Biomedical subjects

M Iwaki

Publications and source records attributed to M Iwaki.

At least 91 records · Page 5Linked to original sources

PubMed1995

Stereoselective binding properties of naproxen glucuronide diastereomers to proteins.

The stability of naproxen glucuronide (NAP-G) diastereomers was investigated in buffer, 0.3% and 3% human serum albumin (HSA) solutions, and human plasma. R-NAP-G was found to be less stable in phosphate buffer than its S-diastereomer, whereas incubation media containing protein in general increased the degradation rate of NAP-G but also caused a change of the stereoselective stability where the R-NAP-G was more stable than S-NAP-G. Reversible binding of NAP-Gs to HSA (0.3%) was investigated and compared with the corresponding properties of naproxen (NAP) enantiomers. NAP-G diastereomers exhibited a considerable and stereoselective affinity to HSA, although less than that observed for the NAP enantiomers. In vitro irreversible binding of NAP-Gs to HSA, human and rat plasma proteins was also investigated. Irreversible binding was higher for R-NAP-G (50 microM) than for S-NAP-G (50 microM) in all incubation media. This stereoselective difference was observed with HSA containing medium as well as in rat and human plasma. Incubation with unconjugated NAP did not lead to irreversible binding. Preincubation of HSA with acetylsalicylic acid (approximately 11 mM) and glucuronic acid (50 mM) decreased the extent of irreversible binding suggesting involvement of lysine residues for covalent binding. Preincubation with S-NAP also decreased the irreversible binding yield.

Animals↗

PubMed1995

Identification by in vitro complementation of regions required for cell-invasive activity of Bordetella pertussis adenylate cyclase toxin.

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein composed of an amino-terminal adenylate cyclase (AC) domain linked to a 1300-residue channel-forming RTX (repeats in toxin) haemolysin. The toxin delivers its AC domain into a variety of eukaryotic cells and impairs cellular functions by catalysing unregulated synthesis of cAMP from intracellular ATP. We have examined toxin activities of a set of deletion derivatives of CyaA. The results indicate that CyaA does not have a dedicated target cell-binding domain and that structural integrity and co-operation of all domains, as well as the post-translational fatty acylation mediated by an accessory protein CyaC, are all essential for target cell association and toxin activity of CyaA. When tested individually, all toxin derivatives were inactive and impaired in the tight association with the target cell surface. However, pairs of constructs with nonoverlapping deletions complemented each other in vitro and exhibited a partially restored cytotoxic activity. This suggests that at least a part of the active toxin may act in the form of dimers or higher oligomers. The complementation analysis revealed that the last 217 residues of CyaA, containing the unprocessed secretion signal, form an autonomous domain essential for toxin activity, and that the region from residue 624 to 780 may be directly involved in delivery of the AC toxin into cells.

Acylation↗

PubMed1995

Percutaneous penetration of fluorescein isothiocyanate-dextrans and the mechanism for enhancement effect of enhancers on the intercellular penetration.

To identify the mechanism involved in the enhancement effect of enhancers on the intercellular penetration of large polar molecules, the skin penetration of fluorescein isothiocyanate (FITC)-dextrans (average molecular weight; 4400, 9400, and 69000 Da) and the lipid removal from the intercellular spaces by enhancers were studied using hairless rat skin. Pretreatment of hairless rat skin with enhancers such as n-octanol (20%), laurocapram (2%), isopropylmyristate (IPM, 20%), oleic acid (5%) and cineol (2%), which are water-immiscible, significantly enhanced the flux of FITC-dextrans, while pretreatment with water-miscible enhancers, i.e. dimethyl sulfoxide (DMSO, 5%) and N-methyl-2-pyrrolidone (NMP) did not increase the flux compared with the control. The penetration of FITC-dextrans was approximately size dependent. n-Octanol, laurocapram, IPM and oleic acid dramatically removed ceramides which are the intercellular lipids, whereas NMP and DMSO partly extracted the sphingolipids. A linear relationship was observed between the flux and removal of ceramides (p < 0.01), indicating that the removal of intercellular lipids would cause dramatic dilations between adherent cornified cells and enhance the penetration through the intercellular pathways. When the penetration of FITC-dextrans through Wistar rat skin was compared with that via hairless rat skin, the steady state flux of FITC-dextrans through Wistar rat skin pretreated with water-immiscible enhancers was 1.2- to 4.9-fold higher, suggesting that the penetration of large polar molecules through follicles may play at least some role in the percutaneous absorption.

