Showing papers in "Xenobiotica in 2004"
TL;DR: Intersystem extrapolation factors (ISEFs) are defined that allow the use of rhCYP data for the in vitro–in vivo extrapolation of human drug CL and the variance that is associated with interindividual variation of CYP abundance due to genetic and environmental effects.
Abstract: 1. Recombinantly expressed human cytochromes P450 (rhCYPs) have been underused for the prediction of human drug clearance (CL). 2. Differences in intrinsic activity (per unit CYP) between rhCYP and human liver enzymes complicate the issue and these discrepancies have not been investigated systematically. We define intersystem extrapolation factors (ISEFs) that allow the use of rhCYP data for the in vitro-in vivo extrapolation of human drug CL and the variance that is associated with interindividual variation of CYP abundance due to genetic and environmental effects. 3. A large database (n = 451) of metabolic stability data has been compiled and used to derive ISEFs for the most commonly used expression systems and CYP enzymes. 4. Statistical models were constructed for the ISEFs to determine major covariates in order to optimize experimental design to increase prediction accuracy. 5. Suggestions have been made for the conduct of future studies using rhCYP to predict human drug clearance.
204 citations
TL;DR: HepG2 cells have both Phase I and II enzymes, which activities and at what levels depend on the source and culture conditions, and therefore, HepG1 cells routinely used in in vitro assays should be characterized for their drug-metabolizing capabilities before any results can be fully interpreted.
Abstract: 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.
162 citations
TL;DR: Radioactivity was widely and rapidly distributed, with the highest levels being found in liver, kidney, lung and gastrointestinal tract, but with only low levels penetrating the brain, and gefitinib was cleared primarily by metabolism.
Abstract: Following oral administration of [14C]-gefitinib to albino and pigmented rats, radioactivity was widely and rapidly distributed, with the highest levels being found in liver, kidney, lung and gastrointestinal tract, but with only low levels penetrating the brain. Levels of radioactivity persisted in melanin-containing tissues (pigmented eye and skin). Binding to plasma proteins was high (86-94%) across the range of species examined and was 91% in human plasma. Substantial binding occurred to both human serum albumin and alpha-1 acid glycoprotein. Following oral and intravenous administration of [14C]-gefitinib, excretion of radioactivity by rat, dog and human occurred predominantly via the bile into faeces, with < 7% of the dose being eliminated in urine. In all three species, gefitinib was cleared primarily by metabolism. In rat, morpholine ring oxidation was the major route of metabolism, leading to the formation of M537194 and M608236 as the main biliary metabolites. Morpholine ring oxidation, together with production of M523595 by O-demethylation of the quinazoline moiety, were the predominant pathways in dog, with oxidative defluorination also occurring to a lesser degree. Pathways in healthy human volunteers were similar to dog, with O-demethylation and morpholine ring oxidation representing the major routes of metabolism.
125 citations
TL;DR: It is demonstrated that CYP-mediated metabolism of lactones is also a common metabolic pathway for statins and that the CYP3A4- mediated metabolism of the lactone forms clearly will need to be taken into account in assessing mechanistic aspects of drug–drug interaction involving statins.
Abstract: To gain a better understanding of the metabolic properties between the open acid and lactone form of HMG-CoA reductase inhibitors (statins), the paper focused primarily on characterizing the metabolic properties of statins. We compared the metabolism of the acid and lactone forms of several statins, including atrovastatin, simvastatin, cerivastatin fluvastatin, pitavastatin and rosuvastatin with respect to metabolic clearance, CYP enzymes involved and drug-drug interactions. A remarkable increase in metabolic clearance was noted for all lactones compared with all acids except for pitavastatin lactone. The metabolic clearances of the atrovastatin, simvastatin, cerivastatin, fluvastatin and rosuvastatin lactones were 73-, 70-, 30-, 7- and 64-fold higher, respectively, than those of the corresponding acids. CYP2Cs were critically involved in the metabolism of cerivastatin, fluvastatin and pitavastatin acids. In contrast, CYP2Cs were not involved in the metabolism of the corresponding lactones and CYP3A4 was mainly involved. Moreover, a substantial difference in the metabolic inhibition of statins was found between acids and lactones. Overall, the study demonstrates that CYP-mediated metabolism of lactones is also a common metabolic pathway for statins and that the CYP3A4-mediated metabolism of the lactone forms clearly will need to be taken into account in assessing mechanistic aspects of drug-drug interaction involving statins.
109 citations
TL;DR: Variability in activity of individual CYP enzymes may influence interindividual sensitivity to the toxic effects of chlorpyrifos and diazinon and both organophosphorothioates were more readily detoxified than bioactivated in all human liver microsome preparations.
