SYNAPSE 5:207-212 (1990)

Pargyline-Sensitive Selective Accumulation of a Radiolabeled MPTP Analog in the Primate Cerebral Cortex and Basal Ganglia S.M.N. EFANGE, H.F. KUNG, D.C. MASH, M. JABIR, J. BILLINGS, J. PABLO, A. DUTTA, AND A. FRESHLER De artments of Radiology and Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota (&M.N.E., A.D., A.F.); Department of Radiology, University of Pennsylvania Hospital, Philadelphia, Pennsylvania (H.F.K., J.B.); Departments of Neurology, Pharmacology and Radiology, University of Miami School of Medicine, Miami, Florida (D.M., M .J., J.P.)

KEY WORDS

[ 12511MHTP,Monoamine oxidase, MHTP, 4-Homo-MPTP

ABSTRACT The distribution of radioiodinated N-methyl-4-(4-hydroxy-3-iodobenzyl)1,2,3,6-tetrahydropyridine(MHTP), an analog of the reportedly nontoxic N-methyl4-benzyl-1,2,3,6-tetrahydropyridine, (4-homo-MPTP1,has been studied in the primate. [ lZ3I]MHTP-derivedradioactivity exhibited a progressive accumulation and prolonged retention within the primate eye. Following iv injection, [12311MHTPrapidly accumulated within the primate brain and was subsequently oxidized to a radiolabeled metabolite. The half-life of [12311MHTP-derivedradioactivity within the primate brain was 50 min. The highest concentrations of radioactivity were found in the caudate-putamen and the frontal, temporal and cingulate cortices; the substantia nigra and inferior olivary nucleus were labeled with medium intensity. Very low concentrations of radiolabel were detected in the cerebellum and white matter. Selective accumulation of [lZ5I]MHTP-derived radioactivity within these structures was blocked by pretreatment with pargyline, suggesting that monoamine oxidase B is involved in the bioactivation of radioiodinated MfiyP. v

INTRODUCTION dopaminergic neurons were found to maintain greater concentrations of MPP+than other regions of the brain otent neurotoxin N-methyl-4-phenyl-l,2,3,6-

The tetrahy ropyridine (MPTP) induces symptoms of Parkinson’s disease in humans and nonhuman primates (Burns et al., 1983; Davis et al., 1979; Langston et al., 1983). The MPTP-induced lesion exhibits striking biochemical and histopathological similarities with idiopathic Parkinsonism and, therefore, provides a useful model for the study of the latter. MPTP-induced central neuropathology is presumed to reflect the summation of several processes, which may be divided into four phases: 1)delivery of MPTP, 2) MAO-catalyzed bioactivation to yield the cationic metabolite and putative neurotoxin MPP+,3) selective accumulation of MPP+by catecholaminergic neurons via the dopamine reuptake system (Javitch et al., 1985a), and 4) the induction of dopaminergic neurotoxicity (for reviews, see Schultz, 1988; Singer et al., 1987a,b; Snyder and D’Amato, 1986). Of these four phases, the last named is also the least understood. However, the selective dopaminergic neurotoxicity of MPTP may be partially explained by the regional distribution of MPP in vivo. Following the administration of radiolabeled MPTP to rodents, high concentrations of radioactivity were found within the caudate-putamen, nucleus accumbens, and locus coeruleus (Lyden et al., 1985; Lyden-Sokolowski et al., 1988).These areas of the brain are known to contain the highest concentrations of catecholamine uptake sites (Javitch et al., 198513). Similarly, in higher primates,

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@ 1990 WILEY-LISS, INC.

(Johannesen et al., 1985; Irwin and Langston, 1985). In the baboon and monkey, high concentrations of radioactivity were found in the basal ganglia following the administration of N-[llC]MPTP (Hartvig et al., 1986; Livni et al., 1986; Moerlein et al., 1986). Selective accumulation of radioactivity was prevented by pretreatment with the monoamine oxidase (MAO) inhibitors pargyline and tranylcypromine, thus implicating MPP+ in the observed regional selectivity (Hartvig et al., 1986; Moerlein et al., 1986). Although radiolabeled MFTP may be useful for mapping dopaminergc innervation in vivo, the toxicity of this compound would severely limit its clinical applicability as a radiotracer. However, MPTP represents a useful prototype for the design of metabolically activated site-specific radiotracers. Such radiotracers would complement the receptor ligands such as fluorodeoxydeoxyglucose (FDG), [18Flfluoro-DOPA, and N3-[11Clmethylspiperone,which have been used for the study of neurodegenerative diseases. In our attempts to develop nontoxic MPTP-like radiotracers, we recogReceived August 8,1989;accepted in revised form October 11,1989

S.M.N. Efange’s present address is Department of Radiology, Box 382 UMHC, University of Minnesota Hospital and Clinics, 420 Delaware Street S.E., Minneapolis, MN 55455.

