Drug Metabolism and Disposition current issue

[SHORT COMMUNICATIONS] Covariation of Human Microsomal Protein Per Gram of Liver with Age: Absence of Influence of Operator and Sample Storage May Justify Interlaboratory Data Pooling

2008-11-18

Scaling of metabolic clearance values from liver microsomal data or recombinantly expressed cytochrome P450 enzymes to predict human hepatic clearance requires knowledge of the amount of microsomal protein per gram of liver (MPPGL). Identification of physiological covariates of MPPGL requires analysis of values from large diverse populations, which necessitates pooling of data from numerous sources. To ensure compatibility between results obtained within and between studies, the impact of interoperator differences and sample storage on values of MPPGL was investigated. With use of triplicate samples from one liver (HL86), no statistically significant difference was detected between values of MPPGL prepared from samples stored at -80°C (23.5 ± 1.2 mg g^-1) and those determined using fresh tissue (21.9 ± 0.3 mg g^-1). Although there was a significant difference in the yield of microsomal protein obtained from another liver sample (HL43) by three different operators (17 ± 1, 19 ± 2, and 24 ± 1 mg g^-1; p = 0.004, analysis of variance), no difference was observed in the estimated MPPGL after application of appropriate correction factors for each operator (28 ± 1, 30 ± 5, and 31 ± 4 mg g^-1). The result provided justification for pooling reported values of MPPGL for use in covariate analysis. Investigation of the relationship between age and MPPGL provided preliminary evidence that MPPGL values increase from birth to a maximum of 40 mg g^-1 [95% confidence interval for the geometric mean (95% CI mean_geo): 37–43 mg g^-1 at approximately 28 years followed by a gradual decrease in older age (mean of 29 mg g^-1 at 65 years; 95% CI mean_geo: 27–32 mg g^-1). Accordingly, appropriate age-adjusted scaling factors should be used in extrapolating in vitro clearance values to clinical studies.

[SHORT COMMUNICATIONS] The Combination of Chemical and Antibody Inhibitors for Superior P450 3A Inhibition in Reaction Phenotyping Studies

Rock, D. A., Foti, R. S., Pearson, J. T. — 2008-11-18

Cytochrome P450 (P450) reaction phenotyping is a key process toward accurately determining the contribution of different P450s to the metabolism of new chemical entities. The significance of P450s to drug disposition has led to the identification of selective chemical and antibody inhibitors for individual P450 enzymes. Despite these advances, the maximal inhibition attainable is limited by the use of inhibitor concentrations that maintain selectivity for the individual P450s. Thus, most commercially available inhibitors produce a maximal inhibition of ~80%. Herein, the combination of chemical plus antibody inhibitors is explored to find whether P450 3A could be selectively and completely (>99%) inhibited by using both inhibitors simultaneously.

[SHORT COMMUNICATIONS] Structural Characterization of Novel Adenine Dinucleotide Phosphate Conjugates of Imatinib in Incubations with Rat and Human Liver Microsomes

Ma, S., Subramanian, R., Xu, Y., Schrag, M., Shou, M. — 2008-11-18

Imatinib, a potent and selective protein tyrosine kinase inhibitor, has been approved for the treatment of chronic myelogenous leukemia and metastatic and unresectable malignant gastrointestinal stromal tumors. In vitro metabolism of imatinib was investigated in rat and human liver microsomes. Besides several oxidative metabolites and an N-desmethyl metabolite, as previous reported, a novel metabolite with a mass addition of 621 atomic mass units to the parent was detected as the major metabolite in the incubations with rat liver microsomes, using NADPH as a cofactor. The analysis of MS² and MSⁿ data revealed that this metabolite corresponded to adenine dinucleotide phosphate (ADP⁺) conjugate of imatinib. The ADP⁺ adduct was scaled up from rat liver microsomal incubations and isolated for NMR analysis. NMR data confirmed and conclusively showed the conjugation had occurred between the pyridine nitrogen of imatinib to the ribose ring of ADP⁺ moiety. The formation of this adduct was enzymatic and required NADP⁺ as a reactant. In addition, ADP⁺ adducts of imatinib N-oxide and desmethyl imatinib were also detected as minor metabolites in the incubations with rat liver microsomes. In contrast, only trace levels of ADP⁺ adducts of imatinib and desmethyl imatinib were detected in the incubations with human liver microsomes. Imatinib-ADP⁺ adducts have been observed only in in vitro studies to date. The physiological role of these adducts is not clear, nor is their in vivo relevance.

[SHORT COMMUNICATIONS] Differential Allelic Expression in Leukoblast from Patients with Acute Myeloid Leukemia Suggests Genetic Regulation of CDA, DCK, NT5C2, NT5C3, and TP53

Jordheim, L. P., Nguyen-Dumont, T., Thomas, X., Dumontet, C., Tavtigian, S. V. — 2008-11-18

mRNA expression levels of certain genes have shown predictive value for the outcome of cytarabine-treated AML-patients. We hypothesized that genetic variants play a role in the regulation of the transcription of these genes. We studied leukoblasts from 82 patients with acute myeloid leukemia and observed various extent and frequency of differential allelic expression in the CDA, DCK, NT5C2, NT5C3, and TP53 genes. Our attempts to identify the causative regulatory single nucleotide polymorphisms by a bioinformatics approach did not succeed. However, our results indicate that genetic variations are at least in part responsible for the differences in overall expression levels of these genes.

