Membrane transporters are integral membrane proteins typically having 12 transmembrane domains. Most of the SLC family transporters consist of 300-800 amino acid residues with a molecular mass of 40-90 kDa, while the corresponding values of ABC family transporters are 1,200-1,500 residues and 140-180 kDa, respectively. Each transporter has a characteristic tissue distribution and subcellular localization. I have isolated cDNAs of various transporters, including oligopeptide transporter PEPT1, monocarboxylic acid transporter MCT1 and organic cation/carnitine transporters (OCTNs), and determined their tissue distribution and subcellular localization. I have also determined the absolute expression levels of transporters to evaluate their relative contributions to drug transport in various tissues. It is important to note that expression levels of transporters can be changed under various physiological conditions and by administration of drugs. Changes in expression level, subcellular localization and functional properties can all be involved in inter-individual differences in drug pharmacokinetics. Transporters are among the key determinants of drug disposition.
It has been reported that inhibition of the P-glycoprotein (P-gp) results in the improved absorption of P-gp substrate in the intestinal tract. In fact, the increased permeability of P-gp substrate across the intestinal epithelium was observed following inhibition of P-gp in in vitro experiments. To develop the formulation containing P-gp inhibitor and P-gp substrate for practical use, it is necessary to know whether the results obtained in the in vitro experiments are reproducible at whole body level. It is also important to find out the regional difference of the P-gp activity in the intestinal tract. In this study, we examined whether verapamil, a specific inhibitor of P-gp, improves the absorption of rhodamine123 (Rho123), a substrate of P-gp, from the jejunum, ileum, and colon of rats using the in situ loop method. The water content in the loop decreased during the experiment, resulting in a significant change of the Rho123 concentration in the loop. Thus, to accurately determine the absorption rate of Rho123, it was necessary to measure the water movement. It was found that there was a regional difference in the water movement, i.e., greatest in colon, followed by ileum. Verapamil did not change the water movement in any intestinal regions. When the concentration of Rho123 in the loop was corrected by water movement, the Rho123 clearance was in the order of ileum (1.15 μL/min/cm), colon (0.83 μL/min/cm) and jejunum (0.47 μL/min/cm). In the presence of verapamil, the Rho123 clearance was significantly increased at jejunum and ileum but not in colon (ileum: 2.08 μL/min/cm, colon: 1.14 μL/min/cm, jejunum: 1.28 μL/min/cm). These results suggest that P-gp inhibits the drug absorption in jejunum and ileum. From these results, it is possible to evaluate the role of P-gp and its regional difference in the in situ experiments. In particular, the inhibition of P-gp results in an increase in absorption of the P-gp substrate limited to jejunum and ileum.
Two research groups have reported the effect of genetic polymorphisms of CYP2C9 and CYP2C19 on the pharmacokinetic parameters of phenytoin in Japanese epileptic patients. We measured the plasma phenytoin concentrations at steady-state in 20 routinely treated Japanese patients, and evaluated the usefulness of genotyping the CYP2C subfamily in predicting plasma concentrations and determining the dosage regimens of phenytoin. The plasma phenytoin concentrations predicted by genotypes of the CYP2C subfamily were well correlated with the observed concentrations in some patients, but not in some patients. The pharmacokinetic parameters (Vmax and Km) in individual patients, which were obtained from population estimates according to Bayes' theorem, showed considerable interindividual variability even among patients with the same genotype. In addition, we assessed the effect of plasma protein binding on the residual interindividual variability in the clearance of phenytoin; however, there was no significant correlation between the unbound fraction and the intrinsic metabolic activity (Vmax/Km). These findings suggested that the mechanism responsible for the large variability in the clearance of phenytoin is not completely resolved, and that we should not overestimate the usefulness of genotyping the CYP2C subfamily in determining the dosage regimens of the drug.
The -1584C/G single nucleotide polymorphism (SNP) in the promoter region of CYP2D6 was suggested to have the potential to influence CYP2D6 activity. In this report, we demonstrated the frequencies of -1584C to G substitution-related alleles, such as CYP2D6*2, CYP2D6*21, CYP2D6*35 and CYP2D6*41, in the Japanese population. The frequencies of CYP2D6*2, *41 and *21 were 0.102, 0.026 and 0.005, respectively. We also showed a relationship between the SNP and other common alleles, CYP2D6*4, *5, *10, *14 and *18. Interestingly, the SNP was detected in all three subjects carrying CYP2D6*14. This finding suggests the -1584G is included in the CYP2D6*14 allele, which is a null-allele characteristic to the Japanese population. This report presents practical information on CYP2D6 alleles that should be considered in the pharmacokinetic study of CYP2D6 substrates in the Japanese population.
