Effect of St. John's Wort extract on intestinal expression of cytochrome P4501A2: studies in LS180 cells.

Correspondence Address:
C S Karyekar
Pharmacokinetics-Biopharmaceutics Laboratory, School of Pharmacy, University of Maryland, Baltimore 21201, USA.
USA

Source of Support: None, Conflict of Interest: None

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PMID: 12215688

BACKGROUND AND AIMS: St. John's Wort (SJW) is known to induce expression and activity of cytochrome P4503A4 (CYP3A4). However, its effects on other cytochrome P450 (CYP) are not well understood. Our objective was to characterise the effect of SJW on the expression of CYP1A2 in the LS180 intestinal cell model. STUDY DESIGN AND METHODS: LS180 cells were cultured in the presence and absence of SJW extract for 48 hours. CYP1A2 protein content was measured by Western blot analysis using monoclonal antibody. Time-dependent expression of CYP1A2 was assessed during exposure to SJW extract for 24 hours and following its removal for another 24 hours. RESULTS: SJW increased the expression of CYP1A2 in the LS180 cells in a concentration dependent manner. The induction was time-dependent, as enzyme levels returned to baseline within 4-8 hours after removal of SJW. CONCLUSIONS: SJW reversibly induces expression of CYP1A2 in LS180 cells. This induction may be responsible for reduced plasma theophylline concentrations upon co-administration of SJW, as reported earlier.

Keywords: Blotting, Western, Cytochrome P-450 CYP1A2, biosynthesis,drug effects,Drug Interactions, Human, Intestinal Mucosa, drug effects,pathology,Plant Extracts, pharmacology,Sensitivity and Specificity, Time Factors, Tumor Cells, Cultured,

in vitro and in vivo studies have evaluated the effects of SJW extract on the expression and activity of hepatic CYP3A4 and the drug efflux protein P-glycoprotein (Pgp).[7],[8],[9],[10],[11] These data suggest that SJW induces CYP3A4 activity, which could explain many of these drug inte-ractions. Additionally, SJW may also induce intestinal Pgp expression and activity and influence pharmacokinetics of drugs such as indinavir and digoxin.[7],[8] Recently, a pharmacokinetic interaction between SJW and theophylline was reported, where plasma concentrations following SJW therapy were decreased.[12] Since theophylline is metabolised mainly by CYP1A2, it is plausible that induction of CYP1A2 by SJW may explain the reduced theophylline concentrations and loss of therapeutic efficacy.

Our aim was to determine the effect of SJW on CYP1A2 expression in an in vitro model of intestinal cells (LS180).

::   Material and method

LS180 cells were purchased from American Tissue Cell Culture (Rockville, MD). SJW extract was kindly gifted by Pure World Chemicals (Hackensack, NJ). This extract was reported to contain 0.3% hypericin and 5% hyperforin content, which was verified by the supplier using HPLC (Lot# OOI-3334 – actual content 0.315% hypericin and 5.61% hyperforin). Relative dilutions were made from the working solution (100x) containing 10 ?M hyperforin and 9.4 ?M hypericin. ?-naphthaflavone (BNF) was purchased from Sigma Chemicals (Fair Lawn, NJ). Monoclonal anti-CYP1A2 antibodies were purchased from Gentest Corporation (Boston, MA) and minimum essential medium (MEM) and foetal bovine serum (FBS) from Gibco (Lenexa, KS).

The LS180 cells were cultured in MEM supplemented with 10% FBS in six well tissue culture plates with 5% CO2 and 95% relative humidity. To examine the effect of SJW on the expression of CYP1A2, cells were treated with increasing concentrations of the extract (1x, 10x, and 100x) for 48 hours. Cells treated in plain medium and BNF (50 ?M), a known inducer of CYP1A2, were used as negative and positive controls, respectively. After 48 hours, the cells were lysed and CYP1A2 content was measured by Western blot analysis.

For the time of exposure experiment, LS180 cells were cultured in the same manner as mentioned above. The cells were then treated with SJW (100x) for 24 hours. Cell lysates were obtained at 4, 8, 12, 18, and 24 hours upon treatment. After 24 hours, cells were treated with SJW free medium for 24 hours and samples were then obtained at 4, 8, 12, 18, and 24 hours and analysed by Western blot.

