Gepotidacin Pharmacokinetics-Pharmacodynamics against Escherichia coli in the One-Compartment and Hollow-Fiber In Vitro Infection Model Systems.

Gepotidacin is a novel, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action with an in vitro spectrum of activity that includes Escherichia coli. Our objectives herein were the following: (i) to identify the pharmacokinetic-pharmacodynamic (PK-PD) index associated with the efficacy of gepotidacin against E. coli; (ii) to determine the magnitude of the above-described PK-PD index associated with various bacterial reduction endpoints for E. coli; and (iii) to characterize the relationship between gepotidacin exposure and on-therapy E. coli resistance amplification. A 24-h one-compartment in vitro infection model was used to investigate the first two study objectives, and a 10-day hollow-fiber in vitro infection model was used to evaluate the third objective. For the dose-fractionation studies (objective i) in which E. coli NCTC 13441 (gepotidacin MIC, 2 mg/liter) was evaluated, gepotidacin free-drug area under the concentration-time curve (AUC) from 0 to 24 h to the MIC (AUC/MIC ratio) was identified as the PK-PD index most closely associated with change in bacterial burden (r2 = 0.925). For the dose-ranging studies (objective ii), in which four E. coli isolates (gepotidacin MIC range, 1 to 4 mg/liter) were studied, the magnitude of the median gepotidacin free-drug AUC/MIC ratio associated with net bacterial stasis and 1- and 2-log10 CFU reductions for the pooled data set was 33.9, 43.7, and 60.7, respectively. For the hollow-fiber in vitro infection model studies (objective iii), in which one isolate (E. coli NCTC 13441; gepotidacin MIC, 2 mg/liter) was evaluated, gepotidacin free-drug AUC/MIC ratios of 275 and greater were sufficient to suppress on-therapy resistance amplification. Together, the data generated from these studies will be useful to support discrimination among candidate dosing regimens for future clinical study.

Relationship between Gepotidacin Exposure and Prevention of On-Therapy Resistance Amplification in a Neisseria gonorrhoeae Hollow-Fiber In Vitro Infection Model.

Multidrug-resistant Neisseria gonorrhoeae has emerged as a threat to global health. The relationship between gepotidacin exposure and prevention of on-therapy amplification of drug-resistant N. gonorrhoeae was examined using a 7-day hollow-fiber in vitro infection model. The study design included both inactive (no-treatment and ciprofloxacin) and active (ceftriaxone) control regimens. Study drug concentration-time profiles were simulated in the in vitro system for a single oral 0.5 g ciprofloxacin dose, a single intramuscular 0.25 g ceftriaxone dose, and single or two (8 to 12 h apart) oral gepotidacin doses ranging from 0.75 to 12 g. The initial bacterial burden inoculated in the model was 106 CFU/ml. The gepotidacin, ciprofloxacin, and ceftriaxone broth MIC values for the challenge isolate (N. gonorrhoeae GSK #8) were 0.5, 2, and 0.002 mg/liter, respectively. Samples were collected for enumeration of total and drug-resistant bacterial populations and drug concentrations. The no-treatment control reached a bacterial density greater than 108 CFU/ml over 24 h and remained consistent over the 7-day study period. The bacterial density in the model system of the ciprofloxacin regimen matched that of the growth control throughout the study duration, while the ceftriaxone regimen sterilized the model system by the end of day 1. For gepotidacin, a full dose-response relationship was observed. While failure was observed for the 0.75-, 1.5-, and 3-g single-dose regimens, all gepotidacin single- or divided-dose regimens totaling at least 4.5 g prevented resistance amplification and sterilized the model system. These data are useful to provide gepotidacin dose selection support for treating patients with gonorrhea infections.

Norepinephrine in Combination with Antibiotic Therapy Increases both the Bacterial Replication Rate and Bactericidal Activity.

We previously demonstrated that the rate and extent of an antimicrobial agent's bactericidal effects were coupled to the bacterial replication rate, the latter of which was modulated with the sodium chloride concentration. Herein, we describe the results from a 24-h one-compartment in vitro infection model study that was designed to demonstrate that an antimicrobial agent's bactericidal effects could be amplified when it is administered with a pharmaceutical agent that increases the bacterial replication rate. The antimicrobial and growth-promoting agents selected were levofloxacin and norepinephrine, respectively. The challenge isolate was Escherichia coli JMI 21711R (levofloxacin MIC, 8 mg/liter). Within the in vitro infection model, a human levofloxacin concentration-time profile (half-life, 7 h) was simulated and the challenge isolate was subjected to an ineffective monotherapy exposure (free-drug area under the concentration-time curve over 24 h divided by the MIC [AUC/MIC] ratio of 6) with and without norepinephrine as a continuous infusion (275 mg/liter). Samples were collected from the model during the course of the study for bacterial density determinations and drug concentration assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS). As expected, the norepinephrine and no-treatment control arms failed immediately, followed by the levofloxacin monotherapy arm, which failed slowly over time. The levofloxacin-epinephrine regimen resulted in a 2-log10 CFU reduction in bacterial density over the first 6 to 8 h of the study, which was followed by regrowth of a highly levofloxacin-resistant subpopulation (MIC, 64 mg/liter). These data demonstrate that increasing the rate of bacterial replication with a pharmaceutical product in combination with antimicrobial therapy represents an opportunity to increase the rate and magnitude of bactericidal effect.

