Brain histamine H 1 receptor occupancy of orally administered antihistamines, bepotastine and diphenhydramine, measured by PET with 11 C-doxepin

Brain histamine H1 receptoroccupancy of orallyadministered antihistamines,bepotastine anddiphenhydramine, measuredby PET with 11C-doxepinManabu Tashiro,1 Xudong Duan,1 Motohisa Kato,3

Masayasu Miyake,1 Shoichi Watanuki,1 Yoichi Ishikawa,2

Yoshihito Funaki,2 Ren Iwata,2 Masatoshi Itoh1 & Kazuhiko Yanai1,3

Divisions of 1Cyclotron Nuclear Medicine and 2Radiopharmaceutical Chemistry, Cyclotron and

Radioisotope Centre, Tohoku University, and 3Department of Pharmacology, Tohoku University

Graduate School of Medicine, Sendai, Miyagi, Japan

CorrespondenceDr Manabu Tashiro, Division of CyclotronNuclear Medicine, Cyclotron andRadioisotope Centre, Tohoku University,6-3 Aoba, Aramaki, Aoba-ku, Sendai,Miyagi 980-8578, Japan.Tel: + 81 22 795 7797Fax: + 81 22 795 7797E-mail: mtashiro@m.tains.tohoku.ac.jp----------------------------------------------------------------------

Keywordsfirst-generation antihistamine,histamine H1 receptor occupancy,placebo-controlled crossover studydesign, positron emission tomography,second-generation antihistamine----------------------------------------------------------------------

Received3 April 2007

Accepted17 January 2008

Published OnlineEarly11 April 2008

WHAT IS ALREADY KNOWN ABOUTTHIS SUBJECT• ‘Bepotastine besilate’ is a novel second-

generation antihistamine developed inJapan and its antiallergic effects have alreadybeen demonstrated by various studies.

• However, only a few clinical studiesregarding its sedative property are available.

• In addition, histamine H1 receptoroccupancy (H1RO) of this new antihistaminehas never been measured by positronemission tomography (PET).

WHAT THIS STUDY ADDS• This paper provides the first measurement

result of cerebral H1RO of bepotastinebesilate (approximately 15%) as determinedby PET.

• This result is in accordance with the clinicalclassification of bepotastine as asecond-generation antihistamine.

• In addition, the relationship betweensubjective sleepiness and cerebral H1RO ofthis second-generation antihistamine isdemonstrated for the first time using aplacebo-controlled crossover study design.

AIMSAntihistamines are frequently used for treating various allergic diseases, butoften induce sedation. The degree of sedation can be evaluated by measuringhistamine H1 receptor occupancy (H1RO) in the brain using positron emissiontomography (PET). The aim was to measure H1RO of bepotastine, a newsecond-generation antihistamine, and to compare it with that ofdiphenhydramine.

METHODSEight healthy male volunteers (mean age � SD 24.4 � 3.3 years) were studiedafter single oral administration of bepotastine (10 mg), diphenhydramine(30 mg) or placebo, by PET imaging with 11C-doxepin in a crossover studydesign. Binding potential ratio and H1ROs were calculated using placebo dataand were compared between bepotastine and diphenhydramine in the anteriorand posterior cingulate gyri (ACG and PCG, respectively), superior and inferiorfrontal cortices (SFC and IFC, respectively), orbitofrontal cortex (OFC), insularcortex (IC), lateral and medial temporal cortices (LTC and MTC, respectively),parietal cortex (PC), occipital cortex (OC) and sensorimotor cortex (SMC). Plasmaconcentration of each antihistamine was measured, and its correlation to H1ROwas examined.

RESULTSH1RO after bepotastine treatment was significantly lower than that afterdiphenhydramine treatment in all cortical regions (P < 0.001). Mean H1ROs ofbepotastine and diphenhydramine were 14.7% and 56.4%, respectively. H1ROsof both bepotastine and diphenhydramine correlated to their respective drugplasma concentration (P < 0.001).

CONCLUSIONOral bepotastine (10 mg), with its relatively low H1RO and thus minimalsedation, has the potential for use as a mildly or slightly sedative antihistaminein the treatment of various allergic disorders.

