Interspecies scaling of excretory amounts using allometry - retrospective analysis with rifapentine, aztreonam, carumonam, pefloxacin, miloxacin, trovafloxacin, doripenem, imipenem, cefozopran, ceftazidime, linezolid for urinary excretion and rifapentine, cabotegravir, and dolutegravir for fecal excretion.

1. Interspecies allometry scaling for prediction of human excretory amounts in urine or feces was performed for numerous antibacterials. Antibacterials used for urinary scaling were: rifapentine, pefloxacin, trovafloxacin (Gr1/low; <10%); miloxacin, linezolid, PNU-142300 (Gr2/medium; 10-40%); aztreonam, carumonam, cefozopran, doripenem, imipenem, and ceftazidime (Gr3/high; >50%). Rifapentine, cabotegravir, and dolutegravir was used for fecal scaling (high; >50%). 2. The employment of allometry equation: Y = aW(b) enabled scaling of urine/fecal amounts from animal species. Corresponding predicted amounts were converted into % recovery by considering the respective human dose. Comparison of predicted/observed values enabled fold difference and error calculations (mean absolute error [MAE] and root mean square error [RMSE]). Comparisons were made for urinary/fecal data; and qualitative assessment was made amongst Gr1/Gr2/Gr3 for urine. 3. Average correlation coefficient for the allometry scaling was >0.995. Excretory amount predictions were largely within 0.75- to 1.5-fold differences. Average MAE and RMSE were within ±22% and 23%, respectively. Although robust predictions were achieved for higher urinary/fecal excretion (>50%), interspecies scaling was applicable for low/medium excretory drugs. 4. Based on the data, interspecies scaling of urine or fecal excretory amounts may be potentially used as a tool to understand the significance of either urinary or fecal routes of elimination in humans in early development.

Antibiotic uptake by cultured Atlantic cod leucocytes and effect on intracellular Francisella noatunensis subsp. noatunensis replication.

The granuloma disease caused by Francisella noatunensis subsp. noatunensis in farmed Atlantic cod has not been successfully treated by use of antibacterials, even when antibacterial resistance testing indicates a sufficient effect. The reason for this treatment failure may be the intracellular existence of the bacteria within immune cells, mainly macrophages. To investigate the effect of antibacterials on intracellular Francisella replication, we established a protocol for the detection of drugs within Atlantic cod immune cells using high-performance liquid chromatography (HPLC). When the uptake and intracellular concentrations of oxolinic acid and flumequine were analysed in isolated adherent head kidney leucocytes (HKLs) by HPLC, we found that uptake was rapid and the intracellular concentrations reflected the extracellular exposure concentrations. To investigate the effect of the antibacterial compounds on intracellular bacterial replication, adherent HKLs experimentally infected with the bacteria were analysed using flow cytometry and intracellular labelling of bacteria by specific antibodies. We found that flumequine did not inhibit intracellular bacterial replication. Unexpectedly, the results indicated that the intracellularly effiacy of the drug was reduced. The HPLC method used proved to be highly applicable for accurate determination of intracellular drug concentrations. When combined with sensitive and specific flow cytometry analyses for identification and measurement of intracellular bacterial replication, we suggest that this approach can be very valuable for the design of antibacterial treatments of intracellular pathogens.

Effects of light regime and oxygen profile on transformation of oxolinic acid in pond sediment.

This study investigated the effects of light (visible light - 5800 lux, 24h) or dark regime and aerobic or anaerobic condition on the decay of added oxolinic acid (OA) at 5, 10 and 20 mg L(-1) in eel pond sediment. An asymptotic decaying exponential model C(t)=C(min)+C(o) × exp (-k × t) was used to facilitate quantitative approach to OA transformation, where C(t) is the concentration of OA after t days, C(min) the estimated level-off concentration of OA residue, C(o) the concentration of added OA and k the decaying coefficient. OA decayed faster under light (C(min)=4.6 mg L(-1)) than under dark (C(min)=7.8 mg L(-1)) and also decayed faster under aerobic (C(min)=4.0 mg L(-1)) than under anaerobic condition (C(min)=8.5 mg L(-1)). C(min) increased with C(o). Sundrying and tilling eel pond bottom should be able to reduce OA residue significantly.

