Assessment of the stereoselective metabolism of methaqualone in man by capillary electrophoresis

Methaqualone (MQ) and its hydroxylated metabolites are quinazoline derivatives that exhibit atropisomerism. As a continuation of our previous work with these compounds (Electrophoresis 2001, 22, 3270-3280), chiral capillary zone electrophoresis with hydroxypropyl-beta-cyclodextrin as buffer additive and multiwavelength absorbance detection is shown to be an effective tool to provide insight into the stereoselectivity of the MQ metabolism. The five major monohydroxy MQ metabolites formed during biotransformation do not show enantiomerization at temperatures up to 85 degrees C. Enzymatic and acidic hydrolysis of urines that were collected after concomitant administration of 250 mg of MQ and 25 mg diphenhydramine (DH) chloride are both shown to provide stereoselective metabolic patterns with 4'-hydroxymethaqualone, the major urinary metabolite, being excreted almost exclusively as a single enantiomer. A stereoselectivity in the formation of 2'-hydroxymethaqualone and 2-hydroxymethaqualone was also observed in vitro using human liver microsomes and preparations containing the cytochrome P450 enzyme (CYP) CYP3A4 only. The presence of DH during incubation with human liver microsomes did not reveal a difference in the metabolic pattern obtained. Furthermore, CYP2D6 and CYP2C19 do not significantly contribute to the metabolism of MQ. This was independently observed in vitro and via analysis of urines of individuals that are either efficient metabolizer phenotypes or poor metabolizer phenotypes for the two polymorphic enzymes. Although interindividual differences in the monitored metabolic patterns were noted, no marked difference could be related to a CYP2D6 or CYP2C19 polymorphism.     

Development of a method to measure methadone enantiomers and its metabolites without enantiomer standard compounds for the plasma of methadone maintenance patients

A liquid chromatography–photodiode array (LC‐PDA) method using a chiral analytical column was developed to determine the plasma levels of enantiomers of methadone and its chiral metabolite, 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), without the standard compounds of R‐form or S‐form enantiomers. This method was established by the characteristics of recombinant cytochrome P‐450 (CYP) isozymes, where CYP2C19 prefers to metabolize R‐methadone and CYP2B6 prefers to metabolize S‐methadone. We incubated the racemic methadone standard with either enzyme for 24 h. We identified the retention times of R‐ and S‐methadone to be around 10.72 and 14.46 min, respectively. Furthermore, we determined the retention times of R‐ and S‐EDDP to be approximately 6.76 and 7.72 min, respectively. No interferences were shown through the retention times of morphine, buprenorphine and diazepam. With the high recovery rate of a solid‐phase extraction procedure, this method was applied in analyzing plasma concentrations of seven methadone maintenance patients where R‐ and S‐methadone and R‐ and S‐EDDP were 233.4 ± 154.9 and 185.9 ± 136.3 ng/mL and 84.4 ± 99.4 and 37.6 ± 22.9 ng/mL, respectively. These data suggest that the present method can be applied for routine assay for plasma methadone and EDDP concentrations for patients under treatment. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro

Ketamine, a phencyclidine derivative, is used for induction of anesthesia, as an anesthetic drug for short term surgical interventions and in subanesthetic doses for postoperative pain relief. Ketamine undergoes extensive hepatic first-pass metabolism. Enantioselective capillary electrophoresis with multiple isomer sulfated β-cyclodextrin as chiral selector was used to identify cytochrome P450 enzymes involved in hepatic ketamine and norketamine biotransformation in vitro. The N-demethylation of ketamine to norketamine and subsequently the biotransformation of norketamine to other metabolites were studied via analysis of alkaline extracts of in vitro incubations of racemic ketamine and racemic norketamine with nine recombinantly expressed human cytochrome P450 enzymes and human liver microsomes. Norketamine was formed by CYP3A4, CYP2C19, CYP2B6, CYP2A6, CYP2D6 and CYP2C9, whereas CYP2B6 and CYP2A6 were identified to be the only enzymes which enable the hydroxylation of norketamine. The latter two enzymes produced metabolic patterns similar to those found in incubations with human liver microsomes. The kinetic data of ketamine N-demethylation with CYP3A4 and CYP2B6 were best described with the Michaelis–Menten model and the Hill equation, respectively. This is the first study elucidating the individual enzymes responsible for hydroxylation of norketamine. The obtained data suggest that in vitro biotransformation of ketamine and norketamine is stereoselective.     

