Jet Nebulization of Prostaglandin E₁ During Neonatal Mechanical Ventilation: Stability, Emitted Dose and Aerosol Particle Size

Abstract

Background

We have previously reported the safety of aerosolized PGE₁ in neonatal hypoxemic respiratory failure. The aim of this study is to characterize the physicochemical properties of PGE₁ solution, stability, emitted dose and the aerodynamic particle size distribution (APSD) of PGE₁ aerosol in a neonatal ventilator circuit.

Methods

PGE₁ was diluted in normal saline and physicochemical properties of the solution characterized. Chemical stability and emitted dose were evaluated during jet nebulization in a neonatal conventional (CMV) or high frequency (HFV) ventilator circuit by a High Performance Liquid Chromatography - Mass Spectrometry method. The APSD of the PGE₁ aerosol was evaluated with a six-stage cascade impactor during CMV.

Results

PGE₁ solution in normal saline had a low viscosity (0.9818 cP) and surface tension (60.8 mN/m) making it suitable for aerosolization. Little or no degradation of PGE₁ was observed in samples from aerosol condensates, the PGE₁ solution infused over 24 h, or the residual solution in the nebulizer. The emitted dose of PGE₁ following jet nebulization was 32–40% during CMV and 0.1% during HFV. The PGE₁ aerosol had a mass median aerodynamic diameter of 1.4 µm and geometric standard deviation of 2.9 with 90% of particles being < 4.0 µm in size.

Conclusion

Nebulization of PGE₁ during neonatal CMV or HFV is efficient and results in rapid nebulization without altering the chemical structure. On the basis of the physicochemical properties of PGE₁ solution and the APSD of the PGE₁ aerosol, one can predict predominantly alveolar deposition of aerosolized PGE₁.

Introduction

Neonatal hypoxemic respiratory failure (NHRF) is usually associated with potentially reversible pulmonary hypertension that causes right-to-left shunting and profound hypoxemia. Inhaled nitric oxide (INO), a selective pulmonary vasodilator (SPV), has revolutionized the treatment of NHRF (1). However, lack of sustained improvement in 30–46% of infants and the need for specialized delivery systems make the treatment expensive and limit availability. Several investigators have explored the use of aerosolized prostaglandin E₁ (PGE₁) as a SPV in patients with respiratory failure to improve oxygenation because of its selective action not only on the pulmonary circulation, but also on well-ventilated lung units resulting in improvement in ventilation perfusion ratio and oxygenation (2–5).

PGE₁ is a crystalline compound stable at room temperature (RT) in the solid state and as a solution in non-aqueous solvents such as ethanol. In isotonic saline (pH 4.5), PGE₁ is stable for more than a month at 0 °C and greater than 90% stable at 37 °C for 2 days (6). Aqueous solutions of PGE₁ show a small, spontaneous dehydration to the significantly less active PGA₁ (Figure 1) within 2 h following dilution from an ethanolic solution at RT (7). While oxidation of PGE₁ at the hydroxyl at C-15 followed by the reduction of the 13,14-double bond, resulting in the formation of 13,14-dihydro-15-keto PGE₁ (Figure 1), is the primary metabolic inactivation step in vivo, the initial oxidation to 15-keto PGE₁ requires strong oxidizing agents in vitro (8)

Figure 1.

Prostaglandin E₁ and its potential degradation products. In vivo metabolism of PGE₁ results in the formation of 13,14-dihydro-15-keto PGE₁. PGA₁ is a non-enzymatic decomposition product of PGE₁. While 15-keto PGE₁ is readily formed from PGE₁ in vivo, its formation in vitro requires strong oxidizing agents.

Although the stability of aqueous PGE1 solutions has been studied extensively, there is little data on the stability of PGE₁ in aerosol following nebulization. Jet nebulization results in a large surface area of the solution being exposed to and becoming saturated with the driving gas (typically oxygen) under high pressure. Under these conditions, it is, at least, theoretically possible that PGE₁ could undergo oxidative degradation resulting in a lower emitted dose. In addition to the chemical stability, aerodynamic particle size distribution (APSD) is an important determinant of pulmonary drug deposition and an essential component of marketing authorization for aerosol formulations. However, PGE₁ aerosol obtained by jet nebulization has not been characterized.

The aim of the present study is to evaluate the chemical stability and emitted dose of PGE₁ following continuous jet nebulization in a neonatal ventilator circuit using the highly sensitive and selective high performance liquid chromatography with mass spectrometry (LC-MS) technique. An additional goal is to characterize the APSD following jet nebulization of PGE₁ in a neonatal ventilator circuit using the low flow MiniHeart nebulizer designed for continuous aerosol delivery to infants.

