Influence of diluent volume of colistimethate sodium on aerosol characteristics and pharmacokinetics in ventilator-associated pneumonia caused by MDR bacteria

Influence of diluent volume of colistimethate sodium on aerosol characteristics and... Abstract Objectives Nebulized colistimethate sodium (CMS) can be used to treat ventilator-associated pneumonia caused by MDR bacteria. The influence of the diluent volume of CMS on aerosol delivery has never been studied. The main objectives of the study were to compare aerosol particle characteristics and plasma and urine pharmacokinetics between two diluent volumes in patients treated with nebulized CMS. Methods A crossover study was conducted in eight patients receiving nebulized CMS every 8 h. After inclusion, nebulization started with 4 million international units (MIU) of CMS diluted either in 6 mL (experimental dilution) or in 12 mL (recommended dilution) of normal saline in a random order. For each diluent volume, CMS aerosol particle sizes were measured and plasma and urine samples were collected every 2 h. Nebulization time and stability of colistin in normal saline were assessed. Results The mass median aerodynamic diameters were 1.4 ± 0.2 versus 0.9 ± 0.2 μm (P < 0.001) for 6 and 12 mL diluent volumes, respectively. The plasma area under the concentration–time curve from 0 to 8 h (AUC0–8) of colistinA+B was 6.6 (4.3–17.0) versus 6.7 (3.6–14.0) μg·h/mL (P = 0.461) for each dilution. The total amount of colistin and CMS eliminated in the urine represented, respectively, 17% and 13% of the CMS initially placed in the nebulizer chamber for 6 and 12 mL diluent volumes (P = 0.4). Nebulization time was shorter [66 (58–75) versus 93 (69–136) min, P = 0.042] and colistin stability was better with the 6 mL diluent volume. Conclusions Nebulization with a higher concentration of CMS in saline (4 MIU in 6 mL) decreases nebulization time and improves colistin stability without changing plasma and urine pharmacokinetics or aerosol particle characteristics for lung deposition. Introduction Colistimethate sodium (CMS) is an inactive pro-drug of colistin (active metabolite) that forms colistin by spontaneous hydrolysis in aqueous solution. Colistin is a complex, multicomponent antibiotic mixture,1 with colistin A and colistin B accounting for ∼85% of this mixture.2–5 The antibiotic effect of colistin A and B is concentration dependent.1 Bergen et al.1,6 additionally described a high correlation between the overall killing effect of Pseudomonas aeruginosa and the ratio of the area under the concentration–time curve (AUC) of plasma concentrations of unbound colistin over MIC. According to experimental models, nebulization of CMS offers the possibility of generating high colistin concentrations at the site of infection.7 In addition, nebulized CMS is often used to treat ventilator-associated pneumonia (VAP) caused by MDR Gram-negative bacteria.8–13 The reported regimens for nebulized CMS range between 6 and 15 million international units (MIU) administered two or three times daily, and pharmacokinetic and clinical studies suggest that high doses of nebulized CMS compensate for colistin loss due to extrapulmonary deposition.12–16 The ‘summary of product characteristics’ recommends dissolving each 1 MIU of CMS with 3 mL of normal saline solution.17 As a result, 12 mL of saline is required when a high dose of 4 MIU is nebulized. Using high dilutions has, however, two disadvantages. First, it can increase nebulization time,18 compromising antibiotic stability and clinical tolerance.19 Second, as the volumes of the nebulizer chambers generally used for nebulization of antibiotics are often <10 mL, the nebulizer chamber needs to be filled at least twice to nebulize 12 mL of CMS. This not only increases nurses’ workload, but also carries the risk of incomplete administration. Decreasing the volume of diluent for the same dose of drug could have the advantage of reducing preparation and delivery time; however, the increased viscosity could change the properties of the aerosol. The impact of CMS concentration on the efficiency of aerosol delivery has never been studied. The aim of this study was to compare aerosol characteristics and pharmacokinetics between two dilutions of CMS in patients with VAP due to MDR Gram-negative bacteria treated with nebulized CMS. To this purpose, we conducted a bench study to determine the CMS particle size and a clinical study to assess the plasma pharmacokinetics and urinary elimination of CMS and colistin following administration of nebulized CMS. Methods From April 2015 to July 2016, a particle size study of the aerosols produced by nebulizers was performed in the laboratory of the Service des Explorations Fonctionnelles de la Respiration, de l’Exercice et de la Dyspnée and a prospective and crossover clinical study was conducted in the multidisciplinary ICU. Concentrations of colistin and CMS were measured in the pharmacology department of La Pitié-Salpêtrière Hospital. In both studies, 6 mL (experimental dilution) and 12 mL (recommended dilution) diluent volumes for 4 MIU of CMS were compared; a vibrating-mesh nebulizer with a maximum fill volume of 6 mL was used for nebulization (Aeroneb® Solo, Galway, Ireland). The study protocol was approved by the Comité de Protection des Personnes (CPP-Ile-de-France VI) and was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Informed consent was obtained from the patients and/or patients’ relatives. Bench study for determination of particle sizes The nebulizer chamber was loaded with 4 MIU of CMS dry powder for nebulization (Colimycin, Sanofi-Aventis, France), diluted in 6 or 12 mL of normal saline solution. The nebulizer was connected to the inspiratory limb of the ventilator circuit, 15 cm away from the Y-piece. During nebulization, the following ventilator settings were used: volume-controlled mode with constant inspiratory flow, respiratory rate of 12 breaths/min, inspiratory/expiratory (I/E) ratio of 50% and end-inspiratory pause representing 20% of the duty cycle. Aerosol particle size was determined using a laser aerosol particle sizer based on light diffraction with a measurement range between 0.3 and 10 μm over 16 channels (Topas Gmbh®, Dresden, Germany). Direct sampling of the aerosol was done under a suction hood (temperature 23–5°C, humidity 39%–46%) at the tip of the endotracheal tube (inner diameter 7.5 mm; Covidien, Ireland) with a sampling flow rate of 3 L/min. Analysis of the aerosol during 10 s runs provided the particle size spectrum and allowed the calculation of the mass median aerodynamic diameter (MMAD or D50) and of the diameter below which 90% of the aerosolized particle sizes are included (D90). This analysis was repeated every 15 min until the nebulizer chamber emptied (a total of 12 measurements per hour) and each dilution was studied three times. The primary endpoint was the aerosol particle size (D50 and D90) produced during nebulization and the second endpoint was the aerosol particle size produced at the start and end time of nebulization. Pharmacokinetic study in patients with VAP The criteria for eligibility were age >18 years and a VAP due to MDR Gram-negative bacteria requiring treatment with nebulized CMS.20 Patients with renal failure requiring renal replacement and patients treated with a combination of nebulized and intravenous CMS were excluded from the study. Creatinine clearance (CLCR) was estimated by measuring the 24 h urine/plasma creatinine ratio. All patients were treated with 4 MIU of CMS nebulized every 8 h (corresponding to 320 mg of CMS). At inclusion, the eight patients were randomized into two fixed blocks of four, one block comprising the 6 mL dilution arm (study started with 6 mL) and the other the 12 mL dilution arm (study started with 12 mL). The solutions of 4 MUI of CMS dissolved in 6 or 12 mL of normal saline were nebulized using a vibrating-mesh nebulizer according to a random order. The CMS solutions were prepared immediately before administration. The nebulizer was connected to the inspiratory limb of a non-humidified ventilator circuit, 15 cm from the Y-piece. During nebulization, mechanical ventilation was performed using a volume-controlled mode with a constant flow, an I/E ratio of 0.5, a respiratory frequency between 14 and 18 breaths/min and a tidal volume of 6–8 mL/kg. A strict coordination between patient and ventilator was required and therefore 2 mg/kg propofol was infused to mitigate patient–ventilator asynchrony. The study protocol is shown in Figure 1. For each dilution, sampling was performed after a minimum of two consecutive days of administration of nebulized CMS with the same dilution. Thereafter, the treatment was switched to the second dilution. Five successive plasma and urine samples were drawn immediately before the administration of CMS and 2, 4, 6 and 8 h after starting nebulization of CMS. All blood samples were collected in tubes containing lithium heparin, and plasma was separated immediately into 5 mL polypropylene vials. Urine samples were collected directly from the proximal part of the bladder catheter every 2 h and urine volume was measured at the time of each urine collection. Plasma and urine samples were immediately stored at –80°C until analysis within a maximum period of 2 months.21 Figure 1. View largeDownload slide Clinical study protocol. PK = pharmacokinetic. Five successive vertical bars = PK study period of urine and plasma sampling at 0, 2, 4, 6 and 8 h after the beginning of CMS nebulization. D = day. Screening day = first day of CMS nebulization. Randomization day = patient randomized to either the 6 mL arm (group of patients starting CMS nebulization with 6 mL diluent volume) or the 12 mL arm (group of patients starting CMS nebulization with 12 mL diluent volume). Figure 1. View largeDownload slide Clinical study protocol. PK = pharmacokinetic. Five successive vertical bars = PK study period of urine and plasma sampling at 0, 2, 4, 6 and 8 h after the beginning of CMS nebulization. D = day. Screening day = first day of CMS nebulization. Randomization day = patient randomized to either the 6 mL arm (group of patients starting CMS nebulization with 6 mL diluent volume) or the 12 mL arm (group of patients starting CMS nebulization with 12 mL diluent volume). The primary endpoint was the plasma AUC from 0 to 8 h (AUC0–8) of colistin A, colistin B, and the sum of colistin A and B (colistinA+B). The secondary endpoints were based on the following parameters: Plasma AUC0–8 of CMS A and CMS B and the sum of CMS A and B (CMSA+B). Renal clearance (CLR) of colistin A, colistin B, CMS A and CMS B. Urinary elimination of colistinA+B and CMSA+B from 0 to 8 h (Qu0–8). Nebulization time. Correlations between plasma trough concentration (C0) of colistinA+B or CMSA+B and CLCR, and between plasma AUC0–8 of colistinA+B or CMSA+B and CLCR measured on the same day. Stability of colistin A and colistin B in normal saline solution. Samples were analysed for colistin