a Department of Medicine and Pathology, Charles R. Drew University of Medicine and Sciences, 1731 East 120th Street, Los Angeles, CA 90059; b Division of Clinical and Basic Immunology, University of California, Irvine, CA, USA
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Abstract |
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Introduction |
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Leucopenia, induced by chemotherapy for cancer, is a primary risk factor for the development of fatal Gramnegative infections. Antibiotic prophylaxis or therapy for such infections in leucopenic patients is often supplemented with granulocyte colony stimulating factor (GCSF) and granulocyte macrophage colony stimulating factor (GMCSF) to stimulate the production of phagocytic cells and enhance phagocytic function.9,10
Trovafloxacin is a long-acting, broad spectrum, new fluoroquinolone effective against various aerobic and anaerobic Gram-positive and Gram-negative organisms. The aims of this study were to determine the in vivo efficacy of trovafloxacin in the treatment of pneumonia caused by K. pneumoniae in an immunocompromised host, and to evaluate whether the addition of rGCSF to trovafloxacin therapy improves outcome. The efficacy of trovafloxacin was compared with ciprofloxacin, a quinolone with a spectrum of activity mainly for Gram-negative organisms, and cephazolin, a cephalosporin used conventionally in the treatment of Klebsiella infections.1113
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Materials and methods |
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The subjects used were 68-week-old male DBA/2 mice (Taconic Laboratories, Germantown, New York, USA). K. pneumoniae American Type Culture Collection (ATCC) 43816 was passaged to mice three times at the beginning of the experiment to maintain virulence. P388 murine leukaemia cells were maintained in vitro as a suspension culture in Rosewell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal calf serum, 100 mg/L penicillin and 50 mg/L streptomycin.
Tumour induction and therapy
One million P388 murine leukaemia cells were injected into the peritoneal cavity of mice. Twenty-four hours after the introduction of P388 leukaemic cells, the mice were treated ip with either daunorubicin (DNR) 10 µg/dose or saline once a day for 3 days.14
Lung infection
Mice were anaesthetized with xylazine (16 mg/kg) and ketamine (80 mg/kg) and infected by intratracheal injection with 40 µl of trypticase soy broth containing 104 colony forming units (cfu) of K. pneumoniae.
Antimicrobial therapy
Therapy was initiated 4 h after intratracheal instillation of K. pneumoniae. The mice were treated with alatrovafloxacin (CP-116, 517-27), the prodrug of trovafloxacin (Pfizer Inc., Groton, CT, USA) or alatrovafloxacin with rGCSF or ciprofloxacin (Bayer, West Haven, CT, USA) or ciprofloxacin with rGCSF or cephazolin (Bristol Myers Squibb, Princeton, NJ, USA) or rGCSF alone. All antibiotics were given into the peritoneal cavity three times a day at 4 hour intervals. See Figure 1 for the experimental design. The doses used for alatrovafloxacin and ciprofloxacin were 30 mg/kg and 20 mg/kg, respectively. The prodrug alatrovafloxacin is readily soluble in water at neutral pH, and 30 mg of this is equivalent to 20 mg of the active compound.11 The dose of rGCSF used was 10 µg/kg. The following controls were used: (i) Tumour-bearing mice that were infected but not treated with any antibiotic. (ii) Tumour-bearing mice infected with K. pneumoniae and treated with rGCSF alone. (iii) Healthy mice infected with K. pneumoniae. The dose of ciprofloxacin and cephazolin used was 20 mg and 2 mg tds, respectively. The dose of antibiotic used was six times that recommended in humans, and was based on the pharmacokinetics of antibiotics in experimental animals.1519 The rGCSF dose was based on previous reports.20,21
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Antibiotic therapy was given for 7 days. The mice were killed by cervical dislocation 2 days after the discontinuation of antibiotics. At autopsy, the lungs were removed aseptically and were homogenized in ground glass homogenizers. Ten-fold serial dilutions of the homogenate were made in saline and 0.1 ml of each dilution was spread on trypticase soy agar plates. This was done in triplicate and the plates were incubated for 24 h at 37°C. Colony counts were performed after incubation. In tumour-bearing mice, trovafloxacin levels in the serum and from the liver, lung and spleen tissues at 1, 4, 8, 24 and 48 h after the last dose were determined by bioassay, using ATCC Escherichia coli 25922 as the test organism.16,22 The limit of detection was 0.25 mg/L for trovafloxacin. Results were expressed as means ± S.D. The MICs of antibiotics for K. pneumoniae used in this study were determined by the broth microdilution method.22 K. pneumoniae was found to be susceptible to trovafloxacin (MIC 0.5 mg/L), ciprofloxacin (MIC 0.25 mg/L) and cephazolin (MIC 12 mg/L). Tumourbearing mice pretreated for 3 days with DNR were subsequently treated with trovafloxacin (20 mg/kg, tds) or ciprofloxacin (20 mg/kg, tds) or saline (controls) for 7 days. Blood samples were collected before initiation of DNR therapy, and on days 3, 5 and 7 post-initiation of DNR therapy. Red blood cells were lysed with Turk solution and white blood cell (WBC) counts were determined using a haemocytometer.
