in-vivo rat striatal 5-ht 4 receptor occupancy using non-radiolabelled...

In-vivo rat striatal 5-HT4 receptor occupancy usingnon-radiolabelled SB207145Ramakrishna Nirogia,b, Vishwottam Kandikerea, Gopinadh Bhyrapunenia, Ramanatha Saralayaa,Devender Reddy Ajjalaa, Raghupathi Reddy Aletia and Mohammed Abdul Rasheedb

aPharmacokinetics and Drug Metabolism and bDepartment of Medicinal Chemistry, Discovery Research, Suven Life Sciences Ltd, Hyderabad, India

Keywords5-HT4 receptor occupancy; LC-MS/MS; rat;SB207145

CorrespondenceRamakrishna Nirogi, Suven Life Sciences Ltd,Serene Chambers, Road -5, Avenue -7,Banjara Hills, Hyderabad – 500 034, India.E-mail: [email protected]

Received August 17, 2012Accepted December 19, 2012

doi: 10.1111/jphp.12030

Abstract

Objectives The objective of the current investigation was to develop a simple,rapid method for determining in-vivo 5-hydroxytryptamine type 4 receptor(5-HT4R) occupancy in rat brain using non-radiolabelled SB207145 as a tracer foraccelerating the drug discovery process.Methods In-vivo tracer optimization studies for tracer dose, survival intervalsand brain distribution profile were carried out in rats. The tracer was pharmaco-logically validated using potent well-characterized 5-HT4R ligands. The brainregional concentrations of tracer (SB207145); plasma and brain concentrations of5-HT4R ligands were quantified using high-performance liquid chromatographycoupled with a tandem mass spectrometric detector (LC-MS/MS).Key findings SB207145 showed a higher specific binding in striatum (1.96 ng/g)and lower binding in cerebellum (0.66 ng/g), which is consistent with findings ofother published 5-HT4R expression studies. Pretreatment with potent 5-HT4

ligands dose-dependently reduced striatal SB207145 concentration and the effec-tive dose to achieve 50% receptor occupancy (ED50) values were 4.8, 2.0, 7.4, 9.9,3.8 and 0.02 mg/kg for GR113808, piboserod, prucalopride, RS67333, TD8954 andPF04995274, respectively.Conclusions Results from the mass spectrometry approach to determine 5-HT4Roccupancy in rat brain are comparable with those reported using radiolabelledscintillation spectroscopy methods. In conclusion, the LC-MS/MS characteriza-tion permits use of tracer at a preclinical stage in high-throughput fashion as wellas characterization of target expression.

Introduction

The 5-hydroxytryptamine type 4 receptor (5-HT4R) is amember of the seven transmembrane-spanning G-protein-coupled family of receptors and is distributed in the centraland peripheral neuronal system.[1–2] The 5-HT4R has a dis-crete localization in the central nervous system (CNS),[2–5]

with highest levels of 5-HT4 mRNA found in the caudateand putamen, and lower levels in the temporal cortex andhippocampus.[6] In the CNS, 5-HT4Rs have been shown tomediate activation of adenylate cyclase in rat hippocampalpreparations[1,7–9] and play an essential role in the memoryprocesses.[5,10] Stimulation of the 5-HT4R evokes the releaseof acetylcholine (ACh) from guinea-pig enteric nerveterminal,[11–13] cortex and hippocampus in rat.[14,15] Thesefindings suggest that 5-HT4R may have a role in cognitivefunctions in preclinical models.[14–20]

A postmortem binding study in Alzheimer’s patientsbrain demonstrated marked loss of 5-HT4Rs in the hip-pocampus.[21] 5-HT4R agonists, when administered aloneor in combination with acetylcholinesterase inhibitors,reversed the muscarinic-receptor-antagonist-induced cog-nitive deficits.[15,16,22–24] 5-HT4R activation also regulatessecretion of non-amyloidogenic soluble form of amyloidprecursor protein (sAPPa) in in-vitro and in-vivo animalmodels.[25–27] sAPPa has potent neuroprotective and neuro-tropic properties and enhances long-term potentiation.[28]

These results support the potential role of 5-HT4R agonistsin both the symptomatic and disease-modifying treatmentof cognitive disorders.

5-HT4R ligands are reported to be effective in cogni-tive enhancement and increase the cortical levels of ACh

bs_bs_banner

And PharmacologyJournal of Pharmacy

Research Paper

© 2013 The Authors. JPP © 2013Royal Pharmaceutical Society 2013 Journal of Pharmacy and Pharmacology, 65, pp. 704–712704

after systemic administration.[14,29] The therapeutic activityof 5-HT4R ligands in preclinical models depends on boththe extent of receptor occupancy and intrinsic efficacy.[30]

5-HT4R agonists with lower intrinsic efficacy require higherlevels of receptor occupancy to be effective in physiologicaland behavioural response (e.g. memory enhancement orACh release).[29,30] In preclinical research, receptor occu-pancy for 5-HT4R ligands has been measured by employingradiolabelled ligands.[29,30] Assessing receptor occupancyusing radiolabelled ligands is hazardous, expensive and timeconsuming. Measuring receptor occupancy by employing anon-radiolabelled ligand would confer a number of advan-tages such as turn-around time (results obtained within aday), no radioactive waste and the ability to collect targetengagement and exposure data from the same animal.[31–35]

