In an attempt to find an end-point for cancer chemotherapy, this study was designed to measure the adenine compounds in the plasma of breast cancer patients using HPLC with a selective reagent for adenine bases. The patients were treated by chemotherapy using cyclophosphamide, methotrexate and 5-fluorouracil. Blood was collected in tubes containing EDTA, the plasma separated by centrifugation and analysed by HPLC. An early peak due to the fluorescent derivative of an unknown compound reacted with bromoacetoaldehyde and its concentration appeared proportional to the chemotherapeutic courses of treatment. The compound in its native state without fluorescent derivatization was efficiently purified by using columns of DEAE-and CM-Sephadex. Its UV spectrum revealed maxima at 271, 280 and 272 nm in solutions of pH 7, pH 3 and pH 12, respectively. The electrophoretograms showed that it was neutral, positively and negatively charged at pH 7, pH 3 and pH 12, respectively. Thin-layer chromatograms showed that it had the same Rf as 2'-deoxycytidine (dCyd) which was confirmed by a positive reaction for deoxyribose. It was concluded that bromoacetoaldehyde formed a weakly fluorescent product with dCyd which gave rise to the early peak in the anion exchange chromatograms. From the calculation of the recovery obtained by the purification process, the cancer patients undertaking more than 12 courses had a dCyd level of approximately 20 mM while the corresponding figure in normal volunteers was less than 1 mM. These results may be useful in assessing the status of the cancer patients.

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The induction of macrophage colony-stimulating factor (M-CSF) in monkey plasma following administration of FK 565 was observed within 2 h of injection peaked at 4 h, and remained high after 24 h. Interleukin-6 (IL-6) and M-CSF levels increased in monkeys treated with FK565, even at doses as low as 0.01 mg/kg. Granulocyte CSF (G-CSF) levels increased slightly following a dose of 1 mg/kg, but granulocyte macrophage CSF (GM-CSF) was not detected at any doses of FK565 studied. To examine the thrombopoietic activity of FK565 in vivo, single doses of drug (0.01, 0.1 or 1.0 mg/kg) were administered i.v. to cynomolgus monkeys or normal mice on day 0. The promotes platelet (PLT) count after FK565 injection decreased transiently on days 1 and 2, and then increased in a dose-dependent manner on day 5 and was still high on day 14. The experiment using anti-PLT antibody showed that the increased PLT count was not simply due to a rebound phenomenon after the transient decrease in PLT. The effect of i.v. FK565 was studied in mice myelosuppressed with a single dose of mitomycin C (MMC) (5.6 mg/kg). The fall in PLT count was suppressed on day 7 by 0.1 and 1.0 mg/kg FK565. Although intact cells or tissues are necessary for an increase in PLT following FK565 treatment, FK565 suppressed the impaired hematopoietic function seen after chemotherapy. FK565 is proposed as a drug to restore reduced neutrophil and platelet counts found in AIDS or cancer therapy.

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An improved method for the purification of human erythropoietin with high in vivo activity from urine was developed. This method involved ion-exchange, gel permeation, affinity chromatography, and reverse-phase chromatography but did not involve any stabilizing procedures. The purified human urinary erythropoietin showed a single broad band with a molecular weight between 37000 and 39000 Da on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and had an in vivo specific activity of 160000 IU/mg comparable to that of human erythropoietin produced in recombinant Chinese hamster ovary cells. We found that omission of the phenol treatment and ethanol precipitation which are usually used in the purification of human urinary erythropoietin greatly improved the biological activity of the final product. Phenol treatment followed by ethanol precipitation did not affect the amino acid composition but decreased the apparent molecular weight and N-acetylglucosamine content of human urinary erythropoietin. These findings suggest that phenol treatment followed by ethanol precipitation does not restore erythropoietin with highly branched sugar chains which would have high in vivo specific activity as reported previously (M. Takeuchi, et al. (1989) Proc. Natl. Acad. Sci. U.S.A., 86, 7819-7822).

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HeLa cells were synchronized by double thymidine-block and allowed to grow after removal of thymidine. Three proteinases, tryptase 17 : 17, proteinase In and late G2 proteinase, were prepared from the HeLa cells harvested at the time when each proteinase appeared in the cell cycle of the cells. All of them were suggested to be trypsin-like serine proteinases, because they hydrolyzed trypsin-specific fluorogenic substrates and their activities were inhibited by benzamidine, soybean trypsin inhibitor, leupeptin, tosyl-L-lysine chloromethan (TLCK) and diisopropylfluorophosphate (DEP). However, the actions of these proteinases on the substrates and inhibitors suggested that they were three different proteinases. They were strongly inhibited by 4-tert-butylphenyl and biphenyl esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, amidinopiperidine-4-acetic and 4-propionic acids, which retard the second DNA synthetic (S) and mitotic (M) phases for 3 h, 4-tert-butylphenyl ester of amidinopiperidin-4-carboxylic acid, which blocks initiation of S phase, the ester of amidinopiperidine-4-butyric acid, which suppresses the second S and M phases, and the esters of trans-4-amidinocyclohexanecarboxylic and 4-propionic acids which inhibit M phase.

