We investigated the specificity of obtained antisera to β-methyldigoxin by the enzyme immunoassay. Three types of hapten-bovine serum albumin (BSA) conjugates were synthesized to obtain high specific antisera to β-methyldigoxin. The haptens were linked to the carrier protein through hemisuccinate at C-3′ and C-3″ positions in the digitoxose chain and at C-12 position in the aglycone. Anti-β-methyldigoxin 3′-hemisuccinate–BSA antiserum showed a low detection limit (0.2 ng/ml) and possessed high specificity for β-methyldigoxin, exhibiting low cross-reactions with digoxigenin bisdigitoxoside (8.3%), dihydrodigoxin (4.8%), digitoxin (1.5%), and digoxigenin monodigitoxoside (0.95%), except for cross-reaction with digoxin (43%). Compared with commercial anti-digoxin antiserum, clinically used to monitor β-methyldigoxin concentration in human serum, cross-reaction data of anti-β-methyldigoxin 3′-hemisuccinate–BSA antiserum showed higher specificity for β-methyldigoxin. The intra-assay and inter-assay variations using this antiserum were less than 6.9% and 8.1%, respectively. The recovery tests were good, within the range of 96.2—104.3%. Phenyl boric acid (PBA) column treatment was effective to rapidly and selectively separate β-methyldigoxin from the mixture of β-methyldigoxin and its metabolites in human serum. The recovery tests of β-methyldigoxin with PBA column in human serum were about 110% and interference of metabolites of β-methyldigoxin was negligible. These results suggest that anti-β-methyldigoxin 3′-hemisuccinate–BSA antiserum and PBA column treatment are useful to more precisely monitor the unchanged type of β-methyldigoxin concentration in human serum.
A one-step fluorescence polarization immunoassay (FPIA) was developed to measure progesterone level using an immunocomplex single reagent (SR), a preequilibrated mixture of antibody and tracer. Several fluorescence-labeled progesterone tracers were synthesized using the combination of two progesterone derivatives, 11α-hemisuccinyloxyprogesterone (P-11HS) and progesterone-3-(O-carboxymethyl)oxime (P-3CMO), and three fluorescence labels, fluoresceinamine isomer I and II, and ethylenediamine fluoresceinthiocarbamyl (EDF). Antiserum was prepared using a progesterone-bovine serum albumin (BSA) imunogen. The influence of the tracer label was significantly different in titer and sensitivity for antibody binding. The best pair of antibody and progesterone tracer was selected for the antigen-antibody reaction. They were the antibody produced from P-11HS-BSA immunogen and P-11HS-EDF tracer. One-step FPIA is a speedy, homogeneous type of immunoassay which needs neither incubation nor separation of free and bound analyte to measure fluorescence polarization. The detection limit of progesterone by SR-FPIA is approximately 2.7 ng/ml with 50 μl samples. The performance characteristics are acceptable for standard curve reproducibility (coefficient of variation (CV): 0.6—6.4%), precision (CV: 3—13%), and mean dilution recovery (95—102%). The total assay time for 10 samples is about 7 min. This immunocomplex SR has proven to be stable compared with the respective solutions of antibody and tracer.
The effects of culture medium and serum components on immunoglobulin (Ig) production by mouse splenocytes were examined. In this study, we showed that culture medium containing charcoal–dextran-treated fetal bovine serum (CDFBS) supported Ig production more efficiently than culture medium containing FBS. In addition, RPMI 1640 medium supported Ig production more efficiently than Dulbecco modified Eagle medium. In addition, an increase in medium IgA production was observed with the increase in both CDFBS and FBS concentrations, whereas an increase in IgM level was observed in the presence of 5% and 10% FBS and 10% CDFBS. Dose-dependent effects of estrogenic compounds on Ig production were also examined. We found that 17β-estradiol induced increases in IgM and IgE levels, but diethylstilbestrol enhanced only the IgE level. On the other hand, tamoxifen and bisphenol A enhanced medium IgM level at physiological concentrations. Among flavonoids, daidzein enhanced IgM and IgE levels at concentrations above 10 μM, and genistein induced a decrease in IgM level and an increase in IgE level at concentrations above 10 μM. In addition, quercetin and luteolin enhanced medium IgE level at all concentrations tested, whereas IgA, IgG, and IgM levels were not affected. These results suggest that environmental estrogens affect Ig production of mouse Splenocytes in a complex and class-specific manner.
