multiple organ dysfunction, the concentration of each antibiotic in the lungs, compared with that in .... syndrome by its prolonged activation of the alternative com-.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1996, p. 819–821 0066-4804/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 40, No. 3
Influence of Experimental Rat Model of Multiple Organ Dysfunction on Cefepime and Amikacin Pharmacokinetics O. MIMOZ,1* A. JACOLOT,2 C. PADOIN,2 J. QUILLARD,3 M. TOD,2,4 K. LOUCHAHI,2 K. SAMII,1 AND O. PETITJEAN2,4 Service d’Anesthe´sie-Re´animation Chirurgicale,1 and Service d’Anatomo-Pathologie,3 Ho ˆpital Biceˆtre, 94270 Le Kremlin-Biceˆtre, and Cre´pit 93, Centre de Recherche en Pathologie Infectieuse et Tropicale,4 and De´partement de Pharmacologie-Toxicologie Hospitalie`re,2 Ho ˆpital Avicenne, 93009 Bobigny, France Received 8 August 1995/Returned for modification 28 September 1995/Accepted 6 December 1995
We adapted an experimental model of multiple organ dysfunction to study the alterations it induces in the pharmacology of cefepime and amikacin. The half-lives of both antibiotics were significantly prolonged because of nonsignificant enhancement of the volume of distribution and reduced renal elimination. In the presence of multiple organ dysfunction, the concentration of each antibiotic in the lungs, compared with that in the lungs of healthy controls, was significantly decreased, despite similar concentrations in plasma, indicating that the application of a standard antibiotic concentration in plasma could lead to underdosage in tissues during the initial days of therapy. Squibb) pharmacokinetics were performed when multiple organ dysfunction was well established, i.e., 12 days after i.p. injection of zymosan, and the results were compared with those of similar studies with healthy rats. Pharmacokinetic parameters. Each rat (five or six per group) received a single 1-ml i.p. injection of either 50 mg of cefepime per kg or 18 mg of amikacin per kg. Multiple blood samples (300 ml) were collected 30, 60, 90, 120, 150, 180, 240, 300, and 360 min after antibiotic injection. Saline (600 ml) was injected intraarterially after collection of each blood sample to restore blood volume. Individual cefepime and amikacin pharmacokinetic parameters were determined. The peak concentration in plasma and the time it took to reach that peak were the experimental values. A noncompartmental model was used to fit the data by weighted least squares with the weighting factor 1/ycalc to determine half-life, volume of distribution in the b phase total body clearance, and the area under plasma concentration-time curve extrapolated to infinity for cefepime and amikacin in healthy rats and rats with multiple organ dysfunction, taking the fraction of the cefepime or amikacin dose absorbed to be equal to 1 (Siphar). Antibiotic concentrations in lung tissue. Six to 10 other rats per group were sacrificed 1, 2, and 3 h after being given a single 1-ml i.p. injection of 50 mg of cefepime per kg and 18 mg of amikacin per kg. Blood was collected to determine the antibiotic concentration in plasma. The lungs were homogenized in 1 ml of saline, and the resulting suspension was centrifuged for 10 min at approximately 1,000 3 g and 38C. The supernatant was collected, and antibiotic levels were assayed. Antibiotic measurements were not corrected for blood contamination because blood represents less than 6% of lung weight and neither antibiotic accumulated in erythrocytes; therefore, the presence of blood does not lead to substantial errors in the evaluation of amikacin or cefepime levels (11). Measurement of antibiotic concentrations. The cefepime concentration was determined by using a modified version of the high-performance liquid chromatography assay described by Barbhaiya et al. (2). The quantification limit of the assay was 1 mg/liter, and the between-days coefficient of variation ranged from 7% at 1 mg/liter to 6% at 50 mg/liter. The amikacin concentration was determined by using an immunoenzyme as-