British Journal of ClinicalPharmacology

DOI:10.1111/j.1365-2125.2008.03143.x

Br J Clin Pharmacol / 65:6 / 811–821 / 811© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd

Introduction

Histamine H1 receptor (H1R) antagonists, or antihistamines,are often used for treating allergic disorders such as sea-sonal rhinitis. Antihistamines mainly act on peripheraltissues,but can induce sedation as a central side-effect.Thisundesirable side-effect is caused by blockade of nervetransmission in the histaminergic neuron system whichprojects from the tuberomammillary nucleus in the poste-rior hypothalamus to almost all cortical areas [1–5]. First-generation antihistamines that can easily penetrate theblood–brain barrier (BBB), such as diphenhydramine andd-chlorpheniramine, tend to occupy a large proportion ofpostsynaptic H1Rs as demonstrated by positron emissiontomography (PET) [1, 6–8]. PET also reveals that second-generation antihistamines (mildly or slightly sedative anti-histamines), such as cetirizine and olopatadine, can slightlypenetrate the BBB and occupy some H1Rs [1, 6, 9, 10]. Userswho take these second-generation antihistamines atdoubled or tripled doses to achieve desired effects maysuffer from dose-related cognitive impairment. Third-generation antihistamines (truly nonsedative anti-histamines),such as fexofenadine and ebastine,hardly pen-etrate the BBB and do not occupy H1Rs even at high doses,as demonstrated by 11C-doxepin PET [9].Thus, the sedativeproperty of antihistamines can be evaluated in terms of H1Roccupancy (H1RO). Such variations in BBB permeability arecaused by various factors, including differences in lipophi-licity, molecular size and actions of drug transporters.

Bepotastine besilate ({d-(S)-4-[4-[(4-chlorophenyl)(2-pyridyl)methoxy]piperidino} butyric acid monobenze-nesulphonate, betotastine besilate, CAS 125602-71-3, TAU-284 or Talion), a new second-generation antihistaminedeveloped in Japan, is now used as an oral tablet for aller-gic rhinitis and chronic urticaria (Figure 1) [11–13].Previousstudies have demonstrated its excellent antiallergic effectscompared with other antihistamines such as ketotifen,ceti-rizine, epinastine and terfenadine [14–18], whereas only afew studies have shown its central effects [18, 19]. Only oneavailable animal behavioural study by Kato and colleagueshas demonstrated that bepotastine is a highly specificdrug to H1R, having no significant binding affinity for his-tamine H3, adrenergic a1, a2, b, dopaminergic D2, seroton-ergic 5HT2, muscarinic or benzodiazepine receptors, and

that it poorly penetrates the BBB [19]. Takahashi andcolleagues first conducted a double-blind, placebo-controlled, crossover study to measure subjective sedationand psychomotor activities following administration ofbepotastine, cetirizine, fexofenadine and olopatadine [18],where bepotastine had the least sedative effect [18].

To date, we have measured H1ROs of various second-generation and third-generation antihistamines, but notthat of bepotastine. It is of great interest to examine H1ROof bepotastine in humans. Thus, the primary aim of thepresent study was to measure subjective sedation andcerebral H1RO of bepotastine and to compare the resultswith those of diphenhydramine, a typical sedative antihis-tamine [20], using a placebo-controlled crossover studydesign that would make the interpretation of resultsclearer and easier by minimizing potential errors due tointersubject variability [10]. Another aim was to determinewhether bepotastine should be classified as a trulynonsedative or mildly sedative antihistamine.

Methods

The present study was approved by the Committeeon Clinical Investigation at Tohoku University GraduateSchool of Medicine, Japan, and was performed in accor-dance with the principles of the Declaration of Helsinki. Allexperiments were performed at the Cyclotron and Radio-isotope Centre, Tohoku University.

Subjects and study designEight male Japanese volunteers (mean age � SD24.4 � 3.3 years), who provided written informed consentafter receiving a detailed description of the study, wererecruited. All subjects were in good health with no clinicalhistory of major physical or mental illness, showed noabnormality in brain magnetic resonance imaging (MRI),and were not receiving any concomitant medication likelyto interfere with the study results. Nicotine, caffeine, grape-fruit and grapefruit juice were not allowed on the test day,and alcohol was not allowed on the test day or the daybefore PET measurement.

All subjects underwent PET measurement after singleoral administration of bepotastine (10 mg), diphenhy-dramine (30 mg) or a lactobacteria preparation (6 mg)used as placebo in a three-way crossover study, withminimum wash-out intervals of 7 days between treat-ments.The lactobacteria preparation has been widely usedas placebo in Japan, and its administration has producedno statistical difference between pre- and post-administration in previous cognitive studies [7, 9, 10, 21].The present PET study was conducted in a single-blindedmanner, and after drug administration each subject wasasked to remain seated comfortably on a sofa. To deter-mine bepotastine and diphenhydramine plasma concen-trations, blood samples were collected from each subject

Cl O

NOH

O

Bepotastine besilate

S OH

O

O

O

Figure 1Chemical structure of bepotastine besilate

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before drug administration and at 0, 60, 120 and 180 minpost administration. Subjective sleepiness of each subjectwas also measured at 0, 60, 120 and 180 min post admin-istration using the Line Analogue Rating Scale (LARS) [22]and the Stanford Sleepiness Scale (SSS) as used in previousstudies [9, 23].

