The promise of pharmacogenomics includes managing patient-to-patient variability, developing better drugs faster and cheaper, and providing better medical care. However, whether there would be cost savings in clinical trials and the emergence of pharmacoeconomic modeling for diagnostic tests is still under consideration. In this talk, I would like to discuss these issues from clinical and discovery aspects of pharmacogenomics. Other ethical concerns including genomic data acquisition, data analysis, sensitivity and specificity, confidentiality and storage of patient sample, will be discussed as well. How to integrate the driven technologies with informatics will be one of the key issues to success.

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A gene encoding a 5' flanking region of human carboxylesterase(CES) has been cloned and characterized. Analysis of genomic lambda clone H2B revealed that the gene spans about 1, 021 bp. This clone include an in-frame initiation codon (ATG) of human liver CES isozyme HU1, and the clone completely overlap the CES HU1 cDNA. The transcription start site was determined by CAP-Site hanting methods. An additional 892 by of DNA from the 5' flanking region of the gene was cloned and sequenced in order to elucidate the structure of the promoter. In this sequence several possible binding sites for transcription factors have been identified, but TATA-box was not present. The putative promoter region was investigated by gel mobility shift assay and the reporter gene assay. The promoter region include HNF-1, CCAAT-Box, GC-box and initiator. We also observed genetic polymorphism of a 5' flanking region of human liver CES by PCR-SSCP analysis and DNA sequence of individual human livers. These results suggest that the individual differences of human liver esterase activity may be, at least in part, dependent on the genetic polymorphism of a 5' flanking region of CES HU1.

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2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic polychlorinated organic pollutants that have been studied. Most of the effects of TCDD are mediated by a cytosolic receptor known to as the aryl hydrocarbon receptor (AhR). Several xenobiotic-metabolizing enzymes, such as cytochrome P450 1A1 (CYP1A1), 1A2 (CYP1A2), UDP-glucuronosyltransferase, NAD(P)H-quinone oxidoreductase, and aldehyde dehydrogenase-3, have the XRE sequence, AhR-ARNT complex binding motif, in the 5'-upstream region of their genes and AhR-mediated induction of gene expression by TCDD is observed. The induction of EROD and MROD activities by TCDD were proved to be mediated by AhR, since TCDD treatment of AhR-null (Ahr-/-) mice, which lack the AhR gene, produced no enhancement of EROD and MROD activities in liver microsomes. Xanthine dehydrogenase (XDH) and xanthine oxidase(XO) activities of Ahr+/+ (C57BL/6J) mice were also increased by TCDD. However, no induction was observed in Ahr-/- mice. Thus, induction of XDH and XO activities was also mediated by AhR. XDH and XO catalyze oxidation of hypoxanthine to xanthine and xanthine to uric acid with concomitant reduction of NAD+ or molecular oxygen. The reaction generates reactive oxygen species, and XO is involved in lipid peroxidation and reperfusion injury. Reactive oxygen species are generated as by-products of the action of XO/XDH. Therefore, the induction of these enzymes by TCDD may contribute significantly to the various toxicities of TCDD. In this study, we showed that XDH/XO activities are induced by TCDD and the inductions are mediated by AhR.

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To evaluate CYP2D6 mutants as drug-metabolizing enzymes, we expressed four single amino acid-substituted mutants (P34S, G42R, R296C and S486T) and two double amino acid-substituted mutants (P34S/S486T and R296C/S486T) in yeast cells. The profiles of P34S, G42R and P34S/S486T in microsomal expression levels and oxidation capacities towards debrisoquine (DB) and bunitrolol (BTL) were much different from those of the wild type, while the behavior of R296C, S486T and R296C/S486T was similar to the wild type in these indices. CYP contents in microsomal and whole cell fractions, which were measured spectrophotometrically and immunochemically, indicated that a part of P34S protein was located in microsomal membranes whereas most of P34S protein was in cytosolic fraction. Enzyme assay revealed that P34S showed much lower activities in DB and BTL 4-hydroxylations than the wild type in terms of nmol/min/mg protein, but similar activities to the wild type in terms of nmol/min/nmol CYP, indicating that P34S, which can anchor to endoplasmic reticulum membrane and hold a prosthetic heme group, retains normal functions. However, G42R did not have normal functions even in terms of nmol/min/nmol CYP; i.e., its Km values for DB and BTL oxidations were much higher than those of the wild type. The results indicate that similar to the substitution of Pro-34 to Ser, that of Gly-42 to Arg results in the decrease in the enzymatic function but in a manner different from P34S.

