Endothelial cell compatibility of azithromycin and erythromycin

H. Vorbacha, C. Armbrustera,*, B. Robibarob, A. Griesmacherc, I. El Menyawic, H. Daxeckerc, M. Raabc and M. M. Müllerc

a Department of Internal Medicine II, Pulmonary Centre, Vienna; b Department of Pulmonary Medicine, University Hospital of Vienna; c Ludwig Boltzmann Institute for Cardiothoracic Surgery Research at the Institute of Laboratory Diagnostics, Kaiser-Franz-Josef-Spital, Vienna, Austria


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Phlebitis is a severe local adverse event related to the use of parenteral macrolides. In order to evaluate the effect of azithromycin and erythromycin on human venous endothelial cells, we set up an in vitro model. The intracellular levels of purine nucleotides, as adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and guanosine 5'-triphosphate (GTP), were measured by means of high-performance liquid chromatography. Incubation of cells with 2 mg/mL azithromycin and erythromycin resulted in a rapid decline of intracellular ATP from 12.5 ± 0.9 nmol/million cells to 4.1 ± 0.3 and 2.6 ± 0.4 nmol/million cells, respectively, after 60 min. In addition, ADP was extensively depleted from 2.1 ± 0.17 nmol/million cells to 0.8 ± 0.09 and 0.8 ± 0.13 nmol/million cells after 60 min. After exposure of 0.5 mg/mL azithromycin and erythromycin, no significant decline of intracellular high-energy phosphate levels occurred after 20 and 60 min. Based on these results, solutions of azithromycin and erythromycin may not be well tolerated and may cause local adverse reactions even if diluted according to the manufacturer's recommendation.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Azithromycin and erythromycin are macrolide antimicrobial agents used widely for treatment of infections caused by Chlamydia pneumoniae, Chlamydia trachomatis, Legionella pneumophila, Mycoplasma hominis, Mycoplasma pneumoniae and Streptococcus pneumoniae. They are generally well tolerated with a low incidence of systemic adverse events. However, local intravenous site reactions have been reported with azithromycin and erythromycin that may interfere with therapy.1,2 The tolerance of intravenously applied antibiotics is usually tested in animal models.3 To provide an alternative test system, an in vitro model was established using human umbilical venous endothelial cells (HUVEC). We investigated the effects of azithromycin and erythromycin, available for intravenous application, on these cultures. By determining the intracellular contents of adenine and guanine nucleotides using the highly sensitive high-performance liquid chromatography (HPLC) method, endothelial metabolism was examined. We measured the intracellular adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP) levels, reflecting energy production of these cells. Guanosine 5'-triphosphate (GTP), which has an important role in DNA/ RNA synthesis, G-protein coupled signal transduction and glycosylation of membrane proteins, was evaluated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cell culture

Endothelial cells were prepared using human umbilical veins from umbilical cords recovered with verbal informed consent. Cells were isolated and cultured according to a modified standard procedure.4 Briefly, fresh human umbilical veins were filled with 0.1% collagenase solution and incubated at 37°C. The veins were then perfused with medium 199 (Sigma, St Louis, MO, USA) containing 20% bovine calf serum (HyClone, Road Logan, UT, USA). Cells were collected from the perfusate by centrifugation (300g, 4°C) and seeded into culture T-75 flasks precoated with human fibronectin (Upstate Biotechnology Inc., Lake Placid, NY, USA). Cells were cultured in medium 199 containing 20% bovine calf serum, 50 000 U/L penicillin– streptomycin (Gibco, Paisley, UK), 50 mg low molecular weight heparin (Sigma) and 15 mg/L H-Neurext (endothelial cell growth supplement; Upstate Biotechnology Inc.). The confluent primary monolayers (c. 8 000 000 cells/flask) were washed and trypsinized. The cell suspensions were transferred into each well of a six well culture plate and cultivated for 4 days. Only cells from these first subcultures were used for the experiments. We used uninfected HUVECs because the causative microorganisms do not usually play a significant role at the site of antibiotic infusions.

Antibiotics

The commercially available preparations of erythromycin (Abbott, Chicago, IL, USA) and azithromycin (Pfizer Inc., New York, NY, USA) were dissolved in 10 mL water for injection and diluted further with 0.9% NaCl.

Incubation with azithromycin and erythromycin

For the experiments the culture medium was removed and the cell layers were washed gently with Dulbecco':s phosphate-buffered saline (Gibco). Thereafter, azithromycin and erythromycin solutions at a concentration of 2, 1 and 0.5 mg/mL were added to the endothelial cells and incubated for 20 or 60 min. Control experiments were performed using 0.9% NaCl. All incubations were carried out in a humidified incubator at 37°C and 5% CO2.

Determination of high-energy phosphates

The energy-rich phosphates were measured by means of HPLC.5 ATP, ADP and GTP were separated by injecting 100 µL of the neutralized supernatant on to a CNU-010 column (Chemcon, Vienna, Austria) using a KH2PO4 gradient. Buffer A consisted of 0.015 mol/L KH2PO4 (pH 3.45), and buffer B of 0.5 mol/L KH2PO4 (pH 3.45). A linear gradient rising from 0% B to 100% B 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 made at a wavelength of 254 nm.

The amount of formed ATP, ADP and GTP was determined by the ratio of its peak area in relation to the corresponding standards measured under the same conditions. The linear range for all three nucleotides was between 0.75 and 30 µmol/L. The results are given as nmol/million cells.