In vivo modulation of immune response and antioxidant defense in Atlantic cod, Gadus morhua following oral administration of oxolinic acid and florfenicol.

Oxolinic acid and florfenicol are the commonly used antibiotics for the treatment of bacterial diseases in Atlantic cod, Gadus morhua. The changes in selected innate humoral immune response of the fish, bacterial proliferation in serum and transcriptional activity of selected immune and antioxidant defense-related genes following oral administration of these antimicrobial compounds were evaluated. Juvenile cod (75-100 g) were fed commercial feed coated with either florfenicol (10 mg kg(-1) fish, active ingredient) or oxolinic acid (20 mg kg(-1) fish, active ingredient) at a ration of 0.5% body weight for 10 days. Whole blood and serum samples were collected on the 10th day of feeding the antibiotics and at 3, 5 and 10 days after their withdrawal. Serum protein was significantly higher in fish at the 10th day post-withdrawal of both antibiotics. Florfenicol-fed fish had lower myeloperoxidase activity at 3 days post-withdrawal, while there were differential effects on alkaline phosphatase activity. Vibrio anguillarum and Aeromonas salmonicida were significantly reduced in the sera of antibiotics-fed fish until the 5th day post-withdrawal. Florfenicol could inhibit V. anguillarum better than oxolinic acid, while A. salmonicida was more susceptible than V. anguillarum upon treatment with both antibiotics. Furthermore, transcriptional profiles of selected genes related to bacterial defense, inflammation and antioxidant defense were dependent on the type of antibiotics that was administered and the time of sampling. These results indicate that oral administration of antibiotics modulates the immune response and antioxidant defense in Atlantic cod and these may, in turn, affect their ability to resist bacterial pathogens.

Study of the evolution and degradation products of ciprofloxacin and oxolinic acid in river water samples by HPLC-UV/MS/MS-MS.

The results of a degradation study of the (fluoro)quinolone antibiotics ciprofloxacin and oxolinic acid in river water samples are presented in this paper. The decomposition of these compounds at ambient temperature was monitored during five months by HPLC-UV, and two consecutive degradation processes (photo- and bio/chemical-degradation) were observed in both cases although with different degradation rates. Ciprofloxacin was completely degraded after 3 months whereas 80% of oxolinic acid remained unaltered after five months of storage. The analysis of the degradation compounds formed was carried out using MS and tandem MS-MS, allowing the identification of four new ciprofloxacin transformation products not previously described in the literature. Possible degradation pathways for this antibiotic in river water are proposed.

Preferential binding of quinolones to DNA with alternating G, C / A, T sequences: a spectroscopic study.

The binding of quinolones, nalidixic acid (Nal), oxolinic acid (Oxo) with double stranded polynucleotides was undertaken by using UV-melting, UV-Vis absorption, fluorescence and CD spectroscopic techniques. The binding of Nal or Oxo to the polynucleotides under low-salt buffer conditions were determined for poly (dA).(dT), poly [d(A-T)], poly (dG).(dC), poly [d(G-C)] and E. coli DNA. The fluorescence data were analyzed using a previously established two step mechanism with two different DNA-Drug complexes [Rajeswari et al., Biochemistry 26, 6825-31 (1987)]. The first complex [DN](1) with a binding constant K(1), is formed where the interactions are 'nonspecific' and complex [DN](2) with a binding constant K(2), is formed where the interactions are "specific" which involve (additional) hydrophobic type of interactions like 'stacking' of the drug and the overall association constant is represented as K(=K(1)K(2)). The order of binding for Nal and Oxo is: poly [d(G-C)] > poly [d(A- T)] > E. coli > poly (dG).(dC) > poly (dA).(dT). Interaction of quinolones seems to be preferential in the alternating G, C or A, T stretches of DNA than those of non-alternating. Within any alternating or non-alternating in DNA sequences the G, C rich sequences have distinctly greater binding than A, T sequences. The overall association constant data (K) reveal higher binding of Oxo to DNA compared to Nal to any given polynucleotide investigated; which also explains the higher antibacterial potency of Oxo. Changes in the absorption difference spectra and in circular dichroic spectra also manifest these results. As the melting temperatures of the polynucleotides were only marginally raised in presence of the quinolone, we rule out the possibility of 'classical intercalation' of the drug. Amino group of guanine facilitates the binding of quinolones and therefore has the greater binding with the DNA. However, poly (dG).(dC) is known to exist in 'A' conformation which is not adopted by quinolones as in the case of poly (dA).(dT). Present results suggest that Nal or Oxo bind to DNA in a non-classical fashion which is partially stacking in nature.

