Tsutomu Irikura

Differences in susceptibility to quinolones of outer membrane mutants of Salmonella typhimurium and Escherichia coli.

The mechanism of penetration of quinolones through the bacterial outer membrane was studied with lipopolysaccharide-deficient and porin-deficient mutants. The data indicated that the lipopolysaccharide layer might form a permeability barrier for hydrophobic quinolones such as nalidixic acid but not for hydrophilic quinolones such as norfloxacin and ciprofloxacin. The results also showed that quinolones with a low relative hydrophobicity appeared to permeate through OmpF porin, whereas quinolones with a low relative hydrophobicity appeared to permeate through OmpF porin, whereas quinolones with a high relative hydrophobicity appeared to permeate through both OmpF porin and phospholipid bilayers.

Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa.

Two genetically distinct classes of norfloxacin-resistant Pseudomonas aeruginosa PAO4009 mutants were isolated spontaneously. Two norfloxacin resistance genes, nfxA and nfxB, were mapped hex-9001 and leu-9005 and between pro-9031 and ilv-9023, respectively, on the P. aeruginosa PAO chromosome. The nfxA gene was shown to be an allele of nalA by transductional analysis with bacteriophage F116L. The nfxB mutant showed a 16-fold increase in resistance to norfloxacin and a slight increase in resistance to nalidixic acid. The nfxB mutant was unique in that it showed hypersusceptibility to beta-lactam and aminoglycoside antibiotics. This mutant had about a threefold-lower rate of norfloxacin uptake than that of the wild-type strain or nfxA mutant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins demonstrated the appearance of a 54,000-dalton protein in the nfxB mutant. These findings suggested that the norfloxacin resistance mechanism in the nfxB mutant might be an alteration in outer membrane permeability to norfloxacin. Images

Isolation and characterization of norfloxacin-resistant mutants of Escherichia coli K-12.

We isolated spontaneous mutants from Escherichia coli K-12 with low-level resistance to norfloxacin. These mutants were classified into the following three types on the basis of their properties: (i) NorA appeared to result for mutation in the gyrA locus for the A subunit of DNA gyrase; (ii) NorB showed low-level resistance to quinolones and other antimicrobial agents (e.g., cefoxitin, chloramphenicol, and tetracycline), and the norB gene was considered to map at about 34 min on the E. coli K-12 chromosome; (iii) NorC was less susceptible to norfloxacin and ciprofloxacin but was hypersusceptible to hydrophobic quinolones such as nalidixic acid and rosoxacin, hydrophobic antibiotics, dyes, and detergents. Susceptibility to bacteriophages and the hydrophobicity of the NorC cell surface also differed from that of the parent strain. The norC gene was located near the lac locus at 8 min on the E. coli K-12 chromosome. Both NorB and NorC mutants had a lower rate of norfloxacin uptake, and it was found that the NorB mutant was altered in OmpF porin and that the NorC mutant was altered in both OmpF porin and apparently in the lipopolysaccharide structure of the outer membrane.

In vitro and in vivo antibacterial activity of AM-833, a new quinolone derivative.

AM-833 showed potent activity against members of the family Enterobacteriaceae, Neisseria spp., and Haemophilus influenzae and good activity against staphylococci, Pseudomonas aeruginosa, and Branhamella catarrhalis. Against these bacteria, its activity was roughly comparable to that of norfloxacin and ofloxacin but was slightly less potent than that of ciprofloxacin. This compound also showed good activity against drug-resistant strains such as methicillin-resistant Staphylococcus aureus and gentamicin-resistant Pseudomonas aeruginosa. The protective effects of a single oral dose of AM-833 on systemic bacterial infections in mice were greater than those of norfloxacin. AM-833 was as effective as ofloxacin and ciprofloxacin against systemic infections with Escherichia coli and Pseudomonas aeruginosa, and it showed somewhat higher activity against staphylococcal infections than did the other quinolones. AM-833 exhibited good prophylactic activity against E. coli infections. AM-833 also proved effective against localized infections such acute pneumonia and ascending urinary tract infections in mice. The excellent therapeutic efficacy of AM-833 against these systemic and local infections may be a result of its good oral absorption and high levels in tissues.

Pharmacokinetics of a new quinolone, AM-833, in mice, rats, rabbits, dogs, and monkeys.

