Metabolism Of Phenacetin Research Articles

Effect of Saccharolactone on CYP-mediated Metabolism of Xenobiotics.

Saccharolactone is used as a β-glucuronidase inhibitor in in vitro microsomal and recombinant uridine diphosphoglucuronosyl transferases (rUGTs) incubations to enhance glucuronide pathway and, thereby, formation of glucuronide metabolites. We investigated its effect on CYP mediated metabolism of drugs (compound-174, phenacetin and quinidine) using human liver microsomes (HLM) supplemented with Phase-1 and Phase-2 co-factors. Compounds were incubated in HLM supplemented with co-factors to assess Phase-1 (NADPH) and Phase-2 (NADPH, alamethicin, saccharolactone and UDPGA) metabolism. CYP phenotype assay for compound-174 was conducted in HLM (± 1-ABT) and human recombinant CYP isoforms. CYP inhibition profile of saccharolactone was also generated in HLM. The metabolism of compound-174, phenacetin and quinidine in HLM significantly decreased in reactions containing additional components like alamethicin, saccharolactone and UDPGA and indicated that the addition of saccharolactone inhibited the metabolism. Phenacetin and quinidine are known substrates of CYP1A2 and CYP3A4 isoforms. The metabolism of compound- 174 was significantly inhibited in the presence of 1-ABT in HLM, and CYP3A4 and CYP2C8 isoforms were found to be the predominant isoforms responsible for its metabolism. Further evaluation of CYP inhibition in HLM indicated saccharolactone to be a strong inhibitor of CYP1A2, 2D6, 3A4 and 2C8 isoforms with IC50 values of less than 4 mM. The findings indicated that saccharolactone being a strong inhibitor of CYP1A2, 2D6, 3A4 and 2C8 isoforms (IC50 < 4 mM), resulted in significant inhibition of the metabolism of compound-174, phenacetin and quinidine in HLM and caution should be exercised in using it with proper titration of the concentrations.

Circadian oscillator NPAS2 regulates diurnal expression and activity of CYP1A2 in mouse liver

We aimed to investigate the potential role of NPAS2 in controlling diurnal expression and activity of hepatic CYP1A2 and to determine the underlying mechanisms. Regulatory effects of NPAS2 on CYP1A2 were determined using Npas2 knockout (Npas2-/-) mice as well as AML-12, Hepa1-6 and HepG2 cells. mRNA and protein levels were detected by reverse transcription-quantitative real-time PCR and western blotting, respectively. In vitro and in vivo CYP1A2 activities were respectively evaluated using the probe substrates phenacetin and theophylline. Transcriptional regulation was investigated using luciferase reporter assays and ChIP-Seq analysis. Loss of Npas2 in mice decreased CYP1A2 expression (at both mRNA and protein levels) and blunted its rhythmicity in the liver. Likewise, Npas2 ablation down-regulated the enzymatic activity of CYP1A2 (probed by metabolism of phenacetin and theophylline) and abrogated its time-dependency. Cell-based assays confirmed that NPAS2 positively regulated CYP1A2 expression. Mechanistic study indicated that NPAS2 trans-activated Cyp1a2 through its specific binding to the −416 bp E-box-like element within the gene promoter. In conclusion, NPAS2 was identified as a key transcriptional regulator of diurnal expression of hepatic CYP1A2 in mice. Our findings have implications for improved understanding of circadian metabolism and chronopharmacokinetics.

Psoralen and isopsoralen from Psoraleae Fructus aroused hepatotoxicity via induction of aryl hydrocarbon receptor-mediated CYP1A2 expression

