Development of a novel conjugatable sunitinib analogue validated through in vitro and in vivo preclinical settings

Article abstract of DOI:10.1016/j.jchromb.2018.05.050

Sunitinib is an oral FDA/EMEA approved multi-targeted tyrosine kinase inhibitor. It possesses anti-angiogenic and antitumor activity against a variety of advanced solid tumors. However, its chemical core does not allow a potential linkage to tumor-homing elements that could eventually enhance its potency. Therefore, a novel linkable sunitinib derivative, designated SB1, was rationally designed and synthesized. The pharmaceutical profile of SB1 was explored both in vitro and in vivo. Mass spectrometry and NMR spectroscopy were utilized for characterization, while MTT assays and LC-MS/MS validated protocols were used to explore its antiproliferative effect and stability, respectively. Cytotoxicity evaluation in three glioma cells showed that SB1 preserved the antiproliferative effect of sunitinib. SB1 was stable in vitro after 24 h incubation in mouse plasma, while both agents exhibited bioequivalent pharmacokinetic characteristics after i.v. administration in Balb/c mice. To evaluate the levels of SB1 in mouse plasma, a novel analytical method was developed and validated in accordance to the US FDA and the EU EMA guidelines. We formulated a novel linkable sunitinib analog exhibiting similar antiproliferative and apoptotic properties with native sunitinib in glioma cell lines. Both SB1 and native sunitinib showed identical in vitro stability in mouse plasma and pharmacokinetics after i.v. administration in Balb/c mice.

Full text of DOI:10.1016/j.jchromb.2018.05.050

Accepted Manuscript
Development of a novel conjugatable sunitinib analogue validated
through in vitro and in vivo preclinical settings
Mohamed A. El Mubarak, Iliana Leontari, Giannopoulou
Efstathia, Eirinaios I. Vrettos, Abdul kadar Shaikh, Siatis E.
Konstantinos, Charikleia Danika, Haralabos P. Kalofonos,
Andreas G. Tzakos, Gregory B. Sivolapenko
PII:
DOI:
Reference:
S1570-0232(18)30405-7
doi:10.1016/j.jchromb.2018.05.050
CHROMB 21214
To appear in:
Journal of Chromatography B
Received date:
Revised date:
Accepted date:
8 March 2018
30 May 2018
31 May 2018
Please cite this article as: Mohamed A. El Mubarak, Iliana Leontari, Giannopoulou
Efstathia, Eirinaios I. Vrettos, Abdul kadar Shaikh, Siatis E. Konstantinos, Charikleia
Danika, Haralabos P. Kalofonos, Andreas G. Tzakos, Gregory B. Sivolapenko ,
Development of a novel conjugatable sunitinib analogue validated through in vitro and in
vivo preclinical settings. Chromb (2017), doi:10.1016/j.jchromb.2018.05.050
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ACCEPTED MANUSCRIPT
Full-Length Research Paper
Development of a novel conjugatable sunitinib analogue validated through in vitro and in
vivo preclinical settings
Mohamed A. El Mubaraka, Iliana Leontaria, Giannopoulou Efstathiac, Eirinaios I. Vrettosb,
Abdul kadar Shaikhb, Siatis E Konstantinosc, Charikleia Danikaa, Haralabos P. Kalofonosc,
Andreas G. Tzakosb, Gregory, B Sivolapenkoa,*
aDepartment of Pharmacy, Laboratory of Pharmacokinetics, University of Patras, Patras, Greece
bSection of Organic Chemistry and Biochemistry, Department of Chemistry, University of
Ioannina, GR-45110, Ioannina, Greece
cClinical Oncology Laboratory, Division of Oncology, Department of Medicine, University of
Patras, 26504, Rio, Greece
*Corresponding author at: Department of Pharmacy, University of Patras, 26504 Rio-Patras,
Greece. Tel. +302610962323, Fax: +302610969955, E-mail: gsivolap@upatras.gr
Abstract
Sunitinib is an oral FDA/EMEA approved multi-targeted tyrosine kinase inhibitor. It possesses
anti-angiogenic and antitumor activity against a variety of advanced solid tumors. However, its
chemical core does not allow a potential linkage to tumor-homing elements that could eventually
enhance its potency. Therefore, a novel linkable sunitinib derivative, designated SB1, was
rationally designed and synthesized. The pharmaceutical profile of SB1 was explored both in
vitro and in vivo. Mass spectrometry and NMR spectroscopy were utilized for characterization,
while MTT assays and LC-MS/MS validated protocols were used to explore its antiproliferative
effect and stability, respectively. Cytotoxicity evaluation in three glioma cells showed that SB1
preserved the antiproliferative effect of sunitinib. SB1 was stable in vitro after 24 h incubation in
mouse plasma, while both agents exhibited bioequivalent pharmacokinetic characteristics after
i.v. administration in Balb/c mice. To evaluate the levels of SB1 in mouse plasma, a novel
analytical method was developed and validated in accordance to the US FDA and the EU EMA
guidelines. We formulated a novel linkable sunitinib analog exhibiting similar antiproliferative
and apoptotic properties with native sunitinib in glioma cell lines. Both SB1 and native sunitinib
showed identical in vitro stability in mouse plasma and pharmacokinetics after i.v. administration
in Balb/c mice.
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Keywords
Sunitinib; sunitinib conjugatable analogue; in vitro cytotoxicity; in vivo pharmacokinetics;
analytical methodology.
