Cytochrome P450 Isozymes Research Articles

Kinetic characterization and identification of the enzymes responsible for the hepatic biotransformation of adinazolam and N-desmethyladinazolam in man.

The kinetics of the N-demethylation of adinazolam to N-desmethyladinazolam (NDMAD), and of NDMAD to didesmethyladinazolam (DDMAD), were studied with human liver microsomes using substrate concentrations in the range 10-1000 microM. The specific cytochrome P450 (CYP) isoforms mediating the biotransformations were identified using microsomes containing specific recombinant CYP isozymes expressed in human lymphoblastoid cells, and by the use of CYP isoform-selective chemical inhibitors. Adinazolam was demethylated by human liver microsomes to NDMAD, and NDMAD was demethylated to DDMAD; the substrate concentrations, Km, at which the reaction velocities were 50% of the maximum were 92 and 259 microM, respectively. Another metabolite of yet undetermined identity (U) was also formed from NDMAD (Km 498 microM). Adinazolam was demethylated by cDNA-expressed CYP 2C19 (Km 39 microM) and CYP 3A4 (Km 83 microM); no detectable activity was observed for CYPs 1A2, 2C9, 2D6 and 2E1. Ketoconazole, a relatively specific CYP 3A4 inhibitor, inhibited the reaction; the concentration resulting in 50% of maximum inhibition, IC50, was 0.15 microM and the inhibition constant, Ki, was < 0.04 microM in five of six livers tested. Troleandomycin, a specific inhibitor of CYP 3A4, inhibited adinazolam N-demethylation with an IC50 of 1.96 microM. The CYP 2C19-inhibitor omeprazole resulted in only partial inhibition (IC50 21 microM) and sulphaphenazole, alpha-naphthoflavone, quinidine and diethyldithiocarbamate did not inhibit the reaction. NDMAD was demethylated by cDNA-expressed CYP 3A4 (Km 220 microM, Hill number A 1.21), CYP 2C19 (Km 187 microM, Hill number A 1.29) and CYP 2C9 (Km 1068 microM). Formation of U was catalysed by CYP 3A4 alone. Ketoconazole strongly inhibited NDMAD demethylation (IC50 0.14 microM) and formation of U (IC50 < 0.1 microM) whereas omeprazole and sulphaphenazole had no effect on reaction rates. These results show that CYP 3A4 is the primary hepatic CYP isoform mediating the N-demethylation of adinazolam and NDMAD. Co-administration of adinazolam with CYP 3A4 inhibitors such as ketoconazole or erythromycin might lead to reduced efficacy, since adinazolam by itself has relatively weak benzodiazepine agonist activity, with much of the pharmacological activity of adinazolam being attributable to its active metabolite NDMAD.

Differential modulation of the expression of important drug metabolising enzymes and transporters by endothelin-1 receptor antagonists ambrisentan and bosentan in vitro

The safety and effectiveness of drugs used to treat chronic diseases critically depend on their propensity to interact with co-administered drugs. Induction of enzymes and drug transporters involved in the clearance and distribution of drugs may critically reduce exposure with their substrates and thus lead to nonresponse. We therefore investigated the impact of the endothelin-1 receptor antagonists bosentan and ambrisentan on the expression of relevant human efflux and uptake transporters and on phase 1 and phase 2 enzymes. LS180 adenocarcinoma cells were treated for four days with bosentan or ambrisentan (1–50μM), the positive control rifampicin, or medium only (negative control). For evaluation of bosentan also HuH-7 human hepatoma cells were used and treated similarly. Gene expression was quantified at the mRNA level by real-time reverse transcription polymerase chain reaction and for some genes also at the protein level by western blot analysis. Comparable to rifampicin, bosentan was a moderate to strong inductor for all cytochrome P450 isozymes and ATP-binding cassette transporters tested, and it also induced organic anion transporting polypeptides. 50μM bosentan up-regulated e.g. CYP3A4 8.5-fold, ABCB1 5.1-fold, and ABCB11 1.9-fold at the mRNA level in LS180 cells. In HuH-7 cells induction was much less pronounced (e.g. CYP3A4 1.9-fold for bosentan). In contrast, ambrisentan only weakly induced some of the genes investigated in LS180 cells. These findings corroborate the in vivo finding that bosentan is much more prone to drug interactions than ambrisentan.

