Bio-organisms possess numerous systems that produce reactive oxygen species (ROS). Severe oxidative stress induces cellular damage that can lead to apoptosis or necrosis, but moderate ROS levels constitute and modulate normal and critical physiological pathways in the regulation of cellular functions, including signaling cascades and transcriptional/post-transcriptional control of gene expression. ROS are also found to mimic some of the physiological stimuli by direct modification of factors or indirect mechanisms via change in the oxidative and reductive status inside/outside cells. This review will describe the biological relevance and essential roles of ROS in animal cells, focusing on signal transduction, gene expression, apoptosis and aging.
A simple and sensitive semi-micro high-performance liquid chromatography (HPLC) was established for determining the serum levels of glycyrrhizin (GL) in humans. Butyl p-hydroxybenzoate was used as the internal standard and serum was deproteinized by methanol. The samples were separated on Capcell Pak C18 UG120 column (150×1.5 mm i.d.; particle size, 5 μm). The detection limit of GL in serum was 100 ng/ml, which enables determination of serum levels of GL after administration of a therapeutic dose. This time-course study suggested that the elimination rate of GL differed between subjects for the same administered dose, although the sample was too small to allow a meaningful comment. In clinical practice, GL is used for its antiviral and anti-inflammatory effects. Excessive administration of GL can induce pseudoaldosteronism; however the optimal GL concentration in serum remains to be determined. The determination method reported here is expected to aid in the safe and efficient use of the drug in clinical practice.
The preparation and antigenic properties of digoxin C-3' and C-3" hemisuccinate-bovine serum albumin (BSA) conjugates are described. The antisera were prepared by immunizing rabbits with each of the digoxin-BSA conjugates, and properties of the antisera were characterized by RIA with 3H-labeled digoxin. The antidigoxin antiserum from immunization with digoxin 3'-hemisuccinate-BSA conjugate possessed high specificity for digoxin, exhibiting fairly low cross-reactions with dihydrodigoxin (2.1%), digoxigenin monodigitoxoside (0.9%), digoxigenin bisdigitoxoside (0.6%), and digoxigenin (0.1%).
Two highly sensitive and specific enzyme-linked immunosorbent assays (ELISAs) for the determination of 7-thyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin (irinotecan) and 7-ethyl-10-hydroxycamp-tothecin (SN-38), an active metabolite of irinotecan, were developed, which are capable of measuring as low as 16 and 160 pg of each drug/ml, respectively.Anti-irinotecan antibody was obtained by immunizing rabbits with irinotecan conjugated with mercapto-succinyl bovine serum albumin (MS.BSA) using N-(4-diazophenyl)maleimide (DPM) as a heterobifunctional coupling agent. An enzyme marker was similarly prepared by coupling irinotecan with horseradish peroxidase (HRP) via DPM. This ELISA for irinotecan was specific for irinotecan and showed almost no cross-reactivity with its active metabolite SN-38. Anti-SN-38 antibody was obtained by immunizing rabbits with SN-38 conjugated with BSA using the N-succinimidyl ester method. An enzyme marker was prepared by coupling SN-38 with HRP employing DPM. The ELISA for SN-38 was specific to SN-38 and showed a very slight cross-reactivity with irinotecan (0.08%). Using the 2 assays, we reconfirmed the rapid metabolite of irinotecan with rat serum. The 2 ELISAs may be a valuable tool for studies of the pharmacokinetics and pharmacodynamics of these drugs.