Non-radiolabelled ligands have been used successfully forvarious targets, including dopamine D2, serotonin 5HT2A,NK1, opiate, histamine H3, neuronal nicotinic ACh a4b2,

nociceptin and kappa opioid receptor occupancy, in pre-clinical studies.[31–37]

SB207145 is a selective 5-HT4R antagonist, has a highaffinity for 5-HT4R (Ki 0.3 nm) and potential ease of 11Clabelling.[38] [11C]SB207145 has been reported as a noveluseful tracer for brain molecular imaging of the5-HT4R.[38–40] Positron emission tomography (PET) studiesin Gottingen minipigs, rats and humans showed that theradiotracer readily entered the brain, giving rise to a hetero-geneous distribution consistent with the known regionalpattern of 5-HT4R expression.[29,38–40] This is the first reportto employ non-radiolabelled SB207145 for mapping the5-HT4R and for measuring in-vivo receptor occupancyof structurally diverse 5-HT4R ligands. This novel approachcould be used to accelerate the screening process to identifyadditional 5-HT4R ligands and/or to link preclinical behav-ioural responses to in-vivo target engagement without theneed for expensive micro-PET analyses. The tracer, and thusthe assay, was pharmacologically validated with known,potent, structurally diverse 5-HT4 ligands includingGR113808, piboserod, prucalopride, RS67333, TD8954 andPF04995274.

Materials and Methods

Chemicals

SB207145, GR113808, piboserod, prucalopride, RS67333,TD8954 and PF04995274 (Figure 1) were synthesized inthe medicinal chemistry laboratory, Suven Life SciencesLtd (Hyderabad, India). All other chemicals used were ofanalytical grade and purchased from Sigma-Aldrich (StLouis, MO, USA). SB207145 was dissolved in salineand water for injection was used as a vehicle forGR113808, piboserod, prucalopride, RS67333, TD8954 andPF04995274.

Animals

Adult male Sprague–Dawley rats, aged 10–14 weeks, 225–300 g, were procured from the National Institute of Nutri-tion (Hyderabad, India). Rats were housed in groups of fourin an air-conditioned and temperature-controlled room(23 � 2°C and 55 � 10% humidity) maintained on a 12-hlight–dark cycle. Rats had free access to food and water andwere introduced to the experimental holding rooms at least2 days before the start of the study to allow adaptation tohousing conditions. The experimental protocol wasapproved by the Institutional Animal Ethics Committee(IAEC) of Suven Life Sciences Ltd. The IAEC were governedby the directions of the Committee for the Purpose ofControl and Supervision of Experiments on Animals(CPCSEA), India (Ethical approval No. PK-PRO-707-13/Dated August 06, 2011).

In-vivo tracer characterization of SB207145

In-vivo tracer optimization

Non-radiolabelled SB207145 was administered intrave-nously (3 mg/kg) through the lateral tail vein to a group ofmale Sprague–Dawley rats to determine the in-vivo braindistribution at 40 min post tracer dose. The isolated brainregions were transferred into pre-labelled tubes. The postweight of the vials was taken. The net weight of the tissueswas obtained from the difference in pre and post weight ofvials and frozen on dry ice until quantification of SB207145.

Male Sprague–Dawley rats (n = 4 at each time point)were treated intravenously with different doses of SB207145(0.5, 1, 2, 3 and 10 mg/kg). Rats were sacrificed at 30 minpost dose of SB207145. Cerebellum and striatum wererapidly dissected out and frozen on dry ice until quantifica-tion of SB207145. Time-dependent regional brain uptakestudy was performed at 5, 10, 20, 30, 40 and 60 min postdose of SB207145 (2 mg/kg, i.v.; n = 4 rats/group/point), andcerebellum and striatum were collected and handled asdescribed above.

In-vivo brain 5-HT4 receptor occupancy

Vehicle or GR113808 (0.03, 0.1, 0.3, 1, 3, 10 and 20 mg/kg,p.o.), piboserod (0.3, 1, 3, 10, 20 and 30 mg/kg, p.o.), pruca-lopride (0.1, 0.3, 1, 3, 10, 20 and 30 mg/kg, p.o.), RS67333(1, 3, 10, 20 and 30 mg/kg, p.o.), TD8954 (1, 3, 10, 20 and30 mg/kg, p.o.) and PF04995274 (0.003, 0.01, 0.03, 0.1, 0.3,1 and mg/kg, s.c.) were administered to male Sprague–Dawley rats (n = 4/group). After 1 h of treatment withcompound or vehicle, rats were restrained and SB207145was administered intravenously (2 mg/kg). The rats weresacrificed 30 min after intravenous injection of SB207145.Cerebellum and striatum were collected and handled asdescribed above; tracer concentrations obtained from these

Ramakrishna Nirogi et al. In-vivo 5-HT4 receptor occupancy

© 2013 The Authors. JPP © 2013Royal Pharmaceutical Society 2013 Journal of Pharmacy and Pharmacology, 65, pp. 704–712 705

regions were used in receptor occupancy calculations. Fromall the rats, 0.5–1 ml of trunk blood was collected intoheparinized 2.0-ml polypropylene tubes. Blood was centri-fuged at 4000 rpm for 10 min at 4°C (Eppendorf AG 22331;Hamburg, Germany). Whole brain (minus cerebellum andstriatum) was homogenized (Glascol tissue homogenizer;London, UK) in cold water by adjusting to 20% w/v. All

plasma and brain samples were stored at -50°C until quan-tification of respective test compounds using LC-MS/MS.

