Selection of QPX7831, an Orally Bioavailable Prodrug of Boronic Acid ß-Lactamase Inhibitor QPX7728.

In recognition of the need for effective oral therapies to treat Gram-negative bacterial infections, efforts were directed toward identifying an oral prodrug of ß-lactamase inhibitor clinical candidate QPX7728. Seventeen prodrugs were synthesized; key properties investigated were rates of cleavage to the active form in vitro, pharmacokinetics across species, and crystallinity. Compound 5-Na (QPX7831 Sodium) emerged with optimal properties across all key attributes.

In Vivo Activity of QPX7728, an Ultrabroad-Spectrum Beta-Lactamase Inhibitor, in Combination with Beta-Lactams against Carbapenem-Resistant Klebsiella pneumoniae.

Resistance to beta-lactams has created a major clinical issue. QPX7728 is a novel ultrabroad-spectrum cyclic boronic acid beta-lactamase inhibitor with activity against both serine and metallo-beta-lactamases developed to address this resistance for use in combination with beta-lactam antibiotics. The objective of these studies was to evaluate the activity of QPX7728 in combination with multiple beta-lactams against carbapenem-resistant Klebsiella pneumoniae isolates in a neutropenic mouse thigh infection model. Neutropenic mice were infected with strains with potentiated beta-lactam MICs of ≤2 mg/liter in the presence of 8 mg/liter QPX7728. Two strains of carbapenem-resistant K. pneumoniae were tested with aztreonam, biapenem, cefepime, ceftazidime, ceftolozane, and meropenem alone or in combination with 12.5, 25, or 50 mg/kg of body weight of QPX7728 every 2 hours for 24 hours. Treatment with all beta-lactams alone either was bacteriostatic or allowed for bacterial growth. The combination of QPX7728 plus each of these beta-lactams produced bacterial killing at all QPX7728 doses tested. Overall, these data suggest that QPX7728 administered in combination with different partner beta-lactam antibiotics may have utility in the treatment of bacterial infections due to carbapenem-resistant K. pneumoniae.

Discovery of Cyclic Boronic Acid QPX7728, an Ultrabroad-Spectrum Inhibitor of Serine and Metallo-ß-lactamases.

Despite major advances in the ß-lactamase inhibitor field, certain enzymes remain refractory to inhibition by agents recently introduced. Most important among these are the class B (metallo) enzyme NDM-1 of Enterobacteriaceae and the class D (OXA) enzymes of Acinetobacter baumannii. Continuing the boronic acid program that led to vaborbactam, efforts were directed toward expanding the spectrum to allow treatment of a wider range of organisms. Through key structural modifications of a bicyclic lead, stepwise gains in spectrum of inhibition were achieved, ultimately resulting in QPX7728 (35). This compound displays a remarkably broad spectrum of inhibition, including class B and class D enzymes, and is little affected by porin modifications and efflux. Compound 35 is a promising agent for use in combination with a ß-lactam antibiotic for the treatment of a wide range of multidrug resistant Gram-negative bacterial infections, by both intravenous and oral administration.

Pharmacodynamics of Meropenem against Acinetobacter baumannii in a Neutropenic Mouse Thigh Infection Model.

Acinetobacter baumannii infections are difficult to treat and have limited treatment options. Carbapenems, including meropenem, are currently considered the first-line agents for the treatment of infections caused by Acinetobacter spp. The percentage of a 24-hour period that the concentration of free drug in plasma is above the MIC (%24-h fT>MIC) to achieve stasis, 1 log CFU, or 2 log CFU of bacterial killing against A. baumannii has not been studied previously for meropenem. The objective of this study was to determine these parameters for meropenem against A. baumannii in a neutropenic mouse thigh infection model. Six A. baumannii clinical isolates with MICs ranging from 0.25 to 16 mg/liter were tested. Meropenem produced a bacteriostatic effect with a %24-h fT>MIC of 7 to 24% and produced 1 log CFU of bacterial killing with a %24-h fT>MIC of 15 to 37%.