A and B by UPLC-MS/MS using a method developed by Bihan et al.12 The method proved to be accurate and precise in the range from 30 to 6000 ng/mL for colistin A and from 15 to 3000 ng/mL for colistin B; the within-day precision (CV %) was lower than 15%. The limit of quantification was 60 ng/mL for colistin A and 30 ng/mL for colistin B.12 The plasma pharmacokinetics of colistin and CMS were evaluated from the concentration–time data at each dilution by a non-compartmental analysis using WinNonlin pharmacokinetic software, version 5.2 (Pharsight Corporation®, Mountain View, CA, USA). Graphical analysis and determination of AUC0–8 were performed using a linear trapezoidal model. Colistin and CMS CLR was estimated using the following formula: CLR=(U×V)/P where U and P are the urine and plasma antibiotic concentrations and V is the 2 h urine volume. The total amount of colistin and CMS eliminated in the urine (Qu0–8) between two nebulizations was estimated by adding the amount eliminated every 2 h. CMS concentrations were calculated indirectly from the difference between colistin concentrations determined after acidic treatment and unhydrolysed colistin concentrations directly determined without acidic treatment as previously described.12,22,23 The stability of colistin A or B was assessed in triplicate at two dilutions: 1 MIU of CMS in 1.5 mL of normal saline (representing 4 MUI/6 mL) and in 3.0 mL of normal saline (representing 4 MUI/12 mL). Each sample was kept at room temperature for 9 h and analysed at 1, 2, 4, 6 and 9 h after reconstitution. Both molecules were considered stable in normal saline when measured signal intensities were within the limits of 95%–105% of the initial signal intensity. Statistical analysis Statistical analysis was performed using Sigmastat, version 3.5 (SystatS®, USA) software. Quantitative variables are presented as mean ± SD for the bench study and median (IQR) for the clinical study. Particle sizes produced with 6 and 12 mL diluent volumes were compared using bilateral unpaired Student’s t-test. Two-way analysis of variance for repeated measures was used to compare changes in particle sizes (D50 and D90) over time using 6 and 12 mL diluent volumes; these parameters were log-transformed. Differences in pharmacokinetic parameters in patients between 6 and 12 mL diluent volumes were evaluated with the Wilcoxon signed-rank test. Correlations between CLCR and C0 or AUC0–8 of colistinA+B or CMSA+B were analysed using the Spearman correlation. A P value of <0.05 was considered statistically significant. Results Aerosol particle sizes Overall, D50 (MMAD) was bigger with 6 mL than with 12 mL of diluent volume (1.4 ± 0.2 versus 0.9 ± 0.2 μm, P < 0.001). At the end of nebulization, 90% of aerosolized particles had a diameter of <3.0 ± 0.6 μm when using a diluent volume of 6 mL. As shown in Figure 2, with a diluent volume of 12 mL, D50 and D90 increased significantly from the beginning to the end of nebulization compared with when a diluent volume of 6 mL was used. The particle sizes were not different between the two diluent volumes at the end of nebulization for D90 (P = 0.741). The nebulization time was significantly shorter with the 6 mL dilution (63 ± 3 versus 90 ± 15 min, P = 0.04). Figure 2. View largeDownload slide Changes in particles sizes of CMS and colistin from the start to the end of one nebulization. The changes in particle sizes over time were analysed using a two-way ANOVA for repeated measures with two factors: effect of diluent volume (6 versus 12 mL) and effect of time (start versus end time of nebulization). The presence of an interaction (P < 0.05) between the two factors indicates a significant increase in particle sizes from the start to the end of nebulization with a diluent volume of 12 mL compared with a diluent volume of 6 mL. Open circles = 12 mL diluent volume. Filled circles = 6 mL diluent volume. T0 = start time of nebulization. T60 = end time of nebulization for 6 mL diluent volume. T75–90 = end time of nebulization for 12 mL diluent volume. Figure 2. View largeDownload slide Changes in particles sizes of CMS and colistin from the start to the end of one nebulization. The changes in particle sizes over time were analysed using a two-way ANOVA for repeated measures with two factors: effect of diluent volume (6 versus 12 mL) and effect of time (start versus end time of nebulization). The presence of an interaction (P < 0.05) between the two factors indicates a significant increase in particle sizes from the start to the end of nebulization with a diluent volume of 12 mL compared with a diluent volume of 6 mL. Open circles = 12 mL diluent volume. Filled circles = 6 mL diluent volume. T0 = start time of nebulization. T60 = end time of nebulization for 6 mL diluent volume. T75–90 = end time of nebulization for 12 mL diluent volume. Colistin and CMS pharmacokinetics Eight patients who fulfilled the inclusion criteria were included: four in each arm of crossover CMS dilution. The clinical characteristics of the patients are summarized in Table 1. CLCR values did not differ between the two conditions studied (P = 0.461). The duration of invasive mechanical ventilation before inclusion was 19 (8–27) days. VAP was caused by P. aeruginosa in seven patients and by Acinetobacter baumannii in one patient. The duration of treatment was 9 (8–11) days; the clinical cure rate was 63% and the ICU mortality was 13%. Table 1. Clinical characteristics of patients Patient Age (years) Gender Weight (kg) Diagnosis at admission Simplified acute physiology II score at admission CLCR 6 mL (mL/min) CLCR 12 mL (mL/min) Inspiratory constant flow delivered by ventilator (L/min) 1 19 male 78 multiple trauma 57 200 197 15.4 2 64 male 70 pneumonia 24 20 27 11.2 3 73 male 82 pneumonia 32 32 20 12.6 4 60 male 84 post-operative liver transplantation 49 60 72 14.4 5 68 female 61 acute respiratory failure 46 68 92 14.0 6 72 male 81 aspiration pneumonia 49 112 105 11.2 7 58 male 100 pericardial tamponade 37 44 41 16.8 8 44 male 140 acute respiratory distress syndrome 33 72 90 19.8 Median (IQR) 62 (51–70) 82 (74–92) 42 (33–49) 64 (38–92) 81 (34–99) 14.2 (11.9–16.1) Patient Age (years) Gender Weight (kg) Diagnosis at admission Simplified acute physiology II score at admission CLCR 6 mL (mL/min) CLCR 12 mL (mL/min) Inspiratory constant flow delivered by ventilator (L/min) 1 19 male 78 multiple trauma 57 200 197 15.4 2 64 male 70 pneumonia 24 20 27 11.2 3 73 male 82 pneumonia 32 32 20 12.6 4 60 male 84 post-operative liver transplantation 49 60 72 14.4 5 68 female 61 acute respiratory failure 46 68 92 14.0 6 72 male 81 aspiration pneumonia 49 112 105 11.2 7 58 male 100 pericardial tamponade 37 44 41 16.8 8 44 male 140 acute respiratory distress syndrome 33 72 90 19.8 Median (IQR) 62 (51–70) 82 (74–92) 42 (33–49) 64 (38–92) 81 (34–99) 14.2 (11.9–16.1) Table 1. Clinical characteristics of patients Patient Age (years) Gender Weight (kg) Diagnosis at admission Simplified acute physiology II score at admission CLCR 6 mL (mL/min) CLCR 12 mL (mL/min) Inspiratory constant flow delivered by ventilator (L/min) 1 19 male 78 multiple trauma 57 200 197 15.4 2 64 male 70 pneumonia 24 20 27 11.2 3 73 male 82 pneumonia 32 32 20 12.6 4 60 male 84 post-operative liver transplantation 49 60 72 14.4 5 68 female 61 acute respiratory failure 46 68 92 14.0 6 72 male 81 aspiration pneumonia 49 112 105 11.2 7 58 male 100 pericardial tamponade 37 44 41 16.8 8 44 male 140 acute respiratory distress syndrome 33 72 90 19.8 Median (IQR) 62 (51–70) 82 (74–92) 42 (33–49) 64 (38–92) 81 (34–99) 14.2 (11.9–16.1) Patient Age (years) Gender Weight (kg) Diagnosis at admission Simplified acute physiology II score at admission CLCR 6 mL (mL/min) CLCR 12 mL (mL/min) Inspiratory constant flow delivered by ventilator (L/min) 1 19 male 78 multiple trauma 57 200 197 15.4 2 64 male 70 pneumonia 24 20 27 11.2 3 73 male 82 pneumonia 32 32 20 12.6 4 60 male 84 post-operative liver transplantation 49 60 72 14.4 5 68 female 61 acute respiratory failure 46 68 92 14.0 6 72 male 81 aspiration pneumonia 49 112 105 11.2 7 58 male 100 pericardial tamponade 37 44 41 16.8 8 44 male 140 acute respiratory distress syndrome 33 72 90 19.8 Median (IQR) 62 (51–70) 82 (74–92) 42 (33–49) 64 (38–92) 81 (34–99) 14.2 (11.9–16.1) As shown in Figure 3, significant inter-individual variability was observed in plasma concentrations of colistinA+B and CMSA+B. However, as shown in Tables 2 and 3, the plasma AUC0–8 for colistinA+B and CMSA+B, the CLR of colistin A and B and CMS A and B, and the amount of colistinA+B and CMSA+B eliminated in the urine between 0 and 8 h did not differ between patients treated with either 6 or 12 mL diluent volumes. The total amount of colistin and CMS eliminated in the urine represented, respectively, 17% and 13% of the CMS initially placed in the nebulizer chamber for the 6 and 12 mL diluent volumes (P = 0.4). There were no correlations between CLCR and Qu0–8 for colistin and CMS. Table 2. Pharmacokinetic parameters of colistin A, B and colistinA+B, and CMS A, B and CMSA+B for 6 and 12 mL diluent volumes Diluent volume (mL) Plasma AUC0–8 (μg·h/mL) P Qu0–8 (mg) P CLR (mL/min) P Colistin A 6 5.4 (3.5–13.7) 0.383 3.7 (2.8–5.2) 0.461 10.5 (4.7–17.6) 0.383 12 5.5 (2.9–11.2) 3.0 (2.2–4.1) 10.1 (3.3–12.9) Colistin B 6 1.2 (0.8–3.3) 0.461 1.1 (0.7–1.4) 0.461 12.4 (6.0–22.8) 0.461 12 1.1 (0.7–2.8) 0.8 (0.6–1.0) 11.9 (3.8–15.0) ColistinA+B 6 6.6 (4.3–17.0) 0.461 4.8 (3.6–6.8) 0.461 12 6.7 (3.6–14.0) 3.8 (2.8–5.1) CMS A 6 50.0 (26.1–56.6) 0.547 25.4 (18.6–50.5) 0.844 12.1 (8.9–16.2) 0.844 12 33.9 (29.2–66.0) 21.2 (14.3–41.6) 7.7 (4.4–14.4) CMS B 6 16.9 (7.8–19.9) 0.195 11.5 (3.1–21.8) 0.844 13.1 (5.7–20.9) 0.742 12 11.2 (7.2–22.7) 8.1 (4.6–15.0) 8.7 (4.6–18.2) CMSA+B 6 67.4 (33.9–76.1) 0.461 35.9 (22.6–72.2) 0.742 12 43.9 (37.5–88.7) 30.9 (19.2–54.6) Diluent volume (mL) Plasma AUC0–8 (μg·h/mL) P Qu0–8 (mg) P CLR (mL/min) P Colistin A 6 5.4 (3.5–13.7) 0.383 3.7 (2.8–5.2) 0.461 10.5 (4.7–17.6) 0.383 12 5.5 (2.9–11.2) 3.0 (2.2–4.1) 10.1 (3.3–12.9) Colistin B 6 1.2 (0.8–3.3) 0.461 1.1 (0.7–1.4) 0.461 12.4 (6.0–22.8) 0.461 12 1.1 (0.7–2.8) 0.8 (0.6–1.0) 11.9 (3.8–15.0) ColistinA+B 6 6.6 (4.3–17.0) 0.461 4.8 (3.6–6.8) 0.461 12 6.7 (3.6–14.0) 3.8 (2.8–5.1) CMS A 6 50.0 (26.1–56.6) 0.547 25.4 (18.6–50.5) 0.844 12.1 (8.9–16.2) 0.844 12 33.9 (29.2–66.0) 21.2 (14.3–41.6) 7.7 (4.4–14.4) CMS B 6 16.9 (7.8–19.9) 0.195 11.5 (3.1–21.8) 0.844 13.1 (5.7–20.9) 0.742 12 11.2 (7.2–22.7) 8.1 (4.6–15.0) 8.7 (4.6–18.2) CMSA+B 6 67.4 (33.9–76.1) 0.461 35.9 (22.6–72.2) 0.742 12 43.9 (37.5–88.7) 30.9 (19.2–54.6) Data are expressed as median (IQR). Table 2. Pharmacokinetic parameters of colistin A, B and colistinA+B, and CMS A, B and CMSA+B for 6 and 12 mL diluent volumes Diluent volume (mL) Plasma AUC0–8 (μg·h/mL) P Qu0–8 (mg) P CLR (mL/min) P Colistin A 6 5.4 (3.5–13.7) 0.383 3.7 (2.8–5.2) 0.461 10.5 (4.7–17.6) 0.383 12 5.5 (2.9–11.2) 3.0 (2.2–4.1) 10.1 (3.3–12.9) Colistin B 6 1.2 (0.8–3.3) 0.461 1.1 (0.7–1.4) 0.461 12.4 (6.0–22.8) 0.461 12 1.1 (0.7–2.8) 0.8 (0.6–1.0) 11.9 (3.8–15.0) ColistinA+B 6 6.6 (4.3–17.0) 0.461 4.8 (3.6–6.8) 0.461 12 6.7 (3.6–14.0) 3.8 (2.8–5.1) CMS A 6 50.0 (26.1–56.6) 0.547 25.4 (18.6–50.5) 0.844 12.1 (8.9–16.2) 0.844 12 33.9 (29.2–66.0) 21.2 (14.3–41.6) 7.7 (4.4–14.4) CMS B 6 16.9 (7.8–19.9) 0.195 11.5 (3.1–21.8) 0.844 13.1 (5.7–20.9) 0.742 12 11.2 (7.2–22.7) 8.1 (4.6–15.0) 8.7 (4.6–18.2) CMSA+B 6 67.4 (33.9–76.1) 0.461 35.9 (22.6–72.2) 0.742 12 43.9 (37.5–88.7) 30.9 (19.2–54.6) Diluent volume (mL) Plasma AUC0–8 (μg·h/mL) P Qu0–8 (mg) P CLR (mL/min) P Colistin A 6 5.4 (3.5–13.7) 0.383 3.7 (2.8–5.2) 0.461 10.5 (4.7–17.6) 0.383 12 5.5 (2.9–11.2) 3.0 (2.2–4.1) 10.1 (3.3–12.9) Colistin B 6 1.2 (0.8–3.3) 0.461 1.1 (0.7–1.4) 0.461 12.4 (6.0–22.8) 0.461 12 1.1 (0.7–2.8) 0.8 (0.6–1.0) 11.9 (3.8–15.0) ColistinA+B 6 6.6 (4.3–17.0) 0.461 4.8 (3.6–6.8) 0.461 12 6.7 (3.6–14.0) 3.8 (2.8–5.1) CMS A 6 50.0 (26.1–56.6) 0.547 25.4 (18.6–50.5) 0.844 12.1 (8.9–16.2) 0.844 12 33.9 (29.2–66.0) 21.2 (14.3–41.6) 7.7 (4.4–14.4) CMS B 6 16.9 (7.8–19.9) 0.195 11.5 (3.1–21.8) 0.844 13.1 (5.7–20.9) 0.742 12 11.2 (7.2–22.7) 8.1 (4.6–15.0) 8.7 (4.6–18.2) CMSA+B 6 67.4 (33.9–76.1) 0.461 35.9 (22.6–72.2) 0.742 12 43.9 (37.5–88.7) 30.9 (19.2–54.6) Data are expressed as median (IQR). Table 3. Plasma AUC0–8 and C0 of colistinA+B and CMSA+B ColistinA+B CMSA+B AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL Patient 1 8.47 7.23 0.61 0.73 23.18 10.95 0.89 0.648 Patient 2 23.74 17.94 2.88 2.13 34.81 34.56 3.49 2.99 Patient 3 21.66 26.38 2.75 3.21 32.95 40.74 3.45 3.86 Patient 4 4.31 3.76 0.472 0.474 77.48 72.96 8.02 7.82 Patient 5 4.81 2.59 0.516 0.367 74.68 40.41 6.52 4.33 Patient 6 4.37 3.41 0.451 0.417 74.68 47.16 6.64 5.67 Patient 7 12.38 10.12 1.59 1.31 141.72 136.63 16.96 13.98 Patient 8 4.17 6.07 0.49 0.64 60.07 104.37 7.52 11.02 Median (IQR) 6.6 (4.3–17.0) 6.7 (3.6–14.0) 0.6 (0.5–2.2) 0.7 (0.4–1.7) 67.4 (33.9–76.1) 43.9 (37.5–88.7) 6.6 (3.5–7.8) 5.0 (3.4–9.4) ColistinA+B CMSA+B AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL Patient 1 8.47 7.23 0.61 0.73 23.18 10.95 0.89 0.648 Patient 2 23.74 17.94 2.88 2.13 34.81 34.56 3.49 2.99 Patient 3 21.66 26.38 2.75 3.21 32.95 40.74 3.45 3.86 Patient 4 4.31 3.76 0.472 0.474 77.48 72.96 8.02 7.82 Patient 5 4.81 2.59 0.516 0.367 74.68 40.41 6.52 4.33 Patient 6 4.37 3.41 0.451 0.417 74.68 47.16 6.64 5.67 Patient 7 12.38 10.12 1.59 1.31 141.72 136.63 16.96 13.98 Patient 8 4.17 6.07 0.49 0.64 60.07 104.37 7.52 11.02 Median (IQR) 6.6 (4.3–17.0) 6.7 (3.6–14.0) 0.6 (0.5–2.2) 0.7 (0.4–1.7) 67.4 (33.9–76.1) 43.9 (37.5–88.7) 6.6 (3.5–7.8) 5.0 (3.4–9.4) a Mean value of C0 at 0 and 8 h. Table 3. Plasma AUC0–8 and C0 of colistinA+B and CMSA+B ColistinA+B CMSA+B AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL Patient 1 8.47 7.23 0.61 0.73 23.18 10.95 0.89 0.648 Patient 2 23.74 17.94 2.88 2.13 34.81 34.56 3.49 2.99 Patient 3 21.66 26.38 2.75 3.21 32.95 40.74 3.45 3.86 Patient 4 4.31 3.76 0.472 0.474 77.48 72.96 8.02 7.82 Patient 5 4.81 2.59 0.516 0.367 74.68 40.41 6.52 4.33 Patient 6 4.37 3.41 0.451 0.417 74.68 47.16 6.64 5.67 Patient 7 12.38 10.12 1.59 1.31 141.72 136.63 16.96 13.98 Patient 8 4.17 6.07 0.49 0.64 60.07 104.37 7.52 11.02 Median (IQR) 6.6 (4.3–17.0) 6.7 (3.6–14.0) 0.6 (0.5–2.2) 0.7 (0.4–1.7) 67.4 (33.9–76.1) 43.9 (37.5–88.7) 6.6 (3.5–7.8) 5.0 (3.4–9.4) ColistinA+B CMSA+B AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL Patient 1 8.47 7.23 0.61 0.73 23.18 10.95 0.89 0.648 Patient 2 23.74 17.94 2.88 2.13 34.81 34.56 3.49 2.99 Patient 3 21.66 26.38 2.75 3.21 32.95 40.74 3.45 3.86 Patient 4 4.31 3.76 0.472 0.474 77.48 72.96 8.02 7.82 Patient 5 4.81 2.59 0.516 0.367 74.68 40.41 6.52 4.33 Patient 6 4.37 3.41 0.451 0.417 74.68 47.16 6.64 5.67 Patient 7 12.38 10.12 1.59 1.31 141.72 136.63 16.96 13.98 Patient 8 4.17 6.07 0.49 0.64 60.07 104.37 7.52 11.02 Median (IQR) 6.6 (4.3–17.0) 6.7 (3.6–14.0) 0.6 (0.5–2.2) 0.7 (0.4–1.7) 67.4 (33.9–76.1) 43.9 (37.5–88.7) 6.6 (3.5–7.8) 5.0 (3.4–9.4) a Mean value of C0 at 0 and 8 h. Figure 3. View largeDownload slide Individual plasma concentration–time profiles (μg/mL) of colistinA+B (top panels) and CMSA+B (bottom panels) for 6 and 12 mL diluent volumes. Figure 3. View largeDownload slide Individual plasma concentration–time profiles (μg/mL) of colistinA+B (top panels) and CMSA+B (bottom panels) for 6 and 12 mL diluent volumes. The plasma C0 of colistinA+B and the C0 of CMSA+B were similar for the two diluent volumes (P = 0.844 and P = 0.383, respectively). Significant negative correlations were found between the C0 of colistinA+B and CLCR (ρ = −0.713, P = 0.002) and between the plasma AUC0–8 of colistinA+B and CLCR (ρ = −0.663, P = 0.005), but there was no correlation between the C0 or plasma AUC0–8 of CMSA+B and CLCR. Nebulization time and stability of colistin A and B The nebulization time in patients was significantly shorter with the 6 mL dilution compared with the 12 mL dilution [66 (58–75) versus 93 (69–136) min, P = 0.042]. As shown in Figure 4, for colistin A, signal losses of 7.2% ± 6.9% and 34.9% ± 0.6% were observed 1 h after reconstitution of CMS with 6 and 12 mL, and 10.6% ± 7.4% and 40.5%± 2.9% after 2 h, respectively. For colistin B, signal losses of 20.8% ± 6.0% and 37.5%± 8.6% were observed 1 h after reconstitution with 6 and 12 mL, and 27.0%± 2.1% and 42.7%± 5.3% after 2 h, respectively. Colistin A and B signal losses at room temperature were greater when CMS was diluted in 12 mL. Less than 40% of the initial signal was detected for colistin A and colistin B after 9 h for both diluent volumes. Figure 4. View largeDownload slide Stability of colistin A (top panel) and colistin B (bottom panel) after reconstitution of 4 MIU in 6 mL (filled circles) or 12 mL (open circles) of 0.9% NaCl. Stability was considered to have been achieved when the measured signal intensities were within the limits of 95%–105% of the initial signal. Figure 4. View largeDownload slide Stability of colistin A (top panel) and colistin B (bottom panel) after reconstitution of 4 MIU in 6 mL (filled circles) or 12 mL (open circles) of 0.9% NaCl. Stability was considered to have been achieved when the measured signal intensities were within the limits of 95%–105% of the initial signal. Discussion The results of this study showed that 4 MIU of CMS diluted in 6 mL of normal saline generated MMADs of <3 μm, which is suitable for distal lung deposition. Plasma pharmacokinetic parameters as well as urinary excretion of colistinA+B and CMSA+B did not differ between the 6 and 12 mL diluent volumes, but nebulization time was shorter and colistin stability was better with the 6 mL diluent volume. This study provides the first quantitative description of the effect of reducing the diluent volume of nebulized CMS for treatment of VAP caused by MDR Gram-negative bacteria. Aerosol particle size is one of the most important variables for optimizing the dose deposited in the lung. Particles >5 μm have a strong tendency to deposit in the ventilator circuit and large airways,24 and the optimal MMAD for distal lung deposition ranges between 0.5 and 3 μm.25 An MMAD of 1.4 μm and 90% of particles <3.0 μm indicate that the dilution of 4 MIU in 6 mL is suitable for distal lung deposition. Another finding supporting the use of this dilution is the stability of particle size during the nebulization period compared with the 12 mL diluent volume. The main limitation of this clinical study is the lack of any analysis of the lung epithelial lining fluid (ELF) to assess how much drug was actually delivered to the distal lung fields. Because of the clinical difficulties involved in sampling ELF in patients every 2 h, we designed the present study to evaluate the differences in the plasma pharmacokinetic parameters of colistinA+B and CMSA+B between the two dilutions. Following CMS nebulization, some of the converted colistin deposited on the distal lung diffuses through the alveolar-epithelium to reach the pulmonary interstitium where colistin exerts its bactericidal activity by binding to bacterial cell membranes. It is known that plasma concentrations of colistin and CMS at the steady state following CMS nebulization are significantly lower than, but correlated to, the lung ELF concentrations.15,16,26 The crossover study made it possible to compare two diluent volumes because it eliminated most of the factors that influence plasma concentrations, such as ventilator settings, lung and kidney function, and inter-subject variability. Indeed, the pharmacokinetic analysis was performed with fewer time points. However, the observation that the plasma concentrations were stable over time and did not exhibit significant peaks suggests a regular diffusion of CMS and colistin through the alveolar–capillary barrier. The lack of any difference in the plasma AUC0–8 between the two diluent volumes suggests strongly that nebulization with 6 mL is as efficient as with 12 mL in terms of colistin lung parenchyma deposition. In agreement with previous studies, high inter-individual variability of plasma CMS and colistin concentrations was observed.14,15 The elimination pathways of colistin are for the most part unknown.27 One explanation for this variability could be the conversion speed of CMS to colistin in plasma, which depends on renal elimination efficiency, as we found a negative correlation between CLCR and colistin plasma concentrations. It is difficult to compare our pharmacokinetic data with previously published studies in which different methodologies, doses and dilutions were used.14,15 In order to maintain the stability of colistin, the urine sample was taken by directly puncturing the proximal part of the bladder catheter.22 It has been reported that 50% of the CMS dose intravenously administered is recovered in the urine within 24 h.28,29 In our study, 13%–17% of the CMS dose initially placed in the nebulizer chamber was recovered in the urine. The similar urinary elimination of CMSA+B and colistinA+B for each of the two dilutions used in this study supports the lack of any difference between the two diluent volumes in terms of lung deposition. Our results confirmed a previous study showing that the stability of CMS and colistin is concentration dependent.19 The instability of CMS solution over time at room temperature indicates that CMS solution should be reconstituted just before administration, and that the duration of nebulization should be limited as much as possible. Furthermore, colistin degradation products can cause pulmonary inflammatory reactions in animals, and may also contribute to human lung injury and induce bronchial hyper-reactivity.30 Therefore, the lower dilution volume may also be advantageous from a safety standpoint.31 The degradation products of colistin over time have not, however, been clearly identified.16,19 A vibrating-mesh nebulizer has been used to assess nebulized antibiotics in most experimental and clinical studies.7,11–13,15 This device has certain advantages compared with a jet nebulizer