Approximately 1% of Orientals and 7 to 10% of Caucasians lack the activity of cytochrome P450 (CYP) 2D6, and these individuals are known as poor metabolizers (PM). On the other hand, approximately 4% of Caucasians are PM of CYP2C19, while its frequencies are 18 to 23% in Orientals. These differences in the frequencies of PM seen in the different ethnic populations are mainly due to the differences in the distribution frequency of variously defective alleles of CYP2D6 and 2C19. Recent progress in molecular biology of CYPs has enabled the mechanism of CYP polymorphism to be elucidated and the polymerase chain reaction was developed for its genotyping. Our previous studies showed that the frequencies of CYP2C19*2, *3, CYP2D6*2, *5, *10, *14, and CYP2C9*3 among the Japanese subjects were 28.7, 13.2, 12.9, 6.2, 38.6, 2.2 and 2.1%, respectively. In this review, we compared the frequencies of these mutant alleles of CYPs seen in the Japanese population with those reported previously for other ethnic populations.

View full abstract

Clinical application of pharmacokinetics/pharmacodynamics (PK/PD) from animal to human, and from normal to disease states was examined. First, we tried to elucidate the factors affecting the hepatic transport of sulfobromophthalein and indocyanine green in chronically intoxicated rats and dogs. Also, we examined the clinical implication of hepatic clearance of indocyanine green, chenodeoxicholi cacid, antipyrine, and galactose in patients with chronic liver diseases. Second, we examined the PK/PD study on β-blockers in order to establish the rational usage in patients. Third, we examined the drug interaction between new quinolones and non-steroidal antinframatic agents, and further, studied electrocardiographically on the adverse effect, QT prolongation in non-arrhythmic agents, terfenadine, ebastine and epinastine. Fourth, the metabolic inhibition of midazolam by itraconazole or ketoconazole was examined in order to evaluate the degree of drug-drug interaction concerning metabolic inhibition in the liver quantitatively, and we succeeded the prediction of increasing ratio in AUC of midazolam in concomitant administration of itraconazole or ketoconazole. Finally, we applied above outcomes to clinical practise using various approaches.

View full abstract

Some evaluation methods and computer analysis programs on personal computers have been developed for pharmacokinetic analysis. Akaike's Information Criterion (AIC) was introduced to statistically select an optimum model for drug disposition. The moment analysis which has bee been used in chromatography and chemical engineer was applied to the evaluation of absorption, distribution, metabolism and excretion (ADME) of drugs. A pharmacokinetic analysis program based on nonlinear least squares, MULTI, was developed on a personal computer in the early age. We have made other analysis program MULTI (RUNGE) where a pharmacokinetic model is defined as ordinal differential equations, MULTI (FILT) based on a Fast Inverse Laplace Transform (FILT) and MULTI (FEM) based on the Finite Element Method (FEM). MULTI2 (BAYES) based on Bayesian least squares and MULTI (ELS) based on extended least squares were developed for the population pharmacokinetics (PPK).

View full abstract

Mercapturic acids (N-acetylcysteine conjugates) are not an ultimate excretion form in many compounds, but are converted to methylthio-containing metabolites, or S-substituted mercapto acetic acid or mercapto lactic acid via mercapto pyruvic acid. Methylthio-containing metabolites are generated by the action of N-deacetylases for mercapturic acids, β-lyase for cysteine conjugates and methyl transferases for thiols. Methylsulfides thus formed are subsequently oxidized to the methylsulfoxides or methylsulfones prior to excretion. In addition to above enzymes, reductases for methyl sulfoxide were also predominantly localized in the liver and kidney. In the formation of S-substituted mercapto acetic acid, likely participation of cysteine conjugate aminotransferase, thio-pyruvic acid oxidase and in some cases thio-lactic acid reductases is indicated.

