Gas phase derivatization of ammonia with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole and its application to urease assay. An ammonia-specific and rapid fluorometric method for determination of ammonia and urease activity was developed. The method is designed to assay ammonia levels or urease activity for the rapid diagnosis of Helicobacter pylori infection. 4-Fluoro-7-nitrobenzo-2-oxa-1,3-diazole was used to derivatize ammonia and 4-amino-7-nitrobenzo-2-oxa-1,3-diazole was analysed by high performance liquid chromatography at an excitation wavelength of 455 nm and an emission wavelength of 520 nm. Derivatization was designed to react with ammonia gas produced in a strong alkaline pH sample. The fluorescent intensity was linear in the range of 0.1-10 mM ammonia per tube when the reaction was carried out for 15 min at 37 degrees C. Urease activity, judged as the amount of ammonia production from urea, could be measured at 25 ng per tube (S/N = 1.5) with Jack bean meal urease. Because of its rapidity, this assay is potentially superior to the current standard method in use in clinical settings.


Effect of tetrahydropyranyladriamycin (THP-ADR) and 4'-epiadriamycin (4'-epi-ADR) on lipid peroxide levels in mice. Lipid peroxide levels were measured in mouse tissues to study their relation to the cardiotoxicity of anthracyclines. The effects of tetrahydropyranyladriamycin (THP-ADR) and 4'-epiadriamycin (4'-epi-ADR), which are less cardiotoxic than adriamycin (ADR), were examined. Neither THP-ADR nor 4'-epi-ADR increased the lipid peroxide levels in the heart, however, both increased reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent lipid peroxidation in mouse liver and lung microsomes, to the same degree as ADR. Therefore, the results obtained in vitro were not the same as those obtained in vivo. Of all of the anthracyclines tested, only THP-ADR increased the lipid peroxide levels in the lung. Thus, THP-ADR may be pulmotoxic. Differences in the activities of glutathione peroxidase and glutathione S-transferase reflected the differences in lipid peroxide levels.


Effect of bacteriohopane-32-ol on lipid metabolism in Hep G2 cells. To investigate the biological activity of the hopane group of pentacyclic triterpenes, the effect of bacteriohopane-32-old (Monol) on lipid synthesis and secretion was determined using Hep G2 cells. Despite its structural similarity to 25-hydroxycholesterol, Monol did not affect free and esterified cholesterol synthesis determined by the incorporation from [14C]acetate. Monol reduced the phospholipid secretion from Hep G2 cells without affecting cellular phospholipid synthesis from [3H]glycerol. It also decreased the secretion of apolipoprotein B. These results suggest that the Monol-induced reduction in phospholipid secretion is due to a decrease in the number of lipoprotein particles secreted from Hep G2 cells.


Influence of time of administration of a Shosaiko-to extract granule on blood concentration of its active constituents. Using a Shosaiko-to extract granule, we investigated the effects of the timing of administration (orally, before and after a meal) on the plasma concentration of its active constituents, glycyrrhizin (GL), baicalin, baicalein and glycyrrhizic acid (GA), a metabolite of GL. The pattern of plasma concentration change of GL differed between the two times of administration, and followed a two-phase pattern when the granules were taken before meals. There was no difference neither in the area under the plasma concentration-time curve (AUC) between the two periods, nor AUC itself. GA showed no difference in plasma concentration pattern, nor was baicalin detected in the plasma following administration by either method. The plasma concentration pattern of baicalein differed between the two timings but followed that of the two-phase pattern in each case. The plasma concentration pattern of active constituents of Shosaiko-to extract granule varied with the time of administration, but there was no significant difference in the maximum plasma concentration or AUC of active constituents depending on the administration. We concluded that the present clinical timing of administration of Shosaiko-to should be determined on the basis of patient compliance and other relevant factors.


Prodrugs of 2',3'-dideoxyinosine (DDI): improved oral bioavailability via hydrophobic esters. Five ester prodrugs of 2'3'-dideoxyinosine (DDI) were synthesized for the purpose of improving oral bioavailability. The prodrugs, acetate (C2-DDI), octanoate (C8-DDI), stearate (C18-DDI), benzoate (Bz-DDI), and hemisuccinate (Suc-DDI) were proved to quantitatively regenerate their parent drug by enzymatic hydrolysis. Though the chemical stability of the prodrugs under acidic conditions was not improved, their solubility in water was significantly decreased by esterification, except for Suc-DDI. Bioavailability was evaluated by oral administration to rats. Two hydrophobic prodrugs (C8-DDI and Bz-DDI) showed higher absolute bioavailability (23.5% and 31.0%, respectively) than did DDI (15.2%), though that of C2-DDI (11.5%) and Suc-DDI (4.5%) was poor.


New antitumor bicyclic hexapeptides, RA-XI, -XII, -XIII and -XIV from Rubia cordifolia. Four new bicyclic hexapeptides, named as RA-XI, -XII, XIII and -XIV were isolated from Rubia cordifolia and showed potent antitumor activities against P-388. The structures were elucidated from spectroscopic and chemical evidences. RA-XII, XIII and -XIV were proved to be unique bicyclic hexapeptidic glucosides.


Synthesis and aldose reductase inhibitory activity of triazine derivatives possessing acetic acid group. N-Acetic acid derivatives of 6-aryl-pyrazolo-triazin-4-ones were synthesized for evaluation as new aldose reductase inhibitors. The intrinsic activity of each compound was assessed by measuring the inhibition of enzymatic activity in an isolated pig lens enzyme preparation. All the prepared compounds exhibited a significant in vitro aldose reductase inhibitory effect (10(-6) M less than or equal to IC50 less than or equal to 10(-4) M). Furthermore, biological activity (log 1/IC50) for most of the data sets could be correlated directly to electronic and steric parameters. Finally, spatial configuration of the most active derivative 6c (IC50 = 2 x 10(-6) M) was compared with that of tolrestat and with pharmacophor requirements of the aldose reductase inhibitor site using a molecular modeling system.


Agents for the treatment of overactive detrusor. III. Synthesis and structure-activity relationships of N-(4-amino-2-butynyl)acetamide derivatives. A series of N-(4-amino-2-butynyl)acetamides were synthesized and examined for their inhibitory activity on detrusor contraction and mydriatic activity as an index of anticholinergic side effect. Among those compounds synthesized, (+)-2-cyclohexyl-N-(4-dimethylamino-2-butynyl)-2-hydroxy-2-phenylacet amide hydrochloride ((+)-13b.HCl), 2-cyclohexyl-2-hydroxy-N-(4-methylamino-2-butynyl)-2-phenylacetamide+ ++ hydrochloride (13c.HCl), N-(4-dimethylamino-2-butynyl)-2,2-diphenyl-2-hydroxyacetamide hydrochloride (14a.HCl), and 2,2-diphenyl-N-(4-ethylamino-2-butynyl)-2-hydroxyacetamide hydrochloride (14b.HCl) showed equipotent inhibitory activity on detrusor contraction to oxybutynin (1) and less mydriatic activity. Further evaluation of these compounds as an agent for the treatment of overactive detrusor has been examined.


Studies on cerebral protective agents. I. Novel 4-arylpyrimidine derivatives with anti-anoxic and anti-lipid peroxidation activities. Novel 4-arylpyrimidine derivatives were synthesized by the oxidation of 4-aryl-1,4-dihydropyrimidines, and their effects on anti-anoxic (AA) activity in mice and anti-lipid peroxidation (ALP) activity in rat brain mitochondria were investigated. Among these compounds, ethyl 6-methyl-2-phenyl-4-(4-pyridyl)-5-pyrimidinecarboxylate (4b) has AA activity (10 mg/kg, i.p.) and ethyl 6-methyl-4-(3-nitrophenyl)-2-phenyl-5-pyrimidinecarboxylate (4f) has ALP activity (73% inhibition at 10(-5) g/ml). The latter compound (100 mg/kg, i.p.) was also effective on arachidonate-induced cerebral edema in rats with comparable potency to that of vitamin E.


Novel benzamides as selective and potent gastrokinetic agents. IV. Synthesis and structure-activity relationships of 2-substituted 4-amino-N-[(4-benzyl-2-morpholinyl)methyl]-5-chlorobenzamides. A new series of 2-substituted 4-amino-N-[(4-benzyl-2-morpholinyl)methyl]-5-chlorobenzamides (4-39) including a few 4-fluorobenzyl analogues were prepared and evaluated for their gastrokinetic activity by determining their effects on the gastric emptying activity of phenol red semisolid meal in rats. The C-2 substituent comprises alkoxy and variously substituted alkoxy groups. Among the derivatives, 4-amino-N-[(4-benzyl-2-morpholinyl)methyl]-2-(n-butoxy)-5-chlorobenza mide (5), its 4-fluorobenzyl (6), and 3-methyl-2-butenyloxy analogues (22) were superior to cisapride and essentially equipotent to the 2-ethoxy analogue (1b, AS-4370 as its citrate) in gastrokinetic activity. These compounds, like AS-4370, had no dopamine D2 receptor antagonistic activity.


Synthesis of the optically active trans-isomers of diltiazem and their cardiovascular effects and Ca-antagonistic activity. Optically active trans-isomers of diltiazem were synthesized and their cardiovascular effects were evaluated in anesthetized dogs and in isolated guinea pig hearts. Both (+)-2 (2R,3S) and (-)-2 (2S,3R) were much less active than diltiazem (1, 2S,3S) with short duration of action. No substantial enantiomeric difference in activity was seen between them. Their Ca-antagonistic activities on Ca(2+)-induced contractions in K(+)-depolarized canine basilar arteries were also examined. Absolute stereochemistry of (+)-2 was determined to be 2R,3S by X-ray crystallographic analysis.


Synthesis and antitumor activity of fused quinoline derivatives. III. Novel N-glycosylamino-indolo[3,2-b]quinolines. Novel indolo[3,2-b]quinolines (1b-k), having a nitro, amino, acetamido, methanesulfonamido, or glycosylamino group at the 2, 7, or 8-position, were prepared and their antitumor activities against P388 leukemia in mice were examined. The 7-galactopyranosylamino derivative (1g) showed the most potent activity (optimal dose = 25 mg/kg, T/C greater than 333%, cure rate 5/6).


Imidazo[1,2-alpha]pyridines. III. Synthesis and bradycardic activity of new 5-imidazo[1,2-alpha]pyridin-6-ylpyridine derivatives. Structural modification of the cardiotonic agent, loprinone (E-1020, 1), suggested by data that it has a less positive chronotropic effect than milrinone (15), led us to find novel bradycardic agents that were structurally different from homoveratryl amine derivatives. Alkyl-oxy, -thio, and -amino derivatives at the 2-position of the pyridine ring of 1 produced bradycardic activity without a significant effect on blood pressure and myocardial contractility. Aryloxy analogues also decreased heart rate, and members with an electron-withdrawing group at the ortho position of the phenyl ring showed higher activity. Replacement of the imidazo[1,2-alpha]pyridine with pyridine resulted in diminished activity. The mechanism of bradycardic activity of these compounds seems to be direct action on the sinus node.


