Product case study Humalog

Humalog (tradename, also known as insulin lispro) was the first recombinant fast-acting insulin 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 insulins (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 alteration significantly 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 unmodified 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 insulin, IGF-1 molecules display a significantly decreased propensity to self-associate. Sequencing studies earlier revealed that the prolineB28-lysineB29 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 prolineB28-lysineB29 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 LysB28-ProB29 proinsulin is expressed in E. coli. Following fermentation and recovery, the proinsulin is treated with trypsin and carboxypeptidase B, resulting in the proteolytic excision of the engineered insulin molecule. It is then purified to homogeneity by a number of high-resolution chromatographic steps. The final product formulation also contains m-cresol (preservative and stabilizer), zinc oxide (stabilizer), glycerol (tonicity modifier) and a phosphate-based buffer 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 administration, as opposed to 50-120 min in the case of regular insulin. Its serum t1/2 is also shorter than that of regular insulin (60 min as opposed to 90 min). Product safety and efficacy 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 potential negative adverse effect is hypoglycaemia. The product was developed and is marketed by Eli Lilly.

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Figure 11.5 Three-dimensional structure of the engineered fast-acting insulin, 'Insulin lispro'. Structural details courtesy of the Protein Data Bank, http://www.rcsb.org/pdb/

which it is least soluble) from 5.4 to a value approaching 7.0. The engineered insulin is expressed in a recombinant E. coli K12 host strain and is produced via the 'proinsulin route' as described previously. The purified product is formulated at pH 4.0, a pH value at which it is fully soluble. Upon s.c. injection, the insulin experiences an increase in pH towards more neutral values and, consequently, appears to precipitate in the subcutaneous tissue. It resolubilizes very slowly and, hence, a greatly prolonged duration of release into the bloodstream is noted. Consequently, a single daily injection supports the maintenance of acceptable basal blood insulin levels, and insulin molecules are still detected at the site of injection in excess of 24 h after administration.

Levemir (tradename) is an alternative engineered long-acting insulin product that gained approval for general medical use in 2004 (Table 11.3). This differs from native insulin in that it is devoid of the threonine B30 residue and (more importantly from a pharmacokinetic perspective) contains a 14-carbon fatty acid residue covalently attached to the side chain of lysine residue B29. This allows the insulin to bind reversibly to albumin, both at the site of injection and in plasma (albumin contains three high-affinity fatty acid binding sites). This, in turn, ensures constant and prolonged release of free insulin, bestowing upon it a prolonged duration of action of up to 24 h. Product manufacture entails initial expression of insulin in engineered Saccharomyces cerevisiae, purification and acylation (attachment of the fatty acid group).

The generation of engineered insulin analogues raises several important issues relating to product safety and efficacy. Alteration of a native protein's amino acid sequence could render the engineered product immunogenic. Such an effect would be particularly significant in the case of insulin, as the product is generally administered daily for life. In addition, alteration in structure could have unintended influences upon pharmacokinetic and/or pharmacodynamic characteristics of the drug. Preclinical and, in particular, clinical evaluations undertaken upon the analogues thus far approved, however, have confirmed their safety and efficacy. The sequence changes introduced are relatively minor and do not seem to elicit an immunological response. Fortuitously, neither have the alterations made affected the ability of the insulin molecule to interact with the insulin receptor, and trigger the resultant characteristic biological responses.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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