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Biomedical subjects

T Hatanaka

Publications and source records attributed to T Hatanaka.

At least 73 records · Page 4Linked to original sources
PubMed1996

Specificity of substrate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes.

1. We evaluated the specificity of 15 substrates and 14 inhibitors of the cytochrome P450s using nine human P450 forms expressed in HepG2 cells using a recombinant vaccinia virus and also in human liver microsomes. 2. Coumarin, 7-ethoxyresorufin, 7-benzyloxyresorufin, tolbutamide, aniline and diazepam were form-selective substrates towards CYP2A6, the CYP1A subfamily, CYP2B6, the CYP2C subfamily, CYP2E1 and the CYP3A subfamily respectively. However, a selective substrate for CYP2D6 was not found among the chemicals tested. 3. SKF-525A inhibited > 40% of the metabolic activity of all substrates tested, and the inhibitory effects differed among P450 forms. Sulphaphenazole, 7,8-benzoflavone, quinidine and troleandomycin were selective inhibitors of the CYP2C subfamily (except CYP2C19), the CYP1A subfamily, CYP2D6 and the CYP3A subfamily respectively. Methoxsalen (CYP2A6 inhibitor) inhibited the metabolic activity of CYP1A2 as well as that of CYP2A6. Diethyldithiocarbamate (CYP2E1 inhibitor) inhibited the metabolic activities of CYP2A6 and CYP2C19 in addition to that of CYP2E1. 4. Our results indicated that substrates and inhibitors reported as P450 selective probes are not necessarily specific for individual human P450 forms. These results may provide useful information regarding human P450 substrates and inhibitors in vitro using human liver microsomal samples.

Blotting, Western↗
PubMed1996

Human liver microsomal diazepam metabolism using cDNA-expressed cytochrome P450s: role of CYP2B6, 2C19 and the 3A subfamily.

1. We have examined the metabolism of diazepam by ten human cytochrome P450 forms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) expressed in HepG2 cells using a recombinant vaccinia virus system. 2. Among the P450 forms tested, diazepam was significantly demethylated by CYP2B6, 2C9, 2C19, 3A4 and 3A5, with 2C19 exhibiting the highest rate at concentrations < 0.1 mM, and hydroxylated only by the latter three enzymes, with 3A5 being the most active. The N-demethylation activity of diazepam by 2C19 at a concentration of 20 microM was six times of that by 3A4. However, that by 2C9 was detected at only a trace level. 3. CYP2C19, 3A4 and 3A5 of the ten human P450s catalysed the 3-hydroxylation of nordiazepam, and 2B6, the 2C subfamily and the 3A subfamily catalysed the N-demethylation of temazepam. CYP3A4 exhibited the highest activity of nordiazepam 3-hydroxylation and temazepam N-demethylation. 4. Diazepam N-demethylation by human liver microsomes correlated with diazepam 3-hydroxylation, but not S-mephenytoin 4'-hydroxylation. 5. Our results suggest that in the human liver, the metabolism of diazepam to nordiazepam is mediated by CYP3A4, which has been reported as the most abundant P450 form in human liver as well as 2C19, which has been reported as a polymorphic enzyme.

Anti-Anxiety Agents↗
PubMed1995

Structure of a new alkaline serine protease (M-protease) from Bacillus sp. KSM-K16.

An alkaline serine protease, M-protease, from Bacillus sp. KSM-K16 has been crystallized. Two morphologically different crystal forms were obtained. Crystal data of form 1: space group P2(1)2(1)2(1), a = 47.3, b = 62.5, c = 75.6 A, V = 2.23 x 10(5) A(3), Z = 4 and V(m) = 2.09 A(3) Da(-1). Crystal data of form 2: space group P2(1)2(1)2(1), a = 75.82 (2), b = 57.79 (2), c = 54.19 (1) A, V = 2.29 (2) x 10(5) A(3), Z = 4 and V(m) = 2.15 A(3) Da(-1). The crystal structure of M-protease in form 2 has been solved by molecular replacement using the atomic model of subtilisin Carlsberg (SBC) which is 60% homologous with M-protease, and refined to the crystallographic R-factor of 0.189 for 7004 reflections with F(o)/sigma(F) > 3 between 7 and 2.4 A resolution. The final model of M-protease contains 1882 protein atoms, two calcium ions and 44 water molecules. The three-dimensional structure of M-protease is essentially similar to other subtilisins of known structure. The 269 C(alpha) positions of M-protease have an r.m.s. difference of 1.06 A with the corresponding positions of SBC. The crystal data of form 2 are close to those of SBC, though the structure determination of form 2 made it clear that it is not isomorphous to the crystal structure of SBC. The deletions of amino acids occur at the residues 36' and 160'-163' compared with SBC (numerals with primes show the numbering for SBC). The deletion of the four residues (160'-163') may significantly affect the lack of isomorphism between M-protease and SBC.

