Efficacy of liposome-encapsulated ciprofloxacin compared with ciprofloxacin and ceftriaxone in a rat model of pneumococcal pneumonia

Martin H. Ellbogen1,2,3,4, Keith M. Olsen5, Martha J. Gentry-Nielsen1,2,3,4 and Laurel C. Preheim1,2,3,4,*

1 Infectious Diseases Section; 2 Veterans Affairs Medical Center, 4101 Woolworth Avenue, Omaha, NE 68105; 3 Creighton University School of Medicine; 4 University of Nebraska College of Medicine; 5 University of Nebraska College of Pharmacy, Omaha, NE, USA

Received 3 July 2002; returned 9 August 2002; revised 20 September 2002; accepted 26 September 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Encapsulation of ciprofloxacin in sterically stabilized liposomes results in a prolonged circulation time and improved pharmacokinetics. Liposome-encapsulated ciprofloxacin was compared with conventional ciprofloxacin and ceftriaxone in a rat model of pneumococcal pneumonia. Male Sprague–Dawley rats were infected transtracheally with type 3 Streptococcus pneumoniae and then treated with intravenous ceftriaxone (100 mg/kg), ciprofloxacin (40 or 80 mg/kg) or liposomal ciprofloxacin (40 or 80 mg/kg) administered once or twice daily for 3 days. White blood counts, development of bacteraemia and mortality were measured for 10 days. Antibiotic concentrations in serum, lung lavage fluid and white blood cells recovered from lung lavage fluid were determined. Liposomal ciprofloxacin concentrations were significantly higher in serum and lavage fluid compared with conventional ciprofloxacin, resulting in greater area under the serum concentration–time curve and maximum serum concentration. Despite these higher concentrations, survival rates were similar between groups treated with equivalent doses of liposomal ciprofloxacin versus ciprofloxacin. When antibiotics were given once daily, ceftriaxone was more effective than either form of ciprofloxacin.

Keywords: liposomal ciprofloxacin, rat pneumonia model, experimental pneumococcal pneumonia, rat model of pneumococcal pneumonia


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae is the most common bacterial cause of adult pneumonia.1 The prevalence of S. pneumoniae isolates resistant to penicillin has increased substantially in the last 10 years.2 Pneumococcal strains with reduced susceptibility to penicillin are often resistant to other ß-lactams, macrolides, trimethoprim–sulfamethoxazole and tetracyclines. Ceftriaxone, an expanded-spectrum cephalosporin, is approved for therapy of pneumococcal infections, including pneumonia, bacteraemia and meningitis. It is effective and widely used for known or suspected infections caused by penicillin-resistant pneumococci.3

For pneumococcal strains with penicillin resistance (MIC >= 2 mg/L), various fluoroquinolones have been recommended as empirical therapy for the treatment of community-acquired pneumonia.4 Most newer quinolone agents have shown enhanced activity against S. pneumoniae. However, the use of ciprofloxacin for pneumococcal pneumonia has been associated with clinical failure and breakthrough bacteraemia.5,6

The effectiveness of some antimicrobial agents may be enhanced by incorporating them into liposomal formulations.7,8 Liposomal encapsulation is known to be an effective method for reducing drug toxicity, prolonging circulation time after intravenous administration and increasing the accumulation of drugs at sites of disease.9,10 Conventional liposomes administered intravenously are rapidly removed by the mononuclear phagocyte system (MPS), particularly the liver and spleen.7,9,11 Thus, they are unable to achieve therapeutic concentrations in tissues outside the MPS. Sterically stabilized or Stealth (Sequus Pharmaceuticals, Menlo Park, CA, USA) liposomes avoid uptake by the MPS, resulting in long blood circulation half-lives.11 These liposomes also accumulate in tumours and tissue sites where there is infection or inflammation. Studies using an experimental model of unilateral Klebsiella pneumoniae pneumonia in rats have shown substantial localization of sterically stabilized liposomes in infected lung tissue.7,9,12