Carbonyl reductase activity for a novel hypnotic, N3-phenacyluridine, was mainly localized in the cytosol fraction of rabbit liver. The enzyme (N3-phenacyluridine reductase) which catalyzes the reduction of N3-phenacyluridine to N3-α-hydroxy-β-phenethyluridine was surified from the cytosolic fraction of rabbit liver by various chromatographic techniques (DEAE-Sephacel, Red Sepharose CL-6B and hydroxylapatite). N3-Phenacyluridine reductase had a minimum molecular weight of 39 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This enzyme required reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor and its optimal pH was 7.5. Flavonoids (quercetin and quercitrin) were potent inhibitors of the enzyme, but pyrazole or harbital had little effect. The apparent Km and Vmax values of the enzyme for the reduction of N3-phenacyluridine were 0.32 mM and 8.7 units/mg protein, respectively. A variety of carbonyl compounds, including N3-phenacyluridine, were effectively reduced by the enzyme. However, the enzyme purified from rabbit liver differs in several respects from known carbonyl reductases in rabbit liver.
An anti mouse sperm monoclonal antibody (A-1) inhibited sperm penetration into the egg zona pellucida and bound to an acrosomal area of sperm. In this study, we examined whether or not the antibody affects the sperm capacitation and the acrosome reaction. Sperm were incubated in modified Krebs-Ringer bicarbonate medium in the presence or absence of the antibody. The capacitation of sperm was assessed by chlortetracycline fluorescence pattern assay. The percentage of capacitated sperm did not increase in the presence of antibody, but increased time-dependently in its absence. The acrosome reaction of the capacitated sprem was induced by the addition of ionophore. The ionophore, however, failed to induce the reaction in the presence of the A-1 antibody. Next, the calcium influx into spermatocytes was examined. The capacitated sperm, preloaded with Fure-2, were treated with ionomycin in the presence or absence of the A-1 antibody. The influx of calcium ions into capacitated spermatozoa was also inhibited by the antibody. Thus a monoclonal antibody, A-1, inhibited the sperm capacitation, acrosome reaction and calcium influx into spermatocytes.
The effect of an aureolic acid, mithramycin (MTM) on multidrug resistance (MDR) was investigated. At a concentration of 0.02-0.1 mg/ml (about 20-90 μM), MTM repressed MDR1 gene transcription of SBC-3/ADM, a MDR-phenotype subline derived from human small cell lung tumor. Under the same conditions, another aureolic acid, chromomycin A3, showed potent cytotoxicity. FACS analysis revealed that 5 μM MTM depleted the P-glycoprotein (Pgp) and lowered the efflux activity of SBC-3/ADM cells. Furthermore, MTM sensitized the cells against adriamycin[○!R]. These results suggest that MTM would be a useful modulator of MDR induced by Pgp.
Apoptosis of NG108-15 neuroblastoma×glioma hydrid cells (NG108-15 cells) is induced by a morphine alkaloid derivative, buprenorphine hydrochloride (Bph). In a previous report, we used various apotosis inhibitors to identify the "death pathway", and found that caspase inhibitors Ac-YVAD-CHO (Ac-Yyr-Val-Ala-Asp-CHO) and Ac-DEVD-CHO (Ac-Asp-Glu-Val-Asp-CHO) did not inhibit this particular apoptosis. Here we tested ZAVD-FMK (Z-Val-Ala-Asp[OMe]-CH2F) and Z-DEVD-FMK (Z-Asp[OMe]-Glu-[OMe]Val-Asp[OMe]-CH2F) for their ability to inhibit Bph-induced NG108-15 apoptosis. These tri- or tetra-peptide caspase inhibitors have a fluoromethyl ketone in their C-terminus instead of an aldehyde, and thus are more permeable than Ac-YVAD-CHO and AC-DEVD-CHO. Our observations DNA ladder formatin, cell morphology changes, and caspase-3 activities all indicated that these cell membrane-permeable caspase inhibitors completely inhibited the apoptosis, providing strong evidince that this apoptosis occurs through the caspase cascade "death pathway." Our previous report also showed that pretreatment of NG108-15 cells with TPCK (N-tosyl-L-phenylalanyl chloromethyl ketone) prevented DNA fragmentation and decreased the cell viability in Bph-induced apoptosis. The comparsion of caspase-3 activities in Bph-induced samples with or without TPCK pretreatment revealed that caspase-3 was activated in both samples. Taken together, these results indicate that the Bph-induced apoptosis of NG-108-15 cells occurs via the conventional caspase-dependent death pathway and that TPCK pretreatment results in a DNA ladder-deficient apoptosis.