A sensitive HPLC method for simultaneous determination of endogenous levels of all-trans- (ATRA), 13-cis- (13cRA), and 9-cis-retinoic acids (9cRA) was applied to serum samples from healthy volunteers and type II diabetes mellitus patients. Levels of 9cRA (around 0.2 ng/ml in both groups) were below the limit of quantification. The concentrations of ATRA and 13cRA were reliably quantified, and the within-day and between-days variances indicated that they were well maintained with little variation. Concentrations of serum ATRA and 13cRA of diabetic patients (ATRA: 1.76±0.54 ng/ml; 13cRA: 1.77±0.39 ng/ml, n=13) were rather lower than those of healthy subjects (ATRA: 2.08±0.53 ng/ml; 13cRA: 2.05±0.26 ng/ml, n=18), but the differences were not significant, except for the sum of ATRA and 13cRA (p=0.033). Interestingly, the serum levels of retinoic acids in diabetic patients correlated positively with the hemoglobin A1c (HbA1c) values (ATRA: r=0.57, p<0.05; 13cRA: r=0.62, p<0.05). The results warrant further studies on the possible involvement of uncontrolled serum retinoic acids levels in the pathogenesis and/or treatment of diabetes mellitus.
We constructed an assay system of a luciferase reporter with p16/INK4a gene transcriptional regulatory domain to identify p16-inducing substances, and found toyocamycin to induce gene expression from the screening of culture fluids of Streptomyces. Toyocamycin is a nucleoside analog, and it increased the p16 mRNA level in human normal fibroblasts or synovial cells as assessed by Northern blot hybridization or real time RT-PCR. It also induced cellular senescence in normal human fibroblasts. The transcriptional regulatory regions of human p16 gene that were responsible for the induction were analyzed using deletion mutants of the transcriptional regulatory region of p16 linked to the luciferase gene. The DNA fragment −111 to +1 bp from the cap site was sufficient to drive toyocamycin-activated transcription of p16/luciferase reporter. Nucleotide sequences within this domain contained the Sp1- and Ets-binding sequences. Mutations were introduced into these sequences, and the Sp1 sequence was found to be critical for the induction, and this notion was confirmed from gel-mobility shift assay.
Apoptotic cells are effectively ingested and removed by phagocytes. This process is dependent on specific recognition by phagocytes of ligands expressed exclusively on apoptotic cells. These ligands, cell surface molecules such as phosphatidylserine (PS), altered sugar chains, and the thrombospondin-binding domain, are expressed following the induction of apoptosis. However, they are not expressed simultaneously, and each factor seems to have a respective role in different phases of apoptosis. In this paper, we classified the apoptotic process into three phases (initial phase, metaphase, and later phase) according to cell viability, the expression of PS, and the change of sugar chains, and studied the role of individual molecules in the phagocytosis of apoptotic cells in each phase. We found that in the initial phase, characterized by an increase in PS expression but no decrease in cell viability, and metaphase, characterized by a decrease in cell viability and a change of cell surface sugar chains, PS, galactose, and the vitronectin receptor play important roles. In the later phase, when each factor is respectively constant, PS and galactose play important roles in phagocytosis, but the vitronectin receptor does not. We suggest that cell surface molecules respectively fulfill their role in the apoptotic process.
β-D-Glucuronyltransferase, which transfers D-glucuronic acid (GlcA) from UDP-GlcA to N-acetyl-D-galactosamine (GalNAc) at the nonreducing end of chondro-pentasaccharide-PA (pyridylamino-), GalNAcβ1-(4GlcAβ1-3GalNAcβ1)2-PA, was purified 339-fold with an 11.0% yield from 2-d-old chick corneas by chromatography on DEAE-Sepharose, WGA-agarose, heparin-Sepharose, and 1st and 2nd UDP-GlcA-agarose (in the presence of Gal) columns. The activity was detected by fluorescence of PA residues of the product. The purified enzyme has an optimum pH of 7.0 (Mes buffer), and much higher activity toward chondro-heptasaccharide-PA than toward the chondro-pentasaccharide-PA, but no activity toward p-nitrophenyl-β-GalNAc. The enzyme activity was almost completely inhibited by GalNAc (20 mM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme fraction showed one band of 38 kDa with many other bands. The amino acid sequence was determined for the tryptic digests of the 38 kDa band protein. The sequences determined showed no homology to those of several β-glucuronyltransferases reported previously. It seems that the enzyme is involved in the elongation of chondroitin sulfate chains in vivo.
