Recombinant DNA technology facilitates not only production of human insulin in microbial systems, but also facilitates generation of insulins of modified amino acid sequences. The major aims of generating such engineered insulin analogues include:
• Identification of insulins with altered pharmacokinetic properties, such as faster-acting or slower-acting insulins.
• Identification of super-potent insulin forms (insulins with higher receptor affinities). This is due to commercial considerations, namely the economic benefits that would accrue from utilizing smaller quantities of insulin per therapeutic dose.
The insulin amino acid residues that interact with the insulin receptor have been identified (A1, A5, A19, A21, B10, B16, B23-25), and a number of analogues containing amino acid substitutions at several of these points have been manufactured. Conversion of histidine to glutamate at the B10 position, for example, yields an analogue displaying fivefold higher activity in vitro. Other substitutions have generated analogues with even higher specific activities. However, increased in vitro activity does not always translate to increased in vivo activity.
Attempts to generate faster-acting insulins have centred upon developing analogues that do not dimerize or form higher polymers at therapeutic dose concentrations. The contact points between individual insulin molecules in insulin dimers/oligomers include amino acids at positions B8, B9, B12-13, B16 and B23-28. Thus, analogues with various substitutions at these positions have been generated. The approach adopted generally entails insertion of charged or bulky amino acids, in order to promote charge repulsion or steric hindrance between individual insulin monomers. Several are absorbed from the site of injection into the bloodstream far more quickly than native soluble (fast-acting) insulin. Such modified insulins could thus be injected at mealtimes rather than 1 h before, and several such fast-acting engineered insulins have now been approved for medical use (Table 11.3). 'Insulin lispro' (tradename 'Humalog') was the first such engineered short-acting insulin to come to market (Box 11.1 and Figure 11.5).
'Insulin Aspart' is a second fast-acting engineered human insulin analogue now approved for general medical use. It differs from native human insulin in that the prolineB28 residue has been replaced by aspartic acid. This single amino acid substitution also decreases the propensity of individual molecules to self-associate, ensuring that they begin to enter the bloodstream from the site of injection immediately upon administration.
A number of studies have also focused upon the generation of longer-acting insulin analogues. The currently used Zn-insulin suspensions, or protamine-Zn-insulin suspensions, generally display a plasma half-life of 20-25 h. Selected amino acid substitutions have generated insulins which, even in soluble form, exhibit plasma half-lives of up to 35 h.
Optisulin or Lantus are the tradenames given to one such analogue that gained general marketing approval in 2000 (Table 11.3). The international non-proprietary name for this engineered molecule is 'insulin glargine'. It differs from native human insulin in that the C-terminal aspargine residue of the A-chain has been replaced by a glycine residue and the P-chain has been elongated (again from its C-terminus) by two arginine residues. The overall effect is to increase the molecule's pI (the pH at which the molecule displays a net overall zero charge and, consequently, at
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