Development of vascular system is a complex process in which generation of cellular components and their organization into a highly hierarchial structure are undertaken in a short period of time. The latter process is further divided into formation of the primitive vascular plexus and remodeling processes. Despite of the increasing list of remodeling molecules, however, we understand only a little how these molecules regulate the process. The major reason for this problem might be an absolute deficit of our knowledge about cell biology of endothelial cells (EC) and mural cells (MC), which is essential for correlating phenotypes displayed at structural level to the phenotype at cellular level.
To overcome this problem, we have developed a method to generate distinct sets of ECs and MCs from ES cells, surface markers defining populations at intermediate stages during differentiation of vascular cells, a monitoring system to assess the response of each EC and MC to various stimuli, methods to deliver genes to EC specifically, and finally a culture system in which an organized architecture is produced through cell-cell interactions. In this symposium, we will describe the latest status quo and also a future potential of our in vitro system. While the system itself is yet far from complete, the present system can address to some questions in cell biology of remodeling. The first question that I addressed using this system is the role of VEGFR3 in vascular remodeling. We will present evidence showing that VEGFR3-signal stablilizes the EC sheet that are agitated by VEGFR2-signal. Moreover, we will also present a method to analyze EC/MC interaction during vasculogenesis.

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Cells are endowed with mechanisms to protect them from chemical insults. One of such mechanisms is metabolism of chemicals that can be either endogenous, naturally-occurring, or synthetic origin. The metabolisms primarily occur in liver that is followed by kidney and intestine. In general, chemicals are first metabolized by P450 (CYP) enzymes and subsequently are conjugated by transferases, called the Phase I and Phase II enzymes, respectively. To maximize metabolic capability, liver cells induce metabolizing enzymes and also drug transporters. Although induction can be regulated at various steps during synthesis of enzyme proteins, gene transcription plays the primary role in the regulation. There are three major mechanisms that mediate drug-inducible gene transcription: Ah receptor (AhR)-mediated, Nrf2-mediated, and nuclear receptor-mediated mechanisms.

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CS-747, a prodrug-type thienopyridine anti-platelet agent, was converted to pharmacologically active metabolites in vitro in isolated rat hepatocytes. The active metabolites had a free sulfhydryl group, and were associated with 2 asymmetric carbons. Of 4 optical isomers of the active metabolites, one optical isomer showed a high activity in inhibiting the platelet aggregation. Other metabolites of CS-747 without having free sulfhydryl group, produced by rat hepatocytes in vitro, were pharmacologically inactive. The active metabolites were also detectable in plasma after oral administration of CS-747 to rats and dogs. In the plasma of rats and dogs after administration of 14C-CS-747, disulfide-type cysteine conjugates of the active metabolites were detected as the most major metabolite. The cysteine conjugates of the active metabolites were not pharmacologically active in vitro while these were active in vivo after intravenous administration to rats, suggesting their conversion to the active metabolite after disulfide reduction. The activity to produce the active metabolite in 9000g supernatant fraction of rat liver was NADPH-dependent and inducible by phenobarbital, suggesting an involvement of cytochrome P450. Experiments using various isoforms of expressed human liver cytochrome P450 indicated that CYP3A4 and CYP2B6 are involved in this metabolic activation.

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The metabolism and hypnotic mechanism of N³-phenacyluridine were studied in mouse. Of the radioactivity, 65% was excreted in urine within 48 hours after i.p. administration of [³H]N³-phenacyluridine. The urinary metabolites N³-phenacyluracil and N³-α-hydroxy-β-phenethyluridine were excreted, isolated and analyzed by mass spectrometry. Racemates of N³-α-hydroxy-β-phenethyluridine were synthesized and both isomers were separated as N³-(S)-(+)- and N³-(R)-(-)-α-hydroxy-β-phenethyluridine by HPLC (CHIRALCEL-OJ column). The reduction process took place with high stereo-selectivity, which gave an alcohol product in the urine with same retention as one of the synthetic isomers separated by HPLC. N³-Phenacyluracil and uridine were identified as minor metabolites. N³-(S)-(+)-α-hydroxy-β-phenethyluridine, but not N³-(R)-(-)-α-hydroxy-β-phenethyluridine, possessed hypnotic activity in mice and potentiated pentobarbital-induced sleep with a similar potency to the parent compound, N³-phenacyluridine. An active metabolite, N³-(S)-(+)-α-hydroxy-β-phenethyluridine, inhibited [³H]N³-phenacyluridine binding to synaptic membrane of bovine thalamus showing 10.2 nM of Ki value, while N³-phenacyluridine, N³-(R)-(-)-α-hydroxy-β-phenethyluridine and racemate had 0.65, 397.4 and 1908 nM of Ki values, respectively. The present study indicates that N³-(S)-(+)-α-hydroxy-β-phenethyluridinea, major metabolite of N³-phenacyluridine, is an active metabolite and bound a novel uridine receptor involving the hypnotic activity.