Measurement of plasma concentrations ofbepotastine and diphenhydraminePlasma concentrations of bepotastine and diphenhy-dramine were measured using liquid chromatography/tandem mass spectrometry (LC/MS/MS) together with anelectrospray ionization method [24]. LC was performed onan Agilent 1100 Series LC instrument (Agilent TechnologiesInc., Santa Clara, CA, USA) equipped with an analyticalcolumn. The MS/MS system was the API 4000 (AppliedBiosystems/MDS Sciex, ON, Canada). The Solid PhaseExtraction (SPE) cartridge (OASIS HLB 3 ml/60 mg; WatersCorp., Milford, MA, USA) was pretreated with 2 ml of metha-nol, 2 ml of water and 2 ml of 0.2 M Na2CO3/HCl buffer(pH 11).

For measurement, an internal standard solution (10 ml)and methanol (10 ml) were added to each plasma sample(50 ml). To the resulting solution, 930 ml of 0.2 M Na2CO3/HCl buffer was added for bepotastine measurement, and1000 ml of 0.1% formic acid containing acetonitrile/methanol (50 : 50, v/v) was added for diphenhydraminemeasurement. The mixture was applied onto the SPE car-tridge after pretreatment as mentioned above.Separationswere carried out on a high-performance liquid chromatog-raphy (HPLC) column [CAPCELL PAK C18 MG II (3 mm)2.0 mmf ¥ 100 mm; Shiseido Co., Ltd, Tokyo, Japan) at aflow rate of 0.2 ml min-1 and at a column temperature of40 °C. The reconstituted extract (5 ml) was injected onto anHPLC system with mobile phases for bepotastine measure-ment including 10 mmol l-1 ammonium acetate and aceto-nitrile of varied concentrations, namely, 32% (0–9 min),70% (9.5–12.5 min) and 32% (12.6–24 min), and withmobile phases for diphenhydramine measurement includ-ing 0.1% heptafluorobutyric acid and acetonitrile of variedconcentrations, namely, 40% (0–7 min), 70% (7.5–10.5 min)and 40% (11–21 min).

Detection of bepotastine was based on fragmentationof the precursor ion (m/z = 389 to product ion m/z = 202with collision energy of 29 eV for bepotastine, andm/z = 256 to product ion m/z = 167 with collision energyof 19 eV for diphenhydramine), and that of the internalstandard was based on fragmentation of the precursor ion(m/z = 389 to product ion m/z = 201 with collision energyof 29 eV for bepotastine, and m/z = 270 to product ionm/z = 181 with collision energy of 17 eV for diphenhy-dramine) in positive multiple reaction monitoring (MRM)mode. Positive ions were detected using an API 4000system at 550 °C nebulizer gas temperature, with 5000 Vion spray voltage, 68.9 kPa (nitrogen) curtain gas and Level4 collision gas for bepotastine, and 206.8 kPa curtain gasand Level 4 collision gas for diphenhydramine. Chromato-graphic data for positive MRM were collected usingAnalyst software (ver. 1.2, Applied Biosystems/MDS Sciex)with cycle times of 1.010 s per cycle for bepotastine and0.5200 s per cycle for diphenhydramine.The lowest detect-able concentration was around 1 ng ml-1 for both antihis-tamines, and some values slightly under the threshold(only for diphenhydramine) were extrapolated. As for vali-dation, the following items were checked for bepotastineand diphenhydramine, respectively: accuracy (100.7% and102.2%), correlation coefficients to standard solutions(r > 0.99 for both), and coefficients of variation (CVs)of three different concentrations (n = 5) (1.7–2.3%,1.8–3.8%).

For examination of the relationship between estimatedbinding potential ratio (BPR) of 11C-doxepin and plasmaconcentration of each antihistamine, the areas under thecurves (AUCs) of bepotastine and diphenhydramine werecalculated for 0–180 min (AUC0-3 h) post administration(Table 1).

PET tracer and image acquisitionDoxepin is one of the tricyclic antidepressants that hasbinding affinity to other receptors such as muscarinicreceptors to some extent. However, its affinity to histamineH1Rs is much higher than to other receptors and is veryhigh compared with other antidepressants [25]. Thus, dox-epin’s affinity to other receptor systems is negligible in this

Table 1Plasma concentrations of bepotastine and diphenhydramine (n = 8)

Bepotastine time (min) Mean (ng ml-1) SEM CV, % Diphenhydramine time (min) Mean (ng ml-1) SEM CV, %

0 0.0 0.0 0.0 0 0.0 0.0 0.060 78.0 17.3 62.9 60 6.2 2.3 104.4

120 71.0 4.6 18.2 120 16.3 2.1 36.4180 82.6 8.4 28.7 180 19.7 1.2 17.0

AUC0-3 h 196.1 24.3 35.0 AUC0-3 h 32.3 4.0 35.3

CV, coefficient of variation; SEM, standard error of mean.

