Objective To establish an LC-MS/MS method for simultaneous determination of hydrocortisone and 6β-hydroxycortisol concentrations in human urine. Methods Hydrocortisone-d4 and 6β-hydroxycortisone-d4 were selected as internal standards(IS), and the urine samples were treated with methanol containing IS for analysis. The chromatographic column was a CORTECS® C18+ column(2.1 mm×50 mm,2.7 μm), and the mobile phase was methanol and water containing 0.1% formic acid. A gradient elution was applied with a flow rate of 0.6 mL·min-1. MS/MS detection was operated in a positive mode by multiple reaction monitoring. The detection ions for quantitative analysis were m/z 363.2→121.1 for cortisol, m/z 367.2→121.1 for hydrocortisone-d4, m/z 379.2→325.2 for 6β-hydroxycortisol, and m/z 383.2→328.2 for 6β-hydroxycortisol-d4, respectively. Results The detecting linearity for hydrocortisone and 6β-hydroxycortisol was good within the range of 1.00~300 ng·mL-1 and 5.00~1500 ng·mL-1, respectively, and the lower limit of quantification was 1.00 ng·mL-1 and 5.00 ng·mL-1, respectively. The precision within and between batches was less than 15.0%, and the accuracy was within the range of -6.9% to 5.7%. The matrix effect was 95.6%~106.0%, and the recovery was 97.2%~108.3%. The urine sample was stable at 25 ℃ for 10 hours, or in 3 circles of freezing and thawing, or at -80 ℃ for 4 months. And the supernatant after preparation was stable at 10 ℃ for 24 hours in the sampler. Conclusion In this study, a sensitive, accurate, convenient, and rapid method for simultaneous determination of hydrocortisone and 6β-hydroxycortisone concentrations in human urine was established, which could be applied for the evaluation of human CYP3A activity.
API 4000质谱仪,配备电喷雾离子源及Analyst1.6.3数据处理系统(美国应用生物公司);1290 Infinity高效液相色谱仪,包括G1316C柱温箱、G4226A多孔板自动进样器和G4220A二元高压泵(美国安捷伦科技有限公司);BP211D 电子天平(德国Sartorius公司,感量:0.01 mg);Stratos离心机(德国Thermo Fisher公司);Mixmate涡旋混匀仪(德国Eppendorf公司)。
1.2 试药
氢化可的松(德国Dr.Ehrenstorfer GmbH公司,批号:41210,纯度:99.8%);6β-羟基氢化可的松(美国Sigma-Aldrich公司,批号:SLBN4942V,纯度≥98%);d4-氢化可的松(加拿大Toronto Research Chemicals公司,批号:5-KSS-11-4,纯度:99%);d4-6β-羟基氢化可的松(加拿大Toronto Research Chemicals公司,批号:1-JMR-119-2,纯度:95%);甲醇为色谱纯(美国Merck公司);甲酸为色谱纯(美国Aladdin公司);超纯水(18.2 MΩ)由Millipore纯水仪制得。
Fig.1
Chromatograms of hydrocortisone,6β-hydroxycortisone and their internal standards in water and urine 1.blank sample;B.quality control sample at medium dose;C.blank urine sample;D.Urine sample containing internal standard
LUOX,LI XM,HU ZY,et al.Evaluation of CYP3A activity in humans using three different parameters based on endogenous cortisol metabolism[J].Acta Pharmacol Sinica,2009,30(9):1323-1329.
Currently, there is considerable debate as to which method is more accurate for measuring the activity of CYP3A in vivo: cortisol 6beta-hydroxylation clearance (Cl(m(6beta))) or the urinary ratio of 6beta-OHF to F (6beta-OHF/F). Furthermore, the value of measuring endogenous levels of cortisol over a 24 h period (AUC(F)) needs to be confirmed. The aim of the present study was to determine which method was most effective at measuring changes in the in vivo activity of CYP3A: AUC(F), Cl(m(6beta)), or 6beta-OHF/F.
WILLIAMS JA,RING BJ,CANTRELL VE,et al.Comparative metabolic capabilities of CYP3 A4,CYP3A5,and CYP3A7[J].Drug Metab Dispos,2002,30(8):883-891.
