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F Komada

Publications and source records attributed to F Komada.

At least 37 records · Page 2Linked to original sources
PubMed1996

Effects of transfection with the Cu, Zn-superoxide dismutase gene on xanthine/xanthine oxidase-induced cytotoxicity in fibroblasts from rat skin.

PURPOSE: The effects of transfection with the human Cu, Zn-superoxide dismutase (hSOD)4 gene on active oxygen-induced cytotoxicity in rat skin fibroblasts (FR) were studied for the purpose of developing the novel delivery system of hSOD using hSOD gene. METHODS: An expression plasmid for hSOD, pRc/RSV-SOD, was constructed and used to transfect FR cells. Xanthine (X)/xanthine oxidase (XO) system were used to generate active oxygen species. The effects of transfection with the hSOD gene on active oxygen-induced cytotoxicity were assessed by comparing the number of surviving cells and the level of lipid peroxidation in host and transformants after exposure to X/XO system. RESULTS: The cellular SOD activity in RSV-SOD cells transfected with pRc/RSV-SOD was significantly increased in comparison with host or RSV cells transfected with the pRc/RSV plasmid containing no hSOD gene as a control. Furthermore, Western blot analysis using an anti-hSOD antibody indicated the production of hSOD in RSV-SOD cells. On the other hand, although the numbers of surviving cells in both host and RSV-SOD cultures after exposure to X/XO system decreased in a time-dependent manner, the decrease in number of surviving RSV-SOD cells was less than that in host cells. In the presence of catalase, the decreases in number of surviving cells in both host and RSV-SOD cultures after exposure to the X/XO system were also less than those in the absence of catalase. However, the decreases in cell survival in RSV-SOD cultures were significantly less than those in host cells in the presence of catalase. Furthermore, the levels of lipid peroxidation in RSV-SOD cells exposed to the X/XO system in the presence or absence of catalase were lower than those in host cells. These results indicated that the increase in cellular SOD activity by transfection with the hSOD gene protects cells from oxidative stress. CONCLUSIONS: Human SOD gene therapy may be useful for treatment of diseases in which oxidative tissue damage is produced.

Analysis of Variance↗
PubMed1996

Pharmaceutical studies for gene therapy: expression of human Cu, Zn-superoxide dismutase gene transfected by lipofection in rat skin fibroblasts.

To evaluate whether lipofection using Lipofectin is suitable for delivering foreign genes into skin fibroblasts as target cells, we performed experiments using human superoxide dismutase (hSOD) and neomycin-resistance (Neo) genes as models in rat skin fibroblasts (FR and primary cells) in vitro. The amounts of DNA used in the lipofection procedure significantly affected the transfection efficiencies, and the optimal amounts were determined for all cells used. However, the efficiencies in rat skin fibroblasts were about 20-fold higher than that in rat lung epithelial-like cells (L2 cells). The differences in plasmid vectors (pRc/RSV-SOD and pRc/CMV-SOD) hardly affected the transfection efficiencies. The amounts of Lipofectin significantly affected the transfection efficiencies, and the optimal amounts were determined for both types of skin fibroblasts. However, cytotoxic effects in both skin fibroblasts were observed with high doses of Lipofectin. On the other hand, with optimal amounts of DNA and Lipofectin, the reporter gene (NeoT) introduced into cells was mainly integrated into the host cell chromosome. Western blot analysis showed the continuous expression of hSOD protein for at least 45 d in skin fibroblasts transfected with the expression plasmid for hSOD by Lipofectin under the optimal conditions, and the cellular SOD activity fluctuated in parallel with the expression of hSOD protein. Differences in the type of cells also affected the expression of hSOD. These results indicate that it is necessary to set up optimal conditions for transfection using Lipofectin for each cell type, and that transfection with Lipofectin under optimal conditions may be an efficient method for introduction of foreign genes into skin fibroblasts for use as a clinical delivery system of therapeutic protein.

Animals↗
PubMed1996

Effect of transfection with a superoxide dismutase expression plasmid on xanthine/xanthine oxidase-induced cytotoxicity in cultured rat lung cells.

