Efficacy of ceftazidime in a mouse model after lethal aerosol exposure to Burkholderia pseudomallei

research design

Female mice placed in the study were at least 6 weeks old upon arrival, weighed between 16 and 20.1 g on the day of exposure, and had no clinical signs of disease at the time of exposure. Mice were randomly assigned to study groups by body weight. A patterned assignment was utilized to select animals in Groups 1, 11, 12, and 13 into subgroups and all test animals into challenge performance order. At least one animal from each test group was selected for each challenge exposure run. 190 female her-BALB/c her mice were exposed to the aerosolized substance. B. Pseudomalray (strain K96243) target 8100 cfu/animal, equivalent to 50 × LD₅₀ (actual average inhaled dose 11,100 cfu; 69 × LD₅₀) for 1 day. He performed 10 aerosol exposures of 19 mice each. Aerosol concentrations for all exposure studies ranged from 7450 cfu/animal to 18,300 cfu/animal (standard deviation 4119 cfu/animal). The mass median aerodynamic diameter of inhaled particles ranges from 1.26 to 1.31 μm (geometric standard deviation 1.47 to 1.56 μm), allowing for lower lung deposition of inhaled particles.^33,34After aerosol exposure, mice were treated with test article (ceftazidime) or control article (sterile saline). Two treatment cohorts were included in the study. In the first cohort, the efficacy of 14-day ceftazidime therapy was examined through assessment of bacterial tissue burden and 60-day survival after treatment discontinuation. Eight different treatment regimens were evaluated (Table 1) consisted of two treatment frequencies: twice daily (BID) and four times daily (QID). Two starting time points: 24 hours after exposure (24 hours) and 48 hours after exposure (48 hours). and two doses: 600 mg/kg/day and 1200 mg/kg/day. The 600 mg/kg/day dose was achieved by administering 300 mg/kg BID or 150 mg/kg QID and the 1200 mg/kg/day dose was achieved by administering 600 mg/kg BID or 300 mg/kg QID. achieved by dosing. Doses were administered approximately every 12 hours for BID administration and approximately every 6 hours for QID administration. To assess disease progression, tissue burden was examined in the kidney, liver, lung, and spleen of all treated mice and a cohort of untreated mice at 24, 36, 48, and 60 hours after exposure. Using a satellite cohort of mice, we performed a pharmacokinetic analysis of his three doses of ceftazidime administered as a single dose approximately 24 hours after exposure. No exposed animals were excluded from survival analysis. No blindfolds or masking were used during the conduct of the study.

test system

Female BALB/c mice, 6–8 weeks old upon arrival, were obtained from Charles River Laboratories (Stone Ridge, NY). General procedures for the care and husbandry of animals can be found in the AAALAC International Recommendations, current requirements described in the Guide for the Care and Use of Laboratory Animals (National Research Council, latest edition), the United States Department of Agriculture through animals. meets the current requirements stated by Conforms to the Revised Welfare Law and Standard Operating Procedures for Testing Facilities. Animal use protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and the US Army’s Office of Animal Care and Use Review (ACURO). Findings are reported according to ARRIVE guidelines.

Goods testing and control

Ceftazidime for Injection, USP (Hospira, Lake Forest, IL) was diluted to 95 mg/mL in Sterile Water for Injection, USP. The dosing solution was further diluted with sterile saline, USP to 15, 30, or 60 mg/mL to achieve doses of 150 mg/kg/dose, 300 mg/kg/dose, and 600 mg/kg/dose, respectively. Animals in the control group received sterile saline. Doses were administered intraperitoneally (IP) in a target volume of 10 mL/kg. Injections were made at contralateral sites during each administration to minimize local tissue damage.

Challenge agent and aerosol exposure

Burkholderia pseudomallei Strain K92643 was used for animal exposure. Strain selection was based on previous internal characterization. Minimum inhibitory concentration (MIC) tests performed internally on strain K96243 yielded his MIC of 1 μg/mL for ceftazidime. Lots used for aerosol exposure were propagated and characterized by the Battelle Biomedical Research Center.fresh B. Pseudomalray Suspensions were prepared by inoculating stock material with LB broth containing 4% glycerol (LBG), incubating at 37 °C (±2 °C), and shaking at 250 rpm for 18–24 h. Did. Dilute the resulting starter culture with sterile LBG to an OD₆₀₀ 0.200 (± 0.05). This culture was then incubated at 37 °C (±2 °C) and 250 rpm for 18–20 h. Cultures were checked for purity after Gram staining. Cultures were then centrifuged at 10,000 rcf for 10 minutes. The resulting pellet was washed and resuspended in phosphate-buffered saline (BSGT) containing gelatin and trehalose. The washing and resuspension steps were repeated a total of two times. Suspension was removed and adjusted with buffer to reach OD.₆₀₀ 2.1 (± 0.1).Aerosol exposure was performed as previously described³⁵Inhaled dose of 8100 cfu equivalent to 50 × LD₅₀aimed at consistency with previous internal model development experiments.