1. Introduction
Sunitinib is an oral FDA/EMEA approved multi-targeted tyrosine kinase inhibitor since 2007. It
has demonstrated anti-angiogenic and direct antitumor activity against a wide variety of
advanced solid tumors. Sunitinib is the standard first-line treatment for metastatic renal-cell
carcinoma (RCC) [1,2] and second-line therapy for patients with advanced gastrointestinal
stromal tumors (GIST) who failed on imatinib [3]. On May 20, 2011, the Food and Drug
Administration (FDA) approved sunitinib for the treatment of progressive well-
differentiated pancreatic neuroendocrine tumors (pNET) in patients with unresectable, locally
advanced or metastatic disease [4]. Currently, sunitinib is also under investigation for the
treatment of other types of cancer [5–7]. Compared to other kinase inhibitors, sunitinib has a
high affinity (i.e. Kd=100 nM) towards a broad range of kinases [8] including: the platelet-
derived growth factor receptors (PDGFR); PDGFR-α, -β and the vascular endothelial growth
factor receptors (VEGFR); VEGFR-1 and-2 [5,6]. The systemic pharmacokinetics of sunitinib
have been characterized in animals [9,10] and humans [11,12]. In accordance with other anti-
tumor agents, a dose-effect association probably also exists for kinase inhibitors [13]. Regarding
sunitinib, it appeared that higher area under the plasma concentration time curves (AUC) of
sunitinib and its active metabolite SU12662, is associated with better outcomes in RCC patients
in terms of response rates, progression-free survival and overall survival [12]. Nevertheless,
higher doses of sunitinib are frequently accompanied by uncontrolled peripheral toxicity,
necessitating dose reductions and interruptions [14]. Sunitinib has been associated with many
side effects including abnormal heart rhythm changes, high blood pressure and hormone
problems (thyroid and adrenal gland problems) [15]. Its usage often results in unusual or
unpleasant taste in the mouth, cough, nausea, vomiting, upset stomach, constipation, dry skin and
changes in skin or hair color. In addition, it is known to cause severe cutaneous toxicities and
cardiotoxicity [14,16,17].
Ideally, a methodology that could be applied to native sunitinib and provide a new molecule with
enhanced cancer-cell recognition, binding and internalization, would pave the way for safer
cancer treatment. Linking sunitinib to small molecules, macromolecules or even more
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complicated structures, which have favorable tumor targeting and affinity properties, would
enhance the population of the cytotoxic agent in tumor cells. However, the chemical structure of
sunitinib does not offer the opportunity for conjugation that would lead to an optimal anti-tumor
agent.
Along these lines, we designed and synthesized a novel linkable sunitinib analogue that allows
the quick and easy cross-linking with various molecules containing different characteristic
groups that could augment the therapeutic profile of sunitinib. Also we explored the in vitro and
in vivo properties of this sunitinib analogue. The newly reported modified molecule is a
carboxylic acid analogue of sunitinib, designated SB1, where the diethylamine moiety of the
native sunitinib has been replaced by a carboxylic acid group. The introduction of the carboxylic
group in the core of sunitinib provides the opportunity for the facile engagement of sunitinib
with molecules with different structures that can eventually increase the tumor homing and
cancer-cell internalization properties of the parent compound. The carboxylic acid of SB1 was
rationally selected from a wide pool of different available functional groups as it could lead to an
ester or amide linked conjugate. The design was based on FDA-approved conjugates, like
Mylotarg, or conjugates in phase II and III clinical trials, like AEZS-108 and ANG1005 [18],
that utilize such linking moieties. Ester and amide bonds are often invoked due to the elevated
levels of esterases and amidases in cancer cells.
Furthermore, we investigated whether this structural modification on sunitinib’s core, altered the
in vitro cytotoxic profile with respect to the parent drug. For this set of experiments, we used
three glioblastoma cell lines. We chose this model because the growth of glioblastoma cells is
tightly related with VEGF expression, a key-target molecule of sunitinib. Recent evidence
support the efficacy of a therapy targeted VEGF/VEGFR [19–21]. To evaluate whether possible
differences exist in the pharmacokinetic profile between SB1 and native sunitinib we conducted
in vivo animal studies. To achieve this we developed a sensitive, accurate and validated LC-
MS/MS analytical method for the detection of SB1 and native sunitinib in mouse plasma. The
method has been validated in agreement with the Guidance for Industry, as specified by the FDA
and EMEA [22,23] under ISO17025 criteria.
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2. Experimental
2.1. Reagents and chemicals
SB1 and native sunitinib have been synthesized with greater than 98% purity by the Laboratory
of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina,
Greece. All the chemicals for the synthesis were of the best grade commercially available (Acros
Organics, Sigma Aldrich, Alfa-Aesar, Fluka and Merck) and were used without further
purification. All the solvents were purified according to standard procedures; dry solvents were
obtained according to the methods described in the literature and stored over molecular sieves.
Analytical thin-layer chromatography was performed on glass plates coated with silica gel
1
containing calcium sulfate binder. The H NMR and 13C NMR spectra were recorded on a
1
Bruker AV-250 (250 MHz) spectrometer in DMSO-d6 and at 298 K. Chemical shifts for H
NMR spectra are reported as δ units of parts per million (ppm) and relative to the signal of
DMSO (δ 2,50 multi). Multiplicities are given as follows: s (singlet); d (doublet); dd (doublet of
doublets); t (triplet); bs (broad singlet); m (multiplet). Coupling constants are reported as J values
in Hz. ESI-MS spectra were measured on a Bruker EVOQ™ Elite ER LC-TQ system. Methanol,
acetonitrile, ethanol, isopropanol, N, N-dimethylsulfoxide (DMSO) and ammonium formate (LC-
MS grade) were purchased from Sigma-Aldrich. Erlotinib was used as internal standard (IS)
(Cayman, Island). Formic acid and hydrochloric acid were purchased from Merck (Germany).