Capillary electrophoretic investigation of the enantioselective metabolism of propafenone by human cytochrome P-450 SUPERSOMES: Evidence for atypical kinetics by CYP2D6 and CYP3A4

An enantioselective CE method was used to identify the ability of CYP450 enzymes and their stereoselectivity in catalyzing the transformation of propafenone (PPF) to 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF). Using in vitro incubations with single CYP450 enzymes (SUPERSOMES), 5OH-PPF is shown to be selectively produced by CYP2D6 and N-dealkylation is demonstrated to be mediated by CYP2D6, CYP3A4, CYP1A2, and CYP1A1. For the elucidation of kinetic aspects of the metabolism with CYP2D6 and CYP3A4, incubations with individual PPF enantiomers and racemic PPF were investigated. With the exception of the dealkylation in presence of R-PPF only, which can be described by the Michaelis-Menten model, all CYP2D6-induced reactions were found to follow autoactivation kinetics. For CYP3A4, all NOR-PPF enantiomer formation rates as function of PPF enantiomer concentration were determined to follow substrate inhibition kinetics. The formation of NOR-PPF by the different enzymes is stereoselective and is reduced significantly when racemic PPF is incubated. Clearance values obtained for CYP3A4 dealkylation are stereoselective whereas those of CYP2D6 hydroxylation are not. This paper reports the first investigation of the PPF hydroxylation and dealkylation kinetics by the CYP2D6 enzyme and represents the first report in which enantioselective CE data provide the complete in vitro kinetics of metabolic steps of a drug.     

Characterization of the stereoselective metabolism of thiopental and its metabolite pentobarbital via analysis of their enantiomers in human plasma by capillary electrophoresis.

Using capillary zone electrophoresis (CZE) with a 75 mM phosphate buffer at pH 8.5 containing 5 mM hydroxypropyl-gamma-cyclodextrin (OHP-gamma-CD) as chiral selector, the separation of the enantiomers of thiopental and its oxybarbiturate metabolite, pentobarbital, is reported. Enantiomer assignment was performed via preparation of enantiomerically enriched fractions using chiral recycling isotachophoresis (rITP) processing of racemic barbiturates and analysis of rITP fractions by chiral CZE and circular dichroism spectroscopy. Thiopental and pentobarbital enantiomers in plasma were extracted at low pH using dichloromethane and extracts were reconstituted in acetonitrile or 10-fold diluted, achiral running buffer. The stereoselectivity of the thiopental and pentobarbital metabolism was assessed via analysis of 12 plasma samples that stemmed from patients undergoing prolonged or having completed long-term racemic thiopental infusion. The data obtained revealed a modest stereoselectivity with R-(+)-thiopental/S-(-)-thiopental and R-(+)-pentobarbital/S-(-)-pentobarbital plasma ratios being < 1 (P < 0.05 compared to data obtained with racemic controls) and > 1 (P < 0.001), respectively. The total S-(-)-thiopental plasma concentration was found to be on average about 24% higher compared to the concentration of R-(+)-thiopental, whereas the total R-(+)-pentobarbital plasma level was observed to be on average 29% higher compared to the S-(-)-pentobarbital concentration.     

Efficient nitro-aldol reaction using SmI2: a new route to nitro alcohols under very mild conditions

A novel method to obtain racemic 1-nitroalkan-2-ols by reaction of bromonitromethane with a variety of aldehydes and promoted by SmI2 is reported. On the basis of these results, the chiral version has also been performed with chiral N,N-dibenzyl amino aldehydes, affording the corresponding enantiopure 3-amino-1-nitroalkan-2-ols with good stereoselectivity.     

Site-Directed Mutagenesis of Cytochrome P450 2D6 and 2C19 Enzymes: Expression and Spectral Characterization of Naturally Occurring Allelic Variants

Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.     

Rapid genotyping of CYP2D6, CYP2C19 and TPMT polymorphisms by primer extension reaction in a dipstick format

In recent years an increasing amount of interest has been directed at the study and routine testing of polymorphisms responsible for variations in drug metabolism. Most of the current methods involve either time-consuming electrophoresis steps or specialized and expensive equipment. In this context, we have developed a rapid, simple and robust method for genotyping of CYP2D6*3, CYP2D6*4, CYP2C19*2, CYP2C19*3 and TPMT*2 single nucleotide polymorphisms (SNP). Genomic DNA is isolated from whole blood and the segments that span the SNP of interest are amplified by PCR. The products are subjected directly (without purification) to two primer extension (PEXT) reactions (three cycles each) using normal and mutant primers in the presence of biotin-dUTP. The PEXT primers contain a (dA)₃₀ segment at the 5′ end. The PEXT products are detected visually by a dry-reagent dipstick-type assay in which the biotinylated extension products are captured from immobilized streptavidin on the test zone of the strip and detected by hybridization with oligo(dT)-functionalized gold nanoparticles. Patient samples (76 variants in total) were genotyped and the results were fully concordant with those obtained by direct DNA sequencing.     

