a Departments of Pathology and Laboratory Medicine, and b Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4283, USA
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Abstract |
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Introduction |
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Materials and methods |
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Twenty-two low passage strains of Legionella spp. that had been isolated in our laboratory, comprised 15 strains of L. pneumophila (11 serogroup 1, and one each of serogroups 2, 4, 6 and 9); two strains each of Legionella micdadei, Legionella longbeachae and Legionella dumoffii; and one strain of Legionella bozemanii. These 22 strains had previously been used in other studies,69 including L. pneumophila F889 and F2111, which have been studied extensively in cell and animal models of L. pneumophila infection.913 Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were used as the control organisms for susceptibility testing. Inocula for susceptibility testing were made by growing legionellae on morpholinepropanesulphonic acid (MOPS)-buffered charcoal yeast extract medium supplemented with 0.1% -ketoglutarate (BCYE
), and non-legionellae were grown on commercial trypticase soy agar containing 5% sheep blood (BBL, Sparks, MD, USA).14 Incubation was at 35°C in humidified air for 2448 h.
Antimicrobials
BMS-284756 (Bristol-Myers Squibb Pharmaceuticals, New Brunswick, NJ, USA), levofloxacin (Daiichi Pharmaceutical Co., Tokyo, Japan), azithromycin (Pfizer Laboratories, Groton, CT, USA) and erythromycin (Abbott Pharmaceuticals, Abbott Park, IL, USA) standard powders were obtained from their respective manufacturers. For susceptibility studies, BMS-284756 was dissolved in 5% dextrose for injection USP; subsequent dilutions were made in tissue culture medium M199 (Gibco-BRL, Grand Island, NY, USA), MuellerHinton broth or buffered yeast broth, as appropriate. BMS-284756 administered to guinea pigs was dissolved in 90% Capmul MCM [a mixture of monoglycerides and diglycerides of capyrilic and capric acid in glycerol (Abitec Corp., Janesville, WI, USA)] in water acidified with 42 mM (final concentration) d,l-lactic acid USP. Levofloxacin was dissolved in sterile water for injection USP. Erythromycin was first dissolved in 95% ethanol, then diluted in water or tissue culture medium M199, as appropriate. Azithromycin standard powder was dissolved in 95% ethanol, then in M199 medium or water, as appropriate. Levofloxacin and azithromycin commercial preparations for iv administration were prepared according to the manufacturer's instructions, and were diluted so that the volume administered was 1 mL. The BMS-284756 solutions used for the guinea pig studies were 10 or 15 g/L, and were prepared within 24 h of administration.
Antimicrobial susceptibility testing
Microbroth dilution susceptibility testing was performed as described previously using n-(2-acetamido)-2-aminoethanesulphonic acid (ACES)-buffered yeast extract broth supplemented with 0.1% -ketoglutarate (BYE
) (Legionella) or MuellerHinton broth (non-Legionella), with a final volume of 100 µL and final bacterial concentration of 5 x 105 cfu/mL.15
Growth inhibition in alveolar macrophages
Guinea pig pulmonary alveolar macrophages were harvested and purified as described previously.6 The final concentration of macrophages was c. 105 cells/well. Incubation conditions for all macrophage studies were 5% CO2 in air at 37°C.
L. pneumophila F889 and F2111 grown overnight on BCYE agar were used to infect the macrophages. Approximately 104 bacteria were added to each well. Bacteria were incubated with macrophages for 1 h in a shaking incubator, and then for 1 day in stationary culture, as described previously.6 One set of replicate wells was washed three times with tissue culture medium (500 µL), then sonicated at low energy (setting 2.5, 0.5 inch probe, 50% duty cycle, 10x 1 s pulses repeated once; Heat Systems-Ultrasonics Model 380, Farmingdale, NY, USA) to release intracellular bacteria, which were quantified by growth on BCYE
agar. Antimicrobials were then added to the washed non-sonicated wells; wells with no antimicrobial served as growth controls. The infected tissue cultures were then incubated for 2 days, after which supernatant samples were taken for quantitative culture. The antimicrobials were then removed by washing, and the experiment continued for four more days, with daily quantification of L. pneumophila in well supernatants. All experiments were carried out in duplicate or triplicate, and quantitative plating was done in duplicate. All wells were observed microscopically each day to detect macrophage infection and to roughly quantify numbers of macrophages in the wells. BMS-284756 macrophage toxicity control wells contained macrophages, tissue culture medium and antimicrobial, but no bacteria. Previous studies have demonstrated a lack of macrophage toxicity caused by erythromycin, azithromycin or levofloxacin.6,10,15,16 In this system there is no extracellular growth of L. pneumophila, so all increases in supernatant bacterial concentration are the result of intracellular growth.
