Journal of Infectious Diseases Advance Access published April 22, 2014 1 

Interaction of Drug- and Granulocyte-Mediated Killing of Pseudomonas

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aeruginosa in a Murine Pneumonia Model

GL Drusano, Weiguo Liu, Steven Fikes, Ryan Cirz, Nichole Robbins, Stephanie Kurhanewicz, Jaime Rodriquez, David Brown, Dodge Baluya, Arnold Louie

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University of Florida, Achaogen, San Francisco, Ca

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Corresponding Author: GL Drusano, M.D. Professor and Director, Institute for Therapeutic Innovation, University of Florida, 6550 Sanger Road, Lake Nona, FL 32827,

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(407) 313 7060, [email protected]

© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of  America. All rights reserved. For Permissions, please e‐mail: [email protected]

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Institute for Therapeutic Innovation, Department of Medicine, College of Medicine



Abstract Background: Granulocyte kill of bacterial pathogens is a saturable process, as

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previously demonstrated. There is virtually no quantitative information about how granulocytes interact with antimicrobial chemotherapy for bacterial cell kill.

Methods: We performed a dose-ranging study with the aminoglycoside plazomicin against Pseudomonas aeruginosa ATCC27853 in a granulocyte-replete murine

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doses 5 times over 24 hours, mimicking a human daily administration profile).

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Pharmacokinetic profiling was performed in plasma and Epithelial Lining Fluid. All samples were simultaneously analyzed with a population model. Mouse cohorts were

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treated for 24 hours; other cohorts treated with the same therapy were observed for another 24 hours off therapy, allowing delineation of the therapeutic effect necessary to

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reduce bacterial burden below the half-saturation point. Results: There was a clear point identified for the WBCK50 (half-saturation point) and was 2.45x106 + 6.84x105 CFU/g. Higher plazomicin exposures reduced bacterial burden to 2 Log10(CFU/g) to allow the granulocytes to contribute optimally to bacterial clearance.

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pneumonia model. Plazomicin was administered in a humanized fashion (decrementing



Introduction Ventilator-Requiring Hospital-Acquired Bacterial Pneumonia (VRHABP) caused

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by Pseudomonas aeruginosa remains a difficult therapeutic problem for clinicians. While antimicrobial therapy can be optimized (1), the relationship between the interaction of antibiotics and the bacterial cell killing that is mediated by granulocytes is unclear.

We have recently demonstrated that bacterial cell killing by granulocytes is a

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evaluations, antibiotics were not employed. Granulocytes unaided by antibiotics were

below the half-saturation point.

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able to mediate substantial bacterial kill as long as the organism burden remained

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In this evaluation, we employed the new aminoglycoside plazomicin in a granulocyte-replete murine pneumonia model (3) to examine the interaction of

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granulocytes with antimicrobial therapy. We initiated a pneumonia with a dense bacterial burden at therapy start and performed a “humanized” dose ranging experiment (4). Other treated cohorts of animals were observed for 24 hours off therapy to examine

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the impact of granulocyte kill on residual bacterial burden.

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Methods

Microorganism. Pseudomonas aeruginosa strain ATCC 27853 was employed. The

MIC determination was by CLSI microbroth methodology for plazomicin (5).

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saturable process (2, 3) in both murine thigh and pneumonia models. In both these



PK studies. To correlate the doses of drug administered to mice with measures of exposure, pharmacokinetic (PK) studies were conducted in separate cohorts of mice

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with Pseudomonas aeruginosa pneumonia. For plazomicin (6), we administered the

drug on a humanized dosing scheme administered five times over a 24 hr interval. The humanization scheme was derived from earlier single-dose pharmacokinetic studies performed with plazomicin (data not shown). The fraction of the total daily (mg/kg of

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doses were administered at 2, 6, 10, 16 and 22 hrs after bacterial challenge. Plazomicin

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total daily doses of 5.5, 10.9, 14.6, 18.2, 21.9, 43.8, 87.5, 175 and 225 mg/kg/day were evaluated.

