Wataru Takasaki

Inhibition of in vitro metabolism of simvastatin by itraconazole in humans and prediction of in vivo drug-drug interactions

AbstractPurpose. To evaluate an interaction between simvastatin and itraconazole in in vitro studies and to attempt a quantitative prediction of in vivo interaction in humans. Methods. The inhibitory effect of itraconazole on simvastatin metabolism was evaluated using human liver microsomes and the Ki values were calculated for the unbound drug in the reaction mixture. A physiologically-based pharmacokinetic model was used to predict the maximum in vivo drug-drug interaction. Results. Itraconazole competitively inhibited the metabolism of simvastatin to M-1 and M-2 with Ki values in the nM range. The area under the curve (AUC) of simvastatin after concomitant dosing with itraconazole was predicted to increase ca. 84-101-fold compared with that without administration of itraconazole. Taking into consideration the fact that this method predicts the maximum interaction, this agrees well with the clinical observation of a 19-fold increase. A similar prediction, based on the Ki value without taking into account the drug adsorption to microsomes, led to an underevaluation of the interaction. Conclusions. It was demonstrated that the competitive inhibition of CYP3A4-mediated simvastatin metabolism by itraconazole is the main cause of the drug interaction and that a Ki value corrected for drug adsorption to microsomes is the key factor in quantitatively predicting the maximum in vivo drug interactions.

Immunoaffinity purification of 11-dehydro-thromboxane B2 from human urine and plasma for quantitative analysis by radioimmunoassay

11-Dehydro-thromboxane B2 is now considered to be a reliable parameter of thromboxane A2 formation in vivo. An immunoaffinity purification method was developed for radioimmunoassay of this compound contained in human urine and plasma. Monoclonal anti-11-dehydro-thromboxane B2 antibody was prepared and coupled to BrCN-activated Sepharose 4B. Human urine or plasma was applied to a disposable column of the immobilized antibody. After the column was washed with water, 11-dehydro-thromboxane B2 was eluted with methanol/water (95/5) with a recovery of more than 90%. The purified extract was subjected to a radioimmunoassay utilizing 11-[3H]dehydro-thromboxane B2 methyl ester and the monoclonal anti-11-dehydro-thromboxane B2 antibody. The detection range of the assay was 10-600 fmol (IC50 = 90 fmol). The cross-reactivities of the antibody with thromboxane B2, 2,3-dinor-thromboxane B2, and other arachidonate metabolites were less than 0.05%. These compounds were efficiently separated from 11-dehydro-thromboxane B2 by the immunoaffinity purification. This procedure also allowed the separation of 11-dehydro-thromboxane B2 from unidentified urinary and plasma substances which interfered with the radioimmunoassay. Validity of the results obtained by the radioimmunoassay was confirmed by GC/MS employing selected ion monitoring for quantification.

Characterization of phenotypes in Gstm1-null mice by cytosolic and in vivo metabolic studies using 1,2-dichloro-4-nitrobenzene.

Glutathione S-transferase Mu 1 (GSTM1) has been regarded as one of the key enzymes involved in phase II reactions in the liver, because of its high expression level. In this study, we generated mice with disrupted glutathione S-transferase Mu 1 gene (Gstm1-null mice) by gene targeting, and characterized the phenotypes by cytosolic and in vivo studies. The resulting Gstm1-null mice appeared to be normal and were fertile. Expression analyses for the Gstm1-null mice revealed a deletion of Gstm1 mRNA and a small decrease in glutathione S-transferase alpha 3 mRNA. In the enzymatic study, GST activities toward 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) in the liver and kidney cytosols were markedly lower in Gstm1-null mice than in the wild-type control. Gstm1-null mice had GST activities of only 6.1 to 21.0% of the wild-type control to DCNB and 26.0 to 78.6% of the wild-type control to CDNB. After a single oral administration of DCNB to Gstm1-null mice, the plasma concentration of DCNB showed larger AUC0–24 (5.1–5.3 times, versus the wild-type control) and higher Cmax (2.1–2.2 times, versus the wild-type control), with a correspondingly lower level of glutathione-related metabolite (AUC0–24, 9.4–17.9%; and Cmax, 9.7–15.6% of the wild-type control). In conclusion, Gstm1-null mice showed markedly low ability for glutathione conjugation to DCNB in the cytosol and in vivo and would be useful as a deficient model of GSTM1 for absorption, distribution, metabolism, and excretion/toxicology studies.

Generation and functional characterization of mice with a disrupted glutathione S-transferase, theta 1 gene.

