JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 JPETThis Fast article Forward. has not been Published copyedited and on formatted. June 3, The 2004 final asversion DOI:10.1124/jpet.103.064782 may differ from this version.
JPET #64782
SL25.1131, a new, reversible and mixed inhibitor of Downloaded from
MAO-A and MAO-B: Biochemical and behavioral profile jpet.aspetjournals.org
N Aubin, P Barneoud, C Carter, D Caille, N Sontag, C Marc, J Lolivier, A Gardes, C
Perron, A Le Kim, T Charieras, M Pandini, P Burnier, F Puech, S Jegham, P George, B at ASPET Journals on September 24, 2021
Scatton and O Curet
Central Nervous System Research Department, Sanofi-Synthélabo Recherche
Bagneux, France.
1
Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
Running title: SL25.1131, a new reversible and mixed MAO-A/B inhibitor.
Corresponding author
Pascal Barneoud, CNS Research Department, Sanofi-Synthélabo Recherche, 31 avenue
Paul-Vaillant Couturier, 92220 Bagneux, France.
e- mail: [email protected]
Number of: Downloaded from
Text pages: 26
Tables: 3 jpet.aspetjournals.org Figures: 9
References: 38
Word in the abstract: 263 at ASPET Journals on September 24, 2021
Word in the introduction: 756
Word in the discussion: 1490
Abbreviations:
MAOI, monoamine oxidase inhibitor; MAO, monoamine oxidase; NE, norepinephrine;
DA, dopamine; 3-MT, 3-methoxytyramine; NMN, normetanephrine; 5-HT, serotonin;
DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid; 5-HIAA,
5-hydroxyindolacetic acid; DHBA, 3,4-dihydroxybenzylamine; TYR, tyramine; L-5-HTP,
L-5-hydroxytryptophan; PEA, phenylethylamine; HPLC, high-performance liquid
chromatography; SBP, systolic blood pressure; 6-OHDA, 6-hydroxydopamine; MPTP,
1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine; COMT; Catechol O-methyl-transferase,
Kiapp; substrate-dependent Ki.
Recommended section assignment: Neuropharmacology
2 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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Abstract
SL25.1131 is a new, non-selective and reversible monoamine oxidase (MAO) inhibitor,
belonging to a oxazoloquinolinone series. In vitro studies showed that SL25.1131 inhibits
rat brain MAO-A and MAO-B with IC50's of 6.7 nM and 16.8 nM and substrate-
dependent Ki (Kiapp) of 3.3 nM and 4.2 nM, respectively. In ex vivo conditions, the oral
administration of SL25.1131 induced a dose-dependent inhibition of MAO-A and
MAO-B activities in the rat brain with ED50’s of 0.67 mg/kg and 0.52 mg/kg, Downloaded from respectively. In the rat brain, duodenum and liver, the inhibition of MAO-A and MAO-B
by SL25.1131 (3.5 mg/kg, p.o.) was reversible and the recovery of MAO-A and MAO-B
activities was complete 16 h after administration. SL25.1131 (3.5 mg/kg, p.o.) increased jpet.aspetjournals.org
tissue levels of DA, NA, 5-HT and decreased levels of their deaminated metabolites,
DOPAC, HVA and 5-HIAA. In mice, SL25.1131 induced a dose-dependent potentiation
of 5-HTP-induced tremors and PEA-induced stereotypies with ED50’s of 0.60 mg/kg and at ASPET Journals on September 24, 2021
2.8 mg/kg, p.o., respectively. SL25.1131 was able to re-establish normal striatal
dopaminergic tone and locomotor activity in MPTP-lesioned mice. In addition, when
co-administered with L-DOPA, SL25.1131 increased the available DA in the striatum
and the duration of L-DOPA-induced hyperactivity. The duration of the effect of
L-DOPA on circling behavior in 6-OHDA-lesioned rats was also increased. The
neurochemical profile of SL25.1131 demonstrates that this compound is a mixed, potent
and reversible MAO-A/B inhibitor in vitro, in vivo and ex vivo. SL25.1131 has
therapeutic potential as a symptomatic treatment during the early phase of Parkinson's
disease, and as an adjunct to L-DOPA therapy during the early and late phases of the
disease.
3 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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Introduction
Monoamine oxidase (MAO) deaminates monoamine neurotransmitters as well as
exogenous amines. Two isoforms of MAO have been described in mammalian tissue,
namely MAO-A and MAO-B, which exhibit different substrate profiles, respond
differently to selective inhibitors and are present in different cellular and subcellular
locations. MAO-A preferentially deaminates serotonin (5-HT), and norepinephrine (NE)
and is selectively inhibited by clorgyline (Johnston, 1968), whereas MAO-B is selectively Downloaded from inhibited by L-deprenyl and preferentially deaminates phenylethylamine (PEA) and
benzylamine (Knoll and Magyar, 1972). Dopamine (DA) and tyramine (TYR) are
metabolized by both enzyme isoforms (for review see Strolin-Benedetti and Dostert, jpet.aspetjournals.org
1985).
The first generation of irreversible and non-selective MAO inhibitors displayed
antidepressant activity but induced serious side effects including hepatotoxicity, at ASPET Journals on September 24, 2021
orthostatic hypotension, and most importantly the "cheese effect" characterized by an
acute hypertensive crisis when given in combination with foods containing TYR (Bieck
and Antonin, 1989). Because both isoforms of MAO metabolize TYR and are present in
the intestine, it was assumed that after selective MAO-A inhibition, TYR could be
deaminated by MAO-B. However, the next generation of irreversible and selective MAO-
A inhibitors (i.e. clorgyline) also induced hypertension when associated with TYR. The
lack of tyramine effect with L-deprenyl, a selective and irreversible MAO-B inhibitor
suggests that the tyramine effect with clorgyline may be related to the irreversible nature
of the inhibition of MAO-A and not to a non-selective inhibition of both isoforms of
MAO. Indeed, the cheese effect, while still present, is less problematic with reversible
MAO-A inhibitors such as befloxatone (Curet et al, 1998) and moclobemide (Da Prada et
al, 1989). While reversibility of MAO-A inhibition is associated with a better safety
4 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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profile, there is no evidence that MAO inhibitors need to be selective for therapeutic
effect. Although each MAO isoform shows amine selectivity, each can assume the
function of the other when one is inhibited (Butcher et al, 1990). This suggests that non-
selective MAO inhibitors might have broader therapeutic effects than selective MAO-A
or MAO-B inhibitors. These should be reversible inhibitors to minimise their potential
side effects.
Parkinson’s disease (PD) is a progressive neurodegenerative disorder of the Downloaded from mesotelencephalic dopaminergic system. The major clinical symptom, i.e. the inability to
initiate voluntary movements, can be relieved by Levodopa (L-DOPA) (Cotzias et al,
1969). However, a major problem of L-DOPA therapy is the maintenance of a good jpet.aspetjournals.org
responsiveness to the drug during long-term therapy (Agid et al, 1995). The success of
long-term L-DOPA therapy in PD is compromised by the almost invariable intrusion of
dyskinesias during the periods of the day when medication is at its most effective (Agid at ASPET Journals on September 24, 2021
et al, 1995). After treatment with the drug for more than 5 or 6 years, a majority of
patients develop response fluctuations, dyskinesia or dystonia (Lang and Lozano, 1998a,
b). There is therefore a need for novel symptomatic treatments, which could alleviate
motor dysfunction in PD at an early stage, and consequently delay the use of L-DOPA. In
addition, when associated with L-DOPA treatment in PD at a late stage, this treatment
should reduce the fluctuation in the effects of L-DOPA by lengthening its duration of
action and therefore diminish its undesirable effects. The irreversible MAO-B inhibitor
selegiline (Deprenyl) was developed for the treatment of PD (Birkmayer et al, 1983), and
there is some evidence that selegiline monotherapy reduces the “wearing off” akinesia
and delays the need for L-DOPA therapy in mild, previously untreated PD by 8 to 9
months (Parkinson study group, 1996a). The onset of disability sufficient to require
L-DOPA therapy was also delayed in patients who received the reversible MAO-B
5 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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inhibitor lazabemide (Parkinson study group, 1996b). However, MAO-A inhibitor can
also deaminate dopamine oxidatively and the reversible MAO-A inhibitor moclobemide
has demonstrated therapeutic potential in PD (Sieradzan et al, 1995). Thus, drugs that
inhibit both MAO-A and -B may be more efficacious than selective MAO-A or MAO-B
inhibitors in PD (Fahn and Chouinard, 1998).