Statistical analysis
Chi-squared (2) test was used to compare the differences between the different treatment groups.
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Results |
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Results are shown in Figure 2 and Table I
. Infection with K. pneumoniae was associated with high mortality rates in untreated control mice. Treatment with trovafloxacin, but not with ciprofloxacin or cephazolin was associated with prolongation of survival in mice; the survival rates of mice treated with trovafloxacin, ciprofloxacin or cephazolin were 80%, 23% and 35%, respectively. Recombinant GCSF did not further improve the survival rate of mice treated with trovafloxacin, but it did prolong the survival of mice treated with ciprofloxacin. The survival rate of mice treated with ciprofloxacin plus rGCSF was lower than that of mice treated with trovafloxacin alone or trovafloxacin plus rGCSF. As expected, treatment with rGCSF alone did not prolong the survival of mice (data not shown). Trovafloxacin was also found to be more effective than ciprofloxacin or cephazolin in clearing K. pneumoniae lung infection. The lung cultures were negative for K. pneu-moniae in mice treated with trovafloxacin alone or trovafloxacin plus rGCSF whereas cultures were positive in 43% of the animals treated with ciprofloxacin or cephazolin. The therapeutic efficacy of ciprofloxacin in combination with rGCSF was similar to that of trovafloxacin alone (the cure rates were 100%).
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Antibiotic therapy modestly increased the survival rate but failed to clear the infection (Table II). The survival rate of mice treated with trovafloxacin (28%) was not substantially different from that of mice treated with ciprofloxacin (18%). Recombinant GCSF increased slightly the efficacy of trovafloxacin in improving the survival rate (from 28% to 43%) and in eliminating infection (cure rate from 2% to 8%). Microbiological evaluation in survivors showed that there was a log decrease in K. pneumoniae counts in the lung tissue of animals treated with trovafloxacin or trovafloxacin plus rGCSF, when compared with saline-treated controls.
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In mice without tumours, trovafloxacin was effective in protecting against death as well as in clearing the lung infection (Table II). Among the infected controls, the survival rate was 0% (data not shown), whereas it was 100% among those treated with trovafloxacin or ciprofloxacin.
In vivo effect of trovafloxacin on WBC count
Experiments were done to determine (i) whether DNR therapy is associated with the development of leucopenia in the animal model and (ii) the effect the quinolone antibiotic therapy might have on WBC counts (leucopenia) in mice pretreated with DNR. It can be seen from the results in Figure 3 that DNR caused a mild and transient decrease in leucocyte count in tumour-bearing mice. Trovafloxacin increased the WBC counts in tumour-bearing animals pretreated with DNR. In contrast, no such increase in WBC count was observed in ciprofloxacin-treated animals. These results suggest that trovafloxacin but not ciprofloxacin, may reverse transient decreases in leucocyte count in tumour-bearing mice.
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Results are shown in Figure 4. Following the last ip injection of trovafloxacin in tumour-bearing mice, the mean peak serum antibiotic concentration was 26.2 ± 32.4 mg/L which fell to a mean of 7.5 ± 2.5 mg/L at 4 h. At 8 h, the serum levels were 3.2 ± 1.8 mg/L and were barely detectable at 24 and 48 h after the last injection. Trovafloxacin concentrations in the tissues of liver, lung and spleen were high. The concentration of trovafloxacin in the lungs of tumour-bearing mice was 17.5 ± 4.9 mg/L at 1 h, which was about 35 times the MIC of trovafloxacin of the K. pneumoniae isolate used in this experiment. The concentration of trovafloxacin in the lung was high for about 4 h (17.1 ± 4.3 mg/L). It then fell to 1.8 ± 1.4 mg/L at 8 h and was undetectable at 24 and 48 h.
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Discussion |
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This study demonstrates clearly that (i) trovafloxacin is more effective than ciprofloxacin or cephazolin in the treatment of K. pneumoniae pneumonia in immunocompromised mice (tumour-bearing mice receiving conventional cancer chemotherapy) and (ii) therapy with trovafloxacin is as effective as ciprofloxacin in the treatment of lung infection with K. pneumoniae in immunocompetent mice (without tumours).
