Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms

The present study investigated the effects of Radix Astragali (RA) and Radix Rehmanniae (RR), the major components of an anti-diabetic foot ulcer herbal formula (NF3), on the metabolism of model probe substrates of human CYP isoforms, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, which are important in the metabolism of a variety of xenobiotics. The effects of RA or RR on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6β-hydroxylase) activities were investigated using pooled human liver microsomes. NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.98mg/ml) and CYP3A4 (IC(50)=0.76mg/ml), with K(i) of 0.67 and 1.0mg/ml, respectively. With specific human CYP2C9 and CYP3A4 isoforms, NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.86mg/ml) and CYP3A4 (IC(50)=0.88mg/ml), with K(i) of 0.57 and 1.6mg/ml, respectively. Studies on RA or RR individually showed that RR was more important in the metabolic interaction with the model CYP probe substrates. RR dose-dependently inhibited the testosterone 6β-hydroxylation (K(i)=0.33mg/ml) while RA showed only minimal metabolic interaction potential with the model CYP probe substrates studied. This study showed that RR and the NF3 formula are metabolized mainly by CYP2C9 and/or CYP3A4, but weakly by CYP1A2, CYP2D6 and CYP2E1. The relatively high K(i) values of NF3 (for CYP2C9 and CYP3A4 metabolism) and RR (for CYP3A4 metabolism) would suggest a low potential for NF3 to cause herb-drug interaction involving these CYP isoforms.     

Enzyme kinetic and molecular docking studies on the metabolic interactions of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from Halenia elliptica D. Don, with model probe substrates of human cytochrome P450 enzymes

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb-drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC(50)=1.06μM) and CYP2C9 (IC(50)=3.89μM), minimal inhibition on CYP3A4 (IC(20)=11.94μM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the K(i) values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12μM, 2.00μM and 95.03μM, respectively. HM-1 competitively inhibited testosterone 6β-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.     

Development and evaluation of an efficient HPLC/MS/MS method for the simultaneous determination of pseudoephedrine and cetirizine in human plasma: application to phase-I pharmacokinetic study

A sensitive, simple and highly selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and evaluated to determine simultaneously the concentrations of pseudoephedrine and cetirizine in human plasma. The chief benefit of the present method is the minimal sample preparation, as the procedure is only one-step protein precipitation. Two drugs were separated on a C(8) column and analyzed by LC/MS/MS using positive electrospray ionisation (ESI). The method had a chromatographic run time of 12.0 min and a linear calibration curve over the concentration range of 1.0-800 ng/ml for pseudoephedrine and 1.0-400 ng/ml for cetirizine, respectively. The lower limit of quantification of the two drugs was 1.0 ng/ml, respectively. The intra- and inter-batch precisions were less than 9.7%. The method described herein has been first used to reveal the pharmacokinetic characters in healthy Chinese volunteers treated with oral administration of different dosages of cetirizine dihydrochloride and controlled-released pseudoephedrine hydrochloride compound tablet, and approached the influence of a standard meal on the extent and rate of absorption of the combination tablet.     

Interaction of serum proteins with CYP isoforms in human liver microsomes: inhibitory effects of human and bovine albumin,alpha-globulins,alpha-1-acid glycoproteins and gamma-globulins on CYP2C19 and CYP2D6

