Effect of kynurenine 3-hydroxylase inhibition on the dyskinetic and antiparkinsonian responses to...

Effect of Kynurenine 3-Hydroxylase Inhibition on theDyskinetic and Antiparkinsonian Responses to Levodopa in

Parkinsonian Monkeys

Pershia Samadi, MSc,1,2 Laurent Gregoire, BSc,1 Arash Rassoulpour, BSc,3 Paolo Guidetti, PhD,3

Emanuela Izzo, PhD,4 Robert Schwarcz, PhD,3 and Paul J. Bedard, MD, PhD1,2*

1Centre de Recherche en Neurosciences, Centre Hospitalier Universitaire de Quebec, Quebec, Canada2Departement de Medicine, Universite Laval, Quebec, Canada

3Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, Maryland, USA4Newron Pharmaceuticals, Bresso, Italy

Abstract: Homeostatic interactions between dopamine andglutamate are central to the normal physiology of the basalganglia. This relationship is altered in Parkinsonism and inlevodopa-induced dyskinesias (LID), resulting in an upregu-lation of corticostriatal glutamatergic function. Kynurenicacid (KYNA), a tryptophan metabolite with antagonist ac-tivity at ionotropic glutamate receptors and the capability toinhibit glutamate release presynaptically, might therefore beof therapeutic value in LID. To evaluate this hypothesis, weused a pharmacological tool, the kynurenine 3-hydroxylaseinhibitor Ro 61-8048, which raises KYNA levels acutely. Ro61-8048 was tested in MPTP cynomolgus monkeys with astable parkinsonian syndrome and reproducible dyskinesias

after each dose of levodopa. Serum and CSF concentrationsof KYNA and its precursor kynurenine increased dose-dependently after Ro 61-8048 administration, alone or incombination with levodopa. Coadministration of Ro 61-8048 with levodopa produced a moderate but significantreduction in the severity of dyskinesias while maintainingthe motor benefit. These results suggest that elevation ofKYNA levels through inhibition of kynurenine 3-hydroxy-lase constitutes a promising novel approach for managingLID in Parkinson’s disease. © 2005 Movement DisorderSociety

Key words: dyskinesia; glutamate; kynurenic acid; MPTP;Parkinson’s disease

Parkinson’s disease (PD) results from the death ofdopaminergic neurons in the substantia nigra pars com-pacta. Loss of these neurons leads to striatal dopaminedeficiency, which is responsible for the major symptomsof PD.1 Replacement of striatal dopamine by its biopre-cursor L-dopa alleviates most of these symptoms byactivation of striatal dopamine receptors, which are pri-marily located on medium spiny output neurons.2,3 How-ever, long-term L-dopa therapy is frequently associatedwith the development of abnormal involuntary move-

ments termed dyskinesias.4,5 Although the neurochemi-cal abnormalities responsible for L-dopa–induced dyski-nesias (LID) are not well understood,6 an impairedcortico–basal ganglia–thalamo–cortical loop is believedto be critical for triggering these motor complications.7

Dopamine and glutamate interact closely on the samedendritic spines of medium spiny striatal efferents.8 Un-der physiological conditions, glutamatergic afferentsfrom the cerebral cortex and the thalamus activate theseneurons directly, whereas dopamine serves as a modula-tor of glutamatergic excitation.9–11 This scenario is dis-rupted in LID due to changes in dopamine receptorfunction resulting from pulsatile and intermittent expo-sure to dopamine.12,13 In turn, these changes alter thefunction of glutamatergic afferents to the striatum.14–18

Both ionotropic [N-methyl-D-aspartate (NMDA), kai-nate, and �-amino-3-hydroxy-5-methyl-4-isoxazolepro-pionic acid (AMPA)] and metabotropic (mGluR1 to 8)

*Correspondence to: Dr. Paul J. Bedard, Centre de Recherche enNeurosciences, Le Centre Hospitalier Universitaire de Quebec, CHULRC-9800, 2705 Boulevard Laurier, Quebec, G1V4G2, Canada.E-mail: [email protected]

Received 20 August 2004; Revised 26 November 2004; Accepted 2December 2004

Published online 13 June 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mds.20596

Movement DisordersVol. 20, No. 7, 2005, pp. 792–802© 2005 Movement Disorder Society

