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Home , Dopamine transporter, Ethylphenidate, Serotonin transporter, Tropane

Carbon-i i-d-threo- Binding to Transporter in Baboon

Yu-Shin Ding, Joanna S. Fowler, Nora D. Volkow, Jean Logan, S. John Gatley and Yuichi Sugano

Brookhaven National Laboratory, Upton, New York; and Department of Psychiatry, SUNY Stony Brook@Stony Brook@NY

children (1). MP is also used to treat (2). The The more active d-enantiomer of methyiphenidate (dI-threo psychostimulant properties of MP have been linked to its methyl-2-phenyl-2-(2-piperidyl)acetate, Ritalin)was labeled with binding to a site on the , resulting in lic (t1,@:20.4 mm) to characterize its binding, examine its spec inhibition of dopamine and enhanced levels of ificftyfor the dopamine transporter and evaluate it as a radio synaptic dopamine. tracer forthe presynapticdopaminergicneuron. Methods PET We have developed a rapid synthesis of [11C]dl-threo studies were canied out inthe baboon. The of methylphenidate ([“C]MP)to examine its pharmacokinet r1c]d-th@O-msth@ha@idate @f'1C]d-thmo-MP)weremeasured ics and pharmacological profile in vivo and to evaluate its and compared with r1cY-th@o-MP and with fts racemate ff@1C]fl-thmo-meth@1phenidate,r1c]MP). Nonradioact,ve meth suitability as a radiotracer for the presynaptic ylphenidate was used to assess the reveralbilityand saturability (3,4). These first PET studies of MP in the baboon of the binding. GBR 12909, 3@3-(4-iodophenyI)-2-car and brain demonstrated the saturable [1‘C]MP boxylic acid methyl ester (fi-Cfl), tomoxetine and binding to the dopamine transporter in the baboon brain were used to assess the binding specificity. Results The ratio and its sensitivity to dopamine neuron degeneration in between radioactivityinthe sttiatum and that inthe cerebellum Parkinson's disease. Although these results are promising (ST/CB)after injection of r1c]d-th@-MP was higherthan that in terms of labeled MP as a for the presynaptic for r1c]MP and r1cv-th@o-MP (3.3 for d-, 2.2 for racemic and dopaminergic neuron, the use of dl-threo-methylphenidate, 1 .1 for I- in the same baboon). Most of the striatal binding of the form which is marketed, is not ideal because it is a [11CJd-threo-MP was displaceable by injection of nonradioactive MP. Pretreatment with nonradioactive MP (0.5 mg/kg), mixture of enantiomers. It has been shown that d-threo-MP GBR12909 (1.5 mg/kg) and R11-55(0.3 mg/kg) markedly re is more potent in the induction of locomotor activity and duced striatalbut not cerebellaruptake ofr1C]d-threo-MP. Inall has a higher affinity for the dopamine transporter than cases, the ST/CBafterpretreatmentwas reduced byabout 60% l-threo-MP (5). The potency (IC50) of dl-threo-MP in dis compared to 43% for r1C]MP. The ratios of distribution vol placing [3H]dl-threo-MP from striatal synaptosomal mem umes at steady-state forthe ST/GBforthe three separate stud brane binding sites is 0.21 pM compared to 0.088 pM for ies in the same baboon were reduced by about 50%, as com the d-enantiomer and 1.2 pM for the l-enantiomer (6). The pared with 37% for r1C]MP. In contrast, pretreatment with relative affinities of racemic MP and its individual enanti tomoxetine(3.0 mg/kg) or citalopram (2.0 mg/kg) did not change omers to monoamine transporters have been reported r1cw-th@O-MP kinetics; the ST/GB after pretreatment was similar to that for the control. Conclusion: These results dam (5, 7,8) (Table 1). Thus, while [11C}d-threo-MP would be onstrate the saturable, reversibleand specificbindingof r 1C]d- predicted to be a promising PET ligand, [11C]l-threo-MP thmo-MP to the dopamine transporter in the baboon brain, would be expected to contribute to nonspecific binding and suggesting that r1Gw-th@-MP willbe a useful PETtracer for to the absorbed radiation burden. Additionally, while it has the presynaptic dopaminergicneuron in lMnghuman brain. been demonstrated that the two enantiomers have a similar after intravenous (but not oral) administra Key Words: carbon-i 1-d-thmo methylphenidate; dopamine transporter@stereoselectMty; dopaminergk@neuron tion (9,10), the use of [11C}d-threo-MP,rather than the , is preferred because it would allow mea J NuciMed1995;36@98-2305 surement of an arterial input function from a single labeled compound, thus facilitating quantitation. We report here the characterization of [11C]d-threo-MP binding in baboon brain which include the following: mea (MP, Ritalin) is the drug of choice surement of regional brain uptake and clearance; an assess for the treatment of attention deficit hyperactivity disorder ment of the reproducibility of repeated measures; the effect (ADHD), which is estimated to affect 2%—5%of school age of pharmacological interventions with various drugs to as sess if the binding is saturable and specific. The saturability of the binding was assessed with unlabeled methylpheni ReceivedNov.8, 1994;revisionaccepted Apr.8, 1995. Forcorrespondenceor reprintscontactVu-Shin1@ng,PhD,Departmentof date. An assessment of the specificity of [‘1C]d-threo-MP Chemistrj,Bldg.555,BrookhavenNationalLaboratory,Upton,NY11973. for the presynaptic dopaminergic neuron was determined

