Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin.

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test.

novel 6'-n-aminoglycoside acetyltransferase AAC(6')-Iaj from a clinical isolate of Pseudomonas aeruginosa.

Pseudomonas aeruginosa NCGM1588 has a novel chromosomal class 1 integron, In151, which includes the aac(6')-Iaj gene. The encoded protein, AAC(6')-Iaj, was found to consist of 184 amino acids, with 70% identity to AAC(6')-Ia. Escherichia coli transformed with a plasmid containing the aac(6')-Iaj gene acquired resistance to all aminoglycosides tested except gentamicin. Of note, aac(6')-Iaj contributed to the resistance to arbekacin. Thin-layer chromatography revealed that AAC(6')-Iaj acetylated all aminoglycosides tested except gentamicin. These findings indicated that AAC(6')-Iaj is a functional acetyltransferase that modifies the amino groups at the 6' positions of aminoglycosides and contributes to aminoglycoside resistance of P. aeruginosa NCGM1588, including arbekacin.

Uptake of aminoglycoside antibiotics into brush-border membrane vesicles and inhibition of (Na+ + K+)-ATPase activity of basolateral membrane.

The effects of aminoglycoside antibiotics on plasma membranes were studied using rat renal basolateral and brush-border membrane vesicles. 3',4'-Dideoxykanamycin was bound to the basolateral membrane and brush-border membrane vesicles. They had a single class of binding sites with nearly the same constant, and the basolateral membrane vesicles had more binding sites than those of the brush-border membrane. Dideoxykanamycin B was transported into the intravesicular space of brush-border membrane vesicles, but not into that of basolateral membrane vesicles. The (Na+ + K+)-ATPase activity of the plasma membrane fraction prepared from the kidney of rat administered with dideoxykanamycin B intravenously decreased significantly. Aminoglycoside antibiotics entrapped in the basolateral membrane vesicles inhibited (Na+ + K+)-ATPase activity, but those added to the basolateral membrane vesicles externally failed to do so. The activity of (Na+ + K+)-ATPase was non-competitively inhibited by gentamicin. It is thus concluded that aminoglycoside antibiotics are taken up into the renal proximal tubular cells across the brush-border membrane and inhibit the (Na+ + K+)-ATPase activity of basolateral membrane. This inhibition may possibly disrupt the balance of cellular electrolytes, leading to a cellular dysfunction, and consequently to the development of aminoglycoside antibiotics' nephrotoxicity.

Inhibition of alkaline phosphatase activity and D-glucose uptake in rat renal brush-border membrane vesicles by aminoglycosides.

The binding of aminoglycoside antibiotics to, and their effects on, the plasma membrane were studied using isolated rat renal brush-border membrane vesicles. Dibekacin was noted to bind with brush-border membrane vesicles having a single class of many binding sites. 3H-labeled dibekacin binding was inhibited competitively by unlabeled dibekacin, gentamicin or amikacin. The inhibition constants obtained from the Dixon plots followed the order of gentamicin approximately equal to dibekacin greater than amikacin. The alkaline phosphatase activity of brush-border membrane vesicles was inhibited by gentamicin significantly, as was also observed by a histochemical study. Sodium-dependent D-glucose uptake by brush-border membrane vesicles was significantly inhibited by the addition of gentamicin.

Dibekacin intrarenal distribution characteristics and renal cortical elution kinetics. Comparison with gentamicin and tobramycin.

Dibekacin (3',4-dideoxykanamycin B) is a new semisynthetic aminoglycoside developed in Japan and one which has found wide clinical acceptance in that country. The antibacterial activity of this compound indicates that it is relatively similar to gentamicin. Since it appears that the intrarenal distributional characteristics and renal cortical kinetics of aminoglycosides provide some predictive information concerning the clinical incidence of nephrotoxicity, we designed a series of pharmacokinetic studies in healthy mongrel dogs which would define such kinetic information for dibekacin and would contrast the results with similar studies for gentamicin and tobramycin. The renal cortical kinetics of dibekacin, as developed in these studies, show that in a canine model the behavior of dibekacin is similar to that of gentamicin and significantly different from tobramycin. Dibekacin and gentamicin show reproducibly higher renal cortical tissue concentrations than tobramycin in both the acute infusion studies and multiple dosing studies. The results suggest that dibekacin may possess the same inherent nephrotoxic potential as that of gentamicin. In order to show any difference in clinical toxicity between gentamicin and dibekacin, a very extensive randomized, double-blind, prospective clinical trial of efficacy and toxicity will be needed.