Previous studies have shown that the pharmacokinetic parameters of aminoglycosides are profoundly modified in intensive care unit patients, but little is known about other classes of antibiotics, such as b-lactams (16). In this study, we adapted an experimental model described by Goris et al. (5) by injecting smaller doses of zymosan to evaluate the multiple organ dysfunction-induced pharmacokinetic modifications of a new blactam, cefepime, in plasma and lungs. Amikacin was also studied to compare the pharmacokinetic modifications induced by our model with those observed in humans and thus to determine whether it could constitute a valuable model. Experimental multiple organ dysfunction. Twenty Wistar rats were randomly assigned to a zymosan group or a healthy group (10 per cohort). Each rat in the former group received an intraperitoneal (i.p.) injection of 0.5 mg of zymosan (Sigma) per g suspended in liquid paraffin to induce a chronic release of inflammatory mediators, which resulted in maximal multiple organ dysfunction 12 days later. Previous studies showed that all of the surviving animals 12 days after zymosan challenge improved until complete recovery. Because zymosan induced acute peritonitis during the first 3 days after its administration, 0.1 mg of imipenem-cilastatin (Merck Sharp and Dohme-Chibret) per g was administered simultaneously to diminish the initial mortality due to overwhelming infection. No i.p. injection was given to rats in the healthy group which served as a control. To simulate further the human situation, all rats received i.p. injections of 0.9 M saline equal to 0.025 ml/g on days 0, 1, 2, 9, 10, and 11 following randomization. Clinical conditions were recorded daily, and on day 12, all surviving rats were anesthetized and bled by aortic puncture to determine biological parameters, and the lungs, kidneys, spleen, liver, and a part of the digestive tract were removed and weighted. Relative organ weights were calculated by using the formula (organ weight/body weight) 3 100. After fixation with 4% formaldehyde, microscopic sections of these organs were prepared for staining with hematoxylin-eosin-saffron and examined (n 5 six per group). Studies of cefepime and amikacin (Bristol-Myers
* Corresponding author. Phone: (33) 1 45213441. Fax: (33) 1 45212875. 819
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TABLE 1. Comparison of relative organ weights between healthy rats and those with multiple organ dysfunction Median relative organ wt (range) Organ
Lung Liver Kidney Spleen
Healthy rats (n 5 10)
Rats with multiple organ dysfunction (n 5 9)
P value
0.4 (0.3–0.4) 3.3 (2.8–3.7) 0.6 (0.5–0.6) 0.2 (0.2–0.4)
0.7 (0.4–0.9) 4.0 (3.6–4.7) 0.7 (0.6–0.8) 0.7 (0.5–1.1)
0.002 0.002 0.009 0.001
say (EMIT assay). The limit of quantification was 1 mg/liter, and the coefficients of variation were below 8% over the entire range of measurement. The biological matrices used for highperformance liquid chromatography and EMIT standards were bovine plasma for plasma samples and water for tissue samples. Statistical analysis. Results are expressed as medians with ranges. Data from healthy rats and rats with multiple organ dysfunction were compared by using the Mann-Whitney U test. A P value below 0.05 was considered to be significant. From days 1 to 4, all rats in the zymosan group showed symptoms of illness. They became lethargic and anorexic, hyperventilated, and had ruffled fur and diarrhea. Epistaxis and bleeding conjunctivae were also observed. From days 4 to 8, the rats improved, becoming very active, gaining weight, and no longer hemorrhaging or having diarrhea. Between days 9 and 12, their general condition deteriorated again, with increasing hyperventilation, tachycardia, and loss of weight. All control rats remained healthy throughout the experimental period and progressively gained weight. One zymosan group rat died on day 10. The remaining 19 rats were sacrificed on day 12 for biological and histological studies. Premortem examination showed that healthy rats gained significantly more weight during the study period than did rats with multiple organ dysfunction (7.8 versus 1.5%; P , 0.002). Postmortem examination of rats with multiple organ dysfunction showed signs of extensive acute peritonitis, edematous lungs, pale liver, and large spleen, whereas postmortem examination results of healthy rats were normal. As shown in Table 1, in the presence of multiple organ dysfunction, the relative weights of each organ was significantly increased, indicating increased water content. The results of blood analyses are summarized in Table
