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T Yokoi

Publications and source records attributed to T Yokoi.

At least 91 records · Page 5Linked to original sources
PubMed1999

A new variant CYP2D6 allele (CYP2D6*21) with a single base insertion in exon 5 in a Japanese population associated with a poor metabolizer phenotype.

Two poor metabolizer individuals of debrisoquine were identified among 215 healthy Japanese by a phenotyping test. Analysis of the CYP2D6 gene from one of two poor metabolizers, who was not homozygous for the previously described CYP2D6 variant alleles (CYP2D6*3, CYP2D6*4, CYP2D6*5 and CYP2D6*18), showed that this individual was heterozygous for a new allele, CYP2D6/C8 (CYP2D6*21). CYP2D6*21 had a single cytosine insertion at position 2661 in exon 5. This cytosine insertion generated a stop codon at the 17 bp downstream of this insertion site. A method to detect this allele was established with an allele specific-polymerase chain reaction. This method showed that another one of two poor metabolizers also possessed CYP2D6*21 allele heterozygously. In 318 healthy Japanese, five individuals carried this allele, heterozygously (0.81%, 5/636 chromosomes). Based on the present and our previous data, the poor metabolizer frequency in Japanese was estimated to be 0.39%, which accounted for approximately 45% of the individuals phenotyped as poor metabolizers by in-vivo tests.

Alleles↗
PubMed1999

Detection of three genetic polymorphisms in the 5'-flanking region and intron 1 of human CYP1A2 in the Japanese population.

Interindividual variability of the activity of CYP1A2 may be expected to affect cancer susceptibility, since the enzyme is capable of activating several carcinogens. In the present study, we found three new polymorphisms in the 5'-flanking region (CYPIA2/B) and intron 1 (CYPIA2/C and CYP1A2/D) of CYP1A2 in Japanese by using polymerase chain reaction-single strand conformation polymorphism. We developed methods to detect these polymorphisms by polymerase chain reaction-restriction fragment length polymorphism and performed a population study (159 subjects) to estimate the frequencies of the alleles. The frequencies of the CYP1A2/A (adenine), CYP1A2/B (thymine-deleted), CYP1A2/C (guanine) and CYP1A2/D (adenine) variants were 21.1, 42.0, 8.2 and 61.3%, respectively. The results of family study supported the idea that these CYP1A2 genotypes are inherited with an autosomal codominant transmission.

Cytochrome P-450 CYP1A2↗
PubMed1999

Synthesis of pyrazinone ring-containing opioid mimetics and examination of their opioid receptor-binding activity.

Cyclization of dipeptidyl chloromethyl ketones gave 6-(4-aminobutyl)-3-carboxyethyl-5-methyl-2(1H)-pyrazinone, 3-(4-aminobutyl)-6-carboxyethyl-5-methyl-2(1H)-pyrazinone, and 3,6-bis(4-aminobutyl)-5-methyl-2(1H)-pyrazinone, which were inserted into the enkephalin sequence to give opioid mimetics. Thus, it was confirmed that a pyrazinone ring can be easily inserted into a peptide sequence in order to evaluate structural components required for biologically active peptides.

Chromatography, High Pressure Liquid↗
PubMed1999

A new CYP2A6 gene deletion responsible for the in vivo polymorphic metabolism of (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride in humans.

(+)-Cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) is a newly developed drug as a platelet-activating factor receptor antagonist. The disposition of SM-12502 was investigated in plasma from 28 healthy Japanese volunteers after a single i.v. administration of SM-12502. Three of 28 subjects were phenotyped as poor metabolizers (PMs). Genomic DNAs from three extensive metabolizers or three PMs of SM-12502 were analyzed by Southern blot analysis with CYP2A6 cDNA as a probe. DNAs from three PMs digested with SacI and SphI showed novel restriction fragment length polymorphisms (RFLPs); one type without 4.5- and 2.6-kb fragments and a weak density of a 6.4-kb fragment (E-type), and the other type without 7.1- and 5.5-kb restriction fragments (C'-type) as compared with three extensive metabolizers, respectively. The deletional restriction fragments specific to three PMs in SacI- and SphI-RFLPs were identified as CYP2A6. Using polymerase chain reaction-RFLP analyses of the gene from the three PMs, we found that the exon 1, exon 8, and exon 9 in CYP2A6 were absent. A new RFLP characterized by SacI and SphI was found to be due to the entire gene deletion of the three exons and was associated with the decreased metabolism of SM-12502. This study demonstrates a new deletional allele in the human CYP2A6 gene responsible for the poor metabolic phenotype of SM-12502.