The effects of serum proteins on the in vitro hydroxylation pathways of mephenytoin (CYP2C19) and debrisoquine (CYP2D6) were studied to enhance the predictability of in vivo drug metabolism from in vitro assays. Both CYP substrates are known to be weakly bound to albumin and the applicability of the free drug hypothesis was further appraised. Since bovine serum albumin (BSA) is used widely in in vitro assays, a comparison between human and bovine proteins was made. Four major serum proteins were studied: albumin, alpha1-acid glycoprotein (AGP), alpha- and gamma-globulins. Human serum albumin (HSA) inhibited both CYP activities about 20% more than BSA. The addition of human alpha-globulins, but not the bovine protein, resulted in marked reduction of 86% and 41% in CYP2C19 and CYP2D6 activities, respectively. This reduction of activity was strikingly greater than the fraction bound (14 and 22%, respectively). The inhibition was of the competitive type and the Ki values of human alpha-globulins on CYP2C19 and CYP2D6 were found to be 0.45% (4.5 mg/ml) and 3.5% (35 mg/ml), respectively. The effect of both human and bovine gamma-globulins on CYP isoforms was negligible. The Ki values of human and bovine AGP for CYP2C19 were 1.84% (420 microM) and 0.93% (210 microM), respectively. For HSA, human alpha-globulins and human and bovine AGP, the strongly decreased CYP activities in vitro cannot be explained by the free drug hypothesis. A direct interaction of these serum proteins with CYP enzymes is postulated. Differential effects of bovine and human serum proteins and CYP specific inhibition were observed.     

Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 μg/ml) decreased dextromethorphan O-demethylation in vitro (IC(50)=23.3 μg/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 μg/ml) also decreased CYP2D6 activity (IC(50)=265.8 μg/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity (IC(50)=35.4 μM), compared to quinidine, a specific CYP2D6 inhibitor (IC(50)=0.9 μM). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with IC(20) of 40.8 μM, 16.5 μM and 61.4 μM, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K(i) values of 4.23 μg/ml and 2.53 μM, respectively, compared to quinidine, K(i)=0.41 μM. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phe120 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.     

Polysaccharide peptides from Coriolus versicolor competitively inhibit model cytochrome P450 enzyme probe substrates metabolism in human liver microsomes

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy or health supplement in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited rat CYP2C11-mediated tolbutamide 4-hydroxylation and in human CYP2C9. In this study, the effects of the water extractable fraction of PSP on the metabolism of model CYP1A2, CYP2D6, CYP2E1 and CYP3A4 probe substrates were investigated in pooled human liver microsomes. PSP (1.25-20μM) dose-dependently decreased CYP1A2-mediated metabolism of phenacetin to paracetamol (IC(50) 19.7μM) and CYP3A4-mediated metabolism of testosterone to 6β-hydroxytestosterone (IC(20) 7.06μM). Enzyme kinetics studies showed the inhibition of CYP1A2 activity was competitive and concentration-dependent (K(i)=18.4μM). Inhibition of testosterone to 6β-hydroxytestosterone was also competitive and concentration-dependent (K(i)=31.8μM). Metabolism of dextromethorphan to dextrorphan (CYP2D6-mediated) and chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1-mediated) was only minimally inhibited by PSP, with IC(20) values at 15.6μM and 11.9μM, respectively. This study demonstrated that PSP competitively inhibited the CYP1A2- and CYP3A4-mediated metabolism of model probe substrates in human liver microsomes in vitro. The relatively high K(i) values for CYP1A2 and CYP3A4 would suggest a low potential for PSP to cause herb-drug interaction related to these CYP isoforms.     

Rapid and simultaneous determination of nifedipine and dehydronifedipine in human plasma by liquid chromatography-tandem mass spectrometry: Application to a clinical herb-drug interaction study

