Contribution of Oxazolidinones to the Efficacy of Novel Regimens Containing Bedaquiline and Pretomanid in a Mouse Model of Tuberculosis

New regimens based on two or more novel agents are sought to shorten or simplify treatment of tuberculosis (TB). Pretomanid (PMD) is a nitroimidazole in phase 3 trials that has significant bactericidal activity alone and in combination with bedaquiline (BDQ) and/or pyrazinamide (PZA). We previously showed that the novel combination of BDQ+PMD plus the oxazolidinone sutezolid (SZD) had sterilizing activity superior to that of the first-line regimen in a murine model of TB. The present experiments compared the activity of different oxazolidinones in combination with BDQ+PMD with or without PZA in the same model. The 3-drug regimen of BDQ+PMD plus linezolid (LZD) had sterilizing activity approaching that of BDQ+PMD+SZD and superior to that of the first-line regimen. The addition of PZA further enhanced activity. Reducing the duration of LZD to 1 month did not significantly affect the activity of the regimen. Halving the LZD dose or replacing LZD with RWJ-416457 modestly reduced activity over the first month but not after 2 months. AZD5847 and tedizolid also increased the bactericidal activity of BDQ+PMD, but they were less effective than the other oxazolidinones. These results provide optimism for safe, short-course oral regimens for drug-resistant TB that may also be superior to the current first-line regimen for drug-susceptible TB.     

Verapamil Increases the Bactericidal Activity of Bedaquiline against Mycobacterium tuberculosis in a Mouse Model

Bedaquiline is a newly approved drug for the treatment of multidrug-resistant tuberculosis, but there are concerns about its safety in humans. We found that the coadministration of verapamil with subinhibitory doses of bedaquiline gave the same bactericidal effect in mice as did the full human bioequivalent bedaquiline dosing. Adding verapamil to bedaquiline monotherapy also protected against the development of resistant mutants in vivo. The adjunctive use of verapamil may permit use of lower doses of bedaquiline to be used and thereby reduce its dose-related toxicities in tuberculosis patients.     

Comparative Efficacy Evaluation of Dicationic Carbazole Compounds, Nitazoxanide, and Paromomycin against Cryptosporidium parvum Infections in a Neonatal Mouse Model

The efficacies of dicationic carbazole compounds, nitazoxanide (NTZ), and paromomycin were evaluated against the AUCp1 isolate of Cryptosporidium parvum by using a neonatal mouse model. Compounds were solubilized or suspended in deionized water and administered orally by gavage to neonatal mice at a constant dose rate on days 0 to 5 (treatment started on day 0). Dose rates varied for individual carbazole compounds but ranged from 0.65 to 20 mg/kg of body weight. NTZ was tested at 100 and 150 mg/kg, and paromomycin was tested at 50 mg/kg. Efficacies were determined by comparing numbers of oocysts present in treated versus control mice at necropsy examination on day 6. Demonstrable efficacy was observed for several carbazole compounds, based on significant reductions in the numbers of oocysts recovered from treated mice versus control mice. Compounds 1, 7, and 10 (19.0 mg/kg) reduced oocyst passage in treated mice to less than 5% of that in control mice. Treatment with compounds 6, 8, and 9 (17.0 mg/kg) resulted in reductions of oocyst output to less than 10% of that in controls. Although they were not comparable in efficacy to compounds 1, 6, 7, 8, 9, and 10, treatment with other carbazole compounds resulted in statistically significant reductions in oocyst output in treated versus control mice. Compound 1 retained efficacy resulted in reduction of oocyst output to approximately 6% of that in controls when the dose was reduced to 5 mg/kg. Further reductions in the dose rate resulted in considerable reductions in anticryposporidial activity. Likewise, the efficacies of compounds 9 and 10 were reduced substantially when the doses were lowered to one-half the screening dose. Paromomycin yielded excellent activity (reduction of oocyst output to <2% of that in controls) at a dose of 50 mg/kg. NTZ yielded moderate efficacy as powder and injectable formulations administered at 100 mg/kg orally (reduction of oocyst output to 42 and 26% of that in controls, respectively). Oral administration of the injectable formulation of NTZ at a dose of 150 mg/kg resulted in improved efficacy (oocyst output, <5% of that in controls).     

