Jan Markussen

The mechanism of protraction of insulin detemir, a long-acting, acylated analog of human insulin.

AbstractPurpose. Insulin detemir has been found in clinical trials to be absorbed with very low variability. A series of experiments were performed to elucidate the underlying mechanisms. Methods. The disappearance from an injected subcutaneous depot and elimination studies in plasma were carried out in pigs. Size-exclusion chromatography was used to assess the self-association and albumin binding states of insulin detemir and analogs. Results. Disappearance T50% from the injection depot was 10.2 ± 1.2 h for insulin detemir and 2.0 ± 0.1 h for a monomeric acylated insulin analog. Self-association of acylated insulin analogs with same albumin affinity in saline correlated with disappearance rate and addition of albumin to saline showed a combination of insulin detemir self association and albumin binding. Intravenous kinetic studies showed that the clearance and volume of distribution decreased with increasing albumin binding affinity of different acylated insulin analogs. Conclusions. The protracted action of detemir is primarily achieved through slow absorption into blood. Dihexamerization and albumin binding of hexameric and dimeric detemir prolongs residence time at the injection depot. Some further retention of detemir occurs in the circulation where albumin binding causes buffering of insulin concentration. Insulin detemir provides a novel principle of protraction, enabling increased predictability of basal insulin.

Soluble, fatty acid acylated insulins bind to albumin and show protracted action in pigs.

SummaryWe have synthesized insulins acylated by fatty acids in the ε-amino group of LysB29. Soluble preparations can be made in the usual concentration of 600 nmol/ml (100 IU/ml) at neutral pH. The time for 50% disappearance after subcutaneous injection of the corresponding TyrA14(125I)-labelled insulins in pigs correlated with the affinity for binding to albumin (r=0.97), suggesting that the mechanism of prolonged disappearance is binding to albumin in subcutis. Most protracted was LysB29-tetradecanoyl des-(B30) insulin. The time for 50% disappearance was 14.3±2.2 h, significantly longer than that of Neutral Protamine Hagedorn (NPH) insulin, 10.5±4.3 h (p<0.001), and with less inter-pig variation (p<0.001). Intravenous bolus injections of LysB29-tetradecanoyl des-(B30) human insulin showed a protracted blood glucose lowering effect compared to that of human insulin. The relative affinity of LysB29-tetradecanoyl des-(B30) insulin to the insulin receptor is 46%. In a 24-h glucose clamp study in pigs the total glucose consumptions for LysB29-tetradecanoyl des-(B30) insulin and NPH were not significantly different (p=0.88), whereas the times when 50% of the total glucose had been infused were significantly different, 7.9±1.0 h and 6.2±1.3 h, respectively (p<0.04). The glucose disposal curve caused by LysB29-tetradecanoyl des-(B30) insulin was more steady than that caused by NPH, without the pronounced peak at 3 h. Unlike the crystalline insulins, the soluble LysB29-tetradecanoyl des-(B30) insulin does not elicit invasion of macrophages at the site of injection. Thus, LysB29-tetradecanoyl des-(B30) insulin might be suitable for providing basal insulin in the treatment of diabetes mellitus.

Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks

Insulin is thought to elicit its effects by crosslinking the two extracellular α-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.

Mechanism of protracted metabolic effects of fatty acid acylated insulin, NN304, in dogs: retention of NN304 by albumin

Aims/hypothesis. The provision of stable, reproducible basal insulin is crucial to diabetes management. This study in dogs examined the metabolic effects and interstitial fluid (ISF) profiles of fatty acid acylated insulin, LysB29-tetradecanoyl, des-(B30) human insulin (NN304). Methods. Euglycaemic clamps were carried out under inhalant anaesthesia during equimolar intravenous infusions (3.6 pmol · min–1· kg–1 for 480 min) of human insulin or NN304 (n = 8 per group). Results. Steady-state total NN304 (albumin-bound and unbound) was considerably higher in plasma compared with human insulin (1895 ± 127 vs 181 ± 10 pmol/l, p < 0.001) and increased in interstitial fluid (163 ± 14 vs 106 ± 9 pmol/l, p < 0.01). The halftime for appearance of NN304 in interstitial fluid was slower than human insulin (92 vs 29 min, p < 0.001). Yet, equivalency of action was shown for glucose turnover; steady-state glucose uptake (Rd) of 7.28 ± 0.55 and 6.76 ± 0.24 mg · min–1· kg–1 and endogenous glucose production of 0.11 ± 0.12 and 0.22 ± 0.03 mg · min–1· kg–1 (p > 0.40; NN304 and human insulin, respectively). Similar to interstitial fluid, half times for Rd and endogenous glucose production were delayed during NN304 infusion (162 vs 46 min and 80 vs 31 min, respectively; p < 0.01 vs human insulin). Conclusion/interpretation. Firstly equivalency of steady-state action is found at equimolar physiologic infusions of human insulin and NN304. Secondly NN304 binding to plasma albumin results in slower NN304 appearance in the interstitial compartment compared with human insulin. Thirdly the delay in appearance of NN304 in interstitial fluid may not in itself be a source of the protracted action of this insulin analogue. The protracted effect is due primarily to albumin binding of the insulin analogue NN304. [Diabetologia (1999) 42: 1254–1263]

Fatty acid acylated insulins display protracted action due to binding to serum albumin

The insulin example demonstrates that it is possible to alter pharmacokinetics and dynamics. By careful choice of ligand and site of substitution it is possible to provide the molecule with the desired characteristics without loosing potency. In this example the following characteristic of insulin has been obtained: 1. A prolonged acting insulin analogue which can be formulated as a neutral soluble preparation 2. Insulin has been provided with albumin binding properties 3. Improved duration of action compared to NPH insulin 4. Improved dynamic profile without sharp peeks of activity, i.e. smoother action