Acute Inhibition of Cardiac Monoamine Oxidase A after Tobacco Smoke Inhalation: Validation Study of [C]Befloxatone in Rats Followed by a Positron Emission Tomography Application in Baboons

The in vivo characteristics of [C]befloxatone were assessed in myocardium of rats and monkeys. A complete multicompartmental model was developed to quantify monkey cardiac monoamine oxidase A (MAO-A) binding sites using positron emission tomography (PET) and was applied to assess the acute effects of inhalation of tobacco smoke. Unknown compounds contained in tobacco smoke inhibit brain MAO. In vitro, befloxatone inhibits selectively, competitively, and reversibly MAO-A in human tissues. [C]Befloxatone (1.85 MBq) was i.v. injected into rats. Animals were sacrificed, dissected, and samples were assessed for radioactivity. Another group of rats was pretreated with clorgyline (10 mg/kg i.v.). Monkeys were injected with [C]befloxatone (222–370 MBq), and the chest was imaged with PET for 2 h. Presaturation and displacement experiments were performed using unlabeled befloxatone. For quantification of myocardial binding sites (Bmax), [ C]befloxatone was first injected as a tracer dose (2.7–9.3 nmol) and 20 min later injected as a mixture of labeled and unlabeled befloxatone (labeled, 10.3–41.9 nmol; unlabeled, 407–765 nmol). In rodents, cardiac uptake was high (3.39 0.5% injected dose/g tissue) and strongly inhibited (80%) by clorgyline. In monkeys, administration of unlabeled befloxatone displaced 85% of cardiac radioactivity. Bmax was found to be 208 13 pmol ml 1 tissue. Inhalation of tobacco smoke decreased Bmax: 150 6.2 pmol ml , whereas nicotine did not. [C]Befloxatone allows a good visualization of the heart. Cardiac MAO-A Bmax was quantified and a clear effect of acute inhalation of tobacco smoke was evidenced. Therefore, a single cigarette can interfere with the cardiac turnover of catecholamines. MAO (monoamine: oxidoreductase deaminating EC 1.4.3.4) plays a key role in the metabolism of endogenous amines and xenobiotics. MAO has been subdivided into MAO-A and MAO-B subtypes according to substrate specificity (MAO-A: 5-hydroxytryptamine, norepinephrine, epinephrine, and serotonin; and MAO-B: phenylethylamine and benzylamine). MAO inhibitors are widely used for treatment of depression and tobacco addiction or tobacco weaning (Robinson 2002; Villegier et al., 2003). Therefore, in vivo study of MAO using PET has mainly focused on neuropsychiatric disorders or tobacco addiction. Smoking is a major public health problem. Although several pharmacological properties of nicotine are known, tobacco smoke contains about 4000 chemical compounds with unknown properties. Smokers have reduced levels of brain monoamine oxidase (Fowler et al., 1996a,b). Little in vivo data are available about myocardial MAO. In the heart, MAO-A is abundant (Saura et al., 1992). Active reuptake (uptake-1) metabolism of norepinephrine by MAO and by catechol-O-methyltransferase are the major ways for ending the action of the neurotransmitters (Eisenhofer et al., 2004). In vivo MAO studies with PET involve the use of an inhibitor of the enzymes. The prototype inhibitors (“suicide inactivators”, which engender covalent bonds with the active site of the enzyme and induce irreversible inhibition) used in PET studies are [C]deprenyl and [C]clorgyline (Fowler et al., 1996a,b), which inhibit MAO-B and MAO-A, respectively. They have been successfully used in humans with PET to demonstrate the inhibition of brain MAO-A and MAO-B in smokers. But these two inhibitors are known to exhibit a strong deuterium effect; therefore, their synthesis needs to have the deuterated precursors (Fowler et al., 1995). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.085704. ABBREVIATIONS: MAO, monoamine oxidase; PET, positron emission tomography; i.d., injected dose; ANOVA, analysis of variance. 0022-3565/05/3141-431–436$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 314, No. 1 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 85704/3038969 JPET 314:431–436, 2005 Printed in U.S.A. 431 at A PE T Jornals on A ril 8, 2017 jpet.asjournals.org D ow nladed from Among the second generation of reversible, highly selective MAO-A inhibitors, befloxatone inhibited competitively MAO-A in several human and rodent tissues with Ki values ranging from 1.9 to 3.6 nM (Curet et al., 1996). Its corresponding values for MAO-B were 270 to 900 nM (Curet et al., 1996). The C-radiolabeled befloxatone (Dollé et al., 2003a,b) has been developed to study brain MAO-A and was fully characterized in baboons (Bottlaender et al., 2003). In whole-body scans, we have observed that both the human and baboon hearts were clearly seen (unpublished data). Therefore, pharmacological characterization of befloxatone cardiac uptake was performed in rats and in baboons. A compartmental model was applied to PET data to determine the left ventricular density of binding sites and to assess their acute changes induced by inhalation of tobacco smoke. As a consequence of a partial inhibition of cardiac MAO-A activity, the clearance of catecholamine could be reduced, altering the sympathetic tone and potentially contributing to some deleterious effects of smoking. Therefore, the PET study of MAO in the heart seems clinically relevant. Materials and Methods Radiosynthesis of [C]Befloxatone Befloxatone [(5R)-5-(methoxymethyl)-3-[4-[(3R)-4,4,4-trifluoro-3hydroxybutoxy] phenyl]-2-oxazolidinone] was labeled with C from the ring-opened precursor (R)-1-methoxy-3-[[4-[(3R)-4,4,4-trifluoro3-hydroxybutoxy]-phenyl]amino]-2-propanol using no-carrier-added [C]phosgene (Dollé et al., 2003a,b). Typically, starting from a 44.4 GBq cyclotron-produced [C]CH4 batch, 5.55 to 11.10 GBq of [C]befloxatone with a radiochemical and chemical purity of more than 99% were routinely obtained within 20 min of radiosynthesis (including high-pressure liquid chromatography purification) with specific radioactivities of 18.5 to 74.0 GBq/ mol.