Contractile activity of porcine neuromedin U-25 and various neuromedin U-related peptide fragments on isolated chicken crop smooth muscle. Contractile activity of porcine neuromedin U-25 (p-NMU-25) and various neuromedin U (NMU) peptide fragment amides was examined on chicken crop smooth muscle preparation. The relative activity (expressed as RA value) of p-NMU-25 to porcine neuromedin U-8 (p-NMU-8) was 5.51 +/- 0.09, and p-NMU-25 (15-25) was the most potent fragment with an RA value of 7.78 +/- 0.05. All C-terminal 11-peptide amides of rat, rabbit, and frog NMU peptides retained activity about three-fold higher than the corresponding C-terminal 8-peptide amides. The peptide segment Asn15-Arg-Arg17 of p-NMU-25, as well as the corresponding positions of various NMU peptides: Ser13-Gly-Gly15 of rat NMU and Ser15-Arg-Gly17 of rabbit and frog NMUs, appeared to be involved in the structural requirements for increased contractile activity in the assay system.


The mutagenic constituents of Rubia tinctorum. Twenty compounds were isolated from the roots of Rubia tinctorum which are used as a commercial source of madder color. Among these compounds, mollugin (1), 1-hydroxy-2-methylanthraquinone (2), 2-ethoxymethylanthraquinone(11), rubiadin (13), 1,3-dihydroxyanthraqunone (14), 7-hydroxy-2-methylanthraquinone (16), lucidin (17), 1-methoxymethylanthraquinone (18) and lucidin-3-O-primeveroside (19) showed mutagenicity with Salmonella typhimurium TA 100 and/or TA 98. Since the mutagenic compounds isolated are anthraquinone derivatives with the exception of compound 1, structure-mutagenicity relationships of the anthraquinones were also studied. The results suggested that the greatest activity is exhibited by 1,3-dihydroxyanthraquinones possessing methyl or hydroxylmethyl group on carbon 2.


Selected ion monitoring for the determination of bromovalerylurea in human plasma. A gas chromatography-selected ion monitoring procedure with chemical ionization is described for the determination of bromovalerylurea (BVU) in human plasma. BVU was extracted with ether after addition of 2-bromo-2-methylpropylurea as an internal standard. The lower limit of BVU quantification by this method was 2 ng/0.1 ml plasma volume. This procedure was used to determine the sequential plasma levels of BVU in a human volunteer following a single oral dose of a commercial analgesic.


Production and specificity of anti-22-oxacalcitriol antisera. 22-Oxacalcitriol 3-hemiglutarate, a haptenic derivative of 22-oxacalcitriol, was synthesized to obtain a specific antibody for use in radioimmunoassay. Three antisera were elicited in rabbits against the hapten conjugated with bovine serum albumin, and their specificity was examined by cross-reaction study. One of the antisera was found to be satisfactorily specific, and expected to provide a radioimmunoassay useful for the pharmacokinetic study of 22-oxacalcitriol.


Colorimetric assay for lysozyme using Micrococcus luteus labeled with a blue dye, Remazol brilliant blue R, as a substrate. Micrococcus luteus (M. lysodeikticus) labeled with Remazol brilliant blue R (blue ML) was prepared as a novel substrate for the colorimetric assay of lysozyme. The treatment of the labeled substrate with lysozyme resulted in the release of soluble blue products which can be easily measured spectrophotometrically at 600 nm. The blue color was most efficiently released at pH 7 and ionic strength of 0.2 on incubation with hen lysozyme at 40 degrees C. A new colorimetric method for the assay of lysozyme using this substrate was developed. The assay system gave a linear dose-response curve, and as little as 0.1 microgram of human lysozyme (1 microgram/ml, 100 microliters) can be detected. The present method is more convenient and reproducible than the conventional lysozyme assay with bacterial cells. Application of the system to the determination of lysozyme in human serum is described.


Solubilization of limulus test reactive material(s) from Candida cells by murine phagocytes. Solubilization of limulus test reactive materials from Candida was examined in the presence or absence of phagocytic cells. Solubilized limulus test reactive materials (LTRM) were detected in culture supernatant, and hot water and sodium hydroxide extracts of the acetone dried cells of Candida parapsilosis. Suspensions of Candida cells also reacted with limulus test, and LTRM were released from the acetone dried cells by serum treatment. After treatment of the acetone dried cells with polymorphonuclear leucocytes (PMN) or macrophages (M phi), a significant amount of LTRM was solubilized. Significant amounts of LTRM were also released by PMN during treatment of live and growing C. parapsilosis. The reactivity of LTRM was completely inhibited by the addition of excess amount of purified (1----3)-beta-D-glucan, suggesting LTRM from Candida cells as described above would contain (1----3)-beta-D-glucan. These results suggested that LTRM during fungal infection would come from the extracellular water soluble polysaccharide fraction as well as the insoluble cell wall fraction solubilized by the action of phagocytes.


Biopolymers from marine invertebrates. XIII. Characterization of an antibacterial protein, dolabellanin A, from the albumen gland of the sea hare, Dolabella auricularia. An antibacterial factor, dolabellanin A, was purified from the albumen gland of a sea hare, Dolabella auricularia. Purified dolabellanin A was a glycoprotein of 250 kilodaltons consisting of 4 subunits, and showed both antibacterial and antineoplastic activities. The two activities were lost in parallel on heating and at low and high pH. This factor was half-maximally active for gram-positive and -negative bacteria at 0.018-0.48 microgram/ml, and its action was not bactericidal but bacteriostatic. Dolabellanin A did not induce morphological elongation of bacteria or the release of adenosine triphosphate, but it completely inhibited the syntheses of deoxyribonucleic acid (DNA) and ribonucleic acid by E. coli within 6 min. These results suggest that dolabellanin A, which is found in a marine invertebrate, the sea hare, is a new antibacterial protein, and that it exerts its action by inhibiting nucleic acid synthesis, as does a DNA-inhibiting chemotherapeutic drug.


Oily drug carriers in cancer chemotherapy. II. Preparation of viscous ethyl oleate for intraarterial infusion therapy and its disposition in rats and hamsters. Viscous ethyl oleate (VEO) was prepared as an oil drug carrier by the addition of aluminum stearate or ethyl cellulose. Since the rate of shear of VEO containing aluminum stearate was greatly and nonlinearly changed against the shearing stress compared to that containing ethyl cellulose, the latter was used for subsequent microvascular and organ distribution experiments in rats and hamsters. For infusion into the carotid artery in hamsters, neat ethyl oleate (EO, 4cP) or VEOs of various apparent viscosities (40, 80, 120 cP-VEOs) embolized the vascular system in the cheek pouch, although arrival time to the site where the embolization was observed and the embolization period differed depending on the type of oily drug carrier. For infusion into the hepatic artery in rats, however, only 120 cP-VEO embolized the vascular system in the liver. After infusion of the oily drug carrier containing 3H-oleic acid into the artery of hamster cheek pouch and rat liver, 30-50% of the radioactivity was gradually eliminated within 48 h, whereas about 80% of the dose was rapidly eliminated after infusion to rat stomach and kidney. In addition, the amount of 120 cP-VEO remaining in each organ 48 h after infusion was higher than those of EO and 40 and 80 cP-VEOs. Histological observation after infusion in rat liver revealed that 120 cP-VEO slowly migrated from the artery or arteriole to the sinusoidal capillary region. These results suggest that 120 cP-VEO can be used as a drug carrier because of its function of vascular embolization and high retention in a targeted tissue.


Thermodynamic aspects of fatty acids binding to human serum albumin: a microcalorimetric investigation. Thermodynamic parameters have been evaluated for the binding interaction between human serum albumin (HSA) and unbranched fatty acids (FFA) on the basis of a flow microcalorimetric measurement at pH 7.4 and 37 degrees C by computer-fitting to single- and two-class binding models. The heat of binding increased exothermically with increasing alkyl chain length. FFA with nine or less carbons bound to only one class of binding sites (n = 2) with a binding constant (K) of 10(4) M-1. FFA with ten or more carbons bound to the first class of binding sites with high affinity K in the older of 10(5) to 10(6) M-1, and to the second class with a lower affinity and high capacity. The free energy change of the first class of binding sites (delta G1) became more negative as the chain length of FFA was increased. The enthalpy change per mol of FFA (delta H) decreased at the rate of -7.47 kJ.mol-1.CH-1(2) to a minimum at C9 and then increased due to the hydrophobicity of alkyl chains. Compensation analysis for the i th class of HSA molecule by plotting molar changes of enthalpy (delta Hmi) against entropy (delta Smi) and free energy (delta Gmi) indicates two distinct binding sites. The first class (i = 1) of the long-chain FFA on HSA is an entropy-driven reaction associated with nearly constant values of delta Hm1 (-43.0 +/- 4.8 kJ.mol-1), slightly negative values of delta Sm1 (-47.4 less than or equal to delta Sm1 less than or equal to -8.1 J.mol-1.K-1) and -delta Gm1 values, increasing with increasing alkyl chain length. The second class (i = 2) of the long-chain FFA may lie in the same region as the binding sites of the short- and medium-chain FFA with a linear relationship between delta Hmi-delta Smi.


Effect of glycyrrhizinate on dissolution behavior and rectal absorption of amphotericin B in rabbits. The effects of dipotassium glycyrrhizinate (GLYK) on the dissolution behavior and bioavailability of amphotericin B (AMB) were investigated. The mixtures of AMB and GLYK were prepared at different molar ratios by lyophilization. Lyophilization resulted in amorphous AMB either alone or in the mixture. Dissolution rates of AMB of the mixtures were markedly faster than that of lyophilized AMB alone, which was followed by a decrease of dissolution. The initially-enhanced dissolution rate was likely to be due to the improvement of surface wettability of drug particles with GLYK rather than the amorphous state of AMB. A phase solubility study of AMB with GLYK indicated that the increasing solubility was caused by micellar solubilization. The in vitro release rate of AMB from suppositories containing the lyophilized mixtures was significantly accelerated by increasing the amount of GLYK. The rectal absorption of AMB from suppositories containing either the drug alone, a physical mixture or a lyophilized mixture was studied using rabbits. The absorption of the mixture (AMB/GLYK = 1/9) was about 35 times greater in the area under the serum concentration-time curve (0-24 h) than that of lyophilized AMB alone. These results suggest that GLYK is useful for improving the dissolution property of AMB and the bioavailability of the drug incorporated in suppositories.


Laser diffraction estimation of particle size distribution of slightly water-soluble drugs coexisting with additives: application to solid dosage forms. A simple and quantitative evaluation method for particle size distribution (fx(r)) of slightly water-soluble drugs dispersed in an aqueous medium together with other water-insoluble additives was developed using a laser diffraction method. The particle size distribution function of the powder mixture, (f(r)), was assumed as f(r) = phi x.fx(r) + phi a.fa(r), where phi is the volume fraction of each component dispersed in a measurement medium and fa(r) is the distribution function of another water-insoluble additive "a". In order to calculate fx(r) from f(r), it is necessary to know the density of drug and additive in the measurement medium, d(x) and d(a), but this is difficult to determine since particles usually swell in the medium. Thus, a method was developed to use their relative value, delta a (= da/dx). As a practical application, oxolinic acids (OA) of three sizes (OA-S (about 2 microns), OA-M (about 7 microns) and OA-L (about 24 microns)) were used as model drugs. delta a values were determined for various additives using the mixture of OA-S and each additive. Then, using delta as, fx(r) of OA-M or OA-L in the mixture containing OA-M or OA-L and additives was calculated from the f(r) experimentally determined for the mixture. They agreed well with their original distributions. The method was applied to some dosage forms, and the results obtained had good correlation with those from turbidity, wet sieving or dissolution test.