View full abstract

Protocols for evaluating drug interaction caused by enzyme inhibition were established by the Drug Interaction Database Working Group of the Human and Animal Bridge Discussion Group (HAB). Basically, procedures for measurements of the inhibition constant (Ki) of the test article were methodically investigated. 7-Ethoxyresorufin (CYP1A1&2), coumarin (CYP2A6), 7-benzyloxyresorufin (CYP2B6), tolbutamide (CYP2C8&9), S-mephenytoin (CYP2C19), bufuralol (CYP2D6), chlorzoxazone (CYP2E1), nifedipine (CYP3A4) and testosterone (CYP3A4) were used as specific substrates for various isoforms of human liver cytochrome P450 (P450). At least 3 concentrations of the test article were appropriately selected in experiment on concentration-dependency of enzyme inhibition, and used in measurement of apparent Ki value by Dixon plot. In Ki measurements, the P450 substrate was added to the assay system at 3 different concentrations (Km, 1/2 Km and 2 Km). To correct the apparent Ki value of the test article by the binding to the liver microsomes, free concentration of the test article in the system for Ki measurements was determined most conveniently by ultracentrifugation. In this HAB-protocol, method to judge the mechanismbased inhibitor by detecting increased inhibition by the test article after preincubation in the presence of NADPH was also included. Magnitude of drug interaction (R) was estimated according to the equation: R=1+I/Ki, where I represents the concentration of the test article, most preferably the C_max value either in the peripheral or the portal blood.

View full abstract

In the present study, we examined the genetic polymorphism in drug transporters, using organic cation/carnitine transporter OCTN2 as a model transporter. Since OCTN2 plays a role in carnitine transport by mediating especially a renal reabsorption of carnitine from urine, free carnitine concentrations in blood and urine were used for phenotyping of the genetic mutation of OCTN2. From general population (n=973), the phenotyping resulted in 46 people with significantly low blood free carnitine concentration. Among them 36 people and 69 normal people were further examined for their free carnitine concentration and OCTN2 genes. Only from the group that showed a low carnitine blood concentration, three kinds of heterozygous mutations of OCTN2 gene that show functional loss were found. Furthermore, based on the phenotype observed by systemic carnitine deficiency patients and their family, many type of mutations in OCTN2 were found with functional loss. So, phenotyping of OCTN2 gene by blood and urinary concentration of carnitine is a efficient way to identify the polymorphism of OCTN2 that shows functional alteration. Since OCTN2 was suggested to be involved in renal excretion of organic cations such as tetraethylammonium, genetic polymorphisms that cause functional alteration in carnitine transport may change the pharmacokinetics of organic cations. However, the OCTN2-mediated transport mechanism in carnitine transport is different from that of organic cations in terms of sodium ion dependency, the functional alterations of carnitine transport activity by genetic polymorphism are not necessarily cause the same effect on the pharmacokinetics of organic cations. In addition to OCTN2, we found genetic polymorphism in organic anion transporter OATP-C, which is an important transporter for hepatic excretion of anionic drugs in human. So, we need further functional analysis of various alleles of transporter genes for the better understanding of inter-individual differences of pharmacokinetic characteristics of drugs.

View full abstract

Renal organic anion transporters, OAT-K1 and OAT-K2, are expressed specifically in the brushborder membranes of the proximal straight tubules. Both of them transported structurally unrelated compounds such as antitumor drug methotrexate, various endogenous organic anions (thyroid hormones, taurocholic acid and conjugated steroids), and an antiretroviral drug zidovudine, which possesses no typical anionic moiety. In addition, OAT-K1 and OAT-K2 showed bidirectional transport of substrates. Moreover, methotrexate efflux via these transporters was stimulated in the presence of extracellular folic acid-derivatives, suggesting that they could serve as anion exchangers to enhance the apical efflux of methotrexate.
In the state of renal failure caused by 5/6 nephrectomy, the mRNA expression levels of OAT-K1 and OAT -K2 were significantly diminished, and the renal clearance of methotrexate was markedly decreased compared with that in sham-operated rats. Furthermore, additional folinic acid treatment resulted in a significant increase in methotrexate renal clearance in sham-operated rats, but not in 5/6 nephrectomized rats. These findings suggested that the decreased expressions of OAT-K1 and OAT-K2 were attributed to the longer exposure to methotrexate and invalidated folinic acid rescue in 5/6 nephrectomized rats.
In conclusion, OAT-K1 and OAT-K2 may play important roles in the renal handling of hydrophobic anionic drugs.

