Zhi-Ying Yang

Fluorinated benzamide neuroleptics—III. Development of (S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-[18F]fluoropropyl)-2,3-dimethoxybenzamide as an improved dopamine D-2 receptor tracer

We have prepared five new analogs (n-propyl, iso-propyl, allyl, n-butyl, and iso-butyl) of the dopamine D-2 receptor antagonist, FPMB which result from modifications of the ethyl group at the pyrrolidine nitrogen in FPMB. As expected, all new derivatives showed higher apparent lipophilicity (log kw), with iso-butyl being the most lipophilic (log kw = 2.52), followed by the allyl derivative (log kw = 2.43). The allyl group showed the largest increase in affinity (from 0.26 nM for the ethyl substituent to 0.03 nM for the allyl substituent, almost 10-fold), followed by the n-propyl substituent which showed approximately five-fold better affinity than did the ethyl substituent. Radiosynthesis of (S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-[18F]fluoropropyl)-2, 3-dimethoxybenzamide ([18F]fallypride) was carried out by nucleophilic substitution reaction of (S)-N-[(1-allyl-2-pyrrolidinyl) methyl]-5-(3-tosyloxypropyl)-2,3-dimethoxybenzamide with no carrier added 18F-. [18F]Fallypride was obtained in approximately 20-40% yields (EOS/EOB, decay corrected) in specific activities of 900-1700 Ci/mmol after reverse phase HPLC purification in 60 min from EOB. High striatal uptake (upto 2.5% injected dose/g) of [18F]fallypride in rats was observed with striatal/cerebellar ratios of 17, 42, 63 and 122 at 30, 60, 90 and 120 min post-injection, respectively. PET experiments with [18F]fallypride in a cebus monkey showed a brain uptake of 0.10% injected dose/cc. In rhesus monkeys [18F]fallypride showed rapid specific uptake in the striata (0.04-0.06% injected dose/cc) with striata/cerebellum ratios of approx. 3.0 at 14 min, 5.0 at 35 min and 8 at 70 min post-injection. Specifically bound [18F]fallypride was displaced with haloperidol (1 mg/kg) with a half-life of 18 min in the rhesus monkey.

Preliminary assessment of extrastriatal dopamine d-2 receptor binding in the rodent and nonhuman primate brains using the high affinity radioligand, 18F-fallypride

We have identified the value of 18F-fallypride [(S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-[18F]fluoropropyl)-2, 3-dimethoxybenzamide], as a dopamine D-2 receptor radiotracer for the study of striatal and extrastriatal receptors. Fallypride exhibits high affinities for D-2 and D-3 subtypes and low affinity for D-4 (3H-spiperone IC50s: D-2 = 0.05 nM [rat striata], D-3 = 0.30 nM [SF9 cell lines, rat recombinant], and D-4 = 240 nM [CHO cell lines, human recombinant]). Biodistribution in the rat brain showed localization of 18F-fallypride in striata and extrastriatal regions such as the frontal cortex, parietal cortex, amygdala, hippocampus, thalamus, and hypothalamus. In vitro autoradiographic studies in sagittal slices of the rat brain showed localization of 18F-fallypride in striatal and several extrastriatal regions, including the medulla. Positron emission tomography (PET) experiments with 18F-fallypride in male rhesus monkeys were carried out in a PET VI scanner. In several PET experiments, apart from the specific binding seen in the striatum, specific binding of 18F-fallypride was also identified in extracellular regions (in a lower brain slice, possibly the thalamus). Specific binding in the extrastriata was, however, significantly lower compared with that observed in the striata of the monkeys (extrastriata/cerebellum = 2, striata/cerebellum = 10). Postmortem analysis of the monkey brain revealed significant 18F-fallypride binding in the striata, whereas binding was also observed in extrastriatal regions such as the thalamus, cortical areas, and brain stem.

