Mapping human brain monoamine oxidase A and B with 11C-labeled suicide inactivators and PET.

The regional distributions of monoamine oxidase (MAO) types A and B have been identified in human brain in vivo with intravenously injected 11C-labeled suicide enzyme inactivators, clorgyline and L-deprenyl, and positron emission tomography. The rapid brain uptake and retention of radioactivity for both 11C tracers indicated irreversible trapping. The anatomical distribution of 11C paralleled the distribution of MAO A and MAO B in human brain in autopsy material. The corpus striatum, thalamus, and brainstem contained high MAO activity. The magnitudes of uptake of both [11C]clorgyline and L-[11C]deprenyl were markedly reduced in one subject treated with the antidepressant MAO inhibitor phenelzine. A comparison of the brain uptake and retention of the 11C-labeled inactive (D-) and active (L-) enantiomers of deprenyl showed rapid clearance of the inactive enantiomer and retention of the active enantiomer within MAO B-rich brain structures, in agreement with the known stereoselectivity of MAO B for L-deprenyl. Prior treatment with unlabeled L-deprenyl prevented retention of L-[11C]deprenyl. Thus, suicide enzyme inactivators labeled with positron emitters can be used to quantitate the distribution and kinetic characteristics of MAO in human brain structures.

Mapping brain neuroleptic receptors in the live baboon.

An experimental strategy for external detection of specific neuroleptic receptors in living brain using positron emission transaxial tomography (PETT) and [11C]spiroperidol was applied to the mapping of brain neuroleptic receptors in the live baboon. A double injection of [11C]spiroperidol with an intervening time interval for carbon-11 decay and an intervening dose of (+)-butaclamol to block specific neuroleptic receptors produced two sets of PETT scans which were subtracted to produce a three-dimensional map of relative regional binding of neuroleptic receptors in the baboon brain. Sixty-five percent of the total radioactivity in the striatum was bound to neuroleptic receptors at 65 min after injection.

Serial [18F]N-methylspiroperidol PET studies to measure changes in antipsychotic drug D-2 receptor occupancy in schizophrenic patients.

An indirect approach to the relationship among drug dose, plasma level, and the competition between a labeled neuroleptic drug [18F]N-methylspiroperidol (18F-NMS) for binding sites in striatal tissue in normal and schizophrenic subjects is described. The slope of the line plotting the ratio of activity in the striatum (As) to activity in the cerebellum (Ac) versus time up to 5 hr postinjection of 18F-NMS is taken as a marker of site occupancy. An inverse relation between labeled competitor uptake and drug plasma level has been demonstrated for the classes of antipsychotic drug studied. Striatal uptake studies showed a progressive increase in all subjects following drug withdrawal up to 156 hr postwithdrawal. Uptake and clearance of 18F-NMS in cerebellar tissue was not appreciably affected by antipsychotic medication or drug withdrawal.

Synthesis and adrenergic activity of benzimidazole bioisosteres of norepinephrine and isoproterenol.

The concept of bioisosterism between benzimidazole and catechol was applied to the design and synthesis of benzimidazole analogues of norepinephrine, (R,S)-1-[5(6)-benzimidazolyl]-2-aminoethanol (2), and of isoproterenol, (R,S)-1-[5(6)-benzimidazolyl]-2-isopropylaminoethanol (4). Compound 2 was shown to be a partial bioisostere of norepinephrine, with direct agonist activity at the alpha-adrenergic receptor. The ED50 for 2 in contracting the guinea pig isolated aortic strip was determined to be 8.0 x 10(-6) M. Compound 4 was shown to be a partial bioisostere of isoproterenol, with direct activity as a beta-adrenergic agonist. The ED50 values for positive chronotropic and inotropic effects of 4 on the isolated guinea pig atrial preparation were determined to be 6.2 x 10(-6) and 3.8 x 10(-6) M, respectively. The ED50 for 4 on the isolated guinea pig tracheal preparation was determined to be 1.6 x 10(-6) M. These results indicate that 4 shows greater selectively for the beta-2 adrenergic receptor than does isoproterenol. The chemical stability of benzimidazole, compared with that of catechol, suggests that benzimidazole bioisosteres of catecholamines may be of value as adrenergic drugs.

No-carrier-added N-(3-[18F]fluoropropyl)spiroperidol: biodistribution in mice and tomographic studies in a baboon.

