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SCIENCE CHINA Life Sciences, Volume 60, Issue 2: 215-224(2017) https://doi.org/10.1007/s11427-015-4911-7

In vitro and in vivo evaluation of cucurbitacin E on rat hepatic CYP2C11 expression and activity using LC-MS/MS

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  • ReceivedJul 3, 2015
  • AcceptedJul 27, 2015
  • PublishedSep 9, 2015

Abstract

This study explored the effects of cucurbitacin E (CuE), a bioactive compound from Cucurbitaceae, on the metabolism/ pharmacokinetic of tolbutamide, a model CYP2C9/11 probe substrate, and hepatic CYP2C11 expression in rats. Liquid chromatography-(tandem) mass spectrometry (LC-MS/MS) assay was used to detect tolbutamide as well as 4-hydroxytolbutamide, and then successfully applied to the pharmacokinetic study of tolbutamide in rats. The effect of CuE on CYP2C11 expression was determined by western blot. CuE (1.25–100 μmol L-1) competitively inhibited tolbutamide 4-hydroxylation (CYP2C11) activity only in concentration-dependent manner with a Ki value of 55.5 μmol L-1in vitro. In whole animal studies, no significant difference in metabolism/pharmacokinetic of tolbutamide was found for the single pretreatment groups. In contrast, multiple pretreatments of CuE (200 µg kg-1 d-1, 3 d, i.p.) significantly decreased tolbutamide clearance (CL) by 25% and prolonged plasma half-time (T1/2) by 37%. Moreover, CuE treatment (50–200 µg kg-1 d-1, i.p.) for 3 d did not affect CYP2C11 expression. These findings demonstrated that CuE competitively inhibited the metabolism of CYP2C11 substrates but had no effect on rat CYP2C11 expression. This study may provide a useful reference for the reasonable and safe use of herbal or natural products containing CuE to avoid unnecessary drug-drug interactions.


Funded by

National Natural Science Foundation of China(81301908)

Science and Technology Commission of Shanghai Municipality(13ZR1412600,14DZ2270100)


Acknowledgment

Acknowledgements This work was supported by the National Natural Science Foundation of China (81301908), and the Science and Technology Commission of Shanghai Municipality (13ZR1412600, 14DZ2270100).


Interest statement

Compliance and ethics The authors declare that they have no conflict of interest.


References

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  • Figure 1

    Chemical structure of cucurbitacin E.

  • Figure 2

    Representative chromatograms after multiple reaction monitoring (MRM) of tolbutamide and 4-hydroxytolbutamide in rat plasma. A, A blank plasma sample. B, A blank plasma spiked with tolbutamide, 4-hydroxytolbutamide and IS at lower limit of quantification (LLOQ) sample. C, A plasma sample collected at 60 min after caudal intravenous administration of 10 mg kg-1 tolbutamide in rats.

  • Figure 3

    (Color online) Inhibition of CYP2C11 activity by CuE in rat liver microsomes, presented as 4-hydroxytolbutamide/tolbutamide ratios. Results were x¯ ±SE of six rats. Statistical significance was determined by analysis of variance. **, P<0.01; ***, P<0.001.

  • Figure 4

    (Color online) Primary Lineweaver-Burk plot (A) and the secondary plot for Ki (B) in the inhibition of CYP2C11-mediated tolbutamide 4-hydroxylase by various concentrations of CuE (5–50 μmol L-1) in rat liver microsomes. Tolbutamide was used at concentrations of 25, 50, 75 and 100 μmol L-1. Each data point represents the mean of six rats.

  • Figure 5

    (Color online) Concentration-time profiles of (A) tolbutamide and (B) 4-hydroxytolbutamide after a single administration of CuE (50–200 μg kg-1, i.p.) after a standard dose of tolbutamide (10 mg kg-1, i.v.). Results were x ¯±SE of 5–8 rats. *, P<0.05; **, P<0.01; ***, P<0.001.

  • Figure 6

    (Color online) Concentration-time profiles of (A) tolbutamide and (B) 4-hydroxytolbutamide after a 3 d administration of CuE (50–200 μg kg-1 day-1, i.p.) after a standard dose of tolbutamide (10 mg kg-1, i.v.). Results were x ¯±SE of 5–8 rats. *, P<0.05; **, P<0.01; ***, P<0.001.