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Twenty one types of genetic polymorphisms in CYP2D6 gene were determined in liver DNA of Japanese and Caucasians and compared these CYP2D6 genotypes with CYP2D6 protein levels and bufuralol 1'- and 6-hydroxylation activities in liver microsomes of these human samples. We detected 11 types of CYP2D6 genetic polymorphisms and classified these humans into 17 genotypes; 7 types were found in the Japanese and 13 types in the Caucasian. CYP2D6* 10B, but not CYP2D6*10A, was the most frequent in mutation at 34.6% in the Japanese, whereas in Caucasians, CYP2D6 polymorphisms including CYP2D6*4A, *4D, *4E, *4L *3, *9, and *M12 (frequencies at 6.8, 3.4, 4.5, 9.1, 2.3, 2.3, 4.5%) respectively, were detected. A Caucasian having homozygous CYP2D6*3/*3 had a protein with slower gel mobility (immunoblotting with anti-CYP2D6 antibody) and a very low activity for bufuralol 1'-hydroxylation. Five Caucasian samples with CYP2D6*4A/*4A, *4A/*4L, or *4D/*4L had no measurable CYP2D6 protein and very low bufuralol 1'-hydroxylation activities. Seven Japanese subjects with CYP2D6*10B/*IOB had CYP2D6 protein at levels of -20% of those present in humans with CYP2D6*1 and *2 and catalyzed bufuralol 1'-hydroxylation at low rates. These results support the view that CYP2D6*3, *4A, *4D, and *4L are major genotypes in producing poor metabolizer phenotypes in CYP2D6 in Caucasians, whereas CYP2D6*10B is a major causes in decreasing CYP2D6 protein expression and catalytic activities in Japanese. We also determine three types of CYP2E1 genetic polymorphisms, namely RsaI/PstI-, DraI-, and MspI-types, and compared these genotypes with levels of CYP2E1 and activities of 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation in liver microsomes from these human samples. The results obtained collectively indicated that RsaI/PstI-, DraI-, and MspI-types of CYP2E1 polymorphisms may not cause significant alterations in protein expression and enzyme catalytic activities of CYP2E1 enzyme in human livers.

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Previous studies revealed that while rat CYP2B1/2 are highly inducible by phenoharhital (PB), guinea pig CYP2B18A is expressed constitutively and has low inducible nature following PB treatment. To solve the low inducibility in guinea pigs. we cloned and sequenced CYP2B18A gene. Since the intron structures of the CYP2B2 gene have not yet been determined, we sequenced this gene as well as the 2B18A gene. Comparison of gene upstream between the two genes revealed that guinea pigs lack in the phenoharhital-responsive unit (PBRU) which is demonstrated to play a crucial role in PB-mediated induction of the CYP2B2. This observation seems to support the low-inducihility of CYP2B18A. Further, NF-κB and RBP-Jκ binding elements, which have been reported to mediate suppression of CYP2B2 gene, were not found in the CYP2B18A gene, supporting highly constitutive expression of this gene. To clarify the mechanism underlying PB-mediated induction, we analyzed further the structure of the CYP2B2 gene in the Qdj:SD rats that lack in response to PB-mediated induction of this protein. As the results, neither large deletion nor insertion was detected in the CYP2B2 gene of this mutant rats. In addition, no mutation was detected in the PBRU region and all exons. These results suggested that impaired induction of CYP2B2 in Qdj:SD rats is due either to mutation at the region different from the PBRU or exons, or defect in traps-acting factor(s).