Combination treatment with meropenem plus levofloxacin is synergistic against Pseudomonas aeruginosa infection in a murine model of pneumonia.

BACKGROUND: Meropenem plus levofloxacin treatment was shown to be a promising combination in our in vitro hollow fiber infection model. We strove to validate this finding in a murine Pseudomonas pneumonia model. METHODS: A dose-ranging study with meropenem and levofloxacin alone and in combination against Pseudomonas aeruginosa was performed in a granulocytopenic murine pneumonia model. Meropenem and levofloxacin were administered to partially humanize their pharmacokinetic profiles in mouse serum. Total and resistant bacterial populations were estimated after 24 hours of therapy. Pharmacokinetic profiling of both drugs was performed in plasma and epithelial lining fluid, using a population model. RESULTS: Meropenem and levofloxacin penetrations into epithelial lining fluid were 39.3% and 64.3%, respectively. Both monotherapies demonstrated good exposure responses. An innovative combination-therapy analytic approach demonstrated that the combination was statistically significantly synergistic (α = 2.475), as was shown in the hollow fiber infection model. Bacterial resistant to levofloxacin and meropenem was seen in the control arm. Levofloxacin monotherapy selected for resistance to itself. No resistant subpopulations were observed in any combination therapy arm. CONCLUSIONS: The combination of meropenem plus levofloxacin was synergistic, producing good bacterial kill and resistance suppression. Given the track record of safety of each agent, this combination may be worthy of clinical trial.

Pharmacodynamic evaluation of the activities of six parenteral vancomycin products available in the United States.

A recent report found that generic parenteral vancomycin products may not have in vivo efficacies equivalent to those of the innovator in a neutropenic murine thigh infection model despite having similar in vitro microbiological activities and murine serum pharmacokinetics. We compared the in vitro and in vivo activities of six of the parenteral vancomycin products available in the United States. The in vitro assessments for the potencies of the vancomycin products included MIC/minimal bactericidal concentration (MBC) determinations, quantifying the impact of human and murine serum on the MIC values, and time-kill studies. Also, the potencies of the vancomycin products were quantified with a biological assay, and the human and mouse serum protein binding rates for the vancomycin products were measured. The in vivo studies included dose-ranging experiments with the 6 vancomycin products for three isolates of Staphylococcus aureus in a neutropenic mouse thigh infection model. The pharmacokinetics of the vancomycin products were assessed in infected mice by population pharmacokinetic modeling. No differences were seen across the vancomycin products with regard to any in vitro evaluation. Inhibitory sigmoid maximal bacterial kill (Emax) modeling of the relationship between vancomycin dosage and the killing of the bacteria in mice in vivo yielded similar Emax and EC50 (drug exposure driving one-half Emax) values for bacterial killing. Further, there were no differences in the pharmacokinetic clearances of the 6 vancomycin products from infected mice. There were no important pharmacodynamic differences in the in vitro or in vivo activities among the six vancomycin products evaluated.

Analysis of combination drug therapy to develop regimens with shortened duration of treatment for tuberculosis.

RATIONALE: Tuberculosis remains a worldwide problem, particularly with the advent of multi-drug resistance. Shortening therapy duration for Mycobacterium tuberculosis is a major goal, requiring generation of optimal kill rate and resistance-suppression. Combination therapy is required to attain the goal of shorter therapy. OBJECTIVES: Our objective was to identify a method for identifying optimal combination chemotherapy. We developed a mathematical model for attaining this end. This is accomplished by identifying drug effect interaction (synergy, additivity, antagonism) for susceptible organisms and subpopulations resistant to each drug in the combination. METHODS: We studied the combination of linezolid plus rifampin in our hollow fiber infection model. We generated a fully parametric drug effect interaction mathematical model. The results were subjected to Monte Carlo simulation to extend the findings to a population of patients by accounting for between-patient variability in drug pharmacokinetics. RESULTS: All monotherapy allowed emergence of resistance over the first two weeks of the experiment. In combination, the interaction was additive for each population (susceptible and resistant). For a 600 mg/600 mg daily regimen of linezolid plus rifampin, we demonstrated that >50% of simulated subjects had eradicated the susceptible population by day 27 with the remaining organisms resistant to one or the other drug. Only 4% of patients had complete organism eradication by experiment end. DISCUSSION: These data strongly suggest that in order to achieve the goal of shortening therapy, the original regimen may need to be changed at one month to a regimen of two completely new agents with resistance mechanisms independent of the initial regimen. This hypothesis which arose from the analysis is immediately testable in a clinical trial.

Interaction of drug- and granulocyte-mediated killing of Pseudomonas aeruginosa in a murine pneumonia model.