The human cytochromes P450 (P450) CYP3A contribute to the biotransformation of 50% of oxidatively metabolized drugs. The predominant hepatic form is CYP3A4, but recent evidence indicates that CYP3A5 contributes more significantly to the total liver CYP3A than was originally thought. CYP3A7 is the major fetal form and is rarely expressed in adults. To compare the metabolic capabilities of CYP3A forms for 10 substrates, incubations were performed using a consistent molar ratio (1:7:9) of recombinant CYP3A, P450 reductase, and cytochrome b5. A wide range of substrate concentrations was examined to determine the best fit to kinetic models for metabolite formation. In general, K(m) or S(50) values for the substrates were 3 to 4 times lower for CYP3A4 than for CYP3A5 or CYP3A7. For a more direct comparison of these P450 forms, clearance to the metabolites was determined as a linear relationship of rate of metabolite formation for the lowest substrate concentrations examined. The clearance for 1'-hydroxy midazolam formation at low substrate concentrations was similar for CYP3A4 and CYP3A5. For CYP3A5 versus CYP3A4, clearance values at low substrate concentrations were 2 to 20 times lower for the other biotransformations. The clearance values for CYP3A7-catalyzed metabolite formation at low substrate concentrations were substantially lower than for CYP3A4 or CYP3A5, except for clarithromycin, 4-OH triazolam, and N-desmethyl diltiazem (CYP3A5 - CYP3A7). The CYP3A forms demonstrated regioselective differences in some of the biotransformations. These results demonstrate an equal or reduced metabolic capability for CYP3A5 compared with CYP3A4 and a significantly lower capability for CYP3A7.
YIN OQ,SHIX,TOMLINSONB,et al.Interindividual and intraindividual variability of the urinary 6beta-Hydroxycortisol/Cortisol ratio in Chinese subjects:implications of its use for evaluating CYP3A activity[J].J Clin Pharmacol,2004,44(12):1412-1417.
The present study determined the interindividual and intrandividual variability of the urinary 6beta-hydroxycortisol/cortisol ratio, a useful marker for CYP3A induction and inhibition in Chinese subjects. The study consisted of 2 parts. In part I, 82 healthy male Chinese subjects underwent 3 study sessions, each separated by a 1-week interval. In part II, 20 subjects who initially completed part I underwent another 3 sessions over a period of 3 to 4 months. During each session, a first-morning urine specimen was collected from each subject for the quantification of urinary concentrations of cortisol and 6beta-hydroxycortisol. There were no significant differences in the mean 6beta-hydroxycortisol/cortisol ratios among the 3 sessions (P > .05, 1-way analysis of variance) for both part I and part II of the study. A normal distribution of the 6beta-hydroxycortisol/cortisol ratio was observed (P = .849, Kolmogorov-Smirnov test). This ratio varied 30-fold (range, 0.76-23.23) among the study subjects. The mean intraindividual variabilities during the short (3-week) and long (3- to 4-month) periods were 30.9% +/- 17.5% and 32.2% +/- 17.1%, respectively. The genetic fraction contributing to the observed variability in the 6beta-hydroxycortisol/cortisol ratio was estimated to be 0.91. The genetic component is likely to contribute significantly to the variability of the 6beta-hydroxycortisol/cortisol ratio, and such variability should be considered when the ratio is used to evaluate CYP3A induction or inhibition in a given ethnic population.
WOOLSEY SJ,BEATON MD,CHOI YH,et al.Relationships between endogenous plasma biomarkers of constitutive cytochrome P450 3A activity and single-time-point oral midazolam microdose phenotype in healthy subjects[J].Basic Clin Pharmacol Toxicol,2016,118(4):284-291.
