Journal of Antimicrobial Chemotherapy (2000) 45, 533-535
© 2000 The British Society for Antimicrobial Chemotherapy
Brief reports |
Endothelial cell compatibility of trovafloxacin and levofloxacin for intravenous use
a Second Medical Department/Pulmologisches Zentrum Vienna, Sanatoriumstrasse 2, A-1140 Vienna; b Department of Pulmonary Medicine, University Hospital of Vienna; c Institute of Laboratory Diagnostics, Kaiser Franz Josef Hospital, Vienna, Austria
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Abstract |
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Levofloxacin and trovafloxacin have excellent activity against a variety of Gram-positive and Gram-negative organisms resistant to the established agents. One local side-effect closely related to the use of parenteral fluoroquinolones is phlebitis. To evaluate the effect of trovafloxacin and levofloxacin on endothelial cell viability, intracellular levels of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), guanosine 5'-triphosphate (GTP) and guanosine 5'-diphosphate (GDP) levels were measured using high-performance liquid chromatography. Trovafloxacin at concentrations of 2 and 1 mg/mL reduced the intracellular ATP content from 12.5 ± 1.7 to 1.9 ± 0.3 nmol/106 cells and 9.3 ± 0.8 nmol/106 cells, respectively, within 60 min. In addition, ADP, GTP and GDP levels were extensively depleted. Levofloxacin at concentrations of 5 and 2.5 mg/mL led to a significant ATP decline from 12.5 ± 1.7 to 2.3 ± 0.2 nmol/106 cells and 10.3 ± 0.9 nmol/106 cells, respectively, within 60 min. These data indicate that infusions of high doses of trovafloxacin or levofloxacin are not compatible with maintenance of endothelial cell function. Commercial preparations have to be diluted and should be administered into large veins.
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Introduction |
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Second-generation fluoroquinolones (trovafloxacin, levofloxacin) have an extended spectrum of activity compared with older-generation fluoroquinolones.1,2 However, local reactions at the site of infusion after intravenous administration of fluoroquinolones may interfere with therapy, as has been reported previously for ciprofloxacin and fleroxacin.1,3
The intravenous compatibility of antibiotic solutions has usually been evaluated in animal models.4 To test the tolerance of intravenous trovafloxacin and levofloxacin, an in vitro system was devised using human umbilical endothelial cells (HUVEC).
Intracellular purines which reflect DNA/RNA synthesis, energy production and signal transduction were investigated. Levels of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), guanosine 5'-triphosphate (GTP) and guanosine 5'-diphosphate (GDP) were measured using high-performance liquid chromatography (HPLC).
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Materials and methods |
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Endothelial cells were prepared using human umbilical veins. Cells were isolated and cultured according to a modified standard procedure. 5,6 Commercial preparations of trovafloxacin (Pfizer Ltd, Kent, UK) and levofloxacin (Hoechst AG, Frankfurt am Main, Germany) were diluted with 0.45% NaCl or 0.9% NaCl to yield final concentrations of 2.0 mg/mL and 5.0 mg/mL, respectively.
For the experiments the culture medium was removed and the cell layers were washed with Dulbecco's phosphate buffered saline (Gibco, Paisley, UK). Trovafloxacin and levofloxacin at final concentrations of 2.0 mg/mL, 1.0 mg/ mL, 0.5 mg/mL and 5.0 mg/mL, 2.5 mg/mL, 1.0 mg/mL, respectively, were added to the endothelial cells and incubated for 20 or 60 min. All incubations were carried out in a humidified incubator at 37°C and 5% CO2.
Purines were measured using HPLC.7 ATP, ADP, GTP and GDP were separated by injecting 100 µg of the neutralized supernatant on to a CNU-010 column (Chemcon, Vienna, Austria) using a KH2PO4 gradient. Buffers consisted of 0.015 mol/L KH2PO4 pH 3.45 and 0.5 mol/L KH2PO4 pH 3.45. A linear gradient rising from 0% to 100% 0.5 mol/L buffer in 40 min was used with a total running time of 60 min and an equilibrium delay of 8 min. The flow rate was 1.2 mL/min and the detection was performed at a wavelength of 254 nm.
Amounts of ATP, ADP, GTP and GDP were quantified by determining the ratio of peak areas in relation to corresponding standards.
The linear range for all four nucleotides was between 0.75 and 30 µmol/L. Results are given as nmol/106 cells.
Data of 12 different experiments are expressed as mean ± s.d. The statistical significance was determined by means of the Mann–Whitney U test. P < 0.001 was considered significant.
