Biomedical subjects

K Chiba

Publications and source records attributed to K Chiba.

At least 127 records · Page 7Linked to original sources

PubMed1999

Role of human CYP2B6 in S-mephobarbital N-demethylation.

The role of cytochrome P-450s (CYPs) in S-mephobarbital N-demethylation was investigated by using human liver microsomes and cDNA-expressed CYPs. Among the 10 cDNA-expressed CYPs studied (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), only CYP2B6 could catalyze S-mephobarbital N-demethylation. The apparent K(m) values of human liver microsomes for S-mephobarbital N-demethylation were close to that of cDNA-expressed CYP2B6 (about 250 microM). The N-demethylase activity of S-mephobarbital in 10 human liver microsomes was strongly correlated with immunodetectable CYP2B6 levels (r = 0.920, p<.001). Orphenadrine (300 microM), a CYP2B6 inhibitor, inhibited the N-demethylase activity of S-mephobarbital in human liver microsomes to 29% of control activity. Therefore, it appears that CYP2B6 mainly catalyzes S-mephobarbital N-demethylation in human liver microsomes.

Anticonvulsants↗

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↗

PubMed1998

A new culture system to study the metabolism of the intervertebral disc in vitro.

STUDY DESIGN: This study determined whether entrapment of a rabbit intervertebral disc in alginate gel helped to promote the retention of normal metabolic activities by the nucleus pulposus and anulus fibrosus in tissue culture. OBJECTIVES: To establish an in vitro culture system to study the metabolism of the intervertebral disc as a whole integral organ. SUMMARY OF BACKGROUND DATA: In vitro studies of the metabolism of intervertebral discs have been scarce because of the difficulties involved in maintaining the integrity of the tissues, especially that of the nucleus pulposus, in culture medium. METHODS: Rabbit intervertebral discs were embedded in alginate gel and maintained in culture for as long as 1 month. At weekly intervals, experiments were performed to measure the rate of proteoglycan synthesis and to characterize proteoglycans newly synthesized by cells in the anulus fibrosus and nucleus pulposus. In addition, at these same time intervals, the contents of sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, and collagen in these two intervertebral disc tissues were measured to evaluate tissue integrity. Intervertebral discs cultured in medium alone were used as controls and analyzed in parallel. RESULTS: The anulus fibrosus and nucleus pulposus of intervertebral discs cultured in alginate gel sustained a higher rate of proteoglycan synthesis and maintained a higher content of extracellular matrix components than the respective controls at all times. CONCLUSIONS: This new alginate tissue culture system should prove useful for studying the metabolism of whole intervertebral discs.

Alginates↗

PubMed1998

Comparative studies on the catalytic roles of cytochrome P450 2C9 and its Cys- and Leu-variants in the oxidation of warfarin, flurbiprofen, and diclofenac by human liver microsomes.

S-Warfarin 7-hydroxylation, S-flurbiprofen 4'-hydroxylation, and diclofenac 4'-hydroxylation activities were determined in liver microsomes of 30 humans of which 19 were wild-type (Arg144.Ile359), 8 were heterozygous Cys (Cys144.Ile359), and 3 were heterozygous Leu (Arg144.Leu359) allelic variants of the cytochrome P450 2C9 (CYP2C9) gene. All of the human samples examined contained P450 protein(s) immunoreactive with anti-CYP2C9 antibodies in liver microsomes. Individuals with the Cys144 allele of CYP2C9 had similar, but slightly lower, activities for the oxidations of these substrates than those of wild-type CYP2C9. One of the three human samples heterozygous for the Leu359 allele had very low Vmax and high Km values for the oxidation of three substrates examined, while the other two individuals gave kinetic parameters comparable to those seen in the wild-type and Cys144 CYP2C9. Reverse transcriptase-polymerase chain reaction analysis, however, showed that all of the three human samples with the heterozygous Leu359 variant were found to express both Ile359 and Leu359 variants at relatively similar extents in liver RNA of three humans. These results suggest that the Cys144 variant of CYP2C9 catalyzes the CYP2C9 substrates at rates comparative to, but slightly lower than, those of wild-type CYP2C9, while the Leu359-allelic variant has slower rates for the oxidation of these drug substrates. Activities for the oxidation of these CYP2C9 substrates in humans with heterozygous Leu359 allele is likely to be dependent on the levels of expression of each of the wild- and Leu-variants in the livers. However, one of the humans with a heterozygous Leu allele was found to have very low activities towards the oxidation of CYP2C9 substrates. The basis of this defect in catalytic functions towards CYP2C9 substrates is unknown.

