A highly sensitive ELISA for the determination of (s)-9-dimethylaminomethyl-10-hydroxy-camptothecin (topotecan) capable of measuring as low as 80 pg/ml was developed. Anti-topotecan antibody was obtained by immunizing rabbits with topotecan conjugated with bovine serum albumin using diazotized m-aminobenzoic acid as a cross-linker. Enzyme labeling of topotecan with β-D-galactosidase was performed by utilizing another cross-linker, N-(4-diazophenyl)maleimide. The specificity of this ELISA appears to be primarily toward the lactone moiety of topotecan, showing a very slight cross-reactivity with the lactone opened-ring of topotecan. The values for the topotecan concentrations detected using this assay were comparable with those detected using HPLC. There was a good correlation between the values determined by the two methods. These findings suggest that this ELISA can detect the natural amounts of the lactone form. Using this assay, drug levels were easily determined in the blood and urine of rats for 5 h after i.v. administration of topotecan at a single dose of 1 mg/kg. The sensitivity and specificity of the ELISA should provide a useful tool for developing pharmacokinetic and pharmacodynamic studies of topotecan.

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An enzyme immunoassay (EIA) has been developed for determination of 6-amino-5-chloro-1-isopropyl-2-(4-methyl-1-piperazinyl) benzimidazole (KB-6806), a novel 5-HT₃ receptor antagonist. Anti-KB-6806 antiserum was elicited against the KB-6806-bovine serum albumin (BSA) conjugate prepared by a diazo coupling reaction through the inherent 6-amino group. β-Galactosidase-labeled 6-amino-5-chloro-1-isobutyl-2-(4-methyl-1-piperazinyl)benzimidazole was similarly prepared by diazo coupling reaction as an enzyme-labeled antigen with a hapten heterologous combination of antiserum. The modification at the 4-methyl group of the piperazine moiety of KB-6806 significantly decreased the binding affinity to the antibody. This method could quantitate KB-6806 in dog plasma in the concentration range of 0.078-10 ng/ml with good accuracy and precision. The EIA method has been successfully applied to the determination of KB-6806 in plasma after intravenous administration of KB-6806 to dogs.

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When isolated mouse fat pads were incubated with orthovanadate (vanadate) or insulin for up to 4 h, the leptin secretion into the medium was decreased by vanadate and increased by insulin. Propranolol, a nonspecific antagonist of β-adrenergic receptors, bupranorol, a specific antagonist of β₃-adrenergic receptor, and H-89, an inhibitor of cAMP-dependent protein kinase (PKA) all inhibited the decrease by vanadate to various extents. In contrast, no inhibition was observed with specific antagonists of β₁- and β₂-adrenergic receptors or with inhibitors of protein kinase C and Ca/calmodulin kinase. Short-term incubation of the fat pads with vanadate showed a transient increase in the cellular cAMP content; this increase was inhibited by propranolol and bupranolol. Vanadate had no effect on the incorporation of [³H]-leucine into proteins of the fat pads with a 4-h incubation, although insulin stimulated the incorporation. The decreasing effect of vanadate on the leptin secretion seems to be independent of the regulation of protein synthesis. These results suggest that vanadate decreases the leptin secretion through mechanisms involving the increase in cellular cAMP content via β₃-adrenergic receptor, probably leading to the activation of PKA.

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Aromatase is a cytochrome P-450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione (AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the ability of AD isomers, 4-en-6-one 1a, 5-en-4-one 2a, and 5-en-7-one 3a, competitive inhibitors of aromatase with an A, B-ring enone structure to serve as a substrate, we incubated the three inhibitors separately with human placental aromatase in the presence of NADPH in air. The metabolites were analyzed as the methoxime-trimethylsilyl ethers by gas chromatography-mass spectrometry. All of the inhibitors were found to be oxygenated with aromatase to produce the corresponding 19-hydroxy derivatives 1b, 2b, and 3b with rates of 2.0, 51, and 0.3 pmol/min/mg protein, respectively. Only in the experiment with the 5-en-4-one steroid 2a, the production of the 19-oxo metabolite 2c was detected with a rate of 3.1 pmol/min/mg protein. The 19-oxygenation of steroid 2a, the best substrate for aromatase among the three, was kinetically determined to give the V_max value of 40 pmol/min/mg protein and the K_m value of 1.43 μM, respectively. The results reveal that a good inhibitor of aromatase is not essentially a good substrate for the enzyme in a series of the A, B-ring enone steroids.