Permeability of insulin entrapped in liposome through the nasal mucosa of rabbits. The permeability of liposome entrapping insulin through the nasal mucosa of rabbit has been studied and compared with the permeability of insulin solution with or without pretreatment by sodium glycocholate (GC). Insulin entrapped in liposome was not detected in the receiver cell using the diffusion cells with the nasal mucosa. On the other hand, permeability of insulin entrapped in liposome increased after the pretreatment of GC. The phospholipids which result from liposomes, were not observed in the receiver. Also, the GC remaining in the nasal mucosa was measured. Considering the mechanism of permeation of insulin entrapped in liposome through the nasal mucosa, the GC remaining in the nasal mucosa may cause the lysis of liposomes.


Decomposition of linear dodecylbenzenesulfonate by simultaneous treatment with ozone and ultraviolet irradiation: rapid disappearance of formed mutagens. The decomposition products and mutagenic activity in Salmonella typhimurium strains TA98, TA100 and TA104 in the presence and absence of S9 mix of linear dodecylbenzenesulfonate (DBS) in aqueous solution after ozone treatment alone or simultaneous treatment with ozone and ultraviolet (UV) irradiation (ozone/UV treatment) were investigated. The decomposed DBS solutions after these treatments for 4 h were mutagenic for strains TA98, TA100 and TA104 both with and without S9 mix, but this mutagenicity disappeared rapidly during further ozone/UV treatment. Mutagenicity of the decomposed solution of DBS, however, was not substantially decreased by treatment with ozone alone. Formaldehyde and glyoxal were identified as the decomposition products of DBS in water by high-performance liquid chromatography after treatment with 2,4-dinitrophenylhydrazine. Although these two compounds were mutagenic for strain TA104 both with and without S9 mix, they disappeared after further ozone/UV treatment but not after ozone treatment alone. These results indicate that ozone/UV treatment is an effective procedure for purifying drinking water.


Synthesis, structure and antitumor activity of a water-soluble platinum complex, (1R,3R,4R,5R)-(-)-quinato(1R,2R-cyclohexanediamine)platinum (II). The reaction of dihydroxo(1R,2R-cyclohexanediamine)platinum(II) with (-)-quinic acid gave a water soluble complex, (-)-quinato(1R,2R-cyclohexanediamine)platinum(II). The crystal structure of the complex was determined by X-ray analysis. The data indicate a chelation of the alpha-hydroxycarboxylic acid part of quinic acid to platinum(II). The complex shows moderate antitumor activity against murine leukemia L1210 at high doses (T/C x 100 = 179% at a dose of 200 mg/kg).


Synthesis and biological activities of 1-[2-(dimethylamino)ethyl]- and 1-[3-(dimethylamino)propyl]-substituted 3-methyl-1,8-dihydrocycloheptapyrazol-8-ones and related compounds. 3-Methyl-1,8-dihydrocycloheptapyrazol-8-ones (2a-c), prepared from 3-acetyltropolones (1a-c), were treated with diazomethane, methyl iodide, dimethyl sulfate, and diethyl sulfate to give 1- and 2-alkylated compounds. The 1,8-dihydrocycloheptapyrazol-8-one (2a) also reacted with 2-(dimethylamino)ethyl, 2-(diethylamino)ethyl, 3-(dimethylamino)propyl, and 2,3-dihydroxypropyl chloride to afford the corresponding 1-substituted products. A preliminary study was made of the biological activities of some of the obtained compounds.


Studies on angiotensin converting enzyme inhibitors. VI. Synthesis and angiotensin converting enzyme inhibitory activities of the dicarboxylic acid derivative of imidapril and its diastereoisomers. All possible diastereoisomers of the dicarboxylic acid (10a), the biologically active form of imidapril (1), were synthesized, and their inhibitory activity against angiotensin converting enzyme (ACE) was examined. The in vitro ACE inhibitory activity of these compounds greatly depended on the configurations of the three asymmetric carbons in each molecule. The (S,S,S) isomer (10a) showed much more potent activity than the others.


Effects of anti-epileptic drugs on the L-tryptophan binding to human serum albumin. Effects of four anti-epileptic drugs (AED; phenobarbital, phenytoin, carbamazepine and sodium valproate) on the L-tryptophan binding to human serum albumin were studied. Among these drugs examined, only sodium valproate inhibited the binding even within the concentrations of its therapeutic range, and the Klotz plotting analysis revealed that the inhibition was competitive. The results of examinations with sera from epileptic patients medicated with these AED and drug-free normal controls also suggested that the protein binding ratios of L-tryptophan were decreased in the blood plasma of some patients with the high valproate concentrations and the low albumin contents.


Application of poly-L-lysine to purification of leukocyte cathepsin G by affinity chromatography. Poly-L-lysine with molecular masses of 3.3-290 kDa increased the amidolytic activities of leukocyte elastase and cathepsin G at low concentration, but had little effect on the activities of pancreatic elastase, alpha-chymotrypsin, plasmin and thrombin. Highly purified cathepsin G was obtained from column of EAH Sepharose 4B or Suc-L-Tyr-D-Leu-D-Val-pNA-Sepharose (affinity chromatography) by elution with poly-L-lysine solution (0.4 mg/ml, molecular weight (MW.) 290000 or 2.2 mg/ml, MW. 3300). Leukocyte elastase, adsorbed to Suc-L-Tyr-D-Leu-D-Val-pNA-Sepharose, was not eluted with poly-L-lysine solution. The amino acid composition of purified cathepsin G has been determined.


Biopolymers from marine invertebrates. XII. A novel cytolytic factor from a hermit crab, Clibanarius longitarsus. Bioactive polymers were sought in marine arthropoda and a novel cytolytic factor was found in a hermit crab, Clibanarius longitarsus. The partially purified factor showed activity in fractions corresponding to a molecular weight of about 10 kilodaltons on a Sephadex G-75 column. This cytolytic factor was halfmaximally active for tumor cells at 0.13-0.66 micrograms/ml and for normal cells at 1.9-82 micrograms/ml. Tumor lysis by the factor was time dependent and was complete within 12 h. This bioactive polymer was labile on heating, at low and high pH.


Bacteriostatic effect of 4,7-dicyanobenzofurazan due to inactivation of 2,3-dihydroxyisovalerate dehydratase. As part of our research on benzofurazans (BZs), we have reported the bacterioses of BZs in Escherichia coli, which may be due to O2-. produced within E. coli in the presence of dioxygen (O2). Incubation of E. coli with 4,7-dicyanobenzofurazan (1) lowered the 2,3-dihydroxyisovalerate dehydratase activity detectable in extracts from these cells. Addition of branched chain amino acids such as valine and leucine protected E. coli from growth inhibition by compound 1, though it could not protect E. coli from the damage by paraquat (PQ). Addition of Fe(III)-tris[N-(2-pyridylmethyl)-2-aminoethyl]amine (Fe-TPAA), a novel superoxide dismutase mimic, protected the dehydratase in a dose-dependent manner, which confirms that inactivation of the dehydratase is largely due to production of O2-.. The possibility was discussed that the bacteriostatic effect of compound 1 is due to the inactivation of 2,3-dihydroxyisovalerate dehydratase.


Pharmacokinetics of brain natriuretic peptide in rats. The pharmacokinetics of rat brain natriuretic peptide (rBNP) was compared with that of alpha-rat atrial natriuretic peptide (alpha-rANP) in rats. After intravenous infusion in rats (600 pmol min-1kg-1 for 2 min), the disappearance of plasma rBNP was 4-fold slower than that of alpha-rANP. The estimated mean plasma clearance rates for rBNP and alpha-rANP were 45.9 ml min-1kg-1 and 74.4 ml min-1kg-1, respectively. The affinity of rBNP for the clearance receptor or degradation enzyme was considered to be lower than that of alpha-rANP.


Site-specific cleavage of glycated human serum albumin in the presence of iron. Nonenzymatically glycated human serum albumin was incubated with ferric ion at 37 degrees C in 0.2 M phosphate buffer, pH 7.4, up to 30 days. In the incubation mixture, amino acids, tyrosine and phenylalanine, were detected, suggesting site specific cleavage of glycated human serum albumin.


pH-inducible beta-glucosidase and beta-glucuronidase of intestinal bacteria. beta-Glucosidase and beta-glucuronidase of human and rat fecal bacteria were induced by cultivation in alkaline media although their growths were not affected. When a bacterium isolated from human feces producing each enzyme was cultured in a medium at pH 5 for 12-15 h and then adjusted to pH 8, beta-glucosidase and beta-glucuronidase were induced 9.2-fold and 11.5-fold, respectively.


The enantioselective metabolism of p-cymene in rabbits. p-Cymene (1) was metabolized in rabbits and the following four optically active metabolites, 2-(p-tolyl)-1-propanol (3': R/S = 65:35), 2-(p-tolyl)propanoic acid (5': R/S = 0:100), p-(2-hydroxy-1-methylethyl)benzoic acid (6': R/S = 91:9) and p-(1-carboxyethyl)benzoic acid (8': R/S = 30:70), were isolated in addition to three optically inactive metabolites, 2-(p-tolyl)-2-propanol (2), p-isopropylbenzoic acid (4'), and p-(1-hydroxy-1-methylethyl)benzoic acid (7'). The presumed metabolic pathways of p-cymene in rabbits were confirmed by the administration of the intermediate metabolites (2, 3', 4', and 5'). The enantiomeric ratios of the metabolites, 3' and 6', suggested that omega-hydroxylations of the isopropyl group in 1 and 4' occurred preferentially at the pro-S methyl group. In the metabolism of 1, the S-isomers are predominant in the propanoic acid derivatives, but the R-isomers are rich in the propanol derivatives. It is of interest that the metabolism of 4', however, produced predominantly the corresponding propanol derivative (6'; R/S = 91:9) and propanoic acid derivative (8'; R/S = 80:20) possessing the same R-configuration. Some optically active p-cymene derivatives were also synthesized as standard compounds.


Preparation and antitumor activity of 2''-O-, 3''-O- and 2'',3''-di-O-substituted derivatives of etoposide. The 2''-O-, 3''-O- and 2'',3''-di-O-substituted derivatives (4a--p) of etoposide were prepared by nucleophilic substitution of 4'-O-benzyloxycarbonyletoposide (2) followed by deprotection. Controlled reaction (a limited amount of reagents and low temperature) was required for preparing the mono-O-substituted derivatives. In terms of ED125 values, doses which show 125% of T/C against P388 leukemia in mice, both the 2''-O-acetate (4a, ED125 = 0.18 mg/kg) and 3''-O-acetate (4b, 0.23 mg/kg) were nearly as active as etoposide (1, 0.19 mg/kg), while the 2'',3''-di-O-acetate (4c, 1.9 mg/kg) was somewhat less potent. In the replacement with other substituents, antitumor activity of the 2''-O-substituted derivatives was affected much more by the difference of the substituents as compared with that of the corresponding 3''-O-substituted derivatives. In the 2'',3''-di-O-substituted derivatives, the activity was decreased additively on the substituents.