Ethnopharmacological relevancePsoraleae Fructus (PF), a traditional Chinese medicine, has long been used to treat diseases such as cancer, osteoporosis and leukoderma. Psoralen and isopsoralen are main bioactive ingredients of PF with anti-tumor, anti-inflammatory, estrogen-like neuroprotection, etc., meanwhile they are also representative hepatotoxic components of PF. Hepatic CYP1A2 has been reported to be the important metabolic enzymes involved in psoralen and isopsoralen-induced hepatotoxicity. However, the relationship between the hepatotoxicity and CYP1A2 expression, and the underlying mechanism of regulating CYP1A2 expression remain unclear. Aim of studyThe aim of this study was to explore the associated mechanism between psoralen or isopsoralen induced hepatotoxicity and activated aryl hydrocarbon receptor (AhR)-mediated transcriptional induction of CYP1A2 in vitro and in vivo. Materials and methodsPsoralen and isopsoralen at different doses were treated on HepG2 cells (10, 25, 50, 100, 200 μM for 2, 12, 24, 36, 48 h) and mice (20, 80, 160 mg/kg for 3, 7, 14 days) for different time, to assess the correlation of induced hepatotoxicity and CYP1A2 mRNA and protein expression in vivo and in vitro, as well as the effect on CYP1A2 enzyme activity evaluated by phenacetin metabolism. In addition, the potential mechanism of the regulation of CYP1A2 expression mediated by AhR was explored through nucleocytoplasmic shuttling, immunofluorescence, cellular thermal shift assay and molecular docking, etc. ResultsPsoralen and isopsoralen induced cytotoxicity in HepG2 cells, and hepatomegaly, biochemicals disorder and tissue pathological impairment in mice, respectively in dose- and time-dependent manners. Simultaneously accompanied with elevated levels of CYP1A2 mRNA and protein in the same trend, and the CYP1A2 activity was remarkably inhibited in vitro but significantly elevated overall in vivo. Besides, psoralen and isopsoralen bound to AhR and activated translocation of AhR from the cytoplasm to the nucleus, leading to the transcriptional induction of target gene CYP1A2. ConclusionsHepatotoxicities in HepG2 cells and mice aroused by psoralen and isopsoralen were related to the induction of CYP1A2 expression and activity, whose underlying mechanism might be psoralen or isopsoralen activated AhR translocation and induced increase of CYP1A2 transcriptional expression. Hopefully, these finding are conductive to propose an alert about the combined usage of psoralen or isopsoralen and AhR ligands or CYP1A2 substrates in clinical practice.

Development of a method to determine cytochrome P450 1A2, 2C9, 2D6 and 3A4 activity sheep hepatic microsomes

IntroductionEx vivo studies of human fetal hepatic drug metabolism are uncommon as it requires access to functional liver tissue and therefore raises practical and ethical concerns. Large animal models provide an alternative opportunity to study changes in cytochrome P450 (CYP) activity in the mother and fetus during pregnancy. We aimed to develop methods to determine the activity of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in sheep hepatic microsomes. MethodsWe identified optimal conditions to determine the activity of CYP1A2 (using the probe drug phenacetin), CYP2C9 (diclofenac), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam) by varying techniques for microsome extraction, probe drug concentration, incubation time and microsome concentration. The specificity of each probe drug was assessed by determining the rate of metabolism when specific CYP enzyme inhibitors were included in the reaction. ResultsThe optimum incubation time and probe drug concentration was six hours with 5 μM phenacetin (CYP1A2), four hours with 10 μM diclofenac (CYP2C9), 30 min with 1 μM of midazolam (CYP3A4) and 10 min with 1 μM dextromethorphan (CYP2D6). For both CYP2D6 and CYP3A4 reactions required 20 μg of microsomal protein, whereas for CYP1A2 and CYP2C9, reactions required 40 μg of microsomal protein. Metabolism of phenacetin, dextromethorphan and midazolam was reduced by specific enzyme inhibitors, but the specific CYP2C9 inhibitor sulfaphenazole did not substantially inhibit diclofenac metabolism. DiscussionThis study identifies the optimal conditions for determining CYP activity in maternal sheep hepatic microsomes. In doing so, we have developed a standardised protocol for assessment of microsomal activity of CYP3A4, CYP1A2 and CYP2D6, but we were unable to optimise conditions for assessment of CYP2C9. This approach can be applied to investigate the impact of pregnancy complications on maternal and fetal hepatic drug metabolism.