Animals↗

PubMed1995

Inhibition kinetics of theophylline metabolism by mexiletine and its metabolites.

To further characterize the mode of drug interaction between theophylline (TP) and mexiletine (ME), in vitro kinetic studies were carried out using rat liver microsomes and 9000 x g supernatant. The kinetic study revealed that the Km value and Vmax/Km ratio for the metabolic conversion of TP to 1,3-dimethyluric acid (1,3-DMU) were the second lowest and the highest, respectively, of four metabolic pathways. Thus, the rank of efficiency of the oxidative metabolism by microsomal cytochrome P-450 (P-450) isozymes was TP to 1,3-DMU > TP to 1-methylxanthine (1-MX) > TP to 3-MX > 1,3-DMU to 1-methyluric acid, suggesting that the isozyme metabolizing TP would have a higher affinity for the oxidation at the 8-position in TP molecules than at the 1- and 3-positions. Lineweaver-Burk plots showed that the conversion of TP to 3-MX and to 1,3-DMU was inhibited competitively by ME and its metabolites, and that the pathway of TP to 1-MX was inhibited noncompetitively. In consideration of the Ki values calculated, it seems probable that deamino-p-hydroxy ME (DApHME) might be the most potent inhibitor of the metabolic pathways of TP, and that the rank order of inhibition is approximately DApHME > p-hydroxy ME > deamino-hydroxymethyl ME > ME > hydroxymethyl ME, with some exceptions. The mechanism of the interaction between TP and ME is probably due to the metabolic antagonism in the liver, and TP, ME and their metabolites share some of the same metabolic pathways, mediated by P-450 isozymes.

Animals↗

PubMed1995

Comparison of the in vitro skin penetration of propiverine with that of terodiline.

This study was designed to clarify the relationship between the properties of propiverine and skin penetration, and to compare the in vitro penetration characteristics of propiverine and terodiline through rat skin. Propiverine in both hydrochloride and free forms penetrated across the skin extremely slowly, with a 2.6 times higher flux in the hydrochloride than that in the free base, in the absence of enhancers. Various enhancers failed to enhance the penetration of propiverine hydrochloride, whereas the same agents slightly increased the flux of the free form, these being due to the slow release rate of the free form from the gel formulations, an extremely high lipophilicity (log Poct/water > 4.97), much less solubility (0.141 mg/ml) and a large partition capacity of the drug to skin components. Terodiline in both forms was able to rapidly penetrate through the skin, even in the absence of enhancers, with 20.2 and 9.8 times higher fluxes respectively, than the corresponding forms of propiverine. The high penetration characteristics of terodiline would be due to a suitable lipophilicity, low binding property as well as the structural masking from the binding to the epidermal components. Propiverine hydrochloride penetrated through the stratum corneum 4.4 times and viable skin 3.1 times higher than through full-thickness skin, while the fluxes of terodiline through the stratum corneum and viable skin were similar to each other, with high penetration rates for each form.

Administration, Cutaneous↗

PubMed1995

[Induction and properties of nitric oxide synthase in rat retinal pigment epithelial cells in culture].

