- Research Article
- Open Access
Identification and characterization of in vivo, in vitro and reactive metabolites of vandetanib using LC–ESI–MS/MS
© The Author(s) 2018
- Received: 28 January 2018
- Accepted: 19 September 2018
- Published: 24 September 2018
- N-methyl piperidine
- In vivo metabolites
- In vitro metabolites
- Cyano conjugates
Vandetanib (ZD6474) is an available orally inhibitor of vascular endothelial growth factor receptor 2 (VEGFR) . VEGFR has gained great importance as pharmacologic targets as a Tyrosine kinase receptors . Vandetanib, on 6 April 2011, was approved by the FDA for the treatment of patients suffered from symptomatic or progressive medullary thyroid cancer with unresectable, locally advanced, or metastatic disease. It was considered the first drug approved for this case. The trade name of vandetanib was Caprelsa tablets (AstraZeneca Pharmaceuticals LP). Sudden death and QT prolongation of the are severe side effects for vandetanib .
Metabolism is detoxification process of xenobiotics and endogenous compounds by transforming into more hydrophilic compounds to allow excretion outside the body. Drug metabolism work is an essential step in the process of drug discovery, and is usually the factor that evaluate the degree of given drug success to take the approval and to reach the market . Drug metabolism research is done through in vitro and in vivo techniques. In vivo metabolism was performed through the single dose administration of vandetanib to rat using oral gavage followed by gathering of urine samples, at specific time intervals, that contain the drugs and their possible metabolites. In vitro techniques include drugs incubation with various types of in vitro preparations (e.g. hepatocytes and liver microsomes) separated from rats then sample processing and analysis using chromatographic techniques.
Phase I metabolism either in vitro or in vivo of cyclic tertiary amines generates oxidative metabolites including: α-carbonyl formation, ring opening metabolites, N-oxygenation, ring hydroxylation and N-dealkylation. Metabolites are often less toxic than parent molecules, but occasionally undergo bioactivation forming unstable reactive intermediates that considered more toxic in comparison to parent molecules [5–7]. Reactive metabolites can covalently bind to proteins, which is considered the initiating step in the process of drug-induced organ toxicities [8, 9].
N-methyl piperidine ring is a part of vandetanib chemical structure that is considered a cyclic tertiary amine. Drugs that contain cyclic tertiary amine group are able to form iminium intermediates which are hard nucleophiles [10–12]. GSH or its derivatives are not the appropriate as capturing agent for hard nucleophiles while potassium cyanide (KCN) is the best agent for trapping these reactive intermediate including iminium ion _ENREF_7  resulted in stable adducts formation which can be characterized, separated and detected using LC–MS/MS [13, 14].
Since bioactivation is often considered the central reason for observed side effects including phototoxicity and prolongation of QT interval [3, 15], we tested the reactive metabolites formation by incubation of vandetanib with 1.0 mM KCN. Upon literature review, N-demethyl vandetanib, vandetanib N-oxide and glucuronide conjugate were found in plasma, urine, and feces . The full mechanism of bioactivation of vandetanib is not yet reported.
List of chemicals and materials
LC Laboratories (MA, USA)
Acetonitrile (ACN, HPLC-grade), ammonium formate (NH4COOH), poly ethylene glycol 300 (PEG 300), dimethyl sulfoxide (DMSO), potassium cyanide (KCN) and formic acid (HCOOH)
Eurostar Scientific Ltd. (Liverpool, UK)
Water (HPLC grade)
Milli-Q plus purification instrument (USA)
The experimental animal care center at King Saud University (KSA)
Vandetanib (20 µmol/mL) was incubated at with RLMs (1.0 mg/mL), NADPH (1.0 mmol/mL) and K/Na phosphate buffer (50 mmol/mL, pH 7.4) containing MgCl2 (3.3 mmol/mL). Incubation was done at thermostatted shaking water bath (37 °C) for 60 min before the reactions were quenched using two mL of ACN (ice-cold). The incubation mixtures were centrifuged at 14,000 rpm for 12 min then the supernatants were collected then subjected to dryness under a stream of N2. Samples residues were reconstituted in mobile phase (95% solvent A and 5% solvent B). The same steps were repeated using a trapping agent (KCN at 1.0 mmol/mL) to capture reactive intermediates forming adducts.
In vivo metabolism of vandetanib
Six male Sprague–Dawley rats of average weight (340 g) and 4 weeks of age were brought from animal house of King Saud University (Riyadh, KSA). Each rat was housed in special metabolism cage that was placed in animal care facility in a 12-h light/dark cycle (7:00–19:00). Rats had free access to standard water and animal food. Rats were maintained in metabolism cages for 72 h before study starting. Vandetanib was formulated in special solution (5% Tween 80, 4% DMSO, 30% PEG 300, HPLC H2O) to allow dispersion of vandetanib. Each rat received a calculated vandetanib depending on its weight.
