Glucagon analogues compatible with prandial insulin co-infusion

Abstract

A stabilized glucagon analogue containing an additional modification that attenuates its molar hormonal activity and prolongs its duration of action such that its activity and duration more closely match that of a prandial insulin analogue than is characteristic of wild-type glucagon. A pharmaceutical formulation containing said glucagon analogue and a prandial insulin analogue for the treatment of diabetes mellitus.

Description

GLUCAGON ANALOGUES COMPATIBLE WITH PRANDIAL INSULIN COINFUSIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 563,056 filed on March 8, 2024, the disclosure of which is expressly incorporated herein.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety is a computer-readable nucleotide / amino- acid sequence listing submitted concurrently herewith and identified as follows: XML file named “PCT Sequence Listing 420851. xml”, created on March 3, 2025.TECHNICAL FIELD

[0003] The present disclosure relates to methods of modifying a bioactive peptide to impair its activity and delay its clearance. More specifically, the present disclosure relates to a class of glucagon peptide analogues that exhibit three properties: (a) resistance to fibrillation at neutral pH, (b) reduced or suboptimal hormonal signaling activity, and (c) modest or suboptimal binding to serum albumin.BACKGROUND

[0004] Engineering non-standard proteins, including therapeutic agents and vaccines, can have broad medical and societal benefits. Naturally occurring peptides and proteins — as encoded in the genomes of human beings, other mammals, vertebrate organisms, invertebrate organisms, or eukaryotic cells in general, may have evolved to function optimally within a cellular context but may be suboptimal for therapeutic applications. Analogues of such peptides and proteins may exhibit improved biophysical, biochemical, or biological properties. Protein and / or peptide analogues can be used to achieve enhanced activity and / or more rapid action. Endogenous hormones bind to cognate receptors to regulate vertebrate metabolism, including in humans. The design of more rapidly acting hormone analogues or hormone analogues that are more resistant to fibrillation (a major route of physical degradation of pharmaceutical formulations) than the respective wild-type hormones would be beneficial.

[0005] The administration of insulin has long been established as a treatment for diabetes mellitus. A major goal of conventional insulin replacement therapy in patients with diabetes mellitus is tight control of the blood-glucose concentration to prevent its excursion above orbelow the normal range characteristic of healthy human subjects. Excursions above the normal range are associated with an increased long-term risk of microvascular disease, including retinopathy, blindness, and renal failure. Hypoglycemia in patients with diabetes mellitus is a frequent complication of insulin replacement therapy and, when severe, can lead to significant morbidity (including altered mental status, loss of consciousness, seizures, and death). Indeed, fear of such complications poses a major barrier to efforts by patients (and physicians) to obtain rigorous control of blood glucose concentrations (i.e. , excursions within or just above the normal range), and in patients with long-established Type 2 diabetes mellitus, such efforts (“tight control”) may lead to increased mortality. In addition to the above consequences of severe hypoglycemia (designated neuroglycopenic effects), mild hypoglycemia may activate counter- regulatory mechanisms, including over-activation of the sympathetic nervous system leading to anxiety and tremulousness (symptoms designated adrenergic). Patients with diabetes mellitus may not exhibit such warning signs, however, defining a condition known as hypoglycemic unawareness. The absence of symptoms of mild hypoglycemia increases the risk of major hypoglycemia and its associated morbidities and mortality. Multiple and recurrent episodes of hypoglycemia are also associated with chronic cognitive decline, a proposed mechanism underlying the increased prevalence of dementia in patients with long-standing diabetes mellitus. There is, therefore, an urgent need for new diabetes treatment technologies that would treat or reduce the risk of hypoglycemia while preventing upward excursions in blood-glucose concentration above the normal range.

[0006] Diverse technologies have been developed to treat or mitigate the threat of hypoglycemia in patients treated with insulin. Foundational to all such efforts is the education of the patient (and also members of his or her family) regarding the symptoms of hypoglycemia and, following the recognition of such symptoms, the urgency of the need to ingest food or liquid that is rich in glucose, sucrose, or another rapidly digested form of carbohydrate; an example is provided by orange juice supplemented with sucrose (cane sugar). This baseline approach has been extended by the development of specific diabetes-oriented products, such as squeezable tubes containing an emulsion containing glucose in a form that can be rapidly absorbed through the mucous membranes of the mouth, throat, stomach, and small intestine. “Rescue” preparations of the counter-regulatory hormone glucagon, provided as a powder, have likewise been developed in a form amenable to rapid dissolution and subcutaneous injection as an emergency treatment of severe hypoglycemia. Glucagon rescue kits typically contain the hormone as a powder because aqueous solutions of wild-type glucagon exhibit exquisitesusceptibility to fibrillation, which inactivates the hormone. Stabilized forms of glucagon, including glucagon analogues containing multiple amino-acid substitutions (including unnatural substitutions), have long been sought to enable the development of rescue pens containing an aqueous pharmaceutical formulation for immediate treatment of severe hypoglycemia. In the last decade, stabilized forms of glucagon have been explored as dual Glucagon / GLP-1 receptor agonists, as a treatment for obesity, motivating also design of glucagon-only agonists for coadministration with GLP- 1.

[0007] The risk of hypoglycemia has motivated innovations in technologies for continuous glucose monitoring (CGM) and in the control algorithms that connect such monitors to insulin pumps. Such an algorithm may halt subcutaneous injection of insulin and trigger an audible alarm when hypoglycemic readings of the interstitial glucose concentration are encountered. Such a device-based approach has led to the recent FDA approval of closed-loop systems in which the pump and monitor are combined with a computer-based algorithm as an “artificial pancreas.” A key metric for closed-loop performance is time in range (TiR), the fractional time in euglycemia and not in hypoglycemia or hyperglycemia. Efforts to optimize TiR have led to the development of bihormonal pumps with control algorithms that coordinate subcutaneous injection of either insulin or glucagon. This technology has, in turn, motivated interest in glucagon analogue formulations sufficiently stable to maintain activity and fluid-flow properties for 3-7 days in the reservoir of a bihormonal pump at ambient temperatures. In such devices, automated injection of a bolus of glucagon, in essence, provides a routine “rescue” from actual or predicted hypoglycemia.

