Phannacogenomics is a rapidly emerging field focused on understanding the genetic factors underlying drug action and applying this information to improve the clinical use and development of pharmaceutical products. In clinical practice, the understanding of how genetic variation leads to variable drug effects can be used to prescribe drugs selectively to individuals in regimens and doses that are most likely to be safe and effective. In drug development, knowledge of genetic variation that may affect drug action can be used to reduce the cost and complexity of clinical trials, increase the success rate of achieving regulatory approval, and achieve approvals for new classes of diagnostic and therapeutic products.

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Gene sequensing and gene expression are of particular significance in pharmacogenomics. Sequencing is used to locate polymorphisms, and monitoring of gene expression can provide clue about the genomic response to disease and treatment. Then, we used gene expression profile analysis to identify therapeutic target genes by studying change of gene expression in animal models of oxidative stress and hypoxia. Oxidative stress and hypoxia are important in the pathogenesis of various diseases. We found novel drug target candidates in oxidative stress and hypoxia through this pharmacogenomic strategy. Our results suggest that the pharmacogenomics have the potential for strategy to define novel drug targets in various diseases.

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trans-Cinnamaldehyde (CA) which is an α, β-unsaturated aldehyde, one of the most widely used flavoring agents in the world, is mutagenic in Salmonella, and is an inducer of the skin inflammation in rodents and in the human. In rat liver cytosol, the glutathione (GSH) conjugation of CA proceeded at a much higher rate than did its NADH-dependent reduction. CA was found to be a good substrate for the rat (r) liver class Mu GSH S-transferases (rGSTs) M2-2, M1-2, and MI-1 in decreasing order. The glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was irreversibly and highly 4(S)-selectively inactivated by the enantiomers of racemic 4-hydroxy2(E)-nonenal (4-HNE), a reactive product released from biomembranes by lipid peroxidation in cells. IC₅₀ of 4(R)HNE for GAPDH was 3.6-fold higher than that of the 4(S)-enantiomer. In rat liver cytosol, the 4-HNE was detoxified highly 4(S)-selectively by GSH conjugation and 4(R)-selectively by NADH-dependent reduction mediated by alcohol dehydrogenase (ADH). In the cytosol, however, the GSH conjugation of 4(R)-HNE proceeded at a much higher rate than did its ADH-mediated reduction. The minor GST isoform, A4-4, in the rat liver played a major role in the cytosolic (S)-selective GSH conjugation. The catalytic efficiency, kcat/Km, of purified rGSTA4-4 was 4-fold higher for 4(S)-HNE than for 4(R)-HNE. Because of its much smaller Km than that of 4(R)-HNE, 4(S)-HNE was preferentially detoxified by rGSTA4-4 when racemic 4-HNE was used as a substrate.

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Troglitazone, a new oral antidiabetic drug, is reported not to be oxidized by cytochrome P450 (P450 or CYP) enzymes. However, cDNA-expressed CYP2C8 and CYP3A4 were active in catalyzing formation of a quinone-type metabolite. Intensity of inhibitory effects of specific P450 inhibitors and antiP450 antibodies on the quinone-type metabolite formation depended on human liver samples and their P450 status. Azelastine, an antiallergy drug, was N-demethylated by CYP2D6, CYP3A4 and CYP1 A2 in human liver microsomes biphasically. Human intrinsic clearance was predictable from kinetic parameters using cDNA-expressed P450 enzymes. The results suggest that different P450 enzymes in human liver have major roles in quinone-type metabolite formation from troglitazone and azelastine N-demethylation and that the hepatic contents of these P450 forms determine which P450 enzymes play the major roles in drug metabolism in individual humans.