Evaluation of d-amphetamine effects on the binding of dopamine D-2 receptor radioligand, 18F-fallypride in nonhuman primates using positron emission tomography

We have investigated the ability of dopamine to compete with the binding of the high affinity dopamine D2 receptor positron emission tomography (PET) radioligand, 18F‐fallypride. In vitro dissociation of 18F‐fallypride with dopamine in rat striatal homogenates exhibited a dissociation rate, koff, of 1.76 × 10−2 min−1 while the association rate constant, kon, was found to be 5.30 × 108 M−1 min−1. This resulted in a dissociation constant, KD of 33 pM for 18F‐fallypride. For in vivo studies, we investigated the effects of reserpine and d‐amphetamine treatment on 18F‐fallypride in an attempt to study competition of endogenous dopamine with the radioligand at the receptor sites in rats and monkeys. PET experiments with 18F‐fallypride in two male rhesus monkeys were carried out in a PETT VI scanner. In control experiments, rapid specific uptake of 18F‐fallypride in the striata was observed (0.05–0.06% injected dose (ID)/g) while nonspecifically bound tracer cleared from other parts of the brain. Striata/cerebellum ratios for 18F‐fallypride were approximately 8 at 80 min postinjection, respectively. The monkeys received various doses (0.25 to 1.50 mg/kg) of d‐amphetamine (AMPH) pre‐ and postinjection of the radioligand. There was a decrease of specifically bound 18F‐fallypride as well as evidence of an enhanced clearance of specifically bound 18F‐fallypride after administering AMPH in the two monkeys. The dissociation rates, koff, of 18F‐fallypride without AMPH was <10−4 min−1 but after 25 min preadministration of AMPH (1 mg/kg), it was 4.1 × 10−3 min−1 and after 17, 45 and 90 min postadministration of AMPH (1 mg/kg) it was 3.6 × 10−3 to 4.0 × 10−3 min−1. Lower doses of AMPH (0.25 mg/kg) had a reduced effect on the binding of 18F‐fallypride. No effect was seen until about 30 minutes after the injection of AMPH. Studies with various doses indicated that 18F‐fallypride has a maximum response at doses of 0.75–1.50 mg/kg, with an approximately 16%/hour reduction in binding. These results indicate that AMPH stimulated release of endogenous dopamine reduces the specific binding of 18F‐fallypride. Synapse 27:1–13, 1997.

18F-desmethoxyfallypride: A fluorine-18 labeled radiotracer with properties similar to carbon-11 raclopride for pet imaging studies of dopamine D2 receptors

We have developed (S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-18F-fluoropropyl)-2- methoxybenzamide (18F-desmethoxyfallypride) as a fluorine-18 radiotracer with properties analogous to that of 11C-raclopride. In vitro experiments in rat brain homogenates showed an association rate constant of 2.16 x 10(8) M(-1)min(-1) and a dissociation rate constant of 0.073 min(-1). High striatal uptake (up to 0.08% injected dose/cc) of 18F-desmethoxyfallypride in rhesus monkeys was observed in PET experiments. The radiotracer cleared from the striata with a dissociation rate of 1.80 x 10(-2) min(-1). Striatum to cerebellum ratios peaked at 3.0 in 30 min after which they decreased steadily. Intravenously administered haloperidol displaced specifically bound 18F-desmethoxyfallypride with a koff of 0.058 min(-1). Synaptic dopamine released by the treatment of the monkeys with d-amphetamine increased the dissociation rate of 18F-desmethoxyfallypride to 0.83 min(-1) thus reducing specifically bound 18F-desmethoxyfallypride by 56% over a period of 42 mins compared to a reduction of only 20% in controls during this time period. The sensitivity of 18F-desmethoxyfallypride towards competition with dopamine should make this radiotracer useful in PET studies to evaluate in vivo pharmacological effects of various agents that alter levels of endogenous dopamine.

Monoamine oxidase A inhibition by fluoxetine: an in vitro and in vivo study.