[0008] The same concern for the acute and chronic complications of hypoglycemia has motivated interest in “smart” insulin delivery technologies based on glucose-responsive materials or insulin analogues. For more than three decades, there has been the development of glucose-responsive macromolecular complexes, polymers, and hydrogens for co-administration with an insulin analogue or modified insulin molecule, such that the rate of release of the hormone from the subcutaneous depot depends on interstitial glucose concentration. Such systems in general contain a glucose-responsive polymer, gel, or other encapsulation material; they may also require a derivative of insulin, such that the modification enables the binding of the hormone to the above material. An increase in the ambient concentration of glucose in the interstitial fluid at the site of subcutaneous injection may displace the bound insulin or insulin derivative either by competitive displacement of the hormone or by physical-chemical changes in the properties of the polymer, gel, or other encapsulation material. The goal of such systemsis to provide an intrinsic autoregulation feature to the encapsulated or gel-coated subcutaneous depot such that the risk of hypoglycemia is mitigated through delayed release of insulin when the ambient concentration of glucose is within or below the normal range. To date, no such glucose-responsive systems are in clinical use.

[0009] A fundamentally different therapeutic approach envisions co-formulation and coinjection of glucagon and a prandial insulin analogue. This approach in principle would exploit a physiologic switch in the liver’s relative hormone responsiveness (between glucagon and insulin) as a function of glycemia. Under hyperglycemic conditions, insulin signaling predominates, whereas under hypoglycemic conditions, glucagon predominates. The proof of concept, demonstrating hypoglycemia protection when exogenous insulin and glucagon are coadministered is known. Unfortunately, this approach is confounded in practice by (a) the intrinsic stability of the glucagon molecule, (b) the greater molar activity of glucagon relative to insulin, and (c) the briefer duration of action of glucagon relative to the duration of prandial insulin analogues. Whereas a variety of molecular approaches to the stabilization of the glucagon molecule have been disclosed, the confounding factors limit the utility of this approach for the treatment of patients with diabetes mellitus.

[0010] The present disclosure is directed at solving this problem via the modification of a stabilized glucagon analogue to calibrate its activity and duration of action relative to a prandial insulin analogue.SUMMARY

[0011] The present disclosure includes one or more of the features recited in the appended claims and / or the following features which, alone or in any combination, may comprise patentable subject matter.

[0012] The development of stabilized glucagon analogues is medically beneficial. The present disclosure is directed to the development of a stabilized glucagon analogue that is “suboptimal” with respect to molar activity and rapidity of action. By reducing the activity of glucagon and modestly extending its duration of action from about 1 hour to about 3 to 5 hours, the “suboptimal” modifications may enhance the compatibility of the glucagon analogue for coformulation and co-injection with a prandial insulin analogue for the treatment of diabetes mellitus. Such co-formulation and co-injection may protect a patient from hypoglycemia, which is a serious complication of insulin replacement therapy.

[0013] The present disclosure is directed towards the modification of a stabilized glucagonanalogue to calibrate its activity and duration of action relative to a prandial insulin analogue.

[0014] The present disclosure is directed toward the modification of a stabilized glucagon analogue such that its molar activity and duration of action on subcutaneous injection more closely match those of prandial insulin analogues than is characteristic of wild-type glucagon or the parent stabilized glucagon analogue. The present disclosure is directed towards embodiments that may reduce glucagon in vitro and in vivo activities (i.e., potency) to allow for essentially unopposed action of insulin under hyperglycemic conditions while being able to activate glucose production under hypoglycemic conditions. The present disclosure is directed towards embodiments that may extend the duration of glucagon action from less than one hour to about 3 to 5 hours, such that protection from late-post-prandial hypoglycemia is conferred on co-injection of the modified glucagon analogue and prandial insulin analogue in the subcutaneous space.

[0015] The present disclosure is directed to a class of glucagon peptide analogues that may resist fibrillation at neutral pH, exhibit reduced or suboptimal hormonal signaling activity, and exhibit modest or suboptimal binding to serum albumin. The latter two "suboptimal" properties may enhance the compatibility of the glucagon analogue for co-formulation and co-injection with a prandial insulin analogue to confer protection from hypoglycemia 2-5 hours after a meal. In one embodiment, the glucagon analogue is provided by a modification of Lysl2 in dasiglucagon by acylation of its epsilon-amino group; an acyl-chain length of 5-10 carbons to provide weak albumin binding and decreased hormonal activity. These perturbations enhance the compatibility of the glucagon analogue with a co-injected prandial insulin analogue regarding molar activities and duration of action.

[0016] The present disclosure is directed to a method of using rapid-acting glucagon-insulin co-formulation and co-injection for treating diabetes mellitus is described. Such a method reduces the risk of post-prandial hypoglycemia and allows for the effective use of insulin to achieve tight glycemic control.

[0017] According to a first aspect of the present disclosure, a stabilized glucagon analogue comprises a modification designed to jointly (a) impair molar activity by about 2 fold to about 20 fold relative to a wild-type human glucagon and (b) extend the duration of hormonal signaling on subcutaneous injection to about 3 hours to about 5 hours. In some embodiments, the molar activity may be impaired by about 2 fold to about 4 fold, about 4 fold to about 8 fold, about 8 fold to about 12 fold, about 12 fold to about 16 fold, or about 16 fold to about 20 fold,including any amount or range comprised therein.

[0018] In some embodiments, the modification is an acetylation of a Lysine residue by an acetyl moiety at position 12. In some embodiments, the modification is an acetylation of a Lysine residue within a C-terminal extension of the glucagon analogue of lengths 1-10 residues. In some embodiments the acetyl moiety contains from about 5 to about 10 carbon atoms. In some embodiments, the acetyl moiety is connected to the Lysine residue through a spacer element comprises 3 to 10 carbon atoms carbons. In some embodiments, the acetyl moiety further comprises 1 to 3 nitrogen atoms.

[0019] In some embodiments, the modification is a dicarboxylic acid, or a spacer-linked dicarboxylic acid linked to the epsilon amino group of the Lysine residue. In some embodiments, the stabilized glucagon analogue is dasiglucagon (SEQ ID 2). In some embodiments, the stabilized glucagon analogue comprises a side-chain lactam bridge or sidechain disulfide bridge. In some embodiments, the stabilized glucagon analogue is further modified by polyethylene glycol (PEGylation).

