* Department of Pharmacology and Toxicology, Adams Building Room 238, Great King St., University of Otago Medical School, Dunedin, New Zealand;
New Zealand Institute for Crop and Food Research Ltd. and
Department of Psychological Medicine, University of Otago, Dunedin, New Zealand
Received August 17, 2001; accepted November 2, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: St. John's wort; UDPGT; CYP3A; CYP2E1; CYP1A.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the last 23 years, drug interactions linked to the use of SJW have been reported (Ernst, 1999; Fugh-Berman, 2000
; Johne et al., 1999
; Nebel et al., 1999
; Piscitelli et al., 2000
; Ruschitzka et al., 2000
). Many of the case studies and clinical trials report interactions with SJW and drugs that are substrates of cytochrome P4503A (CYP3A). However, there is limited direct evidence to support the theory that these drug interactions have resulted from CYP3A induction. Experimentally, there has only been one in vitro (Moore et al., 2000
) and one in vivo study (Durr et al., 2000
), and two clinical trials (Durr et al., 2000
; Roby et al., 2000
) that have provided evidence of CYP3A induction by SJW. Additionally, there is no evidence that SJW alters CYP450 activity following treatment of less than 7 days duration. Specifically, SJW (280 mg/kg/day, 4 days) failed to increase CYP450 isoforms in the mouse (Bray et al., in press
) and did not change the metabolism of dextramethorphan or alprazolam when administered to healthy volunteers (300 mg, 3 times a day for 3 days; Markowitz et al., 2000
).
Since the majority of the literature to date has reported interactions of SJW with substrates of CYP3A (Ernst, 1999; Fugh-Berman, 2000
; Piscitelli et al., 2000
; Ruschitzka et al., 2000
), information regarding the effect of SJW on the activity of CYP3A is vital. Therefore, the aim of this study was to determine the minimum duration of SJW administration required to alter the catalytic activity and polypeptide levels of hepatic CYP3A in male Swiss Webster mice. Since the function and regulation of CYP3A is highly conserved among mammalian species (Maurel, 1996
), a reasonable comparison will be able to be made between the results obtained in rodents and the existing human data.
A potential interaction between SJW and theophylline, a substrate metabolized by both CYP3A and CYP1A, has also been reported (Nebel et al., 1999) and thus determining the effect of SJW on other isoforms of hepatic CYP450 is also important. Therefore, CYP1A and CYP2E1 were also examined for alterations in catalytic activity and polypeptide levels following SJW administration. Additionally, previous reports have demonstrated that changes in drug conjugation can result in biologically significant drug interactions (Bray and Rosengren, 2001
; Douidar and Ahmed, 1987
). Therefore, it is possible that the drug interactions reported with indinavir, cyclosporine A, and oral contraceptives following SJW ingestion (Ernst, 1999
; Fugh-Berman, 2000
; Piscitelli et al., 2000
; Ruschitzka et al., 2000
) could have resulted from an increase in the rate of drug conjugation. Accordingly, the ability of SJW to alter the catalytic activity of UDP-glucuronosyltransferase (UDPGT), a major conjugation enzyme, was also measured.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
St. John's wort extraction and dosing.
St. John's wort extract HyperiFinTM, containing 0.3% total hypericins (ratio pseudohypericin:hypericin 2.5:1) and 2.3% hyperforin (3.6% hyperforin homologues and adhyperforin) was supplied by Finzelberg GmbH & Co. KG (Andernach, Germany). This SJW preparation was chosen as it has been well characterized (Kurth and Spreemann, 1998). The manufacturer's claims for composition were confirmed by HPLC analyses using a Waters HPLC (Milford, MA) system, and a Merck (Darmstadt, Germany) Lichrospher 100 RP-18 column (250 x 10 mm). SJW was extracted from the silica carrier by dissolution with methanol (9 ml, 10 min ultrasonication) and filtering. The SJW was then dried and dissolved in a corn oil vehicle. Samples were stored at 20°C and thoroughly vortexed before administration. SJW was administered at 280 mg/kg as this dose contains 10 mg/kg of hyperforin, which was previously shown to exhibit antidepressant effects in rats (Chatterjee et al., 1998
; Kaehler et al., 1999
). SJW was also administered at a lower dose (140 mg/kg), which is approximately 10-fold greater than that routinely used in clinical investigations (300 mg, tid).
