1. Assay methods to detect drug interaction in toxicological samples were established by determining cytochrome P450 content and its activity in liver samples. The O-dealkylation reaction of 7-alkoxycoumarin was indicated to reflect changes in the molecular forms of P450s, and the enzyme induction or inhibition in the toxicological samples was easily detected by using the established methods.
  2. During toxicological studies of 450191-S or the sleep inducer rilmazafone, a phenobarbital type-induction of hepatic drug metabolizing enzymes was observed in animals, and the doses required for the induction differed markedly between rats and dogs. Enzyme induction was caused by some specific metabolites of 450191-S, and the plasma concentrations of these metabolites were comparable when the enzyme induction was developed in both animals.
  3. A nonsteroidal antiinflammatory compound 480156-S showed a slight or no effect on microsomal drug metabolizing activity in rats. On the other hand, repeated administration of this compound to humans resulted in a marked decrease in the oxidative metabolism of 480156-S, followed by a marked increase in the plasma concentrations of the compound. When volunteers were given 480156-S followed by several drugs, such as tolubutamide, the plasma clearance was delayed remarkably, indicating a severe drug interaction.
  4. Cytochrome P450 belonging to the CYP2C family was indicated to participate in the oxidative metabolism of 480156-S in both rat and human liver microsomes. The preincubation of microsomes with 480156-S caused a concentration-dependent inhibition of CYP2C-dependent tolubutamide hydroxylation reaction in both rats and humans. There was a marked species difference in the susceptibility to the inhibitory effect of 480156-S, and the concentration required to inhibit rat CYP2C was almost 10 times higher than that required in humans.
  5. The cephem antibiotics having N-methyltetrazolethiol (NMTT) at the 3′-position substituent were demonstrated to inhibit mitochondrial low K_m aldehyde dehydrogenase (ALDH), and produced disulfiram-like (Antabuse) reaction during alcohol metabolism. Pharmacokinetic studies indicated that NMTT released from the antibiotics in bile duct or intestine cause the inhibitory action followed by the development of disulfiram-like reaction.
  6. Attempts had been made to develop new cephem antibiotics lacking the disulfiram-like reaction by changing the chemical structure of 3′-position substituents, and a hydroxyethyltetrazolethiol was found not to inhibit the enzyme. Based on this result, together with the antibacterial activity, we have developed a new oxacephem antibiotic flomoxef (6315-S). Flomoxef showed no disulfiram-like reaction both in rats and human.

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  Recent advances in blood-brain barrier (BBB) research have led to a new understanding of drug transport processes at the BBB. The BBB acts as a dynamic regulatory interface at which nutrients necessary for neural activity are actively taken up into the brain from the blood circulation, and actively excludes metabolites that might interfere with the maintenance of brain homeostasis. Such influx and efflux transport functions at the BBB would also control the concentrations of various drugs in the brain interstitial fluid (ISF), which are an important determinant of the central nervous system (CNS) effects. Thus, direct measurement of the brain ISF concentration of drugs can provide significant information for clarifying the influx and efflux transport functions of drugs across the BBB. Although several experimental techniques have been developed to investigate transport functions across the BBB, in vivo brain microdialysis seems to be one of the most suitable techniques for characterizing the influx and efflux transport functions across the BBB under physiological and pathological conditions. This review covers studies during the past decade, in which the influx and efflux transport of drugs across the BBB was kinetically and mechanistically evaluated by means of the brain microdialysis technique. Some applications of brain microdialysis to studies on neuronal function and neurotherapeutics are also included.

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  The aim of the present work is to develop a convenient and rapid screening system in vitro for intestinal drug absorption mediated by oligopeptide transporter (PepT1). In this study, (1) Transports of cephalexin (CEX) and L-phenylalanine (L-Phe) across Caco-2 monolayers were measured and compared with those of passively transported drugs, (2) Inhibitory effects of various drugs on the transport of [¹⁴C]glycylsarcosine (Gly-Sar) across Caco-2 monolayers were measured and correlated with their in vivo permeability to rat small intestine, (3) Intracellular pH-change induced by co-transport of drugs with proton into Caco-2 cells was monitored by using Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices Corp.). Concentration-dependent transport was observed in Caco-2 monolayers for CEX and L-Phe, although their permeability was relatively low compared to those of passively transported drugs. Inhibitory effects of various drugs including β-lactam antibiotics and angiotensin converting enzyme-inhibitors on the transport of Gly-Sar correlated well with their in vivo permeability to rat small intestine. It was demonstrated that CEX, but not cefazolin, induced gradual decrease in the intracellular pH of Caco-2 cells. The degree of intracellular pH-change caused by various drugs showed a sigmoidal or saturable relationship with their permeability to rat small intestine. These in vitro approaches with Caco-2 cells should be useful to evaluate in vivo intestinal permeability of drugs mediated by PepT1, suggesting a possibility of high throughput screening of drug absorption.