Pharmacokinetics and Pharmacodynamics of Clofazimine in a Mouse Model of Tuberculosis

The antileprosy drug clofazimine has shown potential for shortening tuberculosis treatment; however, the current dosing of the drug is not evidence based, and the optimal dosing is unknown. Our objective was to conduct a preclinical evaluation of the pharmacokinetics and pharmacodynamics of clofazimine in the mouse model of tuberculosis, with the goal of providing useful information on dosing for future studies. Pharmacokinetic parameters were evaluated in infected and uninfected BALB/c mice. Pharmacodynamic parameters were evaluated in Mycobacterium tuberculosis-infected mice that were treated for 12 weeks with one of six different clofazimine dosing regimens, i.e., doses of 6.25, 12.5, and 25 mg/kg of body weight/day and 3 regimens with loading doses. Clofazimine progressively accumulated in the lungs, livers, and spleens of the mice, reaching levels of greater than 50 μg/g in all tissues by 4 weeks of administration, while serum drug levels remained low at 1 to 2 μg/ml. Elimination of clofazimine was extremely slow, and the half-life was dependent on the duration of drug administration. Clofazimine exhibited dose-dependent tissue and serum concentrations. At any dose, clofazimine did not have bactericidal activity during the first 2 weeks of administration but subsequently demonstrated potent, dose-independent bactericidal activity. The antituberculosis activity of clofazimine was dependent on neither the dose administered nor the drug concentrations in the tissues, suggesting that much lower doses could be effectively used for tuberculosis treatment.     

Efficacy of Trovafloxacin against Penicillin-Susceptible and Multiresistant Strains of Streptococcus pneumoniae in a Mouse Pneumonia Model

The increasing emergence of penicillin-resistant and multidrug-resistant strains of Streptococcus pneumoniae will create a serious therapeutic problem in coming years. Trovafloxacin is a novel naphthyridone quinolone with promising activity against S. pneumoniae, including penicillin-resistant strains (MIC for 90% of the isolates tested, 0.25 μg/ml). We compared its in vivo efficacy with that of other fluoroquinolones (ciprofloxacin, temafloxacin, and sparfloxacin) and a reference beta-lactam (amoxicillin) in a model of acute experimental pneumonia. Immunocompetent Swiss mice were infected by peroral tracheal delivery of a virulent, penicillin-susceptible strain (MIC, 0.03 μg/ml); leukopenic Swiss mice were infected with three poorly virulent, penicillin-resistant strains (MICs, 4 to 8 μg/ml) and a ciprofloxacin-resistant strain (MIC, 32 μg/ml). Treatments were started 6 h (immunocompetent mice) or 3 h (leukopenic mice) after infection. Doses ranging from 12.5 to 300 mg/kg were given at 12- or 8-h intervals for 3 days. Trovafloxacin (25 mg/kg) was the most effective agent in vivo against penicillin-susceptible and -resistant strains. Corresponding survival rates were 2- to 4-fold higher than with 50-mg/kg sparfloxacin or temafloxacin and 8- to 16-fold higher than with 100-mg/kg ciprofloxacin. The ratios of the area under the concentration-time curve to the MIC in serum and lung tissue were more favorable with trovafloxacin than with the other quinolones. Efficacy in vivo correlated with pharmacokinetic parameters. Trovafloxacin shows potential for the treatment of infections due to penicillin-susceptible and -resistant S. pneumoniae but appears to be ineffective against a ciprofloxacin-resistant strain.     

Efficacy of the Novel Topical Antimicrobial Agent PXL150 in a Mouse Model of Surgical Site Infections

Antimicrobial peptides have recently emerged as a promising new group to be evaluated in the therapeutic intervention of infectious diseases. This study evaluated the anti-infectious effect of the short, synthetic, broad-spectrum antimicrobial peptide PXL150 in a mouse model of staphylococcal surgical site infections. We found that administration of PXL150, formulated in an aqueous solution or in a hydroxypropyl cellulose gel, significantly reduced the bacterial counts in the wound compared with placebo treatment, warranting further investigations of the potential of this peptide as a novel local treatment of microbial infections.     

