ORIGINAL ARTICLE 

Annals of Nuclear Medicine Vol. 9, No. 3, 119-123, 1995 

Positron emission tomography with 4-[18F]fluoro-L-m-tyrosine in MPTP-induced hemiparkinsonian monkeys 

Nobuaki HAYASE, Katsuyoshi TOMIYOSHI, Kazushige WATANABE, Satoru HORIKOSHI, Takashi SHIBASAKI and Chihiro OHYE 

Department of Neurosurgery and Nuclear Medicine, Gunma University School of Medicine 

PET imaging studies with 4-[18F]fluoro-L-m-tyrosine (FMT) in normal macaca monkeys showed selective accumulations of radioactivity in the striatum with time. In monkeys rendered hemi-parkinsonian by intracarotid infusion of l-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP), FMT uptake was eliminated in the lesioned striatum. FMT-PET studies were able to detect dopaminergic terminals in both normal and hemiparkinsonian monkeys, and clearly showed a reduction in aromatic L-amino acid decarboxylase (AAAD) activities in the MPTP-lesioned striatum. These results show that FMT is promising as a PET tracer for the evaluation of central dopaminergic systems in parkinsonism. 

Key words : PET, Parkinson's disease, fluoro-L-m-tyrosine, MPTP, primates 

INTRODUCTION 

THE MOST PROMINENT NEUROCHEMICAL FEATURE of Parkinson's disease is the dopaminergic depletion in the nigrostriatal pathway. Approximately an 80% reduction in nigrostriatal dopamine content is thought to be necessary before the symptoms arise.l 
PET scan with a dopaminergic probe such as 6-[18F]fluoro-L-DOPA (FDOPA) is an useful technique to use in estimating nigrostriatal dopaminergic function in living brains. Several studies have been performed in normal human subjects,2.3 patients suffering from idio-pathic parkinsonism,3-5 and non-human primates.6,7 Fur-thermore, FDOPA-PET study has been reported as a method for detecting preclinical Parkinson's disease.8 
However, recent lines of evidence strongly indicate disadvantages in the FDOPA-PET study. FDOPA is ex-tensively metabolized in the periphery not only to [18F]fluoro-dopamine by AAAD, but also to 3-o-methyl-6-fluoro-L-DOPA (3-OMFD)9,10 by catechol-o-methyl transferase (COMT). Significant formation of 3-OMFD, which crosses the blood-brain-barrier bidirectionally, makes image contrast and biochemical interpretation of a FDOPA-PET study ambiguous and complicated. 
To circumvent this problem, L-DOPA analogs that are not substrates for COMT but still maintain specificity toward AAAD11 were sought. A fluorinated analog of m-tyrosine is proposed as such a substance.11-15 4_ [18F]Fluoro-L-m-tyrosine is a L-DOPA analog that is essentially involved in L-DOPA metabolic pathways but without 3-0-methylation or extensive peripheral metabo-lism. Recent FMT-PET studies reported improvement in the image contrast compared to that obtained with FDOPA.12,15 
Discovery of the neurotoxin I -methyl-4-phenyl- 1,2,3 ,6-tetrahydropyridine (MPTP) provides a valuable tool to use in investigating the effects of nigrostiatal denervation and its relationship to parkinsonian symptoms in human subjects16 and primates. 17 The infusion of MPTP into one internal carotid artery of primates damages dopaminergic cells of the substantia nigra pars compacta (SNpc) ipsilateral to the side of infusion with little or no apparent damage to the contralateral side, thus producing a unilat-eral parkinsonian or hemiparkinsonian syndrome.18-20 
We demonstrate imaging PET studies with a new dopa-minergic PET tracer, 4-[18F]fluoro-L-m-tyrosine in nor-mal and hemiparkinsonian model monkeys.

