Elke Störmer

Saint John's wort: An in vitro analysis of P-glycoprotein induction due to extended exposure

Chronic use of Saint Johns wort (SJW) has been shown to lower the bioavailability for a variety of co‐administered drugs including indinavir, cyclosporin, and digoxin. Decreases in intestinal absorption through induction of the multidrug resistance transporter, P‐glycoprotein (P‐gp), may explain decreased bioavailability. The present study characterized the response of P‐gp to chronic and acute exposure of SJW and hypericin (HYP, a presumed active moiety within SJW) in an in vitro system. Experiments were performed with 3 to 300 μg ml−1 of methanol‐extracted SJW and 0.03 to 3 μM HYP, representing low to high estimates of intestinal concentrations. In induction experiments, LS‐180 intestinal carcinoma cells were exposed for 3 days to SJW, HYP, vehicle or a positive control (ritonavir). P‐gp was quantified using Western blot analysis. P‐gp expression was strongly induced by SJW (400% increase at 300 μg ml−1) and by HYP (700% at 3 μM) in a dose‐dependent fashion. Cells chronically treated with SJW had decreased accumulation of rhodamine 123, a P‐gp substrate, that was reversed with acute verapamil, a P‐gp inhibitor. Fluorescence microscopy of intact cells validated these findings. In Caco‐2 cell monolayers, SJW and HYP caused moderate inhibition of P‐gp‐attributed transport at the maximum concentrations tested. SJW and HYP significantly induced P‐gp expression at low, clinically relevant concentrations. Similar effects occurring in vivo may explain the decreased bioavailability of P‐gp substrate drugs when co‐administered with SJW.

Decreased plasma levels of amitriptyline and its metabolites on comedication with an extract from St. John's wort ( Hypericum perforatum ).

Extracts of St. John’s wort (Hypericum perforatum) became increasingly popular as easily available remedies for mild to moderate depression. Comedication with hypericum extract was recently shown to drastically reduce plasma concentration of ciclosporin, digoxin, and indinavir. We investigated the possible interaction of hypericum extract LI160 with amitriptyline. Both antidepressants have a high probability of concomitant use. Twelve patients requiring amitriptyline treatment received a single dose of hypericum extract (900 mg) at day 1, continued by a 12-to 14-day treatment with retarded amitriptyline (75 mg twice daily). Then hypericum (900 mg/day) was added for another 14 to 16 days. Steady-state pharmacokinetics of amitriptyline were compared before and after multiple-dose treatment with hypericum extract. Furthermore, comparisons were made for single-dose kinetics of hypericum-extract ingredients hypericin, pseudohypericin, and hyperforin between the first day of concomitant treatment and LI160 alone. Multiple-dose comedication with LI160 led to a statistically significant decrease in the area under the plasma concentration–time curve within one dosing interval of amitriptyline by 22% (p = 0.03) and nortriptyline by 41% (p = 0.002), as well as of all hydroxylated metabolites, except for 10-E-hydroxynortriptyline. Plasma levels of amitriptyline and hydroxylated metabolites gradually decreased, whereas nortriptyline concentrations were already markedly decreased after 3 days of cotreatment with hypericum. Cumulative urinary amounts of amitriptyline and metabolites decreased to the same extent as plasma concentrations upon hypericum comedication. Induction of cytochrome P-450 enzymes or drug transporters (P-glycoprotein) by St. John’s wort extract may explain this pharmacokinetic interaction. Physicians should be aware of this interaction when treating patients with amitriptyline.

Differential modulation of P-glycoprotein expression and activity by non-nucleoside HIV-1 reverse transcriptase inhibitors in cell culture.

AbstractPurpose. This study investigated the effects of the non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTI) nevirapine (NVR), efavirenz (EFV), and delavirdine (DLV) on P-glycoprotein (P-gp) activity and expression to anticipate P-gp related drug-drug interactions associated with combination therapy. Methods. NNRTIs were evaluated as P-gp substrates by measuring differential transport across Caco-2 cell monolayers. Inhibition of P-gp mediated rhodamine123 (Rh123) transport in Caco-2 cells was used to assess P-gp inhibition by NNRTIs. Induction of P-gp expression and activity in LS180V cells following 3-day exposure to NNRTIs was measured by western blot analysis and cellular Rh123 uptake, respectively. Results. The NNRTIs showed no differential transport between the basolateral to apical and apical to basolateral direction. NNRTI transport in either direction was not affected by the P-gp inhibitor verapamil. DLV inhibited Rh123 transport, causing a reduction to 15% of control at 100 μM (IC50 = 30 μM). NVR caused a concentration-dependent induction of P-gp expression in LS180V cells resulting in a 3.5-fold increase in immunoreactive P-gp at 100 μM NVR. Induction attributable to EFV and DLV was quantitatively smaller. NVR significantly reduced cellular uptake of Rh123 into LS180V cells, indicating increased drug efflux due to induced P-gp activity; effects of EFV and DLV were smaller. Acute DLV treatment of LS180V cells previously induced with NVR or ritonavir did not reverse the decreased Rh123 cell accumulation. Conclusions. NNRTIs show differential effects on P-gp activity and expression in vitro. Clinical studies are required to elucidate the clinical importance of potential drug interactions.

MDR1 haplotypes do not affect the steady-state pharmacokinetics of cyclosporine in renal transplant patients.

