CHAPTER 9

Sunitinib Malate
Mohammed Gabr Kassem,* A.F.M. Motiur Rahman,*
and Hesham M. Korashy†

Contents 1. Introduction 364

1.1. Nomenclature 365
1.1.1. Systematical chemical names 365
1.1.2. Nonproprietary names 365
1.1.3. Proprietary names 365
1.2. Formulae 365
1.2.1. Empirical formula, molecular weight,
and CAS number 365
1.2.2. Structural formula [7-9] 365
1.2.3. Smiles 366
1.3. Elemental analysis 366
1.4. Physical properties 366
1.4.1. Appearance 366
1.4.2. Solubility 366
1.4.3. Melting point 366
1.4.4. Stability 366
1.4.5. Dissociation constant 366
1.4.6. Partition coefficient (P) 366
1.5. Uses and applications 366
2. Methods of Preparation 367
2.1. Synthesis of sunitinib 1 367
3. Physical Properties 369
3.1. Spectroscopy 369
3.1.1. Ultraviolet spectroscopy 369
3.1.2. Vibrational spectroscopy 369

* Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Kingdom of
Saudi Arabia
{
Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh,
Kingdom of Saudi Arabia

Profiles of Drug Substances, Excipients, and Related Methodology, Volume 37 # 2012 Elsevier Inc.
ISSN 1871-5125, DOI: 10.1016/B978-0-12-397220-0.00009-X All rights reserved.

363
364 Mohammed Gabr Kassem et al.

3.1.3. Nuclear magnetic

resonance spectrometry 370
3.2. Mass spectrometry 373
3.2.1. Fragmentation pattern of sunitinib 374
4. Methods of Analysis 374
4.1. Chromatographic methods 374
4.1.1. High-performance liquid chromatography
(ultraviolet detection) 374
4.1.2. High-performance liquid
chromatography/mass spectrometry 376
4.1.3. Ultra-performance liquid
chromatography/mass spectrometry 381
4.2. Immunoassay methods 381
4.3. Spectrofluorimetry 382
5. Pharmacology 382
5.1. Pharmacokinetics 383
5.1.1. Absorption 384
5.1.2. Distribution 384
5.1.3. Metabolism and excretion 384
Acknowledgement 385
References 385

1. INTRODUCTION
Sunitinib maleate is a multitargeted tyrosine kinase inhibitor that inhibits
tumor cell proliferation and angiogenesis. It is approved for the treatment
of renal cell carcinoma (RCC) and imatinib-resistant gastrointestinal
stromal tumor (GIST) [1–4]. Its activity in other tumor types (such as
hepatocellular carcinoma or non-small cell lung cancer) is currently
being investigated in numerous clinical trials (see www.clinicaltrials.
gov). Sunitinib is given orally, once daily as a 50-mg capsule over
4 weeks, followed by a 2-week rest period, in repeated 6-week treatment
cycles. At this dose, the most frequently observed adverse effects are
fatigue, hypertension, diarrhea, stomatitis, and hand–foot syndrome [4].
Following oral administration, sunitinib is slowly absorbed from the
gastrointestinal tract, reaching maximum plasma concentrations after
about 6–12h. It is primarily metabolized by CYP 3A4 to its active
N-desethyl metabolite (SU12662) and is subject to presystemic metabo-
lism by this enzyme [5]. In a mass balance study in humans with
14
C-labeled sunitinib, 61% of the radioactive dose was recovered in feces
and 16% in urine. In plasma samples, sunitinib and SU12662 accounted
for 71% and 20.5% of the total radioactivity, respectively [6].
Approximately 95% (90%) of sunitinib (SU12662) is bound to plasma
proteins. Because of the long terminal half-lives of sunitinib and SU12662
 Sunitinib Malate 365

(40–60h, and 80–110h, respectively), steady-state concentrations are not

achieved until 2 weeks of continuously daily dosing [2].

1.1. Nomenclature
1.1.1. Systematical chemical names

N-[2-(Diethylamino)ethyl]-5-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-
indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide
hydrogen (2S)-2-hydroxybutanedioate [7]

N-[2-(Diethylamino)ethyl]-5-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-
indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide [7,8]

5-[5-Fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl] -1H-pyrrole-
3-carboxilic acid (2-diethylaminoethyl)amide [8]

(2S)-2-Hydroxybutanedioic acid-N-[2-(diethylamino)ethyl]-5-[(Z)-(5-
fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-
1H-pyrrole-3-carboxamide (1:1) [9]

1.1.2. Nonproprietary names

Sunitinib maleate

1.1.3. Proprietary names

SutentÒ, Pfizer Inc., SU11248Ò [7–11]

1.2. Formulae
1.2.1. Empirical formula, molecular weight, and CAS number

Sunitinib maleate C26H33FN4O7 532.56 (341031-54-7) [7–9]

Sunitinib base C22H27FN4O2 398.47 (557795-19-4) [7–9]

1.2.2. Structural formula [7-9]

HN H
O
N N
F OH
HO
O
O OH O
N
H

Sunitinib malate
366 Mohammed Gabr Kassem et al.

1.2.3. Smiles
CCN(CC)CCNC(¼¼O)C1¼¼C(C)NC(\C¼¼C2/C(¼¼O)NC3¼¼C2C¼¼C(F)
C¼¼C3)¼¼C1C.O¼¼C(O)CC(O)C(¼¼O)O

1.3. Elemental analysis

Base: C 66.31%, H 6.83%, F 4.77%, N 14.89% [7]
Malate salt: C 58.62%, H 6.22%, F 3.54%, N 10.47%

1.4. Physical properties

1.4.1. Appearance
Malate salt: yellow powder
Base: orange solid [8]

1.4.2. Solubility
Malate salt: Soluble in dimethyl sulfoxide at 40mg/mL and very poorly
soluble in ethanol and water

1.4.3. Melting point

Malate salt: 194–200  C
Base: 189–191  C

1.4.4. Stability
Sunitinib malate is a photosensitive substance [12].

1.4.5. Dissociation constant

pKa of base: 9.30 [13,14].