The effect of sialic acid (N-acetyl neuraminic acid), sialic acid dimer, sialic acid polymers (colominic acid) and sulfated colominic acid on the activity of hyaluronidase, on the dispersion of cumulus cells by mouse sperm and on in vitro mouse fertilization (sperm penetration of zona pelllucida) were evaluated. Bovine testicular hyaluronidase activity was significantly inhibited by colominic acid and sulfated colominic acid, but not by sialic acid and its dimer. The dispersion of cumulus cells from eggs by mouse sperm was also inhibited by colominic acid and sulfated colominic acid. In vitro fertilization of mouse gametes was inhibited by sulfated colominic acid. The IC50 value of sulfated colominic acid-induced inhibition of fertilization was 0.3 mg/ml (ca. 0.9 mM). The value changed from 0.9 mM for cumulus-surronded egg to 1.5 mM for cumulus free-egg. On the other hand, colominic acid showed little or no inhibitory effect on mouse in vitro fertilization at 0.5 mg/ml (ca. 1.6 mM). This antifertility activity by sulfated colominic acid did not appear to be due to an effect on sperm motility or on the oocytes. These results suggest that (1) the cumulus cells surrounding the eggs were dispersed by sperm hyaluronidase, (2) hyaluronidase was inhibited by colominic acid and by sulfated colominic acid, (3) sulfated colominic acid inhibits sperm penetration of zona pellucida by the inhibition of hyaluronidase and/or some enzymes required for mouse gametes fertilization.
The variable region of heavy chain (VH) of human rheumatoid factor (hRF) IgH was connected with the variable region of light chain (VL) with the peptide-linker (GGGSGGGSGGGS) by genetic engineering method and the single-chain FV (scFv) was expressed in E. coli. On design, scFv and scFv (tag) were planned; the latter had a detection marker at the carboxyl-terminal. These scFvs were expressed as inclusion bodies in E. coli, purified in the presence of 8 M urea by gel filtration and renatured to the active form in vitro. As a control, the Fv, non-covalently associated VH and VL fragements, was also constructed. The 3 derivatives showed almost the same binding activity to rabbit-IgG to which hRF is cross-reactive. ScFv (tag) was the most stable against urea among the 3 derivatives.
The anabolic effect of salmon calcitonin (SCT) on skeletal tissue was examined on glucocorticoid-induced osteopenia in female rats (12 weeks old). By the administration of methylprednisolone acetate (MPA : 0.1 mg/kg, s.c., 3 times/week) for 8 weeks, histomorphometrically detectable osteopenia with the characteristics of decreased bone formation and increased bone resorption developed in proximal tibia metaphysis. Serum osteocalcin level was also decreased by MPA treatment. Subsequent treatment with SCT (10 U/kg, s.c., 5 times/week) was found to reverse once developed osteopenia with the return of the osteocalcin level, though rats were still on MPA. Histomorphometry revealed that SCT decelerated bone resorption but augmented bone formation in this osteopenic model. Affer a single injection of SCT (2.5 U/kg-40 U/ke, s.c.), the serum level of parathyroid hormone (PTH) which had a potent anabolic on bone formation increased in a dose-dependent manner. These results indicate that SCT has a stimulating effect on osteoblastic bone formation and this anabolic effect may at least in part be due to its indirect effect to increase endogenous PTH secretion.
