A novel glucagon-like peptide-1/glucagon receptor dual agonist exhibits weight-lowering and diabetes-protective effects.

Glucagon has plenty of effects via a specific glucagon receptor(GCGR) like elevating the blood glucose, improving fatty acids metabolism, energy expenditure and increasing lipolysis in adipose tissue. The most important role of glucagon is to regulate the blood glucose, but the emergent possibilities of hyperglycaemia is exist. Glucagon could also slightly activate glucagon-like peptide-1 receptor(GLP-1R), which lead to blood glucose lowering effect. This study aims to erase the likelihood of hyperglycaemia and to remain the inherent catabolic effects through improving GLP-1R activation and deteriorating GCGR activation so as to lower the bodyweight and show diabetes-protective effects. Firstly, twelve cysteine modified GLP-1/GCGR dual agonists were synthesized (1-12). Then, the GLP-1R/GCGR mediated activation and biological activity in normal ICR mice were comprehensively performed. Compounds substituted by cysteine at positions 22, 23 and 25 in glucagon were observed to be better regulators of the body weight and blood glucose. To prolong the half-lives of derivatives, various fatty side chain maleimides were modified to optimal glucagon analogues. Laurate maleimide conjugate 4d was the most potent. Administration of 1000 nmol/kg 4d once every two days for a month normalized adiposity and glucose tolerance in diet-induced obese (DIO) mice. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were observed. These studies suggest that compound 4d behaves well in lowering body weight and maintaining energy expenditure without a chance of hyperglycaemia, 4d has strong clinical potential as an efficient GLP-1/GCGR agonist in the prevention and treatment of obesity and dyslipidemia.

Pyridyl-alanine as a Hydrophilic, Aromatic Element in Peptide Structural Optimization.

Glucagon (Gcg) 1 serves a seminal physiological role in buffering against hypoglycemia, but its poor biophysical properties severely complicate its medicinal use. We report a series of novel glucagon analogues of enhanced aqueous solubility and stability at neutral pH, anchored by Gcg[Aib16]. Incorporation of 3- and 4-pyridyl-alanine (3-Pal and 4-Pal) enhanced aqueous solubility of glucagon while maintaining biological properties. Relative to native hormone, analogue 9 (Gcg[3-Pal6,10,13, Aib16]) demonstrated superior biophysical character, better suitability for medicinal purposes, and comparable pharmacology against insulin-induced hypoglycemia in rats and pigs. Our data indicate that Pal is a versatile surrogate to natural aromatic amino acids and can be employed as an alternative or supplement with isoelectric adjustment to refine the biophysical character of peptide drug candidates.

Development of a highly stable, nonaqueous glucagon formulation for delivery via infusion pump systems.

Despite a vigorous research effort, to date, the development of systems that achieve glucagon stability in aqueous formulations (without reconstitution) has failed to produce any clinical candidates. We have developed a novel, nonaqueous glucagon formulation based on a biocompatible pharmaceutical solvent, dimethyl sulfoxide, which demonstrates excellent physical and chemical stability at relatively high concentrations and at high temperatures. This article reports the development of a novel, biocompatible, nonaqueous native human glucagon formulation for potential use in subcutaneous infusion pump systems. Data are presented that demonstrate physical and chemical stability under presumed storage conditions (>2 years at room temperature) as well as "in use" stability and compatibility in an Insulet's OmniPod(®) infusion pump. Also presented are results of a skin irritation study in a rabbit model and pharmacokinetics/pharmacodynamics data following pump administration of glucagon in a diabetic swine model. This nonaqueous glucagon formulation is suitable for further clinical development in pump systems.

Optimization of the native glucagon sequence for medicinal purposes.