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receptors are present in the striatum19–21 and are con-ceivably involved in the pathophysiology of LID. In-deed, both NMDA and AMPA receptors are upregulatedin the putamen of PD patients with LID.16 Further sup-porting an increase in glutamatergic tone, an increase inthe size of cortical afferent fibers and the number ofcorticostriatal synapses, as well as motor cortex hyper-activation, have been reported in dyskinetic PD patientsand monkeys.22–24 Moreover, systemic administration ofNMDA receptor antagonists prevents or reduces LIDwithout affecting the antiparkinsonian response to L-dopain MPTP parkinsonian monkeys.25–27 In the clinicalrealm, amantadine, budipine, and memantine, all drugswith significant potency as NMDA receptor antagonists,can improve LID in PD patients.28–30 Taken together,these studies suggest that heightened glutamate receptoractivity could play a role in the pathophysiology of LID,and interventions that reduce glutamatergic activity holdpromise for the treatment of LID.15,16,31,32

Two metabolites of the kynurenine pathway of trypto-phan degradation [quinolinic acid (QUIN) and kynurenic

acid (KYNA)] have opposing actions at the NMDA recep-tor. Thus, QUIN is a selective but rather weak NMDAreceptor agonist, whereas KYNA, at low micromolar con-centrations, acts as a competitive antagonist at the glycinecoagonist site of the receptor.33–35 At higher concentrations,KYNA can also reduce glutamatergic function by inhibitingAMPA and kainate receptors.33,36 In addition, KYNA in-hibits the �7 subtype of the nicotinic acetylcholine(�7nACh) receptor, and this effect is in part responsible forthe observed KYNA-induced reduction in striatal glutamaterelease.37 Interestingly, the levels of KYNA and its imme-diate bioprecursor kynurenine (KYN) are reduced in thebrain of L-dopa–treated patients with end-stage PD,38,39

suggesting that an increase in KYNA formation mightprovide therapeutic benefits in LID. We decided to test thishypothesis by using Ro 61-8048, a potent inhibitor ofkynurenine 3-hydroxylase, which is known to raise KYNAlevels after systemic administration in rodents in vivo.40 Asillustrated in Figure 1, inhibition of this pivotal enzymediverts kynurenine pathway metabolism toward the forma-tion of KYNA.35,37 The objective of this study was to

FIG. 1. Kynurenine pathway in mam-malian cells: effect of kynurenine 3-hy-droxylase inhibition.

KYNURENINE 3-HYDROXYLASE INHIBITION 793

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examine the biochemical and behavioral effects of systemicRo 61-8048 administration in MPTP parkinsonian mon-keys, which show a stable parkinsonian syndrome andexhibit reproducible dyskinesias after L-dopa administra-tion.

MATERIALS AND METHODS

Experimental Animals

All experiments were performed with four to six adultfemale cynomolgus monkeys (Macaca fascicularis)weighing 2.9 to 5 kg. The animals were handled inaccordance with the NIH Guide for the Care and Use ofLaboratory Animals, and all procedures were approvedby the Institutional Animal Care Committee of LavalUniversity. The animals were housed in separate obser-vation cages in a temperature-controlled room (23 �1°C) with a 12-hour light/dark cycle (lights on 6:00AM–6:00 PM). They were fed once daily in the afternoonwith pellets and fruits and had free access to water.Before the experiments, all monkeys were exposed tosubcutaneous (s.c.) injections of the neurotoxin MPTP atweekly intervals (2–3 mg per injection) until a bilateralstable parkinsonian syndrome developed (i.e., unchangedparkinsonian score of 8 or more, over at least 1 month).41

The total dose of MPTP and the time necessary to obtainsustained parkinsonian features varied from 9 to 23.5 mgand from 4 to 32 weeks, respectively. Each animal wasthen treated orally (p.o.), once daily, with L-dopa/benserazide (Prolopa) for a few months until dyskinesiaswith a predominantly choreic nature developed.41 Sub-sequently, dyskinesias were elicited acutely by adminis-tering L-dopa/benserazide. To maintain priming and forcomfort, the animals received Prolopa two or three timesper week.

Chemicals and Drugs

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)and all other fine chemicals were purchased from SigmaChemical (St. Louis, MO). Ro 61-8048 was synthesized byNiKem Research Srl according to Rover and colleagues.40

The powder was suspended in 0.1% (v/v) Tween 80/sterilewater, homogenized in a glass homogenizer, and adminis-tered as a homogeneous suspension. The maximum volumethus administered by oral gavage (p.o.) was 40 ml. As acontrol, the same volume of a 0.1% (v/v) Tween 80/sterilewater suspension was administered p.o.