Abstract: The cytochrome P450 (CYP)-mediated biotransformation of the organophosphorothioate insecticides chlorpyrifos and diazinon was investigated. Rates of desulphuration to the active oxon metabolite (chlorpyrifos-oxon and diazinon-oxon) and dearylation to non-toxic hydrolysis products were determined in human liver microsome preparations from five individual donors and in recombinant CYP enzymes. Chlorpyrifos and diazinon underwent desulphuration in human liver microsome with mean Km = 30 and 45 microM and V(max) = 353 and 766 pmol min(-1) mg(-1), respectively. Dearylation of these compounds by human liver microsome proceeded with Km = 12 and 28 microM and V(max) = 653 and 1186 pmol min(-1) mg(-1), respectively. The apparent intrinsic clearance (V(max)/Km) of dearylation was 4.5- and 2.5-fold greater than desulphuration for chlorpyrifos and diazinon, respectively. Recombinant human CYP2B6 possessed the highest desulphuration activity for chlorpyrifos, whereas CYP2C19 had the highest dearylation activity. In contrast, both desulphuration and dearylation of diazinon were catalysed at similar rates, in the rank order CYP2C19 > CYP1A2 > CYP2B6 > CYP3A4. Both organophosphorothioates were more readily detoxified (dearylation) than bioactivated (desulphuration) in all human liver microsome preparations. However, the role of individual CYP enzymes in these two biotransformation pathways varied according to the structure of the organophosphorothioate, which was reflected in different activation/detoxification ratios for chlorpyrifos and diazinon. Variability in activity of individual CYP enzymes may influence interindividual sensitivity to the toxic effects of chlorpyrifos and diazinon.
92 citations
TL;DR: Increased expression of CYP3A isoforms and of P-gp occurs with 3 days of exposure to RIT in rats, and might contribute to drug interactions involving RIT (and other antiretroviral agents) in humans.
Abstract: 1. The consequences of extended exposure to the human immunodeficiency viral protease inhibitor ritonavir (RIT) on the expression and function of CYP3A isoforms in the liver and in enteric mucosal cells, and on the expression of the efflux transport protein P-glycoprotein (P-gp) in enteric mucosa and in brain microvessel endothelial cells, were evaluated in rat. Dexamethasone (DEX), a known inducer of CYP3A and P-gp in rodents, served as a positive control. 2. Male CD-1 rats received RIT (20 mg kg(-1)), DEX (80 mg kg(-1)) or vehicle by oral/duodenal gavage once daily for 3 days. 3. Compared with vehicle control, CYP3A activity in liver microsomes (intrinsic clearance for triazolam hydroxylation in vitro) was increased by a factor of 2-4 by RIT, and by 10-14-fold by DEX. Similar increases were observed in expression of immunoactive CYP3A protein. Overall, maximum reaction velocity and immunoactive protein were highly intercorrelated (r2 = 0.89). Both RIT and DEX also increased function and expression of enteric CYP3A, although to a more modest extent (about 1.7-fold for RIT, about 3.3-fold for DEX). 4. Enteric P-gp expression was equally induced (by 2.8-fold) by both RIT and DEX. P-gp expressed in brain microvessel endothelial cells was increased by a factor of 1.3 by both compounds. 5. Thus, increased expression of CYP3A isoforms and of P-gp occurs with 3 days of exposure to RIT in rats. Qualitatively similar changes occur in human cell culture models and in clinical studies, and might contribute to drug interactions involving RIT (and other antiretroviral agents) in humans.
75 citations
TL;DR: In this article, the authors characterize mouse gender and strain differences in the metabolism of commonly used human cytochrome (CYP) P450 probe substrates (phenacetin, coumarin, 7-ethoxy-4-trifluoromethyl coumarlin, amiodarone, paclitaxel, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, chlorzoxazone, p-nitrophenol, testosterone and lauric acid) were
Abstract: The aim was to characterize mouse gender and strain differences in the metabolism of commonly used human cytochrome (CYP) P450 probe substrates. Thirteen human CYP probe substrates (phenacetin, coumarin, 7-ethoxy-4-trifluoromethyl coumarin, amiodarone, paclitaxel, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, chlorzoxazone, p-nitrophenol, testosterone and lauric acid) were used in activity measurements. The metabolism of the probe substrates was compared in liver microsomes from male and female NMRI, CBA, C57bl/6, 129/SvJ and CD1 strains. The expression of proteins identified on Western blots with commonly available antibodies selective for specific human and rat CYP enzymes were compared in the different mouse strains. Males had higher metabolism than corresponding females for phenacetin O-deethylation (human marker for CYP1A2 activity), and a high correlation was found between phenacetin activity and immunoreactivity in Western blots produced with rat CYP1A2 antibodies. Protein detected by antibodies cross-reacting with human CYP2B6 and rat CYP2B1/2 antibodies was female specific except for the 129/SvJ strain, where it was absent in both genders. Females generally had a higher metabolism of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation (human markers for CYP2D activity). Bufuralol 1'-hydroxylation correlated with a female-dominant mouse CYP, which was detected with antibodies against rat CYP2D4. p-Nitrophenol 2-hydroxylation correlated better than chlorzoxazone 6-hydroxylation with the protein detected with antibodies against rat CYP2E1, indicating that p-nitrophenol is a more specific substrate for mouse CYP2E1.
62 citations
TL;DR: The disposition of temozolomide in rats was similar to that observed in humans and the distribution pattern of radioactivity was gender independent.