BACKGROUND: Glucagon is a life-saving medication used in the treatment of hypoglycemia. It possesses poor solubility in aqueous buffers at or near physiological pH values. At low and high pH, at which the peptide can be formulated to concentrations of a milligram or more per milliliter, the chemical integrity of the hormone is limited, as evidenced by the formation of multiple degradation-related peptides. Consequently, the commercial preparation is provided as a lyophilized solid with an acidic diluent and directions for rendering it soluble at the time of use. Any unused material is recommended for disposal immediately after initial use. METHODS: A set of glucagon analogs was prepared by solid-phase peptide synthesis to explore the identification of a glucagon analog with enhanced solubility and chemical stability at physiological pH. The physical properties of the peptide analogs were studied by solubility determination, high-performance chromatography, and mass spectral analysis. The biochemical properties were determined in engineered human embryonic kidney cell line 293 (HEK293) cells that overexpressed either the human glucagon or glucagon-like peptide-1 (GLP-1) receptors linked to a luciferase reporter gene. RESULTS: We observed the previously characterized formation of glucagon degradation products upon incubation of the peptide in dilute acid for extended periods or elevated temperature. Lowering the isoelectric point of the hormone through the substitution of asparagine-28 with aspartic acid significantly increased the solubility at physiological pH. Similarly, the C-terminal extension (Cex) of the hormone with an exendin-based, 10-residue, C-terminal sequence yielded a peptide of dramatically enhanced solubility. These two glucagon analogs, D28 and Cex, maintained high potency and selectivity for the glucagon receptor relative to GLP-1 receptor. CONCLUSIONS: Glucagon presents unique structural challenges to the identification of an analog of high biological activity and selectivity that also possesses sufficient aqueous solubility and stability such that it might be developed as a ready-to-use medicine. The glucagon analogs D28 and Cex demonstrated all of the chemical, physical, and biochemical properties supportive of further study as potential clinical candidates for treatment of hypoglycemia.

Design, synthesis and crystallization of a novel glucagon analog as a therapeutic agent.

Glucagon and glucagon-like peptide 1 (GLP-1) are drugs or drug candidates for the treatment of metabolic diseases such as diabetes and obesity. The native hormones have pharmacological deficiencies such as short half-life and poor solubility. A novel glucagon receptor agonist named glucagon-Cex has been designed, synthesized and crystallized. This peptide was highly soluble under physiological conditions and crystallized readily. The crystal diffracted X-rays to 2.2 A resolution and the diffraction was consistent with space group P23, with unit-cell parameters a = b = c = 48.20 A, alpha = beta = gamma = 90.0 degrees. The crystals were suitable for a full structural determination to reveal the conformational differences between glucagon-Cex and the native hormone.

Advancements in the anti-diabetes chemotherapeutics based on amino acids, peptides, and peptidomimetics.

Diabetes Mellitus (DM) is a highly prevalent chronic disease. Recent years have witnessed development of many new oral drugs; novel insulin analogs and their delivery systems for the treatment of patients with either type-1 or type-2 DM. The impetus for developing new antidiabetic drugs comes from the unmet need of pharmacological tools that allow diabetic patients to achieve recommended glucose control targets by precise, safe and effective ways. The number of people afflicted with DM worldwide has increased considerably in recent years and is projected to increase dramatically over the next decades. In the recent times, design and synthesis of bioactive peptides and peptidomimetics has undergone a paradigm shift. Non-proteinogenic amino acids, peptides and peptidomimetics are emerging as novel drug candidates for the treatment of various diseases and/or disorders. This review mainly discusses the advancements in the usage of unnatural amino acids, peptides and peptidomimetics as potential therapeutic agents for the treatment of DM.

Structural requirements of the N-terminal region of GLP-1-[7-37]-NH2 for receptor interaction and cAMP production.

A series of GLP-1-[7-36]-NH(2) (tGLP-1) and GLP-1-[7-37] analogs modified in position 7, 8, 9 and 36, have been designed and evaluated on murine GLP-1 receptors expressed in RIN T3 cells for both their affinity and activity. Ten of the synthesized peptides were found full agonists with activities superior or at least equal to that of the native hormone. Five of them were investigated for their plasmatic stability and the most stable, [a(8)-desR(36)]GLP-1-[7-37]- NH(2) (Compound 8), evaluated in vivo in a glucose tolerance test which confirmed a clearly longer activity than that of the native hormone. We also performed circular dichroism study and propose a hypothetical structural model explaining the most part of observed activities of GLP-1 analogs on RIN T3 cells.