[0020] In some embodiments, the modification is a C-terminal domain comprises an albumin binding domain that confers binding to serum albumin. In some embodiments, the albumin-binding domain is modified to weaken binding to albumin to calibrate clearance of the glucagon analogue is 3 to 5 hours. In some embodiments, the C-terminal domain is derived from albumin-binding domains such as GA3, ABD035, or ABDCon. In some embodiments, the glucagon analogue moiety is derived from dasiglucagon.

[0021] According to a second aspect of the present disclosure, a pharmaceutical composition comprises a glucagon analogue as described above with a prandial insulin analogue. In some embodiments, the glucagon analogue is calibrated so that a duration of action of the glucagon analogue matches that of the prandial insulin analogue.

[0022] According to a third aspect of the present disclosure, a method of treating a patient afflicted with hypoglycemia, comprises administering a physiologically effective amount of the pharmaceutical composition described above.

[0023] According to a fourth aspect of the present disclosure, a method of treating a patient with diabetes mellitus, comprises administering a physiologically effective amount of the pharmaceutical composition described above.BRIEF DESCRIPTION OF DRAWINGS

[0024] The following description accompanies the drawing(s), all given by way of nonlimiting examples that may be useful to understand how the described method and composition may be embodied.

[0025] Fig. 1 illustrates a physiological glucose-responsive “switch."

[0026] Fig. 2A illustrates the insulin signaling pathway.

[0027] Fig. 2B illustrates the glucagon signaling pathway.

[0028] Fig. 2C illustrates a “cross-talk” paradigm for concurrent glucagon- and insulin signaling in hepatocytes.

[0029] Fig. 3 illustrates the time course of glucagon action.

[0030] Fig. 4 illustrates the in vivo activity of glucagon insulin fusion proteins (FPs) highlight mismatch in timing between glucagon action and insulin action. Activity of FPs was evaluated upon subcutaneous injection in non-diabetic rats: A / A, active insulin and active glucagon moieties; A / I, active insulin and inactive glucagon moieties; FA, inactive insulin and active glucagon moieties; and PI, inactive insulin and inactive glucagon moieties. The dose in each case was 36 nmol / kg dose with n=9-10 rats per group. Initial increase in BGC was observed only on injection of A / A FP (•; 0-20 min) or FA FP (A; 0-45 min), demonstrating dependence on an active glucagon moiety. Effects of FI FP (♦) was indistinguishable from that of diluent alone (X). Activities were measured under hyperglycemic conditions (mean initial BGC ca. 400 mg / dL); error bars represent SEM.

[0031] Fig. 5A illustrates the crystal structure of fatty acids bound to human serum albumin. Fig. 5B is a schematic illustration of acylated hormones bound to circulating albumin as a long- lived intravenous depot. The degree of protracted action depends on the strength of binding to albumin.

[0032] Fig. 6 shows the primary structure of human glucagon. The conserved Lysine residue at position 12 is highlighted in red (circled).

[0033] Fig. 7 shows an alternative site of acylation in an extended glucagon analogue.

[0034] Fig. 8 illustrates data obtained from dog studies of glucagon and albumin pre-bound acylated derivative of dasiglucagon. (a-c) Control studies of wild-type glucagon at doses of (a) 150 ng / kg, (b) 300 ng / kg and (c) 600 ng / kd. (d) Acylated acylated analogue containing a C8- carbon fatty diacid bound to Lysl2 via a polar space element (SEQ ID 4) at a dose of 3.6 pg / kg.DETAILED DESCRIPTION

[0035] Certain exemplary embodiments will now be described to provide an overall understanding of the principles of the structure, function, manufacture, and use of the devices and methods disclosed herein. One or more examples of these embodiments are illustrated in the accompanying drawings. Those skilled in the art will understand that the devices and methods specifically described herein and illustrated in the accompanying drawings are nonlimiting exemplary embodiments and that the scope of the present disclosure is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present disclosure. Accordingly, aspects and features of every embodiment may not be described concerning each embodiment, but those aspects and features apply to the various embodiments unless statements or understandings are to the contrary.

[0036] As used herein, the term “patient” or “user” refers to any subject including mammals and humans. The patient may have a disease or is suspected of having a disease and as such is being treated with a drug. In some instances, the patient is a mammal, such as a human, a premature neonate, neonate, infant, juvenile, adolescent, or adult thereof. In some instances, the term “patient,” as used herein, refers to a human (e.g., a man, a woman, or a child). In some instances, the term “patient,” as used herein, refers to laboratory animals of an animal model study. The patient or subject may be of any age, sex, or combination thereof.

[0037] The term “treating” refers to administering therapy in an amount, manner, or mode effective (e.g., a therapeutic effect) to improve a condition, symptom, disorder, or parameter associated with a disorder, or a likelihood thereof.

[0038] The terms “essentially” or “substantially” as used herein mean to a great or significant extent, but not completely.

[0039] The term “about” as used herein refers to any values, including both integers and fractional components that are within a variation of up to ±10% of the value modified by the term “about.”

[0040] As used herein the term "amino acid" encompasses any molecule containing both amino and carboxyl functional groups, wherein the amino and carboxylate groups are attached to the same carbon (the alpha carbon). The alpha carbon optionally may have one or two furtherorganic substituents. For the purposes of the present disclosure designation of an amino acid (e.g., by reference to the amino acid single-letter code) without specifying its stereochemistry is intended to encompass either the L or D form of the amino acid or a racemic mixture. However, in the instance where an amino acid is designated by its three-letter code and includes a residue number, the D form of the amino acid is specified by the inclusion of a lowercase d before the three-letter code and residue number (e.g., dLysl), wherein the designation lacking the lower case d (e.g., Lysl) is intended to specify the native L form of the amino acid. In this nomenclature, the inclusion of the residue number designates the position of the amino acid in the sequence of the peptide, wherein amino acids that are located within this sequence are designated by positive residue numbers numbered consecutively from the N Terminus. Additional amino acids linked to an analogue of a native peptide either at the N Terminus or through a side chain are numbered starting with 0 and increasing in negative integer value as they are further removed from the native peptide sequence.

[0041] As used herein the term “non-coded amino acid” encompasses any amino acid that is not an L-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Vai, Trp, Tyr.

[0042] As used herein a “bioactive polypeptide” refers to polypeptides that are capable of exerting a biological effect in vitro and / or in vivo.

