Pharmacokinetics and Metabolism of Liposome-Encapsulated 2,4,6-Trihydroxygeranylacetophenone in Rats Using High-Resolution Orbitrap Liquid Chromatography Mass Spectrometry.

2,4,6-trihydroxy-3-geranylacetophenone (tHGA) is a bioactive compound that shows excellent anti-inflammatory properties. However, its pharmacokinetics and metabolism have yet to be evaluated. In this study, a sensitive LC-HRMS method was developed and validated to quantify tHGA in rat plasma. The method showed good linearity (0.5-80 ng/mL). The accuracy and precision were within 10%. Pharmacokinetic investigations were performed on three groups of six rats. The first two groups were given oral administrations of unformulated and liposome-encapsulated tHGA, respectively, while the third group received intraperitoneal administration of liposome-encapsulated tHGA. The maximum concentration (Cmax), the time required to reach Cmax (tmax), elimination half-life (t1/2) and area under curve (AUC0-24) values for intraperitoneal administration were 54.6 ng/mL, 1.5 h, 6.7 h, and 193.9 ng/mL·h, respectively. For the oral administration of unformulated and formulated tHGA, Cmax values were 5.4 and 14.5 ng/mL, tmax values were 0.25 h for both, t1/2 values were 6.9 and 6.6 h, and AUC0-24 values were 17.6 and 40.7 ng/mL·h, respectively. The liposomal formulation improved the relative oral bioavailability of tHGA from 9.1% to 21.0% which was a 2.3-fold increment. Further, a total of 12 metabolites were detected and structurally characterized. The metabolites were mainly products of oxidation and glucuronide conjugation.

Fatal Taxus baccata ingestion with perimortem serum taxine B quantification.

INTRODUCTION: Common yew (Taxus baccata) is a common decorative evergreen shrub with potentially fatal toxicity hallmarked by seizure, arrhythmia and cardiovascular collapse if ingested. Taxine B has been identified as one of the most cardiotoxic taxine alkaloids in Taxus spp, and another alkaloid, 3,5-dimethoxyphenol (3,5-DMP), is used as a marker of ingestion. We present a fatal case of ingestion of yew with perimortem serum and gastric taxine B, and 3,5-DMP concentrations. CASE PRESENTATION: A 22-year-old woman was brought to the emergency department (ED) from a nearby botanical garden after she was found apneic and pulseless after a witnessed generalized tonic clonic seizure. The patient was found to have a wide complex rhythm with persistent cardiovascular collapse and expired despite maximal supportive care in the ED. A baggie of plant material was found on the patient, identified as Taxus baccata. Perimortem serum and gastric samples were analyzed to quantify serum and gastric taxine B and 3,5-DMP concentrations. RESULTS: Perimortem serum showed a 3,5-DMP concentration of 86.9 ng/mL, and taxine B of 80.9 ug/mL. CONCLUSION: We report a perimortem serum and gastric taxine B and 3,5-DMP concentrations in a fatal case of T. baccata toxicity.

Determination of myrtucommulone from Myrtus communis in human and rat plasma by liquid chromatography/tandem mass spectrometry.

Recent studies revealed that the non-prenylated acylphloroglucinol myrtucommulone (MC) from myrtle ( MYRTUS COMMUNIS) potently suppresses the biosynthesis of eicosanoids by direct inhibition of cyclooxygenase-1, microsomal prostaglandin E2 synthase (mPGES)-1, and 5-lipoxygenase at IC50 values in the range of 1 to 29 µM. Moreover, MC showed potent efficacy in animal models of inflammation after intraperitoneal administration. Since the main prerequisite for therapeutic efficacy is sufficient bioavailability, it is important to evaluate whether the concentrations of MC achieved in plasma coincide with the pharmacological active concentrations determined in vitro. For that reason, a sensitive LC/MS/MS method has been developed and validated for the determination of MC in human plasma. This method is based on liquid-liquid extraction of plasma samples with 20 % ethyl acetate in tert-butyl methyl ether using the structurally related acylphloroglucinol hyperforin as the internal standard. Chromatographic separation was achieved on a Gemini C6 Phenyl column using a mixture of acetonitrile/water (85 : 15 v/v) containing 6 mM ammonium formate in a run time of 15 min at a flow rate of 1 mL/min, a column temperature of 40 °C, and an autosampler temperature of 5 °C. Mass spectrometric quantification was carried out in the negative ion mode using electrospray ionization (ESI) and multiple-reaction monitoring (MRM). The most intense [M-H]⁻ MRM transition at m/z 667.4 → m/z 194.9 was used for quantification of MC and the transition at m/z 535.4 to m/z 383.2 was used to monitor hyperforin. The method was linear in the range of 1-100 ng/mL with r > 0.998, an intra- and inter-day RSD of 1.1-8.4 and 7.1-11.8 %, respectively, and a maximum R. E. of 13.8 % at the lowest concentration level. Moreover, cross validation revealed the suitability of the developed LC/MS method for application in rat studies.

