Published papers

The prediction error in the Bayesian analysis program for digoxin was evaluated in Japanese patients, and factors influencing the accuracy were investigated. Serum concentrations of digoxin were monitored two times and were compared with the predicted values obtained by using the Bayesian analysis program. The prediction error at the first time was 43.1%. Although this estimation error was reasonably restored at the second time of monitoring, the prediction error remained at 26.6%. These data suggested that unknown factors not included in the program affected the serum concentration of digoxin. Retrospective research of the digoxin serum concentrations in the patients suggested the coadministration of the drugs, which were the P-glycoprotein modulators, as well as the unexpected alteration of the serum creatinine, were the important factors influencing the prediction of the drug serum concentrations. We next examined the inhibitory effect of quinidine, verapamil and spironolactone on the transcellular transport of digoxin by using human P-glycoprotein overexpressing LLC-GA5-COL150 cells. Quinidine, verapamil and spironolactone could inhibit the transcellular transport of digoxin by 50%. In addition, the reduction of the renal clearance by 50%, which could possibly be caused by this inhibition, led to the increase of 36% in the steady state through concentrations of digoxin in the physiological pharmacokinetic model. In conclusion, the prediction of long-term serum concentration-time profiles of digoxin, based on the Bayesian analysis, will be disturbed by the coadministration of the P-glycoprotein modulators and the unexpected alteration of the serum creatinine.

Purpose. To predict the CYP2C19 genotype-dependence in anti-Helicobacter pylori (H. pylori) therapy when lansoprazole or rabeprazole was used instead of omeprazole as a proton pump inhibitor (PPI). Methods. A comparative pharmacokinetic study with each PPI was designed as an open, randomized, and crossover study of 18 Japanese healthy volunteers who were classified into the homozygous, heterozygous extensive metabolizer and the poor metabolizer based on the CYP2C19 genotype determined by PCR-RFLP method. Each subject received a single oral dose of 20 mg omeprazole, 30 mg lansoprazole, or 20 mg sodium rabeprazole, with at least 1 week washout period between treatments. Plasma concentrations of PPIs and their metabolites were monitored until 12 h after medication. Results. Pharmacokinetic profiles of omeprazole and lansoprazole were well correlated with the CYP2C19 genotype. The heterozygous extensive metabolizer was slightly different from the homozygote, but there was no statistically significant difference. The CYP2C19 genotype dependence found for lansoprazole was not obvious compared with omeprazole. As for rabeprazole, the pharmacokinetic profile was independent of the CYP2C19 genotype. Conclusions. CYP2C19 genotype dependence will be found in the anti-H. pylori therapy even when lansoprazole is used as the PPI.

Isoniazid (INH) is metabolized by polymorphic N-acetyltransferase2 (NAT2). In the present study, the relationship between the NAT2 genotype and the INH acetylator phenotype was examined in Japanese tuberculous patients and compared with healthy subjects. Subjects were classified according to the genotyping into NAT2* 5B (allele4), NAT2*6A (allele3) and NAT2*7B (allele2), using the PCR-RFLP method. Twelve healthy subjects and 7 tuberculous patients participated in the INH acetylator phenotyping study, in which each subject was administered an oral dose of INH, followed by urine sampling for 24 h. Urinary concentrations of INH and N-acetylisoniazid (AcINH) were measured by the HPLC method. The urinary recoveries of INH (% of dose) in healthy subjects in relation to NAT2 genotyping were as follows: 6.4±2.2 in the homozygotes for the wild-type allele, 10.7±2.2 in the compound heterozygotes for the mutant allele, and 38.6±6.4 in the homozygotes for the mutant allele. In the patients study, the findings in the corresponding three groups were 4.0±1.7, 8.8 and 18.3±9.3. Although no significant difference was found because of the lower systemic exposure of INH in patients compared with healthy subjects, there were differences in the disposition kinetics of INH between subjects with and without mutations in the NAT2 gene, and these findings were observed not only in healthy subjects but also in patients who had comedicated drugs and hepatic dysfunctions. The findings indicated that the metabolism of INH by NAT2 is clearly impaired in subjects with mutations in the NAT2 gene, and thus genotyping for three NAT2 point mutations was adequate to predict the metabolism of INH in Japanese tuberculous patients as well as healthy subjects. This NAT2 genotyping could become a useful alternative to TDM for INH.

