Pharmacokinetics and lung delivery of PDDS-aerosolized amikacin (NKTR-061) in intubated and mechanically ventilated patients with nosocomial pneumonia

The purpose of this multicenter (n = 6) trial was to evaluate the pharmacokinetics of PDDS-delivered aerosolized amikacin, combined with intravenous antibiotics, for patients with Gram-negative VAP, HAP or HCAP. Patients were included when they were aged 18 years or older, mechanically ventilated, had nosocomial pneumonia (defined as the presence of a new or progressive infiltrate(s) on chest radiograph and at least two of the following: fever, defined as core temperature >39.0°C or hypothermia, defined as core temperature <35.0°C; leukocyte count ≥10,000/mm³ or ≤4,500/mm³; and new onset of purulent sputum production or respiratory secretions, or a change of sputum characteristics [10, 11]); and a Gram-negative organism was detected by Gram-staining of tracheal aspirates. Non-inclusion criteria were: primary lung cancer or another malignancy metastasized to the lung, known or suspected active tuberculosis, cystic fibrosis, AIDS, or Pneumocystis jiroveci pneumonia; severe hypoxemia (partial pressure of oxygen/fraction of inspired oxygen (FiO₂) ratio <100 mmHg); renal failure (serum creatinine >2 mg/dL or currently on dialysis); immunocompromised status; neutropenia; body mass index of 30 kg/m² or more; severe burns (>40% of total body surface area); refractory septic shock; known respiratory colonization with amikacin-resistant Gram-negative rods; and/or having received amikacin within the preceding seven days. After inclusion, patients received 400 mg of aerosolized amikacin twice daily (800 mg per day) for 7 to 14 days. Every patient's trough serum amikacin concentrations were measured daily. Patients who did not receive three full days of study medication were excluded.

For the study, a specially prepared, preservative-free formulation of amikacin sulfate formulated for inhalation (NKTR-061) was used for aerosolization, not a standard intravenous preparation. This solution contained amikacin sulfate at a concentration of 125 mg/mL; pH and osmolarity were adjusted for inhalation. Prior to starting studies in humans, inhalation toxicology studies were performed to make sure the dose was safe for inhalation.

The Institutional Review Board of each participating center approved the protocol, and informed consent was obtained from patients or their legally authorized representative prior to enrollment.

The PDDS Clinical consists of a nebulizer/reservoir unit, T-piece adapter, air-pressure feedback unit for breath synchronization and a control module (Figure 1a). The nebulizer/reservoir unit, which is breath-synchronized and provides aerosol during the first 75% of inspiration, comprises the OnQ^® aerosol generator and a conical 6.25 mL drug reservoir, which contains the entire dose and requires no refilling. The aerosol generator consists of a proprietary high-frequency vibrating element that creates a rapid pumping of liquid droplets (of 3 to 5 μm) through tapered apertures to form the aerosol. The aerosol-generating process is electronically controlled via the control module. The nebulizer/reservoir unit is connected to the ventilator circuit through a T-piece adapter between the Wye-piece and the endotracheal tube. A cable connects the nebulizer/reservoir to the control module. The air pressure-feedback (for breath-synchronization) unit is connected to the inspiratory limb of the ventilator circuit and to the control module by pressure tubing.

The PDDS is a specialty drug delivery system for single-patient use. It is designed to deliver medication to adult patients on mechanical ventilation. The PDDS nebulizer/reservoir unit operates in phasic, breath-synchronized mode only, providing aerosol during the first 75% of inspiration during mechanical ventilation. This is accomplished by the control module sensing a positive pressure breath through the air pressure feedback tube. Limiting the aerosol formation to the first 75% of the inspiratory cycle enhances efficiency of delivery and minimizes exhaled aerosol. The duration of nebulization is dependent upon the patient's minute ventilation. The aerosol delivery time varies between 45 and 60 minutes [12]. The PDDS is an investigational device and is not commercially available.

During nebulization, patients had to receive positive-pressure ventilation (i.e., pressure-control or volume-assist control modes). A heat-moisture exchanger or heated humidifier could be used with the device. For aerosolization, 3.2 mL of amikacin sulfate was added in the reservoir.

Aerosols were continued after extubation, the nebulizer/reservoir unit was attached to a reservoir unit with a mouthpiece, one-way valves and an expiratory filter (Figure 1b). In this configuration, the aerosol was generated continuously (not only during inspiration), and nebulization of the dose was completed in approximately 15 to 20 minutes in a previous study [12].

Fifteen to 30 minutes after the end of the first aerosolized dose given on day 3, all patients underwent fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) in an infection-involved zone, as previously described [13]. After premedication with intravenous sedatives and a short-acting paralytic agent if needed (left to the discretion of the treating physician), the FiO₂ was adjusted to 95% or more. The fiberoptic bronchoscope was advanced to the bronchial orifice selected on the basis of the radiographic infiltrate location. BAL was performed by instilling a total of at least 120 mL of sterile, non-bacteriostatic saline. The liquid recovered after the first aliquot was discarded, and the remaining BAL fluids were filtered through sterile gauze and pooled. The time between BAL onset and the total recovery of the six aliquots was kept as short as possible to minimize free diffusion of solutes, particularly urea, through the alveolar epithelium during the procedure. The entire procedure was well tolerated by all the patients. All efforts were made to keep the BAL specimen processing time as short as possible. BAL fluid samples were frozen and stored at -35°C until analyzed, i.e., determinations of ELF volume (V_ELF) and amikacin concentration.