Abstract: 1. Temozolomide, an imidazotetrazine derivative, is a cytotoxic alkylating agent of broad-spectrum antitumour activity. The absorption, metabolism, distribution and excretion of temozolomide have been investigated in male and female Sprague–Dawley and Long–Evans rats following single oral or intravenous dose administration of 200 mg m−2 non-radiolabelled or 14C-radiolabelled temozolomide. The distribution of 14C-temozolomide was also evaluated by whole-body autoradiography in male Sprague–Dawley rats. Plasma concentrations of temozolomide and its active metabolite 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) were determined by high-performance liquid chromatography (HPLC) with ultraviolet detection. Plasma, urine and faeces were profiled by HPLC with radiochemical detection.2. Temozolomide was rapidly and extensively (>90%) absorbed and widely distributed in tissues. The distribution pattern of radioactivity was gender independent. Penetration into the brain following oral or intravenous administ...
57 citations
TL;DR: The use of the three main in vivo metabolites as additional substrates enabled a more comprehensive metabolic pathway to be constructed and this has been valuable in supporting the more limited data available from the human in vivo study.
Abstract: The in vitro metabolism of gefitinib was investigated by incubating [14C]-gefitinib, as well as M537194, M387783 and M523595 (the main metabolites of gefitinib observed in man), at a concentration of 100 microM with human liver microsomes (4 mg ml(-1)) for 120 min. These relatively high substrate and microsomal protein concentrations were used in an effort to generate sufficient quantities of metabolites for identification. HPLC with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of a large number of metabolites. Although 16 metabolites were identified, metabolism was restricted to three regions of the molecule. The major pathway involved morpholine ring-opening and step-wise removal of the morpholine ring and propoxy side chain. O-demethylation of the quinazoline methoxy group was a quantitatively less important pathway, in contrast to the clinical situation, where O-desmethyl gefitinib (M523595) is the predominant plasma metabolite. The third metabolic route, oxidative defluorination, was only a minor route of metabolism. Some metabolites were formed by a combination of these processes, but no metabolism was observed in other parts of the molecule. Incubation of gefitinib produced ten identified metabolites, but the use of the three main in vivo metabolites as additional substrates enabled a more comprehensive metabolic pathway to be constructed and this has been valuable in supporting the more limited data available from the human in vivo study.
57 citations
TL;DR: The findings show novel and unexpected effects of these phytochemicals on the expression of human hepatic biotransformation enzymes that play key roles in chemical-induced carcinogenesis.
Abstract: 1. The naturally occurring compounds curcumin (CUR), 3,3'-diindolylmethane (DIM), isoxanthohumol (IXN), 8-prenylnaringenin (8PN), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) protect animals against chemically induced tumours. Putative chemoprotective mechanisms include modulated expression of hepatic biotransformation enzymes. However, few, if any, studies have used human primary cells as test models. 2. The present study investigated the effects of these phytochemicals on the expression of four carcinogenesis-relevant enzymes--cytochrome P450 (CYP)1A1 and 1A2, NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S-transferase A1 (GSTA1)--in primary cultures of freshly isolated human hepatocytes. 3. Quantitative RT-PCR analyses demonstrated that CYP1A1 was up-regulated by PEITC and DIM in a dose-dependent manner. CYP1A2 transcription was significantly activated following DIM, IXN, 8PN and PEITC treatments. DIM exhibited a remarkably effective induction response of CYP1A1 (474-, 239- and 87-fold at 50, 25 and 10 microM, respectively) and CYP1A2 (113-, 70- and 31-fold at 50, 25 and 10 microM, respectively), that was semiquantitatively reflected in protein levels. NQO1 expression responded to PEITC (11 x at 25 microM), DIM (4.5 x at 50 microM) and SFN (5 x at 10 microM) treatments. No significant effects on GSTA1 transcription were seen. 4. The findings show novel and unexpected effects of these phytochemicals on the expression of human hepatic biotransformation enzymes that play key roles in chemical-induced carcinogenesis.
56 citations
TL;DR: Functional interferences provide new insight for understanding how xenobiotics might exert adverse effects, and second how physiopathological stimuli affect xenobiotic metabolism.
Abstract: 1. Recent investigations on nuclear receptors and other transcription factors involved in the regulation of genes encoding xenobiotic metabolizing and transport systems reveal that xenobiotic-dependent signalling pathways are embedded in, and establish functional interactions with, a tangle of regulatory networks involving the glucocorticoid and oestrogen receptors, the hypoxia-inducible factor, the vitamin D receptor and other transcription factors/nuclear receptors controlling cholesterol/bile salt homeostasis and liver differentiation. 2. Such functional interferences provide new insight, first for understanding how xenobiotics might exert adverse effects, and second how physiopathological stimuli affect xenobiotic metabolism.
TL;DR: The model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance and provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance.
Abstract: 1: The aim was to investigate whether precision-cut rat tissue slices could be used to predict metabolic drug clearance in vivo. To obtain a complete picture, slices not only from liver, but also from lung, kidney, small intestine and colon were included. 2: The metabolic clearances of 7-ethoxycoumarin, 7-hydroxycoumarin, testosterone, methyltestosterone and warfarin were determined by measuring the disappearance of these compounds during incubation with slices prepared from liver, lung, kidney, small intestine and colon. 3: The total in vitro metabolic clearance was determined by adding the individual in vitro organ clearances from the slices. Prediction based on the in vitro clearance was within an order of magnitude to the corresponding in vivo values. Interestingly, the relative contribution of extrahepatic metabolic clearance of the studied compounds to total clearance was remarkably high, ranging from 35 to 72% of the total metabolic clearance. 4: It is concluded that the model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance. In addition, it provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance.