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nized, as have others, that the various processes culmiPreparation of [ 12311MHTP nating in MPTP-induced dopaminergic toxicity repreTo 100 ~1 of an ethanol solution containing 0.32 pmol sent a loose aggregate of biochemical events and not a of N-methyl- 444-hydroxybenzyl)-l,2,3,6-tetrahydropytightly coupled biochemical sequence. Consequently, we ridine, the following were added in the stated order: postulated that, with suitable structural modification of glacial acetic acid (100 p1),5.4 mCi of NalZ3Iin 200 pl of MPTP, neurotoxicity may be eliminated or substan- 0.1 N NaOH and chloramine-T (0.33 pmol in 20 pl of tially diminished while the attributes of bioactivation water). The vial was capped and shaken for 90 min. The and selective neuronal uptake are maintained. MPTP reaction was then quenched with 200 ~1 of 5% aq soanalogs with such properties would be potentially use- dium bisulfite. The resulting mixture was made alkaful as radiolabeled biological probes for mapping cate- line with solid sodium bicarbonate, and later treated cholaminergic innervation. with water (300 p1) and a saturated solution of sodium Our initial attempts to identify such probes were chloride (200 11.1).The resulting mixture was extracted guided by a report that the MPTP homologue N-methyl- with methylene chloride (3 x 1.0 ml), and the combined 4-benzyl-l,2,3,6-tetrahydropyridine ($-horno-MFTP),re- organic extracts were dried over anhydrous Na2SO4. portedly an excellent substrate of MAO, failed to exhibit The solution was subsequently concentrated under a in vivo dopaminergic toxicity in mice (Youngster et al., stream of air to give a residue (4.84mCi), which was 1987).We have since shown that, in the rodent, another dissolved in ethanol (200 p1) and purified on a C18 homolog N-methyl-4-(4-h~droxy-3-[12511iodobenzyl~-l, column (15%5 mM phosphate buffer, 85% methanol; 2,3,64etrahydropyridine ([ 2511MHTP)undergoes MAO- flow rate 1 ml/min). In this system, the retention time catalyzed bioactivation to yield a radiolabeled metabo- was 6 min. The eluate collected yielded 3.5 mCi. The lite that selectively accumulates within the locus coer- eluate was concentrated at 50°C under a stream of air to uleus (Efange et al., 1989).In this paper, we describe the yield a residue, which was treated with a saturated disposition of [12511MHTPin the primate. solution of sodium chloride and reextracted with dichloromethane (3 x 1.0 ml). The organic extracts were MATERIALS AND METHODS dried over sodium sulfate and concentrated to a residue, N-methyl-4-(4-hydroxybenzyl)-l,2,3,6-tetrahydro-which was dissolved in isotonic saline. Radiochemical pyridine and N-methyl-4-(4-hydroxy-3-iodobenzyl)-l, purity as determined by HPLC was greater than 95%. 2,3,6-tetrahydropyridinewere synthesized in our labo- This material was then used for biological evaluation. ratory and characterized by spectroscopic methods and [1251]MHTPwas synthesized in a similar manner. Howelemental analysis (see Fig. 1).Details of their synthesis ever, the reaction time was 30 Inin. will be provided elsewhere. Pargyline hydrochloride Planar imaging and tissue distribution study was obtained from Sigma chemical company (St. Louis, MO) and ketamine hydrochloride was obtained from A dose of 3 mCi of [1231]MHTP and 75 pCi of Parke-Davis. (S)-N-[l-ethyl-2-pyrrolidinyl)]methyl-2[12511MHTPin 0.25 ml of isotonic saline was adminishydroxy-3-iodo-6-methoxybenzamide ([1231]IBZM)was tered iv to a 25-year-old 8 kg female Aeteles Geoffrey synthesized by a procedure reported earlier (Kunget al., monkey sedated with ketaminc!. Planar anterior imag1989). ing was begun simultaneously using a Picker Dyna camera equipped with an all-purpose collimator. The followingimaging sequence was employed.t = 0-6 min, 30 frameslmin; t = 6-66min, 1 frameimin; t = 80-120 min, 1 frame/min. The brain was flagged, and the counts in this region were plotted against time to generate a time activity curve for the brain. At the end of the dynamic imaging sequence, static images of the chest and abdominal region were also acquired. At 2 hr 40 min postinjection, the animal was sacrificed with an overdose of sodium pentobarbital and immediately decapitated. The brain was carefully removed, weighed (123.1 g), and w,ashedwith cold isotonic saline. Sagittal slices near the midline were obtained. These were used for static imaging. These images were obtained with an all-purpose iind pinhole collimator. The slices were subsequently frozen and processed for autoradiography. The rest of the carcass was kept in a cold room and the remaining organs were harvested 16 hr following decapitation. At this time, the organs were collected whole, rinsed with distilled water, dried with absorbent aper, and weighed (liver, 126.72 g; lungs, 60.79 g; Eeart, 29.14 g; kidneys, 22.91 g; spleen, 4.45g). Small portions of these organs were subsequently collected, weighed, and counted in a Nuclear Chicago model 1185 125 gamma counter for 1231.Four days later, these same samples were counted for 1251.Suitable dilutions of the original dose were used as standards. Fig. 1. Structure of I'2311/l'Z511MHTPand MPTP.