Monoamine oxidase A (MAO‐A) inhibition was investigated both in vitro and in vivo in rat brains by using the radioligand, 18F‐fluoroclorgyline (N‐[3‐(2′,4′‐dichlorophenoxy)‐2‐18F‐fluoropropyl]‐N‐methylpropargylamine). In vitro binding affinities of six compounds, clorgyline, Ro 41–1049, deprenyl, fluoxetine, norfluoxetine and citalopram, were studied. Fluoxetine and norfluoxetine showed in vitro affinities of 36.5 and 68 μM for MAO‐A, respectively. Fluoxetine and norfluoxetine also significantly inhibited (more than 20%) the binding of the radioligand in vivo while citalopram and deprenyl showed very poor affinities in vitro for MAO‐A and had no effect in vivo. The in vivo effects of the various drugs were directly comparable to their in vitro affinities for binding to MAO‐A as seen in the correlation plot of percent control in vivo binding of 18F‐fluoroclorgyline and binding affinity, ‐log IC50 (R2 = 0.979). An acute dose of 20 mg/kg of fluoxetine inhibited binding of 18F‐fluoroclorgyline by more than 20%, while lower doses had some significant effects. These results provide evidence on the in vitro and in vivo inhibition of monoamine oxidase A by fluoxetine. Synapse 31:285–289, 1999.

Evaluation of the binding of the radiolabeled antidepressant drug, 18F-fluoxetine in the rodent brain: an in vitro and in vivo study

We have developed 18F-fluoxetine as a radiotracer analog of the antidepressant drug fluoxetine (Prozac). In vitro saturation experiments of 18F-fluoxetine were carried out on rat midbrain tissue and citalopram was used for measuring nonspecific binding. A saturation curve for the binding of 18F-fluoxetine was not obtained. Even when fluoxetine (10 microM) was used for measurements of nonspecific binding, a saturation curve was difficult to obtain. Other compounds, such as deprenyl, clorgyline, amphetamine, and reserpine were also not able to reduce the binding of 18F-fluoxetine. Ex vivo autoradiographic experiments with 18F-fluoxetine did not reveal any specific uptake in various brain regions. In vivo administration of 18F-fluoxetine in rats showed similar uptake in all the brain regions with little regional selectivity. A subcellular analysis of rat brain tissue after intravenous (IV) administration of 18F-fluoxetine indicated significant amounts of binding in mitochondria and synaptosomes. In summary, in vitro experiments with 18F-fluoxetine indicate little specific binding. Binding to the serotonin transporter was not identifiable. High nonspecific binding of the tracer resulting from its subcellular nature in the brain masks the ability to detect binding to the serotonin uptake sites in vivo. These findings indicate that a large portion of the binding of 18F-fluoxetine in rat brains is subcellular and clears slowly out of the cells. Other sites, such as monoamine oxidase, may also play a significant role in the action of fluoxetine.

Evaluation of monoamine oxidase B inhibition by fluoxetine (Prozac): an in vitro and in vivo study.

Inhibition of monoamine oxidase B was investigated both in vitro and in vivo in rats by using the radioligand, N-(6-[18F]fluorohexyl)-N-methylpropargylamine ([18F]FHMP). Binding affinities of five compounds, deprenyl, clorgyline, fluoxetine, norfluoxetine and citalopram were studied. Fluoxetine and norfluoxetine showed affinities of 17 and 13 microM for monoamine oxidase B, respectively. Acute doses of fluoxetine and norfluoxetine (20 mg/kg) also significantly inhibited (10 to 15%) the binding of the radioligand in vivo while citalopram showed lower affinity (140 microM) for monoamine oxidase B and little effect in vivo. The in vivo effects of the various drugs were directly comparable to their in vitro affinities for binding to monoamine oxidase B in the correlation plot of percent control in vivo binding of [18F]FHMP and binding affinity, -logIC50 (R2 = 0.989). These results provide evidence for a potential role of monoamine oxidase B inhibition in the therapeutic effects of Prozac.