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Human intestinal microsomes were found to catalyze dealkylation and hydroxylation of an antihistamine ebastine and the former was shown to be mediated by intestinal CYP3A. Ebastine hydroxylase was purified from monkey intestinal microsomes and it was identified as a CYP4F isoform by the amino acid sequence. CYP4F cDNAs were then cloned from human intestine and expressed in yeast. A novel CYP4F (4F12) only exhibited the high activity of the hydroxylation, however, its actual contribution in human microsomes was only partial (about 20%) when judged by immunoinhibition study using anti-CYP4F antibody. From a marked inhibition of ebastine hydroxylation by arachidonic acid, CYP2J2 was speculated to involve in the hydroxylation. In fact, anti-CYP2J antibody inhibited 70% of ebastine hydroxylation. The finding above that human intestinal CYP2J and CYP4F as well as CYP3A play an important role in the drug metabolism extends the concept of studies of the drug metabolism in the intestine, the organ being enrolled in the first pass effect.

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Capecitabine, an orally administered triple prodrug of 5-FU shows tumor-preferential exposure of 5-FU by being sequentially metabolized to 5-FU by three enzymes, which show relatively specific organ expression. To investigate the mechanism of tumor-preferential exposure of 5-FU after oral administration of capecitabine, a physiologically based pharmacokinetic model describing the pharmacokinetic behaviors of capecitabine and its metabolites including 5-FU in humans was constructed. The factors that have the greatest influence on the pharmacokinetics of 5-FU after administration of capecitabine were clarified by sensitivity analyses. The sensitivity analysis demonstrated the exposure of tumor tissue to 5-FU depends mainly on the activity of both thymidine phosphorylase (producing enzyme of 5-FU from the precursor, 5'-DFUR) and DPD (eliminating enzyme of 5-FU) in tumor tissue, as well as blood flow rate in tumor tissue with saturation of DPD activity resulting in higher 5-FU AUC in tumor tissue. The therapeutic index of capecitabine was found to be at least 17 times greater than that of other fluoropyrimidine, including doxifluridine, the prodrug of 5-FU, and 5-FU over their respective clinical dose ranges.

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In the drug discovery stage, in vitro metabolism studies such as metabolic stability, species differences, identification of metabolic enzymes, inhibition and/or induction of metabolic enzyme using with materials derived from human tissues will be presentated.

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In past years, we experienced some incidents which realize the significance of human in vitro metabolism. In a case of 156-S, this drug caused drug-drug interactions in human clinical study but not observed in rats. Differences between CYP2C enzymes in human and rat were observed in vitro later, and confirmed recently by computational 3D-model analysis. After 156-S and terfenadine incidents, we established human in vitro test system for drug-drug interactions. Rilmazafone was an example that human in vitro studies revealed not to be affected by CYP inhibitors since hydrolysis pathway is the major elimination route. And, in a certain project, in vitro metabolism of compounds with human liver microsomes injected us an insight to chose appropriate lead compound from four candidates. As mentioned above, human in vitro studies give us a lot of important information. As future aspects, human hepatocyte system covers more comprehensive metabolic mechanisms, and useful to postulate metabolites formed in vivo. Further, computational 3D-model analysis of drug metabolizing enzymes will be a powerful tool to estimate metabolic stability and possibility of enzyme inhibition.