[0043] As used herein, a peptide or polypeptide is intended to encompass peptides / polypeptides that have modified amino- and / or carboxy termini. For example, an amino-acid sequence designating the standard amino acids is intended to encompass standard amino acids at the N- and C Terminus as well as modified amino acids, such as a corresponding C -terminal amino acid modified to comprise an amide group in place of the terminal carboxylic acid.

[0044] As used herein an “acylated” amino acid is an amino acid comprising an acyl group that is non-native to a naturally occurring amino acid, regardless of the means by which it is produced. Exemplary methods of producing acylated amino acids and acylated peptides are known in the art; these include acylating an amino acid before inclusion in the peptide or chemical acylation of the peptide following its complete synthesis. In some embodiments the acyl group causes the peptide to have one or more of (i) a prolonged half-life in circulation, (ii) a delayed onset of action, (iii) an extended duration of action, (iv) an improved resistance to proteases and (v) increased or decreased potency at insulin receptor isoforms.

[0045] As used herein, an “alkylated” amino acid is an amino acid containing an alkyl group that is non-native to a naturally occurring amino acid, regardless of how it is produced. Exemplary methods of producing alkylated amino acids and alkylated peptides are known in the art; these include alkylating an amino acid before inclusion in the peptide or chemical alkylation of the peptide following its synthesis. Without being held to any particular theory, it is believed that alkylation of peptides will achieve similar, if not the same, effects as acylation of the peptides, e.g., a prolonged half-life in circulation, a delayed onset of action, an extended duration of action, an improved resistance to proteases and increased or decreased potency.

[0046] As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate-buffered saline (PBS) solution, water, emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.

[0047] As used herein the term "pharmaceutically acceptable salt" encompasses salts of compounds that retain the biological activity of the parent compound and that are not biologically or otherwise undesirable. Many of the compounds disclosed herein are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0048] As used herein, the term "hydrophilic moiety" encompasses any compound that is readily water-soluble or readily absorbs water, and that are tolerated in vivo by mammalian species without toxic effects (i.e., are biocompatible). Examples of hydrophilic moieties include polyethylene glycol (PEG), polylactic acid, polyglycolic acid, a polylactic-polyglycolic acid copolymer, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyl methacrylate, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose and co-polymers thereof, as well as natural polymers including, for example, albumin, heparin, and dextran.

[0049] As used herein, the term "treating" includes alleviation of the symptoms associated with a specific disorder or condition and / or preventing or eliminating said symptoms. For example, as used herein the term "treating diabetes" (or “treating DM”) will refer in general to maintaining blood-glucose concentrations near normal levels and may include increasing or decreasing blood-glucose concentrations depending on a given situation

[0050] As used herein, an "effective" amount or a "therapeutically effective amount" of an insulin analogue refers to a nontoxic but sufficient amount of an insulin analogue to provide the desired effect. For example, one desired effect would be the prevention or treatment of hyperglycemia. The amount that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, mode of administration, nutritional status and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0051] As used herein, the term "parenteral" means not through the alimentary canal but by some other route such as intranasal, inhalation, subcutaneous (SQ), intramuscular, intraspinal, or intravenous (IV).

[0052] As used herein, the term "derivative" is intended to encompass chemical modification to a compound (e.g., an amino acid), including chemical modification in vitro, e.g., by introducing a group in a side chain in one or more positions of a polypeptide, e.g., a nitro group in a tyrosine residue or iodine in a tyrosine residue, or by conversion of a free carboxylic group to an ester group or to an amide group, or by converting an amino group to an amide by acylation, or by acylating a hydroxy group rendering an ester, or by alkylation of a primary amine rendering a secondary amine or linkage of a hydrophilic moiety to an amino-acid side chain. Other derivatives are obtained by oxidation or reduction of the side chains of the aminoacid residues in the polypeptide.

[0053] As used herein, the term "identity" relates to the similarity between two or more sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100 to achieve a percentage. Thus, two copies of exactly the same sequence have 100% identity, whereas two sequences that have amino-acid deletions, additions, or substitutions relative to one another have a lower degree of identity. Those skilled in the art will recognize that several computer programs, such as those that employ algorithms such as BLAST (Basic Local Alignment Search Tool, Altschul et al. (1993) J. Mol. Biol. 215:403-410), are available for determining percent sequence identity.

[0054] As used herein, an amino-acid “modification” refers to a substitution of an amino acid, or the derivation of an amino acid by the addition and / or removal of chemical groups to / from the amino acid, and includes substitution with any of the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids.Commercial sources of atypical amino acids include Sigma-Aldrich (Milwaukee, WI), ChemPep Inc. (Miami, FL), and Genzyme Pharmaceuticals (Cambridge, MA). Atypical amino acids may be purchased from commercial suppliers, synthesized de novo, or chemically modified or derivatized from naturally occurring amino acids.

[0055] As used herein, an amino acid "substitution" refers to the replacement of one aminoacid residue by a different amino-acid residue.

[0056] As used herein, the general term "polyethylene-glycol chain" (or "PEG chain") encompasses mixtures of condensation polymers of ethylene oxide and water, in a branched or straight chain, represented by the general formula H(OCH2CH2)nOH, wherein n is at least 2. "Polyethylene-glycol chain" (or "PEG chain") is used in combination with a numeric suffix to indicate the approximate average molecular weight thereof. For example, PEG-5,000 refers to polyethylene-glycol chain having a total molecular weight average of about 5,000 Daltons.

[0057] As used herein, the term "pegylated" and like terms include any compound that has been modified from its native state by linking a polyethylene-glycol chain to the compound. A "pegylated polypeptide" is a polypeptide that has a PEG chain covalently bound to the polypeptide.

[0058] As used herein, a "linker" is a bond, molecule, or group of molecules that binds two separate entities to one another. Linkers may provide for optimal spacing of the two entities or may further supply a labile linkage that allows the two entities to be separated from each other. Labile linkages include photocleavable groups, acid-labile moieties, base-labile moieties, and enzyme-cleavable groups.

[0059] As used herein, the term "patient" without further designation is intended to encompass any warm-blooded vertebrate domesticated animal (including for example, but not limited to livestock, horses, cats, dogs, and other pets) and humans.

[0060] The term “isolated” as used herein means having been removed from its natural environment. In some embodiments, the analogue is made through recombinant methods wherein the analogue is isolated from the host cell, which typically may be a bacterial cell, yeast cell, inset cell, or mammalian cell.