A unique type II topoisomerase mutant that is hypersensitive to a broad range of cleavage-inducing antitumor agents.

Bacteriophage T4 provides a useful model system for dissecting the mechanism of action of antitumor agents that target type II DNA topoisomerases. Many of these inhibitors act by trapping the cleavage complex, a covalent complex of enzyme and broken DNA. Previous analysis showed that a drug-resistant T4 mutant harbored two amino acid substitutions (S79F, G269V) in topoisomerase subunit gp52. Surprisingly, the single amino acid substitution, G269V, was shown to confer hypersensitivity in vivo to m-AMSA and oxolinic acid [Freudenreich, C. H., et al. (1998) Cancer Res. 58, 1260-1267]. We purified this G269V mutant enzyme and found it to be hypersensitive to a number of cleavage-inducing inhibitors including m-AMSA, VP-16, mitoxantrone, ellipticine, and oxolinic acid. While the mutant enzyme did not exhibit altered DNA cleavage site specificity compared to the wild-type enzyme, it did display an apparent 10-fold increase in drug-independent DNA cleavage. This suggests a novel mechanism of altered drug sensitivity in which the enzyme equilibrium has been shifted to favor the cleavage complex, resulting in an increase in the concentration of cleavage intermediates available to inhibitors. Mutations that alter drug sensitivities tend to cluster within two specific regions of all type II topoisomerases. Residue G269 of gp52 lies outside of these regions, and it is therefore not surprising that G269V leads to a unique mechanism of drug hypersensitivity. We believe that this mutant defines a new category of type II topoisomerase mutants, namely, those that are hypersensitive to all inhibitors that stabilize the cleavage complex.

The looped domain organization of the nucleoid in histone-like protein defective Escherichia coli strains.

We have investigated the major Escherichia coli histone-like proteins (H-NS, HU, FIS, and IHF) as putative factors involved in the maintenance of the overall DNA looped arrangement of the bacterial nucleoid. The long-range architecture of the chromosome has been studied by means of an assay based on in vivo genomic fragmentation mediated by endogenous DNA gyrase in the presence of oxolinic acid. The fragmentation products were analysed by CHEF electrophoresis. The results indicate that in vivo a large fraction of the bacterial chromatin constitutes an adequate substrate for the enzyme. DNA fragments released upon oxo-treatment span a size range from about 1000 kb to a limit-size of about 50 kb. The latter value is in excellent agreement with the average size reported for bacterial chromosomal domains. The DNA gyrase-mediated fragmentation does not appear to be significantly altered in strains depleted in histone-like proteins as compared to an E. coli wild type strain. This suggests that these proteins may not represent critical determinants for the maintenance of the supercoiled loop organisation of the E. coli chromosome.

gyrB-225, a mutation of DNA gyrase that compensates for topoisomerase I deficiency: investigation of its low activity and quinolone hypersensitivity.

The B subunit of DNA gyrase (GyrB) consists of a 43 kDa N-terminal domain, containing the site of ATP binding and hydrolysis, and a 47 kDa C-terminal domain that is thought to play a role in interactions with GyrA and DNA. In cells containing a deletion of topA (the gene encoding DNA topoisomerase I) a compensatory mutation is found in gyrB. This mutation (gyrB-225) results in a two amino acid insertion in the N-terminal domain of GyrB. We found that cells containing this mutation are more sensitive than wild-type cells to quinolone drugs with respect to bacteriostatic and lethal action. We have characterised the mutant GyrB protein in vitro and found it to have reduced DNA supercoiling, relaxation, ATPase, and cleavage activities. The mutant enzyme is up to threefold more sensitive to quinolones than wild-type. The mutation also increases the affinity of GyrB for GyrA and DNA, while the affinity of quinolone for the enzyme-DNA complex is unaffected. We propose that the loss in activity is due to misfolding of the GyrB-225 protein, providing an example in which misfolding of one protein, DNA gyrase, suppresses a deficiency of another, topoisomerase I. The increased quinolone sensitivity is proposed to be a consequence of an altered conformation of the protein that renders quinolones better able to disrupt, rather than generate, gyrase-drug-DNA complexes.