Animals.
Male Swiss Webster mice (7 weeks of age, 22 g) were purchased from AgResearch (Hamilton, NZ). Mice were housed in plastic cages on shredded paper bedding and had constant access to rodent diet and water. They were maintained at 2124°C on a 12 h light/dark cycle in an approved animal care facility. Mice were dosed with St. John's wort extract (140 mg/kg or 280 mg/kg, po), or corn oil vehicle (5 ml/kg, po) for 1, 2, or 3 weeks. Necropsies were performed 24 h following the end of the treatment period. Positive control groups consisted of dexamethasone (75 mg/kg, ip) administered for 4 days, ß-naphthoflavone (80 mg/kg, ip) administered for 3 days, phenobarbital (80 mg/kg, ip) administered for 3 days, and acetone (4.8 mg/kg, po) administered 16 h prior to necropsy. There were 8 mice in each of the treatment groups.
Evaluation of hepatic injury.
Immediately after euthanasia with carbon dioxide, blood was collected from the inferior vena cava and placed on ice. Plasma alanine aminotransferase (ALT) activity was measured as an indicator of hepatic injury and was determined kinetically with a commercially available kit. The results are expressed as international units per liter (IU/l).
CYP450 Assays
Hepatic microsomes were prepared by differential centrifugation (Guengerich, 1989) and the protein was immediately determined by the bicinchoninic acid method (Smith et al., 1985
). Microsomes were stored at 80°C until utilized in the following catalytic assays.
CYP1A catalytic activity.
Ethoxyresorufin O-deethylation was used as a selective probe for changes in the catalytic activity of CYP1A (Ryan and Levin, 1990) and was performed as described previously (Bray et al., 2001
). Results are expressed as nmol/mg/min.
CYP2E1 catalytic activity.
p-Nitrophenol hydroxylation was used as a selective probe for changes in the catalytic activity of CYP2E1 (Koop et al., 1985) and was performed as described previously (Bray and Rosengren, 2001
). Results are expressed as nmol/mg/min.
CYP3A catalytic activity.
Erythromycin N-demethylation was used as a selective probe for changes in the catalytic activity of CYP3A (Wrighton et al., 1985) and was performed as described previously (Bray et al., 2001
). Results are expressed as nmol/mg/min.
Conjugation Assays
p-Nitrophenol conjugation.
The catalytic activity of UDPGT was determined by measuring p-nitrophenol glucuronidation (Fowler et al., 1994). The reaction mixture contained 1 mg microsomal protein, 0.25 M sodium phosphate buffer, pH 7.1, 3.5 mM UDPGA and 0.15 mM pNP in a final volume of 0.7 ml. The samples were preincubated in a shaking water bath for 2 min at 37°C and the reaction was initiated by the addition of UDPGA. After 10 min the reaction was terminated by the addition of 2 ml 10% TCA and the samples were centrifuged. After centrifugation, 0.05 ml 10 N NaOH was added to 1 ml of the resulting supernatant and the absorbance at 400 nm was read immediately. Results are expressed as nmol/mg/min.
Electrophoresis and Western blotting.
Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described (Laemmli, 1970). Briefly, 10 µg of microsomal protein was loaded onto wells of a 10% polyacrylamide gel. CYP1A, CYP2E1, and CYP3A polypeptide levels were quantified by Western immunoblotting as described (Towbin et al., 1979
). Nitrocellulose membranes were incubated with rabbit-antirat primary antibody (either CYP1A, CYP2E1, or CYP3A), and the intensity of the bands was determined as described previously (Bray and Rosengren, 2001
).