The pharmacokinetics of AM-833 [6,8-difluoro-1-(2-fluoroethyl)-1, 4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid] were studied in mice, rats, rabbits, dogs, and monkeys by reversed-phase high-performance liquid chromatography. AM-833 was rapidly and completely absorbed from the digestive tracts of mice, rats, and dogs. About half of AM-833 bound to rat and dog serum proteins. Drug levels in lung, spleen, liver, and kidney tissues of rats and dogs were greater than the respective levels in serum but lower in brain tissue. Drug levels in tissues declined with the decrease in levels in serum. AM-833 penetrated rapidly and well into inflammatory exudate of rats. Elimination half-lives in serum were species dependent, ranging from 1.57 h in rabbits to 9.42 h in dogs. Profiles of drug levels in serum were dose related over a single dose range from 2 to 40 mg/kg and not modified significantly during multiple dosing in dogs. Unchanged AM-833 was excreted in urine and bile in both rats and dogs. The metabolism of AM-833 was suggested by evidence that 24-h total recovery of unchanged AM-833 in urine and bile accounted for about half of the intravenous dose in rats.

Comparative Activities of AM-715 and Pipemidic and Nalidixic Acids Against Experimentally Induced Systemic and Urinary Tract Infections

Dose for dose, AM-715 was at least five times as active as pipemidic acid and nalidixic acid against systemic and urinary bladder-kidney infections in mice.

Gas chromatographicmass spectrometric determination of amitriptyline and its major metabolites in human serum

A gas chromatographic--electron-impact ionization mass spectrometric method has been developed for the determination of amitriptyline (AMT) and its metabolites, nortriptyline (NT), 10-hydroxyamitriptyline (10-OH-AMT) and 10-hydroxynortriptyline (10-OH-NT) in human serum. The lower limit of detection was 2 ng/ml for all compounds except 5 ng/ml for 10-OH-NT. The calibration curves for AMT and 10-OH-AMT were linear up to 100 ng/ml, and up to 200 ng/ml for NT and 10-OH-NT. The accuracy of the assay in terms of coefficient of variation was less than 7%. The extraction efficiency was almost quantitative for all compounds except 60% for 10-OH-NT. Using this method, human serum samples which had been collected after oral administration of a single 50-mg dose of AMT were analyzed. Ratios of the conjugation of each metabolite were estimated, including AMT.

[Experimental nephrotic hyperlipidemia induced in rats by daunorubicin and effects of KCD-232[4-(4'-chlorobenzyloxy)benzyl nicotinate] on lipid metabolism].

Mechanisms for hypercholesterolemia and hypertriglyceridemia and the effects of KCD-232, a new hypolipidemic agent, on them were studied in male Wistar rats with daunorubicin (DR)-induced nephrosis. Single intravenous injection of DR dose-dependently increased urinary protein loss and serum lipid levels (0,3,6 and 12 mg/kg). Twenty-four days after the injection of DR (6 mg/kg), serum cholesterol (Ch) and triglyceride (TG) levels markedly increased and free fatty acid level tended to decrease with no effects on liver lipid levels. Hepatic Ch synthesis from [14C]acetate in vitro increased by 2.1-fold, while exogenous Ch absorption slightly decreased. The clearance of intravenously injected [3H]Ch from the circulation was delayed. Hepatic fatty acid (FA) synthesis also increased by 2.7-fold, and hepatic TG lipase activity tended to decrease. KCD-232 improved the hypercholesterolemia and hypertriglyceridemia of DR-treated rats. The drug inhibited the elevated hepatic Ch synthesis and exogenous Ch absorption and thus improved the delayed Ch clearance from the circulation. KCD-232 markedly inhibited the elevated hepatic FA synthesis and stimulated both hepatic FA oxidation and lipoprotein lipase activity from the epididymal adipose tissue of the nephrotic rats. These results suggest that 1. DR-induced hypercholesterolemia is due to both an increased Ch synthesis in the liver and delayed clearance of Ch from the circulation; 2. DR-induced hypertriglyceridemia is caused by both an increased hepatic FA synthesis and depressed TG hydrolysis in the circulation; 3. KCD-232 improves the hypercholesterolemia by inhibiting the elevated Ch synthesis and Ch absorption from the gut; and 4. KCD-232 improves the hypertriglyceridemia by inhibiting the elevated hepatic FA synthesis and by stimulating both hepatic FA oxidation and TG hydrolysis activity in the circulation.

The Effects of Substituted Phenyl Glycosides and Corresponding Thioglycosies on the Growth of Transplanted Tumor in Mice

Forty substituted phenyl glycosides and corresponding thioglycosides were synthesized and tested the tumor growth effect against sarcoma 180 with these compounds. Most of the aromatic thioglycosides markedly increased the tumor growth in weight. It seems that compound XIV or XVII possessed the highest activity among the compounds examined. One of most active substances accelerate a growth of the body weight in young mice and a regeneration of the partial hepatectomy in rats. On the other hand, these compounds have no immunosuppressive or activating effect.