Guinea pig pneumonia model
Hartley strain male guinea pigs, 300 g in weight, were used as described previously.13 All animals were observed for illness 1 week before infection; in the case of those used for the treatment study, temperatures and weights were obtained during the pre-infection period. The guinea pigs were infected with L. pneumophila serogroup 1, strain F889, administered by the intratracheal route as described previously.13 The inocula (300 µL) contained 7.6 x 106 and 6.3 x 106 cfu for the pharmacokinetic and treatment studies, respectively. Approval for all animal studies was obtained from the University of Pennsylvania Institutional Animal Care and Use Committee.
Pharmacokinetic studies
Two studies were performed with two different BMS-284756 formulations. In the first study plasma and lung were sampled as described previously, and in the second study only plasma was sampled.17 In the first study, BMS-284756 was solubilized in 5% dextrose in water (pH 3.8 with 85% d,l-lactic acid USP), and was given as a single ip injection 10 mg/kg, 4 g/L to guinea pigs (mean weight 322 g) with experimentally induced L. pneumophila pneumonia 1 day after infection.17 At timed intervals after injection, anaesthetized animals in groups of two or three were exsanguinated by removal of heart blood under direct vision, followed by lung removal. Heart blood was transferred immediately to K-EDTA tubes (Vacutainer; Becton-Dickinson, Rutherford, NJ, USA) and placed at 5°C. Within 1 h, the plasma was separated by centrifugation at 5000g at 5°C for 10 min, and then stored frozen at 70°C until processed further. Following removal, lungs were rinsed in phosphate-buffered saline, drained on sterile cotton gauze, weighed and ground in a measured volume of high-performance liquid chromatography (HPLC) grade water; the final volume of the homogenate was measured to determine the lung weight per volume of final homogenate. Controls included guinea pig lung homogenate and plasma that had been collected identically from normal guinea pigs given identical anaesthesia, but no antimicrobial. Subsequently the specimens were thawed and 200 µL was mixed with 750 µL acetonitrile containing internal standard, T-3811-IS-01 (Toyama Chemical Co., Tokyo, Japan). The specimens were then vortex-mixed vigorously, and the supernatants collected by centrifugation. The supernatants were dried under N2 at 40°C, and the dried specimens frozen at 70°C, until shipped to the Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb, on dry ice for assay.
In the second study uninfected guinea pigs were given a single ip dose of BMS-284756 10100 mg/kg solubilized in Capmul MCM. Only plasma samples were obtained for analysis. The frozen plasmas were sent directly to Bristol-Myers Squibb without acetonitrile precipitation or addition of an internal standard, and were processed there using the same method as described above. For each assay, normal lung and plasma were seeded with BMS-284756 to serve as drug extraction controls.
Drug assay
BMS-284756 was assayed in plasma and lung homogenate by HPLC with tandem mass spectrometry at the Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb.
Chromatographic separation was achieved with gradient elution on an Inertsil 5 µm, ODS-2, 50 x 2.0 mm column (ANSYS Technologies, Inc., Lake Forrest, CA, USA). The mobile phase was 0.1% formic acid in water and 0.1% formic acid in acetonitrile/tetrahydrofuran (90:10 v/v), pumped at a flow rate of 0.3 mL/min. Detection was by positive electrospray tandem mass spectrometry. The selected reaction monitoring transitions monitored were m/z 427m/z 380 for the internal standard. Dried plasma and lung residues prepared as described above were reconstituted in 0.1% formic acid and 20 µL was injected into the HPLC system. Peak area ratios of analyte to internal standard were calculated for all samples.
BMS-284756 calibrators were prepared in pooled guinea pig plasma over the concentration range 0.01010 mg/L. The calibration curve was calculated using quadratic regression weighted by 1/x. Assay precision and accuracy were determined by quintuplicate analysis of plasma seeded with BMS-284756 (0.03, 5.00 and 8.00 mg/L). Within-run and between-run coefficients of variation for the assay were <5% and <8%, respectively. The average accuracy of control samples was within ±7% of the target concentrations.