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The mammalian carboxyle sterase comprise a multigene family, the gene products of witch are localized in the endoplasmic reticulumo f many tissues. These enzymes efficiently catalyze the hydrolysis of a variety of drugs or prodrugs containing ester- and amide-bond to the respective free acids and alcohol. Since ester derivatives of therapeutic agents have been in use as prodrugs, carboxylesterase are a major determinant of the pharmacokinetic behavior of most prodrugs, and the activity can be influenced either by direct interactions of a variety of compounds either directly or at the level of enzyme regulation. On the other hand, mammalian carboxylesterases represent a multigene family the products of which are localized in the endoplasmic reticulum of many tissues. Among various tissues of animals, the highest hydrolase activity towards various substrates is found in the liver. These hydrolase activities are present in a number of tissues in addition to the liver. Humans also express carboxylesterase in liver, small intestine, brain, kidney, lung, and plasma. Since a significant number of drugs are metabolized by carboxylesterase, altering the activity of this enzyme expressed in each tissue has important clinical implications.. However little is known about the differences in structure and hydrolytic capability of carboxylesterase isozymes expressed in each tissues of humans. In the present study, we have undertaken the structural characterization of carboxylesterase isozymes expressed in human liver, small intestine and brain to learn more about the molecular basis responsible for their functional differences.

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Interaction between cytochrome P450 (P450) and other sorts of drug metabolizing enzymes was studied by affinity chromatography with CYP1A1-conjugated column. Solubilized rat liver microsomes were charged onto the column and the eluate was analyzed by immunoblotting. The result showed that the CYP1A1 column effectively trapped microsomal epoxide hydrolase (mEH) and UDP-glucuronosyltransferase (UGT) as well as NADPH-P450 reductase. Non-drug metabolizing enzymes such as calnexin and protein disulfide isomerase did not show any affinity to the column. Neither BSA- nor glycine-conjugated column could trap microsomal protein. These results strongly suggest the specific interaction between P450 and mEH/UGT. When estimated using stereo-selective hydrolysis for styrene oxide as the index, the function of rat mEH was enhanced by co-incubation with CYP1A1, 2B1 and 2C11. The same picture was also seen for the interaction between dog mEH and CYP2B11/3A12. These results suggest that P450 interacts with mEH and UGT to facilitate a series of multistep metabolic conversion.

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A cDNA of CYP2D29 was cloned from Japanese monkey (JM) livers. Its deduced amino acid sequence exhibited 96% homology to that of human CYP2D6, which was higher than the values of Cynomolgus monkey CYP2D17 (93%) and Marmoset CYP2D19 (91%). CYP2D29 protein was expressed in yeast cells, and the microsomal oxidation activities towards debrisoquine (DB), bunitrolol (BTL) and bufuralol (BF) were determined, and compared with those of recombinant CYP2D6, and of JM and human liver microsomes (Ms). For DB 4-hydroxylation, recombinant CYP2D29 and CYP2D6 showed similar Km and Vmax values, whereas the Km value of JM liver Ms was 20 times that of recombinant CYP2D29. JM liver microsomal DB 4-hydroxylase was markedly inhibited by quinidine and anti-rat CYP2D2 antibody. A similar tendency in the kinetic parameters was observed in BTL 4-hydroxylation, but Km values for BF 1″-hydroxylation were similar between recombinant CYP2D29 and JM liver Ms. These results suggest that the oxidation of DB and BTL in JM livers is mediated not only by CYP2D29 but also by another CYP(2D) enzyme. The usefulness of the monkey as a model of human in drug metabolism study will be discussed.

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A rationale is described to explain that the urinary 6β-hydroxycortisol/cortisol (6β-OHF/F) ratio does not appropriately reflect in vivo CYP3A activity (phenotype). To evaluate CYP3A-mediated 6β-hydroxylation of cortisol in vivo, “fractional metabolic clearance” specific for the 6β-hydroxylation was calculated by measuring both urinary 6β-hydroxycortisol and plasma cortisol after administering stable isotopically labelled cortisol to humans. It is shown that the urinary 6β-OHF/F ratio is a function of metabolic clearance and renal clearance of cortisol. The observed intra- and interindividual variabilities of the renal clearance indicate that the urinary ratio is not appropriate for the CYP3A phenotyping. We propose a safe and reliable CYP3A-phenotyping.

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The membrane transport processes of drugs are critical issues to determine their absorption, distribution and elimination. Recently, various drug transporters have been identified and characterized. In the small intestine, a peptide transporter PEPT1 mediates the absorption of various peptide-like drugs including β-lactam antibiotics as well as amino acid ester compounds lacking peptide bond. In the kidney, the basolateral organic ion transporters OAT1, OCT1 and OCT2 are mediate renal distribution of hydrophilic anionic and cationic drugs, respectively. The OAT1 was indicated to be a potential interaction site between methotrexate and nonsteroidal antiinflamatory drugs, and the brush-border type OAT-K1/K2 could be a target transporterf or methotrexate-leucovorine rescue therapy. The pharmacokinetic roles of P-glycoprotein (MDR1) have been clarified in various tissues. The renal P-glycoprotein was revealed to be an interaction site between digoxin and clarithromycin or itraconaxzole. In addition, the intestinal P-glycoprotein was suggestedto act as an absorptive barrier for tacrolimus in recipients of liver and small bowel transplantation.

