Neutropenic Thigh Infection Model Research Articles

Optimization of the Central α-Amino Acid in Cystobactamids to the Broad-Spectrum, Resistance-Breaking Antibiotic CN-CC-861.

Cystobactamids have a unique oligoarylamide structure and exhibit broad-spectrum activity against Gram-negative and Gram-positive bacteria. In this study, the central α-amino acid of the cystobactamid scaffold was modified to address the relevance of stereochemistry, hydrogen bonding and polarity by 33 derivatives. As demonstrated by three matched molecular pairs, l-amino acids were preferred over d-amino acids. A rigidification to a six-membered system stabilized the bioactive conformation for the on-target Escherichia coli gyrase, but did not improve antimicrobial activity. Compound CN-CC-861, carrying a propargyl side chain, had more than 16-fold lower minimal inhibitory concentration (MIC) values against Enterococcus faecalis, Staphylococci and Acinetobacter strains, compared to known analogues. Moreover, CN-CC-861 retained activity against multidrug-resistant enterococci, displayed strong bactericidal activity, moderate-low frequencies of resistance and in vivo efficacy in a neutropenic thigh infection model with E. coli. Overall, the findings will guide the design of new promising structures with higher activities and broader spectrum.

Identification of coumarin – benzimidazole hybrids as potential antibacterial agents: Synthesis, in vitro and in vivo biological assessment, and ADMET prediction

The direct-linked coumarin-benzimidazole hybrids, featuring aryl and n-butyl substituents at the N1-position of benzimidazole were synthesized through a Knoevenagel condensation reaction. This reaction involved the condensation of 1,2-diaminobenzene derivatives with coumarin-3-carboxylic acids in the presence of polyphosphoric acid (PPA) at 154 °C. The in vitro antibacterial potency of the hybrid molecules against different gram-positive and gram-negative bacterial strains led to the identification of the hybrids 6m and 6p with a MIC value of 6.25 μg/mL against a gram-negative bacterium, Klebsiella pneumonia ATCC 27736. Cell viability studies on THP-1 cells demonstrated that the compounds 6m and 6p were non-toxic at a concentration of 50 µM. Furthermore, in vivo efficacy studies using a murine neutropenic thigh infection model revealed that both compounds significantly reduced bacterial (Klebsiella pneumonia ATCC 27736) counts (more than 2 log) compared to the control group. Additionally, both compounds exhibited favorable physicochemical properties and drug-likeness characteristics. Consequently, these compounds hold promise as lead candidates for further development of effective antibacterial drugs.

Membrane-targeting, ultrashort lipopeptide acts as an antibiotic adjuvant and sensitizes MDR gram-negative pathogens toward narrow-spectrum antibiotics

Globally, infections due to multi-drug resistant (MDR) Gram-negative bacterial (GNB) pathogens are on the rise, negatively impacting morbidity and mortality, necessitating urgent treatment alternatives. Herein, we report a detailed bio-evaluation of an ultrashort, cationic lipopeptide ‘SVAP9I’ that demonstrated potent antibiotic activity and acted as an adjuvant to potentiate existing antibiotic classes towards GNBs. Newly synthesized lipopeptides were screened against ESKAPE pathogens and cytotoxicity assays were performed to evaluate the selectivity index (SI). SVAP9I exhibited broad-spectrum antibacterial activity against critical MDR-GNB pathogens including members of Enterobacteriaceae (MIC 4–8 mg/L), with a favorable CC50 value of ≥100 mg/L and no detectable resistance even after 50th serial passage. It demonstrated fast concentration-dependent bactericidal action as determined via time-kill analysis and also retained full potency against polymyxin B-resistant E. coli, indicating distinct mode of action. SVAP9I targeted E. coli's outer and inner membranes by binding to LPS and phospholipids such as cardiolipin and phosphatidylglycerol. Membrane damage resulted in ROS generation, depleted intracellular ATP concentration and a concomitant increase in extracellular ATP. Checkerboard assays showed SVAP9I's synergism with narrow-spectrum antibiotics like vancomycin, fusidic acid and rifampicin, potentiating their efficacy against MDR-GNB pathogens, including carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO critical priority pathogen. In a murine neutropenic thigh infection model, SVAP9I and rifampicin synergized to express excellent antibacterial efficacy against MDR-CRAB outcompeting polymyxin B. Taken together, SVAP9I's distinct membrane-targeting broad-spectrum action, lack of resistance and strong in vitro andin vivopotency in synergism with narrow spectrum antibiotics like rifampicin suggests its potential as a novel antibiotic adjuvant for the treatment of serious MDR-GNB infections.