BACKGROUND: Killing of bacterial pathogens by granulocytes is a saturable process, as previously demonstrated. There is virtually no quantitative information about how granulocytes interact with antimicrobial chemotherapy to kill bacterial cells. METHODS: We performed a dose-ranging study with the aminoglycoside plazomicin against Pseudomonas aeruginosa ATCC27853 in a granulocyte-replete murine pneumonia model. Plazomicin was administered in a humanized fashion (ie, administration of decrementing doses 5 times over 24 hours, mimicking a human daily administration profile). Pharmacokinetic profiling was performed in plasma and epithelial lining fluid. All samples were simultaneously analyzed with a population model. Mouse cohorts were treated for 24 hours; other cohorts treated with the same therapy were observed for another 24 hours after therapy cessation, allowing delineation of the therapeutic effect necessary to reduce the bacterial burden to a level below the half-saturation point. RESULTS: The mean bacterial burden (±SD) at which granulocyte-mediated kill was half saturable was 2.45 × 10(6) ± 6.84 × 10(5) colony-forming units of bacteria per gram of tissue (CFU/g). Higher levels of plazomicin exposure reduced the bacterial burden to <5 log10 CFU/g, allowing granulocytes to kill an additional 1.0-1.5 log CFU/g over the subsequent 24 hours. CONCLUSIONS: For patients with large bacterial burdens (eg, individuals with ventilator-requiring hospital-acquired pneumonia), it is imperative to kill ≥2 log10 CFU/g early after treatment initiation, to allow the granulocytes to contribute optimally to bacterial clearance.

Impact of meropenem in combination with tobramycin in a murine model of Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa pneumonia remains a difficult therapeutic problem. Optimal doses and modes of administration of single agents often do not result in acceptable outcomes. Further, emergence of resistance occurs frequently in this setting with single-agent chemotherapy. The purpose of these experiments was to evaluate combination chemotherapy with meropenem plus tobramycin for P. aeruginosa in a murine pneumonia model. Neutropenia was induced by cyclophosphamide. Pharmacokinetics of meropenem and tobramycin were determined using a population pharmacokinetic approach. Both drugs were given at 4-h intervals. Meropenem was administered as total daily doses of 30 to 600 mg/kg of body weight, while tobramycin doses ranged from 50 to 400 mg/kg. Combination therapy evaluated all combinations of 50, 100, and 150 mg/kg/day of tobramycin doses with 60 or 300 mg/kg/day of meropenem. Total and drug-resistant organisms were enumerated. Meropenem alone had a near-maximal effect at 60 mg/kg/day (3.18 log10 [CFU/g] kill from stasis). The time > MIC in epithelial lining fluid (ELF) at this dose was 35.25% of 24 h. For tobramycin alone, the near-maximal effect was at 150 mg/kg/day and the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC ratio) in ELF was 240.3. Resistance suppression occurred at an ELF AUC/MIC ratio of 110.6. For combination therapy, the near-maximal effect was reached at 60 mg/kg/day and 50 mg/kg/day of meropenem and tobramycin, which produced a 35.25% time > MIC in ELF and an ELF AUC/MIC ratio of 80.1. The interaction was additive. All combination regimens suppressed resistance. Combination therapy produced additive drug interaction and suppressed all resistance amplification. It is likely that optimal therapy for Pseudomonas aeruginosa pneumonia will involve a combination of agents.

Resistance emergence mechanism and mechanism of resistance suppression by tobramycin for cefepime for Pseudomonas aeruginosa.

The panoply of resistance mechanisms in Pseudomonas aeruginosa makes resistance suppression difficult. Defining optimal regimens is critical. Cefepime is a cephalosporin whose 3' side chain provides some stability against AmpC ß-lactamases. We examined the activity of cefepime against P. aeruginosa wild-type strain PAO1 and its isogenic AmpC stably derepressed mutant in our hollow-fiber infection model. Dose-ranging studies demonstrated complete failure with resistance emergence (both isolates). Inoculum range studies demonstrated ultimate failure for all inocula. Lower inocula failed last (10 days to 2 weeks). Addition of a ß-lactamase inhibitor suppressed resistance even with the stably derepressed isolate. Tobramycin combination studies demonstrated resistance suppression in both the wild-type and the stably derepressed isolates. Quantitating the RNA message by quantitative PCR demonstrated that tobramycin decreased the message relative to that in cefepime-alone experiments. Western blotting with AmpC-specific antibody for P. aeruginosa demonstrated decreased expression. We concluded that suppression of ß-lactamase expression by tobramycin (a protein synthesis inhibitor) was at least part of the mechanism behind resistance suppression. Monte Carlo simulation demonstrated that a regimen of 2 g of cefepime every 8 h plus 7 mg/kg of body weight of tobramycin daily would provide robust resistance suppression for Pseudomonas isolates with cefepime MIC values up to 8 mg/liter and tobramycin MIC values up to 1 mg/liter. For P. aeruginosa resistance suppression, combination therapy is critical.