Research Center for Infectious Diseases, Laval University, Quebec City, Quebec, Canada G1V 4G2
Received 8 October 2004; returned 8 December 2004; revised 24 January 2005; accepted 2 February 2005
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Abstract |
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Methods: CD1 mice were infected with 107 cfu of Streptococcus pneumoniae. Treatments (intraperitoneal) with IL-10 (1 µg per mouse), ceftriaxone (20 mg/kg) or the combination of IL-10 + ceftriaxone were initiated 18 h after infection. Groups of mice were sacrificed at several time points from 5 min to 24 h after initiation of therapy. Ceftriaxone was quantified in blood and lungs using a microbiological assay. Additional groups of mice received a second dose of IL-10 at 36 h post-infection. Survival rates were recorded over 14 days.
Results: The clearance of ceftriaxone was significantly reduced in infected mice compared with that in non-infected animals (P < 0.01), whereas AUC, mean residence time, t1/2 and AUClung/AUCserum were significantly enhanced (P < 0.01, 0.01, 0.05, 0.05). Co-administration of IL-10 with ceftriaxone in infected animals further retained ceftriaxone in the bloodstream and reduced its volume of distribution at steady state and the ratio of AUClung/AUCserum. IL-10 alone did not modify significantly the pharmacokinetics of ceftriaxone in blood and lungs of non-infected animals.
Conclusions: The results suggest that pulmonary infection, and therapy with IL-10, both affect the pharmacokinetics of ceftriaxone. Indeed, administration of IL-10 + ceftriaxone improved the survival rate of mice (P < 0.001 compared with therapy with ceftriaxone alone). IL-10 should be considered as an adjunctive therapy to antibiotics against severe infections.
Keywords: Streptococcus pneumoniae , IL-10 , inflammation
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Introduction |
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As multiresistance of virulent strains to common antibiotics emerges worldwide, it is likely that antitoxin antibodies and immunomodulator drugs capable of enhancing host resistance or preventing physiological disturbances and inflammatory injuries will prove useful in the future. Indeed, co-administration of antibiotics and cytokines has already been considered.9 Granulocyte-colony stimulating factor (G-CSF) showed limited efficacy against pneumonia in animal models and clinical trials.10,11 Interleukin-10 (IL-10) has been reported to either improve or worsen the outcome of infectious and non-infectious diseases in various experimental conditions.1214 IL-10 administered early in the course of infection hampered host defence and worsened the outcome of murine pneumococcal pneumonia (in the absence of antibiotic therapy).15 In contrast, IL-10 given at later stages of infection prevented severe inflammation and lung oedema, and facilitated bacterial clearance in mice treated with ceftriaxone.16 Whether protection by IL-10 was also mediated through alteration of the pharmacokinetics of ceftriaxone remained unverified. Understanding the pharmacokinetics of antibiotics in infected subjects co-treated with IL-10 is not only of theoretical interest but of therapeutic significance.
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Materials and methods |
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Pneumonia was induced by intranasal inoculation of 107 cfu of S. pneumoniae serotype 3 to immunocompetent female (1820 g) CD1 Swiss mice (Charles River, St-Constant, Canada), as previously described.16,17 All experimental procedures were approved by the Laval University Committee for Protection of Animals and were conducted in accordance with guidelines of the Canadian Council on Animal Care.
Treatments
Treatments were initiated 18 h after infection with placebo (0.1 mL) or with a single intraperitoneal dose of 1 µg of recombinant murine IL-10 per mouse (Schering-Plough, Kenilworth, NJ, USA), a single intraperitoneal dose of 20 mg ceftriaxone per kg of body weight (Hoffmann-La Roche Limited, Mississauga, Canada), or the combination of IL-10 + ceftriaxone. Uninfected animals were also used as controls. The MIC of ceftriaxone for the S. pneumoniae strain was < 0.016 mg/L.