Synthesis and evaluation of iodinated benzamide derivatives as selective and reversible monoamine oxidase B inhibitors. A new series of iodinated analogues of N-(2-aminoethyl)benzamide was synthesized and evaluated for inhibitory potency and specificity toward monoamine oxidase type-B (MAO-B). Among them, N-(2-aminoethyl)-2-chloro-4-iodobenzamide hydrochloride (2d) showed high inhibitory potency and selectivity against MAO-B. The type of MAO-B inhibition by 2d was non-competitive and the inhibition constant (Ki) was 0.80 microM. Strong and selective in vivo MAO-B inhibition by 2d was also confirmed. The brain MAO-B inhibition by 2d was reversible and the enzyme activity completely returned to the control value 24 h after administration. Compound 2d was, therefore, considered to be a candidate for advanced development as a radioiodinated ligand that may be useful for functional MAO-B studies in the living brain using single photon emission computer tomography.


Positron-emitting N-[18F]fluoroalkyl and [18F]fluoropyrrolidinyl analogues of eticlopride as potential in vivo radioligands for dopamine D2 receptors. N-Fluoroalkyl and 4-fluoropyrrolidinyl eticlopride analogues with high affinity toward central nervous system dopamine D2 receptors in vitro were labelled with positron emitting fluorine-18 (t1/2 = 110 min), and their in vivo biodistribution was investigated in rats. N-[18F]Fluoro-ethyl and -propyl eticlopride derivatives showed poor in vivo selectivity in the rat brain. On the other hand, 4-[18F]fluoropyrrolidinyl eticlopride exhibited almost constant and relatively high striatal concentration. The striatal/cerebellar radioactivity ratio, which corresponds to the ratio of a brain D2 receptor-rich to poor region, gradually increased to 5.2-6.4, 90 min after the injection. The striatal accumulation was selectively inhibited by pre-injection of haloperidol, a dopamine D2 antagonist, without affecting accumulation in other tissues. Thus, the selective striatal accumulation of 4-[18F]fluoropyrrolidinyl eticlopride in striatal tissue appears to be due to the specific binding to dopamine D2 receptors.


Novel uracil derivatives: newly synthesized centrally acting agents. A series of 1-amino-5-substituted uracils and their 4-thio or 2,4-dithio substituted analogues were synthesized and assayed for anti-conflict activity in rats and anesthetic activity in mice. 1-Amino-5-halogenouracils 3b-e, 1-amino-4-thiouracil (9a), and 1-amino-5-halogeno-4-thiouracils 9c, d showed both anti-conflict and anesthetic activities. The most active compound was 1-amino-5-chloro-4-thiouracil (9d) which showed anxiolytic activity at 2 mg/kg of oral administration (p.o.) on a modified Geller-Seifter conflict schedule. Its minimum effective dose (MED) was lower than that of diazepam. The 50 percent effective dose (ED50) for anesthetic activity in mice of the compound (9d) was 32.9 mg/kg, p.o.


Synthesis of trans-4-aminomethylcyclohexanecarbonyl-L- and -D-phenylalanine-4-carboxymethylanilide and examination of their inhibitory activity against plasma kallikrein. Based on studies of structure-activity relationship, trans-4-aminomethylcyclohexanecarbonyl-L-phenylalanine-4-carbox ymethylanilide (Tra-Phe-APAA) was designed as a selective plasma kallikrein inhibitor and synthesized. Tra-Phe-APAA inhibited plasma kallikrein with a Ki value of 0.81 microM, while it inhibited glandular kallikrein, plasmin, urokinase, factor Xa and thrombin with Ki values of greater than 500, 390, 200, greater than 500, and greater than 500 microM, respectively. However, its stereoisomer, Tra-D-Phe-APPA did not exhibit any detectable inhibitory activity against the above enzymes.


Synthesis and antiarrhythmic activity of 2,2-dialkyl-1'-(N-substituted aminoalkyl)-spiro-(chroman-4,4'-imidazolidine)-2',5'-diones. A novel series of 2,2-dialkyl-1'-(N-substituted aminoalkyl)-spiro-[chroman-4,4' -imidazolidine]-2',5'-diones was synthesized and evaluated for antiarrhythmic activity in chloroform- or/and aconitine-induced ventricular arrhythmia in mice. Among these compounds, (-)-6-chloro-2,2-dimethyl-1'-[3-(4-hydroxypiperidino)propyl] -spiro-[chroman-4,4'-imidazolidine]-2',5' -dione was found to be more effective than reference agents and was selected for further development.


Effects of Shosaikoto (kampo medicine) on lipid metabolism in macrophages. We investigated effects of Shosaikoto treatment on cholesterol metabolism in macrophages. Although macrophages, harvested from mice treated with Shosaikoto, took up a small amount of control low density lipoprotein (LDL) (thiobarbituric acid-reactive substance (TBA-RS) value was 0.27 pmol/mg of protein) as control macrophages, they took up more LDL modified with CuSO4 (TBA-RS value was 6.12 pmol/mg of protein) than control macrophages. Degradation of both control LDL and oxidized LDL was enhanced in Shosaikoto treated macrophages. In the presence of control LDL or in the absence of LDL, incorporation of [3H]oleic acid into chlesteryl oleate was significantly reduced in Shosaikoto treated macrophages. This suggests that acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in macrophages was partly inhibited by Shosaikoto treatment. On the other hand, in the present of oxidized LDL, cholesteryl ester accumulated in Shosaikoto treated macrophages as much as in controls. However, cholesteryl oleate efflux from macrophages in the presence of high density lipoprotein (HDL) was enhanced in Shosaikoto treated macrophages. These result indicate that Shosaikoto facilitates oxidized LDL catabolism in macrophages, resulting in the augmentation of oxidized LDL uptake and the elimination of cholesterol from macrophages by HDL. These Shosaikoto effects may prevent foam cell formation and the progression of atherosclerotic lesions.


Conjugated 1 beta-hydroxycholic acid in the urine of newborns and pregnant women measured by radioimmunoassay using antisera raised against N-(1 beta-hydroxycholyl)-2-aminopropionic acid-bovine serum albumin conjugate. Anti-tauro 1 beta-hydroxycholic acid antisera were prepared by immunizing rabbits with N-(1 beta,3 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholan-24-oyl)-2- aminopropionic acid-bovine serum albumin (BSA) conjugate. The antisera raised had high affinity (1.25-1.46 x 10(9) M-1) and specificity for conjugated 1 beta-hydroxycholic acid; cross-reactivity for glyco 1 beta-hydroxycholic acid was 100% and that for the glycine and taurine conjugates of other 1 beta-hydroxylated bile acids ranged from 11.30 to 0.23%. Urinary concentrations of conjugated 1 beta-hydroxycholic acid were determined by radioimmunoassay in newborns, 0-20 d after birth, in amounts ranging from 0.29 to 18.51 micrograms/ml and in women in late pregnancy (less than 1.13 micrograms/ml), as well as in normal women (less than 0.12 microgram/ml).


A radiometric assay method for aromatase activity using [1 beta-3H]16 alpha-hydroxyandrostenedione. [1 beta-3H]16 alpha-Hydroxyandrostenedione (16 alpha-OHA) (715 mCi/mmol) was prepared from commercially available [1 beta-3H]androstenedione (A) by the microbiological method with Streptomyces roseochromogenes and its structure and purity were determined by chromatographic and reverse isotope dilution methods. When [1 beta-3H]16 alpha-OHA was incubated with human placental microsomes and reduced nicotinamide adenine dinucleotide phosphate (NADPH), 3H2O-release into the medium was dependent upon protein concentration and incubation time. An apparent Km and Vmax of the microsomal aromatase for the [1 beta-3H]substrate were 650 nM and 34 pmol/min/mg protein, respectively. In this assay, aromatase activity could be determined as low as 0.1 nmol estrogen formation/min/mg protein. 3-Deoxyandrostenedione, a potent competitive inhibitor of the A aromatization, also blocked the 16 alpha-OHA aromatization in a competitive manner with Ki of 15 nM.


Preparation of acetylmercapto-3-carboxypropanoyl insulins using preparative high-performance liquid chromatography on an anion-exchange column. A method for the preparation of insulin derivatives which have protected sulfhydryl group(s) at definite site(s) on the molecule is described. Porcine insulin reacts with S-acetylmercaptosuccinic anhydride to afford four species of insulin derivatives that have 2 (or 3)-acetylmercapto-3-carboxypropanoyl group(s) at i) Gly(A1), ii) Gly(A1) and Phe(B1), iii) Gly(A1) and Lys(B29), and iv) Gly(A1), Phe(B1) and Lys(B29) positions. The derivatives are efficiently separated in a preparative scale by anion-exchange high-performance liquid chromatography on a TSKgel DEAE-2SW column. The four derivatives are all readily deacetylated with hydroxylamine to give the corresponding sulfhydryl insulin derivatives.


Catabolism of hemoglobin-haptoglobin complex in microsome subfractions. After internalization of hemoglobin-haptoglobin complex (Hb-Hp) via receptor-mediated endocytosis (RME) into liver parenchymal cells, organelles containing the complex distribute in the microsome fraction (Ms). Prior to the catabolism, Hb-Hp dissociates symmetrically into two 82,000-dalton (82 kDa) subunits. In the present investigation, the first event of Hb-Hp metabolism in Ms were further examined after [3H-heme, 14C-glogin]Hb-Ho or [125I-Hb]Hp injection to rats. Shortly after the internalization of Hb-Hp, this complex in Ms was intact. At 60 min after injection, radioactive materials of Ms extracted by freezing and thawing (F&T) with yield of 15% were composed of Hb-Hp, 82 kilodaltons (kDa) subunits and Hb metabolites with a ratio of 1:6:13. The heme metabolites were identified as [3H]bilirubin by high performance liquid chromatography (HPLC). The ratio of Hb-Hp/82 kDa subunits/Hb metabolites in microsome residue of the F&T was 40:8:1. The radioactivity in Ms at 60 min localized microsomes subfraction except Golgi light fraction. In electron microscope radioautography of microsome subfraction using [125I]Hb-Hp, silver grains were observed over or within morphologically heterogenous vesicles, e.g. vesicles containing very low density lipoprotein (VLDL) particles with appendage like multi-vesicular body (MVB) or compartment of uncoupling of receptor and ligand (CURL) in Goligi light and intermediate fractions. These studies suggest that Hb-Hp internalized by RME is dissociated symmetrically into two 82 kDa subunits in organelles of Ms, and that organelles with MVB or CURL-like structures are associated with Hb-Hp metabolism.