Quantification of SB207145 in brain tissues

SB207145 concentrations in different brain regions werequantified using a liquid chromatography-tandem mass

OO

N

NHS

OO

O

O

ClNH

NO

O

O

NHN

O

N

N

O

OCl

NH2

H3C

H2N

H2N H3CCH3

CH3

CH3

HN

NH

N

N

O

O

O

O

N

O

OHNO

NO

O

O

OMe

Cl

O

NN

(a) SB207145

(c) Piboserod (d) Prucalopride

(f) TD8954(e) RS67333

(g) PF04995274

(b) GR113808

Figure 1 Chemical structures of SB207145, GR113808, piboserod, prucalopride, RS67333, TD8954 and PF04995274.

Ramakrishna Nirogi et al.In-vivo 5-HT4 receptor occupancy


spectrometric system consisting of HPLC equipped withLC-ADVP binary pump, A DGU 20A5 degasser and a SILHTc auto sampler equipped with a CTO-10 AS VP ther-mostated column oven. Separation was achieved by using aZorbax XDB C18 column with dimensions of 2.1 ¥ 50 mm(5 mm) at a temperature of 50°C. Mass spectrometer detec-tion was performed by using an API-4000 triple quadrupoleinstrument (AB Sciex, Concord, ON, Canada) in multiplereaction monitoring modes. An electronically triggered six-port switching valve (Valco Instruments, Houston, TX,USA) was used for diverting/introducing flow into theLC-MS/MS system.

The mobile phase consisted of solvent A (0.1% formicacid in water) and solvent B (100% acetonitrile) with a flowrate of 0.3 ml/min. The gradient programme started with5% acetonitrile 95% buffer and was maintained up to0.5 min. In a step gradient at 0.51 min acetonitrile waschanged to 90% and remained the same up to 1.5 min.At 1.51 min acetonitrile was changed to 5% forre-conditioning of the column until the end of the run(4 min). Total flow was diverted to waste up to 1.5 min byusing a valco valve; flow was introduced into the mass spec-trometric source from 1.51 min to 4 min. The retentiontime of the SB207145 was found to be 2.5 min.

Upon fragmentation it produced fragments of m/z 212.1,156.1 and 112.1 (Figure S1 supplemental material). A mul-tiple reaction monitoring transition of 341.1 → 112.1 pro-duced the highest intensity with stable response (Figure S1supplemental material). Mass spectrometric detection wasperformed under positive ionization mode with turbo ionspray interface. Data was acquired with Analyst 1.6 versionsoftware (AB Sciex).

The frozen tissues were allowed to thaw to room tem-perature, four volumes (w/v) of acetonitrile were added toeach tube containing a tissue. These samples were homog-enized using a sonic dismembrator (Branson Sonifier modelmicro SD-150D; Danbury, CT, USA), and homogenizedsamples were centrifuged for 10 min at 10 000 rpm (Eppen-dorf 5417R; Westbury, NY, USA). The supernatant fractionswere transferred into labelled vials and a 5-ml volume wasinjected into the chromatographic system for analysis. Astandard stock solution of SB207145 (1 mg/ml) was pre-pared in methanol. Working solutions for calibration wereprepared by appropriate dilution in water–methanol(50 : 50, v/v). Calibration curves were generated in therange of 0.1–10 ng/g by adding known quantities ofworking solutions to a series of brain tissue samples fromthe untreated rats and processed as described above.

The extraction efficiency of SB207145 was determined inbrain tissue samples from untreated rats. SB207145 solutionwas added to each sample to obtain a final concentration of0.3 or 3 ng/g. Six tissue samples were used at each concen-tration. In parallel, six aqueous standards were prepared

equivalent to 0.3 ng/g or 3 ng/g. Spiked tissue samples wereextracted as described above and the SB207145 levels in theextracts were measured by LC-MS/MS. Extraction efficiencywas measured by comparing the peak area obtained fortissue extracted samples with that of aqueous samples.

Quantification of analytes

Calibration standards were prepared by spiking 5 ml ofanalyte solution to the 45 ml of plasma or 20% brainhomogenate and mixing with 5 ml of internal standard solu-tion (Table S1, supplemental material). All samples wereextracted by protein precipitation using 200 ml ofacetonitrile. The mixture was vortexed and centrifuged at10 000 rpm for 10 min at 10°C. The supernatant fractionwas transferred into pre-labelled vials and injected into thechromatographic system. The details of the chromato-graphic and mass spectrometer conditions are given in thesupplemental material (Table S1).

Receptor occupancy calculations anddata analysis

In the current methodology, a well-established ratiomethod was used to calculate the percentage 5-HT4R occu-pancy from each rat. SB207145 concentration ratio in thestriatum to the cerebellum was calculated, whereby thestriatum tracer concentration was represented by the areawith rich 5-HT4R expression and the cerebellum tracer con-centration was represented by the area with little or noexpression of 5-HT4R. This method assumed that the non-specific binding in the striatum was similar to the nonspe-cific binding determined in the cerebellum. Hence thespecific binding of the tracer was determined by taking thedifference in the SB207145 concentration between the stria-tum and cerebellum.