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Amino acid substitutions were examined to increase the stability of the mutant human lysozyme C77/95A by filling the cavity created by this mutation. To modulate the cavity with hydrophobic amino acids or by the formation of a hydrogen bond, five amino acid-substituted mutants, C77AC95L, C77AC95I, C77LC95A, C77IC95A and C77/95S, were designed and constructed based on computer graphics investigations for stabilizing the mutant protein. The values of the melting temperatures, T_m, at pH 3.0 of the two mutant proteins C77LC95A and C77/95S were increased by 2.9°C and 2.3°C, respectively, as compared to that of C77/95A. The C77IC95A and C77AC95L proteins showed almost the same stability as C77/95A. The increase in the stability of the proteins might be explained by the filling of the cavity space around positions 77 and 95 with the side residue of Leu77 in C77LC95A, and by the formation of a hydrogen bond between Ser77 and Ser95 in C77/95S. On the other hand, the substitution with isoleucine at 95 (C77AC95I) decreased the stability. The activities of the five mutant proteins against the synthetic substrate, p-nitrophenyl tetra-N-acetyl-β-chitopentaoside, were higher than that of the wild-type human lysozyme, while the lytic activities against M. lysodeikticus were decreased in C77LC95A and C77IC95A, and increased in C77AC95L.

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Intravenous administration of the carboxamide-methylated light chain (G₁L) from human serum IgG induced tumor necrosis factor (TNF), one of the inflammatory cytokines, in the serum of mice. Human serum IgG, however, showed no induction of TNF in the serum. G₁L showed a potent anti-inflammatory effect in carrageenin-induced paw edema, and the anti-inflammatory effect of G₁L was significantly blocked by the concomitant administration of anti-TNF antiserum, but TNF alone had no anti-inflammatory effect.

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The effects of allopurinol and 4-aminopyrazolo [3, 4-d] pyrimidine (4APP) on adenine-induced renal injury in mice were examined. Plasma urea nitrogen (UN) and creatinine levels increased after the oral administration of adenine to mice. However, plasma UN and creatinine levels decreased inversely with the dose of 4APP when a different dosage of 4APP was administered together with adenine. Yet 4APP did not have any effect on the UN or creatinine levels when 4APP was administered after adenine administration. Plasma UN and creatinine levels increased in the allopurinol-administered group as in the adenine-administered group. Moreover, from light microscopic observation by hematoxylin-eosin staining, microvacuolic changes in the proximal tubuli were detected in the mouse kidney in the adenine-administered group, and epithelial cell loss, degeneration and microvacuolic changes in the proximal tubuli were observed in the mouse kidney in the adenine-and-allopurinol-administered group. However, there were no changes in the proximal tubuli in the mouse kidney in the adenine-and-4APP-administered group. These findings suggested that 4APP inhibits the action of adenine in the mouse kidney.

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The effects of various metal chelators on endothelin (ET)-converting enzyme (ECE) activity were examined in vitro. Three chelators, 2, 3-dimercapto-1-propanol (DMP), toluene-3, 4-dithiol (TDT) and 8-mercaptoquinoline (8-MQ), were found to dose-dependently inhibit ECE activity, but this inhibition was much weaker compared with EDTA. In the presence of Zn²⁺, the inhibitory activity of all these compounds, including EDTA, was abolished. The addition of Ca²⁺ and Mg²⁺ markedly attenuated the inhibitory activity of EDTA, but the other three chelators were still able to inhibit ECE. ECE, once inactivated by EDTA or 8-MQ, was reactivated by the addition of divalent cations such as Zn²⁺ and Mn²⁺. These compounds also inhibited angiotensin-converting enzyme activity in a manner similar to the inhibition exhibited towards ECE. Chelate-titration indicated that DMP, TDT and 8-MQ chelate Zn²⁺ but not Ca ²⁺ and Mg²⁺. These results suggest that the ECE inhibition exhibited by these compounds is mainly attributable to their chelating activities. The metal-selective chelating activity by DMP, TDT and 8-MQ may contribute to the retention of ECE inhibition in the presence of Ca²⁺ and Mg²⁺.