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Recently, molecular cloning methodologies have enabled us to clone the cDNA of transporters, and characterize the transport activity using cDNA transfectants. It is necessary to examine in future studies the contribution of each transporter to the overall disposition of some drugs. The contribution of a transporter can be evaluated using selective inhibitors. In this study, we focused on organic anion transporters such as organic anion transporting polypeptides (OATPs; rOatp1 ?? 3) and organic anion transporters (OATs; rOat1 ?? 3). Stable cDNA transfectants have been established using LLC-PK1 cells as host and inhibition studies for the uptake of their typical ligands were carried out. Digoxin is a specific inhibitor for rOatp2, while cimetidine and benzylpenicillin (PCG) are selective inhibitors for rOat3. Taurocholate has similar affinity for rOatp1 ?? 3, but quite low affinity for rOat1 ?? 3. p-Aminohippurate (PAH) is a inhibitor for rOat1 and rOat3, but not for rOat2 (Ki>1 mM) and rOatp1 ?? 3. Using these inhibitors, we have evaluated the contribution of rOatp2 on the blood brain barrier, and that of rOat3 to the uptake of PCG and PAH, and pravastatin into the choroid plexus and kidney, respectively, has been evaluated. Estradiol 17β glucuronide is rapidly eliminated from the brain (elimination half life is approximately 20 min). By examining inhibitory effect of digoxin, the contribution of rOatp2 has been estimated at most 40% and additional uptake system is present on the blood-brain barrier. The uptake of PCG and PAH, and pravastatin into the choroid plexus and kidney is mainly accounted for by rOat3.

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Hepatobiliary transport of three new drug candidates were investigated to evaluate the contribution of membrane transporters on their pharmacokinetics. Compound A is an anionic cyclopentapeptide endothelin receptor antagonist, Compound B is an anionic endothelin antagonist (non-peptide), and Compound C is a neutral indolocarbazole anticancer agent. In rats, all three compounds were excreted predominantly into bile without oxidative metabolism. Compounds A, B and C were taken up extensively by isolated rat hepatocytes with Km values of 9.5, 5.7 and 8.9 μM and Vmax values of 517, 564 and 1600 pmol/min/10⁶cells, respectively. The uptake was ATP-dependent for Compounds A and B but not for C. In spite of discrete chemical structures, uptake mechanisms of Compounds A and B are analogous judging from their inhibition profile by various compounds. Compounds A and B were taken up into rat canalicular membrane vesicles (CMV) in an ATP-dependent manner. Contribution of cMOAT (MRP2/ABCC2) is important for canalicular transport of Compounds A and B because their ATP-dependent uptakes into CMV prepared from EHBR were insignificant.

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In vitro cell culture system is one of useful tools for analyzing function of the blood brain barrier (BBB). We have established conditionally immortalized endothelial cell lines from the transgenic rat and mouse harboring temperature sensitive SV40 large T antigen gene. These culture cells expressed GLUT-1 and mdrIa and their growth depended on the expression of is SV40 T antigen in cells. Using these cell lines together with in vivo analyzing system such as brain efflux index method, we revealed following new BBB functions: 1. Amino acid transport involved in osmoregulation. 2. Stereoselective acidic amino acid transport. 3. Efflux transport system of neurotransmitter, GABA. 4. Efflux transport system of neurotransmitter metabolites, homovanillic acid, and effect of uremic toxin on the efflux system. 5. Supply creatine as energy depot to brain. These evidences suggested that the BBB is not only a supporting system but also a protecting system for maintaining the central nervous system.