Animal treatment study
Guinea pigs surviving the surgery necessary to administer the Legionella were randomized into four treatment groups 1 day after infection. Starting on that day, treatment was given once daily at 9 a.m. Eighteen animals received BMS-284756 10 mg/kg ip in Capmul MCM for 5 days; another 12 animals received azithromycin 15 mg/kg, ip for 2 days; 12 animals received levofloxacin 10 mg/kg ip for 5 days; and 12 control animals received saline solution 1 mL for 5 days. Dosing schedules were designed to emulate expected peak serum concentrations in humans, as determined by pharmacokinetic studies in the animals and published studies in humans, without regard to differing drug clearances in the two species.15,18,19 Animal weights and temperatures were taken before drug administration on all treatment days, except day 1 when they were taken 2 h after drug administration. Necropsies and quantitative lung cultures were performed on all animals that died. All animals surviving for 12 days post-infection were killed with pentobarbital. Necropsies, lung histopathology and quantitative lung cultures were performed on half of the lowest weight survivors from all treatment groups.13 The lower limit of detection of L. pneumophila in the lung was c. 100 cfu/g. All animal studies were approved by the University of Pennsylvania Institutional Animal Care and Use Committee.
Statistical analysis
All data analysis was performed with either Prism (version 3.02) or InStat (version 2.01) software (GraphPad, San Diego, CA, USA). Prism was also used to calculate pharmacokinetic parameters. A P value 0.05 was predefined as significant. Lung histological scores and culture results were analysed using one-way analysis of variance (ANOVA), with Bonferroni's multiple comparison test for post-hoc testing. Body weight and temperature comparisons were analysed using the methods specified for each result. Lungs containing no detectable bacteria were arbitrarily assigned a value of 10 cfu/g to calculate and compare group mean values.
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Results |
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The MIC50, MIC90 and MIC range of BMS-284756 for the Legionella spp. tested were 0.008, 0.032 and 0.004 0.064 mg/L. The respective azithromycin values were 0.125, 0.25 and 0.0320.5 mg/L. Levofloxacin values were 0.016, 0.032 and 0.0160.032 mg/L, respectively. MICs of BMS-284756, levofloxacin and azithromycin for strain F889 were 0.008, 0.032 and 0.125 mg/L, respectively; the respective MICs for strain F2111 were 0.008, 0.016 and 0.50 mg/L. The highest BMS-284756 and levofloxacin MICs were obtained for Legionella spp. other than L. pneumophila, L. dumoffii and L. bozemanii, respectively. The highest azithromycin MICs were obtained for two strains of L. pneumophila. No broth medium inhibition of the test drugs was observed. MICs of the three antimicrobials tested for the control S. aureus or E. coli strains using BYE broth were within one doubling dilution of the result obtained using Mueller Hinton broth. A previous study of erythromycin activity against the same Legionella strains, conducted using identical methods, showed that the MIC50, MIC90 and MIC range were 0.125, 0.25 and 0.0640.5 mg/L; values for F889 and F2111 were 0.125 and 0.25 mg/L, respectively.20
Antimicrobial inhibition of intracellular growth
All four antimicrobials tested significantly inhibited both L. pneumophila strains grown in guinea pig alveolar macrophages, but the results differed for the two strains (Figure 1). The activity of all antimicrobials tested was nearly identical for both strains at 0.25 mg/L, and before washout of the antimicrobial. However, after drug removal, erythromycin-treated wells showed rapid regrowth of L. pneumophila. After drug removal, F889 was still markedly inhibited by azithromycin and levofloxacin, but not by BMS-284756. There was enhanced activity of BMS-284756 at a higher concentration (1 mg/L) against F889, but not against F2111. In contrast, levofloxacin and azithromycin (1 mg/L) showed a post-antibiotic effect against both strains for several days. BMS-284756 showed no evidence of macrophage toxicity in drug-only control wells.