Purified water (18.2 MΩ) was prepared using a Milli Q-Integral system (Merck-Millipore,
Watford, UK). All solvents filtered through 0.22 μm filters (Titan Membrane, Merck Millipore).
2.2. Synthetic procedures
The whole synthetic scheme, as well as the spectra for the final SB1 product can be found in
Supporting Information (Fig. S1- S3).
2.2.1. Synthesis of tert-butyl (E)-2-(hydroxyimino)-3-oxobutanoate (2): Tert-butyl 3-
oxobutanoate (1) (0.0126 mol, 1 eq) was dissolved in 5 mL AcOH at 0 oC. A solution of NaNO2
(0.0252 mol, 2 eq) in 3 mL water was added dropwise for a period of about 10 mins and the
resulting mixture was stirred at RT for 1 h. Water (20 mL) was added to the reaction mixture and
the organic layer was extracted with diethyl ether, washed with aq. NaHCO3, dried over Na2SO4,
filtered and the solvent was removed under reduced pressure to afford 2.3 g of expected product
1
(yield: 97.5%) which was used without further purification. H NMR (250 MHz, DMSO-d6,
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298K) δ: d 13.03 (s, 1H), 2.31 (s, 3H), 1.45 (s, 9H). MS (ESI+) m/z: calcd: 187.2; found: 188.1
[M+H]+.
2.2.2. Synthesis of 2-(tert-butyl) 4-ethyl 3,5-dimethyl-1H-pyrrole-2,4-dicarboxylate (3):
Ethyl 3-oxobutanoate (0.01474 mol, 1.2 eq) was dissolved in 20 mL AcOH. Zn dust (0.00933
mol, 0.76 eq) was added and the resulting slurry was stirred at 60 °C for 5 minutes. At this
temperature, a solution of compound (2) (0.01228 mol, 1 eq) in 5 mL AcOH was added drop
wise and the resulting mixture was stirred at 80 °C for 1 h. The reaction was filtered hot, the
filtrate was poured into ice and the precipitated solid was collected through filtration. The solid
was dissolved in diethyl ether, water (20 mL) was added to and the organic layer was separated,
dried over Na2SO4, filtered and the solvent was evaporated under reduced pressure to afford 1.5
1
g of reddish solid (yield: 45.6%) which was used without further purification. H NMR (250
MHz, DMSO-d6, 298K) δ: 11.61 (br, 1H), 4.16 (q, J=7.14, 14.14 Hz, 2H), 2.42 (s, 3H), 2.39 (s,
3H), 1.51 (s, 9H), 1.26 (t, J=7.13 Hz, 3H). MS (ESI+) m/z: calcd: 267.33; found: 268.1 [M+H]+.
2.2.3. Synthesis of ethyl 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylate (4): Compound (3)
(0.0056 mol, 1 eq) was dissolved in 20 mL TFA and stirred at rt for 30 minutes. The reaction
mixture was cooled to 0 °C and trimethyl orthoformate (0.0084 mol, 1.5 eq) was added to and
stirred at rt for 2 h. TFA was evaporated under reduced pressure and 20 mL water was added to
the residue. The solid obtained was filtered, washed with water and dried under high vacuum to
1
afford 1.02 g of brown solid (yield: 93,3%) which was used without further purification. H
NMR (250 MHz, DMSO-d6, 298K) δ: 12.16 (br, 1H), 9.61 (s, 1H), 4.18 (q, J=7.1, 14.14 Hz,
2H), 2.45 (s, 3H), 2.41 (s, 3H), 1.27 (t, J=7.08Hz, 3H). MS (ESI+) m/z: calcd: 195.22; found:
196.3 [M+H]+.
2.2.4. Synthesis of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (5): KOH (0.010
mol, 2 eq) was dissolved in 9 mL water. Compound (4) (0.005 mol, 1 eq) dissolved in 3 mL
o
methanol was added dropwise and the reaction mixture was refluxed at 80 C for 3 h. The
reaction mixture was then cooled to rt and extracted twice with dichloromethane. The aqueous
layer was adjusted to pH 4 with 10N HCl while keeping the temperature below 15 °C (water
added to facilitate the stirring if needed). Τhe solid was collected by filtration, washed with water
and dried under vacuum to afford 721 mg of brown solid (yield: 86.3%) which was used without
1
further purification. H NMR (250 MHz, DMSO-d6, 298K) δ: 12.11 (br, 2H), 9.60 (s, 1H), 2.44
(s, 3H), 2.40 (s, 3H). MS (ESI+) m/z: calcd: 167.16; found: 168.0 [M+H]+.
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2.2.5. Synthesis of (E)-5-fluoro-3-hydrazonoindolin-2-one (7): 5-fluoroindoline-2,3-dione (6)
(3.634 mmol, 1 eq) was dissolved in 9 mL methanol and hydrazine monohydrate (7.267 mmol, 2
eq) was added to it at rt, stirred for 30 min at rt and refluxed for 12 h. The reaction mixture was
cooled to rt and the solid was filtered, washed with methanol and dried under vacuum to afford
1
640 mg of yellow solid (yield: 98.3%) which was used without further purification. H-NMR
(250 MHz, DMSO-d6, 298K) δ: 10.63 (s, 1H), 6.93-6.89 (m, 2H), 6.82-6.68 (m, 2H), 3.37 (s,
2H) MS (ESI+) m/z: calcd: 179.15; found 180.2 [M+H]+.