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Drug transport mechanism across the placenta is still unknown. The trophoblast that form the placental barrier have an important role in controlling the passage of molecules from mother to fetus. In this study, we investigated that dipeptide and folic acid are actively transported by transporters on the trophoblast. The human choriocarcinoma cell line, BeWo, was cultured on tissue culture plates. When the cells reachedc onfluence, drug uptake experiments were performed. The uptake of [¹⁴C]glycylsarcosine ([¹⁴C]Gly-Sar) by BeWo cells was saturated at higher concentrations. The metabolic inhibitors significantly inhibited the uFtake of [¹⁴C]Gly-Sar. In adUtion, various di- or tri-peptides inhibited the uptake of [¹⁴C]Gly-Sar. The uptake of [³H]folic acid was saturable at higher concentrations and inhibited by metabolic inhibitors. Analogs of folic acid, methotrexate and 5-methyltetrahydrofolate, inhibited the uptake of [³H]folic acid by BeWo cells. Kinetic analysis using Lineweaver-Burk plots revealed that methotrexatec ompetitively inhibited the uptake of [³H]folic acid and folic acid competitively inhibited the uptake of [³H]methotrexate. The findings confirm that dipeptide and folic acid are transported by their transporters on BeWo cell monolayers. The dehydroepiandrosteron sulfate (DHEAS) uptake by BeWo cells from basal side of the cells was higher than that from apical side, and the uptake was inhibited by an excess amount of DHEAS. The findings confirm that the carrier of DHEAS exists on the basal membrane of BeWo cells. This workd emonstrated the possibility of using a human monolayer-forming cell line, BeWo, to characterize putative trans-trophoblast transport mechanisms and their potential roles in controlling nutrients and drug across the placental barrier.

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In the present study, we examined the tissue distribution and functional characteristics of a member of organic cation/carnitine transporter family, OCTN1 in human by using membrane vesicles prepared from the HEK293 cells that were stably expressing human OCTN1. Immunohistochemical analysis of mouse OCTN1 in kidney demonstrated that OCTN1 is expressed strongly in epithelial cells of tubular cells in cortex. In epithelial cells, OCTN1 showed differential expression from Na⁺/K⁺-ATPase, which is a basolaterally localized enzyme, and exclusively expressed in the apical membrane of the cells. Membrane vesicles prepared from HEK293 cells that were stably transfected with human OCTN1 showed increased uptake of tetraethylammonium (TEA) compared with that by membrane vesicles form Mock-transfected cells. Uptake of [¹⁴C]TEA was apparently pH dependent and showed overshoot uptake in the presence of outwardly-directed proton gradient. Preloading of unlabeled TEA enhanced the initial uptake of [¹⁴C]TEA. These kinds of and other studies suggested that OCTN1 may be involved in the pH dependent apical membrane transport of organic cations in kidney and it may also functional as the organic cation/organic cation antiporter. Since OCTN1 is expressed in various tissues, OCTN1 may also contribute in the distribution and/or efflux of cationic compounds in those tissues by being energized by proton or cation gradients across the cellular membranes.

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We have proposed the organ surface as a novel application site of drugs, and elucidated the absorption mechanism from the organ surface. In the present study, we examined the drug distribution after application to the rat liver and kidney surface and evaluated possibility of site-selective drug targeting to the organ. We selected phenol red (PR), FITC-dextran (FD-4, Mw 4, 400), and 5-FU as model drugs. The concentration of the model drugs at the administered site after application to the rat liver surface was significantly higher than those in the other two sites. In addition, the concentration of PR and 5-FU in the right kidney after application to the rat right kidney surface was high compared with that in the left kidney, although no significant difference was observed in FD-4. A similar tendency was observed in the urinary excretion rate. Moreover, selectivity advantage of the organ surface application was proven kinetically based on the physiological model. Consequently, drug application to the organ surface could improve availability in the desired site of a new drug such as genome medicine.

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In order to investigate the effect of oxidative stress on the structure, function and pharmacokinetics of human serum albumin (HSA), oxidized HSAs were prepared by treating with chloramine-T (CT) at various concentrations (0, 1, 10, 50mM). Treatment of HSA with high concentrations of CT (10, 50mM) generated a significant increase in reactive carbonyl content while low concentration of CT (1mM) led to selective oxidation of cysteine (Cys) and methionine (Met). Structural changes were observed in spectroscopic studies of oxidized HSAs treated with 10 and 50mM of CT but not with 1mM of CT. Structural damages resulted from such oxidative stresses altered the binding properties as well as reduced the esterase-like activity of HSA. However, these alterations were little for HSA treated with low concentration of CT. These HSAs were labeled with ¹²⁵I and were then intravenously administered to rats. The half-lives of the extensively oxidized-HSAs treated with high CT concentrations decreased significantly whereas the half-life for the HSA treated with low CT concentration was comparable to that of the native HSA. In addition, since the half-life of a mutant, C34S, was also comparable to that of the native HSA, selective oxidation of Cys34 hardly leads to conformational and functional changes of HSA. Thus, the present results suggest that the Met residues, together with Cys, play an important role in the antioxidant property of HSA and that oxidation of the Met residues could be the main cause for the structural and functional changes.

