupling (at least one additional element positioned between the two elements). Thus, the terms “coupled to” and “coupled with” are used synonymously.

[0154] It will be apparent to those skilled in the art that many more modifications beyond those already described are possible without departing from the concept of the invention as herein. Therefore, the subject matter of the invention is not limited beyond the appended claims. Furthermore, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible way that is consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to an element, component, or step in a non-exclusive manner, indicating that the referenced element, component, or step exists, is utilized, or can be combined with other elements, components, or steps not explicitly referenced. Where the specification or claims refer to at least one of those selected from the group consisting of A, B, C.... and N, the text should be interpreted as requiring only one element from the group, and not A plus N or B plus N, etc.

Claims

1. It is a reaction intermediate, Conformation-modified albumin isolated in a solvent, At least one hydrophobic pocket of the albumin is not occupied by non-covalently bound lipids, and the conformation-modified albumin is not occupied by the albumin. Michael Acceptor reagent and A reaction intermediate that includes this intermediate.

2. The reaction intermediate according to claim 1, wherein the isolated conformation-modified albumin is recombinant human albumin.

3. The reaction intermediate according to claim 1, wherein the isolated conformation-modified albumin is at least partially defatted albumin.

4. The reaction intermediate according to claim 1, wherein the solvent is an aqueous solvent and the pH of the solvent is pH < 7.

0.

5. The reaction intermediate according to claim 1, wherein the Michael acceptor comprises a planar acceptor group.

6. The reaction intermediate according to claim 1, wherein the Michael acceptor reagent comprises a maleimide group or a bromomaleimide group.

7. The reaction intermediate according to claim 1, further comprising a linker covalently coupled to a class B G protein-coupled receptor (GPCR) agonist peptide in the Michael acceptor reagent.

8. Chemically modified albumin, wherein the Cys ３４ The sulfur atom of the albumin is covalently bonded to the chiral carbon atom of the coupling group, such that the albumin is Cys ３４ A chemically modified albumin in which the chiral atom is modified with a coupling group and the chiral atom has a preferred stereochemical configuration.

9. The chemically modified albumin according to claim 8, wherein the preferred stereochemical configuration is preferred in a ratio of at least 70:

30.

10. The chemically modified albumin according to claim 8, wherein the preferred stereochemical configuration is preferred in a ratio of at least 90:

10.

11. The chemically modified albumin according to claim 8, wherein the albumin is human albumin and optionally a recombinant.

12. The chemically modified albumin according to claim 8, wherein the albumin is a conformationally modified albumin.

13. The chemically modified albumin according to claim 8, wherein the albumin is in an aqueous solvent having a pH < 7.

0.

14. The aforementioned albumin, unreacted Cys ３４ Unreacted Cys ３４ The chemically modified albumin according to claim 8, wherein the ratio of albumin having a group to chemically modified albumin is at least 1:1.

2.

15. The chemically modified albumin according to claim 8, wherein the coupling group is covalently coupled to the linker.

16. The chemically modified albumin according to claim 15, wherein the linker is covalently coupled to a class B G protein-coupled receptor (GPCR) agonist peptide.

17. A chemically modified albumin, wherein the albumin is Cys ３４ A chemically modified albumin in which the albumin is modified with a Michael addition conjugate, wherein the Michael addition conjugate is resistant to retro-Michael addition reactions.

18. The chemically modified albumin according to claim 17, wherein the Michael addition conjugate comprises a hydrophilic linker.

19. The chemically modified albumin according to claim 18, wherein the hydrophilic linker is covalently bound to a class B G protein-coupled receptor (GPCR) agonist peptide.

20. The chemically modified albumin according to claim 17, wherein at least one hydrophobic pocket of the albumin is not occupied by a non-covalently bonded lipid.

21. The chemically modified albumin according to claim 17, wherein the albumin is at least partially degreased albumin.

22. The chiral carbon atom of the Michael addition conjugate is the Cys ３４ The chemically modified albumin according to claim 17, wherein the sulfur forms a covalent bond with the chiral carbon atom, and the chiral carbon atom has a preferred stereochemical configuration.