UNLABELLED: We investigated the production of nitric oxide (NO) and nitric oxide synthase (NOS) by rat retinal pigment epithelial cells (RPE) in culture. METHODS: RPE was isolated from the eyes of 7-10 day-old rats and cultured in Dulbecco's modified Eagle medium with 10% fetal calf serum. NO2- concentration in the culture medium was determined with the Griess reaction. NOS was assayed by measuring 3H citrulline as the reaction product. RT-PCR was performed on the isolated RNA fractions from RPE. RESULTS: Rat RPE produced NOS with the combined stimulation of the bacterial endotoxin and cytokines including interferon gamma. PCR experiments showed that stimulated RPE expressed a gene product corresponding to inducible-type NOS. The enzyme required arginine, NADPH, and tetrahydrobiopterin for the reaction. The specific activity of the crude RPE extract was approximately 270 pmol/min/mg. CONCLUSION: Like bovine RPE, rat RPE produced NOS by the immunological stimulation. Induced NOS and the product NO may play important roles in inflammation of the eye.

Amino Acid Oxidoreductases↗

PubMed1995

Stereoselective reversible binding properties of the glucuronide conjugates of fenoprofen enantiomers to human serum albumin.

The stereoselective binding of fenoprofen enantiomers and fenoprofen glucuronide diastereomers to human serum albumin (HSA) was investigated using an ultrafiltration method. Fenoprofen glucuronides exhibit a considerable and stereoselective affinity to HSA, although less than seen for the parent drug. The (R)-glucuronide shows a higher affinity to HSA than the (S)-diastereomer. With the enantiomers, no significant difference could be detected. Diazepam and probenecid reduced the binding of the glucuronides, as well as that of the fenoprofen enantiomers. These results suggest that parent drug and its glucuronide metabolites occupy the same binding region on the albumin molecule. Both fenoprofen enantiomers, as well as racemic fenoprofen, are capable of reducing the extent of reversibly bound fenoprofen glucuronide.

Animals↗

PubMed1995

Stereoselective disposition of naproxen glucuronide in the rat.

The disposition of R- and S-naproxen glucuronides were investigated after intravenous administration (approximately 1.5 mg/kg) to normal male Sprague-Dawley rats and to rats pretreated with phenylmethylsulfonylfluoride, an inhibitor of esterases. The relative stability of the two glucuronides also was measured in vitro. Both diastereomers were hydrolyzed rapidly in vivo, liberating naproxen, but R-naproxen glucuronide was hydrolyzed faster than the corresponding S-diastereomer. This difference resulted in a larger plasma AUC(Nap):AUC(Nap-G) ratio for the R-glucuronide. There was, however, no marked difference in the apparent clearance of the R- and S-diastereomers. Administration of phenylmethylsulfonylfluoride had no significant effect on the disposition of the two diastereomers. In 0.15 M phosphate buffer (ph 7.4) at 37 degree C, the fastest degradation process for both diastereomers in vitro was acyl migration. Our results show that R-naproxen glucuronide is more labile than S-naproxen glucuronide in vivo and in vitro, and suggest that hydrolysis, rather than biliary excretion, is the major process leading to elimination of R-naproxen glucuronide in vivo in the rat. These results demonstrate that the rat may in certain situations be an inadequate model for studying the disposition of acyl glucuronides and that the metabolic disposition, and possibly toxicities, of diastereomeric metabolites of chiral drugs can be quite different even when the individual diastereomers have similar apparent clearances.

Animals↗

PubMed1994

Simultaneous measurement of nicotinic acid and its major metabolite, nicotinuric acid in urine using high-performance liquid chromatography: application of solid-liquid extraction.

The concentrations of nicotinic acid (NiAc) and nicotinuric acid (NiUAc), a major metabolite of NiAc, were simultaneously determined in urine using solid-phase extraction (cation-exchange extraction) and reversed-phase high-performance liquid chromatography with ultraviolet detection. The intra- and inter-day precision studies showed good reproducibilities: the coefficients of variations were less than 8.1% for NiAc and 8.8% for NiUAc. The calibration curves were linear (r2 > 0.9934) in the concentration range 10-1000 micrograms/ml. The removal of endogenous interferences in urine by solid-phase extraction presented here is superior to the pretreatment protocols reported previously by other workers. The method was used in a preliminary pharmacokinetic study in rats after intravenous administration of NiAc (5 and 15 mg/kg).