Wood creosote, a mixture of guaiacol, creosol and related compounds, has long been used as an antidiarrheal agent. The goal of our study was to investigate the antisecretory effect of wood creosote and to compare it to the effect of loperamide, a synthetic opioid widely used in the treatment of diarrhea. Experiments were performed in rat jejunal and colonic mucosal sheets, mounted in modified Ussing chambers. Active electrogenic transport was monitored electrically as short circuit current (Isc) and hypersecretory responses were induced by acetylcholine (ACh). Neither loperamide nor wood creosote had any significant effect on basal Isc, when added to the serosal bathing solutoin at concentrations of 0.1-50 μg/ml. In contrast, under hypersecretory conditions, both agents showed concentration-dependent (0.1-100 μg/ml) antisecretory effects inhibiting ACh-induced responses in the jejunum and colon. However, the effects suggest regional differences, with loperamide being most potent in the jejunum, while wood creosote showed equal potency in both jejunum and colon. Based upon these in vitro findings, we conclude that like loperamide, the antidiarrheal action of wood creosote is due, at least in part, to its antisecretory activity.
Since amphiphilic drugs are known to interact with biomembranes, we investigated local vessel damage and thrombosis which might be brought about by intravenous dosing using chlorpromazine (CPZ) as a representative compound. CPZ-induced hemolysis was suppressed by an increase in sucrose concentration in the medium, characterizing this hemolysis to be colloid-osmotic lysis, which includes the enhancement of membrane phospholipid fluidity and consequent small pore formation in the membranes. This was supported by the observation that hemolysis by filipin, not featuring the stage of small pore formation, was not affected by sucrose. [14C]Glucose-entrapping liposomes were degraded by CPZ, and this degradation was enhanced by an increase in the intravesicle glucose concentration. These results indicated that the compound could induce colloid-osmotic lysis in erythrocytes and artifical membrane vesicles. CPZ also injured cultured porcine aortic endothelial cells (PAEC), as evidenced by lactate dehydrogenase (LDH) leakge. This injury was also suppressed by increase in sucrose concentration in the medium, suggesting that colloid-osmotic lysis again occurred. When rats were intravenously injected with CPZ, local endothelial cell (EC) injury and associated thrombus formation were observed, suggesting that CPZ's action was also evident in vivo. To our knowledge, this is the first finding which suggests that an intravenoulsy dosed amphiphilic drug can injure local ECs based on a colloid-osmotic lysis mechanism leading to thrombosis.
We examined the differentiation-inducing effects of extracts of 49 wild plants, 25 types of seaweed and 26 mushrooms in Akita on the human leukemia cell line HL60 and a B16 mouse melanoma-derived sub-clone with high differentation capability (B16 2F2). Differentiation inducers of HL60 cells such as rethinoic acid, showed no effects on the differentiation of B16 2F2 cells. Furthermore, chemical compounds known to be inducers of B16 cells, did not induce differentiation of HL60 cells. Screening tests showed that the differentiation of HL60 cells was induced by extracts of 28 wild plants, 10 types of seaweed and 2 mushrooms, and melanogenesis of B16 2F2 cells was increased by extracts of 21 wild plants, 8 types of seaweed and 7 mushrooms. All of the alcoholic extracts of plants belonging to the subfamily Cichorioideae of the family Compositae caused cell differentiation of the melanoma cell line. The extracts of Chinese dandelion root, also inhibited cell growth and induced melanogenesis of B16 2F2 cells. We isolated the active compound from ethanol extracts of the crude drug. Chemical and physical data for the active compound were identical with those for lupeol, a lupane-type triterpene.