Efficacy of Enrofloxacin in a Mouse Model of Sepsis

We examined the efficacy of enrofloxacin administered by 2 different routes in a mouse model of sepsis. Male CD1 mice were infected with a bioluminescent strain of enteropathogenic Escherichia coli and treated with enrofloxacin either by injection or in drinking water. Peak serum levels were evaluated by using HPLC. Mice were monitored for signs of clinical disease, and infections were monitored by using bioluminescence imaging. Serum levels of enrofloxacin and the active metabolite ciprofloxacin were greater in the group treated by injection than in controls or the groups treated by administration in drinking water. Survival of the group treated with enrofloxacin injection was greater than that of controls and groups treated with enrofloxacin in the drinking water. Bioluminescence in the group treated with enrofloxacin injection was less than that in the groups treated with oral administration at 12 h and in the groups treated orally and the control group at 16 h. According to these findings, we recommend the use of injectable enrofloxacin at 5 mg/kg SC for mice with systemic infections.Abbreviation: MIC, minimal inhibitory concentrationSepsis is defined as the systemic inflammatory response to the presence of bacterial infection.⁶ It affects approximately 750,000 people annually in the United States, with a mortality rate of 30% to 50% and a financial burden of US$16.7 billion dollars.¹ Septic shock is persistent hypotention with hypoperfusion abnormalities or organ dysfunction secondary to sepsis and kills 10 times more people than myocardial infarction in the United States.^7,38 Bacterial isolates from patients with gram-negative infections most commonly include Escherichia coli, Klebsiella species, or Enterobacter species.⁶ Due to the widespread and severe nature of this disease, this is an active area of scientific research.^14,17Enrofloxacin is a frequently used antibiotic approved for use in dogs, cats, cattle, and pigs in the United States. It is a fluoroquinolone antibiotic that leads to bacterial cell death through the inhibition of topoisomerase II, which controls supercoiling of bacterial DNA.³³ The bactericidal effect is dependent on the concentration of antibiotic present in tissues,³⁵ both of the parent compound and of the active metabolite ciprofloxacin.¹⁶ Enrofloxacin has been studied in a wide range of laboratory animal species including bovines,^16,26,27 dogs,^5,36 frogs,^20,25 horses,⁴¹ macaques,⁴ marmosets,⁴⁴ mice,^{22,32,34,36,40,49} rabbits,¹⁹ rats,⁸ sheep,¹⁵ and swine.^15,37,52 Published doses in veterinary formularies range from 2.5 to 20 mg/kg for enteral and parenteral bolus dosing and for rodents at 0.05 to 0.2 mg/mL in drinking water.^11,45,46 In laboratory animal medicine, fluoroquinolone antibiotics are sometimes administered as a therapeutic agent to populations of animals. One example of this practice is for eradication of an opportunistic pathogen such as Pneumocysitis carinii in genetically modified mice.³² In addition, fluroquinolones are used on a large scale prophylactically in research protocols involving bone marrow transplantation in mice.³⁹ When large groups of animals require treatment, the route of administration can affect the personnel time required to conduct the research.Bioluminesence imaging is a noninvasive imaging modality that has been used to study cell trafficking, tumor development, gene expression, gene therapy, inflammation and infection, protein–protein interactions, and protein stability and function in laboratory rodents.¹³ Bioluminescent imaging systems use specialized charge-coupled cameras to capture low amounts of light. Organisms can be genetically engineered to express firefly luciferase or other types of luciferase enzymes, which emit light when the organisms are provided an exogenous substrate, such as luciferin, in the presence of ATP and oxygen.²⁹ In addition, specialized bacteria have been engineered to carry genes for both the luciferase and the substrate, a long-chain fatty aldehyde, and thus do not require an exogenous source of substrate.⁴⁷ These types of modified bacteria have been used to study the progression and treatment of bacterial infections of the gastrointestinal tract,^10,21 wounds,^23,47 and surgical implants.^30,51,53 Advantages of bioluminescence imaging include the ability to assess the spatial and temporal distribution of bacteria after intervention in the same animal, thus reducing animal numbers.The purpose of the current study was to refine a model of sepsis by evaluating the efficacy of different routes of administration of prophylactic antibiotics. This work was part of a larger study evaluating the pathogenesis of hospital-acquired infection in patients receiving blood transfusions during concurrent antibiotic therapy. With an effective model system, research results can be translated to treatment of human diseases. Serial noninvasive optical imaging of a bioluminescently engineered strain of enteropathogenic Escherichia coli was used as a biomarker of bacterial infection in the presence of mitigating drug regimens. We hypothesized that there would be no difference in the severity of infection, evaluated as photons per second emitted from a region of interest over the abdomen, between animals that received enrofloxacin via different routes of administration in this model of sepsis. Our goal was to establish an effective method of preventing or treating gram-negative sepsis through antibiotic administration that minimized animal handling and manipulation, promoted animal wellbeing, and maximized the information obtained from the animals used in our research.     