We inserted human Cu, Zn-superoxide dismutase (hSOD) cDNA into the eukaryotic expression plasmid (pRc/CMV) under the control of the cytomegalovirus promoter. The hSOD expression plasmid (pRc/CMV-SOD) was transfected in L2 cells by mean of lipofection. The intracellular SOD activity in pRc/CMV-SOD transfected cells (CMV-SOD cells) was about twice that in host cells. However the level of extracellular SOD activity was similar in CMV-SOD and host cells. When exposed to xanthine (X)/xanthine oxidase (XO) to generate active oxygen species, significantly more CMV-SOD cells than host cells survived. The production of lipid peroxidation in host cells significantly increased in the presence of X/XO, but that in CMV-SOD cells did not change. Thus, transfection with SOD gene effectively prevented X/XO-induced cytotoxicity. The results indicated that increasing the level of intracellular SOD activity protected cells against extracellular superoxide anion stress.

Animals↗
PubMed1996

Effect of transfection with superoxide dismutase expression plasmid on superoxide anion induced cytotoxicity in cultured rat lung cells.

Human Cu, Zn-superoxide dismutase (hSOD) cDNA was inserted into a eukaryotic expression plasmid (pRc/CMV) under the control of a cytomegalovirus promoter. The hSOD expression plasmid (pRc/CMV-SOD) was transfected into L2 cells by means of lipofection. The integration of the hSOD gene in genomic DNAs in the cells transfected with pRc/CMV-SOD plasmid was examined by Southern blotting using hSOD cDNA as the probe. However, Southern blots of host cells (without transfection) and CMV cells (pRc/CMV plasmid transfection) indicated no hybridization of hSOD cDNA. Western blots indicated that hSOD was expressed in CMV-SOD cells. The SOD activity in CMV-SOD cells was about twice that in host and CMV cells. Furthermore, this SOD activity in CMV-SOD cells was enhanced for 60 d after the selection of cell clones. After exposure to paraquat and catalase, about 90% of the CMV-SOD cells survived compared with the untreated controls, whereas about 60% of the host cells survived. The production of lipid peroxidation in host cells increased significantly after exposure to both paraquat and catalase, whereas that in CMV-SOD cells did not change. The correlation between the surviving cells and lipid peroxidation was inverse. These results indicated that transfection with the SOD gene was effective against superoxide anion induced cytotoxicity.

Animals↗
PubMed1996

Effect of clarithromycin on the bioavailability of cyclosporin in rats.

This study was conducted to determine the effect of clarithromycin (CAM) on the bioavailability of cyclosporin (CYA) in rats, and to compare its effect with that of erythromycin (EM). The area under the blood CYA concentration-time curve (AUCi.v.) values after intravenous administration of CYA (2 mg/kg) in combination with CAM or EM (100 mg/kg, p.o.) were significantly increased compared with those of CYA alone, suggesting that there was metabolic inhibition of CYA in the liver by CAM or EM. The time to reach the peak concentration after oral administration of CYA (10 mg/kg) tended to be longer with increasing doses of both CAM and EM (10 and 100 mg/kg, p.o.). Each AUCp.o. value for the CAM or EM coadministration group, except the EM (100 mg/kg) coadministration group (about 77% increase), was comparable to that for the CYA alone group. Both CAM and EM (10 and 100 mg/kg, p.o.) were shown to delay gastric emptying in a dose-dependent manner. The gastric emptying in the group treated with CAM (100 mg/kg) was significantly lower than that with EM (100 mg/kg). It is suggested that CAM as well as EM might affect the oral bioavailability of CYA by inhibiting its metabolism and simultaneously by changing the gastrointestinal motility in rats. Thus, caution is recommended when administering CYA concomitantly with CAM to humans.

Administration, Oral↗
PubMed1995

Population pharmacokinetics of theophylline. III. Premarketing study for a once-daily administered preparation.