2298 The Journal of NuclearMedicine•Vol.36 •No. 12 •December 1995 TABLE I pling. For each paired study, two tracer doses of [‘‘CJd-threo-MP Binding of Methyiphenidate to Monoamine Transporters (5—9mCi in 3 ml saline, 0.017—0.03j.@mole(4—[email protected])per injection; i.v.) was administered with a 2—3-hrtime period between doses to Dopamine Norepinephnne test the reproducibility of measurements or to examine the effects rWVd@threo@MP*276520,000I-threo-MP'360280023,000dI@threo@MPt390190015,000TransportBinding (Ki, of drug pretreatment on the binding of [‘‘CJd-threo-MP.The same scanning protocol was performed as described for [1‘C]dl-threo MP(4). Arterialbloodsamplingandplasmaassayforthe presence of unchanged labeled MP were carried out following the same nM)d-thieo-MP@1300190!-threo-MP@1(lCso, procedure as reported for [‘‘C]dl-threo-MP.Vital signs, including heart and respiratory rate, were monitored and recorded through 1,0001200 out the study.

*Becauseof a limitedamount of material,these are the results of a Drug Prefreatment single experiment(10 concentrations of compound, int,iplicate).Forthe The pharmacological profile of [‘‘C]d-threo-MPbinding in ba dopamine, norepinephrineand serotonintransporters, [@H]WlN35,428, boon was determined by carrying out a baseline PET study and [@H]nisoxetlne and [@H], respectively, were used with mem then pretreating with an intravenous injection of the following branespreparedfromthe ,frontalcortexand brainstemofrat drugs at the pharmacological doses prior to the second injection of brain,respectively,as described by Pan at al. (7). [‘1C]d-threo-MP:dl-threo-MP (0.5 mg/kg, 24 mm prior); GBR tAdapted from Ritzat al. (8). 12909(1.5 mg/kg, 23 and 45 mm prior); p3-CIT(0.32 mg/kg, 90 mm @AdaptedfromPatrickat al. (5). prior); tomoxetine (3.0 mg/kg, 20 mm prior); and citalopram (2.0 mg/kg, 30 and 120 mm prior). The timing for drug administration was chosen for maximum drug uptake at the time of tracer admin by pretreatment with GBR 12909 (a selective dopamine istration. This information was available from PET studies for uptake blocker) (11), 3/3-(4-iodophenyl)tropane-2-carbox GBR 12909(17) and dl-threoMP (4). Citalopramhas been la ylic acid methyl ester (/3-CIT) (an inhibitor of both the beled with “Cand tissue distribution performed in mice (18). dopamine and serotonin transporters) (12), tomoxetine (a Pretreatment times of 30 mm and 2 hr were chosen. The timing for selective uptake inhibitor) (13) or cita /3-CIT was obtained from a SPED' study with [‘231]13-CIT(12). lopram (a selective serotonin uptake inhibitor) (14). A The timing for tomoxetine was based on the report of Kleven et al. chase experiment with unlabeled MP was also performed to (13). further verify binding reversibility and specificity. The time Chase Expetiment course of unchanged tracer in arterial plasma was also To investigatethe reversibilityand specificityof [‘‘C]d-threo measured and used to calculate the steady-state distribu MP binding,unlabeledMP (0.5mg/kg)wasinjected15mm after tion volume in brain (15). the second tracer dose of [‘‘C]d-threo-MP.

