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

Summary

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.

Abstract

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.

Introduction

Biomedicines such as peptides, oligonucleotides, and antibodies are considered to have potential application as novel therapeutic agents for refractory central nervous system disorders that currently have no curative therapy. However, because most biomedicines are water-soluble macromolecules, delivery from the blood into the brain via intravenous or oral administration is extremely difficult due to impedance of the blood-brain barrier (BBB).

In recent years, intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that avoids the BBB^1,2,3,4,5. However, there have been relatively few reports regarding the quantitative analysis of nose-to-brain pathway delivery⁶. Moreover, there have been virtually no reports on established optimal administration conditions and dosing regimens, such as volume, times, time-periods, and speed, for investigations of nose-to-brain delivery. The aforementioned deficiencies can be ascribed to the following reasons: (i) an optimal method of intranasal administration for mice has yet to be established, and (ii) intranasal administration by pipetting, which is generally used, is typically characterized by interindividual variation among animals due to mucociliary clearance (MC), thereby often leading to underestimations of the actual nose-to-brain delivery potential of a particular drug.

Inhalation anesthesia using isoflurane (initiation: 4%, maintenance: 2%) with an inhalation mask for rodents has gained widespread use, with the aim of reducing or eliminating the pain associated with surgery performed on experimental animals. The use of masks makes it relatively straightforward to perform typical drug administration in experimental animals under inhalation anesthesia via the subcutaneous, intraperitoneal, and intravenous routes. However, in the case of intranasal administration, the mask needs to be temporarily removed from the animals for drug administration. With maintenance under 2% isoflurane, animals typically awaken rapidly from inhalation anesthesia. When the administration volume per dose is large, this could cause the drug solution to flow from the nasal cavity into the esophagus, and therefore a single large dose may need to be broken down into multiple smaller doses for intranasal administration to small animals. As intranasal administration necessitates mask removal for repeated administration and sufficient time for sustained nasal cavity delivery, there is a high probability that mice would awaken from anesthesia during the administration procedure. This makes it very difficult to perform intranasal administration under a stable anesthetic state, and probably contributes to the observed interindividual variation of nose-to-brain delivery among rodents.

In this study, we therefore developed two novel methods of stable intranasal administration under inhalation anesthesia, which impose minimal physical stress on the experimental animals. For the first method, we used a temporarily openable mask that enables intranasal administration during inhalation anesthesia. The openable part of the mask incorporates a silicone plug that can be used in accordance with administration timing to facilitate stable intranasal administration using a pipette. For the second method, a cannula was surgically inserted to pass from the esophagus into the nasal cavity, and a syringe pump was then attached to this so that the drug solution could be directly and reliably delivered into the nasal cavity under stable inhalation anesthesia. This method can enhance the delivery of drugs into the brain via the nose-to-brain route, because by substantially minimizing the effects of MC, drug retentively in the nasal cavity would be improved. In addition, we describe a method for quantitatively evaluating drug distribution levels (% for the injected dose/g brain) in the brain using radio-labeled [¹⁴C]-inulin [molecular weight (MW): 5,000] as a model substrate of water-soluble macromolecules.

Protocol

This animal study (#AP17P004) was performed in accordance with the guidelines approved by the Nihon University Animal Care and Use Committee (Tokyo, Japan). This study (#17-0001) was approved by the Radioisotope Center of the School of Pharmacy, Nihon University.

1. Animals Used for Intranasal Administration Under Inhalation Anesthesia

1. House the experimental mice in stainless-steel cages under a 12-h light/dark cycle (light on 8:00 AM–8:00 PM), with a controlled temperature maintained at 23 ± 1°C, humidity of 50% ± 10%, and ad libitum access to food and water.
2. Prior to experimentation, anesthetize the mice via inhalation of 2% isoflurane, following initiation at a concentration of 4%. Confirm the requisite level of anesthetization by checking for the disappearance of surface righting.  

2. Preparation of the Administration Solution

1. Prepare an administration solution of [¹⁴C]-inulin (50 μM, 0.5 μCi/mL per mouse) by diluting in phosphate-buffered saline, and store at 4 °C until use.

3. Intranasal Administrations for Mice

1. Micropipette method using a temporarily openable inhalation mask (Figure 1)
   Note: This technique is a modification of the intranasal administration protocol using a micropipette established by Frey et al.³
   1. Fix mice in the supine position on a corkboard by taping their limbs under inhalation anesthesia with 2% isoflurane (Figure 1A).
   2. Administer a total volume of 25-μL administration solution to each mouse at 30-s intervals, via 1- to 2-μL doses alternatively administered into the left and right nostrils while the mice are fixed under inhalation anesthesia (Figure 1B and C).
   3. Note: Waste anesthetic gas is scavenged by active means (fume hood, hard-ducted biosafety cabinet, vacuum, etc.) when the  mask is opened and by passive means(waste anesthetic gas cannister)  when closed 
2. Reverse cannulation method from the airway side through the esophagus (Figure 2)
   Note: This technique is a modification of the intranasal absorption protocol for rats established by Hirai et al.⁷
   1. Fix mice in the supine position on a corkboard by taping their limbs under inhalation anesthesia with 2% isoflurane.
   2. Hair in the neck is shaved and prepped via betadine or chlorhexidine application followed by an alcohol rinse.
   3. Expose the trachea and esophagus by expanding the skin below the throat with forceps after making a small incision (1.5 cm) with scissors.
   4. Make an incision (1 mm) in the trachea using scissors.
   5. Insert a cannula (internal diameter: 0.58 mm, exterior diameter: 0.965 mm) to a length of 1.2 cm and attach the opposite end of the cannula to the inside of the inhalation mask.
   6. Make an incision (1 mm) in the esophagus using scissors, insert a cannula (internal diameter: 0.28 mm, exterior diameter: 0.61 mm) to a length of 1.4 cm toward the posterior part of the nasal cavity, and ligate it (Figure 2A and B).
      Note: Procedures 3.2.2 to 3.2.4 were carried out under a stereoscopic microscope at ×10 magnification.
   7. Attach a needle (27G × 1/2) to a 1-mL syringe filled with an administration solution and connect to a programmable micro-syringe pump.
   8. Connect above needle to the cannula that had been inserted into the esophagus at 3.2.5 (Figure 2C).
   9. Administer a total volume of 25 μL [¹⁴C]-inulin solution at a constant rate (5 μL/min) (Figure 2C and 2D).

