Brief Article Cite This: Mol. Pharmaceutics 2018, 15, 1371−1376
Pulmonary Delivery of Ceftazidime for the Treatment of Melioidosis in a Murine Model Sara I. Ruiz,†,‡ Larry E. Bowen,†,‡,§ Mark M. Bailey,*,§ and Cory Berkland∥,⊥ †
United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702, United States Laulima Government Solution, Honolulu, Hawaii 96814, United States § PneumoDose, LLC, Alexandria, Virginia, 22314 United States ∥ Department of Chemical & Petroleum Engineering, The University of Kansas, Lawrence, Kansas 66047, United States ⊥ Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States ‡
ABSTRACT: Burkholderia pseudomallei, the etiological agent responsible for melioidosis, exhibits a great public health toll in its endemic regions. The elevation of B. pseudomallei to a Tier I select agent underscores the urgent need for effective therapeutics and preventatives. The current treatment regimen for melioidosis is suboptimal, requiring an intensive phase of intravenous antibiotic followed by months of oral antibiotics. Inhaled antibiotics are a promising avenue to pursue for pulmonary diseases, including melioidosis, since this mode of delivery mimics the likely exposure route and can provide high drug doses directly to the infected tissue. Ceftazidime was delivered via a nose-only system to BALB/c mice challenged with B. pseudomallei. Mice treated with nebulized ceftazidime became symptomatic but survived until study end, which was comparable to those treated intraperitoneally. Upon necropsy, bacteria remained within the spleens of the majority of the experimental animals. The effectiveness of nebulized ceftazidime warrants additional studies to improve the treatment regimen and to test as a prophylactic therapy against B. pseudomallei. KEYWORDS: B. pseudomallei, ceftazidime, melioidosis, murine model, aerosol
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INTRODUCTION Current treatments for melioidosis are suboptimal because of the necessity to deliver intravenous antibiotics every 8 h, often for several weeks, and due to the long duration of oral treatment following the initial intensive phase. The complex treatment regimen is a result of the low oral bioavailability of antibiotics effective against B. pseudomallei, which negates the possibility of addressing an outbreak with oral antibiotics alone.1−4 The current antibiotic regimen also regularly fails, leading to chronic infection in 5−25% of the infected population.5 Furthermore, B. pseudomallei is now a Tier 1 select agent and risk of exposure as an aerosolized bioterrorism agent is of concern. In this setting, an ideal therapy should be rapidly deployable as a prophylactic or therapeutic agent and able to be delivered directly to the site of infection to neutralize the pathogen prior to the onset of disease symptoms. Ceftazidime has a low minimum inhibitory concentration (MIC) (approximately 2 μg/mL) against B. pseudomallei.2 The drug can be dissolved in buffered water at high concentration and exhibits preference for the aqueous phase (Log P = −1.60), which suggests that the drug may persist in the hydrophilic mucus of the respiratory tract after inhalation.6 In addition to favorable physicochemical properties, published studies provide clues supporting the efficacy of inhaled ceftazidime. Cazzola et al. demonstrated that intramuscular injection of ceftazidime yielded concentrations in the bronchial mucosa above the MIC, © 2018 American Chemical Society
but the drug was diluted significantly when it crossed into the epithelial lining fluid.7 Additionally, van’t Veen et al. showed that the antimicrobial activity of ceftazidime was not adversely affected by lung surfactant, indicating that direct delivery to the lungs may be a viable delivery method.8 Our previous study demonstrated that inhaled ceftazidime was retained in the lungs of mice for up to 6 h post aerosol delivery.9 The efficacy of nebuilized ceftazidime against an aerosolized lethal challenge of B. pseudomallei as a postexposure prophylactic was examined. Female BALB/c mice were administered treatment 12 h postchallenge via either a noseonly inhalation exposure system or intraperitoneally. Mice were euthanized on days 1, 3, and 9 postexposure with the lung, liver, and spleen homogenized and plated to detect bacteria. Bacteremia was monitored throughout the study, with all mice being euthanized 21 days postexposure with the lung, liver, and spleen homogenized and plated.