1.4.6. Partition coefficient (P)

For the water–octanol system: log P¼2.5 [15]

1.5. Uses and applications

Sunitinib is an oral multitargeted tyrosine kinase inhibitor with antiangio-
genic and antitumor activities attributable to the inhibition of several related
tyrosine kinase receptors, including vascular endothelial growth factor
receptors (VEGFRs) types 1 and 2 (FLT1 and FLK1/KDR), platelet-derived
growth factor receptors (PDGFR-and PDGFR-b), stem cell factor receptor
(c-KIT), and Fms-related tyrosine-3 receptor (FLT3), which are implicated in
tumor proliferation, angiogenesis, and metastasis [1,4,5,16,17].
 Sunitinib Malate 367

2. METHODS OF PREPARATION

2.1. Synthesis of sunitinib 1 [18–21]

The discovery route to sunitinib 1, as described by Sun and coworkers,
commenced with a two-step synthesis of 5-fluorooxindole 10 starting
from the corresponding 5-fluoroisatin [22,23]. Heating a neat mixture of
5-fluoroisatin and hydrazine hydrated to 110  C effected a Wolff–Kishner
reduction and ring opening to give acyl hydrazine. The crude material
was subjected to an intramolecular acylation reaction by exposure to
aqueous HCl at room temperature to afford the oxindole 10 in 73%
yield over two steps (Scheme 9.1).
The requisite pyrrole subunit was prepared using the Knorr pyrrole
synthesis (Scheme 9.2) [23,24]. tert-Butyl acetoacetate 2 was reacted with
sodium nitrite in acetic acid to furnish oxime 3. Exposure of this interme-
diate to ethyl acetoacetate 4 under reductive cyclization conditions using
zinc and acetic acid afforded the tetra-substituted pyrrole 5 in 65% yield
over two steps. Selective hydrolysis of the tert-butyl ester, followed by
decarboxylation, was accomplished by stirring 5 in HCl and ethanol to
provide intermediate 6 in 87% yield. The unsubstituted position of pyr-
role 6 was then formulated in quantitative yield by treatment with Vils-
meier reagent generated from DMF and POCI3 in dichloromethane.
Hydrolysis of the ethyl ester using aqueous potassium hydroxide in
methanol provided the key intermediate 17 in 94% yield, thus setting
the stage for the introduction of the amide side chain and the final
coupling reaction with oxindole 10. The CDI-mediated amidation of
carboxylic acid 7 with diamine 8 afforded the desired amide 9. With
sufficient quantities of 9 in hand, the synthesis could be completed via
condensation of 9 with 5-fluorooxindole (10) to furnish the sunitinib free
base 1 in 88% yield.
Sunitinib was also synthesized using the route described below
(Scheme 9.3). Treatment of diketene with N,N-diethylethylenediamine in
tert-butyl methyl ether furnished â-ketoamide 12 in excellent yield [25].
Oxime 3, derived from tert-butyl acetoacetate, was treated with amide 12
in the presence of zinc and acetic acid according to the classic Knorr
pyrrole formation conditions, which led to pyrrole 14. The a-free pyrrole
thus obtained 14 was treated with chloromethylene dimethylammonium

O H
F F N F
NH2NH2.H2O NH2 HCl, H2O
O O
111 °C O 73 %, 2 steps N
N NH2
H 10 H

SCHEME 9.1 Synthesis of 5-fluorooxindole 10.

368 Mohammed Gabr Kassem et al.

O O
O O O O OEt
HN
NaNO2 4O O
O O
HOAc Zn/HOAc, O
2 NOH 3 71% (2 steps) O
5

OEt HN OH 1. CDI, THF

HCl, EtOH HN 1. DMF, POCl3,

2. KOH O 2. H2N NH2

87 % O
6 3. HCl O 8
93 % 7

H N
H F HN N
NEt2
HN N O
N O
10 H F
O
O
N 80 % from 7 1
N
Et2N 9 H

H N
HN N

L-malic acid O OH
F COOH
1-Butanol HOOC
O 1-malate
H2O N
H

SCHEME 9.2 Synthesis of sunitinib malate 1-malate via condensation of amide 9 and
5-fluorooxindole 10.

O 3, Zn, HOAc, 53 %,
t-BuOMe O O or
NEt2
+ H2N N
8 rt N 3, H2, Pd/C, 45 psig,
O 11 93 % H 12 65–70 ⬚C, 77 %

H N H Cl
H N –
H2SO4 HN N Cl
HN N
MeOH 14 N
t-BuO + 15
13 O
O 100 %
O CH3CN

H N
HN N
H F
N
HN N O
O
N 10 F
H H
O O
1
N 16 KOH, 74 % N
+ H
–
Cl

SCHEME 9.3 Synthesis of sunitinib free base 1 from Vilsmeier adduct 16 and 5-fluor-
ooxindole 10.
 Sunitinib Malate 369

chloride 15 in acetonitrile to form the Vilsmeier adduct 16 in situ [26].

Addition of 5-fluorooxindole (10) and KOH to the reaction mixture at
this stage afforded the desired product 1.

3. PHYSICAL PROPERTIES

3.1. Spectroscopy
3.1.1. Ultraviolet spectroscopy
The ultraviolet/visible (UV/VIS)-absorption spectrum of sunitinib was
recorded, and the wavelength of the maximum absorption peak (lmax) was
noted. The absorption spectrum of sunitinib in ethanol was scanned from
200 to 600nm, using a UV/VIS spectrometer (Varian Cary 50 spectropho-
tometer). As shown in Fig. 9.1, the lmax of sunitinib is located at 430nm.

3.1.2. Vibrational spectroscopy

3.1.2.1. Infrared absorption spectroscopy of sunitinib base The principal
peaks in the IR spectrum (NaBr pellet sampling) of sunitinib base were
reported at 3298, 3230, 2968, 1676, 1627, 1590, 1544, 1498, and 1334cm1 [18].