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  Recently, we proposed that singlet oxygen (¹O₂) plays an essential role in microsomal cytochrome P450 (P450)-dependent p-hydroxylation of aniline and O-deethylation of 7-ethoxycoumarin. We then examined whether the role of ¹O₂ is general in the P450-dependent substrate oxidations. In the present study, we examined ω- and (ω-1)-hydroxylations of lauric acid, O-demethylation of p-nitroanisole, and N-demethylation of aminopyrine in rat liver microsomes. The addition of β-carotene and NaN₃ significantly suppressed these reactions in a concentration-dependent manner, and ¹O₂ during the reactions was detected by ESR spin-trapping using 2,2,6,6-tetramethyl-4-piperidone (TMPD) as a ¹O₂-spin trapping reagent, where the addition of ¹O₂ quenchers, SKF-525A as a P450 inhibitor, or p-nitroanisole decreased ESR signal intensities due to TMPD-¹O₂ adduct. Next, we examined the effect of ¹O₂ quenchers on P450-dependent reactions in the human liver microsomes, and ¹O₂ was also indicated to be an active species in substrate hydroxylations and dealkylations such as nifedipine oxidation by CYP3A4. On the basis of the results, we concluded that ¹O₂ is an essentially important active oxygen species in both rat and human P450-dependent substrate oxidations.

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  Species difference in nisoldipine oxidation activities was investigated using small intestinal microsomes of rats, guinea pigs, dogs, monkeys and humans. The oxidation activities were estimated by measuring metabolites formation (BAY o 3199 and BAY r 9425) of nisoldipine. For the preparation of small intestinal microsomes of various animal species, the effect of protease inhibitors was preliminarily investigated. The formation of BAY o 3199 significantly increased in the rat small intestinal microsomes prepared with trypsin inhibitor. Using the trypsin inhibitor-treated small intestinal microsomes of various animals, metabolic intrinsic clearances (CL_{int, in vitro}) for BAY o 3199 and BAY r 9425 formations were estimated based on an Eadie-Hofstee plot. The total CL_{int,in vitro} estimated by the sum of CL_{int, in vitro} for both formations in the small intestines of all species was much lower than that in the liver. There was a marked species difference in the nisoldipine oxidation activities in the small intestines, with the rank order being humans=monkeys>dogs>rats>guinea pigs, versus the following order in the liver: rats>monkeys=guinea pigs>humans>dogs. The formations of both BAY o 3199 and BAY r 9425 in the human intestinal microsomes were inhibited by pretreatment with troleandomycin (TAO) and antiserum against CYP3A4. Similar inhibition profile by TAO was obtained from the monkey intestinal microsomes. These results implied that monkeys would be a good predictor of human small intestinal metabolism for CYP3A4 substrates.

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  Using a microtiter plate (MTP) assay consists of recombinant cytochromes P450 and fluorescent probes, we evaluated inhibitory effects of commercially available model-compounds, 18 typical substrates and 8 selective inhibitors, on nine cytochromes P450 (CYPs) activities. The IC₅₀ values obtained from the assay were used to estimate inhibition constant (Ki) values, assuming competitive inhibition. The Ki values calculated from IC₅₀ (the Ki_-cal) with the MTP assay using recombinant CYPs were compared with the Ki values (the Ki_-rep), reported for human liver microsomes (HLM). Regarding all the inhibitory effects of the 26 test compounds on each CYP activity, a good correlation (r²=0.7306) was found between Ki_-cal and Ki_-rep.
  The inhibitory patterns of some compounds on the five major CYP isoforms were estimated, using the MTP assay with the preincubation method. Furafylline and erythromycin, both mechanism based inhibitors, strongly inhibited CYP1A2 and CYP3A4 activity, respectively and their inhibitory effects increased depending on the preincubation time. In contrast, the inhibitory effects of phenacetin, diclofenac, S-mephenytoin, dextromethorphan, bufuralol and terfenadine, typical substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, respectively, on each recombinant CYP activity decreased after preincubation.
  Therefore, the MTP assay is a useful high throughput screening method to evaluate inhibitory effects of new drug candidates on 9 CYP isoforms in HLM. In addition, the MTP assay with the preincubation method might be beneficial to estimate inhibitory patterns on CYP isoforms of new drug candidates and to estimate main CYP isoforms responsible for metabolism of these compounds.

