Effect of Tacrolimus Formulation (Prolonged-Release vs Immediate-Release) on Its Susceptibility to Drug-Drug Interactions with St. John's Wort.

Tacrolimus is metabolized by cytochrome P450 3A (CYP3A) and is susceptible to interactions with the CYP3A and P-glycoprotein inducer St. John's Wort (SJW). CYP3A isozymes are predominantly expressed in the small intestine and liver. Prolonged-release tacrolimus (PR-Tac) is largely absorbed in distal intestinal segments and is less susceptible to CYP3A inhibition. The effect of induction by SJW is unknown. In this randomized, crossover trial, 18 healthy volunteers received single oral tacrolimus doses (immediate-release [IR]-Tac or PR-Tac, 5 mg each) alone and during induction by SJW. Concentrations were quantified using ultra-high performance liquid chromatography coupled with tandem mass spectrometry and non-compartmental pharmacokinetics were evaluated. SJW decreased IR-Tac exposure (area under the concentration-time curve) to 73% (95% confidence interval 60%-88%) and maximum concentration (Cmax ) to 61% (52%-73%), and PR-Tac exposure to 67% (55%-81%) and Cmax to 69% (58%-82%), with no statistical difference between the 2 formulations. The extent of interaction appeared to be less pronounced in volunteers with higher baseline CYP3A4 activity and in CYP3A5 expressors. In contrast to CYP3A inhibition, CYP3A induction by SJW showed a similar extent of interaction with both tacrolimus formulations. A higher metabolic baseline capacity appeared to attenuate the extent of induction by SJW.

The phytoestrogens daidzein and equol inhibit the drug transporter BCRP/ABCG2 in breast cancer cells: potential chemosensitizing effect.

PURPOSE: The soy isoflavone genistein has been described to up-regulate breast cancer resistance protein (BCRP) and, thus, enhance chemoresistance in breast cancer cells. The aim of this work was to assess the effect of long- and short-term incubation with daidzein, the second most abundant soy isoflavone and its metabolite equol on the expression and activity of P-glycoprotein, multidrug resistance-associated proteins 1 and 2 (MRP1 and MRP2) and BCRP in breast cancer cells. METHODS: MCF-7 and MDA-MB-231 cells were treated with phytoestrogen concentrations within the range achieved in individuals with a high isoflavone intake. Transporter expression was evaluated at protein and mRNA level through western blot and qRT-PCR, respectively. Transporter activity was determined using doxorubicin, mitoxantrone and carboxy-dichlorofluorescein as substrates. RESULTS: Daidzein (5 µM) up-regulated MRP2- and down-regulated MRP1 protein expressions in MCF-7 and MDA-MB-231 cells, respectively. Both effects were ER-dependent, as determined using the antagonist ICI 182,780. The decrease in MRP1 mRNA in MDA-MB-231 cells indicates a transcriptional mechanism. On the contrary, MRP2 induction in MCF-7 cells takes place post-transcriptionally. Whereas changes in the transporter expression had a minor effect on the transporter activity, acute incubation with daidzein, R-equol and S-equol led to a strong inhibition of BCRP activity and an increase in the IC50 of BCRP substrates. CONCLUSIONS: In contrast to previous reports for genistein, daidzein and equol do not provoke a major up-regulation of the transporter expression but instead an inhibition of BCRP activity and sensitization to BCRP substrates.

Herb⁻Drug Interaction Potential of Anti-Borreliae Effective Extracts from Uncaria tomentosa (Samento) and Otoba parvifolia (Banderol) Assessed In Vitro.