Due to high basal interindividual variation in cytochrome P450 3A (CYP3A) activity and susceptibility to drug interactions, there has been interest in the application of efficient probe drug phenotyping strategies, as well as endogenous biomarkers for assessment of in vivo CYP3A activity. The biomarkers 4β-hydroxycholesterol (4βHC) and 6β-hydroxycortisol (6βHCL) are sensitive to CYP3A induction and inhibition. However, their utility for the assessment of constitutive CYP3A activity remains uncertain. We investigated whether endogenous plasma biomarkers (4βHC and 6βHCL) are associated with basal CYP3A metabolic activity in healthy subjects assessed by a convenient single-time-point oral midazolam (MDZ) phenotyping strategy. Plasma 4βHC and 6βHCL metabolic ratios (MRs) were analysed in 51 healthy adult participants. CYP3A activity was determined after administration of an oral MDZ microdose (100 μg). Simple linear and multiple linear regression analyses were performed to assess relationships between MDZ oral clearance, biomarkers and subject covariates. Among study subjects, basal MDZ oral clearance, 4βHC and 6βHCL MRs ranged 6.5-, 10- and 13-fold, respectively. Participant age and alcohol consumption were negatively associated with MDZ oral clearance (p = 0.03 and p = 0.045, respectively), while weight and female sex were associated with lower plasma 4βHC MR (p = 0.0003 and p = 0.032, respectively). Neither 4βHC nor 6βHCL MRs were associated with MDZ oral clearance. Plasma 4βHC and 6βHCL MRs do not relate to MDZ single-time-point metabolic phenotype in the assessment of constitutive CYP3A activity among healthy individuals.
STREETMAN DS,BERTINO JS J,NAFZIGER AN.Phenotyping of drug-metabolizing enzymes in adults:a review of in-vivo cytochrome P450 phenotyping probes[J].Pharmacogenetics,2000,10(3):187-216.
Cytochrome P450 phenotyping provides valuable information about real-time activity of these important drug-metabolizing enzymes through the use of specific probe drugs. Despite more than 20 years of research, few conclusions regarding optimal phenotyping methods have been reached. Caffeine offers many advantages for CYP1A2 phenotyping, but the widely used caffeine urinary metabolic ratios may not be the optimal method of measuring CYP1A2 activity. Several probes of CYP2C9 activity have been suggested, but little information exists regarding their use, largely due to the narrow therapeutic index of most CYP2C9 probes. Mephenytoin has long been considered the standard CYP2C19 phenotyping probe, but problems such as sample stability and adverse effects have prompted the investigation of potential alternatives, such as omeprazole. Several well-validated CYP2D6 probes are available, including dextromethorphan, debrisoquin and sparteine, but, in most cases, dextromethorphan may be preferred due to its wide safety margin and availability. Chlorzoxazone remains the only CYP2E1 probe that has received much study. However, questions concerning phenotyping method and involvement of other enzymes have impaired its acceptance as a suitable CYP2E1 phenotyping probe. CYP3A phenotyping has been the subject of numerous investigations, reviews and commentaries. Nevertheless, much controversy regarding the selection of an ideal CYP3A probe remains. Of all the proposed methods, midazolam plasma clearance and the erythromycin breath test have been the most rigorously studied and appear to be the most reliable of the available methods. Despite the limitations of many currently available probes, with continued research, phenotyping will become an even more valuable research and clinical resource.
Chan SW,XIAOY,HUM,et al.Associations of the CYP-3A5*3 and CYP3A4*1G polymorphisms with the pharmacokinetics of oral midazolam and the urinary 6β-hydroxycortisol/cortisol ratio as markers of CYP3A activity in healthy male Chinese[J].J Clin Pharm Ther,2016,41(5):552-558.
The oral plasma clearance of midazolam and the ratio of 6β-hydroxycortisol (6β-OHF) to cortisol (F) in urine are two potential markers for evaluating CYP3A activity in vivo. We assessed the influence of two common CYP3A polymorphisms on the pharmacokinetics of oral midazolam and urinary ratio of 6β-OHF/F in healthy Chinese.
LIU YT,HAO HP,LIUCX,et al.Drugs as CYP3A probes,inducers,and inhibitors[J].Drug Metab Rev,2017,39(4):699-721.