TL;DR: The pharmacokinetics and oral bioavailability of S-4 indicate that it is an excellent candidate for clinical development and rapidly absorbed, slowly cleared, and has a moderate volume of distribution in rats.
Abstract: 1: S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide (also known as S-4) is a non-steroidal selective androgen receptor modulator demonstrating tissue-selective androgenic and anabolic effects. The purpose of the present study was to examine the systemic pharmacokinetics, elimination and oral bioavailability of S-4 in rats. 2: Thirty-five male Sprague-Dawley rats weighing approximately 250 g were randomly assigned to one of seven treatment groups. Intravenous doses of 0.5, 1, 10, and 30 mg kg(-1) were given via a jugular catheter. Oral doses of 1, 10 and 30 mg kg(-1) were administered via gavage. Plasma concentrations were determined using a validated high-performance liquid chromatography or by a high-performance liquid chromatography/mass spectrometry method. 3: Clearances ranged between 1.0 and 2.1 ml min(-1) kg(-1) and varied with dose. The volume of distribution was approximately 0.448 l kg(-1) in all treatment groups. Oral bioavailability was also dose dependent, with the lower doses showing complete oral bioavailability. The half-life of S-4 over the dose range tested was between 2.6 and 5.3 h. 4: It was demonstrated that S-4 is rapidly absorbed, slowly cleared, and has a moderate volume of distribution in rats. The pharmacokinetics and oral bioavailability of S-4 indicate that it is an excellent candidate for clinical development.
TL;DR: The results suggest that glibenclamide is mainly metabolized by CYP3A4, whereas lansoprazole is metabolizing by both CYP2C19 and CYP 3A4 in human liver microsomes.
Abstract: 1. The identification and relative contributions of human cytochrome P450 (CYP) enzymes involved in the metabolism of glibenclamide and lansoprazole in human liver microsomes were investigated using an approach based on the in vitro disappearance rate of unchanged drug.2. Recombinant CYP2C19 and CYP3A4 catalysed a significant disappearance of both drugs. When the contribution of CYPs to the intrinsic clearance (CLint) of drugs in pooled human microsomes was estimated by relative activity factors, contributions of CYP2C19 and CYP3A4 were determined to be 4.6 and 96.4% for glibenclamide, and 75.1 and 35.6% for lansoprazole, respectively.3. CLint of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CLint of lansoprazole significantly correlated with CYP2C19 and CYP3A4 marker activities in human liver microsomes from 12 separate individuals. Effects of CYP-specific inhibitors and anti-CYP3A serum on the CLint of drugs in pooled human liver microsomes reflected the relative contributi...
TL;DR: Unlike the uptake pattern previously observed for albendazole, the parent TCBZ and its sulphoxide and sulphone metabolites showed a similar ability to penetrate into the trematode parasite.
Abstract: Triclabendazole (TCBZ) is an halogenated trematodicidal benzimidazole compound extensively used in veterinary medicine. It is active against immature and adult stages of the liver fluke Fasciola hepatica. Free and conjugated TCBZ metabolites have been identified in the bile of treated sheep. The experimental aims were to characterize the in vitro patterns of TCBZ biotransformation both in the animal host (sheep liver microsomes) and target parasite (F. hepatica microsomal preparation); and to compare the ex vivo diffusion of TCBZ parent drug and its oxidized metabolites (TCBZ sulphoxide [TCBZSO], TCBZ sulphone [TCBZSO2], and TCBZ-hydroxy derivatives) into F. hepatica. Additionally, the octanol-water partition coefficients for TCBZ and all its metabolites were estimated as an indicator of the relationship between drug lipophilicity and diffusion into the target parasite. Drug/metabolites concentrations were quantified by HPLC after sample clean up and a solvent-mediated chemical extraction. Sheep liver microsomes metabolized TCBZ into its sulphoxide and sulphone metabolites after 30 min of incubation. The rate of TCBZ sulphoxidation in the liver was significantly greater (p < 0.01) than that observed for the sulphonation of TCBZSO. The trematode parasite oxidized TCBZ into its sulphoxide metabolite after 60 min of incubation at a metabolic rate of 0.09 nmol min(-1) mg protein(-1). TCBZ and all its oxidized metabolic products were recovered from F. hepatica as early as 15 min after their ex vivo incubation in a Kreb's Ringer Tris buffer. However, the diffusion of the hydroxy-derivatives into the fluke was lower than that observed for TCBZ, TCBZSO and TCBZSO2. There was a high correlation (r=0.82) between drug lipophilicity (expressed as octanol-water partition coefficients) and drug availability measured within the parasite. Unlike the uptake pattern previously observed for albendazole, the parent TCBZ and its sulphoxide and sulphone metabolites showed a similar ability to penetrate into the trematode parasite. Understanding the relationship between TCBZ metabolism, the relative pharmacological potency of its metabolic products and their ability to reach the target parasite may be critical to optimize its flukicidal activity, particularly when TCBZ resistant flukes have been already isolated in the field.
TL;DR: In the rat, the enzyme system hydrolysing levetiracetam is not induced by phenobarbital, at least under the experimental conditions used herein, whereas the enzymeSystem(s) involved in the other metabolic pathways is induced.
