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EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Formulation and Device Selection for the Generation of Pharmaceutical Aerosols
NOTE: Formulation and device selection depend on the individual drug&#39;s physicochemical properties to be aerosolized.
<ol>
	<li>Nebulized liquid aerosols.
	<ol>
		<li>Dissolve the drug in 100 mL of water or physiologic saline.</li>
		<li>Load a 100 mL syringe with the drug solution and place the syringe into the syringe pump with a flow rate set at 1 mL/min.</li>
		<li>Connect the syringe pump to the jet nebulizer and purge the air from the feed line leading to the nebulizer.</li>
		<li>Connect the pressurized air source to the jet nebulizer and set the airflow meter to 10 L/min.</li>
		<li>Insert the jet nebulizer into the pre-separator. The pre-separator connects the nebulizer to the central aerosol plenum of the inhalation unit (Figure 1). 
		NOTE: Many drug compounds have limited aqueous solubility and are better formulated as dry powder aerosols. If a stable suspension can be made from a micronized mass median aerodynamic diameter (MMAD &#60; 5 &#956;M) compound, it may be used with the jet nebulizer. Caution should be used as the suspension may clog the nebulizer. Nebulizer feed concentrations from 1 mg/mL for potent compounds (like the bronchodilator ipratropium) to 40 mg/mL suspensions (for less potent compounds like salbutamol sulfate) have been used. The syringe pump feed rate is set at 1 mL/min for a practical reason: to allow the aerosol concentration equilibration period and 45-min-long exposure to be completed without the need to reload the syringe.</li>
	</ol>
	</li>
</ol>
2. Aerosol Exposure Experiment Setup
<ol>
	<li>Measure the drug concentration (by collecting aerosol on an absolute filter) and particle size distribution (by collecting aerosol using a cascade impactor) of the aerosol that enters the inhalation unit post pre-separator/cyclone. Use these parameters, the animal&#39;s minute ventilation, body weight, and exposure time, to estimate the drug dose deposited in the lungs.
	<ol>
		<li>Weigh the absolute filter and record the filter weight. Place the filter into the filter holder and assemble the filter holder. Connect the inlet of the absolute filter holder to a central aerosol plenum sample port and the outlet to a vacuum source set to sample the aerosol at a flow rate of 1 L/min for the experiment.&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: The mass of the drug on the filter after sampling for 45 min can be in the sub-microgram range and/or be mixed with lactose, NaCl salt, or another vehicle. A microbalance that reads to 0.1 micrograms is necessary. The filter must be equilibrated and weighed in a humidity-controlled environment to get an accurate weight of the drug deposited on the filter. The weight of the drug on the filters can only be used in dose calculation if there is no vehicle in the formulation or the vehicle is water. When there is a vehicle in the formulation other than water, the weight of the substance on the filter only gives an estimated starting point for further drug content analysis by high-performance liquid chromatography (HPLC).</li>
		<li>Weigh and record the weight of the 7 cascade impactor stage filters and one final &#34;tailpiece&#34; filter. Place a filter on each of the seven stages of the cascade impactor and assemble the Mercer cascade impactor. Connect the inlet of the cascade impactor to a central aerosol plenum sample port and the outlet to a vacuum source set to sample the aerosol at the flow rate the cascade impactor is calibrated at (usually 0.5 or 1 L/min) for the duration of the experiment.</li>
		<li>Monitor the aerosol content of the inhalation unit with the real-time aerosol monitor (see Table of Materials and Reagents) to confirm the aerosol generators are functional and producing a stable aerosol throughout the experiment. Connect the inlet of the real-time aerosol monitor to a central aerosol plenum sample port and the outlet to a vacuum source set to sample the aerosol at a flow rate of 1 L/min for the experiment. 
		NOTE: The real-time aerosol monitor signal is reported in &#956;g/L but is calibrated for road dust and must be recalibrated for each drug aerosol to yield correct aerosol concentration values. Calibration is unnecessary when using the monitor to confirm aerosol concentration&#39;s presence or absence and temporal stability.</li>