TABLE 2. Comparison of blood analysis results of healthy rats and rats with multiple organ dysfunction Median (range) Biological parameter
Healthy rats (n 5 10)
PaO2 (mm Hg) 92 (82–103) Glycemia (mmol/liter) 9.1 (7.2–11.3) Creatinine (mmol/liter) 54 (38–58) Alkaline phosphatase 92 (69–114) (U/liter) Bilirubin (U/liter) 7 (4–19) 3.0 (1.6–8.2) Leukocytes (109/liter) Polymorphonuclear leuko- 37 (16–61) cytes (%) Lymphocytes (%) 59 (36–78) Hematocrit (%) 41 (39–46) 710 (532–852) Thrombocytes (109/liter)
Rats with multiple organ dysfunction (n 5 9)
P value
77 (65–90) 7.0 (4.3–9.1) 51 (43–55) 127 (85–150)
0.01 0.003 0.5 0.003
12 (4–14) 5.4 (3.7–8.2) 60 (55–72)
0.2 0.02 0.004
35 (22–42) 37 (35–38) 1,263 (1,086–1,486)
0.003 0.001 0.001
TABLE 3. Pharmacokinetic parameters measured in plasma after i.p. injection of 50 mg of cefepime per kg or 18 mg of amikacin per kg into healthy rats or rats with multiple organ dysfunction Median (range) Drug and parametera
Healthy ratsb
Rats with multiple organ dysfunctionc
P value
Cefepime Cmax (mg/liter) Tmax (min) Vb (liters/kg) CLT (ml/min z kg) t1/2b (h) AUC (h z mg/liter)
54 (39–83) 30 (30–60) 0.4 (0.4–0.5) 7.2 (6.3–7.3) 0.7 (0.6–0.8) 116 (113–131)
78 (60–94) 30 (30–30) 0.5 (0.4–0.8) 6.7 (4.3–9.6) 0.9 (0.7–1.0) 123 (96–195)
0.17 0.21 0.27 0.29 0.05 0.37
Amikacin Cmax (mg/liter) Tmax (min) Vb (liters/kg) CLT (ml/min z kg) t1/2b (h) AUC (h z mg/liter)
18 (15–41) 30 (30–30) 0.6 (0.3–0.8) 8.7 (4.3–9.8) 0.9 (0.9–1) 35 (31–70)
24 (22–27) 30 (30–30) 0.7 (0.5–0.9) 5.3 (3.5–7.2) 1.2 (1–2.4) 57 (42–86)
0.34 1 0.37 0.15 0.01 0.15
a Cmax, peak concentration in plasma; Tmax, time to peak concentration in plasma; Vb, volume of distribution in the b phase; CLT, total body clearance; t1/2b; half-life during the b phase; AUC, area under the plasma concentrationtime curve. b There were seven healthy rats in the cefepime group and six in the amikacin group. c There were six rats each with multiple organ dysfunction in the cefepime and amikacin groups.
2. Experimentally induced multiple organ dysfunction generated several microscopic modifications in different organs. Lung specimens showed thickening of alveolar walls, edema, and infiltrates of mononuclear cells. Kidney specimens exhibited focal necrosis of proximal tubules and edema. Spleens were depleted of lymphoid tissue, while the numbers of megakaryocytes and erythroblasts were increased. Liver specimens showed engorged veins, inflammatory lymphocyte infiltrates, and hyperplasia of Kupffer cells. The wall of the small intestine specimen was infiltrated by mononuclear cells. Cefepime and amikacin pharmacokinetic parameters in healthy animals were similar to those previously reported (Table 3). For both antibiotics, the presence of multiple organ dysfunction significantly increased the antibiotic elimination half-lives because of nonsignificant enhancement of the volume of distribution and reduced renal elimination (Table 3). Despite similar concentrations of the two antibiotics in plasma in both study groups, their concentrations in the lungs were significantly lower in rats with multiple organ dysfunction 1 and 2 h after administration (Table 4). No antibiotic was detected in the lungs 3 h after injection. Experimental models of multiple organ dysfunction in small laboratory animals generally utilize a bacterial or endotoxin challenge (7, 12, 17). The mortality induced by these models is high. Recently, Goris et al. (5) developed an experimental animal model of multiple organ dysfunction that we adapted by using a lower dose of zymosan to stimulate the human clinical syndrome by its prolonged activation of the alternative complement pathway and macrophages induced by an i.p. injection of zymosan into the rats (5). Twelve days after zymosan injection, the clinical, biochemical, and microscopic alterations in our model closely resembled those reported in critically ill septic patients and in other animal models of bacterium or endotoxin-induced multiple organ dysfunction (5). Importantly, mortality in our model was relatively low in comparison with that observed in previously reported models. Modifica-
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TABLE 4. Concentrations of cefepime or amikacin in plasma and lungs measured 1, 2, and 3 h after antibiotic administration in healthy rats and rats with multiple organ dysfunction Time after drug administration (h) and site