Nifedipine (NIF), a calcium channel antagonist, is metabolized primarily by cytochrome P450 (CYP3A4) to dehydronifedipine (DNIF). As such, NIF is often used as a probe drug for determining CYP3A4 activity in human studies. A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated to simultaneously determine NIF and DNIF in human plasma using nitrendipine as the internal standard (IS). After extraction of the plasma samples by ether-n-hexane (3:1, v/v), NIF, DNIF and the IS were subjected to LC/MS/MS analysis using electro-spray ionization (ESI). Chromatographic separation was performed on a Hypersil BDS C(18) column (50 mm x 2.1 mm, i.d., 3 microm). The method had a chromatographic running time of approximately 2.5 min and linear calibration curves over the concentrations of 0.5-100 ng/mL for NIF and DNIF. The recoveries of the one-step liquid extraction method were 81.3-89.1% for NIF and 71.6-80.4% for DNIF. The lower limit of quantification (LLOQ) of the analytical method was 0.5 ng/mL for both analytes. The intra- and inter-day precision was less than 15% for all quality control samples at concentrations of 2, 10, and 50 ng/mL. The validated LC/MS/MS method has been successfully used to study pharmacokinetic interactions of NIF with the herbal antidepressant St. John's wort in healthy volunteers. These results indicated that the developed LC/MS/MS method was efficient with a significantly shorter running time (2.5 min) for NIF and DNIF compared to those methods previously reported in the literature. The presented LC/MS/MS method had acceptable accuracy, precision and sensitivity and was used in a clinical pharmacokinetic interaction study of NIF with St. John's wort, a known herbal inducer of CYP3A4. St. John's wort was shown to induce NIF metabolism with increased plasma concentrations of DNIF.     

Quantification of dextromethorphan and metabolites: a dual phenotypic marker for cytochrome P450 3A4/5 and 2D6 activity

A sensitive and selective liquid chromatographic procedure using fluorimetric detection was developed to quantify dextromethorphan (DTM), 3-methoxymorphinan (3MM), dextrorphan (DT), 3-hydroxymorphinan (3OH) and two internal standards, codeine (COD) and ethylmorphine (ETM), in urine. Precision and accuracy of the assay were determined over a concentration range of 5–3200 ng/ml urine for DTM, 5–400 ng/ml urine for 3MM, 400–40 000 ng/ml urine for DT and 200–16 000 ng/ml urine for 3OH, by assaying freshly prepared calibration standards and replicates of six quality control (QC) samples on separate days. All of the inter-day and intra-day coefficients of variation (C.V.s) were less than 20% except for a low QC for 3MM. The inter-day and intra-day accuracies were less than 20% for the low QCs, less than 15% for the medium QCs and less than 12% for the high QCs, for all compounds. The limit of quantification (LOQ) was 2 ng/ml urine for DTM and 3MM, 250 ng/ml urine for DT, and 100 ng/ml urine for 3OH. Absolute recovery was 76% for DTM, 74% for 3MM, 77% for DT, 46% for 3OH, 73% for ETM, and 57% for COD. The frequency distribution of the CYP2D6 metabolic ratio (DTM/DT) illustrated a bimodal distribution whereas, the CYP3A metabolic ratio (DTM/3MM) exhibited a unimodal distribution in overnight urine samples of volunteers who ingested 30 mg dextromethorphan hydrobromide. The CYP2D6 metabolic ratio significantly correlated with 3MM/3OH (r=0.82) and DTM/3OH (r=0.95) but did not correlate with the CYP3A metabolic ratio (r=0.27).     

Simple chromatography method for simultaneous determination of dextromethorphan and its main metabolites in human plasma with fluorimetric detection

Dextromethorphan, the innocuous non-narcotic antitussive agent, is the most widely used probe drug to assess CYP2D6 function both in vivo and in vitro. For this reason a simple and selective high performance liquid chromatography method with fluorimetric detection for simultaneous quantitation of dextromethorphan, and its main metabolites in human plasma was developed and validated. The method involved a simple and rapid protein precipitation protocol, using a mixture of ZnSO(4) and methanol. The analysis was performed on a 3 microm, C(18) Tracer Excel 15 cm x 0.4 cm i.d. column by gradient elution in which Mobile phase A consisted of potassium dihydrogen phosphate buffer (pH = 3, 0.01 M):methanol:tetrahydrofuran (68.5:31:0.5), and mobile phase B consisted of methanol:tetrahydrofuran (93.25:6.75). Linear calibration curves were obtained in the range of 10-500 ng/ml for dextromethorphan, dextrorphan and hydroxymorphinan. The limit of quantitation (LOQ) was 10 ng/ml for each compound. The maximum within and between days precisions were 7.4 and 7.8%, respectively. The accuracies at four different concentration levels ranged from 88.2 to 111.5%. The recoveries were between 88.0 and 108.6%. The assay method was successfully applied to determine dextromethorphan metabolic ratio after an oral dose of 30 mg of dextromethorphan hydrobromide.     