N-terminal His(7)-modification of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity.

Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His(7)-modified analogue of GLP-1, N-pyroglutamyl-GLP-1, as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC(50) values 32.9 and 6.7 nM, respectively) compared with native GLP-1 (IC(50) 0.37 nM). Similarly, both analogues stimulated cAMP production with EC(50) values of 16.3 and 27 nM respectively compared with GLP-1 (EC(50) 4.7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5.6 mM glucose (P<0.05 to P<0.001) similar to native GLP-1. Both analogues (25 nM/kg body weight) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body weight) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biologically potent forms of GLP-1. The particularly powerful antihyperglycaemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes.

[Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16-0.52 nm) and IC50s (4.3-8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)2]incretins > or = [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.

NN-2211 Novo Nordisk.

Novo Nordisk A/S, under license from Scios Inc, is developing NN-2211, a stable analog of the naturally occurring peptide hormone glucagon-like peptide 1 (GLP-1), which stimulates insulin release in response to increases in blood sugar levels, for the potential treatment of type 2 diabetes.

Development of potent glucagon antagonists: structure-activity relationship study of glycine at position 4.

We examined the functional role of glycine at position 4 in the potent glucagon antagonist [desHis(1), Glu(9)]glucagon amide, by substituting the L- and D-enantiomers of alanine and leucine for Gly(4) in this antagonist. The methyl and isobutyl side-chain substituents were introduced to evaluate the preference shown by the glucagon receptor, if any, for the orientation of the N-terminal residues. The L-amino acids demonstrated only slightly better receptor recognition than the D-enantiomers. These results suggest that the Gly(4) residue in glucagon antagonists may be exposed to the outside of the receptor. The enhanced binding affinities of analogs 1 and 3 compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide, may have resulted from the strengthened hydrophobic patch in the N-terminal region and/or the increased propensity for a helical conformation due to the replacement of alanine and leucine for glycine. Thus, as a result of the increased receptor binding affinities, antagonist activities of analogs 1-4 were increased 10-fold compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide. These potent glucagon antagonists have among the highest pA(2) values of any glucagon analogs reported to date.

A new approach to search for the bioactive conformation of glucagon: positional cyclization scanning.

In search for the bioactive conformation of glucagon, "positional cyclization scanning" was used to determine secondary structures of glucagon required for maximal interaction with the glucagon receptor. Because glucagon is flexible in nature, its bioactive conformation is not known except for an amphiphilic helical conformation at the C-terminal region. To understand the conformational requirement for the N-terminal region that appears to be essential for signal transduction, a series of glucagon analogues conformationally constrained by disulfide or lactam bridges have been designed and synthesized. The conformational restrictions via disulfide bridges between cysteine i and cysteine i + 5, or lactam bridges between lysine i and glutamic acid i + 4, were applied to induce and stabilize certain corresponding secondary structures. The results from the binding assays showed that all the cyclic analogues with disulfide bridges bound to the receptor with significantly reduced binding affinities compared to their linear counterparts. On the contrary, glucagon analogues containing lactam bridges, in particular, c[Lys(5), Glu(9)]glucagon amide (10) and c[Lys(17), Glu(21)]glucagon amide (14), demonstrated more than 7-fold increased receptor binding affinities than native glucagon. These results suggest that the bioactive conformation of glucagon may adopt a helical conformation at the N-terminal region as well as the C-terminal region, which was not evident from earlier biophysical studies of glucagon.

Biological activities of glucagon-like peptide-1 analogues in vitro and in vivo.