Materials and Methods

This in vitro study was exempt from IRB review as no human or animal subjects were involved.

Drug Preparation

PGE₁ (500 µg/ml of ethanol) was diluted in normal saline to a final concentration of ~20 µg/ml. Physicochemical properties of the PGE₁ solution were characterized. Freshly diluted solutions of PGE₁ were dispensed by the pharmacy just prior to the start of continuous aerosolization and every 24 hours thereafter for 72 hours. Samples were collected at 0 and 24 hours from the dispensed solution and stored at −80 °C.

Mechanical ventilation

Conventional mechanical ventilation (CMV) was initiated with a pressure-limited ventilator (V.I.P. Bird®, Viasys® Healthcare) at a breath rate of 42 bpm, FiO₂ of 1.0, peak inspiratory pressure of 20 cm H₂O, end expiratory pressure of 4 cm H₂O and duty cycle of 35% to simulate clinical use in moderate to severe NHRF. High frequency ventilation (HFV) was initiated with the SensorMedics 3100A High Frequency Oscillatory Ventilator (Viasys® Healthcare) at a mean airway pressure of 20 cm H₂O, amplitude of 35, frequency of 10 Hz and duty cycle of 33%. Gas flow in the heated humidified ventilator circuit was 8 and 28 LPM for CMV and HFV, respectively. A 3.5 mm ID endotracheal tube (ETT) connected to the ventilator was kept open to a closed glass container (capacity 25 ml) to collect the condensate (Figure 2). A nebulizer was placed in the inspiratory limb of the ventilator circuit ~50 cm from the ETT for CMV and ~35 cm from the ETT for HFV. Condensed aerosol in the glass container at the end of ETT was transferred at regular intervals to clean glass vials and stored at −80 °C. Samples were collected every 30 min for the first 6 h followed by every 6 h for the next 66 hours during CMV. For HFV experiments, condensate samples were collected every 30 min for 8 hours.

Figure 2.

Ventilator and nebulizer circuit. The star represents the place where the cascade impactor was connected with an adaptor.

Administration of Continuous aerosol

Aerosols of PGE₁ were generated with a jet nebulizer (low flow, MiniHeart, Westmed Inc., Tucson, AZ) driven by blended oxygen at a flow rate of 2 LPM. At the start of aerosol therapy, the nebulizer chamber was primed with 2 ml of PGE₁ solution for aerosolization followed by continuous delivery into the nebulizer chamber at 4 ml/h. Residual solution in the nebulizer was sampled at the end of the experiment.

LC-MS setup for resolution and identification of PGE₁ and its potential degradation products

At the time of analyses, the samples were thawed, diluted with HPLC mobile phase and placed in the autosampler that was maintained at 0–5 °C. Samples were routinely injected using autoinjector and the typical injection volume was 10 µl. Resolution of PGE₁ and its potential degradation product PGA₁ along with the less likely oxidation product 15-keto PGE₁ was achieved by the HPLC using Synergi, Hydro-RP column (150×2 mm, 4 µ, 80 Å, Phenomenex, Torrance, CA) with acetonitrile:water:acetic acid (45:55:0.1 v/v) as mobile phase at a flow rate of 400 µl/min on Alliance 2695 liquid chromatograph equipped with an autoinjector (Waters Corporation, Milford, MA). The eulate was monitored by MS/MS on a Micromass QuattroLC controlled by Masslynx software (Waters Corporation, Milford, MA). Optimum mass spectrometry conditions for monitoring PGE₁, PGA₁, and 15-keto PGE₁ were as follows: Needle voltage, 2.8 kV; Cone voltage, 20 V; solvent block temperature, 150 °C; desolvation temperature, 300 °C; Collision voltage 16 kV, and collision gas pressure 3.4×10⁻⁴ mBar. Multiple Reaction Monitoring (MRM) method was employed to detect PGE₁ (m/z 353→235), 15-keto PGE₁ (m/z 351→333), and PGA₁ (m/z 335→235) (Figure 3).

Figure 3.

Mass spectral fragmentation pattern of prostaglandin E₁ and its metabolites.