Adult↗
PubMed1999

Inhibitory effects of azelastine and its metabolites on drug oxidation catalyzed by human cytochrome P-450 enzymes.

Azelastine, an antiallergy and antiasthmatic drug, has been reported to be metabolized mainly to desmethylazelastine and 6-hydroxyazelastine in mammals. In the present study, the inhibitory effects of azelastine and its two metabolites on human cytochrome P-450 (CYP) isoform-dependent reactions were investigated to predict the drug interactions of azelastine using microsomes from human B-lymphoblast cells expressing CYP. The specific activities for human CYP isoforms included: 7-ethoxyresorufin O-deethylation (CYP1A1), phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), 7-benzyloxyresorufin O-dealkylation (CYP2B6), S-warfarin 7-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and testosterone 6beta-hydroxylation (CYP3A4). In almost all the activities, desmethylazelastine exhibited stronger inhibition than azelastine and 6-hydroxyazelastine. Desmethylazelastine, but not azelastine and 6-hydroxyazelastine, uncompetitively inhibited CYP2B6 activity (Ki = 32.6 +/- 4.8 microM). Azelastine, desmethylazelastine, and 6-hydroxyazelastine competitively inhibited CYP2C9 activity (Ki = 13. 9 +/- 1.8, 15.0 +/- 3.1, and 17.0 +/- 4.1 microM, respectively), CYP2C19 activity (Ki = 21.9 +/- 2.2, 7.3 +/- 1.6, and 9.3 +/- 1.6 microM, respectively), and CYP2D6 activity (Ki = 1.2 +/- 0.1, 1.5 +/- 0.2, and 3.0 +/- 0.5 microM, respectively). Azelastine and desmethylazelastine competitively inhibited CYP3A4 activity (Ki = 23. 7 +/- 4.6 and 13.2 +/- 2.3 microM). 6-Hydroxyazelastine interfered with the determination of testosterone 6beta-hydroxylation by HPLC. CYP1A2, CYP2A6, and CYP2E1 activities were not significantly inhibited by azelastine and the two metabolites. Among the human CYPs tested, the inhibitory effects of azelastine and its two metabolites were the most potent on human CYP2D6. In consideration of the Ki values and the concentration of azelastine and desmethylazelastine in human livers after chronic oral administration of azelastine, the possibility of in vivo drug interaction of azelastine and other drugs that are mainly metabolized by CYP2D6 was suggested although it might not cause critical side effects. The inhibition of CYP2C9, CYP2C19, and CYP3A4 activity by azelastine and its two metabolites might be clinically insignificant.

Anti-Allergic Agents↗
PubMed1999

Involvement of CYP2E1 as A low-affinity enzyme in phenacetin O-deethylation in human liver microsomes.