Chromatography of beta-glucuronidase from bovine liver. A study of the enzyme binding sites of prepared adsorbents. beta-Glucuronidase from bovine liver was adsorbed to the adsorbents prepared with CH-Sepharose 4B and either the competitive inhibitor or its analogs such as p-aminophenyl 1-thio-beta-D-glucuronic acid, -glucoside, -galactoside, and N-acetyl glucosaminide. The adsorbed enzyme was eluted at 0.1 or 0.5 M NaCl by a stepwise gradient. Chromatography of the enzyme was also performed by using the adsorbents prepared with Epoxy-activated Sepharose 6B and amine compounds or other compounds. In order to see whether the hydroxyl groups of the sugar parts in the ligand are necessary for the adsorption of the enzyme, chromatography was performed by using the adsorbents prepared with sugar derivatives as the ligand. As a result, it was found that beta-glucuronidase had an affinity for adsorbents prepared with either acetyl derivatives or methoxy derivatives of glycosides and CH-Sepharose 4B. From the results of elution of the enzyme with NaCl from adsorbents having amide bonding, it was clarified that the affinity of the enzyme for adsorbents without glycosides in the ligands correlated with acidity of the amide in the adsorbents. Hydrogen bond chromatography was performed with the prepared adsorbents. The enzyme was adsorbed under a high concentration of ammonium sulfate, and the elution of the adsorbed enzyme from adsorbents was examined by the degradation of salt. The enzyme was most easily eluted from aminoethyl 1-thio-beta-D-glucuronic acid-CH Sepharose 4B at 0.9 M ammonium sulfate and at 0.5 M concentration of the salt with p-aminophenyl 1-thio-beta-D-glucuronic acid-CH Sepharose 4B.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of calphobindin II (annexin VI) on procoagulant and anticoagulant activities of cultured endothelial cells. Effects of human placental calphobindin II (CPB-II) on the protein C activation and prothrombin activation on the cell surface of cultured calf pulmonary arterial endothelial cells have been investigated. CPB-II inhibited thrombin generation by factor Xa bound to the surface of the cultured endothelial cells in a dose-dependent manner. The amount (IC50) of CPB-II causing the inhibition at 50% was estimated to be approximately 10 nM. CPB-II was found to be ineffective, however, in the protein C activation by thrombin-thrombomodulin (TM) complex on the cell surface. Assay using purified TM revealed that CPB-II was able to exhibit the inhibitory potency for the protein C activation exclusively in the reconstituted system with negatively charged phospholipids. These results suggest that the neutral phospholipids participate in the protein C activation through the thrombin-TM system on the endothelial cell surface. The ability of CPB-II to inhibit procoagulant activity without affecting anticoagulant activity on the cultured endothelial cells is probably related to its potential physiological function, while it is able to exert various degrees of influence upon these activities in blood coagulation by interacting with negatively charged phospholipids in vitro.


Enzymatic synthesis of glucoside derivatives of validamine and valienamine. alpha- And beta-glucoside derivatives of validamine and valienamine were prepared by enzymatic transglucosidation using alpha- and beta-glucosidase of Rhodotorula lactosa. The structures of these derivatives have been elucidated by 13C- and 1H-nuclear magnetic resonance spectral analysis. Thus, 7-alpha-glucoside, 7-alpha-isomaltoside, and 4-alpha-glucoside of validamine and 7-alpha-glucoside, 7-alpha-isomaltoside, 4-alpha-glucoside, and 4-alpha-isomaltoside of valienamine were obtained from maltose and validamine or valienamine using alpha-glucosidase. 7-beta-glucoside, 2-beta-glucoside, and 4-beta-glucoside of validamine or valienamine were obtained from cellobiose and validamine or valienamine using beta-glucosidase. These derivatives were tested for alpha-glucosidase inhibitory activity on rat small intestinal glycosidases.


Mechanisms of pharmacokinetic interaction between propranolol and quinidine in rats. In order to study the mechanism of propranolol-quinidine interaction, the effects of quinidine on propranolol pharmacokinetics were examined in male Wistar rats. The concurrent oral administration of quinidine (10 mg/kg) markedly increased the plasma concentration of propranolol (2.5 mg/kg), and the area under the propranolol concentration-time curve increased about 3.6-fold. These results are consistent with previous observations in man and indicate the possible usefulness of the male Wistar rat as an animal model for investigating the mechanisms of the drug interaction. When propranolol was given intravenously, a concurrent administration of quinidine increased the apparent distribution volume of propranolol, mainly by decreasing its plasma protein binding. However, the systemic clearance of propranolol was not significantly altered by quinidine. Thus, quinidine increased the availability of oral propranolol from 13.8 +/- 2.2 to 44.2 +/- 4.6% (p less than 0.01). Furthermore, quinidine delayed the elimination of propranolol from the isolated perfused rat liver. These results indicate that quinidine reduces the presystemic elimination of propranolol in the liver, thereby increasing its systemic availability after oral administration.


Increase in water permeability of negatively charged liposomal membrane by local anesthetics. Effect of the local anesthetics dibucaine, tetracaine, lidocaine and procaine on the water permeability of phospholipid membrane was examined using liposomes composed of bovine heart cardiolipin and egg yolk phosphatidylcholine in a molar ratio of 2/98 by monitoring the osmotic shrinkage of liposomes in hypertonic glucose solution at pH 7.3 and 30 degrees C. These local anesthetics greatly accelerated the water permeability by destabilizing the membrane structure. The effect was found to be governed by the hydrophobicity of the anesthetics. There was also a significant correlation between the membrane destabilizing actions and the anesthetic activities.


Bioavailability of morphine in rabbits after rectal administration of suppository containing controlled release morphine tablet. Two kinds of sustained release morphine suppositories have been prepared; one is an oleaginous base suppository (MSC) containing a controlled release morphine tablet (MST: MS Contin), and the other is a hollow-type suppository (MSCH) containing MST and morphine powder packed in its hollow space. In vitro release tests and in vivo rectal absorption experiments in rabbits were performed. The profiles of morphine release from MST and MSC in vitro were similar, and revealed that suppository bases had no effect on the release profile of morphine from the preparation. Morphine release from MSCH was rapid in the early phase, and then enclosed morphine was slowly and continuously released from MST. Phamacokinetics of morphine from the suppository were compared with the orally administered MST, and it was found that there was no difference in the maximum plasma concentration (Cmax) and the peak time (Tmax) between MSC and MST, but the mean residence time (MRT) of MSC was approximately three times longer than that of MST, and the extent of bioavailability (BA) of MSC was significantly larger than that of MST (71.6 +/- 14.2% and 11.9 +/- 4.0%, respectively). Cmax can be altered arbitrarily by changing the morphine content in the hollow space of MSCH. As in the case of MSC, the plasma concentration of morphine from MSCH was maintained. It is concluded from the above results that MSC is a satisfactory sustained release morphine suppository for the treatment of cancer pain, administering it twice a day, and that MSCH is effective due to its fast analgesic effect and sustained release nature not only for cancer pain but also for surgical operations.


Effects of fatty acids, fatty amines and propylene glycol on rat stratum corneum lipids and proteins in vitro measured by fourier transform infrared/attenuated total reflection (FT-IR/ATR) spectroscopy. Fourier transform infrared/attenuated total reflection (FT-IR/ATR) spectroscopy was used to examine the effect of fatty acids, fatty amines and propylene glycol (PG) on the molecular mobility of rat stratum corneum lipids and keratinized proteins, using a hydrophobic solute, indomethacin, and a polar solute, 5- and 6-carboxyfluorescein (CF). Treatment of the skin with either oleic acid or oleylamine resulted in significant CH2 C-H asymmetric stretching band shifts and broadening. The extent of spectral alteration varied with the chemical structure of the penetrant. The penetrants increased the lipophilic indomethacin flux and shortened the lag times through the skin in vitro. The plot of frequency changes vs. indomethacin flux or lag time demonstrated a linear relationship, thus indicating that spectral alteration in CH2 C-H stretching regions of stratum corneum lipids may provide a reliable index for characterizing penetrants. The data also showed that the hydrophilic group which attached to the CH2 group in the penetrant molecules did not play a part in the membrane permeability enhancing action. Oleic acid and oleylamine appeared to induce a conformational alteration of the keratinized proteins from alpha-helix to beta sheet. Such alteration was also observed with PG treatment. Accumulation of CF was significantly increased by the PG pretreatment of the skin, thus suggesting that PG-induced protein conformational changes could be related to the enhancement of CF accumulation.


Assay of cell surface-bound immunoliposomes using monoclonal antibody reactive with a cross-linking reagent. A monoclonal antibody (mAb) reactive with a crosslinking reagent, N-(m-maleimidobenzoyl)dipalmitoylphosphatidylethanolamine (mMBPE), in liposomes was produced from a hybridoma clone established by a fusion between P3X63Ag8.653 mouse myeloma cells and spleen cells from a BALB/c mouse hyperimmunized with the antibody-coated liposomes containing mMBPE. Using this mAb (termed AL-6), the quantity of immunoliposomes bound on target tumor cells was assessed by flow cytofluorometry. The results obtained using fluorescein isothiocyanate-coupled AL-6 allowed the enumeration not only of the immunoliposomes bound on all tumor cells but also those on individual target tumor cells. The relevance of this assay method was confirmed by a comparison with another assay method of cell-bound liposomes using immunoliposomes containing carboxyfluorescein in the vesicles.


Evaluation of enteric coated tablet sensitive to pancreatic lipase. I. In vitro disintegration test. We designed a new enteric coated preparation which is pH independent and functions by pancreatic lipase activity in the duodenum. Triolein (TO) and trilaurin (TL) were selected as lipase sensitive components and ethylcellulose (EC) was used as the support film for TO and TL. Tablets (330 mg, d = 10 mm) containing a model drug, sulfamethizole (SMZ), were coated with 1% each of TO, TL and EC solution by the fluidized bed coating technique. Disintegration tests were carried out in the media including JPXI 1st fluid (pH 1.2, JP-1), 2nd fluid (pH 6.8, JP-2) and JP-2 with gall powder and pancreatic lipase (JP-2-GL). The lag time of disintegration of the tablet (TOTL-Tab) coated 5-7 mg/tab with TO, TL and EC was about 10 min and all of the tablets disintegrated completely within 30 min in JP-2-GL. However, in the other media, which did not contain lipase, TOTL-Tab did not disintegrate for at least 2 h. It was confirmed that TO and TL in the coating film were digested by lipase. In addition, the tensil strength of the film decreased quickly after incubation in JP-2-GL. These results suggest that the application of TO, TL and EC to tablet coating is useful for an enteric release preparation sensitive to pancreatic lipase, even if patients have low gastric acidity or are taking antacids.


Percutaneous absorption of ketoprofen from acrylic gel patches containing d-limonene and ethanol as absorption enhancers. The percutaneous absorption of ketoprofen (KPF) from gel patches containing d-limonene and ethanol was investigated in rats. Plasma levels of KPF varied with the kind of polymers which constitute the gel patch, and the highest level was observed when the copolymer of ethylacrylate (EA) and diethyleneglycolmethacrylate (DEGMA) was used as a vehicle. The amount of KPF permeating through the rat skin from the gel patch was well correlated with that of ethanol. Permeations were enhanced with increase in the amount of d-limonene distributed from the vehicle to the skin tissue. The amount of d-limonene accumulated in the skin varied greatly with the kind of polymers; the highest accumulation was observed with the EA-DEGMA copolymer, and decreased with increasing affinity of d-limonene to the polymers. The reason EA-DEGMA copolymer showed the highest percutaneous absorption of KPF from gel patches containing d-limonene may be the hydrophilic nature of this polymer which showed the lowest affinity to d-limonene.


