Evaluation of the inhibitory and induction potential of YM758, a novel If channel inhibitor, for human P450-mediated metabolism.

This study was designed to examine the in vitro metabolism of YM758, a novel cardiovascular agent, and to evaluate its potential to cause drug interactions and induction of CYP isozymes. After incubation with pooled human liver microsomes, YM758 was converted to two major metabolites (AS2036313-00, and YM-394111 or YM-394112). The formation of AS2036313-00, and YM-394111 or YM-394112 were mediated by CYP2D6 and CYP3A4, respectively, which was elucidated by using a bank of human liver microsomes and recombinant CYP enzymes in combination with the utilization of typical substrates and inhibitors. The Ki values of YM758 for midazolam, nifedipine, and metoprolol metabolism ranged from 59 to 340 microM, being much higher than the YM758 concentration in human plasma. The formation of AS2036313-00, and YM-394111 or YM-394112 was inhibited by quinidine and ketoconazole with Ki values of 140 and 0.24 microM, respectively, which indicates that YM758 metabolism may be affected by coadministration of strong CYP2D6 and 3A4 inhibitors in vivo, given the clinical plasma concentrations of quinidine and ketoconazole. After human hepatocytes were exposed to 10 microM YM758, microsomal activity and mRNA level for CYP1A2 were not induced while those for CYP3A4 were slightly induced. The tested concentration was much higher than that in human plasma, which suggests that the induction potential of YM758 is also negligible.

Effects of cocaine and its oxidative metabolites on mitochondrial respiration and generation of reactive oxygen species.

Cocaine is capable of producing severe hepatocellular necrosis in laboratory animals and humans. The mechanism of cocaine hepatotoxicity is not well understood, but appears to result from the actions of one or more N-oxidative metabolites of cocaine. Mitochondria have been proposed as critical cellular targets for cocaine toxicity, and previous studies have found depressed mitochondrial respiration and increased mitochondrial generation of reactive oxygen species (ROS) in animals treated with cocaine. To examine the potential role of cocaine N-oxidative metabolites in these effects, mitochondrial respiration and ROS generation were examined in isolated mouse mitochondria treated with cocaine and its N-oxidative metabolites-norcocaine, N-hydroxynorcocaine, and norcocaine nitroxide. Cocaine, in concentrations of 0.25 or 0.5 mM, had no effect on state 3 respiration, state 4 respiration, respiratory control ratio (RCR), or ADP/O ratio. Norcocaine (0.5 mM) inhibited state 3 respiration, and N-hydroxynorcocaine (0.5 mM) inhibited both state 3 and state 4 respiration. Norcocaine nitroxide had the greatest effect on mitochondrial respiration; the lower concentration (0.25 mM) completely inhibited both state 3 and state 4 respiration. Preincubation of mitochondria with cocaine or metabolites increased the inhibitory effect of norcocaine and N-hydroxynorcocaine, but not cocaine. Cocaine, norcocaine, and N-hydroxynorcocaine (0.1 mM) had no effect on ROS generation during state 3 respiration, and cocaine and norcocaine decreased ROS generation under state 4 conditions. Norcocaine nitroxide interfered with the fluorescence ROS assay and could not be assessed. The results suggest that the effects of cocaine on mitochondrial respiration are due to its N-oxidative metabolites. Inhibition of mitochondrial respiration by the N-oxidative metabolites of cocaine may be the underlying cause for observed ATP depletion and subsequent cell death.

Immunochemical detection of hepatic cocaine-protein adducts in mice.

Cocaine is capable of producing hepatic necrosis in laboratory animals and humans. Studies in mice indicate that N-oxidative metabolism of cocaine is required for hepatotoxicity and have suggested that toxicity may result from the adduction of proteins by cocaine-reactive metabolites. To aid in identifying protein targets for cocaine-reactive metabolites, an antibody was raised in rabbits immunized with cocaine linked via the tropane nitrogen to a carrier protein (bovine serum albumin). Hepatic proteins from cocaine-treated mice (ICR males, 50 mg of cocaine/kg of body weight, ip) and saline-treated controls were prepared from whole liver homogenate or following subcellular fractionation, and Western blot analyses of hepatic proteins using this antibody were conducted following one- and two-dimensional SDS-PAGE. Analysis of liver homogenate from cocaine-treated mice revealed major protein targets with approximate molecular masses of 20 kDa (pI = 6.0), 44 kDa (two proteins with pI's of 5.0 and 7.0), 52-54 kDa (pI = 4.5), and 64 kDa (pI = 5.5). These specific protein targets were shown to be localized in the mitochondria and microsomes. Several minor bands of immunoreactivity were also seen in mice treated with cocaine, but not in saline-treated controls. Pretreatment of mice with the P450 inhibitor SKF 525A diminished or eliminated the formation of these cocaine-protein adducts. Liver sections from cocaine-treated mice immunostained using the antibody indicated the presence of cocaine-adducted proteins in the centrilobular and midzonal regions of the lobule, corresponding to areas of hepatocyte swelling and necrosis. This study indicates that reactive metabolites from cocaine bind to discrete proteins in specific regions of the liver, consistent with a role for protein adduction in cocaine hepatotoxicity.