Encapsulation of ciprofloxacin by sterically stabilized liposomes results in a prolonged circulation time and greatly increases the ciprofloxacin area under the concentration–time curve (AUC) (L. Guo, Sequus Pharmaceuticals, Inc., personal communication). Distribution of these liposomes to sites of infection may result in higher tissue AUC and more effective therapy. Our study, therefore, compared the effectiveness and pharmacokinetics of liposome-encapsulated ciprofloxacin, conventional ciprofloxacin and ceftriaxone for the treatment of pneumococcal pneumonia in a rat model.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimentally induced pneumococcal pneumonia

Male Sprague–Dawley rats (Charles River/Sasco, Kingston, NY, USA) weighing 225–250 g were anaesthetized with ether, and a small incision was made in the skin overlying the trachea.13 After exposing the trachea by blunt dissection, a 20 gauge catheter was inserted into the mainstem bronchus. Ten times the lethal dose50 (3 x 107 cfu) of type 3 S. pneumoniae (ATCC 6303) suspended in 0.3 mL of phosphate-buffered saline (PBS) was injected into the lungs, followed by 0.3 mL of air. The wound was closed with sterile metal clips.

Antibiotic treatment

Treatment experiments included four study groups receiving ciprofloxacin (Cipro; Bayer Pharmaceuticals, West Haven, CT, USA), sterically stabilized liposome-encapsulated ciprofloxacin (Sequus Pharmaceuticals), ceftriaxone (Rocephin, Roche Laboratories, Nutley, NJ, USA) or free liposome (control). Ciprofloxacin and liposomal ciprofloxacin were administered either once or twice daily at a dose of 40 or 80 mg/kg, whereas ceftriaxone was administered only once daily at a dose of 100 mg/kg (Table 1). These doses were chosen based on the manufacturer’s recommendation for community-acquired pneumonia, of administering intravenous ciprofloxacin 1200 mg/day in humans or 17.14 mg/kg/day in a 70 kg patient. Using a correspondent surface area–dosage conversion factor of seven from man to rat, the equivalent human dose in the rat would be 120 or 17.14 mg/kg/day. Thus, doses both above and below the usual human dose ranging from 40 to 160 mg/kg/day were administered. Antibiotics were administered for 3 days via tail vein injection beginning 18 h post-infection. The MIC of the study antibiotics for the infecting strain was 1.5 mg/L of ciprofloxacin and 0.016 mg/L of ceftriaxone, as determined by Etest.


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Table 1.  Antibiotic treatment schedule
 
Peripheral white blood cell (WBC) counts and blood cultures

Blood was obtained using aseptic puncture of foot veins under ether anaesthesia.14 Cell counts were performed with a haemocytometer at baseline and on days 1, 3, 5 and 10 after infection. WBC differential cell counts were performed on stained slides (Diff-Quik; Baxter Scientific Products, McGraw Park, IL, USA). Quantitative blood cultures were also performed on days 1, 3, 5 and 10 by a plate count technique.

Survival studies

Based on the results of our earlier experiments, rats were infected transtracheally with 10 times the expected lethal dose50 of type 3 pneumococci (3 x 107 cfu).15 Mortality was observed for 10 days post-infection.

Antibiotic sampling

Additional rats not used in mortality studies were infected and treated as above with liposomal ciprofloxacin 80 mg/kg, ciprofloxacin 80 mg/kg or ceftriaxone 100 mg/kg. All antibiotics were administered once daily. Serum samples for determination of pharmacokinetic and pharmacodynamic parameters were obtained at 1, 3, 5, 12 and 24 h (immediately before the next dose) after the second dose of antibiotic in one group of rats. All sera were frozen at –70°C for subsequent antibiotic assays. A second group of rats was killed by exsanguination 1 h after the second dose of antibiotic for determination of lung antibiotic concentration. The lungs and trachea were removed immediately, and a 20-gauge catheter was positioned in the carina. Cold PBS was injected into the lungs in 8 mL aliquots and recovered by gravity drainage until a final volume of 50 mL was collected.15 Aliquots of bronchoalveolar lavage fluid (BALf) were cultured. Cells were collected by centrifugation, re-suspended in PBS and counted with a haemocytometer. Differential cell counts were performed on Diff-Quik-stained cytospin cell preparations.

