Heparin is an anionic macromolecular drug. It has been widely used as an anticoagulant, and numerous efforts to clarify the mechanism of its disposition in the body have been made to help expand its clinical applications, using its newly found biological activities, as well as to further improve its use in anticoagulant therapy. It has now been shown that heparin is taken up extensively not only by Kupffer cells but also by parenchymal cells in the liver, the major distribution organ, and a receptor-mediated endocytotic mechanism, which is shared by heparin analogs and various anionic macromolecules, is responsible for heparin uptake in both types of cells. Although the characteristics of the receptors for heparin in both cells have lots of similarities to those of scavenger receptors, the receptors in parenchymal cells do not accept acetylated low density lipoprotein (Ac-LDL) as a ligand, which is the only striking difference between them and major scavenger receptors. Although the receptors in Kupffer cells, which accept Ac-LDL as a ligand, may belong to class A scavenger receptors, this remains to be established. We therefore conclude at present that it is likely that novel scavenger-like receptors for heparin (heparin receptors) or unidentified scavenger receptors are responsible for heparin uptake in the liver.
1-Aminobenzotriazole (ABT) is widely used as a non-specific inhibitor of animal cytochrome P450 (CYP). In the present study, the inhibitory effect of ABT was investigated on drug oxidations catalyzed by human CYP isoforms. This inhibitory effect was compared with that of SKF-525A, another non-specific inhibitor, and ketoconazole, a potent inhibitor of CYP3A. Bacurovirus-expressed recombinant human CYP isoforms were used as an enzyme source. The specific activities for human CYP isoforms are: phenacetin O-deethylation, for CYP1A2; diclofenac 4'-hydroxylation, for CYP2C9; S-mephenytoin 4'-hydroxylation, for CYP2C19; bufuralol 1'-hydroxylation, for CYP2D6; chlorzoxazone 6-hydroxylation, for CYP2E1; testosterone 6β-hydroxylation, nifedipine oxidation, and midazolam 1'-hydroxylation, for CYP3A4. ABT inhibited both CYP1A2-dependent activity (Ki=330 μM) and CYP2E1-dependent activity (Ki=8.7 μM). In contrast, SKF-525A weakly inhibited CYP1A2-dependent activities (46% inhibition at 1200 μM) and CYP2E1-dependent activities (65% inhibition at 1000 μM). ABT exhibited the highest Ki value for CYP2C9-dependent diclofenac 4'-hydroxylation among those determined by this assay (Ki=3500 μM). Moreover, SKF-525A showed strong inhibition of CYP2D6-dependent bufuralol 1'-hydroxylation (Ki=0.043 μM). Ketoconazole inhibited all tested drug oxidations, however, its inhibitory effect on CYP1A2-dependent activities was very weak (50% inhibition at 120 μM). ABT, SKF-525A, and ketoconazole showed different selectivity and had a wide range of Ki values for the drug oxidations catalyzed by human CYP enzymes. Therefore, we conclude that inhibitory studies designed to predict the contribution of CYP enzymes to the metabolism of certain compounds should be performed using multiple CYP inhibitors, such as ABT, SKF-525A, and ketoconazole.
We previously established a method for assessing in vivo drug-metabolizing capacity by pharmacokinetic estimation of the quantity of cytochrome P450 (CYP) in vivo (PKCYP test), in which an apparent liver-to-blood free concentration gradient in vivo (qg) is introduced (Matsunaga et al., Jpn. J. Hosp. Pharm., 26: 492-504 (2000)). This method was applied to estimate the amount of CYP2C11 in rats treated with carbon tetrachloride (CCl4-treated rats). In this study, we estimated the amount of CYP1A2 in CCl4-treated rats by using acetanilide and caffeine as a probe and a model drug, respectively. In CCl4-treated rats, the total body clearance (CLtot) of acetanilide and caffeine was about one-fifth and one-eighth of that in control rats, respectively. In CCl4-treated rats, the amount of CYP1A2 was predicted as 0.60±0.06 nmol/kg from the clearance of acetanilide mediated by CYP1A2. Moreover, the clearance of caffeine mediated by CYP1A2 in CCl4-treated rats was estimated as 0.47±0.05 mL/min/kg by using the predicted amount of CYP1A2. The observed value was 0.44±0.03 mL/min/kg, and the predicted value was within the 95% confidence interval of the observed value. In conclusion, we have demonstrated that the PKCYP test can also be applied for estimating the amount of CYP1A2 in CCl4-treated rats.
