Effect of kanglaite on rat cytochrome P450

Abstract

Context: Kanglaite (KLT) is an oily substance extracted from Coix lacryma-jobi Linn. (Cramineae) and has been proved to significantly improve the life span and quality of life of patients, when combined with chemotherapy, radiotherapy, or surgery.

Objective: The purpose of this study was to find out whether KLT influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4) by using cocktail probe drugs in vivo.

Materials and methods: A cocktail solution at a dose of 5?mL/kg, which contained phenacetin (20?mg/kg), bupropion (20?mg/kg), tolbutamide (5?mg/kg), omeprazole (20?mg/kg), and midazolam (10?mg/kg), was given as oral administration to rats treated with 7?d intraperitoneal injection of KLT. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0 (SPPS Inc., Chicago, IL).

Results: In the experiment, there was a statistically significant difference in the t_1/2, C_max, AUC_(0–∞), and CL for phenacetin, bupropion, tolbutamide, omeprazole, and midazolam. Our study showed that treatment with multiple doses of KLT had induction effect on rat CYP1A2, while CYP2B6, CYP2C9, CYP2C19, and CYP3A4 enzyme activities had been inhibited after multiple doses of KLT treatment.

Conclusions: KLT can either induce or inhibit activities of CYP. Therefore, caution is needed when KLT is co-administration with some CYP substrates in clinic, which may result in herb–drug interactions.     

Potential of pranlukast and zafirlukast in the inhibition of human liver cytochrome P450 enzymes

1.?The potential of zafirlukast to inhibit several human cytochrome P450 enzymes is well known. However, pranlukast, a structural analogue of zafirlukast, has not been studied. Accordingly, the inhibitory potential of pranlukast was evaluated and compared with that of zafirlukast, a known CYP2C9 inhibitor, in in vitro microsomal incubation studies.

2.?Both pranlukast and zafirlukast showed moderate inhibition of CYP2C9-catalysed tolbutamide 4-methylhydroxylation, competitively inhibiting tolbutamide 4-methylhydroxylation with estimated mean K_i values of 3.82 ± 0.50 and 5.86 ± 0.08?μM, respectively.

3.?Pranlukast had no effect on CYP2C19-catalysed S-mephenytoin 4′-hydroxylation or CYP3A4-catalysed midazolam 1-hydroxylation. However, zafirlukast showed minor inhibition of these reactions. Neither pranlukast nor zafirlukast inhibited CYP1A2-catalysed phenacetin O-deethylation, CYP2D6-catalysed dextromethorphan O-demethylation or CYP2E1-catalysed chlorzoxazone 6-hydroxylation.

4.?The results suggest that like zafirlukast, pranlukast also has the potential moderately to inhibit CYP2C9-catalysed tolbutamide 4-methylhydroxylation. Therefore, the inhibitory potential of pranlukast should be considered when it is co-administered with CYP2C9 substrates with narrow therapeutic ranges (e.g. S-warfarin, phenytoin).     

Pharmacokinetics behaviors of l-menthol after inhalation and intravenous injection in rats and its inhibition effects on CYP450 enzymes in rat liver microsomes

Abstract

1. l-Menthol, as a kind of monocyclic terpene, is widely used in inhalation formulations, food and tobacco. The purpose of this study was to investigate the pharmacokinetic behavior of l-menthol as well as its influence on the activities of cytochrome P450 enzymes.

2. The pharmacokinetic behaviors of l-menthol after inhalation (50?mg/kg) and intravenous injection (10?mg/kg) were investigated. A rat liver microsomal model was adopted to elucidate the inhibitory effect of l-menthol on CYP1A2, CYP2C11, CYP2D1/2, CYP2D4, CYP2E1 and CYP3A1 using phenacetin, tolbutamide, omeprazole, dextromethorphan, chlorzoxazone and testosterone as probe drugs, respectively.

