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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 Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00938 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on February 1, 2018

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Molecular Pharmaceutics

Pulmonary Delivery of Ceftazidime for the Treatment of Melioidosis in a Murine Model Sara I. Ruiz1,2, Larry E. Bowen1,2,3, Mark M. Bailey3*, Cory Berkland4,5

1

United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD

2

Laulima Government Solution, Honolulu, HI

3

PneumoDose, LLC, Alexandria, VA

4

The University of Kansas, Department of Chemical & Petroleum Engineering, Lawrence, KS

5

The University of Kansas, Department of Pharmaceutical Chemistry, Lawrence, KS

*

Corresponding Author:

107 S West Street #574 Alexandria, VA 22314-2891 [email protected] Phone: 703-951-3814 Keywords: B. pseudomallei, ceftazidime, melioidosis, murine model, aerosol

Disclaimer: 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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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 sub-optimal, 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.

TABLE OF CONTENTS GRAPHIC

B. pseudomallei

Nebulized ceftazidime 12 hours

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INTRODUCTION Current treatments for melioidosis are sub-optimal because of the necessity to deliver intravenous antibiotics every 8 hours, 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 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 intramuscular injection of ceftazidime

yielded concentrations in the bronchial mucosa above the MIC, but the drug was diluted significantly when it crossed into the epithelial lining fluid [7]. Additionally, van’t Veen, et al. showed the antimicrobial activity of ceftazidime was not adversely affected by lung surfactant, indicating 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 hours post aerosol delivery [9].

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The efficacy of nebuilized ceftazidime against an aerosolized lethal challenge of B. pseudomallei as a post-exposure prophylactic was examined.

Female BALB/c mice were

administered treatment 12 hours post-challenge via either a nose-only inhalation exposure system or intraperitoneally. Mice were euthanized on days 1, 3, and 9 post-exposure 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 post-exposure with the lung, liver and spleen homogenized and plated.

EXPERIMENTAL SECTION Research was conducted under an IACUC approved protocol in compliance with the Animal Welfare Act, PHS Policy, and 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 micro-isolator tops with 12 hour light/12 hour 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.

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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 0.1mL of blood was collected via the submandibular vein into a microisolator tube (Wampole Laboratories) on days 0, 12, 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 7x106CFU/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 minutes 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 300mOsmol/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.

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Treatment. Mice received treatment starting at 12 hours post-challenge either intraperitoneally (IP) or by nose-only inhalation, which continued every 12 hours 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 distilled water. The exposure system plenum, nebulizer, and sampler flows were input. Each pre-exposure test was five minutes 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 0.8 g/mL sodium carbonate and connected to the aerosol generation, conditioning, and delivery line. Exposure system administrative and operating parameters were entered. A single, pre-weighed 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 minutes. Following a five-minute wash cycle, the mice were removed from the plenum, passed out of the biosafety cabinet, and transported back to their housing room. A single

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filter sample was collected for 45 minutes 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 five minutes. 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 minutes. Following a five-minute 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) x 35 min = 3.8 mg. APS samples were collected for the entirety of each of the initial seven treatments to determine the particle size

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distribution of the aerosolized test article (MMAD, GSD). Each APS sample was collected for five seconds. At the end of each exposure, the inhalation exposure system was flushed with aerosolized distilled water for five minutes. 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 articles 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 plated on sheep blood agar plates (Remel). The plates were incubated at 37°C for 24 hours and colonies enumerated.

Statistical Analyses. Aerosolized treatment was compare to IP treatment using a two-tailed Ttest assuming unequal variances.

RESULTS B. pseudomallei Challenge. Target dose range for the aerosol challenge was 150 ± 50 LD50. The calculated actual dose received was 114 LD50 which resulted in a uniformly lethal dose to

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BALB/c acute model of melioidosis [24].

The reported spray factor (ratio of aerosol

concentration to nebulizer concentration) was 3.58x10-7.

Ceftazidime Treatment Aerosol Dose. Inhaled dose was calculated as the product of the aerosol concentration at the breathing zone of the mice on the plenum, minute volume, and exposure duration. Minute volume was calculated from experimentally derived data from BALB/c mice in a double chamber plethysmograph [10, 11]. The double chamber plethysmograph is similar in style and function to the nose-only restraints tubes used for this study. Inhaled dose for the vehicle group was 0.23 mg. The target test article inhaled dose was 1.8 mg. The observed test article inhaled dose was 1.73 ± 0.43 mg. Inhaled dose data are presented in Table 1.

