Target site penetration of fosfomycin in critically ill patients

Christian Joukhadar1,2,*, Nikolas Klein1, Peter Dittrich3, Markus Zeitlinger1, Alexander Geppert2, Keso Skhirtladze1, Martin Frossard4, Gottfried Heinz2 and Markus Müller1

1 Department of Clinical Pharmacology, Division of Clinical Pharmacokinetics, University of Vienna Medical School, Allgemeines Krankenhaus, Waehringer Guertel 18–20, A-1090 Vienna; 2 Department of Cardiology, Division of Intensive Care Medicine, University of Vienna Medical School, Vienna; 3 Department of Pharmacology, Karl-Franzens University, Graz; 4 Department of Emergency Medicine, University of Vienna Medical School, Vienna, Austria

Received 14 September 2002; returned 13 December 2002; revised 2 January 2003; accepted 3 February 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective: The present study was undertaken to investigate the target site penetration properties of fosfomycin, an antibiotic particularly suitable for treatment of soft tissue infections (STIs) in critically ill patients.

Methods and results: The study population included nine patients with sepsis. Penetration of fosfomycin into the interstitial space fluid of skeletal muscle was measured using the microdialysis technique, following a single intravenous administration of 8.0 g of fosfomycin to patients. The median (range) fosfomycin area under the concentration versus time profile for plasma and skeletal muscle were 673 (459–1108) and 477 (226–860) mg·h/L (P < 0.011), respectively. Interstitial maximum concentrations were lower than plasma values (P < 0.029). Median fosfomycin concentrations in the interstitium and plasma exceeded 70 mg/L throughout the observation period of 4 h and covered MICs for Streptococcus pyogenes, Staphylococcus aureus and Pseudomonas aeruginosa. Simulation of bacterial growth inhibition of S. pyogenes, based on tissue concentration data, confirmed the bactericidal properties of fosfomycin described in previous studies.

Conclusion: Fosfomycin concentrations in muscle interstitium and plasma exceeded the MICs for a range of clinically relevant pathogens in critically ill patients. Thus, fosfomycin exhibits a tissue pharmacokinetic profile, which appears to offer an alternative to other broad-spectrum antibiotics in intensive care patients suffering from STI.

Keywords: sepsis, target site, human, microdialysis


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Severe soft tissue infections (STIs) may lead to serious or life-threatening complications, requiring appropriate antibiotic therapy1 and often intensive care management combined with surgical intervention. However, despite the use of potent antibiotics and proper surgical intervention, the management of STIs caused by group A Streptococcus strains still remains a difficult task, indicated by mortality rates ranging from 20% to 100%.2 Notably, the occurrence of deleterious complications in group A Streptococcus and Staphylococcus aureus infections, such as necrotizing fasciitis, myositis and streptococcal/staphylococcal toxic shock syndrome, contributes substantially to morbidity and mortality in critically ill patients. Thus, armed with a knowledge of tissue drug penetration properties, the conscientious physician has to empirically select the appropriate antibiotic for the initial antimicrobial therapy.

Fosfomycin is a bactericidal broad-spectrum antibiotic that is not structurally related to other classes of antimicrobial agents. Fosfomycin was demonstrated to exert high in vitro activity against a range of Gram-positive bacteria such as Streptococcus pyogenes and S. aureus and some Gram-negative bacteria such as Pseudomonas aeruginosa.3,4 In addition, it was reported that concentrations of fosfomycin in the soft tissues of healthy volunteers are almost identical to corresponding plasma levels.5 These favourable drug characteristics have made fosfomycin a frequently administered antibiotic in the initial empirical therapy of patients with sepsis and/or STI throughout the European Union. However, in the USA, fosfomycin is approved by the Food and Drug Administration (FDA) only for the treatment of uncomplicated cystitis.

The achievement of appropriate target site concentrations of antibiotics is of particular relevance as this may be regarded as essential to eradicate the relevant pathogen and is suggested to be associated with clinical outcome.68 Recent data, however, strongly suggest that concentrations of antibiotics in the interstitium of soft tissues might be ineffective in septic patients, despite adequate plasma concentrations. This was attributed to an impaired transcapillary antibiotic transfer to the target site.913 Thus, tissue penetration of most available antibiotics in the setting of sepsis is poor.

Based on this observation, the present study was carried out to measure the microbiologically active concentrations of fosfomycin in skeletal muscle interstitium and to relate these concentrations to corresponding plasma levels.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The protocol was approved by the local ethics committee and was carried out in accordance with the Declaration of Helsinki and current revisions of the Good Clinical Practice (GCP) Guidelines of the European Commission. The study met the criteria set by the local ethics committee for patients who were unable to give written consent because of incapacity.

