Cytochrome P450 (CYP) 1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A are major factors involved in the metabolism of clinically prescribed drugs. Because the time course after drug treatment discontinuation has received little attention, we aimed to clarify the chronological changes of rifampicin-induced CYP enzyme activities after rifampicin discontinuation. Thirteen volunteers took 450 mg of rifampicin once daily, and the cocktail method, which uses caffeine, losartan, omeprazole, dextromethorphan, and midazolam as CYP-specific probes, was repeatedly used for the evaluation of CYP levels. Concentrations of probes and metabolites were determined by liquid chromatography-tandem mass spectrometry. Seven-day rifampicin administration increased CYP2C19 and CYP3A enzyme activities. The induced CYP2C19 and CYP3A activities remained elevated at 4 days after rifampicin discontinuation and returned to baseline levels 8 days after rifampicin discontinuation. CYP1A2 and CYP2D6 enzyme activities showed no significant changes, and CYP2C9 enzyme activity was increased with rifampicin administration, with a tendency toward statistical significance. Drug interactions can occur even after rifampicin discontinuation.

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A "cocktail" approach, which involves simultaneous administration of multiple CYP-specific probes, concurrently detects the activity of multiple CYP enzymes. We developed and validated a rapid and selective LC-MS/MS method for determining the plasma concentrations of 5 CYP probe drugs and metabolites (caffeine/paraxanthine, CYP1A2 substrate; losartan/losartan carboxylic acid (E3174), CYP2C9 substrate; omeprazole/5-hydroxyomeprazole, CYP2C19 substrate; dextromethorphan/dextrorphan, CYP2D6 substrate; and midazolam/1′-hydroxymidazolam, CYP3A4 substrate) by single-step extraction, followed by a single LC-MS/MS run. An Ostro 96-well plate was used for extraction of CYP substrates and metabolites from human plasma and urine. Following optimization of the chromatographic conditions, all the peaks were well separated, and retention times ranged between 4.4 and 11.7 min. The total run time for a single injection was within 13 min. The accuracy and precision values suggested that the assay had high accuracy and reliability in plasma and urine samples. No significant matrix interference was observed. To demonstrate the efficacy of this method, plasma and urine concentrations of 5 CYP probe substrates and their metabolites were determined after simultaneous oral administration of 5 drugs to 4 healthy volunteers. All the substrates and metabolites were detected over an 8 h period, and the plasma concentrations of each substrate at 8 h after administration were above the lower limit of quantification. Urine concentrations of drugs and their metabolic ratio were evaluated after the administration. In conclusion, the advantage of our cocktail approach is that it enables in vivo assessment of the activity of various drug-metabolizing enzymes in a single assay. Key words cytochrome P450; cocktail; LC-MS/MS; healthy volunteerThe CYP system, which constitutes the major drug-metabolizing machinery in humans, catalyzes the biotransformation of xenobiotics and has been reported to process over 90% of the currently marketed drugs.1,2) The drug-metabolizing activities of different CYP isoforms show high inter-and intraindividual variations; age, gender, genetics, environmental factors, dietary components, and endogenous mediators contribute to this variability. [3][4][5] In addition, certain medications can induce or inhibit the activity of drug-metabolizing enzymes, resulting in drug-drug interactions. Variation in CYP activity results in variation in the pharmacokinetics of its substrates, which in turn may alter the pharmacodynamics of these drugs. Thus, predicting the pharmacokinetics and pharmacodynamics of the drug substrates of CYP is important for predicting potential drug interactions and for CYP substrate dose optimization.A "cocktail" approach concurrently detects the activity of multiple CYP enzymes and involves simultaneous administration of multiple CYP-specific probes followed by the evaluation of their pharmacokinetics in a single experiment. Since the Pittsburgh cocktail was first introduced in 1997, 6) several cocktail...

A procedure was developed for the quantitative determination of chafuroside A, a flavone C-glycoside with potent anti-inflammatory activity, and its regioisomer chafuroside B, as well as isovitexin and vitexin, by selected reaction monitoring liquid chromatography-tandem mass spectrometry (SRM LC-MS/MS) analysis. This method was successfully applied to commercial leaves of green tea, houji tea, oolong tea, and black tea. High levels of chafurosides A and B were found in oolong tea leaves that had been heated at >140 degrees C. Next, their precursors, prechafurosides A and B, were isolated from methanol extract of oolong tea leaves prepared from Shizu 7132, Camellia sinensis (L.) O. Kuntze, by partition with n-butanol and H2O and chromatography on Diaion SP-825, Sephadex LH-20, and ODS C-18, guided by assay of chafuroside formation. Prechafurosides A and B gave chafurosides A and B, respectively, in good yields when heated at 160 degrees C for 0.5 h. Solvolysis of prechafurosides A and B with pyridine and dioxane quantitatively afforded isovitexin and vitexin, respectively. On the basis of these results and physicochemical data (MS, UV, and NMR), prechafurosides A and B were concluded to be new flavone C-glycoside sulfates, isovitexin-2''-sulfate and vitexin-2''-sulfate, respectively.