Glutathione S-transferase (GST) theta 1 (GSTT1) has been regarded as one of the key enzymes involved in phase II reactions because of its unique substrate specificity. In this study, we generated mice with the disrupted Gstt1 gene (Gstt1-null mice) by gene targeting and analyzed the metabolic properties in cytosolic and in vivo studies. The resulting Gstt1-null mice failed to express the Gstt1 mRNA and GSTT1 protein by reverse transcriptase-polymerase chain reaction analysis and two-dimensional fluorescence difference gel electrophoresis/mass spectrometry analysis, respectively. However, the Gstt1-null mice appeared to be normal and were fertile. In an enzymatic study using cytosolic samples from the liver and kidney, GST activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), dichloromethane (DCM), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was markedly lower in Gstt1-null mice than in the wild-type controls, despite there being no difference in GST activity toward 1-choloro-2,4-dinitrobenzene between Gstt1-null mice and the wild-type controls. Gstt1-null mice had GST activity of only 8.7 to 42.1% of the wild-type controls to EPNP, less than 2.2% of the wild-type controls to DCM, and 13.2 to 23.9% of the wild-type controls to BCNU. Plasma BCNU concentrations after a single i.p. administration of BCNU to Gstt1-null mice were significantly higher, and there was a larger area under the curve5–60 min (male, 2.30 times; female, 2.28 times, versus the wild-type controls) based on the results. In conclusion, Gstt1-null mice would be useful as an animal model of humans with the GSTT1-null genotype.

Stereoselective determination of the active metabolites of a new anti-inflammatory agent (CS-670) in human and rat plasma using antibody-mediated extraction and high-performance liquid chromatography

The main metabolites of (+-)-2-[4-(2-oxocyclohexylidenemethyl)phenyl]propionic acid (CS-670), a new pro-drug anti-inflammatory agent of the 2-arylpropionic acid type, have one or two chiral centres arising from reduction of the oxocyclohexylidene moiety in addition to an original chiral centre in the propionic acid moiety. To determine these metabolites stereoselectively, antibody-mediated extraction was investigated as a stereoselective clean-up method prior to chiral HPLC. Immunoglobulin G, which recognizes each stereoisomeric cyclohexanol moiety, was coupled to cyanogen bromide-activated Sepharose 4B to prepare re-usable immobilized antibody, and its specificity was improved by examination of a washing process after charging of samples. Plasma extracted with the immobilized antibody column was derivatized with a chiral reagent to separate the enantiomers of the propionic acid moiety by HPLC. This newly developed analytical method clarified the stereoselective biotransformation of the pro-drug to pharmacologically active forms in humans and rats related to reduction of the oxocyclohexylidene moiety and chiral inversion in the propionic acid moiety.

Methemoglobinemia Induced by 1,2-Dichloro-4-nitrobenzene in Mice with a Disrupted Glutathione S-Transferase Mu 1 Gene

A specific substrate to Mu class glutathione S-transferase (GST), 1,2-dichloro-4-nitrobenzene (DCNB), was administered to mice with a disrupted GST Mu 1 gene (Gstm1-null mice) to investigate the in vivo role of murine Gstm1 in toxicological responses to DCNB. A single oral administration of DCNB at doses of 500 and 1000 mg/kg demonstrated a marked increase in blood methemoglobin (MetHB) in Gstm1-null mice but not in wild-type mice. Therefore, Gstm1-null mice were considered to be more predisposed to methemoglobinemia induced by a single dosing of DCNB. In contrast, 14-day repeated-dose studies of DCNB at doses up to 600 mg/kg demonstrated a marked increase in blood MetHB in both wild-type and Gstm1-null mice. However, marked increases in the blood reticulocyte count, relative spleen weight, and extramedullary hematopoiesis in the spleen were observed in Gstm1-null mice compared with wild-type mice. In addition, microarray and quantitative reverse transcription-polymerase chain reaction analyses in the spleen showed exclusive up-regulation of hematopoiesis-related genes in Gstm1-null mice. These changes were considered to be adaptive responses to methemoglobinemia and attenuated the higher predisposition to methemoglobinemia observed in Gstm1-null mice in the single-dose study. In toxicokinetics monitoring, DCNB concentrations in plasma and blood cells were higher in Gstm1-null mice than those in wild-type mice, resulting from the Gstm1 disruption. In conclusion, it is suggested that the higher exposure to DCNB due to Gstm1 disruption was reflected in methemoglobinemia in the single-dose study and in adaptive responses in the 14-day repeated-dose study.