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The roles of human P450 enzymes in the oxidation of xenobiotics have been shown to be determined by a) the ratio of V_max to K_m of individual P450 forms and b) the levels of expression of individual P450 forms In liver microsomes of human samples. We examined which P450 enzymes are most important in the 4-deethylation of 7-ethoxycoumarin and 6-hydroxylation of chlorzoxazone, two model reactions suggested to be catalyzed by several P450 enzymes, in liver microsomes of different human samples. Kinetic parameters for these two reactions were determined in 14 forms of recombinant human P450 expressed in baculovirus systems. Genetic polymorphisms In P450 genes are also important in understanding basis of different susceptibilities of individuals towards numerous xenobiotic chemicals. We examined the frequency distribution of CYP2A6 genetic polymorphisms in liver DNA of 39 Japanese and 43 Caucasians in the newly-developed two-step PCR method with modifications and the differences in CYP2A6-dependent catalytic activities were determined and compared in liver microsomes of individuals genotyped for CYP2A6 gene.

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To validate the usefulness of recombinant CYP enzymes in metabolic studies of optically active drugs, enantiomer selectivity was examined for oxidation of optically active R-blokers by rat CYP2D enzymes (CYP2D1, -2D2, -2D3 and -2D4) and human CYP2D6 expressed in yeast cells, and was compared with the profile of liver microsomes (Ms). In bunitrolol (BTL) 4hydroxylation rate, selectivity of [(+)-BTL<(-)-BTL] was obtained for rat liver Ms, whereas a reversed selectivity was observed for CYP2D2 that was only one BTL 4-hydroxylase among the rat CYP2D enzymes examined. In propranolol (PL) 4-, 5 and 7-oxidation and N-desisopropylation, rat liver Ms exhibited selectivity of [R(+)-PL>S(-)-PL] in a low PL concentration range. CYP2D2 having the highest PL oxidation activities among the rat CYP2D enzymes showed selectivity of [R(+)PL<S(-)-PL] only for PL 4-hydroxylation while selectivity for other indices was the same between rat liver Ms and the recombinant CYP2D2. Human liver Ms yielded selectivity of [R(+)-PL>S(-)-PL] in a low PL concentration range, whereas recombinant CYP2D6 did not show clear selectivity. PL oxidation by human CYP2D6 showed biphasic kinetics. Kinetics of racemic bufuralol (BUF) were monophasic for rat CYP2D2 and human CYP2D6 but biphasic for rat CYP2D4. Considering these results, different enantioselectivity between liver Ms and recombinant CYP2D enzymes and biphasic kinetics for recombinant CYP enzymes may be caused by different microenvironments surrounding CYP enzymes between liver and yeast cells.

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To clarify the mechanism of phenobarbital-mediated increase in the CYP2B subfamily, we tried to detect target(s) of the inducer using azido-PB (AZPB), a photoaffinity ligand, as the probe. Immunoblot experiments with anti-AZPB antibody showed that binding of AZPB to proteins in rat liver microsomes the molecular weight of which is around 50 kDa is specifically competed by PB. Liver cytosol did not have any such proteins. When purified P450s were used as the target for AZPB, the binding was competed by PB most markedly in the CYP2B subfamily. The binding of AZPB to purified CYP2B2 was effectively lowered by 2, 2', 4, 4', 5, 5'-hexachlorobiphenyl, one of the potent inducers of the CYP2B subfamily, as well as PB. The other inducers for other subfamilies such as dexamethasone and β-naphthotlavone were without the effect. These results support the idea that the target of inducers mediating CYP2B induction is the CYP2B itself. To search the endogenous mediators which play a role in the CYP2B induction, we examined the role of steroid hormones and certain sterols in cultured rat hepatocytes. As the results, testosterone and the metabolites did not have ability to induce CYP2B proteins and to suppress PB-mediated induction. On the other hand, estradiol caused marked induction of the CYP2B 1/2. The role of estradiol was partially supported by experiments using aromatase-deficient mice: constitutive expression of the Cyp2b 10 in the knockout mice is less than the wild-type mice. The role of cholesterol and androstenol in the regulation of the rat CYP2B will also be presented.