Concentrations of antibiotics

All sample matrixes containing antibiotic were assayed by high-performance liquid chromatography (HPLC) techniques.

Ceftriaxone assay

The concentrations of ceftriaxone in BALf, alveolar macrophage (AM) cells and plasma were assayed by an HPLC assay with UV detection as previously described.16 The method was validated for linearity, precision, accuracy and specificity in our laboratory. All samples were prepared prior to injection on the column. Briefly, following freeze–thaw cycles of the AM cells, acetonitrile containing the internal standard (cefalexin) was added to AM cells and plasma for deproteination, and then vortexed, followed by centrifugation. An aliquot of 50 µL of the supernatant was injected onto the column. The BALf samples were prepared by centrifugation and then filtered through a 0.22 µ filter prior to extraction and injection onto the column. Separation was performed with a C18 reverse phase column (70 x 4.6 mm; particle size 3 µm; Phenomix, Torrance, CA, USA). The mobile phase consisted of 24 mM hexadecyl trimethylammonium bromide-phosphate buffer (pH 7.0), acetonitrile (43:5:52, v/v/v), at a flow rate of 1.5 mL/min at a temperature of 35°C. Quality control samples were included in each analytical sequence to confirm accuracy and precision of the assay. The assay was linear over the range 0.10–100 mg/L with the limit of quantification set at 10 ng/mL. Samples that exceeded the 100 mg/L concentration were diluted and re-injected on the column. The inter- and intra-day coefficients of variation were determined for each matrix: 7.7% and 3.8% for plasma, 8.0% and 5.2% for AM cells, and 9.8% and 7.8% for BALf.

Ciprofloxacin assay

All ciprofloxacin (including liposomal ciprofloxacin) concentrations in BALf, AM and plasma were determined by adapting a previously published HPLC assay with fluorescent detection and using moxifloxacin as an internal standard.17,18 The stationary phase consisted of a C18 column (250 x 4 mm; Phenomix) heated to 50°C with a dry column heater (Shimadzu). Separation was performed via an HPLC system with a fluorescent detector set at 290 nm (Shimadzu). Before extraction, the AM cells were exposed to freeze–thaw cycles to release intracellular drug. Following the extraction procedure (acetonitrile), supernatants of all matrixes containing the internal standard were injected onto the column proceeded by a guard column. The mobile phase consisted of 0.04 M H3PO4/acetonitrile/terbutylammonium hydroxide/0.005 M dibutyl amine phosphate reagent at pH 2.2. The lower range of quantification was 0.1 ng/mL and was linear over the range of 0.01–20 mg/L for all matrixes. To determine the accuracy and precision of the assay, quality control standards were prepared and run separately from the standard curves. The intra- and inter-day coefficients of variation at 1.0 mg/L were 4.5% and 5.9% for plasma, 6.7% and 8.6% for AM, and 5.6% and 7.8% for BALf.

Estimation of epithelial lining fluid (ELF) volumes and determination of drug concentration in AM cells and ELF

Albumin and urea concentrations in plasma and BALf were determined by previously described methods.19 The volume of ELF contained in each BALf sample was determined by the urea dilution method and results confirmed by application of the same method with albumin dilution.20 Urea diffuses freely throughout the body with the concentration in plasma equal to that in other body fluids. The ELF volume (VELF) is estimated by the following relationship: VELF = VBAL x (UreaBAL/Ureaplasma), where VBAL is the volume of BALf, UreaBAL is the urea concentration in the BALf and Ureaplasma is the concentration of urea in the plasma. The concentration of antibiotic in the ELF (AbxELF) was determined by: AbxELF = AbxBAL/VELF, where AbxBAL is the total amount of drug in each BALf sample, as determined by HPLC analysis.