Statistical analysis.
Individual groups were analyzed using a 2-way ANOVA coupled with the Student-Newman-Keuls post-hoc test with p < 0.05 as the minimum requirement for a statistically significant difference.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dose-response and time-course experiments demonstrated that SJW failed to alter the catalytic activity or polypeptide levels of any of the CYP450 isoforms examined when administered for up to 2 weeks. Additionally, there was no change in UDPGT catalytic activity following all doses of SJW. However, after 3 weeks of administration, SJW increased the catalytic activity and polypeptide levels of both CYP3A (a 2-fold increase in catalytic activity and a 6-fold increase in immunoreactive protein) and CYP2E1 (2-fold increase in catalytic activity and a 2.5-fold increase in immunoreactive protein), regardless of the dose administered.
Previous studies in humans, which examined the effect of SJW on CYP3A activity, have shown no change in this parameter when 7 healthy volunteers were administered SJW (300 mg, tid) for less than 7 days (Markowitz et al., 2000). However, when SJW (300 mg) was taken by 13 human subjects 3 times a day for 14 days, CYP3A4 was increased as measured by urinary 6-ß-hydroxycortisol/cortisol ratios (Roby et al., 2000
). The results showed that SJW increased this ratio from a baseline of 7.1 ± 4.5 to 13 ± 4.9 (Roby et al., 2000
). This increase was seen in all but 1 subject and the increase in 6-ß-hydroxycortisol/cortisol ratios ranged from 0.14 to 2.59-fold (Roby et al., 2000
). Additionally, Durr et al. (2000) reported a 1.4-fold increase in the functional activity of CYP3A and a 1.4-fold increase in intestinal P-glycoprotein expression following SJW (300 mg, tid) administration for 2 weeks in 8 healthy male volunteers. Therefore, it appears from these 2 clinical investigations and our results that repeated dosing for extended periods is required before changes in CYP450 activity are observed. However, the clinical studies performed to date are limited as they analyzed sample sizes of less than 15 individuals.
One rodent study has demonstrated that SJW (1 g/kg, po), when administered to rats for 14 days, resulted in a 2.5-fold increase in the expression of hepatic CYP3A and a 3.8-fold increase in the expression of intestinal P-glycoprotein, as determined by Western immunoblotting (Durr et al., 2000). Interestingly, these results were tissue-specific as SJW had no effect on the expression of intestinal CYP3A or hepatic P-glycoprotein. This study is limited by the fact that it was necessary to house the rats in the dark throughout the experiment to prevent phototoxicity. Therefore, the extremely high dose of SJW has tempered the significance of these results.
One study in primary human hepatocytes has demonstrated that both SJW and hyperforin induced CYP3A4 mRNA via activation of the orphan nuclear pregnane X receptor (PXR; Moore et al., 2000). Activation of PXR was achieved in CV-1 cells transfected with expression plasmids for human PXR. Three commercial SJW extracts (775 µg/ml) and hyperforin (1 µM) were tested in this system. All 4 displayed a 6 to 7-fold increase in the activation of PXR (Moore et al., 2000
). Further studies revealed that the 3 SJW preparations and hyperforin induced CYP3A4 mRNA expression in primary human hepatocytes. The results showed that all 4 treatments produced an increase in CYP3A4 mRNA levels following a 30 h incubation period (Moore et al., 2000
). Since pure hyperforin was as effective as SJW in the activation of PXR and CYP3A4 mRNA, the authors concluded that hyperforin was responsible for much, if not all, of this activity. However, no indication was given as to whether the level of hyperforin in the 3 SJW extracts was equivalent to the dose of isolated hyperforin tested.