Binding of 3',4'-dideoxykanamycin B to ATP and its related compounds.

The interactions of aminoglycoside, 3',4'-dideoxykanamycin B(DKB) with ATP and its related compounds were investigated. ATP, ADP, cyclic AMP and FAD bound to the DKB-conjugated Sepharose 4B column. The binding of DKB to ATP was also confirmed by equilibrium gel filtration. In the acidic pH region, the fluorescence of nucleotides was quenched by DKB. The Stern-Volmer plots showed that the molar ratios of the complexes were 1:1. The apparent stability constant was dependent on the number of the phosphate groups of nucleotides and was in the order of ATP greater than ADP greater than AMP.

Structures of enzymatically modified products of arbekacin by methicillin-resistant. Staphylococcus aureus.

Only a limited number of strains of methicillin-resistant Staphylococcus aureus (MRSA) moderately resistant to arbekacin (ABK) have been isolated clinically. Three inactivated products of ABK have been obtained by reaction with excess amounts of a crude enzyme preparation extracted from an ABK-resistant MRSA strain (MIC, 25 micrograms/ml). The 2"-O-phosphate was the major product together with small amounts of the 6'-N-acetate and the double modification product. The structures of these modification products were determined by MS and NMR spectral analyses.

Enzymatic 2'-N-acetylation of arbekacin and antibiotic activity of its product.

Aminoglycoside antibiotics (AGs) with a free 2'-amino group were subjected to enzymatic N-acetylation using a cell free extract that contained an aminoglycoside 2'-N-acetyltransferase, AAC (2'), derived from a kasugamycin-producing strain of Streptomyces kasugaensis. TLC and antibiotic assay of the incubated reaction mixtures revealed that a modified compound retaining substantial antibiotic activity was formed from arbekacin (ABK), while modification of the other AGs resulted in the marked decrease in antibiotic activity. Structure determination following isolation from a large scale reaction mixture showed the modified ABK to be 2'-N-acetyl ABK. In addition, 2',6'-di-N-acetyl ABK was formed as a minor product. The same conversion also occurred with dibekacin (DKB) resulting in the formation of 2'-N-acetyl DKB and 2',6'-di-N-acetyl DKB. MIC determination showed antibacterial activity (1.56 approximately 3.13 micrograms/ml) of 2'-N-acetyl ABK against a variety of organisms. By contrast, 2'-N-acetyl DKB showed no substantial antibiotic activity. We believe 2'-N-acetyl ABK has the highest and broadest antibacterial activity, compared with known N-acetylated AGs.

Ribosomal proteins S9 and L6 participate in the binding of [3H]dibekacin to E. coli ribosomes.

The degrees of binding of [3H]dibekacin to LiCl-treated cores of E. coli ribosomes were reduced by increasing LiCl concentrations. The 1.15 M LiCl core lost 70 approximately 80% of the original binding capacity. The antibiotic attachment to the 1.15 M LiCl core was restored by reconstitution with the split proteins (SP), which were obtained by the treatment of 70S ribosomes with LiCl at concentrations of 0.8 approximately 1.15 M. The basic proteins, split off during the transition from 0.4 M LiCl core to 0.8 approximately 1.15 M LiCl core, seemed to be involved in the drug binding. SP0.4 approximately 1.15, which was obtained by the treatment of the 0.4 M LiCl core with 1.15 M LiCl, was fractionated by CM-Sephadex C-25 column chromatography, and each fraction was assayed for protein composition and the capability of restoring the ability of the 1.15 M LiCl core to bind the drug. Of ribosomal proteins eliminated with 1.15 M LiCl, the addition of either S9 or L6 alone to the 1.15 M LiCl core was observed to restore approximately 50% of the binding as compared to the 70S ribosome alone, and both proteins restored about 70% of the binding. The results suggested that ribosomal proteins S9 and L6 were involved in the attachment of [3H]dibekacin to the ribosome. The antibiotic binding to the 70S ribosome and 1.15 M LiCl core reconstituted with S9 and L6 was considerably inhibited by unlabelled dibekacin or kanamycin, and partially inhibited by gentamicin or neomycin, but was not significantly affected by streptomycin or viomycin.

Pharmacokinetics of dibekacin in children with cystic fibrosis.