Pharmacokinetics/pharmacodynamics

Liposomal ciprofloxacin, ciprofloxacin and ceftriaxone serum concentrations versus time data were analysed via WinNonlin Software, Standard Edition, Version 1.5 (Scientific Consulting, Inc., Cary, NC, USA). Pharmacokinetic parameters were estimated using a non-compartmental extravascular dose model with the AUC calculated over the dosage interval (0–24 h) by the trapezoidal rule. Pharmacodynamic parameters were calculated by dividing the MIC for S. pneumoniae (ceftriaxone 0.016 mg/L, ciprofloxacin 1.5 mg/L and liposomal ciprofloxacin 1.5 mg/L) into the AUC (AUC/MIC) and Cmax (Cmax/MIC). The time serum concentration exceeded the MIC (T > MIC) was calculated from the Cmax, half-life and respective MIC of each antibiotic.

Statistical analysis

Mortality statistics were compared by Fisher’s exact test. WBC parameters and mean pharmacokinetic and pharmacodynamic parameters were compared by one-way ANOVA, with post-hoc comparisons by Tukey’s and Newman–Keuls tests, respectively. The level of significance was set at P < 0.05 for all analyses.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peripheral WBC and differential cell counts

Mean peripheral WBC counts were similar on days 1, 3, 5 and 10 for groups receiving any antibiotic regardless of dosing schedule (Figure 1a shows data for once-daily therapy). WBC counts were significantly lower on day 1 of therapy for all groups (P < 0.05 for rats treated once daily and P < 0.008 for rats treated twice daily). Although WBC counts were lower in the liposomal control groups as the study progressed, this difference was not statistically significant because of the small numbers that survived in this group. The mean percentage of polymorphonuclear leucocytes (PMNL) was also similar between treatment groups (Figure 1b). Percentage PMNL was significantly increased above baseline on day 5 for rats in all treatment groups receiving once-daily therapy (treatment ended on day 3). By day 10, WBC and PMNL counts had returned to baseline values for surviving rats in all treatment groups.



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Figure 1. WBC counts (a) and percentage of PMNL (b) in peripheral blood samples obtained by foot-stick on groups of rats receiving 3 days of once-daily therapy with ciprofloxacin (CIP), liposomal ciprofloxacin (LIP-CIP), free liposome (LIP) or ceftriaxone (CFT). Data are means ± S.E.M. (black bars, CIP 80 mg/kg; striped bars, LIP-CIP 80 mg/kg; white bars, LIP; grey bars, CFT 100 mg/kg). *Value significantly lower than on days 0 and 5 (P < 0.05); §value significantly higher than on days 0 and 3 (P < 0.05); **value significantly higher than on day 0 (P < 0.05); {dagger}value for single surviving rat; {ddagger}no surviving rats in LIP-control group. n = 10 for (a) and (b).

 
WBC, PMNL and bacteria in BALf

The most unexpected difference was found for mean WBC in BALf (Figure 2a), which was significantly lower on day 2 of therapy for liposomal ciprofloxacin than for ceftriaxone or ciprofloxacin (P < 0.03). The mean percentage of PMNL recovered in BALf did not vary significantly between groups (Figure 2b). Bacterial counts in BALf on day 2 of therapy were highest for rats treated with ciprofloxacin (Figure 2c). Rats treated with liposomal ciprofloxacin had slightly lower counts, but still averaged 6 x 104 cfu/mL of lavage fluid. In sharp contrast, there were no organisms isolated from BALf obtained from rats treated with ceftriaxone.



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Figure 2. WBC counts (a), percentage of PMNL (b) and numbers of S. pneumoniae (c) in BALf from groups of rats receiving 3 days of once-daily therapy with ciprofloxacin (CIP), liposomal ciprofloxacin (LIP-CIP), free liposome (LIP) or ceftriaxone (CFT). Data are means ± S.E.M. (black bars, CIP 80 mg/kg; striped bars, LIP-CIP 80 mg/kg; grey bars, CFT 100 mg/kg) (n = 3–4). *Value significantly lower than for other two groups (P < 0.03); **value significantly higher than for (LIP-CIP) (P = 0.04); {dagger}value is 0, significantly lower than for other two groups (P < 0.0001).

