Identification and characterization of in vivo, in vitro and reactive metabolites of vandetanib using LC-ESI-MS/MS

Abstract

Vandetanib (Caprelsa tablets, VNT) is an orally inhibitor of vascular endothelial growth factor receptor 2. The current research reports the characterization and identification of in vitro, in vivo and reactive intermediates of VNT. In vitro metabolites of VNT were performed by incubation with rat liver microsomes (RLMs). Extraction of vandetanib and its in vitro metabolites from the incubation mixtures were done by protein precipitation. In vivo metabolism was done by giving one oral dose of vandetanib (30.8 mg/kg) to Sprague Dawley rats in metabolic cages by using oral gavage. Urine was gathered then filtered at certain time intervals (0, 6, 12, 18, 24, 48, 72, 96 and 120 h) from vandetanib dosing. A similar volume of ACN was added to each collected urine sample. Both layers (organic and aqueous) were injected into liquid chromatography electro spray ionization tandem mass spectrometry (LC-ESI-MS/MS) to detect in vivo vandetanib metabolites. N-methyl piperidine ring of vandetanib is considered a cyclic tertiary amine that undergoes metabolism forming iminium intermediates that are very reactive toward nucleophilic macromolecules. Incubation of vandetanib with RLMs in the presence of 1.0 mM KCN was made to check reactive metabolites as it is usually responsible for noticeable idiosyncratic toxicities including phototoxicity and QT interval prolongation. Four in vivo phase I, one in vivo phase II metabolites, six in vitro phase I metabolites and four cyano conjugates of vandetanib were detected by LC-MS/MS. In vitro and in vivo phase I metabolic reactions were N-oxide formation, N-demethylation, α-carbonyl formation and α-hydroxylation. In vivo phase II metabolic reaction was direct conjugation of vandetanib with glucuronic acid. All metabolic reactions occurred in N-methyl piperidine of vandetanib which causes toxicity and instability of vandetanib.

Keywords: Cyano conjugates; In vitro metabolites; In vivo metabolites; N-methyl piperidine; Vandetanib.

Fig. 1

EIC of MIP at m/z 475 showing two peaks; vandetanib (50.3 min) and VA475 (54.8 min) (a), PI mass spectrum of vandetanib (b) and PI mass spectrum of VA475 at m/z 475 (c)

Scheme 1

Proposed CID of vandetanib

Scheme 2

Proposed CID of VA475

Fig. 2

EIC of MIP at m/z 461 showing one peak (VA461) at 49.7 min (a) and PI mass spectrum of VA461 at m/z 461 (b)

Scheme 3

Proposed CID of VA461

Fig. 3

EIC of MIP at m/z 489 showing two peaks: VA489a (66.8 min) and VA489b (67.9 min) (a), PI mass spectra of VA489a (b) and VA489b at m/z 489 (c)

Scheme 4

Proposed CID of VA489a

Scheme 5

Proposed CID of VA489b

Fig. 4

PI chromatogram of MIP at m/z 491 showing two peaks: VA491a (57.1 min) and VA491b (67.4 min) (a), PI mass spectra of VA491a (b) and VA491b at m/z 491 (c)

Scheme 6

Proposed CID of VA491a

Scheme 7

Proposed CID of VA491b

Fig. 5

PIs mass spectrum of VB486

Scheme 8

Proposed CID of VB486

Fig. 6

PI chromatogram of MIP at m/z 500 showing two peaks: VB500a (68.4 min) and VB500b (75.9 min) (a), PI mass spectra of VB500a (b) and VB500b (c)

Scheme 9

Proposed CID of VB500a

Scheme 10

Proposed CID of VB500b

Fig. 7

PI chromatogram of MIP at m/z 502 showing one peak (VB502) at 77.1 min (a), PI mass spectrum of VB502 (b)

Scheme 11

Proposed CID of VB502

Scheme 12

Bioactivation mechanism of piperidine ring of vandetanib

Fig. 8

Overlayed PI chromatograms of vandetanib (58.3 min) and VC475 (68.6 min) (a), VC461 (57.2 min) (b) and VC491a (56.5 min) and VC491b (67.0 min) (c)

Fig. 9

PI mass spectrum of VC651

Scheme 13

Proposed CID of VC651

Fig. 10

Proposed in vitro metabolites, in vivo metabolites and cyano conjugates of vandetanib