Both isoniazid (INH) and cefazolin (CEZ) can have serious adverse effects on the central nervous system (CNS), causing seizures. In this study, we investigated the effect of INH on the pharmacodynamics of CEZ-induced seizures in rats. Male Wistar rats pretreated with INH (150 mg/kg i.p.) or saline received an intravenous infusion of CEZ at 3.2 g/h/rat until the onset of seizures, then samples of cerebrospinal fluid (CSF), blood (for serum), and brain were obtained immediately. The administration of INH was associated with a reduction in the total dose of CEZ required to produce seizures. The concentrations of CEZ in serum, brain, and CSF in INH-treated rats at the onset of seizures were significantly lower than those in control rats. In rats coadministered with pyridoxine (150 mg/kg s.c.), the concentration of CEZ in CSF at the onset of seizures was significantly higher than that in rats administered INH only. These results suggest that INH potentiates the sensitivity of the CNS to CEZ-induced seizures, and that the increased sensitivity is associated with the inhibition of vitamin B6 metabolism by INH.
Chimeric mice with near-completely humanized liver were constructed by transplantating hepatocytes from a Japanese and Caucasian donor. In the present study, we investigated the induction of human CYP1A2 and CYP3A4 mRNA in a primary culture of the cryopreserved chimeric mouse hepatocytes. β-naphthoflavone (β-NF) and rifampicin (Rif) were used as typical cytochrome P450 (CYP) inducers for CYP1A2 and CYP3A4, respectively. Analysis was performed by the real-time reverse-transcription polymerase chain reaction method. CYP1A2 mRNA in the primary culture of chimeric mouse hepatocytes in mice No. 1, 2, and 3 was significantly increased 3.8-, 6.3-, and 3.3-fold by 5 μM β-NF exposure, respectively, compared with the 0.1% DMSO treated control (p<0.01). CYP3A4 mRNA in the primary culture of chimeric mouse hepatocytes in mice No. 1, 2, and 3 was significantly increased 8.4-fold (p<0.001), 2.2-fold (p<0.01), and 2.3-fold (p<0.05) by 50 μM Rif exposure, respectively, compared with the 0.1% DMSO treated control. The present study demonstrated that a primary culture of cryopreserved hepatocytes from chimeric mice with humanized liver could be used for evaluating the induction of drug metabolizing enzymes in human. This in vitro method may be a useful method for screening the induction potency of new drug candidates on drug metabolizing enzymes.
The metabolism of a novel dual antagonist for α4β1/α4β7 integrin, TR-14035, and the role of polymorphic enzyme responsible for this metabolism were investigated. Human liver microsomes catalyzed the NADPH-dependent metabolism of TR-14035 to a primary metabolite, O-desmethyl TR-14035. This formation was completely blocked by both sulfaphenazole, a selective CYP2C9 inhibitor, and CYP2C9 antibody, whereas potent inhibitors selective for other CYPs exhibited little effects. Of 12 recombinant CYPs examined, O-desmethyl metabolite was principally formed by CYP2C9. CYP1A1, an extrahepatic enzyme, also had this activity (about one-fourth of CYP2C9). Utilizing recombinant CYP2C9*1, Km and Vmax/Km values of 23.3 μM and 0.284 μL/min/pmol CYP2C9, respectively, were obtained for the O-desmethyl formation, which were quite similar to those in CYP2C9*2 enzyme. In contrast, Vmax/Km value in recombinant CYP2C9*3 was approximately one-sixth of CYP2C9*1 and *2. In agreement, kinetics studies using human liver microsomes with CYP2C9*1/*1, *2/*2 and *3/*3 genotypes revealed that the Vmax/Km value in *2/*2 microsomes was comparable to that in wild type microsomes, in contrast, that in *3/*3 microsomes was reduced. These results demonstrate CYP2C9 is a primary enzyme mediating the O-desmethylation of TR-14035 in human liver. In homozygotes of CYP2C9*3, the metabolic clearance of TR-14035 should be decreased compared with homozygotes of CYP2C9*1 or 2.