The following equation was used to calculate the 5-HT4Roccupancy:

% Occupancy Ratio Ratiot v= × − −( ) −( )[ ]{ }100 1 1 1 (1)

Where, Ratiot is the ratio of SB207145 concentrationsbetween the striatum and cerebellum in individual rats pre-treated with standard 5-HT4 ligands and Ratiov indicates themean ratio of SB207145 concentrations measured betweenthe striatum and cerebellum of vehicle-treated rats. Themean concentration ratio (striatum to the cerebellum) ofSB207145 in vehicle-pretreated rats represents 0% occu-pancy. With increasing occupancy of the receptor by the testcompound, the concentration of SB207145 in the totalbinding region decreases to the concentration in the non-specific binding region and the resulting ratio alsodecreases. At 100% occupancy there are no vacant receptorsto which SB207145 can bind in the striatum. In this case the



SB207145 binding in the striatum (total binding) and cer-ebellum (nonspecific binding) is same and the ratio equalsone. Receptor occupancy was calculated by linear extrapola-tion between the ratio establishing 0% and 100% occu-pancy. Receptor occupancy curves were fitted using thesigmoidal dose–response equation. GraphPad Prism soft-ware (GraphPad, San Diego, CA, USA) was used to calculateeffective dose required to achieve 50% receptor occupancy(ED50) value and the brain and plasma concentrationrequired to achieve 50% receptor occupancy (EC50) value.

Statistical analysis

The data were expressed as mean � SEM (n = 4/group).Statistical analysis was performed by one-way analysis ofvariance followed by Dunnett’s test for receptor occupancyand regional distribution. The threshold for statistical sig-nificance was set at P < 0.05. Statistical analysis was carriedout using Graph Pad Prism (Version 4).

Results

In-vivo tracer characterization of SB207145

In-vivo tracer optimization

SB207145 gained ready access to the brain with higheruptake in receptor-rich tissue than in other brain substruc-

tures (Figure 2). High levels of tracer were observed in thestriatum (1.96 � 0.07 ng/g, **P < 0.01 vs cerebellum), mod-erate levels were observed in the hippocampus (1.74 �

0.09 ng/g, **P < 0.01) and frontal cortex (1.06 � 0.03 ng/g,*P < 0.05) with much lower levels in the thalamus(0.93 � 0.08 ng/g, *P < 0.05), brain stem (0.85 � 0.14 ng/g)and cerebellum (0.66 � 0.02 ng/g). Concentrations meas-ured in all the brain regions were within the limits of quan-tification. The mean extraction efficiency of SB207145 was94 � 2% at the concentrations used in the assay.

A non-dose-dependent increase in SB207145 concentra-tion was observed across brain regions at intravenous dosesof 0.5, 1, 2, 3 and 10 mg/kg, (Table 1). The total (striatum)to nonspecific (cerebellum) ratio of SB207145 wasdecreased with increasing dose (Table 1). The tracer dose(2 mg/kg) was selected based on the ability to detect thelowest cerebellar concentration which could be reliablymeasured using the LC-MS/MS.

Maximum brain uptake of SB207145 was found in thestriatum at 5 min post dose and declined gradually withtime (Figure 3). The striatum to cerebellum concentrationratio increased with time, and at 30 min post dose theSB207145 concentration ratio was found to be ~3.5, andmaintained to 40 min (Figure 3). The concentration ratiobetween striatum and cerebellum at 30 min post dose wasconsistent between the studies, and was found to be3.85 � 0.46 (mean � SEM) from 15 different experiments.

Cereb

ellum

Stria

tum

Fronta

l corte

x

Hippoca

mpus

Thala

mus

Hypoth

alam

us

Brain

stem

0.0

0.5

1.0

1.5

2.0

2.5

SB20

7145

(n

g/g

)

Figure 2 In-vivo brain regional distribution of SB207145 (3 mg/kg,i.v., at 40 min) in male Sprague–Dawley rats (mean � SEM, n = 4/group). *P < 0.05 and **P < 0.01, compared with the cerebellumvalues.

0 10 20 30 40 50 600.0

0.5

1.0

1.5

2.0Striatum (ST) Cerebellum (CB)ST/CB ratio

0

5

10

15

Time (min)

SB20

7145

(n

g/g

)

ST/CB

ratio

Figure 3 Time response in-vivo binding of SB207145 in striatum andcerebellum after an intravenous administration of SB207145 (2 mg/kg)in rats (mean � SEM, n = 4/ group).

Table 1 SB207145 concentrations in the rat striatum and cerebellum at 30 min post dose

Rat brain regions

Dose (mg/kg, i.v.)

0.5 1 2 3 10

Striatum (ng/g) 0.63 � 0.03 0.89 � 0.08 1.28 � 0.03 1.84 � 0.21 4.01 � 0.08Cerebellum (ng/g) 0.14 � 0.01 0.22 � 0.01 0.37 � 0.01 0.73 � 0.07 1.90 � 0.14Ratio (striatum/cerebellum) 4.50 � 0.18 4.05 � 0.08 3.46 � 0.05 2.52 � 0.08 2.11 � 0.03

Data are presented as means � SEM, n = 4/group.