Liver toxicity from norcocaine nitroxide, an N-oxidative metabolite of cocaine.

The oxidative metabolism of cocaine to norcocaine nitroxide has been postulated to be essential for cocaine hepatotoxicity. The hepatic effects of norcocaine nitroxide have never been evaluated in vivo, however. In this study mice were administered norcocaine nitroxide i.p., and hepatotoxicity was assessed using serum alanine aminotransferase activities and microscopic examination of liver tissue. Hepatotoxicity of norcocaine nitroxide was dose-related; significant injury was detectable at doses of 20 to 30 mg/kg i.p., and severe hepatocellular necrosis was observed at doses of 40 and 50 mg/kg. Elevated serum alanine aminotransferase activities peaked between 12 and 18 hr after norcocaine nitroxide treatment. Electron microscopy revealed the presence of pronounced changes in cell morphology as early as 30 min after the norcocaine nitroxide dose. Pretreatment of mice with phenobarbital had no effect on the magnitude of hepatic injury but shifted the intralobular site of necrosis from the midzonal to the periportal region. Pretreatment with diazinon, an esterase inhibitor, increased norcocaine nitroxide-induced liver damage, whereas each of the P450 inhibitors SKF 525A, cimetidine, troleandomycin, ketaconazole and chloramphenicol significantly diminished norcocaine nitroxide hepatotoxicity. The results indicate that norcocaine nitroxide is hepatotoxic and suggest the involvement of P450 enzymes.

Synthesis and messenger ribonucleic acid expression of apolipoproteins E and A-I by the bovine corpus luteum during the estrous cycle and pregnancy.

In an attempt to characterize proteins secreted by the corpus luteum, explant cultures of luteal slices from cows taken on Days 3, 7, 11, 14, 17, and 19 of the estrous cycle, and Days 17, 88, 180, and > 240 of pregnancy were incubated with H-leucine for 24 h. Proteins in luteal-conditioned medium were separated by two-dimensional PAGE, transferred to polyvinylidene fluoride membrane, and subjected to N-terminal amino acid microsequencing. Microsequence analysis revealed that the bovine corpus luteum synthesized and released de novo synthesized apolipoproteins (Apo) E and A-I in culture during the estrous cycle and pregnancy. Release of Apo E was observed only on Day 3 of the estrous cycle. Release of Apo A-I was observed on Days 11, 14, 17, and 19 of the estrous cycle, and on all days of pregnancy examined. To demonstrate the presence of the appropriate mRNA and characterize the temporal relationship for these identified proteins, total RNA was isolated from corpora lutea on Days 2, 3, 7, 16, 17, and 20 of the estrous cycle, and on Days 17, 90, 170, 180, and 272 of pregnancy, and submitted to Northern and dot blot analysis. Apo E mRNA was expressed only on Days 2-3 of the estrous cycle and was not expressed on the other days of the cycle or during pregnancy. A single Apo E mRNA transcript about 1.0 kilobase (kb) in size was observed. Expression of Apo A-I mRNA was detected on all days of the estrous cycle and pregnancy examined. Apo A-I cDNA hybridized with a single mRNA transcript about 1.0 kb in size. Apo A-I mRNA levels did not differ among days of the estrous cycle, although higher levels of Apo A-I mRNA were observed during later stages of pregnancy. Serum concentrations of Apo A-I and progesterone were correlated across the estrous cycle but not during the prepartum period or after parturition. This study demonstrates for the first time that the corpus luteum synthesizes Apo E and Apo A-I and expresses their respective mRNAs. The pattern of expression of Apo E and Apo A-I mRNAs paralleled that of de novo synthesis of their respective proteins after incubation of luteal tissue with [H]leucine. The role of luteal apolipoproteins may involve an autocrine/paracrine function influencing luteal development, tissue remodeling, and steroidogenesis.