3.1.2.2. Infrared absorption spectroscopy of sunitinib malate The infrared

absorption spectrum of sunitinib is shown in Fig. 9.2 and was obtained on
a Shimadzu infrared spectrophotometer using a NaCl plate. The principal
peaks were observed at 3350, 2943, 2831, 1650, and 1031cm1.

1.0

0.8

0.6
l max = 430 nm
Abs

0.4
l max = 265 nm

0.2

0.0

200 300 400 500 600

Wavelength (nm)

FIGURE 9.1 UV spectra of sunitinib malate (4.0mM solution in ethanol).

370 Mohammed Gabr Kassem et al.

120

100

80 l max = 1650
l max = 2831

l max = 2943
%T

60

40
l max = 3350

20
l max = 1031

3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600

FIGURE 9.2 Infrared spectroscopy of sunitinib malate.

TABLE 9.1 Infrared spectroscopic data for sunitinib malate in NaCl plate

Entry Bond Absorption peaks (lmax cm1) Appearance

1 CF 1031 Strong

2 NHC¼¼O 1650 Normal
3 CH (alkyl) 2831 Strong
4 HC¼¼CH (aryl) 2943 Strong
5 OH (acid), NH 3350 Broad

Assignments for the major absorption bands are provided in Table 9.1.
Reported principal peaks were at 3298, 3230, 2968, 1676, 1627, 1590, 1544,
1498, 1334cm1 [18].

3.1.3. Nuclear magnetic resonance spectrometry

1
H and 13C nuclear magnetic resonance (NMR) spectra of sunitinib were
obtained with a Bruker 500MHz NMR spectrometer. Chemical shifts
were expressed in parts per million (ppm) (Table 9.2) with respect to the
tetramethylsilane reference signal for 1H and 13C NMR (Figs. 9.3 and 9.4,
respectively).

3.1.3.1. 1H NMR spectrum 1H NMR (500MHz, DMSO-d6): d 13.73 (s, 1H,

NH), 10.93 (s, 1H, NH), 10.80–10.70 (br, 1H, OH, maleate), 7.77 (d, 1H,
J¼8.0Hz), 7.73 (s, 1H, olefinic H), 7.72 (d, 1H, J¼8.0Hz), 6.93 (d, 1H, J¼8.0
TABLE 9.2 Comparative study of 1H NMR spectra for sunitinib malate with literature

Chemical shifta (sunitinib malate)

Entry (500MHz, DMSO-d6) Chemical shiftb (400MHz, CDCl3) Chemical shiftc (300MHz, CDCl3)

1 13.73 (s, 1H, NH) – 13.31 (s, 1H, NH)

2 10.93 (s, 1H, NH) – 8.90 (s, 1H, NH)
3 10.80–10.70 (br, 1H,  OH, malate) –
4 7.77 (d, 1H, J¼8.0Hz) 7.75 (dd, J¼9.4, 2.5Hz, 1H), 7.14 (dd, 1H, J¼8.8, 2.2Hz)
5 7.73 (s, 1H, olefinic H) 7.71 (s, 1H) 7.22 (s, 1H, olefinic H)
6 7.72 (d, 1H, J¼8.0Hz) 7.43 (t, J¼5.6Hz, 1H) 6.85 (td, 1H, J¼8.8 2.2Hz)
7 6.93 (d, 1H, J¼8.0Hz) 6.92 (td, J¼9.1, 2.5Hz, 1H) 6.78 (dd, 1H, J¼8.1, 4.4Hz)
8 6.87 (d, 1H, J¼8.0Hz) 6.84 (dd, J¼8.5, 4.6Hz, 1H) 6.72 (t, 1H, J¼4.4Hz)
9 4.01 (s, 1H, malate) – –
10 3.48 (br, 2H) 3.41–3.36 (m, 2H) 3.54 (q, 2H, J¼5.2Hz)
11 2.96 (s, 6H) 2.65–2.58 (m, 6H) 2.71 (t, 2H, J¼5.9Hz), 2.64 (q, 4H, J¼6.6Hz)
12 2.56 (q, 1H, malate)
13 2.34 (q, 1H, malate)
14 2.51 (s, 3H) 2.47 (s, 3H) 2.55 (s, 3H)
15 2.44 (s, 3H) 2.43 (s, 3H) 2.31 (s, 3H)
14 1.14 (t, J¼7.3Hz, 6H) 1.07 (t, J¼7.1Hz, 6H) 1.05 (t, 6H, J¼7.4Hz)
a 1
H NMR was taken in DMSO-d6.
b
Reference [18].
c
Reference [20].
 372

14

210
1.00 13.737

13

200 190
180
12
176.081
172.113

170
169.542
165.158

11
1.38 10.931

FIGURE 9.4
FIGURE 9.3
159.133

160
157.271

13
10
136.806 1.56
134.544

150 140
130.173
127.107

9
127.032

130
125.812
Mohammed Gabr Kassem et al.

124.800
119.927 7.779

120
8
114.890 7.763

Expended area 105–177 ppm

112.543 3.10
112.348 7.731
110.058 7.709
109.991

ppm
6.939

7
106.066

110 100
105.860 2.00 6.923
6.860

90
6

80
70
66.385 5
4.009
3.476

60
50.541
46.676 2.970
4

40.914 1.02
2.958

50
39.968
39.802 2.12 2.575
39.635 2.559

40
3

39.468 6.11 2.544

39.301
39.134 8.94 2.511

30
38.968 2.468
35.150 1.22
2

H NMR spectra of sunitinib malate in DMSO-d6.

2.449

C NMR spectra of sunitinib malate in DMSO-d6.

20
2.366
13.419
10.618 2.343

10
9.752 6.31 2.335
1

1.144
1.078
1.063
0
 Sunitinib Malate 373

Hz), 6.87 (d, 1H, J¼8.0Hz), 4.01 (s, 1H, malate), 3.48 (br, 2H), 2.96 (s, 6H),
2.56 (q, 1H, malate), 2.34 (4, 1H, malate), 2.51 (br, 2H), 1.14 (s, 6H)ppm.