L-dopa methyl ester (25–40 mg/kg; Sigma) was dis-solved in 1 ml of 0.9% sterile saline and administered s.c.This was always accompanied by an s.c. injection ofbenserazide (50 mg per monkey in 1 ml of 0.9% sterilesaline; Hoffmann-La Roche, Montreal, Canada). This

mixture is termed “L-dopa” throughout this article. Dos-ages refer to the amount of L-dopa methyl ester admin-istered. Prolopa (Hoffmann-La Roche), a mixture of 100mg L-dopa and 25 mg benserazide, was administeredorally where indicated.

Blood and Cerebrospinal Fluid (CSF) Sampling

In pilot experiments, blood samples were taken beforeand 3 hours after treatment with Prolopa. Subsequently,blood samples were obtained before and 90 minutes and3 hours after each treatment with Ro 61-8048. In ordernot to interfere with the behavioral observations, bloodwas only obtained before and 3 hours after the simulta-neous treatment of Ro 61-8048 and L-dopa. In one study,both blood and CSF samples were obtained 2 hoursbefore and 3 hours after the administration of Ro 61-8048 (100 mg/kg). CSF sampling, either at lumbar orcervical locations, was performed under light ketamine/xylazine anesthesia. Venous blood (5 ml per monkey)was collected in a vacutainer tube. Blood was then cen-trifuged (10 min, 1,000 g) at 4°C. Both serum and CSFsamples were frozen and stored at �80°C until the day ofthe assay.

Behavioral Analyses

Drugs were always administered in the morning of theday of the experiment. Spontaneous behaviors of theanimals were assessed through a one-way screen andscored every 15 minutes using the parkinsonian disabil-ity and dyskinesia scales described below. Animals wereassessed undisturbed for 3 hours or until the drug effecthad subsided (during the on state). An observer who wasunaware of the experimental protocol assessed the be-havioral response to the simultaneous administration ofL-dopa and Ro 61-8048.

Dyskinesias were evaluated in face, neck, trunk, arms,and legs as follows: none � 0, mild � 1, moderate � 2,and severe � 3. These were based on assessment of theamplitude, interference with normal motor activity, andfrequency of abnormal movements according to a vali-dated rating scale.41,42 The total dyskinesia score was thesum of the ratings for all body segments (maximal score,21). The mean dyskinesia score for each animal wascalculated as the average of all scores obtained for 3hours or for the total duration of the drug effects.

The parkinsonian syndrome was evaluated for eachanimal before and after the treatments according to anestablished scale.41,42 This included assessment of thefollowing parameters: (1) posture: normal � 0, flexedintermittent � 1, flexed constant � 2, crouched � 3; (2)mobility: normal � 0, mild reduction � 1, moderatereduction � 2, severe reduction � 3; (3) climbing:

794 P. SAMADI ET AL.


present � 0, absent � 1; (4) gait: normal � 0, slow � 1,very slow � 2, very slow with freezing � 3; (5) groom-ing: present � 0, absent � 1; (6) vocalization: present �0, absent � 1; (7) social interactions: present � 0,absent � 1; (8) tremor: absent � 0, mild action tremor �1, moderate action tremor � 2, resting tremor � 3. Themaximal disability score was 16. The mean parkinsonianscore for each animal was the average of all scoresobtained for 3 hours or for the total duration of the drugeffects. The minimum parkinsonian score was the mini-mum score observed during 3 hours or for the totalduration of the drug effects.

Locomotor activity was quantified using an electronicmotility monitoring system (Datascience, St. Paul, MN).Using radiowave frequency, a probe around the neck ofeach animal transmitted the signal to a receiver that wasfixed on each cage. This receiver was connected to acomputer and provided a motility count every 5 minutes.

Biochemical Analyses

KYN and KYNA Determination.

