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In colorectal surgery, it is currently accepted that antibiotic prophylaxis is necessary for members of the family Enterobacteriaceae and anaerobic bacteria, especially Bacteroides fragilis. Abdominal surgery with a class II risk involves clean contaminated procedures, with a rate of infection without prophylactic antibiotherapy between 10 and 20%. The effectiveness of prophylaxis depends upon the achievement of adequate concentrations of antimicrobial agents in the tissues (5, 9, 11). These concentrations should be maintained during the whole surgical procedure and require an easy route of drug administration. The aim of the present study was to evaluate plasma and epiploic-fat drug concentrations of pefloxacin and its metabolite (norfloxacin) obtained after a single injection of 800 mg of pefloxacin (intravenously [i.v.]) (4, 7, 8). Pefloxacin is a quinolone derivative which possesses a broad-spectrum antibacterial activity. It is active against most aerobic gram-negative rods, including enterobacteria (MIC at which 90% of isolates are inhibited [MIC₉₀], 1 µg/ml). For Staphylococcus aureus and streptococci, including enterococci, MICs are between 0.06 and 8 µg/ml and between 2 and 4 µg/ml, respectively. In this study pefloxacin was administered with metronidazole, which is active against Bacteroides. The metronidazole concentrations were not determined because the patients received a metronidazole treatment beforehand.

Approval from the Institutional Ethical Committee was obtained, and all patients gave informed consent. Ten patients (six men and four women) were included; their mean age was 61.6 ± 18.4 years, with a range of 26 to 78 years. All patients underwent a scheduled colorectal surgery lasting for over 2 h.

Patients already under therapy with an antibiotic other than metronidazole during the 3 days before and those with hepatic insufficiency or who had a history of previous intolerance to pefloxacin were excluded from the study.

A single dose of 800 mg of pefloxacin (i.v.) was infused at a constant rate starting during the 30 min after the induction of anesthesia.

Five milliters of blood and 1 cm³ of omentum were collected at 30 min (H₀), at 60 min, and then every 30 min, following the end of infusion until closure of the peritoneum. A control blood sample was taken before the pefloxacin perfusion.

Plasma samples were obtained after centrifugation of blood (6,700 × g) for 10 min (E80 centrifuge; Jouan, Saint Nazaire, France) and stored at 35°C until analysis. Epiploic-fat samples were taken by the surgeons at the same time as plasma samples and were frozen. Plasma and tissue antibiotic concentrations were determined by high-performance liquid chromatography (HPLC).

Pefloxacin was extracted from plasma after addition of 5 µg of an internal standard agent (4844P, an analog of pefloxacin). Four milliliters of dichloromethane and 100 µl of phosphate buffer (pH 7.4) were added to 500 µl of plasma. The mixture was agitated for 10 min and then centrifuged (10 min, 5,300 × g). Organic phase (3.5 ml) was collected and transferred to a new tube. Four milliliters of dichloromethane was added again to the aqueous phase. After centrifugation, the supernatant was eliminated and the organic phase was added to the solution previously collected. The organic phase was evaporated at 60°C. The residue was diluted with 100 µl of mobile phase, and 20 µl was injected into the HPLC column.

Epiploic-fat samples were processed as follows: each sample (500 mg) was kept at 4°C after the addition of dichloromethane (4 ml), and 5 µg of an internal reference agent (4844P) was added and mixed by using an automatic grinder (Ultra Turrax; Ika-Werk, Stauffen, Germany). The mixture was collected and then centrifuged (10 min, 5,300 × g).

A liquid-liquid extraction with a solution of sodium hydroxide (0.1 N) was performed. Four milliliters of sodium hydroxide (0.1 N) was added to the dichloromethane. The mixture was agitated for 10 min and centrifuged (5 min, 5,300 × g).