Studies support a role for glucagon-like peptide 1 (GLP-1) as a potential treatment for diabetes. However, since GLP-1 is rapidly degraded in the circulation by cleavage at Ala(2), its clinical application is limited. Hence, understanding the structure-activity of GLP-1 may lead to the development of more stable and potent analogues. In this study, we investigated GLP-1 analogues including those with N-, C-, and midchain modifications and a series of secretin-class chimeric peptides. Peptides were analyzed in CHO cells expressing the hGLP-1 receptor (R7 cells), and in vivo oral glucose tolerance tests (OGTTs) were performed after injection of the peptides in normal and diabetic (db/db) mice. [D-Ala(2)]GLP-1 and [Gly(2)]GLP-1 showed normal or relatively lower receptor binding and cAMP activation but exerted markedly enhanced abilities to reduce the glycemic response to an OGTT in vivo. Improved biological effectiveness of [D-Ala(2)]GLP-1 was also observed in diabetic db/db mice. Similarly, improved biological activity of acetyl- and hexenoic-His(1)-GLP-1, glucagon((1-5)-, glucagon((1-10))-, PACAP(1-5)-, VIP(1-5)-, and secretin((1-10))-GLP-1 was observed, despite normal or lower receptor binding and activation in vitro. [Ala(8/11/12/16)] substitutions also increased biological activity in vivo over wtGLP-1, while C-terminal truncation of 4-12 amino acids abolished receptor binding and biological activity. All other modified peptides examined showed normal or decreased activity in vitro and in vivo. These results indicate that specific N- and midchain modifications to GLP-1 can increase its potency in vivo. Specifically, linkage of acyl-chains to the alpha-amino group of His(1) and replacement of Ala(2) result in significantly increased biological effects of GLP-1 in vivo, likely due to decreased degradation rather than enhanced receptor interactions. Replacement of certain residues in the midchain of GLP-1 also augment biological activity.

Design and synthesis of conformationally constrained glucagon analogues.

Glucagon was systematically modified by forming lactam bridges within the central region of the molecule to give conformationally constrained cyclic analogues. Six cyclic glucagon analogues have been designed and synthesized. They are c[Asp(9),Lys(12)][Lys(17,18), Glu(21)]glucagon-NH(2) (1), c[Asp(9),Lys(12)]glucagon-NH(2) (2), c[Lys(12),Asp(15)]glucagon-NH(2) (3), c[Asp(15), Lys(18)]glucagon-NH(2) (4), [Lys(17)-c[Lys(18), Glu(21)]glucagon-NH(2) (5), and c[Lys(12),Asp(21)]glucagon-NH(2) (6). The receptor binding potencies and receptor second messenger activities were determined by radio-receptor binding assays and adenylate cyclase assays, respectively, using rat liver plasma membranes. Most interestingly, analogues 1, 2, 3, and 4 were antagonists of glucagon stimulated adenylate cyclase activity, whereas analogues 5 and 6 were partial agonists in the functional assay. All of the cyclic analogues were found to have reduced binding potencies relative to glucagon. The structural features that might be responsible for these effects were studied using circular dichroism spectroscopy and molecular modeling. These results demonstrated the significant modulations of both receptor binding affinity and transduction (adenylate cyclase activity) that can accompany regional conformational constraints even in larger polypeptide ligands. These studies suggest that the entire molecular conformation, including the flexible middle portion, is important for molecular recognition and transduction at the hepatic glucagon receptor.

GLP-1-analogues resistant to degradation by dipeptidyl-peptidase IV in vitro.

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and improves glycemic control in type 2 diabetes. In serum the peptide is degraded by dipeptidyl peptidase IV (DPP IV). The resulting short biological half-time limits the therapeutic use of GLP-1. DPP IV requires an intact alpha-amino-group of the N-terminal histidine of GLP-1 in order to perform its enzymatic activity. Therefore, the following GLP- analogues with alterations in the N-terminal position 1 were synthesized: N-methylated- (N-me-GLP-1), alpha-methylated (alpha-me-GLP-1), desamidated- (desamino-GLP-1) and imidazole-lactic-acid substituted GLP-1 (imi-GLP-1). All GLP-1 analogues except alpha-me-GLP-1 were hardly degraded by DPP IV in vitro. The GLP-1 analogues showed receptor affinity and in vitro biological activity comparable to native GLP-1 in RINm5F cells. GLP-1 receptor affinity was highest for imi-GLP-1, followed by alpha-me-GLP-1 and N-me-GLP-1. Only desamino-GLP-1 showed a 15-fold loss of receptor affinity compared to native GLP-1. All analogues stimulated intracellular cAMP production in RINm5F cells in concentrations comparable to GLP-1. N-terminal modifications might therefore be useful in the development of long-acting GLP-1 analogues for type 2 diabetes therapy.