Dose-dependent pharmacokinetics of glycyrrhizin in rats. The dose-dependent pharmacokinetics of glycyrrhizin (GLZ) was investigated by measuring drug disappearance from plasma and biliary excretion in rats. The decline in plasma concentration was biexponential after an i.v. dose of 5, 10, 20, or 50 mg/kg. Dosage, however, had a marked effect on the pharmacokinetics, with a greater-than-proportional increase in area under the plasma concentration curve (AUC) at doses of 20 and 50 mg/kg, even though the increase was proportional at doses of 5 and 10 mg/kg. There was also a significant increase of the steady-state distribution volume (Vdss), as well as significant decreases in total body (CLtot) and biliary (CLB) clearances, at 20 and 50 mg/kg from those at 5-10 and 5-20 mg/kg, respectively. The AUC, Vdss, and renal clearance (CLR) at a given dose showed no significant difference between rats with and without bile fistulas. The plasma unbound fraction (fp) (0.006-0.026) increased with increasing plasma GLZ concentration over the observed range (2-900 micrograms/ml). No significant change in Vdss for unbound GLZ was observed between the doses, indicating that the distribution of GLZ into tissues is not changed by an increase in dose. On the other hand, a dose dependency in CLtot for unbound GLZ was observed and confirmed to be attributed to dose dependency in CLB for unbound GLZ since there was no significant difference in CLR or metabolic clearance for unbound GLZ between the doses.(ABSTRACT TRUNCATED AT 250 WORDS)


Studies on natural antioxidants in citrus species. I. Determination of antioxidative activities of citrus fruits. The antioxidative activities of twenty types of citrus fruits were investigated with a screening method which is based on rat liver microsomal lipid peroxidation induced by dihydronicotinamide adenine dinucleotide phosphate (NADPH) and adenosine diphosphate (ADP). The activities of the exocarp were greater than those of the sarcocarp and the activities from immature fruits (collected in July-August) were greater than those from mature fruits. The strongest antioxidative activity was found in ponkan (Citrus reticulata Blanco) collected in July.


Direct injection method for quantitation of delta-aminolevulinic acid in urine by high-performance liquid chromatography. A highly sensitive and simple method for determining delta-aminolevulinic acid (ALA) in urine was established, using direct injection of urine into a high-performance liquid chromatographic column, with fluorometric detection after post-column derivatization with o-phthalaldehyde (OPA). The recovery of ALA was about 100% and ALA was completely separated on an ion exchange column (retention time, 38 min). The detection limit for ALA was 10 pmol (S/N = 2). The mean levels of urinary ALA of 10 healthy volunteers, 4 patients with acute intermittent porphyria, and 2 workers occupationally exposed to lead were 0.76, 5.25, and 23.54 mg/l, respectively. Because of its simplicity, the method is considered to be suitable for routine analysis of urinary ALA in the clinical laboratory.


Enzyme linked immunosorbent assay for beta-endorphin in human plasma. We established a highly sensitive and specific double-antibody enzyme linked immunosorbent assay for beta-endorphin (beta-EP). For competitive reactions, the beta-EP-antibody was incubated with beta-EP standard (or sample) and beta-D-galactosidase-labeled beta-EP (delayed addition). Free and antibody-bound labeled antigen were separated by using an anti-rabbit immunoglobulin G coated immunoplate. The enzyme activity on the plate was fluorometrically determined. The minimal detection limit was approximately 0.4fmol/well (10 pmol/l). Using this assay system, beta-EP-like immunoreactivity (-LI) in human plasma was determined. The level of beta-EP-LI in extracted human plasma from 6 normal subjects was 2.44 +/- 0.68 pmol/l. High performance liquid chromatography analysis of the plasma of a normal subject revealed a single immunoreactive form which eluted with the same retention time as that of synthetic beta-EP.


Antitumor activity of Hypsizigus marmoreus. I. Antitumor activity of extracts and polysaccharides. Antitumor activity of Hypsizigus marmoreus, one of the most popular Japanese edible mushrooms, was investigated. The aqueous and methanol extracts were tested against allogeneic tumor, solid sarcoma 180 and syngeneic tumor, Meth A fibrosarcoma. The aqueous extract was highly active in inhibiting growth of solid sarcoma 180, but not as much for Meth A fibrosarcoma. Fractionation of antitumor substances of the aqueous extract isolated four polysaccharides. Chemical analysis revealed one of them to be beta-(1-3)-glucan with a remarkable inhibitory effect against tumor-growth of sarcoma 180.


Dose-dependent enzyme suppression in spleen induced by GM1 (monosialoganglioside 1) administration to mice. Our previous studies suggested that the administration of exogenous gangliosides to the body modulates enzymatic networks in the brain. In the present study, we tested whether that is the case with another organ, spleen. By testing the dose response relationship, we found that there is a optimum dose for the effect of enzymatic modulation of GM1 (monosialoganglioside 1) administration. Although the optimum level varied depending on each of the examined hydrolytic enzymes, it usually fell in the range around 50 micrograms/kg body weight. The findings led us to conclude that the enzyme-modulating actions of gangliosides come not merely from the bizarre actions in vivo of high molecular exogenous substances.


Preparations of cyclic sulfoxide derivatives and their evaluation as transdermal penetration enhancers. Novel cyclic sulfoxides, such as 2-octyl, 2-dodecyl and 2-hexadecyltetrahydrothiophene-1-oxide were prepared by the alkylation of tetrahydrothiophene-1-oxide. Additionally, 2-methyl, 2-ethyl and 2-propyl-5-dodecyltetrahydro-thiophene-1-oxide were conducted by further alkylation. Their enhancing activity on the penetration of indomethacin through rabbit skin was evaluated in in vitro experiments, and the effect of the alkyl length on the enhancing activity was discussed. Among the 2-alkyl-tetrahydrothiophene-1-oxides, the compounds containing dodecyl and hexadecyl groups promoted a much greater penetration of the drug through the skin than the compound containing an octyl group. A stronger effect was observed in the experiment using 2-dodecyl-5-alkyltetrahydrothiophene-1-oxide, as compared with the that of 2-dodecyl-tetrahydrothiophene-1-oxide. The substitution of the alkyl groups to the next position of the sulfoxide group seemed to make the enhancing activities large.


Species and organ differences of sulphate conjugation of p-nitrophenol in liver and platelets. Sulphate conjugation of p-nitrophenol (p-NP) in the liver and platelet cytosol of guinea pigs, rabbits and dogs were studied. The dependency of phenol sulphotransferase (PST) activity on p-NP concentration in the liver of guinea pigs and rabbits and in the platelets of guinea pigs were similar to that reported for the liver (Mizuma et al., J. Pharmacobio-Dyn., 6, 851 (1983)) and platelets (Nakamura et al., J. Pharm. Pharmacol., 42, 207 (1990)) of rats. There was one peak of PST activity on p-NP at the concentration of 1 to 10 microM, and the PST activity was increased again with an increase of p-NP concentration above the original concentration. On the other hand, a peak in PST activity on p-NP at the concentration of 1 to 10 microM was not observed in the platelets of rabbits and dogs. These results indicated species and organ differences in PST activity on p-NP in liver and platelets. The biphasic activities of the PST and p-NP in platelets and liver of rat and guinea pig were similar to that reported in humans (Reiter et al., Naunyn-Schmiedeberg's Arch. Pharmacol., 324, 140 (1983)).


[Relationship between various stromal cells in long-term bone marrow culture]. In the present studies, the relationship and special junctions between various stromal cells in long term bone marrow culture were reported. The possible significance of these signs vis-a-vis the hemopoietic microenvironment were discussed. Fibronectin and thrombospondin were detected on the substratum and surface of the stromal cells using immunogold techniques. These adhesion proteins on the stromal cells might be important in cell-cell and cell-stroma connection.


[Normal radiographic appearance of the distal ends of the radius and ulna--a correlative radiographic and histologic slab study of the wrists in 50 infants]. Based on a correlative radiographic and histologic slab study of the wrists in 50 infants who died of unrelated diseases, the author's chief conclusions are as follow: 1) On the wrist radiograph of the infant, bone bark in the Ranvier's groove may appear as a "thorn-like" bony process on the margins of the metaphysis of the radius and ulna. 2) The radiographic appearance of the provisional zone of calcification at the distal ends of the radius and ulna are variable on radiographs of normal wrists, and familiarity with these variations precludes incorrect diagnosis. 3) The locations of the partially convex part of the provisional zone of calcification on the radiograph of the wrist correspond to where the vessels in the epiphyseal cartilage are located.


[A study on the role of immunosuppression in the pathogenesis of brucellosis]. An immunosuppressive effect was seen in murine brucellosis as detected by using plaque forming cell (PFC) and 3H-labelled lymphocyte blastogenesis transformation (LBT) assays. The immunosuppression PFC could last until 11 months after infection, while that of LBT was only seen at 3 months after infection. This immunosuppression could be relieved by using immunomodulators such as levamisole, bestatin, IL-2 and polyporus umbellatus. IL-2 and polyporus umbellatus could reverse the suppression to normal levels. Thus, this study provided evidence that immunosuppression is a component of pathogenesis of brucellosis.


[Synthesis and cloning of the whole human erythropoietin (EPO) gene]. A 600 bp synthetic erythropoietin (EPO) gene encoding all 166 amino acids of the EPO protein and 27 amino acids of the signal peptide has been constructed. The whole gene was divided into three large fragments consisting of a total of 32 oligonucleotides. These oligonucleotides were synthesized by the solid-phase phosphor-amidite method and ligated into three large fragments. These latter three were separately cloned into vector M13mp19 and then transformed into E. coli JM 103. Positive clones were screened with 32P-labeled probes. The sequences of the fragments were confirmed by DNA sequencing, and the sequence of the whole synthetic EPO gene was confirmed by enzymatic digestion and sequencing. The results indicated that the nucleotide sequence of the synthetic EPO gene is identical to that of the original.


[Specific killing of human leukemic T-cells by a monoclonal antibody coupled to the cytotoxin from Chinese cobra]. The immunotoxin (IT) was prepared by conjugating cytotoxin from Chinese Cobra (Naja naja Atra) venom with monoclonal antibody (McAb) Wu71 directed to human T-cells. First, calcium ions were used to suppress the cytolytic reaction while cell antigen was allowed to react with McAb. Then magnesium ions and the chelating agent EGTA were used to abolish the calcium inhibition. With this treatment, the IT showed high cytotoxicity for the leukemic cell line CEM, which was antigen positive (82.4% of the cells were killed at a concentration of 0.5 x 10(-6) mol/L of IT), but very little cytotoxicity for the antigen negative cell line Raji (23.3% cells were killed at the same concentration). Under scanning electron microscopy, it could be seen that the cell membranes of CEM were broken by IT, and the cells had died. The results suggest that the McAb Wu71 plus cytotoxin IT has potential application in leukemia therapy.


[Risk factors analysis of leukemia and aplastic anemia in China. Chinese Epidemiologic Study Group of Leukemia and Aplastic Anemia]. Based on the incidence survey of leukemia and aplastic anemia (AA) from 1986 to 1988, Case control studies (1257 new leukemia cases and 339 new AA cases) were carried out according to the type of leukemia and AA in order to better understand the epidemiologic characteristics of the diseases. Controls were matched randomly (age, sex and ethnic group) from the same population. The data were analyzed with the conditional Logistic multi-regression model and calculated on an IBM-PC/XT. The risk factors of M2a were found to be X-rays, antipyretics, benzene, pesticides and bimolane; that of M3 was chloramphenicol; that of M5 was X-rays; and that of other ANLLs was phenylbutazone. The risk factors of ALL were chloramphenicol, phenylbutazone and family members with cancer; those of CML were X-rays and hepatitis; those of CLL were chloramphenicol and benzene; those of AAA were antipyretics and hepatitis; and that of CAA ws X-rays.


[Pulmonary impedance plethysmographic features of obliterative pulmonary hypertension]. Pulmonary impedance plethysmography (PIP) has been used for the diagnosis of cor pulmonale secondary to COPD for about ten years. But the PIP features of obliterative pulmonary hypertension (OPH) have not yet been reported. We carried out a comparative study between PIP and hemodynamics determined by catheterization, in which 32 cases of OPH were studied. The results showed that the PIP features of OPH included a delayed delta Zmax peak, and the QZM interval had a positive linear correlation with pulmonary arterial pressure (r = 0.67, P less than 0.0001). According to electric field theory, it was supposed that this phenomenon might be due to narrowed pulmonary arterioles which limit the blood flow.