Serum and/or CSF was thawed and diluted (1:50 and1:10, respectively; v/v) in ultrapure water. A 300 �laliquot of the diluted sample was acidified with 75 �l of6% perchloric acid. After centrifugation (10 min, 12,000g), an aliquot of the supernatant was diluted (1:1, v/v)with HPLC mobile phase (200 mM zinc acetate and3.5% acetonitrile, pH 6.2). Two hundred �l aliquotswere subjected to HPLC analysis using a 3 �m C18

HR80 reverse-phase column (80 � 4.6 mm; ESA,Chelmsford, MA) at a flow rate of 1 ml per minute. In theeluate, KYN was detected in-line by UV absorbance at365 nm. KYNA was determined fluorimetrically using aPerkin-Elmer LC 240 Fluorescence Detector (Perkin-Elmer, Beaconsfield, U.K.; excitation wavelength, 344nm, emission wavelength, 398 nm). The retention timesof KYN and KYNA under these conditions were approx-imately 4 and 5 minutes, respectively. Data were re-corded using an HP 3390A integrator (Hewlett-Packard,Palo Alto, CA).

Quinolinic Acid Determination.

Serum and/or CSF was thawed, and 2.5 �l aliquotswere added to a glass tube. Fifty microliters of internalstandard (100 nM 3,5-pyridinedicarboxylic acid) and 50�l of 62.5 mM tetrabutylammonium hydrogen sulfatewere added, and the mixture was lyophilized overnight.The samples were then reacted with 25 �l of methylenechloride containing 7.5% of diisopropylethylamine and3% pentafluorobenzyl bromide (PFB-Br) at 60–65°C for15 minutes. Subsequently, 50 �l of decane and 750 �l of

water were added. The samples were thoroughly mixed,and the decane phase was removed. The gas chromatog-raphy (GC) and the electron capture negative ionizationmass spectrometry (MS) system (ThermoFinnigan, SanJose, CA) was a Trace GC coupled with a Trace MSquadrupole mass spectrometer. Chromatographic separa-tion and GC/MS analysis was achieved using a 30 m

FIG. 2. Parkinsonian (Park) score and locomotor activity followingthe systemic administration of Ro 61-8048 on 3 consecutive days. Fourmonkeys were used in the study. A: Mean and minimum parkinsonianscores 3 hours after each treatment. Data are mean � SEM. There wasno significant difference in parkinsonian symptoms between the threedoses of Ro 61-8048 and vehicle treatment (nonparametric Friedman’stest followed by multiple comparisons based on Friedman rank sums).B: Total mobility counts were recorded individually during 2 hoursafter Ro 61-8048 administration. Data are mean � SEM. There were nosignificant treatment effects (ANOVA for repeated measures followedby Fisher’s probability of least significant differences).



Rtx-5MS capillary column (0.25 mm i.d., 0.25 �m filmthickness, Restek) with helium as a carrier gas. A split/splitless injection port (1 �l injection volume) was usedat 220°C. The temperature program was as follows:155°C for 1.25 minutes, 40°C/minutes to 275°C, 10°C/minutes to 320°C, 1 minutes at 320°C. The ion sourcetemperature was 220°C. Selected ion monitoring wasperformed by recording the signal of the characteristic(M-PFB) ion, a di-PFB derivative with an m/z ratio of346.43

Statistical Analysis

The mean parkinsonian and dyskinesia scores ob-tained for each animal during 3 hours or the total dura-tion of the effect of a given drug were analyzed using thenonparametric Friedman’s test followed by multiplecomparisons based on Friedman rank sums or the Wil-coxon matched-pairs signed-rank test. For locomotor ac-tivity, total mobility counts were recorded for each ani-mal up to 3 hours following a given treatment. Data forall monkeys were then compared using an analysis ofvariance (ANOVA) for repeated measures followed byFisher’s probability of least significant differences(PLSD). Biochemical data were analyzed using the non-parametric Friedman’s test followed by PLSD.

RESULTS

Behavioral Analyses

Ro 61-8048 Administration.

Compared with vehicle treatment, Ro 61-8048 admin-istration (10, 30, or 100 mg/kg, administered on 3 con-secutive days) did not have any effect on the animals’parkinsonian score or locomotor activity (Fig. 2).

Simultaneous Administration of L-Dopa andRo 61-8048.

