Codexis may 08.qxp

Hydroxylation reactions with P450 enzymes
Hydroxylation reactions catalysed using P450 enzymes have a range ofapplications in drug metabolite production and lead diversificationFacilitating the identification of toxic metabolites early in the drug development process and enabling the diversification of identification and preliminary toxicity and lead compounds through the generation of a activity testing poses significant challenges.
broad range of hydroxylated derivatives may Chemical synthesis has been traditionally synthetic, redesigned gene and formulated lead to the discovery of safer, more potent employed to produce metabolites, but often with all the components necessary for a fully the synthetic route requires many steps and resulting in a low yield and a high cost in compounds including drugs are metabolised both time and materials. An alternative to Commonly in drug metabolism the first step lyophilised preparation, the HCB is easier to ship, store, and handle, and because it is their cost, batch-to-batch variability in activity, and restriction on availability limit components for activity, the user need only drugs frequently fail not due to problems add water and the drug to be metabolised.
with the drug candidate itself but due to problems arising from the metabolites.4 For microsomes from alternative animal sources have been used as in in vitro alternative to guidance that strongly recommends that all parallel to determine which is the active early in the drug development process.5 It is enzyme for metabolism of a given drug.
highly desirable that such characterisation Once identified, the most active cytochrome testing. Often, once the site of hydroxylation or other modification is identified, a drug can be redesigned to improve its activity and/or half-life. A common strategy is to metabolites. Table 1 shows the reaction of Human Cytochrome Biocatalysts
used in the production of key metabolites.
use of microsomes or in vivo systems can be problematic metabolite and/or extending the reactions are all complete within 6 hours.
advantage of the use of recombinant human production of the 4-hydroxy derivative of P450 enzymes is the ability to generate a desired metabolite in a one-step reaction.
complete within 1 hour, with a quantitative display biological activities superior to or conversion of drug to the desired metabolite. different from that of the drug from which system is complex, requiring at a minimum they are derived. The identification of these Going beyond human
so-called ‘active metabolites’ can lead to reductase component to be functional. The drug metabolites
safer, more active drug substances. A well- reductase requires a reduced nicotinamide cofactor, typically NADPH, and this cofactor converted into ‘active metabolites’ has led incorporate cytochrome P450-like reactions into lead diversification strategies. In order have all the biological activity of terfenadine patients, leading to its approval in 1996 by activity, fewer drug-drug interactions and reduced toxicity, the ability to diversify a improved drug in place of the original drug 28 sp2 May 2008
and sustain a longer reaction time, resulting metabolism in human liver microsomes.
in a much higher final titre of hydroxylated and CYP1A2 as N-desisopropylase. Drug Metab Dispos 1994; 22(6):909–915.
different hydroxylated derivatives can be new product that simplifies the production throughput screening for lead optimisation: of a wide range of hydroxylated derivatives.
a rational approach. Current Opinion in Drug Discovery and Development 2000; 3, Plate contains 96 variants of the bacterial 5. Guidance for Industry, Safety Testing of as P450-BM3. The distinctive feature of the Fexofenadine. Drugs 1998; 55(2): 269- and the reductase component are contained 7. Ueng Y.F., Yu H.J., Lee C.H., Peng C., Jan W.C., Ho L.K., Chen C.F., Don M.J. Journal separate reductase enzyme is required. By expense of the drug development process.
of Chromatography A 2005; 1076, 103- creating a platform of variants based on the metabolites early in the drug development 8. Hubl U., Stevenson D.E. Enzyme and format, Codexis has simplified the process process and enabling the diversification of Microbial Technology 2001; 29, 306-311.
lead compounds through the generation of a broad range of hydroxylated derivatives may lead to the discovery of safer, more potent Kambourakis, and David Rozzell of Codexis.
target compound, and the useful reactions clinical trials. sp2
REFERENCES
enzymes provides important benefits in both drugs, and diseases. Mol Interv. 2003; hydroxylated derivatives of lead compounds.
information
related to metabolism and chemical toxicity.
substrates, leading to better scalability.
variant for the hydroxylation of diclofenac.
50-fold higher concentration of diclofenac % cconversion metabolite(s) Reaction ttime Internet Links:
Table 1. Metabolite production of selected drugs using Human Cytochrome Biocatalysts
and a BM3 mutant from the Codexis MicroCyp plate. Each reaction was performed with a
P450 biocatalyst concentration of 1 µM (1000 nmol / litre), in the presence of the
corresponding P450 reaction mix, at 30°C with agitation to promote oxygen transfer to the
reaction solution. The final column also represents biocatalyst productivity as milligrams
of metabolite(s) produced per µmol of P450 biocatalyst.
May 2008 sp2 29

Source: http://www.che.caltech.edu/groups/fha/publications/Biocatalysis_May_2008-1.pdf