View full abstract

The purpose of the present study was to characterize the transport mechanism of basic fibroblast growth factor (bFGF) through the blood-brain barrier (BBB). Following internal carotid artery perfusion, ¹²⁵I-bFGF was transcytosed through the BBB from blood into brain parenchyma. The binding of ¹²⁵I-bFGF to the isolated bovine brain capillaries was saturated with a dissociation constant of 40 nM. This binding was significantly inhibited by unlabeled peptide, poly-L-lysine, heparin and glycosaminoglycans with a sulfate residue. RT -PCR analysis revealed mRNA of perlecan, FGF receptors (FGFR1 and FGFR2) in the conditionally immortalized mouse brain capillary endothelial cell lines (TM-BBB). The internalization of ¹²⁵I-bFGF in TM-BBB was temperature-dependent and concentration-dependent, and showed a dependence on medium osmolarity. This internalization was significantly inhibited by the treatment of heparinase, sodium chlorate and antibody specific to mouse perlecan, a heparan sulfate proteoglycan. These results are consistent with the conclusion, 1) ¹²⁵I-bFGF is transcytosed through the BBB, 2) internalization of ¹²⁵I-bFGF into brain capillary endothelial cells is, in part, brought about by an endocytosis mechanism that is triggered by binding to HSPG.

View full abstract

Troglitazone, a new oral antidiabetic drug, has been reported to cause idiosyncratic hepatitis in certain individuals. The mechanism for the hepatic failure was investigated with comparison between troglitazone and its metabolites and other thiazolidinediones, pioglitazone and rosiglitazone. Hepatic toxicity did not appear in rats in troglitazone-administered study after modifications of sulfotransferase and glucuronosyltrasferase activities and glutathione concentration. Oxidation pathway of troglitazone to a quinine-type metabolite was catalyzed mainly by CYP2C8 and CYP3A4 in human liver microsomes. Inhibitory effects of troglitazone and its metabolites on drug oxidation activities of human P450s would not be potent, based on its low blood concentrations and high protein binding ratio. An autoimmune antibody against aldolase B was identified in two patients with troglitazone-induced idiosyncratic hepatitis. However, this antibody was also detected in some other hepatic diseases, in spite of no cases in health control subjects. Treatment of HepG2 cell lines with troglitazone and a quinone type-metabolite showed time and concentration-dependent cytotoxicity. Troglitazone induced apoptotic cell death in HepG2 cells characterized by internucleosomal DNA fragmentation and nuclear condensation. Taking these results into consideration, the causal factor (s) for idiosyncratic hepatitis in human remained unclear.

View full abstract

An overdose of acetaminophen results in liver injury in experimental animals and human. It is initiated by metabolic activation by cytochrome P450 enzymes into the reactive N-acetyl p-benzoquinone imine metabolite followed by its covalent binding to liver target proteins. However factors other than the covalent binding contribute to the severity of the toxicity and involvement of activated Kupffer cells and infiltrated macrophages has been demonstrated. These liver nonparenchymal cells produce and release the inflammatory mediators, reactive oxygen species, nitric oxide and cytokines, which are suggested to be involved in the liver injury. Liver mRNAs of interleukin-6 (IL-6), IL-10 and IL-11, which are classified as anti-inflammatory cytokines and are considered to suppress inflammatory tissue injury, increased markedly after the acetaminophen administration. Acetaminophen-induced liver injury was exaggerated in mice deficient in IL6 (IL-6-/ -mice) and IL-10/ -mice as compared with corresponding wild-type mice. Administration of recombinant human IL-11 protein prior to acetaminophen blocked the liver injury. Immunoblot analysis of liver homogenate indicated no significant effect of the perturbation of the cytokines on formation of acetaminophen-protein adduct. These results demonstrated protective role of these anti-inflammatory cytokines against the hepatotoxicity of acetaminophen, which is not due to effect on the metabolic activation. It also suggested involvement of proinflammatory cytokines in the pathogenesis of acetaminophen-induced liver injury, because they are regulated by anti-inflammatory cytokines.

View full abstract