3. The plasma concentration reached the C_max within 1.0?h (inhalation) and descended with the T_1/2 of 8.53 and 6.69?h for inhalation and i.v. administration, respectively. IC₅₀ for inhibition of l-menthol on CYP 450 enzymes were 4.35?μM for 2D4, 8.67?μM for 1A2, 13.02?μM for 3A1, 14.78?μM for 2D1/2, 234.9?μM for 2C11 and 525.4?μM for 2E1, respectively.

4. The results illustrate the pharmacokinetic process of l-menthol in rats and provide information for further rational applications. l-Menthol had moderate inhibitions on CYP2D4 and 1A2, which might affect the disposition of medicines primarily dependent on these pathways.     

Development and validation of probe drug cocktails for the characterization of CYP450-mediated metabolism by human heart microsomes

1. The objective of our study was to develop and validate a cocktail approach to allow the simultaneous characterization of various CYP450-mediated oxidations by human heart microsomes for nine probe drug substrates, namely, 7-ethoxyresorufin, bupropion, repaglinide, tolbutamide, bufuralol, chlorzoxazone, ebastine, midazolam and dodecanoic acid.

2. The first validation step was conducted using recombinant human CYP450 isoenzymes by comparing activity measured for each probe drug as a function of (1) buffer used, (2) selectivity towards specific isoenzymes and (3) drug interactions between probes. Activity was all measured by validated LC-MSMS methods.

3. Two cocktails were then constituted with seven of the nine drugs and subjected to kinetic validation. Finally, all probe drugs were incubated with human heart microsomes prepared from ventricular tissues obtained from 12 patients undergoing cardiac transplantation.

4. Validated cocktail #1 including bupropion, chlorzoxazone, ebastine and midazolam was used to characterize CYP2B6-, 2E1-, 2J2- and 3A5-mediated metabolism in human hearts.

5. Cocktail #2 which includes bufuralol, 7-ethoxyresorufin and repaglinide failed the validation step. Substrates in cocktail #2 as well as tolbutamide and dodecanoic acid had to be incubated separately because of their physico-chemical characteristics (solubility and ionization) or drug interactions.

6. Activity in HHM was the highest towards ebastine, chlorzoxazone and tolbutamide.     

Effect of codeine on CYP450 isoform activity of rats

Context: Codeine, also known as 3-methylmorphine, is an opiate used to treat pain, as a cough medicine and for diarrhoea. No study on the effects of codeine on the metabolic capacity of CYP enzyme is reported.

Objective: In order to investigate the effects of codeine on the metabolic capacity of cytochrome P450 (CYP) enzymes, a cocktail method was employed to evaluate the activities of CYP2B1, CYP2D1, CYP1A2, CYP3A2 and CYP2C11.

Materials and methods: Sprague–Dawley rats were randomly divided into codeine group (low, medium, high) and control group. The codeine group rats were given 4, 8, 16?mg/kg (low, medium, high) codeine by continuous intragastric administration for 14?days. Five probe drugs bupropion, metroprolol, phenacetin, midazolam and tolbutamide were given to rats through intragastric administration, and the plasma concentrations were determined by UPLC-MS/MS.

Results and conclusion: The pharmacokinetic parameters of bupropion and metroprolol experienced obvious change with AUC_(0-t), C_max increased and CL decreased for bupropion in medium dosage group and midazolam low dosage group. This result indicates that the 14?day-intragastric administration of codeine may inhibit the metabolism of bupropion (CYP2B1) and midazolam (CYP3A2) in rat. Additional, there are no statistical differences for albumin (ALB), alkaline phosphatase (ALP), creatinine (Cr) after 14 intragastric administration of codeine, while alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (UA) increased compared to control group. The biomedical test results show continuous 14?day-intragastric administration of codeine would cause liver damage.     

Effects of notoginsenoside R1 on CYP1A2, CYP2C11, CYP2D1, and CYP3A1/2 activities in rats by cocktail probe drugs

Context: Notoginsenoside R1 (NGR1) is the main component with cardiovascular activity in Panax notoginseng (Burk.) F. H. Chen, an herbal medicine that is widely used to enhance blood circulation and dissipate blood stasis.