23. The chemically modified albumin according to claim 22, wherein the preferred stereochemical configuration is preferred in a ratio of at least 70:

30.

24. Cys ３４ A method of modifying albumin, To provide or prepare conformation-modified albumin in a solvent, wherein at least one hydrophobic pocket of the conformation-modified albumin is not occupied by non-covalently bonded lipids, and the pH of the solvent is pH < 7.

0. The aforementioned Cys ３４ The sulfur atom is reacted with the coupling group to produce the modified albumin. Methods that include...

25. The method according to claim 24, wherein the conformation-modified albumin is modified by at least partial degreasing.

26. The method according to claim 24, wherein the pH of the solvent is 4.0 to 6.

0.

27. The method according to claim 24, wherein the reaction step is carried out in equimolar ratios between the conformation-modified albumin and the coupling group.

28. The method according to claim 24, wherein the modified albumin is retro-Michael resistant to the covalently bonded coupling group.

29. The method according to claim 24, wherein the reaction step is given sterically priority.

30. The method according to claim 24, wherein the reaction step is sterically selective.

31. The method according to claim 24, wherein the coupling group comprises a planar Michael acceptor group.

32. The method according to claim 31, wherein the planar Michael acceptor group comprises a maleimide group or a bromomaleimide group.

33. The method according to claim 31, wherein the coupling group is further covalently coupled to a class B G protein-coupled receptor (GPCR) agonist peptide.

34. Cys ３４ Cys that reacts with the Michael addition reagent ３４ A method for reducing the retro-Michael addition reaction in a modified albumin, comprising The preparation of conformation-modified albumin in a solvent such that at least one hydrophobic pocket of the conformation-modified albumin is not occupied by non-covalently bonded lipids, wherein the solvent has a pH of < 7.

0. The aforementioned Cys ３４ The sulfur atom is reacted with the coupling group of the Michael addition reagent, thereby producing the modified albumin. Methods that include...

35. The method according to claim 34, wherein the conformation-modified albumin is modified by at least partial degreasing.

36. The method according to claim 34, wherein the pH of the solvent is 4.0 to 6.

0.

37. The method according to claim 34, wherein the reaction step is carried out in equimolar ratios between the conformation-modified albumin and the coupling group.

38. The method according to claim 34, wherein the retro-Michael addition reaction is reduced by at least 50%.

39. The method according to claim 34, wherein the Michael addition reagent comprises a linker covalently coupled to a class B G protein-coupled receptor (GPCR) agonist peptide.

40. The method according to claim 34, wherein the Michael addition reagent has a structure according to any one of SEQ ID NOs: 1 to 471.

41. A method for producing a class B G protein-coupled receptor (GPCR) agonist fusion protein, To provide or produce conformation-modified albumin, The GPCR agonist peptide is added to the conformation-modified albumin via the Michael addition reaction. ３４ To make them share coupling Includes, A method wherein the coupling step is performed at pH < 7.

0.

42. The method according to claim 41, wherein the conformation-modified albumin is modified by at least partial degreasing.

43. The method according to claim 41, wherein the coupling step is performed using albumin in a molar excess relative to the GPCR agonist peptide.

44. The method according to claim 41, wherein the pH is 4.0 to 6.

0.

45. The method according to claim 41, wherein the GPCR agonist peptide comprises a linker having a planar Michael acceptor group.

46. The method according to claim 45, wherein the planar Michael acceptor group comprises a maleimide group or a bromomaleimide group.

47. The method according to claim 45, wherein the Michael addition reaction produces a sterically preferred chiral carbon atom in the Michael acceptor group.

48. The method according to claim 45, wherein the Michael addition reaction produces a sterically selective chiral carbon atom in the Michael acceptor group.

49. The method according to claim 45, wherein the GPCR agonist peptide has a structure according to any one of SEQ ID NOs: 1 to 471.

50. A method for producing a retro-Michael addition-resistant class B G protein-coupled receptor agonist (GPCR) fusion protein, To provide or produce conformation-modified albumin, The GPCR agonist peptide is added to the conformation-modified albumin via the Michael addition reaction. ３４ To make them share coupling A method comprising the following, wherein the coupling step is performed at pH < 7.