Animal Models in Regulatory Breakpoint Determination: Review of New Drug Applications of Approved Antibiotics from 2014-2022.

We sought to better understand the utility and role of animal models of infection for Food and Drug Administration (FDA)-approved antibiotics for the indications of community-, hospital-acquired-, and ventilator-associated bacterial pneumonia (CABP, HABP, VABP), complicated urinary tract infection (cUTI), complicated intra-abdominal infection (cIAI), and acute bacterial skin and structural infections (ABSSSIs). We reviewed relevant documents from new drug applications (NDA) of FDA-approved antibiotics from 2014-2019 for the above indications. Murine neutropenic thigh infection models supported the choice of a pharmacokinetic-pharmacodynamic (PKPD) target in 11/12 NDAs reviewed. PKPD targets associated with at least a 1-log bacterial decrease were commonly considered ideal (10/12 NDAs) to support breakpoints. Plasma PK, as opposed to organ specific PK, was generally considered most reliable for PKPD correlation. Breakpoint determination was multi-disciplinary, accounting at minimum for epidemiologic cutoffs, non-clinical PKPD, clinical exposure-response and clinical efficacy. Non-clinical PKPD targets in combination with probability of target attainment (PTA) analyses generated breakpoints that were consistent with epidemiologic cutoffs and clinically derived breakpoints. In 6/12 NDAs, there was limited data to support clinically derived breakpoints, and hence the non-clinical PKPD targets in combination with PTA analyses played a heightened role in the final breakpoint determination. Sponsor and FDA breakpoint decisions were in general agreement. Disagreement may have arisen from differences in the definition of the optimal PKPD index or the ability to extrapolate protein binding from animals to humans. Overall, murine neutropenic thigh infection models supported the reviewed NDAs by providing evidence of pre-clinical efficacy and PKPD target determination, and played, in combination with PTA analysis, a significant role in breakpoint determination for labeling purposes.

Indole-based aryl sulfides target the cell wall of Staphylococcus aureus without detectable resistance.

Sulfur-containing classes of the scaffold "Arylthioindoles" have been evaluated for antibacterial activity; they demonstrated excellent potency against methicillin-resistant Staphylococcus aureus (MRSA) as well as against vancomycin-resistant strains and a panel of clinical isolates of resistant strains. In this study, we have elucidated the mechanism of action of lead compounds, wherein they target the cell wall of S. aureus. Further, S. aureus failed to develop resistance against two lead compounds tested in a serial passage experiment in the presence of the compounds over a period of 40 days. Both the compounds demonstrated comparable in vivo efficacy with vancomycin in a neutropenic mice thigh infection model. The results of these antibacterial activities emphasize the excellent potential of thioethers for developing novel antibiotics and may fill in as a target for the adjustment of accessible molecules to develop new powerful antibacterial agents with fewer side effects.

Naturally Derived Malabaricone B as a Promising Bactericidal Candidate Targeting Multidrug-Resistant Staphylococcus aureus also Possess Synergistic Interactions with Clinical Antibiotics.

The emergence of multidrug-resistant (MDR) superbugs underlines the urgent need for innovative treatment options to tackle resistant bacterial infections. The clinical efficacy of natural products directed our efforts towards developing new antibacterial leads from naturally abundant known chemical structures. The present study aimed to explore an unusual class of phenylacylphenols (malabaricones) from Myristicamalabarica as antibacterial agents. In vitro antibacterial activity was determined via broth microdilution, cell viability, time-kill kinetics, biofilm eradication, intracellular killing, and checkerboard assays. The efficacy was evaluated in vivo in murine neutropenic thigh and skin infection models. Confocal and SEM analyses were used for mechanistic studies. Among the tested isolates, malabaricone B (NS-7) demonstrated the best activity against S. aureus with a favorable selectivity index and concentration-dependent, rapid bactericidal killing kinetics. It displayed equal efficacy against MDR clinical isolates of S. aureus and Enterococci, efficiently clearing S. aureus in intracellular and biofilm tests, with no detectable resistance. In addition, NS-7 synergized with daptomycin and gentamicin. In vivo, NS-7 exhibited significant efficacy against S. aureus infection. Mechanistically, NS-7 damaged S. aureus membrane integrity, resulting in the release of extracellular ATP. The results indicated that NS-7 can act as a naturally derived bactericidal drug lead for anti-staphylococcal therapy.

