ANTIMICROBLAL AGENTS AND CHEMOTHERAPY, OCt. 1992, p. 2099-2103
Vol. 36, No. 10
0066-4804/92V102099-05$02.00/0 Copyright © 1992, American Society for Microbiology
Penetration of Cefpodoxime Proxetil in Lung Parenchyma and Epithelial Lining Fluid of Noninfected Patients C. MULLER-SERIEYS,1 C. BANCAL,2'3 M. C. DOMBRET,2'3 P. SOLER,4 G. MURCIANO,5 M. AUBIER ,23 AND E. BERGOGNE-BEREZINl*
Department of Microbiology, 1 Department of Pneumology,2 Institut National de la Sante et de la Recherche Medical Unites 226 and 482,4 and Thoracic and Cardiac Surgical Unit,' Bichat-Claude Bernard University Hospital, 46 Rue Henri-Huchard, 75877 Paris Cedex 18, France Received 23 April 1991/Accepted 2 June 1992
The pulmonary disposition of cefpodoxime was studied in 12 patients with pulmonary opacities after a single oral dose of 260 mg of cefpodoxime-proxetil, which is equivalent to 200 mg of cefpodoxime. Blood and lung tissue samples were collected during surgery, and bronchoalveolar lavage was carried out 3 h (group A) or 6 h (group B) after drug administration. Urea was used as an endogenous marker for measurement of the volume of epithelial lining fluid (ELF). Concentrations were measured by using a microbiological assay. The mean concentrations of cefpodoxime in plasma, ELF, and lung tissue were, respectively, 1.85 0.82 mg/liter, 0.22 + 0.13 mg/liter, and 0.89 + 0.80 mg/kg of body weight in group A and 1.40 1.25 mg/liter, 0.12 0.14 mg/liter, and 0.84 0.61 mg/kg in group B. Concentrations in lung parenchyma 6 h after dosing were at least equal to or above the MICs for 90%o of the strains of most organisms commonly found in respiratory tract infections, whereas data for ELF suggest levels of drug insufficient to inhibit bacteria.
Cefpodoxime-proxetil (RU 51807; CS 807) is a new oral prodrug of a cephalosporin derivative, cefpodoxime. This broad-spectrum cephalosporin is active in vitro against both gram-negative and gram-positive bacteria (7, 18), especially those usually responsible for respiratory infections, such as Streptococcus pneumoniae, Haemophilus influenzae, and Branhamella (Moraxella) catarrhalis. However, the in vitro activity seems to be insufficient to predict therapeutic efficacy in pulmonary infections, and it has been postulated that the efficacy of a drug depends also on its pharmacokinetics and distribution within the respiratory tree (1, 15). The aim of the present study was therefore to evaluate the penetration of cefpodoxime in lung tissue and epithelial lining fluid (ELF) in patients undergoing pulmonary surgery.