0.

51. The method according to claim 50, wherein the GPCR agonist peptide has a structure according to any one of SEQ ID NOs: 1 to 471.

52. The method according to claim 50, wherein the conformation-modified albumin is modified by at least partial degreasing.

53. The method according to claim 50, wherein the coupling step is performed using albumin in a molar excess relative to the GPCR agonist peptide.

54. The method according to claim 50, wherein the pH is 4.0 to 6.

0.

55. The method according to claim 50, wherein the GPCR agonist peptide comprises a linker having a planar Michael acceptor group.

56. The method according to claim 55, wherein the planar Michael acceptor group comprises a maleimide group or a bromomaleimide group.

57. The method according to claim 55, wherein the Michael addition reaction produces a sterically preferred chiral carbon atom in the Michael acceptor group.

58. The method according to claim 55, wherein the Michael addition reaction produces a sterically selective chiral carbon atom in the Michael acceptor group.

59. A resin-immobilized cysteine-reactive class B G protein-coupled receptor (GPCR) agonist peptide, A GPCR agonist peptide containing Lys amino acids, (a) The linker is covalently bonded via an amide bond formed by the epsilon amino group of the Lys amino acid, (b) A GPCR agonist peptide containing a Lys amino acid, which is covalently bonded to a synthetic resin having a spacer via amide bonds formed by distal amino groups in the spacer. A resin-immobilized cysteine-reactive class B G protein-coupled receptor (GPCR) agonist peptide containing this peptide.

60. The linker is MPa, (bromo)MPa, AEEA, AEEA-MPa, AEEA-(bromo)MPa, (AEEA) ２ -MPA, (AEEA) ２ -(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA) ２ -OA-MPA, and (AEEEA) ２ An agonist peptide according to claim 59, selected from the group consisting of -OA-(bromo)MPA.

61. The agonist peptide according to claim 59, wherein the synthetic resin comprises a polystyrene resin having the distal amino group tricyclic spacer.

62. The agonist peptide according to claim 61, wherein the synthetic resin comprises Ramage resin.

63. The agonist peptide according to claim 61, wherein the substitution level of the resin is about 0.4 to 0.6 mmol / gram.

64. The agonist peptide according to claim 61, wherein the synthetic resin is crosslinked with divinylbenzene by about 1%.

65. The agonist peptide according to claim 61, wherein the GPCR agonist peptide has a structure according to any one of SEQ ID NOs: 1 to 471.

66. A peptide solid synthesis intermediate, The first amino acid of the peptide is covalently coupled to the C-terminus of the peptide via an amide bond, comprising a Ramage resin. The first amino acid is further covalently coupled to the linker via an amide bond in the side chain of the first amino acid. A peptide solid synthesis intermediate comprising the linker containing a coupling group.

67. The intermediate according to claim 66, wherein the first amino acid is lysine.

68. The intermediate according to claim 66, wherein the linker is selected from the group consisting of AAA-MPA, AAA-AAA-MPA, AAA-OA-MPA, and AAA-OA-AAA-MPA.

69. The intermediate according to claim 66, wherein the coupling group includes a planar Michael acceptor group.

70. The intermediate according to claim 69, wherein the planar Michael acceptor group is a maleimide group or a bromomaleimide group.

71. A method for synthesizing a peptide containing a linker having a coupling group, To provide a synthetic resin having a spacer containing a distal amino group, The method involves covalently coupling a first amino acid to the resin using the distal amino group, wherein the first amino acid has a protected reactive group in its side chain, and the covalent coupling is performed. Using solid-phase synthesis, multiple Fmoc-protected amino acids are sequentially attached to the first amino acid to obtain a protected peptide. The method involves selectively deprotecting the protected reactive group in the side chain and reacting the reactive group with a linker, wherein the linker contains a second protected reactive group. The second protected reactive group in the linker is selectively deprotected, and the second reactive group is reacted with the coupling group. The protected peptide is deprotected, the deprotected peptide is cleaved from the resin, thereby forming the peptide containing the linker having the coupling group. A method of synthesis that includes this.