 
Bacteria in blood and BALf

All rats treated once or twice daily with liposomal control were bacteraemic by day 5 post-infection (Figure 3a and b). In contrast, by day 3 of therapy, none of the rats treated with ceftriaxone remained bacteraemic. Ceftriaxone was the only therapy that sterilized the blood in all rats treated. The groups treated with twice-daily liposomal ciprofloxacin 80 mg/kg, twice-daily ciprofloxacin 40 and 80 mg/kg or once-daily ceftriaxone had significantly lower rates of bacteraemia (P < 0.05) than the liposomal control group (Figure 3a). Rates of bacteraemia were similar between liposomal ciprofloxacin and ciprofloxacin at the same doses. When therapies were administered once daily (Figure 3b), ceftriaxone was more effective in clearing bacteraemia than all other therapies (P < 0.01). No other significant variations were noted among groups treated with once-daily therapy.



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Figure 3. Bacteraemia with twice-daily therapy (a) (n = 10) and with once-daily therapy (b) (n = 9–10). *Bacteraemia significantly lower than for the free liposome (LIP) group (P < 0.05); **bacteraemia significantly lower than for all other groups (P < 0.01). Filled circles, ciprofloxacin (CIP) 40 mg/kg; open circles, liposomal ciprofloxacin (LIP-CIP) 40 mg/kg; filled squares, CIP 80 mg/kg; open squares, LIP-CIP 80 mg/kg; filled triangles, ceftriaxone 100 mg/kg/(once a day); crosses, LIP-control. Rx, duration of antibiotic therapy.

 
Survival studies

All rats treated with liposomal control died, whereas all rats treated once daily with ceftriaxone survived. Survival after twice-daily therapy with ciprofloxacin or liposomal ciprofloxacin at 40 or 80 mg/kg was significantly higher than for rats in the liposomal control group (Figure 4a). Ten day survival with twice-daily liposomal ciprofloxacin was identical to conventional ciprofloxacin at equivalent doses. There was 80% survival among rats treated with 80 mg/kg of either preparation. Only half the rats treated twice daily with 40 mg/kg of either liposomal ciprofloxacin or ciprofloxacin survived, a rate significantly lower than that for the group treated once daily with ceftriaxone (P < 0.035).



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Figure 4. Survival with twice-daily therapy (a) (n = 10) and once-daily therapy (b) (n = 9–10). *Survival significantly higher than for the free liposome (LIP) group (P < 0.001); **survival significantly higher than for the LIP group (P < 0.05), but lower than for the ceftriaxone (CFT) group (P < 0.035); §survival on day 6 significantly greater for the liposomal ciprofloxacin (LIP-CIP) (80 mg/kg) group than for the LIP-CIP (40 mg/kg) and LIP groups (P< 0.05); §§survival significantly greater for the CFT group than for all other groups (P < 0.01). Filled circles, ciprofloxacin (CIP) 40 mg/kg; open circles, LIP-CIP 40 mg/kg; filled squares, CIP 80 mg/kg; open squares, LIP-CIP 80 mg/kg; filled triangles, CFT 100 mg/kg (once a day); crosses, LIP-control. Rx, duration of antibiotic therapy.

 
Among groups of rats treated once daily, survival at day 10 was <50% for all groups treated with any ciprofloxacin preparation (Figure 4b). This was significantly lower (P < 0.01) than survival for the ceftriaxone-treated group. On day 6, survival with liposomal ciprofloxacin 80 mg/kg was significantly greater than the liposomal control group and the liposomal ciprofloxacin 40 mg/kg group (P < 0.05). However, by the end of the study there were no significant differences among groups treated once or twice daily with any form of ciprofloxacin.