Neutropenic Rat Thigh Infection Model for Evaluation of the Pharmacokinetics/Pharmacodynamics of Anti-Infectives.

The neutropenic mouse infection model is extensively used to characterize the pharmacokinetics/pharmacodynamics (PK/PD) of anti-infective agents. However, it is difficult to evaluate agents following intravenous (i.v.) infusions using this model. Furthermore, in many drug discovery programs, lead identification and optimization is performed in rats, and pharmacology is performed in mice. Alternative models of infection are needed for robust predictions of PK/PD in humans. The rat is an alternative model of infection which can overcome the shortcomings of the mouse model. However, the rat neutropenic thigh infection (NTI) model has not been adequately characterized for evaluation of the PK/PD of anti-infectives. The aim of this study was to characterize the PK/PD of ciprofloxacin against bacterial pathogens in a rat NTI model. We studied the PK/PD relationships of ciprofloxacin against wild-type Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae in neutropenic Wistar rats following administration of 10, 30, and 100 mg/kg as single intravenous boluses and 30- and 60-min infusions. The PK/PD of ciprofloxacin against all four pathogens was AUC/MIC dependent and independent of the duration of administration at 10, 30, and 100 mg/kg. At human-equivalent rat doses, the PK/PD targets of ciprofloxacin achieved in rats for microbiological cure were similar to those reported in human patients. The neutropenic rat thigh infection model can be used to evaluate anti-infective agents intended to be administered as infusions in the clinic, and it complements the mouse model, increasing the robustness of PK/PD predictions in humans. IMPORTANCE Many antibiotics are administered as intravenous infusions in the clinic, especially in intensive care units. Anti-infective drug discovery companies develop clinical candidates that are intended to be administered as i.v. infusions in the clinic. However, there are no well-characterized models with which they can evaluate the PK/PD of the candidates following i.v. infusions. The neutropenic rat thigh infection model reported in this study helps in evaluating anti-infective agents that are intended to be administered as i.v. infusions in the clinic. The rat model is useful for simulating the clinical conditions for i.v. infusions for treatment of infections, such as acute bacterial skin and skin structure, lung, and urinary tract infections. This model is predictive of efficacy in humans and can serve as an additional confirmatory model, along with the mouse model, for determining the proof of concept and for making robust predictions of efficacy in humans.

In vitro and in vivo anti-inflammatory, antibacterial and pharmacokinetic properties of baicalein

Baicalein is a bioactive flavone originally isolated from the roots of Scutellaria baicalensis, Scutellariala teriflora and Oroxylum indicum. The in vitro and in vivo anti-inflammatory and antibacterial properties of baicalein and pharmacokinetics after its single intramuscular administration were studied in Wistar rats. The in vitro anti-inflammatory activity of baicalein (10, 50 and 100 µM) was tested for its ability to inhibit the COX-2 enzyme by measuring PGE2 levels and determination of nitric oxide (NO) production in lipopolysaccharide (LPS) treated RAW 264.7 macrophage cells, in which baicalein was found to have significant inhibition of NO and PGE2 production in RAW 264.7 macrophage cells as compared with the LPS control group. The in vivo anti-inflammatory activity of baicalein (200 mg/kg) was assessed using the carrageenan-induced rat paw oedema model, following intramuscular injection. A significant percentage of inhibition of oedema volume was observed when compared with the carrageenan control group. In vitro and in vivo antibacterial activities of baicalein were determined by the micro broth dilution technique and neutropenic rat thigh infection model, wherein baicalein did not show any antibacterial property. Concentrations of baicalein were determined in rat plasma by high performance liquid chromatography (HPLC) after a single intramuscular administration at a dose of 200 mg/kg body weight, in which the mean peak plasma drug concentration (Cmax) of 0.77 ± 0.02 μg/mL was achieved at 0.08 h. The mean elimination half-life (t½β), the apparent volume of distribution (Vd(area)), total body clearance (Cl(B)) and mean residence time (MRT) were observed as 0.63 ± 0.06 h, 601.03 ± 28.18 L/kg, 677.39 ± 35.36 L/h/kg and 0.76 ± 0.06 h, respectively. Conclusively, in the present study, baicalein did not show in vitro or in vivo antibacterial property, but proved to have good anti-inflammatory activity. The available anti-inflammatory drugs have proved to have side effects in veterinary and human therapeutics. In this situation, baicalein may become an effective alternative to non-steroidal anti-inflammatory drugs and should also be studied in target animal species. Further research should be carried out to improve the solubility and bioavailability of baicalein through injectable routes.