in terms of reduced residual volume and increased amount of aerosol delivery.32 A recent bench study using a vibrating-mesh nebulizer failed to find any difference in bronchodilator lung dose among different dilutions.33 Although the optimal dosing of nebulized CMS in terms of efficacy and toxicity needs to be defined by further clinical trials,15,34 the current clinical use of nebulized CMS for treating pneumonia tends to administer high doses to overcome extrapulmonary loss and optimize colistin lung deposition.11,13 For a vibrating-mesh nebulizer with a maximum fill volume of 6 mL, the use of a 6 mL diluent volume reduces nursing workload and nebulization time while increasing colistin stability. As a consequence, it could improve treatment efficiency and potentially reduce side effects.26,35,36 Our study is in line with recent expert opinion that strongly suggests focusing clinical investigations on achieving continuous improvements in nebulization techniques before implementing large clinical Phase III trials.26,34,37 It should be pointed out that the results obtained in our study cannot be generalized to drug delivery by jet or ultrasonic nebulizers. Jet nebulizers tend to have high residual volumes of drug remaining in the chamber at the end of nebulization. Hence, using an increased fill volume of a more dilute solution could decrease the amount of drug trapped in the dead volume and thereby increase the aerosol delivery.18 In conclusion, using a vibrating-mesh nebulizer for CMS delivery results in shorter nebulization time and better colistin stability with 4 MUI CMS in 6 mL normal saline. CMS and colistin pharmacokinetics are not influenced by reducing dilution. The aerosol particle size increases with the more concentrated solution, but the range remains in limits that do not impair distal lung deposition. These findings may contribute to improving clinical practice and optimizing the technique for CMS nebulization during clinical trials. Acknowledgements We thank all the ICU nurses, in particular Mr Jean-Baptiste Cartier, for their support and participation in this study. Funding All support was provided from institutional and/or departmental sources. Transparency declarations None to declare. References 1 Bergen PJ , Bulitta JB , Forrest A et al. Pharmacokinetic/pharmacodynamic investigation of colistin against Pseudomonas aeruginosa using an in vitro model . Antimicrob Agents Chemother 2010 ; 54 : 3783 – 9 . Google Scholar CrossRef Search ADS PubMed 2 Orwa JA , Govaerts C , Busson R et al. Isolation and structural characterization of colistin components . J Antibiot (Tokyo) 2001 ; 54 : 595 – 9 . Google Scholar CrossRef Search ADS PubMed 3 Suzuki T , Fujikawa K. Studies on the chemical structure of colistin. IV. Chemical structure of colistin B . J Biochem 1964 ; 56 : 182 – 9 . Google Scholar CrossRef Search ADS PubMed 4 Suzuki T , Hayashi K , Fujikawa K. Studies on the chemical structure of colistin. III. Enzymatic hydrolysis of colistin A . J Biochem 1963 ; 54 : 412 – 8 . Google Scholar CrossRef Search ADS PubMed 5 Suzuki T , Hayashi K , Fujikawa K et al. The chemical structure of polymyxin E: the identities of polymyxin E1 with colistin A and of polymyxin E2 with colistin B . J Biochem 1965 ; 57 : 226 – 7 . Google Scholar CrossRef Search ADS PubMed 6 Bergen PJ , Li J , Nation RL. Dosing of colistin—back to basic PK/PD . Curr Opin Pharmacol 2011 ; 11 : 464 – 9 . Google Scholar CrossRef Search ADS PubMed 7 Lu Q , Girardi C , Zhang M et al. Nebulized and intravenous colistin in experimental pneumonia caused by Pseudomonas aeruginosa . Intensive Care Med 2010 ; 36 : 1147 – 55 . Google Scholar CrossRef Search ADS PubMed 8 Ehrmann S , Roche-Campo F , Bodet-Contentin L et al. Aerosol therapy in intensive and intermediate care units: prospective observation of 2808 critically ill patients . Intensive Care Med 2016 ; 42 : 192 – 201 . Google Scholar CrossRef Search ADS PubMed 9 Sole-Lleonart C , Rouby JJ , Blot S et al. Nebulization of antiinfective agents in invasively mechanically ventilated adults: a systematic review and meta-analysis . Anesthesiology 2017 ; 126 : 890 – 908 . Google Scholar CrossRef Search ADS PubMed 10 Sole-Lleonart C , Rouby JJ , Chastre J et al. Intratracheal administration of antimicrobial agents in mechanically ventilated adults: an international survey on delivery practices and safety . Respir Care 2016 ; 61 : 1008 – 14 . Google Scholar CrossRef Search ADS PubMed 11 Abdellatif S , Trifi A , Daly F et al. Efficacy and toxicity of aerosolised colistin in ventilator-associated pneumonia: a prospective, randomised trial . Ann Intensive Care 2016 ; 6 : 26. Google Scholar CrossRef Search ADS PubMed 12 Bihan K , Lu Q , Enjalbert M et al. Determination of colistin and colistimethate levels in human plasma and urine by high-performance liquid chromatography-tandem mass spectrometry . Ther Drug Monit 2016 ; 38 : 796 – 803 . Google Scholar CrossRef Search ADS PubMed 13 Lu Q , Luo R , Bodin L et al. Efficacy of high-dose nebulized colistin in ventilator-associated pneumonia caused by multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii . Anesthesiology 2012 ; 117 : 1335 – 47 . Google Scholar CrossRef Search ADS PubMed 14 Athanassa ZE , Markantonis SL , Fousteri MZ et al. Pharmacokinetics of inhaled colistimethate sodium (CMS) in mechanically ventilated critically ill patients . Intensive Care Med 2012 ; 38 : 1779 – 86 . Google Scholar CrossRef Search ADS PubMed 15 Boisson M , Jacobs M , Grégoire N et al. Comparison of intrapulmonary and systemic pharmacokinetics of colistin methanesulfonate (CMS) and colistin after aerosol delivery and intravenous administration of CMS in critically ill patients . Antimicrob Agents Chemother 2014 ; 58 : 7331 – 9 . Google Scholar CrossRef Search ADS PubMed 16 Boisson M , Grégoire N , Cormier M et al. Pharmacokinetics of nebulized colistin methanesulfonate in critically ill patients . J Antimicrob Chemother 2017 ; 72 : 2607 – 12 . Google Scholar CrossRef Search ADS PubMed 17 Résumé des Caractéristiques du Produit: Colimycine. http://agence-prd.ansm.sante.fr/php/ecodex/rcp/R0271322.htm. Agence nationale de sécurité du médicament et des produits de santé, 2016 . 18 Hess D , Fisher D , Williams P et al. Medication nebulizer performance. Effects of diluent volume, nebulizer flow, and nebulizer brand . Chest 1996 ; 110 : 498 – 505 . Google Scholar CrossRef Search ADS PubMed 19 Wallace SJ , Li J , Rayner CR et al. Stability of colistin methanesulfonate in pharmaceutical products and solutions for administration to patients . Antimicrob Agents Chemother 2008 ; 52 : 3047 – 51 . Google Scholar CrossRef Search ADS PubMed 20 American Thoracic Society; Infectious Diseases Society of America . Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia . Am J Respir Crit Care Med 2005 ; 171 : 388 – 416 . CrossRef Search ADS PubMed 21 Dudhani RV , Nation RL , Li J. Evaluating the stability of colistin and colistin methanesulphonate in human plasma under different conditions of storage . J Antimicrob Chemother 2010 ; 65 : 1412 – 5 . Google Scholar CrossRef Search ADS PubMed 22 Gobin P , Lemaitre F , Marchand S et al. Assay of colistin and colistin methanesulfonate in plasma and urine by liquid chromatography-tandem mass spectrometry . Antimicrob Agents Chemother 2010 ; 54 : 1941 – 8 . Google Scholar CrossRef Search ADS PubMed 23 Ma Z , Wang J , Gerber JP et al. Determination of colistin in human plasma, urine and other biological samples using LC-MS/MS . J Chromatogr B Analyt Technol Biomed Life Sci 2008 ; 862 : 205 – 12 . Google Scholar CrossRef Search ADS PubMed 24 Brain JD , Valberg PA. Deposition of aerosol in the respiratory tract . Am Rev Respir Dis 1979 ; 120 : 1325 – 73 . Google Scholar PubMed 25 Folkesson HG , Westrom BR , Karlsson BW. Permeability of the respiratory tract to different-sized macromolecules after intratracheal instillation in young and adult rats . Acta Physiol Scand 1990 ; 139 : 347 – 54 . Google Scholar CrossRef Search ADS PubMed 26 Ehrmann S , Chastre J , Diot P et al. Nebulized antibiotics in mechanically ventilated patients: a challenge for translational research from technology to clinical care . Ann Intensive Care 2017 ; 7 : 78. Google Scholar CrossRef Search ADS PubMed 27 Grégoire N , Aranzana-Climent V , Magréault S et al. Clinical pharmacokinetics and pharmacodynamics of colistin . Clin Pharmacokinet 2017 ; 56 : 1441 – 60 . Google Scholar CrossRef Search ADS PubMed 28 Couet W , Grégoire N , Marchand S et al. Colistin pharmacokinetics: the fog is lifting . Clin Microbiol Infect 2012 ; 18 : 30 – 9 . Google Scholar CrossRef Search ADS PubMed 29 Garonzik SM , Li J , Thamlikitkul V et al. Population pharmacokinetics of colistin methanesulfonate and formed colistin in critically ill patients from a multicenter study provide dosing suggestions for various categories of patients . Antimicrob Agents Chemother 2011 ; 55 : 3284 – 94 . Google Scholar CrossRef Search ADS PubMed 30 McCoy KS. Compounded colistimethate as possible cause of fatal acute respiratory distress syndrome . N Engl J Med 2007 ; 357 : 2310 – 1 . Google Scholar CrossRef Search ADS PubMed 31 FDA Drug Safety Podcasts—Colistimethate (Marketed as Coly-Mycin M and Generic Products). https://www.drugs.com/fda-alerts/733-5015.html. 32 Ari A , Atalay OT , Harwood R et al. Influence of nebulizer type, position, and bias flow on aerosol drug delivery in simulated pediatric and adult lung models during mechanical ventilation . Respir Care 2010 ; 55 : 845 – 51 . Google Scholar PubMed 33 Berlinski A , Willis JR. Albuterol delivery by 4 different nebulizers placed in 4 different positions in a pediatric ventilator in vitro model . Respir Care 2013 ; 58 : 1124 – 33 . Google Scholar CrossRef Search ADS PubMed 34 Rello J , Sole-Lleonart C , Rouby JJ et al. Use of nebulized antimicrobials for the treatment of respiratory infections in invasively mechanically ventilated adults: a position paper from the European Society of Clinical Microbiology and Infectious Diseases . Clin Microbiol Infect 2017 ; 23 : 629 – 39 . Google Scholar CrossRef Search ADS PubMed 35 Lu Q , Yang J , Liu Z et al. Nebulized ceftazidime and amikacin in ventilator-associated pneumonia caused by Pseudomonas aeruginosa . Am J Respir Crit Care Med 2011 ; 184 : 106 – 15 . Google Scholar CrossRef Search ADS PubMed 36 Westerman EM , Le Brun PP , Touw DJ et al. Effect of nebulized colistin sulphate and colistin sulphomethate on lung function in patients with cystic fibrosis: a pilot study . J Cyst Fibros 2004 ; 3 : 23 – 8 . Google Scholar CrossRef Search ADS PubMed 37 Rello J , Rouby JJ , Sole-Lleonart C et al. Key considerations on nebulization of antimicrobial agents to mechanically ventilated patients . Clin Microbiol Infect 2017 ; 23 : 640 – 6 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Influence of diluent volume of colistimethate sodium on aerosol characteristics and pharmacokinetics in ventilator-associated pneumonia caused by MDR bacteria

Loading next page...