MATERIALS AND METhODS Assay of Carbon-11-d-thmo-Methylphenidate in Plasma Unchanged F'‘C]d-threo-MPin plasma was determined by a Unlabeled dl-threo-MP - HC1 and ritalinic acid were provided solid-phase extraction method as described for [‘‘C]MP(4). by Ciba-Geigy Corp., Summit, NJ. The individual enantiomers (d-threo-MP and l-threo-MP) were prepared in our laboratoiy Image and Data Malysis accordingto a previouslypublishedmethodfor preparativesepa Regionsof interest (ROIs) on baboon brain were drawn di ration of dl-threo-MP using (R)-(—)and (S)-(+) 1,1'-binaphthyl rectly on the PET scans as described previously (16). The striatal 2-2'-diylhydrogenphosphateas the resolvingreagents(5). Cita ROIsweredrawnin twosequentialplanesat the levelof the genu lopram and tomoxetine were provided by Lundbeck (Sweden) and of the corpus callosum. The thalamic ROl was drawn in the lower Eli Lilly (Indianapolis, IN), respectively, and GBR12909 was pur plane from which the striatal regions were obtained. The brain chased from Research Biochemicals Inc., Natick, MA. stem ROIs were drawn on the slice immediately inferior to the Synthesis of Carbon-11-d-threo-Methylphenidate level of the thalamic region. The thalamic ROl was drawn across Carbon-11-d-threo-MP was prepared in two steps: 0-methyl the midline to include both the right and left sides. Cerebellar ation of the N-protected d-threo-ritaliniic acid derivative with ROIs were drawnin the plane that intersectedthe middleof the cerebellum and ROIs were obtained in the left and right hemi [1 ‘C]H31 followed by hydrolysis. The total synthesis time was 40 mm, with an average specific activity of 1.5 Ci/@mole(EOB) and spheres. radiochemical (>98%) and enantiomeric purity (99%) (3). Time-activity data (%ID/cc) at 60 mm for tissue ‘‘Cconcentra tion were used to calculate the striatum-to-cerebellum ratio. Time PETStudies of @arbon-11-d-threo-MethyIphenidate activitycurvesfor ‘‘Cand the timecourseof unchangedtracer in Baboon Studies. Two adult female baboons (Papio anubis) were plasma were used to calculate the distribution volume in the in used in six paired PET studies over an 8-mo period with at least 4 striatal,thalamic,cerebellarand midbrainregionsusinga graph wk betweenstudies.The baboonwas anesthetizedand prepared ical analysis method for reversible systems (Logan plots) as pre for PET studies as described previously (16). Animals were mi viously described (15). The ratio of the distribution volume in tially anesthetized with an intramuscular injection of striatum to that in the cerebellum was used as the model param hydrochloride (10 mg/kg), intubated and transported to the PET eter to calculate dopamine transporter availability at baseline and facility. They were maintained on oxygen, nitrous oxide and isoflu after drug interventions. rane throughout the study. Catheters were placed in an antecubital The distribution volume provides a measure of binding that is a vein for radiotracer injection and femoral artery for blood sam linear function of transporter availability given by:

Garbon-11-d-th,@o-MethyIphenidate in Baboon Brain •Ding at al. 2299 0.06 3.5

@ 0.05 3.0 H) E @ 0.04 :@ 2.5 0 .0 .@ 0.03 c@ 2.0 U E ? 0.02 ! 1.5

0.01

0 0.50 0 10 20 30 40 50 60 70 0.0 10 20 30 40 50 60 70 TIme (mln.) TIme (mm.) FIGURE1. Time-actMtycurvesfor11Cinthe st,iatum(solkicir FIGURE2. Ratiosofstriatum-to-cerebellumfor[11C]d-threo-MP cbs), (solid squares) and cerebellum (open squares) of (diamonds), r1c]MP (triangles)and r1cy-th@-Mp (circles) in the baboon brain after injection of r1GJd-th@-Mp. Curves represent same baboon (Brie). an average of six different runs in the same baboon (Brie).