2.2.6. Synthesis of 5-fluoroindolin-2-one (8): A mixture of sodium ethoxide (0.0225 mol, 5 eq)
and compound (7) (0.0045 mol, 1 eq) in 8.5 mL ethanol was refluxed for 6 h. The reaction
mixture was cooled to rt, poured slowly into ice and the pH was adjusted to 1 using 4N HCl. The
solid obtained was filtered, washed with water and dried under vacuum to afford 502 mg of
1
reddish solid (yield: 73.6%) which was used without further purification. H-NMR (250 MHz,
DMSO-d6, 298K) δ: 10.36 (s, 1H), 7.08–7.10 (m, 1H), 6.96–7.01 (m, 1H), 6.76–6.79 (m, 1H),
3.49 (s, 2H). MS (ESI+) m/z: calcd: 151.14; found: 151.3 [M+H]+.
2.2.7. Synthesis of (Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-
pyrrole-3-carboxylic acid (9): A mixture of compound (8) (2.40 mmol, 1 eq), compound (5)
(2.28 mmol, 0.95 eq) and piperidine (2 drops) in 20 mL ethanol was refluxed for 3 h. The
precipitated solid was filtered, washed with ethanol and dried under high vacuum. The solid
collected was again refluxed in ethanol for 1 h. The solid was filtered, washed with ethanol and
dried under high vacuum to afford 542 mg of yellow solid (yield: 79,2%) which was used
1
without further purification. H-NMR (250 MHz, DMSO-d6, 298K) δ: 13.69 (s, 1H), 12.1 (br,
1H), 10.90 (s, 1H), 7.75 (d, J=9.6 Hz, 1H), 7.72 (s, 1Η), 6.92 (ddd, J=8.5 Hz, 2.5, 1H), 6.84 (m,
J=3.6, 1H), 2.54 (s, 3H), 2.49 (s, 3H). MS (ESI+) m/z: calcd: 300.29; found: 300.2 [M+H]+.
2.2.8. Synthesis of ethyl (Z)-3-(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-
1H-pyrrole-3-carboxamido)propanoate (10): To the solution of compound (9) (0.599 mmol, 1
eq), EDCI.HCl (1.199 mmol, 2 eq), HOBt (0.719 mmol, 1.2 eq) and β-alanine ethyl ester
hydrochloride (0.719 mmol, 1.2 eq) in 6 mL dry DMF, dry NEt3 (2.397 mmol, 4 eq) was added
at 0 °C and the resulting mixture was stirred at rt for 24 h. Brine (400 mL) was added and the
precipitated solid was collected by filtration, washed with water and used immediately for the
1
final reaction. H-NMR (250 MHz, DMSO-d6, 298K) δ: 13.67 (s, 1H), 10.88 (s, 1H), 7.75 (dd,
J=2.4, 6.9 Hz, 1H), 7.71 (s, 1H), 7.69 (d, 5.6 Hz, 1H), 6.92 (dt, J=2.4, 9.4 Hz, 1H), 6.83 (dd,
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J=4.5, 8.4 Hz, 1H), 4.08 (q, J=7.1, 14.25 Hz, 2H), 3.44 (m, 2H), 2.57 (m, 2H), 2.41 (s, 3H), 2.39
(s, 3H), 1.19 (t, J=7.14 Hz, 3H). MS (ESI+) m/z: calcd: 399.42; found: 400.3 [M+H]+.
2.2.9.
3-(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-
carboxamido) propanoic acid (11): To a solution of (10) (0.117 g, 1 eq) in 11 mL of methanol,
6 mL of 4N solution of LiOH in H2O was added drop wise and the resulting mixture was stirred
at room temperature for 48 h. The mixture was neutralized to pH 7 by the addition of conc. HCl
and then methanol was removed under reduced pressure. The residue was adjusted to pH 3 with
8N HCl and the precipitated solid was filtered, washed with water and dried well to obtain 70 mg
1
of the final SB1 as an orange solid (yield: 64,6%). H-NMR (250 MHz, DMSO-d6, 298K) δ:
13.66 (s, 1H), 10.89 (s, 1H), 7.75 (dd, J=2.0, 7.0 Hz, 1H), 7.70 (s, 1H), 7.67 (t, J=5.4 Hz, 1H),
6.98-6.80 (m, 2H), 3.44 (m, 2H), 2.48 (t, J=1.8 Hz, 2H), 2.41 (s, 3H), 2.39 (s, 3H). MS (ESI+)
m/z: calcd: 371.37; found: 372.4 [M+H]+.
2.3. In vitro experiments
2.3.1. Cell culture
The in vitro experiments were performed using U87, LN18 and M059K glioblastoma cell lines.
All cell lines were purchased by American Type Culture Collection (ATCC). Cells were cultured
in DMEM supplemented with 2 mM L-glutamine. The medium for U87 and M059K cells
contained 10% fetal bovine serum (FBS) (working medium 1 hereafter) and the medium for
LN18 contained 5% FBS (working medium 2 hereafter). All mediums were supplemented with
100 U mL-1 penicillin-streptomycin and 50 μg mL-1gentamycin and cultured at 37 C, 5% CO2
and 100% humidity. The tested agents; SB1and sunitinib were diluted in water otherwise DMSO
was used to a final concentration of 0.5%. In case of dose-dependent experiments, the tested
concentrations were: 0, 1, 2, 5, 10, 50 and 100 μM. All the other experiments were performed
using the IC50 value as was extrapolated from proliferation curves.
2.3.2. MTT assay (2D cell proliferation assay)
To determine whether the tested agents affect cell proliferation, the 3-[4,5-dimethylthiazol-2-yl]-
2,5-dimethyltetrazolium bromide (MTT) assay was used, as previously described [24]. Briefly,
cells were seeded at a density of 8 x103 cells/well in 48-well plates and cells treated as described
above. Forty-eight hours after agents’ application, MTT solution (5 mg/mL in PBS) was added at
a volume equal to 1/10, to each well and incubated for 2 h, at 37 °C. The medium was removed
and 100 μL acidified isopropanol (0.33 mL HCl in 100 mL isopropanol) was added to each well
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in order to solubilize the dark blue formazan crystals. The solution was transferred to 96-well
plates and was immediately read in a microplate reader (Tecan, Sunrise, Magellan 2) at a
wavelength of 570 nm using reference wavelength 620 nm.