Phenacetin O-deethylation (POD) exhibits biphasic kinetics in human liver microsomes. Although cytochrome P-450 (CYP) 1A2 is responsible for the high-affinity component of POD, the enzyme(s) that catalyzes the low-affinity reaction is still unknown. We examined the roles of human CYPs in POD by using human liver microsomes and recombinant CYPs from baculovirus-infected insect cells. Of the recombinant CYPs studied, CYP1A2 showed the highest POD activity. CYP1A1, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 also showed POD activity at 500 microM phenacetin. K(M) values of recombinant CYP1A2 and CYP2E1 (28 +/- 2 microM and 785 +/- 125 microM, respectively) were similar to those of the high- and low-affinity components of POD in pooled human liver microsomes (15 +/- 5 and 894 +/- 189 microM, respectively). Fluvoxamine (10 microM) and anti-CYP1A2 antibodies potently inhibited POD activity at 500 microM phenacetin in pooled human liver microsomes to 22.8 and 34.2% of controls, respectively. CYP2E1 inhibitors diethyldithiocarbamate and aniline also reduced POD activity. The combination of fluvoxamine (10 microM) and aniline (1 mM) further inhibited the residual POD activity not inhibited by fluvoxamine alone. Microsomal POD activity in 12 human livers in the absence of fluvoxamine was correlated with immunoquantified CYP1A2 levels (r = 0.961, p <.001) and, in the presence of 10 microM fluvoxamine, was correlated with immunoquantified CYP2E1 levels (r = 0.589, p <.01) or chlorzoxazone 6-hydroxylase activity (r = 0.823, p <.001). These results suggest that CYP2E1 is responsible for the low-affinity component of POD in human liver microsomes.

Analgesics, Non-Narcotic↗
PubMed1999

Enhancement of cytochrome P-450 3A4 catalytic activities by cytochrome b(5) in bacterial membranes.

Activities of testosterone, nifedipine, and midazolam oxidation by recombinant cytochrome P-450 (P-450) 3A4 coexpressed with human NADPH-P-450 reductase (NPR) in bacterial membranes (CYP3A4/NPR membranes) were determined in comparison with those of other recombinant systems and of human liver microsomes with high contents of CYP3A4. Growth conditions for Escherichia coli transformed with the bicistronic construct affected expression levels of CYP3A4 and NPR; an excess of NPR over P-450 in membrane preparations enhanced CYP3A4-dependent testosterone 6beta-hydroxylation activities of the CYP3A4/NPR membranes. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4/NPR membranes after addition to either bacterial membranes or purified enzymes. NPR was observed to enhance catalytic activity when added to the CYP3A4/NPR membranes, either in the form of bacterial membranes or as purified NPR (in combination with cholate and b(5)). Apparent maximal activities of testosterone 6beta-hydroxylation in CYP3A4/NPR membranes were obtained when the molar ratio of CYP3A4/NPR/b(5) was adjusted to 1:2:1 by mixing membranes containing each protein. Testosterone 6beta-hydroxylation, nifedipine oxidation, and midazolam 4- and 1'-hydroxylation activities in CYP3A4/NPR membranes plus b(5) systems were similar to those measured with microsomes of insect cells coexpressing CYP3A4 with NPR and/or of human liver microsomes, based on equivalent CYP3A4 contents. These results suggest that CYP3A4/NPR membrane systems containing b(5) are very useful models for prediction of the rates for liver microsomal CYP3A4-dependent drug oxidations.

Anesthetics, Intravenous↗
PubMed1999

Noninvasive quantification of cerebral blood flow using 99mTc-ECD and SPECT.

UNLABELLED: The aim of this study was to develop a simple, noninvasive method for quantifying regional cerebral blood flow (rCBF) using 99mTc-ethyl cysteinate dimer (ECD) by a single SPECT scan and single venous sampling. METHODS: Using a three-compartment model, we introduced the regional brain fractionation index (BFI), Cb(Ts)/integral of 0-Ts Ca(tau)dtau [Ca(t), arterial input; Cb(t), brain activity]. Regional BFI obtained at the optimum time Ts (min) was converted to rCBF using an exponential function, which was obtained by analyzing the relationship between regional BFI and rCBF (= F) obtained by the standard 133Xe inhalation SPECT method. The integral of the concentration of 99mTc-ECD in arterial blood corrected for physical decay [Ca(t)] in BFI was estimated from a single venous blood sample obtained at the optimum time Tv using the regression line obtained by analyzing the relationship between the integral of Ca(t) and venous sample data. The data come from three groups of patients. The first group of patients (n = 16) underwent a complete 99mTc-ECD BFI study with measurement of Ca(t) and dynamic SPECT scanning, as well as a 133Xe inhalation study to measure rCBF The results were used to analyze the relationship between regional BFI and rCBF (obtained with 133Xe) and to determine the optimum time Ts for obtaining BFI. Data from the second group of patients (n = 15) were used to analyze the relationship between the integral of Ca(t) and venous sample data and to determine the optimum time Tv for one-point venous blood sampling. Finally, the third group of patients (8 patients, 10 studies) was used to validate the current method by comparing the results with 133Xe inhalation SPECT. RESULTS: Regional BFI obtained at time Ts = 20 min showed good agreement (r = 0.907; a = 0.552, b = 0.962) with rCBF. The venous sample data obtained at time Tv = 6 min showed a good correlation (r = 0.988) with BFI. In comparing rCBF values thus obtained and those obtained by the 133Xe method, we found a good correlation (r = 0.917, slope = 1.01). CONCLUSION: The proposed method has three advantages: (a) accurate quantification of rCBF without underestimation in the high flow range, (b) simplicity and noninvasiveness and (c) the ability to use any type of SPECT camera for the study.