Two potential radioligands, no-carrier-added (NCA) N-(2-[18F]fluoroethyl)spiroperidol (3) and N-(3-[18F]fluoropropyl)spiroperidol (4) have been synthesized for PET imaging of dopamine receptors in humans. Compounds 3 and 4 were synthesized by N-alkylation of spiroperidol with NCA 1-bromo-2-[18F]-fluoroethane (2b), 1-[18F]fluoro-3-iodopropane (2c) and 1-bromo-3-[18F]fluoropropane (2d) respectively. The biodistribution of 4 in mice showed that the mouse brain uptake of radioactivity was similar to that of [18F]-N-methylspiroperidol (1.1% of the administered dose), but the activity in bone (femur) increased with time. The kinetic distribution of compound 4 in baboon brain was similar to that of [18F]-N-methylspiroperiodol, and the striatal accumulation of radioactivity was also blocked stereoselectively by butaclamol. The ratio of striatum to cerebellum radioactivities at 3 hr after injection was 5.9. Analysis of the metabolic stability of 4 in mouse brains for 1 hr indicated that, like [18F]-N-methylspiroperidol, it is relatively stable to metabolic transformation in the central nervous system. These results suggest that compound 4 may be a useful radioligand for PET studies of the dopamine receptor in humans.

Syntheses and specific activity determinations of no-carrier-added (NCA) F-18-labeled butyrophenone neuroleptics--benperidol, haloperidol, spiroperidol, and pipamperone.

A general method for the syntheses of no-carrier-added (NCA) 18F-labeled butyrophenone neuroleptics--benperidol, haloperidol, spiroperidol, and pipamperone is described. These 18F-labeled neuroleptic drugs are synthesized by a multistep synthesis in an overall radiochemical yield of 10-20% at end of bombardment (EOB) in a synthesis time of 90 min from EOB. The sequence involves the synthesis of NCA p-[18F]fluorobenzonitrile from NCA [18F]-fluoride and p-nitrobenzonitrile using the rapidly converted to gamma-chloro-p-[18F]fluorobutyrophenone which is alkylated with appropriate amines to give NCA 18F-labeled benperidol, haloperidol, spiroperidol, and pipamperone. The final product is purified by preparative high performance liquid chromatography (HPLC). The 18F solution used in the synthesis as determined by ion chromatography contains 15.3 +/- 9.0 nmol of stable fluoride. The specific activities of the resulting butyrophenone neuroleptics were determined to be 3 Ci/mumol (at EOB) (range 1-6 Ci/mumol) as determined by radioreceptor assay and HPLC assay.

No-carrier-added fluorine-18-labeled N-methylspiroperidol: synthesis and biodistribution in mice.

No-carrier-added fluorine-18- (18F) labeled N-methylspiroperidol (4) was synthesized from four different substrates: p-nitrobenzonitrile (1), cyclopropyl p-nitrophenyl ketone (2A), p-cyclopropanoyl-N,N,N-trimethylanilinium iodide (2B) and p-cyclopropanoyl-N,N,N-trimethylanilinium perchlorate (2C) using the nucleophilic aromatic substitution reaction. Radiochemical yield, synthesis time, experimental simplicity, and specific activity were compared. In addition, factors which influence the yield of the nucleophilic aromatic substitution were studied. Based on these studies, the synthesis of 4 from 2A maximizes product specific activity and experimental simplicity and provides 4 in 10-15% radiochemical yield [based on [18F-] with a mass of less than 2 nmol and a specific activity of greater than 10 Ci/mumol (EOB)]. The synthesis of 4 from 8-[4-(4-nitrophenyl)-4-oxobutyl]-3-methyl-1-phenyl-1,3,8-triazaspiro+ ++ [4.5]decan-4-one (5) and Cs[18F] using the nucleophilic aromatic substitution reaction gave unacceptably low and erratic yields. The biodistribution of 4 in mice showed a maximum brain uptake of 1.1% of the administered dose at 5 min and declined to approximately 0.6% at 120 min.

Improved delineation of human dopamine receptors using [18F]-N-methylspiroperidol and PET.