[SHORT COMMUNICATIONS] Nonenzymatic Formation of a Novel Hydroxylated Sulfamethoxazole Derivative in Human Liver Microsomes: Implications for Bioanalysis of Sulfamethoxazole Metabolites

2008-11-18

Sulfamethoxazole is metabolized by microsomal CYP2C9 to a hydroxylamine that is thought to be responsible for the relatively high incidence of hypersensitivity reactions associated with the drug. Accurate quantification of the hydroxylamine requires the loss of metabolite through autoxidation to be blocked with ascorbate. In this study, a partly nonenzymatically generated arylhydroxylated derivative of sulfamethoxazole was identified by liquid chromatography/mass spectrometry in incubations of human liver microsomes, and it was found to coelute with the isomeric hydroxylamine under the conditions of three published high-performance liquid chromatography (HPLC) assays. Partial inhibition of the aryl hydroxylation by 1-aminobenzotriazole suggested some involvement of cytochrome P450. However, the formation of this compound was ascorbate-dependent, and it was enhanced by the addition of Fe²⁺/EDTA and inhibited by desferrioxamine but not by mannitol. These findings are consistent with the phenol being generated via an Fe²⁺/ascorbate/O₂-oxygenating system that does not involve hydroxyl radicals. It was also produced by H₂O₂/ascorbate. Because the compound shares close chromatographic similarities with the hydroxylamine metabolite, it is possible that previous studies may have inaccurately characterized or quantified sulfamethoxazole metabolism.

[ARTICLES] CYP2C9-Catalyzed Metabolism of S-Warfarin to 7-Hydroxywarfarin in Vivo and in Vitro in Chimeric Mice with Humanized Liver

Inoue, T., Nitta, K., Sugihara, K., Horie, T., Kitamura, S., Ohta, S. — 2008-11-18

Chimeric mice having humanized livers were constructed by transplantation of human hepatocytes. In this study, we investigated whether these mice have a capacity for drug metabolism similar to that of humans by examining hydroxylation of S-warfarin, which is predominantly metabolized to S-7-hydroxywarfarin, catalyzed by CYP2C9, in humans but not mice. The 7-hydroxylating activity of chimeric mouse liver microsomes toward S-warfarin was approximately 10-fold higher than that of control (urokinase-type plasminogen activator-transgenic severe combined immunodeficient) mice. The 7-hydroxylase activity of chimeric mouse liver microsomes was markedly inhibited by sulfaphenazole, as was that of human liver microsomes, whereas the activity of control mice was unaffected. The CYP2C isoform in chimeric mouse liver was also confirmed to be the human isoform, CYP2C9, by immunoblot analysis. In the present in vivo study, the level of S-7-hydroxywarfarin in plasma of chimeric mice was approximately 7-fold higher than that in control mice, in agreement with the in vitro data. Thus, the CYP2C isoform in chimeric mice functions in vivo and in vitro as a human isoform, CYP2C9. These results suggest that chimeric mice with humanized liver could be useful for predicting drug metabolism in humans, at least regarding CYP2C9-dependent metabolism.

[ARTICLES] Design, Data Analysis, and Simulation of in Vitro Drug Transport Kinetic Experiments Using a Mechanistic in Vitro Model

2008-11-18

The use of in vitro data for quantitative predictions of transporter-mediated elimination in vivo requires an accurate estimation of the transporter Michaelis-Menten parameters, V_max and K_m, as a first step. Therefore, the experimental conditions of in vitro studies used to assess hepatic uptake transport were optimized regarding active transport processes, nonspecific binding, and passive diffusion (P_dif). A mechanistic model was developed to analyze and accurately describe these active and passive processes. This two-compartmental model was parameterized to account for nonspecific binding, bidirectional passive diffusion, and active uptake processes based on the physiology of the cells. The model was used to estimate kinetic parameters of in vitro transport data from organic anion-transporting peptide model substrates (e.g., cholecystokinin octapeptide deltorphin II, fexofenadine, and pitavastatin). Data analysis by this mechanistic model significantly improved the accuracy and precision in all derived parameters [mean coefficient of variations (CVs) for V_max and K_m were 19 and 23%, respectively] compared with the conventional kinetic method of transport data analysis (mean CVs were 58 and 115%, respectively, using this method). Furthermore, permeability was found to be highly temperature-dependent in Chinese hamster ovary (CHO) control cells and artificial membranes (parallel artificial membrane permeability assay). Whereas for some compounds (taurocholate, estrone-3-sulfate, and propranolol) the effect was moderate (1.5–6-fold higher permeability at 37°C compared with that at 4°C), for fexofenadine a 16-fold higher passive permeability was seen at 37°C. Therefore, P_dif was better predicted if it was evaluated under the same experimental conditions as V_max and K_m, i.e., in a single incubation of CHO overexpressed cells or rat hepatocytes at 37°C, instead of a parallel control evaluation at 4°C.

