Evaluation of Time-Sensitive Nebulization Mode Converter

Study Design and Participants This study was designed to evaluate the performance of the proposed nebulizer mode converter using both in-vitro and ex-vivo models. The study involved healthy adult volunteers who provided informed consent before participating in the study. The aim was to assess drug delivery efficiency and medication wastage when using conventional jet nebulizers (JNs) compared to the modified nebulization system incorporating the mode converter. Each participant received the inhaled medication via both modes of nebulization-continuous jet nebulization and intermittent nebulization facilitated by the mode converter to allow for a direct comparison of drug deposition efficiency and aerosol delivery. Experimental Models Two experimental setups were utilized: an in-vitro model employing a breathing simulator and an ex-vivo model involving human volunteers. The in-vitro model used a high-fidelity breathing simulator programmed to replicate a standardized adult respiratory pattern. The simulator was set to an inhalation-to-exhalation (I:E) ratio of 1:2, a respiratory rate of 20 breaths per minute, and a tidal volume of 500 mL. The inhaled drug dose, residual drug volume within the nebulizer chamber, and deposition of salbutamol sulfate within the delivery tubing (connection tube used to connect the jet nebulizer with the filter, which is used for collection of delivered aerosols) were measured to assess efficiency. In the ex-vivo model, each volunteer inhaled the aerosolized medication while an inhalation filter was placed between the nebulizer and the subject's airway to collect the delivered drug. As with the in-vitro model, residual drug amounts within the nebulizer chamber and salbutamol sulfate deposition within the delivery tubing were also measured. This setup enabled direct assessment of the proportion of medication that reached the subject's respiratory tract under both continuous and intermittent nebulization conditions. To ensure the reliability of results, each nebulization mode underwent ten repeated runs in both models. A jet nebulizer of the same commercial model was used for all in-vitro and ex-vivo experiments to maintain consistency and eliminate variability associated with differences between individual nebulizers. Experiments were conducted under normal ambient laboratory conditions, with room temperature maintained at 22-24 °C and relative humidity between 40-55%. The experimental model setup was designed to evaluate the performance of the nebulization mode converter in comparison to conventional continuous nebulization. The system consisted of a standard jet nebulizer (Planet Health Wheeze nebulizer, Planet Health, Vapo Healthcare Co., Ltd, China), a breathing simulator (5600i, Michigan Instruments, USA), and an intermittent aerosol converter device (EGPO-SES, EG/P/2025/361).\[1\] Three distinct configurations were examined to assess aerosol generation and delivery under different operating conditions (Figure 1). In the ex-vivo model, the intermittent aerosol converter did not rely on flow-triggered breath sensing. Instead, the inspiratory and expiratory durations of each subject were measured using the Certifier FA Plus and used to set a time-based cycling pattern in which the converter switched on during the subject's inhalation interval (allowing passage of compressed air to the nebulizer head) and off during the exhalation interval. Both inhalation and exhalation times were fully adjustable by the subject, who could reset these intervals at any time to match changes in their spontaneous breathing pattern. The device also produced a fine click sound when transitioning between inhalation and exhalation phases, providing an audible cue that helped the subject maintain synchronization with the intermittent aerosol release. In the clinical (ex-vivo) model, the inspiratory-to-expiratory (I:E) ratio was determined using the Certifier FA Plus high-flow analyzer (TSI Incorporated, USA). This device provides real-time respiratory measurements, including inspiratory time, expiratory time, and overall breathing cycle parameters. It was used to precisely determine the I:E ratio in volunteers receiving nebulized therapy, ensuring standardized respiratory conditions across all participants. To ensure accuracy and consistency, the I:E ratio was measured multiple times for each participant, with the means of the most stable readings being used for data analysis. These measurements were critical in evaluating the synchronization of aerosol generation with the inhalation phase when using the nebulization mode