Difficulties in dopamine transporter radioligand PET analysis: the example of LBT-999 using [18F] and [11C] labelling: part II: Metabolism studies.

INTRODUCTION: LBT-999, (E)-N-(4-fluorobut-2-enyl)-2ß-carbomethoxy-3ß-(4'-tolyl)nortropane, has been developed for PET imaging of the dopamine transporter. [(18)F]LBT-999 PET studies in baboons showed a lower brain uptake than [(11)C]LBT-999 and a high bone uptake, suggesting the presence of interfering metabolites. Therefore, in vitro and in vivo metabolism of these radiotracers was investigated. METHODS: Rat and human liver microsomal incubations, baboon plasma and rat brain extracts were analyzed by radio-HPLC and LC-MS-MS. RESULTS: In vitro experiments demonstrated the formation by P450s of five polar metabolites. The main routes of LBT-999 metabolism proposed were N-dealkylation, tolyl-hydroxylation and dealkylation plus tolyl-hydroxylation. In vivo in baboons, [(18)F]LBT-999 was rapidly converted into a [(18)F]hydroxylated metabolite likely oxidized in plasma into a [(18)F]carboxylic acid and into unlabeled N-dealkyl-LBT-999. The latter was detected in baboon plasma and in rat brain by LC-MS-MS. The time course of unchanged [(18)F]LBT-999 decreased rapidly in plasma and was higher than that of [(11)C]LBT-999 due to the formation of unlabeled N-dealkyl-LBT-999. In rats, striatum-to-cerebellum ratios of [(18)F]LBT-999, [(18)F]hydroxylated and [(18)F]acidic metabolite were 20, 4.2 and 1.65, respectively, suggesting a possible accumulation of the hydroxylated compound in the striatum. CONCLUSION: P450s catalyzed the formation of dealkylated and hydroxylated metabolites of LBT-999. In baboons, an extensive metabolism of [(18)F]LBT-999, with formation of unlabeled N-dealkyl-LBT-999, [(18)F]fluorobutenaldehyde (or its oxidation product) and [(18)F]hydroxy-LBT-999 able to penetrate the brain, prevented an easy and accurate estimation of the input function of the radiotracer. CYP3A4 being the main P450 involved in the metabolism of LBT-999, a similar pathway may occur in humans and confound PET quantification.

In vitro metabolism studies of (18)F-labeled 1-phenylpiperazine using mouse liver S9 fraction.

The in vitro metabolism of 1-(4-[(18)F]fluoromethylbenzyl)-4-phenylpiperazine ([(18)F]1) and 1-(4-[(18)F]fluorobenzyl)-4-phenylpiperazine ([(18)F]2) was investigated using mouse liver S9 fraction. Results were compared to those of in vivo metabolism using mouse blood and bone and to in vitro metabolism using mouse liver microsomes. Defluorination was the main metabolic pathway for [(18)F]1 in vitro and in vivo. Based on TLC, HPLC and LC-MS data, [(18)F]fluoride ion and less polar radioactive metabolites derived from aromatic ring oxidation were detected in vitro, and the latter metabolites were rapidly converted into the former with time, whereas only the [(18)F]fluoride ion was detected in vivo. Similarly, the in vitro metabolism of [(18)F]2 using either S9 fraction or microsomes showed the same pattern as the in vivo method using blood; however, the radioactive metabolites derived from aromatic ring oxidation were not detected in vivo. These results demonstrate that liver S9 fraction can be widely used to investigate the intermediate radioactive metabolites and to predict the in vivo metabolism of radiotracers.

Inhibition of enzymes of drug metabolism in rat liver by ultrasound and correction by heparin.

After 6 days following the local effect (during operation) of ultrasound (2 Wt/cm2, 1 min) the microsomal fraction showed decreased total content of cytochromes P-450 (P-450), rate of NADPH oxidation, activity of NADPH-cytochrome P450 reductase and P450 IIE1 (aniline as substrate) by 40, 28, 16 and 42 %, respectively. In addition, after 12 days the activities of P450 IIIA1 (ethylmorphine as substrate) and cytosolic sulphobromophthalein glutathione transferase (SBPh-GT) were decreased by 59 and 26 %. The administration of heparin (intramuscularly, 250 ED/kg, in a day, 3 and 6 times) exerted a normalizing effect. The P450 concentration, NADPH oxidation rate and P450 IIB1 activity (amidopyrine as substrate), IIE1 and IIIA1, SBPh-GT and 1-chloro-2,4-dinitrobenzene-GT in microsomes and cytosol exceeded the corresponding values in untreated animals by 31, 40, 68, 224, 68, 42, 24 and 36 %. The administration of heparin to control animals (intramuscularly, 250 and 500 mg/kg, in a day, 5 times) essentially unaffected both the monooxygenase, glucuro- and glutathione-conjugating systems and the elimination of antipyrine (substrate of preferably P-450 IA2) and SBPh (substrate of GT) from rat blood plasma. The experimental results provide evidence for a possible role of endogenous heparin in maintaing the optimal level of the activities of the enzyme systems of xenobiotics microsomal oxidation and conjugation in liver injury. One of the most important functions of the liver is its ability to execute biotransformation of a wide range of xenobiotics and some endogenous substances [1]. The activities of the enzyme systems catalyzing these reactions are under a sophisticated regulatory control. Among the natural factors capable of changing the function of enzymes involved in the xenobiotic biotransformation are vitamins [2], phospholipids [3], hormones [4] and many others. We studied the effect of heparin on the activities of the monooxygenase, glucuro- and glutathione transferase systems of the intact and ultrasound-treated rat liver. The significance of this study consists in the elucidation of a putative participation of heparin in the control of the activities of the enzyme system of xenobiotic biotransformation in the intact liver and under membranous pathology of the organ.