SL25.1131 [3(S),3a(S)-3-methoxymethyl-7-[4,4,4-trifluorobutoxy]-3,3a,4,5-tetrahydro-
1H-oxazolo[3,4-a]quinoline-1-one] is an oxazoloquinolinone derivative (Figure 1) which Downloaded from belongs to a "fourth generation" of MAO inhibitors which combine reversibility and
mixed inhibitory activity towards both MAO-A and MAO-B isoenzymes.
The aim of the present study was to establish the neurochemical and behavioral profile of jpet.aspetjournals.org
SL25.1131 in rodents. In addition, the relative safety of SL25.1131 was compared to that
of irreversible mixed MAO-A/B-inhibitors (phenelzine and nialamide) by evaluating the
tyramine pressor effect in the rat. Finally, biochemical and behavioral techniques were at ASPET Journals on September 24, 2021
used to evaluate the therapeutic potential of SL25.1131 in rodent models of PD during the
early phase of the lesion, and as an adjunct to L-DOPA therapy during the late phase of
the lesion.
6 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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Materials and methods
Animals
Studies on MAO activities used male Sprague-Dawley rats (OFA, Iffa-Credo, l’Arbresle,
France) weighing 100-200 g. Interaction studies with tyramine were carried out on male
Sprague-Dawley rats (OFA, Iffa-Credo) weighing 330-664 g on the day of dosing. Male
OF1 mice (Iffa-credo) weighing 20-30 g were used for the potentiation of PEA-induced
effects and the potentiation of L-5HTP-induced effects. The other behavioral studies used Downloaded from male OFA rats and male C57Bl/6 mice (Iffa-Credo), weighing 180-200 g and 16-25 g,
respectively, at the start of the experiment. The MPTP studies used male C57Bl/6 mice
(Iffa-Credo), weighing 20-25 g at the start of the experiment. Rats were housed in groups jpet.aspetjournals.org
of four in polypropylene cages (37 x 37 x 18 cm), and mice were housed in groups of
8-10 in polypropylene cages (22 x 10 x 8 cm). All animals were housed in a controlled
environment (light/dark cycles of 12 h with lights on from 7:00 a.m. to 7:00 p.m., at ASPET Journals on September 24, 2021
temperature of 21 ± 2°C) with food and water ad libitum. All experimental procedures
complied with international guidelines and French legislation.
Chemicals and drugs
Dopamine (DA), norepinephrine (NE), dihydroxyphenylacetic acid (DOPAC),
homovanillic acid (HVA), 3-methoxytyramine (3-MT), normetanephrine (NMN),
5-hydroxyindolacetic acid (5-HIAA), 3,4-dihydroxybenzylamine (DHBA),
serotonin (5-HT, 5-hydroxytryptamine), phenelzine sulfate, L-DOPA, benserazide,
PEA hydrochloride, tyramine hydrochloride, desmethylimipramine (DMI),
apomorphine hydrochloride, 6-OHDA and 2,5-diphenyloxazole (PPO) were supplied by
Sigma (St Louis, MO, U.S.A.). L-5-hydroxytryptophan (L-5-HTP) was obtained from
Fluka (Mulhouse, France) and ketamine hydrochloride from Parke Davis (Detroit, MI).
7 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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Tolcapone was obtained from Roche (Suisse). SL25.1131 was synthesized by the
Medicinal Chemistry Department of Sanofi-Synthelabo Research (France). 5-hydroxy
[side chain-2-14C] tryptamine creatinine sulfate (specific activity: 1.8-2.2 GBq/mmol,
[14C]-5-HT) was supplied by Amersham (Buckinghamshire, U.K.).
Phenylethylamine hydrocloride-[ethyl-1-14C] (specific activity: 1.5-2.2 Gbq/mmol,
[14C]PEA), was supplied by New England Nuclear (NEN) Life Sciences Products
(Boston, MA, U.S.A.). 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) was Downloaded from obtained from Research Biochemicals International (Natick, MA, U.S.A.). Imalgene
500® was obtained from Merial (France). Toluene, EDTA (disodium salt of
ethylenediaminetetraacetic acid) and ethyl acetate were purchased from Labosi jpet.aspetjournals.org
(Osi, Paris, France), and Biofluor from NEN Life Sciences Products. The analytical-grade
buffers NaH2PO4, 2 H2O and Na2HPO4, 12 H2O, perchloric acid and sucrose were
purchased from Merck (Darmstadt, Germany). All other reagents were standard at ASPET Journals on September 24, 2021
laboratory reagents of analytical grade.
Determination of MAO-A and MAO-B activities
MAO activities were determined as previously described by Curet et al (1996).
In vitro experiments. For IC50 determination, rat forebrains were homogenized in 20
volumes of buffer (0.25 M sucrose, 10 mM sodium-phosphate buffer, pH 7.4) at 4°C
(final concentration 5 mg of tissue/assay). Briefly, 100 µl of homogenates were
preincubated for 20 min at 37°C with various concentrations of inhibitor in a total volume
of 400 µl. After this preincubation period, the reaction was started by the addition of
[14C]-5-HT as the specific MAO-A substrate (final concentration 125 µM,
specific activity: 1 µCi/µmol) or [14C]-PEA as the specific MAO-B substrate
(final concentration 8 µM, specific activity: 10 µCi/µmol). The final volume of
8 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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incubation was 500 µl in buffer (0.25 M sucrose, 10 mM sodium-phosphate buffer,
pH 7.4). The incubation times were 5 min for MAO-A and 1 min for MAO-B. The
reaction was stopped by the addition of 200 µl of 4 M HCl. Deaminated metabolites were
extracted by vigorous shaking for 10 min in 7 ml of toluene/ethyl acetate (v/v). Following
extraction, the aqueous phase was frozen with liquid nitrogen and the organic layer was
poured into a scintillation vial to which 10 ml of toluene containing 0.4% (wt/v)
2.5 diphenyloxazol (PPO) was subsequently added. After 5 min of agitation, radioactivity Downloaded from was measured in a scintillation spectrometer (LS-1801, LS-1701, Beckmann, Irvine, CA,
U.S.A.). Blank values were obtained by adding HCl prior the substrate. MAOs activities
were corrected for the efficiencies of extraction of deaminated metabolites into the jpet.aspetjournals.org
14 14 toluene/ethyl acetate phase (for [ C]-PEA, 0.95 and for [ C]-5-HT, 0.77). IC50 values
were calculated from inhibition curves and Ki values were determined from the Cheng
Prussof equation (Ki = IC50/(1 + S/Km)) assuming competitive inhibition of MAO-A and at ASPET Journals on September 24, 2021
MAO-B with a Km of 125 µM for 5-HT and 8 µM for PEA.
For substrate-dependent Ki (Kiapp) MAO-A determination, rat forebrain was
homogenised in buffer (0.25 M sucrose, 10 mM sodium phosphate buffer, pH 7.4, 100
mg of tissue/ml) and then incubated at 37°C for 15 min in the presence of mofegiline
(2 pmol/mg of tissue), a selective MAO-B inhibitor. After this period, the homogenates
were diluted (final concentration 1.82 mg/assay) and preincubated with various
concentrations (0.625 to 7.5 nM) of SL25.1131 during 60 min. After this period [14C]-5-
HT (final concentration 100 µM, specific activity: 1 µCi/µmol) was added and incubated
for 15 min. For substrate-dependent Ki (Kiapp) MAO-B determination, rat forebrain was
homogenised in buffer (0.25 M sucrose, 10 mM sodium phosphate buffer, pH 7.4, 100
mg of tissue/ml) and then incubated at 37°C for during 15 min in the presence of
clorgyline (2 pmol/mg of tissue), a selective MAO-A inhibitor. After this period,
9 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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homogenates were diluted (final concentration 1 mg/assay) and preincubated with various
concentrations (0.75 to 12 nM) of SL25.1131 during 120 min. After this period [14C]-
PEA (final concentration 4 µM, specific activity: 20 µCi/µmol) was added and incubated
for 3 min. The reaction was stopped and radioactivity was determined as previously
described.
