Indirect SPECT Imaging Evaluation for Possible Nose-to-Brain Drug Delivery Using a Compound with Poor Blood–Brain Barrier Permeability in Mice

Single-photon emission computed tomography (SPECT) imaging using intravenous radioactive ligand administration to indirectly evaluate the time-dependent effect of intranasal drugs with poor blood-brain barrier permeability on brain drug distributions in mice was evaluated. The biodistribution was examined using domperidone, a dopamine D2 receptor ligand, as the model drug, with intranasal administration at 0, 15, or 30 min before intravenous [123I]IBZM administration. In the striatum, [123I]IBZM accumulation was significantly lower after intranasal (IN) domperidone administration than in controls 15 min after intravenous [125I]IBZM administration. [123I]IBZM SPECT was acquired with intravenous (IV) or IN domperidone administration 15 min before [123I]IBZM, and time–activity curves were obtained. In the striatum, [123I]IBZM accumulation was clearly lower in the IN group than in the control and IV groups. Time–activity curves showed no significant difference between the control and IV groups in the striatum, and values were significantly lowest during the first 10 min in the IN group. In the IN group, binding potential and % of receptor occupancy were significantly lower and higher, respectively, compared to the control and IV groups. Thus, brain-migrated domperidone inhibited D2R binding of [123I]IBZM. SPECT imaging is suitable for research to indirectly explore nose-to-brain drug delivery and locus-specific biological distribution.


Introduction
Almost all potential therapies are greatly restricted from entering the brain because the blood-brain barrier (BBB) is highly successful at protecting the brain from the entry of drugs [1,2]. P-glycoprotein (P-gp) is considered one of the most important efflux transporters located at the BBB, and it protects the brain from chemicals and toxins. For this 2 of 10 reason, many drugs have an affinity for P-gp and drugs administered intravenously cannot accumulate in the brain due to the BBB. Nose-to-brain (N2B) drug delivery is an attractive route for direct drug delivery from the nose to the brain that bypasses the BBB [3]. Neotrofin, a P-gp substrate, administered intracerebroventricularly in the lateral cerebral ventricle is inhibited from being excreted from the brain when combined with verapamil, a P-gp inhibitor [4]. Thus, P-gp substrate drugs may temporarily accumulate in the brain via N2B drug delivery because they are excreted from the brain when they are recognized by P-gp in the brain. However, it remains unclear whether P-gp-mediated efflux has an effect on time-dependent drug accumulation in the brain. Therefore, it is useful to establish a method for accurate evaluation of the effect of intranasal P-gp substrate drugs.
Radionuclide imaging using positron emission tomography (PET), or single-photon emission computed tomography (SPECT), enables direct, non-invasive measurement of functional proteins (e.g., receptors, transporters) in a living organism. A previous PET study evaluated brain uptake of 11 C-labeled CH 3 -Orexin A after intranasal delivery in rodents and nonhuman primates. However, these imaging methods have issues that include the technical difficulty of labeling an evaluation compound with a radioisotope, a high likelihood of changes in properties of an evaluation compound by labeling, as well as uncertainties in detecting labeled metabolites rather than a labeled intact (nonlabeled) compound. In addition to these issues, if the above method is applied to N2B drug delivery, there is great difficulty in achieving rigorous detection of very slight and sensitive brain uptake of a radiolabeled evaluation compound because of extreme differences in radioactivity between the nasal cavity and the brain [5].
Instead of the above method of direct evaluation of a radiolabeled compound in the brain by PET, indirect evaluation using brain PET imaging of specific receptor occupancy has been performed to examine the time-dependent effect of naloxone administered intranasally on mu opioid receptor occupancy in nonhuman primates and human subjects, with intravenous injection of [ 11 C]carfentanil as a PET tracer ligand into the receptor [6,7]. Since the naloxone used in these studies is highly nasally absorbable, BBB-permeable, and does not activate efflux transporter P-gp [8,9], it is possible that the results of receptor occupancy with nasal naloxone in these studies are based on brain naloxone distributions via the BBB route rather than the N2B route. Although brain PET imaging with a 11 C-labeled compound of receptor occupancy enables the use of an intact evaluation compound and includes evaluation of its pharmacodynamics in the brain, the volume of 11 C-labeled compounds injected into the subject is usually high because 11 C-labeled compounds in new radiopharmaceuticals have a very short radioactive half-life and low radioactive concentration. In this study, we demonstrated a SPECT imaging method that uses intravenous radioactive ligand administration to indirectly evaluate the time-dependent effect of intranasal drugs with poor BBB permeability on brain drug distribution in mice. To evaluate brain drug distribution in mice via only the N2B route, the present mouse study used domperidone, a dopamine D2 receptor (D2R) antagonist, which has been reported to have only peripheral effects due to its poor BBB permeability caused by active efflux transport of P-gp [10,11], as a model drug for N2B delivery. In addition, (S)-(-)-3iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)-methyl]benzamide ([ 123 I]IBZM), a D2R ligand with known high BBB permeability, was selected as the radiopharmaceutical to indirectly evaluate brain domperidone distribution by competing D2R binding using SPECT imaging.