Quantitative Prediction of Mechanism-Based Inhibition Caused by Mibefradil in Rats

It was previously demonstrated that mibefradil, which shows mechanism-based inhibition in humans, also caused drug-drug interactions (DDIs) with midazolam (MDZ) in rats. In this study, we aimed to quantitatively predict the DDIs observed in rats using a physiologically based pharmacokinetic (PBPK) model from in vitro inactivation parameters. For more precise predictions, contribution ratios of cytochrome P450 (P450) isozymes involved in MDZ metabolism and inactivation parameters of mibefradil against each isozyme were incorporated in the predictive model. The evaluation of metabolic rate using recombinant P450s suggested that CYP3A2 and CYP2C11 contributed to 89 and 11% of MDZ metabolism, respectively. Inactivation studies of mibefradil against the two isozymes showed that the maximal inactivation rate constants (k(inact)) were considerable in both isozymes (0.231-0.565 min(-1)), whereas the inhibitor concentration producing half the k(inact) (K(I, app)) of CYP3A2 (0.263-0.410 μM) was a good deal lower than that for CYP2C11 (6.82-11.4 μM). As a result of predicting the DDIs using the PBPK model, predicted increases in areas under the concentration-time curve of MDZ with coadministration of mibefradil (284 and 510% at 6 and 12 mg/kg mibefradil, respectively) closely corresponded to the observed values (226 and 545%, respectively). From those results, it was thought that the construction of a predictive model for DDIs using the PBPK model in detail would enable us to quantitatively predict in vivo DDIs from in vitro data. This approach to predict DDIs on the basis of the contributing isozymes would be important for predicting clinical DDIs of drugs metabolized by multiple enzymes.

Inhibition of pyrene biotransformation by piperonyl butoxide and identification of two pyrene derivatives in Lumbriculus variegatus (Oligochaeta)

Using the freshwater annelid Lumbriculus variegatus (Oligochaeta), the presence of cytochrome P450 (CYP) isozymes was investigated by analyzing metabolites of the polycyclic aromatic hydrocarbon (PAH) pyrene in treatments with and without the CYP inhibitor piperonyl butoxide (PBO). The results show a low biotransformation capability of L. variegatus (7% of total pyrene body burden as metabolites at 168 h). Addition of PBO resulted in a significant reduction of metabolites, suggesting the presence of a CYP in L. variegatus. Besides 1-hydroxypyrene, three peaks representing unknown metabolites were detected in LC-FLD (liquid chromatography with fluorescence detection) chromatograms of L. variegatus. Deconjugations showed that sulfonation and glucosidation are involved in the formation of these unknowns. Further studies with the time of flight mass analyzer provided the identification of the glucose-sulfate conjugate of 1-hydroxypyrene. The same metabolites were detected in the solvent-nonextractable fraction by incubation of the tissue residues with proteinase K, suggesting that part of these metabolites are bound to proteins. Overall, the slow biotransformation of pyrene by L. variegatus (involving CYP) supports the use of this species in standard bioaccumulation tests; however, the tissue-bound metabolite fraction described in the current study deserves further investigation for its toxicity and availability to upper trophic levels through diet.

Efficient Generation of Hepatoblasts From Human ES Cells and iPS Cells by Transient Overexpression of Homeobox Gene HEX

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed α-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.

Increased Expression of P-Glycoprotein Is Associated with Doxorubicin Chemoresistance in the Metastatic 4T1 Breast Cancer Model

Development of drug resistance is one of the major causes of breast cancer treatment failure. The goal of this study was to understand the chemoresistance mechanism using the highly metastatic 4T1 breast cancer model, which emulates stage IV breast cancer in humans. The metastatic 4T1 breast cancer cell line treated with either doxorubicin or 5-FU showed a concentration-dependent reduced cell proliferation, with induced G2-phase growth arrest (doxorubicin) or G1-phase growth arrest (5-FU). Doxorubicin treatment partially suppressed the multiorgan metastasis of 4T1 breast cancer cells in the lung, heart, liver, and bone, compared with either 5-FU or cyclophosphamide. We isolated and characterized 4T1 breast cancer cells from doxorubicin-resistant metastatic tumors (cell line 4T1-R). Multiorgan metastasis of drug-resistant 4T1 breast tumors was totally resistant to doxorubicin treatment. Our results indicate that doxorubicin is localized exclusively in the cytoplasm of resistant 4T1 breast cancer cells and that it cannot reach the nucleus because of increased nuclear expression of P-glycoprotein. Pretreatment of doxorubicin-resistant 4T1-R breast cancer cells with verapamil, a general inhibitor of P-glycoprotein, increased nuclear translocation of doxorubicin and cellular cytotoxicity. Thus, impaired nuclear translocation of doxorubicin due to increased expression of P-glycoprotein is associated with doxorubicin resistance of highly metastatic 4T1 breast cancer.

