Aerosolized, Gas-Phase, Intravitreal Methotrexate Reduces Proliferative Vitreoretinopathy in a Randomized Trial in a Porcine Model.

Purpose: To determine the effectiveness of aerosol-delivered methotrexate (AD-MTx) in a large-animal (porcine) model of proliferative vitreoretinopathy (PVR). Design: Prospective, randomized, interventional, double-masked, controlled, large-animal study with predetermined clinical and histopathologic outcome criteria. Controls: Half of the pigs were randomly assigned to receive an identical volume of aerosol-delivered normal saline (AD-NS) using identical delivery systems and treatment intervals. Methods: Proliferative vitreoretinopathy was surgically induced in 16 pigs (8 males and 8 females), randomly assigned to receive 2 doses (group A) or 3 doses (group B) of either AD-MTx (1.6 mg/0.4 ml) or normal saline (AD-NS). Group A pigs were euthanized at week 2 (n = 8), and group B pigs were euthanized at week 3 (n = 8). Masked clinical PVR scores (0-6) by a vitreoretinal surgeon and histopathology PVR scores (0-8) by a masked ophthalmic pathologist were used to determine outcomes. Main Outcome Measures: The mean, combined clinical and histopathology scores (both anterior and posterior) were used to determine the overall treatment effect between the groups. Results: The mean masked score (± standard deviation) when all grading end points (clinical + histopathology) were combined was a mean of 8.0 ± 2.3 in the AD-MTx group versus a higher 9.9 ± 2.0 in the AD-NS control group (P = 0.05). The clinical score was 3.88 ± 1.2 in the AD-MTx group versus 4.63 ± 1.6 in the AD-NS group (P = 0.16). The histopathology score for anterior PVR was 2.5 ± 0.8 in the AD-MTx group versus 2.5 ± 0.5 in the AD-NS group (P = 0.50), and the posterior PVR was 1.63 ± 1.6 in the AD-MTx group versus 2.75 ± 1.3 in the AD-NS group (P = 0.07). When the frequency of methotrexate dosing in group A (2 doses) was compared with that in group B (3 doses), the mean score was 8.75 versus 9.13 (P = 0.38), respectively, suggesting an insignificant difference. Conclusions: After surgical induction of PVR in an aggressive, high-risk, large-animal model, AD-MTx reduced posterior PVR formation compared with AD-NS. Additional dosing at week 3 did not improve the outcomes. No difference in anterior PVR formation was noted with intervention. This novel drug delivery system has implications for PVR reduction and warrants further investigation. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Aerosol Analysis via Electrostatic Precipitation-Electrospray Ionization Mass Spectrometry.

Electrospray ionization (ESI) is the preferred mode of ion generation for mass analysis of many organic species, as alternative ionization techniques can lead to appreciable analyte fragmentation. For this reason, ESI is an ideal method for the analysis of species within aerosol particles. However, because of their low concentrations (∼10 µg/m(3)) in most environments, ESI has been applied sparingly in aerosol particle analysis; aerosol mass spectrometers typically employ analyte volatilization followed by electron ionization or chemical ionization, which can lead to a considerable degree of analyte fragmentation. Here, we describe an approach to apply ESI to submicrometer and nanometer scale aerosol particles, which utilizes unipolar ionization to charge particles, electrostatic precipitation to collect particles on the tip of a Tungsten rod, and subsequently, by flowing liquid over the rod, ESI and mass analysis of the species composing collected particles. This technique, which we term electrostatic precipitation-ESI-MS (EP-ESI-MS), is shown to enable analysis of nanogram quantities of collected particles (from aerosol phase concentrations as low as 10(2) ng m(-3)) composed of cesium iodide, levoglucosan, and levoglucosan within a carbon nanoparticle matrix. With EP-ESI-MS, the integrated mass spectrometric signals are found to be a monotonic function of the mass concentration of analyte in the aerosol phase. We additionally show that EP-ESI-MS has a dynamic range of close to 5 orders of magnitude in mass, making it suitable for molecular analysis of aerosol particles in laboratory settings with upstream particle size classification, as well as analysis of PM 2.5 particles in ambient air.

In Vitro Evaluation of a Device for Intra-Pulmonary Aerosol Generation and Delivery.

