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.

This method can help answer key questions in the pharmaceutical sciences field about intranasal administration and pharmacokinetics and pharmacology of drugs of interest. The main advantage of this technique is that it can be used for the quantitative evaluation of nose-to-brain delivery drug candidates and inhalation anesthesia with minimal stress to the animals. The implications of this technique extends towards the therapy of central nervous system diseases as it contributes to the development of drug delivery technologies via the nose-to-brain route.

Demonstrating the procedure will be Mitsuyoshi Fukuda, a PhD student from my laboratory. For intranasal delivery via micropipette in an isolated radio isotope facility. Tape and anesthetize mouse to a cork board in the supine position, and administer 25 microliters of carbon-14 inulin solution in one to two microliter doses, alternatively, into the right and left nostrils of the animal.

For reverse cannulation delivery from the airway side through the esophagus, place the anesthetized mouse under a dissecting microscope, and make a 1.5 centimeter skin incision over the throat. Use forceps to expand the incision until the trachea is exposed, and make a one millimeter incision in the exposed trachea. Insert a cannula 1.2 centimeters to the pre-marked position into the incision, and attach the opposite end of the cannula to the inside of an inhalation mask.

After this, use forceps to expose the esophagus from under the trachea. Use scissors to make a one millimeter incision in the esophagus and insert a second cannula 1.4 centimeters to the pre-marked position toward the posterior end of the nasal cavity. It is important to insert cannula to an appropriate length according to the weight of the experimental animal and to adjust the length of the cannula in other ones.

Ligate the esophageal cannula and attach a 27-gauge needle to a one milliliter syringe filled with an administration solution connected to a programmable microsyringe pump. Then deliver 25 microliters of carbon-14 inulin at a constant five microliter per minute rate until the entire volume of solution has been administered. A slow but constant diffusion rate is important for the retention of as much drug solution as possible within the nasal cavity.

To quantify the amount of carbon-14 inulin that crossed the blood-brain barrier 30 minutes after the inulin administration, use scissors to carefully open the cranium of each experimentally-treated animal from the medulla oblongata side, and use a microspatula to carefully scoop out the brains. Place each brain on a piece of saline-moistened filter paper in a Petri dish on ice as it is harvested. And use a saline-soaked cotton swab to clean any blood from the surface of each brain.

Next, quickly but carefully, dissect the brains into olfactory bulb, cerebrum, and medulla oblongata, plus pons sections. And place the brain samples in tissue solubilizer for one hour at 50 degrees Celsius, followed by the addition of 10 microliters of liquid scintillation cocktail. To determine the radioactivity of the applied solution, transfer a 25 microliter aliquot of the administration solution, dissolved in the scintillation cocktail, to a scintillation vial, and measure the disintegrations per minute of the carbon-14 radioactivity within the brain sample and the applied solution in a liquid scintillation counter.

Under inhalation anesthesia, no experimental inter-individual variation is observed among intranasally-drug delivered animals. When the esophageal reverse cannula nasal cavity administration method is used, significantly higher levels of carbon-14 inulin are observed in the olfactory bulb, cerebrum, and medulla oblongata, than via micropipette delivery. Moreover, higher carbon-14 inulin is detected in the olfactory bulb and the medulla oblongata, both of which are prominently-evolved in the nose-to-brain pathway, than in the cerebrum.

After its development, this technique paved the way for researchers in the field of nose-to-brain delivery to explore the bioactivity of large molecule therapeutic agents in the central nervous system.