Brain and Nasal Cavity Anatomy of the Cynomolgus Monkey: Species Diﬀerences from the Viewpoint of Direct Delivery from the Nose to the Brain

pharmaceutics

Communication Brain and Nasal Cavity Anatomy of the Cynomolgus Monkey: Species Diﬀerences from the Viewpoint of Direct Delivery from the Nose to the Brain

Toshiyasu Sakane 1,*, Sachi Okabayashi 2, Shunsuke Kimura 3, Daisuke Inoue 4 , Akiko Tanaka 1 and Tomoyuki Furubayashi 1 1 Department of Pharmaceutical Technology, Kobe Pharmaceutical University, Motoyamakita-machi 4-19-1, Higashinada, Kobe, Hyogo 658-8558, Japan; [email protected] (A.T.); [email protected] (T.F.) 2 New Drug Research Center Inc., Minatojimaminami-machi 7-1-14, Chuo-ku, Kobe, Hyogo 650-0047, Japan; [email protected] 3 Department of Pharmacokinetics, Faculty of Pharmaceutical Sciences, Doshisha Women’s College of Liberal Arts, Kodo, Kyotanabe, Kyoto 610-0395, Japan; [email protected] 4 College of Pharmaceutical Sciences, Ritsumeikan University, Noji-Higashi 1-1-1, Kusatsu, Shiga 525-8577, Japan; [email protected] * Correspondence: [email protected]; Tel./Fax: +81-78-441-7530

Received: 29 November 2020; Accepted: 15 December 2020; Published: 18 December 2020

Abstract: Based on structural data on the nasal cavity and brain of the cynomolgus monkey, species diﬀerences in the olfactory bulb and cribriform plate were discussed from the viewpoint of direct delivery from the nose to the brain. Structural 3D data on the cynomolgus monkey skull were obtained using X-ray computed tomography. The dimensions of the nasal cavity of the cynomolgus monkey were 5 mm width 20 mm height 60 mm depth. The nasal cavity was very narrow and × × the olfactory region was far from the nostrils, similar to rats and humans. The weight and size of the monkey brain were 70 g and 55 mm width 40 mm height 70 mm depth. The olfactory bulb × × of monkeys is plate-like, while that of humans and rats is bulbar, suggesting that the olfactory area connected with the brain of monkeys is narrow. Although the structure of the monkey nasal cavity is similar to that of humans, the size and shape of the olfactory bulb are diﬀerent, which is likely to result in low estimation of direct delivery from the nose to the brain in monkeys.

Keywords: nose to brain delivery; monkey; species diﬀerence; nasal cavity; olfactory bulb; cribriform plate

1. Introduction The eﬃcient delivery of drugs to the brain is very important for drug therapy for brain disorders such as Parkinson’s and Alzheimer’s disease, multiple sclerosis, and autism spectrum disorder. In general, the blood–brain barrier (BBB) strictly inhibits the entry of hydrophilic drugs into the brain from systemic circulation after intravenous and oral administration. The BBB is a continuous cerebral vasculature with endothelial cells connected by tight junctions formed by cell adhesion proteins, including occludin. Endocytosis activity is very low in the cerebral vasculature [1]. These features result in the failure of brain disorder treatment by hydrophilic and high molecular weight drugs such as peptides. Some reviews have summarized strategies to overcome poor systemic drug delivery to the brain [2]. One of them is a direct delivery route from the nasal cavity. Small drugs, peptides, and even genes and cells were reported to be delivered directly to the brain after nasal application in rats.