New low molar mass organosiloxanes with unusual ferroelectric properties

A new low molar mass chiral organosiloxane mesogen and its racemic analogue have been synthesized and their mesomorphic and ferroelectric properties investigated. The chiral derivative, denoted A*B, exhibits one tilted enantiotropic ferroelectric smectic C mesophase over a broad temperature range, with very high tilt angles and moderate spontaneous polarization (36° and 19 nC cm^-2 at 20°C). The achiral siloxane derivative, denoted A*B, exhibits one broad enantiotropic smectic C phase. Preliminary electro-optic measurements indicate that the spontaneous polarization is weakly dependent on temperature between 10°C and 50°C, the latter being the S*_c to isotropic phase transition. The tilt angle and layer spacing are temperature independent, and current response times of less than 200 μs were measured at 25°C for fields of 10 V μ^-1. These results are discussed in comparison with those for side chain polymer liquid crystal structures and other low molar mass ferroelectric materials.     

Identification of cytochrome P450 isoforms involved in the metabolism of artocarpin and assessment of its drug–drug interaction

Artocarpin isolated from an agricultural plant Artocarpus communis has shows anti‐inflammation and anticancer activities. In this study, we utilized recombinant human UDP‐glucuronosyltransferasesupersomes (UGTs) and human liver microsomes to explore its inhibitory effect on UGTs and cytochrome p450 enzymes (CYPs). Chemical inhibition studies and screening assays with recombinant human CYPs were used to identify if CYP isoform is involved in artocarpin metabolism. Artocarpin showed strong inhibition against UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, CYP2C8 and CYP3A4. In particular, artocarpin exhibited competitive inhibition against CYP3A4 and noncompetitive inhibition against UGT1A3 and UGT1A7. The half inhibition concentration values for CYP3A4, UGT1A3 and UGT1A7 were 4.67, 3.82 and 4.82 μm , and the inhibition kinetic parameters for them were 0.78, 2.67 and 3.14 μm , respectively. After artocarpin was incubated in human liver microsomes and determined by HPLC, we observed its main metabolites (M1 and M2). In addition, we proved that CYP2D6 played the key role in the biotransformation of artocarpin in human liver microsomes. The result of molecular docking further confirmed that artocarpin interacted with CYP2D6, CYP2C8 and CYP3A4 through hydrogen bonds. This study provided preliminary results for further research on artocarpin or artocarpin‐containing herbs.     

In vitro interaction cocktail assay for nine major cytochrome P450 enzymes with 13 probe reactions and a single LC/MSMS run: analytical validation and testing with monoclonal anti-CYP antibodies

A sensitive and rugged LC/MSMS method was developed for a comprehensive in vitro metabolic interaction screening assay with N-in-1 approach reported earlier. A cocktail consisting of ten cytochrome P450 (CYP)-selective probe substrates with known kinetic, metabolic and interaction properties in vivo was incubated in a pool of human liver microsomes, and metabolites of melatonin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), amodiaquine (CYP2C8) tolbutamide (CYP2C9), omeprazole (CYP2C19 and CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1), midazolam (CYP3A4) and testosterone (CYP3A4) were simultaneously analysed with a single LC/MSMS run. Altogether, 13 metabolites and internal standard phenacetin were analysed in multiple reaction mode. Polarity switching mode was utilized to acquire negative ion mode electrospray data for hydroxychlorzoxazone and positive ionization data for the rest of the analytes. Fast gradient elution was applied, giving total injection cycle of 8 min. The method was modified for two different LC/MSMS systems, and was validated for linear range, detection limit, accuracy and precision for each metabolite. In addition, cocktail inhibition system was further tested using monoclonal anti-CYP antibodies as inhibitors for each probe reaction.     