The aim of the present study is to investigate the effects of long-term Hypericum perforatum treatment on spatial learning and memory in rats. Hypericum preparation (HP) standardized to 0.3% hypericin content was administered orally for 9 weeks in doses of 4.3 and 13 μg/kg corresponding to therapeutic dosages in humans of 0.3 and 0.9 mg of total hypericins daily. A Morris water maze paradigm was used. The mean escape latency over 4 d for the Control group (21.9 s) and HP 4.3 group (21.7 s) was significantly greater than the latency of the HP 13 group (15.8 s). In the probe trial on day 5, the HP 13 group crossed the correct annulus in the SE quadrant more often (4.5) than the other groups: Con (2.4) and HP 4.3 (3.1). After completion of the behavioral experiment, the regional brain concentrations of monoamines and metabolites were estimated in selected brain regions, i.e. prefrontal cortex, hippocampus and hypothalamus. Analysis of variance (ANOVA) demonstrated significant differences in the content of monoamines and metabolites between the treatment groups compared to the Control. The increased 5-hydroxytryptamine (5-HT) levels in the prefrontal cortex correlated positively with the retention of spatial memory. These findings show that the long-term administration of Hypericum perforatum can improve learning and spatial memory with significant changes in the content of monoamines in several brain regions.
The effects of sodium nitroprusside (SNP) in rat cerebellar granular cell culture were investigated in the present study. All doses of the SNP (10, 25, 50, 100, 250, 500 μM) were able to induce cell death compared with control values (p<0.001 for all groups tested). Interestingly enough, a nonlinear dose–response curve was obtained for SNP-induced neurotoxicity. We also investigated the possible neuroprotective effects of nimodipine and dantrolene, alone or in combination. Both drugs failed to prevent neuronal cell death at the doses tested, either alone or in combination. Despite the fact that the most effective dose was a dantrolene concentration of 10 μM with SNP 500 μM and a concentration of 1 μM with SNP 50 μM, the differences were insignificant statistically. According to our results, SNP-induced cerebellar toxicity appears to be an independent reaction from L-type or endoplasmic reticulum calcium currents.
We previously reported that the retinoic acid (RA) insensitivity of RARβ induction is a general feature of human colon cancer cells (Biochem. Pharmacol., 59: 485–496, 2000). In the present investigation, we analyzed potential transcriptional defects associated with the expression of the RARβ gene in colon cancer cells. Transfection of reporter constructs containing the RARβ gene promoter as well as truncated fragments of the promoter showed a significant induction of reporter activity by RA treatment in RA-sensitive HCT-15 cells, but not in RA-resistant DLD-1 cells. The results suggest that the transcriptional defect of RARβ expression may not be due to the presence of a specific cis-element in the RARβ promoter. Next we examined whether coactivators and corepressors of nuclear receptors were involved in the RA sensitivity of colon cancer cells. Transfection of coactivators such as CREB binding protein (CBP) and p300 up-regulated the RA-responsive element present in the RARβ promoter (βRARE) in DLD-1 cells up to the level in HCT-15, while coexpression of the nuclear receptor corepressor (NCoR) suppressed the βRARE activity in HCT-15 cells. The expression level of CBP protein was consistently higher in HCT-15, while that of NCoR and Sin3A was higher in DLD-1 cells. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) increased both basal and RA-induced βRARE activity in DLD-1, indicating that histone deacetylase is involved in the regulation of RARβ gene expression. Taken together, our results show that differential function of coactivators and corepressors may determine the level of RARβ induction that may mediate retinoid action in colon cancer cells.
We previously reported that a human analogue of pulmonary surfactant protein-C (SP-C), SP-CL16(6—28), with 23 residues was the most active analogue in a reconstituted lipid mixture and had the shortest chain among the poly-leucine-analogues examined. There has been little research on the chemical components of synthetic lung surfactants (SLSs). In the present study, we attempted to compare SLS with various phospholipids in surface activity. That is, SP-CL16(6—28) plus various phosphatidylglycerols (PG) were tested for surface activity in a Langmuir–Wilhelmy surface balance (WSB) apparatus and pulsating bubble surfactmeter (PBS). Further, SLSs were examined for biological properties using an animal model of surfactant deficiency, infant respiratory distress syndrome (IRDS), in vivo. Palmitoyl-oleoyl-phosphatidylglycerol (POPG)-SLS exhibited minimum and maximum surface tensions of 1.7 mN/m and 28.6 mN/m in WSB and 8.5 mN/m and 36.2 mN/m in PBS, respectively. Moreover, in the IRDS model, POPG-SLS remarkably improved the lung volume (LV) of a premature lagomorph fetus at LV30 cmH2O and LV5 cmH2O. That is, a significant improvement equal to the LV of a full-term fetus was observed. The level of LV exhibited respiratory improvement equivalent to surfactant-TA. SLS seemed comparable in surface activity with Surfacten® (Surfactant-TA), a modified surfactant preparation which has been used for the treatment of RDS.