time between cefpodoxime administration and fiber-optic bronchoscopy, which was performed 3 and 6 h after drug administration for groups A and B, respectively. Venous-blood samples were collected into heparinized Vacutainers just before dosing and at the same time as bronchoalveolar lavage (BAL) and lung tissue sampling. Plasma was immediately separated from the blood by centrifugation at +4°C and frozen at -80°C until assay. BAL was done at 3 or 6 h depending on the group. The procedure used was as follows. Bronchoscopy was performed under topical anesthesia with lidocaine. After routine inspection of the respiratory tract and aspiration of tracheobronchial secretions and before any biopsy, the bronchoscope tip was wedged into a subsegmental bronchus of the right middle lobe. Lavage was performed by infusing 50 ml of sterile 0.9% saline solution through the aspiration port and collecting it via the same port into a plastic trap by using wall suction. Three 50-ml volumes (150 ml) were used. The time required to perform the BAL procedure did not exceed 2 min for each patient, and samples were immediately frozen and transported to the laboratory. The first aliquot was discarded because of bronchial contamination; further aliquots were filtered through sterile gauze and pooled. A 1-ml portion was taken for cell enumeration with a Malassez cell, cell identification with cytocentrifuge preparations (Cytospin; Shandon Southern Instruments, Sewickley, Pa.), and staining with the Wright-Giemsa stain (6). Lavage fluid was then centrifuged at +4°C (600 x g, 10 min) to separate cells from the fluid components. A fraction of supernatant fluid was removed for determination of urea, glucose, and albumin contents, and the rest was lyophilized and stored at -80°C until assay. Eight days after the BAL procedure, thoracotomy was performed in the same patients. Before surgery, all the patients received orally the same single dose of cefpodoxime as was administered before the BAL procedure, i.e., 200 mg of cefpodoxime. A peripheral piece of macroscopically normal tissue (2 to 3 g) was obtained at a predetermined time during the operative procedure. Adherent blood was gently removed with gauze, and the piece of tissue was
MATERIALS AND METHODS Patients. Twelve patients (10 male and 2 female; mean age, 54 + 3 years; mean weight, 63 + 9.5 kg) who underwent fiber-optic bronchoscopy for diagnostic purposes for pulmonary opacities were included in the study. Excluded were patients with known allergies to cephalosporins, impairment of renal function (serum creatinine level of >150 ,mol liter-1) or hepatic functions (alanine amino transferase, aspartate amino transferase, or bilirubin greater than twice the upper limit of normal), and active lung infection and patients who had received another antimicrobial therapy in the two previous weeks and/or been treated with gastric plasters (e.g., cinetidine, ranitidine sucralfate). The experimental protocol was approved by the local hospital ethics committee, and informed consent was granted by the patients prior to enrollment in the study. Patient characteristics are shown in Table 1. Study design. Each patient swallowed two tablets of cefpodoxime-proxetil (130 mg per tablet; equivalent to 200 mg of cefpodoxime) with 150 ml of water after fasting. Patients were randomized into two groups, A and B, according to the *
Corresponding author. 2099
2100
MULLER-SERIEYS ET AL.
ANTimICROB. AGENis CHFmoTHER. TABLE 1. Demographic characteristics of study patients
Patient no.
Age (yr)
Wt (kg)
Sexa
Amt of tobacco used
(yr-pack)
Medical history
1 2 3 4 5 6 7 8 9 10 11 12
66 50 64 52 56 67 62 63 36 64 29 45
67 51 70 75 63 49 66 50 65 52 65 79
F M M M M F M M M M M M
0 45 40 30 40 50 40 80 10 90 15 63
Metastasis of kidney adenocarcinoma Adenocarcinoma Adenocarcinoma Squamous-cell carcinoma Large-cell carcinoma Squamous-cell carcinoma Squamous-cell carcinoma Squamous-cell carcinoma Localized sequelae of tuberculosis Squamous-cell carcinoma Localized bronchial dystrophy Oat cell carcinoma
a F, female; M, male.