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There are wide inter-individual variations in the levels of P450 isoforms whose expression is affected by genetical factors, environmental chemicals, foods, and drugs. The elimination of drugs depends on the levels of P450 isoforms and it is important to know P450 levels for predicting phamacokinetics of drugs. Human P450s expressed in cultured cells and antibodies against these P450s are now available. We can easily investigate drug-metabolism by human P450. However, it is difficult to know levels of P450 in human tissues. We are trying to develop non-invasive assessment of human drug metabolizing enzyme levels in vivo and have developed the method for measurement of P450 mRNA in peripheral lymphocyte by quantitative PCR. Diabetes induced CYP2E1 mRNA inhuman lymphocytes several times. Typical inducer for CYP3A4, rifampicin induced CYP3A4 mRNA in lymphocytes 2 to 5 times. CYP4B1 mRNA was barely detected in lymphocytes of normal subjects but its expression was high in bladder tumor patients.

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Species difference in nisoldipine metabolism was kinetically investigated to identify an animal model which can be predictable to the first pass metabolism in human intestine. Intestinal microsomes were prepared using a buffer containing 0.5 mg/ml trypsin inhibitor to prevent a loss of P450 activity during the preparation. The intrinsic clearance (Vmax/Km) estimated from the formation of two primary metabolites in the small intestinal microsomes was higher in monkeys, followed by humans, dogs, rats and guinea pigs. Noted that the CLint estimated in rat being a conventional animal was lower than that in humans. Some P450 inducers such as DEX were orally administered to rats to produce a mimic condition toward human small intestinal activity. Although DEX was the most potent inducer, the activity was only induced by a factor of 2. Since it is known that there are strain differences in metabolism activity and induction effect, the metabolic activity in several strains in addition to Wistar was also investigated. DA rats provided the highest small intestinal activity, and its activity increased by DEX was comparable to the CLint in human. The corresponding hepatic activity was similar in magnitude in all strains with and without DEX treatment. The small intestinal and hepatic activities in DEX-treated rats were inhibited by ketoconazole, CYP 3A inhibitor, and these estimated IC5o values suggested that CYP 3A would mainly be a responsible isoform for the small intestinal metabolism in DEX-treated DA rats.

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A CYP3A1 gene, which encodes testosterone 613-hydroxylase in rats, is isolated and analyzed. This gene encodes an active P450 form also corresponding to CYP3A23 ¹⁾. The promoter region of CYP3A1 gene contains three binding sites (A, B and C site) for liver nuclear proteins similar to CYP3A2 gene^{1, 2)}. Both CYP3A1 and CYP3A2 genes are transactivated by HNF-4 through A sites, in COS-1 cells co-transfected with a HNF-4 expression vector³⁾. The HNF-4-mediated activation of CYP3A1 gene is suppressed by ARP-1 and/or EAR-3 through B site without competition with HNF-4. The B site is known as a binding site for PXR-RXR heterodimer⁴⁾. The PXR-RXR binding site also exist in CYP3A4 genes⁵⁾ but its position differs from that of CYP3A1 gene. The homology of the 5' region from -1 by to -180 by is low between two genes. A CYP3A4-reporter fused gene is not activated in the presence of inducer, rifampicin, in HepG2 cells, which expresses native HNF-4 and PXR, although the CYP3A4-reporter fused gene is activated by ARP-1 and/or EAR-3. These results suggest, in addition to PXR response, that the different induction profile CYP3A1 and CYP3A4 may be arisen from species-specific transcriptional regulation by nuclear factors.

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Interindividual differences in the pharmacokinetics of venlafaxine, a new antidepressant, were shown during early clinical trials in Japan. Venlafaxine is metabolized mainly by CYP2D6 to an active metabolite, O-desmethylvenlafaxine (ODV). We have found that the CYP2D6*10 genotype influenced the pharmacokinetics of venlafaxine in Japanese population. We have recently expressed the CYP2D6*10A, *10C, *1(wild) and other two variants in yeast cell. The CO-reduced difference spectra of the variants conteining amino acid substitution Pro34->Ser resulted in a dominant absorption at 420nm as well as 450nm, suggesting that the substitution caused a unstabl form proteine. In addition, exchange of the proline residue to serine at same position in rat CYP2D1 also increased P420, denatured P450. The apparent Km values of CYP2D6* 10A, CYP2D6* 10C and CYP2D6* 1(wild) for bufuralol 1'-hydroxylation were 6.39, 63.1 and 1.31μM, and for venlafaxine O-demethylation 83.6, 82.2 and 9.91 p.M, respectively. These findings suggest that the decreased disposition of venlafaxine in vivo was caused not only instability but also increased Km value of CYP2D6* 10. We proposed that these enzymes in vitro may be used as a tool for predicting the effect of the CYP2D6*10 genotypes on human in vivo pharmacokinetics.