ence in the striatum-to-cerebellum ratio: 3.3 for [1‘C]d DV = K11k2(1+ B@,ssfK@) Eq. 1 threo-MP, 2.2 for [1'C]MP and 1.1 for [“C]l-threo-MP(Fig. for regions containing transporter sites characterized by an equi 2). librium dissociation constant K@(K@= KdfNS; 1NSmeans free The average distribution volumes of the striatum and fraction of tracer in tissue) and transporter concentration B@. K, cerebellum for baseline studies of [“CJd-threo-MP, and k2are the plasma-to-tissueand the tissue-to-plasmatransport [“C]MPand [“C]l-threo-MPfor baboons Brie and Angel constant,respectively.For regionswithno transporter,the distri are presented in Table 2. The distribution volume for the bution volume is given by: striatum decreased in the order d > dl > 1.The ratio of the DV = K,/k2. Eq. 2 distribution volumes (striatum-to-cerebellum) averaged 2.4 for baboon Brie and 2.2 for baboon Angel. Although we A parameterproportionalto freetransporterconcentrationcanbe observed high intersubject variability in the absolute values obtained from Equations 1 and 2 giving: for the striatal and the cerebellar distribution volumes B@/K@= [DVROI/DVCBI 1, Eq. 3 (compare Brie and Angel), the distribution volume ratios for the d and 1 enantiomers for the two baboons were where K@,and k2 include the free fraction of tracer in tissue. similar. The cerebellar values for the d and 1 isomers dif Equations 1 and 2 are based on classical compartmental analysis in fered from one another (p < 0.02). It is noted that the which the effects of CBF and capillary permeability are implicitly included in the parameters K1 and k2. distribution volume for both the striatum and cerebellum Changes in the distribution volume ratio for a ROl to that in the was smaller for the 1isomer than the d isomer, which may cerebellum after drug treatment were considered significant if they be due to pharmacokinetic factors such as plasma were greater than 3 s.d.s from the average distribution volume binding. ratio at baseline. The average distribution volumes for three different ba

RESULTS TABLE 2 Kinetics of Brain Regional Activities Stereoselectivity of Carbon-i 1-Methylphenidate In each baseline study, brain activity distributed hetero Binding in Baboon Brain geneously, with the highest uptake occurring in striatum. BaboonTracerDV@DVCBDVS-IIDVCBBrieC1C]d-thmo Peak uptake (average 0.05% ID/cc) occurred 5—15mm postinjection for the striatum, 5—10mm for the thalamus ±3.44 ±1.77 ±0.20 and 3—5mm for the cerebellum (Fig. 1). The half-times for (n = 8) clearance from peak uptake for [11CJd-threo-MP (n = 12) r1c@'[email protected]@12.3322.19 ±1.7113.7812.28 ±1.502.451.82 ± = 5) were 70 ±10, 35 ±5 and 25 ±5 mm for the striatum, ±0.7810.80 ±0.67(n1.140.01(n± thalamus and cerebellum, respectively, compared to 60 ±5, 3)[email protected] = 35 ±5, 25 ±5 mm for [“C]MP(n = 8) (4). The striatum [email protected](n±0.751 1.7 ±0.202.18 ± to-cerebellum ratio was about 2.8 for baboon Angel and 3.3 = 3) for baboon Brie at 60 mm. Comparative studies of three 1)DV 1.17 (n = “C-labeledMP tracers (individual enantiomers and race mate) on the same baboon (Brie) demonstrated a differ = distributionvolume;ST = striatum;CB = cerebellum.

2300 The Journal of NuclearMedicine•Vol.36 •No. 12 •December 1995 TABLE 3 Average Distribution Volumes ±s.d. for Different Brain Regions

BaboonStriatumThalamusMidbrainCerebellumBrie 1.78Angel(n = 8)33.61 ±3.4415.78 ±1.7815.33 ±1.8613.78 ± 0.46Carm(n = 2)25.54 ±1.0614.25 ±1.3513.26 ±0.661 1.67 ± (n = 1)27.4114.7615.2213.73

