This study was supported in part by the Private University Research Branding Project from MEXT; a Grant-in-Aid for Scientific Research (C) (17K08249 [to T.K. and T.S.]) from the Japan Society for the Promotion of Science (JSPS); a grant for cooperative research from the Hamaguchi Foundation for the Advancement of Biochemistry [to T.S.], and the Takeda Science Foundation [to T.K.]. We thank Mr. Yuya Nito and Ms. Akiko Asami for their valuable technical assistance in conducting the experiments.

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
<ol>
	<li>House the experimental mice in stainless-steel cages under a 12-h light/dark cycle (light on 8:00 AM&#8211;8:00 PM), with a controlled temperature maintained at 23 &#177; 1&#176;C, humidity of 50% &#177; 10%, and ad libitum access to food and water.</li>
	<li>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.&#160;&#160;</li>
</ol>
2. Preparation of the Administration Solution
<ol>
	<li>Prepare an administration solution of [14C]-inulin (50 &#956;M, 0.5 &#956;Ci/mL per mouse) by diluting in phosphate-buffered saline, and store at 4 &#176;C until use.</li>
</ol>
3. Intranasal Administrations for Mice
<ol>
	<li>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
	<ol>
		<li>Fix mice in the supine position on a corkboard by taping their limbs under inhalation anesthesia with 2% isoflurane (Figure 1A).</li>
		<li>Administer a total volume of 25-&#956;L administration solution to each mouse at 30-s intervals, via 1- to 2-&#956;L doses alternatively administered into the left and right nostrils while the mice are fixed under inhalation anesthesia (Figure 1B and C).</li>
		<li>Note:&#160;Waste anesthetic gas is&#160;scavenged by active means&#160;(fume hood, hard-ducted biosafety cabinet, vacuum, etc.) when the&#160; mask is opened and by passive&#160;means(waste anesthetic gas cannister)&#160; when closed&#160;</li>
	</ol>
	</li>
	<li>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
	<ol>
		<li>Fix mice in the supine position on a corkboard by taping their limbs under inhalation anesthesia with 2% isoflurane.</li>
		<li>Hair in the neck&#160;is shaved and&#160;prepped via betadine or chlorhexidine application followed by an alcohol rinse.</li>
		<li>Expose the trachea and esophagus by expanding the skin below the throat with forceps after making a small incision (1.5 cm) with scissors.</li>
		<li>Make an incision (1 mm) in the trachea using scissors.</li>
		<li>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.</li>
		<li>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 &#215;10 magnification.</li>
		<li>Attach a needle (27G &#215; 1/2) to a 1-mL syringe filled with an administration solution and connect to a programmable micro-syringe pump.</li>
		<li>Connect above needle to the cannula that had been inserted into the esophagus at 3.2.5 (Figure 2C).</li>
		<li>Administer a total volume of 25 &#956;L [14C]-inulin solution at a constant rate (5 &#956;L/min) (Figure 2C and 2D).</li>
	</ol>
	</li>
</ol>
4. Quantitative Experiment Using Radio-Labeled Water-Soluble Macromolecules ([14C]-inulin)
<ol>
	<li>Decapitate the experimental mice under anesthesia&#160;and open up their craniums, using scissors and from the side of the medulla oblongata, while taking care not to damage the brains.</li>
	<li>Extract carefully the whole brain by scooping using a micro-spatula from cranium.</li>
	<li>Place a filter paper moistened with saline solution on a Petri dish that is stored on ice.</li>
	<li>Place the extracted brains on the moistened filter paper.</li>
	<li>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.</li>
	<li>Dissect brains rapidly, and divide them into three parts: the olfactory bulb, cerebrum, and medulla oblongata (including the pons).</li>
	<li>Place brain samples in tissue solubilizer at 50 &#176;C for 1 h.</li>
	<li>Add 10 &#956;L of the liquid scintillation cocktail to the brain samples.</li>
	<li>Transfer a 25 &#956;L aliquot of the administration solution dissolved in scintillation cocktail to a scintillation vial to determine the radioactivity of the applied solution.</li>
	<li>Measure the disintegrations per minute of [14C] radioactivity in the brain sample ([14C] Xbrain) and the applied solution ([14C] XIN dose) in a liquid scintillation counter equipped with an appropriate crossover correction for 3H and 14C.</li>
</ol>
5. Data Analysis
<ol>
	<li>Calculate the drug distribution levels (%) for the injected dose (ID%) using the following equation: 
	ID%&#8260;g brain = ([14C] Xbrain/[14C] XIN dose) &#215; 100, 
	where Xbrain (dpm/g brain) is the amount of [14C]-inulin measured in the brain tissue and XIN dose (dpm/25 &#956;L solution) is the concentration of the [14C]-inulin in the solution used for intranasal administration.</li>
</ol>