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We have successfully predicted in vivo drug metabolic clearances in human liver from in vitro data obtained using human liver samples. The magnitude of differences between in vivo intrinsic hepatic clearances (CLint, in vivo) and in vitro intrinsic metabolic clearances (CLint, in vitro) calculated from the in vitro metabolic data with human liver microsomes was less than 3-fold for about 60 % of the 50 metabolic reactions catalyzed by cytochrome P450 (CYP). In particular, the predictability was good for those metabolic pathways mediated mainly by CYP3A4. Furthermore, as far as all the 9 metabolic pathways we studied were concerned, the values of CLint, in vitro obtained using recombinant human CYP isozymes were in good agreement with those obtained using human liver microsomes, irrespective of the CYP isozymes involved. This finding suggests that recombinant human CYP isozymes are a good substitute for human liver microsomes as far as predicting in vivo metabolic clearance is concerned.

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A gene encoding a 5' flanking region of human liver carboxylesterase(CES) has been isolated and characterized. Analysis of genomic lambda clone H2A revealed that the gene spans about 1, 001 bp. The 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 hantind 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 no TATA-box was present. The putative promoter region was investigated by gel mobility shift assay and the reporter gene assay. The promoter region include CCAAT-Box, GC-box and initiater. 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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Human phenol sulfotransferase (TS-PSULT) is known to be polymorphically distributed in tissues such as platelets and livers. Two polymorphic sites (²¹³Arg/His and ²²³Met/Val) were found on a TS-PSULT gene encoding a major hepatic form, STIA3. The ²¹³Arg/His polymorphism, but not ²²³Met/Val, was found also with Japanese population (n=143). Recombinant ²¹³Arg (frequent)-type ST1A3 was more stable than ²¹³His (less frequent), while catalytic activity difference was within 2-fold. These results suggested the properties of ²¹³His-type enzyme may explain low platelet activities in homozygous ²¹³His individuals.

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In this study, we determined the frequencies of CYP2D6 mutant alleles in 162 normal Japanese subjects and 37 human liver microsomes using PCR methods. Moreover, we examined the metabolic activity for bufuralol (BL) 1'-hydroxylation, desipramine (DMI) 2-hydroxylation and dextromethorphan (DM) O-demethylation using 37 human liver microsomes genotyped. The results showd that the most frequentry occurring mutant allele in our Japanese subject was CYP2D6*10, followed by *2, *5 and *14 with frequencies of 38.6 %, 13.0%, 6.2% and 2.2 %, respectively. The frequencies of CYP2D6 mutant alleles in 37 human liver microsomes were consistent with that of normal Japanese subjects. In vitro study, human liver microsomes with CYP2D6*10/*10 genotypes showed the catalitic activity of one third of the *1/*1 genotypes for BL F-hydroxylation, DMI 2-hydroxylation and DM 0demethylation. Among 37 human liver microsomes studied, only one CYP2D6*5 homozygote (*51*5) was identified. The mean metabolic ratio of human liver microsomes with CYP2D6*51*5 genotype predicting poor metabolizer (PM) showed lowest activity in 37 subjects. The CYP2D6 protein contents in human liver microsomes with CYP2D6*5l*10 and *10l*10 genotypes showed half of the *1/*1 genotypes using immunoassay, whereas CYP2D6 mRNA with*10/*10 genotypes was 2.2-fold higher than that of the *1/*1 genotypes (p < 0.05). Therefore, the difference between CYP2D6*10/*10 and *1/*1 genotypes in catalytic activity appear to be explained by the instability of *10 protein.
In conclusion, the most frequent mutant alleles of CYP2D6 in Japanese subjects is CYP2D6*10, which is associated with a decreased activity for BL 1'-hydroxylation, DMI 2-hydroxylation and DM O-demethylation. In addition, CYP2D6*5 and *14 are the major mutant alleles responsible for 83 % of the PM phenotypes in Japanese subjects.