Samento (extract from Uncaria tomentosa) and Banderol (extract from Otoba parvifolia) have been demonstrated to have anti-inflammatory and antimicrobial properties, e.g., against different morphological forms of Borrelia burgdorferi. However, there is hardly any data on the pharmacological safety of these two herbal medicines. This in vitro study aimed at scrutinizing their possible characteristics as perpetrators in pharmacokinetic herbal⁻drug interactions. Inhibition of cytochrome P450 enzymes (CYPs) was quantified by commercial kits and inhibition of drug transporters by use of fluorescent probe substrates. Induction was quantified by real-time RT-PCR and activation of pregnane x receptor (PXR) and aryl hydrocarbon receptor (AhR) by reporter gene assays. Organic anion transporting polypeptide 1B1 (OATP1B1) (IC50 = 0.49 ± 0.28%) and OATP1B3 (IC50 = 0.65 ± 0.29%) were potently inhibited by Banderol, but only weakly by Samento. CYP3A4 was inhibited about 40% at a Samento concentration of 1%. Samento significantly induced mRNA expression of CYP2J2, UGT1A3, UGT1A9, ABCB1, and SLCO1B1 and strongly activated PXR, but hardly AhR. In conclusion, the perpetrator profiles of Samento and Banderol for herb⁻drug interactions completely differ. Clinical studies are strongly recommended to clarify whether the effects observed in vitro are of clinical relevance.

The phytoestrogen genistein enhances multidrug resistance in breast cancer cell lines by translational regulation of ABC transporters.

Breast cancer is the most frequent malignancy in women. Multidrug resistance due to overexpression of ABC drug transporters is a common cause of chemotherapy failure and disease recurrence. Genistein (GNT) is a phytoestrogen present in soybeans and hormone supplements. We investigated the effect of GNT on the expression and function of ABC transporters in MCF-7 and MDA-MB-231 breast cancer cell lines. Results demonstrated an induction at the protein level of ABCC1 and ABCG2 and of ABCC1 in MCF-7 and MDA-MB-231, respectively. MCF-7 cells showed a concomitant increase in doxorubicin and mitoxantrone efflux and resistance, dependent on ABCG2 activity. ABCC1 induction by GNT in MDA-MB-231 cells modified neither drug efflux nor chemoresistance due to simultaneous acute inhibition of the transporter activity by GNT. All inductions took place at the translational level, as no increment in mRNA was observed and protein increase was prevented by cycloheximide. miR-181a, already demonstrated to inhibit ABCG2 translation, was down-regulated by GNT, explaining translational induction. Effects were independent of classical estrogen receptors. Results suggest potential nutrient-drug interactions that could threaten chemotherapy efficacy, especially in ABCG2-expressing tumors treated with substrates of this transporter.

The effect of induction of CYP3A4 by St John's wort on ambrisentan plasma pharmacokinetics in volunteers of known CYP2C19 genotype.

To evaluate the impact of CYP2C19 polymorphisms on ambrisentan exposure and to assess its modification by St. John's wort (SJW), 20 healthy volunteers (10 CYP2C19 extensive, four poor and six ultrarapid metabolizers) received therapeutic doses of ambrisentan (5 mg qd po) for 20 days and concomitantly SJW (300 mg tid po) for the last 10 days. To quantify changes of CYP3A4 activity, midazolam (3 mg po) as a probe drug was used. Ambrisentan pharmacokinetics was assessed on days 1, 10 and 20, and midazolam pharmacokinetics before and on days 1, 10, 17 and 20. At steady state, ambrisentan exposure was similar in extensive and ultrarapid metabolizers but 43% larger in poor metabolizers (p < 0.01). In all volunteers, SJW reduced ambrisentan exposure and the relative change (17-26%) was similar in all genotype groups. The extent of this interaction did not correlate with the changes in CYP3A activity (midazolam clearance) (rs = 0.23, p = 0.34). Ambrisentan had no effect on midazolam pharmacokinetics. In conclusion, SJW significantly reduced exposure with ambrisentan irrespective of the CYP2C19 genotype. The extent of this interaction was small and thus likely without clinical relevance.

Influence of St. John's wort on the steady-state pharmacokinetics and metabolism of bosentan.