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Br J Clin Pharmacol / 65:6 / 813

imaging study, as also confirmed by experiments usinghistamine H1R knock-out mice, where doxepin binding inthe brain was nearly zero [26]. Thereafter, 11C-doxepinhas been used to evaluate the distribution of histamineH1Rs. 11C-doxepin kinetics in plasma and the brain arenot affected by the sedative antihistamine d-chlorpheniramine using arterial sampling data combinedwith metabolite analysis [6].

In the present study, 11C-doxepin was prepared by11C-methylation of desmethyl doxepin with 11C-methyltriflate as described previously [10, 27]. 11C-doxepin radio-chemical purity was >99%, and its specific radioactivity atthe time of injection was 120.9 � 80.55 GBq mmol-1

(3268 � 2177 mCi mmol-1). 11C-doxepin-containing salinesolution was intravenously injected into each subject at90 min post administration, a time close to Tmax of bothantihistamines (1.2 h for bepotastine and 2–3 h for diphen-hydramine). The injected dose and cold mass of 11C-doxepin were 135.4 � 19.83 MBq (3.660 � 0.536 mCi) and1.587 � 0.895 nmol, respectively, and the radiological dosewas calculated based on a previous study on radiologicalexposure [28].

Approximately 60 min after 11C-doxepin injection, thesubjects were positioned on the coach of the PET scanner(SET2400W; Shimadzu Co., Kyoto, Japan) for transmissionscan (6 min) and emission scan in the three-dimensional(3D) mode lasting for 15 min (70–85 min post injection of11C-doxepin) in a similar fashion to our previous work [10,29]. PET brain images from a 15-min-long emission scanwere corrected for scattering based on a previous study[30] and for tissue attenuation using post-injection trans-mission scan data according to previous work [31]. Brainimages were reconstructed with a filtered back-projectionalgorithm, with the aid of a supercomputer SX-7 at theInformation Synergy Centre, Tohoku University, Sendai,Japan. The brain images were then normalized by plasmaradioactivity at 10 min post injection to yield imagesreflecting distribution volume (DV) based on our staticscan protocol reported previously [10, 32].Validation usingvenous sampling instead of arterial sampling was carriedout by another group, giving no difference betweenvenous and arterial sampling at 10 min post injection (M.Senda, personal communication, 23 August 2007).

Three brain images of each subject, following oraladministration of bepotastine, diphenhydramine andplacebo, were coregistered to an identical stereotaxic braincoordinate system using an MRI-T1 image of each subject,with the aid of Statistical Parametric Mapping (SPM2,Wellcome Department, UK) software package [33]. MRIimages were obtained with a 1.5-T magnetic resonance(MR) scanner (HiSpeed, Ver. 9.1; General Electric Inc., WI,USA) at Sendai Seiryo Clinic (miyagi, Japan). T1-weightedimages (Vascular TOF SPGR: TR/TE 50/2.4 ms, FA 45°,number of excitations 1, matrix size 256 ¥ 256, spatialresolution: x, y, z = 0.86, 0.86, 20.0 mm, respectively) werecollected from all subjects.

Regions of interest (ROIs) were first placed on the fol-lowing brain regions on the T1 images that had preciseanatomical information, i.e. anterior and posterior cingu-late gyri (ACG and PCG, respectively), prefrontal cortices(PFC), orbitofrontal cortex (OFC), insular cortex (IC), tempo-ral cortex (TC), parietal cortex (PC), occipital cortex (OC),primary sensorimotor cortex (SMC), thalamus, striatum,midbrain, pons, and cerebellum. ROI was defined for eachcortical region by two to five circles with a diameter of7.6 mm for each hemisphere in four to five consecutivebrain transaxial slices, as indicated in Figure 2A. For thethalamus, striatum, pons and midbrain, the margin of eachregion was traced in MRI T1 images. An averaged valuefrom all ROIs was used as a representative value of eachregion. Information on ROI location was automaticallytransferred to the coregistered three PET images reflectingDV, and the binding potential ratio (BPR) was calculated foreach region using the following equation: BPR = [(DV ofeach region - DV of cerebellum)/DV of cerebellum] [8, 9].Finally, H1ROs of bepotastine and diphenhydramine werecalculated for each cortical region using the followingequation: H1RO = [(BPR of placebo - BPR of antihistamine)/BPR of placebo] ¥ 100. BPR brain images were also createdby applying the same equation to each DV brain image[8–10, 34, 35]. BPR brain images were analysed statisticallyon a voxel-by-voxel basis using SPM2 [33], following spatialnormalization and smoothing using the same methodas in our previous work. Differences in parameter valuesbetween bepotastine, diphenhydramine and placebo(control) were statistically examined, and regional maximaof statistical significance (P < 0.001) were projected ontosurface-rendered MRI-T1 standard brain images. Preciselocations of statistically significant regions were identifiedwith the Co-Planar Stereotaxic Atlas [36].