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CYP2A6 is known as an enzyme responsible for the metabolism of coumarin. As its toxicological significance, aflatoxin B₁ and NNK are metabolically activated by this CYP to mutagens. In our recent studies, we found that a newly developed drug, SM-12502, was a specific substrate of CYP2A6, and 3 out of 28 Japanese were poor metabolizers (PM) of this drug. Genomic analysis of CYP2A6 gene in the PM indicated that an existence of a new CYP2A6 gene variant which lacks the entire region of open reading frame for the enzyme. The frequency of the individual who possess the mutation homozygously was estimated as 3-4% in Japanese. During the course of investigation of the frequency, we found an another CYP2A6 variant whose 60 by region in the 3'-untranslated region was substituted by the corresponding sequences of CYP2A7 gene. In this study, we investigated the frequency of these polymorphic alleles as well as reported v1 (CYP2A6*2) variant among different race.

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Valproic acid is an anticonvulsant drug used for the treatment of epilepsy, but it shows fetal toxicity, including teratogenicity. In the present study, to elucidate the transport mechanisms of valproic acid across the blood-placental barrier, we carried out the transcellular transport and uptake experiments with human placental choriocarcinoma epithelial cells (BeWo) in culture. Transcellular transport of [³H]valproic acid across BeWo cell monolayer was unidirectional transport from maternal to fetal circulation. Uptake of [³H]valproic acid by cells was temperature and pH-dependent, and increased at acidic pH. Futhermore, various experiments are carring to evaluate the transport mechanisms of organic anion drugs across the blood-placental barrier.

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We recently developed the retinal capillary endothelial cell lines (TR-iBRB) from transgenic rat harboring temperature-sensitive SV 40 large T-antigen gene. The purpose of the present study is to characterize the transport functions such as L-cystine and L-lactate as are substrates for system xc and MCT, respectively, in TR-iBRB cells. TR-iBRB expressed P-glycoprotein encoded mdrl aand 1 b mRNA. RT-PCR analysis revealed MCT1 and MCT2 mRNA in TR-iBRB. The uptake of [¹⁴C]-L-lactate was a pH-dependent, temperature-dependent, and concentration-dependent manner with a Michaelis constant (Km) of 1.7 mM. It was inhibited by monocarboxylates but not by dicarboxylates. The uptake of [¹⁴C]-L-cystine by TR-iBRB was a concentration-dependent mannerwith a Km of 9.2μM. It was inhibited by substrates orinhibitors of system xc such as L-glutamic acid, L-α-aminoadipic acid, L-homocysteic acid, and L-quisqualic acid, but not by Laspartic acid, neutral and basic amino acids. L-Cystine uptake was increased by diethyl malete (DEM) treatment. L-Cystine uptake increased by DEM was inhibited by substrates and inhibitors of system xc, suggesting induction of system xc in TR-iBRB. Pending in vivo analysis, TR-iBRB is a useful in vitro model to elucidate transport functions at iBRB. These transport functions in iBRB may contribute the regulation of pharmacokinetics in retina.

