Short-Term Inhibition of Translation by Cycloheximide Concurrently Affects Mitochondrial Function and Insulin Secretion in Islets from Female Mice.

Since glucose stimulates protein biosynthesis in beta cells concomitantly with the stimulation of insulin release, the possible interaction of both processes was explored. The protein biosynthesis was inhibited by 10 µM cycloheximide (CHX) 60 min prior to the stimulation of perifused, freshly isolated or 22 h-cultured NMRI mouse islets. CHX reduced the insulinotropic effect of 25 mM glucose or 500 µM tolbutamide in fresh but not in cultured islets. In cultured islets the second phase of glucose stimulation was even enhanced. In fresh and in cultured islets CHX strongly reduced the content of proinsulin, but not of insulin, and moderately diminished the [Ca2+]i increase during stimulation. The oxygen consumption rate (OCR) of fresh islets was about 50% higher than that of cultured islets at basal glucose and was significantly increased by glucose but not tolbutamide. In fresh, but not in cultured, islets CHX diminished the glucose-induced OCR increase and changes in the NAD(P)H- and FAD-autofluorescence. It is concluded that short-term CHX exposure interferes with the signal function of the mitochondria, which have different working conditions in fresh and in cultured islets. The interference may not be an off-target effect but may result from the inhibited cytosolic synthesis of mitochondrial proteins.

Functional characterization of the defective CYP2C9 variant CYP2C9*18.

Cytochrome P450 2C9 (CYP2C9) is one of the most important drugs metabolizing enzymes and accounts for the metabolism of about 13%-17% of clinical drugs. Like other members in CYP2 family, CYP2C9 gene exhibits great genetic polymorphism among different races and individuals. CYP2C9*18 is one CYP2C9 allelic variant identified in a Southeast Asian population and is estimated to cause the amino acid substitutions of I359L and D397A in CYP2C9 enzyme simultaneously. Limited by the low expression level in bacteria and COS-7 cells, no valuable enzyme kinetics have been reported on this CYP2C9 variant. In this study, the baculovirus-based system was used for the high expression of recombinant CYP2C9 s in insect cells. As a result, together with I359L substitution, D397A could significantly decrease the protein expression of CYP2C9.18 in insect cells, although substitution of D397A alone had no effect on the expression of CYP2C9 in vitro. As compared with that of wild-type enzyme, both CYP2C9.18 variant and D397A variant could decrease more than 80% of the catalytic activity of CYP2C9 enzyme toward three probe substrates, suggesting that caution should be exercised when patients carrying CYP2C9*18 taking medicines metabolized by CYP2C9 enzyme with a narrow therapeutic window.

Contribution of the human cytochrome P450 2C subfamily to the metabolism of and the interactions with endogenous compounds including steroid hormones.

The objectives of this study were as follows: 1) to compare the metabolic activities of endogenous compounds and their effects on dopamine formation and hydroxylation of steroid hormones, mediated by human cytochrome P450 (CYP), including CYP2C9.1 and CYP2C19, as well as the variants CYP2C9.2 (Arg144Cys) and CYP2C9.3 (Ile359Leu); and 2) to assess the effects of steroid hormones on the activities of CYP2C9.1, CYP2C9.2, and CYP2C19 to estimate the contribution of the CYP2C subfamily to metabolism and drug-drug interactions of endogenous compounds. Dopamine formation from p -tyramine and 6ß- and 21- (for progesterone) hydroxylation of testosterone, cortisol, and progesterone by CYP2C9 variants, CYP2C19, CYP2D6, and CYP3A4 were determined using HPLC. The effects of steroid hormones such as testosterone, cortisol, and progesterone on tolbutamide methyl hydroxylation mediated by CYP2C subfamily members were investigated. Only CYP2D6 catalyzed dopamine formation. The 6ß-hydroxylation activities of testosterone, cortisol, and progesterone catalyzed by CYP2C9 variants and CYP2D6 were less than 5% of those by CYP3A4. Although cortisol did not inhibit tolbutamide methyl hydroxylation catalyzed by CYP2C9.1, CYP2C9.2, or CYP2C19 and testosterone did not inhibit CYP2C19 activity, the reactions catalyzed by CY2C9.1 and CYP2C9.2 were inhibited by testosterone. The inhibition of progesterone by CYP2C19 was stronger than that by CYP2C9.1 and CYP2C9.2. CYP2C9.1 and CYP2C19 noncompetitively and competitively inhibited tolbutamide methyl hydroxylation with inhibition constants of 43.2 µM and 1.03 µM, respectively. Clinical interactions among endogenous compounds would vary within the CYP2C subfamily, although the contribution of the CYP2C subfamily may be of minor importance for dopamine formation and the detoxification (6ß-hydroxylation) of endogenous steroid hormones.