In-vivo brain 5-HT4 receptor occupancy

Pretreatment with 5-HT4R ligands GR113808, piboserod,prucalopride, RS67333, TD8954 and PF04995274 dose-dependently decreased the concentrations of SB207145 inthe rat striatum compared with the vehicle-treated group.Plot of percentage 5-HT4R occupancy versus dose levelsproduced a dose–response occupancy curve for GR113808,piboserod, prucalopride, RS67333, TD8954 andPF04995274 (Figure 4). The calculated relative ED50 (mg/kg) value for each curve is listed in Table 3. The sigmoidaldose–response curve revealed that PF-04995274 was themost potent among all tested compounds, and required a0.02 mg/kg dose to occupy 50% of the 5-HT4Rs (Table 3). Adose-dependent increase in striatum 5-HT4R occupancyand brain and plasma concentration was observed forGR113808, piboserod, prucalopride, RS67333, TD8954 andPF04995274 (Figures 4 and 5). GR113808, RS67333 andPF04995274 were demonstrated to have 1.5- to 3-foldhigher brain concentrations compared with plasma.However, the brain concentrations of piboserod, prucalo-pride and TD8954 were low when compared with plasmaconcentrations (cb/cp < 1). The calculated EC50 (plasma orbrain concentration at 50% occupancy) for each compoundis shown in Table 3.

Discussion

5-HT4Rs are widely distributed in the CNS. 5-HT4R activa-tion regulates the release of ACh and secretion of sAPPa inpreclinical animal studies. 5-HT4R agonists play a potentialrole in the symptomatic and disease-modifying treatmentof cognitive disorders and have received considerable inter-est in current CNS drug discovery programmes. The mainobjective of this investigation was to accelerate the drug

screening step by developing a simple, rapid and reliablemethod for determining the 5-HT4 in-vivo receptor occu-pancy in rat brain. In this study we have demonstrated forthe first time use of non-radiolabelled SB207145 for meas-uring receptor occupancy of 5-HT4R ligands in rat brain.

Many of the 5-HT4 ligands were evaluated as positronemission tomography or single emission computed tomo-graphy tracers in human studies. SB207145 is a selectivepotent 5-HT4R antagonist with suitable kinetics for quanti-fication of the 5-HT4R and potential ease of 11C label-ling.[38,41,42] [11C]SB207145 has been used for brainmolecular imaging of the 5-HT4R with PET in Gottingenminipigs and humans.[38–40] PET studies in Gottingen mini-pigs, rats and humans showed that the radiotracer readilyentered the brain, giving rise to a heterogeneous distribu-tion consistent with the known regional pattern of 5-HT4Rexpression.

[29,38–40] 11C-SB207145 is currently the only PETradioligand available for imaging the 5-HT4R in vivo in theCNS of humans. These properties encouraged us to selectSB207145 as a tracer for assessing the 5-HT4R occupancy.

Preliminary study of rat in-vivo brain regional distribu-tion showed that SB207145 underwent substantial uptake inthe brain. SB207145 had shown higher levels in the brainregions expressing 5-HT4R sites and lower levels for otherreceptor sites, thus demonstrating the good selectivity ofSB207145 for the 5-HT4R. A high level of specific binding(3–4 fold) was observed in the striatum in comparison withthe cerebellum. An appreciable total to nonspecific (stria-tum to the cerebellum) concentration ratio was observedfor SB207145 (ª3.5), and rapid elimination was observed in

0.1 1 10 100–25

0

25

50

75

100

125 GR113808, p.o. Piboserod, p.o. Prucalopride, p.o.TD8954, p.o.RS67333, p.o. PF04995274, s.c.

*

**

Dose (mg/kg)

% 5

-HT 4

rec

epto

r o

ccu

pan

cy

0.001 0.01 0.1 1 100

25

50

75

100

**

**** ** ** **

Figure 4 In-vivo 5-HT4R occupancy (%) in the rat striatum afteradministration of GR113808, piboserod, prucalopride, RS67333 andTD8954 at 1 h post dose (mean � SEM, n = 4/group). Inset graph rep-resents the in-vivo 5-HT4R occupancy of PF04995274 *P < 0.05 and**P < 0.01, compared with vehicle control values.

0.1 1 10 100 1000 10000–25

0

25

50

75

100

125

150

PF04995274, s.c.

RS67333, p.o.

GR113808, p.o.

Piboserod, p.o.

Prucalopride, p.o.

TD8954, p.o.

Plasma (ng/ml)

% 5

-HT 4

rec

epto

r o

ccu

pan

cy

1 10 100 1000–25

0

25

50

75

100

125

Brain (ng/g)

Figure 5 In-vivo 5-HT4R occupancy (%) versus 5-HT4R ligands levels inthe plasma (ng/ml) and brain (ng/g). The x-axis represents the plasma(ng/ml) and brain concentration (ng/g, inset) at 90 min post dose. They-axis represents the percentage 5-HT4R occupancy in the rat striatum(mean � SEM, n = 4/group). Inset graph represents the occupancyversus brain exposure of 5-HT4R ligands.



the cerebellum. The regional distribution pattern ofSB207145 in the rat brain reflected the known distributionof the 5-HT4R and is consistent with previous reportsfor binding to [11C]SB207145 in Gottingen minipigs andhumans.[38–41] Based on this preliminary outcome, the stria-tum (high density of 5-HT4R) and cerebellum (low densityof 5-HT4R) were chosen for further studies. The SB207145dose was selected based on the cerebellar concentration,which could be reliably quantified using LC-MS/MS. Anadequate survival interval is required for the tracer to enterinto the brain, bind to the target and unbound tracer toeliminate from the brain. To determine the proper tracertreatment time, a time-course brain distribution study wasperformed in rats. The maximal signal-to-noise ratio wasobserved at 30 min post dose. At 30–40 min SB207145exhibited lower cerebellar concentration levels when com-pared with the striatum.