Animals
All applicable institutional guidelines of Kanazawa University for the care and use of animals were followed. All procedures were performed in accordance with the ethical standards of Kanazawa University (Animal Care Committee of Kanazawa University, AP-173851) and with international standards for animal welfare and institutional guidelines. The mice used in the study (ddY, males, 28.5 ± 0.9 g, 29 mice in total) were obtained from Japan SLC, Tokyo, Japan. Tracheal cannulation according to the method of Hirai et al. [13] was performed in all mice to increase N2B drug delivery during the intranasal administration of domperidone. The drug solution was administered into the nasal cavity via the tracheal catheter. The section of trachea to the lung was also cannulated to maintain spontaneous breathing.

Biological Distribution Study after an Intravenous Dose of [ 125 I]IBZM with an Intranasal Domperidone or Saline Dose
All procedures in mice were conducted under isoflurane inhalation anesthesia. Domperidone diluted with saline was intranasally administered at a dose of 2 μg/g body weight and at a volume of 20 μL, using a micro-syringe via the tracheal catheter. Saline was intranasally administered as the control in the same manner. Approximately 200 kBq

Animals
All applicable institutional guidelines of Kanazawa University for the care and use of animals were followed. All procedures were performed in accordance with the ethical standards of Kanazawa University (Animal Care Committee of Kanazawa University, AP-173851) and with international standards for animal welfare and institutional guidelines. The mice used in the study (ddY, males, 28.5 ± 0.9 g, 29 mice in total) were obtained from Japan SLC, Tokyo, Japan. Tracheal cannulation according to the method of Hirai et al. [13] was performed in all mice to increase N2B drug delivery during the intranasal administration of domperidone. The drug solution was administered into the nasal cavity via the tracheal catheter. The section of trachea to the lung was also cannulated to maintain spontaneous breathing.

Biological Distribution Study after an Intravenous Dose of [ 125 I]IBZM with an Intranasal Domperidone or Saline Dose
All procedures in mice were conducted under isoflurane inhalation anesthesia. Domperidone diluted with saline was intranasally administered at a dose of 2 µg/g body weight and at a volume of 20 µL, using a micro-syringe via the tracheal catheter. [ 125 I]IBZM, cardiac blood was collected under isoflurane inhalation anesthesia, and the animals were euthanized and quickly fractionated into the olfactory bulb, cerebral cortex, striatum, hippocampus, and cerebellum under ice-cold conditions. The wet tissues were then weighed, and the radioactivity in the weighed tissue samples was measured using a gamma counter (AccuFLEX γARC-7010; Hitachi). Data are expressed as the %injected dose per g wet tissue (%ID/g tissue).
Pharmaceutics 2022, 14, x FOR PEER REVIEW 4 of 10 [ 125 I]IBZM were injected into the tail vein of mice at 0, 15, and 30 min after intranasal domperidone administration, or at 0 min after intranasal saline administration (n = 4 at each time point, 16 mice in total) (Figure 2). At 30 min after the intravenous dose of [ 125 I]IBZM, cardiac blood was collected under isoflurane inhalation anesthesia, and the animals were euthanized and quickly fractionated into the olfactory bulb, cerebral cortex, striatum, hippocampus, and cerebellum under ice-cold conditions. The wet tissues were then weighed, and the radioactivity in the weighed tissue samples was measured using a gamma counter (AccuFLEX γARC-7010; Hitachi). Data are expressed as the %injected dose per g wet tissue (%ID/g tissue). intranasal group, n = 4; for each administration). After mice are sacrificed, brain anatomy is immediately examined, and then radioactivity is measured.