Dosages and pharmacokinetics of dibekacin were studied in a group of 15 children with cystic fibrosis (CF) and a control group of 9 children. The mean dosage regimens of dibekacin were respectively equal to 2.19 mg/kg and 2.16 mg/kg in the CF and non-CF patients. The mean peak serum values for the CF and non-CF groups were respectively equal to 5.3 micrograms/ml. Comparison of pharmacokinetic parameters (i.e. half-life, distribution volume, total body clearance and area under the curve) showed no significant difference between the two groups (p greater than 0.05).

Pharmacokinetics of dibekacin after intramuscular administration in man.

Pharmacokinetic behavior of dibekacin after intramuscular administration was studied in nine healthy volunteers by cross over administrations of three dosage levels (1 mg(pot.)/kg, 1.5 mg(pot.)/kg and 2 mg(pot.)/kg). Drug concentrations in serum and urinary recoveries were measured and those data were analysed pharmacokinetically. The serum levels of this drug were in proportion to dosage, and the pharmacokinetic parameters were almost the same through the three dosage levels. Then it was considered that the pharmacokinetic behaviors of dibekacin were almost the same within the range of these dosage levels. No significant difference was found between the body clearances calculated from the data of serum levels, and the renal clearances calculated from the data of urinary recovery and serum levels. Therefore it is considered that the elimination of the drug from blood is almost due to renal excretion.

[Pharmacokinetics of arbekacin in dogs. I. Serum concentration and urinary excretion].

A new aminoglycoside antibiotic, arbekacin (HBK) was intramuscularly and intravenously administered to dogs in order to study its pharmacokinetics in comparison to amikacin (AMK). The results obtained are summarized as follows. Serum concentrations of HBK were well correlated with dose levels. The dose-serum concentration relationship with HBK was similar to other aminoglycoside antibiotics. Biological half-lives of HBK and AMK were both about 1 hour in dogs. This was also similar to other aminoglycoside antibiotics. There was no significant difference in peak serum concentrations between 1 hour intravenous infusion and intramuscular injection of HBK at 2 mg/kg in dogs. Repetitive administration of HBK to dogs at 2 mg/kg twice a day for 14 days did not affect its serum concentration and biological half-life. Urinary excretion of HBK in dogs in 24 hours after administration accounted for about 80-90%.

[Pharmacokinetics of arbekacin in dogs. II. The distribution of arbekacin in tissues].

A new aminoglycoside antibiotic, arbekacin (HBK) was intramuscularly and intravenously administered to Beagle dogs at a dose of 10 mg/kg to study its distribution into various tissues. The results obtained are summarized as follows. Biological half-lives of HBK in serum after intramuscular and intravenous injection were 1.15 hours and 1.00 hour after administration, respectively. These half-lives were similar to the results obtained in previous studies. Maximum concentrations of HBK in tissues and biological fluids after intramuscular injection were the highest in kidney followed by vagina, serum, urinary bladder, uterus, ovarium, lung, parotid gland, trachea, tonsil, gall bladder, pericardiac fluid, thymus, muscle, heart, liver, mandibular gland, bile, aqueous humor, pancreas, cerebrospinal fluid and brain, in this order. Maximum concentrations were found at 4 hours in trachea, aqueous humor and cerebrospinal fluid, and at 2 hours in pericardiac fluid. In other tissues and biological fluids, they were obtained at 0.5-1 hour. Maximum concentrations in tissues and biological fluids after 1 hour intravenous injection were the highest in kidney followed by serum, ovarium, vagina, lung, parotid gland, urinary bladder, uterus, spleen, thymus, pericardiac fluid, gall bladder, trachea, tonsil, heart, liver, pancreas, muscle, mandibular gland, bile, aqueous humor, cerebrospinal fluid and brain in this order. Maximum concentrations were found at 4 hours in trachea, cerebrospinal fluid and bile, and at 2 hours in pericardiac fluid. In other tissues and biological fluids, they were obtained at the end of the infusion. Maximum concentrations in these biological fluids after both intramuscular and intravenous injection were similar to serum concentrations except kidney, trachea, and other biological fluids.(ABSTRACT TRUNCATED AT 250 WORDS)

[Studies of dibekacin eye-drops. Intraocular penetration (author's transl)].