The substrate-dependent Ki (Kiapp) and enzyme concentration (e) were calculated by
non-linear regression analysis (RS1, Vax computer) with the following equation: Downloaded from a=1-((I+Kiapp+e)-sqr((I+Kiapp+e)2-(4xIxe)))/(2xe)
(Boudier and Bieth, 1989) where a represents the fraction of free enzyme, e (nM) is the
concentration of MAO-A (in the present study e = 2.5 nM) or MAO-B (in the present jpet.aspetjournals.org
study e = 0.6 nM), I (nM) is the total concentration of SL25.1131. Ki is expressed in nM.
Ex vivo experiments. The animals were treated with compounds and decapitated at at ASPET Journals on September 24, 2021
specified times after treatment. The different tissues (rat whole brain, frontal cortex,
striatum, liver and duodenum) were dissected out, rapidly frozen and kept at -80°C until
MAO-A and MAO-B determination. MAO-A and MAO-B activities were assayed as
previously described with minor modifications. Whole forebrain, frontal cortex and
striatum homogenates (100 µl, final concentration 5 mg of tissue/assay, homogenisation
buffer: 0.25 M sucrose, 10 mM sodium phosphate buffer, pH 7.4) were incubated for
5 min with 400 µl of [14C]-5-HT (final concentration 125 µM, specific activity:
1 µCi/µmol) or 1 min with 400 µl of [14C]-PEA (final concentration 8 µM,
specific activity: 10 µCi/µmol) previously brought to 37°C. Liver homogenates (100 µl,
final concentration 1 mg of tissue/assay, homogenisation buffer: 0.25 M sucrose, 10 mM
sodium phosphate buffer, pH 7.4) were incubated for 6 min with 400 µl of [14C]-5-HT
(final concentration 125 µM, specific activity: 1 µCi/µmol) or 1 min with 400 µl of
10 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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[14C] PEA (final concentration 8 µM, specific activity: 10 µCi/µmol) previously brought
to 37°C. Duodenum homogenates (100 µl, final concentration 5 mg of tissue/assay,
homogenisation buffer: 0.25 M sucrose, 10 mM sodium phosphate buffer, pH 7.4) were
incubated for 5 min with 400 µl of [14C]-5-HT (final concentration 125 µM, specific
activity: 1 µCi/µmol) or 2.5 min with 400 µl of [14C]-PEA (final concentration 8 µM,
specific activity: 10 µCi/µmol) previously brought to 37°C. The reaction was stopped and
radioactivity was determined as previously described. Downloaded from
Assay of brain catecholamines, 5-HT and their related metabolites
Animals were killed by decapitation at specified times after oral administration of jpet.aspetjournals.org
SL25.1131. Striata and frontal cortex were dissected out, frozen, weighed and stored at
-80 °C until analysis. NE, 5-HT, DA, HVA, DOPAC, NMN, 3-MT and 5-HIAA were
quantified in the supernatant by high-pressure liquid chromatography with at ASPET Journals on September 24, 2021
electrochemical detection and were assayed as described previously (Aubin et al, 1998).
Interaction with tyramine in awake rats
Arterial pressure was directly measured in awake freely moving rats (n = 11-20) prepared
with indwelling cannulas in the carotid artery as described previously (Caille et al, 1996).
MAOIs were studied at equipotent pharmacological doses, i.e, at least 3 times the oral
ED50 values determined in the rat L-5-HTP potentiation-test (see table 3). Pre-treatment
time for MAOIs was chosen to produce a maximal peripheral inhibition of MAO.
Tyramine responses were expressed as the maximal changes in ∆SBP maximal, in
millimeters of mercury, the most sensitive parameter for the evaluation of a hypertensive
effect, calculated vs. the control value (just before the tyramine (12 mg/kg, p.o.)
administration). Data were expressed as mean ± sem.
11 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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Unilateral lesion of the mesotelencephalic dopaminergic system in rats
The mesotelencephalic system was lesioned unilaterally by two injections of 6-OHDA
(6 µg in 1.5 µl; vehicle: 0.9% saline and 0.01% ascorbic acid) in the left medial forebrain
bundle (stereotaxic coordinates relative to bregma: AP = 0/-1, ML = +1.6, V = -8 from
dura, incisor bar at +5), as described previously (Barneoud et al., 2000). Rats were
screened for the presence of a total dopaminergic lesion by examining their circling Downloaded from response to 50 µg/kg apomorphine hydrochloride (see rotation activity section). Rats that
made fewer than 150 contralateral rotations over 45 min were excluded.
jpet.aspetjournals.org
Behavioral testing
Y-Maze Activity. Locomotor and rearing activities in mice were determined in a
symmetrical Y-maze as described by Marks et al (1985). The maze consisted of three at ASPET Journals on September 24, 2021
arms 26 cm long, 6.1 cm wide and 15 cm high. Each arm was subdivided into two equal
sections. The mice were placed individually in the center of the maze, and movements
from one section to another were counted over a 4-min period.
L-5-HTP-induced tremors. The potentiation of L-5-HTP was assessed according to the
method of Lessin (1959). Groups of mice received p.o. injections of the test compounds
or the vehicle, followed 60 min later by L-5-HTP (100 mg/kg, i.p.). This dose did not
induce tremors in control animals. Immediately after receiving L-5-HTP, mice were
placed individually in clear polystyrene cages (22 x 10 x 8 cm). Thirty min after
L-5-HTP, they were individually observed for 30 sec for the presence of generalised
tremor, which was scored as either present or absent. The activity of the test compounds
12 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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was expressed as the dose which produced generalized tremors in 50% of the animals
(ED50) as calculated by log-probit analysis of the linear part of the dose-response curve.
PEA-induced stereotypies. The potentiation of PEA was assessed according to the
method of Braestrup et al (1975). After transfer from the animal quarters to the
experimental room, mice were placed individually in clear polystyrene cages
(22 x 10 x 8 cm). Different groups of mice (10 per treatment) were treated with oral doses Downloaded from of the test compounds or the vehicle, 60 min before an i.p. injection of 25 mg/kg of PEA.
This dose produced no effect in animals receiving vehicle pre-treatment. Fifteen min after
administration of PEA, they were observed for 2.5 min for the presence or absence of jpet.aspetjournals.org
stereotyped behavior (rapid repeated lateral head movements, sniffing and licking). The
activity of the test compounds was expressed as the dose which produced stereotyped
behavior in 50% of the animals (ED50) as calculated by log-probit analysis (Utistat at ASPET Journals on September 24, 2021
version 2.01) of the linear part of the dose-response curve.
L-DOPA-induced activity. MPTP-treated mice were placed individually in clear
polystyrene cages (22 x 10 x 8 cm) and were observed individually for 30 sec for the
presence or absence of locomotor activity, rearing or stereotypies. Mice were recorded as
positive if at least one of these behaviors was observed.
L-DOPA-induced rotation. Rats were tested in an automated rotameter (Med Associates
Inc., Vermont) and the number of full 360° turns per 5 min interval was recorded
automatically by microcomputer. Three weeks after surgery, animals were treated with
L-DOPA and tested in rotometers and the number of full turns per min was recorded
during a 480 min period. Rotations in the ipsilateral and contralateral directions were
13 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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counted separately, and the analyses were based on the net scores (contralateral minus
ipsilateral rotations); a positive score indicated that animals exhibited a net contralateral
bias, whereas a negative score indicated a net ipsilateral bias.
Drug treatments
SL25.1131, tolcapone, L-DOPA and benserazide were suspended in 0.5% (wt/v)
methocel gel in sterile water with the addition of 0.05% Tween 80. Control groups Downloaded from received the vehicle used for SL25.1131 (0.5% methocel gel plus 0.05% Tween 80).