External standard method of quantitation was used for the assay of PGE₁ content in the condensates of nebulized PGE₁ solutions. For this method of quantitation, gravimetric standards prepared from crystalline PGE₁ (obtained from Cayman Chemical Company, Ann Arbor, MI) were used. An accurately weighed sample of PGE₁ (1–1.5 mg) was dissolved in reagent grade acetonitrile to give a 1 mg/ml solution. This stock solution was diluted to 100 µg/ml in the HPLC mobile phase described above. A 50 µl aliquot of this diluted stock was further diluted with 950 µl of the HPLC mobile phase to give the highest concentration standard, 5 µg/ml, for the standard curve. This standard was serially diluted to give standards of concentration 0.05, 0.1, 0.5, and 1 µg/ml. A five point standard curve was constructed by injecting 10 µl of each of the standards.

Statistical Analyses

Descriptive statistical analyses were used to summarize interval scale sample characteristics. The volume output from the nebulizer, PGE₁ concentration and cumulative emitted dose of PGE₁ were plotted against time to evaluate trends during the experiment. The linear mixed model procedure was used to assess the change in cumulative emitted dose of PGE₁ considering effects from the repeated measurement of these variables and their changes over time. Two-tailed significance level was set at 0.05. Statistical analyses were performed using the SPSS® statistical package, version 15.0.1 (SPSS Inc., Chicago, IL, USA) and SAS/STAT® software, Version 9.1.3 (SAS Institute Inc., Cary, NC, USA).

Aerodynamic Particle Size Distribution

PGE₁ solution for aerosolization was prepared from synthetic PGE₁ and aerosolized in the conventional mechanical ventilation circuit as described above. Aerosol samples were taken at the beginning of the endotracheal tube (Figure 2). The aerosol particle size was determined using a 6-stage cascade impactor (QCM Cascade Impactor, California Instruments, Model IMPAQ AS-6) with cutoff stages at greater than 8, 4, 2, 1, 0.5, 0.3, and a filter at 0.25 µm. The IMPAQ AS-6 operates at a flow rate of 12.5 liters/minute and is suitable for the determination of aerosol APSD in mechanically ventilated neonates in whom the ventilator flow rate of 8–10 LPM closely approximates the flow rate in the cascade impactor. It is equipped with a diaphragm type vacuum pump capable of generating a maximum vacuum of 24 inHg. PGE₁ deposited on each stage of the impactor at the end of 5 seconds was collected on glass slides. The glass slides were rinsed with 0.5mL of normal saline solution. The extraction efficiency of the PGE₁ from the impactor plates was > 95%. The filter was rinsed with 2 mL of normal saline solution. All tests were done at room temperature and under ambient conditions of humidity. Each cutoff stage solution was analyzed by LC-MS for PGE₁ and its potential degradation products (PGA₁, 15-keto-PGE₁) as described above. The official method described in the U.S. Pharmacopoeia to determine MMAD and GSD from cascade impactor data involves manual plotting of the cumulative percent of mass smaller than the stated diameter (ordinate) versus diameter (abscissa), on log probability paper (9). As manual plotting of the cascade impactor data is tedious, the use of linear regression with conversion of cumulative percent smaller than the stated diameter into standard deviation units (ordinate) and the logarithm of the effective cutoff diameters (abscissa) have been suggested as a reasonable approach. For this report, the cascade impactor data was plotted by both methods, but only the latter has been presented.

Results

The surface tension of the PGE₁ solution, measured with an optical contact angle tensiometer at a temperature of 299 K (78.8°F; 26° C) and an ambient pressure of 1 atmosphere, was 60.8 ±0.3 mN/m. The viscosity, measured using a Canon Fenske Routine viscometer under similar conditions, was 0.9815 and 0.9820 centipoise (cP) in two different runs.

LC-MS resolution and identification of PGE₁ and its potential degradation products

Baseline resolution of PGE₁ and its potential degradation product PGA₁ along with the less likely oxidation product 15-keto PGE₁ was achieved by HPLC (Figure 4). Retention times of PGE₁, 15-keto PGE₁, and PGA₁ were 2.2, 3.3, and 5.9 min, respectively, under the standard LC-MS conditions described above. Intra-day variation of the retention times is less than 0.1 min and inter-day variation is less than 0.3 min. Typical peak widths at half height were about 25 s.

Figure 4.

LC-MS chromatograms of a mixture of prostaglandin E₁ and its potential metabolites (prostaglandin A₁ and 15-keto prostaglandin E₁). The three panels represent PGE₁, 15-keto PGE₁, and PGA₁ from top to bottom. A small peak observed at the same retention time as PGE₁ (2.25 min) in the PGA₁ chromatogram (bottom panel) is the PGA₁ derived from PGE₁ in the ion source of the mass spectrometer.