3.1.3.2. 13C NMR spectrum Unreported 13C NMR data of sunitinib malate
are as follows: 13C NMR (125MHz, DMSO-d6): d 176.08 (malate), 172.11
(malate), 169.54, 165.16, 159.13, 157.27, 136.80, 134.54, 130.17, 127.10,
127.03, 125.81, 124.80, 119.93, 114.89, 112.54, 112.35, 110.06, 109.99,
106.07, 105.86, 66.38 (malate), 50.54, 46.68, 40.91 (malate), 35.16, 13.42,
10.62, and 9.75ppm.

3.2. Mass spectrometry

The mass spectral analysis of sunitinib was carried out with an Agilent
6320 Ion Trap LC/MS system by infusion of 2mg of sunitinib solution in
1:1 acetonitrile/water without a column. Smart fragmentor and automatic
optimization were performed to obtain the spectra and cause fragmenta-
tion. Source parameters were as follows: temperature was 350  C, gas flow
was 12L/min, and the nebulizer was set at 60psi. Figure 9.5 shows the
mass spectrum for the parent compound (m/z 398); Scheme 9.4 and
Figs. 9.6–9.8 show the detailed mass fragmentation pattern interpretation
of the drug substance. The reported m/z was 399.2204 (MþHþ),
C22H27FN4O2þH (theoretical 399.2196) [27].
Sunitinib malate shows 398.7 (m/z) as a metastable ion peak (Fig. 9.5).

H HN H
HN
N N
N N
F
F O
O MS O
O
. C4H6O5 N
N H
H

Molecular weight: 532.56 m/z 398.7

398.7

317.7
0
300 350 400 450 500 550 600 650 m/z

FIGURE 9.5 MS spectra for fragment sunitinib malate molecular weight 532.56 in
positive mode gave m/z 398.47 [M-malate]þ.
374 Mohammed Gabr Kassem et al.

HN H HN H
N N N N
F F
O MS O MS
2
O O
N . C4H6O5 N
H H
MW 532.56 MW 398.47

HN H HN HN
+
N
F F
O O MS3 O
O and O + O + and
N N N
H H H
MW 326.34 MW 283.28 MW 264.28
3
MS

HN HN +
HN
+ + +
+ and + and + and
+ O +
+ N
N N
MW 247.27 MW 219.26 MW 126.13 MW 94.13

SCHEME 9.4 Fragmentation of sunitinib malate.

3.2.1. Fragmentation pattern of sunitinib

MS2 scan of the metastable ion peak gives two fragments at 398.7 (m/z, by
loss of diethylamine) and 282.7 (m/z, by loss of N,N-diethyethylene
diamine), respectively (see Fig. 9.6).
MS3 scan of the fragment at 398.7 (m/z) also gave 282.7 (m/z, by loss of
ethylamine) (see Fig. 9.7).
MS3 scan of the 284.7 (m/z) gave the number of fragments majoring
264.7 (m/z), 247.8 (m/z), 217.8 (m/z), 128.4 (m/z), and 94.6 (m/z), respec-
tively (Fig. 9.8).

4. METHODS OF ANALYSIS

4.1. Chromatographic methods

4.1.1. High-performance liquid chromatography (ultraviolet detection)
There are a few high-performance liquid chromatography (ultraviolet
detection) (HPLC/UV) methods published to determine sunitinib, either
as a single component or simultaneously with its metabolite. In the
method developed by Blanchet et al. [28], after a liquid–liquid extraction
with ethyl acetate, sunitinib and ranitidine (the internal standard (IS)) are
separated on cyanopropyl column using a simple binary mobile phase of
ammonium acetate buffer (20mM, pH 6.8):acetonitrile (55:45, v/v). Sam-
ples were eluted isocratically at a flow rate of 1mL/min throughout the
10-min run. A dual wavelength mode was used, with ranitidine
 Sunitinib Malate 375

+MS2(398.7), 0.5min #28

6 HN H
+
N
F
HN H O
O
N N N m/z 325.9
F H
O 2 325.9
O MS
5
Intens.⫻10

N and
4 H m/z 398.7
HN

F
O
O +
N
H m/z 282.7
2
282.7

0
50 100 150 200 250 300 350 m/z

FIGURE 9.6 MS2 spectra for fragment 398.7 (m/z) in positive mode gave 325.9 (m/z) and
282.7 (m/z).

monitored at 255nm and sunitinib at 431nm. The calibration was linear

over the range of 20–200ng/mL. Inter- and intraday coefficients of varia-
tion were less than 7%. This method is sensitive, accurate, and selective. It
has been successfully implemented to monitor trough sunitinib concen-
trations in plasma samples (n¼39) from 14 unselected cancer patients
treated with the recommended once-daily dose of 50mg or less. It has
been concluded that this method can be used in routine clinical practice to
monitor plasma sunitinib concentrations in cancer patients treated with
once-daily administration.
Etienne-Grimaldi et al. [12] developed an original, simple, HPLC assay
with UV detection, allowing the simultaneous measurement of sunitinib
and SU12662 to be performed in plasma after a single organic extraction.
This simple and robust analytical assay could be helpful in the setting of
pharmacokinetically based dose adaptation for sunitinib. In addition, a
stability study of sunitinib and SU12662 in different light exposure con-
ditions was presented. HPLC analysis was performed on an ODS column,
and UV detection was monitored at 369nm (run time: 15min). The IS was
vandetanib. The mobile phase was composed of 60% ammonium formate
20mM (pH 3.25, adjusted with formic acid) and 40% acetonitrile. The flow
rate was 0.8mL/min. This assay was selective and sensitive enough (limit
of detection: 1ng/mL) to quantify minimal concentrations at steady
state (Css min) of sunitinib and SU12662 in treated patients.
376 Mohammed Gabr Kassem et al.