Animals↗

PubMed1994

Pharmacokinetics of indomethacin octyl ester (prodrug) and indomethacin produced from the prodrug.

A prodrug of indomethacin, indomethacin octyl ester (IM-OE), was synthesized and its pharmacokinetics was investigated in rat. To describe the time course of the plasma indomethacin and IM-OE after intravenous (iv) and oral administrations, a pharmacokinetic model with four compartments was developed. Indomethacin rapidly appeared in plasma after iv administration of IM-OE and declined in a monoexponential manner, with a rapid decline and low plasma levels of IM-OE. The plasma concentrations of indomethacin after oral administration of IM-OE were much lower than those after oral administration of indomethacin. The high concentrations of IM-OE compared with indomethacin were detected in liver 3 h after oral dosing of the prodrug, although IM-OE was not detected in plasma. A good fit was obtained between the observed and calculated curves based on the model, which includes a conversion rate constant of IM-OE to indomethacin for both iv and oral dosings of IM-OE. Additionally, the model could successfully describe the plasma concentration versus time profiles after indomethacin dosings.

Administration, Oral↗

PubMed1994

Mechanism of the enhancement effect of n-octyl-beta-D-thioglucoside on the transdermal penetration of fluorescein isothiocyanate-labeled dextrans and the molecular weight dependence of water-soluble penetrants through stripped skin.

To clarify the mechanism of the enhancing effect of n-octyl-beta-D-thioglucoside (OTG), which acts as a potent enhancer for skin penetration of peptides and water-soluble penetrants, the in vitro penetration of macromolecules [fluorescein isothiocyanate-labeled dextrans (FTIC-dextrans)] was evaluated with hairless rat skin and stripped skin. The FITC-dextrans (MW, 4400, 9600, and 69,000 Da, referred to as FD-4, FD-10, and FD-70, respectively) penetrated more easily in the presence of OTG (1.5%), with high fluxes equivalent to those through stripped skin. This result indicated that the enhancement effect of OTG on the penetration of macromolecules through the stratum corneum was extensive, and the barrier function of the corneum was nearly eliminated by the OTG treatment. OTG significantly solubilized the stratum corneum proteins and ceramides during the initial time stage. Scanning electron microscopic observations demonstrated that OTG treatment dramatically changed the cell membrane (i.e., exfoliation of cell membranes and dissociation of adherent cornified cells), suggesting a significant disturbance of the cohesive laminae and barrier functions. The extent of dissociation of cell membranes increased with treatment time, without significant changes in the cell junctions. These results clarify that the enhancement mechanism of OTG was different from that of laurocapram and other lipophilic enhancers. The permeability of polar solutes with differing molecular sizes (MW, 180-69,000 Da) through stripped skin was size dependent (r = 0.997, p < 0.001). However, the viable epidermis and dermis restricted the penetration of macromolecules, such as FD-70.

Animals↗

PubMed1994

Scanning electron microscopy of the egg of Mansonia dives (Diptera: Culicidae).

The egg of Mansonia dives (Schiner) is described by means of scanning electron micrographs and compared with other species described in previous studies. The egg of Ma. dives differs from those of Ma. uniformis (Theobald), Ma. indiana (Edwards), Ma. annulifera (Theobald), Ma. annulata (Leicester), and Ma. bonneae (Edwards) in that it has a tuberculated anterior cup. A key for identification of the eggs of the six Mansonia species, based on electron microscopy, is presented.

Animals↗

PubMed1994

Catalytic nature of tyrosine hydroxylase in bovine ocular tissues.