Evaluation of cytochrome P450-based drug metabolism in hemorrhagic shock rats that were transfused with native and an artificial red blood cell preparation, Hemoglobin-vesicles.

Hemoglobin-vesicles (Hb-V) are being developed as red blood cell (RBC) substitutes. In this study, we report on quantitative and qualitative alterations of hepatic cytochrome P450 (CYPs) and the pharmacokinetics of CYP-metabolizing drugs, with a focus on four CYP isoforms (CYP1A2, CYP2C11, CYP2E1 and CYP3A2), after Hb-V resuscitation from a massive hemorrhage. The results of proteome analysis and western blot data indicate that resuscitation with both Hb-V and packed RBC (PRBC) resulted in a decrease in the protein levels of CYPs. Along with a decrease in the protein expression of CYPs, pharmacokinetic studies showed that the elimination of CYP-metabolizing drugs was prolonged in the Hb-V and PRBC resuscitation groups. It is also noteworthy that the CYP-metabolizing drugs in the Hb-V resuscitation group was retained for a longer period compared to the PRBC resuscitation group, and this is attributed to the CYP isoforms having a lower metabolic activity in the Hb-V resuscitation group than that for the PRBC resuscitation group. These findings suggest that resuscitation with Hb-V after a massive hemorrhage has a slight but not clinically significant effect on drug metabolism via CYPs in the liver due to decreased protein levels and the metabolic activity with respect to the CYPs.

An identification and functional evaluation of a novel CYP2C9 variant CYP2C9*62.

Warfarin is the most commonly used anticoagulant in the clinical treatment of thromboembolic diseases. The dose of warfarin varies significantly within populations, and the dose is closely related to the genetic polymorphisms of the CYP2C9 and VKORC1 genes. In this study, a new CYP2C9 nonsynonymous mutation (8576C > T) was detected after the genetic screening of 162 patients took warfarin. This mutation, named as the new allele CYP2C9*62, can result in an arginine to cysteine amino acid substitution at position 125 of the CYP2C9 protein (R125C). When expressed in insect cells, the protein expression of CYP2C9.62 was significantly lower than that of the wild-type, and its metabolic activity was also significantly decreased after the addition of three typical CYP2C9 probe drugs, suggesting that the new mutant can dramatically affect the metabolism of CYP2C9 drugs in vitro.

Green analytical method for the simultaneous analysis of cytochrome P450 probe substrates by poly(N-isopropylacrylamide)-based temperature-responsive chromatography.