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This study aimed to examine molecular mechanisms responsible for the metabolic fate of S-nitrosoglutathione (GSNO) in endothelial cells. After addition of 1 mM GSNO in culture medium, concentration of S-nitrosothiols (RSNO) significantly decreased with concomitant accumulation of nitrite (NO₂^-) only in the presence of human endothelial cells (ECV304), while no change in RSNO decomposition and NO₂^- accumulation was observed in case of S-nitrosocysteine. Bathocuproine disulfonic acid (BCS), a chelator for Cu(I), prevented the cell-mediated decomposition of RSNO and accumulation of NO₂^-. Chelator for Cu(II), Fe(II), or Fe(III); inhibitors of γ-glutamyltranspeptidase; or a superoxide quenching enzyme had no effect on the cell-mediated degradation of RSNO and accumulation of NO₂^-. These results indicate that cellular Cu(I) would play a major role in the conversion of GSNO into NO₂^-. We recently demonstrated that human glyoxalase I (Glo I) interacts with GSNO in vitro and within cells. When Glo I interacts with GSNO, Glo I is inactivated and is chemically modified with pI alteration on 2D gels. So, we examined effect of Cu(I) chelation on the Glo I response. As a result, chelation of cellular Cu(I) by BCS enhanced the inactivation and chemical modification of Glo I by GSNO. The Glo I response could be detected when the cells were exposed to GSNO at 10 μM, corresponding to the concentration of RSNO under physiological conditions, with pretreatment of BCS. Metal chelators for copper and iron ions had no effect on the sensitivity of Glo I to an nitric oxide (NO) radical donor. These results indicate that chelation of cellular Cu(I) potentiates the sensitivity of Glo I to GSNO. The observation in the present study implicates that intracellular levels of GSNO might be elevated, accompanying with stabilization of extracellular RSNO.

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In order to quantify fragmented DNA extracted from apoptotic cells, we devised a separation method which condenses fragmented DNA into a small band, separating it from larger-size DNA with agarose gel electrophoresis. Calf thymus DNA and standard fragmented DNA were loaded onto 1.0% gel for 0.5, 1.0, 1.5 and 2.0 cm length, and onto 0.7, 1.0, 1.5 and 2.0% of gels for 1 cm length. DNA was then extracted from gel slices with the UltraClean^TM 15 DNA Purification Kit, and estimated by measuring fluorescence intensity using Hoechst No.33258 dye. DNA recovery from the gel showed constant values regardless of the amount of loaded DNA up to 1 μg/assay, and a plot of loaded DNA amounts vs. the DNA amount yielded resulted in a strait line in any gel concentration used. Our results show the best conditions to estimate DNA fragmentation rates in apoptotic cells in which fragmented DNA was separated from thymus DNA by loading on 1.0% gel for 1.0 cm length. We used our method to estimate fragmentation rates in DNA fractions extracted from apoptotic human cervical fibroblast, amnion epithelial and chorion laeve trophoblast cells by stimulation with actinomycin D. The results show that DNA fragmentation rates in these cells were consistent with the electrophoretic patterns of the DNA samples shown by their photographs.

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We have recently found that L-glutamate suppresses morphological changes of astrocytes induced by amyloid β protein, adenosine 3', 5'-cyclic monophosphate or phorbol ester in culture. To test the possibility that L-glutamate affects organization of the cytoskeleton, we investigated its effect on morphological changes induced by disruption of actin filaments with cytochalasin B. Cultured rat cortical astrocytes exhibited flat, polygonal morphology in the absence of stimulation, and changed into process-bearing stellate cells following treatment with cytochalasin B (50 μM). L-Glutamate strongly suppressed the stellation induced by cytochalasin B. The effect of L-glutamate was mimicked by D- and L-aspartate and transportable glutamate uptake inhibitors. These results suggest that glutamate transporter activity leads to cytoskeletal actin organization in astrocytes.