Microbiological and inflammatory status at initiation of treatment
To ensure reproducibility of our model,16 health status, bacterial growth and inflammation were verified in seven infected (and seven non-infected) animals that were sacrificed (by CO2 inhalation) at initiation of treatments (18 h post-infection). Body weight, serum creatinine, bacterial counts in blood and lungs, leucocyte counts in bronchoalveolar lavage (BAL) fluid, pulmonary vascular permeability and lung wet/dry weight ratio were quantified as previously described, in similarly processed samples.16
Pharmacokinetic study
The pharmacokinetics of ceftriaxone were studied simultaneously, over a 24 h period, in infected and non-infected mice that were treated 18 h post-infection with a single dose of ceftriaxone (20 mg/kg of body weight) and one dose of 1 µg of IL-10 (or placebo) per mouse. At each time-point of 5, 15, 30 and 45 min and 1, 2, 4, 8 and 24 h after the injection of ceftriaxone, four mice per group were killed by CO2 and immediately exsanguinated by cardiac puncture. Sera were obtained for determination of antibiotic levels. Lungs and heart were removed together and weighed. Residual blood was cleared from lungs by perfusing 2 mL of sterile saline into the right ventricle using a sterile syringe. Blood-free lungs were then homogenized with a Potter-Elvehjem homogenizer in 1 mL of potassium phosphate buffer (50 mM, pH 6.0) at 4°C. Tissues were centrifuged at 3000 g for 30 min and supernatants were used to quantify ceftriaxone. Ceftriaxone concentrations in sera and lung tissues were determined by agar well bioassay, using antibiotic medium No. 1 (Becton Dickinson) and Escherichia coli ATCC 39188 as the test organism. Standard curves of ceftriaxone (in the range 0.05200 mg/L) were prepared in normal sera and lung homogenates from untreated mice. All samples and standards were tested in triplicate. The limit of detection of the bioassay was 0.05 mg/L, the intra-day coefficient of variation for standard concentrations tested was < 6.2% and the inter-day coefficient of variation was < 6.7%.
Pharmacokinetic analysis
Non-compartmental analysis was applied for the pharmacokinetic calculations. The elimination rate constant (kel) was first estimated from the slope obtained by least-squares regression analysis for the terminal portion of the concentration versus time curve. The elimination half-life (t1/2) was then calculated according to the formula t1/2=ln2/kel. The area under the concentrationtime curve (AUC) was calculated by the trapezoidal rule. The total mean residence time (MRT) was calculated as MRT=AUMC/AUC. The AUMC was the area under the concentrationtime versus time curve. Total serum clearance (CL) was calculated as dose/AUC. The volume of distribution at steady state (Vss) was determined by the equation Vss=MRT x CL. The degree of penetration into lung tissue was determined by comparison of the area under the concentrationtime curve for the lung tissue with that for the serum.
Efficacy study
To further investigate the influence of IL-10 in pneumococcal pneumonia, groups of 12 mice were infected and treated with ceftriaxone and IL-10 as described above, with the exception that a second dose of 1 µg of IL-10 was administered 36 h after infection. Survival rates were recorded over 14 days. The experiment was repeated twice.
Statistical analyses
Statistical analysis of the difference between groups (for all parameters tested including pharmacokinetic data, microbiology, inflammatory and physiological status, except for survival rates) was performed by analysis of variance using a least squares method (StatView SE + Graphics, Abaccus Concepts Inc., Berkeley, CA, USA). If the F test indicated a difference, group comparisons were performed by Fisher's protected least significant difference test. Survival rates on day 14 of infection were compared by 2 test. All data are presented as means ± SD.
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Results |
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Body weight (18.3 ± 1.1 g versus 17.9 ± 1.3 g) and serum creatinine (11 ± 2.1 µmol/L versus 13 ± 10 µmol/L) did not differ significantly between non-infected and infected animals, respectively, at initiation of the pharmacokinetic study. At that time point (18 h post-infection), lethargy, ruffled fur and hunched appearance characterized infected mice. Bacterial counts in lungs reached 6.3 log10 cfu. Intense pulmonary leucocyte infiltration was observed in BAL fluid of those mice (180 000 cells/mL), whereas leucocyte counts remained < 200 cells/mL in non-infected animals (P < 0.001). Significant enhancement of pulmonary vascular permeability was noted (index 2.5 ± 0.5 units) compared with that in non-infected mice (1.6 ± 0.3 units) (P < 0.05). Lung wet/dry ratio did not differ significantly at that time (5.1 ± 0.3 units versus 4.8 ± 0.2 units). No bacteria were detected in the bloodstream of animals with pneumonia at the initiation of the pharmacokinetic study.