Involvement of CYP3A4/5 and CYP2D6 in the metabolism of aconitine using human liver microsomes and recombinant CYP450 enzymes

Aconitine (AC), a famous major Aconitum alkaloid, has effective antirheumatic function with high toxicity. The aim of our study was to in-depth investigate cytochrome P450 isozymes (CYPs) involved in aconitine metabolism in vitro. We used human liver microsomes (HLMs) as well as recombinant CYPs to investigate the metabolism pathways of aconitine by liquid chromatography–tandem mass spectrometry. Fluvoxamine maleate, gemfibrozil, amiodarone hydrochloride, omeprazole, quinidine, diethyldithiocarbamic acid and ketoconazole were successfully applied as test inhibitors for CYP1A2, CYP2C8, CYP2C9, CYP2C19*1, CYP2D6*1, CYP2E1 and CYP3A4/5 in HLMs, respectively. Six CYP-mediated metabolites were found and characterized in human liver microsomes and eight recombinant CYP isoforms. The inhibitor of CYP 3A had a strong inhibitory effect, the inhibitors of CYP 2C9, 2C8 and CYP2D6 had little inhibitory effects, whereas CYP2C19, 1A2 and 2E1 had no obvious inhibitory effects on AC metabolism. Hydroxylation and di-demethylation of aconitine were conducted by human recombinant CYP 3A5 and 2D6, dehydrogenation was only processed by CYP3A4/5, and the main CYP isoforms metabolizing aconitine to demethyl-aconitine and N-deethyl-aconitine were CYP3A4/5 and CYP2D6. In conclusion, aconitine can be transformed into at least six CYP-mediated metabolites in HLMs, CYP 3A4/5 and 2D6 were the most important CYP isoforms responsible for the de-methylation, N-deethylation, dehydrogenation, and hydroxylation of aconitine.

Cytochrome P450 isozyme protein verified in the skin of southern hemisphere humpback whales (Megaptera novaeangliae): Implications for biochemical biomarker assessment

Large mysticete whales represent a unique challenge for chemical risk assessment. Few epidemiological investigations are possible due to the low incidence of adult stranding events. Similarly their often extreme life-history adaptations of prolonged migration and fasting challenge exposure assumptions. Molecular biomarkers offer the potential to complement information yielded through tissue chemical analysis, as well as providing evidence of a molecular response to chemical exposure. In this study we confirm the presence of cytochrome P450 reductase (CPR) and cytochrome P450 isoenzyme 1A1 (CYP1A1) in epidermal tissue of southern hemisphere humpback whales (Megaptera novaeangliae). The detection of CYP1A1 in the integument of the humpback whale affords the opportunity for further quantitative non-destructive investigations of enzyme activity as a function of chemical stress.

The glyoxalase system as an example of a cellular maintenance pathway with relevance to aging

