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. 2023 Dec 11:13:1297281.
doi: 10.3389/fcimb.2023.1297281. eCollection 2023.

Development and validation of a rabbit model of Pseudomonas aeruginosa non-ventilated pneumonia for preclinical drug development

Affiliations

Development and validation of a rabbit model of Pseudomonas aeruginosa non-ventilated pneumonia for preclinical drug development

Emmanuelle Gras et al. Front Cell Infect Microbiol. .

Abstract

Background: New drugs targeting antimicrobial resistant pathogens, including Pseudomonas aeruginosa, have been challenging to evaluate in clinical trials, particularly for the non-ventilated hospital-acquired pneumonia and ventilator-associated pneumonia indications. Development of new antibacterial drugs is facilitated by preclinical animal models that could predict clinical efficacy in patients with these infections.

Methods: We report here an FDA-funded study to develop a rabbit model of non-ventilated pneumonia with Pseudomonas aeruginosa by determining the extent to which the natural history of animal disease reproduced human pathophysiology and conducting validation studies to evaluate whether humanized dosing regimens of two antibiotics, meropenem and tobramycin, can halt or reverse disease progression.

Results: In a rabbit model of non-ventilated pneumonia, endobronchial challenge with live P. aeruginosa strain 6206, but not with UV-killed Pa6206, caused acute respiratory distress syndrome, as evidenced by acute lung inflammation, pulmonary edema, hemorrhage, severe hypoxemia, hyperlactatemia, neutropenia, thrombocytopenia, and hypoglycemia, which preceded respiratory failure and death. Pa6206 increased >100-fold in the lungs and then disseminated from there to infect distal organs, including spleen and kidneys. At 5 h post-infection, 67% of Pa6206-challenged rabbits had PaO2 <60 mmHg, corresponding to a clinical cut-off when oxygen therapy would be required. When administered at 5 h post-infection, humanized dosing regimens of tobramycin and meropenem reduced mortality to 17-33%, compared to 100% for saline-treated rabbits (P<0.001 by log-rank tests). For meropenem which exhibits time-dependent bactericidal activity, rabbits treated with a humanized meropenem dosing regimen of 80 mg/kg q2h for 24 h achieved 100% T>MIC, resulting in 75% microbiological clearance rate of Pa6206 from the lungs. For tobramycin which exhibits concentration-dependent killing, rabbits treated with a humanized tobramycin dosing regimen of 8 mg/kg q8h for 24 h achieved Cmax/MIC of 9.8 ± 1.4 at 60 min post-dose, resulting in 50% lung microbiological clearance rate. In contrast, rabbits treated with a single tobramycin dose of 2.5 mg/kg had Cmax/MIC of 7.8 ± 0.8 and 8% (1/12) microbiological clearance rate, indicating that this rabbit model can detect dose-response effects.

Conclusion: The rabbit model may be used to help predict clinical efficacy of new antibacterial drugs for the treatment of non-ventilated P. aeruginosa pneumonia.

Keywords: Pseudomonas aeruginosa; meropenem; non-ventilated pneumonia model; preclinical efficacy model validation; rabbit model; tobramycin.

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Conflict of interest statement