placed in a sterile vial, quickly frozen at -80°C, and stored until assay. The timing of this sampling depended on the group to which the patient had been assigned. Sample preparation. Prior to microbiological assay, lyophilized powder of BAL fluid was suspended in 1 ml of distilled water. After the assay had been carried out, the concentration of cefpodoxime was expressed as micrograms per milliliter of epithelial lining fluid. Pieces of lung parenchyma were weighed to reach 1 g of tissue, which was ground in 2 ml of distilled water by sonication (Ultraturrax U25; Bioblock Laboratory) and then shaken on ice for 45 min to extract the antibiotic. The tissue homogenate obtained was centrifuged at 1,500 x g for 10 min at +4°C, and a second extraction of antibiotic was made by using the sediment, which was suspended in 2 ml of distilled water, shaken on ice for 30 min, and centrifuged at 1,500 x g for 10 min. The concentration of cefpodoxime in lung was measured in two successive supernatant fractions by bioassay. The blood contamination was measured in each supernatant by absorption spectrophotometry of oxyhemoglobin (13). The concentrations of cefpodoxime in tissue due to the blood contamination were calculated by using the Roncoroni formula as follows: tissue contamination = (amount of hemoglobin in supernatant/amount of hemoglobin in blood) x S x K x (100 - hematocrit/100), where Kis the dilution factor and S is the concentration of cefpodoxime in plasma. Thus, intratissular concentration was the observed concentration minus the concentration of contamination in tissue. Use of urea to determine the volume of ELF recovered by BAL. Because of its free diffusion through the body, urea was chosen as an endogenous marker of the ELF dilution, as previously described by Rennard et al. (10). The volume of ELF recovered by BAL was calculated as follows: volume of ELF recovered (ml) = (concentration of urea in BAL [mg/ml] x volume of BAL [ml])/concentration of urea in plasma (mg/ml). The urea content was determined by using a commercially available kit (65 UV; Sigma Chemical Co., St. Louis, Mo.). The albumin concentrations in BAL fluid and plasma were determined by an enzyme-linked immunoassay (10). The concentration of cefpodoxime in ELF was obtained by dividing the total amount of recovered drug by the calculated volume of ELF. Cefpodoxime assay. Cefpodoxime concentrations in plasma, ELF, and lung parenchyma were determined by using a standard agar diffusion microbiological method. The test organisms used were Morganella morganii 313 UC3 (Roussel-Uclaf Laboratories) with antibiotic medium 2 (Dif-
co Laboratories) for samples with expected levels of >0.5 mg of cefpodoxime per liter and Proteus rettgeni UC2 (9) with antibiotic medium 1 (Difco) for samples with expected levels of 0.05) between the concentrations measured in both conditions (3.23 ± 0.09 and 0.84 + 0.03 versus 3.16 ± 0.09 and 0.87 ± 0.03 mg/liter in the presence of standards in equine serum and human serum, respectively). Thus, standards were prepared with half antibiotic-free equine serum (Pasteur Diapostics) and half phosphate buffer (pH 6.8) for plasma samples. For ELF assays, standards were prepared with phosphate buffer (pH 6.8). Controls were lyophilized liquid recovered from BAL samples taken from patients not treated with antibiotics because saline solution has no antibacterial activity. For lung assays, the antimicrobial activities of the supernatants of samples from the first four patients were calculated by reading the zones of inhibition against the zones for a supernatant of human antibioticfree lung fluids and buffer standards. Because of the absence of any significant difference in the antibiotic concentration in the lung with a supernatant or buffer curve, subsequent samples were assayed against buffer standards. Statistical evaluation. All data are presented as means standard deviations. Statistical values, including results reported as below the lower limit of detection by the assay, were calculated by using the lowest detectable value obtained in that assay. A value of 0.05 or less was considered
significant. RESULTS Characterization of cell population and volume of ELF recovered by BAL. The volume of ELF recovered by BAL was 34.4 + 13.7 ml, with a mean number of cells of 317,000 + 324,000/ml. The differential cell count revealed 84% 12% macrophages, 9.6% _ 11% lymphocytes, and 6% _
DISPOSITION OF CEFPODOXIME-PROXETIL IN HUMAN LUNG
VOL. 36, 1992
2101
TABLE 2. Concentrations of cefpodoxime in plasma, ELF, and lung tissue and ratios of concentrations in lung tissue and ELF to those in plasma Group (treatment time) and patient no.
