Article Figures & Data
Figures
- Scheme 1.
Metabolism of S-efavirenz catalyzed by CYP2B6.
- Fig. 1.
Primary and secondary metabolism of S-efavirenz catalyzed by coexpressed wild-type CYP2B6.1, P450 oxidoreductase, and cytochrome b5. Formation of 8-hydroxyefavirenz (8-OH-EFV) (●) and 8,14-dihydroxyefavirenz (8,14-diOH-EFV) (▲). Results are the mean ± S.D. of triplicate determinations. (A) Metabolism over the substrate range 0.25–40 μM. The solid line represents predicted concentrations on the basis of parameters from nonlinear regression using the Hill equation. The dotted line represents predicted concentrations on the basis of parameters from nonlinear regression using the Michaelis-Menten equation. The inset shows an Eadie-Hofstee plot for 8-hydroxyefavirenz formation. The solid line represents predicted values on the basis of parameters from nonlinear regression using the Hill equation. (B) Metabolism over the substrate range 0.25–100 μM, showing substrate inhibition. The solid line represents predicted concentrations on the basis of parameters from analysis using cooperative substrate binding and substrate inhibition with cooperativity in the inhibitory mode (LiCata model). The inset shows an Eadie-Hofstee plot for 8-hydroxyefavirenz formation. The solid line represents predicted concentrations on the basis of parameters from nonlinear regression using the LiCata model.
- Fig. 2.
Metabolism of S-efavirenz and R-efavirenz to 8-hydroxyefavirenz at therapeutic concentrations. Asterisks denote rates significantly different from wild-type (P < 0.05). Not shown are results for CYP2B6.16 and CYP2B6.18, which had negligible activity.
- Fig. 3.
8-Hydroxyefavirenz formation from S-efavirenz (●) and R-efavirenz (▲) catalyzed by CYP2B6 variants, POR.1, and cytochrome b5. Solid lines represent predicted concentrations on the basis of parameters from nonlinear regression using the Hill equation. Parameter estimates are in Table 3.
- Fig. 4.
S-Efavirenz hydroxylation by CYP2B6 variants, POR.1, and cytochrome b5 showing positive cooperativity and substrate inhibition. Solid lines represent predicted concentrations on the basis of parameters from nonlinear regression using model of cooperative substrate binding with substrate inhibition and cooperative inhibitor binding. Parameter estimates are in Table 4.
- Fig. 5.
Stereoselectivity of efavirenz metabolism. Shown is formation of 8-hydroxyefavirenz from S-efavirenz (●) and R-efavirenz (▲) by CYP2B6.1. The solid line represents predicted concentrations on the basis of parameters from nonlinear regression using the Hill equation for 8-hydroxylation of S-efavirenz and R-efavirenz. The inset compares predicted concentrations for R-efavirenz hydroxylation on the basis of parameters from nonlinear regression using the Hill equation (solid line) and Michaelis-Menten equation (dotted line).
Tables
CYP2B6 Allele Variant cDNA Sequence Mutation Protein Sequence Mutationa Allele Frequency (%) CYP2B6*1 Wild-type Wild-type CYP2B6*4 rs2279343 785A>G K262R 2-4 Ca CYP2B6*5 rs3211371 1459C>T R487C 12 Ca CYP2B6*6 rs3745274, rs2279343 516G>T, 785A>G Q172H/K262R 33 Af, 28 Ca CYP2B6*7 rs3745274, rs2279343, rs3211371 515G>T, 785A>G, 1459C>T Q172H/K262R/R487C 3 Ca CYP2B6*9 rs3745274 516G>T Q172H CYP2B6*16 rs2279343, rs28399499 785A>G, 983T>C K262R/I328T 6.9 Af CYP2B6*17 rs33973337, rs33980385, rs33926104 76A>T, 83A>G, 85C>A, 86G>C T26S/D28G/R29T 6.3 Af CYP2B6*18 rs28399499 983T>C I328T 9.4 Af CYP2B6*19 rs34826503 516G>T, 785A>G, 1006C>T Q172H/K262R/R336C 1.6 Af CYP2B6*26 rs3826711, rs2279343, rs3745274 499C>G, 516G>T, 785A>G P167A/Q172H/K262R 1.3 As Af, African; As, Asian; Ca, Caucasian.