Abstract: 1: The pharmacokinetics and metabolism of 14C-levetiracetam, a new anti-epileptic agent, in mouse, rat, rabbit and dog after a single oral dose were investigated. Moreover, the in vitro hydrolysis of levetiracetam to its major carboxylic metabolite by rat tissue homogenates was investigated to identify tissues involved in the production of the metabolite. Data are also presented on the induction of the enzyme(s) involved in levetiracetam hydrolysis in the rat. 2: Levetiracetam was rapidly and almost completely absorbed. The unchanged drug accounted for a very high percentage of plasma radioactivity. Levetiracetam did not bind to plasma proteins. Although brain radioactivity concentrations were lower than those of whole blood at early time points, brain-to-blood ratios increased over time. The predominant route of elimination of total 14C was excretion via urine, accounting for about 81, 93, 87 and 89% of the dose in the mouse, rat, rabbit and dog, respectively. Consequently, levetiracetam was poorly metabolized. It was submitted in vivo to hydrolysis and/or oxidation. Hydrolysis of the amide function of levetiracetam produced the corresponding acid. However, levetiracetam could also be oxidized at positions 3 and 4 of the 2-oxopyrrolidine ring. Finally, the compound and the corresponding acid metabolite could be oxidized at position 5 of the 2-oxopyrrolidine ring and then hydrolysed with the opening of the ring. 3: All the investigated rat tissues (liver, kidney, lung, brain, small intestine mucosa) had the potential to produce the acid metabolite. By contrast, the acid was undetectable following incubation of levetiracetam with buffer alone or heat-denaturated liver fractions. 4: No marked species or sex differences were observed in the absorption, disposition and metabolism of levetiracetam. 5:The hydrolysis of levetiracetam is carried out by an enzymatic process characterized by a broad tissue distribution. In the rat, the enzyme system hydrolysing levetiracetam is not induced by phenobarbital, at least under the experimental conditions used herein, whereas the enzyme system(s) involved in the other metabolic pathways is induced.
TL;DR: Using in vitro metabolic interaction data, human in vivo pharmacokinetic interactions involving EP could be predicted nearly quantitatively.
Abstract: 1: The inhibition kinetics of eplerenone (EP) 6beta-hydroxylation by 10 drugs were determined in vitro using human liver microsomes. Inhibition factors were calculated from in vitro inhibition constant (Ki) and three different inhibitor Cmax values (liver Cmax of total and unbound inhibitor, and maximum influx concentration of inhibitor into the liver). Subsequently, the inhibition factors were compared with available pharmacokinetic data derived from clinical interaction trials conducted by Pfizer involving EP and these drugs. EP was also evaluated for its effect on the metabolism of 10 drugs in vitro, and again the in vitro data were compared with results from the clinical trials. 2: The Ki values for the inhibition of EP 6beta-hydroxylation by cisapride, cyclosporine, digoxin, erythromycin, fluconazole, ketoconazole, midazolam, saquinavir, simvastatin and verapamil were 2.90, 1.24,>75.0, 9.50, 59.0, 0.160, 8.10, 0.546, 6.23 and 13.3 microM, respectively. Among the three methods, inhibition factors (Rb) calculated using the Ki and estimated liver Cmax values of the unbound drug were best correlated with the in vivo area under the curve-fold increases of EP in humans. The Rb values for the drugs listed above were 1.04, 1.69, 1.00, 2.17, 2.24, 4.90, 1.00, 1.82, 1.01 and 1.04, respectively, and the in vivo area under the curve-fold increases of EP by these drugs were 1.04, 1.16, 0.930, 2.87, 2.24, 5.39, 1.00, 2.07, 1.03 and 1.98, respectively. 3: EP did not have any significant effects on the drugs tested in vitro or in the clinic. 4: Using in vitro metabolic interaction data, human in vivo pharmacokinetic interactions involving EP could be predicted nearly quantitatively. The lack of effects of EP on the pharmacokinetics of other drugs in man was also suggested in the in vitro data.
TL;DR: Concentrations of radioactivity in plasma exceeded gefitinib throughout the profile, indicating the presence of circulating metabolites in both rat and dog, similar to that observed in man.
Abstract: The pharmacokinetics of gefitinib and its metabolites in rat and dog were investigated in preclinical studies conducted to support the safety evaluation and clinical development of gefitinib, the first EGFR tyrosine kinase inhibitor approved for the treatment of non-small-cell lung cancer. Following intravenous dosing (5 mg kg(-1), gefitinib plasma half-life was 3-6h in rats and dogs, although studies using a more sensitive HPLC-MS assay produced longer estimates of half-life (7-14h). In these studies, plasma clearance was high (male rat: 25 ml min(-1) kg(-1); female rat: 16 ml min(-1) kg(-1); male dog: 16 ml min(-1) kg(-1)), as was the volume of distribution (8.0-10.41 kg(-1) in rat; 6.31 kg(-1) in dog), and exposure in female rats was double that in males. Following administration of [14C]-gefitinib, concentrations of radioactivity in plasma exceeded gefitinib throughout the profile, indicating the presence of circulating metabolites in both rat and dog. An HPLC-MS assay was developed to measure concentrations of gefitinib and five potential metabolites in plasma. All five metabolites were detected in the rat, but at levels much lower than gefitinib. In the dog, exposure to gefitinib and M523595 was similar, with much lower concentrations of M537194 and only trace levels of the other metabolites. This profile of metabolites is similar to that observed in man.