Evaluation of fluorescence- and mass spectrometry-based CYP inhibition assays for use in drug discovery

The potential for metabolism-related drug-drug interactions by new chemical entities is assessed by monitoring the impact of these compounds on cytochrome P450 (CYP) activity using well-characterized CYP substrates. The conventional gold standard approach for in vitro evaluation of CYP inhibitory potential uses pooled human liver microsomes (HLM) in conjunction with prototypical drug substrates, often quantified by LC-MS/MS. However, fluorescent CYP inhibition assays, which use recombinantly expressed CYPs and fluorogenic probe substrates, have been employed in early drug discovery to provide low-cost, high-throughput assessment of new chemical entities. Despite its greatly enhanced throughput, this approach has been met with mixed success in predicting the data obtained with the conventional gold standard approach (HLM+LC-MS). The authors find that the predictivity of fluorogenic assays for the major CYP isoforms 3A4 and 2D6 may depend on the quality of the test compounds. Although the structurally more optimized marketed drugs yielded acceptable correlations between the fluorogenic and HLM+LC-MS/MS assays for CYPs 3A4, 2D6, and 2C9 (r2 = 0.5-0.7; p < 0.005), preoptimization, early discovery compounds yielded poorer correlations (r2 < or = 0.2) for 2 of these major isoforms, CYPs 3A4 and 2D6. Potential reasons for the observed differences are discussed.     

Validation of a liquid chromatography-mass spectrometry method to assess the metabolism of dextromethorphan in rat everted gut sacs

A rapid, sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method was developed for the simultaneous assay of dextromethorphan and its metabolites in tissue culture medium and its intestinal metabolism studied with the rat everted gut sac model. The method was validated in the concentration range of 0.1-2.5 microM (27.1 ng/mL-0.677 microg/mL) for dextromethorphan and 0.005-0.5 microM for dextrorphan and 3-methoxymorphinan (1.28 ng/mL-0.128 microg/mL) and 3-hydroxymorphinan (1.22 ng/mL-0.122 microg/mL). The limits of quantification (LOQ) were 0.0025 microM (12.5 fmoles, 3.4 pg, 5 microL injected) for dextromethorphan; 0.0025 microM for dextrorphan, 3-methoxymorphinan (24.9 fmoles, 6.4 pg injected), and 3-hydroxymorphinan (25.1 fmoles, 6.1 pg injected) with 10 microL injected. The detection of dextrorphan and 3-methoxymorphinan showed that both the P450 isoforms CYP3A and 2D were active in the intestinal mucosa and metabolised dextromethorphan during its passage across the mucosa.     

Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats

A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1'-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4'-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, -20 degrees C), three freeze-thaw cycles and autosampler (24 h, 4 degrees C) stability. The stability of urine samples was also prepared with and without beta-glucuronidase incubation (37 degrees C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.     

Simultaneous analysis of cytochrome P450 probes-dextromethorphan, flurbiprofen and midazolam and their major metabolites by HPLC-mass-spectrometry/fluorescence after single-step extraction from plasma