The development of resistance to current antifungal therapeutics drives the search for effective new agents. The fact that some acetophenone-derived Mannich bases had shown antifungal activities in our previous studies led us to design and synthesize acetophenone-derived bis Mannich bases, B1—B5, bis(β-aroylethyl)methylamine hydrochlorides, to evaluate their antifungal activity. These bis Mannich bases were then converted to the corresponding piperidinols, C1—C5, which are structural isomers of bis derivatives, 3-aroyl-4-aryl-1-methyl-4-piperidinol hydrochlorides, to see alterations in biological activity. A stability study of B1 and C1 was also carried out to estimate whether they alkylate the thiols. All compounds studied have shown antifungal activity, especially against dermatophytes (Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, and Microsporum canis), in the concentration range studied (2—128 μg/ml). The activity was especially apparent against T. tonsurans. All compounds had at least equal antifungal activity compared with the reference compound amphotericin-B against T. tonsurans. Bis Mannich bases were generally found to be more potent compounds than their structural isomer piperidinols. The results of our stability studies suggest that thiol alkylation may contribute to the antifungal activity of the Mannich bases synthesized. Even though all compounds showed antifungal activity against dermatophytes, bis Mannich bases B1, B2, B4, and B5 appear to have potential for developing novel antifungal agents against dermatophytes.
Plasma lipid levels and lipoprotein lipase (LPL) are known to follow circadian rhythms in rats. However, very little information is available on the variations in respiratory quotient (RQ) during the 24-h period in rats. The aims of this study were to provide an overall view of the effects of circadian rhythm on RQ and to determine the relationship of LPL and RQ with metabolic parameters in these animals. Male rats were fed ad libitum and were kept under a 12 : 12-h light–dark cycle. Rats were killed every 2 h over a 24-h period for measurement of metabolic parameters and tissue LPL activity. The RQ was measured every 4 h over the same 24-h period. The gastric contents increased during the dark phase and decreased during the light phase. For the metabolic parameters, circadian rhythms were detected for plasma glucose, triglycerides, high-density lipoprotein cholesterol and non esterified free fatty acids, but not for plasma total cholesterol or phospholipids. The RQ and adipose tissue LPL activity increased during the dark phase, while skeletal muscle LPL activity decreased during this phase. The RQ was inversely correlated with skeletal muscle LPL activity (r=−0.880) and positively correlated with adipose tissue LPL activity (r=0.937). These results appear to show that rats tend toward consumption of fat by accelerating fat oxidation, resulting in suppression of fat accumulation in the light phase, while tending toward fat accumulation by the suppression of fat oxidation in the dark phase.
Effects of peripheral administration of 5-HT (5-hydroxytryptamine, serotonin) on hyperphagia induced by 2-deoxy-D-glucose(2-DG) were studied in rats. It was found that 5-HT i.p. reduced 2-DG-elicited feeding in rats dose-dependently. The 5-HT-induced hypophagia was antagonized by the 5-HT2A receptor antagonist, ketanserin. It is known that 2-DG induces glucoprivation, resulting in hyperphagia and hyperglycemia. However, 5-HT did not affect hyperglycemia induced by 2-DG. These results suggest that peripheral injection of 5-HT reduces 2-DG-induced hyperphagia mediated by the peripheral 5-HT2A receptor and that its effects are not due to enhancement of hyperglycemia.
Previously, we reported that (±)-IA but not DES produces O2− spontaneously in PBS. We are interested in the possibility that these compounds might produce active oxygen species under mild cell culture conditions. On incubation of RAW 264.7 cells with (±)-IA, the signal of 5,5′-dimethyl-1-pyrroline-N-oxide (DMPO)-OH adducts increased but no more than the additive effect. However, stimulation of RAW cells with LPS and INF-γ enhanced the formation of DMPO-OH adducts slightly more than the additive effect, especially when the concentration of (±)-IA increased. In the case of DES, the spectra of DMPO-OH adducts did not increase concentration-dependently in the absence of RAW 264.7 cells, however in their presence, they increased concentration-dependently, especially when these cells were stimulated with LPS and IFN-γ. The results were interpreted to mean that DES would have a higher oxidation potential than (±)-IA, not be oxidized to semiquinoes spontaneously, and therefore not produce DMPO-OH adducts in the absence of RAW cells. In their presence, DES might be easily oxidized to semiquinones by the reaction with O2− produced from RAW 264.7 cells.