4. Quantitative Experiment Using Radio-Labeled Water-Soluble Macromolecules ([¹⁴C]-inulin)

1. Decapitate the experimental mice under anesthesia and open up their craniums, using scissors and from the side of the medulla oblongata, while taking care not to damage the brains.
2. Extract carefully the whole brain by scooping using a micro-spatula from cranium.
3. Place a filter paper moistened with saline solution on a Petri dish that is stored on ice.
4. Place the extracted brains on the moistened filter paper.
5. Wipe off blood adhering to the surface of the brain with a cotton swab moistened with saline solution to eliminate at least the influence of [¹⁴C]-inulin in the blood on the surface of the brain.
6. Dissect brains rapidly, and divide them into three parts: the olfactory bulb, cerebrum, and medulla oblongata (including the pons).
7. Place brain samples in tissue solubilizer at 50 °C for 1 h.
8. Add 10 μL of the liquid scintillation cocktail to the brain samples.
9. Transfer a 25 μL aliquot of the administration solution dissolved in scintillation cocktail to a scintillation vial to determine the radioactivity of the applied solution.
10. Measure the disintegrations per minute of [¹⁴C] radioactivity in the brain sample ([¹⁴C] X_brain) and the applied solution ([¹⁴C] X_{IN dose}) in a liquid scintillation counter equipped with an appropriate crossover correction for ³H and ¹⁴C.

5. Data Analysis

1. Calculate the drug distribution levels (%) for the injected dose (ID%) using the following equation:
   ID%⁄g brain = ([¹⁴C] X_brain/[¹⁴C] X_{IN dose}) × 100,
   where X_brain (dpm/g brain) is the amount of [¹⁴C]-inulin measured in the brain tissue and X_{IN dose} (dpm/25 μL solution) is the concentration of the [¹⁴C]-inulin in the solution used for intranasal administration.

Results

Figure 3 shows the [¹⁴C]-inulin levels (ID%⁄g brain) in the olfactory bulb (A), cerebrum (B), and medulla oblongata (C) obtained using the two types of intranasal administration assessed in the present study. Intranasal administration using the pipette method enabled delivery of [¹⁴C]-inulin into the brain using openable inhalation masks (Figure 1). Under inhalation anesthesia, the quantitative results revea...

Discussion

The nose-to-brain delivery of drugs is expected to have a pronounced effect on central nervous system disorders because this pathway represents a direct transportation route that bypasses the BBB. Three different nose-to-brain pathways have been reported to date⁸. The first is the olfactory nerve pathway, which passes from the olfactory mucosa in the nasal mucosa to the forebrain via the olfactory nerve. The second is the trigeminal nerve pathway, which passes from the respiratory mucosa in the na...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ddY mouse	Japan SLC, Inc.		Male, 4-6 weeks, 20-30 g
Isoflurane	Pfizer	v002139
Isoflurane setup	SHINANO manufacturing CO. LTD.	SN-487-OTAir, SN-489-4
Isoflurane mask	SHINANO manufacturing CO. LTD.		For small rodents
Isoflurane mask (openable type)	SHINANO manufacturing CO. LTD.		Special orders
Anesthesia Box	SHINANO manufacturing CO. LTD.	SN-487-85-02
Animal experiments scissors-1	NATSUME SEISAKUSHO CO., LTD.	B-27H
Animal experiments scissors-2	NATSUME SEISAKUSHO CO., LTD.	B-13H
Tweezers-1	FINE SCIENCE TOOLS Inc.	11272-30	Dumont #7 Dumoxel
Tweezers-2	NATSUME SEISAKUSHO CO., LTD.	A-12-1
Cannula tube (PE-50)	Becton, Dickinson and Company.	5069773	I.D.: 0.58 mm, O.D.: 0.965 mm
Cannula tube (SP-10)	NATSUME SEISAKUSHO CO., LTD.	KN-392	I.D.: 0.28 mm, O.D.: 0.61 mm
Shaver	MARUKAN, LTD.	DC-381
Stereoscopic microscope	Olympus Corporation	SZ61
Needle 27G 1/2 in 13 mm	TERUMO CORPORATION	NN-2738R
1 mL syringe	TERUMO CORPORATION	SS-01T
Syringe pump	Neuro science	NE-1000
Cellulose membrane	Toyo Roshi Kaisya, Ltd.	00011090
Micro spatula	Shimizu Akira Inc.	91-0088
Micropipette (0.5-10 uL)	Eppendorf AG	Z368083
Pipette chip	Eppendorf AG	0030 000.811
Tape	TimeMed Labeling System, Inc.	T-534-R	For fixing mouse
[14C]-Inulin	American Radiolabeled Chemicals Inc.	ARC0124A	0.1 mCi/mL
EtOH	Wako Pure Chemical Industries, Ltd.	054-00461
Liquid scintillation counter	Perkin Elmer Life and Analytical Sciences, Inc	Tri-Carb 4810TR