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The functions of leukocyte-derived chemotaxin 2 (LECT2), a novel liver-specific protein, are not well defined, especially after hepatic injury. The changes in expression of LECT2 mRNA were investigated after concanavalin A (Con A)-induced hepatic injury in mice. Serum glutamate pyruvate transaminase (s-GPT) activity and the percentage of liver DNA fragmentation, an indicator of hepatic apoptosis, increased 8 h after intravenous administration of Con A (13 mg/kg). Expression of LECT2 mRNA was reduced from 8—24 h after injection of Con A, but was detected again 48 h after recovery from hepatic injury. Expression of tumor necrosis factor (TNF)-α and interferon (IFN)-γ mRNA was observed in liver 2 h after Con A injection. Z-Val-Ala-Asp(OMe)-CH₂F (Z-VAD-FMK), a caspase inhibitor, was administered to mice to investigate whether LECT2 was involved in apoptosis of liver cells after Con A injection. Z-VAD-FMK inhibited s-GPT activity and DNA fragmentation in the liver 8 h after Con A-induced hepatic injury, but did not prevent the reduction of LECT2 mRNA, or induction of TNF-α and IFN-γ mRNA expression. When the relation between expression of LECT2, TNF-α and IFN-γ mRNAs was examined 8 h after Con A injection in wild-type or immunodeficient (nu^-/nu^-) mice, the increase in TNF-α and IFN-γ mRNA expression was found to be closely related to a reduction in LECT2 mRNA expression. These results suggest that the reduction in expression of LECT2 mRNA is not directly involved in apoptosis and may be inversely related to the expression of TNF-α and/or IFN-γ mRNA in the injured liver.

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The effect of the light schedule on toxic interactions between propranolol and disopyramide were studied in chick embryos. Fertilized eggs of White Leghorns were incubated under dark conditions and investigated, on two occasions, under light conditions or under dark conditions. Propranolol, with and without disopyramide, was injected into the air sac of fertilized eggs on the 16th day of incubation. Electrocardiograms (ECGs) were recorded 0 to 60 min after the injection. After the injection of propranolol with disopyramide, the heart rate was significantly decreased compared with the injection of propranolol alone under light conditions. In addition, this toxic interaction between propranolol and disopyramide was more severe under dark conditions than under light conditions. These findings indicate that manipulation of the light schedule has a marked influence on the toxic interaction between propranolol and disopyramide in chick embryos.

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The human estrogen receptor (hER) exists as two subtypes, hER α and hER β, that differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activities of soy isoflavones after digestion with enteric bacteria in competition binding assays with hER α or hER β protein, and in a gene expression assay using a yeast system. The estrogenic activities of these isoflavones were also investigated by the growth of MCF-7 breast cancer cells. Isoflavone glycoside binds weakly to both receptors and estrogen receptor-dependent transcriptional expression is poor. The aglycones bind more strongly to hER β than to hER α. The binding affinities of genistein, dihydrogenistein and equol are comparable to the binding affinity of 17 β-estradiol. Equol induces transcription most strongly with hER α and hER β. The concentration required for maximal gene expression is much higher than expected from the binding affinities of the compounds, and the maximal activity induced by these compounds is about half the activity of 17 β-estradiol. Although genistin binds more weakly to the receptors and induces transcription less than does genistein, it stimulates the growth of MCF-7 cells more strongly than does genistein.