2.4. Instrumentation
2.4.1. Chromatographic conditions
Chromatographic analyses were performed on a Waters HPLC system (Alliance HT 2795)
equipped with a temperature-controlled autosampler and a degasser. Chromatographic analysis
was achieved using a Kinetex C8 column 50 mm × 2.1 mm, 2.6 μm, proguard, 2.1 to 4.6 mm
(Phenomenex). The mobile phase was consisted of ammonium formate 5 mM, pΗ = 3.0 (solvent
A) and 0.1% formic acid in acetonitrile/methanol (90/10 v/v) (solvent B). A gradient elution was
started with 10% of solvent B, then increased with a linear increment to 50% until 0.5 min and
maintained for 1.0 min. The elution was then decreased to 10% of solvent B in 1.5 min, and this
composition was maintained until the end of the run (7 min). The column temperature was
maintained at 40 oC throughout all the experiments, whereas the sample temperature was kept at
15 oC. Fifty microliters volume of injection was delivered with a flow rate 0.25 mL min-1.
2.4.2. Mass spectrometric conditions
Micromass Quattro Micro tandem MS system (Waters, Milford, MA, USA), was used for
monitoring the analytes. The mass spectrometer was operated in the positive ion mode, using the
electrospray source. The tuning parameters of SB1, sunitinib and IS, were optimized by infusing
a solution containing 500 ng mL-1 of the analytes with a flow rate of 30 μL min-1 to the mass
spectrometer. The analytes were detected by monitoring the precursor → product ion transition
using Multiple Reaction Mode (MRM) scan mode with 500 ms dwell time for each transition.
The selected transitions m/z were → 372.5>283, 399.5>283 and 394>278 for the SB1, sunitinib
and IS, respectively. The source temperature was 100 °C, desolvation temperature was 300 °C,
desolvation gas flow was 500 L h-1 and cone gas flow was 50 L h-1 were estimated. The capillary
voltage was set at 3.5 kV, while the cone voltage values for SB1, sunitinib and the IS at 20, 29
and 40 V, respectively was optimized. The multiplier was set at 650 V and argon was used as the
collision gas. Lastly, the optimized collision energy of SB1, sunitinib and IS was chosen 17 eV,
27 eV and 30 eV respectively. All data were acquired using the MassLynxv. 4.0 software.
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2.5. Preparation of calibrators and spiked controls
Standard stock solutions of SB1, sunitinib and IS at a concentration of 5 mg mL-1 were prepared
by dissolving in DMSO. Therefore, these solutions were subsequently used to prepare working
solutions (WS) of SB1 and sunitinib (0.025-20 μg mL-1), as well as IS (5 μg mL-1). Calibration
standards (CS) and Quality control (QC) samples were prepared by spiking appropriate amounts
of WS into the blank plasma. Final concentrations of the calibration standards were 0.25, 1, 10,
25, 50, 100 and 200 ng mL-1 and IS was 50 ng mL-1, while the QCs were 5, 75 and 150 ng mL-1.
Calibration standards and QCs were prepared fresh on the day of extraction. All the stock
solutions (WS, CS and QCs) were immediately stored at −20 °C.
2.6. Sample treatment
All plasma samples were thawed at room temperature, vortexed and centrifuged at 2500g for 10
o
minutes at approximately 4 °C. Three hundred microliter of acetonitrile (4 C) were added to an
aliquot of 100 μL of SB1 and sunitinib spiked plasma samples (10 L aliquot of each working
solution of analytes, 10 L of IS working solution, 80 L blank plasma) in order to facilitate the
precipitation of plasma proteins. The solutions were subsequently vortexed for one min and
centrifuged at 10.000g for 10 min (4 °C). The supernatants were transferred into another tube
and the samples were dried on a Centrivap Cold Trap concentrator (Labconco, USA) for ~ 3 h.
The dried extracts were reconstituted in 1 mL of acetonitrile: 5 mM ammonium formate (20:80
v/v) pH 1.5, filtered through RC 0.22 μm filters (Phenomenex) and finally 50 μL of the solution
were injected into the chromatographic system.
2.7. Stability assay
Stability studies of the spiked mouse plasma with SB1 and sunitinib as described above, were
performed under three conditions: (a) after remaining for a week in the refrigerator (4–8 °C), (b)
after 3 freeze-thaw cycles (freeze at −20 °C – thaw at room temperature) and c) after remaining
for 6 months at −20 °C at low (5 ng mL-1), medium (75 ng mL-1) and high (150 ng mL-1)
concentration levels.
Two different concentrations of 5 and 50 ng mL-1 were used to assess the proteolytic stability of
SB1 ex-vivo in mouse plasma, after incubation at 37 °C. Mouse plasma (900 L) were spiked
with 100 L of SB1 (5 and 50 μg mL-1) respectively, gently shaken at 300rpm in a thermo mixer
over a period of 24 h. Following SB1 degradation, the incubation was terminated at specified
time points 0, 0.5, 1, 2, 4, 12 and 24 h by using the same procedure above as shown in 2.5.
