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Review ArticleArticle

Efavirenz Metabolism: Influence of Polymorphic CYP2B6 Variants and Stereochemistry

Pan-Fen Wang, Alicia Neiner and Evan D. Kharasch
Drug Metabolism and Disposition October 2019, 47 (10) 1195-1205; DOI: https://doi.org/10.1124/dmd.119.086348
Pan-Fen Wang
Department of Anesthesiology, Duke University School of Medicine, Durham, North Carolina (P.-F.W., E.D.K.) and Department of Anesthesiology, Washington University in St. Louis, St. Louis, Missouri (A.N.)
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Alicia Neiner
Department of Anesthesiology, Duke University School of Medicine, Durham, North Carolina (P.-F.W., E.D.K.) and Department of Anesthesiology, Washington University in St. Louis, St. Louis, Missouri (A.N.)
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Evan D. Kharasch
Department of Anesthesiology, Duke University School of Medicine, Durham, North Carolina (P.-F.W., E.D.K.) and Department of Anesthesiology, Washington University in St. Louis, St. Louis, Missouri (A.N.)
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Figures

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  • Scheme 1.
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    Scheme 1.

    Metabolism of S-efavirenz catalyzed by CYP2B6.

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    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.
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    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.
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    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.
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    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.
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    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

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    TABLE 1

    CYP2B6 variants

    CYP2B6 AlleleVariantcDNA Sequence MutationProtein Sequence MutationaAllele Frequency (%)
    CYP2B6*1Wild-typeWild-type
    CYP2B6*4rs2279343785A>GK262R2-4 Ca
    CYP2B6*5rs32113711459C>TR487C12 Ca
    CYP2B6*6rs3745274, rs2279343516G>T, 785A>GQ172H/K262R33 Af, 28 Ca
    CYP2B6*7rs3745274, rs2279343, rs3211371515G>T, 785A>G, 1459C>TQ172H/K262R/R487C3 Ca
    CYP2B6*9rs3745274516G>TQ172H
    CYP2B6*16rs2279343, rs28399499785A>G, 983T>CK262R/I328T6.9 Af
    CYP2B6*17rs33973337, rs33980385, rs3392610476A>T, 83A>G, 85C>A, 86G>CT26S/D28G/R29T6.3 Af
    CYP2B6*18rs28399499983T>CI328T9.4 Af
    CYP2B6*19rs34826503516G>T, 785A>G, 1006C>TQ172H/K262R/R336C1.6 Af
    CYP2B6*26rs3826711, rs2279343, rs3745274499C>G, 516G>T, 785A>GP167A/Q172H/K262R1.3 As
    • Af, African; As, Asian; Ca, Caucasian.

    • ↵a All CYP2B6 variants result in missense mutations.

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    TABLE 2

    8-Hydroxyefavirenz and 8,14-dihydroxyefavirenz formation in low substrate concentration range

