Improved Aerosol Delivery System for Intubated Animals

Aerosol administration of medication to animals is much more complicated than in humans due to particle size in relationship...


Introduction
Testing medication on animals is a key final step before beginning testing in humans. Because of genetic similarity, nonhuman primate (NHP) is the preferred animal for both efficacy and toxicology testing in genetic replacement therapeutic programs such as lung gene therapy for cystic fibrosis. For parental delivery, dosing is simple in that all the medication is delivered. For enteral medication, bioavailability comes into play but can usually be predicted from the dose administered and the blood level achieved.
However, when targeting pulmonary delivery for an agent expected to stay largely in the lungs, predictable dosing can be a challenge.

One option of direct delivery to the lungs was the Penn Century
Microsprayer [1,2] which is no longer made or available. Another option has been the Aero Probe™ made by Trudell Medical International (also currently not commercially available) which has shown promise in the airway delivery of lung gene therapy vectors in an intubated rabbit model [3]. This multilumen catheter can be centered in the endotracheal tube of a deeply anesthetized animal which can be ventilated by the pulses of air from the controller which nebulizes a liquid extruded from the center lumen. The ensuing droplets are in the order of 8-10 µm in diameter and have been shown to effectively transfer genetic material to the airway [3].
However, droplets of this size may be too large to penetrate deeply and if alveolar delivery is desired, alternatives may be necessary.
Various nebulizing systems have been proposed for both spontaneously breathing animals [4] using a face mask and for intubated animals. MacLoughlin and colleagues [4] duplicated breathing patterns of NHPs and them mimicked this pattern with a breath simulator. In the in vitro set up using a vibrating mesh nebulizer on a t piece there was no endotracheal tube or face mask connection between the delivery device and the "inspiratory" filter and they estimated that they would delivery a mean of 32% of the charge dose (range 24-48%) Particle size was not considered and any material arriving on the "inspiratory" filter was considered as delivered to the lungs. Unfortunately, in other studies, the delivery efficiency is relatively low, in the order of 2%, and variable [1].
While this is not a problem for inexpensive drugs, it can be a serious drawback for expensive hard to make products such as those used for lung gene therapy. Furthermore, dose ranging studies require a reasonable degree of predictability of delivery from the device utilized and variability can be considerable in spontaneously breathing animals [2]. The purpose of this communication is to describe an efficient and predictable delivery system for use in intubated non-human primates (NHP) as a prelude to preclinical studies in a lung gene therapy program for cystic fibrosis.

Materials and Methods
The nebulizer chosen was the Aero Eclipse™ II Breath Actuated Nebulizer (AE II BAN) made by Trudell Medical International (London, Ontario). Based on the previous experience ventilating rabbits via the controller for the Aeroprobe, it was decided to ventilate the NHPs from the flow from the AE II BAN driven by the original controller for the AeroProbe, the LABneb™CCU (CCU), which was available from previous studies although is also not currently commercially available. This is a 50-psi source with a flow of approximately 8 L/min through the nebulizer. The CCU can be programmed to deliver pulses of varying duration which controls the inspired volume. The AE II BAN is connected to a t-piece that leads directly to the connections for a 3.5 mm endotracheal tube (ET). The right angle of the "t is directed upwards and connected directly onto the expiratory filter and to another t-piece ( Figure 1  AeroEclipse II Breath Actuated Nebulizer™ (Trudell Medical International).

LABneb™Catheter Control Unit (CCU).
For the in vitro set up, an electrostatic filter was placed at the end of the endotracheal tube that would normally have gone into the NHP. In the in vivo setting, the anesthetized NHP is held in the sitting position with the nebulizer above so that any rain out of aerosol tends to run into the lungs by gravity and is preferentially directed to the dependent lobes. In the in vitro setting, some rain out may be lost at the connector to the inspiratory filter although virtually all aerosol that arrives at the filter is captured. Expiration is through another electrostatic filter that is used for quantification of recovery in the in vitro setting but also to prevent environmental contamination from biological active viral vectors in the in vivo setting. The total "dead space" from where the AE II BAN connects to the filter on the inspiratory limb is 74 mL (measured by water displacement) with the inspiratory filter which would not be present in vivo having a dead space of 24 mL. The filter exits into a lung simulator with settings of either "infant" or "child." The lung simulator was an Ing Mar Medical ASL 5000 (Pittsburg, PA). Prior to charging the nebulizer, the pulse duration is adjusted to achieve