Pharmacokinetics of ceftriaxone
The concentrationtime curves of ceftriaxone in serum and lung tissues are shown in Figures 1 and 2. Their derived pharmacokinetic parameters determined for infected and non-infected animals are summarized in Tables 1 and 2. After reaching similar peak levels, serum concentrations of ceftriaxone in infected mice (untreated or treated with IL-10) declined more slowly than those in uninfected mice. CL values were significantly lower in infected mice (P < 0.01). Indeed, serum t1/2, AUC and MRT of ceftriaxone for infected mice reached two-fold greater values than those for healthy controls (P < 0.05, 0.01 and 0.01, respectively). IL-10 enhanced serum AUC of ceftriaxone in infected mice, but only the group receiving ceftriaxone alone showed a higher Vss value in infected mice than in normal mice. IL-10 did not modify significantly the pharmacokinetic data of ceftriaxone in the blood and lungs of non-infected animals, thus excluding potential interactions between both drugs or interference of IL-10 with protein binding of ceftriaxone.
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Survival rates
All untreated animals died by day 5 post-infection (Figure 3). IL-10 therapy alone did not prevent death. Ceftriaxone therapy achieved a 54% survival rate. The addition of IL-10 to ceftriaxone achieved a 95% survival rate, thus providing significantly better protection than that afforded by ceftriaxone alone (P < 0.001).
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Discussion |
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Evaluation of the pathophysiological status of animals confirmed the reproducibility of our model, as identical bacterial growth and similar magnitude of inflammation (leucocyte infiltration into the lungs and high pulmonary vascular permeability) to that previously reported16 were still observed in infected mice.
Streptococcal infection altered the serum kinetics of ceftriaxone, as shown by enhanced AUC and Vss, prolonged elimination half-life and delayed clearance. Our results are consistent with clinical reports that show a greater volume of distribution and a longer half-life of drugs in infected patients compared with healthy controls.3,25 Critically ill patients, particularly those with severe sepsis, often have a reduced effective circulating volume, in part due to generalized increased capillary permeability and peripheral oedema.3 Albumin leak (tissue/plasma ratio) secondary to microvascular injury also increases in animals suffering from Pseudomonas pneumonia and sepsis.26 The enhanced capillary permeability observed in our experiment (with a potential reduction in renal and biliary plasma flow) may thus have contributed to alter the clearance of ceftriaxone in infected mice.
Ceftriaxone in lungs was recovered at higher concentrations in infected animals than in healthy controls. The high pulmonary levels might result from enhanced permeability of the pulmonary capillary bed upon the action of bacterial virulence factors and of inflammatory and vasoactive mediators released by host cells. The tissue distribution of antibiotics has long been a matter of interest in the literature.27 Indeed, our data suggest that tissue penetration and distribution of drugs in pneumonia cannot be predicted from those reported in studies with healthy subjects. In fact, microdialysis is an appropriate method to measure the tissue concentrations of antibiotics in infectious and inflammatory diseases.
Whether pneumonia or sepsis can modify the pharmacokinetic parameters of ceftriaxone by altering its protein binding in mice has not been documented (the protein binding of ceftriaxone in healthy mice has been reported to vary from 8596%).28
Obviously, for an agent such as ceftriaxone with a free concentration of 5%, slight changes in protein binding would have significant effects. Data for rabbits show no changes in sepsis, but these data were for cephalosporins with moderate protein binding.29
Our results also show that anti-inflammatory therapy with IL-10 further modifies pulmonary and blood levels of antibiotics. There is considerable evidence that IL-10 modulates capillary permeability in various inflammatory conditions.30,31 IL-10 in our model was previously shown to reduce capillary permeability, haemorrhages and leucocyte infiltration into the lungs.16 It is likely that by reducing pulmonary inflammation, IL-10 protected lungs and contributed to the high survival rate of mice. In addition, the present study shows that IL-10 helps maintain ceftriaxone in the bloodstream, and may reduce its volume of distribution and the ratio of AUClung/AUCserum compared with infected animals that receive only ceftriaxone therapy. The persistence of high concentrations of ceftriaxone in blood supports our previous findings that IL-10 therapy is associated with a low rate of sepsis in this animal model.16 Our results also suggest that pulmonary levels of drugs do not necessarily correlate with blood levels. Modulation of pulmonary capillary permeability by IL-10 did influence tissue uptake of ceftriaxone. In that context, resistant strains that display high MICs could necessitate sustained high levels of drugs in lungs.
IL-10 holds enormous potential for the treatment of infectious, inflammatory and autoimmune disorders. It is suggested that the therapeutic potential of IL-10 to selectively ameliorate human infectious and inflammatory processes can be realized through a careful selection of the clinical conditions in which patients are undergoing concomitant treatment with antimicrobial regimens.32 Verification of antibiotic pharmacokinetics during simultaneous administration of IL-10 in human volunteers and infected patients should be made.
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Acknowledgements |
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