INTRODUCTION

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Keywords: microdialysis; extracellular space; antiinfective agents; respiratory tract infections

Community-acquired pneumonia accounts for 600,000 hospitalizations per year in the United States and is the sixth leading cause of death and the most common cause of death due to infectious diseases in the United States (1). Similarly, ventilator-associated pneumonia in critically ill patients is a frequent cause of death from nosocomial infections (2), with an estimated prevalence that ranges from 10 to 65% (3, 4). Although the introduction of antimicrobial therapy has substantially improved the outcome of respiratory tract infections (5, 6) in both outpatients and critically ill patients, there is a remarkable number of cases in which antimicrobial therapy fails to be effective. Some therapeutic failures are attributed to drug- resistant pathogens (7). In the case of susceptible microorganisms, however, the reasons for an adverse outcome often remain unclear.

One possible explanation for therapeutic failure relates to an inability of the antibiotic to sufficiently penetrate into the target site (8). Although it has commonly been believed that most antibiotics display almost complete tissue-to-plasma equilibria, several reports have indicated that target site drug levels may be substantially lower than corresponding plasma levels (8). Suboptimal target site concentrations of antibiotics may have important clinical implications as it is a potential explanation for therapeutic failure in many cases (9, 10) and it is conceivable that subinhibitory concentrations in tissues may also trigger bacterial resistance (11).

Antibiotic failures have been attributed to impaired target site penetration for a wide range of clinical situations (12), including respiratory tract infections (15). Thus, measurement of antimicrobial concentrations in lung tissue could lead to a critical reappraisal of current antimicrobial dosing guidelines. To date, drug concentrations at the site of infection in the respiratory tract have been studied by assaying whole lung tissue, sputum, respiratory secretions, and pleural fluid (18) and by sampling bronchoalveolar lavage or epithelial lining fluid (22). However, at least two major methodological problems hampered a correct interpretation of data derived from these techniques: (1) longitudinal pharmacokinetic data were derived only from interindividual data pooling because it is impossible to measure antimicrobial concentrations in lung tissue continuously; and (2) finding a correlation between concentrations in the sample and at the exact anatomical site of action was difficult or impossible.

We have therefore developed a novel approach to measure drug levels in human lung tissue in vivo. In contrast to previous approaches, this enables the continuous measurement of unbound drug concentrations in the interstitial space fluid, the relevant target site for most bacterial infections (26). For this purpose, time versus concentration profiles of a model -lactam antibiotic, cefpirome, were measured in lung interstitial fluid by microdialysis probes.

	METHODS

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The study was approved by the local ethics committee. All patients were given a detailed description of the study and their written informed consent was obtained. The study was performed in accordance with the Declaration of Helsinki and the Good Clinical Practice Guidelines of the European Commission.

The study population included five male patients undergoing elective lung operations for pulmonary tumors (age 53 to 66 yr; weight 60 to 94 kg; height 174 to 180 cm) without recent lung infections.

Experimental Design

The principles and details of microdialysis have been described previously (27). In this study we used a special, custom-made flexible microdialysis probe (Metalant AB, Stockholm, Sweden) with a total length of 50 cm to allow for pulmonal movement during respiration (Figure 1). The tip of the probe was made of a 0.6 × 50-mm polyether sulphone membrane with a molecular weight cutoff of 15,000 D; the in vitro recovery was 78% and 43% at flow rates of 1.5 (patients 1-3) and 4.0 µl/min (patients 4 and 5), respectively.

View larger version (9K): [in this window] [in a new window]   Figure 1.   Schematic illustration of the functional principle of the microdialysis probe. The probe is perfused with a physiological solution by means of a pump. The solution enters the probe via the inlet tubing and at the probe tip (arrow) unbound analytes diffuse across the membrane out of the tissue into the probe. The fluid, which is now called dialysate, leaves the probe via the outlet tubing and is collected by fractions in small cups for subsequent chemical analysis.