Activity of Aerosolized Levofloxacin against Burkholderia cepacia in a Mouse Model of Chronic Lung Infection.

Burkholderia cepacia complex is an opportunistic pathogen capable of causing chronic pulmonary infections. These studies were conducted to demonstrate the activity of aerosolized levofloxacin in a chronic mouse lung infection model caused by B. cepacia isolates from patients with cystic fibrosis. Treatment with aerosolized levofloxacin for 4 days produced at least 1 log CFU of bacterial killing against all strains tested, suggesting possible utility in the treatment of lung infections caused by B. cepacia isolates.

Activity of Meropenem-Vaborbactam against Pseudomonas aeruginosa and Acinetobacter baumannii in a Neutropenic Mouse Thigh Infection Model.

We have evaluated the activity of meropenem-vaborbactam against clinical isolates of Pseudomonas aeruginosaandAcinetobacter baumannii in a neutropenic mouse thigh infection model. Data show that meropenem-vaborbactam regimens equivalent to 3-h infusions every 8 h with 2 g meropenem and 2 g vaborbactam produced bacterial killing against strains with MICs of 2 to 16 mg/liter and suggests that this combination may have utility in the treatment of infections caused by P. aeruginosa and A. baumannii.

Pharmacodynamics of Minocycline against Acinetobacter baumannii in a Rat Pneumonia Model.

Minocycline is currently approved in the United States for the treatment of infections caused by susceptible isolates of Acinetobacter spp. The objective of these studies was to determine the minocycline exposures associated with an antibacterial effect against Acinetobacter baumannii in a rat pneumonia model. Rats received minocycline doses as 30-min intravenous infusions. In the rat pneumonia model, six clinical isolates of A. baumannii with MICs ranging from 0.03 to 4 mg/liter were studied. In this model, minocycline produced a bacteriostatic effect with a free 24-h area under the concentration-time curve (AUC)/MIC ratio of 10 to 16 and produced 1 log of bacterial killing with a free 24-h AUC/MIC of 13 to 24. These exposures can be achieved with the current FDA-approved dosage regimens of intravenous minocycline.

Pharmacokinetics/Pharmacodynamics of Vaborbactam, a Novel Beta-Lactamase Inhibitor, in Combination with Meropenem.

Vaborbactam is a novel beta-lactamase inhibitor with activity against important beta-lactamases, in particular, serine carbapenemases, and is currently approved in combination with meropenem as Vabomere for the treatment of complicated urinary tract infections, including pyelonephritis. This combination is highly active against Gram-negative pathogens, especially Klebsiella pneumoniae carbapenemase (KPC)-producing carbapenem-resistant Enterobacteriaceae The objective of these studies was to evaluate vaborbactam pharmacokinetics (PK) and pharmacodynamics (PD) relationships for efficacy in a neutropenic mouse thigh infection model, as well as in an in vitro hollow-fiber infection model, in combination with a fixed exposure of meropenem using KPC-containing strains of Enterobacteriaceae For both models, the meropenem dosage regimen was designed to simulate a 2-g dose administered every eight hours (q8h) by 3-h infusion. Vaborbactam dosage regimens were designed to produce a wide range of 24-h areas under the concentration-time curves (AUCs) in the thigh infection model. However, for the hollow-fiber model, the AUCs were limited to values of 192, 320, or 550 mg · h/liter. In both the animal and in vitro models, the PK-PD parameter that best described the antibacterial activity of vaborbactam, when administered in combination with meropenem at exposures equivalent to 2 g dosed q8h by 3-h infusion in humans, was the 24-h free vaborbactam AUC/meropenem-vaborbactam (with vaborbactam at 8 mg/liter) MIC ratio. The magnitude of this ratio for bacteriostasis was 9 to 12 and the magnitude to observe a 1-log kill was 18 to 38. In addition, a magnitude greater than 24 suppressed the development of resistance in the in vitro hollow-fiber model.