converter. The recorded I:E ratios were then used to compare aerosol deposition efficiency between continuous and intermittent nebulization modes. Each volunteer received the medication with two nebulization modes: the first is the continuous mode without using the nebulization mode converter, and the second is the intermittent mode through incorporation of the nebulization mode converter, as shown in Figure 1. By integrating the Certifier FA Plus into the study protocol, precise and reproducible respiratory parameters were obtained, enhancing the validity of the ex-vivo experimental results. This approach ensured that the study accurately simulated clinical conditions, providing a robust assessment of the nebulization mode converter's effectiveness in optimizing aerosol therapy. Nebulization Procedure For each experimental run, 1 mL of salbutamol sulfate respirator solution (Farcolin, 6050 μg/mL; Pharco Pharmaceuticals, Egypt) was placed inside the nebulizer chamber. The breathing simulator and ventilator were turned on 30 seconds before nebulizer activation to establish a stable and reproducible respiratory cycle. The nebulizers were operated under standardized conditions until the sputtering phase, which signified the near-complete aerosolization of the drug solution. During ex-vivo experiments, volunteers inhaled the medication through both the conventional jet nebulizer mode and the modified mode with the converter in separate sessions, allowing for comparative analysis of drug deposition. Drug Collection and Analysis Following nebulization, all inhalation filters and nebulizer chambers were systematically rinsed with 90% acetonitrile (v/v) to recover any residual drug content. The collected samples were then subjected to sonication to ensure complete extraction of salbutamol sulfate. Drug quantification was performed using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The HPLC system employed was the Agilent 1260 Infinity Diode Array Detector VL (G131SD, Agilent, USA), configured to detect salbutamol sulfate at a wavelength of 225 nm. A mobile phase composed of a 90:10 (v/v) acetonitrile-to-water mixture containing 0.1% phosphoric acid was used, with a flow rate of 1 mL/min. The separation was achieved using a ZORBAX Eclipse Plus C18 column (25 × 4.6 mm, Agilent, USA), with an injection volume of 100 μL. The detection sensitivity was optimized with a lower limit of detection (LOD) of 0.35 μg/mL and a lower limit of quantification (LOQ) of 2.55 μg/mL, ensuring precise and accurate measurements of delivered and residual drug amounts.\[2, 3\] Breathing Simulator Settings To replicate adult breathing conditions, a Michigan Instruments 5600i breathing simulator (USA) was employed. The simulator was programmed with an inhalation-to-exhalation ratio of 1:2, a respiratory rate of 20 breaths per minute, and a tidal volume of 500 mL. These settings ensured standardized and reproducible conditions across all experimental runs. This controlled experimental approach allowed for a thorough and scientifically rigorous evaluation of aerosol drug delivery efficiency between conventional jet nebulization and the modified intermittent nebulization system facilitated by the mode converter. Ethical considerations: The current study has been ethically reviewed and approved by the Research Ethical Committee (FM-BSU REC), Faculty of Medicine, Beni-Suef University, under Approval No: FMBSUREC/01092024. Study Design and Participants The present study employed a combined in-vitro aerodynamic characterization model and an in-vivo clinical pharmacokinetic model to evaluate the aerosol delivery performance of a conventional jet nebulizer with and without integration of the Breath-Synchronized Nebulization Mode Converter (NMC). The in-vitro aerodynamic characterization model was designed to assess aerosol particle deposition characteristics under simulated physiological breathing conditions. Aerosol performance was evaluated using an Andersen Cascade Impactor Mark II (ACI, Copley Scientific, UK) integrated with a custom-designed breathing simulation platform capable of reproducing synchronized inspiratory and expiratory phases. The in-vivo clinical pharmacokinetic model was conducted to evaluate pulmonary bioavailability and effective lung deposition associated with both nebulization modes. The clinical study included asthmatic patients who received aerosolized salbutamol sulfate using conventional continuous nebulization and breath-synchronized intermittent nebulization in a randomized crossover design. Pulmonary bioavailability was assessed using urinary salbutamol excretion collected 30 minutes after