The brain uptake of [18F]-N-methylspiroperidol, a butyrophenone neuroleptic with high selectivity for the dopamine receptor, has been measured in three normal human volunteers using positron emission tomography for times up to 12 hr postinjection. These studies demonstrated two unique findings concerning the in vivo distribution of this neuroleptic: (a) it is tightly bound to dopamine D-2 receptors in the caudate-putamen brain regions, and (b) these regions are the only large brain structures which exhibit appreciable long-term retention. In addition, radioactivity clears rapidly from plasma, and the percentage of unchanged [18F]-N-methylspiroperidol in plasma declines rapidly. These results suggest that this compound binds irreversibly to dopamine D-2 receptors, and that there are few if any dopamine D-2 receptors in the human frontal cortex. These studies emphasize not only the importance of characterizing neurotransmitter receptors in living human brain using a ligand labeled with a positron emitting nuclide of sufficiently long half-life to allow monitoring of brain radioactivity distribution for several hours after the injection of radioligand, but also of accurately determining the amount of unchanged tracer in plasma for tracer kinetic modeling.

Synthesis and biodistribution of no-carrier-added [1-11C]putrescine.

No-carrier-added [1-11C]putrescine was synthesized in 20% radiochemical yield in a synthesis time of 50 min by the Michael addition of potassium [11C]cyanide to acrylonitrile followed by reduction of the [11C]dinitrile with borane-methyl sulfide complex. Biodistribution in mice at 5, 30, and 60 min showed low uptake in normal brain tissue.

Brain alpha-adrenergic receptors: comparison of [3H]WB 4101 binding with norepinephrine-stimulated cyclic AMP accumulation in rat cerebral cortex.

The ability of a series of adrenergic agents to displace the binding to brain membranes of [3H]WB 4101, a potent alpha-adrenergic antagonist (WB 4101 = 2-[2-(2,6-dimethoxyphenoxy)ethylaminomethyl]-1,4-benzodioxane hydrochloride), has been compared with the potency of these agents in stimulating or inhibiting the alpha-adrenergic component of cyclic AMP accumulation in rat cerebral cortex slices. [3H]WB 4101 rapidly bound to a high affinity site (KD = 2.7 nM) in membranes from cerebral cortex. Binding came to equilibrium by 2 min at 37 degrees C and was rapidly reversed in the presence of phentolamine. The potencies of adrenergic agents (WB 4101 greater than phentolamine greater than naphazoline) in displacing binding of [3H]WB 4101 were comparable to the potencies of these agents as inhibitors of the alpha-adrenergic component of norepinephrine-stimulated cyclic AMP accumulations. Phenoxybenzamine, clonidine, chlorpromazine and haloperidol were about 10--30 times more potent in inhibiting cyclic AMP accumulation than in displacing [3H]WB 4101 binding. The potency of classical alpha-adrenergic agonists in displacing WB 4101 (epinephrine greater than norepinephrine greater than methoxamine) correlated with the ability of these agonists to increase cyclic AMP levels. Overall a significant correlation (r = 0.87, P less than 0.005) was found between WB 4101 binding and alpha-adrenergically mediated cyclic AMP accumulation in brain. Several ligands bind to specific sites in brain membranes with alpha-adrenergic receptor properties. The identification of these binding sites as receptors depends on a correlation of binding with a known alpha-adrenergic receptor-mediated response in brain. These data demonstrating that WB 4101 correlates with norepinephrine-stimulated cyclic AMP accumulation suggest that WB 4101 may bind to the membrane receptor sites mediating the alpha-adrenergic accumulation of cyclic AMP in rat cerebral cortex.

Carbon-11 labelled ketamine-synthesis, distribution in mice and PET studies in baboons.

No-carrier-added (NCA)[11C](+/-)-ketamine (2a) and its enantiomers (+)-2b and (-)-2c were synthesized by methylation of the corresponding norketamine (1a-c) with [11C]H3I in an overall radiochemical yield of 20% (EOB) with specific activities of 0.35-0.45 Ci/mumol at EOB in a synthesis time of 40 min from EOB. Compound 2a was metabolized rapidly in mouse brain and labeled metabolites appeared in baboon plasma. PET studies of compounds 2a-c in a baboon showed that influx of compounds 2a-c into the brain was high for the first few min but radioactivity then declined rapidly. Although the retention of radioactivity in the baboon striatum was not significantly different for 2a-c 20 min post-injection, graphical analysis of time-activity data for each enantiomer and for the racemate in baboon striatum suggested that (+)-ketamine may interact with receptors slightly more effectively than its (-)-enantiomer or racemate. However, due to its rapid metabolism in the brain and a similar uptake in the striatum and cerebellum, [11C]ketamine may not be an ideal tracer for studying NMDA receptor with PET.

Species differences in [11C]clorgyline binding in brain.