Pharmacokinetic studies

Mean serum Cmax (mg/L) was highest for liposomal ciprofloxacin 80 mg/kg, intermediate for ciprofloxacin 80 mg/kg and lowest for ceftriaxone 100 mg/kg (Table 2). Serum Cmax was significantly higher for liposomal ciprofloxacin than for conventional ciprofloxacin (P < 0.01). Liposomal ciprofloxacin also achieved significantly higher concentrations in BALf than conventional ciprofloxacin (P < 0.01). Concentrations between the ciprofloxacin groups were similar in WBC obtained by bronchoalveolar lavage. Levels of ceftriaxone were essentially undetectable in both BALf and in WBC obtained by BAL. Liposomal ciprofloxacin resulted in significantly (P < 0.01) higher serum, AUC and BALf concentrations compared with either ciprofloxacin or ceftriaxone. The pharmacodynamic concentration-dependent parameters (AUC/MIC and Cmax/MIC) were highest for ceftriaxone, but liposomal ciprofloxacin had significantly greater values (P < 0.01) than conventional ciprofloxacin. The non-concentration-dependent pharmacodynamic parameter T > MIC demonstrated no difference between the ciprofloxacin formulations (P > 0.05), but ceftriaxone concentrations were greater than the MIC for a significantly longer time (P < 0.01). The calculated half-life for each antibiotic was 0.98 h (ciprofloxacin), 0.72 h (liposomal ciprofloxacin) and 1.03 h (ceftriaxone).


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Table 2.  Pharmacokinetic and pharmacodynamic parameters of ciprofloxacin (CIP) 80 mg/kg/day, liposomal ciprofloxacin (LIP-CIP) 80 mg/kg/day or ceftriaxone (CFT) 100 mg/kg/day in chow-fed rats
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many respiratory bacterial pathogens are susceptible in vitro to ciprofloxacin. However, susceptibilities of S. pneumoniae to ciprofloxacin are unpredictable since MIC90s usually range from 0.78 to 6.2 mg/L.2 In a mouse protection model of S. pneumoniae peritonitis, the in vivo efficacy of ciprofloxacin was dependent on the peak concentration/MIC ratio being at least 10.6.21 These levels are not often obtained with this drug against the pneumococcus in clinical practice. Although cures of pneumococcal pneumonia with ciprofloxacin have been reported, superinfections with S. pneumoniae during ciprofloxacin therapy are well documented.4 As a result of its unreliable activity against S. pneumoniae, ciprofloxacin is not recommended as empirical therapy for community-acquired pneumonia.6

Liposomes have been used successfully as carriers of antimicrobial agents in models of bacterial, protozoal, fungal and viral infections.7,8,11,22 Initially, studies utilized ‘classical’ liposomes composed of natural phospholipids and cholesterol. These preparations accumulated rapidly in cells of the MPS but did not otherwise distribute widely in tissue. Antimicrobial agents entrapped in these liposomes have been used successfully in infections in MPS tissues caused by facultative or obligate intracellular pathogens. Ciprofloxacin encapsulated in large unilamellar vesicles demonstrated efficacy in an in vivo Salmonella typhimurium model.22 However, liposomal encapsulation of ciprofloxacin10 and sparfloxacin23 did not enhance activity in the therapy of experimental Mycobacterium avium infection.

Sterically stabilized liposomes have been developed by incorporation of polyethylene glycol polymers conjugated to distearoyl phosphatidylethanolamine (PEG-DSPE).11 These PEGylated liposomes avoid uptake by the MPS, resulting in enhanced blood circulation times. Substantial localization of sterically stabilized liposomes in infected lung tissue has also been observed.7,9 In a rat model of K. pneumoniae pneumonia, the therapeutic effect of ceftazidime and gentamicin was increased by encapsulation with sterically stabilized liposomes, and one dose of liposomal ceftazidime was found to be as effective as a continuous 2 day infusion of free ceftazidime.9