TL;DR: Data suggest that polymorphically expressed CYP2D6 is the enzyme mainly responsible for MeOPP O-demethylation, a novel designer drug studied in male Wistar rats.
Abstract: 1. The in vivo metabolism of 1-(4-methoxyphenyl)piperazine (MeOPP), a novel designer drug, was studied in male Wistar rats.2. MeOPP was mainly O-demethylated to 1-(4-hydroxyphenyl)piperazine (4-HO-PP) in addition to degradation of the piperazine moiety.3. O-demethylation, the major metabolic step, was studied with cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes in pooled human liver microsomes (pHLM) and in single donor human liver microsomes with CYP2D6 poor metabolizer genotype (PM HLM).4. CYP2D6 catalysed O-demethylation with apparent Km and Vmax values of 48.34 ± 14.48 µM and 5.44 ± 0.47 pmol min−1 pmol−1 CYP, respectively. pHLM catalysed the monitored reaction with an apparent Km = 204.80 ± 51.81 µM and Vmax = 127.50 ± 13.25 pmol min−1 mg−1 protein.5. The CYP2D6-specific chemical inhibitor quinidine (1 and 3 µM) significantly inhibited 4-HO-PP formation by 71.9 ± 4.8% and by 98.5% ± 0.5%, respectively, in incubation mixtures with pHLM and 200 µM MeOPP.6. O-demethylation was significantly l...
TL;DR: The findings provide a coherent explanation for the inter-individual variability in the pharmacokinetics of CYP1A2 substrate drugs in dogs.
Abstract: The pharmacokinetics of YM-64227 (4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]-pyrimidine-2-(1H)-one), a novel and selective phosphodiesterase type 4 inhibitor, was characterized in beagle dogs Based on the plasma parent drug to major hydroxylated metabolite ratio, 21 dogs were phenotyped as 16 extensive metabolizers (EM) and five poor metabolizers (PM) Nucleotide sequences of CYPs 1A2, 2B11, 2C21, 2D15, 2E1 and 3A12 were investigated in the EM and PM dogs A CYP1A2 1117 C>T single nucleotide polymorphism was found, which resulted in an amino acid change from an Arg codon to a stop codon at position 373 All dogs phenotyped as PM were T/T homozygous, whereas EMs were C/C homozygous and C/T heterozygous In Western blotting of liver microsomes, CYP1A protein expression was detected in the C/C and C/T types, but not in the T/T type Of 65 dogs genotyped using genome DNA, the frequencies of the C and T alleles were 061 and 039, respectively, suggesting approximately 15% of the dogs would not express the CYP1A2 protein The findings provide a coherent explanation for the inter-individual variability in the pharmacokinetics of CYP1A2 substrate drugs in dogs
TL;DR: The pharmacokinetic parameters determined help to explain the observed differences in the relative amounts of OH-PCBs in humans and other mammals exposed to environmental PCBs.
Abstract: 1. Hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs) are, depending on their structure, strongly retained in mammalian, fish and bird blood. This is due to strong, though reversible, binding to the thyroxine binding and transporting protein transthyretin. 2,3,3′,4′,5-Pentachloro-4-biphenylol (4-OH-CB 107) and 2,2′,3,4′,5,5′,6-heptachloro-4-biphenylol (4-OH-CB 187) are two of five major OH-PCB congeners in human plasma.2. The relative amounts of OH-PCB congeners vary between species and also between human populations, in spite of similar PCB congener patterns, and may depend on different pharmacokinetic parameters of the OH-PCBs. In the present study, the pharmacokinetic parameters of 4-OH-CB 107 and 4-OH-CB 187 were determined in the rat after a single intravenous dose of 1 µmol kg−1. Plasma samples were analysed by gas chromatography/mass spectrometry.3. 4-OH-CB 107 had a half-life of 3.8 days; 4-OH-CB 187 had a half-life of 15 days. Volumes of distribution were 0.07 and 0.11 l kg−1, respec...
TL;DR: The overall degradation reaction rates for this series of compounds have been modelled successfully using Hammett substituent constants, computational chemistry-derived partial atomic charges and the experimentally determined carbonyl carbon 13C-NMR chemical shifts of the benzoic acids and their ethyl and glucuronide esters.
Abstract: 1. In a previously reported study, a number of 4-substituted benzoic acid acyl glucuronides were synthesized and their degradation rates determined using nuclear magnetic resonance (NMR) spectroscopy. It was shown that this reaction was strongly influenced by the nature of the substituent at the 4-position of the benzoyl moiety.2. The overall degradation reaction rates for this series of compounds have been modelled successfully using Hammett substituent constants, computational chemistry-derived partial atomic charges and the experimentally determined carbonyl carbon 13C-NMR chemical shifts of the benzoic acids and their ethyl and glucuronide esters.3. The primary contribution to reactivity is the scale of the electron-donating or -withdrawing effect of the substituent; however, additional contributions such as steric parameters must also be considered when modelling reactions outside a single chemical series.4. The derived property–reactivity relationships should find utility in medicinal chemistry effo...