		<li>Set the process control parameters (airflow, vacuum, pressure, aerosol generator power) to the required values that depend on the number of animals connected to the inhalation unit. The inhalation unit and aerosol generators are continuously controlled/monitored by a computerized process control/data acquisition system (DACO) supplied by the manufacturer (see Table of Materials and Reagents). The airflow rate into the inhalation unit should be minimally approximately 2 times the total minute ventilation rate of all animals in the inhalation unit to avoid a buildup of CO2.</li>
	</ol>
	</li>
	<li>Load mice into nose-only restrainers before exposing them to aerosol in the nose-only inhalation unit. Also load restraint stress control animals into restrainers to breathe room air. 
	NOTE: A dose/response experiment consists of multiple groups of mice exposed to aerosol for different amounts of time. Exposure time controls each group&#39;s dose during a dose/response experiment.
	<ol>
		<li>Angle the restraining tube towards the ceiling while trying to load the animals, as they tend to run upward, trying to escape. Pointing the tubes down while loading will encourage turning around and escaping out the back of the tube. Ensure the mouse&#39;s nose is oriented into the pointed end of the tube and fix the variable-position plunger into the back end of the restrainer. 
		NOTE: The plunger is fashioned to allow the mouse&#39;s tail to protrude from the holder, which enables the mouse to regulate its body temperature while in the restrainer.</li>
		<li>Adjust the plunger to allow the mice to rotate but not turn head to tail to ensure the aerosol is inhaled.</li>
		<li>Continuously monitor the mice while in the restraining tubes. After positioning the plunger, small mice (&#60;20 grams) often attempt to turn head to tail in the tubes and adopt a U-position where they have trouble breathing. This turning behavior is most prevalent in the first 5 minutes of restraint; after that, the mice rarely attempt to turn head to tail.</li>
	</ol>
	</li>
</ol>
3. Aerosol Delivery
<ol>
	<li>Insert stoppers to plug the delivery ports of the inhalation unit and activate the aerosol generator, compressed air flow controller, and inhalation unit vacuum pump from within the process control software.</li>
	<li>Once the readings from the real-time aerosol monitor demonstrate the aerosol concentration has to come to equilibrium (~30 min,&#160;Figure 2), begin removing stoppers and inserting the nose-only restraining tubes containing mice into the inhalation unit. Repeat until all mice exposed to the drug are connected to the inhalation unit. 
	NOTE: In the example experiment, the total airflow supplied to the inhalation unit via the aerosol generator and dilution air is set to provide a 0.5 L/min flow rate to each animal exposure port in use. For example, a 15 L/min total airflow is enough to supply each of the 30 ports in the inhalation unit. This is much more airflow than required by the minute ventilation of the mice, but a larger airflow is necessary to supply the energy (pressure drop across the aerosol generator) to produce (atomize/de-agglomerate) the aerosol.</li>
	<li>Once all animals are loaded into the exposure unit, turn on the vacuum sampling pumps connected to the absolute filter and cascade impactor using the process control software.</li>
	<li>When all exposures are completed, turn off the aerosol generator and remove the remaining mice from the inhalation unit.&#160; 
	NOTE: Animals will be handled in a change station hood or by personnel wearing a facemask. After the conclusion of aerosol delivery, the mice are removed from the tubes and sanitized after each use.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Nose-only exposure inhalation unit</td>
			<td>TSE systems</td>
			<td>700100-KNES-040-ss</td>
			<td>Custom configurations available</td>
		</tr>
		<tr>
			<td>DACO data acquisition system</td>
			<td>TSE systems</td>
			<td>700400-PRO-C-D/1</td>
			<td></td>
		</tr>
		<tr>
			<td>MC One Jet Mill</td>
			<td>Jetpharma</td>
			<td>DEC MicroJet 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Turbula Mixer</td>
			<td>GlenMills Inc</td>
			<td>T2F</td>
			<td></td>
		</tr>
		<tr>
			<td>Micronized Lactose</td>
			<td>DFE Pharma</td>
			<td>Lactohale 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydraulic press</td>
			<td>Specac</td>
			<td>GS15011</td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade impactor filters</td>
			<td>Pall Life Sciences</td>
			<td>7219</td>
			<td>Emfab filter</td>
		</tr>
		<tr>
			<td>Absolute filters</td>
			<td>Whatman</td>
			<td>10370302</td>
			<td>5 cm diameter</td>
		</tr>
		<tr>
			<td>Real time aerosol monitor 
			Microdust Pro Monitor</td>
			<td>Casella</td>
			<td>CEL-712</td>
			<td></td>
		</tr>
		<tr>
			<td>Ipratropium bromide</td>
			<td>Spectrum Chemical</td>
			<td>I1178</td>
			<td>pre-micronized</td>
		</tr>
		<tr>
			<td>flexiVent FX1 system</td>
			<td>scireq</td>
			<td>FV-FXCS</td>
			<td></td>
		</tr>
	</tbody>
</table>