Activity of Meropenem-Vaborbactam in Mouse Models of Infection Due to KPC-Producing Carbapenem-Resistant Enterobacteriaceae.

Meropenem-vaborbactam (Vabomere) is highly active against Gram-negative pathogens, especially Klebsiella pneumoniae carbapenemase (KPC)-producing, carbapenem-resistant Enterobacteriaceae The objective of these studies was to evaluate the efficacy of meropenem alone and in combination with vaborbactam in mouse thigh and lung infection models. Thighs or lungs of neutropenic mice were infected with KPC-producing carbapenem-resistant Enterobacteriaceae, with meropenem MICs ranging from ≤0.06 to 8 mg/liter in the presence of 8 mg/liter vaborbactam. Mice were treated with meropenem alone or meropenem in combination with vaborbactam every 2 h for 24 h to provide exposures comparable to 2-g doses of each component in humans. Meropenem administered in combination with vaborbactam produced bacterial killing in all strains tested, while treatment with meropenem alone either produced less than 0.5 log CFU/tissue of bacterial killing or none at all. In the thigh model, 11 strains were treated with the combination of meropenem plus vaborbactam (300 plus 50 mg/kg of body weight). This combination produced from 0.8 to 2.89 logs of bacterial killing compared to untreated controls at the start of treatment. In the lung infection model, two strains were treated with the same dosage regimen of meropenem and vaborbactam. The combination produced more than 1.83 logs of bacterial killing against both strains tested compared to untreated controls at the start of treatment. Overall, these data suggest that meropenem-vaborbactam may have utility in the treatment of infections due to KPC-producing carbapenem-resistant Enterobacteriaceae.

Activity of Meropenem-Vaborbactam against Carbapenem-Resistant Enterobacteriaceae in a Murine Model of Pyelonephritis.

The recently approved combination of meropenem and vaborbactam (Vabomere) is highly active against Gram-negative pathogens, especially Klebsiella pneumoniae carbapenemase (KPC)-producing, carbapenem-resistant Enterobacteriaceae We evaluated the efficacy of meropenem-vaborbactam against three clinically relevant isolates in a murine pyelonephritis model. The data indicate that the combination of meropenem and vaborbactam significantly increased bacterial killing compared to that with the untreated controls. These data suggest that this combination may have utility in the treatment of complicated urinary tract infections due to KPC-producing, carbapenem-resistant Enterobacteriaceae.

Activity of Simulated Human Dosage Regimens of Meropenem and Vaborbactam against Carbapenem-Resistant Enterobacteriaceae in an In Vitro Hollow-Fiber Model.

The objective of these studies was to evaluate the exposures of meropenem and vaborbactam that would produce antibacterial activity and prevent resistance development in carbapenem-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae strains when tested at an inoculum of 108 CFU/ml. Thirteen K. pneumoniae isolates, three Enterobacter cloacae isolates, and one Escherichia coli isolate were examined in an in vitro hollow-fiber model over 32 h. Simulated dosage regimens of 1 to 2 g of meropenem with 1 to 2 g of vaborbactam, with meropenem administered every 8 h by a 3-h infusion based on phase 1 or phase 3 patient pharmacokinetic data, were studied in the model. A dosage of 2 g of meropenem in combination with 2 g of vaborbactam was bactericidal against K. pneumoniae, E. cloacae, and E. coli strains, with meropenem-vaborbactam MICs of up to 8 mg/liter. When the vaborbactam exposure was adjusted to the levels observed in patients enrolled in phase 3 trials (24-h free AUC, ∼550 mg · h/liter, versus 320 mg · h/liter in the phase 1 studies), 2 g of meropenem with 2 g of vaborbactam was also bactericidal against strains with meropenem-vaborbactam MICs of 16 mg/liter. In addition, this level of vaborbactam also suppressed the development of resistance observed using phase 1 exposures. In this pharmacodynamic model, exposures similar to 2 g of meropenem in combination with 2 g of vaborbactam administered every 8 h by a 3-h infusion in phase 3 trials produced antibacterial activity and suppressed the development of resistance against carbapenem-resistant KPC-producing strains of Enterobacteriaceae.

