Ciprofloxacin enhances T cell function by modulating interleukin activities.

Ciprofloxacin (CIP) is a quinolone carboxylic acid derivative with a broad spectrum of antibacterial activity. CIP (0.1-30 micrograms/ml) enhanced DNA synthesis of mouse spleen cells and human peripheral blood lymphocytes (PBL) that had been activated with T cell mitogens or with alloantigens. In addition, CIP increased the amount of IL-2 found in the supernatants of phytohaemagglutinin (PHA)-stimulated human PBL. The presence of CIP in the medium (0.3-10 micrograms/ml) increased the levels of IL-1 found in the culture supernatants of adherence-enriched mouse macrophages, human monocyte/macrophages and a human monocytic cell line stimulated with lipopolysaccharide. In contrast there was no effect of CIP on the release of IL-1 by freshly isolated human monocytes or by cells of the keratinocyte line, A431. CIP alone had no influence on the basal release of IL-2 by NOB-1 cells, a T cell line that responds to IL-1 with an increase in IL-2 synthesis, but, in combination with recombinant IL-1, CIP significantly enhanced the release of IL-2 by these cells. The results of this study suggest that CIP modulates the immune response at two levels--the production of IL-2 by activated T cells and the production of IL-1 by activated monocyte/macrophages. However, CIP did not affect the primary antibody response in vitro or in vivo against sheep erythrocytes and ovalbumin respectively. Thus the enhancing action of ciprofloxacin on the immune system appears to be restricted to T cell function and macrophage/T cell interactions.

Bactericidal activity of ciprofloxacin, norfloxacin and ofloxacin in serum and urine after oral administration to healthy volunteers.

A 200 mg oral dose of ciprofloxacin, norfloxacin or ofloxacin was administered to six healthy male volunteers in a three way cross-over study in order to examine the kinetics in humans in relation to the bactericidal activity in serum and urine. Serum concentrations for ciprofloxacin were similar to norfloxacin and lower than ofloxacin. Despite this fact, ciprofloxacin showed the highest serum bactericidal titers compared to norfloxacin and ofloxacin when serum-resistant Escherichia coli C14 was used as a test organism. These results correlate with observations from timekill curve studies in human whole blood, where ciprofloxacin showed superior bactericidal activity compared to norfloxacin or ofloxacin. The amounts of unchanged drug excreted in urine (48 h period) were found to be 35%, 24% and 77% for ciprofloxacin, norfloxacin and ofloxacin respectively, indicating different excretion kinetics. The volumes of urine excreted in the different collection periods were comparable for the three drugs tested. Mean urine concentrations for ciprofloxacin were higher during the 0 to 4 h collection periods, whereas ofloxacin was excreted into the urine over a longer time period. Measurements of urine bactericidal activity showed that ciprofloxacin had the highest titers during the early collection periods, whereas the prolonged excretion of ofloxacin did not result in higher urine bactericidal titers, compared to ciprofloxacin.

Oral ciprofloxacin treatment for Salmonella typhimurium infection of normal and immunocompromised mice.

Oral treatment of Salmonella typhimurium infection with ciprofloxacin was compared with conventional chemotherapy with ampicillin or chloramphenicol in normal (CFW1) and immunocompromised (C57BL/6) mice. Administration of the antibiotics for 12 days reduced the number of bacteria in livers and the mortality of C57BL/6 mice significantly. Ciprofloxacin was considerably more effective than ampicillin in prolongation of the mean survival time of these mice. Similar to conventional chemotherapeutic agents, ciprofloxacin did not prevent fatal disease in most C57BL/6 mice when the treatment lasted 12 days only. On the other hand, ciprofloxacin cured lethal S. typhimurium illness in immunocompromised mice after long-term oral chemotherapy for 26 days at a dosage of 100 mg/kg twice a day. This was not achieved by either ampicillin or chloramphenicol. In normal mice, 12 days of therapy with ciprofloxacin was sufficient for a significant decrease in both the number of viable bacteria in livers and the mortality of lethally infected mice. The results provide a basis for an alternative antibiotic treatment by the oral route in immunocompromised hosts with systemic infections.

Efficacy of ciprofloxacin in stationary-phase bacteria in vivo.