The encapsulation of ciprofloxacin in sterically stabilized liposomes greatly enhances its AUC. The antibiotic releases from the circulating liposomes for an extended period of time, increasing the ciprofloxacin AUC from 2.14 to ~470 mg/L (L. Guo, Sequus Pharmaceuticals, Inc., personal communication). In our study, liposomal ciprofloxacin efficacy and pharmacokinetics were compared with conventional ciprofloxacin and ceftriaxone in a rat model of pneumococcal pneumonia. Liposomal ciprofloxacin had significantly higher peak concentrations in serum and BALf than conventional ciprofloxacin. The pharmacodynamic parameters AUC/MIC and Cmax/MIC were likewise markedly improved with liposomal ciprofloxacin, although there were no differences in T > MIC. The serum half-lives of the antibiotics were similar, although liposomal ciprofloxacin was associated with a shorter half-life. Twice-daily dosing significantly improved the outcome for groups receiving any form of ciprofloxacin, but once-daily dosing with liposomal ciprofloxacin resulted in no demonstrable survival benefit compared with conventional ciprofloxacin. Among the once-daily therapies, ceftriaxone was clearly more effective. Ceftriaxone had significantly greater time above the MIC than either ciprofloxacin or liposomal ciprofloxacin, which is consistent with previously described data.

Among the many ways to describe the relationship between antibiotic concentration and efficacy, the AUC/MIC ratio has been advocated to quantify differences in antimicrobial effects among the fluoroquinolones. Forrest et al.24 found that the best predictor of both clinical and microbiological cure of Gram-negative pneumonia with intravenous ciprofloxacin was an AUC/MIC ratio >125. A recent study proposes that the AUC/MIC ratio might be the best predictor of the comparative antimicrobial effects of different quinolones.25 Another group has found that a different pharmacodynamic parameter, the ratio of the peak plasma concentration to the MIC (Peak/MIC), might be a useful predictor of clinical and microbiological outcomes with the fluoroquinolones.26 In vitro data suggest that fluoroquinolones must achieve an AUC/MIC ratio of >30 for bacterial eradication of S. pneumoniae;27,28 however, it appears that these measures of pharmacodynamics may not be applicable to liposomal formulations of fluoroquinolones. Despite the theoretical advantages in the pharmacodynamics of liposomal ciprofloxacin, which include a marked increase in the AUC/MIC and the Cmax/MIC, it proved no more efficacious than conventional ciprofloxacin in terms of rat survival and bacterial killing in serum and BALf. Despite the large AUC/MIC ratios achieved by liposomal ciprofloxacin, it failed to improve efficacy over conventional ciprofloxacin. The precise reasons for the failure of liposomal ciprofloxacin to produce enhanced in vivo efficacy are uncertain, but several explanations can be proposed. Although there were no substantial differences seen in the numbers of peripheral blood WBC and PMNL among the various groups in this study, treatment with liposomal ciprofloxacin resulted in significantly reduced WBC counts in BALf. Liposomal encapsulation of ciprofloxacin may potentially inhibit WBC recruitment into the lungs, which may counteract the antimicrobial effects of the drug. Ciprofloxacin and liposomal ciprofloxacin both achieved pharmacodynamic parameters (AUC/MIC and Cmax/MIC) that should have resulted in efficacy. Owing to the limited number of samples and the subsequent pharmacokinetic modelling, it is possible that the first serum sample was obtained before complete distribution of ciprofloxacin. The high Cmax concentrations are suggestive of this sampling problem. Although ciprofloxacin T > MIC is not generally considered to be a good predictor of efficacy, our values (6.9 h or 28% of the interval) were considerably less than that observed in a mouse model of infection (72% of the interval).21 The AUC and Cmax ratios observed in this study for both liposomal and conventional ciprofloxacin suggest that the antibiotics should have been effective. A short post-antibiotic effect previously reported for ciprofloxacin may have influenced efficacy. A more frequent dosing of both ciprofloxacin dosage regimens may have alleviated this problem.