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In our previous manuscripts, it was clarified that direct delivery from the nose to the brain (DDNB) enables two peptide drugs, oxytocin (MW: 1008 Da) [3] and CPN-116 (MW: 835 Da) [4] to act on the brain. Han et al. [5] showed that the concentration of plasmid DNA (7.2 kbase) in the brain after nasal administration was 10 times higher than the serum concentration, while after intravenous administration it was two orders of magnitude lower in the brain than that in the serum. After nasal application of plasmid DNA encoding β-galactosidase, a significantly higher expression was observed. According to Danielyan et al. [6], after mesenchymal stem cells and T406 human glioma cells stained with fluorescent dye are administered nasally, they can be observed in the thalamus, hippocampus, and cerebral cortex. After nasal administration of 3 105 mesenchymal stem cells, 1000 cells were × observed in the whole brain, indicating that 3% were successfully delivered. Similar results were described by Yu-Taeger et al. [7] and Galeano et al. [8]. In primary research, mice and rats have typically been used for in vivo studies on DDNB. However, the anatomy of the nasal cavity and brain and their connections in humans are different from those in animals. The data derived from animal studies should be carefully interpreted for extrapolation to humans. At the preclinical stage of the drug development process, monkeys are usually used to evaluate the pharmacokinetics and efficacy of drug candidates. The nasal cavities and brains of monkeys are expected to be more similar to those of humans than those of rats and mice. Invasive treatment is impossible in human studies, but determination of drug concentrations in the monkey brain is feasible. However, no manuscript has reported on the anatomy of the monkey brain and nasal cavity, while information on those of humans and rats is available [9–12]. In this study, to better understand the anatomy of the monkey nasal cavity, brain, and olfactory bulb, 3D data on the head of a cynomolgus monkey were obtained using X-ray computed tomography. Together with observations of the brain and the olfactory bulb, the anatomy of the brain and the nasal cavity of humans, monkeys, and rats was compared, and the influence of species differences on DDNB is discussed.

2. Materials and Methods The skull and brain of the female monkey (5 years old) were provided by the New Drug Research Center, Inc. (Kobe, Japan). The head was taken from the monkey euthanized after another animal study by intramuscular ketamine and intravenous sodium pentobarbital followed by exsanguination. The animal study (study No.: 18905) was approved on 24 October 2018 by the Animal Care Council at the New Drug Research Center, Inc. (approval No.: 181019A). The study was conducted in accordance with relevant national legislation. The acquisition of 3D data of the monkey skull was outsourced to JMC Corporation (Yokohama, Kanagawa, Japan), in which an industrial nanofocus CT scanner, Phoenix Nanotom M (GE Measurement & Control, Tokyo, Japan) was used. Images were obtained under the setup of the CT scanner, as indicated below. Voxel size: 0.04 mm 0.04 mm × Image number: 2000 Accelerating voltage: 140 kV Filament current: 120 mA

The 3D data were visualized with VGSTUDIO MAX 3.2 (Volume Graphics GmbH, Heidelberg, Germany).

3. Results

3.1. Structure and Size of the Cynomolgus Monkey Nasal Cavity Figure1 shows the head of the cynomolgus monkey and the position criteria (0 mm) for the horizontal, coronal, and sagittal sections. Pharmaceutics 2020, 12, 1227 3 of 8 Pharmaceutics 2020, 12, x FOR PEER REVIEW 3 of 8

Pharmaceutics 2020, 12, x FOR(1)horizontal PEER REVIEW section at 0 mm 3 of 8 (3) (1)horizontal section at 0 mm (3) (2)

(2)

(1)

(1) (2)coronal section at 0 mm (3)sagittal section at 0 mm (2)coronal section at 0 mm (3)sagittal section at 0 mm