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Drug extraction controls and HPLC assay of the drug dosing solution gave the expected values, confirming the accuracy of the assays. The two pharmacokinetic studies used the same single ip dose of 10 mg/kg, but different drug formulations. In the first study using BMS-284756 4 g/L dissolved in acidified 5% dextrose in water, mean plasma concentrations were 0.5 and 0.3 mg/L at 0.5 and 1 h post-dose, respectively. Respective lung concentrations at the same time points were 0.8 and 0.3 mg/kg. Starting at 1 h post-dose the lung and plasma concentrations were nearly identical at each time point, and became undetectable (<0.01 mg/L or mg/kg) 34 h post-dose. The elimination plasma half-lives (tHs) for plasma and lung were 0.36 and 0.62 h, respectively. Because tissue concentrations were lower than expected, the study was repeated using a different formulation designed to enhance drug solubility and peritoneal absorption. Uninfected guinea pigs were given BMS-284576 15 g/L in Capmul MCM. Mean BMS-284756 plasma concentrations for this formulation were 1.86 and 0.74 mg/L at 0.5 and 1 h post-injection, respectively. At 2 h postinjection the plasma concentration was 0.19 mg/L. The tH for this formulation was 0.47 h, and the AUC024 was 1.71 mgh/L. Higher doses (30 and 100 mg/kg) of BMS-284756 in Capmul MCM proved fatal within 1 h of injection, and resulted in unconsciousness before death; toxicity was attributed to the larger volumes of Capmul vehicle used at these higher doses (data not shown). A 5 day study of Capmul MCM administration alone (0.24 mL) to three healthy animals for 5 days produced transient unconsciousness on day 1 and weight loss, but no peritoneal irritation; the animals regained weight after cessation of the Capmul administration (data not shown).
Therapy in guinea pigs
Sixteen of 18 guinea pigs treated with BMS-284756 survived for 12 days post-infection, as did 11 of 12 azithromycin-treated and 12 of 12 levofloxacin-treated animals (P > 0.5 by 2 test). This was in contrast to 100% deaths in the 12 guinea pigs receiving saline alone, a highly significant difference from the outcome of the three treatment groups (P < 0.0001,
2 test) (Figure 2
). Necropsies of the two BMS-284756-treated animals that died before day 12 showed that both had peritonitis, which was most severe in the right lower quadrant, the site of drug injection. The lungs from these animals were much smaller, and less consolidated, than lungs from saline-treated animals; histological examination showed little residual lung consolidation (data not shown). All 16 BMS-284756-treated guinea pigs that survived to day 12 had necropsy evidence of healed peritonitis, manifested exclusively by intra-abdominal adhesions in the right lower quadrant. No azithromycin-, levofloxacin- or saline-treated animal had peritonitis at necropsy. A study of three uninfected animals that received Capmul MCM alone daily for 5 days at the same volume as was given to the BMS-284756-treated animals showed that no animals developed peritonitis. The azithromycin-treated animal that died on day 12 had evidence of colitis, with lungs appearing normal on gross and histological examination.
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Discussion |
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Our in vitro susceptibility results show that BMS-284756 has lower MICs than levofloxacin for Legionella, and that both drugs have considerably lower MICs than azithromycin. These results are in concordance with previous studies.3,4 No interpretive breakpoints are available for BMS-284756, although the measured MICs for Legionella are well below expected serum levels.19 The azithromycin and levofloxacin measured MICs are below the NCCLS breakpoints for these drugs when testing other bacteria. However, extracellular MICs can be unreliable for predicting drug efficacy for the treatment of Legionnaires' disease, making it difficult to interpret MICs even when antimicrobial breakpoints are established for other genera.12
BMS-284756 inhibited the growth of intracellular L. pneumophila, and at higher concentration (1 mg/L) had bactericidal activity against one of two strains tested. This is in contrast to the solely bacteriostatic activity of erythromycin against the same strains. We have shown previously in this model that erythromycin never demonstrates intracellular bactericidal activity, even at extracellular concentrations as high as 5 mg/L.16 BMS-284756 was less active against intracellular L. pneumophila than were levofloxacin or azithromycin at the same concentrations. Both levofloxacin and azithromycin are among the most active antimicrobial agents of their class against intracellular L. pneumophila.15,16
BMS-284756 was very rapidly cleared from guinea pig plasma, a feature of most drugs studied in the guinea pig model. In contrast to the tH of c. 0.5 h in the guinea pig, the value for humans dosed with a single oral dose ranges from 12.6 to 15.4 h.19 The peak BMS-284756 plasma concentrations in guinea pigs were about 30% of those obtained in humans receiving a single oral 400 mg dose, and the guinea pig AUC024 was only about 2% of that observed for humans.19 Unfortunately, higher BMS-284756 plasma and lung concentrations could not be achieved in guinea pigs because of the poor aqueous solubility of the drug, and toxicity of the Capmul MCM vehicle prevented the use of higher doses.