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2.8. Pharmacokinetic studies
The kinetics of both SB1 and sunitinib had been studied in Balb/c mice. Overall, 36 adult (3-4
weeks’ old) mice (weighted 25+2 g) had been provided by the Animal House facilities of the
University of Patras. Eighteen (18) mice were injected i.v. (tail vein) with 3 mg/kg SB1, whereas
another group of 18 mice was administered 3 mg/kg native sunitinib (i.v., tail vein). A group of
3 mice was euthanized, and then was sacrificed at predetermined time points: 3, 5, 15, 30, 45 and
60 min. Blood samples were collected via cardiac puncture and immediately transferred in
heparin tubes. The entire experimentation process was carried out in accordance to the European
Communities Council Directive (86/609/EEC) and the Greek National Laws (Animal Act, PD
56/13). All samples were immediately centrifuged at 2500g (4 °C) for 10 min and then stored at
−20 ◦C until analysis. The levels of SB1 and of sunitinib were measured in mouse plasma by LC-
MS/MS.
Winnonlin 5.0.1 was employed to estimate pharmacokinetic parameters utilizing non-
compartmental and two-compartmental analysis model. The area under the plasma
concentration-time curve (AUC) was calculated by the linear trapezoidal rule. In addition,
elimination half-life (t1/2), clearance (CL), Tmax, Cmax and apparent volume of distribution (Vd)
were calculated.
2.9. Method validation
The method was validated according to the bioanalytical method validation guidelines of the US
Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) [22,23]
under ISO17025 criteria for (1) linearity, (2) precision (repeatability and intermediate precision),
(3) accuracy and (4) stability.
3. Results
3.1. In vitro studies
The biological properties of SB1 were investigated in order to verify that the modifications
performed to native sunitinib did not alter its cytotoxic effect. It was found that SB1 exerted an
antiproliferative activity in U87, LN18 and M059K glioma cells as shown in Fig. 1 (A). The IC50
values of SB1 were greater in U87 and LN18 cell lines compared to the IC50 values of native
sunitinib, but they were similar in the case of M059K cells (Fig. 1 (C)). The cytotoxic profile of
the modified sunitinib is similar to that of native sunitinib (Fig. 1 (B)) for one cell line and very
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close for the other two cell lines revealing that the chemical modification applied on native
sunitinib sustained its antiproliferative capacity.
3.2. Method development
3.2.1 Optimization of chromatographic conditions
Chromatographic conditions, especially the composition of the mobile phase, the column type,
the flow rate and the oven temperature were optimized through several trials. It was found that a
gradient elution of a mixture of ammonium formate 5 mM, pΗ = 3.0 (solvent A) and 0.1%
formic acid in acetonitrile/methanol (90/10 v/v) (solvent B) could achieve this purpose, and
therefore was finally adopted as the mobile phase. The Kinetex C8 50 mm × 2.1 mm, 2.6 μm,
o
with a proguard, 2.1 to 4.6 mm (Phenomenex) column operated at 40 C produced a good peak
shape and response, even at the lowest concentration level of the analytes. Herein, the protein
precipitation (PP) technique was employed for sample preparation. PP is helpful in producing a
clean sample and also minimizing the experimental costs. Among the different solvents
investigated alone and in combination for their suitability, acetonitrile was found to be optimal as
it produced a clean chromatogram for the blank sample and yielded the highest recovery for the
analytes from plasma (Fig. 2). A good internal standard must mimic the analyte during extraction
and compensate for any analyte on the column. Erlotinib was found to be the optimal choice for
the quantification of SB1 and native sunitinib, due to its similar extraction characteristics,
retention time, stability and detector response as the analytes.
3.2.2. Isomerization
Sunitinib is an analog of SU5416 (semaxinib) and exhibits light-induced isomerization due to the
presence of a double bond between 2-oxindol and the pyrrole ring. Rotation around this double
bond leads to the existence of two isomers: the E-isomer and Z-isomer [25]. However, when
sunitinib is added into a solution in light, it adopts the E-isomer, which is an inactive form of
sunitinib [26]. Here, we report a suitable method for the determination of SB1 and native
sunitinib in mouse plasma (Fig. 2). In the current work, the isomerization was avoided by
adjusting the pH of the samples and mobile phase at 2.15 and 3.0, respectively. Furthermore, the
temperature of the autosampler adjusted at 15 oC, was of critical importance.
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3.3. Method Validation
3.3.1. Linearity and Sensitivity
Linearity was calculated using the IS method. Calibration standards of SB1 and native sunitinib
concentrations (0.250-200 ng mL-1) were prepared and assayed. The peak area ratios of SB1 and
native sunitinib to the IS against the nominal concentration of calibration standards were used for
plotting the calibration curve. The curve was found to be linear for this range of concentration
with coefficient of determination ≥ 0.999 (Table 1). The sensitivity was measured by using the
lower limit of quantification (LLOQ = 0.25 ng mL-1) of the analytes compared to its blank
plasma sample. It is important to note that the peak area of blank samples should not be more
than 20% of the mean peak area of LLOQ of analytes [23]. The %RSD and %RE were found to
be ≤ 6.51% and ≤ 3.23%, respectively. These data further confirmed that the analytical method
was able to quantify the LLOQ with high accuracy and sensitivity (Fig. 2).
3.3.2. Precision and accuracy
The intra- and inter- day accuracy and precision were determined using spiked samples at three
different concentrations (low = 5 ng mL-1, medium = 75 ng mL-1and high =150 ng mL-1) with
five replicates for each concentration (Table 2). The accuracy (expressed as % relative error) was
found to be ≤ 3.45 and the precision (expressed as a relative standard error) was found to be ≤
5.87 (Table 2). The % coefficient of variation (RE and RSD) should be within 10%.
3.3.3. Selectivity and carry over
Selectivity was evaluated by analyzing mouse blank plasma samples from six different sources
in order to test the interference during the analysis. No significant interference was observed.