HN H HN
+
N
F F
O MS3 O
O O +
N N
H H
m/z 325.9 m/z 282.7

Intens. +MS3(398.7®325.9), 0.6 min #19

2000

1500

1000 282.7

500

175.5 267.7

50 100 150 200 250 300 350 m/z

3
FIGURE 9.7 MS spectra for fragment 398.7 (m/z) in positive mode gave 282.7 (m/z).

4.1.2. High-performance liquid chromatography/mass spectrometry

Several coupled high-performance liquid chromatography/mass spec-
trometry (LC-MS/MS) methods have been reported to determine suniti-
nib, either as a single drug or in combination with other tyrosine kinase
inhibitors and/or its metabolite SU12662 in plasma [29–34].
Minkin et al. [29] developed and validated an assay using LC-MS/MS
for the determination of sunitinib in human plasma. The assay’s lower
limit of quantitation (LLOQ) is 0.2ng/mL, with a simple sample prepara-
tion procedure and a rapid chromatographic run time of 3min. Sample
preparation involved a liquid–liquid extraction by the addition of 0.2mL
of plasma with 4.0mL tert-butyl-methyl ether extraction solution contain-
ing 25ng/mL of the IS clozapine. Separation of compounds was achieved
on a C18 (502.1mm i.d., 3.5mm) analytical column using a mobile phase
 Sunitinib Malate 377

+MS2(282.7), 0.7 min #33

1200

HN HN HN
F 3 +
1000 O O MS O O and + and
+ O
N N
H m/z 264.7 N
H m/z 282.7 m/z 247.8
HN
+ HN
800 and + and +
N
N
m/z 217.8 m/z 128.4 m/z 94.6
Intens.

600

247.8

128.4
400 264.7
217.8
150.6

199.5
106.5 176.7
200

226.3 284.8
94.6 190.4 300.3
163.5 324.0

50 100 150 200 250 300 350 m/z

FIGURE 9.8 MS3 spectra for fragment 282.7 m/z in positive mode.

consisting of acetonitrile/water (65:35, v/v) containing 0.1% formic acid

and isocratic flow at 0.15mL/min for 3min. The analytes were monitored
by tandem-mass spectrometry with electrospray positive ionization. The
mass spectrum of sunitinib showed a protonated molecular ion [MHþ] at
m/z 399.0. The major fragment observed was at m/z 283.0, which was
selected for subsequent monitoring in the third quadrupole. The mass
spectrum of the IS, clozapine, showed a [MHþ] at m/z 327.0, and the high
collision energy gave a major product ion at m/z 270.0. Linear calibration
curves in human plasma were generated over the range of 0.2–500ng/mL
with values for the coefficient of determination of exceeding 0.9950.
Within- and between-day precision and accuracy were less than or
equal to 10%. The method was applied to the quantitation of sunitinib
in plasma samples from a patient receiving daily oral therapy with
sunitinib.
A fast, sensitive, universal, and accurate LC-MS/MS method for the
determination of four different tyrosine kinase inhibitors (TKIs) from
biological materials was developed by Honeywell et al. [30]. Utilizing a
simple protein precipitation with acetonitrile, a 20-mL sample volume of
biological matrixes can be extracted at 4  C with minimal effort. After
centrifugation, the sample extract is introduced directly onto the LC-
MS/MS system without further cleanup and assayed across a linear
378 Mohammed Gabr Kassem et al.

range of 1–4000ng/mL. Chromatography was performed using a Dionex

Ultimate 3000 with a Phenomenex prodigy ODS3 (2.0100mm, 3mm)
column and eluted at 200mL/min with a tertiary mobile phase consisting
of 20mM ammonium acetate/acetonitrile/methanol (2.5:6.7:8.3%). Injec-
tion volumes varied from 0.1 to 1mL depending on the concentration of
the drug observed. Samples were observed to be stable for a maximum of
48h after extraction when kept at 4  C. Detection was performed using a
turbo-spray ionization source and mass spectrometric positive multiple
reaction monitoring (þMRM) mode for gefitinib (447.1 and 127.9 m/z),
erlotinib (393.9 and 278.2 m/z), sunitinib (399.1 and 283.1 m/z), and
sorafenib (465.0 and 251.9 m/z) at an ion voltage of þ3500V. The accuracy,
precision, and limit of quantification (LOQ) from cell culture medium
were as follows: gefitinib: 100.23.8%, 11.2nM; erlotinib: 101.63.7%,
12.7nM; sunitinib: 100.84.3%, 12.6nM; sorafenib: 93.93.0%, 10.8nM,
respectively. This was reproducible for plasma, whole blood, and
serum. The method was observed to be linear between the LOQ and
4000ng/mL for each analyte. Effectiveness of the method was illustrated
with the analysis of samples from a cellular accumulation investigation
and determination of steady-state concentrations in clinically treated
patients.
Haouala et al. [31] developed a liquid chromatography-tandem-mass
spectrometry method (LC-MS/MS), requiring 100mL of plasma for the
simultaneous determination of the six major inhibitors of TKI kinases
imatinib, nilotinib, dasatinib, sunitinib, sorafenib, and lapatinib. Plasma
is purified by protein precipitation, and the supernatant is diluted in
ammonium formate 20mM (pH 4.0). Reverse-phase chromatographic
separation of TKIs is obtained using a gradient elution of 20mM ammo-
nium formate, pH 2.2 and acetonitrile containing 1% formic acid, fol-
lowed by rinsing and re-equilibration to the initial solvent composition
up to 20min. Imatinib-d8 was used as the IS.
Analyte quantification, using matrix-matched calibration samples, is
performed by electrospray ionization (ESI)-triple quadrupole mass spec-
trometry by selected reaction monitoring detection using the positive
mode. The method was validated according to FDA recommendations,
including assessment of extraction yield, matrix effects variability
(<9.6%), overall process efficiency (87.1–104.2%), as well as TKIs short-
and long-term stability in plasma. The method is precise (interday CV%:
1.3–9.4%), accurate (9.2 to þ9.9%), and sensitive (lower limits of quanti-
fication comprised between 1 and 10ng/mL). This is the first broad-range
LC-MS/MS assay covering the major currently in-use TKIs. It is an
improvement over previous methods in terms of convenience (a single
extraction procedure for six major TKIs, reducing significantly the analyti-
cal time), sensitivity, selectivity, and throughput. It may contribute to filling
the current knowledge gaps in the pharmacokinetics/pharmacodynamics
 Sunitinib Malate 379