72. The method according to claim 71, wherein the synthetic resin is Ramage resin.

73. The method according to claim 71, wherein the first amino acid is lysine.

74. The linker is MPa, (bromo)MPa, AEEA, AEEA-MPa, AEEA-(bromo)MPa, (AEEA) ２ -MPA, (AEEA) ２ -(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA) ２ -OA-MPA, and (AEEEA) ２ The method according to claim 71, selected from the group consisting of -OA-(bromo)MPA.

75. The method according to claim 71, wherein the reactive group in the linker comprises a planar Michael acceptor group.

76. The method according to claim 75, wherein the planar Michael acceptor group is a maleimide group or a bromomaleimide group.

77. The method according to claim 71, wherein the protected peptide comprises a class B G protein-coupled receptor (GPCR) agonist peptide.

78. A class B G protein-coupled receptor (GPCR) agonist peptide having a reactive linker, A GPCR agonist peptide containing the Lys amino acid, wherein a linker is covalently bonded via an amide bond formed by the epsilon-amino group of the Lys amino acid. Includes, The linker further comprises a thiol-reactive group, and the GPCR agonist peptide has a structure according to any one of SEQ ID NOs: 1 to 471. A class B G protein-coupled receptor (GPCR) agonist peptide with a reactive linker.

79. The agonist peptide according to claim 78, wherein the thiol-reactive group comprises a planar Michael acceptor group.

80. The agonist peptide according to claim 78, wherein the thiol-reactive group comprises a maleimide group or a bromomaleimide group.

81. The agonist peptide according to claim 78, wherein the linker is selected from the group consisting of AAA-MPA, AAA-AAA-MPA, AAA-OA-MPA, and AAA-OA-AAA-MPA.

82. A class B G protein-coupled receptor (GPCR) agonist peptide having a reactive linker, GPCR agonist peptides containing Lys amino acids, A linker covalently bonded to the GPCR agonist peptide via an amide bond formed by the epsilon-amino group of the Lys amino acid and the carboxyl group of the linker, the linker further comprising a maleimide group Includes, The GPCR agonist peptide has a structure according to one of SEQ ID NOs: 1 to 471. A class B G protein-coupled receptor (GPCR) agonist peptide with a reactive linker.

83. The linker is MPa, (bromo)MPa, AEEA, AEEA-MPa, AEEA-(bromo)MPa, (AEEA) ２ -MPA, (AEEA) ２ -(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA) ２ -OA-MPA, and (AEEEA) ２ An agonist peptide according to claim 78, selected from the group consisting of -OA-(bromo)MPA.

84. A class B G protein-coupled receptor (GPCR) agonist fusion protein, Cys ３４ Conformation-modified albumin containing amino acids, GPCR agonist peptides containing Lys amino acids, A linker that covalently couples the conformation-modified albumin to the GPCR agonist peptide Includes, (a) The linker is covalently bonded to the GPCR agonist peptide via an amide bond formed by the epsilon amino group of the Lys amino acid, (b) The linker is the chiral carbon atom of the coupling group in the linker and the Cys ３４ A class B G protein-coupled receptor (GPCR) agonist fusion protein that is covalently bound to the albumin via a covalent bond with a sulfur atom in an amino acid.

85. The agonist fusion protein according to claim 84, wherein the conformation-modified albumin is at least partially delipidated albumin.

86. The agonist fusion protein according to claim 84, wherein the chiral carbon atoms are in a sterically preferred configuration.

87. The agonist fusion protein according to claim 84, wherein the linker is retro-Michael resistant.

88. The agonist fusion protein according to claim 84, wherein the fusion protein is retro-Michael-resistant albenatide (AB-013-AEEA-succinimide (SEQ ID NO: 2)-albumin).

89. The agonist fusion protein according to claim 84, wherein the fusion protein is retro-Michael-resistant albenatidex (AB-014-AEEA-succinimide (SEQ ID NO: 185)-albumin).

90. The agonist fusion protein according to claim 84, wherein the fusion protein is retromichael-resistant albudiptide (AB-029-AEEA-succinimide (SEQ ID NO: 7)-albumin).