SPECT Imaging after an Intravenous Dose of [ 123 I]IBZM with an Intranasal or Intravenous Dose of Domperidone
All procedures in mice were conducted under isoflurane inhalation anesthesia (Figure 2). Domperidone diluted with saline was administered intravenously into the tail vein (n = 3; one mouse died) or intranasally (n = 4) using a micro-syringe via the tracheal catheter at a dose of 2 μg/g body weight and volume of 20 μL. As a control, saline was intranasally administered to the mice (n = 5) in the same manner. Furthermore, 14.0 ± 1.6 MBq [ 123 I]IBZM diluted with saline was intravenously administered into the tail vein 15 min after the intranasal dose (IN groups) and intravenous dose (IV groups) of domperidone or saline, and SPECT acquisition started immediately after the dose of [ 123 I]IBZM, at 5 min/frame for 90 min using a VECTor + system (MILabs, Utrecht, The Netherlands). SPECT images were obtained and reconstructed using the ordered subset expectation maximization method with 16 subsets and 6 iterations, including scatter and attenuation correction. The voxel size was set to 0.8 × 0.8 × 0.8 mm 3 . Post-reconstruction smoothing filtering was applied using a 1.0-mm Gaussian filter. Image displays were obtained using the medical image data analysis software programs Pmod (ver. 3.7, PMOD Technologies LLC, Zurich, Switzerland) and Amide (exe. 1.0.4-1). Time-activity curves (TACs) for the striatum and cerebellum were generated from the SPECT images, and coronal images were displayed as similar section images. Specific binding was calculated by subtracting SUVmean in the cerebellum from that in the striatum. Binding potential (BP) and % of receptor occupancy Normal ddY mice are intravenously or intranasally administered 2 µg/g body weight/20 µL domperidone (control, n = 5; intravenous group, n = 3 (one mouse died); intranasal group, n = 4; for each administration). After mice are sacrificed, brain anatomy is immediately examined, and then radioactivity is measured.

SPECT Imaging after an Intravenous Dose of [ 123 I]IBZM with an Intranasal or Intravenous Dose of Domperidone
All procedures in mice were conducted under isoflurane inhalation anesthesia ( Figure 2). Domperidone diluted with saline was administered intravenously into the tail vein (n = 3; one mouse died) or intranasally (n = 4) using a micro-syringe via the tracheal catheter at a dose of 2 µg/g body weight and volume of 20 µL. As a control, saline was intranasally administered to the mice (n = 5) in the same manner. Furthermore, 14.0 ± 1.6 MBq [ 123 I]IBZM diluted with saline was intravenously administered into the tail vein 15 min after the intranasal dose (IN groups) and intravenous dose (IV groups) of domperidone or saline, and SPECT acquisition started immediately after the dose of [ 123 I]IBZM, at 5 min/frame for 90 min using a VECTor + system (MILabs, Utrecht, The Netherlands). SPECT images were obtained and reconstructed using the ordered subset expectation maximization method with 16 subsets and 6 iterations, including scatter and attenuation correction. The voxel size was set to 0.8 × 0.8 × 0.8 mm 3 . Post-reconstruction smoothing filtering was applied using a 1.0-mm Gaussian filter. Image displays were obtained using the medical image data analysis software programs Pmod (ver. 3.7, PMOD Technologies LLC, Zurich, Switzerland) and Amide (exe. 1.0.4-1). Time-activity curves (TACs) for the striatum and cerebellum were generated from the SPECT images, and coronal images were displayed as similar section images. Specific binding was calculated by subtracting SUV mean in the cerebellum from that in the striatum. Binding potential (BP) and % of receptor occupancy (%RO) using SUV mean in the equilibrium state were calculated in the striatum and cerebellum [14].