Result
The Table 1 shows the individual data points for all experiments. demonstrating the effect of the larger volume delivered for the same apparatus dead space. Individual data are given in the Table   1. The particle size distribution showed a mass median diameter (MMD) of 2.07 µm with geometric standard deviation of 1.57.
During dismantling of the second infant run, a drop of nebulisate spilled from the inspiratory filter casing. It was taken up with a blotter but inadvertently added to the connectors rather than to the inspiratory filter assay. This and a slightly lower output for this run resulted in a lower deposition.

Discussion
The results would suggest that there was not only a relatively efficient delivery of the initial charge but also that there was While the efficiency of this system is less than that of the no longer available Penn Century Microsprayer [1,2], the preliminary study in an NHP showed wide spread distal deposition of dye whereas nuclear medicine studies with the Penn Century Microsprayer suggest very proximal deposition [1]. This diffuse distribution is the result of a very small particle size distribution (MMD 2 µm) coupled with the larger ventilator volumes compared with normal tidal breathing. With normal breathing, the majority of the inspired volume goes to the mid lung zones [5]  There are some limitations to this study. It is recognized that rain out in the ET will end up in the dependent areas of the lungs through gravity, a feature that was obvious in the preliminary NHP study and will contribute to non-uniform aerosol distribution.
In the in vitro set up, some of this rain out will end up on the inspiratory filter. The assumption that whatever was captured on the inspiratory filter represents aerosol that would deposit in the lung is not entirely valid. Deposition of rain out in the ET has already been recognized but some of the aerosol that would end up in the large airways in vivo at the end of expiration would be washed out at the start of inspiration but in the in vitro model, this would be captured on the inspiratory filter. However, such losses would be offset by the lower dead space in vivo since the 24 mL of additional dead space from the inspiratory filter would not be there.
The increased ratio of deposition on the inspiratory filter to that on the expiratory filter with larger ventilator volumes means that the effect of apparatus dead space would be greater in smaller animals. Another was the lung model that was used. In this device, expiration is retarded to mimic the normal physiological post inspiration, inspiratory muscle activity which maintains lung volume during expiration to enhance gas exchange. This results in a sinusoidal pattern of both inspiration and expiration where inspiration is shorter than expiration. However, when deeply anesthetized, this pattern is lost with expiration becoming a rapid emptying of the lungs and the expiratory phase becoming shorter [6]. Because of the intent to ventilate the NHP with the nebulizer delivery system, the level of anesthesia necessary to prevent the animal from fighting the "ventilator" was deep and maintained and expiration was much more rapid that that seen in the in vitro model used. The biggest issue with this is that it greatly increased the time required to go to "dryness" because it required an artificial expiratory pause. This was not appreciated initially and failure to allow for complete expiration of the first "child" model led to a recording of a higher peak pressure than that seen later when a greater pause was used. In summary, this experimental aerosol delivery system designed for animals in the 3 to 10-kilogram range would appear to be more efficient than other systems described.
The use with larger animals may be possible but the inspiratory flow is limited at 133 mL/second so there would be a limit as to the volume that could be achieved in a reasonable inspiratory time.
The particle size distribution is such that widespread peripheral deposition would be anticipated. The system is relatively simple and could provide a valuable resource to investigators should it become available for more widespread use.

Author Disclosure Statement
Dr Coates has done non remunerated consulting work with 4D Molecular Therapeutics and has done collaborative research with Trudell Medical International. The study was sponsored by 4D Molecular Therapeutics and Trudell Medical International.

Author Contribution
Drs Coates and Johnson developed the concept based on a successful preliminary in vivo study. Ms Doyle developed the interface between the nebulizer and associated connectors and the breath simulator as well as doing the HPLC analysis. Mr Coultes was responsible for modifying the nebulizer to prevent aerosol leaks when pressurized. Mr Nagel oversaw all laboratory work and made the particle size measurements. All authors assisted in the preparation of the manuscript.