Group A (3 h) 1 2 3 4 5 6
Mean SD Group B (6 h) 7 8 9 10 11
12 Mean SD
Concn (mg/liter) on day 1 in:
Plasma
ELF
2.70 2.10 2.65 0.35 2.00 1.28 1.85 0.82
0.40 0.20 0.24 0.02 0.33 0.12 0.22 0.13
0.54 1.89 0.55 0.58 0.86 4.00 1.40 1.25
0.02 0.28 0.02 0.02 0.02 0.34 0.12 0.14
Concn (mg/liter) on day 8 in:
Ratio of concn in ELF/plasma (%)
7.4% neutrophils. The percentage of bronchial epithelial cells was
Vol. 36, No. 10
0066-4804/92V102099-05$02.00/0 Copyright © 1992, American Society for Microbiology
Penetration of Cefpodoxime Proxetil in Lung Parenchyma and Epithelial Lining Fluid of Noninfected Patients C. MULLER-SERIEYS,1 C. BANCAL,2'3 M. C. DOMBRET,2'3 P. SOLER,4 G. MURCIANO,5 M. AUBIER ,23 AND E. BERGOGNE-BEREZINl*
Department of Microbiology, 1 Department of Pneumology,2 Institut National de la Sante et de la Recherche Medical Unites 226 and 482,4 and Thoracic and Cardiac Surgical Unit,' Bichat-Claude Bernard University Hospital, 46 Rue Henri-Huchard, 75877 Paris Cedex 18, France Received 23 April 1991/Accepted 2 June 1992
The pulmonary disposition of cefpodoxime was studied in 12 patients with pulmonary opacities after a single oral dose of 260 mg of cefpodoxime-proxetil, which is equivalent to 200 mg of cefpodoxime. Blood and lung tissue samples were collected during surgery, and bronchoalveolar lavage was carried out 3 h (group A) or 6 h (group B) after drug administration. Urea was used as an endogenous marker for measurement of the volume of epithelial lining fluid (ELF). Concentrations were measured by using a microbiological assay. The mean concentrations of cefpodoxime in plasma, ELF, and lung tissue were, respectively, 1.85 0.82 mg/liter, 0.22 + 0.13 mg/liter, and 0.89 + 0.80 mg/kg of body weight in group A and 1.40 1.25 mg/liter, 0.12 0.14 mg/liter, and 0.84 0.61 mg/kg in group B. Concentrations in lung parenchyma 6 h after dosing were at least equal to or above the MICs for 90%o of the strains of most organisms commonly found in respiratory tract infections, whereas data for ELF suggest levels of drug insufficient to inhibit bacteria.
Cefpodoxime-proxetil (RU 51807; CS 807) is a new oral prodrug of a cephalosporin derivative, cefpodoxime. This broad-spectrum cephalosporin is active in vitro against both gram-negative and gram-positive bacteria (7, 18), especially those usually responsible for respiratory infections, such as Streptococcus pneumoniae, Haemophilus influenzae, and Branhamella (Moraxella) catarrhalis. However, the in vitro activity seems to be insufficient to predict therapeutic efficacy in pulmonary infections, and it has been postulated that the efficacy of a drug depends also on its pharmacokinetics and distribution within the respiratory tree (1, 15). The aim of the present study was therefore to evaluate the penetration of cefpodoxime in lung tissue and epithelial lining fluid (ELF) in patients undergoing pulmonary surgery.