Statistical Analysis
Data are presented as means and standard deviation. After normality testing using the Kolmogorov-Smirnov test for statistical analysis of TACs and specific binding, and the Shapiro-Wilk test for analysis of BP and %RO, p values were calculated using Welch's t-test for comparisons between two groups and analysis of variance. Tukey's test was used for comparisons among the three groups. All analyses were conducted using GraphPad Prism 8 statistical software (GraphPad Software, Inc., La Jolla, CA, USA). A P value of less than 0.05 was considered indicative of a significant difference.

Results
The appropriate intranasal dose schedule of domperidone was assessed prior to the SPECT study. In the biological distribution study, we decided to sacrifice the mice 30 min after [ 125 I]IBZM injection because the ratio of striatum to cerebellum was highest in each brain tissue 30 min after an intravenous dose of [ 125 I]IBZM. Table 1 shows the biological distributions (%ID/g) for each brain tissue and organ. Compared with the controls that received saline instead of domperidone, a significant decrease in accumulation of [ 125 I]IBZM was observed only for the striatum, which expresses high levels of D2R [15], in the group that was dosed with intranasal domperidone at 15 Figure 5 shows the specific binding value-time curves for the striatum in the three groups, which were calculated using the SUV mean values for the striatum and cerebellum. Specific binding value at approximately the first 20 min was significantly lower in the IN group compared with the other two groups. The equilibrium state of the specific binding value-time curve for the striatum was achieved at 25-35 min in all three groups. The mean BP values in the equilibrium state were 1.27, 1.08, and 0.67 for the control group, IV group, and IN group, respectively; the BP value was significantly lower in the IN group than in the other two groups ( Table 2). The mean %RO value was significantly higher in the IN group than in the IV group (Table 2).
Specific binding value at approximately the first 20 min was significantly lower in the IN group compared with the other two groups. The equilibrium state of the specific binding value-time curve for the striatum was achieved at 25-35 min in all three groups. The mean BP values in the equilibrium state were 1.27, 1.08, and 0.67 for the control group, IV group, and IN group, respectively; the BP value was significantly lower in the IN group than in the other two groups ( Table 2). The mean %RO value was significantly higher in the IN group than in the IV group (Table 2).

Discussion
In the present study, a SPECT imaging method was demonstrated in mice to indirectly evaluate whether temporal accumulation of drugs with an affinity for P-gp occurred in the brain via N2B drug delivery. Domperidone, a D2R ligand that is well-known to be poorly BBB permeable, was used as the model drug for N2B delivery. The mouse animal model was appropriate for N2B delivery because the mouse nose has a high ratio of olfactory region to nasal mucosa, and a surgical procedure was performed to place the tip of a catheter in the nasal cavity for intranasal drug administration. A rodent study is appropriate for new drug screening tests by N2B delivery for the reasons of cost-effectiveness and convenience of use in comparison with nonhuman primates and other animals. It is important to synthesize radioactive ligands with high radioactive concentration to ensure a small injection volume. The radioactive concentration of [ 125/123 I]IBZM used in this study