As a whole, the in vivo results demonstrate that SJW when administered for 23 weeks increases CYP3A (in mice, rats and humans) and intestinal P-glycoprotein/Mdr1 (in rats and humans). However, the increases in the catalytic activity of CYP3A are quite modest in both the rodent and human investigations and range from 0.14 to 2.5-fold. These studies all administered SJW preparations that contained 0.3% hyperforin. Therefore, a comparison of the results can be made despite the lack of specific data with regard to the CYP3A induction potential of isolated hyperforin.
CYP1A catalytic activity and polypeptide levels were unchanged following 3 weeks of SJW administration in mice. Since the results obtained for CYP3A catalytic activity in the mouse closely resembles those reported from clinical investigations, it is possible that CYP1A activity following SJW will be similar in both species. If so, the interactions between SJW and theophylline reported by Nebel et al. (1999) would be mediated through CYP3A induction only. However, clinical investigations have not examined CYP1A activity following SJW and this must be conducted before conclusions are made regarding SJW's effect on this enzyme in humans.
There have been no previous reports of SJW altering CYP2E1 activity, but this study has demonstrated that the catalytic activity and polypeptide levels of CYP2E1 were increased in the mouse following 3 weeks of SJW administration. While the clinical significance of drug interactions with CYP2E1 is less important than with CYP3A, it is relevant to characterize changes in CYP2E1 as overexpression of this isoform has been shown to increase reactive oxygen species and elicit toxicity in the absence of added toxicant (Nieto et al., 1999; Wu and Cederbaum, 2001
). Moreover, CYP2E1 activity can also be induced by changes in normal physiological parameters such as fasting and diabetes (Hong et al., 1987
; Ioannides et al., 1996
). Therefore, it is possible that SJW may interact with both disease states and substrates for CYP2E1 such as acetaminophen, chloroform, and vinyl chloride (Brady et al., 1989
; Raucy et al., 1989
).
In conclusion, this is the first in vivo study to demonstrate changes in CYP450 isoforms following moderate doses of SJW. While no single preclinical rodent study can predict with certainty clinical drug interactions, our results in conjunction with clinical studies (Durr et al., 2000; Roby et al., 2000
) demonstrate that SJW use for 23 weeks increases CYP3A catalytic activity and polypeptide levels. Additionally, SJW did not alter CYP1A or UDPGT activity in the mouse, while CYP2E1 catalytic activity and polypeptide levels was increased 2-fold. No other rodent or human study has examined these parameters. Therefore, these results provide a framework for elucidating the species-specific response of CYP3A, CYP2E1, and CYP1A to SJW. Since SJW has increased CYP3A in multiple species, future studies should focus on determining the specific constituent(s) responsible for this effect.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brady, J. F., Li, D., Ishizaki, H., Lee, M., Ning, S. M., Xiao, F., and Yang, C. S. (1989). Induction of cytochromes P450IIE1 and P450IIB1 by secondary ketones and the role of P450IIE1 in chloroform metabolism. Toxicol. Appl. Pharmacol. 100, 342349.[ISI][Medline]
Bray, B. J., Brennan, N. J., Perry, N. B., Menkes, D. B., and Rosengren, R. J. (in press). Short term treatment with St. John's wort extract, hypericin or hyperforin fails to induce CYP450 isoforms in the Swiss Webster Mouse. Life Sci.