Amino Acid Substitution↗

PubMed1998

Linkage of mutant alleles of CYP2C18 and CYP2C19 in a Japanese population.

The coincidence of mutated alleles of CYP2C18 and CYP2C19 was studied in 154 Japanese subjects. The mutant alleles of CYP2C18 studied were CYP2C18m1 (T204 --> A substitution in exon 2) and CYP2C18mFR (A-460 --> T substitution in the 5'-flanking region), and those of CYP2C19 were CYP2C19m1 (G689 --> A substitution in exon 5) and CYP2C19m2 (G636 --> A substitution in exon 4). They were identified by polymerase chain reaction and restriction fragment length polymorphism. The results indicate that genotypes of CYP2C18m1 and CYP2C18mFR are completely coincident with those of CYP2C19m2 and CYP2C19m1, respectively. The finding suggests that the mutations of CYP2C18 and CYP2C19 examined in the present study are very closely linked with each other (i.e. CYP2C18m1 vs CYP2C19m2 and CYP2C18mFR vs CYP2C19m1), at least in a Japanese population.

Alleles↗

PubMed1998

FTY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats. II. FTY720 prolongs skin allograft survival by decreasing T cell infiltration into grafts but not cytokine production in vivo.

FTY720, a novel immunosuppressant, prolonged the survival of WKAH skin allografts transplanted into MHC-incompatible F344 rats. In this allograft model, the median survival time of the control group was 7 days, whereas those of the groups given FTY720 orally at 0.1 mg/kg and cyclosporin A (CsA) at 10 mg/kg were 10.5 and 11 days, respectively. In contrast, FTY720 (0.1 mg/kg) combined with CsA (10 mg/kg) synergistically prolonged allograft survival with a median survival time exceeding 70 days. To elucidate the mechanisms of this remarkable synergistic effect, mRNA expressions of IL-2 and IFN-gamma and that of CD3 (delta-chain), which reflects T cell infiltration, in allografts were temporally analyzed using a semiquantitative PCR method. In WKAH skin allografts, mRNA levels of IL-2, IFN-gamma, and CD3 were increased as compared with isograft controls, peaking on days 4 to 5. CsA (10 mg/kg) significantly inhibited elevations of IL-2 and IFN-gamma mRNA, while slightly inhibiting that of CD3 mRNA in allografts. On the contrary, FTY720 (0.1 mg/kg) markedly inhibited the elevation of CD3 mRNA, while slightly inhibiting those of IL-2 and IFN-gamma mRNA. FTY720 (0.1 mg/kg) combined with CsA (10 mg/kg) almost completely suppressed the intragraft expressions of mRNA for IL-2, IFN-gamma, and CD3. Immunohistochemical staining and flow cytometric analysis also confirmed that FTY720 decreased T cell infiltration into allografts. From these results, the synergistic effect of FTY720 combined with CsA on prolongation of allograft survival is presumably based on the respective inhibitions of T cell infiltration and cytokine production in grafts.

Animals↗

PubMed1998

FTY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats. I. FTY720 selectively decreases the number of circulating mature lymphocytes by acceleration of lymphocyte homing.