Statistical analysisDifferences in subjective sleepiness and BPR betweenbepotastine, diphenhydramine and placebo were exam-ined using ANOVA followed by multiple comparison withBonferroni correction. The difference in H1RO betweenbepotastine and diphenhydramine was examined usingpaired Student’s t-test. The relationship between plasmadrug concentration (AUC) and H1RO was examined usingPearson’s correlation test. A probability of P < 0.05 wasconsidered statistically significant. All statistical examina-tions were performed using SPSS for Windows 13.0.1(Japanese version).

Results

Plasma concentrations of bepotastineand diphenhydramineMean plasma concentrations and AUCs of bepotastine anddiphenhydramine are shown in Table 1. The peak mean

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plasma concentration of bepotastine ranged from 60 to180 min post administration because, in five of the eightsubjects, it peaked at 60 min post administration, whereasin the other three subjects it peaked at 120 or 180 min postadministration. Mean plasma concentration of diphenhy-dramine was maximal from 120 to 180 min post adminis-tration (Table 1).

Subjective sleepiness

Results of mean subjective sleepiness are shown inFigure 3. Mean subjective sleepiness of diphenhydraminepeaked at 120–180 min post administration and that ofbepotastine at 120 min post administration. Both LARSand SSS manifested similar patterns. Multiple comparison

Figure 2Binding potential ratio (BPR) images of 11C-doxepin in the human brain (A) and results of voxel-by-voxel comparison (B). BPR of 11C-doxepin was calculatedin healthy male subjects (n = 8) by positron emission tomography following oral administrations of placebo (left), bepotastine (10 mg, middle) or diphen-hydramine (30 mg, right), and their magnetic resonance imaging-T1 images (far right), demonstrated in the transaxial (top), coronal (middle) and sagittal(bottom) sections for each treatment condition, were compared (A). White circles in the transaxial images indicate the regions of interest (ROIs). The brainimage of each subject was transformed to fit stereotaxic brain space (spatial normalization) and was averaged across each drug condition to generate themean images displayed (A).The images demonstrate that diphenhydramine treatment results in BPR significantly lower than those of other drug conditions(B). Height threshold of voxel values was set at P < 0.001 and extent threshold was set at 10 voxel minimum. Results were not corrected for multiplecomparisons.There are no areas with significantly lower BPR after bepotastine treatment compared with those after placebo treatment (‘bepotastine 10 mg< placebo’ in the left columns). In contrast, red colour shows areas with significantly lower BPR after diphenhydramine treatment compared with those afterplacebo treatment (‘diphenhydramine 30 mg < placebo’ in the right columns). In both columns, significant areas are demonstrated in four aspects, namely,left and right medial (L. MED and R. MED) and right and left lateral (R. LAT and L. LAT) aspects (P < 0.001, uncorrected, using SPM2) (B)

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following repeated ANOVA demonstrated that subjectivesleepiness following diphenhydramine administration wassignificantly stronger (P < 0.001) than that of both bepo-tastine and placebo at 120 and 180 min post administra-tion, and that of bepotastine was not significantly differentfrom that of placebo (Figure 3).

Brain distribution of 11C-doxepinRadioactivity distribution patterns of 11C-doxepin areshown in Figure 2A. The mean BPR image, averaged fromeight subjects, following bepotastine treatment wassimilar to that following placebo treatment in an individualsubject. Namely, high radioactivity was observed in ACG,PCG, PFC, OFC, IC,TC, PC, OC and SMC following both treat-ments, whereas the radioactivity distribution pattern fol-

lowing diphenhydramine treatment was much lower thanthat following the other two treatments (Figure 2A).

Comparison of parametric BPR images(bepotastine vs. diphenhydramine)Using SPM2 on a voxel-by-voxel basis, parametric brainBPR images following bepotastine or diphenhydraminetreatment were statistically compared with those follow-ing placebo treatment. In Figure 2B, red areas show brainregions where BPRs were significantly lower (P < 0.001) fol-lowing diphenhydramine treatment than those followingplacebo treatment (Figure 2B, right; Table 3). ACG, PFC andTC demonstrated significantly lower BPR after diphenhy-dramine treatment than after placebo treatment (Table 2).On the other hand, SPM analysis showed no brain areaswhere BPR was significantly lower after bepotastine treat-ment than after placebo treatment (Figure 2B, left).

ROI-based comparison of BPR and H1ROBPRs in H1R-rich regions such as ACG, PFC, IC,TC, PC and OCwere evaluated based on ROI analysis (Figure 4A). BPRs fol-lowing bepotastine treatment were only slightly differentfrom those following placebo treatment. However, BPRsfollowing diphenhydramine treatment were significantlylower than those following placebo or bepotastine treat-ment (P < 0.001) for all cortical regions. In the thalamus,striatum, pons and midbrain, there was no significant dif-ference in BPRs between diphenhydramine, bepotastineand placebo treatments.