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Cytochrome P450s including in the CYP3A subfamily are involved in metabolism of clinically relevant substrates as well as numerous endogenous compounds. CYP3A forms are induced by the pretreatment with many drugs, but show the different induction profile between experimental animals and human. We have been studied the activation mechanism of rat CYP3A1/2 gene expression. Level of the transcriptional activation, however, varied among cultured-cell lines. These results prompted us to study in vivo induction mechanism of human CYP3A4 gene expression by use of adenovirous expression vector (AdCYP3A4-362), which was constructed with a proximal promoter region (-362 to +11) of the CYP3A4 gene and a reporter gene. Induction level of CYP3A4 gene expression differed not only among drugs but also among cultured-cell lines. In Hep G2 cells, dexamethasone and clotrimazole induced the gene expression, but rifampicin did not. In contrast, rifampicin induced the gene expression in LS174T cells. Therefore AdCYP3A4-364 was injected into ddy mouse and measured the reporter activity and also mouse liver CYP3A activity. Induction profile of CYP3A4 gene expression in mouse liver agreed well with that of mouse CYP3A activity. A mutation study of a receptor binding region (ER-6) showed the diminished induction level of CYP3A4 gene expression, but not of mouse CYP3A activity. These results strongly suggest that in vivo study of the gene transcriptional activation is very important to assess the induction profile of CYP3A4 gene expression by drugs.

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The purpose of this study was to clarify the mechanism(s) responsible for the growth hormone (GH)-induced expression of the CYP2C12 gene. To identify a functional GH-responsive element (GHRE) in vivo, we performed the direct injection of promoter-luciferase chimeric genes into female rat livers. The results showed that the luciferase activity was decreased to approximately 20% by the deletion of the sequence between nucleotides -4213 and -4161. Within this region, two copies of a possible GHRE were present. The sequence of the GHRE was overlapped with that of an interferon-γ-activated sequence (GAS), known to be recognized by the signal transducer and activator of transcription (STAT) proteins. In fact, a super shift assay showed that STAT5 was capable of binding to the core sequence of the GHRE. Furthermore, a luciferase assay with reporter plasmids, which lacks HNF4 or HNF6-binding sites, revealed that the GH-stimulated expression of the CYP2C12 gene was regulated cooperatively by STAT5, HNF-4, HNF-6 and the factor(s) which binds to the elements, 2C12-I (-4095 to -4074) and 2C12-II (-4072 to -4045). The cooperative regulation by STAT5 and the liver-enriched transcription factors account for the GH-dependent and the liver-specific expression of the CYP2C12 gene in female rats.

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In the present study, we studied 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 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, many type of mutations in OCTN2 were found with functional loss. Accordingly, organic cations that are transported by OCTN2 are suggested to exhibit altered pharmacokinetic behavior in the people who have the heterozygousity. This is the first demonstration of genetic polymorphism in organic cation transporters found in normal population.

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We isolated cDNAs coding rat organic anion transporters, OAT-K1 and OAT-K2, expressed specifically in the brush-border membranes of the proximal straight tubules. In this study, we have examined the physiological roles of OAT-K1 and OAT-K2 in the urinary excretion of anionic drugs with the in vitro and in vivo experiments.
Methotrexate efflux via these transporters in the stable transfectants was stimulated in the presence of extracellular organic anions, 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 In addition, the renal clearance of methotrexate was markedly decreased in 5/6 nephrectomized rats, compared with that in sham-operated rats. Moreover, 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.

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Background & Aims: One approach to the development of targeted cancer chemotherapy exploits increased uptake of the agent into neoplastic cells. In this scenario, higher concentrations of the agent in cancer cells are responsible for differential killing while the low concentration in normal human cells decreases side effects. The aim of this study is to isolate an organic anion transporter which is weakly in normal, but abundantly expressed in cancer cells, to deliver the anti-cancer drugs to the cells.
Methods: A human liver cDNA library was screened with LST-1 cDNA as a probe. Northern blot analyses were performed using the isolated cDNA (termed LST-2). A LST-2 specific anitbody was raised and immunohistochemical analyses were performed. Xenopus oocyte expression system was used for functional analysis. We also established a permanent cell line which consistently expresses LST-2 to examine the relationship between MTX uptake and sensitivity.
Results: The islated cDNA, LST-2 has 79.7 %.of overall homology with human LST-1. LST-2 exclusively expressed in the liver and its immunoreactiviy was highest at the basolateral membrane of the hepatocytes around the central vein. Although its weak expression in the liver, LST-2 is abundantly expressed in the gastric, colon and pancreatic cancers. On the other hand, the LST-1 was only detected in a hepatic cell line. LST-2 transports methotrexate in a saturable and dose dependent manner. Introduction of the LST-2 gene into mammalian cells potentiates sensitivity to methotrexate.
Conclusion: LST-2 is a one of the prime candidate molecule for determining methotrexate sensitivity and may be a good target to deliver anti-cancer drugs to the gastrointestinal cancers.