 
/lp/ou_press/influence-of-diluent-volume-of-colistimethate-sodium-on-aerosol-5f07DyoD9K
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
ISSN
0305-7453
eISSN
1460-2091
D.O.I.
10.1093/jac/dky044
Publisher site
See Article on Publisher Site

Abstract

Abstract Objectives Nebulized colistimethate sodium (CMS) can be used to treat ventilator-associated pneumonia caused by MDR bacteria. The influence of the diluent volume of CMS on aerosol delivery has never been studied. The main objectives of the study were to compare aerosol particle characteristics and plasma and urine pharmacokinetics between two diluent volumes in patients treated with nebulized CMS. Methods A crossover study was conducted in eight patients receiving nebulized CMS every 8 h. After inclusion, nebulization started with 4 million international units (MIU) of CMS diluted either in 6 mL (experimental dilution) or in 12 mL (recommended dilution) of normal saline in a random order. For each diluent volume, CMS aerosol particle sizes were measured and plasma and urine samples were collected every 2 h. Nebulization time and stability of colistin in normal saline were assessed. Results The mass median aerodynamic diameters were 1.4 ± 0.2 versus 0.9 ± 0.2 μm (P < 0.001) for 6 and 12 mL diluent volumes, respectively. The plasma area under the concentration–time curve from 0 to 8 h (AUC0–8) of colistinA+B was 6.6 (4.3–17.0) versus 6.7 (3.6–14.0) μg·h/mL (P = 0.461) for each dilution. The total amount of colistin and CMS eliminated in the urine represented, respectively, 17% and 13% of the CMS initially placed in the nebulizer chamber for 6 and 12 mL diluent volumes (P = 0.4). Nebulization time was shorter [66 (58–75) versus 93 (69–136) min, P = 0.042] and colistin stability was better with the 6 mL diluent volume. Conclusions Nebulization with a higher concentration of CMS in saline (4 MIU in 6 mL) decreases nebulization time and improves colistin stability without changing plasma and urine pharmacokinetics or aerosol particle characteristics for lung deposition. Introduction Colistimethate sodium (CMS) is an inactive pro-drug of colistin (active metabolite) that forms colistin by spontaneous hydrolysis in aqueous solution. Colistin is a complex, multicomponent antibiotic mixture,1 with colistin A and colistin B accounting for ∼85% of this mixture.2–5 The antibiotic effect of colistin A and B is concentration dependent.1 Bergen et al.1,6 additionally described a high correlation between the overall killing effect of Pseudomonas aeruginosa and the ratio of the area under the concentration–time curve (AUC) of plasma concentrations of unbound colistin over MIC. According to experimental models, nebulization of CMS offers the possibility of generating high colistin concentrations at the site of infection.7 In addition, nebulized CMS is often used to treat ventilator-associated pneumonia (VAP) caused by MDR Gram-negative bacteria.8–13 The reported regimens for nebulized CMS range between 6 and 15 million international units (MIU) administered two or three times daily, and pharmacokinetic and clinical studies suggest that high doses of nebulized CMS compensate for colistin loss due to extrapulmonary deposition.12–16 The ‘summary of product characteristics’ recommends dissolving each 1 MIU of CMS with 3 mL of normal saline solution.17 As a result, 12 mL of saline is required when a high dose of 4 MIU is nebulized. Using high dilutions has, however, two disadvantages. First, it can increase nebulization time,18 compromising antibiotic stability and clinical tolerance.19 Second, as the volumes of the nebulizer chambers generally used for nebulization of antibiotics are often <10 mL, the nebulizer chamber needs to be filled at least twice to nebulize 12 mL of CMS. This not only increases nurses’ workload, but also carries the risk of incomplete administration. Decreasing the volume of diluent for the same dose of drug could have the advantage of reducing preparation and delivery time; however, the increased viscosity could change the properties of the aerosol. The impact of CMS concentration on the efficiency of aerosol delivery has never been studied. The aim of this study was to compare aerosol characteristics and pharmacokinetics between two dilutions of CMS in patients with VAP due to MDR Gram-negative bacteria treated with nebulized CMS. To this purpose, we conducted a bench study to determine the CMS particle size and a clinical study to assess the plasma pharmacokinetics and urinary elimination of CMS and colistin following administration of nebulized CMS. Methods From April 2015 to July 2016, a particle size study of the aerosols produced by nebulizers was performed in the laboratory of the Service des Explorations Fonctionnelles de la Respiration, de l’Exercice et de la Dyspnée and a prospective and crossover clinical study was conducted in the multidisciplinary ICU. Concentrations of colistin and CMS were measured in the pharmacology department of La Pitié-Salpêtrière Hospital. In both studies, 6 mL (experimental dilution) and 12 mL (recommended dilution) diluent volumes for 4 MIU of CMS were compared; a vibrating-mesh nebulizer with a maximum fill volume of 6 mL was used for nebulization (Aeroneb® Solo, Galway, Ireland). The study protocol was approved by the Comité de Protection des Personnes (CPP-Ile-de-France VI) and was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Informed consent was obtained from the patients and/or patients’ relatives. Bench study for determination of particle sizes The nebulizer chamber was loaded with 4 MIU of CMS dry powder for nebulization (Colimycin, Sanofi-Aventis, France), diluted in 6 or 12 mL of normal saline solution. The nebulizer was connected to the inspiratory limb of the ventilator circuit, 15 cm away from the Y-piece. During nebulization, the following ventilator settings were used: volume-controlled mode with constant inspiratory flow, respiratory rate of 12 breaths/min, inspiratory/expiratory (I/E) ratio of 50% and end-inspiratory pause representing 20% of the duty cycle. Aerosol particle size was determined using a laser aerosol particle sizer based on light diffraction with a measurement range between 0.3 and 10 μm over 16 channels (Topas Gmbh®, Dresden, Germany). Direct sampling of the aerosol was done under a suction hood (temperature 23–5°C, humidity 39%–46%) at the tip of the endotracheal tube (inner diameter 7.5 mm; Covidien, Ireland) with a sampling flow rate of 3 L/min. Analysis of the aerosol during 10 s runs provided the particle size spectrum and allowed the calculation of the mass median aerodynamic diameter (MMAD or D50) and of the diameter below which 90% of the aerosolized particle sizes are included (D90). This analysis was repeated every 15 min until the nebulizer chamber emptied (a total of 12 measurements per hour) and each dilution was studied three times. The primary endpoint was the aerosol particle size (D50 and D90) produced during nebulization and the second endpoint was the aerosol particle size produced at the start and end time of nebulization. Pharmacokinetic study in patients with VAP The criteria for eligibility were age >18 years and a VAP due to MDR Gram-negative bacteria requiring treatment with nebulized CMS.20 Patients with renal failure requiring renal replacement and patients treated with a combination of nebulized and intravenous CMS were excluded from the study. Creatinine clearance (CLCR) was estimated by measuring the 24 h urine/plasma creatinine ratio. All patients were treated with 4 MIU of CMS nebulized every 8 h (corresponding to 320 mg of CMS). At inclusion, the eight patients were randomized into two fixed blocks of four, one block comprising the 6 mL dilution arm (study started with 6 mL) and the other the 12 mL dilution arm (study started with 12 mL). The solutions of 4 MUI of CMS dissolved in 6 or 12 mL of normal saline were nebulized using a vibrating-mesh nebulizer according to a random order. The CMS solutions were prepared immediately before administration. The nebulizer was connected to the inspiratory limb of a non-humidified ventilator circuit, 15 cm from the Y-piece. During nebulization, mechanical ventilation was performed using a volume-controlled mode with a constant flow, an I/E ratio of 0.5, a respiratory frequency between 14 and 18 breaths/min and a tidal volume of 6–8 mL/kg. A strict coordination between patient and ventilator was required and therefore 2 mg/kg propofol was infused to mitigate patient–ventilator asynchrony. The study protocol is shown in Figure 1. For each dilution, sampling was performed after a minimum of two consecutive days of administration of nebulized CMS with the same dilution. Thereafter, the treatment was switched to the second dilution. Five successive plasma and urine samples were drawn immediately before the administration of CMS and 2, 4, 6 and 8 h after starting nebulization of CMS. All blood samples were collected in tubes containing lithium heparin, and plasma was separated immediately into 5 mL polypropylene vials. Urine samples were collected directly from the proximal part of the bladder catheter every 2 h and urine volume was measured at the time of each urine collection. Plasma and urine samples were immediately stored at –80°C until analysis within a maximum period of 2 months.21 Figure 1. View largeDownload slide Clinical study protocol. PK = pharmacokinetic. Five successive vertical bars = PK study period of urine and plasma sampling at 0, 2, 4, 6 and 8 h after the beginning of CMS nebulization. D = day. Screening day = first day of CMS nebulization. Randomization day = patient randomized to either the 6 mL arm (group of patients starting CMS nebulization with 6 mL diluent volume) or the 12 mL arm (group of patients starting CMS nebulization with 12 mL diluent volume). Figure 1. View largeDownload slide Clinical study protocol. PK = pharmacokinetic. Five successive vertical bars = PK study period of urine and plasma sampling at 0, 2, 4, 6 and 8 h after the beginning of CMS nebulization. D = day. Screening day = first day of CMS nebulization. Randomization day = patient randomized to either the 6 mL arm (group of patients starting CMS nebulization with 6 mL diluent volume) or the 12 mL arm (group of patients starting CMS nebulization with 12 mL diluent volume). The primary endpoint was the plasma AUC from 0 to 8 h (AUC0–8) of colistin A, colistin B, and the sum of colistin A and B (colistinA+B). The secondary endpoints were based on the following parameters: Plasma AUC0–8 of CMS A and CMS B and the sum of CMS A and B (CMSA+B). Renal clearance (CLR) of colistin A, colistin B, CMS A and CMS B. Urinary elimination of colistinA+B and CMSA+B from 0 to 8 h (Qu0–8). Nebulization time. Correlations between plasma trough concentration (C0) of colistinA+B or CMSA+B and CLCR, and between plasma AUC0–8 of colistinA+B or CMSA+B and CLCR measured on the same day. Stability of colistin A and colistin B in normal saline solution. Samples were analysed for colistin