We performed a comparative study on tyrosine hydroxylase (TH), a rate-limiting enzyme in catecholamine biosynthesis, in two ocular tissues, the retina and the iris-ciliary body. Immunoblotting analysis and gel filtration study suggested no significant difference in their subunit structure and their oligomeric form. The optimal pH of TH was 6.0 in retina and 6.3 in iris-ciliary body. However, at the physiological pH (7.25), the retinal TH had only 15% of the maximum activity while TH in the iris-ciliary body had 70% of the maximum activity. In hydroxylapatite chromatography, both extracts showed different elution profiles; the major TH activity in retina appeared earlier (200 mM, phosphate concentration) than that in iris-ciliary body (300 mM). When these enzymes were phosphorylated by catalytic subunit of cAMP-dependent protein kinase, most of the activity shifted to the later peak in both enzymes. Also, the activity of dephosphorylated TH in iris-ciliary body shifted to the earlier peaks. These results indicate that native TH in retina is basically less phosphorylated and thus exists in a less activated form than that in iris-ciliary body.

Animals↗

PubMed1994

Percutaneous absorption of physiologically active peptides, ebiratide and elcatonin, in rats.

This study was designed to evaluate the percutaneous absorption of physiologically active peptides, ebiratide (a behaviorally potent adrenocorticotropic analog) and elcatonin (a hypocalcemic peptide) in an attempt to develop an efficient transdermal therapeutic system for the treatment of diseases. The [125I]ebiratide penetration through rat skin from gel formulations could be described fairly well by a zero-order kinetic profile. Skin penetration was the greatest when EDTA, n-octyl-beta-D-thioglucoside (OTG, 1.5%) and taurocholate (1.0%) were combined in a gel formulation. The order of flux was: EDTA, OTG and taurocholate (formulation 3) > OTG and taurocholate (formulation 2) > glucosyl-beta-cyclodextrin and OTG (formulation 4). When the transdermal systems of [125I]ebiratide prepared using a corresponding gel formulation were applied to rat abdomen, the plasma levels of radioactivity after formulations 3 and 2 were much higher than those after formulation 1 without enhancers, and the radioactivity was observed in the brain, although in a very small quantity. The hypocalcemic effect of elcatonin was measured in vivo after application of the transdermal systems. The plasma calcium levels decreased comparatively rapidly and low levels were maintained for a long period, indicating the effectively percutaneous absorption of elcatonin. Formulation 7 containing D-limonen and taurocholate as enhancers and/or inhibitors showed much higher hypocalcemic effect than two other formulations combined with laurocapram or N,N-diethyl-m-toluamide, consequently giving the highest pharmacological availability (8.7 +/- 1.0%). These results clearly demonstrated that the peptides were effectively absorbed through rat skin in the presence of enhancers.

Administration, Cutaneous↗

PubMed1994

Effect of food on propranolol oral clearance and a possible mechanism of this food effect.

To better define the mechanism of the increased plasma concentration of propranolol (PL) after meals, the effect of the dietary constitution of a meal on the kinetics of PL and beta-naphthoxylactic acid (NLA), a main metabolite, after administration of the drug, was investigated in rats. Additionally, the hepatic uptake of PL and cytochrome P-450 (P-450) content and uridine 5'-diphosphoglucuronyltransferase (UDPGT) activity in liver were measured after glucose intake. As a result, protein (skim milk) intake slightly, but not significantly, increased the area under the plasma concentration-time curve (AUC) and bioavailability of PL, with a slight increase (16%) in hepatic blood flow, and enhanced PL metabolism to NLA. Soybean oil and fatty acid intake significantly decreased the bioavailability of PL, while glucose intake dramatically decreased the hepatic uptake of PL and P-450 content at high glucose levels, resulting in a decrease in the plasma PL concentration at the initial time period and in the inhibition of a metabolic conversion to NLA. Thus, a possible mechanism involved in the effect of food on PL bioavailability could have been due largely to the decreased microsomal P-450 content and hepatic uptake of PL after glucose intake, but only partly to the increased hepatic blood flow after protein intake.

Administration, Oral↗

PubMed1994

Physicochemical and hydrolytic characteristics of phenytoin derivatives.