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Briefly, granulocyte-replete mice were infected with P. aeruginosa ATCC 27853 via the intranasal route. Two hours later, the humanized regimens for the total daily

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doses of plazomicin were administered to different cohorts of mice. Plasma and bronchoalveolar lavage (BAL) fluid were collected from three sacrificed mice per time point (and dosage) at 10 time points over the 24-h PK study. Plasma and BAL fluid were stored at -80°C until they were assayed for drug content by liquid chromatography (LC) and dual mass spectrometry (MS-MS) (LC/MS-MS). The pre-diluted concentration of

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antibiotic in the BAL fluid (epithelial lining fluid or ELF) was calculated by comparing the ratio of the amounts of urea measured in simultaneously collected plasma and BAL

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fluid.

Murine pneumonia model. The model and detailed methods have been previously described (7). All animal experimentation was approved by the local institutional animal care and use committee (IACUC). A mouse pneumonia model previously described (7)

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body weight) dose at each injection was 0.555, 0.238, 0.127, 0.047 and 0.033. These



was used. Female, 24- to 26-g, outbred Swiss-Webster mice (Taconic Farms, Taconic, NY) were provided water and food ad libitum. Anesthetized mice were infected via the

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intranasal route with a 20-μl volume using 2 x 107 CFU of P. aeruginosa. The bacterial

inoculum was confirmed by quantitative cultures. Two hours after bacterial inoculation, and just prior to therapy initiation, five mice were sacrificed for baseline quantitative cultures of lung homogenates.

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untreated control group (cohorts 19 and 20). Twenty-six hours after infection, therapy

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was stopped and one untreated control group and 9 cohorts of treated mice were humanely sacrificed. The other control group and 9 treated cohorts were followed to

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hour 50. At this time, these groups were humanely sacrificed.

The tissues were homogenized and washed with normal saline solution to

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prevent drug carryover. Homogenates were then quantitatively cultured onto drug-free agar to characterize the effect of each regimen on the total bacterial population. After incubation of the plates at 35°C for 48 h, colonies were enumerated. Means and standard deviations for the quantitative culture samples were calculated.

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Plazomicin and Urea Assays in Plasma and BAL. Mouse plasma samples

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(0.050 ml) were deproteinated with acetonitrile (0.150 ml). The samples were centrifuged, and an aliquot of the supernatant (0.050 ml) was transferred into an appropriately labeled autosampler vial containing 1.00 ml of high-pressure liquid chromatography (HPLC)-grade water. Samples were analyzed by high-pressure liquid chromatography–tandem mass spectrometry for plazomicin concentrations. Mouse BAL

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Eighteen animal cohorts were administered plazomicin. There was also an



fluid samples (0.050 ml) were diluted with 0.100 ml of HPLC-grade water and were also analyzed by high-pressure liquid chromatography–tandem mass spectrometry for

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plazomicin concentration determinations. The LC/MS-MS system was comprised of

a Shimadzu Prominence HPLC system and an Applied Biosystems/MDS Sciex API5000 LC/MS-MS system.

regression curves) was 2.15 – 8.35% for plasma. For BAL fluid, these values ranged

for plasma and 5.85 - 9.77% for BAL.

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from 1.54 – 9.17%. For urea, the range of % coefficient of variation was 3.63 – 10.5%

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Mathematical Models. As mentioned above, we performed a separate pharmacokinetic study in infected mice. This model has been described previously (7). It is presented

Eqn (1) Eqn (2)

dX1 ⁄ dt = -X1 · Ka

dX2 ⁄ dt = X1 · Ka - [(CL ⁄ F ⁄ Vc ⁄ F) + K23] · X2 + K32 · X3 dX3 ⁄ dt = K23 · X2 - K32 · X3

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Eqn (3)

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below:

Eqn (4)

dX4/dt

= K24 · X2 - K42 · X4

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The plasma concentration equals X2/(Vc/F), and the ELF concentration equals X4/VELF. The relationship between drug exposure and cell kill as well as the cell kill

attributable to granulocytes was also evaluated (see below). Eqn (5)

PlazoKill = (X(4)/VELF))H/(C50-Plazo-KillH+(X(4)/VELF)H

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For plazomicin, the % coefficient of variation over the range of 0.1-100 mg/l (2



Eqn (6)

d(CFU/g)/dt = (Kgmax · (1-(CFU/g /Popmax)) · CFU/g – Kkillmax · PlazoKill · CFU/g -(KkillWBC · CFU/g)/(WBCKill50 + CFU/g)) · CFU/g

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X1 – X4 represent the amounts of plazomicinn in the absorption compartment (site of injection), Central Compartment (rapidly exchangeable volume, closely

associated with blood), the “rest of the mouse” and the Epithelial Lining Fluid volume, respectively; Vc = Volume of the Central Compartment; CL/F = Apparent Plasma

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constants; VELF is the Volume of the Epithelial Lining Fluid (ELF) compartment; Ka is the

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first order absorption rate constant; Kgmax is the first order growth rate constant; POPMAX is the maximal size of the bacterial population; Kkillmax is the maximal kill rate

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constant for plazomicin therapy; PlazoKill is the sigmoid Emax function for cell kill in which C50-Plazo-Kill is the plazomicin concentration at which the plazomicin-attributable kill is half-

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maximal and H is Hill’s constant; KkillWBC is the maximal kill rate attributable to granulocytes; WBCKill50 is the bacterial burden at which granulocyte-mediated kill is halfsaturable; IC(5) is the initial bacterial burden at therapy initiation. In the system output, CFU/g is Log10 transformed.

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The BigNPAG program described by Leary et al. (8) was used for population modeling. The inverse of the estimated observation variance was employed for

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weighting. Bayesian estimates were obtained using the “population-of-one” utility in BigNPAG. Model evaluation was performed by predicted-observed plots. The mean weighted error served as the measure of bias. The bias-adjusted weighted mean squared error served as the measure of precision.

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Clearance; K23, K32, K24 and K42 are first order intercompartmental transfer rate



Simulation was performed employing the ADAPT V package of programs (9).

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Results

MIC to Plazomicin and Mutational Frequency to Resistance (6) for ATCC 27853:

The plazomicin MIC value for the challenge strain was 2 mg/L determined in triplicate.

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(-5.59 Log10[CFU/ml]) - 1/426,580 colonies (-5.63 Log10[CFU/ml]).

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Murine Pharmacokinetics of Plazomicin: The mean and median parameter vectors as well as the estimates of standard deviations of the parameter values for the

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population are shown in Table 1. By simulation, the fractional penetration (AUCELF/AUCPLASMA) for plazomicin into ELF was 0.876.

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The model fit the data acceptably well. For the pre-Bayesian (population) estimation, the plasma concentration Predicted-Observed plot had a regression line of: Observed = 1.400 x Predicted – 1.336 ; r2 = 0.983 ; Bias = 1.770 ; Precision = 96.75.

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For the ELF concentrations, the Predicted-Observed plot had a regression line of: Observed =1.940 x Observed - 1.940; r2 = 0.934 ; Bias = -0.304 ; Precision = 2.465.

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For the Bayesian (individual) analysis, the plasma concentration Predicted-Observed plot had a regression line of: Observed = 1.315 x Predicted – 0.306 ; r2 = 0.994 ; Bias = -0.922 ; Precision = 9.878. For the ELF concentrations, the Predicted-Observed plot had a regression line of:

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The mutational frequency to plazomicin resistance was 1/389,045 colonies



Observed =1.083 x Observed + 0.0613; r2 = 0.998 ; Bias = -0.181 ; Precision = 0.719. The median estimates were employed for fitting drug exposure and WBC impact

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on colony counts over time in the second pharmacodynamic population analysis.