[0061] The term “purified” as used herein encompasses the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment; in practice, this means having been increased in purity as a result of being separated from other components of the originalcomposition. The term "purified polypeptide" is used herein to describe a polypeptide that has been separated from other compounds including, but not limited to nucleic-acid molecules, lipids, and carbohydrates.

[0062] The present disclosure is directed to the utility of modifying a bioactive peptide to impair peptide activity and delay peptide clearance. Surprisingly, such impairment can improve the compatibility of the bioactive peptide with co-formulated partner proteins or peptides in a dual pharmaceutical formulation. Glucagon and insulin are examples if such therapeutic peptides or proteins.

[0063] In one embodiment, the present disclosure is directed to a physiologic switch in the liver’s relative hormone responsiveness (between glucagon and insulin) as a function of glycemia. Under hyperglycemic conditions, insulin signaling predominates, whereas under hypoglycemic conditions, glucagon predominates. Fig. 1 illustrates a physiological glucoseresponsive “switch." Co-injection of insulin and glucagon exploits a physiological switch in hepatic hormone responsiveness. Under hyperglycemic conditions, insulin activity prevails, whereas glucagon overpowers insulin under hypoglycemic conditions.

[0064] The potential utility of this switch could in principle be enhanced by “cross-talk” between the respective insulin- and glucagon signaling pathways in the liver. Fig. 2 illustrates the “cross-talk” paradigm for concurrent glucagon- and insulin signaling in hepatocytes. Fig. 2A shows the insulin signaling pathway. Fig. 2B shows the glucagon signaling pathway. Fig. 2C shows the cross-talk between pathways, rationalizing why glucagon signaling may enhance insulin sensitivity (Panels are adapted from: Habegger, K. M. Cross Talk between Insulin and Glucagon Receptor Signaling in the Hepatocyte. Diabetes 71, 1842-1851 (2022).) Fig. 3 shows the intrinsic stability of the glucagon molecule, the greater molar activity of glucagon relative to insulin, and the shorter duration of action of glucagon relative to the duration of prandial insulin analogues. These aspects complicate the use of this physiological switch (the panel is reproduced from: Castle, J.R., El Youssef, I., Branigan, D., Newswanger, B., Strange, P., Cummins, M., Shi, L., and Prestrelski, S. Comparative Pharmacokinetic / Pharmacodynamic Study of Liquid Stable Glucagon Versus Lyophilized Glucagon in Type 1 Diabetes Subjects. I. Diabetes Sci. Technol. 10(5): 1101-7 (2016); see also Ulrike Hbvelmann, U., Bysted, B.V., Mouritzen, U., Macchi, F., Larners, D., Kronshage, B., Mpller, D.V., Heise, T. Pharmacokinetic and Pharmacodynamic Characteristics of Dasiglucagon, a Novel Soluble and Stable Glucagon Analog. Diabetes Care 41 (3): 531 -537 (2018)). Pharmacodynamics (red; right-hand axis) and pharmacokinetics (purple; left-hand axis) of glucagon (or non-acylated / non-PEGylated glucagon analogues) following subcutaneous injection via two devices (solid lines, pump; dashed lines; GlucaGen hypo-kit, Novo-Nordisk). Note waning of glucagon action after ca. 45 min and negligible activity after 2 hours — a mismatch in timing relative to onset of late post-prandial hypoglycemia.

[0065] Fig. 4 illustrates the in vivo activity of glucagon insulin fusion proteins (FPs) highlight mismatch in timing between glucagon action and insulin action. Activity of FPs was evaluated upon subcutaneous injection in non-diabetic rats: A / A, active insulin and active glucagon moieties; A / I, active insulin and inactive glucagon moieties; FA, inactive insulin and active glucagon moieties; and FI, inactive insulin and inactive glucagon moieties. The dose in each case was 36 nmol / kg dose with n=9-10 rats per group. Initial increase in BGC was observed only on injection of A / A FP (•; 0-20 min) or FA FP (A; 0-45 min), demonstrating dependence on an active glucagon moiety. Effects of FI FP (♦) was indistinguishable from that of diluent alone (X). Activities were measured under hyperglycemic conditions (mean initial BGC ca. 400 mg / dL); error bars represent SEM. The pharmacodynamic mismatch is illustrated in PCT WO2022173807A3 and is incorporated by reference herein.

[0066] Fig. 8 illustrates dog studies of glucagon and albumin pre-bound acylated derivative of dasiglucagon. Figs. 5a-5c illustrate control studies of wild-type glucagon at doses of (5a) 150 ng / kg, (5b) 300 ng / kg and (5c) 600 ng / kd. Fig. 5d illustrates data from using a acylated analogue containing a C8-carbon fatty diacid bound to Lysl2 via a polar space element (Ns(S)- (22,42-dicarboxy-10,19,24-trioxo-3,6,12,L5-tetraoxa-9,l 8,23-triazadotetracontan- l-oyl); SEQ ID 4) at a dose of 3.6 ng / kg. The mass of the dog was about 30 kg. The dog studies were performed following subcutaneous injection of glucagon or the acylated glucagon analogue between the shoulder blades. The injection volume was 8 ml containing 100 pg peptide. The peptide was dissolved in a filtered buffer containing 3% human serum albumin and 70 ppm polysorbate-20 (Tween) in 5 mM sodium phosphate (pH 7.4) and 140 mM NaCl.

[0067] The glucagon molecule contains 29 residues and binds to a G-protein-coupled receptor (GPCR). Crystal structures of complexes between glucagon or glucagon analogues and the glucagon receptor have defined the hormone’s mode of binding and key hormone- receptor contacts. Specific residues are indicated by the amino-acid type (typically in standard three-letter (or one letter) code; e.g., Lys (K) and Ala (A) indicate Lysine and Alanine) followed by the residue number. For example, Histidine at position 1 , which is critically required for activity, is designated Hisl (or Hl). The insulin molecule contains two chains, an A chain, containing 21 residues, and a B chain containing 30 residues. The mature hormone is derivedfrom a longer single-chain precursor, designated proinsulin. Specific residues in the insulin molecule are indicated by the amino-acid type (typically also in standard three-letter code) and in superscript the chain (A or B) and position in that chain. For example, Alanine at position 14 of the B chain of human insulin is indicated by AlaB14; and likewise, Lysine at position B28 of insulin lispro (the active component of Humalog; Eli Lilly and Co.) is indicated by LysB28. Insulin binds to a disulfide-linked dimeric receptor tyrosine kinase with chains (a )2, where the a and p chains are processed from a single biosynthetic precursor. The a subunit is extracellular and contains the insulin-binding sites, whereas the P subunit is transmembrane; the latter both contributes to the extracellular “legs” of the receptor and contains the intracellular tyrosinekinase domain (one per P subunit).

