Disposable Dosators Intended for Dry Powder Delivery to Mice

Summary

Pharmaceutical dry powder development necessitates reliable in vivo testing, often using a murine model. Device technology for accurately and reproducibly delivering dry powder aerosols to mice is restricted. This study presents disposable dosators for pulmonary drug delivery at mouse-relevant doses, aiding initial proof-of-concept research.

Abstract

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus metering and dosing, and a propellant-free dispersal mechanism. To develop pharmaceutical dry powder aerosol products, robust in vivo testing is essential. Typically, initial studies involve using a murine model for preliminary evaluation before conducting formal studies in larger animal species. However, a significant limitation in this approach is the lack of suitable device technology to accurately and reproducibly deliver dry powders to small animals, hindering such models' utility. To address these challenges, disposable syringe dosators were developed specifically for intrapulmonary delivery of dry powders in doses appropriate for mice. These dosators load and deliver a predetermined amount of powder obtained from a uniform bulk density powder bed. This discrete control is achieved by inserting a blunt needle to a fixed depth (tamping) into the powder bed, removing a fixed quantity each time. Notably, this dosing pattern has proven effective for a range of spray-dried powders. In experiments involving four different model spray-dried powders, the dosators demonstrated the ability to achieve doses within the range of 30 to 1100 µg. The achieved dose was influenced by factors such as the number of tamps, the size of the dosator needle, and the specific formulation used. One of the key benefits of these dosators is their ease of manufacturing, making them accessible and cost-effective for delivering dry powders to mice during initial proof-of-concept studies. The disposable nature of the dosators facilitates use in animal procedure rooms, where cleaning and refilling reusable systems and weighing materials is inconvenient. Thus, developing disposable syringe dosators has addressed a significant hurdle in murine dry powder delivery for proof-of-concept studies, enabling researchers to conduct more accurate and reproducible preliminary studies in small animal models for pulmonary drug delivery.

Introduction

The use of dry powder inhalers (DPIs) for pulmonary drug delivery has garnered significant interest over the past three decades due to the global phase-out of chlorofluorocarbon propellants^1,2. DPIs offer numerous benefits over other pulmonary delivery systems, such as metered dose inhalers and nebulizers, including formulation stability, portability, ease of use, and propellant-free dispersal mechanisms². However, before moving DPI products toward clinical translation, several preclinical studies must be conducted, many of which are initially completed using a murine model. Nevertheless, technologies available to deliver dry powders accurately and reproducibly to small animals are limited.

Common methods to deliver dry powders to small animals, such as mice, include passive inhalation^3,4,5,6,7 and direct administration^{8,9,10,11,12,13}. Passive inhalation typically requires a custom chamber that utilizes large doses of spray-dried powder to prepare a sufficient aerosol cloud. As mice are obligate nose breathers¹⁴, delivery by passive inhalation requires the powder to travel through the nose and throat to reach the lungs, necessitating the maintenance of an aerosol cloud with sufficient particle aerodynamic properties^7,8. While a useful technique that is more physiologically relevant than direct delivery due to inhalation as a result of normal breathing¹⁴, it may not be suitable for initial studies where powder mass is limited.

Alternatively, a number of intratracheal delivery devices for direct dry powder delivery have been reported^{8,9,10,11,12,13}. Intratracheal devices bypass the nose and throat, delivering the powder directly to the lungs and allowing for finer control over the delivered dose¹⁴. Additionally, some devices, especially those prepared using a tamping loading procedure⁹, can be prepared with smaller quantities, which is an important consideration for initial proof-of-concept studies. The lack of universally available intratracheal delivery devices has hindered their potential for use, limiting availability and leading to interlaboratory differences¹⁴. In this study, we propose a simple, inexpensive, disposable dosator for intratracheal delivery that can be utilized for proof-of-concept murine studies in the development of dry powder aerosols.

Protocol

All animal experiments were conducted in accordance with the Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center. Healthy female BALB/c mice, ~6-8 weeks old, were administered the dry powder content of one dosator by intrapulmonary aerosol delivery for a pharmacokinetic study using spectinamide 1599 dry powders⁹. The animals were obtained from a commercial source (see Table of Materials).