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The cytotoxicity of diesel exhaust particles (DEPs) toward human leukemic promyelocytic cells HL-60 was examined. DEPs were toxic and cytotoxicity increased in a dose-dependent manner. All cells died with 750 μg/ml DEPs in culture media. Apoptosis occurred in HL-60 cells exposed to DEPs. The cytotoxicity of DEP extracts with organic solvents was much lower than those of DEPs and organic solvent-washed residual DEPs. HL-NR6 cells, an HL-60 variant cell line, having higher superoxide dismutase and catalase activities than HL-60 cells, were more resistant to DEP cytotoxicity. When preincubated with the fluorescent probe diacetoxymethyl 6-carboxy-2', 7'-dichlorodihydrofluorescinate diacetate and then exposed to DEPs, HL-60 cells emitted green fluorescence under blue illumination, indicating that reactive oxygen species were generated within the cells. The DEP cytotoxicity correlated inversely with the cellular concentration of reduced glutathione (GSH), which had been attenuated with L-buthionine-(R,S)-sulfoximine, a γ-glutamylcysteine synthetase inhibitor, and was lowered with ethyl reduced glutathionate, a GSH carrier across biomembranes. Further, DEPs themselves decreased the cellular concentration of GSH in a dose-dependent manner. The α-tocopherol model compound 2,2,5,7,8-pentamethylchroman-6-ol decreased DEP cytotoxicity, while α-tocopherol had no effect. In addition, quinacrine, an endocytosis inhibitor, decreased DEP cytotoxicity. These results show that DEPs are cytotoxic and suggest that the cytotoxicity results from generation of reactive oxygen species by DEPs which have been incorporated into cells.

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Entandrophragma angolense is a medicinal plant used in folk medicine against several diseases including peptic ulcer. Methyl angolensate was isolated from E. angolense by recrystallization from methanol. The needle-like crystals were characterized and tested on isolated rabbit jejunum, guinea pig ileum and the rat fundus strip. The compound was also evaluated on the gastrointestinal transit in mice. The results showed that the compound exerted significant concentration dependent inhibition of smooth muscle and reduced the propulsive action of the gastrointestinal tract in mice. The relaxation observed did not attenuate acetylcholine and histamine induced contractions, but was found to inhibit contractions induced by serotonin. It is therefore suggested that methyl angolensate may exert its activity on gastrointestinal smooth muscle via serotonergic mechanisms.

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The effects of the soy isoflavone glycoside, daidzin, genistin, and glycitin on bone loss and lipid metabolism in ovariectomized (ovx) rats were compared with those of estrone. Thirty-six 11-week-old female Sprague-Dawley rats were assigned to six groups, sham-operated, ovx, ovx+glycitin, ovx+daidzin, ovx+genistin, and ovx+estrone and fed matched amounts of a commercial calcium-deficient diet for 4 weeks. Throughout this period, daidzin, genistin or glycitin (25, 50 or 100 mg/kg/d) was given orally using a stomach tube, or estrone (7.5 μg/kg/d) was administered subcutaneously. Daidzin, genistin and glycitin significantly prevented bone loss in ovx rats at a dose of 50 mg/kg/d, like estrone. At this dose glycitin and daidzin also prevented ovx-induced uterine atrophy and increases in body weight gain, abdominal fat, serum total cholesterol and triglyceride, and urinary excretion of pyridinoline and deoxypyridinoline with statistical significance, like estrone. On the other hand, genistin prevented ovx-induced uterine atrophy only at a dose of 100 mg/kg, but did not block any other change of ovx rats at a dose of 50 or 100 mg/kg. These findings indicate that daidzin, glycitin, and genistin are effective in preventing bone loss and the former two compounds are effective in reversing the unfavorable changes of lipid metabolism in this model. It is suggested that the preventive effect of daidzin or glycitin on bone loss in ovx rats is due to suppression of bone turnover, as in the case of estrone, but genistin has a different mechanism of action from the other compounds. Soy isoflavone glycosides may represent a potential alternative therapy in the treatment of bone loss and lipid metabolism abnormality in ovarian hormone-deficient women.