The granuloma pouch model in mice infected with Escherichia coli or Pseudomonas aeruginosa was used to investigate the bactericidal effect of ciprofloxacin in vivo on bacteria in the stationary growth phase. Ciprofloxacin caused a rapid decline in the number of colony-forming units (cfu) of E. coli shortly after initiation of therapy (40 mg/kg, intraperitoneally). Ciprofloxacin was more effective than norfloxacin or pefloxacin and comparable in efficacy to ofloxacin. The drugs penetrated well into the pouch exudate, exceeding the minimal inhibitory concentrations (MICs) of the infecting organisms. The concentrations of pefloxacin or ofloxacin were higher than those of norfloxacin or ciprofloxacin. Ciprofloxacin also showed good killing effects in pouches infected with one strain of P. aeruginosa (ICB 7453, MIC of 0.06 micrograms/ml). However, with another P. aeruginosa strain (ICB 7933), which has a MIC of 0.5 micrograms/ml, killing of stationary cells in vivo was not very pronounced. Electron microscopic evaluation of the pouch exudate revealed that phagocytosed and non-phagocytosed E. coli cells were severely damaged in comparison with untreated control cells. The earliest ultrastructural changes could be observed 15 minutes after initiation of therapy. The results demonstrate that ciprofloxacin is effective in mice for the treatment of a local inflammatory abscess harboring a stationary population of E. coli or P. aeruginosa. This specific kind of killing occurs in vivo when drug concentrations are at least eight to 10 times higher than the MIC.

Antibacterial activity of the metabolites of ciprofloxacin and its significance in the bioassay.

The antibacterial activity of the metabolites of ciprofloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3- quinolinecarboxylic acid, Bay o 9867; designated tradename: Ciprobay) M1, M2, M3 and M4 was tested with the agar dilution method against various Gram-positive and Gram-negative bacteria in comparison to ciprofloxacin, norfloxacin and nalidixic acid. The results show that M1 had only a weak antibacterial activity comparable to nalidixic acid, whereas M2 was significantly less active. M3, which is one of the main metabolites in urine has a broad antibacterial activity but was less active than ciprofloxacin or norfloxacin. M4 which is a very minor metabolite of ciprofloxacin was the most active compound with minimal inhibitory concentrations for strains of Escherichia coli or Klebsiella pneumoniae in the range of norfloxacin, whereas with staphylococci the antibacterial activity was comparable to ciprofloxacin. Possible interactions between ciprofloxacin and the metabolites in the bioassay system, using Escherichia coli (ICB 4004) were studied, to explain discrepancies between the microbiological assay and the HPLC-method reported in the literature. It could be demonstrated that under conditions where the concentration of ciprofloxacin exceeds or equals the concentration of the metabolites or mixtures of them no increase in the inhibition zones for ciprofloxacin could be observed, which would have led to false high values for ciprofloxacin in the bioassay system. From these data we conclude that the antibacterial activity of the metabolites in biological specimens, e.g. urine, does not influence the bioassay results.

Effect of ciprofloxacin on stationary bacteria studied in vivo in a murine granuloma pouch model infected with Escherichia coli.

A granuloma pouch model in mice was used to investigate the effect of ciprofloxacin in vivo on cells of Escherichia coli (Neumann) under stationary growth conditions. The animals were treated up to three times intraperitoneally with 2.5, 10 or 40 mg/kg ciprofloxacin 24 h after infection. The numbers of viable bacteria in the pouch exudate were determined over a period of 24 h. A rapid decline of more than 1 logarithmic unit of the number of colony forming units was observed after 2-4 h with all treatment schedules. The effect on stationary cells was more pronounced with the high dose of ciprofloxacin and also dependent on the frequency of treatment. Ciprofloxacin penetrated well into the pouch exudate and reached concentrations of 2.08 +/- 0.16 microgram/ml and 0.1 +/- 0.05 microgram/ml 2 h after treatment with 40 and 2.5 mg/kg, respectively. The results of this study demonstrate that ciprofloxacin is effective in the treatment of a local inflammatory abscess in mice harbouring a stationary population of E. coli.

Evaluation of the in vitro bactericidal action of ciprofloxacin on cells of Escherichia coli in the logarithmic and stationary phases of growth.

Cells of Escherichia coli Neumann and E. coli KL16 were suspended in phosphate-buffered saline pH 7.4 and allowed to reach stationary growth conditions. Ciprofloxacin was added at different concentrations, and time-kill curves were constructed. It could be demonstrated that the number of viable cells was reduced quickly by several logs for E. coli Neumann, whereas a weak and slow killing effect was observed with E. coli KL16. When ciprofloxacin or norfloxacin was added to logarithmically growing cultures of E. coli Neumann or E. coli KL16, no principal differences in the killing rate for the two strains could be observed. Ciprofloxacin, however, was more bactericidal than norfloxacin. It was also demonstrated that the bactericidal action of ciprofloxacin on cells in the stationary growth phase was better at pH 7.4 than at pH 8.6. This dependence is different from that observed in MIC studies, in which the MIC were lower at pH 8.0 than at pH 7.2. It was also found that the bactericidal action of ciprofloxacin or norfloxacin on cells of E. coli Neumann in the stationary phase of growth could not be reduced by the addition of chloramphenicol, whereas under conditions of logarithmic growth the rapid killing effect of ciprofloxacin was reduced in the presence of chloramphenicol.