The absolute bioavailability (BA) of ciprofloxacin and fleroxacin were evaluated in 19 adult Nigerin male volunteers. Subjects meeting the selection criteria were randomized to recetive treatment either with fleroxacin (200 mg-i.v. and 200 mg oral dose) or ciprofloxacin (200 mg-i.v. and 250 mg oral dose). The i.v./oral or oral/i.v. switch was made after a one week washout period. Blood and nrine samples were collected at pre-determined time intervals over a 72 h period for analysis of drug levels.Following intravenous administration the maximum serum concentration (Cmax) was 2.7±1.06 mg/l for ciprofloxacin and 0.99±0.41 mg/l for fleroxacin; the area under the blood level curve (AUC) was 8.82±3.19 mg·h/l with ciprofloxacin and 8.52±3.83 mg·h/l with fleroxacin. Follwing oral administration the Cmax was 1.52±0.94 mg/l with ciprofloxacin and 0.57±0.08 mg/l with fleroxacin; the AUC was 9.87±4.10 and 7.55±1.42 mg·h/l, respectively. The absolute BA following oral administration was found to be 0.79±0.47 for ciprofloxacin and 1.01±0.78 for fleroxacin. When these BA results were corrected for renal clearance (Clr) and eliminiation half-life (t1/2) the values were reduced to 0.37±0.17 and 0.31±0.18, respectively, for ciprofloxacin and 0.53±0.23 and 0.99±0.38, respectively, for fleroxacin. Only 38% with ciprofloxacin and 59% with fleroxacin, of the administered dose, was excreted unchanged following oral administration.More work, however, needs to be done on ciprofloxacin to support and/or confirm the above findings. Fleroxacin, on the hand, exhibited a different trend from that observed in the literature with respect to Cmax and AUC where the values observed in this study were 3-4 fold lower than expected following identical doses, whilst on the other hand the observed BA profile in this study was consistent with literature trends.
This paper studied the effect of intranasal inoculation of Lactobacillus fermentum, a microorganism belonging to the normal flora of the mouse pharynx, on the respiratory tract of mice. Optimal temporary colonization in different areas of the tract was obtained through administration of 4 times a dose of 5×107 CFU. L. fermentum remained in the trachea and bronchia up to the 7th day after inoculation. Re-inoculation of lactobacilli on the 10th day produced a transient colonization of the respiratory tract. Histologial modifications produced in the trachea were mainly observed as an increased lymphocyte population at sub-mucosa level on the 4th day after inoculation. There was an increased number of activated macrophages in cytological sildes of lung tissues on days 2 and 4. Re-inoculation also produced stimulation of the G2 macrophages on days 12, 14 and 17. From a histological point of view there were no other important changes in the organs studies. These suggest stimulation of the immune system, especially that of the mucosal surfaces, after intranasal administration of L. fermentum in the experimental model employed. Stimulation was reflected in tracheal lymphocyte proliferation and increased lung macrophage population which have to be further studied in more detail.
Ten nucleoside analogues with anti-herpes or anti-HIV activity were investigated for their transport into the cerebrospinal fluid (CSF) following intravenous administration in rats. The novel anti-herpes agent 1-β-D-arabinofuranosyl-2-thio-5-fluorocytosine (5F-araSC) showed the highest CSF/plasma concentration ratio (>20%), while that of acyclovir (ACV) was very low (<5%). A linear relationship was observed between the partition coefficient (chloroform/water) and CSF/unbound plasma concentration in 6 of 9 agents. The exceptions were DDI, AZT and ACV, which showed much lower concentrations in the CSF than expected from their hydrophobicity and protein binding activities. The effects of probenecid treatment on the CSF and plasma concentrations were measured with continuous intravenous administration of ACV, AZT, araC and 5F-araSC. Probenecid markedly increased the CSF concentrations of ACV and AZT, although the effect was minimal in araC and 5F-araSC. These results may provide useful information for molecular design of nucleoside analogues with better transport to the brain.