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The present study was aimed to quantify the expression of mRNA of drugt ransporters, including PepT1, mrp2 and mdr1a in the small intestine and to examine the correlations between the expression and the transport activity. Variation in expression levels of transporters along the proximal-distal axis of the intestine was examined. Small intestine of starved and fed rats were removed and divided in eight segments and total RNAs isolated. In fed rats, yield of total RNA was constant among each segment, while the increase was observed in the upper part of small intestine of starved rats. Complementary DNA was prepared by reverse transcription of total RNA of each segment and real-time PCR was performed to quantify mRNA expression of various drug transporters. Expression of PepT1 mRNA was lower in upper segment than that in lower segment. In starved rats, PepT1 expression was increased significantly in the middle part of the intestine. As for mrp2, the expression was higher in upper segment and lower in lower segment and the increase in the expression by the starvation was observed in each segment of the intestine. The mdr1a expression was increased gradually along the proximal-distal axis of the small intestine and there was no difference between starved and fed rats. The actual number of PepT1 mRNA expressed in each segment was approximately 10-fold greater than that of mrp2 or mdrla. The intestinal absorption of cefadroxil (CDX) in each segment at pH6.0 was measured using Ussing chamber. Permeability coefficient of CDX was higher in the ileum and lower in the duodenum. In the starved rat, permeability coefficient of CDX in jejunum was significantly increased compared to fed rats. There was good correlation between PepT1 mRNA expression level and permeability coefficient of CDX under both fed and starved conditions. It is suggested that change in physiological and/or pharmacological state of the intestinal tissue may affect the expression and function of various transporters and thereby altars disposition of drugs.

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To develop the absorption enhancing formulation with local toxicity low enough for the practical use, we have investigated the effect of amino acids on the local toxicity and the absorption enhancement caused by sodium laurate (C12), used as an absorption enhancer. As a result, the absorption of phenol red (PR), an unabsorbable marker, was enhanced significantly by applying 10 mM C12 and the addition of amino acids did not change this absorption-enhancing effect of C12 in in situ colon loop study. On the other hand, although C 12 significantly increased the elution of phospholipids, proteins, and lactate dehydrogenase from colon, which are biological markers for local toxicity, amino acids significantly attenuated this mucosal injury caused by C12. In the present study, we tried to elucidate the mechanism involved in the protective effects of amino acids against mucosal injury caused by C12, and HSP70 was suggested to be involved in the cytoprotection by L-glutamine. Furthermore, the attenuation of intracellular Ca²⁺level and of histamine release, which are enhanced by C12, were also suggested to be mechanisms behind the cytoprotective action of amino acids.

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Inter-laboratory or inter-membrane variations in drug permeability across Caco-2 monolayers are considered to be one of the most critical problems in drug absorption screening performed in pharmaceutical companies. We have already reported that the variations in drug permeability found in Caco-2 or MDCK monolayers are basically due to the differences in the paracellular permeability through cell junctions. In this paper, paracellular permeability of various drugs was calculated according to the pore-theory proposed by Renkin¹⁾, then their permeability to the standard membrane having the defined pore size of the paracellular pathway was re-calculated. Through this mathematical correction process, inter-laboratory or inter-membrane variations in drug permeability could be minimized, giving the standardized permeability of all drugs. The experimental conditions such as pH of the transport medium influence the permeability of weak-electrolyte drugs²⁾. Assuming that the pH of the apical medium mainly affects the transcellular transport of drugs, drug permeability of defined pH condition was estimated from the concentration of unionized molecule of drugs. The results obtained in this research are quite important to predict the oral drug absorption in humans from the in vitro study with Caco-2 or other cell monolayers.

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DDS technology required for an effective in vivo gene transfer depends on target disease. Important features are target cell-specificity, level, duration and cell number of gene expression following in vivo application. These features are determined not only by the ability of vectors used, but also by the nature of target cells, administration route, biodistribution, and interaction with components in the body. In this presentation, we report our approaches to improve in vivo gene transfer in two target cells: hepatocytes and myocytes. Targeted delivery of pDNA to hepatocytes was achieved by using galactose as a targeting ligand, but the level of gene expression might not be high enough even after boosting the expression with fusogenic peptides. Combined use of physical stimuli could be a solution. Muscle cells are easily transfected by direct injection of viral or nonviral vectors. However, direct injection results in gene expression only in limited cells adjacent to the injection site. For treatments of muscular dystrophies, gene expression in widespread myocytes is needed. To this end, gene delivery to myocytes in the diaphragm and hind legs was examined through intravascular injection of naked pDNA.