Human cytochrome P450 (CYP) 3A subfamily members (mainly CYP3A4 and CYP3A5) mediate the metabolism of approximately half all marketed drugs and thus play a critical role in the drug metabolism. A huge number of studies on CYP3A-mediated drug metabolism in humans have demonstrated that CYP3A activity exhibits marked ethnic and individual variability, in part because of altered levels of CYP3A4 expression by various environmental factors and functionally important polymorphisms present in CYP3A5 gene. Accumulating evidence has revealed that CYP3A4 and CYP3A5 have a significant overlapping in their substrate specificity, inducers and inhibitors. Therefore, it is difficult to define their respective contribution to drug metabolism and drug-drug interactions. Furthermore, P-glycoprotein and CYP3A are frequently co-expressed in the same cells and share a large number of substrates and modulators. The disposition of such drugs is thus affected by both metabolism and transport. In this review, we systematically summarized the frequently used CYP3A probe drugs, inducers and inhibitors, and evaluated their current status in drug development and research.
PENG CC,TEMPLETONI,THUMMEL KE,et al.Evaluation of 6β-hydroxycortisol,6β-hydroxycortisone,and a combination of the two as endogenous probes for inhibition of CYP3A4 in vivo[J].Clin Pharmacol Ther,2011,89(6):888-895.
An endogenous probe for CYP3A activity would be useful for early identification of in vivo cytochrome P450 (CYP) 3A4 inhibitors. The aim of this study was to determine whether formation clearance (CL(f)) of the sum of 6β-hydroxycortisol and 6β-hydroxycortisone is a useful probe of CYP3A4 inhibition in vivo. In human liver microsomes (HLMs), the formation of 6β-hydroxycortisol and 6β-hydroxycortisone was catalyzed by CYP3A4, and itraconazole inhibited these reactions with half maximal inhibitory concentration (IC(50))(,u) values of 3.1 nmol/l and 3.4 nmol/l, respectively. The in vivo IC(50,u) value of itraconazole for the combined CL(f) of 6β-hydroxycortisone and 6β-hydroxycortisol was 1.6 nmol/l. The greater inhibitory potency in vivo is probably due to circulating inhibitory itraconazole metabolites. The maximum in vivo inhibition was 59%, suggesting that f(m,CYP3A4) for cortisol and cortisone 6β-hydroxylation is ~60%. Given the significant decrease in CL(f) of 6β-hydroxycortisone and 6β-hydroxycortisol after 200-mg and 400-mg single doses of itraconazole, this endogenous probe can be used to detect moderate and potent CYP3A4 inhibition in vivo.
LUOX,LIX,HUZ,et al.Evaluation of CYP3A activity in humans using three different parameters based on endogenous cortisol metabolism[J].Acta Pharmacologica Sinica,2009,30(9):1323-1329.
Currently, there is considerable debate as to which method is more accurate for measuring the activity of CYP3A in vivo: cortisol 6beta-hydroxylation clearance (Cl(m(6beta))) or the urinary ratio of 6beta-OHF to F (6beta-OHF/F). Furthermore, the value of measuring endogenous levels of cortisol over a 24 h period (AUC(F)) needs to be confirmed. The aim of the present study was to determine which method was most effective at measuring changes in the in vivo activity of CYP3A: AUC(F), Cl(m(6beta)), or 6beta-OHF/F.
JUIFPE,BOEHLERM,DONAZZOLOY,et al.A pharmacokinetic drug-drug interaction study between selexipag and midazolam,a CYP3A4 substrate,in healthy male subjects[J].Eur J Clin Pharmacol,2017,73(9):1121-1128.
In vitro data showed that selexipag and its active metabolite (ACT-333679) have an inductive effect on CYP3A4, CYP2B6, and CYP2C9 at concentrations approximately 100-fold higher than the maximum plasma concentration (C max) measured under steady-state conditions. In order to confirm in vivo the lack of induction at the enterocyte level, we assessed the effect of selexipag on midazolam, a substrate of hepatic and intestinal CYP3A4.
RIVORY LP,SLAVIEROK,SEALE JP,et al.Optimizing the erythromycin breath test for use in cancer patients[J].Clin Cancer Res,2000,6(9):3480-3485.
