mapping the cerebral monoamine oxidase type a : positron...

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Mapping the Cerebral Monoamine Oxidase Type A : Positron

Emission Tomography Characterisation of the Reversible Selective

Inhibitor [11C]Befloxatone.

Michel BOTTLAENDER, Frédéric DOLLÉ, Ilonka GUENTHER, Dimitri ROUMENOV,

Chantal FUSEAU, Yann BRAMOULLE, Olivier CURET#, Jamir JEGHAM#,

Jean-Louis PINQUIER#, Pascal GEORGE#, Heric VALETTE.

CEA, Service Hospitalier Frédéric Joliot, DSV/DRM, 4, place du général Leclerc 91406 ORSAY, France. #Sanofi-Synthelabo Recherche, 92225 BAGNEUX, France

Copyright 2003 by the American Society for Pharmacology and Experimental Therapeutics.

JPET Fast Forward. Published on February 11, 2003 as DOI:10.1124/jpet.102.046953This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on February 11, 2003 as DOI: 10.1124/jpet.102.046953

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Running title: Befloxatone in vivo by PET Address for correspondence:

Michel Bottlaender

4, place du général Leclerc

91401 ORSAY, France

Tel : 33 1 69 86 77 12

Fax : 33 1 69 86 77 49

email : [email protected]

Text pages : 21 pages (including abstract and references)

Tables : 2

Figures :6

References : 38

Abstract : 236 words

Introduction : 624 words

Discussion : 1220 words

Neuropharmacology section.

Abbreviations:

PET: Positron Emission Tomography; MAO: Monoamine Oxidase; MAOI: Monoamine

Oxidase Inhibitor; MRI: Magnetic Resonance Imaging; ROI: Region of Interest; DV:

Distribution Volume; ID: Injected dose; TAC: time activity curve.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on February 11, 2003 as DOI: 10.1124/jpet.102.046953

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ABSTRACT

Befloxatone is a competitive and reversible inhibitor of MAO-A (MAOI-A). The aim of

the study was to characterize the in vivo properties of [11C]befloxatone, and to validate its

use as a ligand for the study of MAO-A by positron emission tomography (PET).

PET studies were performed in baboons after i.v. injection of [11C] befloxatone (551 ±

70 MBq, i.e.14.9 ± 1.9 mCi). [11C]befloxatone enters rapidly in the brain with a maximum

uptake at 30 min. Brain concentration of the tracer is high in thalamus, striatum, pons and

cortical structures (1.5-1.8 % of injected dose per 100 ml of tissue) and lower in

cerebellum (1.07 % ID/100ml). Non saturable uptake, obtained after a pre-treatment with a

high dose of non-labelled befloxatone (0.4 mg/kg), is very low and represents only 3% of

the total uptake. Brain uptake of [11C]befloxatone is not altered by a pre-treatment of a

high dose with lazabemide (0.5 mg/kg i.v.), a selective MAOI-B, but is completely

blocked by a pre-treatment with moclobemide (MAOI-A, 10 mg/kg). This confirms, in

vivo, the selectivity of befloxatone for type A MAO. [11C]befloxatone brain radioactivity

was displaced by administration of unlabelled befloxatone (30 min after the tracer

injection). The displacement of the tracer from its binding sites is dose dependent with an

ID50 of 0.02 mg/kg for all studied structures.

These results indicate that [11C]befloxatone will be an excellent probe for the study of

MAO-A in humans using PET.

Key Words : Monoamine oxidase; Positron Emission Tomography; in vivo; befloxatone;

monkey, pharmacology.


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Monoamine oxidases (EC 1.4.3.4.; MAO) are flavoproteins integrated to outer

mitochondrial membranes. They oxidatively deaminate neurotransmitters and xenobiotic

amines. The enzyme exists in two isoforms, MAO-A and MAO-B which differ in primary

structure, substrate specificity and inhibitor sensitivity. In the brain MAO-A oxidises mainly

serotonin, norepinephrine and dopamine and is found primarily in catecholaminergic

neurones, whereas MAO-B oxidises phenethylamine and dopamine and is mainly localised in

serotoninergic neurones and glial cells. Both forms are important for regulation of

monoaminergic transmission. Fluctuation in functional MAO activity may be associated with

human diseases such as Parkinson’s and Alzheimer’s diseases, depression and certain

psychiatric disorders (Brunner et al., 1993). Moreover, it was shown that heavy smokers

possess lower brain MAO-A and MAO-B levels (Fowler et al., 1996a; Fowler et al., 1996b)

and that their inhibition facilitates smoking cessation (Berlin et al., 1995).