Bromate, an inorganic oxyhalide disinfection by-product, is known to cause kidney damage, haemolysis and methaemoglobinemia. In potassium bromate (KBrO3)-treated mice (1.2 mmol/kg), elevation of methaemoglobin (MetHb) concentration in blood was observed simultaneously with an elevation of the NO concentration and attenuation of glutathione peroxidase (GPx) activity. Renal oxidative stress and kidney damage were also confirmed in the KBrO3-treated mice. A pre-administered GPx-mimic ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) dose-dependently diminished the KBrO3-induced changes in MetHb concentration and GPx activity. Renal oxidative stress and kidney damage caused by the KBrO3 administration were also dose-dependently suppressed by ebselen. On the other hand, ebselen did not suppress the KBrO3-induced elevation of the NO concentration. KBrO3-induced methaemoglobinemia, renal oxidative stress and kidney damage, consequently, seemed to result from the attenuation of GPx activity. Besides, the enhancement of NO production was not likely to be a result but a cause for the KBrO3-induced attenuation of GPx activity. In in vitro experiments, oxidation of human oxy-haemoglobin (HbO2) to MetHb was observed in a reaction mixture containing HbO2 and an NO donor, NOC-7 (1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene) or SIN-1 (3-(4-morpholinyl)sydnonimine), and this oxidation was inhibited by the NO scavenger carboxy-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide). However, no MetHb formation was observed in a reaction mixture containing HbO2 and KBrO3. These results suggest that KBrO3-induced methaemoglobinemia results from the reduction of GPx activity in blood by the KBrO3-induced increases in superoxide, NO and ONOO−.
Hypericum androsaemum is a medicinal plant species containing many polyphenolic compounds, namely flavonoids and phenolic acids. Since polyphenolic compounds have high antioxidant potential, the ability of H. androsaemum infusion to act as a scavenger of reactive oxygen species (superoxide radical, hydroxyl radical and hypochlorous acid) was investigated. Superoxide radical was generated by the xanthine/xanthine oxidase and phenazine methosulphate/NADH systems. The infusion-mediated prevention of nitroblue tetrazolium reduction by the superoxide radical was used as the measured endpoint. Hydroxyl radical was generated by the Fe3+-EDTA/ascorbate Fenton system, and assayed by evaluating deoxyribose degradation using the thiobarbituric acid method. Hypochlorous acid scavenging activity was tested by measuring the inhibition of hypochlorous acid-induced 5-thio-2-nitrobenzoic acid oxidation to 5,5′-dithiobis(2-nitrobenzoic acid). The tested infusion mainly exhibited a potent scavenging effect on superoxide radicals (although a noncompetitive inhibitory effect on xanthine oxidase was also observed). The infusion also acted as a moderate scavenger of hydroxyl radicals and hypochlorous acid. A phytochemical study of the infusion was also undertaken, and nine phenolic compounds were identified.
Lippia citriodora is an herbal species which contains several flavonoids and phenolic acids. In view of the pharmacological interest in natural phenolic compounds as antioxidants, this study examined the superoxide radical, hydroxyl radical and hypochlorous acid scavenging activities of L. citriodora infusion. Superoxide radical was generated either in an enzymatic or in a chemical system, and scavenging ability was assessed by the inhibition of nitroblue tetrazolium reduction. Hydroxyl radical was generated by the reaction of an iron-EDTA complex with H2O2 in the presence of ascorbic acid, and was assayed by evaluating deoxyribose degradation. Hypochlorous acid scavenging activity was tested by measuring the inhibition of 5-thio-2-nitrobenzoic acid oxidation. The results demonstrate that this infusion has a potent superoxide radical scavenging activity and a moderate scavenging activity of hydroxyl radical and hypochlorous acid. The chemical composition of the lyophilized infusion was also determined in an attempt to establish its relationship with the antioxidant activity found in the present study.
To evaluate the antithrombotic activities of puerarin and daidzin from the rhizome of Pueraria lobata, in vitro and ex vivo inhibitory activities of these compounds and their metabolite, daidzein, were measured. These compounds inhibited ADP- and collagen-induced platelet aggregation. Daidzein was the most potent. However, when puerarin and daidzin were intraperitoneally administered, their antiaggregation activities were weaker than when these compounds were administered orally. When in vivo antithrombotic activities of these compounds against collagen and epinephrine were measured, these compounds showed significant protection from death due to pulmonary thrombosis in mice. To evaluate the antiallergic activity of puerarin, daidzin, and daidzein, their inhibitory effects on the release of β-hexosaminidase from RBL 2H3 cells and on the passive cutaneous anaphylaxis (PCA) reaction in mice were examined. Daidzein exhibited potent inhibitory activity on the β-hexosaminidase release induced by DNP-BSA and potently inhibited the PCA reaction in rats. Daidzein administered intraperitoneally showed the strongest inhibitory activity and significantly inhibited the PCA reaction at doses of 25 and 50 mg/kg with inhibitory activity of 37 and 73%, respectively. The inhibitory activity of intraperitoneally administered daidzein was stronger than those of intraperitoneally and orally administered puerarin and daidzin. Therefore we believe that puerarin and daidzin in the rhizome of Pueraria lobata are prodrugs, which have antiallergic and antithrombotic activities, produced by intestinal microflora.