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The effects of metal chelators on endothelin (ET)-converting enzyme (ECE) activity in vivo were examined. Three compounds, (2, 3-dimercapto-1-propanol (DMP), toluene-3, 4-dithiol (TDT) and 8-mercaptoquinoline (8-MQ)), which inhibited ECE in in vitro studies, exhibited inhibitory activity towards big ET-1-induced sudden death in mice, while EDTA did not. Similar results were obtained in big ET-1-induced hypertension. Big ET-1-induced hemoconcentration was inhibited by pretreatment with 8-MQ or EDTA but not with DMP or TDT. The elevation of immunoreactive ET-1 (IR-ET-1) in plasma after administration of big ET-1 was inhibited by pretreatment with the three compounds but not by EDTA. On the other hand, no chelator inhibited the elevation of IR-ET-1 in lung tissue after injection of big ET-1. Taking into consideration the in vitro results, more selective chelating activity of the compounds towards Zn²⁺ rather than Ca²⁺ and Mg²⁺ may contribute to the inhibition of big ET-1-induced responses in vivo. The ET-1 formation involved in big ET-1-induced hemoconcentration may have different physiological characteristics from that involved in big ET-1-induced sudden death or hypertension.

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The acute effects of an alcohol extract of Crocus sativus L. (CS-extract) were studied on learning and memory in step through (ST) and step down (SD) tests in normal as well as in learning-and memory-impaired mice. A single oral administration of CS-extract had no effects on memory registration, consolidation or retrieval in normal mice. CS-Extract reduced the ethanol-induced impairment of memory registration both in ST and SD tests and the ethanol-induced impairment of memory retrieval in SD test. CS-Extract decreased the motor activity (MA) and prolonged the sleeping time induced by hexobarbital. These results suggested that CS-extract ameliorates the impairment effects of ethanol on learning and memory processes, and possesses a sedative effect.

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The inducibility of chromosomal aberration was studied using five mammalian topoisomerase inhibitors in Chinese hamster lung fibroblastic cells (CHL) in two protocols involving 6 and 24 h treatment, with respect to the mechanism of chromosomal aberration. In the 6 h treatment plus 18 h recovery culture (6 h treatment), a high incidence of chromosome-type aberration was observed with three mammalian topoisomerase II (topo-II) inhibitors : etoposide, adriamycin and mitoxantrone, which stabilize the cleavable complex of topo-II and DNA. In the 24 h continuous treatment (24 h treatment), these chemicals induced mainly chromatid-type aberrations. The chromosome-type aberrations which appeared as a result of the 6 h treatment were primarily induced, because the majority of aberrant cells induced by the inhibitors remained in the first metaphase, as determined by the 5-bromodeoxyuridine labelling method. This also indicates that the cells were sensitive to these topo-II inhibitors at the G₁-S boundary phase. Aclarubicin, however, which does not form the cleavalbe complex but is an antagonist against topo-II, and camptothecin, an inhibitor of topoisomerase I, mainly induced chromatid-type aberrations, irrespective of the duration of treatment. These results suggest that the differential induction of chromosomal aberrations by topoisomerase inhibitors reflects different mechanisms of inhibition by the topoisomerases, and that DNA double strand breakages promoted by the stabilization of the cleavable complex formed by topo-II are closely associated with the induction of chromosome-type aberration.

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λDNA strand breaks were easily induced in a reaction system involving alloxan with reduced glutathione (GSH) in the presence of FeCl₃ in a HEPES-NaOH buffer, pH 7.4. Increasing concentrations of FeCl₃ in the reaction system caused DNA strand breaks in a concentration-dependent fashion, suggesting that iron is required to induce the DNA strand breaks. Catalase, scavengers of hydroxyl radicals (HO·) and iron-chelators almost completely inhibited the DNA strand breaks, but superoxide dismutase (SOD) did not do so, suggesting that the HO·, formed by a Fenton-type reaction, was the species responsible for the DNA strand breaks. The addition of FeCl₃ to the solution containing DNA caused the formation of a DNA-Fe (III) complex, in which Fe (III) was reduced by an alloxan radical (HA·) but not by a superoxide radical. Only when apotransferrin was added to the reaction mixtures before the addition of FeCl₃, were both the DNA strand breaks and the reduction of Fe (III) strongly inhibited. These results suggest that the Fe (III) bound to DNA catalyzes the DNA strand breaks which may be caused by the generation of site-specific HO· via an HA·-dependent Fenton-type reaction.

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The present study was carried out as an approach from intracellular Ca²⁺ to clarify the preventive effects of a traditional Chinese medicine, Sho-saiko-to (Kampo prescription, TJ-9), against various metabolic disorders during endotoxemia. In this experiment, we estimated the cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) in liver single cells using a photonic microscope system. The [Ca²⁺]_i in a single liver cell of endotoxin (6 mg/kg, i.p.)-injected mice was greater at 18 h post-intoxication than that in the control, whereas the administration of endotoxinin to TJ-9 (500mg/kg/d, p.o.)-pretreated mice resulted in a markedly lower level of [Ca²⁺]_i than that in endotoxin-treated mice. In the mice pretreated with TJ-9, the Ca²⁺-ATPase activity in liver plasma membrane 18 h after endotoxin injection was markedly increased as compared to that in the endotoxin-treated mice. By contrast, the Ca²⁺-ATPase activity in liver mitochondria was lower in endotoxemic mice pretreated with TJ-9 than in mice given endotoxin alone. State 3 respiration and the respiratory control index (RCI), which are the parameters of mitochondrial function, were 41% and 35% lower, respectively, in the liver of mice given endotoxin than those levels in the control. However, the levels of state 3 and RCI in endotoxin-TJ-9-treated mice were markedly increased as compared to those of the endotoxin-treated mice. These findings suggest the protective effect of TJ-9 in the damage of liver mitochondrial function in endotoxin-poisoned mice. Kampo prescription Sho-saiko-to may protect some of the the various metabolic disorders caused by the change in Ca²⁺-mobility in the ischemic state of tissues during endotoxemia.