time between cefpodoxime administration and fiber-optic bronchoscopy, which was performed 3 and 6 h after drug administration for groups A and B, respectively. Venous-blood samples were collected into heparinized Vacutainers just before dosing and at the same time as bronchoalveolar lavage (BAL) and lung tissue sampling. Plasma was immediately separated from the blood by centrifugation at +4°C and frozen at -80°C until assay. BAL was done at 3 or 6 h depending on the group. The procedure used was as follows. Bronchoscopy was performed under topical anesthesia with lidocaine. After routine inspection of the respiratory tract and aspiration of tracheobronchial secretions and before any biopsy, the bronchoscope tip was wedged into a subsegmental bronchus of the right middle lobe. Lavage was performed by infusing 50 ml of sterile 0.9% saline solution through the aspiration port and collecting it via the same port into a plastic trap by using wall suction. Three 50-ml volumes (150 ml) were used. The time required to perform the BAL procedure did not exceed 2 min for each patient, and samples were immediately frozen and transported to the laboratory. The first aliquot was discarded because of bronchial contamination; further aliquots were filtered through sterile gauze and pooled. A 1-ml portion was taken for cell enumeration with a Malassez cell, cell identification with cytocentrifuge preparations (Cytospin; Shandon Southern Instruments, Sewickley, Pa.), and staining with the Wright-Giemsa stain (6). Lavage fluid was then centrifuged at +4°C (600 x g, 10 min) to separate cells from the fluid components. A fraction of supernatant fluid was removed for determination of urea, glucose, and albumin contents, and the rest was lyophilized and stored at -80°C until assay. Eight days after the BAL procedure, thoracotomy was performed in the same patients. Before surgery, all the patients received orally the same single dose of cefpodoxime as was administered before the BAL procedure, i.e., 200 mg of cefpodoxime. A peripheral piece of macroscopically normal tissue (2 to 3 g) was obtained at a predetermined time during the operative procedure. Adherent blood was gently removed with gauze, and the piece of tissue was
MATERIALS AND METHODS Patients. Twelve patients (10 male and 2 female; mean age, 54 + 3 years; mean weight, 63 + 9.5 kg) who underwent fiber-optic bronchoscopy for diagnostic purposes for pulmonary opacities were included in the study. Excluded were patients with known allergies to cephalosporins, impairment of renal function (serum creatinine level of >150 ,mol liter-1) or hepatic functions (alanine amino transferase, aspartate amino transferase, or bilirubin greater than twice the upper limit of normal), and active lung infection and patients who had received another antimicrobial therapy in the two previous weeks and/or been treated with gastric plasters (e.g., cinetidine, ranitidine sucralfate). The experimental protocol was approved by the local hospital ethics committee, and informed consent was granted by the patients prior to enrollment in the study. Patient characteristics are shown in Table 1. Study design. Each patient swallowed two tablets of cefpodoxime-proxetil (130 mg per tablet; equivalent to 200 mg of cefpodoxime) with 150 ml of water after fasting. Patients were randomized into two groups, A and B, according to the *
Corresponding author. 2099
2100
MULLER-SERIEYS ET AL.
ANTimICROB. AGENis CHFmoTHER. TABLE 1. Demographic characteristics of study patients
Patient no.
Age (yr)
Wt (kg)
Sexa
Amt of tobacco used
(yr-pack)
Medical history
1 2 3 4 5 6 7 8 9 10 11 12
66 50 64 52 56 67 62 63 36 64 29 45
67 51 70 75 63 49 66 50 65 52 65 79
F M M M M F M M M M M M
0 45 40 30 40 50 40 80 10 90 15 63
Metastasis of kidney adenocarcinoma Adenocarcinoma Adenocarcinoma Squamous-cell carcinoma Large-cell carcinoma Squamous-cell carcinoma Squamous-cell carcinoma Squamous-cell carcinoma Localized sequelae of tuberculosis Squamous-cell carcinoma Localized bronchial dystrophy Oat cell carcinoma
a F, female; M, male.