Normally, the absence of interfering components is accepted where the response is less than 20%
of the lower limit of quantification for the analyte and 3% for the internal standard. Carry-over
was assessed by injecting blank samples after injecting a high concentration sample, at the upper
limit of quantification. The response of the blank sample at the retention time of the analyte was
<20% of the corresponding peak area of the LLOQ sample. Therefore, the carry-over is
considered acceptable (Fig. 2).
3.3.4. Recovery and matrix effect
Recovery was found to be in the range of 97.0–101.2% for SB1, 93.8–100.8% for native
sunitinib and 104.8% for IS. After the extraction procedures, the recoveries were consistent and
precise between analytes and the IS. Matrix effect was evaluated by analyzing mouse blank
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plasma samples from six different donors in order to test the interference during the analysis. The
matrix effect was assessed by comparing the signals of analytes in plasma with those of analytes
dissolved in mobile phase at the same concentration. The matrix effects were 94.3%, 99.8% and
97.7% for SB1; and 97.5%, 101.6% and 103.4% for sunitinib at three QC concentrations.
The matrix effects were 92.0% for IS. IS normalized matrix effects were 95.6%, 100.1% and
98.6% for SB1; and 101.7%, 104.3% and 103.5% for sunitinib at three QC concentrations.
Negligible matrix effects were observed, indicating consistent suppression of ionization. The
high precision, accuracy, recovery and low matrix effects seen in this assay can be attributed, at
least in part, to the similar structures and behavior of the IS (Fig. 2).
3.3.5. Stability assay
Plasma bench-top (after one week), refrigerated (at 4 C and -20 C) and freeze-thaw stability
results are presented in the Supporting Information (Tables S1-S4). Precision (%RSD < 6.50)
and accuracy (RE < 3.09%) values show an excellent stability of SB1 and sunitinib under the
tested conditions. Indeed, recovery reached ≥ 95 ± 5% and precision and accuracy are lower than
15% [23].
3.3.6. Proteolytic stability
The data obtained for proteolytic stability following time point of 0, 0.5, 1, 2, 4, 12 and 24
h incubation of SB1 in mouse plasma are depicted in Fig. 3. Interestingly, over 98% of the
compound remained completely intact even after 24 h of incubation. This remarkable resistance
of SB1 to proteolysis confers a distinctive advantage in the therapeutic use of the compound.
3.4. Pharmacokinetic studies
The validated method for quantitation of SB1 and native sunitinib in mouse plasma was applied
to pharmacokinetic studies, after a single i.v. administration of 3 mg kg-1 SB1 and native
sunitinib. The in vivo pharmacokinetic parameters were determined by using non-compartmental
and two-compartmental analysis: AUC0-inf was determined by the trapezoidal rule up to the
infinity measured concentration. The maximum concentration (Cmax) of SB1 and native sunitinib
was 213.04 ± 0.79 and 216.14 ± 2.93 ng mL-1, respectively. SB1 and native sunitinib showed an
elimination half-life (t1/2) of 69.01 and 70.81 min, an apparent volume of distribution (Vd) of
358.83 and 360.73 mL and a clearance (CL) of 3.60 and 3.53 mL min-1, respectively (Table 3).
Pharmacokinetic parameters from two-compartmental analysis: AUC0-inf of SB1 and native
sunitinib was 18288.22 and 176207.65 respectively, while the Tmax of both agents was 0.33 min.
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The Cmax was 333.64 ± 1.08 and 318.86 ± 1.34 ng mL-1, t1/2 of 77.45 and 72.07 min, Vd of
173.63, 182.34 mL and CL of 3.28 and 3.41 mL min-1, respectively (Table 4).
From the pharmacokinetic data, it was found that SB1 is identical to native sunitinib (p ≥ 0.05).
Meanwhile, the apparent volume of distribution, the clearance and the AUC indicate that the
structural modification of sunitinib did not affect the pharmacokinetic profile of the molecule in
mouse plasma (Fig. 4).
4. Discussion and Conclusions
Despite extensive preclinical and clinical research, glioblastoma remains among the most
aggressive malignancies. Because glioblastomas are highly vascular tumors, therapies that target
angiogenesis have attracted the interest if scientists and several strategies have been tested
[21,27] i.e. combination of tyrosine kinase inhibitors such sunitinib with new molecules or the
discovery of miRNAs that facilitate the effect of sunitinib in glioblastoma cells. In this paper, the
approach that was applied was the chemical modification of sunitinib in a way that will be able
to be conjugated to tumor homing elements. The sunitinib analogue (SB1) was synthesized in
order to serve as a core molecule that would allow its efficient linkage with other molecules,
which could potentially enhance the tumor localization and specificity of sunitinib. In SB1 a
carboxylic acid group was installed in the core of sunitinib so that it could serve both as a linking
position with tumor targeting elements, but also, after its delivery inside cells and release, the
resulted more polar compound, with respect to the parent sunitinib molecule, to be potentially
trapped inside the targeted cell.
The in vitro and in vivo biological properties of SB1 were explored, with respect to the native
sunitinib. In vitro comparative experiments were conducted in three glioblastoma cell lines and
showed that SB1 preserved the high antiproliferative effect of the native sunitinib (Fig. 1). In
addition, after single i.v. administration in Balb/c mice, we found that both agents exhibit equal
pharmacokinetic characteristics and comparable elimination half-life, volume of distribution,
total body clearance and area under the curve time/plasma concentration (Fig. 4, Table 3).
Moreover, it was found that SB1 remained completely intact to in vitro proteolysis after 24 h
incubation in mouse plasma (Fig. 3).
In order to determine the concentration of the SB1 for the in vitro stability and the in vivo kinetic
studies, a new LC-MS/MS method has been developed. The analytical method was validated in
accordance to the US FDA and the EU EMA guidelines. Linearity was found within a range
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concentration of 0.250 and 200 ng mL-1 with coefficient of determination of ≥ 0.999 (Table 1).