So the dose for rat was 30.8 mg/kg. Rats were given a single calculated dose of vandetanib. One rat was used as a control and was given solvent without vandetanib. Oral gavage was used for vandetanib dosing to rats. Urine samples were collected after draining into compartments fixed to metabolism cages before vandetanib dosing as control sample and at specific time periods (6, 12, 18, 24, 48, 72, 96 and 120 h) following vandetanib dosing. Filtration of Urine samples was done using 0.45 µm syringe filters for discarding of particulate matters in the urine. A similar volume of ACN was added to each collected urine sample and then the resulted mixture was shaken by vortexing for 1 min. After storing the mixture at 4 °C overnight, two solvent layers (upper organic layer and lower aqueous layer) were formed. Both layers were evaporated until dryness under stream of N2 and reconstituted respectively in 1 mL of mobile phase and transferred to HPLC Agilent vials for LC–MS/MS analysis. Control urine samples obtained from rats before drug dosing were done in the same way described for sample purification method. These samples were analyzed by LC–MS/MS to obtain control chromatograms.
Optimized parameters of the established LC–MS/MS methodology
Agilent 1200 (Agilent Technologies, CA, USA)
Agilent 6410 QqQ (Agilent Technologies, CA, USA)
Mobile phase (gradient)
Aqueous phase: 10 mM Ammonium formate in H2O (pH: 4.1 using Formic acid)
Positive electrospray ionization (ESI)
Organic phase: ACN (0.1% Formic acid)
Drying gas: N2 gas
Pressure (55 psi)
Flow rate (12 L/min)
Flow rate: 0.2 mL/min
Elution time: 90 min
Agilent eclipse plus C18 Column
ESI temperature: 350 °C
Capillary voltage: 4000 V
Internal diameter (mm)
N2 (high purity)
Particle size (μm)
Product ion (PI) and full mass scan and
22 ± 2 °C
Mass Hunter software
Vandetanib, in vivo, in vitro and reactive metabolites
Fragmentor voltage (V)
Post time 15
Collision energy (eV)
Identification of in vitro metabolites, in vivo metabolites and cyano conjugates of vandetanib
Extracted ion chromatograms (EICs) for the vandetanib proposed metabolites were used to identify metabolites in the total ion chromatogram (TIC) of ether RLMs incubation extract or urine extract. CID of proposed metabolites molecular ion peaks (MIP) of was performed in the collision cell to get product ion (PI) mass spectra. Structures of metabolites were done by reconstructing the product ions. In vivo vandetanib-related metabolites were concentrated in the organic layer while endogenous components of the urine and highly polar metabolites were located in the aqueous layer.
Identification of in vitro phase 1 vandetanib metabolites
Phase I metabolites of Vandetanib using MS scan and PI scan
Major daughter ions
Proposed conjugate composition
Previously detected (reference)
V + H
V − CH2 + H
N-demethylation and α oxidation
V − CH2 + O + H
V + O − 2H + H
N-demethylation and 2 α oxidation
V − CH2 + 2O + H
V + O + H
V + O + H
Identification of vandetanib and VA475 metabolite
Identification of VA461 metabolite
Identification of VA489 metabolite
Identification of VA91a and VA491b metabolite
Characterization of vandetanib reactive metabolites
Vandetanib cyano conjugates
Postulated conjugate composition
α Cyano addition and N-demethylation
V − CH3 + CN
α Cyano addition
V + CN
N-demethylation, α oxidation and α Cyano addition
V − CH3 + CN + O
484.3, 361.3, 287.4, 203.1
N-demethylation, α hydroxylation and α Cyano addition
V − CH3 + CN + OH
Identification of VB486 cyano conjugate of vandetanib
Identification of VB500a and VB500b cyano conjugates of vandetanib
Identification of VB502 cyano conjugate of vandetanib
Bioactivation mechanism of vandetanib
Identification of vandetanib in vivo metabolites
PI mass spectra comparison between control urine samples with urine extracts as well as PI comparison of vandetanib and proposed metabolites (Table 3) permitted the identification of four in vivo phase I and one phase II metabolites. Metabolic reactions for in vivo phase I metabolites were proposed to be N-oxide formation, N-demethylation and α-hydroxylation while for phase II metabolites were the result of N-conjugation of vandetanib with glucuronic acid. In vivo vandetanib phase I metabolites are previously mentioned in in vitro vandetanib phase I metabolism.
Excretion of vandetanib and its in vivo metabolites in rat urine
Phase II vandetanib in vivo metabolites: glucuronic acid conjugates
MWA AAK, HWD, and SMA established the experimental design. MWA run the research. MWA, HWD, SMA and AAK analyzed the data. HWD, NSA and MWA wrote the first draft of the manuscript. SMA, AAK and NSA contributed in editing the language of the manuscript. SMA, HWD and AAK follow up the research steps. NSA made proofreading of the manuscript. All authors read and approved the final manuscript.
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at the King Saud University for funding this work through the Research Group Project No. RGP-322.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
Animal Care Center Guidelines at College of Pharmacy of King Saud University were followed. Use Committee and Local Animal Care of King Saud University approved maintenance of rats.
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