TL;DR: The presence of some circulating factors such as cytokines, nitric oxide, parathyroid hormones and increased intracellular calcium play a role in the regulation of DMEs in renal diseases.
Abstract: 1. The activity of drug-metabolizing enzymes (DMEs) in extrahepatic organs is highest in the kidneys. Generally, the kidneys contain most, if not all, of the DMEs found in the liver. Surprisingly, some of these DMEs show higher activity in the kidneys than in the liver. 2. Most of the renal DMEs are localized in the cortex of the kidneys, especially in the proximal tubules. DMEs are also found in the distal tubules and collecting ducts. 3. Renal diseases such as acute and chronic renal failure and renal cell carcinoma alter the regulation of both hepatic and extrahepatic phase I and II DMEs. Changes in the expression of these DMEs seem to be tissue and species specific. 4. Generally, there is significant down-regulation of most of the phase I and a few of phase II DMEs at the protein, mRNA and activity levels. Unfortunately, the mechanisms leading to the alteration in DMEs in renal diseases remain unclear, although many theories have been made. 5. The presence of some circulating factors such as cytokines, nitric oxide, parathyroid hormones and increased intracellular calcium play a role in the regulation of DMEs in renal diseases.
TL;DR: Because S-etodolac was metabolized more rapidly than R-etdolac in human cryopreserved hepatocytes, the stereoselectivities of UGT1A9 for etodolacs substantially influenced the overall metabolism of S- and R-ETodolaf in man.
Abstract: 1. In vitro metabolic studies with etodolac were performed. S- and R-etodolac were converted to the acylglucuronide and hydroxylated metabolites by UDP-glucuronosyltransferase (UGT) and cytochrome P450 in microsomes. However, the stereoselectivities of UGT and P450 for the isomers were opposite. S-etodolac was glucuronidated preferentially than R-etodolac by UGT. In contrast, R-etodolac was hydroxylated preferentially than S-etodolac by P450. 2. Of several human P450 enzymes, CYP2C9 had the greatest activity for hydroxylation of R-etodolac. Sulfaphenazole, an inhibitor of CYP2C9, and anti-CYP2C9 antibody inhibited the hydroxylation of R-etodolac in human liver microsomes. CYP2C9 therefore contributes to the stereoselective hydroxylation of R-etodolac. 3. Of several human UGT enzymes, UGT1A9 had the greatest activity for glucuronidation of S-etodolac. Propofol and thyroxine, inhibitors of UGT1A9, inhibited the glucuronidation of S-etodolac in human liver microsomes. Therefore, UGT1A9 is mainly responsible for the stereoselective glucuronidation of S-etodolac. 4. Because S-etodolac was metabolized more rapidly than R-etodolac in human cryopreserved hepatocytes, the stereoselectivities of UGT1A9 for etodolac substantially influenced the overall metabolism of S- and R-etodolac in man.
TL;DR: It was concluded that monkeys given midazolam orally could be useful models for predicting drug–drug interactions in man based on CYP3A enzyme inhibition.
Abstract: 1. Anti-human cytochrome P450 (CYP) 3A4 antiserum completely inhibited midazolam metabolism in monkey liver microsomes, suggesting that midazolam was mainly metabolized by CYP3A enzyme(s) in monkey liver microsomes. 2. Midazolam metabolism was also inhibited in vitro by typical chemical inhibitors of CYP3A, such as ketoconazole, erythromycin and diltiazem, and the apparent K(i) values for ketoconazole, erythromycin and diltiazem were 0.127, 94.2 and 29.6 microM, respectively. 3. CYP3A inhibitors increased plasma midazolam concentrations when midazolam and CYP3A inhibitors were co-administered orally. However, the pharmacokinetic parameters of midazolam were not changed by treatment with CYP3A inhibitors when midazolam was given intravenously. This suggests that CYP3A inhibitors modified the first-pass metabolism in the liver and/or intestine, but not systemic metabolism. 4. The drug-drug interaction responsible for CYP3A enzyme(s) inhibition was observed when midazolam and inhibitors were co-administrated orally. Therefore, it was concluded that monkeys given midazolam orally could be useful models for predicting drug-drug interactions in man based on CYP3A enzyme inhibition.
TL;DR: PPC metabolism appears overall less influenced by CYP2C9 genotype compared with other oral anticoagulants and it may thus be a valuable alternative for therapeutic antICOagulation of patients expressing CYP 2C9 variant alleles.
Abstract: The effect of cytochrome P450 (CYP) 2C9 polymorphisms on the stereoselective biotransformation of the oral anticoagulant phenprocoumon (PPC) to inactive, monohydroxylated metabolites was studied in vitro and in vivo.In human liver microsomes, the (S)-7-hydroxylation — being the major metabolic pathway — was significantly compromised in a gene–dose-dependent manner in samples expressing the CYP2C9*2 or CYP2C9*3 allele. The CYP2C9*3/*3 genotype corresponded to an almost fourfold lower (S)-7-hydroxylation rate than CYP2C9*1/*1 (wild-type).The intrinsic clearance of human recombinant CYP2C9*2 and CYP2C9*3 for the (S)-7-hydroxylation was 28.9 and 50.9% lower than of CYP2C9*1, respectively.The area under the plasma concentration–time curve (AUC) of PPC metabolites after oral intake of 12 mg racemic PPC was significantly lower in volunteers expressing the CYP2C9*2 or CYP2C9*3 allele. Increasing plasma AUC metabolic ratios (parent compound/metabolite) in CYP2C9*2 and CYP2C9*3 variant allele carriers were found fo...