Administration, Inhalation↗
PubMed1999

Oxidation of troglitazone to a quinone-type metabolite catalyzed by cytochrome P-450 2C8 and P-450 3A4 in human liver microsomes.

Troglitazone, a new oral antidiabetic drug, is reported to be mostly metabolized to its conjugates and not to be oxidized by cytochrome P-450 (P-450) enzymes. Of fourteen cDNA-expressed human P-450 enzymes examined, CYP1A1, CYP2C8, CYP2C19, and CYP3A4 were active in catalyzing formation of a quinone-type metabolite at a concentration of 10 microM troglitazone, whereas CYP3A4 had the highest catalytic activity at 100 microM substrate. In human liver microsomes, rates of the quinone-type metabolite formation (at 100 microM) were correlated well with rates of testosterone 6beta-hydroxylation (r = 0.98), but those at 10 microM troglitazone were not correlated with any of several marker activities of P-450 enzymes. Quercetin efficiently inhibited quinone-type metabolite formation (at 10 microM troglitazone) in human samples that contained relatively high levels of CYP2C, whereas ketoconazole affected these activities in liver microsomes in which CYP3A4 levels were relatively high. Anti-CYP2C antibodies strongly inhibited quinone-type metabolite formation (at 10 microM troglitazone) in CYP2C-rich human liver microsomes (by approximately 85%); the intensity of this effect depended on the human samples and their P-450 status. The results suggest that in human liver both CYP2C8 and CYP3A4 have major roles in quinone-type metabolite formation and that the hepatic contents of these two P-450 forms determine which P-450 enzymes play major roles in individual humans. CYP3A4 may be expected to play a role in formation of quinone-type metabolite from troglitazone even at a low concentration in humans.

Aryl Hydrocarbon Hydroxylases↗
PubMed1999

Azelastine N-demethylation by cytochrome P-450 (CYP)3A4, CYP2D6, and CYP1A2 in human liver microsomes: evaluation of approach to predict the contribution of multiple CYPs.

Azelastine, an antiallergy and antiasthmatic drug, has been reported to be mainly N-demethylated to desmethylazelastine in humans. In the present study, Eadie-Hofstee plots of azelastine N-demethylation in human liver microsomes were biphasic. In microsomes from human B-lymphoblast cells, recombinant cytochrome P-450 (CYP)2D6 and CYP1A1 exhibited higher azelastine N-demethylase activity than did other CYP enzymes. On the other hand, recombinant CYP3A4 and CYP1A2 as well as CYP1A1 and CYP2D6 in microsomes from baculovirus-infected insect cells were active in azelastine N-demethylation. The K(M) value of the recombinant CYP2D6 (2.1 microM) from baculovirus-infected insect cells was similar to the K(M) value of the high-affinity (2.4+/-1.3 microM) component in human liver microsomes. On the other hand, the K(M) values of the recombinant CYP3A4 (51.1 microM) and CYP1A2 (125.4 microM) from baculovirus-infected insect cells were similar to the K(M) value of the low-affinity (79.7+/-12.8 microM) component in human liver microsomes. Bufuralol inhibited the high-affinity component, making the Eadie-Hofstee plot in human liver microsomes monophasic. Azelastine N-demethylase activity in human liver microsomes (5 microM azelastine) was inhibited by ketoconazole, erythromycin, and fluvoxamine (IC(50) = 0.08, 18.2, and 17.2 microM, respectively). Azelastine N-demethylase activity in microsomes from twelve human livers was significantly correlated with testosterone 6beta-hydroxylase activity (r = 0.849, p<.0005). The percent contributions of CYP1A2, CYP2D6, and CYP3A4 in human livers were predicted using several approaches based on the concept of correction with CYP contents or relative activity factors (RAFs). Our data suggested that the approach using RAF(CL) (RAF as the ratio of clearance) is most predictive of the N-demethylation clearance of azelastine because it best reflects the observed N-demethylation clearance in human liver microsomes. Summarizing the results, azelastine N-demethylation in humans liver microsomes is catalyzed mainly by CYP3A4 and CYP2D6, and CYP1A2 to a small extent (in average, 76.6, 21.8, and 3.9%, respectively), although the percent contribution of each isoform varied among individuals.