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We have studied intestinal metabolism and transport, which is considered as sequential (1) or competitive (2) process in absorption (Scheme 1). (1) Disaccharide (maltose, cellobiose, lactose) conjugates of p-nitrophenol were hydrolyzed to p-nitrophenyl β-glucosides (p-NPβglc) on the mucosal side. p-NPβglc was transported by Na⁺/glucose cotransporter (SGLT1). Transport clearance of p-NPβglc formed from cellobiose and lactose conjugates of p-NP were higher than that from maltose or of p-NPβglc itself. These results suggest that SGLT1 may be cooperatively coupled with lactase/phloridzin hydrolase catalizing hydrolysis of cellobiose and lactose conjugates. There might be cooperative relationship between peptidase and H⁺/oligopeptide cotransporter or amino acid transporter as well. (2) Kyotorphin (KTP) was too unstable in intestine to be absorbed. KTP appeared on the serosal side in the presence of peptidase inhibitors. Meanwhile, cyclic KTP was stable in intestine to be absorbed. Absorption clearance of cyclic KTP was higher than the overall transport clearance of KTP, which was calculated according to the metabolic inhibition model. These results indicate that metabolism degradation and membrane transport are competitive. Unless drug is stabilized against metabolic enzyme, intestinal absorption of drug can not be improved even if membrane transport is increased.

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Gamma aminobutyric acid (GABA) and acidic amino acids act as the predominant suppressive and excitatory neurotransmitter in the brain, respectively, and are widely distributed over the central nervous system (CNS). For GABA transport, the initial uptake rate of [³H]-GABA by TM-BBB was sodium- and chloride-dependent as well as being concentration-dependent. Its initial uptake was inhibited by the several GABA transporters inhibitors. RT-PCR analysis showed that GAT2/BGT1 was predominantly expressed in the GAT isoforms. For acidic amino acid transport, the efflux rate constants of [³H]L-Asp and [³H]L-Glu from brain across the BBB were 0.207 and 0.0346 min^-1, respectively. [³H]D-Asp was not eliminated from the brain over a 20-min period. The initial uptake rate of [³H]L-Asp by TM-BBB was sodium- dependent as well as being concentration-dependent. Its initial uptake was not observed the self-inhibition at 600 μM unlabeled compounds, therefore, [³H]L-Asp was mainly transported via any other transporter rather than EAAT1 ?? 3 at the BBB. These BBB transport may partially regulate the brain distribution and control their brain interstitial fluid concentration by supporting both glial and neuronal transporters.

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This study was carried out to investigate the transport mechanism of basic fibroblast growth factors (bFGF) through blood-brain barriers (BBB). Following an internal carotid artery perfusion, ¹²⁵I-bFGF was transcytosed through the BBB from blood into brain parenchyma. The saturable binding of ¹²⁵I-bFGF to the isolated bovine brain capillaries (K_d = 40 nM, and B_max = 200 pmol/mg) was significantly inhibited by unlabelled bFGF, poly-L-lysine, heparin and glycosaminoglycans with a sulfate residue. The heparin-resistant binding of ¹²⁵I-bFGF to the conditionally immortalized mouse brain capillary endothelial cell lines (TM-BBB) was saturable and showed significant dependence on temperature and medium osmolarity. This binding was effectively inhibited in cells treated with heparinase, sodium chlorate and an antibody specific to perlecan, a HSPG. Finally, RT-PCR analysis revealed expression of perlecan, and FGF receptors (FGFR1 and FGFR2) mRNA in TM-BBB. These results are consistent with the conclusions, 1) ¹²⁵I-bFGF is transcytosed through the BBB, and 2) transport of ¹²⁵I-bFGF through the BBB may be, in part, ascribed to endocytosis mechanism that is triggered by binding to HSPG.