Coadministration of Ro 61-8048 (30 or 100 mg/kg)and L-dopa (25 mg/kg) resulted in a significant reductionof the dyskinetic response to L-dopa on 3 consecutivedays. Compared to results obtained with L-dopa alone,dyskinesias were reduced by approximately 20% by the

FIG. 3. Dyskinetic response to L-dopa (25 mg/kg) alone (solid bars) orin conjunction with increasing doses of Ro 61-8048 (hatched bars) on3 consecutive days. Doses of Ro 61-8048 (in mg/kg) are given insidethe bars. Dyskinesia scores were obtained during 3 hours after treat-ment. Data are mean � SEM (n � 4). **P � 0.01 vs. respectivecontrols (L-dopa alone; nonparametric Friedman’s test followed bymultiple comparisons based on Friedman rank sums).

FIG. 4. Time course of the dyskinetic response to L-dopa (25 mg/kg) alone (solid squares) or in conjunction with increasing doses of Ro 61-8048(open circles) on 3 consecutive days. Each data point illustrates the average dyskinetic response, obtained during a 15-minute observation period. Dataare the mean � SEM (n � 4). *P � 0.05 (two-by-two comparisons for each 15-minute period; Wilcoxon matched-pairs signed-rank test).



addition of 30 or 100 mg/kg of Ro 61-8048. No sucheffect was observed when 10 mg/kg Ro 61-8048 wascoadministered with L-dopa (Fig. 3). The time course ofdyskinetic responses is illustrated in Figure 4 (P � 0.05;two-by-two comparisons for each 15-minute period;Wilcoxon matched-pairs signed-rank test). Notably, co-administration of Ro 61-8048 and L-dopa did not affectthe improvement in parkinsonian score and locomotoractivity induced by L-dopa alone. All these drug treat-ments were significantly different from vehicle treatment(Fig. 5).

L-Dopa Administration Preceded by TreatmentWith Ro 61-8048.

Acute administration of Ro 61-8048 (100 mg/kg) 90minutes before L-dopa (30–40 mg/kg) once a week for 3weeks reduced the dyskinetic response to L-dopa byapproximately 20% (Fig. 6A). The time courses of dys-kinesias induced by L-dopa alone and by Ro 61-8048 �L-dopa are shown in Figure 6B (P � 0.05; two-by-twocomparisons for each 15-minute period; Wilcoxonmatched-pairs signed-rank test). Pretreatment with Ro61-8048 did not affect the improvement in parkinsonianscore and locomotor activity induced by L-dopa alone.All these drug treatments were significantly differentfrom controls (Fig. 6 C,D).

Biochemical Analyses

Effect of L-Dopa on Serum KYN and KYNA Levels.

Serum levels of KYN and KYNA were determined infour animals before and 3 hours after a dose of Prolopa.This treatment did not cause significant changes in thelevels of either metabolite [basal values: 6.49 � 0.71 �M(KYN) and 102 � 20 nM (KYNA); 3 hours: 5.98 � 0.70�M (KYN) and 85 � 13 nM (KYNA); Wilcoxonmatched-pairs signed-rank test; Z � �1.83, P 0.07,KYN; Z � �1.47, P 0.14, KYNA].

Effect of Ro 61-8048 Alone or in Combination WithL-Dopa on Serum Levels of KYN, KYNA, andQUIN.

As illustrated in Figure 7, Ro 61-8048 administrationsignificantly increased the serum levels of KYN 3 hoursafter the administration of 30 or 100 mg/kg Ro 61-8048.Serum levels of KYNA were significantly increased bothat 90 minutes and 3 hours after 10, 30, or 100 mg/kg Ro61-8048 (n � 4).

Coadministration of the two highest doses of Ro 61-8048 (30 and 100 mg/kg) with L-dopa (25 mg/kg) causedsignificant dose-dependent increases in KYN and KYNAlevels in the serum 3 hours after drug treatment (n � 4).

No significant differences in the increase in KYN andKYNA levels were observed between Ro 61-8048 ad-ministration alone or in combination with L-dopa.

No changes in serum QUIN levels were detected 3hours after the coadministration of Ro 61-8048 and L-dopa (25 mg/kg). Thus, basal QUIN levels were 708 �

FIG. 5. Antiparkinsonian effect of L-dopa (25 mg/kg) alone or inconjunction with increasing doses of Ro 61-8048 on 3 consecutive days(n � 4). A: Mean and minimum parkinsonian (Park) scores at 3 hours.Data are mean � SEM. There was no difference between animalsreceiving L-dopa alone and animals receiving Ro 61-8048 � L-dopa.However, all drug effects were significantly different from vehicle(P � 0.01; nonparametric Friedman’s test followed by multiple com-parisons based on Friedman rank sums). B: Locomotor activity during2.5 hours after each treatment. Data are mean � SEM. Animalsreceiving Ro 61-8048 � L-dopa did not show significant further im-provement in locomotor activity compared to animals treated withL-dopa alone (P 0.05, ANOVA for repeated measures). All treatmenteffects were significantly different from vehicle (P � 0.01; ANOVAfor repeated measures followed by Fisher’s probability of least signif-icant differences).