Positron Emission Tomography (PET) is a non invasive imaging technique allowing the

study of metabolism, receptors and, in the present case, enzymes in living human brain

(Fowler et al., 1987a; Pappata et al., 1996). Few radiotracers have been developed for MAO-

A PET studies such as [11C]clorgyline and [11C]harmine (Fowler et al, 1987a; 1996b; 2000;

Bergström et al., 1997a; Bergström et al., 1997b; Bergström et al., 1997c). However, both

tracers present some drawbacks. A recent study show that [11C]clorgyline present an

unexplained species difference in that [11C]clorgyline was not retained in baboon brain in

contrast to results in human (Fowler et al., 2001). [11C]harmine is extensively metabolised in

plasma therefore the input function determination (unchanged compound) is subject to large

errors (Bergström et al. 1997a). Brofaromine was the first reversible MAO-A inhibitor

labelled for PET (with 11C), but the brain uptake could not be significantly reduced by a pre-

treatment with either irreversible (clorgyline) or reversible (moclobemide) MAO-A inhibitors

(Ametamey et al., 1996).


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Befloxatone, an oxazolidinone derivative, is a selective MAO-A reversible inhibitor with

potential antidepressant properties (Curet et al., 1994; Rovei et al., 1994; Wouters et al.,

1999). The biochemical and pharmacological profiles of befloxatone were extensively studied

in rats and in human tissues (Curet et al., 1996;Caille et al., 1996). These studies revealed

that befloxatone is a selective, competitive, specific and reversible MAO-A inhibitor in rat as

well in human tissues. Befloxatone is very potent at inhibiting in vitro MAO-A activity (Ki =

2 nM vs 150 nM for MAO-B), more potent than the other reversible MAO-A inhibitors

(including moclobemide, brofaromine, harmaline). The specificity of befloxatone for MAO-A

was confirmed against other neurotransmitter and transporter systems. In vivo, befloxatone

increases brain levels of norepinephrine, dopamine and serotonin and decreases the levels of

corresponding deaminated metabolites (dihydroxyphenylacetic acid and 5-hydroxyindolacetic

acid (Curet et al., 1996). Befloxatone is much more potent (10 to 500 fold) than other

reversible or irreversible MAO-A inhibitors in classical anti-depressant tests in rodents

(Caille et al., 1996). Befloxatone displays a higher activity in test involving MAO-A (with

ED50 of about 0.1 to 0.2 mg/kg p.o. or 0.07 mg/kg i.v.) than in test involving MAO-B (ED50

of about 58 mg/kg p.o.) (Caille et al., 1996).

Due to its selective and reversible MAO-A inhibition, befloxatone possesses a good safety

profile particularly regarding the observation of tyramine induced pressor effect and the drug

interactions (Caille et al., 1996; Rosenzweig et al., 1998).

All the biochemical and pharmacological characteristics of the drug suggest that

befloxatone is an excellent candidate for the PET study of the MAO-A in human. Moreover,

the structure of the molecule allows to label it with carbon-11, a positron emitter isotope,

using phosgene.


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This work aimed to characterise the properties of the drug in living brain and to determine

if [11C]befloxatone is a suitable radioligand to study the MAO-A by PET in vivo. This

preclinical study was performed in non-human primate using PET.


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METHODS

Animals

All animal use procedures were in strict accordance with the recommendations of the EEC

(86/609/CEE) and the French National Committee (décret 87/848) for the care and use of

laboratory animals. Twelve PET experiments were carried out on 4 male Papio anubis

baboons (mean weight 14.4 ± 5 kg). Each animal was allowed to rest for a period of at least 2

weeks between PET studies.