Effects of Dendrobium huoshanense on expressions and activities of hepatic microsomal cytochrome P450s in mice

This study was carried out to investigate the effect of oral administration of Dendrobium huoshanense on the expressions and activities of hepatic microsomal cytochrome P450s in mice, and to provide a reference for the evaluation of drug-drug interactions between D. huoshanense and clinical drugs. The C57BL/6 mice were randomly divided into blank control group, D. huoshanense low dose group (crude drug 1.25 g·kg⁻¹), D. huoshanense high dose group (crude drug 7.5 g·kg⁻¹), and phenobarbital positive control group (0.08 g·kg⁻¹). Each group was intragastrically administered with drugs for 2 weeks. The mice were sacrificed and their liver microsomes were prepared. The expressions of major subtypes of P450 enzyme were determined by Western blot and the probe drugs were used to detect the enzyme activities of P450 subtypes with protein expression changes. Western blot analysis showed that the protein expressions of CYP1A1, CYP1A2 and CYP2B in liver tissues were up-regulated in D. huoshanense-treated group. In vitro enzyme activity tests showed that there were no significant difference in metabolism of 7-ethoxyresorufin (a probe drug for CYP1A1) and bupropion (a probe drug for CYP2B) between D. huoshanense group and control group. The metabolism of phenacetin (a probe drug for CYP1A2) showed a statistical difference in rate Vmax, and it was significantly increased by approximately 20% in D. huoshanense group as compared with the blank control group, and the clearance CLint in treated group was also increased by about 32%. Therefore, oral administration of D. huoshanense had no effects on the activities of most hepatic P450 enzymes in mice, with no drug-drug interaction related to the P450 enzyme system in most clinical drugs theoretically. However, oral administration of D. huoshanense may accelerate the metabolism of CYP1A2-catalyzed drugs, which needs to be considered in clinical practice.

Modulation of Rat Hepatic CYP1A and 2C Activity by Honokiol and Magnolol: Differential Effects on Phenacetin and Diclofenac Pharmacokinetics In Vivo.

Honokiol (2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol) and magnolol (4-Allyl-2-(5-allyl-2-hydroxy-phenyl)phenol) are the major active polyphenol constituents of Magnolia officinalis (Magnoliaceae) bark, which has been widely used in traditional Chinese medicine (Houpu Tang) for the treatment of various diseases, including anxiety, stress, gastrointestinal disorders, infection, and asthma. The aim of this study was to investigate the direct effects of honokiol and magnolol on hepatic CYP1A and 2C-mediated metabolism in vitro using rat liver microsomes and in vivo using the Sprague-Dawley rat model. Honokiol and magnolol inhibited in vitro CYP1A activity (probe substrate: phenacetin) more potently than CYP2C activity (probe substrate: diclofenac): The mean IC50 values of honokiol for the metabolism of phenacetin and diclofenac were 8.59 μM and 44.7 μM, while those of magnolol were 19.0 μM and 47.3 μM, respectively. Notably, the systemic exposure (AUC and Cmax) of phenacetin, but not of diclofenac, was markedly enhanced by the concurrent administration of intravenous honokiol or magnolol. The differential effects of the two phytochemicals on phenacetin and diclofenac in vivo pharmacokinetics could at least be partly attributed to their lower IC50 values for the inhibition of phenacetin metabolism than for diclofenac metabolism. In addition, the systemic exposure, CL, and Vss of honokiol and magnolol tended to be similar between the rat groups receiving phenacetin and diclofenac. These findings improve our understanding of CYP-mediated drug interactions with M. officinalis and its active constituents.

Long-term human primary hepatocyte cultures in a microfluidic liver biochip show maintenance of mRNA levels and higher drug metabolism compared with Petri cultures.