To further clarify the pharmacokinetic characteristics of phenytoin (DPH) and its derivatives, DPH-1-methylnicotininate (MNDPH), valeroyl DPH (VADPH) and valproyl DPH (VPDPH), in plasma and brain, we have investigated their physicochemical properties and protein binding characteristics. Additionally, the hydrolytic conversion of these derivatives to DPH was also studied using small intestine, liver and brain tissues, as well as rat plasma. The log partition coefficient (PC) values of all derivatives were much higher than that of DPH. Judging from their pKa values (5.68 and 5.91 for VADPH and VPDPH, respectively) and pH-solubilities, VADPH and VPDPH were acidic compounds, while MNDPH was basic. These data indicated that most fractions of VADPH and VPDPH existed as an ionized form (these fractions existed in an ionized form, 0.98 and 0.97, respectively) at physiological pH, whereas MNDPH existed as a unionized form under the same conditions. Rosenthal or Scatchard plots of the binding data of DPH and its derivatives to both rat plasma protein and bovine serum albumin (BSA) exhibited straight lines over their concentration ranges used, indicating that DPH and its derivatives have a single binding site on the protein. The binding potencies (K or n.Pt value) of the derivatives to both proteins were much greater than that of DPH. No DPH produced from VADPH and VPDPH was found in the biological fluids over a period of 24 h. However, the hydrolysis of MNDPH to DPH was observed in plasma and the tissues used, with the most rapid hydrolysis in the small intestine, and the hydrolysis rate constant in plasma was ca. 20-fold greater than that in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

The first case of equine motor neuron disease in Japan.

A 9-year-old male horse showed emaciation, weakness and trembling and was euthanatized. Histopathological examinations revealed loss, swelling and chromatolysis of motor neurons throughout the spinal ventral horns, axonal degeneration of the ventral spinal roots. Eosinophilic cytoplasmic inclusions were distributed in degenerated spinal ventral neurons. Ultrastructurally, the inclusions consisted of aggregations of granular dense material and a few vesicles. They reacted positively with polyclonal antibody against ubiquitin. The present case was diagnosed as equine motor neuron disease, which has recently been reported in North America and the United Kingdom.

Animals↗

PubMed1994

The role of nitric oxide synthase in endotoxin-induced uveitis: effects of NG-nitro L-arginine.

PURPOSE: To evaluate the role of nitric oxide synthase (NOS) in endotoxin-induced uveitis (EIU). METHODS: EIU was caused in Lewis rats by injecting lipopolysaccharide (LPS) into the foot pad, and the effects of an NOS inhibitor, NG-nitro L-arginine (NA), was comparatively studied by the simultaneous administration of NA and LPS. Total NOS and inducible NOS (iNOS) activities were differentially assayed in the anterior segment of the eye in EIU with and without NA treatment. The effects of NA on EIU were also evaluated by clinical manifestation, histology, and protein concentration in the aqueous humor. RESULTS: In untreated rats, there was no significant iNOS activity. With the EIU model, iNOS activity showed a marked increase in the anterior segment of the eye, reaching a maximum 9 hours after LPS injection (10,850 +/- 1,650 cpm/mg protein, mean +/- SEM). NA reduced the iNOS activity 9 hours after injection to 2,400 +/- 90 cpm/mg protein (P < 0.001). Aqueous humor protein concentration in the EIU model was 10.6 +/- 0.75 mg/ml, and the cell number was 216 +/- 12 cells/microliters. NA significantly reduced these factors to 4.25 +/- 0.48 mg/microliters for the protein concentration (P < 0.0005) and 25 +/- 6 cells/ml for the cell number (P < 0.0005). Histologic studies showed less prominent infiltration of polymorphonuclear cells in the anterior uvea than for conventional EIU. CONCLUSIONS: Induction of iNOS may play a key role in the pathogenesis of EIU. Because inhibition of iNOS activity reduced the inflammatory response, suppression of NO formation may inhibit the development of EIU.

Amino Acid Oxidoreductases↗