After starting the first day 3 aerosol, blood was drawn to measure serum amikacin concentrations at 30 minutes, and 1, 3, 6, 9, 12 and 24 hours, and cumulative urine samples, 0 to 12 and 12 to 24 hours, were collected to determine amikacin excretion via the kidneys. Serum creatinine levels were determined daily in each center's laboratory, according to local practices. Tracheal aspirates were collected on day 3 after the first aerosol and during the following 24 hours. Although tracheal suctioning was routinely performed by the nurses, tracheal aspirates collection was not compulsorily requested in the protocol and thus not performed in all patients: only 19 had tracheal aspirates collection for amikacin concentration determination. Moreover, because tracheal aspirates were collected as part of routine care, they were collected at different times for each patient. All samples were frozen and stored at -35°C until analyzed.

The determination of amikacin concentrations in serum, tracheal aspirates and BAL, and urea levels in serum and BAL were performed by MEDTOX Laboratories (Saint Paul, MN, USA). All methods were pre-validated according to current Food and Drug Administration guidelines.

As previously described [14, 15], the V_ELF was evaluated using urea as an endogenous marker of ELF dilution. Because urea diffuses easily and rapidly throughout the body, ELF and plasma urea concentrations are the same. In this setting, knowing the urea concentration in plasma and the urea quantity in a lavage sample enables V_ELF to be calculated, as follows: V_ELF = (BAL volume × (urea) in BAL)/(urea) in plasma, where (urea) is the urea concentration. Once the recovered V_ELF is known, then any acellular component concentration (e.g., amikacin) can be calculated from it. The urea contents of BAL fluid samples were determined using a commercially available kit (Abbott Clinical Chemistry Urea Nitrogen Kit; Abbott Diagnostics, Abbott Park, IL, USA), and subsequently validated for analyzing urea in BAL. The urea content in corresponding serum samples was determined using the same kit without modification of the methodology as specified by the manufacturer.

Serum samples drawn on day 3 were analyzed for amikacin over a concentration range of 200 to 500 ng/mL using an high performance liquid chromatography-mass spectrometry (HPLC-MS)/MS-based methodology. This methodology was used because commercial techniques for measuring amikacin were not sensitive enough to measure the expected serum levels in this study. Serum samples were mixed with internal standard (tobramycin) and 800 μL of 2% trichloroacetic acid and 200 μL of acetonitrile. Samples were then centrifuged and filtered through C18 extraction cartridges. The sample effluent was then analyzed using a 100 × 2.1 mm Betasil C18 column (Thermo Scientific, Waltham, MA, USA) and a mobile phase starting at 80% 1.5 mM heptafluorobutyric acid and 14% methanol and 6% water. The mobile phase was changed stepwise to a final composition of 80% methanol 20% water over the course of two minutes.

Amikacin was monitored using the specific fragmentation reactions produced under electronspray ionization - mass spectrometry (ESI-MS)/MS conditions on an ABI-Sciex 5000 triple quadrupole mass spectrometer (Applied Biosystem, Foster City, CA, USA). Amikacin was quantified by summing the transitions 586.2>425.2, 586.2>163.1 and 586.2>264.2.

Furthermore, trough serum amikacin concentrations before the morning nebulization were determined daily during the treatment period. Dosages were performed at each center using the kits available locally, with detection thresholds differing from one site to another. When the concentration was below the detection threshold, the latter was arbitrarily given as the value.

The BAL amikacin concentration was analyzed using a commercially available Syva^® Emit^® kit (Siemens Healthcare Diagnostics, Deerfield, IL, USA), designed for the analysis of amikacin in human serum. The methodology was modified to allow the analysis of amikacin in BAL over a concentration range of 2.50 to 50.00 μg/mL by simply preparing assay calibrators and quality-control samples in BAL fluid; no further modification of the assay procedure was required. This methodology was validated by performing an analytical method validation in full accordance to Food and Drug Administration guidelines and current bioanalytical industry practice.

The maximum serum amikacin concentration after the first dose on day 3 was defined as C_max, with the time to C_max defined as T_max. The area under the serum amikacin concentration-time curve after the first dose (AUC_{0-12 hour}) was calculated from the experimental data points obtained after the first dose on day 3 (30 minutes, and 1, 3, 6, 9 and 12 hours) using the trapezoidal method.

To determine amikacin absorption during the study period, amikacin concentrations were measured in the two day 3 urine collections, which reflected the quantity of each 12-hour dose absorbed via inhalation.

Because day 3 tracheal aspirates were not collected at specific time points, the 24-hour collection time was divided into four equivalent six-hour periods and then all results obtained during the corresponding period were pooled. The first period (H1 to H6) corresponds to the first six hours following the first day 3 aerosol, the second (H7 to H12) to the next six hours (before the second aerosol of the day), the third (H13 to H18) to the six hours following the second day 3 nebulization, and the fourth (H19 to H24) to the last six hours of the day, before the next aerosol.

All results are expressed as medians (interquartile range (IQR)), unless specified otherwise.