Objective: The objective of this study is to investigate NGR1's effects on CYP1A2, CYP2C11, CYP2D1, and CYP3A1/2 activities in rats in vivo through the use of the Cytochrome P450 (CYP450) probe drugs.

Materials and methods: After pretreatment with NGR1 or physiological saline, the rats were administered intraperitoneally with a mixture solution of cocktail probe drugs containing caffeine (10?mg/kg), tolbutamide (15?mg/kg), metoprolol (20?mg/kg), and dapsone (10?mg/kg). The bloods were then collected at a set of time-points for the ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) analysis.

Results: NGR1 was shown to exhibit an inhibitory effect on CYP1A2 by increased caffeine C_max (43.13%, p?<?0.01) and AUC_0???∞ (40.57%, p?<?0.01), and decreased CL/F (62.16%, p?<?0.01) in the NGR1-treated group compared with those of the control group, but no significant changes in pharmacokinetic parameters of tolbutamide, metoprolol, and dapsone were observed between the two groups, indicating that NGR1 had no effects on rat CYP2C11, CYP2D1, and CYP3A1/2.

Discussion and conclusion: When NGR1 is co-administered with drugs that are metabolized by CYP1A2, the pertinent potential herb–drug interactions should be monitored.     

Use of a cocktail of probe substrates for drug-metabolizing enzymes for the assessment of the metabolic capacity of hepatocyte preparations

1.?A cocktail of the following probe substrates for human drug-metabolizing enzymes was used to characterize hepatocyte preparations: phenacetin (for CYP1A2), diclofenac (CYP2C9), diazepam (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4/5) and 7-hydroxycoumarin (for glucuronidation and sulphation).

2.?The cocktail was incubated with cryopreserved human, dog or minipig hepatocytes or with freshly prepared rat hepatocytes. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an Open Access environment that allowed less experienced MS operators to login, submit and analyse sample sets using predefined settings without the immediate attendance of an experienced analyst. Intrinsic clearances (CL_int) were calculated from the disappearance of the compounds from the incubations.

3.?Initially, the cocktail used for human, rat and dog hepatocyte incubations contained 7-ethoxycoumarin instead of 7-hydroxycoumarin. However, 7-ethoxycoumarin had an inhibitory effect on the metabolism of phenacetin.

4.?The highest CL_int estimated with human and dog hepatocytes was observed for 7-hydroxycoumarin. For rat and minipig hepatocytes, the highest CL_int was observed for bufuralol. In incubations with dog and minipig hepatocytes, the lowest CL_int was seen with diclofenac, whereas for human and rat hepatocytes, the lowest value was observed with diazepam and phenacetin, respectively.

5.?When the cocktail was incubated together with human hepatocytes and 1?μM ketoconazole, the CL_int of midazolam was decreased to about 7.5% of the control value, whereas the metabolism of the other cocktail compounds was virtually unaffected by this CYP3A inhibitor.

6.?It is suggested that a cocktail of specific human probe substrates for drug-metabolizing enzymes can be used routinely for the determination of the metabolic capacity of hepatocyte preparations in order to ensure the quality and reproducibility of experiments. Moreover, a cocktail of specific probe substrates can also be a useful tool for studies on enzyme inhibition.     

Effects of Guanxinning injection on rat cytochrome P450 isoforms activities in vivo and in vitro

Abstract

1. We aimed to investigate the regulatory effects of Guanxinning injection (GXNI) on activities of cytochrome P1A2 (CYP1A2), CYP2C11, CYP2D1 and CYP3A1/2 by probe drugs in rats in vivo and in vitro.

2. GXNI-treated and blank control groups were administered GXNI and physiological saline by caudal vein for 14 days consecutively, then they were given the probe drugs of caffeine (10?mg/kg), tolbutamide (10?mg/kg), metoprolol (20?mg/kg) and dapsone (10?mg/kg) by intraperitoneal injection. The blood samples were collected at different times for ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. Changes of the pharmacokinetics parameters between the GXNI-treated and the blank control groups were used to evaluate the effects of GXNI on the four CYP450 isoforms in rats in vivo. After blood collection, the livers of rats were taken and made microsomes for in vitro tests. The relevant metabolites of phenacetin, tolbutamide, dextromethorphan and testosterone were analyzed quantitatively by high-performance liquid chromatography (HPLC) after microsome incubation. The statistical differences between the two groups were observed to detect the effects of GXNI on the four CYP450 isoforms in rats in vitro.