Discovery of a Cyclic Boronic Acid ß-Lactamase Inhibitor (RPX7009) with Utility vs Class A Serine Carbapenemases.

The increasing dissemination of carbapenemases in Gram-negative bacteria has threatened the clinical usefulness of the ß-lactam class of antimicrobials. A program was initiated to discover a new series of serine ß-lactamase inhibitors containing a boronic acid pharmacophore, with the goal of finding a potent inhibitor of serine carbapenemase enzymes that are currently compromising the utility of the carbapenem class of antibacterials. Potential lead structures were screened in silico by modeling into the active sites of key serine ß-lactamases. Promising candidate molecules were synthesized and evaluated in biochemical and whole-cell assays. Inhibitors were identified with potent inhibition of serine carbapenemases, particularly the Klebsiella pneumoniae carbapenemase (KPC), with no inhibition of mammalian serine proteases. Studies in vitro and in vivo show that RPX7009 (9f) is a broad-spectrum inhibitor, notably restoring the activity of carbapenems against KPC-producing strains. Combined with a carbapenem, 9f is a promising product for the treatment of multidrug resistant Gram-negative bacteria.

Innate immune protection against infectious diseases by pulmonary administration of a phospholipid-conjugated TLR7 ligand.

Pulmonary administration of Toll-like receptor (TLR) ligands protects hosts from inhaled pathogens. However, systemic side effects induced by TLR stimulation limit clinical development. Here, a small-molecule TLR7 ligand conjugated with phospholipid, 1V270 (also designated TMX201), was tested for innate immune activation and its ability to prevent pulmonary infection in mice. We hypothesized that phospholipid conjugation would increase internalization by immune cells and localize the compound in the lungs, thus avoiding side effects due to systemic cytokine release. Pulmonary 1V270 administration increased innate cytokines and chemokines in bronchial alveolar lavage fluids, but neither caused systemic induction of cytokines nor B cell proliferation in distant lymphoid organs. 1V270 activated pulmonary CD11c+ dendritic cells, which migrated to local lymph nodes. However, there was minimal cell infiltration into the pulmonary parenchyma. Prophylactic administration of 1V270 significantly protected mice from lethal infection with Bacillus anthracis, Venezuelan equine encephalitis virus and H1N1 influenza virus. The maximum tolerated dose of 1V270 by pulmonary administration was 75 times the effective therapeutic dose. Therefore, pulmonary 1V270 treatment can protect the host from different infectious agents by stimulating local innate immune responses while exhibiting an excellent safety profile.

Levofloxacin reduces inflammatory cytokine levels in human bronchial epithelia cells: implications for aerosol MP-376 (levofloxacin solution for inhalation) treatment of chronic pulmonary infections.

Inflammation resulting from chronic bacterial infection in the lung contributes to long-term pulmonary complications in chronic pulmonary infections such as cystic fibrosis. Aerosol administration of levofloxacin as in the form of the investigational formulation MP-376 results in higher concentrations in lung tissues that are higher than those that can be attained with oral or intravenous dosing of levofloxacin. The objective of this study was to evaluate the effect of high concentrations of levofloxacin achieved with aerosol administration of MP-376 on proinflammatory cytokine secretion by immortalized human bronchial epithelia cells in vitro. Additionally, we investigated the potential mechanisms of the immunomodulatory effect of levofloxacin. In vitro studies in human lung epithelial cell lines showed that levofloxacin led to a dose-related reduction in IL-6 and IL-8 concentrations, with 300 µg mL(-1) resulting in the reduction of levels of IL-6 by fourfold and IL-8 by twofold (P<0.05); in contrast, tobramycin increased IL-6 levels by 50%, but had no effect on IL-8. Levofloxacin treatment did not affect the cytokine mRNA level and nuclear factor-κB-dependent promoter activity. These findings suggest that high concentrations of levofloxacin obtained in pulmonary tissues following the administration of aerosol MP-376 may provide additional benefits in patients with chronic pulmonary infections that are independent of its antibacterial properties.