Consideration of conformational transitions and racemization during process development of recombinant glucagon-like peptide-1.

Physicochemical characterization of dry, excipient-free recombinant glucagon-like peptide-1 (rGLP-1) indicates the conformation and purity of the bulk peptide is dependent on the purification scheme and the in-process storage and handling. The recombinant peptide preparations were highly pure and consistent with the expected primary structure and bioactivity. However, variations in solubility were observed for preparations processed by different methods. The differences in solubility were shown to be due to conformational differences induced during purification. A processing scheme was identified to produce rGLP-1 in its native, soluble form, which exhibits FT-IR spectra, consistent with glucagon-like peptide-1 synthesized by solid-state peptide synthesis. rGLP-1 was also found to undergo base-catalyzed amino acid racemization. Racemization can impact the yield and impurity profile of bulk rGLP-1, since the peptide is exposed to alkali during its purification. A combination of enzymatic digestion using leucine aminopeptidase (which cleaves N-terminal L-amino acids >> D-amino acids) and matrix-assisted laser desorption ionization mass spectrometry was used to identify racemization as a degradation pathway. The racemization rate increased with increasing temperature and base concentration, but decreased with increasing peptide concentration. The racemized peptides were shown to be less bioactive than rGLP-1.

Structure-activity studies of hydrophobic amino acid replacements at positions 9, 11 and 16 of glucagon.

We have designed and synthesized eight compounds 2-9 which incorporate neutral, hydrophobic amino acid residues in positions 9, 11 and 16 of the glucagon molecule: (2) [desHis1, Val9. Ile11,16] glucagon amide, (3) [desHis1, Val9,11,16] glucagon amide, (4) [desHis1, Val9, Leu11,16]glucagon amide, (5) [desHis1, Nle9, Ile11,16]glucagon amide, (6) [desHis1, Nle9, Val11,16] glucagon amide, (7) [desHis1,-Nle9, Leu11,16] glucagon amide, (8) [desHis1, Val9, Leu11,16, Lys17,18, Glu21] glucagon amide and (9) [desHis1, Nle9, Leu11,16, Lys17,18, Glu21] glucagon amide. The effect of neutral, hydrophobic residues at positions 9, 11 and 16 led to good binding to the glucagon receptor. Compared to glucagon (IC50 = 1.5 nM), analogues 2-9 were found to have IC50 values of 6.0, 6.0, 11.0, 9.0, 2.5, 2.8, 6.5 and 7.0 nM, respectively. When these compounds were tested for their ability to block adenylate cyclase (AC) activity, they were found to be antagonists having no stimulation of adenyl cyclase, with pA2 values of 6.15, 6.20, 6.30, 7.25, 6.10, 7.30, 6.25 and 7.25, respectively.

Roles of aspartic acid 15 and 21 in glucagon action: receptor anchor and surrogates for aspartic acid 9.

The discovery of aspartic acid at position 9 in glucagon to be a critical residue for transduction has spurred renewed efforts to identify other strategic residues in the peptide sequence that dictate either receptor binding or biological activity. It also became apparent from further studies that Asp9 operates in conjunction with His1 in the activation mechanism that follows binding to the glucagon receptor. Indeed, it was later demonstrated that the protonatable histidine imidazole is important for transduction. It is likely that the interaction of a positively charged histidine 1 with a negatively charged aspartic acid 9 might be part of the triggering step at the molecular level. Two other aspartic acid residues in glucagon are capable of assuming a similar role, namely that of contributing to an electrostatic attraction with histidine via a negative carboxylate. These studies were conducted to investigate the role of aspartic acid 15 and 21 in glucagon action. Evidence reported here, gathered from 31 replacement analogs, supports the idea that in the absence of the requisite carboxyl group at position 9, histidine utilizes Asp21 or Asp15 as a compensatory site. Asp15 was also found to be indispensable for binding and may serve to tether the hormone to the receptor protein at the binding site. It is also demonstrated that these new findings promote the design of better glucagon antagonists.