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Limonene is one of the most simple cyclic monoterpenes, and two enantiomers, d- and l-limonene occur due to the chiral carbon at 4-position. Cyclization of GPP into limonene is catalyzed by the limonene synthase, and some l-limonene synthase cDNAs have already been cloned from several species of plants, mainly from Labiatae family. However, the d-limonene synthase gene has not yet been obtained, therefore, no information is available on the molecular mechanism of stereochemical regulation in limonene formation. To resolve this, we cloned the d-limonene synthase gene (dLMS) from Schizonepeta tenuifolia (Labiatae) by a reverse genetic approach, and we found that both d- and l-limonene synthase share similar features such as a transit peptide, an arginine rich domain, and a metal cation binding site in their structures. Here, we report on the cloning of dLMS, and the putative stereochemical regulation mechanism is discussed based on the comparison of the deduced amino acid sequence of dLMS with those of known l-limonene synthases.

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The effect of glutathionesulphonic acid (N-(N-γ-L-glutamyl-L-β-sulphoalanylglycine, GSO₃H), which is one of the minor metabolites of glutathione (GSH), on the pharmacokinetics of verapamil hydrochloride (verapamil·HCl) and tegafur was investigated in rats. GSO₃H was concomitantly used as sodium salt (GSO₃Na). No significant change by the concomitant use of GSO₃Na was recognized in the pharmacokinetics parameters of verapamil·HCl and tegafur, and plasma elimination of both substances was not affected by GSO₃Na. The tissue-to-plasma concentration ratio (K_p) of verapamil·HCl in the lung 5 min after its administration under concomitant use of GSO₃Na rose significantly, however, this effect disappeared 120 min after administration. No significant change was recognized in other organs. On the other hand, a significant difference of K_p of tegafur under a steady state concentration of GSO₃Na was not recognized in any organs. It seemed that the elevation of a lipid solubility (oil water partition coefficient) of verapamil·HCl by the concomitant use of GSO₃Na was related to the increase of the K_p value of verapamil·HCl in the lung. The partition coefficient of GSO₃Na itself decreased when it was used concomitantly with verapamil·HCl.

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The permeability of insulin across Xenopus pulmonary membrane and the effects of various absorption enhancers on insulin permeability were examined using an in vitro Ussing chamber technique. Absorption enhancers used in this study were sodium caprate (NaCap), sodium glycocholate (NaGC), sodium salicylate (NaSal) and ethylenediaminetetraacetic acid disodium salt (EDTA). The permeability of insulin across Xenopus pulmonary membrane significantly increased in the presence of NaCap and NaGC, while EDTA and NaSal did not enhance the permeability. In addition, the enhancing effect of NaGC increased as the concentrations of these enhancers increased. Transmembrane resistance (Rm) of Xenopus lung was markedly decreased in the presence of these enhancers, and NaCap showed a greater effect on Rm than NaGC. Furthermore, the amount of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) released from the apical side of the Xenopus pulmonary membrane increased in the presence of these enhancers. These results indicate that NaCap and NaGC improve the pulmonary absorption of insulin, but they are toxic to the pulmonary membrane. These findings suggest that this method is useful for estimating the permeability characteristics of peptides across the pulmonary membrane and for evaluating the effects of various additives on their permeability and their membrane toxicity.

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The aim of this present study was to investigate the in vitro transdermal iontophoretic delivery of three diclofenac salts—diclofenac sodium (DFS), diclofenac potassium (DFP), and diclofenac diethylammonium (DFD). A series of physicochemical and electrical variables which might affect iontophoretic permeation of diclofenac salts was studied. Application of 0.3 mA/cm² current density significantly increased the transdermal flux of diclofenac salts as compared to passive transport. The iontophoretic enhancement increased in the order of DFS>DFP>DFD. The permeability coefficient of diclofenac salts all decreased with increasing donor concentration during iontophoresis. The addition of buffer ions and salt ions such as NaCl, KCl, and C₄H₁₂ClN reduced the permeation of diclofenac salts due to competition. However, this effect was lesser for DFD than for DFS and DFP. Comparing the various application modes of iontophoresis, the discontinuous on/off mode showed lower but more constant flux than the continuous mode.