placed in a sterile vial, quickly frozen at -80°C, and stored until assay. The timing of this sampling depended on the group to which the patient had been assigned. Sample preparation. Prior to microbiological assay, lyophilized powder of BAL fluid was suspended in 1 ml of distilled water. After the assay had been carried out, the concentration of cefpodoxime was expressed as micrograms per milliliter of epithelial lining fluid. Pieces of lung parenchyma were weighed to reach 1 g of tissue, which was ground in 2 ml of distilled water by sonication (Ultraturrax U25; Bioblock Laboratory) and then shaken on ice for 45 min to extract the antibiotic. The tissue homogenate obtained was centrifuged at 1,500 x g for 10 min at +4°C, and a second extraction of antibiotic was made by using the sediment, which was suspended in 2 ml of distilled water, shaken on ice for 30 min, and centrifuged at 1,500 x g for 10 min. The concentration of cefpodoxime in lung was measured in two successive supernatant fractions by bioassay. The blood contamination was measured in each supernatant by absorption spectrophotometry of oxyhemoglobin (13). The concentrations of cefpodoxime in tissue due to the blood contamination were calculated by using the Roncoroni formula as follows: tissue contamination = (amount of hemoglobin in supernatant/amount of hemoglobin in blood) x S x K x (100 - hematocrit/100), where Kis the dilution factor and S is the concentration of cefpodoxime in plasma. Thus, intratissular concentration was the observed concentration minus the concentration of contamination in tissue. Use of urea to determine the volume of ELF recovered by BAL. Because of its free diffusion through the body, urea was chosen as an endogenous marker of the ELF dilution, as previously described by Rennard et al. (10). The volume of ELF recovered by BAL was calculated as follows: volume of ELF recovered (ml) = (concentration of urea in BAL [mg/ml] x volume of BAL [ml])/concentration of urea in plasma (mg/ml). The urea content was determined by using a commercially available kit (65 UV; Sigma Chemical Co., St. Louis, Mo.). The albumin concentrations in BAL fluid and plasma were determined by an enzyme-linked immunoassay (10). The concentration of cefpodoxime in ELF was obtained by dividing the total amount of recovered drug by the calculated volume of ELF. Cefpodoxime assay. Cefpodoxime concentrations in plasma, ELF, and lung parenchyma were determined by using a standard agar diffusion microbiological method. The test organisms used were Morganella morganii 313 UC3 (Roussel-Uclaf Laboratories) with antibiotic medium 2 (Dif-
co Laboratories) for samples with expected levels of >0.5 mg of cefpodoxime per liter and Proteus rettgeni UC2 (9) with antibiotic medium 1 (Difco) for samples with expected levels of 0.05) between the concentrations measured in both conditions (3.23 ± 0.09 and 0.84 + 0.03 versus 3.16 ± 0.09 and 0.87 ± 0.03 mg/liter in the presence of standards in equine serum and human serum, respectively). Thus, standards were prepared with half antibiotic-free equine serum (Pasteur Diapostics) and half phosphate buffer (pH 6.8) for plasma samples. For ELF assays, standards were prepared with phosphate buffer (pH 6.8). Controls were lyophilized liquid recovered from BAL samples taken from patients not treated with antibiotics because saline solution has no antibacterial activity. For lung assays, the antimicrobial activities of the supernatants of samples from the first four patients were calculated by reading the zones of inhibition against the zones for a supernatant of human antibioticfree lung fluids and buffer standards. Because of the absence of any significant difference in the antibiotic concentration in the lung with a supernatant or buffer curve, subsequent samples were assayed against buffer standards. Statistical evaluation. All data are presented as means standard deviations. Statistical values, including results reported as below the lower limit of detection by the assay, were calculated by using the lowest detectable value obtained in that assay. A value of 0.05 or less was considered
significant. RESULTS Characterization of cell population and volume of ELF recovered by BAL. The volume of ELF recovered by BAL was 34.4 + 13.7 ml, with a mean number of cells of 317,000 + 324,000/ml. The differential cell count revealed 84% 12% macrophages, 9.6% _ 11% lymphocytes, and 6% _
DISPOSITION OF CEFPODOXIME-PROXETIL IN HUMAN LUNG
VOL. 36, 1992
2101
TABLE 2. Concentrations of cefpodoxime in plasma, ELF, and lung tissue and ratios of concentrations in lung tissue and ELF to those in plasma Group (treatment time) and patient no.
Group A (3 h) 1 2 3 4 5 6
Mean SD Group B (6 h) 7 8 9 10 11
12 Mean SD
Concn (mg/liter) on day 1 in:
Plasma
ELF
2.70 2.10 2.65 0.35 2.00 1.28 1.85 0.82
0.40 0.20 0.24 0.02 0.33 0.12 0.22 0.13
0.54 1.89 0.55 0.58 0.86 4.00 1.40 1.25
0.02 0.28 0.02 0.02 0.02 0.34 0.12 0.14
Concn (mg/liter) on day 8 in:
Ratio of concn in ELF/plasma (%)
7.4% neutrophils. The percentage of bronchial epithelial cells was