<ol>
	<li>Sakane, T., Yamashita, S., Yata, N., Sezaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transnasal+delivery+of+5-fluorouracil+to+the+brain+in+the+rat.">Transnasal delivery of 5-fluorouracil to the brain in the rat.</a> Journal of Drug Targeting. 7 (3), 233-240 (1999).</li><li>Illum, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+drugs+from+the+nasal+cavity+to+the+central+nervous+system.">Transport of drugs from the nasal cavity to the central nervous system.</a> European Journal of Pharmaceutical Science. 11 (1), 1-18 (2000).</li><li>Hanson, L. R., Frey, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+bypasses+the+blood-brain+barrier+to+target+therapeutic+agents+to+the+central+nervous+system+and+treat+neurodegenerative+disease.">Intranasal delivery bypasses the blood-brain barrier to target therapeutic agents to the central nervous system and treat neurodegenerative disease.</a> BMC Neuroscience. 9 (Suppl 3), S5 (2008).</li><li>Chapman, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+treatment+of+central+nervous+system+dysfunction+in+humans.">Intranasal treatment of central nervous system dysfunction in humans.</a> Pharmaceutical Research. 30 (10), 2475-2484 (2012).</li><li>Kanazawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+non-invasive+drug+delivery+system+to+the+brain+for+brain+diseases+therapy.">Development of non-invasive drug delivery system to the brain for brain diseases therapy.</a> Yakugaku-Zasshi. 138 (4), 443-450 (2018).</li><li>Kozlovskaya, L., Abou-Kaoud, M., Stepensky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+drug+delivery+to+the+brain+via+nasal+route.">Quantitative analysis of drug delivery to the brain via nasal route.</a> Journal of Controlled Release. 189, 133-140 (2014).</li><li>Hirai, S., Yashiki, T., Matsuzawa, T., Mima, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorption+of+drugs+from+the+nasal+mucosa+of+rat.">Absorption of drugs from the nasal mucosa of rat.</a> International Journal of Pharmaceutics. 7 (4), 317-325 (1981).</li><li>Lochhead, J. J., Thorne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+biologics+to+the+central+nervous+system.">Intranasal delivery of biologics to the central nervous system.</a> Advances in Drug Delivery Reviews. 64 (7), 614-628 (2011).</li><li>Lalatsa, A., Schatzlein, A. G., Stepensky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+to+deliver+peptide+drugs+to+the+brain.">Strategies to deliver peptide drugs to the brain.</a> Molecular Pharmaceutics. 11 (4), 1081-1093 (2014).</li><li>Kanazawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+delivery+of+small+interfering+ribonucleic+acid+and+drugs+through+intranasal+administration+with+nano-sized+polymer+micelles.">Brain delivery of small interfering ribonucleic acid and drugs through intranasal administration with nano-sized polymer micelles.</a> Medical Devices. 8, 57-64 (2015).</li><li>Kanazawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+nose-to-brain+delivery+of+hydrophilic+macromolecules+with+stearate-+or+polyethylene+glycol-modified+arginine-rich+peptide.">Enhancement of nose-to-brain delivery of hydrophilic macromolecules with stearate- or polyethylene glycol-modified arginine-rich peptide.</a> International Journal of Pharmacology. 530 (1-2), 195-200 (2017).</li><li>Kamei, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+an+enhanced+nose-to-brain+delivery+of+insulin+on+mild+and+progressive+memory+loss+in+the+senescence-accelerated+mouse.">Effect of an enhanced nose-to-brain delivery of insulin on mild and progressive memory loss in the senescence-accelerated mouse.</a> Molecular Pharmaceutics. 14 (3), 916-927 (2017).</li><li>Suzuki, T., Oshimi, M., Tomono, K., Hanano, M., Watanabe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+transport+mechanism+of+pentazocine+across+the+blood-brain+barrier+using+the+in+situ+rat+brain+perfusion+technique.">Investigation of transport mechanism of pentazocine across the blood-brain barrier using the in situ rat brain perfusion technique.</a> Journal of Pharmaceutical Science. 91 (11), 2346-2353 (2002).</li></ol>

The authors have nothing to disclose.

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...

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 BBB1,2,3,4,5. 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.

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

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.

Figure 3 shows the [14C]-inulin levels (ID%&#8260;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...

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

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 ...

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19.7K Views.  Nihon University. 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.