High-performance liquid chromatography (HPLC) is the most common analytical method practiced in various fields and used for analysis of almost all drug compounds in the pharmaceutical industries. During drug development, an evaluation of potential drug interaction with cytochrome P450 (CYP) is essential. A "cocktail" approach is often used in drug development to evaluate the effect of a drug candidate on multiple CYP enzymes in a single experiment. So far, simultaneous analysis of multiple CYP substrates, which have greatly different structure and physicochemical properties, has required organic solvents and mobile phase gradient methods. However, despite the recent emphasis on environmental protection, analytical methods that use only aqueous solvents without the use of organic solvents for separation have not been studied well. This study sought to develop the simultaneous analysis of multiple CYP substrates by using poly(N-isopropylacrylamide) (PNIPAAm)-based temperature-responsive chromatography with only aqueous solvents and isocratic methods. Good separation of multiple CYP substrates was achieved without using organic solvents and any gradient methods by temperature-responsive chromatography utilizing a P(NIPAAm-co-n-butyl methacrylate (BMA))- and P(NIPAAm-co-N-acryloyl L-tryptophan methyl ester (L-Trp-OMe))-grafted silica column. Overall, PNIPAAm-based temperature-responsive chromatography represents a remarkably simple, versatile, and environmentally friendly bioanalytical method for CYP substrates and their metabolites.

Calycosin Influences the Metabolism of Five Probe Drugs in Rats.

BACKGROUND: Calycosin (CAL), a type of O-methylated isoflavone extracted from the herb Astralagusmembranaceus (AM), is a bioactive chemical with antioxidative, antiphlogistic and antineoplastic activities commonly used in traditional alternative Chinese medicine. AM has been shown to confer health benefits as an adjuvant in the treatment of a variety of diseases. AIM: The main objective of this study was to determine whether CAL influences the cytochrome P450 (CYP450) system involved in drug metabolism. METHODS: Midazolam, tolbutamide, omeprazole, metoprolol and phenacetin were selected as probe drugs. Rats were randomly divided into three groups, specifically, 5% Carboxymethyl cellulose (CMC) for 8 days (Control), 5% CMC for 7 days + CAL for 1 day (single CAL) and CAL for 8 days (conc CAL), and metabolism of the five probe drugs evaluated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: No significant differences were observed for omeprazole and midazolam, compared to the control group. T max and t1/2 values of only one probe drug, phenacetin, in the conc CAL group were significantly different from those of the control group (T max h: 0.50±0.00 vs 0.23±0.15; control vs conc CAL). C max of tolbutamide was decreased about two-fold in the conc CAL treatment group (conc vs control: 219.48 vs 429.56, P<0.001). CONCLUSION: Calycosin inhibits the catalytic activities of CYP1A2, CYP2D6 and CYP2C9. Accordingly, we recommend caution, particularly when combining CAL as a modality therapy with drugs metabolized by CYP1A2, CYP2D6 and CYP2C9, to reduce the potential risks of drug accumulation or ineffective treatment.

Influences of cytochrome b5 expression and its genetic variant on the activity of CYP2C9, CYP2C19 and CYP3A4.

The objective of the present study was to investigate the effects of cytochrome b5 (cytb5) on the drug metabolism catalyzed by CYP2C9, CYP2C19 and CYP3A4. Activities of CYP2C9, CYP2C19, and CYP3A4 were determined by using the prototypical substrates tolbutamide, omeprazole and midazolam, respectively. Cytb5 protein and mRNA contents showed large inter-individual variations with 11- and 6-fold range, respectively. All of three P450s showed an increased activity in proportion to the amount of cytb5 expression. Particularly, CYP3A4 showed the strongest correlation between cytb5 protein amount and the activity, followed by CYP2C9 and CYP2C19. The putative splicing variant, c.288G>A (rs7238987) was identified and was screened in 36 liver tissues by direct DNA sequencing. Liver tissues having a splicing variant exhibited unexpected sizes of cytb5 mRNA and a decreased expression tendency of cytb5 protein compared to the wild-type. A decreased activity in the metabolism of the CYP2C19 substrate omeprazole was observed in liver tissues carrying the splicing variant when compared to the wild-type Cytb5 (P < 0.05). The present results propose that different expression of cytb5 can cause variations in CYP mediated drug metabolism, which may explain, at least in part, the inter-individual difference in drug responses in addition to the CYP genetic polymorphisms.