Previous studies from this laboratory have shown that levofloxacin 10 mg/kg in guinea pigs results in a drug Cmax of 3.1 mg/L and AUC024 of 4.4 mgh/L, c. 50% and 9% of the respective values for humans given levofloxacin 500 mg orally.15 The dose of azithromycin given in this study to guinea pigs yields a Cmax of 0.9 mg/L, and AUC024 of 7 mgh/L, c. 235% and 270%, respectively, of values for humans given a single 500 mg oral dose.18 The significance of azalide or quinolone AUC024 measurements is unknown for the animal model of L. pneumophila pneumonia, or human Legionnaires' disease, because of the intracellular location of the pathogen and the lack of detailed studies on intracellular pharmacokinetics.
In the animal model of L. pneumophila pneumonia, BMS-284756 was not significantly different from azithromycin or levofloxacin in prolonging survival. BMS-284756 therapy resulted in a decreased mortality rate when compared with saline treatment, and also substantial clearing of the bacterium from the lungs. However, the bacterial lung counts at the end of therapy were significantly higher than those observed for azithromycin and levofloxacin. It is unclear whether the higher bacterial lung counts are due to reduced clearance during therapy or relapse. The lack of any detectable bacterial lung count of the BMS-284756-treated animal that died on day 8 suggests that the higher bacterial counts at the end of therapy were due to relapse. Whatever the reason for higher bacterial counts at the end of therapy, the root cause is probably drug concentrations that were too low during therapy as a result of a suboptimal dosing regimen.
Azithromycin-treated animals that had necropsies had no detectable residual lung counts, and a trend toward reduced lung consolidation when compared with the other treatment groups. In addition the azithromycin-treated animals had significantly lower body temperatures during the first days of treatment. These findings are consistent with a prolonged post-antibiotic effect, or intracellular killing, as observed in the macrophage study, and previously by us.16 In addition the lower body temperature and reduced lung consolidation may be related to the proposed non-antibiotic anti-inflammatory effect of this drug.21,22 The clinical significance of such findings is unknown.
Takahata and colleagues5 reported recently on the efficacy of BMS-284756 in a guinea pig model of L. pneumophila pneumonia. They reported that the drug (5 mg/kg/day for 5 days) was more effective than ciprofloxacin in terms of mortality and bacterial clearance 1 day after cessation of therapy. This dose of BMS-284756 resulted in an AUC0 of 1 mgh/L, in contrast to the value of 1.7 mgh/L obtained by us for a higher dosage and different route of administration. The guinea pig ciprofloxacin AUC0
was 0.69 mgh/L, c. 36% of the levels obtained in humans. The shorter period of drug-free observation, smaller numbers of animals in each treatment group and low dose of the comparator provide good explanations for any apparent differences between that study and the results presented here.
Despite the presence of higher lung bacterial counts at the end of BMS-284756 therapy than for the other active therapies, histological studies showed no significant difference between levofloxacin and BMS-284756 treatment group lungs. This finding could be consistent with an inflammatory response that lags behind bacterial relapse, with insensitivity of histological findings, or a type of chronic bacterial persistence that does not elicit an acute inflammatory response.
The weight loss, hypothermia and transient neurotoxicity observed in the BMS-284756-treated animals are due to the Capmul MCM vehicle, rather than to BMS-284756. This conclusion is based on the findings of neurotoxicity and weight loss in healthy animals given Capmul MCM alone. The peritonitis was most likely chemical in nature and due to BMS-284756, as animals administered Capmul MCM alone did not develop peritonitis. Peritonitis caused by BMS-284756 is not of concern in humans as the drug will not be administered by the intraperitoneal route.
The guinea pig model of L. pneumophila pneumonia is an effective predictor of drug efficacy for Legionnaires' disease, especially when combined with supportive evidence of good in vitro and intracellular antimicrobial activity.13 All three of these parameters as determined in this study indicate that BMS-284756 might be an effective agent for the treatment of Legionnaires' disease. Improved BMS-284756 drug pharmacokinetics in humans indicate that the drug might be more effective in man than the guinea pig model suggests. Further investigations of the use of BMS-284756 for the treatment of Legionnaires' disease are indicated.
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Acknowledgements |
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Notes |
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References |
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Received 9 April 2001; returned 26 June 2001; revised 25 July 2001; accepted 24 August 2001