Soluble, prolonged-acting insulin derivatives. I. Degree of protraction and crystallizability of insulins substituted in the termini of the B-chain

Abstract

Hydrophilic insulins, more positively charged than human insulin at neutral pH, have been prepared by substitution with basic amino acids at the termini of the B-chain and by blocking the C-terminal carboxyl group of the B-chain. The isoelectric pH of the insulin is thereby moved from 5.4 towards physiological levels. Slightly acid solutions of derivatives, in which charge has been added in the C-terminus of the B-chain, have a prolonged action in vivo, in particular if the carboxyl group is blocked. It is found that the prolonged-acting hydrophilic insulins crystallize instantly when the pH is adjusted to 7. The prolonged action is ascribed to this readiness to crystallization combined with a low solubility, which may be further decreased by increased concentration of zinc ions. Hydrophobic insulins have a prolonged action independent of the site of substitution even if the derivative is soluble at physiological pH. Some derivatives were prepared from porcine insulin by tryptic transpeptidation. N-terminal B-chain substituted insulins were prepared by alkylation of a biosynthetic single-chain insulin precursor, followed by tryptic transpeptidation rendering the double chain insulin derivative. The observed blood glucose lowering in the rabbits implies that neither N- nor C-terminal B-chain substitution results in substantial deterioration of biological potency. An index for the degree of protraction based on the blood glucose data is used to compare the insulins.

Binding of phenol to R6 insulin hexamers*

“…This is even true for pharmaceutical preparations consisting of acidic solutions of the hormone, because it is believed the material will precipitate predominantly in the form of crystals after neutralization in the subcutaneous depot. 37 Crystal stability is mainly governed by interactions between neighboring hexamers, but solvent and phenol molecules may also be important. Our crystal structure of A21Gly-B31,B32Arg 2 insulin (HOE 901) 10 revealed the presence of a seventh phenol molecule, in addition to the six bound at the dimer/ dimer interfaces in the vicinity of residues A6, A11, and B5.…”

“…[37][38][39] Our own efforts (Seipke et al, unpublished) resulted in the development of A21Gly-B31,B32Arg 2 insulin, which in healthy volunteers exhibits an activity profile extending at least 24 h from the time of injection. 40 This insulin, coded HOE 901 and being developed by Hoechst Marion Roussel, is in phase III clinical trials at this time.…”

Insulin analogues and their potential in the management of diabetes mellitus

“…The first line of research has been to change the isoelectric point (i. e. the pH value at which insulin is least soluble and precipitates) towards neutrality by substituting and adding single amino acids to the molecule of human insulin by means of the rDNA technology. The resultant insulin preparation is soluble in acid solution but precipitates as micro-crystals after injection in the subcutaneous tissue where the pH is neutral [126]. The second principle for protracting a soluble insulin is a modification which promotes the binding to a serum carrier with prolonged half-life, such as albumin [127].…”

Glucose-lowering Effect of Insulin Degludec is Independent of Subcutaneous Injection Region

“…In contrast to other basal insulin preparations, which form crystals or precipitate upon SC injection [12][13][14][15], IDeg stays in solution and forms stable multi-hexamers in the SC tissue from which IDeg monomers gradually separate, producing a slow absorption into the circulation with low day-to-day variability [11,16,17]. IDeg has a considerably longer half-life after SC administration than insulin glargine (25 vs. 12 h) [18] and exhibits a duration of action longer than 42 h [11,16].…”