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Results |
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Incubation of cells with 2 mg/mL trovafloxacin resulted in a rapid decrease in intracellular ATP content to 8.4 ± 0.6 nmol/106 cells or 1.9 ± 0.3 nmol/106 cells after an exposure time of 20 or 60 min (Tables I and II
), respectively. In addition, ADP, GTP and GDP content declined. Experiments with trovafloxacin at concentrations of 0.5 mg/mL resulted in no significant decline in intracellular high energy phosphates (Tables I and II).
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Levofloxacin at a concentration of 5 mg/mL reduced intracellular ATP levels to 9.3 ± 0.5 nmol/106 cells and to 2.3 ± 0.2 nmol/106 cells after 20 and 60 min, respectively. No significant decline in purine content was observed at a levofloxacin concentration of 1 mg/mL (Tables I and II
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Discussion |
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In vitro studies with cultured human cells have proved to be a promising method for predicting toxicity and for clarifying mechanisms of toxicity.8
Since levofloxacin is available at a concentration of 5 mg/mL and trovafloxacin is recommended at a concentration of 2 mg/mL by the manufacturer, these high doses were used in our experiments in order to simulate possible clinical concentrations at the site of infusion. Similar concentrations may be reached by retrograde intravenous pressure infusion techniques. We have shown that these concentrations led to considerable endothelial cell damage. These findings may explain the clinical observations of local reactions at the site of infusion related to intravenous application of trovafloxacin and levofloxacin.
The data summarized in Tables I and II indicate that a detrimental effect measurable after 20 and 60 min occurs only when trovafloxacin and levofloxacin concentrations of 2 mg/mL and 5 mg/mL, respectively, are used, whereas a dilution to 0.5 mg/mL or 1 mg/mL renders the solutions more compatible to endothelial cells. Incubation of cells with 1 mg/mL trovafloxacin or 2.5 mg/mL levofloxacin resulted in a decrease in intracellular ATP, ADP and GTP levels only after an exposure time of 60 min.
The tolerance of intravenously applied antibiotics has usually been tested in animal models.4 By using human venous endothelial cells for testing antibiotic solutions, we introduce a valuable alternative model.
Our data clearly demonstrate that high doses of trovafloxacin and levofloxacin are not compatible with the maintenance of endothelial cell integrity. Commercial preparations of these fluoroquinolones have to be diluted and applied slowly into large veins in order to prevent phlebitis, until a switch from intravenous to oral therapy is possible.
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Notes |
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* Corresponding author. Tel/Fax: +43-1-877-58-20; E-mail: vorbach{at}austriaone.at
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References |
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1 . North, D. S., Fish, D. N. & Redington, J. J. (1998). Levofloxacin, a second-generation fluoroquinolone. Pharmacotherapy 18, 915–35.[Web of Science][Medline]
2 . Alghasham, A. A. & Nahata, M. C. (1999). Trovafloxacin: a new fluoroquinolone. Annals of Pharmacotherapy 33, 48–60.[Abstract]
3 . Hooper, D. C. & Wolfson, J. S. (1993). Quinolone Antimicrobial Agents, 2nd edn. American Society for Microbiology, Washington, DC.
4 . Guay, D. R. P., Patterson, D. R., Seipman, N. & Craft, J. C. (1993). Overview of the tolerability profile of clarithromycin in preclinical and clinical trials. Drug Safety 8, 350–64.[Web of Science][Medline]
5 . Jaffe, E. A., Nachman, R. L., Becker, C. G. & Minck, C. R. (1973). Culture of human endothelial cells derived from umbilical veins. Journal of Clinical Investigation 52, 2745–56.
6 . Jaffe, E. A., Hoyer, L. W. & Nachman, R. L. (1973). Synthesis of antihaemophilic factor antigen by cultured human endothelial cells. Journal of Clinical Investigation 52, 2757–64.
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Griesmacher, A., Weigel, G., Seebacher, G. & Müller, M. M. (1997). IMP-dehydrogenase inhibition in human lymphocytes and lymphoblasts by mycophenolic acid and mycophenolic acid glucuronide. Clinical Chemistry 43, 2312–7.
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Vorbach, H., Robibaro, B., Armbruster, C., Atteneder, M., Reiter, M., Hlousek, M. et al. (1999). Endothelial cell compatibility of clindamycin, gentamicin, ceftriaxone and teicoplanin in Bier's arterial arrest. Journal of Antimicrobial Chemotherapy 44, 275–7.
Received 19 July 1999; returned 13 October 1999; revised 1 November 1999; accepted 13 November 1999
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