Evaluation of cynomolgus monkey pregnane X receptor, primary hepatocyte, and in vivo pharmacokinetic changes in predicting human CYP3A4 induction.

Monkeys have been proposed as an animal model to predict the magnitude of human clinical drug-drug interactions caused by CYP3A4 enzyme induction. To evaluate whether the cynomolgus monkey can be an effective in vivo model, human CYP3A4 inducers were evaluated both in vitro and in vivo. First, a full-length pregnane X receptor (PXR) was cloned from the cynomolgus monkey, and the sequence was compared with those of rhesus monkey and human PXR. Cynomolgus and rhesus monkey PXR differed by only one amino acid (A68V), and both were highly homologous to human PXR (approximately 96%). When the transactivation profiles of 30 compounds, including known inducers of CYP3A4, were compared between cynomolgus and human PXR, a high degree of correlation with EC(50) values was observed. These results suggest that cynomolgus and human PXR respond in a similar fashion to these ligands. Second, two known human CYP3A4 inducers, rifampicin and hyperforin, were tested in monkey and human primary hepatocytes for induction of CYP3A enzymes. Both monkey and human hepatocytes responded similarly to the inducers and resulted in increased RNA and enzyme activity changes of CYP3A8 and CYP3A4, respectively. Lastly, in vivo induction of CYP3A8 by rifampicin and hyperforin was shown by significant reductions of midazolam exposure that were comparable with those in humans. These results show that the cynomolgus monkey can be a predictive in vivo animal model of PXR-mediated induction of human CYP3A4 and can provide a useful assessment of the resulting pharmacokinetic changes of affected drugs.

The effect of St. John's wort on the pharmacokinetics, metabolism and biliary excretion of finasteride and its metabolites in healthy men.

The aim of this study was to investigate what the consequences of induced drug metabolism, caused by St. John's wort (SJW, Hypericum perforatum) treatment, would have on the plasma, biliary and urinary pharmacokinetics of finasteride and its two previously identified phase I metabolites (hydroxy-finasteride and carboxy-finasteride). Twelve healthy men were administered 5mg finasteride directly to the intestine via a catheter with a multi-channel tubing system, Loc-I-Gut, before and after 14 days SJW (300mg b.i.d, hyperforin 4%) treatment. Bile samples were withdrawn via the Loc-I-Gut device from the proximal jejunum. LC-ESI-MS/MS was used to analyze finasteride and its metabolites in plasma, bile and urine. HPLC-UV was used to analyze hyperforin in plasma. The herbal treatment significantly reduced the peak plasma concentration (C(max)), the area under the plasma concentration-time curve (AUC(0-24h)) and the elimination half-life (t(1/2)) of finasteride. The geometric mean ratios (90% CI) were 0.42 (0.36-0.49), 0.66 (0.56-0.79) and 0.54 (0.48-0.61), respectively. Finasteride was excreted unchanged to a minor extent into bile and urine. Hydroxy-finasteride was not detected in plasma, bile or urine. Carboxy-finasteride was quantified in all three compartments and its plasma pharmacokinetics was significantly affected by SJW treatment. Hyperforin concentration in plasma was 21+/-7ng/ml approximately 12h after the last dose of the 14 days SJW treatment. In conclusion, SJW treatment for 2 weeks induced the metabolism of finasteride and caused a reduced plasma exposure of the drug. New knowledge was gained about the biliary and urinary excretion or the drug and its metabolites.