Mucosal adjuvant activity of cholera toxin requires Th17 cells and protects against inhalation anthrax.

Cholera toxin (CT) elicits a mucosal immune response in mice when used as a vaccine adjuvant. The mechanisms by which CT exerts its adjuvant effects are incompletely understood. We show that protection against inhalation anthrax by an irradiated spore vaccine depends on CT-mediated induction of IL-17-producing CD4 Th17 cells. Furthermore, IL-17 is involved in the induction of serum and mucosal antibody responses by CT. Th17 cells induced by CT have a unique cytokine profile compared with those induced by IL-6 and TGF-beta, and their induction by CT requires cAMP-dependent secretion of IL-1beta and beta-calcitonin gene-related peptide by dendritic cells. These findings demonstrate that Th17 cells mediate mucosal adjuvant effects of CT and identify previously unexplored pathways involved in Th17 induction that could be targeted for development of unique mucosal adjuvants.

Efficacy of aerosol MP-376, a levofloxacin inhalation solution, in models of mouse lung infection due to Pseudomonas aeruginosa.

Progressive respiratory failure due to Pseudomonas aeruginosa is the leading cause of morbidity and mortality in patients with cystic fibrosis. The pulmonary delivery of antimicrobial agents provides high concentrations of drug directly to the site of infection and attains pharmacokinetic-pharmacodynamic indices exceeding those which can be achieved with systemic dosing. MP-376 is a new formulation of levofloxacin that enables the safe aerosol delivery of high concentrations of drug to pulmonary tissues. In vivo studies were conducted to demonstrate the efficacy of MP-376 in models of mouse pulmonary infection. The superiority of aerosol dosing over systemic dosing was demonstrated in models of both acute and chronic lung infection. In a model of acute lung infection, aerosol treatment with MP-376 once or twice daily reduced the lung bacterial load to a greater extent than aerosol tobramycin or aztreonam did when they were administered at similar or higher doses. The bacterial killing by aerosol MP-376 observed in the lung in the model of acute pulmonary infection translated to improved survival (P < 0.05). In a model of chronic pulmonary infection, aerosol MP-376 had antimicrobial effects superior to those of aztreonam (P < 0.05) and effects similar to those of tobramycin (P > 0.05). In summary, these data show that aerosol MP-376 has in vivo activity when it is used to treat acute and chronic lung infections caused by P. aeruginosa.

Structure-activity relationship studies of a novel series of anthrax lethal factor inhibitors.

We report on the identification of a novel small molecule inhibitor of anthrax lethal factor using a high-throughput screening approach. Guided by molecular docking studies, we carried out structure-activity relationship (SAR) studies and evaluated activity and selectivity of most promising compounds in in vitro enzyme inhibition assays and cellular assays. Selected compounds were further analyzed for their in vitro ADME properties, which allowed us to select two compounds for further preliminary in vivo efficacy studies. The data provided represents the basis for further pharmacology and medicinal chemistry optimizations that could result in novel anti-anthrax therapies.

ClpX contributes to innate defense peptide resistance and virulence phenotypes of Bacillus anthracis.