Cell lysis was accomplished by adding 200 ?l lysis buffer containing Tris buffer, EDTA, Triton X-100, glycerol, sodium vandate, and benzamidine. The cell lysates were centrifuged and the supernatant was diluted with sample buffer. The proteins were separated on a 15 % SDS-polyacrylamide gel and transferred to a PVDF membrane. The membrane was incubated overnight in 1:1000 dilution of monoclonal CYP1A2 antibody followed by anti-mouse secondary antibody for 1 hour. A constitutional protein in cells (? tubulin) was also developed as loading control. The proteins were visualised using electrochemo-luminisence reagents. The protein bands were scanned and quantified by densitometric image-Q scanning software.

::   Results

Exposure of LS180 cells to SJW extract resulted in a concentration dependent increase in the expression of CYP1A2 [Figure - 1]. Protein expression increased by 2, 30, and 90% at 1x, 10x, and 100x SJW concentrations, respectively, compared to baseline [Figures:1,2]. Marked induction of CYP1A2 was observed in the presence of BNF (positive control).

[Figure - 3] demonstrates the expression of CYP1A2 in LS180 cells at various time points upon exposure to SJW extract and after its removal. There was a time dependent increase in the expression of CYP1A2 up to 24-hours. After removal of SJW, there was rapid reversal of the CYP1A2 expression to baseline within 4 - 8 hours.

::   Discussion

SJW is widely used for treating depression, mood swings and anxiety. Since this herbal product is not regulated by federal drug approval agencies, self-medication with SJW may result in the potential for drug interactions. Due to its natural origin it is largely considered to be safe and free of side effects. However, co-administration with many conventional medications can result in treatment failure or side effects.[6] Recent studies have shown that SJW induces expression and activity of CYP3A4 in the liver and likely induces Pgp in the intestine.[9],[10] Whether SJW has the capacity to induce CYP enzymes other than CYP3A4 is unclear.

Our results suggest that SJW induces CYP1A2 expression in an in vitro model of intestinal cells (LS180). These results may explain a previous report that showed a reduction in plasma theophylline concentrations upon co-administration of the extract observed in a woman taking theophylline along with SJW.[12]

The LS180 cell line used in these experiments is a derivative of human colon cancer cells with constitutional and inducible expression of various CYP enzymes.[9] CYP3A4 induction has been demonstrated in these cells by drugs such as rifampicin, verapamil, erythromycin, which have also shown to induce CYP3A4 in humans.[13] Additionally, this cell line was used to study the induction of CYP1A2 after treatment with 3-methylcholanthrene, known inducer of the enzyme.[14] As noted in [Figure - 1], there was up to 90% induction of CYP1A2 protein by SJW. According to Biber et al, the maximum hyperforin plasma levels reached following a single dose of SJW is approximately 0.3 mM.[15] However, it is possible that the intestinal mucosa is exposed to much higher concentrations of hyperforin (> 10 mM) compared to plasma concentrations. We realize that a limitation of our study is that SJW concentrations used may not represent the concentrations obtained in vivo, and that the effect of metabolites on CYP1A2 expression was not investigated. These limitations may partly explain the recent observations of Wang et al, where the effect of SJW on CYP1A2, 2C9, 2D6 and 3A4 in healthy volunteers was assessed using in vivo probes.[11] The study demonstrated no apparent induction of the CYP1A2 as measured by caffeine pharmacokinetics. These results are discordant with our findings, and could be explained by differences in SJW content among products, or other components of the extract that may be responsible for enzyme induction. In some patients, there was a trend towards lower Cmax and higher caffeine metabolism suggesting that enzyme induction may have occurred in a subset of patients. A recent report by Bray et al failed to show induction of CYP1A2 in liver microsomes upon exposure to SJW (four-day IP administration) in male Swiss Webster mice.[16] However, it is unlikely that the intraperitoneal route of administration would alter intestinal CYP1A2 content, which was not assessed in the study. Moreover, although most extracts are standardized for hypericin or hyperforin content, further research is required to identify other possible extract components that may contribute to enzyme induction.

We also evaluated the relationship between CYP1A2 expression and duration of exposure to SJW. Induction of CYP1A2 upon treatment with SJW and subsequent disappearance upon removal of the extract strongly suggests a direct causal relationship. In addition, the rapid decline in CYP1A2 expression after removal of the extract suggests that this induction is transient. The time required for enzyme levels to return to baseline in vitro (4-8 hours) may be reflective of the conditions in vivo. In this case, it may be possible to minimise SJW-drug interactions by allowing sufficient time between doses.