relationships of the latest TKIs developed after imatinib and better define
their therapeutic ranges in different patient populations in order to evalu-
ate whether a systematic TDM-guided dose adjustment of these anticancer
drugs could contribute to minimize the risk of major adverse reactions and
to increase the probability of efficient, long-lasting, therapeutic response.
A high-performance LC-MS/MS method was developed and vali-
dated by Haznedar [32] for measurement of sunitinib and SU12662 con-
centrations in rat plasma samples. The samples were prepared for
analysis either by an ethyl acetate extraction (used in only a few initial
studies) or after protein precipitation (in test tubes or 96-well plates). The
assay used either propranolol or [2H10]-sunitinib as an IS. The concentra-
tion range of either 1–2000 or 0.1–200ng/mL was validated using a
Keystone BetaBasic-18 column (4.69100mm, 5L) for LC-MS/MS anal-
ysis (Shimadzu Series 10 ADVP HPLC system and Perkin-Elmer Sciex
API-3000 triple quadrupole mass spectrometer). The gradient mobile
phases were 10mM ammonium acetate in methanol/water (10:90) with
0.1% formic acid (A) and 10mM ammonium acetate in methanol/water
(90:10) with 0.1% formic acid (B). The flow rate was 0.8mL/min, which
was reduced to 0.3mL/min before introduction into the mass spectrome-
ter. The mass spectrometer was operated using ionization and scan
modes of positive ion electrospray and MRM, respectively. Each analyte
was detected by tandem MS by monitoring specific molecular ion frag-
ment ion transitions: m/z 399.1 and 325.9 for sunitinib, 371.1 and 283.1 for
SU12662, and 360.1 and 116.0 for propranolol. Calibration curves were
constructed by plotting the analyte/IS peak area ratios against the analyte
concentrations. A weighted linear regression (1/C2) was used to calculate
the concentrations of sunitinib and SU12662 in the study and QC samples.
This assay method demonstrated acceptable selectivity/specificity, line-
arity, and intra- and interassay precision and accuracy. The blank matrix
also showed no significant interference or carryover on the LC-MS/MS
system. The LLOQ was 0.1ng/mL for both sunitinib and SU12662.
Alternatively, a Symmetry Shield C8 column (2.1 9 50mm, 3.5 l) was
used to perform the chromatographic separation. The mobile phase was
15mM ammonium formate buffer solution (pH 3.25)/acetonitrile (75:25,
v/v), with a flow rate of 0.35mL/min. The mass spectrometer was oper-
ated using MRM (m/z for sunitinib and SU12662 were as described above;
m/z 409.1, 325.9 for IS) and in a positive ion mode. The lower and upper
limits of quantitation were 0.07 and 241ng/mL for sunitinib and 0.07 and
220ng/mL for SU12662. This method also showed acceptable assay spec-
ificity/selectivity, linearity, sensitivity, precision, accuracy, and stability.
Radioactivity in rat and monkey blood, plasma, urine, and fecal samples
was measured by liquid scintillation counting (Packard 1900 or 2100 TR
Liquid Scintillation Counter). The counting efficiencies were calculated
by the external standard method, using a series of quenched standards
380 Mohammed Gabr Kassem et al.

(supplied by Packard), generating a calibration curve. The purpose of

these extensive nonclinical studies was to assess pharmacokinetics and
dispositional properties of sunitinib and its primary active metabolite
(SU12662). Sunitinib was administered in single and repeat oral doses in
mice, rats, and monkeys. Assessments were made using LC-MS/MS
methods, radioactive assays, and quantitative whole body autoradiogra-
phy. Sunitinib was readily absorbed with good oral bioavailability and
linear kinetics at clinically-relevant doses. SU12662 plasma levels were
less than those of sunitinib in mice and monkeys, but greater in rats.
Sunitinib was extensively distributed with moderate-to-high systemic
clearance and eliminated primarily into feces. Single- and repeat-dosing
kinetics were similar. A prolonged half-life allowed once-daily dosing,
enabling adequate systemic exposure with limited-to-moderate accumu-
lation. In multiple-dose studies with cyclic dosing, drug plasma concen-
trations cleared from one cycle to the next. Sunitinib exhibited
advantageous pharmacokinetic and dispositional properties in nonclini-
cal species, translating into favorable properties in humans.
A LC-MS/MS-based method combined with protein precipitation,
liquid–liquid extraction, and solid-phase extraction techniques was
developed by Zhou et al. [33] for the determination of sunitinib in
mouse plasma, brain tumor, and normal brain tissue, respectively. The
instrument was operated under the MRM mode using ESI in the positive
ion mode. Separation was achieved on a 502.0mm Luna 3mm C8 column
(Phenomenex Inc., Torrance, CA, USA) with a pre-column of the same
material. The sample solutions (10mL) were injected, and the analytes
were eluted using acetonitrile/1mM ammonium acetate containing
0.1% acetic acid (28:72, v/v) at a flow rate fixed at 0.3mL/min. The
isocratic separation run was completed within 3.2min at 30  C. Camp-
tothecin was used as the IS. A good linear relationship with correlation
coefficients greater than 0.99 was achieved over concentration ranges of
1.37–1000ng/mL for plasma and 4.12–1000ng/g for the normal brain and
brain tumor. The LOQs for sunitinib in mouse plasma, brain tumor, and
normal brain tissue are 1.37ng/mL, 4.12ng/g, and 4.12ng/g, respec-
tively. The reproducibility of the LC-MS/MS method is reliable, with
the intra- and interday precision being less than 15% and accuracy within
15%. The established method was successfully applied to the character-
ization of sunitinib disposition in the brain and brain tumor as well as its
systemic pharmacokinetics in a murine orthotopic glioma model.
In a thesis having the title ‘‘Pharmacokinetic/Pharmacodynamic Mod-
eling and Simulation of Biomarker Response to Venlafaxine and Sunitinib
Administration’’ [33], Sunitinib and SU12662 concentrations in human
plasma were determined by HPLC coupled with mass spectrometry (LC-
MS/MS, Applied Biosystems/MDS Sciex API5000 LC-MS/MS). This
work was performed by Dr. Martina Kinzig at the Institute for Biomedical
 Sunitinib Malate 381