For infants born with respiratory distress syndrome (RDS), liquid bolus delivery of surfactant administered through an endotracheal tube is common practice. While this method is generally effective, complications such as transient hypoxia, hypercapnia, and altered cerebral blood flow may occur. Aerosolized surfactant therapy has been explored as an alternative. Unfortunately, past efforts have led to disappointing results as aerosols were generated outside the lungs with significant pharyngeal deposition and minimal intrapulmonary instillation. A novel aerosol generator (Microjet™) is evaluated herein for intrapulmonary aerosol generation within an endotracheal tube and tested with Curosurf and Infasurf surfactants. Compared with other aerosol delivery devices, this process utilizes low air flow (range 0.01-0.2 L/min) that is ideal for limiting potential barotrauma to the premature newborn lung. The mass mean diameter (MMD) of the particles for both tested surfactants was less than 4 µm, which is ideal for both uniform and distal lung delivery. As an indicator of phospholipid function, surfactant surface tension was measured before and after aerosol formation; with no significant difference. Moreover, this device has an outside diameter of <1mm, which permits insertion into an endotracheal tube (of even 2.0 mm). In the premature infant where intravenous access is either technically challenging or difficult, aerosol drug delivery may provide an alternative route in patient resuscitation, stabilization and care. Other potential applications of this type of device include the delivery of nutrients, antibiotics, and analgesics via the pulmonary route.

PK10453, a nonselective platelet-derived growth factor receptor inhibitor, prevents the progression of pulmonary arterial hypertension.

The platelet-derived growth factor (PDGF) signaling pathway has been found to be activated in human pulmonary arterial hypertension (PAH) and in animal models of the disease. Our study tested the hypothesis that a novel, nonselective inhaled PDGF receptor inhibitor, PK10453, would decrease pulmonary hypertension both in the rat monocrotaline (MCT) model and the rat MCT plus pneumonectomy (MCT+PN) model of PAH. PK10453, delivered by inhalation for 4 (D4)- and 8 (D8)-minute exposures 3 times a day for 2 weeks, decreased right ventricular systolic pressure (RVSP) in both the rat MCT and rat MCT+PN models: RVSP was 80.4 ± 2.6 mmHg in the vehicle MCT group (n = 6), 44.4 ± 5.8 mmHg in the D4 MCT group (n = 6), and 37.1 ± 4.5 mmHg in the D8 MCT group (n = 5; P < 0.001 vs. vehicle); RVSP was 75.7 ± 7.1 mmHg in the vehicle MCT+PN group (n = 9), 40.4 ± 2.7 mmHg in the D4 MCT+PN group (n = 10), and 43.0 ± 3.0 mmHg in the D8 MCT+PN group (n = 8; P < 0.001). In the rat MCT+PN model, continuous telemetry monitoring of pulmonary artery pressures also demonstrated that PK10453 prevented the progression of PAH. Imatinib given by inhalation was equally effective in the MCT model but was not effective in the MCT+PN model. Immunohistochemistry demonstrated increased activation of the PDGFß receptor compared to the PDGFα receptor in neointimal and perivascular lesions found in the MCT+PN model. We show that imatinib is selective for the PDGFα receptor, whereas PK10453 has a lower half-maximal inhibitor concentration (IC50) for inhibition of kinase activity of both the PDGFα and PDGFß receptors compared to imatinib. In conclusion, PK10453, when delivered by inhalation, significantly decreased the progression of PAH in the rat MCT and MCT+PN models. Nonselective inhibition of both the PDGFα and PDGFß receptors may have a therapeutic advantage over selective PDGFα receptor inhibition in PAH.

Distribution of aerosols in murine obliterative bronchiolitis lungs by fluorescent imaging.

UNLABELLED: ABSTRACT Background: Obliterative bronchiolitis (OB) is a major obstacle to the success of lung transplantation and is also a serious complication of hematopoietic stem cell transplant. It has few therapeutic options and respiratory delivery of potential therapeutic drugs is hindered by the narrowed and occluded airways. METHODS: OB was induced in mice using an established protocol and lung function was assessed by plethysmograph. Mice were exposed to four different aerosols of aluminum phthalocyanine tetrasulfonic acid (AlPCS) that ranged in concentration and median particle size distribution (0.2-4.0 µm). The fluorescent intensity and number of pixels were measured for the trachea and lobes at two different compressional thicknesses. With analysis of the fluorescent intensity, the concentration and attenuation coefficient were estimated for each lobe and the trachea as well as individual pixels. The latter allowed generation of images reflective of the concentration. RESULTS: Lungs/trachea from OB mice had lower deposition, which correlated with lung function measurements, and apparent greater variability in the intensity compared to controls. The estimated lung volumes measured by plethysmograph were not different between the OB group and controls; however, total inflational lung capacity was reduced in OB mice. CONCLUSIONS: Despite the variability in disease induction, there is a clear link between aerosol deposition and lung function, which was revealed by fluorescent imaging. The modulation of aerosol deposition in lungs with restrictive airway disease underscores the importance of tailoring aerosolization to optimize drug delivery.