Dose-dependent 5-HT4R occupancy was observed forGR113808, piboserod, prucalopride, RS67333, TD8954 andPF04995274. All tested exogenous 5-HT4 ligands dose-dependently decreased SB207145 5-HT4R binding in the

striatum without affecting the cerebellar concentration,which presents as an area with poor 5-HT4R density(Table 2). Hence the concentration found in the cerebellumwas considered to result from nonspecific binding ofSB207145 and it may not affect the absolute receptoroccupancy. The in-vitro affinity profile revealed thatPF04995274, piboserod, TD8954 and GR113808 were morepotent than prucalopride and RS67333, which is consistentwith calculated ED50 values for the respective compounds(Table 3). Receptor occupancy by PF04995274 and pibose-rod was saturated at higher dose levels. Prucalopride wasfound to be less potent in vivo as compared withPF04995274 and piboserod, which is consistent with brainand plasma exposure versus percentage 5-HT4R occupancy.The calculated ED50 values for 5-HT4R occupancy in thestriatum were found to be 4.75, 1.99, 7.43, 9.86, 3.77 and0.02 for GR113808, piboserod, prucalopride, RS67333,TD8954 and PF04995274, respectively (Table 3). The ED50

values of prucalopride and PF-04995274 were comparablewith reported ED50 values of 0.01 and 8 mg/kg, respectivelyusing radiolabelled tracer.[29,43] GR113808, RS67333 andPF04995274 demonstrated better brain penetration pro-perties compared with TD8954. All six 5-HT4R ligandsshowed a dose-dependent increase in the brain and plasmaconcentration.

Conclusions

In conclusion, the current LC-MS/MS-based method devel-oped in the brain homogenate using SB207145 had an ana-lytical sensitivity of 0.1 ng/g (with a signal-to-noise ratio of�10) and could detect the lowest tracer levels observed inthe cerebellum, which displays a low density of 5-HT4Rs.The non-radiolabelled SB207145 was successfully employedfor assessing the in-vivo receptor occupancy for 5-HT4Rsin the rat by using an LC-MS/MS-based method. Thetechnique presented in this article followed a robustand methodical approach in characterizing a potent non-radiolabelled ligand into a tracer, which could potentially be

Table 2 SB207145 concentrations in the striatum and cerebellumfrom rats treated with PF04995274 or vehicle

Dose group(mg/kg)

SB207145 brain regionalconcentration (ng/g)

% 5HT4

receptoroccupancyCerebellum Striatum

Vehicle 0.36 � 0.08 1.41 � 0.15 0.00 � 9.26PF04995274 (s.c.)

0.003 0.38 � 0.05 1.34 � 0.12 12.91 � 6.410.01 0.39 � 0.02 1.16 � 0.09** 31.94 � 5.580.03 0.38 � 0.01 0.85 � 0.08** 57.50 � 4.590.1 0.38 � 0.03 0.76 � 0.15** 66.05 � 3.340.3 0.39 � 0.01 0.65 � 0.05** 77.63 � 1.611.0 0.37 � 0.04 0.57 � 0.07** 80.79 � 3.353.0 0.38 � 0.03 0.51 � 0.13** 87.83 � 1.25

Data are presented as means � SEM, n = 4/group. *P < 0.05 and**P < 0.01, compared with vehicle control values.

Table 3 In-vitro and in-vivo potency of 5-HT4 ligands

Compound5-HT4,hKi (nM)f

% Receptoroccupancy(ED50, mg/kg)

Plasmaconcentration(EC50, nM)

Brainconcentration(EC50, nM)

Brain/plasmaconcentrationratio

GR113808 0.53a 4.8 � 0.5 101 � 12 126 � 24 1.4 � 0.1Piboserod 0.10c 2.0 � 0.2 135 � 16 32 � 8 0.2 � 0.01Prucalopride 25.10d 7.4 � 0.9 407 � 28 176 � 22 0.4 � 0.02RS67333 6.50b 9.9 � 0.7 14 � 3 21 � 3 1.1 � 0.1TD8954 0.40d 3.8 � 0.3 935 � 79 107 � 9 0.1 � 0.01PF04995274 0.30e 0.02 � 0.004 3 � 1 5 � 1 2.0 � 0.2

hKi, affinity for human 5-HT4Rs, ED50, effective dose required to achieve 50% receptor occupancy, EC50, effective concentration required to achieve50% receptor occupancy. Data are presented as means � SEM, n = 4/group. aData from[44]. bData from[45]. cData from[46]. dData from[47]. eDatafrom[43]. fValues are means.



employed for assessing 5-HT4R ligands in the preclinicalstage. This simple and rapid method allows the generationof occupancy curves for a wide range of compounds at mul-tiple dose levels along with the brain and plasma concentra-tion curves from the same set of animals. Thus, the processof candidate selection during the early stage of drug devel-opment can be accelerated using this reliable method.

Declarations

Conflict of interest

All the research work was carried out in Pharmacoki-netics and Drug Metabolism laboratories, Discovery

research; all the authors are employees of Suven LifeSciences Ltd.

