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Research ArticleArticle

Piperine Is a Mechanism-Based Inactivator of CYP3A

Tiantian Cui, Qian Wang, Xiaoxiao Tian, Kehan Zhang, Ying Peng and Jiang Zheng
Drug Metabolism and Disposition February 2020, 48 (2) 123-134; DOI: https://doi.org/10.1124/dmd.119.088955

Article Figures & Data

Figures

  • Figure1
  • Scheme 1.
    Scheme 1.

    Proposed pathways for the generation of reactive intermediate(s) and NAC conjugates derived from PPR.

  • Fig. 1.
    Fig. 1.

    Quick screening of time-dependent inhibition of cytochrome P450 enzymes by PPR. PPR at concentrations of 0 (■) or 80 (◆) μΜ, human liver microsomes (0.5 mg protein/ml), and NADPH (1.0 mM) were incubated at 37°C for 0, 5, 10, and 20 minutes. The residual enzymatic activities at 0 minute were normalized to 100%. Data represent the mean ± S.D. (n = 3). Comparisons were made using an unpaired t test. * indicates the significance of the remaining enzyme activity of experimental groups with respect to that of control at the same time point (20 minutes) of incubations. Levels are considered significant at *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

  • Fig. 2.
    Fig. 2.

    (A) Time- and concentration-dependent inhibition of CYP3A by PPR. PPR at concentrations of 0 (■), 5 (●), 10 (▲), 20 (▼), 40 (◆), 60 (★), and 80 (○) μM was incubated with human liver microsomes (0.5 mg protein/ml) fortified with NADPH (1.0 mM) at 37°C for 0, 5, 10, and 20 minutes. (B) NADPH-dependent inactivation of CYP3A4 by PPR. Human liver microsomes (0.5 mg/ml) were incubated with vehicle (■) or PPR (80 μM) in the absence (▼) or presence (★) of NADPH. (C) Wilson’s plot. kobs values were calculated from the slope of the regression lines shown in (A). Data represent the mean ± S.D. (n = 3). * indicates the significance of the remaining enzyme activity of experimental groups with respect to that of control at the same time point (20 minutes) of incubations. $ indicates the significance of the remaining enzyme activity of experimental groups at various incubation times, compared with that at 0 minute under the same incubation condition. # indicates the significance of the remaining enzyme activity in the presence of NADPH, compared with that in the absence of NADPH under the same incubation condition. Levels are considered significant at *P < 0.05; **/$$/##P < 0.01; ***P < 0.001; ns, not significant.

  • Fig. 3.
    Fig. 3.

    Effect of competitive inhibitor on inactivation of CYP3A by PPR. Human liver microsomes (0.5 mg protein/ml) were incubated with vehicle (■) or PPR (80 μM) supplemented with ketoconazole at concentrations of 0 (★), 0.01 (◆), 0.1 (▲), or 1.0 (●) μM. Data represent the mean ± S.D. (n = 3). * indicates the significance of the remaining enzyme activity of experimental groups with respect to that of control at the same time point (20 minutes) of incubations. Levels are considered significant at *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 4.
    Fig. 4.

    Partition ratio of CYP3A4 inactivation by PPR. CYP3A4 (50 nM) was incubated with PPR at designed concentrations (0, 2, 5, 10, 20, 40, 60, 80, or 100 μM). The extrapolated P + 1 value was determined from the point of intersection to the abscissa. Data represent the mean ± S.D. (n = 3).

  • Fig. 5.
    Fig. 5.

    Chemical structure and 1H NMR chemical shifts of DM-PPR.

  • Scheme 2.
    Scheme 2.

    Proposed mechanisms for the formation of NAC conjugates M3–M8.

  • Fig. 6.
    Fig. 6.

    (A) Time- and NADPH-dependent inhibition of CYP3A by DM-PPR (80 μM). Human liver microsomes (0.5 mg protein/ml) were incubated with vehicle (■), or DM-PPR (80 μM) in the absence (▲) or presence (◆) of NADPH at 37°C for 0, 5, 10, and 20 minutes. (B) Human liver microsomes (0.5 mg protein/ml) were incubated with vehicle (■), DM-PPR (80 μM) (◆), or PPR (80 μM) (★) in the presence of NADPH. The residual enzymatic activities at 0 minute were normalized to 100%. Data represent the mean ± S.D. (n = 3). * indicates the significance of the remaining enzyme activity of experimental groups with respect to that of control at the same time point (20 minute) of incubations. (A) # indicates the significance of the remaining activity in the presence of NADPH, compared with that in the absence of NADPH under the same incubation condition; (B) # indicates the significance of the remaining activity of the incubation containing DM-PPR (80 μM), compared with that of PPR (80 μM) in the presence of NADPH under the same conditions. ##P < 0.01; and ***P < 0.001 are considered significantly different; ns, not significant.