Patients

Sepsis was classified as outlined in the ACCP/SCCM Consensus Conference Committee guidelines.14 Ten patients were enrolled in the study. Severity of illness required sedation and mechanical ventilation in all patients. None of the patients underwent haemofiltration or haemodialysis before or during the study period. Conventional therapies were not changed during the study period, except for moderate modifications in haemodynamic support with vasopressors and inotropic drugs necessary to maintain baseline mean arterial blood pressure. The patient’s demographic, haemodynamic and laboratory data are presented in Table Go.


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Table 1.  Demographic, haemodynamic, laboratory parameters and APACHE II scores of the patient population (n = 9)
 
Measurement of fosfomycin concentrations

Total fosfomycin levels in plasma and unbound fosfomycin concentrations in microdialysates were determined by a previously published and modified gas chromatography method.15 Fosfomycin was quantified with a calibration curve prepared from blank serum spiked with concentrations of 10–600 mg/L. The relationship of peak area of fosfomycin/peak area of the internal standard ratio and the concentrations of fosfomycin were linear with a correlation coefficient of 0.9936. The coefficients of variation (CV) were 9.7% at 10 mg/L, 8.7% at 200 mg/L and 3.1% at 600 mg/L (n = 6). The limit of detection was 1 mg/L. Intra-day and inter-day CVs were <7%.

In vivo pharmacokinetic (PK)/in vitro pharmacodynamic (PD) simulation

Based on PK data obtained from in vivo experiments, we simulated the time versus concentration profile of fosfomycin in the interstitial space fluid in an in vitro setting in order to generate a PK/PD model, which allows for the description of the antibacterial activity of antibiotics at the target site.16

In brief, Mueller–Hinton broth (Mikrobiologie Mueller–Hinton Bouillon; Merck, Darmstadt, Germany) enriched with 5% horse blood (Hasch Pferdeblut; Hasch Medizinischer Labor Bedarf, Vienna, Austria) was kept in a water bath at 37°C and was inoculated with a strain of S. pyogenes (ATCC 19615) at an approximate inoculum of 5 x 105 cfu/mL. Subsequently, the time versus fosfomycin concentration profiles obtained in vivo from the interstitial fluid were simulated in vitro by changing fosfomycin concentrations in medium by adding the appropriate volume of Mueller–Hinton broth at 20 min intervals. Samples to assess bacterial counts and controls were drawn at pre-defined time points.

In vitro susceptibility tests

The MIC of fosfomycin was determined by a two-fold serial Mueller–Hinton broth microdilution method. The ATCC 19615 S. pyogenes strain was pre-cultured overnight on Columbia agar plates (Columbia + 5% sheep blood; bioMérieux, France) and then introduced into Mueller–Hinton broth mixed with 5% horse blood and containing fosfomycin and glucose 6-phosphate, at an inoculum of ~5 x 105 cfu/mL. The lowest concentration of fosfomycin that inhibited visible bacterial growth after incubation for 20 h at 37°C was determined as the MIC.

Organisms

A strain of ATCC 19615 S. pyogenes with a MIC of 32 mg/L was chosen for the present in vitro experiments. Bacteria were stored in liquid nitrogen at –196°C until used.

Study protocol

Blood samples were obtained from an inserted plastic cannula to monitor blood concentrations of fosfomycin at 20 min intervals. Microdialysis experiments were carried out as described previously.17,18 In brief, a commercially available microdialysis probe (CMA 10; Microdialysis AB, Stockholm, Sweden) with a molecular weight cut-off of 20 kDa, an outer diameter of 0.5 mm and a membrane length of 16 mm was inserted into the skeletal muscle of the thigh by puncture with a 20 gauge intravenous plastic cannula. The steel mandrin was removed, the appropriate site of the probe checked by aspiration and the dialysis probe inserted into the plastic cannula. The microdialysis system was connected and perfused with Ringer’s solution at a flow rate of 1.5 µL/min. This was carried out by use of a precision-pump (Precidor; Infors AG, Basle, Switzerland). After a 30 min baseline sampling period, 8.0 g of fosfomycin dry powder (Fosfomycin ‘Biochemie’, Trockenstechampulle; Biochemie, Vienna) was reconstituted with 200 mL of sterile water just before dosing and administered to patients over a period of ~20 min. Sampling of microdialysates and plasma samples was continued at 20 min intervals for up to 4 h. All samples were stored at –80°C until analysis.