Figure 1. Morphology of the head of the cynomolgus monkey and the position criteria (0 mm) for the Figure 1.Figure Morphology 1. Morphology of the of head the head of the of the cynomolgus cynomolgus monkeymonkey and and the the position position criteria criteria (0 mm) (0 for mm) the for the (1) horizontal (blue), (2) coronal (red), and (3) sagittal (green) sections. (1) horizontal(1) horizontal (blue), (blue), (2) coronal (2) coronal (red), (red), and and (3 (3)) sagittal sagittal (green) (green) sections. sections. FigureFigure 22 shows horizontalhorizontal sections sections at at 2 mm2 mm intervals intervals from from the bottomthe bott ( om8 mm)(−8 mm) to the to top the (+ top10 mm) (+10 − Figuremm)of the of2 nasal showsthe nasal cavity, horizontal cavity, which iswhich very sections narrow.is very at Thenarrow. 2 paranasalmm Theintervals sinusparanasal was from conﬁrmed sinus the wasbo tott confirmed beom a large(−8 mm) round-shaped to be to a thelarge top (+10 mm) ofround-shaped spacethe nasal just beside cavity, space the just which nasal beside cavity. is thevery Thenasal narrow. sections cavity. from The sectionsparanasal8 to +10 from mm sinus − indicate8 to +10was that mm confirmed the indicate height that ofto the bethe a large − heightnasal of cavity the nasal is approximately cavity is approximately 20 mm. According 20 mm. toAccording the section to atthe section6 mm, theat − bottom6 mm, the of thebott nasalom of round-shaped space just beside the nasal cavity. The sections from− −8 to +10 mm indicate that the cavity is wide (4 mm width). With increasing horizontal position, the nasal cavity becomes narrower. height theof the nasal nasal cavity cavity is wide is (4approximately mm width). With 20 increa mm.sing According horizontal to position, the section the nasal at − cavity6 mm, becomes the bo ttom of narrower.The sections The atsections+2 mm, at+ +24 mm, and+4 mm,+6 mmand demonstrate+6 mm demonstrate that the that structure the structure of the upper of the part upper of thepart the nasal cavity is wide (4 mm width). With increasing horizontal position, the nasal cavity becomes ofnasal the nasal cavity cavity is complicated. is complicated. This complicationThis complication is likely is duelikely to due nasal to turbinates. nasal turbinates. narrower. The sections at +2 mm, +4 mm, and +6 mm demonstrate that the structure of the upper part of the nasal cavity is complicated. This complication+2 mm +4 mmis likely due+6 mm to nasal+8 turbinates. mm +10 mm

+2 mm +4 mm +6 mm +8 mm +10 mm sagittal section + sagittal section 0 mm 0 mm + - 0 mm 0 mm - −-22 mm -4−4 mm mm − -66 mm − -88 mm FigureFigure 2. 2. SerialSerial horizontal horizontal sections sections (2 (2 mm mm interv intervals)als) of the cynomolguscynomolgus monkeymonkey skull. skull.

FigureFigure 3 showsshows coronal coronal sections sections at 3 at mm 3 intervalsmm intervals from the from nostril the to nostril the pharynx. to the Complexpharynx. structures Complex due to the nasal turbinates are observed in the six sections from 9 to +6 mm. structures due to the nasal turbinates− -2are2 mm observed in-4−4 themm mm six sections − − -66 mm from − 9 to +6− -88 mmmm. Figure 4 shows sagittal sections of the nasal cavity at 1 mm intervals from the nasal septum (0 Figure 2. Serial horizontal sections (2 mm intervals) of the cynomolgus monkey skull. mm) to the side wall (+5 mm). These sections show that the width of the nasal cavity is 5 mm. It is noteworthy that a space extends from the brain to the upper right part of the nasal cavity (indicated Figureby white 3 shows circles). coronal The space sections is visible at in 3the mm 0 mm intervals and +1 mm from sections, the nostril but disappears to the pharynx.in the +2 mm Complex structuressection, due suggesting to the nasal that turbinates it is very narrow are observed (less than 2in mm the wide). six sections from −9 to +6 mm. Figure 4 shows sagittal sections of the nasal cavity at 1 mm intervals from the nasal septum (0 mm) to the side wall (+5 mm). These sections show that the width of the nasal cavity is 5 mm. It is noteworthy that a space extends from the brain to the upper right part of the nasal cavity (indicated by white circles). The space is visible in the 0 mm and +1 mm sections, but disappears in the +2 mm section, suggesting that it is very narrow (less than 2 mm wide).

Pharmaceutics 2020, 12, 1227 4 of 8 Pharmaceutics 2020, 12, x FOR PEER REVIEW 4 of 8