This retrospective study investigated the impact of MDR1 haplotypes derived from the single‐nucleotide polymorphisms (SNPs) 2677G>T (exon 21) and 3435C>T (exon 26) on the pharmacokinetics of cyclosporine in 98 renal transplant patients. Based on SNPs 2677 and 3435, four different haplotypes and nine different genotypes were identified in the study sample. Frequencies of SNPs, genotypes, and haplotypes were in agreement with previously reported values. Cyclosporine pharmacokinetics were characterized using a 2‐hour AUC (AUC0–12), trough concentrations (C0), and blood concentrations 2 hours after cyclosporine administration (C2). No significant differences in dose‐corrected AUC0–12, C0, or C2 values were observed between carriers of different SNP variants and genotypes (Kruskal‐Wallis test), as well as between carriers and noncarriers of each haplotype (Mann‐Whitney U test). Carriers of haplotype 12 (2677G and 3435T), which has previously been associated with increased digoxin AUC values, had a median AUC0–12 of 18.9 μg•h•L−1 (range: 9.0–35.2) compared to 17.5 μg•h•L−1 (range: 7.5–37.1) in the noncarrier group. It was concluded that MDR1 haplotypes derived from the SNPs 2677G>T (exon 21) and 3435C>T (exon 26) are not associated with cyclosporine pharma‐ cokinetics in renal transplant patients.

Determination of hyperforin, hypericin, and pseudohypericin in human plasma using high-performance liquid chromatography analysis with fluorescence and ultraviolet detection

Hyperforin, hypericin and pseudohypericin are the main ingredients of St. Johns wort extract, which is available over the counter for treatment of mild to moderate depression. To facilitate clinical studies we developed two sensitive HPLC methods for determination of hypericin/pseudohypericin and hyperforin, respectively, in human plasma samples. The achieved limits of quantitation of 0.25 ng/ml for hypericin and pseudohypericin and 10 ng/ml for hyperforin were low enough to allow determination of pharmacokinetic parameters of the substances. Following liquid-liquid extraction of human plasma the samples were separated by isocratic reversed-phase HLPC and analyzed using fluorimetric detection for hypericin/pseudohypericin and UV detection for hyperforin.

Simultaneous determination of celecoxib, hydroxycelecoxib, and carboxycelecoxib in human plasma using gradient reversed-phase liquid chromatography with ultraviolet absorbance detection.

A new HPLC method for the simultaneous determination of celecoxib, carboxycelecoxib and hydroxycelecoxib in human plasma samples has been developed. Following a solid-phase extraction procedure, the samples were separated by gradient reversed-phase HLPC (C(18)) and quantified using UV detection at 254 nm. The method was linear over the concentration range 10-500 ng/ml. The intra-assay variability for the three analytes ranged from 4.0 to 12.6% and the inter-assay variability from 4.9 to 14.2%. The achieved limits of quantitation (LOQ) of 10 ng/ml for each analyte allowed the determination of the pharmacokinetic parameters of the analytes after administration of 100 mg celecoxib.

P‐glycoprotein Interactions of Nefazodone and Trazodone in Cell Culture

This study investigated the effects of nefazodone (NFZ) and trazodone (TZD) on P‐glycoprotein (P‐gp) activity and expression in cell culture. NFZ and TZD showed no differential transport between the basolateral to apical and apical to basolateral direction across Caco‐2 cell monolayers. Transport in either direction was not affected by verapamil. NFZ was a potent inhibitor (IC50 = 4.7 M) of rhodamine123 (Rh123)B to A transport across Caco‐2 cell monolayers, while TZD had minimal effect. Following 72‐hour exposure of LS180V cells to NFZ and TZD (10 M), a twofold increase in immunoreactive P‐gp was observed. Rh123 accumulation into these cells was reduced to 65% and 74% of control by NFZ and TZD (10 M), respectively. It was concluded that differential rates of transport of NFZ and TZD in Caco‐2 cells were not evident. However, NFZ is an inhibitor of P‐gp activity at clinically relevant in vivo concentrations and may have the potential to increase bioavailability of coadministered compounds that are substrates for transport. Concentrations of NFZ and TZD achieved in the intestine after chronic oral dosing may induce P‐gp expression and reduce absorption of coadministered drugs.

Rapid and simple method for the analysis of nateglinide in human plasma using HPLC analysis with UV detection.

Nateglinide (NA) is a novel oral mealtime glucose regulator, recently approved for the treatment of type II diabetes mellitus. To facilitate clinical studies investigating the dependence of NA elimination on the genotype of cytochrome P450 isoenzymes, we developed a rapid HPLC method for determination of NA in human plasma samples. The validated limit of quantitation (LOQ) of 0.1 microg/ml is low enough to allow determination of pharmacokinetic parameters of the substance. The intra-assay coefficients of variation (CV) ranged from 1.6 to 12.9% at NA concentrations of 0.5-7.5 microg/ml. The inter-assay variation for the same plasma concentrations ranged from 3.8 to 8.4%. The calibration was linear in the range of 0.1-20 microg/ml. For the quantitation of NA, only 50 microl of plasma were needed. Following protein precipitation in human plasma, the samples were separated by isocratic reversed phase HLPC and analyzed using ultraviolet detection at 210 nm. Sample preparation time and analysis time are both short and allow rapid analysis of large sample sets.

Übersicht zu Interaktionsstudien mit Johanniskrautextrakten

Obwohl Johanniskrautpraparate weltweit uber viele Jahre und in groβer Haufigkeit eingenommen wurden, liegen Berichte zu Arzneimittelwechselwirkungen erst seit kurzem vor [24, 30, 38, 51]. Die zum Teil deutlichen Effekte einer Johanniskrautgabe auf komedizierte Arzneimittel mundeten in eine Vielzahl von Studien, die sich mit der klinischen Relevanz und den zugrundeliegenden Mechanismen der Interaktionen beschaftigten. Der vorliegende Beitrag mochte diese neuen Erkenntnisse und Studienergebnisse zusammenfassen und sowohl uber das Spektrum als auch uber mogliche Mechanismen der Interaktionen mit Johanniskraut Auskunft geben.