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EXPERIMENTAL SECTION Research was conducted under an IACUC approved protocol in compliance with the Animal Welfare Act, PHS Policy, and Received: Revised: Accepted: Published: 1371
October 26, 2017 December 22, 2017 January 24, 2018 January 24, 2018 DOI: 10.1021/acs.molpharmaceut.7b00938 Mol. Pharmaceutics 2018, 15, 1371−1376
Brief Article
Molecular Pharmaceutics
were entered. A single, preweighed 47 mm TissueQTZ filter (PALL Corporation) was loaded into the filter sampler and attached to the plenum. Five mice were loaded into the restraint tubes. Following transport to the exposure suite, they were passed into the biosafety cabinet and attached to the plenum. Plenum integrity was confirmed by briefly plugging the passive dilution HEPA filter and observing a maximum negative pressure signal from the digital pressure transmitter. The vehicle inhalation exposure duration was 40 min. Following a 5 min wash cycle, the mice were removed from the plenum, passed out of the biosafety cabinet, and transported back to their housing room. A single filter sample was collected for 45 min and weighed to determine the vehicle aerosol concentration (mg/L) and inhaled dose (mg). A continuous aerodynamic particle sizer (APS) sample was collected during the inhalation exposure and wash periods to determine the mass median aerodynamic diameter (MMAD, μm) and geometric standard deviation (GSD). After the wash cycle, the inhalation exposure system was flushed with aerosolized distilled water for 5 min. For test article inhalation exposures, the Mini-HEART Nebulizer was filled with 20 mL of 75 mg/mL Tazicef (ceftazidime for injection, USP) (Hospira Worldwide, Inc.). A preweighed 47 mm TissueQTZ filter was loaded into the filter sampler and attached to the plenum for each exposure. The filter sampler flow rate was metered with a critical orifice with a nominal flow rate of 1.042 L/min. Mice were loaded into the restraint tubes and transported to the exposure suite, where they were passed into the biological safety cabinet and attached to the plenum. Plenum integrity was confirmed by briefly plugging the passive dilution high efficiency particulate absorbing (HEPA) filter and observing a maximum negative pressure signal from the digital pressure transducer. Test article inhalation exposure durations ranged from 30 to 40 min. Following a 5 min wash cycle, the mice were removed from the plenum, passed out of the biological safety cabinet, and transported back to their housing room. A single filter sample was collected during each exposure. The mass of aerosol collected on each filter was corrected for the estimated mass of vehicle. The estimated vehicle mass was calculated by multiplying the fraction of observed vehicle mass to collection time from the vehicle exposure to the time for each test article exposure, e.g., (4.9 mg/45 min) × 35 min = 3.8 mg. APS samples were collected for the entirety of each of the initial seven treatments to determine the particle size distribution of the aerosolized test article (MMAD, GSD). Each APS sample was collected for 5 s. At the end of each exposure, the inhalation exposure system was flushed with aerosolized distilled water for 5 min. Total nominal plenum exhaust volumetric flow for the vehicle exposure was 12.0 L/min. The filter sampler volumetric flow rate was metered using a critical orifice with a volumetric flow rate of 1.141 L/min. Radial mixer volumetric flow was 5.119 L/min, and the nebulizer volumetric flow rate was 5.926 L/min. For test article inhalation exposures, the total nominal plenum exhaust volumetric flow rate ranged from 12.0 to 14.0 L/min. The mean filter sampler volumetric flow rate was 1.219 ± 0.259 L/min. The mean radial mixer volumetric flow rate was 5.113 ± 0.012 L/min. Tissue Processing. Following necropsy, the lung, liver, and spleen were weighed. Tissues were then homogenized in 10% w/v saline (Moltox), and the homogenate was plated on sheep
other Federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International, and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011. Animals. Fifty female BALB/c mice, 6−8 weeks old, were randomly sorted and divided into 10 separate groups, consisting of 5 mice each. Mice were caged with microisolator tops with 12 h light/12 h dark cycles. Feed and water were provided ad libitum. All mice were implanted with IPPT microchips (BioMedic Data Systems, Inc.) prior to start of study for identification and temperature measurement. Animal Handling. Mice were observed twice a day following challenge. Mice were individually assessed by weight, temperature, appearance, provoked behavior, and natural behavior. Animals scoring higher than or equal to 8 were euthanized via barbiturate overdose followed by cervical dislocation. Approximately 0.1 mL of blood was collected via the submandibular vein into a microisolator tube (Wampole Laboratories) on days 0, 12, and 14 and at time of euthanasia. Aerosol Challenge. An overnight culture of B. pseudomallei was grown in GTB at 37 °C, shaking to stationary phase. The culture was diluted to a starting concentration of 7 × 106 CFU/ mL to yield a target dose of 150 ± 50 LD50. Animals were transferred to wire mesh cages and placed in a whole-body aerosol chamber within a class III biological safety cabinet. Mice were exposed to the aerosol created by a three-jet Collison nebulizer operated at a nominal flow rate of 7.5 L/min for 10 min while the chamber was maintained at a constant flow rate of 19.5 L/min. The aerosolization was performed at ambient temperature and humidity. The generated aerosol was sampled with an all-glass impinger (AGI) operated at a nominal flow rate of 6 L/min and analyzed by plating and quantifying the colony forming units (CFU) to determine the inhaled dose. Ceftazidime Preparation. Prior to each treatment, a vial of ceftazidime (Hospira Worldwide, Inc.) was reconstituted in aqueous buffer vehicle (Quality Biological) for a final concentration of 75 mg/mL with an estimated osmolality of 300 mOsmol/kg. The reported pH range for the reconstituted solution is 5 to 8. Vehicle consisted of 0.8 g/mL sodium carbonate (GFS Chemicals) in sterile water. Treatment. Mice received treatment starting at 12 h postchallenge either intraperitoneally (ip) or by nose-only inhalation, which continued every 12 h for 20 total treatments. Regardless of treatment, all mice were given a target dose of 100 mg/kg. Prior to each vehicle and test article inhalation exposure, a pre-exposure system compliance and stability test was conducted. The plenum ports were sealed with port plugs and empty restraint tubes. Plenum integrity was confirmed by briefly plugging the passive dilution HEPA filter and observing a maximum negative pressure signal from the digital pressure transmitter. The Mini-HEART Nebulizer was filled with 20 mL of distilled water. The exposure system plenum, nebulizer, and sampler flows were input. Each pre-exposure test was 5 min in duration. During the test, all system compressed air and vacuum flow rates and plenum pressure, temperature, and relative humidity were confirmed. For vehicle inhalation exposure, the Mini-HEART Nebulizer was filled with 20 mL of 0.8 g/mL sodium carbonate and connected to the aerosol generation, conditioning, and delivery line. Exposure system administrative and operating parameters 1372
DOI: 10.1021/acs.molpharmaceut.7b00938 Mol. Pharmaceutics 2018, 15, 1371−1376
Brief Article
Molecular Pharmaceutics blood agar plates (Remel). The plates were incubated at 37 °C for 24 h and colonies enumerated. Statistical Analyses. Aerosolized treatment was compare to ip treatment using a two-tailed t test assuming unequal variances.