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In this study, we focused on the transport characteristics of tolbutamide and glibenclamide to investigate transport mechanism from blood to brain and/or from brain to blood across Blood-Brain Barrier by using in vitro experiments. We examined the transport of [¹⁴C]tolbutamide and [³H]glibenclamide in Pgp (mdrla, mdrlb, MDR1)-expressing cell lines and a MRP1-expressing cell line to investigate the efflux mechanisms of these drugs. We found that Pgp transports glibenclamide but not tolbutamide, and contributes to the low brain distrubution of glibenclamide, whereas a different efflux mechanism is involved in the case of tolbutamide. We also investigated the uptake of tolbutamide from the blood site into brain endothelial cells using MBEC4 cell line. The uptake of [¹⁴C]tolbutamide by MBEC4 cells is saturable, dependent on temperature and energy. The uptake coefficient increased markedly with decreasing pH of the external medium from neutral to acidic. Replacement of chloride with sulfate or gluconate significantly increased the initial uptake of [¹⁴C]tolbutamide, while replacement with nitrate significantry decreased it. Moreover, the upteke was significantly increased by valinomycin and reduced by an anion-exchange inhibitor, DIDS. The initial uptake of [¹⁴C]tolbutamide was significantly reduced by several sulfa drugs, probenecid, sulfinpylazone, salicylic acid and valproic acid. These results indicate the existence of a pH and membrane-potential-dependent anion exchange system for concentrative uptake of tolbutamide and various organic anions in MBEC4 cells. These findings suggested that BBB transport of organic anions such as tolbutamide is regulated by two transport systems which are influx and efflux transport systems.

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Among six members of MRP (multidrug resistance-associated protein) family proteins (MRPI-6), the function of MRP 1 and 2 has been well characterized. We have already cloned MRP3 as an inducible transporter in liver of mutant rats (EHBR) whose cMOAT is hereditarily defective. In this study, we examined the function of MRP3 using membrane vesicles prepared from rat MRP3-transfected LLC-PK1 cells. Although glucuronide conjugates are good substrates for MRP3, the substrate specificity of MRP3 differs from that of MRP1 and 2 in that glutathione conjugates are poor substrates for MRP3. In addition, MRP3 accepted both non-sulfated and sulfated bile salts as substrates. As MRP3 is highly expressed in intestinal tissues, the function and expression of MRP family proteins in intestinal tissues were investigated using Caco-2 cells as an in vitro model. DNP-SG and estradiol 17β-D-glucuronide were taken up by BBMV prepared from Caco-2 in an ATP-dependent manner. Northern blot analysis indicated high expression of human MRP2 and 3 and low expression of MRP1 and 5 in cultured Caco-2 cells.

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We have isolated a novel liver-specific organic anion transporter, LST-1, that is expressed exclusively in the human, rat and mouse liver. LST-1 is a new gene family located between the organic anion transporter family and prostaglandin transporter. LST-1 transport taurocholate (Km; 13.6 μM) in a sodium independent manner. LST-1 also shows broad substrate specificity. It transports conjugated steroids (dehydroepiandrosterone sulfate, estradiol-17β-glucuronide and estrone-3-sulfate), eicosanoids (prostaglandin E2, thromboxane B2, leukotriene C4, leukotriene E4), and thyroid hormones (thyroxine, Km; 3.0 μM and triiodothyronine, Km; 2.7 μM), reflecting hepatic multispecificity. LST-I is probably the most important transporter in the human liver for clearance of bile acids and organic anions since hepatic levels of another organic anion transporter, OATP is very low. This is also the first report of the human molecule which transports thyroid hormones.

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In the present study, we characterized novel Na+-dependent carnitine transporter family, OCTNs from human and mouse. At present, we isolated two and three members of OCTN family from human and mouse, respectively. OCTN1 and 2 are present in various tissues, including kidney, while mouse OCTN3 was strongly detected in testis and weakly in kidney. By immunohistochemical analysis in kidney, mouse OCTNs were distributed commonly in luminal membrane of tubular epithelial cells, while other subcellular distribution seemed to be distinct among members. Most of human and mouse OCTNs exhibited multifunctionality by transporting both of carnitine and organic cation, TEA. Furthermore, sodium ions were essential for carnitine transport by human and mouse OCTN1 and 2. Interestingly, sodium ions were not essential for carnitine transport by mouse OCTN3. Furthermore, TEA transport was sodium independent. Some of mutations of human OCTN2 lost both of carnitine and TEA transport activity and others lost only carnitine transport activity with normal TEA transport activity. These observations suggested that carnitine and TEA have partially common functional site on OCTN2 and are transported by distinct but mutually coupled mechanism.