    S-Efavirenz8-Hydroxyefavirenz8,14-diOH-efavirenzEmbedded ImageEmbedded Image
    µMpmol/min per picomolespmol/min per picomoles%%
    0.2500.014 ± 0.0100100
    0.50.001 ± 0.0010.055 ± 0.046298
    1.250.055 ± 0.0090.209 ± 0.0332179
    2.50.332 ± 0.0480.460 ± 0.1824258
    5.01.46 ± 0.290.405 ± 0.0787822
    102.43 ± 0.100.505 ± 0.1448317
    204.03 ± 0.280.201 ± 0.095955
    • View popup
    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 Embedded Image 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 VariantS-8-Hydroxyefavirenz Formation from S-EfavirenzR-8-Hydroxyefavirenz Formation from R-Efavirenz
    VmaxEmbedded ImagenClmaxVmaxEmbedded ImagenClmax
    pmol/min per picomolesμMml/min per nanomolespmol/min per picomolesμMml/min per nanomoles
    CYP2B6.14.2 ± 0.27.7 ± 0.62.1 ± 0.30.270.57 ± 0.0916.1 ± 4.41.5 ± 0.30.019
    CYP2B6.4a4.5 ± 0.15.9 ± 0.32.5 ± 0.20.390.78 ± 0.067.0 ± 1.11.2 ± 0.10.071
    CYP2B6.53.3 ± 0.17.3 ± 0.71.4 ± 0.10.250.44 ± 0.026.5 ± 0.81.8 ± 0.30.034
    CYP2B6.63.4 ± 0.111.8 ± 0.51.8 ± 0.10.150.41 ± 0.0514.5 ± 3.81.2 ± 0.20.018
    CYP2B6.71.5 ± 0.15.0 ± 0.31.5 ± 0.10.160.14 ± 0.016.3 ± 1.31.6 ± 0.40.011
    CYP2B6.91.7 ± 0.18.1 ± 1.01.2 ± 0.10.130.13 ± 0.0313.5 ± 5.61.3 ± 0.30.006
    CYP2B6.16b0.19 ± 0.010.012 ± 0.002
    CYP2B6.174.4 ± 0.29.3 ± 0.81.7 ± 0.20.240.38 ± 0.079.2 ± 4.301.2 ± 0.30.055
    CYP2B6.18b0.20 ± 0.010.009 ± 0.001
    CYP2B6.19c3.4 ± 0.113.4 ± 0.91.5 ± 0.10.130.65 ± 0.3331 ± 281.2 ± 0.40.013
    CYP2B6.261.5 ± 0.15.0 ± 0.31.8 ± 0.10.150.21 ± 0.014.2 ± 0.71.9 ± 0.40.025
    • ↵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.

    • View popup
    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.

    VmaxKnClmaxKiVi
    pmol⋅min/pmolμMμMpmol/min per picomoles
    CYP2B6.17.4 ± 2.417 ± 81.4 ± 0.30.2547 ± 100.0001 ± 0.42
    CYP2B6.46.2 ± 1.09 ± 21.7 ± 0.30.3553 ± 80.0002 ± 0.44
    CYP2B6.54.1 ± 0.411 ± 21.2 ± 0.10.2575 ± 105.8e−5 ± 0.42
    CYP2B6.63.6 ± 0.213 ± 11.7 ± 0.10.1484 ± 161.46 ± 0.43
    CYP2B6.72.7 ± 1.016 ± 121.0 ± 0.20.1746 ± 114.5e−5 ± 0.14
    CYP2B6.92.8 ± 0.721 ± 100.9 ± 0.10.1357 ± 94.4e−5 ± 0.15
    CYP2B6.175.5 ± 0.513 ± 21.4 ± 0.10.2371 ± 82.8e−5 ± 0.44
    CYP2B6.193.5 ± 0.214 ± 21.5 ± 0.10.13173 ± 4487.1e−5 ± 21
    CYP2B6.262.1 ± 0.49 ± 41.2 ± 0.30.1556 ± 115.7e−5 ± 0.18
    • View popup
    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., 2006Ariyoshi et al., 2011Zhang et al., 2011Xu et al., 2012Radloff et al., 2013Watanabe et al., 2018This Study (Clmax)
    Expression systemE. coliSf9E. coliSF9COS-1HEKT. ni
    CYP2B6.1100100100100100100100
    CYP2B6.417014296122144
    CYP2B6.51388310992
    CYP2B6.650204918326656
    CYP2B6.715616659
    CYP2B6.93317248
    CYP2B6.178589
    CYP2B6.193748
    CYP2B6.2618356
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Drug Metabolism and Disposition: 47 (10)
Drug Metabolism and Disposition
Vol. 47, Issue 10
1 Oct 2019
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Review ArticleArticle

CYP2B6 Efavirenz Metabolism Genetics and Stereoselectivity

Pan-Fen Wang, Alicia Neiner and Evan D. Kharasch
Drug Metabolism and Disposition October 1, 2019, 47 (10) 1195-1205; DOI: https://doi.org/10.1124/dmd.119.086348

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Review ArticleArticle

CYP2B6 Efavirenz Metabolism Genetics and Stereoselectivity

Pan-Fen Wang, Alicia Neiner and Evan D. Kharasch
Drug Metabolism and Disposition October 1, 2019, 47 (10) 1195-1205; DOI: https://doi.org/10.1124/dmd.119.086348
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