Pharmacodynamics of Plazomicin and Impact of Granulocytes on Cell Kill of P.

aeruginosa ATCC27853 in Murine Lung: The colony counts over time are displayed

No animals survived to hour 50 in the no-treatment controls or in the lowest

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dosing group for plazomicin. The other lower dosing groups (10.9 and 14.6 mg/kg/day) displayed net growth between hours 26 and 50. This transitioned to stasis at

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intermediate doses (18.2 and 21.9 mg/kg/d) and, at higher doses (>43.8 mg/kg/d) there was net cell kill between hours 26 and 50, even though there was no further plazomicin dosing after hour 20 and plazomicin concentrations were under 0.61 mg/L in plasma

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and 0.56 mg/L in ELF at hour 26 (MIC = 2.0 mg/L). The point estimates of the pharmacodynamic model parameters are displayed in Table 2, along with their dispersions. Again, the fit of the model to the data was acceptable. For the pre-Bayesian regression, the predicted-observed plot had a

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regression line of :

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Observed = 1.178 x Predicted – 1.200 ; r2 = 0.747; Bias = 0.011 ; Precision = 3.339. For the Bayesian (individual) estimation, the predicted-observed regression was : Observed = 1.104 x Predicted - 0.724; r2 = 0.896 ; Bias = 0.079 ; Precision = 1.402. For this output, the p value was 104 CFU/ml of BAL fluid. This is the resultant number after dilution by the BAL fluid. Previous investigations have shown that this dilution factor is on the order of 30-100 fold (15), as demonstrated

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by urea dilution between plasma and BAL fluid. Consequently, the bacterial burden after BAL in a documented case of VRHABP is greater than 3x105 CFU/ml. This is at the

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bottom of the range for definition of the condition. All other patients will have greater burdens. Given our identified half saturation point, attainment of early, adequate chemotherapy and its subsequent salutary effect becomes understandable. Zaccard et al (16) examined bilateral BAL in patients with suspected VRHABP.

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There were 134 samples from patients with P. aeruginosa, Enterobacter spp, K. pneumonia, Acinetobacter spp and Serratia marcescens. Correcting for the 30 fold or

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greater dilution (not done in the original Zaccard paper), the bacterial baseline burdens were distributed as seen in Table 3. Greater than a majority of patients had a baseline bacterial burden that meets or exceeds the half-saturation point seen in our studies. Fully one quarter of patients are in the near full saturation range. We would speculate

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the explanation for these clinical findings.

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that this observation is a major reason that these patients are so difficult to treat successfully. In many, the granulocytes are essentially taken out of the equation by the

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size of the bacterial burden. This large burden also markedly increases the likelihood of resistance emergence, as in many instances, the organism number will exceed the

inverse of the mutational frequency to resistance. Rapid emergence of resistance has been documented during therapy in randomized trials for this condition, particularly for

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adequate antimicrobial therapy has a salutary impact on patient outcome. Aggressive,

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adequate therapy will reduce the bacterial burden below the half-saturation point and allow granulocytes to increase bacterial clearance. Rapid clearance of the bacterial

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infection may result in better clinical outcomes. Alvarez-Lerma demonstrated that in patients with Hospital-Acquired Bacterial pneumonia (19), early, adequate antimicrobial therapy significantly lowered attributable mortality, the number of complications per

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patient and episodes of shock.

The results of this investigation are concordant with this publication (19), but also places quantitation around this finding. The half-saturation point (WBCK50) and its 95% confidence interval (CI) as estimated here was 2.45 x 106 CFU/g (mean), 2.68 x 106

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CFU/g (median) and a 95% CI of 1.11 x 106 – 3.79 x 106 CFU/g.