1-octanol/water distribution coefficient of oxolinic acid: influence of pH and its relation to the interaction with dissolved organic carbon.

The distribution of oxolinic acid (OA) between 1-octanol and buffers at a broad range of pH values was studied and found to decrease with increasing pH. The distribution coefficient to dissolved organic carbon (DOC), log DDOC, was estimated and compared with an experimentally derived log DDOC, showing the experimental value to be almost three orders of magnitude higher. Because only the neutral molecule is assumed to distribute to 1-octanol, the interaction with DOC is considered to be electrostatic in character.

Behavioural and neurochemical evidence that the antimicrobial agent oxolinic acid is a dopamine uptake inhibitor.

The antimicrobial agent oxolinic acid, injected i.p. in mice, induced a dose dependent increase in locomotor activity. This stimulation culminated at the 32 mg/kg dose and became smaller for higher doses (64-128 mg/kg). When opposed to increasing doses (50-100-200 microg/kg i.p.) of haloperidol (D2 dopamine receptor antagonist), the stimulant locomotor effect of 32 mg/kg oxolinic acid was not significantly reversed. On the contrary increasing doses (7.5-15-30 microg/kg s.c.) of SCH 23390 (D1 dopamine receptor antagonist) inhibited the stimulant locomotor effect. In mice made completely akinetic by a pretreatment with reserpine (4 mg/kg s.c., 18 h before testing), dexamphetamine (2 mg/kg s.c.) reversed this akinesia and even displayed a stimulant activity, similar to that observed in mice not treated by reserpine. On the contrary, oxolinic acid (32 mg/kg) did not reverse the reserpine induced akinesia and even opposed the reversion induced by dexamphetamine. In a synaptosomal fraction prepared from striatum of rats, oxolinic acid inhibited the 3H dopamine uptake with an IC50 = 4.3+/-0.6 x 10(-6) M. Finally, in mice injected i.v. with a tracer dose of 3H WIN 35428 (1 microCi) (a dopamine uptake blocker), 32 mg/kg oxolinic acid, i.p. administered, reduced by about 50% the specific binding of the radioligand to striatal dopamine carriers. It is concluded that the stimulant locomotor effect of oxolinic acid depends on the blockade of the neuronal dopamine uptake complex.

Determination and confirmation of identities of flumequine and nalidixic, oxolinic, and piromidic acids in salmon and shrimp.

A previously published liquid chromatographic (LC) method for determining residues of flumequine (FLU) and nalidixic (NAL), oxolinic (OXO), and piromidic (PIR) acids in catfish tissue was applied to salmon and shrimp muscle. Identities of all 4 residues in salmon and shrimp were confirmed by gas chromatography/mass spectrometry (GC/MS). The tissue is homogenized with acetone, the acetone extract is defatted with hexane, and the quinolones are extracted into chloroform. The extract is further purified by first partitioning into base and then back-extracting from a solution acidified to pH 6.0. Analytes are determined by LC with simultaneous UV and fluorescence detection. Muscle tissue was fortified with each quinolone at 5, 10, 20, 40, and 80 ng/g. Average recoveries and relative standard deviations (RSDs) for salmon, which represent an average of the 5 levels for each analyte, ranged from 75.9 to 90.8% and from 2.25 to 6.40%, respectively. Average recoveries and RSDs for shrimp ranged from 81.3 to 91.2% and from 7.34 to 10.7%, respectively. Identities of OXO, FLU, NAL, and PIR were confirmed in extracts of salmon and shrimp tissue fortified at 10 ng/g by determination of decarboxylated quinolones by GC/MS. Four diagnostic ions were monitored for OXO, FLU, and PIR, and 5 ions were monitored for NAL. All ion relative abundances were within 10% of those calculated for standard decarboxylated quinolones. Optimum conditions for decarboxylation and GC/MS confirmation are given.

Effects of transcription and translation on gyrase-mediated DNA cleavage in Escherichia coli.