Octanoate is taken up by the brain and converted in astrocytes to glutamine through the TCA cycle after β-oxidation. Consequently, [111C]octanoate might serve as a useful positron emission tomography (PET) probe for studying cerebral oxidative metabolism and/or astroglial functions. The present study attempted to evaluate the utility of using [111C]octanoate as a PET tracer for imaging and evaluating the pathopysiology of ischemic stroke. We used a canine model of thromboembolic stroke. Five male beagle dogs were implanted with an indwelling catheter in the left internal carotid artery. A single autologous blood clot was injected into the left internal carotid artery through the catheter. The brain distribution of [111C]octanoate and cerebral blood flow (CBF) were determined 24 h after insult using a high resolution PET scanner. Post mortem brain regions unstained with 2, 3, 5-triphenyltetrazolium chloride (TTC) were defined as infarcts. In the region of an infarct, accumulation of [111C]octanoate decreased concurrently with CBF reduction. In contrast, normal accumulation of [111C]octanoate was observed in ischemic but vital regions, suggesting that an increased accumulation of [111C]octanoate relative to CBF takes place in these regions. In conclusion, [111C]octanoate accumulated in ischemic but vital retions, indicating that [111C]octanoate is potentially useful PET tracer for imaging and evaluating the pathophysiology of ischemic stroke.
Falconesones A and B are new type of yellow compound extracted from ascomycetous fungi, Emericella falconensis. Falconensone A p-bromophenylhydrazone and falconensone A dioxime are derivatives of falconensone A which have also been synthesized recently. The structural similarity of the falconensones to α-tocopherol (vitamin E) led us to investigate whether falconensones exhibit antioxidant activity. These studies found that falconensone A p-bromophenylhydrazone and falconensone A dioxime scavenge α, α-diphenyl-β-picrylhydrazyl (DPPH) radical in a 1 : 1 ratio in contrast to vitamin E, where a 1 : 2 ratio relative to DPPH radicals was observed. In addition, linoleic acid peroxidation initiated by hydroxyl radicals was diminished by falconensone A p-bromophenylhydrazone to a greater extent than by vitamin E, and lipid peroxidation in rat liver microsome was reduced byfalconensone A dioxime and falconensone A p-bromophenylhydrazone. In contrast, falconensone A and falconensone B, the 4'-nor-methyl derivative of falconensone A, showed much lower activity or were inactive in scavenging radicals. These results suggest that falconensone A p-bromophenylhydrazone and falconensone A dioxime, may be useful new antioxidant agents, wherein the bromophenyl and hydroxy residues of falconensone A may be important for antioxidant activity. Based on these results, derivatives of falconensone A appear to be effective antioxidants that may have clinical utility for diseases treated with vitamin E.
Hinokitiol (β-thujaplicin), β-dolabrin and γ-thujaplicin isolated from Thujopsis dolabrata SIEB, et ZUCC. var hondai MAKINO showed antifungal activities against all of the wood-rotting fungi examined. The antifungal activity of three compounds on Daedalea dickinsii IFO-4979 was especially strong, their mininum inhibitory concentration (MIC) values being 0.2 μg/ml. Their antifungal activities on D. dickinsii IFO-4979 were as high as that of amphotericin B used as a positive control. Three compounds had strong insecticidal activities on Tyrophagus putrescentiae [50%-lethal concentration (LC50 : g/m2) 0.25 in hinokitiol, 0.02 in β-dolabrin and γ-thujaplicin]. Their insecticidal activities were higher than that of N, N-diethyl-m-toluamide (DEET, LC50 : 1.46 g/m2) used as a positive control. Three compopunds also showed strong insecticidal activity on Coptotermes formosanus [LC50 (g/m2) 0.07 in hinokitiol, 0.05 in β-dolabrin and γ-thujaplicin], although their insecticidal activities were much lower than that of commerical chloropyrifos (LC50 : 0.00016 g/m2).
Each of the four arginine residues in the HM-1 killer toxin was replaced by alanine using site-directed mutagenesis. The polymerase chain reaction (PCR)-constructed mutant gene was successfully expressed in HM-1 toxin resistant Saccharomyces cereviasiae. Among four HM-1 toxin analogues, R82A HM-1 toxin and R86A HM-1 toxin lost killer activity, while R61A HM-1 toxin and R85A HM-1 toxin retained activity. These results strongly indicate the importance of the arginine residues at positions 82 and 86 which are located in the C-terminal region of the HM-1 toxin for the action of killer activity.