↵a All CYP2B6 variants result in missense mutations.
- TABLE 2
8-Hydroxyefavirenz and 8,14-dihydroxyefavirenz formation in low substrate concentration range
S-Efavirenz 8-Hydroxyefavirenz 8,14-diOH-efavirenz 
µM pmol/min per picomoles pmol/min per picomoles % % 0.25 0 0.014 ± 0.010 0 100 0.5 0.001 ± 0.001 0.055 ± 0.046 2 98 1.25 0.055 ± 0.009 0.209 ± 0.033 21 79 2.5 0.332 ± 0.048 0.460 ± 0.182 42 58 5.0 1.46 ± 0.29 0.405 ± 0.078 78 22 10 2.43 ± 0.10 0.505 ± 0.144 83 17 20 4.03 ± 0.28 0.201 ± 0.095 95 5 - TABLE 3
Kinetic parameters for 8-hydroxyefavirenz formation from efavirenz enantiomers
Wild-type CYP2B6 and all variants were coexpressed with wild-type POR.1 and cytochrome b5. Results (Vmax and
and n) are the parameter estimate and S.E. of the estimate, determined by nonlinear regression analysis of the Hill equation, over the substrate concentration range 0.25–40 μM S-efavirenz and 0.11–45 μM R-efavirenz.CYP2B6 Variant S-8-Hydroxyefavirenz Formation from S-Efavirenz R-8-Hydroxyefavirenz Formation from R-Efavirenz Vmax n Clmax Vmax n Clmax pmol/min per picomoles μM ml/min per nanomoles pmol/min per picomoles μM ml/min per nanomoles CYP2B6.1 4.2 ± 0.2 7.7 ± 0.6 2.1 ± 0.3 0.27 0.57 ± 0.09 16.1 ± 4.4 1.5 ± 0.3 0.019 CYP2B6.4a 4.5 ± 0.1 5.9 ± 0.3 2.5 ± 0.2 0.39 0.78 ± 0.06 7.0 ± 1.1 1.2 ± 0.1 0.071 CYP2B6.5 3.3 ± 0.1 7.3 ± 0.7 1.4 ± 0.1 0.25 0.44 ± 0.02 6.5 ± 0.8 1.8 ± 0.3 0.034 CYP2B6.6 3.4 ± 0.1 11.8 ± 0.5 1.8 ± 0.1 0.15 0.41 ± 0.05 14.5 ± 3.8 1.2 ± 0.2 0.018 CYP2B6.7 1.5 ± 0.1 5.0 ± 0.3 1.5 ± 0.1 0.16 0.14 ± 0.01 6.3 ± 1.3 1.6 ± 0.4 0.011 CYP2B6.9 1.7 ± 0.1 8.1 ± 1.0 1.2 ± 0.1 0.13 0.13 ± 0.03 13.5 ± 5.6 1.3 ± 0.3 0.006 CYP2B6.16b 0.19 ± 0.01 0.012 ± 0.002 CYP2B6.17 4.4 ± 0.2 9.3 ± 0.8 1.7 ± 0.2 0.24 0.38 ± 0.07 9.2 ± 4.30 1.2 ± 0.3 0.055 CYP2B6.18b 0.20 ± 0.01 0.009 ± 0.001 CYP2B6.19c 3.4 ± 0.1 13.4 ± 0.9 1.5 ± 0.1 0.13 0.65 ± 0.33 31 ± 28 1.2 ± 0.4 0.013 CYP2B6.26 1.5 ± 0.1 5.0 ± 0.3 1.8 ± 0.1 0.15 0.21 ± 0.01 4.2 ± 0.7 1.9 ± 0.4 0.025 ↵a CYP2B6.4 (only) showed substrate inhibition with R-efavirenz. Results in the table for CYP2B6.4and R-efavirenz were from the Hill equation and the substrate concentration range 0.11–18 μM. Data were also analyzed with the model for substrate inhibition (LiCata model, x = 3) over the substrate concentration range 0.11–45 μM, yielding Vmax = 0.90 ± 0.18, K = 9.0 ± 3.5, n = 1.1 ± 0.1, Clmax = 0.74, Vi = 0.09 ± 0.19, and Ki = 38 ± 11 μM.