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It has been reported that antipsychotic drugs such as phenothiazine derivatives, dibenzyl derivatives and others with calmodulin antagonism induce cataracts, which are generically called phenothiazine-induced cataracts. In the ophthalmologic field, the mechanism of cataractogenesis by such drugs is a problem urgently requiring a solution. This paper reports the relationship between the properties of such drugs and cataract formation. In this experiment, phenothiazine derivatives (chlorpromazine, trifluoperazine, prochlorperazine and perphenazine), dibenzyl derivatives (imipramine and amitriptyline), and other drugs (cyproheptadine and calmidazolium) were investigated. As a result, it was clarified that phenothiazine derivatives and calmidazolium have high lipophilicity, strong inhibition activity against phosphodiesterase, and a large permeability constant, and that those drugs induce high levels of Ca²⁺ accumulation in the lens. It was also revealed that those drugs were distributed only at the peripheral region of the lens after they had penetrated into the lens. From these findings, we inferred that the cataract formation may be caused by lens protein aggregation followed by the inactivation of calmodulin-dependent Ca-ATPase and subsequent Ca²⁺ accumulation in the lens. Furthermore, we have been convinced that it is necessary to lower at least the lipophilicity of these drugs to suppress the side effect of antipsychotic drugs inducing cataractic formation.

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The macrocyclic polyamines, cyclen and cyclam, and their derivatives have been tested for inhibitory activity against the cytopathogenic effect (CPE) of human immunodeficiency virus type 1 strain HTLV-III_B (HIV-1_IIIB) on CD4⁺ human lymphoblastoma MT-4 cells. Cyclam and its derivatives were complexed with a variety of transition metal ions Ni^II, Zn^II, Cu^II, Fe^III and Co^III. The divalent metal complexes effected lower toxicity and greater anti-HIV-1 activity, while the trivalent metal complexes had no effect on HIV-1-dependent CPE. When dimerized, the anti-HIV activity of monomers was significantly enhanced. A potent inhibition of CPE by biscyclam was transiently observed 4 d after the virus infection, but was not seen at 6 d due to severe toxicity. The toxicity of biscyclam, referred to as delayed toxicity, could be overcome by a metal complexation. The strain specificities of biscyclams were further studied by testing their effects on syncytium formation between HIV-infected and uninfected human acute lymphoblastic leukemia MOLT-4 cells. The 50% inhibitory concentrations of biscyclams against HIV-2_GH-1-dependent syncytium formation were less than one hundredth those for the other HIV strains (HIV-1_IIIB, HIV-1_RF and HIV-1_SF-2).

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A number of hydroxychalcones were synthesized to evaluate their protective effects against oxidative cell damage and the production of superoxide anion. The hydroxychalcones which have a 3, 4-dihydroxycinnamoyl structure were potent inhibitors of lipid peroxidation in rat liver microsomes. In particular, we found that 2', 4', 3, 4-tetrahydroxychalcone (3) exhibited a potent inhibitory effect on H₂O₂-induced hemolysis due to an antioxidant effect. In addition, this compound strongly inhibited CCl₄-induced cytotoxicity in primary cultured hepatocytes and substantially decreased the production of superoxide anion by rat peritoneal exudate macrophages.

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Little information is available concerning the neurotoxicity of ioversol injected in animals. The effects of ioversol and other contrast media as well as vehicle control on the blood-brain barrier (BBB) were examined by injection into the left carotid artery of Mongolian gerbils 1 min prior to the i.v. injection of [8-¹⁴C] dopamine. The animals were sacrificed 1 min after the i.v. injection to prepare the frontal brain sections following the stereotaxic atlas. No uptake of the labeled substance was observed when the vehicle control and ioversol were used, suggesting that exposure to ioversol does not damage the BBB. Both meglumine sodium amidotrizoate and iohexol caused damage to the BBB, as shown in the cerebral cortex, brain stem, and hippocampus sections in the left semisphere. Iopaidol induced a much less but significant effect. Our results suggest that ioversol is an useful contrast medium.