Ocular tissue levels of dibekacin (DKB) were studied in rabbits after instillation of 0.3% DKB eye-drops five times every 5 minutes. (1) In normal eyes, DKB levels were determined in all of the outer parts of eye and some of inner parts such as aqueous humor and vitreous body. Such levels were relatively higher than those of gentamicin. (2) In cauterized eyes by NaOH tissue levels were several times higher than those obtained in normal eyes. (3) Considering these results and MIC levels against both Gram-positive and Gram-negative bacteria such as Pseudomonas aeruginosa or Staphylococcus aureus, it is suggested that 0.3% DKB eye-drops will show effectiveness in clinical use.

[Fundamental studies of combination of antibiotics, especially on pharmacokinetics. III. Combination of ampicillin and dibekacin (author's transl)].

1. Pharmacokinetics was investigated when ampicillin (ABPC) (50 mg/kg) and dibekacin (DKB) (5 mg/kg), in combination with pre- or post-treatment, were intravenously injected to the rat or rabbit. 2. The antibiotics in the samples were separated by the paper-electrophoretic technique and then concentrations were determined by the cup thin-layer plate method using Bacillus subtilis ATCC 6633 as the test organism. 3. The biological half-life of DKB was prolonged in pretreatment with ABPC and that of ABPC was shortened in pretreatment with DKB. An initial level of ABPC was elevated. Similar tendency was observed in both of the rat and rabbit. 4. Urinary excretion rates of both antibiotics in the combining group tended to decrease compared with the single administration group. 5. Binding of ABPC to serum proteins was competitively inhibited by DKB. Binding of DKB to serum proteins increased.

[Fundamental and clinical studies on intravenous drip infusion of dibekacin in the pediatric field].

Dibekacin (DKB), an antibiotic of aminoglycoside group, was administered at 4 different dosages of 0.5, 1.0, 1.5 and 2.0 mg/kg as intravenous drip infusion taking 30 minutes or 1 hour. For each dose level, 3 cases each were used out of 24 boys from 1 year and 1 month to 14 years and 7 months of age, and serum concentrations as well as urinary concentrations and recovery rate were determined. After removed of 4 cases unassessable of therapeutic efficacy, 7 cases consisting of 1 case of chronic bronchitis, 1 case of lung abscess and 5 cases of urinary tract infections were treated with DKB at a mean daily dosage of 3.3 mg/kg in 2 or 3 divided doses as intravenous drip infusion taking 30 minutes or 1 hour. The mean treatment period was 7 days. The clinical and bacteriological results were analyzed in these cases and for analysis of side effects drop out cases were also included. The following results were obtained. Following 30 minutes intravenous drip infusion of DKB at 0.5, 1.0, 1.5 and 2.0 mg/kg, the serum concentration peaked at the end of infusion for all dose levels. The highest peak concentration of 9.17 mcg/ml was obtained for the dose level of 2.0 mg/kg. The highest dosage with which serum concentration does not exceed concentrations of 10 to 12 mcg/ml was found to be 2.0 mg/kg. The mean highest serum concentrations obtained were 1.65, 3.49, 5.40 and 8.67 mcg/ml for the dosages of 0.5, 1.0, 1.5 and 2.0 mg/kg, respectively, and the mean AUCs determined by the two-compartment model were 2.99, 6.04, 10.5 and 14.2 mcg X hr/ml, respectively, showing dose response relation in terms of peak concentration and AUC among groups. The mean T1/2 values for each dosage were 1.55, 1.54, 1.77 and 2.03 hours, respectively, with a longer tendency in T1/2 for the dose level of 2.0 mg/kg with unknown cause. When 0.5, 1.0, 1.5 and 2.0 mg/kg of DKB were infused taking 1 hour, the peak of serum concentration appeared also at the end of the infusion. The highest concentration was obtained with 2.0 mg/kg and it was 7.02 mcg/ml. Considering from the concentrations obtained for 0.5 mg/kg and 1.0 mg/kg groups the highest dosage at which the serum concentration does not exceed 10 to 12 mcg/ml was estimated to be 2.5 mg/kg.(ABSTRACT TRUNCATED AT 400 WORDS)

[Absorption, distribution, and excretion of arbekacin after intravenous and intramuscular administration in rats].

