Synthetic insulin
In the vial, insulin glargine is mixed with a preservative, metacresol, to lengthen the shelf-life. Glycerol is added, possibly to increase surface tension to aid injection. Varying quantities of hydrochloric acid and sodium hydroxide are included to balance to pH 4, as well as water for injection (a pharmaceutical term for highly purified, deionized water). The addition of zinc chloride forms Zn 2+ ions, about which insulin hexamers form (11). These hexamers, after injection, precipitate in the neutral subcutaneous space. This results in a more protracted action as it takes a longer time for insulin monomers to dissociate from the depot and be absorbed into the bloodstream. In comparison to NPH insulin, this causes a longer and flatter time-action profile of less variation. This increases the predictability of the insulin, resulting in amore practical basal insulin for background use. In clinical studies where subjects with type 1 and type 2 diabetes received either once daily doses of insulin glargine or once- or twice- daily doses of NPH, those given glargine were shown to have significantly reduced risks of nocturnal hypoglycaemia (reviewed in reference 11). The manufacture of insulin glargine is achieved through genetic modification of Escherichia coli bacteria. Plasmids are cut open with specific restriction enzymes at specific places in the DNA sequence. The DNA sequence for the insulin glargine molecule is then ‘ sewn in ’ using specific ligase enzymes. The plasmids act as vectors and are reinserted into the E. coli . The transgenic bacteria are then grown in large fermentation tanks which have various environmental controls to ensure both that the bacteria grow very rapidly, and so produce large amounts of insulin glargine, but also that no intruder organism may grow as well (14). After a set period of time, the mixture is ‘ harvested ’ . The insulin glargine is removed by a filtration and purification process and is subsequently combined with the other chemicals that make up the insulin in a vial for injection. The next basal insulin analogue to be produced using recombinant DNA technology was insulin detemir. Despite having a similar action to insulin glargine, detemir has a significantly different structure. The amino acid at position B30, threonine, is omitted, and a 14-carbon myristoyl fatty acid is attached to lysine at position B29 (11). As with insulin glargine, insulin detemir is dissolved in a solution of glycerol, phenol, and metacresol, which all act as preservatives. The source of Zn 2+ ions for insulin hexamers to form around comes not from zinc chloride but from zinc acetate, which, as a weak acid, buffers changes in pH so avoids drastic changes in solubility of the insulin upon injection. The remainder of the solution is made up of sodium chloride, disodium phosphate dihydrate, hydrochloric acid, sodium hydroxide and water for injections (15). The ratios of these are particularly important in a solution of insulin detemir as it is essential that the insulin remains at a neutral pH of 7 from in the vial to injection. At the subcutaneous site of injection, the myristoyl fatty acid tail allows the molecules to bond as dihexamers, each hexamer about a single Zn 2+ ion. Subsequently, whilst still in the subcutaneous tissue, the myristoyl groups bind to albumin, pulling the dihexamers apart so as to be absorbed as a protein-insulin complex into the blood. This reaction between the dihexamers and albumin can only occur in neutral pH and is key to the more stable time-action profile of the insulin (11). Further reversible binding to albumin in circulating blood acts to buffer changes in absorption rate caused by differences in local blood flow at the injection site, decreasing within-patient pharmacodynamic variability in comparison to insulin glargine (16). The large-scale pharmaceutical manufacture of insulin detemir is similar to that of insulin glargine, except that its DNA sequence is expressed in a strain of yeast called Saccharomyces cerevisiae , and acylation is performed after initial purification of the unmodified insulin molecule from the fermentation broth (15).
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