3. The in vivo and in vitro results demonstrated that GXNI could induce CYP1A2 activity in rats, but had no significant effects on CYP2C11, CYP2D1 and CYP3A1/2.     

丹参-红花药对配伍前后对大鼠肝药酶亚型CYP1A2、CYP2E1和CYP3A4活性的影响

目的 研究丹参、红花药对配伍前后对大鼠肝药酶亚型CYP1A2、CYP2E1和CYP3A4活性的影响。方法 分别选用咖啡因、氯唑沙宗和咪达唑仑作为CYP1A2、CYP2E1和CYP3A4的探针药物。将大鼠随机分为4组,即空白对照组、丹参（1.2 g生药/kg）组、红花（0.4 g生药/kg）组、丹参（1.2 g生药/kg）+红花（0.4 g生药/kg）组,按上述剂量ig给药7 d。于末次给药后30 min,尾iv探针药物咖啡因、氯唑沙宗和咪达唑仑溶液,在不同的时间点取血进行检测;以甲硝唑为内标,采用HPLC法检测探针药物咖啡因、氯唑沙宗和咪达唑仑的量,评价各药物组对大鼠CYP3A4、CYP2E1和CYP1A2活性的影响。结果 与空白对照组比较,丹参组咖啡因、氯唑沙宗和咪达唑仑的清除率（CL）有所增强,曲线下面积（AUC）减少,其半衰期（t_1/2）有减少趋势,但差异均不显著;红花组咖啡因和氯唑沙宗的CL有所降低,但差异不显著,咪达唑仑的CL显著降低（P<0.01）,氯唑沙宗的AUC增加,但差异不显著,咖啡因和咪达唑仑的AUC明显增加（P<0.05、0.01）;丹参+红花组咖啡因和氯唑沙宗的CL明显降低（P<0.05）,曲线下面积（AUC）明显增加（P<0.05）,其t_1/2有延长趋势,但差异不显著。结论 丹参、红花配伍后对CYP450亚型CYP1A2和CYP2E1有抑制作用,这可能是丹参、红花配伍协同增效的作用机制之一。     

HPLC 法同时测定大鼠血浆中 CYP450酶探针药物氯唑沙宗、甲苯磺丁脲和咪达唑仑浓度

目的：建立同时测定大鼠血浆中细胞色素 P450（CYP450）酶探针药物氯唑沙宗、甲苯磺丁脲和咪达唑仑浓度的高效液相色谱法。方法色谱柱采用 Diamonsil C18（250 mm ×4．6 mm,5μm）,流动相为甲醇与磷酸二氢铵缓冲液（61∶39,V ／V）,流速1 mL·min －1,检测波长230 nm,带宽4 nm,柱温35℃,以地西泮为内标,同时检测氯唑沙宗、甲苯磺丁脲和咪达唑仑的大鼠血浆药物浓度。结果氯唑沙宗、甲苯磺丁脲和咪达唑仑均在0．1～50 mg·L －1（r ＝0．9995）的范围内线性关系良好;低、中、高3个浓度下,3种探针药物精密度试验中相对标准偏差为4．78％～9．78％,回收率为92．71％～109．79％。结论该方法操作简便,灵敏度高,重现性好,符合生物样品分析要求,适用于同时测定3种 CYP450酶亚型探针药物氯唑沙宗、甲苯磺丁脲和咪达唑仑的大鼠血浆药物浓度,可为 CYP450酶活性测定提供分析方法学参考。     

Direct and metabolism‐dependent cytochrome P450 inhibition assays for evaluating drug–drug interactions