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The cytotoxicity of maltosyl-α-cyclodextrin (G₂-α-CyD) and maltosyl-β-cyclodextrin (G₂-β-CyD) toward Caco-2 cells was compared with that of natural α-cyclodextrin (α-CyD), β-cyclodextrin (β-CyD) and γ-cyclodextrin (γ-CyD). The degree of increase in cytotoxicity was dependent on the CyD’s type and the concentration: the cytotoxicity of CyDs at the same concentration increased in the order of γ-CyD<G₂-β-CyD<G₂-α-CyD<<α-CyD, although β-CyD could not be compared to other CyDs because of low solubility in water. α-CyD decreased transepithelial electrical resistance (TEER) and increased the apical-to-basolateral (AP-to-BL) transport of [³H]mannitol, a paracellular transport marker, in a concentration-dependent manner, suggesting that α-CyD decreased the integrity of Caco-2 cell monolayers. In addition, α-CyD increased the AP-to-BL transport of rhodamine 123, a transcellular transport marker, under the experimental conditions being independent of P-glyco-protein. In contrast, G₂-α-CyD, G₂-β-CyD and γ-CyD had slight effect on both TEER and the transport of mannitol and rhodamine 123 even at relatively high concentrations up to 150 mM. The inability of G₂-α-CyD and G₂-β-CyD to effect TEER and the transport of mannitol and rhodamine 123 could be explained by the findings that these maltosylated CyDs released only a small amount of membrane constituents from Caco-2 cell monolayers and interacted only weakly with monolayers composed of L-α-dipalmitoylphosphatidylcholine (DPPC) formed on water. These results indicate that G₂-α-CyD has less cytotoxicity and less disturbing ability toward Caco-2 cell monolayers than α-CyD, and G₂-β-CyD has, at least, comparable cytotoxicity to β-CyD toward them. Thus, from the safety point of view, highly water-soluble G₂-α-CyD and G₂-β-CyD may be particularly useful in various pharmaceutical formulations.

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The prediction error in the Bayesian analysis program for digoxin was evaluated in Japanese patients, and factors influencing the accuracy were investigated. Serum concentrations of digoxin were monitored two times and were compared with the predicted values obtained by using the Bayesian analysis program. The prediction error at the first time was 43.1%. Although this estimation error was reasonably restored at the second time of monitoring, the prediction error remained at 26.6%. These data suggested that unknown factors not included in the program affected the serum concentration of digoxin. Retrospective research of the digoxin serum concentrations in the patients suggested the coadministration of the drugs, which were the P-glycoprotein modulators, as well as the unexpected alteration of the serum creatinine, were the important factors influencing the prediction of the drug serum concentrations. We next examined the inhibitory effect of quinidine, verapamil and spironolactone on the transcellular transport of digoxin by using human P-glycoprotein overexpressing LLC-GA5-COL150 cells. Quinidine, verapamil and spironolactone could inhibit the transcellular transport of digoxin by 50%. In addition, the reduction of the renal clearance by 50%, which could possibly be caused by this inhibition, led to the increase of 36% in the steady state through concentrations of digoxin in the physiological pharmacokinetic model. In conclusion, the prediction of long-term serum concentration-time profiles of digoxin, based on the Bayesian analysis, will be disturbed by the coadministration of the P-glycoprotein modulators and the unexpected alteration of the serum creatinine.

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Dissolution rate is considered an important factor affecting absorption and efficacy after the oral administration of tolbutamide. Since in many cases traditional Chinese medicines, including Sho-saiko-to (TJ-9, Xiao Chaihu Tang), are taken with other drugs, it is likely that the dissolution and absorption of concomitant drugs in the gastrointestinal tract are influenced by the presence of traditional Chinese medicines. In this study, the effects of TJ-9 on the in vitro dissolution of tolbutamide were examined. We carried out the dissolution test of tolbutamide in the absence or presence of traditional Chinese medicines (Kakkon-to, TJ-1; Hachimi-jio-gan, TJ-7; Chorei-to, TJ-40; Shakuyaku-kanzo-to, TJ-68; TJ-9; Glycyrrhizae Radix, GR; glycyrrhizin, GL) by using a pH 1.2 dissolution medium. Tolbutamide was determined by HPLC assay. The moment parameters, i.e., mean dissolution time (MDT), and the dissolution rate constant up to 20 min (k_d) were estimated from the dissolution profiles on the basis of the first-order kinetics. Preparations containing GR, namely TJ-1, TJ-9 and TJ-68, significantly reduced the k_d and increased the MDT of tolbutamide, while TJ-7 and TJ-40 had no effect on the early dissolution profile of tolbutamide. The extent of decrease in the k_d in the presence of TJ-1, TJ-9 and TJ-68 was dependent on their GR contents. Similar inhibitory effects on the dissolution rate of tolbutamide were observed when GR alone was added to the test medium. In addition, GL, a major constituent of GR, induced a 50% increase in MDT and a 30% decrease in k_d. The above results indicate that Chinese traditional preparations containing GR have an inhibitory effect on the in vitro dissolution of tolbutamide, which is derived from GL in the preparations.

