815-P: Enhanced Disulphide Bond Stability Contributes to the Once Weekly PD Profile of Insulin Icodec

Abstract

Insulin icodec is a novel basal insulin analogue with a long half-life (196 hours) that is in clinical development for the treatment of diabetes with once weekly dosing. Insulin icodec was developed based on the hypothesis that lowering the insulin receptor affinity in the presence of a strong HSA-binding moiety would result in a long half-life, provided that non-receptor mediated clearance is diminished. The observation that insulin icodec retains its full potency over weeks has been attributed to low non-insulin receptor mediated clearance as well as to limited degradation in circulation, due to the amino acid modifications in insulin icodec that confer proteolytically-stabilization. Here, we report a previously undescribed insulin clearance mechanism, resulting in splitting insulin molecules into A- and B-chains by a thiol-disulfide exchange reaction. We found that the amino acid substitution of Tyr in A14 to Glu provided a major stabilization of insulin against such degradation while only minor improvements were introduced by the Tyr in B16 to His and Phe in B25 to His substitutions. These results correlate with improvements in the folding stability and likely reflect reduced exposure of insulin icodec's disulfide bonds to the thiol-disulfide exchange reaction. Even though insulin icodec's amino acid substitutions significantly limit its susceptibility to this new clearance mechanism, some free B-chain has been observed in plasma samples from minipigs and people with type 2 diabetes. In summary, we have identified thiol-disulfide exchange reactions as an additional insulin clearance mechanism and minimizing insulin icodec's susceptibility to this process has significantly contributed to its long PD, making it suitable for once weekly dosing. Disclosure F.Hubalek: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. C.Cramer: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. H.Helleberg: Employee; Novo Nordisk A/S. E.Johansson: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. G.Schluckebier: Employee; Novo Nordisk. D.B.Steensgaard: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk. J.Sturis: Employee; Novo Nordisk A/S, Other Relationship; Novozymes, Stock/Shareholder; Novo Nordisk A/S. T.Kjeldsen: Employee; Novo Nordisk, Stock/Shareholder; Novo Nordisk.