Animals↗
PubMed1999

Genetic polymorphism of CYP2D6 in the Japanese population.

The frequencies of CYP2D6 mutations in a Japanese population were investigated. Individuals were classified into three groups: control individuals, cancer patients and Parkinsonians. Genotyping for CYP2D6*3, CYP2D6*4 and CYP2D6*18 was carried out using the polymerase chain reaction, and that for CYP2D6*5 was also carried out using XbaI restriction fragment length polymorphism. The frequencies of the CYP2D6*3, CYP2D6*4, CYP2D6*5 and CYP2D6*18 mutant alleles were 0%, 0.77%, 4.10% and 0.53% in more than 256 Japanese control individuals, respectively. Based on these data, the population frequency of the CYP2D6 poor metabolizer phenotype was estimated to be 0.29%. The distribution of the four mutated alleles was not significantly different between control individuals and cancer patients or Parkinsonians.

Alleles↗
PubMed1999

Characterization of a novel variant (S145C/L311V) of 3alpha-hydroxysteroid/dihydrodiol dehydrogenase in human liver.

Human liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (DD) is involved in the metabolism of steroid hormones and polycyclic aromatic hydrocarbons, and is also responsible for the reduction of ketone-containing drugs. To account for the interindividual difference in the activity, we isolated and characterized clones for the human liver enzymes. The sequence of the cDNA clone coding for the variant differed from that coding for the wild-type DD by two nucleotides (substitutions of C with G at positions 434 and 931) which caused two amino acid replacements, Ser145 to Cys (S145C) and Leu311 to Val (L311V). The heterologous expression of the variant mRNA was confirmed in four of 31 liver samples from Japanese by an allele-specific polymerase chain reaction. The effects of the mutations on the catalytic properties were examined with the recombinant enzymes expressed in Escherichia coli. The introduction of S145C/L311V double mutations resulted in three- to five-fold decreased activities for xenobiotic and steroidal substrates, whereas no significant change was observed by an introduction of the S145C mutation alone. The results substantiate the existence of polymorphic forms for human liver DD, and also suggest the importance of the residue at position 311 for substrate binding to the enzyme.

Base Sequence↗
PubMed1999

Interobserver errors in anthropometry.

To present basic information on the interobserver precision and accuracy of 32 selected anthropometric measurement items, six observers measured each of 37 subjects once in two days. The data were analyzed by using ANOVA, and mean absolute bias, standard deviation of bias, and mean absolute bias in standard deviation unit were used as measures of bias. By comparing the results of the two days, the effects of the practice on measurement errors were also investigated. Variance was overestimated by more than 10% in five measurements. Interobserver error variance and random error variance were highly correlated with each other. Measures of the bias were significantly correlated with interobserver and especially with random error variances. The interobserver errors were drastically reduced on the second day in the measurement items in which the causes of the interobserver errors could be specified. It was speculated that even when the definitions of the landmarks and measurement items were clear, the ambiguity in the practical procedures in locating landmarks, applying instruments, and so on, permitted each observer to develop his or her own measurement technique, and it in turn caused interobserver errors. To minimize interobserver and random errors, the standardization of measurement technique should be extended to the details of the practical procedures.