FTY720, given i.v. or orally at 0.03 mg/kg or more, significantly prolonged skin allograft survival in a dose-dependent manner and showed more potent immunosuppressive activity than cyclosporin A (CsA) or tacrolimus (FK506) in MHC-incompatible rat strains of WKAH donors and F344 recipients. However, unlike CsA or FK506, FTY720 up to 1000 nM did not affect IL-2 production in allogeneic MLC. Within 3 to 24 h after a single oral administration of FTY720 at 0.1 to 1 mg/kg, the number of lymphocytes in the rats was markedly decreased in the peripheral blood and thoracic duct lymph and partially in spleen. By contrast, the number of lymphocytes in peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer's patches (PP) was significantly increased at the same time. Intravenous transfusion of calcein-labeled rat lymphocytes into rats revealed that FTY720 significantly accelerated lymphocyte homing to PLN, MLN, and PP, dose dependently. Since FTY720-induced lymphocyte homing was completely blocked by simultaneous treatment of the calcein-labeled lymphocytes with mAbs against CD62L, CD49d, and CD11a before the transfusion, the acceleration of lymphocyte homing by FTY720 appears to be mediated by lymphocyte-homing receptors. These findings indicate that FTY720 sequesters circulating mature lymphocytes into PLN, MLN, and PP by acceleration of lymphocyte homing and thereby decreases the number of lymphocytes in peripheral blood, thoracic duct lymph, and spleen. Based on these observations, sequestration of circulating mature-lymphocytes is presumed to be a main mechanism of the immunosuppressive activity of FTY720.

Animals↗

PubMed1998

Synthesis of a new class of camptothecin derivatives, the long-chain fatty acid esters of 10-hydroxycamptothecin, as a potent prodrug candidate, and their in vitro metabolic conversion by carboxylesterases.

Five (20S)-10-hydroxycamptothecin derivatives carrying the long-chain fatty acid esters were prepared for the development of a new class of prodrug-type agents. In vitro experiments using three kinds of purified carboxylesterase isozymes from the liver microsomes of rat, pig, and human demonstrated that these derivatives were efficiently metabolized by enzymes compared with CPT-11.

Animals↗

PubMed1998

ER-34122, a novel dual 5-lipoxygenase/cyclooxygenase inhibitor with potent anti-inflammatory activity in an arachidonic acid-induced ear inflammation model.

OBJECTIVE AND DESIGN: To investigate the effect of ER-34122, a novel pyrazole derivative, on 5-lipoxygenase (LOX) and cyclooxygenase (COX) metabolite production in vitro, ex vivo and in vivo. MATERIAL: In vitro, lysate of rat basophilic leukemia cells, the microsome fraction of sheep seminal vesicles, human polymorphonuclear leukocytes, human synovial cells, and human monocytes. Ex vivo and in vivo, male Balb/c mice or SD rats. TREATMENT: In ex vivo study, ER-34122 (0.03-1 mg/kg) was orally administered 1 h before withdrawal of blood samples. In carrageenin-induced paw edema, ER-34122 (3-100 mg/kg) and indomethacin (1-10mg/kg) were orally administered 1 h before carrageenin injection. In arachidonic acid-induced ear inflammation, ER-34122 (0.3-10mg/kg), zileuton (10-100mg/kg) and indomethacin (0.3-3mg/kg) were orally administered 1 h before arachidonic acid application. METHODS: 5-Hydroxyeicosatetraenoic acid and other eicosanoids were determined by using an HPLC method and enzyme immunoassay, respectively. Rat hind paw edema and mouse ear edema were assessed by measuring paw volume and ear thickness, respectively. Myeloperoxidase (MPO) activity and eicosanoid content of the ear tissue were also determined. RESULTS: ER-34122 inhibited both LOX and COX product generation in vitro, and ex vivo. ER-34122 and indomethacin inhibited carrageenin-induced paw edema formation. In the arachidonic acid-induced ear inflammation, ER-34122 inhibited inflammatory responses (edema formation and MPO accumulation) as well as eicosanoids (LTB4, LTC4 and PGE2) generation. A representative LOX inhibitor, zileuton, also inhibited these inflammatory responses, while a COX inhibitor, indomethacin, did not suppress them though it completely inhibited PGE2 generation. CONCLUSIONS: The anti-inflammatory characteristics of ER-34122 are considered to be superior to those of COX inhibitors such as indomethacin, because in addition to its inhibitory activity on the COX pathway, ER-34122 inhibits LOX products generation, as revealed by the inhibition of edema formation or polymorphonuclear leukocyte infiltration in the arachidonic acid-induced ear inflammation model.