In a recent issue of AGING, Scheckhuber et al. studied the effect of methylglyoxal (MG) metabolism on growth and lifespan in P. anserina [1]. Methylglyoxal (2-oxopropanal) is generated enzymatically by three types of enzymes (methylglyoxal synthase, cytochrome P450 isozyme and amine oxidase) and non-enzymatically as a side product of glycolysis from dihydroxyacetone phosphate and glyceraldehyde-3-phosphat. MG reacts by Maillard reaction with lysine and arginine residues of proteins and thus leads to advanced glycosylation end products (AGE) [2]. It is removed enzymatically via the glyoxalase system, which consists of two enzymes [3]. On a glucose rich media, overexpression of only one of the two enzymes, PaGlo1, led to a reduction of lifespan, while overexpression of PaGlo1 together with PaGlo2, increased lifespan. A situation reminiscent of the superoxide dismutase (SOD), catalase system that is involved in the detoxification of the superoxide radical. There, SOD converts the superoxide radical into hydrogen peroxide, which is then removed by catalase or glutathione peroxidase. Also in that case it has been observed that overexpressing only the first component (SOD) is detrimental instead of increasing resistance to oxidative stress [4,5,6]. In both cases an apparently simply reaction system displays phenotypes that are surprising and can be better understood when modelled mathematically in a systems biological context. Such an approach showed that an alternative reaction pathway is required for the superoxide radical to explain the experimental findings [7] and thus provided a deeper understanding of the possible mechanism. Similarly, also the glyoxalase system might benefit from a modelling approach to test if the hypothesis that the negative effects of oxerexpressing PaGlo1 alone are mediated via a reduction of free GSH, is plausible. It has also be shown that the mutagenic action of methylglyoxal involves the generation of free oxygen radicals [8]. Since GSH is also involved in the detoxification of reactive oxygen species, its decline would be detrimental for MG degradation as well as the removal of ROS that are generated by MG. Numerical values are of critical importance for the construction of kinetic models and the absence of such values is often a serious obstacle. However, in erythrocytes the steady state concentrations and kinetic properties of the glyoxalase system have been determined [9,10], which opens the way for a systems biological approach. The work of Scheckhuber [1] thus provides interesting results for further investigations of a cellular maintenance pathway with relevance to aging.

Characterization of Highly Efficacious Allosteric Agonists of the Human Calcium-Sensing Receptor

We discovered structurally novel human calcium-sensing receptor (CaSR) allosteric agonists and compared their pharmacology to phenylalkylamine calcimimetics. 1-Benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol (AC-265347) activated CaSR signaling in cellular proliferation and phosphatidylinositol (PI) hydrolysis assays with potencies of 30 and 10 nM, respectively. (S)-1-Benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol) [(S)-AC-265347], the S-enantiomer of AC-265347, was approximately 10- to 20-fold more potent than (R)-1-benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol) [(R)-AC-265347]. The phenylalkylamines cinacalcet and calindol had activity similar to that of AC-265347 in cellular proliferation assays but less activity in PI assays. All compounds had reduced activity when extracellular Ca(2+) was removed, indicating that they cooperate with Ca(2+) to activate CaSRs, and all activated CaSR isoforms with the N-terminal extracellular domain deleted, indicating that they interact with the transmembrane domains. In both cases, AC-265347 and therefore (S)-AC-265347 were significantly more efficacious than the phenylalkylamines. Mutations E837A(7.39) and I841A(7.43) strongly reduced phenylalkylamine-induced signaling, but not AC-265347- or (S)-AC-265347-induced signaling, suggesting different modes of binding. AC-265347 and (S)-AC-265347 stimulated significantly greater responses than cinacalcet or calindol at each of four loss-of-function human polymorphic CaSR variants. AC-265347 did not inhibit the CYP2D6 cytochrome P450 isozyme, unlike cinacalcet, which is a potent CYP2D6 inhibitor. In rats, AC-265347, (S)-AC-265347, and (R)-AC-265347 each reduced serum parathyroid hormone (PTH) with a rank order potency correlated with their in vitro potencies. AC-265347 and (S)-AC-265347 also reduced plasma ionizable calcium ([Ca(2+)](o)). AC-265347 was orally active, and its plasma concentrations correlated well with its effects on serum PTH. Thus, these highly efficacious CaSR allosteric agonists represent leads for developing therapeutic agents with potential advantages over existing therapies.

In Vitro Metabolism of 17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin in Human Liver Microsomes

The objective of this study was to investigate the oxidative metabolism pathways of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), a geldanamycin (GA) derivative and 90-kDa heat shock protein inhibitor. In vitro metabolic profiles of 17-DMAG were examined by using pooled human liver microsomes (HLMs) and recombinant CYP450 isozymes in the presence or absence of reduced GSH. In addition to 17-DMAG hydroquinone and 19-glutathionyl 17-DMAG, several oxidative metabolites of 17-DMAG were detected and characterized by liquid chromatography-tandem mass spectrometry. Different from previously reported primary biotransformations of GA and GA derivatives, 17-DMAG was not metabolized primarily through the reduction of benzoquinone and GSH conjugation in HLMs. In contrast, the primary biotransformations of 17-DMAG in HLMs were hydroxylation and demethylation on its side chains. The most abundant metabolite was produced by demethylation from the methoxyl at position 12. The reaction phenotyping study showed that CYP3A4 and 3A5 were the major cytochrome P450 isozymes involved in the oxidative metabolism of 17-DMAG, whereas CYP2C8, 2D6, 2A6, 2C19, and 1A2 made minor contributions to the formation of metabolites. On the basis of the identified metabolite profiles, the biotransformation pathways for 17-DMAG in HLMs were proposed.