[ARTICLES] Quantitative Tissue and Gene-Specific Differences and Developmental Changes in Nat1, Nat2, and Nat3 mRNA Expression in the Rat

2008-11-18

Human N-acetyltransferase 1 (NAT1) and 2 (NAT2) are important phase II enzymes involved in the biotransformation of xenobiotics. In toxicity and carcinogenicity studies, functional polymorphism of rat N-acetyltransferase is considered a model for similar human variability. To accurately quantitate expression of the three rat N-acetyltransferases, we developed sensitive, specific assays for Nat1, Nat2, and Nat3 mRNAs. In male F344 rats, tissue-specific expression varied over a limited range for both Nat1 (~19-fold) and Nat2 (~30-fold), with the highest expression of both genes in colon. Expression of Nat3 mRNA was at least 2 to 3 orders of magnitude less than that of Nat1 or Nat2. Comparison of Nat1 and Nat2 mRNA expression in bladder, colon, liver, and lung of male and female F344 rats detected no significant gender-specific difference. In Sprague-Dawley and F344 rats ranging in age from neonate to mature adult, colon showed a >10-fold increase in Nat2 during the first postnatal month that did not correlate with changes in Nat1. In contrast, Nat2 showed no developmental change in Sprague-Dawley or F344 liver as Nat1 increased modestly. These measures of rat Nat expression confirm that Nat3 expression is negligible and that Nat1 and Nat2 are the primary determinants of arylamine acetylation activity in all tissues tested. The findings demonstrate differential tissue-specific and developmental regulation of the rat Nat1 and Nat2 genes and contribute to more complete understanding of tissue-, gender-, and development-specific expression patterns of the cognate N-acetyltransferase genes of humans and other species.

[ARTICLES] Systemic Functional Expression of N-Acetyltransferase Polymorphism in the F344 Nat2 Congenic Rat

Hein, D. W., Bendaly, J., Neale, J. R., Doll, M. A. — 2008-11-18

Rat lines congenic for the rat N-acetyltransferase 2 [(RAT)Nat2] gene were constructed and characterized. F344 (homozygous Nat2 rapid) males were mated to Wistar Kyoto (homozygous Nat2 slow) females to produce heterozygous F1. F1 females were then backcrossed to F344 males. Heterozygous acetylator female progeny from this and each successive backcross were identified by rat Nat2 genotyping and mated with F344 rapid acetylator males. After 10 generations of backcross mating, heterozygous acetylator brother/sister progeny were mated to produce the homozygous rapid and slow acetylator Nat2 congenic rat lines. p-Aminobenzoic acid (selective for rat NAT2) and 4-aminobiphenyl N-acetyltransferase activities were expressed in all tissues examined (liver, lung, esophagus, stomach, small intestine, colon, pancreas, kidney, skin, leukocytes, and urinary bladder in male and female rats and in breast of female and prostate of male rats). NAT2 expression in rat extrahepatic tissues was much higher than that in liver. In each tissue, activities were Nat2-genotype-dependent, with the highest levels in homozygous rapid acetylators, intermediate levels in heterozygous acetylators, and lowest in homozygous slow acetylators. Sulfamethazine (selective for rat NAT1) N-acetyltransferase activities were observed in all tissues examined in both male and female rats except for breast (females), bladder, and leukocytes. In each tissue, the activity was Nat2 genotype-independent, with similar levels in homozygous rapid, heterozygous, and homozygous slow acetylators. These congenic rat lines are useful for investigating the role of NAT2 genetic polymorphisms in susceptibility to cancers related to arylamine carcinogen exposures.

[ARTICLES] Functional Characterization of 17 CYP2D6 Allelic Variants (CYP2D6.2, 10, 14A-B, 18, 27, 36, 39, 47-51, 53-55, and 57)

2008-11-18

Cytochrome P450 2D6 (CYP2D6) is an enzyme of potential importance for the metabolism of drugs used clinically, and it exhibits genetic polymorphism with interindividual differences in metabolic activity. To date, 21 CYP2D6 allelic variants have been identified in the Japanese population. The aim of this study was to investigate the functional characterization of CYP2D6 variants identified in Japanese subjects. Wild-type CYP2D6 and its variants, namely, CYP2D6.2, CYP2D6.10, CYP2D6.14A, CYP2D6.14B, CYP2D6.18, CYP2D6.27, CYP2D6.36, CYP2D6.39, CYP2D6.47, CYP2D6.48, CYP2D6.49, CYP2D6.50, CYP2D6.51, CYP2D6.53, CYP2D6.54, CYP2D6.55, and CYP2D6.57 were transiently expressed in COS-7 cells, and enzymatic activities of the CYP2D6 variant proteins were characterized using bufuralol and dextromethorphan. Functional characterization of 17 CYP2D6 variants revealed an absence of enzyme activity in four (CYP2D6.14A, CYP2D6.36, CYP2D6.47, and CYP2D6.57), low activity in eight (CYP2D6.10, CYP2D6.14B, CYP2D6.18, CYP2D6.49, CYP2D6.50, CYP2D6.51, CYP2D6.54, and CYP2D6.55), and high activity in one (CYP2D6.53) compared with the wild type. Analysis of CYP2D6 variant proteins can be useful for predicting CYP2D6 phenotypes and could be applied to personalized drug therapy.

[ARTICLES] Purification and Characterization of Flavin-Containing Monooxygenase Isoform 3 from Rat Kidney Microsomes