Towards Translation of PqsR Inverse Agonists: From In Vitro Efficacy Optimization to In Vivo Proof-of-Principle.

Pseudomonas aeruginosa (PA) is an opportunistic human pathogen, which is involved in a wide range of dangerous infections. It develops alarming resistances toward antibiotic treatment. Therefore, alternative strategies, which suppress pathogenicity or synergize with antibiotic treatments are in great need to combat these infections more effectively. One promising approach is to disarm the bacteria by interfering with their quorum sensing (QS) system, which regulates the release of various virulence factors as well as biofilm formation. Herein, this work reports the rational design, optimization, and in-depth profiling of a new class of Pseudomonas quinolone signaling receptor (PqsR) inverse agonists. The resulting frontrunner compound features a pyrimidine-based scaffold, high in vitro and in vivo efficacy, favorable pharmacokinetics as well as clean safety pharmacology characteristics, which provide the basis for potential pulmonary as well as systemic routes of administration. An X-ray crystal structure in complex with PqsR facilitated further structure-guided lead optimization. The compound demonstrates potent pyocyanin suppression, synergizes with aminoglycoside antibiotic tobramycin against PA biofilms, and is active against a panel of clinical isolates from bronchiectasis patients. Importantly, this in vitro effect translated into in vivo efficacy in a neutropenic thigh infection model in mice providing a proof-of-principle for adjunctive treatment scenarios.

Pharmacokinetics and Pharmacodynamics (PK/PD) of Corallopyronin A against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a World Health Organization’s high priority pathogen organism, with an estimated > 100,000 deaths worldwide in 2019. Thus, there is an unmet medical need for novel and resistance-breaking anti-infectives. The natural product Co-rallopyronin A (CorA), currently in preclinical development for filariasis, is efficacious against MRSA in vitro. In this study, we evaluated the pharmacokinetics of CorA after dosing in mice. Furthermore, we determined compound concentrations in target compartments, such as lung, kidney and thigh tissue, using LC-MS/MS. Based on the pharmacokinetic results, we evaluated the pharmacodynamic profile of CorA using the standard neutropenic thigh and lung infection models. We demonstrate that CorA is effective in both standard pharmacodynamic models. In addition to reaching effective levels in the lung and muscle, CorA was detected at high levels in the thigh bone. The data presented herein encourage the further exploration of the additional CorA indications treatment of MRSA- and methicillin-sensitive S. aureus- (MSSA) related infections.

Influence of tramadol on bacterial burden in the standard neutropenic thigh infection model

The neutropenic thigh infection model is one of the standard models in pharmacokinetic/ pharmacodynamic (PK/PD) characterization of novel antibacterials which are urgently needed due to the rise of antimicrobial resistance. The model enables to investigate PK/PD parameters crucial for translation of animal results towards humans. However, the neutropenic thigh infection model can result in moderate to severe discomfort of the animals, especially when high inocula are used. Tramadol has been proven to reduce pain effectively. This study investigates if tramadol influences the bacterial burden in the primary organ, the thighs, and organs affected by secondary seeding. Therefore, several strains of the ESKAPE pathogens, namely S. aureus, P. aeruginosa, K. pneumoniae, E. coli, A. baumannii and E. faecalis were examined. It was shown that tramadol did not influence the bacterial burden neither in thighs nor in organs affected by secondary seeding for the strains of E. faecalis, S. aureus, P. aeruginosa, K. pneumoniae and E.coli tested here, whereas secondary seeding seemed to be affected by tramadol for the tested strain of A. baumannii. Consequently, it was demonstrated that tramadol is an option to reduce discomfort in the untreated group for the strains of five out of the six tested ESKAPE pathogens and, thereby, contributes to the refinement of one of the standard PK/PD models.

Pyrvinium pamoate potentiates levofloxacin against levofloxacin-resistant Staphylococcus aureus.