Single-drop liquid-phase microextraction for the determination of hypericin, pseudohypericin and hyperforin in biological fluids by high performance liquid chromatography.

The analysis of hypericin, pseudohypericin (collectively called in this study hypericins) and hyperforin in biological fluids is reported using single-drop liquid-phase microextraction in conjunction with HPLC-UV-fluorescence detection. A new option for analysis of the active principle constituents in biological samples is proposed, reducing the steps required prior to analysis. There are several parameters which determine the mass transfer such as the extraction solvent, drop and sample volumes, extraction time and temperature, pH and ionic strength, stirring rate and depth of needle tip in the bulk solution. These parameters were chosen to optimize the performance in the current study. The method was validated with respect to precision, accuracy and specificity. The intra-day precision values were below 2.3% for the high concentration level of control samples and 6.2% for the low level. The respective inter-day precision values were calculated to be below 4.4 and 7.1%, respectively, for the two concentration levels. Accuracy of the method, calculated as relative error, ranged from -2.6 to 7.0%. It was demonstrated that as long as the extraction procedure is consistently applied, quantitative analysis is performed accurately and reproducibly in human urine and plasma samples. Limits of quantitation (LOQs) in urine were calculated to be 3, 6 and 12 ng/ml for pseudohypericin, hypericin and hyperforin, respectively. Slightly higher limits were measured in plasma, i.e. 5, 12 and 20 ng/ml, for the respective analytes.

Hyperforin plasma level as a marker of treatment adherence in the National Institutes of Health Hypericum Depression Trial.

BACKGROUND: A previously reported clinical trial of Hypericum perforatum (St John's wort) in depression did not demonstrate efficacy. We assessed treatment adherence by measuring plasma hyperforin and evaluated the possible impact of adherence on study results. METHODS: Outpatients with major depression (N = 340) were randomized to an 8-week trial of H. perforatum (900-1500 mg/d), sertraline (50-100 mg/d) as active comparator, or placebo. Plasma was available from 292 patients (86% of randomized). Samples from the placebo and H. perforatum groups were assayed for hyperforin, and samples from the sertraline group for sertraline/N-desmethyl-sertraline. RESULTS: Of the 104 patients randomized to placebo, 18 (17%) had detectable plasma hyperforin. Of the 97 patients randomized to H. perforatum, 17 (17%) had no detectable plasma hyperforin. All the assayed sertraline patients (N = 91) had plasma sertraline/N-desmethyl-sertraline. The clinical trial conclusions remained unchanged when only patients with plasma assay consistent with random assignment were included in the analyses. CONCLUSIONS: One of every 6 patients assigned to placebo had plasma hyperforin, and 1 of every 6 patients assigned to H. perforatum had no detectable plasma hyperforin. The finding underscores the difficulty of enforcing treatment adherence in clinical trials of preparations that are readily available in the community.

High-performance liquid chromatography measurement of hyperforin and its reduced derivatives in rodent plasma.

A reverse-phase high-performance liquid chromatography method was developed for the determination of hyperforin and its reduced derivatives octahydrohyperforin and tetrahydrohyperforin in rodent plasma. The procedure includes solid-phase extraction from plasma using the Baker 3cc C8 cartridge, resolution on the Symmetry Shield RP8 column (150 mm x 4.6 mm, i.d. 3.5 microm) and UV absorbance detection at 300 nm. The assay was linear over a wide range, with an overall coefficient of variation less than 10% for all compounds. The precision and accuracy were within acceptable limits and the limit of quantitation was sufficient for studies preliminarily assessing the disposition of tetrahydrohyperforin and octahydrohyperforin in the mouse and rat.

Simultaneous determination of hypericin and hyperforin in human plasma with liquid chromatography-tandem mass spectrometry.