We developed methods for evaluating the ntial inhibition of human cytochrome P450 (CYP) enzymes, including CYP1A2, CYP2A6, CYP2B6, CYP2 C9, CYP2 C19, CYP2D6, CYP2E1 and CYP3A4, using pooled human liver microsomes (HLMs). The CYP inhibition assay used substrate cocktail sets [set A: phenacetin for CYP1A2, coumarin for CYP2A6, (S)‐(+)‐mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4; set B: bupropion for CYP2B6, tolbutamide for CYP2C9, chlorzoxazone for CYP2E1, and testosterone for CYP3A4] with quantitation by liquid chromatography–tandem mass spectrometry. A direct inhibition assay was performed with the substrate cocktails without β‐nicotinamide adenine dinucleotide phosphate (NADPH) pre‐incubation, and a metabolism‐dependent inhibition (MDI) assay was performed after 30 min of pre‐incubation with NADPH in HLMs. MDI was identified based on the half‐maximal inhibitory concentration (IC₅₀) shifts. The IC₅₀ values of the direct inhibitors determined using the probe substrate cocktails were in good agreement with previously reported values. Eight metabolism‐dependent inhibitors including furafylline, 8‐methoxypsoralen, tienilic acid, ticlopidine, fluoxetine, paroxetine, disulfiram and verapamil against CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, respectively, resulted in significant IC₅₀ shifts (≥2.5‐fold) after pre‐incubation. Thus, these CYP inhibition assays are considered to be useful tools for evaluating both direct inhibition and MDI at an early stage of the drug discovery and development process. Copyright © 2011 John Wiley & Sons, Ltd.     

Evaluation of the impact of waterpipe tobacco smoke exposure on the activity and expression of rat hepatic CYP450: a pharmacokinetic study

Abstract

Waterpipe smoke contains many toxic constituents that can alter drug pharmacokinetics. This study assessed the effect of waterpipe smoke exposure on the activity and expression of CYP450 enzymes in rats. Animals (n?=?10/group) were exposed to either waterpipe smoke or side-stream cigarette smoke for 1?h/day (6 days/week) for 31 days, or fresh air (control). An intragastric cocktail solution containing three probe drugs, phenacetin, chlorzoxazone and testosterone was administered to assess the activity of CYP1A2, CYP2E1 and CYP3A, respectively. Serum concentrations were determined using LC-MS/MS and the pharmacokinetic parameters were calculated. The mRNA expression of hepatic enzymes was also quantified. Waterpipe and cigarette smoke exposure did not significantly alter the pharmacokinetics of phenacetin, chlorzoxazone and testosterone. For example, the clearance and drug exposure values were comparable among groups for all probe drugs. Additionally, there was no significant effect of waterpipe and cigarette smoke on mRNA expression of hepatic CYP1A2, CYP2E1 and CYP3A2. The results demonstrate that waterpipe smoke exposure had no effect on the functional expression of three key CYP450 isoforms in rats. Future research is required with longer exposure periods to waterpipe smoke. Such work serves to enhance current understanding of effect of waterpipe smoke exposure on pharmacokinetics.     

Effect of single‐walled carbon nanotubes on cytochrome P450 activity in human liver microsomes in vitro

Single‐walled carbon nanotubes (SWCNTs) are made from a rolled single sheet of graphene with a diameter in the nanometer range. SWCNTs are potential carriers for drug delivery systems because antibodies or drugs can be loaded on their surface; however, their effect on the activities of cytochrome P450 (CYP) remains unclear. The aim of this study was to investigate the effect of two kinds of SWCNTs with different lengths (FH‐P‐ and SO‐SWCNTs) on human CYP activity. In addition, other nano‐sized carbon materials, such as carbon black, fullerene‐C₆₀, and fullerene‐C₇₀ were also evaluated to compare their effects on CYP activities. Ten CYP substrates (phenacetin, coumarin, bupropion, paclitaxel, tolbutamide, S‐mephenytoin, dextromethorphan, chlorzoxazone, midazolam, and testosterone) were used. Testosterone 6β‐hydroxylation and midazolam 1′‐hydroxylation, which are catalysed by both CYP3A4 and CYP3A5 in liver microsomes, were decreased by 25% and 45%, respectively, in the presence of 0.1 mg/ml SO‐SWCNT. Dextromethorphan O‐demethylation, which is catalysed mainly by CYP2D6, was decreased by 40% in the presence of SO‐SWCNT. Other CYP activities, however, were not attenuated by SO‐SWCNT. FH‐P‐SWCNT, carbon black, fullerene‐C₆₀, and fullerene‐C₇₀ at 0.1 mg/ml had no effect on CYP activities. The K_i values for testosterone 6β‐hydroxylation, midazolam 1′‐hydroxylation, and dextromethorphan O‐demethylation in liver microsomes were 136, 34, and 56 μg/ml, respectively. SO‐SWCNT was determined to be a competitive inhibitor of CYP3A4, CYP3A5, and CYP2D6. These results suggest that the effect of SO‐SWCNT differs among CYP isoforms, and that the inhibition potency depends on the physicochemical properties of the nanocarbons.     