The standard curve showed less than 5% variation in the peak areas of three injections of each standard (Figure 5). Standard curves run at the beginning and end of each set of experimental samples (~23–30 samples) were comparable indicating consistency of mass spectrometer response over the duration of the experiment. In addition, quality control standards injected for every 10 sample injections, gave a response identical to the standard curve indicating lack of instrument response drift. Under the standard conditions the Limit of Detection (LOD) for both PGE₁ and its potential metabolites (PGA₁ and 15-keto PGE₁) is 0.0025 µg/ml and the Limit of Quantitation (LOQ) is 0.03 µg/ml.

Figure 5.

Typical standard curve of prostaglandin E₁ analyzed by LC-MS. The standards range from 0.05 to 5 µg/ml (0.05, 0.1, 0.25, 0.5, 1, and 5 µg/ml); 10 µl of each standard was injected on to the HPLC column. Thus the actual amount of PGE₁ injected on to the column ranges from 0.5 to 50 ng. Each data point represents the average of three independent chromatographic runs.

Stability and Emitted Dose

Four experiments were performed with continuous jet nebulization in a CMV circuit for 72 hours and two with HFV for 8 hours. All experiments were performed in the Neonatal Intensive Care Unit at the Children’s Hospital of Michigan at ambient temperature and relative humidity.

Stability of PGE₁ following jet nebulization during mechanical ventilation

No significant degradation of PGE₁ was observed in samples collected from the dispensed PGE₁, residual solution in nebulizer, or condensate at the ETT during either mode of ventilation (Figure 6). Average PGA₁ levels in the PGE₁ solution dispensed by the pharmacy were 2.0±0.5% of the PGE₁ levels at baseline and increased 0.2% over 24 hours (2.2±0.7%). Average PGA₁ levels in the condensate samples were 2.7±0.9% of the PGE₁. To rule out the possibility of degradation of PGE₁ to some other unforeseen product, several of the condensate as well as nebulizer samples were scanned between m/z 150–450 in the full scan mode in addition to the MRM mode for the three compounds (i.e. PGE₁, 15-keto PGE₁, and PGA₁). No other molecular species was detected in the Total Ion Chromatograms of the full scans (Figure 7).

Figure 6.

Representative LC-MS chromatograms from dispensed PGE₁ solution (a), residual solution in nebulizer during HFV (b), condensate collected during CMV (c) and condensate collected during HFV (d).

Figure 7.

Total Ion Chromatogram (right panel) of a typical condensate scanned from m/z 150–450 along with MRM scans (left panel) for PGE₁ (top), 15-keto PGE₁ (second from top), and PGA₁ (third from top). The large peak at the void volume in the Total Ion Chromatogram is due to the sodium chloride present in the condensate sample.

Concentration of PGE₁ in dispensed PGE₁ solution, and residual solution in nebulizer

Concentration of PGE₁ in the solution dispensed by the pharmacist was determined at the start of infusion and 24 hours later. There was high correlation between PGE₁ concentration at baseline and 24 hours later (r=0.891, p<0.05) in six paired samples (Table 1) suggesting chemical stability. Residual volume in the nebulizer was < 3 ml during CMV and ~10 ml during HFV. PGE₁ concentration in the solution collected from the nebulizer during HFV was comparable to that of the infused solution (Table 4, p>0.1) indicating little degradation of PGE₁.

Table 1.

Concentration of PGE₁ at the beginning and end of a 24 h period in the sample solution that was infused into the nebulizer.

Sample	PGE1 (µg/ml)
	at 0 h	at 24 h
Syringe 1	20.9	19.8
Syringe 2	15.4	15.4
Syringe 3	20.9	20.9
Syringe 4	18.7	17.6
Syringe 5	17.6	18.7
Syringe 6	22.0	20.9

Table 4.

Emitted dose of PGE₁.