Discussion
In the present study, a SPECT imaging method was demonstrated in mice to indirectly evaluate whether temporal accumulation of drugs with an affinity for P-gp occurred in the brain via N2B drug delivery. Domperidone, a D2R ligand that is well-known to be poorly BBB permeable, was used as the model drug for N2B delivery. The mouse animal model was appropriate for N2B delivery because the mouse nose has a high ratio of olfactory region to nasal mucosa, and a surgical procedure was performed to place the tip of a catheter in the nasal cavity for intranasal drug administration. A rodent study is appropriate for new drug screening tests by N2B delivery for the reasons of cost-effectiveness and convenience of use in comparison with nonhuman primates and other animals. It is important to synthesize radioactive ligands with high radioactive concentration to ensure a small injection volume. This result corresponds closely to the result of the SPECT image analysis for the intranasal domperidone group, in which the accumulation of [ 123 I]IBZM in the striatum was lower than that in the intranasal saline group. Conversely, the lack of a significant decrease in [ 125 I]IBZM accumulation in the striatum for the pretreatment group of 30 min with intranasal domperidone compared with the control may mean that the distribution of domperidone in the brain via the N2B route is transient due to complex factors, including the effects of P-gp expressed in endothelial cells lining the brain capillaries, as well as the olfactory mucosa [16,17]. In support of this hypothesis, a recent similar study was conducted to assess the N2B delivery of quinidine as a poor BBB-permeable drug in rats. This experiment demonstrated the possibility that quinidine, a P-gp substrate, could be transported into the striatum by intranasal administration in a pretreatment with the P-gp specific inhibitor [18].
The transport pathways in N2B drug delivery include the intracellular route (axonal transport), the paracellular route, the transcellular route through olfactory epithelium, and the trigeminal nerve route through respiratory epithelium [19,20]. Although we could not clarify the detailed mechanism of N2B delivery of domperidone in this study, we consider that the main pathway for nasal transport of domperidone is the paracellular route through the lamina propria of olfactory epithelium. In fact, fluorophore-conjugated dextran, a non-directional tracer, has been shown to be easily and quickly transported into the lamina propria of olfactory epithelium [21,22]. However, further experiments are necessary to clarify in more detail the mechanism of transport of domperidone into the brain.
In the present study, TACs ( Figure 4) and specific binding value-time curves ( Figure 5) of [ 123 I]IBZM for the striatum were significantly lower in the intranasal domperidone group compared with the intranasal saline and intravenous domperidone groups. In addition, BP and %RO values were significantly lower in the intranasal domperidone group compared with the intravenous domperidone group. From the perspective of ligand-receptor binding competition, these results also indicate that the intact (non-labeled) intranasal domperidone could be transported to the brain via the N2B pathway and compete with binding of [ 123 I]IBZM in the striatum.
As a limitation, the striatum, which has the highest expression of D2R and the highest accumulation of [ 123 I]IBZM SPECT imaging in the mouse brain, was analyzed. Because the brain size is small, and the resolution of SPECT images is low, it was difficult to set volumes of interest for each brain region. Imaging studies using rats and monkeys, which have larger brain areas than mice, are needed. In addition, it is necessary to confirm the dosedependent increase in %RO of domperidone by IN administration and to confirm detailed brain region-selective %RO changes in rats and primates, which have larger brain sizes than mice, in the future. In addition, whether P-gp substrates other than domperidone, such as amisulpride and sulpiride, can also be evaluated for brain migration needs to be examined.

Conclusions
Indirect SPECT imaging evaluation using intravenous [ 123 I]IBZM administration could evaluate the time-dependent effect of intranasal domperidone in a mouse study and may be a suitable method for use in N2B drug delivery research to explore time-dependent brain uptakes and locus-specific biological distribution of such drugs as the P-gp substrate domperidone and other poorly BBB-permeable agents. Funding: The present study was funded in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Nos. 18K19508 and 21H02865) and Shin Nippon Biomedical Laboratories, Ltd.

Institutional Review Board Statement:
The study was conducted in accordance with the ethical standards of Kanazawa University (Animal Care Committee of Kanazawa University, AP-173851) and with international standards for animal welfare and institutional guidelines.

Informed Consent Statement: Not applicable.
Data Availability Statement: The authors confirm that the data supporting the findings of this study are available within the article.