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Multidrug resistance-associated protein 2 (MRP2), along with MRPI, transports glutathione and glucuronide conjugates as well as non-conjugated organic anions. In the present study, we examined the role of charged amino acids in the transmembrane domains of rat Mrp2, conserved among MRP families, by means of sitedirected mutagenesis. A single amino acid mutation from Lys to Met at 325 (K325M), from Arg to Leu at 586 (R586L) and from Asp to Asn at 329 (D329N) resulted in a marked reduction in the transport activity of glutathione-S conjugates (2, 4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene C₄ (LTC₄)) without affecting that of the subsequent metabolites of LTC₄ (LTD₄, LTE₄ and LTF₄) or glucuronide conjugates (17β-estradiol 17-β-D-glucuronide (E₂17βG) and E3040-glucuronide). In contrast to the reduced affinity for DNP-SG and LTC₄, the affinity for E₂17βG was increased several-fold in these mutants, suggesting that amino acids at 325 and 586 may play an important role in distinguishing between glutathione and glucuronide conjugates. Furthermore, R1096L acquired the ability to transport TC, which is not transported by Mrp2. Since Mrp3, which transports TC, has a neutral amino acid at 1084 corresponding to this position, the charge-dependence on the transport ability of TC was further investigated in both Mrp2 and Mrp3. In the case of Mrp2, substitution of Arg at 1096 by Lys (R1096K) and Val (RIO96V) also resulted in the acquisition of the ability to transport TC. In contrast, substitution of Leu at 1084 of rat Mrp3 by Lys (L1084K_Mrp3) resulted in the loss of transport activity for both E₂17βG and TC, although transport activity for these compounds was unaffected by substitution by Val (L1084V_Mrp3) or Met (L1084M_Mrp3). This suggests that the charged moieties at 1096 of Mrp2 and 1084 of Mrp3 do not simply govern the transport ability for TC; Arg at 1096 acts as an obstacle to the transport of TC in rat Mrp2, whereas a neutral amino acid at the corresponding position in Mrp3 (Leu at 1084) is required for transport of both TC and E₂17βG.

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It is well-known that only free drug in blood is concerned with pharmacological activity. In case a therapeutic drug is administered with another displacer, which may displace competitively at the binding site of serum protein, the free fraction of this drug would be increased and consequently produce a high biological activity. So if the binding of radiopharmaceuticaltso serum proteinc an be inhibited by displacers with high protein binding affinity, the total clearance and tissue distribution of this tracer would be enhanced. In this study, the interaction between ¹²³I-labeled N-isopropyl-p-iodoamphetamine (¹²³I-IMP) and several binding displacers was evaluated to improve cerebral imaging in rats and a monkey. The serum protein binding of ¹²³I-IMP was evaluated by ultrafiltration. The free fraction rate of ¹²³I-IMP was increased up to 1.2 times of control with albumin displacers. 6-Methoxy-2-naphtylacetic acid (6MNA), a clinically available HSA site II displacer, was selected for following studies. The rat biodistribution showed more rapid clearance of ¹²³I-IMP with 6MNA loading. In scintigraphic study in rats, cerebral accumulation was slightly accelerated. It was also observed hasten cerebral accumulation in monkey dynamic scintigraphy. Indeed, 6MNA treatment increased free ¹²³I-IMP in monkey serum study. Therefore, rapid and high cerebral accumulation was achieved. The displacement method could easily be applied to human study. The displacement of ¹²³I-IMP binding on serum protein could shorten imaging time and reduce radiation dose for patients. Furthermore, in other radiopharmaceuticals with high protein binding, the competitive displacement on specific binding sites can control their tissue distribution and kinetics in clinical application.