Rabbit dehydrogenase/reductase SDR family member 11 (DHRS11): Its identity with acetohexamide reductase with broad substrate specificity and inhibitor sensitivity, different from human DHRS11.

Human dehydrogenase/reductase SDR family member 11 (DHRS11) has been recently reported to be an NADP+-dependent 3(17)ß-hydroxysteroid dehydrogenase, and its orthologs are predicted in genomic analyses of various animals. Among them, the amino acid sequence of predicted rabbit DHRS11 shares 92% identity with that of human DHRS11 and matches peptide sequences (composed of total 87 amino acids) of rabbit heart acetohexamide reductase (RHAR) previously reported. However, the physiological role of RHAR remains unknown, because its known substrates are only acetohexamide and 1,4-naphthoquinone. To elucidate whether the two rabbit enzymes are identical, we have isolated the cDNA for rabbit DHRS11, which was abundantly detected in the brain, heart, kidney and intestine by RT-PCR. The recombinant rabbit DHRS11 reduced acetohexamide and 1,4-naphthoquinone, and was inhibited by tolbutamide and phenobarbital (RHAR-specific inhibitors), demonstrating its identity with RHAR. Rabbit DHRS11 also reduced α-dicarbonyl compounds, aldehydes and aromatic ketones (acetylbenzenes and acetylpyridines), and exhibited 3(17)ß-hydroxysteroid dehydrogenase activity. It was competitively inhibited not only by tolbutamide and phenobarbital, but also more potently by several non-steroidal anti-inflammatory drugs such as diclofenac and sulindac. The broad substrate specificity and inhibitor sensitivity were different from those of human DHRS11, which did not reduce aliphatic aldehydes and aromatic ketones despite its higher 3(17)ß-hydroxysteroid dehydrogenase activity, and was insensitive to tolbutamide, phenobarbital and diclofenac. The site-directed mutagenesis of Thr163 and Val200 in human DHRS11 to the corresponding residues (Gly and Leu, respectively) in rabbit DHRS11 suggested that these residues are pertinent to the differences in properties of rabbit and human DHRS11s.

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.

Predicted contributions of cytochrome P450s to drug metabolism in human liver microsomes using relative activity factor were dependent on probes.

Contributions of cytochrome P450 (CYP450) isoforms to drug metabolism are often predicted using relative activity factor (RAF) method, assuming RAF values were independent of probe. We aimed to report probe-dependent characteristic of RAF values using CYP3A4 or CYP2C9 probes. Metabolism of four CYP3A4 probes (testosterone, midazolam, verapamil and atorvastatin) and three CYP2C9 probes (tolbutamide, diclofenac and S-warfarin) in human liver microsomes (HLM) and cDNA-expressed recombinant CYP450 (Rec-CYP450) systems were characterized and RAFCL value was estimated as ratio of probe intrinsic clearance in HLM to that in Rec-CYP450. CYP450i contributions to metabolic reaction of a probe were predicted using other probes and compared with data from specific inhibitions. Contributions of CYP3A4 and CYP2C9 to metabolism of deoxypodophyllotoxin and nateglinide were also predicted. RAF values were dependent on probes, leading to probe-dependently predicted contributions. Predicted contributions of CYP3A4 to formations of 6ß-hydroxytestosterone, 1'-hydroxymidazolam, norverapamil, ortho-hydroxyatorvastatin and para-hydroxyatorvastatin using other probes were 47.46-219.46%, 21.62-98.87%, 186.49-462.44%, 21.87-101.15% and 53.62-247.97%, respectively. Predicted contributions of CYP3A4 and CYP2C9 to nateglinide metabolism were 8.18-37.84% and 36.08-94.04%, separately. In conclusion, CYP450i contribution to drug metabolism in HLM estimated using RAF approach were probe-dependent. Therefore, contribution of each isoform must be confirmed by multiple probes.