Co-Expression of Recombinant Human CYP2C9 with Human Cytochrome P450 Reductase in Protease Deficient S. cerevisiae Strain at a Higher Scale Yields an Enzyme of Higher Specific Activity

Cytochrome P450 (CYP450) isozymes play an important role in the study of drug metabolism and drug discovery. A number of reports are available that describe recombinant expression of CYP450 isozymes. In this paper, human CYP2C9 and human cytochrome P450 reductase cDNAs were cloned and expressed in Premas proprietary yeast episomal and integrative vectors respectively under the influence of GAL1 promoter. Yeast cells were grown and induced at optimal parameters to make microsomal membranes. Isolated microsomal membranes were analyzed for CYP2C9 and cytochrome P450 reductase activity, CYP2C9 content and inhibition properties. We report heterologous expression of human CYP2C9 along with human cytochrome P450 reductase in protease deficient S. cerevisiae at a 5 litre scale resulting in high yields (8-10 nmols/litre) of enzyme with higher specific activity (2-3 fold higher). This yields a superior enzyme and makes it amenable to miniaturization of screening assays with concomitant lowering of costs.

In vitro effects of natural prenyloxycinnamic acids on human cytochrome P450 isozyme activity and expression

Previous studies demonstrated that natural prenyloxyphenylpropanoid derivatives have potent biological properties like anti-cancer effects in vitro and in vivo. Additionally they are extremely safe and associated with low toxicity, making them excellent candidates as chemopreventive agents. However, so far only little is known about possible interactions with isoforms of cytochrome P450 (CYPs) being involved in the metabolism of xenobiotics and representing a major site for drug-drug interactions. The aim of this study was to evaluate the effects of selected natural prenyloxyphenylpropanoids (prenyloxycinnamic acids) on expression and activity of some major CYPs and on the activity of the major drug efflux transporter P-glycoprotein (P-gp). Inhibition of CYP3A4, CYP2C19, and CYP2D6 was quantified using commercially available kits. P-gp inhibtion was quantified by calcein assay. Induction of CYP mRNA (CYP3A4, CYP2C19, CYP2C9, and CYP2B6) was measured in LS180 cells by quantitative real-time reverse transcriptase polymerase chain reaction using the LightCycler technology. Only boropinic acid revealed substantial inhibition of CYPs, especially of CYP2C19 (IC₅₀ = 31±5μM). This compound also had the most pronounced effect on CYP mRNA expression among the prenyloxycinnamic acids tested. However all but 4'-isopentenyloxy-p-coumaric acid revealed inducing effects on CYPs with different induction profiles. P-gp was only significantly inhibited by 4'-geranyloxyferulic acid. This was the first study demonstrating modulating effects of prenyloxycinnamic acids on CYP activity and expression and on P-gp activity. The results suggest that boropinic acid is most prone to drug-drug interactions at the level of CYPs, whereas 4'-isopentenyloxy-p-coumaric acid does not modulate CYP activity and expression.

Oxidative DNA damage and reporter gene mutation in the livers of gpt delta rats given non-genotoxic hepatocarcinogens with cytochrome P450-inducible potency

Previous reports have proposed that reactive oxygen species resulting from induction of cytochrome P450 (CYP) isozymes might be involved in the modes of action of hepatocarcinogens with CYP-inducible potency. In the present study, we investigated 8-hydroxydeoxyguanosine (8-OHdG) levels, in vivo mutagenicity and glutathione S-transferase placental form (GST-P)-positive foci in the livers of gpt delta rats treated with piperonyl butoxide (PBO) or phenobarbital (PhB) for 4 and 13 weeks. Significant elevations in Cyp 1A1 and Cyp 1A2 mRNA levels after PBO treatment, and in Cyp 2B1 mRNA levels after PBO or PhB treatment, appeared together with remarkable hepatomegaly through the experimental period. Time-dependent and statistically significant increases in 8-OHdG levels were observed in the PBO treatment group along with significant increases in proliferating cell nuclear antigen (PCNA)-positive hepatocytes at 4 weeks, while no increase in 8-OHdG levels was found in PhB-treated rats. No changes in mutant frequencies of gpt and red/gam (Spi(-)) genes in liver DNA from PBO- or PhB-treated rats were observed at 4 or 13 weeks. A 13-week exposure to either PBO or PhB did not affect the number and area of GST-P-positive hepatocytes. CYP 1A1 and 1A2 induction may be responsible for elevated levels of 8-OHdG in PBO-treated rats. However, neither GC:TA transversions nor deletion mutations, typically regarded as 8-OHdG-related mutations, were observed in any of the treated rats. We conclude that reactive oxygen species, possibly produced through CYP catalytic pathways, likely induced genomic DNA damage but did not give rise to permanent gene mutation.