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Cationic liposome-mediated transfection is a useful method for in vitro and in vivo gene transfer. However, there is little information on the relationship between physicochemical and structural properties of DNA-cationic liposome complexes and their transfection efficiency. In this study, we have systemically studied the relationship between various properties of DNA-cationic liposome complexes and their transfection efficiency in vivo and in vitro. Our results demonstrated that for both in vitro and in vivo transfection, structural and physicochemical characteristics of the complexes were significantly related to their transfection efficiency. In in vitro transfection, the charge ratio between DNA and cationic liposome significantly affects the release of plasmid DNA complexed with the cationic liposomes from the complexes, which is an important determinant of the optimal charge ratio. For in vivo intravenous transfection, Chol-containing complexes with a higher transfection activity showed a stable lamellar structure and was more stable to the interaction with erythrocytes. DOPE-containing complexes, a less efficient vector, induced the fusion between erythrocytes due to its fusogenic property and lost their transfection activity. These results indicate that the interaction with erythrocytes is an important factor for intravenous gene delivery.

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Plasma concentration and vasodilating effect after i.v. bolus injection of stereoisomeric organic nitrates were evaluated. Pharmacokinetics of mononitrates were analyzed with a linear 1-compartment model. The distribution volumes were identical but systemic clearances were different among stereoisomers. The concentration data after dinitrate administration could be described based on a 2-compartment model for dinitrate distribution, with elimination only from central compartment via metabolism to mononitrate, and then mononitrate-dependent metabolic clearance was estimated. In the vasodilation by mononitrate administered intravenously, the maximum effect was not observed. The difference from baseline level of mean arterial pressure (ΔMAP) was related to plasma concentration with a log-linear model, and an identical slope and different intercepts among isomeric mononitrates were obtained. The pharmacological effect following dinitrate dosing were analyzed a sigmoidal Emax model assuming additive effect of dinitrate and mononitrate. Although almost the same Hill's constant and maximum effect values were estimated, EC50 values were different among stereoisomers. The clearance and EC50 values of stereoisomers with nitrate group at the exo position were generally higher than those at the endo position. It suggests that stereostructure of organic nitrates controls the vasodilator potency and duration of action.

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Stereoselective debromination of R, S-(α-bromoisovaleryl)urea (R, S-BIU) to isovalerylurea (IVU) in rats in vivo and in vitro was shown. IVU was isolated and identified in the blood of rats dosed R, S-BIU. When S- or R-BIU was intraperitoneally dosed to rats, the amounts of IVU from S-BIU was higher than that from R-enantiomer. The amounts of IVU from S-BIU was enhanced by the pretreatment of rats with phenobarbital. The debromination of BIU by rat liver slices showed the similar stereoselective debromination and was stimulated by the pretreatment of rats with phenobarbital. In contrast, the stereoselective debromination of BIU by rat blood and rat cecum contents was not observed. Human liver microsomes exhibited the debrominating activity, but the activity was markedly varied among samples. When the debromination activity was examined using recombinant CYPs expressed human CYPs in human B limphoblastoid cells, CYP2A6 exhibited the highest activity, and followed by CYP2B6, 2D6 and 3A4. In these cases, the stereoselective debromination toward S-BIU was also observed.

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We have developed a strategy for the quantitative prediction of drug-induced Parkinsonism based on receptor-occupancies of the domapine D-1 receptor (DRD1), the dopamine D-2 receptor (DRD2) and the muscarinic acetylcholine receptor (mAChR). The clinical risk of an individual prescription to induce Parkinsonism can be quantitatively predicted by this strategy from the set of pharmacokinetic data in humans and the affinities of each drug for DRD1, DRD2 and mAChR. The inter-individual variation of the risk may be also predictable by using this strategy if several individual factors, such as hepatic and renal functions, genetic polymorphisms of metabolic enzymes and receptors etc., are properly incorporated. This strategy can be applied onto a computer-aided system to select the best prescription for a patient.