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A methanolic extract (CM-ext) from Corydalis tuber (Corydalis turtschaninovii BESSER forma yanhusuo Y. H. CHOU et C. C. HSU) has been screened for activity in experimental models of inflammation. CM-ext (200 or 500 mg/kg, p.o.) inhibited an increase in vascular permeability in mice induced by acetic acid, and reduced acute paw edema in rats induced by compound 48/80 or carrageenin. CM-ext suppressed the development of adjuvant-induced edema in arthritic rats. CM-ext and its alkaloidal components, dehydrocorydaline, d-glaucine and l-tetra-hydrocoptisine inhibited compound 48/80-induced histamine release from peritoneal mast cells of rats. Since these substances from C. tuber were found to be effective in both the acute and chronic phases of inflammation, the crude drug C. tuber can be considered to exert anti-inflammatory activity.

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Various crude drugs were examined for their tyrosinase inhibitory activity. Marked activity was observed in Chouji and Yakuchi extracts and the active substances in these extracts were identified as eugenol and yakuchinone A, respectively. Other vanillyl compounds such as ferulic acid, curcumin and yakuchinone B also had higher activities than eugenol or yakuchinone A and inhibited the enzyme competitively. The presence of the hydroxyl group at the 4 position of the aromatic ring of the cinnamoyl moiety and the α, β-unsaturated carbonyl conjugated with an aromatic ring in these substances may play important roles in the competitive inhibition of tyrosinase.

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This study was performed to determine the active constituents of the root of Panax ginseng C. A. MAYER in the amelioration of ethanol-induced impediment of brain growth in the neonatal stage. To establish an animal model of the brain growth impediment caused by ethanol, ethanol (6 g/kg s.c.) was administered to rat pups on postnatal day 6, which corresponded to the third trimester of pregnancy for humans. Brain weight, especially cerebellar weight, was significantly reduced in the ethanol-exposed pups. In contrast, neither separation from dams nor pentobarbital treatment affected brain weight. A saponin fraction of ginseng extract prevented this ethanol-induced reduction of brain weight. Some ginseng saponins including ginsenosides Rg₁, Rb₂, Rd, Rf and Re effected stimulated a potent recovery of cerebellum growth in this animal model.

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To evaluate the effect of introducing a saccharide moiety to poly (amino acids) on tissue distribution, several glycoconjugates of ε-(2-methoxyethoxyacetyl)-poly (L-lysine) of three molecular weights were synthesized using an octylene spacer between the sugar and polymer chain. Methoxyethoxyacetylation of the ε-amino group of the lysine unit in poly (L-lysine) was useful for avoiding nonspecific distribution to many tissues as the result of cationic charges. The tissue-targeting ability of each saccharide moiety was considered as the actual amount changed in each tissue caused by saccharide modification. Galactose terminated saccharides such as galactose, lactose and N-acetylgalactosamine accumulated exclusively in the liver, probably by the hepatic receptor. These conjugates could therefore be good carriers for a drug delivery system to the liver. On the other hand, the mannosyl and fucosyl conjugates were preferentially delivered to the reticuloendothelial systems such as those in the liver, spleen and bone marrow. In particular, fucosyl conjugates accumulated more in the bone marrow than in the spleen. Xylosyl conjugates accumulated mostly in the liver and lung. Generally, the accumulated amount in the target tissue increased with increasing molecular weight and an increased number of saccharides on one molecule of polymer.

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The elimination of dexamethasone sodium m-sulfobenzoate, DMSB, following intravitreal injection, was measured in rabbit vitreous body under in vivo and in vitro conditions. The rate of elimination in vivo was appreciably greater than that in vitro, indicating that the in vivo data include not only the elimination due to metabolism/degradation in the vitreous humor, but also the elimination through the surrounding tissues such as the posterior aqueous humor, the retina/choroid/sclera membrane, and the lens. A general mathematical model based on Fick's second law of diffusion was developed for describing the pharmacokinetics of the intravitreal injection of DMSB. The model parameters were independently determined from a set of in vitro experiments. The in vivo data of elimination of DMSB following intravitreal injection agreed with the profiles calculated from the mathematical model, together with the model parameters determined from the in vitro experiments. The present in vivo/in vitro correlation, with the help of computer simulation, can be used for optimizing the therapeutic systems of intravitreal drug delivery.

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The effects of the force of contraction in the gastrointestinal (GI) tract and of food on drug release were investigated. Two dosage forms were administered orally to beagle dogs. Dosage form A was a matrix in which drug release was affected by the force of contraction in the GI tract, and dosage form B was not affected by the contraction force. The in vitro dissolution curves from dosage forms A and b using the paddle-beads method were similar to in vivo drug release, with the respective release of up to at least 50%. The in vitro release by means of the paddle-beads method was characterized by the in vivo drug release. The in vivo drug release profile from dosage form A was affected by food. A 1 : 1 relationship between in vivo and in vitro release was observed up to 6 h in those dogs under the fed condition, but only up to 2.5 h in those under the fasted condition. In the earlier stage of oral administration there was no difference in drug release profile between dogs of the fed and fasted conditions ; however, the drug release rate in the fed condition was faster than that in the fasted condition 2-4 h after administration. It was thus assumed that in the fed condition, drug release from dosage form A was affected by gastro-duodenal transit time or by the different environment in the distal parts of the GI tract rather than by the force of contraction.