The failure of ciprofloxacin and cephazolin therapy in the treatment of febrile granulocytopenic cancer patients with lymphomas and solid tumours and in granulocytopenic animals has been reported.2326 Although cephazolin is effective against Klebsiella pneumoniae in vitro,25,27 it is totally ineffective for treatment of Klebsiella infections in neutropenic rats.26 The cumulative mortality among neutropenic female rats infected with K. pneumoniae has been reported to be 24% when treated with 40 mg/kg cephazolin and only improved (to 34%) when amikacin (8 mg/kg) was added.26 Our data on ciprofloxacin therapy and cephazolin concur with these reports. In this study, ciprofloxacin when given alone, at a dose of tds 20 mg/kg (equivalent to 400600 mg/day human adult dose) protected only 18% of the tumour-bearing mice in the first experiment and 26% in the latter. Similarly, cephazolin was found to be effective in only 35% of mice with cancer. In contrast, ciprofloxacin was found to be very effective in eliminating the same infection in normal (non-tumour-bearing) mice.
Multiple factors contribute to the final outcome of any antibiotic in eliminating infection and prevention of death owing to infection. These factors include susceptibility of the organism to the drug, access and retention of the drug at the infected site, dosing regimen, tissue and serum pharmacokinetics and, more importantly, the status of the host immune response. Trovafloxacin levels in the serum and lung tissue were high and remained high for about 8 h; indeed the trovafloxacin level reached was 35 x MIC for the organism used in this study for the first 4 h and 5 x MIC or more for 8 h or longer (Figure 4). Excellent tissue concentrations of trovafloxacin were also achieved in the liver (5060 x MIC) and in the spleen (2070 x MIC). In previous studies this group has reported similarly high ciprofloxacin concentrations in lung tissue when compared with serum levels.16 Thus both trovafloxacin and cipro-floxacin achieve high therapeutic tissue concentrations in the lung. In this study, both antibiotics sterilized the lungs in the normal immunocompetent (non-tumour-bearing) mice infected with K. pneumoniae and all mice (100%) recovered on either ciprofloxacin or trovafloxacin therapy.
Ciprofloxacin and cephazolin were ineffective in sterilizing the lungs in immunocompromised animals. These observations suggest that host defences are important when the efficacy of antibiotics, used alone, is considered in the immunocompromised host. The fact that trovafloxacin is highly effective in eliminating klebsiella infection in both immunocompetent and immune-suppressed animals suggests that this drug may provide a potentially significant advance in antimicrobial therapy for infections in cancer patients.
The reasons for the superior efficacy of trovafloxacin when compared with both ciprofloxacin and cephazolin in eliminating K. pneumoniae in tumour-bearing mice, treated with daunorubicin, are not known. This group has shown previously that trovafloxacin at 1 and 5 mg/L was more effective in a model of intracellular infection with Salmonella typhimurium and Staphylococcus aureus than ciprofloxacin in human polymorphonuclear leucocytes and promonocytic U937.28,29 This study has shown that trovafloxacin, but not ciprofloxacin, accelerated the recovery from leucopenia induced by daunorubicin. Furthermore, trovafloxacin alone was shown to be as effective as trovafloxacin plus GCSF in protecting the animals from lethal infection as well as in sterilizing the lungs. Taken together, these findings would suggest that trovafloxacin, at therapeutically achievable levels, may either directly or indirectly augment host phagocytic cell defence mechanisms and help eliminate infections in tumour-bearing mice that received cancer chemotherapy.
In this study, it was observed that trovafloxacin, like ciprofloxacin and cephazolin, failed to cure the K. pneumoniae lung infection or to prolong survival in mice with untreated tumour. The reason(s) for this failure is not known. It is known that cancer is associated with profound immunosuppression and that elimination of infection requires intact host defences.
In conclusion, trovafloxacin, a recently developed trifluoroquinolone, was found to be highly effective in the treatment of K. pneumoniae lung infection in tumour- bearing mice rendered leucopenic by daunorubicin therapy. Trovafloxacin was more effective than cephazolin or ciprofloxacin with or without the addition of rGCSF. There are obvious differences in the overall therapeutic efficacy between ciprofloxacin and trovafloxacin in treatment of infections associated with cancer. Whether this remarkable difference would be substantiated in future clinical studies of cancer patients remains to be determined.
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Acknowledgments |
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Notes |
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References |
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Received 26 January 1999; returned 19 April 1999; revised 16 August 1999; accepted 23 August 1999