OBJECTIVE: We assessed the effect of St. John's wort (SJW) on bosentan pharmacokinetics at steady-state in different CYP2C9 genotypes in healthy volunteers. METHODS: Nine healthy extensive metabolizers of CYP2C9 and 4 poor metabolizers received therapeutic doses of bosentan (125 mg q.d. on study day 1; 62.5 mg b.i.d. on study day 2, 125 mg b.i.d. on study days 3 - 20) for 20 days and SJW (300 mg t.i.d.) concomitantly for the last 10 days. Bosentan pharmacokinetics was assessed on days 1, 10, and 20. Concurrently, we repeatedly quantified changes of CYP3A activity using low dosed midazolam (3 mg p.o.) as a probe drug. RESULTS: Due to auto-induction of its metabolism, Cl/F increased by 67%, thus significantly lowering bosentan exposure (AUC) to 60% after 10 days of bosentan administration (n = 13, p < 0.05). Concurrently, midazolam clearance (CYP3A activity) increased by 224% (n = 13, p < 0.05) and further increased after SJW by 374% compared to baseline (n = 13, p < 0.05). SJW increased midazolam clearance by 47% (n = 13, p < 0.05) but failed to alter bosentan exposure and clearance consistently. No significant differences in bosentan exposure and clearance changes were observed in CYP2C9 poor metabolizers. CONCLUSION: SJW increased CYP3A activity but had no consistent effect on bosentan clearance. However, inter-individual changes of the interaction were large, suggesting that close monitoring of bosentan effects may be advisable. The contribution of CYP2C9 to this interaction seems to be minor.

Effect of the CYP3A inhibitor ketoconazole on the PXR-mediated induction of CYP3A activity.

PURPOSE: The aim of this clinical study was to investigate a previously proposed mechanism of ketoconazole-mediated inhibition of cytochrome P450 3A (CYP3A) induction. METHODS: A two-phase, randomized, cross-over, open, mono-centre trial was carried out. Participants received ketoconazole and St John's wort for 8 days to study the proposed suppression of St John's wort-mediated induction of CYP3A at the transcriptional level. In the second phase, we studied the inhibitory effect of a single dose of ketoconazole directly at the enzyme level during CYP3A induction by St John's wort. Midazolam served as a marker substance of CYP3A activity using an established limited sampling strategy. RESULTS: After 8 days of simultaneous ketoconazole and St John's wort administration, CYP3A-mediated midazolam metabolism was strongly inhibited (81 % decrease in clearance). Following the induction of CYP3A with St John's wort (6.6-fold increase in clearance on day 8), a single dose of ketoconazole strongly inhibited midazolam metabolism to the same degree (82 % decrease in clearance in relation to baseline). An induction of midazolam metabolism was observed after discontinuation of both drugs in both study phases. These results apparently contradict the in vitro results where ketoconazole showed an inhibitory effect on the transcription of CYP3A genes. CONCLUSIONS: Ketoconazole is a strong inhibitor of CYP3A, also when used concomitantly with St John's wort. In therapeutic doses it does not inhibit pregnane X receptor-mediated induction of CYP3A in vivo.

Modulators of very low voriconazole concentrations in routine therapeutic drug monitoring.

