in black and white
Main menu
Share a book About us Home
Biology Business Chemistry Computers Culture Economics Fiction Games Guide History Management Mathematical Medicine Mental Fitnes Physics Psychology Scince Sport Technics

biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
Previous << 1 .. 168 169 170 171 172 173 < 174 > 175 176 177 178 179 180 .. 292 >> Next

• 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 which would accrue from utilizing smaller quantities of insulin per therapeutic dose.
The insulin amino acid residues which 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, e.g. conversion of histidine to glutamate at the B10 position yields an analogue displaying five-fold 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 which 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 8.3). Insulin Lispro was the first such engineered short-acting insulin to come to market (Box 8.3 and Figure 8.8).
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 trade names given to one such analogue that gained general marketing approval in 2000 (Table 8.3). The international non-proprietary name (inn) 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 b-chain has been elongated (again from its C-terminus) by two arginine residues. The overall effect is to increase the molecule’s isoelectric point (pI, the pH at which the molecule displays a net overall zero charge and consequently 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 previously described. 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 more than 24 h after administration.
Box 8.3. Insulin Lispro
Insulin Lispro was the first recombinant fast-acting insulin analogue to gain marketing approval (Table 8.3). It displays an amino acid sequence identical to native human insulin, with one alteration — 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 as the change in sequence disrupts the formation of inter-chain hydrophobic interactions critical to self-association.
Insulin Lispro was developed by scientists at Eli Lilly (which, along with Novo Nordisk, are the world’s largest producers of therapeutic insulins). The rationale underlying the sequence alteration was rooted in studies, not of insulin, but of insulin-like growth factor-1 (IGF-1; Chapter 7). The latter displays a strong structural resemblance to proinsulin, with up to 50% of amino acid residues within the IGF-1 A- and B-domains being identical to those found in comparable positions in the insulin A- and B-chains. When compared to 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.
Previous << 1 .. 168 169 170 171 172 173 < 174 > 175 176 177 178 179 180 .. 292 >> Next