Caspase-Based PET for Evaluating Pro-Apoptotic Treatments in a Tuberculosis Mouse Model

Purpose

Despite recent advances in antimicrobial treatments, tuberculosis (TB) remains a major global health threat. Mycobacterium tuberculosis proliferates in macrophages, preventing apoptosis by inducing anti-apoptotic proteins leading to necrosis of the infected cells. Necrosis then leads to increased tissue destruction, reducing the penetration of antimicrobials and immune cells to the areas where they are needed most. Pro-apoptotic drugs could be used as host-directed therapies in TB to improve antimicrobial treatments and patient outcomes.

Procedure

We evaluated [¹⁸F]-ICMT-11, a caspase-3/7-specific positron emission tomography (PET) radiotracer, in macrophage cell cultures and in an animal model of pulmonary TB that closely resembles human disease.

Results

Cells infected with M. tuberculosis and treated with cisplatin accumulated [¹⁸F]-ICMT-11 at significantly higher levels compared with that of controls, which correlated with levels of caspase-3/7 activity. Infected mice treated with cisplatin with increased caspase-3/7 activity also had a higher [¹⁸F]-ICMT-11 PET signal compared with that of untreated infected animals.

Conclusions

[¹⁸F]-ICMT-11 PET could be used as a noninvasive approach to measure intralesional pro-apoptotic responses in situ in pulmonary TB models and support the development of pro-apoptotic host-directed therapies for TB.

     

Pharmacokinetic and Pharmacodynamic Evaluation of AZD5847 in a Mouse Model of Tuberculosis

AZD5847, a novel oxazolidinone with an MIC of 1 μg/ml, exhibits exposure-dependent killing kinetics against extracellular and intracellular Mycobacterium tuberculosis. Oral administration of AZD5847 to mice infected with M. tuberculosis H37Rv in a chronic-infection model resulted in a 1.0-log₁₀ reduction in the lung CFU count after 4 weeks of treatment at a daily area under the concentration-time curve (AUC) of 105 to 158 μg · h/ml. The pharmacokinetic-pharmacodynamic parameter that best predicted success in an acute-infection model was an AUC for the free, unbound fraction of the drug/MIC ratio of ≥20. The percentage of time above the MIC in all of the efficacious regimens was 25% or greater.     

Optimization of Pyrrolamides as Mycobacterial GyrB ATPase Inhibitors: Structure-Activity Relationship and In Vivo Efficacy in a Mouse Model of Tuberculosis

Moxifloxacin has shown excellent activity against drug-sensitive as well as drug-resistant tuberculosis (TB), thus confirming DNA gyrase as a clinically validated target for discovering novel anti-TB agents. We have identified novel inhibitors in the pyrrolamide class which kill Mycobacterium tuberculosis through inhibition of ATPase activity catalyzed by the GyrB domain of DNA gyrase. A homology model of the M. tuberculosis H37Rv GyrB domain was used for deciphering the structure-activity relationship and binding interactions of inhibitors with mycobacterial GyrB enzyme. Proposed binding interactions were later confirmed through cocrystal structure studies with the Mycobacterium smegmatis GyrB ATPase domain. The most potent compound in this series inhibited supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC₅₀) of <5 nM, an MIC of 0.03 μg/ml against M. tuberculosis H37Rv, and an MIC₉₀ of <0.25 μg/ml against 99 drug-resistant clinical isolates of M. tuberculosis. The frequency of isolating spontaneous resistant mutants was ∼10⁻⁶ to 10⁻⁸, and the point mutation mapped to the M. tuberculosis GyrB domain (Ser208 Ala), thus confirming its mode of action. The best compound tested for in vivo efficacy in the mouse model showed a 1.1-log reduction in lung CFU in the acute model and a 0.7-log reduction in the chronic model. This class of GyrB inhibitors could be developed as novel anti-TB agents.     