[0068] In some embodiments, a conservative amino acid substitution is an exchange within one of the following five groups:I. Small aliphatic, nonpolar, or slightly polar residues:Ala, Ser, Thr, Pro, Gly;II. Polar, negatively charged residues and their amides:Asp, Asn, Glu, Gin, cysteic acid and homocysteic acid;III. Polar, positively charged residues:His, Arg, Lys; Ornithine (Om)IV. Large, aliphatic, nonpolar residues:Met, Leu, He, Vai, Cys, Norleucine (Nle), homocysteineV. Large, aromatic residues:Phe, Tyr, Trp, acetyl phenylalanine

[0069] One embodiment of the present disclosure is directed to a class of glucagon analogues comprising multiple amino-acid substitutions. The amino acid substitution may be a conservative amino acid substitution. The amino acid substitution may be a natural or unnatural amino acid substitution introduced to protect the peptide from fibrillation.

[0070] One embodiment of the present disclosure is directed to a class of glucagon analogues comprising side-chain / side-chain tethers (or “staples”) that reduce or preserve bioactivity. In some embodiments, the bioactivity may be reduced or preserved while the peptide hormone’s susceptibility to fibrillation is reduced.

[0071] In some embodiments, the glucagon analogues may either comprise a conservedLysine at position 12 (as in wild- type glucagon) or, if this Lysine is lacking, such a Lysine may be substituted at position 12.

[0072] One embodiment of the present disclosure is directed towards the modification of a stabilized glucagon analogue to calibrate its activity and duration of action relative to a prandial insulin analogue. Such calibration requires impairing its intrinsic molar activity by perturbing the hormone -receptor interface and impairing its clearance by conferring low-affinity binding to serum albumin. Fig. 5A illustrates a crystal structure of fatty acids bound to human serum albumin (Protein Databank entry 1E7E; Bhattacharya, A.A., Grune, T., Curry, S. Crystallographic Analysis Reveals Common Modes of Binding of Medium and Long-Chain Fatty Acids to Human Serum Albumin. J. Mol. Biol. 303:721 (2000)). Fig. 5B shows an illustration of acylated hormones bound to circulating albumin as a long-lived intravenous depot. Degree of protracted action depends on strength of binding to albumin.

[0073] In one embodiment, a conserved Lysine residue at position 12 of the hormone may be acylated (Fig. 6). In one embodiment, a glucagon peptide analogue may be extended at its C-terminus by 1-10 residues containing a Lysine residue as site of acetylation (for example, a tripeptide extension as shown in Fig. 7). Fig. 7 shows an optional placement of C-terminal tripeptide extension with Lys32 preceded by non-basic residues X and Y (residues 30 and 31). The new C terminus may optionally be amidated. Residue 30 may be Gly, Ser, Ala, Asp, or Glu and residue 31 may be Ser, Ala, or Gly.

[0074] In one embodiment, rapid-acting glucagon-insulin co-formulation and co-injection is used to treat diabetes mellitus. In some embodiments, acylation of the epsilon-amino group of Lysl2 may impair activity and confer binding to serum albumin, thereby delaying clearance. In some embodiments, acylated versions of stabilized glucagon analogues may be tunable to optimize their compatibility with prandial insulin analogues for co-formulation and co-injection into the subcutaneous space for the safer and more effective treatment of diabetes mellitus. The disclosed glucagon analogues and glucagon analogue-insulin analogue co-infusion systems would thus meet medical needs not optimally addressed by current technologies.

[0075] EXEMPLARY EMBODIMENTS

[0076] SEQ ID 1 is the sequence of wild-type human glucagon. The conserved Lysine is at position 12.

[0077] SEQ ID 1 : His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser- Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr.

[0078] In one embodiment, the present disclosure id directed to SEQ ID 2. SEQ ID 2 is the sequence of dasiglucagon. The seven sites of modification are at positions 16, 17, 20, 21, 24, 27, at 28.

[0079] SEQ ID 2: His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-aib- Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Tip-Leu-Glu-Ser-Thr.

[0080] In one embodiment, the present disclosure is directed to SEQ ID 3. SEQ ID 3 is an analogue of dasiglucagon wherein the a-aminobutyric acid (Aib) ordinarily at position 16 is substituted by Serine.

[0081] SEQ ID 3 : HSQGTFTSDYSKYLDSARAEEFVKWLEST.

[0082] In one embodiment, the present disclosure is directed to SEQ ID 4. SEQ ID 4 is an analogue of dasiglucagon wherein the a-aminobutyric acid (Aib) ordinarily at position 16 is substituted by Serine and wherein an acylated Lysine [K*] at position 12 denotes Lys[Ns(S)- (22,42-dicarboxy- 10, 19,24-trioxo-3 ,6, 12,15 -tetraoxa-9, 18,23-triazadotetracontan- 1 -oyl) ] connected via a peptide bond to a C8 fatty diacid.

[0083] SEQ ID 4: HSQGTFTSDYSK[K*]YLDSARAEEFVKWLEST

[0084] In one embodiment, the present disclosure is directed to SEQ ID 5. SEQ ID 5 is an analogue of dasiglucagon containing a three-residue C-terminal extension (EGK; italics) wherein the C-terminus is amidated.

[0085] SEQ ID 5: HSQGTFTSDYSKYLD[Aib]ARAEEFVKWLEST-EGK-NH2

[0086] In one embodiment, the present disclosure is directed to SEQ ID 6. SEQ ID 6 is an analogue of dasiglucagon wherein the a- aminobutyric acid (Aib) ordinarily at position is substituted by Serine and wherein an acylated Lysine [K*] at the C-terminus of a three-residue extension (EGK; italics) denotes Lys[Ne(S)-(22,42-dicarboxy-10,19,24-trioxo-3,6,12,15- tetraoxa-9,18,23-triazadotetracontan- 1 -oyl)] connected via a peptide bond to a C8 fatty diacid.