Journal Article↗
PubMed1995

Oxygen transport and hemodynamics during retrograde whole-body perfusion.

The changes in oxygen transport and hemodynamics during retrograde whole-body extracorporeal perfusion (retro-ECC) were studied in six mongrel dogs. Oxygen consumption during retro-ECC, in which the blood flow rate was set at 25% and 50% of the flow during antegrade extracorporeal perfusion (ante-ECC), respectively, was relatively high compared with that during ante-ECC. These changes were caused by an increase in the oxygen extraction ratio to 71.5% +/- 8.2% and 51.2% +/- 12.4% during retro-ECC/25% and retro-ECC/50%, respectively. Thus, tissue perfusion was apparently well maintained by retrograde perfusion on the basis of the oxygen transport data. However, central venous pressure increased markedly to 29.5 +/- 11.6 mmHg and 56.2 +/- 24.5 mmHg during retro-ECC/25% and retro-ECC/50%, respectively, because of massive venous congestion caused by insufficient arterial return of perfused blood. The great venous compliance and increased systemic vascular resistance were the main causes of circulatory failure during retro-ECC. The risk of serious complications owing to the venous congestion must be considered during retrograde perfusion, especially during the clinical application of retrograde cerebral perfusion.

Animals↗
PubMed1995

Chlorzoxazone is metabolized by human CYP1A2 as well as by human CYP2E1.

Chlorzoxazone, a muscle-relaxing drug, is metabolized by carbon-hydroxylation at position 6. Chlorzoxazone has been suggested as an in vivo probe for CYP2E1. We studied the specificity of such a substrate using vaccinia virus expressed human P450 forms and the effect of inhibitors for chlorzoxazone metabolism by human liver microsomes. The 6-hydroxylation of chlorzoxazone was mediated by CYP1A2 as well as by CYP2E1. The Km value of CYP1A2 and CYP2E1 for the reaction was 5.69 microM and 232 microM, respectively. However, the Vmax value of CYP2E1 for the reaction was approximately 8.5-fold higher than that of CYP1A2. The CYP1A inhibitor, alpha-naphthoflavone, as well as the CYP2E1 inhibitor, diethyldithiocarbamate, decreased chlorzoxazone 6-hydroxylation at a low substrate concentration by human liver microsomes. Our results raise questions about the suitability of chlorzoxazone as an in vivo probe for hepatic CYP2E1 activity. In human liver microsomal samples, the Km = 40 microM was different from either the Km of CYP1A2 or CYP2E1. We think that this discrepancy is due to the co-expression of similar levels of CYP1A2 and CYP2E1 in human liver. Furthermore, it is suggested that the role of CYP2E1 in 6-hydroxychlorzoxazone formation at the physiological chlorzoxazone concentration of 30-60 microM is almost the same when compared to that of CYP1A2.

Benzoflavones↗
PubMed1995

[Study on baths with crude drug. II.: the effects of coptidis rhizoma extracts as skin permeation enhancer].