The population pharmacokinetic parameters for a once-daily administered preparation, Uniphyl were estimated from data collected in the premarketing clinical trial. Altogether, 2772 serum theophylline concentrations were obtained from 131 normal subjects and 306 patients suffering from chronic asthma or chronic obstructive pulmonary disease who participated in the phase I, II, and III clinical trials in Japan. The serum concentration profile was described by a linear one-compartment model with first-order absorption. The factors affecting the pharmacokinetics of this drug were examined by the likelihood ratio test using a nonlinear mixed effect model (NONMEM). The first-order absorption rate constant (Ka) for a 200-mg tablet in a fasting condition was obtained as 0.0773 (1/h), which was smaller than the elimination rate constant (0.168 1/h), indicating the flip-flop characteristic of this preparation. Food indigestion increased the Ka by 17% and the absorption lag time by 5-fold but did not affect the extent of absorption. The 400-mg tablet showed a Ka value 19%, smaller than the 200-mg tablet. Children not older than 15 years showed 58% longer absorption lag time. The inter-individual variability in Ka was 19%, suggesting small variability in the vivo release process. The total body clearance was related to hepatic function, smoking habits, and age. Furthermore, clearance decreased in association with the severity of illness. The findings obtained here are useful not only for the initial dosage adjustment for patients with a variety of backgrounds but also for doses individualization based on serum concentration monitoring with or without the Bayesian feedback method.

Adolescent↗
PubMed1994

Intratracheal delivery of peptide and protein agents: absorption from solution and dry powder by rat lung.

Proteins of high molecular weight and low lipophilicity must be administered parenterally to achieve the desired therapeutic blood levels. We investigated the absorption of peptide and protein agents by rat lung following their intratracheal administration, expressing it as percent bioavailability. An aqueous solution and/or a dry powder of calcitonin, insulin, thyrotropin stimulating hormone (TSH), follicle stimulating hormone (FSH), and human chorionic gonadotropin (HCG) was delivered into the exposed trachea of anesthetized rats, and blood was sampled from the jugular vein at specified intervals. The bioavailabilities of TSH, FSH, and HCG delivered in a solution of neutral pH were 2.5, 2.3, and 0.2 %, respectively. Transpulmonary absorption of a solution of these agents, administered with a surfactant or under acidic conditions, was 2-30 times greater than the values obtained in controls. The bioavailabilities of calcitonin, insulin, TSH, FSH, and HCG, given intratracheally as a dry powder, were 11.5, 6.5, 1.6, 0.6, and 0.1%, respectively. Following intratracheal administration, we noted a negative association between molecular weight and bioavailability. The intratracheal route may thus be useful for delivering peptide and protein agents.

Absorption↗
PubMed1994

Effect of epidermal growth factor on Cu, Zn-superoxide dismutase expression in cultured fibroblasts from rat skin.

The effects of epidermal growth factor (EGF) on Cu, Zn-superoxide dismutase (SOD) in cultured fibroblasts from rat skin exposed to superoxide anions were studied. Cross-linking of [125I]hEGF using disuccinimidyl suberate and immunoblot analysis using anti-EGF receptor antibody to crude plasma membrane fractions of fibroblasts showed that a 170 kDa EGF receptor protein was present on the membrane, as in A431 cells which over express a specific EGF receptor. The cytosolic SOD enzyme activity in fibroblasts exposed to superoxide anions 24 h after treatment with EGF plus nafamostat (NM), a potent protease inhibitor, was increased 1.6-fold compared to control-treated cells. Treatment with either EGF or NM alone, evoked little increase in SOD enzyme activity. The increase in Cu, Zn-SOD protein levels corresponded to the increase in cytosolic SOD enzyme activity in fibroblasts. The Cu, Zn-SOD mRNA level in fibroblasts treated with EGF plus NM at 3 and 6 h was higher than that of the control. Additionally, levels of [125I]hEGF degradation products released into the medium from fibroblasts exposed to superoxide anions were significantly reduced in the presence of NM. These results suggest that the stabilization of EGF by NM in culture is an important factor in the expression of its effects, and that EGF induces Cu, Zn-SOD expression by accelerating transcription of the Cu, Zn-SOD gene in cells, resulting in their protection from the effects of superoxide anion radicals.

Animals↗
PubMed1993

Cytoprotective effects of epidermal growth factor (EGF) ointment containing nafamostat, a protease inhibitor, on tissue damage at burn sites in rats.