  • Figure 7

    Effects of CuE on CYP2C11 expression in the rat. Rats received 3 d pretreatment according to the following groups. A, Control. B, Carbamazepine (60 mg kg-1 d-1, i.p.). C, CuE (50 µg kg-1 d-1, i.p.). D, CuE (100 µg kg-1 d-1, i.p.). E, CuE (200 µg kg-1 d-1, i.p.). Data were expressed as the x¯±SE of six rats. *, P<0.05.

  • Table 1   Pharmacokinetics of tolbutamide (10 mg kg, i.v.) in rats after a single administration of CuE (50–200 μg kg, i.p.)

    Parameters

    Control

    Fluconazole

    60 mg kg-1

    CuE treatment

    50 μg kg-1

    100 μg kg-1

    200 μg kg-1

    T1/2 (min)

    217.36±18.97

    544.19±19.32***

    245.23±11.21

    242.43±25.34

    263.32±12.65

    Cinitial (μg mL-1)

    80.19±6.99

    75.47±2.18

    74.29±5.15

    69.64±4.20

    76.95±4.08

    AUC 0-T (min μg mL-1)

    13,867.31±782.34

    18,701.70±754.69**

    14,680.75±370.82

    14,622.76±616.34

    14,111.75±385.60

    AUC 0-∞ (min μg mL-1)

    20,208.64±1,725.75

    51,559.50±2,291.20***

    23,176.09±784.79

    23,549.27±2,430.00

    23,216.93±782.26

    Vd (mL kg-1)

    153.90±5.19

    152.86±6.61

    152.44±3.59

    148.51±3.29

    163.74±6.20

    CL (mL min-1 kg-1)

    0.51±0.04

    0.20±0.01***

    0.43±0.01

    0.44±0.04

    0.43±0.01

    MRT (min)

    139.23±2.84

    164.17±0.95***

    145.31±1.84

    144.77±2.62

    146.11±1.07

    Fluconazole (60 mg kg-1, i.p.) was used as a positive control for enzyme inhibition studies. Results were x ¯±SE of 5–8 animals. **, P<0.01; ***, P<0.001.

  • Table 2   Pharmacokinetics of tolbutamide (10 mg kg, i.v.) in rats after multiple pretreatments with CuE (50–200 μg kg d, i.p.) for 3 d

    Parameters

    Control

    Carbamazepine

    60 mg kg-1 d-1, 3 d

    CuE treatment

    50 μg kg-1 d-1,

    3 d

    100 μg kg-1 d-1,

    3 d

    200 μg kg-1 d-1,

    3 d

    T1/2 (min)

    182.26

    ±18.24

    118.09

    ±13.21*

    215.60

    ±19.55

    242.49

    ±21.74

    250.29

    ±21.85*

    Cinitial (μg mL-1)

    68.71

    ±4.55

    62.55

    ±3.23

    68.27

    ±5.84

    71.99

    ±3.89

    72.71

    ±3.29

    AUC 0-T (min μg mL-1)

    12,227.19

    ±669.35

    9,644.59

    ±658.73*

    13,056.35

    ±1,079.53

    13,463.53

    ±817.00

    14,111.86

    ±920.50

    AUC 0- (min μg mL-1)

    16,484.26

    ±1,546.06

    11,011.48

    ±996.78*

    19,669.24

    ±2,433.98

    21,120.10

    ±2,196.18

    22,190.16

    ±2,172.22

    Vd (mL kg-1)

    158.91

    ±9.07

    154.35

    ±10.07

    162.51

    ±6.67

    167.64

    ±4.25

    164.66

    ±8.16

    CL (mL min kg-1)

    0.64

    ±0.07

    0.93

    ±0.09**

    0.56

    ±0.07

    0.51

    ±0.06

    0.48

    ±0.04*

    MRT (min)

    136.00

    ±4.79

    118.00

    ±3.37*

    140.17

    ±3.10

    140.71

    ±2.06

    145.93

    ±2.76

    Carbamazepine (60 mg kg-1 d-1, i.p., 3 d) was used as a positive control for enzyme induction studies. Results were x ¯±SE of 5–8 animals. *, P<0.05; **, P<0.01.

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