Drugs

Befloxatone ((5R)-5-(methoxymethyl)-3-[4-[(3R)-4,4,4-trifluoro-3-

hydroxybutoxy]phenyl] -2-oxazolidinone), moclobemide and lazabemide were provided by

Synthélabo, Bagneux, France. All drugs were dissolved in a mixture of physiological saline /

EtOH / propanediol (98 / 2 / 2, v:v:v) and were injected intravenously.

Befloxatone was labelled with carbon-11 (t1/2 : 20.4 minutes) using [11C]phosgene

(Landais and Crouzel 1987; Link et al., 1997) and the corresponding ring-opened precursor

(R)-1-methoxy-3-[[4-[(3R)-4,4,4-trifluoro-3-hydroxybutoxy]phenyl]amino]-2-propanol

(provided by Synthélabo, Bagneux, France). Details of the radiosynthesis have been

published elsewhere (Dolle et al., 1999; 2003). Typically, 5.55-9.25 GBq (150 to 250 mCi )

of [11C]Befloxatone with a radiochemical- and chemical purity of more than 99% were

routinely obtained within 25 minutes of radiosynthesis (including HPLC purification). Final

formulation as an i.v. injectable pharmaceutical was performed as follows : (1) HPLC

solvent removal by evaporation ; (2) taking up the residue in 5 ml of physiological saline ;

(3) filtration on a 0.22 µm Millipore filter.


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Magnetic Resonance Imaging

A magnetic resonance imaging (MRI) examination was performed for each baboon in

order to provide detailed anatomical images corresponding to the PET slices. The

examinations were performed with a 1.5 Tesla SIGNA system (General Electric, Milwaukee,

WI) and a custom-made receive-only coil was used in proximity to the baboon’s head to

provide a high sensitivity. The animal was anaesthetised by i.m. injection of a mixture of

ketamine (15 mg/kg, Ketalar, Park-Davis, France) and xylazine (1.5 mg/kg, Rompun, Bayer,

France) (Banknieder et al., 1978) and positioned using a stereotaxic headholder so that the

stereotaxic reference plane was aligned with a laser beam figuring the axial reference plane of

the MR scanner. The imaging protocol used a T1-weighted inversion-recovery sequence in 3D

mode and a 256x192 matrix over 124 slices of 1.5 mm in thickness.

Positron Emission Tomography data acquisition

PET studies were performed with a high resolution tomograph (ECAT 953B/31; CTI PET

system, Knoxville, TN), which allowed reconstruction of 31 slices every 3.3 mm with spatial

and axial resolution of 5.7 mm and 5.0 mm, respectively (Bendriem, 1991). Animals were

anaesthetised using isoflurane/nitrous-oxide mixture (1%/66%), controlled by an Ohmeda

ventilator (Ohmeda OAV 7710, Madison, WI) with 33% oxygen. The tidal volume was

adjusted to achieve stable end-tidal carbon dioxide tension between 38-40 mmHg. The

baboon’s head was fixed in a headholder and positioned in the scanner gantry for axial plane

acquisition. A transmission scan (68Ge rods, 15 minutes) was recorded to correct for γ-ray

attenuation.

The image acquisition started (T0 min) at the intravenous injection of [11C]befloxatone

(551 ± 70 MBq, i.e.14.9 ± 1.9 mCi; specific radioactivity : 12.5 ± 3.3 GBq/µmol, i.e. 339 ±

89 mCi/µmol) and lasted 120 min (T120 min). Thirty-three images were acquired with scan


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duration starting from 30 sec and increasing up to 10 min during the experiment.Three types

of PET experiments were designed. Control experiments were performed to define the

cerebral time activity curves (TAC) of [11C]befloxatone (n=2). Saturation experiments, in

which all MAO-A or MAO-B sites were saturated, were performed by administration of a

treatment dose of appropriate cold drug before the tracer injection. MAO-A sites were

saturated by befloxatone (0.4 mg/kg; n=2) or moclobemide (10 mg/kg; n=1), MAO-B sites

were saturated by lazabemide (0.5 mg/kg; n=1). Displacement experiments were performed

by i.v. administration, 30 min after the tracer injection (T30 min), of a dose of unlabelled

befloxatone. Six displacement experiments were performed with increasing doses of

befloxatone (0.004, 0.02, 0.04, 0.04, 0.1, 0.4 mg/kg i.v.).