boons for the striatum, thalamus, midbrain and cerebellum produce a significant change in the distribution volume are given in Table 3. The average distribution volume was ratios in any brain region. greatest in the striatum with lower values in the thalamus, In a chase experiment, [“C]d-threo-MPbinding proved midbrain and cerebellum. to be reversible as the injection of nonradioactive MP at 15 Time-activity curves for the striatum and cerebellum at mm induced a displacement of striatal activity to a level baseline and after treatment with a-CIT are shown in Fig similar to the cerebellar activity. As a result, the striatum ure 3A and the corresponding plots for the striatum-to to-cerebellum ratio dropped significantly and approached cerebellum ratio are shown in Figure 3B. f3-CIT markedly one. In contrast, it steadily increased during that period in reduced the striatal but not cerebellar binding of [“C]d the control experiment (Fig. 4), indicating that, at 15 mm, threo-MP, demonstrating the saturable and specific binding most of the striatal activity was associated with saturable of [1'C]d-threo-MP to the dopamine transporter in the sites. brain. For MP pretreatment, the striatum-to-cerebellum The results of the assay for unchanged tracer in baboon ratio also approached one. For GBR 12909 pretreatment, plasma after intravenous injection of [“C]d-threo-MPwere the ratio was reduced to 1.4—1.5. similar to that for [11C]MP (Fig. 5), which are consistent The average and individual ratios for baseline studies with other existing MP pharmacokinetic data that MP is and individual values for drug treatment studies are pre rapidly metabolized and the two enantiomers have a similar sented in Table 4. The standard deviations for the average bioavailability after intravenous (but not oral) administra distribution volume ratios were used to assess the signifi tion (9,10,19—21). cance of drug-induced changes. We considered changes greater than 3 s.d.s from the baseline average to be signif icant. The ratios of distribution volume for the striatum to DISCUSSION cerebellum were reduced by 54%, 50% and 55% for unla A number of trends characterize today's chiral drug in beled MP, GBR 12909 and p3-CIT,respectively. A param dustry. Perhaps the most important one is that a steady, eter proportional to free transporter concentration (B,,,,,,/ rising flow of single-isomer forms of chiral drugs in the U.S. K@)also showed a large change after pretreatment, with and world markets is creating an increasing demand for 91%, 79% and 100% reduction for unlabeled MP, GBR enantiomeric intermediates and bulk active compounds, as 12909 and @3-CIT,respectively (data not shown). In con well as enantioselective technology and services (22). The trast, pretreatment with citalopram and tomoxetine did not driving force is that single enantiomers can be more selec

0.06

0.05 I 0.04 @‘ 0.03

0.02

0.01

0.50 0.0 10 20 30 40 50 60 70 80 0.0 10 20 30 40 50 60 70 80 TIme (mm.) Time (mln.)

FiGURE3. (A@T,me-actMtycurvesforC1G]d-threo-MPinstriatum(circles),thalamus(squares)andcerebellum(triangles)of baboon brain before (solid)and after (open) 13-CIrpretreatment striatum-to-cerebellum ra@osare presented for the same data in Figure3B (angle). @ There is marked change in striatal uptake and in the striatum-to-cerebellum ratio (B)after pretreatment with (3-dr.

Carbon-11-d-threo-Methylphenidatein Baboon Brain•Dingat al. 2301 TABLE 4 Distribution Volume Ratks (R0VCerebellum) for Carbon-i 1-d-thmo-MP for Baseline (Run 1) and Drug Treatment (Run 2) @ Study average Drugtreathent DV@/DVce % DV@/DVcB % Ch@flge* DVMB/DVCB % Change* 0.05AngelBrie (n = 6)none2.43 ±0.241 .17 ±0.111 .09 ± 0.04Cairn(n = 2)none2.19 ±0.041 .22 ±0.091 .14± 1)none2.01.081.11Saturabil@rtyBrieBaseline(n =

1.03—10DA MP2.46 [email protected] 1.06—151.14 transporterBrieBaseline

1.13—14BrieBaselineGBR;45 mm2.75 [email protected] .26 .09—4AngelBaselineGBR;23mm2.66 1.345@t1 1.20—51.14 1

1.00—10BrieBaselinef3-CIT2.16 [email protected] 1.03—111.11 .07 0.94-10NE j3-Cfl2.22 1.00@SSt1 0.96-101.05 transporterBrieBaseline .05 1.124GarniBaselineTomoxetine2.24 2.10—111 1.0501.08 .08 1.1105HT Tomoxetine2.00 1.90—101 1.1351.11 TransporterAngelBaseline