1. Preparation of the dosator and the filling components

1. Trim the plastic luer portion of a 2.54 cm (1 inch) blunt stainless-steel needle (21-25 G) using either a precision sectioning saw (see Table of Materials), or a belt sander until 2-3 mm of the plastic luer remains (Figure 1A and Figure 2A).
   NOTE: If a belt sander is used, the stainless-steel needle may need to be cleaned using a smaller needle or wire to remove the possible obstructions created.
2. Cut off the tip (1-1.5 cm) of a 0.6 mL conical centrifuge tube. Fill the tip of the tube with 30-35 mg of powder.
   ​NOTE: See Representative Results for the details of the example powders used for the present study. The powder aerosol performance should be evaluated prior to use in this application following standard methodology as described in USP General Chapter <601> (see Table of Materials).
3. If storing and/or transporting the powder, use the tube cap (cut off) to close the vial. Seal with paraffin film to minimize powder exposure to ambient moisture if storing and/or transporting.

2. Loading and assembling dosators

1. Tamp the trimmed stainless-steel needle into the powder bed in the 0.6 mL conical centrifuge tube tip as many times as needed to achieve the desired dose (Figure 2B). Gently wipe the sides of the stainless-steel needle with a low-lint wiper to remove any excess powder (Figure 3).
2. Gently insert the loaded stainless-steel needle into a 3.81 cm (1.5 inch) polypropylene or 5.08 cm (2 inch) polytetrafluoroethylene (PTFE) needle (16-20 G) (see Table of Materials) to avoid dislodging any powder (Figure 1B,C and Figure 2C).

3. Actuating dosators

1. Draw back a disposable syringe to the desired volume, which may vary based on application.
   NOTE: For intrapulmonary administration in mice, 0.15-0.6 mL is typically appropriate^8,9.
2. Attach the syringe to the luer lock on the polypropylene or PTFE needle (Figure 2D).
3. Insert the needle end of the dosator into the desired target. For analyzing powder content and reproducibility, insert the needle through a perforated rubber septum or paraffin film into a vial containing a small amount (e.g., 1-5 mL) of water and/or organic solvent (e.g., ethanol), with solvent identity and volume dependent on active pharmaceutical ingredient (API) physical characteristics and the quantification method.
   1. For delivery to mice, insert the needle up to the first bronchial bifurcation of the trachea of anesthetized mice following established protocols^9,15.
4. Depress the syringe forcefully, expelling the powder out of the device into the collection vial (Figure 2E).
   NOTE: The same technique must be followed for delivering the powder to the murine lungs.
5. For analyzing content and reproducibility from the collection vial, utilize an appropriate analytical method for the specific API, such as UV-Visible (UV-Vis) spectrophotometry or high-performance liquid chromatography (HPLC).

Results

The aerosol performance of various spray-dried powders was established prior to use in this study. The aerodynamic particle size distribution (APSD) was described by the mass median aerodynamic diameter (MMAD), representing the size that divides the distribution in two at the 50^th percentile (d₅₀), and the geometric standard deviation (GSD), reflecting the breadth of the distribution. The GSD is defined by the square root of the aerodynamic diameter at the 80^th percentile divided by ...

Discussion

As mice are obligate nose breathers, delivery via passive inhalation for initial proof-of-concept studies makes efficiency and dose estimation challenging as the powder must pass the nose and throat in a manner dependent on particle properties and powder dispersion efficiency^7,8,14. The use of the dosators developed herein bypasses the nose and throat, with the dosator inserted to the first bronchial bifurcation

Materials

Name	Company	Catalog Number	Comments
0.6 mL microcentrifuge tubes	Fisher Scientific	05-408-120
Analytical balance	Mettler Toledo	AR1140	Any analytical balance with sufficient range can be used
Blunt stainless-steel needle, 1 inch, 21 G	McMaster-Carr	75165A681
Blunt stainless-steel needle, 1 inch, 22 G	McMaster-Carr	75165A683
Blunt stainless-steel needle, 1 inch, 25 G	McMaster-Carr	75165A687
Disposable syringe with luer lock (1 mL)	Fisher Scientific	14-823-30	3-mL syringes can also be used
Female BALB/c mice	Charles River, Wilmington, MA, USA
High-performance cascade impactor	Next Generation Impactor	Apparatus 5
Lab film (e.g., Parafilm)	Fisher Scientific	S37440
Low-lint wiper (e.g., Kimwipes)	Kimberly-Clark Professional	34133
Low-resistance dry powder inhaler	RS01 mod 7
Polypropylene needle, 1.5 inch, 16 G	McMaster-Carr	6934A111
Polypropylene needle, 1.5 inch, 18 G	McMaster-Carr	6934A53
Polypropylene needle, 1.5 inch, 20 G	McMaster-Carr	6934A55
Precision sectioning saw	TedPella	812-300	Belt sander can be used as an alternative
PTFE needle, 2 inch, 20 G	McMaster-Carr	75175A694
USP General Chapter <601>			http://www.uspbpep.com/usp31/v31261/usp31nf26s1_c601.asp