Authors BZ, LC, CS, AD and BS are or were employed by AstraZeneca. Author BD previously received funding from AstraZeneca, Pfizer, Merck, Arsanis, ContraFect, and Integrated Biotherapeutics for preclinical studies using earlier iterations of the rabbit model of non-ventilated pneumonia. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Representative lung sections showing semi-quantitative scoring of the severity of hemorrhage, edema, exudate/fibrin deposits, diffuse PMN infiltrate, multifocal aggregated and altered PMNs, and PMNs in bronchi. Each lung section was scored according to the following scale: A = 0, absent; B = 1, mild; C = 2, moderate; D = 3, severe. For hemorrhage, arrows show red blood cells, and (A) absent, (B) scarce and located near the alveolar septa, (C) responsible for a mild and diffuse infiltration with patchy aggregates, and (D) diffuse massive infiltrate covering >50% of the lung section. For edema, arrows point to edema, and (A) absent, (B) mild streaked edema, (C) abundant edema filling partially the alveoli in 30-50% of the lung section, (D) abundant edema entirely filling the alveoli in >50% of the lung section. For exudate/fibrin, arrows point to fibrin, (A) absent, (B) mild fibrin exudate mixed with edema, (C) moderate fibrin exudate covering 30-50% of the lung section, (D) patchy fibrin exudate covering >50% of the lung section. For diffuse PMN infiltrates, arrows point to PMN, and (A) absent, (B) scarce PMN infiltrate with large unscathed areas, (C) mild PMN infiltrate covering the majority of the section with spared areas, (D) massive intra-alveolar PMN infiltrate covering >50% of the lung section. For multifocal aggregated, altered PMNs, arrows point to PMNs, (A) absent, (B) scarce small PMN aggregates, (C) moderately aggregated and altered PMN, (D) multiple big PMN aggregates covering the greater part of the lung section. For PMNs in bronchi, arrows point to PMNs, (A) absent, (B) PMN underlining the bronchiole/bronchi wall, (C) occupying >50% of the bronchiole/bronchi wall, (D) fulfilling the entire bronchiole/bronchi section.
Figure 2
Figure 2
Longitudinal pathological changes in the lungs of rabbits with P. aeruginosa-induced non-ventilated pneumonia. Gross and histological changes of representative lungs harvested at (A) pre-determined time points of 3, 4, 5, 6, 10 h after endobronchial instillation with live Pa6206, or at 21 h after endobronchial instillation with live Pa6206 when rabbit developed respiratory failure, (B) 5 h after endobronchial instillation with LRS, and (C) 5 h or 96 h after endobronchial instillation with UV-killed Pa6206. Semi-quantitative scoring of the severity of hemorrhage, edema, exudate/fibrin, diffuse PMN infiltrate, multifocal altered PMN aggregates, and bronchiolitis are provided in Table 1 and Figure 3 .
Figure 3
Figure 3
Natural history of P. aeruginosa-induced non-ventilated pneumonia in rabbits. (A) Lung weight/body weight (LW/BW) ratios, and (B–D) bacterial burden in lung, spleen and kidneys increased over time and peaked at the time of death in the 36 rabbits that were challenged by endobronchial instillation with 1.1 x 108 CFU of live Pa6206. Of these, 30 Pa6206-challenged rabbits were euthanized at predetermined time points of 3, 4, 5, 6, 10 h post-infection (6 rabbits for each time point). The remaining 6 live Pa6206-challenged rabbits were euthanized when they exhibited signs of respiratory distress and had blood lactate >10 mmol/L—their terminal endpoints (TE) were between 13 and 23 h post-infection. Seven control control rabbits were included for comparison: 1 control rabbit was euthanized at 5 h after endobronchial instillation with lactated Ringer’s solution (LRS); and 3 control rabbits each were euthanized at 5 h or 96 h, respectively, after endobronchial instillation with UV-killed Pa6206 (6.6 x 108 CFUs irradiated by ultraviolet light for 30 minutes). No bacteria were cultured from lung, spleen and kidneys from the seven control rabbits.
Figure 4
Figure 4
Blood counts and plasma IL-8 are consistent with acute respiratory distress syndrome and sepsis in the rabbit natural history study. (A) Red blood cells, RBC; (B) platelets; (C) white blood cells, WBC; (D) lymphocytes; (E) neutrophils; (F) monocytes; (G) eosinophils; (H) basophils; and (I) plasma interleukin-8, IL-8 (CXCL-8). Linear test for trend was used to evaluate whether means for each parameter increase (or decrease) systematically over the seven sampling time points, namely, pre-infection (0), 3, 4, 5, 6, and 10 h post-infection, and terminal endpoint between 13 and 23 h post-infection when rabbits were euthanized for development of respiratory failure when their lactate exceeded 10 mmol/L.
Figure 5
Figure 5
Evolution of clinically relevant biomarkers of acute respiratory distress syndrome in the rabbit natural history study. (A) PaO2; (B) PaCO2; (C) pH; (D) bicarbonate; (E) base excess; (F) lactate; (G) potassium, K+; (H) creatinine; (I) blood urea nitrogen, BUN; (J) alanine aminotransferase, ALT; (K) amylase; and (L) glucose. Linear test for trend was used to evaluate whether means for each parameter increase (or decrease) systematically over the seven sampling time points, namely, pre-infection (0), 3, 4, 5, 6, and 10 h post-infection, and terminal endpoint between 13 and 23 h post-infection when rabbits were euthanized for development of respiratory failure when their lactate exceeded 10 mmol/L.
Figure 6
Figure 6
Human-equivalent meropenem and tobramycin dosing regimens protected against lethal infection in the rabbit non-ventilated pneumonia model with P. aeruginosa. (A, D) Kaplan-Meier survival curves, (B, E) LW/BW ratio, and (C, F) log10(CFU/organ). For the first validation study (A–C), rabbits were randomized at 5 h post-infection with 2.0 x 108 CFU Pa6206 for treatment with normal saline (NS; 0.9% NaCl) IV q8h or tobramycin (TOB) 5 mg/kg IV q8h at 5 h post-infection (12 rabbits per experimental group). For the second validation study (D-F), rabbits were randomized at 5 h post infection (h post-infection) with 1.0 x 108 CFU Pa6206 for treatment with normal saline IV q2h, meropenem (MEM) 80 mg/kg IV q2h, or tobramycin (TOB) 2.5 mg/kg IV once at 5 h post-infection and then normal saline IV q2h (12 rabbits per experimental group). One rabbit in the meropenem group died at 9 h post-infection, which was about 3 minutes after IV injection of the second dose of meropenem; this rabbit did not show any sign of respiratory distress sepsis or respiratory failure immediately prior to antibiotic injection. One-sided log-rank (Mantel-Cox) test was used to test the hypothesis that survival of animals treated with saline is shorter than survival of those treated with meropenem or tobramycin, using P<0.025 (significance level of 0.05 divided by two different comparisons) being considered statistically significant to account for multiple comparisons using Bonferroni method. LW/BW ratio and bacterial densities for saline-treated animals were compared to those of each of the other two treatment groups by nonparametric one-way analysis of variance (ANOVA) with Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Filled symbols represent data from dead animals, and open symbols represent data from surviving animals that were euthanized at 96 h post-infection. X-marked symbols represent data from male rabbits, whereas those not marked with an X represent data from female rabbits. Statistical significance abbreviations: * for P<0.05; ** for P<0.01; *** for P<0.001.

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