+3 mm +6 mm +9 mm +12 mm

0 mm - + Pharmaceutics 2020, 12, x FOR PEER REVIEW 4 of 8

+3 mm +6 mm +9 mm +12 mm 0 mm

0 mm sagittal- section+

0 mm −-33 mmmm −-66 mm −-99 mm −-1212 mmmm FigureFigure 3. Serial 3. Serial coronal coronal sections sections (3 (3 mm mm interval intervals)s) ofof the the cynomolgus cynomolgus monkey monkey skull. skull. sagittal section Figure4 shows sagittal sections+1 m ofm the nasal+2 cavity mm at 1 mm+3 m intervalsm from+4 m them nasal septum+5 mm (0 mm) to the side wall (+5 mm). These sections show that the width of the nasal cavity is 5 mm. It is noteworthy that a space extends from the brain to the upper right part of the nasal cavity (indicated by white circles). The space is visible− in-33 mmmm the 0 mm and −-66 mm+1 mm sections, −-99 mm but disappears −-1212 mmmm in the +2 mm section, suggestinghorizontal section that it is very narrow (less than 2 mm wide). + Figure 3. Serial coronal sections (3 mm intervals) of the cynomolgus monkey skull. +1 mm +2 mm +3 mm +4 mm +5 mm 0 mm 0 mm

horizontal section +Figure 4. Serial sagittal sections (1 mm intervals) of the cynomolgus monkey skull.

3.2. Size0 mm of the Nasal Cavity and Relative Position0 with mm the Brain

The left part of Figure 5 shows the shape and size of the nasal cavity. As demonstrated by the structural data above, the dimensions of the nasal cavity are 5–6 mm width, 20 mm height, and 50 FigureFigure 4. 4. SerialSerial sagittal sagittal sections sections (1 (1 mm mm intervals) intervals) of the cynomolgus monkeymonkey skull.skull. mm depth. These data also indicate that the olfactory region, the area around the cribriform plate connected3.2.3.2. Size Size toof of thethe the Nasal Nasalolfactory Cavity Cavity bulb, and and Relative Relativeis 40 mm Position Position away with from the Brain the nostril. Efficient delivery of drugs to the olfactory area using a normal spraying device is likely difficult. On the right side of Figure 5, the TheThe left left part part of of Figure Figure 5 showsshows thethe shapeshape andand sizesize ofof thethe nasal nasal cavity. cavity. As As demonstrated demonstrated by by the the sagittalstructuralstructural cross-section data data above, of the the dimensionsdimensionsmonkey skull of of the theis nasal shownasal cavity n.cavity The are aredissected 5–6 5–6 mm mm width, brain width, 20 was mm 20 placedmm height, height, andat the 50and mmoriginal 50 positionmmdepth. depth. to Theseshow These datathe locationsdata also indicatealso ofindicate the that nasal the that olfactory cavity the olfact region,andory brain. theregion, areaAs shown aroundthe area in the around the cribriform photo, the cribriformthe plate brain connected is plate located at theconnectedto upper the olfactory right to the position bulb, olfactory is 40 of mmbulb, the awaynasal is 40 from cavity.mm theaway nostril. from E theﬃcient nostril. delivery Efficient of drugs delivery to the of olfactory drugs to area the olfactoryusing a normal area using spraying a normal device spraying is likely di deviceﬃcult. is On like thely right difficult. side of On Figure the 5right, the sagittalside of cross-sectionFigure 5, the sagittalof the monkeycross-section skull of is shown.the monkey The dissectedskull is show brainn. wasThe placeddissected at the brain original was placed position at tothe show original the locations of the nasal cavity and brain. As shown in the photo, the brain is located at the upper right position to show the locations of the nasal cavity and brain. As shown in the photo, the brain is located position of the nasal cavity. at the upper right position of the nasal cavity.

Figure 5. Scheme representing the structure and dimensions of the nasal cavity (A) and the locations of the nasal cavity and brain in the skull (B).

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+3 mm +6 mm +9 mm +12 mm

0 mm - +

0 mm

sagittal section

−-33 mmmm −-66 mm −-99 mm −-1212 mmmm Figure 3. Serial coronal sections (3 mm intervals) of the cynomolgus monkey skull.

+1 mm +2 mm +3 mm +4 mm +5 mm

horizontal section +

0 mm 0 mm

Figure 4. Serial sagittal sections (1 mm intervals) of the cynomolgus monkey skull.

3.2. Size of the Nasal Cavity and Relative Position with the Brain The left part of Figure 5 shows the shape and size of the nasal cavity. As demonstrated by the structural data above, the dimensions of the nasal cavity are 5–6 mm width, 20 mm height, and 50 mm depth. These data also indicate that the olfactory region, the area around the cribriform plate connected to the olfactory bulb, is 40 mm away from the nostril. Efficient delivery of drugs to the olfactory area using a normal spraying device is likely difficult. On the right side of Figure 5, the sagittal cross-section of the monkey skull is shown. The dissected brain was placed at the original positionPharmaceutics to show2020, 12 the, 1227 locations of the nasal cavity and brain. As shown in the photo, the brain is located5 of 8 at the upper right position of the nasal cavity.