8

*I

1

I

[

IIMHTP

CH3

MPTP

BASAL GANGLIA-SELECTIVERADIOMELED MPTP ANALOG

The percentage doselg values were calculated from the tissue radioactivity and the standards. The percentage doselor an values were then obtained from the percentage oseig and the corresponding organ weights. Radioactivity in the whole brain was estimated from an average of four sagittal slices taken near the midline. These were considered to represent the majority of brain regions. In vivo autoradiographic studies of [12511MHTP Control study Following sedation with ketamine (40 mg im), a male cynomolgus monkey (3 kg; between 1 and 4 years old) received an iv injection of [12511MHTP(12.4 mCi). The animal was sacrificed after 60 min with an overdose of pentobarbital (65 mg). After decapitation, the brain was carefully removed and sectioned into two hemispheres. One hemisphere was dissected and counted in a gamma counter to determine the amount of radioactivity present within the brain. The percent of the injected dose in the whole brain was calculated by comparison with suitable dilutions of the injected dose. The other hemisphere was immediately frozen in powdered dry ice. Thirty-micrometer sagittal sections were cut from this latter hemisphere at -18°C on a cryostat microtome (Hackermright).Tissue sections for autoradiography were mounted on acid-washed slides, and dried at room temperature for 3-5 min. Autoradiograms were generated by apposing tissue sections to I3H1Ultrafilm (LKB, Sweden) or to Dupont NMB film with suitable brain paste standards. Following exposure, sections were subjected t o Nissl staining to delineate cytoarchitectural landmarks. Autoradiograms were inspected by projection planimetry and compared to cytoarchitectural charts of Nissl-stained sections to reveal cytoarchitectural boundaries of the accumulated radioactivity. Computer-assisted scanning densitometry was utilized to convert the autoradiographic density to pseudocolor images. Pargyline pretreatment and comparison with [123111BZM While under ketamine anesthesia, a male cynomolgus monkey (3.25 kg; between 1 and 4 years old) received an ip injection of gargyline hydrochloride (50 mgl kg). After 60 min, [I 511MHTP (4.0 mCi) and the dopamine D, receptor ligand [123111BZM(2.5 mCi) were coadministered by iv injection. The animal was sacrificed by an overdose of pentobarbital 60 min after the injection of the radiotracers. The brain was quickly removed from the skull, weighed, and sectioned into two hemispheres. One hemisphere was used for regional dissection studies, as described above, to determine the percentage of the injected radioactivity within the brain. The other hemisphere was frozen and subsequently used for autoradiography and Nissl staining in parallel localization studies with [1251]MHTP.

