Imaging of VMAT2 binding sites in the brain by (18)F-AV-133: the effect of a pseudo-carrier.

OBJECTIVES: Recently, 9-[(18)F]fluoropropyl-(+)-dihydrotetrabenazine ((18)F-AV-133) was reported as a new vesicular monoamine transporter (VMAT2) imaging agent for diagnosis of Parkinson's disease (PD). To shorten the preparation of (18)F-AV-133 and to make it more widely available, we evaluated a simple, rapid purification with a solid-phase extraction method (SPE) using an Oasis HLB cartridge instead of high pressure liquid chromatography (HPLC). The SPE method produced doses containing a pseudo-carrier, 9-hydroxypropyl-(+)-dihydrotetrabenazine (AV-149). METHODS: To test the possible side effects of this pseudo-carrier, comparative dynamic PET scans of the brains of normal monkeys (2 each) and uni-laterally 6-OH-dopamine-lesioned PD monkeys (2 each) were performed using (18)F-AV-133 doses prepared by either SPE (containing pseudo-carrier) or HPLC (containing no pseudo-carrier). Autoradiographs of post mortem monkey brain sections were evaluated to confirm the relative (18)F-AV-133 uptake in the PD monkey brains and the effects of the pseudo-carrier on VMAT2 binding. RESULTS: The radiochemical purity of the (18)F-AV-133, whether prepared by SPE or by HPLC, was excellent (>99%). PET scans of normal and PD monkey brains showed an expected reduction of VMAT2 in the lesioned areas of the striatum. It was not affected by the presence of the pseudo-carrier, AV-149 (maximally 250 µg/dose). The reduced uptake in the striatum of the lesioned monkey brains was confirmed by autoradiography. Ex vivo inhibition studies of (18)F-AV-133 binding in rat brains, conducted with increasing amounts of AV-149, suggested that at the highest concentration (3.5mg/kg) the VMAT2 binding in the striatum was only moderately blocked (20% reduction). CONCLUSIONS: The pseudo-carrier, AV-149, did not affect the (18)F-AV-133/PET imaging of VMAT2 binding sites in normal or uni-laterally lesioned monkey brains. The new streamlined SPE purification method will enable (18)F-AV-133 to be widely available for routine clinical application in determining changes in monoamine neurons for patient with movement disorders or other psychiatric illnesses.

Simple, rapid and reliable preparation of [¹¹C]-(+)-α-DTBZ of high quality for routine applications.

[¹¹C]-(+)-α-DTBZ has been used as a marker of dopaminergic terminal densities in human striatum and expressed in islet beta cells in the pancreas. We aimed to establish a fully automated and simple procedure for the synthesis of [¹¹C]-(+)-α-DTBZ for routine applications. [¹¹C]-(+)-α-DTBZ was synthesized from a 9-hydroxy precursor in acetone and potassium hydroxide with [¹¹C]-methyl triflate and was purified by solid phase extraction using a Vac tC-18 cartridge. Radiochemical yields based on [¹¹C]-methyl triflate (corrected for decay) were 82.3% ± 3.6%, with a specific radioactivity of 60 GBq/µmol. Time elapsed was less than 20 min from end of bombardment to release of the product for quality control.

A simple synthesis of [11C]dihydrotetrabenazine (DTBZ).

[11C]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9-[11C]methoxy-10 -methoxy-1,2,3,4,6,7,- hexahydro-11bH-bezo[alpha]-quinolizine) ([11C]DTBZ) was synthesized by reacting the 9-hydroxy precursor in DMSO with gas-phase [11C]methyl iodide on a column of alumina impregnated with KOH. The reaction was instantaneous at room temperature. This column was then connected to the inlet of a short column containing basic alumina. Elution with cyclohexane removed radioactive contaminants. The radioactive product was then eluted with a few milliliters ether containing 1% ethanol. The [11C]DTBZ was obtained in isolated yields of > 200 mCi and specific activities > 1600 Ci/mmol.

Absolute configuration of (+)-alpha-dihydrotetrabenazine, an active metabolite of tetrabenazine.

Chiral column liquid chromatography and enantiospecific enzymatic hydrolysis were utilized to separate the enantiomers of alpha- and beta-dihydrotetrabenazine and alpha-9-O-desmethyldihydrotetrabenazine, three benzo[a]quinolizines derived from the amine-depleting drug tetrabenazine. An X-ray crystal structure analysis of (-)-alpha-9-O-desmethyldihydrotetrabenazine gave an absolute structure of that compound as the 2S, 3S, 11bS isomer. Therefore, (-)-alpha-dihydrotetrabenazine also has the 2S, 3S, 11bS absolute configuration. (+)-alpha-Dihydrotetrabenazine, the single biologically active isomer from the metabolic reduction of tetrabenazine, thus has the absolute configuration of 2R, 3R, 11bR. For further in vitro and in vivo studies of the vesicular monoamine transporter, it is now possible to use the single enantiomer of radiolabeled alpha-dihydrotetrabenazine.

Purification of a [3H]dihydrotetrabenazine-binding protein from bovine adrenal medulla.

A high affinity binding site for [3H]dihydrotetrabenazine is thought to be present on the monoamine transport protein from chromaffin granules. We describe a procedure for purification of this binding activity from frozen bovine adrenal tissue, and we partially characterize the purified preparation. Binding activity solubilized with sodium cholate and soybean lecithin was fractionated on wheat germ lectin-Sepharose, phenyl-Sepharose, Mono Q, and hydroxylapatite. Denaturing electrophoresis of the purified binding activity, followed by silver staining, revealed a single broad band centered at an apparent molecular weight of 85,000. This preparation bound [3H]dihydrotetrabenazine with an apparent dissociation constant of 2.7 nM and had a site density of 10 nmol/mg. Treatment of the purified protein with neuraminidase reduced the apparent molecular weight by 9000, indicating the presence of terminal sialic acids on the oligosaccharide portion of this molecule.