Novick, R. M., Elfarra, A. A. — 2008-11-18

Rats are a common animal model for metabolism and toxicity studies. Previously, the enzymatic properties of rat flavin-containing monooxygenase (FMO) 1 purified from hepatic and renal microsomes and that of FMO3 purified from hepatic microsomes were characterized. This study investigated the physical, immunological, and enzymatic properties of FMO3 purified from male rat kidney microsomes and compared the results with those obtained with isolated rat liver FMO3. Renal FMO3 was purified via affinity columns based on the elution of l-methionine (Met) S-oxidase activity and reactivity of the eluted proteins with human FMO3 antibody. In general, Met S-oxidase-specific activity was increased 100-fold through the purification steps. The resulting protein had similar mobility (~56 kDa) as isolated rat liver FMO3 and cDNA-expressed human FMO3 by SDS-polyacrylamide gel electrophoresis. When the isolated kidney protein band was subjected to trypsin digestion and matrix-assisted laser desorption ionization/time of flight mass spectral analysis, 34% of the sequence of rat FMO3 was detected. The apparent K_m and V_max values for rat kidney FMO3 were determined using the known FMO substrates Met, seleno-l-methionine, S-allyl-l-cysteine (SAC), and methimazole (N-methyl-2-mercaptoimidazole). The stereoselectivity of the reactions with Met and SAC were also examined using high-performance liquid chromatography. The obtained kinetic and stereoselectivity results were similar to those we obtained in the present study, or those previously reported, for rat liver FMO3. Taken together, the results demonstrate many similar properties between rat hepatic and renal FMO3 forms and suggest that renal FMO3 may play an important role in kidney metabolism of xenobiotics containing sulfur and selenium atoms.

[ARTICLES] Metabolism of 5-Isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (BMS-645737): Identification of an Unusual N-Acetylglucosamine Conjugate in the Cynomolgus Monkey

2008-11-18

5-Isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (BMS-645737) is a potent and selective vascular endothelial growth factor receptor-2 antagonist. In this study, liquid chromatography/tandem mass spectrometry and NMR were used to investigate the biotransformation of BMS-645737 in vitro and in the cynomolgus monkey, dog, mouse, and rat. Metabolic pathways for BMS-645737 included multistep processes involving both oxidation and conjugation reactions. For example, the 2-methyl-1H-pyrrolo moiety underwent cytochrome P450-catalyzed hydroxylation followed by oxidation to a carboxylic acid and then conjugation with taurine. Alternatively, the 5-methyl-1,3,4-oxadiazol-2-yl moiety was metabolized by hydroxylation and then conjugation with sulfate. The pyridin-5-yl group underwent direct glucuronidation in hepatocytes (dog, monkey, human) and conjugation with N-acetylglucosamine in the monkey. Conjugation with glutathione and processing along the mercapturic acid pathway was a minor metabolic pathway in vivo, although BMS-645737 did not form conjugates in the presence of glutathione-supplemented liver microsomes. Other minor biotransformation pathways included oxidative dehydrogenation, dihydroxylation, and hydrolytic opening of the oxadiazole ring followed by either deacetylation or hydrolysis of the resulting diacyl hydrazide. Whereas previous studies have shown the formation of N-acetylglucosamine conjugates of alcohols, arylamines, and other small molecules, this report describes the biotransformation of a heterocyclic aromatic amine via direct conjugation with N-acetylglucosamine.

[ARTICLES] An Assessment of Drug-Drug Interactions: The Effect of Desvenlafaxine and Duloxetine on the Pharmacokinetics of the CYP2D6 Probe Desipramine in Healthy Subjects

2008-11-18

A number of antidepressants inhibit the activity of the cytochrome P450 2D6 enzyme system, which can lead to drug-drug interactions. Based on its metabolic profile, desvenlafaxine, administered as desvenlafaxine succinate, a new serotonin-norepinephrine reuptake inhibitor, is not expected to have an impact on activity of CYP2D6. This single-center, randomized, open-label, four-period, crossover study was undertaken to evaluate the effect of multiple doses of desvenlafaxine (100 mg/day, twice the recommended therapeutic dose for major depressive disorder in the United States) and duloxetine (30 mg b.i.d.) on the pharmacokinetics (PK) of a single dose of desipramine (50 mg). A single dose of desipramine was given first to assess its PK. Desvenlafaxine or duloxetine was then administered, in a crossover design, so that steady-state levels were achieved; a single dose of desipramine was then coadministered. The geometric least-square mean ratios (coadministration versus desipramine alone) for area under the plasma concentration versus time curve (AUC) and peak plasma concentrations (C_max) of desipramine and 2-hydroxydesipramine were compared using analysis of variance. Relative to desipramine alone, increases in AUC and C_max of desipramine associated with duloxetine administration (122 and 63%, respectively) were significantly greater than those associated with desvenlafaxine (22 and 19%, respectively; P < 0.001). Duloxetine coadministered with desipramine was also associated with a decrease in 2-hydroxydesipramine C_max that was significant compared with the small increase seen with desvenlafaxine and desipramine (-24 versus 9%; P < 0.001); the difference between changes in 2-hydroxydesipramine AUC did not reach statistical significance (P = 0.054). Overall, desvenlafaxine had a minimal impact on the PK of desipramine compared with duloxetine, suggesting a lower risk for CYP2D6-mediated drug interactions.

[ARTICLES] Involvement of Intestinal Uptake Transporters in the Absorption of Azithromycin and Clarithromycin in the Rat

2008-11-18

Macrolide antibiotics azithromycin (AZI) and clarithromycin (CLARI) are large molecular weight compounds and are substrates for apically polarized efflux transporters such as P-glycoprotein, which can potentially restrict intestinal absorption. However, despite these undesired physicochemical and biopharmaceutical properties, AZI and CLARI exhibit moderate to excellent p.o. bioavailability in preclinical species and humans. Intestinal uptake transporters, such as organic anion transporting polypeptides (OATPs), can facilitate the uptake of drugs that are substrates and hence increase p.o. absorption. The present study was designed to determine whether the intestinal Oatps are involved in absorption of these macrolides. AZI or CLARI was dosed p.o. to Sprague-Dawley rats after p.o. administration with vehicle or rifamycin SV (RIF), an OATP inhibitor. The p.o. exposures of AZI and CLARI were reduced 65 and 45%, respectively, when coadministered with an optimized RIF regimen. The p.o. RIF had no affect on the total blood clearance of these macrolides and most likely did not cause induction of metabolizing enzymes and/or transporters. Therefore, the results suggest that inhibition of an RIF-sensitive uptake transporter such as Oatp along the rat gastrointestinal tract was responsible for reduced p.o. exposure of AZI and CLARI. In addition, AZI and CLARI caused inhibition of taurocholate uptake in rat Oatp1a5-transfected Madin-Darby canine kidney cell monolayers. The in vitro and in vivo results suggest that the intestinal Oatps are involved in the p.o. absorption of AZI and CLARI in the rat.

