Andrew Mark Vick

Engineering and characterization of the long‐acting glucagon‐like peptide‐1 analogue LY2189265, an Fc fusion protein

Glucagon‐like peptide‐1 (GLP‐1) receptor agonists are novel agents for type 2 diabetes treatment, offering glucose‐dependent insulinotropic effects, reduced glucagonemia and a neutral bodyweight or weight‐reducing profile. However, a short half‐life (minutes), secondary to rapid inactivation by dipeptidyl peptidase‐IV (DPP‐IV) and excretion, limits the therapeutic potential of the native GLP‐1 hormone. Recently, the GLP‐1 receptor agonist exenatide injected subcutaneously twice daily established a novel therapy class. Developing long‐acting and efficacious GLP‐1 analogues represents a pivotal research goal. We developed a GLP‐1 immunoglobulin G (IgG4) Fc fusion protein (LY2189265) with extended pharmacokinetics and activity.

pI-shifted insulin analogs with extended in vivo time action and favorable receptor selectivity

A long-acting (basal) insulin capable of delivering flat, sustained, reproducible glycemic control with once daily administration represents an improvement in the treatment paradigm for both type 1 and type 2 diabetes. Optimization of insulin pharmacodynamics is achievable through structural modification, but often at the expense of alterations in receptor affinity and selectivity. A series of isoelectric point (pI)-shifted insulin analogs based on the human insulin sequence or the GlyA21 acid stable variant were prepared by semi-synthetic methods. The pI shift was achieved through systematic addition of one or more arginine (Arg) or lysine (Lys) residues at the N terminus of the A chain, the N terminus of the B chain, the C terminus of the B chain, or through a combination of additions at two of the three sites. The analogs were evaluated for their affinity for the insulin and IGF-1 receptors, and aqueous solubility under physiological pH conditions. Notably, the presence of positively charged amino acid residues at the N terminus of the A chain was consistently associated with an enhanced insulin to IGF-1 receptor selectivity profile. Increased IGF-1 receptor affinity that results from Arg addition to the C terminus of the B chain was attenuated by cationic extension at the N terminus of the A chain. Analogs 10, 17, and 18 displayed in vitro receptor selectivity similar to that of native insulin and solubility at physiological pH that suggested the potential for extended time action. Accordingly, the in vivo pharmacokinetic and pharmacodynamic profiles of these analogs were established in a somatostatin-induced diabetic dog model. Analog 18 (A0:Arg, A21:Gly, B31:Arg, B32:Arg human insulin) exhibited a pharmacological profile comparable to that of analog 15 (insulin glargine) but with a 4.5-fold more favorable insulin:IGF-1 receptor selectivity. These results demonstrate that the selective combination of positive charge to the N terminus of the A chain and the C terminus of the B chain generates an insulin with sustained pharmacology and a near-native receptor selectivity profile.

In Vivo and In Vitro Characterization of Basal Insulin Peglispro: A Novel Insulin Analog

The aim of this research was to characterize the in vivo and in vitro properties of basal insulin peglispro (BIL), a new basal insulin, wherein insulin lispro was derivatized through the covalent and site-specific attachment of a 20-kDa polyethylene-glycol (PEG; specifically, methoxy-terminated) moiety to lysine B28. Addition of the PEG moiety increased the hydrodynamic size of the insulin lispro molecule. Studies show there is a prolonged duration of action and a reduction in clearance. Given the different physical properties of BIL, it was also important to assess the metabolic and mitogenic activity of the molecule. Streptozotocin (STZ)-treated diabetic rats were used to study the pharmacokinetic and pharmacodynamic characteristics of BIL. Binding affinity and functional characterization of BIL were compared with those of several therapeutic insulins, insulin AspB10, and insulin-like growth factor 1 (IGF-1). BIL exhibited a markedly longer time to maximum concentration after subcutaneous injection, a greater area under the concentration-time curve, and a longer duration of action in the STZ-treated diabetic rat than insulin lispro. BIL exhibited reduced binding affinity and functional potency as compared with insulin lispro and demonstrated greater selectivity for the human insulin receptor (hIR) as compared with the human insulin-like growth factor 1 receptor. Furthermore, BIL showed a more rapid rate of dephosphorylation following maximal hIR stimulation, and reduced mitogenic potential in an IGF-1 receptor–dominant cellular model. PEGylation of insulin lispro with a 20-kDa PEG moiety at lysine B28 alters the absorption, clearance, distribution, and activity profile receptor, but does not alter its selectivity and full agonist receptor properties.