Bray, B. J., Goodin, M. G., Inder, R. E., and Rosengren, R. J. (2001). The effect of retinol on hepatic and renal drug-metabolising enzymes. Food Chem. Toxicol. 39, 19.[ISI][Medline]
Bray, B. J., and Rosengren, R. J. (2001). Retinol potentiates acetaminophen-induced hepatotoxicity in the mouse: Mechanistic studies. Toxicol. Appl. Pharmacol. 173, 129136.[ISI][Medline]
Chatterjee, S. S., Bhattacharya, S. K., Wonnemann, M., Singer, A., and Muller, W. E. (1998). Hyperforin as a possible antidepressant component of hypericum extracts. Life Sci. 63, 499510.[ISI][Medline]
Douidar, S. M., and Ahmed, A. E. (1987). A novel mechanism for the enhancement of acetaminophen hepatotoxicity by phenobarbital. J. Pharmacol. Exper. Ther. 240, 578583.[Abstract]
Durr, D., Stieger, B., Kullak-Ublick, G. A., Rentsch, K. M., Steinert, H. C., Meier, P. J., and Fattinger, K. (2000). St John's Wort induces intestinal P-glycoprotein/MDR1 and intestinal and hepatic CYP3A4. Clin. Pharmacol. Ther. 68, 598604.[ISI][Medline]
Ernst, E. (1999). Second thoughts about safety of St. John's wort. Lancet 354, 20142016.[ISI][Medline]
Fowler, B., Kleinow, K., Squibb, K., Lucier, G., and Hayes, A. (1994). Organelles as tools in toxicology. In Principles and Methods of Toxicology (A. W. Hayes, Ed.), pp. 12011230. Raven Press, New York.
Fugh-Berman, A. (2000). Herb-drug interactions. Lancet 355, 134138.[ISI][Medline]
Guengerich, F. (1989). Enzyme assay and purification. In Principles and Methods of Toxicology (A. W. Hayes, Ed), pp. 777814. Raven Press, New York.
Holzl, J., and Ostrowski, E. (1987). Johanniskraut (Hypericum perforatum L.) HPLC-analyse der wichtigen inhaltsstoffe und deren variabilitat in einer population. Dtsch. Apoth. Ztg. 127, 12271230.
Hong, J. Y., Pan, J. M., Gonzalez, F. J., Gelboin, H. V., and Yang, C. S. (1987). The induction of a specific form of cytochrome P 450 (P 450j) by fasting. Biochem. Biophys. Res. Comm. 142, 10771083.[ISI][Medline]
Ioannides, C., Barnett, C., Irizar, A., and Flatt, P. (1996). Expression of Cytochrome P450 proteins in disease. In Cytochromes P450: Metabolic and Toxicological Aspects (C. Ioannides, Ed.), pp. 301327. CRC Press, Boca Raton, FL.
Jensen, A. G., Hansen, S. H., and Nielsen, E. O. (2001). Adhyperforin as a contributor to the effect of Hypericum perforatum L. in biochemical models of antidepressant activity. Life Sci. 68, 15931605.[ISI][Medline]
Johne, A., Brockmoller, J., Bauer, S., Mauer, A., Langheinrich, M., and Roots, I. (1999). Pharmacokinetic interaction of digoxin with an herbal extract from St John's wort (Hypericum perforatum). Clin. Pharmacol. Ther. 66, 338345.[ISI][Medline]
Josey, E. S., and Tackett, R. L. (1999). St John's Wort: A new alternative for depression? Int. J. Clin. Pharmacol. Ther. 37, 111119.[ISI][Medline]
Kaehler, S. T., Sinner, C., Chatterjee, S. S., and Philippu, A. (1999). Hyperforin enhances the extracellular concentrations of catecholamines, serotonin and glutamate in the rat locus coeruleus. Neurosci. Lett. 262, 199202.[ISI][Medline]
Koop, D. R., Laethem, C. L., and Tierney, D. J. (1985). The utility of p-nitrophenol hydroxylation in P450IIE1 analysis. Drug Metab. Rev. 20, 54152.
Kurth, H., and Spreemann, R. (1998). Phytochemical characterization of various St. John's Wort extracts. Adv. Ther. 15, 117128.[ISI][Medline]
Laakmann, G., Schule, C., Baghai, T., and Kieser, M. (1998). St. John's wort in mild to moderate depression: The relevance of hyperforin for clinical efficacy. Pharmacopsychiatry 31(Suppl. 1), 5459.[ISI][Medline]
Laemmli, U. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[ISI][Medline]
Markowitz, J. S., DeVane, C. L., Boulton, D. W., Carson, S. W., Nahas, Z., and Risch, S. C. (2000). Effect of St. John's wort (Hypericum perforatum) on cytochrome P-450 2D6 and 3A4 activity in healthy volunteers. Life Sci. 66, 133139.[ISI][Medline]
Maurel, P. (1996). The CYP3 family. In Cytochromes P450: Metabolic and Toxicological Aspects (C. Ioannides, Ed.), pp. 241270. CRC Press, Boca Raton, FL.