%

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influx of radioactivity peaked within 2 min and was followed by a monoexponential efflux phase. The halflife of radioactivity in the brain was 50 min (data not shown). Static images of the monkey head showed a pronounced accumulation of radioactivity within the eyes of the monkey at 1hr postinjection (data not shown), which was not evident in the earlier images (Fig. 2). Such time-dependent intense accumulation of radioactivity in the primate eye has also been reported in studies using radiolabeled MPTP (Hartvig et al., 1986). At 160 min following the administration of the radiotracer, the brain (1.88%), liver (2.41%),and kidneys (0.50%)contained the highest levels of radioactivity (Table 1).In contrast, the heart contained very low levels of radioactivity. Pargyline pretreatment In the cynomolgus monkey, the brain was found to contain 1.44%of the injected dose of radioactivity at 60 min postinjection. This value decreased to 0.25% with pargyline pretreatment. However, 3.47% of injected [1231]IBZMwas found in the monkey brain after pargyline pretreatment. This value is comparable to a value of 3.71%reported earlier in an untreated monkey (Kung et al., 1989). In vivo autoradiographic studies In vivo accumulation of radioactivity 60 min after injection of [lZ3I]MHTPrevealed regional variations consistent with the known distribution of dopaminergic presynaptic markers. In rostra1 brain areas, the highest accumulation was observed within the caudate and putamen (Fig. 3A). The striatum (caudate and putamen) receives a dense plexus of dopaminergic innerva-

RESULTS Planar dynamic imaging and .. tissue biodistribution studies In the planar dynamic imaging study, a rapid accu2. Planar anterior image of an Aeteles Geoffrey monkey followmulation of radioactivity was observed in the Aeteles ingFig. injection of ['231]MHTP.The image shows significant accumulation Geoffrey monkey brain followingthe administration of a of ['231]MHTP-derived radioactivity within the monkey brain. Image is and [1231]MHTP(data not shown). The a sum of five 1minute images (from 25 to 29 mid. mixture of [12511-

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S.M.N. EFANGE ET AL. TABLE I. Tissue distribution of radroactivity at 160 min following iu injection of [1231]-and [Iz5I] MHTP into a monkey [l231]

Organ'

Percent dose/g

Liver Spleen Kidneys Heart Lung Muscle Brain

0.015 0.002

0.017 0.002 0.005 0.002 0.012

MHTP~ Percent dosdorgan 1.86

0.01 0.38 0.07 0.28

[lZ51]M H T P ~

Percent dose/g

Pvrcent dose/organ

0.019 0.002 0.003 0.003

2.41 0.01 0.50 0.10 0.33

0.006 0.002

1.53

1.88

'Organ weights: liver, 126.72 g; spleen, 4.46 g; kidneys, 22.90 g; heart, 29.14 g; lung, 60.79 g; brain, 123.4 g. :Counted 2 days after the experiment. .'Counted 4 days after the experiment.

tion originating from cell bodies that are located in the substantia nigra. Moderate radiotracer activity was observed over the substantia nigra at this level (Fig. 3A). Within the cerebral cortex, the orbitofrontal, temporal, and cingulate gyri exhibited increased radiotracer uptake. The cerebellum, brainstem, thalamus, and occiptial lobe had low activity levels. Radiotracer uptake in myelinated fiber tracts such as the corpus callosum, anterior commisure, and internal capsule was substantially lower than in gray matter. This pattern was completely reversed by pargyline retreatment (Fig. 3B). The blockade of monamine oxi ase B by pargyline resulted in radiotracer accumulation only within myelinated structures in brain. This observation suggests that the bioactivation of [ 12311MHTP results in the selective accumulation of radioactivity within those brain areas that contain a dense plexus of dopaminergic innervation. DISCUSSION As revealed by dynamic gamma scintigraphy, [lZ3Iland [lZ5I]MHTPreadily enter the brain. Tissue distribution studies indicate a 1.88%brain retention of radioactivity at 160 min postradiotracer injection. This value contrasts sharply with a 0.1% retention found in the rat brain at 120 min postinjection (Efange et al., 1989). However, the half-life of [lz3I1MHTP-derivedradioactivity (50min) in the primate brain is much shorter than that reported for MPP+ (Markey et al., 1984). Although the liver, kidneys, and brain contained the highest levels of radioactivity at the end of the scintigraphic study, a progressive accumulation of radioactivity was observed within the eyes over the course of the imaging study. Accumulation within this organ was evident within 50 min postinjection; such accumulation has been reported by other groups using radiolabeled MPTP in the monkey (Hartvig et al., 1986) and is presumed to reflect high retinal MA0 activity and trapping of metabolites by melanin-containing dopaminergic neurons of the retina (D'Amato et al., 1987; Lyden et al., 1985). In contrast to the rodent heart, which maintains levels of [ 1251]MHTP-derivedradioactivity comparable to those of the brain (Efange et al., 1989),very little radioactivity was retained in the monkey heart. This difference cannot be readily explained, and is all the more puzzling given both the high myocardial MA0 activity and the reported high retention of MPP' by the primate heart (Langston et al., 1984).