[Effect of d-catechin on carbon tetrachloride- and d-galactosamine-induced cytotoxicity in primary cultured rat hepatocytes]. The effects of d-catechin (d-CTC) on carbon tetrachloride- and d-galactosamine (d-Ga1N)-induced cytotoxicity in primary cultured rat hepatocytes were examined. After 1.5 h preincubation, d-CTC was added at doses of 0.1-5.0 mg/ml to culture medium together with 10 mmol/L CCl4 or 5 mmol/L d-GalN, respectively. GOT, GPT and LDH levels were measured 1.5 h after treatment. The results showed that d-CTC at doses of 0.6 to 5.0 mg/ml could protect the hepatocytes against the toxic effects of CCl4 and d-GalN. At the higher doses (2.5-5.0 mg/ml), d-CTC showed weak inhibition of LDH and GPT activities, but these did not influence its anti-hepatotoxic activity.


[Acute effect of small dosages of somatostatin analogue SMS201-995 on gallbladder contractility in normal adults]. Recently, a long-acting, somatostatin analogue, SMS201-995(SMS), has been developed and extensively investigated. It has been proved to be remarkably effective in treating many diseases, including active acromegaly. We observed the effect of SMS on gallbladder contractility in normal adults using the fatty meal test. Our results show that gallbladder contractility was inhibited significantly for 6 hours after the subcutaneous injection of SMS in dosages of 50, 25, 12.5, and 5 micrograms. We conclude that it is not practical to avoid gallbladder disfunction during long-term SMS treatment by decreasing the dosages of SMS.


[YAG laser treatment in anterior segment disease of the eye]. Three hundred and fifty-two eyes (230 patients) with various diseases were treated with the first China-made YAG laser therapy instruments. The success rate was 100%. Two hundred and seventy-six eyes with primary angle closed glaucoma were treated by peripheral iridotomy. Postoperatively, the IOP was lowered by 0.267 kPa (2.43 mmHg). The dose and frequency of medication could then be lowered. The closed chamber angle index improved. Forty-nine eyes with membranous cataract were treated by posterior capsulotomy: Visual acuity was improved by 95%. The main complications during or immediately after operation were as follows: transient elevation of IOP, hyphema, anterior uveitis and corneal edema. All of these were well controlled.


[Preparation of liposomes entrapping plasmid DNA and linear DNA]. Liposomes entrapping plasmids pSV2-neo DNA, pUC18-ras DNA, pSV2-neo-ras DNA and linear DNA were prepared. The liposomes were composed of DOPC/Chol/OA (4:4:3) and pH-sensitive DOPE/Chol/OA (4:4:3), respectively. The efficiency of DNA entrapment was about 50%. Gel electrophoresis analysis showed: Liposome-entrapped DNA was not digested by DNase; The entrapped DNA molecules were intact and stable for at least 5-6 months at 4 degrees C. During preparation of pH-sensitive liposome, the pH must be kept at 8.0.


[Primary culture of rat pituitary cells in serum-free medium]. A method for primary culturing of rat anterior and intermediate/posterior pituitary cells in complete serum-free defined medium (CSFM) is described. Dispersed pituitary cells were prepared by using a multiple enzyme digestion system. Insulin, transferrin and serum albumin were essential additives for maintenance of primary rat pituitary cells in CSFM. Morphological immunocytochemical and RIA studies during three weeks' culture indicated that beta-endorphin-like immunoreactivity (beta-End-IR) was contained in and released from pituitary cells. The secretion rate remained almost constant for 2 weeks after the 5th day in culture. This method provides an ideal in vitro model for studying the effects of various factors on the synthesis and release of beta-endorphin in pituitary cells and the mechanism of regulation of other target gland hormones.


[Ageing suppresses the enhancement of T cell mitogenesis by opioid peptides and enkephalins increase phagocytosis of murines macrophage]. The opioid peptides methionine enkephalin (M-ENK) and beta-endorphin (beta-END) (1 x 10(-5)-1 x 10(-11) mg/ml) were investigated for their effect on the PHA (1:500, 1:750 or 1:1000) induced proliferative response of old and young Wistar rat splenic lymphocytes in vitro. The different effects in young and old rats on proliferative response to PHA were determined. The results showed that MENK and beta-END significantly enhanced the proliferative response to PHA in young rats, while enhancement by M-ENK and beta-endorphin (PHA 1:750, 1:1000) was not observed in old rats. The PHA-induced proliferative response was 30%-40% lower in old rats than in young rats. Our results suggest an altered response to neuro-immunomodulation with age. The in vitro effect of ENKs on phagocytosis was also studied. The results indicated that LENK (10(-4)-10(-6) mg/ml) and MENK (10(-2)-10(-4) mg/ml) could stimulate the phagocytosis of peritoneal macrophages from Balb/c mice.


[Clinical and instrumental evaluation of erectile impotence: a proposal of a diagnostic protocol]. There has been increasing interest in recent years in patients complaining of erectile dysfunction. This has prompted research and the development of diagnostic procedures aimed at allowing increasingly rapid characterization of the type of impotence involved (psychogenic or organic) and thus the most effective treatment. In this preliminary study, we present our study methodology in patients with erectile dysfunction, emphasising in particular the diagnostic procedures best suited to revealing the presence of an organic cause in the pathogenesis of impotence.


[Surgical treatment of common bile duct calculosis: a comparative critical evaluation of long-term results of sphincterotomy and choledochotomy approaches]. From an analysis of operations performed by the Authors between 1974 and 1988, sphincterotomy emerged as the operation of choice given the small percentage of complications compared to the higher percentage of complications that arose with choledochotomy with external biliary derivation, in which use of the Kehr tube is hardly ever directly implicated. However the results of choledochotomy shown also demonstrate a possible improvement through better intraoperative procedures or by complementary endoscopic sphincterotomy where indicated; hence this procedure with external biliary deviation, according to the Authors, merits reevaluation.


[A rare cause of intestinal occlusion: Morgagni-Larrey hernia]. Authors report a case of Morgagni-Larrey obstructed hernia recently observed. After remembering pathogenetic hypothesis and pathological findings, they discuss clinical patterns, diagnostic tools and differential diagnosis. Authors suggest to consider surgical repair as a choice treatment, also for asymptomatic patients.


The continuing medical education needs of anaesthetists. Learning needs assessment is the term applied to the process of identifying or diagnosing a learner's educational needs. It is the foundation of a systematic continuing medical education (CME) programme. Needs assessment has been identified as the most pressing problem of medical education directors in North America. Furthermore, the CME learning needs, interests or motivations of anaesthetists have never been studied. The amount of time and effort required for needs assessment is probably a major deterrent to this activity. The investigators adopted simple and straightforward means of assessing the "perceived learning needs" and topic interests of anaesthetists. Questionnaires were sent by mail to anaesthetists practicing in teaching and non-teaching hospitals in the Toronto area. The questionnaire presented a list of CME content areas. The respondents were asked to indicate on scale of 1 to 10 their Current Expertise, Ideal-Desired Expertise, and Interest-Motivation levels for each content area. Need Score for each content area was calculated by taking the difference between Ideal and Current Expertise responses. A total of 101/305 anaesthetists (29%) responded to the survey. Most of the respondents had been in anaesthesia practice for less than ten years. Regional nerve block, acute pain control, and medicolegal considerations received high overall ranks in both the need and interest categories. Paediatric anaesthesia, anaesthesia for trauma surgery and thoracic anaesthesia had top ranks among the subspecialty fields. Regional anaesthesia techniques received higher need and interest ranks than intravenous and inhalational techniques. The learning needs of anaesthetists of a large urban centre have been identified, and this information is useful to CME planners.


[Biomechanical and clinical interpretation of firearm wounds. General problems. V. The propaedeutic ABC of terminal ballistics]. Modern portable firearms, whether for military or civilian use, present substantially different features as instruments for striking, wounding and killing compared to those used in the past on account both of their intrinsic characteristics as thermo-chemico-ballistic machines, guaranteeing extra, more easily achievable performance and of the characteristics of the bullets used. The factors responsible for this difference, which consists essentially in an unprecedented wounding capability, are, in the military field, the enormous amount of research which only now is beginning to yield a bare minimum of concrete results in terms of futuristic forms and devastating performance, amongst other things because it is easier to achieve, and, in the "civilian" field, the maniacal search for an unlikely definitive wound, based on the unusual nature of the cartridge and on sophisticated training.


[Rupture of the cervical esophagus. Apropos of a case of perforation by a foreign body]. The Authors describe a case of perforation of the cervical oesophagus by a foreign body (dental prosthesis). After outlining the clinical and instrumental elements which may be useful in the diagnosis, the Authors go on to tackle the topic of the most appropriate choice of therapy in the course of foreign-body perforation and in perforation of the oesophagus in general.


Perioperative effects of oral ketorolac and acetaminophen in children undergoing bilateral myringotomy. Prophylactic administration of analgesics before surgery can decrease the intraoperative anaesthetic requirement and decrease pain during the early postoperative period. In a double-blind, placebo-controlled study involving 90 healthy ASA physical status I or II children undergoing bilateral myringotomy, we compared the postoperative analgesic effects of oral acetaminophen and ketorolac, when administered 30 min before induction of anaesthesia. Patients were randomized to receive saline (0.1 ml.kg-1), acetaminophen (10 mg.kg-1) or ketorolac (1 mg.kg-1) diluted in cherry syrup to a total volume of 5 ml. Anaesthesia was induced and maintained with halothane and nitrous oxide via a face mask. Postoperative pain was assessed by a blinded observer using an objective pain scale. The three study groups were similar with respect to demographic data, duration of anaesthesia and surgery, induction behaviour, oxygen saturation, incidence of postoperative emesis and, recovery times. The ketorolac group had lower postoperative pain scores and required less frequent analgesic therapy in the early postoperative period compared with the acetaminophen and placebo groups. In contrast, there were no differences in pain scores or analgesic requirements between the acetaminophen and the placebo groups. We conclude that the preoperative administration of oral ketorolac, but not acetaminophen, provided better postoperative pain control than placebo in children undergoing bilateral myringotomy.


Analgesic efficacy and safety of a caudal bupivacaine-fentanyl mixture in children. The analgesic efficacy and safety of a single caudal injection of a bupivacaine-fentanyl mixture was investigated in this prospective, controlled, triple-blinded study of 34 children, aged 1-11 yr and of ASA physical status I-II undergoing urological surgery. After induction of anaesthesia and before surgery, the children were randomly assigned to receive a caudal injection of 1.0 ml.kg-1 bupivacaine 0.125% with epinephrine 1:400,000 and either fentanyl 1.0 microgram.kg-1 in 1.0 ml of normal saline or 1.0 ml of normal saline. After completion of surgery, patients were assessed in the recovery room for six hours from the time of the caudal injection and for a further 18 hr on the ward. While in the recovery room arterial oxygen saturation and respiratory rate were monitored continuously and recorded hourly together with end-tidal carbon dioxide, pain and sedation scores. Other complications were also recorded. While on the ward, pain and sedation scores, respiratory rate and side effects were recorded every two hours. Postoperative analgesia was provided by intravenous morphine. Analgesic requirements were recorded for the 24-hr study period. Pain and sedation scores did not differ between groups. Respiratory depression or hypoxia did not occur. The incidences of other side effects did not differ. There were no differences in the numbers of patients requiring morphine within eight hours, the time to first morphine administration or the total morphine requirements. We conclude that a single caudal injection of a bupivacaine-fentanyl mixture with epinephrine administered prior to surgery, while safe, offers no advantage over an injection of bupivacaine 0.125% with epinephrine for paediatric urological surgery.