PEA, L-5-HTP, 6-OHDA and MPTP were dissolved in physiological saline and the doses
refer to the salt. For all other drugs, the doses always refer to the free base and are jpet.aspetjournals.org
expressed in mg/kg of body weight. Drugs were administered in a volume of 5 and
10 ml/kg in rats and mice, respectively. Tolcapone and SL25.1131 were administered
orally; L-DOPA and benserazide were administered intraperitoneally. MPTP was at ASPET Journals on September 24, 2021
administered in saline subcutaneously.
Statistical analysis
IC50, ED50 and confidence intervals were calculated with a RS1 procedure
(Utistat-dose-effect). When appropriate, drug effects were assessed by comparing drug-
treated animals with control animals using either a student's t test for single comparison
or an analysis of variance (one or two factors) followed by the Dunnett’s (homogenous
variances) or the Bonferroni-Holm (heterogeneous variances) tests (Utistat, RS1,
Everstat) or Newman-Keuls or a nonparametric Kruskal-Wallis test. The
L-DOPA-induced hyperactivity in MPTP-lesioned mice was subjected to a Chi-square
analysis and Fisher’s exact test.
14 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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Results
Inhibition by SL25.1131 of MAO-A/B activity in rat brain homogenates in vitro
In vitro, SL25.1131 induced a potent and non-selective inhibition of MAO-A and
MAO-B activities in rat brain homogenates with IC50's (confidence interval within
brackets) of 6.7 [6.0-7.9] nM and 16.8 [14.9-18.9] nM, respectively (table 1).
For purposes of comparison, IC50’s of reversible selective MAO-A inhibitors including
befloxatone, cimoxatone, harmaline, moclobemide, brofaromine BW137OU87 and Downloaded from toloxatone, and selective MAO-B inhibitors including lazabemide (reversible) and
L-deprenyl (irreversible) (Curet et al, 1996, 1998), are also presented in table 1.
With low affinity inhibitors, the amount of compound needed to inhibit enzymatic jpet.aspetjournals.org
activity is relatively large compared with the amount of enzyme. Consequently, the
amount of inhibitor bound to the enzyme represents a negligible fraction and the
concentration of free inhibitor can be taken as equal to the total concentration of inhibitor. at ASPET Journals on September 24, 2021
Under these conditions, the Michaelis equation can be used.
In contrast, with very efficacious inhibitors (Ki values of the order of 10-9M) including
SL25.1131, the concentration of inhibitor and enzyme may be of the same order. In this
case, the binding of the inhibitor to the enzyme reduces the concentration of free inhibitor
and the equation, which is used, requires modification of IC50 or Ki parameters, which
can not be calculated absolutely (Morrison, 1969). In addition, standard experimental
conditions for determination of IC50 requires optimisation (preincubation period). For
these reasons, apparent Ki (Kiapp), a parameter which takes into account the MAO-A and
MAO-B concentrations (see materials and methods) has been calculated.
In rat brain, Kiapp of SL25.1131 was 3.31 ± 0.07 nM for MAO-A and 4.18 ± 0.06 nM for
MAO-B. Control activities for MAO-A and MAO-B were 170 and 58 pmole/min/mg
tissue, respectively.
15 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
Effect of SL25.1131 on MAO-A and MAO-B activities in rat brain ex vivo
SL25.1131, 1 h after oral administration, induced a dose-dependent inhibition of MAO-A
and MAO-B activities in the rat brain with ED50’s of 0.67 [0.52-0.82] and 0.52 [0.40-
0.64] mg/kg, respectively (table 2). A dose of 10 mg/kg (p.o.) of SL25.1131 was
necessary for the full inhibition of MAO-A and –B activities in rat and mouse brain
(result not shown). Downloaded from
For purposes of comparison, ED50’s of selective MAO-A inhibitors including befloxatone
(reversible), moclobemide (reversible) and clorgyline (irreversible), selective MAO-B
inhibitors including lazabemide (reversible) and L-deprenyl (irreversible), and jpet.aspetjournals.org
irreversible mixed MAO inhibitors including tranylcypromine, phenelzine, pargyline and
nialamide (Curet et al, 1998), are also presented in table 2.
Time course of the effect of systemic administration of SL25.1131 (3.5 mg/kg, p.o.), at ASPET Journals on September 24, 2021
administered 1, 2, 4, 8, 16 and 24 h before decapitation, on MAO-A and MAO-B
activities in rat frontal cortex, striatum, liver and duodenum is shown in figure 2.
SL25.1131 (3.5 mg/kg, p.o.) induced a maximal inhibition of MAO-A (-80% to -85%)
and MAO-B (~-95%) at 1 h after administration. The inhibition of both isoforms of MAO
was fully reversible and the recovery of MAO-A and MAO-B activities was complete at
16 h.
Effects of SL25.1131 on the levels of monoamines and their metabolites in the rat
frontal cortex and striatum
As shown in figure 3, SL25.1131 (3.5 mg/kg, p.o.), in the rat frontal cortex, induced a
maximal increase of NE (35%), 5-HT (100%) and NMN (250%) levels and a maximal
16 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
decrease of DOPAC (-70%) and HVA (-55%) levels 1 h after administration. In this
region, levels of 5-HIAA were not significantly modified.
A similar pattern was observed in the striatum where a substantial increase of DA (25%),
5-HT (40%) and 3-MT (150%) levels and a significant decrease of DOPAC (-80%),
HVA (-75%) and 5-HIAA (-20%) levels were observed 1 h after dosing. In both regions,
the levels of monoamines and their metabolites returned to control values between 8 and
24 h. Downloaded from
Potentiation of L-5-HTP and PEA-induced behaviors by SL25.1131
Systemic administration of L-5-HTP induces tremors in mice due to its 5-HT releasing jpet.aspetjournals.org
effect. Likewise, systemic administration of PEA induces hyperactivity and stereotyped
behavior in mice due to its DA releasing effect. Because L-5-HTP and PEA are
selectively deaminated by MAO-A, and MAO-B, respectively, a mixed inhibitor of these at ASPET Journals on September 24, 2021
two enzymes should potentiate the behavioral effects of both L-5-HTP and PEA. The
potentiating effects of oral administration of SL25.1131 on L-5-HTP-induced tremors in
rat and in mice and PEA-induced stereotypies in mice are shown in table 3.
The ED50 (confidence interval within brackets) for potentiation of L-5-HTP-induced
tremors by SL25.1131 is 0.61 [0.55–0.69] mg/kg, p.o. and 0.60 [0.55–0.67] mg/kg, p.o.,
in rat and in mice, respectively. In mice, the ED50 (confidence interval within brackets)
for potentiation of PEA-induced stereotypies by SL25.1131 is 2.8 [2.7-3.0] mg/kg, p.o..
For purposes of comparison, ED50’s of a variety of reference compounds (Curet et al,
1998), are also presented in table 3, demonstrating the specificity of the
L-5-HTP-induced tremors and PEA-induced stereotypies for MAO-A, and MAO-B
activities, respectively.
17 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
Interaction of SL25.1131 with tyramine in the awake rat
As shown in figure 4, after pre-treatment (60 min) with SL25.1131 (3.3 mg/kg, p.o.),
tyramine (12 mg/kg, p.o.) did not alter SBP (∆ SBP maximal = 22 ± 6 compared to 20 ± 8
mm Hg in the vehicle-treated group).
For purposes of comparison, the effects of other reversible MAO-A inhibitors including
befloxatone, moclobemide and brofaromine and irreversible mixed MAO inhibitors
including nialamide and phenelzine (Caille et al, 1996), are also presented in figure 4. Downloaded from The tested doses of MAOIs were determined in accordance with their relative in vivo
activity in rats, i.e, at least 3 times the oral ED50 values determined in the rat L-5-HTP
potentiation-test (table 3). Pre-treatment time for MAOIs was chosen to produce a jpet.aspetjournals.org
maximal peripheral inhibition of MAO; 60 min for SL25.1131 (3.3 mg/kg, p.o.), 45 min
for befloxatone (0.5 mg/kg, p.o.), 30 min for moclobemide (5 mg/kg, p.o.), 60 min for
brofaromine (21 mg/kg, p.o.), nialamide (60 mg/kg, p.o.) and phenelzine (48 mg/kg, at ASPET Journals on September 24, 2021
p.o.).
