Investigating the in vitro metabolism of fipexide: characterization of reactive metabolites using liquid chromatography/mass spectrometry

Abstract

The in vitro metabolism of the nootropic drug fipexide was studied using different liquid chromatography/mass spectrometry (LC/MS) techniques. This drug has been withdrawn from the market due to toxic effects. No previous reports have investigated the possible involvement of reactive metabolites in the toxicity of fipexide. The hydrolysis of this drug leads to the formation of two potentially toxic species, 3,4-methylenedioxybenzylpiperazine (MDBP) and 4-chlorophenoxyacetic acid (4-CPA). Here, we investigate the in vitro metabolism of fipexide in human, rat, mouse and dog, as well as of MDBP and 4-CPA in human and rat, while focusing on the formation of reactive metabolites. A combination of LC/MS analyses on a hybrid quadrupole-linear ion trap instrument and accurate mass data from QqTOF measurements was employed for the characterization of these metabolites. Microsomal metabolites of fipexide were MDBP, 4-CPA, fipexide N-oxide or hydroxyl, demethylenated fipexide and other minor ones, all of which were investigated by tandem mass spectrometry. Reactive metabolites were detected using several trapping procedures with small molecules such as glutathione, its ethyl ester derivative and N-acetylcysteine. The demethylenated metabolite, a catechol, formed its corresponding ortho-quinone, which readily reacts with these nucleophiles. MDBP was studied in a similar manner, due to its ability to form an analogous catechol. Because of its acidic nature, 4-CPA was assessed for possible acylglucuronide and acyl-CoA thioester metabolites, which could also be involved in bioactivation pathways. Several important metabolites were identified as potential mediators of toxicity via protein binding.

Induced Chiroptical Changes of a Water‐Soluble Cryptophane by Encapsulation of Guest Molecules and Counterion Effects

“…The next test compound was one that has recently been reported to form GSH adducts in microsomal and hepatocyte incubations 26, 27. This nootropic drug was designed and used as a treatment for cognitive disorders,28, 29 but was quickly withdrawn from the market due to severe and unexplained toxicity 30, 31.…”

“…This nootropic drug was designed and used as a treatment for cognitive disorders,28, 29 but was quickly withdrawn from the market due to severe and unexplained toxicity 30, 31. Since then, in vitro studies have shown the possibility of linking this toxicity to the formation of reactive metabolites26 and covalent binding to proteins 27. The reactive metabolite identified in microsomal incubations is the catechol derivative of fipexide, which can easily be further oxidized to a quinone intermediate and subsequently attacked by GSH.…”

Investigating the in vitro metabolism of veratridine: Characterization of metabolites and involved cytochrome P450 isoforms

“…Examples of such drugs include phenytoin [27] and tamoxifen [28]. The catechols are unstable and very difficult to detect without additional agents, such as superoxide dismutase (SOD) or ascorbate, to inhibit any further oxidation into their corresponding ortho-quinones [29]. In the present study, protonated molecule ions at m/z 646 and 642, loss of 28 Da (m/z 674 → 646) and 32 Da (m/z674 → 642) from veratridine were detected and the peaks were weak (data not shown).…”

Phenyl group participation in rearrangements during collision-induced dissociation of deprotonated phenoxyacetic acid

“…Furthermore, CID of phenoxyacetate, p ‐chlorophenoxyacetate and deprotonated efaproxiral gave a second product ion which was in each case attributed to decarboxylation. The structures of the product ions were assigned as the corresponding aryloxymethanide ions (ArylO–CR 2 – , R = H or Me) consistent with heterolytic cleavage of the C α –CO 2 – bond .…”