A selective and sensitive method for the simultaneous determination of hypericin and hyperforin--the two main active ingredients of St. John's Wort (SJW) extract--in human plasma depending on liquid/liquid-extraction and LC/MS/MS detection has been developed, validated after specifying the stability of the photosensitive hypericin in plasma samples during light exposure and applied to samples of a patient. After extraction with ethyl acetate/n-hexane in the darkness, sample extracts were chromatographed isocratically within 6 min on a Kromasil RP-18 column. The analytes were detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source. The limit of quantification was 0.05 ng/mL for hypericin and 0.035 ng/mL for hyperforin. The accuracy of the method varied between 101.9 and 114.2% and the precision ranged from 4.7 to 15.4% (S.D., batch-to-batch) for both analytes. The method was linear at least between 0.05 and 10 ng/mL for hypericin and between 0.035 and 100 ng/mL for hyperforin. Using this method hypericin and hyperforin were determined successfully in a patient over seven days following discontinuation of exposure with therapeutic doses of St. John's Wort extract.

Undeclared exposure to St. John's Wort in hospitalized patients.

AIM: The herbal medicine St. John's Wort (SJW) causes substantial decreases in the plasma concentrations of a range of co-administered drugs. Therefore, we evaluated the extent of systemic exposure to hyperforin and hypericin, two of the main constituents of SJW, in patients on admission and during hospital stay, and compared the results with known use of SJW as documented in the drug chart and detected in additional interviews. METHODS: One hundred and fifty patients aged > or = 18 years and admitted, between August 2000 and February 2002, to an internal medicine ward of a large German university hospital were included. Hyperforin and hypericin was determined in plasma by a sensitive liquid chromotography/mass spectometry (LC/MS/MS) method. To assess undeclared use of SJW the data were compared to information obtained from drug charts and from up to three interviews that had a particular focus on intake of herbal medicines and self-medication during hospitalization. RESULTS: Hyperforin was detected in 12 patients (plasma concentration on the first day of hospitalization = 12-100 ng ml(-1) in five patients and < 3 ng ml(-1) in seven), and hypericin in five patients (0.5-4.3 ng ml(-1)). Nine patients (6%) were taking/had taken SJW without the knowledge of the medical team and the pharmacist, who conducted the additional interviews, and 11 (7.3%) were taking/had taken SJW without the knowledge of the medical team alone. Seven of these patients were treated concurrently with drugs that can interact with SJW. CONCLUSIONS: Unrecognized use of SJW is frequent and may have an important influence on the effectiveness and safety of drug therapy during hospital stay.

Determination of phloroglucinol in human plasma by high-performance liquid chromatography-mass spectrometry.

A sensitive and selective liquid chromatographic method coupled with mass spectrometry (LC-MS) was developed for the quantification of phloroglucinol in human plasma. Resorcinol was used as internal standard, with plasma samples extracted using ethyl acetate. A centrifuged upper layer was then evaporated and reconstituted with mobile phase. The reconstituted samples were injected into a C(18) XTerra MS column (2.1 x 100 mm) with 3.5-microm particle size. The analytical column lasted for at least 500 injections. The mobile phase was 15% acetonitrile (pH 3.0), with flow-rate at 200 microl/min. The mass spectrometer was operated in negative ion mode with selective ion monitoring (SIM). Phloroglucinol was detected without severe interferences from plasma matrix when used negative ion mode. Phloroglucinol produced a parent molecule ([M-H](-)) at m/z 125 in negative ion mode. Detection of phloroglucinol in human plasma was accurate and precise, with quantification limit at 5 ng/ml. This method has been successfully applied to a study of phloroglucinol in human specimens.

Determination of phloroglucinol in human plasma by gas chromatography-mass spectrometry.

A specific and sensitive method has been developed for the determination of phloroglucinol in plasma; it involves an optimized procedure for blood sampling designed to minimize the in vitro oxidation of the molecule, and gas chromatography-mass spectrometry after silylation of the compound. The method allowed a reliable determination of phloroglucinol in plasma. The precision and accuracy of the assay, reported as coefficients of variation, were below 15%. Using a plasma sample of 0.25 ml, the limit of quantitation was 5 ng/ml with a precision of 17.4%, which is sensitive enough for pharmacokinetic studies. Stability studies under different conditions revealed that ascorbic acid limits the degradation of phloroglucinol in plasma during storage at freezer temperatures.