H1RO following diphenhydramine or bepotastine treat-ment was also calculated using H1RO after placebo treat-ment as baseline (0%) (Figure 4B). Cortical mean H1ROfollowing bepotastine treatment was approximately14.7% and that following diphenhydramine treatmentwas approximately 56.4%. H1ROs following bepotastinetreatment were significantly lower than those followingdiphenhydramine treatment (P < 0.001) in all cortical

0

20

40

60

pre 60 min 120 min 180 min

Sub

ject

ive

slee

pine

ss (

LA

RS

)

* ** *

Figure 3Subjective sleepiness evaluated using the Line Analogue Rating Scale(LARS). Eight healthy subjects were studied following oral administrationof bepotastine (BEP, 10 mg), diphenhydramine (DIP, 30 mg) or placebo(PLA). *P < 0.001 by ANOVA followed by multiple comparison with Bonfer-roni correction. Error bars represent interindividual variability (SEM). BEP,( ); DIP, ( ); PLA, ( )

Table 2Binding potential ratios and histamine H1 receptor occupancies in placebo, bepotastine and diphenhydramine conditions

RegionsBPRPla BPRBEP BPRDIP 95% CI H1ROBEP H1RODIP 95% CI C.E. C.E.Mean SD Mean SD Mean SD Pla-DIP BEP-DIP Mean SD Mean SD DIP-BEP BEP DIP

Anterior cingulate gyrus (ACG) 0.78 0.22 0.66 0.11 0.33 0.09 0.28, 0.64 0.24–0.42 14.3 11.9 57.7 8.9 33.1, 53.8 0.54 -0.30Posterior cingulate gyrus (PCG) 0.85 0.18 0.71 0.09 0.40 0.09 0.28, 0.61 0.18–0.43 14.8 12.3 52.0 9.7 24.1, 50.2 0.51 -0.57

Prefrontal cortex (PFC) 0.65 0.16 0.55 0.10 0.27 0.10 0.26, 0.51 0.20–0.37 14.2 12.2 59.3 12.2 31.7, 58.5 0.66 -0.28Orbitofrontal cortex (OFC) 0.60 0.20 0.50 0.13 0.24 0.08 0.23, 0.51 0.18–0.35 15.4 11.8 60.5 6.9 34.1, 56.2 -0.01 -0.28

Insular cortex (IC) 0.80 0.18 0.66 0.09 0.36 0.09 0.29, 0.57 0.21–0.37 15.9 9.9 54.0 8.0 28.7, 47.4 0.52 -0.29Temporal cortex (TC) 0.61 0.18 0.51 0.09 0.27 0.11 0.22, 0.48 0.21–0.28 14.4 12.6 57.2 11.0 30.6, 55.0 0.04 -0.20

Parietal cortex (PC) 0.54 0.17 0.44 0.13 0.21 0.12 0.22, 0.43 0.17–0.30 16.0 16.9 62.8 16.5 30.6, 62.9 0.34 0.16Occipital cortex (OC) 0.49 0.10 0.43 0.06 0.27 0.08 0.15, 0.31 0.11–0.22 11.0 12.8 46.3 13.5 23.5, 47.0 0.31 0.06

Somatosensory cortex (SMC) 0.44 0.14 0.36 0.10 0.19 0.09 0.13, 0.38 0.06–0.29 16.5 15.9 57.6 21.6 18.9, 63.3 0.78 -0.18Mean 0.64 0.14 0.54 0.12 0.28 0.07 14.7 1.6 56.4 5.0 0.48 -0.19

Results of statistical evaluation (P-values) are shown in Figure 3. BEP, bepotastine; BPR, binding potential ratio; C.E., correlation efficient of H1 RO to plasma concentration of eachantihistamine; 95% CI, 95% confidence interval; DIP, diphenhydramine; H1RO, histamine H1 receptor occupancy; Pla, placebo; SD, standard deviation.

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regions. These data demonstrate that BPR following bepo-tastine treatment is substantially higher than that follow-ing diphenhydramine treatment in all cortical regionsstudied.

Relationships between subjective sleepiness,plasma drug concentration and H1ROSubjective sleepiness (AUC of LARS curve) did not signifi-cantly correlate to plasma concentration of bepotastine(r = 0.04), but did correlate well to plasma concentration ofdiphenhydramine (r = 0.72). H1ROs following bepotastineadministration significantly correlated to plasma concen-tration of bepotastine in ACG, PCG, PFC, IC and SMC,whereas those following diphenhydramine administrationwere all negatively correlated (Table 2). A similar trendwas observed between cortical mean H1RO and plasmaconcentration of both antihistamines (Figure 5A,B).However, when the baseline data are plotted together,mean H1RO due to diphenhydramine tended to increaserapidly with diphenhydramine concentration, whereasthat due to bepotastine gradually increased with bepotas-tine concentration (Figure 5A,B). H1RO following bepotast-ine administration did not significantly correlate tosubjective sleepiness (r = 0.01) (Figure 5D), whereas thatfollowing diphenhydramine administration negatively cor-related to subjective sleepiness when the baseline datawere plotted together (Figure 5C).