Funding

This research received no specific grants from any fundingagency in the public, commercial or not-for profit sectors.

Acknowledgement

The authors acknowledge the support received from MrVenkateswarlu Jasti, CEO, Suven Life Sciences Ltd, Hydera-bad, India.

References

1. Bockaert J et al. Pharmacologicalcharacterization of 5-hydroxytryptamine4 (5-HT4) recep-tors positively coupled to adenylatecyclase in adult guinea-pig hippocam-pal membranes: effect of substitutedbenzamide derivatives. Mol Pharmacol1990; 37: 408–411.

2. Eglen RM et al. Central 5-HT4 recep-tors. Trends Pharmacol Sci 1995; 16:391–398.

3. Bonaventure P et al. Mapping of sero-tonin 5-HT4 receptor mRNA andligand binding sites in the postmortem human brain. Synapse 2000;36: 35–46.

4. Domenech T et al. Identification andcharacterization of serotonin 5-HT4receptor binding sites in human brain:comparison with other mammalianspecies. Brain Res Mol Brain Res 1994;21: 176–180.

5. Waeber C et al. [3H]-GR113808 labels5-HT4 receptors in the human andguinea-pig brain. Neuroreport 1993; 4:1239–1242.

6. Medhurst AD et al. QuantitativemRNA analysis of five C-terminalsplice variants of the human 5-HT4receptor in the central nervous systemby TaqMan real time RT-PCR. MolBrain Res 2001; 90: 125–134.

7. Dumuis A et al. A nonclassical5-hydroxytryptamine receptor posi-tively coupled with adenylate cyclasein the central nervous system. MolPharmacol 1988; 34: 880–887.

8. Markstein R et al. Pharmacologicalcharacterisation of 5-HT receptorspositively coupled to adenylyl cyclasein the rat hippocampus. NaunynSchmiedebergs Arch Pharmacol 1999;359: 454–459.

9. Torres GE et al. Cyclic AMP andprotein kinase A mediate 5-hydroxytryptamine type 4 receptorregulation of calcium-activated potas-sium current in adult hippocampalneurons. Mol Pharmacol 1995; 47:191–197.

10. Ullmer C et al. Distribution of 5-HT4receptor mRNA in the rat brain.Naunyn Schmiedebergs Arch Pharma-col 1996; 354: 210–212.

11. Kilbinger H, Wolf D. Effect of 5-HT4receptor stimulation on basal andelectrically evoked release of acetyl-choline from guinea pig myentericplexus. Naunyn-Schmiedeberg’s ArchPharmacol 1992; 345: 270–275.

12. Matsuyama S et al. Identification ofputative 5-hydroxytryptamine 4receptors in guinea pig stomach: theeffect of TSK159, a novel agonist, ongastric motility and acetylcholinerelease. J Pharmacol Exp Ther 1996;276: 989–995.

13. Takada K et al. Regional difference incorrelation of 5-HT4 receptor distri-bution with cholinergic transmissionin the guinea pig stomach. Eur J Phar-macol 1999; 374: 489–494.

14. Consolo S et al. 5-HT4 receptorstimulation facilitates acetylcholinerelease in rat frontal cortex. Neurore-port 1994; 5: 1230–1232.

15. Mohler EG et al. VRX-03011, a novel5-HT4 agonist, enhances memoryand hippocampal acetylcholine efflux.Neuropharmacology 2007; 53: 563–573.

16. Fontana DJ et al. The effects ofnovel, selective 5-hydroxytryptamine(5-HT)4 receptor ligands in rat spatialnavigation. Neuropharmacology 1997;36: 689–696.

17. Lamirault L, Simon H. Enhancementof place and object recognitionmemory in young adult and old ratsby RS 67333, a partial agonist of5-HT4 receptors. Neuropharmacology2001; 41: 844–853.

18. Moser PC et al. SL65.0155, a novel5-hydroxytryptamine(4) receptorpartial agonist with potent cognitio-nenhancing properties. J PharmacolExp Ther 2002; 302: 731–741.

19. Yamaguchi T et al. Evidence for 5-HT4receptor involvement in the enhance-ment of acetylcholine release byp-chloroamphetamine in rat frontalcortex. Brain Res 1997a; 772: 95–101.

20. Yamaguchi T et al. Facilitation of ace-tylcholine release in rat frontal cortexby indeloxazine hydrochloride:involvement of endogenous serotoninand 5-HT4 receptors. NaunynSchmiedebergs Arch Pharmacol 1997b;356: 712–720.

21. Reynolds GP et al. 5-Hydroxytryptamine (5-HT)4 recep-tors in post mortem human braintissue: distribution, pharmacology andeffects of neurodegenerative diseases.Br J Pharmacol 1995; 114: 993–998.



22. Cachard-Chastel M et al. Prucaloprideand donepezil act synergisticallyto reverse scopolamine- inducedmemory deficit in C57Bl/6j mice.Behav Brain Res 2008; 187: 455–461.

23. Fontana DJ et al. Stereoselective effectsof (R)- and (S)-zacopride on cognitiveperformance in a spatial navigationtask in rats. Neuropharmacology 1996;35: 321–327.

24. Galeotti N et al. Role of 5-HT4 recep-tors in the mouse passive avoidancetest. J Pharmacol Exp Ther 1998; 286:1115–1121.