Evaluation of an in-capillary approach for performing quantitative cytochrome P450 activity studies

An automated in-capillary assay requiring very small quantities of reagents was developed for performing in vitro cytochrome P450 (CYP450) drug metabolism studies. The approach is based on the following: (i) hydrodynamic introduction of nanoliter volumes of substrate and enzyme solutions in the sandwich mode, within a capillary; (ii) mixing the reagents by diffusion across the interfaces between the injected solutions; (iii) collection of the capillary content at the end of the in-capillary assay; and (iv) off-line analysis of the incubation mixture by ultrahigh pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). After optimizing the injection sequence of the reagents, the in-capillary approach was applied to the quantitative determination of the kinetics of drug metabolism reactions catalyzed by three CYP450 isozymes involved in human drug metabolism: CYP1A2, CYP2D6, and CYP3A4. It was demonstrated that this in-capillary method was able to provide similar kinetic parameters for CYP450 activity (e.g., Michaelis constants and turnover values) as the classical in vitro method, with a drastic reduction of reagent consumption.

Cytochrome P450-mediated hepatic metabolism of new fluorescent substrates in cats and dogs

This study aimed to investigate the biotransformation of cat liver microsomes in comparison to dogs and humans using a high throughput method with fluorescent substrates and classical inhibitors specific for certain isozymes of the human cytochrome P450 (CYP) enzyme family. The metabolic activities associated with CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A were measured. Cat liver microsomes metabolized all substrates selected for the assessment of cytochrome P450 activity. The activities associated with CYP3A and CYP2B were higher than the activities of the other measured CYPs. Substrate selectivity could be demonstrated by inhibition studies with α-naphthoflavone (CYP1A), tranylcypromine/quercetine (CYP2C), quinidine (CYP2D), diethyldithiocarbamic acid (CYP2E) and ketoconazole (CYP3A) respectively. Other prototypical inhibitors used for characterization of human CYP activities such as furafylline (CYP1A), tranylcypromine (CYP2B) and sulfaphenazole (CYP2C) did not show significant effects in cat and dog liver microsomes. Moreover, IC50-values of cat CYPs differed from dog and human CYPs underlining the interspecies differences. Gender differences were observed in the oxidation of 7-ethoxy-4-trifluoromethylcoumarin (CYP2B) and 3-[2-(N, N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (CYP2D), which were significantly higher in male cats than in females. Conversely, oxidation of the substrates dibenzylfluorescein (CYP2C) and 7-methoxy-4-trifluoromethylcoumarin (CYP2E) showed significant higher activities in females than in male cats. Overall CYP-activities in cat liver microsomes were lower than in those from dogs or humans, except for CYP2B. The presented difference between feline and canine CYP-activities are useful to establish dose corrections for feline patients of intensively metabolized drugs licensed for dogs or humans.

An open-label, randomized, single-center, two-period, phase I, crossover study of the effect of zibotentan (ZD4054) on the pharmacokinetics of midazolam in healthy male volunteers