The erythromycin breath test (EBT) is a putative in vivo probe for drug metabolism by cytochrome P450 3A4 (CYP3A4). Because many anticancer drugs are metabolized by this system, we sought to further develop the EBT as a tool for predicting the clearance, in cancer patients, of drugs metabolized by CYP3A4. Sixteen adult patients with incurable cancer were studied. The EBT was performed on day 1 and breath sampled after the i.v. injection of 4 microCi of 14C-erythromycin. The breath 14CO2 flux (CERt) was estimated at 11 time points over 2 h. On day 2, the EBT was repeated midway through a 10-min infusion of 100 mg of erythromycin lactobionate, and the plasma pharmacokinetics of erythromycin were determined. The infusion of 100 mg of erythromycin did not modify the EBT results significantly. The values of the conventional EBT parameter CER20 min obtained on day 1 were comparable for most subjects (0.03-0.06% dose/min), with the exception of an individual receiving the known CYP3A4 inducers dexamethasone and phenytoin who returned a value of 0.14% dose/min. There was no significant correlation between any of the conventional EBT parameters and erythromycin clearance. However, two parameters reflecting early emergence of breath radioactivity (1/TMAX and CER3 min/CERMAX) correlated significantly with erythromycin clearance (P = 0.005 and 0.006, respectively). Novel parameters derived from the EBT are significantly correlated with the clearance of erythromycin even in the presence of confounding factors, such as metastatic liver disease, altered protein binding, and comedication. These parameters may enable dose optimization of cytotoxics metabolized by CYP3A4.
KURNIKD,WOOD AJ,WILKINSON GR.The erythromycin breath test reflects P-glycoprotein function independently of cytochrome P450 3A activity[J].Clin Pharmacol Ther,2006,80(3):228-324.
The erythromycin breath test (ERBT) has been widely used as a phenotypic measure of cytochrome P450 (CYP) 3A activity in individuals, as well as its modulation by inhibitors or inducers. However, it is not entirely clear what this measure actually reflects because, in addition to CYP3A, animal studies suggest that P-glycoprotein is also involved in erythromycin's hepatic disposition. Thus studies were undertaken to determine the effect of tariquidar, a potent P-glycoprotein inhibitor that does not affect CYP3A activity, on the ERBT and on the CYP3A-mediated metabolism of midazolam, a non-P-glycoprotein substrate.
LEEJ,KIM AH,YIS,et al.Distribution of exogenous and endogenous CYP3A markers and related factors in healthy males and females[J].AAPS J,2017,19(4):1196-1204.
Cytochrome P450 (CYP) 3A is an important drug-metabolizing enzyme in humans. Assessing CYP3A activity is necessary for predicting therapeutic outcomes or the potential adverse events of various therapeutics. This study sought to evaluate the distribution of endogenous and exogenous markers reflecting hepatic CYP3A activity and related factors affecting its activity in healthy male and female. Each subject was given a single 1 mg dose of midazolam intravenously. Pharmacokinetics, pharmacometabolomics, and pharmacogenomics analyses were performed to evaluate CYP3A activity. Urinary and plasma steroids were quantified with gas chromatography coupled with triple-quadrupole mass spectrometry (GC-MS), and the concentrations of midazolam and its metabolites were quantified with liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS). A total of 100 subjects completed this study. Midazolam clearance (MDZ CL) and the metabolic ratio (MDZ MR) were significantly correlated with 6β-OH-cortisol/cortisol and 6β-OH-cortisone/cortisone. MDZ CL, 6β-OH-cortisol/cortisol, and 6β-OH-cortisone/cortisone decreased with increasing age (Pearson r = -0.333, -0.329, and -0.528, respectively; P < 0.05). When the markers were compared according to sex, MDZ CL and 6β-OH-cortisol/cortisol showed significant difference between sexes. However, MDZ CL was higher in female group than male group and 6β-OH-cortisol/cortisol was higher in male group than female group. No significant differences in markers were found when comparing progesterone levels. Our results indicate that both exogenous and endogenous markers showed decreased CYP3A activity with increasing age, which suggested that age could be a factor that significantly influences CYP3A activity.
OHNOM,YAMAGUCHI,SAIKIK,et al.Specific determination of urinary 6beta-hydroxycortisol and cortisol by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry[J].J Chromatogr B,2000,746(1):95-101.