and Pharmaceutical Research (IMBP) under the supervision of Prof.

Dr. Fritz Sörgel. The method used d5-sunitinib as an IS which was synthe-
sized by Elsinghorst [21]. The response from calibration standards of
sunitinib and SU12662 was linear from 0.06 to 100ng/mL. The LLOQ
for plasma samples was 0.06ng/mL for both analytes. Coefficients of
variation (CV) expressing precision and relative errors (RE) expressing
analytical accuracy ranged from 1.0% to 5.3% and þ0.9% to þ5.6% for
sunitinib and from 2.9% to 3.9% and 2.2% to þ3.9% for SU12662,
respectively.

4.1.3. Ultra-performance liquid chromatography/mass spectrometry

Simultaneous determination of nine TKIs in plasma samples by 96-well
solid-phase extraction and ultra-performance LC-MS/MS published by
Bouchet et al. [35]. The described chromatography was performed on a
Waters Acquity-UPLCÒ system with BEH C18-502.1mm column, under
a gradient of ammonium formate–acetonitrile. An Acquity–TQDÒ system
with ESI was used for detection. Samples were prepared by solid-phase
extraction (OasisÒ MCX mElution), and eluate was injected in the system.
Calibration curves ranged from 10 to 5000ng/mL for imatinib, its metabo-
lite, nilotinib, lapatinib, erlotinib and sorafenib, and from 0.1 to 200ng/mL
for dasatinib, axitinib, gefitinib, and sunitinib. Peaks of each compound
(retention time from 0.76 to 2.51min) were adequately separated. The mean
relative extraction recovery was in the range of 90.3–106.5% thanks to the
use of stable isotopes (imatinib-d8, nilotinib-Mþ4, dasatinib-Mþ6, and
sorafenib-Mþ4) as ISs. There was no significant ion suppression observed
at the respective TKI retention times. This rapid, sensitive, and specific
UPLC-MS/MS method is able to perform simultaneous quantification of
nine TKIs in human plasma and usable for routine TDM.

4.2. Immunoassay methods

Establishment of a homogeneous time-resolved fluorescence immunoas-
say method for high-throughput screening of protein TKIs was reported
by Li and coworkers [36]. Specific fluorescence signals at 670 and 612nm
were measured by multifunctional microplate reader when the fluores-
cence was emitted through a resonance energy transfer between fluores-
cent materials (XL-665 and Eu3þ). The inhibitory activity of sunitinib, a
standard PTK inhibitor, on vascular endothelia growth factor receptor
2 (VEGFR-2) kinase activity was investigated. A homogeneous time-
resolved fluorescence immunoassay was established for high-throughput
screening of PTK inhibitor. In this system, the concentrations of VEGFR-2,
adenosine triphosphate (ATP), and polypeptide substrate were 5ng/mL,
100mm/L, and 50mm/L, respectively. Sunitinib inhibited VEGFR-2 kinase
activity with an IC50 value of 86.7nmol/L, which was close to the values
382 Mohammed Gabr Kassem et al.

tested using the other method. The homogeneous time-resolved fluores-

cence immunoassay can be easily used for high-throughput screening of
PTK inhibitors.

4.3. Spectrofluorimetry
Arıcı et al. [37] described a validated spectrofluorimetric method for the
determination of sunitinib malate, using a dye complexation approach. The
method was developed for the indirect determination of sunitinib malate in
bulk substance and in pharmaceutical preparations. The complex formed in
this reaction system was regarded as the ion-association complex between
the drug cation and Eosin Y. This method has the advantages of simplicity,
sensitivity, and reproducibility and is useful and convenient for quality
control and routine determination of drugs in pharmaceutical dosage
forms where precision, time, and cost effectiveness of analytical methods
are important. The dye complexation spectrofluorimetric quantification of
sunitinib malate was performed by monitoring the emission of the free dye
at 800nm, following its excitation at 350nm. The described method was fully
validated as to the analytical parameters of accuracy, linearity, specificity,
limit of detection, limit of quantification, precision, reproducibility,
and robustness. The described method displayed linearity over the concen-
tration range of 0.08–5.00mg/mL. The limit of detection was found to be
0.041mg/mL (7.69108 M), and the limit of quantitation was 0.850mg/mL
(1.59106 M). Defined parameters of the method (such as medium pH,
dissolved oxygen concentration, temperature, dye concentration., incuba-
tion time, etc.) were varied systematically to check robustness. Reproduc-
ibility was tested by applying the proposed method to the assay of sunitinib
malate using the same operational conditions, namely in two different
laboratories at different elapsed time and using different instruments.
Results obtained from lab-to-lab variations were found to be reproducible,
as RSD values did not exceed 2%.