Purpose. A combination of proton pump inhibitors and antimicrobials has been applied as an anti-Helicobacter pylori (H. pylori) therapy. Omeprazole, one of the proton pump inhibitors, is metabolized by CYP2C19, which exhibits genetic polymorphism. It was reported previously that the overall anti-H, pylori efficacy can be related to the CYP2C19 genotype. The main aim of the present study was to obtain a rational explanation for the relationship between the overall anti-H. pylori efficacy and the CYP2C19 genotype. Methods. Six healthy volunteers were classified as extensive metabolizers and poor metabolizers, according to their CYP2C19 genotypes. Plasma concentrations and intragastric pH were monitored prior to and until 24 h after the administration of 20 mg omeprazole. The stability of amoxicillin, clarithromycin, and metronidazole was examined using buffer solutions with monitored intragastric pH, and their remaining percentage in the intragastric space was simulated. Results. The poor metabolizers, classified by the CYP2C19 geno-types, showed the higher effectiveness in anti-H pylori therapy, via the higher plasma concentration of omeprazole and the higher intragastric pH, and possibly the higher stability of antimicrobials in the higher intragastric pH. Conclusions. CYP2C19 genotyping is a very useful method to determine the effective and safe dosage regimen including the selection of the dual and triple therapy in anti-H. pylori therapy.

The role of the sugar chain on the fibrin affinity property of tissue plasminogen activator (t-PA) was investigated using two variants of wild type t-PA (WT t-PA I and WT t-PA II) and mutant type t-PA (mt-PA ; Gln117 t-PA I and Gln117 t-PA II), whose sugar chains have different structures. In terms of fibrin affinity, Gln117 t-PA was higher than WT t-PA ; moreover, Type II was higher than Type I. Bindings mediated via finger domain (F mode) and kringle 2 domain (K2 mode) were distinguished using ε-amino caproic acid (EACA). Consequently, F mode and K2 mode bindings were inhibited by the sugar chains at Asn117 and 184, respectively. These results were assumed to be due to the steric hindrance of the sugar chains.

We have studied the influence of Gly-Ala-Arg peptide at the N-terminus and the oligosaccharide at Asn184 on the clearance of tissue plasminogen activator (t-PA). In order to intensify the influence of these structural features, Gln117 t-PA, which is a mutant tissue plasminogen activator (mt-PA) expressed in mouse C127 cells, was used for the investigation. It is altered to remove a high mannose type oligosaccharide by the mutation of an amino acid from Asn117 to Gln. We isolated 4 variants of Gln117 t-PA by cation- exchange chromatography, which are abbreviated as S-I, S-II, L-I and L-II. These variants originated from the heterogeneity of the peptide chains (S- chain, 527 amino acids, L-chain, 530 amino acids) and oligosaccharide (Type I, 2 oligosaccharides, Type II, 1 oligosaccharide). Pharmacokinetics of these variants were investigated after single intravenous administration to male rats at a dose of 250 μg/kg. Significant differences in pharmacokinetic parameters were observed among these variants, but there was no considerable difference in fibrin clot lysis time (FCLT) activity. Gly-Ala-Arg peptide at the N-terminus increased the CL(t), whereas the oligosaccharide at Asn184 decreased the CL(t). Moreover, the effects of the N-terminal peptide and the oligosaccharide on the CL(t) were independent of each other. Our study with Gln117 t-PA revealed the role of the N-terminal peptide found in the L-chain produced during the processing of t-PA precursor.