Cytochrome P450 enzymes catalyze oxidative metabolism of most pharmaceutical compounds. Consequently dextromethorphan, flurbiprofen, midazolam and other compounds are commonly used as probe substrates to evaluate cytochrome P450 function in humans. A "cocktail" approach employing simultaneous administration of two or more of the probe substrates has been used by various investigators in recent years. An analytical strategy to simultaneously extract and analyze dextromethorphan, flurbiprofen and midazolam and their major metabolites (dextrorphan, 4'-hydroxy-flurbiprofen and 1'-hydroxy-midazolam) by HPLC-MS/fluorescence was developed and is described here. The three probe substrates and their major metabolites were extracted simultaneously by means of a solid-phase (Bond Elut Certify cartridges) extraction procedure from 200 microl of pig plasma. The extraction efficiency was more than 79.5% for each of the six analytes. The extracted compounds were chromatographically separated on a Luna C8(II) column (50 mm Lx3 mm ID) in a single run of 20 min and analyzed by either fluorescence (flurbiprofen and 4'-hydroxy-flurbiprofen) or selective ion monitoring (dextromethorphan, dextrorphan, midazolam and 1'-hydroxy-midazolam) with positive electrospray ionization. The limit of quantification was 2.5 ng/ml for midazolam and 5 ng/ml for the other five analytes. The assay was precise and accurate (error: -9.1 to 12.1) with total CVs of 13.9% or better for each of the 6 analytes. This method was used to analyze concentrations of the three probes and their metabolites in plasma after intravenous administration to a healthy pig.     

Determination of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in human serum using liquid chromatography-tandem mass spectrometry

Concentrations of mono(2-ethylhexyl)phthalate (MEHP), and di(2-ethylhexyl)phthalate (DEHP), in serum of healthy volunteers were determined by high performance liquid chromatography (HPLC) with tandem mass spectrometry (LC/MS/MS). The serum was extracted with acetone, followed by hexane extraction under acidic conditions, and then applied to the LC/MS/MS. Recoveries of 20 ng/ml of MEHP and DEHP were 101+/-5.7 (n=6) and 102+/-6.5% (n=6), respectively. The limits of quantification (LOQ) of MEHP and DEHP in the method were 5.0 and 14.0 ng/ml, respectively. The concentration of MEHP in the serum was at or less than the LOQ. The concentration of DEHP in the serum was less than the LOQ. Contaminations of MEHP and DEHP from experimental reagents, apparatus and air during the procedure were less than the LOQ and were estimated to be <1.0 and 2.2+/-0.6 ng/ml, respectively. After subtraction of the contamination, the net concentrations of MEHP and DEHP in the serum were estimated at or <5 and <2 ng/ml, respectively. To decrease contamination by DEHP, the cleanup steps and the apparatus and solvent usage were minimized in the sample preparation procedures. The high selectivity of LC/MS/MS is the key for obtaining reliable experimental data from in the matrix-rich analytical samples and for maintaining a low level contamination of MEHP and DEHP in this experimental system. This method would be a useful tool for the detection of MEHP and DEHP in serum.     

Investigation of cytochrome P450 1A2 and 3A inhibitory properties of Danshen tincture

Danshen (Salvia miltiorrhiza Bunge) as a famous Traditional Chinese medicine is widely used in the treatment of cardiovascular and cerebrovascular diseases in the world. Danshen tincture (DT), extracted from Danshen root with a mixture of water and alcohol, is a commonly used preparation method for human consumption. The aim of this study was to investigate the effects of DT on the cytochrome P450 (CYP) 1A2 and 3A activities by human and rat liver microsomes. Effects of DT were assessed with use of Danshen ethanolic extract (DEE) and selective substrates, markers of CYP activities. DEE (0.5-10 μg/ml) competitively inhibited human and rat liver microsomal CYP1A2 activity with inhibition constant (K(i)) values at 3.40 and 5.16 μg/ml, respectively. At the same time, DEE (2.5-20 μg/ml) not only noncompetitively inhibited human liver microsomal CYP3A4/5 activity with a K(i) of 11.9 μg/ml, but also competitively inhibited rat liver microsomal CYP3A1/2 activity with a K(i) of 52.1 μg/ml. The data indicate that DEE inhibited the metabolism of CYP1A2 and 3A substrates in human and rat liver in vitro with different mode of inhibition. This study may be helpful for clinical application of Danshen tincture.     