  • Fig. 7.
    Fig. 7.

    MS characterization of metabolites M1 and M2. Extracted ion (m/z 449.0/320.0 for M1 and M2) chromatograms obtained from LC-MS/MS analysis of incubations containing PPR, human liver microsomes, and NAC in the absence (A) or presence of NADPH (B). (C) Extracted ion chromatograms obtained from LC-MS/MS analysis of synthetic M1 and M2. (D) MS/MS spectra of M1 and M2 generated in microsomal incubations. (E) MS/MS spectra of synthetic M1 and M2.

  • Fig. 8.
    Fig. 8.

    MS characterization of DM-PPR–derived NAC conjugates M3-M6. Extracted ion (m/z 435.0/221.0 for M3-M6) chromatograms obtained from LC-MS/MS analysis of incubations containing PPR, human liver microsomes, and NAC in the absence (A) or presence of NADPH (B). (C) Extracted ion chromatograms obtained from LC-MS/MS analysis of synthetic M3–M6. (D) MS/MS spectra of M3–M6 generated in microsomal incubations. (E) MS/MS spectra of synthetic M3–M6.

  • Fig. 9.
    Fig. 9.

    MS characterization of DM-PPR–derived NAC conjugates M7 and M8. Extracted ion (m/z 437.0/181.0 for M7 and M8) chromatograms obtained from LC-MS/MS analysis of incubations containing PPR, human liver microsomes, and NAC in the absence (A) or presence of NADPH (B). (C) Extracted ion chromatograms obtained from LC-MS/MS analysis of synthetic M7 and M8. (D) MS/MS spectra of M7 and M8 generated in microsomal incubations. (E) MS/MS spectra of synthetic M7 and M8.

  • Fig. 10.
    Fig. 10.

    P450 enzymes responsible for the generation of M3–M6 in vitro. (A) Rates of M3–M6 formation in incubations containing PPR, individual recombinant P450 enzymes, NAC, and NADPH. (B) Effects of selective P450 inhibitors on the generation of M3–M6. PPR was incubated with human liver microsomes, NAC, and NADPH, in the absence (control) or presence of individual P450 enzyme inhibitors. Data represent the mean ± S.D. (n = 3). *, #, $, and % indicate the significance of the amount of M3–M6 produced in experimental groups compared with that of the control, respectively. Comparisons were made using unpaired t test. */#/$/%P < 0.05; **/##/$$/%%P < 0.01; ***/###/$$$/%%%P < 0.001 are considered significantly different.

  • Fig. 11.
    Fig. 11.

    P450 enzymes responsible for the generation of M7 and M8 in vitro. (A) Rates of M7 and M8 formation in incubations containing PPR, individual recombinant P450 enzymes, NAC, and NADPH. (B) Effects of selective P450 inhibitors on the generation of M7 and M8. PPR was incubated with human liver microsomes, NAC, and NADPH in the absence (control) or presence of individual P450 enzyme inhibitors. Data represent the mean ± S.D. (n = 3). * and # indicate the significance of the amount of M7 and M8 produced in experimental groups, compared with that of the control, respectively. Comparisons were made using unpaired t test. */#P < 0.05; **P < 0.01; ###P < 0.001 are considered significantly different.

Tables

  • TABLE 1

    Effects of K3Fe(CN)6 on the inactivation of CYP3A

    Data represent the mean ± S.D. (n = 3). The percentage of CYP3A activity was determined by calculation of the ratio of enzyme activity of each sample after 0 or 20 minute incubation with PPR vs. that of the corresponding control sample without PPR treatment. % Reversed stands for the contribution of KFC to the recovered activity of CYP3A.

    SystemCYP3A
    % Control (0 min)% Control (20 min)
    −KFC50.7 ± 2.940.5 ± 2.6
    +KFC86.3 ± 8.480.2 ± 15.7
    % Reversed/39.7

    • KFC, potassium ferricyanide.

Additional Files

  • Data Supplement

    • Supplemental Data -

      Supplementary Figure 1 -  1H NMR spectrum of synthetic DM-PPR.

      Supplementary Figure 2 -  Characterization of PPR-cysteine adduct.

      Supplementary Figure 3 - Mass spectrometric characterization of DM-PPR-derived NAC conjugates M3-M6 and M7-M8. 

      Supplementary Table 1 - Mass spectrometric data acquired from high-resolution mass spectrometric analysis of synthetic DM-PPR. 

      Supplementary Methods
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Research ArticleArticle

Inactivation of CYP3A by Piperine

Tiantian Cui, Qian Wang, Xiaoxiao Tian, Kehan Zhang, Ying Peng and Jiang Zheng
Drug Metabolism and Disposition February 1, 2020, 48 (2) 123-134; DOI: https://doi.org/10.1124/dmd.119.088955
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