91. The agonist fusion protein according to claim 84, wherein the fusion protein is retromichael-resistant albutide (AB-044-AEEA-succinimide (SEQ ID NO: 223)-albumin).

92. A pharmaceutical composition, Class B G protein-coupled receptor (GPCR) agonist fusion protein combined with an aqueous carrier Includes, The aforementioned GPCR fusion protein, ３４ The material comprises a conformation-modified albumin containing an amino acid, a GPCR agonist peptide containing a Lys amino acid, and a linker that covalently couples the conformation-modified albumin to the GPCR agonist peptide. (a) The linker is covalently bonded to the GPCR agonist peptide via an amide bond formed by the epsilon amino group of the Lys amino acid, (b) The linker is the chiral carbon atom of the coupling group in the linker and the Cys ３４ It is covalently bonded to the albumin via a covalent bond with the sulfur atom in the amino acid, A pharmaceutical composition wherein the aqueous carrier has a pH of < 7.

0.

93. The pharmaceutical composition according to claim 92, wherein the GPCR agonist peptide and the linker have a structure according to any one of SEQ ID NOs: 1 to 471.

94. The linker is MPa, (bromo)MPa, AEEA, AEEA-MPa, AEEA-(bromo)MPa, (AEEA) ２ -MPA, (AEEA) ２ -(bromo)MPA, AEEEA-OA-MPA, AEEEA-OA-(bromo)MPA, AEEA-OA-AEEA-MPA, AEEA-OA-AEEA-(bromo)MPA, (AEEEA) ２ -OA-MPA, and (AEEEA) ２ A pharmaceutical composition according to claim 92, selected from the group consisting of -OA-(bromo)MPA.

95. The pharmaceutical composition according to claim 92, wherein the aqueous carrier has a pH of 4.0 to 6.

0.

96. The composition is unreacted Cys ３４ The pharmaceutical composition according to claim 92, further comprising albumin having an amino acid.

97. The pharmaceutical composition according to claim 92, wherein the composition is formulated for injection or intranasal administration.

98. The pharmaceutical composition according to claim 92, wherein the composition contains the GPCR agonist peptide at a concentration of 1.5 mg / mL or less.

99. The pharmaceutical composition according to claim 92, further comprising metformin and / or thiazolidinedione.

100. The pharmaceutical composition according to claim 92, wherein the GPCR agonist fusion protein is retromichael-resistant albenatide (AB-013-AEEA-succinimide (SEQ ID NO: 2)-albumin).

102. The pharmaceutical composition according to claim 92, wherein the GPCR agonist fusion protein is retromichael-resistant albenatidex (AB-014-AEEA-succinimide (SEQ ID NO: 185)-albumin).

103. The pharmaceutical composition according to claim 92, wherein the GPCR agonist fusion protein is retromichael-resistant albudiptide (AB-029-AEEA-succinimide (SEQ ID NO: 7)-albumin).

104. The pharmaceutical composition according to claim 92, wherein the GPCR agonist fusion protein is retromichael-resistant albutide (AB-044-AEEA-succinimide (SEQ ID NO: 223)-albumin).

105. A pharmaceutical composition, It contains a class B G protein-coupled receptor (GPCR) agonist fusion protein combined with an aqueous carrier. The aforementioned GPCR fusion protein is a Michael adduct product of conformation-modified albumin and a linker further covalently bonded to the GPCR agonist peptide. The aforementioned fusion protein is retro-Michael-tolerant. Pharmaceutical composition.

106. The pharmaceutical composition according to claim 105, wherein the composition is formulated for injection or intranasal administration.

107. The pharmaceutical composition according to claim 105, wherein the composition contains the GPCR agonist peptide at a concentration of 1.5 mg / mL or less.

108. The pharmaceutical composition according to claim 105, wherein the preparation has a pH of 4.0 to 6.

0.

109. Unresponsive Cys ３４ The pharmaceutical composition according to claim 105, further comprising albumin having an amino acid.

110. The pharmaceutical composition according to claim 105, wherein the Michael adduct is a sterically preferred or sterically selective Michael adduct.

111. The pharmaceutical composition according to claim 105, wherein the GPCR agonist peptide and the linker have a structure according to any one of Sequence ID No. 1 to 471.