When epidermal growth factor (EGF) ointment containing a protease inhibitor, nafamostat (NM), was applied to burn sites in rats, the superoxide dismutase (SOD) enzyme activity and protein content increased 45% and 60%, respectively, at these sites 1 d after the burns compared with the control ointment. Following treatment with EGF plus NM (EGF + NM) ointment, messenger RNA for SOD also increased, to about 1.6 times that of the control at 1 d after the burn, indicating that this ointment stimulates SOD synthesis at burn sites in vivo. In contrast, following treatment with EGF + NM ointment, the content of heat shock protein (HSP 70) in the burned tissue decreased to about 70% of the control value 1 d after the burn. These findings suggest that EGF + NM ointment alleviated tissue damage at burn sites at an early stage, and that this was related to the stimulation of SOD synthesis and reduced HSP 70 levels. We also examined the effects of SOD ointment on wound healing at burn sites. A dose-dependent increase in the dry weight of granulation tissue at wound sites 3 d after the burn was observed following the application of this ointment. These results suggest that SOD may play an important role in wound healing after burns.

Animals↗
PubMed1993

Systemic effects of epidermal growth factor (EGF) ointment containing protease inhibitor or gelatin in rats with burns or open wounds.

The systemic effects of epidermal growth factor (EGF) ointment containing nafamostat (NM), gabexate, or gelatin was studied in rats with burns or open wounds. At 1 d after burn, plasma epinephrine, cortisol, and glutamic-oxalacetic transaminase (GOT) levels were elevated, but treatment with EGF plus NM (EGF+NM) ointment significantly suppressed the increase in these levels. Further, there was no loss of body weight in the open wound model following treatment with EGF+NM ointment, while loss of body weight occurred in animals in which EGF ointments without NM were applied. Increases in plasma epinephrine 1 d after open wound formation were also suppressed by the application of EGF+NM ointment. Treatment with EGF ointment containing gabexate (GX) or gelatin (GL) ameliorated changes in body weight that occurred after open wound formation, while loss of body weight in animals with open wounds occurred following the application of ointment base, EGF ointment, GX ointment, or GL ointment. The present study thus indicates that the topical application of EGF ointment containing a protease inhibitor has ameliorative systemic effects.

Animals↗
PubMed1993

Improved bioavailability of para-boronophenylalanine by cyclodextrin complexation.

This study was undertaken to develop an oral dosage form for para-boronophenylalanine (BPA) plus cyclodextrin (CD) for use in the thermal neutron capture therapy for malignant melanoma. Powders of the BPA and CD complexes were obtained in a molar ratio of 1:2. X-ray diffraction of the BPA-CD complexes showed halo patterns that indicated that each complex was in a new solid state as an amorphous compound. The enhancement of BPA solubility by glucosyl (G1)- and maltosyl (G2)-alpha-CD was greater than that with the other CDs. The isolation rate of BPA from its complex was different for each BPA-CD complex. The bioavailability of BPA in rats was improved with oral administration of the BPA-alpha-CD, G1-alpha-CD, and G2-alpha-CD complexes. In contrast, a complex of BPA and dimaltosyl (G2G2) or G2-beta-CD, which had low release rate and low solubility, did not improve the bioavailability of BPA. These results indicate that the solubility and release rate of BPA from a complex in solution are important for the bioavailability of BPA after oral administration of BPA-CD complexes.

Animals↗
PubMed1993

Gamma/delta T-cell lymphoma with hepatosplenomegaly: report of a case.

We describe the case of a patient with peripheral gamma/delta T-cell lymphoma (T-ML) with hepatosplenomegaly, generalized lymphadenopathy, and bone marrow involvement. A 44-year-old man had lymphoma, which became clinically apparent 2 months after the onset of myositis and insulin-dependent diabetes mellitus. A cervical lymph node biopsy specimen showed diffuse infiltration by large neoplastic cells with vascular proliferation. The neoplastic cells expressed the T-cell receptor (TCR)delta chain detected by TCR delta 1 and delta-TCS1, CD3, CD30, CD45RO, and epithelial membrane antigen, but not the TCR beta chain detected by beta F1, CD1a, CD2, CD4, CD5, CD7, CD8, CD25, HLA-DR, and terminal deoxynucleotidyl transferase. The cells had a clonal rearrangement of TCR gamma chain gene and a germ-line configuration of immunoglobulin heavy chain gene and TCR beta chain gene. Despite chemotherapy, the patient died of refractory lymphoma 4 months after diagnosis. Examination at autopsy revealed that the main hepatic and splenic neoplastic infiltration sites were the portal area and white pulp, respectively. Our patient differed from those with gamma/delta T-ML with hepatosplenic involvement reported previously with respect to the hepatic and splenic neoplastic infiltration patterns and the presence of lymphadenopathy.