Animals↗

PubMed1998

An immunosuppressive regimen using FTY720 combined with cyclosporin in canine kidney transplantation.

FTY720 induces apoptosis, specifically in lymphocytes, and prolongs allograft survival in rats and dogs. The purpose of this study was to define an effective range of FTY720 doses that could be combined with a suboptimal dose (10 mg/kg) of cyclosporin for canine kidney allograft recipients. The combination significantly prolonged allograft survival in all groups receiving FTY720 at a dose of 0.1, 0.3, 1.0, or 3.0 mg/kg. None of the recipients died due to notable side effects of the drug. In peripheral blood, the number of lymphocytes was extremely low, whereas the percentage of granulocytes increased during FTY720 administration. No significant difference in cyclosporin trough levels was observed between the cyclosporin-alone group and the combination groups. We conclude from the present study that FTY720 has a potent effect at an extremely low dose and a wide therapeutic window when combined with cyclosporin in canine kidney transplants.

Animals↗

PubMed1998

Multiple regression analysis of the factors influencing the results of expansive open-door laminoplasty for cervical myelopathy due to ossification of the posterior longitudinal ligament.

We conducted a multiple regression analysis on the surgical results (minimum follow-up of 5 years) of 55 patients who underwent expansive open-door laminoplasty (open-door ELAP) for cervical myelopathy due to ossification of the posterior longitudinal ligament (OPLL) in order to determine statistically the impact of factors influencing the results. Duration of myelopathy was found to be a significant factor indicating poor results, while progression of ossification and age at the time of surgery were significant factors indicating deterioration. Three other factors (preoperative severity of myelopathy, degree of spinal canal expansion and changes of the curve indices before and after surgery) did not have a significant influence on the results. To improve the surgical results, it is thus essential to operate on patients as soon as they develop symptoms of myelopathy. There exists a certain limitation in posterior decompression for OPLL including open-door ELAP until prevention of the progression of ossification becomes possible.

Adult↗

PubMed1998

Detection of TNF alpha and Fas ligand mRNA within synovial mononuclear cells by fluorescence in-cell labeling PCR (FICL-PCR).

T cells that infiltrate the synovial lesions of rheumatoid arthritis may play a key role in its pathogenesis. To learn more about their functional nature, we determined the frequency of synovial T cells that harbored the TNF alpha and Fas ligand transcript by a technique, called Fluorescence In-Cell Labeling Polymerase Chain Reaction (FICL-PCR). The mRNA of interest was detected in fixed cells by the incorporation during PCR of a fluorescein-12-dUTP label following an initial reverse transcription PCR step. Using this technique the CD3 transcript was detected in the T leukemic cell line, MOLT-4, with calculated sensitivity and specificity values of 91% and 100%, respectively. The percentage mean (+/-S.D.) of TNF alpha mRNA positive cells and Fas ligand mRNA positive cells in peripheral blood mononuclear cells from 12 rheumatoid arthritis patients were 5.1+/-2.3% and 4.8+/-3.1%, respectively. The percentage mean (+/-S.D.) of TNF alpha mRNA positive cells and Fas ligand mRNA positive cells among synovial mononuclear cells from six rheumatoid arthritis patients was 16.8+/-8.3% and 10.8+/-1.8%, respectively. This result indicates that the cytotoxic T cells expressing TNF alpha accumulate in rheumatoid arthritic lesions where they may play a pathogenic role.

Arthritis, Rheumatoid↗

PubMed1998

Inhibitory effects of antiarrhythmic drugs on phenacetin O-deethylation catalysed by human CYP1A2.