	CMV				HFV
	Expt.1	Expt.2	Expt.3	Expt.4	Expt.5	Expt.6
Mean Conc. of PGE1 in infused soln. (µg/ml)a	20.4±0.8	16.5±1.6	20.9±1.2	17.2±1.5	23.7	25.4
Mean Conc. of PGE1 in condensate (µg/ml)b	11.9±5.6	9.0±3.3	10.9±3.5	8.3±2.9	0.26±0.07	0.19±0.04
Dilution factor	1.6	1.7	1.9	2.1	92	137
Volume of PGE1 infused (ml/hr)	4	4	4	4	4	4
Volume of PGE1 collected at ETT (ml/hr)	2.7±1.0	2.8±1.1	2.5±1.6	3.3±1.3	0.42±0.11	0.79±0.18
% Emitted dosec	40	39	32	40	0.1	0.1
PGE1 in residual solution in the nebulizer (µg/ml)	n/d	n/d	n/d	n/d	23.9	26.6

^aAverage of three batches of PGE₁ (each infused for 24 h) used over 72 h in experiments 1–4. Experiments 5 & 6 were conducted with one batch each of PGE₁ for 8 h.

^bData from Table 2 and Table 3.

^cratio of total PGE₁ collected in the condensate versus the infused amount per hour.

^d no significant residual volume collected in the nebulizer for CMV experiments to quantify PGE₁.

Emitted dose of PGE₁ following continuous aerosol delivery in a humidified ventilator circuit

Condensate at the end of the ETT was collected at 23 time points during CMV over 72 h and 16 time points during HFV over 8 h (Table 2 and Table 3). The condensate recovered represented ~50–71% of the infused volume (4 ml/h) in the experiments using CMV and 10–20% of the infused volume in experiments using HFV. Concentration of PGE₁ in the condensate collected at the ETT was lower than that in the dispensed PGE₁ solution. Condensate volume and PGE₁ concentration reached the mean values for the experiment by the second hour in all experiments. Over the 72 hour experiments, variability in the form of reciprocal cyclical trends were noted in the condensate volume and PGE₁ concentration with periods of increased volume output being associated with decreased PGE₁ concentration. We suspect that this variability may result from fluctuations in ambient temperature and relative humidity in the NICU, inherent variability in the performance of the nebulizers over time, and/or inaccuracies in sample collection although every precaution was taken at every step to keep experimental conditions constant. The emitted dose of PGE₁ in each of the six experiments was calculated from the condensate volume and concentration of PGE₁ for each sample and expressed as a percent of the nominal dose infused into the nebulizer chamber (Table 4). The emitted dose was 32–40% of the nominal dose for CMV and 0.1% for HFV. The cumulative dose of delivered PGE₁ at the ETT end of the circuit showed a statistically significant linear increase with time indicating consistent drug delivery (Figure 8).

Table 2.

Volume and Concentration of PGE₁ in the condensates collected at the end of endotracheal tube during CMV

Time (h)	Expt. 1		Expt. 2		Expt. 3		Expt. 4
	Vol. (ml)	PGE1 (µg/ml)	Vol. (ml)	PGE1 (µg/ml)	Vol. (ml)	PGE1 (µg/ml)	Vol. (ml)	PGE1 (µg/ml)
0.5	0.0	0.0	0.0	0.0	0.3	6.6	0.7	6.6
1.0	0.5	6.6	1.3	7.7	0.2	8.8	1.3	6.6
1.5	1.2	12.1	0.9	8.8	0.9	8.8	1.6	7.7
2.0	1.4	14.3	2.1	8.7*	2.3	11.0	2.2	8.8
2.5	1.1	16.5	1.7	9.9	1.3	9.9	1.2	8.8
3.0	1.4	16.5	1.5	11.0	2.6	12.1	3.2	8.8
3.5	2.6	17.6	2.0	11.0	2.9	9.9	3.1	7.7
4.0	1.7	17.6	1.8	8.8	0.6	15.4	2.0	8.8
4.5	2.0	14.3	2.0	7.7	1.5	14.3	2.1	9.9
5.0	1.6	17.6	1.4	9.9	2.8	14.3	2.6	9.9
5.5	1.4	17.6	1.3	9.9	1.7	12.1	0.8	9.9
6.0	1.1	17.6	1.6	12.1	0.7	11.0	1.8	11.0
12	11.2	16.5	18.3	4.4	14.0	15.4	17.0	7.7
18	19.9	6.6	21.2	4.4	10.0	5.5	14.0	3.3
24	20.6	7.7	21.0	11.0	16.4	5.5	21.2	9.9
30	16.9*	9.5*	16.3*	9.2*	16.1	5.5	18.8	2.2
36	13.6	16.5	22.2	4.4	17.8	15.4	18.2	11.0
42	20.2	3.3	8.6	6.6	8.2	15.4	9.0	7.7
48	21.0	7.7	21.1	12.1	14.4	4.4	14.8	12.1
54	17.0	5.5	19.4	11.0	17.8	12.1	18.2	13.2
60	15.6	16.5	17.2	12.1	9.2	11.0	17.8	3.3
66	15.4	11.0	4.2	12.1	2.0	12.1	17.0	11.0
72	16.0	4.4	11.4	14.3	4.8	13.2	21.2	4.4
Mean±SDa	2.7±1.0	11.9±5.6	2.8±1.1	9.0±3.3	2.5±1.6	10.9±3.5	3.3±1.3	8.3±2.9

^*Missing sample due to broken vial. Data estimated using mean imputation.