Bacillus anthracis is a National Institute of Allergy and Infectious Diseases Category A priority pathogen and the causative agent of the deadly disease anthrax. We applied a transposon mutagenesis system to screen for novel chromosomally encoded B. anthracis virulence factors. This approach identified ClpX, the regulatory ATPase subunit of the ClpXP protease, as essential for both the hemolytic and proteolytic phenotypes surrounding colonies of B. anthracis grown on blood or casein agar media, respectively. Deletion of clpX attenuated lethality of B. anthracis Sterne in murine subcutaneous and inhalation infection models, and markedly reduced in vivo survival of the fully virulent B. anthracis Ames upon intraperitoneal challenge in guinea pigs. The extracellular proteolytic activity dependent upon ClpX function was linked to degradation of cathelicidin antimicrobial peptides, a front-line effector of innate host defense. B. anthracis lacking ClpX were rapidly killed by cathelicidin and alpha-defensin antimicrobial peptides and lysozyme in vitro. In turn, mice lacking cathelicidin proved hyper-susceptible to lethal infection with wild-type B. anthracis Sterne, confirming cathelicidin to be a critical element of innate defense against the pathogen. We conclude that ClpX is an important factor allowing B. anthracis to subvert host immune clearance mechanisms, and thus represents a novel therapeutic target for prevention or therapy of anthrax, a foremost biodefense concern.

Anthrax toxins inhibit neutrophil signaling pathways in brain endothelium and contribute to the pathogenesis of meningitis.

BACKGROUND: Anthrax meningitis is the main neurological complication of systemic infection with Bacillus anthracis approaching 100% mortality. The presence of bacilli in brain autopsies indicates that vegetative bacteria are able to breach the blood-brain barrier (BBB). The BBB represents not only a physical barrier but has been shown to play an active role in initiating a specific innate immune response that recruits neutrophils to the site of infection. Currently, the basic pathogenic mechanisms by which B. anthracis penetrates the BBB and causes anthrax meningitis are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Using an in vitro BBB model, we show for the first time that B. anthracis efficiently invades human brain microvascular endothelial cells (hBMEC), the single cell layer that comprises the BBB. Furthermore, transcriptional profiling of hBMEC during infection with B. anthracis revealed downregulation of 270 (87%) genes, specifically key neutrophil chemoattractants IL-8, CXCL1 (Gro alpha) and CXCL2 (Gro beta), thereby strongly contrasting hBMEC responses observed with other meningeal pathogens. Further studies using specific anthrax toxin-mutants, quantitative RT-PCR, ELISA and in vivo assays indicated that anthrax toxins actively suppress chemokine production and neutrophil recruitment during infection, allowing unrestricted proliferation and dissemination of the bacteria. Finally, mice challenged with B. anthracis Sterne, but not the toxin-deficient strain, developed meningitis. CONCLUSIONS/SIGNIFICANCE: These results suggest a significant role for anthrax toxins in thwarting the BBB innate defense response promoting penetration of bacteria into the central nervous system. Furthermore, establishment of a mouse model for anthrax meningitis will aid in our understanding of disease pathogenesis and development of more effective treatment strategies.

In vivo efficacy of a phosphodiester TLR-9 aptamer and its beneficial effect in a pulmonary anthrax infection model.

Immunostimulatory oligonucleotide (ISS-ODN) used as adjuvants are commonly modified with phosphorothioate (PS). The PS backbone prevents nuclease degradation, but confers undesired side effects, including systemic cytokine release. Previously, R10-60, a phosphodiester (PO) ISS-ODN, was structurally optimized as an intracellular Toll-like receptor-9 agonist. Here intravenous, intradermal and intranasal administration of PO R10-60 elicit local or adaptive immune responses with minimal systemic effects compared to a prototypic PS ISS-ODN in mice. Furthermore, prophylactic intranasal administration of PO R10-60 significantly delayed death in mice exposed to respiratory anthrax comparable to the PS ISS-ODN. The pattern of cytokine release suggested that early IL-1beta production might contribute to this protective effect, which was replicated with recombinant IL-1beta injections during infection. Hence, the transient effects from a PO TLR-9 agonist may be beneficial for protection in a bacterial bioterrorism attack, by delaying the onset of systemic infection without the induction of a cytokine syndrome.