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  Pitavastatin is a potent competitive inhibitor of HMG-CoA reductase. In the current study, to elucidate the hepatobiliary excretion of pitavastatin, we investigated the plasma concentration and biliary excretion of ¹⁴C-pitavastatin in EHBR. We also evaluated the distribution of pitavastatin in mdr1a/b knockout mice by whole body autoradiography and quantitative radioassay. In view of the widespread clinical use of pitavastatin and the importance of drug-drug interaction, the inhibitory effect on Pgp-mediated activation of ATPase was also investigated. No marked difference was observed in the plasma concentration and biliary excretion of radioactivity between SDR and EHBR after dosing of ¹⁴C-pitavastatin. Little radioactive transfer into the brain was detected in mdr1a/b knockout mice and the ATPase activity of human Pgp was negligible in the presence of pitavastatin. Moreover, no inhibitory effect on the Pgp-mediated activation of ATPase by verapamil was found in the presence of pitavastatin over a wide concentration range. These results indicated that a cMOAT and Pgp-mediated transport mechanism did not play a major role in the distribution of pitavastatin.

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  Incubation of 4-hydroxybiphenyl (p-phenylphenol) in the presence of UDP-glucuronic acid (UDPGA) with liver microsomes from male and female dogs produced a more polar metabolite peak than a simultaneously produced peak of 4-hydroxybiphenyl monoglucuronide in the high performance liquid chromatography (HPLC) chromatogram. Tandem mass spectrometry (MS/MS) and two-dimensional nuclear magnetic resonance (NMR) analyses revealed this polar metabolite as a 4-hydroxybiphenyl diglucuronide having a β-D-glucuronopyranosyl-(1→2)-β-D-glucuronopyranosyl moiety, where the two glucuronic acids are connected directly at the 1″→2′ position. Liver microsomes from Sprague-Dawley rat, cynomolgus monkey and human, converted 4-hydroxybiphenyl only to the monoglucuronide, suggesting that there is a dog UDP-glucuronosyltransferase (UGT), with a wider substrate specificity capable of glucuronidating 4-hydroxybiphenyl monoglucuronide to the diglucuronide.

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  St John's wort (SJW) is Hypericum perforatum L., Hypericaceae, a herbaceous perennial plant native to Europe and Asia, and its various preparations are widely used for the treatment of mild-to-moderately severe depressive disorders. With increasingly prevalent use, the interactions with SJW preparations with co-administered drugs have been reported, presumably via MDR1-mediated processes. In this paper, the effects of SJW extract on antiproliferative effects of anticancer drugs and the expression of MDR1 mRNA were examined using HeLa and its MDR1-overexpressing subline. The effects on MDR1-mediated transport were also evaluated using [³H]digoxin and LLC-GA5-COL150 cells, which were established by transfection of human MDR1 cDNA into porcine kidney epithelial LLC-PK₁ cells. The content of hypericin, a presumed active moiety within SJW extract, was determined by HPLC with a photo diode array to be 0.085 (w/w)%, and the effects of hypericin were also evaluated and compared with those of SJW extract. It was concluded that SJW extract reversed the cytotoxicity of paclitaxel and slightly of daunorubicin, down-regulated MDR1 mRNA, and inhibited MDR1-mediated transport, presumably due to other components than hypericin.