Background: Drug repurposing is a viable approach to expediting the tedious conventional drug discovery process, given rapidly increasing bacterial resistance. In this context, we have repurposed pyrvinium pamoate (PP) for its antibacterial activity against Staphylococcus aureus. Methods: US FDA-approved non-antibiotics were screened against clinically relevant bacterial pathogens to identify antibacterials. The hits were further evaluated utilizing a variety of preclinical parameters, following which in vivo efficacy was estimated in isolationand in combination in a murine neutropenic thigh infection model. Result: The screening identified PP exhibiting potent activity against S. aureus along with concentration-dependent killing. PP also showed a post-antibiotic effect of >22h and significantly eradicated preformed S. aureus biofilms and intracellular S. aureus at 1×and 5× MIC, respectively. PP synergized with levofloxacin both in vitro and in vivo, resulting in ∼1.5 and ∼0.5 log10CFU/g reduction against susceptible and resistant S. aureus infections, respectively, as compared with untreated control. Conclusion: Pyrvinium potentiates levofloxacin against levofloxacin-resistant S. aureus.

Pharmacodynamic Thresholds for Beta-Lactam Antibiotics: A Story of Mouse Versus Man.

Beta-lactams remain a critical member of our antibiotic armamentarium and are among the most commonly prescribed antibiotic classes in the inpatient setting. For these agents, the percentage of time that the free concentration remains above the minimum inhibitory concentration (%fT > MIC) of the pathogen has been shown to be the best predictor of antibacterial killing effects. However, debate remains about the quantity of fT > MIC exposure needed for successful clinical response. While pre-clinical animal based studies, such as the neutropenic thigh infection model, have been widely used to support dosing regimen selection for clinical development and susceptibility breakpoint evaluation, pharmacodynamic based studies in human patients are used validate exposures needed in the clinic and for guidance during therapeutic drug monitoring (TDM). For the majority of studied beta-lactams, pre-clinical animal studies routinely demonstrated the fT > MIC should exceed approximately 40–70% fT > MIC to achieve 1 log reductions in colony forming units. In contrast, clinical studies tend to suggest higher exposures may be needed, but tremendous variability exists study to study. Herein, we will review and critique pre-clinical versus human-based pharmacodynamic studies aimed at determining beta-lactam exposure thresholds, so as to determine which targets may be best suited for optimal dosage selection, TDM, and for susceptibility breakpoint determination. Based on our review of murine and clinical literature on beta-lactam pharmacodynamic thresholds, murine based targets specific to each antibiotic are most useful during dosage regimen development and susceptibility breakpoint assessment, while a range of exposures between 50 and 100% fT > MIC are reasonable to define the beta-lactam TDM therapeutic window for most infections.

Minocycline pharmacodynamics against Stenotrophomonas maltophilia in the neutropenic murine infection model: implications for susceptibility breakpoints.

Minocycline displays high susceptibility rates against Stenotrophomonas maltophilia at the current breakpoint of 4 mg/L. However, no pharmacodynamic data are available to guide dosing or justify this breakpoint. The murine neutropenic thigh infection model was utilized to determine minocycline pharmacodynamics against four S. maltophilia through dose ranging and fractionation studies. The efficacy of a human simulated regimen (HSR) of 100 mg IV q12h was tested against 17 isolates with a range of minocycline MICs. Monte Carlo simulation was employed to assess the PTA for achieving defined pharmacodynamic thresholds in critically ill patients. The pharmacodynamic index best correlated with reductions in cfu was fAUC/MIC (R2 = 0.376). The composite fAUC/MIC required for stasis and 1 log10 reduction was 9.6 and 23.6, respectively. The minocycline 100 mg q12h HSR yielded no bacterial reduction at MICs ≥1 mg/L and mixed efficacy at 0.5 mg/L. Monte Carlo simulation of minocycline 200 mg IV q12h achieved the 1 log10 kill threshold with PTAs of 93% and 51.7% at MICs of 0.5 and 1 mg/L, respectively, but 0.1% at the current breakpoint of 4 mg/L. Clinically utilized minocycline dosing regimens fail to reach exposures predicted to be efficacious against S. maltophilia in critically ill patients at the current susceptibility breakpoint.