As shown in figure 4, when SL25.1131 or other reversible MAOIs were given before the
tyramine challenge (12 mg/kg, p.o.), no modification of the effect of tyramine was
observed, whereas the irreversible MAOIs phenelzine (48 mg/kg, p.o.) and nialamide
(60 mg/kg, p.o.) produced a marked potentiation of the pressor effect with tyramine under
the same conditions.
Effects of SL25.1131 on striatal levels of DA, DOPAC, 3-MT and 5-HT in
MPTP-lesioned mice
As shown in figure 5, MPTP (40 mg/kg, s.c.) decreased DA, DOPAC and 3-MT levels by
-90%, -83% and -78%, respectively and was without effect on 5-HT levels.
18 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
In MPTP-lesioned mice, SL25.1131 (10 mg/kg, p.o.) increased 3-MT and 5-HT levels by
400% and 55%, respectively and decreased levels of DOPAC by -86% without
significant modification of DA levels.
Effects of SL25.1131 alone or in combination with L-DOPA on striatal levels of DA,
DOPAC, 3-MT and 5-HT in MPTP-lesioned mice
In MPTP–lesioned mice, different doses of L-DOPA (25, 50, 100 mg/kg, i.p. + Downloaded from benserazide 25 mg/kg, i.p.) increased striatal levels of DA (228%, 243%, 460%),
DOPAC (594%, 852%, 2165%) and 3-MT (119%, 98%, 182%) but decreased levels of
5-HT (-22%, -26%, -51%) (figure 6). Compared to MPTP groups, administration of jpet.aspetjournals.org
different doses of L-DOPA (i.p.) in combination with a dose of SL25.1131 (10 mg/kg,
p.o.) increased the levels of DA (349%, 873%, 591%), 3-MT (1019%, 1205%, 2733%)
and 5-HT (51%, 41%, 32%), (figure 6). These variations were dose-dependent except for at ASPET Journals on September 24, 2021
DA. Furthermore, SL25.1131 had no effect on striatal L-DOPA levels in MPTP-lesioned
mice (data not shown).
Effects of SL25.1131 on MPTP-induced hypoactivity in mice
Forty-eight hour after a single MPTP administration, locomotion was reduced in mice by
30% (Figure 7; F(3,36)=6.59, p=0.001). At the same time, striatal DA levels were
decreased by approximately 80%. It is probable that this hypoactivity is closely related to
the loss of striatal DA terminals, since the co-administration of the re-uptake blocker
(GBR12935) with MPTP counteracts both the penetration of the toxin MPP+ into
dopaminergic neurons and the hypoactivity (results not shown). SL25.1131, administered
1 h before the behavioral evaluation (a period at which the lesion was completed), was
able to re-establish, in lesioned-mice, a locomotion similar to that exhibited by
19 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
vehicle-treated control mice. In addition, SL25.1131 treatment did not change this
behavior in non-lesioned mice.
Effects of SL25.1131 and tolcapone on L-DOPA-induced activity in MPTP-lesioned
mice
L-DOPA treatment increased in a dose-dependent manner the number of animals which
demonstrated general activity (without treatment, mice remained motionless because of Downloaded from the habituation to the cage). The active doses were 25 and 50 mg/kg and the sub-active
dose was 12.5 mg/kg (figure 8A). Pre-treatment with SL25.1131 (10 mg/kg, p.o.)
potentiated the effect of L-DOPA (12.5 mg/kg) (from 20% to 70% responders) whereas jpet.aspetjournals.org
tolcapone (30 mg/kg, p.o.) failed to have any effect (figure 8A). Pre-treatment with
SL25.1131 maintained the therapeutic effect with half the dose of L-DOPA (figure 8A).
Moreover, as illustrated in figure 8B, pre-treatment with SL25.1131 (10 mg/kg, p.o.) or at ASPET Journals on September 24, 2021
tolcapone (30 mg/kg, p.o.) increased the duration of L-DOPA effect at doses of 12 mg/kg
and 25 mg/kg, respectively.
Effects of SL25.1131 and tolcapone on L-DOPA-induced rotations in rats with a
complete 6-OHDA lesion
In 6-OHDA-lesioned rats, the combined administration of L-DOPA (5-20 mg/kg, i.p.)
and benserazide (15 mg/kg, i.p.) induced contralateral circling behavior (figure 9A) and
the L-DOPA treatment increased in a dose-dependent manner the duration of turning
behavior [F(94,3572)=8.23, p<0.001] and the total number of turns [F(2,76)=9.9,
p<0.001]. Administration of either SL25.1131 (10 mg/kg, p.o.) or tolcapone (30 mg/kg,
p.o.) increased the duration (F(270,3060)=4.0 P<0.001) and the total number of turns
(F(3,34)=13.36, p<0.001) induced by a dose of 5 mg/kg of L-DOPA (figure. 9B).
20 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
Combined inhibition of both the MAO and COMT inhibitors dramatically potentiated
L-DOPA-induced turning in comparison to MAO or COMT inhibition alone.
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021
21 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
Discussion
In vitro, with IC50's of 6.7 nM and 16.8 nM and Kiapp's of 3.3 nM and 4.2 nM for rat
brain MAO-A and MAO-B, respectively, SL25.1131 is a potent, mixed and competitive
MAO inhibitor. In contrast to its potent inhibitory effect on MAO-A/B, SL25.1131 is
devoid of any activity (Ki>1µM) at 100 different receptor binding sites studied (CEREP
binding profiles, data not shown). Ex vivo, SL25.1131 induced a dose-dependent and
reversible inhibition of MAO-A and MAO-B activities in rat brain with ED50’s of 0.67 Downloaded from and 0.52 mg/kg, p.o., respectively. Its inhibitory activity is similar to that of
tranylcypromine and SL25.1131 is 10 to 20-fold more potent than phenelzine.
The pattern of the effects of SL25.1131 on monoamine levels and their metabolites is that jpet.aspetjournals.org
expected for a reversible MAO-A/B inhibitor with an increase of tissue levels of
monoamines associated with a decrease of their deaminated metabolites and an increase
of their methylated metabolites. Monoamine levels and metabolites returned to basal at ASPET Journals on September 24, 2021
values 8 to 16 h after dosing. This time-course paralleled those of the inhibition of
MAO-A and MAO-B activities further demonstrating that SL25.1131 is a reversible
MAO inhibitor in vivo.
Because selective MAO-B inhibitors do not affect monoamine levels and their
metabolites in the rat brain, the variations observed after systemic administration of
SL25.1131 are likely mainly due to MAO-A inhibition (Da Prada et al, 1989; Kan et al,
1987; Curet et al, 1996). However, since previous reports demonstrated that monoamines
can also be deaminated by MAO-B when MAO-A is inhibited (Butcher et al, 1990),
variations in monoamine levels following administration of SL25.1131 could be due to
inhibition of both isoforms of MAO.
The behavioral syndromes induced by L-5-HTP or PEA are potentiated by MAO-A and
MAO-B inhibition, respectively (Jacobs, 1976; Ortmann et al, 1980; Olanow et al, 1984)
22 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
and these functional tests can thus be used to evaluate the selectivity of inhibition of
either enzyme isoform in vivo. The ED50 values for inhibition of MAO-A activity and of
L-5-HTP-induced tremors in mice (0.67 and 0.60 mg/kg, p.o., respectively) [tables 2 and
3] are of the same order of magnitude. In addition, SL25.1131 potentiated PEA-induced
stereotypies in mice with an ED50 of 2.8 mg/kg, p.o. [table 3] while decreasing MAO-B
activity with an estimated ED50 of 0.52 mg/kg, p.o. [table 2]. These results confirm the in
vivo activity of SL25.1131 as an inhibitor of MAO-A and MAO-B. Downloaded from In this study, we investigated the interaction of SL25.1131 with oral tyramine effects in
freely moving awake rats according to Caille et al (1996). When SL25.1131 (at a dose
which fully inhibited MAO-A and MAO-B activities) was given 1 h before the tyramine jpet.aspetjournals.org
challenge (12 mg/kg, p.o.), no change in the pressor response induced by tyramine was
observed. A similar lack of tyramine potentiation was observed with reversible MAOIs,
whereas in the same conditions, phenelzine and nialamide, two irreversible and mixed at ASPET Journals on September 24, 2021
MAO inhibitors, increased the tyramine pressor response. These results suggest that, in
our experimental conditions, reversible inhibition of MAO-A/B activities by SL25.1131
provides a better safety profile.