154 nM, and levels following the application of 10, 30,and 100 mg/kg Ro 61-8048 were 744 � 130, 771 � 128,and 656 � 49 nM, respectively (P 0.1; nonparametricFriedman’s test; n � 4).

Effect of Ro 61-8048 on CSF Levels of KYN,KYNA, and QUIN.

Treatment with Ro 61-8048 (100 mg/kg) caused asignificant increase in the CSF levels of KYN andKYNA, but not QUIN, after 3 hours (Table 1). In thesame animals, blood samples were collected before se-dation for CSF sampling and 3 hours after Ro 61-8048administration. Notably, serum KYN and KYNA levelsin these monkeys were not raised as high as expectedfrom the preceding results, possibly due to the effects ofthe ketamine/xylazine combination used to induce anes-thesia for CSF sampling (cf. Fig. 7 and Table 1).

DISCUSSION

The results of the present study indicate that systemicadministration of the kynurenine 3-hydroxylase inhibitorRo 61-8048 has a beneficial effect on LID in parkinso-nian dyskinetic monkeys. Importantly, improvement of

dyskinesias was observed after the joint application ofRo 61-8048 and L-dopa, and the treatment had no effecton the antiparkinsonian efficacy of L-dopa. Parallel bio-chemical analyses showed that Ro 61-8048 increased thelevel of KYN and KYNA in serum and CSF of parkin-sonian animals, and that this effect was not influenced bythe coadministration of L-dopa. The rather moderate yetsignificant reduction of dyskinesias (by about 20%) sug-gests that pharmacological interventions resulting in theincreased formation of KYNA may provide clinical ben-efit as a novel adjuvant in the treatment of motor com-plications in PD.

KYN, the pivotal metabolite of the major tryptophandegradation pathway in mammals, is present in bloodand brain in low micromolar concentrations. At leastthree distinct enzymes, kynurenine 3-hydroxylase,kynureninase, and kynurenine aminotransferase, nor-mally catalyze the breakdown of KYN in mammaliantissues (cf. Fig. 1). In rodents, inhibition of kynurenine3-hydroxylase disturbs the catabolic balance and causesincreases in the concentration of both KYN andKYNA.44,45 This is associated with antidystonic efficacy

FIG. 6. Dyskinetic and antiparkinsonianresponses to L-dopa (30–40 mg/kg) aloneor in combination with Ro 61-8048 (100mg/kg, administered 90 minutes before L-dopa; n � 6). Each bar or curve representsthe mean � SEM of three experimentscarried out once a week for 3 weeks. A:Mean dyskinesia scores. **P � 0.01 vs.L-Dopa alone (nonparametric Friedman’stest followed by multiple comparisonsbased on Friedman rank sums). B: Meandyskinetic responses, obtained every 15minutes, of monkeys treated with L-dopaalone (solid squares) or Ro 61-8048 �L-dopa (open circles). *P � 0.05 (two-by-two comparisons for each 15-minute pe-riod; Wilcoxon matched-pairs signed-ranktest). C: Mean parkinsonian score 3 hoursafter L-dopa. D: Total locomotor activityduring 3 hours after L-dopa. All drug ef-fects in C and D were significantly differ-ent from vehicle-injected controls (P �0.01). No significant differences were ob-served between treatments with L-dopaalone and Ro 61-8048 � L-dopa (C, non-parametric Friedman’s test followed bymultiple comparisons based on Friedmanrank sums; D, ANOVA for repeated mea-sures followed by Fisher’s probability ofleast significant differences).



as recently shown by using Ro 61-8048 in a geneticmodel of dystonia.46 Our biochemical results demon-strated that inhibition of kynurenine 3-hydroxylase byRo 61-804840 also raises serum KYN and KYNA levelsin monkeys in a time- and dose-dependent manner. By 3hours, the two higher doses of Ro 61-8048 used (30 and100 mg/kg) raised the levels of KYNA up to 20-fold, andthese increases were accompanied by substantial eleva-tions in KYN. Notably, coadministration of L-dopa didnot influence these effects of Ro 61-8048. No changes inserum QUIN levels were detected after these treatments,suggesting that the doses of Ro 61-8048 used wereinsufficient to interfere effectively with metabolismalong the QUIN branch of the kynurenine pathway.