Ceftazidime Particle Size Distribution. The particle size distribution (PSD) of the aerosolized test article was determined using an APS. Data were collected for the first seven exposures only. The mean mass median aerodynamic diameter (MMAD) was 2.8 ± 0.7 µm (n = 7) and the mean geometric standard deviation was 3.2 ± 0.7 µm. An insoluble aerosol with a MMAD of 2.8 µm would have an expected deposition fraction of approximately 4% in the pulmonary-alveolar region of the lungs [12]. Although the ceftazidime/vehicle aerosol was soluble, this metric provides a baseline or maximum estimate of pulmonary-alveolar deposition.

All exposure

aerosols were polydisperse (GSD > 1.2). Since the mean 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

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MMADs of 1.0 µm, 2.0 µm, and 3.5 µm are 7%, 8%, and < 4%, respectively. PSD data are shown in Table 1.

Clinical Observations and Survival. The average weight of mice prior to exposure was 18.32 ±1.1 g with no significant difference among the four groups (IP vehicle, nebulized vehicle, IP ceftazidime, and nebulized ceftazidime). Following challenge, mice were observed twice a day for changes in weight, temperature, behavior, and appearance. The overall clinical score was tabulated with each of these categories weighted equally. Mice in the vehicle groups succumbed to infection within 48 hours. Prior to euthanasia, the mice exhibited severe weight loss along with a significant temperature decrease, hunched posture, piloerection and subdued activity aligned with previously published observations [13]. Mice in the treatment groups became symptomatic; however, survival was improved. Both treatment regimens displayed weight loss, hunched posture, and piloerection within two days post-exposure. The weight loss in the mice receiving nebulized treatment was greater than the IP group. Neither group regained their baseline weight during the course of the study, although the IP group began to gain weight at day 5 and aerosol treated mice at day 6 (Figure 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

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injection or via inhalation. IP ceftazidime did, however, result in a slightly lower overall clinical score from approximately day 4-14.

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 post-exposure had bacteria present, with the largest burden present in the lung. By day 3 post-exposure, there was 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 and 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.

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 sub-optimal because they require two phases: an intense

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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]. 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 one day, post-infection 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 two days post-infection 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 surfactant [6] 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 six hours. [9] Here, we demonstrate the

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efficacy of inhaled ceftazidime compared to intraperitoneal injection and a placebo (vehicleonly) 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.

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

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and to test as a prophylactic therapy against B. pseudomallei. Once control of infection and elimination is established, the drug regimen can be evaluated in a therapeutic model.

ACKNOWLEDGEMENTS AND DISCLOSURES 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.

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21. 22.