Fluoroquinolone antibiotics demonstrate dose-dependent pharmacodynamics, but encapsulation of antibiotics by PEG-DSPE-containing liposomes appears to confer dose-independent kinetics.8 Thus, dose-dependent measures of antimicrobial effects, such as the AUC/MIC, may not be applicable. T > MIC may be more important, and in this study liposomal ciprofloxacin achieved the least time above the MIC of all drugs tested. Finally, to obtain a therapeutic effect, degradation of liposomes is needed for release of encapsulated antibiotic.8 The increased concentration of ciprofloxacin in BALf found in our study may not actually represent an increase in bioactive free drug but merely accumulation of liposomally entrapped drug. However, in studies performed with liposomal ciprofloxacin in a rat model of K. pneumoniae pneumonia the ciprofloxacin appeared to be continuously released from the liposome.12 Notably, the MIC of ciprofloxacin for that K. pneumoniae was 15-fold lower than for the pneumococcal strain used in our study. Although measured concentrations of the drug were increased in our model they were insufficient to overcome ciprofloxacin’s inherent lack of activity against S. pneumoniae.

In summary, encapsulation of ciprofloxacin in sterically stabilized liposomes results in significantly higher peak concentrations of drug in serum and BALf. However, in our rat model of pneumococcal pneumonia, the efficacy of liposome-encapsulated ciprofloxacin was similar to conventional ciprofloxacin. Of the once-daily therapies, 3 days of ceftriaxone treatment was more effective in clearing bacteraemia and improving survival than treatment with either form of ciprofloxacin.


    Acknowledgements
 
We are indebted to Mei Yue and Mary U. Snitily for their expert technical assistance. This study was supported in part by a grant from Sequus Pharmaceuticals, Menlo Park, CA, USA.


    Footnotes
 
* Corresponding author. Tel: +1-402-449-0650; Fax: +1-402-977-5601; E-mail: laurel.preheim{at}med.va.gov Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Bartlett, J. G. & Mundy, L. M. (1995). Community-acquired pneumonia. New England Journal of Medicine 333, 1618–24.[Free Full Text]

2 . Sahm, D. F., Karlowsky, J. A., Kelly, L. J., Critchley, I. A., Jones, M. E., Thornsberry, C. et al. (2001). Need for annual surveillance of antimicrobial resistance in Streptococcus pneumoniae in the United States: 2-year longitudinal study. Antimicrobial Agents and Chemotherapy 45, 1037–42.[Abstract/Free Full Text]

3 . Friedland, I. R. & McCracken, G. H., Jr (1994). Management of infections caused by antibiotic-resistant Streptococcus pneumoniae. New England Journal of Medicine 331, 377–82.[Free Full Text]

4 . Bartlett, J. G., Dowell, S. F., Mandell, L. A., File, T. M., Musher, D. M. & Fine, M. J. (2000). Practice guidelines for the management of community-acquired pneumonia in adults. Clinical Infectious Diseases 31, 347–82.[CrossRef][Medline]

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6 . Hooper, D. C. & Wolfson, J. S. (1991). Fluoroquinolone antimicrobial agents. New England Journal of Medicine 324, 384–94.[ISI][Medline]

7 . Bakker-Woudenberg, I. A. J. M., Lokerse, A. F., ten Kate, M. T., Mouton, J. W., Woodle, M. C. & Storm, G. (1993). Liposomes with prolonged blood circulation and selective localization in Klebsiella pneumoniae-infected lung tissue. Journal of Infectious Diseases 168, 164–71.[ISI][Medline]

8 . Bakker-Woudenberg, I. A. J. M., ten Kate, M. T., Stearne-Cullen, L. E. T. & Woodle, M. C. (1995). Efficacy of gentamicin or ceftazidime entrapped in liposomes with prolonged blood circulation and enhanced localization in Klebsiella pneumoniae-infected lung tissue. Journal of Infectious Diseases 171, 938–47.[ISI][Medline]

9 . Bakker-Woudenberg, I. A. J. M., Lokerse, A. F., ten Kate, M. T. & Storm, G. (1992). Enhanced localization of liposomes with prolonged circulation time in infected lung tissue. Biochimica et Biophysica Acta 1138, 318–26.[ISI][Medline]

10 . Leitzke, S., Bucke, W., Borner, K., Muller, R., Hahn, H. & Ehlers, S. (1998). Rationale for and efficacy of prolonged-interval treatment using liposome-encapsulated amikacin in experimental Mycobacterium avium infection. Antimicrobial Agents and Chemotherapy 42, 459–61.[Abstract/Free Full Text]

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