Input function and metabolite studies

During each PET acquisition, arterial blood samples (n=30) were withdrawn from a

femoral artery at designated times. Blood and plasma radioactivity were measured in a γ-

counter and the blood-plasma time-activity curves were corrected for [11C] decay from the

beginning of the PET acquisition (T0 min).

In the control experiments, 8 plasma samples were collected (1, 2, 7, 10, 15, 20, 22.5, 27.5

min after tracer injection) for metabolites analysis, using HPLC. After deproteinisation using

acetonitrile the samples were centrifuged and the supernatant removed and used directly for

the chromatographic evaluation. The data acquisition and analysis were carried out using

Winflow software (version 1.21, JMBS Developments, Grenoble, France). The column

(reverse phase Waters µ Bondapak C18 column, 300 x 7.8 mm, 10 µm; Waters, Milford,

MA) was eluted applying a gradient from 20% acetonitrile in 0.01 M phosphoric acid up to

80% in 5.5 minutes, up to 90% in 7.5 minutes and back to 20% at 7.6 minutes with a total

run length of 10 minutes. The flow rate of the eluent as well as the flow rate of the liquid


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scintillator was maintained at 6 ml/min. Under this conditions befloxatone elutes with a

retention time of 6 minutes while desmethylbefloxatone elutes before with a retention time of

5.3 minutes.

PET data analysis

Regions of interest (ROI) for anatomical structures such as cortex, cerebellum, pons,

thalamus and striata were delineated on MRI images of each baboon and then transferred to

the PET studies of the corresponding animal. The concentration of radioactivity in each ROI

was determined on each sequential scan and expressed as percent of the injected dose per 100

ml of tissue (% ID / 100 ml). Distribution volumes for individual ROI (DV) were calculated

using the graphic analysis developed by Logan et al.(1990) for reversible tracers. This

computation required the uptake PET data from a ROI versus time and and input function

which is the unchanged tracer concentration in arterial plasma.

As the plasma input function depends on the amount of befloxatone administered (see

Results section), the ratio [brain TACs] to [plasma AUC0→120 min] were calculated to allow

comparison between PET experiments.

Brain radioactivity in control experiments correspond to [11C]befloxatone total uptake.

Values from saturation experiments with pre-treatment of high amount of unlabelled

befloxatone represent [11C]befloxatone non-specific uptake. The [11C]befloxatone regional

specific uptake was estimated by subtracting the regional non-specific uptake from the

regional total uptake (Pappata et al., 1988; Persson et al., 1988; Brouillet et al., 1990).

In our experiments, the injected tracer mass was not higher than 61 nmol (47 ± 14 nmol,

n=12), and the tracer uptake in the brain was always less than 0.02 %ID/ml of tissue

(maximal 0.0179 %ID/ml in putamen, see Table I). As, in vitro, MAO-A Bmax is about 400 to

700 pmol/ml of brain tissue in human and monkey (Saura et al., 1992; May et al., 1991), the


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[11C]befloxatone occupied always less than 5% of the MAO-A binding sites. This allows the

11C-labelled tracer to act as a true tracer. Therefore, the percent of [11C]befloxatone

displacement can be considered to represent an index of the MAO-A sites occupied by

befloxatone.

In displacement experiment, the percent [11C]befloxatone displacement was calculated at

T95 min, when the equilibrium between brain and plasma is reached (ratio brain/plasma

radioactivities is constant), using the following equation:

Displacement (%) = 100 × (BC - BD) / BC

where BC represents the specific uptake in control experiments (as defined above). BD is

the specific uptake after displacement, calculated by subtracting the regional non-specific

uptake from the regional residual uptake in displacement experiment.

The logdose-displacement curves were fitted using the logistic model (Microcalc

Origin). The results are expressed as mean ± standard deviation.


