PurposeBasal insulins with improved kinetic properties can potentially be produced using acylation by fatty acids that enable soluble, high-molecular weight complexes to form post-injection. A series of insulins, acylated at B29 with fatty acids via glutamic acid spacers, were examined to deduce the structural requirements.MethodsSelf-association, molecular masses and hexameric conformations of the insulins were studied using size exclusion chromatography monitored by UV or multi-angle light scattering and dynamic light scattering, and circular dichroism spectroscopy (CDS) in environments (changing phenol and zinc concentration) simulating a pharmaceutical formulation and changes following subcutaneous injection.ResultsWith depletion of phenol, insulin degludec and another fatty diacid–insulin analogue formed high molecular mass filament-like complexes, which disintegrated with depletion of zinc. CDS showed these analogues adopting stable T3R3 conformation in presence of phenol and zinc, changing to T6 with depletion of phenol. These findings suggest insulin degludec is dihexameric in pharmaceutical formulation becoming multihexameric after injection. The analogues showed weak dimeric association, indicating rapid release of monomers following hexamer disassembly.ConclusionsInsulins can be engineered that remain soluble but become highly self-associated after injection, slowly releasing monomers; this is critically dependent on the acylation moiety. One such analogue, insulin degludec, has therapeutic potential.

Oral administration of therapeutic peptides is hindered by poor absorption across the gastrointestinal barrier and extensive degradation by proteolytic enzymes. Here, we investigated the absorption of orally delivered semaglutide, a glucagon-like peptide-1 analog, coformulated with the absorption enhancer sodium N-[8-(2-hydroxybenzoyl) aminocaprylate] (SNAC) in a tablet. In contrast to intestinal absorption usually seen with small molecules, clinical and preclinical dog studies revealed that absorption of semaglutide takes place in the stomach, is confined to an area in close proximity to the tablet surface, and requires coformulation with SNAC. SNAC protects against enzymatic degradation via local buffering actions and only transiently enhances absorption. The mechanism of absorption is shown to be compound specific, transcellular, and without any evidence of effect on tight junctions. These data have implications for understanding how highly efficacious and specific therapeutic peptides could be transformed from injectable to tablet-based oral therapies.

We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å . DILP5 shares the basic fold of the insulin peptide family (T conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel ␤-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel ␤-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.The ligands and receptors of the insulin peptide family constitute an ancient metazoan signaling system that plays a crucial pleiotropic role in cell growth, metabolism, reproduction, and longevity (1-7).The mammalian insulin receptor belongs to the family of receptor-tyrosine kinases and is composed of two ␣ subunits and two ␤ subunits linked together by disulfide bonds (for review, see Refs. 4 and 8 -10). The existence of a homologue of the mammalian insulin receptor in Drosophila melanogaster (DIR) 2 was suggested in 1985 by Petruzzelli et al. (11), who identified a glycoprotein of 350 -400 kDa that binds bovine insulin specifically with moderate affinity (15 nM). The cDNA sequence of the DIR is remarkably similar to that of the mammalian insulin and IGF-I receptors (with 33% sequence identity) except for substantial N-and C-terminal extensions (12, 13).In evolution, there is a single receptor from Cnidarians up to and including Amphioxus (Branchiostoma californiense), the phylum closest to vertebrates (for review see Refs. 1, 4 -6). In vertebrates, gene duplications resulted in three related receptors; that is, the insulin receptor, the type I IGF receptor, and the orphan insulin receptor-related receptor (1, 5).In humans, members of the insulin peptide family include insulin, the insulin-like growth factors I and II, and seven relaxin-related peptides (for review, see Ref. 14). The same basic fold is shared for all molecules in the superfamily whose structure is known; the B domain contains a single ␣-helix that lies across the two ␣-helices of the A domain (15) and two canonical disulfide bridges that connect the A-and Bchains, whereas an intrachain disulfide bridge is present in the A-chain.The D. melanogaster genome contains seven insulin-like genes that are expressed in a highly tissue-and stage-specific patterns, dilp1-7 (16). dilp2 is the most related to human insulin with 35% sequence identity, whereas dilp5 has 27.8% identity (16).So far, the structures of only two invertebrate insulin-like peptides have been determined by N...

PurposeTo study the self-association states of insulin degludec and insulin aspart alone and combined in pharmaceutical formulation and under conditions simulating the subcutaneous depot.MethodsFormulations were made of 0.6 mM degludec at 3 and 5 Zn/6 insulin monomers, and 0.6 mM aspart (2 Zn/6 insulin monomers). Self-association was assessed using size-exclusion chromatography (SEC) monitored by UV and orthogonal reverse-phase chromatography.ResultsSimulating pharmaceutical formulation, degludec eluted as dihexamers, whereas aspart eluted as hexamers and monomers. Combining degludec at low zinc with aspart increased dihexamer content, indicating hybrid hexamer formation. At high zinc concentration, however, there was no evidence of this. Simulating the subcutaneous depot by removing preservative, degludec eluted as multihexamers and aspart as monomers. Aspart was incorporated into the multihexamer structures when combined with degludec at low zinc, but there was no such interaction with high-zinc degludec. SEC using progressively diluted concentrations of phenol and m-cresol showed that dissociation of aspart into monomers occurs before the formation of degludec multihexamers.ConclusionInsulins degludec and aspart can be combined without forming hybrid hexamers, but this combinability is dependent on zinc and preservative concentration, and requires that degludec is fully dihexameric before addition of aspart.

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