5. PHARMACOLOGY

Sunitinib malate (SutentÒ, SU11248) is an oral, multitargeted TKI that

specifically inhibits vascular endothelial growth factor receptors 1,
2 and 3 (VEGFR1, -2 and -3, respectively), platelet-derived growth factor
receptor alpha and beta (PDGFR-a and -b), FLT3 colony-stimulating
factor receptor type 1 (CSF-1R) and the receptor encoded by the ret
proto-oncogene (RET) [38,39]. Sunitinib is approved for first line treat-
ment of metastatic renal cell carcinoma (mRCC) and imatinib-resistant
metastatic GIST [5,40,41]. Sunitinib is metabolized by cytochrome P450
(CYP) 3A4 to in active metabolites [42].
 Sunitinib Malate 383

Clinical pharmacokinetics of sunitinib show a high inter-patient varia-

bility (approximately 40%), which is largely unexplained [5]. This could
result in supra- or sub-therapeutic sunitinib levels leading to toxicity or
inefficacy, respectively. Since sunitinib is predominantly metabolized by
CYP3A4, variability in the activity of this enzyme may explain a consid-
erable proportion of the observed inter-patient variability in sunitinib
pharmacokinetics.
It is thought that sunitinib is neither an inhibitor nor an inducer of
CYP-enzymes and therefore the drug is considered not prone to drug-
drug and drug-food interactions, while other TKIs (e.g. imatinib, erlotinib,
gefitinib) appear to be substrates and/or inhibitors of several CYP-
enzymes in vivo and in vitro [1,33,43–47]. For sunitinib, in vivo confirma-
tory studies to define an effect of sunitinib on CYP-enzymes are lacking.
Moreover, recently it was shown in an in vitro study that sunitinib is a
substrate for and an inhibitor of the transporter proteins ATP-binding
cassette (ABC) ABCG2 and to some extent ABCB1, which may also lead to
drug–drug interactions [48].
Mechanism of Action. Sunitinib malate is a small molecule that
inhibits multiple receptor tyrosine kinases (RTKs), some of which are
implicated in tumor growth, pathologic angiogenesis, and metastatic
progression of cancer. Sunitinib was evaluated for its inhibitory activity
against a variety of kinases (>80 kinases) and was identified as an inhibi-
tor of platelet-derived growth factor receptors (PDGFRa and PDGFRb),
vascular endothelial growth factor receptors (VEGFR1, -2, and -3), stem
cell factor receptor (KIT), Fms-related tyrosine-3 receptor (FLT3), CSF-1R,
and the glial cell line-derived neurotrophic factor receptor [48]. Sunitinib
inhibition of the activity of these RTKs has been demonstrated in bio-
chemical and cellular assays, and inhibition of function has been demon-
strated in cell proliferation assays. The primary metabolite exhibits
similar potency compared to sunitinib in biochemical and cellular assays.
Sunitinib inhibited the phosphorylation of multiple RTKs (PDGFRb,
VEGFR2, KIT) in tumor xenografts expressing RTK targets in vivo and
demonstrated inhibition of tumor growth or tumor regression and/or
inhibited metastases in some experimental models of cancer. Sunitinib
demonstrated the ability to inhibit growth of tumor cells expressing
dysregulated target RTKs (PDGFR, RET, or KIT) in vitro and to inhibit
PDGFRb- and VEGFR2-dependent tumor angiogenesis in vivo [49].

5.1. Pharmacokinetics
Pharmacokinetic investigations in both healthy volunteers and cancer
patients have shown that following a single oral dose, peak plasma
sunitinib concentrations occur between 6 and 12h post dose [49]. In
addition, sunitinib and SU12662 have previously been shown to display
384 Mohammed Gabr Kassem et al.

linear pharmacokinetics and have prolonged half-lives of approximately

40 and 80h, respectively [50,51]. Sunitinib is well absorbed [51], its bio-
availability is not affected by food intake [49,50,52], and no significant
changes in pharmacokinetics are observed with repeat versus single
dosing [49].
No differences in pharmacokinetics have been observed between
healthy volunteers and cancer patients in individual studies [49,53].
However, when analyzed across multiple studies, factors such as patient
status, age, gender, race, body weight, and clinical performance status
may affect the pharmacokinetics of sunitinib in individuals, resulting in
increased or decreased exposure to sunitinib, SU12662, or total drug.

5.1.1. Absorption
The maximum plasma concentration of sunitinib is achieved within 6–12
h, and the absolute bioavailability is unknown. The drug may be taken
with or without food, since food only has a marginal effect on the expo-
sure [52]. The inter-patient variability is large [5]. A recent case report
describes a significant decrease in sunitinib exposure area-under-the-
curve (AUC) in an obese patient, which might indicate that body mass
index has a pronounced effect on drug exposure and might thereby
explain partly the large inter-patient variability [54].

5.1.2. Distribution
The apparent volume of distribution (Vd/F) for sunitinib was 2230L. In
the dosing range of 25–100mg, the area under the plasma concentration–
time curve and Cmax increases proportionately with dose. The terminal
half-lives of sunitinib and its primary active metabolite are approximately
40–60 and 80–110h, respectively [5]. Binding of sunitinib and its primary
active metabolite to human plasma protein in vitro was 95% and 90%,
respectively, with no concentration dependence in the range of 100–4000
ng/mL. With repeated daily administration, sunitinib accumulates 3- to
4-fold, while the primary metabolite accumulates 7- to 10-fold. Steady-
state concentrations of sunitinib and its primary active metabolite are
achieved within 10–14 days.

5.1.3. Metabolism and excretion

Sunitinib is primarily metabolized by CYP3A4 to produce its primary
active metabolite, SU12662, which is further metabolized by CYP3A4 into
inactive metabolites [52]. The primary active metabolite comprises 23–37%
of the total exposure. Data on additional enzymes involved in the metabo-
lism are still unknown.
Sunitinib is primarily eliminated with the feces (61%), with renal
elimination accounting for only 16% of the administered dose. However,
there are no studies on the pharmacokinetics in patients with serious
 Sunitinib Malate 385

hepatic or renal insufficiency [42]. Additionally in a case report describing

two hemodialyzed patients on sunitinib therapy, the plasma concentra-
tion of the drug and its major metabolite at steady-state were comparable
to patients with normal renal function [55].

ACKNOWLEDGEMENT
This work was supported by the College of Pharmacy Research Center, King Saud
University.

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