Phenotyping of CYP450 in human liver microsomes using the cocktail approach

The cocktail approach is an advantageous strategy used to monitor the activities of several cytochromes P450 (CYPs) in a single test to increase the throughput of in vitro phenotyping studies. In this study, a cocktail mixture was developed with eight CYP-specific probe substrates to simultaneously evaluate the activity of the most important CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and the CYP3A subfamily. After cocktail incubation in the presence of human liver microsomes (HLMs), the eight selected substrates and their specific metabolites were analyzed by ultra-high-pressure liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry. Qualitative and quantitative data were simultaneously acquired to produce an overview of the extended phase I biotransformation routes for each probe substrate in the HLMs and to generate phenotypic profiles of various HLMs. A comparison of the cocktail strategy with an individual substrate assay for each CYP produced similar results. Moreover, the cocktail was tested on HLMs with different allelic variants and/or in the presence of selective inhibitors. The results were in agreement with the genetic polymorphisms of the CYPs and the expected effect of the alterations. All of these experiments confirmed the reliability of this cocktail assay for phenotyping of the microsomal CYPs.     

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography for the analysis of methadone and its metabolites in serum of heroin addicts

Methadone (MET) metabolism has been largely demonstrated with a high inter-individual variability and, therefore, quantification of MET is very important for therapeutic drug monitoring. A cation-selective exhaustive injection and sweeping MEKC (CSEI-Sweeping) was first developed to analyze MET and its two metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP), in human serum. After pretreatment, the samples were electrokinetically injected into capillary (10 kV, 500s) and swept by the separation phosphate buffer (100 mM, pH 4.0) containing 20% tetrahydrofuran and 100 mM SDS at -15 kV. The LODs were 200 pg/mL for MET and EMDP, and 400 pg/mL for EDDP. Ten volunteers were administered MET (5.0-120.0 mg/day) orally for 84 days and serum samples were taken after the daily dose of MET (days 1, 2, 7, 14, 28, 56 and 84) individually. This method was used for monitoring MET and its metabolites in heroin addicts and for pharmacokinetic investigations.     

Corydaline inhibits multiple cytochrome P450 and UDP-glucuronosyltransferase enzyme activities in human liver microsomes

Corydaline is a bioactive alkaloid with various antiacetylcholinesterase, antiallergic, and antinociceptive activities found in the medicinal herb Corydalis Tubers. The inhibitory potential of corydaline on the activities of seven major human cytochrome P450 and four UDP-glucuronosyltransferase enzymes in human liver microsomes was investigated using LC-tandem MS. Corydaline was found to inhibit CYP2C19-catalyzed S-mephenytoin-4'-hydroxylatoin and CYP2C9-catalyzed diclofenac 4-hydroxylation, with K(i) values of 1.7 and 7.0 mM, respectively. Corydaline also demonstrated moderate inhibition of UGT1A1-mediated 17b-estradiol 3-glucuronidation and UGT1A9-mediated propofol glucuronidation with K(i) values of 57.6 and 37.3 mM, respectively. In the presence of corydaline, CYP3A-mediated midazolam hydroxylation showed a decrease with increasing preincubation time in a dose-dependent manner with K(i) values of 30.0 mM. These in vitro results suggest that corydaline should be evaluated for potential pharmacokinetic drug interactions in vivo due to potent inhibition of CYP2C19 and CYP2C9.     

A sensitive and high‐throughput LC‐MS/MS method for inhibition assay of seven major cytochrome P450s in human liver microsomes using an in vitro cocktail of probe substrates

A sensitive and high‐throughput LC‐MS/MS method was established and validated for the simultaneous quantification of seven probe substrate‐derived metabolites (cocktail assay) for assessing the in vitro inhibition of cytochrome P450 (CYP) enzymes in pooled human liver microsomes. The metabolites acetaminophen (CYP1A2), hydroxy‐bupropion (CYP2B6), n‐desethyl‐amodiaquine (CYP2C8), 4′‐hydroxy‐diclofenac (CYP2C9), 4′‐hydroxy‐mephenytoin (CYP2C19), dextrorphan (CYP2D6) and 1′‐hydroxy‐midazolam (CYP3A4/5), together with the internal standard verapamil, were eluted on an Agilent 1200 series liquid chromatograph in <7 min. All metabolites were detected by an Agilent 6410B tandem mass spectrometer. The concentration of each probe substrate was selected by substrate inhibition assay that reduced potential substrate interactions. CYP inhibition of seven well‐known inhibitors was confirmed by comparing a single probe substrate assay with cocktail assay. The IC₅₀ values of these inhibitors determined on this cocktail assay were highly correlated (R² > 0.99 for each individual probe substrate) with those on single assay. The method was selective and showed good accuracy (85.89–113.35%) and between‐day (RSD <13.95%) and within‐day (RSD <9.90%) precision. The sample incubation extracts were stable at 25 °C for 48 h and after three freeze–thaw cycles. This seven‐CYP inhibition cocktail assay significantly increased the efficiency of accurately assessing compounds’ potential inhibition of the seven major CYPs in drug development settings. Copyright © 2014 John Wiley & Sons, Ltd.     