Human primary hepatocytes were cultivated in a microfluidic bioreactor and in Petri dishes for 13 days. mRNA kinetics in biochips showed an increase in the levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6, HNF4a, SULT1A1, UGT1A1 mRNA related genes when compared with post extraction levels. In addition, comparison with Petri dishes showed higher levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6 related genes at the end of culture. Functional assays illustrated a higher urea and albumin production over the period of culture in biochips. Bioreactor drug metabolism (midazolam and phenacetin) was not superior to the Petri dish after 2 days of culture. The CYP3A4 midazolam metabolism was maintained in biochips after 13 days of culture, whereas it was almost undetectable in Petri dishes. This led to a 5000-fold higher value of the metabolic ratio in the biochips. CYP1A2 phenacetin metabolism was found to be higher in biochips after 5, 9 and 13 days of culture. Thus, a 100-fold higher metabolic ratio of APAP in biochips was measured after 13 days of perfusion. These results demonstrated functional primary human hepatocyte culture in the bioreactor in a long-term culture. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro studies of berberine metabolism and its effect of enzyme induction on HepG2 cells

Ethnopharmacological relevanceBerberine (BER) and BER-original herbal medicines have a variety of pharmacological functions and have been widely used in clinical. However, its effect of enzyme induction on cytochrome P450 (CYP) in human hepatocytes is unknown. Material and methodMetabolism of berberine and its effect on main metabolic enzymes in HepG2 cell in vitro was investigated. Cocktail probe drugs, mRNA expression and protein expression were used to evaluate the metabolism potency. Meanwhile, an UPLC–MS/MS method was validated for the analysis of BER and four probe drugs in HepG2 cell. ResultBER significantly increased the metabolism of midazolam, phenacetin and tolbutamide by inducing the CYP1A2 and 3A4 enzyme in a dose-dependent manner, the mRNA and protein expression of CYP1A2 and 3A4 were increased by berberine at 1000ng·mL−1. The activity of CYP1A2 and 3A4 could be induced by BER more than 500ng·mL−1 in HepG2 cell, which was confirmed by the increase of its mRNA and protein expression. ConclusionBER increases the metabolism of cocktail drugs such as midazolam, phenacetin and tolbutamide by increasing the mRNA and protein expression of CYP1A2 and 3A4.

Impact of age on hepatic cytochrome P450 of domestic male Camborough-29 pigs.

Swine is not only an important species in veterinary medicine research but also a popular animal model for human drug discovery. It is valuable to understand the impact of pig age on abundance and activity of porcine hepatic cytochrome P450 (CYP450). Liver microsomes were prepared from Camborough-29 intact male pigs at the age of 1day and 2weeks and the castrated male pigs at the age of 5, 10, and 20weeks. Hepatic CYP450 content in the liver microsomes was measured using a UV/visible spectroscopic method. The activities of CYP450s were evaluated by metabolism of phenacetin, coumarin, tolbutamide, bufuralol, chlorzoxazone, and midazolam. The porcine hepatic CYP450 content increased with age with a plateau between age 2 and 5weeks. Activities of all CYP450 enzymes increased with age of pigs too. The bufuralol 1'-hydroxylase showed the highest hepatic activities compared with other CYP enzymes at all ages of pigs. The average activities at the age of 20weeks were about five times higher than those at the age of 5weeks for most of the CYP enzymes. With compensation of the ratio of liver to body weights, the overall CYP450 metabolism capability of the pigs may be peaked around ages of 10 to 20weeks. Those findings suggest that metabolism can be significantly different in growing phase of pigs and that the age may be an important factor in porcine medicine evaluation and pig model development.

Inhibition of Baicalin on Metabolism of Phenacetin, a Probe of CYP1A2, in Human Liver Microsomes and in Rats