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We have investigated a quantitative prediction of in vivo human hepatic clearance from in vitro experiments. In vivo intrinsic clearance (CL_{int, in vivo}) were compared with in vitro intrinsic clearance (CL_{int, in vitro}) from in vitro metabolism data obtained by using liver microsomes focusing on P450 metabolism in rats, dogs and humans. The scaling factor (CL_{int, in vivo}/CL_{int, in vitro}) were different among the model compounds, while most of the animal scaling factors were within 2-fold of the values in humans. When human CL_{int, in vitro} values were compared with the actual CL_{int, in vivo}, correlations were not necessarily good. By contrast, using human CL_{int, in vitro} corrected with the animal scaling factor yielded better predictions of CL_{int, in vivo} that were mostly within 2-fold of actual values. Further, CL_{int, in vivo} were compared with CL_{int, in vitro} from in vitro metabolismd ata obtained by using rat fresh and human cryopreserved hepatocytes. Human CL_{int, in vitro} corrected with the rat scaling factor yielded good predictions of CL_{int, in vivo} that were mostly less than several-fold of actual values. This method thus provides a more reliable prediction of human hepatic clearance and will be useful in drug discovery.

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Recombinant wild-type and mutant human serum albumins were produced using a yeast expression system. We investigated, by ultrafiltration, the high affinity binding of two representative site II-ligands, namely ketoprofen(KP) and diazepam(DZ). According to the crystal structure of HSA, the residues of ⁴¹⁰Arg and ⁴¹¹Tyr protrude into the center of site II (in subdomain 3A), and the binding results showed that the guanidino moiety of ⁴¹⁰Arg the phenolic oxygen and the aromatic ring of ⁴¹¹Tyr are important for KP binding. The guanidino moiety probably interacts electrostatically with the carboxyl group of KP, the phenolic oxygen could make a hydrogen-bond to the ketogroup of the ligand, and the aromatic ring may participate in a specific stacking interaction with one of or both of the aromatic rings of KP. By contrast, ⁴¹⁰Arg is not important for DZ binding. In addition to its unique ligand binding properties, HSA also possesses an esterase-like activity, and studies with p-nitrophenyl acetate as a substrate showed that although ⁴¹⁰Arg is important, the enzymatic activity of albumin is much more dependent on the presence of ⁴¹¹Tyr. In order to characterize the warfarin (WF) binding site, ¹⁹⁹Lys, ²¹⁴Trp, ²¹⁸Arg and ²⁴²His were mutated. At pH 7.4, the affinity of H242Q and K199A were found to be about 6 and 4 times higher as compared to that of wild-type HSA, respectively. However, the affinity of R218H and W214A for WF were about 3 and 2 times reduced.These results show that the side chain of ²¹⁴Trp and ²¹⁸Arg are important for WF binding. In contrast, t he side chains of ¹⁹⁹Lys and ²⁴²His interfere with binding. Finally, the effect of the N-B transition on WF binding will be discussed.

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The mechanisms underlying nephrotoxicity, specifically proximal tubular acidosis and albuminuria, induced by cisplatin (CDDP) were investigated. In the kidney, Na⁺/H⁺ exchanger and vacuolar (V)-H⁺-ATPase are two major H⁺ excretion systems located in the brush-border membrane. In brush-border membrane vesicles isolated from CDDP-treated rats, the activity of V-H⁺-ATPase, but not Na⁺/H⁺ exchanger, decreasedm arkedly. In OK cells, CDDP treatment induced intracellular acidification. Thus, the inhibition of V-H⁺-ATPase would be involved in the proximal tubular acidosis induced by CDDP. V-H⁺-ATPase, as well as chloride channel ClC-5, in the endosomal membrane is important for the endocytic system. In OK cells, endocytosis of albumin was inhibited by CDDP, as well as by inhibitors for V-H⁺-ATPase and ClC-5. In vivo, CDDP administration to rats induced albuminuria, with maximum effect observed at 2-3 days after administration. Such a pattern was comparable with the inhibition of CDDP on V-H⁺-ATPase. Similar pattern was also observed for the urinary excretion of vitamin D binding protein, which is specifically taken up by the proximal tubular cells via megalin-mediated endocytosis. Taken together, the inhibition of V-H⁺-ATPase by CDDP would result in not only proximal tubular acidosis but also albuminuria (proteinuria).