AIMS: The aim of the study was to clarify whether the pharmacokinetic interaction between theophylline and mexiletine is mediated by inhibition of CYP1A2 and to assess the possible interaction potential of other antiarrhythmic drugs with drugs metabolized by CYP1A2. METHODS: The inhibitory effects of mexiletine and 10 antiarrhythmic drugs on phenacetin O-deethylation, a marker reaction of CYP1A2, were studied using human liver microsomes and cDNA-expressed CYP1A2. RESULTS: Propafenone and mexiletine inhibited phenacetin O-deethylation with IC50 values of 29 and 37 microM, respectively. Disopyramide, procainamide and pilsicainide produced negligible inhibition of phenacetin O-deethylation (IC50 >1 mM). Amiodarone, bepridil, aprindine, lignocaine, flecainide and quinidine inhibited phenacetin O-deethylation in a concentration-dependent manner, although the inhibitory effects were relatively weak with IC50 values ranging from 86 to 704 microM. Propafenone and mexiletine selectively abolished the high-affinity component of phenacetin O-deethylation in human liver microsomes. In addition, propafenone and mexiletine inhibited phenacetin O-deethylation catalysed by cDNA-expressed CYP1A2. CONCLUSIONS: These data suggest that, among the antiarrhythmic drugs studied, propafenone and mexiletine are relatively potent inhibitors of CYP1A2, which may cause a drug-drug interaction with drugs metabolized by CYP1A2.

Anti-Arrhythmia Agents↗

PubMed1998

FTY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats, III. Increase in frequency of CD62L-positive T cells in Peyer's patches by FTY720-induced lymphocyte homing.

FTY720, a novel immunosuppressant, sequesters circulating mature lymphocytes, especially T cells, within lymph nodes and Peyer's patches by accelerating lymphocyte homing, and thereby causes lymphocyte depletion in the blood. The FTY720-induced acceleration of lymphocyte homing appears to be mediated by lymphocyte homing receptors including CD62L, CD49d/beta7, and CD11a/CD18. In this study, expressions of CD62L, CD49d and CD11a on T cells in the peripheral blood, lymph nodes and Peyer's patches were analysed by flow cytometry in rats given FTY720 (1 mg/kg) orally. FTY720 markedly decreased the number of peripheral blood T cells, while not affecting CD62L, CD49d and CD11a expressions at 1-3 hr after administration. In contrast, both the frequency of CD62L-positive T cells and intensity of CD62L expression on T cells were increased in Peyer's patches but not lymph nodes at 3 hr after administration of FTY720. CD49d and CD11a expressions on T cells were unaffected by FTY720 in both Peyer's patches and lymph nodes at the same point in time. On the other hand, analysis of lymphocyte homing with calcein-labelled lymphocytes and anti-CD62L monoclonal antibody (mAb) confirmed that FTY720 predominantly increased CD62L-dependent lymphocyte homing to Peyer's patches. These findings indicate that FTY720 increases the frequency of CD62L-positive T cells by accelerating CD62L-predominant homing in Peyer's patches.

Animals↗

PubMed1998

Heteroduplex analysis: a useful screening method for glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSDIa), also known as von Gierke disease, is the most common and severe disease of glycogenoses and is caused by a deficiency of glucose-6-phosphatase (G6Pase) and transmitted by an autosomal recessive trait. The encoding gene of G6Pase is composed of only five exons and each exon is short. With heteroduplex analysis (HDA) method, we analyzed the genomic DNA from a patient diagnosed with GSDIa and from her parents. Exons II and IV of the patient showed heteroduplex bands. The mother had a heteroduplex band of exon II, and the father had a heteroduplex band of exon IV. In a mini-slab electrophoresis, exons II and IV of the patient did not show clear heteroduplex bands, but they appeared broader than the others, which made us suspect that they were heteroduplex bands. HDA is an easy and simple method and can verify mutant homozygous DNA fragments by adding wild-type DNA. We think that HDA may be a very useful screening method for the detection of novel genomic mutation in GSDIa in large-scale and mini-slab electrophoresis.