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The renal disposition characteristics of superoxide dismutase (SOD) and its derivatives, including macromolecular conjugates with polyethylene glycol and carboxymethyl-dextran, a cationized derivative, and glycosylated derivatives with galactose and mannose, were studied in the isolated perfused rat kidney. Renal disposition processes, such as glomerular filtration, tubular reabsorption, and uptake from the capillary side, were quantitatively determined by single-pass indicator dilution experiments under filtering and nonfiltering kidney conditions. Native SOD had a high glomerular filtration rate (40% of that of inulin) and was effectively reabsorbed in the tubules, while no significant uptake was observed from capillary side. Macromolecular conjugates showed restricted glomerular filtration due to an increase in molecular size. Cationization of SOD greatly enhanced its association with the tissue, not only from the luminal side but also from the capillary side, based upon electrostatic interaction. Galactosylated and mannosylated SOD showed reduced tubular reabsorption and increased exposure of the luminal surface to the enzyme. In addition, a small but significant uptake of mannosylated SOD from the capillary side was observed. This uptake was dose-dependent and completely inhibited by mannan, suggesting that mannose receptor-mediated endocytosis existed in the capillary side of the kidney. Thus, we can manipulate the renal disposition profiles of SOD by changing its physicochemical or biological properties through chemical modification.

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Rectal absorption of ozagrel (a selective thromboxane synthetase inhibitor) from suppositories was studied in rabbits. Two kinds of suppositories were prepared, one was from ozagrel powder (OPS, ozagrel powder suppository), and the other from ozagrel tablet (OTS, ozagrel tablet suppository). Ozagrel from OPS was absorbed quickly with a T_max value of 26.3±7.5 min, and the peak plasma level was significantly higher than that involving intravenous infusion or oral dose (50.3±6.7 vs. 32.8±5.4 or 9.9±1.2 μg/ml), indicating that OPS may be a useful dosage form rather than injection. OTS was absorbed less rapidly than OPS, but the AUCs of both suppositories were similar. Because OTS was prepared using an ozagrel tablet, it is fairly easy to get an equal content in each suppository. Therefore, OTS is not only an experimentally interesting dosage form, like OPS, but is also a practical preparation for clinical use.

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Quantitative relationship between chemical structure and oral bioavailability of 188 non-congeneric organic medicinals were studied to construct an expert system for predicting pharmacokinetic properties of organic chemicals. The compounds studied were classified into three groups : non-aromatics, aromatics, and heteroaromatics. Their oral bioavailability data observed in human adults were allotted into three ratings, and the relationships with chemical structure were analyzed using fuzzy adaptive least-squares. Quantitative relationship models formulated for the three structure groups gave significant information about factors influencing bioavailability, and were statistically reliable in both recognition and leave-one-out prediction despite the diversity and complexity of the structures of the compounds investigated.

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The pharmacokinetics of recombinant human insulin-like growth factor-I (rhIGF-I) was examined in normal and hypophysectomized (Hypox) rats after i.v. administration. The plasma concentration of rhIGF-I administered i.v. (0.32, 1.0 and 3.2 mg/kg) declined biexponentially in both normal and Hypox rats. Half-lives of the β-phase were not significantly different among the doses examined in both animal groups, but were shorter in Hypox rats. In Hypox rats, the values of the area under the plasma concentration-time curve, the mean residence time, the variance of residence time and the apparent volume of distribution at steady-state decreased, while the total body clearance (CL_total) increased. The distribution of rhIGF-I after i.v. administration (1.0 mg/kg) was examined in normal rats. High distribution to the kidney was observed at early time points (5 min and 1 h), but no significant distribution was found in other tissues. The ligation of renal vasculature greatly reduced the CL_total, suggesting that the kidney is the main elimination organ. In spite of the rapid distribution of rhIGF-I to the kidney, the urinary excretion of intact rhIGF-I was negligible. Thus, the metabolism of rhIGF-I in the kidney was examined in vitro, and the results showed extensive metabolism in the brush border and lysosomal fractions of tubular cells. In the plasma of normal rats, rhIGF-I formed the 50 kDa complex first, and the 150 kDa complex was formed slowly. However, since unbound rhIGF-I and the 50 kDa complex were eliminated rapidly through the kidney, most of the rhIGF-I in the plasma was present as the 150kDa complex later on. The lack of 150kDa complex in the plasma of Hypox rats might be the reason why the CL_total of rhIGF-I is greater in Hypox rats than in normal rats.