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Knox, C., et al., DrugBank 3.0: a comprehensive resource for 'omics' research on drugs. Nucleic Acids Res, 2011. 39(Database issue): p. D1035-41. Thibault, F.M., et al., Antibiotic susceptibility of 65 isolates of Burkholderia pseudomallei and Burkholderia mallei to 35 antimicrobial agents. J Antimicrob Chemother, 2004. 54(6): p. 1134-8. Wishart, D.S., et al., DrugBank: a knowledgebase for drugs, drug actions and drug targets. Nucleic Acids Res, 2008. 36(Database issue): p. D901-6. Wishart, D.S., et al., DrugBank: a comprehensive resource for in silico drug discovery and exploration. Nucleic Acids Res, 2006. 34(Database issue): p. D668-72. Limmathurotsakul, D., et al., Risk factors for recurrent melioidosis in northeast Thailand. Clin Infect Dis, 2006. 43(8): p. 979-86. Hansch, C., A. Leo, and D.H. Hoekman, Exploring QSAR.: Hydrophobic, electronic, and steric constants. 1995: American Chemical Society. Cazzola, M., et al., Pulmonary penetration of ceftazidime. J Chemother, 1995. 7(1): p. 50-4. van 't Veen, A., et al., Influence of pulmonary surfactant on in vitro bactericidal activities of amoxicillin, ceftazidime, and tobramycin. Antimicrob Agents Chemother, 1995. 39(2): p. 329-33. Ruiz, S.I., et al., Formulation and Characterization of Nanocluster Ceftazidime for the Treatment of Acute Pulmonary Melioidosis. J Pharm Sci, 2016. 105(11): p. 3399-3408. DeLorme, M.P. and O.R. Moss, Pulmonary function assessment by whole-body plethysmography in restrained versus unrestrained mice. J Pharmacol Toxicol Methods, 2002. 47(1): p. 1-10. Flandre, T.D., P.L. Leroy, and D.J. Desmecht, Effect of somatic growth, strain, and sex on doublechamber plethysmographic respiratory function values in healthy mice. J Appl Physiol (1985), 2003. 94(3): p. 1129-36. Schlesinger, R.B., Comparative deposition of inhaled aerosols in experimental animals and humans: a review. J Toxicol Environ Health, 1985. 15(2): p. 197-214. Leakey, A.K., G.C. Ulett, and R.G. Hirst, BALB/c and C57Bl/6 mice infected with virulent Burkholderia pseudomallei provide contrasting animal models for the acute and chronic forms of human melioidosis. Microb Pathog, 1998. 24(5): p. 269-75. Schweizer, H.P., Mechanisms of antibiotic resistance in Burkholderia pseudomallei: implications for treatment of melioidosis. Future Microbiol, 2012. 7(12): p. 1389-99. Ibrahim, B.M., et al., Challenges and advances in the development of inhalable drug formulations for cystic fibrosis lung disease. Expert Opin Drug Deliv, 2011. 8(4): p. 451-66. Hoppentocht, M., et al., Developments and strategies for inhaled antibiotic drugs in tuberculosis therapy: a critical evaluation. Eur J Pharm Biopharm, 2014. 86(1): p. 23-30. West, T.E., et al., Murine pulmonary infection and inflammation induced by inhalation of Burkholderia pseudomallei. Int J Exp Pathol, 2012. 93(6): p. 421-8. Gauthier, Y.P., et al., Study on the pathophysiology of experimental Burkholderia pseudomallei infection in mice. FEMS Immunol Med Microbiol, 2001. 30(1): p. 53-63. 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): p. 96-9. Sookpranee, M., et al., Multicenter prospective randomized trial comparing ceftazidime plus cotrimoxazole with chloramphenicol plus doxycycline and co-trimoxazole for treatment of severe melioidosis. Antimicrob Agents Chemother, 1992. 36(1): p. 158-62. Wilson, W.J., et al., Immune Modulation as an Effective Adjunct Post-exposure Therapeutic for B. pseudomallei. PLoS Negl Trop Dis, 2016. 10(10): p. e0005065. 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): p. 77-81.



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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): p. 460-4.


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Table 1. Dosimetry of ceftazidime delivery.

Mean MMAD (μm)

Stdev (μm)

Mean GSD

Stdev

Treatment #

Inhaled Dose (mg)

1 2 3 4 5 6 7* 8 9 10 11 12 13 14 15 16 17 18 19 20

2.41 1.31 1.19 1.42 1.99 1.73 0.71 1.23 2.04 2.38 1.35 1.75 1.73 2.02 1.88 2.15 2.00 1.77 1.92 1.53

3.05 2.55 2.28 3.84 3.12 3.18 1.77 NA NA NA NA NA NA NA NA NA NA NA NA NA




*Sample collection error

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Figure 1. Bacterial burden at euthanasia.

1000000

800000

CFU/gram of Tissue

IP Ceftazidime Nebulized Ceftazidime

600000

400000

200000

Day 1

17

Day 3

Day 9


Day 28

Spleen

Liver

Lung

Spleen

Liver

Lung

Spleen

Liver

Lung

Spleen

Liver

0 Lung

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 2. Average weight for each treatment group.*

Treatment End

22

20

Weight (mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


18

IP Vehicle IP Ceftazidime Nebulized Vehicle Nebulized Ceftazidime

16

14 0

5

10

15

Day Post-Exposure *All mice treated with control vehicle were euthanized on day 3 post-exposure. *Half error bars shown for clarity in treatment groups.

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Figure 3. Average clinical scores for each treatment group.*

10

Treatment End IP Vehicle

8 IP Ceftazidime 6

Score

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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4

2

0 0

5

10

15

Day Post-Exposure *Half error bars shown for clarity in treatment groups.

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20

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Figure 4. Average temperature for each treatment group.*

Treatment End 40

38

Temperature (oC)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


36

34 IP Vehicle IP Ceftazidime Nebulized Vehicle Nebulized Ceftazidime

32

30 0

5

10

15

Day Post-Exposure

20


20


Figure 5. Average weight of spleen at time of euthanasia.

0.7 0.6

Weight (grams)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.5 0.4 0.3 0.2 0.1 0

21


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Pulmonary Delivery of Ceftazidime for the ... - ACS Publications

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