No. of rats, median cefepime concna (range) Healthy rats
Rats with multiple organ dysfunction
No. of rats, median amikacin concna (range) Healthy rats
Rats with multiple organ dysfunction
1 Plasma Lungs
10, 57 (21–82) 9, 76 (40–97) 10, 23 (8–28) 9, 13 (7–15)b
10, 26 (20–39) 9, 22 (16–36) 10, 4 (3–6) 9, 2 (2–2)b
Plasma Lungs
10, 35 (26–38) 8, 29 (20–38) 10, 9 (3–13) 8, 3 (2–6)b
10, 9 (7–16) 10, ,1
8, 13 (10–16) 8, ,1
Plasma Lungs
6, 19 (9–28) 6, ,1
6, 5 (3–7) 6, ,1
6, 7 (5–9) 6, ,1
2
3 6, 13 (9–18) 6, ,1
a Concentrations in plasma are in milligrams per liter. Concentrations in lung tissue are in micrograms per gram. b P , 0.05 for rats with multiple organ dysfunction versus healthy rats for the antibiotic given.
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13). Since b-lactams and aminoglycosides are distributed in the total extracellular water, the proportion of fluid containing antibiotics was increased and the apparent antibiotic level in the whole tissue should have increased, but the opposite was observed, suggesting that antibiotic concentrations in the extracellular space were decreased in animals with multiple organ dysfunction. Decreased blood flow to the tissues, as observed in humans and other experimental models (12, 17), could explain, at least in part, the results observed. Our finding that in the presence of multiple organ dysfunction, concentrations of both antibiotics in the lungs were significantly decreased, despite their similar concentrations in plasma, suggests that the recommendation of a standard antibiotic concentration in plasma could lead to suboptimal concentrations in tissues during the initial days of therapy. These pharmacokinetic alterations need to be confirmed, and their effects in an experimental model of infection must be evaluated. We thank R. J. A. Goris and Monique Jansen for help with the adaptation of the zymosan model and P. Norwood for rereading the manuscript. We thank Bristol-Myers Squibb (France) for supporting this work. REFERENCES
tions of cefepime and amikacin pharmacokinetics induced by our experimental multiple organ dysfunction were comparable, with significantly increased half-lives and nonsignificant enhancement of the volume of distribution. Expanded volumes of distribution and prolonged half-lives of aminoglycosides and some b-lactams (including cefepime) have already been reported for critically ill subjects (1, 3, 6, 9, 10, 15, 16). The increase in the volume of distribution is largely explained by the enhancement of extracellular fluid following endothelial damage and tissue edema (4, 13). The slight increase in the volume of distribution in our model compared with that observed in humans could be explained by the lack of various factors affecting pharmacokinetic parameters, i.e., massive volume expansion, mechanical ventilation, positive end-expiratory pressure, fever, old age, etc., which could not be reproduced in our model (8, 14). The addition of an experimental infection, which could modify per se the pharmacokinetic parameters of both antibiotics and emphasize the modifications observed (especially enhancement of the volume of distribution could be expected), needs to be addressed. The variability of the pharmacokinetic parameters observed in animals with multiple organ dysfunction was not greater than in healthy rats, which is different from reported findings on humans. However, intensive care unit patients do not form a homogeneous population and the gravity of the illness varies from one patient to another. By contrast, with the same dose of zymosan the intensity of inflammatory system stimulation was similar among all of the animals, and this can explain the lack of great variability among pharmacokinetic parameters in rats with multiple organ dysfunction. Concentrations of both antibiotics in the lungs were significantly lower in rats with multiple organ dysfunction than in healthy rats, despite similar concentrations measured in plasma. Even if we were unable to confirm that antibiotic concentrations in the extracellular fluid proportionally decrease with regard to antibiotic concentrations in whole tissue, in light of our results we can assume it. Multiple organ dysfunction induces an increase in the water content of organs (Table 1) that is located primarily in the extracellular space (4,
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