Adult↗
PubMed1998

In vivo detection of mutations induced by aflatoxin B1 using human CYP3A7/HITEC hybrid mice.

CYP3A7-M10 mouse is a transgenic mouse carrying human CYP3A7 cDNA, in which CYP3A7 is expressed in the small intestine but not in the kidney. HITEC mouse is a transgenic mouse developed to detect mutagenic potency of various chemicals in vivo. The M10/HITEC mouse was established by crossmating of these two strains of mice. When a 9,000 x g supernatant fraction prepared from the small intestine was added to an incubation mixture for Ames test with Salmonella typhimurium TA98 strain to examine the mutagen-producing activity from aflatoxin B1 (AFB1), the mutagen-producing activities of the 9, 000 x g supernatant fraction from the small intestine was found to be 1.7-fold higher in the M10/HITEC mice than in HITEC mice. Such a difference in the capacity to activate AFB1 was not seen with the 9, 000 x g supernatant fraction from the kidney from both strains of mice. Male M10/HITEC mice of 8 weeks old were treated with a single i.p. injection of AFB1 ( 8 mg/kg body weight). The mutation of the introduced rpsL gene in the genomic DNA from the small intestine and the kidney was analyzed. The mutation frequency in the small intestine of M10/HITEC mice was significantly higher (p<0.05) than that of HITEC mice, while the mutation frequency in both strains was similar in the kidney. These results provide the first evidence for the toxicological function of CYP3A7 in vivo.

Aflatoxin B1↗
PubMed1998

Measurement of local cerebral blood flow by magnetic resonance imaging: in vivo autoradiographic strategy using 17O-labeled water.

Local cerebral blood flow (LCBF) was measured in cats by magnetic resonance imaging (MRI), using 17O-labeled water (H[2]17O) as a tracer. Cat brain images were obtained using a 256 x 240 matrix fast spin-echo sequence with a total acquisition time of 124 seconds per image. Intravenous injection of H(2)17O and sampling of arterial blood were simultaneously performed during the MRI scan. Injection of H(2)17O over 40 seconds caused a transient decrease in brain signal intensity, from which the changes in H(2)17O concentration of brain tissue were calculated. The concentration of H(2)17O in arterial blood was measured directly by 17O nuclear magnetic resonance (NMR) spectroscopy, and used as an input function for the calculation of LCBF. LCBF was successfully determined using an in vivo autoradiographic strategy in a total of three cats. In one, LCBF measurements were also performed under hypercapnic conditions, and LCBF maps of the cat brain during normocapnia and hypercapnia were constructed. These LCBF maps reflected well the changes in LCBF induced by hypercapnia, which indicated the validity of the used method.

Animals↗
PubMed1998

Expression of Rab3A in the cortical region in mouse metaphase II eggs.

Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in regulated exocytosis at presynapses. At fertilization, sperm-egg fusion induces cytosolic calcium mobilization and cortical granule (CG) exocytosis in the egg. However, it is not yet clear whether Rab3A is involved in this process. We previously reported that Rabphilin-3A functions in calcium-dependent CG exocytosis. Rabphilin-3A is known to bind with Rab3A, Rab3B, and Rab3C. In this study, we clarified which member of the Rab3 was expressed in mouse metaphase II eggs. Messenger RNA encoding Rab3A but not Rab3B or Rab3C, was detected in unfertilized metaphase II eggs by RT-PCR. Rab3A protein was also detected in unfertilized metaphase II eggs by immunoblot analysis. Next the expression and the localization of Rab3A in eggs at various stages of development were determined by immunofluorescence analysis using confocal laser scanning microscopy. Rab3A protein was specifically distributed in the cortical region in eggs from before fertilization to the two-cell stage. However, it was not detected at the three- or four-cell stage 40 hr after fertilization. These results suggest that Rab3A may function with Rabphilin-3A in CG exocytosis.

Animals↗