Baicalin has been used as mainly bioactive constituent of about 100 kinds of traditional Chinese medicines in Chinese pharmacopoeia. The effect of baicalin on cytochrome P450 should be paid more attention because baicalin was used widely. The aim of this study was to investigate whether baicalin could inhibit CYP1A2 in pooled human liver microsomes (HLMs) and in rats in vivo and the gene polymorphisms could affect inter-individual variation in IC50 in 28 human livers. Phenacetin was used as probe of CYP1A2. Kinetic parameter of CYP1A2 and IC50 of baicalin on CYP1A2 to each sample were measured and the common CYP1A2 polymorphisms (−3860G>A and −163C>A) were genotyped. The results showed that baicalin exhibited a mixed-type inhibition in pooled HLMs, with a Ki value of 25.4 µM. There was substantial variation in Km, Vmax, CLint of CYP1A2 and IC50 of baicalin on CYP1A2 (3∼10-fold). The range was from 26.6 to 114.8 µM for Km, from 333 to 1330 pmol·min−1·mg−1protein for Vmax and from 3.8 to 45.3 µL·min−1·mg−1 protein for CLint in HLMs (n = 28). The Mean (range) value of IC50 in 28 HLMs was 36.3 (18.9 to 56.1) µM. The genotypes of −3860G>A and −163C>A had no significant effect on the inhibition of baicalin on CYP1A2. The animal experiment results showed that baicalin (450 mg/kg, i.v.) significantly decreased the Cmax and CL of phenacetin, and increased C60 min, t1/2, Vd and AUC (P<0.05). There were significant correlations between percentage of control in C60 min, t1/2, CL, AUC of phenacetin and Cmax of baicalin in 11 rats (P<0.05). Protein binding experiments in vitro showed that baicalin (0–2000 mg/L) increased the unbound phenacetin from 14.5% to 28.3%. In conclusion, baicalin can inhibit the activity of CYP1A2 in HLMs and exhibit large inter-individual variation that has no relationship with gene polymorphism. Baicalin can change the pharmacokinetics of phenacetin in rats.

English

The aim of this study was to investigate the utility of the phenacetin metabolism test in evaluating hepatic reserve function prior to hepatectomy for the prevention of postoperative liver dysfunction. Fifty-six patients with hepatocellular carcinoma had undergone hepatectomy. Patients were classified into group I (normal group, n = 37) and group II (with peak total bilirubin &gt;53.8 &mu;mol/L for 7 days after hepatectomy, n = 19) based on the levels of total bilirubin after hepatectomy. The receiver operating characteristic (ROC) analysis was made to assess the hepatic reserve function to predict liver dysfunction of the patients after hepatectomy. Hepatic reserve function was evaluated by phenacetin metabolism test; the ratio of plasma total paracetamol to phenacetin at 2 h after oral 1.0 g phenacetin. There were no significant differences in preoperative variables or intraoperative findings except the ratio of plasma total paracetamol to phenacetin. ROC analysis showed that the sensitivity and specificity of the ratio of plasma total paracetamol to phenacetin &le;1.2 were 85.4 and 72.9%, respectively for predicting liver dysfunction of the patients after hepatectomy. The ratio of plasma total paracetamol to phenacetin correlated with the temporary postoperative liver dysfunction (P = 0.008). Phenacetin metabolism test before hepatectomy appears to provide direct and reliable measure of hepatic reserve function, thus helping in surgical decision making regarding the extent of hepatectomy and in the prevention of the occurrence of postoperative liver dysfunction.&nbsp; &nbsp; Key words: Phenacetin metabolism test, hepatic reserve function, hepatectomy, liver Dysfunction

Possibility of Decrease in CYP1A2 Function in Patients With End‐Stage Renal Disease

Propranolol, the substrate of cytochrome P450 (CYP) 1A2 and CYP2D6, has been reported to be in high concentrations in end-stage renal disease (ESRD) patients. This has been thought to be due to the decrease in the nonrenal clearance of propranolol. The objective of this study is to elucidate the reason for the decrease in nonrenal clearance in ESRD patients. CYP1A2 and CYP2D6 activities were estimated by the phenacetin O-deethylation and methoprolol O-demethylation methods, respectively. Pooled normal serum and pooled uremic serum were deproteinized by methanol in order to exclude high-molecular-weight compounds. We selected as candidate inhibitors: uremic toxins such as 3-indoxyl sulfate, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, indole-3-acetic acid, and hippuric acid, and xanthine derivatives such as allantoin, uric acid, and xanthine. In this study, uremic serum was found to inhibit the CYP1A2-mediated metabolism of phenacetin to acetaminophen in a concentration-dependent and competitive manner. Xanthine also inhibited the metabolism of CYP1A2. On the other hand, uremic serum and the four uremic toxins did not inhibit the CYP2D6-mediated metabolism of metoprolol to O-demethylmetoprolol. In conclusion, this study suggests that the increase of the bioavailability of propranolol in ESRD is partly induced by the inhibition of the hepatic metabolism of CYP1A2 by xanthine in the uremic serum.

