There are two kinds of β-oxidation systems of fatty acids in mitochondria and peroxisomes in animal liver cells. These β-oxidation systems may play different physiological roles in the cell. Peroxisomal β-oxidation system has been demonstrated to participate in the catabolism of intarcellular acyl compounds such as very long chain fatty acids, long chain dicarboxylic acids and bile acid precursors in addition to fatty acids. The difference of functions between mitochondrial and peroxisomal β-oxidation systems is mainly due to the difference of characteristics of enzymes participating in the β-oxidation in both organella. We have studied the β-oxidation of xenobiotic acyl compounds and found that the peroxisomal β-oxidation is involved in the chain-shortening of acyl side chains of several compounds. In the present review, the author describes the comparison between peroxisomal and mitochondrial β-oxidation of phenylfatty acids (PFAs), oxidative chain shortening of N-(α-methylbenzyl)azelaamic acid (C9) as a specific substrate for the peroxisomal β-oxidation system, application of C9 which is a specific substrate for peroxisomal β-oxidation system for diagnosis of peroxisome disorders and participation of peroxisomal β-oxidation system in the metabolic activation of prodrugs, YNK-01, by peroxisomal β-oxidation system.
Nine different antibodies against P450 isoforms were prepared using purified cytochrome P450s (P450) expressed in E. coli. Purified isozymes were injected into rabbits to raise specific antibody. The resulting antibodies were characterized for their specificity and sensitivity through each particular P450 enzyme-mediated probe reaction. Anti-CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 antibodies proved to be strong immunoinhibitors with inhibitory effects specific to their corresponding antigen. Antiserum derived from the CYP2C19-immunized rabbits was reacted with CYP2C9 as well as CYP2C19 and immunoabsorbed with membrane-bound CYP2C9 expressed in E. coli. Antibody specific for CYP2C19 was obtained. Anti-CYP2C19 together with the anti-CYP2C8 and anti-CYP2C9 can be very useful for determining the contribution of a particular P450 in the metabolism of a drug. The developed inhibitory antibodies will serve as in vitro-specific tools for evaluating the quantitative contribution of individual P450 enzymes to drug metabolism.
In the present study, human cytochrome P450 (CYP) forms involved in producing the primary metabolites of NE-100 were identified. Major metabolites of NE-100 in human liver microsomes (HLM) were N-depropylation of NE-100 (NE-098), p-hydroxylation of phenyl group of NE-100 (NE-152), m-hydroxylation of phenyl group of NE-100 (NE-163) and O-demethylation of NE-100 (NE-125). Judging from the correlation and inhibition studies, NE-125 and NE-152+163mix formations were predominantly mediated by CYP2D6 and NE-098 formation was mediated by multiple CYP forms at a low NE-100 concentration (0.1 μM) in the HLM. According to relative activity factor (RAF) approaches, all these reactions were predominantly catalyzed by CYP2D6 at a substrate concentration assuming a plasma level of NE-100 (Km>>S) in case of the human liver. Depending on the increase in NE-100 concentrations, the rate of contribution for NE-098 and NE-152+163mix formations increased in CYP3A4, although the predominant contribution of CYP2D6 for NE-125 formation did not change. In human intestinal microsomes (HIM), NE-100 was mainly metabolized to NE-098 and NE-152+163mix by CYP3A4. The intrinsic clearance for their formations in HIM was 3.2 and 14.9 times less than those in HLM, respectively, and no formation of NE-125 was observed in HIM. These results strongly suggest that CYP2D6 is the predominant form for NE-100 metabolism in the human liver in in vivo conditions (Km>>S) and the liver plays a more important role than does the small intestine in the first pass metabolism.
To elucidate the mechanism of the interaction of basic drugs with Na+-dependent L-alanine absorption from the small intestine, we investigated the effect of imipramine on the transport of L-alanine via system B0, which is thought to be one of the main Na+-dependent systems for intestinal absorption of short-chain neutral amino acids, including L-alanine. The uptake of L-alanine by cells that express hATB0 (human amino acid transporter B0) was inhibited in the presence of imipramine. The Eadie-Hofstee plot showed that the inhibition was not competitive with the substrate (L-alanine) but competitive with Na+. When rat intestinal brush border membrane vesicles were used, several basic drugs had inhibitory effects. Inhibition was also observed in intestinal absorption evaluated by an in situ single-pass perfusion technique. However, the potencies of inhibition were different. The potency of inhibition was dependent on the lipophilicity of the drugs.
Hitherto three variant forms of ABCG2 have been documented on the basis of their amino acid moieties (i.e., Arg, Gly, and Thr) at the position 482. In the present study, we have generated those variants of ABCG2 by site-directed mutagenesis and expressed them in Sf9 insect cells. The apparent molecular weight of the expressed ABCG2 variants was 130, 000 under non-reductive conditions, whereas it was reduced to 65, 000 by treatment with mercaptoethanol. It is suggested that ABCG2 exists in the plasma membrane of Sf9 cells as a homodimer bound through cysteinyl disulfide bond(s). Both ATPase activity and drug transport of ABCG2 variants were examined by using plasma membrane fractions prepared from ABCG2-overexpressing Sf9 cells. The ATPase activity of the plasma membrane expressing ABCG2 (Gly-482) was significantly enhanced by prazosin. In contrast, ABCG2 (Arg-482) transports [3H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly-482 and Thr-482). It is strongly suggested that the amino acid moiety at the position of 482 is critical for the substrate specificity of ABCG2.
Objective: To describe the factors affecting pharmacokinetics of telmisartan, an angiotensin II receptor antagonist, a population pharmacokinetic (PPK) model has been developed based upon the data collected from healthy volunteers and hypertensive patients. Methods: A total of 1566 plasma samples were collected from 20 healthy volunteers and 129 hypertensive patients, together with the demographic background. The data were analyzed by the NONMEM program using two-compartment model with first-order absorption. The robustness of the obtained PPK model was validated by the bootstrapping resampling method. Results: The oral clearance (CL/F) was found to be associated with age, dose and alcohol consumption, but neither related to serum creatinine nor smoking history. The volume of distribution for the central compartment was related to age and dose, and the volume of distribution for the peripheral compartment was related to body weight and gender. The absorption rate constant (Ka) and the absorption lag time were described as function of dose. The CL/F decreased with advanced age. The CL/F decreased and Ka increased with higher dose, reflecting the super-proportional increase in the plasma levels of telmisartan. The AUC and Cmax values predicted by the present PPK model were well consistent with the observed values. The means of parameter estimates obtained with 200 bootstrap replicates were within 95-111% of the final parameter estimates from the original data set. Conclusion: A PPK model for telmisartan developed here well described the individual variability and exposure, and robustness of the model has been validated by the bootstrapping method.