completion of nebulization (USAL 0.5), a validated surrogate marker of pulmonary drug deposition following inhalation therapy. Experimental Models The aerodynamic performance of aerosolized salbutamol sulfate generated by the conventional jet nebulizer and the jet nebulizer integrated with the Nebulization Mode Converter (NMC) was evaluated using an Andersen Cascade Impactor Mark II (ACI, Copley Scientific, UK) operated at a calibrated flow rate of 15 L/min. \[4\] To simulate physiologically relevant respiratory conditions, a custom-designed breathing simulation platform was incorporated into the experimental setup. The simulator consisted of two independently controlled pumps connected to an automated timing-control unit: a vacuum pump to simulate the inspiratory phase, and a positive-pressure air pump to simulate the expiratory phase. The transition between inspiratory and expiratory phases was controlled electronically using a programmable timing module configured according to predefined inspiratory-to-expiratory (I: E) ratios representative of spontaneous tidal breathing patterns. During the inspiratory phase, negative pressure generated by the vacuum pump created directional airflow through the nebulizer-mouthpiece assembly toward the cascade impactor, simulating pulmonary aerosol inhalation. During the expiratory phase, the inspiratory pathway was interrupted while the expiratory pump generated reverse airflow to simulate exhaled breathing conditions. For the intermittent nebulization arm, the Nebulization Mode Converter synchronized aerosol generation exclusively with the inspiratory phase by controlling compressed air delivery to the nebulizer chamber. Aerosol production was automatically interrupted during simulated exhalation, thereby minimizing aerosol loss during the expiratory period. In contrast, the conventional nebulization mode continuously generated aerosol throughout both inspiratory and expiratory phases, independent of the breathing cycle. This experimental configuration enabled dynamic evaluation of aerosol generation under breathing conditions closely resembling clinical respiratory patterns rather than static continuous-flow testing. High-performance liquid chromatography Method: Following aerosol collection, the stages and collection plates of the Andersen Cascade Impactor were rinsed with 90% acetonitrile (v/v) and subjected to sonication to ensure complete recovery of deposited salbutamol sulfate. Quantitative analysis of salbutamol was subsequently performed using a validated ultraviolet high-performance liquid chromatography (UV-HPLC) method utilizing an Agilent 1260 Infinity HPLC system (Agilent Technologies, USA), as previously described in earlier studies. \[2, 3, 5, 6\] To ensure experimental consistency and minimize device-related variability, the same commercial model of jet nebulizer was used throughout all in-vitro investigations. All experiments were conducted under standardized laboratory conditions, with ambient temperature maintained between 22 and 24 °C. The clinical model: The clinical pharmacokinetic model included 36 asthmatic patients who provided written informed consent prior to participation in the study. The study was designed to compare pulmonary bioavailability achieved using conventional continuous jet nebulization with that obtained using a Breath-Synchronized Nebulization Mode Converter integrated with the same jet nebulizer system. Ethical considerations: The current study has been ethically reviewed and approved by the Research Ethical Committee (FM-BSU REC), Faculty of Medicine, Beni-Suef University, under Approval No: FMBSUREC/01092024. Each participant received aerosolized salbutamol sulfate using both nebulization modes in a randomized crossover design. The two intervention arms included: (1) conventional continuous nebulization without incorporation of the Nebulization Mode Converter, and (2) breath-synchronized intermittent nebulization using the Nebulization Mode Converter integrated with the jet nebulizer. Each nebulization mode was administered on separate study days (day 1 and day 3) with random assignment of the initial intervention sequence and an adequate washout period between sessions to minimize carryover effects. This crossover design enabled direct within-subject comparison of pulmonary drug bioavailability and aerosol delivery performance between both nebulization modes while minimizing intersubject variability. Prior to aerosol administration, the inspiratory-to-expiratory (I: E) ratio for each participant was determined using a Certifier FA Plus high-flow analyzer (TSI Incorporated, USA). This system provides