Pharmacokinetic changes of unbound theophylline are due to plasma protein binding displacement and CYP1A2 activity inhibition by baicalin in rats

Ethnopharmacological relevanceBaicalin is one of the major bioactive constituents of Scutellariae Radix, the root of Scutellariae baicalensis Georgi and possesses a wide variety of pharmacological properties. Aim of the studyTo elucidate the effect of baicalin on the pharmacokinetics of theophylline in rats, focusing on plasma protein binding displacement and inhibition effect on CYP1A2 in vivo and in vitro. Materials and methodsThe study was a randomized, three-period crossover design. Nine rats were given saline (control) or 450mg/kg baicalin (dosage regimen A or B). Dosage regimen A was administered once at 0h. Dosage regimen B was divided into three dosages (225,112.5, 112.5mg/kg) and was given at 0, 2 and 4h, respectively. Then theophylline (5mg/kg, i.v.) was administered immediately. The effect of baicalin on CYP1A2 activity was determined by metabolism of phenacetin in vitro and plasma protein binding of theophylline was determined by ultrafiltration. ResultsCmax decreased from (12.4±1.6) to (8.7±0.9) and (8.6±2.0)mg/L, T1/2 increased by 116 and 96%, Vd increased by 51 and 49% for total theophylline in rats treated with dosage regimen A and B of baicalin, respectively. Cmax was significantly increased, Vd decreased by 43 and 29% for unbound theophylline in rats treated with dosage regimen A and B of baicalin, respectively (P<0.01). T1/2 of unbound theophylline increased by 104% only in rats treated with dosage regimen B. No significant effects on the CL and AUC of both total and unbound theophylline were observed in the rats treated with dosage regimen A, but the CL decreased and AUC increased for total theophylline and CL decreased for unbound theophylline in the group treated with dosage regimen B (P<0.05). Correlation analysis showed that the mean unbound theophylline (%) and mean baicalin concentration was in good correlation (P<0.01). Baicalin decreased metabolism of phenacetin and exhibited a mixed-type inhibition in rat liver microsomes, with a Ki value of 88.1μM in vitro. Moreover baicalin was a competitive displacer of theophylline from plasma protein in vitro. ConclusionsThe changes in Cmax, T1/2, CL and AUC of theophylline due to baicalin may be attributed to two mechanisms, plasma protein binding displacement and CYP1A2 activity inhibition.

Preferred Binding Orientations of Phenacetin in CYP1A1 and CYP1A2 Are Associated with Isoform-Selective Metabolism

Human cytochromes P450 1A1 and 1A2 play important roles in drug metabolism and chemical carcinogenesis. Although these two enzymes share high sequence identity, they display different substrate specificities and inhibitor susceptibilities. In the present studies, we investigated the structural basis for these differences with phenacetin as a probe using a number of complementary approaches, such as enzyme kinetics, stoichiometric assays, NMR, and molecular modeling. Kinetic and stoichiometric analyses revealed that substrate specificity (k(cat)/K(m)) of CYP1A2 was approximately 18-fold greater than that of CYP1A1, as expected. Moreover, despite higher H₂O₂ production, the coupling efficiency of reducing equivalents to acetaminophen formation in CYP1A2 was tighter than that in CYP1A1. CYP1A1, in contrast to CYP1A2, displayed much higher uncoupling, producing more water. The subsequent NMR longitudinal (T₁) relaxation studies with the substrate phenacetin and its product acetaminophen showed that both compounds displayed similar binding orientations within the active site of CYP1A1 and CYP1A2. However, the distance between the OCH₂ protons of the ethoxy group (site of phenacetin O-deethylation) and the heme iron was 1.5 Å shorter in CYP1A2 than in CYP1A1. The NMR findings are thus consistent with our kinetic and stoichiometric results, providing a likely molecular basis for more efficient metabolism of phenacetin by CYP1A2.