^aCondensate volume averages are computed as ml/h.

Table 3.

Volume and Concentration of PGE₁ in the condensates collected at the end of endotracheal tube during HFV

Time (h)	Expt. 5		Expt. 6
	Vol. (ml)	PGE1 (µg/ml)	Vol. (ml)	PGE1 (µg/ml)
0.5	0.10	0.22	0.20	0.12
1	0.15	0.27	0.32	0.10
1.5	0.18	0.32	0.40	0.14
2	0.28	0.28	0.44	0.19
2.5	0.17	0.18	0.43	0.19
3	0.18	0.20	0.40	0.18
3.5	0.19	0.18	0.28	0.21
4	0.20	0.16	0.38	0.21
4.5	0.18	0.20	0.33	0.19
5	0.32	0.25	0.39	0.19
5.5	0.25	0.24	0.46	0.19
6	0.27	0.42	0.36	0.18
6.5	0.23	0.30	0.50	0.20
7	0.21	0.26	0.58	0.22
7.5	0.25	0.28	0.43	0.19
8	0.18	0.38	0.41	0.25
Average	0.42±0.11	0.26±0.07	0.79±0.18	0.19±0.04

^a Condensate volume averages are computed as ml/h.

Figure 8.

Cumulative emitted dose of PGE₁ during CMV. Solid line represents best-fitting line for the data. Dotted lines represent 95% confidence intervals of the individual observations.

Aerodynamic Particle Size Distribution

Cascade impactor analysis was performed on 5 different samples and the results of the five trials averaged. LC-MS chromatographic profile of samples collected from cascade impactor revealed no detectable degradation of PGE₁ (data not shown). The distribution of drug captured in the cascade impactor showed a deposition peak at 1.0 µm (35%) (Table 5). Ninety percent of the particles were <4.0 µm in diameter and 88% of the particles <2.0 µm. The cumulative percent of mass less than the stated diameter, was plotted on the ordinate after conversion to units of standard deviation (z scores) versus the logarithm of the particle diameter on the abscissa to obtain a rational approximation of the log probability graph, the official method described in the USP for determination of particle size (Figure 9) (9, 10). A logarithmic regression curve was fitted to determine the particle size at 50% of the accumulated deposition (mass median aerodynamic diameter, MMAD). Geometric standard deviation (GSD) was calculated as the MMAD divided by the particle size at 16% deposition. The PGE₁ aerosol had a MMAD of 1.4 µm with a GSD of 2.9. R² for this relationship was greater than 0.9 in this series of experiments. On the basis of the low MMAD, and large proportion (90%) of particles <4.0µm, one can predict predominantly alveolar deposition of aerosolized PGE₁.

Table 5.

Aerosol Particle Size Distribution

Cascade impactor stage (µm)	Depositiona (%)	Cumulative depositiona (%)
0.25	5.5	5.5
0.3	8.5	14.0
0.5	24.5	38.6
1	35.1	73.7
2	14.3	88.1
4	2.1	90.1
> 8	9.9	100

^aCalculated from the PGE₁ values obtained by LC-MS

Figure 9.

Cumulative Log-Probability Plot of particle size distribution. The best-fit line is from logarithmic linear regression.

Discussion

Targeted delivery of drugs to the site of action is preferable not only for enhanced efficacy but also to reduce systemic exposure and potential toxicity. Intravenous PGE₁, a potent vasodilator used empirically in the treatment of NHRF, is associated with systemic hypotension and worsening of oxygenation due to increased venous admixture (11–15). This has led investigators to explore the delivery of PGE₁ directly to the lungs as an inhalation, thus minimizing systemic effects and achieving selective pulmonary vasodilation (2–4). In this study, we have demonstrated for the first time that PGE₁ can be nebulized safely during neonatal mechanical ventilation. The nebulizer set up is simple to use and results in rapid nebulization of the drug without altering the chemical structure of PGE₁. We have also characterized, for the first time, the APSD specific to PGE₁ aerosol generated using the low flow MiniHeart jet nebulizer. The pulmonary deposition of aerosolized medications depends on several nebulizer and patient related factors of which APSD is perhaps the most important (16). For convenience, many studies in the past have used salbutamol or tracer materials to characterize the APSD for a given nebulizer (16, 17). However, nebulizer performance is affected by the solution used. Therefore it is important to characterize the aerosol for a particular drug-nebulizer combination (18).