Whole blood collection and pharmacokinetic analysis

Cardiac blood sampling was performed as the final procedure. Prior to collection, mice were anesthetized with a mixture of ketamine (80–100 mg/kg) and xylazine (5–10 mg/kg).A bacteremia specimen was collected on K.₃Place in an EDTA tube and keep at room temperature until processing. Pharmacokinetic (PK) samples were collected on K.₃Place in an EDTA tube and store on damp ice until processing. Plasma was collected and sterile filtered using a 0.2 micron PES filter. Samples were stored in a freezer set to maintain −80 °C until analysis.

Ceftazidime was extracted from plasma and quantified using an in-house developed high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method. This is a sensitive, high-throughput method utilizing protein precipitation extraction followed by strong cation exchange (SCX) LC–MS/MS. Shimadzu Prominence XR Series HPLC and AB Sciex Triple Quad 5500 mass spectrometer in positive ionization mode (ESI⁺). Chromatographic separations were performed using an Agilent Zorbax 300-SCX 5µ, 2.1 × 150 mm column with a gradient mobile phase consisting of 95:5 25 mM ammonium formate:acetonitrile (v:v) (A) and 70:30 25 mM ammonium formate. and obtained. : Add 500 mM ammonium formate (B) to acetonitrile at a flow rate of 0.5 mL/min and set the column oven temperature to 30 °C. Ceftazidime pentahydrate obtained from the United States Pharmacopeia (USP) was used to prepare solutions of standards and quality control samples prepared in mouse plasma. Cefepime hydrochloride, a chemical analog obtained from USP, was prepared as an internal standard. Addition of internal standards allowed reproducible quantification as ceftazidime and cefepime were monitored using multiple reaction monitoring (MRM) at 547.0/468.0 and 481.0/86.0, respectively. Regression analysis was evaluated by analyzing a series of calibration standards at eight concentrations. The lower limit of quantification for ceftazidime was determined to be 50 ng/mL in plasma using a sample size of 100 µL.

Free plasma concentrations were adjusted using an estimated protein binding of 26% and 15% in mice and humans, respectively.³⁶PK and exposure parameters were estimated using noncompartmental analysis with Phoenix WinNonlin software (Certara LP, Princeton, NJ). The following acceptance criteria were used to assess concentration-time profiles. (1) Coefficient of determination (r²(2) the time of the last observed concentration was greater than 3 half-lives; and (3) AUC_∞ The extrapolated area was less than 20%. Mouse data were fitted to WinNonlin’s compartmental model and used to simulate concentrations of PK/PD target attainment.

Tissue collection and bacterial enumeration

At necropsy, aliquots of kidney, liver, lung, and spleen were aseptically harvested from each mouse, weighed and analyzed using the gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Tissue homogenates were serially diluted in triplicate and spread on LB agar (LBGA) containing 4% glycerol or Columbia blood agar. Spread plates were placed in an incubator set to maintain 37 °C for an average of 58 hours (41.4–93.1 hours). Plates were counted and cfu/gram tissue was calculated for statistical analysis.

statistical analysis

Statistical analysis was performed using SAS (version 9.4) and R (version 3.6.3) on a 64-bit platform. To determine group size, we performed a power analysis based on a single one-sided Bauchreux test performed with a 5% type I error rate to determine survival statistics between single treatment and control groups. detected a statistically significant difference. Assuming a 5% survival rate in the control group, a power of 88% with 10 animals per treatment group is considered, assuming a probability of survival in the treatment group of at least 60%. An additional 2 animals included in the 48 h post-exposure group allowed statistical significance to be maintained in the event of premature death. Percentages of surviving animals and exact 95% confidence intervals were calculated for each group in the efficacy cohort. A one-tailed Bauschroux test was used to compare survival between each treatment group and the saline control group (group 2) at study end, treatment end, and 30 days after treatment discontinuation. A two-tailed Bauschroux test was used to compare survival rates between each pair of time points within each group. A Bonferroni-Holm multiple comparison procedure was used to control for an overall type I error rate of 5% in each set of tests. A log-rank test was used to test for significant differences in time to death between each treatment group and the saline control group. A Bonferroni-Holm multiple comparison procedure was used to control for an overall type I error rate of 5% across all tests. Kaplan-Meier curves for each group were plotted to visually represent survival and time-to-death data. For quantitative assessment of bacterial load in tissues, we calculated geometric means and 95% confidence intervals for each tissue type by group. An analysis of variance (ANOVA) model was fitted to the radix-10 log-transformed bacterial load to assess the effect of treatment on tissue bacterial load. The mean bacterial load of each treatment group was statistically compared to the mean bacterial load of the saline control group (group 2) within the model for each tissue type. Dunnett’s multiple comparison procedure was used to control for an overall Type I error rate of 5% across all tests per tissue.