Comparative metabolism of xenobiotic chemicals by cytochrome P450s in the nematode Caenorhabditis elegans.

We investigated the metabolic capabilities of C. elegans using compounds whose metabolism has been well characterised in mammalian systems. We find that similar metabolites are produced in C. elegans as in mammals but that C. elegans is deficient in CYP1-like metabolism, as has been seen in other studies. We show that CYP-34A9, CYP-34A10 and CYP-36A1 are the principal enzymes responsible for the metabolism of tolbutamide in C. elegans. These are related to the mammalian enzymes that metabolise this compound but are not the closest homologs suggesting that sequence comparison alone will not predict functional conservation among cytochrome P450s. In mammals, metabolite production from amytryptiline and dextromethorphan is dependent on specific cytochrome P450s. However, in C. elegans we did not find evidence of similar specificity: the same metabolites were produced but in small amounts by numerous cytochrome P450s. We conclude that, while some aspects of cytochrome P450 mediated metabolism in C. elegans are similar to mammals, there are differences in the production of some metabolites and in the underlying genetics of metabolism.

Novel in vitro dynamic metabolic system for predicting the human pharmacokinetics of tolbutamide.

Liver metabolism is commonly considered the major determinant in drug discovery and development. Many in vitro drug metabolic studies have been developed and applied to understand biotransformation. However, these methods have disadvantages, resulting in inconsistencies between in vivo and in vitro experiments. A major factor is that they are static systems that do not consider the transport process in the liver. Here we developed an in vitro dynamic metabolic system (Bio-PK metabolic system) to mimic the human pharmacokinetics of tolbutamide. Human liver microsomes (HLMs) encapsulated in a F127'-Acr-Bis hydrogel (FAB hydrogel) were placed in the incubation system. A microdialysis sampling technique was used to monitor the metabolic behavior of tolbutamide in hydrogels. The measured results in the system were used to fit the in vitro intrinsic clearance of tolbutamide with a mathematical model. Then, a PBPK model that integrated the corresponding in vitro intrinsic clearance was developed to verify the system. Compared to the traditional incubation method, reasonable PK profiles and the in vivo clearance of tolbutamide could be predicted by integrating the intrinsic clearance of tolbutamide obtained from the Bio-PK metabolic system into the PBPK model. The predicted maximum concentration (Cmax), area under the concentration-time curve (AUC), time to reach the maximum plasma concentration (Tmax) and in vivo clearance were consistent with the clinically observed data. This novel in vitro dynamic metabolic system can compensate for some limitations of traditional incubation methods; it may provide a new method for screening compounds and predicting pharmacokinetics in the early stages, supporting the development of compounds.

Organic Anion Transporter 2 Mediates Hepatic Uptake of Tolbutamide, a CYP2C9 Probe Drug.

Tolbutamide is primarily metabolized by CYP2C9, and, thus, is frequently applied as a clinical probe substrate for CYP2C9 activity. However, there is a marked discrepancy in the in vitro-in vivo extrapolation of its metabolic clearance, implying a potential for additional clearance mechanisms. The goal of this study was to evaluate the role of hepatic uptake transport in the pharmacokinetics of tolbutamide and to identify the molecular mechanism thereof. Transport studies using singly transfected cells expressing six major hepatic uptake transporters showed that tolbutamide is a substrate to organic anion transporter 2 (OAT2) alone with transporter affinity [Michaelis-Menten constant (Km)] of 19.5 ± 4.3 µM. Additionally, OAT2-specific transport was inhibited by ketoprofen (an OAT2 inhibitor) and 1 mM rifamycin SV (pan inhibitor), but not by cyclosporine and rifampicin (OAT polypeptides/Na+-taurocholate cotransporting polypeptide inhibitors). Uptake studies in primary human hepatocytes confirmed the predominant role of OAT2 in the active uptake with significant inhibition by rifamycin SV and ketoprofen, but not by the other inhibitors. Concentration-dependent uptake was noted in human hepatocytes with active transport characterized by Km and Vmax values of 39.3 ± 6.6 µM and 426 ± 30 pmol/min per milligram protein, respectively. Bottom-up physiologically based pharmacokinetic modeling was employed to verify the proposed role of OAT2-mediated hepatic uptake. In contrast to the rapid equilibrium (CYP2C9-only) model, the permeability-limited (OAT2-CYP2C9 interplay) model better described the plasma concentration-time profiles of tolbutamide. Additionally, the latter well described tolbutamide pharmacokinetics in carriers of CYP2C9 genetic variants and quantitatively rationalized its known drug-drug interactions. Our results provide first-line evidence for the role of OAT2-mediated hepatic uptake in the pharmacokinetics of tolbutamide, and imply the need for additional clinical studies in this direction.