Selectivity and specificity of LC-MS method for PGE₁ quantitation

PGE₁ is usually measured by HPLC after derivatization with a chromophore (19). While this method is effective at high concentrations (~500 µg/ml), it is not suitable for the present study where the concentration in the final sample is expected to be <25–30 µg/ml. Moreover, the UV-HPLC method is not suitable for the identification of unknown degradation products that could be produced during nebulization in a positive pressure ventilator circuit with high FiO₂. HPLC resolution of PGE₁ and its potential degradation products combined with mass spectrometric identification and quantitation used in this study offers unsurpassed selectivity and excellent sensitivity with a LOD of 0.0025 µg/ml, LOQ of 0.03 µg/ml and a dynamic range of 0.03–5 µg/ml. This technique can be further optimized to detect lower concentrations of PGE₁ if necessary.

Stability of PGE₁ during nebulization

Despite exposure to high oxygen tension during nebulization, PGE₁ is stable as evidenced by the absence of any degradation products in the total ion chromatograms of the condensate samples other than PGE₁ and PGA₁ (Figure 7). The molecular weight range of the scan was chosen to encompass molecular species that could result from the addition of several molecules of oxygen under the hyperbaric conditions of the experiment as well as smaller fragments that could result from the oxidative degradation of PGE₁.

Emitted dose of PGE₁

We used a modification of the wet nebulization method for aerosol collection because it is relatively accurate and avoids the drying or reconstitution steps involved in filter paper collection which may mask or add to changes occurring during the aerosolization process (20–23). The condensate collected at the ETT represented ~50–71% of the volume infused into the nebulizer during CMV and 10–20% of the infused volume during HFV. Variations in condensate volumes over time reflect inherent variability of nebulizers, changes in ambient conditions, angulation of ventilator tubing, and evaporation. Similar variability has been reported by other investigators (21, 24). However, inspite of variations in volume, there was consistent delivery PGE₁ over time.

The concentration of PGE₁ in the condensate collected at the ETT was lower than that in the dispensed PGE₁ solution. The lower concentration of PGE₁ in the condensate reflects dilution due to condensation of water vapor from the humidified gas flowing through the ventilator at a high rate. The water content of gas with 100% humidity at 35.7 °C is estimated to be 41 mg/L (25, 26). At a flow rate of 8 LPM through CMV and 28 LPM through HFV, the volume of water in the form of vapor is ~28 and ~78 ml/h, respectively. This water contributes to dilution of the condensate resulting in lower PGE₁ concentration. As expected, this dilution is significantly more pronounced in the HFV experiments because of the higher gas flow rate. After adjustment for the dilution factor, the emitted dose is nearly 100%.

The emitted dose of 32–40% during CMV and 0.1% during HFV in the current report is an under-estimate for several reasons. First, it was not possible to collect the entire aerosol at the ETT because of the high gas flow rate in the ventilator as discussed above. Second, we did not collect the aerosol adhering to the ventilator circuit or entering the expiratory tubing. Third, the closed glass container is a poor substitute for the respiratory system of the neonate with intrinsic compliance, branching airways, numerous alveolar interfaces and alveolar-capillary gradient. Fourth, the heated and humidified ventilator circuit in this report may have decreased the emitted dose (21, 27, 28). Although additional measures such as a condenser or negative pressure in the circuit have been used to enhance aerosol recovery, we tried to simulate the clinical condition as closely as possible (23). Underestimation of delivered dose raises the concern of potential overdose in neonates undergoing aerosol therapy with the set up described in this report. However, this was not substantiated by animal toxicity studies with inhaled PGE₁ (unpublished data) and the Phase I/II study of inhaled PGE₁ in neonatal hypoxemic respiratory failure (5)

The emitted dose in this report is lower than that reported in adult (57–81%) but higher than that reported in pediatric ventilation studies (0.3–10%) (17, 21, 29–32). These discrepancies are related to differences in delivery systems, administration techniques and nebulizer design (28).