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To develop liver cell-specific liposomal drug carrier systems, we synthesized novel glycosylated cholesterol derivatives, cholesten-5-yloxy-N-(4-((l-imino-2-β-D-thioglycosylethyl)amino)alkyl) formamide. These glycosylated cholesterols were incorporated into liposomes consisting of DSPC and cholesterol. Mean particle size of all prepared liposomes were about 90 nm. After intravenous injection, these glycosylated liposomes were rapidly taken up by the liver. Fractionation of liver cells revealed that galactosyalted liposomes were taken up mainly by liver parenchymal cells while mannosyalted liposomes by liver non-parenchymal cells. Inhibition studies with galactosylated and mannosylated bovine serum albumin suggested that galactosylated and mannosylated liposomes might be recognized by asialoglycoprotein receptor and mannose receptor, respectively. Then, we studied in vivo distribution of prostaglandin E₁ (PGE₁) and probucol associated with galactosylated liposomes. Liver accumulation of these drugs were significantly higher than that of non-modified liposomes and drug itself. Moreover, PGE₁ associated with galactosylated liposomes was effective for acute hepatitis induced by intraperitoneal injection of carbon tetrachloride.

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The purpose of this investigation is to quantitatively describe the pharmacoldnetics of theophylline (TP) and valproic acid (VPA) in guinea pig plasma, cerebrospinal fluid (CSF) andtear using a basic physiological model, and to characterize the correlation between tear and CSF concentrations on guinea pigs and patients. The time courses of TP and VPA concentrations in CSF and tear were similar to those of free concentrations in plasma and could be described by a basic physiological model in which the CSF and tear compartments are independently connected with the plasma compartments (free drugs) based on the apparent diffusion clearances. The CSF concentrations of TP and VPA at the steady state of both guinea pigs and patients could be predicted by the tear concentrations of them using the resulting pharmacokinetic parameters. These results indicated that the measurement of drug concentrations in tear which can be collected non-invasively is a very useful method for therapeutic drug monitoring of TP and VPA.

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We iiave evaluated the absorption of ME3229, an ester-type prodrug of a hydrophilic glycoprotein IIb/IIIa antagonist, ME3277. Although Log D value and permeability across Caco-2 cell monolayer of ME3229 became high enough for us to expect good oral absorption, less than 10% of dose was absorbed in rats. To clarify this discrepancy, we have separately evaluated the disappearance rate from the gut lumen (VI), hydrolysis rate in the gut lumen before absorption (Vdeg), uptake rate into the enterocytes (Vuptake) and appearance rate into the mesenteric vein (V2), by using single-pass perfusion technique. The data suggested that ME3229 permeated the apical membrane and was taken up into the enterocytes at the reasonable rate for its lipophilicity, but only a small part reached the mesenteric vein mainly due to the efflux of the metabolites formed in the enterocytes into the lumen. Transport characteristics of ME3277 across rat intestinal tissue suggested the existence of an active efflux system to pump out the metabolite(s) of ME3229.

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It has been reported that orally active cephalosporin antibiotics are absorbed from the small intestine via the oligopeptide transporter (PepTl). In the present study, methods have been established to predict oral absorption of cephalosporins using the in vitro data obtained with rat intestinal brush border membrane vesicles (BBMV) or Caco-2 cells. Uptake into BBMV or Caco-2 cells via PepTl was estimated by subtracting the uptake at 4°C from that at 25 or 37°C, which was well correlated with the extent of oral absorption (F) according to the intestinal absorption model reported by Amidon at al. The present method gave fairly good prediction of oral absorption of cephalosporins (Tablel, Figs. 1 and 2).

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A novel methods of assessing the extent of bioavailability (EBA) or pharmacological availability (PA) of Arg-vasopressin (AVP), disopyramide (DP) and insulin (INS) from pharmacological data were presented using an integrated pharmacokinetic pharmacodynamic (PK-PD) model. The PK-PD relationship of AVP was influenced by intra-vascular input rate of drug, and the EBA obtained by the anti-diuretic effect following LV bolus study was significantly underestimated. However, good prediction of EBA of DP by QT prolongation of I.V. bolus injection was resulted, because the PK-PD relationship of DP was maintained in the range of dosing rate investigated. Using the relationship between EC₅₀ for hyperglycemic effect and infusion rate of INS, the pharmacological effect of INS could be described by the model. From these results, we concluded that EBA or PA was reasonably predicted by a PK-PD model, provided that appropriate intra-vascular dosing rate as a reference formulation, are available.