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RESULTS

Blood and plasma time activity curves: After [11C]befloxatone a rapid phase of

distribution in blood (about 5 min), the decline in radiotracer plasma concentration was fitted

by a monoexponential function (Intercept = 0. 14 % ID / 100 ml plasma, T½ = 56.8 min in

control experiment and Intercept = 0.83 % ID / 100 ml plasma, T½ = 86.6 min in saturation

experiments). As shown in Figure 1, the [11C]befloxatone plasma TACs showed a marked

difference between control experiments and saturation experiments (pre-injection of high

amount of unlabelled befloxatone). After the distribution phase, the plasma radioactive

concentration was 8-fold higher in the saturation experiments than in the control experiments

(0.6 ± 0.2 vs 0.07 ± 0.03 % ID / 100 ml respectively at T30 min). Similarly, in displacement

experiments, the administration of unlabelled befloxatone at T30 min induced an increase of

radiotracer plasma concentration. In these experiments, the plasma radioactive level,

measured at 90 min (one hour after cold drug administration), was correlated with the amount

of injected cold drug (r=0.994; p<0.001).

In all experiments (n=12), the blood to plasma concentration ratio was constant during the

two hours of the experiments (0.9 ± 0.06) consistent with a similar kinetic of

[11C]befloxatone in both blood and plasma.

Metabolite analysis: [11C]befloxatone was extracted from plasma with an extraction

efficiency of 96%. After tracer injection, [11C]befloxatone was relatively stable in vivo, since

78% of the radioactivity in plasma at T30 min represented unchanged befloxatone (inset in

Figure 1). The detectable metabolite has a shorter retention time (2.1 min) indicating a more

hydrophilic compound. No peak was detectable at the retention time of the

desmethylbefloxatone (5.3 min).

Cerebral distribution of [11C]befloxatone : Tomographic images obtained after i.v.

[11C]befloxatone display an heterogeneous distribution of radioactivity (Figure 2). A high


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uptake was found in basal ganglia, thalamus, pons and cortical structures, while cerebellum

and white matter present lower uptake (Table I). Similar results were found using the

calculated DV in the different structures : high DV values (19-23 mL/g) in he basal ganglia,

thalamus and cortex and low values (10-11 mL/g) in cerebellum and white matter (Table I).

The DV values in the different brain structures are strongly correlated with the uptake values

obtained directly from the PET image (30 min after tracer injection) (r = 0.83, p<0.001).

Cerebral time activity curves : After i.v. [11C]befloxatone, radioactivity was detected early

in the brain (in the first 30-sec PET image), and increased rapidly. In brain structures with a

high uptake, the radioactivity reached maximal values at 30 min (Table I), and then decreased

slowly until the end of the experiments (Figure 3). The tracer TACs was slightly different in

structures with lower uptake such as cerebellum and white matter. In these structures the

maximal uptake was reached in less than 5 min and the washout was faster (Figure 3).

Saturability of [11C]befloxatone brain uptake : Pre-injection of unlabelled befloxatone

(0.4 mg/kg) before the radiotracer prevents the tracer from accumulating in the brain (Figure

4). The radioactive concentrations were very low and identical in all structures (0.25 ± 0.035

% DI/100ml). After correction of the plasma input function, the non-specific uptake

represents less than 5 % (from T45 min) of the total uptake.

Selectivity of [11C]befloxatone brain uptake : Brain uptake of [11C]befloxatone was

blocked by pre-treatment with the MAO-A specific inhibitor moclobemide (10 mg/kg; Figure

4). Pre-injection of the MAO-B specific inhibitor lazabemide (0.5 mg/kg) did not induce

significant change of the [11C]befloxatone brain uptake compared to that observed in control

experiments (Figure 4).

Reversibility of [11C]befloxatone brain uptake : Increasing doses of unlabelled

befloxatone were administered 30 min after the tracer injection (T30 min) in separate

experiments. Figure 5 shows that, compared with control cerebral TACs of [11C]befloxatone,


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administration of befloxatone produced a wash-out of the radioactivity. The new equilibrium

between brain and plasma was reached at T95 min i.e. 60 min after the unlabelled

befloxatone administration.