Rocuronium (ORG 9426) neuromuscular blockade at the adductor muscles of the larynx and adductor pollicis in humans. The effects of rocuronium, 0.25 or 0.5 mg.kg-1, were measured simultaneously on the adductor muscles of the larynx and adductor pollicis in 14 adult patients. Anaesthesia was induced and maintained with propofol and fentanyl. Tracheal intubation was performed without muscle relaxants. The recurrent laryngeal and ulnar nerves were both stimulated supramaximally, at the notch of the thyroid cartilage and at the wrist respectively, using train-of-four stimulation. The laryngeal response was evaluated by measuring the pressure change in the cuff of a tracheal tube positioned between the vocal cords. Onset time, intensity of blockade and duration of action were less at the larynx than at the adductor pollicis. After rocuronium, 0.25 mg.kg-1, the onset time (interval between injection and maximal T1 blockade) was 1.6 +/- 0.1 min and 3.0 +/- 0.3 min (mean +/- SEM) at the laryngeal muscles and adductor pollicis, respectively (P less than 0.01 between muscles). Maximum blockade was 37 +/- 8% and 69 +/- 8%, respectively (P less than 0.05), and time to 90% T1 recovery was 7 +/- 1 min and 20 +/- 4 min, respectively (P less than 0.05). With 0.5 mg.kg-1, the onset time was also more rapid at the vocal cords (1.4 +/- 0.1 min) than at the adductor pollicis (2.4 +/- 0.2 min, P less than 0.001). Maximum blockade was 77 +/- 5% and 98 +/- 1%, respectively (P less than 0.01), and time to 90% T1 recovery was 22 +/- 3 min and 37 +/- 4 min, respectively (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


Carbon dioxide absorption and gas exchange during pelvic laparoscopy. Twelve ASA physical status I-II patients undergoing pelvic laparoscopy for infertility were enrolled in a study to quantify the effects of CO2 insufflation and the Trendelenburg position on CO2 elimination and pulmonary gas exchange, and to determine the minute ventilation required to maintain normocapnia during CO2 insufflation. Measurements of O2 uptake (VO2), CO2 elimination (VCO2), minute ventilation (VE), FIO2, and respiratory exchange ratio (RQ) were made during three steady states: control (C) taken after 15 min of normoventilation but before CO2 insufflation, after 15 min (L1) and 30 min (L2) of hyperventilation during CO2 insufflation. The FIO2 was controlled at 0.5 and arterial blood gases were used to calculate the oxygen tension-based indices of pulmonary gas exchange. After 15 min and 30 min of CO2 insufflation, the volume of CO2 absorbed from the peritoneal cavity was estimated at 42.1 +/- 5.1 and 38.6 +/- 6.6 (SEM) ml.min-1 respectively, increasing CO2 elimination through the lungs by about 30%. Hyperventilation of the lungs by a 20-30% increase in minute ventilation maintained normocapnia. Despite the CO2 pneumoperitoneum and Trendelenburg position, there was no impairment of pulmonary oxygen exchange as estimated by (A-alpha)DO2. This study demonstrated that a 30% increase in minute ventilation, achieved by increasing tidal volume to more than 10 ml.kg-1, is sufficient to eliminate the increased CO2 load and maintain normal pulmonary O2 exchange during pelvic laparoscopy.


Exposure of operating room personnel to nitrous oxide during paediatric anaesthesia. This study was undertaken to quantify the exposure of operating room staff to nitrous oxide during routine paediatric otolaryngeal surgery and to determine the influence of the method of induction of anaesthesia on this exposure. The nitrous oxide exposure of the anaesthetist, the surgeon and the circulating nurse were measured, using body-worn passive atmospheric samplers, during twelve routine paediatric otolaryngeal surgical lists. During six of the lists an inhalational technique, with nitrous oxide, oxygen and halothane, was used for the induction of anaesthesia. During the other six lists anaesthesia was induced using intravenous thiopentone. In all cases, anaesthesia was maintained using nitrous oxide, oxygen and halothane. Regardless of the induction technique used, the mean nitrous oxide exposures of the anaesthetist, the surgeon and the nurse all exceeded the maximum level of 25 ppm.hr-1 recommended by the United States National Institute for Occupational Safety and Health (NIOSH). The use of an intravenous technique for the induction of anaesthesia reduced the nitrous oxide exposure of the anaesthetist and the circulating nurse. This suggests that, although the use of an intravenous induction may reduce exposure to nitrous oxide, the NIOSH recommendations for maximum exposure of operating room personnel to nitrous oxide are currently unattainable in practice.


Reliability of auscultation in positioning of double-lumen endobronchial tubes. Auscultation is a well-established technique to confirm the position of double-lumen endobronchial tubes (DLTs). However, some authors have recommended that fibreoptic bronchoscopy (FOB) is also indicated. The aims of this study were to determine first if bronchoscopy after blind placement of DLTs improved positioning; and second if preoperative bronchoscopy could detect difficult intubation. Twenty-four patients undergoing aortic or lung surgery were studied. After intubation with a single-lumen tube, an initial FOB was performed by an independent observer to check the airway anatomy. Then, the single-lumen tube was replaced by a DLT using a classical "blind" intubation method. Subsequent FOB was performed first by the independent observer to record the DLT position and next by the investigators for improvement or correction of their positioning under visual control. Fibreoptic bronchoscopy after blind placement of DLTs resulted in repositioning 78% left-sided DLTs and 83% right-sided DLTs. Preoperative bronchoscopy did not always detect an airway abnormality which might lead to difficult positioning of the DLTs. In conclusion, auscultation is an unreliable method of confirming the position of DLTs and should be followed by fibreoptic bronchoscopy.


Methylparaben and propylparaben do not alter cerebral blood flow in humans. In vitro studies suggest that the preservatives methylparaben and propylparaben included in some multidose vials of succinylcholine are the cerebral vasodilators responsible for the increases in intracranial pressure (ICP) documented after succinylcholine administration. To test this hypothesis, we measured cerebral blood flow (CBF) and cerebral blood flow velocity (CBFV) with inhaled 133Xenon and transcranial Doppler respectively in healthy humans before and after the intravenous administration of methylparaben and propylparaben. We found no change in either CBF or CBFV after the paraben injections and therefore conclude that it is unlikely that the rise in ICP seen with succinylcholine is caused by cerebral arterial vasodilatation from the preservatives methylparaben and propylparaben.


Perioperative haemotherapy: I. Indications for blood component transfusion. The practice of transfusion medicine has undergone substantial change over the last decade. Much of the impetus for the change has come from the isolation of human immunodeficiency virus (HIV) and the linkage of HIV transmission to blood transfusion. The purpose of this paper is to collate and review the literature relating to the indications for blood transfusion and provide recommendations for the appropriate utilization of blood products. Peer-reviewed and published studies and reviews relating to aspects of clinical blood transfusion were identified through computer searches and searching of the bibliographies of identified articles. Emphasis was placed on the literature published within the last decade and particularly in the years 1985-91. Material was chosen which was of proved clinical importance and in which findings were consistent among different investigators or different centres. Less emphasis was placed on material reporting new findings of uncertain clinical relevance or findings that were not consistent with majority reports. It is concluded that the only indication for red cell transfusion is to increase the oxygen carrying capacity of the blood and that an adjustment downwards in the haemoglobin concentration at which blood is transfused (transfusion trigger) from the traditional level of 100 g.L-1 is supported by the physiological and clinical data. Perioperative haemoglobin concentrations of 80 g.L-1 are acceptable in otherwise healthy young patients. The transfusion trigger should be adjusted upwards from this in medically compromised patients and in the elderly (greater than 60 yr). Fresh frozen plasma (FFP) is only indicated when there are documented deficiencies of coagulation factors. Platelet concentrates (PC) are indicated for the treatment of clinical coagulopathy resulting from thrombocytopaenia or platelet dysfunction. Routine or prophylactic administration of either FFP or PC after cardiopulmonary bypass or during resuscitation from haemorrhage is not indicated.


Epidural anaesthesia for caesarean section in an achondroplastic dwarf. This report describes the anaesthetic management of an 18-yr-old achondroplastic dwarf who presented for elective Caesarean section. Epidural anaesthesia was performed without technical difficulty using 8 ml carbonated lidocaine 2% with epinephrine 1:200,000. Although the skeletal abnormalities of achondroplasia have been cited as contraindications to the use of epidural anaesthesia, clinical experience does not support this contention. Previous reports have described technical difficulties in these patients, such as dural puncture and inability to advance the catheter into the epidural space, but no serious complications resulted and epidural anaesthesia was successful on subsequent attempts. The existing literature on the anaesthetic management of achondroplasia for Caesarean section is reviewed and considerations are presented concerning the choice of local anaesthetic, the epidural test dose, and dose titration.


Anaesthetic considerations in the child with Menkes' syndrome. The author presents and discusses the anaesthetic implications of a four-month-old infant with Menkes' syndrome who required tracheostomy. Menkes' syndrome is an X-linked recessive disorder of copper absorption and metabolism. Defective processing of copper results in abnormalities of several enzyme systems leading to severe dysfunction of multiple organ systems. Due to the progressive nature of this disorder and its severe effects on several different organ systems, most importantly the central nervous system, these children frequently require anaesthetic care during imaging procedures such as MRI or during various surgical operations. The high prevalence of seizure disorders, gastroesophageal reflux with the risk of aspiration, and airway complications related to poor pharyngeal muscle control are of concern to the anaesthetist. In addition, defective collagen formation, similar to that seen in Ehlers-Danlos syndrome, may be present. Identification of these associated conditions during the preoperative examination will guide the selection of appropriate, safe anaesthetic care for these children.


Development of a computerized database for the study of anaesthesia care. To record, tabulate and report problems associated with anaesthesia, we have developed an information collection system and computer software to follow all patients attended by an anaesthetist at a teaching hospital in Canada. For the last 15 mo, data for 17,000 patients have been collected and the system is ongoing. Data collection is from three sources: carbonless copies of the handwritten Operating Room (OR) and Post Anaesthetic Care Unit (PACU) records, other hospital databases, and postoperative visits. Adverse events (observations which differ from specific physiological variables, or require an intervention and do not normally occur during the routine conduct of anaesthesia), are defined directly on each OR and PACU record. These events are recorded when they occur by the attending anaesthetist or the PACU nurse. All data are verified by a research nurse and an anaesthetist. Computer software, developed from DBase IV, is used to track 95 individual items on preoperative status and anaesthetic technique and another possible 1,450 selections for drugs, physicians, airways, surgical procedures and events for each patient. Data are analyzed with SAS software and reports generated to link the casemix and process with outcome. Comparison of data entered into the computer programme to a retrospective chart review revealed discrepancies of less than 0.5%. Collection, verification and computer entry takes five minutes per patient and the on-going cost is estimated at $4 per patient record. Analysis of the information collected in this database has been useful for research of adverse outcome following anaesthesia, resident expertise profiles, and the administrative management of an anaesthesia department.