Adult↗

PubMed1998

Synthesis and structure--antibacterial activity relationships of 7-(3-amino-1-propynyl and 3-amino-1-propenyl)quinolones.

7-(3-Amino-1-propynyl)-1-cyclopropyl-6,8-difluoro-1,4-dihydro-4- oxoquinoline-3-carboxylic acid (7a) and some related compounds (7b-f, 8a, b, 9) were prepared via palladium(0)-catalyzed cross-coupling reaction of 7-iodoquinolone 12 with acetylenic compounds and their antibacterial activity was tested. The methylene homologue (7d) and the N-methyl derivative (7e) of 7a showed essentially the same activity as that of 7a. Addition of methyl group(s) to C'-3 of 7a (giving 7b, c) reduced the activity. The hydrogenation of 7a to (Z)-3-amino-1-propenyl (8a), (E)-3-amino-1-propenyl (8b) and 3-amino-1-propyl (9) compounds retained or enhanced the activity of 7a. Among the compounds prepared, 8a was the most active, but was less active than ciprofloxacin (1). In order to get insight into structure-activity relationships, the spatial distribution of the amino groups of 7a, 8a, b, and 9 was examined by means of computer-aided molecular modeling.

Anti-Infective Agents↗

PubMed1998

7-(2-Aminomethyl-1-azetidinyl)-4-oxoquinoline-3-carboxylic acids as potent antibacterial agents: design, synthesis, and antibacterial activity.

2-Aminomethyl-1-azetidinyl, -1-pyrrolidinyl, and -1-piperidinyl groups were designed as novel C-7 substituents for potential antibacterial quinolone agents. Of the three substituents, the 2-aminomethyl-1-azetidinyl group (compound 12a) was found to be the most favorable for enhancing the activity of the 6,8-difluoroquinoline molecule 12. Therefore the 2-aminomethyl-1-azetidinyl group was introduced into a variety of quinolines (giving 24-26a, and 28a) and naphthyridines (giving 31a and 32a). Through optical resolution of 1-benzylazetidine-2-carboxamide (19) and chiral synthesis of its R-isomer, both enantiomers of 2-aminomethyl-1-azetidinyl quinolines 12a and 24-26a were also prepared. The most active of all the compounds was 5-amino-6,8-difluoroquinoline (R)-26a. The activity of (R)-26a was more potent than those of the corresponding 1-piperazinyl derivative (3) and sparfloxacin (1), and was comparable to those of the corresponding 3-amino-1-pyrrolidinyl (4), 3-aminomethyl-1-pyrrolidinyl (5), and 3-amino-1-azetidinyl (6) derivatives.

4-Quinolones↗

PubMed1998

[Genetic polymorphism of the CYP2C subfamily].

Cytochromes P450 (CYP) are a superfamily of hemoproteins that metabolize various foreign compounds. The human CYP2C subfamily is one of the subfamilies of the CYP2 family and it consists of four members of CYP isoforms, CYP2C8, CYP2C9, CYP2C18 and CYP2C19. A well-characterized genetic polymorphism occurs in CYP2C19, which is associated with the 4'-hydroxylation of S-mephenytoin. There are two phenotypes, extensive metabolizers and poor metabolizers (PM) of mephenytoin. The frequency of PM in the Japanese population is 20%, while only 3% of Caucasians are PM of mephenytoin. Two defective alleles, designated as CYP2C19*2 and CYP2C19*3, have been described, and the latter mutation has been detected only in Oriental populations. Recently, an allelic variant of CYP2C9 that causes substitution of Leu359 for Ile359 has been shown to be associated with the decreased metabolic clearance of various therapeutic agents including warfarin, tolbutamide and phenytoin. The frequency of this variant allele in the Japanese population is 2%, while those of the Caucasians are 6-9%. Although the role of CYP2C18 in the drug metabolism remains obscure, we have recently found that defective alleles of CYP2C19*3 and CYP2C18m1 are completely linked, suggesting that PM of CYP2C19 with CYP2C19*3 alleles is a PM of CYP2C18 and vice versa.

Alleles↗