Meier, B. (2001). Comparing phytopharmaceuticals: The example of St. John's Wort. Adv. Ther. 18, 3546.[ISI][Medline]
Moore, L. B., Goodwin, B., Jones, S. A., Wisely, G. B., Serabjit-Sing, C. J., Willson, T. M., Collins, J. L., and Kliewer, S. A. (2000). St. John's wort induces hepatic drug metabolism through activation of the pregnane X receptor. Proc. Natl. Acad. Sci. U.S.A. 97, 75007502.
Nebel, A., Schneider, B. J., Baker, R. K., and Kroll, D. J. (1999). Potential metabolic interaction between St. John's Wort and theophylline. Ann. Pharmacother. 33, 502.
Nieto, N., Friedman, S. L., Greenwel, P., and Cederbaum, A. I. (1999). CYP2E1-mediated oxidative stress induces collagen type I expression in rat hepatic stellate cells. Hepatology 30, 987996.[ISI][Medline]
Piscitelli, S. C., Burstein, A. H., Chaitt, D., Alfaro, R. M., and Falloon, J. (2000). Indinavir concentrations and St. John's wort. Lancet 355, 547548.[ISI][Medline]
Raucy, J. L., Lasker, J. M., Lieber, C. S., and Black, M. (1989). Acetaminophen activation by human liver cytochromes P450IIE1 and P450IA2. Arch. Biochem. Biophys, 271, 270283.[ISI][Medline]
Roby, C. A., Anderson, G. D., Kantor, E., Dryer, D. A., and Burstein, A. H. (2000). St John's Wort: Effect on CYP3A4 activity. Clin. Pharmacol. Ther. 67, 451457.[ISI][Medline]
Ruschitzka, F., Meier, P. J., Turina, M., Luscher, T. F., and Noll, G. (2000). Acute heart transplant rejection due to Saint John's wort. Lancet 355, 548549.[ISI][Medline]
Ryan, D. E., and Levin, W. (1990). Purification and characterization of hepatic microsomal cytochrome P-450. Pharmacol. Ther. 45, 153239.[ISI][Medline]
Singer, A., Wonnemann, M., and Muller, W. E. (1999). Hyperforin, a major antidepressant constituent of St. John's wort, inhibits serotonin uptake by elevating free intracellular Na+1. J. Pharmacol. Exp. Ther. 290, 13631368.
Smith, P., Kohn, R., Hermanson, G., Mallia, A., Gartner, F., Provenzano, M., Fujimoto, E., Goeke, N., Olsen, B., and Klank, D. (1985). Measurement of protein using bicinchoninic acid. Annu. Biochem. 150, 7685.
Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 43504354.[Abstract]
Wills, R. B. H., Bone, K., and Morgan, M. (2000). Herbal products: Active constituents, modes of action and quality control. Nutr. Res. Rev. 13, 4777.[ISI]
Wrighton, S. A., Schuetz, E. G., Watkins, P. B., Maurel, P., Barwick, J., Bailey, B. S., Hartle, H. T., Young, B., and Guzelian, P. S. (1985). Demonstration in multiple species of inducible hepatic cytochromes P450 and their mRNAs related to the glucocorticoid-inducible cytochrome P450 of the rat. Mol. Pharmacol. 28, 312321.[Abstract]
Wu, D., and Cederbaum, A. I. (2001). Removal of glutathione produces apoptosis and necrosis in HepG2 cells overexpressing CYP2E1. Alcohol Clin. Exp. Res. 25, 619628.[ISI][Medline]