Before closing the chest at the end of the conventionally performed lung surgery, a 1.2 × 45-mm venous cannula (Venflon, Becton Dickinson, Helsingborg, Sweden) was inserted percutaneously through the chest wall near the thoracotomy. The tip of the flexible microdialysis catheter was then led into the thoracic cavity through this cannula. Employing a gutter-like 1.4-mm slit needle (supplied with the CMA-60 microdialysis set, CMA, Sweden), the tip of the microdialysis probe was inserted into the reinflated lung under visual control (Figure 2). The slit needle was then retracted and peeled off the tube of the microdialysis catheter and the probe was perfused with degassed Ringer's solution. After final inspection for air leakage or bleeding at the insertion site the chest was closed conventionally. Patients were extubated in the operation theater. The pharmacokinetic study started when conscious and spontaneously breathing patients arrived in the recovery room. Cefpirome (Cefrom 2 g, Usiphar, Compiegne, France) was administered intravenously over a period of 5 min after baseline measurements. Sampling of dialysates and plasma and chemical analysis of cefpirome were performed as previously described in detail by our group (28, 30).

View larger version (115K): [in this window] [in a new window]   Figure 2.   Intraoperative view of the open chest with a microdialysis probe (arrow) inserted into the lung tissue.

Calculation and Data Analysis

Data are presented as means and standard error of the mean (SEM). Data were processed with MS Excel for Windows, SPSS for Windows and Kinetika (Version 2.0.2; INNAPHASE, Philadelphia, PA). The time-versus-cefpirome concentration profiles for plasma and lung interstitial fluid were measured and the following key pharmacokinetic parameters were determined: area under the concentration curve (AUC), AUC_plasma/AUC_tissue ratio, maximum drug concentration (C_max), time to maximum drug concentration (T_max), and half-life time of the -phase (T_1/2).

	RESULTS

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Figure 3A shows that the mean concentration of cefpirome in lung interstitial fluid rose rapidly over 40 min, then fell at a slightly slower rate than did plasma concentrations, so that plasma and lung interstitial fluid concentrations were the same 200 min after the dose. Figure 3B shows the same data as that in Figure 3A, plotted as the plasma:tissue drug concentration ratio. Key pharmacokinetic characteristics are shown in Table 1. In lung interstitial fluid, the AUC was approximately two-thirds of that in plasma and the maximum drug concentration was approximately 40% of plasma levels. The time taken to achieve the maximum drug concentration was 10 times longer in lung interstitial fluid than in plasma.

View larger version (9K): [in this window] [in a new window]   View larger version (10K): [in this window] [in a new window]   Figure 3.   Mean concentration versus time profile in plasma and in lung interstitial fluid (A) and plasma:tissue ratio versus time profile (B) of cefpirome after postoperative administration of a single intravenous dose of 2 g cefpirome to patients undergoing lung surgery due to a neoplasm (n = 5, data are presented as means + SEM). The dotted line represents the calculated mean unbound plasma concentration of cefpirome considering protein binding of ~ 10% (30). Plasma; lung interstitial fluid.

Cefpirome levels exceeded minimum inhibitory concentration (MIC₉₀) values for the potential respiratory pathogens Streptococcus pneumoniae (MIC₉₀ 0.03 µg/ml [31]), Klebsiella species (MIC₉₀ 0.25 µg/ml [32]), Staphylococcus aureus (MIC₉₀ 2 µg/ml [32]), and Pseudomonas aeruginosa (MIC₉₀ 8 µg/ml [32]) during the observational period of 240 min. If these results are extrapolated to longer periods, the cefpirome concentrations in lung interstitial fluid exceed the MIC₉₀ for Pseudomonas aeruginosa and Staphylococcus aureus for 350 and 550 min, respectively. The time above MIC₉₀ exceeds 12 h for Streptococcus pneumoniae and Klebsiella species.

Chest X-ray immediately after surgery and 24 h later revealed regular postsurgical patterns in all patients. Discharge from hospital was safely possible in all patients after a mean of 6 d. Adverse events or clinical complications related to the microdialysis probes were not observed.