A and B by UPLC-MS/MS using a method developed by Bihan et al.12 The method proved to be accurate and precise in the range from 30 to 6000 ng/mL for colistin A and from 15 to 3000 ng/mL for colistin B; the within-day precision (CV %) was lower than 15%. The limit of quantification was 60 ng/mL for colistin A and 30 ng/mL for colistin B.12 The plasma pharmacokinetics of colistin and CMS were evaluated from the concentration–time data at each dilution by a non-compartmental analysis using WinNonlin pharmacokinetic software, version 5.2 (Pharsight Corporation®, Mountain View, CA, USA). Graphical analysis and determination of AUC0–8 were performed using a linear trapezoidal model. Colistin and CMS CLR was estimated using the following formula: CLR=(U×V)/P where U and P are the urine and plasma antibiotic concentrations and V is the 2 h urine volume. The total amount of colistin and CMS eliminated in the urine (Qu0–8) between two nebulizations was estimated by adding the amount eliminated every 2 h. CMS concentrations were calculated indirectly from the difference between colistin concentrations determined after acidic treatment and unhydrolysed colistin concentrations directly determined without acidic treatment as previously described.12,22,23 The stability of colistin A or B was assessed in triplicate at two dilutions: 1 MIU of CMS in 1.5 mL of normal saline (representing 4 MUI/6 mL) and in 3.0 mL of normal saline (representing 4 MUI/12 mL). Each sample was kept at room temperature for 9 h and analysed at 1, 2, 4, 6 and 9 h after reconstitution. Both molecules were considered stable in normal saline when measured signal intensities were within the limits of 95%–105% of the initial signal intensity. Statistical analysis Statistical analysis was performed using Sigmastat, version 3.5 (SystatS®, USA) software. Quantitative variables are presented as mean ± SD for the bench study and median (IQR) for the clinical study. Particle sizes produced with 6 and 12 mL diluent volumes were compared using bilateral unpaired Student’s t-test. Two-way analysis of variance for repeated measures was used to compare changes in particle sizes (D50 and D90) over time using 6 and 12 mL diluent volumes; these parameters were log-transformed. Differences in pharmacokinetic parameters in patients between 6 and 12 mL diluent volumes were evaluated with the Wilcoxon signed-rank test. Correlations between CLCR and C0 or AUC0–8 of colistinA+B or CMSA+B were analysed using the Spearman correlation. A P value of <0.05 was considered statistically significant. Results Aerosol particle sizes Overall, D50 (MMAD) was bigger with 6 mL than with 12 mL of diluent volume (1.4 ± 0.2 versus 0.9 ± 0.2 μm, P < 0.001). At the end of nebulization, 90% of aerosolized particles had a diameter of <3.0 ± 0.6 μm when using a diluent volume of 6 mL. As shown in Figure 2, with a diluent volume of 12 mL, D50 and D90 increased significantly from the beginning to the end of nebulization compared with when a diluent volume of 6 mL was used. The particle sizes were not different between the two diluent volumes at the end of nebulization for D90 (P = 0.741). The nebulization time was significantly shorter with the 6 mL dilution (63 ± 3 versus 90 ± 15 min, P = 0.04). Figure 2. View largeDownload slide Changes in particles sizes of CMS and colistin from the start to the end of one nebulization. The changes in particle sizes over time were analysed using a two-way ANOVA for repeated measures with two factors: effect of diluent volume (6 versus 12 mL) and effect of time (start versus end time of nebulization). The presence of an interaction (P < 0.05) between the two factors indicates a significant increase in particle sizes from the start to the end of nebulization with a diluent volume of 12 mL compared with a diluent volume of 6 mL. Open circles = 12 mL diluent volume. Filled circles = 6 mL diluent volume. T0 = start time of nebulization. T60 = end time of nebulization for 6 mL diluent volume. T75–90 = end time of nebulization for 12 mL diluent volume. Figure 2. View largeDownload slide Changes in particles sizes of CMS and colistin from the start to the end of one nebulization. The changes in particle sizes over time were analysed using a two-way ANOVA for repeated measures with two factors: effect of diluent volume (6 versus 12 mL) and effect of time (start versus end time of nebulization). The presence of an interaction (P < 0.05) between the two factors indicates a significant increase in particle sizes from the start to the end of nebulization with a diluent volume of 12 mL compared with a diluent volume of 6 mL. Open circles = 12 mL diluent volume. Filled circles = 6 mL diluent volume. T0 = start time of nebulization. T60 = end time of nebulization for 6 mL diluent volume. T75–90 = end time of nebulization for 12 mL diluent volume. Colistin and CMS pharmacokinetics Eight patients who fulfilled the inclusion criteria were included: four in each arm of crossover CMS dilution. The clinical characteristics of the patients are summarized in Table 1. CLCR values did not differ between the two conditions studied (P = 0.461). The duration of invasive mechanical ventilation before inclusion was 19 (8–27) days. VAP was caused by P. aeruginosa in seven patients and by Acinetobacter baumannii in one patient. The duration of treatment was 9 (8–11) days; the clinical cure rate was 63% and the ICU mortality was 13%. Table 1. Clinical characteristics of patients Patient Age (years) Gender Weight (kg) Diagnosis at admission Simplified acute physiology II score at admission CLCR 6 mL (mL/min) CLCR 12 mL (mL/min) Inspiratory constant flow delivered by ventilator (L/min) 1 19 male 78 multiple trauma 57 200 197 15.4 2 64 male 70 pneumonia 24 20 27 11.2 3 73 male 82 pneumonia 32 32 20 12.6 4 60 male 84 post-operative liver transplantation 49 60 72 14.4 5 68 female 61 acute respiratory failure 46 68 92 14.0 6 72 male 81 aspiration pneumonia 49 112 105 11.2 7 58 male 100 pericardial tamponade 37 44 41 16.8 8 44 male 140 acute respiratory distress syndrome 33 72 90 19.8 Median (IQR) 62 (51–70) 82 (74–92) 42 (33–49) 64 (38–92) 81 (34–99) 14.2 (11.9–16.1) Patient Age (years) Gender Weight (kg) Diagnosis at admission Simplified acute physiology II score at admission CLCR 6 mL (mL/min) CLCR 12 mL (mL/min) Inspiratory constant flow delivered by ventilator (L/min) 1 19 male 78 multiple trauma 57 200 197 15.4 2 64 male 70 pneumonia 24 20 27 11.2 3 73 male 82 pneumonia 32 32 20 12.6 4 60 male 84 post-operative liver transplantation 49 60 72 14.4 5 68 female 61 acute respiratory failure 46 68 92 14.0 6 72 male 81 aspiration pneumonia 49 112 105 11.2 7 58 male 100 pericardial tamponade 37 44 41 16.8 8 44 male 140 acute respiratory distress syndrome 33 72 90 19.8 Median (IQR) 62 (51–70) 82 (74–92) 42 (33–49) 64 (38–92) 81 (34–99) 14.2 (11.9–16.1) Table 1. Clinical characteristics of patients Patient Age (years) Gender Weight (kg) Diagnosis at admission Simplified acute physiology II score at admission CLCR 6 mL (mL/min) CLCR 12 mL (mL/min) Inspiratory constant flow delivered by ventilator (L/min) 1 19 male 78 multiple trauma 57 200 197 15.4 2 64 male 70 pneumonia 24 20 27 11.2 3 73 male 82 pneumonia 32 32 20 12.6 4 60 male 84 post-operative liver transplantation 49 60 72 14.4 5 68 female 61 acute respiratory failure 46 68 92 14.0 6 72 male 81 aspiration pneumonia 49 112 105 11.2 7 58 male 100 pericardial tamponade 37 44 41 16.8 8 44 male 140 acute respiratory distress syndrome 33 72 90 19.8 Median (IQR) 62 (51–70) 82 (74–92) 42 (33–49) 64 (38–92) 81 (34–99) 14.2 (11.9–16.1) Patient Age (years) Gender Weight (kg) Diagnosis at admission Simplified acute physiology II score at admission CLCR 6 mL (mL/min) CLCR 12 mL (mL/min) Inspiratory constant flow delivered by ventilator (L/min) 1 19 male 78 multiple trauma 57 200 197 15.4 2 64 male 70 pneumonia 24 20 27 11.2 3 73 male 82 pneumonia 32 32 20 12.6 4 60 male 84 post-operative liver transplantation 49 60 72 14.4 5 68 female 61 acute respiratory failure 46 68 92 14.0 6 72 male 81 aspiration pneumonia 49 112 105 11.2 7 58 male 100 pericardial tamponade 37 44 41 16.8 8 44 male 140 acute respiratory distress syndrome 33 72 90 19.8 Median (IQR) 62 (51–70) 82 (74–92) 42 (33–49) 64 (38–92) 81 (34–99) 14.2 (11.9–16.1) As shown in Figure 3, significant inter-individual variability was observed in plasma concentrations of colistinA+B and CMSA+B. However, as shown in Tables 2 and 3, the plasma AUC0–8 for colistinA+B and CMSA+B, the CLR of colistin A and B and CMS A and B, and the amount of colistinA+B and CMSA+B eliminated in the urine between 0 and 8 h did not differ between patients treated with either 6 or 12 mL diluent volumes. The total amount of colistin and CMS eliminated in the urine represented, respectively, 17% and 13% of the CMS initially placed in the nebulizer chamber for the 6 and 12 mL diluent volumes (P = 0.4). There were no correlations between CLCR and Qu0–8 for colistin and CMS. Table 2. Pharmacokinetic parameters of colistin A, B and colistinA+B, and CMS A, B and CMSA+B for 6 and 12 mL diluent volumes Diluent volume (mL) Plasma AUC0–8 (μg·h/mL) P Qu0–8 (mg) P CLR (mL/min) P Colistin A 6 5.4 (3.5–13.7) 0.383 3.7 (2.8–5.2) 0.461 10.5 (4.7–17.6) 0.383 12 5.5 (2.9–11.2) 3.0 (2.2–4.1) 10.1 (3.3–12.9) Colistin B 6 1.2 (0.8–3.3) 0.461 1.1 (0.7–1.4) 0.461 12.4 (6.0–22.8) 0.461 12 1.1 (0.7–2.8) 0.8 (0.6–1.0) 11.9 (3.8–15.0) ColistinA+B 6 6.6 (4.3–17.0) 0.461 4.8 (3.6–6.8) 0.461 12 6.7 (3.6–14.0) 3.8 (2.8–5.1) CMS A 6 50.0 (26.1–56.6) 0.547 25.4 (18.6–50.5) 0.844 12.1 (8.9–16.2) 0.844 12 33.9 (29.2–66.0) 21.2 (14.3–41.6) 7.7 (4.4–14.4) CMS B 6 16.9 (7.8–19.9) 0.195 11.5 (3.1–21.8) 0.844 13.1 (5.7–20.9) 0.742 12 11.2 (7.2–22.7) 8.1 (4.6–15.0) 8.7 (4.6–18.2) CMSA+B 6 67.4 (33.9–76.1) 0.461 35.9 (22.6–72.2) 0.742 12 43.9 (37.5–88.7) 30.9 (19.2–54.6) Diluent volume (mL) Plasma AUC0–8 (μg·h/mL) P Qu0–8 (mg) P CLR (mL/min) P Colistin A 6 5.4 (3.5–13.7) 0.383 3.7 (2.8–5.2) 0.461 10.5 (4.7–17.6) 0.383 12 5.5 (2.9–11.2) 3.0 (2.2–4.1) 10.1 (3.3–12.9) Colistin B 6 1.2 (0.8–3.3) 0.461 1.1 (0.7–1.4) 0.461 12.4 (6.0–22.8) 0.461 12 1.1 (0.7–2.8) 0.8 (0.6–1.0) 11.9 (3.8–15.0) ColistinA+B 6 6.6 (4.3–17.0) 0.461 4.8 (3.6–6.8) 0.461 12 6.7 (3.6–14.0) 3.8 (2.8–5.1) CMS A 6 50.0 (26.1–56.6) 0.547 25.4 (18.6–50.5) 0.844 12.1 (8.9–16.2) 0.844 12 33.9 (29.2–66.0) 21.2 (14.3–41.6) 7.7 (4.4–14.4) CMS B 6 16.9 (7.8–19.9) 0.195 11.5 (3.1–21.8) 0.844 13.1 (5.7–20.9) 0.742 12 11.2 (7.2–22.7) 8.1 (4.6–15.0) 8.7 (4.6–18.2) CMSA+B 6 67.4 (33.9–76.1) 0.461 35.9 (22.6–72.2) 0.742 12 43.9 (37.5–88.7) 30.9 (19.2–54.6) Data are expressed as median (IQR). Table 2. Pharmacokinetic parameters of colistin A, B and colistinA+B, and CMS A, B and CMSA+B for 6 and 12 mL diluent volumes Diluent volume (mL) Plasma AUC0–8 (μg·h/mL) P Qu0–8 (mg) P CLR (mL/min) P Colistin A 6 5.4 (3.5–13.7) 0.383 3.7 (2.8–5.2) 0.461 10.5 (4.7–17.6) 0.383 12 5.5 (2.9–11.2) 3.0 (2.2–4.1) 10.1 (3.3–12.9) Colistin B 6 1.2 (0.8–3.3) 0.461 1.1 (0.7–1.4) 0.461 12.4 (6.0–22.8) 