Evaluation of the effects of Mitragyna speciosa alkaloid extract on cytochrome P450 enzymes using a high throughput assay

The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE) on human recombinant cytochrome P450 (CYP) enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC(50)) values of 0.78 μg/mL and 0.636 μg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC(50) of 39 μg/mL, and weak inhibition was detected for CYP2C19. The IC(50) of CYP2C19 could not be determined, however, because inhibition was <50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6), ketoconazole (CYP3A4), tranylcypromine (CYP2C19) and furafylline (CYP1A2) were ACCESSused as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.     

The stereoselective α-alkylation of chiral β-hydroxy esters and some applications thereof

The stereoselectivity of the α-alkylation of chiral β-hydroxy ester is discussed. The configuration of the alkylated product was proved chemically (Scheme 2). A one pot aldol-alkylation reaction was developed leading stereoselectively to racemic (s*,s*)-α-alkyl-β-hydroxy ester (Scheme 3,4). Baker's yeast reduction of 2-alkyl-3-keto ester led to valuable chiral (2RS,3S)-intermediates, which were converted via the corresponding dianion to compounds with a chiral quaternary C atom (Scheme 6). Synthetic applications of the above findings are shown in the synthesis of various chiral compounds (Scheme 8 and 9).     

卡那霉素作为手性选择剂的毛细管电泳手性药物分离研究

建立了一种以天然易得的卡那霉素为手性添加剂，用毛细管区带电泳法快速分离市售对乙肝有良好治疗效果的药物联苯双脂衍生物的方法，拓宽了毛细管电泳中手性选择剂的范围，通过实验研究了卡那霉素、甲醇 含量PH值，磷酸盐缓冲体系和硼硝缓冲体系对手性分离的影响，以及三种有机溶剂（甲醇、乙晴、异丙醇）添加剂对手性分离的影响，结果表明，在含有3％卡那霉素，30mol/L，硼砂缓冲体系（PH＝8．0）添加30％异丙醇是最佳的分离条件。     

Fast chiral separations using sulfated beta-cyclodextrin and short-end injection in capillary electrophoresis

The general applicability of sulfated beta-cyclodextrin as chiral selector and short-end injection in capillary electrophoresis (CE) as a powerful screening tool for fast and efficient chiral separation of Ormeloxifene enantiomers and racemic Ormeloxifene analogues is demonstrated. Using the short-end injection procedure, all of the 16 racemic compounds studied were successfully separated with high efficiencies and with analysis times of less than 1.2 min. Furthermore, long-end injections of eight analogues named C1-C8 afforded separations with extremely high efficiencies. A statistical evaluation of the resolution values obtained in short-end and long-end injections of compounds C1-C8 showed that the sensitivity of the CE method towards structural changes in the studied molecules is intact when the chiral analysis is performed with short-end injection compared to conventional long-end injection.     

Synthesis of novel cyclohexanediol-derived chiral phosphite ligands and their application in the Cu-catalyzed conjugate addition of organozinc to cyclic enones

A series of novel chiral diphosphite ligands have been synthesized from (1R,2R)-trans-1,2-cyclohexanediol, (1S,2S)-trans-1,2-cyclohexanediol, racemic trans-1,2-cyclohexanediol and chlorophosphoric acid diary ester, and were successfully employed in the Cu-catalyzed asymmetric 1,4-conjugate addition of diethylzinc to cyclohexenone with up to 99% ee. It was found that ligand 1,2-bis[(R)-1,1'-binaphthyl-2,2'-diyl]phosphitecyclohexanediol 6a derived from racemic diol skeleton can show similar catalytic performance compared with ligand (1R,2R)-bis[(R)-1,1'-binaphthyl-2,2'-diyl]phosphitecyclohexanediol 6a' derived from enantiopure startingmaterial. A significant dependence of stereoselectivity on the type of enone and the ring size of the cyclic enone was observed. Moreover, the configuration of the products was mainly determined by the configuration of the binaphthyl moieties of diphosphite ligands in the 1,4-addition of cyclic enones.