Calculations for microdialysis experiments

Recovery values for fosfomycin were obtained from a previous study.5 Interstitial concentrations were calculated by the following equation:

Interstitial concentration (mg/L) =

100 x (concentrationdialysate/mean recovery %)

Pharmacokinetic calculations

Plasma protein binding of fosfomycin has been reported to be negligible.19 Thus, total plasma fosfomycin concentrations were assumed to constitute unbound fosfomycin. PK analysis was carried out using commercially available software (Kinetica 3.0; Innaphase Sarl, Paris, France). The area under the concentration–time curve for plasma and interstitium was calculated from non-fitted data by employing the trapezoidal rule. The following PK parameters were determined: maximum concentration (Cmax), time to maximum concentration (tmax) and area under the concentration–time curve (AUC). The volume of drug distribution at steady-state (Vdss) and clearance (CL) were calculated for plasma in patients by use of standard formulae as follows: CL = dose/AUCtotal; Vdss = CL x MRT, where MRT represents the mean residence time of a molecule.

Main PK data are summarized in Table Go. The AUCmuscle/AUCplasma ratios were calculated as a measure of drug penetration into the peripheral compartments. Although 10 patients were enrolled in the present study, the malfunction of one microdialysis probe rendered the PK profile of fosfomycin in one patient invalid. Therefore, only nine patients were eligible for data analysis.


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Table 2.  Main PK parameters calculated for the study population
 
Statistical calculations

For statistical comparison of PK parameters between plasma and interstitium, Wilcoxon tests were carried out as parameters were non-normally distributed. Data are presented as medians (range) and means ± S.E.M. A P value <0.05 was considered significant.


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 Materials and methods
 Results
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 References
 
Human experiments

Figure 1 shows time versus unbound fosfomycin concentration profiles in plasma and the interstitium of skeletal muscle in patients (n = 9), following single intravenous administration of 8.0 g over ~20 min.



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Figure 1. Time versus fosfomycin concentration profiles for plasma (circles) and skeletal muscle (squares) in patients (n = 9) following single intravenous administration of 8.0 g of fosfomycin over a period of ~20 min. Data are depicted as means ± S.E.M.

 
The main PK parameters for plasma and the interstitium of skeletal muscle are presented in Table Go. The median fosfomycin AUC0–4 values were 673 mg·h/L (range 459–1108) for plasma and 477 mg·h/L (range 226–860) for skeletal muscle (P < 0.011). Median Cmax values were lower in interstitium than in plasma (P < 0.029).

Laboratory experiments: PK/PD simulation

By using the terminal plasma elimination half-life of 3.8 h for fosfomycin, as calculated in our in vivo experiments (Table Go), the in vitro bacterial growth simulation experiments were extrapolated from 4 to 6.3 h. The time–kill curves of S. pyogenes are presented in Figure 2. S. pyogenes strains, which were not exposed to fosfomycin concentrations, served as controls and showed an ~2 log10 increase in bacterial counts per mL after 6.3 h. In contrast, bacterial counts of S. pyogenes decreased by 1.67 log10 and 1.81 log10 per mL following exposure to time–concentration profiles of fosfomycin measured in plasma and tissue interstitium, respectively. Bacterial time–kill curves for plasma and tissue were carried out in triplicate.



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Figure 2. Time–kill curves of S. pyogenes following incubation to plasma (black triangles) and tissue (white triangles) time–concentration profiles derived from in vivo experiments depicted in Figure 1. Bacterial killing was simulated over a period of 6.3 h. Bacterial growth control curve is presented as circles. Data are depicted as means ± S.E.M.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Failure of antimicrobial therapy to eradicate the relevant pathogen in patients has been commonly attributed to the development of resistance of pathogens to distinct antimicrobial agents. However, this universal concept does not explain therapeutic failure in the case of infections caused by bacteria proven susceptible to the administered antibiotic. Another explanation for therapeutic failure may relate to impaired transcapillary drug transfer of the pharmacologically active, unbound fraction of the antibiotic into tissue interstitium.913,19 In order to evaluate the penetration properties of fosfomycin into the soft tissue interstitium in septic patients, we used microdialysis, a minimally invasive clinical technique.1618

In the present study, we were able to demonstrate that fosfomycin concentrations in muscle interstitium fully equilibrate with plasma within 80 min in critically ill patients. The excellent tissue penetration property of fosfomycin was also reflected by the high killing rate of S. pyogenes at the target site, demonstrated by the use of a PK/PD simulation (Figure 2).