MATERIALS AND METHODS 

4- [18F]Fluoro-L-m-tyrosine (FMT) was synthesized by an acetylhypofluorite method as described earlier.21.22 The HPLC-purified product was determinded as more than 98% chemical pure. 
Scanning was performed with a SHR2000 positron tomograph (Hamamatsu Photonics, Co. Ltd., Japan), 23 which has a reconstructed spatial resolution of 3 .5 mm full width at half-maximum (FWHM) at the center of the image. The slice thickness is 4.5 mm FWHM, and seven equally spaced parallel planes with a 6.5 mm interval from center to center are obtained simultaneously. Attenuation correction was performed by means of a transmission scan with a 68Ga source. Four monkeys (Macaca fuscata) weighing 6.3-9.0 kg were anesthetized with ketamine (20 mg/kg, i.m.) and catheterized in a femoral artery for blood samplings and in a vein for an injection of FMT. General anesthesia was maintained throughout the study with pentobarbital ( 10 mg/kg, i.v. every hour). For repro-ducible positioning, we developed a special animal chair equipped with ear bars and a head fixation device referred to as a stereotaxic device. The animal was fixed on the chair to slice parallel to the canthomeatal line. The laser beam on the PET scanner projected an external landmark of the monkey's head to confirm the place to slice. 
FMT was administered intravenously (5-6 mCi; specific activity, 1-2 Ci/nmol) following carbidopa pre-treatment (5 mg/kg, i.v., 60 min before FMT administra-tion). Dynamic image aquisitions were performed for 2 hours immediately after tracer administration. The full study consisted of a sequence of twelve 2-min frames, nine 4-min frames and six 1O-min frames. This procedure yielded dynamic information on tracer accumulations in brain tissues. Following the last dynamic frame, a static image acquisition was performed for 30 min. Changes in regional radioactivity with time were calculated for the striatum and cerebellum. 
Arterial blood samples were collected as follows: every 15 sec for the 3rd min, then 5, 7, 10, 15, 20, 30, 40, 60, 100 and 160 min after the tracer injection. Plasma samples (300 ul) were separated by centrifugation at 1000 x G. The [18F]radioactivity of each sample was measured with a well counter (Aloka Universal Scaler, model TDC-501). 
After baseline FMT-PET studies, two monkeys re-ceived MPTP infusions and were scanned after MPTP treatment (between 2 and 5 months). MPTP administra-tion was performed according to the method described in a previous report.24 Under general anesthesia with ketamine (20 mg/kg, i.m.) and pentobarbital ( 10 mg/kg, i.v.), a dose of 0.2 mg/kg MPTP in 50 ml heparinized saline was infused directly into the right internal carotid artery over 20 min. The animals displayed persistent signs of left-sided flexed posture, bradykinesia, and no tremor, and developed a preference to use the right limbs and to turn spontaneously toward the right side. Apomorphine (0.1 mg/kg, i .m. ) reversed these hemiparkinsonian syndromes and produced rotation in the direction contralateral to the side of infusion of MPTP. The parkinsonian signs and symptoms remained for at least 6 months. No behavior recovery was observed. 

RESULTS 

PET imaging studies in normal monkeys displayed selec-tive accumulations of 18F after FMT administration with time in the striatum. Bilateral striatums were clearly demarcated on the PET images (Fig. 1 , left). After MPTP treatment, accumulation of 18F in the infused side of the striatum was eliminated, compared with the uninfused side (Fig. 1, right). Low nonspecific binding and little apparent defluorination of FMT were found, as there was little accumulation of radioactivity in bone structures. 
The decay-corrected accumulation time course in nor-mal monkeys showed rapid accumulation at the striatal structure (Fig. 2A). The ratio of radioactivity in the striatum for FMT to that in the cerebellum increased with time. After MPTP infusion, uptake of radioactivity in the MPTP-lesioned striatum was reduced to the level of non-specific distribution in the cerebellum (Fig. 2B). The amount of 18F in each region of interest was expressed as the average cpm/pixel within the region normalized to the number of mCi of FMT injected. The time course of radioactivity in the arterial plasma showed fast clearance of tracers from circulating plasma (Fig. 3). 
Figure 4 shows the striatum/cerebellum accumulation ratio; the striatal activity over the period 120-150 min after administration of FMT divided by corresponding cerebellar activity . The mean striatum/cerebellum ratio in normal monkeys reached 3.2. In hemiparkinsonian mon-keys, the lesioned-striatum to cerebellum ratio was 1.3; but on the other hand, the contralateral striatum to cerebel-lum ratio reached 3.9. MPTP infusion significantly re-duced the accumulation of FMT in the striatum. 