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The RT-PCR analysis revealed that the relative resistance of dendritic cells (DCs) to adenovirus vectors (Ad)-mediated gene transfer is due to the absence of Coxsackie-adenovirus receptor (CAR) expression, and that DCs expressed adequate av-integrins. Therefore, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob can efficiently transduce and express high levels of the LacZ gene into DCs. The gene delivery by fiber-mutant Ad was more efficient than that by conventional Ad in both murine DC lines and normal human DCs (NHDC). Furthermore, NHDC transduced with fiber-mutant Ad and conventional Ad at 8000-vector particles/cell resulted in a 70-fold difference in β-galactosidase activity. These results demonstrated that the gene delivery and expression efficiency of transgenes by α_v-integrin-targeted Ad was more efficient than by conventional Ad on CAR deficient cells. On the other hand, we have developed regulatable Ad that allow gene expression to be tightly controlled by low concentrations of tetracyclines. As an example of the potential clinical utility of this technology, we highlight our results obtained in an immunotherapy model for prostate cancer with a tetracycline-regulatable Ad expressing the cytokine interleukin-12. Ad with tetracycline-regulatable gene expression provide new opportunities and improved safety for gene therapy applications in humans.

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We constructed a novel RNA-protein hybrid ribozyme that have the site-specific cleavage activity of the hammerhead ribozyme and the unwinding activity of the endogenous RNA helicase. This hybrid ribozyme leads to extremely efficient cleavage of any target mRNA regardless of the secondary structure of the RNA. Since the novel hybrid ribozymes can attack any site within mRNA, libraries were made of the hybrid ribozymes with randomized binding arms and introduced into cells. This procedure made it possible to readily identify the relevant genes associated with phenotype in the apoptosis pathway. This application of a randomized library of hybrid ribozymes represents a simple powerful method for identification of genes associated with specific phenotypes in the post genome era.

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[in Japanese]

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We investigated whether or not the liver dysfunction in patientst reated with troglitazone (TG) is related to polymorphism in UDP-glucuronosyltransferases (UGTs), which are partially responsiblef or TG metabolism. In the toxicologicasl tudy of TG using Gunn rats, which are hereditarily deficient in the UGT1 family, the metabolic profile of TG in Gunn rats was very similar to that of Wistar rats, the parent strain of Gunn rats, and no sign of the liver dysfunction was observed in both rats. UGTs involved in TG glucuronidation in rats and humans have been characterized. The experiments using liver microsomes of Gunn rats suggestedt hat the enzymer esponsible for glucuronidation in rats was UGT2B2, an androsterone UGT, by inhibition studies. In humans, contribution of UGT1A1 was estimatedto be about 30% of the total TG glucuronidation by UGTs, using human liver microsomes and recombinant UGTs. Other UGT1 and UGT2 enzymes seem to be responsible for the remaining 70% of TG glucuronidation. The multiplicity of UGTs involved in TG glucuronidation in humans may even allow patients lacking bilirubin UGT (UGT1A1) activity to metabolize TG to the glucuronide. These observation suggest that the polymorphism of UGT is not the reason for the liver dysfunction induced by the TG treatment.

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The 43 member companies of the Japan Pharmaceutical Manufactures Association established Pharma SNP Consortium (PSC), a non-profit organization, which will implement research projects over three years between 2000-2003 with a total budget of 1 billion yen. The following are concrete research themes of the PSC: Standardization to collect samples from human donors; informed consent, protection of privacy (anonymity, information management system, etc.), ethics committee, and procedures (blood sampling, storage, etc.). And the analysis on gene polymorphisms (SNPs) related with pharmacokinetics; to identify SNPs of approximately 180 pharmacokinetics related genes, which are drug metabolizing enzymes and drug transporters, to analyze the allelic frequency of SNPs on a general Japanese population group composed of about 800 individuals, to generate a database for the analysis of SNPs allelic frequency, and to express varia is in insect cells, analyze their function and examine the influence on the drug responses.
It is hoped that the PSC's plan will contribute to the development of the healthcare by constructing a common basis for the drug response analysis.