Influence of pH and human urine on the antibacterial activity of ciprofloxacin, norfloxacin and ofloxacin.

The influence of pH on the antibacterial activity of ciprofloxacin, norfloxacin and ofloxacin was studied in broth and pooled human urine by microdilution susceptibility tests. Selected strains of E. coli, Staphylococcus aureus and Pseudomonas aeruginosa were used as test organisms. The results show that cultivation at pH 5.7 in urine increased the MIC values for all three quinolones 8, 16 and 32-fold compared with broth at pH 7.1. Killing curves show that in urine with 10 mcg/ml ciprofloxacin, rapid killing of E. coli and Pseudomonas aeruginosa occurred, whereas ofloxacin and especially norfloxacin were less effective.

Assessment of the in vitro and in vivo activity of ciprofloxacin measured against current standards of therapy.

The bactericidal activity of ciprofloxacin in active human serum was investigated using serum-resistant Escherichia coli C14 and Pseudomonas aeruginosa 220 as test organisms. In 100% or 80% human serum the growth rate of E. coli C14 was lower than in broth. This also influenced the killing rate of ciprofloxacin. Ciprofloxacin was more active in serum than norfloxacin or ofloxacin and killed Pseudomonas aeruginosa 220 much more rapidly than azlocillin or tobramycin. Rapid killing was also observed in vivo in the blood of intraperitoneally infected mice, ciprofloxacin being superior to norfloxacin, ofloxacin and sisomicin. In mouse protection studies with E. coli Neumann as infecting organism, the dose-effect ratio showed that, despite extremely low blood concentrations, ciprofloxacin had a full therapeutic effect at a dose of 2.5 mg/kg p.o. or less than or equal to 0.5 mg/kg s.c., whereas cefotaxime, despite similar low MIC values in vitro, had to be administered at a dose of 20 mg/kg s.c. in order to achieve 100% survival. These data indicate that ciprofloxacin is effective at low concentrations both in vitro and in vivo.

Influence of ciprofloxacin on the ultrastructure of gram-negative and gram-positive bacteria.

Killing curves were performed in pooled active human serum using serum resistant Escherichia coli C14, Pseudomonas aeruginosa 220 and Staphylococcus aureus 25151 as test organisms. Ciprofloxacin (a quinoline-carboxylic acid derivative, Bay o 9867) was added in concentrations of 0.025-4.0 micrograms/ml. Samples were taken at different times to determine the number of viable bacteria and to prepare specimens for transmission electron microscopy. The following drug concentration-dependent results were obtained: In the early phase of the bactericidal process in Escherichia coli C14 distinct loosenings of the peripheral cytoplasm occurred. With Pseudomonas aeruginosa 220 osmiophilic condensations were detected in the less dense areas where the bacterial DNA may be located. At low concentrations, elongation of gram-negative bacterial cells was observed, whereas at higher concentrations rupture and lysis occurred. In staphylococci, severe disturbances of septum formation and surface deformations could be found; in the final stage proteolysis and empty envelopes were observed.

The in vitro and in vivo activity of ciprofloxacin.

The antibacterial activity of ciprofloxacin (Bay o 9867) was compared with those of norfloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, cefaclor, sisomicin and cefotaxime in in vitro and mouse protection studies. Approximately 300 clinical isolates of clinically important gram-positive and gram-negative species were used. The median MICs of ciprofloxacin against gram-positive and gram-negative bacteria ranged from less than or equal to 0.015-1 mg/l. Ciprofloxacin was 2-8 fold more active than norfloxacin and 100-fold more active than nalidixic acid. It also had a wider spectrum of activity against gram-positive organisms including even enterococci. No cross-resistance was observed between ciprofloxacin and beta-lactam antibiotics or aminoglycosides. Only acidic pH conditions decreased its activity. Ciprofloxacin showed rapid bactericidal action against organisms in both the logarithmic and stationary growth phases. In mouse protection studies (intraperitoneal infection) ciprofloxacin was significantly more effective than norfloxacin, ampicillin, trimethoprim-sulfamethoxazole, and also showed excellent activity against Pseudomonas infections.