Nicotine and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Binding and Access Channel in Human Cytochrome P450 2A6 and 2A13 Enzymes

Cytochromes P450 (CYP) from the 2A subfamily are known for their roles in the metabolism of nicotine, the addictive agent in tobacco, and activation of the tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Although both the hepatic CYP2A6 and respiratory CYP2A13 enzymes metabolize these compounds, CYP2A13 does so with much higher catalytic efficiency, but the structural basis for this has been unclear. X-ray structures of nicotine complexes with CYP2A13 (2.5 Å) and CYP2A6 (2.3 Å) yield a structural rationale for the preferential binding of nicotine to CYP2A13. Additional structures of CYP2A13 with NNK reveal either a single NNK molecule in the active site with orientations corresponding to metabolites known to form DNA adducts and initiate lung cancer (2.35 Å) or with two molecules of NNK bound (2.1 Å): one in the active site and one in a more distal staging site. Finally, in contrast to prior CYP2A structures with enclosed active sites, CYP2A13 conformations were solved that adopt both open and intermediate conformations resulting from an ∼2.5 Å movement of the F to G helices. This channel occurs in the same region where the second, distal NNK molecule is bound, suggesting that the channel may be used for ligand entry and/or exit from the active site. Altogether these structures provide multiple new snapshots of CYP2A13 conformations that assist in understanding the binding and activation of an important human carcinogen, as well as critical comparisons in the binding of nicotine, one of the most widely used and highly addictive drugs in human use.

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 (IC50 19.7μM) and CYP3A4-mediated metabolism of testosterone to 6β-hydroxytestosterone (IC20 7.06μM). Enzyme kinetics studies showed the inhibition of CYP1A2 activity was competitive and concentration-dependent (Ki=18.4μM). Inhibition of testosterone to 6β-hydroxytestosterone was also competitive and concentration-dependent (Ki=31.8μM). Metabolism of dextromethorphan to dextrorphan (CYP2D6-mediated) and chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1-mediated) was only minimally inhibited by PSP, with IC20 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 Ki values for CYP1A2 and CYP3A4 would suggest a low potential for PSP to cause herb–drug interaction related to these CYP isoforms.

Phenacetin O-Deethylation is a Useful Tool for Evaluation of Hepatic Functional Reserve in Rats with CCl4-Induced Chronic Liver Injury

Mortality of liver resection is as high as 3.1% to 25% in patients with chronic liver disease. Evaluation of hepatic functional reserve is critical for the prediction of risk of postoperation death. Phenacetin O-deethylation is a marker reaction of cytochrome P4501A2 (CYP1A2) activity. In this study, our aim is to investigate whether phenacetin O-deethylation is a useful tool for the evaluation of hepatic functional reserve in rats with chronic liver injury. Rat model for chronic liver injury was established by subcutaneous administration of 50% CCl(4), 1 mL/kg twice per week for 12 wk. Hepatic CYP1A2 activity, content, and mRNA expression were determined (n = 10). Effects of 15%, 30%, and 45% hepatectomy on phenacetin O-deethylation were evaluated in the rats (n = 5 in each group). Additionally, the correlation of risk of death after 70% hepatectomy with phenacetin O-deethylation was studied in 27 rats with chronic liver injury. Compared with normal controls, CYP1A2 activity, content, and mRNA expression decreased 33%, 60%, and 50% in the rats with chronic liver injury (P < 0.05), respectively. Following the increasing of liver-resected size, CYP1A2 activity decreased proportionally (r(s) = -0.877, P < 0.05). Six of 27 rats with chronic liver injury died within 7 d after 70% hepatectomy. Phenacetin metabolism was impaired more severely in 6 rats that died than in 21 living rats (P < 0.05). Phenacetin O-deethylation is a useful tool for the evaluation of hepatic functional reserve in the rats with CCl(4)-induced chronic liver injury.