1103. Minocycline (MIN) Pharmacodynamics (PD) against Stenotrophomonas maltophilia (STM) in a Neutropenic Murine Thigh Infection Model

BackgroundAntibiotic treatment options for serious STM infections are limited. MIN displays in vitro activity against STM; however, limited data supports optimal dosing for STM. Herein, we employed the murine neutropenic thigh infection model to assess MIN PD against STM.MethodsFour clinical STM isolates with MIN MICs 0.25 – 1 mg/L were included. Both thighs of neutropenic ICR mice were inoculated with bacterial suspensions of 107 colony forming units (CFU)/mL. Mice received uranyl nitrate on Day -3 to provide predictable renal impairment. Two hours after inoculation, MIN or control was administered subcutaneously. Pharmacokinetic (PK) studies of 2.5, 25, 50, and 100 mg/kg were conducted. Previously reported protein binding of 78.1% was used to define free exposure. Dose ranging studies were conducted on all STM to assess in vivo activity over a range of MIN exposures. MIN total daily doses (TDD) of 10, 20, and 50 mg/kg were fractionated q24h, q12h, and q6h against a single STM to determine the PD index best correlated with reductions in CFU/mL. Efficacy was measured in log10CFU/thigh at 24h compared with 0h controls. Composite CFU data were fitted to an Emax model to determine the fAUC/MIC exposure for stasis and 1 log10 reduction.ResultsMIN PK was linear up to 50 mg/kg and well described by a 1 compartment model with first order absorption and elimination. Mean PK parameters across the linear range were: Vd, 2.97 L/kg; K01, 10.62 1/h; and K10, 0.35 1/h. Mean ± SD bacterial burden at 0h across all isolates was 6.17±0.20 log10CFU/thigh. In 24h controls, bacterial growth was 7.90±0.85 log10CFU/thigh. A dose response was observed across all isolates using TDD of 2-300 mg/kg. PD indices correlated with CFU reductions as follows: fAUC/MIC (R2=0.613), fCmax/MIC (R2=0.590), and %fT >MIC (R2=0.504). The fAUC/MIC needed for stasis and 1 log10 reduction at 24h was 9.6 and 23.6, respectively. ConclusionThese are the first data describing MIN PD against STM. Against these STM, MIN fAUC/MIC was the PD index best correlated with CFU reductions. The exposure thresholds defined in this study will be useful in designing optimal MIN dosing regimens for treating STM infections and re-assessment of the current susceptibility breakpoint. The study was funded under FDA Contract 75F40120C00171.Disclosures David P. Nicolau, PharmD, Abbvie, Cepheid, Merck, Paratek, Pfizer, Wockhardt, Shionogi, Tetraphase (Other Financial or Material Support, I have been a consultant, speakers bureau member, or have received research funding from the above listed companies.) Joseph L. Kuti, PharmD, Allergan (Speaker’s Bureau)BioMérieux (Consultant, Research Grant or Support, Speaker’s Bureau)Contrafect (Scientific Research Study Investigator)GSK (Consultant)Merck (Research Grant or Support)Paratek (Speaker’s Bureau)Roche Diagnostics (Research Grant or Support)Shionogi (Research Grant or Support)Summit (Scientific Research Study Investigator)