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The human genome project has been almost completed in 2000, and a number of postgenomic studies will be conducted in the next decade. Efficacy and adverse events following any kind of therapy including drug administration would be predicted from genetic characteristics of individuals. In this presentation, we will introduce our investigations concerning 1) CYP2C19 genotype-related efficacy in anti-H.pylori therapy, 2) NAT2 genotype-related PK of procainamide, isoniazide and sulfasarazine, 3) MDR1 genotype-related digoxin PK, and 4) β2-adrenergic receptor genotype-related desensitization to β2-agonist.

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PK/PD-based concepts can be applied to optimization of experimental design and may also be used for extrapolating foreign clinical data. Although the use of appropriate biomarkers and its linkage to clinical endpoint is essential in late clinical trials, robust linkage is not always needed in early clinical studies. Infrastructure of facilities and education of PK/PD concepts should mostly be needed in Japanese circumstances.

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The Caco-2 human-epithelial cell line has been widely used as a tissue-culture model for permeability measurements to predict human oral absorption in the drug discovery stage. In my presentation I will address some of the strategic applications of the Caco-2 model. Physicochemical properties affect the permeability. For example, permeability tends to be underestimated in hydrophilic compounds. For determining the permeability of poorly soluble drugs, some solubilizing reagents or bovine serum albumin can be used. The expression of some carrier-mediated transport systems has been confirmed on Caco-2 cell monolayers; therefore, we should pay attention to evaluating compounds that may be substrates of the transporters. The pH of the medium also affects the result of this assay. Transport studies with an apical pH value at 6.0 or 6.5 showed better prediction of in vivo drug absorption in human. For compounds that are substrates of P-glycoprotein (Pgp), the use of a Pgp inhibitor resulted in a better estimate of absorption in humans. The results suggest that compounds can be ranked according to how well they are absorbed; namely, those with Papp less than 1×10^-6cm/s (poorly absorbed), between 1×10^-6cm/s and 1×10^-5cm/s (moderately absorbed), and greater than 1×10^-5cm/s (well absorbed). The data of the Caco-2 assays are sometimes different, however, from those in different laboratories due to fluctuations in permeability resulting from passages and culture conditions, even when using the same clone. For high throughput screening, the permeability in 96-well Caco-2 assay demonstrates a good correlation with known human absorption for a variety of compounds. This is comparable to a 24-well system. Automation machines can be successfully applied for Caco-2 assays. Several in silicostudies for the prediction of Caco-2 permeability have also been conducted. A database system to which scientists easily access should be developed in collaboration with information-technologyg roups.

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It is important to predict and evaluate drug metabolism in drug discovery, because there are many pharmacokinetic problems arising from drug metabolism. Particularly, construction of the rapid and reliable prediction. evaluation methods is important in this study. The drug metabolism items that should be investigated are raised as follows: (1) hepatic clearance, (2) metabolite profile, (3) CYP identification, (4) CYP inhibition, (5) enzyme induction. In this presentation, I will discuss the construction and application of the prediction evaluation methods for hepatic clearance, metabolic profile and CYP inhibition in drug discovery.

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For an entry of a candidate having the suitable oral absorption in a development study, we have developed the methods that were high-throughput for the measurement of pKa, solubility, lipophilicity and permeability. On the development of methods, a platform on analysis operation like 96well format was effective for working some examination methods for evaluating physico-chemical properties systematically. And We have carried out evaluation of their physico-chemical properties with examinations of biological activity simultaneously in the study for lead optimization.

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Although advancements in combinatorial chemistry and high-throughput screening (HTS) have been very effective at producing “lead compounds” against a particular target, these compounds do not necessarily demonstrate acceptable DMPK properties concerning oral absorption, metabolic stability and CYP inhibition. In vitro DMPK studies introducing higher throughput capabilities are very useful but costly in the lead optimization process, and they might have not resulted in an overall increased efficiency in the drug development process. Recent trends in lead optimization are to use virtual screening to generate a drug candidate's DMPK profile. Here we present our experiences about the application of in silico techniques to predict the intestinal absorption, plasma protein binding and CYP inhibition. Applying in silico DMPK analysis early in the drug development cycle should save time and money in alliance with experimentally based methods including HTS, because this enables the virtual redesign of candidates in lead optimization. The sophisticated HTS systems and the integrated in silico approach are set to become partners, each compensating for the weakness of the other, in bringing new drug candidates to market.

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