The aim of the second part of the present study was to evaluate the therapeutic potential
of SL25.1131 on biochemical and behavioral deficits in rodent models of early and late
PD. Consistent with previous reports (Heikkila et al, 1984; Sundstrom et al, 1987),
systemic administration of MPTP markedly decreased striatal levels of DA, DOPAC and
3-MT without affecting 5-HT. Administration of SL25.1131 in MPTP-treated mice failed
to increase DA but increased 5-HT and 3-MT and decreased DOPAC levels. Because
3-MT levels can be considered as an index of DA release in the synaptic cleft,
normalization of 3-MT levels in MPTP-lesioned mice treated with SL25.1131 suggests
that DA function in the synaptic cleft recovered in these mice. In support of this
23 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
hypothesis, hypoactivity exhibited by the MPTP-lesioned mice, a functional consequence
of DA depletion, disappeared when SL25.1131 was administered. These biochemical and
behavioral data support the possibility that SL25.1131 may ameliorate symptoms related
to DA depletion in PD patients.
The behavioral experiments showed that SL25.1131 markedly potentiated
L-DOPA-induced activity in MPTP-lesioned mice, both prolonging hyperactivity and
increasing the number of responding animals. The effect of COMT inhibition was less Downloaded from marked. Administration of tolcapone increased the duration of the L-DOPA-induced
hyperactivity without affecting the number of responders to L-DOPA. In 6-OHDA-
lesioned rats, SL25.1131 and tolcapone also increased the duration of the effect of jpet.aspetjournals.org
L-DOPA treatment by prolonging rotational behavior. In addition, combined inhibition of
both MAOs and COMT dramatically potentiated the duration of L-DOPA-induced
turning in comparison to MAO or COMT inhibitions only. Our results are in accordance at ASPET Journals on September 24, 2021
with those Heeringa et al (1997) who demonstrated that inhibition of both
MAO-A (Ro41-1049) and MAO-B (R19-6327) potentiated the duration of L-DOPA
rotations. Taken together, these results demonstrate that inhibition of MAO-A and
MAO-B by SL25.1131 increased DA transmission in MPTP or 6-OHDA-lesioned
animals alone and in combination with L-DOPA.
L-DOPA remains the most effective treatment for PD even if it is associated with a
number of problems (i.e. motor fluctuations and dyskinesias) after only five to seven
years of use. Many clinicians favour delaying the introduction to L-DOPA until
symptoms are severe enough to warrant its use (Fahn, 1996), particularly in younger
patients, and DOPA-sparing strategies are often used, mainly using other
antiparkinsonian drugs first and keeping the dosage of L-DOPA as low as possible when
it is utilized. MAO mixed-inhibitors, which reduce the metabolism of DA, could alleviate
24 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
the motor dysfunctions in PD patients when administered alone (Fahn and Chouinard,
1998). As we demonstrated here, the reversible mixed MAO inhibitor SL25.1131
increased striatal dopaminergic transmission in partial MPTP-lesioned mice, the levels of
the extracellular DA metabolite 3-MT reaching normal values, and re-established in the
lesioned mice a locomotion similar to that exhibited by control mice. The treatment of
patients with early PD with selegiline could delay the introduction of L-DOPA by about 9
months (Parkinson Study Group, 1996a). To provide possible benefit by inhibition of Downloaded from both MAO types A and B, Fahn and Chouinard (1998) offered patients with mild PD not
requiring symptomatic therapy the choice to be treated with tranylcypromine. The authors
found a lower rate of reaching end-point (the time when symptomatic treatment is jpet.aspetjournals.org
needed) in tranylcypromine-treated patients compared to selegiline-treated patients,
suggesting that inhibiting both MAO-A and –B may have merit as a symptomatic
treatment in early PD. at ASPET Journals on September 24, 2021
As PD progresses, the use of L-DOPA becomes increasingly necessary. Whether the
motor complications seen with chronic L-DOPA therapy in patients with PD are actually
caused by long-term L-DOPA therapy or simply reflect the progression of the disease is
unknown. Nevertheless, the frequency of motor fluctuations and dyskinesias could result
in part from the pulsatile stimulation of receptors caused by intermittent doses of standard
L-DOPA formulations (Chase et al, 1993). However, more frequent smaller doses may
cause more off time and dose failures (Metman et al, 1997), and alternative formulations
and routes of delivery developed to lengthen the relatively short half-life of conventional
preparations of L-DOPA are difficult to use because of reduced bioavailability (Koller
and Pahwa, 1994) and their pharmacokinetic profiles (Hughes, 1997). Thus, drugs
designed to extend the duration of the response to L-DOPA should regulate these
fluctuations. COMT inhibitors such as tolcapone and entacapone have been used and they
25 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
can prolong the action of an individual dose of L-DOPA and significantly reduce «off»
time (Kurth et al, 1997). However, they also increase L-DOPA side-effects such as
dyskinesias or hallucinations, perhaps because they enhance the level of cerebral
L-DOPA. By blocking the degradation of DA, the reversible mixed MAO inhibitor
SL25.1131 potentiated the increase in striatal dopaminergic transmission induced by
L-DOPA in MPTP-lesioned mice without changing the level of cerebral L-DOPA (see
results section). In addition, it potentiated L-DOPA-response in animal models of PD, Downloaded from reducing by half the dose of L-DOPA necessary for their therapeutic effect and increasing
the duration of this effect. Because our compound is devoid of activity on L-DOPA
metabolism, we are tempted to speculate that, if co-administered with a low dose of jpet.aspetjournals.org
L-DOPA in early PD, it would stabilize the L-DOPA fluctuations at the start of the
treatment and it would reduce the frequency of the future L-DOPA side-effects
(i.e. dyskinesias and motor fluctuations). at ASPET Journals on September 24, 2021
In conclusion, the neurochemical profile of SL25.1131 demonstrates that this compound
is a mixed, potent and reversible MAO-A and MAO-B inhibitor in vitro, ex vivo and in
vivo, which exhibits in animals a better safety index than irreversible mixed MAOIs.
SL25.1131 was able to re-establish normal striatal dopaminergic tone and locomotor
activity in MPTP-lesioned mice. In addition, when co-administered with L-DOPA,
SL25.1131 optimised the effect of L-DOPA by increasing the available DA in the
striatum and the duration of its effect. A study in non-human primate would be envisaged
to reinforce our data in rodents. Thus, SL25.1131 has therapeutic potential as a
symptomatic treatment during the early phase of the PD, and as an adjunct to L-DOPA
therapy during the early and late phases of the disease.
26 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
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Acknowledgments
The authors thank Dr. D. Sanger for critically reading the manuscript. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021
27 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
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33 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
Legends of Figures
Figure 1. Chemical structure of SL25.1131 [3(S),3a(S)-3-methoxymethyl-7-[4,4,4-
trifluorobutoxy]-3,3a,4,5-tetrahydro-1H-oxazolo[3,4-a]quinoline-1-one.
Figure 2. Time-course of the effects of systemic administration of SL25.1131 on the ex
vivo activity of MAO-A () and MAO-B () in the rat frontal cortex, striatum, liver and Downloaded from duodenum. Animals were sacrificed at various times after the administration of
SL25.1131 (3.5 mg/kg, p.o.) and MAO-A and MAO-B activities were measured in
homogenates. The results are expressed as percentage of variation vs. respective controls. jpet.aspetjournals.org
Each point represents the mean value ± sem, n=6 animals per group, *p<0.05 compared
to respective control. Control activities (nanomoles/min/g of tissue) were
(MAO-A/MAO-B): frontal cortex 214 ± 6/67 ± 3; striatum, 200 ± 6/67 ± 2; at ASPET Journals on September 24, 2021
liver, 478 ± 10/315 ± 8 and duodenum 166 ± 8/45 ± 3 compared to control rats.