The displacement measured at T95 min was dose-dependent, the lowest dose (0.004

mg/kg) produced non-detectable displacement, the highest dose (0.4 mg/kg) completely

displaced the specifically bound [11C]befloxatone (Table II). The relationship between log-

doses of befloxatone and [11C]befloxatone displacements was roughly sigmoidal (Figure 6).

After non-linear fitting of the dose-displacement data, ID50 could be estimated for each brain

structures studied and was about 0.020 mg/kg.


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DISCUSSION

The present study shows that, befloxatone, labelled with a positron emitter (Carbon 11) is

an excellent tool for PET assessment of MAO-A binding sites as demonstrated by

displacement and saturation experiments. In vivo, [11C]befloxatone penetrates and

accumulates rapidly in the brain, displays a high specific uptake which can be rapidly and

dose-dependently reversed. [11C]befloxatone presents a high selectivity for the isoform A of

MAO. These results, obtained in vivo, confirm the biochemical and pharmacological profile

of befloxatone found in rodent and in human tissues (Curet et al., 1996; Caille et al., 1996).

[11C]befloxatone was rapidly distributed in the body after i.v. administration, as shown by

the distribution phase of the radioactivity in plasma. During the elimination phase, the

[11C]befloxatone plasma concentration was very low when a tracer dose is administered. This

concentration was dramatically increased (Figure 1) when a high amount of MAO-A inhibitor

(befloxatone or moclobemide) was administered before the tracer. This is due to the presence

of high level of MAO-A sites in several peripheral organs such as the liver, kidney,

myocardium and duodenum (Saura et al., 1996). Indeed, the saturation of theses sites by the

pre-treatment dose of MAO-A inhibitors, reduces the distribution volume of the

[11C]befloxatone injected and subsequently increase its plasma concentration. This feature

was also found in human using an irreversible MAO-A specific ligand [11C]harmine, after a

seven days treatment with a MAO-A inhibitors (Bergström et al., 1997c). It is therefore

obvious that brain TACs of [11C]befloxatone, which are highly dependent of the plasma input

function, have to be corrected by this parameter (see Methods section).

[11C]Befloxatone at a tracer dose, was relatively stable in vivo. The only radiolabelled

metabolite detected at T30 min in the plasma accounted for about 20% of the total plasma

radioactivity. The short retention time of this compound indicates a low lipophilicity

suggesting a poor brain penetration of this compound.


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The distribution of radioactivity measured in vivo by PET in different brain regions

(Figure 2) paralleled the areas of MAO-A concentration determined in vitro in primate brain

either by histochemistry (Westlund et al., 1985) or autoradiography (Saura et al., 1992).

Similar results were found in man, using the irreversible MAO-A PET ligand, [11C]clorgyline

(Fowler et al., 1987). As in in vitro studies, we did not find any region that was devoid of

MAO-A and significant radioactivity was even detectable in white matter.

The noradrenergic system is thought to be the main compartment of MAO-A in most

species examined including monkey, and human brain. Autoradiographical, histochemical

studies and transcript expression of MAO-A has been clearly imaged most abundant in the

human locus cœruleus (3 to 5 times higher than in cortex) and, to a lesser extend in the

interpedoncular nuclei (Luque et al., 1996; Saura et al., 1992; Willoughby et al., 1988;

Westlund et al., 1988). In PET studies, due to the small size of these structures and the

limited resolution of the technique, it is not possible to clearly identify these nuclei within the

pons. Our so called "pons" region which contains these nuclei displayed a high radioactivity.

However, compared to the values we obtained in the cortex, the activity in the pons was not

as high as expected from in vitro studies. This can be explained by the fact that, due to their

small size, compared to the resolution of the PET camera, the nuclei are more sensitive than

larger structures to partial volume effect. Thus PET measured activity in these nuclei is

underestimated when compared to structures such as cortical regions and even basal ganglia.