[ARTICLES] Aryl Hydrocarbon Receptor-Dependent Induction of Flavin-Containing Monooxygenase mRNAs in Mouse Liver

2008-11-18

Flavin-containing monooxygenases (FMOs) are important in detoxication but generally are considered not to be inducible by xenobiotics. Our recent microarray studies revealed induction of FMO2 and FMO3 mRNAs by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in liver of mice with wild-type aryl hydrocarbon receptor (AHR) but not in Ahr-null mice. The aim of the present study was to delineate mechanisms of FMO regulation. In adult male mice, basal FMO3 mRNA is low but was induced 6-fold at 4 h and 6000-fold at 24 h. The ED50 was approximately 1 µg/kg for FMO2 and FMO3, similar to that for the classic AHR-regulated gene, Cyp1a1. In adult female mice basal FMO3 mRNA is high and was not induced at 4 h but was elevated 8-fold at 24 h. FMO5 mRNA was significantly down-regulated by TCDD in both male and female adult mice. Juvenile mice show no sex difference in response to TCDD; FMO3 was induced 4 to 6-fold by TCDD in both sexes. Chromatin immunoprecipitation demonstrated recruitment of AHR and aryl hydrocarbon nuclear translocator proteins to Fmo3 regulatory regions, suggesting that induction by TCDD is a primary AHR-mediated event. Although FMO2 and FMO3 mRNAs were highly induced by TCDD in adult males, overall FMO catalytic activity increased only modestly. In contrast to the striking up-regulation of FMO2 and FMO3 in mouse liver, TCDD has little effect on FMO mRNA in rat liver. However, FMO2 and FMO3 mRNAs were highly induced in transgenic mice that express wild-type rat AHR, indicating that lack of induction in rat is not due to an incompetent AHR in this species.

[ARTICLES] A Double Transgenic Mouse Model Expressing Human Pregnane X Receptor and Cytochrome P450 3A4

2008-11-18

Cytochrome P450 3A4 (CYP3A4), the most abundant human cytochrome P450 in liver, participates in the metabolism of ~50% of clinically used drugs. The pregnane X receptor (PXR), a member of the nuclear receptor superfamily, is the major activator of CYP3A4 transcription. However, because of species differences in response to PXR ligands, it is problematic to use rodents to assess CYP3A4 regulation and function. The generation of double transgenic mice expressing human PXR and CYP3A4 (TgCYP3A4/hPXR) would provide a solution to this problem. In the current study, a TgCYP3A4/hPXR mouse model was generated by bacterial artificial chromosome transgenesis in Pxr-null mice. In TgCYP3A4/hPXR mice, CYP3A4 was strongly induced by rifampicin, a human-specific PXR ligand, but not by pregnenolone 16-carbonitrile, a rodent-specific PXR ligand. Consistent with CYP3A expression, hepatic CYP3A activity increased ~5-fold in TgCYP3A4/hPXR mice pretreated with rifampicin. Most antihuman immunodeficiency virus protease inhibitors are CYP3A substrates and their interactions with rifamycins are a source of major concern in patients coinfected with human immunodeficiency virus and Mycobacterium tuberculosis. By using TgCYP3A4/hPXR mice, human PXR-CYP3A4-mediated rifampicin-protease inhibitor interactions were recapitulated, as the metabolic stability of amprenavir, nelfinavir, and saquinavir decreased 52, 53, and 99%, respectively, in the liver microsomes of TgCYP3A4/hPXR mice pretreated with rifampicin. In vivo, rifampicin pretreatment resulted in an ~80% decrease in the area under the serum amprenavir concentration-time curve in TgCYP3A4/hPXR mice. These results suggest that the TgCYP3A4/hPXR mouse model could serve as a useful tool for studies on CYP3A4 transcription and function in vivo.

[ARTICLES] Confirmation That Cytochrome P450 2C8 (CYP2C8) Plays a Minor Role in (S)-(+)- and (R)-(-)-Ibuprofen Hydroxylation in Vitro