The complete amino acid sequences of [2Fe–2S] ferredoxins from Scopolia japonica and Lycium chinense have been determined by automated Edman degradation of the entire Cm-proteins and of the peptides obtained by enzymatic digestions. These two ferredoxins exhibited only 2—7 differences in the amino acid sequence when compared to the Datura-ferredoxins (D. stramonium, D. metel, and D. arborea), and especially only 2 or 3 differences compared to D. arborea. On the contrary, 8—19 differences were observed among the other solanaceous ferredoxins. This suggests that S. japonica and L. chinense are closely related taxonomically to Datura plants, especially to D. arborea.
The MeOH extract of the roots of Inula helenium showed a high inhibitory activity for cell growth against MK-1, HeLa and B16F10 cell lines. Significant activity was found in the hexane-soluble fraction. From the hexane-soluble fraction, seven sesquiterpenes, namely, one germacrane (4β,5α-epoxy-1(10),11(13)-germacradiene-8,12-olide), one elemane (igalane), and five eudesmanes (alantolactone, isoalantolactone, 11α,13-dihydro-alantolactone, 11α,13-dihydro-isoalantolactone, 5-epoxyalantolactone) were isolated. In vitro antiproliferative activities of the isolates against MK-1, HeLa and B16F10 cells are reported.
The predictability of serum vancomycin (VCM) concentrations by means of the Bayesian method was evaluated to establish whether the method can be used to select safe and effective VCM treatment regimens for individual patients. Serum VCM concentrations at the trough and 2 h after the end of infusion (peak) were measured. Pharmacokinetic parameters were calculated for VCM dosage regimens based on a two-compartment model with the Bayesian method, using the Japanese population pharmacokinetic parameters estimated by Yasuhara et al. (1998). The predictive performance for serum VCM concentrations and the dosage regimens were analyzed using two points of serum VCM concentration in 41 patients whose serum creatinine and age were in the ranges of 0.4—4.6 mg/dl and 24—92 years, respectively. Although the predicted values for trough and peak VCM concentrations were slightly lower than measured VCM concentrations, the predictive performance was generally good. There were no differences among the groups classified by serum creatinine or age. An examination of predicted data that differed markedly from the measured serum VCM concentrations indicated that a larger difference in volume of distribution at the steady state (Vdss) calculated from serum VCM concentrations at the beginning and revision of dosage regimens resulted in a poorer correlation of predicted values and measured values. This finding indicates that therapeutic drug monitoring should be conducted frequently, and the dosage regimen revised accordingly, in the case of patients who may have a change of Vdss of VCM, for example, due to a complication such as heart failure or edema.
The effect of a TEI enhancer mixed system consisting of triethanolamine (T), ethanol (E) and isopropyl myristate (IPM) on the skin permeation of acidic, basic and neutral drugs were evaluated in vitro using excised hairless rat skin. The binary enhancer system consisting of IPM and ethanol (EI) produced marked improvement on the penetration of all the drugs tested. When T was added to the EI system, a greater enhancing effect was found only on acidic drugs with a carboxyl group, compared with the flux in the EI system. On addition of another amine to the EI system, instead of T, mefenamic acid (MA), which exhibited the highest enhancing effect of the model drugs, showed an approximately 14—180 times greater flux than when delivered by the EI system. On simultaneous application of isosorbide dinitrate (ISDN) with MA in the TEI system, the flux of MA increased on increasing the T concentration in the TEI system, while, the flux of ISDN, a neutral drug, was unaffected by the T concentration. Application of MA in the EI system after pretreatment of the TEI system showed that the residual amount of T in the skin plays an important role in the skin permeation of MA. Furthermore, at a fixed concentration of MA, the flux of MA increased on increasing the T concentration in the TEI system, while the flux of E remained unchanged. Finally, the infrared spectrum of MA with amine in the E solution indicated that the carboxyl group of MA was ionized. These results demonstrated that the formation of an ion pair between MA and T, but not the effect of T on the skin, may be responsible for the enhanced skin permeation of MA using the TEI system.