[11C]Clorgyline selectively binds to MAO A in the human brain. This contrasts with a recent report that [11C]clorgyline (in contrast to other labeled MAO A inhibitors) is not retained in the rhesus monkey brain [4]. To explore this difference, we compared [11C]clorgyline in the baboon brain before and after clorgyline pretreatment and we also synthesized deuterium substituted [11C]clorgyline (and its nor-precursor) for comparison. [11C]Clorgyline was not retained in the baboon brain nor was it influenced by clorgyline pretreatment or by deuterium substitution, contrasting to results in humans. This suggests a species difference in the susceptibility of MAO A to inhibition by clorgyline and represents an unusual example of where the behavior of a radiotracer in the baboon brain does not predict its behavior in the human brain.

[18F]-N-Methylspiroperidol: the radioligand of choice for PETT studies of the dopamine receptor in human brain.

N-Methylspiroperidol, the amide N-methyl analogue of the neuroleptic spiroperidol, was radiolabeled with fluorine-18, and its distribution in the baboon brain was studied using positron emission transaxial tomography. Stereospecific binding was demonstrated in the striatum (but not in the cerebellum) by pretreatment with (-)- or (+)-butaclamol. The kinetic distribution was similar to that of [18F]spiroperidol, but the absolute striatal uptake (in percent of administered dose) was at least two-fold higher. Analysis of baboon blood at 10 min after injection indicated that less than half of the radioactivity in the plasma was due to unchanged radioligand. Analysis of the metabolic stability of [18F]-N-methylspiroperidol in rat brain for 4 hr indicated that, like [18F]spiroperidol, it is very stable to metabolic transformation in the rat central nervous system. Striatal uptake and retention in the rat was five-fold higher for [18F]-N-methylspiroperidol than for [18F]spiroperidol. These results suggest that [18F]-N-methylspiroperidol is an ideal choice for studies of the dopamine receptor in humans.

Denervation-induced changes in alpha and beta adrenergic receptors of the rat submandibular gland.

Denervation resulted in a marked increase in the beta adrenergic response and isoproterenol-stimulated cyclic adenosine 3':5'-monophosphate accumulation of dispersed cells prepared from a rat submandibular gland. This increase in beta adrenergic response was paralleled by an increase in the density of beta adrenergic receptors in membranes prepared from these glands. Denervation also produced a moderate increase in alpha adrenergic receptor density in membranes prepared from whole glands. However, the alpha adrenergic response in cells, epinephrine-induced release of potassium, appeared unaltered by denervation. Furthermore, membranes prepared from denervated dispersed cells did not show the increase in alpha adrenergic receptor density seen in membranes from an intact denervated gland. These data demonstrate that removal of noradrenergic nerve terminals affects alpha and beta adrenergic receptors differently. While it is clear that beta adrenergic membrane receptors participate in denervation-induced supersensitivity, the changes in alpha adrenergic membrane receptors are more complex and may not contribute to the supersensitivity seen after denervation. On the basis of competitive binding studies, the adrenergic receptors in membranes from intact submandibular glands were subclassified as beta-1 and alpha-2. Denervation did not alter the binding characteristics of the alpha-2 receptors in this gland, demonstrating that alpha-2 adrenergic membrane receptors can be postsynaptic in this adrenergically innervated tissue.

Synthesis and biodistribution of 2-deoxy-2-[18F]fluoro-D-glucopyranosyl [18F]fluoride in mice.

The synthesis and biodistribution of 2-deoxy-2-[18F]fluoro-D-glucopyranosyl [18F]fluoride (4 approximately) in mice are described. Reaction of 3,4,6-tri-O-acetyl-D-glucal (1 approximately) with [18F]F2 in freon-11 at -78 degrees C gives 2-deoxy-2-[18F]fluoro-3,4,6-tri-O-acetyl-alpha-D-glucopyranosyl [18F]fluoride (2 approximately) and 2-deoxy-2-[18F]fluoro-3,4,6-tri-O-acetyl-beta-D-mannopyranosyl [18F]fluoride (3 approximately). Partial hydrolysis of 2 approximately with NaOMe/MeOH gives compound 4 approximately in a radiochemical yield of approximately 28% (based on 18F recovered) and in a synthesis time of 70 min from EOB. The biodistribution of compound 4 approximately in mice suggests that it defluorinates in vivo to give 2-deoxy-2-[18F]fluoro-D-glucose (5 approximately) and [18F]fluoride as metabolites.