In-vitro metabolism of glycyrrhetinic acid by human and rat liver microsomes and its interactions with six CYP substrates

Objectives Glycyrrhetinic acid is the main metabolite of glycyrrhizin and the main active component of Licorice root. This study was designed to investigate the in‐vitro metabolism of glycyrrhetinic acid by liver microsomes and to examine possible metabolic interactions that glycyrrhetinic acid may have with other cytochrome P450 (CYP) substrates. Methods Glycyrrhetinic acid was incubated with rat liver microsomes (RLM) and human liver microsomes (HLM). Liquid chromatography tandem mass spectrometry was used for glycyrrhetinic acid or substrates identification and quantification. Key findings The K_m and V_max values for HLM are 33.41 µm and 2.23 nmol/mg protein/min, respectively; for RLM the K_m and V_max were 24.24 µm and 6.86 nmol/mg protein/min, respectively. CYP3A4 is likely to be the major enzyme responsible for glycyrrhetinic acid metabolism in HLM while CYP2C9 and CYP2C19 are considerably less active. Other human CYP isoforms have minimal or no activity toward glycyrrhetinic acid. The interactions of glycyrrhetinic acid and six CYP substrates, such as phenacetin, diclofenac, (S)‐mephenytoin, dextromethorphan, chlorzoxazone and midazolam were also investigated. The inhibitory action of glycyrrhetinic acid was observed in CYP2C9 for 4‐hydroxylation of diclofenac, CYP2C19 for 4′‐hydroxylation of (S)‐mephenytoin and CYP3A4 for 1′‐hydroxylation of midazolam with half maximal inhibitory concentration (IC50) values of 4.3‐fold, 3.8‐fold and 9.6‐fold higher than specific inhibitors in HLM, respectively. However, glycyrrhetinic acid showed relatively little inhibitory effect (IC50 > 400 µm ) on phenacetin O‐deethylation, dextromethorphan O‐demethylation and chlorzoxazone 6‐hydroxylation. Conclusions The study indicated that CYP3A4 is likely to be the major enzyme responsible for glycyrrhetinic acid metabolism in HLM while CYP2C9 and CYP2C19 are considerably less active. The results suggest that glycyrrhetinic acid has the potential to interact with a wide range of xenobiotics or endogenous chemicals that are CYP2C9, CYP2C19 and CYP3A4 substrates.     

Sodium tanshinone IIA sulfonate and its interactions with human CYP450s

1.Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA, a famous Chinese medicine used for many years to treat cardiovascular disorders. However, the role of cytochrome P450 (CYP) enzymes in the metabolism of STS was unclear. In this study, we screened the main CYPs for the metabolism of STS and studied their interactions in vitro.

2.Seven CYPs were screened for the metabolism of STS by human liver microsomes (HLMs) or recombinant CYP isoforms. To determine the potential of STS to affect CYP-mediated phase I metabolism in humans, phenacetin (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), metoprolol (CYP2D6), chlorzoxazone (CYP2E1), S-Mephenytoin (CYP2C19), and midazolam (CYP3A4) were used as the respective probe substrates. Enzyme kinetic studies were performed to investigate the mode of inhibition of the enzyme–substrate interactions.