Figure 3. Time-course of the effects of systemic administration of SL25.1131 on the
tissue levels of monoamines and their deaminated and methylated metabolites in the
cerebral cortex and striatum of the rat. Animals were sacrificed at various times after the
administration of SL25.1131 (3.5 mg/kg, p.o.). The results are expressed as percentage of
variation vs. respective controls. Each point represents the mean value ± sem, n=6
animals per group. *p<0.05 compared to respective control. Controls levels (ng/g) in rat
frontal cortex: NE, 231 ± 9; NMN, 14 ± 2; 5-HT, 350 ± 13; 5-HIAA, 205 ± 5; DOPAC,
33 ± 8; HVA, 45 ± 5 and in striatum: DA, 7660 ± 154; 3-MT, 353 ± 27; 5-HT, 512 ± 28;
5-HIAA, 584 ± 25; DOPAC, 981 ± 54; HVA, 802 ± 48.
34 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
Figure 4: Effects of SL25.1131 (3.3 mg/kg, p.o.) on the tyramine (12 mg/kg, p.o.)-
induced pressor effect in freely moving rats in comparison with those of reference
MAOIs (Caille et al, 1996). Doses correspond to at least to three times the ED50 value in
the L-5-HTP-potentiation test in the rat (table 3). Pre-treatment times were 60 min for
SL25.1131 (3.3 mg/kg, p.o.), 45 min for befloxatone (0.5 mg/kg, p.o.), 30 min for
moclobemide (5 mg/kg, p.o.), 60 min for brofaromine (21 mg/kg, p.o.), nialamide (60
mg/kg, p.o.) and phenelzine (48 mg/kg, p.o.). Results were expressed as the maximal Downloaded from changes in SBP maximal, in millimeters of mercury]. Each point represents the mean
value ± sem, n=10-20. *p<0.05 compared to vehicle group (Student's t test).
jpet.aspetjournals.org
Figure 5. Ex vivo effects of SL25.1131 (10 mg/kg, p.o.) on tissue levels of DA, DOPAC,
3-MT and 5-HT in striatum of C57Bl/6 mice lesioned by a single injection of MPTP
(40 mg/kg, s.c.) 8 days before. The animals were sacrificed 1 h after the administration of at ASPET Journals on September 24, 2021
SL25.1131. Control group was injected with appropriate vehicles. MPTP group received
MPTP and the appropriate vehicle. The results are expressed as percentage of variation
vs. controls (mean ± sem, n=6). Controls levels (ng/g of frozen tissue) were: DA, 15207 ±
820; DOPAC, 1705 ± 337; 3-MT, 906 ± 94 and 5-HT, 733 ± 78. ∗ p<0.05 vs. control
(Newman-Keuls one way), • p< 0.05 vs. MPTP.
Figure 6. Ex vivo effect of different doses of L-DOPA (25, 50, 100 mg/kg,
i.p.)/benserazide (25 mg/kg, i.p.) alone or in combination with SL25.1131 (10 mg/kg,
p.o.) on tissue levels of DA, DOPAC, 3-MT and 5-HT in the striatum of C57Bl/6 mice
lesioned by a single injection of MPTP (40 mg/kg, s.c.) 8 days before. The animals were
sacrificed 1 h after the administration of SL25.1131 and 30 min after the administration
of L-DOPA/benserazide. The control group was injected with appropriate vehicles. The
35 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
MPTP group received MPTP and the appropriate vehicle. The results are expressed as
percentage change vs. controls (mean ± sem, n=6). Control levels (ng/g of frozen tissue)
were: DA, 14796 ± 419; DOPAC, 3327 ± 260; 3-MT, 1105 ± 69 and 5-HT, 725 ± 59. ∗
p<0.05 vs. respective control (Anova two way).
Figure 7. Effects of SL25.1131 on MPTP-induced hypoactivity in mice. Mice were
injected with MPTP (40 mg/kg, s.c.) or saline, and their motor behavior was studied 2 Downloaded from days later, and 60 min after SL25.1131 (10 mg/kg, p.o.) treatment (8 animals per group)
or vehicle. Mice were subdivided into four groups according to the treatments they
received; saline + vehicle (n=16); saline + SL25.1131 (n=8); MPTP + vehicle (n=8); jpet.aspetjournals.org
group MPTP + SL25.1131 (n=8). Results are expressed as mean number of crossings
(mean ± sem) ** p<0.01.
at ASPET Journals on September 24, 2021
Figure 8. Effects of SL25.1131 and tolcapone on L-DOPA-induced hyperactivity in
MPTP-lesioned mice. Mice were injected with MPTP (40 mg/kg, s.c.) and their activity
was studied 7 days later. A) L-DOPA dose-response curve. B) L-DOPA dose-duration
relationship. Mice were subdivided into groups according to the treatment they received;
vehicle + vehicle with L-DOPA (6, 12, 25 and 50 mg/kg, i.p., n=12); vehicle +
SL25.1131 with L-DOPA (3, 6, 12 and 25 mg/kg, i.p., n=12); tolcapone + vehicle with L-
DOPA (6, 12, 25 and 50 mg/kg, i.p., n=12). Benserazide (25 mg/kg, i.p.) was added to L-
DOPA. SL25.1131 and tolcapone were administered 30 min and 90 min before L-DOPA,
respectively. The behaviors were studied 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h after
L-DOPA treatment. Results are expressed as mean time (mean ± sem), * p<0.05 **
p<0.01 versus the «vehicle + vehicle» group
36 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #64782
Figure 9. Effect of SL25.1131 (10 mg/kg, p.o.) and tolcapone (30 mg/kg, p.o.) alone or in combination on L-DOPA-induced rotations in complete 6-OHDA-lesioned rats. A)
Dose dependent effect of L-DOPA. One week after the apomorphine test, different doses of
L-DOPA + benserazide (15 mg/kg) were administered i.p. and rotation rate was measured over a 240-min-period. Three groups of rats (n=9 each) were treated with the three doses of
L-DOPA (5, 10, and 20 mg/kg, i.p.), each rat having received the three doses after three weeks of testing. B) Effects of SL25.1131 and tolcapone co-administered with a low dose
(5 mg/kg, i.p.) of L-DOPA. Animals were subdivided into four groups according to the Downloaded from treatment they received before the administration of a low dose of L-DOPA (5 mg/kg + benserazide 15 mg/kg, i.p.); group V + V: vehicle + vehicle (n=8); group V + SL: vehicle +
SL25.1131 (n=11); group T + V: tolcapone + vehicle (n=8); group T + SL: Tolcapone + jpet.aspetjournals.org
SL25.1131 (n=11); SL25.1131 and tolcapone were administered 60 min and 90 min before
L-DOPA, respectively. Rotation rate was measured over a 450-min-period. Results are expressed as mean rotation rate (mean ± sem). at ASPET Journals on September 24, 2021
37 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
Table 1: In vitro MAO-A and MAO-B inhibition by SL25.1131 and various MAO inhibitors in rat brain homogenates. The confidence intervals are shown in brackets
*Data from Curet et al, 1996, 1998. Downloaded from
IC50 (nM) Compounds B/A MAO-A MAO-B jpet.aspetjournals.org SL25.1131 6.7 [6.0-7.9] 16.8 [14.9-18.9] 2.5 Befloxatone* 3.8 [3.6-4.1] 300 [275-361] 79 Cimoxatone* 2.7 [2.6-2.9] 90 [83-99] 33
Harmaline* 12 [10-14] >1000 >83 at ASPET Journals on September 24, 2021 Moclobemide* 23000 [21000-26000] >10000 >0.5 Brofaromine* 15 [14-16] >1000 >67 BW 1370U87* 50 [46-54] >1000 >20 Toloxatone* 3260 [2860-3660] 39000 [31000-50500] >12 L-Deprenyl* 970 [864-1000] 4.6 [3.9-5.5] <0.005 Lazabemide* >10000 30 [25-39] <0.003
JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
Table 2: Ex vivo MAO-A and MAO-B inhibition by SL25.1131 in rat brain homogenates: comparison with other MAO inhibitors. The confidence intervals are shown in brackets
*Data from Curet et al, 1998. Downloaded from
ED (mg/kg, p.o.) 50 jpet.aspetjournals.org Compounds B/A MAO-A MAO-B
SL25.1131 0.67 [0.52-0.82] 0.52 [0.40-0.64] 0.8 Befloxatone* 0.02 [0.01-0.04] 1.2 [0.9-1.8] 60 at ASPET Journals on September 24, 2021 Moclobemide* 1 [0.8-1.2] >120 >120 Clorgyline* 3.4 [3.8-3.0] >20 >6 Tranylcypromine* 0.5 [0.43-0.56] 0.24 [0.21-0.27] 0.5 Phenelzine* 6.2 [5.5-7.4] 12 [8.4-15.5] 2 Pargyline* 9.3 [8.2-10.5] 1.4 [1.3-1.7] 0.15 Nialamide* 13 [11-15] 57 [47-68] 4.4 L-Deprenyl* >30 1.5 [1.6-1.4] <0.05 Lazabemide* >1 0.065 [0.04-0.11] <0.065
JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
Table 3: In vivo potentiation of L-5-HTP in rat and in mice and PEA-induced effects by
SL25.1131 in mice: comparison with other MAO inhibitors. The confidence intervals are shown in brackets
*Data from Caille et al, 1996.