Figure 5. SchemeScheme representing thethe structurestructure andand dimensions dimensions of of the the nasal nasal cavity cavity (A ()A and) and the the locations locations of Pharmaceuticsofthe the nasal nasal cavity2020 cavity, 12 and, xand FOR brain brain PEER in in the REVIEW the skull skull (B ).(B). 5 of 8

3.3.3.3. Size Size of theof the Brain Brain PhotosPhotos of of the the monkey monkey brain brain taken taken from from di differentﬀerent angles angles are are shown shown inin FigureFigure6 6.. TheThe weight of the thebrain brain is is 70 70 g. g. The The dimensions dimensions of of the the brain brain are are 45 45 mm mm height, height, 55 55 mm mm width, width, and and 70 70 mm mm depth. depth. The Thewhite white circles circles indicate indicate the the right right olfactory bulb. InIn thethe frontfrontview, view, the the olfactory olfactory bulb bulb has has a plate-likea plate-like structure.structure. In In the the bottom bottom view, view, the the long long olfactory olfactory tract tract connecting connecting the the olfactory olfactory bulb bulb to to the the brain brain is is shown.shown. The The appearance appearance of theof the olfactory olfactory bulb bulb is diisﬀ differenterent from from that that of ratsof rats and and humans. humans.

FigureFigure 6. Photos6. Photos of theof the cynomolgus cynomolgus monkey monkey brain brai takenn taken from from three three diﬀ differenterent angles. angles.

4. Discussion4. Discussion MonkeysMonkeys have have been been used used for for some some relevant relevantin vivoin vivostudies. studies. According According to Kumarto Kumar et al.,et al., after after nasal nasal sprayingspraying of progesterone,of progesterone, the concentration the concentration in the cerebrospinalin the cerebrospinal fluid (CSF) fluid in female (CSF) rhesusin female monkeys rhesus wasmonkeys significantly was significantly higher than higher that after than intravenous that after in injection,travenous although injection, there although were there similar were plasma similar concentrationsplasma concentrations [13]. A prostaglandin [13]. A prostaglandin D agonist D exhibited agonist moreexhibited potent more pharmacological potent pharmacological activity (sleep-inducingactivity (sleep-inducing effect) in monkeys effect) after in nasalmonkeys administration after nasal than administration after intravenous than administration after intravenous [14]. In theadministration cynomolgus [14]. monkey, In the nasally cynomolgus administered monkey, interferon- nasallyβ administered1b reached levels interferon- one orderβ1b of reached magnitude levels higherone inorder the olfactoryof magnitude bulb and higher trigeminal in the nerveolfactory than bulb in other and peripheraltrigeminal tissues. nerve than The brainin other distribution peripheral of interferon-tissues. Theβ1b brain was distribution visualized by of autoradiography interferon-β1b was [15]. visualized Iwasaki et al.by investigatedautoradiography species [15]. diff Iwasakierences et in DDNBal. investigated [16]. They species used thedifferences ratio Kp,in in DDNB/Kp,iv as[16]. an They index used of DDNB the ratio and K clarifiedp,in/Kp,iv that as an the index delivery of DDNB of sixand compounds clarified tothat the the olfactory delivery tract of six and compounds the rest of theto the brain olfactory after nasal tract administrationand the rest of tothe monkeys brain after wasnasal higher administration than that in rats,to monkeys while the was uptake higher by than the that olfactory in rats, bulb while and the trigeminal uptake by nerve the olfactory was lower. bulb Theand authors trigeminal suggested nerve that was no lower. mechanism The authors offers suggested better delivery that no in monkeys.mechanism A offers rare case better of adelivery human in pharmacokineticmonkeys. A rare study case was of reported a human by Bornpharmacokinetic et al. [17]. MSH study/ACTH(4–10), was reported vasopressin, by Born and et insulin al. [17]. wereMSH/ACTH(4–10), nasally applied tovasopressin, human volunteers, and insulin and thewere peptide nasally concentrations applied to human in the volunteers, lumbar CSF and and the peptide concentrations in the lumbar CSF and serum were measured. There were much higher concentrations of MSH/ACTH(4–10) in the CSF after a 5 mg dose. Serum levels after 1 mg and 5 mg doses were similar to those in the placebo group. Two cranial nerves connected to the nasal cavity, the olfactory and trigeminal nerves, are involved in the direct pathway from the nose to the brain [18]. The olfactory nerve extends from the olfactory bulb through small holes in the cribriform plate to the olfactory epithelium in the nasal cavity. Olfactory receptors are expressed at the terminal membranes of olfactory neurons. The role of the olfactory nerve is to transmit information on smells captured by olfactory receptors. The pathway in which the olfactory nerve is involved is called the olfactory route, through which it is feasible to deliver drugs to the cerebrum. In contrast, the trigeminal nerve extends from the pons to the nasal cavity. The route of the trigeminal nerve from the pons through the skull to the nasal cavity is much longer than that of the olfactory nerve. Drugs can be delivered directly to the pons and cerebellum through the trigeminal route. For this pathway, it is not yet clear how or through which route drugs are transported to the pons and cerebellum. Because the targets of many drugs acting on the brain are located in the cerebrum, this research focuses on the olfactory pathway.