The anticonvulsant agent phenytoin (5,5-diphenylhydantoin) is mainly excreted as 5-(4′-hydroxyphenyl)-5-phenylhydantoin (4′-HPPH) O-glucuronide in humans. Previously, we demonstrated that the glucuronidation of 4′-HPPH is catalyzed by multiple UDP-glucuronosyltransferases (UGTs) of UGT1A1, UGT1A4, UGT1A6, and UGT1A9. Since 4′-HPPH may be bioactivated to a reactive metabolite by peroxidase, the glucuronidation in considered to be a detoxification pathway. In the present study, we investigated the relationship between the extent of interindividual variability in the urinary excretion levels of 4′-HPPH and its O-glucuronide and genotyping of CYP2C9, CYP2C19, UGT1A1, UGT1A6, and UGT1A9. 4′-HPPH and its glucuronide in urine samples from 15 patients to whom phenytoin was administered were measured by liquid chromatography-tandem mass spectrometry. When the molar ratio of 4′-HPPH O-glucuronide/4′-HPPH was calculated as an index of glucuronidation, a large interindividual variability (11 fold) was observed in the 15 patients. Phenytoin is metabolized to 4′-HPPH by CYP2C9 and CYP2C19 in which there are genetic polymorphisms. Although 5 patients were genotyped as heterozygotes of mutated alleles of CYP2C9 or CYP2C19 genes, no relationship with the interindividual difference in the total excretion levels of 4′-HPPH and its O-glucuronide was observed. The UGT1A1*6, UGT1A1*28, UGT1A1*60 and UGT1A6*2 alleles were found in 1, 3, 6, and 8 patients, respectively. Although there was no relationship between the genetic polymorphisms of UGT1As and the interindividual difference in the 4′-HPPH glucuronidation, the large interindividual variability of 4′- HPPH glucuronidation may contribute to interindividual differences in toxic reactions to phenytoin.
Nineteen genetic variations, including 11 novel ones, were found in exon 1 and its flanking region of the UDP-glucuronosyltransferase (UGT) 1A4 gene from 256 Japanese subjects, consisting of 60 healthy volunteers, 88 cancer patients and 108 arrhythmic patients. These variations include -217T>G and -36G>A in the 5′-flanking region, 30G>A (P10P), 127delA (43fsX22; frame-shift from codon 43 resulting in the termination at the 22nd codon, codon 65), 175delG (59fsX6), 271C>T (R91C), 325A>G (R109G), and 357T>C (N119N) in exon 1, and IVS1+1G>T, IVS1+98A>G and IVS1+101G>T in the following intron. Among them, 127delA and 175delG can confer early termination of translation, resulting in an immature protein that probably lacks enzymatic activity. Variation IVS1+1G>T is located at a splice donor site and thus may lead to aberrant splicing. Since we did not find any significant differences in the frequencies of all the variations among the three subject groups, the data were analyzed as one group. The allele frequencies of the novel variations were 0.006 for IVS1+101G>T, 0.004 for 30G>A (P10P) and 357T>C (N119N), and 0.002 for the 8 other variations. In addition, the two known nonsynonymous single nucleotide polymorphisms (SNPs), 31C>T (R11W) and 142T>G (L48V), were found at 0.012 and 0.129 frequencies, respectively. The SNP 70C>A (P24T), mostly linked with 142T>G (L48V) in German Caucasians, was not detected in this study. Sixteen haplotypes were identified or inferred, and some haplotypes were confirmed by cloning and sequencing. It was shown that most of 142T>G (L48V) was linked with -219C>T, -163G>A, 448T>C (L150L), 804G>A (P268P), and IVS1+43C>T, comprising haplotype *3a; haplotype *4a harbors 31C>T (R11W); 127delA (43fsX22) and 142T>G (L48V) were linked (haplotype *5a); 175delG (59fsX6) was linked with 325A>G (R109G) (*6a haplotype); and -219C>T, -163G>A, 142T>G (L48V), 271C>T (R91C), 448T>C (L150L), 804G>A (P268P), and IVS1+43C>T comprised haplotype *7a. Our results provide fundamental and useful information for genotyping UGT1A4 in the Japanese and probably Asian populations.
The correct authors' affiliation of Kazuo Komamura, Shiro Kamakura, Masafumi Kitakaze and Hitonobu Tomoike is the Division of Cardiology, National Cardiovascular Center, Osaka, Japan We apologize for this error.
Wrong:Division of Cardiology, Okayama University, Okayama, Japan
Right:Division of Cardiology, National Cardiovascular Center, Osaka, Japan