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The effects of eight prospective absorption enhancers on the nasal mucosa in rabbit have been assessed using an in vitro Ussing chamber technique. Sodium taurodihydrofusidate (STDHF), sodium deoxycholate (DC), polyoxyethylene-9-lauryl ether (BL-9), lysophosphatidylcholine (LPC) and sodium dodecyl sulfate (SDS) were found to possess relatively high protein leaching activity, while sodium glycocholate (GC), sodium taurocholate (TC) and EDTA had relatively low activity. The permeation of fluorescein isothiocyanate-labeled dextran (FD, M.W. 9400) as a model drug across the nasal mucosa was found to be greater in the presence of these enhancers. Their enhancement ratio was found to be in the order of BL-9>STDHF>SDS>LPC>DC>EDTA>GC>TC, which correlated with the protein leaching activity. The differences in protein leaching and enhancement ratio dependent on the magnitude of change of membrane resistance (△Rm), indicating that these enhancers damaged the membrane and increased FD permeation. △Rm thus appears to be a useful indicator by which one can estimate nasal mucosa damage by the enhancers.

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The pharmacokinetics of zonisamide was studied using routine therapeutic drug monitoring data from 68 epileptic patients. The 266 serum concentration data at steady-state after repetitive oral administration were analyzed using the nonlinear mixed effects model (NONMEM) program designed for estimation of population pharmacokinetic parameters. A one-compartment model with dose-dependent clearance was used for the pharmacokinetic analysis of zonisamide. The volume of distribution (V) was estimated to be 1.27 l/kg in a typical 33-kg patient, assuming that the bioavailability of orally administered zonisamide is 100%. The maximal daily dose to be cleared (V_max) and the concentration giving half maximal clearance (a Michaelis-Menten constant) was 27.6 mg/d/kg and 45.9 μg/ml, respectively. The parameter of a power function of weight to adjust V and V_max was estimated to be 0.741. In addition, V_max for zonisamide appears to be 13% increased in patients receiving carbamazepine concurrently. The population pharmacokinetic parameters of zonisamide will be useful for designing dosage regimens in epileptic patients.

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Profiles of absorption versus drug molecular weight and absorption versus drug lipophilicity were investigated in both the small and large intestines of rats by an in situ loop method. The absorption-molecular weight profiles examined using different-sized polyethylene glycols (PEGs) were different between the small and large intestines ; the large-intestinal absorption of PEGs with molecular weights larger than 300 was poor, while PEGs with molecular weights up to 600 were relatively well absorbed in the small intestine. It is suggested that the paracellular route for drug penetration in the large intestine is restricted more than in the small intestine. The absorption-lipophilicity profiles were also examined in various regions (loops of 6 cm) of rat intestine using three acylsalicylic acids, acetyl-, propionyl-and butyrylsalicylic acids. The absorption rates of the acylsalicylic acids were different in the intestinal regions : the jejunum>the ileum>the colon>the rectum. In each region, the absorption rate increased with the drug lipophilicity. However, it was shown that the absorption rates in the small intestine tended to reach a ceiling at the high lipophilicity. To confirm this tendency, the absorption rates of acetaminophen and indomethacin were compared in the four intestinal regions. The absorption rates of highly lipophilic indomethacin were similar in the large and small intestines, while intermediately lipophilic acetaminophen was more rapidly absorbed in the small intestine than in the large intestine. A thicker unstirred water layer adjacent to the small-intestinal mucosa would be one of the factors which cause such varying absorption-lipophilicity profiles.

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A direct radioimmunoassay for the determination of E6123, a novel antagonist of platelet activating factor (PAF) receptor, was developed in order to study the pharmacokinetics at low dose. This procedure used [³H] E6123 as the radioligand and an antiserum obtained from rabbits immunized with the hapten covalently bound to bovine serum albumin. M1B, one of the main metabolites of E6123, exhibited cross-reactivity with antisera. But this metabolite had no effect on measurements of E6123, because the amount of M1B in plasma radioactivity after administration of [¹⁴C] E6123 to dogs and monkeys was low. The sensitivity limit of this assay was 25 pg/ml of plasma when 0.1ml of plasma was used and the assay showed good accuracy and high precision. The validity of the radioimmunoassay was demonstrated by comparative analysis of a number of samples after oral and intravenous administration (1.0 mg/kg) by HPLC-UV method (r=0.972-0.984, slope=1.0314-1.2143). The pharmacokinetics of E6123 was studied at a dose of 30 μg/kg. After intravenous administration, the plasma concentration-time curves in all species fitted a two-compartment model and the terminal half-lives in guinea pigs, dogs and monkeys (both poor and extensive metabolizers) were 4.77, 1.71, 5.34 and 1.07 h, respectively. After oral administration, the maximum plasma concentrations were obtained within 0.83-3.00 h and the half-life for each animal was almost the same as that after intravenous administration. The mean bioavailabilities of E6123 in guinea pigs, dogs and monkeys (poor and extensive metabolizers) were 106.9, 45.7, 59.1 and 22.8%, respectively.