Estimation of Interindividual Variability of Pharmacokinetics of CYP2C9 Substrates in Humans.

The activity of metabolic enzymes varies across individuals and populations. Activity varies even among individuals sharing the same genotype. Genetic polymorphisms in CYP2C9 cause significant interindividual variability in the metabolism of its substrates. However, the variability of CYP2C9 intrinsic hepatic clearance (CLint,h,CYP2C9) among subjects of the same genotype has not been reported. In this study, we estimated the coefficient of variation (CV) for the intrinsic hepatic clearance of tolbutamide by CYP2C9 for each CYP2C9 genotype using previously reported area under the blood concentration curve (AUC) and oral clearance (CLoral) values in a Monte Carlo simulation with a dispersion model. The CVs for tolbutamide CLint,h,CYP2C9 were estimated to be 18.1%, 23.9%, 25.4%, 22.3%, 13.0%, and 19.8% for CYP2C9*1/*1, *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3, respectively. These values are smaller than those previously reported for CYP2D6*1/*1 (43%), CYP2C19*1/*1 (66%), and CYP3A4 (33%). These CV values were used to predict AUC and CLoral variability of other CYP2C9 substrates, which are also substrates of other CYP isoforms. Then, the estimated CVs were consistent with those reported in previous studies of genotyped and ungenotyped subjects. Our estimates of CLint,h,CYP2C9 variability together with the variabilities of other isoforms are useful for predicting the AUC variability of CYP2C9 substrates.

Effect of Temperature on Tolbutamide Binding to Glycated Serum Albumin.

Glycation process occurs in protein and becomes more pronounced in diabetes when an increased amount of reducing sugar is present in bloodstream. Glycation of protein may cause conformational changes resulting in the alterations of its binding properties even though they occur at a distance from the binding sites. The changes in protein properties could be related to several pathological consequences such as diabetic and nondiabetic cardiovascular diseases, cataract, renal dysfunction and Alzheimer's disease. The experiment was designed to test the impact of glycation process on sulfonylurea drug tolbutamide-albumin binding under physiological (T = 309 K) and inflammatory (T = 311 K and T = 313 K) states using fluorescence and UV-VIS spectroscopies. It was found in fluorescence analysis experiments that the modification of serum albumin in tryptophanyl and tyrosyl residues environment may affect the tolbutamide (TB) binding to albumin in subdomain IIA and/or IIIA (Sudlow's site I and/or II), and also in subdomains IB and IIB. We estimated the binding of tolbutamide to albumin described by a mixed nature of interaction (specific and nonspecific). The association constants Ka (L∙mol-1) for tolbutamide at its high affinity sites on non-glycated albumin were in the range of 1.98-7.88 × 104 L∙mol-1 (λex = 275 nm), 1.20-1.64 × 104 L∙mol-1 (λex = 295 nm) and decreased to 1.24-0.42 × 104 L∙mol-1 at λex = 275 nm (T = 309 K and T = 311 K) and increased to 2.79 × 104 L∙mol-1 at λex = 275 nm (T = 313 K) and to 4.43-6.61 × 104 L∙mol-1 at λex = 295 nm due to the glycation process. Temperature dependence suggests the important role of van der Waals forces and hydrogen bonding in hydrophobic interactions between tolbutamide and both glycated and non-glycated albumin. We concluded that the changes in the environment of TB binding of albumin in subdomain IIA and/or IIIA as well as in subdomains IB and IIB influence on therapeutic effect and therefore the studies of the binding of tolbutamide (in diabetes) to transporting protein under glycation that refers to the modification of a protein are of great importance in pharmacology and biochemistry. This information may lead to the development of more effective drug therapy in people with diabetes.