[0087] SEQ ID 6: HSQGTFTSDYSKYLDSARAEEFVKWLESTEG[K*]

[0088] In one embodiment, the present disclosure is directed to SEQ ID 7. SEQ ID 7 is an analogue of dasiglucagon wherein the a-aminobutyric acid (Aib) ordinarily at position is substituted by Serine and wherein an acylated Lysine [K*] at the C-terminus of a three-residue extension (EGK; italics) denotes Lys[Ns(S)-(22,42-dicarboxy-10,19,24-trioxo-3,6,12,15- tetraoxa-9,18,23-triazadotetracontan-l-oyl)] connected via a peptide bond to a C8 fatty diacid.

[0089] SEQ ID 7: H[Aib]QGTFTSDYSKYLDSARAEEFVKWLESTEG[K*]-NH2

[0090] In one embodiment, the present disclosure is directed to SEQ ID 8. SEQ ID 8 is an analogue of dasiglucagon containing a lactam bridge between the side chains of Lysine substituted at position 13 (bold) and a Glutamic Acid substituted at position 17, wherein the a- aminobutyric acid (Aib) ordinarily at position is substituted by Serine, wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0091] SEQ ID 8: HSQGTFTSDYSKKLDSEAAEEFVKWLEST

[0092] In one embodiment, the present disclosure is directed to SEQ ID 9. SEQ ID 9 is an analogue of glucagon containing a lactam bridge between the side chains of Lysine substituted at position 13 (bold) and a Glutamic Acid substituted at position 17 wherein a Lysine is available at either at position 12 or at the C-terminus of a three-residue extension (EEK) for modification by an acylated moiety.

[0093] SEQ ID 9: HSQGTFTSDYSKKLDSERAQDFVQWLM[Aib28]TEEK

[0094] In one embodiment, the present disclosure is directed to SEQ ID 10. SEQ ID 10 is an analogue of glucagon containing u-aminobutyric acid (Aib) at positions 2 and 28 wherein a Lysine is available at either at position 12 or at the C-terminus of an amidated four-residue extension (EGSK) for modification by an acylated moiety.

[0095] SEQ ID 10: H[Aib]QGTFTSDYSKYLD[Aib]RRAQDFVQWLM[Aib]TEGSK- NH2

[0096] In one embodiment, the present disclosure is directed to SEQ ID 11. SEQ ID 11 is an analogue of dasiglucagon containing a-aminobutyric acid (Aib) at position 2, wherein the Aib ordinarily at position 16 is substituted by Serine, wherein the Threonine (T) ordinarily at position 5 is substituted by Alanine (A), wherein the Glutamic Acid (E) ordinarily at position 21 is substituted by Ala (A), wherein a 8-residue C-terminal extension permits sortase-mediated ligation, and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0097] SEQ ID 11 : H[Aib]QGAFTSDYSKYLDS ARAEAFVKWLESTEGLPETGGS

[0098] In one embodiment, the present disclosure is directed to SEQ ID 12. SEQ ID 12 is an analogue of dasiglucagon containing a-aminobutyric acid (Aib) at position 2, wherein the Aib ordinarily at position 16 is substituted by Serine (S), wherein the Glutamic Acid (E) ordinarily at position 21 is substituted by Ala (A), wherein a 8-residue C-terminal extension permits sortase-mediated ligation (GLPETGGS) and wherein a Lysine is available at position12 for modification by an acylated moiety.

[0099] SEQ ID 12: H[Aib]QGTFTSDYSKYLDSARAEAFVKWLESTEGLPETGGS

[0100] In one embodiment, the present disclosure is directed to SEQ ID 13. SEQ ID 13 is an analogue of dasiglucagon containing a-aminobutyric acid (Aib) at position 2, wherein the Threonine (T) ordinarily at position 5 is substituted by Ala (A; bold), wherein the Aib ordinarily at position 16 is substituted by Serine (S), wherein a 9-residue C-terminal extension permits sortase-mediated ligation (EGLPETGGS) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0101] SEQ ID 13: H[Aib]QGAFTSDYSKYLDSARAEEFVKWLESTEGLPETGGS

[0102] In one embodiment, the present disclosure is directed to SEQ ID 14. SEQ ID 14 is an analogue of dasiglucagon wherein the Glycine (G) ordinarily at position 4 is substituted by Ala (A) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0103] SEQ ID 14: HSQATFTSDYSKYLD[Aib]ARAEEFVKWLEST

[0104] In one embodiment, the present disclosure is directed to SEQ ID 15. SEQ ID 15 is an analogue of dasiglucagon wherein the Threonine (T) ordinarily at position 5 is substituted by Ala (A) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0105] SEQ ID 15: HSQGAFTSDYSKYLD[Aib]ARAEEFVKWLEST

[0106] In one embodiment, the present disclosure is directed to SEQ ID 16. SEQ ID 16 is an analogue of dasiglucagon wherein the Threonine (T) ordinarily at position 7 is substituted by Ala (A) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0107] SEQ ID 16: HSQGTFASDYSKYLD[Aib]ARAEEFVKWLEST

[0108] In one embodiment, the present disclosure is directed to SEQ ID 17. SEQ ID 17 is an analogue of dasiglucagon wherein the Tyrosine (Y) ordinarily at position 7 is substituted by Ala (A) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0109] SEQ ID 17: HSQGTFTSDYSKALD[Aib]ARAEEFVKWLEST

[0110] In one embodiment, the present disclosure is directed to SEQ ID 18. SEQ ID 18 is an analogue of dasiglucagon wherein the Aib ordinarily at position 7 is substituted by Serine (S) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0111] SEQ ID 18: HSQGTFTSDYSKYLDS ARAEEFVKWLEST

[0112] In one embodiment, the present disclosure is directed to SEQ ID 19. SEQ ID 19 is an analogue of dasiglucagon wherein the Arginine (R) ordinarily at position 18 is substituted by Ala (A) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0113] SEQ ID 19: HSQGTFTSDYSKYLD[Aib]AAAEEFVKWLEST

[0114] In one embodiment, the present disclosure is directed to SEQ ID 20. SEQ ID 20 is an analogue of dasiglucagon wherein the Glutamic Acid (E) ordinarily at position 21 is substituted by Ala (A) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0115] SEQ ID 20: HSQGTFTSDYSKYLD[Aib]ARAEAFVKWLEST