real-time respiratory measurements, including inspiratory time, expiratory time, respiratory cycle duration, and breathing pattern characteristics. The device was used to accurately characterize individual respiratory profiles and to optimize synchronization of aerosol generation with the inspiratory phase during breath-synchronized intermittent nebulization. To ensure measurement precision and reproducibility, respiratory parameters were assessed repeatedly for each participant, and the mean of the most stable recordings was used for analysis. These measurements were essential for accurate temporal alignment between aerosol release and the inspiratory phase throughout nebulization sessions. Each participant received aerosolized salbutamol sulfate consisting of 1 mL Farcolin® solution containing 6050 µg salbutamol sulfate diluted with 2 mL normal saline placed within the nebulizer chamber. Aerosol therapy was administered using two nebulization modes: (1) conventional continuous nebulization without incorporation of the Nebulization Mode Converter, and (2) breath-synchronized intermittent nebulization using the Nebulization Mode Converter integrated with the jet nebulizer system. Both nebulization modes were administered on separate study days with an adequate washout period between sessions to minimize carryover effects. Sample size estimation was performed using G\*Power software (version 3.1, Heinrich Heine University, Düsseldorf, Germany). The calculation was based on findings from a previously published ex vivo study evaluating the Nebulization Mode Converter, which demonstrated significantly improved salbutamol delivery during intermittent nebulization compared with conventional continuous nebulization.\[7\] Because the previous experimental model demonstrated a very large effect size under controlled laboratory conditions, a conservative large clinical effect size (Cohen's dz = 0.90) was selected for estimation of the current in-vivo crossover study to account for anticipated variability in respiratory patterns and urinary pharmacokinetic measurements. Using a paired-samples t-test, with a two-sided alpha level of 0.05 and a statistical power of 80%, the minimum required sample size was estimated to be 15 participants. To improve statistical robustness and compensate for potential variability and dropouts, 36 asthmatic patients were ultimately recruited. Participants were instructed to empty their bladders 15 minutes prior to aerosol administration to minimize the influence of residual urinary salbutamol on pharmacokinetic assessment. Urine samples were subsequently collected 30 minutes after completion of nebulization, and total urine volume was recorded for each participant. Collected samples were stored at -20 °C until analysis. Urinary salbutamol sulfate was extracted using a solid-phase extraction (SPE) technique prior to quantitative analysis. Salbutamol concentrations were then determined using a validated high-performance liquid chromatography (HPLC) method performed on an Agilent 1260 Infinity system (Agilent Technologies, USA), as previously described in published analytical methodologies.\[8-10\] Early urinary salbutamol excretion at 30 minutes (USAL 0.5) was used as a validated surrogate marker of pulmonary bioavailability and lower respiratory tract drug deposition following inhalation therapy. Statistical Analysis Statistical analysis was performed using IBM SPSS Statistics software (version 26.0; IBM Corp., Armonk, NY, USA). Continuous variables were expressed as mean ± standard deviation (SD), whereas categorical variables were presented as frequencies and percentages. Normality of data distribution was assessed using the Shapiro-Wilk test in addition to visual inspection of histograms and Q-Q plots. Because the study utilized a paired crossover design in which each participant underwent both continuous and breath-synchronized intermittent nebulization, within-subject comparisons were performed using paired-samples t-tests for normally distributed variables. Non-parametric data, when applicable, were analyzed using the Wilcoxon signed-rank test. Aerodynamic characterization parameters, including Fine Particle Dose (FPD ≤3 µm and ≤5 µm), Fine Particle Fraction (FPF% ≤3 µm and ≤5 µm), and Mass Median Aerodynamic Diameter (MMAD), were compared between nebulization modes. In the clinical pharmacokinetic model, urinary salbutamol excretion at 30 minutes (USAL 0.5) was analyzed as a surrogate marker of pulmonary bioavailability and lower respiratory tract deposition. All statistical tests were two-tailed, and a p-value \<0.05 was considered statistically significant.