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serum were measured. There were much higher concentrations of MSH/ACTH(4–10) in the CSF after a 5 mg dose. Serum levels after 1 mg and 5 mg doses were similar to those in the placebo group. Two cranial nerves connected to the nasal cavity, the olfactory and trigeminal nerves, are involved in the direct pathway from the nose to the brain [18]. The olfactory nerve extends from the olfactory bulb through small holes in the cribriform plate to the olfactory epithelium in the nasal cavity. Olfactory receptors are expressed at the terminal membranes of olfactory neurons. The role of the olfactory nerve is to transmit information on smells captured by olfactory receptors. The pathway in which the olfactory nerve is involved is called the olfactory route, through which it is feasible to deliver drugs to the cerebrum. In contrast, the trigeminal nerve extends from the pons to the nasal cavity. The route of the trigeminal nerve from the pons through the skull to the nasal cavity is much longer than that of the olfactory nerve. Drugs can be delivered directly to the pons and cerebellum through the trigeminal Pharmaceuticsroute. For 2020 this, 12 pathway,, x FOR PEER it is REVIEW not yet clear how or through which route drugs are transported to the pons 6 of 8 and cerebellum. Because the targets of many drugs acting on the brain are located in the cerebrum, thisFigure research 7 shows focuses the on location the olfactory of the pathway. olfactory bulb and cribriform plate in the monkey skull. The sagittal sectionFigure7 at shows 1 mm the is shown location on of the the left olfactory side. The bulb connection and cribriform between plate the in thenasal monkey cavity skull.and brain is indicatedThe sagittal by sectiona white at square. 1 mm is On shown the onright the side, left side. the Thesection connection in the betweenwhite square the nasal is expanded. cavity and The narrowbrain space, is indicated indicated by aby white a white square. dashed On the line, right is shown side, the in section the skull. in the Based white on square its shape, is expanded. the narrow The narrow space, indicated by a white dashed line, is shown in the skull. Based on its shape, the narrow space fits the olfactory bulb, as the inset photo shows. The cribriform plate is located between the space ﬁts the olfactory bulb, as the inset photo shows. The cribriform plate is located between the olfactory bulb and the nasal cavity. The thickness of the cribriform plate is likely 3–5 mm. The size of olfactory bulb and the nasal cavity. The thickness of the cribriform plate is likely 3–5 mm. The size of the cribriformthe cribriform plate plate is is a a determinantdeterminant of theof the eﬃciency efficiency of DDNB. of DDNB. Judging Judging from the shapefrom ofthe the shape olfactory of the olfactorybulb, thebulb, area the of thearea cribriform of the platecribriform of the monkeyplate of is the likely monkey small, leading is likely to thesmall, underestimation leading to the underestimationof DDNB. of DDNB.

olfactory bulb

1 cm 1 cm

sagittal section at 1 mm cribriform plate FigureFigure 7. Location 7. Location of the of the olfactory olfactory bulb bulb and and cribrifo cribriformrm plate plate in in the the cynomolgus cynomolgus monkey monkey skull. skull.