The time-course pattern of the frequency of micronucleated hepatocytes in vivo after partial hepatectomy (PH) was studied in mice using N-nitrosodimethylamine (DMN), N-nitrosodiethylamine (DEN), and 1, 2-dimethylhydrazine (DMH), which are rodent liver carcinogens with potent clastogenic activity in the liver. With all chemicals, production of micronucleated hepatocytes was not clearly observed at 3 d after PH, but was clear 4 or 5 d after PH. We propose that it is preferable to perform a preliminary assay prior to the main assay when estimating the clastogenic potential of certain chemicals towards hepatocytes in vivo.
Cichorium intypbus contains two 1β-hydroxyeudesmanolides, magnolialide and artesin, together with several constituents. Magnolialide inhibits the growth of several tumor cell lines and appears to induce differentiation of human leukemia HL-60 and U-937 cells to monocyte/macrophage-like cells. Another 1β-hydroxyeudesmanolide, artesin, and other consitutents were inactive. The content of magnolialide was shown to be highest in the leaves of Inje cultivar among the cultivars investigated in this study.
The neurotoxicity associated with tacrolimus is one of the major limitations for its administration after organ transplantation. This study investigated the correlation between neurotoxicity and the intracerebral concentration of tacroliums. Rats were given one of three doses of tacrolimus (5, 10, and 20 mg/kg/d) orally twice a day for 2 weeks and neurotoxic events were observed. The rats were sacrificed on either day 7 or 14. The trough values of the whole blood and the corresponding intracerebral concentrations were than measured. None of the rats receiving dosage of 5 mg/kg/d showed any neurotoxic symptoms throughout the two-week test period. In rats receiving a dosage of 10 mg/kg/d, however, all seven surviving rats presented tremors or seizures during the second week. In rats receiving a dosage of 20 mg/kg/d, 40% of the rats presented tremors or seizures during the first week. The threshold concentration of tacrolimus in the brain resulting in neurotoxic events was therefore estimated as approximately 700 ng/g. At concentrations over this threshold value, the intensity of the neurological event increases with the concentrations of tacrolimus in the brain. Using a linear correlation between the whole blood and intracerebral concentrations (r=0.967) or tacroliums the pharmacological threshold for the whole blood trough level was estimated as approximately 20 ng/ml, which falls into the same value reported for the incidental threshold of neurotoxicity in renal transplant recipients [Bottiger et al., Br. J. Clin. Pharmacol., 48, 445-448 (1999)]. Therefore, it is suggested that the rat is a good animal model to quantitatively evaluate the risk of neurotoxicity associated with tacrolimus in human, and that frequent measurement of whole blood tacrolimus concentrations is important for predicting and preventing neurotoxic events.
We report here the antisense effect of phosphodiester oligdoeoxynucleotide (D-ODN) using fusogenic liposomes (FL) as its carrier. FL has envelope proteins of the Sendai virus within its membrane and introduces its contents directly and efficiently into cytosol by means of the virus-cell fusion mechanism. Using antisense (AS) D-ODN 15-mer complementary to the c-myc proto-oncogene mRNA, including the translation initiation codon site, we analyzed the growth of HL-60 cells by [3H]-thymidine uptake. AS-ODNs encapsulated in FL inhibited the growth by about 70% that of the control HL-60 cells at 2.48 μM. It contrast, sense and scramble D-ODNs encapsulated in FL showed no effect of the growth of HL-60 cells at the same concentration. Even at 50 μM, free form D-ODNs did not show any effect. These results suggest that FL is potentially a useful delivery vehicle for oligonucleotide-based therapeutics, and that D-ODN may be a likely candidate for oligodeoxynucleotides when an efficient delivery system is used.