3.STS inhibited the activity of CYP3A4 in a dose-dependent manner in the HLMs and CYP3A4 isoform. Other CYP isoforms, including CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on the metabolism of STS.

4.The results suggested that STS primarily inhibits the activities of CYP3A4 in vitro, and STS has the potential to perpetrate drug–drug interactions with other CYP3A4 substrates.     

Simultaneous and comprehensive in vivo analysis of cytochrome P450 activity by using a cocktail approach in rats

A cocktail approach can detect the activities of multiple cytochrome P450 (CYP) isoforms following the administration of multiple CYP‐specific substrates in a single experiment. This study aimed to develop a simultaneous and comprehensive in vivo analysis of CYP activity in rats. The rats received an oral administration of losartan (10 mg/kg) and omeprazole (40 mg/kg). Caffeine (1 mg/kg), dextromethorphan (10 mg/kg) and midazolam (10 mg/kg) were administered 15 min later. In the drug‐interaction phase, the rats were treated orally with dexamethasone (80 mg/kg) 24 h before, or with ketoconazole (10 mg/kg), fluvoxamine (100 mg kg) or fluconazole (10 mg/kg) 1 h before the administration of cocktail drugs. The concentrations of the drugs and their metabolites were determined by LC/MS/MS. Plasma concentrations of five CYP substrates and their metabolites were simultaneously evaluated after the oral drug administration. Fluvoxamine and fluconazole significantly increased the C_max and AUC of caffeine, and the AUC of omeprazole and midazolam. Dexamethasone significantly increased C_max and AUC of losartan, while it decreased the C_max of midazolam. Ketoconazole showed no significant effect on the pharmacokinetic parameters of the tested drugs. In conclusion, a cocktail approach was developed for simultaneous and comprehensive analysis of the activities of multiple CYP isoforms in rats. In this approach, the effects of inhibitors and an inducer of various CYP isoforms were examined. Although further studies are necessary to predict the effects in humans, this approach may be expected to serve as a convenient method for detecting drug–drug interactions in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

Phase I and II enzyme characterization of two sources of HepG2 cell lines

1.?The metabolism by HepG2 cell from two sources (M1, M2) of 12 substrates is reported: ethoxyresorufin, ethoxycoumarin, testosterone, tolbutamide, chlorzoxazone, dextromethorphan, phenacetin, midazolam, acetaminophen, hydroxycoumarin, p-nitrophenol and 1-chloro-2,4-dinitrobenzene (CDNB), and a pharmaceutical compound, EMD68843.

2.?Activities varied markedly. Some were present in M1 (CYP1A, CYP2C9, CYP2E1) but absent in M2. M1 had a more complete set of Phase I enzymes than M2. CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A activities were present at levels similar to human hepatocytes. Phase II metabolism differed between M1 and M2. M1 conjugated hydroxycoumarin and p-nitrophenol to glucuronides only, whereas M2 produced sulfates. Glutathione conjugation of CDNB metabolism was 10-fold higher in M1 than in M2, but was still much lower than in human hepatocytes. CYP2E, CYP2C, CYP2B6 and CYP3A (but not CYP1A, glucuronyl S-transferase or S-transferase) were inducible in M1. Metabolites of EMD68843, produced by induced (but not uninduced) M1 were the same as those produced in human hepatocytes.

3.?In conclusion, HepG2 cells have both Phase I and II enzymes, which activities and at what levels depend on the source and culture conditions. Therefore, HepG2 cells routinely used in in vitro assays should be characterized for their drug-metabolizing capabilities before any results can be fully interpreted.     

Investigation of selective inhibitory effects of glycyrol on human CYP 1A1 and 2C9

1.?Glycyrol is a coumarin derivative isolated from the roots of Glycyrrhiza uralensis called Gamcho in Korea and commonly used as a sweetener in oriental medicine. Glycyrol shows several biological activities, including anti-oxidative, anti-inflammatory, antibacterial, anti-angiogenic, and anti-allergenic properties. Although there have been studies on the biological effects of glycyrol, the inhibitory effects of glycyrol on cytochrome P450 (CYP) activities have not been investigated.