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The age-dependent characteristics of transdermal permeation of ethyl nicotinate (EN) and its metabolism to nicotinic acid (NA) were examined in rats at the ages of a fetus at 21 d, 3, 10, 50, 270 and 360 d. Skin transport in vitro was investigated using mounted skin in side-by-side diffusion chambers, and flux of EN and NA was determined. With developing and aging in rats, EN flux from EN-saturated solution declined gradually, however, NA flux increased drastically at 10 and 50 d. To elucidate the mechanism of this age-dependent escalation of NA flux, a hydrolysis study was performed using skin homogenate, and the Michaelis-Menten parameters (V_max and K_m) of EN were evaluated. V_max and V_max/K_m ratio showed the same tendency with NA flux/total (EN+NA) flux ratio, suggesting that skin esterases in rats are developed gradually after birth, then increase markedly and become steady in the adult period. On the other hand, the affinity parameter, K_m, was almost the same among all ages. Moreover, metabolic saturation of esterase during the transdermal process occurred in all ages, and maximal NA flux and EN concentration in the donor compartment for the maximal NA flux were also affected by age. These findings indicated that the discrepancy in transdermal profiles of EN among the ages tested was dominantly due to the difference in the development of esterase in the growth process.

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Granisetron (GRN) is widely used for patients with various cancers who suffer from chemotherapy-induced vomiting and nausea. The pharmacokinetics of GRN has not been fully evaluated in such patients, however, and its dosage regimen is still controversial. In this study, we determined GRN levels in serum and urine from lung cancer patients and children suffering from cancer after intravenous infusion. In lung cancer patients, the inter-individual variations in t_1/2β, area under the concentration-time curve (AUC), and Vd_β were relatively smaller than expected from previous reports on healthy subjects, while t_1/2β was prolonged more than 5-fold in healthy subjects. Urinary excretion of unchanged GRN in lung cancer patients was ca. 15% of dose, consistent with previous reports, and one individual demonstrated an even higher urinary excretion (ca. 45%). The pharmacokinetic parameters of GRN in child cancer patients varied markedly among individuals, and some child patients had smaller t_1/2β than adult patients. In these cases, GRN should be administered at shorter intervals. These results suggested that a pharmacokinetic study of GRN was necessary for planning a dosage regimen and managing chemotherapy-induced vomiting and nausea.