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A study was conducted to explore whether a positive correlation between serum and salivary concentrations of the well-known antiepileptic drug, valproic acid (VPA), in epileptic patients could be explained by facilitated diffusion. The total concentration in saliva (C_s) would be related to the apparent ratio (R_app=100·C_s/C_t) of C_s to the total concentration in serum (C_t) as follows : C_s=A·R_app+B. This equation can be illustrated with microcomputer-simulated figures by assuming a process of facilitated diffusion for the transport of VPA into saliva from blood by the mechanism of monocarboxylic acid absorption through the intestinal brush-border membrane vesicles. The above equation has been proved to be valid when applied to the data reported separately by Gugler and coworkers and by Nitsche and Mascher, who evaluated the pharmacokinetics of VPA. Moreover, we can estimate the serum concentration with the salivary concentration using the above equation.

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The capability of β-alanyl-L-histidinato zinc (AHZ) to increase alkaline phosphatase activity in the femoral diaphysis from elderly rats was investigated. The femoral-diaphyseal tissues were removed from weanling (3-week-old) and elderly (10-month-old) female rats. Bone tissues were cultured in Dulbecco's modified Eagle medium (high glucose, 4.5%) supllemented with antibiotics and bovine serum alubumin. Among various other bone-stimulating factors (AHZ ; 10^-5M, zinc sulfate ; 10^-4M, sodium fluoride ; 10^-3M, insulin ; 10^-8M, and β-estradiol ; 10^-9M), AHZ had a potent effect on increasing alkaline phosphatase activity in the diaphyseal tissues from both rat groups. In the bone tissues from elderly rats, the effect was concentration dependent (10^-7-10^-5M). At 10^-5M the effect of AHZ was seen for a longer time during 72-h culture, although the zinc sulfate (10^-5M) effect was no longer. The effect of AHZ to increase bone alkaline phosphatase activity was completely abolished by the presence of cycloheximide (10^-6M). AHZ thus appears able to directly stimulate alkaline phosphatase activity dependent on protein synthesis in the bone tissues from elderly rats.

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We investigated the age-dependency of serum levels of 9 kinds of proteases. The results showed that the enzymatic activities corresponding to urokinase, plasmin, and thrombin, all involved in blood clotting and fibrinolysis, were inversely correlated with age. This suggests that there is some similarity between the normal process of aging and the pathologic process of Alzheimer's disease. Compared with our previous data on Alzheimer patients, the present results indicate that some derangement in the aging process is involved in the pathogenetic mechanisms of Alzheimer's disease.

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The expression in Escherichia coli of a tet (K) gene, which originated in Staphylococcus aureus, was studied. The minimum inhibitory concentration (MIC) of tetracycline (TC) for E. coli cells that carried the tet (K) gene was only slightly higher than that for the recipient cells. This result indicated that the level of expression of the tet (K) gene in E. coli was very low. Insertion of a lac promoter into the upstream region of the tet (K) gene resulted in a slight increase in the MIC, from 12.5 to 50 μg/ml in the presence of isopropyl-β-D-thiogalactopyronoside. An altered tet (K) gene, in which the initiation codon and the ribosome-binding site (RBS) were changed from TTG to ATG and from GAGG to GGAGG, respectively, and in which the distance between the RBS and the initiation codon was increased from 4 to 11 bases, was associated with high-level resistance to TC, with a MIC of 200 μg/ml. The MIC resembles that associated with expression of the tetA (B) gene of E. coli. These results indicate that the barrier to expression of the tet (K) gene in E. coli is located at the initiation of translation.

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The inhibitory effect of NaCl coadministration on the reabsorption of Li⁺ in the salivary ducts and the renal tubules was investigated in beagle dogs. Following the bolus intravenous administration of LiCl (0.145 meq/kg), 50 ml of NaCl solution (100, 200 meq/l) was administered orally each hour, and parotid saliva (Pr) and mandibular-sublingual saliva (MS) were collected under the continuous stimulation of salivation with citric acid. The renal clearance of Li⁺ increased with the increased concentration of administered NaCl solution. This could be a result of the competitive inhibition of the tubular reabsorption of Li⁺ by Na⁺. The salivary clearance of Li⁺ also tended to increase by the administration of NaCl. These results suggest the existence of a reabsorption process of Li⁺ in the salivary glands similar to that in the renal tubule. However, when the administered NaCl was increased from 100 to 200 meq/l, the renal clearance was further increased, but the salivary clearance was not. This suggests that the transport capacity of Li⁺ reabsorption is relatively small in the salivary glands, compared with that in the renal tubule, and that the inhibitory effect of NaCl on Li⁺ reabsorption had already reached a maximum level when 100 meq/l NaCl was administered.

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In the present experiment, we found that inhaling lavender oil vapour blocked pentetrazol-and nicotine-induced convulsion and electroshock convulsion in mice.

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