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Examining Figure 1 and Table 3 shows that near-maximal granulocyte-mediated

bacterial cell kill requires that the bacterial burden be reduced by chemotherapy to the range of less than or equal to 105 CFU/g. Table 3 shows that a 2 Log10(CFU/g) kill early

in therapy will attain this end in a relatively high fraction of patients. Consequently, when therapeutic regimens are being designed for patients with VRHABP, the minimal

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Pseudomonas aeruginosa (17, 18). Finally, this also provides insight into why early

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pharmacodynamic target (e.g. as seen in an animal model or from a hollow fiber system evaluation) should be a 2 Log10(CFU/g) kill. It would also be important to also attain

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resistance suppression. For the latter, particularly with P. aeruginosa infections,

combination therapy may be the best path forward to attain these goals (20 – 22).

These findings set the stage to perform a clinical trial that will validate this hypothesis.

Finally, duration of therapy remains an issue for these patients. Chastre et al (23)

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therapy for 401 patients with Hospital-Acquired Bacterial Pneumonia requiring

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ventilation. There were no mortality differences seen between groups. The one major difference seen was in the subgroup of patients infected with non-fermenting Gram-

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negative bacilli (e.g. P. aeruginosa, Acinetobacter spp). Here there was a statistical difference in recurrence rate in favor of the longer therapy (40.6% vs 25.4%; difference,

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15.2%, 90% CI, 3.9%-26.6%). However, among patients who developed recurrent infections, multi-resistant pathogens emerged less frequently in those who had received 8 days of antibiotics (42.1% vs 62.0% of pulmonary recurrences, P = 0.04). We have performed this particular experiment with a single isolate of

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Pseudomonas aeruginosa. It may be that other isolates might respond differently, particularly tolerant organisms or strains that are particularly slow growing, as two

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possible features that might have an impact on the findings. Further experimentation is needed.

By identifying a pharmacodynamic target for the therapy of these patients of > 2

Log10(CFU/g) bacterial kill, we can hopefully reduce the number of recurrences, shorten

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performed a randomized, double-blind trial comparing 8 versus 15 days of antimicrobial

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therapy and decrease resistance emergence. Having the help of granulocytes in

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bacterial clearance is key to attaining these outcomes.

Footnote Page:

Acknowledgements: This work was supported by R01AI079578 and RO1AI079729,

Therapeutic Innovation. The content is solely the responsibility of the authors and does

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not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health.

disclose.

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Corresponding Author:

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Ryan Cirz, Ph.D. is an employee of Achaogen. No other author has any conflicts to

GL Drusano, M.D.

Professor and Director

Institute for Therapeutic Innovation

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University of Florida 6550 Sanger Road

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Lake Nona, FL 32827 (407) 313 7060

[email protected]

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grants from the National Institute of Allergy and Infectious Diseases to the Institute for

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References:

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2. Drusano GL, B Vanscoy, W Liu, S Fikes, D Brown, A Louie. Saturability of

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Antimicrob Agents Chemother. 2011; 55:2693-2695.

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3. Drusano GL, C Fregeau, W Liu, DL Brown, A Louie. Impact of Burden on Granulocyte Clearance of Bacteria in a Mouse Thigh Infection Model. Antimicrob

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Agents Chemother. 2010;54:4368-4372.

4. M Deziel, H. Heine, A Louie, M Kao, WR Byrne, J Bassett, L Miller, K Bush, M

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Kelley, GL Drusano. Identification of effective antimicrobial regimens for use in humans for the therapy of Bacillus anthracis infections and post-exposure prophylaxis. Antimicrob Agents Chemother 2005;49:5099-5106.

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5. Clinical and Laboratory Standards Institute. 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved

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standard. CLSI publication M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA. 6. Zhanel GG, CD Lawson, S Zelinitsky, B Findlay, F Schweizer, H Adam, A Walkty, E Rubinstein, AS Gin, D Hoban, JP Lynch, JA Karlowsky. Comparison of the

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Granulocyte Kill of Pseudomonas aeruginosa in a Murine Model of Pneumonia.