Effect of Dimethyl Sulfoxide on In Vitro Cytochrome P4501A2 Mediated Phenacetin O-Deethylation in Human Liver Microsomes

In this study, we report the effect of dimethyl sulfoxide (DMSO), acetonitrile, and methanol on the CYP1A2-mediated metabolism of phenacetin in human liver microsomes. Phenacetin O-deethylation is the preferred probe reaction for CYP1A2, in which the metabolite, acetaminophen, is quantified using liquid chromatography-tandem mass spectrometry. DMSO was found to inhibit CYP1A2-mediated phenacetin O-deethylation even at low concentrations (0.1%). Acetonitrile did not significantly change the phenacetin O-deethylation activity at concentrations up to 2%. There was no effect on the phenacetin O-deethylation when methanol was present at levels up to 2%. We found that the DMSO level should be kept lower than 0.05% because a concentration of 0.1% strongly affected the metabolism of phenacetin. These findings should be taken into consideration when designing in vitro metabolism studies, especially studies in which metabolism of the investigational compound needs to be evaluated, which would confound the results. The findings from this study indicate that methanol is the suitable solvent and has no significant effects on CYP1A2-mediated phenacetin O-deethylation.

A Physiologically Based Pharmacokinetic Model Characterizing Mechanism-based Inhibition of CYP1A2 for Predicting Theophylline/Antofloxacin Interaction in both Rats and Humans

Clinical studies have revealed that some fluoroquinolones may cause severe adverse effects when co-administered with substrates of CYP1A2. Our previous study showed antofloxacin (ATFX) was responsible for mechanism-based inhibition (MBI) of the metabolism of phenacetin in rats. In the clinical setting, ATFX is likely to be administrated with theophylline (TP), which is mainly metabolized by CYP1A2. The aim of the present study was to investigate the possible mechanism of TP/ATFX interaction. In vitro studies showed that the inhibitory effect of ATFX on the formation of three TP metabolites depended on NADPH, the pre-inhibition time, and ATFX concentration, i.e., factors which characterize MBI. In vivo studies demonstrated that single-dose ATFX (20 mg/kg) did not affect the pharmacokinetic behavior of TP, but multidose ATFX (20 mg/kg b.i.d. for 7.5 days) significantly increased the AUC of TP, decreased the amount of three TP metabolites in urine, and suppressed hepatic microsomal activity. A physiologically based pharmacokinetic (PBPK) model characterizing MBI of the three TP metabolites was developed for predicting TP/ATFX interaction in rats; this model was further extrapolated to humans. The predicted results were in good agreement with observed data. All the results indicated that ATFX was responsible for MBI of the metabolism of TP, and the PBPK model characterizing MBI may give good prediction of TP/ATFX interaction.

Evaluating human liver reserve function by measuring serum concentrations of phenacetin and its metabolites

To evaluate human liver reserve function (LRF) by a simple and efficient method for measuring serum concentrations of phenacetin and its metabolites. Overall 20 patients with liver cirrhosis (Child-Pugh score ≥ 7, aged 48-79 years), 30 healthy young volunteers (aged 18-40 years), and 20 healthy elderly volunteers (aged 61-80 years) were enrolled. All participants received a single oral dose of 0.5 g phenacetin. Liquid chromatography tandem mass spectrometry was used to determine the serum concentrations of phenacetin and its metabolites, including acetaminophen, acetaminophen glucuronide and acetaminophen sulfate. The serum concentration of phenacetin was significantly higher in cirrhotic patients than those in either of the healthy volunteer groups (P < 0.001). It was higher in healthy elderly volunteers than that in healthy young ones but there was no statistically significant difference (P > 0.05) between them. The serum concentrations of acetaminophen, acetaminophen glucuronide and acetaminophen sulfate were significantly lower in cirrhotic patients than in the healthy controls (P < 0.001). The serum concentrations of these three metabolites in healthy elderly volunteers were lower than those in healthy younger volunteers but again, there was no statistical significant difference (P > 0.05). The serum concentration of acetaminophen in healthy male volunteers was significantly higher than that in the women (P < 0.05). Monitoring cytochrome P450 1A2 (CYP450 1A2)-mediated phenacetin metabolism is a simple and efficient method for evaluating human LRF. This method would warrant further validation in a large cohort clinical study.