0.461 12 1.1 (0.7–2.8) 0.8 (0.6–1.0) 11.9 (3.8–15.0) ColistinA+B 6 6.6 (4.3–17.0) 0.461 4.8 (3.6–6.8) 0.461 12 6.7 (3.6–14.0) 3.8 (2.8–5.1) CMS A 6 50.0 (26.1–56.6) 0.547 25.4 (18.6–50.5) 0.844 12.1 (8.9–16.2) 0.844 12 33.9 (29.2–66.0) 21.2 (14.3–41.6) 7.7 (4.4–14.4) CMS B 6 16.9 (7.8–19.9) 0.195 11.5 (3.1–21.8) 0.844 13.1 (5.7–20.9) 0.742 12 11.2 (7.2–22.7) 8.1 (4.6–15.0) 8.7 (4.6–18.2) CMSA+B 6 67.4 (33.9–76.1) 0.461 35.9 (22.6–72.2) 0.742 12 43.9 (37.5–88.7) 30.9 (19.2–54.6) Diluent volume (mL) Plasma AUC0–8 (μg·h/mL) P Qu0–8 (mg) P CLR (mL/min) P Colistin A 6 5.4 (3.5–13.7) 0.383 3.7 (2.8–5.2) 0.461 10.5 (4.7–17.6) 0.383 12 5.5 (2.9–11.2) 3.0 (2.2–4.1) 10.1 (3.3–12.9) Colistin B 6 1.2 (0.8–3.3) 0.461 1.1 (0.7–1.4) 0.461 12.4 (6.0–22.8) 0.461 12 1.1 (0.7–2.8) 0.8 (0.6–1.0) 11.9 (3.8–15.0) ColistinA+B 6 6.6 (4.3–17.0) 0.461 4.8 (3.6–6.8) 0.461 12 6.7 (3.6–14.0) 3.8 (2.8–5.1) CMS A 6 50.0 (26.1–56.6) 0.547 25.4 (18.6–50.5) 0.844 12.1 (8.9–16.2) 0.844 12 33.9 (29.2–66.0) 21.2 (14.3–41.6) 7.7 (4.4–14.4) CMS B 6 16.9 (7.8–19.9) 0.195 11.5 (3.1–21.8) 0.844 13.1 (5.7–20.9) 0.742 12 11.2 (7.2–22.7) 8.1 (4.6–15.0) 8.7 (4.6–18.2) CMSA+B 6 67.4 (33.9–76.1) 0.461 35.9 (22.6–72.2) 0.742 12 43.9 (37.5–88.7) 30.9 (19.2–54.6) Data are expressed as median (IQR). Table 3. Plasma AUC0–8 and C0 of colistinA+B and CMSA+B ColistinA+B CMSA+B AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL Patient 1 8.47 7.23 0.61 0.73 23.18 10.95 0.89 0.648 Patient 2 23.74 17.94 2.88 2.13 34.81 34.56 3.49 2.99 Patient 3 21.66 26.38 2.75 3.21 32.95 40.74 3.45 3.86 Patient 4 4.31 3.76 0.472 0.474 77.48 72.96 8.02 7.82 Patient 5 4.81 2.59 0.516 0.367 74.68 40.41 6.52 4.33 Patient 6 4.37 3.41 0.451 0.417 74.68 47.16 6.64 5.67 Patient 7 12.38 10.12 1.59 1.31 141.72 136.63 16.96 13.98 Patient 8 4.17 6.07 0.49 0.64 60.07 104.37 7.52 11.02 Median (IQR) 6.6 (4.3–17.0) 6.7 (3.6–14.0) 0.6 (0.5–2.2) 0.7 (0.4–1.7) 67.4 (33.9–76.1) 43.9 (37.5–88.7) 6.6 (3.5–7.8) 5.0 (3.4–9.4) ColistinA+B CMSA+B AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL Patient 1 8.47 7.23 0.61 0.73 23.18 10.95 0.89 0.648 Patient 2 23.74 17.94 2.88 2.13 34.81 34.56 3.49 2.99 Patient 3 21.66 26.38 2.75 3.21 32.95 40.74 3.45 3.86 Patient 4 4.31 3.76 0.472 0.474 77.48 72.96 8.02 7.82 Patient 5 4.81 2.59 0.516 0.367 74.68 40.41 6.52 4.33 Patient 6 4.37 3.41 0.451 0.417 74.68 47.16 6.64 5.67 Patient 7 12.38 10.12 1.59 1.31 141.72 136.63 16.96 13.98 Patient 8 4.17 6.07 0.49 0.64 60.07 104.37 7.52 11.02 Median (IQR) 6.6 (4.3–17.0) 6.7 (3.6–14.0) 0.6 (0.5–2.2) 0.7 (0.4–1.7) 67.4 (33.9–76.1) 43.9 (37.5–88.7) 6.6 (3.5–7.8) 5.0 (3.4–9.4) a Mean value of C0 at 0 and 8 h. Table 3. Plasma AUC0–8 and C0 of colistinA+B and CMSA+B ColistinA+B CMSA+B AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL Patient 1 8.47 7.23 0.61 0.73 23.18 10.95 0.89 0.648 Patient 2 23.74 17.94 2.88 2.13 34.81 34.56 3.49 2.99 Patient 3 21.66 26.38 2.75 3.21 32.95 40.74 3.45 3.86 Patient 4 4.31 3.76 0.472 0.474 77.48 72.96 8.02 7.82 Patient 5 4.81 2.59 0.516 0.367 74.68 40.41 6.52 4.33 Patient 6 4.37 3.41 0.451 0.417 74.68 47.16 6.64 5.67 Patient 7 12.38 10.12 1.59 1.31 141.72 136.63 16.96 13.98 Patient 8 4.17 6.07 0.49 0.64 60.07 104.37 7.52 11.02 Median (IQR) 6.6 (4.3–17.0) 6.7 (3.6–14.0) 0.6 (0.5–2.2) 0.7 (0.4–1.7) 67.4 (33.9–76.1) 43.9 (37.5–88.7) 6.6 (3.5–7.8) 5.0 (3.4–9.4) ColistinA+B CMSA+B AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL AUC0–8 (μg·h/mL) 6 mL AUC0–8 (μg·h/mL) 12 mL C0a (μg/mL) 6 mL C0a (μg/mL) 12 mL Patient 1 8.47 7.23 0.61 0.73 23.18 10.95 0.89 0.648 Patient 2 23.74 17.94 2.88 2.13 34.81 34.56 3.49 2.99 Patient 3 21.66 26.38 2.75 3.21 32.95 40.74 3.45 3.86 Patient 4 4.31 3.76 0.472 0.474 77.48 72.96 8.02 7.82 Patient 5 4.81 2.59 0.516 0.367 74.68 40.41 6.52 4.33 Patient 6 4.37 3.41 0.451 0.417 74.68 47.16 6.64 5.67 Patient 7 12.38 10.12 1.59 1.31 141.72 136.63 16.96 13.98 Patient 8 4.17 6.07 0.49 0.64 60.07 104.37 7.52 11.02 Median (IQR) 6.6 (4.3–17.0) 6.7 (3.6–14.0) 0.6 (0.5–2.2) 0.7 (0.4–1.7) 67.4 (33.9–76.1) 43.9 (37.5–88.7) 6.6 (3.5–7.8) 5.0 (3.4–9.4) a Mean value of C0 at 0 and 8 h. Figure 3. View largeDownload slide Individual plasma concentration–time profiles (μg/mL) of colistinA+B (top panels) and CMSA+B (bottom panels) for 6 and 12 mL diluent volumes. Figure 3. View largeDownload slide Individual plasma concentration–time profiles (μg/mL) of colistinA+B (top panels) and CMSA+B (bottom panels) for 6 and 12 mL diluent volumes. The plasma C0 of colistinA+B and the C0 of CMSA+B were similar for the two diluent volumes (P = 0.844 and P = 0.383, respectively). Significant negative correlations were found between the C0 of colistinA+B and CLCR (ρ = −0.713, P = 0.002) and between the plasma AUC0–8 of colistinA+B and CLCR (ρ = −0.663, P = 0.005), but there was no correlation between the C0 or plasma AUC0–8 of CMSA+B and CLCR. Nebulization time and stability of colistin A and B The nebulization time in patients was significantly shorter with the 6 mL dilution compared with the 12 mL dilution [66 (58–75) versus 93 (69–136) min, P = 0.042]. As shown in Figure 4, for colistin A, signal losses of 7.2% ± 6.9% and 34.9% ± 0.6% were observed 1 h after reconstitution of CMS with 6 and 12 mL, and 10.6% ± 7.4% and 40.5%± 2.9% after 2 h, respectively. For colistin B, signal losses of 20.8% ± 6.0% and 37.5%± 8.6% were observed 1 h after reconstitution with 6 and 12 mL, and 27.0%± 2.1% and 42.7%± 5.3% after 2 h, respectively. Colistin A and B signal losses at room temperature were greater when CMS was diluted in 12 mL. Less than 40% of the initial signal was detected for colistin A and colistin B after 9 h for both diluent volumes. Figure 4. View largeDownload slide Stability of colistin A (top panel) and colistin B (bottom panel) after reconstitution of 4 MIU in 6 mL (filled circles) or 12 mL (open circles) of 0.9% NaCl. Stability was considered to have been achieved when the measured signal intensities were within the limits of 95%–105% of the initial signal. Figure 4. View largeDownload slide Stability of colistin A (top panel) and colistin B (bottom panel) after reconstitution of 4 MIU in 6 mL (filled circles) or 12 mL (open circles) of 0.9% NaCl. Stability was considered to have been achieved when the measured signal intensities were within the limits of 95%–105% of the initial signal. Discussion The results of this study showed that 4 MIU of CMS diluted in 6 mL of normal saline generated MMADs of <3 μm, which is suitable for distal lung deposition. Plasma pharmacokinetic parameters as well as urinary excretion of colistinA+B and CMSA+B did not differ between the 6 and 12 mL diluent volumes, but nebulization time was shorter and colistin stability was better with the 6 mL diluent volume. This study provides the first quantitative description of the effect of reducing the diluent volume of nebulized CMS for treatment of VAP caused by MDR Gram-negative bacteria. Aerosol particle size is one of the most important variables for optimizing the dose deposited in the lung. Particles >5 μm have a strong tendency to deposit in the ventilator circuit and large airways,24 and the optimal MMAD for distal lung deposition ranges between 0.5 and 3 μm.25 An MMAD of 1.4 μm and 90% of particles <3.0 μm indicate that the dilution of 4 MIU in 6 mL is suitable for distal lung deposition. Another finding supporting the use of this dilution is the stability of particle size during the nebulization period compared with the 12 mL diluent volume. The main limitation of this clinical study is the lack of any analysis of the lung epithelial lining fluid (ELF) to assess how much drug was actually delivered to the distal lung fields. Because of the clinical difficulties involved in sampling ELF in patients every 2 h, we designed the present study to evaluate the differences in the plasma pharmacokinetic parameters of colistinA+B and CMSA+B between the two dilutions. Following CMS nebulization, some of the converted colistin deposited on the distal lung diffuses through the alveolar-epithelium to reach the pulmonary interstitium where colistin exerts its bactericidal activity by binding to bacterial cell membranes. It is known that plasma concentrations of colistin and CMS at the steady state following CMS nebulization are significantly lower than, but correlated to, the lung ELF concentrations.15,16,26 The crossover study made it possible to compare two diluent volumes because it eliminated most of the factors that influence plasma concentrations, such as ventilator settings, lung and kidney function, and inter-subject variability. Indeed, the pharmacokinetic analysis was performed with fewer time points. However, the observation that the plasma concentrations were stable over time and did not exhibit significant peaks suggests a regular diffusion of CMS and colistin through the alveolar–capillary barrier. The lack of any difference in the plasma AUC0–8 between the two diluent volumes suggests strongly that nebulization with 6 mL is as efficient as with 12 mL in terms of colistin lung parenchyma deposition. In agreement with previous studies, high inter-individual variability of plasma CMS and colistin concentrations was observed.14,15 The elimination pathways of colistin are for the most part unknown.27 One explanation for this variability could be the conversion speed of CMS to colistin in plasma, which depends on renal elimination efficiency, as we found a negative correlation between CLCR and colistin plasma concentrations. It is difficult to compare our pharmacokinetic data with previously published studies in which different methodologies, doses and dilutions were used.14,15 In order to maintain the stability of colistin, the urine sample was taken by directly puncturing the proximal part of the bladder catheter.22 It has been reported that 50% of the CMS dose intravenously administered is recovered in the urine within 24 h.28,29 In our study, 13%–17% of the CMS dose initially placed in the nebulizer chamber was recovered in the urine. The similar urinary elimination of CMSA+B and colistinA+B for each of the two dilutions used in this study supports the lack of any difference between the two diluent volumes in terms of lung deposition. Our results confirmed a previous study showing that the stability of CMS and colistin is concentration dependent.19 The instability of CMS solution over time at room temperature indicates that CMS solution should be reconstituted just before administration, and that the duration of nebulization should be limited as much as possible. Furthermore, colistin degradation products can cause pulmonary inflammatory reactions in animals, and may also contribute to human lung injury and induce bronchial hyper-reactivity.30 Therefore, the lower dilution volume may also be advantageous from a safety standpoint.31 The degradation products of colistin over time have not, however, been clearly identified.16,19 A vibrating-mesh nebulizer has been used to assess nebulized antibiotics in most experimental and clinical studies.7,11–13,15 This device