The urinary 6beta-hydroxycortisol to cortisol ratio is believed to be a noninvasive index of cytochrome P450 3A activity. For precise assessment of the ratio in human urine, we have developed a reversed-phase high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry method. The selective method was accurate and reproducible with intra- and inter-day precision of variation coefficients of less than 8%. The 6beta-hydroxycortisol to cortisol ratio ranged from 3.0 to 12.4 in healthy Japanese 24-h urine. With the recent popularization of LC-MS, our LC-MS method will be advantageous to detect human in vivo CYP3A activity for clinical investigation and routine measurement in various laboratories.
BARRETTYC,AKINSANYAB,CHANG SY,et al.Automated on-line SPE LC-MS/MS method to quantitate 6beta-hydroxycortisol and cortisol in human urine:Use of the 6beta-hydroxycortisol to cortisol ratio as an indicator of CYP3A4 activity[J].J Chromatogr B Analyt Technol Biomed Life Sci,2005,821(2):159-165.
A sensitive method for quantitation of urinary 6beta-hydroxycortisol (6beta-HC) and cortisol using on-line SPE and LC-MS/MS was developed and validated. Human urine samples were injected directly onto an on-line solid phase extraction apparatus, Prospekt-2, followed by HPLC separation and electrospray triple quadrupole LC-MS/MS detection. The inter-day precision for the 6beta-HC:cortisol ratio was 7-9%. The lower limit of quantitation was 1 and 0.2 ng/mL for 6beta-HC and cortisol, respectively. Using the method we observed a diurnal variation on the 6beta-HC:cortisol ratio in healthy volunteers with the maximal ratio observed in the 2-10 pm urine collection period.
LUTZU,BITTNERN,UFERM,et al.Quantification of cortisol and 6 beta-hydroxycortisol in human urine by LC-MS/MS,and gender-specific evaluation of the metabolic ratio as biomarker of CYP3A activity[J].J Chromatogr B Analyt Technol Biomed Life Sci,2010,878(1):97-101.
Drug-drug and food-drug interactions are often due to an inhibition or induction of drug-metabolizing cytochrome P450 (CYP) enzymes and may result in non-response or adverse reactions. Hence, phenotypic biomarkers of CYP activity appear as useful tools for individualized pharmacotherapy. The metabolic ratio (MR) of the concentration of 6beta-hydroxycortisol (6beta-OHC) to cortisol (MR 6beta-OHC/cortisol) in human urine had been proposed as an endogenous marker for CYP3A activity. Here, we report on the improvement of published LC-MS/MS methods for the simultaneous quantification of cortisol and 6beta-OHC, using on-line sample cleanup by column switching and isotope-labeled analogues as internal standards. [(2)H(2)]6beta-OHC was prepared by incubation of human recombinant CYP3A4 with commercially available [(2)H(2)]cortisol. Analytical sensitivity could be increased about 10-fold. The first morning urine of 69 female and 27 male healthy volunteers was analyzed for cortisol and 6beta-OHC. Concentrations ranged from 1.0 to 142 and 24 to 670ng/mL, respectively. Individual MR 6beta-OHC/cortisol varied more than 20-fold and we were able to show for the first time for a Caucasian population significantly higher MR values in females as compared to males. This non-invasive biomarker for CYP3A activity lends itself for the study of genetic differences as well as enzyme induction or inhibition in the clinical setting without the need of using a probe drug.
MASAYAT,MAKOTOT,YASUHIROM,et al.Acyl glucuronidation of fluoroquinolone antibiotics by the UDP-glucuronosyltransferase 1A subfamily in human liver microsomes[J].Drug Metab Dispos,2005,33(6):803-811.