112. A pharmaceutical composition, It contains a class B G protein-coupled receptor (GPCR) agonist fusion protein combined with an aqueous carrier. The aforementioned GPCR fusion protein is a Michael adduct product of albumin and a linker further covalently bonded to a GPCR agonist peptide. The above composition contains unreacted Cys ３４ It further contains albumin which amino acids are present, A pharmaceutical composition wherein the aqueous carrier has a pH of < 7.

0.

113. The unreacted Cys ３４ The pharmaceutical composition according to claim 112, wherein the ratio of albumin having amino acids to albumin to which the linker and the GPCR agonist peptide are bound is at least 0.3:1.

0.

114. The unreacted Cys ３４ The pharmaceutical composition according to claim 112, wherein the albumin having an amino acid is a conformation-modified albumin.

115. The unreacted Cys ３４ The pharmaceutical composition according to claim 112, wherein the albumin having amino acids and the albumin to which the linker and the GPCR agonist peptide are bound are recombinant human albumin.

116. The pharmaceutical composition according to claim 112, wherein the GPCR agonist peptide and the linker have a structure according to any one of SEQ ID NOs: 1 to 471.

117. A pharmaceutical composition, The solution comprises a pharmaceutically acceptable liquid carrier in which a GPCR agonist peptide is covalently coupled to albumin via a linker, and a class B G protein-coupled receptor (GPCR) agonist fusion protein is combined with the GPCR agonist peptide, A pharmaceutical composition wherein the pharmaceutically acceptable liquid carrier substantially does not contain an unbound GPCR agonist peptide.

118. The pharmaceutical composition according to claim 117, wherein the GPCR agonist fusion protein is retromichael-resistant albenatide (AB-013-AEEA-succinimide (SEQ ID NO: 2)-albumin).

119. The pharmaceutical composition according to claim 117, wherein the GPCR agonist fusion protein is retro-Michael-resistant albenatidex (AB-014-AEEA-succinimide (SEQ ID NO: 185)-albumin).

120. The pharmaceutical composition according to claim 117, wherein the GPCR agonist fusion protein is retromichael-resistant albudiptide (AB-029-AEEA-succinimide (SEQ ID NO: 7)-albumin).

121. The pharmaceutical composition according to claim 117, wherein the GPCR agonist fusion protein is retromichael-resistant albutide (AB-044-AEEA-succinimide (SEQ ID NO: 223)-albumin).

122. The pharmaceutical composition according to claim 117, wherein the amount of unbound GPCR agonist peptide is 1% or less of the amount of the GPCR agonist peptide covalently coupled to albumin via the linker.

123. The pharmaceutical composition according to claim 117, wherein the amount of unbound GPCR agonist peptide is 0.1% or less of the amount of the GPCR agonist peptide covalently coupled to albumin via the linker.

124. A method for manufacturing a pharmaceutical composition, To provide a pharmaceutically acceptable carrier with a pH < 7.0, The pharmaceutically acceptable carrier includes a retro-Michael-resistant G protein-coupled receptor (GPCR) agonist fusion protein to which a GPCR agonist peptide is covalently bound. Includes, A method for producing a pharmaceutical composition in which the GPCR agonist peptide is present in the composition at a concentration of 1.5 mg / mL or less.

125. The method according to claim 124, wherein the composition is formulated for injection or intranasal administration.

126. The method according to claim 124, wherein the GPCR agonist fusion protein is retro-Michael-resistant albenatide (AB-013-AEEA-succinimide (SEQ ID NO: 2)-albumin).

127. The method according to claim 124, wherein the GPCR agonist fusion protein is retro-Michael-resistant albenatidex (AB-014-AEEA-succinimide (SEQ ID NO: 185)-albumin).

129. The method according to claim 124, wherein the GPCR agonist fusion protein is retromichael-resistant albumin (AB-029-AEEA-succinimide (SEQ ID NO: 7)-albumin).

130. The method according to claim 124, wherein the GPCR agonist fusion protein is retromichael-resistant albutide (AB-044-AEEA-succinimide (SEQ ID NO: 223)-albumin).

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