Very low voriconazole concentrations are commonly observed during therapeutic drug monitoring. Possible mechanisms include inappropriate dose selection, rapid metabolism (as a result of genetic polymorphisms or enzyme induction), and also nonadherence. We aimed to develop a method to distinguish between rapid metabolism of and nonadherence to voriconazole by quantification of voriconazole metabolites. In addition, the relevance of common genetic polymorphisms of CYP2C19 was assessed. In a retrospective study, samples with voriconazole concentrations 0.2 µg/mL or less in routine therapeutic drug monitoring (as quantified by high-performance liquid chromatography) were evaluated. Voriconazole and its N-oxide metabolite were quantified in residual blood using a highly sensitive liquid chromatography-tandem mass spectroscopy method (lower limit of quantitation = 0.03 µg/mL). Genetic polymorphisms of CYP2C19 were determined by real-time polymerase chain reaction using the hybridization probe format and the polymerase chain reaction-random fragment length polymorphism format. A total of 747 routine therapeutic drug monitoring plasma/blood samples of 335 patients treated with systemic voriconazole were analyzed and in 18.7% of all samples, voriconazole concentrations 0.2 µg/mL or less were found. In 32 samples (30 patients) with adequate dosage and timing of blood withdrawal, nonadherence was strongly suspected in seven patients because voriconazole-N-oxide concentrations were below 0.03 µg/mL, which was not observed in a reference group of 51 healthy volunteers with controlled drug intake. In 10 patients, of whom EDTA blood was available, the ultrarapid metabolizer genotype (CYP2C19*1\*17, CYP2C19*17\*17) was found in 80% and its prevalence was significantly higher as compared to a reference group (P = 0.02). In conclusion, quantification of voriconazole-N-oxide allowed for detection of suspected nonadherence in one of four patients with very low voriconazole concentrations. In the remaining patients, ultrarapid metabolism resulting from the CYP2C19*17 polymorphism appears to play a major role. Thus, in the case of voriconazole therapy failure, both nonadherence and genetic factors have to be considered.

Involvement of drug transporters in the synergistic action of FOLFOX combination chemotherapy.

FOLFOX is a cytostatic drug combination for adjuvant treatment of colorectal cancer (CRC) consisting of 5-fluorouracil (5-FU), leucovorin, and oxaliplatin. The mechanism of synergistic interaction of these drugs is poorly understood and little is known concerning the role of drug transporters and the impact of oxaliplatin metabolites oxalate and dichloro-diaminocyclohexane platinum. We therefore investigated the influence of FOLFOX components on drug transporter expression by quantitative real-time polymerase chain reaction and on the efficacy of each FOLFOX component by proliferation assay in the CRC model cell line LS180. Control experiments with transporter over-expressing cell lines were used to assess the significance of important transporters for the cytostatic activity of FOLFOX components. Moreover, we assessed the pharmacological contribution of the oxalato-ligand to the effect of oxaliplatin. FOLFOX components led to several alterations in expression of drug transporters. For instance, 5-FU significantly suppressed ATP7B and human organic cation transporter 2 and increased multidrug resistance-associated protein (MRP) 2 mRNA expression (5.8-fold). This was accompanied by a significant sensitisation to oxaliplatin. Over-expression of certain ABC-transporters (BCRP/ABCG2, MRP2/ABCC2 or MRP3/ABCC3) was demonstrated to be beneficial for the efficacy of oxaliplatin. The results obtained indicate that both down- and up-regulations of drug transporters could favour synergistic action of this drug combination. Moreover, oxaliplatin metabolite oxalate seems to positively modulate oxaliplatin's action as elucidated by median effect analysis. In conclusion, we propose as one mechanism for FOLFOX synergism the 5-FU mediated suppression of ATP7B, the over-expression of glutathione exporters such as MRP2/ABCC2 and the decrease of glutathione levels by oxalate.

Reduction of cytotoxicity of the alkaloid emetine through P-glycoprotein (MDR1/ABCB1) in human Caco-2 cells and leukemia cell lines.

The cytotoxicity of the alkaloid emetine was determined in six human cell lines that differ in the expression of ABC transporters, such as multiple drug resistance protein 1 (MDR1/ABCB1) and multidrug resistance associated protein 1 (MRP1/ABCC1). Emetine reveals a substantial cytotoxicity due to apoptosis that is inversely correlated with the expression of MDR1. Confluent Caco-2 cells with high MDR1 activity and the MDR1 over-expressing leukemia cell line CEM/ADR5000 are more resistant towards emetine (EC (50) 250 microM and 2 microM, respectively) than cells with a low expression of MDR1 (Jurkat cells, CCRF-CEM cells, HL-60 cells) or cells which over-express MRP1 (HL-60/AR) (EC (50) between 0.05 microM for CCRF-CEM and 0.17 microM for Jurkat cells). Apparently emetine is a substrate for MDR1 but not for MRP1. Furthermore, emetine is able to up-regulate the expression of MDR1 as shown IN VITRO by real-time PCR and transport activity studies.