Background: Zibotentan (ZD4054) is an oral, specific endothelin A receptor antagonist presently under investigation for the treatment of hormone-resistant prostate cancer. Preclinical in vitro studies suggest that zibotentan has the potential to act as a time-dependent inhibitor of the cytochrome P450 isozyme 3A4 (CYP3A4) metabolic pathway. In clinical practice, it is likely that zibotentan will be coadministered with drugs metabolized by this pathway; the potential exists, therefore, that zibotentan-induced drug interactions could occur. Objectives: The primary objective of this study was to evaluate the effect of zibotentan on the pharmaco-kinetics of a clinically relevant dose of midazolam in healthy volunteers. Secondary objectives were to evaluate exposure to zibotentan, ensure the safety of the healthy volunteers dosed, and investigate the effect of zibotentan on the pharmacokinetics of the midazolam metabolites 1-hydroxy midazolam and 4-hydroxy midazolam. The potency of zibotentan as a CYP3A4 inhibitor was also assessed. Methods: This was an open-label, randomized, singlecenter, 2-period, Phase I, crossover study. Volunteers were randomized in a 1:1 ratio to 1 of 2 cohorts. In cohort 1, volunteers received a single dose of midazolam 7.5 mg on day 1 (treatment A) of a 2-day study period. After a minimum 7-day washout period, volunteers received zibotentan 10 mg once daily on days 1 through 7, plus a single dose of midazolam 7.5 mg on day 6 (treatment B) of a 7-day study period. In cohort 2, volunteers received treatment B followed by treatment A, with a minimum 7-day washout period between treatments. AUC 0−∞ and C max data were expressed as geometric least squares mean ratios and 90% CIs for midazolam + zibotentan:midazolam. A moderate interaction between midazolam and zibotentan was predefined to have occurred if the upper 90% CI of the ratio was >1.5. Adverse events (AEs) were assessed according to the National Cancer Institute's Common Terminology Criteria for Adverse Events version 3. AE data were assessed based on information provided by the volunteer, through open-ended and nonleading verbal questions to the volunteer at each visit, and through observation by the investigational team, other care providers, or relatives. Results: Six volunteers (all white) were included in each cohort (cohort 1, mean [SD] age, 48 [7] years; mean weight, 74 [6] kg; cohort 2, mean age, 51 [11] years; mean weight, 75 [13] kg). Steady-state levels of zibotentan, achieved over 7 days, increased the midazolam AUC 0−∞ by 1.2-fold compared with midazolam alone. The upper limits of the 90% CIs for the AUC 0−∞ and C max ratios were below the predefined level of 1.5 (1.37 and 1.32, respectively). Zibotentan had no apparent effect on the pharmacokinetics of 1-hydroxy midazolam and 4-hydroxy midazolam. Fatigue was reported in 11 volunteers (92%) receiving midazolam monotherapy and 10 (83%) receiving midazolam combined with zibotentan. Headache was reported in all 12 volunteers after zibotentan monotherapy. Conclusions: In this population of healthy male volunteers, once-daily zibotentan 10 mg increased the AUC 0−∞ of midazolam 1.2-fold; however, the treatment ratio was below the predefined limit for clinical significance. Zibotentan was well tolerated when given alone or in combination with midazolam. The results indicate that once-daily zibotentan 10 mg acted as a weak inhibitor of the CYP3A4 pathway. ClinicalTrials. gov identifier: NCT00709553.

Estimating Pediatric Doses of Drugs Metabolized by Cytochrome P450 (CYP) Isozymes, Based on Physiological Liver Development and Serum Protein Levels

We established a method for estimating pediatric doses of drugs metabolized by cytochrome P450 (CYP) isozymes, using the free fraction of drug in plasma (fu), serum protein level (P), liver volume (LV), and CYP activity (Vmax/Km) as indices of physiological and biochemical development in children up to 15 years old. This method allows the child/adult dose ratio (D(C)/D(A))=child/adult oral clearance ratio (CL((PO)(C))/CL((PO)(A))) of drugs mainly metabolized in the liver to be estimated by the following equation: [formula: see text]. Major metabolism of drugs was ascribed to CYP1A2 for theophylline and caffeine, and CYP1A2 and CYP2D6 for propranolol and mexiletine. For theophylline and caffeine, CL((PO)(C))/CL((PO)(A)) calculated from the child/adult body surface area ratio (BSA ratio) and the value calculated by our method were compared, using CL((PO)(C))/CL((PO)(A)) calculated from the clearance ratio based on population pharmacokinetics (PPK ratio) as a reference. For all drugs, pediatric doses calculated from the Crawford equation and our equation were compared, with predetermined doses as the reference. For theophylline and caffeine, the relative accuracy of our method was significantly higher than that of BSA-based estimation when the PPK ratio was used for reference. For theophylline, caffeine, and propranolol, the relative accuracy of our method was significantly higher than that of BSA-based estimation when predetermined doses were used for reference. These findings indicate the validity of our method which considers the physiological and biochemical development (i.e., fu, P, LV, and CYP activity) for pediatric dose estimation.