B

In our earlier studies of [lz5I1MHTPin the rodent, we have shown that [lz5I1MHTP-derivedradioactivity selectively accumulates within the locus coeruleus (Efange et al., 1989).Furthermore, this accumulation is inhibited by pretreatment with pargyline (Efange et al., 1989).In contrast to the pattern of distribution observed in the rodent, the present study reveals selective accumulation of [1251]MHTP-derivedradioactivity within the caudate-putamen, substantia nigra, and certain limbic cortices of the monkey brain. Pretreatment of a monkey with pargyline completely blocked the regional selective accumulation described above. With pargyline pretreatment, white matter structures exhibit greater concentrations of radioactivitj than grey matter. This distribution pattern is characteristic of neutral lipophilic tracers such as [lz5I]MHTP. Taken together, these results suggest that NL40 is involved in the conversion of [lZ5I]MHTPinto a radiolabeled metabolite that selectively accumulates within the caudate putamen and limbic cortex. This view is consistent with the large decrease in whole brain retention of [12511MHTPderived radioactivity associated with pargyline pretreatment. Thus the trapping suggested by the scintigraphic studies may reflect central MA0 activity. Based on the above evidence and the structural analogy between MPTP and [12511MHTP(see Fig. 11,it may be postulated that the [lz5I1MHTP-derivedmetabolite selectively accumulates within dopamine neurons via the dopamine reuptake system. Clearly, the comparison with the dopamine D2 ligand [I 23111BZMshows that its accumulation within the caudate and putamen is by a

Fig. 3. Pseudocolor images through the caudate and putamen of a c omolgus monkey illustrating the regional accumulation of I'[ IMHTP-derived radioactivity in s a g i t t a l slide-mounted sections. A Sixty minutes after an iv injection of 1 'IIMHTP, a marked accumulation of the radiotracer was observed throughout the basal ganglia (BG).Moderate activity was observed within the substantia nigra (SN) a t this level. In the cerebral cortex, high radiotracer activity was also seen over the orbitofrontal, temporopolar, and cingulate gyri. Dorsal to the cerebellum (CB), the occipital lobe sliows levels of radioactivity that correspond t o those seen in the CB. Background levels were observed over white matter structures, which appear as purple pseudocolor codes (low activity) in this autoradiogram. B: Pargyline pretreatment results in a virtual reversal in the accumulation pattern for ['2'IlMHTP-derived radioactivity. The basal ganglia were completely devoid of labeling, whereas white matter-containing areas now appear a s red pseudocolor codes (high density) in the autoradiogram.

BASAL GANGLIA-SELECTIVE RADIOLABELED MPTP ANALOG

Figure 3.

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S.M.N. EFANGE ET AL.

different mechanism. It may be argued that the selective accumulation of the radiolabeled MHTP metabolite within the primate caudate and putamen simply reflects high MA0 activity within these structures. This view would be consistent with the selective accumulation of [lz5I1MHTP-derivedradioactivity within the rodent locus coeruleus (Efange et al., 19891, a structure known to contain high concentrations of NLAO (Willoughby et al., 1988a). However, the intense labeling of the primate limbic cortex, a region known to contain relatively low MAO-B concentrations (Willoughby et al., 1988b), fails to support the above position. Furthermore, the high level of cortical accumulation of the [12511MHTP-derivedmetabolite sharply contrasts with the uniformly low level of accumulation of MPP' within this region (Javitch et al., 198513; Markey et al., 1984).In this connection, it should be noted that MPP+, following its production by MA0 within astroglia, is known to diffuse out of these cells (Brooks et al., 1989; Mytilineou and Friedman, 1988; Schinelli et al., 1988).Thus the metabolite need not be trapped at its locus of production. Evidently, although ['2511MHTP and MPTP may be structurally similar, their biological profiles are sufficiently dissimilar to warrant further investigation. Finally, a satisfactory explanation for the species-dependent regional distribution of ['2511MHTPderived radioactivity cannot be readily advanced a t this time.

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