The inhibitory effects of nicardipine, nifedipine and itraconazole on P- glycoprotein-mediated transport of [3H]digoxin were examined using LLC-PK1 and LLC-GA5-COL150 cells, a porcine kidney epithelial LLC-PK1 cell line transformed with MDR1 cDNA from man which results in overexpression of P- glycoprotein on the apical membrane. Basal-to-apical transport of [3H]digoxin in LLC-GA5-COL150 cells was higher than in LLC-PK1 cells; apical-to-basal transport was markedly lower in LLC-PK1 cells and even lower in LLC-GA5-COL150 cells. This is consistent with the possibility that [3H]digoxin is transported by P-glycoprotein. Co-administration of nicardipine or itraconazole markedly inhibited the basal-to-apical transport of [3H]digoxin in LLC-GA5-COL150 cells, and apical-to-basal transport also increased. The effect of nifedipine was less marked than that of nicardipine or itraconazole. Intracellular accumulation of [3H]digoxin after apical application in LLC-GA5-COL150 cells was 2.3 times less than in LLC-PK1 cells, and was increased by the addition of nicardipine or itraconazole, consistent with their inhibitory effects on transcellular transport. Following basal application of [3H]digoxin, its intracellular accumulation in LLC-GA5-COL150 cells was, unexpectedly, comparable with that in LLC-PK1 cells, and was hardly affected by the addition of nicardipine or itraconazole. In conclusion, it has been shown that nicardipine and itraconazole inhibited transport of digoxin, which is presumably mediated by P-glycoprotein. This explains their effects observed in clinical use.

Faropenem (FRPM) is an oral penem antibacterial medicine with a high stability to beta-lactamase which has a broad antibacterial spectrum and a strong antibacterial activity to gram positive bacteria, and gram negative bacteria, except for Pseudomonas aeruginosa and anaerobic bacteria. The sale of faropenem was started in 1997. Based on the plasma faropenem concentration vs. time data obtained from clinical trials using healthy adult volunteers and patients, and a post-marketing clinical study using advanced age patients, the population pharmacokinetic analysis of a total of 20 studies was performed. The concentration data of 1,065 points from 218 patients whose creatinine clearances were measured (85 points/12 patients) or estimated (980 points/206 patients) from plasma concentration levels was used for the analysis. A one-compartment model with first-order absorption was used as a pharmacokinetic model where the absorption lag-time was assumed. Among the population pharmacokinetic parameters, the apparent total body clearance (CL), apparent distribution volume (V), first-order absorption rate constant (ka) and absorption lag-time (tlag) showed great inter-subject variation. In the final model, creatinine clearance (CLcr) was chosen as the most influential covariate against CL and CL was well correlated with CLcr when CLcr was less than 80 mL/min. The inter-subject variability in CL was explained by considering CLcr as a cofactor. The apparent distribution volume increased in the geriatric patients. Both ka and tlag were dependent on meals in all patients. Based on these results, the population pharmacokinetic model of faropenem was made by taking these covariates into consideration. From the results of population pharmacokinetics analysis, the prolongation of the elimination half-life (t1/2) of faropenem was suggested in the patients with decreased kidney function as previously reported.

The drug-metabolizing enzyme thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathiopurine, which are used as immunosuppressants and in the treatment of acute lymphoblastic leukemia and rheumatoid arthritis. TPMT enzymatic activity is a polymorphic trait, and poor metabolizers may develop life-threatening bone marrow failure. To avoid such adverse effects, the TPMT enzymatic activity in patients' red blood cells (RBCs) is routinely measured prior to thiopurine administration in a limited number of oncology clinics. In the present study, we took advantage of a highly sensitive and specific automated denaturing high-performance liquid chromatography (dHPLC) technique that not only detects known polymorphic alleles, but also identifies previously uncharacterized sequence variants. We developed a dHPLC-based protocol to analyze the entire coding region and validated the protocol to detect all 16 previously described variant alleles. We further analyzed the entire coding region of the TPMT gene in 288 control samples collected worldwide and identified two novel amino acid substitutions Arg163Cys (487C>T) and Arg226Gln (677G>A) within exons 7 and 10, respectively. The clinical application of this comprehensive screening system for examining the entire TPMT gene would help to identify patients at risk for bone marrow failure prior to 6-mercaptopurine therapy. © Mary Ann Liebert, Inc.