Skin permeation and its enhancing activity of Coptidis Rhizoma (Coptis japonica Makino) were studied in regard to its application as a bath agent. As a result, methanol extracts and three alkaloids (berberine, coptisine, and palmatine isolated from Coptidis Rhizoma) enhanced effectively the skin permeation of 5-fluorouracil, which is taken as a hydrophilic permeant. Furthermore, it was observed that diffusion coefficient is almost constant on the skin permeation of 5-fluorouracil and was also observed that these three kinds of alkaloids did not penetrate through the skin, but adsorbed into the skin. These results suggest that these three protoberberine type alkaloids increase the concentration of polar drugs in the skin and enhance the skin permeation similarly to surfactants.

Animals↗
PubMed1994

Detection of the products of polymerase chain reaction by an ELISA system based on an ion sensitive field effect transistor.

An ELISA system was developed using a pH sensitive ISFET (pH-FET) as a detector, a pipette tip as a solid phase, and urease as a detecting enzyme. Double stranded PCR products with digoxigenin and biotin at both terminals were obtained by using digoxigenin- and biotin-labeled primers. 1 microliters of the PCR solution was directly introduced into the end part of a pipette tip coated with anti-digoxigenin antibody. Biotin-labeled PCR products captured at the solid phase were detected with avidin-urease, of which the activity was measured by a pH-FET in a pH-measuring cell containing urea solution. The assay was used to detect HTLV-I provirus gene integrated in the genome of a human MT-1 cell, and it was found that 100 pg of the genomic DNA of MT-1 cell was specifically detectable after 35 cycles of PCR. Also the detection limit of the present ELISA system itself was determined by using known amounts of purified PCR product labeled with digoxigenin and biotin, and it was found that 10 amol of the labeled DNA in 1 microliter of sample was detectable.

Base Sequence↗
PubMed1994

An application of the hydrodynamic pore theory to percutaneous absorption of drugs.

Skin permeability of drugs was evaluated based on the hydrodynamic pore theory. Four polar solutes were used, with differing molecular sizes--ethylene glycol, 1,3-butylene glycol, antipyrine and sucrose--and isosorbide dinitrate was also selected as a lipophilic drug. The skin permeations of solvent (D2O) and one of these drugs were measured simultaneously under various osmotic pressures to calculate the reflection coefficient. The clearance of isosorbide dinitrate was independent of the solvent flux, whereas a linear relationship was obtained between the solvent flux and the clearance of each hydrophilic drug except for sucrose. The reflection coefficient of the hydrophilic drugs increased with increasing molecular radius. These results suggest that the convective flow contributes significantly to the total skin permeability of hydrophilic drugs and that the extent of contribution decreases with increasing molecular size of the drugs. The pore radius of the skin barrier could be estimated from the reflection coefficient of the hydrophilic drugs and the resulting value was compared with that for the other absorption sites, jejunum, rectum, and nose. The apparent water influx was also compared to assess the volume occupied by the pores. The pore radius and apparent influx of skin were lower than those for the other absorption sites, which is apparently one reason for low skin permeability of drugs, especially hydrophilic drugs.

Animals↗
PubMed1994

Cardiovascular changes during continuous hyperthermic peritoneal perfusion.

Changes in blood temperature, hemodynamics, and oxygen transport were evaluated in 11 patients during continuous hyperthermic peritoneal perfusion (CHPP), a technique in which the peritoneal cavity is perfused continuously with heated solution to treat intraperitoneal cancer. CHPP was undertaken 46.8 min after the resection of cancer. Blood temperature, measured with a thermistor of a pulmonary artery catheter, reached 39.2 degrees C. Heart rate increased to 100.7 +/- 21.4 bpm (mean +/- SD) and the cardiac index to 4.61 +/- 0.80 L.min-1.m-2. Mean arterial pressure decreased to 75.5 +/- 10.8 mm Hg and systemic vascular resistance index to 1239 +/- 394 dynes.s.cm-5.m2. Oxygen consumption greatly increased to 139.1 +/- 35.2 mL.min-1.m-2, concurrently with a smaller increase in oxygen delivery to 619.7 +/- 83.7 mL.min-1.m-2 and a slight increase in oxygen extraction. Pulmonary oxygenation capacity was also disturbed. Although these cardiovascular changes were mainly due to systemic hyperthermia, other changes may be caused by splanchnic heating, abdominal distention, and pharmacologic action of methoxamine, propranolol, diltiazem, and fentanyl.