To determine the transport mechanism of sulpiride in an in vitro model of the human intestine, we investigated the transepithelial transport of this agent in Caco-2 cells. The transepithelial transport and intracellular accumulation of sulpiride were measured using Caco-2 cell monolayers cultured on a permeable membrane. The transepithelial transport of sulpiride in Caco-2 cells showed temperature dependence, and the transport was enhanced at weakly acidic pH on the apical side. These results demonstrate that the transepithelial transport of sulpiride is carrier mediated. To identify the drug transporter species that take part in the transepithelial transport of sulpiride, we examined the effects with the addition and preloading with specific substrates and inhibitors of various drug transporters. The results obtained from these examinations indicated that the apical-to-basolateral transport of sulpiride is mediated by the peptide transporter PEPT1, organic cation transporters OCTN1 and OCTN2 on the apical membrane, and the basolateral peptide transporter on the basolateral membrane. The basolateral-to-apical transport is mediated by the basolateral peptide transporter and organic cation transporter OCT1 on the basolateral membrane and by P-glycoprotein on the apical membrane. A decrease in the absorption of sulpiride may occur in coadministration protocols involving PEPT1-, OCTN1-, and OCTN2-transported drugs. Coadministration using the P-glycoprotein-transported drugs, in contrast, may enhance the absorption of sulpiride.
The influx transport mechanism of pentazocine (PTZ) at the blood-brain barrier (BBB) was investigated in rats using the carotid injection technique. The uptake kinetics of PTZ into the rat brain exhibited saturability, which occurred by both nonsaturable and carrier-mediated transport processes. The in vivo kinetic parameters were estimated as follows: the maximal uptake rate (Jmax), 3.6±1.2 μmol/min/g brain and the apparent Michaelis constant (Kt), 3.7±1.7 mM for the saturable component of PTZ into the brain, and the nonsaturable uptake rate constant (Kd), 0.06±0.04 ml/min/g brain. The uptake of PTZ by the brain was strongly inhibited by lidocaine, imipramine and propranolol, and also by H1-antagonists such as mepyramine, diphenhydramine. In addition, narcotic-antagonist analgesic (buprenorphine, butorphanol or eptazocine) and an opioid antagonist (naloxone) significantly inhibited PTZ transport. These results suggest that PTZ permeates into the brain via a carrier-mediated transport system, which may widely recognize the cationic drugs.
In the present study, we examined whether polymorphisms in the ATP-binding cassette (ABC) transporter genes, MDR1, MRP1 and MRP2, were associated with their respective mRNA expression levels in duodenal enterocytes of 13 healthy Japanese volunteers. MDR1 genotypes of T-129C, G2677(A,T) and C3435T, MRP1 genotypes of G128C, C218T, G2168A and G3173A, and MRP2 genotypes of C-24T, G1249A, C2302T, C2366T and G4348A were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or direct sequencing. Mutations T-129C, G2677(A,T) and C3435T of MDR1 gene were found at allele frequencies of 2/26, 16/26 and 12/26, respectively. Mutations G2168A of the MRP1 gene and C-24T of the MRP2 gene were also found at allele frequencies of 1/26 and 6/26, respectively, whereas other mutations were not detected in MRP1 and MRP2 genes. The relative concentrations (mean±S.E.) of MDR1 mRNA to villin mRNA were 0.38±0.15, 0.56±0.14 and 1.13±0.42 in the subjects with C/C3435, C/T3435 and T/T3435, respectively, which supported the lower serum concentrations of digoxin after single oral administration in the subjects with the mutant T-allele at position 3435. Genetic collaboration between positions 3435 and 2677 was suggested, and those in G/G2677, G/(A,T)2677 and T/(A,T)2677 were 0.16±0.05, 1.10±0.40, and 0.63±0.16, respectively (p=0.107). However, there was no remarkable effect of the G2168A of the MRP1 gene or of C-24T of the MRP2 gene on the relative MRP1 or MRP2 mRNA concentrations, respectively.
Conjugates of mitomycin C (MMC) with estradiol bezoate and estradiol via glutaric acid (EB-glu-MMC and E-glu-MMC, respectively) were examined for their antitumor activities against P388 leukemia and sarcoma 180. EB-glu-MMC and E-glu-MMC were suspended in 10% (v/v) propylene glycol in saline and administered intraperitoneally to mice bearing P388 leukemia intraperitoneally or to mice bearing sarcoma 180 subcutaneously. The antitumor effect against P388 leukemia was greater in the order MMC>E-glu-MMC>EB-glu-MMC, and only the former two compounds significantly increased life span. On the other hand, EB-glu-MMC and E-glu-MMC showed suppression of sarcoma 180 growth at higher doses close to or better than MMC. In the mixture of 1/15 M phosphate buffer (pH 7.4, ionic strength (μ) adjusted to 0.3 with NaCl)–propylene glycol (9 : 1, v/v) at 37 °C, MMC was released much more slowly from EB-glu-MMC suspension than from E-glu-MMC suspension. With regard to chemotherapy against sarcoma 180, both conjugates were considered to supply MMC slowly but effectively at higher doses.