DISCUSSION 

The cerebral uptake of 18F after FMT administration was stereoselective and showed a persistent regional accu-mulation in the striatum. This selective retention of radio-activity is in agreement with the high concentration of dopaminergic terminals in the striatal region. 
m-Tyrosine is thought to cross the blood brain barrier, not to be the substrate for COMT,25 and to be decarbox-ylated26 to give m-tyramine which could act as a false dopamine neurotransmitter.27 FMT was probably decar-boxylated to 4- [ 18F]fluoro-L-m-tyramine by AAAD, similar to that reported in the case of m-tyrosine.12,28 HPLC analysis of rat striatal radioactivity revealed that at 30 min after FMT injection, most of the activity was associated with 4-fluor0-3-hydroxyphenylacetic acid (FPAC).12 As the transformation of FMT to FPAC essentially depends@on AAAD at nerve terminals, specific localization of 18F in FMT-PET closely reflects AAAD activity in the striatum. 
The decay-corrected accumulation time course curves showed differences between the phannacokinetic behavior in the striatum and the rest of the brain. The striatal distribution of FMT in normal monkeys was relatively rapid, and increased lineally over time at 2 hours, com-pared to the gradual decrease in non-specific accumula-tion in the cerebellum. Despite the fact that the clearance of tracers from circulating plasma was fast, the accumu-lation of 18F in the striatum gradually rose. As sulfo-conjugated 4-fluoro-3-hydroxyphenyl-ethylamine (FMA), which is major peripheral metabolite of FMT,12 is not likely to cross the blood-brain-barrier, the specific-to-nonspecific accumulation ratio increases. The striatum/ cerebellum ratio of radioactivity more than tripled in 2 hours. In contrast to FDOPA-PET,7,8,10 significant and specific time dependent accumulation of 18F activity oc-curred after FMT administration. 
MPTP treatment produced almost complete de-struction (> 95%) of dopaminergic cells20 and resulted in significant ipsilateral reduction in dopamine. Striatal dopamine (DA) levels and AAAD activity30 on the MPTP-treated side of hemiparkinsonian monkeys decreased by more than 95% and 94%, respectively, in comparison with the non-treated side or non-treated monkeys. In the present FMT-PET studies, the reduction of 18F retention in the MPTP-treated striatum was clearly visualized. The uptake of radioactivity in the MPTP-lesioned striatum was reduced to the level of non-specific distribution in the cerebellum. This reduction in 18F retention is closely related to depletion of AAAD activity. However, it is premature to conclude that there is a quantitative correla-tion between 18F retention and AAAD activity in MPTP-hemiparkinsonian monkey without metabolic analysis. 
We showed the capability of PET employing FMT to visualize nigrostriatal dopaminergic functions in vivo, and also revealed a significant reduction in 18F accumu-lation in the MPTP-lesioned striatum. It is mandatory to clarify the metabolic pathway of FMT and its metabolites to establish a quantitative kinetic model of FMT. Never-theless, the present results suggest that an FMT-PET study can localize and quantitate AAAD activity in these terminals. The development of quantitative methods for the analysis of FMT uptake data will contribute a signifi-cant advance in the performance of FMT-PET studies. 

ACKNOWLEDGMENTS 

We thank Mr. T. Ishihara for operating the cyclotron (BC17 10, Japan Steel Works, Ltd.). Carbidopa was kindly provided by Sankyo Pharmacy. Co. Ltd. 

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