Adult↗
PubMed1992

A role for protein kinase C in the growth of human erythroid progenitor cells.

We searched for a possible role for protein kinase C in the growth of human erythroid progenitor cells, using pharmacologic approaches. Two protein kinase C inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine, dose-dependently inhibited the growth of immature erythroid progenitor cells (BFU-E) induced by interleukin 3 (IL-3) plus erythropoietin (Ep) or granulocyte macrophage colony-stimulating factor (GM-CSF) plus Ep whereas a weaker analog of H-7, N-(2-guanidinoethyl)-5-isoquinoline sulfonamide (HA-1004), had no effect on the number of BFU-E. These three compounds had no effect on the growth of mature erythroid progenitor cells (CFU-E) stimulated by Ep. The culture of accessory cell-depleted bone marrow demonstrated that the effects of these compounds on colony formation do not appear to be mediated by accessory cells. The potential of these compounds to inhibit the GM-CSF-dependent growth of KG-1 cells correlated well with the extent of their inhibitor of protein kinase C activities from KG-1 cells. Thus, the protein kinase C system is apparently involved in the growth of BFU-E, supported by IL-3 or GM-CSF. The growth signal for CFU-E transduced by Ep may be achieved through other systems.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Inhibition of platelet aggregation by the cAMP-phosphodiesterase inhibitor, cilostamide, may not be associated with activation of cAMP-dependent protein kinase.

We examined the involvement of cAMP-dependent protein kinase (A kinase)2 in the inhibition by cilostamide, a specific inhibitor of the low Km cAMP-phosphodiesterase (PDE), on 9,11-epithio-11,12-methanothromboxane A2 (STA2)-induced platelet aggregation. For comparative purposes, the PGE1 analogue, 17S-20-dimethyl-trans-delta 2-PGE1 (OP-1206) was used. OP-1206 (IC50 = 18 +/- 0.55 nM) and cilostamide (IC50 = 40 +/- 4.5 nM) were both potent inhibitors of the platelet aggregation induced by STA2 (1 microM). OP-1206 and cilostamide dose-dependently inhibited elevations in intracellular free Ca2+ ([Ca2+]i) caused by STA2. OP-1206 caused an almost complete inhibition of Ca2+ mobilization, but cilostamide did not prevent the STA2-induced elevation in [Ca2+]i to the same extent as OP-1206, even at a high concentration (greater than 200 nM). Cilostamide did not increase the cAMP level at concentrations (5-100 nm) which affected STA2-induced aggregation. OP-1206 significantly increased cAMP contents in platelets, and the degree of aggregation inhibition by OP-1206 appears to be related to the size of increase in cAMP. OP-1206 increased phosphorylation of the 50,000 mol. wt vasodilator-stimulated phosphoprotein, at concentrations of 7.9-79 nM, which inhibited aggregation induced by STA2. Cilostamide treatment resulted in a marginal increase in the 50,000 mol. wt phosphorylation at concentrations (10-100 nM) which completely inhibited the STA2-induced aggregation. (8R*, 9S*, 11S*)-(-)-9-Hydroxy-9-n-hexyloxy-8-methyl-2,3,9,10- tetrahydro-8,11-epoxy-1H, 8H, 11H-2, 7b, 11a-triazadibenzo(a,g)-cycloocta(c,d,e)trinden-1-one (KT-5720), a specific inhibitor of A kinase, not only reversed the inhibition by OP-1206 of STA2-induced platelet aggregation, but also inhibited the OP-1206-induced protein phosphorylation. However, the inhibition by cilostamide of STA2-induced aggregation was not prevented by pretreatment with KT-5720. Inhibition of the STA2-induced aggregation by OP-1206 may be associated with cAMP-dependent protein phosphorylation, while cilostamide may have inhibitory effects on STA2-induced platelet activation through mechanisms other than the activation of A kinase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

Expression of three major protein kinase C isozymes in various types of human leukemic cells.