GSD was significantly greater than the target of 2.5, the data was further analyzed to determine the contribution of particles in relevant size ranges in which the deposition fraction is well described for rodents. The mean percentages of particles collected smaller than 1.0 μm, 2.0 μm, and 3.5 μm were 76.1 ± 9.1%, 91.9 ± 13.0%, and 99.5 ± 0.4%, respectively. The estimated deposition fractions for insoluble particles with MMADs of 1.0 μm, 2.0 μm, and 3.5 μm are 7%, 8%, and 1.2). Since the mean
2). Weight loss is a key indicator of infection in the melioidosis mouse model, since fever is rarely observed during infection (unpublished data), and clinical scoring is subjective. Overall clinical scores that took into account percentage weight loss, behavior, and appearance showed a similar pattern to weight loss (Figure 3). Temperatures among both groups were relatively consistent throughout the study (Figure 4). Upon cessation of treatment, both groups began to lose weight, suggesting that neither treatment was able to completely clear the infection. The temperature and weight data were not statistically different regardless if ceftazidime was given via ip injection or via inhalation. Ip ceftazidime did, however, result in 1373
DOI: 10.1021/acs.molpharmaceut.7b00938 Mol. Pharmaceutics 2018, 15, 1371−1376
Brief Article
Molecular Pharmaceutics
Bacterial Burden. Throughout the study, bacterial presence was monitored in the blood and tissue (lung, liver, and spleen) of each experimental group. Control animals were bacteremic at the time of euthanasia. Treatment groups were only bacteremic at day 21, indicating that although ceftazidime was able to control the infection, it was unable to completely eliminate the bacteria (data not shown). All collected tissues on day 1 postexposure had bacteria present, with the largest burden present in the lung. By day 3 postexposure, there were no detectable bacteria in any of the ip treated animals. Bacteria were present in the aerosol treated mice, although at lower values than previously observed. The largest burden was in the spleen, which persisted until end of study. As can be observed in Figure 1, the ip treated mice showed an increase in bacteria at day 9 that continued until study end. Nebulized ceftazidime treatment yielded statistically higher amounts of bacteria in the lung and spleen on day 3. The spleen at end of study for the aerosol treated mice was indicative of a chronic infection, with noticeable pockets of bacteria, although the weight of spleens was statistically indistinguishable (Figure 5). The general appearance and weight of the other tissues throughout the study were not statistically different.
Figure 2. Average weight for each treatment group. All mice treated with control vehicle were euthanized on day 3 postexposure. Half error bars shown for clarity in treatment groups.
Figure 3. Average clinical scores for each treatment group. Half error bars shown for clarity in treatment groups.
Figure 5. Average weight of spleen at time of euthanasia.
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DISCUSSION The biphasic treatment model of meliodiosis is employed to address the rapid dissemination of the bacteria and ability to establish a chronic infection.14 Current treatment regimens for melioidosis are suboptimal because they require two phases: an intense intravenous antibiotic phase, followed by several months of oral antibiotic therapy for complete bacterial eradication. In the context of developing a medical countermeasure against B. pseudomallei as a biological threat agent, this is an inefficient therapeutic option because it would be difficult to deliver intravenous antibiotics to a large population of infected individuals. Thus, more portable and easy to administer therapies are desirable. In addition, patient compliance with the 20 week therapeutic course is important for decreasing the risk of relapse.5
Figure 4. Average temperature for each treatment group. Half error bars shown for clarity in treatment groups.
a slightly lower overall clinical score from approximately day 4 to 14. 1374
DOI: 10.1021/acs.molpharmaceut.7b00938 Mol. Pharmaceutics 2018, 15, 1371−1376
Molecular Pharmaceutics
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The advent of inhalable antibiotics as a treatment option for tuberculosis and in cystic fibrosis patients underscores the opportunity to utilize this technology for other pulmonary diseases.15,16 For treatment of B. pseudomallei infection, a high level of retention of antibiotic in the lungs is desired. Following aerosol exposure, a permissive mouse model found pulmonary lesions within 1 day postinfection, concentrated at the alveolar and perivascular region.17 B. pseudomallei dissemination from the initial site of infection is rapid and was observed to occur as soon as 2 days postinfection in mouse models.18 However, prior to dissemination, rapid replication occurred within the lungs of challenged mice.17 In addition, patients had a higher percentage of unfavorable outcomes the earlier bacteria was detected in blood samples.19 It is imperative, therefore, to intervene early and halt dissemination from the initial site of infection. Previous studies have demonstrated that ceftazidime is likely to be retained in the hydrophilic mucus of the respiratory tract, and that its therapeutic activity is not adversely affected by lung surfactant6 Thus, direct delivery of antibiotic to the lungs may be an efficacious therapy, either alone or as an adjunct. Previously, we showed that inhaled ceftazidime persists in the lungs of mice for up to 6 h.9 Here, we demonstrate the efficacy of inhaled ceftazidime compared to intraperitoneal injection and a placebo (vehicle-only) control. Efficacy of nebulized ceftazidime delivered to the lungs was comparable to intraperitoneal injection, delayed the onset of death, and reduced symptoms in infected animals. Although neither nebulized ceftazidime nor ip injection completely eliminated infection, these results suggest that, with further optimization, inhaled ceftazidime may be a promising treatment against acute pulmonary melioidosis. As is the case with current melioidosis treatment regimens, oral antibiotics are required as an adjunct therapy for complete bacterial eradication.20 Thus, pairing nebulized ceftazidime with oral antibiotic therapy may lead to complete elimination of infection. This was recently demonstrated in a therapeutic model of infection in which BALB/c mice intranasally challenged with B. pseudomallei were administered tolfenamic acid and ceftazidime. Animals receiving combination therapy survived infection and had significantly lower bacterial load within examined tissues.21 This phenomenon has also been described using traditional antibiotics.22,23 This therapeutic model will be examined in future work.