Gyrase-mediated DNA cleavage on plasmid DNAs was measured in Escherichia coli treated with oxolinic acid. On pBR322 DNA, gyrase cleavage sites were concentrated in the region between the 3'-ends of the tetA and bla genes. The preferential cleavage in this region was dependent on RNA transcription and the divergent orientation of these two transcription units. The enhanced gyrase cleavage also required translation; chloramphenicol treatment or the insertion of a translation terminator within the 5'-proximal region of the tetA gene abolished the enhanced cleavage. We suggest that the enhanced gyrase cleavage may reflect the changes in local DNA supercoiling during RNA transcription as gyrase cleavage in vitro was shown to be sensitive to the supercoiling state of DNA. The effects of transcription and translation on gyrase cleavage can best be explained by the twin-supercoiled-domain model of transcription (Liu, L. F., and Wang, J. C. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7024-7027).

[Absorption, distribution, excretion, and metabolism of 14C-miloxacin in female rats (author's transl)].

Absorption, distribution, excretion, and metabolism of 5,8-dihydro-5-methoxy-8-oxo-2H-1, 3-dioxolo[4,5-g]quinoline-7-carboxylic acid(miloxacin), a new antimicrobial agent, were studied in female rats by using 14C-miloxacin which was administered orally to the animals in a dose of 50 mg/kg. 14C-Miloxacin was absorbed rather fast and the radioactivity of 14C distributed widely in a variety of tissues. Peak concentrations of 14C in serum and tissues occurred 1 to 2 hr after dosing, and were approximately 60 micrograms equivalent of miloxacin per ml or g in serum, liver and kidney. Excretion of 14C in urine and feces was fast, and recoveries of 14C during 48 hr period were approximately 30% in urine and 60% in feces. Concentrations of intact 14C-miloxacin were higher in serum and kidney while lower in liver. Major metabolites in excreta were the demethoxy derivative (M-1) and the glucuronide of miloxacin; and as minor metabolites five other metabolites were identified. As sex differences, the following facts were observed; concentrations of 14C in serum and tissues were generally 1.2 to 1.6 times higher in female rats those in male rats, and the capacity to metabolize miloxacin, especially in the glucuronic acid conjugation, was rather lower in female rats than that in male rats.

Absorption and excretion of miloxacin in mice, rats, and dogs.

Miloxacin, a synthetic antibacterial agent structurally related to oxolinic acid, has a broad spectrum of activity in vitro against gram-negative bacteria and considerable activity in vivo against infections with these bacteria. These observations led to studies on the absorption and excretion of miloxacin in mice, rats, and dogs after administration of a single oral dose. Studies on oxolinic acid have been included for comparison. Peak serum levels of miloxacin, attained 1 h after administration of 20, 50, and 100 mg/kg to rats and dogs, were approximately 20, 40, and 60 micrograms/ml, respectively. Peak levels in mice receiving the same dose were 15, 60, and 80 micrograms/ml at 0.5 h. Peak serum levels of oxolinic acid were attained 0.5 to 1 h later than the above times at comparable doses and were one-half to one-fourth those of miloxacin. Urinary recovery of miloxacin at the above doses ranged from 3.2 to 6.5% during the 24-h posttreatment period. Recoveries of oxolinic acid were one-half to one-fifth those of miloxacin. At a 50-mg/kg dose, rats excreted 4.6% of the miloxacin in bile in the 20-h posttreatment period.

Mode of action of a new nalidixic acid derivative, AB206.

A new chemotherapeutic agent, AB206, shows potent antibacterial activity against gram-negative bacteria, including most of the nalidixic acid-resistant strains tested. It strongly inhibits deoxyribonucleic acid (DNA) synthesis in Escherichia coli, but only slightly inhibits ribonucleic acid and protein synthesis. Its activity on DNA synthesis in vivo and in vitro is greater than that of nalidixic acid. AB206 also strongly inhibits in vivo DNA synthesis in nalidixic acid-susceptible and -resistant clinical isolates of Proteus and Serratia. AB206 shows high penetrability into E. coli cells, as demonstrated by antibacterial activity with or without ethylenediaminetetraacetic acid, inhibition of in vivo and in vitro DNA synthesis, and uptake of the drug into cells, as compared to nalidixic acid. It appears that the high antibacterial activity of AB206 may be explained both by its potent inhibitory action against DNA synthesis and also by its high penetrability into bacterial cells.