We examined the levels of protein kinase C (PKC) activity and the expressions of its three major isozymes, designated types I (gamma), II (beta), and III (alpha), in the cytosol and particulate fractions of cells from patients with acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL), in an attempt to elucidate the cell type- or lineage-specific expression of these isozymes. The levels of PKC activities in the cytosol and particulate fractions from AML cells were higher than those from ALL or CLL cells. The average PKC activities of AML cells, ALL cells, and CLL cells were 18.7, 12.2, and 11.3 pmol/min/10(8) cells, respectively, in the cytosol fractions and 4.4, 3.1, and 2.6 pmol/min/10(8) cells, respectively, in their particulate fractions. M1 cells (French-American-British classification) and AML cells with T-lymphocyte-associated surface antigens, such as CD2 and CD7, had significantly lower PKC activities among AML cells. Immunoblot analyses using monoclonal antibodies against each isozyme revealed that all three isozymes were broadly distributed on leukemic cells with considerable variability in the level of expression. All lymphoid leukemic cells expressed PKC-gamma in the cytosol fractions, albeit a minor component; however, this type was observed in cells from only half the number of AML patients. Those AML cells with cytosolic PKC-gamma usually expressed lymphoid surface antigens, such as CD2, CD7, and CD19. On the other hand, cytosolic PKC-beta and PKC-alpha were commonly observed in all types of leukemic cells. AML cells expressed these two types at almost equal levels, but in lymphoid cells, expressions of PKC-beta were usually more abundant than those of PKC-alpha. These data suggest that AML cells with lymphoid antigens might have a lower PKC activity but more predominant expression of cytosolic PKC-gamma than the usual AML cells.

Adult↗
PubMed1991

Design of 9-beta-D-arabinofuranosyladenine (ara-A) transdermal delivery system for animal studies: regulation of drug concentration in vivo.

Transdermal delivery systems of 9-beta-D-arabinofuranosyladenine (ara-A), having controlling membranes of various permeabilities, were developed and applied to Azone-pretreated hairless mouse abdominal skin. It was confirmed that the blood concentrations of ara-A and its metabolite 9-beta-D-arabinofuranosylhypoxanthine (ara-H) in hairless mice are controlled by the permeability of the controlling membrane in the transdermal patch. Furthermore, these blood concentrations were found to closely agree with the values obtained from theoretical model calculations. Finally, but importantly, the "micropharmacokinetic" behavior of ara-A in cutaneous tissue could also be predicted. These results suggest that the transdermal patch may be employed in dermal and transdermal ara-A efficacy studies in the treatment of cutaneous herpes virus infections in hairless mice.

Administration, Cutaneous↗
PubMed1991

Enhanced bioavailability of subcutaneously injected insulin by pretreatment with ointment containing protease inhibitors.

The present study was undertaken to develop an ointment preparation containing a protease inhibitor for stabilizing subcutaneously injected insulin. The ointment containing the protease inhibitor, gabexate mesilate or nafamostat mesilate, was applied to the skin around the insulin injection site. Three results were obtained. First, gabexate and nafamostat inhibited insulin degradation in subcutaneous tissue homogenates in vitro. Second, after application of gabexate or nafamostat ointment, an appreciable amount of gabexate or nafamostat appeared in the subcutaneous tissue of rats or hairless mice and their concentrations were comparable to those seen in the in vitro experiment. Third, insulin degradation at the subcutaneous injection site in the rat was depressed after pretreatment with gabexate or nafamostat ointment. Pretreatment with gabexate or nafamostat ointment increased the plasma immunoreactive insulin (IRI) levels and the hypoglycermic effect of insulin in healthy volunteers. These results indicate that gabexate or nafamostat ointments stabilize subcutaneously injected insulin.

Administration, Topical↗