1.13—2.2BrieBaselineCttalopram;2hr2.22 2.18—21.28 1.27—11.16

Citalopram;30 mm2.222.303.61.10 1.1321.04 1.116

lbe%changeis[(run2 —run1@run1]x 100. tDr@ treatmentchanged the valueof the DVratioby more than 3 s.d. fromthe average of the baselinevaluesforthat animal. Dv = distributionvolume;ST = striatum;GB= cerebellum;N = thalamus;MB = midbrain;DA= dopamine;NE= norepinephrine;5HT= serotonin.

tively effective. Methylphenidate is a central nervous sys racemic form. It has been shown, however, that the phar tem widely used to treat children with attention macological activity resides almost entirely in the d-threo deficit hyperactivity disorder and is marketed as dl-threo enantiomer (5). The rapid synthesis we have developed to obtain enantiomerically pure “C-labeledd-threo-MP allows

4.0

3.5 E @ 3.0 .0 @ 2.5 0 E 2.0

@. 1.5 Cl) 1.0

0.50 0 10 20 30 40 50 60 70 Time (mm.)

FiGURE 4. Effect of MP on the st,iatum-to-cerebellum ratio for C1G]d-th@eo-MP(Brie).Methylphen@ate (0.5 mg/kg, i.v.)was in jected 15 mm @n@on of C1C)d-thieo-MP and induced a dis FiGURE5. Percentageof unchangedtracerinbaboonplasma plecement of r1G)d-th@-MP uptake in the striatum, resulting in the after intravenous injectionof [11C]d-threo-MP(circles)and r1GIMP reductkn of the striatum-to-cerebellum ratio. (squares).