The organic phase was eliminated, the aqueous phase was neutralized at pH 7.4 by addition of concentrated trichloracetic acid, and 4 ml of dichloromethane was added. The tubes were agitated (10 min) and then centrifuged (5 min, 5,300 × g). The aqueous phase was eliminated, and the organic phase was evaporated at 60°C. After the evaporation, the residue was diluted with 100 µl of mobile phase and 20 µl was injected into the HPLC column. The mobile phase was a mixture of acetonitrile and citrate buffer adjusted to pH 4.8 (85:15, vol/vol), and the pumping rate was 1.0 ml/min.

The extraction recoveries of pefloxacin were 92.3% ± 5.0% and 61.3% ± 16.5% for plasma and fat, respectively. The extraction recoveries of norfloxacin were 80.9% ± 7.0% and 69.4% ± 14.0% for plasma and fat, respectively. The extraction recoveries were 80.5% ± 4.4% of internal standard for both plasma and fat.

The antibiotic concentrations in plasma and epiploic fat were analyzed by HPLC assay with a model 112 pump (Gold system; Beckman, San Ramon, Calif.), a 100 RP-18 endcapped Lichrosphere column (5 µm, 125 by 4 mm) coupled to a 100 RP-18 Lichrosphere precolumn (5 µm, 25 by 4 mm; Merck), and a spectrofluorometric detector (330 and 418 nm).

The parameters concerning the validation of the HPLC method for pefloxacin and norfloxacin (linearity, repeatability, and reproducibility) are in Tables 1 and 2. The lower limits of quantification were 0.02 and 0.025 µg/ml for pefloxacin and norfloxacin, respectively.

Plasma and fat antibiotic concentrations were plotted against time. The areas under the plasma and fat during concentration-time curves (AUCs) were calculated from 0 to 150 min by using the trapezoidal rule. Evaluation of tissue penetration was performed by using two parameters: the rate of penetration at each time period (percent) calculated by the epiploic-fat drug concentration-to-plasma drug concentration ratio and the mean ratio of the fat AUC to the plasma AUC. The results are expressed as means and ranges for demographic data and means and standard deviations for antibiotic concentrations and AUCs. Plasma pefloxacin concentrations decreased by 40% from H₀ to 150 min (Table 3). Epiploic-fat pefloxacin concentrations were stable during the whole procedure and remained higher than 1 µg/g. Plasma norfloxacin concentrations are shown in Table 4 and were low and stable throughout the operation. Great individual variability could be observed. The rate of penetration of pefloxacin increased from 20 to 40% in 90 min before decreasing to 31% at the end of the operation.

The mean values of fat AUC and plasma AUC were 5.24 ± 2.65 and 16.37 ± 3.34 mg × h liter¹, respectively. The ratio of the fat AUC to the plasma AUC was 32%.

Epiploic-fat norfloxacin concentrations are not presented since they were below the limit of quantification.

The goal of prophylaxis was to reach and maintain blood and tissue drug concentrations at levels over the MIC that inhibit growth of contaminating agents throughout the surgical procedure (3). In the present study plasma pefloxacin concentrations were comparable to those obtained by Barre et al. (2). When we compared the plasma and tissue pefloxacin concentrations with the MIC₉₀ for enterobacteria (1 mg/liter), especially Escherichia coli (MIC₉₀ is 0.125 mg/liter) (1, 10), we obtained during the operation concentrations in tissue always higher than the MIC₉₀ and observed that no patients after the operation presented infection.

The rate of penetration of pefloxacin into epiploic fat was about 32% during the operation. This value of diffusion was about the same than that obtained by Daschner et al. for ciprofloxacin with a single infusion of 100 mg (6). In that study, the rate of diffusion was about 34% but the conditions were different because Daschner et al. evaluated the tissue pharmacokinetics of ciprofloxacin after i.v. administration prior to urological surgery.

The N-desmethyl derivate (norfloxacin) has the same in vitro activity as pefloxacin (MIC₉₀ is 0.5 mg/liter) (2). In this study plasma norfloxacin concentrations were low and unlikely to be clinically relevant.

In conclusion, the infusion of 800 mg of pefloxacin at the induction of anesthesia allowed tissue drug concentrations sufficient to protect the patient from potential contamination by enterobacteria during the operation.