The increase in KYN and KYNA levels seen follow-ing inhibition of kynurenine 3-hydroxylase was paral-leled by a moderate but significant reduction of L-dopa–induced dyskinesias. Interestingly, Ro 61-8048 did notinfluence the antiparkinsonian effectiveness of L-dopa inthis coadministration paradigm. Since serum KYNA lev-els started to rise 90 minutes after the application of Ro61-8048, a second experiment was performed to test thebehavioral outcome when Ro 61-8048 was given 90minutes before L-dopa. In this pretreatment scenario, too,the kynurenine 3-hydroxylase inhibitor reduced the dys-kinetic response to L-dopa without diminishing antipar-kinsonian efficacy. These findings suggested a possiblecausal relationship between elevated KYNA levels andthe observed antidyskinetic effect.

Because of its polar structure and the lack of specifictransport mechanisms, KYNA is largely prevented fromentering the brain from the circulation. In contrast, pe-ripheral KYN readily penetrates through the blood–brainbarrier using the large neutral amino acid carrier. In thebrain, KYN is avidly accumulated by glial cells, whichalso harbor the various enzymes of the kynurenine path-way. Kynurenine aminotransferases, which are preferen-tially contained in astrocytes,47 are responsible for theirreversible transamination of KYN to KYNA, and

FIG. 7. Effect of Ro-61-8048 alone or in combination with L-dopa (25mg/kg) on serum KYN and KYNA concentrations in four MPTPparkinsonian monkeys. Increasing doses of Ro 61-8048 were admin-istered on 3 consecutive days. Blood samples were taken before (whitebars) and 90 minutes (gray bars) and 3 hours (black bars) after eachtreatment. Data are mean � SEM. **P � 0.005 and ***P � 0.001 vs.corresponding basal levels; †P � 0.05 and ††P � 0.01 vs. correspond-ing levels at 90 minutes after treatment (nonparametric Friedman’s testfollowed by Fisher’s probability of least significant differences).

TABLE 1. Kynurenine, kynurenic acid, and quinolinic acid levels in serum and cerebrospinal fluid ofparkinsonian monkeys treated with Ro 61-8048

2 hours before treatment 3 hours after treatment

KYN (�M) KYNA (nM) QUIN (nM) KYN (�M) KYNA (nM) QUIN (nM)

Serum 7.01 � 0.85 123 � 23 1,232 � 175 7.83 � 0.44 418 � 36a 1,344 � 127CSF 0.11 � 0.01 5.3 � 1.8 137 � 9 0.25 � 0.5a 7.3 � 2.4a 131 � 26

Values are expressed as mean � SEM of six animals.CSF was obtained under light ketamine/xylazine anesthesia. Ro 61-8048 (100 mg/kg) was administered 2 hours after the first

CSF sampling.aP 0.05 versus basal levels (Wilcoxon matched pairs signed-rank test).KYN, kynurenine; KYNA, kynurenic acid; QUIN, quinolinic acid.



newly formed KYNA promptly enters the extracellularcompartment.48 These events probably account for theincrease in both KYN and KYNA levels in the CSF ofparkinsonian monkeys after the systemic administrationof Ro 61-8048. However, since it is not known whetherperipherally administered Ro 61-8048 penetrates theblood–brain barrier, the observed increases in CSF KYNand KYNA levels may have also been caused by inhibi-tion of brain kynurenine 3-hydroxylase by the drug.