ED 50 (mg/kg, p.o.) Downloaded from Rat Mice PEA/ L-5-HTP mice Compounds L-5-HTP L-5-HTP PEA jpet.aspetjournals.org Tremor Tremor Stereotypy SL25.1131 0.61 [0.55-0.69] 0.60 [0.55-0.67] 2.8 [2.7-3.0] 4.6 Befloxatone* 0.15 [0.11-0.21] 0.21 [0.18-0.24] 58 [55-62] 276
Moclobemide* at ASPET Journals on September 24, 2021 1.6 [1.1–2.1] 0.73 [0.63-0.84] 1.8 [1.3-2.3] 2.5 Brofaromine* 6.9 [4.2–9.5] 2.1 [1.4-3.2] 50% at 200 Tranylcypromine* 16 [14-18] 1.6 [1.6-1.7] 0.8 [0.8-0.81] 0.5 Nialamide* 20 [15–29] 5.2 [4.9-5.7] 5.7 [5.3-6.1] 1.1 Phenelzine* 12.3 [11.5-13.1] 2.6 [2.1-3.2] 0.2 L-Deprenyl* >128 3.3 [2.6-4.2] <0.025 Lazabemide* >128 0.5 [0.4-0.5] <0.0039
JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
CF3 O Downloaded from
N CH O 3 jpet.aspetjournals.org O O at ASPET Journals on September 24, 2021
Figure 1 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
FRONTAL CORTEX STRIATUM 120 120
100 100 * * 80 80
60 * 60 * * * % CONTROLS % 40 CONTROLS % 40 * * * 20 * 20 * *
* Downloaded from * * * 0 0 0 5 10 15 20 25 0 5 10 15 20 25 TIME (h) TIME (h)
LIVER DUODENUM 120 120 jpet.aspetjournals.org
100 100
* 80 80 *
60 * * 60
* * at ASPET Journals on September 24, 2021 % CONTROLS % % CONTROLS % 40 40 * * MAO-A * * * 20 * 20 MAO-B * * * * 0 * 0 0 5 10 15 20 25 0 5 10 15 20 25 TIME (h) TIME (h)
Figure 2 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
Frontal cortex 350 * 5-HT 300 * 5-HIAA NE 250 DOPAC HVA * 200 * NMN
* Downloaded from
Contrrol 150 * * * *
% * 100 * * 50 * * jpet.aspetjournals.org * * 0 5 10 15 20 25 Time (hr) at ASPET Journals on September 24, 2021 Striatum 350 5-HT 300 5-HIAA * DA 250 DOPAC * HVA 200 * 3-MT * 150 * * * * % Control % 100 * * * 50 * * * * * 0 * 0 5 10 15 20 25 Time (hr)
Figure 3 delta SBP max (mm Hg) 10 20 30 40 50 60 70 80 0 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. Figure 4 JPET FastForward.PublishedonJune3,2004asDOI:10.1124/jpet.103.064782 Vehic. SL25.1131 (3.3) reversible MAOIs reversible Beflox. (0.5) Moclo. (5.0) Brofar. (21.0) irreversible MAOIs irreversible (60.0) Nial. * Phenel. (48.0) *
(mg/kg, p.o.)
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 24, 2021 24, September on Journals ASPET at JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
DA 5-HT 120 200 i 100 150 80 60 100
40 Downloaded from 50 %OFCONTROL 20 * %OFCONTROL 0 0 jpet.aspetjournals.org DOPAC 3-MT 120 140 100 120 i at ASPET Journals on September 24, 2021 80 100 60 80 60 40 40
%OFCONTROL * *
20 %OFCONTROL i 20 0 0
Control MPTP (40 mg/kg, s.c.) SL25.1131 (10 mg/kg, p.o.) + MPTP (40 mg/kg, s.c.)
Figure 5 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
250 250 DA 5-HT 200 200 * * * * 150 150 Downloaded from 100 100
50 50 % OF CONTROL OF % % OF CONTROL OF % * 0 0 Control 25 50 100 25 50 100 Control 25 50 100 25 50 100
L-DOPA (mg/kg,ip)+ benserazide(25 mg/kg,ip) L-DOPA (mg/kg,ip)+ benserazide(25 mg/kg,ip) jpet.aspetjournals.org
SL25.1131 SL25.1131 (10 mg/kg, p.o) (10 mg/kg, p.o) MPTP (40 mg/kg, sc) MPTP (40 mg/kg, sc)
500 1000 DOPAC 3-MT *
400 at ASPET Journals on September 24, 2021 750 300 500 200 * * 250 100 % OF CONTROL% * * * * OF % CONTROL * 0 0 Control 25 50 100 25 50 100 Control 25 50 100 25 50 100 L-DOPA (mg/kg,ip)+ benserazide(25 mg/kg,ip) L-DOPA (mg/kg,ip)+ benserazide(25 mg/kg,ip)
SL25.1131 SL25.1131 (10 mg/kg, p.o) (10 mg/kg, p.o)
MPTP (40 mg/kg, sc) MPTP (40 mg/kg, sc)
Figure 6 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
75 NS
60 ** ** Downloaded from
45 crossings of jpet.aspetjournals.org 30 number
15 at ASPET Journals on September 24, 2021
0 V SL V SL
MPTP
Figure 7 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
A
100 Vehicle 80 Tolcapone 30mg/kg, p.o. SL25.1131 10mg/kg, p.o.
60 Downloaded from
40 Active animals (%) animals Active 20 jpet.aspetjournals.org
0
1 10 100
B L-DOPA (mg/kg, i.p.) at ASPET Journals on September 24, 2021 4 Vehicle Tolcapone 30mg/kg, p.o. ** SL25.1131 10mg/kg, p.o. 3 * 2 **
1 Duration of effect (hr) of effect Duration
0
3 6 12 25 50 L-DOPA (mg/kg, i.p.)
Figure 8 JPET Fast Forward. Published on June 3, 2004 as DOI: 10.1124/jpet.103.064782 This article has not been copyedited and formatted. The final version may differ from this version.
100 L-DOPA (5 mg/kg, i.p.) + benserazide (15 mg/kg, i.p.) L-DOPA (10 mg/kg, i.p) + benserazide (15 mg/kg, i.p.) 80 L-DOPA (20 mg/kg, i.p.) + benserazide (15 mg/kg, i.p.)
60
40 Downloaded from (turns /5 min.) 20
Contralateral Rate Rotation 0 0 100 200 300 400 jpet.aspetjournals.org Time (Min.)
vehicle + vehicle vehicle + tolcapone (30 mg/kg, p.o.)
at ASPET Journals on September 24, 2021 100 vehicle + SL25.1131(10 mg/kg, p.o.) tolcapone (30 mg/kg, p.o.) + SL25.1131 (10 mg/kg, p.o.) 80
60
40 (turns /5min.) 20
Contralateral Rate Rotation 0 0 100 200 300 400 Time (Min.)
Figure 9