Simultaneous determination of steroid composition of human testicular fluid using liquid chromatography tandem mass spectrometry

A rapid, sensitive, and specific method using liquid chromatography tandem mass spectrometry (LC/MS/MS) has been developed for simultaneous determination of testosterone (T), dihydrotestosterone (DHT), estradiol (E2), and 5alpha-androstan-3alpha, -17beta-diol (3alpha-Diol) within human testicular fluid. Sample pretreatment involved a one-step extraction with diethyl ether. The analytes were separated on a Waters X-Terra C18 (150 mm x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (70:30, v/v) containing 0.1% formic acid using isocratic flow at 0.15 ml/min for 8 min. The column effluent was monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 0.1-50 ng/ml for T, 0.02-1 ng/ml for DHT, 0.05-2 ng/ml for E2, and 0.2-10 ng/ml for 3alpha-Diol, with values for the coefficient of determination of >0.99. The overall extraction efficiency was greater than 86% for T, 75% for DHT, 66% for E2, and 60% for 3alpha-Diol. The values for within-day and between-day precision and accuracy were <15%. We measured each of the four steroids in testicular sample volumes of only 20 microl, obtained by percutaneous testicular aspiration. The mean intratesticular testosterone concentration found by LC/MS/MS, 572 +/- 102 ng/ml, was similar to that previously obtained by radioimmunoassay (RIA). The mean intratesticular estradiol concentration was 15.7 +/- 2.3 ng/ml, which also correlated well with RIA measurement. Both DHT and 3alpha-Diol were below the limits of detection by RIA, but could be measured accurately by LC/MS/MS. In conclusion, LC/MS/MS represents a sensitive and accurate means by which to measure four separate steroids within small volume samples of testicular fluid.     

Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.     

Heterologous Expression of Human Cytochromes P450 2D6 and CYP3A4 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and Their Functional Characterization

This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17α sequence were inserted into a pCWori⁺ vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K_m and V_max values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.     

High-performance liquid chromatographic assays for bufuralol 1'-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver

Bufuralol, debrisoquine, and dextromethorphan are three prototype substrates of the common genetic deficiency of oxidative drug metabolism in man known as debrisoquine/sparteine-type polymorphism. We describe assays for the in vitro metabolism of (+)- and (-)-bufuralol, debrisoquine, and dextromethorphan in human liver microsomes and reconstituted purified cytochrome P-450 isozymes. These assays combine nonextractive sample preparation by precipitation of protein with perchloric acid with reversed-phase inorganic ion-pair HPLC and fluorescence detection. The minimal detectable levels of the major metabolites formed are 1'-hydroxybufuralol, 0.1 ng/ml; 4-hydroxydebrisoquine, 0.8 ng/ml; and dextrorphan, 0.1 ng/ml. Formation of these metabolites is linear for at least 45 min and between 1 and 100 micrograms of microsomal protein. Comparative kinetic analysis of the three monooxygenase reactions in human liver microsomes revealed an apparent biphasicity of (+)- and (-)-bufuralol 1'-hydroxylation and dextromethorphan O-demethylation but monophasic formation of 4-hydroxydebrisoquine in the substrate concentration range (less than 1 mM) studied. These data, in combination with those obtained by purified human cytochrome P-450 isozymes indicate the involvement of the same enzyme in the metabolism of all three substrates investigated. However, additional and distinct activities contribute to the metabolism of (+)- and (-)-bufuralol and dextromethorphan.     

Determination of scopolamine in human serum and microdialysis samples by liquid chromatography–tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC–MS–MS) method with a rapid and simple sample preparation was developed for the determination of scopolamine in biological fluids. Scopolamine and the internal standard atropine in serum samples were extracted and cleaned up by using an automated solid phase extraction method. Microdialysis samples were directly injected into the LC–MS system. The mass spectrometer was operated in the multi reaction monitoring mode. A good linear response over the range of 20 pg/ml to 5 ng/ml was demonstrated. The accuracy for added scopolamine ranged from 95.0 to 104.0%. The lower limit of quantification was 20 pg/ml. This method is suitable for pharmacokinetic studies.