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This report investigates the pharmacokinetics of nimustine (ACNU), cytosine arabinoside (Ara-C), and methotrexate (MTX) in cerebrospinal fluid (CSF) during CSF perfusion chemotherapy. A 47-year-old Japanese man with spinal cord, cerebellum and brain stem dissemination of oligo-astrocytoma received nine courses of CSF perfusion chemotherapy with ACNU, Ara-C, and MTX. A CSF perfusion chemotherapy solution was perfused via an Ommaya reservoir in the ventricle, and was discharged by drainage though another Ommaya reservoir in the lumbar spinal canal. CSF samples via Ommaya reservoirs in the lumbar spinal canal were obtained during the fifth and eighth courses of treatment. The concentrations of ACNU and Ara-C in CSF were measured by HPLC, and the MTX concentrations by fluorescence polarization immunoassay. In the fifth course of treatment, a CSF injection chemotherapy solution, consisting of 5 mg of ACNU dissolved in 20 ml of artificial CSF, was injected over a few minutes using the Ommaya reservoir. Next, a CSF perfusion chemotherapy solution, consisting of 10 mg of Ara-C and 5 mg of MTX dissolved in 100 ml of artificial CSF, was perfused over 2 h. In the eighth course of treatment, a CSF perfusion chemotherapy solution, consisting of 5 mg of ACNU, 10 mg of Ara-C and 5 mg of MTX dissolved in 100 ml of artificial CSF, was perfused over 2 h. In both treatments, the highest concentrations of Ara-C and MTX in CSF were observed 1 or 2 h after the end of perfusion, with the values of each drug being similar. The CSF AUCs of Ara-C and MTX in each treatment were of similar values. Although the highest concentration of ACNU in CSF was observed in the fifth treatment 1 h after injection (an injection chemotherapy of ACNU plus a perfusion chemotherapy of Ara-C and MTX), the concentration of ACNU in CSF was undetectable in the eighth treatment (a perfusion chemotherapy of ACNU, Ara-C and MTX). We were successful in administering all anticancer drugs, and reaching a level of over 1.0 μg/ml concentration in CSF of the lumbar spinal canal, using an injection chemotherapy of ACNU plus a perfusion chemotherapy of Ara-C and MTX; this was done even though the drugs, in particular ACNU, underwent some perfusion-period dependent decomposition.

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The objective of this study was to investigate the correlation between in vitro and in vivo liposome-complement interactions. Third component of the complement (C3) fragments associated with hydrogenated egg phosphatidylcholine (HEPC)-based liposomes in vivo and complement-dependent destabilization in vitro were determined as an indication of liposome-complement interaction in vivo and in vitro, respectively. C3 flagments on the liposomes were detected in both rats and guinea pigs. Pretreatment with K76COOH (K76), a complement inactivating agent, reduced the binding of C3 fragments. These findings indicated that the liposomes remarkably activated the complement system in both animals in vivo. Interestingly, significant complement-dependent liposome destabilization was observed in rat serum, but not in guinea pig serum, indicating that the liposomes activated the complement system in rats, but not in guinea pigs in vitro. Taken together, it is apparent that in vitro complement activation by the liposomes is not in agreement with in vivo complement activation in ginea pigs. This discrepancy in the liposome-complement interaction would suggest the need for further investigation to utilize the information obtained from the liposome-complement interaction to predict in vivo behavior of the liposomes.

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The participation of reactive oxygen species (ROS) in virus inactivation by 1,9-dimethylmethylene blue (DMMB) phototreatment in stroma-free hemoglobin (SFH) was investigated with the use of scavengers, quenchers and enhancer. Virus (R17 bacteriophage) photoinactivation by either activated monomer or dimer DMMB was suppressed by sodium azide (singlet oxygen quencher) and promoted by the substitution of H₂O for deuterium oxide (D₂O), which is known to prolong the lifespan of singlet oxygen. There was no or little effect of mannitol (hydroxyl radical scavenger) and superoxide dismutase (superoxide scavenger) on the photoinactivation. Similar experiments were conducted to investigate the mechanism of methemoglobin (Met-Hb) formation by the activated monomer of DMMB. There was little effect of the singlet oxygen quencher, histidine, or the enhancer, D₂O, on Met-Hb formation. However, rutin, which inhibits not only singlet oxygen but also other ROS, and mannitol supressed the formation of Met-Hb by activated monomer. The addition of superoxide dismutase (SOD) did not inhibit the formation. In contrast to the activity of the DMMB monomer, that of the dimer was inhibited by histidine and enhanced by D₂O. The addition of neither mannitol nor SOD affected Met-Hb formation by activated dimer. These results collectively suggest that virus photoinactivation by the activated monomer and dimer of DMMB as well as Met-Hb formation by the activated dimer proceed via a singlet oxygen mediated pathway. In contrast, singlet oxygen may play a less important role in Met-Hb formation by the activated monomer.

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