The significant difference in the emitted dose during CMV and HFV is a reflection of the inherent differences in principles of operation of the two ventilators; limited understanding of aerosol deposition during HFV and consequent inability to accurately assess drug delivery during HFV by in vitro methods (33–35). It is remarkable that condensate was obtained as soon as 30 min after the onset of nebulization during HFV and that PGE₁ was present in the condensate at this time. In the only other report of aerosol therapy during HFV, Dijk et al. have shown similar deposition (~10%) following jet nebulization of comparable doses of surfactant during CMV and HFV (36). Clinical experience also suggests that inhaled drugs like albuterol, and inhaled nitric oxide are effective during CMV and HFV at comparable doses.

Aerodynamic Particle Size Distribution

Physicochemical properties of drug solution may affect particle size and consequently lung deposition (37). It has previously been shown that during jet nebulization, the droplet-size is inversely proportional to viscosity (in the range 0.5 – 20 cP). However, increase in viscosity is associated with a decrease in aerosol output. Thus, the least viscous fluids produce aerosols with the highest respirable fraction. Although a lower surface tension has been reported to be associated with increased aerosol output, this has not been confirmed by other investigators. The PGE₁ solution prepared in 0.9% normal saline in this study had a viscosity comparable to that of water and surface tension lower than that of water making it ideal for generating aerosols.

Particle sizes from nebulizers are classified according to aerodynamic diameter which accounts for both the density and irregular shape of drug particles and more accurately predicts the behavior of the aerosol (18). The proportion of the aerosol that can penetrate beyond terminal bronchioles to gas exchange regions is called the respirable fraction (RF) (38). The RF in adults and older children consists of particles < 5 µm in diameter, although recent studies suggest that a particle size closer to 2.5 µm is more accurate in predicting alveolar deposition especially in infants (16, 39, 40). In this study, the PGE₁ aerosol had a high respirable fraction with 90% of the particles < 4.0 µm and 88% of the particles < 2.0 µm.

Despite the important findings described in this study, there are potential deficiencies. Limitations of our study include the relative inefficiency of the aerosol collecting system and our inability to mimic the complex anatomy of the neonatal respiratory system and the effect of disease state on aerosol drug delivery. Although we have evaluated for the first time the APSD of the PGE₁ aerosol generated by jet nebulization, the effects of other nebulizer-, ventilator- and patient-related factors need to be investigated further. It is important to recognize that cascade impactor data may not reflect in vivo conditions (18, 41–43). The hygroscopic particles of most therapeutic aerosols are subject to evaporation in the cascade impactor, while the particles delivered into the lungs are exposed to a much more humid environment thus increasing their size and altering their aerodynamic behavior (41). Moreover, drug concentration and particle size can change over the course of nebulization; this was not evaluated in the study. However, earlier studies have shown that there is little variation in aerosol particle size during continuous nebulization despite changes in temperature, drug concentration, viscosity and surface tension (37, 41).

Conclusions

Our study is unique in several aspects. We have shown for the first time that PGE₁ can be nebulized safely and delivered efficiently in a neonatal CMV or HFV circuit. The nebulizer set up is simple to use and results in rapid nebulization of the drug without altering the chemical structure of PGE₁. In addition, we have developed a LC-MS method for the quantification of PGE₁ in physiological saline that is sensitive, reproducible, selective for the analyte, and has a wide dynamic range of quantitation. The emitted dose determined during CMV and HFV is probably an underestimation. Better aerosol capturing techniques are needed to more accurately determine emitted dose. We have also demonstrated that the APSD of PGE₁ following jet nebulization in a neonatal ventilator circuit is characterized by a small MMAD and large GSD with majority of the partcles < 4 µm in size that would favor alveolar deposition. Further studies are needed to evaluate actual lung deposition in vivo following jet nebulization of PGE₁, effect of nebulizer-, ventilator-, and patient-related factors on drug delivery, its relation to response and linkage with receptor sites.

ABBREVIATIONS

APSD

aerodynamic particle size distribution

CMV

conventional mechanical ventilation

ETT

endotracheal tube

FIO₂

Fractional inspired oxygen concentration

GSD

geometric standard deviation

HFV

High frequency ventilation

HPLC

High performance liquid chromatography

INO

inhaled nitric oxide

LC-MS

liquid chromatography - mass spectrometry

MRM

Multiple reaction monitoring

MMAD

mass median aerodynamic diameter

NHRF

neonatal hypoxemic respiratory failure

PGE₁

prostaglandin E₁

RF

respirable fraction

SPV

selective pulmonary vasodilator