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Brief Article
AUTHOR INFORMATION
Corresponding Author
*107 S West Street #574, Alexandria, VA 22314-2891. E-mail:
[email protected]. Phone: 703-951-3814. ORCID
Cory Berkland: 0000-0002-9346-938X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
The authors gratefully acknowledge funding from NSF EPSCoR in the State of Kansas. The authors also acknowledge David Dyer, Samantha Baker, Jeanean Ghering, and Christopher Jensen for laboratory technical support. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army or the Department of Defense.
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CONCLUSIONS
An urgent need exists to develop antibiotic therapies against B. pseudomallei, since there is no effective vaccine. Previous experimentation has shown that protection from an aerosol exposure is more difficult to achieve than utilizing other common delivery methods such as oral or intravenous antibiotics. The ability of nebulized ceftazidime to delay death and control symptoms warrants further exploration as an adjunct therapy. Future experimentation is needed to assess the efficacy of nebulized treatment with ceftazidime to improve the treatment regimen and to test as a prophylactic therapy against B. pseudomallei. Once control of infection and is established, the drug regimen can be more deeply evaluated in a therapeutic model. 1375
DOI: 10.1021/acs.molpharmaceut.7b00938 Mol. Pharmaceutics 2018, 15, 1371−1376
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Molecular Pharmaceutics (16) Hoppentocht, M.; et al. Developments and strategies for inhaled antibiotic drugs in tuberculosis therapy: a critical evaluation. Eur. J. Pharm. Biopharm. 2014, 86 (1), 23−30. (17) West, T. E.; et al. Murine pulmonary infection and inflammation induced by inhalation of Burkholderia pseudomallei. Int. J. Exp. Pathol. 2012, 93 (6), 421−8. (18) Gauthier, Y. P.; et al. Study on the pathophysiology of experimental Burkholderia pseudomallei infection in mice. FEMS Immunol. Med. Microbiol. 2001, 30 (1), 53−63. (19) Tiangpitayakorn, C.; et al. Speed of detection of Burkholderia pseudomallei in blood cultures and its correlation with the clinical outcome. Am. J. Trop. Med. Hyg. 1997, 57 (1), 96−99. (20) Sookpranee, M.; et al. Multicenter prospective randomized trial comparing ceftazidime plus co-trimoxazole with chloramphenicol plus doxycycline and co-trimoxazole for treatment of severe melioidosis. Antimicrob. Agents Chemother. 1992, 36 (1), 158−62. (21) Wilson, W. J.; et al. Immune Modulation as an Effective Adjunct Post-exposure Therapeutic for B. pseudomallei. PLoS Neglected Trop. Dis. 2016, 10 (10), e0005065. (22) Ulett, G. C.; et al. A comparison of antibiotic regimens in the treatment of acute melioidosis in a mouse model. J. Antimicrob. Chemother. 2003, 51 (1), 77−81. (23) Feterl, M.; et al. Activity of tigecycline in the treatment of acute Burkholderia pseudomallei infection in a murine model. Int. J. Antimicrob. Agents 2006, 28 (5), 460−4.
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DOI: 10.1021/acs.molpharmaceut.7b00938 Mol. Pharmaceutics 2018, 15, 1371−1376