has certain advantages compared with a jet nebulizer in terms of reduced residual volume and increased amount of aerosol delivery.32 A recent bench study using a vibrating-mesh nebulizer failed to find any difference in bronchodilator lung dose among different dilutions.33 Although the optimal dosing of nebulized CMS in terms of efficacy and toxicity needs to be defined by further clinical trials,15,34 the current clinical use of nebulized CMS for treating pneumonia tends to administer high doses to overcome extrapulmonary loss and optimize colistin lung deposition.11,13 For a vibrating-mesh nebulizer with a maximum fill volume of 6 mL, the use of a 6 mL diluent volume reduces nursing workload and nebulization time while increasing colistin stability. As a consequence, it could improve treatment efficiency and potentially reduce side effects.26,35,36 Our study is in line with recent expert opinion that strongly suggests focusing clinical investigations on achieving continuous improvements in nebulization techniques before implementing large clinical Phase III trials.26,34,37 It should be pointed out that the results obtained in our study cannot be generalized to drug delivery by jet or ultrasonic nebulizers. Jet nebulizers tend to have high residual volumes of drug remaining in the chamber at the end of nebulization. Hence, using an increased fill volume of a more dilute solution could decrease the amount of drug trapped in the dead volume and thereby increase the aerosol delivery.18 In conclusion, using a vibrating-mesh nebulizer for CMS delivery results in shorter nebulization time and better colistin stability with 4 MUI CMS in 6 mL normal saline. CMS and colistin pharmacokinetics are not influenced by reducing dilution. The aerosol particle size increases with the more concentrated solution, but the range remains in limits that do not impair distal lung deposition. These findings may contribute to improving clinical practice and optimizing the technique for CMS nebulization during clinical trials. Acknowledgements We thank all the ICU nurses, in particular Mr Jean-Baptiste Cartier, for their support and participation in this study. Funding All support was provided from institutional and/or departmental sources. Transparency declarations None to declare. References 1 Bergen PJ , Bulitta JB , Forrest A et al. Pharmacokinetic/pharmacodynamic investigation of colistin against Pseudomonas aeruginosa using an in vitro model . Antimicrob Agents Chemother 2010 ; 54 : 3783 – 9 . Google Scholar CrossRef Search ADS PubMed 2 Orwa JA , Govaerts C , Busson R et al. Isolation and structural characterization of colistin components . J Antibiot (Tokyo) 2001 ; 54 : 595 – 9 . Google Scholar CrossRef Search ADS PubMed 3 Suzuki T , Fujikawa K. Studies on the chemical structure of colistin. IV. Chemical structure of colistin B . J Biochem 1964 ; 56 : 182 – 9 . Google Scholar CrossRef Search ADS PubMed 4 Suzuki T , Hayashi K , Fujikawa K. Studies on the chemical structure of colistin. III. Enzymatic hydrolysis of colistin A . J Biochem 1963 ; 54 : 412 – 8 . Google Scholar CrossRef Search ADS PubMed 5 Suzuki T , Hayashi K , Fujikawa K et al. The chemical structure of polymyxin E: the identities of polymyxin E1 with colistin A and of polymyxin E2 with colistin B . J Biochem 1965 ; 57 : 226 – 7 . Google Scholar CrossRef Search ADS PubMed 6 Bergen PJ , Li J , Nation RL. Dosing of colistin—back to basic PK/PD . Curr Opin Pharmacol 2011 ; 11 : 464 – 9 . Google Scholar CrossRef Search ADS PubMed 7 Lu Q , Girardi C , Zhang M et al. Nebulized and intravenous colistin in experimental pneumonia caused by Pseudomonas aeruginosa . Intensive Care Med 2010 ; 36 : 1147 – 55 . Google Scholar CrossRef Search ADS PubMed 8 Ehrmann S , Roche-Campo F , Bodet-Contentin L et al. Aerosol therapy in intensive and intermediate care units: prospective observation of 2808 critically ill patients . Intensive Care Med 2016 ; 42 : 192 – 201 . Google Scholar CrossRef Search ADS PubMed 9 Sole-Lleonart C , Rouby JJ , Blot S et al. Nebulization of antiinfective agents in invasively mechanically ventilated adults: a systematic review and meta-analysis . Anesthesiology 2017 ; 126 : 890 – 908 . Google Scholar CrossRef Search ADS PubMed 10 Sole-Lleonart C , Rouby JJ , Chastre J et al. Intratracheal administration of antimicrobial agents in mechanically ventilated adults: an international survey on delivery practices and safety . Respir Care 2016 ; 61 : 1008 – 14 . Google Scholar CrossRef Search ADS PubMed 11 Abdellatif S , Trifi A , Daly F et al. Efficacy and toxicity of aerosolised colistin in ventilator-associated pneumonia: a prospective, randomised trial . Ann Intensive Care 2016 ; 6 : 26. Google Scholar CrossRef Search ADS PubMed 12 Bihan K , Lu Q , Enjalbert M et al. Determination of colistin and colistimethate levels in human plasma and urine by high-performance liquid chromatography-tandem mass spectrometry . Ther Drug Monit 2016 ; 38 : 796 – 803 . Google Scholar CrossRef Search ADS PubMed 13 Lu Q , Luo R , Bodin L et al. Efficacy of high-dose nebulized colistin in ventilator-associated pneumonia caused by multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii . Anesthesiology 2012 ; 117 : 1335 – 47 . Google Scholar CrossRef Search ADS PubMed 14 Athanassa ZE , Markantonis SL , Fousteri MZ et al. Pharmacokinetics of inhaled colistimethate sodium (CMS) in mechanically ventilated critically ill patients . Intensive Care Med 2012 ; 38 : 1779 – 86 . Google Scholar CrossRef Search ADS PubMed 15 Boisson M , Jacobs M , Grégoire N et al. Comparison of intrapulmonary and systemic pharmacokinetics of colistin methanesulfonate (CMS) and colistin after aerosol delivery and intravenous administration of CMS in critically ill patients . Antimicrob Agents Chemother 2014 ; 58 : 7331 – 9 . Google Scholar CrossRef Search ADS PubMed 16 Boisson M , Grégoire N , Cormier M et al. Pharmacokinetics of nebulized colistin methanesulfonate in critically ill patients . J Antimicrob Chemother 2017 ; 72 : 2607 – 12 . Google Scholar CrossRef Search ADS PubMed 17 Résumé des Caractéristiques du Produit: Colimycine. http://agence-prd.ansm.sante.fr/php/ecodex/rcp/R0271322.htm. Agence nationale de sécurité du médicament et des produits de santé, 2016 . 18 Hess D , Fisher D , Williams P et al. Medication nebulizer performance. Effects of diluent volume, nebulizer flow, and nebulizer brand . Chest 1996 ; 110 : 498 – 505 . Google Scholar CrossRef Search ADS PubMed 19 Wallace SJ , Li J , Rayner CR et al. Stability of colistin methanesulfonate in pharmaceutical products and solutions for administration to patients . Antimicrob Agents Chemother 2008 ; 52 : 3047 – 51 . Google Scholar CrossRef Search ADS PubMed 20 American Thoracic Society; Infectious Diseases Society of America . Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia . Am J Respir Crit Care Med 2005 ; 171 : 388 – 416 . CrossRef Search ADS PubMed 21 Dudhani RV , Nation RL , Li J. Evaluating the stability of colistin and colistin methanesulphonate in human plasma under different conditions of storage . J Antimicrob Chemother 2010 ; 65 : 1412 – 5 . Google Scholar CrossRef Search ADS PubMed 22 Gobin P , Lemaitre F , Marchand S et al. Assay of colistin and colistin methanesulfonate in plasma and urine by liquid chromatography-tandem mass spectrometry . Antimicrob Agents Chemother 2010 ; 54 : 1941 – 8 . Google Scholar CrossRef Search ADS PubMed 23 Ma Z , Wang J , Gerber JP et al. Determination of colistin in human plasma, urine and other biological samples using LC-MS/MS . J Chromatogr B Analyt Technol Biomed Life Sci 2008 ; 862 : 205 – 12 . Google Scholar CrossRef Search ADS PubMed 24 Brain JD , Valberg PA. Deposition of aerosol in the respiratory tract . Am Rev Respir Dis 1979 ; 120 : 1325 – 73 . Google Scholar PubMed 25 Folkesson HG , Westrom BR , Karlsson BW. Permeability of the respiratory tract to different-sized macromolecules after intratracheal instillation in young and adult rats . Acta Physiol Scand 1990 ; 139 : 347 – 54 . Google Scholar CrossRef Search ADS PubMed 26 Ehrmann S , Chastre J , Diot P et al. Nebulized antibiotics in mechanically ventilated patients: a challenge for translational research from technology to clinical care . Ann Intensive Care 2017 ; 7 : 78. Google Scholar CrossRef Search ADS PubMed 27 Grégoire N , Aranzana-Climent V , Magréault S et al. Clinical pharmacokinetics and pharmacodynamics of colistin . Clin Pharmacokinet 2017 ; 56 : 1441 – 60 . Google Scholar CrossRef Search ADS PubMed 28 Couet W , Grégoire N , Marchand S et al. Colistin pharmacokinetics: the fog is lifting . Clin Microbiol Infect 2012 ; 18 : 30 – 9 . Google Scholar CrossRef Search ADS PubMed 29 Garonzik SM , Li J , Thamlikitkul V et al. Population pharmacokinetics of colistin methanesulfonate and formed colistin in critically ill patients from a multicenter study provide dosing suggestions for various categories of patients . Antimicrob Agents Chemother 2011 ; 55 : 3284 – 94 . Google Scholar CrossRef Search ADS PubMed 30 McCoy KS. Compounded colistimethate as possible cause of fatal acute respiratory distress syndrome . N Engl J Med 2007 ; 357 : 2310 – 1 . Google Scholar CrossRef Search ADS PubMed 31 FDA Drug Safety Podcasts—Colistimethate (Marketed as Coly-Mycin M and Generic Products). https://www.drugs.com/fda-alerts/733-5015.html. 32 Ari A , Atalay OT , Harwood R et al. Influence of nebulizer type, position, and bias flow on aerosol drug delivery in simulated pediatric and adult lung models during mechanical ventilation . Respir Care 2010 ; 55 : 845 – 51 . Google Scholar PubMed 33 Berlinski A , Willis JR. Albuterol delivery by 4 different nebulizers placed in 4 different positions in a pediatric ventilator in vitro model . Respir Care 2013 ; 58 : 1124 – 33 . Google Scholar CrossRef Search ADS PubMed 34 Rello J , Sole-Lleonart C , Rouby JJ et al. Use of nebulized antimicrobials for the treatment of respiratory infections in invasively mechanically ventilated adults: a position paper from the European Society of Clinical Microbiology and Infectious Diseases . Clin Microbiol Infect 2017 ; 23 : 629 – 39 . Google Scholar CrossRef Search ADS PubMed 35 Lu Q , Yang J , Liu Z et al. Nebulized ceftazidime and amikacin in ventilator-associated pneumonia caused by Pseudomonas aeruginosa . Am J Respir Crit Care Med 2011 ; 184 : 106 – 15 . Google Scholar CrossRef Search ADS PubMed 36 Westerman EM , Le Brun PP , Touw DJ et al. Effect of nebulized colistin sulphate and colistin sulphomethate on lung function in patients with cystic fibrosis: a pilot study . J Cyst Fibros 2004 ; 3 : 23 – 8 . Google Scholar CrossRef Search ADS PubMed 37 Rello J , Rouby JJ , Sole-Lleonart C et al. Key considerations on nebulization of antimicrobial agents to mechanically ventilated patients . Clin Microbiol Infect 2017 ; 23 : 640 – 6 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Journal of Antimicrobial ChemotherapyOxford University Press

Published: Feb 28, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off