In conclusion, exposure of LS180 cells to SJW extract resulted in reversible induction of CYP1A2. This model is well suited to study the herb-drug interactions and CYP1A2 expression.

::   Acknowledgments

The authors would like to thank Dr. Paul Shapiro (University of Maryland) for assisting in conduction of the Western blots and Dr. Kan He and Dr. Qunyi Zheng, Pure World Inc (South Hackensack, NJ) for their kind gift of the St. John’s Wort extract.

 

:: References

1.	Lin JH, Lu AY. Inhibition and induction of cytochrome P-450 and clinical implications. Pharmacotherapy 1998;18:84-112.
2.	Kolars JC, Awni WM, Merion RM, Watkins PB. First-pass metabolism of cyclosporin by gut. Lancet 1995;338:1488-90.
3.	Gomez DY, Wacher VJ, Tomalanovich SJ, Benet LZ. Effect of ketoconazole on intestinal metabolism and bioavailability of cyclsopsorine. Clin Pharmacol Ther 1995;58:15-9.
4.	Smith DA, Abel SM, Hyland R, Jones BC. Human cytochrome P450s: selectivity and measurement in vivo. Xenobiotica 1998;28:1095-128.
5.	Nahrstedt A, Butterweck V. Biologically active and other chemical constituents of the herb of Hypericum perforatum L. Pharmacopsy-chiat 1997;30:129-34.
6.	Izzo AA, Ernst E. Interactions between herbal medicines and prescribed drugs: a systematic review. Drugs 2001;61:2163-75.
7.	Durr D, Stieger B, Kullal-Ublick GA, Rentsch KM, Steinert HC, Meier PJ, et al. St. John’s Wort induces intestinal P-glycoprotien/MDR1 and intestinal and hepatic CYP3A4. Clin Pharmacol Ther 2000;68:598-604.
8.	Hennessy M, Kelleher D, Spiers JP, Barry M, Kavanagh P, Back D, et al. St John’s Wort increases expression of P-glycoproetin: implications for drug interactions. Br J Clin Pharmacol 2002;53:75-82.
9.	Roby CA, Anderson GD, Kantor E, Dryer DA, Burstein AH. St. John’s Wort: Effect on CYP4A3 activity. Clin Pharmacol Thera 2000;67:451-57.
10.	Moore LB, Goodwin B, Jones SA, Wisley GB, Singh CJ, Wilson TM, et al. St. John’s Wort induces hepatic drug metabolism through activation of pregnane X receptor. PNAS 2000;97:7500-02.
11.	Wang Z, Gorski C, Hamman MA, Huang S, Lesko LJ, Hall SD. The effects of St John’s wort (hypericum perforatum) on human cytochrome P450 activity. Clin Pharmacol Ther 2001;70:317-26.
12.	Nebel A, Schneider BJ, Baker RK, Kroll DJ. Potential metabolic interaction between St. John’s Wort and theophylline. Ann Pharmacother 1999;33:502.
13.	Scheutz E, Beck W, Scheutz J. Modulators of p-glycoprotein and CYP3A are co-ordinately up-regulate these proteins in human colon cancer cells. Mol Pharmacol 1996;49:311-18.
14.	Li W, Harper PA, Tang BK, Okey AB. Regulation of cytochrome P450 enzymes by aryl hydrocarbon receptor in human cells: CYP1A2 expression in the LS180 colon carcinoma cell line after treatment with 2,2,8-tetrachlorodibenzo-p-digoxin or 3-methylcholanthrene. Biochem Pharmacol 1998;56:599-612.
15.	Biber A, Fischer H, Romer A, Chatterjee SS. Oral bioavailability of hyperforin from hypericum extracts in rats and human voluenteers. Pharmacopsychiatry 1998;31:36-43.
16.	Bray BJ, Brennan NJ, Perry NB, Menkes DB, Rosengren RJ. Short term treatment with St. John’s wort, hypericin or hyperforin fails to induce CYP450 isoforms in the Swiss Webster mouse. Life Sci 2002; 70:1325-35.

    Figures

[Figure - 1], [Figure - 2], [Figure - 3]

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