Absorption, distribution, and excretion of arbekacin (HBK) were studied in rats after intravenous or intramuscular administration of HBK at a dose of 10 mg/kg or 20 mg/kg. Elimination half-lives of HBK were 0.69 hour for bolus intravenous administration, 0.55 hour for constant rate intravenous infusion, and 0.57 hour for intramuscular administration. Cumulative urinary excretions within 24 hours after administration were 74.7% of the dose for bolus intravenous administration, and 79.1% of the dose for intramuscular administration. No significant difference was observed in the cumulative urinary excretions between the 2 administration routes. Cumulative biliary excretions within 24 hours after administration were around 0.1% of doses regardless administration routes, bolus intravenous or intramuscular administration. The tissue or organ distribution of HBK after bolus intravenous administration was similar to that after intramuscular administration. The drug was distributed most abundantly into the kidney followed by plasma and the lung. The distribution of the drug into the liver was the least among the 6 tissues or organs examined in this study. The protein binding of HBK was studied by an equilibrium dialysis method at three different concentrations of HBK, 5, 10, and 20 micrograms/ml. Binding ratios of HBK to human serum, human serum albumin, and rat serum were less than 15%.

[Clinical studies on dibekacin for infectious diseases following intramuscular and intravenous drip administration. Concentration in infected tissues and clinical responses (author's transl)].

An antibiotic drug of aminoglycoside group, dibekacin (DKB) for parental use was used in 48 patients hospitalized due to acute or subacute infection of abdominal organs: 36 appendicitis, 9 cholecystitis and 3 others. DKB in a dose of 100 mg was given intramuscularly in 38 cases, and in 10 cases was given intravenously by single or drip infusion before the operation. The materials of A-bile, B-bile, wall of gallbladder, the appendix wall, ascites with pus and serum were taken during the operation. DKB concentration was measured by bioassay method with Bacillus Subtilis ATCC 6633 strain. With a few marked exceptions, DKB concentration in B-bile were higher than those in A-bile. DKB concentrations in gallbladder wall and appendix wall were directly proportional to the degree of pathological changes of inflammation. DKB concentrations in infected tissues after intravenous drip infusion, they were higher relatively than those after intramuscular administration. DKB concentrations in serum after intravenous drip infusion reached to peak immediately the end of infusion, and in the infected tissue they reached to peak at the same time and stayed for a relatively long time, then they were declined slowly. For the therapeutic purpose, DKB was given to the 6 patients with acute peritonitis of the above cases. DKB in a dose of 100 mg were administered by intravenous drip infusion for 2 hours, twice in a day for 3 - 10 days. Clinical response was excellent in 2 cases, good in 3 cases, fair in 1 case and poor in none. No adverse effect was observed. Therefore, it was supposed that DKB could be used safely by intravenous drip infusion.

[Concentration of dibekacin sulfate in bone (author's transl)].

The concentrations in serum, cancellous and cortical bones of dibekacin sulfate were measured in 16 cases. Dibekacin sulfate rapidly penetrates into bone. The peak concentration in serum and cancellous bone is reached within 1 hour after intramuscular injection and the concentration in cancellous bone parallels that in serum. The concentration in cortical bone increases with time. Cancellous bone has higher concentration than cortical bone. Dibekacin sulfate is suitable for prevention of infection in orthopaedic surgery, because it maintains a bactericidal level in bone for enough time.

[Aerosol administration of dibekacin--the sputum concentration].

Serum concentrations of dibekacin (DKB), sisomicin (SISO) and gentamicin (GM) were measured in 3 rabbits after intratracheal administration through the transtracheal teflon tube. The peak serum levels (average) were 106 micrograms/ml (administrated 100 mg DKB for injection), 148 micrograms/ml (administrated 100 mg DKB solution), 166 micrograms/ml (administrated 100 mg SISO solution) and 80 micrograms/ml (administrated 80 mg GM solution). Serum concentrations and urine excretions of DKB were measured in 3 volunteers after aerosol administration using ultrasonic nebulizer. The peak serum levels (average) were 4.6 micrograms/ml (administrated 100 mg DKB for injection) and 3.1 micrograms/ml (administrated 100 mg DKB solution). The urine excretions (average) were 3.7 mg and 4.3 mg respectively during 6 hours. Before and after administration of DKB aerosol the spirogram and flow-volume curve were examined in the volunteers. But the examinations showed no changes. Sputum concentrations were measured in 1 patient with chronic bronchobronchiolitis after administration of DKB aerosols using the ultrasonic nebulizer. The highest sputum concentration was acquired immediately after nebulization and the sputum levels decreased gradually while time passed. Six patients with the lower respiratory tract infections were treated with DKB aerosol therapy and the utility rate was 80%.