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), Cmax 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.

In vitro Phase I- and Phase II-Drug Metabolism in The Liver of Juvenile and Adult Göttingen Minipigs.

PURPOSE: In view of pediatric drug development, juvenile animal studies are gaining importance. However, data on drug metabolizing capacities of juvenile animals are scarce, especially in non-rodent species. Therefore, we aimed to characterize the in vitro biotransformation of four human CYP450 substrates and one UGT substrate in the livers of developing Göttingen minipigs. METHODS: Liver microsomes from late fetal, Day 1, Day 3, Day 7, Day 28, and adult male and female Göttingen minipigs were incubated with a cocktail of CYP450 substrates, including phenacetin, tolbutamide, dextromethorphan, and midazolam. The latter are probe substrates for human CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. In addition, the UGT multienzyme substrate (from the UGT-GloTM assay), which is glucuronidated by several human UGT1A and UGT2B enzymes, was also incubated with the porcine liver microsomes. RESULTS: For all tested substrates, drug metabolism significantly rose postnatally. At one month of age, 60.5 and 75.4% of adult activities were observed for acetaminophen and dextrorphan formations, respectively, while 35.4 and 43.2% of adult activities were present for 4-OH-tolbutamide and 1'-OH-midazolam formations. Biotransformation of phenacetin was significantly higher in 28-day-old and adult females compared with males. CONCLUSIONS: Maturation of metabolizing capacities occurred postnatally, as described in man.

Development of a Specific Substrate-Inhibitor Panel (Liver-on-a-Chip) for Evaluation of Cytochrome P450 Activity.

We developed a cytochrome P450 substrate-inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC-mass-spectroscopy. The selected specific substrate-inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.

Influence of High-fat and High-cholesterol Diet on Major CYP Activities in the Liver.

We previously reported that a high-fat and high-cholesterol (HFHC) diet for 12 weeks induced non-alcoholic steatohepatitis (NASH) and influenced major CYP subtype gene expression levels and activities in a mouse model. In the present study, we determined the effects of the HFHC diet on CYP expression levels and activities prior to the establishment of NASH. When male C57BL/6J mice were fed the HFHC or a normal chow diet (Control) ad libitum for 4 weeks, body weights were significantly lower, whereas liver weights and hepatic lipid contents were significantly higher in the HFHC group than in the Control group. Under these conditions, hepatic microsomal luciferin-H (human CYP2C9 substrate) hydroxylation activity was significantly lower in the HFHC group than in the Control group. In order to investigate drug efficacy in mice fed the HFHC diet, an intraperitoneal glucose tolerance test was conducted with or without a pretreatment with tolbutamide (a CYP2C substrate) after 4 weeks of feeding. The plasma glucose-lowering effects of tolbutamide were attenuated in the HFHC group even though luciferin-H hydroxylation activity was suppressed in this group. The reason for this discrepancy was attributed to the mRNA expression levels of Cyp2c44 being lower and those of Cyp2c29 and Cyp2c66, which are involved in the metabolism of tolbutamide, being higher in the HFHC group than in the Control group. These results indicate that the expression of Cyp2c in the liver is influenced by the HFHC diet prior to the establishment of NASH and its regulation differed among the subtypes examined.