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Troglitazone, a new oral antidiabetic drug, has been reported to cause idiosyncratic hapatitis in certain individuals. The mechanism for hepatic failure was investigated with comparison between troglitazone and its metabolites and other thiazolidinedions. Oxidation pathway of troglitazone to a qunone-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 CYPs were not potent. Autoimmune antigen was identified in patients with idiosyncratic hepatitis. Hepatic toxicity did not appear in troglitazone treated rats after modifications of sulfotransferase, gluclonosyl-trasferase, or glutathione S-transferase activities. 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. Taking these results into consideration, the causal factor(s) for idiosyncratic hepatitis in human remained unclear.

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Liver injury induced by an overdose of acetaminophen (APAP) has been known to be initiated by covalent binding of the reactive N-acetyl-p-benzoquinone imine metabolite to liver target proteins, but factors other than the covalent binding contribute to the severity of the toxicity. The present study focused on the role of cytokines, which are released mainly from liver nonparenchymal cells, on APAP hepatotoxicity in mice. Liver mRNAs of interleukin-6 (IL-6), IL-10 and IL-11, which are considered to suppress inflammatory tissue injury, increased markedly after the administration of hepatotoxic doses of APAP, as did serum levels of the expressed cytokine proteins. Hepatotoxicity of APAP was exaggerated in mice deficient in IL-6 (IL-6 -/- mice) and IL-10 -/- mice as compared with corresponding wild-type mice. Administration recombinant human IL-11 protein prior to the APAP challenge blocked the liver injury. These results demonstrated the protective role of these cytokines against the APAP hepatotoxicity. Increases in liver mRNAs of tumor necrosis factor-α and IL-1β were observed in the APAP-challenged IL-10 -/-mice, but not in IL-6 -/- mice, suggesting that these proinflammatory cytokines are involved in the initiation of APAP-induced liver injury in mice lacking IL-10.

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My research has been supported by many many peoples including my professors and collaborators. What I could done in the research varied from basic to clinical ones will be described briefly. Instead of showing over all my research. I would like to introduce how big support I have obtained and an example of project which I could perform for cancer patients.

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The complete sequencing of the human genome will bring the paradigm shift in drug discovery and development and new diagnosis and treatment of disease. Genetic determinants of drug response include polymorphisms that affect drug absorption, distribution (targeting), metabolism, excretion and receptor activation. Knowing the main genetic determinants of drug response allows us to design drugs acting at target molecules (proteins/genes) critical for disease progression, and to select the most appropriate drug. Under such circumstances, pharmaceutical scientists (particularly, scientists in DMPK (drug metabolism and pharmacokinetic) ) should have further responsibility to apply appropriately the new scientific outputs obtained in the structural and functional analyses of genomics to the discovery and development of new medicines. Taking my efforts to develop the extrapolation methods of pharmacokinetic properties based on in vitro measurements with the use of human tissues and enzymes/transporters expression systems as examples, I would like to share with you my idea on what kind of efforts we DMPK scientists should make in the new millennium. It is also unanimously important for us to educate younger generation so that they can have flexible spirits of challenging new research area and of creating the new areas of science not sticking to the conventional scientific concepts and tools.

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Although passive diffusion, which depends on the lipid solubility, has been believed to be an exclusive mechanism for the membrane transport of many compounds, we have demonstrated that several drugs are transported into or out from the cells by the tissue-specific transporters in intestinal and renal epithelial cells, hepatocytes and brain capillary endothelial cells which form the blood-brain barrier. During these studies, we found the genetic polymorphism in drug transporters, using organic cation/carnitine transporter OCTN2. Only from the volunteers that showed low carnitine blood concentration, several kinds of heterozygous mutations of OCTN2 gene that show functional loss were found. Furthermore, based on the phenotype observed by systemic carnitine deficiency, many types of mutations in OCTN2 were found with functional loss for carnitine and several organic cations. Accordingly, organic cations that are transported by OCTN2 are suggested to have altered pharmacokinetic behavior in the people who have an allele heterozygously. This is the first demonstration of genetic polymorphism in organic cation transporters found in normal population. Such pharmacogenomic study is a rapidly emerging field focused on understanding the genetic factors underlying individual pharmacokinetics of drugs among patients and applying this information to improve the clinical use and development of pharmaceutical products.

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