Molecular engineering of safe and efficacious oral basal insulin

Abstract

Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing.

Fig. 1. Influence of half-life on peak to trough in steady state in dogs with once daily dosing.

The simulations were based on data for OI338 tested in dogs and the PK profiles were normalized to the area-under-the-curve (AUC) for a 24-h period. Absorption and volume of distribution was kept constant and clearance was changed dependent of the elimination half-life.

Fig. 2. Structure and main biological properties of insulin analogues.

a Structures of the insulin analogues; note that R in HI, OI106, OI189 is hydrogen (i.e., chemically unmodified insulin) and that R in OI216, OI338 and OI320 is the chemical modification of B29 (see full drawing of chemical modification); Note that R is identical for OI216, OI338 and OI320 as illustrated in 2a) (all analogues are desB30). Insulin residues that are modified are A14 (Native Tyr, modified Glu), B25 (Native Phe), modified His), B27 Thr, modified by deletion (des), B30 Thr), modified by deletion (des). b Stability of the insulin analogues towards proteolytic degradation in rat duodenum extract. The degradation half-life relative to human insulin is shown (mean +/− SD from a number of independent experiments as indicated. c Receptor binding affinities of the insulin analogues relative to human insulin (HI = 100%) in the presence of 1.5% human serum albumin (HSA). Data are presented as mean values +/− SD from a number of independent experiments as indicated. d Potency of oral insulin analogues relative to human insulin on whole-body glucose disposal during different constant molar intravenous (IV) infusion rates to steady state (see the “Methods” section) under euglycemic clamp in healthy and conscious rats (circles), dogs (triangles) and pigs (squares), (confidence intervals is indicated and summaried in Table 1).

Fig. 3. Pharmacokinetic properties of oral insulin analogues.

a Pharmacokinetic profiles of OI106 (triangles), OI216 (squares) and OI338 (circles) after single oral dosing in healthy and conscious dogs (n = 8); the plotted concentrations are exposure divided by the oral dose in nmol/kg for each insulin analogue. Coefficient of variation, CV, for the AUC, was 75–100%. b Pharmacokinetic profiles of in OI338 after IV (2 nmol/kg, n = 8); peroral (105 nmol/kg, n = 8) and SC (4 nmol/kg, n = 4) administrations in dogs. CV for AUC was around 10% after IV and SC administration and ~100% after oral dosing. Note the fast absorption profile after oral administration (closed circles). After reaching C_max the oral pharmacokinetic profile almost mimics that of an IV administration (see also Table 1). c Bioavailability of different insulin analogues after oral administration in GIPET®I tablets to dogs (mean +/− SD from a number of independent experiments. d Individual 12-h pharmacokinetic profiles (day 1, 7, 14) of OI338 after multiple oral daily dosing in eight dogs (each dog one colour). e Coefficient of variation of bioavailability based on area under the curves in Fig. 3d), multiple dose study. Intra/inter-individual CV based on the mean of the group was reduced from 100 to 40% after treatment to steady state.

Fig. 4. Hyperinsulinemic, euglycemic clamp experiment.

A representative dose response plot (GIR = glucose infusion rate) from a 5-h hyperinsulinemic, euglycemic clamp experiment in healthy and conscious rats performed to determine the potency of OI320 (black circles) relative to human insulin (white circles). The dots represent the average GIR during the last 60 min of the experiment in each rat (n = 6 rats per dose (group); independent experiments). Similar dose response studies were performed in healthy and conscious pigs and dogs as indicated in (Fig. 2d) and; see Table 1 for 95% confidence interval.

Fig. 5. Schematic overview of insulin levels, distribution kinetics and the kinetic/dynamic relationship.

a–c Schematic overview of insulin levels (arrow width) and distribution kinetics upon a oral dosing of fast acting insulin (e.g. human insulin), b oral dosing of a plasma protracted insulin (e.g. OI338 and OI320), and c SC dosing of a long acting insulin, which is protracted SC (e.g. insulin glargine or insulin degludec, or OI338/OI320 dosed SC). d The pharmacodynamic time course (GIR = glucose infusion rate required to maintain euglycemia) of orally dosed OI320 and pharmacokinetic exposure during hyperinsulinemic euglycemic clamp in healthy and conscious dogs. Note the long lag time of pharmacodynamic effect in respect to exposure. The CV for the GIR AUC was in the range 45–80% and for the pharmacokinetic AUC the CV was 45–100%.

Fig. 6. Assessment of hypoglycaemia after overdosing in dogs.

Plasma glucose lowering, and dynamics of glucose turnover obtained from an over-dose study with OI320 performed in healthy and conscious mongrel dogs on day four after once daily IV dosing with a maintenance dose of OI320 (40 pmol/kg/min for 45 min) for three days. The three days of pre-treatment was performed to mimic a steady-state situation. a The hypoglycaemic response in dogs to 45 min intraportal infusion of IO320 in three different doses (40 (black squares), 80 (black triangles) or 120 (black circles) pmol/kg/min) (n = 5 for each doses; independent experiments; mean +/− S.E.M.) or human insulin (white circles) at 24 pmol/kg/min (n = 3; independent experiments; mean +/− S.E.M.; independent experiments). The dose of human insulin was set to 60% of the maintenance dose of OI320 (40 pmol/kg/min) in order not to induce too severe hypoglycaemia. b The dynamics of glucose appearance (Ra, hepatic glucose production) and d glucose disappearance (Rd, glucose uptake mainly by muscle and fat). c Changes in glucose clearance. The vertical lines in Fig. 6a–d indicate the portal insulin infusion period from 0–45 min.