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next generation aminoglycoside plazomicin to gentamicin, tobramycin and amikacin. Expert Review of Anti-infective Therapy 2012;10:459-473, 2012.

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delineation of exposure targets. Antimicrob Agents Chemother. 2011; 55:3406-3412.

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8. Leary R, Jelliffe R, Schumitzky A, Van Guilder M. 2001. An adaptive grid

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13. Piskin N, H Aydemir, N Oztoprak, D Akduman, F Comert, F Kokturk, G Celebi. Inadequate treatment of ventilator-associated and hospital-acquired pneumonia: risk

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K23

K32

K24

K42

Units

L

L/H

H-1

H-1

H-1

H-1

Mean

0.0189

0.0301

11.09

10.69

6.120

13.74

Median

0.0071

0.0109

9.060

13.92

4.292

SD

0.0164

0.0219

2.221

3.555

2.297

VELF

Ka

L

H-1

0.0045

28.04

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CL/F

0.0023

28.86

2.147

0.0025

1.775

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15.45

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Vc = Volume of the Central Compartment; CL/F = Apparent Plasma Clearance; K23, K32, K24 and K42 are first order intercompartmental transfer rate constants; VELF is the Volume of the Epithelial Lining Fluid (ELF) compartment; Ka is the first order absorption rate constant

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Vc

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Table 1: Pharmacokinetic Parameter Values and their Dispersions

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Table 2: Parameter Values and Dispersions for the Pharmacodynamic Model

-1

-1

C50

H

POPMAX

IC5 CFU

H

H

mg/L

--

CFU

Mean

0.523

2.578

15.32

16.77

9.08x10

Median

0.057

1.256

16.94

18.49

9.86x10

SD

1.370

2.305

5.408

2.277

2.23x10

9

-1

7

8.50x10 9

7

5.60x10 9

WBCK50

7

2.87x10

H

CFU/g

0.730

2.45x10

0.354

2.68x10

1.007

6.84x10

6 6 5

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Kgmax is the first order growth rate constant; POPMAX is the maximal size of the bacterial population; Kkillmax is the maximal kill rate constant for plazomicin therapy; PlazoKill is the sigmoid Emax function for cell kill in which C50-Plazo-Kill is the plazomicin concentration at which the plazomicin-attributable kill is half-maximal and H is Hill’s constant; KkillWBC is the maximal kill rate attributable to granulocytes; WBCKill50 is the bacterial burden at which granulocyte-mediated kill is half-saturable; IC(5) is the initial bacterial burden at therapy initiation. In the system output, CFU/g is Log10 transformed.

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Units

Kkill-WBC

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Kkill

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Kg

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Table 3: Dilution-transformed Colony Counts from Patients with Gram-negative Pneumonia Documented by Bronchalveolar Lavage. After Zaccard et al. J Clin Microbiol. 2009;47:2918-2924

>3x105 >3x106 >3x107 Total 49 (36.6%) 50 (37.3%) 35 (26.1%) 134 (100%) ______________________________________________________________________

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Dilution corrections were made using the urea dilution seen in patients with VentilatorRequiring Hospital-Acquired Bacterial Pneumonia. (Lodise et al. Antimicrob Agents Chemother. 2011;55:1606-1610); Units are in colonies/ml of ELF

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Downloaded from http://jid.oxfordjournals.org/ at Universidade de BrasÃ-lia on May 6, 2014

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Figure 1: Colony counts of Pseudomonas aeruginosa ATCC 27853 in the lungs of mice. Cohorts were sacrificed at Baseline (2 hrs post-challenge), at hour 26 and at hour 50. The no-treatment controls and the lowest dose plazomicin-treated group had no survivors at hour 50.

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

Killing of bacterial pathogens by granulocytes is a saturable process, as previously demonstrated. There is virtually no quantitative information abou...
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