	DISCUSSION

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With a few notable exceptions such as heparin, most drugs do not exert their effects within the plasma compartment but in defined target tissues into which drugs have to distribute from the central compartment. Unfortunately, a complete and lasting equilibration between blood and tissue cannot always be taken for granted. This notion is corroborated for various disease states by a large body of literature (8). Due to the strong scientific rationale of obtaining drug levels from the site of action rather than from the bloodstream, clinical tissue distribution studies are also increasingly encouraged by regulatory agencies in drug development (33, 34).

In the case of respiratory tract infections, several techniques have been developed based on assaying whole lung tissue, sputum, respiratory secretions, and pleural fluid (18). However, whole lung drug concentrations may serve only as weak predictors of clinical efficacy, as these represent an average of several different tissue compartments. Sputum concentrations may vary greatly because of technical difficulties with sampling, sputum pooling, and blood contamination. The use of bronchoalveolar lavage has provided the opportunity to discriminate between intra- and extracellular sites representative of pneumonia (22) by sampling the epithelial lining fluid. However, epithelial lining fluid is obtained by bronchoscopy and therefore longitudinal pharmacokinetic data can be derived only by interindividual data pooling.

In the present set of experiments, we have developed a novel microdialysis-based approach for measuring drug concentrations in lung interstitial fluid. One important advantage of the present approach relates to its ability to continuously measure drug concentrations in the interstitial space fluid. The principle of microdialysis is based on the presence of a gradient between two fluid compartments along a membrane. One fluid compartment is the dialysate fluid and the other is the interstitial fluid, to which the probe is exposed. Although the probe is also in contact with alveoli, membrane exchange will not take place with alveolar air, but only with a fluid phase. Because the majority of infections are localized within the extracellular space, the interstitial space fluid is considered an important target site for antimicrobial chemotherapy (26). Cefpirome was used as a model drug in the present study because its pharmacokinetic properties in plasma and tissues are well characterized (28, 35) and it is a standard drug for the treatment of respiratory tract infections (39). One potential shortcoming with this technique is that the antibiotic concentrations in the interstitial fluid of normal lungs may not reflect those in the interstitial fluid of lungs in patients with pneumonia.

The main finding of the present study was that in vivo microdialysis in the human lung is feasible and enables online measurement of lung tissue concentrations in spontaneously breathing patients. Unbound cefpirome concentrations in lung interstitial fluid were 66% of corresponding total plasma values with a mean AUC_plasma/AUC_tissue ratio of ~ 1.5. Given that approximately 10% of cefpirome is bound to plasma proteins (30), the present results corroborate the view that cefpirome shows a favorable tissue penetration profile. Lung concentrations of cefpirome exceeded the MIC₉₀ values of most relevant bacteria, including strains of Streptococcus pneumoniae and Klebsiella species. However, for bacteria with an MIC₉₀ > 8 µg/ml, complete coverage will be achieved only for 350 min postdosing. These data are similar to data from previous studies on the distribution of cefpirome into other human tissues (36, 37).

Overall, the procedure was well tolerated in patients undergoing lung surgery. The probes were left in place for 4 h and no complications were observed. The removal of the microdialysis probes caused only mild pain to the patients, which was far less than removal of the standard drain. Closed chest microdialysis of the lung in spontaneously breathing patients has several advantages compared with the current methodological armamentarium for measuring drug concentrations in the lung: (1) microdialysis probes can be implanted during routine surgery without major time expense; (2) continuous sampling is possible for several hours; (3) measurements can be performed under physiological and pathological conditions; and (4) bedside monitoring of target site antibiotic concentrations becomes possible as technical means for immediate analysis become available.

In summary, closed chest microdialysis is a feasible and safe method to measure drug concentrations in the human lung in vivo. The present data also corroborate the use of cefpirome as a valuable agent in the treatment of lung infections with most extracellular bacteria.