Adult↗
PubMed1994

Kinetic analysis of neuromuscular blockade. I. Relationship between twitch depression and stimulation frequency after d-tubocurarine administration.

The degree of twitch depression induced by nondepolarizing neuromuscular blocking drugs is known to be dependent on the stimulation frequency employed. Train-of-four (TOF) stimulations with different frequencies (0.67, 1.33 and 2.0 Hz) were delivered to a sciatic nerve of a rat and series of four twitch heights of a tibialis anterior muscle were measured after d-tubocurarine (d-TC) administration. With a decrease of stimulus interval, twitch heights were intensely depressed. We hypothesized that the oservations are due to the changes of released amount of neuromuscular transmitter, acetylcholine, dependent on stimulus interval, and a pharmacokinetic/pharmacodynamic model based on the hypothesis was proposed. The model allowed simultaneous fitting of the twitch height depression after d-TC administration. It also could give a rationale to the fact that TOF stimulation at 2.0 Hz is a more sensitive monitoring method of neuromuscular function than single twitch stimulation (0.1-0.2 Hz).

Acetylcholine↗
PubMed1994

Kinetic analysis of neuromuscular blockade. II. Train-of-four fade induced by d-tubocurarine and alpha-bungarotoxin.

The degree of train-of-four (TOF) fade, i.e., the reduction of the fourth to the first twitch height in a train, induced by d-tubocurarine (d-TC) and alpha-bungarotoxin (alpha-BX) was investigated. The fade induced by d-TC was pronounced in comparison with that by alpha-BX, and the difference was analyzed using a kinetic model. Based on the assumptions: (1) Acetylcholine (ACh) binds to the nicotinic receptor and evokes twitch response. (2) The amount of released ACh is dependent on stimulus interval. (3) d-TC interacts competitively with the receptor. (4) alpha-BX interacts irreversibly with the receptor. It was suggested that the fade by d-TC and alpha-BX can be explained by the difference of the receptor occupancy by ACh which was caused by different interaction mechanisms of the two muscle relaxants with receptors.

Acetylcholine↗
PubMed1994

In vitro-in vivo correlation of percutaneous absorption: isosorbide dinitrate and morphine hydrochloride.

The potential of an in vitro skin preparation as a model for predicting in vivo percutaneous absorption of drugs was examined. In vitro and in vivo skin permeation data for two model drugs with different lipophilicity, isosorbide dinitrate (ISDN) and morphine hydrochloride (MPH), were compared using pharmacokinetic techniques. In vitro permeation data published previously were analyzed based on a single pathway model, and permeation parameters were obtained. The disposition parameters were estimated from the plasma concentration profiles after i.v. administration. The plasma concentrations after topical application were then simulated using the obtained permeation and disposition parameters, and the values were compared with the corresponding observed ones. Although the simulated plasma concentration curves were not greatly different from those observed, there were some differences in the time course-pattern. Causes for these in vitro-in vivo differences were discussed.

Animals↗
PubMed1994

[The usefulness of transcutaneous gas monitoring during hemorrhagic shock; discrepancy between the two transcutaneous gas tensions of anterior thorax and femur].

We measured transcutaneous gas tensions of both anterior thorax and femur. The differences between the two transcutaneous gas tensions were compared, as well as the relationship between the two transcutaneous and mixed venous blood gas tensions, in 10 dogs during hemorrhagic shock. The changes in femoral transcutaneous gas tensions correlated better with the changes in mixed venous gas tensions. The correlation between the two transcutaneous gas tensions were fairly good (oxygen tensions; r = .827, carbon dioxide tensions; r = .867). However, the discrepancy between the two gas tensions became greater in severe shock. Hence, oxygen tensions became smaller and carbon dioxide tensions became greater on femur than on anterior thorax. This indicates the possibility of "maldistribution of blood flow", which has already been detected between vital organs and skin, also exists between peripheral and central skin. Therefore, transcutaneous gas tensions should be monitored at peripheral skin, where gas tensions show greater changes and reflect systemic perfusion precisely than at central skin, during shock.