These data are of particular relevance as previous findings revealed that the equilibration process between plasma and tissue interstitium may be incomplete or substantially prolonged for several antibiotics in intensive care patients.913 This has been attributed mainly to the presence of interstitial oedema. Oedemas are the result of a decreased transcapillary colloid osmotic gradient,911 fluid overload and an increased volume of distribution found in septic patients. In addition, an alteration of blood flow, especially in the micro-circulation due to sepsis20 and the administration of vasopressors might affect tissue penetration of antibiotics.

In contrast to findings in post-operative7 and septic patients,9,11 rapid tissue equilibration of fosfomycin was observed following a single dose in the present patient population. Almost complete plasma to tissue equilibration was also observed previously in healthy volunteers.5 Fosfomycin’s favourable tissue penetration characteristics might be attributed to its relatively low molecular weight of only 182, which is approximately three-fold lower than that of ß-lactams administered in previous trials.9,11 This low molecular weight may facilitate penetration across capillary pores. Other drug characteristics of fosfomycin, which have been reported to substantially affect tissue penetration, are low plasma protein binding,18 high hydrophilicity5 and a volume of distribution that is comparable to the total extracellular body water (Table Go).

In addition to these pharmacological explanations, the severity of sepsis may also have significant impact on the penetration process of antibiotics into soft tissues. This becomes evident as adjustments of fluid replacement and vasopressive catecholamines depend mainly on arterial blood pressure measurements in septic patients. Vasopressive catecholamines increase arterial blood pressure, but decrease capillary recruitment and microcirculatory blood flow at peripheral sites. Thus, not only macro- but also microcirculatory blood flow may be important determinants of antibiotic tissue penetration, as recently reported in patients suffering from peripheral arterial occlusive disease.21

Differences in the ability of antibiotics to penetrate into the target site may determine bacterial eradication and clinical outcome.6,7 Optimal bacterial killing by fosfomycin and ß-lactams will be achieved when the time period exceeding the MIC for the relevant pathogen (t > MIC) is maximized.68 Effective bacterial killing can be expected from the first antibiotic dosage when the pathogen’s MIC is covered for at least 60–70% of the dosing interval.22 However, fosfomycin and ß-lactams lack any significant post-antibiotic effect on Gram-negative bacteria, and thus it appears reasonable that trough levels should exceed relevant MICs throughout the duration of antibiotic therapy in ‘difficult-to-treat’ pathogens.

Despite the favourable tissue penetration properties of fosfomycin, monotherapy with fosfomycin is limited because of the rapid development of resistant strains. Thus, in clinical practice other antibiotics such as aminoglycosides,23,24 ß-lactams24 or fluoroquinolones are co-administered with fosfomycin. These combinations were shown to act synergically on antibacterial activity3,4 and helped to avoid the development of rapid resistance to fosfomycin.

Simulations of time–kill profiles of bacteria for fosfomycin alone (as depicted in Figure 2) underestimate bacterial eradication in a clinical setting. However, the PK/PD simulation allows for the quantification of anti-infective activity of antimicrobial agents in the interstitium of soft tissues, which is the target site of many extracellular infections.16,25,26 The data gained from these experiments confirm previous findings of appropriate activity of fosfomycin against clinically relevant bacteria.3,5,27

In our study population, median fosfomycin concentrations in interstitium and plasma never dropped below 70 mg/L during the observation period of 4 h. Taking a plasma and tissue half-life of ~3.5 h (Table Go) for fosfomycin into account, target site concentrations will reach ~35 mg/L, 8 h after drug administration. Thus, the present study provides circumstantial evidence that a dosage of 8 g fosfomycin twice a day (the currently recommended dose in severe infections) might be insufficient to eradicate less susceptible bacteria with MIC90s of ~35 mg/L, unless a synergically acting antibiotic is co-administered. In these cases, it appears advisable to select a dosage that is expected to maintain adequate antibacterial concentrations at the infectious site throughout the dosing interval. This might be achieved by increasing the daily dosage, by shortening the dosing interval or by administration of fosfomycin using continuous perfusion devices.

In conclusion, the present study has shown that effective concentrations of fosfomycin are attained in the muscle interstitium and plasma for a range of clinically relevant pathogens. These findings support the use of fosfomycin as an alternative antimicrobial agent in the therapy of severe STIs in critically ill patients.


    Acknowledgements
 
We are indebted to our study nurses Petra Zeleny and Edith Lackner for their essential contributions to the study.


    Footnotes
 
* Corresponding author. Tel: +43-1-40400-2981; Fax: +43-1-40400-2998; E-mail: christian.joukhadar{at}univie.ac.at Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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