Several lines of evidence suggest that a reduction inglutamatergic tone may provide clinical improvement inLID and may also be of benefit in the prevention ofabnormal movements induced by prolonged administra-tion of dopamine receptor agonists. Thus, chronic pulsa-tile L-dopa administration causes motor cortex hyperac-tivation in PD patients, who have reduced dopamine-buffering capacity.24 This in turn leads to theconcomitant hyperactivity of corticostriatal glutamater-gic projections, which play a crucial role in the inductionof dyskinesias.15,16,26,49 Moreover, NR1/NR2B NMDAand AMPA receptors are upregulated in the putamen ofPD patients with motor complications,16 and these alter-ations are believed to be critically involved in the patho-physiology of dyskinesias.16,26,50 Accordingly, adjunc-tive treatment with glutamate receptor antagonists causesimprovement in LID,25,27,28,51 and coadministration of anNR1A/NR2B-selective NMDA receptor antagonist withL-dopa prevents the induction of dyskinesias in drug-naive parkinsonian monkeys.26

KYNA normally inhibits the glycine coagonist site ofthe NMDA receptor with an IC50 in the low micromolarrange.33–35 Since brain KYNA levels were not measuredhere, and since CSF determinations do not accuratelyreflect synaptic concentrations, it is not clear if the Ro61-8048–induced elevations in KYNA formation in thepresent study were sufficient to affect NMDA receptorsdirectly. Although even modest increases in brain KYNAcan attenuate excitatory synaptic function,45,52,53 mecha-nisms other than direct NMDA receptor blockade maytherefore have contributed to the Ro 61-8048–inducedreduction in LID described here.54–56 For example, sys-temic administration of Ro 61-8048 or local perfusionwith very low concentrations (30–100 nM) of KYNApotently inhibits glutamate release in the rat striatum, andthis effect cannot be prevented by NMDA receptorblockade.37 It may be mediated by the �7 subtype of thenicotinic acetylcholine (�7nACh) receptor, which is se-lectively inhibited by low concentrations of KYNA.54 Infact, since activation of presynaptic �7nACh receptors isassociated with enhanced glutamatergic transmis-sion,57,58 the improvement of dyskinesias by Ro 61-8048may be related in part to KYNA-induced �7nACh re-

ceptor-mediated presynaptic inhibition of glutamate re-lease.

In spite of the fact that a significant reduction in LIDwas observed when L-dopa was applied together with Ro61-8048, several mechanisms may have obscured evengreater efficacy of the kynurenine 3-hydroxylase inhibi-tor. First and for unknown reasons, systemic L-dopaadministration acutely reduces extracellular levels ofKYNA in the brain.59 Second, both L-dopa and KYNenter the brain through the same carrier, namely, thelarge neutral amino acid transporter (F. Moroni, personalcommunication), so that the simultaneous administrationof the two drugs may have limited the ability of KYN topenetrate into the brain and serve as a precursor of brainKYNA. Third, L-dopa therapy in rodents results in aselective increase in the astrocytic glutamate transporterprotein GLT1 in the striatum,60 allowing abnormallylarge amounts of glutamate to be taken up into the cell.Since brain KYNA synthesis takes place almost exclu-sively in astrocytes,35,61 and since glutamate is an effec-tive inhibitor of one of KYNA’s major biosyntheticenzymes,61 L-dopa treatment may indirectly interferewith the transamination of KYN to KYNA in the brainand thus diminish the efficacy of Ro 61-8048. Finally,L-dopa treatment was recently shown to decrease theexpression of striatal nACh receptors,62 and this effect,too, may have compromised the effectiveness of elevatedbrain KYNA. Additional studies using a dopamine re-ceptor agonist in the place of L-dopa could clarify severalof these issues.

In conclusion, the present study provides both behav-ioral and biochemical evidence in support of the hypoth-esis that pharmacological interventions resulting in in-creased KYNA formation could be a useful adjuvant inthe treatment of drug-induced motor complications inPD. Such treatments will counteract the KYNA deficitthat exists in the brain of L-dopa–treated Parkinson’sdisease patients.39 Through presynaptic inhibition of glu-tamate release and/or blockade of upregulated NMDAreceptors, elevated KYNA levels may then offset thehyperglutamatergic tone, which presumably underliesLID. Notably, this novel pharmacological approach mayoffer advantages compared to antidyskinetic drugs withNMDA receptor channel blocking activity such as aman-tadine, which are currently in clinical use as adjuvants toL-dopa.63–65

NOTE ADDED IN PROOF

In support of the present study, we recently showedthat nanomolar concentratiosn of KYNA significantlyreduce the extracellular levels of dopamine in the ratstriatum.66



Acknowledgments: This study was supported by NewronPharmaceuticals and a grant from the Canadian Institute ofHealth Research (to P.J.B.), as well as a scholarship from leFonds de la Recherche en Sante du Quebec (to P.S.). We thankSteve Brochu for excellent technical assistance and MoniqueBaron for administrative help.

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