2.?We investigated the inhibitory effects of glycyrol on the activities of CYP isoforms using a cocktail of probe substrates in pooled human liver microsome (HLM) and human recombinant cDNA-expressed CYPs. Glycyrol strongly inhibited CYP1A-mediated phenacetin O-deethylation and CYP2C9-mediated diclofenac 4′-hydroxylation in HLMs, which were the result of competitive inhibition as revealed by a Dixon plot. In addition, glycyrol showed selective inhibition of CYP1A1- and CYP1A2-catalyzed phenacetin O-deethylase activity with a half-maximal inhibitory concentration of (IC₅₀) 1.3 and 16.1?μM in human recombinant cDNA-expressed CYP1A1 and CYP1A2, respectively.

3.?Glycyrol decreased CYP2C9-catalyzed diclofenac 4′-hydroxylation activity with IC₅₀ values of 0.67?μM in human recombinant cDNA-expressed CYP2C9. This is the first investigation of competitive inhibitory effects on CYP1A1 and CYP2C9 in HLMs.     

混合探针法评价白香丹胶囊对大鼠体内CYP450酶活性的影响

目的：建立同时测定大鼠血浆中6种CYP450探针药物的方法,并采用Cocktail法研究白香丹胶囊对大鼠CYP450酶活性的影响。方法：选用茶碱（CYP1A2）、氯唑沙宗（CYP2E1）、甲苯磺丁脲（CYP2C6）、右美沙芬（CYP2D2）、奥美拉唑（CYP2D1）、咪达唑仑（CYP3A2）作为探针药物。大鼠随机分为对照组和给药组,给药组每天灌胃白香丹胶囊0.675 g·kg-1,对照组灌胃等量生理盐水,连续给药14 d。第15天均灌胃给予6个探针药物,于不同时间点眼眶取血,采用LC-MS/MS法测定各探针药物浓度,计算药动学参数并进行组间t检验。结果：与对照组相比,给药组茶碱（P<0.01）、氯唑沙宗（P<0.01）、甲苯磺丁脲（P<0.05）代谢显著加快;右美沙芬、奥美拉唑、咪达唑仑代谢无显著性差异。结论：白香丹胶囊对大鼠 CYP1A2有中强诱导作用,对CYP2E1、CYP2C6有弱诱导作用。     

A convenient five-drug cocktail for the assessment of major drug metabolizing enzymes: a pilot study

AIMS: To assess the feasibility of administering at the same time low doses of five probe drugs, metoprolol (25 mg), chlorzoxazone (250 mg), tolbutamide (250 mg), dapsone (100 mg) and caffeine (100 mg) to determine simultaneously the activities of CYP2D6, CYP2E1, CYP2C9, CYP3A4, CYP1A2, N-acetyltransferase-2 and xanthine oxidase. METHODS: Ten healthy young non-smoking males received the following drugs or combinations of drugs over a 5-week period: week 1) metoprolol; 2) tolbutamide; 3) caffeine, chlorzoxazone and dapsone; 4) caffeine, chlorzoxazone, dapsone and metoprolol; 5) caffeine, chlorzoxazone, dapsone, metoprolol and tolbutamide. The drugs were self-administered at bedtime and urine was collected for the following 8 h. RESULTS: Mean molar phenotypic ratios obtained after administering metoprolol (mean change of -11%) or tolbutamide (mean change of -0.3%) alone, were not significantly different from those obtained when other drugs were co-administered (P > 0.05). The mean within-subject coefficients of variation were 33%, 18%, 22%, 13%, 16%, 13% and 5% for CYP3A4, CYP2D6, CYP2C9, CYP2E1, CYP1A2, N-acetyltransferase 2 and xanthine oxidase metabolic ratios, respectively. No significant interactions (P > 0.5) were observed during the simultaneous administration of various combinations of the five probe drugs. CONCLUSIONS: We propose that this cocktail, composed of five widely available drugs, constitutes a promising means of simultaneously determining the activities of the major CYP enzymes in large populations.