Chemoprevention of 1,2-dimethylhydrazine-induced colonic preneoplastic lesions in Fischer rats by 6-methylsulfinylhexyl isothiocyanate, a wasabi derivative

The preventive effects of dietary exposure to a wasabi derivative 6-methylsulfinylhexyl isothiocyanate (6-MSITC) during the initiation and post-initiation phases on the development of 1,2-dimethylhydrazine (DMH)-induced colonic aberrant crypt foci (ACF), and β-catenin-accumulated crypts (BCAC) were investigated in male F344 rats. To induce ACF and BCAC, rats were given four weekly subcutaneous injections of DMH (40 mg/kg body weight). The rats also received diets containing 200 or 400 ppm 6-MSITC during the initiation or post-initiation phases. The experiment was terminated 12 weeks after the start. DMH exposure produced a substantial number of ACF (323.8±69.7/colon) and BCAC (3.80±1.05/cm(2)) at the end of the study. Dietary administration of 6-MSITC at a dose of 400 ppm during the initiation phase caused a significant reduction in the total number of ACF (52% reduction, P<0.0001), larger ACF (4 or more crypt ACF) (58% reduction, P<0.001) and BCAC (76% reduction, P<0.00001). The dietary exposure to 6-MSITC significantly reduced the size (crypt multiplicity) of BCAC during both initiation and post-initiation treatment when compared to group 1 treated with DMH alone. Immunohistochemically, 6-MSITC administration lowered the proliferating cell nuclear antigen labeling index in ACF and BCAC. In addition, protein levels of hepatic cytochrome P-450 isozymes at 24 h after 6-MSITC exposure were significantly suppressed (P<0.01). The results indicated that 6-MSITC exerted chemopreventive effects in the present short-term colon carcinogenesis bioassay, through alterations in cell proliferation activity and drug metabolizing enzyme levels.

Effect of a high cornstarch diet on hepatic cytochrome P450 2C and 3A activity and progesterone half-life in dairy cows

In the cow, inadequate concentrations of progesterone during gestation may lead to an abrupt termination of pregnancy. The primary organ involved in progesterone catabolism is the liver, which contains an abundance of cytochrome P450 isozymes (EC 1.14.14.1; mixed-function monooxygenases). The objectives of the current experiment were to determine the effect of feeding 2 isoenergetic and isonitrogenous diets, formulated to cause divergent insulin secretion, on hepatic cytochrome P450 2C (CYP2C) and 3A (CYP3A) activity as well as the resulting biological half-life of progesterone. Twenty-two Holstein cows averaging 80±7 d in milk were randomly assigned to either a high cornstarch diet or a high fiber diet in a crossover experimental design consisting of two 14-d periods. Dry matter intake, milk yield, milk lactose yield, and milk lactose percentage were similar between the 2 diets. Milk fat yield and milk fat percentage were higher in cows fed the high fiber diet, whereas milk protein yield tended to be higher and milk protein percentage was higher in cows fed the high cornstarch diet. Energy balance tended to be improved by 57% in cows consuming the high cornstarch diet. Insulin concentrations at the time of liver biopsy (3.16±0.04h post-feeding) were increased by 44% in cows consuming the high cornstarch diet compared with cows consuming the high fiber diet. Cytochrome P450 2C activity was decreased by 45%, whereas CYP3A activity tended to be lowered by 34% in cows consuming the high cornstarch diet. Cytochrome P450 2C mRNA expression tended to be decreased by 21% in cows fed the high cornstarch diet, whereas CYP3A mRNA expression was not different between the dietary treatments. The fractional rate constant of progesterone decay was not different between the 2 diets; however, the half-life of progesterone tended to be longer in cows fed the high cornstarch diet compared with cows fed the high fiber diet (85 vs. 64min, respectively). In summary, cows consuming the high cornstarch diet had increased insulin concentrations and lower hepatic CYP2C and CYP3A activity and tended to have a longer progesterone half-life compared with cows consuming the high fiber diet. Feeding diets that stimulate insulin secretion could alter progesterone clearance during lactation, when dairy cows have increased rates of progesterone inactivation because of high energy demands and increased DMI.