Recently, a new type of interaction has been reported in which fruit juices diminish oral drug bioavailability through inhibition of organic anion-transporting polypeptide (OATP). In this study, we aimed to clarify the duration of OATP inhibition by grapefruit juice (GFJ), and to compare it with the duration of GFJ-induced inhibition of cytochrome P450 (CYP) 3A4 activity. Seven healthy volunteers were enrolled in this open-label, single-sequence study. They were orally administered celiprolol (100 mg) and midazolam (15 µg/kg) with water on the control day. Three days later, they ingested GFJ (200 mL) 3 times a day for 3 d. On day 1, the same drugs were administered with GFJ. On days 3 and 7, the same drugs were administered with water. Pharmacokinetics of both drugs were evaluated on each trial day. The peak plasma concentration (C max ) and the area under the plasma concentration-time curve from 0 to 8 h (AUC 0-8 ) of celiprolol significantly decreased on day 1, and the mean ratios of these values and the corresponding control-day values were 0.18 and 0.25, respectively. The C max and AUC 0-8 returned to the control levels on days 3 and 7. In contrast, AUC 0-8 of midazolam were higher on days 1 and 3 than on the control day (mean ratio, 2.12 and 1.47, respectively). The AUC 0-8 returned to the control level on day 7. In conclusion, results of this study indicated that the OATP inhibition caused by GFJ dissipated faster than GFJ-mediated alterations in CYP3A4 activity, which were sustained for at least 48 h.Key words organic anion-transporting polypeptide; cytochrome P450; grapefruit juice Food and medication are often taken together. However, certain foods interact with drugs by altering mechanisms that are important determinants of systemic drug availability. In particular, grapefruit juice is known to alter the pharmacokinetics of over 30 prescription drugs by affecting their bioavailability.1-3) The mechanism of this interaction is inhibition of intestinal cytochrome P450 (CYP) 3A4 activity and increased systemic exposure of CYP3A4 substrates. [4][5][6][7][8] In 2012, the United States Food and Drug Administration issued a press release to warn consumers that the intake of grapefruit juice along with medication could cause dangerous side effects. 9)Recently, a new type of interaction has been reported in which fruit juices diminish oral drug bioavailability through inhibition of the organic anion-transporting polypeptide (OATP) uptake transporter, which is a family of membrane solute carrier (SLC) transporters. OATP influences the intestinal absorption of several drugs in clinical use. [10][11][12][13][14] Grapefruit juice decreases the plasma concentrations of some drugs including β-adrenoceptor blocking agents and antihistamines by this mechanism. For example, grapefruit juice was reported to reduce the area under the plasma concentration-time curve (AUC) value for fexofenadine to a mean value of 58% of that when the drug was administered with the corresponding volume of water.10) Furthermore, celiprolol, ...

A cocktail approach can detect the activities of multiple cytochrome P450 (CYP) isoforms following the administration of multiple CYP-specific substrates in a single experiment. This study aimed to develop a simultaneous and comprehensive in vivo analysis of CYP activity in rats. The rats received an oral administration of losartan (10 mg/kg) and omeprazole (40 mg/kg). Caffeine (1 mg/kg), dextromethorphan (10 mg/kg) and midazolam (10 mg/kg) were administered 15 min later. In the drug-interaction phase, the rats were treated orally with dexamethasone (80 mg/kg) 24 h before, or with ketoconazole (10 mg/kg), fluvoxamine (100 mg kg) or fluconazole (10 mg/kg) 1 h before the administration of cocktail drugs. The concentrations of the drugs and their metabolites were determined by LC/MS/MS. Plasma concentrations of five CYP substrates and their metabolites were simultaneously evaluated after the oral drug administration. Fluvoxamine and fluconazole significantly increased the Cmax and AUC of caffeine, and the AUC of omeprazole and midazolam. Dexamethasone significantly increased Cmax and AUC of losartan, while it decreased the Cmax of midazolam. Ketoconazole showed no significant effect on the pharmacokinetic parameters of the tested drugs. In conclusion, a cocktail approach was developed for simultaneous and comprehensive analysis of the activities of multiple CYP isoforms in rats. In this approach, the effects of inhibitors and an inducer of various CYP isoforms were examined. Although further studies are necessary to predict the effects in humans, this approach may be expected to serve as a convenient method for detecting drug-drug interactions in rats.

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