Discussion

Recently, molecular imaging techniques have beenactively applied in clinical pharmacology research [1, 8–10,37]. One of the primary aims of such work is to evaluate therelationship between doses of therapeutic drugs and theiradverse reactions in terms of changes in ‘endophenotypes’in the brain [38]. The primary aim of the present studywas to clarify whether bepotastine belongs to second-generation antihistamines in terms of H1RO. Another aim

Table 3Regions with significantly lower specific binding following diphenhydramine treatment compared with those following placebo treatment

Regions Brodmann’s area Hemisphere x, y, z (mm) Cluster size T-value Z-value P-value

Superior temporal gyrus 22 R 54–56 16 29 543 11.55 6.41 <0.001Medial temporal gyrus 21 L -58-12-12 S.C. 11.41 6.37 <0.001

Precuneus 7 R 26–58 48 S.C. 11.08 6.29 <0.001Medial temporal gyrus 21 L -48 4–32 32 8.14 5.41 <0.001

Superior frontal gyrus 10 L -24 60 12 104 7.53 5.18 <0.001Superior frontal gyrus 8 L -28 24 50 38 7.07 5 <0.001

Medial frontal gyrus 10 R 28 54 14 1342 8.53 5.55 <0.001Medial frontal gyrus 9 R 42 6 40 S.C. 8.46 5.52 <0.001

Medial frontal gyrus 6 R 24 4 58 S.C. 8.98 5.7 <0.001Inferior frontal gyrus 46 L -40 44 12 38 8.96 5.69 <0.001

Cluster size is represented by the number of voxels (voxel size: 2.0 ¥ 2.0 ¥ 2.0 mm3). S.C., the same cluster as above. Results are not corrected for multiple comparisons.

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Figure 4Region of interest (ROI)-based analyses of binding potential ratios (BPR)(A) and histamine H1 receptor occupancy (H1RO) (B) in the cortex. ROImeasurements were performed in the anterior and posterior cingulategyri (ACG and PCG, respectively), prefrontal cortex (PFC), orbitofrontalcortex (OFC), insular cortex (IC), temporal cortex (TC), sensorimotor cortex(SMC), parietal cortex (PC) and occipital cortex (OC) after treatments withplacebo (PLA), bepotastine (BEP) and diphenhydramine (DIP). Compari-son of BPRs shows differences in the sedative properties of the threedrugs (A). H1ROs due to BEP and DIP are shown, taking H1RO due toplacebo as 0% (B). *P < 0.001, ANOVA followed by multiple comparisonwith Bonferroni correction. Error bars represent interindividual variability(SEM). PLA, (�); BEP, ( ); DIP, (�)

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was to determine whether bepotastine should be classi-fied as a truly nonsedative or mildly sedative antihista-mine, as previously discussed in the Consensus Group onNew Generation Antihistamines (CONGA) [3].

In the present study, H1ROs following a single oraladministration of bepotastine (10 mg) or diphenhy-dramine (30 mg) were 14.7 and 56.4%, respectively. Therelatively high H1RO due to diphenhydramine agrees withthose of other reported first-generation antihistamines [1,6, 38]. Single oral administration of d-chlorpheniramine(2 mg) achieved 50–77% H1RO, and this high H1RO wasassociated with high prevalence of sleepiness and cogni-tive decline [6, 8, 39]. On the other hand, bepotastine’s lowH1RO value suggests that this antihistamine can be classi-fied into the category of second-generation antihista-mines [6, 8, 9, 39–42, 38].

Thus, whether bepotastine can be classified as trulynonsedative or not is an additional point of clinical impor-tance. In the present study, subjective sleepiness follow-ing bepotastine administration (10 mg) was negligible(Figure 3).This finding seems to be in accordance with thatof Takahashi and colleagues [18], where bepotastine

(10 mg) induced slight subjective sedation among healthyvolunteers. In their study, the best performance wasachieved following bepotastine treatment in a word-processing task compared with those following cetirizine(10 mg), fexofenadine (60 mg) or olopatadine (5 mg) treat-ment [18]. Another study [11] has demonstrated thatresults of serial arithmetic tests following oral administra-tion of bepotastine (2.5, 5, 10 and 20 mg) showed nosignificant differences from those following placebo treat-ment. These clinical studies tried to demonstrate thenonsedative property of bepotastine, but they failed toinclude an active placebo (sedative antihistamine) toprove sensitivity of the selected tasks as recommended inthe CONGA guideline [3]. An additional animal study [19]has demonstrated that bepotastine manifested a sedativeprofile similar to that of cetirizine and terfenadine. As awhole, these results suggest the low liability of bepotastineto produce sedative side-effects at a therapeutic dose of10 mg. Considering the significant correlation betweenH1RO and plasma drug concentration (AUC) found in thepresent study (Figure 5B), bepotastine may be classified asa mildly sedative antihistamine.