Animals↗
PubMed1994

The excitability of blink reflexes in patients with neurologic and psychiatric disorders.

The blink reflex and its recovery curve were studied in 12 patients with neurologic and psychiatric disorders, i.e., juvenile Parkinson's disease (case 1), Gilles de la Tourette's syndrome (case 2), anorexia nervosa (case 3), mild developmental delay (cases 4 and 5), a meningomyelocele with winking spasms (case 6), Parkinson's disease (cases 7-10) and OPCA (cases 11 and 12). The cases were divided into 4 groups on the basis of the results, (1) hyperexcitability of facial motoneurons only (case 6), (2) hyperexcitability of facial motoneurons and brainstem interneurons (case 1 and 2), (3) hyperexcitability of brainstem interneurons only (cases 4, 5 and 7-12) and (4) hypoexcitability of both sides (case 3). Therefore, as to the excitability an abnormal pattern of blink reflexes could be a neurophysiologic marker of some neurologic and psychiatric disorders.

Adolescent↗
PubMed1994

[Short-term intensive consolidation chemotherapy in the treatment of acute nonlymphocytic leukemia].

Thirty-four adults with AML were treated with conventional remission induction chemotherapy consisting of Ara-C and daunorubicin. The median age was 55 years. Thirty (88%) patients showing complete remission (CR) were treated with four courses of intensive consolidation chemotherapy: course 1 with 7 days Ara-C and 4 days of mitoxantrone; course 2 and 7 days Ara-C, 5 days of etoposide, vincristine day 10 and vinblastine day 12 (A-Triple-V); course 3 with 7 days Ara-C and 3 days of aclacinomycin; course 4 with 7 days Ara-C and 3 days of daunorubicin. Then patients were observed without further therapy until relapse. The median duration of relapse-free survival for patients < 60 years of age was 13 months, with 49% patients projected to continue first CR at 52 months. In contrast, only 19% of patients 60 years or older were projected to be in CR at 22 months. Most patients experienced significant side effects including fever, liver dysfunction, pneumonia and septicemia during consolidation therapies. Short-term intensive consolidation therapy appeared to be efficacious for patients < 60 years of age. The results in older individuals were worse than expected, and the use of G-CSF was suggested to improve this problem.

Aclarubicin↗
PubMed1993

The blink reflex in neonates with a subsequent poor outcome.

We examined maturational changes in the electrical blink reflex in 11 handicapped children, i.e., 4 cases of developmental delay, 4 of cerebral palsy, 2 of congenital hydrocephalus, and 1 of congenital cytomegalovirus infection. The developmental delay and cerebral palsy cases were all born at 25-36 weeks' gestation. In all cases, prolonged latency, low amplitude, or the absence of the late component of the blink reflex was observed in the follow-up study. These results indicate that prematurity and some congenital impairment mainly influence the maturation of the long-loop reflex arc in the blink reflex.

Blinking↗
PubMed1993

Interaction of subtilisin BPN' and recombinant Streptomyces subtilisin inhibitors with substituted P1 site residues.

Kinetic analysis was performed on the interaction between subtilisin BPN' and recombinant species of a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), of which the P1 site amino acid residue, Met73, was replaced by site-directed mutagenesis. The inhibitor constant, Ki, was determined from the residual enzyme activity by using a peptide substrate. The rate constant of binding, kon, and the rate constant of dissociation, koff, were determined from a progress curve of the substrate hydrolysis in the presence of the inhibitor by using newly derived equations. A recombinant SSI in which Met73 was replaced by Ile showed an affinity (1/Ki) toward subtilisin BPN' of only about 7% of that of the wild-type SSI, and the kinetic analysis revealed that the increase of koff was responsible for this difference. The affinity of other SSI mutants in which Met73 was replaced by Glu or Asp decreased significantly as pH became increasingly alkaline. The decrease in the affinity of these recombinants was due to the decrease of kon rather than the increase of koff. Stopped-flow studies revealed that the binding reaction was reconcilable with a two-step mechanism, and the kinetic parameters for each step were obtained for the binding of the enzyme and recombinant SSIs.

Bacterial Proteins↗