Fluorouracil (5-FU) prodrug tegafur (FT) is used widely for treating cancer patients. It has been reported that CYP2A6 and thymidine phosphorylase (TP) are involved in the formation of 5-FU from FT. In this study, the relative contribution of cytochrome P450 (P450) to the formation of 5-FU from FT was assessed using human liver 9000 × g supernatant (S9) and hepatocytes, which contain both enzymes. Intrinsic clearances of 5-FU formation from FT by P450 (NADPH dependent) and TP (NADPH independent) in human liver S9 were 1.36 and 0.169 μL/min/mg protein, respectively. The formation of 5-FU from FT in human liver S9 was inhibited over 82% by 8-methoxypsoralen, a CYP2A6-selective inhibitor. The formation of 5-FU from FT was also evaluated in human hepatocytes, cells that not only exhibit P450 and TP activity but also have anabolic capacity. The results indicated that CYP2A6 played a major role in 5-FU formation, which was consistent with the results using human liver S9. Factors that can affect the level of CYP2A6 activity in patients, e.g., genetic polymorphism, should be considered when using FT for chemotherapy.
The present study was undertaken to characterize the transport of (3-methyl-His2) thyrotropin-releasing hormone ([3H]MeTRH) across the blood-brain barrier in mice and the effects of thyrotropin-releasing hormone (TRH) and its analogues (taltirelin and montirelin) on the transport and brain distribution. Integration plot analysis was used to calculate the influx clearance (CLin) of [3H]MeTRH after intravenous (i.v.) injection in mice. Furthermore, the capillary depletion method was performed to determine whether [3H]MeTRH crossed the blood-brain barrier. The effects of TRH and its analogues on the brain distribution of [3H]MeTRH were also examined by co-injection with the radioligand. The brain distribution of [3H]MeTRH and [14C]sucrose increased with the time after i.v. injection in mice, and the level of [3H]MeTRH was significantly higher than that of [14C]sucrose 5 min after the injection. The CLin value of [3H]MeTRH was significantly higher than that of [14C]sucrose, and the value of [3H]MeTRH was reduced by co-injection with unlabeled MeTRH. Also, capillary depletion showed that [3H]MeTRH was distributed largely in the brain parenchyma and this distribution was significantly inhibited by co-injection of TRH and montirelin but not taltirelin. The present study indicates that the transport of [3H]MeTRH into the brain may be via a saturable process.
The purpose of this study was to investigate the distribution of Grepafloxacin (GPFX), a new quinolone antimicrobial agent, in the lung epithelial lining fluid (ELF) and the alveolar macrophage (AM) in rats, which are potential infection sites in respiratory tract infections. We also aimed to clarify the mechanism governing the transferability of GPFX into the alveolus compartment from a kinetic point of view. The AUC ratios of ELF/plasma and AM/plasma after the oral administration of GPFX were 5.69 ± 1.00 and 352 ± 57, respectively, which were several-fold greater than those of ciprofloxacin (CPFX). Pharmacokinetic analyses of time profiles of GPFX concentrations in ELF and AM revealed that the influx clearance from plasma to ELF across the alveolar barrier is 5-fold greater than the efflux clearance from ELF. In addition, the permeability of GPFX across the cultured AM cell membrane was 7-fold and 11-fold greater than that of levofloxacin (LVFX) and CPFX, respectively. The extent of intracellular binding to AM cells (expressed as a constant (α)) was the greatest for GPFX, followed by CPFX and LVFX. There was a significant correlation between the α value and the partitioning to the immobilized artificial membrane (IAM) column, which consists of phospholipid residues covalently bound to silica. These results suggest that GPFX is highly distributed in ELF and AM, and that the high transferability of GPFX into ELF may be attributable to the existence of asymmetrical transport across the alveolar barrier. In addition, it was suggested that both rapid permeability across the AM cell membrane and avid binding to the membrane phospholipids may be responsible for the high accumulation of GPFX in AM.
We sequenced all exons and exon-intron junctions of the flavin-containing monooxygenase 3 (FMO3) gene from 27 Japanese individuals who are trimethylaminuria volunteers judged by self-reported analysis. We found two novel single nucleotide polymorphisms (SNPs) (21246 T>A and 21265 C>T) causing amino acid substitutions (Asp198Glu and Arg205Cys in exon 5), respectively. The Asp198Glu allele also presented together with known SNPs (20852 C>T in exon4, 20960_20962 CTT deletion, 21115 G>A in intron 4, and 21243_21244 TG deletion in exon 5) in the same allele of the FMO3 gene to form a novel haplotype. These sequences are as follows: 1) SNP, 030609Fujieda019; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-TTCGGGCTG(TG/-)AT/AATTGCCACAGAA-3'. 2) SNP, 030609Fujieda020; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-ACAGAACTCAGCC/TGCACAGCAGAAC-3'.