Statistical analysis

Data from eight different experiments are expressed as mean ± s.d. The statistical significance was determined by means of Mann–Whitney U-test. P < 0.001 (*) was considered to be significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Effects of 2 mg/mL azithromycin and erythromycin on HUVEC

A 60 min incubation of cells with 2 mg/mL azithromycin or erythromycin resulted in a rapid decrease of intracellular ATP to 4.1 ± 0.3 and 2.6 ± 0.4 nmol/million cells, respectively (TableGo). In addition, ADP was significantly decreased indicating a depletion of the ATP/creatine phosphate (CP) regeneration system.


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Table. Total amount of intracellular purines in nmol/106 cells (mean ± s.d.)
 
GTP levels were stable during the first 20 min of exposure to 2 mg/mL azithromycin or erythromycin but declined extensively after 60 min (TableGo).

Effects of 1 mg/mL azithromycin and erythromycin on HUVEC

After incubation with 1 mg/mL azithromycin or erythromycin, the decrease of ATP/ADP and GTP levels after 60 min was less pronounced (TableGo). Intracellular high-energy triphosphates and the corresponding diphosphates did not decline after 20 min.

Effects of 0.5 mg/mL azithromycin and erythromycin on HUVEC

Purine nucleotide profiles were not affected during exposure to 0.5 mg/mL azithromycin or erythromycin. Even after 60 min of exposure ATP, ADP and GTP levels showed no significant difference compared with controls, indicating that no cellular damage occurred.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In vitro studies with HUVEC have proved to be a promising method for predicting toxicity and for clarifying mechanisms of toxicity.6,7 As previously shown for glycopeptide antibiotics and for high-dose therapy of locally applied antimicrobial agents, our in vitro model of HUVEC provides an alternative to animal models.7,8 However, in vitro models have some limitations, such as lacking the dilution factor of blood flow washing away antibiotic solutions from the site of the infusion in vivo.

To investigate cellular impairment in response to azithromycin and erythromycin, intracellular purine content was measured after incubation with different concentrations of these macrolides. As shown in the TableGo, 1 mg/mL azithromycin and erythromycin led to an ATP decline compared with untreated HUVEC. This decrease, when combined with the data given in the TableGo, can be considered as reversible since ATP levels can be restored by the action of creatine kinase using ADP as substrate and CP as phosphate donor. However, severe ATP depletion is irreversible and leads to endothelial cell death.9 Azithromycin and erythromycin at a dose of 2 mg/mL result in an ATP depletion that does not seem to be reversible via the CP/ADP regeneration system because of the strong decrease of intracellular ADP. The depletion of intracellular GTP might support this consideration and reflect a functional and structural alteration. A dilution to 0.5 mg/mL rendered the solutions more compatible to HUVEC and showed no significant difference in the purine nucleotide profiles compared with controls.

These data are in line with clinical observations that the occurrence of phlebitis could be reduced by diluting the commercially available preparations of erythromycin to a final concentration of 1 mg/mL.2,10 The importance of this in vitro study should be underlined, since no data exist on the influence of azithromycin on human endothelial cells.

In conclusion, based on our data we would like to draw clinicians' attention to the possibility that solutions of these macrolides may not be well tolerated, even if diluted according to the manufacturer's recommendation.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported by a grant from the Austrian Society of Chemotherapy.


    Notes
 
* Correspondence address. Mantlergasse 23/2/12 A-1130 Vienna, Austria. Tel/Fax: +43-1-877-58-20; E-mail: christine.armbruster{at}gmx.at Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Whitman, M. S. & Tunkel, A. R. (1992). Azithromycin and clarithromycin: overview and comparison with erythromycin. Infection Control and Hospital Epidemiology 12, 357–68.

2 . Guay, D. R. (1996). Macrolide antibiotics in pediatric infectious diseases. Drugs 51, 515–36.[ISI][Medline]

3 . Guay, D. R. P., Patterson, D. R., Seipman, N. & Minck, C. R. (1993). Overview of the tolerability profile of clarithromycin in preclinical and clinical trials. Drug Safety 8, 350–64.[ISI][Medline]

4 . Jaffe, E. A., Nachman, R. L., Becker, C. G. & Minck, C. R. (1973). Culture of human endothelial cells derived from umbilical veins: identification by morphology and immunologic criteria. Journal of Clinical Investigation 52, 2745–56.[ISI][Medline]

5 . 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.[Abstract/Free Full Text]

6 . Griesmacher, A., Weigel, G., David, M., Schimke, J. & Müller, M. M. (1992). Influence of oxygen radicals generating agents on eicosanoid metabolism of human endothelial cells. Thrombosis Research 65, 721–31.[ISI][Medline]

7 . 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.[Abstract/Free Full Text]

8 . Robibaro, B., Vorbach, H., Weigel, G., Weihs, A., Hlousek, M., Presterl, E. et al. (1998). Endothelial cell compatibility of glycopeptide antibiotics for intravenous use. Journal of Antimicrobial Chemotherapy 41, 297–300.[Abstract]

9 . Pearson, J. D. & Gordon, J. L. (1985). Nucleotide metabolism by endothelium. Annual Review of Physiology 47, 617–27.[ISI][Medline]

10 . Brittain, D. C. (1987). Erythromycin. Medical Clinics of North America 71, 1147–54.[ISI][Medline]

Received 27 July 2001; returned 5 October 2001; revised 24 October 2001; accepted 26 October 2001