Evaluation of inhibitory potencies for compounds inhibiting P-glycoprotein but without maximum effects: f2 values.

In cell culture systems with aqueous buffers, concentration-response curves to lipophilic inhibitors are difficult to establish because plateau effects (Imax) are often not reached because of limited drug solubility. Consequently, the inhibitory potency of a compound will not be definable using IC50 values (concentration exerting 50% of Imax). Since alternative potency measures f2 values, the concentrations required to double baseline signals have been proposed. Using both methods, we reevaluated the concentration-response curves of calcein assays with 78 compounds in three different cell culture systems and found a close correlation between both methods (r(s) = 0.93-0.99, p < or = 0.0028). These findings suggest that f2 values are a valuable alternative to define rank orders of highly lipophilic inhibitors as a basis for the prediction of pharmacological interaction properties in clinical settings. Although it was only tested for inhibition of P-glycoprotein, it seems likely that this method may be transferred to other assays with other proteins.

Opposite effects of short-term and long-term St John's wort intake on voriconazole pharmacokinetics.

OBJECTIVES: Constituents of St John's wort (SJW) in vivo induce the cytochrome P450 (CYP) isozymes 3A4, 2C9, and 2C19 but in vitro were shown to inhibit them. This study investigates both short- and long-term effects of SJW on the antifungal voriconazole, which is metabolized by these enzymes. METHODS: In a controlled, open-label study, single oral doses of 400 mg voriconazole were administered to 16 healthy men stratified for CYP2C19 genotype before and on day 1 and day 15 of concomitant SJW intake (300 mg LI 160 3 times daily). Plasma and urine concentrations of voriconazole were determined by liquid chromatography with mass-spectrometric detection. RESULTS: During the initial 10 hours of the first day of SJW administration, the area under the voriconazole plasma concentration-time curve was increased by 22% compared with control (15.5 +/- 6.84 h . microg/mL versus 12.7 +/- 4.16 h . microg/mL, P = .02). After 15 days of SJW intake, the area under the plasma concentration-time curve from hour 0 to infinity was reduced by 59% compared with control (9.63 +/- 6.03 h . microg/mL versus 23.5 +/- 15.6 h . microg/mL, P = .0004), with a corresponding increase in oral voriconazole clearance (CL/F) from 390 +/- 192 to 952 +/- 524 mL/min (P = .0004). The baseline CL/F of voriconazole and the absolute increase in CL/F were smaller in carriers of 1 or 2 deficient CYP2C19*2 alleles compared with wild-type individuals (P < .03). CONCLUSIONS: Coadministration of SJW leads to a short-term but clinically irrelevant increase followed by a prolonged extensive reduction in voriconazole exposure. SJW might put CYP2C19 wild-type individuals at highest risk for potential voriconazole treatment failure.

Extracts and kavalactones of Piper methysticum G. Forst (kava-kava) inhibit P-glycoprotein in vitro.

Root extracts from kava-kava (Piper methysticum G. Forst) are clinically used for the treatment of anxiety and restlessness. Due to reported cases of liver toxicity, kava-kava extracts were withdrawn from the market in several countries in 2002. Because the efflux transporter P-glycoprotein (P-gp) is involved in the absorption, distribution, and excretion of many drugs and often participates in drug-drug interactions, we studied the effect of a crude kava extract and the main kavalactones kavain, dihydrokavain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin on the P-gp-mediated efflux of calcein-acetoxymethylester in the P-gp-overexpressing cell line P388/dx and the corresponding cell line P388. The crude extract and the kavalactones showed a moderate to potent inhibitory activity with f2) (concentration needed to double baseline fluorescence) values of 170 microg/ml and 17 to 90 microM, respectively. The f2 value of yangonin could not be determined due to its higher lipophilicity. In conclusion, our results for the first time demonstrate P-gp-inhibitory activity of kava-kava and its components in vitro.