Therapeutic hormones - Pharmaceutical Biotechnology: Concepts and Aapplications - page 304

Box 11.1
Product case study: Humalog
Humalog (tradename, also known as insulin lispro) was the fi rst recombinant fast-acting insu-
lin analogue to gain marketing approval (in 1996). It is indicated for the treatment of diabetes
mellitus, for the control of hyperglycaemia and is used in conjunction with long-acting insu-
lins (see main text). It is administered subcutaneously. The product displays an amino acid
sequence identical to native human insulin with one alteration, i.e. an inversion of the natural
proline-lysine sequence found at positions 28 and 29 of the insulin B-chain. This simple alter-
ation signifi cantly decreased the propensity of individual insulin molecules to self-associate
when stored at therapeutic dose concentrations. The dimerization constant for insulin lispro is
300 times lower than that exhibited by unmodifi ed human insulin. Structurally, this appears
to occur because the change in sequence disrupts the formation of interchain hydrophobic
interactions critical to self-association.
The rationale underlining the sequence alteration was rooted in studies not of insulin, but
of IGF-1 (Chapter 10). The latter displays a strong structural resemblance to proinsulin, with
up to 50 per cent of amino acid residues within the IGF-1 A- and B-domains being identical to
those found in comparative positions in the insulin A- and B-chains. When compared with in-
sulin, IGF-1 molecules display a signifi cantly decreased propensity to self-associate. Sequenc-
ing studies earlier revealed that the proline B28 -lysine B29 sequence characteristic of insulin is
reversed in IGF-1. It was suggested that if this sequence difference was responsible for the
differences in self-association propensity, then inversion of the proline B28 -lysine B29 sequence
in insulin would result in its decreased self-association. Direct experimentation proved this
hypothesis accurate.
Insulin lispro is manufactured commercially in a manner quite similar to the 'proinsulin'
route used to manufacture native recombinant human insulin. A synthetic gene coding for
Lys B28 -Pro B29 proinsulin is expressed in E. coli. Following fermentation and recovery, the pro-
insulin is treated with trypsin and carboxypeptidase B, resulting in the proteolytic excision of
the engineered insulin molecule. It is then purifi ed to homogeneity by a number of high-resolu-
tion chromatographic steps. The fi nal product formulation also contains m -cresol (preservative
and stabilizer), zinc oxide (stabilizer), glycerol (tonicity modifi er) and a phosphate-based buf-
fer and is presented in vial, cartridge and injector pen formats.
Humalog has proven equipotent to regular human insulin, but its effect is more rapid and of
shorter duration. After s.c. administration it displays similar bioavailability to regular insulin
(typically 55-77 per cent). Peak serum levels are usually recorded 30-90 min after administra-
tion, as opposed to 50-120 min in the case of regular insulin. Its serum t 1/2 is also shorter than
that of regular insulin (60 min as opposed to 90 min). Product safety and effi cacy have been
established in several trials, an earlier major trial being an open label, crossover study of 1008
patients with type-1 diabetes and 722 patients with type-2 diabetes. Predictably, the major po-
tential negative adverse effect is hypoglycaemia. The product was developed and is marketed
by Eli Lilly.