The brain uptake of [11C]befloxatone showed that the tracer freely crosses the blood-brain

barrier and binds rapidly to its target sites with a maximum at 30 min. This is consistent with

studies in rodents showing that maximal inhibition of MAO-A brain activity is obtained at

30-60 min after befloxatone administered p.o.(Curet et al., 1996). The very low residual

[11C]befloxatone uptake (less than 5% of the total uptake) obtained after injection of a high

dose of unlabelled befloxatone indicates that the most part of the brain radioactivity is


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specifically bound to MAO-A. Moreover, pre-treatment with another selective MAO-A

inhibitor, moclobemide (Da Prada et al., 1989), has the same effect. Only a low residual

radioactivity was observed, confirming that [11C]befloxatone binds in vivo with a high

specificity to the MAO-A.

To test, in vivo, the MAO isoform selectivity of [11C]befloxatone, a high dose of

lazabemide, a selective MAO-B inhibitor (Haefely et al., 1990), was injected before

[11C]befloxatone. The lazabemide dose (0.5 mg/kg) was chosen high enough to saturate

MAO-B isoform (Bench et al., 1991). In our study, in spite of this high dose of lazabemide,

the brain uptake of [11C]befloxatone was not decreased (Figure 4). This results demonstrates

that in vivo [11C]befloxatone binds with a high specificity and selectivity to MAO-A. This

confirms the high selectivity of befloxatone in vivo for MAO-A as found in the in vitro and

ex vivo enzyme inhibition studies of befloxatone (Curet et al., 1996) as well as in the in vivo

pharmacological profile(Caille et al., 1996).

Biochemical studies have shown that befloxatone binds to MAO-A in a reversible and

competitive manner in rat and human tissues (Curet et al., 1996). To verify these

characteristics in vivo, competition experiments were performed with increasing doses of

unlabelled befloxatone administered after the tracer injection (at T30 min). We showed that

befloxatone displaces the specifically bound [11C]befloxatone in all brain structures. The

radioactivity washout was very rapid, in that a new equilibrium between brain and plasma

was achieved after 30 min. This confirms that, in vivo, the MAO-A befloxatone binding is

rapidly reversible.

[11C]Befloxatone was displaced in a dose dependent manner confirming the reversible

interaction of befloxatone with MAO-A demonstrated in vitro (Curet et al., 1996; Wouters et

al., 1999). In our study we found that the estimated dose of befloxatone needed to displace

half of the specifically bound radioactivity in brain is very low (about 0.02 mg/kg). The high


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affinity of befloxatone for MAO-A (Ki = 2.5 nM), associated with an extensive brain

penetration may explain these results. Befloxatone was shown to be very potent in inhibiting

the rat brain MAO-A activity (increase brain concentration of monoamines ED50 = 0.06

mg/kg, p.o.; decrease monoamine metabolites ED50 = 0.03 mg/kg, p.o.)(Curet et al., 1996).

In conclusion, the in vivo properties of befloxatone may bring new insights for the

therapeutic use of befloxatone. The knowledge of the brain pharmacokinetics of befloxatone

may be of great interest for a better control of the intensity and the duration of the enzyme

inhibition. This, together with the determination of the dose of befloxatone that inhibits its

target, may lead to a better definition of the therapeutical doses. Moreover, the competitivity

of befloxatone binding to its target, demonstrated in vivo, tend to minimise the interaction

with tyramine absorbed with the food and thus to avoid the cheese effect in patients, a major

side effect of irreversible MAO inhibitors. All these features may provide a significant

improvement in the therapeutic use of befloxatone.

The present report shows that in vivo [11C]befloxatone enters readily the brain and binds to

MAO-A in a potent, selective, and reversible manner. Moreover, [11C]befloxatone present a

very low non-saturable uptake and is metabolised rather slowly. All these biochemical

characteristics suggest that [11C]befloxatone is a unique tool for the in vivo study of MAO-A

brain.

AKNOWLEDGMENTS:

We thank C. Jouy and F. Sergent for their help and outstanding care of the non-human

primate colony.


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LEGENDS

Figure 1: [11C]befloxatone plasma TACs in control (n ; n=2) and saturation experiments

with befloxatone (l ; n=2). Values are expressed in % ID / 100ml of plasma. The

inset represent the percent of non metabolised [11C]befloxatone in plasma during

the control experiments.

Figure 2: PET axial brain slices obtained 30 min after i.v. [11C]befloxatone and

superimposed with the MRI image. Radioactive concentration is reported in color.