TL;DR: It is concluded that precision-cut rat lung slices, of 600 µm thickness, can be maintained metabolically viable in culture for some 8 h, but after this point a marked decline in metabolic activity was noted.
Abstract: 1. The principal objective was to evaluate the functional and structural integrity of precision-cut rat lung slices in culture over 72 h. 2. Lung slices metabolized 7-ethoxycoumarin in a time-dependent fashion, the major metabolites being the sulphate and glucuronide of 7-hydroxycoumarin with very low levels of the free compound. Prior treatment of rats with beta-naphthoflavone elevated markedly the rate of metabolism. The optimum slice thickness, as exemplified by the metabolism of 7-ethoxycoumarin, was about 600 microm. 3. Lung slices retained metabolic viability towards 7-ethoxycoumarin for 8 h, but after this point a marked decline in metabolic activity was noted. However, very low levels of activity were still evident following a 72 h incubation. 4. Morphological examination of lung slices revealed nuclear degeneration and loss of tissue architecture following 24h incubation. When cellular integrity was assessed using lactate dehydrogenase, a time-dependent leakage was evident with maximum loss occurring within 24h; longer incubations did not result in further leakage. 5. It is concluded that precision-cut rat lung slices, of 600 microm thickness, can be maintained metabolically viable in culture for some 8 h.
TL;DR: The identity of the human metabolites of ketoprofen (2-(3-benzoylphenyl)-propanoic acid) excreted via urine was investigated after a single oral dose of the racemic drug.
Abstract: The identity of the human metabolites of ketoprofen (2-(3-benzoylphenyl)-propanoic acid) excreted via urine was investigated after a single oral dose of the racemic drug. Drug metabolites were concentrated and partially purified from urine using solid-phase extraction chromatography before separation and identification by directly coupled HPLC-MS and HPLC-NMR. The metabolites identified were the ester glucuronides of the parent drug and its phase I metabolites, 2-[3-(3-hydroxybenzoyl)phenyl]-propanoic acid, 2-[3-(4-hydroxybenzoyl)phenyl]-propanoic acid and 2-[3-(hydroxy(phenyl)methyl)phenyl]-propanoic acid, the latter formed by reduction of the ketone group of ketoprofen. In addition, two novel minor metabolites were identified as the ether glucuronides of 2-[3-(3-hydroxybenzoyl)phenyl]-propanoic acid and 2-[3-(4-hydroxybenzoyl)phenyl]-propanoic acid. These conjugates were all observed as diastereoisomeric pairs of unequal proportions. Purification of these metabolites by preparative chromatography allowed stereochemistry assignments. Metabolites were quantified by 1H-NMR spectroscopy after spectral simplification achieved by hydrolysis of the conjugates.
TL;DR: Level of glycolic acid, if formed in vivo, following exposures to similar concentrationsof ethylene glycol, would be lower in humans than in rabbits and rats, according to the results of the present study.
Abstract: 1. The metabolism of [1,2-14C]-ethylene glycol and [1,2-14C]-glycolic acid was studied in vitro using precision-cut tissue slices prepared from the livers of female Sprague—Dawley rats, New Zealand white rabbits and humans. The time-course for production of metabolites formed from ethylene glycol at concentrations from 3 to 40 mM was determined to compare quantitatively the differences between species in the rates and amounts of formation of glycolic acid, the presumed developmental toxicant of ethylene glycol. The rates of metabolism of glycolic acid to glyoxylic acid at concentrations from 0.05 to 16 mM by liver tissue from the different species were also determined. The apparent VmaJKm for the metabolic conversions of ethylene glycol to glycolic acid and for glycolic acid to glyoxylic acid in liver tissue from the different species were obtained.2. There were qualitative differences in the metabolic profiles and quantitative differences in the formation of glycolic acid between the mammalian liver syst...
TL;DR: There is the possibility of drug-drug interactions when prescribing DA-8159 concomitantly with known inhibitors or inducers of CYP3A4, and drug–drug interactions would not be predicted on the basis of DA- 8159 inhibiting the metabolism of coadministered drugs.
Abstract: 1. The purpose of this paper was to characterize cytochrome P450 (CYP) enzymes involved in N-dealkylation of a new oral erectogenic, DA-8159 to DA-8164, a major circulating active metabolite, in human liver microsomes and to investigate the inhibitory potential of DA-8159 on CYP enzymes.2. CYP3A4 was identified as the major enzyme responsible for DA-8159 N-dealkylation to DA-8164 based on correlation analysis and specific CYP inhibitor and antibody-mediated inhibition study in human liver microsomes, and DA-8159 metabolism in cDNA expressed CYP enzymes. There is the possibility of drug-drug interactions when prescribing DA-8159 concomitantly with known inhibitors or inducers of CYP3A4.3. DA-8159 was found to be only a very weak inhibitor of eight major CYPs (1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4), the largest inhibition occurring against CYP2D6 (IC50 67.7 μM) in human liver microsomes. Drug–drug interactions would not be predicted on the basis of DA-8159 inhibiting the metabolism of coadministered drugs.