Enzyme immunoassay of human plasma 11-dehydrothromboxane B2

11-Dehydrothromboxane B2 (11-dhTXB2) is a proposed marker compound for thromboxane A2 formed in vivo. An enzyme immunoassay was established for determination of the plasma concentration of this compound. The assay was based on a horseradish peroxidase-linked immunoassay utilizing polyclonal anti-11-dhTXB2 antibody obtained from a rabbit, and enabled determination of 11-dhTXB2 in the range of 2 to 500 pg/tube with an IC50 of 36 pg. The cross-reactivities with TXB2, 2,3-dinor-TXB2 and other prostanoids were less than 0.05%. Validity of the enzyme immunoassay was confirmed by a radioimmunoassay utilizing a monoclonal antibody. The plasma 11-dhTXB2 was immunoaffinity-purified by one step using an immobilized monoclonal antibody. The mean plasma level of 11-dhTXB2 in six male volunteers was 4.0 +/- 0.3 pg/ml by this enzyme immunoassay.

Enzyme-linked immunosorbent assay of pravastatin, a HMG-CoA reductase inhibitor, in human plasma.

An enzyme-linked immunosorbent assay (ELISA) was developed for sensitive and specific determination of pravastatin (PS) sodium, a HMG-CoA reductase inhibitor. Preparation of immunogens to obtain antisera was carried out using chemically modified PS; beta-alanine derivative of PS (for ELISA-1) and 5-deoxy- PS (for ELISA-2) were linked to bovine serum albumin via its terminal carboxylic acid by the N-succinimidyl ester method, to avoid intramolecular lactonization of PS. Enzyme-labeled antigens were prepared similarly by coupling with horseradish peroxidase, and were used by homogeneous combination of antisera. The enzymic activity was determined using a microtiter plate coated with second antibody and tetramethylbenzidine as a chromogenic substrate. Both of the ELISA systems enabled the determination of PS in a range of 5 to 500 pg/well, with an IC50 of 36 to 130 pg/well. Cross-reactivties with main metabolites in plasma, which differed from PS in decaline moiety, were less than a few percent. When ELISA-1 was applied to the determination of PS in human plasma directly after dilution with the ELISA buffer, the detection limit and the intra-assay coefficient (5 ng/ml of PS) were 500 pg/ml and 4.5%, respectively. Further, ELISA-1 was validated by gas chromatography-mass spectrometry with the determination of PS in human plasma after oral administration at a dose of 10 mg/body.

Novel derivatization and immunoextraction to improve microanalysis of 11-dehydrothromboxane B2 in human urine.

A new, highly selective procedure for the determination of 11-dehydrothromboxane B2 (11-dehydro-TXB2) in human urine is described. Following the addition of [19,19,20,20-2H4]11-dehydro-TXB2 as an internal standard, samples were extracted with an affinity column of anti-11-dehydro-TXB2. Conversion of the immunoextracted 11-dehydro-TXB2 into its 1-methyl ester-11-n-propylamide-9,12,15-tris-dimethylisopropylsilyl ether derivative was followed by gas chromatography-selected-ion monitoring. The mass spectrum of the 11-dehydro-TXB2 derivative was dominated by the base peak ion of [M-C3H7]+ at m/z 698, which accounted for more than 10% of the total ion current. A typical result showed that the immunoaffinity purification procedures provided an extremely clean alternative to more conventional methods of chromatographic fractionation, and that interfering substances from the urine matrix were almost entirely eliminated during the microanalysis.

Evaluation of hepatic glutathione transferase Mu 1 and Theta 1 activities in humans and mice using genotype information.

We investigated the impact of glutathione transferases Mu 1 (GSTM1)- and glutathione transferase Theta 1 (GSTT1)-null genotypes on hepatic GST activities in humans and compared the results with those of Gstm1- and Gstt1-null mice. In liver with GSTM1/Gstm1-null genotype, GST activity toward p-nitrobenzyl chloride (NBC) was significantly decreased in both humans and mice. In addition, in liver with GSTT1/Gstt1-null genotype, GST activity toward dichloromethane (DCM) was significantly decreased in both humans and mice. Therefore, null genotypes of GSTM1/Gstm1 and GSTT1/Gstt1 are considered to decrease hepatic GST activities toward NBC and DCM, respectively, in both humans and mice. This observation shows the functional similarity between humans and mice for GSTM1 and GSTT1 toward some substrates. In the case of NBC and DCM, Gst-null mice would be relevant models for humans with GST-null genotype. In addition, decreases in GST activities toward 1,2-dichloro-4-nitrobenzene, trans-4-phenyl-3-buten-2-one, and 1-chloro-2,4,-dinitrobenzene were observed in Gstm1-null mice, and a decrease in GST activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane was observed in Gstt1-null mice. However, an impact of GST-null genotypes on GST activities toward these substrates was not observed in humans. In the case of these mouse-specific substrates, Gst-null mice may be relevant models for humans regardless of GST genotype, because GST activities, which are higher in wild-type mice than in humans, were eliminated in Gst-null mice. This study shows that comparison of hepatic GST activities between humans and mice using genotype information would be valuable in using Gst-null mice as human models.