Metabolism recombinant cytochrome

Cerep evaluates hits, leads, and new compounds from the metabolic point of view using liver microsomes and recombinant cytochrome P450s (275). The results of these metabolic screening studies offer insight to the rate of metabolic pathways, in vivo pharmacokinetic properties, and drug-drug interactions. 

Giraud C, Tran A, Rey E, Vincent J, Treluyer JM, Pons G. In vitro characterization of clobazam metabolism by recombinant cytochrome P450 enzymes importance of CYP2C19. Drug Metab Dispos 2004 32(ll) 1279-86. 

Predictive models can be better produced when recombinant cytochrome data are available, an experimental technique which may increase the probability of obtaining consistent and predictive models, since one protein is involved in the metabolic reaction. The most accurate data to describe the rate and affinity of the ligand towards an enzyme are the kinetic parameters Vmax and Km. Nevertheless, the calculation of these parameters is time consuming. A less precise parameter is the determination of the compound percentage remaining in a cytochrome incubation after a certain period of time. These metabolic data are less accurate and can only be used to classify the compounds in a metabolic system as stable or unstable. This type of data was the basis for a predictive model of metabolic stability towards CYP3A4 [35]. 

Patki, K.C., von Moltke, L.L. and Greenblatt, D.J. (2003) In vitro metabolism of midazolam, triazolam, nifedipine, and testosterone by human liver microsomes and recombinant cytochromes P-450 role of CYP3A4 and CYP3A5. Drug Metabolism and Disposition The Biological Fate of Chemicals, 31, 938-944. 

Shou, M., Krausz, K. W., Gonzalez, F. J., and Gelboin, H. V. (1996) Metabolic activation of the potent carcinogen dibenzo[a,l]pyrene by human recombinant cytochrome P450, lung and liver microsomes. Carcinogenesis 17, 2429-2433. 

Recombinant cytochrome P450 2D18 metabolism of dopamine and arachidonic acid. J. Pharmacol. Exp. Ther. 294, 1120-1130. 

Stringer RA, Strain-Damerell C, Nicklin P, Houston JB. Evaluation of recombinant cytochrome P450 enzymes as an in vitro system for metabolic clearance predictions. Drug Metab Dispos 2009 37(5) 1025-1034. 

In this context, the applications of PyMS in biotechnology are of particular interest. The technique has been used to detect and measure the level of metabolites produced in cell culture, such as indole produced by cultured Escherichia coli cells grown in different types of media, and to predict the yield of recombinant protein in culture systems, such as E. coli producing recombinant cytochrome B5 or a-interferon. An analogous application is the use of PyMS to profile drugs and their metabolic breakdown products in serum (e.g., cyclosporin, vancomycin), or to profile microbial isolates for novel drug-producing strains. 

In another study by Godin et al. (2007), the rat and human CYP450 isoforms and rat serum esterase that metabolized deltamethrin and esfenvalerate were identified. Table 12 gives the Vmax and kinetic parameters for the metabolism of deltamethrin and esfenvalerate by rat and human recombinant cytochrome P450s. 

Trubetskoy, O.V., Gibson, J.R., and Marks, B.D. 2005. Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes. J. Biomol. Screen. 10 56. 

Several kinetic parameters can be measured on different experimental systems to account for the interaction of a compound with CYPs. For example when studying the metabolic stability of a compound, it could be measured in a recombinant CYP system, in human liver microsomes, in hepatocytes and so on. Each system increases in biological complexity. Although in the recombinant CYP system only the cytochrome under consideration is studied, in the case of the human liver microsomes, there is a pool of enzyme present that includes several CYPs, and finally in the hepatocyte cell system, metabolizing enzymes play an important role in the metabolic compound stability. In addition, transport systems are also present that could involve recirculation or other transport phenomena. The more complex the experimental system, the more difficult it is to extract information on the protein/ligand interaction, albeit it is closer to the in vivo real situation and therefore to the mechanism that is actually working in the body. 

Kinetic parameters of metabolism of fluorinated analogues of propanolol by a cytochrome enzyme (recombinant CYP1A2) have been determined. They clearly indicate that the A/-dealkylation was almost tenfold lower for the A/-CH2CF3... 

Kinetic parameters of metabolism of fluorinated analogues of propanolol by cytochrome enzyme (recombinant CYP1A2) have been determined. They clearly indicate that the A-dealkylation process was 10-fold lower for the N—CH2CF3 compound with respect to propanolol itself. Hydroxylation of the naphthalene ring process is not observed in the case of propanolol but it becomes the major process with the fluoro analogue (Figure 3.13)." The same decreased metabohsm trend has also been observed with lower pKa values for CYP2D6 cytochrome enzyme." ... 

Acceptable drug metabolism and interaction profiles based on microsomal and recombinantly expressed cytochrome P450 isoenzyme studies Recent addition Toxicogenomic data for early assessment of alteration in expression of RNA transcripts (and therefore proteins) related to toxic responses so that modification of safer and more potent variants of NME could be made and identified early... 

Crewe HK et al (2002) Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes formation of the 4-hydroxy, 4 -hydroxy and N-desmethyl metabolites and isomerization of trans-4-hydroxytamoxifen. Drug Metab Dispos 30 869-874... 

Cytochrome b5 affects the kinetics of drug metabolism by certain CYP enzymes hence, coexpression of this microsomal hemoprotein (together with NADPH-CYP reductase) can affect the catalytic efficiency of certain recombinant CYP enzymes (76,109). For example, the presence of cytochrome b5 tends to increase Fmax for reactions catalyzed by CYP3 A4, whereas it tends to decrease Km for reactions catalyzed by CYP2E1. In both cases, cytochrome b5 increases Vmax/Km, which is a measure of in vitro intrinsic clearance. The fact that some commercially available recombinant CYP enzymes are expressed with cytochrome b5 while others are not complicates the interpretation of results of studies performed with recombinant human CYP enzymes. 

Figure 27 Metabolism of DB289 (antiparasitic prodrug) by a panel of recombinant human CYP enzymes. Abbreviation CYP, cytochrome P450.

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