[0116] In one embodiment, the present disclosure is directed to SEQ ID 21. SEQ ID 21 is an analogue of dasiglucagon wherein the Tryptophan (W) ordinarily at position 25 is substituted by Ala (A) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0117] SEQ ID 21 : HSQGTFTSDYSKYLDXARAEEFVKALEST

[0118] In one embodiment, the present disclosure is directed to SEQ ID 22. SEQ ID 22 is an analogue of dasiglucagon wherein the Glutamic Acid (E) ordinarily at position 25 is substituted by Ala (A) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0119] SEQ ID 22: HSQGTFTSDYSKYLD[Aib]ARAEEFVKWLAST

[0120] In one embodiment, the present disclosure is directed to SEQ ID 23. SEQ ID 23 is an analogue of dasiglucagon wherein the Aib ordinarily at position 16 is substituted by Serine (S), In one embodiment, the present disclosure is directed to SEQ ID 23. SEQ ID 23 wherein the Glutamic Acid (E) ordinarily at position 21 is substituted by Ala (A) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0121] SEQ ID 23: HSQGTFTSDYSKYLDSARAEAFVKWLEST

[0122] In one embodiment, the present disclosure is directed to SEQ ID 24. SEQ ID 24 is an analogue of dasiglucagon wherein the Aib ordinarily at position 16 is substituted by Serine (S), wherein a 9-residue C-terminal extension permits sortase-mediated ligation (EGLPETGGS) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0123] SEQ ID 24: HSQGTFTSDYSKYLDSARAEEFVKWLESTEGLPETGGS

[0124] In one embodiment, the present disclosure is directed to SEQ ID 25. SEQ ID 25 is an analogue of dasiglucagon wherein the Aib ordinarily at position 16 is substituted by Serine (S), wherein the Glutamic Acid (E) ordinarily present at position 21 is substituted by Alanine (A), wherein a 9-residue C-terminal extension permits sortase-mediated ligation (EGLPETGGS) and wherein a Lysine is available at position 12 for modification by an acylated moiety.

[0125] SEQ ID 25: HSQGTFTSDYSKYLDSARAEAFVKWLESTEGLPETGGS

[0126] The figures provided herein are not necessarily to scale, although a person skilled in the art will recognize instances where the figures are to scale and / or what a typical size is when the drawings are not to scale. While in some embodiments movement of one component is described with respect to another, a person skilled in the art will recognize that other movements are possible. Additionally, a number of terms may be used throughout the disclosure interchangeably but will be understood by a person skilled in the art. Further, to the extent features, sides, or steps are described as being “first’" or “second,’" such numerical ordering is generally arbitrary, and thus such numbering can be interchangeable. Still further, in the present disclosure, like-numbered components of various embodiments generally have similar features when those components are of a similar nature and / or serve a similar purpose. Lastly, the present disclosure includes some illustrations and descriptions that include prototypes, bench models, or experimental design. A person skilled in the art will recognize how to rely upon the present disclosure to integrate the techniques, systems, devices, and methods provided for into a product in view of the present disclosures.

[0127] While the concepts of the present disclosure are susceptible to various modifications and alternative forms, specific exemplary embodiments of the disclosure have been shown by way of example. It should be understood, however, that there is no intent to limit the concepts of the present disclosure to the particular disclosed forms; the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims. Although this disclosure refers to specific embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the subject matter set forth in the accompanying claims.

Claims

CLAIMS:

1. A stabilized glucagon analogue comprising a modification designed to jointly (a) impair molar activity by about 2 fold to about 20 fold relative to wild-type human glucagon and (b) extend a duration of hormonal signaling on subcutaneous injection to about 3 hours to about 5 hours.

2. The glucagon analogue of claim 1, wherein the modification is an acetylation of a Lysine residue by an acetyl moiety at position 12 or a Lysine residue within a C- terminal extension of the glucagon analogue of lengths 1-10 residues.

3. The glucagon analogue of claim 2, wherein the acetyl moiety contains from about 5 to about 10 carbon atoms.

4. The glucagon analogue of claim 2, wherein the acetyl moiety is connected to the Lysine residue through a spacer element comprises 3 to 10 carbon atoms carbons.

5. The glucagon analogue of claim 4, wherein the acetyl moiety further comprises 1 to 3 nitrogen atoms.

6. The glucagon analogue of claim 1, wherein the modification is a dicarboxylic acid or a spacer-linked dicarboxylic acid linked to the epsilon amino group of the Lysine residue.

7. The glucagon analogue of claim 1 , wherein the stabilized glucagon analogue is dasiglucagon (SEQ ID 2).

8. The glucagon analogue of claim 1, wherein the stabilized glucagon analogue comprises a side-chain lactam bridge or side-chain disulfide bridge.

9. The glucagon analogue of claim 1, wherein the stabilized glucagon analogue is further modified by polyethylene glycol (PEGylation).

10. The glucagon analogue of claim 1, wherein the modification is a C-terminal domain comprises an albumin binding domain that confers binding to serum albumin.

11. The glucagon analogue of claim 10, wherein the albumin-binding domain is modified to weaken binding to albumin to calibrate clearance of the glucagon analogue is 3 to 5 hours.

12. The glucagon analogue of claim 10, wherein the C-terminal domain is derived from albumin-binding domains selected from GA3, ABD035, and ABDCon.

13. The glucagon analogue of claim 2, wherein glucagon analogue moiety is derived from dasiglucagon.

14. A pharmaceutical composition comprising a glucagon analogue of claim 1 with a prandial insulin analogue.

15. A method of treating a patient afflicted with hypoglycemia, comprising administering a physiologically effective amount of the composition of claim 14.

16. A method of treating a patient with diabetes mellitus, comprising administering a physiologically effective amount of the composition of claim 14.

17. A stabilized glucagon analogue comprising a modification designed (a) resist fibrillation at neutral pH, (b) exhibit reduced or suboptimal hormonal signaling activity, and (c) exhibit modest or suboptimal binding to serum albumin.

18. The glucagon analogue of claim 17, wherein the modification is an acetylation of a Lysine residue by an acetyl moiety at position 12 or a Lysine residue within a C- terminal extension of the glucagon analogue of lengths 1-10 residues.

19. The glucagon analogue of claim 17, wherein the modification comprises changes in at positions 16, 17, 20, 21, 24, 27, at 28.

20. The glucagon analogue of claim 17, wherein the modification is calibrated so that a duration of action of the stabilized glucagon analogue matches that of a prandial insulin analogue.

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