A high uptake was found in basal ganglia, thalamus, pons and cortical structures,

while cerebellum and white matter present lower uptake

Figure 3: [11C]befloxatone TACs in several brain structures, in a control experiment

(radiotracer is injected i.v. at T0 min). Radioactivity in plasma is also displayed.

Values are expressed in % ID / 100ml of tissue.

Figure 4: [11C]befloxatone TACs in the frontal cortex obtained in the control (n ; n=2) and

saturation with befloxatone l ; n=2) experiments and compared to TACs obtained

after pre-treatment with the MAO-A specific inhibitor moclobemide (10 mg/kg; �

) and the MAO-B specific inhibitor lazabemide (0.5 mg/kg; ��9DOXHV�DUH�3(7�

data corrected by the plasma AUC0→120.

Figure 5: [11C]befloxatone TACs in the frontal cortex obtained in 4 separates displacement

experiments (open diamonds) as compared to the control (n) and saturation with

befloxatone (l) experiments. To make the figure more readible, data are

normalised to the value at 30 min

Figure 6: . Dose-displacement relationship in four brain structures. Experimental data (l)

are fitted using the logistic model (line).


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0 20 40 60 800

1

2

3

10

15

SaturationControl

Rad

ioac

tivity

(%

ID

/ 10

0 m

l)

Time (min)

0 5 10 15 20 25 300

20

40

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Perc

ent o

f to

tal r

adio

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Time (min)

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0 30 60 90 1200.0

0.3

0.6

0.9

1.2

1.5

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Frontal cortex

Thalamus

Cerebellum

PlasmaRad

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(%

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Lazabemide

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a A

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0 30 60 90 1200.0

0.3

0.6

0.9

1.2

Saturation0.4 mg/kg

0.1 mg/kg0.04 mg/kg

0.02 mg/kg

Control

Befloxatone

[11

C]B

eflo

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[11C

]Bef

loxa

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dis

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20

40

60

80

100

Thalamus

0.01 0.1 1

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Table I: [11C]befloxatone distribution in baboon’s brain. Comparison of [11C]befloxatone

uptake obtained 30 min after i.v. administration and DV determined on the two

hours experiments (n=2). Uptake values are expressed in percent of injected dose

per 100 ml of tissue (%ID/100ml tissue). Values are mean and standard deviation.

Brain structure Uptake ± std. dev.

(%ID/100ml)

DV ± std. dev.

(mL/g)

Putamen 1.79 ± 0.081 22.4 ± 3.31

Caudate nucleus 1.52 ± 0.008 19.0 ± 4.10

Thalamus 1.66 ± 0.062 20.2 ± 3.93

Pons 1.67 ± 0.150 16.0 ± 3.60

Occipital cortex 1.53 ± 0.210 19.0 ± 1.85

Frontal cortex 1.53 ± 0.240 23.0 ± 2.13

Temporal cortex 1.48 ± 0.251 21.5 ± 3.20

Cerebellum 1.07 ± 0.050 9.7 ± 0.94

White matter 0.91 ± 0.214 11.0 ± 0.42


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Table II : Befloxatone-induced dose-dependent displacements of [11C]befloxatone in all brain

regions studied. Percentage of [11C]befloxatone displacement were determined 95

min after tracer injection (see Methods section). Results for each dose were

calculated from one PET experiment.

Befloxatone

dose

[11C]befloxatone displacements (%)

(mg/kg) Putamen

Caudate

Nucleus Thalamus Pons

Frontal

Cortex

Temporal

Cortex Cerebellum

White

matter

0.004 -11.8 -6.8 -8.9 -7.7 -2.7 -4.8 2.2 -7.6

0.020 47.5 50.4 51.0 51.6 52.4 53.5 66.7 55.1

0.040 77.3 74.8 78.5 71.0 76.3 79.4 77.9 79.1

0.040 83.9 84.8 85.5 81.7 85.5 86.7 83.8 82.6

0.100 92.1 92.9 93.6 92.6 92.5 93.2 92.8 91.2

0.400 99.8 100.1 100.6 102.0 99.8 99.3 99.6 99.4


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