The new analytical method proved precise, accurate, specific and could serve as a simple, rapid
and sensitive way for the detection of SB1 (Tables 1 and 2). Moreover, utilizing the same
methodology for the detection of the reference molecule (native sunitinib) in mouse plasma, the
lower level of detection of sunitinib was improved (0.250 ng mL-1) as compared to the current
bibliography [24-27] (Table 1, Fig. 2).
In conclusion, we rationally designed and constructed an analogue of native sunitinib that
exhibited a similar in vitro anti-tumor effect, as well as in vivo pharmacokinetic profile in Balb/c
mice compared to native sunitinib. Therefore, SB1 can substitute native sunitinib, with the
advantage that it can be easily conjugated to tumor homing carriers and other bespoke molecules.
Therefore, SB1 could constitute the initial impetus for the formulation of a novel type of stable
prodrugs aiding towards the development of more effective and safer versions of sunitinib by
possessing enhanced tumor-targeting properties.
5. Funding
This research project was funded by the “DEDEVAP”, is cofounded by the European Union
(ERDF) and the Greek State under the "SYNERGASIA" action of the OPERATIONAL
PROGRAMME “COMPETITIVENESS II”
6. Acknowledgments
M.A.E.M would like to thank Anastasios Giannou Medical Doctor at University of Patras,
Greece, for supporting my research with his help in the animal experiments.
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Figure 1. The effect of SB1 and native sunitinib in cell proliferation of glioma cell lines. The dose
response curves of SB1 (A) and native sunitinib (B) in U87, LN18 and M059K cell lines were estimated
48 h after agents’ applications to cells. The IC50 values (C) were extrapolated by the aforementioned
curves.
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Figure 2. Representative chromatograms of: (A) Mobile phase (1) and blank mouse plasma (2, 3). (B)
Mouse plasma spiked with SB1, native sunitinib and erlotinib at concentration level 0.25 ng mL-1.
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Table 1
Linearity data (line equation with a standard calibration curve) of SB1 and native sunitinib spiked in
mouse plasma (n = 5 for each concentration level)
Substance
Concentration
(ng mL-1)
Relative
error
LLOD
LLOQ
Line equation
r2
(ng mL-1) (ng mL-1)
SB1
0.25 – 200.00
0.25 – 200.00
0,0218*x+0,011
0,0245*x-0,031
0.999
0.999
0.085
0.085
0.25
0.25
3.23
2.47
Sunitinib
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Table 2
(%) Recovery, relative standard deviation (% RSD) and relative error (RE, %) in different days of SB1
and native sunitinib spiked in mouse plasma (n = 5 at each concentration level)
Theoretical
concentrations
(ng mL-1)
Between-day
(%) Recovery
Within-day
Analyte
Level
RSD
(%)
R.E
(%)
(%)Recovery RSD
(%)
RE
(%)
Low
5.00
75.00
150.00
98.89
98.04
97.03
5.87
2.79
3.74
1.11
0.96
1.97
97.19
97.55
98.34
3.42
5.70
2.95
1.49
3.45
3.15
SB1 Medium
High
Low
Sunitinib Medium
High
5.00
75.00
150.00
97.93
97.44
99.56
4.98
3.25
2.63
2.06
1.62
1.77
97.58
99.89
98.51
4.09
3.25
2.79
1.99
2.78
3.35
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Figure 3. Proteolytic stability of spiked SB1 in mouse plasma at time points 0, 0.30, 1, 2, 4, 12 and 24 h
incubation at 37oC at concentration levels 5 and 50 ng mL-1. The data represent the mean for three
independent experiments.
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Table 3
Non-compartmental pharmacokinetic parameter of SB1 and native sunitinib after a single intravenous
administration (3 mg kg-1) in mice (n = 3).
Analyte
AUC
AUMC
T1/2
(min)
69.01
70.81
Vd
CL
(mL min-1)
3.60
(ng . min mL-1)
16648.51
(ng . min mL-1)
1661945.31
1724874.77
(mL)
SB1
358.83
360.73
Sunitinib
16990.98
3.53
AUC0-inf, the area under the plasma concentration–time curve to time last; AUMC0-inf, the area under the first
moment curve; t1/2, half-life; Vz, apparent volume of distribution; CL, clearance rate;
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Table 4
Two-compartmental pharmacokinetic parameter of SB1 and native sunitinib after a single intravenous
administration (3 mg kg-1) in mice (n = 3).
Analyte
AUC
AUMC
T1/2
Vd
CL
Cmax
Tmax
Css
(ng . min mL-1) (ng . min mL-1)
(min)
77.45
72.07
(mL) (mL min-1) (ng mL-1) (min) (ng mL-1)
SB1
18288.22
17607.65
1994323.40
1787332.02
173.36
182.34
3.28
3.41
333.64
318.86
0.33
0.33
55418.85
53356.51
Sunitinib
AUC0-inf, the area under the plasma concentration–time curve to time last; AUMC0-inf, the area under the first
moment curve; t1/2, half-life; Vd, apparent volume of distribution; CL, clearance rate;
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Figure 4. Concentration–time profiles of SB1 and native sunitinib in mouse plasma at different time
points after single intravenous administration of 3 mg kg-1.
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ACCEPTED MANUSCRIPT
Highlights
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Development of a novel linkable SB1 analogue of native sunitinib.
The biological properties of SB1 was investigated in vitro to verify the
modifications of native sunitinib.
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A validated LC-MS/MS method for SB1 and native sunitinib in mouse plasma
were developed.
The validated method was applied to the pk analysis and stability assay of SB1 in
mouse plasma.
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