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,

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, ane....

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