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Figure 5Relationship between mean H1 receptor occupancy (H1RO), plasma concentration and subjective sleepiness following administrations of bepotastine anddiphenhydramine. Relationship between mean H1RO (across brain regions) and plasma concentrations was examined for diphenhydramine (A) andbepotastine (B). Plasma concentrations of the two antihistamines are presented as area under the curve (AUC). H1RO of diphenhydramine rapidly increaseswith plasma concentration, whereas H1RO of bepotastine gradually increases with plasma concentration. Error bars represent intraindividual variability (SD).Dotted curves reflect the estimated curves of relationship between plasma concentration and the H1RO analysed by the Michaelis–Menten equation (A andB). Subjective sleepiness (presented as AUC of line-analogue rating scale curve: AUC_LARS) demonstrates mild correlation to mean H1RO due to diphen-hydramine (C), whereas subjective sleepiness due to bepotastine demonstrates no correlation to mean H1RO due to bepotastine (D)

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818 / 65:6 / Br J Clin Pharmacol

In general, H1RO is used as an index of BBB permeabil-ity, but it could also be affected by gut absorption thatcan raise plasma drug concentration. At the molecularlevel, variation in BBB permeability has been determinedby factors such as lipophilicity, molecular size and dif-ferent actions of various drug transporters includingP-glycoprotein (P-gp), an efflux pump expressed in capil-lary endothelial cells in the BBB [10]. Many lipophilic first-generation antihistamines are absorbed in full amount inthe gut and can freely enter the brain tissue, whereas gutabsorption and brain penetration of second-generationantihistamines tend to be reduced. For fexofenadine, asubstrate of P-gp, both gut absorption and BBB perme-ability are highly reduced because of its low membranepermeability and high action of P-gp. For bepotastine,also a substrate of P-gp, the same extent of reduction asthat of fexofenadine is observed [43]. The gradualincrease in H1RO with plasma bepotastine concentration,suggesting its relatively high membrane permeability(Figure 5B), may be associated with the action of P-gp inthe BBB. On the other hand, gut absorption of bepotast-ine tends to be much higher than that of fexofenadine,presumably because of bepotastine’s higher membranepermeability in the upper part of the small intestinewhere P-gp expression is low [43]. Such difference isone of the possible reasons for explaining the dif-ference between bepotastine and fexofenadine. An-other reason is that fexofenadine is transported notonly by P-gp but also by the organic anion protein trans-porter family, further reducing its absorption in the gut[44, 45].

The limitations of the present method are as follows.The short scanning PET protocol would be useful espe-cially in conducting a placebo-controlled crossover studybecause of the enormous mental and physical stress ofvolunteers observed in a previous study, where they wererequested to complete four sets of 100-min-long PETexaminations [34]. It is therefore important to develop ashort scan protocol, although such protocols wouldinclude a larger amount of noise. Users of the PET systemshould consider these limitations. We note here that notexact values but, rather, approximations of DV and BP weremeasured in this study.

We were interested in the effects of an acute singledose of an antihistamine, and we planned to start PETscanning at the time point near Tmax for each drug, but thetracer injection for diphenhydramine condition seemed tobe slightly early since the plasma concentration ofdiphenhydramine was still increasing (Table 1). Therefore,H1RO due to diphenhydramine might give slightly differ-ent values when measured in the equilibrium state. Thismight be the reason why H1RO following diphenhy-dramine treatment did not correlate positively to theplasma concentration of diphenhydramine, contrary toour expectation based on a previous study usingd-chlorpheniramine [8].

In summary, we have examined H1RO of bepotastine(10 mg; 14.7%) and compared it with that of diphenhy-dramine (30 mg; 56.4%) given as a single oral administra-tion, using PET in a placebo-controlled crossover study. It issuggested that oral administration of bepotastine (10 mg),with its low H1RO and minimal sedation effects, is useful fortreating various allergic disorders. As for the dose depen-dency of its sedative effects,another cognitive study involv-ing an active placebo is needed in order to draw a definitiveconclusion. The dose dependency of H1RO should also beexamined by PET measurements at higher doses.

This work was in part supported by Grants-in-Aid for ScientificResearch (nos. 17390156 for K.Y. and 16790308 for M.T.) fromthe Japan Society of Promotion of Science (JSPS) and the Min-istry of Education, Culture, Sports, Science and Technologyin Japan, as well as by a grant from the Japan Society ofTechnology (JST) on research and education in ‘molecularimaging’. We thank the volunteers of the PET study and MrsKazuko Takeda for her care of them. We also thank TANABER&D SERVICE Co., Ltd (Osaka, Japan) for technical support inmeasuring the plasma concentration of antihistamines.

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