FigureFigure 8 shows8 shows a schematic a schematic representation representation ofof thethe locations locations of of the the nasal nasal cavity cavity and and brain brain in rats, in rats, monkeys,monkeys, and and humans. humans. In Inrats, rats, walk walkinging quadrupedally, quadrupedally, the the nasal nasal cavity cavity is locatedis located in front in front of the of brainthe brain and the spinal cord extends horizontally. In contrast, in humans, walking bipedally, the nasal cavity and the spinal cord extends horizontally. In contrast, in humans, walking bipedally, the nasal cavity is translocated below the brain and the spinal cord extends vertically downward. The shift in the is translocated below the brain and the spinal cord extends vertically downward. The shift in the position of the nasal cavity relative to the brain likely represents evolutionary changes between rats positionand humans.of the nasal The relativecavity relative position to of thethe nasalbrain cavity likely in represents the monkey, evolutionary walking bipedally changes at some between times rats and humans.and quadrupedally The relative at others, position is possibly of the nasal an intermediate cavity in the between monkey, rats walking and humans. bipedally at some times and quadrupedally at others, is possibly an intermediate between rats and humans.

Figure 8. Changes in the relative locations of the nasal cavity, olfactory bulb, and brain in rats, monkeys, and humans.

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Figure 7 shows the location of the olfactory bulb and cribriform plate in the monkey skull. The sagittal section at 1 mm is shown on the left side. The connection between the nasal cavity and brain is indicated by a white square. On the right side, the section in the white square is expanded. The narrow space, indicated by a white dashed line, is shown in the skull. Based on its shape, the narrow space fits the olfactory bulb, as the inset photo shows. The cribriform plate is located between the olfactory bulb and the nasal cavity. The thickness of the cribriform plate is likely 3–5 mm. The size of the cribriform plate is a determinant of the efficiency of DDNB. Judging from the shape of the olfactory bulb, the area of the cribriform plate of the monkey is likely small, leading to the underestimation of DDNB.

olfactory bulb

1 cm 1 cm

sagittal section at 1 mm cribriform plate Figure 7. Location of the olfactory bulb and cribriform plate in the cynomolgus monkey skull.

Figure 8 shows a schematic representation of the locations of the nasal cavity and brain in rats, monkeys, and humans. In rats, walking quadrupedally, the nasal cavity is located in front of the brain and the spinal cord extends horizontally. In contrast, in humans, walking bipedally, the nasal cavity is translocated below the brain and the spinal cord extends vertically downward. The shift in the position of the nasal cavity relative to the brain likely represents evolutionary changes between rats Pharmaceuticsand humans.2020 The, 12, relative 1227 position of the nasal cavity in the monkey, walking bipedally at some times7 of 8 and quadrupedally at others, is possibly an intermediate between rats and humans.

Figure 8.8. ChangesChanges in in the the relative relative locations locations of the of nasalthe nasa cavity,l cavity, olfactory olfactory bulb, and bulb, brain and in rats,brain monkeys, in rats, andmonkeys, humans. and humans.

5. Conclusions The anatomy of the monkey nasal cavity is similar to that of humans and rats. The nasal cavity is narrow, and the olfactory region is away from the nostril. However, the olfactory bulb is diﬀerent. The shape of the monkey olfactory bulb is plate-like, while that of humans and rats is bulbar. The diﬀerence suggests the small area of the olfactory region, possibly resulting in the underestimation of DDNB as compared to humans.

Author Contributions: Conceptualization, T.S.; methodology, S.O.; software, S.K.; validation, S.K.; formal analysis, A.T.; investigation, A.T.; resources, S.O.; data curation, D.I.; writing—original draft preparation, T.S.; writing—review and editing, T.F.; visualization, D.I.; supervision, T.S.; project administration, T.S.; funding acquisition, T.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. Dr. Sachi Okabayashi is the assistant manager of New Drug Research Center Inc. New Drug Research Center Inc. had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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