2302 The Journal of NuclearMedicine•Vol.36 •No. 12 •December 1995 us to characterize its binding, to compare it with the 1- The binding specificity of [“C]d-threo-MPto dopamine enantiomer and racemic mixture, to examine its specificity transporters in the striatum was further supported by the to dopaminergic and to evaluate it as a radiotracer lack of blocking effect after pretreatment with tomoxetine for the presynaptic dopaminergic neuron. or citalopram (inhibitors of the norepineprine and seroto nm transporter, respectively) (Table 4). This result is con Binding cmaractenstics sistent with a report that the distribution of d-threo MP is In our previous study using [‘‘C]MP(dl-threo racemic inhibited by (which inhibits both dopamine and form), we demonstrated the specific binding to dopamine norepinephrine transport) but not by (norepi transporters as well as the sensitivity to degeneration of the nephrine ) (26). It also supports the pre dopaminergic neurons in early Parkinson's disease (4). vailing view that the stimulant effects of MP and related Comparative studies of [“C]d-threo-MPand [“CJMPin the drugs are related more consistently to their inhibitory ac same baboon (Brie) demonstrated a large difference in the tions on the dopamine transporter than on the norepineph ratio of specific-to-nonspecific binding, as indicated by stri rine or the . atal versus cerebellar uptake: 3.3 for [“C]d-threo-MPand A numberof PET and SPEC!' radioligandsthat bind to only 2.2 for [“C]MP(Fig. 2). This is consistent with the the neuronal dopamine transporter in vivo and have been potency in displacing [3H]dl-threo-MP from striatal synap shown to be sensitive to the loss of dopamine transporters tosomal membrane binding sites—IC50corresponding to (either in vivo or in vitro with tritium- or ‘@I-labeledtrac 0.088 pM for d-threo-MP and 0.21 pM for dl-threo-MP (6). ers) have been reported, including [“C}(27— The ability of [1'CJd-threo-MP to label dopamine trans 29), [“C] and [3H]cocaine (30—32), [‘8F]GBR porter was primarily demonstrated by blocking studies with 13119and [3H]GBR 12935 (33,34) and “C-and [3H]WIN various monoamine uptake inhibitors. GBR 12909 is a 35428 (35—39)and ‘@I-and ‘@I-labeledcocaine analogs selective inhibitor of dopamine uptake. The IC50of GBR (12,40—44).In comparison with these ligands, [“C]MPhas 12909 to inhibit uptake of radiolabeled monoamines into several attractive features for PET, including its high up rat synaptosomes is 1 nM for dopamine, 170 nM for 5-HT take and reversibility in the brain, which facilitates kinetic and 440 nM for NE (11). GBR 12909 inhibited [“C]d modeling (15). The fact that MP is an approved drug threo-MP uptake in the striatum but not in the thalamus or expedites approval for human studies. It also makes it cerebellum, which is consistent with [“C]d-threo-MPbind possible to use unlabeled MP to assess saturability of bind ing to dopamine transporters in the striatum. A decrease of ing in . The present study demonstrates a higher 50% in the distribution volume ratio in the striatum to that ratio for specific versus nonspecific binding for [“C]d in the cerebellum (DVsTIDV@, Table 4), which corre threo-MP, the ratio of striatum-to-cerebellum after pre @ sponds to + 1, was observed after pretreatment treatment with unlabeled MP and GBR 12909was reduced with GBR 12909. If one quantifies the percent change in by about 60% for [“C]d-threo-MP(data not shown) com @ in the striatum with GBR 12909, this corresponds pared to 43% for [“C]MP.The ratio of distribution vol to 78%. The failure to totally block MP binding by GBR umes for the striatum-to-cerebellum for these two separate 12909 was probably related to a single dose being used studies was reduced by 54% and 50%, respectively, for (insufficient dose) as well as to differences in the binding [“C]d-threo-MP(Table 4) compared to 37% and 38% for patterns within the dopamine transporter between these [“CJMP(4) Thus, [“C]d-threo-MPis characterized by a two drugs (23,24). In fact, when we pretreat with the co higher striatal uptake with higher striatal-to-cerebellar ra caine analog 13-CIT (0.3 mg/kg i.v.), the distribution volume tios, higher degree of the specificity to dopamine trans in the striatum for [“C]d-threo-MPis reduced almost to the porter and the reversibility which facilitates kinetic model distribution volume value of the cerebellum (Table 4). It ing. has been reported that a-CIT binds to both dopamine and 5-HT transporters with in vitro IC50, 1.6 nM for displacing Evaluating Dopaminergic Neuron Loss [3H]CFTfromdopaminetransportersitesinmonkeystri In anticipation of the use of [“CJd-threo-MPto evaluate atum and 3.8 nM for displacing [3H]paroxetine from 5-HT to loss of dopaminergic neurons in Parkinson's disease, we transporter sites in rat cortical membranes (25). The per examined the potential effect of elevated striatal dopamine @ cent change in in striatum with @3-CITtreatment produced by L-DOPA therapy on radiotracer binding. We corresponds to 97%—100%, which is consistent with the found that treatment with L-DOPA (50 mg/kg) plus specific binding of the tracer to dopamine transporter in the benserazide, an aromatic amino acid decarboxylase inhibi striatum. We observed a 5%—15%change in the distribu tor (5 mg/kg) did not change the binding of [“CJd-threo-MP tion volume ratio (DV@fDV@) for the thalamus with in the baboon brain (45). This result suggests that the 13-CIT, GBR 12909 and MP. The variability in the response, sensitivity of [“C]d-threo-MPto changes in dopamine con however, as well as the fact that it was small (less than 3 s.d. centration is low and the uptake would not be affected by from the average of the baseline measures) is only sugges administration of L-DOPA in Parkinson patients. These tive of a trend. A similar small reduction was observed with PET studies as well as a previous PET studywith [“C]MP the distribution volume ratio for the midbrain after /3-CIT (4) suggest that [“CJd-threo-MPmay be sensitive to the and MP treatment. loss in dopamine neurons associated with normal aging and

