Testosterone CYP3A4/5/7 substrate

FIGURE 4.20 Structures of the CYP3A4 substrates, erythromycin, nifedipine, testosterone, and midazolam, and their metabolites. 

For substrates exhibiting non-Michaelis-Menten kinetics (e.g., several CYP3A4 substrates), a wider range of substrate concentrations may be required to accurately determine reaction kinetics. CYP3A4 should be measured with at least two substrates, one exhibiting positive cooperativity (e.g., testosterone) and one exhibiting autoinhibition (e.g., midazolam). 

Figure 22 Examples of enzyme kinetic plots used for determination of Km and Vmax for a normal and an allosteric enzyme Direct plot [(substrate) vs. initial rate of product formation] and various transformations of the direct plot (i.e., Eadie-Hofstee, Lineweaver-Burk, and/or Hill plots) are depicted for an enzyme exhibiting traditional Michaelis-Menten kinetics (coumarin 7-hydroxylation by CYP2A6) and one exhibiting allosteric substrate activation (testosterone 6(3-hydroxylation by CYP3A4/5). The latter exhibits an S-shaped direct plot and a hook -shaped Eadie-Hofstee plot such plots are frequently observed with CYP3A4 substrates. Km and Vmax are Michaelis-Menten kinetic constants for enzymes. K is a constant that incorporates the interaction with the two (or more) binding sites but that is not equal to the substrate concentration that results in half-maximal velocity, and the symbol n (the Hill coefficient) theoretically refers to the number of binding sites. See the sec. III.C.3 for additional details.

Recently, Chu et al. reported an ultra-fast LC/MS method for analysis of cytochrome P450 3A4 and 2D6 inhibition assaysd Testosterone and dextromethorphan were used as the specific substrates for CYP3A4 and CYP 2D6, respectively. LC/MS analyses were performed on a Sciex API 3000 mass spectrometer equipped with a Shimadzu LC-lOAdvp pump and a PE 200 autosampler. A Phenomenex Luna CIS (4.6x30 mm) column was used along with very steep gradients. Each sample analysis was completed in 0.5 min. 

Figure 13 Effect of substrate incubation time on the metabolism-dependent inhibition of CYP3A4/5 by mibefradil. Mibefradil (0.01 to 10 pM) was examined as a direct-acting and metabolism-dependent inhibitor with either a 0- or 15-minute preincubation with NADPH followed by either a 5- or 30-minute incubation with testosterone (100 pM). In both cases, the IC50 value after a 15-minute preincubation with NADPH was approximately 0.07 pM. However, the IC50 value for direct inhibition (0-minute preincubation) varied nearly fourfold depending on the length of the substrate incubation, with a longer substrate incubation period diminishing the apparent impact of metabolism-dependent inhibition due to increased inactivation during the substrate incubation.

Galetin A, Clarke SE, Houston JB. Multisite kinetic analysis of interactions between prototypical CYP3A4 subgroup substrates midazolam, testosterone and nifedipine. Drug Metab Dispos 2003 31 1108-1116. 

Figure 8.19 The substrate, testosterone, (in red) is shown within the active site of CYP3A4. Hydrogen bonds are dashed iines. The heme structure is shown in the bottom piane. (From DFV Lewis, Univ Surrey. Figure is Fig 6 from Xenobiotica 34 549-569, 2004.)...

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