Synthetic linear and cyclic glucagon antagonists.

The synthesis and biological activities of seven new glucagon analogues are reported. The design of compounds 2-5 is based on potent antagonists recently reported from this laboratory, where we have focused on modifications in the N-terminal region. In this report we have concentrated specifically on modifications to histidine-1. In addition we have prepared two cyclic compounds 7 and 8, related to a linear in vivo antagonist [Glu9]glucagon, reported by Merrifield (Unson et al. (1987) Proc. Natl. Acad. Sci. USA 84, 4083-4087). The N-terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue (S)-5,6,7,8-tetrahydro-5-oxoimidazo(1,5-c)pyrimidine-7-carboxylic acid (Toc), desaminohistidine (dHis) and 3-(4-nitrobenzyl)histidine. The structures of the new compounds are as follows. [Toc1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (2); [Toc1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon amide (3); [3-(4-nitrobenzyl)His1,D-Phe4,Tyr5,Arg12,Lys17,18,G lu21]glucagon (4); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (5); [dHis1,Glu9]glucagon (6); (desHis1)[Glu9,Lys12]glucagon amide (7); (desHis1)-[Glu9,Lys12,Asp15]glucagon amide (8). The binding potencies of the linear analogues, as expressed a percentage of glucagon binding, are 2.6 (2), 0.13 (3), 0.8 (4), 0.8 (5), 2.2 (6). Both cyclic analogues 7 and 8 show biphasic binding curves. The IC50 values for 7 at the high and low affinity sites are 1.5 and 167 nM, respectively (IC50 of glucagon = 1.3 nM). The IC50 values for 8 at the high and low affinity sites are 4.7 and 3451 nM, respectively. The cyclic analogues are characterized by fast atom bombardment mass spectrometry of endoproteinase ASP-N digests. The specificity of the enzyme used in these studies enables differentiation of isomers of the cyclic glucagon analogues which differ only in the position of cyclic amide bond. Analogues 2, 3 and 5-8 are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase (AC) system. Analogue 4 is a partial agonist (5.7% compared to glucagon) of AC. Introduction of unusual amino acids which do not contain a primary alpha-amino group such as Toc at the N-terminus is expected to increase in vivo metabolic stability by protecting against degradation by aminopeptidases.

Biological activities of des-His1[Glu9]glucagon amide, a glucagon antagonist.

Hyperglycemia in diabetes mellitus is generally associated with elevated levels of glucagon in the blood. A glucagon analog, des-His1[Glu9]glucagon amide, has been designed and synthesized and found to be an antagonist of glucagon in several systems. It has been a useful tool for investigating the mechanisms of glucagon action and for providing evidence that glucagon is a contributing factor in the pathogenesis of diabetes. The in vitro and in vivo activities of the antagonist are reported here. The analog bound 40% as well as glucagon to liver membranes, but did not stimulate the release of cyclic AMP even at 10(6) higher concentration. However, it did activate a second pathway, with the release of inositol phosphates. In addition, the analog enhanced the glucose-stimulated release of insulin from pancreatic islet cells. Of particular importance were the findings that the antagonist also showed only very low activity (less than 0.2%) in the in vivo glycogenolysis assay, and that at a ratio of 100:1 the analog almost completely blocked the hyperglycemic effects of added glucagon in normal rabbits. In addition, it reduced the hyperglycemia produced by endogenous glucagon in streptozotocin diabetic rats. Thus, we have an analog that possesses properties that are necessary for a glucagon antagonist to be potentially useful in the study and treatment of diabetes.