65. In Vivo Efficacy of Human Simulated Minocycline (MIN) against Stenotrophomonas maltophilia (STM)

BackgroundThe current susceptibility breakpoint for MIN against STM is 4mg/L, yielding >99% of isolates susceptible. Unfortunately, there are limited pre-clinical and clinical data to support this breakpoint for STM. The purpose of this study was to evaluate the efficacy of a MIN human simulated regimen (HSR) against STM across a wide range of MICs in the murine neutropenic thigh model.MethodsClinical STM with modal MIN MICS of 0.25-8mg/L were included. Confirmatory pharmacokinetic (PK) studies were performed in infected neutropenic mice to develop a MIN HSR providing an area under the curve (AUC) and maximum concentration (Cmax) exposure similar to MIN 100mg intravenous (IV) q12h at steady-state based on PK parameters from critically ill adult patients. The murine neutropenic thigh infection model was utilized to examine the antibacterial effects of the confirmed MIN HSR against 17 STM. Both thighs of neutropenic ICR mice were inoculated with bacterial suspensions of 107 colony forming units (CFU)/mL. Two hours after inoculation, the MIN HSR was administered subcutaneously (SC) over 24h. Control mice received normal saline. Efficacy was measured as the change in log10CFU/thigh at 24h compared with 0h controls.ResultsMIN 22, 10, 14, and 10mg/kg dosed SC at 0, 6, 12, and 18h best recapitulated the human Cmax and AUC profile. Mean ± standard deviation bacterial burden at 0h across all isolates was 6.03±0.32 log10CFU/thigh. Bacterial growth was 1.35±0.68 log10CFU/thigh in 24h controls. Six of 7 isolates (86%) with MIC ≤ 0.5mg/L achieved 1-log kill with MIN HSR (-1.44±1.37 log10CFU/thigh). All STM with MIC ≥ 1mg/L experienced bacterial growth (1.18±0.79 log10CFU/thigh) (Figure).Figure. Efficacy of a minocycline human simulated exposure of 100mg intravenous Q12h in the murine neutropenic thigh model against 17 clinical Stenotrophomonas maltophilia isolates ConclusionThese data describe the in vivo efficacy of a MIN HSR with exposures similar to MIN 100mg IV q12h in critically ill adults. Lack of antibacterial activity against STM with MICs ≥ 1mg/L justifies a reassessment of the current susceptibility breakpoint. The study was funded under FDA Contract 75F40120C00171. Disclosures David P. Nicolau, PharmD, Abbvie, Cepheid, Merck, Paratek, Pfizer, Wockhardt, Shionogi, Tetraphase (Other Financial or Material Support, I have been a consultant, speakers bureau member, or have received research funding from the above listed companies.) Joseph L. Kuti, PharmD, Allergan (Speaker’s Bureau)BioMérieux (Consultant, Research Grant or Support, Speaker’s Bureau)Contrafect (Scientific Research Study Investigator)GSK (Consultant)Merck (Research Grant or Support)Paratek (Speaker’s Bureau)Roche Diagnostics (Research Grant or Support)Shionogi (Research Grant or Support)Summit (Scientific Research Study Investigator)

Levofloxacin pharmacodynamics against Stenotrophomonasmaltophilia in a neutropenic murine thigh infection model: implications for susceptibility breakpoint revision.

Levofloxacin displays in vitro activity against Stenotrophomonas maltophilia (STM); however, current susceptibility breakpoints are supported by limited data. We employed the murine neutropenic thigh infection model to assess levofloxacin pharmacodynamics against STM. Twenty-six clinical STM were studied using the neutropenic murine thigh infection model. Human simulated regimens (HSR) of levofloxacin 750 mg q24h were administered over 24 h. Efficacy was measured as the change in log10 cfu/thigh at 24 h compared with 0 h. Composite cfu data were fitted to an Emax model to determine the fAUC/MIC needed for stasis and 1 log10 reduction at 24 h. Monte Carlo simulation was performed to determine PTA. Levofloxacin MICs ranged from 0.5-8 mg/L. Mean bacterial burden at 0 h was 6.21 ± 0.20 log10 cfu/thigh. In the 24 h controls, bacterial growth was 1.64 ± 0.66 log10 cfu/thigh. In isolates with levofloxacin MICs ≤1, 2 and ≥4 mg/L, changes in bacterial density following levofloxacin HSR were -1.66 ± 0.89, 0.13 ± 0.97 and 1.54 ± 0.43 log10 cfu/thigh, respectively. The Emax model demonstrated strong agreement between fAUC/MIC and change in bacterial density (R2 = 0.82). The fAUC/MIC exposure needed for stasis and 1 log10 reduction was 39.9 and 54.9, respectively. PTAs for the 1 log10 reduction threshold were 95.8, 72.2, and 26.6% at MICs of 0.5, 1 and 2 mg/L, respectively. These are the first data to describe fAUC/MIC thresholds predictive of cfu reductions for levofloxacin against STM. Due to poor in vivo efficacy and PTA at MICs ≥2 mg/L, reassessment of the current susceptibility breakpoint is warranted.