2008-11-18

Various groups have sought to determine the impact of CYP2C8 genotype (and CYP2C8 inhibition) on the pharmacokinetics (PK) of ibuprofen (IBU) enantiomers. However, the contribution of cytochrome P450 2C8 (CYP2C8) in human liver microsomes (HLMs) has not been reported. Therefore, in vitro cytochrome P450 (P450) reaction phenotyping was conducted with selective inhibitors of cytochrome P450 2C9 (CYP2C9) and CYP2C8. In the presence of HLMs, sulfaphenazole (CYP2C9 inhibitor), and anti-CYP2C9 monoclonal antibodies (mAbs) inhibited (73–100%) the 2- and 3-hydroxylation of both IBU enantiomers (1 and 20 µM). At a higher IBU concentration (500 µM), the same inhibitors were less able to inhibit the 2-hydroxylation of (S)-(+)-IBU (32–52%) and (R)-(-)-IBU (30–64%), whereas the 3-hydroxylation of (S)-(+)-IBU and (R)-(-)-IBU was inhibited 66 to 83 and 70 to 89%, respectively. In contrast, less inhibition was observed with montelukast (CYP2C8 inhibitor, ≤35%) and anti-CYP2C8 mAbs (≤24%) at all concentrations of IBU. When (S)-(+)-IBU and (R)-(-)-IBU (1 µM) were incubated with a panel of recombinant human P450s, only CYP2C9 formed appreciable amounts of the hydroxy metabolites. At a higher IBU enantiomer concentration (500 µM), additional P450s catalyzed 2-hydroxylation (CYP3A4, CYP2C8, CYP2C19, CYP2D6, CYP2E1, and CYP2B6) and 3-hydroxylation (CYP2C19). When the P450 reaction phenotype and additional clearance pathways are considered (e.g., direct glucuronidation and chiral inversion), it is concluded that CYP2C8 plays a minor role in (R)-(-)-IBU (<10%) and (S)-(+)-IBU (~13%) clearance. By extension, one would not expect CYP2C8 inhibition (and genotype) to greatly affect the pharmacokinetic profile of either enantiomer. On the other hand, CYP2C9 inhibition and genotype are expected to have an impact on the PK of (S)-(+)-IBU.

[ARTICLES] Pharmacokinetics, Distribution, Metabolism, and Excretion of Deferasirox and Its Iron Complex in Rats

2008-11-18

Deferasirox (Exjade, ICL670, CGP72670) is an iron-chelating drug for p.o. treatment of transfusional iron overload in patients with β-thalassemia or sickle cell disease. The pharmacokinetics and disposition of deferasirox were investigated in rats. The animals received single intravenous (10 mg/kg) or p.o. (10 or 100 mg/kg) doses of ¹⁴C-radiolabeled deferasirox. Biological samples were analyzed for radioactivity (liquid scintillation counting, quantitative whole-body autoradioluminography), for deferasirox and its iron complex [high-performance liquid chromatography (HPLC)/UV], and for metabolites (HPLC with radiodetection, liquid chromatography/mass spectrometry, ¹H and ¹³C NMR, and two-dimensional NMR techniques). At least 75% of p.o.-dosed deferasirox was absorbed. The p.o. bioavailability was 26% at the 10 mg/kg dose and showed an overproportional increase at the 100 mg/kg dose, probably because of saturation of elimination processes. Deferasirox-related radioactivity was distributed mainly to blood, excretory organs, and gastrointestinal tract. Enterohepatic recirculation of deferasirox was observed. No retention occurred in any tissue. The placental barrier was passed to a low extent. Approximately 3% of the dose was transferred into the breast milk. Excretion of deferasirox and metabolites was rapid and complete within 7 days. Key clearance processes were hepatic metabolism and biliary elimination via multidrug resistance protein 2. Deferasirox, iron complex, and metabolites were excreted largely via bile and feces (total ≥90%). Metabolism included glucuronidation at the carboxylate group (acyl glucuronide M3) and at phenolic hydroxy groups, as well as, to a lower degree, cytochrome P450-catalyzed hydroxylations. Two hydroxylated metabolites (M1 and M2) were administered to rats and were shown not to contribute substantially to iron elimination in vivo.

[ARTICLES] 2-Diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) Revisited: Comparative Cytochrome P450 Inhibition in Human Liver Microsomes by SKF525A, Its Metabolites, and SKF-Acid and SKF-Alcohol

Franklin, M. R., Hathaway, L. B. — 2008-11-18

When incubated with human liver microsomes, 2-diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) undergoes cytochrome P450 (P450)-dependent oxidative N-deethylation to the secondary amine metabolite 2-ethylaminoethyl-2,2-diphenylvalerate (SKF8742). P450-selective inhibitors indicated CYP3As catalyzed this reaction, and the deethylation rate correlated best with the CYP3A activity across a range of human liver microsomes. SKF525A and its metabolite and primary amine analog all inhibited CYP2B6-, CYP2C9-, CYP2C19-, CYP2D6-, and CYP3A-selective reactions to varying degrees but had little effect on CYP1A2, CYP2A6, and CYP2E1 reactions. Only the inhibition of CYP3A showed major enhancement when the inhibitors were preincubated with NADPH-fortified microsomes, and the extent of metabolic intermediate (MI) complex formation approximated typical CYP3A content. Two "lost with time" SKF525A derivatives devoid of the ethylamine moiety, 2,2-diphenylpropylethanol (SKF-Alcohol) and 2,2-diphenylpropylacetic acid (SKF-Acid) did not form an MI complex and were identified as selective inhibitors of CYP2C9. Although without detectable metabolism, their CYP2C9 inhibition fitted best with a competitive mechanism. Thus, not all the human P450s are inhibited by SKF525A and related compounds, and the mechanisms contributing to those that are inhibited vary with the isoform. P450 MI-complex formation only seems to play a role with CYP3As.