Distribution of aerosols in mouse lobes by fluorescent imaging.

Better methods are needed to quantify the distribution of drug among the airways of the lungs of small animals to facilitate the development of agents that can target specific airways. Mice were exposed to aerosols of aluminum phthalocyanine tetrasulfonic acid (AlPCS) that ranged in concentration and size (0.2-2.8 µm). The trachea and lobes were removed and placed between glass slides, and fluorescent images were obtained at two different compression thicknesses. The intensity, normalized by the area, exposure time, and thickness, was then plotted as a function of compression thickness, from which the concentration and attenuation coefficient were estimated for each lobe and then for each pixel of the image. The latter was then used to generate an image reflective of the concentration. The lobe volume, concentration, and tissue attenuation of AlPCS was consistent among the lobes. The deposition fraction increased with decreasing particle size. The network of lines in the concentration image indicated that connective tissue has a lower concentration. The central airways were clearly evident in the images of mice exposed to the very small and large aerosols. This approach provides a rapid, economical means to obtain high resolution images of mouse lungs from which detailed analysis of the distribution of deposited aerosol particles can be obtained.

Measurement of the distribution of aerosols among mouse lobes by fluorescent imaging.

Lung samples were prepared to investigate the perturbing effects of light absorption for quantifying the fluorescence signal of aluminum phthalocyanine tetrasulfonic acid (AlPCS). Standard solutions of known concentration and depth were imaged with different exposure times and analyzed. The intensity was found to be a linear function of concentration, depth, exposure time, and area. Mice were exposed to an aerosol of AlPCS with a mass median aerodynamic diameter of 390 nm and geometric standard deviation of 1.8. Images of intact lung lobes and lung homogenates were obtained and then analyzed to allow quantifying the concentration of AlPCS among the lung lobes and trachea. For the distribution of aerosols, the results indicate that the concentration was uniform among the different lobes. Combining the quantitative analysis of the concentration with image analysis of the area/thickness, the mass deposited in each lobe was readily determined. This approach provides a quantitative means to determine the selectivity of drug delivery to mouse lower respiratory tract.

High-frequency ultrasonic atomization for drug delivery to rodent animal models - optimal particle size for lung inhalation of difluoromethyl ornithine.

A high-(8-MHz) and a low-(1.7-MHz) frequency ultrasonic transducer were compared for delivering aerosols to mouse lung. The aerosol concentration (mass of dry particles/volume of air) rose nonlinearly with solution concentration of difluoromethyl ornithine for both transducers. The particle size was linear with the cube root of the solution concentration, and the slope of the low-frequency transducer was 8 times greater than that of the high-frequency transducer. The deposition fraction assessed by the assayed mass in the lung relative to the calculated inhaled mass was found to decline exponentially with particle size. The lower-frequency transducer provided a higher dose despite a lower deposition fraction, but the high-frequency transducer was more efficient and provides a more selective deposition in the lower respiratory tract while operating with significantly less demands on aerosol drying.

Intraocular nanoparticle drug delivery: a pilot study using an aerosol during pars plana vitrectomy.

PURPOSE: To describe a method of drug delivery to the retina via aerosolized nanoparticles in the gas phase during the gas-exchange stage of vitrectomy in a porcine model. METHODS: An ultrasonically atomized and dried sodium fluorescein aerosol was produced with a concentration of 12 ng/mL and a mass median particle size of 407 nm. Eighteen porcine eyes were randomly divided into six groups and subjected to standard three-port pars plana vitrectomy. After the air-fluid exchange and during the gas exchange, the eyes were exposed to the aerosol, either as a steady flow through the chamber (3.6 microg/min) or as a single fill (50 ng) at three exposure times (three eyes/time point). RESULTS: The flow-through delivery mode provided a relatively uniform deposition of aerosol on the inner surface of the retina, and longer delivery time led to an increase in the quantity deposited, with greater than 40 ng total deposition by 10 minutes. The single-fill method had uniform deposition but lower total delivery, approximately 10 ng by 60 minutes. Modeling of the data suggests that deposition in the flow-through mode is successful if the vitreous chamber contents are well mixed. The single-fill delivery was described by diffusion in a quiescent state. CONCLUSIONS: This study demonstrates a novel method of drug delivery to the posterior pole by using aerosolized nanoparticles that may be used in the gas phase of vitrectomy. Therapeutic applications include antimetabolites for modulation of proliferative vitreoretinopathy, antimicrobial agents for endophthalmitis, antiangiogenic compounds for vasoproliferative disorders, corticosteroid delivery, and other pharmacotherapies directed at the retina and choroid.