25. Cachard-Chastel M et al. 5-HT4receptor agonists increase sAPPalphalevels in the cortex and hippocampusof male C57BL/6j mice. Br J Pharma-col 2007; 150: 883–892.

26. Lezoualc’h F, Robert SJ. The serotonin5-HT4 receptor and the amyloid pre-cursor protein processing. Exp Geron-tol 2003; 38: 159–166.

27. Robert SJ et al. The human serotonin5-HT4 receptor regulates secretion ofnon-amyloidogenic precursor protein.J Biol Chem 2001; 276: 44881–44888.

28. Turner PR et al. Roles of amyloid pre-cursor protein and its fragments inregulating neural activity, plasticityand memory. Prog Neurobiol 2003; 70:1–32.

29. Johnson DE et al. The 5-hydroxytryptamine 4 receptor ago-nists prucalopride and PRX-03140increase acetylcholine and histaminelevels in the rat prefrontal cortex andthe power of stimulated hippocampalq oscillations. JPET 2012; 341: 681–691.

30. Shen F et al. 5-HT4 receptor agonistmediated enhancement of cognitivefunction in vivo and amyloid precur-sor protein processing in vitro: a phar-macodynamic and pharmacokineticassessment. Neuropharmacology 2011;61: 69–79.

31. Barth VN et al. Comparison of ratdopamine D2 receptor occupancy fora series of antipsychotic drugs meas-ured using radio labeled or nonlabeled

raclopride tracer. Life Sci 2006; 78:3007–3012.

32. Chernet E et al. Use of LC/MS toassess brain tracer distribution in pre-clinical, in vivo receptor occupancystudies: dopamine D2, serotonin2Aand NK-1 receptors as examples. LifeSci 2005; 78: 340–346.

33. Need AB et al. In vivo rat brain opioidreceptor binding of LY255582 assessedwith a novel method using LC/MS/MSand the administration of threetracers simultaneously. Life Sci 2007;81: 1389–1396.

34. Nirogi R et al. In vivo receptor occu-pancy assay of histamine H3 receptorantagonist in rats using non-radiolabeled tracer. J PharmacolToxicol Methods 2012; 65: 115–121.

35. Nirogi R et al. Rat thalamic a4b2 neu-ronal nicotinic acetylcholine receptoroccupancy assay using LC–MS/MS.J Pharmacol Toxicol Methods 2012; 65:136–141.

36. Pike VW et al. Synthesis and Evalua-tion of Radioligands for ImagingBrain Nociceptin/Orphanin FQPeptide (NOP) Receptors with Posi-tron Emission Tomography. J MedChem 2011; 54: 2687–2700.

37. Mitch CH. Discovery of aminobenzy-loxyarylamides as K opioid receptorselective antagonists: application topreclinical development of a K opioidreceptor antagonist receptor occu-pancy tracer. J Med Chem 2011; 54:8000–8012.

38. Gee AD et al. Synthesis and evaluationof [11C]SB207145 as the first in vivoserotonin 5-HT4 receptor radioligandfor PET imaging in man. Curr Radiop-harm 2008; 1: 110–114.

39. Kornum BR et al. Evaluation of thenovel 5-HT4 receptor PET ligand[11C]SB207145 in the Gottingenminipig. J Cereb Blood Flow Metab2009; 29: 186–196.

40. Madsen K et al. Mass dose effectsand in vivo affinity in brain PETreceptor studies a study of cerebral5-HT4 receptor binding with

[11C]SB207145. Nucl Med Biol 2011;38: 1085–1091.

41. Marner L et al. Kinetic modeling of11C-SB207145 binding to 5-HT4receptors in the human brain in vivo.J Nucl Med 2009; 50: 900–908.

42. Marner L et al. Brain imaging of sero-tonin 4 receptors in humans with[11C] SB207145-PET. Neuroimage2010; 50: 855–861.

43. Grimwood S et al. Translational recep-tor occupancy for the 5-HT4 partialagonist PF-04995274 in rats, non-human primates and healthy volun-teers. Alzheimers Dement 2011; 7(4Suppl.) S653.

44. Curtet S et al. New arylpiperazinederivatives as antagonists of thehuman cloned 5-HT4 receptor iso-forms. J Med Chem 2000; 43: 3761–3769.

45. Mialet J et al. Pharmacological charac-terization of the human 5-HT4(d)receptor splice variant stably expressedin Chinese hamster ovary cells. Br JPharmacol 2000; 131: 827–835.

46. Wardle KA et al. Selective and func-tional 5-hydroxytryptamine4 receptorantagonism by SB 207266. Br J Phar-macol 1996; 118: 665–670.

47. Beattie D et al. The pharmacology ofTD-8954, a potent and selective5-HT4 receptor agonist with gastroin-testinal prokinetic properties. FrontPharmacol 2011; 2: 25.

Supporting InformationAdditional Supporting Information maybe found in the online version of thisarticle:

Table S1 Chromatography and analyticalmethod details for GR113808, piboserod,prucalopride, RS67333, TD8954 andPF04995274 used in brain and plasmaexposure measurement.Fig. S1 Full-scan positive ion turboIon-spray product ion mass spectra and theproposed patterns of fragmentation ofSB207145.



in-vivo rat striatal 5-ht 4 receptor occupancy using non-radiolabelled...

Documents