[ARTICLES] Interindividual Variation in Relative CYP1A2/3A4 Phenotype Influences Susceptibility of Clozapine Oxidation to Cytochrome P450-Specific Inhibition in Human Hepatic Microsomes

Zhang, W. V., D'Esposito, F., Edwards, R. J., Ramzan, I., Murray, M. — 2008-11-18

The atypical antipsychotic drug clozapine (CLZ) is effective in a substantial number of patients who exhibit treatment-resistance to conventional agents. CYP1A2 is generally considered to be the major enzyme involved in the biotransformation of CLZ to its N-demethylated (norCLZ) and N-oxygenated (CLZ N-oxide) metabolites in liver, but several studies have also implicated CYP3A4. The present study assessed the interplay between these cytochrome P450s (P450s) in CLZ biotransformation in a panel of hepatic microsomal fractions from 14 individuals. The relative activity of P450s 1A2 and 3A4 in microsomes was found to be a major determinant of the relative susceptibility of norCLZ formation to inhibition by the P450-selective inhibitors fluvoxamine and ketoconazole. In contrast, the activity of CYP3A4 alone was correlated with the susceptibility of CLZ N-oxide formation to inhibition by these agents. These findings suggest that both P450s may be dominant CLZ oxidases in patients and that the relative activities of these enzymes may determine clearance pathways. In vivo assessment of CYP1A2 and CYP3A4 activities, perhaps by phenotyping approaches, could assist the optimization of CLZ dosage and minimize pharmacokinetic interactions with coadministered drugs.

[ARTICLES] Activation of the Aryl Hydrocarbon Receptor by the Calcium/Calmodulin-Dependent Protein Kinase Kinase Inhibitor 7-Oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic Acid (STO-609)

Monteiro, P., Gilot, D., Langouet, S., Fardel, O. — 2008-11-18

This study was designed to analyze the effects of the Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid) toward the aryl hydrocarbon receptor (AhR) pathway because Ca²⁺/calmodulin-dependent protein kinase (CaMK) I, known as a downstream CaMKK effector, has been recently shown to contribute to the AhR cascade. STO-609 failed to alter up-regulation of the AhR target CYP1A1 in response to the potent AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. STO-609, used at a 25 µM concentration known to fully inhibit CaMKK activity, was surprisingly found to markedly induce CYP1A1 expression and activity by itself in MCF-7 cells; it similarly up-regulated various other AhR target genes in human macrophages. STO-609-related CYP1A1 induction was prevented by chemical inhibition or small interfering RNA-mediated knockdown expression of AhR. Moreover, STO-609 was demonstrated to physically interact with the ligand-binding domain of AhR, as assessed by TCDD binding competition assay, and to induce AhR translocation to the nucleus. As already reported for AhR agonists, STO-609 triggered the increase of [Ca²⁺]_i and activation of CaMKI, whose inhibition through the use of the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester or the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), respectively, prevented STO-609-mediated CYP1A1 activity induction. Taken together, these results demonstrate that the CaMKK inhibitor STO-609 can act as an AhR ligand and, in this way, fully activates the Ca²⁺/CaMKI/AhR cascade. Such data, therefore, make unlikely any contribution of CaMKK activity to the AhR pathway and, moreover, suggest that caution may be required when using STO-609 as a specific inhibitor of CaMKKs.

[ARTICLES] Lacteal Secretion, Fetal and Maternal Tissue Distribution of Dasatinib in Rats

2008-11-18

Dasatinib [N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide; BMS-354825] is a potent and broad-spectrum kinase inhibitor used for the treatment of chronic myeloid leukemia and Philadelphia chromosome positive (Ph⁺) acute lymphoblastic leukemia. Dasatinib exhibited extensive lacteal secretion in Sprague-Dawley rats following a single p.o. dose of [¹⁴C]dasatinib (10 mg/kg, 300 µCi/kg). Radioactivity was detected through 72 h postdose, with a milk/plasma area under concentration-time curve from 0 to infinity (AUC_0-inf) ratio of approximately 25. The majority of the total radioactivity in milk was attributed to unchanged dasatinib. After a single dose of [¹⁴C]dasatinib to pregnant Sprague-Dawley rats at gestation day 18, radioactivity was extensively distributed in maternal tissues. The radioactivity detected by tissue excision or quantitative whole-body autoradiography was highest in adrenal gland, mammary tissue, lungs, kidneys, liver, and placenta. Compared with maternal tissues, a relatively low level of radioactivity was detected in fetal tissues. The concentrations of dasatinib-equivalents in fetal liver and kidneys were <13% of the respective maternal organs. The C_max of dasatinib-equivalents in fetal blood was approximately 39% of that in maternal blood; however, the AUC values were comparable. Fetal brain/blood ratios of C_max and AUC_0-inf were approximately 1.58 and 1.48, respectively, which were much greater than the maternal ratios of 0.12 and 0.13. In summary, dasatinib was extensively distributed in maternal tissues and secreted into milk, but its penetration into the adult brain was limited. Transporters may be involved in mediating dasatinib distribution in the adult rat, whereas in the fetus, tissue and blood exposures were similar, suggesting that distribution in the fetus is predominantly mediated by diffusion.

[ARTICLES] Structural Determinants of Substrate Specificity Differences between Human Multidrug Resistance Protein (MRP) 1 (ABCC1) and MRP3 (ABCC3)

Grant, C. E., Gao, M., DeGorter, M. K., Cole, S. P. C., Deeley, R. G. — 2008-11-18