↵b CYP2B6.16 and CYP2B6.18 rates were measured at a fixed substrate concentration of 40 μM S- and R-efavirenz.
↵c For R-efavirenz hydroxylation by CYP2B6.19, an alternative Michaelis-Menten model of linear regression analysis at low substrate concentrations generated a ratio of Vmax/Km = 0.019.
- TABLE 4
Kinetic parameters for 8-hydroxyefavirenz formation from S-efavirenz
Results (Vmax, K, n, Ki, and Vi) are the parameter estimates and S.E. of the estimate, determined by nonlinear regression analysis using a model of cooperative substrate binding with substrate inhibition and cooperative inhibitor binding over the substrate concentration range 0.25–100 μM.
Vmax K n Clmax Ki Vi pmol⋅min/pmol μM μM pmol/min per picomoles CYP2B6.1 7.4 ± 2.4 17 ± 8 1.4 ± 0.3 0.25 47 ± 10 0.0001 ± 0.42 CYP2B6.4 6.2 ± 1.0 9 ± 2 1.7 ± 0.3 0.35 53 ± 8 0.0002 ± 0.44 CYP2B6.5 4.1 ± 0.4 11 ± 2 1.2 ± 0.1 0.25 75 ± 10 5.8e−5 ± 0.42 CYP2B6.6 3.6 ± 0.2 13 ± 1 1.7 ± 0.1 0.14 84 ± 16 1.46 ± 0.43 CYP2B6.7 2.7 ± 1.0 16 ± 12 1.0 ± 0.2 0.17 46 ± 11 4.5e−5 ± 0.14 CYP2B6.9 2.8 ± 0.7 21 ± 10 0.9 ± 0.1 0.13 57 ± 9 4.4e−5 ± 0.15 CYP2B6.17 5.5 ± 0.5 13 ± 2 1.4 ± 0.1 0.23 71 ± 8 2.8e−5 ± 0.44 CYP2B6.19 3.5 ± 0.2 14 ± 2 1.5 ± 0.1 0.13 173 ± 448 7.1e−5 ± 21 CYP2B6.26 2.1 ± 0.4 9 ± 4 1.2 ± 0.3 0.15 56 ± 11 5.7e−5 ± 0.18 - TABLE 5
Summary of reported 8-hydroxylation of S-efavirenz
Relative activities are shown as percentage of CYP2B6 variants Clint (Clmax for this study) compared with the wild type, on the basis of data in Table 3.
Bumpus et al., 2006 Ariyoshi et al., 2011 Zhang et al., 2011 Xu et al., 2012 Radloff et al., 2013 Watanabe et al., 2018 This Study (Clmax) Expression system E. coli Sf9 E. coli SF9 COS-1 HEK T. ni CYP2B6.1 100 100 100 100 100 100 100 CYP2B6.4 170 142 96 122 144 CYP2B6.5 138 83 109 92 CYP2B6.6 50 20 49 183 266 56 CYP2B6.7 156 166 59 CYP2B6.9 33 172 48 CYP2B6.17 85 89 CYP2B6.19 37 48 CYP2B6.26 183 56
In this issue
CYP2B6 Efavirenz Metabolism Genetics and Stereoselectivity