a technique to generate and deliver drug aerosols directly to mouse lungs

Source: Phillips, J. E. et al., <a href="https://app.jove.com/v/55454">Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the generation of drug aerosols and the targeted nose-only delivery of these aerosols to mice using an inhalation unit equipped with an aerosol generator. The mouse is restrained in a nose-only restrainer and connected to the inhalation unit, which delivers the drug directly into the mouse&#39;s nasal cavity. Subsequently, the drug passes through the trachea and enters the lungs.

<img alt="Figure 1" src="/files/ftp_upload/22246/22246_Figure1.jpg" /> 
Figure 1: Inhalation unit with dry powder aerosol generator attached.&#160;The nebulizer used to generate aerosol from liquid formulations is shown on the right. 1) Wright dust feed, 2) cyclone, 3) syringe pump, 4) jet nebulizer inserted into pre-separator, 5) dilution air mixer. When delivering nebulized aerosols, the dust feed, and cyclone are replaced by the syringe pump, jet nebulizer/pre-sep...

A Technique to Generate and Deliver Drug Aerosols Directly to Mouse Lungs

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with a nose-only inhalation unit connected to an aerosol generator assembly.
Connect the generator to a pressurized air source. Next, connect a syringe containing a therapeutic drug solution, mounted on an infusion pump set to a desired flow rate.
Start the airflow and drug flow to initiate aerosol generation.
The compressed airflow breaks the drug solution into tiny droplets, generating micron-sized drug aerosols.
The drug aerosols pass through the generator into the connected inhalation unit.
Now, place a conscious mouse in a nose-only restrainer with its nose oriented into the tube&#39;s pointed end. Fix the plunger.
When the aerosol concentration in the inhalation unit reaches equilibrium, insert the restrainer carrying the mouse into the inhalation unit.
The restrainer facilitates aerosol exposure directly to the murine nasal cavity.
The murine nasal cavity allows the aerosols to pass through the trachea, enabling their direct delivery to the lungs.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

128 Views. Source: Phillips, J. E. et al., Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System. J. Vis. Exp. (2017) This video demonstrates the generation of drug aerosols and the targeted nose-only delivery of these aerosols to mice using an inhalation unit equipped with an aerosol generator. The mouse is restrained in a nose-only restrainer and connected to the inhalation unit, which delivers the drug directly into the mouse's nasal...

Video: A Technique to Generate and Deliver Drug Aerosols Directly to Mouse Lungs - Concept

Development of a Nose-only Inhalation Toxicity Test Chamber That Provides Four Exposure Concentrations of Nano-sized Particles

Using a numerical analysis based on computerized fluid dynamics, a nose-only inhalation toxicity chamber with four different exposure concentrations is designed and validated for flow field uniformity and cross-contamination among the exposure ports for each concentration. The designed flow field values are compared with the measured values from exposure ports located horizontally and vertically. For this purpose, nanoscale sodium chloride particles are generated as test particles and introduced to the inhalation chamber to evaluate the cross-contamination and concentration maintenance among the chambers, for each concentration group. The results indicate that the designed multiconcentration inhalation chamber can be used in animal inhalation toxicity testing without cross-contamination among concentration groups. Moreover, the designed multiconcentration inhalation toxicity chamber can also be converted to a single-concentration inhalation chamber. Further testing with gas, organic vapor, or non-nanoscale particles will ensure the use of the chamber in the inhalation testing of other test articles.

A nose-only inhalation toxicity chamber capable of testing inhalation toxicity at four different exposure concentrations was designed and validated for flow field uniformity and cross-contamination between the exposure ports for each concentration. Here, we present a protocol to confirm that the designed chamber is effective for inhalation toxicity testing.

Using a numerical analysis based on computerized fluid dynamics, a nose-only inhalation toxicity chamber with four different exposure concentrations is ...

JoVE Journal

Engineering

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...

Medicine

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

A Technique to Generate and Deliver Drug Aerosols Directly to Mouse Lungs

Transcript

A Technique to Generate and Deliver Drug Aerosols Directly to Mouse Lungs

Development of a Nose-only Inhalation Toxicity Test Chamber That Provides Four Exposure Concentrations of Nano-sized Particles

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intratracheal Administration of Dry Powder Formulation in Mice