Multidrug resistance proteins (MRPs) are members of the "C" branch of the ATP-binding cassette transporter superfamily. Human MRP1 transports a wide range of natural product drugs and structurally diverse conjugated and unconjugated organic anions. Its closest relative is MRP3. Despite their structural similarity, the homologs differ substantially in their substrate specificity. It is noteworthy that MRP1 transports glutathione (GSH) and GSH conjugates and displays GSH-stimulated transport of a number of unconjugated and conjugated compounds. In contrast, MRP3 does not transport GSH and is a poor transporter of GSH conjugates. However, both proteins transport glucuronide conjugates, such as 17β-estradiol 17-(β-d-glucuronide). We have constructed a series of MRP1/MRP3 hybrids and used them to identify a region of MRP1 that is critical for binding and transport of GSH conjugates such as leukotriene C₄ (LTC₄). Substitution of this region encompassing transmembrane helices 8 and 9 and portions of cytoplasmic loops 4 and 5 of MRP1 with the equivalent region of MRP3 eliminated LTC₄ transport. Transport of other substrates was either unaffected or enhanced. We identified three residues in this region: Tyr⁴⁴⁰, Ile⁴⁴¹, and Met⁴⁴³, mutation of which differentially affected transport. It is noteworthy that substitution of Tyr⁴⁴⁰ with Phe, as found in MRP3, reduced LTC₄ and GSH-stimulated estrone-3-sulfate transport without affecting transport of other substrates tested. The mutation increased the K_m for LTC₄ 5-fold and substantially reduced photolabeling of MRP1 by both [³H]LTC₄ and the GSH derivative, azidophenacyl-[³⁵S]GSH. These results suggest that Tyr⁴⁴⁰ makes a major contribution to recognition of GSH and the GSH moiety of conjugates such as LTC₄.

[ARTICLES] Key Residues Controlling Phenacetin Metabolism by Human Cytochrome P450 2A Enzymes

DeVore, N. M., Smith, B. D., Urban, M. J., Scott, E. E. — 2008-11-18

Cytochrome P450s (P450s) metabolize a large number of diverse substrates with specific regio- and stereospecificity. A number of compounds, including nicotine, cotinine, and aflatoxin B₁, are metabolites of the 94% identical CYP2A13 and CYP2A6 enzymes but at different rates. Phenacetin and 4-aminobiphenyl were identified as substrates of human cytochromes P450 1A2 and 2A13 but not of CYP2A6. The purpose of this study was to identify active site amino acids that are responsible for CYP2A substrate specificity using phenacetin as a structural probe. Ten amino acid residues that differ in the CYP2A13 and CYP2A6 active sites were exchanged between the two enzymes. Phenacetin binding revealed that the six substitution, CYP2A13 S208I, A213S, F300I, A301G, M365V, and G369S decreased phenacetin affinity. Although incorporation of individual CYP2A13 residues into CYP2A6 had little effect on this enzyme's very low levels of phenacetin metabolism, the combination of double, triple, and quadruple substitutions at positions 208, 300, 301, and 369 increasingly endowed CYP2A6 with the ability to metabolize phenacetin. Enzyme kinetics revealed that the CYP2A6 I208S/I300F/G301A/S369G mutant protein O-deethylated phenacetin with a K_m of 10.3 µM and a k_cat of 2.9 min^-1, which compare very favorably with those of CYP2A13 (K_m of 10.7 µM and k_cat of 3.8 min^-1). A 2.15 Å crystal structure of the mutant CYP2A6 I208S/I300F/G301A/S369G protein with phenacetin in the active site provided a structural rationale for the differences in phenacetin metabolism between CYP2A6 and CYP2A13.

[ARTICLES] N-(4-[2-(1,2,3,4-Tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine Carboxamide (GF120918) As a Chemical ATP-Binding Cassette Transporter Family G Member 2 (Abcg2) Knockout Model to Study Nitrofurantoin Transfer into Milk

Wang, L., Leggas, M., Goswami, M., Empey, P. E., McNamara, P. J. — 2008-11-18

Genetic knockout mice studies suggested ATP-binding cassette transporter family G member 2 (ABCG2)/Abcg2 translocates nitrofurantoin at the mammary-blood barrier, resulting in drug accumulation in milk. The purpose of this study was to establish the role of Abcg2 in nitrofurantoin accumulation in rat milk using N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) as a "chemical knockout" equivalent. The inhibitory effect of GF120918 was verified in Madin-Darby canine kidney II cells stably expressing rat Abcg2 with Hoechst 33342 and nitrofurantoin flux in Transwells. Nitrofurantoin was infused (0.5 mg/h) in the absence and presence of GF120918 (10 mg/kg in dimethyl sulfoxide) to Sprague-Dawley lactating female rats using a balanced crossover design. Administration of GF120918 increased nitrofurantoin concentration in serum (from 443 ± 51 to 650 ± 120 ng/ml) and decreased concentration in milk (from 18.1 ± 0.9 to 1.9 ± 1.2 µg/ml), resulting in corresponding mean values for milk to serum concentration ratio (M/S) of 41.4 ± 19.1 versus 3.04 ± 2.27 in the absence and presence of GF120918 (p < 0.05), respectively. There was a decrease in systemic clearance with GF120918 (2.8 ± 0.5 l/h/kg) compared with vehicle controls (4.1 ± 0.5 l/h/kg; p < 0.05). Western blot analysis revealed good expression of Abcg2 and no P-glycoprotein (P-gp) expression in mammary gland, whereas immunohistochemistry confirmed the apical expression of Abcg2 in lactating mammary gland epithelia. Nitrofurantoin active transport into rat milk can be inhibited by GF120918 resulting in a 10-fold lower M/S. Although GF120918 inhibits both Abcg2 and P-gp, the high expression of Abcg2 and the absence of detectable P-gp expression in lactating mammary gland validate an important role for Abcg2 in nitrofurantoin accumulation in rat milk. GF120918 is particularly useful as a rat chemical knockout model to establish ABCG2's role in drug transfer into milk during breastfeeding.