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  Novel single nucleotide polymorphisms (SNPs) were found in introns 1, 3, 4, 5, and 7 of the gene encoding cytochrome (CYP) 2D6 in 90 Japanese subjects. Some of these SNPs were associated with C2850T and/or C100T, which are key SNPs for CYP2D6^*2 and CYP2D6^*10, respectively.
  The SNPs in the CYP2D6 gene (ACCESSION NUMBER, M33388) were designated as follows: SNP, MPJ6_2D6010 (IVS1-138C>G) associated with C2850T; SNP, MPJ6_2D6012 (IVS1-41T>G) associated with C100T, C2850T and G4180C; SNP, MPJ6_2D6016 (IVS3+32A>G); SNP, MPJ6_2D6017 (IVS4-17C>G); SNP, MPJ6_2D6018 (IVS4-17C>T); SNP, MPJ6_2D6020 (IVS5+46G>A); SNP, MPJ6_2D6021 (IVS5-49T>C); SNP, MPJ6_2D6023 (IVS7+40A>C) associated with both C100T, C2850T and G4180C; SNP, MPJ6_2D6024 (IVS7+64T>C); and SNP, MPJ6_2D6025 (IVS7-9C>T) associated with C2850T. These SNPs are convenient for allele typing and should be further investigated for their potential effects on transcription, such as splicing.

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  We discovered a novel single nucleotide polymorphism (SNP) at position 325 (G325A) in exon 5 of the multidrug-resistance 1 (MDR1) gene in a study of 37 healthy Japanese subjects.
Details are as follows. SNP, 020614Honda001; GENE NAME, human P-glycoprotein (MDR1); ACCESSION NUMBER, M29427; LENGTH, 25 bases; 5′-ATGAATCTGGAGG/AAAGACATGACCA-3′.
This SNP is expected to cause an amino acid substitution (Glu108Lys). In this study, one homozygote and one heterozygote for G325A were identified.

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  We sequenced all nine exons and exon-intron junctions of the CYP2A6 gene from 33 Japanese and 28 Caucasians. We found twenty one single nucleotide polymorphisms (SNPs) including four SNPs causing amino acid substitutions, one silent SNP in exon 5, one SNP in a 5′-flanking region, four SNPs in a 3′-untranslated region, and eleven SNPs in introns. The four mutations (13G>A and 86G>A in exon 1, and 2134A>G and 2161C>T in exon 4) causing amino acid substitutions (Gly⁵Arg, Ser²⁹Asn, Lys¹⁹⁴Glu, and Arg²⁰³Ser), respectively, were as follows: SNP, 020719Kiyotani004; GENE NAME, CYP2A6; ACCESSION NUMBER, NG_000008.4; LENGTH, 25 base; 5′-ATGCTGGCCTCAG/AGGATGCTTCTGG-3′. SNP, 020719Kiyotani005; GENE NAME, CYP2A6; ACCESSION NUMBER, NG_000008.4; LENGTH, 25 base; 5′-AGCAGAGGAAGAG/ACAAGGGGAAGCT-3′. SNP, 020719Kiyotani011; GENE NAME, CYP2A6; ACCESSION NUMBER, NG_000008.4; LENGTH, 25 base; 5′-CGCTTTGACTATA/GAGGACAAAGAGT-3′. SNP, 020719Kiyotani012; GENE NAME, CYP2A6; ACCESSION NUMBER, NG_000008.4; LENGTH, 25 base; 5′-CTGTCACTGTTGC/TGCATGATGCTAG-3′. New alleles having these SNPs were designated as CYP2A6^∗13-CYP2A6^∗16.

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  Three novel single nucleotide polymorphisms (SNPs) were found in the UDP-glucuronosyltransferase (UGT) 1A10 gene from 24 Japanese patients with various cancers who were administered the anti-tumor drug, irinotecan (CPT-11). The detected SNPs were as follows:
  1) SNP, MPJ6_U1A003; GENE NAME, UGT1A10; ACCESSION NUMBER, AF297093; LENGTH, 25 bases; 5′-CAGATGCCATGAC/TTTTCAAGGAGAG-3′
  2) SNP, MPJ6_U1A004; GENE NAME, UGT1A10; ACCESSION NUMBER, AF297093; LENGTH, 25 bases; 5′-CCTAGAAATAGCC/TTCTGAAATTCTC-3′
  3) SNP, MPJ6_U1A030; GENE NAME, UGT1A10; ACCESSION NUMBER, AF297093; LENGTH, 25 bases; 5′-GGTTGTAGTCATG/ACCAGAGGTGAGT-3′
  All the three SNPs were located in exon 1 and their frequencies were all 0.021. Among these SNPs, MPJ6_U1A003 and U1A030 resulted in amino acid alterations, T202I and M59I, respectively. The third SNP, MPJ6_U1A004, introduced a synonymous amino acid change (A231A).

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