Carbon-i 1-d-threo-Methyiphenidate in Baboon Bran •Ding at al. 2303 may also be a marker of dopamine transporters in neuro 7. Pan D, GatleySi, DeweySL,et al. Bindingof brominesubstitutedanalogs degenerative diseases such as Parkinson's. A particularly of methylphenidateto monoaminetransporters.EurI Pharmaco!1994;264: 177—182. important characteristic of such a radiotracer is high sen 8. Ritz MC, Lamb Ri, Goldberg SR, Kuhar Mi. Cocaine receptors on dopa sitivity to early or presymptomatic changes that may iden mine transporters are related to self-administrationof cocaine. Science tify subjects at risk who may optimally benefit from therapy. 1987;237:1219—1223. 9. SrinivasN, HubbardJ, KorchinskiE, MidhaK. Stereoselectiveurinary We view reversibility of [“C]d-thi@o-MPbinding as a par pharmacokineticsofd@!-threo-methylphenidateanditsmajormetabolitein ticularly important characteristic that facilitates quantifica humans.I PharmSci 1992;81:747—749. tion relative to irreversibly trapped tracers which may cx 10. SrinivasN, HubbardJ, KorchinskiE, MidhaK Enantioselectivepharmaco kinetics of d,l-threo-methylphenidate in humans. Phann Res 1993;10:14—21. hibit flow-limited binding. 11. AndersonPH.The dopamineuptakeinhibitorGBR 12909:selectivityand molecularmechanismof action.Eurl Pharmaco!1989;166:493—504. CONCLUSION 12. LaruelleM,BaldwinRM,MalisonRT,et al. SPECFimagingof dopamine andserotonintransporterswith[‘@Il@-CIT:pharmacologicalcharacteriza We have characterized [“C]d-threo-MPbinding in the tionof brainuptakein nonhumanprimates.Synapse1993;13:295—309. baboon brain. The availability of this labeled drug makes it 13. KlevenMS,AnthonyEW,WoolvertonWL.Pharmacologicalcharacteriza tion of the discriminativestimuluseffectsof cocainein rhesusmonkey.I possible to compare the pharmacokinetics with its racemic Phannaco! Er@@Ther 1990@,254:312—317. form ([“CJMP)in the human brain. The observation of 14. HyttelJ.Citalopram;pharmacologicalprofileof a specificserotoninuptake higher specific-to-nonspecific binding and selectivity to do inhibitor with activity. Plvg Neuropsychopharmaco! Bid Pay pamine transporters and reversibility suggests that this may chiatry 1982;6:277—295. 15. Logan i, Fowler iS, Volkow ND, et al. Graphical analysis of reversible be a potentially useful tracer to evaluate the degeneration radioligandbindingfrom time-activitymeasurementsappliedto [N-―C of dopaminergic neurons and the associated loss of nerve methyl]-(-).cocainePET studiesin humansubjectsI CerebBloodF!ow terminals in the striatum. This study supports further eval Metab 1990;10:740—747. 16. DeweySL,MacGregorRR,BrodieID, et al.Mappingmuscarinicreceptors uation of [‘1C]d-threo-MPas a PET radiotracer for the in human and baboon brain using [N-―C-methyl]benztropine. 1990; presynaptic dopaminergic neuron in human brain. Studies 5:213—223. are now underway to determine the sensitivity of 17. KoeppeR, KilbournM, FreyK, Penneyi, HakaM, KuhID. Imagingand kinetic modeling of [‘8F]GBR12909, a dopamine uptake inhibitor. I Nuc! d-threo-MPto neuronallossoccurringin normalagingand Med 1990;31:720. @ in neurodegenerative disease and to monitor the effect of 18. HumeS, Pascali PikeV, et a! Citalopramlabelingwithcarbon-li and drugs on the rate of dopaminergic neuron loss. evaluation in rat as a potential for in vitro PET studies of 5-HT re-uptakesites.InslAppi InsinimB 1991;3:339—351. 19. ChanYM,SoldinSJ,SwansonJM,DeberCM,Thiessenii, MacleodS.Gas ACKNOWLEDGMENTS chromatographic/mass spectrometric analysis of methylphenidate (Ritalin) in serum. CIitzBiochem 1980;13:266-272. This research was carried out at Brookhaven National Labora 20. Hungund BL, Perrl JM, Hurwic MJ, Sverd i, Winsberg BG. Pharmacoki tory under contract DE-AQ@2-76CH00016with the U.S. Depart netics of methylphenidate in hyperkinetic children. Br I C!in Pharmacol ment of Energyand supportedby its Officeof Health and Envi 1979;8:571—576. ronmental Research. Additional support was provided by National 21. WarginW, PatrickK, Kilts C, GualtieriT, et al. Pharmacokineticsof Institutesof Health grantsNS-15380and NS-15638and National methylphenidate in man, rat and monkey. I Pharmaco! Erp Ther 1983;226: 382—386. Institute of Drug Abuse grant DA-06278. The authors are grateful [email protected]@Chemicaland EnRjneethzgNews1994;Sept.,38-72. to CIBAGeigyfor providingsamplesof dl-threo-MPand ritalinic 23. KilbournMR. In vivobindingof [‘F]GBR13119to the brain dopamine acid and to Ivy Carroll and Michael Kuhar for providingthe uptake system.Lifr Sci 1988;42:1347—1353. /3-CIT. 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