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Charcoal hemoperfusion is a very effective treatment for removing drugs with a small volume of distribution in cases of overdoses. However, it is not capable of clearing generally high protein bound drugs. We successfully treated a phenytoin overdosed patient with charcoal hemoperfusion despite a relative high protein binding percentage (90.8 ± 0.5 %) was maintained before, during and after the procedure. In addition, we examined the in vitro protein binding of phenytoin in the presence of activated charcoal. The bound phenytoin was found to dissociate from plasma proteins and subsequently became adsorbed to the activated charcoal. There are two equilibria, one, an interaction between drug and activated charcoal and the other, an interaction between drug and plasma protein are competing processes. Considering that phenytoin is bound to albumin with a large number of binding sites (n=6) and a small binding constant (K = 6 × 10³ M^-1), the extent of adsorption to activated charcoal may depend on the magnitude of the binding constant of the drug to plasma proteins. Therefore, we measured the plasma concentrations of the drugs in arterial blood entering the charcoal column (A) from overdosed patients treated with charcoal hemoperfusion and those in venous blood exiting the column (V) to determine the extraction efficiency, (A-V)/A . Based on these results, we try to define a guideline for the treatment of drug overdose using the protein binding percentage and distribution volume of the drug.

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Real-time analysis of R-84760 in blood was achieved by a combination of microdialysis (MD) and tandem mass spectrometry (MS/MS). With this technique, the analyte collected in real time from MD is separated by MS/MS, without any subsequent chromatographic separation. The R-84760 concentration was obtained from the plateau part of the ion intensities or the corrected values using an internal standard, after immersing the MD probe into the dialysis solution containing the drug for a definite time. Since contamination of the ion source was prevented by using an organic solvent for the perfused solution, it was possible to establish a stable analysis method. For a MD membrane of length 4 mm, the R-84760 concentration in saline was linear over the range 5 to 541.1 ng/ml (R²=0.9998). Moreover, the R-84760 concentration in rat whole blood was linear over the range 24.9 to 1868.9 ng/ml (R²=0.9995). As this method allowed the measurement of free drug concentration in rat blood, this analysis was also able to provide data needed for determining the protein-binding ratio. The protein-binding ratio obtained from the calibration curve in saline and rat whole blood was 87-90%, which was close to the result obtained by another analysis method. The concentration profile of R-84760 in blood as obtained by the MD-MS/MS method correlated well with the concentration profile in plasma, which was simultaneously monitored by LC-MS/MS.

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Among vanadium's wide variety of biological functions, insulin-mimetic effect is the most interesting and important. Recently, vanadyl ion (+4 oxidation state of vanadium) and its complexes have been shown to normalize the blood glucose levels of streptozotocin-induced diabetic rats (STZ-rats). During our investigations, vanadylmethylpicolinate complex (VO-MPA) was found to exhibit higher insulin-mimetic activity and less toxicity than other complexes. Electron spin resonance (ESR) is capable of measuring the paramagnetic species in biological samples. We have developed the in vivo blood circulation monitoring (BCM)-ESR method to analyze the signals due to stable organic radicals in real time. In the present study, we applied this method to elucidate the global disposition of paramagnetic vanadyl species. In addition, the distribution of vanadium to several organs was investigated by neutron activation analysis (NAA). ESR spectra due to the presence of vanadyl species were obtained in the circulating blood, and their pharmacokinetic parameters were estimated using compartment models. The results indicate that vanadyl species are distributed considerably to the peripheral tissues. The exposure of vanadyl species in the blood was found to be enhanced by VO-MPA treatment. Vanadium accumulated in the bone was suggested to relate with the long-term effective character of VO-MPA. Given these results, we concluded that the pharmacokinetic character of vanadyl species is closely related with the structure and antidiabetic activity of the vanadyl compounds.

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