Acyl glucuronidation is an important metabolic pathway for fluoroquinolone antibiotics. However, it is unclear which human UDP-glucuronosyltransferase (UGT) enzymes are involved in the glucuronidation of the fluoroquinolones. The in vitro formation of levofloxacin (LVFX), grepafloxacin (GPFX), moxifloxacin (MFLX), and sitafloxacin (STFX) glucuronides was investigated in human liver microsomes and cDNA-expressed recombinant human UGT enzymes. The apparent Km values for human liver microsomes ranged from 1.9 to 10.0 mM, and the intrinsic clearance values (calculated as Vmax/Km) had a rank order of MFLX > GPFX > STFX > > LVFX. In a bank of human liver microsomes (n = 14), the glucuronidation activities of LVFX, MFLX, and STFX correlated highly with UGT1A1-selective beta-estradiol 3-glucuronidation activity, whereas the glucuronidation activity of GPFX correlated highly with UGT1A9-selective propofol glucuronidation activity. Among 12 recombinant UGT enzymes, UGT1A1, 1A3, 1A7, and 1A9 catalyzed the glucuronidation of these fluoroquinolones. Results of enzyme kinetics studies using the recombinant UGT enzymes indicated that UGT1A1 most efficiently glucuronidates MFLX, and UGT1A9 most efficiently glucuronidates GPFX. In addition, the glucuronidation activities of MFLX and STFX in human liver microsomes were potently inhibited by bilirubin with IC50 values of 4.9 microM and 4.7 microM, respectively; in contrast, the glucuronidation activity of GPFX was inhibited by mefenamic acid with an IC50 value of 9.8 microM. These results demonstrate that UGT1A1, 1A3, and 1A9 enzymes are involved in the glucuronidation of LVFX, GPFX, MFLX, and STFX in human liver microsomes, and that MFLX and STFX are predominantly glucuronidated by UGT1A1, whereas GPFX is mainly glucuronidated by UGT1A9.
HIROMIS,MIYUKIK,SHINOBUU,et al.Use of endogenous cortisol 6β-hydroxylation clearance for phenotyping in vivo CYP3A activity in women after sequential administration of an oral contraceptive(OC)containing ethinylestradiol and levonorgestrel as weak CYP3A inhibitors[J].Steroids,2014,87:137-144.
The present study was undertaken to evaluate the time courses of in vivo cytochrome P450 3A (CYP3A) inhibition in four healthy women after sequential administration of an oral contraceptive (OC) containing ethinylestradiol and levonorgestrel, using 6β-hydroxylation clearance of endogenous cortisol (CLm(6β)) as a new index for CYP3A phenotyping. The 6β-hydroxylation clearance (CLm(6β)) was followed every 2h from 9:00 or 11:00 to 17:00 on days 0 (baseline), 1, 2, 21, and 28 during a single menstrual cycle. The serum concentrations of endogenous estradiol and progesterone were also measured. The time course data of CLm(6β) clearly demonstrated 43-64% inhibition of CYP3A activity in women taking a low daily dose of the OC for 21days. The average CLm(6β) levels that were suppressed by the OC in four women were extremely low (0.60-1.23mL/min) compared with the normal CLm(6β) range (1.5-3.5mL/min) that was obtained from 49 healthy subjects in our previous study. The in vivo inhibitory potencies (43-64%) obtained in this study were stronger than expected from reported in vitro studies (∼20%). Furthermore, it would take at least seven days to return to the baseline activity of CYP3A after discontinuation of the OC. The results presented here should provide important information on the inhibitory effect of OC on the CYP3A activities in women, which are involved in the metabolism of a number of drugs with a narrow therapeutic range.
Interindividual and intraindividual variability of the urinary 6beta-Hydroxycortisol/Cortisol ratio in Chinese subjects:implications of its use for evaluating CYP3A activity
Relationships between endogenous plasma biomarkers of constitutive cytochrome P450 3A activity and single-time-point oral midazolam microdose phenotype in healthy subjects
Associations of the CYP-3A5*3 and CYP3A4*1G polymorphisms with the pharmacokinetics of oral midazolam and the urinary 6β-hydroxycortisol/cortisol ratio as markers of CYP3A activity in healthy male Chinese
Specific determination of urinary 6beta-hydroxycortisol and cortisol by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry
Automated on-line SPE LC-MS/MS method to quantitate 6beta-hydroxycortisol and cortisol in human urine:Use of the 6beta-hydroxycortisol to cortisol ratio as an indicator of CYP3A4 activity
Quantification of cortisol and 6 beta-hydroxycortisol in human urine by LC-MS/MS,and gender-specific evaluation of the metabolic ratio as biomarker of CYP3A activity
Use of endogenous cortisol 6β-hydroxylation clearance for phenotyping in vivo CYP3A activity in women after sequential administration of an oral contraceptive(OC)containing ethinylestradiol and levonorgestrel as weak CYP3A inhibitors
, 杨雨晴