N-(6-18F-Fluorohexyl)-N-Methylpropargylamine: A Fluorine-18-Labeled Monoamine Oxidase B Inhibitor for Potential Use in PET Studies

We have synthesized N-(6-18F-fluorohexyl)-N-methylpropargylamine (18F-FHMP) as a positron emission tomography (PET) radiotracer for monoamine oxidase B (MAO-B). The radiosynthesis was carried out by a fluorine-for-bromine substitution in 30-40% radiochemical yield in specific activities of 1-2 Ci/micromol. Selectivity for MAO-B was demonstrated by the high affinity of (R)-deprenyl (IC50 = 6.8 nM) and lower affinity of clorgyline (IC50 = 1.2 microM) for the inhibition of 18F-FHMP binding in vitro in rat brain homogenates. In vitro autoradiographic studies in rat brain slices showed localization of 18F-FHMP in regions such as the ependyma of the lateral ventricle, dorsal raphe, area postrema, and other regions such as the cerebellum. The specific binding observed in the autoradiograms was displaced by preincubation with (R)-deprenyl. In in vivo experiments, the uptake of 18F-FHMP in the rat brains was high (0.10-0.20% injected dose/g). The binding of 18F-FHMP in the rat brain correlated with the general distribution of MAO-B and was displaced completely by preadministration of 10 microM (R)-deprenyl. These results suggest that 18F-FHMP is a potential PET radiotracer for MAO-B for use in in vitro and in vivo experiments.

Synthesis, radiosynthesis, and biological evaluation of fluorinated thienylcyclohexyl piperidine derivatives as potential radiotracers for the NMDA receptor-linked calcium ionophore

Various thienylcyclohexyl piperidine (TCP) derivatives were synthesized and evaluated as potential candidates for use as radiotracers for the in vivo study of the NMDA receptor ion-channel. Modification of the thienylcyclohexylpiperidine was accomplished by substituting its piperidine ring either with other cycloamine rings or N-alkyl-substituted amines, and these two classes of TCP derivatives were synthesized using two different methods. The compounds exhibited affinities ranging from 65 nM up to micromolar in competition assays for the receptor ion-channel labeled with 3H-(+)-MK-801 in rat brain homogenates. Radiosynthesis of 1-[1-(2-thienyl)-4-([18F]fluoro)-cyclohexyl]-1,2,5, 6-tetrahydropyridine was carried out by nucleophilic substitution reaction of 1-[1-(2-thienyl)-4-tosyloxycyclohexyl]-1,2,5,6-tetrahydropyridine with no carrier added 18F-, and the yield was approximately 5-10% (decay corrected) in specific activities of 500-1000 Ci/mmol after reverse-phase HPLC purification. The tracer showed good uptake in rat brains after i.v. injection (approx. 0.10% injected dose/g at 30 min. p.i.). However, the specific uptake in receptor-rich regions (striata, hippocampus, frontal cortex, and parietal cortex) improved only marginally with time compared to cerebellum. Three hours postinjection, parietal cortex showed a maximum ratio of 1.9. Preliminary PET experiment with this radiotracer in a rhesus monkey showed good uptake in the brain regions. However, little retention of the radiotracer was observed in the receptor-rich regions.

DEVELOPMENT OF FLUORINE-18 RADIOPHARMACEUTICALS FOR DOPAMINE NEURORECEPTORS

Fluorine-18 offers good radionuclide characteristics for incorporation into positron radiopharmaceuticals. Development of fluorine-18 radiopharmaceuticals for neuroreceptor imaging by positron emission tomography (PET) is a challenge that most of us in the PET community have encountered. The magnitude of the problem is due to the multidisciplinary nature of issues that a radiochemist faces, such as drug design, organic syntheses, in vitro pharmacology, radiolabeling methods, in vivo pharmacology, in vivo stability and radiotracer modeling methods in order to achieve the successful application of a radiopharmaceutical in meaningful human studies. The radiochemist is therefore forced to anticipate and take into account in the design process the potential hurdles that might be encountered upon arrival at each step. Fluorine is becoming a more common element that is incorporated into biologically interesting molecules within the pharmaceutical industry. When such fluorinated lead compounds from the pharmaceutical industry are available, development of a fluorine-18 radiopharmaceutical is somewhat simplified, and then largely depends upon radiolabeling methods, in vivo pharmacology, in vivo stability and radiotracer modeling methods. However, there remain a large number of potentially useful pharmaceuticals which are of interest to PET researchers that do not possess a fluorine atom. Therefore as a question of investment versus outcome should researchers, particularly radiochemists in the PET community, embark on new drug development rather than use only what is available from the pharmaceutical industry.