In Vivo Efficacy of Daptomycin against Methicillin-Resistant Staphylococcus aureus in a Mouse Model of Hematogenous Pulmonary Infection

Daptomycin is inactivated by pulmonary surfactant, but its effectiveness in hematogenous pulmonary infection has been poorly studied. The potential therapeutic application was evaluated in a methicillin-resistant Staphylococcus aureus (MRSA) hematogenous pulmonary infection mouse model. Compared with control results, daptomycin improved survival (P < 0.001) and decreased the number of abscesses and bacteria in the lungs (P < 0.01). Daptomycin may be an effective therapeutic option for MRSA hematogenous pulmonary infection.     

Protective Efficacy of a Sulfated Sialyl Lipid (NMSO3) against Human Rotavirus-Induced Diarrhea in a Mouse Model

Antiviral activity of sulfated sialyl lipid (NMSO3) against human rotavirus (RV) was examined in vitro and in vivo. NMSO3 inhibited the replication of four major serotypes (G1 to G4) of human rotavirus with a low 50% effective concentration of 1 to 5 microg/ml and 50% cytotoxic concentration of 153 microg/ml when determined by plaque assays with MA104 cells. Exposure of NMSO3 to HCl (pH 2.0) for 30 min exhibited no loss of anti-RV activity. Time-of-addition experiments revealed that NMSO3 inhibited the adsorption of four serotypes of RV to MA104 cells. Furthermore, an assay of virus binding with radiolabeled RVs revealed that NMSO3 inhibited the binding of virus to MA104 cells, suggesting that NMSO3 may bind to VP4 and/or VP7. Prophylactic oral administration of NMSO3 (10 microg three times per day, 4 days) to five suckling mice starting 30 min before inoculation of MO strain (3 x 10(6) PFU/mouse) prevented the development of diarrhea. Four of five mice showed no stool or brown formed stool, and only one mouse showed brown soft stool, while water treatment caused watery diarrhea in all five mice. The mean titer of antibody to RV in mice which received NMSO3 at 10 microg three times per day for 4 days was significantly lower than that of untreated, infected mice. NMSO3 is a promising candidate for the prophylactic treatment of human RVs.     

Efficacy of AiiM,an N-Acylhomoserine Lactonase,against Pseudomonas aeruginosa in a Mouse Model of Acute Pneumonia

Quorum sensing (QS) in Pseudomonas aeruginosa regulates the production of many virulence factors and plays an important role in the pathogenesis of P. aeruginosa infection. N-acyl homoserine lactones (AHL) are major QS signal molecules. Recently, a novel AHL-lactonase enzyme, AiiM, has been identified. The aim of this study was to evaluate the effect of AiiM on the virulence of P. aeruginosa in a mouse model of acute pneumonia. We developed a P. aeruginosa PAO1 strain harboring an AiiM-expressing plasmid. The production of several virulence factors by the AiiM-expressing strain was examined. Mice were intratracheally infected with an AiiM-expressing PAO1 strain. Lung histopathology, bacterial burden, and bronchoalveolar lavage (BAL) fluid were assessed at 24 h postinfection. AiiM expression in PAO1 reduced production of AHL-mediated virulence factors and attenuated cytotoxicity against human lung epithelial cells. In a mouse model of acute pneumonia, AiiM expression reduced lung injury and greatly improved the survival rates. The levels of proinflammatory cytokines and myeloperoxidase activity in BAL fluid were significantly lower in mice infected with AiiM-expressing PAO1. Thus, AiiM can strongly attenuate P. aeruginosa virulence in a mammalian model and is a potential candidate for use as a therapeutic agent against P. aeruginosa infection.     

小鼠嗜酸粒细胞性支气管炎模型与哮喘模型的蛋白组学差异表达分析

【目的】探讨小鼠嗜酸粒细胞性支气管炎模型（EB模型）与哮喘模型的蛋白组学差异表达。【方法】EB模型小鼠（实验组）、哮喘模型小鼠（哮喘组）及对照组小鼠各4只,取肺组织提取总蛋白质,采用基质辅助电离解析飞行时间质谱对蛋白质进行序列分析,并比较三组肺组织蛋白差异。【结果】双向电泳图像显示三组肺组织蛋白表达比较差异有显著性（P＜0．05）,经质谱检测肽质量指纹谱与标准分子量、等电点对照分析鉴定出20个蛋白,分别为谷胱甘肽-S-转移酶 M1（GSTM1）、热休克蛋白 B1（HSPB1）等,其中 GSTM1在哮喘组表达下调、HSPB1在哮喘组表达上调。【结论】GSTM1、HSPB1等可能参与气道高反应性的发生机制。