The purpose of this study was to develop a method for the measurement of the cell kinetics of spleen lymphocytes using the ROSA 26 transgenic mouse ubiquitously expressing β-galactosidase (β-gal). Spleen lymphocytes were isolated from ROSA 26 mice and intravenously inoculated into C57BL/6 mice under normal conditions and inflammatory conditions following lipopolysaccharide (LPS) treatment. Spleen lymphocyte accumulation in tissues was determined as a measurement of β-gal activity. Spleen lymphocytes isolated from ROSA 26 mice have β-gal activities of 1.45×10−4 pg per cell. A good correlation between β-gal activities and cell numbers was obtained (r2=0.999) over the range 1×103 to 1×107 cells, corresponding to 70 fg to 350 pg β-gal activity. Spleen lymphocytes (4×107 cells) were intravenously inoculated into normal mice and subsequently each tissue was isolated and the corresponding β-gal activity measured. Spleen lymphocyte accumulation was relatively high in the spleen and lymph nodes. The accumulated spleen lymphocyte cell number was 1.39×107 cells/g spleen and 5.45×107 cells/g lymph node 1 h and 6 h after inoculation, respectively, and this remained constant up to 24 h. In the lung, lymphocyte accumulation was 3.98×107 cells/g tissue 10 min after inoculation then gradually fell to 7.09×105 cells/g tissue after 24 h. In addition, the femoral muscle following intramuscular injection of LPS showed a high accumulation of spleen lymphocytes, whereas the untreated and contralateral femoral muscle had the same level as the background. In conclusion, spleen lymphocytes isolated from ROSA 26 mice can be used to measure β-gal activity and the sensitivity is relatively high over the 70 fg to 350 pg range. This suggests that cells isolated from the ROSA 26 mouse can be applied to the study of cell kinetics.
Bile acids are believed to play a role in the etiology of colorectal cancer, and high fecal excretion of secondary bile acids was correlated with increased incidence of colon cancer. Recently, it was also reported that there is an increase in plasma of the secondary bile acid, deoxycholic acid in men with colorectal adenomas. Since deoxycholic acid is formed in the colon and absorbed into the portal systemic circulation, it was suggested that the blood concentration of this bile acid reflects the level of exposure of colonic cells to deoxycholic acid. The objective of this study was to investigate whether plasma deoxycholic acid level represents the fecal content of this bile acid in several animal species with different bile acid composition and deoxycholic acid contribution to the bile acid pool. Eight rabbits, hamsters, guinea pigs, and rats were used in this study. Blood samples and feces were collected on days 1, 3, 5 and 7. Bile samples were obtained only on day 7. The plasma, fecal and biliary bile acids were analyzed by gas chromatography-mass spectrometry. Bile acid composition and deoxycholic acid content varied greatly between the animal species studied. There was a variation in the concentration of total bile acids in the plasma and feces obtained at different times during the experiments, however, the bile acids profile remained constant throughout the study. The data obtained shows that although plasma bile acid profile was not similar to fecal bile acids profile, however, there was a significant correlation between the level of plasma and fecal deoxycholic acid. Plasma deoxycholic acid concentration might be a reliable biomarker for the degree of exposure of colon cells to this bile acid, and may be useful in further studies on the role of secondary bile acids in colon carcinogenesis.
A novel anti-tumor agent, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one dihydrochloride (TAS-103), effectively inhibits both topoisomerase I and II activities. To enhance anti-tumor efficacy and to reduce the side effects of the agent, liposomalization of TAS-103 was performed. TAS-103 was effectively entrapped in liposomes by a remote-loading method, and was stable at 4 °C and in the presence of 50% serum. To evaluate the anti-tumor efficacy of liposomal TAS-103, the growth inhibition against Lewis lung carcinoma cells in vitro and the therapeutic efficacy against solid tumor-bearing mice in vivo were examined. Liposomal TAS-103 showed strong cytotoxic effect against Lewis lung carcinoma cells in a dose dependent manner and effectively suppressed solid tumor growth accompanying longer survival time of tumor-bearing mice in comparison with the mice treated with free TAS-103. These results suggest that liposomal TAS-103 is useful for cancer therapy.
The 70% methanol soluble fraction from a licorice acetone extract was found to inhibit cell proliferation in human monoblastic leukemia U937 cells by inducing apoptosis. Separation by the methods including preparative HPLC provided us with an active compound, which was identified as licocoumarone. Several lines of evidence indicated that licocoumarone induced apoptosis in U937 cells. Thus, licocoumarone is suggested to be potentially useful as a natural anti-cancer agent.