Pharmacodynamic evaluation of meropenem, cefepime, or aztreonam combined with a novel β-lactamase inhibitor, nacubactam, against carbapenem-resistant and/or carbapenemase-producing Klebsiella pneumoniae and Escherichia coli using a murine thigh-infection model

BackgroundCarbapenem-resistant Enterobacterales (CRE) and carbapenemase-producing Enterobacterales (CPE) are difficult to treat and are a serious public health threat. Nacubactam (NAC) is a novel non-β-lactam diazabicyclooctane β-lactamase inhibitor with in vitro activity against some Enterobacterales expressing classes of β-lactamases. MethodsThe antimicrobial efficacy of meropenem (MEM), cefepime (FEP), and aztreonam (ATM), each in combination with NAC, were assessed in vitro and in vivo against Klebsiella pneumoniae and Escherichia coli. Ten isolates, including CRE and/or CPE with β-lactamase genes, were used in this study. The relationship between phenotype and in vivo efficacy was assessed in a murine neutropenic thigh-infection model. Efficacy was determined by the change in bacterial quantity. ResultsThe results of the in vitro study showed the minimum inhibitory concentrations of the combination of NAC with either MEM, FEP, or ATM in a 1:1 ratio were 2 to >128-fold lower than those of MEM, FEP, or ATM alone against CRE+ isolates. In addition, combinations of β-lactams and NAC administered in the murine thigh-infection model showed greater efficacy against CRE+/CPE+, CRE+/CPE-, and CRE-/CPE+ isolates harboring various β-lactamase genes (IMP-1, IMP-6, KPC, DHA-1, or OXA-48) compared with MEM, FEP, ATM, and NAC alone. ConclusionMEM, FEP, or ATM in combination with NAC showed potent in vivo antimicrobial activity in a murine thigh-infection model caused by K. pneumoniae and E. coli, including CRE and/or CPE isolates. These findings indicate that these combinations of β-lactams and NAC are potential candidates for the treatment of CRE and/or CPE infections.

Synergistic Effect of a Pleuromutilin Derivative with Tetracycline against Streptococcus suis In Vitro and in the Neutropenic Thigh Infection Model.

Tetracycline (TET) has been widely used in the treatment of Streptococcus suis (S. suis) infection. However, it was found that the efficacy of many antibiotics in S. suis decreased significantly, especially tetracycline. In this study, GML-12 (a novel pleuromutilin derivative) was used in combination with TET against 12 S. suis isolates. In the checkerboard assay, the TET/GML-12 combination exhibited synergistic and additive effects against S. suis isolates (n = 12). In vitro time-killing assays and in vivo therapeutic experiments were used to confirm the synergistic effect of the TET/GML-12 combination against S. suis strains screened based on an FICI ≤ 0.5. In time-killing assays, the TET/GML-12 combination showed a synergistic effect or an additive effect against three isolates with a bacterial reduction of over 2.4-log10 CFU/mL compared with the most active monotherapy. Additionally, the TET/GML-12 combination displayed potent antimicrobial activity against four isolates in a mouse thigh infection model. These results suggest that the TET/GML-12 combination may be a potential therapeutic strategy for S. suis infection.

Pharmacokinetics-Pharmacodynamics of Enmetazobactam Combined with Cefepime in a Neutropenic Murine Thigh Infection Model.

Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae are classified as critical priority pathogens, with extended-spectrum β-lactamases (ESBLs) as principal resistance determinants. Enmetazobactam (formerly AAI101) is a novel ESBL inhibitor developed in combination with cefepime for empirical treatment of serious Gram-negative infections in settings where ESBLs are prevalent. Cefepime-enmetazobactam has been investigated in a phase 3 trial in patients with complicated urinary tract infections or acute pyelonephritis. This study examined pharmacokinetic-pharmacodynamic (PK-PD) relationships of enmetazobactam, in combination with cefepime, for ESBL-producing isolates of Klebsiella pneumoniae in 26-h murine neutropenic thigh infection models. Enmetazobactam dose fractionation identified the time above a free threshold concentration (fT > CT ) as the PK-PD index predictive of efficacy. Nine ESBL-producing isolates of K. pneumoniae, resistant to cefepime and piperacillin-tazobactam, were included in enmetazobactam dose-ranging studies. The isolates encoded CTX-M-type, SHV-12, DHA-1, and OXA-48 β-lactamases and covered a cefepime-enmetazobactam MIC range from 0.06 to 2 μg/ml. Enmetazobactam restored the efficacy of cefepime against all isolates tested. Sigmoid curve fitting across the combined set of isolates identified enmetazobactam PK-PD targets for stasis and for a 1-log10 bioburden reduction of 8% and 44% fT > 2 μg/ml, respectively, with a concomitant cefepime PK-PD target of 40 to 60% fT > cefepime-enmetazobactam MIC. These findings support clinical dose selection and breakpoint setting for cefepime-enmetazobactam.